Synthesis of a-amanitin and its derivatives

The present invention relates to the chemical synthesis of a-amanitin and its derivatives. The present invention also relates to intermediate products of the a-amanitin synthesis.

Description

The objective of the present invention is to provide means and methods to chemically synthesize amanitin or derivatives thereof. This objective is attained by the subject-matter of the independent claims of the present specification.

Terms and definitions

Amino acid sequences are given from amino to carboxyl terminus. Capital letters for sequence positions refer to L-amino acids in the one-letter code (Stryer, Biochemistry, 3 ^rd ed. p. 21). Lower case letters for amino acid sequence positions refer to the corresponding D- or (2R)-amino acids.

The term“protecting group” in the context of the present specification relates to a moiety covalently attached to a functional group (particularly the carboxylic acid moiety, the amino moiety or the hydroxyl moiety of the molecules discussed herein) that can be selectively attached to the functional group and selectively removed without affecting the integrity or chiral orientation of the carbon backbone of the molecule the protecting group is attached to, nor cleaving particular other protecting groups attached to other protecting groups attached to the molecule.

The term“deprotection agent” in the context of the present specification relates to an agent which is able to cleave a certain protecting group. The skilled person is able to select the deprotection agent according to the protecting group. The conditions under which the protecting group is cleavable constitute the deprotection agent, e.g. if the protecting group is cleavable under acidic conditions, then the deprotection agent is an acid.

The term“preactivated carboxylic group” in the context of the present specification relates to a carboxylic moiety being reacted into an active ester susceptible for the nucleophilic attack of an amine group in order to form a peptide bond.

The term“preactivated amino group” in the context of the present specification relates to an amino group being reacted into a N-trimethylsilyl amine with increased nucleophilicity to attack a carboxylic acid moiety in order to form a peptide bond.

A comprehensive review of modern protecting group chemistry, particularly as it pertains to the compounds disclosed herein, is available in Peter G. M. Wuts, Greene's Protective

Groups in Organic Synthesis, 5th Edition, Wiley 2014. US 6693178 B2 - "Protecting groups useful in the synthesis of polysaccharides, natural products, and combinatorial libraries" and US 20160024143 A1 - "Deprotection method" are incorporated herein by reference.

Standard convention of organic chemistry, by which a non-designated position in a formula is deemed to be a saturated carbon, is followed herein.

A first aspect of the invention relates to a method for preparation of a compound of formula

(I ox)

(I ox)

wherein

a) a compound of formula (llox)

wherein

X and Y are H, or Y is OH and X is OR ^PGP wherein R ^PGP is a protecting group for phenolic OH groups, particularly a phenolic OH-protecting group not acid- or alkali-labile, more particularly cleavable under reductive conditions, most particularly benzyl (Bn) or

X and Y are selected from F, Cl, Br, and I,

particularly X and Y are H or Y is OH and X is OR ^PGP

Z and W are H, or

Z is OH and W is OR ^PGOH , wherein R ^PGOH is a protecting group for hydroxyl-groups, particularly a hydroxyl-protecting group cleavable with fluoride ions, more particularly TBS, TMS, TES, TBDPS, TIPS, or disiloxane, most particularly TBS, is reacted with a peptide bond forming reagent,

particularly with a coupling reagent selected from a carbodiimide, an imidazolinium reagent, a phosphonium salt, an organo-phosphorous reagent, an uranium salt, a pyridinium reagent, and a phosphonic acid,

more particularly with HATU [1-[bis(dimethylamino)methylene]-1 H-1 ,2,3-triazolo[4,5- b]pyridinium 3-oxide hexafluorophosphate], COMU, HBTU, TBTU, TOMBU, COMBU, or HCTU,

in a reaction step (a1),

and for Y being OH and/or Z being OH, the compound is reacted with a deprotection agent removing R ^PGP and/or R ^PGOH ,

or wherein

b) the compound of formula (II)

wherein

X, Y, Z and W have the same meanings as defined above, is reacted with a peptide bond forming reagent, particularly with HATU

in a reaction step (a2),

yielding a compound of formula (I)

wherein the sulfur atom is subsequently oxidized,

i. using manganese ions, more particularly the compound is reacted with a compound of formula (XXII)

and with Mn(OTf)2 and H2O2,

ii. using PPO, dibenzyolperoxide, tert-butyl peroxybenzoate, or lauroyl peroxide; or

iii. using iodine and oxygen;

in a reaction step (b2),

and for Y being OH and/or Z being OH, the compound is reacted with a deprotection agent removing R ^PGP and/or R ^PGOH , particularly for R ^PGP with reductive conditions and for R ^PGOH with fluoride ions,

to yield the compound characterized by (I ox). For cyclisation, the amide-NFh of compound (II) or (llox) does not need to be protected. No significant side reactions were observed without protecting group.

In certain embodiments, the oxidation of the sulfur atom is performed using manganese ions. In certain embodiments, the chemoselective oxidation of the sulfur atom is performed using a compound of formula (XXII)

with Mn(0Tf)2 and H2O2.

In certain embodiments, the chemoselective oxidation of the sulfur atom is performed using PPO (Phthaloyl peroxide), dibenzyolperoxide, tert-butyl peroxybenzoate, or lauroyl peroxide. Preparation of PPO is described in (S. Gan, J. Yin, Y. Yao, Y. Liu, D. Chang, D. Zhu, L. Shi, Org. Biomol. Chem. 2017, 15, 2647-2654.).

In certain embodiments, the oxidation of the sulfur atom is performed with mCPBA (meta- chloroperoxybenzoic acid) in isopropanol/ethanol (8:3).

In certain embodiments, the oxidation of the sulfur atom is performed with an oxaziridinium salt as described in (Rio et al, Org. Lett. 2007, 9,12, 2265-2268).

In certain embodiments, the oxidation of the sulfur atom is performed with non-enantio- selective agents or simply with oxygen or hydrogenperoxide.

In certain embodiments, the oxidation of the sulfur atom is performed with iodine and oxygen. A second aspect relates to a method for preparation of a compound of formula (I)

wherein a compound of formula (II)

wherein

X and Y are H, or

Y is OH and X is OR ^PGP wherein R ^PGP is a protecting group for phenolic OH groups, particularly a phenolic OH-protecting group not acid- or alkali-labile, more particularly cleavable under reductive conditions, most particularly benzyl or

X and Y are selected from F, Cl, Br, and I,

particularly X and Y are H, or Y is OH and X is OR ^PGP ,

Z and W are H, or

Z is OH and W is OR ^PGOH , wherein R ^PGOH is a protecting group for hydroxyl-groups, particularly a hydroxyl-protecting group cleavable with fluoride ions, more particularly TBS is reacted with a coupling reagent selected from a carbodiimide, an imidazolinium reagent, a phosphonium salt, an organo-phosphorous reagent, an uronium salt, a pyridinium reagent, and a phosphonic acid,

particularly with a peptide bond forming reagent, more particularly with HATU, COMU, HBTU, TBTU, TOMBU, COMBU, or HCTU in a reaction step (a),

and for Y being OH and/or Z being OH, the compound is reacted with a deprotection agent removing R ^PGP and/or R ^PGOH in a reaction step (b)

to yield the compound characterized by (I).

In certain embodiments, a compound of formula (III)

and a compound of formula (IV) or (IVox)

wherein

R ^NHB is an amino protecting group, particularly an amino protecting group cleavable under alkaline conditions, more particularly Fmoc, or an amino protecting group cleavable with hydrogenolysis, particularly Cbz, most particularly R ^NHB is Fmoc

W and X have the same meaning as outlined above,

wherein

the amino-group of (IV) or (IVox) is preactivated, particularly with MSA, and preactivated (IV) or preactivated (IVox) and (III) are reacted with a peptide bond forming reagent, particularly with HATU, or

the amino-group of (IV) or (IVox) is preactivated, particularly with MSA, and the carboxyl- group of compound (III) is preactivated, particularly with an O-PFP-ester, O-PCP-ester, or OSu-ester, and preactivated (IV) or preactivated (IVox) and preactivated (III) are reacted in a reaction step (c), and the compound is reacted with a deprotection agent removing R ^NHB in a reaction step (d), particularly with a base if R ^NHB is Fmoc, or with hydrogenolysis if R ^NHB is Cbz, more particularly with Et2lMH, tris-2-amino-ethylamin, DBU, morpholine, or piperidine if R ^NHB is Fmoc,

to yield the compound characterized by (II) or (I I ox).

For coupling compounds (II) and (IV) or (I Vox), the acid-COOH group of compound (IV) or (IVox) does not need to be protected. No significant side reactions were observed without protecting group.

In certain embodiments, a compound of formula (IV)

wherein

Rcoox is a carboxyl-protecting group, particularly /Butyl,

R ^NHX is an amino-protecting group, particularly Teoc,

X has the same meaning as outlined above,

wherein the sulfur atom is subsequently oxidized, particularly

i. using manganese ions, more particularly the compound is reacted with a compound of formula (XXII)

and with Mn(OTf)2 and H2O2,

ii. using PPO, dibenzyolperoxide, tert-butyl peroxybenzoate, or lauroyl peroxide; or iii. using iodine and oxygen;

and with Mn(OTf)2 and H2O2 in a reaction step (d2),

and the compound is reacted with a deprotection agent removing R ^coox and R ^NHX , particularly with a strong acid, more particularly at a pH of -3 to 2, most particularly with 80- 95% TFA,

to yield the compound characterized by (IVox).

In certain embodiments, the oxidation of the sulfur atom is performed using manganese ions. In certain embodiments, the chemoselective oxidation of the sulfur atom is performed using a compound of formula (XXII)

with Mn(0Tf)2 and H2O2.

In certain embodiments, the chemoselective oxidation of the sulfur atom is performed using PPO (Phthaloyl peroxide), dibenzyolperoxide, tert-butyl peroxybenzoate, or lauroyl peroxide. Preparation of PPO is described in (S. Gan, J. Yin, Y. Yao, Y. Liu, D. Chang, D. Zhu, L. Shi, Org. Biomol. Chem. 2017, 15, 2647-2654.).

In certain embodiments, the oxidation of the sulfur atom is performed with mCPBA (meta- chloroperoxybenzoic acid) in isopropanol/ethanol (8:3).

In certain embodiments, the oxidation of the sulfur atom is performed with an oxaziridinium salt as described in (Rio et al, Org. Lett. 2007, 9,12, 2265-2268).

In certain embodiments, the oxidation of the sulfur atom is performed with non-enantio- selective agents or simply with oxygen or hydrogenperoxide.

In certain embodiments, the oxidation of the sulfur atom is performed with iodine and oxygen. In certain embodiments, a compound of formula (V)

wherein

R ^NHF is an amino protecting group, particularly an amino protecting group cleavable with fluoride ions or strong acids, more particularly Teoc,

R¥OA j _{S a} carboxyl-protecting group, particularly a carboxyl-protecting group cleavable under strongly acidic conditions, more particularly tert- butyl,

X has the same meaning as outlined above,

is reacted with a coupling reagent selected from a carbodiimide, an imidazolinium reagent, a phosphonium salt, an organo-phosphorous reagent, an uronium salt, a pyridinium reagent, and a phosphonic acid,

particularly a peptide bond forming reagent, more particularly with T3P, HATU, COMU,

HBTU, TBTU, TOMBU, COMBU, or HCTU, in a reaction step (e),

and the compound is reacted with a deprotection agent removing R ^NHF and R ^COOA in a reaction step (f), particularly with TFA,

to yield the compound characterized by (IV).

In certain embodiments, a compound of formula (VI)

and a compound of formula (VII)

wherein

R ^NHA is an amino protecting group, particularly an amino protecting group cleavable under acidic conditions, more particularly Boc,

RCOOA RNHF _anc| x _ave the same meaning as outlined above,

wherein compound (VI) is

preactivated with a peptide bond forming reagent, particularly with HATU, COMU, HBTU, TBTU, TOMBU, COMBU, or HCTU, followed by a reaction with the silylated compound (VII), or

is preactivated as in OSu-ester, followed by a reaction with the compound (VII)

in a reaction step (g),

and the compound is reacted with a deprotection agent removing R ^NHA in a reaction step (h), particularly with acidic conditions, more particularly at a pH of -3 to 0, even more particularly with HCI or p-toluenesulfonic acid, most particularly with 2 M HCI in Dioxan,

to yield the compound characterized by (V).

In certain embodiments, a compound of formula (VIII)

and a compound of formula (IX)

wherein

R ^COOZ j _{S a} carboxyl- protecting group, particularly a carboxyl-protecting group cleavable with Zn, more particularly Tee, or R ^cooz is H, R ^COOA , R ^N HF R ^N HA _anc| x (-, _{ave t(} -, _{e same} meaning as outlined above, are reacted in a reaction step (i), and if R ^cooz is a carboxyl-protecting group, the compound is reacted with a deprotection agent removing R ^cooz in a reaction step 0, particularly with Zn, to yield the compound characterized by (VI).

A protection group strategy was applied that relies on acid stability. Decreasing pH values were used for deprotection. First, the Tee group of tryptophan (R ^cooz of compound VIII) was removed under reductive conditions using Zn with mildly acidic pH. Afterwards, the Boc group of cysteine (R ^NHA of compound IX) was removed with p-toluenesulfonic acid. Last,

Teoc (R ^NHF ) and tert-butyl (R ^COOA ) of compound (V) were removed concomitantly with 95%TFA.

In certain embodiments, a compound of formula (X)

a la (XI)

and a compound of formula (XII)

wherein

R ^Pep is an active ester, particularly O-pentafluorophenol or OSu-ester,

R ^NHB is an amino protecting group, particularly an amino protecting group cleavable under alkaline conditions, more particularly Fmoc,

are reacted with solid phase peptide synthesis in a reaction step (k), wherein the carboxyl- group of compound (XII) may be protected,

to yield the compound characterized by (III). A third aspect relates to a method for preparation of a compound of formula (XIII), (XIIIC), (XI I IN), or (XIIICN)

(XIIICN)

wherein a compound of formula (XIV)

wherein

R ^coos is a carboxyl-protecting group, particularly a carboxyl-protecting group cleavable with silylating agents, more particularly tert- butyl,

R ^NHZ is an amino protecting group, particularly an amino protecting group cleavable under alkaline conditions, more particularly Fmoc, or an amino protecting group cleavable under reductive conditions, more particularly trifluoroacetyl;

is reacted with Osmium(IV)-oxide in a reaction step (I), particularly in CHCI ₃ /H ₂ O, and optionally, the compound is reacted with a deprotection agent removing R ^NHR and/or R ^coos in a reaction step (m), particularly with silylating agents for R ^coos and reductive conditions or alkaline conditions for R ^NHR , more particularly with TMSOTf and Lutidine for R ^coos and/or sodium borohydride for R ^NHZ [if R ^NHZ is trifluoroacetyl] or alkaline conditions for R ^NHZ [if R ^NHZ is Fmoc],

to yield the compound characterized by (XIII), (XI 11C), (XIIIN) , or (XIIICN).

When the compound of formula (XIII) is used in the synthesis of a-amanitin or its derivatives, either the deprotection of the amino-protecting group or of the carboxyl- protecting group is omitted, as shown in formula (XI 11 C) or (XIIIN). In addition, the OH-groups of compound (XIII) are protected before proceeding with the synthesis.

The oxidation with Osmium(IV)-oxide is particularly stereoselective (2.5: 1) in CHCI3/H2O. An Upjohn-dihydroxylation protocol is employed. Only the solvent influences the stereoselectivity here, as in e.g. fBuOH/hhO mainly the opposite diastereomer of compound (XIII) is produced.

A fourth aspect relates to a method for preparation of a compound of formula (XV)

wherein a compound of formula (XVI)

and a compound of formula (XVII) or (XVI Is)

wherein

R ^NHR an amino protecting group cleavable under reductive conditions, more particularly trifluoroacetyl,

R ^COOA is a carboxyl-protecting group, particularly a carboxyl-protecting group cleavable under strongly acidic conditions, more particularly tert- butyl,

are reacted with [(p-cymene)RuCl2]2 in a reaction step (n) yielding the compound (XXIII) or

(XXIV) the compound (XXVI) is reacted with a deprotection agent removing R ^COOA in a reaction step (o), and is reacted with acylase in a reaction step (p), or

the compound (XXIII) is reacted with a deprotection agent removing R ^COOA in a reaction step (o), and is reacted with a deprotection agent removing R ^NHR in a reaction step (q), particularly with reductive conditions, more particularly with sodium borohydride,

to yield the compound characterized by (XV).

The reaction with acylase in reaction step (p) removes the protecting group R ^NHR .

In certain embodiments, the compound of formula (XXIII) is directly employed in the synthesis of the compound of formula (XIII), (XIIIC), or (XI 11 N) without a deprotection step in between.

The chiral compound (XV) may be gained directly from the reaction with [(p-cymene)RuCl2]2. The reaction is particularly stereoselective when compound (XVI Is) is employed. Then, compound (XXIII) is gained and no acylase step is necessary. Stereoselectivity is improved compared to methods known from literature (e.g. A. Bayer, U. Kazmaier, Org. Lett. 2010, 12, 21 , 4960-4963).

Otherwise, when using compound (XVII), compound (XXIV) in an 1 :1 mixture of the (R,R) and the (S,S) diastereomers is gained. From this mixture, the correct diastereomer

[compound (XV)] is gained with the use of acylase.

A fifth aspect relates to a method for preparation of a compound of formula (XVIII)

wherein a compound of formula (XIX)

and a compound of formula (XX)

wherein

R ^PGP is a protecting group for phenolic OH groups, particularly a phenolic OH-protecting group not acid- or alkali-labile, more particularly cleavable under reductive conditions, most particularly benzyl,

are reacted with Ni ²⁺ in a reaction step (r)

to yield the compound characterized by (XVIII).

A sixth aspect relates to a method for preparation of a compound of formula (lox), wherein a compound of formula (I)

wherein the sulfur atom is oxidized,

i. using manganese ions, more particularly the compound is reacted with a

compound of formula (XXII)

and with Mn(OTf)2 and H2O2,

ii. using PPO, dibenzyolperoxide, tert-butyl peroxybenzoate, or lauroyl peroxide; or

iii. using iodine and oxygen;

yielding the compound (lox).

In certain embodiments, the oxidation of the sulfur atom is performed using manganese ions. In certain embodiments, the chemoselective oxidation of the sulfur atom is performed using a compound of formula (XXII)

with Mn(OTf)2 and H2O2.

In certain embodiments, the chemoselective oxidation of the sulfur atom is performed using PPO (Phthaloyl peroxide), dibenzyolperoxide, tert-butyl peroxybenzoate, or lauroyl peroxide. Preparation of PPO is described in (S. Gan, J. Yin, Y. Yao, Y. Liu, D. Chang, D. Zhu, L. Shi, Org. Biomol. Chem. 2017, 15, 2647-2654.).

In certain embodiments, the oxidation of the sulfur atom is performed with mCPBA (meta- chloroperoxybenzoic acid) in isopropanol/ethanol (8:3).

In certain embodiments, the oxidation of the sulfur atom is performed with an oxaziridinium salt as described in (Rio et al, Org. Lett. 2007, 9,12, 2265-2268).

In certain embodiments, the oxidation of the sulfur atom is performed with non-enantio- selective agents or simply with oxygen or hydrogenperoxide.

In certain embodiments, the oxidation of the sulfur atom is performed with iodine and oxygen.

In certain embodiments, the method according to the third aspect is applied for the method of the first aspect. Compound (X) can be obtained from compound (XIII).

In certain embodiments, the method according to the fourth aspect is applied for the method of the third aspect. Compound (XIV) can be obtained from compound (XV).

In certain embodiments, the method according to the fifth aspect is applied for the method of the first aspect. Compound (VIII) can be obtained from compound (XVIII).

A seventh aspect of the invention relates to a method for preparation of a compound of formula (XXIII) or (XXIIlox)

and a compound of formula (X)

w have the same meanings as defined above,

wherein the amino-group of (IV) or (IVox) is preactivated, particularly with MSA, and preactivated (IV) or preactivated (IVox) and (X) are reacted with a peptide bond forming reagent, particularly with HATU, COMU, HBTU, TBTU, TOMBU, COMBU, or HCTU, more particularly with COMU, in a reaction step (s) to yield the compound (XXIII) or (XXIIlox), respectively.

An eighth aspect of the invention relates to a method for preparation of a compound of formula (XXVI) or (XXVIox)

wherein a compound of formula (XXVIII) or (XXVIllox), respectively,

(XXVIII)

( XXVI 11 ox)

and a compound of formula (XXV)

wherein

X, W, and R ^NHB have the same meanings as defined above,

R ^NHB2 is an amino-protecting group, particularly an amino-protecting group cleavable under acidic conditions, more particularly Boc;

R ^COOY is a carboxyl-protecting group, particularly fluorenylmethyl or benzyl, more particularly fluorenylmethyl;

wherein (IV) or (I Vox) and (XXV) are reacted with a peptide bond forming reagent, particularly with HATU, COMU, HBTU, TBTU, TOMBU, COMBU, or HCTU, in a reaction step (t) to yield the compound (XXVI) or (XXVI ox), respectively.

A ninth aspect of the invention relates to a method for preparation of a compound of formula (XXVII) or (XXVI lox)

(XXVI I ox) wherein

a compound of formula (IV) or (IVox),

and a compound of formula (X)

wherein X, W, and R ^NHB have the same meanings as defined above,

wherein the amino-group of (IV) or (IVox) is preactivated, particularly with MSA, and preactivated (IV) or preactivated (IVox) and (X) are reacted with a peptide bond forming reagent, particularly with COMU, in a reaction step (s) to yield the compound (XXIII) or (XXI 11 ox), respectively,

and subsequently compound (XXIII) or (XXIIlox) and compound (XXV) are reacted with a peptide bond forming reagent, particularly with HATU, COMU, HBTU, TBTU, TOMBU, COMBU, or HCTU

in a reaction step (u) to yield the compound (XXVII) or (XXVI lox), respectively;

or

compound (XXIII) or (XXIIlox) and compound (XXV) are reacted with a peptide bond forming reagent, particularly with HATU, COMU, HBTU, TBTU, TOMBU, COMBU, or HCTU in a reaction step (u) to yield the compound (XXVII) or (XXVI lox), respectively;

or

a compound of formula (XXVIII) or (XXVIllox), respectively,

(XXVIllox)

and a compound of formula (XXIX)

wherein

X, W, and R ^NHB have the same meanings as defined above,

R ^NHB2 is an amino-protecting group, particularly an amino-protecting group cleavable under acidic conditions, more particularly Boc;

wherein R ^COOY is a carboxyl-protecting group, particularly fluorenylmethyl or benzyl, more particularly fluorenylmethyl;

wherein (XXVIII) or (XXVIllox) and (XXV) are reacted with a peptide bond forming reagent, particularly with HATU, COMU, HBTU, TBTU, TOMBU, COMBU, or HCTU,

in a reaction step (t) to yield the compound (XXVI) or (XXVI ox), respectively

and subsequently compound (XXVI) or (XXVIox) and compound (X) are reacted with a peptide bond forming reagent, particularly with HATU, in a reaction step (v) to yield the compound (XXVII) or (XXVI lox), respectively

or

compound (XXVI) or (XXVIox) and compound (X) are reacted with a peptide bond forming reagent, particularly with HATU, in a reaction step (v) to yield the compound (XXVII) or (XXVI I ox), respectively.

A tenth aspect of the invention relates to a method for preparation of a compound of formula (I) or (lox), wherein a compound of formula (XXVII) or (XXVIlox) prepared according to the ninth aspect

(XXVI I ox)

is reacted with a coupling reagent selected from a carbodiimide, an imidazolinium reagent, a phosphonium salt, an organo-phosphorous reagent, an uronium salt, a pyridinium reagent, and a phosphonic acid,

particularly a peptide bond forming reagent, more particularly with T3P, HATU, COMU, HBTU, TBTU, TOMBU, COMBU, or HCTU,

to yield compound (I) or (lox).

A further aspect relates to a compound of the general formula (I)

wherein

Y is H and Z is H,

Y is H and Z is OH,

Y is OH and Z is H

Y is F, Cl, I or Br and Z is OH, or

Y is F, Cl, I or Br and Z is H;

particularly Y and Z are independently selected from OH and H. A further aspect relates to a compound of the general formula (II)

wherein

X is H and W is H,

X is OH and W is OH,

X is H and W is OH,

X is OH and W is H,

X is F, Cl, I or Br, and W is OH, or X is F, Cl, I or Br, and W is H;

particularly X and W are independently selected from OH and H.

A further aspect relates to a compound of the general formula (llox)

wherein

X is H and W is H,

X is OH and W is OH,

X is H and W is OH,

X is OH and W is H,

X is F, Cl, I or Br, and W is OH, or

X is F, Cl, I or Br, and W is H;

particularly X and W are independently selected from OH and H.

A further aspect relates to a compound of the general formula (IVox)

wherein

X is H or OH, or

X is F, Cl, I or Br

particularly X is selected from OH and H. A further aspect relates to a compound of the general formula (XXVII I)

wherein

X is H and W is H,

X is OH and W is OH,

X is H and W is OH,

X is OH and W is H,

X is F, Cl, I or Br, and W is OH, or

X is F, Cl, I or Br, and W is H;

particularly X and W are independently selected from OH and H.

A further aspect relates to a compound of the general formula (XXVIl lox)

(XXI 11 ox)

wherein

X is H and W is H,

X is OH and W is OH,

X is H and W is OH,

X is OH and W is H,

X is F, Cl, I or Br, and W is OH, or X is F, Cl, I or Br, and W is H;

particularly X and W are independently selected from OH and H.

A further aspect relates to a compound of the general formula (XXVI)

wherein

- X is H and W is H,

- X is H and W is OH,

- X is OH and W is H,

X is F, Cl, I or Br, and W is OH, or

- X is F, Cl, I or Br, and W is H,

particularly X is H and W is H, or X is H and W is OH, or X is OH and W is H

A further aspect relates to a compound of the general formula (XXVIox)

I ox)

wherein

X is H and W is H,

X is OH and W is OH,

X is H and W is OH, - X is OH and W is H,

- X is F, Cl, I or Br, and W is OH, or

- X is F, Cl, I or Br, and W is H,

particularly X and W are independently selected from OH and H.

The invention is further illustrated by the following examples and figures, from which further embodiments and advantages can be drawn. These examples are meant to illustrate the invention but not to limit its scope.

Brief description of the figures

Fig. 1 Synthesis of the Fmoc-protected (2S,3R,4R)-4,5-dihydroxyisoleucine derivatives via regioselective Ru-catalyzed allylic alkylation after Kazmaier et al. (Kazmaier et ai, Chem.

Eur. J. 2004, 20 ,10484-10491) and Sharpless dihydroxylation.

Fig. 2 Chiral GC MS chromatogram of the allylic alkylation product 5, derivatized for GC-MS by methylation of the C-terminus.

Fig. 3 Synthesis of the (S)-configured allylic carbonate (S)-4 suitable for the following asymmetric allylic alkylation reaction (Sharpless et al., J. Am. Chem. Soc. 1987, 109, 5765- 5780., E. Balmer et al., J. Chem. Soc., Perkin Trans.1 1993, 399-400.).

Fig. 4 Synthesis of Fmoc-protected (2S,3R,4R)-4,5-dihydroxyisoleucine derivatives via asymmetric regioselective Ru-catalyzed allylic alkylation (A. Bayer, U. Kazmaier, Org. Lett. 2010, 12, 21 , 4960-4963) and Upjohn-dihydroxylation.

Fig. 5 Synthesis of the Fmoc-protected (2S,3R,4R)-4,5-dihydroxyisoleucine derivative 13 by Tfa-deprotection using NaBH4 (shortcut 1).

Fig. 6 Synthesis of the Fmoc-protected (2S,3R,4R)-4,5-dihydroxyisoleucine derivative 13 by direct Sharpless dihydroxylation of fully protected didehydroisoleucine 5 after allylic alkylation

(shortcut 2).

Fig. 7 Chiral GC MS chromatogram of the asymmetric allylic alkylation product, derivatized for GC MS by methylation of the C-terminus.

Fig. 8 a) Synthesis of tridentate ligand (S)-28[1 ,2] b) Synthesis of (S)-6-hydroxytryptphan derivative 33 by dynamic kinetic resolution with a chiral tridentate ligand (Zhou et al., Angew. Chem. Int. Ed Engl. 2014, 53, 7883-7886; Nian et al., Angew. Chem. Int. Ed Engl. 2015, 54, 12918-12922).

Fig. 9 Synthesis of the tryptathionine building blocks.

Fig. 10 Synthesis of H-Gly-lle-Gly-OH (45)

Fig. 11 Synthesis of Fmoc-Asn-Hyp-DHIL(TBS) ₂ -OH (48). Fig. 12 Assembly of the peptide building blocks affording a-amanitin (61) and amaninamide (62).

Fig. 13a Dipeptide synthesis of FhN-Asn-Hyp-OFm.

Fig. 13b Alternative route for a-amanitin (61) and amanin amide (62).

Examples

Example 1: Strategy for the synthesis of (2S,3R.4R)-4.5-dihvdroxyisoleucine derivatives 9 step synthesis via Ruthenium-catalyzed allylic alkylation

The (2S,3R,4R)-4,5-dihydroxyisoleucine derivative 13 was synthesized in 9 steps (Fig. 1) using glycine tert- butyl ester as starting material, which was /V-terminally protected quantitatively in the first step by trifluoroacetylation of the amino group. The fully protected glycine derivative 8 was then submitted to regioselective Ruthenium-catalyzed asymmetric allylic alkylation after Kazmaier et al. (Kazmaier et ai, Chem. Eur. J. 2004, 20 ,10484-10491) The alkylating reagent was a terminal alkene (4) bearing tert- butyl carbonate as leaving group, easily accessible by Boc-protection of the racemic allylic alcohol 3 using B0C2O and NaH. The allylic alkylation reaction led to the mainly anti-directed formation of a fully protected didehydroisoleucine derivative 5 with a diastereomeric ratio (dr) of 90:10, calculated by submission of 5 to chiral GC MS after fBu-deprotection of the carboxylic moiety and methylation using TMSCHN2 (Fig. 2).

Separation of the desired L-configured enantiomers from the remaining D-configured enantiomers was performed by enzymatic kinetic resolution using the enzyme Acylase I from Aspergillus melleus. Previous deprotection of the f-butyl protecting group was inevitable in order for the enzymatic reaction to take place. The resulting didehydroisoleucine 7 was then Fmoc- protected at the /V-terminus and refurnished with the fBu-protecting group at the C- terminus prior to the asymmetric dihydroxylation of the terminal double bond. While Fmoc was the protecting group of choice in order to submit the final derivative to SPPS, the tBu protecting group proved to be essential in order to avoid the formation of a highly stable lactone during the dihydroxylation reaction and the subsequent separation of diastereomers using column chromatography on silica gel. The asymmetric dihydroxylation of the fully protected didehydroisoleucine derivative 9 was performed in a biphasic system of water and CHCI3, which led to the formation mainly of the 2S,3R,4R-configured dihydroxyisoleucine (10). Separation of all four diastereomers was easily achieved by a purification step on silica gel at this stage. Finally, the hydroxy groups of the side chain were protected prior to deprotection of the C-terminus. The protecting group of choice was the TBS-protecting group, allowing the mild tBu cleavage in quantitative yield using an excess of TMSOTf in the final step affording the final Fmoc-protected (2S,3R,4R)-4,5-dihydroxyisoleucine derivative. The overall yield after 9 steps was calculated to be ~7%.

Asymmetric synthesis strategies via regioselective Ruthenium-catalyzed allylic alkylation and possible shortcuts

In order to achieve a higher overall yield of the synthesis route described in the previous section a chiral alkylating reagent during the allylic alkylation reaction was used (Fig. 3). Employing a chiral transfer during the allylic alkylation, the desired (2S,3S)-configured didehydroisoleucine is preferably formed. The chiral allylic alcohol but-3-en-2-ol (S)-3 was synthesized according to published literature procedures via Sharpless epoxidation of (£)- crotyl alcohol 18 followed by in situ tosylation of the hydroxy group affording epoxide 19. The chiral carbonate (S)-4 was then formed by reductive elimination using Nal and a Zinc- Copper-couple after Balmer et al. followed by Boc-protection of the hydroxyl group.

The overall synthesis strategy for the enantiomerically pure Fmoc-protected (2S,3R,4R)-4,5- dihydroxyisoleucine derivative (Fig. 4) followed the same route as described in the previous section. The only difference was the use of the chiral allylic carbonate (S)-4 during the Ruthenium-catalyzed allylic alkylation which resulted in the formation of the fully protected (2S,3S)-didehydroisoleucine 5 with an enantiomeric excess of 98% due to a chiral transfer of the alkylating reagent (Fig. 7).

The diastereomeric ratio (dr) was calculated to be 86: 14 towards the (2R,3S)-diastereomer and 99: 1 towards the (2S,3R)-configured diastereomer. The former was separated conveniently by the following acylase reaction which led to the formation of a enantiomerically pure didehydroisoleucine 7 with a dr of 99: 1. Because of the enantiomeric purity the asymmetric dihydroxylation also resulted in a higher yield as there were only two diastereomers that needed separation afterwards instead of four. The overall yield starting from glycine tert- butyl ester (1) after 9 steps was calculated to be 17-21 %.

The high enantiomeric excess of 5 also made it possible to take two shortcuts resulting in a higher yield (Fig. 5 and 6).

One shortcut was the direct Tfa-deprotection of didehydroisoleucine 5 followed by Fmoc- protection. This way, the tBu-cleavage and reattachment was omitted resulting in the formation of the dihydroxylation substrate within two steps instead of four. The enantiomeric excess of 13 following shortcut 1 was calculated by chiral HPLC and resulted to be 95%. The overall yield was calculated to be 24-29%.

A second shortcut was the direct Sharpless dihydroxylation of the allylic alkylation product 5 followed by the protection of the side chain with the TBS protecting groups. The diastereomers were separated by column chromatography on silica gel after cleavage of the Tfa protecting group using LiOH and Fmoc-protection of the C-terminus (11). The enantiomeric excess of 13 following shortcut 2 was calculated by chiral HPLC and resulted to be 70%. Both shortcuts enable the synthesis of Fmoc-4,5-dihydroxyisoleucine in 7 steps instead of 9 and provided higher overall yields.

Example 2: Strategy for the synthesis of (S)-6-hydroxytryptophan derivatives

The (S)-6-hydroxytryptophan derivative 33 was synthesized in four steps, starting with an alkylation of the commercially available 6-benzoxyindol (29) using L-serine and acetic anhydride in acetic acid, which leads to the racemic A/-acetyl-6-benzoxytryptophan (30) in moderate yields (Blaser, et ai, Tetrahedron Lett.2008, 2795-2798). After deacetylation with 40% NaOH in MeOH/dioxane the racemic 6-benzoxytryptophan (31) was obtained, which was submitted to a dynamic kinetic resolution following a protocol from Zhou et ai. and Nian et al. (Zhou et ai, Angew. Chem. Int. Ed Engl. 2014, 53, 7883-7886; Nian et ai, Angew. Chem. Int. Ed Engl. 2015, 54, 12918-12922). Therefore, the tridentate ligand (S)-28 was synthesized in two steps according to the literature (Zhou et ai., Angew. Chem. Int. Ed Engl. 2014, 53, 7883- 7886; Nian et al., Angew. Chem. Int. Ed Engl. 2015, 54, 12918-12922). The racemic 6- benzoxytryptophan (31) was treated with the ligand (S)-28, K2CC>3 and Ni(NC> ₃ ) _2* 6H ₂ 0 as a nickel source, which gave the Ni(ll)-complex 32. The diastereomeric ratio of >99% was determined by chiral HPLC with a CHIRALPAK AD-H column (hexane/isopropanol = 55/45, l = 280 nm, 0.8 mL/min). After separation of the two diastereoisomers by silica gel column chromatography, the absolute stereochemistry was undoubtedly determined by single-crystal X-ray diffraction. Disassembly of the complex 32 under acidic conditions resulted in the target enantiomerically pure (S)-6-hydroxytryptophan derivative 33 (Fig. 8).

3-(6-Benzoxy- 1H-indol-3-yl)-2-acetylaminopropionic acid (30):

¹ H NMR (400 MHz, DMSO-de): d (ppm) = 1.79 (s, 3 H) 2.92 (dd, J=14.68, 8.66 Hz, 1 H) 3.09 (dd, J= 14.68, 4.89 Hz, 1 H) 4.42 (td, J= 8.22, 5.14 Hz, 1 H) 5.09 (s, 2 H) 6.72 (d, J=6.27 Hz, 1 H) 6.89 (d, J=2.01 Hz, 1 H) 6.98 (d, J=2.01 Hz, 1 H) 7.27 - 7.33 (m, 1 H) 7.35 - 7.41 (m, 3 H) 7.42 - 7.48 (m, 2 H) 8.1 1 (d, J= 7.78 Hz, 1 H) 10.64 (s, 1 H).

¹³ C NMR (100 MHz, DMSO-de): d (ppm) = 173.59, 169.20, 154.45, 137.68, 136.67, 128.39, 127.62, 127.48, 122.27, 121.85, 1 18.78, 1 10.00, 109.21 , 95.95, 69.49, 52.98, 27.22, 22.42.

HRMS (ESI): m/z calc fur C20H20N2O4 (M+H) ⁺ 353.1496, found 353.1487.

(S)-3-amino-3-(6-(benzoxy)-1H-indol-3-yl)propanoic acid - Schiff Base Complex (32):

1 H NMR (500 MHz, CDCb): d (ppm) = 1.27 - 1.39 (m, 1 H) 1.61 - 1.74 (m, 2 H) 1.76 - 1.88 (m,

1 H) 2.00 - 2.13 (m, 1 H) 2.71 - 3.05 (m, 4 H) 3.23 (dd, J=14.65, 3.97 Hz, 1 H) 4.01 (d, J=12.51

Hz, 1 H) 4.20 (t, J=4.65 Hz, 1 H) 5.00 (s, 2 H) 6.55 (d, J=2.44 Hz, 1 H) 6.62 - 6.74 (m, 2 H)

6.79 (s, 1 H) 6.88 (d, J=1.83 Hz, 1 H) 7.03 (dd, J=9.31 , 2.44 Hz, 1 H) 7.12 (d, J= 8.70 Hz, 1 H) 7.16 - 7.33 (m, 7 H) 7.34 - 7.57 (m, 5 H) 8.09 (d, J=9.31 Hz, 3 H) 8.38 (br. s., 1 H) 8.80 (d, J=1.68 Hz, 1 H).

13C NMR (100 MHz, CDCI3): d (ppm) =179.92, 156.24, 141.28, 137.77, 137.47, 135.19, 134.01 , 133.58, 133.51 , 132.64, 131.17, 130.41 , 130.04, 129.64, 129.32, 128.84, 128.13, 127.73, 125.83, 124.16, 123.53, 123.36, 120.68, 110.92, 110.18, 96.86, 71.78, 70.98, 63.47, 58.66, 30.95, 22.83.

HRMS (ESI): m/z calc^sHssChl UNiCU (M+H) ⁺ 835.1150, found 835.1159.

The dr was determined by chiral HPLC with a CHIRALPAK AD-H column

(hexane/isopropanol = 55/45, l = 280 nm, 0.8 mL/min). t _R (major diastereomer) = 13.29 min, >99:1 dr, t _R (minor diastereomer) = 17.28 min.

(S)-6-benzoxytryptophan (33):

HRMS (ESI): m/z calc. C ₁₈ H ₁₈ N ₂ 0 ₃ (M+H) ⁺ 311.1390, found 311.1391.

Example 3: Synthesis of a-amanitin and amaninamide Synthesis of the peptide building blocks

The thioether building units 40 and 41 were readily established by treatment of a fully protected L-cystine derivative (35) with sulfuryl chloride. Cleavage of the disulfide afforded the highly reactive sulfenyl chloride monomer 36, which in the following step is susceptible for an electrophilic aromatic substitution (SsAr) either solely /V-terminally protected or fully protected 6-hydroxytryptophan and tryptophan derivative 38 and 39. The use of the TCE-protecting group at the C-terminus helped to suppress the formation of undesired side-products with residual sulfuryl chloride from the sulfenyl chloride formation, but was not imperative for the reaction to take place (Fig. 9).

The tripeptide building block H-Gly-lle-Gly-OH (45) was synthesized in solution phase by first synthesizing a /V-terminally Cbz- and C-terminally Bn-protected tripeptide, followed by simultaneous Cbz- and Bn-deprotection by hydrogenolysis using H ₂ and Pd/C as catalyst. (Fig. 10).

The Fmoc-Asn-Hyp-DHIL(TBS) ₂ -OH tripeptide 48 was synthesized on solid phase using the CTC-resin. The use of Fmoc-Asn-OPfp (47) during the coupling of asparagine with no protecting group at the side chain suppressed the formation of the dehydration product (a tripeptide containing Fmoc^-cyanoalanine) to the extent of only 10% (Fig. 11).

A C-terminally 9-Fluorenylmethyl ester-protected dipeptide building block 66 was synthesized by esterification of frans-/V-(Boc)-4-hydroxy-L-proline (63) with 9-fluorenylmethanol affording fully protected 4-hydroxy-L-proline 64. Boc-deprotection under acidic conditions, followed by coupling with Boc-Asn-OH using EDC and HOBt and repeated Boc-deprotection under acidic conditions led to the formation of dipeptide building block 66 with no formation of the dehydration side product present.

Assembly of the peptide building blocks towards (S)-Deoxy-(0)-Benzyl a-amanitin and (S)- Deoxy amaninamide.

First, monocyclic thioethers 55 and 56 were synthesized in order to obtain the bicyclic structures of (S)-Deoxy (O)-benzyl-a-amanitin and (S)-Deoxy amaninamide (Fig. 12). In order to do so, the thioether building blocks 51 and 52 were deprotected using Zn and AcOH in DMF, transformed into an active ester using L/,L/ ^' -disuccinimidyl carbonate, followed by coupling of the C- and /V-terminally deprotected tripeptide building block 45. After deprotection with p-toluoenesulfonic acid or 2 M HCI, the peptides were cyclized with T3P and DIPEA in DMF/DCM within 3 h. Afterwards, monocyclic pentapeptides 53 and 54 were deprotected using 80% TFA in DCM and coupled to tripeptide 48 using a protocol activating not only the carboxylic function of the tripeptide by an active ester forming agent, but also the amino group of monocyclic pentapeptides by a silylating agent. Octapeptides 57 and 58 were then /V-terminally Fmoc-deprotected and cyclized using HATU in DMF. The TBS protecting groups were cleaved from the DHIL residue by treatment of the peptides with 1 M TBAF for 2h.

A second pathway leading to the formation of (S)-Deoxy-(0)-benzyl-a-amanitin and (S)- Deoxy amaninamide was the direct coupling of DHIL-derivative 13 to the fully deprotected monocyclic pentapeptides 55 and 56 by using a silylating agent for the /V-terminus of the pentapeptides and an active ester forming agent for the carboxylic function of the DHIIe derivative, analogous to the method described above. Monocyclic hexapeptides 67 and 68 could then be coupled to the C-terminally Fm-protected dipeptide building block 66 leading to the formation monocyclic octapeptides 69 and 70. The final cyclization was then performed after simultaneous Fmoc and Fm-cleavage and subsequent TBS-deprotection from the DHIL residue.

Sulfide oxidation affording a-amanitin and amaninamide

The asymmetric oxidation of the tryptathionine moiety leading to (O)-Benzyl-a-amanitin and amaninamide (62) was achieved by using a manganese complex as a catalyst with a porphyrine inspired chiral ligand, following a protocol reported by Gao et al (D. Wen, L. Jun, S. Gao, Org. Lett. 2013, 15, 22, 5658-61). Hydrogenolysis with H2 and Pd/C of (O)-Benzyl-a- amanitin in THF afforded the natural product a-amanitin (61) after 30 min of reaction time. Materials and methods tert-Butyl (2,2,2-trifluoroacetyl)glycinate (2):

To a solution of glycine tert- butyl ester hydrochloride (10.0 g, 137 mmol, 1.00 eq) in MeOH (150 ml) triethylamine (17.4 ml, 125 mmol, 2.10 eq) was added dropwise. After stirring for 5 min ethyl trifluoroacetate (16.4 ml, 137 mmol, 2.3 eq) was added and the mixture was stirred for 16 h at room temperature during which time a clear solution formed. Then, the reaction mixture was concentrated under reduced pressure and the resulting residue acidified with 2 N HCI before being extracted with EtOAc (3x100 ml). The organic layers were combined, then washed with sat. NaHCC>3 (2x100 ml), distilled H2O (2x100 ml) and brine (2x100 ml) and dried over MgSCU. The solvent was removed in vacuo to give the product (2) as a yellow oil (13.5 g, quant.).

¹ H NMR (CDC -d ¹ , 400 MHz): d = 1.50 (s, 9 H), 4.02 (d, J= 5.02 Hz, 2 H), 6.89 ppm (br s, 1 H).

¹³ C NMR (CDCb-d ¹ , 100 MHz): d = 27.91 , 41.94, 83.52, 115.60 (q, J= 287.28 Hz), 157.06 (q, J=39.60 Hz), 167.28 ppm.

HRMS (ESI): m/z calculated: C ₈ H ₁₂ F ₃ N0 ₃ (M-H)- 226.0686, found 226.0693.

But-3-en-2-yl tert- butyl carbonate (4):

4

But-3-en-2-ol (1.50 g, 20.8mmol, 1.00 eq) was added slowly to a solution of NaH (1.50 g, 62.4 mmol, 3.00 eq) in dry THF (40 ml) at 0°C. Then B0C2O (5.9 g, 27 mmol, 1 3eq) was added in portions over 10 min under vigorous stirring at this temperature. The reaction mixture was allowed to warm to room temperature overnight and then diluted with Et ₂ 0 after 16h of vigorous stirring. Excess sodium hydride was quenched by the slow addition of water. The resulting mixture was then extracted with diethyl ether (3x50 ml). The combined organic extracts were washed with brine (1x50 ml), dried over MgSCU and concentrated under reduced pressure. After purification by column chromatography on silica gel (hexane/EtOAc, 10: 1) the product (4) was obtained as a colourless liquid (3.6 g, 20.3 mmol, quant.).

¹ H NMR (CDCb-d ¹ , 400 MHz): d = 1.36 (d, J = 6.53 Hz, 3 H), 1.49 (s, 9 H), 5.13 - 5.17 (m, 2 H), 5.25 - 5.31 (m, 1 H), 5.83 - 5.91 ppm (m, 1 H).

¹³ C NMR (CDCb-d ¹ , 100 MHz): d = 19.72, 27.48, 73.72, 81.62, 1 15.78, 137.78, 137.17, 152.53 ppm.

HRMS (ESI): m/z calculated: C ₉ H ₁₆ 0 ₃ (M+H) ⁺ 173.1172, found 173.1 171. tert-Butyl 3-methyl-2-(2,2,2-trifluoroacetamido)pent-4-enoate (Trifluoroacetyl 4,5- didehydroisoleucine tert-butyl ester) (5):

LHMDS (1 M in THF, 11 mmol, 2.5eq) was added slowly to a solution of trifluoroacetyl glycine te/f-butylester (2, 1.0 g, 4.4 mmol, 1.5 eq) in dry THF (12 ml) at -78°C. After stirring for 10 min a solution of dried ZnCb (720 g, 5.30 mmol, 1.20eq) in dry THF (6 ml) was added at this temperature and stirring was continured for another 30 min at -78°C. Meanwhile a solution was prepared from [(p-cymene)RuCl ₂ ] ₂ (37 mg, 0.06mmol, 0.02eq) and triphenylphosphine (32 mg, 0.12mmol, 0.04eq) in dry THF (6ml) and stirred for 10 min at room temperature. Then, the allylic carbonate (4) (517 mg, 3.00 mmol, 1.00eq) was added and the resulting solution was added to the chelated enolate at -78°C. The reaction mixture was allowed to warm to room temperature overnight. After diluting with EtC> ₂ (100 ml) the reaction mixture was hydrolyzed by addition of 1 M KHSCU until the precipitate was fully dissolved in the organic layer. Then, the layers were separated, the aqueous layer was extracted with diethyl ether (3x50 ml) and the combined organic layers were washed with brine (100ml) and dried over MgSCU. The solvent was removed in vacuo and the crude product was purified by column chromatography on silica gel (hexane/EtOAc, 10: 1), which afforded the product (5) as a colourless oil (740 mg, 88%).

¹ H NMR (CDCb-d ¹ , 400 MHz): 6 = 1.10 (d, J= 7.03 Hz, 3 H), 1.49 (s, 9 H), 2.82 - 2.89 (m, 1 H), 4.48 - 4.54 (m, 1 H), 5.07 - 5.20 (m,2 H), 5.70 (s, 1 H), 6.70 ppm (d, J= 7.53 Hz, 1 H).

¹³ C NMR (CDCb-d ¹ , 100 MHz): d = 15.75, 27.97, 40.21 , 56.71 , 83.35, 1 17.15 (q, J= 273.2 Hz), 117.46, 136.78, 157.03 (q, J=39.60 Hz), 168.89 ppm. 3-methyl-2-(2,2,2-trifluoroacetamido)pent-4-enoic acid (Trifluoroacetyl 4,5- didehydroisoleucine) (6):

Fully protected didehydroisoleucine (5) (1.0 g, 3.6 mmol, 1.0eq) wasdissolved in a solution of 95% TFA in DCM. After stirring for 2 h at roomtemperature the solvent was evaporated in vacuo to afford the trifluoroacetylated didehydroisoleucine (6) (800 mg, quant.) as a white solid.

¹ H-NMR (400 MHz, CDCIs): d (ppm) = 6.63 (br, 1 H), 5.66 - 5.77 (m, 1 H) 5.19 - 5.27 (m, 2 H), 4.66 (q, J=4.2 Hz, 1 H), 2.90 - 2.99 (m, 1 H), 1.15 (d, J=7.04 Hz, 3 H)

¹³ C-NMR (100 MHz, CDCIs): d (ppm) = 175.37, 157.67, 136.37, 1 18.37, 117.00, 56.43, 39.56, 16.32

HRMS (ESI): m/z calc for CsHgFsNOs (M-H) 224.0529, found 224.0536. (2S,3S)-2-amino-3-methylpent-4-enoic acid (4,5-Didehydroisoleucine) (7)

To a solution of the racemic trifluoroacetylated didehydroisoleucine 6 (700 mg, 3.10 mmol, 1.00 eq) in Sorensen phosphate buffer (15 ml, pH 7.5) 4 M KOH (777 pi, 3.10 mmol, 1.00 eq) and acylase I from Aspergillus melleus (300 mg) was added. After 6 h at 36°C the reaction mixture was filtered through a Amicon Ultra centrifugal filter unit (cut off 10 kDa). The resulting the amino acid buffer mixture was then submitted to the subsequent Fmoc protection without any further purification.

HRMS (ESI): m/z calc for C ₆ HI I N0 ₂ (M+H) ⁺ 130.0863, found 130.0858. Fmoc-4, 5-didehydroisoleucine (8)

FmocHt

To a stirring solution of 4,5-didehydroisoleucine 8 in Sorensen phosphate buffer (15 ml) was added a solution of Fmoc-OSu (823 g, 2.44 mmol, 1.05 eq) in acetone (10ml). After stirring for 16 h the reaction mixture was diluted with 50 ml water, acidified to pH 2 with 1 N HCI and extracted with ethyl acetate (3x30 ml). The combined organic layers were washed with brine, dried over MgSCU and concentrated under reduced pressure. The crude product was purified by column chromatography on silica gel (1 % MeOH/DCM) to obtain the Fmoc didehydroisoleucine 8 as a white solid (810 mg, 75%, 2 steps).

¹ H-NMR (400 MHz, DMSO-d ₆ ): d (ppm) = 8.49 (br, 1 H), 7.68 (d, J= 7.5 Hz, 2 H), 7.44 - 7.54 (m, 2 H), 7.32 (t, J= 7.02 Hz, 2 H), 7.23 (t, J= 7.02 Hz, 2 H), 5.45 - 5.73 (m, 1 H), 5.18 - 5.32 (m, 1 H), 5.01 - 5.17 (m, 1 H), 4.30 - 4.37 (m, 2 H), 4.12- 4.19 (m, 1 H), 2.39- 2.54 (m, 1 H), 2.44 - 2.52 (m, 1 H), 1.60 (d, J= 6.0 Hz, 2 H), 1.05 (d, J= 7.0 Hz, 1 H)

¹³ C-NMR (100 MHz, DMSO-d ₆ ): d (ppm) = 177.01 , 156.04, 143.58, 141.45, 137.20, 130.67, 127.93, 127.02, 125.20, 124.13, 120.18, 1 17.60, 67.46, 58.04, 53.33, 47.25, 39.65, 35.09

HRMS (ESI): m/z calc for C21 H21 NO4 (M+H) ⁺ 374.1363, found 374.1360.

Fmoc-4,5-didehydroisoleucine tert-butyl ester (9)

BF3*OEt2 (1.69 ml, 13.7 mmol, 6.0 eq) was added to a stirring solution of Fmoc- protected 4,5- didehydroisoleucine (8) (800 mg, 2.28 mmol, 1.00 eq) in 10 ml ¾uOAc. The reaction mixture was stirred at rt for 5 min, then cooled to 0°C and neutralized with saturated NaHCCh. The reaction mixture was then extracted with EtOAc (3*50 ml), washed with 1 M HCI (3x20ml) and brine (2x20ml) and dried over MgSCU. After evaporation of the solvent under reduced pressure the crude product was purified by column chromatography on silica gel (hexane/EtOAc, 10: 1) to obtain the fully protected 4,5-didehydroisoleucine (9) as a colourless oil (650 mg, 70%). ¹ H NMR (400 MHz, CDC ): d (ppm) = 1.07 - 1.14 (m, 3 H), 1.49 (s, 9 H), 2.74 - 2.85 (m, 1 H), 4.21 - 4.34 (m, 2 H), 4.39 (m, J= 6.90, 6.90 Hz, 2 H), 5.08 - 5.18 (m, 2 H), 5.24 (s, 1 H), 5.68 - 5.79 (m 1 H), 7.33 (t, J= 7.53 Hz, 2 H), 7.41 (t, J= 7.53 Hz, 2 H), 7.61 (d, J= 7.53 Hz, 2 H), 7.78 (d, J= 7.53 Hz, 2 H)

¹³ C NMR (100 MHz, CDCI3): d (ppm) = 15.66, 27.75, 40.07, 46.86, 58.02, 66.69, 81.93, 1 16.32, 119.64, 124.81 , 126.72, 127.36, 137.45, 140.97, 143.56, 155.92, 170.27

HRMS (ESI): m/z calc for C25H29NO4 (M+H) ⁺ 430.1989, found 430.1982.

Fmoc-4,5-dihydroxyisoleucine tert-butyl ester (10)

A/-methylmorpholine-/\/-oxide (287 mg, 2.45 mmol, 1.30 eq) was added to a stirring solution of 9 and potassium osmate dihydrate (45.2 mg, 122 pmol, 0.05eq) in 15ml of a 4: 1 mixture of CHCI ₃ and water and stirred for 20 min at rt. Then, Fmoc- protected 4,5-dihydroxyisoleucine tert- butyl ester (10) (1.00 g, 2.45 mmol, 1.00 eq) was added to the biphasic mixture. The resulting mixture was stirred at rt for 16 h and diluted with 100 ml DCM. Afterwards, saturated sodium metabisulfite solution (20ml) was added and extracted with DCM (3x10 ml) after being stirred for 30 min. the combined organic phases were dried over NaSCU and evaporated under reduced pressure. The crude product was purified by column chromatography on silica gel (0.6% MeOH/DCM to 1.0% MeOH/DCM gradient) to obtain the 4,5-dihydroxyisoleucine derivative 10 as white crystals (439 mg, 40%).

¹ H NMR (CDCh-d ¹ , 400 MHz): d = 1.00 (d, J= 7.03 Hz, 3 H), 1.50 (s, 9 H), 1.94 - 2.04 (m, 3 H), 3.57 (dd, J=10.79, 3.01 Hz, 1 H), 3.72 (t, J=9.50 Hz, 1 H), 3.76 - 3.82 (m, 1 H), 4.18 - 4.27 (m, 2 H), 4.37 - 4.47 (m, 2 H), 5.90 (d, J=8.28 Hz, 1 H), 7.33 (t, J= 7.03 Hz, 2 H), 7.41 (t, J= 7.28 Hz, 2 H), 7.61 (d, J= 7.28 Hz, 2 H), 7.77 ppm (d, J= 7.53 Hz, 2 H)

¹³ C NMR (CDC -d ¹ , 100 MHz): d = 10.41 , 27.99, 38.32, 47.14, 57.85, 64.71 , 67.18, 71.70, 82.74, 119.96, 125.02, 127.06, 127.71 , 141.27, 143.63, 156.82 ppm

HRMS (ESI): m/z calc for C25H31 NO6 (M+H) ⁺ 442.2224, found 442.2222. tert-butyl(2S,3R,4R)-2-((((9H-fluoren-9-yl)methoxy)carbonyl) amino)-4,5-bis((tert- butyldimethylsilyl) oxy)-3-methylpentanoate (11)

TBSO

TBSO

FmocH

O

11

Under a nitrogen atmosphere te/f-butyldimethylsilyl chloride (546 g, 3.62 mmol, 8.0 eq) was added to a stirring solution of Fmoc-4,5-dihydroxyisoleucine tert- butyl ester (10, 200 mg, 452 pmol, 1.00 eq) in 4 ml of a 1 : 1 mixture of dry DMF and pyridine. Then, DMAP (8.3 mg, 68 pmol, 0.15 eq) was added and the resulting mixture was stirred for 24 h at rt. Afterwards, the reaction mixture was diluted with 50 ml EtOAc and washed with 1 M HCI (3x20ml) and brine (2x20ml), dried over MgSCU and evaporated under reduced pressure. The crude product was purified by column chromatography on silica gel (hexane/EtOAc, 19: 1) to obtain the fully protected 4,5- dihydroxyisoleucine (9) as a colourless oil (271 mg, 90%).

¹ H NMR (CDCb-d ¹ , 500 MHz): d = 0.06 - 0.09 (m, 6 H), 0.1 1 (s, 3 H), 0.17 (s, 3 H), 0.90 - 0.95 (m, 18 H), 1.00 (d, J= 7.02 Hz, 3 H), 1.49 (s, 9 H), 2.35 - 2.44 (m, 1 H), 3.44 (dd, J=9.69, 8.32 Hz, 1 H), 3.52 - 3.57 (m, 1 H), 3.82 - 3.86 (m, 1 H), 4.19 - 4.26 (m, 2 H), 4.39 (d, J= 7.02 Hz, 2 H), 5.99 (d, J=8.24 Hz, 1 H), 7.30 (t, J= 7.78 Hz, 2 H), 7.40 (t, J=7.40 Hz, 2 H), 7.63 (dd, J= 9.77, 7.78 Hz, 2 H), 7.76 (d, J= 7.63 Hz, 2 H)

¹³ C NMR (CDCb-d ¹ , 126 MHz): -5.50, -5.38, -4.64, -4.09, 10.40, 18.05, 18.26, 25.87, 25.90, 28.04, 35.51 , 47.27, 59.12, 64.18, 66.80, 73.74, 81.38, 1 19.88, 125.20, 126.98, 127.55, 141.28, 144.02, 144.17, 156.46, 171.22

HRMS (ESI): m/z calculated: CsyHsgNOeSh M+H)* 670.3953, found 670.3945.

(2S,3R,4R)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)- 4,5-bis((tert- butyldimethylsilyl)oxy)-3-methylpentanoic acid (13)

A solution of fully protected 4,5-dihydroxyisoleucine 11 (100 mg, 149 pmol, 1.0 eq) was dissolved in 2 ml dry DCM, treated with 2,6 lutidine (173 pi, 1.49 mmol, 10.0 eq) and cooled to 0°C. Then, TMSOTf (135 pi, 746 pmol, 5.0 eq) was added and the reaction mixture was allowed to warm to room temperature overnight. The solution was diluted with Et2<D (10ml) followed by the addition of Sorensen phosphate buffer (pH=7; 5ml). Afterwards, the pH of the mixture was adjusted to 2 by the dropwise addition of NaHS0 ₄ -solution (10%). The phases were separated and the aqueous phase was extracted with Et ₂ 0 (3x10ml). The combined organic phases were washed with brine, dried over NaS04 and evaporated under reduced pressure. The crude product was purified by column chromatography on silica gel (1% MeOH/DCM) to furnish the final product (13) as a colourless oil (90.5 mg, quant.).

¹ H NMR (CDC -d ¹ , 500 MHz): d = 0.02 - 0.14 (m, 12 H), 1.04 (d, J= 7.02 Hz, 3 H), 2.36 - 2.47 (m, 1 H), 3.55 (t, J=8.24 Hz, 1 H), 3.58 - 3.64 (m, 1 H), 3.79 - 3.88 (m, 1 H), 4.24 (t, J= 6.79 Hz, 1 H), 4.37 - 4.49 (m, 3 H), 6.10 (d, J= 7.32 Hz, 1 H), 7.31 (t, J=7.48 Hz, 2 H), 7.40 (t, J= 7.32 Hz, 2 H), 7.61 (t, J= 7.63 Hz, 2 H), 7.76 (d, J=7.48 Hz, 2 H)

¹³ C NMR (CDCb-d ¹ , 126 MHz): -5.48, -5.41 , -4.74, -4.07, 11.07, 18.00, 18.25, 25.79, 25.87, 37.01 , 47.23, 57.77, 64.68, 67.00, 73.97, 1 19.95, 125.05, 125.12, 127.03, 127.64, 141.33, 143.86, 156.43

HRMS (ESI): m/z calculated: CssHsi NOeSh (M+H) ⁺ 614.3328, found 614.3324. ((2S,3S)-3-methyloxiran-2-yl) methyl 4-methylbenzenesulfonate ((S,S)-19)

A flame dried flask was charged with 10 g of crushed activated 3 A molecular sieves and flushed with nitrogen for several minutes. Then, DCM (200 ml) was added and the flask was cooled to -20°C. (+)-Diisopropyl tartrate (DIPT) (1.75 g, 7.49 mmol, 0.06 eq), crotyl alcohol (9.00 g, 125 mmol, 1.00 eq) and Ti(0/Pr) ₄ (1.77 g, 6.24 mmol, 0.05 eq) were added sequentially at this temperature. The resulting mixture was stirred for 15 min at -20°C, then a solution of tert-butyl hydroperoxide (TBHP, 5 M in DCM) (50.0 ml, 150 mmol, 2.00 eq) was added dropwise. The reaction mixture was stirred for 2 h at this temperature. Careful quenching of the excess TBHP was carried out by careful addition of trimethyl phosphite (22.0 ml, 187 mmol, 1.50 eq) at -20°C after which trimethyl amine (26.1 ml, 187 mmol, 1.50 eq), DMAP (1.83 g, 15.0 mmol, 0.12 eq) and a solution of p-toluenesulfonyl chloride (23.8 g, 125 mmol, 1.00 eq) in DCM (100ml) was added sequentially. The temperature was raised to -10°C and the reaction mixture stirred for 36 h. Afterwards the mixture was filtered through a pad of Celite and washed with DCM. The filtrate was then washed with 10 % tartaric acid (2x100ml), saturated NaHCOs (2x100ml) and brine (2x100ml). The organic phase was dried over MgS0 ₄ and evaporated under reduced pressure. The crude product was purified by column chromatography on silica gel (hexane /EtOAc, 2:1) and recrystallized (Et ₂ 0/hexane) in order to afford the tosylate (S,S-19))) as white needles (20.2 g, 67%). ¹ H NMR (CDC -d ¹ , 400 MHz): d = 1.30 (d, J= 5.27 Hz, 3 H), 2.46 (s, 3 H), 2.87 - 2.94 (m, 1 H), 2.92 (s, 1 H), 3.98 (dd, J=1 1.42, 5.90 Hz, 1 H), 4.18 (dd, J=11.42, 3.89 Hz, 1 H), 7.36 (d, J=8.28 Hz, 2 H), 7.80 ppm (d, J=8.53 Hz, 2 H).

¹³ C NMR (CDCb-d ¹ , 100 MHz): d = 16.95, 21.64, 52.77, 55.45, 70.03, 127.94, 129.89, 132.70, 145.06 ppm.

HRMS (ESI): m/z calculated: Cn H ₁₄ 0 ₄ S (M+H) ⁺ 243.0686, found 243.0678.

(S)-But-3-en-2-ol ((S)-3):

A solution of tosylate (S,S)-19 in dry THF (20ml) was added to a suspension of Zinc-Copper- couple (6g) and dry Nal (22.3 g, 149 mmol, 3.00 eq) in dry THF (150ml). The resulting suspension was stirred for 2h at 70°C, cooled to room temperature and filtered through a pad of silica. Afterwards the THF-butenol mixture was distilled under reduced pressure (200 mbar, 100°C) while the collecting flask was cooled to -78°C (dry ice). The THF-butenol solution was then submitted to the next step without further purification.

(S)-But-3-en-2-yl tert-butyl carbonate ((S)-4):

The butenol-THF solution of cooled t , 145 mmol, 3.00 eq) was carefully added under a nitrogen atmosphere in small portions. Then B0C2O (11.7 g, 53.4 mmol, 1.1 eq) was added in portion-wise over 10 min under vigorous stirring at this temperature. The reaction mixture was allowed to warm to room temperature overnight and then diluted with Et2<D after 16h of vigorous stirring. Excess sodium hydride was quenched by the slow addition of water. The resulting mixture was then extracted with diethyl ether (3x50 ml). The combined organic extracts were washed with brine (1x50 ml), dried over MgS0 ₄ and concentrated under reduced pressure. After purification by column chromatography on silica gel (hexane/EtOAc, 10: 1) the product (S)-4 was obtained as a colourless liquid (6.25 g, 75% two steps). 3-(6-Benzoxy- 1H-indol-3-yl)-2-acetylaminopropionic acid (30):

A suspension of L-serine (942 mg, 8.96 mmol, 4.00 eq.) in AC ₂ O (4.02 mL, 42.6 mmol, 9.5 eq.) and AcOH (22 mL) was stirred for 16 h at r.t. 6-benzoxyindol 29 was added and after a reaction time of 2 h at 75°C the solvent was removed under reduced pressure. After the residue was taken up in water (50 mL) and the pH was adjusted to 11 , the aqueous layer was washed with MTBE (2x50 mL) and acidified to pH = 3. The aqueous layer was extracted with EtOAc (4 x 50 mL), dried over Na ₂ SC> ₄ and the solvent was removed under reduced pressure. The crude product was recrystallized in MeOH, which gave compound 30 as a light brown solid (620 mg, 82%).

(S)-3-amino-3-(6-(benzoxy)-1H-indol-3-yl)propanoic acid - SchiffBase Complex (32):

A suspension of 30 (1.03 g, 3.32 mmol) in 40% NaOH (hhO/MeOHv/v = 1 :1 , 14 mL/mmol) was stirred at 110°C for 4 h, neutralized with cone. HCI to pH =7 and the solvent was removed in vacuo. The crude product was dissolved in MeOH (20 mL/mmol) and Ni(OAc) _2* 6 H ₂ O (966 mg, 3.32 mmol) and (S)-28 (1.78 g, 3.65 mmol) were added followed by K ₂ CC> ₃ (2.09 g, 15.1 mmol). The resulting mixture was refluxed for 16 h and the precipitate was filtered of and washed with DCM. The solvent of the filtrate was removed in vacuo and the crude product was suspended in DCM, washed with H ₂ O and dried with Na ₂ SC> ₄ . After removal of the solvent in vacuo the crude product was purified with DCM/MeOH (v/v = 40:1) to give the product 32 as an orange solid (2.44 g, 88%). (S)-6-benzoxytryptophan (33):

To a solution of 32 (180mg, 0.51 mmol) in MeOH was added 6 M HCI (15 mL) The solution was stirred for 45 min at 70°C and cone. NH ₄ 0H-solution was added until pH=7 was reached. The aqueous layer was washed with EtOAc (2 x 15 mL), and the pH was adjusted to 10. The precipitate was centrifuged and washed two times with water (pH = 10), to give (S)-6- benzoxytryptophan as a white solid.

(S)-3-(6-(benzyloxy)-1H-indol-3-yl)-2-(((2-(trimethylsily l)ethoxy)carbonyl)amino)propanoic acid (33a)

Triethylamine (446 mI_ g, 3.22 mmol, 2.00 eq.) and TeocOSu (626 mg, 2.42 mmol, 1.50 eq.) was added successively to a solution of L-6-benzoxytryptophan (33, 0.5 g, 1.61 mmol, 1.00 eq.) in DMF (20 ml_). The reaction mixture was stirred at r.t. for 2 h, then concentrated under reduced pressure .The aqueous layer was carefully acidified to pH = 4 by dropwise addition of 1 M HCI and extracted with EtOAc (3 x 100 ml_). The organic phase was washed with brine (2 x 100 ml_), dried over Na2SC>4 and evaporated under reduced pressure to afford the product 33a as a white solid (665 mg, 91 %).

HRMS (ESI): m/z calc for C ₁₇ H ₂₄ N ₂ 0 ₄ Si (M+H) ⁺ 455.1997, found 455.1999.

((2-(trimethylsilyl)ethoxy)carbonyl)-L-tryptophan (37a)

Triethylamine (11.3 ml_ g, 80.9 mmol, 1.5 eq.) and TeocOSu (18.2 g, 70.0 mmol, 1.30 eq.) was added successively to a solution of L-tryptophan (37, 1 1.0 g, 53.9 mmol, 1.00 eq.) in a 1 : 1 mixture of dioxane/water (200 ml_). The reaction mixture was stirred at r.t. for 2 h, then concentrated under reduced pressure .The aqueous layer was carefully acidified to pH = 4 by dropwise addition of 1 M HCI and extracted with EtOAc (3 x 100 ml_). The organic phase was washed with brine (2 x 100 ml_), dried over Na2S04 and evaporated under reduced pressure to afford the product 37a as a white solid (18.2 g, 97%).

HRMS (ESI): m/z calc for C ₁₇ H ₂₄ N ₂ 0 ₄ Si (M+H) ⁺ 349.1578, found 349.1582.

2,2,2-trichloroethyl ((2-(trimethylsilyl)ethoxy)carbonyl)-L-tryptophanate (38)

To a solution of /V-Teoc protected L-tryptophane (37a, 2.80 g, 8.04 mmol, 1.00 eq.) in DCM (32 ml_) at 0°C was added DMAP (147 mg, 1.21 mmol, 0.15 eq.) and EDC*HCI (2.00 g, 10.4 mmol, 1.30 eq.) successively . After stirring at 0°C for 10 min 2,2,2-trichloroethanol (1.54 ml_, 16.1 mmol, 2.00 eq.) was added and the solution was stirred for 2 h at r.t. The reaction mixture was diluted with DCM (100 ml_), washed with 0.5 M HCI (2 x 50 ml_), sat. NaHCC>3 solution (50 ml_) and brine (50 ml_). After drying over NaSCU and removal of the solvent under reduced pressure the crude product was purified by column chromatography on silica gel (1 % MeOH/DCM) to afford compound 37a as a pale yellow solid (3.51 g, 91 %).

HRMS (ESI): m/z calc for C^HasChl hCUSi (M+H) ⁺ 479.0722, found 479.0721.

2,2,2-trichloroethyl (S)-3-(6-(benzyloxy)-1H-indol-3-yl)-2-(((2-(trimethylsilyl)e thoxy)carbonyl)- amino)propanoate (39)

To a solution of compound 33a (1.0 g, 2.2 mmol, 1.0 eq.) in DCM (8.8 mL) at 0°C was added DMAP (40.3 mg, 0.329 mmol, 0.15 eq.) and EDC*HCI (548 mg, 2.86 mmol, 1.30 eq.) successively. After stirring at 0°C for 10 min 2,2,2-trichloroethanol (0.42 mL, 4.4 mmol, 2.00 eq.) was added and the solution was stirred for 2 h at r.t. The reaction mixture was diluted with DCM (50 mL), washed with 0.5 M HCI (2 x 25 mL), sat. NaHCC>3 solution (25 mL) and brine (25 mL). After drying over Na2SC>4 and removal of the solvent under reduced pressure the crude product was purified by column chromatography on silica gel (0.5% MeOH/DCM) to afford compound 39 as a yellow oil (1.1 g, 85%).

HRMS (ESI): m/z calc for CasHsiCbNaOsSi (M+H) ⁺ 585.1141 , found 585.1139. Synthesis of (N-Boc) 2-cystine-(OtBu) 2 (35)

Boc

35

A solution of L-cystine-(OtBu)2 (34, 10 g, 24 mmol, 1.0 eq.) in a 1 :1 mixture of hhO/dioxane (240 mL) was treated with NaHCC>3 (8.06 g, 96.0 mmol, 4.00 eq.) and B0C2O (10.1 mL, 47.0 mmol, 2.00 eq.) and the reaction mixture was stirred for 16 h at r.t. The reaction mixture was concentrated under reduced pressure and the aqueous layer was extracted with EtOAc (3 x 120 mL). The organic layer was washed with brine (100 mL), dried over Na2SC>4 and the solvent was removed under reduced pressure to afford 35 (13.2 g, 24.0 mmol, quant.) as a pale yellow solid.

HRMS (ESI): m/z calc for C24H44N2O8S2 (M+H) ⁺ 553.2612, found 553.2615. tert-butyl S-(6-(benzyloxy)-3-((S)-3-oxo-3-(2,2,2-trichloroethoxy)-2-(( (2-(trimethylsilyl)ethoxy) carbonyl)amino)propyl)-1H-indol-2-yl)-N-(tert-butoxycarbonyl )-L-cysteinate (40)

40

To a solution of (A/-Boc)2-L-Cystin-(OtBu)2 (35, 900 mg, 1.63 mmol, 1.00 eq.) in CHCI3 (16.3 ml_) was added SO2CI2 (263 mI_, 3.26 mmol, 2.00 eq.). After the reaction mixture was stirred for 1 h at r.t. the solvent was removed under reduced pressure. The residue was redissolved in CHCI3 (16.3 ml_) and cooled to 0°C and added to an ice cold solution of 39 (800 mg, 1.67 mmol, 1.00 eq.) and NaHCC>3 (420 mg, 5.00 mmol, 3.00 eq.) in CHCI3 (16.7 ml_) dropwise over a periode of 10 min. Afterwards the reaction mixture was stirred for 15 min at 0°C and 1 h at r.t.. The organic layer was washed with H2O (10 mL) and sat. NaHCC>3-solution (10 mL). After drying of the organic layer with Na2SC>4 and removal of the solvent under reduced pressure the crude product of 40 was used in the next step without further purification.

HRMS (ESI): m/z calc for CssHsaChNsOgSSi (M+H) ⁺ 860.2332, found 860.2323. tert-butyl N-(tert-butoxycarbonyl)-S-(3-((S)-3-oxo-3-(2,2,2-trichloroet hoxy)-2-(((2- (trimethylsilyl)ethoxy)carbonyl)amino)propyl)-1H-indol-2-yl) -L-cysteinate (41 )

To a solution of (A/-Boc)2-L-Cystin-(OtBu)2 (39, 5.06 g, 9.15 mmol, 1.00 eq.) in CHCI3 (92 mL) was added SO2CI2 (1.48 mL, 18.3 mmol, 2.00 eq.). After the reaction mixture was stirred for 1 h at r.t. the solvent was removed under reduced pressure. The residue was redissolved in CHCI3 (92 mL), cooled to 0°C and added dropwise to an ice cold solution of 38 (4.4 g, 9.17 mmol 1.00 eq.) and NaHCCh (2.31 g, 27.5 mmol, 3.00 eq.) in CHCI3 (92 mL) over a periode of 10 min. Afterwards the reaction mixture was stirred for 15 min at 0°C and 1 h at r.t. The organic layer was washed with H2O (2 x 100 mL) and sat. NaHCCh-solution (2 x 80 mL). After drying of the organic layer with Na2SC>4 and removal of the solvent under reduced pressure the crude product of 41 was used in the next step without further purification.

HRMS (ESI): m/z calc for Csi HUeChNsOsSSi (M+H) ⁺ 754.1913, found 754.1917.

(S)-3-(6-(benzyloxy)-2-(((R)-3-(tert-butoxy)-2-((tert-but oxycarbonyl)amino)-3-oxopropyl)thio)- 1H-indol-3-yl)-2-(((2-(trimethylsilyl)ethoxy)carbonyl)amino) propanoic acid (49):

Boc

A solution of tryptathionine derivative 39 (1.63 mmol, 1.00 eq.) in DMF (8.4 ml_) was treated with CH3COOH (0.8 ml_) and zinc (3.51 g, 53.6 mmol, 33.0 eq.) for 2 h at 45°C. The reaction mixture was filtered over Celite and the solvent was removed under reduced pressure. The crude product was dissolved in EtOAc (50 ml_) and washed with 10% KHSO ₄ solution (2 x 25 ml_) and brine (2 x 25 ml_). After drying over NaaSCU and removing of the solvent under reduced pressure, the crude product was purified by C18 reverse phase chromatography (ACN/H2O 50% to 100% gradient) to give compound 49 as a yellow solid (840 mg, 83% over 2 steps).

HRMS (ESI): m/z calc for CseHsi NsOgSSi (M+H) ⁺ 730.3183, found 730.3188. (S)-3-(2-(((R)-3-(tert-butoxy)-2-((tert-butoxycarbonyl)amino )-3-oxopropyl)thio)-1H-indol-3-yl)- 2-( ((2-(trimethylsilyl) ethoxy)carbonyl) amino) propanoic acid ( 50)

A solution of tryptathionine derivative 38 (9.15 mmol, 1.00 eq.) in DMF (40 ml_) was treated with CH3COOH (4 ml_) and zinc (20.0 g, 302 mmol, 33.0 eq.) for 2 h at 45°C. The reaction mixture was filtered over Celite and the solvent was removed under reduced pressure. The crude product was dissolved in EtOAc (200 mL) and washed with 10% KHSO ₄ solution (2 x 50 mL) and brine (2 x 50 mL). After drying over NaaSCL and removing of the solvent under reduced pressure, the crude product was purified by C18 reverse phase chromatography (ACN/H2O 50% to 100% gradient) to afford compound 50 as a yellow oil (5.0 g, 88%. over 2 steps).

HRMS (ESI): m/z calc for Csi HUeChNsOsSSi (M+H) ⁺ 624.2769, found 624.2775.

( ( benzyloxy) carbonyl) glycyl-L-isoleucine (44)

To a solution of Cbz-glycine (42, 10.0 g, 32.7 mmol, 1.00 eq.) in acetone (100 mL) was added a suspension of L-isoleucine (4.71 g, 35.9 mmol, 1.10 eq.) and NaHCC>3 (8.23 g, 87.9 mmol, 3.00 eq.) in water (100 mL). The reaction mixture was stirred at r.t. for 3 h and concentrated under reduced pressure .The aqueous layer was carefully acidified to pH = 4 by dropwise addition of 1 M hid and extracted with EtOAc (3 x150 mL). The organic phase was then washed with brine (2 x 100 mL), dried over Na2S04 and evaporated under reduced pressure to afford the product 44 as a colourless oil (10.1 g, 96%). HRMS (ESI): m/z calc for C^HaaNaOs (M+H) ⁺ 323.1601 , found 323.1606.

Glycyl-L-isoleucylglycine (45) :

Dipeptide 44 (10.1 g, 31.3 mmol, 1.00 eq.) and benzyl glycinate (8.21 g, 40.7 mmol, 1.30 eq.) were dissolved in dry DMF (125 ml_). Then, COMU (17.4 g, 40.7 mmol, 1.30 eq.) and DIPEA (12.6 ml_, 72.1 mmol, 3.00 eq.) were added at 0°C. The reaction mixture was allowed to warm to r.t. overnight and diluted with EtOAc (300 ml_) afterwards. After washing with a solution of 10% KHSCU-solution (2x100 ml_) the fully protected tripeptide precipitated in the organic phase. The organic phase was cooled to 4°C for 4 h in order for the peptide to precipitate, then the precipitate was filtered and washed with cold EtOAc. The precipitate was redissolved in a 1 : 1 mixture of water and THF (260 ml_). Pd/C (1 g) was added to the solution after degassing with N2 for 30 min. Then, the reaction mixture was degassed with hydrogen for 1 h. After vigorous stirring at room temperature under 1.0 atm of hydrogen overnight, the catalyst was filtered through a pad of Celite. Afterwards, the mixture was concentrated under reduced pressure to obtain the product 45 as a white solid (5.71 g, 74 %)

HRMS (ESI): m/z calc for C10H19N3O4 (M+H) ⁺ 246.1448, found 246.1440.

Synthesis of pentapeptide 51:

A solution of thioether building block 49 (1 11 mg, 0.14 mmol, 1.00 eq.) in AcN (0.7 ml_) was treated with collidine (37 mI_, 0.27 mmol, 2.0 eq) and L/,L/’-disuccinimidyl carbonate (39 mg, 0.15 mmol, 1.1 eq.) and stirred for 1 h at r.t.. A solution of tripeptide 45 (44 mg, 0.18 mmol, 1.3 eq) in a 1 :4 mixture of AcN/hhO (1 mL) was added and the reaction mixture was stirred for 2 h at r.t.. Afterwards, the mixture was diluted with EtOAc (20 mL), 10% KHS0 ₄ -solution (20 mL) was added and the aqueous layer was extracted with EtOAc (2 x 20 mL). The organic layer was washed with brine (2 x 20 mL), dried over Na ₂ S0 ₄ and evaporated under reduced pressure which afforded pentapeptide 51 as a yellow solid (115 mg, 90%).

HRMS (ESI): m/z calc for C ₄₆ H ₆₈ N ₆ 0 ₁₂ SSi (M+H) ⁺ 957.4458, found 957.4457.

Synthesis of pentapeptide 52:

A solution of tryptathionine building block 50 (2.0 g, 2.5 mmol, 1.0 eq.) in AcN (10 ml_) was treated with collidine (659 pl_, 4.95 mmol, 2.00 eq) and L/,L/’-disuccinimidyl carbonate (697 mg, 2.72 mmol, 1.10 eq.) and stirred for 1 h at r.t.. A solution of tripeptide 45 (790 mg, 3.22 mmol, 1.30 eq.) in a 1 :4 mixture of AcN/hhO (18 ml_) was added and the reaction mixture was stirred for 2 h at r.t.. Afterwards, the mixture was diluted with EtOAc (100 mL), 10% KHSCU-solution (20 mL) was added and the aqueous layer was extracted with EtOAc (2x50 mL). The organic layer was washed with brine (2 x 50 mL), dried over Na ₂ S0 ₄ and evaporated under reduced pressure which afforded pentapeptide 52 as a yellow solid (2.15 g, 93%).

HRMS (ESI): m/z calc for (M+H) ⁺ CseHsiCIsNeOnS 851.4039, found 851.4058.

Fully protected cyclic pentapeptide 53 :

Pentapeptide 51 (151 mg, 0.180 mmol, 1.00 eq.) was dissolved in 1 ml_ of a solution of p-toluenesulfonic acid in THF (1.8 M) and was stirred for 4 h at r.t. Then, the reaction mixture was neutralized by the addition of DIPEA (320 pl_, 1.84 mmol, 10 eq) and diluted with DCM (180 ml_). Afterwards, DIPEA (60.2 pL, 354 pmol, 2.00 eq.) and T3P (50% in EtOAc, 210 pL, 354 pmol, 0.34 eq.) were added. After the solution was stirred for 16 h at r.t. 2/3 of the the solvent was concentrated under reduced pressure. The organic phase was washed with 10% KHSCU-solution (20 ml_), sat. NaHCC>3-solution (20 ml_), water (20 ml_) and brine (20 ml_). The organic layer was dried over Na2SC>4 and the solvent was removed under reduced pressure. The crude product was purified by C18 reverse phase chromatography (AcN/hhO 50% to 100% gradient) to afford cyclic pentapeptide 53 as a yellow solid (82 mg, 70%)

HRMS (ESI): m/z calc for C^ HssNeOgSSi (M+H) ⁺ 839.3828, found 839.3839.

Fully protected cyclic pentapeptide (54):

Pentapeptide 52 (700 mg, 0.822 mmol, 1.00 eq.) was dissolved in 10 ml_ of 2 M hid in dioxane and stirred for 40 min at r.t. Then, the reaction mixture diluted with 40 ml_ of dioxane and the solvent was evaporated under reduced pressure. The precipitate was dissolved in 8 ml_ DMF and diluted with 82 ml_ DCM. Afterwards, DIPEA (279 mI_, 1.64 mmol, 2.00 eq.) and T3P (50% in EtOAc, 977 mI_, 1.64 mmol, 2.00 eq.) were added. After the solution was stirred for 5 h at r.t., 1/3 of the the solvent was concentrated under reduced pressure. The organic phase was washed with 10% KHS0 ₄ -solution (20 ml_), sat. NaHCC>3-solution (20 ml_), water (20 ml_) and brine (20 ml_). The organic layer was dried over Na ₂ S0 ₄ and the solvent was removed under reduced pressure. The crude product was purified by C18 reverse phase chromatography (AcN/FhO 50% to 100% gradient) to afford cyclic pentapeptide 54 as a yellow solid (420 mg, 72%).

HRMS (ESI): m/z calc for C ₃₄ H ₅₂ N ₆ 0 ₈ SSi (M+H) ⁺ 733.3409, found 733.3409. Fully deprotected monocyclic pentapeptide 55 :

Monocyclic Pentapeptide 53 (125 mg, 0.17 mmol, 1.00 eq.) was stirred in TFA/DCM/TIPS (8:15:0.5) for 2 h at r.t. The solvent was removed under reduced pressure and the crude product was purified by C18 reverse phase chromatography (AcN/FhO 20% to 100%) to afford the fully deprotected monocyclic pentapeptide 55 as a white powder (100 mg, quant.). HRMS (ESI): m/z calc for CsiHssNeOyS (M+H) ⁺ 639.2595, found 639.2590.

Fully deprotected monocyclic pentapeptide 56:

Monocyclic Pentapeptide 54 (250 mg, 0.34 mmol, 1.00 eq.) was stirred in TFA/DCM/TIPS (8:15:0.5) for 2 h at r.t. The solvent was removed under reduced pressure and the crude product was purified by C18 reverse phase chromatography (AcN/hhO 10% to 30%) to afford the fully deprotected monocyclic pentapeptide 56 as a white powder (200 mg, quant.).

HRMS (ESI): m/z calc for C^H _t NeOeS (M+H) ⁺ 533.2177, found 533.2188. Tri peptide (48):

HRMS (ESI): m/z calc for C ₄₂ H ₆₄ N ₄ 0 ₁ oSi ₂ (M+H) ⁺ 841.4233, found 841.4253.

Monocyclic octapeptide 57:

A solution of fully deprotected monocyclic pentapeptide 55 (50.0 g, 0.078 mmol, 1.00 eq.) and MSA (13.8 pL, 0.86 mmol, 1.1 eq.) in DMA (1.5 mL) was stirred for 2 h at 50°C. Separately, a solution of Fmoc-Asn-Hyp-DHIIe(TBS)2-OH (98.8 mg, 0.12 mmol, 1.50 eq.), COMU (36.9 mg, 0.086 mmol, 1.10 eq.) and DIPEA (15.0 pL, 0.083 mmol, 1.10 eq.) in DMA (0.4 mL) was stirred for 30 min at 0°C. The silylated monocyclic peptide was then added to the activated tripeptide and stirred for 1 h at 0°C then at 35°C for 3 h. Et2lMH (82.0 pL, 0.078 mmol, 10.0 eq.) was added and stirred for 1 h at r.t. The solvent was removed under reduced pressure and the crude product was purified using preparative HPLC (Sunfire Prep C18 OBD 10pm, 50x150 mm column, gradient A) to afford octapeptide 57 as a white solid (65.5 mg, 68%).

HRMS (ESI): m/z calc for CssHgoNhoOnSSh (M+H) ⁺ 1239.5970, found 1239.5980. Monocyclic octapeptide 58:

A solution of fully deprotected monocyclic pentapeptide 56 (40.0 g, 0.075 mmol, 1.00 eq.) and MSA (12 pL, 0.075 mmol, 1.00 eq.) in DMA (1.5 mL) was stirred for 2 h at 50°C. Similtaneously, a solution of Fmoc-Asn-Hyp-DHIIe(TBS)2-OH (95 mg, 0.11 mmol, 1.50 eq.), COMU (35.0 mg, 0.083 mmol, 1.10 eq.) and DIPEA (14.0 pL, 0.083 mmol, 1.10 eq.) in DMA (0.4 mL) was stirred for 30 min at 0°C. The silylated monocyclic peptide was then added to the activated tripeptide and stirred for 1 h at 0°C then at 35°C for 3 h. Et2lMH (77 pL, 0.75 mmol, 10 eq.) was added and stirred for 2 h at r.t. The solvent was removed under reduced pressure and the crude product was purified using preparative HPLC (Sunfire Prep C18 OBD 10pm, 50x150 mm column, gradient A) to afford octapeptide 58 as a white powder (70 mg, 68%).

HRMS (ESI): m/z calc for Csi H^NhoO SSh (M+H) ⁺ 1 133.5551 , found 1 133.5549.

Monocyclic octapeptide 59:

A solution of TBAF in THF (1 M, 0.52 mL, 10.0 eq) was added to a solution of octapeptide 57 (70 mg, 62 mmol, 1.0 eq) in THF (61.8 mL) and stirred for 2 h at r.t.. The solvent was evaporated in vacuo and the crude product purified by C18 reverse phase chromatography

(AcN/H20 5% to 30%) to afford the product 59 as a white solid (45 mg, 85%). HRMS (ESI): m/z calc for C ₄₆ H ₆₂ N ₁ o0 ₁₄ S (M+H) ⁺ 1011.4240, found 1001.4247.

Monocyclic octapeptide 60:

A solution of TBAF in THF (1 M, 0.62 ml_, 10.0 eq) was added to a solution of octapeptide 58 (70 mg, 62 mmol, 1.0 eq) in THF (0.62 mL) and stirred for 2 h at r.t.. The solvent was evaporated in vacuo and the crude product purified by C18 reverse phase chromatography (AcN/H20 5% to 60%) to afford the product 61 as a white solid (49 mg, 88%).

HRMS (ESI): m/z calc for CsgHseNhoO S (M+H) ⁺ 905.3822, found 905.38113.

2-((9H-fluoren-9-yl) methyl) 1-(tert- butyl) (2S, 4R)-4-hydroxypyrrolidine- 1, 2-dicarboxylate ( 64):

A solution of /V-Boc- protected (2S,4R)-4-hydroxyproline 63 (5.0 mg, 22 mmol, 1.0 eq.) in DMF (20 mL) was added dropwise to a solution of 9-Fluorenemethanol (8.5 mg, 43 mmol, 2.0 eq.), EDC*HCI (8.3 g, 43 mmol, 2.0 eq.) and DMAP (396 mg, 3.24 mmol, 0.150 eq.) in DCM (220 mL). The reaction mixture was stirred at r.t. for 2 h. Then, 10% KHSCU solution (50 mL) was added. The organic phase was washed with brine (50 mL) and dried over NaSCU. Afterwards, the solvent was removed under reduced pressure and the crude product was purified by column chromatography on silica gel (hexane/ethyl acetate = 1 : 1) to afford compound 64 as a white solid (5.0 g, 56%).

(9H-fluoren-9-yl)methyl (2S,4R)-4-hydroxypyrrolidine-2-carboxylate (64a):

64 (5.0 g, 22 mmol, 1.0 eq.) was treated with 4 M HOI in Dioxane (30 mL) at r.t. for 1 h. Afterwards, the solvent was evaporated under reduced pressure to afford the product (64a) as a white solid (3.7 g, quant.).

HRMS (ESI): m/z calc for C ₁₉ H ₁₉ N0 ₃ (M+H) ⁺ 310.1438, found 310.1426.

(9H-fluoren-9-yl)methyl (2S,4R)-1-((tert-butoxycarbonyl)-L-asparaginyl)-4-hydroxypyr rolidine- 2-carboxylate (65)

/V-Boc-protected asparagine (1.7 g, 7.3 mmol, 1.5 eq.), 64a (1.5 g, 4.8 mmol, 1.0 eq.), EDC*HCI (1.4 g, 7.3 mmol, 1.5 eq.) and HOBt*H ₂ 0 (1.5 g, 9.7 mmol, 2.0 eq.) were dissolved in DMF (72 mL) and stirred at r.t. for 16 h. The reaction mixture was diluted with EtOAc (200 mL). Then, 10% KHSCU solution (50 mL) was added. The organic phase was washed with 10% KHSO4 solution (50 mL) and brine (2 x 50 mL). After drying over NaSCU and removal of the solvent under reduced pressure the crude product was purified by C18 reversed phase chromatography (AcN/H2020% to 70%) to afford dipeptide 65 as a white powder (1.6 g, 78%). HRMS (ESI): m/z calc for C28H33N3O7 (M+H) ⁺ 524.2391 , found 524.2396.

(9H-fluoren-9-yl) methyl (2S,4R)-1-(L-asparaginyl)-4-hydroxypyrrolidine-2-carboxylate (66)

65 (1.0 g, 1.9 mmol, 1.0 eq.) was treated with 4 M HOI in Dioxane (30 mL) at r.t. for 1 h. Afterwards, the solvent was evaporated under reduced pressure to afford the product (66) as a white solid (870 mg, quant.).

HRMS (ESI): m/z calc for C ₂₃ H ₂₅ N ₃ 0 ₅ (M+H) ⁺ 424.1866, found 424.1858.

Monocyclic hexapeptide 67:

A solution of fully deprotected monocyclic pentapeptide 55 (42 mg, 0.66 mmol, 1.00 eq.) and MSA (11.6 pL, 0.723 mmol, 1.10 eq.) in DMA (2 mL) was stirred for 2 h at 50°C. Simultaneously, a solution of Fmoc-DHIIe(TBS)2-OH (13, 52 mg, 0.85 mmol, 1.30 eq.), COMU (36 mg, 0.85 mmol, 1.30 eq.) and DIPEA (15 pL, 0.85 mmol, 1.30 eq.) in DMA (0.4 mL) was stirred for 30 min at 0°C. The silylated monocyclic peptide was then added to the activated dihydroxyisoleucine derivative and stirred for 1 h at 0°C then at r.t. overnight. Afterwards, the mixture was diluted with EtOAc (50 mL) and washed with 10% KHSCU solution (3 x 5 mL). The organic phase was washed with brine (2 x 20 mL), dried over NaSCL and evaporated under reduced pressure. The crude product of 67 was used in the next step without any further purification.

HRMS (ESI): m/z calc for Ce^syNyO^SSh (M+H) ⁺ 1234.5745, found 1234.5745. Monocyclic hexapeptide 68:

A solution of fully deprotected monocyclic pentapeptide 56 (100 g, 0.188 mmol, 1.00 eq.) and MSA (33.2 pL, 0.207 mmol, 1.10 eq.) in DMA (4 ml_) was stirred for 2 h at 50°C. Simultaneously, a solution of Fmoc-DHIIe(TBS)2-OH (13, 149 mg, 0.244 mmol, 1.30 eq.), COMU (104 mg, 0.244 mmol, 1.30 eq.) and DIPEA (42.5 pL, 0.244 mmol, 1.30 eq.) in DMA (1.25 ml_) was stirred for 30 min at 0°C. The silylated monocyclic peptide was then added to the activated dihydroxyisoleucine derivative and stirred for 1 h at 0°C then at r.t. overnight. Afterwards, the mixture was diluted with EtOAc (100 mL) and washed with 10% KHSCU solution (3 x 10 mL). The organic phase was washed with brine (2 x 25 mL), dried over NaSCU and evaporated under reduced pressure. The crude product of 68 was used in the next step without any further purification.

HRMS (ESI): m/z calc for CsyHsi NyOnSSh (M+H) ⁺ 1128.5326, found 1 128.5316.

Monocyclic octapeptide 69:

Crude monocyclic hexapeptide 67 (0.66 mmol, 1.0 eq.), dipeptide 66 (42 mg, 0.10 mmol, 1.50 eq.) were dissolved in DMF (1.5 mL). Then, DIPEA (17.3 mL, 0.10 mmol, 1.50 eq.) and HATU (38 mg, 0.10 mmol, 1.5 eq) were added at 0°C. The reaction mixture was allowed to warm to r.t. overnight and concentrated under reduced pressure. The crude product was purified by C18 reversed phase chromatography (AcN/hhO 60% to 100%) to fully protected octapeptide 69 as a white solid (60 mg, 55% over two steps).

HRMS (ESI): m/z calc for CeyHnoNioOieSSb (M+H) ⁺ 1639.7433, found 1639.7404.

Monocyclic octapeptide 70:

Crude monocyclic hexapeptide 68 (0.188 mmol, 1.00 eq.), dipeptide 68 (1 19 mg, 0.282 mmol, 1.50 eq.) were dissolved in DMF (3 ml_). Then, DIPEA (49.1 ml_, 0.282 mmol, 1.50 eq.) and HATU (108 mg, 0.282 mmol, 1.50 eq) were added at 0°C. The reaction mixture was allowed to warm to r.t. overnight and concentrated under reduced pressure. The crude product was purified by C18 reversed phase chromatography (AcN/H20 60% to 100%) to fully protected octapeptide 70 as a white solid (170 mg, 59% over two steps).

HRMS (ESI): m/z calc for CeoHiwNioOisSSb (M+H) ⁺ 1533.7015, found 1533.7004.

Monocyclic octapeptide 71:

Monocyclic octapeptide 69 (15 mg, 10.3 pmol, 1.00 eq.) was dissolved in DMF (0.1 ml_). Et2lMH (10.8 mI_, 0.103 mmol, 10 eq.) was added and stirred for 2 h at r.t. The solvent was removed under reduced pressure and the precipitate was redissolved in THF (0.2 ml_). Then, a solution of TBAF in THF (1 M, 0.10 ml_, 10 eq) was added and the reaction mixture was stirred for 4 h at r.t.. The solvent was evaporated in vacuo and the crude product purified by C18 reverse phase chromatography (ACN/H2O 5% to 70%) to afford the product 71 as a white solid (8 mg, 77%).

HRMS (ESI): m/z calc for C ₄₆ H ₆₂ N ₁ o0 ₁₄ S (M+H) ⁺ 1011.4240, found 1001.4241.

Monocyclic octapeptide 72.

Monocyclic octapeptide 69 (20.0 mg, 14.8 pmol, 1.00 eq.) was dissolved in DMF (0.15 ml_). Et2NH (15.5 mI_, 0.148 mmol, 10 eq.) was added and stirred for 2 h at r.t. The solvent was removed under reduced pressure and the precipitate was redissolved in THF (0.3 ml_). Then, a solution of TBAF in THF (1 M, 0.15 ml_, 10 eq) was added and the reaction mixture was stirred for 4 h at r.t.. The solvent was evaporated in vacuo and the crude product purified by C18 reverse phase chromatography (ACN/H2O 5% to 30%) to afford the product 72 as a white solid (10 mg, 75%).

HRMS (ESI): m/z calc for CsgHseNioOisS (M+H) ⁺ 905.3822, found 905.3810. (S)-Deoxy-(0)-benzyl-a-amanitin (61a) :

Monocyclic octapeptide 59 or 71 (20.0 mg, 19.2 pmol, 1.00 eq.) was dissolved in DMF (19 ml_). Then, DIPEA (6.71 pl_, 38.5 p ol, 2.00 eq.) and HATU (4.98 mg, 38.5 pmol, 2.00 eq) were added at 0°C. The reaction mixture was allowed to warm to r.t. overnight and concentrated under reduced pressure. The crude product was purified using preparative HPLC (Sunfire Prep C18 OBD 10pm, 50x150 mm column, gradient C) to afford (O)-Benzyl-a-amanitin (61- a, 13 mg, 68%) as a white powder.

HRMS (ESI): m/z calc for C ₄₆ H ₆ oN ₁₀ 0 ₁₃ S (M+H) ⁺ 993.4135, found 993.4145.

(S)-Deoxyamaninamide (72a) :

Monocyclic octapeptide 60 or 72 (20.0 mg, 21.4 pmol, 1.00 eq.) was dissolved in DMF (21 ml_). Then, DIPEA (7.47 mI_, 42.9 pmol, 2.00 eq.) and HATU (16.3 mg, 42.9 pmol, 2.00 eq) were added at 0°C. The reaction mixture was allowed to warm to r.t. overnight and concentrated under reduced pressure. The crude product was purified using preparative HPLC (Sunfire Prep C18 OBD 10pm, 50x150 mm column, gradient B) to afford (S)-Deoxyamaninamide (72a, 14 mg, 74%) as a white powder. HRMS (ESI): m/z calc for C39H54N10O12S (M+H) ⁺ 887.3716, found 887.3718.

(O)-Benzyl-a-amanitin (61b):

The prophyrine derived ligand (22 pg, 0.45 pmol, 0.3 eq.) and MnOTf2 (16 pg, 0.45 pmol, 0.3 eq.) were dissolved in DCM (1 ml_) and stirred for 3 h at r.t. Then Octapeptide 61 a (1.5 mg, 1.5 p ol, 1.0 eq.) dissolved in DMF (500 pL), AcOH (0.21 pL, 3.8 pmol, 2.5 eq.) and H2O2 (0.1 1 pL, 4.5 pmol, 3.0 eq.) were added. The reaction mixture was cooled down to 0°C and was stirred for 16 h at 0°C. The solvent was removed under reduced pressure and the crude product was used in the next step without further purification.

HRMS (ESI): m/z calc for C ₄₆ H ₆ oN ₁ o0 ₁₄ S (M+H) ⁺ 1009.4084 found 1009.41 18. Amaninamide (62):

The prophyrine derived ligand (73 pg, 1.5 pmol, 0.3 eq.) and MnOTf2 (53 pg, 1.5 pmol, 0.3 eq.) were dissolved in DCM (1 ml_) and stirred for 3 h at r.t. Then Octapeptide 72 (5 mg, 5 pmol, 1.0 eq.) dissolved in DMF (500 pL), AcOH (0.700 pL, 12.7 pmol, 2.5 eq.) and H ₂ 0 ₂ (0.37 pL, 15.0 pmol, 3.0 eq.) were added. The reaction mixture was cooled down to 0°C and was stirred for 16 h at 0°C. The solvent was removed under reduced pressure and the crude product was purified using preparative HPLC (Sunfire Prep C18 OBD 10pm, 50x150 m column, gradient D) to afford amaninamide 62 as a white powder.

HRMS (ESI): m/z calc for C ₃₉ H ₅₄ N ₁₀ O ₁₃ S (M+H) ⁺ , found. a-Amanitin (61):

The crude product of 61 b was dissolved in THF/H20 (2: 1) and Pd/C (1 mg) was added. The reaction mixture was flushed with N2 for 10 min, then with H2 for 10 min and was stirred for 2 h at r.t. The solvent was removed under reduced pressure and the crude product was purified using preparative HPLC (Sunfire Prep C18 OBD 10pm, 50x150 mm column) to afford cr- Amanitin (61) as a white powder.

HRMS (ESI): m/z calc for C39H54N10O14S (M+H) ⁺ 919.3614 found 919.3614.

Preparative HPLC purification gradients:

Gradient A: 0-25 min 40%-60% B, 25-35 min 100% B; 35-40 min 40%B

0.1 % formic acid in water (Solvent A) and 0.1 % formic acid in ACN (Solvent B).

Gradient B: 0-30 min 10%-30% B, 30-40 min 100% B; 40-50 min 10%B

0.1 % formic acid in water (Solvent A) and 0.1 % formic acid in ACN (Solvent B).

Gradient C: 0-60 min 5%-50% B, 60-65 min 100% B; 65-70 min 5%B

0.1 % formic acid in water (Solvent A) and 0.1 % formic acid in ACN (Solvent B).

Solid-phase peptide synthesis

Automated or manual solid-phase peptide synthesis was performed in 50 pmol scale. Loading: To a 10 ml syringe reactor with frit and cap 1g of tritylchloride polystyrene (TCP) resin (0.9 mmol/g) were added and 7 ml drydichloromethane(DCM) . For resin loading with the first amino acids, the resin was pre-swollen for 10 min and the solvent was removed by evaporation in vacuum. A mixture of the amino acids (0.6 mmol) and 3 equivalents of N,N-diisopropylamine (DIPEA) dissolved in 5 ml dry DCM was added to the resin. The syringe was agitated for 30 min at room temperature. The solution was removed and the resin was washed (2x 5ml N,N- dimethylformamide(DMF), 2x 5 mIDCM). Capping of non-reacted functional groups of the resin was performed with DCM, methanol and DIPEA 80: 15:5 (2x 10 ml, 10min). After washing (5x 5 ml DMF), Fmoc-removal was achieved with DMF/piperidine (4: 1 , 5 ml, 1x 2 min, 1x 20 min). After final washing (2x 5 ml DMF, 1x 5ml methanol, 3x 5 ml DCM), the resin was dried in vacuo. Coupling of Fmoc protected amino acids: To 200 mg of the resin (~0.5mmol/g), a 0.25M solution of the amino acid in DMF (2.5 eq relative to resin loading) was added. After addition of a 0.5M solution of DIPEA in DMF (2.5eq) and a 0.25 M solution ofO-(benzotriazoM-yl)- N,N,N’,N’-tetramethyluronium tetrafluoroborate TBTU in DMF (2.5 eq), the reaction solution was mixed for 15min. A second coupling was performed for 15min. Fmoc removal: DMF/piperidine (4: 1 , 2.5ml) was added to the resin and mixed for 2.5min. The procedure was repeated 4 times. The resin was washed with DMF (6x 2.5 ml). After the final coupling cycle, the resin was washed with DCM (6x 2 ml). Cleavage: After addition of the cleavage cocktail (DCM/HFIP 4: 1 , the syringe was shaken for 30min. The solution was transferred to a flask and the solvent was removed in vacuo.. Further instructions can be found in (Amblard M, Fehrentz JA, Martinez J, Subra G. Mol Biotechnol. 2006 Jul;33(3):239-54).

Abbreviations

BF3*Et20: boron trifluoride etherate

Bn: benzyl

Boc: tert-butyloxycarbonyl

BMIM-PF ₆ : 1-butyl-3-methylimidazolium hexafluorophosphate

Cbz: benzyloxycarbonyl

COMBU: 4-{[1 ,3-Dimethyl-2,4,6-trioxotetrahydropyrimidin-

5(6/-/)ylidenaminooxy](dimethylamino)methylen}morpholin-4 -iumhexafluorophosphate

COMU: (1-Cyano-2-ethoxy-2-oxoethylidenaminooxy)dimethylamino-morph olino-carbenium- hexafluorophosphate

[(p-cymene) RuChb: (cymene)ruthenium dichloride dimer

DCM: dichloromethane

DHIL: dihydroxy-isoleucine

DIPEA: N,N-diisopropylethylamine DMA: dimethylacetamide

DMF: dimethylformamide

(DHQD)2PHAL: hydroquinidine 1 ,4-phthalazinediyl diether

Fm - 9-Fluorenylmethyl

Fmoc: fluorenylmethyloxycarbonyl

Fmoc-OSu: 9-Fluorenylmethyl N-succinimidyl carbonate

HATU: 1-[Bis(dimethylamino)methylene]-1 H-1 ,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate; Hexafluorophosphate Azabenzotriazole Tetramethyl Uronium

HBTU: 2-(1 /-/-Benzotriazol-1-yl)-1 , 1 ,3,3-tetramethyluronium-hexafluorophosphate

HCTU: 2-(6-Chlor-1 /-/-benzotriazol-1-yl)-1 , 1 ,3,3-tetramethylaminium-hexafluorophosphate

Hyp: trans-4-hydroxy-proline

LHMDS: lithium bis(trimethylsilyl)amide

MSA: N-Methyl-N-trimethylsilylacetamid

NMO: 4-methylmorpholine 4-oxide

PPh3: triphenylphosphine

PPO: Phthaloyl peroxide

T3P: 2,4,6-tripropyl-1 ,3,5,2,4,6-trioxatriphosphorinane-2,4,6-trioxide

TBAF: tetra-n-butylammonium fluoride

tBuOAc: tert-butyl-acetate

TBS: tert.-butyldimethylsilyl

TBTU: 2-(1 H-Benzotriazole-1-yl)-1 , 1 ,3,3-tetramethyla iniu tetrafluoroborate

Tee: trichloroethyl

Teoc: 2-(Trimethylsilyl)ethoxycarbonyl

TFA: trifluoroacetic acid

THF: tetrathydrofuran

TMSOTf: trimethylsilyl trifluoromethanesulfonate

TOMBU: N-{[1 ,3-Dimethyl-2,4,6-trioxotetrahydropyrimidin-5(6H)- ylidenaminooxy](dimethylamino)methylen}-N-methylmethanaminiu mhexafluorophosphate