The present invention relates to the synthesis of peptides by enzymatic coupling of oligopeptide fragments, and to the synthesis of oligopeptide fragments containing groups suitable for the enzymatic synthesis of oligopeptides therefrom.

Peptides, in particular oligopeptides have many applications, for instance as pharmaceutical, food or feed ingredient, agrochemical or cosmetic ingredient.

In recent years the scalable synthesis of peptides has gained significant importance, as such products have been increasingly investigated and marketed in the pharmaceutical, cosmetics and nutrition industry.

For the purpose of this invention, with peptides is meant any chain of two or more amino acids, linked to each other by so-called peptide bonds. For the purpose of this invention, with 'oligopeptides' is meant a peptide comprising 2-200 amino acids.

It is known that oligopeptides can be chemically synthesized in solution or on a solid phase via highly optimized processes. However, there are still some limitations in chemical peptide synthesis especially on a large scale. For instance, oligopeptides longer than 10-15 amino acids are difficult to synthesize on a solid phase because they tend to form tertiary structures (by so-called "hydrophobic collapse") making peptide elongation very troublesome so that a large excess of reagents and amino acid building blocks is needed. Additionally the purification of the final product is often cost-inefficient due to the presence of significant amounts of different oligopeptides of similar length.

Therefore, oligopeptides longer than 10 amino acids are often synthesized by a combination of solid phase synthesis of protected oligopeptide fragments which are subsequently chemically condensed in solution, e.g. a 10 + 10 condensation to make an oligopeptide of 20 amino acids.

A major drawback of chemically protected oligopeptide fragment condensation is that upon activation of the C-terminal amino acid residue racemisation occurs, except when C-terminal Gly or Pro residues are used. Therefore, the chemically protected oligopeptide fragment condensation strategy is limited to using C- terminally activated Gly and Pro residues, or one has to deal with a very difficult purification process due to the formation of undesired diastereoisomers.

In contrast, enzyme-catalyzed oligopeptide couplings are completely devoid of racemisation and have several other advantages over chemical oligopeptide synthesis. For industrial application, an enzymatic oligopeptide synthesis concept based on a kinetic approach, i.e. using an activated carboxy component, is most attractive. The other approach, the so-called thermodynamic approach, is unattractive in practise because the equilibrium in peptide synthesis is usually on the side of the starting compounds, the yields are generally low and the reactions are relatively slow so that much enzyme is needed (see for instance N. Sewald and H.-D. Jakubke, in:

"Peptides: Chemistry and Biology", 1 ^st reprint, Ed. Wiley-VCH Verlag GmbH, Weinheim 2002).

For application of the kinetic approach, there are specially designed ester moieties known in the literature that are particularly recognized by certain enzymes, for instance the so-called "substrate mimetics", as reviewed by F. Bordusa et ai m Current Protein and Peptide Science 2002, 3, 159-180. In this approach, the inactivated amino acid or peptide has a C-terminal ester moiety which resembles a specific amino acid to such an extent that an enzyme which is selective for that specific amino acid rapidly reacts with any amino acid bearing the ester moiety. A good example is the use of 4-guanidinophenyl (Gp) esters as discovered by F. Bordusa et al. which resemble Arg to such an extent that trypsin, which is in its hydrolytic properties specific for Arg-X sequences (in which X stands for any proteinogenic amino acid), can couple almost any C-terminus (bearing a Gp ester) to various amino acid and peptide nucleophiles (see, for instance M. Thormann et a/ in Biochem. 1999, 38, 6056). Disadvantages of the substrate mimetic approach are that substrate mimetics (such as Gp esters) require a laborious multi-step chemical synthesis which is difficult to scale up and by which often racemisation of the amino acid occurs; the substrate mimetics are also instable, and thus difficult to handle on a large scale and their solubility in aqueous solution is low.

Another method using the kinetic approach is based on the use of peptide C-terminal carbamoylmethyl (Cam) esters which can be enzymatically coupled to peptide nucleophiles using the serine endopeptidase Subtilisin A, as described by Ye et al. Tetrahedron, 2005, 61, 5933. The challenge associated with this approach is the preparation of the peptide C-terminal Cam-esters, in which the peptide C-terminal carboxyl group serves as a nucleophile in the reaction with a strong electrophile (such as bromoacetamide). However S-containing amino acids or unprotected tyrosine cannot be used with this method, and, additionally, in the case of longer peptides, the reactions become sluggish resulting in low yields of the resulting Cam esters.

The purpose of the present invention is therefore to make available a versatile, mild and racemization-free method for the preparation of oligopeptide C- terminal activated esters. More in particular, the present invention provides an excellent method for the racemisation-free synthesis of oligopeptide C-terminal enol esters of oligopeptide fragments and the use thereof in the synthesis of larger oligopeptides.

The present invention relates to a method for the enzymatic synthesis of an oligopeptide, comprising the coupling of

i) at least one oligopeptide enol ester of 2 or more amino acids,

comprising a C-terminal enol ester represented by the formula

-C(0)-0-CR ¹ =CH-R ² wherein each R ¹ and R ² independently represents a hydrogen atom or an optionally substituted alkyl group or an optionally substituted cycloalkyl group or an optionally substituted alkenyl group or an optionally substituted aryl group,

with the C-terminal enol ester having been prepared by reacting the C-terminal oligopeptide carboxylate group with an alkyne derivative of the formula R ¹ - C≡C-R ² catalysed by a soluble transition metal catalyst system comprising a transition metal of group 8-10, which optionally contains one or more ligands and a counterion, and

wherein the oligopeptide enol ester optionally comprises an A/-terminal protecting group,

with

ii) an amino acid or oligopeptide nucleophile,

comprising an A/-terminal amine group, and

optionally comprising a C-terminal protecting group,

which enzymatic coupling is carried out in an organic solvent or an organic solvent mixture comprising 1 vol% or less water relative to the total amount of liquids in which the enzymatic coupling reaction predominantly takes place, in the presence of a serine endopeptidase.

Ruthenium-catalyzed addition of A/-protected amino acids to propyne has been described by Ruppin et al. [Tetrahedron Letters, 29 (42), p. 5365-8, 1988], however, this reaction resulted in the Markovnikov isopropenyl esters, rather than in an anti-Markovnikov product as obtained according to the present invention. No indication was given about how the anti-Markovnikov compounds of the present invention could be prepared.

Doucet et. al. [J. Chem. Soc, Chem. Commun. p. 850-1 , 1993] describe the synthesis of Z-enol esters of N-protected amino acids (like alanine and phenylalanine) catalyzed by [bis(diphenylphosphino)alkane]bis(2- methylpropenyl)ruthenium complexes. However, when the inventors applied the conditions as described by Doucet et al. to the preparation of the C-terminal enol esters of dipeptides instead of amino acids, severe racemisation of the C-terminal amino acid residue of the dipeptide occurred. It is generally known that oligopeptides are significantly more prone to racemisation on their C-terminal amino acid residue than single amino acids.

Lumbroso et. al. [Org. Lett. p. 5498-1 , 2010] describe the semi- selective synthesis of Z-enol esters of N-protected amino acids (alanine and valine) catalyzed by bis(1 ,5-cyclooctadiene)rhodiumchloride complexes in THF at 1 10°C, under which conditions significant racemisation of the C-terminal amino acid of oligopeptides occurs.

None of the above documents relates to the preparation of C-terminal enol esters of N-protected oligopeptides and the use of these enol esters in the enzymatic coupling with amino acid or oligopeptide nucleophiles to obtain larger oligopeptides.

For the purpose of the present invention with "enzymatic peptide synthesis" is meant the protease catalysed reaction between an activated peptide C- terminal ester (i.e., the acyl donor) and a peptide amine nucleophile (i.e., the acyl acceptor) to form a new peptide amide bond.

For the purpose of the invention an optionally substituted alkyl group means a linear or branched alkyl group that may carry one or more substituents such as halogen atoms, N0 ₂ , CN, primary, secondary or tertiary amine groups, ester groups, amide groups, ketone groups, aldehyde groups, functional groups containing a S or P atom, or cycloalkyl, alkenyl or aryl groups which may themselves also carry one or more substituents.

For the purpose of the invention an optionally substituted cycloalkyl group means a cycloalkyl group that may carry one or more substituents such as halogen atoms, N0 ₂ , CN, primary, secondary or tertiary amine groups, ester groups, amide groups, ketone groups, aldehyde groups, functional groups containing a S or P atom, or alkyl, cycloalkyl, alkenyl or aryl groups which may themselves also carry one or more substituents.

For the purpose of the invention an optionally substituted alkenyl group means an alkenyl group that may carry one or more substituents such as halogen atoms, N0 ₂ , CN, primary, secondary or tertiary amine groups, ester groups, amide groups, ketone groups, aldehyde groups, functional groups containing a S or P atom, or alkyl, cycloalkyl, alkenyl or aryl groups which may themselves also carry one or more substituents.

For the purpose of the invention an optionally substituted aryl group means an aryl group that may carry one or more substituents such as halogen atoms, N0 ₂ ,CN, primary, secondary or tertiary amine groups, ester groups, amide groups, ketone groups, aldehyde groups, functional groups containing a S or P atom, or alkyl, cycloalkyl, alkenyl or aryl groups which may themselves also carry one or more substituents.

If both groups R ¹ and R ² are not a hydrogen atom and are not identical, a mixture of two oligopeptide enol esters may be formed during the Ru- catalyzed enol ester formation reaction which may both be coupled enzymatically to the amino acid or oligopeptide nucleophile.

In a usual and preferred embodiment of the invention, however, the R ¹ group equals a hydrogen atom.

Even more preferably, the R ¹ group equals a hydrogen atom and the R ² group is selected from the group of optionally substituted C1-12 alkyl, optionally substituted C _3- i ₂ cycloalkyl, optionally substituted C _2- i ₂ alkenyl, or optionally substituted C ₅ -18 aryl groups.

Particularly good results have been obtained with R ¹ being a hydrogen atom and R ² being an unsubstituted linear alkyl chain, such as methyl, ethyl, n-propyl, n-butyl, n-pentyl, or n-hexyl, or an alkyl group containing an amide functionality within the chain or at the terminus of the chain, such as CH ₂ NHCOCH ₃ , CH ₂ NHCOPh,

CH ₂ NHCOCH ₂ Ph, (CH ₂ ) ₃ CONH ₂ , or (CH ₂ ) ₂ CONH ₂ . Most preferably, R ¹ is a hydrogen atom and R ² is n-butyl, CH ₂ NHCOCH ₃ , (CH ₂ ) ₃ CONH ₂ , or (CH ₂ ) ₂ CONH ₂ .

In the case of R ¹ being a hydrogen atom and R ² not being a hydrogen atom, the resulting oligopeptide C-terminal enol ester represented by the formula C(O)- 0-CH=CH-R ² can be an E-enol ester or a Z-enol ester, i.e. the resulting enol ester can possess the E or Zgeometry. For the purpose of the invention, with a E-enol ester is meant that the carboxyl functionality and the R ² group are on opposite sides of the double bond of the enol ester, whereas with a Z-enol ester is meant that the carboxyl functionality and the R ² group are on the same side of the double bond of the enol ester. Both the E- and Z-isomers of oligopeptide enol esters may be suitable substrates in the enzymatic coupling with amino acid or oligopeptide nucleophiles.

For the purpose of the present invention an ' -terminal protecting group" means that an A/-terminal amine function is provided with a protective group, generally substantially protecting the amine group from being coupled to the carboxylic function of another molecule. Suitable A/-terminal 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 A/-protecting groups include carbamate or acyl type protecting groups, for instance 'Cbz'

(benzyloxycarbonyl), 'Boc' (tert-butyloxycarbonyl), 'For' (formyl), 'Fmoc' (9- fluorenylmethoxycarbonyl), 'PhAc' (phenacetyl) and 'Ac' (acetyl). The groups For, PhAc and Ac may be introduced and cleaved enzymatically using the enzymes Peptide Deformylase, PenG acylase or Acylase, respectively. Chemical cleavage methods are generally known in the art. Another example of a suitable A/-protecting group is pyroglutamate.

For the purpose of the present invention an "amino acid or oligopeptide nucleophile" means an amino acid or oligopeptide containing an N- terminal amine group that can take part in the enzymatic peptide coupling reaction.

For the purpose of the present invention a "C-terminal protecting group" means that a C-terminal carboxylic function is provided with a protective group, generally substantially protecting the carboxyl group from being coupled to an amine group of another molecule. The C-terminal protective group may be a C-terminal ester whereby the C-terminal carboxyl group is at least substantially protected from being coupled to an amine under peptide synthesis conditions used. A t-alkyl group is a commonly used protective group. The C-terminal protective group may also be a C- terminal carboxy-amide. A primary carboxy amide is a commonly used protective group. The C-terminal protective group may also be a hydrazide, a carbamoyl- hydrazide or a thioester. The C-terminal protection may be temporary or permanent, the latter meaning that this protective moiety is part of the desired end product. The oligopeptide enol esters and the amino acid and oligopeptide nucleophiles according to the present invention, which have amino acid side chains containing functional groups that may possibly react in the method according to the present invention (i.e. hydroxyl, carboxylic acid, primary or secondary amine (including indole and guanidino), thiol or carboxyamide functionalities), may be either fully protected, partially protected or unprotected on these functional groups.

In the context of the invention with "amino acid side chain" is meant a side chain of any proteinogenic or non-proteinogenic amino acid.

Proteinogenic amino acids are the amino acids that are encoded by the genetic code. Proteinogenic amino acids include: alanine (Ala), valine (Val), leucine (Leu), isoleucine (lie), serine (Ser), threonine (Thr), methionine (Met), cysteine (Cys), asparagine (Asn), glutamine (Gin), tyrosine (Tyr), tryptophan (Trp), glycine (Gly), aspartic acid (Asp), glutamic acid (Glu), histidine (His), lysine (Lys), arginine (Arg), proline (Pro) and phenylalanine (Phe).

Non-proteinogenic amino acids may in particular be selected amongst

D-amino acids, phenylglycine, DOPA (3,4-dihydroxy-L-phenylalanine), beta-amino acids, 4-fluoro-phenylalanine, or C ^a -alkylated amino acids.

Suitable catalyst systems for use in the preparation of the oligopeptide C-terminal enol ester according to the invention are catalyst systems comprising a transition metal of group 8-10 optionally comprising one or more ligands and a counter ion.

Preferably, the transition metal is chosen from the group of

Ruthenium, Iridium, Palladium and Rhodium, most preferably Ruthenium.

Particularly good results have been obtained using ruthenium-based catalyst systems and phosphorus-based ligands.

In a particular embodiment of the method according to the present invention the ruthenium-based catalyst systems for use in the preparation of the oligopeptide C-terminal enol ester comprise at least one bidentate phosphine ligand. Particularly good results have been obtained when the phosphines are electron poor, such as in the case where both P-atoms are directly attached to one or more aromatic groups. In this embodiment of the invention, and if the R ¹ group is a hydrogen atom and the R ² group is not a hydrogen atom, the enol ester formation reaction yields predominantly or exclusively the Z-enol ester. Even better results have been obtained using a bidentate phosphine ligand in which the two phosphorus atoms are separated by a bridge containing 4 or 5 atoms, preferably by a bridge containing 4 carbon atoms. Even more preferably, 1 ,4-bis(diphenylphosphino)butane (dppb), (+)(S,S)-2,3-0- isopropylidene-2,3-dihydroxy-1 ,4-bis(diphenylphosphino)butane ((+)-DIOP) or (-)(R,R)- 2,3-0-isopropylidene-2,3-dihydroxy-1 ,4-bis(diphenylphosphino)butane ((-)DIOP) are used as the bidentate phosphine ligand. Most preferably, (+)-DIOP is used as the bidentate phosphine ligand.

dppb (-)DIOP {+)DIOP

In a particular embodiment of the method according to the present invention the catalyst system is prepared in situ from a metal precursor and a ligand.

In a particular embodiment of the method according to the present invention the catalyst system is prepared in situ from Ru(COD)(methallyl) ₂ and a bidentate phosphorus ligand. In principle, the methallyl residues can be replaced by any other coordinating residues such as alkenes or acids. Particularly good results have been obtained replacing the methallyl residues with trifluoroacetic acid.

Suitable solvents for use in the metal-based catalysed preparation of the oligopeptide C-terminal enol esters according to the present invention are alcohols, ethers and halogenated hydrocarbons. Suitable alcohols are lower alcohols, such as methanol, ethanol, 1 -propanol, and 2-propanol. Suitable ethers are for instance diethyl ether, diisopropyl ether, tetrahydrofuran, methyl-tetrahydrofuran and methyl-tert-butyl ether. Suitable halogenated hydrocarbons are tetrachloromethane, chloroform, dichloromethane, dichloroethane, trichloroethane and tetrachloroethane. The metal- based catalysed preparation of the oligopeptide C-terminal enol esters may preferably be performed under inert and anhydrous conditions.

Suitably, the temperature at which this reaction is carried out will be at least 0°C, more preferably at least 20°C, more preferably at least 40°C, and at most 100°C, preferably at most 80°C, more preferably at most 60°C. A suitable temperature range is between 0 and 100°C, more preferably between 20 and 80°C, even more preferably between 40 and 60°C. Preferably the amount of alkyne used is between 0.8 and 10 equivalents based on the peptide C-terminal carboxylic acid, more preferably between 0.9 and 5 equivalents, even more preferably between 1 and 2 equivalents. In principle, the amount of catalyst used is determining the rate of the peptide C-terminal enol ester formation reaction and can be chosen from a broad range. Preferably the amount of catalyst used is between 0.01 and 20 mol%, more preferably between 0.1 and 10 mol%, even more preferably between 0.5 and and 5 mol%.

In the method of the invention the coupling of the oligopeptide enol ester with the amino acid or oligopeptide nucleophile, is catalysed by a serine endopeptidase (E.C. 3.4.21 ). In principle any serine endopeptidase capable of catalyzing the coupling reaction can be used. When referring to a serine endopeptidase from a particular source, recombinant serine endopeptidases 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.

Preferably, the serine endopeptidase used in the method of the invention is a subtilisin. In the enzyme classification system the class for subtilisins is E.C.3.4.21.62.

Various subtilisins are known in the art, see e.g. US 5,316,935 and the references cited therein. Such subtilisins may be used in the method according to the invention.

Examples of organisms from which a subtilisin used in the method of the invention may be derived include Trichoderma species, such as from Trichoderma reesei; Rhizopus species, such as from Rhizopus oryzae; Bacillus species, such as from Bacillus licheniformis, Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus clausii, Bacillus lentus, Bacillus alkalophilus, Bacillus halodurans; Aspergillus species, such as from Aspergillus oryzae or Aspergillus niger, Streptomyces species, such as from Streptomyces caespitosus or Streptomyces griseus; Candida species; fungi; Humicola species; Rhizoctonia species; Cytophagia; Mucor species; and animal tissue, in particular from pancreas, such as from porcine pancreas, bovine pancreas or sheep pancreas.

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) subtilisins in a method 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 enzyme may have improved properties, for instance with respect to one or more of the following aspects: substrate scope, activity, stability, organic solvent resistance, temperature profile,

synthesis/hydrolysis ratio and side reaction profile.

In a preferred method subtilisin A is used to catalyse the enzymatic coupling reaction. Subtilisin A is a commercially available subtilisin from Novozymes and has been found particularly advantageous with respect to condensing the coupling partners to give the desired peptide product with a good yield in a relatively short time.

Alcalase® is a suitable source for subtilisin A. This product is available from Novozymes (Bagsvaerd, Denmark). Alcalase® is a cheap and industrially available proteolytic enzyme mixture produced by Bacillus licheniformis (containing subtilisin A as a major enzyme component).

Commercially available enzymes, such as Alcalase®, may be provided by the supplier as a liquid, in particular an aqueous liquid. In such case, the enzyme is preferably first isolated from undesired liquid, for instance excess water or alcohols that cause an undesired side-reaction. This may suitably be accomplished by precipitation, usually followed by separation of the solid from the liquid, and/or drying. Precipitation may be accomplished using an alcohol, such as iert-butanol. In case another alcohol is used, care should be taken that such alcohol does not interfere adversely with the coupling reaction.

In a preferred embodiment, the enzyme is used in an immobilized form. At least in some embodiments this may result in an increased yield of

synthesised oligopeptide after a relatively short reaction time. Particularly good results have been obtained with Alcalase cross-linked enzyme aggregates (Alcalase-CLEAs) or with Alcalase immobilized on solid particles such as Alcalase-lmibond, Alcalase- Epobond, Alcalase-immozyme or Alcalase-Dicalite. Immobilization of the enzyme also may allow easy recovery of the enzyme after the enzymatic coupling reaction, so that it can be recycled and repeatedly used in consecutive enzymatic coupling reactions.

It is possible to carry out the enzymatic coupling reaction in an inert organic solvent. Some examples of suitable solvents are for instance N,N- dimethylformamide (DMF), /V-methyl-pyrrolidinone (NMP), A/JV-dimethylacetamide (DMA), dimethylsulphoxide (DMSO), acetonitrile, a hydrocarbon such as toluene, a halogenated hydrocarbon, such as dichloromethane, 1 ,2-dichloroethane or chloroform, an ether, such as methyl-tert-butyl ether (MTBE), tetrahydrofuran (THF), 2-methyl- tetrahydrofuran (Me-THF) or 1 ,2-dimethoxyethane, or a (halogenated) alcohol, such as 2,2,2-trifluoroethanol (TFE) or a mixture of these organic solvents. Preferably, the enzymatic coupling reaction may be carried out in an organic solvent or organic solvent mixture comprising MTBE, THF, Me-THF, 1 ,2-dimethoxyethane, dichloromethane, 1 ,2- dichloroethane, TFE, DMF, NMP, DMA or DMSO. Most preferably, the enzymatic coupling reaction may be carried out in an organic solvent or organic solvent mixture comprising THF, a mixture of THF with DMF or NMP or DMA or DMSO, MTBE, a mixture of MTBE with DMF or NMP or DMA or DMSO, dichloromethane or a mixture of dichloromethane with DMF or NMP or DMA or DMSO.

The enzymatic coupling reaction is typically 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.

With substantially non-aqueous is meant that the reaction medium is free of water or contains a minor amount of water, i.e. an amount of 1 vol% or less water, based on the total volume of liquids in the reaction medium. 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 volume of liquids in the phase wherein the enzymatic coupling reaction (at least predominantly) takes place.

A desired upper limit for the water concentration in the enzymatic coupling reaction depends on the concentrations of oligopeptide ester and oligopeptide nucleophile, on the specific enzyme, the solvent used, the nature of the peptide to be synthesised (e.g. the size of the peptide and the sequence of the amino acids), the desired final conversion and the desired reaction rate.

Preferably, the water concentration in the enzymatic coupling reaction is 1 .0 vol% or less, more preferably 0.5 vol% or less, even more preferably 0.1 vol% or less, and most preferably 0.05 vol% or less, whilst still retaining substantial desired enzyme activity.

No lower limit for the water concentration in the enzymatic coupling reaction is presented here, because the minimal amount of water that may need to be present is below the detection limits of well known analytical methods, such as Karl- Fischer titration. In an advantageous embodiment, water that is released by the enzyme may be removed continuously or intermittently. In principle, the water removal may be accomplished in a manner known in the art. Good results have been achieved using molecular sieves. The addition of various amounts of molecular sieves to the enzymatic coupling reaction allows the variation of the water concentration below its detection limit. Too low a water concentration, for instance obtained by the addition of a large amount of molecular sieves, may in some cases lead to gradual (partial) enzyme deactivation during the coupling reaction. The man skilled in the art can easily determine the optimal water concentration for a certain enzymatic coupling reaction by variation of the amount of molecular sieves. Very suitable for the water removal is evaporation, such as azeotropic removal using vacuum or distillation. Good results have in particular been achieved using a Soxhlet apparatus. In such a Soxhlet apparatus water is continuously removed from the reaction mixture by coevaporation with the solvent or solvent mixture wherein the enzymatic coupling reaction takes place, and the recondensing mixture of the solvent or solvent mixture containing water is led through a dropping funnel which is filled with a drying agent, so that dry solvent or solvent mixture returns to the reaction mixture.

In case of (partial) enzyme deactivation during the enzymatic coupling reaction, the (partly) deactivated enzyme may be completely or almost completely reactivated by rehydration, for instance by stirring the (partly) deactivated enzyme in an aqueous solution. This reactivation may allow repeated use of the enzyme in consecutive coupling reactions. In some cases, in particular with lyophilized enzymes, the enzyme needs to be hydrated before the enzymatic coupling in order to get sufficient catalytic activity. In case of non-immobilized enzymes such a hydration may be performed by stirring the enzyme in aqueous solution followed by precipitation, for instance with a water-miscible organic solvent such as te/t-butanol. In case of immobilized enzymes such a hydration may be performed by washing with an aqueous solution, followed by washing with one or more organic solvents, for instance with a water-miscible organic solvent such as tert-butanol and subsequently a water- immiscible solvent such as MTBE.

In particular, the method of the invention allows coupling of an oligopeptide enol ester to an amino acid or oligopeptide nucleophile, without needing a large excess of one of the coupling partners based on the other coupling partner in order to obtain the synthesised peptide in an acceptable yield within a relatively short time. The molar ratio of the oligopeptide enol ester to the amino acid or oligopeptide nucleophile usually is chosen in the range of 2:1 to 1 :4, in particular in the range of 1 :1 to 1 :3, preferably in the range of 1 :1 to 1 :2, more preferably 1 :1 to 1 :1 .5, even more preferably 1 :1 to 1 :1.2.

In principle the pH used in the enzymatic coupling reaction (in as far as a pH exists in the chosen reaction medium) may be chosen within wide limits, as long as a pH is chosen at which the enzyme shows sufficient activity. Such a pH is usually known for the enzyme to be used and may be based on its known hydrolytic activity in an aqueous solution, or can be routinely determined, making use of a known substrate for the enzyme under known reaction conditions. 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 the pH may be 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 organic solvent-soluble acids, such as acetic acid and formic acid.

In principle, the temperature used in the enzymatic coupling reaction is not critical, as long as a temperature is chosen at which the enzyme(s) used show sufficient activity and stability. Such a temperature is usually known for the enzyme(s) to be used or can be routinely determined, making use of a known substrate for the enzyme(s) under known reaction conditions. Generally, the temperature may be at least 0 °C, in particular at least 15 °C or at least 25 °C. In particular if one or more enzyme(s) originating from a thermophilic organism are used, the temperature may preferably be at least 35 °C. A desired maximum temperature depends upon the enzyme(s). In general such maximum temperature is known in the art, e.g. indicated in a product data sheet in case of commercially available enzyme(s), or can be determined routinely based on common general knowledge. The temperature is usually 70°C or less, in particular 60°C or less or 50 °C or less. However, in particular if one or more enzyme(s) from a thermophilic organism are used, the temperature may be chosen higher, for example up to 90°C.

Optimal temperature conditions for the enzymatic coupling reaction can easily be identified for a specific enzyme by a person skilled in the art through routine experimentation based on common general knowledge. For instance, for subtilisin, in particular subtilisin A (e.g. in Alcalase®), the temperature may

advantageously be in the range of 25-60°C.

The invention will now be illustrated by the following examples without being limited thereto.

Experimental section

General

Unless stated otherwise, chemicals and reagents were purchased from commercial sources.

The metal-catalysed oligopeptide C-terminal enol ester formation reactions under inert atmosphere were carried out with standard Schlenk techniques. Syntheses which are sensitive to oxidation and humidity were performed under nitrogen or argon atmosphere using dry and degassed solvents. Two different protocols were used to degas the applied solvents: A) by subjecting the frozen solvent (liquid nitrogen) in appropriate Schlenk tubes to vacuum (0.02 mbar). After melting of the solvent the Schlenk tube was refilled with argon. This procedure was repeated 3 times. B) in a Schlenk tube with septum and cannula by passing a stream of argon through the solvent. The Schlenk tube was placed in an ultrasonic bath for 20 to 30 minutes, depending on the volume of solvent used.

Alcalase-CLEA OM was obtained from CLEA technologies (Delft, the

Netherlands).

All commercial solvents were stored on activated molecular sieves. For that purpose 3A or 4A molecular sieves were activated at oil pump vacuum (0.02 mbar) at 200°C for 2 days. Prior to storage on activated molecular sieves, toluene and acetonitrile were dried over aluminium oxide, tetrahydrofuran over sodium and benzophenone, methanol over magnesium turnings and iodine, ethanol over sodium and diethyl phthalate and dichloromethane over phosphorus pentoxide P ₄ Oi ₀ and subsequently over CaH ₂ . NMR spectra were recorded on a Bruker A VANCE III: 300.36 MHz- ¹ H-NMR, 75.5 MHz- ¹³ C-NMR or Varian lnova-500: 500 MHz- ¹ H-NMR, 125 MHz- ¹³ C-NMR. Chemical shifts δ are referenced to residual protonated solvent signals as internal standard. Analytical HPLC was performed using one of the following methods using a Shimadzu Nexera apparatus containing a Poroshell 120 SB-C18 (2.7 μηη, Agilent) column 100x3 mm at 40°C. The flow rate was 0.7 mL/min. Solvent A: H ₂ 0 + 0.01 vol% HCOOH; Solvent B: MeCN. The elution progamme was from A/B = 90/10 at t = 0 to A/B = 0:100 at t = 5 min. The ESI MS was run in the positive and negative mode. UV detection was performed at 210 nm.

To determine the e.e. (enantiomeric excess) of an amino acid, peptide samples were hydrolyzed in 6M hydrochloric acid at 95°C over a time span of 16h. Subsequently the reaction mixtures were evaporated in vacuo using a rotary evaporator to remove most of the acid. The residue was then dissolved in water and the pH adjusted to approximately 9 using 0.4M potassium tetraborate buffer pH 10.5.

Analysis of UV-active amino acids (phenylalanine) was directly done without derivatization on an Astec Chirobiotic column (250x4.6 mm) using a

water/methanol mixture (40/60, v/v) at a flow rate of 0.7 mL/min, a temperature of 25°C and a detection wavelength of 210 nm. The retention times were: 8.2 min (L-Phe) and 12.1 min (D-Phe).

To analyze the amount of racemization of a C-terminal amino acid other than phenylalanine firstly the enolesters were hydrolysed with 6N DCI in D ₂ 0 during 16 hrs at 85°C followed by an esterification reaction with 2-3N HCI in 2-propanol for 1 h at 1 10 °C and finally an acylation with pentafluoropropionic anhydride at 150 °C for 10 min. The amount of epimerization was determined via GC-MS (Agilent

Technologies 5975C inert MSD with Triple Axis Detector) using a Chirasil-L-Val column using the following parameters: Initial temp: 50 °C, Maximum temp: 200 °C, Initial time: 5.00 min Equilibration time: 0.50 min, Rate (4 °C/min), Final temp 200, Final time 5.00 min. Electron impact (El, 70 eV) HRMS spectra were recorded on a Waters GCT Premier equipped with direct insertion (Dl) and GC (HP GC7890A). HRMS spectra recorded with MALDI-TOF mass spectrometry were performed on a Micromass TofSpec 2E Time-of-Flight Mass Spectrometer.

Analytical thin layer chromatography was performed using TLC-plates from Merck (TLC aluminium foil, silica gel 60 F254). Generally, the spots were visualized using a UV lamp (λ = 254, 366 nm) or by treatment with different staining reagents (listed below) followed by heating. CAM-solution: 2.0 g cer(IV)-sulfate, 50.0 g ammonium molybdate and 50 ml. cone. H ₂ S0 ₄ in 400 ml. water. Potassium

permanganate: 3.0 g potassium permanganate, 20.0 g K ₂ C0 ₃ , 300 ml. of a 5 % aqueous NaOH solution.

Preparative flash column chromatography was performed using silica gel 60 from ACROS Organics (35-70 μηη particle size). The mass of silica gel used was 100 times (w/w) the amount of dry crude product. The length of columns used differ from 10 to 40 cm. The Rf-values are listed in the experimental section, the following abbreviations for the eluents were used: EE (ethyl acetate), CH (cyclohexane), DCM (methylene chloride) and MeOH (methanol). Melting points are uncorrected and were determined with the apparatus "Mel-Temp® ¹ from Electrothermal wit an integrated microscopical support. The temperature was measured with a mercury-in-glass thermometer. The determination of specific rotations was performed on a polarimeter 341 from Perkin Elmer. A sodium vapor lamp (589 nm) was used as source of monochromatic light.

A/-(2-propynyl)-acetamide was prepared by acylation of

propargylamine in dichloromethane in the presence of acetic anhydride, triethyl amine and DMAP according to A. Hashmi et al., Org. Lett. 2004, 6, 4391-4394). Hex-5- ynamide and pent-4-ynamide were synthesized according to P. Jacobi et al., J. Org. Chem. 1997, 62, 2907-2916.

Preparation of ruthenium catalyst systems

-(2-methylallyl)(dppb)ruthenium(ll)

Bis-(2-methylallyl)(dppb)ruthenium(ll)

An oven-dried Schlenk-tube with a magnetic stirring bar was charged with bis-(2-methylallyl)(1 ,5-cyclooctadiene)ruthenium(ll) (500 mg, 1.57 mmol, 1 .0 eq) and dppb (669 mg, 1.57 mmol, 1 .0 eq.) under an argon atmosphere. After evacuation and refilling with argon, dry n-hexane (8.5 ml.) was added. After heating to 55 °C in a preheated oil bath, a brownish solution was formed. Stirring was continued for another 5 h at this temperature while the yellow product precipitated. Using an inert Schlenk filter funnel, the precipitate was collected and washed with cold and dry n-hexane (3 χ 5 ml_). The solid was dried under vacuum (0.02 mbar) for several hours yielding the product as a yellowish to greenish powder (765 mg, 1.20 mmol, 77 %). -(2-methylallyl)((+)-DIOP)rutheni

Bis-(2-methyia)IyiM{+J-D!OP)ruthenium(li)

An oven-dried Schlenk-tube with a magnetic stirrer was charged with bis-(2-methylallyl)(1 ,5-cyclooctadiene)ruthenium(ll) (450 mg, 1.41 mmol, 1 .0 eq) and (+)-DIOP (717 mg, 1.41 mmol, 1.0 eq) under argon atmosphere. After evacuation and refilling with argon, dry n-hexane (8.5 ml.) was added. After heating to 55 °C in a preheated oil bath, a brownish solution was formed. Stirring was continued for another 5 h at this temperature while the yellow product precipitated. Using an inert Schlenk filter funnel, the precipitate was collected and washed with cold and dry n-hexane (3 χ 5 ml_). The solid was dried under vacuum (0.02 mbar) for several hours yielding the product as a yellowish to greenish powder (877 mg, 1.24 mmol, 88 %).

Bis-(2-methylallyl)((+)-DIOP)ruthenium(ll) [Ru(TFA) ₂ (dppb)]

A solution of bis-(2-methylallyl)(dppb)ruthenium(ll) (200 mg, 0.31 mmol, 1.00 eq) in 6 ml. toluene in an oven-dried Schlenk-tube with a magnetic stirrer was treated dropwise at -78°C with trifluoroacetic acid (48.8 μΙ_, 0.63 mmol, 2.02 eq). The reaction mixture was stirred for 1 h at -78°C and then heated to ambient temperature. During warming up a rapid color change from orange to brown occurred and a precipitate was formed. After stirring at ambient temperature for 15 min, the solvent was evaporated in vacuo and the product [Ru(TFA) ₂ (dppb)] was washed with n- hexane (1 x2 ml_). The solid was dried under vacuum (0.02 mbar) for several hours yielding the product as a yellowish to greenish powder (156 mg, 0.21 mmol, 66 %).

[Ru( -DIOP)]

bis-(2-methylallyl)((+)-DIOP)ruthenium(ll) [Ru(TFA) ₂ ((+)-DIOP)]

A solution of bis-(2-methylallyl)((+)-DIOP)ruthenium(ll) (71 mg, 0.10 mmol, 1.00 eq) in 6 ml. toluene in an oven-dried Schlenk-tube with a magnetic stirrer was treated dropwise at -78°C with trifluoroacetic acid (15.4 μΙ_, 0.20 mmol, 2.00 eq). The reaction mixture was stirred for 1 h at -78°C and then heated to ambient temperature. During warming up a rapid color change from orange to brown occurred and a precipitate was formed. After stirring at ambient temperature for 15 min the solvent was evaporated in vacuo and the product [Ru(TFA) ₂ ((+)-DIOP)] washed with n- hexane (1 *2 mL). The solid was dried under vacuum (0.02 mbar) for several hours yielding the product as a green powder (50 mg, 0.06 mmol, 61 %).

Example 1. Ligand screening for the synthesis of Z-Ala-Markovnikov-enolester, Z-Ala-Z-anti-Markovnikov-enolester and Z-Ala-E-anti-Markovnikov-enolester

135 mg Z-Ala-OH (600 μηιοΙ, 1 .00 eq) together with 2.6 mg

(24.1 μηηοΙ, 0.04 eq) sodium carbonate were added to an oven-dried Schlenk tube under argon and were then suspended in 400 μΙ_ degassed CHCI ₃ . In the meantime in another oven-dried Schlenk tube 3.7 mg (6.0 μηηοΙ, 0.01 eq) ((p-cumene)RuCI ₂ )2 (I) together with 0.02 eq of various ligands were dissolved in 400 μΙ_ degassed CHCI ₃ under argon and stirred for 30 min to form the catalyst of this reaction. The catalyst solution was transferred to the colourless suspension of Z-alanine and sodium carbonate, the Schlenk tube was washed with degassed CHCI ₃ (4 x 400 μΙ_) and this solution was also added to the reaction mixture. 90 μΙ_ (780 μηηοΙ, 1.30 eq) degassed 1-hexyne were added to the coloured suspension and the reaction mixture was stirred at 50 °C in a preheated oil bath under argon. The conversion was monitored via thin- layer chromatography and the isomers were quantified by GC-MS.

) Markovnikov enol ester

£-anf -Markovnikov enol ester

Z-anf -Markovnikov enol ester

diprpx (14)

This table shows that various phosphorus ligands can be used for the catalyzed Markovnikov enol ester formation and that bidentate phosphine ligands favour the formation of Z-anti-Markovnikov enol esters.

Example 2. Screening of solvents for the ruthenium-catalyzed anti-Markovnikov enol ester formation of Z-Leu-Phe-OH using the bis-(2- methylallyl)(dppb)ruthenium(ll) catalyst system.

All reactions were carried out under argon in an oven-dried Schlenk vessel containing a magnetic stirring bar. Under a flow of argon the vessel was charged with Z-Leu-Phe-OH (100 mg, 0.24 mmol, 1 .0 eq), bis-(2- methylallyl)(dppb)ruthenium(ll) (6.2 mg, 9.7 μηηοΙ, 0.04 eq) and dry solvent (0.5 mL) degassed prior to use. If necessary the materials were dissolved by heating to reflux temperature for a few seconds and then the vessel was immediately cooled and immersed in an oil bath preheated to 40°C. Then degassed 1-hexyne (34 μΙ_, 0.29 mmol, 1.2 eq) was added to the reaction mixture with a Hamilton syringe and the mixture was stirred at 40°C for the indicated time. Samples were withdrawn and analyzed by HPLC-MS analysis.

In all cases, the Z-anti-Markovnikov enol ester was the only product.

The Z-anti-Markovnikov enol ester was purified (entry 8 and 15) using flash column chromatography: (16 g silica gel, 22 ^χ 1.5 cm, cyclohexane/ethyl acetate = 5:1 (v/v), R _f = 0.30)

Yield entry 15: 0.109 g (91 %)

Content of D-Phe-OH: <0.1 %

HRMS: [MNa] ⁺ : calculated: 517.2678 found: 517.2716

WD = -13.1 (c = 0.5 in chloroform)

mp = 72-73°C

¹ H-NMR (300 MHz, CDCI ₃ ): δ = 0.90-0.92 (m, 9H, -CH ₂ -CH ₃ , -CH-(CH ₃ ) ₂ ), 1 .26-1.72 (m, 7H, -NH-CH-CH ₂ -CH-(CH ₃ ) ₂ , -CH ₂ -CH-(CH ₃ ) ₂ , -C0 ₂ -CH=CH-CH ₂ -(CH ₂ ) ₂ -CH ₃ ),

2.04-2.1 1 (m, 2H, -CH=CH-CH ₂ -CH ₂ -), 3.15-3.17 (m, 2H, C ₆ H ₅ -CH ₂ -CH-NH), 4.17-4.22 (m, 1 H, NH-CH(CH ₂ -CH(CH ₃ ) ₂ )-CO-NH), 4.92-4.99 (m, 2H, CO-NH-CH(CH ₂ -Ph)-C0 ₂ -, -C0 ₂ -CH=CH-CH ₂ -), 5.06-5.15 (m, 3H, HN-C0 ₂ -CH ₂ -C ₆ H ₅ , .NH-), 6.51 (d, 1 H, ³ J(H,H) = 7.2 Hz, -NH-), 6.97 (d, 1 H, ³ J(H,H) = 6.3 Hz, C0 ₂ -CH=CH-CH ₂ ), 7.09-7.36 (m, 10H, H _AT ).

¹³ C-NMR (75 MHz, CDCI ₃ ): δ = 14.0 (CH ₃ -(CH ₂ ) ₃ -CH=CH-0 ₂ C), 22.1 , 22.3, 23.0, 24.2, 24.8, 31.3 (Caiiphatic), 38.1 (C ₆ H ₅ -CH ₂ -CH-NH-), 41.4 (NH-CH-CH ₂ -CH-(CH ₃ ) ₂ ), 53.2, 53.6 (2 x NH-CH), 67.3 (HN-C0 ₂ -CH ₂ -C ₆ H ₅ ), 1 15.8 (C0 ₂ -CH=CH-CH ₂ -), 127.4 (C _Ar ), 128.2 (2 x CAT), 128.4 (C _Ar ), 128.7 (2 χ C _Ar ), 128.8 (2 χ C _Ar ), 129.4 (2 χ C _Ar ), 133.6 (C0 ₂ -CH=CH-CH ₂ -), 135.5 (C _q , _Ar ), 136.3 (C _q , _Ar ), 156.2 (NH-C0 ₂ -CH ₂ -C ₆ H ₅ ), 168.7 (C _q ), 171 .9 (Cq).

The table shows that various solvents can be used for the catalyzed anti-Markovnikov enol ester formation, but there is a preference for using ethers and primary and secondary alcohols, in particular for using 2-propanol. Furthermore, the purified product can be obtained in high yield.

Example 3. Ruthenium-catalyzed anti-Markovnikov enol ester formation of Z-Leu- Phe-OH using different phosphorous ligands

All reactions were carried out under argon in a previously oven-dried

Schlenk vessel containing a magnetic stirring bar. Under a flow of argon the vessel was charged with bis-(2-methylallyl)(cycloocta-1 ,5-diene)ruthenium(ll) (3.1 mg, 9.7 μηηοΙ, 0.04 eq) and different bidentate phosphine ligands (9.7 μηηοΙ, 0.04 eq) and dry THF (0.25 ml.) degassed prior to use. The solution was stirred at 40°C for 1 h under argon. Then, Z-Leu-Phe-OH (100 mg, 0.24 mmol, 1.00 eq) was added and the Schlenk vessel was rinsed with dry THF (0.25 ml.) degassed prior to use, under inert conditions.

Degassed 1-hexyne (34 μΙ_, 0.29 mmol, 1.20 eq) was finally added with a Hamilton syringe to the solution and the mixture was stirred at 40°C for the indicated time.

Samples were taken and analyzed by HPLC-MS analysis.

Z-L-Leu-L-Phe-Markovnikov-enolester

-26-

Z-Anti-Markovnikov-enolester/

Conversion after

Entry Ligand E-Anti-Markovnikov-enolester/

24h [%]

Markovnikov-enolester

1 16 43 93/-/6

2 17 18 41 /-/59

3 18 57 29/-/71

4 19 97 98/-/2

5 20 57 84/-/16

6 21 43 53/-/47

7 22 51 91/-/9

8 23 77 25/-/75

9 24 54 93/-/7

10 25 22 19/-/81

1 1 26 40 78/-/22

12 27 5 nd

13 28 46 97/-/3

14 29 15 83/-/17

15 30 78 99/-/1

16 31 18 64/-/36

17 32 57 98/-/2

18 33 10 nd

19 34 5 nd

20 35 56 99/-/1

21 36 17 78/-/22

22 37 21 75/-/25

23 38 8 nd

24 (-)-39 98 96/-/4

25 (+)-39 99 >99/-/<1

This table shows that various bidentate phosphine ligands can be used for the catalyzed enol ester formation. It also appears that in particular ligands 19, 30, (-)39 and (+)39 which all have a bridge of 4 carbon atoms between the P atoms, give a very high conversion and give almost exclusively the Z-anti-Markovnikov enol ester products.

Example 4. Ruthenium catalyzed Z-anti-Markovnikov enol ester formation of Z- Leu-Phe-OH using the Ru-catalyst system Bis-(2-methylallyl)((+)- DIOP)ruthenium(N), 1-hexyne and different solvents

All reactions were carried out under argon in an oven-dried Schlenk vessel containing a magnetic stirring bar. Under a flow of argon the vessel was charged with Z-Leu-Phe-OH (0.25 mmol, 1.0 eq), Ru-catalyst system Bis-(2- methylallyl)((+)-DIOP)ruthenium(ll) (7.1 mg, 0.01 mmol, 0.04 eq) and dry solvent (0.5 ml.) degassed prior to use. If necessary the materials were dissolved by heating to reflux temperature for a few seconds and then the vessel was immediately cooled and immersed in an oil bath preheated to 40°C. Then degassed 1-hexyne (0.3 mmol, 1.2 eq) was added to the reaction mixture with a Hamilton syringe and the mixture was stirred at 40°C for the indicated time. The reactions were monitored by taking samples and analyzing them using HPLC-MS. After the appropriate conversion had been reached, the solution was concentrated to dryness. The tawny solid residue was then redissolved in dichloromethane, the solution evaporated to dryness in the presence of the same quantity of silica gel and finally subjected to chromatographic purification. Fractions containing the product were pooled and concentrated in vacuo. The resulting product was finally dried at 0.02 mbar. In all cases only the formation of Z-anti- Markovnikov enol esters could be observed. Analytical data was identical to that of example 2.

This table shows that using the Ru-catalyst system Bis-(2-methylallyl)((+)- DlOP)ruthenium(ll) higher reaction rates can be obtained as well as reduced degrees of epimerization of the C-terminal amino acid, compared to using the Ru-catalyst system Bis-(2-methylallyl)(dppb)ruthenium(ll) as shown in Example 2. The table further shows that when using alcohols or ethers as the solvent, epimerisation of the C- terminal amino acid can be reduced to a minimum.

Example 5. Ruthenium-catalyzed enol ester formation of Z-Leu-Phe-OH using various amide containing alkynes in combination with Bis-(2- methylallyl)(dppb)ruthenium(ll) or Bis-(2-methylallyl)((+)-DIOP)ruthenium(ll) ligands

All reactions were carried out under argon in an oven-dried Schlenk vessel containing a magnetic stirring bar. Under a flow of argon the vessel was charged with Z-Leu-Phe-OH (0.25 mmol, 1.0 eq), Ru-catalyst system (7.1 mg, 0.01 mmol, 0.04 eq) and dry solvent (0.5 ml.) degassed prior to use. The materials were dissolved by heating to reflux temperature for a few seconds and then the vessel was immediately cooled and immersed in an oil bath preheated to 40°C. The alkyne (0.3 mmol, 1.2 eq) was added to the reaction mixture and the mixture was stirred at 40°C. The course of the reaction was monitored by taking samples and analyzing them using HPLC-MS. After the appropriate conversion had been reached the solution was concentrated to dryness. The tawny solid residue was then redissolved in

dichloromethane, the solution evaporated to dryness in the presence of the same quantity of silica gel and finally subjected to a chromatographic purification. Fractions containing the product were pooled and concentrated in vacuo. The product was finally dried at 0.02 mbar.

Using hex-5-ynamide in combination with Bis-(2-methylallyl)((+)- DlOP)ruthenium(ll) ligand:

In both cases only the Z-anti-Markovnikov enol ester was formed.

Using A-(2-propynyl)-acetamide in combination with Bis-(2- methylallyl)(dppb)ruthenium(ll) ligand:

In all cases only the Z-anti-Markovnikov enol ester was formed.

Z-Leu-Phe-Z-anti-Markovnikov enol ester product was purified (entry 1 ) by flash column chromatography: (16 g silica gel, 15 x 2 cm, cyclohexane/ethyl acetate = 1 :5. For analytical data see below.

Using N-(2-propynyl)-acetamide, N-(2-propynyl)-benzamide and N-(2-propynyl)- phenylacetamide in combination with Bis-(2-methylallyl)((+)-DIOP)ruthenium(ll) ligand:

The numbers in brackets corresponding to by-products (assuming an equivalent extinction factor as the desired product)

In all cases the Z-anti-Markovnikov enol ester was formed, along with by-products which probably resulted from a subsequent addition of the amide in the enol ester of the desired reaction product on the excess alkyne. Purification of the product and analytical data was identical to that described above.

The Z-Leu-Phe-Z-anti-Markovnikov enol ester product was purified (entry 1 ) by flash column chromatography: (16 g silica gel, 15 x 2 cm, cyclohexane/ethyl acetate = 1 :5 (v/v), R _f = 0.26)

Yield: 92 mg (75 %)

Content of D-Phe-OH: 0.1 %

HRMS: [MNa] ⁺ : calculated: 532.2424

found: 532.2449

[a]p = -19.5 (c = 0.5 in chloroform)

mp = 149°C

1H-NMR (300 MHz, CDCI ₃ ): δ = 0.90-0.92 (m, 6H, -CH-(CH ₃ ) ₂ ), 1.43-1.52 (m, 1 H, -CH ₂ - CH-(CH ₃ ) ₂ ), 1.57-1 .66 (m, 2H, -NH-CH-CH ₂ -CH-(CH ₃ ) ₂ ), 1 .94 (s, 3H, CH ₃ -CO-NH-CH ₂ - CH=CH-C0 ₂ -), 3.13 (d, 2H, ³ J(H,H) = 6.0 Hz, C ₆ H ₅ -CH ₂ -CH-NH), 3.71-3.81 (m, 2H, CH ₃ -CO-NH-CH ₂ -CH=CH-C0 ₂ -), 4.19-4.21 (m, 1 H, NH-CH(CH ₂ -CH(CH ₃ ) ₂ )-CO-NH), 4.87 (pseudo q, 1 H, ³ J(H,H) = 6.6 Hz, 7.2 Hz, CO-NH-CH(CH ₂ -Ph)-C0 ₂ -), 5.03-5.08 (m, 3H, -C0 ₂ -CH=CH-CH ₂ + HN-C0 ₂ -CH ₂ -C ₆ H ₅ ), 5.37 (d, 1 H, ³ J(H,H) = 8.1 Hz, -NH-), 5.92 (pseudo s, 1 H, -NH-), 6.86 (d, 1 H, ³ J(H,H) = 7.6 Hz, -NH-), 7.04 (d, 1 H, ³ J(H,H) = 6.0 Hz, C0 ₂ -CH=CH-CH ₂ ), 7.13 (d, 2H, ³ J(H,H) = 6.6 Hz, H ), 7.23-7.33 (m, 8H, H ). ¹³ C-NMR (75 MHz, CDCI ₃ ): δ = 22.0, 23.0, 23.2, 24.8 (C _a i _ip hatic), 33.8 (CH ₃ -CO-NH-CH ₂ - CH=CH-C0 ₂ -), 37.9 (C ₆ H ₅ -CH ₂ -CH-NH-), 41.1 (NH-CH-CH ₂ -CH-(CH ₃ ) ₂ ), 53.6, 53.8 (2 x NH-CH), 67.3 (HN-C0 ₂ -CH ₂ -C ₆ H ₅ ), 1 1 1.2 (C0 ₂ -CH=CH-CH ₂ -), 127.4 (C _Ar ), 128.1 (2 χ CA _T ), 128.4 (C _Ar ), 128.7 (2 χ C _Ar ), 128.8 (2 χ C _Ar ), 129.4 (2 χ C _Ar ), 135.6 (C0 ₂ -CH=CH- CH ₂ -), 136.1 (C _q , _Ar ), 136.2 (C _q , _Ar ), 156.4 (NH-C0 ₂ -CH ₂ -C ₆ H ₅ ), 168.0 (C _q ), 171 .4 (C _q ), 172.3 (Cq). Using pent-4-ynamide and hex-5-ynamide in combination with Bis-(2- methylallyl)((+)-DIOP)ruthenium(ll) ligand:

In both cases 98% of the product was the desired Z-anti-Markovnikov enol ester, and 2% consisted of a by-product resulting from an addition of the amide of the desired enol ester on the excess alkyne. Entry 1 was purified using flash column chromatography: (15 g silica gel, 22 ^χ 1.5 cm, cyclohexane/ethyl acetate = 1 :5 (v/v), R _f = 0.12)

Yield: 103.8 mg (82 %)

Content of D-Phe-OH: <0.1 %

HRMS: [MNa] ⁺ : calculated: 532.2424

found: 532.2463

[α] _η = +1 1.2 (c = 0.5 in chloroform) ¹ H-NMR (300 MHz, CDCI ₃ ): δ = 0.92-0.94 (m, 6H, -CH(-CH ₃ ) ₂ ), 1.45-1.65 (m, 3H, HN- CH-CH ₂ -CH(-CH ₃ ) ₂ , -CH ₂ -CH(-CH ₃ ) ₂ ), 2.15-2.39 (m, 4H, H ₂ N-CO-CH ₂ -CH ₂ -, H ₂ N-CO- CH ₂ -CH ₂ -), 3.15 (d, 2H, ³ J(H,H) = 5.8 Hz, CeHg-ChU-CH-NH), 4.24- 4.29 (m, 1 H, NH- CH-CO-NH), 4.88-4.99 (m, 2H, CO-NH-CH-C0 ₂ -, C0 ₂ -CH=CH-CH ₂ -), 5.09 (s, 2H, HN- C0 ₂ -CH ₂ -C ₆ H ₅ ), 5.40-5.68 (m, 3H, -C0 ₂ -NH-CH-, -CHz-CO-NhU), 6.99-7.01 (m, 2H, C0 ₂ -CH=CH-CH ₂ - + -CH-CO-NH-CH-C0 ₂ -), 7.12-7.34 (m, 10H, H ).

1 ³ C-NMR (75 MHz, CDCI ₃ ): δ = 20.7 (C0 ₂ -CH=CH-CH ₂ -), 22.0, 23.0 (2 ^χ C, CH(- CH ₃ ) ₂ ), 24.8 (-CH ₂ -CH(-CH ₃ ) ₂ ), 35.2, 37.9 (2 ^χ C, C ₆ H ₅ -CH ₂ -CH-NH-, NH ₂ -CO-CH ₂ - CH ₂ -), 41 .4 (NH-CH-CH ₂ -CH-(CH ₃ ) ₂ ), 53.6, 53.9 (2 ^χ NH-CH), 67.2 (HN-C0 ₂ -CH ₂ - C ₆ H ₅ ), 1 13.3 (C0 ₂ -CH=CH-CH ₂ -), 127.2 (2 ^χ C _Ar ), 128.2 (C _Ar ), 128.4 (C _Ar ), 128.7 (2 ^χ CA _T ), 128.7 (2 ^χ C _Ar ), 129.7 (2 ^χ C _Ar ), 135.1 (C0 ₂ -CH=CH-CH ₂ -), 135.8 (C _q , _Ar ), 136.3 (C _q ,Ar), 156.4 (NH-C0 ₂ -CH ₂ -C ₆ H ₅ ), 168.1 (C _q ), 172.3 (C _q ), 174.9 (C _q ).

Entry 2 was purified using flash column chromatography: (16 g silica gel, 22 ^χ 1.5 cm, cyclohexane/ethyl acetate = 1 :5 (v/v), R _f = 0.31 )

Yield: 0.1 10 g (0.210 mmol, 84 %), off-white gum-like solid

Content of D-Phe-OH: <0.1 %

HRMS: [MH] ⁺ : calculated: 524.2761

found: 524.2740

HRMS: [MNa] ⁺ : calculated: 546.2580

found: 546.2576

[a] _p = -1.5 (c = 0.5 in chloroform)

[α] _η = -35.2 (c = 0.5 in methanol)

¹ H-NMR (300 MHz, CDCI ₃ ): δ = 0.82-0.84 (m, 6H, -CH(-CH ₃ ) ₂ ), 1.36-1.70 (m, 5H, H ₂ N- CO-CH ₂ -CH ₂ - CH ₂ -CH=CH-0 ₂ C-, HN-CH-CH ₂ -CH(-CH ₃ ) ₂ , -CH ₂ -CH(-CH ₃ ) ₂ ), 1 .87-1.96 (m, 2H, H ₂ N-CO-CH ₂ -CH ₂ -), 2.01-2.12 (m, 2H, C0 ₂ -HC=CH-CH ₂ -CH ₂ -), 3.00-3.13 (m, 2H, C ₆ H ₅ -CH ₂ -CH-NH), 4.17-4.27 (m, 1 H, NH-CH(CH ₂ -CH(CH ₃ ) ₂ )-CO-NH), 4.79-4.91 (m, 2H, CO-NH-CH(CH ₂ -Ph)-C0 ₂ -, C0 ₂ -CH=CH-CH ₂ -), 5.02 (s, 2H, HN-C0 ₂ -CH ₂ - C ₆ H ₅ ), 5.33 (d, 1 H, ³ J(H,H) = 7.9 Hz, -CH-CO-NH-CH-), 5.52 (br. s, 1 H, -CH ₂ -CO-NH ₂ ), 5.71 (br. s, 1 H, -CH ₂ -CO-NH ₂ ), 6.94 (d, 1 H, ³ J(H,H) = 6.1 Hz, C0 ₂ -CH=CH-CH ₂ -), 7.04- 7.27 (m, 1 1 H, ΗΑ _γ + -CH-CO-NH-CH(CH ₂ -Ph)-C0 ₂ -).

¹³ C-NMR (75 MHz, CDCI ₃ ): δ = 22.1 , 23.0 (2 ^χ C, CH(-CH ₃ ) ₂ ), 23.7, 24.4 (C0 ₂ -CH=CH- CH ₂ -CH ₂ , C0 ₂ -CH=CH-CH ₂ -CH ₂ ), 24.8 (-CH ₂ -CH(-CH ₃ ) ₂ ), 34.4, 38.1 (C ₆ H ₅ -CH ₂ -CH- N H-, N H ₂ -CO-CH ₂ -CH ₂ -), 41 .5 (N H-CH-CH ₂ -CH-(CH ₃ ) ₂ ), 53.5, 53.7 (2 ^χ N H-CH), 67.2 (H N-C0 ₂ -CH ₂ -C ₆ H ₅ ), 1 14.4 (C0 ₂ -CH=CH-CH ₂ -), 127.2 (C _AR ), 128.1 (2 ^χ C _AR ), 128.4 (CA _T ), 128.7 (2 ^χ C _AR ), 128.7 (2 ^χ C _AR ), 129.5 (2 ^χ C _AR ), 134.8 (C0 ₂ -CH=CH-CH ₂ -), 135.8 (C _q)A r), 136.3 (C _q)A r), 156.3 (N H-C0 ₂ -CH ₂ -C ₆ H ₅ ), 168.2 (C _Q ), 172.3 (C _Q ), 175.4 (C _Q ).

Final conclusion: the tables in Example 5 show that many different amide containing alkynes can be used to obtain a very efficiently catalysed

esterification reaction. Example 6. Ruthenium-catalyzed enol ester formation using the [Ru(TFA) ₂ ((+)- DIOP)] ligand.

The reaction was carried out under argon in an oven-dried Schlenk vessel containing a magnetic stirring bar. Under a flow of argon the vessel was charged with Z-Leu-Phe-OH (0.24 mmol, 1.0 eq), [Ru(TFA) ₂ ((+)-DIOP)] (8.0 mg, 9.7 μηηοΙ, 0.04 eq) and dry 2-propanol (0.5 ml.) degassed prior to use. The material was dissolved by heating to reflux temperature for a few seconds and then the vessel was immediately cooled and immersed in an oil bath preheated to 40°C. 1-hexyne (0.29 mmol, 1.2 eq) was added to the reaction mixture and the mixture was stirred at 40°C. The course of the reaction was monitored by taking samples and analyzing them using HPLC-MS. After 3 h the solution was concentrated to dryness. The tawny solid residue was then redissolved in dichloromethane, the solution evaporated to dryness in the presence of the same quantity of silica gel and finally subjected to a chromatographic purification. Fractions containing the product were pooled and concentrated in vacuo. The product was finally dried at 0.02 mbar.

Yield after flash chromatography: 109 mg (91 %)

Characterization data for this compound are reported in example 2. Example 7. Ruthenium-catalyzed enol ester formation of different di-, tri- and tetrapeptides using 1-hexyne in combination with the Bis-(2-methylallyl)((+)- DIOP)ruthenium(M) ligand.

All reactions were carried out under argon in an oven-dried Schlenk vessel containing a magnetic stirring bar. Under a flow of argon the vessel was charged with Z-Peptide-OH (0.25 mmol, 1.0 eq), Ru-catalyst system Bis-(2- methylallyl)((+)-DIOP)ruthenium(ll) (7.1 mg, 0.01 mmol, 0.04 eq) and dry 2-propanol (0.5 mL) degassed prior to use. If necessary the materials were dissolved by heating to reflux temperature for a few seconds and then the vessel was immediately cooled and immersed in an oil bath preheated to 40°C. Then degassed 1-hexyne (0.3 mmol, 1.2 eq) was added to the mixture using a Hamilton syringe and the mixture was stirred at 40°C. The course of the conversion was monitored by taking samples and analyzing them with HPLC. After the appropriate conversion was reached, the solution was concentrated in vacuo. The tawny solid residue was then redissolved in

dichloromethane, the solution concentrated in vacuo in the presence of the same quantity of silica gel and finally subjected to chromatographic purification. All fractions containing the product were pooled and concentrated in vacuo. The product was finally dried at 0.02 mbar. In all cases the Z-anti-Markovnikov enol ester was the only reaction product.

Nore: in some cases the reaction was performed on a slightly different scale. The Prot- peptide-OH amount is then indicated and all other amounts should be adapted with the same ratio.

R = -(CH ₂ ) ₃ CH ₃ [1 a]

R = -(CH ₂ ) ₃ CONH ₂ [1 b]

Prot = Boc (entry 7), Z for all other entries

Ethanol was used as the solvent instead of 2-propanol; After 3 h at a conversion

65%, a second amount (equal to the first amount) of Ru catalyst system was added; ; ^c n.d. = not determined. This table shows that different peptide sequences and lengths can be used in the catalysed enol ester formation reaction. It should also be noted that also side-chain unprotected serine, tyrosine and methionine residues can be used without side product formation.

Entry 1 was purified using flash column chromatography: (12 g silica gel, 15 x 1 .5 cm, cyclohexane/ethyl acetate = 4:1 (v/v), R _f = 0.26)

Yield: 93.4 mg (92 %)

Content of D-Ala-OH: 0.1

HRMS: [MH] ⁺ : calculated: 419.2546

found: 419.2543

HRMS: [MNa] ⁺ : calculated: 441 .2365

found: 441 .2366

WD = -17.4 (c = 0.5 in chloroform) ¹ H-NMR (300 MHz, CDCI ₃ ): δ = 0.88-0.95 (m, 9H, -CH ₂ -CH ₃ , -CH-(CH ₃ ) ₂ ), 1 .28-1.74 (m, 7H, -CH-CH ₂ -CH-(CH ₃ ) ₂ , -CH ₂ -CH-(CH ₃ ) ₂ , -CH _{2 alky} , _chain ), 1.44 (d, 3H, ³ J(H,H) = 6.9 Hz, CONH-CH-CH ₃ ), 2.10-2.17 (m, 2H, -CH=CH-CH ₂ -CH ₂ -), 4.22-4.26 (m, 1 H, NH-CH- CO-NH), 4.65 (pseudo p, 1 H, ³ J(H,H) = 6.9, 7.2, 7.5 Hz, CO-NH-CH-CH ₃ ), 4.95 (pseudo q, 1 H, ³ J(H,H) = 6.6, 7.5 Hz, C0 ₂ -CH=CH-CH ₂ -), 5.1 1 (s, 2H, HN-C0 ₂ -CH ₂ - C ₆ H ₅ ), 5.23 (br d, 1 H, ³ J(H,H) = 8.1 Hz, C0 ₂ -NH-CH-CH ₂ -), 6.57 (br d, 1 H, ³ J(H,H) = 6.9 Hz, -CH-CO-NH-CH-), 6.97 (d, 1 H, ³ J(H,H) = 6.3 Hz, C0 ₂ -CH=CH-CH ₂ ), 7.30-7.34 (m,

¹³ C-NMR (75 MHz, CDCI ₃ ): δ = 14.0 (CH ₃ -C _a i _ky i chain), 18.3 (CH ₃ -CH-NH-CO-), 22.1 , 22.3, 23.1 24.2, 24.8, 31.3 (C _a i _ip hatic), 41.7 (NH-CH-CH ₂ -CH-(CH ₃ ) ₂ ), 48.1 (CH ₃ -CH-NH-

CO-), 53.5 (NH-CH), 67.3 (HN-C0 ₂ -CH ₂ -C ₆ H ₅ ), 1 15.7 (C0 ₂ -CH=CH-CH ₂ -), 128.2 (2 ^χ

CA _T ), 128.4 (C _Ar ), 128.7 (2 ^χ C _Ar ), 133.9 (C0 ₂ -CH=CH-CH ₂ -), 136.3 (C _q , _Ar ), 156.3 (NH-

C0 ₂ -CH ₂ -C ₆ H ₅ ), 170.1 (Cq), 171 .9 (C _q ).

Entry 2 (performed with 0.75 mmol of Z-Leu-Ala-OH) was purified using flash column chromatography: (43 g silica gel, 40 x 1.8 cm, cyclohexane/ethyl acetate = 1 :7 (v/v), R _f = 0.25)

Yield: 297.0 mg (89 %),

Content of D-Ala-OH: 0.2 %

HRMS: [MNa] ⁺ : calculated: 470.2267

found: 470.2254

[αβ ⁵ = -13.4 (c = 0.5 in chloroform)

mp = 128-139°C

¹ H-NMR (300 MHz, CDCI ₃ ): δ = 0.92 (d, 6H, ³ J(H,H) = 6.2 Hz, -CH ₂ -CH-(CH ₃ ) ₂ ), 1.43 (d, 3H, ³ J(H,H) = 7.1 Hz, NH-CH-CH ₃ ), 1.48-2.47 (m, 9H, -CH ₂ -CH-(CH ₃ ) ₂ , -CH ₂ -CH- (CH ₃ ) ₂ , HC=C-CH ₂ -CH ₂ -, -CH ₂ -CH ₂ -CH ₂ -CO-NH ₂ , -CH ₂ -CH ₂ -CH ₂ -CO-NH ₂ ), 4.20-4.40 (m, 1 H, -C0 ₂ -NH-CH-CO), 4.60 (pseudo p, 1 H, ³ J(H,H) = 7.4 Hz, NH-CH-CH ₃ ), 4.96 (pseudo q, 1 H, ³ J(H,H) = 6.9, 7.5 Hz, C0 ₂ -CH=CH-CH ₂ ), 5.09 (s, 2H, HN-C0 ₂ -CH ₂ - C ₆ H ₅ ), 5.50 (d, 1 H, ³ J(H,H) = 8.3 Hz, -C0 ₂ -NH-CH-CO), 5.83 (br s, 1 H, -CH ₂ -CO-NH ₂ ), 5.90 (br s, 1 H, -CH ₂ -CO-NH ₂ ), 7.02 (d, 1 H, ³ J(H,H) = 6.2 Hz, C0 ₂ -CH=CH-CH ₂ ), 7.17 (d, 1 H, ³ J(H,H) = 6.8 Hz, NH-CH-CH ₃ ), 7.33 (s, 5H, ΗΑ _Γ )-

¹³ C-NMR (75 MHz, CDCI ₃ ): δ = 18.0 (NH-CH-CH ₃ ), 22.1 , 23.1 (-CH-(CH ₃ ) ₂ ), 23.8, 24.5 (2 x C, C0 ₂ -CH=CH-CH ₂ -CH ₂ -, C0 ₂ -CH=CH-CH ₂ -CH ₂ -), 24.8 (CH ₂ -CH-(CH ₃ ) ₂ ), 34.5 (- CH ₂ -CH ₂ -CO-NH ₂ ), 41 .7 (CH ₂ -CH-(CH ₃ ) ₂ ), 48.4 (NH-CH), 53.5 (NH-CH), 67.2 (HN- C0 ₂ -CH ₂ -C ₆ H ₅ ), 1 14.4 (C0 ₂ -CH=CH-CH ₂ ), 128.1 (2 x C _Ar ), 128.3 (C _Ar ), 128.7 (2 x C _Ar ), 135.1 (C0 ₂ -CH=CH-CH ₂ ), 136.3 (C _q,Ar ), 156.4 (HN-C0 ₂ -CH ₂ -C ₆ H ₅ ), 169.8 (C _q ), 172.4 (C _q ), 175.6 (Cq).

Entry 3 was purified using flash column chromatography: (16 g silica gel, 15 x 2 cm, cyclohexane/ethyl acetate = 6:1 (v/v), R _f = 0.20)

Yield: 1 15.0 mg (93 %)

Content of D-Leu-OH: 0.3 %

HRMS: [MNa] ⁺ : calculated: 517.2678

found: 517.2681

[αβ ⁵ = -1.5 (c = 0.5 in chloroform)

¹ H-NMR (300 MHz, CDCI ₃ ): δ = 0.90-0.91 (m, 9H, -CH ₂ -CH ₃ , -CH-(CH ₃ ) ₂ ), 1 .25-1.64 (m, 7H; found: 9H, H-N-CH-CH ₂ -CH-(CH ₃ ) ₂ , -CH ₂ -CH-(CH ₃ ) ₂ , -C0 ₂ -CH=CH-CH ₂ - (CH ₂ ) ₂ -CH ₃ ), 2.13 (pseudo q, 2H, ³ J(H,H) = 7.0 Hz, -CH=CH-CH ₂ -CH ₂ -), 3.02-3.16 (m, 2H, C ₆ H ₅ -CH ₂ -CH-NH), 4.42-4.46 (m, 1 H, NH-CH(CH ₂ -CH(CH ₃ ) ₂ )-CO-NH), 4.60-4.67 (m, 1 H, CO-NH-CH(CH ₂ -Ph)-C0 ₂ -), 4.95 (pseudo q, ³ J(H,H) = 7.4, 6.6 Hz, C0 ₂ -

CH=CH-CH ₂ -), 5.09 (s, 2H, HN-C0 ₂ -CH ₂ -C ₆ H ₅ ), 5.31 (d, 1 H, ³ J(H,H) = 6.5 Hz, C0 ₂ - NH-CH-CH ₂ -), 6.22 (d, 1 H, ³ J(H,H) = 7.4 Hz, -CH-CO-NH-CH-), 6.95 (d, 1 H, ³ J(H,H) = 6.3 Hz, C0 ₂ -CH=CH-CH ₂ ), 7.17-7.34 (m, 10 H, H^)-

¹³ C-NMR (75 MHz, CDCI ₃ ): δ = 14.0 (CH ₃ -(CH ₂ ) ₃ -CH=CH-0 ₂ C), 22.1 , 22.2, 22.6, 24.1 , 24.8, 31.2 (C _a | _iphatic ), 38.3 (C ₆ H ₅ -CH ₂ -CH-NH-), 41.5 (NH-CH-CH ₂ -CH-(CH ₃ ) ₂ ), 50.8, 56.1 (2 x NH-CH), 67.2 (HN-C0 ₂ -CH ₂ -C ₆ H ₅ ), 1 15.5 (C0 ₂ -CH=CH-CH ₂ -), 127.2 (C _Ar ), 128.1 (2 x CA _T ), 128.3 (C _Ar ), 128.6 (2 ^χ C _Ar ), 128.8 (2 ^χ C _Ar ), 129.4 (2 ^χ C _Ar ), 133.7 (C0 ₂ -CH=CH-CH ₂ -), 136.1 (C _q , _Ar ), 136.2 (C _q , _Ar ), 155.9 (NH-C0 ₂ -CH ₂ -C ₆ H ₅ ), 169.7 (C _q ), 170.6 (Cq).

Entry 4 (performed with 0.50 mmol of Z-Phe-Leu-OH) was purified using flash column chromatography: (30 g silica gel, 25 x 2.5 cm, cyclohexane/ethyl acetate = 1 :3 (v/v), R _f = 0.22)

Yield: 240.5 mg (92 %)

Content of D-Leu-OH: 0.1 %

HRMS: [MNa] ⁺ : calculated: 546.2580

found: 546.2588

[a] _p = -5.3 (c = 0.5 in chloroform) ¹ H-NMR (300 MHz, CDCI ₃ ): δ = 0.91 (d, 6H, ³ J(H,H) = 5.6 Hz, -CH(-CH ₃ ) ₂ ), 1 .51-1.84 (m, 5H, H ₂ N-CO-CH ₂ -CH ₂ -, H-N-CH-CH ₂ -CH(-CH ₃ ) ₂ , -CH ₂ -CH(-CH ₃ ) ₂ ), 2.04-2.26 (m, 4H, H ₂ N-CO-CH ₂ -CH ₂ - and HC=C-CH ₂ -CH ₂ -), 3.07 (d, 2H, ³ J(H,H) = 6.5 Hz, C ₆ H ₅ -CH ₂ - CH-NH), 4.56- 4.65 (m, 2H, NH-CH-CO-NH + CO-NH-CH-C0 ₂ ), 4.96 (pseudo q, 1 H, ³ J(H,H) = 6.6, 7.5 Hz, C0 ₂ -CH=CH-CH ₂ -), 5.07 (s, 2H, NH-C0 ₂ -CH ₂ -C ₆ H ₅ ), 5.50 (d, 1 H, ³ J(H,H) = 8.1 Hz, -CH-CO-NH-CH- or -C0 ₂ -NH-CH-), 5.75 and 5.80 (br. s, 2H, -CH ₂ - CO-NH ₂ ), 6.89 (d, 1 H, ³ J(H,H) = 7.2 Hz, -CH-CO-NH-CH- or -C0 ₂ -NH-CH-), 7.01 (d, 1 H, ³ J(H,H) = 6.0 Hz, C0 ₂ -CH=CH-CH ₂ -), 7.16-7.35 (m, 10H, ^).

1 ³ C-NMR (75 MHz, CDCI ₃ ): δ = 22.1 , 22.8 (2 x C, CH(-CH ₃ ) ₂ ), 23.9, 24.5 (2 x C, C0 ₂ - CH=CH-CH ₂ -CH ₂ , C0 ₂ -CH=CH-CH ₂ -CH ₂ ), 24.9 (-CH ₂ -CH(-CH ₃ ) ₂ ), 34.5, 38.6 (2 x C, C ₆ H ₅ -CH ₂ -CH-NH-, NH ₂ -CO-CH ₂ -CH ₂ -), 41.4 (NH-CH-CH ₂ -CH-(CH ₃ ) ₂ ), 51 .1 , 56.1 (2 x NH-CH), 67.2 (HN-C0 ₂ -CH ₂ -C ₆ H ₅ ), 1 14.2 (C0 ₂ -CH=CH-CH ₂ -), 127.2 (C _Ar ), 128.1 (2 χ CA _T ), 128.4 (C _Ar ), 128.7 (2 χ C _Ar ), 128.8 (2 χ C _Ar ), 129.5 (2 x C _Ar ), 135.0 (C0 ₂ -CH=CH- CH ₂ -), 136.2 (C _q ,Ar), 136.4 (C _q , _Ar ), 156.2 (NH-C0 ₂ -CH ₂ -C ₆ H ₅ ), 169.2 (C _q ), 171 .1 (C _q ), 175.5 (Cq).

Entry 5 (performed with 0.10 mmol of Z-Phe-Leu-Ala-OH) was purified using flash column chromatography: (10 g silica gel, 14 ^χ 1 .5 cm, cyclohexane/ethyl acetate = 3:1 (v/v), R _f = 0.13)

Yield: 49.0 mg (87 %)

Content of D-Ala-OH: 0.1 %

HRMS: [MNa] ⁺ : calculated: 588.3049

found: 588.3037

[αβ ⁵ = -23.5 (c = 0.5 in chloroform)

mp = 98°C

¹ H-NMR (300 MHz, CDCI ₃ ): δ = 0.87-0.93 (m, 9H, -CH ₂ -CH ₃ , -CH(-CH ₃ ) ₂ ), 1 .29-1.71 (m, 10H, H-N-CH-CH ₂ -CH-(CH ₃ ) ₂ , -CH ₂ -CH-(CH ₃ ) ₂ , -CH _{2 alky} , _chain , CONH-CH-CH ₃ ), 2.1 1 - 2.18 (m, 2H, HC=CH-CH ₂ -CH ₂ -), 3.08 (d, 2H, ³ J(H,H) = 6.6 Hz, C ₆ H ₅ -CH ₂ -CH-NH), 4.41 -4.48 (m, 2H, NH-CH), 4.61 (p, 1 H, ³ J(H,H) = 7.2 Hz, NH-CH-CH ₃ ), 4.95 (pseudo q, 1 H, ³ J(H,H) = 6.3, 7.5 Hz, C0 ₂ -CH=CH-CH ₂ -), 5.07 (s, 2H, HN-C0 ₂ -CH ₂ -C ₆ H ₅ ), 5.33 (d, 1 H, ³ J(H,H) = 6.3 Hz, -CO-NH-), 6.41 (d, 1 H, ³ J(H,H) = 6.5 Hz, -CO-NH-), 6.71 (d, 1 H, ³ J(H,H) = 5.4 Hz, -CO-NH-), 6.98 (d, 1 H, ³ J(H,H) = 6.3 Hz, C0 ₂ -CH=CH-CH ₂ -), 7.15-7.37 (m, 10H, h _r ). C-NMR (75 MHz, CDCI ₃ ): δ = 14.0 (CH ₃ -C _a i _ky i chain), 18.2 (CH3-CH-NH-CO-), 22.1 , 22.3, 23.0 24.2, 24.7, 31.3 (C _a i _ip hatic), 38.2 (C ₆ H ₅ -CH ₂ -CH-NH-), 41.0 (NH-CH-CH ₂ -CH- (CH ₃ ) ₂ ), 48.1 , 51 .8, 56.3 (3 χ NH-CH), 67.4 (HN-C0 ₂ -CH ₂ -C ₆ H ₅ ), 1 15.7 (C0 ₂ -CH=CH- CH ₂ -), 127.3 (CA _T ), 128.2 (2 x C _Ar ), 128.4 (C _Ar ), 128.7 (2 x C _Ar ), 128.9 (2 x C _Ar ), 129.4 (2 x C _Ar ), 134.0 (C0 ₂ -CH=CH-CH ₂ -), 136.1 (C _q , _Ar ), 136.2 (C _q , _Ar ), 156.2 (NH-C0 ₂ -CH ₂ - C ₆ H ₅ ), 170.0 (Cq), 171 .1 (C _q ), 171 .2 (C _q ).

Entry 6 (performed with 0.20 mmol of Z-Phe-Leu-Ala-OH) was purified using flash column chromatography: (12 g silica gel, 15 ^χ 1.5 cm, cyclohexane/ethyl acetate = 1 :15 (v/v), R _f = 0.31 )

Yield: 102.2 mg (86 %)

Content of D-Ala-OH: 0.1 %

HRMS: [MNa] ⁺ : calculated: 617.2951

found: 617.2950

[α] _η = -13.3 (c = 0.5 in chloroform)

mp = 186-187°C

¹ H-NMR (300 MHz, CDCI ₃ ): δ = 0.85-0.86 (m, 6H, -CH(-CH ₃ ) ₂ ), 1.40-1.53 (m, 4H, -CH ₂ - CH(-CH ₃ ) ₂ , CONH-CH-CH ₃ ), 1 .62-1.85 (m, 4H, H ₂ N-CO-CH ₂ -CH2-, HN-CH-ChU-CH^ CH ₃ ) ₂ , 2.06-2.29 (m, 4H, H ₂ N-CO-CH ₂ -CH ₂ - and HC=CH-CH ₂ -CH ₂ -), 3.06 (d, 2H, ³ J(H,H) = 6.1 Hz, C ₆ H ₅ -CH ₂ -CH-NH), 4.40-4.55 (m, 3H, NH-CH), 4.95 (pseudo q, 1 H, ³ J(H,H) = 6.6, 7.5 Hz, C0 ₂ -CH=CH-CH ₂ -), 5.05 (s, 2H, HN-C0 ₂ -CH ₂ -C ₆ H ₅ ), 5.40 (d, 1 H, ³ J(H,H) = 7.1 Hz, -CO-NH-), 5.89 (bs, 1 H, -CH ₂ -CO-NH ₂ ), 6.02 (bs, 1 H, -CHz-CO-NhU), 6.82 (d, 1 H, ³ J(H,H) = 7.1 Hz, -CO-NH-), 7.01 -7.08 (m, 2H, C0 ₂ -CH=CH-CH ₂ -, -CO- NH-), 7.15-7.37 (m, 10H, Η^).

1 ³ C-NMR (75 MHz, CDCI ₃ ): δ = 17.7 (CH ₃ -CH-NH-CO-), 22.9, 23.1 (2 x C, CH(-CH ₃ ) ₂ ), 23.8, 24.5 (2 x C, C0 ₂ -CH=CH-CH ₂ -CH ₂ , C0 ₂ -CH=CH-CH ₂ -CH ₂ ), 24.8 (-CH ₂ -CH(- CH ₃ ) ₂ ), 34.6, 38.2 (2 x C, C ₆ H ₅ -CH ₂ -CH-NH-, NH ₂ -CO-CH ₂ -CH ₂ -), 41 .8 (NH-CH-CH ₂ - CH-(CH ₃ ) ₂ ), 48.6, 51.1 , 56.1 (3 x NH-CH), 67.4 (HN-C0 ₂ -CH ₂ -C ₆ H ₅ ), 1 14.2 (C0 ₂ - CH=CH-CH ₂ -), 127.3 (C _Ar ), 128.2 (2 x C _Ar ), 128.5 (C _Ar ), 128.7 (2 x C _Ar ), 128.9 (2 x C _Ar ), 129.4 (2 x C _Ar ), 135.2 (C0 ₂ -CH=CH-CH ₂ -), 136.1 (C _q , _Ar ), 136.1 (C _q , _Ar ), 156.4 (NH-C0 ₂ - CH ₂ -C ₆ H ₅ ), 169.5 (Cq), 171 .3 (C _q ), 171.6 (C _q ), 175.7 (C _q ).

Entry 7 was purified using flash column chromatography: (16 g silica gel, 22 ^χ 1.5 cm, cyclohexane/ethyl acetate = 3:1 (v/v), R _f = 0.29) Yield: 1 10.0 mg (86 %)

Content of D-Tyr-OH: 0.2 %

HRMS: [MNa] ⁺ : calculated: 533.2628

found: 533.2591

[( ] _D = -2.5 (c = 0.5 in chloroform)

mp = 126°C

¹ H-NMR (300 MHz, CDCI ₃ ): δ = 0.91 (t, 3H, ³ J(H,H) = 8.7 Hz, -CH ₂ -CH ₃ ), 1.25-1 .40 (m, 13H, -C(CH ₃ ) ₃ , -CH ₂ -(CH2) ₂ -CH ₃ ), 2.12 (pseudo q, 2H,_ ³ J(H,H) = 6.3, 6.9 Hz, -CH=CH- CH2-CH2-), 3.02 (m, 4H, CeHg-ChU-CH-NH, HO-C ₆ H ₄ -CH ₂ -CH-NH ), 4.35 (m, 1 H, NH- CH-CO-NH or CO-NH-CH-C0 ₂ -), 4.86 (pseudo q, 1 H, ³ J(H,H) = 5.4, 7.5 Hz, NH-CH- CO-NH or CO-NH-CH-CO2-), 4.95 (pseudo q, 1 H, ³ J(H,H) = 6.3, 7.5 Hz, C0 ₂ -CH=CH- CH ₂ -), 5.06 (d, 1 H, ³ J(H,H) = 6.2 Hz, C0 ₂ -NH-CH-CH ₂ - or -CH-CO-NH-CH-), 6.40 (s, 1 H, C ₆ H ₅ -OH), 6.53 (d, 1 H, ³ J(H,H) = 7.1 Hz, -CH-CO-NH-CH- or C0 ₂ -NH-CH-CH ₂ -), 6.66 (d, 2H, ³ J(H,H) = 8.3 Hz, H _Ar ), 6.83 (d, 2H, ³ J(H,H) = 8.4 Hz, H _Ar ), 6.94 (d, 1 H, ³ J(H,H) = 6.3 Hz, C0 ₂ -CH=CH-CH ₂ ), 7.15-7.30 (m, 5 H, ΗΑ _Γ ).

¹³ C-NMR (75 MHz, CDCI ₃ ): δ = 14.0 (CH ₃ -C _a i _ky i _chain ), 22.3, 24.3 (C _a ii _Ph atic), 28.4 (- C(CH ₃ ) ₃ ), 31.4 (Caiip _h a _f c), 37.3, 38.4 (2 C, C ₆ H ₅ -CH ₂ -CH-NH-, HO-C ₆ H ₄ -CH ₂ -CH-NH-), 53.4, 55.8 (2 x C, NH-CH), 80.7 ((-C(CH ₃ ) ₃ ), 1 15.7, 1 15.9 (3 x C, C0 ₂ -CH=CH-CH ₂ -, C _Ar,Tyr ), 126.9 (C _q , _Ar ), 127.2 (C _Ar ), 128.8 (2 x C _Ar ), 129.4 (2 x C _Ar ), 130.5 (2 x C _Ar ), 133.6 (C0 ₂ -CH=CH-CH ₂ -), 136.4 (C _q , _Ar ), 155.4, 155.7 (2 x C, NH-C0 ₂ -CH ₂ -C ₆ H ₅ ,

(C _q , _Ar,Tyr ), 168.6 (C _q ), 171.2 (C _q ).

Entry 8 was purified using flash column chromatography: (10 g silica gel, 14 ^χ 1.5 cm, cyclohexane/ethyl acetate = 2:1 (v/v), R _f = 0.26)

Yield: 83 mg (76 %)

Content of D-Ser-OH: 0.1 %

HRMS: [MNa] ⁺ : calculated: 457.2314

found: 457.2359

M _D ^{= +} 5-4 (c = 0.5 in chloroform)

mp = 148-149°C

¹ H-NMR (300 MHz, CDCI ₃ ): δ = 0.88-0.93 (m, 6H, CH ₂ -CH ₂ -CH ₂ -CH ₃ , -CH(-CH ₃ )-CH ₂ - CH ₃ ), 0.97 (d, 3H, ³ J(H,H) = 6.7 Hz, -CH(-CH ₃ )-CH ₂ -CH ₃ ), 1 .10-1.43 (m, 6H, CH ₂ -CH ₂ - CH ₂ -CH ₃ , CH ₂ -CH ₂ -CH ₂ -CH ₃ , -CH(-CH ₃ )-CH2-CH ₃ ), 1 .56 (br s, 1 H), 1 .81 -1.92 (m, 1 H, - CH(-CH ₃ )-CH ₂ -CH ₃ ), 2.08-2.19 (m, 2H, C0 ₂ -CH=CH-CH ₂ -CH ₂ -), 3.92-4.02 (m, 2H, NH- CH-CH ₂ -OH), 4.07 (pseudo t, 1 H, ³ (H, H) = 7.8, 7.5 Hz, NH-CH-CH-(-CH ₃ )-CH ₂ -CH ₃ ), 4.76-4.79 (m, 1 H, NH-CH-CH ₂ -OH), 4.93-5.14 (m, 3H, C0 ₂ -CH=CH-CH ₂ , HN-C0 ₂ -CH ₂ - C ₆ H ₅ ), 5.53 (d, 1 H, ³ J(H,H) = 8.2 Hz, HN-CH-CH(-CH ₃ )-CH ₂ -CH ₃ ), 7.00 (d, 1 H, ³ J(H,H) = 6.3 Hz, C0 ₂ -CH=CH-CH ₂ ), 7.05 (d, 1 H, ³ J(H, H) = 7.8 Hz, NH-CH-CH ₂ -OH), 7.28-7.37 (m, 5H, H _Ar ).

¹³ C-NMR (75 MHz, CDCI ₃ ): δ = 1 1 .3 14.0, 15.6 (CH ₂ -CH ₂ -CH ₂ -CH ₃ , -CH(-CH ₃ )-CH ₂ - CH ₃ , -CH(-CH ₃ )-CH ₂ -CH ₃ ), 22.3, 24.3, 25.0 (3 x C, CH ₂ -CH ₂ -CH ₂ -CH _3, CH ₂ -CH ₂ -CH ₂ - CH _3, -CH(-CH ₃ )-CH ₂ -CH ₃ ), 31 .3 (CH ₂ -CH ₂ -CH ₂ -CH ₃ ), 37.4 (-CH(-CH ₃ )-CH ₂ -CH ₃ ), 54.7, 60.1 (2 x C, HN-CH-), 62.9 (NH-CH-CH ₂ -OH), 67.4 (HN-C0 ₂ -CH ₂ -C ₆ H ₅ ), 1 16.0 (C0 ₂ - CH=CH-CH ₂ ), 128.2 (2 x C _Ar ), 128.4 (C _Ar ), 128.7 (2 x C _Ar ), 133.9 (C0 ₂ -CH=CH-CH ₂ ), 136.2 (C _q ,Ar), 156.9 (HN-C0 ₂ -CH ₂ -C ₆ H ₅ ), 167.7 (C _q ), 171 .9 (C _q ).

Entry 9 was purified using flash column chromatography: (15 g silica gel, 20 x 1 .5 cm, cyclohexane/ethyl acetate = 1 :10 (v/v), R _f = 0.22)

Yield: 98.9 mg (78 %)

Content of D-Pro-OH: 0.3 %

HRMS: [MNa] ⁺ : calculated: 530.2267

found: 530.2245

M _D ^{= +} 6-6 (c = 0.5 in chloroform)

1 H-NMR (300 MHz, CDCI ₃ ): δ = 1 .59-2.39 (m, 10H, C]i-CH ₂ -CH ₂ -CO-N H ₂ , -CH ₂ -CH ₂ - CH ₂ -CO-NH ₂ , -CH ₂ -CH ₂ -CH ₂ -CO-N H ₂ , -CH ₂ -CH ₂ -CH ₂ -N-CH-C0 ₂ , -CH ₂ -CH2-CH ₂ -N- CH-C0 ₂ ), 2.89-3.13 (m, 2H, C ₆ H ₅ -CH ₂ -CH-NH-), 3.23-3.71 (m, 2H, -CH ₂ -CH ₂ -CH ₂ -N- CH-C0 ₂ ), 4.49-4.53 (m, 1 H, -CH ₂ -CH ₂ -CH ₂ -N-CH-C0 ₂ ), 4.70 (pseudo q, 1 H, ³ J(H,H) = 8.1 , 6.9 Hz, C ₆ H ₅ -CH ₂ -CH-NH-), 4.93-5.10 (m, 3H, C0 ₂ -CH=CH-CH _2, HN-C0 ₂ -CH ₂ - C ₆ H ₅ ), 5.41 (br s, 1 H, -CH ₂ -CO-NH ₂ ), 5.50 (d, 1 H, ³ J(H, H) = 8.1 Hz, C ₆ H ₅ -CH ₂ -CH-NH- ), 6.08 (br. s, 1 H, -CH ₂ -CO-NH ₂ ), 7.06 (d, 2H, ³ J(H, H) = 6.2 Hz, C0 ₂ -CH=CH-CH ₂ ), 7.22-7.37 (m, 10H, ΗΙΑ _Γ ).

¹³ C-NMR (75 MHz, CDCI ₃ ): δ = 23.8, 24.1 , 25.2 (3 x C, CH ₂ -CH ₂ -CH ₂ -CO-NH ₂ , -CH ₂ - CH ₂ -CH ₂ -CO-NH ₂ , -CH ₂ -CH ₂ -CH ₂ -N-CH-C0 ₂ ), 29.1 (-CH ₂ -CH ₂ -CH ₂ -N-CH-C0 ₂ ), 34.4, 38.8 (2 x C, C ₆ H ₅ -CH ₂ -CH-NH, -CH ₂ -CH ₂ -CO-N H ₂ ), 47.2 (-CH ₂ -CH ₂ -CH ₂ -N-CH-C0 ₂ ), 59.1 (-CH ₂ -CH ₂ -CH ₂ -N-CH-C0 ₂ ), 67.0 (C ₆ H ₅ -CH ₂ -CH-NH-), 1 14.4 (C0 ₂ -CH=CH-CH ₂ ), 127.2 (C _Ar ), 128.2 (2 x C _Ar ), 128.3 (C _Ar ), 128.7 (4 x C _Ar ), 129.8 (2 x C _Ar ), 135.3 (C0 ₂ - CH=CH-CH ₂ ), 135.9, 136.4 (2 x C _q,Ar ), 155.9 (HN-C0 ₂ -CH ₂ -C ₆ H ₅ ), 169.3 (C _q ), 170.8

(Cq), 175.6 (Cq). Entry 10 was purified using flash column chromatography: (15 g silica gel, 20 x 1 .5 cm, cyclohexane/ethyl acetate = 1 :3 (v/v), R _f = 0.26)

Yield: 106.0 mg (83 %)

Content of D-Val-OH: 0.2 %

HRMS: [MNa] ⁺ : calculated: 532.2424

found: 532.2412

[( ] _D = -4.1 (c = 0.5 in chloroform)

¹ H-NMR (300 MHz, CDCI ₃ ): δ = 0.89 (pseudo t, 6H, ³ J(H,H) = 6.9, 7.2 Hz, -CH(-CH ₃ ) ₂ ), 1.62-1.75 (m, 2H, H ₂ N-CO-CH ₂ -CH ₂ -), 1.99-2.26 (m, 5H, H ₂ N-CO-CH ₂ -CH ₂ -, HC=C- CH ₂ -CH ₂ -, -CH(-CH ₃ ) ₂ ), 2.99-3.13 (m, 2H, C ₆ H ₅ -CH ₂ -CH-NH), 4.53 (dd, 1 H, ³ J(H,H) = 8.8, 5.4 Hz, H-N-CH-CH(-CH ₃ ) ₂ ) 4.58-4.65 (m, 1 H, NH-CH-CO-NH), 4.97 (pseudo q, ³ J(H,H) = 7.8, 6.3 Hz, 1 H, C0 ₂ -CH=CH-CH ₂ -), 5.07 (s, 2H, C ₆ H ₅ -CH ₂ -C0 ₂ -NH), 5.57 (d, 1 H, ³ J(H,H) = 7.7 Hz, -NH-CH-), 5.69 (br s, 1 H, -CH2-CO-NH ₂ ), 5.93 (br s, 1 H, -CH ₂ - CO-NH ₂ ), 6.95 (d, 1 H, ³ J(H,H) = 6.2 Hz, C0 ₂ -CH=CH-CH ₂ -), 7.08 (d, 1 H, ³ J(H,H) = 8.0 Hz, -NH-CH-), 7.16-7.38 (m, 10H, ΗΑ _Γ ).

¹³ C-NMR (75 MHz, CDCI ₃ ): δ = 18.0, 18.9 (-CH-(CH ₃ ) ₂ ), 24.0, 24.5 (2 x C, C0 ₂ - CH=CH-CH ₂ -CH ₂ -, C0 ₂ -CH=CH-CH ₂ -CH ₂ -), 31.5 (-CH-(CH ₃ ) ₂ ), 34.5 (C ₆ H ₅ -CH ₂ -CH- NH), 38.7 (-CH ₂ -CH ₂ -CO-NH ₂ ), 56.2, 57.7 (NH-CH-), 67.2 (HN-C0 ₂ -CH ₂ -C ₆ H ₅ ), 1 14.1 (C0 ₂ -CH=CH-CH ₂ ), 127.1 (C _Ar ), 128.1 (2 x C _Ar ), 128.3 (C _Ar ), 128.7 (2 x C _Ar ), 128.8 (2 x CA _T ), 129.4 (2 x C _Ar ), 134.8 (C0 ₂ -CH=CH-CH ₂ ), 136.3 (C _q,Ar ), 136.5 (C _q,Ar ), 156.2 (HN- C0 ₂ -CH ₂ -C ₆ H ₅ ), 168.1 (C _q ), 171 .3 (C _q ), 175.5 (C _q ).

Entry 1 1 was purified using flash column chromatography: (15 g silica gel, 20 x 1.5 cm, cyclohexane/ethyl acetate = 4:1 (v/v), R _f = 0.26)

Yield: 1 1 1.0 mg (87 %)

Content of D-Met-OH: 0.7 %

HRMS: [MNa] ⁺ : calculated: 535.2242

found: 535.2215

M _D ^{= +} 1 -0 ( ^{c =} 0-5 in chloroform)

mp = 108-109°C

¹ H-NMR (300 MHz, CDCI ₃ ): δ = 0.90 (t, 3H, ³ J(H,H) = 7.1 Hz, -CH ₂ -CH ₃ ), 1.28-1 .41 (m, 5H, -CH ₂ -CH ₂ -CH ₂ -CH ₃ , -CH ₂ -CH ₂ -CH ₂ -CH ₃ ), 1.91-2.20 (m, 7H, -CH ₂ -CH ₂ -S-CH ₃ , - CH ₂ -CH ₂ -S-CH ₃ , -CH=CH-CH ₂ -CH ₂ -), 2.41 (t, 2H, ³ J(H,H) = 7.3 Hz, -CH ₂ -CH ₂ -S-CH ₃ ), 2.97-3.26 (m, 2H, C ₆ H ₅ -CH ₂ -CH-NH), 4.45 (pseudo q, 1 H, ³ J(H,H) = 7.2, 6.9 Hz, C ₆ H ₅ - CH ₂ -CH-NH-), 4.71 -4.77 (m, 1 H, -CO-NH-CH-C0 ₂ -), 4.96 (pseudo q, 1 H, ³ (H,H) = 7.5, 6.3 Hz, -CH=CH-CH ₂ -CH ₂ -), 5.09 (s, 2H, -HN-C0 ₂ -CH ₂ -C ₆ H ₅ ), 5.29 (d, 1 H, ³ J(H,H) = 4.8 Hz, C ₆ H ₅ -CH ₂ -CH-NH-), 6.54 (d, 1 H, ³ J(H,H) = 7.6 Hz, -CO-NH-CH-C0 ₂ -), 6.96 (d, 2H, ³ J(H,H) = 6.3 Hz, C0 ₂ -CH=CH-CH ₂ -CH ₂ -), 7.17-7.38 (m, 10H, H ).

1 ³ C-NMR (75 MHz, CDCI ₃ ): δ = 14.0 (CH ₃ -C _a i _k yi chain), 15.5 (CH ₂ -S-CH ₃ ), 22.3, 24.3,

29.8, 31.3, 31 .5 (5 x C, -CH ₂ -C _a i _ky i chain), 38.4 (C ₆ H ₅ -CH ₂ -CH-NH), 51.6, 56.3 (2 x C, NH- CH), 67.4 (HN-C0 ₂ -CH ₂ -C ₆ H ₅ ), 1 15.9 (-CH=CH-CH ₂ -CH ₂ -), 127.3 (C _Ar ), 128.2 (2 x C _Ar ), 128.4 (C _Ar ), 128.7 (2 x C _Ar ), 128.9 (2 x C _Ar ), 129.5 (2 x C _Ar ), 133.8 (-CH=CH-CH ₂ -CH ₂ - ), 136.2 (2 x C _q ,Ar), 156.0 (HN-C0 ₂ -CH ₂ -C ₆ H ₅ ), 168.8 (C _q ), 170.9 (C _q ).

Example 7: Enzymatic peptide coupling using Z-anti-Markovnikov enol esters

Two tripeptides were prepared, i.e. Z-Leu-Phe-Phe-NH ₂ and Z-Leu- Ala-Phe-NH ₂ . For the pretreatment of the enzyme, Alcalase-CLEA OM (0.8 g) was washed under inert conditions with dry tert-butanol (3 x 5 mL; 50°C) under a gentle stream of argon in a funnel with a sintered glass disc. Tert-butanol was removed by suction filtration taking care that the tert-butanol never became too cold to solidify. The enzyme was washed with dry MTBE (3 x 5 mL) under a gentle stream of argon. The solvent was removed by suction-filtration. The enzyme was dried under vacuum (20 mbar) for 30 min at room temperature.

Z-Leu-Phe-Z-anti-Markovnikov-enolester or Z-Leu-Ala-Z-anti-

Markovnikov-enolester (0.02 mmol, 1.0 eq), H-Phe-NH ₂ (4.9 mg, 0.03 mmol, 1.5 eq) and pretreated Alcalase-CLEA OM (50 mg) were placed in a 5 mL vial. Then dry THF or toluene (1 mL) was added with a syringe and subsequently 3A molecular sieves (beads, 0.5 g) were added. The 5 mL vials were sealed and the reactions were carried out at 50°C and 150 rpm using a shaker (Infors Multitron AJ 1 12). After 18 h of shaking, the reaction mixtures were analyzed by HPLC-MS.

Z-L-Leu-L-Ala-L-Phe-NH ₂

Z-L-l_eu-L-Ala-Z-anf/-Markovnikov-eno

This table shows that the Z-anti-Markovnikov enol esters are excellent substrates in serine endopeptidase catalysed peptide synthesis.

Example 8: Enzymatic synthesis of Z-Leu-Phe-Phe-NH ₂ under Soxhlet conditions

For the pretreatment of the enzyme Alcalase-CLEA OM all manipulations were conducted under a gentle stream of argon. First 0.3 g Alcalase- CLEA OM was washed with distilled water (10 mL) under inert conditions in a funnel with a sintered glass disc and with dry te/t-butanol (3 x 4 mL; 50°C). Tert-butanol was removed by suction filtration under argon, taking care that the tert-butanol never became too cold to solidify. Finally, the enzyme was washed with dry MTBE (3 x 4 mL). The solvent was removed by suction filtration under argon and then the enzyme was dried under vacuum (20 mbar) for 30 min at room temperature. A reflux condenser, a 10 ml. dropping funnel with pressure release, a 10 ml. two-necked flask and a magnetic stirring bar were assembled, dried under vacuum with a heat gun, and flushed with argon. The two-necked flask was charged with pretreated Alcalase-CLEA OM (100 mg) and dry THF (2 ml_, dried over sodium). The 3A molecular sieves (beads, 1.5 g) were placed into the dropping funnel together with a plug of cotton to prevent the molecular sieves from falling into the reaction solution. The reflux condenser was connected to a cryostat containing a cooling agent at -15°C. The magnetic stirrer was adjusted to 50 rpm and the oil bath was heated to 50°C. After adjusting the reduced pressure to enable reflux conditions (270 mbar), the reaction mixture was refluxed for 1 h in the presence of Alcalase-CLEA OM, allowing the recondensing solvent to flow through the molecular sieves back to the reaction flask. Afterwards the pressure was adjusted by using argon to atmospheric pressure. Z-Leu-Phe-Z-anf/-Markovnikov-enolester based on 1-hexyne (19.8 mg, 0.04 mmol, 1.0 eq) and H-Phe-NH ₂ (9.9 mg, 0.06 mmol, 1.5 eq) were added under a gentle stream of argon and the reaction was continued under reduced pressure at 50°C and magnetic stirring (50 rpm). After 17 h the reaction mixture was diluted with DMSO (HPLC-grade) until all precipitated compounds were in solution. The enzyme was removed by filtration (0.2 μηη, Pall) and the resulting solution was subjected to HPLC analysis, which indicated that 75% of the enol ester had been converted to the desired tripeptide and no side-products had been formed.

This result shows that Soxhiet conditions can be successfully applied for the enzymatic peptide synthesis reaction using enol esters. Example 9: Synthesis of Z-Leu-Phe-Phe-NH ₂ using different amide containing enol esters as starting material

The same reaction conditions as for Example 7 were used.

This table shows that different amide containing enol esters can be used as substrate for serine endopeptidase catalyzed peptide synthesis and that the enol esters with R = CH=CHCH ₂ NHCOCH ₃ CH=CH(CH ₂ ) ₃ CONH ₂ , and CH=CH(CH ₂ ) ₂ CONH ₂ are preferred.

Example 10: Enzymatic synthesis of Z-Leu-Phe-Phe-NH ₂ using Soxhlet conditions

For the pretreatment of the enzyme Alcalase-CLEA OM all manipulations were conducted under air. First 0.8 g Alcalase-CLEA OM was washed with distilled water (20 mL) in a funnel with a sintered glass disc. Alcalase-CLEA OM was washed with dry tert-butanol (4 x 5 mL; 30°C). Tert-butanol was removed by suction filtration, taking care that tert-butanol never became too cold to solidify. Finally, the enzyme was washed with dry MTBE (4 5ml_). The solvent was removed by suction filtration. The enzyme was dried under vacuum (20 mbar) for 30 min at ambient temperature.

A reflux-condenser, a 10 mL dropping funnel with pressure release, a 25 mL two- necked flask and a magnetic stirring bar were assembled, dried under vacuum with a heat gun, and flushed with argon. Under argon the 3A molecular sieves (1 .5 g) were placed into the dropping funnel together with a cotton plug to prevent the molecular sieves from falling into the reaction solution. The reflux condensor was connected to a cryostat containing a cooling agent at -15°C. The two-necked flask was charged with Alcalase-CLEA OM (150 mg), H-Phe-NH ₂ (14.8 mg, 0.09 mmol, 1 .5 eq) and Z-Leu- Phe-Z-anti-Markovnikov-enol ester based on hex-5-ynamide (31 .4 mg, 0.06 mmol, 1 .0 eq) in 3 mL of THF. The magnetic stirrer was adjusted to 100 rpm and the oil bath was heated to 50°C. Immediately the reduced pressure was adjusted to 270 mbar to enable reflux conditions, allowing the recondensing solvent to flow through the molecular sieves back to the reaction flask. 0.9 mL DMSO was added after 23 h before measuring the conversion by HPLC-MS. TH F was removed in vacuo using a rotary evaporator. Afterwards DMSO was removed in vacuo (0.02 mbar). Product Z-Leu-Phe- Phe-NH ₂ partially precipitated from THF. The product remaining dissolved in THF was purified by flash column chromatography (DCM/MeOH 20:1 ) yielding Z-Leu-Phe-Phe- NH ₂ in a combined yield of 30.4 mg (91 %) (22.4 mg 67% precipitated, 8.0 mg 24% isolated by flash column chromatography).

R _f = 0.15 (dichloromethane/methanol = 20: 1 (v/v))

Content of D-Phe-OH: <0.1 %

HRMS: [MNa] ⁺ : calculated: 581 .2740

found: 581 .2767

[O |D = -17.3 (c = 0.5 in DMSO)

mp = 182-183°C

¹ H-NMR (300 MHz, DMSO-c ₆ ): δ = 0.78-0.83 (m, 6H, -CH(-CH ₃ ) ₂ ), 1 .24-1 .49 (m, 3H, HN-CH-CH2-CH(-CH ₃ ) ₂ , -CH ₂ -CH(-CH ₃ ) ₂ ), 2.73-3.02 (m, 4H, 2 x C ₆ H ₅ -CH ₂ -CH-NH), 3.97 (m, 1 H, C0 ₂ -N H-CH-CO-NH), 4.44-4.49 (m, 2H, CO-NH-CH-CO-NH-, CO-NH- CH-CO-NH ₂ ), 5.01 (s, 2H, HN-C0 ₂ -CH ₂ -C ₆ H ₅ ), 7.09-7.34 (m, 18H, ^, 3 x NH), 7.92 (d, 1 H, ³ J(H, H) = 7.0 Hz, NH), 8.09 (d, 1 H, ³ J(H, H) = 7.2 Hz, NH).

1 ³ C-NMR (75 MHz, DMSO-c ₆ ): δ = 22.4, 22.9, 24.1 (3 x C, CH(-CH ₃ ) ₂ , -CH ₂ -CH(- CH ₃ ) ₂ ), 37.4, 37.6 (2 x C, C ₆ H ₅ -CH ₂ -CH-NH-), 40.7 (NH-CH-CH ₂ -CH-(CH ₃ ) ₂ ), 53.2, 53.6, 53.7 (3 x N H-CH), 65.4 (H N-C0 ₂ -CH ₂ -C ₆ H ₅ ), 126.1 (C _AR ), 126.2 (C _AR ), 127.6 (2 x CA _T ), 127.7 (C _AR ), 127.9 (2 x C _AR ), 128.0 (2 x C _AR ), 128.3 (2 x C _AR ), 129.1 (2 x C _AR ), 129.2 (2 x CA _T ), 137.0 (C _Q , _AR ), 1 37.5 (C _Q , _AR ), 1 37.7 (C _Q , _AR ), 155.8 (N H-C0 ₂ -CH ₂ -C ₆ H ₅ ), 1 70.6 (Cq), 171 .9 (Cq), 172.5 (C _q ).

This example shows that high product yields can be obtained for the enzymatic coupling reaction using Soxhiet conditions.