Field of the Invention

The present invention relates to reagents for use in site-selective modification of proteins. Methods of conjugating proteins to a second entity using a chemoenzymatic method with a sortase enzyme are also described.

Background of the Invention

The development of recombinant protein technology has provided large scale, commercial production of a plethora of recombinant proteins, products for a variety of applications. However, many applications require the recombinant proteins to be further modified by conjugation to a modifying agent in order to optimize function. For example, protein- plasma half-life and/or immunogenicity may be improved by conjugation of the protein to biologically benign polymers such as polyethylene glycol (PEG), polyglutamate or polyvinylpyrolidone.

For further applications, the recombinant protein may represent a single function of a multi-functional product. There are many examples of such products in the art, including protein-drug conjugates for targeted drug delivery; protein-label conjugates for targeted imaging; protein-protein conjugates wherein each of the constituent recombinant proteins provides a specific function, for example, antibody-toxin conjugates have been developed for targeted anticancer products, or antibody-directed enzyme therapy. In yet other examples, there may be a requirement for a recombinant protein to be conjugated to living cells. Such products may have use in targeted stem cell therapies.

Some methods of modifying proteins are not site-selective and therefore modification occurs at one or more sites of the protein where there is suitable functionality for modification to occur. This suitable functionality may occur in or close to the site required for protein function and therefore modification results in reduced protein functional performance or inactivation of the protein. Furthermore, often modification using non- site-selective methods results in a mixture of modified proteins where modification occurs at different sites in the protein or a combination of different sites in the protein.

The advantages of site-selective modification methods include modification at a site distal to the site responsible for protein function and therefore the modified protein retains functional performance and modification at a single controlled site results in the production of a single product which has advantages in quality, safety and registrability of the modified protein product. Current coupling approaches for site-selective conjugation are based on the introduction of unique functional groups such as ketones and azides into proteins not present in natural amino acids. They can be incorporated into proteins by chemical modification of a protein's N-terminus or C-terminus, unnatural amino acid mutagenesis or by the use of enzymes that transfer prosthetic groups to proteins.

An example of site-selective protein modification has been described in US 5,824,784 in which the N terminus of a recombinant protein can be selectively modified with a modifying agent that contains an aldehyde moiety. In order for the conjugation to selectively modify the N-terminus of the protein, the reductive amination which achieves the conjugation must be carried out at a sufficiently acidic pH to ensure that only the lysine side chain epsilon amino groups are protonated.

Another method involves the use of the Staphylococcus aureus enzyme Sortase A (WO 2010/087994). By inclusion of the LPXTG motif in a recombinant protein and by providing a label or second protein with the required complimentary polyglycine nucleophile, the Sortase enzyme will under some circumstances provide the conjugation of the two component moieties.

However, these described methods face practical limitations in terms of feasibility, scalability and efficacy. Summarv of the Invention

The present invention is predicated in part on the discovery that protein modification can be achieved in good yield by sortase conjugation of a LPXTG modified protein with a functionalized GGG peptide moiety, for example, a GGG peptide moiety comprising a spacer group or steric hinderance near the GGG moiety and a functional group for further attachment of the peptide to another moiety, comprising a complimentary functional group.

In a first aspect, there is provided a method of preparing a compound of formula (II): wherein

A is a protein, a peptide, a label, a radioisotope, a small molecule drug, a targeting molecule, a nanoparticle, a cell, a dendrimer, a photosensitizer, DNA, R A, a carbohydrate, a contrast agent, a catalyst, a lipid or a virus particle,

X _a is selected from a leucine, serine, asparagine, valine, isoleucine, tyrosine and glutamine residue;

selected from a proline, alanine, serine and glycine residue;

X _c is any amino acid residue;

X _d is selected from a threonine, arginine, serine, alanine and glycine residue;

G is a glycine residue;

Xi is absent or is an amino acid residue or a peptide of 2-10 amino acid residues; X ₂ is absent or is an amino acid residue with a reactive functional group in its side chain;

Yi is absent or is a spacer group;

Zi is a reactive functional group; and

n is 0 or an integer of 1 to 4;

said method comprising contacting a compound of formula (I):

Ri-HN-G(G) _n -XiX ₂ YiZi (I) wherein

Ri is hydrogen or an amino protecting group; and

G, Xi X ₂ , Yi, Z _\ and n are defined above;

with a compound of formula (III):

(A)— X _a X _b X _c X _d X _e -X ₃

(III) wherein A, X _a , Xt _> , X _c and Xd are defined above and X _e is selected from a glycine, alanine, asparagine, serine or valine residue and X ₃ is absent, is a purification tag or when A is a protein or peptide, X ₃ is optionally a bond to A;

in the presence of a sortase enzyme. another aspect of the invention, there is provided a compound having formula (IV): wherein G is a glycine residue;

Ri is hydrogen or an amino protecting group;

Xi is absent or is an amino acid residue or a peptide of 2-10 amino acid residues;

X ₂ is an amino acid residue with a reactive functional group in its side chain;

Yi is a spacer group which is attached to X ₂ through the reactive functional group in the side chain of X ₂ ;

Z| is a reactive functional group; and

n is 0 or an integer of 1 to 4. yet another aspect of the invention there is provided a method of preparing a compound formula (V):

(V) wherein G is a glycine residue;

each Ri is independently selected from hydrogen or an amino protecting group; each Xi is independently absent or is an amino acid residue or a peptide of 2-10 amino acid residues;

each X ₂ is independently absent or is an amino acid residue with a reactive functional group in its side chain;

each Yi is independently absent or is a spacer group;

each Y ₂ is independently absent or is a linker group;

B is a protein, a peptide, a label, a radioisotope, a small molecule drug, a targeting molecule, a nanoparticle, a cell, a dendrimer, a polymer, a photosensitizer, DNA, RNA, a carbohydrate, a contrast agent, a catalyst, a lipid or a virus particle;

s is an integer from 1 to 100; and

n is 0 or an integer of 1 to 4.

said method comprising reacting a compound of formula (I) described above with a compound of formula (VI): wherein B, Y ₂ and s are defined above and Z ₂ is a reactive functional group complementary to Z\ of formula (I).

In a further aspect of the invention, there is provided a kit comprising a compound of formula (I) and a sortase enzyme.

Brief Description of the Figures

Figure 1 shows labeling efficiency of site-selective modified proteins in which an scFv antibody with an LPETG tag prepared in Example 1 is conjugated using a sortase enzyme to a variety of GGG peptides comprising a reactive function Z _\ , as described in Example 3. Efficiency of the protein-GGG peptide conjugation is shown as a percentage conversion. The GGG peptides used in the sortase conjugation were selected from GGGWWSS - PEG4-N3, GGGWWK-PEG4-N3, GGGK-PEG4-N3 and GGGWWGA-pG, where pG is a propargyl glycine. The Sortase A mediated conjugation of scFv-LPETG with GGG-eGFP is provided for comparison.

Figure 2 is a graphical representation of optimization of reaction conditions for Sortase A mediated conjugation of Lissamine Rhodamine B-LPETGGHHHHHH with GGGWWSSK-PEG ₄ -functionalized particles. Figure 2A shows optimization of concentration of Sortase A enzyme at 0.1 g/L. Figure 2B shows optimization of concentration of Lissamine Rhodamine B-LPETGGHHHHHH at 0.1 g L. Figure 2C shows optimization of incubation time at 1 hour. Figure 2D shows optimized sortase mediated reaction with 0.1 g/L sortase, 0.1 g/L Lissamine Rhodamine B- LPETGGHHHHHH, 1 hour incubation time, where white bar is particles alone, single hatch bar is reaction with unfunctionalized particles and Lissamine Rhodamine B- LPETGGHHHHHH, double hatch bar is unfunctionalized particles, Lissamine Rhodamine B-LPETGGHHHHHH and Sortase A, grey bar is GGGWWSSK-PEG ₄ -coated particles and Lissamine Rhodamine B-LPETGGHHHHHH and the black bar is GGGWWSSK- PEG ₄ -coated particles, Lissamine Rhodamine B-LPETGGHHHHHH and Sortase A. Figure 3 is a graphical representation of human in vitro thrombi incubated with sortase coupling between scFv-LPETG-GGG modified (PVPON _A i _k )s core shell particles with DL800 label as imaged by near infrared imaging. The thrombi that was untreated (black bar) had fluorescence set to one. Thrombi treated with uncoated (PVPONAII S core shell particles (single hatch bar), scFv(-) coated capsules (double hatch bar), scFv(+) coated capsules (grey bar) and blocked thrombi treated with scFv(+) coated capsules are shown. Strong specific binding was observed with the unblocked thrombi treated with scFv(+) coated particles (scFv(+) = anti-GPIIb/IIIa scFv, scFv(-) = mutated scFv).

Figure 4 is a graphical presentation of binding of non-targeted CHO cells (black bars) and scFv-coupled CHO cells (white bars) to activated platelets immobilized on coverslips. There is significant specific binding of scFv-coupled cells to a layer of activated platelets. The numbers of bound scFv-cells were significantly higher than those of control non- targeted cells.

Description of the Invention

Definitions

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, preferred methods and materials are described. For the purposes of the present invention, the following terms are defined below.

Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

The articles "a" and ' W are used herein to refer to one or to more than one (i.e. to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

As used herein, the term "about" refers to a quantity, level, value, dimension, size, or amount that varies by as much as 30%, 20%, or 10% to a reference quantity, level, value, dimension, size, or amount.

As used herein, the term "peptide" refers to two or more naturally occurring or non- naturally occurring amino acids joined by peptide bonds. Generally, peptides will range from about 2 to about 80 amino acid residues in length, usually from about 5 to about 60 amino acid residues in length and more usually from about 2 to about 40 amino acid residues in length. The peptide may also be a retro-inverso peptide. The peptide may contain a-amino acid residues, β-amino acid residues, D-amino acid residues, L-amino acid residues, naturally occurring amino acid residues or non-naturally occurring amino acid residues.

The amino acid may also be further substituted in the a-position or the β-position with a group selected from -Ci-C ₆ alkyl, -(CH ₂ ) _n CORi, -(CH ₂ ) _n R ₂ , -P0 ₃ H, -(CH ₂ ) _n heterocyclyl or -(CH ₂ ) _n aryl where Ri is -OH, -NH ₂ , -NHC]-C ₃ alkyl, -OCi-C ₃ alkyl or -Ci-C ₃ alkyl and R ₂ is -OH, -SH, -SC,-C ₃ alkyl, -OC,-C ₃ alkyl, -C ₃ -C, ₂ cycloalkyl, -NH ₂ , -NHC _r C ₃ alkyl or -NHC(C=NH)NH ₂ and where each alkyl, cycloalkyl, aryl or heterocyclyl group may be substituted with one or more groups selected from -OH, -NH ₂ , -NHCi-C ₃ alkyl, -OC]-C ₃ alkyl, -SH, -SC,-C ₃ alkyl, -C0 ₂ H, -C0 ₂ C,-C ₃ alkyl, -CONH ₂ or -CONHCi-C ₃ alkyl.

Amino acid structure and single and three letter abbreviations used throughout the specification are defined in Table 1 , which lists the twenty naturally occurring amino acids which occur in proteins as L-isomers.

Table 1

(1) (2)

Amino Acid Three-letter One-letter Structure of side chain

Abbreviation symbol (R)

Alanine Ala A -CH ₃

Arginine Arg R -(CH ₂ ) ₃ NHC(=N)NH ₂

Asparagine Asn N -CH ₂ CONH ₂

Aspartic acid Asp D -CH ₂ C0 ₂ H

Cysteine Cys C -CH ₂ SH

Glutamine Gin Q -(CH ₂ ) ₂ CONH ₂

Glutamic acid Glu E -(CH ₂ ) ₂ C0 ₂ H Glycine Gly G -H

Histidine His H -CH ₂ (4-imidazolyl)

Isoleucine He I -CH(CH ₃ )CH ₂ CH ₃

Leucine Leu L -CH ₂ CH(CH ₃ ) ₂

Lysine Lys K -(CH ₂ ) ₄ NH ₂

Methionine Met M -(CH ₂ ) ₂ SCH ₃

Phenylalanine Phe F -CH ₂ Ph

Proline Pro P see formula (2) above for structure of amino acid

Serine Ser S -CH ₂ OH

Threonine Thr T -CH(CH ₃ )OH

Tryptophan Tip W -CH ₂ (3-indolyl)

Tyrosine Tyr Y -CH ₂ (4-hydroxyphenyl)

Valine Val V -CH(CH ₃ ) ₂

The term "a-amino acid" as used herein, refers to a compound having an amino group and a carboxyl group in which the amino group and the carboxyl group are separated by a single carbon atom, the a-carbon atom. An a-amino acid includes naturally occurring and non-naturally occurring L-amino acids and their D-isomers and derivatives thereof such as salts or derivatives where functional groups are protected by suitable protecting groups. The a-amino acid may also be further substituted in the a-position with a group selected from -Ci-C ₆ alkyl, -(CH ₂ ) _n CORi, -(CH ₂ ) _n R ₂ , -P0 ₃ H, -(CH ₂ ) _n heterocyclyl or -(CH ₂ )„aryl where Ri is -OH, -NH ₂ , -NHCi-C ₃ alkyl, -Od-C ₃ alkyl or -C _r C ₃ alkyl and R ₂ is -OH, -SH, -SC,-C ₃ alkyl, -OC,-C ₃ alkyl, -C ₃ -Ci ₂ cycloalkyl, -NH ₂ , -NHCi-C ₃ alkyl or -NHC(C=NH)NH ₂ and where each alkyl, cycloalkyl, aryl or heterocyclyl group may be substituted with one or more groups selected from -OH, -NH ₂ , -NHCj-C ₃ alkyl, -OC,-C ₃ alkyl, -SH, -SCi-C ₃ alkyl, -C0 ₂ H, -C0 ₂ C,-C ₃ alkyl, -CONH ₂ or -CONHC ₁ -C ₃ alkyl. As used herein, the term "β-amino acid' refers to an amino acid that differs from an a-amino acid in that there are two (2) carbon atoms separating the carboxyl terminus and the amino terminus. As such, β -amino acids with a specific side chain can exist as the R or 5 enantiomers at either of the a (C2) carbon or the β (C3) carbon, resulting in a total of 4 possible isomers for any given side chain. The side chains may be the same as those of naturally occurring a-amino acids (see Table 1 above) or may be the side chains of non-naturally occurring amino acids (see Table 2 below).

Furthermore, the β-amino acids may have mono-, di-, tri- or tetra-substitution at the C2 and C3 carbon atoms. Mono-substitution may be at the C2 or C3 carbon atom. Di-substitution includes two substituents at the C2 carbon atom, two substituents at the C3 carbon atom or one substituent at each of the C2 and C3 carbon atoms. Tri-substitution includes two substituents at the C2 carbon atom and one substituent at the C3 carbon atom or two substituents at the C3 carbon atom and one substituent at the C2 carbon atom. Tetra-substitution provides for two substituents at the C2 carbon atom and two substituents at the C3 carbon atom. Suitable substituents include -Ci-C ₆ alkyl, -(CH ₂ )„CORi, -(CH ₂ ) _n R ₂ , -PO3H, -(CH ₂ ) _n heterocyclyl or -(CH ₂ )„aryl where R, is -OH, -NH ₂ , -NHCi-C ₃ alkyl, -OCi-C ₃ alkyl or -C,-C ₃ alkyl and R ₂ is -OH, -SH, -SCi-C ₃ alkyl, -OC,-C ₃ alkyl, -C ₃ -Ci ₂ cycloalkyl, -NH ₂ , -NHC,-C ₃ alkyl or -NHC(C=NH)NH ₂ and where each alkyl, cycloalkyl, aryl or heterocyclyl group may be substituted with one or more groups selected from -OH, -NH ₂ , -NHCi-C ₃ alkyl, -OC C ₃ alkyl, -SH, -SCi-C ₃ alkyl, -C0 ₂ H, -C0 ₂ d-C ₃ alkyl, -CONH ₂ or -CONHC,-C ₃ alkyl.

Other suitable β-amino acids include conformationally constrained β-amino acids. Cyclic β-amino acids are conformationally constrained and are generally not accessible to enzymatic degradation. Suitable cyclic β-amino acids include, but are not limited to, cis- and trans-2-aminocyclopropyl carboxylic acids, 2-aminocyclobutyl and cyclobutenyl carboxylic acids, 2-aminocyclopentyl and cyclopentenyl carboxylic acids, 2-aminocyclohexyl and cyclohexenyl carboxylic acids and 2-amino-norbornane carboxylic

Suitable derivatives of β-amino acids include salts and may have functional groups protected by suitable protecting groups.

The term "non-naturally occurring amino acid' as used herein, refers to amino acids having a side chain that does not occur in the naturally occurring L-a-amino acids listed in Table 1. Examples of non-natural amino acids and derivatives include, but are not limited to, use of norleucine, 4-amino butyric acid, 4-amino-3 -hydroxy- 5 -phenylpentanoic acid, 6-aminohexanoic acid, t-butylglycine, norvaline, phenylglycine, ornithine, citrulline, sarcosine, 4-amino-3-hydroxy-6-methylheptanoic acid, 2-thienyl alanine and/or D-isomers of amino acids. A list of unnatural amino acids that may be useful herein is shown in Table 2.

Table 2 Non-conventional Code Non-conventional Code amino acid amino acid a-aminobutyric acid Abu L-N-methylalanine Nmala

<x-am i no-a-methy lbutyrate Mgabu L-N-methylarginine Nmarg aminocyclopropane- Cpro L-N-methylasparagine Nmasn carboxylate L-N-methylaspartic acid Nmasp aminoisobutyric acid Aib L-N-methylcysteine Nmcys aminonorbornyl- Norb L-N-methylglutamine Nmgln carboxylate L-N-methylglutamic acid Nmglu cyclohexylalanine Chexa L-N-methylhistidine Nmhis cyclopentylalanine Cpen L-N-methylisoleucine Nmile

D-alanine Dal L-N-methylleucine Nmleu

D-arginine Darg L-N-methyllysine Nmlys

D-aspartic acid Dasp L-N-methylmethionine Nmmet

D-cysteine Dcys L-N-methylnorleucine Nmnle

D-glutamine Dgln L-N-methylnorvaline Nmnva

D-glutamic acid Dglu L-N-methylornithine Nmorn

D-histidine Dhis L-N-methylphenylalanine Nmphe

D-isoleucine Dile L-N-methylproline Nmpro

D-leucine Dleu L-N-methylserine Nmser

D-lysine Dlys L-N-methylthreonine Nmthr

D-methionine Dmet L-N-methyltryptophan Nmtrp

D-ornithine Dorn L-N-methyltyrosine Nmtyr D-phenylalanine Dphe L-N-methylvaline Nmval

D-proline Dpro L-N-methylethylglycine Nmetg

D-serine Dser L-N-methyl-t-butylglycine Nmtbug

D-threonine Dthr L-norleucine Nle

D-tryptophan Dtrp L-norvaline Nva

D-tyrosine Dtyr a-methyl-aminoisobutyrate Maib

D-valine Dval a-methyl-aminobutyrate Mgabu

D-ct-methylalanine Dmala a-methylcyelohexylalanine Mchexa

D-a-methylarginine Dmarg a-methylcylcopentylalanine Mcpen

D-ot-methylasparagine Dmasn a-methyl-a-napthylalanine Manap

D-a-methylaspartate Dmasp a-methylpenicillamine Mpen

D-a-methylcysteine Dmcys N-(4-aminobutyl)glycine Nglu

D-a-methylglutamine Dmgln N-(2-aminoethyl)glycine Naeg

D-a-methylhistidine Dmhis N-(3-aminopropyl)glycine Norn

D-a-methylisoleucine Dmile N-amino-a-methylbutyrate Nmaabu

D-a-methylleucine Dmleu a-napthylalanine Anap

D-a-methyllysine Dmlys N-benzylglycine ^■ Nphe

D-a-methylmethionine Dmmet N-(2-carbamylethyl)glycine Ngln

D-a-methylornithine Dmorn N-(carbamylmethyl)glycine Nasn

D-a-methylphenylalanine Dmphe N-(2-carboxyethyl)glycine Nglu

D-a-methylproline Dmpro N-(carboxymethyl)gIycine Nasp

D-a-methylserine Dmser N-cyclobutylglycine Ncbut

D-a-methylthreonine Dmthr N-cycloheptylglycine Nchep

D-a-methyltryptophan Dmtrp N-cyc lohexylglyc ine Nchex

D-a-methyltyrosine Dmty N-cyclodecylglycine Ncdec

D-a-methylvaline Dmval N-cy lcododecylglyc ine Ncdod

D-N-methylalanine Dnmala N-cyclooctylglycine Ncoct

D-N-methylarginine Dnmarg N-cyclopropylglycine Ncpro

D-N-methylasparagine Dnmasn N-cycloundecylglycine Ncund

D-N-methylaspartate Dnmasp N-(2,2-diphenylethyl)glycine Nbhm

D-N-methylcysteine Dnmcys N-(3,3-diphenylpropyl)glycine Nbhe

D-N-methylglutamine Dnmgln N-(3-guanidinopropyl)glycine : Narg

D-N-methylglutamate Dnmglu N-( 1 -hydroxyethyl)glycine Nthr D-N-methylhistidine Dnmhis N-(hydroxyethyl))glycine Nser

D-N-methylisoleucine Dnmile N-(imidazolylethyl))glycine Nhis

D-N-methylleucine Dnmleu N-(3-indolylyethyl)glycine NmYp

D-N-methyllysine Dnmlys N-methyl-y-aminobutyrate Nmgabu N-methylcyclohexylalanine Nmchexa D-N-methylmethionine Dnmmet

D-N-methylornithine Dnmorn N-methylcyclopentylalanine Nmcpen

N-methylglycine Nala D-N-methylphenylalanine Dnmphe

N-methylaminoisobutyrate Nmaib D-N-methylproline Dnmpro

N-(l -methylpropyl)glycine Nile D-N-methylserine Dnmser N-(2-methylpropyl)glycine Nleu D-N-methylthreonine Dnmthr

D-N-methyltryptophan ϋηιηίφ N-(l-methyIethyl)glycine Nval

D-N-methyltyrosine Dnmtyr N-methylnapthylalanine Nmanap

D-N-methylvaline Dnmval N-methylpenicillamine Nmpen γ-aminobutyric acid Gabu N-(/j-hydroxyphenyl)glycine Nhtyr L-f-butylglycine Tbug N-(thiomethyl)glycine Ncys

L-ethylglycine Etg penicillamine Pen

L-homophenylalanine Hphe L-a-methylalanine Mala

L-a-methylarginine Marg L-a-methylasparagine Masn

L-a-methylaspartate Masp L-a-methyl-/-butylglycine Mtbug L-a-methylcysteine Mcys L-methylethylglycine Metg

L-a-methylglutamine Mgln L-a-methylglutamate Mglu

L-a-methylhistidine Mhis L-a-methylhomophenylalanine Mhphe

L-a-methylisoleucine Mile N-(2-methylthioethyl)glycine Nmet

L-a-methylleucine Mleu L-a-methyllysine Mlys L-a-methylmethionine Mmet L-a-methylnorleucine Mnle

L-a-methylnorvaline Mnva L-a-methylornithine Morn

L-a-methylphenylalanine Mphe L-a-methylproline Mpro

L-a-methylserine Mser L-a-methylthreonine Mthr

L-a-methyltryptophan Μί L-a-methyltyrosine Mtyr L-a-methyl valine Mval L-N-methylhomophenylalanineNmhphe

N-(N-(2,2-diphenylethyl) Nnbhm N-(N-(3,3-diphenylpropyl) Nnbhe carbamylmethyl)glycine carbamylmethyl)glycine l-carboxy-l-(2,2-diphenyl- Nmbc propargyl glycine pG

ethylamino)cyclopropane

As used herein, the term "amino acid residue with a bulky side chain" refers to amino acid residues that have side chains with branched or cyclic substituents. Examples of amino acid residues with a bulky side chain include tryptophan, tyrosine, phenylalanine, homophenylalanine, leucine, isoleucine, histidine, a-methyltryptophan, oc-methyltyrosine, a-methylphenylalanine, a-methylleucine, a-methylisoleucine, a-methylhistidine, cyclopentylalanine, cyclohexylalanine and naphthylalanine.

As used herein, the term "amino acid residue with a reactive functional group in its side chain" refers to an amino acid residue that has a side chain that bears a functional group that is able to react with another functional group. Suitable functional groups include amino groups, carboxylic acid groups, thiol groups and hydroxyl groups. Examples of amino acid residues with a reactive functional group in their side chains include lysine, ornithine, glutamic acid, aspartic acid, serine, threonine, cysteine, oc-methyllysine, cc- methyloraithine, a-methylglutamic acid, a-methylaspartic acid, a-methylserine, a- methylcysteine and a-methylthreonine.

The term "pharmacokinetic modifying polymer" as used herein refers to a polymer that alters pharmacokinetic properties of the molecule in which it is incorporated. For example, a polymer that increases solubility of the compound in which it is incorporated or a polymer that increases the plasma half life of the molecule in which it is incorporated or a polymer which assists in targeting the molecule in which it is incorporated to a particular site in the body. Suitable pharmacokinetic modifying polymers include, but are not limited to, polyethylene glycol (PEG), polypropylene glycol (PPG), polyethyleneoxide (PEO), poly(alkyloxazolines) such as poly(ethyloxazoline) (PEOX), polyvinylpyrrolidone (PVPON), polylysine, polyglutamic acid, polymethacrylic acid and polypropacrylic acid. As used herein, the term "sortase recognition tag' ^' ' refers to the amino acid sequence recognized by the sortase enzyme that enables conjugation with the G(G) _n motif. For example, the sortase recognition tag for Sortase A from S. aureus is LPXTG (SEQ ID NO:l).

The term "alkyl" as used herein refers to straight chain or branched saturated hydrocarbon groups. Suitable alkyl groups include, but are not limited to methyl, ethyl, propyl, isopropyl, butyl, isobutyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, dodecyl, tetradecyl, hexadecyl and octadecyl. The term alkyl may be prefixed by a specified number of carbon atoms to indicate the number of carbon atoms or a range of numbers of carbon atoms that may be present in the alkyl group. For example, Ci-C ₃ alkyl refers to methyl, ethyl, propyl and isopropyl.

The term "alkenyl" as used herein refers to straight chain, branched or cyclic hydrocarbon groups having at least one carbon-carbon double bond. The term alkenyl may be prefixed by a specified number of carbon atoms to indicate the number of carbon atoms or a range of numbers of carbon atoms that may be present in the alkenyl group. For example, C ₂ - C ₃ alkenyl refers to ethenyl, propenyl and isopropenyl. Suitable alkenyl groups include, but are not limited to ethenyl, propenyl, isopropenyl, butenyl, isobutenyl, pentenyl, cyclopentenyl, hexenyl, cyclohexenyl, heptenyl, octenyl, nonenyl, decenyl, dodecenyl, tetradecenyl, hexadecenyl and octadecenyl.

The term "alkynyP' as used herein refers to straight chain, branched or cyclic hydrocarbon groups having at least one carbon-carbon triple bond. The term alkynyl may be prefixed by a specified number of carbon atoms to indicate the number of carbon atoms or a range of numbers of carbon atoms that may be present in the alkynyl group. For example, C ₂ - C ₃ alkynyl refers to ethynyl and propynyl. Suitable alkynyl groups include, but are not limited to ethynyl, propynyl, butynyl, pentynyl, hexynyl, heptynyl, cycloheptynyl, octynyl, cyclooctynyl, nonynyl, decynyl, dodecynyl, tetradecynyl, hexadecynyl and octadecynyl. As used herein, the term "alkylene" refers to a linear chain of divalent -(CH ₂ )- groups that link two groups together. Typically an alkylene group has 1 to 20 -(CH ₂ )- groups, especially 1 to 15, 1 to 10, 2 to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4 or 2 to 3 -(CH ₂ )- groups.

As used herein, the term "alkenylene" refers to an alkylene in which two -(CH ₂ )- groups are replaced by at least one -CH=CH- group and which links two groups together. Typically an alkenylene group has 2-20 carbon atoms and may comprise one or more -CH=CH- groups and optionally further -CH ₂ - groups. The alkenylene group may have 2-15, 2-10, 2-9, 2-8, 2-7, 2-6, 2-5, 2-4 or 2-3 carbon atoms including at least one -CH=CH- group.

As used herein, the term "alkynylene" refers to an alkylene in which two -(CH ₂ )- groups are replaced by at least one -C≡C- group and which links two groups together. Typically an alkynylene group has 2-20 carbon atoms and may comprise one or more -C≡C^ groups and optionally further -CH ₂ - groups. The alkynylene group may have 2-15, 2-10, 2-9, 2-8, 2-7, 2-6, 2-5, 2-4 or 2-3 carbon atoms including at least one -C≡C- group.

The term "aryl" as used herein, refers to a C ₆ -Ci2 aromatic cyclic hydrocarbon groups in which at least one ring is aromatic such as phenyl, naphthyl, biphenyl and tetrahydronaphthyl .

The term "cycloalky as used herein, refers to cyclic hydrocarbon groups. Suitable cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl, cycloundecyl and cyclododecyl.

The term "heterocyclyF as used herein refers to 5 or 6 membered saturated, partially unsaturated or aromatic cyclic hydrocarbon groups in which at least one carbon atom has been replaced by N, O or S. Optionally, the heterocyclyl group may be fused to a phenyl ring. Suitable heterocyclyl groups include, but are not limited to pyrrolidinyl, piperidinyl, pyrrolyl, thiophenyl, furanyl, oxazolyl, imidazolyl, thiazolyl, isoxazolyl, pyrazolyl, isothiazolyl, pyridinyl, quinolinyl, isoquinolinyl, indolyl, benzofuranyl, benzothiophenyl, oxadiazolyl, tetrazolyl, triazolyl and pyrimidinyl. The term "heteroaryl" as used herein, represents a stable monocyclic, bicyclic or tricyclic ring of up to 7 atoms in each ring, wherein at least one ring is aromatic and at least one ring contains from 1 to 4 heteroatoms selected from the group consisting of O, N and S. When more than one ring is present the rings may be fused. The heteroaryl group may also include a carbonyl group attached to an unsaturated carbon in the ring system. Examples of suitable heteroaryl groups include pyrrolyl, furanyl, thienyl, pyrazolyl, imidazolyl, 1 ,2,4-triazolyl, 1,2,3-triazolyl, isoxazolyl, oxazolyl, thiazolyl, isothiazolyl, oxadiazolyl, oxatriazolyl, pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, azepinyl, oxepinyl,. thiepinyl, diazepinyl, coumaranyl, benzofuranyl, isobenzofuranyl, benzothienyl, indolyl, indolinyl, isoindolyl, benzimidazolyl, benzisoxazolyl, benzoxazolyl, benzisoxazolyl, benzothiazolyl, 1-benzopyranyl, 2-benzopyranyl, benzopyran-2-on-yl, benzopyran-l-on-yl, quinolinyl, tetrahydroquinolinyl, isoquinolinyl, quinazolinyl, cinnolinyl, quinoxalinyl, tetrahydroquinoxalinyl, naphthyridinyl, acridinyl, carbazolyl, xanthenyl, phenazinyl, phenothiazinyl, phenoxazinyl, l,4-benzodiazepin-2-on-yl, 1,5- benzodiazepin-2-on-yl, 1 ,4-benzodiazepin-2,5-dion-yl, pyrrolo[2, 1 -c][l ,4]benzodiazepine- 5,1 1-dion-yl, l,4-benzothiazepin-5-on-yl, 5,l l-dihydro-benzo[e]pyrido[3,2-6][l,4]- diazepin-6-on-yl, chromonyl, pyranocoumarinyl, 3,4-dihydroquinoxalin-2-on-yl, quinazolinonyl, quinazolindionyl, imidazoquinoxalinyl, 2,3-dihydrospiro[indene-l,4'- piperidine] and spiro[indoline-3,4'-piperidine]. As used herein, a "hydroxylamine" is a moiety that includes a -O-NH- group. Either or both of the oxygen and amine may be substituted.

The term "hydrazine" is a moiety that includes a -N-N- group, where either or both nitrogen atoms may be substituted. The term "oxo" as used herein refers to a substituent that is =0. An oxo substituent may be substituted on a saturated carbon atom in a ring or alkyl chain to form a carbonyl group.

As used herein, the term "thioester" refers to a moiety that includes a -C(=0)S-R group, where the R group is a substituent such as an alkyl group, an alkenyl group, cycloalkyl group or aryl group. The carbon atom may also be substituted.

The term "complementary reactive functional group" as used herein refers to a reactive functional group that is included in a compound or molecule and which may be paired up with another reactive functional group to undergo a particular reaction following methodologies that have been well described in the art. For example, when the reactive functional group is an alkyne group, the complementary functional group is an azide and when the reactive functional group is an azide, the complementary functional group is an alkyne, together these groups undergo a 1,3-dipolar cycloaddition reaction. Other examples include, but are not limited to, a reactive functional group being a hydroxylamihe or hydrazine and the complementary group being an aldehyde or ketone, or vice versa, where the groups react together to form oximes or hydrazones; the reactive functional group may be an amino group and the complementary group a carboxylic acid, or vice versa, where the two groups undergo amide formation; the reactive functional group may be a thiol group and the complementary group may be an alkene, or vice versa, and the two groups undergo a thiol-ene reaction or the reactive functional group may be a thioester and the complementary group a cysteine or equivalent thereof, or vice versa, and the two groups undergo native chemical ligation.

In one aspect, the present invention provides a method of preparing a compound of formula (II): -X _e X _b X _c dG(G)n- i ₂ YiZi

(Π) wherein A is a protein, a peptide, a label, a radioisotope, a small molecule drug, a targeting molecule, a nanoparticle, a cell, a dendrimer, a photosensitizer, DNA, RNA, a carbohydrate, a contrast agent, a catalyst, a lipid or a virus particle,

X _a is selected from a leucine, serine, asparagine, valine, isoleucine, tyrosine and glutamine residue;

Xb is selected from a proline, alanine, serine and glycine residue;

X _c is any amino acid residue;

Xd is selected from a threonine, arginine, serine, alanine and glycine residue;

G is a glycine residue;

Xi is absent or is an amino acid residue or a peptide of 2-10 amino acid residues; X ₂ is absent or is an amino acid residue with a reactive functional group in its side chain;

Y] is absent or is a spacer group;

Zi is a reactive functional group; and

n is 0 or an integer of 1 to 4;

said method comprising contacting a compound of formula (I):

R,-HN-G(G) _n -XiX ₂ Y ₁ Z, wherein

Ri is hydrogen or an amino protecting group; and

G, Xi X ₂ , Yi, Z _\ and n are defined above;

with a compound of formula (III) :

(A)- X _a XbX _c XdX _E "^3 (III) wherein A, X _a , Xb, X _c and Xd are defined above and X _e is selected from a glycine, alanine, asparagine, serine or valine residue and X ₃ is absent, is a purification tag or when A is a protein or peptide, X ₃ is optionally a bond to A;

in the presence of a sortase enzyme. In this aspect of the invention, the compound of formula (I) described above is contacted with a group A having a sortase recognition tag in the presence of a sortase enzyme. The sortase recognition tag is recognized by the sortase enzyme which then ligates the tag with the G(G) _n portion of formula (I).

The sortase enzyme useful in the method of the invention is any sortase that recognizes the recognition tag present in formula (III). Sortase enzymes have been classified into four classes, designated A, B, C and D based on sequence alignment and phylogenetic analysis (Dramsi et al., Res. Microbiol., 2005, 156(3):289-97). Sortase enzymes may be derived from any bacterial species or strain, such as Gram positive bacterial species. Different sortase enzymes may recognize different sortase recognition tags and the sortase recognition tag used in formula (III) will correspond to the sortase recognition tag of the sortase enzyme used in the method.

In some embodiments, the sortase enzyme is a Sortase A erizyme, which recognizes the recognition tag:

XaXbXcXdXe

in which

X _a is leucine or isoleucine, especially leucine;

Xb is proline or glycine, especially proline;

X _c is any amino acid residue, especially glutamic acid, aspartic acid, alanine, asparagine, lysine or arginine, most especially glutamic acid;

Xa is threonine or alanine; and

Xe is glycine or alanine.

In a particular embodiment, the recognition tag is LPX _C TG (SEQ ID NO:l) where X _c is any amino acid residue, especially glutamic acid, aspartic acid, alanine, asparagine, lysine or arginine, most especially glutamic acid. In some embodiments, the sortase enzyme is a Sortase B enzyme, which recognizes the recognition tag:

X _a XbXcX_Xe

in which

X _a is asparagine;

X _b is proline, serine or alanine, especially proline;

X _c is any amino acid residue, especially glutamic acid, aspartic acid or lysine;

Xa is threonine or serine; and

X _e is asparagine, glycine or serine.

In some embodiments, the sortase enzyme is a Sortase C enzyme which recognizes the recognition tag:

LPXcTG

in which X _c is any amino acid residue.

In some embodiments, the sortase enzyme is a Sortase D enzyme which recognizes the recognition tag:

X _a XbXcXdXe

in which

X _a is asparagine or leucine;

Xb is alanine or proline;

X _c is any amino acid residue, especially glutamic acid, serine, histidine and asparagine;

Xd is threonine; and

X _e is glycine or alanine.

Sortase enzymes may be derived from a number of Gram positive bacteria including Staphylococcus aureus, Bacillus anthracis, Enterococcus faecalis; Listeria monocytogenes, Streptococcus gordonii, Streptococcus pneumoniae, Streptococcus sanquinis, Streptococcus suis, Streptococcus agalactiae, Streptococcus pyrogenes, Streptococcus mutans and Actinomyces naeslundii. In a particular embodiment, the sortase enzyme is Sortase A from Staphylococcus aureus and the recognition of X _a X _b X _c XdXe is LPX _C TG (SEQ ID NO:l), especially LPETG (SEQ ID NO:380). ^"

X ₃ is absent or is a purification tag such as His ₆ , cMyc protein, Hemagglutinan (HA), FLAG-tag, HSV-tag or V5-tag, especially His ₆ . This can be particularly helpful in purification of the reaction product as unreacted formula (III) and the G-X ₃ released in the sortase mediated reaction may be readily removed by chromatography such as affinity chromatography. In some embodiments, when A is a protein or peptide, the sortase recognition tag is attached to the C-terminus of the protein or peptide. In other embodiments, the sortase recognition tag is located within the peptide or protein.

In some embodiments, it has been observed that the sortase mediated reaction is more efficacious if the sortase recognition tag is located at or near the C-terminus of the protein or peptide. For example, the sortase recognition tag or sortase recognition tag and purification tag are located at the C-terminus.

In some embodiments, when A is a protein or peptide which requires a free C-terminal carboxy group for activity, X ₃ is a covalent bond to A. In these embodiments, the sortase recognition tag is located in an accessible loop within the protein or peptide. By "accessible loop" is meant that the recognition tag appears in the sequence of the protein or peptide which is on the surface of the tertiary structure of the protein or peptide and is accessible to both the peptide of formula (I) having the G(G) _n sequence and the sortase enzyme. Although the sortase enzyme will cleave the recognition tag, the protein or peptide may have sufficient tertiary structure, such as disulfide bonds, to maintain the protein intact.

A is a protein, a peptide, a label, a radioisotope, a small molecule drug, a targeting molecule, a nanoparticle, a cell, a dendrimer, a photosensitizer, a virus particle, a carbohydrate, a contrast agent, a catalyst, a lipid, a DNA molecule or an RNA molecule; especially a protein or peptide, more especially a protein, more especially an antibody or a fragment thereof.

The protein or peptide may be any protein or peptide that is useful when conjugated to another moiety, such as another peptide or protein, a label, a radioisotope, a small molecule drug, a targeting molecule, a nanoparticle, a cell, a dendrimer, a polymer, a photosensitizer, a virus particle, a carbohydrate, a contrast agent, a catalyst, a lipid, a DNA molecule or an RNA molecule, or may be useful conjugated to a solid surface. The protein or peptide may be biologically active, for example, an antibody or antibody chain (heavy or light chain), or a portion comprising an immunoglobulin domain (constant or variable domain), a cell proliferation factor, an apoptosis factor, an extracellular signaling molecule, a receptor, an angiogenesis factor or a cell interaction factor. In some embodiments, the antibody is IgG, IgM, IgA or IgE or a fragment thereof. In some embodiments, the antibody is a polyclonal or monoclonal antibody and may be chimeric, humanized or bispecific. The antibody may be selected for its interaction with a specific antigen. Examples of suitable antibodies include, but are not limited to, muromonab, abciximab, rituximab, daclizumab, basiliximab, palivizumab, infliximab, trastuzumab, gemtuzumab, alemtuzumab, ibritumomab, adelimumab, omalizumab, tositumomab, efalizumab, cetuximab, bevacizumab, natalizumab, ranibizumab, panitumumab, eculizumab, certolizumab, pexelizumab, ipilimumab, zanolimumab, denosumab, adalimumab, belimumab and motauizumab. In some embodiments, the protein or peptide is an antibody fragment, such as Fab, Fab', F(ab)'2, F(ab)'3, Dab, Fv, single chain Fv (scFv) fragment scFv-Fc (scFv)2, intrabodies (Kontermann, Methods, 2004, 34:163-170), diabodies, triabodies or tetrabodies, especially a scFv fragment. Examples of suitable antibody fragments include, but are not limited to, anti-ED-B-scFv (extra domain B fibronectin), anti-LIBS GPIIb/IIIa scFv (platelet integrin), anti-E-selectin scFv, anti-P-selectin scFv, anti-Mac- 1 scFv (leukocyte integrin), 59D8 (fibrin), MA- 15CF, A33-scFv, anti-y-SM scFv, ch-TNT-3 Fab and L49 scFv. Examples of other proteins and peptides include proteins and peptide involved in recognition of other proteins and peptides, including, but are not limited to protein kinases such as mitogen activated protein (MAP) kinase, and kinases that directly or indirectly phosphorylate MAP kinase, Januse kinase (JAKI) and cyclin dependent kinases, epidermal growth factor (EGF) receptor, platelet-derived growth factor (PDGF) receptor, fibroblast- derived growth factor (FGF) receptor, insulin receptor and insulin-like growth factor (IGF) receptor, protein phosphatases such as PTPIB, PP2A and PP2C, GDP/GTP binding proteins such as Ras, Raf, ARF, Ran and Rho, GTPase activating proteins (GAF), guanidine nucleotide exchange factors (GEFs), proteases such as caspase 3, 8 and 9, peroxidases, ubiquitin ligases such as MDM2 and E3 ubiquitin ligases, acetylation and methylation proteins such as p300/CBP and histone acetyl transferase, tumor suppressor proteins such as p53, cytokines such as interleukins (IL-1, IL-lra, IL-2, IL-10, IL-11, IL-6), interferons (IFN-a, IFN-β), erythropoietin (EPO), thrombopoietin (TPO), TGF-β, and chemokines (CCL-1 to CCL-28, a-chemokines, β-chemokines, lymphotactin-a, lymphotactin-β and fractalkine), and their receptors such as G-protein coupled receptors, haemopoeitic growth factor, tumor necrosis factors (TNF).

Other suitable proteins include DARPins (designed ankyrin repeat proteins), which are small, single domain proteins that can be selected to bind to a given target protein with high affinity and specificity (Stumpp et al, Drug Discovery Today, 2008, 13:695-701).

In some embodiments, the protein may be an albumin, such as human serum albumin, and this modifies the pharmacokinetic properties of the conjugate.

Suitable labels include but are not limited to fluorescent labels, phosphorescent labels, chemiluminescent labels, streptavadin, biotin, poly(histidine) or a radioactive label.

Suitable small molecule drugs may be any drugs that may be delivered in a more targeted manner or may be more soluble when part of a conjugate. For example, the small molecule drug may be toxic to normal cells as well as diseased cells or microbial cells and conjugation allows targeting to the diseased or microbial cell reducing toxicity to normal host cells. The drug may be a poorly soluble drug that has no or low bioavailability, especially oral bioavailability and which for solubility reasons is difficult to formulate for parenteral administration, and the conjugate improves solubility and bioavailability. Suitable small molecule drugs include diabetes therapies such as biguanides, sulfonylureas, glitazones and insulin; anti-obesity drugs such as orlistat, rimonabant and sibutramine; anti-microbial drugs such as antibiotics, anti-fungal agents and anti-protozoal agents, antiviral agents such as Zovirax and Azt; cholesterol lowering agents such as HMG-CoA inhibitors (statins), cardiovascular drugs such as calcium channel blockers, β-blockers, ACE inhibitors and diuretics, analgesics such as opioids and non-steroidal antiinflammatory drugs, anti-convulsant drugs such as benzodiazepines, barbiturates and GABA analogues, anti-histamines such as diphenhydramine and cimetidine; asthma drugs such as salbutamol and fluticasone, cancer therapies such as chemotherapeutic drugs including cisplatin, carboplatin, taxol and related taxane compounds; immune suppressing drugs such as cyclosporine, hormones and hormone analogues such as insulin and thyroxine; vitamins such as vitamin B group compounds, vitamin E, vitamin C and vitamin A; contraceptives, muscle relaxants such as tubocurarine, succinylcholine and pancuronium; sedatives including barbiturates and benzodiazapines; arthritis drugs such as COX2-selective inhibitors; heparin; fXa blockers such as rivaroxaban; and thrombin inhibitors such as argatroban, lepirudin, desirudin and bivalirudin.

Suitable radioisotopes include but are not limited to, ³ H, ⁴⁷ Ca, ^U C, ¹⁴ C, ⁵⁷ Co, ⁵ Co, ^Cu, ¹⁸ F, ⁷ Ga, ⁶⁸ Ga, ^,8 F, ^m In, ^,23 I, ^,24 I, ¹²⁵ I, ^,31 I, ³² P, ⁷⁵ Se, ¹⁵³ Sm, ^,3 N, ²² Na, ²⁴ Na, ¹⁵ 0, ⁸⁹ Sr, "Tc, ²⁰¹ Tl, ¹³³ Xe and ⁹⁰ Y.

Suitable targeting agents are compounds that bind to cell receptors in specific parts of the body such as tissues or organs, or to cell receptors on unwanted cells such as microbial cells or cancer cells. Antibodies or parts thereof may be used to target particular cells or tissues in the body. Another example is folate as folate receptors overexpressed on the surface of some cancer cells. A further example is cytokines and chemokines and their cell surface receptors. Suitable nanoparticles include, but are not limited to, iron oxide particles used in MRI imaging (MPIO) (McAteer et al, Methods Mol. Biol., 201 1, 680:103-15; Guenther et al, Invest. Radiol., 2010, 45(10):586-91 ; von Zur Muhlen et al, Circulation, 2008, 118:258- 67; von Zur Muhlen et al, J. Vase. Res., 2009, 46:6-14; von Zur Muhlen et al, J. Clin. Invest., 2008, 118:1198-207; McAteer et al, Arterioscler. Thromb. Vase. Biol., 2008, 28:77-83; McAteer et al, Nat. Med., 2007, 13(10): 1253-8), LbL click capsules (Leung et al, Small, 2011, 21 Mar. doi:10.1002/smll. 201002258; Chong et al, Langmuir, 2011, 1 Mar, 27(5): 1724-30; Such et al, Chem. Soc. Rev., 2011, 40(1): 19-29; Becker et al, Small, 2010, 6(17): 1836-52), lipid nanoparticles, liposomes, micelles, GdO particles (Faure et al, Small, 2009, 5(22):2565-75; Ou et al, J. Colloid Interface Sci., 2009, 333(2):684-9; Alric et al, J. Am. Chem. Soc, 2008, 130(18):5908-15) and F click capsules.

Suitable cells include, but are not limited to, stem cells, cells from specific tissues or organs in the body, T-regulatory cells or Natural Killer T cells.

Suitable dendrimers include, but are not limited to, polyamidoamine (PAMAM), poly(ethyleneimine) (PEI), polylysine, polyglutamine, poly(etherhydroxylamine) (PEHAM) and poly(propyleneimine) (PPI) dendrimers. Other suitable dendrimers incorporate diamino building units such as analogues of lysine. Architecture of lysine and lysine analogue dendrimers has been described by Denkewalter in US Patent No. 4,289,872. More preferably the dendrimer includes the at least two generations of building units and all contain one or more branches originating from a core molecule. In particular embodiments, the dendrimer is a polylysine dendrimer of two to eight generations.

Suitable photosensitizers, include but are not limited to, porphyrines, chlorophylls and dyes such as aminolevulinic acid, levulinic acid, methyl aminolevulinic acid, photofrin, Visudyne, Foscan, Metvix, Hexvix ^® , Cysview™, Laserphryin, Antrin, Photochlor, Photosens, Photrex, Civira, Visonac, Amphinex and azadipyrromethenes. Suitable virus particles, include but are not limited to, virus particles able to carry DNA for use in gene therapy. Examples of suitable virus particles include, but are not limited to adenovirus, adeno-associated virus, lentivirus, herpes simplex virus, replication-competent vectors, envelope protein pseudotyping of viral vectors and shells from disease causing viruses.

Suitable carbohydrates include carbohydrates that provide cell signalling functions or those that are unique to the cell surfaces of microbes or viruses. Examples include carbohydrates comprising one or more monomers selected from sialic acid, glucose, galactose, mannose and fucose, particularly Sialyl Le and L-rhamnopyranose.

Suitable contrast agents include but are not limited to paramagnetic particles or metal ions including Cr ³⁺ , Mn ²⁺ , Fe ³⁺ , Co ²⁺ , Cu ²⁺ , Pr ³ *, Eu ³⁺ , Gd ³⁺ , Tb ³⁺ , Tb ⁴⁺ , Dy ³ *, Ho ³⁺ and Er ³⁺ . Suitable catalysts include enzymes, such as thrombolytic enzymes and proteases.

Suitable lipids include long chain fatty acids, such as myristic acid, that provide an increase in plasma half-life. Suitable DNA and RNA is any suitable DNA or RNA or fragment thereof that has therapeutic value. Examples include SiRNA (gene silencing), antagomirs (microRNA inhibition) and microRNA (effector RNA).

The GGG peptide moiety is a compound having formula (I): R,-HN-G(G)n-X,X2YiZ, (I) wherein G is a glycine residue;

Ri is hydrogen or an amino protecting group;

Xi is absent or is an amino acid residue or a peptide of 2- 10 amino acid residues; X ₂ is absent or is an amino acid residue with a reactive functional group in its side chain;

Yi is absent or is a spacer group;

Zi is a reactive functional group; and

n is 0 or an integer of 1 to 4.

In some embodiments, n is 0 and only one glycine residue is present at the N-terminus of formula (I). In some embodiments n is 1 and two glycine residues are present at the N- terminus of formula (I). In other embodiments, n is 2 and three glycine residues are present at the N-terminus of formula (I). In some embodiments, n is 3 and four glycine residues are present at the N-terminus of formula (I). In other embodiments, n is 4 and five glycine residues are present at the N-terminus of formula (I). In particular embodiments, n is 2. In some embodiments, Xi is absent. In other embodiments, Xi is an amino acid residue or a peptide of 2-10 amino acid residues in length, especially 2-9, 2-8, 2-7, 2-5, 2-4 or 2-3 amino acid residues in length, most especially 2-5 amino acid residues in length. While Xi may be any amino acid residue or peptide of 2-10 amino acid residues in length, in some embodiments, Xi comprises one or more amino acid residues with a bulky side chain, especially where there is an amino acid residue with a bulky side chain attached to the C- terminal glycine residue of the G(G)„ sequence.

In one embodiment, Xi is a peptide of the sequence:

XaaiXaa ₂ Xaa ₃ Xaa4

wherein Xaai is an amino acid residue having a bulky side chain;

Xaa ₂ is absent or is an amino acid residue having a bulky side chain;

Xaa ₃ is absent or any amino acid residue; and

Xaa4 is absent or any amino acid residue. In some embodiments Xaai is selected from tryptophan, tyrosine, phenylalanine, leucine, isoleucine and histidine, especially tryptophan, tyrosine and phenylalanine, most especially tryptophan. In some embodiments, where Xaa ₂ is present, Xaa ₂ is selected from tryptophan, tyrosine, phenylalanine, leucine, isoleucine and histidine, especially tryptophan, tyrosine and phenylalanine, most especially tryptophan.

In some embodiments, where Xaa ₃ is present, Xaa ₃ is selected from serine, alanine, aspartic acid, methionine, threonine and valine, especially serine or threonine, most especially serine.

In some embodiments, where Xaa4 is present, Xaa ₄ is selected from serine, alanine, aspartic acid, methionine, threonine and valine, especially serine or threonine, most especially serine.

In some embodiments, one or more of Xaai, Xaa ₂ , Xaa ₃ or Xaa4 is absent. For example, in some embodiments, Xaa ₃ and Xaa4 are absent and Xaai and Xaa ₂ make a peptide of two amino acid residues or Xaa4 is absent and Xaai, Xaa ₂ and Xaa ₃ make a peptide of three amino acid residues.

In some embodiments, X, is selected from -W-, -WS-, -WG-, -WA-, -WW-, -WWS-, -WWG-, -WWA-, -WWSS- (SEQ ID NO:2), -WWSG- (SEQ ID NO:3), -WWSA- (SEQ ID NO:4), -WWGS- (SEQ ID NO:5), -WWGG- (SEQ ID NO:6), -WWGA- (SEQ ID NO:7), -WWAS- (SEQ ID NO:8), -WWAG- (SEQ ID NO:9) and -WWAA- (SEQ ID NO: 10).

In some embodiments, X ₂ is absent and Yi or Zj is attached directly to the C-terminus of Xi or the G(G) _n sequence. In other embodiments, X ₂ is an amino acid with a reactive functional group in its side chain and Yi or Z _\ is attached to the C-terminal carboxy group of X ₂ or to the reactive functional side chain group of X ₂ . In some embodiments, X ₂ is selected from lysine, ornithine, aspartic acid and glutamic acid, especially lysine or ornithine, most especially lysine.

Yi may be absent or may be a spacer group that connects Zj to the C-terminus of the G(G)n sequence, the C-terminus of Xi or the C-terminus or reactive functional group of X ₂ . The spacer group may be any sort of spacer group and may provide relief from steric hindrance around the functional group or may be a pharmacokinetic modifying agent.

In some embodiments, the spacer group is a group containing 1-100 carbon atoms covalently connected by single or multiple bonds, wherein one or more carbon atoms are optionally replaced with heteroatoms sch as O, N, S, or P. Examples include linear or branched alkylene, alkenylene or alkynylene groups or divalent cycloalkyl, aryl, heterocyclyl or heteroaryl groups or combinations thereof, wherein one or more carbon atoms in an alkylene, alkenylene or alkynylene group is optionally replaced by a heteroatom selected from O, N, S or P, and wherein each alkylene, alkenylene, alkynylene, cycloalkyl, aryl, heterocyclyl or heteroaryl group is optionally substituted, for example, with one or more alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heterocyclyl, heteroaryl, -N(R ₂ ) ₂ , oxo group, -OR ₂ , -SR ₂ , -COR ₂ , -C0 ₂ R ₂ , -CONH ₂ , -NHC(0)-NH ₂₎ -NH-C(=NH)-NH ₂ , -PO4H3 or -S(0) ₂ N(R ₂ ) ₂ wherein each R ₂ is independently selected from hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroalkyl or heteroaryl.

In some embodiments, the spacer has a molecular weight in the range of 85 Da - 1200 Da. In some embodiments, the spacer is polyethylene glycol (PEG) having a molecular weight between 85 and 1200 Da, for example PEG ₂ , PEG ₄ , PEG ₆ , PEGg, PEG( ₂ or PEG ₂₄ , especially PEG ₄ , PEG ₆ , PEG ₈ or PEG ₁₂ .

In some embodiments, the spacer is a pharmacokinetic modifying agent. In particular embodiments, the pharmacokinetic modifying agent is an oligomer or polymer comprising monomers selected from ethyleneoxy, propyleneoxy, alkyloxazoline such as ethyloxazoline, vinylpyrrolidone and amino acids such as lysine, glutamate and aspartate. In some embodiments the pharmacokinetic modifying agent is selected from polyethylene glycol (PEG), polypropylene glycol (PPG), polyethylene oxide (PEO), a poly(alkyloxazoline) such as poly(ethyloxazoline (PEOX), polyvinylpyrrolidone (PVPON), polylysine, polyglutamate and polyaspartate, especially PEG, PPG, PEOX, polylysine, polyglutamate and polyaspartate, or mixtures thereof, more especially PEG.

In some embodiments, the polymer has a molecular weight in the range of 5 kDa - 100 kDa. In some embodiments, the pharmacokinetic modifying polymer is PEG having a molecular weight between 5 kDa and 60 kDa, for example, 10 kDa, 15 kDa, 20 kDa, 30 kDa or 40 kDa, especially 20 kDa.

In a particular embodiment, X ₂ is a lysine, ornithine, glutamic acid or aspartic acid residue and the spacer, especially PEG, is attached to the ε-amino group of lysine, the δ-amino group of ornithine, the γ-carboxy group of aspartic acid or the δ-carboxy group of glutamic acid, especially where X ₂ is lysine and the spacer is attached to the ε-amino group.

In a particular embodiment, X ₂ is a lysine, ornithine, glutamic acid or aspartic acid residue and the pharmacokinetic modifying agent, especially PEG or a polylysine, polyaspartic acid or polyglutamic acid group, is attached to the ε-amino group of lysine, the δ-amino group of ornithine, the γ-carboxy group of aspartic acid or the δ-carboxy group of glutamic acid, especially where X ₂ is lysine and the modifying agent is attached to the ε-amino group.

In embodiments where the spacer or pharmacokinetic modifying agent is attached to the side chain functional group of X ₂ , the C-terminal carboxy group of X ₂ may optionally have another group attached, for example, a label such as a fluorescent label, a radioactive label or an imaging label.

Zi is a reactive functional group that is capable of reacting with another moiety. In some embodiments, the reactive functional group is a group complementary to a reactive functional group on the moiety to which it is proposed to be attached. For example, Z _\ may react with another functional group via a 1 ,3-cycloaddition reaction, hydrazone formation, oxime formation, amide formation such as an amide formed by native chemical ligation, thio-ene reaction or Diels- Alder reaction.

In some embodiments, Zj is selected from an azide, an alkyne, a hydroxylamine, a hydrazine, an aldehyde, a ketone, an amino group, a carboxylic acid, a thiol group, an alkene, a thioester or a cysteine residue. In particular embodiments, Z _\ is an azide, an alkyne, a hydroxylamine, a hydrazine, an aldehyde, an amino group, a carboxylic acid or a thioester. In some embodiments, the carboxylic acid is not the C-terminal carboxylic acid of the glycine moiety; Xi or X ₂ .

The Zj group may be attached directly to the Yi or the C-terminal carboxy group of Xi or X ₂ or the C-terminal carboxy group of the G(G) _n sequence or may be derived from the terminal group of Yi or the C-terminal carboxy group of the G(G) _n sequence, Xj or X ₂ . Alternatively, Z _\ may be attached to the G(G)„ sequence, Xi, X ₂ or Yi through a linker group.

For example, where the group Yi is a polymer that terminates with a carbbxy group, it may be reduced to an aldehyde group by methods known in the art. Where Yi is a polymer that terminates in an amino group, it may be used directly or modified to an azide group, a thiol group or an alkene by methods known in the art.

Alternatively, the terminal group of Yi or the C-terminal carboxy group of Xi or the G(G)„ sequence may be reacted with a linker group that bears the functional group Z _\ . Suitable linker groups include alkylene groups ((CH ₂ ) ) in which x is an integer from 1 to 10 and in which one or more CH ₂ groups may be replaced by -0-, -NH-, -NH-C(O)-, -C(0)-NH, -S- or -C ₆ H4-.

Suitable Z _\ groups, optionally including a linker, include:

-(CH ₂ ) _X N ₃ , -(CH ₂ ) _x ONH ₂ , -(CH ₂ ) _X N=NH ₂ , -(CH ₂ ) _X -SH, -(CH ₂ ) _x CHO, -(CH ₂ ) _x C(0)SR, -(CH ₂ ) _x C(0)R, -(CH ₂ ) _x alkyne, -(CH ₂ ) _X CH=CH ₂ , -(CH ₂ ) _x NH-C(0)CH(NH ₂ )(CH ₂ SH), -(CH ₂ ) _m NHC(0)(CH ₂ ) _p alkyne, -(CH ₂ ) _m -NHC(0)-(CH ₂ ) _p NHNH ₂ , -(CH ₂ ) _m NHC(0)(CH ₂ ) _p - ONH ₂ , -(CH ₂ ) _m NHC(0)(CH ₂ ) _p -SH, . -(CH ₂ ) _m -NHC(0)(CH ₂ ) _p CHO,

-(CH ₂ ) _ra NHC(0)(CH ₂ ) _p C(0)R, -(CH ₂ ) _m NHC(0)(CH ₂ ) _p C(0)SR, -(CH ₂ ) _m NHC(0)(CH ₂ ) _p - CH=CH ₂ , -(CH ₂ ) _m C(0)NH(CH ₂ ) _p alkyne, -(CH ₂ ) _m C(0)NH-(CH ₂ ) _p NHNH ₂ ,

-(CH ₂ ) _m C(0)NH-(CH ₂ ) _p -ONH ₂ , -(CH ₂ ) _m -C(0)NH-(CH ₂ ) _p SH, -(CH ₂ ) _m - C(0)NH(CH ₂ )pCHO, -(CH ₂ ) _m C(0)NH-(CH ₂ ) _p C(0)R, -(CH ₂ ) _m C(0)NH-(CH ₂ ) _p SR, -(CH ₂ ) _m C(0)(CH ₂ ) _p -CH=CH ₂ , -(CH ₂ ) _q NHCO-C6H ₄ -CHO and -(CH ₂ ) _q NHCOC ₆ H ₄ -C(0)R wherein m is an integer from 1 to 10, m + p = x-1 and q = x-2 and R is an alkyl group or alkyl group substituted with an amide group. Some suitable linker-Zi groups include, but are not limited to:

Ri is hydrogen or an amino protecting group. In some cases where the compound of formula (I) may need to be reacted with another moiety before sortase conjugation of the G(G)„ moiety with an LPXTG (SEQ ID NO:l) motif, the N-terminal amino group of the G(G) _n moiety may need to be protected. Suitable protecting groups are known in the art, such as in Greene and Wutz, Protective Groups in organic synthesis, 3 ^rd Ed., Wiley Interscience, 1999. Commonly used amino protecting groups include, but are not limited to, benzyloxycarbonyl (Z), t-butoxycarbonyl (Boc), 2-(4-biphenylyl)-isopropoxycarbonyl (Bpoc), 9-fluorenylmethoxycarbonyl (Fmoc), triphenylmethyl (trityl, trt) and 2- nitrophenylsulphenyl (Nps), especially those groups which can be cleaved using conditions which do not lead to undue loss of protein function, such as Boc and Fmoc.

In some embodiments, the compound of formula (I) is selected from:

GGGW-PEG ₄ -(CH ₂ ) ₂ N ₃ (SEQ ID NO:l 1),

GGGW-PEG ₄ -(CH ₂ ) ₂ NHC(0)CH ₂ -4-(3,3-difluoro)-cyclooctyne (SEQ ID NO:12), GGGW-PEG ₄ -CH ₂ C≡CH (SEQ ID NO:13),

GGGW-PEG ₄ -(CH ₂ ) ₂ NHC(0)CH ₂ ONH ₂ (SEQ ID NO: 14),

GGGW-PEG ₄ -(CH ₂ ) ₂ NHNH ₂ (SEQ ID NO: 15),

GGGW-PEG -(CH ₂ ) ₂ NHC(0)-C ₆ H ₄ -CHO (SEQ ID NO: 16),

GGGW-PEG -(CH ₂ ) ₂ SH (SEQ ID NO: 17),

GGGW-PEG ₄ -CH ₂ CH=CH ₂ (SEQ ID NO: 18),

GGGW-PEG ₄ -CH ₂ C(0)SCH ₂ C(0)NHCH ₃ (SEQ ID NO: 19),

GGGW-PEG ₄ -(CH ₂ ) ₂ NHC(0)-CH(NH ₂ )CH ₂ SH (SEQ ID NO:20),

GGGW-PEG ₄ -(CH ₂ ) ₂ -Maleimide (SEQ ID NO:21),

GGGK-ePEG ₄ -(CH ₂ ) ₂ N ₃ (SEQ ID NO:22),

GGGK-ePEG ₄ -(CH ₂ ) ₂ NHC(0)CH ₂ -4-(3 ,3 -difluoro)-cyclooctyne (SEQ ID NO:23),

GGG -ePEG ₄ -CH ₂ C≡CH (SEQ ID NO:24),

GGGK-ePEG -(CH ₂ ) ₂ NHC(0)CH ₂ ONH ₂ (SEQ ID NO:25),

GGGK-ePEG ₄ -(CH ₂ ) ₂ NHNH ₂ (SEQ ID NO:26),

GGGK-ePEG ₄ -(CH ₂ ) ₂ NHC(0)-C ₆ H ₄ -CHO (SEQ ID NO:27),

GGGK-EPEG ₄ -(CH ₂ ) ₂ SH (SEQ ID NO:28),

GGGK-ePEG ₄ -CH ₂ CH=CH ₂ (SEQ ID NO:29),

GGGK-ePEG ₄ -CH ₂ C(0)SCH ₂ C(0)NHCH ₃ (SEQ ID NO:30),

GGGK-EPEG ₄ -(CH ₂ ) ₂ NHC(0)-CH(NH ₂ )CH ₂ SH (SEQ ID NO:31 ),

GGGK-ePEG ₄ -(CH ₂ ) ₂ -Maleimide (SEQ ID ΝΟ:32)

GGG-PEG ₄ -(CH ₂ ) ₂ N ₃ (SEQ ID NO:33),

GGG-PEG ₄ -(CH ₂ ) ₂ NHC(0)CH ₂ -4-(3,3-difluoro)-cyclooctyne (SEQ ID NO:34), GGG-PEG ₄ -CH ₂ C≡CH (SEQ ID NO:35),

GGG-PEG ₄ -(CH ₂ ) ₂ NHC(0)CH ₂ ONH ₂ (SEQ ID NO:36),

GGG-PEG ₄ -(CH ₂ ) ₂ NHNH ₂ (SEQ ID NO:37),

GGG-PEG ₄ -(CH ₂ ) ₂ NHC(0)-C ₆ H ₄ -CHO (SEQ ID NO:38),

GGG-PEG ₄ -(CH ₂ ) ₂ SH (SEQ ID NO:39),

GGG-PEG ₄ -CH ₂ CH=CH ₂ (SEQ ID NO:40),

GGG-PEG ₄ -CH ₂ C(0)SCH ₂ C(0)NHCH ₃ (SEQ ID N0:41),

GGG-PEG ₄ -(CH ₂ ) ₂ NHC(0)-CH(NH ₂ )CH ₂ SH (SEQ ID NO:42),

GGG-PEG ₄ -(CH ₂ ) ₂ -Maleimide (SEQ ID NO:43),

GGGWW-PEG ₄ -(CH ₂ ) ₂ N ₃ (SEQ ID NO:44),

GGGWW-PEG ₄ -(CH ₂ ) ₂ NHC(0)CH ₂ -4-(3,3-difluoro)-cyclooctyne (SEQ ID NO:45),

GGGWW-PEG ₄ -CH ₂ C≡CH (SEQ ID NO:46),

GGGWW-PEG ₄ -(CH ₂ ) ₂ NHC(0)CH ₂ ONH ₂ (SEQ ID NO:47),

GGGWW-PEG ₄ -(CH ₂ ) ₂ NHNH ₂ (SEQ ID NO:48),

GGGWW-PEG ₄ -(CH ₂ ) ₂ NHC(0)-C ₆ H ₄ -CHO (SEQ ID NO:49),

GGGWW-PEG ₄ -(CH ₂ ) ₂ SH (SEQ ID NO: 50),

GGGWW-PEG ₄ -CH ₂ CH=CH ₂ (SEQ ID NO:51 ),

GGGWW-PEG ₄ -CH ₂ C(0)SCH ₂ C(0)NHCH ₃ (SEQ ID NO: 52),

GGGWW-PEG ₄ -(CH ₂ ) ₂ NHC(0)-CH(NH ₂ )CH ₂ SH (SEQ ID NO:53), GGGWW-PEG ₄ -(CH ₂ ) ₂ -Maleimide (SEQ ID NO:54),

GGGWWK-ePEG ₄ -(CH ₂ ) ₂ N ₃ (SEQ ID NO:55),

GGGWWK-£PEG ₄ -(CH ₂ ) ₂ NHC(0)CH ₂ -4-(3,3-difluoro)-cyclooctyne (SEQ ID N0-56),

GGGWWK-EPEG ₄ -CH ₂ C≡CH (SEQ ID NO:57),

GGGWWK-8PEG ₄ -(CH ₂ ) ₂ NHC(0)CH ₂ ONH ₂ (SEQ ID NO:58),

GGGWWK-8PEG4-(CH ₂ ) ₂ NHNH ₂ (SEQ ID NO:59),

GGGWWK-ePEG ₄ -(CH ₂ ) ₂ NHC(0)-C ₆ H ₄ -CHO (SEQ ID NO:60),

GGGWW -ePEG ₄ -(CH ₂ ) ₂ SH (SEQ ID NO:61),

GGGWWK-8PEG ₄ -CH ₂ CH=CH ₂ (SEQ ID NO:62),

GGGWWK-ePEG ₄ -CH ₂ C(0)SCH ₂ C(0)NHCH ₃ (SEQ ID NO:63), GGGWWK-EPEG ₄ -(CH ₂ ) ₂ NHC(0)-CH(NH ₂ )CH ₂ SH (SEQ ID NO:64), GGGWWK-ePEG ₄ -(CH ₂ ) ₂ -Maleimide (SEQ ID NO:65),

GGGWWSK-EPEG ₄ -(CH ₂ ) ₂ N ₃ (SEQ ID NO:66),

GGGWWSK-ePEG ₄ -(CH ₂ ) ₂ NHC(0)CH ₂ -4-(3,3-difluoro)-cyclooctyne (SEQ ID NO:67),

GGGWWSK-EPEG ₄ -CH ₂ C≡CH (SEQ ID NO:68),

GGGWWSK-EPEG ₄ -(CH ₂ ) ₂ NHC(0)CH ₂ ONH ₂ (SEQ ID NO:69), .

GGGWWSK-EPEG ₄ -(CH ₂ ) ₂ NHNH ₂ (SEQ ID NO:70),

GGGWWSK-ePEG ₄ -(CH ₂ ) ₂ NHC(0)-C ₆ H4-CHO (SEQ ID N0:71),

GGGWWSK-EPEG ₄ -(CH ₂ ) ₂ SH (SEQ ID NO:72),

GGGWWSK-EPEG ₄ -CH ₂ CH=CH ₂ (SEQ ID NO:73),

GGGWWSK-EPEG ₄ -CH ₂ C(0)SCH ₂ C(0)NHCH ₃ (SEQ ID NO:74),

GGGWWSK-EPEG ₄ -(C¾) ₂ NHC(0)-CH(NH ₂ )CH ₂ SH (SEQ ID NO:75), GGGWWS -ePEG ₄ -(CH ₂ ) ₂ -Maleimide (SEQ ID NO:76),

GGGWWSSK-ePEG ₄ -(CH ₂ ) ₂ N ₃ (SEQ ID NO:77),

GGGWWSSK- PEG ₄ -(CH ₂ ) ₂ NHC(0)CH ₂ -4-(3,3-difluoro)-cyclooctyne (SEQ ID NO:78),

GGGWWSS -ePEG ₄ -CH ₂ C≡CH (SEQ ID NO:79),

GGGWWSSK-ePEG ₄ -(CH ₂ ) ₂ NHC(0)CH ₂ ONH ₂ (SEQ ID NO:80),

GGGWWSS -EPEG ₄ -(CH ₂ ) ₂ NHNH ₂ (SEQ ID N0:81 ),

GGGWWSSK-EPEG ₄ -(CH ₂ ) ₂ NHC(0)-C ₆ H ₄ -CHO (SEQ ID NO:82),

GGGWWSSK-8PEG ₄ -(CH ₂ ) ₂ SH (SEQ ID NO:83),

GGGWWSSK-ePEG ₄ -CH ₂ CH=CH ₂ (SEQ ID NO:84),

GGGWWSSK-8PEG ₄ -CH ₂ C(0)SCH ₂ C(0)NHCH ₃ (SEQ ID NO: 85),

GGGWWSS -ePEG ₄ -(CH ₂ ) ₂ NHC(0)-CH(NH ₂ )CH ₂ SH (SEQ ID NO:86), GGGWWSSK-£PEG -(CH ₂ ) ₂ -Maleimide (SEQ ID NO: 87),

GGGW -ePEG ₄ -(CH ₂ ) ₂ N ₃ (SEQ ID NO:88),

GGGW -ePEG ₄ -(CH ₂ ) ₂ NHC(0)CH ₂ -4-(3,3-difluoro)-cyclooctyne (SEQ ID NO:89),

GGGWK-ePEG ₄ -CH ₂ C≡CH (SEQ ID NO:90),

GGGWK-EPEG ₄ -(CH ₂ ) ₂ NHC(0)CH ₂ ONH ₂ (SEQ ID N0:91 ), GGGWK-8PEG ₄ -(CH ₂ )2NHNH2 (SEQ ID NO:92),

GGGWK-£PEG ₄ -(CH ₂ )2NHC(0)-C ₆ H4-CHO (SEQ ID NO:93),

GGGWK-ePEG ₄ -(CH ₂ ) ₂ SH (SEQ ID NO:94),

GGGWK- _e PEG ₄ -CH ₂ CH=CH ₂ (SEQ ID NO:95),

GGGWK-8PEG ₄ -CH ₂ C(0)SCH ₂ C(0)NHCH ₃ (SEQ ID NO:96),

GGGWK-ePEG ₄ -(CH ₂ ) ₂ NHC(0)-CH(NH ₂ )CH ₂ SH (SEQ ID NO :97),

GGGWK-ePEG ₄ -(CH ₂ ) ₂ -Maleimide (SEQ ID NO:98),

GGW-PEG ₄ -(CH ₂ ) ₂ N ₃ (SEQ ID NO:99),

GGW-PEG ₄ -(CH ₂ ) ₂ NHC(0)CH ₂ -4-(3,3-difluoro)-cyclooctyne (SEQ ID NO: 100), GGW-PEG ₄ -CH ₂ C≡CH (SEQ ID NO: 101 ),

GGW-PEG ₄ -(CH ₂ )2NHC(0)CH ₂ ONH ₂ (SEQ ID NO: 102),

GGW-PEG ₄ -(CH2)2NHNH ₂ (SEQ ID NO: 103),

GGW-PEG ₄ -(CH ₂ ) ₂ NHC(0)-C ₆ H4-CHO (SEQ ID NO: 104),

GGW-PEG ₄ -(CH ₂ ) ₂ SH (SEQ ID NO: 105),

GGW-PEG ₄ -CH ₂ CH=CH ₂ (SEQ ID NO: 106),

GGW-PEG ₄ -CH ₂ C(0)SCH ₂ C(0)NHCH ₃ (SEQ ID NO: 107),

GGW-PEG ₄ -(CH ₂ ) ₂ NHC(0)-CH(NH2)CH ₂ SH (SEQ ID NO: 108),

GGW-PEG ₄ -(CH ₂ ) ₂ -Maleimide (SEQ ID NO: 109),

GGK-8PEG -(CH ₂ )2N ₃ (SEQ ID NO: 110),

GGK-8PEG ₄ -(CH ₂ )2NHC(0)CH ₂ -4-(3,3-difluoro)-cyclooctyne (SEQ ID NO:l 1 1), GGK-8PEG ₄ -CH ₂ C≡CH (SEQ ID NO:l 12),

GGK-8PEG ₄ -(CH2)2NHC(0)CH ₂ ONH ₂ (SEQ ID NO:l 13),

GG -8PEG ₄ -(CH ₂ ) ₂ NHNH ₂ (SEQ ID NO: 114),

GGK-8PEG ₄ -(CH ₂ ) ₂ NHC(0)-C ₆ H ₄ -CHO (SEQ ID NO: 115),

GGK-8PEG ₄ -(CH ₂ )2SH (SEQ ID NO:l 16),

GGK-8PEG ₄ -CH ₂ CH=CH ₂ (SEQ ID NO:l 17),

GGK-8PEG -CH ₂ C(0)SCH ₂ C(0)NHCH ₃ (SEQ ID NO: 1 18),

GGK-8PEG ₄ -(CH ₂ )2NHC(0)-CH(NH2)CH ₂ SH (SEQ ID NO: 119),

GGK-8PEG ₄ -(CH ₂ ) ₂ -Maleimide (SEQ ID NO: 120),

GG-PEG ₄ -(CH ₂ ) ₂ N ₃ (SEQ ID NO:121),

GG-PEG ₄ -(CH ₂ )2NHC(0)CH ₂ -4-(3,3-difluoro)-cyclooctyne (SEQ ID NO: 122), GG-PEG ₄ -CH ₂ C≡CH (SEQ ID NO: 123),

GG-PEG ₄ -(CH ₂ ) ₂ NHC(0)CH ₂ ONH ₂ (SEQ ID NO: 124),

GG-PEG ₄ -(CH ₂ ) ₂ NHNH ₂ (SEQ ID NO: 125),

GG-PEG ₄ -(CH ₂ )2NHC(0)-C ₆ H ₄ -CHO (SEQ ID NO: 126),

GG-PEG ₄ -(CH ₂ ) ₂ SH (SEQ ID NO: 127),

GG-PEG ₄ -CH ₂ CH=CH ₂ (SEQ ID NO: 128),

GG-PEG ₄ -CH ₂ C(0)SCH ₂ C(0)NHCH ₃ (SEQ ID NO: 129),

GG-PEG ₄ -(CH ₂ ) ₂ NHC(0)-CH(NH ₂ )CH ₂ SH (SEQ ID NO: 130),

GG-PEG ₄ -(CH ₂ ) ₂ -Maleimide (SEQ ID NO: 131),

GGWW-PEG ₄ -(CH ₂ ) ₂ N ₃ (SEQ ID NO:132),

GGWW-PEG ₄ -(CH ₂ ) ₂ NHC(0)CH ₂ -4-(3,3-difluoro)-cyclooctyne (SEQ ID NO: 133),

GGWW-PEG ₄ -CH ₂ C≡CH (SEQ ID NO: 134),

GGWW-PEG ₄ -(CH ₂ ) ₂ NHC(0)CH ₂ ONH ₂ (SEQ ID NO: 135),

GGWW-PEG ₄ -(CH ₂ ) ₂ NHNH ₂ (SEQ ID NO:136),

GGWW-PEG ₄ -(CH ₂ ) ₂ NHC(0)-C ₆ H4-CHO (SEQ ID NO: 137),

GGWW-PEG ₄ -(CH ₂ ) ₂ SH (SEQ ID NO: 138),

GGWW-PEG ₄ -CH ₂ CH=CH ₂ (SEQ ID NO: 139),

GGWW-PEG ₄ -CH ₂ C(0)SCH ₂ C(0)NHCH ₃ (SEQ ID NO: 140),

GGWW-PEG ₄ -(CH ₂ ) ₂ NHC(0)-CH(NH ₂ )CH ₂ SH (SEQ ID NO: 141),

GGWW-PEG -(CH ₂ ) ₂ -Maleimide (SEQ ID NO: 142),

GGWW - _e PEG ₄ -(CH ₂ ) ₂ N ₃ (SEQ ID NO: 143),

GGWWK-€PEG ₄ -(CH ₂ ) ₂ NHC(0)CH ₂ -4-(3,3-difluoro)-cyclooctyne (SEQ ID NO: 144),

GGWWK- _E PEG ₄ -CH ₂ C≡CH (SEQ ID NO: 145),

GGWWK-£PEG ₄ -(CH ₂ ) ₂ NHC(0)CH ₂ ONH ₂ (SEQ ID NO: 146),

GGWWK-ePEG ₄ -(CH ₂ ) ₂ NHNH ₂ (SEQ ID NO: 147),

GGWWK-ePEG ₄ -(CH2)2NHC(0)-C ₆ H4-CHO (SEQ ID NO: 148),

GGWWK-ePEG ₄ -(CH ₂ ) ₂ SH (SEQ ID NO: 149),

GGWWK-£PEG ₄ -CH ₂ CH=CH ₂ (SEQ ID NO: 150),

GGWWK-ePEG ₄ -CH ₂ C(0)SCH ₂ C(0)NHCH ₃ (SEQ ID NO: 151), GGWWK-8PEG -(CH ₂ ) ₂ NHC(0)-CH(NH ₂ )CH ₂ SH (SEQ ID NO: 152), GGWWK-ePEG ₄ -(CH ₂ ) ₂ -Maleimide (SEQ ID NO: 153),

GGWWSK-8PEG ₄ -(CH ₂ ) ₂ N ₃ (SEQ ID NO: 154),

GGWWSK-sPEG ₄ -(CH ₂ ) ₂ NHC(0)CH ₂ -4-(3,3-difluoro)-cyclooctyne (SEQ ID NO:155),

GGWWS -ePEG ₄ -CH ₂ C≡CH (SEQ ID NO: 156),

GGWWS -ePEG ₄ -(CH ₂ ) ₂ NHC(0)CH ₂ ONH ₂ (SEQ ID NO: 157),

GGWWSK-8PEG ₄ -(CH ₂ ) ₂ NHNH ₂ (SEQ ID NO: 158),

GGWWSK-8PEG ₄ -(CH ₂ ) ₂ NHC(0)-C ₆ H ₄ -CHO (SEQ ID NO: 159),

GGW WSK-ePEG ₄ -(CH ₂ ) ₂ SH (SEQ ID NO: 160),

GGWWS -8PEG ₄ -CH ₂ CH=CH ₂ (SEQ ID NO: 161),

GGWWS -8PEG ₄ -CH ₂ C(0)SCH ₂ C(0)NHCH ₃ (SEQ ID NO: 162),

GGWWSK-8PEG ₄ -(CH ₂ ) ₂ NHC(0)-CH(NH ₂ )CH ₂ SH (SEQ ID NO: 163), GGWWSK-8PEG ₄ -(CH ₂ ) ₂ -Maleimide (SEQ ID NO: 164),

GGWWSSK-8PEG ₄ -(CH ₂ ) ₂ N ₃ (SEQ ID NO: 165),

GGWWSSK-8PEG ₄ -(CH ₂ ) ₂ NHC(0)CH ₂ -4-(3,3-difluoro)-cyclooctyne (SEQ ID NO: 166),

GGWWSS -ePEG ₄ -CH ₂ C≡CH (SEQ ID NO: 167),

GGWWSSK-8PEG ₄ -(CH ₂ ) NHC(0)CH ₂ ONH ₂ (SEQ ID NO: 168),

GGWWSSK-8PEG ₄ -(CH ₂ ) ₂ NHNH ₂ (SEQ ID NO: 169),

GGWWSSK-8PEG ₄ -(CH ₂ ) ₂ NHC(0)-C ₅ H ₄ -CHO (SEQ ID NO: 170),

GGWWSSK-8PEG ₄ -(CH ₂ ) ₂ SH (SEQ ID NO: 171),

GGWWSSK-ePEG ₄ -CH ₂ CH=CH ₂ (SEQ ID NO: 172),

GGWWSSK-8PEG ₄ -CH ₂ C(0)SCH ₂ C(0)NHCH ₃ (SEQ ID NO: 173),

GGWWSSK-8PEG ₄ -(CH ₂ ) ₂ NHC(0)-CH(NH ₂ )CH ₂ SH (SEQ ID NO: 174),

GGWWSSK-8PEG ₄ -(CH ₂ ) ₂ -Maleimide (SEQ ID NO: 175),

GGWK-ePEG ₄ -(CH ₂ ) ₂ N ₃ (SEQ ID NO: 176),

GGWK-8PEG ₄ -(CH ₂ ) ₂ NHC(0)CH ₂ -4-(3,3-difluoro)-cyclooctyne (SEQ ID NO: 177),

GGWK-ePEG ₄ -CH ₂ C≡CH (SEQ ID O:178),

GGWK-8PEG ₄ -(CH ₂ ) ₂ NHC(0)CH ₂ ONH ₂ (SEQ ID NO: 179), GGWK-EPEG ₄ -(CH ₂ ) ₂ NHNH ₂ (SEQ ID NO: 180),

GGWK-8PEG ₄ -(CH ₂ ) ₂ NHC(0)-C ₆ H4-CHO (SEQ ID NO:181),

GGWK-EPEG ₄ -(CH ₂ ) ₂ SH (SEQ ID NO: 182),

GGWK-ePEG ₄ -CH ₂ CH=CH ₂ (SEQ ID NO: 183),

GGWK-ePEG4-CH ₂ C(0)SCrt ₂ C(0)NHCH ₃ (SEQ ID NO: 184),

GGWK-ePEG ₄ -(CH ₂ ) ₂ NHC(0)-CH(NH ₂ )CH ₂ SH (SEQ ID NO: 185), ^' GGWK-ePEG -(CH ₂ ) ₂ -Maleimide (SEQ ID NO: 186),

GW-PEG ₄ -(CH ₂ ) ₂ N ₃ (SEQ ID NO: 187),

GW-PEG ₄ -(CH ₂ ) ₂ NHC(0)CH ₂ -4-(3,3-difluoro)-cyclooctyne (SEQ ID NO: 188), GW-PEG ₄ -CH ₂ C≡CH (SEQ ID NO: 189),

GW-PEG ₄ -(CH ₂ ) ₂ NHC(0)CH ₂ ONH ₂ (SEQ ID NO: 190),

GW-PEG ₄ -(CH ₂ ) ₂ NHNH ₂ (SEQ ID NO: 191),

GW-PEG ₄ -(CH ₂ ) ₂ NHC(0)-C ₆ H4-CHO (SEQ ID NO: 192),

GW-PEG ₄ -(CH ₂ ) ₂ SH (SEQ ID NO: 193),

GW-PEG ₄ -CH ₂ CH=CH ₂ (SEQ ID NO: 194),

GW-PEG ₄ -CH ₂ C(0)SCH ₂ C(0)NHCH ₃ (SEQ ID NO: 195),

GW-PEG ₄ -(CH ₂ ) ₂ NHC(0)-CH(NH ₂ )CH ₂ SH (SEQ ID NO: 196),

GW-PEG ₄ -(CH ₂ ) ₂ -Maleimide (SEQ ID NO: 197),

G -ePEG ₄ -(CH ₂ ) ₂ N ₃ (SEQ ID NO: 198),

GK-8PEG ₄ -(CH ₂ ) ₂ NHC(0)CH ₂ -4-(3,3-difluoro)-cyclooctyne (SEQ ID NO: 199),

G - _e PEG ₄ -CH ₂ C≡CH (SEQ ID NO:200),

G -ePEG ₄ -(CH ₂ ) ₂ NHC(0)CH ₂ ONH ₂ (SEQ ID NO:201),

G -ePEG ₄ -(CH ₂ ) ₂ NHNH ₂ (SEQ ID NO:202),

GK-ePEG ₄ -(CH ₂ ) ₂ NHC(0)-C ₆ H ₄ -CHO (SEQ ID NO:203),

GK-ePEG ₄ -(CH ₂ ) ₂ SH (SEQ ID NO:204),

GK-ePEG ₄ -CH ₂ CH=CH ₂ (SEQ ID NO:205),

G -£PEG ₄ -CH ₂ C(0)SCH ₂ C(0)NHCH ₃ (SEQ ID NO:206),

GK-ePEG ₄ -(CH ₂ ) ₂ NHC(0)-CH(NH ₂ )CH ₂ SH (SEQ ID NO:207),

GK-EPEG ₄ -(CH ₂ ) ₂ -Maleimide (SEQ ID NO:208),

G-PEG ₄ -(CH ₂ ) ₂ N ₃ (SEQ ID NO:209),

G-PEG ₄ -(CH ₂ ) ₂ NHC(0)CH ₂ -4-(3,3-difluoro)-cyclooctyne (SEQ ID NO:210), G-PEG ₄ -CH ₂ C≡CH (SEQ ID N0:211 ),

G-PEG ₄ -(CH ₂ ) ₂ NHC(0)CH ₂ ONH ₂ (SEQ ID N0:212),

G-PEG ₄ -(CH ₂ ) ₂ NHNH ₂ (SEQ ID N0:213),

G-PEG ₄ -(CH ₂ ) ₂ NHC(0)-C ₆ H ₄ -CHO (SEQ ID NO:214),

G-PEG ₄ -(CH ₂ ) ₂ SH (SEQ ID NO:215),

G-PEG ₄ -CH ₂ CH=CH ₂ (SEQ ID N0:216

G-PEG ₄ -CH ₂ C(0)SCH ₂ C(0)NHCH ₃ (SEQ ID NO:217)

G-PEG -(CH ₂ ) ₂ NHC(0)-CH(NH ₂ )CH ₂ SH (SEQ ID N0:218),

G-PEG ₄ -(CH ₂ ) ₂ -Maleimide (SEQ ID N0:219),

GWW-PEG ₄ -(CH ₂ ) ₂ N ₃ (SEQ ID NO:220),

GWW-PEG ₄ -(CH ₂ ) ₂ NHC(0)CH ₂ -4-(3,3-difluoro)-cyclooctyne (SEQ ID NO:221), GWW-PEG ₄ -CH ₂ C≡CH (SEQ ID NO:222),

GWW-PEG ₄ -(CH ₂ ) ₂ NHC(0)CH ₂ ONH ₂ (SEQ ID NO:223),

GWW-PEG ₄ -(CH ₂ ) ₂ NHNH ₂ (SEQ ID NO:224),

GWW-PEG ₄ -(CH ₂ ) ₂ NHC(0)-C ₆ H ₄ -CHO (SEQ ID NO:225),

GWW-PEG ₄ -(CH ₂ ) ₂ SH (SEQ ID NO:226),

GWW-PEG -CH ₂ CH=CH ₂ (SEQ ID NO:227),

GWW-PEG ₄ -CH ₂ C(0)SCH ₂ C(0)NHCH ₃ (SEQ ID NO:228),

GWW-PEG -(CH ₂ ) ₂ NHC(0)-CH(NH ₂ )CH ₂ SH (SEQ ID NO:229),

GWW-PEG ₄ -(CH ₂ ) ₂ -Maleimide (SEQ ID NO:230),

GWWK-ePEG ₄ -(CH ₂ ) ₂ N ₃ (SEQ ID NO:231),

GWW -£PEG ₄ -(CH ₂ ) ₂ NHC(0)CH ₂ -4-(3,3-difluoro)-cyclooctyne (SEQ ID NO:232),

GWWK-EPEG ₄ -CH ₂ C≡CH (SEQ ID NO:233),

GWW -ePEG ₄ -(CH ₂ ) ₂ NHC(0)CH ₂ ONH ₂ (SEQ ID NO:234),

GWWK-ePEG ₄ -(CH ₂ ) ₂ NHNH ₂ (SEQ ID NO:235),

GWWK- _e PEG ₄ -(CH ₂ ) ₂ NHC(0)-C ₆ H ₄ -CHO (SEQ ID NO:236),

GWWK-ePEG ₄ -(CH ₂ ) ₂ SH (SEQ ID NO:237),

GWWK-EPEG ₄ -CH ₂ CH=CH ₂ (SEQ ID NO:238),

GWW -ePEG ₄ -CH ₂ C(0)SCH ₂ C(0)NHCH ₃ (SEQ ID N0.239),

GWWK-ePEG ₄ -(CH ₂ ) ₂ NHC(0)-CH(NH ₂ )CH ₂ SH (SEQ ID NO:240), G W WK-sPEG ₄ -(CH ₂ ) ₂ -Maleimide (SEQ ID NO:241 ),

GWWS - _e PEG ₄ -(CH ₂ ) ₂ N3 (SEQ ID NO:242),

GWWS -8PEG ₄ -(CH ₂ ) ₂ NHC(0)CH ₂ -4-(3,3-difluoro)-cyclooctyne (SEQ ID NO:243),

GWWSK-8PEG ₄ -CH ₂ C≡CH (SEQ ID NO:244),

GWWSK-ePEG ₄ -(CH ₂ ) ₂ NHC(0)CH ₂ ONH ₂ (SEQ ID NO:245),

GWWS -8PEG ₄ -(CH ₂ ) ₂ NHNH ₂ (SEQ ID NO:246),

GWWS -sPEG ₄ -(CH ₂ ) ₂ NHC(0)-C ₆ H ₄ -CHO (SEQ ID N0.247),

GWWS -EPEG ₄ -(CH ₂ ) ₂ SH (SEQ ID NO:248),

GWWSK-ePEG ₄ -CH ₂ CH=CH ₂ (SEQ ID NO:249),

GWWS -8PEG ₄ -CH2C(0)SCH ₂ C(0)NHCH3 (SEQ ID NO:250),

GWWSK-8PEG ₄ -(CH ₂ ) ₂ NHC(0)-CH(NH ₂ )CH ₂ SH (SEQ ID NO:251),

GWWSK-£PEG ₄ -(CH ₂ ) ₂ -Maleimide (SEQ ID NO:252),

GWWSSK-8PEG ₄ -(CH ₂ ) ₂ N ₃ (SEQ ID N0.253),

GWWSSK-8PEG ₄ -(CH ₂ ) ₂ NHC(0)CH ₂ -4-(3,3-difluoro)-cyclooctyne (SEQ ID NO:254),

GWWSSK-ePEG ₄ -CH ₂ C≡CH (SEQ ID N0.255),

GWWSSK-8PEG ₄ -(CH ₂ ) ₂ NHC(0)CH ₂ ONH ₂ (SEQ ID NO:256),

GWWSS -8PEG ₄ -(CH ₂ ) ₂ NHNH ₂ (SEQ ID NO:257),

GWWSSK-8PEG ₄ -(CH ₂ ) ₂ NHC(0)-C ₆ H4-CHO (SEQ ID NO:258),

GWWSSK-8PEG ₄ -(CH ₂ ) ₂ SH (SEQ ID NO:259),

GWWSSK-8PEG ₄ -CH ₂ CH=CH ₂ (SEQ ID NO:260),

GWWSS -8PEG ₄ -CH ₂ C(0)SCH ₂ C(0)NHCH ₃ (SEQ ID NO:261),

GWWSSK-8PEG ₄ -(CH ₂ ) ₂ NHC(0)-CH(NH ₂ )CH ₂ SH (SEQ ID NO:262),

GWWSSK-8PEG ₄ -(CH ₂ ) ₂ -Maleimide (SEQ ID NO:263),

GW -8PEG ₄ -(CH ₂ ) ₂ N ₃ (SEQ ID NO:264),

GWK-8PEG ₄ -(CH ₂ ) ₂ NHC(0)CH ₂ -4-(3,3-difluoro)-cyclooctyne (SEQ ID NO:265), GWK-8PEG ₄ -CH ₂ C≡CH (SEQ ID NO:266),

GWK-8PEG ₄ -(CH ₂ ) ₂ NHC(0)CH ₂ ONH ₂ (SEQ ID NO:267),

GWK-8PEG ₄ -(CH ₂ ) ₂ NHNH ₂ (SEQ ID N0.268),

GWK-8PEG ₄ -(CH ₂ ) ₂ NHC(0)-C ₆ H4-CHO (SEQ ID NO:269), GWK-ePEG ₄ -(CH ₂ ) ₂ SH (SEQ ID NO:270),

GW -ePEG ₄ -CH ₂ CH=CH ₂ (SEQ ID NO:271 ),

GWK-ePEG ₄ -CH ₂ C(0)SCH ₂ C(0)NHCH ₃ (SEQ ID NO:272),

GW -8PEG ₄ -(CH ₂ ) ₂ NHC(0)-CH(NH ₂ )CH ₂ SH (SEQ ID NO:273),

GWK-£PEG ₄ -(CH ₂ ) ₂ -Maleimide (SEQ ID NO:274),.

GGGWWGA-pG (SEQ ID NO:275),

GGGAGAGAC (SEQ ID NO:276),

GGGWWSSK-sPEG ₄ -SANH (SEQ ID NO:277),

GGGWWSSK-ePEG ₄ -DBCO (SEQ ID NO:278),

GGGWWSSK- _E PEG ₄ -penenoic acid (SEQ ID NO:279),

GGG WSK-ePEG ₄ -(CH ₂ )N ₃ (SEQ ID NO :280),

GGGWSK-£PEG ₄ -(CH ₂ ) ₂ NHC(0)CH ₂ -4-(3,3-difluoro)-cyclooctyne (SEQ ID NO:281),

GGGWS -£PEG ₄ -CH ₂ C≡CH (SEQ ID NO:282),

GGGWSK-8PEG ₄ -(CH ₂ ) ₂ NHC(0)CH ₂ ONH ₂ (SEQ ID NO:283),

GGGWSK-ePEG ₄ -(CH ₂ ) ₂ NHNH ₂ (SEQ ID NO:284),

GGGWSK-ePEG ₄ -(CH ₂ ) ₂ NHC(0)-C ₆ H ₄ -CHO (SEQ ID NO:285),

GGGWSK-sPEG ₄ -(CH ₂ ) ₂ SH (SEQ ID NO.286),

GGGWSK-£PEG ₄ -CH ₂ CH=CH ₂ (SEQ ID NO:287),

GGGWSK-£PEG ₄ -CH ₂ C(0)SCH ₂ C(0)NHCH ₃ (SEQ ID NO:288),

GGGWSK-£PEG ₄ -(CH ₂ ) ₂ NHC(0)-CH(NH ₂ )CH ₂ SH (SEQ ID NO:290),

GGGWSK-£PEG ₄ -(CH ₂ ) ₂ -Maleimide (SEQ ID NO:290),

GGG-EEE-SH (SEQ ID NO:291 ),

GGWE-PEG ₄ -(CH ₂ ) ₂ NHNH ₂ (SEQ ID NO:292),

GGGWSK-(e-NH)-CH ₂ C≡CH (SEQ ID O:293),

GGGG-EEE-PEG ₄ -(CH ₂ ) ₂ N ₃ (SEQ ID N0.294),

GGYK-8PEGi ₂ -(CH ₂ ) ₂ NHCOC ₆ H ₄ CHO (SEQ ID NO:295),

GGYK-8PEG ₆ -(CH ₂ ) ₂ NHCOC ₆ H ₄ CHO (SEQ ID NO:296),

GE-PEG ₄ -(CH ₂ ) ₂ N ₃ (SEQ ID NO:297),

GGSEC (SEQ ID NO:298),

GGGFDK-EKK -(CH ₂ ) ₂ N ₃ (SEQ ID N0.299), GGGFDK-8KKK-(CH ₂ ) ₂ NHC(0)CH2-4-(3,3-difluoro)-cyclooctyne (SEQ ID NO:300),

GGGFDK-eKKK-CH ₂ C≡CH (SEQ ID NO:301),

GGGFDK-eKKK-(CH ₂ ) ₂ NHC(0)CH ₂ ONH ₂ (SEQ ID NO:302),

GGGFDK-eKKK-(CH ₂ ) ₂ NHNH ₂ (SEQ ID NO:303),

GGGFDK-eKKK-(CH ₂ ) ₂ NHC(0)-C ₆ H ₄ -CHO (SEQ ID NO:304),

GGGFDK- _E KK -(CH ₂ ) ₂ SH (SEQ ID NO:305),

GGGFDK-EKK -CH ₂ CH=CH ₂ (SEQ ID NO:306),

GGGFDK-eKKK-CH ₂ C(0)SCH ₂ C(0)NHCH ₃ (SEQ ID NO:307),

GGGFDK-e KK-(CH ₂ ) ₂ NHC(0)-CH(NH ₂ )CH ₂ SH (SEQ ID NO:308), GGGFDK-8KKK-(CH ₂ ) ₂ -Maleimide (SEQ ID NO:309),

GGGWSOm (5PEG ₄ )-(CH ₂ ) ₂ N ₃ (SEQ ID NO:310),

GGGWSOm (8PEG ₄ )-(CH ₂ ) ₂ NHC(0)CH ₂ -4-(3,3-difluoro)-cyclooctyne (SEQ ID NO-.311),

GGGWSOm (5PEG ₄ )-CH ₂ C≡CH (SEQ ID NO:312),

GGGWSOm (8PEG ₄ )-(CH ₂ ) ₂ NHC(0)CH ₂ ONH ₂ (SEQ ID NO:313),

GGGWSOm (5PEG ₄ )-(CH ₂ ) ₂ NHNH ₂ (SEQ ID NO:314),

GGGWSOm (8PEG ₄ )-(CH ₂ ) ₂ NHC(0)-C ₆ H ₄ -CHO (SEQ ID NO:315),

GGGWSOm (5PEG ₄ )-(CH ₂ ) ₂ SH (SEQ ID NO:316),

GGGWSOm (6PEG ₄ )-CH ₂ CH=CH ₂ (SEQ ID NO:317),

GGGWSOm (5PEG ₄ )-CH ₂ C(0)SCH ₂ C(0)NHCH ₃ (SEQ ID NO:318),

GGGWSOm (5PEG ₄ )-(CH ₂ ) ₂ NHC(0)-CH(NH ₂ )CH ₂ SH (SEQ ID NO:319), GGGWSOm (8PEG ₄ )-(CH ₂ ) ₂ -Maleimide (SEQ ID NO:320),

GGGWSOm (5PEG ₄ )-(CH ₂ ) ₂ SCOCH ₃ (SEQ ID NO:321),

GGGE-8PEG ₄ -(CH ₂ ) ₂ N ₃ (SEQ ID NO:322),

GGGE-5PEG ₄ -(CH ₂ ) ₂ NHC(0)CH ₂ -4-(3,3-difluoro)-cyclooctyne (SEQ ID NO:323),

GGGE-5PEG ₄ -CH ₂ C≡CH (SEQ ID NO:324),

GGGE-5PEG ₄ -(CH ₂ ) ₂ NHC(0)CH ₂ ONH ₂ (SEQ ID NO:325),

GGGE-8PEG ₄ -(CH ) ₂ NHNH ₂ (SEQ ID N0.326),

GGGE-5PEG ₄ -(CH ₂ ) ₂ NHC(0)-C ₆ H ₄ -CHO (SEQ ID NO:327), GGGE-8PEG ₄ -(CH ₂ ) ₂ SH (SEQ ID NO:328),

GGGE-8PEG ₄ -CH ₂ CH=CH ₂ (SEQ ID NO:329),

GGGE-8PEG ₄ -CH ₂ C(0)SCH ₂ C(0)NHCH ₃ (SEQ ID NO:330),

GGGE-8PEG ₄ -(CH ₂ ) ₂ NHC(0)-CH(NH ₂ )CH ₂ SH (SEQ ID NO:331),

GGGE-8PEG ₄ -(CH ₂ ) ₂ -Maleimide (SEQ ID NO:332),

GGGE-8PEG ₄ -(CH ₂ ) ₂ SCOCH ₃ (SEQ ID NO:333),

GGWYSOrn-8PEG ₆ -(CH ₂ ) ₂ N ₃ (SEQ ID NO:334),

GGWYSOrn-5PEG ₆ -(CH ₂ ) ₂ NHC(0)CH ₂ -4-(3,3-difluoro)-cyclooctyne (SEQ ID NO:335),

GGWYSOrn-8PEG ₆ -CH ₂ C≡CH (SEQ ID NO:336),

GGWYSOm-8PEG ₆ -(CH ₂ ) ₂ NHC(0)CH ₂ ONH ₂ (SEQ ID NO:337),

GGWYSOrn-8PEG ₆ -(CH ₂ ) ₂ NHNH ₂ (SEQ ID NO:338),

GGWYSOrn-5PEG ₆ -(CH ₂ )2NHC(0)-C ₆ H ₄ -CHO (SEQ ID NO:339),

GGWYSOrn-8PEG ₄ -(CH ₂ ) ₂ SH (SEQ ID NO:340),

GGWYSOrn-8PEG ₆ -CH ₂ CH=CH ₂ (SEQ ID NO:341),

GGWYSOrn-8PEG6-CH ₂ C(0)SCH ₂ C(0)NHCH ₃ (SEQ ID NO:342),

GGWYSOrn-8PEG ₆ -(CH ₂ ) ₂ NHC(0)-CH(NH ₂ )CH ₂ SH (SEQ ID NO:343), GGWYSOrn-8PEG ₆ -(CH ₂ ) ₂ -Maleimide (SEQ ID N0.344),

GGGWE-8PEG ₄ -(CH ₂ )N ₃ (SEQ ID NO:345),

GGGWE-8PEG ₄ -(CH ₂ ) ₂ NHC(0)CH ₂ -4-(3,3-difluoro)-cyclooctyne (SEQ ID NO:346),

GGGWE-8PEG ₄ -CH ₂ C≡CH (SEQ ID NO:347),

GGGWE-8PEG ₄ -(CH ₂ ) ₂ NHC(0)CH ₂ ONH ₂ (SEQ ID NO:348),

GGGWE-8PEG ₄ -(CH ₂ ) ₂ NHNH ₂ (SEQ ID N0.349),

GGGWE-8PEG ₄ -(CH ₂ ) ₂ NHC(0)-C ₆ H ₄ -CHO (SEQ ID NO:350),

GGGWE-8PEG ₄ -(CH ₂ ) ₂ SH (SEQ ID NO:351),

GGGWE-8PEG ₄ -CH ₂ CH=CH ₂ (SEQ ID NO:352),

GGGWE-8PEG ₄ -CH ₂ C(0)SCH ₂ C(0)NHCH ₃ (SEQ ID NO:353),

GGGWE-8PEG ₄ -(CH ₂ ) ₂ NHC(0)-CH(NH ₂ )CH ₂ SH (SEQ ID NO:354), GGGWE-8PEG -(CH ₂ ) ₂ -Maleimide (SEQ ID NO:355),

GGGGK-£PEG ₈ -(CH ₂ )N ₃ (SEQ ID NO:356), GGGG -£PEG ₈ -(CH ₂ ) ₂ NHC(0)CH2-4-(3,3-difluoro)-cyclooctyne (SEQ ID NO:357),

GGGGK-ePEG ₈ -CH ₂ C≡CH (SEQ ID NO:358),

GGGGK-8PEG ₈ -(CH ₂ ) ₂ NHC(0)CH ₂ ONH ₂ (SEQ ID NO:359),

GGGG -8PEG ₈ -(CH ₂ ) ₂ NHNH ₂ (SEQ ID NO:360),

GGGGK-8PEG ₈ -(CH ₂ ) ₂ NHC(0)-C ₆ H ₄ -CHO (SEQ ID NO:361),

GGGGK-8PEG ₈ -(CH ₂ ) ₂ SH (SEQ ID NO:362),

GGGG -ePEG ₈ -CH ₂ CH=CH ₂ (SEQ ID NO: 363),

GGGGK-ePEG ₈ -CH ₂ C(0)SCH ₂ C(0)NHCH ₃ (SEQ ID NO:364),

GGGG -ePEG ₈ -(CH ₂ ) ₂ NHC(0)-CH(NH ₂ )CH ₂ SH (SEQ ID NO:365),

GGGG -ePEG ₈ -(CH ₂ ) ₂ -Maleimide (SEQ ID NO:366),

GGGGGWK-e-EEEPEG ₄ -(CH ₂ ) ₂ N ₃ (SEQ ID NO:367),

GGGGGW -8-EEEPEG ₄ -(CH ₂ ) ₂ NHC(0)CH ₂ -4-(3,3-difluoro)-cyclooctyne (SEQ ID NO:368),

GGGGGWK-e-EEEPEG ₄ -CH ₂ C≡CH (SEQ ID NO:369),

GGGGGW -8-EEEPEG ₄ -(CH ₂ ) ₂ NHC(0)CH ₂ ONH ₂ (SEQ ID NO:370),

GGGGGW -e-EEEPEG ₄ -(CH ₂ ) ₂ NHNH ₂ (SEQ ID NO:371),

GGGGGW -e-EEEPEG ₄ -(CH ₂ ) ₂ NHC(0)-C ₆ H4-CHO (SEQ ID N0:372),

GGGGGW -e-EEEPEG ₄ -(CH ₂ ) ₂ SH (SEQ ID NO:373),

GGGGGWK-e-EEEPEG ₄ -CH ₂ CH=CH ₂ (SEQ ID NO:374),

GGGGGWK-6-EEEPEG ₄ -CH ₂ C(0)SCH ₂ C(0)NHCH ₃ (SEQ ID NO:375), GGGGGWK-E-EEEPEG ₄ -(CH ₂ ) ₂ NHC(0)-CH(NH ₂ )CH ₂ SH (SEQ ID NO:376), GGGGGWK-e-EEEPEG ₄ -(CH ₂ ) ₂ -Maleimide (SEQ ID NO:377),

GGGGGWDDK-e-lipoic acid (SEQ ID N0.378),

GGDK-8-CH(NH ₂ )CH ₂ SH (SEQ ID NO:379),

In one aspect of the present invention are compounds of formula (IV) which are a subset of compounds of formula (I):

R ₁ -HN-G(G) _n -XiX ₂ YiZi wherein G is a glycine residue;

Ri is hydrogen or an amino protecting group;

Xi is absent or is an amino acid residue or a peptide of 2-10 amino acid residues; X ₂ is an amino acid residue with a reactive functional group in its side chain;

Yi is a spacer group which is attached to X ₂ through the reactive functional group in the side chain of X ₂ ;

Z] is a reactive functional group; and

n is 0 or an integer of 1 to 4. Preferred embodiments of X ₂ , Yi, Zi and n are as described for formula (I) above.

In the compounds of formula (IV), X ₂ is an amino acid group which has a reactive functional group in the side chain and Yi is a spacer group attached to the reactive functional group of the side chain of X ₂ . In some embodiments, X ₂ is selected from lysine, ornithine, aspartic acid and glutamic acid.

Particular sequences include those of SEQ ID NOs:22-32, 55-98, 110-120, 143-186, 198-208, 231-274, 277-290, 295, 296, 299-377 and 379. The compounds of formula (I) and formula (IV) may be prepared by methods known in the • art. The peptide sequence G(G)„-XiX ₂ may be prepared by solid phase synthetic methods using Fmoc chemistry as described in Fmoc Solid Phase Synthesis, A practical approach, edited by W. C. Chan, P.D. White, Oxford Press, 2000 or Boc chemistry as described by Schnoltzer et al., Int. J. Peptide Protein Res., 40, 180 (1992). Further reactions to incorporate Yi and Zi may be performed while G(G)„-XiX ₂ is still resin bound. Following deprotection and cleavage from the solid support, the peptides may be purified using high performance liquid chromatography (HPLC).

Alternatively, the peptide sequence G(G) _n -XiX ₂ may be prepared by recombinant DNA technology where a DNA sequence encoding the desired peptide sequence, or its precursor, may be inserted into a suitable vector and protein expressed in a suitable expression system.

Where present, Yi may be incorporated into the peptide by reaction of a terminal functional group on the spacer with the C-terminal carboxylic acid of the G(G) _n -XiX ₂ peptide or with a functional group on the side chain of X ₂ if it is present.

For example, Yi may include a terminal amino group which is able to react with the C- terminal carboxylic acid of G, Xi or X ₂ or a carboxy side chain group of X ₂ , which may be activated by formation of an activated ester by methods known in the art, to form an amide.

Another option is where Y _\ includes a terminal carboxy group, which upon activation is able to react with an amino group in the side chain of X ₂ to form an amide bond.

Yi may be reacted with the peptide G(G)„-XiX ₂ before reaction with another group to introduce Z _\ or Yj may be reacted with another group to introduce Z _\ before reaction with the peptide G(G) _n -X ₁ X ₂ . The Zi group may also be introduced by methods known in the art such as amide formation, nucleophilic substitution, elimination, oxidation and reduction. For example, Z _\ may be bifunctional and include an amine functional group capable of amide formation with the C-terminal carboxy group of G(G) _n or G(G)„-Xi or G(G)n-X]X ₂ or with a terminal carboxy group of Yj. Alternatively where Zi includes a carboxy functional group, this may react with a side chain amine in X ₂ or with a terminal amino group of Yi .

When Zi is an azide, the azide may be introduced onto Yi, Xi, or X ₂ or the C-terminal carboxy group of the G(G)„ sequence by reaction of an aminoalkylN3 group with an activated carboxy group to form an amide or reaction of an activated H0 ₂ CalkylN3 group with a side chain amino group of X ₂ or a terminal amino group of Y^ Alternatively, the azide may be formed in situ if Yi terminates with an alkylhydroxy group by using the method described in Tetrahedron, 2009, 65: 7329. Briefly the hydroxy group may be reacted with mesyl chloride to form a mesylate which can be reacted with sodium azide (NaN ₃ ) to form an azide.

5 When Yi includes a terminal hydroxy group, Z _\ as a thiol group may be introduced by conversion of the terminal hydroxyl group to a thioacetate, which is a thiol precursor, by reaction of the hydroxy group with tosyl chloride to form a tosylate which is then reacted with tetrabutylammonium iodide (TBAI) and potassium thioacetate (KSAc) (WO 05/010481, pi 6).

10

When Yi includes a terminal amino group, Z| as a cysteine residue may be introduced by reacting the carboxyl moiety of a protected cysteine with the amino group of Yi under amide forming conditions. A suitable protected cysteine is N-(t-butoxycarbonyl)- thiazolidone-2-carboxylic acid.

!5

When Yi includes a terminal carboxy group, Zi as a thioester may be introduced by reacting an thiol group, such as N-(methyl)mercaptoacetamide, with the activated carboxy group of Yi. 0 When Yi includes a terminal hydroxy group, Zi as an alkene may be introduced by the method outlined in Angew Chem., 2008, 47, 3192, Supp. p7. Briefly, the hydroxy group is reacted with allylchloride and tetrabutylammonium sulphate in dichloromethane in the presence of aqueous NaOH. 5 For any of the reactions above, functional groups may need to be protected and deprotected in some cases selectively. Suitable protecting groups are known in the art and may be found in, for example, Protective Groups in Organic Synthesis, Greene and Wutz, third edition, Wiley Interscience, 1999. 0 The sortase mediated reaction may be performed on any scale and will depend on the amount of conjugated product required. The reaction mixture is maintained at a temperature suitable for conjugation to occur, for example, between 15°C and 50°C, especially from 23°C to 40°C, more especially 30°C to 40°C, most especially about 37°C.

Suitable ratios of sortase enzyme to compound of formula (III) are between 1 :1 to 1 :1000. In some embodiments, the ratio of enzyme to compound of formula (III) is 1 :1 or greater, 1:2 or greater, 1 :3 or greater, 1:4 or greater, 1 :5 or greater, 1 :6 or greater, 1 :7 or greater, 1 :8 or greater, 1 :9 or greater or 1 : 10 or greater. In some embodiments the ratio of enzyme to compound of formula (III) is about 1 :1 to 1 :2.

In some embodiments, the compound of formula (III) is present in an amount between 1 μΜ and 10 mM, especially 1 μΜ to 5 mM, more especially 1 μΜ to 1 mM, especially about 10 μΜ.

In some embodiments, the compound of formula (I) or formula (IV) is present in a ratio with the compound of formula (III) of between 1 :10 to 10:1. In some embodiments, especially where X ₃ is a purification tag, the ratio of formula (I) or formula (IV) to formula (III) is between 1 :1 and 1 :10, especially 1 :1 to 1 :5, more especially about 1 :3. In some embodiments the amount of compound of formula (I) or formula (IV) is in the range of 1 μΜ and 10 mM, especially 10 μΜ to 5 mM, more especially 10 μΜ to 1 mM.

In some embodiments, the sortase enzyme is present in an amount of between 1 μΜ and 500 μΜ, especially about 5 μΜ to 200 μΜ, or 5 μΜ to 100 μΜ, more especially about 10 μΜ.

The sortase mediated reaction may be conveniently carried out in aqueous media such as water or buffer. In some embodiments, the aqueous media is a buffer that maintains a pH between 6 to 8.5, especially 6.5 to 8.5, 7 to 8.5, 7.5 to 8.5, more especially about 8. One suitable buffer is 50 mM Tris with 150 mM sodium chloride at pH 8. The reaction mixture may also comprise calcium ions preferably in the form of calcium chloride, in an amount between 0.1 mM and 20 mM, especially 0.1 to 15 mM. In particular instances where the group A is an antibody or fragment thereof, the calcium chloride concentration is between 0.1 mM and 5 mM, especially between 0.1 and 1 mM, more especially about 0.5 mM.

The reaction time required may be monitored by chromatographic methods or by SDS- PAGE and the reaction stopped at a desired amount of conjugation product has been produced. Suitable reaction times range between 0.5 hour and 24 hours, especially 0.5 to 15 hours, 0.5 to 10 hours or 1 to 5 hours, especially 1 to 5 hours.

When fluorescent labels, photosensitizers or light sensitive molecules are present in the reaction mixture, the reaction may be carried out in the dark. After completion of the reaction, the mixture may be purified by methods known in the art such as centrifugation followed by chromatography methods such as dialysis ultrafiltration, ultracentrifugation, size exclusion chromatography, affinity chromatography, HPLC or fast protein chromatography (FPLC). In one embodiment, the method of the invention further comprises the step of conjugating two compounds of formula (II) with one another wherein Zi of one compound of formula (II) is a complementary reactive functional group to Zi of the second compound of formula (II). In some embodiments, one Zi is an azide and the other Zi is an alkyne and the conjugation occurs by 1 ,3-cycloaddition reaction further mediated by copper or by strain inherent to the alkyne. In other embodiments, one Z _\ is a hydroxylamine and the other Z _\ is an aldehyde or ketone and an oxime is formed under acid catalysis conditions. In further embodiments, one Zi is a hydrazine and the other Zi is an aldehyde or ketone and a hydrazone is formed under acid catalysis conditions. In another embodiment, one Zi is a carboxylic acid and the other Z _\ is an amine and an amide is formed under amide forming conditions. In yet further embodiments, one Zi is a thiol group and the other Z _\ is an alkene or alkyne and the conjugation occurs by thiol-ene or hydrothiolation reaction where SH is added to the double bond either by radical mediated reaction or where the alkene is polarized by conjugation with an electron withdrawing group as in α,β-unsaturated ester, by Michael addition. In yet other embodiments, one Z _\ is a thioester and the other Zi is a cysteine residue or equivalent thereof and conjugation occurs by native chemical ligation.

In some embodiments, each A is the same. In other embodiments, each A is different and each A may be selected for a complementary function. For example, one A may be a small molecule drug and the other a targeting compound selected to deliver the drug to its site of action. One A may be a contrast agent and the other a targeting agent selected to deliver the contrast agent to an organ being studied. One A may be a nanoparticle or dendrimer and the other a protein, peptide, small molecule drug, contrast agent or radioisotope. In particular embodiments, one A is a protein or peptide, especially an antibody or a fragment thereof.

In yet another aspect of the invention, the compound of formula (I) may be reacted with another entity before sortase conjugation with a compound of formula (III), thereby preparing a compound of formula (V). Thus there is provided a method of preparing a compound of formula (V): wherein G is a glycine residue;

each Ri is independently selected from hydrogen or an amino protecting group; each Xi is independently absent or is an amino acid residue or a peptide of 2-10 amino acid residues;

each X ₂ is independently absent or is an amino acid residue with a reactive functional group in its side chain;

each Yi is independently absent or is a spacer group; each Y ₂ is independently absent or is a linker group;

B is a protein, a peptide, a label, a radioisotope, a small molecule drug, a targeting molecule, a nanoparticle, a cell, a dendrimer, a polymer, a photosensitizer, DNA, RNA, a carbohydrate, a contrast agent, a catalyst, a lipid or a virus particle;

s is an integer from 1 to 100; and

n is 0 or an integer of 1 to 4;

said method comprising reacting a compound of formula (I) described above with a compound of formula (VI): wherein B, Y ₂ and s are defined above and Z ₂ is a reactive functional group complementary to Zj. In some embodiments, Z| is an azide and Z ₂ is an alkyne or vice versa and the conjugation occurs by 1,3-cycloaddition reaction further mediated by copper or by strain inherent to the alkyne. In yet other embodiments, Zi is a hydroxylamine and Z ₂ is an aldehyde or ketone or vice versa and an oxime is formed under acid catalysis conditions. In further embodiments, Zi is a hydrazine and Z ₂ is an aldehyde or ketone or vice versa and a hydrazone is formed under acid catalysis conditions. In another embodiment, Zi is a carboxylic acid and Z ₂ is an amine or vice versa and an amide is formed under amide forming conditions. In yet further embodiments, one Zi is a thiol group and the other Z ₂ is an alkene or alkyne or vice versa and the conjugation occurs by thiol-ene or hydrothiolation reaction where SH is added to the double bond either by radical mediated reaction or where the alkene is polarized by conjugation with an electron withdrawing group as in α,β-unsaturated ester, by Michael addition. In yet other embodiments, one Zi is a thioester and the other Z ₂ is a cysteine residue or equivalent thereof or vice versa and conjugation occurs by native chemical ligation. Y ₂ may be absent or may be a linker that connects Z ₂ to B. The spacer may be any sort of spacer and may provide relief from steric hindrance allowing better reaction between Z and Z ₂ . In some embodiments the spacer group is 1 to 10 atoms to length and may be form example, alkylene groups ((CH ₂ ) _X ) in which x is an integer from 1 to 10 and in which one or more C¾ groups may be replaced by -0-, -NH-, -NH-C(O)-, -C(0)-NH, -S- or -C ₅ H4-.

In some embodiments, B may bear more than one -Y ₂ -Z ₂ group allowing multiple compounds of formula (I) to be attached to the surface of B. In this case, s is greater than 1 , for example 2 to 100 or 2 to 80 or 2 to 65 or 2 to 45 or 2 to 35 or 2 to 20, 2 to 18, 2 to 16, 2 to 14, 2 to 12, 2 to 10, 2 to 8, 2 to 6 or 2 to 4. This embodiment is particularly suitable when B is a nanoparticle, a cell or dendrimer in which the resulting dendrimer, cell or nanoparticle is decorated with multiple copies of the G(G)n-motif.

In some embodiments where s is greater than 1, each Ri may be the same or different. When more than one type of Ri is present, a first type of Ri may be selectively removed to allow a first subset of G(G) _n -moieties on B to be modified with one type of A group by sortase conjugation, removal of. a second type of Ri allows a second subset of G(G)„- moieties on B to be modified with a second type of A group by sortase conjugation. This may be further repeated until all types of Ri have been removed and all G(G)„-moieties modified.

This method may then further comprise the step of sortase mediated conjugation with a compound of formula (III) to form a compound of formula (VII): where B, X _a , Xb, X _c , Xd, G, X _l5 X ₂ , Yi, Y ₂ and s are defined above.

The conditions used in the sortase mediated conjugation are the same as those described above. However, the ratio of the compound of formula (III) and the compound of formula (V) will depend on the number of G(G) _n -motifs that are attached to B in the compound of formula (V) (s) and which are not protected by Ri. When s is 1, the ratio of compound of formula (V) to formula (III) will be in the range of 1 : 10 to 10:1, especially 1 :1 to 1 :10, 1 :1 to 1 :5, more especially 1 :3, particularly where X ₃ is a purification tag. When s is greater than 1, the ratio of formula (V) to formula (III) will be in the order of 1 :5 x s to 1 :10 x s, for example 1 :1 x s to 1 :5 x s, especially about 1 :3 x s.

In some embodiments, A and B are the same. In other embodiments, A and B are different. In some embodiments, each A is independently selected and may be the same as another or a group of other A groups or may be different from another or group of other A groups. In particular embodiments, at least one A is a protein or peptide, especially an antibody or a fragment thereof. In some embodiments, B is a protein or peptide, especially an antibody or a fragment thereof, and may be the same or different from A. In yet another embodiment there is a kit comprising a compound of formula (I) and a sortase enzyme.

In some embodiments, the kit further comprises a compound of formula (III), especially where A in the compound of formula (III) is a protein or peptide. In some embodiments, the sortase enzyme is a Sortase A enzyme, especially a Sortase A enzyme from S. aureus. Optionally the kit further comprises a buffer composition suitable for use in a sortase mediated conjugation reaction.

Applications

The site-selective modifications described herein may have many applications, particularly in the pharmaceutical sciences, for example, in drug delivery, imaging, cell therapy, diagnostics, vaccination, radiotherapy, phototherapy and gene therapy.

The site-selective modifications described herein may be used to provide high purity fusion proteins, where the fusion of proteins is mediated by way of the site specific conjugation described above. For example, a fusion of two antibodies or scFvs with different specificities. Examples include antibody-effector fusions wherein the effector may be a toxin, an interleukin (eg: IL2), a cytokine, an RNAse or perforin. Known methods of producing fusion proteins often have low yield and/or low purity. The methods of the present invention allow each protein to be prepared and modified separately and purified if required, then conjugated with high yield and purity.

The site-selective modifications described herein may be used to attach a protein, such as an antibody, with a nanoparticle such as a dendrimer, a polymer, liposomes, micelles, a microparticle of iron oxide (MPIO) or an LbL click capsule. Fusions with MPIO or a dendrimer-gadolinium complex may provide targeted delivery of the MPIO to a specific site in the body for magnetic resonance imaging (MRI). Fusions with click capsules or polymers may also be used to deliver drugs, genetic material, radiolabels or contrast agents in a targeted manner. When a protein, such as an antibody or a fragment thereof is attached to a cell, such as a stem cell or a NK cell, the conjugate may be used to deliver the cells to a specific site in the body. For example, stem cells could be delivered to a tissue in need of regeneration or NK cells delivered to a tumour. Another application is diagnostic or in vitro testing where an antibody or protein is modified in a site selective manner to facilitate attachment of the protein to a surface or a moiety that directly or indirectly provides a signal for detection. The site-specific attachment of a protein to a surface, for example, by site specific attachment of biotin and subsequent binding to an avidin coated surface, provides a means by which the function of that protein, for example, specific binding of an analyte, may be optionally preserved. The site specific attachment of a moiety that directly or indirectly provides a signal, for example, attachment of a fluorescent moiety, provides a means by which the function of that protein, for example, specific binding of an analyte, may be optionally preserved. Such components are important elements of in vitro diagnostic tools which are used to detect particular analytes. The protein or antibody may be conjugated with a dendrimer or polymer for imaging or drug delivery. For example, the antibody may provide targeting to a particular site in the body and the dendrimer or polymer may carry at least one small molecule drug, photosensitizer, radiolabel or contrast agent. The conjugate may then be used to deliver the drug, photosensitizer, radiolabel or contrast agent to a specific site in the body.

Fusion proteins may be formed between recombinant protein or antibody or fragment thereof and human serum albumin (HAS) (monomelic) or human Fc (dimeric). Such fusion proteins may have stable and improved in vivo pharmacokinetic properties.

Some of the methods of the present invention may also allow reactions to proceed under conditions which are more amenable to drug synthesis, for example, the absence of metal ions that may be traditionally used as catalysts (eg: Cu(I) ions). The methods of the present invention may be used to produce conjugates of virus particles carrying specific genetic material with targeting proteins so that the virus particles are delivered to the cells to which. the genetic material is to be delivered. These conjugates may be particularly useful in gene therapy. The methods of the present invention may also be used to produce conjugates of targeting proteins with enzymes. The targeting protein allows the enzyme to be attached to a cell surface and after delivery of a prodrug to the vicinity of the cells, the enzyme can act on the prodrug to produce the active drug in the site it is required to act. Following this theory, conjugates may be produced by the present invention that are suitable for antibody- directed enzyme prodrug therapy (ADEPT), gene-directed enzyme prodrug therapy (GDEPT), clostridial-directed enzyme prodrug therapy (CDEPT) and polymer-directed enzyme prodrug therapy (PDEPT) (Schellmann et al, Mini-Reviews in Medicinal Chemistry, 2010, 10:887-904). The invention will now be described with reference to the accompanying Figures and Examples. However, it is understood that the particularity of the following description is not to supersede the generality of the preceding description of the invention. EXAMPLES

Example 1: Generation and expression of single chain anti-LIBS with C-terminal LPETG-(His)e motif (scFv(+)) and non-binding single chain mutMA2 with C-terminal LPETG-(His) ₆ motif (scFv(-))

Step 1: Single chain anti-LIBS with C-terminal LPETG-(His) 6 motif

The generation of the anti-LIBS scFv from a hybridoma cell line expressing a monoclonal antibody against LIBS epitopes on GPIIb/IIIa has been described previously (Schwarz et al., J. Pharmacol. Exp. Then, 308, 1002-1011, 2004; Stoll et al, Arterioscler. Thromb. Vase. Biol, 27, 1206-1212, 2007). To express anti-LIBS in insect cells, the scFv was cleaved at Ncol and Notl restriction sites and sub-cloned into a pMT vector system (Invitrogen, USA). To clone LPETG motif into pMT vector for generating the anti-LIBS- LPETG (SEQ ID NO: 380), we designed two complementary primers containing the LPETG-(His) ₆ (SEQ ID NO:381) sequence flanked by 5' Notl and 3' Apal as following: sense strand: 5 ^, -GCGGCCGCTCTGCCGGAAACCGGCGGCGGGCCCA-3 ^, (SEQ ID NO:381), antisense strand: 5'-GGGCCCGCCGCCGGTTTCCGGCAGAGCGGCCGCA- 3' (SEQ ID NO:382) (LPETG sequence underlined). Primers were also designed with A overhangs for sub-cloning into pGEM-T Easy vector (Invitrogen, USA). The primers were annealed to a double strand product, ligated into the pGEM-T Easy vector and transformed into DH5a E.Coli cells (Invitrogen, USA) for amplification of the vectors. pGEM-T Easy- LPETG was then digested with EcoRI and then with Notl and Apl. Subsequently, the amplified LPETG (SEQ ID NO:380) strands were cloned into pMT-anti-LIBS at Notl and Apal restriction sites. The resulting plasmid constructs were then transformed into TGI E.Coli cells (Invitrogen, USA). The transformed cells were grown in LB media containing 100 μg/mL ampicillin and 100 mM glucose at 37°C and the plasmids were purified using Plasmid Maxi Kit (Qiagen, Australia). Drosophila S2 cells (Invitrogen, USA) were transfected with pMT-anti-LIBS-LPETG using a method described by Han et al, Nucleic Acids Res., 24, 4362-4363, 1996. Briefly, cells were diluted to Ι ^χ ΙΟ ⁶ cells/mL and mixed with 80 ng/mL anti-LIB S-LPETG-(His) ₆ (SEQ ID NO:280) DNA preincubated with 250 ng mL dimethyldioctadecylammonium bromide for 30 min. The cells were then cultured in Express Five SFM medium containing 18 mM L-glutamine and 1% penicillin/streptomycin at 28°C in ventilated polycarbonate Erlenmeyer flasks (Corning, Acton, MA, USA) under constant rotation (100 rpm, Bench top Orbital Shaker Incubator, Ratek Instruments, Australia). After two days, protein production was induced by 500 μΜ CuS0 . Six days later, the cell supernatant was collected by centrifugation at 15,000 g for 15 min. The cell supernatant was applied to a chelating Sepharose fast flow column (20 mL bed volume, 5 mL/min flow rate, GE Healthcare, Uppsala, Sweden). The column was washed to baseline with PBS, 0.5 M NaCl and 10 mM imidazole in 50 mM Tris, pH 8.0 to remove non-specifically bound proteins. Elution was carried out with 250 mM imidazole in 50 mM Tris, pH 8.0. Fractions of 50 mL were collected. Fractions containing significant amounts of product were pooled and dialyzed against PBS. A second purification was done with a nickel-based metal affinity chromatography column, Ni-NTA column . (Invitrogen), according to the manufacturer's instruction manual. Fractions of 2 mL were collected and dialysed against PBS. Step 2: Non-binding single chain mutMA2 with C-terminal LPETG-(His) ₆ (SEQ ID NO:384)motif(scFv(-))

The non-binding scFv mutMA2 is based on the blocking antibody MA2 as described in Schwarz, FASEB, 2004, 18:1704-1706 and Schwarz, Circ. Res., 2006, 99:25-33. The sequence of the mutMA2 with LPETG tag with a CDR3 mutation of the RND motif responsible for the binding to GPIIbllla was de novo synthesized by Geneart (Regensburg).

DNA (SEQ ID NO:383)

ATG GAG ACA GAC ACA CTC CTG CTA TGG GTA CTG CTG CTC TGG GTT CCA GGT TCC ACT GGT GAC GCG GCC CAG CCG GCC AGG CGC GCC GTA CGA AGC TTG GTA CCG AGC TCG GAT CCA CTC CAG TGT GGT GGA ATT CTC GCG GAA GTG CAG CTG GTG CAG TCT GGA GCT GAG GTG AAT AAG CCT GGG GCC TCA GTG AAG GTC TCC TGC AAG GCT TCT GGA TAC ACC TTC ACC GGC TAC TAT ATG CAC TGG GTG CGA CAG GCC CCT GGA CAA GGG CTT GAG TGG ATG GGA TGG ATC AAC CCT AAC AGT GGT GGC ACA AAC TAT GCA CAG AAG TTT CAG GGC TGG GTC ACC ATG ACC AGG GAC ACG TCC ATC AGC ACC GCC TAC ATG GAG CTG AGC AGG CTG AGA TCT GAC GAC ACG GCC GTG TAT TAC TGT GCG AGA GGC CGT GCT TTG TAT AAC GCG AAC GAC CGG TCC CCC AAC TGG TTC GAC CCC TGG GGC CAG GGA ACC CTG GTC ACC GTC TCC TCA GGG AGT GCA TCC GCC CCA ACC CTT AAG CTT GAA GAA GGT GAA TTT TCA GAA GCA CGC GTA CAG GCT GTG CTG ACT CAG CCG CCC TCG GTG TCA GTG GCC CCA GGA CAG ACG GCC AGG ATT ACC TGT GGG GGA AAC AAC ATT GGA AGT AAA AGT GTG CAG TGG TAC CAG CAG AAG CCA GGC CAG GCC CCT GTG CTG GTC GTC TAT GAT GAT AGC GAC CGG CCC TCA GGG ATC CCT GAG CGA TTC TCT GGC TCC AAC TCT GGG AAC ATG GCC ACC CTG ACC ATC AGC AGG GTC GAA GCC GGG GAT GAG GCC GAC TAT TAC TGT CAG GTG TGG GAT AGT AGT AGT GAT CAT GTG GTA TTC GGC GGA GGG ACC AAG CTG ACC GTC CTA GGT GGG AAA CCC ATT CCT AAC CCA CTG CTG GGA CTG GAT AGC ACA CTG CCT GAG ACT GGC GGG CTG GAA GAG GCG GCC GCT CGA GGA GGG CCC GAA CAA AAA CTC ATC TCA GAA GAG GAT CTG AAT AGC GCC GTC GAC CAT CAT CAT CAT CAT CAT TGA

Amino Acid Sequence (SEQ ID NO:384)

GDPSWLATMETDTLLLWVLLLWVPGSTGDAAQPARRAVR SLVPSSDPLQCGGILAEVQLVQSGAEVNKPGASV VSCKA SGYTFTGYYMHWVRQAPGQGLEWMGWINPNS GGTNY A Q FQGWVTMTRDTSISTAYMELSRLRSDDTAVYYCARGRA LYNANDRSPNWFDPWGQGTLVTVSSGS AS APTLKLEEGEF SEARVQAVLTQPPSVSVAPGQTARITCGGNNIGS SVQWY QQKPGQAP VLVV YDDSDRPSGIPERFSGSNS GN M AT LT I S R VE AGDEAD Y YCQV WDSSSDHV VFGGGTKLTVLGGKPIPNP LLGLDSTLPETGGLEEAAARGGPEQKLISEEDLNSAVDHH HHHH

The scFv was cloned in the expression vector pET20b (catalogue 69739, Merck) and transformed into Escherichia coli BL21(DE3) Competent Cells (catalogue 69450, Merck ) Bacteria were induced with 0.25 mmol/L isopropyl-D-galactoside (Sigma) and incubated for 16 hours at 200 rpm and 23 °C. The bacteria were lysed with an ice-cold hyperosmotic shock solution (20% sucrose, EDTA, Tris), and scFvs were purified by FPLC-procedure, using metal-affinity chromatography (Ni-NTA column, Superflow Columns, Qiagen catalogue 30622). Elution was done with Elution Buffer (50mM NaH ₂ P0 ₄ , 300mM NaCl, 250mM Imidazol) Fractions with highest protein content were pooled and dialysed against PBS.

Example 2: Preparation of GGG-R compounds

Rhodamine B-LPETGGHHHHHH

Peptides were supplied by various commercial peptide manufacturers and were prepared using peptide synthesis grade reagents including Fmoc-protected amino acids for example Fmoc-propargyl glycine (pG). In a representative example, the sequences were assembled on a Symphony synthesizer (Protein Technologies Inc., USA) using O-Benzotriazol-l-yl- Ν,Ν,Ν',Ν'-tetramethyluroiumhexafluorophosphate (HBTU) diisopropylethylamine (DIEA) activation in Ν,Ν-Dimethylformamide (DMF) with piperidine deprotection of the Fmoc groups. CO-[(CH ₂ ) ₂ 0] ₄ (CH ₂ )2N3 was added manually to sequences as the last coupling using Azido-dPEG®4-NHS ester (Item #: 10501 Quanta Biodesign Ohio USA) activated with DIEA in DMF. In a similar manner, Lissamine Rhodamine B-NH-PEG4-CO was added manually to the sequence as the last coupling following piperidine deprotection of the terminal Fmoc NH protecting group, using Lissamine Rhodamine B sulphonamide- dPEG ₄ -acid (item #10229) activated by O-Benzotriazol-l-yl-Ν,Ν,Ν',Ν*- tetramethyluroiumhexafluorophosphate (HBTU) and diisopropylethylamine (DIEA) in NN-Dimethylformamide (DMF).

The crude peptides were cleaved from the resin support using a mixture of Trifluoroacetic acid (TFA), water, dithiothreitol (DTT) and Triisopropylsilane (TIPS) (ratio 92.5:2.5:2.5:2.5 respectively). Crude cyclic peptides were then purified by RP-HPLC (Waters, USA) using C-18 columns with TFA and aqueous acetonitrile buffers and the sequences confirmed by mass spectrometry (ESI-MS) and amino acid analysis. The peptides prepared were:

Lissamine Rhodamine B-LPETGGHHHHHH (SEQ ID NO:385)

GGGWWSSK-ePEG ₄ -N ₃ (SEQ ID NO:77)

GGGWWK-ePEG ₄ -N ₃ (SEQ ID NO:55)

GGGK-sPEG ₄ -N ₃ (SEQ ID NO:22)

GGG-WW-GA-pG (SEQ ID NO:386)

GGG- AGAGAK-Maleimide (SEQ ID NO:387)

GGG-eGFP was used as a comparative example and produced by the following method: Generation, expression and production of GGG-eGFP

The following primers were designed to introduce the Ndel and Xhol restriction sites and GGG Sortase ligand into pEGFP-Cl vector (Clontech, USA): sense strand 5 ' -C ATATGGGAGGCGGCGGTTC AATGGTGAGCAAGGGCGAG-3 ' (SEQ ID NO:388), antisense strand 5 '-CTCGAGCTTGTACAGCTCGTCCATG-3 ' (SEQ ID NO:389) (GGGWW in bold; Ndel and Xhol underlined, respectively). The amplification of GGG-eGFP sequence was performed by PCR using these primers. The PCR products were then cloned into a pET-20b(+) vector system at the Ndel and Xhol restriction sites. Amplification of the plasmids was done using XL1-B E.Coli cells (Invitrogen, USA) and the plasmid purification was performed using a Plasmid Mini-Prep Kit (Qiagen, Australia). GGG-eGFP was expressed in BL21-DE3 E.Coli (Invitrogen, USA). The cells were cultured in LB media containing 100 μg/mL ampicillin until the OD600 of 0.8 was reached. GGG-eGFP production was induced with 1 mmol/L of isopropyl β-D-l- thiogalactopyranoside (IPTG) for 4 hours at 37°C. Bacteria were then isolated by centrifugation at 4000 rpm for 10 min. Proteins were purified using Ni-NTA column (Invitrogen), according to the manufacturer's protocol. Fractions of 2 mL were collected and dialysed against PBS. Example 3: Sortase A mediated reactions

ScFv mut-MA2 with LPETG tag (SEQ ID NO:384) (30 uM) prepared in Example 1 was incubated with the different GGG substrates prepared in Example 2 (GGGWWSSK-PEG - N ₃ (SEQ ID NO: 77), GGGWWK-PEG ₄ -N3 (SEQ ID NO:55), GGG -PEG ₄ -N ₃ , (SEQ ID NO:22) GGGWWGA-pG (SEQ ID NO:386) and GGG-eGFP) (20 uM) in the presence of Sortase A (10 uM) and CaCl ₂ (0.5 μΜ) at 37 °C for 3 h with gentle shaking. The reaction was performed in Sortase coupling buffer (50 mmol/L Tris, 150 mmol/L NaCl, pH 8.0). The sortase conjugation product was treated in one of two different ways:

A. When reaction was complete, the reaction mixture was passed down a Ni-NTA column (Superflow Columns, Qiagen catalogue 30622) and flushed with PBS (Phosphate Buffered

Saline, 8 g NaCl, 0.2 g KC1, 1.44 g Na ₂ HP0 ₄ , 0.24 g KH ₂ P0 ₄ in 1L, pH 7.6). Unreacted scFv-mutMA2-LPETGH ₆ and the sortase enzyme was retained and the yield of sortase- reacted scFv-mutMA2 in the reaction was determined as total protein recovered in the flow / through using the BCA assay protocol (BCA Protein Assay Kit, Pierce catalogue 23225). This was expressed as a percentage of scFv-mutMA2-LPETGH ₆ (SEQ ID NO: 384) in Figure 1.

B. When reaction was complete, 200 μΐ Ni-NTA resin (Superflow resin, Qiagen catalogue 30410) per 1 mL reaction mixture was added and reaction was agitated at 4 °C for 1 hour. The reaction was centrifuged at lOOg and the supernatant isolated. Unreacted scFv- mutMA2-LPETGH ₆ and Sortase was retained by the resin and the yield of sortase-reacted scFv-mutMA2 in the reaction was determined as total protein recovered in the supernatant using the BCA assay protocol as for A. This was expressed as a percentage of scFv- mutMA2-LPETGH ₆ (SEQ ID NO:384) in Figure 1. Results

The Sortase A mediated conjugation of the scFv antibody-LPETG (SEQ ID NO:384) of Example 1 with the GGGR compounds of Example 2 provided scFv-antibody- LPETGGG-R conjugates with varying efficacy as shown in Figure 1. As can be seen in Figure 1, the reaction of a GGG-tagged protein (GGGeGFP) proceeded with an efficacy of about 50% as did the reaction with GGGKPEG ₄ -N ₃ (SEQ ID NO:22). The sortase mediated reactions of the present invention are at least as efficacious as those of the prior art.

However, use of GGGWW (SEQ ID NO:390) motif improved efficacy to greater than 60% and addition of a KPEG ₄ motif improved efficacy of the sortase reaction to at least 80%.

Example 4: Sortase coupling■ between scFv-LPETG and GGG-modifled (PVPON _A i _k )5/PEG _Alk capsules with DL800 Label

The assembly of alkyne functionalized layer-by-layer (LbL) click capsules is described in Leung et al. (Assembly and Degradation of Low-Fouling Click-Functionalized Poly(ethylene glycol)-Based Multilayer Films and Capsules. Leung MKM, Such GK, Johnston APR, Biswas DP, Zhu Z, Yan Y, Lutz JF, Caruso F. Small. 2011 Mar 21. doi: 10.1002/smll.201002258. [Epub ahead of print]) and Kinnane et al, (Low-Fouling Poly(N- vinyl pyrrolidone) Capsules with Engineered Degradable Properties. Kinnane CR, Such GK, Antequera-Garcia G, Yan Y, Dodds SJ, Liz-Marzan LM, Caruso F, Biomacromolecules 2009, 10, 2839).

General

High-purity (Milli^Q) water with a resistivity greater than 18 ΜΩ cm was obtained from an in-line Millipore RiOs/Origin water purification system. Poly(methacrylic acid) (PMA, 30 wt %, Mw ~ 15000) was purchased from Polysciences (U.S.A.) and used as received.

Si0 ₂ particles (3.25 μπι diameter) were purchased from MicroParticles GmbH as a 5 wt % suspension.

01igo^(ethylene glycol) methyl ether rriethacrylate (OEGMA, Mw = 475 Da, mean degree of polymerization, DP, is 8 to 9), 2-(2-methoxyethoxy)ethyl methacrylate (ME0 ₂ MA), 0-(2-Aminoethyl)-0'-(2-azidoethyl)pentaethylene glycol (azido-PEG-amine, Mw = 350.41 Da) and methyl 2-bromo propionate (MBP) were obtained from Aldrich. OEGMA and ME0 ₂ MA monomers were purified by being passed through a neutral alumina column to remove the inhibitor. The reversible addition-fragmentation chain transfer (RAFT) agent 2- cyanoprop-2-yl dithiobenzoate (CPDB) was prepared using literature procedures. (Macromol. Rapid Commun., 2006, 27:821) 2,2'-Azobisisobutyronitrile (AIBN) was recrystallized from 95% ethanol. Solutions were adjusted to the required pH using a pH meter (Mettler Toledo) with 0.1 M HC1 or 0.1 M NaOH. Unless noted otherwise, all chemicals were purchased from Sigma- Aldrich and used without further purification.

Step 1. Synthesis of Linker

A solution of cystamine dihydrochloride (58 mg, 0.25 mmol) and triethylamine (103 mg, 1.02 mmol) in methanol (2 mL) was added to a solution of azido-dPEG ₄ -NHS ester (200 mg, 0.51 mmol, product #10501 Quanta Biodesign Ohio USA) in dioxane (2 mL). The resulting reaction mixture was stirred at room temperature for 12 h. The white precipitate formed was filtered off and the filtrate was concentrated to dryness. The residue was purified by column chromatography over a silica gel (49:50:1 hexane/diethyl ether/ethanol) to give the desired product [(N,iV-(dithiodiethane-2,l-diyl)bis(l-azido tertaethyleneglycol acetamide), 144 mg, 10.37 mmol, 73% yield] as a pale yellow oil. This product is herein denoted as the bisazide linker. NMR spectra were recorded on a Varian Gemini 2000 spectrometer operating at 400 MHz for Ή NMR and 100 MHz for ¹³ C NMR. Chemical shifts are reported in ppm: ¹³ C NMR (100 MHz, D ₂ 0): 176.7 (CO), 69.7, 69.4, 66.9, 66.1 , 50.3, 46.8, 38.2 (CONHCH ₂ -), 36.8, 36,2, 25.3 (-S-CH ₂ -), 14.2, 8.4. Step 2. Synthesis of Poly(N-vinyl pyrrotidone-ran-propargyl acrylate) or PVPONAII _I

vinylpyrrolidone propargyl acrylate RAFT initiator

Poly(N-vinyl pyrrolidone-r n-propargyl acrylate) was synthesized using the following procedure: propargyl acrylate (7.0 mg, 6.4 ^χ 10-2 mmol), N-vinyl pyrrolidone (0.98 g, 8.9 mmol), the phthalimidomethyl xanthate reversible addition-fragmentation chain transfer (RAFT) agent (12.8 mg, 4.6 x 10-2 mmol), and azobisisobutyronitrile ΑΙΒΝ (0.4 mg, 2.4 x 10-3 mmol) were added to a vial. The synthesis of the RAFT initiator used has been described previously (Postma, A.; Davis, T. P.; Evans, R. A.; Li, G. X.; Moad, G.; O'Shea, M. S. Macromolecules 2006, 39, 5293-5306).

These components were then dissolved in 3 mL of dioxane and transferred to a Schlenk tube. The reaction was then purged of oxygen using four freeze-thaw cycles on a Schlenk line. The Schlenk tube was then sealed and the polymerization was conducted in a constant temperature oil bath at 60 °C for 5 h. After polymerization, the reaction was dialyzed in water for several days to remove excess monomer and then the final product was freeze- dried. Gel permeation chromatography showed that the Mn of the polymer was approximately 19000 with a polydispersity of 1.38.

Step 3. Synthesis ofPVPON/fPMA/PVPON _A i s PMA core-shell particles

Briefly, silica particles (~3 μπι diameter, 5 wt% suspension, 100 μί) were first washed with sodium acetate (NaOAc) solution (50 mM, pH 4) by centrifugation (1000 g, 1 min) and redispersed in 200 μΐ- of NaOAc solution (50 mM, pH 4). A total of three centrifugation/redispersion cycles were conducted. This procedure was used as the standard washing procedure. To form the multilayers, an equal volume of poly(N-vinyl pyrrolidone) (PVPON, w ~ 55 000, 1 g L ^'1 in 50 mM NaOAc, pH 4) was added to the particle suspension for adsorption (15 min) with constant shaking. The particles were then washed via three centrifugation (1000 g, 1 min)/redispersion (200 μί) cycles. After washing, 200 sL of poly(methacrylic acid) (PMA, 1 g L ^"1 in 50 mM NaOAc, pH 4) was adsorbed with constant shaking for 15 min, and the particles were then washed again. Alkyne-functionalized poly(N-vinyl pyrrolidone) (PVPON _A ik, 1 g L ^'1 in 50 mM NaOAc, pH 4) was adsorbed onto the PMA layer (using the same process as described for the previous two layers) to create a PMA PVPON _A i _k bilayer.

This PMA/PVPON _A i _k adsorption procedure was repeated to form PVPON/(PMA/PVPON _A i _k )s/PMA core-shell particles.

HrPECrNj

DyLight 800 NHS-Ester (Thermo Scientific) (0.2 mg) was dissolved in DMSO (20 μΙ_) and mixed with < (2-Aminoethyl)-(9'-(2-azidoethyl)pentaethylene glycol (Aldrich) (2 μΐ,) for 4 h to give DL800-PEG-azido (DL800A _Z ). This solution was used without further purification.

Step 5. Synthesis of PVPON/(PMA/PVPON _Alk )s^PMA core-shell particles with DL800 _Az Label

For targeting studies, the particles were labeled with DL800A _Z for near infrared analysis after the completion of the assembly of partially assembled, PVPON/(PMA/PVPON _A i _k )2 PMA, being an intermediate stage in the synthesis of PVPON/(PMA/PVPON _A i _k )5/PMA particles. Without purification, 0.5 μΐ, of the DL800 _AZ suspension together with sodium ascorbate (4.4 g L ^"1 , 50 μΐ,) and copper sulfate (1.75 g L ^"1 , 50 μΐ) (all in 50 mM NaOAc, pH 4) were added to the particles (in 150 μΐ of 50 mM NaOAc, pH 4) and labeling was allowed to proceed for 1 h. Following extensive washing, three bilayers of PVPON _A i _k PMA were assembled on the DL800 _AZ -labeled particles as described in Step 3. Step 6. Reaction of DL800 _Ar labeled PVPON/(PMA/PVPON _M }JPMA core-shell particles with PEGAI/C-

P(OEGMA-co-T SPeTEGMA) Step 6a. Synthesis of 2-(2-(2-(3-(Trimethylsilyl)prop-2-ynyloxy)ethoxy)ethoxy)ethy l Methacrylate (TMSPgTEGMA) :

Sodium hydride (2.0 g, 50 mmol, 60% dispersion in mineral oil) was slowly added to a THF (50 mL) solution of triethylene glycol (11.6 g, 76 mmol) at 0 °C. The mixture was stirred for 20 min before propargyl bromide (4.2 mL, 38 mmol) was added dropwise. The mixture was then stirred at room temperature for 20 h before THF was removed in vacuum. The residual was dissolved in DCM and then washed successively with saturated sodium hydrogen carbonate (NaHC0 ₃ ) (2 x 50 mL) and water (50 mL). The extracts were dried over magnesium sulfate (MgS0 ₄ ), filtered and concentrated in vacuum. The crude product was purified by column chromatography, eluting with a 2:3 mixture of H-hexane and ethyl acetate (EtOAc) to give triethylene glycol alkyne 2-(2-(2-(prop-2- ynyloxy)ethoxy)ethoxy)ethanol as a light yellow oil (4.5 g, 63%). Ή NMR (400 MHz, CDCI3, tetramethylsilane (TMS)): δ H 4.13 (s, 2H, OCH ₂ C≡CH), 3.61-3.58 (m, 10H, CH ₂ 0), 3.50 (t, 2H, HOCH ₂ ), 2.75 (br, 1 H, HO), 2.38 (s, 1H, CH≡C) ppm; ¹³ C NMR (100 MHz, CDCI3, TMS) δ C 77.2 (C≡CH), 75.6 (C≡CH), 70.4 (CH ₂ 0), 70.3 (CH ₂ 0), 70.1 (CH ₂ 0), 69.6 (CH ₂ 0), 69.2 (CH ₂ 0), 61.5 (HOCH ₂ ), 60.1 (OCH ₂ C≡CH) ppm.

Protection of the triethylene glycol alkyne (3.39 g, 18 mmol) was achieved by mixing with silver chloride (0.24 g, 1.8 mmol) suspended in anhydrous DCM (25 mL), followed by addition of l,8-diazabicyclo[5.4.0]undec-7-ene (DBU) (3.5 g, 23 mmol). The reaction mixture was heated to 40°C and chlorotrimethylsilane (2.8 g, 26 mmol) was added dropwise. After stirring for 24 h at 40°C the mixture was cooled to room temperature and diluted with 200 mL of n -hexane. The organic phase was washed successively with NaHC0 ₃ (2 x 50 mL), 0.1 M HC1 (2 ^χ 50 mL) and water (50 mL). The organic phase was dried over MgS0 ₄ , filtered, and concentrated in vacuum. The crude product was purified by column chromatography, eluting with a 8:1 mixture of n-hexane and EtOAc to give TMS-alkyne triethylene glycol 2-(2-(2-(3-(trimethylsilyl)-prop-2-ynyloxy)ethoxy)- ethoxy)ethanol as a light yellow colorless liquid (2.1 1 g, 45%). Ή NMR (400 MHz, CDCI3, TMS): 6 H 4.13 (s, 2H, OCH ₂ C≡CH), 3.61-3.58 (m, 10H, CH ₂ 0), 3.50 (t, 2H, HOCH ₂ ), 2.15 (br, 1H, HO), 0.21 (s, 9H, Si(CH ₃ ) ₃ ) ppm; ¹³ C NMR (100 MHz, CDC1 ₃ , TMS) δ C 105.5 (CH ₂ C≡C), 91.2 (C≡ CSi), 70.8 (CH ₂ 0), 70.7 (CH ₂ 0), 70.3 (CH ₂ 0), 69.1 (CH ₂ 0), 69.0 (CH ₂ 0), 61.1 (HOCH ₂ ), 60.0 (OCH ₂ C≡CH), 5.2 (SiCH ₃ ) ppm.

MethacryloyI chloride (0.78 mL, 8 mmol) dissolved in DCM (10 mL) was added dropwise over 1 h to a mixture of the TMS-alkyne triethylene glycol (2.0 g, 7.7 mmol) and triethylamine (0.81 g, 8 mmol) in DCM (50 mL) at 0°C. The mixture was then stirred at room temperature for 18 h and filtered to remove triethylamine hydrochloride. The filtrate was washed with saturated NaHC0 ₃ (2 * 50 mL) and water (50 mL). The organic phase was dried over MgS0 ₄ , filtered, and concentrated under reduced pressure. The crude product was purified by column chromatography, eluting DCM to give 2-(2-(2-(3- (trimethylsilyl)prop-2-ynyloxy)ethoxy)-ethoxy)ethyl methacrylate (TMSPgTEGMA) as a light yellow colorless liquid (2.1 g, 88%). 1H NMR (400 MHz, CDC1 ₃ , TMS): δ H 6.15 (s, 1H, CH ₂ =C(CH ₃ )), 5.51 (s, 2H, CH ₂ =C(CH ₃ )), 4.13 (s, 2H, OCH ₂ C≡CH), 3.61-3.58 {m, 10H, CH ₂ 0), 3.50 (/, 2H, HOCH ₂ ) , 2.0 (s, 3H, CH ₂ =C(CH ₃ )), 0.21 (s, 9H, Si(CH ₃ ) ₃ ) ppm; ¹³ C NMR (100 MHz, CDC1 ₃ , TMS) δ C 168.3 (CO), 133.8 (=CCH ₃ ), 131.5 (=CH ₂ ), 106.1 (CH ₂ C≡C), 91.6 (C≡CSi), 70.3 (CH ₂ 0), 70.2 (CH ₂ 0), 70.0 (CH ₂ 0), 69.4 (CH ₂ 0), 69.2 (CH ₂ 0), 61.8 (HOCH ₂ ), 60.2 (OCH ₂ C≡CH), 18.8 (=CCH ₃ ), 4.1 (SiCH ₃ ) ppm.

Step 6b. Synthesis of Alkyne-Functionalized Copolymers Poly(OEGMA-co-PgTEGMA) as PEG _A i _k

Poly(OEGMA-co-TMSPgTEGMA) was prepared by RAFT polymerization. OEGMA (1.0 g, 2.1 mmol), TMSPgTEGMA (70 mg, 0.21 mmol), 2-cyanoprop-2-yl dithiobenzoate (CPDB) (prepared as per Macromol. Rapid Commun. 2006, 27, 821) (7.7 mg, 0.035 mmol), and AIBN (0.7 mg, 0.004 mmol) were dissolved in dioxane (2 mL). The mixture was degassed through freeze-pump-thaw (3*) and then reacted at 70°C for 20 h. The mixture was quenched in liquid nitrogen and diluted with THF before it was precipitated into diethyl ether. This precipitation procedure was repeated three times. The resulting product was dried in vacuum to yield poly(OEGMA-co-TMSPgTEGMA) as a red viscous solid, 0.8 g (74%). GPC-MALLS (THF): M _n = 34 kDa, M M _n = 1.13. The degree of polymerization (DP) of the copolymer was determined to be 85 and the alkyne ratio in the copolymer was 5% from Ή NMR (ratio of peak at 4.1 ppm (2H) to 0.2 ppm (9H).).

Poly(OEGMA-co-TMSPgTEGMA) (0.5 g, 0.43 mmol based on the alkyne-trimethylsiyl groups) and acetic acid (37 μί, 0.65 mmol) were dissolved in THF (10 mL). Argon was bubbled through the solution for 10 min at 0°C. A 0.2 M solution of tetra-n-butyl ammonium fluoride in THF (3.2 mL, 0.65 mmol) was added slowly with a syringe with vigorous stirring. The resulting solution was stirred at room temperature for 12 h and then passed through a neutral alumina column. The solution was then concentrated in vacuum and precipitated into o-hexane. The isolated product was dried in vacuum to yield poly(OEGMA-co-PgTEGMA), herein denoted as PEGAII _C , as a light red viscous solid (0.40 g, 80%) withΉ NMR analysis confirming 100% removal of the TMS-protecting groups. Step 6c. Reaction of DL800 _Ar labeled PVPON/fPMA/PVPON^s PMA core-shell particles with PEGAik

To achieve PEGylation, PEGAII _C (1 g L ^"1 in 150 mM NaOAc, pH 5) was deposited onto the DL800 _A: -labeled PVPON/(PMA/PVPON _A i _k ) ₅ PMA particles according to the procedure described in Step 3 giving PVPON/(PMA/PVPON _A | _k ) ₅ /PMA/PEG _A ik core-shell particles. The particles (in 50 μί, of 150 mM NaOAc, pH 5) were then incubated with the bisazide linker from Step 1 (1 g L ^"1 , 150 μΐ,) in the presence of sodium ascorbate (4.4 g L ^"1 , 50 μί,) and copper sulfate (1.75 g L ^'1 , 50 μί) (all in 150 mM NaOAc, pH 5) overnight for crosslinking.

Step 7. Reaction of GGGWWSSK-PEG ₄ -N3 (SEQ ID NO:77) with DL800 _Ar labeled PVPON/(PMA/PVPON _Mk ) s/PMA/PEG _A i _k core-shell particles.

To a solution of the DL800 _Az -labeled crosslinked particles from Step 6 (in 50 μΐ, of 150 mM NaOAc, pH 5) was added a- mixture of GGGWWSSK-PEG ₄ -N ₃ (SEQ ID NO:77) from Example 2 (0.2 g L ^'1 , 150 μϋ,; i.e., final concentration = 0.1 g L ^"1 ), sodium ascorbate (4.4 g L ^" \ 50 μΐ) and copper sulfate (1.75 g L ^"1 , 50 μΐ/) (all in 150 mM NaOAc, pH 5) for 30 min incubation. The GGGWWSSK-PEG ₄ -N ₃ -functionalized particles were then redispersed in 200 μΐ, of NaOAc (50 mM, pH 4) and five alternating layers of PMA/PVPON, i.e., (PMA/PVPON) ₂ PMA, (all at 1 g L ^'1 in 50 mM NaOAc, pH 4) were deposited as protective capping layers using the procedure described in Step 3.

Step 8. Formation of DL800 _Ar labeled GGG modified (PVPON _AU )s PEG _A i _k capsules I. Following washing, the protected particles (DL800 _A z-labeled) prepared in Step 7 were redispersed in 200 μΐ _^ of NaOAc (50 mM, pH 4). To this suspension were added 200 uL of ammonium fluoride (NH ₄ F, 8 M) and 100 μί, of hydrofluoric acid (HF, 2 M) for core dissolution, followed by three centrifugation (1500 g, 4 min)/redispersion (200 μί) cycles. Caution! Hydrofluoric acid and ammonium fluoride are very toxic. Extreme care should be taken, when handling HF solution, and only small quantities should, be prepared. The capsules were redispersed in 100 \ih of PBS for 12 h to remove PVPON and PMA, and GGGWWSSK-PEG4-N3 (SEQ ID NO:77)-functionalized (PVPON _A | _K )5 PEG _A ik capsules were obtained. Step 9. Sortase coupling between DL800 _Ar labeled GGG modified (PVPON _A ik)s/PEG _A i _k capsules and scFv-LPTEG (SEQ ID NO.-282)

The GGGWWSSK-PEG ₄ -N3 (SEQ ID NO:77)-functionalized capsules were incubated in 300 ί of sortase coupling buffer (50 mM Tris, 150 mM NaCl, pH 8) with Sortase A, scFv-LPETG from Example 1 and CaCl ₂ . Sortase A, scFv-LPETG and CaCl ₂ were added to a final concentration of 0.1 g L ^" \ 0.1 g L ^"1 and 0.5 mM, respectively. The mixture was incubated at 37°C for 1 h with gentle shaking. After conjugation, the scFv-LPETG- functionalized capsules were washed with PBS to remove any unattached antibody. This ligation procedure was applied to immobilize both anti-GPIIb/IIIa scFv (denoted as scFv(+)) and the mutated scFv (denoted as scFv(-)).

Example 5: Sortase-mediated conjugation of Lissamine Rhodamine B- LPETGGHHHHHH (SEQ ID NO.-386) with GGGWWSSK-PEG ₄ -N3 (SEQ ID NO:77) modified (PVPON AH) s core-shell particles

Step 1. Preparation of (PVPONAI S core-shell particles

Briefly, silica particles (~3 μπι diameter, 5 wt% suspension, 100 μΙ_.) were first washed with NaOAc (50 mM, pH 4) by centrifugation (1000 g, 1 min) and redispersed in 200 of NaOAc (50 mM, pH 4). To form the multilayers, an equal volume of PVPON was added to the particle suspension for adsorption (15 min) with constant shaking. The particles were then washed via three centrifugation (1000 g, 1 min)/redispersion (200 μί) cycles. After the first layer, the adsorption process was repeated with five bilayers of PMA/PVPON _A ik (both at 1 g L ^"1 in 50 mM NaOAc, pH 4) to form PVPON/(PMA PVPON _A ik)5 core-shell particles. The particles (in 50 μΐ, of 50 mM NaOAc, pH 4) were then incubated with the bisazide linker from Example 4 (1 g L ^'1 , 150 μί) in the presence of sodium ascorbate (4.4 g L ^"1 , 50 μί) and copper sulfate (1.75 g L ^"1 , 50 μΙ- (all in 50 mM NaOAc, pH 4) overnight for crosslinking. Step 2. Sortase-mediated conjugation of Lissamine Rhodamine B-LPETGGHHHHHH (SEQ ID NO.-284) with GGGWWSSK-PEG4-N ₃ (SEQ ID NO:77) modified (PVPONAlk) 5 core-shell particles

For the immobilization of Lissamine Rhodamine B-LPETGGHHHHHH (SEQ ID NO:385) peptide from Example 2, the crosslinked (PVPON _A II _C )S core-shell particles from Step 1 (in 25 iL of 150 mM NaOAc, pH 5) were incubated with a mixture of GGGWWSSK-PEG ₄ - N ₃ (SEQ ID NO:77) (0.2 g L ^" \ 75 iL; i.e., final concentration = 0.1 g L ^"1 ), sodium ascorbate (4.4 g L ^"1 , 25 μΐ) and copper sulfate (1.75 g L ^" \ 25 μΐ) (all in 150 mM NaOAc, pH 5) for 30 min. The GGGWWSSK-PEG ₄ -N3-functionalized particles were redispersed in 100 μί of PBS for 12 h to remove PVPON and PMA.

To evaluate the optimal concentration of Sortase A for the functionalization, the GGGWWSS -PEG4-N3 (SEQ ID NO:77)-coated particles were incubated in 300 L of sortase coupling buffer (50 mM Tris, 150 mM NaCl, pH 8) with Lissamine Rhodamine B- LPETGGHHHHHH (SEQ ID NO:385), CaCl ₂ and Sortase A. Lissamine Rhodamine B- LPETGGHHHHHH (SEQ ID NO:385) and CaCl ₂ were added to a final concentration of 0.1 g L ^"1 and 0.5 mM, respectively. Sortase A was examined at different final concentrations (0, 0.05, 0.1 and 0.5 g L ^'1 ). The mixture was incubated at 37°C for 1 h with gentle shaking. The same procedure was employed for the investigation of the optimal concentration of Lissamine Rhodamine B-LPETGGHHHHHH (SEQ ID NO:385) and ligation time. Lissamine Rhodamine B-LPETGGHHHHHH (SEQ ID NO:385) was tested at 0.05, 0.1 and 0.5 g L ^"1 in the presence of Sortase A (0.1 g L ^"1 ). For the optimization of ligation time, Lissamine Rhodamine B-LPETGGHHHHHH (SEQ ID NO:385) (0.1 g L ^"1 ) was immobilized on the GGGWWSSK-PEG ₄ -N ₃ -coated particles for 0.5, 1 and 2 h in the presence of Sortase A (0.1 g L ^'1 ). After conjugation, the Lissamine Rhodamine B- LPETGGHHHHHH(SEQ ID NO:385)-functionalized particles were washed with PBS extensively.

Fluorescence intensity of the particles not treated with Lissamine Rhodamine B- LPETGGHHHHHH (SEQ ID NO:385) peptide was set at 1. The results in Figure 2A show that the optimal Sortase A concentration is 0.1 g/L. The optimal concentration of Lissamine Rhodamine B-LPETGGHHHHHH (SEQ ID NO:385) peptide was 0.1 g/L as shown in Figure 2B. The results in Figure 2C show that the optimal incubation time is 1 hour.

A further test of the Sortase mediated conjugation was undertaken using the following conditions. The GGGWWSSK-PEG4-N3 (SEQ ID NO:77)-functionalized and unfunctionalized (PVPONAIIOS core-shell particles were incubated with Lissamine Rhodamine B-LPETGGHHHHHH (SEQ ID NO:385) peptide (0.1 g/L) at 37°C for 1 hour at pH 8 (50 mM Tris, 150 mM NaCl) in the presence or absence of Sortase A (0.1 g/L). Fluorescence intensity of the untreated unfunctionalized capsules was set at 1. The results are shown in Figure 2D showing untreated unfunctionalized capsules (white bar), unfunctionalized particles with Lissamine Rhodamine B-LPETGGHHHHHH (SEQ ID NO:385) peptide (hatched bar), unfunctionalized particles, Lissamine Rhodamine B- LPETGGHHHHHH (SEQ ID NO:385) peptide and Sortase A (double hatched bar), GGG W WS SK-PEG ₄ -N3 (SEQ ID . NO:77)-functionalized particles and Lissamine Rhodamine B-LPETGGHHHHHH (SEQ ID NO:385) (gray bar) and GGGWWSSK-PEG4- N ₃ (SEQ ID NO:77)-functionalized particles, Lissamine Rhodamine B- LPETGGHHHHHH (SEQ ID NO:385) and Sortase A (black bar).

Example 6: targeting of scFv-LPETG (SEQ ID NO:282) conjugated DL800 _Ar labeled (PVPONAik)s^PEGAik capsules to in vitro thrombi

The capsules prepared in Example 4 were tested for binding to in vitro human thrombi.

Blood from a healthy volunteer taking no medication was anti-coagulated with citric acid and centrifuged at 1000 rpm for ten minutes. To every 1 mL of the resulting platelet rich plasma (PRP) was added a mixture of adenosinediphosphate (ADP, moLab GmbH, Langenfeld, Germany) (100 sL, 200 mM in water), actin (Dade Behring, Marburg, Germany) (88 iL) and calcium chloride (25 μί, 1 M). To form thrombi, aliquots (100 μί) of the PRP mixture were incubated at 37°C for 12 min. After washing in PBS (with Ca/Mg), each thrombus was exposed to ~2 ^χ I0 ⁵ DL800 _AZ -labeled scFv(+)- and scFv(-)- functionalized, and unfunctionalized capsules in 500 μί, of PBS (with Ca/Mg) at 37°C for 30 min with gentle mixing. The thrombi were then washed in PBS (with Ca/Mg) three times to remove unbound capsules. The fluorescence intensities of the capsule-bound thrombi were analyzed using a Licor Odyssey near infrared imaging system. (Both scFv(+) and scFv(-) were LPETG (SEQ ID NO:380) encoded. scFv(+) = anti-GPIIb/IIIa scFv; scFv(-) = mutated scFv).

To perform completive binding, the activated GP Hb/IIIa receptors on thrombi were pre- blocked- with scFv(+). Thrombi were exposed to scFv(+) (300 μί, 2 g L ^'1 ) in 500 μΐ, of PBS (with Ca/Mg) for 30 min at 37°C with gently mixing. Following incubation, the blocked thrombi were washed three times in PBS (with Ca Mg) to remove attached antibody before addition of the scFv(+)-functionalized capsules. Incubation, washing and analysis were performed as described above.

As shown Figure 3, strong specific binding of anti-GPIIb/IIIa scFv conjugated particles was observed.

Example 7: Sortase conjugation of ScFv-LIBS-LPETG with GGG modified Cells

Cell-anti-LIBS conjugation and functional assessment CHO cells with surface amines were functionalized with sulfhydryl groups according to the protocol described below and reacted with H ₂ N-GGG-AGAGA-K-Malemide (SEQ ID NO:387) as prepared in Example 2. The GGG-functionalized cells were then conjugated with anti-LIBS-LPETG-scFv using sortase methodology. The protocol is set out below.

Step 1. Introduction of sulfltydryls to cell surface via reaction with primary amines using 2-Iminothiolane or Traut's reagent

CHO cells were trypsinized and washed once with PBS. 1 x 10 ⁶ cells were resuspended in 200 μΐ, of modified PBS buffer with EDTA (PBS without Ca and Mg, 4500 mg/L glucose, 15 mM HEPES, 2 mM EDTA, pH 7.3). Traut's reagent was added at the final concentration of 0.6 mM (16 μg). Cells were incubated for 30 min at room temperature with shaking. After the incubation, cells were washed once with modified PBS with EDTA.

Step 2. Labelling cells with NHrGGG-tags via specific reaction of sulfltydryls on cell surface and maleimide groups on NHz-GGG-maleimide peptides:

1 x 10 ⁶ cells were resuspended in 200 μΐ of modified PBS buffer with EDTA. H ₂ N-GGG- AGAGA-K-Maleimide (SEQ ID NO:387) peptide was added to the final concentration of 12 μΜ (2 μg). The reaction mixture was incubated for 30 min at room temperature with shaking. Cells were then washed once with modified PBS buffer without EDTA (PBS with Ca, Mg, 4.5 g/L glucose, 15 mM HEPES, pH 7.3).

Step 3. Sortase-mediated coupling between anti-LIBS-LPETG and NH ₂ -GGG-groups on cell membrane

1 x 10 ⁶ cells were resuspended in 100 μΐ, of modified PBS buffer without EDTA. Anti- LIBS-LPETG and Sortase A enzyme were added to the final concentration of 10 μΜ and 10 μΜ, respectively. Cells were incubated at 37°C for 1 hour with shaking, then pelleted and washed twice with PBS. The supernatant was collected for evaluation of the coupling efficiency. Non-coupled anti-LIBS was quantified by SDS-PAGE. Step 4. Page Analysis

30 μΐ, of each sample (remaining reaction mixture) and 6 μΐ. of 5X reducing SDS loading buffer were added to 1.5-mL tube and denatured at 96°C for 5 min. 36 uL of each sample was run on SDSPAGE gel in SDS running buffer at 30 mA for 2 hours. The gel was then stained with Coommassie Brilliant Blue for 1 hour and subsequently destained for at least 12 hours with Coommassie destaining solution. The gel was visualised and analysed using a BioRad Gel-Doc system with Quantity One software (Australia).

SDS-PAGE analysis of the reaction mixture revealed that the average coupling efficiency was 10.08 ± 3.15 pg (n=3) of anti-LIBS per CHO cell. ft

- 79 -

Step 5. Anti-LIBS-conjugated cells were assessed in in vitro static adhesion assays for their binding ability to activated platelets.

100 iL of diluted purified platelets was added into each well of a 96-well plate and stimulated with 18 μΜ ADP. After incubating at 37°C for 20 min, wells were washed twice with PBS (Ca, Mg) and then blocked with 100 of 1 % BSA in JNL buffer (pH 7.4) for 30 min at 37°C. After washing twice, platelet-coated wells were incubated with predetermined numbers of either targeting cells (anti-LIBS coupled cells) or non-targeting cells (GGG-cells) in PBS with 0.5% BSA at 37°C for 10 min. Wells were then washed twice with PBS and fixed with Cellfix. DIC/fluorescent images were taken by 1X81 Olympus Fluorescence Microscope and Cell ^A P 1692 (AnalySIS Image Processing) software. Binding particles/cells were quantified using Image J software.

It was found that the binding of the cells was significantly stronger than the control (GGG-) cells as shown in Figure 4. The degree of binding increased with increasing cell numbers in a dose dependant manner.

Step 6. Flow Experiments.

After demonstrating the targeting capability of sortase-coupled cells in a static adhesion assay, anti-LIBS-coupled cells were investigated further in flow experiments Glass capillaries (0.20 x 2.0 mm I.D., 10 cm in length, Vitrotubes™, USA) were coated with 42 μΐ, of 100 μg/mL collagen-1 overnight at 4°C. Capillaries were then blocked with 1% BSA in PBS for 1 hour at 37°C. Blood was taken from healthy donors, citrated and perfused through the capillary at a shear rate of 100 s ^"1 . After 5 min, thrombi with desirable sizes formed. The capillary was then washed by perfusing with PBS (Ca, Mg) for 5 min at a shear rate of 500 s ^"1 until no blood cells were observed. Particles in PBS containing 0.5% BSA (3 x 10 ⁶ particles or cells/mL) was perfused through capillary for 5 min at different shear rates. Movies and images were taken using Olympus Fluorescence Microscope and Cell ^A P 1692 (AnalySIS Image Processing) software. Capillaries were washed with PBS and thrombi were specifically stained with FITC-PAC-1 or CD62P-PE. Binding particles were quantified using Image J software. Thrombus area was estimated by Image-Pro Plus 6.0. After perfusing targeted cells through a platelet-coated capillary at 100 s ^'1 for 5 min, approximately 400 cells/mm ² platelet aggregates were immobilized while less than 10 cells bound to a unit area (mm ² ) of platelet aggregates. When the shear rate increased to 250 s ^"1 , specific targeting was still observed, however the number of bound cells decreased to 150 cells/mm ² platelet aggregate, indicating shear dependent targeting efficacy.

Step 7. Cell Staining with CellTracker™ Green CMFDA

To facilitate visualization, cells were stained with CellTracker™ Green CMFDA by the following method.

CHO cells within 20 passages were cultured in 175-cm ² flask until they reached approximately 95% of confluency. The medium was removed and cells were washed once with PBS. CellTracker™ Green CMFDA (Invitrogen, Australia) dye working solution (4 μΜ in plain DMEM buffer without serum) was added to the flask containing the adherent cells. After incubating for 40 min at 37°C/5% C0 ₂ , the dye working solution was replaced with fresh and prewarmed plain DMEM medium (without serum). The cells were incubated for another 30 min at 37°C/5% C0 ₂ . They were then trypsinized, harvested and washed once with PBS before undergoing the coupling process. Step 8. Intravital Microscopy

Intravital microscopy was used to evaluate the targeting of anti-LIBS-coupled cells to activated platelets in mesenteric veins of mice. 4-5 week old C57BL/6 wild-type mice weighing 15-17 g were anaesthetized by intraperitoneal injection of a ketaminerxylazine mixture (100:20 mg/kg body weight). Mesenteric arteries/veins (diameters 100-150 μπι) were observed with inverted microscope (1X81, Olympus) and the images were recorded with digital B/W camera (XM10, Olympus). The injury of arteries/veins was induced by the micro-drop of 10% FeCl ₃ . When the thrombus started building up, 200 of PBS containing the predetermined amounts of anti-Libs particles was injected into the blood flow system via a jugular vein cannula. DIC and fluorescence images of the thrombus were taken before, during and after the injection. Particles were seen by their TRITC autofluorescence. The number of anti-LIBS-conjugated cells binding on the venous thrombi was significantly higher compared to the control (GGG-tagged) cells, indicating successful in vivo cell targeting with this approach.

Example 8: Synthesis ofGGG-PEGs-fCH^s (SEQ ID N0.391)

a. Synthesis of H ₂ N[(CH ₂ ) ₂ 0] ₃ (CH ₂ ) ₂ N3

8.1 8.2 8.3

8.4

A polyethylene glycol compound having a terminal amino group and a terminal azide was prepared following known methods (Tetrahedron, 2009, 65, 7329). Briefly, tetraethyleneglycol 8.1 was treated with mesyl chloride (MsCl) in dichloromethane and triethylamine at 0°C to produce the tetraethyleneglycol dimesylate 8.2. The dimesylated compound was treated with sodium azide (NaN ₃ ) in ethanol/dimethyl acetamide (DMAc) at reflux to give the diazo compound 8.3. One azo group of the diazo compound was then selectively reduced with triphenylphosphine (PPh ₃ ) in a solution of tetrahydrofuran/ether in the presence of 1M HCl to give the monoazo, monoamino PEG compound 8.4 after chromatography.

Synthesis of GGG-PEG ₃ -(CH ₂ ) ₂ N ₃ (SEQ ID NO:391)

Boc-GGG 8.5 was activated with dicarbodiimide (DCC) in the presence of 1.5 equivalents of N-hydroxysucciniamide (NHS) in DMF then treated with 8.4 from a. to form Boc- GGG-PEG ₃ -(CH ₂ ) ₂ N ₃ 8.6. This compound was then treated with trifluoroacetic acid (TFA) to deprotect the N-terminus and provide GGG-PEG ₃ -(CH ₂ ) ₂ N ₃ 8.7. Example 9: Synthesis o/GGGK-ePEG^CHJM (SEQ ID NO:22)

Using standard solid phase synthesis techniques, FmocGGG-EBoc- 9.1 was synthesised on resin. This compound, while still attached to the resin, was treated with TFA in dichloromethane to remove the Boc protecting group from the ε-amino group of the lysine to give FmocGGG-eNH ₂ -K 9.2. H0 ₂ C-PEG ₄ -N ₃ 9.3 (product #16502 purchased from Quanta Biodesign) was activated by treatment with DCC and NHS in dimethylformamide (DMF) and reacted with the deprotected ε-amino group of the lysine to give 9.4. The compound was then deprotected and cleaved from the resin to provide GGGK-ePEG ₄ -N ₃ 9.5.

Example 10: Synthesis of GGG-PEG4-NHCOCH2-4-(3,3~difluorooctacyclooctyne) (SEQ ID NO:34

2-[4-(3,3-difluorocyclooct-l-yne)]acetic acid 10.1 was activated with DCC and NHS in DMF and treated with Boc-NH-[(CH ₂ ) ₂ 0] ₃ (CH ₂ ) ₂ NH ₂ 10.2 to give BocNH-PEG ₃ - (CH ₂ ) ₂ NHCOCH ₂ -4-(3,3-difluorocyclooct-l-yne) 10.3 which was then treated with TFA to remove the Boc protecting group. The resulting H ₂ N-PEG ₃ -(CH ₂ ) ₂ NHCOCH2-4-(3,3- difluorocyclooct-l-yne) 10.3 was reacted with BocGGG 8.5, which had been activated with DCC in DMF, to give BocGGG-PEG ₃ -(CH ₂ ) ₂ NHCOCH ₂ -4-(3,3-difluorocyclooct-l- yne) 10.4. Finally the N-terminal Boc group was deprotected with TFA to give alkyne 10.5.

Example 11: Synthesis of GGGK-sPEG ₃ -(CH^ ₂ NHCOCHi-4-(3,3-difluorooctacyclooct-

2-[4-(3,3-difluorocyclooct-l-yne)]acetic acid 10.1 was activated with DCC and NHS in DMF and treated with H0 ₂ C-[(CH ₂ ) ₂ 0] ₃ (CH ₂ ) ₂ NH ₂ 11.1. After chromatography, the carboxylic acid group of H0 ₂ C-PEG ₃ -(CH ₂ ) ₂ NHCOCH ₂ -4-(3,3-difluorooctacyclooct-l- yne) 11.2 was activated with DCC and NHS in DMF and reacted with resin ^' bound FmocGGG-eNH ₂ -K-resin 10.2 to produce resin bound GGG -ePEG ₃ -(CH ₂ ) ₂ NHCOCH ₂ - 4-(3,3-difluorooctacyclooct-l-yne) 11.3 which was then removed from the resin as described in Example 9 to provide alkyne 11.4. Example 12: Syn

4-Formylbenzoic acid 12.2 was activated with DCC and NHS in DMF and treated with H ₂ N-[(CH ₂ ) ₂ 0]3(CH ₂ ) ₂ NH ₂ 12.1 to give H ₂ N-PEG ₅ -(CH ₂ ) ₂ NHCO-4-formylbenzene 12.3. The resulting H ₂ N-PEG ₃ -(CH ₂ ) ₂ NHCO-4-formylbenzene 12.3 was reacted with BocGGG 8.5, which had been activated with DCC and NHS in DMF, to give BocGGG-PEG ₃ - (CH ₂ ) ₂ NHCO-4-formylbenzene 12.4. Finally the N-terminal Boc group was deprotected with TFA to give aldehyde 12.5. Example 13: Synthesis of GGGK-ePEG _r (CHi) ₂ NHCO-4-formylbenzene (SEQ ID NO:27)

4-Formylbenzoic acid 12.2 was activated with DCC and NHS in DMF and reacted with H0 ₂ C-[(CH ₂ ) ₂ 0]3(CH ₂ ) ₂ NH2 13.1. After chromatography, the carboxylic acid group of H0 ₂ C-PEG ₃ -(CH ₂ ) ₂ NHCO-4-formylbenzene 13.2 was activated with DCC and NHS in DMF and reacted with resin bound FmocGGG-ENH ₂ -K-resin 9.2 to produce resin bound FmocGGGK-ePEG3-(CH ₂ ) ₂ NHCO-4-formylbenzene 13.3 which was then deprotected and removed from the resin to give aldehyde 13.4. Example 14: Synthesis (SEQ ΪΌ NO:36)

H0 ₂ CCH ₂ ONH-Boc 14.1 was activated with DCC and NHS in DMF and treated with an excess of H ₂ N-[(CH ₂ ) ₂ 0]3(CH2)2NH ₂ 12.1 to give H ₂ N-PEG3-(CH ₂ ) ₂ NHCOCH ₂ ONHBoc 14.2. The resulting H ₂ N-PEG3-(CH ₂ )2NHCOCH ₂ ONHBoc was reacted with BocGGG 8.5, which had been activated with DCC and NHS in DMF, to give BocGGGrPEG ₃ - (CH ₂ ) ₂ NHCOCH ₂ ONHBoc 14.3. Finally the N-terminal Boc groups were deprotected with TFA to give alkoxyamine 14.4.

Example 15: Synthesis (SEQID N0.2S)

H0 ₂ CCH ₂ ONHBoc 14.1 was activated with DCC and NHS in DMF and reacted with H0 ₂ C-[(CH ₂ ) ₂ 0]3(CH ₂ ) ₂ NH ₂ 13.1. After chromatography, the carboxylic acid group of H0 ₂ C-PEG ₃ -(CH ₂ ) ₂ NHCOCH ₂ ONHBoc 15.1 was activated with DCC and NHS in DMF and reacted with resin bound FmocGGG-eNH ₂ -K-resin to produce resin bound FmocGGGK-ePEG ₃ -(CH ₂ ) ₂ NHCOCH ₂ ONHBoc 15.2 which was then deprotected and removed from the resin to provide alkoxyamine 15.3. Example 16: Synthesis of GGG-PEG ₄ (CH^ ₂ SCOCH ₃ (SEQ ID NO:392)

o o -o' ^{v v} A

16.2 16.3

BocNH-[(CH ₂ ) ₂ 0] ₄ (CH ₂ ) ₂ OH 16.1 was treated with tosyl chloride and pyridine in dichloromethane to give BocNH-PEG ₄ -(CH ₂ ) ₂ OTs 16.2. The resulting Boc-NH-PEG ₄ - (CH ₂ ) ₂ OTs 16.2 was reacted with tetrabutylammonium iodide (TBAI) and potassium thioacetate (KSAc) in DMF to give Boc-NH-PEG -(CH ₂ ) ₂ SCOCH ₃ 16.3 and then treated with TFA to provide H ₂ N-PEG ₄ -(CH ₂ ) ₂ SCOCH ₃ 16.4. BocGGG 8.5, was activated with DCC and NHS in DMF and reacted with H ₂ N-PEGHCH ₂ ) ₂ SCOCH ₃ 16.4 to give BocGGG-PEG ₄ -(CH ₂ ) ₂ SCOCH ₃ 16.5. Finally the N-terminal Boc group was deprotected with TFA to give thioacetate 16.6.

tBuOCO-[(CH ₂ ) ₂ 0] ₄ (CH ₂ ) ₂ OH 17.1 was treated with tosyl chloride and pyridine in dichloromethane to give tBuOCO-PEG ₄ -(CH ₂ ) ₂ OTs 17.2. The resulting tBuOCO-PEG ₄ - (CH ₂ ) ₂ OTs 17.2 was reacted with tetrabutylammonium iodide (TBAI) and potassium thioacetate (KSAc) in DMF then the t-Butylester was cleaved with TFA to give H0 ₂ C- PEG ₄ -(CH ₂ ) ₂ SCOCH ₃ 17.3. This compound was activated with DCC and NHS in DMF and reacted with resin bound FmocGGG-eNH ₂ -K(SEQ ID NO:293)-resin 9.2 to produce resin bound FmocGGGK-£PEG ₄ -(CH ₂ ) ₂ SCOCH ₃ (SEQ ID NO:294) 17.4 which was then deprotected and removed from the resin to give thioacetate 17.5. Example 18: Synthesis of GGG-PEG ₄ CH C =CH ₂ (SEQ ID NO:40)

Phth-NH-[(CH ₂ ) ₂ 0]4(CH ₂ ) ₂ 0H 18.1 was treated with allyl chloride and tetrabutylammonium sulphate in the presence of NaOH in dichloromethane to give Phth- NH-PEG ₄ -(CH ₂ ) ₂ 0CH ₂ CH=CH ₂ 18.2. The resulting Phth-NH-PEG4-(CH ₂ ) ₂ OCH2CH=CH ₂ 18.2 was reacted with hydrazine to give H ₂ N-PEG ₄ -(CH ₂ ) ₂ 0CH ₂ CH=CH ₂ 18.3. BocGGG 8.5, was activated with DCC and NHS in DMF and reacted with H ₂ N-PEG ₄ - (CH ₂ ) ₂ 0CH ₂ CH=CH ₂ 18.3 to give BocGGG-PEG4-(CH ₂ ) ₂ 0CH ₂ CH=CH ₂ 18.4. Finally the N-terminal Boc group was deprotected with TFA to give the allylic ether 18.5. (SEQ ID NO:29) 19.1

19.3

19.2

9.2

tBuOCO-[(CH ₂ ) ₂ 0] ₄ (CH ₂ ) ₂ OH 17.1 was treated with allyl chloride and tetrabutylammonium sulphate in the presence of aqueous NaOH in dichlororaethane to give tBuOCO-PEG ₄ -(CH ₂ ) ₂ OCH ₂ CH=CH ₂ 19.1. The resulting tBuOCO-PEG - (CH ₂ ) ₂ OCH ₂ CH=CH ₂ 19.1 was treated with TFA to provide H0 C-PEG ₄ - (CH ₂ ) ₂ OCH ₂ CH=CH ₂ 19.2. The carboxylic acid of 19.2 was activated with DCC and NHS in DMF and reacted with resin bound FmocGGG-eNH ₂ -K-resin 9.2 to produce resin bound FmocGGGK-8PEG ₄ -(CH ₂ ) ₂ OCH ₂ CH=CH ₂ 19.3 which was then deprotected and removed from the resin to provide allylic ether 19.4.

Protected cysteine 20.1 was activated with DCC and NHS in DMF then reacted with Fmoc-NH-[(CH ₂ ) ₂ 0] ₃ (CH ₂ ) ₂ NH ₂ 20.2 to give Fmoc-NH-PEG ₃ -(CH ₂ ) ₂ NHCO(4-N-Boc- thiazoline) 20.3. The resulting Fmoc-HN-PEG ₃ -(CH ₂ ) ₂ NHCO(4-N-Boc-thiazoline) 20.3 was Fmoc deprotected with pyridine in DMF to give H ₂ N-PEG ₃ -(CH ₂ ) ₂ NHCO-(4-N-Boc- thiazoline) 20.4. BocGGG 8.5, was activated with DCC and NHS in DMF in the presence of triethylamine and reacted with H ₂ N-PEG ₃ -(CH ₂ ) ₂ NHCO-(4-N-Boc-thiazoline) 20.4 to give BocGGG-PEG ₃ -(CH ₂ ) ₂ NHCO-(4-N-Boc-thiazoline) 20.5. Finally the Boc groups were deprotected with TFA to provide the protected cysteine 21.6. Example 21: Synth HCOf^thiazoline) (SEQ ID NO:395)

Protected cysteine 20.1 was activated with DCC and NHS in DMF then reacted with H0 ₂ C-[(CH ₂ ) ₂ 0]4(CH ₂ ) ₂ NH ₂ 14.1 to give H0 ₂ C-PEG ₄ -(CH ₂ ) ₂ NHCO(4-N-Boc-thiazoline) 21.1. This compound 21.1 was activated with DCC and NHS in DMF and reacted with resin bound FmocGGG-ENH ₂ -K-resin 9.2 to produce resin bound FmocGGGK-ePEG ₄ - (CH ₂ ) ₂ NHCO-(4-N-Boc-thiazoline) 21.2 which was then deprotected and removed from the resin as described in Example 9 to provide protected cysteine 21.3.

Example 22: Synthesis ofGGG-PEG CHfcCOSC CONHCHs (SEQ ID NO:41)

ft

- 91

Boc-NH-[(CH ₂ ) ₂ 0] ₄ (CH ₂ ) ₂ C0 ₂ H 22.1 was activated with DCC and NHS in DMF then treated with N-(methyl)metcaptoacetamide 22.2 to give thioester 22.3. The Boc group of thioester 23.3 was removed by treatment with TFA to give amine-thioester 22.4. The carboxylic acid of Boc-GGG 8.5 was activated by treatment of DCC and NHS in DMF and then reacted with amine-thioester 22.4 in the presence of triethylamine to give Boc-GGG- PEG ₄ -(CH ₂ ) ₂ COSCH ₂ CONHCH3 22.5. The Boc group of the Boc-amino-thioester 23.5 was then removed by treatment with TFA to give thioester 22.6.

Exam le 23: Synthesis o/GGGK-eP

Amine-thioester 22.4 was reacted with 1 eq of /7-nitrophenylchloroformate and triethylamine in DMF to give -nitrophenylcarbamate 23.1. A solution of p- nitrophenylcarbamate 23.1 was added to a suspension of resin bound Fmoc-GGG-£NH ₂ - K(SEQ ID NO:293)-resin 9.2 in DMF to produce resin bound Fmoc-GGG-K-ePEG ₄ - (CH ₂ ) ₂ CO-SCH ₂ CONHCH ₃ (SEQ ID NO:298) 23.4 which was then deprotected and removed from the resin as described in Example 9 to provide thioester 23.3. .Ί98)

The peptide was synthesized on resin. Fmoc-Lys(Dde)-Wang Resin (909 mg, loading 0.33 mmol/g) was allowed to swell in DMF (20 mL) for 30 min. The DMF was drained and the Fmoc protecting group was removed with 20% piperidine/DMF (20 mL) under N ₂ for 30 min. The resin was then washed with DMF (6x10 mL). The glycine residue was conjugated to the lysine residue by adding Boc-Gly-OH (158 mg, 0.9 mmol), HBTU (324 mg, 0.855 mmol), NMM (204 μί, 1.8mmol) in DMF (10 mL) to the resin. The reaction mixture under an N ₂ atmosphere was placed on a shaker for 30 min and the resin was then washed with DMF (3x10 mL). Kaiser test confirmed the reaction was complete.

The ε-ΝΗ ₂ group of lysine was deprotected by treatment with 5% Hydrazine hydrate/DMF (20 mL) under N ₂ for 30 min. The resin was then washed with DMF (6x10 mL). The deprotected lysine amino group was pegylated by treating the resin with HOOC-PEG ₄ - (CH ₂ ) ₂ N ₃ (263 mg, 0.9 mmol), HATU (324 mg, 0.855 mmol) and NMM (204 μί, 1.8 mmol) in DMF (10 mL). The reaction was allowed to proceed under N ₂ for 120 min. The resin was then washed with DMF (3x10 mL). Kaiser test confirmed the reaction was complete. The resin was washed with MeOH (1x10 mL), DCM (3x10 mL) and MeOH (2x1 mL) and then air-dried (1060 mg).

The above peptide was removed from the resin and deprotected by treating with 10.6 mL of a mixture solution (2% H ₂ 0, 3% Tris, 95% TFA) on a shaker at 25°C for 2 hours. The above mixture was filtered to give a filtrate in a 100 mL centrifuge tube. To the filtrate was added 50 mL diethyl ether to triturate the peptide. The solid was Washed with diethyl ether (5x50 mL) and then dried under vacuum for 12 hours to give the crude peptide (108 mg). The crude peptide (108 mg) was dissolved in water (10 mL) and purified by HPLC using a Global Chromatography 30*250mm SP-120-10-C18-AP and a solvent gradient of 18-28 Solvent A/30min. Purity by HPLC: 94.6% MS: Calc 477.27, found: 477.5.

Column: 30*250mm, SP-120-10-C18-AP

Solvent A: 0.1% Trifluoroacetic in 100% Acetonitrile

Solvent B: 0.1% Trifluoroacetic in 100% Water

Gradient: A B

0.01 min 18% 82%

3 min 28% 72%

30.01 min 100% 0%

35 min Stop

Flow rate: 25.0 mL/min

Wavelength: 220 nm

Volume: 10 mL

HPLC Analysis

Type of machine

Beijing chuangxin LC3000 HPLC

Buffer

Solvent A : 0.1 % Trifluoroacetic in 100% Acetonitrile

Solvent B : 0.1 % Trifluoroacetic in 100% Water

MS conditions:

Type of machine

DaoJing LCMS-2020

Ionization

Probe: ESI, Nebulizer Gas Flow: 1.5L/min, Drying Gas Flow: 12L/min, DL Temperature: 250°C, Block Temperature: 200°C, Detector voltage: 1.Okv. Example 25: Synthesis of GGYK(s-NH)-PEGi ₂ -(CH^ ₂ NHC(0)C ₆ H ₄ CHO (SEQ ID -295)

Boc-GGYK on resin was prepared in a similar manner to Example 24 using Fmoc- Lys(Dde)-Wang resin, Fmoc-Tyr(tBu)-OH Fmoc-Gly-OH and Boc-Gly-OH followed by deprotection of the ε-NHDde protecting group.

The lysine ε-ΝΗ ₂ group was reacted with Fmoc-PEGi ₂ -OH (252 mg, 0.3 mmol) in the presence of HATU (108 mg, 0.285 mmol) and NMM (68 μΐ,, 0.6 mmol) in DMF (10 mL) under an N ₂ atmosphere for 120 min. After washing with DMF (3x10 mL), the Fmoc group was deprotected with 20% piperidine/DMF (20 mL) under an N ₂ atmosphere for 30 min. After washing with DMF (6x10 mL), the resin was suspended in DMF (10 mL) and 4-Carboxybenzaldehyde (45 mg, 0.3 mmol), HATU (108 mg, 0.285 mmol) and NMM (68 μί, 0.6 mmol) was added. The reaction proceeded under N ₂ for 120 min followed by washing with DMF (3x10 mL), MeOH (1x10 mL), DCM (3x10 mL) and MeOH (2x10 mL). The resin was then air-dried (420 mg).

The peptide was then removed from the resin and Boc deprotected as described in Example 24 to give the crude peptide (87 mg) which was purified by HPLC as described in Example 24 and analysed by Mass spectrometry. Purity by HPLC: 91.4% MS: C ₅ 4H ₈ 7B ₆ 0 ₂ i ⁺ : Calc: 1155.59, Found: 1156.3. MS conditions:

Type of machine

• Waters ZQ2000

Ionization

Probe: ESI, Cone: 50v, Capillary: 3.00KV, Extractor: 5v, Desovation Temp: 350, Gas Flow: 350.

Example 26: Synthesis of GGYK(e-NH)-PEG ₆ -(CH ₂ ) ₂ NHC(0)C _{6 4} CHO (SEQ ID

This peptide was prepared in an analogous manner to SEQ ID NO: 295 in Example 25 but using Fmoc-NH-(CH2) ₂ -PEG ₆ C0 ₂ H. Purity by HPLC: 94.9% MS: C ₄₂ H63N ₆ 0,5 ⁺ : Calc: 891.43, Found 891.76. Example 27: Synthesis o/GGWYSOrn(0-NH)-PEG ₆ -(CH2)2NHC(0)CH ₂ ONH ₂ (SEQ ID

Boc-GGWYSOrn-6-NHC(0)PEG ₆ (CH ₂ ) ₂ NH ₂ was synthesized in an analogous manner to that described in Example 25 using Fmoc-Orn(Dde)-Wang resin, Fmoc-Ser(tBu)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Trp(Boc)-OH, Fmoc-Gly-OH, Boc-Gly-OH and HOOCPEG ₆ (CH ₂ ) ₂ NHFmoc. The peptide in DMF (10 mL) was treated with Bis-Boc- amino-oxyacetic acid BOC ₂ -Aoa (163 mg, 0.56 mmol), HATU (213 mg, 0.56 mmol) and NMM (171 μί, 1.5 mmol) under an N ₂ atmosphere for 120 min. The resin was then washed with DMF (3x10 mL) followed by washing with MeOH (1x10 mL), DCM (3x10 mL) and MeOH (2x10 mL) and then air-drying (560 mg). The peptide was then removed from the resin and Boc deprotected as described in Example 24 to give the crude peptide (213 mg) which was purified by HPLC as described in Example 24 using a Boston Crest ODS 5um 100A column and a solvent gradient of 15-40 Solvent A/30min followed by analysis by Mass spectrometry as described in Example 24. Purity by HPLC: 93% MS: C49H ₇ 6N ₁₀ Oi8 ²⁺ : Calc(M+2H) ²⁺ : 546.26: Found: 545.75.

Example 28: Synthesis of GGDK(e-NH)Cys (SEQ ID NO.-379)

(S)-thiazolidine-4-carboxylic acid

Boc-GGDK(e-NH ₂ ) was synthesized in an analogous manner to that described in Example 25 using Fmoc-Lys(Dde)-Wang resin, Fmoc-Asp(OtBu)-OH, Fmoc-Gly-OH and Boc-Gly- OH. Boc-GGDK(8-NH ₂ ) in DMF (10 mL) was treated with Fmoc-thiazolidine-COOH (Fmoc-Thz-OH) (110 mg, 0.31 mmol), HATU (118 mg, 0.31 mmol) and NMM (100 μί, 0.88 mmol) under an N ₂ atmosphere for 120 min then washed with DMF (3x10 mL). The resin was further washed with MeOH (1x10 mL), DCM (3x10 mL) and MeOH (2x10 mL) and then air-dried (580 mg). The peptide was then removed from the resin and Boc deprotected as described in Example 24 to give the crude peptide (103 mg) which was purified by HPLC as described in Example 24 using 4.6x250 mm, Sinochrom ODS-BP-5 column and a solvent gradient of 10-35 Solvent A/30min followed by analysis by Mass spectrometry as described in Example 24. Purity by HPLC: 97% MS: CigH ₃ iN60gS ⁺ : Calc: 491.19, Found: 491.55.

Example 29: Synthesis ofGGGFDK(e-NH)-KKK-C(0)C ₆ H ₄ CHO (SEQ ID NO: 304)

Boc-GGGFDKs-NH ₂ was synthesized in a similar manner as described in Example 24 using Fmoc-Lys(Dde)-Wang resin, Fmoc-Asp(OtBu)-OH, Fmoc-Phe-OH, Fmoc-Gly-OH and Boc-Gly-OH. Boc-GGGFDKe-NH ₂ in DMF (10 mL) was treated with Fmoc- Lys(Boc)-OH (141 mg, 0.3 mmol), HBTU (108 mg, 0.285 mmol) and NMM (68 μΐ, 0.6 mmol) under an N ₂ atmosphere for 30 min and was then washed with DMF (3x10 mL). The Fmoc group was removed using 20% piperidine DMF (20 mL) under an N ₂ atmosphere for 30 min followed by washing with DMF (6x10 mL). This reaction and deprotection was repeated twice more with Fmoc-Lys(Boc)-OH to give GGGFDK(e-NH)- KK . GGGFDK(e-NH)-KKK in DMF (10 mL) was then treated with 4- Carboxybenzaldehyde (45 mg, 0.3 mmol), HATU (108 mg,0.285 mmol) and NMM (68 μί, 0.6 mmol) under an N ₂ atmosphere for 120 min. The resin was then washed with DMF (3x10 mL), followed by washing with MeOH (1x10 mL), DCM (3x10 mL) and MeOH (2x10 mL) and then air-drying (438 mg). The peptide was then removed from the resin and Boc deprotected as described in Example 24 to give the crude peptide (92 mg) which was purified by HPLC as described in Example 24 using a SP-120-10-C18-AP column and the purification gradient is 19-31 Solvent A/30min followed by analysis by Mass spectrometry as described in Example 24. Purity by HPLC: 91.9% MS: C ₅₁ H _7g Ni30i4 ⁺ : Calc: 1096.58, Found: 1097.4.

Example 30: Synthesis of GGGE(d-CONH)-PEG4-(CH ₂ ) ₂ C(0)SCH ₂ C(0)NHCH3 (SEQ ID NO: 330)

Boc-GGGE(a-OtBu)(6-CONH) PEG ₄ C0 ₂ H was synthesized from Fmoc-PEG ₄ -2-Cl-Frt-Cl resin using Fmoc-Glu-OtBu, Fmoc-Gly-OH and Boc-Gly-OH and standard peptide synthesis techniques. The peptide was removed from the resin using 2% TFA DCM (20 mL) under N ₂ for 5 min. The peptide was triturated with diethylether (50 mL) and the solid washed with diethylether (5 x 50 mL) and dried under vacuum to give crude Boc protected peptide (378 mg). The crude peptide in DMF (10 mL) was treated with Z- mercapto-N-methylacetamide (64 mg, 0.56 mmol), HATU (152 mg, 0.4 mmol) and NMM (122 μί, 1.07 mmol) under an N ₂ atmosphere for 12 hours. The peptide was triturated with diethyl ether (150 mL) and the solid washed with diethylether (5 x 50 mL) then dried under vacuum for 12 hours to give crude peptide (252 mg). The peptide was purified by HPLC as described in Example 24 using a VYDAC-C18 5μηι 100A column and a solvent gradient of 10-40 Solvent A/30 min. The peptide was analysed by mass spectrometry as described in Example 24. Purity by HPLC: 95.1% MS: C ₂₅ H ₄₅ N ₆ O _l2 S ⁺ : Calc: 653.28, Found 653.6. N0.321)

Boc-GGGWSOrn(6-NH ₂ ) on resin was synthesized in an analogous manner to Example 25 using Fmoc-Orn(Dde)-Wang resin, Fmoc-Ser(tBu)-OH, Fmoc-Trp(Boc)-OH, Fmoc-Gly- OH and Boc-Gly-OH. Boc-GGGWSOrn(5-NH ₂ ) in DMF (10 mL) was added to S-acetyl- dPEG ₄ -NHS ester (63.22 mg, 0.15 mmol) and NMM (107 μί, 0.6 mmol) under an N ₂ atmosphere for 30 min, followed by washing with DMF (3x10 mL), DCM (3x15 mL) and MeOH (3x 15 mL) and then air-dried (342 mg). GGGWSOrn(6-NH)PEG ₄ -(CH ₂ ) ₂ SAc was removed from the resin and deprotected as described in Example 24 to give the crude peptide (98 mg). The crude peptide was purified by HPLC using a Venusil XBP C18(L) column and a solvent gradient of 24-49 Solvent A/30min as described in Example 24 and analysed by mass spectrometry as described in Example 25. Purity by HPLC: 93.1% MS: C ₃₈ H45N ₆ Oi ₂ S ⁺ : Calc: 883.39, Found: 883.82.

GGGWSOrn(8-NH ₂ ) was synthesized as described in Example 25 using Rink Amide-AM Resin, Fmoc-Orn(Dde)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Trp(Boc)-OH, Fmoc-Gly-OH and Boc-Gly-OH. GGGWSOrn(8-NH ₂ ) in DMF (10 mL) was treated with S-acetyl-dPEG ₄ - NHS ester (63.22 mg, 0.15 mmol) and NMM (107 LL, 0.6 mmol) under an N ₂ atmosphere for 30 min followed by washing with DMF (3x15 mL), DCM (3x15 mL) and MeOH (3x 15 mL) and then air-dried (323 mg). The peptide was removed from the resin and deprotected as described in Example 24 to give the crude peptide (89 mg) which was purified by HPLC as described in Example 24 using a Venusil XBP C18(L) column and a solvent gradient of 24-49 Solvent A/30min. The peptide was analysed by mass spectrometry as described in Example 25. Purity by HPLC: 90.8% MS: C3gH6oN90i3S ⁺ : Calc: 882.40, Found 882.06.

Example 33: Synthesis ofGGGWSK(e-NH)C(0)(CH2) ₂ OCH ₂ OCH (SEQ ID NO:396)

Boc-GGGWS (e-NH ₂ ) was synthesized as described in Example 25 using Fmoc- Lys(Dde)-Wang resin, Fmoc-Ser(tBu)-OH, Fmoc-Trp(Boc)-OH, Fmoc-Gly-OH and Boc- Gly-OH. Boc-GGGWS (e-NH ₂ ) in DMF (10 mL) was treated with Propargyl-dPEG®l- NHS ester (136 mg, 0.6 mmol), HATU (216 mg, 0.57 mmol) and NMM (136 μΐ., 0.2 mmol) under an N ₂ atmosphere for 120 min. The resin was then washed with DMF (3x10 mL), MeOH (1x10 mL), DCM (3x10 mL) and MeOH (2x10 mL) and then air-dried (775 mg). The peptide was removed from the resin and deprotected as described in Example 24 to give the crude peptide (125 mg) which was purified by HPLC using a SP-120-10-C18- AP column and a solvent gradient of 15-25 Solvent A/30min as described in Example 24. The peptide was analysed by mass spectrometry as described in Example 25. Purity by HPLC: 96.7% MS: C ₃ 2H ₄ 5N ₈ Oio ⁺ : Calc: 701.33, Found 701.22.

Example 34: Synthesis of GGGWSK(e-NH)C(0)(CH _{2 2} OCH ₂ C≡CH amide

Boc-GGGWSK(e-NH ₂ ) was synthesized as described in Example 25 using Rink Amide MB HA resin, Fmoc-Lys(Dde)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Trp(Boc)-OH, Fmoc-Gly- OH and Boc-Gly-OH. BOC-GGGWSK(E-NH ₂ ) in DMF (10 mL) was treated with Propargyl-dPEG®l-NHS ester (136 mg, 0.6 mmol), HATU (216 mg, 0.57 mmol) and NMM (136 μ∑, 1.2 mmol) under N ₂ for 120 min. The resin was then washed with DMF (3x10 mL). Kaiser test confirmed the reaction was complete. The resin was washed with MeOH (1x10 mL), DCM (3x10 mL) and MeOH (2x10 mL) and then air-dried (760 mg). The peptide was removed from the resin and deprotected as described in Example 24 to give the crude peptide (110 mg) which was purified by HPLC using a SP-120-10-C18-AP column and a solvent gradient of 25-35 Solvent A/30min and analysed by mass spectrometry as described in Example 24. Purity by HPLC: 93.7% MS: C ₃₂ H46N ₉ 0 ₉ ⁺ : Calc: 700.34, Found 700.80.

Example 35: Synthesis of GGGGK(s-NH)-PEGg-(CH2) ₂ NHCO-thiazolidinone (SEQ ID NO.-397)

BOC-GGGGK(E-NH-PEG ₈ -NH ₂ ) was synthesized as described in Example 25 using Fmoc- Lys(Dde)-Wang Resin, Fmoc-Gly-OH, Boc-Gly-OH and Fmoc-PEG ₈ -OH. Boc- GGGGK(e-NH -PEG ₈ -NH ₂ ) on resin in DMF (10 mL) was added to Fmoc-Thz-OH (110 mg, 0.31 mmol), HATU (118 mg, 0.31 mmol) and NMM (100 μΐ,, 0.88 mmol) under an N ₂ atmosphere for 120 min. The resin was then washed with DMF (3x10 mL) followed by washing with MeOH (1x10 mL), DCM (3x10 mL) and MeOH (2x10 mL) and then air- drying (635mg). The peptide was removed from the resin and deprotected as described in Example 24 to give the crude peptide (286 mg) which was purified by HPLC using a VYDAC-C18 column and a solvent gradient of 20-45 Solvent A/30min and analysed by mass spectrometry as described in Example 25. Purity by HPLC: 93% MS: C ₃₇ H ₆₉ N ₈ Oi6S ⁺ : Calc: 913.45, Found 913.74. Example 36: Synthesis of GGGGWE(S-CONH)PEG4-(CH^2C(0)NHNH ₂ (SEQ ID

Boc-GGGGWE(6-COOH) was synthesized as described in Example 25 using Fmoc- Glu(ODmab)-2cl (Trt) Resin, Fmoc-T (Boc)-OH, Fmoc-Gly-OH and Boc-Gly-OH. Boc- GGGGWE(5-COOH) on resin in DMF (10 mL) was added to Amino-dPEG ₄ -t-Boc- hydrazide (75.89 mg , 0.2 mmol), TBTU (96.3 mg, 0.3 mmol), DIEA (107 μΐ,, 0.6 mmol) under an N ₂ atmosphere for 30 min. The resin was then washed with DMF (3x10 mL). Kaiser test confirmed the reaction was complete. The resin was washed with DMF (3x 15 mL), DCM (3x15 mL) and MeOH (3x 15 mL) and then air-dried (442 mg). The peptide was removed from the resin and deprotected as described in Example 24 to give the crude peptide (105 mg) which was purified by HPLC using a kromasil CI 8-5 column and a solvent gradient of 15-40 Solvent A/30min and analysed by mass spectrometry as described in Example 25. Purity by HPLC: 92.3% MS: C ₃ 5H5 ₅ N ₁ o0 ₁ 3 ⁺ : Calc: 823.39, Found 821.88.

(SEQ ID NO:367)

Boc-GGGGGWK(e-NH ₂ )-on resin was synthesized as described in Example 25 using Fmoc-Lys(Dde)-Wang Resin, Fmoc-Trp(Boc)-OH, Fmoc-Gly-OH and Boc-Gly-OH. The Boc-GGGGGWK(e-NH ₂ )-on resin in DMF (10 mL) was added to Fmoc-Glu(Otbu)-OH (128 mg, 0.3 mmol), HBTU (108 mg, 0.285 mmol), NMM (68 μΐ, 0.6 mmol) under an N ₂ atmosphere for 30 min. The resin was then washed with DMF (3x10 mL) and deprotected with 20% piperidine/DMF (20 mL) under an N ₂ for 30 min. The resin was then washed with DMF (6x10 mL). This reaction was repeated twice more to provide Boc- GGGGGWK-e-NH-EEE-C0 ₂ H. Boc-GGGGGWK-£-NH-EEE-C0 ₂ H on resin in DMF (10 mL) was added to N ₃ -(CH ₂ ) ₂ -PEG ₄ -COOH (88 mg, 0.3 mmol), HATU (108 mg, 0.285 mmol) and NMM (68 \xL, 0.6 mmol) under an N ₂ atmosphere for 120 min. The resin was then washed with DMF (3x10 mL) followed by washes with MeOH (1x10 mL), DCM (3x10 mL) and MeOH (2x10 mL) and then air-dried (476 mg). The peptide was removed from the resin and deprotected as described in Example 24 to give the crude peptide (1 12 mg) which was purified by HPLC using a SP-120-10-C18-AP column and a solvent gradient of 25-35 Solvent A/30min and analysed by mass spectrometry as described in Example 24. Purity by HPLC: 91.7% MS: C ₅₃ H ₇₈ Ni ₇ 0 ₂₂ ⁺ : Calc: 1278.9, Found 1279.4. -NH)-Lipoic acid (SEQ ID NO.-378)

Boc-GGGGGWDDK(e-NH ₂ )-on resin was synthesized as described in Example 25 using Fmoc-Lys(Dde)-Wang Resin, Fmoc-Asp(otBu)-OH, Fmoc-Trp(Boc)-OH, Fmoc-Gly-OH and Boc-Gly-OH. Boc-GGGGGWDDK(e-NH ₂ )-on resin in DMF (10 mL) was added to Lipoic acid (64 mg, 0.31 mmol), HATU (1 18 mg, 0.31 mmol) and NMM (100 fiL, 0.88 mmol) under an N ₂ atmosphere for 120 min. The resin was then washed with DMF (3 10 mL) followed by washing with MeOH (1x10 mL), DCM (3x10 mL) and MeOH (2x10 mL) and then air-drying (745mg). The peptide was removed from the resin and deprotected as described in Example 24 to give the crude peptide (340 mg) which was purified by HPLC using a Boston Crest ODS column and a solvent gradient of 21-46/30min and analysed by mass spectrometry as described in Example 24. Purity by HPLC: 90.3% MS: C ₄ 3H6 ₂ N, ,Oi5S ⁺ : Calc: 1036.39, Found 1034.85. ample 39: Synthesis of G-PEG ₄ -CH ₂ OCH (SEQ ID N0.2U)

Boc-Gly-OH (100 mg, 0.57 mmol) in DMF (10 mL) was added to NH ₂ -PEG ₄ -CH ₂ -C≡CH (100 mg, 0.432 mmol), HBTU (332 mg, 0.875 mmol) and DIEA (110.94 μί 0.86 mmol) and allowed to react for 24 h. Water was added to triturate the peptide which was then treated with 11.7 mL of a mixture solution (2% H ₂ 0, 3% Tis, 95% TFA) on a shaker at 25°C for 2 hours. The mixture was filtered and 50 mL diethyl ether was added to the filtrate to triturate the peptide. The solid was washed with diethyl ether (5x50 mL) and dried under vacuum for 12 hours to give the crude peptide (105 mg). The crude peptide was dissolved in water (10 mL) and purified by HPLC using a Venusil XBP-C18 5um 100 A column and a solvent gradient of 10-35 Solvent A/30min as described in Example 24. Mass spectral analysis was also performed as in Example 24. Purity by HPLC: 93% MS: Ci ₃ H ₂₅ N ₂ 0 ₅ ⁺ : Calc: 289.18, Found 289.25.

Boc-GE-(a-COOH) was synthesized on resin as described in Example 24 using Fmoc-Glu- a-ODmab Wang Resin and Boc-Gly-OH. Boc-GE-(a-COOH) on resin in DMF (10 mL) was added to NH ₂ -PEG ₄ -(CH ₂ ) ₂ -N ₃ (197 mg, 0.9 mmol), HATU (324 mg, 0.855 mmol) and NMM (204 μί, 1.8 mmol) under an N ₂ atmosphere for 12 hours. The resin was then washed with DMF (3x10 mL) followed by washing with MeOH (1x10 mL), DCM (3x10 mL) and MeOH (2x10 mL) and then air-drying (1086 mg). The peptide was removed from the resin and deprotected as described in Example 24 to give the crude peptide (102 mg) which was purified by HPLC using a Galaksil UP-C18 lOum 120A column and a solvent gradient of 17-27/30min and analysed by mass spectrometry as described in Example 24. Purity by HPLC: 92.2% MS: Ci ₅ H ₂ 9N ₆ 0 ₇ ⁺ : Calc: 405.21, Found 405.0. NO:400)

Boc-GGWE-(a-COOH) was synthesized on resin as described in Example 24 using Fmoc- Glu-a-ODmab Wang Resin, Fmoc-Trp(Boc)-OH, Fmoc-Gly-OH and Boc-Gly-OH. Boc- GGWE-(a-COOH) on resin in DMF (10 mL) was treated with NH ₂ -PEG ₄ -(CH ₂ ) ₂ - CONHNH-(Boc) (152 mg, 0.4 mmol), HATU (152 mg, 0.4 mmol) and NMM (122 1.07 mmol) under an N ₂ atmosphere for 2 hours. The resin was then washed with DMF (3x10 mL) followed by washing with MeOH (1x10 mL), DCM (3x10 mL) and MeOH (2x10 mL) and then air-drying (750mg). The peptide was removed from the resin and deprotected as described in Example 24 to give the crude peptide (180 mg) which was purified by HPLC using a VYDAC-C18 5um 100A column and a solvent gradient of 10- 35 Solvent A/30min and analysed by mass spectrometry as described in Example 25. Purity by HPLC: 93.6% MS: CsiFLwNgOn* Calc: 709.35, Found 709.3. e (SEQ ID NO.401)

Aminomethylthiazolidine-3-car oxylic add tert-butyl ester

Boc-GGSE-a-COOH was synthesized as described in Example 24 using Fmoc-Glu-ct- ODmab Wang Resin, Fmoc-Ser(tBu)-OH, Fmoc-Gly-OH and Boc-Gly-OH. Boc-GGSE- a-COOH on resin in DMF (10 mL) was treated with (R)-4-Aminomethylthiazolidine-3- carboxylic acid tert-butyl ester (131 mg, 0.6 mmol), HATU (152 mg, 0.4 mmol) and NMM (122 μί, 1.07 mmol) under an N ₂ atmosphere for 2 hours. The resin was then washed with DMF (3x10 mL) followed by washing with MeOH (1x10 mL), DCM (3x10 mL) and MeOH (2x10 mL) and air-drying (780mg). The peptide was removed from the resin and deprotected as described in Example 24 to give the crude peptide (210 mg) which was purified by HPLC using a V YDAC-C 18 5um 100A column and a solvent gradient of 2-25 Solvent A/30min and analysed by mass spectrometry as described in Example 24. Purity by HPLC: 93.1% MS: Ci ₆ H ₂₉ N ₆ 0 ₇ S ⁺ : Calc: 449.18, Found 449.4.

(4-(1.3-Dioxolan-2-yl)phenyl)methanamine

Boc-GGGDEEE-a-COOH was synthesized as described in Example 24 using Fmoc-Glu- a-ODmab Wang Resin, Fmoc-Asp(OtBu)-OH, Fmoc-Gly-OH and Boc-Gly-OH. Boc- GGGDEEE-a COOH on resin in DMF (10 mL) was treated with (4-(1.3-Dioxolan-2- ^' yl)phenyl)methanamine (162 mg, 0.9 mmol), HATU (324 mg, 0.855 mmol) and NMM (204 μί, 1.8mmol) under an N ₂ atmosphere for 12 hours. The resin was then washed with DMF (3x10 mL) followed by washing with MeOH (1x10 mL), DCM (3x10 mL) and MeOH (2x10 mL) and then air-drying (1170 mg). The peptide was removed from the resin and deprotected as described in Example 24 to give the crude peptide (205 mg) which was purified by HPLC using a Galaksil UP-C18 lOum 120A column and a solvent gradient of 1 1-21 Solvent A/30min and analysed by mass spectrometry as described in Example 24. Purity by HPLC: 92.8% MS: C ₃₃ H ₄₃ N ₈ Oi ₆ ^" : Calc: 807.28, Found 807.6. ID NO:403)

Boc-GGG-EEE-a-COOH was synthesized as described in Example 24 using Fmoc-Glu-a- ODmab Wang Resin, Fmoc-Glu(OtBu)-OH, Fmoc-Gly-OH and Boc-Gly-OH. Boc-GGG- EEE-a-COOH on resin DMF (10 mL) was treated with (Trt) S-CH ₂ CH ₂ -NH ₂ (259 mg, 0.9 mmol), HATU (324 mg, 0.855 mmol) and NMM (204 μΐ, 1.8 mmol) under an N ₂ atmosphere for 12 hours. The resin was then washed with DMF (3x10 mL) followed by washing with MeOH (1x10 mL), DCM (3x10 mL) and MeOH (2x10 mL) and then air- drying (1170 mg). The peptide was removed from the resin and deprotected as described by treatment with 11.7 mL of a mixture solution (2.5% H ₂ 0, 2.5% phenol, 2.5% EDT, 5% methylphenylsulf.de, 87.5% TFA) on a shaker at 25°C for 2 hours. The above mixture was filtered and the filtrate was treated with 50 mL diethyl ether to triturate the peptide. The solid was washed with diethyl ether (5x50 mL) and then dried under vacuum for 12 hours to give the crude peptide (165 mg) which was purified by HPLC as described in Example 24 using a SP-120-10-C18-AP column and a solvent gradient of 20-30/30min. Mass spectral analysis was also performed as described in Example 25. Purity by HPLC: 94.1% MS: C ₂₃ H ₃₈ N ₇ Oi ₂ S ⁺ : Calc: 636.23, Found 636.25. 4)

Boc-GGGG-EEE-a-COOH was synthesized as described in Example 24 using Fmoc-Glu- a-ODmab Wang Resin, Fmoc-Glu(OtBu)-OH, Fmoc-Gly-OH and Boc-Gly-OH.

Boc-GGGG-EEE-a-COOH on resin in DMF (10 mL) was treated with NH ₂ -PEG ₄ -(CH ₂ ) ₂ - N ₃ (197 mg, 0.9 mmol), HATU (324. mg, 0.855 mmol) and NMM (204 μί, 1.8mmol) under an N ₂ atmosphere for 12 hours. The resin was then washed with DMF (3x10 mL) followed by washing with MeOH (1x10 mL), DCM (3x10 mL) and MeOH (2x10 mL) and then air-drying (1150mg). The peptide was removed from the resin and deprotected as described in Example 24 to give the crude peptide (189 mg) which was purified by HPLC using a Galaksil UP-C18 lOum 120A column and a solvent gradient of 14-24 Solvent 1 V.

- 109 -

A/30min and analysed by mass spectrometry as described in Example 25. Purity by HPLC: 90.5% MS: C ₃₁ H ₅ 2Nn0 ₁₆ ⁺ : Calc: 834.36, Found 834.41. NO:40S)

GGGGGWE-EEE-a-COOH was synthesized as described in Example 24 using Fmoc-Glu- a-ODmab Wang Resin, Fmoc-Glu(OtBu)-OH, Fmoc-Trp(Boc)-OH, Fmoc-Gly-OH and Boc-Gly-OH. GGGGGWE-EEE-a-COOH on resin in DMF (10 mL) was treated with NH ₂ -PEG ₄ -(CH ₂ ) ₂ -N ₃ (197 mg, 0.9 mmol), HATU (324 mg, 0.855 mmol) and NMM (204 μί, 1.8 mmol) under an N ₂ atmosphere for 12 hours. The resin was then washed with DMF (3x10 mL) followed by washing with MeOH (1x10 mL), DCM (3x10 mL) and MeOH (2x10 mL) and then air-drying (1250mg). The peptide was removed from the resin and deprotected as described in Example 24 to give the crude peptide (257 mg) which was purified by HPLC using a Galaksil UP-C18 lOum 120A column and a solvent gradient of 18-28 Solvent A 30min and analysed by mass spectrometry as described in Example 25. Purity by HPLC: 95.6% MS: C49H70N15O21 ^" : Calc: 1204.49, Found 1204.86. ID NO:276)

This peptide was prepared and cleaved from the resin according to the standard procedures. The crude peptide (257 mg) was purified by HPLC as described in Example 24 using a Galaksil UP-C18 lOum 120 A column and the solvent gradient was 18-28/30min. Mass spectral analysis was performed as for Example 25.

Purity by HPLC: 100%. MS: C ₂ 2H ₃₈ N ₉ OioS ⁺ : Calc: 620.25, Found: 619.15. Example 48: Synthesis ofGGGWWSSK-fe-NH^PEG^fCH^iNHCOCsH^- =C(CH ₃ ) ₂ (SANH) (SEQ ID NO.-277)

Boc-GGGWWSSK-(e-NH ₂ ) on resin was prepared and the Dde protecting group of the lysine ε-ΝΗ ₂ moiety deprotected according to standard procedures. Boc- GGGWWSS -(E-NH ₂ ) on resin in DMF (10 mL) was treated with Fmoc-PEG ₄ - C0 ₂ H (0.3 mmol), HATU (108 mg, 0.285 mmol), NMM (68 μί, 0.6 mmol) under an N ₂ atmosphere for 120 min followed by washing with DMF (3x10 mL). The Fmoc group was removed with 20% piperidine/DMF (20 mL) and the resin was washed with DMF (6x10 mL). Boc-GGGWWSSK-(e-NH ₂ )PEG ₄ -NH ₂ in DMF (10 mL) and treated with NHS-SANH (0.3 mmol) and NMM (68 iL, 0.6 mmol) under an N ₂ atmosphere for 120 min followed by washing with DMF (3x10 mL), then MeOH (1x10 mL), DCM (3xl0mL) and MeOH (2x10 mL) and then air-dried (400 mg). The peptide was removed from the resin and deprotected as described in Example 24 to give the crude peptide. The crude peptide (257 mg) was purified by HPLC as described in Example 24 using a Galaksil UP-C18 lOum 120A column and a solvent gradient of 15-50 Solvent A 30min. Mass spectral analysis was performed as described in Example 25. Purity by HPLC: 95.4%. MS: CeoHssNiei : Calc: 1285.63, Found: 1285.4. -(e-NH)-PEG ₄ -DBCO (SEQ ID NO.-278)

Boc-GGGWWSSK-(e-NH ₂ ) was prepared and the Dde protecting group of the lysine ε-ΝΗ ₂ moiety deprotected according to standard procedures. Boc- GGGWWSSK-(e-NH ₂ ) in DMF (10 mL) was treated ith DBCO-PEG ₄ -C0 ₂ H (0.3mmol), HATU (108 mg, 0.285 mmol), NMM (68 μΐ, 0.6 mmol) under an N ₂ atmosphere for 120 min followed by washing with DMF (3x10 mL) then MeOH (1x10 mL), DCM(3xl0 mL) and MeOH (2x10 mL) and then air-dried (400 mg). The peptide was removed from the resin and deprotected with 5%H ₂ 0, 95%TFA) at 25°C for 2 hours, followed by filtration, to which 50 mL diethyl ether was added to triturate the peptide. The solid was washed with diethyl ether (5x50 mL) and dried under vacuum for 12 hours to give the crude peptide. The crude peptide (257mg) was purified as described in Example 24 using a Galaksil UP-C18 lOum 120A column and a solvent gradient of 15-50 Solvent A/30min. Mass spectral analysis was carried out as described in Example 25. Purity by HPLC: 95.3%. MS: C ₇ 4H95N _l4 Oi9 ⁺ : Calc: 1483.69, Found 1485.3. Example 50: Synthesis ofGGGWWSSK-(s-NH)-PEG ₄ -Pentenoic acid (SEQ ID NO:279)

Boc-GGGWWSSK-(e-NH ₂ ) was prepared and the Dde protecting group of the lysine ε-ΝΗ ₂ moiety deprotected according to standard procedures. Boc- GGGWWSSK-(e-NH ₂ ) on resin in DMF (10 mL) was treated with Fmoc-PEG ₄ - C0 ₂ H (0.3 mmol), HATU (108 mg, 0.285 mmol), NMM (68 μί, 0.6mmol) under N ₂ for 120 min. The resin was then washed with DMF (3x10 mL) and the Fmoc group removed. The resin in DMF (10 mL) was treated with 4-Pentenoic acid (0.3mmol), HATU (108 mg, 0.285 mmol) and NMM (68 μί, 0.6 mmol) under N ₂ for 120 min followed by with DMF (3x10 mL) then with MeOH (1x10 mL), DCM (3x10 mL) and MeOH (2x10 mL) and then air-dried (400 mg). The peptide was removed from the resin and deprotected as described in Example 48 to give the crude peptide. The crude peptide (257mg) was purified and analysed by mass spectroscopy as described in Example 48. Purity by HPLC: 97.2%. MS:

C56H ₇₉ N ₁₄ 0 _I7 ^" , Calc: 1191.57. Found: 1192.15. (SEQ ID NO:77)

GGGWWSSK-(e-NH ₂ ) was prepared and the Dde protecting group of the lysine ε-ΝΗ ₂ moiety deprotected according to standard procedures. GGGWWSSK-(s-NH ₂ ) on resin in DMF (10 mL) was treated with N ₃ -PEG ₄ -C0 ₂ H (0.3mmol), HATU (108 mg, 0.285 mmol), NMM (68 μί, 0.6mmol) under N ₂ ^' for 120 min. The resin was then washed with DMF (3x10 mL) then with MeOH (1x10 mL), DCM (3x10 mL) and MeOH (2x10 mL) and then air-dried (400 mg). The peptide was removed from the resin, deprotected, purified and analysed by mass spectrometry as described in Example 48. Purity by HPLC: 96.7%. MS: C ₅ iH ₇₃ N,40i ₆ ⁺ : Calc: 1137.53, Found: 1138.58.

Example 52: Recombinant preparation ofLPETG tagged proteins and sortase proteins Cloning and DNA preparation

DNA coding for the protein to be expressed was ordered from Geneart/Invitrogen optimised for the expression host (see table). All constructs were transformed into NEB Turbo Competent E, coli (High Efficiency) (C2984) and grown at 37°C under shaking in the presence of ampicillin (100 μg/mL) overnight. DNA was extracted using QIAprep Spin Miniprep Kit (Qiagen) and digested at 37°C for 3h with the corresponding Enzymes (NEB, see table).

Samples were run on 0.8% agarose gel and digested fragments extracted from the gel using QIAquick Gel Extraction Kit (Qiagen). ^■ Inserts were ligated into the corresponding vectors (see table) over night at 4°C using T4 DNA Ligase (NEB). Ligated vectors were transformed into NEB Turbo Competent E. coli (High Efficiency) (Qiagen) and positive colonies selected by PCR. DNA was prepared using QIAprep Spin Miniprep Kit (Qiagen) for construct to be expressed in E. coli and EndoFree Plasmid Maxi Kit (Qiagen) for constructs to be expressed in S2 insect cells, HEL293 or CHO cells. DNA for E. coli expression was transformed into BL21 Star™ Chemically Competent Cells for expression.

Protein production

in E. coli (BL21DE3*)

Confirmed clones are plated on ampicillin (100 μξ/πιΐ,) containing agar plates and incubated at 37°C overnight. One clone is inoculated with 5 mL of LB broth with ampicillin (100 μg/mL) and grown overnight at 37°C under shaking. The starter culture is transferred to a 1L production culture and expanded until the OD reaches 0.6-0.8 units. Protein production is started by adding 1 mM IPTG for 4h. Bacteria are lysed (BugBuster, Novagen), spun down (15,000 g for 15 min at 4°C) and the dialysed (against PBS) supernatant purified via FPLC (IMAC). in S2 insect cells

Drosophila S2 cells (Invitrogen, USA) are transfected with a mixture of DNA and DDAB. Cells are diluted to 2mill cells/mL and mixed with 80 ng/mL DNA preincubated with 250 ng/mL dimethyldioctadecylammonium bromide for 20 min. The cells are then cultured in Express Five SFM medium containing 18 mmol/L L-glutamine and 1% penicillin/streptomycin at 28°C in ventilated polycarbonate Erlenmeyer flasks (Corning, Acton, MA, USA) under constant rotation (100 rpm, Bench top Orbital Shaker Incubator, Ratek Instruments, Australia). Five days later, the cell supernatant is collected by centrifugation at 15,000 g for 15 min and the dialysed (against PBS) supernatant purified via FPLC (IMAC). in HEK293 and CHO mammalian cells

HEK 293 and CHO cells are transfected using Lipofectamine™ 2000 (invitrogen). Briefly, 30 μg of each DNA were incubated with 75 μg Lipofectamine in 3.6 mL of Opti-MEM® Reduced Serum Medium, GlutaMAX™ (invitrogen). DNA is mixed and added to adherent HE and CH0 cells in a 75 cm ² flask in 18 mL of Opti-MEM® Reduced Serum Medium, GlutaMAX™ (invitrogen). After 5 hours the medium was replaced with Gibco® DMEM (invitrogen) containing 10% FCS. Cells were grown for 48h, supernatant was harvested and FPLC purified (Protein G affinity and FLAG tag purification).

Protein purification

Dialysed supernatant (S2 insect cells or HEK293 cells) and bacterial lysate was purified on an automated FPLC system (Biorad Duoflow) using a 5 mL Co-NTA column (clonetech) at a flow rate of 5 mL per min. Supernatant was applied and the column then washed with 10 column volumes of wash buffer (0.5 mol/L NaCl, 20 mmol/L imidazole in 50 mM Tris, pH 8.0) and protein eluted with elution buffer (0.5 mol/L NaCl, 250 mmol/L imidazole in 50 mM Tris, pH 8.0). Fractions with high protein concentration as measured at 280 nm were pooled and dialysed against PBS (without Ca Mg). A summary of the protein preparation is given in Table 3:

Table 3

Protein fall LPETG Size (kD) Vector Production svstem Restriction enzvmes

SE scFv 35 pSecTag2A HEK293 Notl/EcoRI

SCE5 scFv 35 pSecTag2A HE 293 Notl/EcoRI mutMA2 scFv 36 pSecTag2A HE 293 Notl/EcoRI

IL-11 25 pET20b+ BL21DE3* Ncol/Xhol

ILl-ra 21 pET20b+ BL21DE3* Ncol/Xhol

Streptavidin 17 pET20b+ BL21DE3* Ncol/Xhol eGFP 29 pET20b+ BL21DE3* Ncol/Xhol

Sortase B 22 pET20b+ BL21DE3* Ncol/Xhol

Sortase A 20 pQE30 BL21DE3* BamHI

IgG 528 Vh 53 pEE6.4 CHO/HEK293 Hindlll/Apal

IgG 528 VI 28 pEE12.4 CHO/HEK293 Hindlll/RsrII Peroxidase 38 pAC-M3 S2 cells Notl/Ncol mouse i-Domain 32 pHOG NEB turbo Ncol/Xhol The sequences of the protein constructs are:

Sortase A (SEQ ID NO: 406)

MASSHHHHHHDYDIPTTENLYFQGSQAKPQIPKDKSKVAG YIEIPDADI EPVYPGPATPEQLNRGVSFAEENESLDDQNIS IAGHTFIDRPNYQFTNLKAAK GSMVYFKVGNETRKYKMT SIRDVKPTDVGVLDEQKGKDKQLTLITCDDYNEKTGVWEK RKIFVATEVK

Sortase B (SEQ ID NO:407)

MVVRDELRDLQ LNKDMVGWLTIIDTEIDYPILQSKDNDY YLHHNYKNEKARAGSIFKDYRNTNEFLDKNTIIYGHNMKD GSMFADLRKYLDKDFLVAHPTFSYESGLTNYEVEIFAVYE TTTDFYYIETEFPETTDFEDYLQKVKQQSVYTSNV VSGK D R 11 T L S T C DTE DYEKGR MV IQGKLVT

Peroxidase (SEQ ID NO:408)

AMAMQLTPTFYDNSCPNVSNIVRDTIVNELRSDPRIAASIL RLHFHDCFVNGCDASILLDNTTSFRTEKDAFGNANSARGF PVIDRMKAAVESACPRTVSCADLLTIAAQQSVTLAGGPSW RVPLGRRDSLQAFLDLANANLPAPFFTLPQL DSFRNVGL NRSSDLVALSGGHTFGKNQCRFIMDRLYNFSNTGLPDPTL NTTYLQTLRGLCPLNGNLSALVDFDLRTPTIFDN YYVNL EEQKGLIQSDQELFSSPNATDTIPLVRSFANSTQTFFNAFVE AMDRMGNITPLTGTQGQIRLNCRVVNSNSGKPIPNPLLGL DSTLPETGGLEHHHHHHHH IL-11 (SEQ ID NO:409)

MVNCVCRLVLVVLSLWPDRVVAPGPPAGSPRVSSDPRADL DSAVLLTRSLLADTRQLAAQMRD FPADGDHSLDSLPTLA MSAGTLGSLQLPGVLTRLRVDLMSYLRHVQWLRRAGGPS L TLEPELGALQARLERLLRRLQLLMSRLALPQAAPDQPVI PLGPPASAWGSIRAAHAILGGLHLTLDWAVRGLLLLKTRL G PIPNPLLGLDSTLPETGGLEHHHHHHHH ILl-ra (SEQ ID NO:410)

MVASEAACRPSGKRPCKMQAFRIWDTNQKTFYLRNNQLIA GYLQGPNI LEE LDMVPIDLHSVFLGIHGG LCLSCAKSG DDIKLQLEEVNITDLSKNKEED RFTFIRSEKGPTTSFESAA CPGWFLCTTLEADRPVSLTNTPEEPLIVTKFYFQEDQGKPI PNPLLGLDSTLPETGGLEHHHHHHHH

Streptavidin (SEQ ID NO:411)

MVAEAGITGTWYNQLGSTFIVTAGADGALTGTYESAVGN AESRYVLTGRYDSAPATDGSGTALGWTVAWKNNYRNAHS ATTWSGQYVGGAEARINTQWLLTSGTT E A N A WKSTLVGH DTFTKVKPSAASG PIPNPLLGLDSTLPETGGLEHHHHHHH

H mouse i-Domain (SEQ ID NO:412)

M YLLPTAAAGLLLLAAQPAMAMVLRPPQQFPEALRECPQ QESDIVFLIDGSGSINNIDFQKMKEFVSTVMEQF KS TLF SLMQYSDEFRIHFTFNDFKRNPSPRSHVSPIKQLNGRTKTA SGIR VVRELFHKTNGARENAAKILVVITDGEKFGDPLDY KDVIPEADRAGVIRYVIGVGNAFNKPQSRRELDTIASKPAG EHVFQVDNFEALNTIQNQLQEKIFAIEGTQTGSTSSFEHEM SQEGFSASRGGPEQKLISEEDLNSAVDLPETGGEAAALEHH HHHH

IgG 528 light chain (SEQ ID NO:413)

MKLPVRLLVLMFWIPASSSDVLMTQTPLSLPVSLGDQASIS CRSSQN1VHNNGITYLEWYLQRPGQSPKLLIYKVSDRFSGV PDRFSGSGSGTDFTLKISRVEAEDLGIYYCFQGSHIPPTFGG GTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYP REAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTL S AD YEKHKV YACEVTHQGLSSPVTKSFNRGECLPETGGD YKDDDDK

IgG 528 heavy chain (SEQ ID NO:414)

MGWSCIILFLVATATGAHSQVQLQQSGSEMARPGASVKLP C ASGDTFTSYWMHWVKQRHGHGPEWIGNIYPGSGGTNY AEKFKNKVTLTVDRSSRTVYMHLSRLTSEDSAVYYCTRSG GPYFFDYWGQGTSLTVSSAST GPSVFPLAPSSKSTSGGTA A L G C L VKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLY SLSSVVTVPSSSLGTQTYICNVNHKPSN T K VDKRVEPKSCD KTHTCPPCPAPELL G G PSVFLFPPKPKDTLMISRTPEVTCV VVDVSHEDPEVKFNWYVDGVEVHNA TKPREEQYNSTYR VVSVLTVLHQDWLNG EYKCKVSN ALPAPIEKTISKAKG QPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEW ESNGQPENNYKTTPPVLDSDGSFFLYS LTVDKSRWQQGN VFSCSVMHEALHNHYTQKSLSLSPGKLPETGGDYKDDDDK

SE scFv (SEQ ID NO:415)

DAAQPARRAVRSLVPSSDPLQCGGILEVQLVESGGGLVQP GGSLRLSCAASGFMFSRYAMSWVRQA P G KGPEWVSGISGS GGSTYYADSVKGRFTVSRDNSKNT L Y LQMNSLRAEDTAV YYCARIFTHRSRGDVPDQTSFDYWGQGTLVTVSSGSASAP KLEEGEFSEARVSSELTQDPAVSVALGQTVRITCQGDSLRN FYASWYQQKPGQAPTLVIYGLSKRPSGIPDRFSASSSGNTA SLTITGAQAEDEADYYCLLYYGGGQQGVFGGGTKLTVLRG K P I P N PL LGLDSTLPETGGLEEAAARGGPEQKLISEEDLNS AVDHHHHHH

SCE5 scFv (SEQ ID NO:416)

DAAQPARRAVRSLVPSSDPLQCGGILQVQLQESGGGLVQP GGSLRLSC AASGFMFSRYAMS WVRQAPG GPE WVSGISGS GGSTYYADSVKGRFTVSRDNSKNTLYLQMNSLRAEDTAV YYCARGATYTSRSDVPDQTSFDYWGQGTLVTVSSGSASAP KLEEGEFSEARVSELTQDPAVSVALGQTVRITCQGDSLRNF YASWYQQ PGQAPTLVIYGLSKRPSGIPDRFSASSSGNTAS LTITGAQAEDEADYYCLLY Y.G G G Q Q G V F G G GTKLTVLRG KPIPNPLLGLDSTLPETGGLEEAAARGGPEQ K L I SE E D L N S AVDHHHHHH

Egfp (SEQ ID NO:417)

MVSKGEELFTGVVPILVELDGDVNGHKFSySGEGEGD AT Y G LTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMK QHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTL VNRIELKGIDF EDGNILGHKLEYNYNSHNVYIMADKQKN GIKVNF IRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNH YLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYKL PETGGLEHHHHHH

Example 53: Conjugation of peptides with SE scFv tagged protein and GFP-tagged protein

Sortase reaction

LPETG proteins (60 μΜ) were modified with a peptides (180 μΜ) under Sortase catalysis (180 μΜ) for 5 hours at 37°C under shaking (900 rpm) in the presence of 0.5 mmol/L CaCl ₂ in Tris reaction buffer (50 mmol/L Tris, 150 mmol/L sodium chloride, pH 8). Non- reacted LPETG protein and Sortase were removed by addition of 200 \ih Co-NTA beads (Talon, Clonetech) for 3 h under shaking at 4°C. The beads were spun down and the supernatant containing the modified protein dialysis against PBS (lOkD cut-off) overnight at 4°C to remove non-reacted peptide. Coupling efficacy was determined by protein concentration, SDS-PAGE and MALDI. Functional integrity was determined by flow cytometry.

Determination of protein concentration

A bicinchoninic acid assay (BCA) was used to estimate concentration of the purified, post- dialysed samples. A Pierce BCA protein assay kit from Thermo Scientific (Prod # 23227, Lot # MG159654) was used. Standards were pre-prepared at concentrations of 1, 0.5, 0.25, 0.125, 0.0625, 0.03125, 0.0156 and 0.0075 mg/mL in phosphate buffered saline (without Ca ²⁺ Mg ²⁺ ) from the 2 mg/mL vial of bovine serum albumin (BSA, Pierce #23209) provided in the kit. 4 mL of working reagent (WR) was prepared according to the provided instructions from reagents A and B provided in the kit. 200 uL of WR was added to each well of a Costar 96 well cell culture cluster flat bottom plate (Corning Incorporated). 25 μΐ, of each standard was added to each well. 25 of each unknown sample was added to each well. The plate was placed in a 37°C incubator for 30 minutes and read at 560 nm in a Perkin Elmer Victor microplate reader (Perkin Elmer, 1420 Multilabel Counter, Victor 3 V) and analysed using a Wallac 1420 Workstation software package.

SDS-PAGE

30 μΐ, of each sample and 6 μί, of 5X reducing SDS loading buffer were added to 1.5-mL tube and denatured at 96°C for 5 min. 36 μΐ, of each sample was run on 12 % SDS-PAGE gel in SDS running buffer at 30 mA for 2 hours. The gel was then stained with Coommassie Brilliant Blue for 1 hour and subsequently destained for at least 12 hours with Coommassie destaining solution. The gel was visualised and analysed using a BioRad Gel- Doc system with Quantity One software to confirm protein conjugation had occurred. Protein concentration determination before and after reaction was used to determine % efficacy. The results are given in Tables 4 and 5. Table 4: SE scFv (SEQ ID NO:415) conjugation with peptides

Sortase studies SE

SEQ ID NO Peptide Starting amount Recovered %

22 GGGK-ePEG -N ₃ 1 mg 0.367 37

55 GGGWW -8PEG ₄ -N ₃ 1 mg 0.396 40

77 GGGWWSSK-ePEG ₄ -N ₃ 1 mg 0.393 39

386 GGGWWGA-pG 1 mg 0.329 33

276 GGGAGAGAC 1 mg 0.427 43

277 GGGWWSSK-PEG ₄ -SANH 1 mg 0.289 29

278 GGGWWSSK-PEG4-DBCO 1 mg 0.499 50

279 GGGWWSSK-PEG4-pentenoic acid 1 mg 0.421 42

198 G (e-NH)-PEG ₄ -Azide 1 mg 0.389 39

367 GGGGGWK(£-NH)-EEE-PEG ₄ -N ₃ 1 mg 0.426 43

282 GGGWSK(e-NH)-Alkyne 1 mg 0.405 41

211 G-PEG ₄ -Alkyne 1 I mg 0.396 40

296 GGY (8-NH)-PEG ₆ -Aldehyde 1 L mg 0.313 31

295 GG Y (e-NH)-PEG 1 ₂ - Aldehyde 1 mg 0.291 29

304 GGGFDK(e-NH)- K-Aldehyde 1 1 mg 0.320 32

321 GGGWSOrn(6-NH)-PEG ₄ -SAc 1 I mg 0.339 34

378 GGGGGWDDK(e-NH)-Lipoic acid 1 I mg 0.370 37

379 GGDK(e-NH)-Thiazolidine I mg 0.393 39

362 GGGG (e-NH)-PEGg-Thiazolidine I mg 0.327 33

291 GGG-EEE-SH I mg 0.483 48

292 GGWE-PEG ₄ -hydrazine I mg 0.497 50

293 GGGWSK(e-NH)-Alkyne I mg 0.425 42

333 GGGE(5-CONH)-PEG ₄ -Thioester I mg 0.341 34

337 GGWYSOrn (8-NH)-PEG ₆ -ONH ₂ I mg 0.343 34

404 GGGG EEE-PEG -Azide I mg 0.403 40

297 GE-PEG -Azide 1 mg 0.440 44

298 GGSE-Thiazolidine 1 mg 0.488 49

349 GGGG WE(6-CONH)PEG ₄ hydrazine 1 mg 0.447 45

Table 5: GFP (SEQ ID NO:418) conjugation with peptides

Sortase studies GFP

SEQ ID NO Peptide Starting amount Recovered %

22 GGGK-ePEG ₄ -N ₃ 1 mg 0.87 87

55 GGGWWK-ePEG ₄ -N ₃ 1 mg 0.65 65

77 GGGWWSS -ePEG ₄ -N ₃ 1 mg 0.82 82 Sortase studies GFP

386 GGGWWGA-pG 1 L mg 0.79 79

276 GGGAGAGAC 1 mg 0.79 79

211 GGGWWSSK-PEG4-SANH 1 mg 0.79 79

278 GGGWWSSK-PEG ₄ -DBCO 1 mg 0.83 83

279 GGGWWSSK-PEG ₄ -pentenoic acid 1 i mg 0.83 82

198 GK(E-NH)-PEG ₄ -Azide 1 mg 0.81 80

367 GGGGGWK(e-NH)-EEE-PEG -N ₃ 1 mg 0.86 86

282 GGGWS (8-NH)-Alkyne 1 mg 0.84 84

211 G-PEG ₄ -Alkyne 1 I mg 0.77 77

296 GGY (e-NH)-PEG ₆ -Aldehyde I mg 0.51 51

304 GGGFDK(8-NH)-KKK-Aldehyde 1 I mg 0.76 76

321 GGGWSOra(8-NH)-PEG ₄ -SAc 1 L mg 0.73 73

378 GGGGGWDDK(e-NH)-Lipoic acid 1 1 mg 0.63 63

379 GGDK(s-NH)-Thiazolidine L mg 0.78 78

362 GGGGK(E-NH)-PEG ₈ -Thiazolidine I mg 0.88 87

291 GGG-EEE-SH 1 I mg 0.81 81

292 GGWE-PEG ₄ -hydrazine I mg 0.66 66

293 GGGWSK(£-NH)-Alkyne I mg 0.73 73

333 GGGE(6-CONH)-PEG ₄ -Thioester I mg 0.64 64

337 GGWYSOrn (6-NH)-PEG ₆ -ONH ₂ I mg 0.59 59

404 GGGG EEE-PEG ₄ -Azide I mg 0.69 69

297 GE-PEG ₄ -Azide I mg 0.64 64

298 GGSE-Thiazolidine I mg 0.70 70

349 GGGGWE(8-CONH)PEG ₄ -hydrazine I mg 0.71 71

Example 54: Binding of SE scFv protein conjugates with activated platelets

To confirm that the SE scFv protein was still active, its binding to activated platelets was assessed using FACS studies with anti-His-tag and anti-VS-tag antibodies.

Flow cytometry

Blood (3 mL) from healthy volunteers taking no medication was collected by venepuncture, anticoagulated with citric acid and centrifuged at 150 g for 10 min at room temperature in an Eppendorf 5810 centrifuge. The upper yellow platelet-rich plasma (PRP) layer was removed and diluted 1 :20 times in phosphate buffered saline (PBS, with Ca ²⁺ Mg ²⁺ ) and divided into two aliquots (activated and non-activated platelets). 100 μΐ, of 20 mM adenosine diphosphate (ADP) was added to a 1 mL aliquot to activate the platelets. 50 μΐ, of diluted PRP was added to each F ACS tube.

Negative controls for the experiment were asfollows- Activated platelets (0.8 μΐ. Qiagen, Penta-His, Alexa-Fluor 488 Conjugate)

Activated platelets (10 μΐ, of 1 :10 dilution, Invitrogen, anti-V5 FITC, 1.11 mg/mL, P N 46-0308)

Positive controls were as follows-

Activated platelets (0.8 μΐ, Qiagen, Penta-His, Alexa-Fluor 488 Conjugate plus 0.5 ug/mL unconjugated antibody)

Activated platelets (10 μΐ, of 1 :10 dilution, Invitrogen, anti-V5 FITC, 1.11 mg/mL, P N 46-0308 plus 0.5 μg/mL unconjugated antibody)

Activation controls were as follows-

Non-activated platelets (1 μί PAC-1 FITC, BD Biosciences, Cat. #- 340507, 0.025 mg/mL)

Activated platelets (1 μΐ. PAC-1, BD Biosciences, Cat. #- 340507, 0.025 mg/mL)

Staining was carried out as follows: Unconjugated antibody was added to the positive control tubes to achieve a concentration of 0.5 μg/mL in the tube. Conjugated antibody test samples were added at the same concentration. Samples were then incubated at room temperature for 10 minutes. After primary incubation, PAC-1 FITC was added to the non- activated platelet and activated platelet controls.

Penta-His and anti V5 FITC antibodies were added in the volumes mentioned above to the control tubes as well as to each sample tube so that each test sample was incubated separately with Penta-His and with anti-V5 FITC. Tubes were left to incubate for a further 10 minutes in the dark at room temperature. BD CellFIX was diluted 1 :10 times and 500 μΐ. was added to each FACS tube. Tubes were run on a BD FACSCalibur and analysed using FlowJo (TreeStar) flow cytometry software. The LPETG sortase tag was placed before the His tag and after the V5 tag, so during the conjugation reaction the His tag is cleaved off and the V5 is still attached. As depicted in Table 6 providing mean fluorescence values, all products are negative for the His tag but positive for the V5 tag, confirming successful conjugation to the modifying peptides.

Table 6

SEQ ID NO 1 Conjugate peptide and SE scFv Mean fluorescence

SE alone + anti His or anti V5 216.14 212.59

22 GGGK-ePEG ₄ -N ₃ 7.01 127.66

55 GGGWWK-ePEG ₄ -N ₃ 6.99 122.19

77 GGGWWSSK-ePEG ₄ -N ₃ 7.70 43.94

386 GGGWWGA-pG 8.34 76.32

276 GGGAGAGAC 8.68 210.87

277 GGGWWSSK-PEG ₄ -SANH 6.97 132.15

278 GGGWWSSK-PEG ₄ -DBCO 32.57 157.22

279 GGGWWSSK-PEG ₄ -pentenoic acid 12.54 192.50

198 GK(e-NH)-PEG ₄ -Azide 1 1.06 202.30

367 GGGGGWK(6-NH)-EEE-PEG ₄ -N ₃ 9.86 104.72

282 GGGWS (E-NH)-Alkyne 9.30 208.51

211 G-PEG ₄ -Alkyne 8.35 190.85

296 GGY (e-NH)-PEG ₆ -Aldehyde 8.55 147.72

295 GGYK(E-NH) PEG 12- Aldehyde 8.01 200.51

304 GGGFDK(e-NH)-KKK-Aldehyde 7.51 60.64

321 GGGWSOrn(5-NH)-PEG ₄ -SAc 7.81 216.66

378 GGGGGWDDK(e-NH)-Lipoic acid 7.93 174.38

379 GGDK(e-NH)-Thiazolidine 8.62 103.20

362 GGGG (£-NH)-PEG ₈ -Thiazolidine 6.98 64.64

291 GGG-EEE-SH 7.25 221.26

292 GGWE-PEG ₄ -hydrazine 7.4 121.43

293 GGGWS (e-NH)-Alkyne 6.23 169.90

333 GGGE(5-CONH)-PEG ₄ -Thioester 5.98 225.07

337 GGWYSOrn (5-NH)-PEG ₆ -ONH ₂ 6.68 199.57

404 GGGG EEE-PEG -Azide 6.44 108.42

297 GE-PEG ₄ -Azide 6.28 184.47

298 GGSE-Thiazolidine 6.82 107.82

349 GGGGWE(5-CONH)PEG ₄ -hydrazine 6.60 60.42 Example 55: MALDI studies of protein peptide conjugates

Method protein analysis (CHCA) Linear mode peptide: Samples are mixed 1 :1 with Matrix, 10 mg/mL a-cyano-4-hydroxycinnamic acid (Laser BioLabs, Sophia- Antipolis, France) in 50% Acetonitrile 0.1% TF A) and spotted onto the MALDI target plate. Proteins are analysed in Linear mode with a mass range of lOkDa to 50kDa and a focus mass of 35kDa and 29kDa. Laser shots are fired randomly across the sample well and the summed spectrum consists of spectra collected at the rate of 2500 shots/spectrum. The spectrum was internally calibrated against co-spotted Enolase using 2 point calibrate masses, 46672 and 23336 Da Mass Spectrometry Instrument: MALDI TOF/TOF, model 4700 Proteomics Analyser from Applied Biosystems (Foster City, CA, USA) running Software 4000 Series Explorer version 3.0. The results are shown in Tables 7A and 7B.

Table 7A

SEQ ID NO structure Calc: Calc: measured GFP Measured Measured

GFP+Peptide GFP Hydrolysis vs GFP-Pep vs GFP-Hyd

22 GGGK-ePEG ₄ -N ₃ 589.65 27971.63 27381.98 27887.99 -0.00299914 0.01814437

55 GGGWWK-6PEG ₄ -N ₃ 962.08 28344.06 27381.98 28274.45 -0.00246194 0.03156454

77 GGGWWSS -EPEG ₄ -N ₃ 1 136.31 28518.29 27381.98 28444.46 -0.00259558 0.037352792

386 GGGWWGA-pG 784.84 28166.82 27381.98 28057.89 -0.00388233 0.024089837

276 GGGAGAGAC 619.68 28001.66 27381.98

277 GGGWWSS -PEG ₄ -SANI I 1288 28669.98 27381.98

278 GGGWWSS -PEG ₄ -DBCO 1483.61 28865.59 27381.98

27 GGGWWSSK-PEG ₄ -pentenoic acid 1 193.25 28575.23 27381.98

198 GK(s-NH)-PEG ₄ -Azide 476.54 27858.52 27381.98 27830.75 -0.00099782 0.01612497

367 GGGGGWK(e-NH)-EEE-PEG ₄ -N ₃ 1278.32 28660.3 27381.98 28654.13 -0.00021533 0.044396741

282 GGGWSK(e-NH)-Alkyne (amide) 699.64 28081.62 27381.98 28023.56 -0.00207183 0.022894308

211 G-PEG ₄ -Alkyne 288 27669.98 27381.98 27750.83 0.002913426 0.013291494

296 GGY (£-NH)-PEG ₆ -Aldehyde ; 891.05 28273.03 27381.98 27370.7 -0.032967 -0.00041212

295 GGY (e-NH)-PEG,2-Aldehyde 1155.36 28537.34 27381.98 27363.09 -0.04291365 -0.00069035

295 GGYK(s-NH)-PEG,2-Aldehyde 1155.36 28537.34 27381.98 28421.42 -0.00407861 0.036572416

304 GGGFDK(e-NH)-KKK-Aldehyde 1096.27 28478.25 27381.98 28276.99 -0.00711745 0.03165153

321 GGGWSOrn(5-NH)-PEG -SAc 882.61 28264.59 27381.98 28136.49 -0.00455281 0.026816067

378 GGGGGWDD (8-NH)-Lipoic acid 1036.18 28418.16 27381.98 28278.6 _! -0.00493518 0.031706662

379 GGDK(E-NH)-Thiazolidine 490.69 27872.67 27381.98

362 GGGGK(e-NH -PEG ₈ -Thiazolidine ^" 913.15 28295.13 27381.98 28203.73 -0.00324071 0.029136217

291 GGG-EEE-SH 636 28017.98 27381.98 28566.23 0.019192242 0.041456293

292 GGWE-PEG ₄ -hydrazine 708.09 28090.07 27381.98

293 GGGWS (e-NH)-Alkyne 700.64 28082.62 27381.98

333 GGGE(8-CONH)-PEG ₄ -Thioester 652.99 28034.97 27381.98

337 GGWYSOrn (8-NH)-PEG ₆ -ONH ₂ 1090.17 28472.15 27381.98

404 GGGG EEE-PEG ₄ -Azide 833.13 28215.11 28268.33 0.001882672 0.031354877

297 GE-PEG ₄ -Azide 404.44 27786.42 27836.08 0.001784016 0.01631336

297 GE-PEG ₄ -Azide 404.44 27786.42 28171.05 0.013653378 0,028009961

298 GGSE-Thiazolidine 448 27829.98 28416.13 0.020627369 0.036393063

349 GGGGWE(5-CONH)PEG -hydrazide 822.56 28204.54 28262 0.002033119 0.031 137924

GFP alone 28561.2 28561.2 28561.2 28558.26 -0.00010295 -0.00010295

Table 7B

NO structure MW Calc: Calc: measured SE Measured Measured

SE+Peptide SE Hydrolysis vs SE-Pep vs SE-Hyd

22 GGGK-ePEG ₄ -N ₃ 589.65 32251.93 31662.28 ,

55 GGGWWK- _C PEG ₄ -Nj 962.08 32624.36 31662.28

77 GGGWWSSK-ePEG ₄ -N ₃ 1 136.31 32798.59 31662.28

386 GGGWWGA-pG . 784.84 32447.12 31662.28

276 GGGAGAGAC 619.68 32281.96 31662.28

277 GGGWWSSK-PEG ₄ -SANH 1288 32950.28 31662.28

278 GGG W WS SK-PEG4-DBCO 1483.61 33145.89 31662.28

279 GGGWWSSK-PEG ₄ -pentenoic acid 1 193.25 32855.53 31662.28

198 G (e-NH)-PEG ₄ -Azide 476.54 32138.82 31662.28

367 GGGGGWK(E-NH)-EEE-PEG4-N ₃ 1278.32 32940.6 31662.28

282 GGGWS (e-NH) Alkyne (amide) 699.64 32361.92 31662.28

211 G-PEG ₄ -Alkyne 288 31950.28 31662.28

296 GGYK(e-NH)-PEG ₆ - Aldehyde 891.05 32553.33 ^' < 31662.28

295 GGYK(e-NH)-PEG _I2 -Aldehyde 1 155.36 32817.64 31662.28

295 GGYK(e-NH)-PEG,2-Aidehyde 1 155.36 32817.64 31662.28

304 GGGFDK(e-NH)- K -Aldehyde 1096.27 32758.55 31662.28

32Ϊ GGGWSOrn(6-NH)-PEG ₄ -SAc 882.61 32544.89 31662.28

378 GGGGGWDD (E-NH)-Lipoic acid 1036.18 32698.46 31662.28

379 GGDK(E-NH)-Thiazolidine 490.69 32152.97 i 31662.28

362 GGGGK(E-NH)-PEGg-Thiazol idine 913.15 32575.43 31662.28

291 GGG-EEE-SH 636 32298.28 31662.28

292 GGWE-PEG ₄ -hydrazine 708.09 32370.37 31662.28 32090.52 -0.00872064 0.013344751

293 GGGWS (e-NH)-Alkyne 700.64 32362.92 31662.28 321 15.17 -0.00771442 ; _: 0.014102058

333 GGGE(5-CONH)-PEG ₄ -Thioester 652.99 32315.27 31662.28 32164.84 -0.00467685 0.015624514

337 GGWYSOm (8-NH)-PEG ₆ -ONH ₂ 1090.17 32752.45 31662.28 31798.54 -0.02999855 0.004285102

404 GGGG EEE-PEG ₄ -Azide 833.13 32495.41 31662.28

297 GE-PEG ₄ -Azide 404.44 32066772 31662.28

297 GE-PEG ₄ -Azide 404.44 32066.72 31662.28

298 GGSE-Thiazolidine 448 32110.28 31662.28

349 GGGGWE(5-CONH)PEG ₄ -hydrazide 822.56 32484.84 31662.28

SE alone 35223.04 35223.04 35223.04 34202.68 1 -0.02983275 -0.02983275

SE alone 35223.04 35223.04 35223.04 35579 0.010004778 0.010004778

The Sortase mediated modification of a protein with an LEPTG motif has at least two competing mechanisms, being cleavage at LEPT_G followed by amide bond formation to the N-terminal of the Glycine peptide, or cleavage at LEPT_G followed by hydrolysis The ALDI data presented in Tables 7A and 7B shows the observed molecular weight for proteins modified by Sortase as described in Example 53. The columns A and B represent a calculated ratio: (Observed MW - Calculated MW) ÷ Observed MW for each of the two possible Sortase pathways. In most cases, the Observed MW is more consistent with integration of the modifier peptide into the GFP or SE protein. Example 56: Conjugation of two proteins with complementary functional groups Conjugation reaction

Hydrazine/Aldehvde

Proteins modified with complementing reactive groups (Hydrazine/Aldehyde) were mixed in a molar ratio of 1 : 1, 1 :3 or 3: 1 and reacted in reaction buffer (100 mM citrate, 150mM NaCl, pH6.0) in a final volume of 300 μί, for 12h at 4°C. Successful conjugation was confirmed by SDS gel and FACS (high molecular weight product and binding to activated platelets)

Cu free click (DBCO to N

Proteins modified with complementing reactive groups (DBCO/Azide) were mixed in a molar ratio of 1 :1 and reacted in reaction buffer (PBS without Ca/Mg) for 45 min at RT. Successful conjugation was confirmed by SDS gel and FACS (high molecular weight product and binding to activated platelets)

Cu click (Alkvne to NV)

300 μΐ,, of 1 mg/ml antibody solution (modified with Alkyne containing peptide) was mixed with a 1 :2 molar excess of antibody solution (modified with N ₃ containing peptide) in the presence of CAC (copper/ascorbate/chelate mixture) for 40 min at 4°C. CAC = 10iL each of 1.8 mg/mL Cu in MilliQ, 4.4 mg/mL ascorbate in PBS and 4.89 mg/mL chelate in DMSO. Conjugation was confirmed by SDS PAGE.

NCL (Native Chemical Ligation)

For NCL (Native Chemical Ligation) between Thiazolidine to Thioester, the Thiazolidine is first deprotected with 250 mM Methoxyamine HCl in 50mM Acetate buffer (pH 4) for 2h at 37°C and dialysed against PBS. The product was then reacted in 100 raM phosphate buffer pH 7.0-7.5 in the presence of 2% (v/v) thiophenol and 2% (v/v) benzyl mercaptan overnight at 4°C. Successful conjugation was confirmed by SDS gel and FACS (high molecular weight product and binding to activated platelets)

Conjugation to NIR dve

300 μί of 1 mg/mL modified antibody solution (pG peptide) was mixed with a 1 :3 molar excess of NIR-dye (lumiprobe) in the presence of CAC (copper/ascorbate/chelate mixture) for 40 min at 4°C. Excess dye was removed by 3 wash steps using a 30 spincolumn. CAC = 10 μΐ each of 1.8 mg/mL Cu in MilliQ, 4.4 mg/ml ascorbate in PBS and 4.89 mg/mL chelate in DMSO. Labeling was confirmed by SDS PAGE and imaging on an Odyssey NIR system (Licor Biosystems).

Example 57: Conjugation of SE Scv with GFP

Reaction of two protein conjugates of the invention was undertaken. The protein conjugates were prepared in Example 53.

SE scFv modified with GGGWWSSK-PEG ₄ -SANH (SEQ ID NO:277) was reacted with

GFP modified with GGYK(e-NH)PEG, ₂ - Aldehyde (SEQ ID NO:295)

and

SE scFv modified with GGY (E-NH)PEGI ₂ - Aldehyde (SEQ ID NO:295) was reacted with GFP modified with GGGWWSSK-PEG ₄ -SANH (SEQ ID NO:277)

in molar ratios of 1 :1 and 1 :3. The products were assessed by SDS PAGE and showed successful conjugation with a product of 61 kD. Binding of the SE-GFP conjugates to activated platelets was confirmed using FACS. Results are shown in Table 8.

Table 8: Mean fluorescence in binding to activated platelets

Reaction Ratio GFP mean fluorescence (FL1-H)

SE277 + GFP295 1 :1 6.92

SE277 + GFP295 1 :3 13.73

SE295 + GFP277 1 :1 8.41 SE295 + GFP277 1 :3 18.4

SE + GFP295 1 :3 7.51

SE-GFP conjugates reacted in a ratio of 1 :3 have a higher mean fluorescence compared to the control. Example 58: Conjugation of SE scFv with GFP using click reaction

Reaction of two protein conjugates of the invention was undertaken using click chemistry. The protein conjugates were prepared in Example 53.

SE scFv modified with GK(e-NH)PEG ₄ -(CH ₂ ) ₂ N ₃ (SEQ ID NO: 198) was reacted with GFP modified with GGGWSK(e-NH)CHC≡CH (SEQ ID NO:282)

and

SE scFv modified with GGGWSK(e-NH)CHOCH (SEQ ID NO:282) was reacted with GFP modified with GK(e-NH)PEG ₄ (CH ₂ ) ₂ N ₃ (SEQ ID NO: 198). The reaction was carried out as described in Example 56 with the components presented in a 1 :2 ratio. SDS PAGE showed successful conjugation with a band at 61 kD. Binding of the SE-GFP conjugates to activated platelets was confirmed by FACS. The results are shown in Table 9. Table 9: Mean fluorescence in binding to activated platelets

Reaction Ratio GFP mean fluorescence (FL1-H)

SE198 + GFP282 1 :2 19.58

GFP198 + SE282 1 :2 20.15

SE + GFP282 1 :2 9.64

Example 59: Conjugation of SEscFv with GFP using native chemical ligation

The conjugation of two proteins using native chemical ligation was undertaken. The peptides used were prepared in Example 53.

SE scFv modified with GGGE(6-CONH)-PEG ₄ -thioester (SEQ ID N0.333) and GFP modified with GGDK(e-NH)-thiazolidine (SEQ ID N0.379) and

SE scFv modified with GGDK (e-NH)-thiazolidine (SEQ ID NO:379) and GFP modified with GGGE(5-CONH)PEG ₄ -thioester (SEQ ID NO:333)

and

SE scFv modified with GGGGK(6-NH)-PEG ₈ -thiazolidine (SEQ ID NO:362) and GFP modified with GGGE(5-CONH)-PEG ₄ -thioester (SEQ ID NO:333).

The following reactions were carried out in a 1 :3 or 3:1 ratio:

SE379 : SFP333 3:1

SE379 : SFP333 1 :3

GFP379 : SE333 3:1

GFP379 : SE333 1 :3

SE362 : GFP333 3:1

SE362 : GFP333 1 :3

In all cases, the conjugation product (61 kD) was observed by SDS PAGE indicating successful conjugation.

Example 60: Conjugation of protein and dendrimer

Two dendrimers were prepared as follows:

Dendrimer A: Azidobenzamide-NEOEOENrSU(NPN^irLvslin| iH9.TFA½

Azidobenzamide-NEOEOEN[Su(NPN) ₂ ] [Lys] ₁₆ [BOC] ₃₂

[NH ₂ ]EOEOEN[Su(NPN) ₂ ][Lys]i ₆ [BOC] ₃₂ (prepared as described in WO 2008/017125 Example l(xiii)) was reacted with azidobenzoic acid using standard PyBOP coupling conditions.

Azidobenzamide-NEOEOEN[Su(NPN) ₂ ] [Lys]i ₆ [NH ₂ TFA] ₃₂

A solution of azidobenzamide-NEOEOEN[Su(NPN) ₂ ] [Lys] _!6 [BOC] ₃₂ (190 mg, 25.1 μηιοΐ) in DCM/TFA (2.5 mL / 2.5 mL) was stirred for 3 h then added to an ice-cooled, stirred solution of diethyl ether (15 mL).The off-white precipitate was collected by filtration and washed with diethyl ether, then dissolved in water and freeze dried. DendrimerB: Azidobcnzamid-PEG _l? -NEOEOENrSU(NPN^irLvsl _lft fPEGs7n1^

[NH ₂ ]EOEOEN[Su(NPN) ₂ ][Lys]i ₆ [PEG57o]32 (as described in WO 2008/017125 Example l(xvii)) was reacted with azidobenzamide-PEG| ₂ -C0 ₂ H employing standard PyBOP coupling conditions.

SE scFv modified with GGGWSK(£-NH)-alkyne amide (SEQ ID NO:282) or G-PEG ₄ - Alkyne (SEQ ID NO:211) as described in Example 53. The SE-alkynes were conjugated to Dendrimers A and B using a copper catalyzed click reaction with a molar ratio of SE protein to dendrimer of 1:2. Successful conjugation was confirmed by SDS PAGE - SE-Dendrimer A 41 kD, SE-Dendrimer B 56 kD.

Confirmation that activity of the SE scFv protein was preserved was obtained by assessing binding to activated platelets as described above. Activity was maintained for the conjugates as shown in Table 10.

Table 10: SE scFv dendrimer conjugates mean fluorescence

Product Mean fluorescence (anti V5 antibody)

SE282/Dendrimer A 8.78

SE282/Dendrimer B 5.3

SE211 /Dendrimer A 7.07

SE211 /Dendrimer B 6.31

SE + Dendrimer control 7.78

Example 61: Effect of position of LPETG tag on efficacy of reaction with GGG peptides Sortase A mediated conjugation of GGG peptides and LPETG proteins in which the sortase recognition tag is near the C-terminus: or within the protein was compared.

Three proteins were used, GFP in which the LPETG tag is located 13-9 amino acids from the C-terminus

EKRDHMVLLEFVTAAGITLGMDELYKLPETGGLEHHHHH

(SEQIDNO:418).

Mut MA2 protein in which the LPETG tag is located 37-33 amino acid residues from the C-terminus: LPETGGLEEAAARGGPEQKLISEEDLNSAVDHHHHHH

(SEQ ID NO:419)

and

SCE5/SE protein in which the LPETG tag is located 37-33 amino acid residues from the C-terminus:

LPETGGLEEAAARGGPEQKLISEEDLNSAVDHHHHHH

(SEQ ID NO:420)

The conjugation efficacy was assessed as described in Example 53 and is shown in Table 11.

Table 11: Conjugation efficacy

SEQ ID NO: LPETG tag Starting amount Recovered %

277 GFP 1 mg 0.79 79

279 GFP 1 mg 0.83 82

295 GFP 1 mg 0.63 62

362 GFP 1 mg 0.88 87

277 SE 1 mg 0.289 29

279 SE 1 mg 0.421 42

295 SE 1 mg 0.291 29

365 SE 1 mg 0.327 33

277 mutMA2 1 mg 0.390 . 39

279 mut MA2 1 mg 0.455 46

295 mut MA2 1 mg 0.273 27

365 mut MA2 1 mg 0.470 47

Example 62: Modification of other proteins

Several proteins with LPETG tags were modified with GGYK(£-NH)-PEGi ₂ - Aldehyde (SEQ ID NO:295) or GGGWWSSK-PEG ₄ -SANH (SEQ ID NO:277). The proteins modified were IgG (SEQ ID NO:413), peroxidase (SEQ ID NO:408), streptavidine (SEQ ID NO:411), IL-11(SEQ ID NO:409, IL-lra (SEQ ID NO:410), miD (SEQ ID NO: 412), GFP (SEQ ID NO:417) and SE scFv (SEQ ID NO:415). The efficacy of sortase reaction was determined by assessing protein by BC A. The results are shown in Table 12: Table 12

Protein SEQ ID NO: Amount in reaction % coupling

IL-11 SEQ ID NO:295 170 17

IL-lra SEQ ID NO:295 210 1 1

Strept SEQ ID NO:295 150 15

miD SEQ ID NO:295 300 30

peroxidase SEQ ID NO:295 700 70

IgG SEQ ID NO:295 561 28

GFP SEQ ID NO:277 1 mg 79

SE scFv SEQ ID NO:277 1 mg 29

Example 63: Conjugation with Sortase B

Sortase B studies

Sortase B model substrate MAPAALWVALVFELQLWATGHTGANPQTN was modified with GGYK(e-NH)-PEG, ₂ -Aldehyde (SEQ ID NO:295) under Sortase B catalysis conditions and conjugated to GFP modified with GGGWWSSK-PEG ₄ -SANH (SEQ ID NO:277). Successful conjugation (addition of 4kD to GFP) was demonstrated by SDS PAGE.

Example 64: Synthesis of GWOrn(dNH)-PEG ₄ CH ₂ C≡CH

GWOrn(5NH ₂ ) was synthesised as described in Example 24 using Fmoc-Om(Dde)-Wang Resin, Fmoc-Trp(Boc)-OH and Boc-Gly-OH followed by deprotection of the 5-amino group. GWOrn(5NH ₂ ) in DMF (10 mL) was treated with Alkyne-PEG ₄ -C0 ₂ H (183 mg, 0.6 mmol), HATU (216 mg, 0.570 mmol) and NMM (136 μί, 0.6 mmol) under N ₂ for 120 min. The resin was then washed with DMF (3x10 mL). Kaiser test confirmed the reaction was complete. The resin was washed with MeOH (1x10 mL), DCM (3x10 mL) and MeOH (2x10 mL) and then air-dried (800 mg). The peptide was then removed from the resin and deprotected to give the crude peptide (89 mg) as described in Example 24. The peptide was purified by HPLC using a Galaksil UP-C18 lOum 120A column and a solvent gradient of 30-40 Solvent A/30min as described in Example 24 and analysed by mass spectrometry as described in Example 25. Purity by HPLC: 97.6%. MS: C ₃₂ H48N ₅ Oio ⁺ Calc: 662.34, Found: 661.88.