PEPTIDE CONJUGATES, CONJUGATION PROCESS, AND USES THEREOF

TECHNICAL FIELD

The present invention relates to peptide conjugates, methods for making peptide conjugates, conj ugates produced by the methods, pharmaceutical compositions comprising the conjugates, methods of eliciting immune responses in a subject and methods of vaccinating a subject, uses of the conj ugates for the same, and uses of the conjugates in the ma nufacture of medicaments for the same. The present invention also relates to methods of making compounds useful in the synthesis of peptide conj ugates of the invention and to such componds. BACKGROUND ART

Synthetic peptide vaccines genera lly comprise a synthetic copy of an immunogenic part of protein antigens. This approach to vaccine development has a number of adva ntages, including ease of synthesis, avoidance of potentially toxic biological by-products and straightforward characterisation. A key issue in the development of peptide vaccines is the lack of immunogenicity displayed by peptides as sole vaccine components. It is usua lly necessary to include in the vaccine a n adj uvant, designed to activate components of the innate immune system (e.g . Freund's adjuvant) .

An alternative strategy in peptide vaccine design is to create self-adjuvanting vaccines in which the peptide epitope of interest is covalently linked to a n appropriate adjuvant. Such self-adj uvanting vaccines may have enhanced a ntigen uptake, presentation and dendritic cell maturation compared to simple co-formulation of the antigen with an external adjuvant.

Several self-adjuva nting vaccines have been developed, but preparation of the vaccines can be complicated .

There is an ongoing need for new self-adjuvanting vaccines and new methods of making self-adjuvanting vaccines. It is an object of the present invention to go some way towards meeting these needs; and/or to at least provide the public with a useful choice.

Other objects of the invention may become apparent from the following description which is given by way of example only.

Any d iscussion of documents, acts, materia ls, devices, articles or the like which has been included in the present specification is solely for the purpose of providing a context for the present invention. It is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed before the priority date.

SUMMARY OF TH E INVENTION In a first aspect, the present invention broadly consists in a peptide conj ugate compound of the formula (I) :

(I)

wherein

m and w are each independently a n integer from 0 to 7 and v is an integer from 0 to 5,

provided that:

the sum of m, v, and w is at least 3; and

the sum of m and w is from 0 to 7;

n is 1 or 2;

Zl and Z2 are each independently selected from the group consisting of -0-, - NR-, -S-, -S(O)-, -SO2-, -C(0)0-, -OC(O)-, -C(0)N R-, -NRC(O)-, -C(0)S-, - SC(O)-, -OC(0)0-, -N RC(0)0-, -OC(0)N R-, and -NRC(0)N R-;

Rl, R2, Rx, Ry, R4, R5, R6, and R7 at each instance of m, v, w, a nd n are each independently hyd rogen or Cl-6aliphatic;

R, R3, and R8 a re each independently hydrogen or Cl-6a liphatic;

R9 is hydrogen, Cl-6a liphatic, an amino protecting group, L3-C(0)-, or A2; LI and L2 a re each independently selected from is C5-21aliphatic or C4-

20heteroaliphatic;

L3 is Cl-21aliphatic or C2-20heteroaliphatic;

Al is an amino acid, a peptide, OH, OP1, N H2, or NHP2, wherein PI is a carboxyl protecting group, and wherein P2 is a carboxamide protecting group;

A2 is an amino acid or a peptide;

wherein any a liphatic or heteroaliphatic present in any of R, Rl, R2, R3, R4, R5, R6, R7, R8, R9, Rx, Ry, LI, L2, and L3 is optionally substituted ;

or a pharmaceutically acceptable salt or solvate thereof;

with the proviso that: (1) at least one of R9 a nd Al is a peptide comprising, consisting essentially of, or consisting of an amino acid sequence selected from the group consisting of:

(a) 8 or more contig uous amino acid residues from the sequence

XaaiXaa2Xaa ₃ Xaa4LQQLSLLMWITQXaa22FLPVFLAQPPSGQRR [SEQ ID NO: l ], wherein Xaai is absent or is S, Xaa∑ is absent or is a hydrophilic amino acid, Xaa3 is absent or is a hydrophilic amino acid, a nd Xaa4 is absent or is one or more hydrophilic amino acids,

(b) 8 or more contig uous amino acid residues from the sequence

XaaiXaa2Xaa3LQQLSLLMWITQXaa22FLPVFLAQPPSGQRR [SEQ ID NO: 2], wherein Xaai is absent or is S, Xaa∑ is absent or is a hydrophilic amino acid, a nd Xaa3 is absent or is from one to ten hydrophilic amino acids,

(c) 8 or more contiguous amino acid residues from the sequence

XaaiXaa ₂ LQQLSLLMWITQXaa22FLPVFLAQPPSGQRR [SEQ ID NO: 3], wherein Xaa i is absent or is S, and Xaa∑ is absent or is from one to four hydrophilic amino acids, (d) 8 or more contig uous amino acid residues from the sequence

SKKKKLQQLSLLMWITQXaa ₂ 2FLPVFLAQPPSGQRR [SEQ ID NO:4],

(e) the sequence of a ny one of SEQ ID NOs: 1 to 4,

(f) 8 or more contig uous amino acid residues from the sequence

LQQLSLLMWITQXaa ₂ 2FLPVFLAQPPSGQRR [SEQ ID NO: 5], (g) the sequence of SEQ ID NO: 5,

(h) 8 or more contig uous amino acid residues from the sequence SLLMWITQXaa22FLPVF [SEQ ID NO :6],

(i) the sequence of SEQ ID NO: 6,

(j) 8 or more contig uous amino acid residues from the sequence SKKKKSLLMWITQXaa22 [SEQ ID NO :7],

(k) the sequence of SEQ ID NO: 7,

(I) 8 or more contig uous amino acid residues from the sequence SLLMWITQXaa22 [SEQ ID NO: 8],

(m) the sequence of SEQ ID NO: 8, (n) or any combination of two or more of (a) to (m) above, wherein aan in each sequence is independently any natura lly occurring amino acid except C (for example V, I, or L), and any sequence of 8 or more contiguous amino acid residues from a ny of the above sequences comprises Xaa22; or

(2) m is an integer from 3 to 7, and at least one of R9 and Al is an amino acid or a peptide.

In another aspect, the present invention broadly consists in a peptide conj ugate compound of the formula (I) :

(I)

wherein

m and w are each independently a n integer from 0 to 7 and v is an integer from 0 to 5,

provided that:

the sum of m, v, and w is at least 3; and

the sum of m and w is from 0 to 7;

n is 1 or 2;

Zl and Z2 are each independently selected from the group consisting of -0-, -

NR-, -S-, -S(O)-, -SO2-, -C(0)0-, -OC(O)-, -C(0)N R-, -NRC(O)-, -C(0)S-, - SC(O)-, -OC(0)0-, -N RC(0)0-, -OC(0)N R-, and -NRC(0)N R-;

Rl, R2, Rx, Ry, R4, R5, R6, and R7 at each instance of m, v, w, a nd n are each independently hyd rogen or Cl-6aliphatic;

R, R3, and R8 a re each independently hydrogen or Cl-6a liphatic;

R9 is hydrogen, Cl-6a liphatic, an amino protecting group, L3-C(0)-, or A2; LI and L2 a re each independently selected from is C5-21aliphatic or C4- 20heteroaliphatic;

L3 is Cl-21aliphatic or C2-20heteroaliphatic;

Al is an amino acid, a peptide, OH, OP1, NH2, or NHP2, wherein PI is a carboxyl protecting group, and wherein P2 is a carboxamide protecting group;

A2 is an amino acid or a peptide;

wherein any a liphatic or heteroaliphatic present in any of R, Rl, R2, R3, R4, R5, R6, R7, R8, R9, Rx, Ry, LI, L2, and L3 is optionally substituted ; or a pharmaceutically acceptable salt or solvate thereof;

with the proviso that:

(1) at least one of R9 a nd Al is a peptide comprising, consisting essentially of, or consisting of an amino acid sequence selected from the group consisting of: (a) 8 or more contig uous amino acid residues from the sequence

XaaiXaa2Xaa ₃ Xaa4LQQLSLLMWITQXaa22FLPVFLAQPPSGQRR [SEQ ID NO: l ], wherein Xaai is absent or is S, Xaa∑ is absent or is a hydrophilic amino acid, Xaa3 is absent or is a hydrophilic amino acid, a nd Xaa4 is absent or is one or more hydrophilic amino acids (b) 8 or more contig uous amino acid residues from the sequence

XaaiXaa2Xaa ₃ LQQLSLLMWITQXaa22FLPVFLAQPPSGQRR [SEQ ID NO: 2], wherein Xaai is absent or is S, Xaa∑ is absent or is a hydrophilic amino acid, a nd Xaa3 is absent or is from one to ten hydrophilic amino acids,

(c) 8 or more contiguous amino acid residues from the sequence

XaaiXaa ₂ LQQLSLLMWITQXaa22FLPVFLAQPPSGQRR [SEQ ID NO: 3], wherein Xaa i is absent or is S, and Xaa∑ is absent or is from one to four hydrophilic amino acids,

(d) 8 or more contig uous amino acid residues from the sequence

SKKKKLQQLSLLMWITQXaa22FLPVFLAQPPSGQRR [SEQ ID NO:4,

(e) the sequence of a ny one of SEQ ID NOs: 1 to 4, (f) 8 or more contig uous amino acid residues from the sequence

LQQLSLLMWITQXaa ₂ 2FLPVFLAQPPSGQRR [SEQ ID NO: 5],

(g) the sequence of SEQ ID NO: 5,

(h) 8 or more contig uous amino acid residues from the sequence SLLMWITQXaa22FLPVF [SEQ ID NO :6], (i) the sequence of SEQ ID NO: 6,

(j) 8 or more contig uous amino acid residues from the sequence SKKKKSLLMWITQXaa22 [SEQ ID NO :7],

(k) the sequence of SEQ ID NO: 7,

(I) 8 or more contig uous amino acid residues from the sequence SLLMWITQXaa22 [SEQ ID NO: 8], (m) the sequence of SEQ ID NO: 8,

(n) or any combination of two or more of (a) to (m) above, wherein Xaa∑2 in each sequence is independently any naturally occurring amino acid except C (for example V, I, or L), and any sequence of 8 or more contiguous amino acid residues from a ny of the above sequences comprises Xaa22.

In another aspect, the present invention broadly consists in a peptide conj ugate compound of the formu

(I)

wherein

m and w are each independently a n integer from 0 to 7 and v is an integer from 0 to 5,

provided that:

the sum of m, v, and w is at least 3; and

the sum of m and w is from 0 to 7;

n is 1 or 2;

Zl and Z2 are each independently selected from the group consisting of -0-, - NR-, -S-, -S(O)-, -SO2-, -C(0)0-, -OC(O)-, -C(0)N R-, -NRC(O)-, -C(0)S-, - SC(O)-, -OC(0)0-, -N RC(0)0-, -OC(0)N R-, and -NRC(0)N R-;

Rl, R2, Rx, Ry, R4, R5, R6, and R7 at each instance of m, v, w, a nd n are each independently hyd rogen or Cl-6aliphatic;

R, R3, and R8 a re each independently hydrogen or Cl-6a liphatic;

R9 is hydrogen, Cl-6a liphatic, an amino protecting group, L3-C(0)-, or A2;

LI and L2 a re each independently selected from is C5-21aliphatic or C4- 20heteroaliphatic;

L3 is Cl-21aliphatic or C2-20heteroaliphatic;

Al is an amino acid, a peptide, OH, OPl, N H2, or N H P2, wherein PI is a carboxyl protecting group, and wherein P2 is a carboxamide protecting group;

A2 is an amino acid or a peptide;

wherein any a liphatic or heteroaliphatic present in any of R, Rl, R2, R3, R4, R5, R6, R7, R8, R9, Rx, Ry, LI, L2, and L3 is optionally substituted ; or a pharmaceutically acceptable salt or solvate thereof;

with the proviso that:

(2) m is an integer from 3 to 7, and at least one of R9 and Al is an amino acid or a peptide.

Any of the embod iments or preferences described herein may relate to any of the aspects herein alone or in combination with any one or more embodiments or preferences described herein, unless stated or ind icated otherwise. In various embodiments,

Rl, R2, Rx, Ry, R4, R5, R6, and R7 at each instance of m, v, w, a nd n are each independently hyd rogen, Cl-6a lkyl, C2-6alkenyl, C2-6alkynyl, or C3-6cycloa lkyl;

R, R3, and R8 a re each independently hyd rogen, Cl-6a lkyl, C2-6a lkenyl, C2- 6alkynyl, or C3-6cycloalkyl ;

R9 is hydrogen, Cl-6a lkyl, C2-6alkenyl, C2-6alkynyl, C3-6cycloalkyl, an amino protecting group, L3-C(0), or A2;

LI and L2 a re each independently selected from C5-21a lkyl, C5-21a lkenyl, C5- 21alkynyl, or C4-20heteroa lkyl;

L3 is Cl-21alkyl, C5-21alkenyl, C5-21alkynyl, C3-6cycloalkyl, or C2- 20heteroalkyl;

Al is an amino acid, a peptide, OH, OP1, N H2, or NHP2, wherein PI is a carboxyl protecting group, and wherein P2 is a carboxamide protecting group;

A2 is an amino acid or a peptide;

wherein any a lkyl, alkenyl, a lkynyl, cycloalkyl or heteroalkyl present in any of R, Rl, R2, R3, R4, R5, R6, R7, R8, R9, Rx, Ry, LI, L2, a nd L3 is optionally substituted .

In various embodiments,

Rl, R2, Rx, Ry, R4, R5, R6, and R7 at each instance of m, v, w, a nd n are each independently hyd rogen, Cl-6alkyl, C2-6alkenyl, or C3-6cycloalkyl ;

R, R3, and R8 a re each independently hyd rogen, Cl-6a lkyl, C2-6a lkenyl, or C3-

6cycloalkyl ;

R9 is hydrogen, Cl-6a lkyl, C2-6alkenyl, C3-6cycloalkyl, a n amino protecting group, L3-C(0), or A2;

LI and L2 a re each independently selected from C5-21alkyl, C5-21a lkenyl, or C4- 20heteroalkyl;

L3 is Cl-21alkyl, C5-21alkenyl, C3-6cycloalkyl, or C2-20heteroalkyl ;

Al is an amino acid, a peptide, OH, OP1, N H2, or NHP2, wherein PI is a carboxyl protecting group, and wherein P2 is a carboxamide protecting group; A2 is an amino acid or a peptide;

wherein any a lkyl, a lkenyl, cycloalkyl or heteroalkyl present in any of R, Rl, R2, R3, R4, R5, R6, R7, R8, R9, Rx, Ry, LI, L2, and L3 is optiona lly substituted . In various embodiments,

Rl, R2, Rx, Ry, R4, R5, R6, and R7 at each instance of m, v, w, a nd n are each independently hyd rogen, Cl-6alkyl, or C3-6cycloalkyl;

R, R3, and R8 a re each independently hydrogen, Cl-6a lkyl, or C3-6cycloa lkyl; R9 is hydrogen, Cl-6a lkyl, C3-6cycloalkyl, an amino protecting group, L3-C(0), or A2;

LI and L2 a re each independently selected from C5-21alkyl, C5-21a lkenyl, or C4- 20heteroalkyl;

L3 is Cl-21alkyl, C2-21alkenyl, C3-6cycloalkyl, or C2-20heteroalkyl ;

Al is an amino acid, a peptide, OH, OP1, N H2, or NHP2, wherein PI is a carboxyl protecting group, and wherein P2 is a carboxamide protecting group;

A2 is an amino acid or a peptide;

wherein any a lkyl, a lkenyl, cycloalkyl or heteroalkyl present in any of R, Rl, R2, R3, R4, R5, R6, R7, R8, R9, Rx, Ry, LI, L2, and L3 is optiona lly substituted . In various embodiments,

Rl, R2, Rx, Ry, R4, R5, R6, and R7 at each instance of m, v, w, a nd n are each independently hyd rogen, Cl-6alkyl, or C3-6cycloalkyl;

R, R3, and R8 a re each independently hydrogen, Cl-6a lkyl, or C3-6cycloa lkyl; R9 is hydrogen, Cl-6a lkyl, C3-6cycloalkyl, an amino protecting group, L3-C(0), or A2;

LI and L2 a re each independently selected from is C5-21alkyl or C4- 20heteroalkyl;

L3 is Cl-21alkyl, C3-6cycloalkyl, or C2-20heteroa lkyl;

Al is an amino acid, a peptide, OH, OP1, N H2, or NHP2, wherein PI is a carboxyl protecting group, and wherein P2 is a carboxamide protecting group;

A2 is an amino acid or a peptide;

wherein any a lkyl, cycloalkyl or heteroa lkyl present in any of R, Rl, R2, R3, R4, R5, R6, R7, R8, R9, Rx, Ry, LI, L2, a nd L3 is optionally substituted . In various embodiments, Zl and Z2 are each independently selected from the g roup consisting of -C(0)0-, -C(0)NR-, and -C(0)S-.

In various embodiments, the compound of the formula (I) is a compound of the formula (IA) :

(IA).

In various embodiments, v is from 0 to 4, 0 to 3, or 0 to 2, or v is 0 or 1, for example 0.

In certain embodiments, v is from 0 to 3. In exemplary embodiments, v is 0. In various embodiments, m and w are each independently from 0 to 6, 0 to 5, 0 to 4, 0 to 3, 0 to 2, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 to 2.

In various embodiments, m and w are each independently from 0 to 5.

In certain embodiments, m and w are each independently from 1 to 4.

In various embodiments, m is from 1 to 6, for example from 2 to 6, 1 to 5, or 2 to 5. In various embodiments, m is from 1 to 5. In various embodiments, m is from 1 to 3. In exemplary embodiments, m is 2.

In various embodiments, m is from 3 to 6. In certain embodiments, m is from 3 to 5.

In various embodiments, w is 1 or 2. In exemplary embodiments, w is 1.

In various embodiments, the sum of m and w is from 0 to 6, 0 to 5, 0 to 4, 0 to 3, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 2 to 7, 2 to 6, 2 to 5, 2 to 4, or 2 to 3.

In various embodiments, the sum of m and w is from 2 to 7.

In certain embodiments, the sum of m and w is from 2 to 5.

In exemplary embodiments, the sum of m and w is 3.

In various embodiments, v is from 0 to 3; m and w are each independently from 0 to 5; and the sum of m and w is from 2 to 7.

In various embodiments, v is from 1 or 0; m and w are each independently from 0 to 5; and the sum of m and w is from 2 to 7.

In various embodiments, v is 1 or 0; m and w are each independently from 1 to 4; and the sum of m and w is from 2 to 7. In various embodiments, v is 1 or 0; m and w are each independently from 1 to 4; and the sum of m and w is from 2 to 5.

In certain embodiments, v is 1 or 0; m is from 1 to 6; and w is 1 or 2. In certain embodiments, v is 1 or 0; m is from 1 to 5; and w is 1 or 2. In certain embodiments, v is 0; m is from 1 to 6; and w is 1.

In certain embodiments v is 0 or 1 ; m is from 1 to 3; and w is 1 or 2.

In exemplary embodiments, v is 0; m is from 2 to 5; and w is 1.

In exemplary embodiments, v is 0; m is 2; and w is 1.

In certain embodiments, v is 0; m is from 3 to 5; and w is 1. In exemplary embodiments, n is 1.

In certain embodiments, LI and L2 are each independently C5-21aliphatic, for example C9-21alihpatic, Cl l-21aliphatic, or C11-, C13-, C15-, C17-, or C19-a liphatic.

In certain embodiments, LI and L2 are each independently C5-21alkyl.

In various embodiments, LI and L2 are each independently C9-21alkyl. In yet another embodiment, LI and L2 are each independently Cll-21alkyl.

In various exemplary embodiments, LI and L2 are each independently Cll, C13, C15, C17, or C19alkyl, preferably n-alkyl.

In various specifically contemplated embodiments, LI and L2 are each independently C15alkyl. In various embodiments, LI and L2 each independently comprise a linear chain of 9-21 carbon atoms.

In exemplary embodiments, LI and L2 are each independently linear C15alkyl. In some embodiments, L3 is Cl-21alkyl. In various embodiments, L3 is methyl or linear C15alkyl. In exemplary embodiments, L3 is methyl (that is, R9 is acetyl). In some embod iments, the amino protecting group is Boc, Fmoc, Cbz (carboxybenzyl), Nosyl (o- or p-nitrophenylsulfonyl), Bpoc (2-(4-biphenyl)isopropoxycarbonyl) and Dde (l-(4,4-dimethyl-2,6-dioxohexylidene)ethyl) .

In various embodiments, the amino protecting group is Boc or Fmoc. In some embod iments, the amino protecting group is Fmoc.

In some embod iments, the carboxyl protecting group is terf-butyl, benzyl, or allyl.

In various embodiments, the carboxamide protecting g roup is Dmcp or Trityl.

In various embodiments, Rl a nd R2 at each instance of m are each independently Cl- 6alkyl or hyd rogen. In various specifically contemplated embodiments, Rl and R2 at each instance of m are each hydrogen.

In various embodiments, R3 is Cl-6alkyl or hyd rogen . In various specifically

contemplated embodiments, R3 is hyd rogen.

In various embodiments, R4 a nd R5 at each instance of w are each independently Cl- 6alkyl or hyd rogen, preferably hydrogen. In va rious specifically contemplated embodiments, R4 and R5 at each instance of w are each hydrogen.

In various embodiments, Rx and Ry at each instance of v are each independently Cl- 6alkyl or hyd rogen. In various specifically contemplated embodiments, Rx and Ry at each instance of v are each hydrogen.

In various embodiments, R6 a nd R7 at each instance of n are each independently Cl- 6alkyl or hyd rogen. In various specifically contemplated embodiments, R6 and R7 are each hydrogen.

In various embodiments, R8 is independently Cl-6alkyl or hydrogen. In exemplary embodiments, R8 is hydrogen.

In various embodiments, R9 is Cl-6alkyl, hydrogen, a n amino protecting group, L3- C(O), or A2. In exemplary embodiments, R9 is hydrogen, an a mino protecting group, L3-C(0), or A2.

In various embodiments, R8 is hydrogen and R9 is hydrogen, a n amino protecting group, L3-C(0), or A2.

In various embodiments, R8 a nd R9 are each hydrogen ; or R9 is L3-C(0) or A2. In various exemplary embodiments, R8 is hydrogen and R9 is L3-C(0). In various specifically contemplated embodiments, R9 is L3-C(0), wherein L3 is methyl.

In various embodiments, the compound of formula (I) is a compound of the formula (IF)

(IF).

In various embodiments, the compound of formula (IF) is a compound of the formula (IF- 1):

(IF-1). In various embodiments, the compound of formula (I) is a compound of the formula (IB):

(IB) wherein

k is an integer from 0 to 4 (i.e. k is from 0 to 4 when proviso (1) of the first aspect applies and from 1 to 4 when proviso (2) of the first aspect applies); and

Ra, Rb, and Rc are each independently hydrogen or Cl-6aliphatic.

In various embodiments, the compound of formula (IB) is a compound of the formula (IC):

(IC).

In various embodiments, k is from 0 to 3, 0 to 2, 0 to 1, 1 to 4, 1 to 3, or 1 to 2, or k is 0 or 1. In certain embodiments, k is 0 to 3.

In certain embodiments, k is 0 or 1.

In exemplary embodiments, k is 0.

In certain embodiments k is equal to v.

In certain embodiments k is from 1 to 3. In various, embodiments, Ra, Rb, and Rc are each independently hydrogen, Cl-6alkyl, C2-6alkenyl, C2-6alkynyl, or C3-6cycloalkyl.

In various, embodiments, Ra, Rb, and Rc are each independently hydrogen, Cl-6alkyl, C2-6alkenyl, or C3-6cycloalkyl.

In various, embodiments, Ra, Rb, and Rc are each independently hydrogen, Cl-6alkyl, or C3-6cycloalkyl.

In various embodiments, Ra, Rb, and Rc are each independently selected from hydrogen or Cl-6alkyl, preferably hydrogen. In exemplary embodiments, Ra, Rb, and Rc are each hyd rogen.

In various embodiments, the compound of formula (I) is a compound of the formula (ID- 1) :

(ID-1). In various embodiments, m in the compound of formula (ID-1) is from 3 to 5.

In various embodiments, the compound of the formula (I) is a compound of the formula (ID) :

(ID).

In certain embodiments, the compound is a compound of the formula (ID) wherein LI and L2 are each linear C15alkyl.

In various embodiments, LI and L2 are each independently Cl l-21alkyl; m is 2-5; v is 0; w is 1; Rl and R2 at each instance are each hydrogen; R3 is hydrogen; and R4 and R5 are each hydrogen.

In various embodiments, LI and L2 are each independently Cl l-21alkyl; m is 3-5; v is 0; w is 1; Rl and R2 at each instance are each hydrogen; R3 is hydrogen; and R4 and R5 are each hydrogen.

In various embodiments, LI and L2 are each independently Cl l-21alkyl; m is 2; v is 0; w is 1 ; Rl and R2 at each instance are each hydrogen; R3 is hydrogen; and R4 and R5 are each hydrogen.

In various embodiments, n is 1; R6, R7, and R8 are each hydrogen; and R9 is hydrogen, an amino protecting group, L3-C(0), or A2.

In various embodiments, n is 1; R6, R7, and R8 are each hydrogen; and R9 is hydrogen, an amino protecting group, or L3-C(0), wherein L3 is linear C15alkyl or methyl.

In various embodiments, LI and L2 are each independently Cl l-21alkyl; m is 2-5; v is 0; w is 1; Rl and R2 at each instance are each hydrogen; R3 is hydrogen; R4 and R5 are each hydrogen; n is 1; R6, R7, and R8 are each hydrogen; and R9 is hydrogen, an amino protecting group, or L3-C(0), wherein L3 is linear C15alkyl or methyl.

In various embodiments, LI and L2 are each independently Cl l-21alkyl; m is 3-5; v is 0; w is 1; Rl and R2 at each instance are each hydrogen; R3 is hydrogen; R4 and R5 are each hydrogen; n is 1; R6, R7, and R8 are each hydrogen; and R9 is hydrogen, an amino protecting group, or L3-C(0), wherein L3 is linear C15alkyl or methyl. In various embodiments, LI and L2 are each independently Cl l-21alkyl; m is 2; v is 0; w is 1 ; Rl and R2 at each instance are each hydrogen; R3 is hydrogen; R4 and R5 are each hydrogen; n is 1; R6, R7, and R8 are each hydrogen; and R9 is hydrogen, an amino protecting group, or L3-C(0), wherein L3 is linear C15alkyl or methyl. In various embodiments, the compound of formula (I) has the formula (IE) :

(IE).

In various embodiments, the compound of formula (I) has the formula (IEE):

(IEE).

In various embodiments, the compound of formula (I) has the formula (IE-1):

(IE-1).

In various embodiments, the compound of formula (I) has the formula (IEE-1):

(IEE-1).

In various embodiments, the compound of formula (I) has the formula (IE-2):

(IE-2).

In various embodiments, the compound of formula (I) has the formula (IEE-2):

(IEE-2).

In various embodiments, the compound of formula (I) has the formula (IEE-3) :

(IEE-3).

In various embodiments, the compound of formula (I) has the formula (IEE-4):

(IEE-4).

In various embodiments, the amino acid of the amino acid- or peptide conjugate to which the lipid moieties are conjugated is a cysteine residue.

Those skilled in the art will appreciate that, in certain embodiments, the moieties Ll-Zl- and L2-Z2- may be fatty acid groups, for example fatty acid esters. In various embodiments, the moieties may be saturated or unsaturated fatty acid esters. In some embodiments, the fatty acid is saturated . In various embodiments, the fatty acid is a C4-22 fatty acid . In some embodiments, the fatty acid is a C6-22 fatty acid .

In certain embodiments, the fatty acid is a ClO-22 fatty acid . In certain specifically contemplated embodiments, the fatty acid is a C12-22 fatty acid . In various exemplary embodiments, the fatty acid is a C12, C14, C16, C18, or C20 fatty acid .

In some embod iments, the fatty acid is la uric acid, myristic acid, palmitic acid, stea ric acid, arachic acid, palmitoleic acid, oleic acid, elaidic acid, linoleic acid, a-linolenic acid, and a rachidonic acid .

In various embodiments, the fatty acid is lauric acid, myristic acid, palmitic acid, or stearic acid .

In certa in exemplary embodiments, the fatty acid is palmitic acid (and the moieties Ll- Zl- a nd L2-Z2-are each palmitoyl groups).

In some embod iments, Al is OH, OPl, N H2, or NHP2 or R9 is hydrogen, Cl-6alkyl, C3- 6cycloalkyl, an amino protecting g roup, or L3-C(0) . In some embod iments, Al is OPl or OH or R9 is hydrogen, a n amino protecting group or L3-C(0).

In various embodiments, R9 is hydrogen, an amino protecting group or L3-C(0). In some embodiments, R9 is hydrogen or L3-C(0).

In various embodiments, the compound of formula (I) is a peptide conjugate. In various embodiments, at least one of Al and R9 is a peptide comprising, consisting essentia lly of, or consisting of an amino acid sequence selected from the group consisting of those defined in proviso (1) of the first aspect.

In various embodiments, the peptide comprises, consists of, or consists essentially of a n amino acid seq uence selected from the group consisting of: (a) 8 or more contig uous amino acid residues from the sequence SLLMWITQXaa22FLPVF [SEQ ID NO :6],

(b) the sequence of SEQ ID NO: 6,

(c) 8 or more contig uous amino acid residues from the sequence SKKKKSLLMWITQXaa22 [SEQ ID NO :7], (d) the sequence of SEQ ID NO: 7, (e) 8 or more contig uous amino acid residues from the sequence SLLMWITQXaa22 [SEQ ID NO: 8],

(f) the sequence of SEQ ID NO: 8,

(g) or any combination of two or more of (a) to (f) above. In various embodiments, the peptide comprises, consists of, or consists essentially of a n amino acid seq uence selected from the group consisting of:

(a) 8 or more contig uous amino acid residues from the sequence SKKKKSLLMWITQXaa22 [SEQ ID NO :7],

(b) the sequence of SEQ ID NO: 7, (c) 8 or more contig uous amino acid residues from the sequence SLLMWITQXaa22 [SEQ ID NO: 8],

(d) the sequence of SEQ ID NO: 8, or a ny combination of two or more of (a) to (d) above.

In various embodiments, the peptide comprises, consists of, or consists essentially of an amino acid seq uence selected from the group consisting of:

(a) 8 or more contig uous amino acid residues from the sequence SKKKKSLLMWITQXaa22 [SEQ ID NO :7],

(b) the sequence of SEQ ID NO: 7.

In various embodiments, the peptide comprises, consists of, or consists essentially of an amino acid seq uence selected from the group consisting of:

(a) 8 or more contig uous amino acid residues from the sequence SLLMWITQXaa22 [SEQ ID NO: 8],

(b) the sequence of SEQ ID NO: 8.

In various embodiments, Xaan in each seq uence is independently V, I, or L (that is, each Xaa22 is independently V, I, or L).

In exemplary embodiments, Xaa∑2 in each sequence is V (that is, each Xaa∑2 is V). In various embodiments, m is from 3 to 7 and at least one of Al and R9 is an amino acid or peptide as defined in proviso (2) of the first aspect. In some embodiments, at least one of Al and R9 is a peptide. In various embodiments, Al and/or A2 is an amino acid or a peptide. That is, in various embodiments, Al is an amino acid or a peptide and/or R9 is an amino acid or a peptide.

In some embodiments, Al and/or A2 is a peptide. That is, in various embodiments, Al is a peptide and/or R9 is a peptide.

In one embodiment Al and/or A2 is a peptide comprising an epitope.

In some embodiments, Al and/or A2 is a peptide comprising a peptide epitope. In another embodiment, Al and/or A2 is a peptide, wherein the peptide comprises a peptide epitope.

In some embodiments, Aland/or A2 is a peptide substituted with an epitope.

In some embodiments, the epitope is bound to the peptide via a linker group.

In certain embodiments, Al is a peptide. In certain exemplary embodiments, Al is a peptide and R9 is not A2 (that is, R9 is not an amino acid or a peptide).

In certain exemplary embodiments, Al is a peptide and R9 is hydrogen or L3-C(0), for example Me-C(O).

In various embodiments, the peptide comprises an epitope. In various embodiments, the epitope is a peptide epitope.

In certain embodiments, the epitope is coupled or bound via a linker group.

In various embodiments, the amino acid of the peptide conjugate to which the lipid moieties are conjugated is an N-terminal amino acid residue.

In various embodiments, Al is serine or a peptide comprising serine as the first N- terminal amino acid residue.

In some embodiments, Al is a peptide comprising serine as the first N-terminal amino acid residue. In various embodiments, the peptide conjugate comprises one or more solubilising groups.

In some embod iments, the solubilising group comprises a n amino acid sequence comprising two or more hydrophiiic amino acid residues in the peptide chain.

In various embodiments, the solubilising group is an amino acid sequence comprising a sequence of two or more consecutive hydrophiiic amino acid residues in the peptide chain.

In various embodiments, the two or more hydrophiiic amino acid residues are adjacent to the serine residue.

In some embod iments, Al and/or A2 is a peptide comprising a solubilising group.

In various embodiments, Al and/or A2 is a peptide comprising a solubilising group comprising an amino acid sequence comprising two or more hydrophiiic amino acid residues in the peptide chain.

In certain embodiments, Al is a peptide comprising a solubilising group comprising an amino acid seq uence comprising two or more hydrophiiic amino acid residues in the peptide chain.

In some embod iments, Al is a peptide comprising serine as the first N-terminal amino acid residue and a solubilising group comprising an amino acid sequence comprising two or more hydrophiiic amino acid residues in the peptide chain adjacent to the serine.

In some embod iments, the compound comprises a linker or one or more amino acids thereof. In some embodiments, the peptide comprises a linker or one or more amino acids thereof.

In some embod iments, the peptide comprises a peptide epitope bound via a linker to the amino acid to which the lipid moieties are bound .

In some embod iments, the peptide comprises two or more epitopes.

In some embod iments, the peptide comprises a peptide antigen.

In some embod iments, the linker is an amino acid sequence from about 2 to 20, 2 to 18, 2 to 16, 2 to 14, 2 to 12, 2 to 10, or 2 to 8 amino acids in length.

In some embod iments, the compound of formula (I) comprises 3 or more, 4 or more, or 5 or more contiguous a mino acids. In various embodiments, the peptide conjugate is a lipopeptide.

In some embod iments, the compound of formula (I) is a self adjuvanting peptide.

In some embod iments, Al and/or A2 are each independently a peptide comprising from about 8 to 220, 8 to 200, 8 to 175, 8 to 150, 8 to 125, 8 to 100, 8 to 90, 8 to 80, 8 to 70, 8 to 60, 8 to 50, 8 to 40, 8 to 30, 8 to 25, 8 to 20, or 8 to 15 amino acids. In one exemplary embod iment, Al and A2 are each independently a peptide comprising from about 8 to 60 amino acids.

In other embodiments, Al and/or A2 are each independently a peptide comprising from about 8 to 220, 8 to 200, 8 to 175, 8 to 150, 8 to 125, 8 to 100, 8 to 90, 8 to 80, 8 to 70, 8 to 60, 8 to 50, 8 to 40, 8 to 30, 8 to 25, 8 to 20, or 8 to 15 amino acids.

In other embodiments, Al and/or A2 are each independently a peptide comprising from about 5 to 150, 5 to 125, 5 to 100, 5 to 75, 5 to 60, 5 to 50, 5 to 40, 5 to 30, 5 to 25, 5 to 20, 8 to 150, 8 to 125, 8 to 100, 8 to 75, 8 to 60, 8 to 50, 8 to 40, 8 to 30, 8 to 25, or 8 to 20 amino acids. In some embod iments, Al and/or A2 are each independently a peptide, wherein the peptide comprises 8 to 60 amino acids.

In some embod iments, Al and/or A2 are each independently a peptide comprising or substituted with a peptide epitope, wherein the peptide epitope compises from 8 to 60 amino acids. Suitable peptide epitopes include without limitation those described in WO 2016/103192 filed 22 December 2015, the entirety of which is incorporated herein by reference.

In various embodiments, the peptide comprises, consists essentially of, or consists of one or more EBV LM P2 epitopes. In va rious embodiments, the one or more EBV LMP2 epitopes a re MHCI epitopes. In various embodiments, the peptide comprises one or more EBV LMP2 epitopes selected from the group consisting of a ny one of SEQ ID NOs 84 - 109. In various embodiments, the peptide comprises a peptide comprising or consisting of 8 or more contiguous amino acids from the amino acid sequence of any one of SEQ ID NOs 9 - 83. In va rious embodiments, the peptide comprises a peptide comprising or consisting of 12 or more contiguous amino acids from the amino acid sequence of any one of SEQ ID NOs 9 - 83. In various embodiments, the peptide comprises a peptide comprising or consisting of 15 or more contiguous amino acids from the amino acid sequence of any one of SEQ ID NOs 9 - 83, or comprising or consisting of 20 or more contiguous amino acids from the amino acid sequence of any one of SEQ ID NOs 9 - 83. In various embodiments, the peptide comprises a recombinant peptide comprising or consisting of 12 or more contiguous amino acids from the amino acid sequence of any one of SEQ ID NOs 9 - 83. In various embodiments, the recombinant peptide comprises or consists of 15 or more contiguous amino acids from the amino acid sequence of any one of SEQ ID NOs 9 - 83, or comprises or consists of 20 or more contiguous amino acids from the amino acid sequence of any one of SEQ ID NOs 9 - 83.

In various embodiments, the peptide comprises, consists of, or consists essentially of an amino acid sequence selected from the group consisting of any one of SEQ ID NOs 9 - 83.

In various embodiments, the peptide comprises, consists of, or consists essentially of an amino acid sequence selected from the group consisting of

(a) 8 or more contiguous amino acid residues from the sequence

XaaiXaa ₂ Xaa ₃ Xaa4DRHSDYQPLGTQDQSLYLGLQHDGNDGL [SEQ ID NO:9], wherein Xaai is absent or is S or a hydrophilic amino acid, Xaa∑ is absent or is a hydrophilic amino acid, Xaa3 is absent or is a hydrophilic amino acid, and Xaa4 is absent or is one or more hydrophilic amino acids,

(b) 8 or more contiguous amino acid residues from the sequence

Xa a iXa a 2Xa a 3D RH S DYQ PLGTQDQS LYLG LQHDGNDGL [SEQ ID NO: 10], wherein Xaal is absent or is S or a hydrophilic amino acid, Xaa∑ is absent or is a hydrophilic amino acid, and Xaa3 is absent or is from one to ten hydrophilic amino acids,

(c) 8 or more contiguous amino acid residues from the sequence

XaaiXaa ₂ DRHSDYQPLGTQDQSLYLGLQHDGNDGL [SEQ ID NO: 11], wherein Xaal is absent or is S or a hydrophilic amino acid, and Xaa∑ is absent or is from one to four hydrophilic amino acids,

(d) 8 or more contiguous amino acid residues from the sequence

SKKKKDRHSDYQPLGTQDQSLYLGLQHDGNDGL [SEQ ID NO: 12],

(e) 8 or more contiguous amino acid residues from the sequence

DRHSDYQPLGTQDQSLYLGLQHDGNDGL [SEQ ID NO: 13],

(f) 8 or more contiguous amino acid residues from the sequence

Xaa1Xaa2Xaa3Xaa4SLYLGLQHDGNDGL.PPPPYSPRDDSSQH.YEEA [SEQ ID NO: 14], wherein Xaal is absent or is S or a hydrophilic amino acid, Xaa∑ is absent or is a hydrophilic amino acid, Xaa3 is absent or is a hydrophilic amino acid, and Xaa4 is absent or is one or more hydrophilic amino acids, (g) 8 or more contiguous amino acid residues from the sequence

XaaiXaa ₂ Xaa ₃ SLYLGLQHDGNDGLPPPPYSPRDDSSQHIYEEA [SEQ ID NO: 15], wherein Xaal is absent or is S or a hydrophilic amino acid, Xaa∑ is absent or is a hydrophilic amino acid, and Xaa3 is absent or is from one to ten hydrophilic amino acids, (h) 8 or more contiguous amino acid residues from the sequence

Xaa1Xaa2SLYLGLQHDGNDGL.PPPPYSPRDDSSQH.YEEA [SEQ ID NO: 16], wherein Xaal is absent or is S or a hydrophilic amino acid, and Xaa∑ is absent or is from one to four hydrophilic amino acids,

(i) 8 or more contiguous amino acid residues from the sequence

SKKKKSLYLGLQHDGNDGLPPPPYSPRDDSSQHIYEEA [SEQ ID NO: 17],

(j) 8 or more contiguous amino acid residues from the sequence

SLYLGLQHDGNDGLPPPPYSPRDDSSQHIYEEA [SEQ ID NO: 18],

(k) 8 or more contiguous amino acid residues from the sequence

XaaiXaa ₂ Xaa ₃ Xaa4SDYQPLGTQDQSLYLGLQHDGNDGL [SEQ ID NO: 19], wherein Xaai is absent or is S or a hydrophilic amino acid, Xaa∑ is absent or is a hydrophilic amino acid, Xaa3 is absent or is a hydrophilic amino acid, and Xaa4 is absent or is one or more hydrophilic amino acids,

(I) 8 or more contiguous amino acid residues from the sequence

XaaiXaa ₂ Xaa ₃ SDYQPLGTQDQSLYLGLQHDGNDGL [SEQ ID NO:20], wherein Xaai is absent or is S or a hydrophilic amino acid, Xaa∑ is absent or is a hydrophilic amino acid, and Xaa3 is absent or is from one to ten hydrophilic amino acids,

(m) 8 or more contiguous amino acid residues from the sequence

XaaiXaa ₂ SDYQPLGTQDQSLYLGLQHDGNDGL [SEQ ID NO:21], wherein Xaai is absent or is S or a hydrophilic amino acid, and Xaa∑ is absent or is from one to four hydrophilic amino acids,

(n) 8 or more contiguous amino acid residues from the sequence

SKKKKSDYQPLGTQDQSLYLGLQHDGNDGL [SEQ ID NO:22],

(0) 8 or more contiguous amino acid residues from the sequence

SDYQPLGTQDQSLYLGLQHDGNDGL [SEQ ID NO:23], (p) 8 or more contiguous amino acid residues from the sequence

XaaiXaa2Xaa ₃ Xaa4DRHSDYQPLGTQDQSLYLGLQHDGNDGLPPPPYSPRDDSSQHIYEEA [SEQ ID NO:24], wherein Xaai is absent or is S or a hydrophilic amino acid, Xaa∑ is absent or is a hydrophilic amino acid, Xaa3 is absent or is a hydrophilic amino acid, and Xaa4 is absent or is one or more hydrophilic amino acids,

(q) 8 or more contiguous a mino acid residues from the seq uence

Xaa 1Xaa2Xaa3DRHSDYQPLGTQDQSLYLGLQH DGN DGL.PPPPYSPRDDSSQH.YEEA [SEQ ID NO: 25], wherein Xaai is absent or is S or a hydrophilic amino acid, Xaa∑ is absent or is a hydrophilic amino acid, and Xaa3 is absent or is from one to ten hydrophilic amino acids,

(r) 8 or more contiguous a mino acid residues from the seq uence

Xaa iXaa2DRHSDYQPLGTQDQSLYLGLQHDGN DGLPPPPYSPRDDSSQHIYEEA [SEQ ID NO: 26], wherein Xaai is absent or is S or a hydrophilic amino acid, and Xaa∑ is absent or is from one to four hydrophilic amino acids,

(s) 8 or more contiguous a mino acid residues from the seq uence

SKKKKDRHSDYQPLGTQDQSLYLGLQHDGNDGLPPPPYSPRDDSSQHIYEEA [SEQ ID NO: 27], (t) 8 or more contiguous a mino acid residues from the seq uence

DRHSDYQPLGTQDQSLYLGLQHDGN DGLPPPPYSPRDDSSQHIYEEA [SEQ ID NO: 28],

(u) 8 or more contiguous a mino acid residues from the seq uence

Xaa iXaa2Xaa3Xaa4LLWTLVVLLICSSCSSCPLSKILLARLFLYALALLL [SEQ ID NO : 29], wherein Xaai is absent or is S or a hyd rophilic amino acid, Xaa∑ is absent or is a hydrophilic amino acid, Xaa3 is absent or is a hydrophilic amino acid, and Xaa4 is absent or is one or more hydrophilic amino acids,

(v) 8 or more contiguous a mino acid residues from the seq uence

Xaa iXaa ₂ Xaa3LLWTLVVLLICSSCSSCPLSKILLARLFLYALALLL [SEQ ID NO : 30], wherein Xaa i is absent or is S or a hydrophilic amino acid, Xaa∑ is absent or is a hydrophilic amino acid, and Xaa3 is absent or is from one to ten hydrophilic amino acids,

(w) 8 or more contiguous a mino acid residues from the seq uence

Xaa iXaa ₂ LLWTLVVLLICSSCSSCPLSKILLARLFLYALALLL [SEQ ID NO: 31], wherein Xaa i is absent or is S or a hydrophilic amino acid, and Xaa∑ is absent or is from one to four hydrophilic amino acids, (x) 8 or more contiguous a mino acid residues from the seq uence

SKKKKLLWTLVVLLICSSCSSCPLSKILLARLFLYALALLL [SEQ ID NO: 32],

(y) 8 or more contiguous a mino acid residues from the seq uence

LLWTLVVLLICSSCSSCPLSKILLARLFLYALALLL [SEQ ID NO: 33], (z) 8 or more contiguous a mino acid residues from the seq uence

Xaa iXaa2Xaa ₃ Xaa4LMLLWTLVVLLICSSCSSCPLSKILLARLFLYALALLLLA [SEQ ID NO: 34], wherein Xaai is absent or is S or a hydrophilic amino acid, Xaa∑ is absent or is a hydrophilic amino acid, Xaa3 is absent or is a hydrophilic amino acid, a nd Xaa4 is absent or is one or more hydrophilic amino acids,

(aa) 8 or more contiguous a mino acid residues from the seq uence

Xaa iXaa2Xaa ₃ LMLLWTLVVLLICSSCSSCPLSKILLARLFLYALALLLLA [SEQ ID NO : 35], wherein Xaai is absent or is S or a hyd rophilic amino acid, Xaa∑ is absent or is a hydrophilic amino acid, and Xaa3 is absent or is from one to ten hydrophilic amino acids,

(bb) 8 or more contiguous a mino acid residues from the seq uence

Xaa iXaa ₂ LMLLWTLVVLLICSSCSSCPLSKILLARLFLYALALLLLA [SEQ ID NO: 36], wherein Xaai is absent or is S or a hyd rophilic amino acid, and Xaa∑ is absent or is from one to four hydrophilic amino acids, (cc) 8 or more contiguous a mino acid residues from the seq uence

SKKKKLMLLWTLVVLLICSSCSSCPLSKILLARLFLYALALLLLA [SEQ ID NO: 37],

(dd) 8 or more contiguous a mino acid residues from the seq uence

LMLLWTLVVLLICSSCSSCPLSKILLARLFLYALALLLLA [SEQ ID NO: 38],

(ee) 8 or more contiguous a mino acid residues from the seq uence

Xaa iXaa2Xaa ₃ Xaa4LMLLWTLVVLLICSSCSSCPLSKILL [SEQ ID NO: 39], wherein Xaai is absent or is S or a hydrophilic amino acid, Xaa∑ is absent or is a hydrophilic amino acid, Xaa3 is absent or is a hydrophilic amino acid, and Xaa4 is absent or is one or more hydrophilic amino acids,

(ff) 8 or more contiguous a mino acid residues from the seq uence

Xaa iXaa2Xaa ₃ LMLLWTLVVLLICSSCSSCPLSKILL [SEQ ID NO:40], wherein Xaai is absent or is S or a hydrophilic amino acid, Xaa2 is absent or is a hydrophilic amino acid, and Xaa3 is absent or is from one to ten hydrophilic amino acids,

(gg) 8 or more contiguous a mino acid residues from the seq uence

Xaa iXaa ₂ LMLLWTLVVLLICSSCSSCPLSKILL [SEQ ID NO:41 ], wherein Xaai is absent or is S or a hydrophilic amino acid, and Xaa∑ is absent or is from one to four hydrophilic amino acids,

(hh) 8 or more contiguous a mino acid residues from the seq uence

SKKKKLMLLWTLVVLLICSSCSSCPLSKILL [SEQ ID NO:42], (ii) 8 or more contiguous a mino acid residues from the seq uence

LMLLWTLVVLLICSSCSSCPLSKILL [SEQ ID NO:43],

(jj) 8 or more contiguous a mino acid residues from the seq uence

Xaa iXaa2Xaa ₃ Xaa4LLICSSCSSCPLSKILLARLFLYALALLLLA [SEQ ID NO:44], wherein Xaa i is absent or is S or a hydrophilic amino acid, Xaa∑ is absent or is a hydrophilic amino acid, Xaa3 is absent or is a hydrophilic amino acid, and Xaa4 is absent or is one or more hydrophilic amino acids,

(kk) 8 or more contiguous a mino acid residues from the seq uence

Xaa iXaa2Xaa3LLICSSCSSCPLSKILLARLFLYALALLLLA [SEQ ID NO:45], wherein Xaai is absent or is S or a hydrophilic amino acid, Xaa∑ is absent or is a hydrophilic amino acid, and Xaa3 is absent or is from one to ten hydrophilic amino acids,

(II) 8 or more contiguous a mino acid residues from the seq uence

Xaa 1Xaa2LLICSSCSSCPLSKILLARLFLYALAL.LL.LA [SEQ ID NO:46], wherein Xaai is absent or is S or a hydrophilic amino acid, a nd Xaa∑ is absent or is from one to four hydrophilic amino acids,

(mm)8 or more contiguous a mino acid residues from the seq uence

SKKKKLLICSSCSSCPLSKILLARLFLYALALLLLA [SEQ ID NO :47],

(nn) 8 or more contiguous a mino acid residues from the seq uence

LLICSSCSSCPLSKILLARLFLYALALLLLA [SEQ ID NO:48], (00) 8 or more contiguous a mino acid residues from the seq uence

Xaa iXaa ₂ Xaa3Xaa4LN LTTMFLLMLLWTLVVLLICSSCSSCPLSKILLARLFLYALALLLLASALIA GGSI [SEQ ID NO:49], wherein Xaai is absent or is S or a hydrophilic amino acid, Xaa2 is absent or is a hydrophilic amino acid, Xaa3 is absent or is a hydrophilic amino acid, and Xaa4 is absent or is one or more hydrophilic amino acids, (pp) 8 or more contiguous a mino acid residues from the seq uence

Xaa iXaa2Xaa3LNLTTMFLLMLLWTLVVLLICSSCSSCPLSKILLARLFLYALALLLLASAL IAGGS I [SEQ ID NO: 50], wherein Xaai is absent or is S or a hyd rophilic amino acid, Xaa∑ is absent or is a hydrophilic amino acid, and Xaa3 is absent or is from one to ten hydrophilic amino acids, (qq) 8 or more contiguous a mino acid residues from the seq uence

Xaa iXaa ₂ LNLTTMFLLMLLWTLVVLLICSSCSSCPLSKILLARLFLYALALLLLASALIAG GSI [SEQ ID NO: 51 ], wherein Xaa i is absent or is S or a hydrophilic amino acid, and Xaa2 is absent or is from one to four hydrophilic amino acids, (rr) 8 or more contiguous a mino acid residues from the seq uence

SKKKKLN LTTMFLLMLLWTLVVLLICSSCSSCPLSKILLARLFLYALALLLLASALIAGGSI [SEQ ID NO: 52],

(ss) 8 or more contiguous a mino acid residues from the seq uence

LNLTTMFLLMLLWTLVVLLICSSCSSCPLSKILLARLFLYALALLLLASALIAGGSI [SEQ ID

NO: 53],

(tt) 8 or more contiguous a mino acid residues from the seq uence

Xaa iXaa2Xaa ₃ Xaa4FLLMLLWTLVVLLICSSCSSCPLSKILLARLFLYALALLLLASA [SEQ ID NO: 54], wherein Xaai is absent or is S or a hydrophilic amino acid, Xaa2 is absent or is a hydrophilic amino acid, Xaa3 is absent or is a hydrophilic amino acid, a nd

Xaa4 is absent or is one or more hydrophilic amino acids,

(uu) 8 or more contiguous a mino acid residues from the seq uence

Xaa iXaa2Xaa ₃ FLLMLLWTLVVLLICSSCSSCPLSKILLARLFLYALALLLLASA [SEQ ID NO: 55], wherein Xaai is absent or is S or a hydrophilic amino acid, Xaa∑ is absent or is a hydrophilic amino acid, a nd Xaa3 is absent or is from one to ten hydrophilic amino acids,

(vv) 8 or more contiguous a mino acid residues from the seq uence

Xaa iXaa2FLLMLLWTLVVLLICSSCSSCPLSKILLARLFLYALALLLLASA [SEQ ID NO: 56], wherein Xaai is absent or is S or a hyd rophilic amino acid, and Xaa∑ is absent or is from one to four hydrophilic amino acids,

(ww) 8 or more contiguous a mino acid residues from the seq uence

SKKKKFLLMLLWTLVVLLICSSCSSCPLSKILLARLFLYALALLLLASA [SEQ ID NO: 57],

(xx) 8 or more contiguous a mino acid residues from the seq uence

FLLMLLWTLVVLLICSSCSSCPLSKILLARLFLYALALLLLASA [SEQ ID NO: 58], (yy) 8 or more contiguous a mino acid residues from the seq uence

Xaa iXaa2Xaa3Xaa4LQGIYVLVMLVLLILAYRRRWRRLTVCGGIMFLACVLVLIVDAVLQL SPLL [SEQ ID NO: 59], wherein Xaa i is absent or is S or a hydrophilic amino acid, Xaa∑ is absent or is a hydrophilic amino acid, Xaa3 is absent or is a hydrophilic amino acid, and Xaa4 is absent or is one or more hydrophilic amino acids, (zz) 8 or more contiguous a mino acid residues from the seq uence

Xaa iXaa2Xaa ₃ LQGIYVLVMLVLLILAYRRRWRRLTVCGGIMFLACVLVLIVDAVLQLSPLL [SEQ ID NO: 60], wherein Xaa i is absent or is S or a hydrophilic amino acid, Xaa∑ is absent or is a hydrophilic amino acid, and Xaa3 is absent or is from one to ten hydrophilic amino acids, (aaa)8 or more contiguous amino acid residues from the sequence

XaaiXaa ₂ LQGIYVLVMLVLLILAYRRRWRRLTVCGGIMFLACVLVLIVDAVLQLSPLL [SEQ ID NO:61], wherein Xaai is absent or is S or a hydrophilic amino acid, and Xaa∑ is absent or is from one to four hydrophilic amino acids, (bbb)8 or more contiguous amino acid residues from the sequence

SKKKKLQGIYVLVMLVLLILAYRRRWRRLTVCGGIMFLACVLVLIVDAVLQLSPLL [SEQ ID NO:62],

(ccc) 8 or more contiguous amino acid residues from the sequence

LQGIYVLVMLVLLILAYRRRWRRLTVCGGIMFLACVLVLIVDAVLQLSPLL [SEQ ID NO:63], (ddd)8 or more contiguous amino acid residues from the sequence

XaaiXaa2Xaa ₃ Xaa4SGNRTYGPVFM(C)(S)LGGLLTMVAGAVWLTVMSNTLLSAWILTAGFLI FLIGFA [SEQ ID NO:64], wherein Xaai is absent or is S or a hydrophilic amino acid, Xaa2 is absent or is a hydrophilic amino acid, Xaa3 is absent or is a hydrophilic amino acid, and Xaa4 is absent or is one or more hydrophilic amino acids, (eee) 8 or more contiguous amino acid residues from the sequence

XaaiXaa2Xaa ₃ SGNRTYGPVFM(C)(S)LGGLLTMVAGAVWLTVMSNTLLSAWILTAGFLIFLIG FA [SEQ ID NO:65], wherein Xaai is absent or is S or a hydrophilic amino acid, Xaa∑ is absent or is a hydrophilic amino acid, and Xaa3 is absent or is from one to ten hydrophilic amino acids, (fff) 8 or more contiguous amino acid residues from the sequence

XaaiXaa ₂ SGNRTYGPVFM(C)(S)LGGLLTMVAGAVWLTVMSNTLLSAWILTAGFLIFLIG FA [SEQ ID NO:66], wherein Xaai is absent or is S or a hydrophilic amino acid, and Xaa2 is absent or is from one to four hydrophilic amino acids,

(ggg)8 or more contiguous amino acid residues from the sequence

SKKKKSGNRTYGPVFM(C)(S)LGGLLTMVAGAVWLTVMSNTLLSAWILTAGFLIFLIGF A [SEQ ID NO:67],

(hhh)8 or more contiguous amino acid residues from the sequence

SGNRTYGPVFM(C)(S)LGGLLTMVAGAVWLTVMSNTLLSAWILTAGFLIFLIGFA [SEQ ID NO:68], (iii) 8 or more contiguous amino acid residues from the sequence

XaaiXaa2Xaa ₃ Xaa4SNEEPPPPYEDPYWGNGDRHSDYQPLGTQDQSLYLGLQHDGNDGLPP [SEQ ID NO:69], wherein Xaai is absent or is S or a hydrophilic amino acid, Xaa∑ is absent or is a hydrophilic amino acid, Xaa3 is absent or is a hydrophilic amino acid, and Xaa4 is absent or is one or more hydrophilic amino acids, (jjj) 8 or more contiguous a mino acid residues from the seq uence

Xaa iXaa ₂ Xaa ₃ SN EEPPPPYEDPYWGNGDRHSDYQPLGTQDQSLYLGLQHDGN DGLPP [SEQ ID NO: 70], wherein Xaai is absent or is S or a hydrophilic amino acid, Xaa∑ is absent or is a hydrophilic amino acid, and Xaa3 is absent or is from one to ten hydrophilic amino acids,

(kkk) 8 or more contiguous a mino acid residues from the seq uence

Xaa iXaa ₂ SNEEPPPPYEDPYWGNGDRHSDYQPLGTQDQSLYLGLQHDGN DGLPP [SEQ ID NO: 71 ], wherein Xaai is absent or is S or a hydrophilic amino acid, and Xaa∑ is absent or is from one to four hydrophilic amino acids,

(III) 8 or more contiguous a mino acid residues from the seq uence

SKKKKSN EEPPPPYEDPYWGNGDRHSDYQPLGTQDQSLYLGLQHDGNDGLPP [SEQ ID NO: 72],

(mmm) 8 or more contiguous a mino acid residues from the sequence

SN EEPPPPYEDPYWGNGDRHSDYQPLGTQDQSLYLGLQHDGNDGLPP [SEQ ID NO: 73],

(nnn) 8 or more contiguous a mino acid residues from the seq uence

Xaa iXaa2Xaa ₃ Xaa4GN DGLPPPPYSPRDDSSQHIYEEAGRGSMN PVCLPVIVAPYLFWLAAIAA S [SEQ ID NO: 74], wherein Xaai is absent or is S or a hydrophilic amino acid, Xaa∑ is absent or is a hydrophilic amino acid, Xaa3 is absent or is a hydrophilic amino acid, and Xaa4 is absent or is one or more hydrophilic amino acids,

(ooo) 8 or more contiguous a mino acid residues from the seq uence

Xaa iXaa2Xaa ₃ GNDGLPPPPYSPRDDSSQHIYEEAGRGSMNPVCLPVIVAPYLFWLAAIAAS [SEQ ID NO: 75], wherein Xaa i is absent or is S or a hydrophilic amino acid, Xaa∑ is absent or is a hydrophilic amino acid, and Xaa3 is absent or is from one to ten hydrophilic amino acids,

(ppp) 8 or more contiguous a mino acid residues from the seq uence

Xaa iXaa2GNDGLPPPPYSPRDDSSQHIYEEAGRGSM NPVCLPVIVAPYLFWLAAIAAS [SEQ ID NO: 76], wherein Xaai is absent or is S or a hydrophilic amino acid, and Xaa∑ is absent or is from one to four hydrophilic amino acids,

(qqq) 8 or more contiguous a mino acid residues from the seq uence

SKKKKGNDGLPPPPYSPRDDSSQHIYEEAGRGSMNPVCLPVIVAPYLFWLAAIAAS [SEQ ID NO: 77],

(rrr) 8 or more contiguous amino acid residues from the sequence

GN DGLPPPPYSPRDDSSQHIYEEAGRGSMNPVCLPVIVAPYLFWLAAIAAS [SEQ ID NO: 78], (sss) 8 or more contiguous a mino acid residues from the seq uence

Xaa iXaa2Xaa ₃ Xaa4AAIAASCFTASVSTVVTATGLALSLLLLAAVASSYAAAQRKLLTPVTVLT [SEQ ID NO: 79], wherein Xaa i is absent or is S or a hydrophilic amino acid, Xaa∑ is absent or is a hydrophilic amino acid, Xaa3 is absent or is a hydrophilic amino acid, and Xaa4 is absent or is one or more hydrophilic amino acids,

(ttt) 8 or more contiguous a mino acid residues from the seq uence

Xaa iXaa2Xaa ₃ AAIAASCFTASVSTVVTATGLALSLLLLAAVASSYAAAQRKLLTPVTVLT [SEQ ID NO: 80], wherein Xaai is absent or is S or a hydrophilic amino acid, Xaa∑ is absent or is a hydrophilic amino acid, and Xaa3 is absent or is from one to ten hydrophilic amino acids,

(uuu) 8 or more contiguous a mino acid residues from the seq uence

Xaa iXaa ₂ AAIAASCFTASVSTVVTATGLALSLLLLAAVASSYAAAQRKLLTPVTVLT [SEQ ID NO: 81 ], wherein Xaai is absent or is S or a hydrophilic amino acid, and Xaa∑ is absent or is from one to four hydrophilic amino acids, (vvv) 8 or more contiguous a mino acid residues from the seq uence

SKKKKAAIAASCFTASVSTVVTATGLALSLLLLAAVASSYAAAQRKLLTPVTVLT [SEQ ID NO: 82],

(www) 8 or more contiguous a mino acid residues from the sequence

AAIAASCFTASVSTVVTATGLALSLLLLAAVASSYAAAQRKLLTPVTVLT [SEQ ID NO: 83], (xxx) the sequence of any one of SEQ ID NOs: 9 to 83,

(yyy) 8 or more contiguous a mino acid residues from the seq uence of any one of

ESNEEPPPPY [SEQ ID NO: 84],

SN EEPPPPY [SEQ ID NO: 85],

HSDYQPLGT [SEQ ID NO: 86],

PLGTQDQSL [SEQ ID NO: 87],

PLGTQDQSLY [SEQ ID NO: 88],

LGTQDQSLY [SEQ ID NO: 89],

GTQDQSLYL [SEQ ID NO: 90],

GTQDQSLYL [SEQ ID NO: 91],

GTQDQSLYLG [SEQ ID NO: 92],

QSLYLGLQH [SEQ ID NO: 93],

SLYLGLQHD [SEQ ID NO: 94],

GLQHDGN DGL [SEQ ID NO: 95],

GN DGLPPPPY [SEQ ID NO: 96],

GLPPPPYSP [SEQ ID NO: 97], GLPPPPYSPR [SEQ ID NO: 98],

PRDDSSQHIY [SEQ ID NO: 99],

RDDSSQHIY [SEQ ID NO: 100],

HIYEEAGRG [SEQ ID NO: 101],

ILLARLFLY [SEQ ID NO: 102],

SSCSSCPLSKI [SEQ ID NO: 103],

LLWTLVVLL [SEQ ID NO : 104],

FLYALALLL [SEQ ID NO: 105],

CLGGLLTMV [SEQ ID NO: 106],

LIVDAVLQL [SEQ ID NO: 107],

LTAGFLIFL [SEQ ID NO : 108],

TVCGGIMFL [SEQ ID NO: 109],

(zzz) the sequence of any one of SEQ ID NOs: 83- 109,

(aaaa) or a ny combination of two or more of (a) to (zzz) above. In one exemplary embodiment, the peptide comprises one or more epitopes derived from Latent Membrane Protein 2 (LMP2), for example, from full-length EBV LMP2 (amino acids 1-497). In one specifically contemplated embodiment, the peptide comprises, consists essentia lly of, or consists of an amino acid sequence selected from the group consisting of 8 or more contiguous amino acid residues from any one of SEQ ID NOs: 12, 13, 17, 18, 22, 23, 27, 28, 32, 33, 37, 38, 42, 43, 47, 48, 52, 53, 57, 58, 62, 63, 67, 68, 72, 73, 77, 78, 82, or 83.

In another specifically contemplated embodiment, the peptide comprises, consists essentia lly of, or consists of an amino acid sequence selected from the group consisting of 12 or more contiguous amino acid residues from any one of SEQ ID NOs: 12, 13, 17, 18, 22, 23, 27, 28, 32, 33, 37, 38, 42, 43, 47, 48, 52, 53, 57, 58, 62, 63, 67, 68, 72, 73, 77, 78, 82, or 83.

In another specifically contemplated embodiment, the peptide comprises, consists essentia lly of, or consists of an amino acid sequence selected from the group consisting of 15 or more, 18 or more, 20 or more, or 25 or more contiguous amino acid residues from any one of SEQ ID NOs: 12, 13, 17, 18, 22, 23, 27, 28, 32, 33, 37, 38, 42, 43, 47, 48, 52, 53, 57, 58, 62, 63, 67, 68, 72, 73, 77, 78, 82, or 83.

In one embodiment, the peptide comprises, consists essentially of, or consists of a n amino acid seq uence selected from the group consisting of any one of SEQ ID NOs: 12, 13, 17, 18, 22, 23, 27, 28, 32, 33, 37, 38, 42, 43, 47, 48, 52, 53, 57, 58, 62, 63, 67, 68, 72, 73, 77, 78, 82, or 83. In another specifically contemplated embodiment, the peptide comprises, consists essentia lly of, or consists of an amino acid sequence selected from the group consisting of 15 or more, 18 or more, 20 or more, or 25 or more contiguous amino acid residues from any one of SEQ ID NOs: 9 to 83. In one embodiment, the peptide comprises, consists essentially of, or consists of a n amino acid seq uence selected from the group consisting of any one of SEQ ID NOs: 9 to 83.

In one embodiment, the peptide comprises an amino acid sequence selected from the group consisting of any one of SEQ ID NOs: 84 to 109. In one example, the peptide comprises a n amino acid sequence selected from the group consisting of a ny one of SEQ ID NOs: 84 to 101.

In one embodiment, the peptide comprises an amino acid sequence selected from the group consisting of any two or more of SEQ ID NOs : 84 to 109. In one example, the peptide comprises an a mino acid sequence selected from the group consisting of any two or more of SEQ ID NOs: 84 to 101.

In various embodiments the peptide comprises, consists of, or consists essentially of a n amino acid seq uence selected from the group consisting of

(a) 8 or more contig uous amino acid residues from the sequence

XaaiXaa2Xaa ₃ Xaa4GARGPESRLLEFYLAMPFATPM EAELARRSLAQDAPPL [SEQ ID

NO: 110], wherein Xaai is absent or is S, Xaa∑ is absent or is a hydrophilic amino acid,

Xaa3 is absent or is a hydrophilic amino acid, and Xaa4 is absent or is one or more hyd rophilic amino acids,

(b) 8 or more contig uous amino acid residues from the sequence

XaaiXaa2Xaa ₃ GARGPESRLLEFYLAMPFATPM EAELARRSLAQDAPPL [SEQ ID NO: l l l ], wherein Xaai is absent or is S, Xaa∑ is absent or is a hydrophilic amino acid, and Xaa3 is absent or is from one to ten hydrophilic amino acids,

(c) 8 or more contig uous amino acid residues from the sequence

XaaiXaa ₂ GARGPESRLLEFYLAMPFATPMEAELARRSLAQDAPPL [SEQ ID NO: 112], wherein Xaai is absent or is S, and Xaa∑ is absent or is from one to four hydrophilic amino acids,

(d) 8 or more contig uous amino acid residues from the sequence

SKKKKGARGPESRLLEFYLAMPFATPMEAELARRSLAQDAPPL [SEQ ID NO : 113],

(e) the sequence of a ny one of SEQ ID NOs: 110 to 113, (f) 8 or more contig uous amino acid residues from the sequence

GARGPESRLLEFYLAM PFATPM EAELARRSLAQDAPPL [SEQ ID NO : 114],

(g) the sequence of SEQ ID NO: 114,

(h) 8 or more contig uous amino acid residues from the sequence LAMPFATPM [SEQ ID NO: 115],

(i) the sequence of SEQ ID NO: 115,

(j) 8 or more contig uous amino acid residues from the sequence FATPM EAEL [SEQ ID NO: 116],

(k) the sequence of SEQ ID NO: 116, (I) 8 or more contig uous amino acid residues from the sequence

XaaiXaa ₂ Xaa ₃ Xaa4VPGVLLKEFTVSGNILTIRLTAADHR [SEQ ID NO: 117], wherein Xaai is absent or is S, Xaa∑ is absent or is a hydrophilic amino acid, Xaa3 is absent or is a hyd rophilic amino acid, and Xaa4 is absent or is one or more hydrophilic amino acids,

(m) 8 or more contiguous a mino acid residues from the sequence

XaaiXaa ₂ Xaa ₃ VPGVLLKEFTVSGNILTIRLTAADHR [SEQ ID NO: 118], wherein Xaai is absent or is S, Xaa∑ is absent or is a hydrophilic amino acid, a nd Xaa3 is absent or is from one to ten hydrophilic amino acids,

(n) 8 or more contig uous amino acid residues from the sequence

XaaiXaa ₂ VPGVLLKEFTVSGNILTIRLTAADHR [SEQ ID NO: 119], wherein Xaai is absent or is S, and Xaa∑ is absent or is from one to four hyd rophilic amino acids,

(o) 8 or more contig uous amino acid residues from the sequence

SKKKKVPGVLLKEFTVSGNILTIRLTAADHR [SEQ ID NO : 120],

(p) the sequence of a ny one of SEQ ID NOs: 117 to 120,

(q) 8 or more contig uous amino acid residues from the sequence

VPGVLLKEFTVSGNILTIRLTAADHR [SEQ ID NO: 121],

(r) the sequence of SEQ ID NO: 121,

(s) 8 or more contiguous amino acid residues from the sequence EFTVSGNIL [SEQ ID NO: 122],

(t) the sequence of SEQ ID NO: 122, (u) 8 or more contig uous amino acid residues from the sequence

XaaiXaa2Xaa ₃ Xaa4LQQLSLLMWITQCFLPVFLAQPPSGQRR [SEQ ID NO: 123], wherein Xaai is absent or is S, Xaa∑ is absent or is a hydrophilic amino acid, Xaa3 is absent or is a hydrophilic amino acid, a nd Xaa4 is absent or is one or more hydrophilic amino acids

(v) 8 or more contig uous amino acid residues from the sequence

XaaiXaa2Xaa ₃ LQQLSLLMWITQCFLPVFLAQPPSGQRR [SEQ ID NO: 124], wherein Xaai is absent or is S, Xaa∑ is absent or is a hydrophilic amino acid, and Xaa3 is absent or is from one to ten hydrophilic amino acids, (w) 8 or more contiguous amino acid residues from the sequence

XaaiXaa ₂ LQQLSLLMWITQCFLPVFLAQPPSGQRR [SEQ ID NO: 125], wherein Xaai is absent or is S, and Xaa∑ is absent or is from one to four hydrophilic amino acids,

(x) 8 or more contig uous amino acid residues from the sequence

SKKKKLQQLSLLMWITQCFLPVFLAQPPSGQRR [SEQ ID NO: 126], (y) the sequence of a ny one of SEQ ID NOs: 123 to 126,

(z) 8 or more contig uous amino acid residues from the sequence

LQQLSLLMWITQCFLPVFLAQPPSGQRR [SEQ ID NO: 127],

(aa) the sequence of SEQ ID NO: 127,

(bb) 8 or more contiguous a mino acid residues from the seq uence SLLMWITQCFLPVF [SEQ ID NO : 128],

(cc) the sequence of SEQ ID NO: 128,

(dd) 8 or more contiguous a mino acid residues from the seq uence SLLMWITQC [SEQ ID NO: 129],

(ee) the sequence of SEQ ID NO: 129, (ff) or any combination of two or more of (a) to (ee) above.

In one exemplary embodiment, the peptide epitope is derived from NY-ESO-1. In one specifically contemplated embodiment, the peptide comprises, consists essentially of, or consists of an amino acid sequence selected from the group consisting of 8 or more contiguous amino acid residues from any one of SEQ ID NO: 114, 115, 116, 121, 122, 127, 128, and 129. In one embodiment, the peptide comprises, consists essentially of, or consists of a n amino acid seq uence selected from the group consisting of any one of SEQ ID NO: 114, 115, 116, 121, 122, 127, 128, and 129.

In one embodiment, the peptide comprises, consists essentially of, or consists of a n amino acid seq uence selected from the group consisting of any one of SEQ ID NO: 114, 121, and 127.

In one embodiment, the peptide comprises, consists essentially of, or consists of a n amino acid seq uence selected from the group consisting of any one of SEQ ID NO: 113, 120, and 126. In various embodiments, the peptide comprises, consists essentially of, or consists of one or more ovalbumin protein epitopes. In various embodiments, the one or more ovalbumin protein are M HCI epitopes. In various embodiments, the one or more ovalbumin protein are M HCII epitopes.

In various embodiments, the peptide comprises, consists essentially of, or consists of: (a) 8 or more contig uous amino acid residues from the sequence

XaaiXaa2Xaa ₃ Xaa4KISQAVHAAHAEINEAGRESIIN FEKLTEWT [SEQ ID NO: 130], wherein Xaai is absent or is S, Xaa∑ is absent or is a hydrophilic amino acid, Xaa3 is absent or is a hydrophilic amino acid, a nd Xaa4 is absent or is one or more hydrophilic amino acids (b) 8 or more contig uous amino acid residues from the sequence XaaiXaa2Xaa3

KISQAVHAAHAEINEAGRESIINFEKLTEWT [SEQ ID NO: 131], wherein Xaai is absent or is S, Xaa2 is absent or is a hydrophilic amino acid, and Xaa3 is absent or is from one to ten hyd rophilic amino acids,

(c) 8 or more contiguous amino acid residues from the sequence XaaiXaa2

KISQAVHAAHAEINEAGRESIINFEKLTEWT [SEQ ID NO: 132], wherein Xaai is absent or is S, and Xaa2 is absent or is from one to four hydrophilic amino acids,

(d) 8 or more contig uous amino acid residues from the sequence

SKKKKKISQAVHAAHAEINEAGRESIINFEKLTEWT [SEQ ID NO: 133],

(e) the sequence of a ny one of SEQ ID NOs: 130 to 133, (f) 8 or more contig uous amino acid residues from the sequence

KISQAVHAAHAEINEAGRESIINFEKLTEWT [SEQ ID NO: 134],

(g) the sequence of SEQ ID NO: 134, (h) 8 or more contig uous amino acid residues from the sequence SIIN FEKL [SEQ ID NO: 135],

(i) the sequence of SEQ ID NO: 135,

(j) 8 or more contig uous amino acid residues from the sequence ISQAVHAAHAEINEAGR [SEQ ID NO : 136],

(k) the sequence of SEQ ID NO: 136,

(I) or any combination of any two or more of (a) to (k) above.

In various embodiments, the peptide comprises one or more ovalbumin protein epitopes selected from the g roup consisting of any one of SEQ ID NOs 130 - 136. In va rious embodiments, the peptide comprises a peptide comprising or consisting of 8 or more contiguous amino acids from the amino acid sequence of any one of SEQ ID NOs 130 - 136.

In various embodiments, the peptide comprises, consists of, or consists essentially of an amino acid seq uence selected from the group consisting of any one of SEQ ID NOs 130 - 136.

In various embodiments, the peptide comprises one or more immunodominant A*0200 restricted epitopes derived from the cytomeglovirus (CMV) ppUL83 protein ('N LV peptide') consisting of

(gg) 8 or more contiguous a mino acid residues from the sequence N LVPMVATV [SEQ ID NO: 137],

(hh) the sequence of SEQ ID NO: 137,

(ii) 8 or more contig uous amino acid residues from the sequence CSKKKKNLVPMVATV [SEQ ID NO : 138],

(jj) the sequence of SEQ ID NO: 138. In various embodiments, the peptide comprises, consists essentially of, or consists of an amino acid seq uence selected from the group consisting of 8 or more contiguous amino acids from the amino acid sequence of any one of SEQ ID NOs 9 - 129.

In various embodiments, the peptide comprises, consists essentially of, or consists of one or more amino acid sequences selected from the group consisting of those defined in proviso (1) of the first aspect. In various embodiments, Xaa4 in the sequences referred to herein is absent or is from 1 to 17 hydrophilic amino acids, for example, from 1 to 16, 1 to 15, 1 to 14, 1 to 13, 1 to 12, 1 to 11, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 to 2 hydrophilic amino acids, or is a hydrophilic amino acid.

In one embodiment, the peptide conjugate comprises two or more epitopes, such as two or more peptide epitopes.

In some embodiments, the peptide conjugate comprises an antigenic peptide.

In specifically contemplated embodiments, the peptide is a synthetic peptide. In various embodiments, the compound of formula (I) is an isolated compound of formula (I).

In various embodiments, the compound of formula (I) is a pure, purified or substantially pure compound of formula (I).

In various embodiments, the compound of formula (I) of the invention is a compound selected from the group consisting of compounds 910, 911, 912, 913, 930, 931, and 932 of the Examples herein. In another aspect, the present invention broadly consists in a method of making a peptide conjugate of the formula (IF) or a pharmaceutically acceptable salt or solvate thereof of the present invention, the method comprising :

(A) reacting

an epoxide of the formula (XVI):

(XVI); and

an amino acid-comprising conjugation partner comprising a thiol of the formula

(HI), under conditions effective to conjugate the epoxide and amino acid-comprising conjugation partner and provide a compound of formula (XV):

(XV)

wherein

X10 is L1-Z1-, -OH, -SH, -NHR, HNRC(0)0-, P10-O-, P11-S-, P12-NR-, or P12-NRC(0)0-;

Xll is X10 or -OH, -SH, -NHR, or HNRC(0)0- when X10 is P10-O-, P11-S-, P12- NR-, or P12-NRC(0)0- and said conditions are effective to remove P10, Pll, or P12;

P10, Pll, and P12 are each independently a protecting group;

m, n, LI, Zl, R, Rl, R2, R3, R4, R5, R6, R7, R8, R9, and Al are as defined in the compound of formula (IF) of the invention (including provisos (1) and/or (2) of the first aspect); and

converting the compound of formula (XV) to the peptide-conjugate of the formula (IF) of the invention (including provisos (1) and/or (2) of the first aspect) or a pharmaceutically acceptable salt or solvate thereof by one or more additional synthetic steps:

reacting

an epoxide of the formula (XVI)

(XVI); and

an amino acid-comprising conjugation partner comprising a thiol of the formula (III):

(HI), under conditions effective to conjugate the epoxide a nd amino acid-comprising conjugation partner and provide a compound of formula (XV) :

(XV)

wherein

X10 is L1-Z1-, -OH, -SH, -N HR, HN RC(0)0-, P10-O-, P11-S-, P12-N R-, or P12-N RC(0)0-;

Xl l is X10 or -OH, -SH, -N HR, or HNRC(0)0- when X10 is P10-O-, P11-S-, P12- NR-, or P12-NRC(0)0- and said cond itions are effective to remove P10, Pl l, or P12;

P10, Pl l, and P12 a re each independently a protecting group;

m, n, LI, Zl, R, Rl, R2, R3, R4, R5, R6, R7, R8, R9, and Al a re as defined in the compound of formula (IF) but excluding provisos (1) and (2) of the first aspect; and converting the compound of formula (XV) to an amino acid- or peptide-conjugate of the formula (IF) but excluding provisos (1) and (2) of of the first aspect or a salt or solvate thereof by one or more additional synthetic steps:

(IF); and

coupling the amino acid of the amino acid conjugate or an amino acid of the peptide conjugate to an amino acid or an amino acid of a peptide to provide the peptide- conjugate of formula (IF) of the present invention (including provisos (1) and/or (2) of the first aspect) or pha rmaceutically acceptable salt or solvate thereof.

In various embodiments m is from 2 to 5, 2 to 4, or 2 to 3. In exemplary embodiments, m is 2. In other exemplary embodiments, m is from 3 to 5.

In various embodiments, X10 is Ll-Zl- or -OH, -SH, -NHR, P10-O-, P11-S-, or P12- NR-; and Xl l is X10 or -OH, -SH, or -NHR.

In various embodiments, X10 is L1-Z1-, -OH, or P10-O-; and Xl l is X10 or -OH .

In various embodiments, X10 is L1-C(0)0-, OH, or P10-O-; and Xl l is L1-C(0)0-, P10-O-, or OH . In various embodiments, X10 is L1-C(0)0- or P10-O-; and Xll is L1-C(0)0-, P10-O-, or OH.

In exemplary embodiments, X10 is P10-O-; and Xll is PIO-O- or OH.

In various embodiments, R9 is not hydrogen and/or Al is not OH.

In various embodiments, the amino acid-comprising conjugation partner is a peptide containing conjugation partner comprising 15 or less, 14 or less, 13 or less, 12 or less, 11 or less, 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, or 3 or less amino acid residues.

In various embodiments, the C-terminus of the amino acid comprising conjugation partner is protected with a carboxyl protecting group or a carboxamide protecting group and/or the Na-amino group of the amino acid comprising conjugation partner is protected with an amino protecting group.

In exemplary embodiments, R9 is an amino protecting group. In various embodiments, Al is OPl or NHP2. In certain embodiments, Al is OPl.

In exemplary embodiments, R9 is an amino protecting group and Al is OPl in the amino acid comprising conjugation partner. In various embodiments, the method comprises reacting the epoxide and amino acid- comprising conjugation partner in the presence of an acid, for example a strong acid.

In certain embodiments, the acid comprises hydrochloric acid, sulfuric acid, or a mixture thereof.

In certain embodiments, the acid comprises a lewis acid, for example BF3.

In other embodiments, the method comprises reacting the epoxide and amino acid- comprising conjugation partner under neutral conditions.

In various embodiments, the neutral conditions comprise a protic solvent, such as an alcohol, for example ethanol. In other embodiments, the method comprises reacting the epoxide and amino acid- comprising conjugation partner in the presence of a base, for example a mild base.

In some embod iments, the base is an organic amine, for example triethylamine.

In various embodiments, the method comprises providing the epoxide by reacting an alkene of the formula (XVII) :

(XVII)

and a n oxidant under conditions effective to epoxidise the a lkene.

In various embodiments, the oxida nt is a peroxide, such as an organic peroxide, for example m-chloro peroxybenzoic acid, or a n organic N-oxide, for example pyridine N- oxide.

In various embodiments, the method comprises providing the epoxide by reacting an compound of the formula (XVII-A) wherein LG is a leaving group:

(XVII-A)

and a base under conditions effective for epoxidation.

In various embodiments, the compound of formula (XVII-A) is prepared from L-aspartic acid . In various embodiments, the method further comprises providing a single stereoisomer or a stereoisomerically enriched mixture of the epoxide of formula (XVI).

In various embodiments, providing the single stereoisomer or a stereoisomerically enriched mixture of the epoxide of formula (XVI) comprises resolving a racemic mixture of the epoxide.

In various embodiments, the method comprises provid ing a single stereoisomer or a stereoisomerically enriched mixture of the compound of formula (XVII-A). In various embodiments, the method comprises converting the compound of formula (XV) to an amino acid- or peptide conjugate of the formula (IF-1) or a pharmaceutically acceptable salt or solvate thereof by one or more additional synthetic steps:

(IF-1).

In various embodiments, the one or more synthetic steps comprises converting the hydroxyl group bound to the carbon to which R3 is attached to L2-Z2-.

In various embodiments, the one or more synthetic steps comprises acylating the compound of formula (XV) so as to replace the hydrogen atom of the hydroxyl group bound to the carbon to which R3 is attached with L2-C(0)-.

In various embodiments, Xl l is PIO-O- or OH; and the one or more synthetic steps comprise acylating the compound of formula (XV) so as to replace P10 or the hydrogen atom of the hydroxyl group of Xl l with Ll-C(O)-; and/or acylating the compound of formula (XV) so as to replace the hydrogen atom of the hydroxyl group bound to the carbon to which R3 is attached with L2-C(0)-.

In another aspect, the present invention broadly consists in a compound of the formula

Xll is L1-Z1-, -OH, -SH, -NHR, HNRC(0)0-, P10-O-, P11-S-, P12-N R-, or P12-NRC(0)0-;

P10, Pll, and P12 are each independently a protecting group;

m is an integer from 2 to 6; and

n, LI, Zl, R, Rl, R2, R3, R4, R5, R6, R7, R8, R9, and Al are as defined in the compound of formula (I) of the invention (including provisos (1) and/or (2) of the first aspect) or any embodiment thereof; or a salt or solvate thereof. In another aspect, the present invention broadly consists in a method of making a compound of the formula (XV) or a salt or solvate thereof, the method comprising: reacting

an epoxide of the formula (XVI)

(XVI); and

an amino acid-comprising conjugation partner comprising a thiol of the formula

(III):

(HI),

under conditions effective to conjugate the epoxide and amino acid-comprising conjugation partner and provide a compound of formula (XV):

(XV)

wherein

X10 is L1-Z1-, -OH, -SH, -NHR, HNRC(0)0-, P10-O-, P11-S-, P12-NR-, or P12-NRC(0)0-;

Xll is X10 or -OH, -SH, -NHR, or HNRC(0)0- when X10 is P10-O-, P11-S-, P12- NR-, or P12-NRC(0)0- and said conditions are effective to remove P10, Pll, or P12;

P10, Pll, and P12 are each independently a protecting group;

m, n, LI, Zl, R, Rl, R2, R3, R4, R5, R6, R7, R8, R9, and Al are as defined in the compound of formula (IF) of the invention (including provisos (1) and/or (2) of the first aspect). In another aspect, the present invention broadly consists in the use of a compound of the formula (XV) or (XVI) in the synthesis of a peptide-conjugate of the formula (IF) of the present invention (including provisos (1) and/or (2) of the first aspect) or a

pharmaceutically acceptable salt or solvate thereof. In another aspect, the present invention broadly consists in a method of making a peptide-conjugate of the formula (I) or a pharmaceutically acceptable salt or solvate thereof of the present invention, the method comprising:

(A) reacting

a compound of the formula (XXI):

(XXI); and

an amino acid-comprising conjugation partner comprising a thiol of the formula (III):

(HI),

under conditions effective to conjugate the compound of formula (XXI) and amino acid-comprising conjugation partner and provide a compound of formula (XX):

(XX)

wherein

Rm and Rn are each independently hydrogen, Cl-6alkyl, aryl, or heteroaryl;

LG is a leaving group; and

m, w, v, n, Rx, Ry, Rl, R2, R3, R4, R5, R6, R7, R8, R9, and Al are as defined in the compound of formula (I) of the present invention (including provisos (1) and/or (2) of the first aspect); and

converting the compound of formula (XX) to a peptide conjugate of the formula (I) of the present invention (including provisos (1) and/or (2) of the first aspect) or a pharmaceutically acceptable salt or solvate thereof by one or more additional synthetic steps:

(I); or reacting

a compound of the formula (XXI)

(XXI); a nd

an amino acid-comprising conjugation partner comprising a thiol of the formula

(III) :

(HI),

under conditions effective to conj ugate the compound of formula (XXI) and amino acid-comprising conjugation pa rtner a nd provide a compound of formula (XX) :

(XX)

wherein

Rm a nd Rn are each independently hydrogen, Cl-6alkyl, aryl, or heteroaryl; LG is a leaving group; and

m, w, v, n, Rx, Ry, Rl, R2, R3, R4, R5, R6, R7, R8, R9, a nd Al are as defined in the compound of formula (I) but exclud ing provisos (1) and (2) of the first aspect; and converting the compound of formula (XX) to an amino acid- or peptide conj ugate of the formula (I) but excluding provisos (1) a nd (2) of the first aspect or a salt or solvate thereof by one or more additional synthetic steps:

(I); a nd

coupling the amino acid of the amino acid conjugate or an amino acid of the peptide conjugate to an amino acid or an amino acid of a peptide to provide the peptide- conjugate of formula (I) of the present invention (including provisos (1) and/or (2) of the first aspect) or pharmaceutically acceptable salt or solvate thereof..

In various embodiments, Rm and Rn a re each independently selected from hydrogen, Cl-6alkyl, or aryl.

In certain embodiments, Rm is hydrogen, Cl-6alkyl, or aryl ; and Rn is Cl-6alkyl or a ryl .

In various embodiments, the leaving group is a ha lo (for example chloro, bromo, or iodo) or sulfonate (for example a tosylate or mesylate).

In various embodiments, m and v are such that the compound of formula (XXI) comprises a 5-7-membered cyclic aceta l.

In certain embodiment, the cyclic aceta l is a 6-membered cyclic acetal.

In various embodiments, the cyclic aceta l is a 5-membered cyclic acetal and w is an integer greater than 1.

In various embodiments, m is 2 and v is 1.

In various embodiments, R9 is not hydrogen and/or Al is not OH .

In various embodiments, the amino acid-comprising conjugation partner is a peptide containing conjugation partner comprising 15 or less, 14 or less, 13 or less, 12 or less, 11 or less, 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, or 3 or less amino acid residues.

In various embodiments, the C-terminus of the amino acid comprising conjugation partner is protected with a carboxyl protecting group or a carboxamide protecting group and/or the Na-amino group of the amino acid comprising conjugation partner is protected with an amino protecting group.

In exemplary embodiments, R9 is an amino protecting group.

In various embodiments, Al is OPl or NHP2. In certain embodiments, Al is OPl.

In exemplary embodiments, R9 is an amino protecting group and Al is OPl in the amino acid comprising conjugation partner.

In various embodiments, the method comprises reacting the compound of formula (XXI) and the amino acid-comprising conjugation partner of formula (III) in the presence of a base.

In various embodiments, the base comprises an organic amine, for example

triethylamine, N-methylmorpholine, or collidine.

In various embodiments, the cyclic acetal of formula (XXI) is provided in the form of a single stereoisomer or a stereoisomerically enriched mixture.

In various embodiments, the method comprises converting the compound of formula (XX) to an amino acid- or peptide conjugate of the formula (IA) or a pharmaceutically acceptable salt or solvate thereof by one or more synthetic steps:

In various embodiments, the one or more synthetic steps comprises removing the acetal in the compound of formula (XX) to provide a compound of the formula (XXIII-1):

(XXIII-1).

In various embodiments, wherein Rm is optionally substituted aryl, for example phenyl or methoxy substituted phenyl, the method comprises removing the acetal in the compound of formula (XX) to provide a compound of the formula (XXIII-2) or (XXIII-3):

(XXIII-2)

In various embodiments, the one or more synthetic steps comprise converting the hydroxyl group bound to the carbon to which Rl and R2 are attached in the compound of formula (XXIII-1) to L1-Z1-, and/or converting the hydroxyl group bound to the carbon to which Rx and Ry are attached to L2-Z2.

In various embodmiments, the one or more synthetic steps comprise

converting the hydroxyl group bound to the carbon atom to which Rx and Ry are attached in the compound of formula (XXIII-2) to L2-Z2-, removing the RmRnCH- group to provide a hydroxyl group, and converting the hydroxyl group to Ll-Zl; or

converting the hydroxyl group bound to the cabon to which Rx and Ry are attached in the compound of formula (XXIII-2) to L1-Z1-, removing the RmRnCH- group to provide a hydroxyl group, and converting the hydroxyl group to L2-Z2-.

In various embodiments, converting said hydroxyl group to Ll-Zl- or L2-Z2- comprises acylating so as to replace the hydrogen atom of the hydroxyl group with Ll-C(0)-or L2- C(O)-. In another aspect, the present invention broadly consists in a compound of the formula (XX) :

(XX)

wherein :

Rm a nd Rn are each independently hydrogen, Cl-6alkyl, aryl, or heteroaryl; m and w are each independently a n integer from 0 to 7 and v is an integer from 0 to 5,

provided that:

the sum of m, v, and w is at least 3; and

the sum of m and w is from 0 to 7; and

n, Rx, Ry, Rl, R2, R3, R4, R5, R6, R7, R8, R9, and Al a re as defined in the compound of formula (I) of the present invention (including provisos (1) a nd/or (2) of the first aspect) or any embodiment thereof; or a salt or solvate thereof.

In another aspect, the present invention broadly consists in a method of making a compound of the formula (XX) or a salt or solvate thereof, the method comprising :

(A) reacting

a compound of the formula (XXI) :

(XXI); a nd

an amino acid-comprising conjugation partner comprising a thiol of the formula

(III) :

(III), under conditions effective to conj ugate the compound of formula (XXI) and amino acid-comprising conjugation pa rtner a nd provide a compound of formula (XX) :

(XX)

wherein

Rm a nd Rn are each independently hydrogen, Cl-6alkyl, aryl, or heteroaryl;

LG is a leaving group; and

m, w, v, n, Rx, Ry, Rl, R2, R3, R4, R5, R6, R7, R8, R9, and Al are as defined in the compound of formula (I) of the present invention (including provisos ( 1) a nd/or (2) of the first aspect).

In another aspect, the present invention broadly consists in the use of a compound of the formula (XX) or (XXI) in the synthesis of a peptide-conjugate of the formula (IA) of the present invention (including provisos (1) and/or (2) of the first aspect) or a

pharmaceutically acceptable salt or solvate thereof.

In another aspect, the present invention broadly consists in a method of making a peptide conjugate of the formula (I) or a pharmaceutically acceptable salt or solvate thereof of the present invention, the method comprising : (A) reacting

a first lipid-containing conj ugation partner comprising a carbon-carbon double bond,

a second lipid-containing conj ugation partner comprising a carbon-carbon double bond, and

an amino acid-comprising conjugation partner comprising a thiol

under conditions effective to conjugate the first lipid-containing conj ugation partner and the second lipid-containing conj ugation partner to the amino acid-comprising conjugation partner and provide the peptide-conjugate of formula (I) or salt or solvate thereof,

wherein in the amino acid- or peptide conj ugate the sulfur atom from the thiol of the amino acid-comprising conjugation pa rtner is conjugated to a carbon atom from the carbon-carbon double bond of the first lipid-containing conj ugation partner, and a carbon atom from the carbon-carbon double bond of the first lipid-containing conj ugation partner is conjugated to a carbon atom from the carbon-carbon double bond of the second lipid-conta ining conj ugation partner; or

(B) reacting

a first lipid-containing conjugation partner comprising a carbon-carbon double bond,

a second lipid-containing conj ugation partner comprising a carbon-carbon double bond, and

an amino acid-comprising conjugation partner comprising a thiol

under conditions effective to conjugate the first lipid-containing conj ugation partner and the second lipid-containing conj ugation partner to the amino acid-comprising conjugation partner and provide an amino acid- or peptide-conj ugate,

wherein in the amino acid- or peptide conj ugate the sulfur atom from the thiol of the amino acid-comprising conjugation pa rtner is conjugated to a carbon atom from the carbon-carbon double bond of the first lipid-containing conj ugation partner, and a carbon atom from the carbon-carbon double bond of the first lipid-containing conj ugation partner is conjugated to a carbon atom from the carbon-carbon double bond of the second lipid-conta ining conj ugation partner; and

coupling the amino acid of the amino acid conjugate or an amino acid of the peptide conj ugate to an amino acid or a n amino acid of a peptide to provide the peptide- conjugate of formula (I) or salt or solvate thereof.

In one embodiment, the amino acid-comprising conjuation partner is a peptide- containing conjugation partner, a nd the lipid-containing conjugation partners are coupled to the peptide of the peptide-conta ining conjugation partner. In some embod iments, the lipid-containing conjugation partners are conjugated to the or an amino acid of the amino acid-comprising conjugation partner or the peptide of the peptide-containing conjugation partner.

In certa in embodiments, the lipid-containing conjugation pa rtners are conjugated to the or a n amino acid of the amino acid-comprising conj ugation partner.

Accordingly, in another aspect, the present invention broad ly consists in a method of making a peptide conj ugate of formula (I) or a pharmaceutically acceptable salt or solvate thereof of the present invention, the method comprising reacting

a first lipid-containing conj ugation partner comprising a carbon-carbon double bond,

a second lipid-containing conj ugation partner comprising a carbon-carbon double bond, and peptide-containing conj ugation partner comprising a thiol under conditions effective to conjugate the first lipid-containing conj ugation partner and the second lipid-conta ining conj ugation partner to the peptide-containing conjugation partner and provide the peptide conj ugate of formula (I) or salt or solvate thereof,

wherein in the peptide conj ugate the sulfur atom from the thiol of the peptide- containing conjugation partner is conjugated to a carbon atom from the carbon-carbon double bond of the first lipid-containing conjugation partner, and a carbon atom from the carbon-carbon double bond of the first lipid-containing conj ugation partner is conj ugated to a carbon atom from the carbon-carbon double bond of the second lipid-conta ining conjugation partner.

In various embodiment, the conjugate is a lipopeptide, such that the method is for making a lipopeptide.

In various embodiments, the first and second lipid-containing conj ugation partners have the same structure (that is, the first a nd second lipid-conta ining conj ugation partners are identical).

In various embodiments, the method comprises conjugating the sulfur atom of the thiol to a carbon atom of the carbon-carbon double bond of the first lipid containing conjugation partner and then conj ugating a carbon atom from the carbon-carbon double bond to which the thiol is conjugated to a carbon atom of the carbon-carbon double bond of the second lipid-containing conjugation pa rtner.

In various embodiments, the first lipid-containing conj ugation partner is a compound of the formula (IIA) :

(HA);

the second lipid-containing conjugation pa rtner is a compound of the formula

(IIB) :

(IIB); a nd the amino acid-comprising conjugation partner comprises a structure of the formula (III):

(in); wherein:

when the method is (A), Ra, Rb, Rc, LI, L2, Zl, Z2, Rl, R2, Rx, Ry, R3, R4, R5, R6, R7, R8, R9, Al, k, v, and n are as defined in the compound of formula (IB) of the present invention (including provisos (1) and/or (2) of the first aspect); and

when the method is (B), Ra, Rb, Rc, LI, L2, Zl, Z2, Rl, R2, Rx, Ry, R3, R4, R5, R6, R7, R8, R9, Al, k, v, and n are as defined in the compound of formula (IB) but excluding provisos (1) and (2) of the first aspect.

In various embodiments, the amino acid- or peptide conjugate is a compound of the formula (IB):

wherein :

when the method is (A), Ra, Rb, Rc, LI, L2, Zl, Z2, Rl, R2, Rx, Ry, R3, R4, R5, R6, R7, R8, R9, Al, k, v, and n are as defined in the compound of formula (IB) of the present invention (including provisos (1) and/or (2) of the first aspect); and

when the method is (B), Ra, Rb, Rc, LI, L2, Zl, Z2, Rl, R2, Rx, Ry, R3, R4, R5, R6, R7, R8, R9, Al, k, v, and n are as defined in the compound of formula (IB) but excluding provisos (1) and (2) of the first aspect.

In various embodiments, the lipid containing conjugation partners are in stoichiometric excess to the amino acid-comprising conjugation partner.

In various embodiments, the mole ratio of the lipid containing conjugation partners (combined) to amino acid-comprising conjugation partner is at least 7: 1. In various embodiments, the first lipid-containing conjugation partner is a compound of the formula (IIA-1):

(IIA-1);

the second lipid-containing conjugation partner is a compound of the formula

(IIB):

(IIB);

the amino acid-comprising conjugation partner comprises a structure of the formula (III):

(III); and

the conjugate is a compound of the formula (IC) :

wherein :

when the method is (A), Ra, Rb, Rc, LI, L2, Rl, R2, Rx, Ry, R3, R4, R5, R6, R7, R8, R9, Al, k, v, and n are as defined in the compound of formula (IC) of the present invention (including provisos (1) and/or (2) of the first aspect); and

when the method is (B), Ra, Rb, Rc, LI, L2, Rl, R2, Rx, Ry, R3, R4, R5, R6, R7, R8, R9, Al, k, v, and n are as defined in the compound of formula (IC) but excluding provisos (1) and (2) of the first aspect. In various embodiments, LI is Cll-21alkyl; k is 0-3, preferably 0; and Ra, Rb, and Rc are each hydrogen. In various embodiments, LI is Cl l-21alkyl; k is 1-3; and Ra, Rb, a nd Rc are each hyd rogen.

In various embodiments, L2 is Cl l-21alkyl; v is 0-3, preferably 0; and R3, R4, and R5 are each hydrogen. In various embodiments, n is 1 ; R6, R7, and R8 are each hydrogen ; and R9 is hydrogen, an amino protecting group, L3-C(0), or A2.

In various embodiments, n is 1 ; R6, R7, and R8 are each hydrogen; and R9 is hydrogen, an amino protecting group, or L3-C(0), wherein L3 is linear C15alkyl or methyl .

In various embodiments, the compounds of formula (IIA) a nd (IIB) a re each vinyl palmitate.

In various embodiments, the amino-acid comprising conj ugation partner is cysteine, a protected cysteine (including Na-amine a nd/or carboxyl protected cysteine), or a peptide comprising a cysteine residue (including an Να-amine or carboxyl protected cysteine residue), for example, a n N-terminal cysteine residue (including an Να-amine protected cysteine residue).

In some embod iments, the method comprises reacting vinyl pa lmitate and an Na-amino protected cysteine, such as Fmoc-Cys-OH, Boc-Cys-OH, Fmoc-Cys-OPl, or Boc-Cys-OPl . In some embod iments, the carboxyl group of the Na-amino protected cysteine is protected . In one embodiment, the conditions effective to conjugate the lipid-containing conjugation partners to the amino acid-comprising conjugation pa rtner comprises the generation of one or more free radicals. In one embodiment, the cond itions effective to conj ugate the lipid-containing conj ugation partners to the peptide-conta ining conjugation partner comprises the generation of one or more free radicals. In some embod iments, the generation of one or more free radica ls is initiated therma lly and/or photochemica lly. In certa in embodiments, the generation of one or more free radicals is initiated by the thermal and/or photochemica l degradation of a free radical initiator. In exemplary embod iments, the generation of one or more free radicals is initiated by the thermal degradation of a therma l initiator or the photochemical degradation of a photochemical initiator.

In some embod iments, thermal degradation of the free radica l initiator comprises heating the reaction mixture at a suitable temperature. In some embodiments, the reaction mixture is heated at a temperature is from about 40 °C to about 200 °C, from about 50 °C to about 180 °C, from about 60 °C to about 150 °C, from about 65 °C to about 120 °C, from about 70 °C to about 115 °C, from about 75 °C to about 110 °C, or from about 80 °C to about 100 °C. In other embod iments, the reaction mixture is heated at a temperature of at least about 40 °C, at least about 50 °C, at least about 60 °C, or at least about 65 °C. In one specifically contemplated embodiment, the reaction mixture is heated at a temperature of about 90 °C.

In some embod iments, photochemical degradation of the free rad ical initiator comprises irrad iation with ultraviolet light, preferably having a frequency compatiable with the side chains of naturally occurring amino acids. In a specifically contemplated embodiment, the ultraviolet light has a wavelength of about 365 nm. In exemplary embodiments, photochemical degradation of the free radical initiator is carried out at a bout ambient temperature.

In one specifically contemplated embodiment, the thermal initiator is 2,2'- azobisisobutyronitrile (AIBN) . In one specifically contemplated embodiment, the photoinitiator is 2,2-dimethoxy-2-phenylacetophenone (DMPA).

In certa in embodiments, the reaction is carried out in a liquid med ium. In one embodiment, the liq uid medium comprises a solvent. In one embodiment, the solvent is selected from the g roup consisting of N-methylpyrrolidone (N MP), dimethylsulfoxide (DMSO), Ν,Ν-dimethylformamide (DMF), dichloromethane (DCM), 1,2-dichloroethane, and mixtures thereof. In one specifically contemplated embodiment, the solvent comprises N MP, DMF, DMSO, or a mixture thereof.

In one specifically contemplated embodiment, the solvent comprises DMSO or N MP. In exemplary embodiments, the solvent comprises NMP.

In some embod iments, the reaction is carried out in the presence of one or more additives that inhibit the formation of by-products and/or that improve the yield of or conversion to the desired conj ugate.

In various embodiments, the one or more additive is an extra neous thiol, an acid, an orga nosilane, or a combination of a ny two or more thereof.

In some exemplary embodiments, the extraneous or exogenous thiol is selected from the group consisting of reduced glutathione (GSH), 2,2'-(ethylened ioxy)diethanethiol

(DODT), 1,4-dithiothreitol (DTT), protein, a nd sterically hindered thiols. In a specifically contemplated embodiment, the extra neous or exogenous thiol is DTT. In some embodiments, the extraneous or exogenous thiol is a sterically hindered thiol, for example terf-butyl mercaptan. In various embodiments, the acid additive is a strong inorganic or organic acid . In various embodiments, the acid is a strong organic acid . In various embodiments, the acid is TFA.

In various embodiments, the organosilane is a trialkylsilane, for example TIPS. In some embod iments, the one or more additive is selected from the group consisting of TFA, terf-butyl mercaptan, TIPS, and combinations of any two or more thereof.

In certain embodiments, the one or more add itive is a combination of a n acid and an extraneous thiol, for example TFA and terf-butyl mercaptan.

In other embodiments, the one or more add itive is a combination of an acid and an orga nosilane, for example TFA a nd TIPS.

In other embodiments, the one or more add itive is a combination of an extraneous thiol and a n organosila ne, and optionally a n acid, for example a combination of t-BuSH and TIPS, and TFA.

In some embod iments, the reaction is carried out for a period of time from about 5 minutes to about 48 h, 5 minutes to about 24 h, from about 5 minutes to about 12 hours, from about 5 minutes to about 6 hours, from about 5 minutes to about 3 hours, 5 minutes to 2 hours, or form about 5 minutes to about 1 hour. In exemplary

embodiments, the reaction is carried out for a period of time from about 5 minutes to about 1 h. In some embodiments, the reaction is carried out until one of the conj ugation partners is at least about 70%, 80%, 90%, 95%, 97%, 99%, or 100% consumed.

In certain embodiments, the reaction is carried out under substantially oxygen free conditions.

In various embodiments, the amino acid-comprising conjugation partner is a peptide- containing conjugation partner. In one embodiment, the amino acid-comprising conjugation pa rtner comprises an epitope. In one embodiment, the peptide-conta ining conj ugation partner comprises an epitope, such as a peptide epitope.

In one embodiment, the amino acid-comprising conjugation partner comprises two or more epitopes. In one embodiment, the peptide-containing conjugation partner comprises two or more epitopes.

In one embodiment, the amino acid-comprising conjugation partner consists of a peptide. In one embodiment, the amino acid-comprising conjugation partner consists of a peptide comprising a peptide epitope. In one embod iment, the peptide-containing conjugation partner consists of a peptide. In one embodiment, the peptide-containing conjugation partner consists of a peptide comprising a peptide epitope. In some embod iments, the amino acid-comprising conj ugation partner comprises an epitope bound to the or an amino acid of the conjugation partner. In some

embodiments, the peptide-containing conj ugation partner comprises a n epitope bound to the peptide of the peptide containing conjugation partner. In some embodiments, the epitope is bound to the peptide via linker group. In some embod iments, the amino acid-comprising conj ugation pa rtner comprises a peptide epitope bound to the or an amino acid of the conj ugation partner via a linker group. In some embodiments, the peptide-containing conj ugation partner comprises a peptide epitope bound to the peptide via a linker group.

In some embod iments, the amino acid-comprising conj ugation partner and/or the peptide-containing conjugation partner comprises an antigenic peptide.

In one embodiment, the amino acid-comprising conjugation pa rtner a nd/or peptide conjugate comprises a synthetic peptide. In some embodiments, the synthetic peptide is a peptide prepared by a method comprising solid phase peptide synthesis (SPPS).

In various embodiments, the method comprises coupling the amino acid of the amino acid conjugate or a n amino acid of the peptide conjugate to an amino acid or an amino acid of a peptide to provide a peptide conjugate.

In various embodiments, the method comprises coupling the amino acid of the amino acid conjugate to an amino acid or an amino acid of a peptide to provide a peptide conjugate of the present invention . In various embodiments, the peptide comprises an epitope. In various embodiments, the epitope is a peptide epitope.

In some embod iments, the method further comprises coupling the amino acid of the amino acid conjugate to an amino acid or a peptide to provide a peptide conjugate of the present invention. In some embod iments, coupling a peptide comprises individually coupling one or more amino acids and/or one or more peptides. In some embod iments, the method further comprises coupling the amino acid of the amino acid conjugate or an amino acid of the peptide conj ugate to an a mino acid or a peptide so as to provide a peptide conj ugate of the invention comprising a linker group or one or more amino acids thereof. In some embod iments, the method further comprises coupling an amino acid of the peptide conjugate comprising a linker group or one or more amino acids thereof to an amino acid or a peptide so as to provide a peptide conjugate of the invention comprising a peptide epitope bound to the amino acid to which lipid moieties are conj ugated via a linker g roup. In some embod iments, the amino acid of the peptide conjugate to which the lipid moeities are conjugated is a n N-termina l amino acid residue.

In some embod iments, the method further comprises coupling the amino acid of the amino acid conjugate or an amino acid of the peptide conj ugate to an a mino acid or a peptide so as to provide a peptide conj ugate of the invention comprising a peptide epitope.

In some embod iments, the method further comprises coupling an epitope to the amino acid of the amino acid conj ugate or an amino acid of the peptide conjugate.

In some embod iments, the method further comprises coupling a peptide epitope to the amino acid of the amino acid conjugate or an a mino acid of the peptide conjugate. In some embod iments, the epitope is coupled or bound via a linker group.

In some embod iments, the method further comprises coupling an epitope to the peptide of the peptide conj ugate.

In some embod iments, the method further comprises coupling a peptide epitope to the peptide of the peptide conj ugate. In some embod iments, the epitope is bound to the peptide via a linker group.

In various embodiments, the method is (B) and the amino acid-comprising conjugation partner consists of a n amino acid, for example cysteine (including Na-amino and/or C- terminus protected cysteines).

In various embodiments, the amino acid- or peptide-conjugate compound of formula (I) but exclud ing provisos ( 1) and (2) of the first aspect is an amino acid-conjugate. In some embod iments, Al is OH, OP1, N H2, or NHP2 and/or R9 is hydrogen, Cl-6alkyl, C3-6cycloalkyl, an amino protecting group, or L3-C(0) in the amino acid- or peptide- conjugate compound of formula (I) but excluding provisos (1) and (2) of the first aspect.

In some of such embodiments, Al is OP1 or OH and/or R9 is hydrogen, an a mino protecting group or L3-C(0) in the amino acid- or peptide-conjugate compound of formula (I) but excluding provisos (1) and (2) of the first aspect.

In various of such embodiments, Al is OH, OP1, N H2, or NHP2 a nd R9 is hydrogen, Cl- 6alkyl, C3-6cycloalkyl, an amino protecting group, or L3-C(0) in the amino acid- or peptide-conjugate compound of formula (I) but excluding provisos (1) and (2) of the first aspect.

In various embodiments, Al is OH, or OP1, and R9 is hydrogen, an amino protecting group, or L3-C(0) in the amino acid- or peptide-conj ugate compound of formula (I) but excluding provisos (1) a nd (2) of the first aspect.

In various embodiments, the C-terminus of the amino acid comprising conjugation partner is protected with a protecting group and/or the Na-amino group of the amino acid comprising conjugation partner is protected with a protecting group.

In various embodiments, the carboxyl g roup of the C-terminus of the a mino acid is protected with a carboxyl protecting group or a carboxamide protecting group and/or the Να-amino group of the amino acid is protected with an amino protecting group. In various embodiments, the carboxyl g roup of the C-terminus of the a mino acid is protected with a carboxyl protecting group and/or the Να-amino group of the amino acid is protected with an amino protecting group.

In some embod iments, the carboxyl group of the C-terminus of the peptide is protected with a carboxyl protecting group a nd/or the Να-amino g roup of the peptide is protected with an amino protecting group.

In some embod iments, the amino acid residue comprising the thiol is a terminal amino acid residue. In some embodiments, the amino acid residue comprising the thiol is an N- termina l residue.

In some embod iments, Al and/or R9 is a group other than an amino acid or a peptide, and the method comprises coupling an amino acid or a peptide so as to replace Al and/or R9 with the amino acid or peptide. In some embod iments, Al a group other than an amino acid or a peptide, and the method comprises coupling an amino acid or a peptide so as to replace Al with the amino acid or peptide.

In some embod iments, Al is a OH, OP1, NH2, or N HP2 and/or R9 is hydrogen, an amino protecting group or L3-C(0), a nd the method comprises coupling an a mino acid or a peptide so as to replace Al and/or R9 with the amino acid or peptide.

In some embod iments, Al is a OH, OP1, NH2, or N HP2 and R9 is hyd rogen, an amino protecting group or L3-C(0) and the method further comprises coupling an amino acid or a peptide so as to replace Al and/or R9 with the amino acid or peptide. In some embod iments, coupling a peptide comprises individually coupling one or more amino acids and/or one or more peptides.

In some embod iments, coupling the amino acid or peptide provides a peptide conjugate comprising a peptide epitope. In some embodiments, the coupling the amino acid or peptide provides a peptide conj ugate comprising a linker group or one or more amino acids thereof. In some embod iments, coupling the amino acid or peptide provides a peptide conj ugate comprising a peptide epitope bound to the amino acid to which the lipid moieties are conjugated via a linker group.

In some embod iments, the Na-amino group of the amino acid comprising the thiol to which the lipid moieties are conj ugated is acylated . In some embodiments, R9 in the amino acid comprising conjugation pa rtner comprising the thiol is L3-C(0)-, for example Me-C(O)-.

In certa in embodiments, the method further comprises acylating the Να-amino group of the amino acid of the amino acid conj ugate or the amino acid resid ue of the peptide conjugate to which the lipid moeities are conj ugated . In certain embodiments, the method further comprises acylating the Να-amino group with a C2-20 fatty acid, such as acetyl.

In some embod iments, R9 is hydrogen or an amino protecting g roup, and the method further comprises acylating the amino acid conj ugate or peptide conjugate so as to replace the hydrogen or amino protecting group at R9 with L3-C(0). In some embod iments, acylating the amino acid conjugate or peptide conjugate so as to replace the amino protecting group at R9 with L3-C(0) comprises removing the amino protecting group at R9 to provide a hydrogen at R9. In certain embodiments, the or an amino acid of the amino acid-comprising conjugation partner comprises the thiol. In certain embodiments, an amino acid residue of the peptide of the peptide-conta ining conj ugation partner comprises the thiol .

In certain embodiments, the thiol is the thiol of a cysteine residue. In certain embodiments, the cysteine residue is a terminal resid ue. In certain embodiments, the cysteine residue is an N-termina l residue.

In some embod iments, the amino group of the cysteine residue is acylated.

In one embodiment, the amino group is acylated with a C2-20 fatty acid .

In one exemplary embodiment, the C2-20 fatty acid is acetyl or palmitoyl. In another exemplary embod iment, the C2-20 fatty acid is acetyl .

In some embod iments, the amino acid-comprising conj ugation pa rtner and/or peptide conjugate comprises from 8 to 220, 8 to 200, 8 to 175, 8 to 150, 8 to 125, 8 to 100, 8 to 90, 8 to 80, 8 to 70, 8 to 60, 8 to 50, 8 to 40, 8 to 30, 8 to 25, 8 to 20, or 8 to 15 amino acids. In some embodiments, the peptide-containing conj ugation partner comprises from 8 to 220, 8 to 200, 8 to 175, 8 to 150, 8 to 125, 8 to 100, 8 to 90, 8 to 80, 8 to 70, 8 to 60, 8 to 50, 8 to 40, 8 to 30, 8 to 25, 8 to 20, or 8 to 15 amino acids.

In one exemplary embodiment, the amino acid-comprising conj ugation partner and/or peptide conj ugate comprises a peptide comprising from 8 to 60 amino acids. In one exemplary embod iment, the peptide comprises from 8 to 60 amino acids. In other embodiments, the amino acid-comprising conj ugation partner and/or peptide conjugate comprises from 5 to 220, 8 to 220, 5 to 175, 8 to 175, 8 to 150, 10 to 150, 15 to 125, 20 to 100, 20 to 80, 20 to 60, 25 to 100, 25 to 80, 25 to 60, 30 to 80, 40 to 60, or 50 to 60 amino acids. In other embodiments, the peptide-conta ining conj ugation partner comprises from 5 to 220, 8 to 220, 5 to 175, 8 to 175, 8 to 150, 10 to 150, 15 to 125, 20 to 100, 20 to 80, 20 to 60, 25 to 100, 25 to 80, 25 to 60, 30 to 80, 40 to 60, or 50 to 60 amino acids.

In other embodiments, the amino acid comprising conjugation pa rtner and/or peptide conjugate comprises from 5 to 150, 5 to 125, 5 to 100, 5 to 75, 5 to 60, 5 to 50, 5 to 40, 5 to 30, 5 to 25, 5 to 20, 8 to 150, 8 to 125, 8 to 100, 8 to 75, 8 to 60, 8 to 50, 8 to 40, 8 to 30, 8 to 25, or 8 to 20 amino acids. In other embodiments, the peptide-containing conjugation partner comprises from 5 to 150, 5 to 125, 5 to 100, 5 to 75, 5 to 60, 5 to 50, 5 to 40, 5 to 30, 5 to 25, 5 to 20, 8 to 150, 8 to 125, 8 to 100, 8 to 75, 8 to 60, 8 to 50, 8 to 40, 8 to 30, 8 to 25, or 8 to 20 amino acids. In various embodiments, the amino acid comprising conjugation partner is a short peptide. In some embodiments, the short peptide comprises less than 10, 9, 8, 7, 6, 5, 4, or 3 amino acids.

In one embodiment, the amino acid-comprising conjugation pa rtner a nd/or peptide conjugate comprises one or more solubilising groups. In one embodiment, the peptide- containing conjugation partner comprises one or more solubilising groups.

In certain embodiments, the solubilising group is an amino acid seq uence comprising two or more hydrophilic amino acid residues in the peptide chain. In certain embodiments, the solubilising group is an amino acid sequence comprising a sequence of two or more consecutive hydrophilic amino acid residues in the peptide chain. In one embodiment, the hydrophilic amino acid residues are cationic amino acid residues. In one

embodiment, the cationic amino acid residues are arginine or lysine residues. In one specifically contemplated embodiment, the cationic amino acid residues are lysine residues. In one embod iment, the seq uence comprises from 2 to 20, 2 to 15, 2 to 10, 3 to 7, or 3 to 5 amino acids. In one embodiment, the solubilising group is a tri-, tetra-, penta-, hexa-, or hepta- lysine sequence. In one specifically contemplated embodiment, the solubilising group is a tetralysine sequence.

In some embod iments, the peptide conj ugate and/or amino-acid comprising conjugation partner comprises a serine residue adjacent to the amino acid resid ue to which the lipid moeities are conjugated . In a specifically contemplated embod iment, the peptide of the peptide-containing conjugation partner comprises a serine residue adjacent to the amino acid residue to which the lipid moeities are conjugated . In a n exemplary embodiment, the amino acid residue to which the lipid moeities are conjugated is N-termina l. In a specifically contemplated embodiment, the peptide further comprises a consecutive sequence of two or more hydrophilic amino acid residues adjacent to the serine residue.

In certa in embodiments, the peptide conjugate and/or amino-acid comprising conjugation partner comprises a consecutive sequence of two or more hydrophilic amino acid residues adjacent to the serine residue.

In certain embodiments, the peptide conjugate and/or amino acid-comprising conjugation partner comprises only naturally occuring amino acids. In certa in embodiments, the peptide-containing conj ugation partner comprises only naturally occuring amino acids. In other embod iments, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, or 99% or more of the amino acid residues in the peptide are naturally occuring a mino acids. In other embodiments, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, or 99% or more of the amino acid residues in the peptide conjugate and/or amino acid-comprising conjugation partner a re natura lly occuring amino acids.

In exemplary embodiments, the peptide conjugate and/or amino acid-comprising conjugation partner comprises a peptide comprising a peptide epitope. In exemplary embodiments, the peptide of the peptide-conta ining conjugation partner comprises one or more peptide epitopes.

In various embodiments, the peptide comprises, consists essentially of, or consists of an amino acid seq uence selected from the group consisting of those defined in proviso (1) of the first aspect.

In various embodiments, the peptide comprises, consists essentially of, or consists of one or more EBV LM P2 epitopes. In va rious embodiments, the one or more EBV LMP2 epitopes a re MHCI epitopes. In various embodiments, the peptide comprises one or more EBV LMP2 epitopes selected from the group consisting of a ny one of SEQ ID NOs 84- 109. In va rious embodiments, the peptide comprises a peptide comprising or consisting of 12 or more contiguous amino acids from the amino acid sequence of any one of SEQ ID NOs 9 - 83. In various embod iments, the peptide comprises a peptide comprising or consisting of 15 or more contiguous amino acids from the amino acid sequence of any one of SEQ ID NOs 9 - 83, or comprising or consisting of 20 or more contiguous amino acids from the amino acid sequence of any one of SEQ ID NOs 9 - 83.

In various embodiments, the peptide comprises a recombina nt peptide comprising or consisting of 12 or more contiguous amino acids from the amino acid sequence of any one of SEQ ID NOs 9 - 83. In various embod iments, the recombinant peptide comprises or consists of 15 or more contiguous amino acids from the amino acid sequence of any one of SEQ ID NOs 9 - 83, or comprises or consists of 20 or more contig uous amino acids from the amino acid sequence of any one of SEQ ID NOs 9 - 83.

In one exemplary embodiment, the peptide epitope is derived from NY-ESO-1. In one specifically contemplated embodiment, the peptide comprises, consists essentially of, or consists of an amino acid sequence selected from the group consisting of 8 or more contiguous amino acid residues from any one of SEQ ID NO: 114, 115, 116, 121, 122, 127, 128, a nd 129.

In various embodiments, the peptide comprises, consists essentially of, or consists of one or more NY-ESO-1 epitopes. In various embodiments, the one or more NY-ESO-1 epitopes a re MHCI epitopes. In various embodiments, the the peptide comprises, consists essentially of, or consists of an amino acid sequence selected from the group consisting of 8 or more contig uous amino acid resid ues from any one of SEQ ID NO : 114, 115, 116, 121, 122, 127, 128, and 129. In various embodiments, the peptide comprises a peptide comprising or consisting of 12 or more contiguous amino acids from the amino acid sequence of any one of SEQ ID NO: 114, 115, 116, 121, 122, 127, 128, a nd 129. In various embodiments, the peptide comprises a peptide comprising or consisting of 15 or more contiguous amino acids from the amino acid sequence of any one of SEQ ID NO:

114, 115, 116, 121, 122, 127, 128, and 129, or comprising or consisting of 20 or more contiguous amino acids from the amino acid sequence of any one of SEQ ID NO: 114,

115, 116, 121, 122, 127, 128, and 129. In one specifically contemplated embodiment, the reactive functional groups of the amino acids of the peptide-containing conj ugation partner a re unprotected .

In certain embodiments, one or more reactive functiona l groups of one or more amino acids of the peptide conjugate a re unprotected .

In certain embodiments, one or more reactive functiona l groups of the amino acid of the amino acid conjugate are unprotected.

In certain embodiments, one or more reactive functiona l groups of one or more amino acids of the amino acid-comprising conj ugation partner are unprotected.

In certain embodiments, the amino acid-comprising conjugation partner comprises a peptide, wherein the reactive functional groups of the side chains of the amino acids of the peptide are unprotected, with the exception of any thiols other than the thiol to be reacted .

In certain specifically contemplated embodiments, the reactive functional g roups of the amino acids of the peptide of the peptide-containing conjugation partner are

unprotected . In certain specifically contemplated embodiments, the reactive functional g roups of the amino acids of the peptide of the peptide-containing conjugation partner are

unprotected, with the exception of any thiols other than the thiol to be reacted .

Those skilled in the art will appreciate that the peptide of the peptide conj ugate and/or peptide-containing conjugation partner may, as described herein, be optionally substituted, modified, or bound to various other moieties as described herein to provide the peptide conj ugate a nd/or peptide containing conjugation partner.

In some embod iments, the method comprises synthesising the amino acid sequence of a peptide by solid phase peptide synthesis (SPPS);

coupling the amino acid of an amino acid conjugate or a n amino acid of a peptide conjugate to the solid phase bound peptide by SPPS so as to provide a peptide conj ugate comprising a peptide epitope, a peptide conjugate comprising a linker group or one or more amino acids thereof, or a peptide conj ugate comprising a peptide epitope bound to the amino acid to which lipid moeities are conjugated via a linker group.

In some embod iments, the method comprises

reacting the lipid-conta ining conj ugation partners and a n amino acid-comprising conjugation partner to provide a n amino acid or peptide conj ugate;

synthesising the amino acid sequence of a peptide by solid phase peptide synthesis (SPPS);

coupling the amino acid of the amino acid conjugate or an amino acid of the peptide conj ugate to the solid phase bound peptide by SPPS so as to provide a peptide conjugate comprising a peptide epitope, a peptide conjugate comprising a linker group or one or more amino acids thereof, or a peptide conj ugate comprising a peptide epitope bound to the amino acid to which lipid moeities are conj ugated via a linker group.

In some embod iments, the method further comprises acylating the Na-amino group of the amino acid of the amino acid conj ugate or the amino acid to which the lipid-moieties are conjugated of any one of the peptide conj ugates.

In some embod iments, the method comprises cleaving the peptide conj ugate from the solid phase support.

In some embod iments, the method comprises

synthesising the amino acid sequence of the peptide of the peptide-containing conjugation partner by solid phase peptide synthesis (SPPS); and

reacting the lipid-conta ining conj ugation pa rtners and peptide-containing conjugation partner in accordance with any of the embodiments described herein.

In exemplary embodiments, the method comprises

synthesising the amino acid sequence of the peptide of the peptide-containing conjugation partner by SPPS,

cleaving the peptide from the solid phase support; a nd

reacting the lipid-conta ining conj ugation pa rtners and peptide-containing conjugation partner in accordance with any of the embodiments described herein.

In one embodiment, the peptide-containing conjugation partner is not purified prior to reaction with the lipid-containing conjugation partners. In some embod iments, one or more protecting groups are removed on cleaving the peptide from the solid phase support. In certain embodiments, a ll of the protecting groups present in the peptide are removed.

In one embodiment, the SPPS is Fmoc-SPPS. In some embod iments, the amino acid residue in the peptide of the peptide-containing conjugation partner bea ring the thiol to be reacted is an N-terminal amino acid residue and the method comprises acylating the N -terminal amino g roup prior to cleaving the peptide from the solid phase. In specifically contemplated embod iments, the N-terminal residue is a cysteine resid ue. In one embodiment, the method further comprises separating the peptide conj ugate from the reaction medium and optionally purifying the peptide conjugate.

In another aspect, the present invention broadly consists in a method of making a peptide conj ugate, the method comprising

providing an amino acid- or peptide conj ugate of the formula (I) but exclud ing provisos (1) and (2) of the first aspect or a sa lt or solvate thereof, and

coupling the amino acid of the amino acid conjugate or an amino acid of the peptide conj ugate to an amino acid or a n amino acid of a peptide to provide a peptide conjugate of the formula (I) of the invention (including provisos (1) and/or (2) of the first aspect) or a sa lt or solvate thereof. In various embodiments, the product peptide conjugate is a compound of the formula (I) or a pharmaceutically acceptable salt thereof of the present invention .

In various embodiments, the amino acid of the amino acid conjugate is coupled under conditions that reduce epimerisation at the a-carbon of the amino acid . In various embodiments, the conditions are such that less than about 35, 30, 25, 20, 15, 10, 5, 3, 2, or 1% by mol of the amino acid is epimerised . In va rious embodiments, the conditions that reduce epimerisation comprise the use of PyBOP or BOP as the coupling reagent. In various embodiments, the conditions that reduce epimerisation comprise the use of PyBOP as the coupling reagent. In various embod iments, the conditions comprise the use of PyBOP or BOP; and 2,4,6-trimethylpyridine. In various embodiments, the conditions comprise the use of PyBOP and 2,4,6-trimethylpyridine.

In another aspect, the present invention broadly consists in use of an amino acid- or peptide-conjugate of the formula (I) of the present invention (including provisos (1) and/or (2) of the first aspect) or a salt or solvate thereof in the synthesis of an immunogenic peptide-conj ugate. In various embodiments, the immunogenic peptide conj ugate is a compound of the formula (I) of the present invention or a pharmaceutically acceptable salt thereof.

In another aspect, the present invention broadly consists in a peptide conj ugate of the present invention produced by a method of the present invention. In another aspect, the present invention broadly consists in a composition comprising a peptide conjugate of formula (I) of the present invention or a salt or solvate thereof.

In various embodiments, the composition comprises isolated, pure, purified or substantially purified compound of formula (I) of the present invention or a salt or solvate thereof. In various embodiments, the composition comprises at least about 60, 70, 75, 80, 85, 90, 95, 97, 98, or 99% by weight compound of formula (I) of the present invention or a salt or solvate thereof.

In various embodiments, the composition is free of substantially free of amino acid- or peptide containing compounds other than compounds of formula (I) of the present invention.

In another aspect, the present invention broadly consists in a pharmaceutical composition comprising an effective amount of a peptide conjugate compound of the formula (I) of the present invention or a pharmaceutically acceptable salt or solvate thereof, and a pha rmaceutically acceptable carrier. In various embodiments, the pharmaceutical composition comprises an effective amount of two or more peptide conjugate compounds of the formula (I) of the present invention.

In one embodiment, the pha rmaceutical composition is a n immunogenic composition.

In one embodiment, the pha rmaceutical composition does not include an extrinsic adj uvant. In some embod iments, the pharmaceutical composition is a vaccine.

In one embodiment, the pha rmaceutical composition comprises an effective amount of two or more peptide conjugates of the present invention, for example the pharmaceutical composition comprises an effective amount of three or more peptide conjugates of the present invention. In one embodiment, the pha rmaceutical composition comprises an effective amount of one or more peptide conjugates of the present invention together with one or more peptides described herein, or any combination thereof. For example, the pharmaceutical composition comprises an effective amount of two or more peptide conjugates of the present invention a nd one or more peptides described herein, or an effective amount of one or more peptide conjugates of the present invention and two or more peptides described herein.

In another aspect, the present invention broadly consists in a method of vaccinating or eliciting an immune response in a subject comprising administering to the subject an effective amount of one or more peptide conjugate compounds of the formula (I) of the invention or a pha rmaceutically acceptable salt or solvate thereof, or a n effective amount of a pharmaceutical composition of of the present invention.

In another aspect, the present invention broadly consists in use of one or more peptide conjugate compounds of formula (I) of the present invention or a pharmaceutically acceptable salt or solvate thereof or a pha rmaceutica l composition of the present invention in the manufacture of a medicament for vaccinating or eliciting an immune response in a subject.

In another aspect, the present invention broadly consists in one or more peptide conjugate compounds of the formula (I) of the present invention or a pharmaceutically acceptable salt or solvate thereof or a pha rmaceutica l composition of the present invention for vaccinating or eliciting a n immune response in a subject. In another aspect, the present invention broadly consists in use of one or more peptide conjugate compounds of the formula (I) of the invention or a pharmaceutica lly acceptable salt or solvate thereof or a pha rmaceutica l composition of the present invention for vaccinating or eliciting a n immune response in a subject.

In another aspect, the present invention broadly consists in a method of activating TLR2 in a subject, the method comprising administering to the subject an effective amount of one or more peptide conjugate of the invention or a pharmaceutically acceptable salt or solvate thereof, or a n effective amount of a pharmaceutical composition of the invention.

Use of one or more peptide conj ugate compounds of the invention or a pharmaceutically acceptable salt or solvate thereof or a pharmaceutical composition of the invention in the manufacture of a medicament for activating TLR2 in a subject.

One or more peptide conjugate compounds of the invention or a pharmaceutically acceptable salt or solvate thereof or a pharmaceutical composition of the invention for activating TLR2 in a subject. In various embodiments, the method, use, one or more compounds, or pharmaceutical composition is for eliciting an immune response in a subject.

In various embodiments, the method, use, one or more compounds, or pharmaceutical composition is for vaccinating a subject. In some embodiments, the method comprises the administration of one or more peptides described herein and one or more peptide conjugates of the present invention or two or more peptide conjugates of the present invention, for example one or more peptides in combination with one or more peptide conjugates to the subject.

In some embodiments, one or more peptides described herein and one or more peptide conjugates of the present invention or two or more peptide conjugates of the present invention, for example one or more peptides in combination with one or more peptide conjugates, are used for vaccinating or eliciting an immune response in the subject or in the manufacture of a medicament for vaccinating or eliciting an immune response in the subject. In some embodiment, two or more peptide conjugates are used or administered.

In some embodiments the two or more peptide conjugates, or one or more peptides and one or more peptide conjugates are used or administered simultaneously, sequentially, or separately.

In some embodiments, the subject is in need thereof. Asymmetric centers may exist in the compounds described herein. The asymmetric centers may be designated as (R) or (S), depending on the configuration of substituents in three dimensional space at the chiral carbon atom. All stereochemical isomeric forms of the compounds, including diastereomeric, enantiomeric, and epimeric forms, as well as d-isomers and l-isomers, and mixtures thereof, including enantiomerically enriched and diastereomerically enriched mixtures of stereochemical isomers, are within the scope of the invention.

Individual enantiomers can be prepared synthetically from commercially available enantiopure starting materials or by preparing enantiomeric mixtures and resolving the mixture into individual enantiomers. Resolution methods include conversion of the enantiomeric mixture into a mixture of diastereomers and separation of the

diastereomers by, for example, recrystallization or chromatography, and any other appropriate methods known in the art. Starting materials of defined stereochemistry may be commercially available or made and, if necessary, resolved by techniques well known in the art. The compounds described herein may also exist as conformational or geometric isomers, inlcuding cis, trans, syn, anti, entgegen (E), a nd zusammen (Z) isomers. All such isomers and a ny mixtures thereof are within the scope of the invention.

Also within the scope of the invention are any tautomeric isomers or mixtures thereof of the compounds described . As would be appreciated by those skilled in the art, a wide variety of functiona l groups and other structures may exhibit tautomerism. Exa mples include, but are not limited to, keto/enol, imine/enamine, and thioketone/enethiol tautomerism.

The compounds described herein may also exist as isotopologues a nd isotopomers, wherein one or more atoms in the compounds are replaced with different isotopes.

Suitable isotopes include, for example, H ² H (D), ³ H (T), ¹² C, ¹³ C, ¹⁴ C, ^1S 0, and ¹⁸ 0. Proced ures for incorporating such isotopes into the compounds described herein will be apparent to those skilled in the art. Isotopologues a nd isotopomers of the compounds described herein a re also within the scope of the invention. Also within the scope of the invention are salts of the compounds described herein, including pharmaceutically acceptable salts. Such salts include, acid addition salts, base addition salts, and q uaternary sa lts of basic nitrogen-containing groups.

Acid addition salts can be prepa red by reacting compounds, in free base form, with inorganic or organic acids. Examples of inorganic acids include, but a re not limited to, hydrochloric, hydrobromic, nitric, sulfuric, and phosphoric acid . Examples of orga nic acids include, but are not limited to, acetic, trifluoroacetic, propionic, succinic, glycolic, lactic, malic, tartaric, citric, ascorbic, maleic, fumaric, pyruvic, aspartic, glutamic, stearic, salicylic, metha nesulfonic, benzenesulfonic, isethionic, sulfanilic, adipic, butyric, and pivalic. Base addition salts can be prepa red by reacting compounds, in free acid form, with inorganic or organic bases. Examples of inorganic base addition salts include alkali metal salts, alkaline earth metal salts, and other physiologically acceptable metal salts, for example, aluminium, calcium, lithium, magnesium, potassium, sodium, or zinc salts. Examples of organic base addition salts include amine salts, for example, sa lts of trimethylamine, diethylamine, ethanolamine, d iethanolamine, and ethylenediamine.

Quaterna ry salts of basic nitrogen-containing groups in the compounds may be may be prepared by, for example, reacting the compounds with alkyl halides such as methyl, ethyl, propyl, and butyl chlorides, bromides, and iodides, dia lkyl sulfates such as dimethyl, diethyl, dibutyl, a nd d iamyl sulfates, and the like. The compounds described herein may form or exist as solvates with various solvents. If the solvent is water, the solvate may be referred to as a hyd rate, for example, a mono- hydrate, a d i- hydrate, or a tri-hydrate. All solvated forms and unsolvated forms of the compounds described herein are within the scope of the invention. The general chemical terms used in the formulae herein have their usual mea ning .

The term "aliphatic" is intended to include saturated and unsaturated, nonaromatic, straight cha in, branched, acyclic, and cyclic hydrocarbons. Those skilled in the art will appreciate that aliphatic groups include, for example, alkyl, alkenyl, alkynyl, cycloalkyl, and cycloalkenyl groups, and hybrids thereof such as (cycloalkyl)alkyl, (cycloa lkenyl)alkyl and (cycloalkyl)alkenyl groups. In various embodiments, a liphatic groups comprise from 1-12, 1-8, 1-6, or 1-4 carbon atoms. In some embodiments, a liphatic groups comprise 5-21, from 9-21, or from 11-21 carbon atoms, such as from 11, 13, 15, 17, or 19 carbon atoms. In some embodiments, the aliphatic group is saturated .

The term "heteroaliphatic" is intended to include a liphatic groups, wherein one or more chain and/or ring carbon atoms are independently replaced with a heteroatom, preferably a heteroatom selected from oxygen, nitrogen and sulfur. In some embodiments, the heteroa liphatic is saturated . Examples of heteroaliphatic groups include linear or branched, heteroalkyl, heteroalkenyl, and heteroalkynyl groups.

The term "alkyl" is intended to include saturated straight cha in and branched chain hydrocarbon groups. In some embodiments, alkyl groups have from 1 to 12, 1 to 10, 1 to 8, 1 to 6, or from 1 to 4 carbon atoms. In some embodiments, a lkyl groups have from 5-21, from 9-21, or from 11-21 carbon atoms, such as from 11, 13, 15, 17, or 19 carbon atoms. Examples of straight chain alkyl groups include, but a re not limited to, methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, n-heptyl, and n-octyl. Examples of branched alkyl groups include, but are not limited to, isopropyl, iso-butyl, sec-butyl, tert-butyl, neopentyl, isopentyl, a nd 2,2-dimethylpropyl.

The term "alkenyl" is intended to include stra ight and branched chain alkyl groups having at least one double bond between two carbon atoms. In some embodiments, alkenyl groups have from 2 to 12, from 2 to 10, from 2 to 8, from 2 to 6, or from 2 to 4 carbon atoms. In some embodiments, a lkenyl g roups have from 5-21, from 9-21, or from 11-21 carbon atoms, such as from 11, 13, 15, 17, or 19 carbon atoms. In some embodiments, alkenyl groups have one, two, or three carbon-carbon double bonds. Examples of alkenyl groups include, but are not limited to, vinyl, a llyl, -CH = CH (CH3), -CH = C(CH3)2, - C(CH ₃ ) = CH2, and -C(CH ₃ ) = CH (CH ₃ ). The term "alkynyl" is intended to include straight and branched chain alkyl groups having at least one triple bond between two carbon atoms. In some embodiments, the alkynyl group have from 2 to 12, from 2 to 10, from 2 to 8, from 2 to 6, or from 2 to 4 carbon atoms. In some embodiments, a lkynyl groups have one, two, or three carbon-carbon triple bonds. Examples include, but are not limited to, -C≡CH, -C≡CH3, -CH2C≡CH 3, and -C≡CH ₂ CH(CH ₂ CH ₃ )2.

The term "heteroalkyl" is intended to include alkyl groups, wherein one or more cha in carbon atoms are replaced with a heteroatom, preferably a heteroatom selected from the group consisting of oxygen, nitrogen, a nd sulfur. In some embodiments, the heteroalkyl is saturated . Heteroalkyl groups include, for example, polyethylene glycol groups and polyethylene glycol ether groups, and the like.

The term "cycloalkyi" is intended to include mono-, bi- or tricyclic alkyl groups. In some embodiments, cycloalkyi g roups have from 3 to 12, from 3 to 10, from 3 to 8, from 3 to 6, from 3 to 5 carbon atoms in the ring(s). In some embodiments, cycloalkyi g roups have 5 or 6 ring ca rbon atoms. Examples of monocyclic cycloalkyi g roups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. In some embodiments, the cycloalkyi group has from 3 to 8, from 3 to 7, from 3 to 6, from 4 to 6, from 3 to 5, or from 4 to 5 ring carbon atoms. Bi- and tricyclic ring systems include bridged, spiro, and fused cycloalkyi ring systems. Examples of bi- and tricyclic ring cycloalkyi systems include, but are not limited to, bicyclo[2.1.1]hexanyl, bicyclo[2.2.1 ]heptanyl, adamantyl, and decalinyl.

The term "cycloalkenyl" is intended to include non-a romatic cycloalkyi groups having at least one double bond between two carbon atoms. In some embodiments, cycloa lkenyl groups have one, two or three double bonds. In some embodiments, cycloalkenyl groups have from 4 to 14, from 5 to 14, from 5 to 10, from 5 to 8, or from 5 to 6 carbon atoms in the ring(s). In some embodiments, cycloalkenyl groups have 5, 6, 7, or 8 ring carbon atoms. Examples of cycloalkenyl groups include cyclohexenyl, cyclopentenyl, cyclohexad ienyl, butad ienyl, pentadienyl, a nd hexadienyl .

The term "aryl" is intended to include cyclic aromatic hydrocarbon groups that do not contain any ring heteroatoms. Aryl groups include monocyclic, bicyclic and tricyclic ring systems. Examples of aryl groups include, but are not limited to, phenyl, azulenyl, heptalenyl, biphenyl, fluorenyl, phena nthrenyl, anthracenyl, indenyl, indanyl, pentalenyl, and naphthyl . In some embodiments, aryl groups have from 6 to 14, from 6 to 12, or from 6 to 10 carbon atoms in the ring(s) . In some embodiments, the aryl groups are phenyl or naphthyl. Aryl g roups include a romatic-aliphatic fused ring systems. Examples include, but are not limited to, indanyl and tetrahydronaphthyl . The term "heterocyclyl" is intended to include non-aromatic ring systems containing 3 or more ring atoms, of which one or more is a heteroatom . In some embodiments, the heteroatom is nitrogen, oxygen, or sulfur. In some embodiments, the heterocyclyl group contains one, two, three, or four heteroatoms. In some embodiments, heterocyclyl groups include mono-, bi- a nd tricyclic rings having from 3 to 16, from 3 to 14, from 3 to 12, from 3 to 10, from 3 to 8, or from 3 to 6 ring atoms. Heterocyclyl groups include partially unsaturated and saturated ring systems, for example, imidazolinyl and imidazolidinyl. Heterocyclyl groups include fused and bridged ring systems conta ining a heteroatom, for example, quinuclidyl. Heterocyclyl groups include, but are not limited to, azirid inyl, azetidinyl, azepanyl, diazepa nyl, 1,3-dioxanyl, 1,3-d ioxolanyl, isoxazolid inyl, morpholinyl, piperazinyl, piperidinyl, pyranyl, pyrazolid inyl, pyrrolinyl, pyrrolidinyl, tetra hydrofuranyl, tetrahydrothienyl, thiad iazolidinyl, and trithia nyl.

The term "heteroaryl" is intended to include aromatic ring systems containing 5 or more ring atoms, of which, one or more is a heteroatom. In some embodiments, the heteroatom is nitrogen, oxygen, or sulfur. In some embodiments, heteroaryl groups include mono-, bi- and tricyclic ring systems having from 5 to 16, from 5 to 14, from 5 to 12, from 5 to 10, from 5 to 8, or from 5 to 6 ring atoms. Heteroaryl groups include, but are not limited to, pyrrolyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, thiazolyl, pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl, thiophenyl, benzothiophenyl, furanyl, benzofuranyl, indolyl, azaindolyl (pyrrolopyridinyl), indazolyl, benzimidazolyl,

pyrazolopyridinyl, triazolopyridinyl, benzotriazolyl, benzoxazolyl, benzothiazolyl, imidazopyridinyl, isoxazolopyridinylxanthinyl, gua ninyl, q uinolinyl, isoq uinolinyl, tetra hydroquinolinyl, q uinoxalinyl, and q uinazolinyl. Heteroaryl groups include fused ring systems in which all of the rings are a romatic, for example, indolyl, and fused ring systems in which only one of the rings is aromatic, for example, 2,3-dihyd roindolyl.

The term "halo" or "halogen" is intended to include F, CI, Br, and I.

The term "heteroatom" is intended to include oxygen, nitrogen, sulfur, or phosphorus. In some embodiments, the heteroatom is selected from the group consisting of oxygen, nitrogen, and sulfur. As used herein, the term "substituted" is intended to mea n that one or more hyd rogen atoms in the group indicated is replaced with one or more independently selected suitable substituents, provided that the normal valency of each atom to which the substituent/s are attached is not exceeded, and that the substitution results in a stable compound. In various embodiments, optional substituents in the compounds described herein include but are not limited to halo, CN, N0 ₂ , OH, N H ₂ , N HR10, N R10R20, Cl-

6haloa lkyl, Cl-6haloalkoxy, C(0)NH ₂ , C(O)NHR10, C(O)N R10R20, SO2RIO, OR10, SR10, S(O)R10, C(O)R10, and Cl-6a liphatic; wherein RIO and R20 are each independently Cl- 6aliphatic, for example Cl-6alkyl .

The term "carboxyl protecting group" as used herein is mea ns a group that is capable of readily removed to provide the OH group of a carboxyl group and protects the carboxyl group aga inst undesirable reaction during synthetic proced ures. Such protecting groups are described in Protective Groups in Organic Synthesis edited by T. W. Greene et a l. (John Wiley & Sons, 1999) and 'Amino Acid-Protecting Groups' by Fernando Albericio (with Albert Isidro-Llobet and Mercedes Alva rez) Chemical Reviews 2009 (109) 2455- 2504. Examples include, but a re not limited to, a lkyl a nd silyl g roups, for example methyl, ethyl, terf-butyl, methoxymethyl, 2,2,2-trichloroethyl, benzyl, diphenylmethyl, trimethylsilyl, and terf-butyldimethylsilyl, and the like.

The term "amine protecting group" as used herein means a group that is capable of being readily removed to provide the N H2 group of an amine group and protects the amine group aga inst undesirable reaction during synthetic proced ures. Such protecting groups are described in Protective Groups in Organic Synthesis edited by T. W. Greene et a l. (John Wiley & Sons, 1999) and 'Amino Acid-Protecting Groups' by Fernando Albericio (with Albert Isidro-Llobet and Mercedes Alva rez) Chemical Reviews 2009 (109) 2455- 2504. Examples include, but a re not limited to, acyl and acyloxy groups, for example acetyl, chloroacetyl, trichloroacetyl, o-nitrophenylacetyl, o-nitrophenoxy-acetyl, trifluoroacetyl, acetoacetyl, 4-chlorobutyryl, isobutyryl, picolinoyi, aminocaproyi, benzoyl, methoxy-carbonyl, 9-fluorenylmethoxycarbonyl, 2,2,2-trifluoroethoxycarbonyl, 2- trimethylsilylethoxy-carbonyl, terf-butyloxycarbonyl, benzyloxycarbonyl, p- nitrobenzyloxycarbonyl, 2,4-dichloro-benzyloxycarbonyl, and the like. Further examples include Cbz (carboxybenzyl), Nosyl (0- or p-nitrophenylsulfonyl), Bpoc (2-(4- biphenyl)isopropoxycarbonyl) and Dde (l-(4,4-dimethyl-2,6-dioxohexylidene)ethyl).

The term "carboxamide protecting group" as used herein means a group that is capable of being read ily removed to provide the N H2 group of a carboxamide g roup and protects the carboxamide g roup against undesirable reaction during synthetic procedures. Such protecting groups are described in Protective Groups in Organic Synthesis edited by T. W. Greene et al . (John Wiley & Sons, 1999) and 'Amino Acid-Protecting Groups' by Ferna ndo Albericio (with Albert Isidro-Llobet and Mercedes Alvarez) Chemical Reviews 2009 (109) 2455-2504. Examples include, but are not limited to, 9-xa nthenyl (Xan), trityl (Trt), methyltrityl (Mtt), cyclopropyldimethylcarbinyl (Cpd), and dimethylcyclopropylmethyl (Dmcp) . As used herein, the term "and/or" means "and", or "or", or both.

The term "(s)" following a noun contemplates the singular and plural form, or both. The term "comprising" as used in this specification, includ ing the cla ims, means

"consisting at least in part of". When interpreting each statement in this specification, including the claims, that includes the term "comprising", features other than that or those prefaced by the term may a lso be present. Related terms such as "comprise" and "comprises" are to be interpreted in the same manner. The "containing" is a lso to be interpreted in the same manner.

The invention may also be said broadly to consist in the pa rts, elements and features referred to or indicated in the specification of the application, individ ua lly or collectively, in a ny or all combinations of two or more of said parts, elements or features, and where specific integers are mentioned herein which have known equiva lents in the art to which the invention relates, such known equivalents are deemed to be incorporated herein as if individually set forth.

It is intended that reference to a range of numbers disclosed herein (for exa mple, 1 to 10) also incorporates reference to a ll rational numbers within that range (for example, 1, 1.1, 2, 3, 3.9, 4, 5, 6, 6.5, 7, 8, 9, a nd 10) and also a ny ra nge of rational numbers within that range (for example, 2 to 8, 1.5 to 5.5, and 3.1 to 4.7) and, therefore, a ll subranges of a ll ranges expressly disclosed herein are hereby expressly d isclosed. These are only examples of what is specifically intended and all possible combinations of numerical values between the lowest value and the highest value enumerated are to be considered to be expressly stated in this application in a similar ma nner.

Although the present invention is broad ly as defined above, those persons skilled in the art will appreciate that the invention is not limited thereto and that the invention also includes embod iments of which the following description gives examples.

BRIEF DESCRIPTION OF THE FIGURES The invention will be described with reference to the accompa nying figures in which :

Figure 1 is a graph showing the results of a representative TLR agonism assay in HEK- Blue™-hTLR2 cells using titrated concentrations of agonist constructs : 910 (dotted white bars); 930 (g rey bars) ; 931 (striped bars) ; 932 (square hatched bars); and (R)- Pam2Cys-SK4-SLLMWITQV (black bars). Data presented as mean +/- SD ABS (635nm) values for triplicate wells following background subtraction. Dotted lines ind icate ABS in PBS-only wells.

Figure 2A is a ba r graph showing the results of a huma n TLR2 agonism assay in HEK- Blue™-hTLR2 cells using titrated concentrations of the following agonist constructs: A) (from left to right) : 45a, 45b, 46a, 46b, 47b, 910, 911, 912 and 913 (structures depicted in Table 3); B) (from left to right): 45b, 910 and chain elongated structures 930, 931, and 932 (structures depicted in Table 4).

Figure 3 is a bar graph showing the results of a murine TLR2 agonism assay using titrated concentrations of the following agonist constructs: A) (from left to right): 45a, 45b, 46a, 46b, 47b, 910, 911, 912 and 913 (structures depicted in Table 3); B) (from left to right): 45b, 910 and chain elongated structures 930, 931, and 932 (structures depicted in Table 4).

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides peptide conjugate compounds of the formula (I) as defined herein in the first aspect. The inventors have advantageously found that such conjugates have surprising immunogenic activity.

The peptide conjugate compounds of formula (I) may be prepared using the methods and procedures described herein.

Starting materials and/or intermediates useful in the methods may be prepared using known synthetic chemistry techniques (for example, the methods generally described in Louis F Fieser and Mary F, Reagents for Organic Synthesis v. 1-19, Wiley, New York (1967-1999 ed.) or Beilsteins Handbuch der organischen Chemie, 4, Aufl. Ed. Springer- Verlag Berlin, including supplements (also available via the Beilstein online database)) or, in some embodiments, may be commercially available. Preparation of the compounds may involve the protection and deprotection of various chemical groups. The need for protection and deprotection, and the selection of appropriate protecting groups, can be readily determined by a person skilled in the art. Protecting groups and methods for protection and deprotection are well known in the art (see e.g. T. W. Greene and P. G. M. Wuts, Protective Groups in Organic Synthesis, 3 ^rd Ed., Wiley & Sons, Inc., New York (1999)).

As shown in Scheme Al and described below, compounds of formula (IF) that are compounds of formula (I) wherein w is 1, v is 0, and m is from 2 to 6, preferably 2 or 3 to 5, may be prepared via a method of the present invention involving the conjugation of an epoxide to an amino acid-comprising conjugation partner. Scheme Al : Preparation of compounds of formula (IF) via conj ugation to an epoxide.

The method comprises reacting an epoxide of the formula (XVI) and an amino acid- comprising conjugation partner comprising a thiol of the formula (III) under conditions effective to provide the compound of formula (XV) by conjugation of the thiol to the epoxide.

In one embodiment of the method, method (A), the variables m, n, LI, Zl, R, Rl, R2, R3, R4, R5, R6, R7, R8, R9, and Al in the compound of formula (XV) are as defined in the compound of formula (IF) of the invention (including provisos (1) and/or (2) of the first aspect); and the method further comprises converting the compound of formula (XV) to the compound of formula (IF) of the invention by one or more additional synthetic steps. In this embodiment, the amino acid-comprising conjugation partner may comprise a peptide that corresponds the peptide present in the compound of the formula (IF) of the invention produced by the method. In another embod iment of the method, method (B), the variables m, n, LI, Zl, R, Rl, R2, R3, R4, R5, R6, R7, R8, R9, and Al in the compound of formula (XV) are as defined in the compound of formula (IB) but exclud ing provisos (1) and (2) of the first aspect; and the method further comprises converting the compound of formula (XV) to a compound of formula (IF) but exclud ing provisos (1) and (2) of the first aspect by one or more additional synthetic steps; and coupling the compound to an amino acid or peptide to provide the compound of formula (IF) of the invention (including proviso (1) and/or (2) of the first aspect). In this embodiment, the amino acid-comprising conjugation partner may consist of an amino acid or may comprise a peptide that corresponds to a portion of the peptide present in the compound of formula (IF) of the invention produced by the method . In some embod iments, the amino acid comprising conjugation pa rtner reacted with the epoxide consists of an amino acid, for example an Na-amine protected and/or C-terminus protected cysteine. In other embodiments, the amino acid comprising conj ugation partner comprises a peptide, for example a short peptide. In such embodiments, the amino acid comprising conjugation partner may comprise about 15 amino acid resid ues or less, for example 5, 4, or 3 amino acid residues.

The Na-amino group of the amino acid comprising conjugation partner is preferably protected or otherwise substituted (i.e. is not in the form of a free amine -N H2 group) to prevent reaction d uring the conjugation reaction. The C-terminus of the amino acid comprising conjugation partner may a lso be protected .

X10 in the compound of formula (XVI) may be a protected hyd roxyl, thiol, amine, or carbamate group (P10-O-, P11-S-, P12-N R-, or P12-N RC(0)0-, respectively) from which Ll-Zl- a nd L2-Z2- may subsequently be formed . Where X10 is a protected group, the protecting group may be removed in the conjugation reaction to provide a compound of the formula (XV) wherein Xl l is the correspond ing deprotected group. For example, where X10 is a PIO-O- group conjugation may provide the corresponding hydroxyl group as Xl l in the compound of formula (XV).

The epoxide of formula (XVI) comprises a stereogenic centre at the carbon atom to which R3 is attached . Thus, a sing le stereoisomer of the epoxide or a stereoisomerically enriched mixture of the epoxide may used in the reaction to control the stereochemistry of the carbon atom to which R3 is attached in the compound of formula (XV) and subsequent products formed, including the compound of formula (IF). Va rious methods for providing enantiopure or ena ntioenriched mixtures of epoxides are known in the a rt. In various embodiments, providing the single stereoisomer or a stereoisomerically enriched mixture of the epoxide of formula (XVI) comprises resolving a racemic mixture of the epoxide. For example, resolving a racemic epoxide mixture by kinetic hydrolysis, as described by Jacobsen et al, Science, 1997, 277, 936-938.

The epoxide of formula (XVI) may be provided by reacting an alkene of the formula (XVII) with an oxidant under conditions effective to epoxidise the alkene. Numerous methods for epoxidising alkenes are known in the art. In certain embodiments, the epoxidation is carried out by reacting the a lkene with a peroxide or an organic N-oxide as the oxida nt. Examples of suitable peroxides include organic peroxides, for example m- chloro peroxybenzoic acid . Examples of N-oxides include, for example, pyridine N-oxide and the like. Other suitable oxidants will be apparent to those skilled in the art. The reaction may be carried out in a liquid reaction medium comprising a suitable solvent, for example dichloromethane. Alkenes of the formula (XVII) may be commercially available or prepared from commercially ava ilable precursors using standard synthetic chemistry techniq ues.

Those skilled in the art will appreciate that certain X10 groups may be susceptible to oxidation in the epoxidation reaction, for example when X10 comprises an amine group (which may form an N-oxide) or thioether group (which may form e.g . sulfoxides or sulfones). Such groups may be protected during the reaction to prevent oxidation or reduced back to the desired group at an appropriate point in the synthetic sequence after the epoxidation reaction has been carried out.

Alternativley, the epoxide of formula (XVI) may be prepared by treating a compound of formula (XVII-A), wherein LG is a suitable leaving group such as a ha logen, with a base in a suitable solvent to displace the leaving group as shown in scheme A2.

Scheme A2. Epoxidation via leaving group displacement.

(XVII-A) (XVI)

Compounds of the formula (XVII-A) may be commercia lly available or may be prepared from commercially available precursors. Advantageously, in some embodiments, the compound of formula (XVII-A) may be prepared from a n enantiopure a-amino acid . The epoxidation reaction proceeds stereospecifically with inversion of stereochemistry at the carbon to which R3 is attached.

For example, as shown in scheme A2-1, the compound of formula (XVII-A1), which corresponds to a compound of formula (XVII-A) wherein m is 2 and Rl and R2, and R3, R4, and R5 are hyd rogen, X10 is -OH, and LG is bromo, may be prepared from L-aspartic acid (see Volkmann, R. A. et a l. J. Org. Chem., 1992, 57, 4352-4361).

L-Aspartic acid may be converted to be bromosuccinic acid (AA-1) by, for example, treatment with sodium nitrite and a strong acid such as sulfuric acid, to generate nitrous acid in situ, in the presence of sodium bromide at a temperature from -10 to 0°C. The reaction proceeds stereospecifically with overall retention of stereochemistry.

Reduction of bromosuccinic acid (AA-1) to bromodiol (XVII-A1) may be carried out using a suitable reductant, for example by treatment with borane or borane-dimethyl sulfide complex in THF at -78°C allowing the reaction mixture to warm to room temperature. Epoxidation to provide the compound of formula (XVI-la) may be carried out by reacting bromodiol (XVII-A1) with a base, for example cesium carbonate in dichloromethane at room temperature. As noted above, the reaction proceeds stereospecifcally with overall inversion of stereochemistry.

The opposite enantiomer of epoxide (XVI-la) can be prepared from D-aspartic acid by the same procedure.

Scheme A2-1. Preparation of enantiopure epoxide from L-aspartic acid.

Referring again to Scheme Al, the compound of formula (XV) may be subsequently converted by one or more synthetic steps to compound of the formula (IF) as defined in either method (A) or (B). In the one or more steps, the hydroxyl group bound to the carbon to which R3 is attached is converted to an L2-Z2- group.

If Xl l is not L1-Z1-, then the one or more steps also comprises converting Xll to Ll-Zl- . The Ll-Zl- and L2-Z2- groups may be introduced simultaneously or sequentially in any order.

In certain embodiments, the one or more steps comprises acylating the compound of formula (XV) so as to replace the hydrogen atom of the hydroxyl group bound to the carbon to which R3 is attached with L2-C(0)-.

In exemplary embodiments, X10 is PIO-O- or OH; and Xl l is PIO-O- or OH.

In various embodiments, Xl l is PIO-O- or OH; and the one or more synthetic steps comprise acylating the compound of formula (XV) so as to replace P10 or the hydrogen atom of the hydroxyl group of Xl l with Ll-C(O)-; and/or the hydrogen atom of the hydroxyl group bound to the carbon to which R3 is attached with L2-C(0)-.

In certain embodiments, as shown below in Scheme A3 and described herein, the method comprises reacting an epoxide of formula (XVI-1) bearing a protected hydroxyl group with an amino acid comprising conjugation partner of the formula (III) to provide a compound of the formula (XV-la). Scheme A3 : Preparation of bis-ester conj ugates via epoxide conj ugation.

(IF-1)

The conj ugation reaction may be carried out under acid ic cond itions by reacting the epoxide and thiol in the presence of an acid, for example hydrochloric acid, sulfuric acid, or a mixture thereof. The reaction may be carried out in a liquid reaction medium comprising a suitable solvent, such as dichlorometha ne, at a temperature from about -10 to about 50°C, for example from 0 to 40°C.

The hydroxyl protecting group P10 is selected such that it is removable under the conditions effective for conjugation and is therefore removed during the conjugation reaction to provide the desired diol of formula (XV-la). Suitable protecting groups will be apparent to those skilled in the a rt a nd may include, for example, acid labile silyl protecting groups. Alternatively, the conj ugation reaction may be carried using an epoxide of the formula (XVI) wherein X10 is a hydroxyl group, such as the epoxide of formula (XVI-la).

The diol of the formula (XV-la) may be converted to the compound of formula (IF-1) by reaction with the compounds of formula (VI-1) and (VI), wherein X is OH or a suitable leaving group (for example a ha lide, such as chloro or bromo), under cond itions effective for esterification.

The cond itions effective for esterification depend on the nature of the compound of formula (IV) and/or (VI-1). For example, where X is OH, the reaction may be carried out in the presence of a base, such as DMAP, and activating agent, such as Ν,Ν'- diisopropylcarbod iimide (DIC) in a liquid medium comprising a suitable solvent, such as THF. In various embodiments, the compound of formula (VI) and (VI-1) are identical. For example, the compound of formula (VI) and (VI-1) may each be palmitic acid. In such embodiments, conversion of the diol of formula (XV-la) to the compound of formula (IF- 1) may be accomplished in a single step.

In certain embodiments, different LI and L2 groups may be introduced by reacting the diol with a stoichiometric amount of a compound of formula (VI-1) or (VI) to esterify the more reactive of the two alcohols, and then reacting the resultant ester with the other a compound of formula (VI) or (VI-1) to esterify the second alcohol of the diol.

In other embodiments, the method comprises reacting an epoxide of formula (XVI-1) and an amino acid comprising conjugation partner of the formula (III) to provide a compound of the formula (XV-lb) as shown in Scheme A4 below. In such embodiments, the hydroxyl protecting group P10 is stable and is not removed under the conjugation reaction conditions.

The protected alcohol of the formual (XV-lb) provides ready access to compounds of formula (IF-1) wherein LI and L2 are different. Using the compound of formula (XV-lb) to access such compounds, rather than the diol of formula (XV-la), may be more convenient in certain emboidments, for example where there is poor selectivity between the alcohols of the diol of formula (XV-la).

Scheme A4: Preparation of bis-ester conjugates via the compound of formula (XV-lb).

(IF-1) The β-sulfanylhydroxyl group of the compound of formula (XV-lb) may be acylated with a compound of formula (VI) under conditions effective for esterification to provide protected ester (XVIII), then the protecting group P10 removed to provide the alcohol of formula (XIX). The conditions for removal of the protecting group depend on the protecting group used . For example, dilute HF may be used to remove silyl protecting groups, such as TBDMS, TBDPS, a nd the like. The alcohol of formula (XIX) may then be acylated with a compound of formula (VI-1) under conditions effective for esterification to provide the desired compound of formula (IF-1). Those skilled in the art will appreciate that hyd roxyl groups, for example those in the compounds of formulae (XV-la), (XV-lb), and (XIX), may be converted to various other functiona l groups, such as thiols and amines, to provide access compounds of formula (I) bearing Ll-Zl- and L2-Z2- g roups other tha n esters. For example, the compound of formula (XV-l b) can be used to prepare thioester and amide analogues of the compound of formula (IF-1), as shown below in Scheme A5. To prepare amide ana logue (IF-3), the hydroxyl group in the compound of formula (XV-lb) may first be converted to an azide and then red uced to the corresponding amine. The reaction may be carried out under modified M itsunobu cond itions (e.g. L. Rokhum et al, J. Chem. Sci, 2012, 124, 687-691) using PPh ₃ , I2, imidazole, and NaN ₃ to provide the azide, and then PPh ₃ to reduce azide to the amine. Alternatively, the azide may be obtained by first converting the hyd roxyl group to a suitable leaving group, for example a tosyl or mesyl g roup, and then treating with NaN ₃ . Acylation of the amine with a compound of formula (VI) provides the amide of formula (XVIII-2). The acylation reaction may be carried out by reacting a carboxylic acid of the formula (VI) in the presence of a base, for example DMAP, and an activating agent, for example DIC, in a suitable solvent such as THF. Deprotection of the protecting g roup P10 and esterification of the resulta nt alcohol (XIX-2) provides the compound of the formula (IF-3).

Scheme A5. Preparation of thioesters and amides via the compound of formula (XV-lb).

(XIX-2) (IF-3) Thioester analogue (IF-2) may be prepared by first reacting the compound of formula (XV-lb) under Mitsunobu conditions (e.g. PPh3, diethylazodicarboxylate (DEAD)) and trapping with the desired thioacid of formula (VI-2), for example thiopalmitic acid, to provide the compound of formula (XVIII-l)(see e.g. 0. Schulze et al, Carbohydrate Res., 2004, 339, 1787-1802). Deprotection of the protecting group P10 and esterification of the resultant alcohol (XIX-1) provides the compound of the formula (IF-2).

Thioester and amide analgoues of bis-ester (IF-1) may also be prepared from the compound of formula (XIX), as shown in Scheme A6. The compound of formula (XIX) may be converted to the compound of formula (IF-4) by methods analogous to those described above for the conversion of the compound formula (XV-lb) to the compound of formula (XVIII-1).

Similarly, the compound of formula (XIX) may be converted to the compound of formula (IF-5) by methods analogous to those described above for the conversion of the compound of formula (XV-lb) to the compound of formula (XVIII-2). Scheme A6. Preparation of thioesters and amides via the compound of formula (XIX).

L ¹ -C(0)SH

L ¹ -C(0)X

Further analogues of bis-ester (IF-1) may be prepared by replacing the compound of formula (XIX) in Scheme A6 with a compound of formula (XIX-1) or (XIX-2) and then following the synthetic sequences described .

Numerous other compounds of formula (IF) may be prepared by analogous methods, as will be appreciated by those skilled in the art. Compounds of formula (VI), (VI-1), (VI-2), and (VI-3) may be commercially available or prepared from commercia lly available precursors using standard synthetic chemistry techniq ues.

Compounds of formula (I) may also be prepared by a method of the present invention comprising the conjugation of an amino acid comprising conjugation partner and an acetal, as shown in Scheme Bl .

Scheme Bl. Preparation of compounds of formula (I) via acetal (XXI).

The method comprises reacing an amino acid comprising conj ugation partner of the formula (III) and an acetal of the formula (XXI), wherein LG is a suitable leaving group, under conditions effective to provide a compound of the formula (XX).

In one embodiment of the method, method (A), the variables m, w, v, n, Rx, Ry, Rl, R2, R3, R4, R5, R6, R7, R8, R9, and Al in the compound of formula (XX) are as defined in the compound of formula (I) of the invention (includ ing provisos (1) and/or (2) of the first aspect); and the method further comprises converting the compound of formula (XX) to the compound of formula (I) of the invention by one or more additiona l synthetic steps. In this embodiment, the amino acid-comprising conj ugation partner may comprise a peptide that corresponds the peptide present in the compound of the formula (I) of the invention produced by the method .

In another embodiment of the method, method (B), the variables m, w, v, n, Rx, Ry, Rl, R2, R3, R4, R5, R6, R7, R8, R9, and Al in the compound of formula (XX) are as defined in the compound of formula (I) but excluding provisos (1) a nd (2) of the first aspect; and the method further comprises converting the compound of formula (XX) to a compound of formula (I) but excluding provisos (1) a nd (2) of the first aspect by one or more additional synthetic steps; and coupling the compound to an amino acid or peptide to provide the compound of formula (I) of the invention (including proviso (1) and/or (2) of the first aspect). In this embodiment, the amino acid-comprising conj ugation partner may consist of an amino acid or may comprise a peptide that corresponds to a portion of the peptide present in the compound of formula (I) of the invention produced by the method. In some embod iments, the amino acid comprising conjugation pa rtner reacted with the acetal consists of an amino acid, for example a n Να-amine protected a nd/or C-terminus protected cysteine. In other embodiments, the amino acid comprising conjugation partner comprises a peptide, for example a short peptide. In such embodiments, the amino acid comprising conjugation partner may comprise about 15 amino acid residues or less, for example 5, 4, or 3 amino acid residues.

The Na-amino group of the amino acid comprising conjugation partner is preferably protected or otherwise substituted (i.e. is not in the form of a free amine -N H2 group) to prevent reaction d uring the conjugation reaction. The C-terminus of the amino acid comprising conjugation partner may a lso be protected .

In the reaction, the thiol of the compound of formula (III) displaces the leaving group (LG) in the acetal of formula (XXI). Suitable leaving groups include but are not limited to halo (for example chloro, bromo, or iodo) or sulfonate (for example a tosylate or mesylate). Other suita ble leaving g roups will be apparent to those skilled in the art. The size of the aceta l ring in the compound of formula (XXI) may vary. The acetal ring may comprise from 5 to 7 ring atoms (i.e. may be a 5-7-membered cyclic acetal). In certain embodiments, the cyclic acetal is 6-membered . It will be appreciated that when the cyclic acetal is a 5-membered cyclic acetal, in order to provide a compound of the formula (I), w is at least 2 (such that the sum of m, v, and w is at least 3). The conj ugation reaction may be carried out in the presence of a base. For example, the reaction may be carried out in the presence of organic amine, in a suitable solvent, for example DMF, at a temperature of about 50°C. Suitable organic amines include but a re not limited to triethylamine, N-methylmorpholine, collid ine, and the like.

The compound of formula (XXI) may be provided in stereoisomerically pure form or a stereoisomerically enriched mixture by reacting stereoisomerically pure or a

stereoisomerically enriched mixture of the compound of the compound of formula (XXII). Advantageously, stereoisomerically pure compounds of formula (XXII) are readily commercially available, such as (4fi)- or (4S)-(2,2-dimethyl-l,3-dioxa n-4-yl)-methanol.

Other compounds of formula (XXII) may be prepared by routine methods known in the art. As shown in Scheme Bl-1, a compound of formula (XXII-B), wherein Pg is a suitable hydroxyl protecting group, may be reacted with a compound of the formula (XXII-Cl) to provide the aceta l of formula (XXII-D), which may then be converted to the compound of formula (XXII) by removal of the protecting group Pg . Alternatively, the compound of formula (XXII-B) may be reacted with an acyclic acetal of the formula (XXII-C2), wherein Ro a nd Rp a re each independently Cl-4alkyl. The acetylisation reaction may be carried out using an acid, such as camphorsulfonic acid, in a suitable solvent, such as dichloromethane.

The cond itions for removal of the protecting group Pg, depend on the protecting group used . For example, a silyl ether protecting group, such as TBDMS, may be removed by treatment with a source of fluorine, such as tetrabutylammonium fluoride (TBAF) in suitable solvent, such as THF. See, for example C. R. Reddy et a l, (Tetrahedron Letters, 2010, 51(44) 5840-5842) ; and Sauret-Clad iere et a l (Tetrahedron Asymmetry, 1997, 8(3), 417-423).

Scheme Bl-1. Preparation of compounds of formula (XXII).

Referring again to Scheme Bl, compounds of formula (XXI) may be prepared from compounds of formula (XXII) by reaction with a suitable precursor of the leaving group. For example, tosylate or mesylate leaving groups may be prepared by reaction with tosyl chloride or mesyl chloride in the presence of a base and a suitable solvent, and an iodo leaving group may be prepared by reaction with PPh3 and I2.

The compound of formula (XX) may subseq uently be converted by one or more synthetic steps to a compound of the formula (I) as defined in either method (A) or (B), for example a compound of the formula (IA).

The one or more synthetic steps may comprise removing the acetal to provide a d iol of the formula (XXIII-1). The hydroxyl group bound to the carbon to which Rl and R2 are attached in the compound of formula (XXIII-1) may be converted to L1-Z1-, and/or the hydroxyl group bound to the carbon to which Rx and Ry a re attached may be converted to L2-Z2.

For example, as shown in Scheme B2, the aceta l in the compound of formula (XX) may be removed to provide the diol of formula (XXIII-1) by treatment with an acid such as p- toluene sulfonic acid in a solvent such as dichlorometha ne. The diol of formula (XXIII-1) may be converted to the bis-ester compound of formula (IA) via one or more acylation steps in a manner analogous to that described for the conversion of the compound of formula (XV-la) to the compound of formula (IF-1).

Scheme B2. Preparation of bis-ester conj ugates of formula (IA).

Alternatively, in various embodiments wherein Rm is optionally substituted aryl, for example phenyl or methoxy substituted phenyl, the one or more synthetic steps may comprise removing the acetal to provide a compound of the formula (XXIII-2) or (XXIII- 3). The one or more steps may comprise converting the hydroxyl group bound to the carbon atom to which Rx and Ry are attached in the compound of formula (XXIII-2) to L2-Z2-, removing the RmRnCH- group to provide a hyd roxyl group, and converting the hydroxyl group to Ll-Zl ; or converting the hydroxyl group bound to the cabon to which Rx and Ry a re attached in the compound of formula (XXIII-2) to L1-Z1-, removing the RmRnCH- group to provide a hyd roxyl group, and converting the hydroxyl group to L2- Z2-. Such methods advantageously allows a llow the introduction of d ifferent Ll-Zl and L2-Z2- groups.

As illustrated in Scheme B3, the acetal in the compound of formula (XX) may be removed by, for example, treatment with a suitable reducing agent, for example

diisobutylaluminium hydride (DIBAL). The resulting compound of formula compound of formula (XXIII-2) may then be acylated with the compound of formula (VI) to introd uce the desired L2-C(0)0- group. Removal of the RmRnCH- group to provide the compound of formula (XXV-2) may be carried out by hydrogenolysis (e.g . for a benzyl or p- methoxybenzyl group) or any other suitable method having regard to the nature of RmRnCH- group. The compound of formula (XXV-2) may then be converted to the compound of formula (IA) by acylating with the compound of formula (IV-1). The acylation steps may be carried out as described herein with respect to the preparation of the compound of formula (IF-1). Scheme B3. Bis-ester conjugates via compounds of formula (XXIII-2)

L ² -C(0)X (VI)

It will be apparent to those skilled in the art that compounds of formula (IA) may be prepared from compounds of formula (XXIII-3) by a replacing the compounds of formulae (XXIII-2), (VI) and (VI-1) in Scheme B3 with the compounds of formulae (XXIII-3), (VI-1), and (VI), respectively, and then following the synthetic sequence described.

Hydroxyl groups produced on removal of the acetal or RmRnCH- group, such as those in the compounds formulae (XXIII-1), (XXIII-2), (XXIII-3), and (XXV-2), may be converted to various other functional groups, such as thiols and amines, to provide access compounds of formula (I) bearing other Zl and Z2 groups.

It will be appreciated that amide and thioester analogues of the bis-ester compound of formula (IA) may be prepared by methods analogous to those described above with respect to the amide and thioester analogues of the bis-ester compound of formula (IF- 1).

The present invention also provides a method for preparing compounds of formula (I) of the invention via a thiol-ene reaction. The method comprises reacting a first lipid- containing conjugation partner comprising a carbon-carbon double bond, a second lipid- containing conjugation partner a carbon-carbon double bond, and an amino acid- comprising conjugation partner comprising a thiol, under conditions effective to conjugate the first and second lipid-conta ining conjugation partners to the amino acid- comprising conjugation partner. Each lipid containing conj ugation pa rtner comprises and therefore in the reaction provides to the compound of formula (I) a lipid moiety one comprising LI, the other comprising L2.

The thiol-ene reaction involves the addition of a thiol across a non-aromatic carbon- carbon double bond (i.e. hydrothiolation of the carbon-carbon double bond). The reaction proceeds via a free radical mechanism. There are three distinct phases in the reaction : initiation, coupling, a nd termination. Typica lly, radical generation gives rise to an electrophilic thiyl radical which propagates across the ene group of an a lkene, forming a carbon-centred radical and chain tra nsfer from an additional thiol molecule quenches the radica l on carbon to give the fina l prod uct.

Without wishing to be bound by theory, the inventors believe that in the method of the present invention, the thiol is conjugated to a carbon atom of the carbon-carbon double bond of the first lipid conta ining conjugation partner to form a carbon-centred radical, and that this carbon-centred radical, instead of being quenched, is then conjugated with a carbon atom of the ca rbon-carbon double bond of the second lipid-conta ining conjugation partner. The method thus provides amino acid- and peptide conj ugates in which the sulfur atom from the thiol is conjugated to a carbon atom from the carbon-carbon double bond of the first lipid-containing conj ugation partner, and a carbon atom from the carbon-carbon double bond of the first lipid-containing conj ugation partner is conjugated to a carbon atom from the carbon-carbon double bond of the second lipid-containing conjugation partner.

In one embodiment of the method, method (A), conj ugation of the first and second lipid- containing conjugation partners to the amino acid-comprising conj ugation pa rtner provides the peptide conjugate of the formula (I) of the invention . In this embodiment, the amino acid-comprising conjugation pa rtner may comprise a peptide that corresponds the peptide present in the peptide conj ugate of the formula (I) of the invention produced by the method .

In another embod iment of the method, method (B), conjugation of the first and second lipid-containing conj ugation partners to the amino acid-comprising conj ugation partner provides an amino acid- or peptide-conj ugate (of the formula (I) but excluding provisos (1) and (2) of the first aspect); a nd the method further comprises coupling the amino acid- or peptide-conj ugate to an amino acid or peptide to provide the peptide conjugate of formula (I) of the invention (including proviso (1) and/or (2) of the first aspect). In this embodiment, the amino acid-comprising conjugation partner may consist of an amino acid or may comprise a peptide that corresponds to a portion of the peptide present in the compound of formula (I) of the invention produced by the method.

The first and second lipid containing conjugation partners may be the same or different. Those skilled in the art will appreciate that reacting different lipid containing conj ugation partners at the same time may provide a mixture of (potentially up to four different) conjugates. Accordingly, in certa in exemplary embodiments, the first and second lipid containing conjugation partners are the same.

The thiolene reaction may be regioselective with respect to which carbon atom of the carbon-carbon double bond of the first lipid-containing conj ugation partner is conj ugated to the thiol a nd also with respect to which carbon atom of the carbon-carbon double bond of the second lipid-containing conjugation pa rtner is conjugated to which carbon atom of the carbon-carbon double bond from the first lipid-conta ining conjugation pa rtner. Those skilled in the art will appreciate that various regioisomers may be formed in the reaction.

In certain embodiments, the method comprises reacting a first lipid containing conjugation partner of the formula (IIA) and a second lipid containing conj ugation partner of the formula (IIB) with a thiol containing amino acid comprising conj ugation partner (III) under conditions effective to provide a compound of the formula (IB)

(Scheme CI). When the method is (A), Ra, Rb, Rc, LI, L2, Zl, Z2, Rl, R2, Rx, Ry, R3, R4, R5, R6, R7, R8, R9, Al, k, v, and n are as defined in the compound of formula (IB) of the present invention (including provisos (1) and/or (2) of the first aspect) ; and when the method is (B), Ra, Rb, Rc, LI, L2, Zl, Z2, Rl, R2, Rx, Ry, R3, R4, R5, R6, R7, R8, R9, Al, k, v, and n a re as defined in the compound of formula (IB) but excluding provisos (1) and (2) of the first aspect.

Scheme CI. Preparation of compounds of formula (IB) via a thiolene reaction.

(IB)

The cond itions effective for formation of the compound of formula (IB) may vary. In various embodiments, the conditions effective for formation of the compound of formula (IB) may comprise carrying out the reaction with a stoichiometric excess of lipid containing conjugation partner to thiol, such as a stoichiometric ratio of the lipid containing conjugation partners (IIA) and (IIB) (combined) to amino acid-comprising conjugation partner of at least 7 : 1, for example 8: 1, 9 : 1, 10 : 1, 20: 1, 30: 1, 40: 1, 50 : 1, 60: 1, or 70 : 1. The degree of conversion of the amino acid-comprising conjugation partner to the prod uct compound of formula (IB) may vary. Preferably, at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, or 70% of the amino acid-comprising conjugation partner is converted to the compound of formula (IB) . Conversion may be determined by HPLC.

As noted above, without wishing to be bound by theory, the inventors believe that under such conditions reaction of the a lkene of formula (IA) with the thiol of formula (III) results in the formation of a carbon-centred radical of the formula (X), which is trapped with the second alkene of the formula (IIB), rather than quenched by abstraction of a proton from the thiol of another molecule of the formula (III), to provide the desired amino acid- or peptide conj ugate. The reaction may result in the production of a mixture of stereoisomers as it may not be possible to control or influence the stereochemistry of bond formation between the carbon atom to which R3 is bound and the carbon atom to which Rb and Rc are bound owing to the radica l intermediate generated in the course of the reaction. The reaction typically produces a mixture of epimers with respect to the carbon atom to which R3 is bound.

In certain embodiments, the Zl and Z2 in the lipid conta ining-conj ugation partners are each -C(0)0-, a nd the compound of formula (I) formed in the thiolene method is a compound of formula (IC) as defined herein.

In exemplary embodiments, the thiolene method of the present invention comprises reacting a n amino acid-comprising conj ugation partner comprising a structure of the formula (III) with lipid containing-conjugation partners of the formula (IIA) and (IIB) that are vinyl esters to provide a compound of the formula (ID). The reaction may be carried out, for example as described herein, by irradiating a reaction mixture comprising the amino acid comprising conjugation partner; lipid containing-conjugation pa rtners; a photochemical initiator, such as DMPA. One or more add itives may be included that reduce the formation of by products, such as a sterically hindered thiol (for example tert- butylmercaptan), an acid (for example TFA), or an organosilane (for example

triisopropylsilane), or a combination of a ny two or more thereof. The reaction may be carried out in a suitable solvent, such as NM P, at ambient temperature for a suitable period of time, such as 30 minutes.

The reaction is typically initiated by the generation of one or more free radicals in the reaction mixture. One or more free radicals may be generated in the method by any method known in the art. The free rad icals may be generated thermally and/or photochemically. One or more free radical initiators may be used to initiate the generation of free radicals. Suitable free radical initiators include thermal initiators and photoinitiators.

Free radicals are generated from thermal initiators by heating . The rate of degradation of the thermal initiator and resulting free radical formation depends on the initiator a nd the temperature at which the initiator is heated . Higher temperatures genera lly result in faster decomposition. A person skilled in the art will be able to select an appropriate temperature for heating the initiator without undue experimentation.

Numerous thermal initiators are commercia lly available. Examples of thermal initiators include but a re not limited to terf-amyl peroxybenzoate, 1, 1'- azobis(cyclohexanecarbonitrile), 2,2'-azobisisobutyronitrile (AIBN), benzoyl peroxide, tert-butyl hydroperoxide, tert-butyl peracetate, tert-butyl peroxide, tert-butyl peroxybenzoate, tert-butylperoxy isopropyl carbonate, lauroyl peroxide, peracetic acid, and potassium persulfate. Free radicals may be generated from photoinitiators by irrad iation with lig ht. The frequency of light necessary to induce degradation of the photoinitiators and free rad ical formation depends on the initiator. Many photoinitiators can be initiated with ultraviolet light. Light of a specific wavelength or wavelength ra nge may be used to selectively irrad iate the initiator, where the lipid-conta ining conjugation partners or amino acid-comprising conjugation partner, for example a peptide-containing conj ugation partner, comprises photosensitive groups. In certa in embodiments, a freq uency of about 365 nm is used . Light of this frequency is generally compatible with the side chains of natura lly occurring amino acids.

A wide range of photoinitiators are commercially available. Examples of photoinitiators include but a re not limited to acetophenone, anisoin, anthraquinone, anthraquinone-2- sulfonic acid, benzil, benzoin, benzoin ethyl ether, benzoin isobutyl ether, benzoin methyl ether, benzophenone, 3,3',4,4'-benzophenonetetracarboxylic d ianhydride, 4- benzoylbiphenyl, 2-benzyl-2-(d imethylamino)-4'-morpholinobutyrophenone, 4'- bis(diethylamino)benzophenone, 4,4'-bis(dimethylamino)benzophenone,

camphorquinone, 2-chlorothioxanthen-9-one, dibenzosuberenone, 2,2- diethoxyacetophenone, 4,4'-dihyd roxybenzophenone, 2,2-d imethoxy-2- phenylacetophenone (DMPA), 4-(dimethylamino)benzophenone, 4,4'-dimethylbenzil, 2,5- dimethylbenzophenone, 3,4-dimethylbenzophenone, 4'-ethoxyacetophenone, 2- ethylanthraquinone, 3'-hydroxyacetophenone, 4'-hydroxyacetophenone, 3- hydroxybenzophenone, 4-hydroxybenzophenone, 1-hydroxycyclohexyl phenyl ketone, 2- hydroxy-2-methylpropiophenone, 2-methylbenzophenone, 3-methyl benzophenone, methybenzoylformate, 2-methyl-4'-(methylthio)-2-morpholinopropiophenone, phenanthrenequinone, 4'-phenoxyacetophenone, and thioxanthen-9-one.

A person skilled in the art will be able to select appropriate free radical initiators for use in the method having regard to, for example, the nature of the lipid-containing conjugation partners, amino acid-comprising conjugation partner, for example a peptide- containing conjugation partner, a nd a ny other components present in the reaction mixture. In some embodiments, the initiator is present in the reaction in a stoichiometric ratio relative to the starting material comprising the thiol of from about 20: 1 to about 0.05: 1, from about 10 : 1 to about 0.05 : 1, from about 5 : 1 to about 0.05: 1, from about 3: 1 to about 0.5: 1.

The lipid-conta ining conjugation partners a nd amino acid-comprising conjugation partner, for example a peptide-containing conj ugation partner, may be prepared using known synthetic chemistry techniq ues (for example, the methods generally described in Louis F Fieser and Mary F, Reagents for Organic Synthesis v. 1-19, Wiley, New York (1967-1999 ed. ) or Beilsteins Handbuch der organischen Chemie, 4, Aufl. Ed. Springer-Verlag Berlin, including supplements (also ava ilable via the Beilstein online database)) or, in some embodiments, may be commercially ava ilable. For example, lipid-containing conjugation partner compounds of the formula (IIA-1) may be prepared by reacting a compound of the formula (VI) wherein X is OH or a suitable leaving group with a compound of the formula (VII) wherein Y is H, a metal or metalloid, or acyl (for example, alkylcarbonyl) under conditions effective for esterification (or transesterification where Y is an acyl g roup) (Scheme C2).

Scheme C2. Preparation of compounds of the formula (IIA-1).

Methods for esterification (or transesterification) are well known in the art. For example, when X is chloro and Y is H, the reaction may be carried out in the presence of a base, such as pyridine or triethylamine, in a suitable solvent. The acid chloride may be converted in situ to a more reactive species (e.g. to the corresponding iodide, using sodium iod ide). The temperature at which the reaction is carried out depends on the reactivity of the acid species and the solvent used .

For example, vinyl esters of the formula (IIA-1) may be produced by transesterification with vinyl acetate (itself produced industrially by the reaction of acetic acid and acetylene or acetic acid and ethylene over a suitable catalyst) using an acid or metal catalyst. See, for example, EP0376075A2 and S. K. Karmee, J. Oil Palm Res. , 2012, 1518-1523.

Vinyl esters of the formula (IIA-1) may also be prepa red by the addition a carboxylic acid to a terminal acetylene in the presence of a catalyst (usually a pa lladium or ruthenium complex). See, for example, V. Cadierno, J. Francos, J. Gimeno Organometallics, 2011, 30, 852-862; S. Wei, J. Pedroni, A. Meissner, A. Lumbroso, H . -J. Drexler, D. Heller, B. Breit, Chem. Eur. J. , 2013, 19, 12067-12076. Non-termina l acetylenes may also be reacted . See, for example, N . Tsukada, A. Takahashi, Y. Inoue, Tetrahedron Lett., 2011, 52, 248-250 and M . Rotem, Y. Shvo, J. Organometallic Chem. 1993, 448, 159-204.

Further examples of methods for preparing vinyl esters of formula (IIA-I) include:

reaction of divinylmercury with aromatic and aliphatic acids [see, for example, D. J. Foster, E. Tobler, J. Am. Chem. Soc. 1961, 83, 851 ]; Cu(II)-catalyzed esterification of arene carboxylic acids with trimethoxy(vinyl)sila ne in the presence of AgF [see, for example, F. Luo, C. Pan, P. Qia n, J. Cheng, Synthesis 2010, 2005]; vinyl transfer reactions from vinyl acetate to primary and secondary alcohols, and a lso to carboxylic acids with a catalyst system consisting of 2 mol-% of [AuCI(PPh3)] a nd 2 mol-% of AgOAc [see, for example, A. Nakamura, M . Tokunaga, Tetrahedron Lett. 2008, 49, 3729]; and Ir complex ([Ir(cod)CI]2/P(OMe)3)-catalyzed transvinylation [see, for example, H . Nakagawa, Y. Okimoto, S. Sakaguchi, Y. Ishii, Tetrahedron Lett. 2003, 44, 103]. Other suitable methods for preparing compounds of formula (II-A) will be apparent to those skilled in the art.

Lipid conta ining conj ugation partner compounds of the formula (IIB-1) may be prepared in a n analogous fashion, where the compounds of formula (IIA-1) and (IIB-1) are different. Numerous compounds of formula (VI) a re commercially available. Others may be prepared using standard synthetic chemistry techniques from commercially ava ilable precursors. For example, compounds of formula (VI) wherein X is chloro may be prepared treating the correspond ing carboxylic acid with thionyl chloride in a suitable solvent or mixture of solvents. Similarly, compounds of formula (VII) are also commercially available or may be prepared from commercia lly available precursors using standard synthetic chemistry techniq ues.

The order in which the lipid-containing conj ugation partners and amino acid-comprising conjugation partner, for example a peptide-containing conj ugation partner, and any other components present in the reaction mixture a re introduced into the reaction vessel may vary. The reaction may be carried out as a one-pot procedure.

The ratio of the lipid-conta ining conjugation pa rtners to amino acid-comprising conjugation partner, for example a peptide-containing conj ugation partner, in the reaction may vary. In some embodiments, the mole ratio of the first lipid-containing conjugation partner and second lipid-containing conj ugation pa rtner combined (i.e. in total) to the amino acid-comprising conj ugation partner is at least 7: 1, for example 8 : 1, 9: 1, 10 : 1, 20 : 1, 30 : 1, 40: 1, 50: 1, 60 : 1, or 70 : 1.

The reaction may be carried out at any suitable temperature. In some embodiments, the reaction is carried out at a temperature from about -25 °C to about 200 °C, from about - 10 °C to about 150 °C, from about 0 °C to about 125 °C, from about a mbient temperature to about 100 °C. In some embodiments, the reaction is carried out at a temperature of less than about 200 °C, less than about 175 °C, less than about 150 °C, less than about 125 °C, or less tha n about 100 °C. In some embod iments, the reaction is carried out at a temperature above ambient temperature. In one embodiment, the reaction is carried out at a temperature from 40 to 200 °C, from 50 to 150 °C, from 60 to 100 °C, from 65 to 90 °C, or from 70 to 80 °C. In some embod iments, the reaction is carried out at a temperature g reater tha n 40 °C, greater tha n 50 °C, greater tha n 75 °C, greater tha n 100 °C, or greater tha n 150 °C. The temperature at which the reaction is carried out may depend on how free radicals are generated in the reaction. The temperature used may be selected to control the rate of the reaction. The temperature may be adjusted during the course of the reaction to control the rate of the reaction.

If free radicals are generated thermally (e.g. using a thermal initiator), the reaction will generally be carried out at a temperature above ambient temperature. The temperature will depend on the reactivity of the species from which free radica ls are generated.

If free radicals are generated photochemically the reaction may be carried out, advantageously, at ambient temperature. In certain embodiments, it may be desirable to cool the reaction mixture to slow the rate of reaction or conversely heat the reaction mixture to increase the rate of reaction.

A person skilled in the art will be able to select appropriate temperatures for carrying out the method having regard to the reactivity of the starting materials and other reactants present.

The temperature at which the reaction is carried out may be controlled by heating or cooling the reaction mixture by suitable method known in the art. Heat may be applied to the reaction mixture, for example, using a heat exchanger within the reaction vessel, a heating jacket surrounding the reaction vessel, or by immersing the reaction vessel in a heated liquid (e.g . an oil or sand bath). In certain exemplary embodiments, the reaction mixture is heated by microwave irradiation. The progress of the reaction may be monitored by any suitable means, for example, by thin layer chromatography (TLC) or hig h performance liquid chromatorgraphy (HPLC). The reaction may be allowed to proceed to substantial completion, as monitored by the consumption of at least one of the starting materia ls. In some embod iments, the reaction is a llowed to proceed for a period of time from 1 minute to 7 days, 5 minutes to 72 hours, 10 minutes to 48 hours, 10 minutes to 24 hours. In other embodiments, the reaction is a llowed to proceed for a period of time less than 72 h, less than 48 h, less than 24 h, less than 12 h, less than 6 h, less than 4 h, less tha n 2 h, or less than 1 h.

In some embod iments, the reaction is carried out until at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, at least about 99% of the amino acid-comprising conjugation partner has been consumed. The consumption of starting materials may be monitored by any suitable method, for example, HPLC.

The reaction mixture may be mixed by a ny suitable method known in the art, for example, using a magnetic or mechanical stirrer. The method used may depend on the scale on which the reaction is carried out.

The reaction is generally carried out in a liquid reaction medium. The liquid reaction medium may comprise a solvent. Examples of suitable solvents include N- methylpyrrolidone (NMP), d imethylformamide, dichloromethane, 1,2-dichloroetha ne, chloroform, carbon tetrachloride, water, methanol, ethanol, dimethylsulfoxide, trifluoroacetic acid, acetic acid, acetonitrile, and mixtures thereof.

The solvent may be selected based on the solubility of the starting materia ls and other reactants present, for example the free rad ical initiator. In some embodiments, the lipid- containing conjugation partners a re hydrophobic. The hydrophobicity or hydrophilicity of an amino acid-comprising conj ugation partner may vary depending on, for example, the amino acid seq uence of the peptide of a peptide-containing conjugation partner. The presence of a solubilising group in the peptide-containing conj ugation partner may increase solubility in polar solvents, such as water. A person skilled in the art will be able to select an appropriate solvent without undue experimentation. The reaction may be carried out under substantially oxygen-free cond itions. Oxygen may quench free radica ls formed in the reaction. The reaction mixture may be degassed with an inert gas (e.g . nitrogen or argon) that is substantia lly oxygen-free to remove any dissolved oxygen before free radicals are generated . Alternatively, ind ivid ual components of the reaction mixture may be degassed with inert gas that is substantially oxygen-free prior to being combined in the reaction vessel. The reaction may be carried out under an atmosphere of inert gas that is substantia lly oxygen-free.

The method of the present invention may be carried out at ambient pressure.

An additive that inhibits the formation of undesireable by-products and/or that improves the yield of or conversion to the desired product may be included in the reaction mixture in the thiolene method of the present invention. The one or more additive may be a n extraneous thiol, a n acid, an organosila ne, or a combination of any two or more thereof.

The inventors have found that in some embodiments the inclusion of an extraneous or exogenous thiol as an additive in the reaction mixture reduces the formation of undesirable by products. The extraneous thiol may, in some embodiments, increase the efficiency or conversion of the desired thiolene reaction. Examples of suitable extraneous thiols include but are not limited to reduced glutathione, DODT, DTT, protein, sterically hindered thiols, and the like.

In some embod iments, the extraneous thiol is DTT. In other embodiments, the extra neous thiol is a sterically hindered thiol. Non-limiting examples of a suitable sterically hindered extra neous thiol include terf-butyl mercaptan and 1-methylpropyl mercaptan.

Without wishing to be bound by theory, the inventors believe that in certain

embodiments a n extraneous thiol such as terf-butylmercaptan can provide a proton to quench the radical intermediate formed on propogation of the radical of formula (X) with the alkene of formula (IIB) to provide the desired compound of formula (IB) and the resulting thiyl rad ical can propagate the reaction by generating another mole of thiyl radical from the amino acid comprising conjugation partner of formula (III).

It will be apparent that extra neous thiols may in certain embodiments also be capable of prematurely quenching the reaction by provid ing a proton radical of formula (X). In such embodiments, the extraneous thiol and the amount in which it is used may be selected such that the yield of or conversion to (as determined by HPLC) the compound of formula (IB) is optimised .

In various embodiments, the extraneous thiol is present in the reaction in a

stoichiometric ratio relative to the amino acid comprising conj ugation partner of from about 200 : 1 to about 0.05: 1, 100: 1 to 0.05: 1, 80 : 1 to 0.05 : 1, 60 : 1 to 0.05: 1, 40 : 1 to 0.05: 1, 20 : 1 to about 0.05: 1, 10 : 1 to about 0.5: 1, 5 : 1 to about 1 : 1, or 3 : 1 to about 1 : 1. In certa in embodiments, a sterically hindered thiol such as t-BuSH is present in the reaction in a stoichiometric ratio relative to the amino acid comprising conj ugation partner of from about 100 : 1 to 0.05 : 1, for example about 80 : 1, about 40: 1, or about 3: 1.

The inclusion of an acid in some embodiments may also reduce the formation of undesireable by-products. The acid may be a strong inorga nic acid, for example HCI, or orga nic acid, for example TFA. In certain embodiments, the additive is TFA. Without wishing to be bound by theory, the inventors believe that decreasing the pH of the reaction mixture may result in the protonation of electron rich side cha ins of residues such as lysine, etc. which could otherwise participate in single electron transfers a nd form radica l species in the reaction . In various embodiments, the reaction mixture comprises from about 0.01 to 25, 0.01 to 15, 0.01 to 10, or 1 to 10% v/v acid add itive. In certain embodiments, the reaction mixture comprises from 1-10% v/v TFA, for example 5% v/v TFA.

The inventors have found that in some embodiments including both terf-butyl mercaptan and TFA as additives in the reaction mixture can red uce the the formation of undesirable by products and increase the conversion of starting materia l to the desired product. Accordingly, in certain exemplary embod iments, the reaction mixture comprises a combination of an acid and an exogenous thiol, such as a combination of a strong organic acid and a sterica lly hindered thiol, for example a combination of TFA a nd terf-butyl mercaptan. An orga nosilane may also be included as an additive in the thiolene reaction.

Orga nosilanes are rad ical-based red ucing agents, the activity of which can be modulated by varying the substituents on the silicon atom. In various embodiments, the orga nosilane is a compound of the formula (R ^q )3SiH, wherein Rq at each instance is independently hyd rogen or an orga nic group, for example alkyl or aryl, provided that at least one Rq is not hydrogen. Examples of orga nosilanes include but a re not limited to triethylsilane (TES), triphenylsilane, diphenylsilane, triisopropylsilane (TIPS), and the like. In various embodiments, the organosilane is a trialkylsilane, for example TIPS or TES.

Without wishing to be bound by theory, the inventors believe that, as with an extra neous thiol, in certain embodiments a n organosilane such as TIPS can act as a hydrogen donor to provide the desired compound of formula (IB) and promote propagation of the reaction.

In various embodiments, the organosilane is present in the reaction in a stoichiometric ratio relative to the amino acid comprising conjugation partner of from about 200 : 1 to about 0.05 : 1, 100: 1 to 0.05: 1, 80 : 1 to 0.05: 1, 60: 1 to 0.05: 1, 40: 1 to 0.05: 1, 20 : 1 to 0.05: 1, 10 : 1 to 0.5 : 1, 5: 1 to about 1 : 1, or 3 : 1 to about 1 : 1. In certain embodiments, a trialkylsilane such as TIPS is present in the reaction in a stoichiometric ratio relative to the amino acid comprising conjugation partner of from about 100: 1 to 0.05: 1, for example about 80 : 1 or about 40 : 1. The organosilane may be used as an additive in combination with a n extraneous thiol. Alternatively, the orga nosilane may be used instead of an extra neous thiol. An acid, such as TFA, may a lso be present. The inventors have found that in certain

embodiments using TIPS in the reaction together with TFA but without any extraneous thiol can provide hig her conversion to the desired compound of formula (IB) than when a combination of TIPS, t-BuSH, and TFA are used . The additive is genera lly used in an amount sufficient to minimise the formation of undesirable by products without adversely affecting the reaction or any, optiona l, subsequent steps in the method.

The products formed in the reaction and conversion to the desired product may be determined by, for exa mple, HPLC. The concentration of the lipid-conta ining conj ugation partners and amino acid-compirsing conjugation partner, for example a peptide-containing conj ugation partner, respectively, in the reaction mixture may also affect the reaction. Those skilled in the art will be able to vary the concentration of the lipid-containing conj ugation partners a nd peptide- containing conjugation partner in the reaction mixture to e.g. optimise yield and purity without undue experimentation.

In some embod iments, the starting material comprising the thiol is present in a concentration from about 0.05 mM to about 1 M, from about 0.5 mM to about 1 M, from about 1 mM to about 1 M . In some embodiments, the concentration is at least about 0.05 mM, 0.5 mM, or 1 mM . In some embod iments, the concentration of the starting materials comprising the a lkenes is at least about 0.05 mM, 0.5 mM, or 1 mM .

In some embod iments, the amino acid conj ugate or peptide conjugate is separated from the reaction medium after the reaction a nd optiona lly purified . The conjugate may be separated from the reaction medium using any suitable method known in the art, for example, by precipitation.

In some embod iments, the amino acid or peptide conj ugate is purified after separating it from the reaction med ium. For example, the conjugate may be purified by HPLC using one or more suitable solvents.

The present invention also provides a method of making a peptide conjugate, the method comprising

providing an amino acid- or peptide conj ugate of the formula (I) but exclud ing provisos (1) and (2) of the first aspect or a sa lt or solvate thereof, and coupling the amino acid of the amino acid conjugate or an amino acid of the peptide conj ugate to an amino acid or a n amino acid of a peptide to provide a peptide conjugate of the formula (I) of the invention or a salt or solvate thereof.

The amino acid- or peptide conjugate of the formula (I) but exclud ing provisos (1) and (2) of the first aspect or a salt or solvate thereof may be provided by the methods described herein.

The peptide conjugate prod uced by and/or the peptide-containing conjugation partner and/or the peptides coupled in the methods of the present invention may comprise a synthetic peptide. Synthetic peptides may be prepared using solid phase peptide synthesis (SPPS).

The basic principle for solid phase peptide synthesis (SPPS) is a stepwise addition of amino acids to a growing polypeptide chain anchored via a linker molecule to a solid phase support, typically a resin particle, which allows for cleavage a nd purification once the polypeptide chain is complete. Briefly, a solid phase resin support and a starting amino acid a re attached to one another via a linker molecule. Such resin-linker-acid matrices are commercia lly ava ilable.

The amino acid to be coupled to the resin is protected at its Na-terminus by a chemical protecting group.

The amino acid may a lso have a side-cha in protecting group. Such protecting groups prevent undesired or deleterious reactions from taking place during the process of forming the new peptide bond between the carboxyl g roup of the amino acid to be coupled a nd the unprotected Na-amino g roup of the peptide cha in attached to the resin.

The amino acid to be coupled is reacted with the unprotected Να-amino group of the N- termina l amino acid of the peptide cha in, increasing the chain length of the peptide chain by one amino acid. The carboxyl group of the amino acid to be coupled may be activated with a suitable chemical activating agent to promote reaction with the Να-amino group of the peptide chain . The Na-protecting group of N-terminal amino acid of the peptide chain is then removed in preparation for coupling with the next amino acid residue. This techniq ue consists of many repetitive steps making a utomation attractive whenever possible. Those skilled in the art will appreciate that peptides may be coupled to the Na- amino group of the solid phase bound amino acid or peptide instead of an individua l amino acid, for example where a convergent peptide synthesis is desired.

When the desired sequence of amino acids is achieved, the peptide is cleaved from the solid phase support at the linker molecule. SPPS may be carried out using a continuous flow method or a batch flow method .

Continuous flow permits real-time monitoring of reaction progress via a

spectrophotometer, but has two distinct disadvantages - the reagents in contact with the peptide on the resin are diluted, a nd scale is more limited d ue to physical size constraints of the solid phase resin. Batch flow occurs in a filter reaction vessel a nd is useful beca use reactants are accessible and ca n be added manually or automatically.

Two types of protecting groups are commonly used for protecting the N-alpha-amino terminus: "Boc" (terf-butyloxycarbonyl) and "Fmoc" (9-fluorenylmethyloxycarbonyl). Reagents for the Boc method a re relatively inexpensive, but they are highly corrosive a nd require expensive equipment and more rigorous precautions to be taken. The Fmoc method, which uses less corrosive, althoug h more expensive, reagents is typically preferred .

For SPPS, a wide variety of solid support phases are ava ilable. The solid phase support used for synthesis can be a synthetic resin, a synthetic polymer film or a silicon or silicate surface (e.g . controlled pore glass) suitable for synthesis purposes. Generally, a resin is used, commonly polystyrene suspensions, or polystyrene-polyethyleneglycol, or polymer supports for example polyamide. Exa mples of resins functionalized with linkers suitable for Boc-chemistry include PAM resin, oxime resin SS, phenol resin, brominated Wang resin and brominated PPOA resin . Examples of resins suitable for Fmoc chemistry include amino-methyl polystyrene resins, AMPB-BHA resin, Sieber amide resin, Rink acid resin, Tentagel S AC resin, 2-chlorotrityl chloride resin, 2-chlorotrityl a lcohol resin, TentaGel S Trt-OH resin, Knorr-2-chlorotrityl resin, hydrazine-2-chlorotrityl resin, ANP resin, Fmoc photolable resin, HM BA-M BHA resin, TentaGel S HM B resin, Aromatic Safety Catch resinBAI resin and Fmoc-hydroxylamine 2 chlorotrityl resin. Other resins include PL Cl-Trt resin, PL-Oxime resin and PL-HMBA Resin. Genera lly resins are intercha ngeable.

For each resin appropriate coupling cond itions are known in the literature for the attachment of the starting monomer or sub-unit.

Preparation of the solid phase support includes solvating the support in an appropriate solvent (e.g. dimethylformamide). The solid phase typica lly increases in volume during solvation, which in turn increases the surface area ava ilable to carry out peptide synthesis.

A linker molecule is then attached to the support for connecting the peptide chain to the solid phase support. Linker molecules are generally designed such that eventual cleavage provides either a free acid or amide at the C-terminus. Linkers are generally not resin- specific. Examples of linkers include peptide acids for example 4- hydroxymethylphenoxyacetyl-4'-methylbenzyhydrylamine (HMP), or peptide amides for example benzhydrylamine derivatives, or the hydroxymethylphenoxypropionyl (HMPP) linker.

The first amino acid of the peptide sequence may be attached to the linker after the linker is attached to the solid phase support or attached to the solid phase support using a linker that includes the first amino acid of the peptide seq uence. Linkers that include amino acids are commercially ava ilable.

The next step is to deprotect the Na-amino group of the first amino acid . For Fmoc SPPS, deprotection of the Να-amino group may be ca rried out with a mild base treatment (piperazine or piperidine, for example). Side-chain protecting g roups may be removed by moderate acidolysis (trifluoroacetic acid (TFA), for example). For Boc SPPS, deprotection of the Να-amino group may be ca rried out using for example TFA.

Following deprotection, the amino acid chain extension, or coupling, proceeds by the formation of peptide bonds. This process requires activation of the C-a-carboxyl group of the amino acid to be coupled . This may be accomplished using, for example, in situ reagents, preformed symmetrical anhydrides, active esters, acid halides, or urethane- protected N-carboxyanhydrides. The in situ method allows concurrent activation and coupling . Coupling reagents include carbodiimide derivatives, for example Ν,Ν'- dicyclohexylcarbodiimide or Ν,Ν-d iisopropylcarbodiimide. Coupling reagents also include uranium or phosphonium salt derivatives of benzotriazol. Examples of such uranium and phosphonium salts include H BTU (0-lH-benzotriazole-l-yl)-N,N,N',N '- tetramethyluronium hexafluorophosphate), BOP (benzotriazole-l-yl-oxy-tris- (dimethylamino)-phosphonium hexafluorophosphate), PyBOP (Benzotriazole-l-yl-oxy- tripyrrolidinophosphonium hexafluorophosphate), PyAOP, HCTU (0-(lH-6-chloro- benzotriazole-l-yl)-l, l,3,3-tetramethyluronium hexafluorophosphate), TCTU (O-lH-6- chlorobenzotriazole-l-yl)-l,l,3,3-tetramethyluronium tetrafluoroborate), HATU (0-(7- azabenzotriazol-l-yl)-l, l,3,3-tetramethyluronium hexafluorophosphate), TATU (0-(7- azabenzotriazol-l-yl)-l, l,3,3-tetramethyluronium tetrafluoroborate), TOTU (O- [cyano(ethoxycarbonyl)methyleneamino]-N,N,N ', N"-tetramethyluronium

tetrafluoroborate), and HAPyU (0-(benzotriazol-l-yl)oxybis-(pyrrolidino)-uronium hexafluorophosphate. In some embodiments, the coupling reagent is HBTU, HATU, BOP, or PyBOP.

After the desired a mino acid sequence has been synthesized, the peptide is cleaved from the resin. The conditions used in this process depend on the sensitivity of the amino acid composition of the peptide a nd the side-cha in protecting groups. Generally, cleavage is carried out in an environment containing a plurality of scavenging agents to quench the reactive carbonium ions that orig inate from the protective groups and linkers. Common cleaving agents include, for example, TFA and hydrogen fluoride (HF). In some embodiments, where the peptide is bound to the solid phase support via a linker, the peptide chain is cleaved from the solid phase support by cleaving the peptide from the linker. The cond itions used for cleaving the peptide from the resin may concomitantly remove one or more side-cha in protecting groups.

The use of protective groups in SPPS is well established . Examples of common protective groups include but are not limited to acetamidomethyl (Acm), acetyl (Ac), adama ntyloxy (AdaO), benzoyl (Bz), benzyl (Bzl), 2-bromobenzyl, benzyloxy (BzIO), benzyloxycarbonyl (Z), benzyloxymethyl (Bom), 2-bromobenzyloxycarbonyl (2-Br-Z), tert-butoxy (tBuO), tert-butoxycarbonyl (Boc), terf-butoxymethyl (Bum), tert-butyl (tBu), terf-buthylthio (tButhio), 2-chlorobenzyloxycarbonyl (2-CI-Z), cyclohexyloxy (cHxO), 2,6-dichlorobenzyl (2,6-DiCI-Bzl), 4,4'-d imethoxybenzhydryl (Mbh), l-(4,4-d imethyl-2,6-dioxo- cyclohexylidene)3-methyl-butyl (ivDde), 4-{N -[l-(4,4-d imethyl-2,6-dioxo- cyclohexylidene)3-methylbutyl]-amino) benzyloxy (ODmab), 2,4-dinitrophenyl (Dnp), fluorenylmethoxycarbonyl (Fmoc), formyl (For), mesitylene-2-sulfonyl (Mts), 4- methoxybenzyl (MeOBzl), 4-methoxy-2,3,6-trimethyl-benzenesulfonyl (Mtr), 4- methoxytrityl (Mmt), 4-methylbenzyl (MeBzl), 4-methyltrityl (Mtt), 3-nitro-2- pyridinesulfenyl (Npys), 2,2,4,6,7-pentamethyldihydrobenzofurane-5-sulfonyl (Pbf), 2,2,5,7,8-pentamethyl-chromane-6-sulfonyl (Pmc), tosyl (Tos), trifluoroacetyl (Tfa), trimethylacetamidomethyl (Tacm), trityl (Trt) and xa nthyl (Xa n).

Where one or more of the side chains of the amino acids of the peptide contains functiona l groups, such as for example additional carboxylic, amino, hydroxy or thiol groups, additional protective groups may be necessary. For example, if the Fmoc strategy is used, Mtr, Pmc, Pbf may be used for the protection of Arg ; Trt, Tmob may be used for the protection of Asn and Gin ; Boc may be used for the protection of Trp and Lys; tBu may be used for the protection of Asp, Glu, Ser, Thr and Tyr; and Acm, tBu, tButhio, Trt and Mmt may be used for the protection of Cys. A person skilled in the art will appreciate that there are numerous other suitable combinations. The methods for SPPS outlined above are well known in the art. See, for example, Atherton a nd Sheppard, "Solid Phase Peptide Synthesis: A Practica l Approach," New York: IRL Press, 1989; Stewart and Young : "Solid-Phase Peptide Synthesis 2nd Ed .," Rockford, Illinois: Pierce Chemical Co., 1984; Jones, "The Chemica l Synthesis of Peptides," Oxford : Cla rendon Press, 1994; Merrifield, J. Am. Soc. 85: 2146-2149 (1963); Marglin, A. and Merrifield, R. B. Annu. Rev. Biochem. 39: 841-66 ( 1970); a nd Merrifield R. B. JAMA. 210(7) : 1247-54 (1969); a nd "Solid Phase Peptide Synthesis - A Practical Approach" (W.C. Cha n and P. D. White, eds. Oxford University Press, 2000). Equipment for automated synthesis of peptides or polypeptides is readily commercially ava ilable from suppliers such as Perkin Elmer/Applied Biosystems (Foster City, CA) and may be operated according to the ma nufacturer's instructions. Following cleavage from the resin, the peptide may be separated from the reaction medium, e.g. by centrifugation or filtration. The peptide may then be subsequently purified, e.g . by HPLC using one or more suitable solvents.

Advantageously, the inventors have found that in some embod iments the peptide- containing conjugation partner may be used in the methods of the present invention without purification following cleavage of the peptide from the resin.

The inventors have also advantageously found that in some embodiments the thiolene method of the present invention can be carried out using a peptide-containing conjugation partner, wherein the peptide does not conta in a n Na-amino group protecting group or any side chain protecting groups. The reaction is generally selective for reaction of a thiol and a non-aromatic carbon-carbon double bond .

It may be necessary to protect thiol groups present in the peptide-containing conj ugation partner (e.g . in cysteine residues of the peptide) with a protective group to prevent undesirable competing reactions in the methods of the present invention. The thiol groups may be protected with a protective group that is not removable under the conditions used to remove one or more other protecting groups present in the peptide or to cleave the peptide from the resin.

Typica lly, the peptide will be synthesised using amino acids bearing the appropriate protecting groups. A person skilled in the art will be able to select appropriate protecting groups without und ue experimentation. The amino acid-comprising conjugation pa rtner and/or lipid-containing conjugation partners may comprise one or more unsaturated carbon-carbon bonds in addition to the carbon-carbon double bonds of the lipid containing conjugation partners to be reacted . Those skilled in the art will appreciate that the selectivity of the thiol for the carbon- carbon double bond to be reacted in such embodiments may depend on, for example, the steric and/or electronic environment of the carbon-carbon double bond relative to the one or more additional unsaturated carbon-carbon bonds. In certa in embodiments, the carbon-carbon double bonds to be reacted a re activated relative to any other unsaturated carbon-carbon bonds in the amino acid-comprising conjugation partner and lipid- containing conjugation partner. In certain embodiments, the carbon-carbon double bonds to be reacted are activated relative to any other unsaturated carbon-carbon bonds in the peptide-conta ining conjugation partner a nd lipid-containing conjugation partner.

In some embod iments, the Na-amino group of the amino acid of the amino acid- comprising conjugation partner comprising the thiol is acylated, for example acetylated . In some embod iments, the methods of the present invention may comprise acylating, for example acetylating, the Να-amino group of the amino acid of the amino acid-comprising conjugation partner comprising the carbon-carbon double bond or thiol to be reacted .

Where a peptide-containing conj ugation partner has been synthesised by SPPS, acylation may be carried out prior to or after cleavage from the resin. In some embodiments, the amino acid residue of the peptide-conta ining conjugation pa rtner bearing the thiol to be reacted is an N-termina l amino acid residue, for example cysteine, and the method comprises acylating the N-terminal amino group prior to cleaving the peptide.

In some embod iments, the method further comprises acylating, for example acetylating, the Να-amino group of the amino acid of the amino acid conj ugate or the amino acid residue of the peptide conj ugate to which the lipid moieties are conjugated .

Acylation of the Να-amino group of a n amino acid may be carried out by reacting an amino acid or peptide with a n acylating agent in the presence of base in a suitable solvent, for example DMF. Non-limiting examples of acylating agents include acid halides, for example acid chlorides such as acetyl chloride, and acid anhydrides, for example acetic anhydride. Such agents maybe commercia lly available or may be prepared by methods well known in the a rt. Non-limiting examples of suitable bases include triethylamine, diisopropylethylamine, 4-methylmorpholine, and the like.

In other embodiments, the synthesising the peptide of the peptide-containing conjugation partner comprises coupling an amino acid or a peptide comprising an amino acid that is acylated, for example acetylated, at the Να-amino g roup and comprises the thiol to be reacted to one or more amino acids a nd/or one or more peptides.

In some embod iments, the method comprises coupling the amino acid of the amino acid conjugate to an amino acid or a peptide to provide a peptide conjugate. In some embodiments, the method comprises coupling the amino acid of the amino acid conjugate to an amino acid or peptide bound to a solid phase resin support by SPPS. In some embodiments, the method comprises coupling the amino acid of the amino acid conjugate to a peptide bound to a solid phase resin support by SPPS. The method may comprise synthesising the peptide bound to the solid phase resin support by SPPS. In some embod iments, the method further comprises coupling the amino acid of the amino acid conjugate or an amino acid of the peptide conj ugate to an a mino acid or a peptide so as to provide a peptide conj ugate comprising a peptide epitope. In some embodiments, the peptide to be coupled comprises a peptide epitope. In other embodiments, a peptide epitope is formed on coupling . The coupling may be carried out by SPPS as described herein.

In some embod iments, the method comprises coupling the amino acid of the amino acid conjugate to a peptide bound to a solid phase resin support by SPPS so as to provide a peptide conj ugate comprising a peptide epitope. In one embodiment, the peptide of the peptide conjugate to be coupled is bound to a solid phase resin support, and the method comprises coupling an amino acid of the peptide conj ugate to be coupled to a n amino acid or a peptide so as to provide a peptide conjugate comprising a peptide epitope.

In an alternate embod iment, the method comprises coupling an amino acid of the peptide conj ugate to an amino acid or peptide bound to a solid phase resin support by SPPS so as to provide peptide conjugate comprising a peptide epitope.

In some embod iments, the method further comprises coupling an epitope, for example a peptide epitope, to the amino acid conjugate or peptide conjugate. Where the method comprises coupling a peptide epitope, the coupling may be carried out by SPPS as described herein.

In certa in embodiments, the epitope, for example a peptide epitope, is coupled or bound via a linker group. In certain embodiments, the linker g roup is an amino sequence, for example a sequence of two or more, three or more, or four or more contiguous amino acids. In certa in embodiments, the linker comprises from about 2 to 20, 2 to 18, 2 to 16, 2 to 14, 2 to 12, 2 to 10, 4 to 20, 4 to 18, 4 to 16, 4 to 14, 4 to 12, or 4 to 10 amino acids.

It will be appreciated by those skilled in the art that coupling an amino acid or a peptide to a nother amino acid or peptide as described herein may comprise forming a peptide bond between the Na-terminus of the amino acid or an amino acid of the peptide of one coupling partner and the C-terminus of the amino acid or an amino acid of the peptide of the other coupling partner.

In some embod iments, the method of the present invention comprises synthesising the amino acid seq uence of the peptide of the peptide-conta ining conj ugation partner by SPPS; and reacting the peptide-containing conj ugation partner. In some embod iments, the method of the present invention comprises synthesising the amino acid seq uence of the peptide of the peptide-conta ining conj ugation partner by SPPS; and reacting the lipid-containing conj ugation pa rtners with the peptide-containing conjugation partner. In some embod iments, synthesising the amino acid sequence of the peptide of the peptide-containing conjugation partner by SPPS comprises coupling an amino acid or peptide to a n amino acid or peptide bound to a solid phase resin support to provide the amino acid seq uence of the peptide or a portion thereof. In certain embodiments, the amino acid seq uence of the entire peptide of the peptide-containing conjugation partner is synthesised by SPPS.

The peptide-conta ining conjugation pa rtner may be reacted, for example with the lipid- containing conjugation partners in the thiolene method, while bound to a solid phase resin support. Alternatively, the peptide may be cleaved from the solid phase resin support, and optionally purified, prior to reaction, for example with the lipid-containing conjugation partners.

The peptide conjugate and/or amino acid-comprising conjugation partner, for example a peptide-containing conjugation partner, may comprise one or more solubilising groups. The one or more solubilising groups increase the solubility of, for example, the peptide- containing conjugation partner in polar solvents, such as water. In exemplary embodiments, the solubilising group does not adversely affect the biological activity of the peptide conj ugate.

The presence of a solubilising group may be advantageous for formulation and/or administration of the peptide conjugate as a pharmaceutical composition.

In some embod iments, the solubilising group is bound to the peptide of the peptide conjugate and/or peptide-containing conjugation partner. In some embodiments, the solubilising g roup is bound to the peptide of the peptide-containing conjugation partner. In some embod iments, the peptide of the peptide conjugate and/or the peptide of the peptide-containing partner comprises a solubilising g roup. In some embodiments, the peptide of the peptide-conta ining partner comprises a solubilising group. In some embod iments, the solubilising group is bound to the side cha in of an amino acid in the peptide chain. In some embodiments, the solubilising g roup is bound to the C- or N-terminus of the peptide chain. In some embodiments, the solubilising group is bound between two amino acid residues in the peptide chain. In some embodiments, the solubilising g roup is bound to the Na-amino group of one a mino acid residue in the peptide chain and the carboxyl group of another amino acid residue in the peptide chain. Examples of suitable solubilising groups include, but are not limited to, hyd rophilic amino acid sequences or polyethylene glycols (PEGs).

In one embodiment, the solubilising group is a hydrophilic amino acid sequence comprising two or more hydrophilic amino acid residues in the peptide chain. In some embodiments, the solubilising group is an amino acid sequence comprising a sequence of two or more consecutive hydrophilic amino acid residues in the peptide chain. Such solubilising g roups may be formed by adding each amino acid of the solubilising group to the peptide chain by SPPS.

In another embodiment, the solubilising group is a polyethylene glycol . In some embodiments, the polyethylene glycol is bound to the Na-amino group of one a mino acid residue in the peptide cha in and the carboxyl g roup of a nother amino acid residue in the peptide chain.

In some embod iments, the polyethylene g lycol comprises from about 1 to about 100, about 1 to about 50, about 1 to about 25, about 1 to about 20, about 1 to about 15, about 1 to about 15, about 1 to about 10, about 2 to about 10, or about 2 to about 4 ethylene g lycol monomer units. Methods for coupling polyethylene glycols to peptides are known.

In some embod iments, the peptide conj ugate and/or peptide-containing conjugation partner comprises an a ntigen, for example, an antigenic peptide. In one embod iment, the peptide of the peptide conjugate or peptide-containing conj ugation partner is or comprises a n antigen ; or an antigen is bound to peptide, optiona lly via a linker. In some embodiments, the peptide-containing conj ugation partner comprises a n antigen, for example, an a ntigenic peptide. In one embodiment, the peptide of the peptide- containing conjugation partner is or comprises an a ntigen ; or an antigen is bound to peptide, optionally via a linker.

In one embodiment, the antigen comprises a peptide comprising an epitope. In one embodiment, the peptide comprising an epitope is a glycopeptide comprising an epitope. In one embodiment, the antigen comprises a glycopeptide comprising an epitope.

In some embod iments, the peptide conj ugate and/or peptide-containing conjugation partner comprises an epitope. In some embodiments, the peptide of the peptide conjugate and/or peptide-containing conjugation partner comprises an epitope. In some embodiments, the peptide-containing conj ugation partner comprises a n epitope. In some embodiments, the peptide of the peptide-containing conjugation partner comprises an epitope. In some embod iments, the peptide conj ugate and/or peptide-containing conjugation partner comprises two or more epitopes, for example, the peptide of the peptide conjugate and/or peptide-containing conjugation partner comprises two or more epitopes. In some embod iments, the peptide conj ugate and/or peptide-containing conjugation partner is or comprises a glycopeptide comprising a n epitope. In some embodiments, the peptide of the peptide conjugate and/or peptide-conta ining conjugation partner is a glycopeptide. In some embodiments, the peptide conj ugate and/or peptide-conta ining conjugation partner comprises a glycopeptide comprising an epitope bound to the peptide of the peptide conj ugate and/or peptide-conta ining conj ugation pa rtner. In some embodiments, the peptide-containing conj ugation partner is or comprises a glycopeptide comprising an epitope. In some embodiments, the peptide of the peptide-containing conjugation partner is a glycopeptide. In some embodiments, the peptide-conta ining conjugation partner comprises a glycopeptide comprising an epitope bound to the peptide of the peptide-containing conjugation partner.

In some embod iments, the peptide conj ugate and/or peptide-containing conjugation partner comprises a proteolytic cleavage site. In some embodiments, the peptide of the peptide conj ugate and/or peptide-conta ining conjugation partner comprises a proteolytic cleavage site. In some embod iments, the peptide-containing conj ugation partner comprises a proteolytic cleavage site. In some embod iments, the peptide of the peptide- containing conjugation partner comprises a proteolytic cleavage site.

In some embod iments, the peptide of the peptide conjugate and/or peptide-conta ining conjugation partner comprises one or more linker groups. In some embodiments, the peptide of the peptide-conta ining conj ugation partner comprises one or more linker groups.

In some embod iments, the peptide conj ugate and/or peptide-containing conjugation partner comprises a linker group. In some embodiments, the peptide-conta ining conjugation partner comprises a linker group.

In some embod iments, the peptide conj ugate and/or peptide-containing conjugata ion partner comprises an epitope bound to the peptide of the peptide conj ugate a nd/or peptide-containing conjugation partner via a linker group. In some embodiments, the peptide-containing conjugation partner comprises an epitope bound to the peptide of the peptide-containing conjugation partner via a linker group.

Examples of linker groups include but a re not limited to amino acid sequences (for example, a peptide), polyethylene glycol, alkyl amino acids, a nd the like. In some embodiments, the linker is or comprises a proteolytic cleavage site. In some embodiments, the linker is or comprises a solubilising group.

In some embod iments, the linker is bound between two amino acid residues in the peptide chain. In some embod iments, the linker group is bound to the Na-amino group of one amino acid residue in the peptide conjugate a nd/or peptide-conta ining conjugation partner and the carboxyl group of another a mino acid residue in the peptide-conta ining conj ugation partner. In some embodiments, the linker group is bound to the Na-amino group of one amino acid residue in the peptide-containing conjugation pa rtner and the carboxyl group of a nother amino acid residue in the peptide-conta ining conjugation partner.

In certa in embodiments, the linker group is cleavable in vivo from the amino acids to which it is bound . In certain embodiments, the linker g roup is cleavable by hydrolysis in vivo. In certain embodiments, the linker group is cleavable by enzymatic hydrolysis in vivo. Linker groups may be introduced by any suitable method known in the a rt. The method may further comprise coupling an epitope to the amino acid of the amino acid conjugate or the peptide of the peptide conj ugate. The epitope may be bound via a linker g roup, as described above. In some embodiments, the epitope is a peptide epitope. In some embodiments, the method comprises coupling a glycopeptide comprising an epitope. It will be appreciated that in certain desirable embodiments, the peptide conjugates of the invention maintain appropriate uptake, processing, and presentation by antigen presenting cells. Desirably, the lipid-conta ining conjugate does not interfere with presentation of any antigenic peptide present in the conjugate by a ntigen presenting cells. Confirmation of the identity of the peptides synthesized may be conveniently achieved by, for example, amino acid ana lysis, mass spectrometry, Edman degradation, and the like.

The method of the present invention may further comprise separating the amino acid conjugate from the liquid reaction medium. Alternatively, the method of the present invention may further comprise separating the peptide conj ugate from the liq uid reaction medium . Any suitable separation methods known in the art may be used, for example, precipitation and filtration. The conjugate may be subsequently purified, for example, by HPLC using one or more suitable solvents. The present invention also relates to peptide conjugates made by the methods of the present invention.

The peptide conjugates may be pure or purified, or substantially pure.

As used herein "purified" does not require absolute purity; rather, it is intended as a relative term where the material in question is more pure than in the environment it was in previously. In practice the material has typically, for exa mple, been subjected to fractionation to remove various other components, and the resultant materia l has substantially retained its desired biological activity or activities. The term "substantia lly purified" refers to materials that a re at least about 60% free, preferably at least about 75% free, and most preferably at least about 90% free, at least about 95% free, at least about 98% free, or more, from other components with which they may be associated during ma nufacture.

The term "a-amino acid" or "amino acid" refers to a molecule conta ining both an amino group and a carboxyl group bound to a carbon which is designated the a-carbon. Suitable amino acids include, without limitation, both the D- and L-isomers of the natura lly- occurring amino acids, as well as non-naturally occurring amino acids prepared by orga nic synthesis or other metabolic routes. Unless the context specifically indicates otherwise, the term amino acid, as used herein, is intended to include a mino acid ana logs. In certa in embodiments the peptide-containing conj ugation partner comprises only natural amino acids. The term "naturally occurring amino acid" refers to any one of the twenty amino acids commonly found in peptides synthesized in nature, and known by the one letter abbreviations A, R, N, C, D, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y and V.

The term "amino acid ana log" or "non-naturally occurring amino acid" refers to a molecule which is structura lly similar to a n amino acid and which can be substituted for an amino acid . Amino acid ana logs include, without limitation, compounds which are structurally identical to an amino acid, as defined herein, except for the inclusion of one or more additiona l methylene groups between the amino a nd carboxyl group (e.g ., a- amino β-carboxy acids), or for the substitution of the amino or carboxy group by a similarly reactive group (e.g., substitution of the primary amine with a secondary or tertiary amine, or substitution or the carboxy group with an ester or carboxamide).

Unless otherwise indicated, conventional techniques of molecular biology, microbiology, cell biology, biochemistry and immunology, which are within the skill of the art may be employed in practicing the methods described herein . Such techniques are explained fully in the literature, such as, Molecular Cloning : A Laboratory Manual, second edition (Sambrook et al. , 1989); Oligonucleotide Synthesis (M .J. Gait, ed ., 1984) ; Animal Cell Culture (R.I. Freshney, ed ., 1987); Ha ndbook of Experimenta l Immunology (D. M . Weir & C.C. Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (J.M . Miller & M . P. Calos, eds., 1987); Current Protocols in Molecular Biology (F. M . Ausubel et al., eds., 1987); PCR: The Polymerase Chain Reaction, (Mullis et al., eds., 1994) ; Current

Protocols in Immunology (J. E. Coliga n et al., eds., 1991); The Immunoassay Handbook (David Wild, ed., Stockton Press NY, 1994); Antibodies: A Laboratory Manual (Harlow et al., eds., 1987); and Methods of Immunological Ana lysis (R. Masseyeff, W. H. Albert, and N .A. Sta ines, eds., Weinheim : VCH Verlags gesellschaft mbH, 1993). The term "peptide" and the like is used herein to refer to any polymer of amino acid residues of a ny length. The polymer can be linear or non-linea r {e.g. , branched), it can comprise modified amino acids or amino acid analogs. The term also encompasses amino acid polymers that have been mod ified naturally or by intervention, for example, by disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other modification or manipulation, for example conjugation with labeling or bioactive components.

The inventors have found that peptide conjugates of the present invention have immunological activity.

Cell-mediated immunity is primarily mediated by T-lymphocytes. Pathogenic a ntigens are expressed on the surface of antigen presenting cells (such as macrophages, B- lymphocytes, and dend ritic cells), bound to either major histocompatibility MHC Class I or MHC Class II molecules. Presentation of pathogenic antigen coupled to MHC Class II activates a helper (CD4+) T-cell response. Upon binding of the T-cell to the antigen-MHC II complex, CD4+ T-cells, release cytokines and proliferate. Presentation of pathogenic antigens bound to MHC Class I molecules activates a cytotoxic (CD8+ ) T-cell response. Upon binding of the T-cell to the antigen-MHC I complex, CD8+ cells secrete perforin and other mediators, resulting in target cell death. Without wishing to be bound by any theory, the applicants believe that in certain embodiments an enha nced response by CD8+ cells is achieved in the presence of one or more epitopes recog nised by CD4+ cells.

Methods to assess and monitor the onset or progression of a cell-mediated response in a subject are well known in the art. Convenient exemplary methods include those in which the presence of or the level of one or more cytokines associated with a cell-mediated response, such as those identified herein, is assessed . Similarly, cell-based methods to assess or monitor the onset and progression of a cell-mediated response are amenable to use in the present invention, and may include cell proliferation or activation assays, including assays targeted at identifying activation or expansion of one or more populations of immune cells, such as T-lymphocytes.

In certain embodiments, methods of the invention elicit both a cell-mediated immune response and a humoral response. The humoral immune response is mediated by secreted antibodies produced by B cells. The secreted antibodies bind to antigens presented on the surface of invading pathogens, flagging them for destruction.

Aga in, methods to assess and monitor the onset or progression of a humoral response are well known in the art. These include antibody binding assays, ELISA, skin-prick tests and the like.

Without wishing to be bound by theory, the inventors believe that the peptide conj ugates in some embodiments stimulate Toll like receptors (TLRs).

Toll-like receptors (TLRs) are highly conserved pattern recognition receptors (PRRs) that recog nise pathogen-associated molecular patterns and transmit danger signa ls to the cell (Kawai, T., Akira, S., Immunity 2011, 34, 637-650). TLR2 is a cell-surface receptor expressed on a range of different cell types, including dendritic cells, macrophages and lymphocytes (Coffman, R. L, Sher, A., Seder, R. A., Immunity 2010, 33, 492-503).

TLR2 recognises a wide range of microbia l components includ ing lipopolysaccharides, peptidoglycans and lipoteichoic acid . It is unique amongst TLRs in that it forms heterod imers, with either TLR1 or TLR6; the ability to form complexes with other PRRs may explain the wide range of agonists for TLR2 (Feldmann, M ., Steinman, L., Nature 2005, 435, 612-619). Upon ligand bind ing and heterodimerisation, signa lling takes place via the MyD88 pathway, leading to N FKB activation a nd consequent production of inflammatory and effector cytokines. Di- and triacylated lipopeptides derived from bacterial cell-wa ll components have been extensively studied as TLR2 agonists (Eriksson, E. M . Y., Jackson, D. C, Curr. Prot. and Pept. Sci. 2007, 8, 412-417). Lipopeptides have been reported to promote dendritic cell maturation, causing the up-reg ulation of co-stimulatory molecules on the cell surface a nd enha nced a ntigen-presentation. Lipopeptides have a lso been reported to stimulate macrophages to release cytokines and promote the activation of lymphocytes including B cells and CD8+ T cells.

In some embod iments, the peptide conj ugate has TLR2 agonist activity. S-(2,3- bis(palmitoyloxy)-(2RS)-propyl)-/V-palmitoyl-(R-)-Cys-Lys-Ly s-Lys-Lys-OH (Pam3CSK4) is a potent TLR2 agonist and may be selected as benchmark aga inst which the TLR2 agonism of the peptide conjugate compounds of the invention may be compared . In some embodiments, the peptide conj ugate has a TLR2 agonist potency from 1,000, 100, or 10 fold less than of the potency of Pam3CSK4 to 1,000, 100, or 10 fold more tha n the potency of Pam3CSK4. In some embodiments, the peptide conj ugate has TLR2 agonist activity comparable to Pam3CSK4. In some embodiments, the peptide conjugate has TLR2 agonist activity at least about 50%, about 60%, about 70%, about 80%, about 90% that of Pam3CSK4. In some embodiments, the peptide conjugate has TLR2 agonist activity greater tha n that of Pam3CSK4. For example, in some embodiments, the peptide conjugate has TLR2 agonist activity greater than 100%, 150%, 200%, or 500%. In other embod iments, the peptide conj ugate has TLR2 agonist activity 10 fold or 100 fold greater tha n that of Pam3CSK4. In some embodiments, for example in embodiments where a mod ulated immune response is desirable, the peptide conj ugate has TLR2 agonist activity less tha n that of Pam3CSK4. For example, the peptide conjugate has TLR2 agonist activity less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than about 10%, less than about 1%, or less than 0.1% that of Pa m3CSK4.

In some embod iments, the TLR2 agonist activity is determined using a Hek-Blue™ cell assay (for example, by following a procedure analogous to that described in the

Examples herein). In some embod iments, the TLR2 is murine TLR2 or huma n TLR2. In certain exemplary embodiments, the TLR2 (mTLR2) is human TLR2 (hTLR2).

In some embod iments, the peptide conj ugate has an EC50 for TLR2 agonism (preferably hTLR2) of less than about 500 nM as determined using a HEK-Blue™ cell assay (for example, by following a proced ure analogous to that described in the Examples herein), for example less tha n about 400, 300, 250, 200, 175, 150, 125, 100, 75, 50, 25, 20, 15, 10, 5, 4, 3, 2.5, 2, 1.5, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2 or 0.1 nM, a nd useful ranges may be selected from any two of these values, for example from about 0.1 nM to 500 nM, 0.1 nM to 150 nM, or 0.1 nM to 10 nM . In some embodiments, the peptide conjugate has an EC50 for TLR2 agonism (preferably hTLR2) of at least about 0.01 nM as determined using a HEK-Blue™ cell assay (for example, by following a procedure ana logous to that described in the Examples herein), for exa mple at least 0.05, 0.1, 0.5, 1, 1.5, or 2 nM, a nd useful ranges may be selected from any two of these va lues, for example from about 0.01 nM to 2 nM, 0.01 nM to 1.5 nM, or 0.01 nM to 1 nM . In some embodiments, the peptide of the peptide conj ugate a nd/or peptide-containing conjugation partner comprises a serine amino acid residue adjacent to the amino acid through which the lipid moieties are conjugated to the peptide. In some embodiments, the serine is bound to the C-termini of the amino acid . The presence of the serine amino acid residue in this position may enha nce TLR2 binding .

As will be appreciated by those skilled in the art on read ing this d isclosure, the peptide conjugate may comprise an epitope, including, for example two or more epitopes. The epitope may be coupled or bound to the peptide via a linker group. In some

embodiments, the epitope is a peptide epitope. A person skilled in the art will appreciate that a wide range of peptide epitopes may be employed in the present invention.

Antigens

It will be appreciated that a great ma ny antigens, for example tumour antigens or antigens from various pathogenic organisms, have been characterised and are suitable for use in the present invention. All antigens, whether or not presently characterized, that are capable of eliciting an immune response a re contemplated .

Accordingly, depending on the choice of a ntigen the conjugates of the present invention find application in a wide range of immunothera pies, including but not limited to the treatment and prevention of infectious disease, the treatment and prevention of cancer, and the treatment of viral re-activation during or following immunosuppression, for example in patients who have had bone ma rrow transplants or haematopoietic stem cell transpla nts.

Also contemplated are antigens comprising one or more amino acid substitutions, such as one or more conservative amino acid substitutions.

A "conservative amino acid substitution" is one in which an a mino acid residue is replaced with another residue having a chemically similar or derivatised side chain. Families of amino acid residues having simila r side chains, for example, have been defined in the art. These families include, for example, amino acids with basic side chains (e.g ., lysine, arginine, histidine), acid ic side chains (e.g ., aspartic acid, glutamic acid), uncharged polar side cha ins (e.g ., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g ., ala nine, va line, leucine, isoleucine, proline, phenylalanine, methionine, tryptopha n), beta-branched side chains (e.g ., threonine, va line, isoleucine) and a romatic side cha ins (e.g ., tyrosine,

phenylalanine, tryptophan, histid ine). Amino acid analogs (e.g ., phosphorylated or glycosylated amino acids) are a lso contemplated in the present invention, as are peptides substituted with non-naturally occurring amino acids, including but not limited to N- alkylated amino acids (e.g . N-methyl amino acids), D-amino acids, β-amino acids, a nd v- amino acids. Fragments and variants of antigens are also specifically contemplated .

A "fragment" of a peptide, is a subsequence of the peptide that performs a function that is required for the enzymatic or binding activity and/or provides three dimensiona l structure of the peptide, such as the three d imensiona l structure of a polypeptide. The term "variant" as used herein refers to peptide sequences, includ ing for example peptide sequences different from the specifically identified sequences, wherein one or more amino acid residues is deleted, substituted, or added . Varia nts are naturally- occurring variants, or non-naturally occurring variants. Variants are from the same or from other species and may encompass homologues, paralogues and orthologues. In certain embodiments, variants of peptides including peptides possess biological activities that are the same or similar to those of the wild type peptides. The term "va riant" with reference to peptides encompasses all forms of peptides as defined herein.

Those of skill in the a rt will appreciate that the conj ugates of the present invention are in certain embodiments particula rly suited for stimulating T-cell responses, for example in the treatment of neoplastic diseases, including cancer. Conj ugates of the present invention comprising one or more tumour a ntigens are specifically contemplated. It will be appreciated that tumour antigens contemplated for use in the preparation of peptide conjugates of the invention will generally comprise one or more peptides. In certain embodiments of the invention, including for exa mple pharmaceutical compositions of the invention, one or more additional tumour a ntigens may be present, wherein the one or more tumour a ntigens does not comprise peptide. Tumour antigens are typically classified as either unique antigens, or shared antigens, with the latter group including differentiation antigens, cancer-specific a ntigens, and over-expressed antigens. Examples of each class of antigens are amenable to use in the present invention. Representative tumour a ntigens for use in the treatment, for example immunotherapeutic treatment, or vaccination aga inst neoplastic diseases including cancer, are d iscussed below.

Compounds, vaccines and compositions comprising one or more a ntigens prepared using those methods of immunisation a re specifically contemplated .

In certa in embodiments, the tumour antigen is a peptide-containing tumour antigen, such as a polypeptide tumour a ntigen or glycoprotein tumour antigens. In certa in embodiments, the tumour antigen is a saccharide-conta ining tumour a ntigen, such as a glycolipid tumour antigen or a ganglioside tumour antigen . In certa in embodiments, the tumour a ntigen is a polynucleotide-containing tumour antigen that expresses a polypeptide-containing tumour antigen, for instance, an RNA vector construct or a DNA vector construct, such as plasmid DNA. Tumour antigens appropriate for the use in the present invention encompass a wide variety of molecules, such as (a) peptide-containing tumour antigens, including peptide epitopes (which can range, for example, from 8-20 amino acids in length, a lthough lengths outside this range are also common), lipopolypeptides and glycoproteins, (b) saccharide-containing tumour antigens, includ ing poly-saccharides, mucins, ga ngliosides, glycolipids and g lycoproteins, including and (c) polynucleotides that express antigenic polypeptides. Aga in, those skilled in the art will recog nise that a tumour antigen present in a conjugate or composition of the present invention will typically comprise peptide. However, embodiments of the invention where one or more conj ugates comprises a tumour a ntigen that does not itself comprise peptide, but for example is bound to the amino acid-comprising or peptide-containing conjugation partner, a re contemplated . Similarly, compositions of the invention in which one or more tumour a ntigens that does not itself comprise peptide is present are contemplated .

In certain embodiments, the tumour a ntigens are, for example, (a) full length molecules associated with cancer cells, (b) homologues a nd modified forms of the same, including molecules with deleted, added and/or substituted portions, and (c) fragments of the same, provided said fragments rema in antigenic or immunogenic. In certain

embodiments, the tumour antigens are provided in recombinant form. In certain embodiments, the tumour antigens include, for example, class I-restricted antigens recog nized by CD8+ lymphocytes or class II-restricted antigens recognized by CD4+ lymphocytes. In certain embodiments, tumor a ntigens include synthetic peptides comprising class I-restricted antigens recognized by CD8+ lymphocytes or class II- restricted antigens recognized by CD4+ lymphocytes.

Shared tumour antigens are generally considered to be native, unmutated seq uences that are expressed by tumours due to epigenetic changes that allow de-repression of developmenta lly-repressed genes. Accordingly, shared antigens are typically considered preferable to over-expressed or differentiation-associated antigens because there is no expression in norma l tissues. Also, the same antigens can be targeted in a number of cancer patients. For exa mple, the cancer-testis antigen NY-ESO-1 is present in the majority of patients with ma ny tumours, a nd a sizeable minority of patients with other tumours. In another example, breast d ifferentiation tumour a ntigens NYBR-1 and NYBR- 1.1 are found in a proportion of breast cancer sufferers. Shared tumour antigens thus represent a n attractive target for development.

The use of shared tumour antigens, such cancer-testis a ntigens including NY-ESO-1, CTSP-1, CTSP-2, CTSP-3, CTSP-4, SSX2, and SCP1, and breast cancer antigens NYBR-1 and NYBR-1.1, in conj ugates of the present invention is specifically contemplated herein . In one exemplary embodiment, the peptide of the peptide-conta ining conj ugation pa rtner or of the peptide conjugate comprises one or more epitopes derived from NY-ESO-1. In one embodiment, the peptide comprises one or more epitopes derived from NY-ESO-1 residues 79 - 116. In one embodiment, the peptide comprises one or more epitopes derived from NY-ESO-1 residues 118 - 143. In one embodiment, the peptide comprises one or more epitopes derived from NY-ESO-1 residues 153 - 180.

In one specifically contemplated embodiment, the peptide of the peptide-containing conjugation partner or of the peptide conjugate, comprises, consists essentially of, or consists of an amino acid sequence selected from the group consisting of 8 or more contiguous, 10 or more contiguous, 12 or more contiguous, 15 or more contiguous, 20 or more contiguous, or 25 or more contiguous amino acids from a ny one of SEQ ID NOs: 9 to 28.

In various embodiments, the peptide comprises more that one amino acid sequence selected from the g roup consisting of any one of SEQ ID NOs: 9 to 28. In one embodiment, the peptide comprises one or more amino acid sequences selected from the group consisting of SEQ ID NOs : 12 - 15, 20, 21, and 26-28.

Similarly, the prostate vaccine Sipuleucel-T (APC8015, Provenge™), which comprises the antigen prostatic acid phosphatase (PAP), is present in 95% of prostate cancer cells. At least in pa rt due to this potential for efficacy in a significant proportion of prostate ca ncer sufferers, Sipuleucel-T was approved by the FDA in 2010 for use in the treatment of asymptomatic, hormone-refractory prostate cancer. The use of PAP a ntigen in conjugates of the present invention is specifically contemplated in the present invention.

Unique antigens are considered to be those a ntigens that are unique to an individua l or are shared by a small proportion of cancer patients, a nd typica lly result from mutations leading to unique protein sequences. Representative examples of unique tumour antigens include mutated Ras antigens, and mutated p53 antigens. As will be appreciated by those skilled in the art having read this specification, the methods of the present invention enable the ready preparation of conj ugates comprising one or more unique tumour a ntigens, for example to elicit specific T-cell responses to one or more unique tumour a ntigens, for example in the prepa ration of patient-specific therapies.

Accordingly, representative tumour antigens include, but are not limited to, (a) antigens such as RAGE, BAGE, GAGE and MAGE family polypeptides, for example, GAGE-1, GAGE- 2, MAGE-1, MAGE-2, MAGE-3, MAGE-4, MAGE-5, MAGE-6, and MAGE-12 (which can be used, for example, to address melanoma, lung, head a nd neck, NSCLC, breast, gastrointestinal, and bladder tumours), (b) mutated antigens, for example, p53

(associated with various solid tumours, for exa mple, colorectal, lung, head a nd neck cancer), p21/Ras (associated with, for example, melanoma, pancreatic cancer a nd colorectal cancer), CDK4 (associated with, for example, melanoma), M UM 1 (associated with, for example, mela noma), caspase-8 (associated with, for example, head and neck cancer), CIA 0205 (associated with, for example, bladder cancer), HLA-A2-R1701, beta catenin (associated with, for example, mela noma), TCR (associated with, for example, T- cell non-Hodgkins lymphoma), BCR-abl (associated with, for example, chronic myelogenous leukemia), triosephosphate isomerase, MA 0205, CDC-27, and LDLR-FUT, (c) over-expressed antigens, for example, Galectin 4 (associated with, for example, colorectal cancer), Galectin 9 (associated with, for example, Hodgkin's disease), proteinase 3 (associated with, for example, chronic myelogenous leukemia), Wilm's tumour a ntigen-1 (WT 1, associated with, for example, various leukemias), carbonic anhydrase (associated with, for example, renal cancer), a ldolase A (associated with, for example, lung cancer), PRAM E (associated with, for example, melanoma), HER-2/neu (associated with, for example, breast, colon, lung and ovaria n ca ncer), alpha-fetoprotein (associated with, for example, hepatoma), KSA (associated with, for example, colorectal cancer), gastrin (associated with, for example, pancreatic and gastric cancer), telomerase catalytic protein, MUC-1 (associated with, for example, breast and ovaria n cancer), G-250 (associated with, for example, rena l cell carcinoma), p53 (associated with, for example, breast, colon cancer), and carcinoembryonic antigen (associated with, for example, breast cancer, lung cancer, a nd ca ncers of the gastrointestinal tract such as colorectal cancer), (d) shared a ntigens, for example, melanoma-melanocyte

differentiation antigens such as MART-l/Melan A, gplOO, MC1R, melanocyte-stimulating hormone receptor, tyrosinase, tyrosinase related protein-l/TRPl and tyrosinase related protein-2/TRP2 (associated with, for example, melanoma), (e) prostate associated antigens such as PAP, prostatic serum antigen (PSA), PSMA, PSH-P1, PSM-P1, PSM-P2, associated with for example, prostate cancer, (f) immunog lobulin idiotypes (associated with myeloma and B cell lymphomas, for example), a nd (g) other tumour antigens, such as polypeptide- and saccharide-containing antigens including (i) glycoproteins such as sialyl Tn a nd sialyl Le.sup.x (associated with, for example, breast and colorectal cancer) as well as various mucins; glycoproteins are coupled to a carrier protein (for example, MUC-1 are coupled to KLH) ; (ii) lipopolypeptides (for example, MUC-1 linked to a lipid moiety); (iii) polysaccharides (for example, Globo H synthetic hexasaccharide), which are coupled to a carrier proteins (for example, to KLH), (iv) ga ng liosides such as GM2, GM 12, GD2, GD3 (associated with, for example, brain, lung cancer, melanoma), which also a re coupled to carrier proteins (for example, KLH).

Other representative tumour antigens amenable to use in the present invention include TAG-72, (See, e.g ., U .S. Pat. No. 5,892,020; human carcinoma antigen (See, e.g ., U .S. Pat. No. 5,808,005); TP1 and TP3 a ntigens from osteocarcinoma cells (See, e.g ., U .S. Pat. No. 5,855,866); Thomsen-Friedenreich (TF) antigen from adenocarcinoma cells (See, e.g ., U .S. Pat. No. 5, 110,911); KC-4 antigen from huma n prostrate

adenocarcinoma (See, e.g., U .S. Pat. No. 4,743,543); a human colorectal cancer a ntigen (See, e.g ., U .S. Pat. No. 4,921,789); CA125 a ntigen from cystadenocarcinoma (See, e.g ., U .S. Pat. No. 4,921,790) ; DF3 antigen from human breast carcinoma (See, e.g ., U .S. Pat. Nos. 4,963,484 and 5,053,489); a human breast tumour a ntigen (See, e.g ., U .S. Pat. No. 4,939,240) ; p97 a ntigen of human melanoma (See, e.g ., U. S. Pat. No. 4,918,164); carcinoma or orosomucoid-related antigen (CORA) (See, e.g., U .S. Pat. No. 4,914,021); T and Tn haptens in glycoproteins of human breast carcinoma, MSA breast carcinoma g lycoprotein ; MFGM breast carcinoma antigen; DU-PAN-2 pancreatic carcinoma a ntigen ; CA125 ova rian carcinoma a ntigen ; YH206 lung carcinoma antigen, Alphafetoprotein (AFP) hepatocellula r carcinoma antigen; Carcinoembryonic antigen (CEA) bowel cancer antigen; Epithelia l tumour antigen (ETA) breast cancer antigen; Tyrosinase; the raf oncogene product; gp75; gplOO; EBV-LMP 1 & 2; EBV-EBNA 1, 2 & 3C; HPV-E4, 6, 7; C017-1A; GA733; gp72; p53; proteinase 3; telomerase; and melanoma gangliosides. These and other tumour a ntigens, whether or not presently characterized, a re contemplated for use in the present invention.

In certain embodiments, the tumour antigens are derived from mutated or altered cellula r components. Representative examples of altered cellular components include, but are not limited to ras, p53, Rb, altered protein encoded by the Wilms' tumour gene, ubiquitin, mucin, protein encoded by the DCC, APC, and MCC genes, as well as receptors or receptor-like structures such as neu, thyroid hormone receptor, platelet derived growth factor (PDGF) receptor, insulin receptor, epidermal growth factor (EGF) receptor, and the colony stimulating factor (CSF) receptor. Polynucleotide-containing antigens used in the present invention include polynucleotides that encode polypeptide tumour a ntigens such as those listed above. In certain embodiments, the polynucleotide-containing antigens include, but are not limited to, DNA or RNA vector constructs, such as plasmid vectors (e.g ., pCMV), which are capable of expressing polypeptide tumour a ntigens in vivo. The present invention also contemplates the preparation of conjugates comprising vira l antigens that are capable of stimulating T-cell to elicit effective anti-viral immunity in patients who are or have been immunosuppressed, for example patients who have had bone marrow transplants, haematopoietic stem cell transplants, or are otherwise undergoing immunosuppression. Similarly, a ntigens derived from viruses associated with increased incidence of cancer, or that are reported to be cancer-causing, such as human papillomavirus, hepatitis A virus, and hepatitis B virus, are contemplated for use in the present invention.

For example, in certa in embodiments, the tumour antigens include, but are not limited to, pl5, Hom/Mel-40, H-Ras, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR, Epstein Ba rr virus antigens, huma n papillomavirus (HPV) a ntigens, including E6 and E7, hepatitis B and C virus antigens, human T-cell lymphotropic virus antigens, TSP-180, pl85erbB2, pl80erbB-3, c-met, mn-23H l, TAG-72-4, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras, pl6, TAGE, PSCA, CT7, 43-9F, 5T4, 791 Tgp72, beta-HCG, BCA225, BTAA, CA 125, CA 15-3 (CA 27.29\BCAA), CA 195, CA 242, CA-50, CAM43, CD68\KP1, CO-029, FGF-5,

Ga733 (EpCAM), HTgp-175, M344, MA-50, MG7-Ag, MOV18, N B/70K, NY-CO-1, RCAS1, SDCCAG16, TA-90 (Mac-2 binding protein\cyclophilin C-associated protein), TAAL6, TAG72, TLP, TPS, and the like.

In certa in embodiments, the tumour antigens include viral proteins implicated in oncogenesis, such as antigens from Epstein Ba rr virus, human papillomavirus (HPV), including E6 a nd E7, and hepatitis B a nd C, a nd huma n T-cell lymphotropic virus.

It will be appreciated that such viral proteins, as well as various other vira l proteins can also be targets for T cell activity in, for example, treatment against viral disease. In fact, the present invention may be useful in any infection where T cell activity is known to play a role in immunity (effectively all virus infections and many bacterial infections as well, such as tuberculosis). The infectious d iseases described herein a re provided by way of example only and are in no way intended to limit the scope of the invention. It will be appreciated that the present invention may be useful in the treatment of various other diseases and conditions. Representative antigens for use in vaccination against pathogenic organisms are discussed below. Compounds, vaccines and compositions comprising one or more antigens prepared using those methods of immunisation a re specifically contemplated.

Tuberculosis antigens

It will be appreciated that a great ma ny M. tuberculosis a ntigens have been characterised and a re suitable for use in the present invention. All M. tuberculosis antigens, whether or not presently characterized, that are capable of eliciting an immune response are contemplated .

Exemplary M. tuberculosis antigens suitable for use include early secretary a ntigen target (ESAT) -6, Ag85A, Ag85B (MPT59), Ag85B, Ag85C, MPT32, MPT51, M PT59, MPT63, MPT64, MPT83, MPB5, MPB59, M PB64, MTC28, Mtb2, Mtb8.4, Mtb9.9, Mtb32A, Mtb39, Mtb41, TB10.4, TBIOC, TBl lB, TB12.5, TB13A, TB14, TB15, TB15A, TB16, TB16A, TB17, TB18, TB21, TB20.6, TB24, TB27B, TB32, TB32A, TB33, TB38, TB40.8, TB51, TB54, TB64, CFP6, CFP7, CFP7A, CFP7B, CFP8A, CFP8B, CFP9, CFP10, CFP11, CFP16, CFP17, CFP19, CFP19A, CFP19B, CFP20, CFP21, CFP22, CFP22A, CFP23, CFP23A, CFP23B,

CFP25, CFP25A, CFP27, CFP28, CFP28B, CFP29, CFP30A, CFP30B, CFP50, CWP32, hspX (alpha-crystalline), APA, Tuberculin purified protein derivative (PPD), ST-CF, PPE68, LppX, PstS-1, PstS-2, PstS-3, H BHA, GroEL, GroEL2, GrpES, LHP, 19kDa lipoprotein, 71kDa, RD1-ORF2, RD1-ORF3, RD1-ORF4, RD1-ORF5, RD1-ORF8, RD1-ORF9A, RD1- ORF9B, Rvl984c, Rv0577, Rvl827, BfrB, Tpx. Rvl352, Rvl810, PpiA, Cut2, FbpB, FbpA, FbpC, DnaK, FecB, Ssb, RpIL, FixA, FixB, AhpC2, Rv2626c, Rvl211, Mdh, Rvl626, Adk, ClpP, SucD (Belisle et al, 2005; US 7,037,510; US 2004/0057963; US 2008/0199493; US 2008/0267990), or at least one antigenic portion or T-cell epitope of a ny of the above mentioned antigens. Hepatitis antigens

A number of hepatitis antigens have been characterised and are suitable for use in the present invention. Exemplary hepatitis C antigens include C - p22, El - gp35, E2 - gp70, NS1 - p7, NS2 - p23, NS3 - p70, NS4A - p8, NS4B - p27, NS5A - p56/58, and NS5B - p68, and together with one or more antigenic portions or epitopes derived therefrom are each (whether alone or in combination) suitable for application in the present invention. All hepatitis antigens, whether or not presently characterized, that are capable of eliciting an immune response are contemplated .

Influenza antigens

Many influenza antigens have been characterised and are suitable for use in the present invention. Exemplary influenza antigens suitable for use in the present invention include PB, PB2, PA, any of the hemagglutinin (HA) or neuramimidase (NA) proteins, NP, M, and NS, a nd together with one or more a ntigenic portions or epitopes derived therefrom a re each (whether alone or in combination) suitable for application in the present invention. All influenza a ntigens, whether or not presently characterized, that are capable of eliciting an immune response are contemplated .

Anthrax antigens

A number of B. anthracis antigens have been identified as potential candidates for vaccine development a nd are useful in the present invention. For example, PA83 is one such antigen for vaccine development. Currently, only one FDA licensed vaccine for anthrax is available called "Anthrax Vaccine Adsorbed" (AVA) or BioThrax®. This vaccine is derived from the cell-free supernatant of a non-encapsulated stra in of B. anthracis adsorbed to a luminum adjuvant. PA is the primary immunogen in AVA. Other exemplary anthrax antigens suitable for use in the present invention include Protective antigen (PA or PA63), LF and EF (proteins), poly-gamma-(D-g lutamate) capsule, spore a ntigen (endospore specific components), BcIA (exosporium specific protein), BxpB (spore- associated protein), and secreted proteins. All anthrax antigens together with one or more antigenic portions or epitopes derived therefrom, whether or not presently characterized, that are capable of eliciting an immune response are contemplated.

Tularemia antigens A number of F. tularensis antigens have been identified as potential candidates for vaccine development a nd are useful in the present invention. For example, AcpA and IgIC are antigens suitable for vaccine development. Other exemplary Tularemia antigens suitable for use in the present invention include O-antigen, CPS, outer membrane proteins (e.g . FopA), lipoproteins (e.g . Tul4), secreted proteins and lipopolysaccharide. All tularemia antigens together with one or more antigenic portions or epitopes derived therefrom, whether or not presently cha racterized, that are capable of eliciting an immune response are contemplated .

Brucellosis antigens

A number of B. abortusis antigens have been identified as potential candidates for vaccine development a nd are useful in the present invention. For example, Ompl6 is one such antigen for vaccine development. Other exemplary Brucellosis antigens suitable for use in the present invention include O-antigen, lipopolysaccharide, outer membrane proteins (e.g . Ompl6), secreted proteins, ribosomal proteins (e.g . L7 and L12), bacterioferritin, p39 (a putative periplasmic binding protein), groEL(heat-shock protein), lumazine synthase, BCSP31 surface protein, PAL16.5 OM lipoprotein, catalase, 26 kDa periplasmic protein, 31 kDa Omp31, 28 kDa Omp, 25 kDa Omp, and 10 kDA Om lipoprotein. All brucellosis antigens together with one or more antigenic portions or epitopes derived therefrom, whether or not presently characterized, that are capable of eliciting an immune response are contemplated . Meningitis antigens

A number of N. meningitidis antigens have been identified as potential candidates for vaccine development a nd are useful in the present invention. For example, Cys6, PorA, PorB, FetA, and ZnuD are antigens suitable for vaccine development. Other exemplary Meningitis antigens suitable for use in the present invention include O-antigen, factor H binding protein (fHbp), TbpB, NspA, NadA, outer membra ne proteins, group B CPS, secreted proteins and lipopolysaccharide. All menigitis antigens together with one or more antigenic portions or epitopes derived therefrom, whether or not presently characterized, that are capable of eliciting an immune response are contemplated.

Dengue antigens A number of Flavivirus antigens have been identified as potential candidates for vaccine development to treat dengue fever and a re useful in the present invention. For example, dengue virus envelope proteins El - E4 and the membrane proteins M l - M4 are antigens suitable for vaccine development. Other exemplary dengue antigens suitable for use in the present invention include C, preM, 1, 2A, 2B, 3, 4A, 4B and 5. All dengue antigens together with one or more antigenic portions or epitopes derived therefrom, whether or not presently characterized, that are capable of eliciting an immune response are contemplated .

Ebola antigens

A number of ebola virus antigens have been identified as potential candidates for vaccine development to treat ebola infection and a re useful in the present invention. For example, Filoviridae Za ire ebolavirus a nd Sudan ebolavirus virion spike glycoprotein precursor a ntigens ZEBOV-GP, and SEBOV-GP, respectively, are suitable for vaccine development. Other exemplary ebola antigens suitable for use in the present invention include N P, vp35, vp40, GP, vp30, vp24 and L. All ebola a ntigens together with one or more antigenic portions or epitopes derived therefrom, whether or not presently characterized, that are capable of eliciting an immune response are contemplated.

West Nile antigens

A number of West N ile virus antigens have been identified as potential candidates for vaccine development to treat infection and are useful in the present invention . For example, Flavivirus envelope antigen (E) from West Nile virus (WNV) is a non-toxic protein expressed on the surface of WNV virions (WNVE) and are suitable for vaccine development. Other exemplary WNV antigens suitable for use in the present invention include Cp, Prm, NS1, N S2A, NS2B, NS3, NS4A, NS4B and NS5.

All West N ile antigens together with one or more antigenic portions or epitopes derived therefrom, whether or not presently cha racterized, that are capable of eliciting an immune response are contemplated .

The above-listed or referenced antigens are exemplary, not limiting, of the present invention. The present invention also relates to pharmaceutical composition comprising an effective amount of a peptide conjugate of the present invention or a pha rmaceutically acceptable salt or solvent thereof, and a pharmaceutica lly acceptable carrier.

The pharmaceutical compositions may comprise an effective amount of two or more peptide conj ugates of the invention in combination. In some embodiments, the pharmaceutical compositions may comprise one or more peptide conjugates of the invention and one or more peptides as described herein.

The term "pharmaceutically acceptable carrier" refers to a carrier (adjuvant or vehicle) that may be administered to a subject together with the peptide conjugate of the present invention, or a pharmaceutically acceptable salt or solvent thereof, and a

pharmaceutically acceptable carrier.

Pha rmaceutically acceptable carriers that may be used in the compositions include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, self-emulsifying drug delivery systems (SEDDS) such as d-a-tocopherol polyethyleneglycol 1000 succinate, surfactants used in pharmaceutical dosage forms such as Tweens or other similar polymeric delivery matrices, serum proteins, such as huma n serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, pa rtial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hyd rogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol and wool fat. Cyclodextrins such as α-, β-, a nd γ-cyclodextrin, or chemically modified derivatives such as hydroxyalkylcyclodextrins, including 2- and 3-hydroxypropyl- - cyclodextrins, or other solubilized derivatives may also be advantageously used to enha nce delivery. Oil solutions or suspensions may also contain a long-cha in a lcohol diluent or dispersant, or carboxymethyl cellulose or similar dispersing agents, which are commonly used in the formulation of pha rmaceutica lly acceptable dosage forms such as emulsions and or suspensions. The compositions are formulated to allow for ad ministration to a subject by any chosen route, including but not limited to ora l or parentera l (including topical, subcutaneous, intramuscula r and intravenous) administration.

For example, the compositions may be formulated with an appropriate pharmaceutically acceptable carrier (including excipients, d iluents, auxilia ries, and combinations thereof) selected with regard to the intended route of administration and standa rd pharmaceutical practice. For example, the compositions may be administered orally as a powder, liquid, tablet or capsule, or topically as an ointment, cream or lotion. Suitable formulations may contain additional agents as required, including emulsifying, antioxidant, flavouring or colouring agents, a nd may be adapted for immediate-, delayed-, modified-, sustained-, pulsed- or controlled-release. The compositions may be formulated to optimize bioavailability, immunogenicity, or to maintain plasma, blood, or tissue concentrations within the immunogenic or therapeutic range, includ ing for extended periods. Controlled delivery preparations may also be used to optimize the antigen concentration at the site of action, for example.

The compositions may be formulated for period ic administration, for example to provide continued exposure. Strategies to elicit a beneficial immunological response, for example those that employ one or more "booster" vaccinations, are well known in the art, a nd such strategies may be adopted.

The compositions may be administered via the parentera l route. Examples of parenteral dosage forms include aqueous solutions, isotonic saline or 5% g lucose of the active agent, or other well-known pharmaceutically acceptable excipients. Cyclodextrins, for example, or other sol ubil ising agents well-known to those familia r with the art, can be utilized as pharmaceutical excipients for delivery of the therapeutic agent.

Examples of dosage forms suitable for ora l ad ministration include, but are not limited to tablets, capsules, lozenges, or like forms, or any liquid forms such as syrups, aqueous solutions, emulsions and the like, capable of providing a therapeutically effective amount of the composition. Capsules can contain a ny standard pharmaceutically acceptable materials such as gelatin or cellulose. Tablets can be formulated in accorda nce with conventional procedures by compressing mixtures of the active ingredients with a solid carrier a nd a lubricant. Examples of solid carriers include starch and sugar bentonite. Active ingredients can also be administered in a form of a ha rd shell tablet or a capsule containing a binder, e.g ., lactose or mannitol, a conventional filler, and a tabletting agent.

Examples of dosage forms suitable for transdermal administration include, but are not limited, to tra nsdermal patches, transdermal ba ndages, and the like. Examples of dosage forms suitable for topica l administration of the compositions include any lotion, stick, spray, ointment, paste, cream, gel, etc., whether applied directly to the skin or via an intermed iary such as a pad, patch or the like. Examples of dosage forms suitable for suppository administration of the compositions include any solid dosage form inserted into a bodily orifice particularly those inserted rectally, vaginally and urethrally.

Examples of dosage of forms suitable for injection of the compositions include delivery via bolus such as single or multiple administrations by intravenous injection,

subcutaneous, subdermal, and intramuscular administration or ora l administration .

Examples of dosage forms suitable for depot ad ministration of the compositions and include pellets of the peptide conjugates or solid forms wherein the peptide conjugates are entrapped in a matrix of biodegradable polymers, microemulsions, liposomes or are microencapsulated.

Examples of infusion devices for the compositions include infusion pumps for providing a desired number of doses or steady state administration, and include implantable drug pumps.

Examples of impla ntable infusion devices for compositions include any solid form in which the peptide conj ugates are encapsulated within or dispersed throug hout a biodegradable polymer or synthetic, polymer such as silicone, silicone rubber, silastic or similar polymer.

Examples of dosage forms suitable for transmucosal delivery of the compositions include depositories solutions for enemas, pessaries, tampons, creams, gels, pastes, foams, nebulised solutions, powders and similar formulations containing in addition to the active ingredients such carriers as are known in the art to be appropriate. Such dosage forms include forms suitable for inhalation or insufflation of the compositions, including compositions comprising solutions and/or suspensions in pha rmaceutically acceptable, aqueous, or organic solvents, or mixture thereof a nd/or powders. Tra nsmucosal administration of the compositions may utilize any mucosa l membrane but commonly utilizes the nasal, buccal, vagina l and rectal tissues. Formulations suita ble for nasa l administration of the compositions may be administered in a liquid form, for example, nasal spray, nasal drops, or by aerosol administration by nebulizer, including aqueous or oily solutions of the polymer particles. Formulations may be prepared as aqueous solutions for example in saline, solutions employing benzyl alcohol or other suitable preservatives, absorption promoters to enhance bio-availability, fluorocarbons, and/or other solubilising or dispersing agents known in the art.

Examples of dosage forms suitable for buccal or subling ual administration of the compositions include lozenges, tablets and the like. Examples of dosage forms suitable for opthalmic administration of the compositions include inserts a nd/or compositions comprising solutions and/or suspensions in pha rmaceutica lly acceptable, aqueous, or orga nic solvents.

Examples of formulations of compositions, including vaccines, may be found in, for example, Sweetman, S. C. (Ed .). Ma rtindale. The Complete Drug Reference, 33rd Edition, Pharmaceutical Press, Chicago, 2002, 2483 pp. ; Aulton, M . E. (Ed .)

Pha rmaceutics. The Science of Dosage Form Design. Churchill Livingstone, Edinburgh, 2000, 734 pp. ; and, Ansel, H . C, Allen, L. V. and Popovich, N . G. Pharmaceutical Dosage Forms a nd Drug Delivery Systems, 7th Ed ., Lippincott 1999, 676 pp.. Excipients employed in the manufacture of drug delivery systems are described in various publications known to those skilled in the a rt including, for example, Kibbe, E. H.

Handbook of Pharmaceutical Excipients, 3rd Ed ., American Pharmaceutical Association, Washington, 2000, 665 pp. The USP also provides examples of mod ified-release oral dosage forms, including those formulated as tablets or capsules. See, for example, The United States Pharmacopeia 23/National Formulary 18, The United States Pharmacopeial Convention, Inc., Rockville MD, 1995 (hereinafter "the USP"), which also describes specific tests to determine the drug release capabilities of extended-release and delayed- release tablets a nd capsules. The USP test for d rug release for extended-release and delayed-release a rticles is based on d rug dissolution from the dosage unit against elapsed test time. Descriptions of various test apparatus and procedures may be found in the USP. Further guidance concerning the analysis of extended release dosage forms has been provided by the F. D.A. (See Guidance for Industry. Extended release ora l dosage forms : development, eva luation, and application of in vitro/in vivo correlations. Rockville, MD: Center for Drug Evaluation and Research, Food and Drug Administration, 1997). While the composition may comprise one or more extrinsic adj uvants, advantageously in some embodiments this is not necessary. In some embodiments, the peptide conjugate comprises a n epitope and is self adjuvanting .

The present invention provides a method of vaccinating or eliciting an immune response in a subject comprising administering to the subject an effective amount of a peptide conjugate of the present invention . The present invention also relates to a peptide conjugate of the invention for vaccinating or eliciting an immune response in a subject, and to use of a peptide conjugate of the invention in the ma nufacture of a medicament for vaccinating or eliciting a n immune response in a subject.

The present invention also provides a method of vaccinating or eliciting an immune response in a subject comprising administering to the subject an effective amount of the pharmaceutical composition of the present invention. The present invention also relates to a pharmaceutical composition of the invention for vaccinating or eliciting a n immune response in a subject, and to the use of one or more peptide conjugates of the present invention in the manufacture of a medicament for vaccinating or eliciting an immune response in a subject. The present invention also provides a method of activating TLR2 in a subject comprising administering to the subject an effective amount of one or more peptide conjugate of the invention or a pha rmaceutically acceptable salt or solvate thereof, or a n effective amount of a pha rmaceutica l composition of the invention . The present invention also provides use of one or more peptide conj ugate compounds of the invention or a pharmaceutically acceptable salt or solvate thereof or a pharmaceutical composition of the invention in the manufacture of a medicament for activating TLR2 in a subject and one or more peptide conjugate compounds of the invention or a pha rmaceutically acceptable salt or solvate thereof or a pharmaceutical composition of the invention in a subject for activating TLR2 in a subject. Activating TLR2 can stimulate and/or elicit an immune response in the subject, and, in some embodiments, provide immunity.

The administration or use of one or more peptides described herein a nd/or one or more peptide conj ugates of the present invention, for example one or more peptide described herein in together with one or more peptide conj ugates, for vaccinating or eliciting an immune response in the subject is contemplated herein. Where two or more peptide conjugates, or one or more peptides and one or more peptide conjugates are administered or used, the two or more peptide conjugates, or one or more peptides and one or more peptide conj ugates may be administered or used simulta neously, sequentia lly, or separately.

A "subject" refers to a vertebrate that is a mammal, for example, a human. Mammals include, but are not limited to, humans, fa rm animals, sport anima ls, pets, primates, mice and rats. The subject may be in need of said vaccinating, eliciting an immune response, or activiating TLR2.

An "effective amount" is an amount sufficient to effect beneficial or desired results including clinica l results. An effective amount can be administered in one or more administrations by va rious routes of administration .

The effective amount will vary depending on, a mong other factors, the disease indicated, the severity of the disease, the age and relative health of the subject, the potency of the compound administered, the mode of administration a nd the treatment desired . A person skilled in the art will be able to determine appropriate dosages having rega rd to these any other relevant factors. The efficacy of a composition can be eva luated both in vitro and in vivo. For example, the composition can be tested in vitro or in vivo for its ability to induce a cell-mediated immune response. For in vivo studies, the composition can be fed to or injected into an animal (e.g ., a mouse) and its effects on eliciting an immune response are then assessed. Based on the results, an appropriate dosage ra nge a nd administration route can be determined .

The composition may be administered as a single dose or a multiple dose sched ule. Multiple doses may be used in a primary immunisation schedule and/or in a booster immunisation schedule. In certa in embodiments, eliciting an immune response comprises ra ising or enhancing a n immune response. In exemplary embod iments, eliciting an immune response comprises eliciting a humoral and a cell mediated response.

In certa in embodiments, eliciting an immune response provides immunity.

The immune response is elicited for treating a disease or cond ition . A person skilled in the art will appreciate that the peptide conjugates described herein a re useful for treating a variety of d iseases and conditions, depending, for example, on the nature of epitope.

In some embod iments, the diseases or conditions are selected from those associated with the various antigens described herein.

In some embod iments the disease or cond ition is an infectious disease, cancer, or viral re-activation post-bone marrow transplant or following induction of profound

immunosuppression for any other reason.

The term "treatment", and related terms such as "treating" and "treat", as used herein relates generally to treatment, of a human or a non-huma n subject, in which some desired therapeutic effect is achieved . The therapeutic effect may, for example, be inhibition, reduction, amelioration, ha lt, or prevention of a disease or cond ition.

The compositions may be used to elicit systemic and/or mucosal immunity. Enha nced systemic and/or mucosal immunity may be reflected in an enha nced TH1 and/or TH2 immune response. The enhanced immune response may include a n increase in the prod uction of IgGl and/or IgG2a and/or IgA. EXAMPLES

1. Example 1

This example describes the synthesis of diastereomerically pure amino acid conjugates 6A and 6B. 1.1 Preparation and use of enantiopure epoxides 102A and 102B

Diastereomerically pure amino acid conj ugates 6A and 6B may be prepared using enantiopure epoxide 102A or enantiopure epoxide 102B produced stereospecifically from an enantiomerically pure starting materia l.

Ena ntiopure epoxide 102A and enantiopure epoxide 102B were prepared from L-aspartic acid and D-aspartic acid, respectively, by following the procedure described in Volkmann, R. A. et al. J. Org. Chem., 1992, 57, 4352-4361 for the preparation of (R)-(2- hydroxyethyl)oxirane (102A) from L-aspartic acid .

(S)-2-Bromosuccinic acid

To a solution of sod ium bromide (15.46 g, 150.24 mmol) in 6N H2SO4 (33 mL) at 0 °C was added L-aspartic acid (5.00 g, 37.56 mmol) . To the resultant mixture was added sodium nitrite (3.11 g, 45.07 mmol) portionwise over 90 min. The reaction mixture was allowed to stir at 0 °C for a further 2 h. The mixture was then diluted with H2O (17 mL) and extracted with Et2<D (100 mL). The aq ueous layer was diluted with brine (20 mL) and further extract with Et2<D (3 x 100 mL). The combined organic extracts were dried over anhydrous Na2S04 and concentrated in vacuo to give (S)-2-bromosuccinic acid (2.98 g, 41%) as a white solid. The crude was used in subsequent synthetic steps without further purification. [α]ο ^{19 7} -71.5 (c 0.46 in EtOAc) (lit -73.5 (c 6.0 in EtOAc); δ _Η (400 MHz; DMSO) 12.8 (2H, br s, 2 x CO2H), 4.54 (1H, dd, J = 8.5, 6.4 Hz, H-l ), 3.10 (1H, dd, J = 17.2, 8.6 Hz, H-2), 2.90 (1H, dd, J = 17.1, 6.4 Hz, H-2) ; 5c (100 MHz; DMSO) 171.0 (C, CO2H), 170.1 (C, CO2H ), 40.5 (CH, C-l ), 39.5 (CH ₂ , C-2). Spectroscopic data were consistent with those reported in literature.

(R)-2-Bromosuccinic acid

(fi)-2-Bromosuccinic acid was prepared by following the procedure described above for the preparation of (S)-2-bromosuccinic acid, but using D-aspartic acid instead of L- aspartic acid . [CI ] D ²⁰ - ² +66.5 (c 0.2 in EtOAc) . The rema ining spectroscopic data was identical to that observed for (S)-2-bromosuccinic acid .

(S)-2-Bromo-l,4-butanediol To a solution of (S)-2-bromosuccinic acid (2.98 g, 15.20 mmol) in THF (35 mL) at -78 °C was added Bhb'DMS complex (4.33 mL, 45.61 mmol) dropwise over 90 min. The reaction was a llowed to stir at -78 °C for 2h and then warmed to r.t. and allowed to stir for a further 60 h. The reaction was then cooled to 0 °C and MeOH (15 mL) was added slowly. The mixture was then concentrated in vacuo a nd the residue diluted with MeOH (15 mL). This process was repeated 3 times to give the 2-bromo-l,4-butanediol (2.55 g, quant.) as a yellow oil. The crude was used in subsequent synthetic steps without further purification. [a] _D ^{19 6} -36.8 (c 0.5 in CHC ); δ _Η (400 MHz, CDCb) 4.34 (1 H, dq, J = 7.7, 5.3 Hz, H-2), 3.92-3.78 (4H, m, H-l, H-4), 2.40 (2H, br s, 2 x OH), 2.20-2.06 (2H, m, H-3); 5c (100 MHz; CDCb) 67.1 (CH ₂ , C-l), 60.1 (CH ₂ , C-4), 55.2 (CH, C-2), 37.8 (CH ₂ , C-3). Spectroscopic data were consistent with those reported in literature.

(/?)-2-Bromo-l,4-butanediol

(fi)-2-Bromo-l,4-butanediol was prepared by following the procedure described above for the prepa ration of (S)-2-bromo-l,4-butanediol, but using (fi)-2-bromosuccinic acid instead of (S)-2-bromosuccinic acid . [CI ] D ²¹ - ³ +20.0 (c 0.17 in CHCb). The rema ining spectroscopic data was identical to that observed for (S)-2-bromo-l,4-butanediol.

(/?)-( 2-Hydroxyethyl )oxirane (102A)

To a solution of (S)-2-bromo-l,4-buta nediol (2.31 g, 13.76 mmol) in CH2CI2 (46 mL) at r.t. was added CS2CO3 (8.74 g, 24.77 mmol). The resultant mixture was allowed to stir at r.t. for 72 h. The reaction was then filtered through a pad of Celite ^® and concentrated in vacuo to give (R)-(2-hydroxyethyl)oxirane (102A) as a yellow oil with qua ntitative conversion. The crude material was used in subsequent synthetic steps without further purification. [a] _D ²² - ⁹ +35.0 (c 0.61 in CHCb); δ _Η (400 MHz; CDCb) 3.83-3.79 (2H, m, H- 1), 3.12-3.08 (1H, m, H-3), 2.81 (1 H, dd, J = 4.8, 4.1 Hz, H-4), 2.60 ( 1H, dd, J = 4.8, 2.8 Hz, H-4), 2.03-1.95 (1H, m, H-2), 1.78 (1H, t, J = 5.4 Hz, OH), 1.71 (1H, dq, J =

14.6, 5.9 Hz, H-2) ; 5c (100 MHz; CDCb) 60.0 (CH2, C-l), 50.5 (CH, C-3), 46.5 (CH2, C- 4), 34.6 (CH2, C-2). Spectroscopic data were consistent with those reported in literature.

(S)-(2-hydroxyethyl)oxirane (102B)

(S)-(2-Hyd roxyethyl)oxirane (102B) was prepa red by following the procedure described above for the preparation of (R)-(2-hydroxyethyl)oxirane (102A), but using (R)-2- bromo-l,4-buta nediol instead of (S)-2-bromo-l,4-butanediol. [CI ] D ²² - ⁹ -35.2 (c 0.23 in CHCb). The remaining spectroscopic data was identical to that observed for (S)-2-bromo- 1,4-butanediol .

Preparation of diastereomerically pure 6A

804 103A

201 A 6A

To a stirred solution of disulfide 804 (1.59 g, 2.06 mmol) in CH2CI2 (10 mL) at 0 °C was added zinc powder (0.94 g, 14.42 mmol) and a freshly prepared mixture of methanol, cone, hydrochloric acid and cone, sulfuric acid ( 100 :7 : 1, 5 mL). The resultant mixture was allowed to stir at 0 °C for 30 min after which was added epoxide 102A (0.73 g, 8.24 mmol) . The reaction mixture was allowed to stir at 55 °C or refluxed at 70 °C for 17 h. The mixture was then diluted with CH2CI2 (30 mL), filtered through a pad of Celite ^® and washed with brine (50 mL). The aq ueous layer was extracted with CH2CI2 (3 x 50 mL) and the combined organic extracts were dried over anhydrous MgSCU and concentrated in vacuo. The crude was purified by flash column chromatography (hexanes-EtOAc, 1 : 3) to g ive 103A (1.72 g, 88%) as a colourless oil.

Rf 0.15 (hexanes-EtOAc 1 :3); [a] _D ²⁰ - ² -3.5 (c 0.32 in CHC ); VmaxineatVcm ¹ 3347, 2976, 1703, 1518, 1449, 1413, 1369, 1335, 1249, 1151 ; δ _Η (400 MHz; CDCb) 7.77 (2H, d, J = 7.5, FmocH), 7.61 (2H, d, J = 7.2 Hz, FmocH), 7.40 (2H, t, J = 7.4 Hz, FmocH), 7.32 (2H, t, J = 7.5 Hz, FmocH), 5.81 (1H, d, J = 8.0 Hz, N H), 4.53-4.50 (1H, m, H-l), 4.40 (2H, d, J = 6.8 Hz, FmocCH ₂ ), 4.23 (1H, t, J = 7.0 Hz, FmocCH), 3.94-3.88 (1H, m, H-4), 3.85-3.81 (2H, m, H-6), 3.03 (1H, dd, J = 14.0, 4.2 Hz, H-2), 2.94 (1H, dd, J = 14.3, 6.1 Hz, H-2), 2.82 (1H, dd, J = 14.0, 2.9 Hz, H-3), 2.56 (1H, dd, J = 14.0, 9.0 Hz, H-3), 1.74-1.71 (1H, m, H-5), 1.50 (9H, s, C(CH ₃ ) ₃ ); 5c (100 MHz; CDCb) 169.7 (C, COOtBu), 156.0 (C, C(O)Fmoc), 143.6 (C, Fmoc), 141.0 (C, Fmoc), 127.5 (CH, Fmoc),

126.9 (CH, Fmoc), 125.0 (CH, Fmoc), 120.0 (CH, Fmoc), 82.6 (C, C(CH ₃ ) ₃ ), 69.5 (CH, C- 4), 67.2 (CH2, FmocCH ₂ ), 60.2 (CH2, C-6), 54.5 (CH, C-l), 46.9 (CH, FmocCH), 40.5 (CH2, C-3), 37.5 (CH2, C-5), 35.2 (CH2, C-2), 27.8 (3 x CH ₃ , C(CH ₃ ) ₃ ); HRMS (ESI+ ) [M + Na] ⁺ 510.1921 calc for C2 _S H ₃₃ NNaO _s S 510.1921. Synthesis of compound 6A (Procedure A): To a stirred solution of diol 103A (1.52 g, 3.12 mmol) and palmitic acid (2.40 g, 9.35 mmol) in THF (45 mL) at r.t. was added /V,/V'-diisopropylcarbodiimide (1.93 mL, 12.46 mmol) and 4-dimethyla minopyridine (0.04 g, 0.31 mmol). The reaction mixture was allowed to stir at r.t. for 19 h. The mixture was then filtered through a pad of Celite ^® , diluted with EtOAc (50 mL), washed with 1M aq . citric acid (30 mL) and brine (30 mL) and concentrated in vacuo. The residue was then redissolved in TFA (3 mL) and a llowed to stir at r.t. for 30 min. The reaction mixture was again concentrated in vacuo. The crude was purified by flash column chromatography (hexanes-EtOAc, 9 : 1→ 0 : 1) to g ive the title compound 6A ( 1.98 g, 70%) as a colorless oil. [a] _D ²³ - ⁹ +8.4 (c 0.44 in CHCb); Vmax(neat)/cm ¹ 2922, 2852, 1733, 1525, 1450, 1168, 1110; ^X H NMR (400 MHz, CDCb) δ 7.75 (2H, d, J = 7.5 Hz), 7.61 (2H, d, J = 7.2 Hz), 7.38 (2H, t, J = 7.4 Hz), 7.30 (2H, t, J = 7.4 Hz), 5.89 (1H, d, J = 7.8 Hz), 5.13-5.06 ( 1H, m), 4.69-4.63 (1H, m), 4.39 (2H, d, J = 6.5 Hz), 4.23 (1H, t, J = 7.0 Hz), 4.16-4.06 (2H, m), 3.16 (1H, dd, J = 13.8, 4.0 Hz), 3.03 (1 H, dd, J = 13.8, 6.1 Hz), 2.81-2.70 (2H, m), 2.33-2.26 (4H, m), 2.10-1.89 (2H, m), 1.60-1.58 (4H, m, ), 1.35-1.20 (48H, m), 0.89 (6H, t, J = 6.8 Hz); ¹³ C N MR (100 MHz, CDCb) δ 174.0, 173.6, 156.0, 143.6, 141.2, 127.6, 127.0, 125.1, 120.0, 69.5, 67.3, 60.3, 53.6, 47.0, 36.3, 34.5, 34.3, 34.1, 32.0, 31.8, 29.6, 29.4, 29.3, 29.2, 29.1, 25.0, 22.6, 14.0; HRMS (ESI + ) [M + H] ⁺ 908.6069 calc for Cs^seNOeS 908.6065, [M + Na] ⁺ 930.5888 calc for Cs^ssNNaOsS 930.5875. Synthesis of compound 6A (Procedure B):

Diastereomerically pure diol 103A was also converted to d iastereomerically pure conjugate 6A by following procedures analogous to the representative proced ures described below.

Representative procedure for conversion of 103A to 201A To a stirred solution of diol 103A (0.327 g, 0.67 mmol) a nd pa lmitic acid (0.516 g, 2.01 mmol) in THF (9 mL) at r.t. is added diisopropylcarbodiimide (0.414 mL, 2.68 mmol) and 4-dimethylaminopyrid ine (0.01 g, 0.07 mmol). The reaction mixture is allowed to stir at r.t. for 19 h. The mixture is then diluted with EtOAc (30 mL), filtered through a bed of Celite ^® and concentrated in vacuo. The crude is purified by flash column chromatography (CH2CI2) to g ive 201A as yellow oil.

Representative procedure for conversion of 201A to 6A

A solution of diester 201A (0.35 g, 0.364 mmol) in trifluoroacetic acid (2 mL) is allowed to stir at r.t. for 1 h after which the mixture is concentrated in vacuo. The crude is purified by flash column chromatography (hexanes-EtOAc, 9 : 1→ 0: 1) to give 6A as a colourless oil . Fmoc-Cys-OH is described in the literature: H .-K. Cui, Y. Guo, Y. He, F.-L. Wang, H .-H . Cha ng, Y. J. Wa ng, F.-M . Wu, C.-L. Tian, L. Lu, Angew. Chem. Int. Eng. , 2013, 52(36), 9558-9562.

Preparation of diastereomerically pure 6B

102B 804 103B

Synthesis of compound 103B:

To a stirred solution of disulfide 804 (2.01 g, 2.53 mmol) in CH2CI2 (14 mL) at 0 °C was added zinc powder (1.15 g, 17.51 mmol) and a freshly prepared mixture of methanol, cone, hydrochloric acid and cone, sulfuric acid ( 100 :7 : 1, 7 mL). The resultant mixture was allowed to stir at 0 °C for 30 min after which was added epoxide 102B (0.89 g,

10.11 mmol). The reaction mixture was allowed to stir at 55 °C or refluxed at 70 °C for 17 h. The mixture was then d iluted with CH2CI2 (30 mL), filtered through a pad of Celite ^® and washed with brine (50 mL). The aq ueous layer was extracted with CH2CI2 (3 x 50 mL) and the combined orga nic extracts were dried over anhydrous MgSC>4 and concentrated in vacuo. The crude was purified by flash column chromatography

(hexanes-EtOAc, 3 : 1) to give the 103B (2.17 g, 88%) as a colourless oil.

Rf 0.15 (hexanes-EtOAc 1 : 3) ; [CI] D ²² +8.5 (c O.3 in CHC ); VmaxineatVcm ¹ 3347, 2976, 1703, 1518, 1449, 1413, 1369, 1335, 1249, 1151 ; δ _Η (400 MHz; CDC ) 7.77 (2H, d, J = 7.5 Hz, FmocH), 7.61 (2H, d, J = 7.4 Hz, FmocH), 7.40 (2H, t, J = 7.4 Hz, FmocH), 7.32 (2H, t, J = 7.5 Hz, FmocH), 5.74 (1H, d, J = 7.0 Hz, NH), 4.51-4.47 (1H, m, H-l), 4.42- 4.39 (2H, m, FmocCH ₂ ), 4.24 (1H, t, J = 7.0 Hz, FmocCH), 3.93 (1H, br s, H-4), 3.85- 3.81 (2H, m, H-6), 3.31 (1H, br s, OH-4), 3.00-2.78 (2H, m, H-2), 2.80 (1 H, dd, J = 13.5, 3.2 Hz, H-3), 2.55 (1H, dd, J = 13.8, 8.4, Hz, H-3), 2.36 (1H, br s, OH-6) 1.73 (2H, q, J = 5.3, H-5), 1.50 (9H, s, C(CH ₃ ) ₃ ); HRMS 5c (100 MHz; CDCb) 169.8 (C, COOtBu), 156.1 (C, C(O)Fmoc), 143.8 (C, Fmoc), 141.3 (C, Fmoc), 127.7 (CH, Fmoc),

127.1 (CH, Fmoc), 125.1 (CH, Fmoc), 120.0 (CH, Fmoc), 83.0 (C, C(CH ₃ ) ₃ ), 69.9 (CH, C- 4), 67.2 (CH ₂ , FmocChh), 60.7 (CH ₂ , C-6), 54.7 (CH, C-l), 47.1 (CH, FmocCH), 40.9 (CH ₂ , C-3), 37.6 (CH ₂ , C-5), 35.5 (CH ₂ , C-2), 28.0 (3 x CH ₃ , C(CH ₃ ) ₃ ); HRMS (ESI+ ) [M + Na] ⁺ 510.1921 calc for C26H ₃ 3N NaO _s S 510.1921.

Synthesis of compound 6B (Procedure A): To a stirred solution of diol 103B (1.68 g, 3.44 mmol) and palmitic acid (2.65 g, 10.31 mmol) in THF (50 mL) at r.t. was added /V,/V'-diisopropylcarbodiimide (2.13 mL, 13.75 mmol) and 4-dimethyla minopyridine (0.04 g, 0.34 mmol). The reaction mixture was allowed to stir at r.t. for 19 h. The mixture was then filtered through a pad of Celite ^® , diluted with EtOAc (50 mL), washed with 1M aq . citric acid (30 mL) and brine (30 mL) and concentrated in vacuo. The residue was then redissolved in TFA (3 mL) and a llowed to stir at r.t. for 30 min. The reaction mixture was again concentrated in vacuo. The crude was purified by flash column chromatography (hexanes-EtOAc, 9 : 1→ 0 : 1) to g ive the title compound 6B (2.02 g, 65%) as a colorless oil. [a] _D ²³ - ⁴ -4.0 (c 0.2 in CHCb); Vmax(neat)/cm ¹ 2922, 2852, 1733, 1525, 1450, 1168, 1110; ^X H NMR (400 MHz, CDCb) δ 7.75 (2H, d, J = 7.5 Hz), 7.61 (2H, d, J = 7.4 Hz), 7.39 (2H, t, J = 7.4 Hz), 7.40 (2H, t, J = 7.4 Hz), 5.86 (1H, d, J = 7.5), 5.13-5.03 (1 H, m), 4.72-4.62 (1 H, m), 4.40 (2H, d, J = 6.9 Hz), 4.24 (1H, t, J = 7.0 Hz), 4.11 (2H, t, J = 6.5 Hz), 3.14 (1H, dd, J = 12.9, 3.5 Hz), 3.08 (1H, dd, J = 13.8, 4.4 Hz), 2.82-2.69 (2H, m), 2.31-2.26 (4H, m), 2.10-1.87 (2H, m), 1.65-1.54 (4H, m), 1.33-1.22 (48H, m), 0.89 (6H, t, J = 6.8 Hz); ¹³ C NM R (100 MHz, CDCb) δ 174.1, 173.7, 155.0, 143.7, 141.3, 127.8, 127.1, 125.2, 120.0, 69.7,

67.5, 60.5, 53.7, 47.1, 36.5, 34.8, 34.4, 34.3, 32.2, 32.0, 29.8, 29.7, 29.6, 29.4, 29.2, 25.0, 22.8, 14.1 ; HRMS (ESI + ) [M + H] ⁺ 908.6069 calc for Cs^seNOeS 908.6065, [M + Na] ⁺ 930.5888 calc for Cs^ssNNaOsS 930.5875.

Synthesis of compound 6B (Procedure B): Diastereomerically pure diol 103B was also converted to d iastereomerically pure conjugate 6B by following procedures analogous to the representative procedures described below.

Representative procedure for conversion of 103B to 201B

To a stirred solution of diol 103B (0.327 g, 0.67 mmol) and palmitic acid (0.516 g, 2.01 mmol) in THF (9 mL) at r.t. is added diisopropylcarbodiimide (0.414 mL, 2.68 mmol) and 4-dimethylaminopyrid ine (0.01 g, 0.07 mmol). The reaction mixture is allowed to stir at r.t. for 19 h. The mixture is then diluted with EtOAc (30 mL), filtered through a bed of Celite ^® and concentrated in vacuo. The crude is purified by flash column chromatography (CH2CI2) to g ive 201B as yellow oil. Representative procedure for conversion of 20 IB to 6B

A solution of diester 201B (0.35 g, 0.364 mmol) in trifluoroacetic acid (2 mL) is allowed to stir at r.t. for 1 h after which the mixture is concentrated in vacuo. The crude is purified by flash column chromatography (hexanes-EtOAc, 9 : 1→ 0 : 1 ) to give 6B as a colourless oil .

2. Example 2

This example demonstrates the synthesis of amino acid conjugates from va rious starting materials.

7.1 Synthesis of amino acid conjugate 806 from alcohol 800

Step i

To a stirred solution of 4-pentyn-l-ol 800 (5 mL, 53.72 mmol) in CH2CI2 ( 150 mL) at r.t. was added imidazole (3.66 g, 53.72 mmol) and terf-butyldimethylsilyl chloride (8.10 g, 53.72 mmol). The reaction mixture was a llowed to stir at r.t. for 24 h. The mixture was then diluted with Et2<D (200 mL) and washed with water (3 x 100 mL) and brine ( 100 mL) . The orga nic layer was dried over anhydrous Na2S04 and concentrated in vacuo. The crude was purified by filtration throug h silica gel to give 801 (10.64 g, quant.) as a colourless liq uid. Alkyne 801 was used in subsequent synthetic steps without characterisation.

To a stirred solution of alkyne 801 ( 14.08 g, 70.00 mmol) in hexanes (150 mL) at r.t. was added quinoline ( 11.75 mL, 100.00 mmol) and Lindar's catalyst ( 1.408 g). The reaction mixture was connected to a hh-filled balloon ( 1 atm) and allowed to stir at r.t. for 5 h. The mixture was then filtered through a pad of Celite ^® a nd concentrated in vacuo. The crude product was purified by flash column chromatography (petroleum ether-EtOAc, 9 : 1 ) to give 802 ( 14.09 g, 99%) as a colourless liquid .

R _f 0.88 (petroleum ether-EtOAc 9 : 1) ; δ _Η (400 M Hz; CDC ) 5.82 ( 1 H, ddt, J = 17.0, 10.2, 6.7 Hz, H-4), 5.02 ( 1 H, d, J = 17.1 Hz, H _a -5), 4.95 ( 1 H, d, J = 10.4 Hz, H _b -5), 3.62 (2H, t, J = 6.5 Hz, H-l), 2.10 (2H, q, J = 7.2 Hz, H-3), 1.61 (2H, p, J = 7.0 Hz, H-2), 0.90 (9H, s, SiC(CH3) ₃ ), 0.05 (6H, s, Si(CH ₃ ) ₂ ); 5c(100 MHz; CDC ) 138.6 (CH, C-4), 114.5 (CH ₂ , C-5), 62.6 (CH ₂ , C-l), 32.0 (CH ₂ , C-2), 30.5 (CH ₂ , C-3), 26.0 (3 x CH ₃ , SiC(CH ₃ )3), 18.4 (C, SiC(CH ₃ ) ₃ ), -5.3 (2 x CH ₃ , Si(CH ₃ ) ₂ ). Spectroscopic data were consistent with those reported in literature.

Step Hi

To a stirred solution of alkene 802 (8.646 g, 43.16 mmol) in CH2CI2 (100 mL) at r.t. was added mCPBA (8.191 g, 47.47 mmol). The reaction mixture was allowed to stir at r.t. for 15 h. The mixture was then filtered through Celite®, diluted with Et2<D (100 mL)and washed with sat. aq. NaHC0 ₃ (3 x 100 mL) and brine (100 mL). The organic layer was dried over anhydrous Na2S04 and concentrated in vacuo. The crude product was purified by flash column chromatography (petroleum ether-EtOAc, 9:1) to give 803 (8.09 g, 87%) as a colourless liquid.

R _f 0.51 (petroleum ether-EtOAc 9:1); δ _Η (400 MHz; CDCb) 3.70-3.60 (2H, m, H-l),

2.96-2.92 (1H, m, H-4), 2.75 (1H, dd, J = 5.0, 4.0 Hz, H-5), 2.47 (1H, dd, J = 5.0, 2.8 Hz, H-5), 1.73-1.53 (4H, m, H-2, H-3), 0.89 (9H, s, SiC(CH3) ₃ ), 0.04 (6H, s, Si(CH ₃ ) ₂ ); 5c(100 MHz; CDCb) 62.7 (CH2, C-l), 52.2 (CH, C-4), 47.1 (CH2, C-5), 29.1 (CH2, C-2), 29.0 (CH2, C-3), 25.9 (3 x CH ₃ , SiC(CH ₃ ) ₃ ), 18.3 (C, SiC(CH ₃ ) ₃ ), -5.3 (2 x CH ₃ ,

Si(CH ₃ )2). Spectroscopic data were consistent with those reported in literature.

Step iv

803a

To a stirred solution of racemic epoxide 803 (8.272 g, 38.24 mmol), (R,R)-(+)-/V,/V'- bis(3,5-di-terf-butylsalicylidene)-l,2-cyclohexanediaminocob alt(II) (0.121 g, 0.19 mmol) and glacial acetic acid (0.04 mL, 0.76 mmol) in THF (0.35 mL) at 0 °C was added water (0.38 mL) dropwise. The reaction mixture was allowed to stir at r.t. for 48 h. The mixture was then concentrated in vacuo. The crude product was purified by flash column chromatography (petroleum ether-EtOAc, 9:1) to give 803a (4.12 g, 49%) as a yellow oil.

Rf0.51 (petroleum ether-EtOAc 9:1); [a] _D ²¹ - ⁴ +4.65 (c 1.15 in CHCb); δ _Η (400 MHz;

CDCb) 3.61 (2H, t, J = 6.0 Hz, H-l), 2.93-2.88 (1H, m, H-4), 2.74 (1H, dd, J = 5.0, 4.0 Hz, H-5), 2.46 (1H, dd, J = 5.0, 3.0 Hz, H-5), 1.63-1.46 (4H, m, H-2, H-3), 0.89 (9H, s, SiC(CH3) ₃ ), 0.04 (6H, s, Si(CH ₃ ) ₂ ); 5c (100 MHz; CDC ) 63.0 (CH ₂ , C-l ), 52.3 (CH, C-4), 47.1 (CH ₂ , C-5), 32.6 (CH ₂ , C-2), 32.3 (CH ₂ , C-3), 26.0 (3 x CH ₃ , SiC(CH ₃ ) ₃ ), 18.4 (C, SiC(CH ₃ ) ₃ ), -5.3 (2 x CH ₃ , Si(CH ₃ )2) . Spectroscopic data were consistent with those reported in literature.

Step v

To a stirred solution of disulfide 804 (0.751 g, 0.94 mmol), which is commercia lly available, in CH2CI2 (5 mL) at 0 °C was added zinc powder (0.508 g, 7.78 mmol) a nd a freshly prepa red mixture of methanol, cone, hydrochloric acid and cone, sulfuric acid (100 : 7: 1, 2 mL). The resultant mixture was allowed to stir at 0 °C for 30 min. The mixture was then allowed to stir at 70 °C or 65 °C for 5 min after which was added epoxide 803a (0.839 g, 3.88 mmol) . The reaction mixture was a llowed to stir at 65 °C for 19 h. The mixture was then diluted with EtOAc (50 mL), filtered through a pad of Celite ^® and washed with brine (50 mL). The aqueous layer was extracted with EtOAc (3 x 50 mL) and the combined organic extracts were dried over anhydrous Na2S04 and concentrated in vacuo. The crude product was purified by flash column chromatography (hexanes-EtOAc, 1 : 3) to give 805 (0.568 g, 60%) as a colourless oil .

R _f 0.34 (hexane-EtOAc 1 : 3); [a] _D ^{21 0} -26.7 (c 0.03 in CHCb); VmaxineatVcm ¹ 3321, 2931, 1706, 1532, 1450, 1369, 1248, 1152, 1050; δ _Η (400 MHz; CHCb) 7.76 (2H, d, J = 7.5 Hz, FmocH), 7.61 (2H, d, J = 7.2 Hz, FmocH), 7.40 (2H, t, J = 7.4 Hz, FmocH), 7.31 (2H, t, J = 7.4 Hz, FmocH), 5.90 (1H, d, J = 7.8 Hz, NH), 4.51 (1H, dd, J = 12.3, 5.2 Hz, H-l ), 4.39 (2H, d, J = 7.1 Hz, FmocCH ₂ ), 4.23 (1 H, t, J = 7.1 Hz, FmocCH), 3.73-3.58 (3H, m, H-4, H-7), 3.03 (1 H, dd, J = 13.9, 4.4 Hz, H-2), 2.95 (1H, dd, J = 13.9, 5.7 Hz, H-2), 2.80 (1H, dd, J = 13.6, 2.9 Hz, H-3), 2.53 (1H, dd, J = 13.6, 8.9 Hz, H-3), 1.72- 1.61 (4H, m, H-5, H-6), 1.49 (9H, s, C(CH ₃ ) ₃ )); 5c (100 MHz; CHCb) 169.8 (C, C0 ₂ tBu), 156.1 (C, FmocCO), 143.9 (C, Fmoc), 141.1 (C, Fmoc), 127.9 (CH, Fmoc), 127.2 (CH, Fmoc), 125.3 (CH, Fmoc), 120.1 (CH, Fmoc), 83.2 (C, C(CH ₃ ) ₃ ), 70.1 (CH, C-4), 67.3 (CH2, FmocCH ₂ ), 62.8 (CH2, C-7), 54.7 (CH, C-l ), 47.2 (CH, FmocCH), 41.2 (CH2, C-3), 35.5 (CH2, C-2), 33.4 (CH2, C-5), 29.2 (CH2, C-6), 28.1 (3 x CH ₃ , C(CH ₃ ) ₃ ) ; HRMS

(ESI+) [M + Na] ⁺ 524.2077 calc for C27H ₃₅ N NaO _s S 524.2075.

Step vi

To a stirred solution of diol 805 (0.114 g, 0.243 mmol) and palmitic acid (0.180 g, 0.702 mmol) in THF (3 mL) at r.t. was added /V,/V'-diisopropylcarbod iimide (0.145 ml_, 0.936 mmol) and 4-dimethyla minopyridine (0.011 g, 0.094 mmol). The reaction mixture was allowed to stir at r.t. for 17 h. The mixture was then filtered through a pad of Celite ^® , diluted with EtOAc (30 mL), washed with 1M aq . citric acid (30 mL) and brine (30 mL) and concentrated in vacuo. The residue was then redissolved in TFA (3 mL) and a llowed to stir at r.t for 45 min. The reaction mixture was again concentrated in vacuo. The crude prod uct was purified by flash column chromatography (hexanes-EtOAc, 9: 1→ 0: 1) to give 806 (0.220 g, 98%) as a colourless oil.

Rf 0.15 (petroleum ether-EtOAc 1 : 1) ; [a] _D ²¹ - ³ + 10.0 (c 0.08 in CHCb); Vmaxtneatycnr ¹ 2919, 2851, 1723, 1521, 1521, 1221, 1108, 1054; δ _Η (400 MHz; CHCb) 7.76 (2H, d, J = 7.5 Hz, FmocH), 7.62 (2H, d, J = 7.4 Hz, FmocH), 7.39 (2H, t, J = 7.4 Hz, FmocH), 7.30 (2H, td, J = 11.2, 0.9 Hz, FmocH), 5.78 (1H, d, J = 7.6 Hz, NH), 5.04-4.95 (1H, m, H-4), 4.60 (1 H, dd, J = 12.2, 5.2 Hz, H-l), 4.38 (2H, d, J = 7.2 Hz, FmocCH ₂ ), 4.24 (2H, t, J = 7.1 Hz, FmocCH), 4.13-3.99 (2H, m, H-7), 3.16 (1H, dd, J = 13.9, 4.5 Hz, H-2), 3.04 (1H, dd, J = 14.0, 5.3 Hz, H-2), 2.78-2.70 (2H, m, H-3), 2.34-2.25 (4H, m, 2 x

PamCH2aa lkyl), 1.74-1.56 (8H, m, 2 x Pa mCH2pa lkyl, H-5, H-6), 1.32-1.22 (48H, m, 24 x PamCH ₂ alkyl), 0.88 (6H, t, J = 6.9 Hz, 2 x PamCH ₃ a lkyl); 5c (100 M Hz; CHCb) 174.3 (C, CO2H), 174.0 (C, PamCC ), 173.5 (C, PamCC ), 156.0 (C, FmocCO), 143.7 (C, Fmoc), 141.3 (C, Fmoc), 127.8 (CH, Fmoc), 127.1 (CH, Fmoc), 121.2 (CH, Fmoc), 120.0 (CH, Fmoc), 72.1 (CH, C-4), 67.5 (CH2, FmocCH ₂ ), 63.8 (CH2, C-7), 53.6 (CH, C-l), 47.1 (CH, FmocCH), 36.5 (CH2, C-3), 34.6 (CH2, PamCH ₂ aalkyl), 34.5 (CH2, PamCH ₂ aalkyl), 34.3 (CH2, C-2), 31.9 (2 x CH2, PamCH ₂ alkyl), 29.7-29.2 (21 x CH2, PamCH ₂ alkyl, C-5), 25.0 (2 x CH ₂ , Pa mCH2 a lkyl), 24.6 (CH ₂ , C-6), 22.7 (2 x CH ₂ , PamCH ₂ alkyl), 14.1 (2 x CH ₃ , PamCHsa lkyl); HRMS (ESI+) [M + Na] ⁺ 944.6045 calc for C55H ₈ 7N NaOsS 944.6028.

7.1.2 Synthesis of amino acid conjugate 811 from alcohol 807

Step i To a stirred solution of 5-hexen-l-ol 807 (5.00 mL, 41.64 mmol) in CH2CI2 (150 mL) at r.t. was added imidazole (2.86 g, 43.06 mmol) and terf-butyldimethylsilyl chloride (6.34 g, 42.06 mmol). The reaction mixture was allowed to stir at r.t. for 19 h. The mixture was then diluted with EtOAc (400 mL), washed with water (200 mL) and brine (200 mL), dried over anhyd rous Na2S04 and concentrated in vacuo. The crude product was purified by flash column chromatography (petroleum ether) to give 808 (8.846 g, quant.) as a colourless oil .

R _f 0.90 (petroleum ether-EtOAc 9: 1); δ _Η (400 MHz; CDC ) 5.81 (1H, ddt, J = 17.1, 10.1, 6.7 Hz, H-5), 5.00 (1H, dq, J = 17.2, 1.7 Hz, H _a -6), 4.94 (1 H, d, J = 10.5 Hz, H _b -6), 3.61 (2H, t, J = 6.2 Hz, H-l), 2.06 (2H, q, J = 7.1 Hz, H-4), 1.59-1.50 (2H, m, H-2), 1.47- 1.39 (2H, m, H-3), 0.89 (9H, s, SiC(CH3) ₃ ), 0.05 (6H, s, Si(CH ₃ ) ₂ ); 5c (100 MHz; CDCb) 139.0 (CH, C-5), 114.3 (CH2, C-6), 63.1 (CH2, C-l), 33.5 (CH2, C-4), 32.3 (CH2, C-2), 26.0 (3 x CH ₃ , SiC(CH ₃ ) ₃ ), 25.2 (CH2, C-3), 18.4 (C, SiC(CH ₃ ) ₃ ), -5.3 (2 x CH ₃ ,

Si(CH ₃ )2) . Spectroscopic data were consistent with those reported in literature. Step ii

To a stirred solution of alkene 808 (7.58 g, 35.35 mmol) in CH2CI2 (150 mL) at r.t. was added mCPBA (9.15 g, 53.05 mmol) portionwise. The reaction mixture was allowed to stir at r.t. for 18 h. The mixture was then diluted with Et2<D (200 mL), filtered through Celite ^® , washed with 2M aq . NaOH (200 mL) a nd brine (200 mL), dried over anhydrous Na2SC>4 and concentrated in vacuo. The crude product was purified by flash column chromatography (petroleum ether-EtOAc, 9: 1) to g ive 809 (6.91 g, 85%) as a colourless oil .

Rf 0.60 (petroleum ether-EtOAc 9: 1); δ _Η (400 MHz; CDCb) 3.61 (2H, t, J = 6.0 Hz, H-l), 2.93-2.88 (1H, m, H-5), 2.74 (1H, dd, J = 5.0, 4.0 Hz, H-6), 2.46 (1H, dd, J = 5.0, 3.0 Hz, H-6), 1.63-1.46 (6H , m, H-2, H-3, H-4), 0.89 (9H, s, SiC(CH3) ₃ ), 0.04 (6H, s, Si(CH ₃ ) ₂ ); 5c (100 MHz; CDCb) 63.0 (CH2, C-l), 52.3 (CH, C-5), 47.1 (CH2, C-6), 32.6 (CH2, C-4), 32.3 (CH2, C-2), 26.0 (3 x CH ₃ , SiC(CH ₃ ) ₃ ), 22.3 (CH2, C-3), 18.4 (C, SiC(CH ₃ ) ₃ ), -5.3 (2 x CH ₃ , Si(CH ₃ )2) . Spectroscopic data were consistent with those reported in literature.

Step Hi

809 809a To a stirred solution of racemic epoxide 809 (5.887 g, 25.56 mmol), (R,R)-(+ )-/V,/V'- bis(3,5-d i-terf-butylsalicylidene)-l,2-cyclohexanediaminocoba lt(II) (0.083 g, 0.13 mmol) and g lacial acetic acid (0.03 mL, 0.51 mmol) in THF (0.3 mL) at 0 °C was added water (0.253 mL) dropwise. The reaction mixture was allowed to stir at r.t. for 48 h. The mixture was then concentrated in vacuo. The crude product was purified by flash column chromatography (petroleum ether-EtOAc, 9: 1) to g ive 809a (2.913 g, 49%) as a yellow oil .

R _f 0.60 (petroleum ether-EtOAc 9: 1); [a] _D ²⁰ - ⁴ + 5.0 (c 0.02 in CHCb); δ _Η (400 MHz; CDCb) 3.61 (2H, t, J = 6.0 Hz, H-l), 2.93-2.88 (1H, m, H-5), 2.74 (1H, dd, J = 5.0, 4.0 Hz, H-6), 2.46 (1H, dd, J = 5.0, 3.0 Hz, H-6), 1.63-1.46 (6H, m, H-2, H-3, H-4), 0.89 (9H, s, SiC(CH3) ₃ ), 0.04 (6H, s, Si(CH ₃ ) ₂ ); 5c (100 MHz; CDCb) 63.0 (CH ₂ , C-l), 52.3 (CH, C-5), 47.1 (CH2, C-6), 32.6 (CH ₂ , C-4), 32.3 (CH ₂ , C-2), 26.0 (3 x CH ₃ , SiC(CH ₃ )3), 22.3 (CH ₂ , C-3), 18.4 (C, SiC(CH ₃ )3), -5.3 (2 x CH ₃ , Si(CH ₃ )2). Spectroscopic data were consistent with those reported in literature. Step iv

809a 804 810

To a stirred solution of disulfide 804 (0.500 g, 0.649 mmol) in CH2CI2 (5 mL) at 0 °C was added zinc powder (0.300 g, 4.54 mmol) and a freshly prepared mixture of metha nol, cone, hydrochloric acid and cone, sulfuric acid ( 100 :7 : 1, 2 mL). The resultant mixture was allowed to stir at 0 °C for 30 min . The mixture was then allowed to stir at 65 °C for 5 min after which was added epoxide 809a (0.600 g, 2.60 mmol). The reaction mixture was allowed to stir at 65 °C for 19 h. The mixture was then diluted with EtOAc (50 mL), filtered through a pad of Celite ^® and washed with brine (50 mL). The aqueous layer was extracted with EtOAc (3 x 50 mL) and the combined organic extracts were dried over anhydrous Na2SC>4 and concentrated in vacuo. The crude product was purified by flash column chromatography (hexanes-EtOAc, 4: 1→ 1 : 3) to give 810 (0.553 g, 83%) as a colourless oil .

Rf O.39 (hexane-EtOAc 1 : 3); [a] _D ²¹ - ² -25.0 (c 0.07 in CHCb); VmaxineatVcm ¹ 3343, 2934, 2862, 1705, 1513, 1450, 1369, 1344, 1248, 1152; 5H (400 MHz; CHCb) 7.76 (2H, d, J = 7.5 Hz, FmocH), 7.61 (2H, d, J = 7.0 Hz, FmocH), 7.40 (2H, t, J = 7.4 Hz,

FmocH), 7.30 (2H, td, J = 11.2, 1.1 Hz, FmocH), 5.88 (1H, d, J = 7.8 Hz, NH), 4.52 (1 H, dd, J = 12.5, 5.2 Hz, H-l), 4.39 (2H, d, J = 8.1 Hz, FmocCH ₂ ), 4.23 (1H, t, J = 7.1 Hz, FmocCH), 3.70-3.59 (3H, m, H-4, H-8), 3.03 (1 H, dd, J = 13.7, 4.7 Hz, H-2), 2.94 (1H, dd, J = 13.7, 5.4 Hz, H-2), 2.80 (1H, dd, J = 13.6, 3.4 Hz, H-3), 2.51 (1H, dd, J = 13.4, 8.7 Hz, H-3), 1.60-1.38 (15H, m, H-5, H-6, H-7, C(CH ₃ ) ₃ )); 5c (100 MHz; CHCb) 169.7 (C, C0 ₂ tBu), 156.0 (C, FmocCO), 143.8 (C, Fmoc), 141.3 (C, Fmoc), 127.8 (CH, Fmoc), 127.1 (CH, Fmoc), 125.2 (CH, Fmoc), 120.0 (CH, Fmoc), 83.1 (C, C(CH ₃ ) ₃ ), 69.8 (CH, C- 4), 67.2 (CH ₂ , FmocCH ₂ ), 62.5 (CH ₂ , C-8), 54.6 (CH, C-l), 47.1 (CH, FmocCH), 41.1 (CH ₂ , C-3), 35.8 (CH ₂ , C-5), 35.4 (CH ₂ , C-2), 32.4 (CH ₂ , C-7), 28.0 (3 x CH ₃ , C(CH ₃ ) ₃ ), 21.9 (CH ₂ , C-6) ; HRMS (ESI+ ) [M + Na] ⁺ 538.2226 calc for C28H ₃₇ N NaO _s S 538.2234.

Step v

810 811

To a stirred solution of diol 810 (0.190 g, 0.370 mmol) and palmitic acid (0.284 g, 1.10 mmol) in THF (3 mL) at r.t. was added /V,/V'-diisopropylcarbod iimide (0.226 ml_, 1.47 mmol) and 4-dimethyla minopyridine (0.018 g, 0.147 mmol). The reaction mixture was allowed to stir at r.t. for 17 h. The mixture was then filtered through a pad of Celite ^® , diluted with EtOAc (50 mL), washed with 1M aq . citric acid (30 mL) and brine (30 mL) and concentrated in vacuo. The residue was then redissolved in TFA (3 mL) and a llowed to stir at r.t. for 45 min. The reaction mixture was again concentrated in vacuo. The crude product was purified by flash column chromatography (hexa nes-EtOAc, 9 : 1→ 0: 1) to g ive 811 (0.301 g, quant.) as a colourless oil.

Rf 0.20 (petroleum ether-EtOAc 1 : 1) ; [a] _D ²¹ - ² + 10.0 (c 0.07 in CHCb); VmaxineatVcm ¹ 3331, 2917, 2850, 1728, 1692, 1532, 1467, 1451, 1244, 1221, 1198, 1175; 5H (400 MHz; CHCb) 7.76 (2H, d, J = 7.5 Hz, FmocH), 7.62 (2H, d, J = 7.2 Hz, FmocH), 7.40

(2H, t, J = 7.4 Hz, FmocH), 7.30 (2H, td, J = 11.2, 1.0 Hz, FmocH), 5.82 (1H, d, J = 7.9 NH), 5.03-4.92 (1H, m, H-4), 4.71-4.60 (1H, m, H-l ), 4.40 (2H, d, J = 7.0 Hz,

FmocCH ₂ ), 4.24 (1H, t, J = 7.1 Hz, FmocCH), 4.11-4.00 (2H, m, H-8), 3.15 (1H, dd, J = 13.9, 4.4 Hz, H-2), 3.04 (1H, dd, J = 13.8, 5.8 Hz, H-2), 2.78-2.65 (2H, m, H-3), 2.31 (2H, t, J = 7.6 Hz, PamCH ₂ aalkyl), 2.28 (2H, t, J = 7.6 Hz, PamCH ₂ aalkyl), 1.74-1.55 (8H, m, 2 x Pa mCH2pa lkyl, H-5, H-7), 1.45-1.17 (50H, m, 24 x PamCH ₂ alkyl, H-6), 0.88 (6H, t, J = 6.8 Hz, 2 x PamCHsalkyl); 5c (100 MHz; CHCb) 174.3 (C, CO2H), 174.0 (C, PamC0 ₂ ), 173.9 (C, PamCC ), 156.1 (C, FmocCO), 143.7 (C, Fmoc), 141.3 (C, Fmoc), 127.8 (CH, Fmoc), 127.1 (CH, Fmoc), 125.2 (CH, Fmoc), 120.0 (CH, Fmoc), 72.4 (CH, C- 4), 67.4 (CH2, FmocCH ₂ ), 64.0 (CH2, C-8), 53.6 (CH, C-l), 47.1 (CH, FmocCH), 36.6 (CH2, C-3), 34.6 (CH2, Pa mCH2aa lkyl), 34.5 (CH ₂ , PamCH ₂ aalkyl), 34.4 (CH ₂ , C-2), 32.7 (CH2, C-5), 32.0 (2 x CH2, PamCH ₂ alkyl), 29.7-29.3 (20 x CH ₂ , PamCH ₂ alkyl), 28.3 (CH2, C-7) , 25.0 (2 x CH2, PamCH _2p alkyl), 25.0 (2 x CH ₂ , PamCH _2p alkyl), 22.7 (2 x CH ₂ , PamChha lkyl), 21.7 (CH ₂ , C-6), 14.4 (2 x CH ₃ , PamCH ₃ a lkyl); HRMS (ESI+) [M + Na] ⁺ 958.6239 calc for CseHsgN NaOsS 958.6238.

7.1.3 Synthesis of amino acid conjugate 820 from alkene 814 A) Synthesis of alkene 814 from alcohol 812

Step i

812 813

To a stirred solution of 6-heptyn-l-ol 812 (3.33 mL, 26.75 mmol) in CH2CI2 (80 mL) at r.t. was added imidazole (1.76 g, 27.01 mmol) and terf-butyldimethylsilyl chloride (4.07 g, 27.01 mmol). The reaction mixture was allowed to stir at r.t. for 24 h. The mixture was then diluted with Et2<D (100 mL) a nd washed with water (3 x 100 mL) and brine (100 mL) . The orga nic layer was dried over anhydrous Na2S04 and concentrated in vacuo. The crude product was purified by filtration through silica gel to give alkyne 813 (5.68 g, quant.) as a colourless liquid . Alkyne 813 was used in subsequent synthetic steps without characterisation.

Step ii

813 814

To a stirred solution of alkyne 813 (5.34 g, 25.18 mmol) in hexanes (140 mL) at r.t. was added quinoline (4.18 mL, 35.26 mmol) and Lindar's catalyst (0.53 g). The reaction mixture was connected to a hh-filled balloon (1 atm) and allowed to stir at r.t. for 2 h. The mixture was then filtered through a pad of Celite ^® and concentrated in vacuo. The crude product was purified by flash column chromatography (petroleum ether-EtOAc, 9: 1) to give 814 (5.34 g, quant.) as a colourless liquid .

R _f 0.91 (petroleum ether-EtOAc 9: 1) ; δ _Η (400 M Hz; CDC ) 5.81 (1 H, ddt, J = 17.0, 10.3, 6.7 Hz, H-6), 4.99 (1H, dd, J = 17.0 Hz, H _a -7) 4.93 (1H, dd, J = 10.1 Hz, H&-7), 3.60

(2H, t, J = 6.6 Hz, H-l), 2.05 (2H, q, J = 7.0 Hz, H-5), 1.56-1.31 (6H, m, H-2, H-3, H- 4), 0.89 (9H, s, SiC(CH3) ₃ ), 0.05 (6H, s, Si(CH ₃ ) ₂ ); 5c (100 MHz; CDCb) 139.1 (CH, C-6), 114.2 (CH2, C-7), 63.2 (CH2, C-l), 33.8 (CH2, C-5), 33.7 (CH2, C-4), 28.7 (CH2, C-3), 26.0 (3 x CH ₃ , SiC(CH ₃ ) ₃ ), 25.3 (CH2, C-2), 18.4 (C, SiC(CH ₃ ) ₃ ), -5.3 (2 x CH ₃ ,

Si(CH ₃ )2). Spectroscopic data were consistent with those reported in literature.

B) Synthesis of alkene 814 from alcohol 815 Step i

HO ^" ^ ^ ^ - TBSO

815 816

To a stirred solution of 1,6-hexaned iol (815) (16.00 g, 135.39 mmol) in CH2CI2 (150 mL) at r.t. was added imidazole (9.22 g, 135.39 mmol) and tert-butyldimethylsilyl chloride (20.41 g, 135.39 mmol). The reaction mixture was allowed to stir at r.t. for 19 h. The mixture was then filtered, washed with H2O (100 mL) and brine (100 mL), dried over anhydrous Na2S04 and concentrated in vacuo. The crude product was purified by flash column chromatography (petroleum ether-EtOAc, 4: 1) to g ive 816 (25.13 g, 80%) as a colourless liq uid. Alcohol 816 was used in subsequent synthetic steps without characterisation.

Step ii

TBSO ^' TBSO

816 817

To a stirred solution of alcohol 816 (4.90 g, 21.10 mmol) in CH2CI2 (11 mL) at 0 °C was added dimethylsulfoxide (11.08 mL, 154.05 mmol), ΕΓ.3Ν (14.71 mL, 105.52 mmol) and sulfur trioxide pyridine complex (9.89 g, 63.31 mmol). The reaction mixture was allowed to stir at 0 °C for 30 min. The mixture was then q uenched with water (20 mL) and extracted with EtOAc (2 x 50 mL). The combined organic extracts were washed with water (50 mL) and brine (50 mL), dried over anhydrous Na2S04 and concentrated in vacuo. The crude product was purified by flash column chromatography (petroleum ether-EtOAc, 9 : 1) to give 817 (4.71 g, 97%) as a colourless oil. Aldehyde 817 was used in subsequent synthetic steps without cha racterization.

Step Hi

TBSO TBSO

817 814

To a stirred solution of methyltriphenylphosphonium bromide (4.60 g, 12.89 mmol) in THF (30 mL) at -78 °C was added a solution of n-butyllithium (7.16 mL, 1.8 M, 12.89 mmol) dropwise. The resulta nt mixture was warmed to r.t. a nd a llowed to stir for 1 h . The reaction mixture was then cooled to -78 °C and aldehyde 817 (2.56 g, 11.21 mmol) in THF (6 mL) was added dropwise. The reaction mixture was allowed to stir at -78 °C for 3 h and then warmed to r.t. and a llowed to stir for a further 15 h . The mixture was then quenched with sat. aq . N hUCI (10 mL) and extracted with EtOAc (3 x 70 mL). The combined organic extracts were washed with water (2 x 50 mL) and brine (50 mL), dried over a nhydrous Na2S04 and concentrated in vacuo. The crude product was purified by flash column chromatography (petroleum ether-EtOAc, 99: 1) to g ive 814(2.50 g, 98%) as a colourless liquid .

R _f 0.91 (petroleum ether-EtOAc 9: 1) ; δ _Η (400 M Hz; CDCb) 5.81 (1 H, ddt, J = 17.0, 10.3, 6.7 Hz, H-6), 4.99 (1H, dd, J = 17.0 Hz, H _a -7) 4.93 (1H, dd, J - 10.1 Hz, H&-7), 3.60 (2H, t, J = 6.6 Hz, H-l), 2.05 (2H, q, J = 7.0 Hz, H-5), 1.56-1.31 (6H, m, H-2, H-3, H- 4), 0.89 (9H, s, SiC(CH3) ₃ ), 0.05 (6H, s, Si(CH ₃ ) ₂ ); 5c (100 MHz; CDCb) 139.1 (CH, C-6), 114.2 (CH ₂ , C-7), 63.2 (CH ₂ , C-l), 33.8 (CH ₂ , C-5), 33.7 (CH ₂ , C-4), 28.7 (CH ₂ , C-3), 26.0 (3 x CH ₃ , SiC(CH ₃ ) ₃ ), 25.3 (CH ₂ , C-2), 18.4 (C, SiC(CH ₃ ) ₃ ), -5.3 (2 x CH ₃ ,

Si(CH ₃ ) ₂ ). Spectroscopic data were consistent with those reported in literature.

C) Synthesis of amino acid conjugate 820 from alkene 814

To a stirred solution of alkene 814 (4.30 g, 18.40 mmol) in CH ₂ CI ₂ (40 mL) at r.t. was added mCPBA (4.46 g, 25.84 mmol). The reaction mixture was a llowed to stir at r.t. for 7 h. The mixture was then filtered through Celite ^® , diluted with Et ₂ 0 (60 mL) and washed with sat. aq. NaHC0 ₃ (3 x 100 mL) and brine (100 mL). The orga nic layer was dried over anhydrous Na ₂ SC>4 and concentrated in vacuo. The crude product was purified by flash column chromatography (petroleum ether-EtOAc, 9: 1) to g ive 818 (4.30 g, 96%) as a colourless liq uid.

Rf 0.63 (petroleum ether-EtOAc 9: 1) ; 5H (400 M Hz; CDCb) 3.60 (2H, t, J = 6.5 Hz, H-l), 2.92-2.88 (1H, m, H-6), 2.74 (1H, t, J = 4.5 Hz, H-7), 2.46 (1H, dd, J = 5.0, 2.8 Hz, H- 7), 1.56-1.36 (8H, m, H-2, H-3, H-4, H-5), (9H, s, SiC(CH3) ₃ ), 0.04 (6H, s, Si(CH ₃ ) ₂ ); 5c (100 MHz; CDCb) 63.1 (CH ₂ , C-l), 52.3 (CH, C-6), 47.1 (CH ₂ , C-7), 32.8 (CH ₂ , C-5), 32.5 (CH ₂ , C-2), 26.0 (3 x CH ₃ , SiC(CH ₃ ) ₃ ), 25.8 (CH ₂ , C-4), 25.7 (CH ₂ , C-3), 18.4 (C, SiC(CH ₃ ) ₃ ), -5.3 (2 x CH ₃ , Si(CH ₃ ) ₂ ). Spectroscopic data were consistent with those reported in literature.

Step ii

818 818a

To a stirred solution of racemic epoxide 818 (2.23 g, 9.13 mmol), (R,R)-(+ )-/V,/V'- bis(3,5-d i-terf-butylsalicylidene)-l,2-cyclohexanediaminocoba lt(II) (0.03 g, 0.05 mmol) and g lacial acetic acid (0.01 mL, 0.18 mmol) in THF (0.1 mL) at 0 °C was added water (0.09 mL) dropwise. The reaction mixture was allowed to stir at r.t. for 48 h. The mixture was then concentrated in vacuo. The crude product was purified by flash column chromatography (petroleum ether-EtOAc, 9: 1) to g ive 818a (1.09 g, 49%) as a yellow oil .

R _f 0.63 (petroleum ether-EtOAc 9: 1) ; [a] _D ²¹ - ³ +4.2 (c 0.90 in CHC ) ; δ _Η (400 M Hz; CDCb) 3.60 (2H, t, J = 6.5 Hz, H-l), 2.92-2.88 (1H, m, H-6), 2.74 (1H, t, J = 4.5 Hz, H- 7), 2.46 ( 1H, dd, J = 5.0, 2.8 Hz, H-7), 1.56-1.36 (8H, m, H-2, H-3, H-4, H-5), (9H, s, SiC(CH3) ₃ ), 0.04 (6H, s, Si(CH ₃ ) ₂ ); 5c (100 MHz; CDCb) 63.1 (CH ₂ , C-l), 52.3 (CH, C-6), 47.1 (CH ₂ , C-7), 32.8 (CH ₂ , C-5), 32.5 (CH ₂ , C-2), 26.0 (3 x CH ₃ , SiC(CH ₃ ) ₃ ), 25.8 (CH ₂ , C-4), 25.7 (CH ₂ , C-3), 18.4 (C, SiC(CH ₃ ) ₃ ), -5.3 (2 x CH ₃ , Si(CH ₃ ) ₂ ). Spectroscopic data were consistent with those reported in literature.

Step Hi

NHFmoc

To a stirred solution of disulfide 804 (0.30 g, 0.375 mmol) in CH ₂ CI ₂ (1 mL) at 0 °C was added zinc powder (0.20 g, 3.01 mmol) and a freshly prepa red mixture of methanol, cone, hydrochloric acid and cone, sulfuric acid ( 100 :7 : 1, 1 mL). The resultant mixture was allowed to stir at 0 °C for 30 min after which was added epoxide 818a (0.344 g, 1.13 mmol). The reaction mixture was allowed to stir at 70 °C for 17 h. The mixture was then diluted with EtOAc (30 mL), filtered throug h a pad of Celite ^® and washed with brine (30 mL). The aqueous layer was extracted with EtOAc (3 x 30 mL) and the combined orga nic extracts were dried over a nhydrous MgSCU and concentrated in vacuo. The crude prod uct was purified by flash column chromatography (hexanes-EtOAc, 1 : 3) to give 819 (0.350 g, 88%) as a colourless oil.

Rf 0.4 (hexane-EtOAc 1 : 3) ; [a] _D ²⁰ - ⁸ -20.0 (c 0.03 in EtOAc); VmaxineatVcm ¹ 3365, 3933, 1703, 1514, 1450, 1369, 1343, 1248, 1151, 1046; δ _Η (400 MHz; MeOD) 7.79 (2H, d, J = 7.5 Hz, FmocH), 7.68 (2H, d, J = 7.4 Hz, FmocH), 7.39 (2H, t, J = 7.4 Hz,

FmocH), 7.31 (2H, t, J = 4.7 Hz, FmocH), 4.34 (2H, d, J = 7.1 Hz, FmocCH), 4.28 (1 H, dd, J = 8.2, 5.1 Hz, H-l), 4.23 (1H, t, J = 7.0 Hz, FmocCH ₂ ), 3.72-3.61 (1H, m, H-4), 3.57-3.79 (2H, m, H-9), 3.01 (1H, dd, J = 13.8, 5.0 Hz, H-2), 2.86 (1H, dd, J = 13.7, 8.3 Hz, H-2), 2.69 (1H, dd, J = 13.4, 4.9 Hz, H-3), 2.60 (1H, dd, J = 13.4 , 7.0 Hz, H-3), 1.57-1.34 (17H, m, H-5, H-6, H-7, H-8, C(CH ₃ ) ₃ ); 5c (100 MHz; MeOD) 171.8 (C, C0 ₂ tBu), 158.1 (C, FmocCO), 145.3 (C, Fmoc), 142.6 (C, Fmoc), 128.8 (CH, Fmoc), 128.2 (CH, Fmoc), 126.4 (CH, Fmoc), 121.0 (CH, Fmoc), 83.3 (C, C(CH ₃ ) ₃ ), 71.9 (CH, C- 4), 68.2 (CH ₂ , FmocCH ₂ ), 62.9 (CH ₂ , C-9), 56.5 (CH, C-l), 50.2 (CH, FmocCH), 40.8 (CH ₂ , C-3), 37.3 (CH ₂ , C-5), 35.5 (CH ₂ , C-2), 33.6 (CH ₂ , C-8), 28.3 (3 x CH ₃ , C(CH ₃ )3), 26.9 (CH ₂ , C-7), 26.6 (CH ₂ , C-6) ; HRMS (ESI + ) [M + Na ] ⁺ 552.2390 calc for

C29H ₃ 9N NaO _s S 552.2393.

Step iv

819 820

To a stirred solution of diol 819 (0.168 g, 0.317 mmol) and palmitic acid (0.244 g, 0.951 mmol) in THF (4.6 mL) at r.t. was added /V,/V'-diisopropylcarbod iimide (0.191 ml_, 1.269 mmol) and 4-dimethyla minopyridine (0.016 g, 0.127 mmol). The reaction mixture was allowed to stir at r.t. for 17 h. The mixture was then filtered through a pad of Celite ^® , diluted with EtOAc (30 mL), washed with 1M aq . citric acid (30 mL) and brine (30 mL) and concentrated in vacuo. The residue was then redissolved in TFA (3 mL) and a llowed to stir at r.t. for 45 min. The reaction mixture was again concentrated in vacuo. The crude product was purified by flash column chromatography (hexa nes-EtOAc, 9 : 1→ 0: 1) to g ive 820 (0.301 g, quant.) as a colourless oil.

Rf 0.21 (petroleum ether-EtOAc 1 : 1) ; [a] _D ²⁰ - ⁸ +7.5 (c 0.24 in CHC ) ; VmaxineatVcm ¹

3319, 2919, 2851, 1722, 1521, 1471, 1450, 1221, 1055; δ _Η (400 MHz; CDCb) 7.76 (2H, d, J = 7.6 Hz, FmocH), 7.61 (2H, d, J = 7.3 Hz, FmocH), 7.40 (2H, t, J = 7.7 Hz, FmocH), 7.30 (2H, td, J = 11.2, 1.1 Hz, FmocH), 5.82, (1 H, d, J = 7.7 Hz, NH), 5.00- 4.94 (1 H, m, H-4), 4.64 (1H, dd, J = 12.3, 5.6 Hz, H-l ), 4.40 (2H, d, J = 7.1 Hz, FmocCH ₂ ), 4.24 (1H, t, J = 7.1 Hz, FmocCH), 4.10-4.00 (2H, m, H-9), 3.14 (1H, dd, J = 13.8, 4.3 Hz, H-2), 3.04 (1H, dd, J = 13.8, 5.6 Hz, H-2), 2.76-2.67 (2H, m H-3), 2.31 (2H, t, J = 7.6 Hz, Pa mCH2aa l kyl), 2.28 (2H, t, J = 7.6 Hz, PamCH ₂ aalkyl), 1.65-1.56 (8H, m, 2 x Pa mCH2pa lkyl, H-8, H-5), 1.39-1.18 (52H, m, 24 x PamCH ₂ alkyl, H-6, H-7), 0.88 (6H, t, J = 6.9 Hz, 2 x PamCH ₃ alkyl); 5c (100 MHz; CDCb) 174.4 (C, CO2H), 156.1 (C, FmocCO), 143.7 (C, Fmoc), 141.3 (C, Fmoc), 127.8 (CH, Fmoc), 127.1 (CH, Fmoc),

125.2 (CH, Fmoc), 120.0 (CH, Fmoc), 72.4 (CH, C-4), 67.5 (CH2, FmocCH ₂ ), 64.2 (CH2, C-9), 53.6 (CH, C-l), 47.1 (CH, FmocCH), 36.5 (CH2, C-3), 34.6 (CH2, C-2), 34.3 (2 x CH2, Pa mCH2aa lkyl), 33.0 (CH ₂ , C-5), 31.9 (2 x CH ₂ , PamCH ₂ alkyl) 29.7-28.4 (21 x CH ₂ , PamCH ₂ a lkyl, C-8), 25.5 (CH2, C-7), 25.0 (2 x CH2, PamCH _2p alkyl), 24.8 (CH2, C-6), 22.7 (2 x CH2, PamCH ₂ alkyl), 14.1 (2 x CH ₃ , PamCH ₃ alkyl); HRMS (ESI+) [M + Na ] ⁺

972.6358 calc for C57H ₉ iN NaOsS 972.6392.

3. Example 3 This example investigates human TLR2 agonism of compounds of the present invention compared with a conjugate of a known TLR2 agonist ((R)-Parri2Cys).

3.1 Peptide conjugate synthesis

3.1.1 Synthesis of compounds 910, 911, 912, and 913 Procedure A

Peptide conj ugates of the invention 910, 911, 912 and 913 (depicted in Table 1) comprising the peptide sequence SKKKKSLLMWITQV [SEQ ID No: 139] were prepared as described and depicted below (Scheme 1).

The peptide sequence SKKKKSLLMWITQV [SEQ ID No: 139] includes an immunogenic peptide epitope (underlined), which is an analogue of sequence derived from the NY- ESO-1 protein (NY-ESO-1 157-165) and may be used to stimulate human NY-ESO-1- specific CD8+ T-cells as described in Chen, J-L et a l. The Journal of Immunology, 2000, 165, 948-955.

The SKKKK solubilising tag is believed to improve handling a nd ease the purification of the potentially lipophilic peptide.

900: R = CH ₃ (CH ₂ ) ₁₄ ; R ^a = Fmoc 910: R = CH ₃ (CH ₂ ) ₁₄ ; R ^a = H; * = R configuration

□ i"

901: R = CH ₃ (CH ₂ ) ₁₄ ; R ^a = H 911: R = CHJCHd-u ; R ^a = Ac; * = R configuration 902: R = CH ₃ (CH ₂ ) ₁₄ ; R ^a = Ac 912: R = CH ₃ (CH ₂ ) ₁₄ ; R ^a = H; * = S configuration

913: R = CH ₃ (CH ₂ ) ₁₄ ; R ^a = Ac; * = S configuration

Scheme 1. (i) Iterative Fmoc-SPPS; (ii) (R)- or (S)- bis-pamitoylated Fmoc-Cys-OH 6, PyBOP, collidine, DMF; (iii) 20% piperidine/DMF; (iv) AC2O/DMF/ N-Methylmorpholine; (v) TFA/EDT/water.

The desired peptide sequence was synthesised using standard iterative Fmoc Solid-Phase Peptide Synthesis techniq ues on a Tribute peptide synthesiser (Protein Technologies Internationa l, Tucson, AZ). A typical deprotection and coupling cycle carried out on a 0.1 mmol scale entailed removal of the Fmoc protecting group from the resin-bound amino-acid using two treatments of 20% piperidine in DMF (4mL x 5min) then washing the resin with DMF. In a separate vessel the Fmoc amino acid (0.5mmol) and coupling agent (1- [bis(dimethylamino)methylene]-lH-l,2,3-triazolo[4,5-b]pyridi nium 3-oxid

hexafluorophosphate (HATU), 0.45mmol) were dissolved in DMF (1.5 mL) and base (4- methylmorpholine (NMM), 1 mmol) added. After mixing for 1 minute, this solution was transferred to the resin, which was agitated at room temperature (RT) for 1 hour, drained and washed. Reverse phase (RP)-HPLC was carried out using a Dionex Ultimate 3000 HPLC system, with UV detection at 210nm or 225nm. For semi-preparative purifications, a peptide sample was injected into a reverse-phase Phenomenex Gemini C18 column (5μ, 110A; 10x250mm) equilibrated in a suitable mixture of eluent A (water/0.1% TFA) and eluent B (MeCN/0.1%TFA) then an increasing gradient of eluent B was generated to elute the constituent components. Analytical HPLC was performed similarly, using a Phenomenex Gemini C18 column (3μ, 110A; 4.6x150mm). Low-resolution mass spectra were obtained using an Agilent Technologies 6120 Quadrapole mass spectrometer. After coupling the penultimate amino acid residue, the resin-bound peptide chain was then derivatised with the desired diastereomer of amino acid conjugate 6 using BOP (benzotriazole-l-yl-oxy- tris-(dimethylamino)-phosphonium hexafluorophosphate) and collidine in DMF. The conditions for coupling of the amino acid conjugate reduce the propensity of the a-carbon of the amino acid to epimerise on activation. The amino acid conjugate (0.1 mmol) and BOP (0.095 mmol) were combined and dissolved in DMF (1.0 mL). Neat 2,4,6- trimethylpyridine (0.2 mmol) was added. After mixing for 30 seconds the solution was transferred to 0.02 mmol of resin, which was then agitated for 60 minutes, drained and washed (DMF) to afford 900.

The Fmoc group was then removed using 20% piperidine in DMF to provide 901.

The terminal amino group could then be acetylated using Acetic anhydride in DMF to afford 902. Resin (0.01 mmol) was suspended in DMF (2mL) acetic anhydride (0.1 mL) and /V-methylmorpholine (0.1 mL) were added, and the mixture agitated for 5 minutes. The resin was then drained and washed extensively with DMF.

Peptide 901 was cleaved from the resin to provide the peptide conjugate 910 with the R configuration at the indicated position and R ^a = H or the peptide conjugate 912 with the S configuration at the indicated position and R ^a = H (Scheme 1). Peptide 902 was cleaved from the resin to provide the peptide conjugate 911 with the R configuration at the indicated position and R ^a = Acetyl or the peptide conjugate 913 with the S configuration at the indicated position a nd R ^a = Acetyl (Scheme 1) . Resin (0.01 mmol) in 1.0 mL of trifluoroacetic acid containing 5% (v/v) 2,2'-(ethylened ioxy)dietha nethiol was agitated at room temperature for 2 hours. The supernata nt was then drained through a sinter into chilled diethyl ether (lOmL). The resin was then washed with a further 1 mL of TFA, which was also added to the ether. The precipitated material was pelleted by centrifugation a nd the pellet washed once with ether (5mL) before being dissolved in 1 : 1 MeCN/Water (+0.1%tfa) a nd lyophilised .

Purification of 910, 911, 912 and 913 was performed by semi-preparative HPLC using a Phenomenex Gemini C18 (5μ, 110A) 10x250mm column with eluent A being water (+0.1%tfa) and eluent B being MeCN (+0.1%tfa). After injection of the crude peptide sample on to the column the following grad ient was generated : 5%B to 50%B over 1.5 minutes followed by 50%B to 100%B over 23.5 minutes at a flow of 4mL/min. The desired product material collected on elution from the column and freeze-dried .

910: m/z (ESI) 1179.9 [M + 2H ⁺ ]. HPLC ana lysis: Column : Phenomenex Gemini C18 (3μ, 110A, 4.6 x 150 mm); eluent A, water/0.1%TFA; eluent B: MeCN/0.1%TFA; gradient: 5- 100%B over 25 min @ 1 mL/min. Retention time: 22.9 mins.

911 : m/z (ESI) 1201.0 [M + 2H ⁺ ]. HPLC ana lysis: Column : Phenomenex Gemini C18 (3μ, 110A, 4.6 x 150 mm); eluent A, water/0.1%TFA; eluent B: MeCN/0.1%TFA; gradient: 5- 100%B over 25 min @ 1 mL/min. Retention time: 24.3 mins. 912 : m/z (ESI) 1179.9 [M + 2H ⁺ ]. HPLC ana lysis: Column : Phenomenex Gemini C18 (3μ, 110A, 4.6 x 150 mm); eluent A, water/0.1%TFA; eluent B: MeCN/0.1%TFA; gradient: 5- 100%B over 30 min @ 1 mL/min. Retention time: 22.8 mins.

913 : m/z (ESI) 1200.9 [M + 2H ⁺ ]. HPLC ana lysis: Column : Phenomenex Gemini C18 (3μ, 110A, 4.6 x 150 mm); eluent A, water/0.1%TFA; eluent B: MeCN/0.1%TFA; gradient: 5- 100%B over 30 min @ 1 mL/min. Retention time: 24.1 mins.

No. Structure

911 SKKKKSLL W ITQV

Table 1: Structures of peptide conjugates 910-913. All structures comprise the peptide sequence SKKKKSLLMWITQV [SEQ ID No: 139]

Procedure B

Peptide conj ugates of the invention 910, 911, 912 and 913 (depicted in Table 1) were also prepa red by the following alternative procedure.

Fmoc-amino acids were supplied with the following side-chain protection : Fmoc-Ala-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Cys(Trt)-OH, Fmoc- Gln(Trt)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Gly-OH, Fmoc-His(Trt)-OH,

Fmoc-Ile-OH, Fmoc-Leu-OH,Fmoc-Lys(Boc)-OH, Fmoc-Met-OH,Fmoc-Phe-OH, Fmoc-Pro- OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Tyr(OtBu)-OH, Fmoc-Va l-OH .

Purification was performed using semi-preparative Reverse Phase (RP) High-Performa nce Liquid Chromatography (HPLC) using a column and running a gradient as described per procedure for each peptide. Both semi-preparative a nd a nalytical RP-HPLC were conducted using a Dionex U ltimate 3000 HPLC system with the described solvent system. The purest fractions were combined a nd lyophilised . Liquid chromatography-mass spectrometry (LC-MS) chromatog rams were acquired on an Ag ilent 1120 Compact LC system with a Hewlett Packard series 1100 MSD mass spectrometer.

Preparation of peptidyl resin 390 comprising amino acid residue: H ₂ N-SKKKKSLLMWITQV ,0.

Tentagel resin

Tentagel resin (0.581 g, 0.25 mmol scale) was swollen in DMF (6 mL) for 10 min. Fmoc- L-Val-OCH2-pC _s H ₄ -OCH2CH2C02H (0.155 g, 0.30 mmol) and HATU (0.114 g, 0.30 mmol) were dissolved in DMF (3 mL) a nd activated with /V-methylmorpholine (0.055 mL, 0.50 mmol) for 1 min. The resultant mixture was added to the resin and agitated for 1 h at r.t.. The resin was d ra ined a nd washed with DM F (2 x 3 mL). The resin was then treated with 20% v/v piperidine in DM F (2 mL) and agitated for 10 min at r.t.. The resin was drained and the process repeated . The resin was drained and washed with DMF (4 x 3 mL) . The resin was transferred to a Tribute ^® automated peptide synthesiser reaction vessel . The peptide was then elongated using a Tribute ^® automatic peptide synthesiser. Automated synthesis was performed by alternating cycles Fmoc-AA-OH coupling and Fmoc deprotection. The resin was washed with DMF (5 x 4 mL) prior to addition of the coupling mixture, which consisted of Fmoc-AA-OH (5 eq .) and HATU (4.75 eq .) in DMF (2.5 mL) and 2M /V-methylmorpholine in DM F ( 1.5 mL). Upon completion of coupling after ag itation for 1 h at r.t., the resin was drained and washed with DMF (5 x 4 mL). The resin was treated with AC2O (4 mL) and agitated for 10 min at r.t.. The resin was drained, washed with DMF (5 x 4 mL), treated with 20% v/v piperidine in DMF (4 mL) and ag itated for 10 min. The resin was drained and the process was repeated . The next cycle of washing, coupling and deprotection was repeated until all amino acids in the sequence were coupled. The resin was then washed with DMF (2 x 4 mL) and CH2CI2 (2 x 4 mL) to give the peptidyl resin 390 (0.957 g, 0.261 mmol g ¹ loading).

Synthesis of lipidated peptides 910, 911, 912 and 913:

Peptidyl resin 390 (0.038 g, 0.01 mmol scale) was swollen in DMF (2 mL) for 10 min . Fmoc-(S)-/7omoPa m2Cys-OH (6B), for the preparation of 910 and 911, or Fmoc-(fi)- /7omoParri2Cys-OH (6A), for the preparation of 912 and 913, (0.045 g, 0.5 mmol) and PyBOP (0.025 g, 0.0475 mmol) were dissolved in DMF (1 mL) and activated with 2,4,6- collidine (0.013 mL, 0.10 mmol) for 1 min. The resultant mixture was added to the resin and agitated for 1 h at r.t. to give the protected peptidyl resin. The resin was drained and washed with DMF (3 x 2 mL), a subsequent N inhydrin test was negative. The resin was then treated with 20% v/v piperidine in DMF (2 mL) and agitated for 10 min at r.t.. The resin was drained and the process repeated. The resin was dra ined, washed with DM F (3 x 2 mL) a nd CH2CI2 (3 x 2 mL). The resin was then either (a), for the preparation of 911 and 913, treated with AC2O (2 mL) and agitated for 10 min at r.t., then drained and washed with CH2CI2 (2 mL); or (b), for the preparation of 910 and 913, drained and washed with CH2CI2 (2 mL). The peptidyl resin was cleaved and precipitated as follows to give the crude peptide. The resin-bound peptide was treated with a cleavage cocktail of 5% DODT:TFA and agitated for 3 h at r.t.. The resin was separated from the cleavage cocktail solution by filtration, and the filtrate was treated with cold diethyl ether to precipitate the crude peptide followed by centrifugation at 4000 rpm for 5 min. The supernata nt was discarded a nd the pellet washed with diethyl ether and centrifugation was repeated once more. Upon discarding the supernatant, the resulting peptide pellet was dried under a flow of N2 and lyophilised from MeCN : H ₂ 0 (1 : 1) + 0.1% TFA.

The crude peptide was purified by semi-preparative RP-HPLC on a Phenomenex Gemini C18 column (5 μ, 110 A, 10.0 x 250 mm) running a gradient of 5-50% (45% MeCN per min) and then 50-100% (2% MeCN per min) MeCN in H2O + 0.1% TFA at r.t. The purified peptides 910, 911, 912, and 913 were obtained as amorphous solids.

910: (6 mg, 22% with >98% purity) . Rt 22.9 min on a Phenomenex Gemini C18 3μ 110A 4.6 x 150 mm column using a 5-100% MeCN : H ₂ 0 + 0.1% TFA, 3.8% MeCN per min gradient; LRMS (ESI+) [M + 2H] ²⁺ 1179.8 calc for C117H210N21O24S2 1179.9.

911: (3 mg, 12% with >98% purity) . Rt 24.2 min on a Phenomenex Gemini C18 3μ 110A 4.6 x 150 mm column using a 5-100% MeCN : H ₂ 0 + 0.1% TFA, 3.8% MeCN per min gradient; LRMS (ESI+) [M + 2H] ²⁺ 1201.1 calc for C119H212N21O25S2 1201.3.

912: (5 mg, 26% with >98% purity) . Rt 22.7 min on a Phenomenex Gemini C18 3μ 110A 4.6 x 150 mm column using a 5-100% MeCN : H ₂ 0 + 0.1% TFA, 3.8% MeCN per min gradient; LRMS (ESI+) [M + 2H] ²⁺ 1179.8 calc for C117H210N21O24S2 1179.9.

913: (3 mg, 12% with >98% purity) . Rt 24.2 min on a Phenomenex Gemini C18 3μ 110A 4.6 x 150 mm column using a 5-100% MeCN : H ₂ 0 + 0.1% TFA, 3.8% MeCN per min gradient; LRMS (ESI+) [M + 2H] ²⁺ 1201.1 calc for C119H212N21O25S2 1201.3. 3.1.2 Synthesis of compounds 930, 931, and 932

Procedure A

Peptide conj ugates of the invention 930, 931 and 932 (depicted in Table 2) comprising the peptide sequence SKKKKSLLMWITQV [SEQ ID No: 139] were prepared as described and depicted below (Scheme 2). resin + linker

H ₂ N- SKKKKSLLMWITQV - amino acid

920: R = CH ₃ (CH ₂ ) ₁₄ ; R ^a = Fmoc 930: R = CH ₃ (CH ₂ ) ₁₄ ; m = 2

921:R = CH ₃ (CH ₂ ) ₁₄ ;R ^a = H 931:R = CH ₃ (CH ₂ ) ₁₄ ;m = 3

932: R = CH ₃ (CH ₂ ) ₁₄ ; m = 4

Scheme 2. (i) Iterative Fmoc-SPPS; (ii) (R)-bis-pamitoylated Fmoc-Cys-OH 806 (m or 811 (m = 3) or 820 (m=4), PyBOP, collidine, DMF; (iii) 20% piperidine/DMF; (iv) TFA/EDT/ water.

The desired peptide sequence was synthesised using standard iterative Fmoc SPPS techniques as described above.

After coupling the penultimate amino acid residue, the resin-bound peptide chain was then derivatised with either conjugate 806, 811 or 820 using PyBOP (benzotriazol-l-yl- oxytripyrrolidinophosphonium hexafluorophosphate) and collidine in DMF. The conditions for coupling of the amino acid conjugate reduce the propensity of the a-carbon of the amino acid to epimerise on activation. The amino acid conjugate (0.1 mmol) and BOP (0.095 mmol) were combined and dissolved in DMF (1.0 ml_). Neat 2,4,6- trimethylpyridine (0.2 mmol) was added. After mixing for 60 seconds the solution was transferred to 0.02 mmol of resin, which was then agitated for 60 minutes, drained and washed (DMF) to afford 920 (m = 2, 3, or 4).

The Fmoc group was then removed using 20% piperidine in DMF to provide 921 (m = 2, 3, or 4).

Peptide 921 (m = 2, 3, or 4) was cleaved from the resin to provide the peptide conjugate 930 (m = 2) with the R configuration at the indicated position (Scheme 2), the peptide conjugate 931 (m = 3) with the R configuration at the indicated position, or peptide conjugate 932 (m = 4) with the R configuration at the indicated position. Resin (0.01 mmol) in 1.0 mL of trifluoroacetic acid containing 5% (v/v) 2,2'- (ethylenedioxy)diethanethiol was agitated at room temperature for 2 hours. The supernatant was then drained through a sinter into chilled diethyl ether (lOmL). The resin was then washed with a further 1 mL of TFA, which was also added to the ether. The precipitated material was pelleted by centrifugation and the pellet washed once with ether (5mL) before being dissolved in 1:1 MeCN/Water ( + 0.1%tfa) and lyophilised. Purification of 930, 931 and 932 was performed by semi-preparative HPLC using a Phenomenex Gemini C18 (5μ, 110A) 10x250mm column with eluent A being water (+0.1%tfa) and eluent B being MeCN (+0.1%tfa). After injection of the crude peptide sample on to the column the following grad ient was generated : 5%B to 45%B over 3 minutes followed by 45%B to 65%B over 16 minutes at a flow of 4mL/min. The desired prod uct material collected on elution from the column and freeze-dried .

930: m/z (ESI) 1186.6 [M + 2H ⁺ ]. HPLC ana lysis: Column : Phenomenex Jupiter C18 (5μ, 300A, 4.6 x 150 mm); eluent A, water/0.1%TFA; eluent B: MeCN/0.1%TFA; gradient: 5- 95%B over 30 min @ 1 mL/min. Retention time: 25.3 mins. 931 : m/z (ESI) 1193.6 [M + 2H ⁺ ]. HPLC ana lysis: Column : Phenomenex Jupiter C18 (5μ, 300A, 4.6 x 150 mm); eluent A, water/0.1%TFA; eluent B: MeCN/0.1%TFA; gradient: 5- 95%B over 30 min @ 1 mL/min. Retention time: 25.7 mins.

932 : m/z (ESI) 1200.8 [M + 2H ⁺ ]. HPLC ana lysis: Column : Phenomenex Jupiter C18 (5μ, 300A, 4.6 x 150 mm); eluent A, water/0.1%TFA; eluent B: MeCN/0.1%TFA; gradient: 5- 95%B over 30 min @ 1 mL/min. Retention time: 26.2 mins.

No. Structure

ITQV

Table 2: Structures of peptide conjugates 930-932. All structures comprise the peptide sequence SKKKKSLLMWITQV [SEQ ID No: 139]

Procedure B Peptide conj ugates of the invention 930, 931 and 932 (depicted in Table 2) were a lso prepared by the following alternative proced ure.

Fmoc-amino acids, purification, and LCMS were as set forth in Procedure B of section 3.1.1 of this Example. Synthesis of lipidated peptides 930, 931, and 932:

Peptidyl resin 390, prepared as set forth in Procedure B of section 3.1.1 of this Example, (0.083 g, 0.02 mmol scale) was swollen in DMF (2 mL) for 10 min. Amino acid 806 (0.092 g, 0.10 mmol), 811 (0.093 g, 0.10 mmol), or 820 (0.095 g, 0.10 mmol), for the preparation of 930, 931, or 932, respectively, and BOP (0.042 g, 0.095 mmol) were dissolved in DMF (2 mL) and activated with collidine (0.026 mL, 0.20 mmol) for 1 min. The resulta nt mixture was added to the resin a nd agitated for 1 h at r.t. to give the protected peptidyl resin . The resin was dra ined and washed with DMF (3 x 2 mL), a subsequent N inhydrin test was negative. The resin was then treated with 20% v/v piperidine in DMF (2 mL) a nd ag itated for 10 min at r.t.. The resin was drained and the process repeated. The resin was drained, washed with DMF (3 x 2 mL) and CH2CI2 (3 x 2 mL) .

The peptidyl resin was cleaved and precipitated as follows to give the crude peptide. The resin-bound peptide was treated with a cleavage cocktail of 5% DODT:TFA and agitated for 3 h at r.t.. The resin was separated from the cleavage cocktail solution by filtration. All volatiles were removed using a flow of N2 a nd the resulting residue was lyophilised from MeCN : H20 (1 : 1) + 0.1% TFA.

The crude peptide was purified by semi-preparative RP-HPLC on a Phenomenex Gemini C18 column (5 μ, 110 A, 10.0 x 250 mm) running a gradient of 5-50% (45% MeCN per min) and then 50-100% (2% MeCN per min) MeCN in H2O + 0.1% TFA at r.t.. The purified peptides 930, 931, and 932 were obtained as amorphous solids.

930: (8 mg, 17% with >98% purity). Rt 20.9 min on a Phenomenex Gemini C18 3μ 110A 4.6 x 150 mm column using a 5-100% MeCN : H ₂ 0 + 0.1% TFA, 3.8% MeCN per min gradient; LRMS (ESI+) [M + 2H] ²⁺ 1187.1 calc for C118H213N21O24S2 1186.6.

931: (9 mg, 19% with >98% purity) . Rt 25.7 min on a Phenomenex Gemini C18 3μ 110A 4.6 x 150 mm column using a 5-100% MeCN : H ₂ 0 + 0.1% TFA, 3.8% MeCN per min gradient; LRMS (ESI+) [M + 2H] ²⁺ 1194.1 calc for C119H215N21O24S2 1193.6.

932 : (9 mg, 19% with >98% purity). Rt 25.7 min on a Phenomenex Gemini C18 3μ 110A 4.6 x 150 mm column using a 5-100% MeCN : H ₂ 0 + 0.1% TFA, 3.8% MeCN per min gradient; LRMS (ESI+) [M + 2H] ²⁺ 1201.1 calc for C120H217N21O24S2 1200.8. 3.1.3 (R)-Pam2Cys-SK ₄ -SLLMWITQV

Peptide conj ugate (R)-Pam2Cys-SK4-SLLMWITQV was prepared using methods analogous to those described herein above. 3.2 Toll-like Receptor 2 (TLR2) agonism using Hek-Blue cells

HEK-Blue™ Detection medium and HEK-Blue™-hTLR2 cells were purchased from

Invivogen. HEK-Blue™ cells express endogenous levels of huma n (h)TLRl and hTLR6, and exhibit co-transfection of hTLR2 and the reporter gene SEAP (secreted embryonic alka line phosphatase). The SEAP reporter gene is under the control of the IFN-β minimal promoter fused to five AP-1 a nd five N FKB binding sites. Cells were cultured according to manufacturer's instructions.

On the day of the assay, TLR agonists : 910; 930; 931 ; 932; (R)-Pam 2Cys-SK4-

SLLMWITQV; or PBS (negative control) were plated in 20μΙ of endotoxin free water in a 96-well plate. All constructs tested were produced in-house as described above. HEK- Blue™-hTLR2 cells were resuspended at ~2.78xl0 ⁵ cells/ml in HEK-Blue™ Detection medium and 180μΙ of the cell suspension added to each well (~5xl0 ⁴ cells) to give final agonist concentrations as indicated across a 7-logio dilution series (10 ^s M to 10 ^~12 M). Cells were incubated for 16h at 37°C in 5% CO2. SEAP expression was quantified using an EnSpire plate reader (PerkinElmer) at 635nM . Data presented as mean +/- SD ABS (635nm) values for triplicate wells following background subtraction.

Results

The results are shown in Figure 1. All constructs analysed exhibited hTLR2 agonism at concentrations ra nging from 10 ^s to 10 ^~n M . Constructs 930 (grey ba rs), 931 (striped bars), and 932 (square hatched bars) exhibited comparable human TLR2 agonism to 910 (dotted white bars) and to Pam 2Cys-SKKKK-SLLMWITQV (black bars) at each concentration (10 ^s to 10 ^~n M), demonstrating that homologation extension does not inhibit binding to, or signalling through human TLR2 by diacylated lipopeptide constructs. 4. Example 4

This example investigates human TLR2 agonism of compounds of the present invention compared with conj ugates of known TLR2 agonists ((R)-Pam2Cys, (S)-Pam2Cys, (R)- Pam3Cys, and (S)-Pam3Cys). 4.1 Peptide conjugates

The peptide conjugates tested are listed in Tables 3 and 4 below. Peptide conj ugates 910, 911, 912, 913, 930, 931, and 932 were prepared as described above in Example 3. Peptide conjugates 45a, 45b, 46a, 46b, 47a, 47b were prepa red using methods ana logous to those described herein above. 4.2 Toll-like Receptor 2 (TLR2) agonism using Hek-Blue cells

Human TLR2 agonism by the peptide conjugates was investigated using H EK-Blue™- hTLR2 cells and Hek-Blue™ Detection medium (Invivogen) across an 8-logio dilution series (10 ⁹ fM to 10 ² fM). Agonists were diluted and incubated with HEK-Blue™-hTLR2 cells for 16h, and well a bsorbance (ABS) then determined at 655nm using an Ultrama rk™ microplate reader (BioRad). EC50 (nM) values were determined by non-linea r regression curve fit of normalised ABS (655nm) va lues using Prism 7 software (GraphPad).

Results

EC50 va lues were determined for ?omoPam2Cys constructs 910, 911, 912 and 913 and compared with the EC50 values for corresponding Pam2Cys constructs 45a and 45b, N- acetylated Pam2Cys constructs 46a and 46b and Pam3Cys constructs 47a and 47b

(Table 3). The results for the hTLR agonism assay are shown in Figure 2A. All constructs exhibited hTLR2 agonism. HomoPam2Cys constructs demonstrated comparable activity to the corresponding Pam2Cys and Pam3Cys constructs. (S)- ?omoPam2Cys constructs 912 and 913 showed improved activity over the (S)-Pam2Cys constructs 45a and 46a as well as comparable activity to the (R)-Parri3Cys construct 47b.

No. Structure ECso (nM)

22.959b C _1fi H _ai ^O O^NH

O C15H31

O 46.1562 ¹ Y O O^O NH 2

C15H31

Table 3: Structures of peptide conjugates 45a, 45b, 46a, 46b, 47a, 47b, 910-913 and EC so for human TLR2 agonism as determined by HEK-Blue™ assay. X = -SKKKKSLLMWITQV [SEQ ID No: 139].

ECso values were then determined for chain extended constructs 930, 931 and 932 and compared with the EC50 values for ?omoPam2Cys construct 910 and corresponding Pam2Cys construct 45b (Table 4). The hTLR agonism assay results are shown in Figure 2B. All constructs analysed exhibited significant hTLR2 agonism. Constructs 930 and 931 had similar EC50 values to 910 and 45b. Construct 932 exhibited slightly reduced activity compared with construct 910.

No. Structure ECso (nM)

Table 4: Structures of peptide conjugates 45b, 910, 930-932 and ECso for human TLR2 agonism as determined by HEK-Blue™ assay. X = -SKKKKSLLMWITQV

[SEQ ID No: 139] .

5. Example 5 This example investigates murine TLR2 agonism of compounds of the present invention compared with conj ugates of known TLR2 agonists ((R)-Pam2Cys, (S)-Pam2Cys, (R)- Pam3Cys, and (S)-Pam3Cys).

5.1 Peptide conjugates

The peptide conjugates tested are listed in Tables 3 and 4 above. Peptide conjugates 910, 911, 912, 913, 930, 931, and 932 were prepared as described above in Example 3. Peptide conjugates 45a, 45b, 46a, 46b, 47a, 47b were prepa red using methods ana logous to those described herein above.

5.2 Toll-like Receptor 2 (TLR2) agonism using Hek-Blue cells

Murine TLR2 agonism by the peptide conjugates was investigated using HEK-Blue™- mTLR2 cells and Hek-Blue™ Detection medium (Invivogen) across an 8-logio dilution series (10 ⁹ fM to 10 ² fM) as described above in Example 4.

Results

Results for homoPam2Cys constructs 910, 911, 912 and 913 compared with correspond ing Pam2Cys constructs 45a and 45b, N-acetylated Pam2Cys constructs 46a and 46b and Pam3Cys constructs 47a and 47b (structures depicted in Table 3) are presented in Figure 3A. All constructs exhibited murine TLR2 agonism.

Results for cha in extended constructs 930, 931 and 932 compared with ?omoPam2Cys construct 910 and corresponding Pam2Cys construct 45b (structures depicted in Table 4) a re presented in Figure 3B. All constructs analysed exhibited murine TLR2 agonism. It is not the intention to limit the scope of the invention to the abovementioned examples only. As would be appreciated by a skilled person in the art, many va riations are possible without departing from the scope of the invention.