WO2009047354A1 - Chemo-enzymatic synthesis of a c-terminal thioester of an amino acid or a peptide - Google Patents

Abstract

The present invention relates to a method for synthesising a C-terminal thioester of an N-protected amino acid or an optionally N-protected oligopeptide, comprising reacting a thiol with a compound selected from optionally N-protected amino acids, amino acid C-terminal esters (other than the ester to be synthesised) and optionally N-protected oligopeptide C-terminal esters (other than the ester to be synthesised) in the presence of a hydrolytic enzyme, thereby forming the thioester.

Description

CHEMO-ENZYMATIC SYNTHESIS OF A C-TERMINAL THIOESTER OF AN AMINO ACID OR A PEPTIDE

The invention relates to a method for synthesising a C-terminal thioester of an amino acid or a peptide.

C-terminal thioesters of amino acids or peptides are useful in peptide synthesis, for instance for synthesising peptides that may be used as a pharmaceutical, a food or feed ingredient, an agrochemical or as an ingredient for a cosmetic product. In nature, thioesters are used in non-ribosomal peptide synthesis (see, for instance, Marahiel, M.A., Chem. Rev., 1997, 97, 2651 ). Additionally, thioesters of amino acids or peptides can serve as substrates for enzymatic coupling (see, for instance, Lin, H. et al, Chemistry & Biology, 2004, 1 1 , 1635) or as reagent in chemical ligation strategies such as i) the Staudinger ligation (see, for instance, Nilsson, B. L. et al, J. Am. Chem. Soc, 2003, 125, 5268); ii) Native chemical ligation (NCL) (see, for instance, Dawson, P. E., Muir, T.W., Clark-Lewis, I. and Kent, S. B. H., Science, 1994, 266, 776 and Dawson, P. E. and Kent, S. B., Annu. Rev. Biochem., 2000, 69, 923); iii) in case of trimethoxybenzyl thioesters in thioacid/(sulfonyl)azide amidation reactions (Shangguan, N. et al, J. Am. Chem. Soc, 2003, 125, 7754). Additionally, peptide thioesters can be very mildly converted to the corresponding aldehyde or ketone (Fukuyama et al, Aldrichimica acta, 2004, 37, 3, 87) that find application in oxime and hydrazone ligation strategies (Borgia, J.A. et al, TIBTECH, 18, 2000, 243).

Chemoselective (bio)conjugation or ligation reactions are of high interest since they facilitate the chemical synthesis of large and complex biomolecules such as proteins and peptides. As indicated above, Kent and co-workers developed the NCL. In this method polypeptides are synthesized by the condensation of unprotected or partially protected segments, one containing a C-terminal thioester and the other containing an N-terminal cysteine residue. The reaction usually takes place under aqueous (physiological) conditions at about neutral pH to give a near quantitative yield of a single linked product, as shown below. Trans thioesteπfication

The potential of NCL for the introduction of non-proteinogenic amino acids and biophysical tags into peptides and proteins as well as preparation of post- translationally modified proteins has recently been reviewed by David et. al. (in "David, R., Richter, MP. and Beck-Sickinger, A.G., Eur. J. Biochem., 2004, 271 , p. 663").

According to David et. al., the bottleneck in NCL is the generation of peptide C-terminal thioesters, of which the synthesis in solution requires activation of the C-terminus of the peptide for reaction with a suitable thiol. In order to all prevent side reactions, the activated peptide fragment should be completely protected. This limits its solubility and thus makes this method impractical to use.

Usually, C-terminal peptide thioesters are synthesized by solid-phase peptide synthesis (SPPS) methods. SPPS is routinely carried out by either of the following two procedures: the Boc (f-butyloxycarbonyl)/benzyl method (Merrifield) and the Fmoc (9-fluorenylmethoxycarbonyl)/ft-Bu (Sheppard) method. Solid phase synthesis is in general not attractive for production on a large scale.

In the Boc/benzyl method ΛT-deprotection is achieved by treatment with trifluoro acetic acid (TFA). Cleavage from the resin and side chain deprotection relies on harsh conditions (in HF), which makes the method not suitable for the synthesis of acid-sensitive peptides and derivatives such as phospho-peptides (Huse, M., et al., J. Am. Chem. Soc, 2000, 122, p. 8337) and glyco-peptides (Shin et al. J. Am.Cham Soc, 1999, 121 , p1 1684)

The Fmoc/f-Bu method offers complete orthogonality in the Λ/° and side chain protection with Fmoc and f-butyl respectively, since the former is cleaved under alkaline conditions by treatment with piperidine and the latter can be removed under acidic conditions by treatment with TFA. Although the Fmoc/f-Bu method offers more flexibility for modification of the peptide chain, its application to the synthesis of base-labile peptide thioesters is rather limited. Consequently, most of the strategies to obtain peptide thioesters have used the Boc benzyl strategy (Hacken, T. M., Griffin, J. H., Dawson, P.E., Proc. Natl. Acad. Sci. USA, 1999, 10068). Various attempts have been made in the synthesis of peptide

C-terminal thioesters using the Fmoc/f-Bu method. These methods mostly rely on adapting the cleavage cocktail to make it compatible with C-terminal peptide thioesters (Li, X.Q., Kawakmi, T. and Aimoto, S., Tetrahedron Lett., 1998, 39, p. 8669), cleavage of a partially protected precursor peptide, followed by conversion to the corresponding C-terminal peptide thioester in solution (Futaki, S., Sogawa, K., Maruyama, J., Asahara, T. and Niwa, M., Tetrahedron Lett., 1997, 38, p. 6237) and employing special linkers such as the backbone amide linker (BAL) (Alsina, J., Yokum, T. S., Albericio, F. and Barany, G., J. Org. Chem., 1999, 64, p. 8761 ) or the N-acyl sulfonamide linker (Shin, Y., Winans, K.A., Backes, B.J., Kent, S. B. H., Ellman, J.A. and Bertozzi, C. R., J. Am. Chem. Soc, 1999, 121 , p. 11684). However, none of these Fmoc/ ^fBu methods mentioned are as robust as Boc/Benzyl, i.e. the effectivity of these methods tends to be easily affected by relatively small changes in the reaction conditions. Moreover, the need for special linkers, resins and/or complicated procedures remains a general limitation (see e.g. Sewing, A., Hilvert, D., Angew. Int. Ed., 40, 2001 , 3395). In addition, in general chemical methods of peptide synthesis may cause racemisation, especially when activated esters and/or strong bases are used.

Tan et al. (ChemBioChem 2007, 8, 1512-1515) propose an enzymatic approach, requiring a transesterification reaction (using an alkyl ester as a substrate) in an aqueous environment. A specifically engineered subtilisin is needed which shows an increased synthesis/hydrolysis ratio. Relatively high hydrolysis rates are observed.

In an alternative approach for the C-terminal thioester synthesis of peptides, ribosomal fermentation is applied using a bacterial expression system based on the intein mediated self-splicing mechanism of precursor proteins (Muir, T. W., Sondhi, D. and Cole, P.A., Proc. Natl. Acad. Sci. USA 1998, 95, p. 6705.). Although, in principle, peptide thioesters can be obtained in high yields and purities, this method is only efficient for large peptides and is restricted in the choice of the amino acid residue at the C-terminus of the C-terminal peptide thioester (Muralidharan, V., Muir. T. W., Nature Methods, 3, 2006, 429). Additionally, the introduction of non-proteinogenic amino acids and biophysical tags other than isotopically labeled amino acids, is usually not possible using ribosomal fermentation for the synthesis of peptides.

Further, peptide synthesis based on intein splicing is generally not feasible on an industrial scale, at least not in an efficient manner.

To gain more synthetic flexibility, methods for the selective conversion of unprotected peptides to their corresponding peptide C-terminal thioesters are desirable. Such methods would make the conversion to the C-terminal peptide - A -

thioester independent of the method for production of the peptide sequence. This means that the synthesis of peptide C-terminal thioesters is, for instance, not restricted by the base-sensitivity of the thioester moiety and thus allows for a variety of strategies to obtain peptide C-terminal thioesters. It has now been found possible to prepare a C-terminal thioester of an amino acid or a peptide by esterifying an N-protected amino acid or an optionally N-protected peptide or by transesterifying a C-terminal ester or thioester of an N- protected amino acid or optionally N-protected peptide with a thiol using a specific bio-catalyst. Accordingly, the present invention relates to a method for synthesising a C-terminal thioester of an N-protected amino acid or an optionally N-protected peptide, comprising reacting a thiol with a compound selected from N-protected amino acids, optionally N-protected peptides, N-protected amino acid C-terminal esters, optionally N-protected peptide C-terminal esters, N-protected amino acid C-terminal thioesters - other than the thioester to be synthesised - and optionally N-protected peptide C-terminal thioesters - other than the thioester to be synthesised - in the presence of a hydrolytic enzyme (E. C. 3).

In case the thioester is an N-protected C-terminal thioester, the N-protective may thereafter be removed, if desired. The term "ester" is used herein solely for oxo-esters, unless specified otherwise.

When referred to the term thiol, a salt of the thiol, in particular a salt of a monovalent cation and the deprotonated thiol, is meant to be included.

The compound that is to be reacted with the thiol may hereinafter be referred to as the 'substrate'.

The term 'transthioesterification' is used for the conversion of an ester into a thioester or a first thioester into a second thioester.

A peptide used in a method of the invention may be an oligopeptide or a polypeptide. For purpose of this invention, with "peptide" is meant any chain of two or more amino acids. For purpose of this invention, with "oligopeptide" is meant a peptide based on 2-200 amino acids, in particular based on 2-100, more in particular based on 2-50 amino acids, preferably any linear chain of 2-200 amino acids, more preferably of 2-100, 2-50 , 2-20 or 2-10 amino acids. The term "polypeptide" is in particular used for peptide based on more amino acids than an oligopeptide, as defined herein. A protein can be a single peptide molecule or a complex comprising a plurality of peptide molecules (which may be covalently or non covalently linked).

The chemo-enzymatic synthesis of C-terminal amino acid thioester or peptide thioesters allows for great flexibility in peptide synthesis.

For instance, the invention may be employed using an amino acid or peptide (or ester/thioester thereof) of which the side-chains or at least the majority thereof is unprotected. This allows fine-tuning of its solubility.

Further, peptides synthesized by a variety of chemical (and biological) methods can be used to obtain their corresponding C-terminal peptide thioesters. These C-terminal peptide thioesters may in particular be used in native chemical ligation for coupling a peptide to, amongst others, another peptide, to a microarray (e.g. for screening for a biological function of a peptide, such as binding to a specific biomolecule; or for diagnostic purposes); to a multivalent scaffold (i.e. a moiety with a plurality of binding sites for peptides, for instance a dendrimer, a (hyperbranched) polymer or another molecule comprising a plurality of functional groups to which the peptide can be coupled). See e.g. Yeo, D. S., Srinivasan, R., Chen, G.Y. and Yao, S. Q., Chemistry, 2004, 10, p. 4664 or Baal et al, Angew. Chem. Int. ed., 44, 2005, 5052.

Further, in principle, any amino acid (or ester thereof) or oligopeptide (or ester thereof) can be converted into a thioester. The amino acid or oligopeptide can be a proteinogenic or a non-proteinogenic amino acid or oligopeptide. Proteinogenic amino acids are the amino acids that may be found in proteins and that are coded for by the standard genetic code. The proteinogenic amino acids are glycine, L-alanine, L-valine, L-leucine, L-isoleucine, L-serine, L-threonine, L-methionine, L-cysteine, L-asparagine, L-glutamine, L-tyrosine, L-tryptophan, L-aspartic acid, L-glutamic acid, L-histidine, L-lysine, L-arginine, L-proline and L-phenylalanine.

Examples of non-proteinogenic amino acids include D-stereo isomers of proteinogenic amino acids, phenylglycine and 4-fluoro-phenylalanine.

It is surprising that the (trans)thioesterification reaction can efficiently be accomplished with a hydrolytically active enzyme. Even though some hydrolytically active enzymes have been shown to have some catalytic activity with respect to hydrolysing C-terminal thioesters, it is surprising that the thermodynamically unfavourable opposite thioesterification reaction, or the transesterification of an ester to a thioester, can take place efficiently. It is particularly surprising that a hydrolytically active enzyme is capable of catalysing the transthioesterification of an ester to a thioester. Such activity has not been described previously. From the capability of a hydrolytic enzyme, known to be capable of catalysing the hydrolysis of an ester, the skilled person would have no reason to expect a catalytic activity with respect to the transthioesterification of an ester to a thioester.

Moreover, it is surprising that such a hydrolytic enzyme has good activity, for sufficient time to allow substantial (trans)thioesterification of a substrate in the presence of the thiol, since thiols are known to be capable of deactivating and/or destabilising at least a number of enzymes. The inventors contemplate that in particular an enzyme without any cystein units in the enzyme sequence, or at least without any cystein units in an hydrolytically active site of the enzyme, may advantageously be used.

It is especially surprising that a conversion to the thioester of more than 45 % may be realised within a reasonable time span, for instance within about two days or within about a week. More in particular it is surprising that a conversion of at least 50 % or at least 70 % is possible within a reasonable time span. At least in a number of embodiments, if desired, the conversion may be at least 80 %, at least 90 %, or at least 95 %.

The method of the invention is further advantageous in that it is highly regioselective. Thus, also in case the substrate comprises one or more carboxylic acid side chain moieties, it is possible to selectively thioesterify a C-terminal carboxylic acid side chain moieties, whereas the carboxylic side chains are not thioesterified in a detectable amount, or at least to a lesser extent. Examples of more carboxylic acid side chain moieties are free carboxylic acids, amides and carboxylic acid esters.

Further, in accordance with the invention racemisation as a result of forming the thioester, usually is substantially avoided. It is further advantageous that the method of the invention can be carried out with a high conversion, whilst the amino acid (ester) or peptide (ester) and the thiol are in a liquid phase.

The term 'hydrolytic enzyme' is used herein for enzymes from the classification group E. C. 3. As an hydrolytic enzyme, in principle any hydrolytic enzyme capable of catalysing the (trans)thioesterification under the reaction conditions - in particular in the presence of a thiol - alone or in a combination of enzymes can be used. In particular, the hydrolytic enzyme may be an enzyme classifiable in EC 3.1 (enzymes acting on ester bonds) or as a peptidases (E. C. 3.4).

Preferably, one or more hydrolytic enzymes are used selected from the group of lipases, carboxylic ester hydrolases (E. C. 3.1.1 ), thioester hydrolases (E. C. 3.1.2) and peptidases (E. C. 3.4).

A lipase may in particular be used for thioesterifying an aliphatic amino acid, or a C-terminal aliphatic amino acid residue, for instance (N-terminal protected) alanine or a C-terminal alanine residue. A particularly preferred lipase is a lipase from Candida, especially a Candida antarctica lipase, of which lipases Candida antarctica lipase B (CAL-B) is particularly preferred.

In particular a peptidase (E. C. 3.4) may be used. Preferred peptidases are peptidases selected from the group of serine-type carboxypeptidases (E. C. 3.4.16), metallocarboxypeptidases (E. C. 3.4.17), cysteine-type carboxypeptidases (E. C. 3.4.18), serine endopeptidases (E. C. 3.4.21 ), cysteine endopeptidases (E. C. 3.4.22), aspartic endopeptidases (E. C. 3.4.23) and metalloendopeptidases (E. C. 3.4.24), in particular from serine endopeptidases (E.C. 3.4.21 ).

A serine endopeptidase may in particular be selected from the group of trypsin (E.C. 3.4.21.4), α-chymotrypsin (E.C. 3.4.21.1 ) and hydrolytically active mutants thereof.

In particular good results have been achieved with a subtilisin (E.C. 3.4.21.62), such as subtilisin Carlsberg.

The hydrolytic enzyme may be used in any form. For example, the hydrolytic enzyme may be used - for example in the form of a dispersion, emulsion, a solution or in immobilized form (for instance loaded on a support, e.g. a particulate or monolithic carrier material) - as crude enzyme, as a commercially available enzyme, as an enzyme further purified from a commercially available preparation, as an enzyme obtained from its source by a combination of known purification methods, in whole (optionally permeabilized and/or immobilized) cells that naturally or through genetic modification possess hydrolytic activity, or in a lysate of cells with such activity. The hydrolytic enzyme may be obtained or derived from any organism, in particular from an animal, plant, bacterium, a mould, a yeast or fungus. It will be clear to the average person skilled in the art that use can also be made of mutants of naturally occurring (wild type) enzymes with hydrolytic activity in the process according to the invention. Mutants of wild-type enzymes can for example be made by modifying the DNA encoding the wild-type enzymes using mutagenesis techniques known to the person skilled in the art (random mutagenesis, site-directed mutagenesis, directed evolution, gene shuffling, etc.) so that the DNA encodes an enzyme that differs by at least one amino acid from the wild-type enzyme or so that it encodes an enzyme that is shorter compared to the wild-type and by effecting the expression of the thus modified DNA in a suitable (host) cell. Mutants of the hydrolytic enzyme may have improved properties, for instance with respect to one or more of the following aspects: selectivity towards the substrate, activity, stability, solvent resistance, pH profile, temperature profile, substrate profile.

When referred to an enzyme from a particular source, recombinant enzymes originating from a first organism, but actually produced in a (genetically modified) second organism, are specifically meant to be included as enzymes from that first organism. Examples of organisms from which the enzyme may be derived include Trichoderma sp, such as from Trichoderma reesei; Rhizopus sp., such as from Rhizopus oryzae; Bacillus sp, such as from Baccillus licheniformis, Bacillus subtilis Bacillus amyloliquefaciens, Bacillus clausii, Bacillus lentus, Bacillus alkalophilus, Bacillus halodurans; Aspergillus sp., such as from Aspergillus oryzae or Aspergillus niger, Streptomyces sp., such as from caespitosus Streptomyces or Streptomyces griseus; Candida sp.; fungi; Humicola sp; Rhizoctonia sp.; Cytophagia; Mucor sp.; and animal tissue, in particular from pancreas, such as from porcine pancreas, bovine pancreas or sheep pancreas.

As indicated above, a preferred enzyme is subtilisin. Various subtilisins are known in the art, see e.g. US 5,316,935 and the references cited therein.

Also a hydrolytic enzyme as described in WO 2007/082890 may be used, such as BsubpNBE or CAL-A.

As indicated above, subtilisin Carlsberg is a particularly suitable enzyme for use in accordance with the invention. It has surprisingly been found that the reaction can efficiently be carried out by using Alcalase®, available from Novozymes (Bagsvaerd, Denmark). Alcalase is a cheap and industrially available proteolytic enzyme mixture produced by Bacillus licheniformis (containing subtilisin Carlsberg as a major enzyme component). From an experiment with pure subtilisin, the inventors deducted that subtilisin is an active enzyme in a method of the invention.

Commercially available enzyme, such as Alcalase, may be provided by the supplier as a liquid, in particular an aqueous liquid.

Another preferred subtilisin is subtilisin A, which is available from Novozymes. Other suitable hydrolytic enzymes may be selected from the group of the following commercially available products, and functional analogues of such enzymes.

Novozymes (Bagsvaerd, Denmark) offers ovozyme, liquanase, Alcalase, Alcalase-ultra® (in particular effective at alkaline pH), duramyl, esperase, kannase, savinase, savinase ultra, termamyl, termamyl ultra, novobate, polarzyme, neutrase, novoline, pyrase, novocor (bacterial alkaline proteases).

Proteinase-K is available from New England Biolabs, Ipswich (MA), USA).

Novo Nordisk Biochem North America lnc (Franklinton NC, USA) offers Protease Bacillus species (Esperase 6.0 T; Savinase 6.0 T), Protease Bacillus subtilis (Neutrase 1.5 MG), Protease Bacillus licheniformis (Alcalase 3.0 T).

Amano International Enzyme Co (Troy, Va, USA) offers Protease Bacillus subtilis (Proleather; Protease N) and Protease Aspergillus oryzae (Prozyme 6).

In case the enzyme is provided as an aqueous liquid, the enzyme is preferably first isolated from undesired liquid. Such liquid in particular includes water and alcohols that may be detrimental to the reaction (which may compete with the thiol in the reaction). In particular, it is preferred to isolate the enzymes from water, primary alcohols and secondary alcohols, if present.

This may suitably be accomplished by precipitating and/or drying. Precipitation may be accomplished using an alcohol, such as f-butanol, a thiol used in the method of the invention, another thiol or another organic liquid, e.g. an ether which ether may be used as reaction medium wherein the (trans)thioesterification can be carried out. In case an alcohol or another thiol than the thiol used for the (trans)thioesterification, is used for the precipitation of the enzyme, care should be taken that the alcohol or thiol is inert with respect to the (trans)thioesterification reaction in a method of the invention or is at least substantially removed, before carrying out such reaction.

The enzyme may thereafter by resuspended in a suitable liquid, for instance a thiol used in the method of the invention or inert medium, i.e. a medium which does not react with the carboxylic moiety to be (trans)thioesterified, yet wherein the (trans)thioesterification can be carried out). Such media are described in more detail, below.

The amount of enzyme used may be chosen within wide limits, depending upon its catalytic activity under reaction conditions, the desired conversion and the desired reaction time. Usually, the amount is at least 0.001 wt. %, at least 0.01 wt. %, at least 0.1 wt. %, at least 1 wt. %, at least 10 wt. % or at least 20 wt. %, based on the weight of the substrate. For practical reasons the amount is usually 1000 wt. % or less, in particular 750 wt. % or less, 500 wt. % or less, 200 wt. % or less, 100 wt. % or less, or 25 wt. % or less, based on the weight of the substrate.

The amino acid (which is N-protected, at least during the reaction), the amino acid C-terminal ester (which is N-protected, at least during the reaction), the optionally N-protected oligopeptide, or the optionally N-protected oligopeptide C-terminal ester may in particular be represented by a compound of formula I.

Herein P represents H or an N-terminal protecting group. Preferably, P represents an N-terminal protecting group, because it is in general desired to avoid a side-reaction such as competition of the terminal amino group with the thiol. In particular, in case n is 1 , the N-protective group is generally needed to allow the (trans)thioesterification to proceed well. In principle, the integer n can have any value of 1 or more. In particular, n can be at least 2, at least 5, at least 10, at least 15, or at least 20. The integer n can be 200 or less, 100 or less, 75 or less, 60 or less, 30 or less, 20 or less, or 10 or less.

Each R^A and each R^B independently represent H, or an organic moiety preferably an amino acid side chain. Thus, it is not required that R^A is the same in all n amino acid units. Similarly, it is not required that R^B is the same in all n amino acid units.

X represents a hydrogen, a cation - preferably a monovalent cation, such as Na⁺ or K⁺ - or an organic moiety. Within the context of the present invention the term "organic moiety" is in particular meant to include linear or branched, optionally substituted alkyl, alkenyl or alkynyl groups; optionally substituted cycloalkyl groups which optionally have one or more unsaturated, exocyclic or endocyclic, carbon carbon bonds; optionally substituted aryl groups; and optionally substituted aralkyl groups. Alkyl groups may in particular comprise 1-12 carbons. Alkenyl or alkynyl groups may in particular comprise 2-12 carbons. Cycloalkyl groups or aryl groups may in particular comprise 4-12 carbons. Aralkyl groups may in particular comprise 5-13 carbons. An alkyl, alkenyl or alkynyl group optionally comprises one or more heteroatoms in a chain thereof. A cycloalkyl, aryl or aralkyl group optionally comprises one or more heteroatoms in a ring thereof. A heteroatom may in particular be selected from the group of S, O and N. If substituted, an organic moiety may in particular be substituted with optionally protected, functional groups comprising one or more heteroatoms, which heteroatoms may in particular be selected from the group of O, A and N. The functional group may in particular be selected from unprotected or protected hydroxy groups, unprotected or protected thio groups and unprotected or protected amine groups.

Suitable organic moieties for X in a compound of Formula I may in particular be activating groups, Ae. groups facilitating thioesterification. Examples of activating groups are optionally substituted phenyl, optionally substituted benzyl and n-alkyl groups, such as methyl, ethyl or n-propyl. Further, suitable organic moieties may be selected from protecting groups, Ae. groups that protect the C-terminus of an amino acid or peptide during the chemical or enzymatic formation of a peptidic bond. Examples of activating groups are f-alkyl esters such as f-butyl.

In a particularly preferred method of the invention, X represents a hydrogen, methyl or benzyl. The use of an N-protected amino acid, or an optionally

N-protected peptide (in case of a compound according to formula I: X=H) is specifically preferred.

Suitable N-protecting groups are those N-protecting groups which can be used for the synthesis of (oligo)peptides. Such groups are known to the person skilled in the art. Examples of suitable N-protecting groups include carbonyl type protective groups, for instance Z (benzyloxycarbonyl), Boc (f-butyloxycarbonyl), For (formyl) and PhAc (phenacetyl). The groups For or PhAc may be introduced and cleaved enzymatically using the enzymes Peptide Deformylase and PenG acylase, respectively. Chemical cleavage methods are generally known in the art. In the context of the invention with 'amino acid side chain' is meant any proteinogenic or non-proteinogenic amino acid side chain. The reactive groups in the amino acid side chains may be protected by amino acid side chain protecting groups or may be unprotected.

The thiol may form part of a molecule bound to a support, such that the resultant thioester is immobilised on the support or the thiol may be a free molecule. In particular, the thiol may be represented by the formula Y-S-Z, wherein Z presents hydrogen or a cation, preferably a monovalent cation, in particular Na+ or K+, and wherein Y represents hydrogen or an organic moiety. In case Y is an organic moiety, Y may in particular be an organic moiety as defined above. In particular the thiol may be selected from the group of benzyl mercaptane (BnSH), 2, 4, 6 trimethoxy- benzylmercaptane methyl mercaptane, ethyl mercaptane, 3-mercaptopropionic acid ethylester, optionally P-protected diphenylphosphinomethyl mercaptane, and N-acetyl- cysteamine. In case Y comprises an aryl group it is preferred that the -SZ is not directly attached to the aryl group of Y. E.g., in case Y comprises an aralkyl group, -SZ is preferably attached to the alkyl function.

Optionally P-protected diphenylphosphinomethyl mercaptane may advantageously be used for providing a thioester that can be used in a Staudinger ligation.

N-acetyl-cysteamine may in particular be used to prepare a thioester that can serve as an intermediate in the preparation of a cyclic peptide.

In a particularly preferred method of the invention, the thiol is selected from benzyl mercaptane and 3-mercaptopropionic acid ethylester. These have been found particularly suitable for providing a thioester that can for instance be used in a native chemical ligation. Usually, a method of the invention is carried out using a reaction medium wherein the thiol for the (trans)thioesterification is used in molar excess relative to the substrate. The thiol may even be the major solvent. The reaction medium is usually essentially free from other thiols or alcohols that may be detrimental to the reaction (which may compete with the thiol in the reaction). In practice a trace amount of other thiols or alcohols may be present, for instance less than 1 wt. %, in particular less than 0.5 wt. %, more in particular less than 0.1 wt. %, based on the total weight of liquids in the phase wherein the reaction takes place. One or more other solvents that may be present include inert organic solvents, such as ethers, for instance methyl tert-butyl ether (MTBE); tetrahydrofuran (THF); aromatic hydrocarbons, such as toluene; and polar aprotic solvents, such as N, N dimethyl formamide (DMF),

N-methylpyrrolidinone (NMP), dimethyl sulfoxide (DMSO), acetone, dimethoxyethane, acetonitrile and the like.

A method of the invention is usually carried out under substantially non-aqueous conditions. As the skilled person will understand, a small amount of water may be desired, depending upon the enzyme, to enable the enzyme to properly perform its catalytic activity.

The reaction medium usually contains less than 10 wt. % water, based on the total weight of liquids in the phase wherein the reaction takes place. The reaction medium may be dispersed in a second liquid phase or another liquid phase may be dispersed in the reaction medium. In case of a dual or multiphase system, the specified water content is based on the weight of liquids in the phase wherein the (trans)thioesterification reaction (at least predominantly) takes place.

In particular - at least at the beginning of the thioesterification reaction - the water concentration may be less than 4 wt. %. Advantageously, a method may be carried out in a phase containing - at least at the beginning of the thioesterification reaction - less than 2 wt. % water, in particular 1 wt. % or less water, more in particular 0.5 wt. % or less water, for instance about 0.2 wt. % or less water, whilst still retaining substantial desired enzyme activity and a low, or even undetectable undesired hydrolysis.

For a good catalytic activity the presence of a trace of water, e.g. of at least 0.01 wt. %, based on the liquid phase, may be desired. In particular, the water concentration may be at least 0.02 wt. % or at least 0.05 wt. %.

In principle the pH used may be chosen within wide limits. It may in particular be chosen to be about neutral. If desired, alkaline or acidic conditions may be used, depending on the enzyme. If desired, the pH may be adjusted using an acid and/or a base or buffered with a suitable combination of an acid and a base. Suitable acids and bases are in particular those soluble in the reaction medium, e.g. from the group of ammonia and alcohol-soluble acids, e.g. acetic acid and formic acid. The pH of the reaction may be controlled by using an automated pH-stat system. Optimal pH conditions can easily be identified by a person skilled in the art through routine experimentation.

In principle the temperature used is not critical, as long as the enzyme shows substantial activity. Generally, the temperature may be at least 0 ⁰C, in particular at least 15 ⁰C. A desired maximum temperature depends upon the enzyme. In general such maximum temperature is known in the art, e.g. indicated in a product data sheet in case of a commercially available enzyme, or can be determined routinely based on common general knowledge and the information disclosed herein. The temperature is usually 70⁰C or less, in particular 60⁰C or less or 50 °C or less. In particular if a thermophilic hydrolytic enzyme is used, the temperature may be chosen relatively high, for instance in the range of 40 to 90⁰C.

Optimal temperature conditions can be identified for a specific enzyme by a person skilled in the art through routine experimentation, based on common general knowledge and the information disclosed herein. For instance, for subtilisin, in particular subtilisin Carlsberg (e.g. in Alcalase) the temperature may advantageously be in the range of 25-60⁰C.

In an advantageous method, an alcohol or water that is formed, in particular during (trans)esterification, may be removed continuously or intermittently. In principle removal may be accomplished in a manner known in the art. Good results have in particular been achieved using molecular sieves. Also very suitable for the removal is evaporation, such as azeotropic removal using vacuum or distillation. As indicated above a C-terminal amino acid or peptide thioester obtained in accordance with the invention may be used in peptide synthesis. Accordingly, the invention further relates to the use of a thioester obtained in a method according to the invention in the manufacture of an oligopeptide or polypeptide. A chemical method, an enzymatic method or a combination may be used. In particular, chemical ligation, may be used. The chemical ligation may in particular be a native chemical ligation. The chemical ligation may in principle be accomplished in a manner known per se, e.g. as described in "Dawson, P. E., Muir, T. W., Clark-Lewis, I. and

Kent, S. B. H., Science, 1994, 266, p. 776", "Dawson, P.E. and Kent, S. B., Annu. Rev.

Biochem., 2000, 69, p. 923, or Hacken, T.M., Griffin, J. H., Dawson, P. E., Proc. Natl.

Acad. Sci. USA, 1999, 10068 The invention will now be illustrated by the following examples.

EXAMPLES

General Unless stated otherwise, chemicals were obtained from commercial sources and used without further purification. Benzylmercaptane (BnSH) was obtained from Sigma Aldrich. ¹H NMR spectra were recorded on a Bruker Avance 300MHz NMR (300.1 MHz) spectrometer and chemical shifts are given in ppm (δ) relative to CDCI₃ (7.26 ppm) or D₂O (4.79 ppm). Thin layer chromatography (TLC) was performed on pre-coated silica gel 60 F₂₅₄ plates (Merck); spots were visualized using UV light, ninhydrin or CI₂/TDM (N,N,N',N'-tetramethyl-4,4'-diaminodiphenylmethane).

Unless stated otherwise, 3A molsieves (8 to 12 mesh, Acros) were activated under reduced pressure at 200⁰C and f-butanol (^fBuOH) was stored on these molsieves. ^fBuOH was pre-heated to a liquid (45°C) before use. Column chromatography was carried out using silica gel, Merck grade 9385 60 A. Analytical HPLC was performed on an HP1090 Liquid Chromatograph with a reversed-phase column (Inertsil ODS-3, C18, 5μm, 150 * 4.6 mm) at 40⁰C using either an isocratic mixture of 25% eluent A (0.05 vol% methanesulphonic acid in H₂O) and 75% eluent B (0.05 vol% methanesulphonic acid in CH₃CN) during 25 min at a flow rate of 1 mL/min (method 1 ) or a linear gradient of 95% eluent A (0.05 vol% methanesulphonic acid in H₂O) to 98% eluent B (0.05 vol% methanesulphonic acid in CH₃CN) in 25 min at a flow rate of 1 mL/min (method 2). UV detection was performed at 220 nm using a UV-VIS 204 Linear spectrometer. HPLC-MS experiments were performed on an SCIEX API 150 UV-LC-MS system (Applied Biosystems, MSD-SCIEX, Canada), which consists of a binary pump, degasser, autosampler, column oven, diode-array detector and a time- of-flight-MS. The ESI-MS was run in positive mode, with the following conditions: m/z 100-900, 10V declustering potential, 1.005 cycl/sec, 350 ⁰C drying gas temperature, 10 L N₂/min drying gas and 5 kV capillary voltage. The UV detection was performed at 210 nm. The exact mass was determined using an internal reference to recalibrate the m/z axis for each measurement. The samples were directly introduced into the ESI by injection of 5 μL into the eluent flow of 0.5 mL/min. (linear gradient of 95% eluent A (0.01 vol% methanesulphonic acid in H₂O) to 98% eluent B (0.01 vol% methanesulphonic acid in CH₃CN) in 25 min).

Preparation ofAlcalase: 10 mL of Alcalase (Novozymes, Alcalase^® 2.5 L, type DX, PLN04810) was precipitated with 25 mL of 'BuOH, vortexed for 5 min and centrifuged at 4500 rpm for 10 min. The supernatant was discarded and the previously described procedure was repeated twice using ^fBuOH and twice using MTBE, providing a precipitated residue of Alcalase, comprising subtilisin Carlsberg. Preparation of Alcalase-CLEA method A: 1 g lyophilized crosslinked Alcalase-CLEA (CLEA-technologies, 650 AGEU/g, 3.5 wt% water) was suspended in 10 mL ^fBuOH and crushed with a spatula, isolated by filtration and resuspended once more in 10 mL ^fBuOH and isolated by filtration. This procedure was repeated twice with 10 mL portions of MTBE.

Preparation of Alcalase-CLEA method B: 1 g lyophilized crosslinked Alcalase-CLEA (CLEA-technologies, 650 AGEU/g, 3.5 wt% water) was suspended in 10 mL ^fBuOH and crushed with a spatula and isolated by filtration.

Preparation of Alcalase-CLEA method C: 300 mg lyophilized crosslinked Alcalase-CLEA (CLEA-technologies, 650 AGEU/g, 3.5 wt% water) was suspended in 5 mL MTBE and molsieves (3 A, 100 mg) were added. The mixture was shaken at 50⁰C for 16 h and the enzyme was subsequently isolated by filtration.

Preparation of Alcalase-CLEA method D: 300 mg lyophilized crosslinked Alcalase-CLEA (Codexis, 580 U/g), was suspended in 5 mL MTBE and molsieves (3 A, 100 mg) were added. The mixture was shaken at 50⁰C for 16 h and the enzyme was subsequently isolated by filtration. An immobilized form of Candida antarctica lipase-B (Novozymes, trade name: Novozym^® 435) was used without further purification.

EXAMPLE 1 : Synthesis of Z-Phe-SBn starting from Z-Phe-OMe

Chemical synthesis of the Z-Phe-SBn (reference example)

To an ice-cold solution of Z-Phe-OH (359 mg, 1.2 mmol) in CH₂CI₂ (50 mL) was consecutively added 234 mg (1.02 equiv) 1-(3-dimethylaminopropyl)-3- ethylcarbodiimide methiodide (EDCI), BnSH (117 μL, 1 equiv) and a catalytic amount (15 mg) of 4-dimethylaminopyridine. The reaction mixture was stirred for 16 h at ambient temperature and the solvent was removed in vacuo. The residue was purified by column chromatography (eluent: CH₂CI₂; R_f = 0.39;) and the resulting product (352 mg, 87%) was obtained as a clear oil that solidified on standing. R_t = 5.75 min (HPLC method 1 ). ¹H-NMR (CDCI₃, 300 MHz) 5 7.4-7.0 (m, Ph-H, 15H), 5.10 (br s, PhCH₂O + NH, 3H), 4.77 (m, αCH, 1 H), 4.12 (s, PhCH₂S, 2H), 3.13 (m, βCH₂, 2H).

Enzymatic reaction (invention)

iransthio- estenfication O .O ,- moisieves 3A ■[ H ^s

Z-Phe-Ofvte BnSH Z-Phe-SBn

Experiment a

Dry Alcalase (100 mg) was added to a mixture of Z-Phe-OMe (50 mg, 0.16 mmol), BnSH (3 mL, 160 equiv) and molsieves 3A (100 mg). The reaction mixture was stirred for 48 h at 20°C and HPLC indicated that the conversion to the thioester was >95% (as estimated from peak areas).

Experiment b Alcalase-CLEA (300 mg, prepared according to method A) was added to a mixture of Z-Phe-OMe (50 mg, 0.16 mmol), BnSH (3 mL, 160 equiv) and molsieves (3A) (100 mg). The reaction mixture was stirred for 48 h at 50°C and HPLC indicated that the conversion to the thioester was >95% (as estimated from peak areas).

Experiment c

Dry Alcalase (100 mg) was added to a mixture of Z-Phe-OMe (50 mg, 0.16 mmol), BnSH (3 mL, 160 equiv) and molsieves 3A (100 mg). The reaction mixture was stirred for 48 h at 50⁰C and HPLC indicated that the conversion to the thioester was >95% (as estimated from peak areas). Experiment d

Alcalase-CLEA (300 mg, prepared according to method A) was added to a mixture of Z-Phe-OMe (50 mg, 0.16 mmol), BnSH (189 μl_, 10 equiv) and molsieves (3 A; 100 mg) in MTBE (3 ml_). The reaction mixture was stirred for 48 h at 50⁰C and HPLC indicated that the conversion to the thioester was >95% (as estimated from peak areas). Subsequently, the reaction mixture was filtered over Decalite and the filtrate was concentrated in vacuo. The residue was purified by column chromatography (eluent: dichloromethane; R_f = 0.39) and the resulting product (47 mg, 72%) was obtained as a clear oil that solidified on standing. R_t = 5.75 min (HPLC method 1 ). The ¹ H-NMR spectrum (CDCI3) corresponded to that of the chemically synthesized reference compound Z-Phe-SBn.

Experiment e

Dry Alcalase (100 mg) was added to a mixture of Z-Phe-OMe (50 mg, 0.16 mmol), BnSH (189 μL, 10 equiv) and molsieves 3A (100 mg) in MTBE (3 mL). The reaction mixture was stirred for 48 h at 50⁰C and HPLC indicated that the conversion to the thioester was >95% (as estimated from peak areas).

Experiment f Alcalase-CLEA (300 mg, prepared according to method A) was added to a mixture of Z-Phe-OMe (50 mg, 0.16 mmol), BnSH (189 μL, 10 equiv) and molsieves (3A) (100 mg) in acetone (3 mL). The reaction mixture was stirred for 48 h at 50°C and HPLC indicated that the conversion to the thioester was 93% (as estimated from peak areas).

Experiment q

Alcalase-CLEA (300 mg, prepared according to method A) was added to a mixture of Z-Phe-OMe (50 mg, 0.16 mmol), BnSH (189 μL, 10 equiv) and molsieves (3A) (100 mg) in 1 ,2-dimethoxyethane (3 mL). The reaction mixture was stirred for 48 h at 50⁰C and HPLC indicated that the conversion to the thioester was >95% (as estimated from peak areas). EXAMPLE 2: Synthesis of Z-Phe-S-(CH_?)_?-CO_?Et from Z-Phe-OMe

Chemical synthesis of the Z-Phe-S-(CH?)7-CO?Et (reference example)

To an ice-cold solution of Z-Phe-OH (359 mg, 1.2 mmol) in CH₂CI₂ (50 ml_) was consecutively added EDCI (234 mg, 1.02 equiv), 3-mercaptopropionic acid ethyl ester (152 μl_, 1 equiv) and a catalytic amount of DMAP (15 mg). The reaction mixture was stirred for 16 h at ambient temperature. Thereafter, the solvent was removed in vacuo. The residue was purified by column chromatography (eluent: CH₂CI₂; R_f = 0.29) and the resulting product (234 mg, 47%) was obtained as a clear oil. Rf = 3.71 min (HPLC method 1 ). ¹H-NMR (CDCI₃) 5 7.24-7.03 (m, Ph-H, 10H), 5.15

(d, J = 8.6 Hz, (CO)NH, 1 H), 5.00 (s, PhCH₂O, 2H), 4.64 (m, αCH, 1 H), 4.06 (q, J = 7.1 Hz, OCH₂CH₃ 2H), 3.03 (m, CHCH₂Ph + SCH₂CH₂, 4H), 2.50 (m, SCH₂CH₂, 2H), 1.18 (t, J = 7.1 Hz₁ OCH₂CH₃).

Enzymatic reaction (invention)

Ax ^''J transihso-

O f ^""^"■"^■ ssterificatfon i H ii

imo sisves 3A :: i

Z-Pfw-OMe Z-Phe-S-<CH₂)₂COEt

Alcalase-CLEA (300 mg, prepared according to method A) was added to a mixture of Z-Phe-OMe (50 mg, 0.16 mmol), 3-mercaptopropionic acid ethyl ester (203 μL, 10 equiv) and molsieves (3 A, 100 mg) in MTBE (3 mL). The reaction mixture was stirred for 48 h at 50⁰C and HPLC-MS indicated that the conversion to the thioester was 73% (as estimated from peak areas).

EXAMPLE 3: Synthesis of Z-Phe-SEt from Z-Phe-OMe

2-Phs-OMe EtSH Z-Phe-SEt

Alcalase-CLEA (150 mg, prepared according to method A) was added to a mixture of Z-Phe-OMe (25 mg, 0.08 mmol), ethyl mercaptane (59 μL, 10 equiv) and molsieves (3 A, 100 mg) in 1 ,2-dimethoxyethane (1.5 ml_). The reaction mixture was stirred for 48 h at 50⁰C and HPLC indicated that the conversion to the thioester was 64% (as estimated from peak areas). R_t = 21.43 min (HPLC method 2).

EXAMPLE 4: Synthesis of Z-AIa-S-TMOB from Z-AIa-OMe trsnsthio- estefiϋcation

Z-AIa-OMe TMOB-SH Z-ASa-SBn

Alcalase-CLEA (150 mg, prepared according to method A) was added to a mixture of Z-AIa-OMe (1 1 mg, 0.05 mmol), 2,4,6-trimethoxybenzyl mercaptane (TMOB-SH) (50 mg, 5 equiv) and molsieves (3 A, 100 mg) in MTBE (1.5 mL). The reaction mixture was stirred for 96 h at 50⁰C and HPLC indicated that the conversion to the thioester was >95% (as estimated from peak areas). R_t = 21.51 min (HPLC method 2).

EXAMPLE 5: Synthesis of Z-Trp-SBn from Z-Trp-OMe

BnSH Z-Trp-SBr! Alcalase-CLEA (150 mg, prepared according to method A) was added to a mixture of Z-Trp-OMe (28 mg, 0.08 mmol), BnSH (94 μL, 10 equiv) and molsieves (3A) (100 mg) in MTBE (1.5 mL). The reaction mixture was stirred for 48 h at 50°C and HPLC-MS indicated that the conversion to the thioester was 20% (as estimated from peak areas). R_t = 22.88 min (HPLC method 2).

EXAMPLE 6: Synthesis of Z-Lys(Boc)-SBn from Z-Lys(Boc)-OMe

Z-LyS(BoC)-OMe BrtSH Z-Lys(Bocj-SBti

Alcalase-CLEA (150 mg, prepared according to method A) was added to a mixture of Z-Lys(Boc)-OMe (32 mg, 0.08 mmol), BnSH (94 μl_, 10 equiv) and molsieves (3A) (100 mg) in 1 ,2-dimethoxyethane (1.5 ml_). The reaction mixture was stirred for 48 h at 50⁰C and HPLC-MS indicated that the conversion to the thioester was 75% (as estimated from peak areas). R_t = 22.86 min (HPLC method 2).

EXAMPLE 7: Synthesis of Z-AIa-SBn from Z-AIa-OMe trartsthto- estefifi cast on O j f^:'^{" ~}T:

Z-AIa-OMe BnSH Z-AIa-SBn

Alcalase-CLEA (150 mg, prepared according to method A) was added to a mixture of Z-AIa-OMe (19 mg, 0.08 mmol), BnSH (94 μL, 10 equiv) and molsieves (3A) (100 mg) in MTBE (1.5 mL). The reaction mixture was stirred for 48 h at 50⁰C and HPLC indicated that the conversion to the thioester was 91 % (as estimated from peak areas). R₁ = 20.77 min (HPLC method 2).

EXAMPLE 8: Synthesis of Z-Phe-Phe-SBn from Z-Phe-Phe-OMe

+ MeOH

Alcalase-CLEA (300 mg, prepared according to method A) was added to a mixture of Z-Phe-Phe-OMe (74 mg, 0.16 mmol), BnSH (189 μL, 10 equiv) and molsieves (3 A, 100 mg) in 1 ,2-dimethoxyethane (3 mL). The reaction mixture was stirred for 48 h at 50°C and HPLC-MS indicated that the conversion to the thioester was 48% (as estimated from peak areas). R_t = 24.17 min (HPLC method 2).

EXAMPLE 9: Synthesis of Z-AIa-AIa-SBn from Z-AIa-AIa-OMe transthjβ- P : _H O βϋtenficatjαn O : _H

Is ! H ¹I : : ^; nwfelβves 3A : : μ ^|!

Z-Aϊa-Ala-OMiS 8n3H Z-A(a-Afa-S8π

Experiment a Alcalase-CLEA (150 mg, prepared according to method A) was added to a mixture of Z-AIa-AIa-OMe (25 mg, 0.08 mmol), BnSH (94 μL, 10 equiv) and molsieves (3A) (100 mg) in MTBE (1.5 mL). The reaction mixture was stirred for 48 h at 50⁰C and HPLC-MS indicated that the conversion to the thioester was 94% (as estimated from peak areas). R_t = 18.86 min (HPLC method 2).

Experiment b Candida antarctica lipase B (Novozym 435 (70 mg)) was added to a mixture of Z-AIa-AIa-OMe (25 mg, 0.08 mmol), BnSH (82 μL, 10 equiv) and molsieves (3 A, 100 mg) in MTBE (1.5 mL). The reaction mixture was stirred for 24 h at 50⁰C and HPLC indicated that the conversion to the thioester was 66% (as estimated from peak areas). R, = 18.87 min (HPLC method 2).

EXAMPLE 10: Synthesis of Boc-Ala-Ala-Phe-SBn from Boc-Ala-Ala-Phe-OMe »3flStRI0- ββsstlββπnfifieea«Ssko>n«

Boc-Aia-Aia-Phe-O-Wβ BnSM So; -ASa -ASa-PiW-SEn

Alcalase-CLEA (150 mg, prepared according to method A) was added to a mixture of Boc-Ala-Ala-Phe-OMe (34 mg, 0.08 mmol), BnSH (94 μL, 10 equiv) and molsieves (3A) (100 mg) in 1 ,2-dimethoxyethane (1.5 mL). The reaction mixture was stirred for 48 h at 50°C and HPLC indicated that the conversion to the thioester was 63% (as estimated from peak areas). R_t = 21.05 min (HPLC method 2).

EXAMPLE 1 1 : Synthesis of Z-Phe-SBn starting from Z-Phe-OH

eβfeiificafion iS ^ i SH mo ;te^■■t;eves ^" 3*A ^{+ Mβ0H}

Z -P⁽W-OH Bf)SH Z-Phe-SBn

Alcalase-CLEA (400 mg, prepared according to method B) was added to a mixture of Z-Phe-OH (50 mg, 0.16 mmol), BnSH (3 mL, 160 equiv) and molsieves (3 A, 100 mg). The reaction mixture was stirred for 24 h at 50⁰C and HPLC indicated that the conversion to the thioester was 75% (as estimated from peak areas). R_t = 4.89 min (HPLC method 1 ). EXAMPLE 12: Synthesis of Z-AIa-AIa-SBn starting from Z-AIa-AIa-OH sum

H '• WOiSiOVOS : H '

Z-ASa-AIa-OH BnSH Z-AIa-AIa-SBn

Experiment a

Alcalase-CLEA (300 mg, prepared according to method C) was added to a mixture of Z-AIa-AIa-OH (20 mg, 0.07 mmol), BnSH (82 μl_, 10 equiv) and molsieves (3A) (100 mg) in THF (1.5 ml_). The reaction mixture was stirred for 24 h at 50⁰C and HPLC-MS indicated that the conversion to the thioester was 88% (as estimated from peak areas). R_t = 18.87 min (HPLC method 2).

Experiment b

Novozym 435 (70 mg) was added to a mixture of Z-AIa-AIa-OH (24 mg, 0.08 mmol), BnSH (82 μL, 10 equiv) and molsieves (3 A, 100 mg) in MTBE (1.5 mL). The reaction mixture was stirred for 48 h at 50⁰C and HPLC-MS indicated that the conversion to the thioester was 77% (as estimated from peak areas). R₁ = 18.60 min (HPLC method 2).

EXAMPLE 13: Synthesis of Fmoc-Glu-Val-Pro-Ala-SBn starting from

Fmoc-Glu-Val-Pro-Ala-OH

O ^ OS- OH

O H O \

C o ^Q

O H Λ N

\ \ ^H 0 \

F mos 3 "-Vs! Pro Ma OH 1 Ab <Α Sn Alcalase-CLEA (300 mg, prepared according to method D) was added to a mixture of Fmoc-Glu-Val-Pro-Ala-OH (5 mg, 8 μmol), BnSH (900 μL, 1 100 equiv) and molsieves (3 A, 100 mg) in THF (1 mL). The reaction mixture was stirred for 24 h at 50°C and HPLC-MS indicated that the conversion to the thioester was 54% (as estimated from peak areas). R_t = 20.49 min (HPLC method 2). EXAMPLE 14: Synthesis of Z-AIa-SBn from Z-AIa-OH

Z =AIa OH BnSH I AU> SBn

Novozym 435 (70 mg) was added to a mixture of Z-AIa-OH (18 mg, 0.08 mmol), BnSH (82 μl_, 10 equiv) and molsieves (3 A, 100 mg) in MTBE (1.5 ml_). The reaction mixture was stirred for 24 h at 50⁰C and HPLC-MS indicated that the conversion to the thioester was 100% (as estimated from peak areas). R_t = 20.60 min (HPLC method 2).

EXAMPLE 15: Synthesis of Z-GIv-SBn from Z-GIv-OH thio- ,-^

O e&Eertficέtton O f' ϊ|

~._N ^ J^ - OH + ~_N , ^■- *^■ ^- ,K _'-•, S. Λv ) i j H ' moisiev^βs 3A [ i H '

Z-Gfy-OH BnSH Z GSy SBn Novozym 435 (70 mg) was added to a mixture of Z-GIy-OH (17 mg, 0.08 mmol), BnSH (82 μL, 10 equiv) and molsieves (3 A, 100 mg) in MTBE (1.5 mL). The reaction mixture was stirred for 24 h at 50⁰C and HPLC indicated that the conversion to the thioester was 100% (as estimated from peak areas). R_t = 19.64 min (HPLC method 2).

EXAMPLE 16: Synthesis of Z-Pro-SBn from Z-Pro-OH

O _^ ,OH t»Mo- O N S_x -^

O ^v" esteriϊieatiors O ^

\ / moteifeve& 3A ( \ ,

2-Prø-OH BfiSH Z Pro SBn

Novozym 435 (70 mg) was added to a mixture of Z-Pro-OH (20 mg, 0.08 mmol), BnSH (82 μL, 10 equiv) and molsieves (3 A, 100 mg) in MTBE (1.5 mL). The reaction mixture was stirred for 48 h at 50°C and HPLC-MS indicated that the conversion to the thioester was 57% (as estimated from peak areas). R_t = 22.19 min (HPLC method 2). EXAMPLE 17: Synthesis of Z-Met-SBn from Z-Met-OH

p

! H

Z-iVUsf-OH BnSH 2-Met-SBπ

Novozym 435 (70 mg) was added to a mixture of Z-Met-OH (23 mg, 0.08 mmol), BnSH (82 μl_, 10 equiv) and molsieves (3 A, 100 mg) in MTBE (1.5 ml_). The reaction mixture was stirred for 48 h at 50⁰C and HPLC-MS indicated that the conversion to the thioester was 78% (as estimated from peak areas). R_t = 22.08 min (HPLC method 2).

EXAMPLE 18: Synthesis of Z-AIa-SEt from Z-AIa-OH

Z-A!a-OH BSH Z-AIa-SEt Novozym 435 (70 mg) was added to a mixture of Z-AIa-OH (18 mg, 0.08 mmol), BnSH (60 μL, 10 equiv) and molsieves (3 A, 100 mg) in MTBE (1.5 mL). The reaction mixture was stirred for 24 h at 50⁰C and HPLC indicated that the conversion to the thioester was 98% (as estimated from peak areas). R_t = 17.97 min (HPLC method 2).

EXAMPLE 19: Synthesis of Z-AIa-SEt from Z-AIa-OMe transthio- esierification + — SH mo -iS(6ves ^*3A ^{+ M90H}

Z-AIa-OMe EtSH 2-AIa-SEt

Novozym 435 (70 mg) was added to a mixture of Z-AIa-OMe (19 mg, 0.08 mmol), BnSH (60 μL, 10 equiv) and molsieves (3 A, 100 mg) in 1 ,2-dimethoxyethane (1.5 mL). The reaction mixture was stirred for 24 h at 50°C and HPLC indicated that the conversion to the thioester was 98% (as estimated from peak areas). R_t = 17.97 min (HPLC method 2). EXAMPLE 20: Synthesis of Z-AIa-SBn from Z-AIa-OMe

Z Ate OMa BnSH Z Ala SBn

Novozym 435 (70 mg) was added to a mixture of Z-AIa-OMe (19 mg, 0.08 mmol), BnSH (1.5 ml_, 160 equiv) and molsieves (3 A, 100 mg). The reaction mixture was stirred for 24 h at 50⁰C and HPLC-MS indicated that the conversion to the thioester was >95% (as estimated from peak areas). R_t = 20.69 min (HPLC method 2).

Claims

1. Method for synthesising a C-terminal thioester of an N-protected amino acid or an optionally N-protected peptide, comprising reacting a thiol with a compound selected from N-protected amino acids, optionally N-protected peptides,

N-protected amino acid C-terminal esters, optionally N-protected peptide C-terminal esters, N-protected amino acid C-terminal thioesters - other than the thioester to be synthesised - and optionally N-protected peptide C- terminal thioesters - other than the ester to be synthesised - in a liquid phase comprising less than 4 wt. % water using a hydrolytic enzyme as a catalyst for said reacting.

2. Method according to claim 1 , wherein the thiol is represented by the formula Y-S-Z, wherein Z is hydrogen or a cation, preferably a monovalent cation; and Y is hydrogen or an organic moiety, preferably an optionally substituted linear or branched alkyl group, an optionally substituted cycloalkyl group, an optionally substituted aryl group or an optionally substituted aralkyl group, which organic moiety may contain one or more heteroatoms.

3. Method according to claim 2, wherein Y is an optionally substituted linear or branched alkyl group, an optionally substituted cycloalkyl group, or an optionally substituted aralkyl group in which aralkyl group the -S-Z is attached to the alkyl function of the aralkyl group.

4. Method according to any of the preceding claims, wherein the thiol is selected from the group of benzyl mercaptane, methyl mercaptane, ethyl mercaptane, 3-mercaptopropionic acid ethylester, optionally P-protected diphenylphosphinomethyl mercaptane, 2,4,6-trimethoxy-benzylmercaptane and N-acetyl-cysteamine.

5. Method according to claim 4, wherein the thiol is selected from the group of benzyl mercaptane and 3-mercaptopropionic acid ethylester.

6. Method according to any of the preceding claims, wherein the enzyme is a peptidase selected from the group of serine-type carboxypeptidases, metallocarboxypeptidases, cysteine-type carboxypeptidases, serine endopeptidases, cysteine endopeptidases, aspartic endopeptidases and metalloendopeptidases, in particular from serine endopeptidases.

7. Method according to claim 6, wherein the serine endopeptidase is a subtilisin, preferably subtilisin Carlsberg.

8. Method according to any of the claims 1-5, wherein the enzyme is a lipase, preferably a lipase from Candida, more preferably a Candida antarctica lipase, such as Candida antarctica lipase B.

9. Method according to any of the preceding claims, wherein the reaction is carried out in a liquid phase comprising in particular 2 wt. % or less water, more in particular 1 wt. % or less water, based on total liquids of the liquid phase wherein the reaction takes place.

10. Method according to any of the preceding claims, wherein the compound with which the thiol is reacted is selected from the group of N-protected amino acids and optionally N-protected peptides.

1 1. Method according to any of the preceding claims, wherein the compound with which the thiol is reacted is selected from the group of N-protected amino acid C-terminal esters, and optionally N-protected peptide C-terminal esters.

12. Method for preparing a C-terminal thioester of an amino acid or a peptide, wherein an N-protected C-terminal thioester synthesised in a method according to any of the preceding claims is deprotected.

13. Use of a thioester obtained in a method according to any of the preceding claims in the manufacture of an oligopeptide or polypeptide.

14. Use according to claim 13, wherein the oligopeptide or polypeptide is manufactured using chemical ligation, preferably native chemical ligation.

15. Use according to claim 13, wherein the oligopeptide or polypeptide is manufactured using enzymatic ligation.