WO2014067746A1 - Process for the enzymatic formation of amide bonds - Google Patents

Abstract

The present invention is concerned with processes for the enzymatic formation of amide bonds. More specifically the invention is concerned with the formation of amide bonds between amino acid esters acylated on the amino terminus to nicotinic acid or isonicotinic acid and amino compounds with the aid of proteases. The invention provides processes for amide formation employing amino acid esters, connected to nicotinoyl- or isonicotinoyl-groups via an amide bond on the amino terminus, that are more efficient than processes for amide formation employing amino acid esters carrying standard protecting groups such as benzoyl or Z on their amino terminus. In one aspect therefore, the present invention, further, relates to the protease mediated synthesis of organogellant compounds (OG) as depicted below wherein L is a linking moiety of molecular weight from 14 g/mol to 500 g/mol, one of X¹, X² is nitrogen and the other is carbon, and wherein R¹ are sidechain substituents.

Description

Process for the Enzymatic Formation of Amide Bonds

The present invention is concerned with the formation of amide bonds with the aid of proteases in general. One aspect of the present invention, further, relates to the protease mediated synthesis of organogellant compounds (OG) as depicted below

wherein L is a linking moiety of molecular weight from 14 g/mol to 500 g/mol, one of X¹, X² is nitrogen and the other is carbon, and wherein R¹ are sidechain substituents.

Organogellant compounds as depicted above are known in the art to serve as gellants to thicken liquid compositions. Such gellants have, for example, been described in WO 201 1 /1 12912 A1 and WO 201 1 /1 12887 A1 .

Organogellant compounds also termed organogellants herein, in general are used to provide structure and a pleasant texture to liquid consumer products such as, for example, liquid detergent compositions. Furthermore, organogellants can be used to stabilize other components within such compositions such as, for example, enzymes and bleaches. However, organogellants need to be selected carefully for their respective application in order to prevent incompatibilities between organogellant and other components as well as unwanted side effects such as clouding.

Organogellants of the present invention offer significant advantages over other gellants currently in use, such as being compatible with a broad range of consumer products as well as not affecting product clarity.

Synthetic access to organogellants is described in WO 201 1/1 12887 A1 . However, the syntheses described therein are expensive and time-consuming. Accordingly, there is a need in the field for cheaper and faster access to organogellants.

Significant efforts have been devoted in the past to the development of methods for amide bond-formation with the aid of proteases, also termed as "reverse proteolysis" (cf. e.g. Bordusa F (2002) Chem Rev (102) 4817-4867). Typically in a reverse proteolysis reaction a carboxyester compound is reacted with an amino compound in the presence of a protease which catalyses formation of the amide bond. Importantly, if the carboxyester compound itself contains an amino function, as in the case of amino acid esters, care has to be taken to protect or acylate this amino group in order to prevent polymerization of the amino acid.

As presented herein, it was now found that the choice of the acyl-group on the amino terminus of amino acid esters has significant influence on the coupling yield of protease mediated amide bond formig reactions employing such amino-acylated amino acid esters.

More specifically, it was found that amino acid esters connected via an amide bond on their amino terminus to nicotinic acid (pyridine-3-carboxylic acid )or isonicotinic acid (pyridine-4-carboxylic acid ) result in better yields during protease mediated coupling to amino compounds than amino acid esters carrying other moieties on their amino terminus such as for example typical protecting groups Benzoyl- or Z- (Carboxybenzyl).

Protease mediated amide bond formation employing amino acid esters connected via an amide bond on their amino terminus to nicotinic acid or isonicotinic acid, accordingly, provides efficient access to organogellants and peptides.

Accordingly, the present invention provides processes for the manufacture of compounds according to formula I

(I)

comprising reacting a compound of formula II with a compound of formula III in a solvent S, at a Temperature Θ, in the presence of a protease P, wherein compound II is defined as compound II = T or compound II = Q, with

L

H₂N NH₂

(II = T)

and

(II = Q) and

compound (III) is defined as

wherein

M is selected from

, when compound II = T; and

, when compound II = Q;

R¹ and R² are independently selected from hydrogen atom, Ci-C₄ alkyl, Ci-C₄ hydroxyalkyl, Ci-C₄ thioether, C6-C20 aryl, C7-C20 alkylaryl, C7-C20

alkylhydroxyaryl, C₄-C2o alkyl heteroaryl, Ci-C₄ alkyl-C(O)Y; one of X¹, X² is nitrogen, the other is carbon;

L is selected from C2-C20 alkyl, C6-C20 aryl, C7-C20 alkylaryl;

X and Y are independently selected from -OH, -NH₂, -NHR³, -NR³R⁴;

R³ and R⁴ are independently selected from C1-C6 alkyl and C₇-C20 alkylaryl; n is selected from 0, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20;

R^A is selected from C2-C20 alkyl, C6-C20 aryl, C7-C20 alkylaryl. The following definitions are used in the context of the present invention: An alkyl is a linear, branched, or cyclic hydrocarbon chain. It may also be a combination of linear, branched, and cyclic hydrocarbon chains. A C_n-C_m alkyl is an alkyl having n to m carbon atoms.

An aryl is an aromatic hydrocarbon. An aryl may be monocyclic or polycyclic. In the case of polycyclic aryls, the individual aromatic rings may be fused or may be connected by single carbon-carbon bonds. Examples of suitable aryls are phenyl, biphenyl, naphtyl, anthryl, or phenanthryl. A C_n-C_m aryl is an aryl having n to m carbon atoms.

A heteroaryl is an aromatic hydrocarbon that contains 1 to 4 heteroatoms, preferably 1 to 2 heteroatoms. Heteroatoms are independently selected from nitrogen, oxygen, sulfur. A heteroaryl may be monocyclic or polycyclic. A heteroaryl may be attached to the main molecule through any of its carbon or nitrogen atoms. A C_n-C_m heteroaryl is a heteroaryl having n to m carbon atoms and 1 to 4 heteroatoms.

An alkylaryl is an aryl that is substituted with one or more alkyls. An alkylaryl may be attached to the remainder of the molecule through any of its alkyl or aryl carbon atoms. A C_n-C_m alkylaryl contains n to m carbon atoms.

An a Iky I heteroaryl is a heteroaryl that is substituted with one or more alkyls. The alkyl substituents may be attached to the heteroaryl through any of the carbon- or heteroatoms of the heteroaryl. The alkylheteroaryl group may be attached to the remainder of the molecule through any of the alkyl carbon atoms and/or the heteroaryl carbon- or heteroatoms.

A hydroxyalkyl is an alkyl carrying one or more hydroxyl groups. A C_n-C_m

hydroxyalkyl group contains n to m carbon atoms.

A thioether is a moiety wherein two alkyls are linked by a thioether bond. A C_n-C_m thioether group contains n to m carbon atoms in total. The thioether group may be attached to the remainder of the molecule through any of its carbon atoms.

An alkylhydroxyaryl is an alkylaryl, carrying hydroxyl groups on any of the aryl carbon atoms. The alkylhydroxyaryl group may be attached to the remainder of the molecule through any of its alkyl and/or aryl carbon atoms. A C_n-C_m alkylhydroxyaryl contains n to m carbon atoms.

An alkyl-C(O)Y is an alkyl carrying a C(O)Y-group, wherein C(O) is a carbonyl function and Y is selected from -OH, -NH₂, -NHR³, -NR³R⁴; and wherein R³ and R⁴ are independently selected from C1-C6 alkyl and C7-C20 alkylaryl. A C_n-C_m alkyl- C(O)Y contains n to m carbon atoms within the carbonyl-bound alkyl excluding the carbonyl carbon atom itself.

The process of the present invention is performed in a solvent S. Suitable solvents S are mixtures of from 90% (v/v) to 99.9% (v/v) aprotic organic solvent and 10% (v/v) to 0.1 % (v/v) aqueous buffer at a pH between pH 6.5 and pH 8.5.

Suitable aprotic organic solvents comprise dichloromethane, methyl terf-butyl ether, tetrahydrofuran, acetonitrile, 1 ,4-Dioxane, ethylene glycol dimethyl ether, methyl isobutyl ketone, terf-Butanol, methyl ethyl ketone, acetone or mixtures thereof. In the context of the present invention terf-Butanol is considered as an aprotic organic solvent.

A suitable buffer in the context of the present invention is phosphate buffer. Other suitable buffering agents are well known in the art. Suitable buffering agents comprise TAPSO (3-[N-Tris(hydroxymethyl)methylamino]-2-hydroxypropanesulfonic Acid), HEPES (4-2-hydroxyethyl-1 -piperazineethanesulfonic acid), TES (2- {[tris(hydroxymethyl)methyl]amino}ethanesulfonic acid), MOPS (3-(N- morpholino)propanesulfonic acid). Preferably, the buffers of the present invention contain 1 mM of calcium chloride. Further, preferably, the buffers of the present invention contain 1 %(w/v) of urea.

The process of the present invention is performed at a temperature Θ. Suitable temperatures Θ are temperatures selected in relation to ΘΟΡΤ, the temperature optimum of the protease P employed. ΘΟΡΤ, the temperature optimum of a protease P is the temperature where the protease is most efficient at cleaving amide bonds in aqueous buffer. The temperature optimum ΘΟΡΤ is known for a number of proteases and can thus be determined from the literature by a person of skill. Alternatively, the temperature optimum ΘΟΡΤ of a protease P can be determined by a person of skill in the art with experiments regarding the temperature dependence of the velocity of proteolysis reactions involving model substrates of the respective protease. Suitable temperatures Θ for performing the processes of the present invention are selected in the range from (ΘΟΡΤ - 50°C) to (ΘΟΡΤ + 10°C) with the proviso that the reaction mixture used for performing the process should be in the liquid state at the

temperature Θ selected.

The process of the present invention is performed with the aid of a protease P. The protease P can be any protease that is capable of forming the corresponding amide bond under the reaction conditions of the process of the present invention. In order to determine suitability of a protease for use in the process of the present invention a person of skill in the art can for example perform an experiment analogous to examples 2, 3.1 1 or 3.12 as presented herein. As shown experimentally (cf. Example 2) serine proteases and cysteine proteases are well suited for performing the process of the present invention, while aspartic acid proteases and metalloproteases are less/not suitable for performing the process of the present invention. Therefore, in a preferred embodiment of the present invention the protease P is a serine protease or a cysteine protease. In another preferred embodiment of the present invention the protease P is a subtil isin-like serine protease (subtilisin-like serine proteases are defined in Siezen RJ and Leunissen JAM (1997) Protein Science 6: 501-523). In another preferred embodiment of the present invention the protease P is selected from Achromopeptidase from Achromobacter lyticus, Ficin from fig tree latex, Papain from papaya latex, Protease (Subtilisin Carlsberg) from Bacillus licheniformis, Alcalase CLEA (Subtilisin) from Bacillus licheniformis, Protease from Streptomyces griseus, Proteinase K from Tritirachium album, Trypsin from bovine pancreas, a- Chymotrypsin from bovine pancreas, Clostripain from Clostridium histolyticum, Protease P "Amano" 6SD from Aspergillus melleus. In another preferred embodiment of the present invention the protease P is Subtilisin, i.e. EC 3.4.21 .62 according to IUBMB nomenclature. Compounds according to formula (ll=T) are commercially available, compounds according to formula (ll=Q) and formula (III) are accessible via standard peptide chemistry well known to a person of skill in the art.

Reaction products according to formula (I) can be isolated from the reaction mixture according to standard procedures well known to a person of skill. An exemplary procedure would be performed as follows: An amount of water about equal to the volume of the reaction mixture is added to the reaction mixture and the pH is adjusted to 1 -2 by addition of cone. HCI. The bulk of organic solvents is distilled off. The aqueous phase is then separated and washed with isopropyl acetate. The organic phases are discarded. The aqueous phase is concentrated and an amount of isopropyl acetate about equal to the volume of the concentrated aqueous phase is added and the pH is adjusted to 10-1 1 by addition of NaOH solution. Subsequently phase separation is performed at 70 °C. The organic phase is separated and washed with water at 70 °C. The organic phases are discarded. The combined aqueous phases are cooled to 0 °C and the precipitate is filtered off, washed with cold isopropyl acetate and dried at 60 °C in vacuo.

In a preferred embodiment of the present invention compound II is selected from compound II = T with

(II = T),

and

L is selected from C2-C20 alkyl, C6-C20 aryl, C7-C20 alkylaryl.

In another preferred embodiment of the present invention compound II is selected from compound II = T with

(II = T),

and

L is selected from C6-C12 linear alkyl, 1 ,4-dimethylcyclohexyl, xylene.

In another preferred embodiment of the present invention compound II is selected from compound II = T with compound II = T selected from the following list of diamines

In another preferred embodiment of the present invention compound II is selected from compound II = T with

(II = T),

and

L is selected from C2-C20 alkyl.

In another preferred embodiment of the present invention R^A is selected from C2-C20 alkyl, C7-C20 alkylaryl.

In another preferred embodiment of the present invention R^A is selected from C2-C20 alkyl.

In another preferred embodiment of the present invention R¹ is selected from hydrogen atom, Ci-C₄ alkyl, Ci-C₄ thioether, C6-C20 aryl, C7-C20 alkylaryl.

In another preferred embodiment of the present invention R¹ is selected from a hydrogen atom, an n-butyl group, a f-butyl group, a propyl group, a cyclopropyl group, an ethyl group, or one of the side chains of the amino acids alanine, valine, leucine, isoleucine, methionine, phenylalanine, tyrosine, tryptophan, serine, threonine, glutamine, asparagine. In another particularly preferred embodiment, R¹ is selected from one of the side chains of amino acids alanine, valine, leucine, isoleucine, or phenylalanine. Here, the expression "side chain" refers to the substituent group attached to the a-carbon atom of an α-amino acid. For the above- mentioned amino acids, the side chains are methyl, isopropyl, isobutyl, sec-butyl, 2- thiomethyl-ethyl, benzyl, 4-hydroxybenzyl, 3-methylindol, hydroxymethyl, 1 - hydroxyethyl, carboxamidoethyl, carboxamidomethyl.

In another preferred embodiment of the present invention R¹ is selected from Ci-C₄ alkyl.

In another preferred embodiment of the present invention the solvent S is selected from S1 = mixture of 90% (v/v) to 99.9% (v/v) aprotic organic solvent and 10% (v/v) to 0.1 % (v/v) aqueous buffer at a pH between pH 6.5 and pH 8.5, S2 = mixture of 90% (v/v) to 99.9% (v/v) aprotic organic solvent and 10% (v/v) to 0.1 % (v/v) aqueous buffer at a pH between pH 7 and pH 8, S3 = mixture of 90% (v/v) to 99.9% (v/v) acetonitrile and 10% (v/v) to 0.1 % (v/v) aqueous buffer at a pH between pH 6.5 and pH 8.5, S4 = mixture of 90% (v/v) to 99.9% (v/v) 1 ,4-Dioxane and 10% (v/v) to 0.1 % (v/v) aqueous buffer at a pH between pH 6.5 and pH 8.5, S5 = mixture of 90% (v/v) to 99.9% (v/v) ethylene glycol dimethyl ether and 10% (v/v) to 0.1 % (v/v) aqueous buffer at a pH between pH 6.5 and pH 8.5, S6 = mixture of 90% (v/v) to 99.9% (v/v) tert- Butanol and 10% (v/v) to 0.1 % (v/v) aqueous buffer at a pH between pH 6.5 and pH 8.5.

In another preferred embodiment of the present invention the solvent S is selected from S7 = mixture of 95% (v/v) to 99% (v/v) aprotic organic solvent and 5% (v/v) to 1 % (v/v) aqueous buffer at a pH between pH 6.5 and pH 8.5, S8 = mixture of 95% (v/v) to 99% (v/v) acetonitrile and 5% (v/v) to 1 % (v/v) aqueous buffer at a pH between pH 6.5 and pH 8.5, S9 = mixture of 95% (v/v) to 99% (v/v) 1 ,4-Dioxane and 5% (v/v) to 1 % (v/v) aqueous buffer at a pH between pH 6.5 and pH 8.5, S10 = mixture of 95% (v/v) to 99% (v/v) ethylene glycol dimethyl ether and 5% (v/v) to 1 % (v/v) aqueous buffer at a pH between pH 6.5 and pH 8.5, S1 1 = mixture of 95% (v/v) to 99% (v/v) terf-Butanol and 5% (v/v) to 1 % (v/v) aqueous buffer at a pH between pH 6.5 and pH 8.5.

In another preferred embodiment of the present invention the solvent S is selected from S12 = mixture of 95% (v/v) to 99% (v/v) aprotic organic solvent and 5% (v/v) to 1 % (v/v) aqueous buffer at a pH between pH 7 and pH 8, S13 = mixture of 95% (v/v) to 99% (v/v) acetonitrile and 5% (v/v) to 1 % (v/v) aqueous buffer at a pH between pH 7 and pH 8, S14 = mixture of 95% (v/v) to 99% (v/v) 1 ,4-Dioxane and 5% (v/v) to 1 % (v/v) aqueous buffer at a pH between pH 7 and pH 8, S15 = mixture of 95% (v/v) to 99% (v/v) ethylene glycol dimethyl ether and 5% (v/v) to 1 % (v/v) aqueous buffer at a pH between pH 7 and pH 8, S16 = mixture of 95% (v/v) to 99% (v/v) terf-Butanol and 5% (v/v) to 1 % (v/v) aqueous buffer at a pH between pH 7 and pH 8.

In a preferred embodiment of the present invention the temperature Θ is selected in the range from (ΘΟΡΤ - 30°C) to (ΘΟΡΤ + 5°C) with the proviso that the reaction mixture used for performing the process should be in the liquid state at the

temperature Θ selected. In another preferred embodiment of the present invention the temperature Θ is selected in the range from (ΘΟΡΤ - 10°C) to (ΘΟΡΤ + 5°C) with the proviso that the reaction mixture used for performing the process should be in the liquid state at the temperature Θ selected. In another preferred embodiment of the present invention the temperature Θ is selected as 37°C.

In a preferred embodiment of the present invention the protease P is a serine protease or a cysteine protease. In another preferred embodiment of the present invention the protease P is a subtil isin-like serine protease (subtilisin-like serine proteases are defined in Siezen RJ and Leunissen JAM (1997) Protein Science 6: 501-523). In another preferred embodiment of the present invention the protease P is selected from Achromopeptidase from Achromobacter lyticus, Ficin from fig tree latex, Papain from papaya latex, Protease (Subtilisin Carlsberg) from Bacillus licheniformis, Alcalase CLEA (Subtilisin) from Bacillus licheniformis, Protease from Streptomyces griseus, Proteinase K from Tritirachium album, Trypsin from bovine pancreas, a-Chymotrypsin from bovine pancreas, Clostripain from Clostridium histolyticum, Protease P "Amano" 6SD from Aspergillus melleus. In another preferred embodiment of the present invention the protease P is Subtilisin, i.e. EC 3.4.21 .62 according to lUBMB nomenclature.

In another preferred embodiment of the present invention compound II is selected from compound II = T with

(II = T),

and with

M selected from

and with

L selected from C2-C20 alkyl, C6-C20 aryl, C7-C20 alkylaryl, and with

R^A selected from C2-C20 alkyl, C6-C20 aryl, C7-C20 alkylaryl, and with

R¹ selected from hydrogen atom, Ci-C₄ alkyl, Ci-C₄ thioether, C6-C20 aryl, C7-C20 alkylaryl,

and with

one of X¹, X² is nitrogen, the other is carbon,

and with

the solvent S is S1 = mixture of 90% (v/v) to 99.9% (v/v) aprotic organic solvent and 10% (v/v) to 0.1 % (v/v) aqueous buffer at a pH between pH 6.5 and pH 8.5,

and with

the temperature Θ selected in the range from θ = (ΘΟΡΤ - 50°C) to Θ = (ΘΟΡΤ + 10°C) with the proviso that the reaction mixture used for performing the process should be in the liquid state at the temperature Θ selected,

and with

the protease P is a serine protease or a cysteine protease.

In another preferred embodiment of the present invention compound II is selected from compound II = T with

(II = T),

and with

M selected from

and with

L selected from C2-C20 alkyl, C6-C20 aryl, C7-C20 alkylaryl, and with

R^A selected from C2-C20 alkyl, C6-C20 aryl, C7-C20 alkylaryl, and with

R¹ selected from hydrogen atom, Ci-C₄ alkyl, Ci-C₄ thioether, C6-C20 aryl, C7-C20 alkylaryl,

and with

one of X¹ , X² is nitrogen, the other is carbon,

and with

the solvent S is S1 = mixture of 90% (v/v) to 99.9% (v/v) aprotic organic solvent and 10% (v/v) to 0.1 % (v/v) aqueous buffer at a pH between pH 6.5 and pH 8.5,

and with

the temperature Θ selected in the range from θ = (ΘΟΡΤ - 50°C) to Θ = (ΘΟΡΤ + 10°C) with the proviso that the reaction mixture used for performing the process should be in the liquid state at the temperature Θ selected,

and with

the protease P is a subtil isin-like serine protease.

In another preferred embodiment of the present invention compound II is selected from compound II = T with

(II = T),

and with M selected from

and with

L selected from C2-C20 alkyl,

and with

R^A selected from C2-C20 alkyl, C6-C20 aryl, C7-C20 alkylaryl, and with

R¹ selected from hydrogen atom, Ci-C₄ alkyl, Ci-C₄ thioether, C6-C20 aryl, C7-C20 alkylaryl,

and with

X¹ is carbon and X² is nitrogen,

and with

the solvent S selected from S2 = mixture of 90% (v/v) to 99.9% (v/v) aprotic organic solvent and 10% (v/v) to 0.1 % (v/v) aqueous buffer at a pH between pH 7 and pH 8, S3 = mixture of 90% (v/v) to 99.9% (v/v) acetonitrile and 10% (v/v) to 0.1 % (v/v) aqueous buffer at a pH between pH 6.5 and pH 8.5, S4 = mixture of 90% (v/v) to 99.9% (v/v) 1 ,4- Dioxane and 10% (v/v) to 0.1 % (v/v) aqueous buffer at a pH between pH 6.5 and pH 8.5, S5 = mixture of 90% (v/v) to 99.9% (v/v) ethylene glycol dimethyl ether and 10% (v/v) to 0.1 % (v/v) aqueous buffer at a pH between pH 6.5 and pH 8.5, S6 = mixture of 90% (v/v) to 99.9% (v/v) terf-Butanol and 10% (v/v) to 0.1 % (v/v) aqueous buffer at a pH between pH 6.5 and pH 8.5,

and with

the temperature Θ selected in the range from θ = (ΘΟΡΤ - 50°C) to Θ = (ΘΟΡΤ + 10°C) with the proviso that the reaction mixture used for performing the process should be in the liquid state at the temperature Θ selected,

and with

the protease P is a serine protease or a cysteine protease.

In another preferred embodiment of the present invention compound II is selected from compound II = T with

(II = T),

and with

M selected from

R¹ O

and with

L selected from C2-C20 alkyl,

and with

R^A selected from C2-C20 alkyl, C6-C20 aryl, C7-C20 alkylaryl, and with

R¹ selected from hydrogen atom, Ci-C₄ alkyl, Ci-C₄ thioether, C6-C20 aryl, C7-C20 alkylaryl,

and with

X¹ is carbon and X² is nitrogen,

and with

the solvent S selected from S2 = mixture of 90% (v/v) to 99.9% (v/v) aprotic organic solvent and 10% (v/v) to 0.1 % (v/v) aqueous buffer at a pH between pH 7 and pH 8, S3 = mixture of 90% (v/v) to 99.9% (v/v) acetonitrile and 10% (v/v) to 0.1 % (v/v) aqueous buffer at a pH between pH 6.5 and pH 8.5, S4 = mixture of 90% (v/v) to 99.9% (v/v) 1 ,4- Dioxane and 10% (v/v) to 0.1 % (v/v) aqueous buffer at a pH between pH 6.5 and pH 8.5, S5 = mixture of 90% (v/v) to 99.9% (v/v) ethylene glycol dimethyl ether and 10% (v/v) to 0.1 % (v/v) aqueous buffer at a pH between pH 6.5 and pH 8.5, S6 = mixture of 90% (v/v) to 99.9% (v/v) terf-Butanol and 10% (v/v) to 0.1 % (v/v) aqueous buffer at a pH between pH 6.5 and pH 8.5,

and with

the temperature Θ selected in the range from θ = (ΘΟΡΤ - 50°C) to Θ = (ΘΟΡΤ + 10°C) with the proviso that the reaction mixture used for performing the process should be in the liquid state at the temperature Θ selected,

and with

the protease P is a subtil isin-like serine protease.

In another preferred embodiment of the present invention compound II is selected from compound II = Q wit

= Q).

and with

M selected from

and with

R^A selected from C2-C20 alkyl, C6-C20 aryl, C7-C20 alkylaryl, and with

R¹ and R² independently selected from hydrogen atom, Ci-C₄ alkyl, Ci- C₄ hydroxyalkyl, Ci-C₄ thioether, C6-C20 aryl, C7-C20 alkylaryl, C7-C20 alkylhydroxyaryl, C₄-C2o alkyl heteroaryl,

and with

one of X¹ , X² is nitrogen, the other is carbon,

and with

X selected from -OH, -NH₂, -NHR³, -NR³R⁴;

and with

R³ and R⁴ independently selected from C1-C6 alkyl and C₇-C20 alkylaryl; and with

n selected from 0, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10;

and with

the solvent S is S1 = mixture of 90% (v/v) to 99.9% (v/v) aprotic organic solvent and 10% (v/v) to 0.1 % (v/v) aqueous buffer at a pH between pH 6.5 and pH 8.5,

and with

the temperature Θ selected in the range from θ = (ΘΟΡΤ - 50°C) to Θ = (ΘΟΡΤ + 10°C) with the proviso that the reaction mixture used for performing the process should be in the liquid state at the temperature Θ selected,

and with

the protease P is a serine protease or a cysteine protease.

In another preferred embodiment of the present invention compound II is selected from compound II = Q wit

(II = Q)

and with

M selected from

and with

R^A selected from C2-C20 alkyl, C6-C20 aryl, C7-C20 alkylaryl, and with

R¹ and R² independently selected from hydrogen atom, Ci-C₄ alkyl, Ci- C₄ hydroxyalkyl, Ci-C₄ thioether, C6-C20 aryl, C7-C20 alkylaryl, C7-C20 alkylhydroxyaryl, C₄-C2o alkyl heteroaryl,

and with

one of X¹ , X² is nitrogen, the other is carbon,

and with

X selected from -OH, -NH₂, -NHR³, -NR³R⁴;

and with

R³ and R⁴ independently selected from C1-C6 alkyl and C7-C20 alkylaryl; and with

n selected from 0, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10;

and with

the solvent S is S1 = mixture of 90% (v/v) to 99.9% (v/v) aprotic organic solvent and 10% (v/v) to 0.1 % (v/v) aqueous buffer at a pH between pH 6.5 and pH 8.5,

and with

the temperature Θ selected in the range from θ = (ΘΟΡΤ - 50°C) to Θ = (ΘΟΡΤ + 10°C) with the proviso that the reaction mixture used for performing the process should be in the liquid state at the temperature Θ selected,

and with

the protease P is a subtil isin-like serine protease.

In another preferred embodiment of the present invention compound II is selected from compound II = Q wit

= 0),

and with

M selected from

and with

R^A selected from C2-C20 alkyl, C6-C20 aryl, C7-C20 alkylaryl, and with

R¹ and R² independently selected from hydrogen atom, Ci-C₄ alkyl, Ci- C₄ hydroxyalkyl, Ci-C₄ thioether, C6-C20 aryl, C7-C20 alkylaryl, C7-C20 alkylhydroxyaryl, C₄-C2o alkylheteroaryl,

and with

X¹ is carbon and X² is nitrogen,

and with

X selected from -OH,

and with

n selected from 0, 1 , 2, 3,

and with

the solvent S selected from S2 = mixture of 90% (v/v) to 99.9% (v/v) aprotic organic solvent and 10% (v/v) to 0.1 % (v/v) aqueous buffer at a pH between pH 7 and pH 8, S3 = mixture of 90% (v/v) to 99.9% (v/v) acetonitrile and 10% (v/v) to 0.1 % (v/v) aqueous buffer at a pH between pH 6.5 and pH 8.5, S4 = mixture of 90% (v/v) to 99.9% (v/v) 1 ,4- Dioxane and 10% (v/v) to 0.1 % (v/v) aqueous buffer at a pH between pH 6.5 and pH 8.5, S5 = mixture of 90% (v/v) to 99.9% (v/v) ethylene glycol dimethyl ether and 10% (v/v) to 0.1 % (v/v) aqueous buffer at a pH between pH 6.5 and pH 8.5, S6 = mixture of 90% (v/v) to 99.9% (v/v) terf-Butanol and 10% (v/v) to 0.1 % (v/v) aqueous buffer at a pH between pH 6.5 and pH 8.5,

and with

the temperature Θ selected in the range from θ = (ΘΟΡΤ - 50°C) to Θ = (ΘΟΡΤ + 10°C) with the proviso that the reaction mixture used for performing the process should be in the liquid state at the temperature Θ selected,

and with

the protease P is a serine protease or a cysteine protease.

In another preferred embodiment of the present invention compound II is selected from compound II = Q wit

(N = Q).

and with

M selected from

and with

R^A selected from C2-C20 alkyl, C6-C20 aryl, C7-C20 alkylaryl, and with

R¹ and R² independently selected from hydrogen atom, Ci-C₄ alkyl, Ci- C₄ hydroxyalkyl, Ci-C₄ thioether, C6-C20 aryl, C7-C20 alkylaryl, C7-C20 alkylhydroxyaryl, C₄-C2o alkyl heteroaryl,

and with

X¹ is carbon and X² is nitrogen,

and with

X selected from -OH,

and with

n selected from 0, 1 , 2, 3,

and with

the solvent S selected from S2 = mixture of 90% (v/v) to 99.9% (v/v) aprotic organic solvent and 10% (v/v) to 0.1 % (v/v) aqueous buffer at a pH between pH 7 and pH 8, S3 = mixture of 90% (v/v) to 99.9% (v/v) acetonitrile and 10% (v/v) to 0.1 % (v/v) aqueous buffer at a pH between pH 6.5 and pH 8.5, S4 = mixture of 90% (v/v) to 99.9% (v/v) 1 ,4- Dioxane and 10% (v/v) to 0.1 % (v/v) aqueous buffer at a pH between pH 6.5 and pH 8.5, S5 = mixture of 90% (v/v) to 99.9% (v/v) ethylene glycol dimethyl ether and 10% (v/v) to 0.1 % (v/v) aqueous buffer at a pH between pH 6.5 and pH 8.5, S6 = mixture of 90% (v/v) to 99.9% (v/v) terf-Butanol and 10% (v/v) to 0.1 % (v/v) aqueous buffer at a pH between pH 6.5 and pH 8.5,

and with

the temperature Θ selected in the range from θ = (ΘΟΡΤ - 50°C) to Θ = (ΘΟΡΤ + 10°C) with the proviso that the reaction mixture used for performing the process should be in the liquid state at the temperature Θ selected,

and with

the protease P is a subtilisin-like serine protease.

In another preferred embodiment of the present invention R² is selected from a hydrogen atom or one of the side chains of amino acids alanine, valine, leucine, isoleucine, methionine, phenylalanine, tyrosine, tryptophan, serine, threonine, glutamine, asparagine. In another particularly preferred embodiment, R² is selected from one of the side chains of amino acids alanine, valine, leucine, isoleucine, or phenylalanine. Here, the expression "side chain" refers to the substituent group attached to the a-carbon atom of an α-amino acid. For the above-mentioned amino acids, the side chains are methyl, isopropyl, isobutyl, sec-butyl, 2-thiomethyl-ethyl, benzyl, 4-hydroxybenzyl, 3-methylindol, hydroxymethyl, 1 -hydroxyethyl,

carboxamidoethyl , carboxam idomethyl .

Examples

1 . HPLC analysis

The amount of ester substrates, mono- and di-amide products and hydrolysis byproducts were measured by HPLC using a C18 column and acetonitrile /phosphate buffer pH2.3 as solvent.

2. Enzyme screening via hydrolysis

10U of enzyme preparation was weighed in 1 .5ml vials and dissolved in 848μΙ phosphate buffer pH 7.5 (containing 250mM potassium phosphate and 1 mM calcium chloride) or phosphate buffer pH 2.5 (containing 250mM potassium phosphate, 500mM citric acid and 1 mM calcium chloride). 152μΙ solution of 100mM Isonicotinoyl- L-valine methylester or 100 mM Z-L-valine methylester, dissolved in acetonitrile was added and the vials were shaken for 22h at 25°C or 37°C. Conversion of

Isonicotinoyl-L-valine methylester to Isonicotinoyl-L-valine and of Z-L-valine methylester to Z-L-valine was analysed after 22 hours by HPLC.

Enzyme Protease Type Reaction Conversion Conversion parameter to to

IsonicotinoyI- Z-L-valine

L-valine [mM] [mM]

Achromopeptidase Serine protease pH7.5 / 80.1 63,4 from Achromobacter 37°C

lyticus

Ficin from fig tree Cysteine pH7.5 / 98.3 95,8 latex protease 37°C

Papain from papaya Cysteine pH7.5 / 91 .0 62,5 latex protease 25°C

Protease from Aspartatic acid pH2.5 / 4.9 3,0 Aspergillus saitoi protease 37°C

Protease (Subtil isin Serine protease pH7.5 / 100 93,5 Carlsberg) from 37°C

Bacillus licheniformis

Alcalase CLEA Serine protease pH7.5 / 100 98,5 (Subtilisin) from 37°C

Bacillus licheniformis

Protease from Serine protease pH7.5 / 89.0 61 ,6 Streptomyces 37°C

griseus (Streptogrisin B)

Proteinase K from Serine protease pH7.5 / 99.6 91 ,3 Tritirachium album 37°C

Trypsin from bovine Serine protease pH7.5 / 22.6 3,5 pancreas 25°C

a-Chymotrypsin from Serine protease pH7.5 / 91 .6 88,5 bovine pancreas 25°C

Clostripain from Cysteine pH7.5 / 27.6 7,3

Clostridium protease 25°C

histolyticum

Pepsin from porcine Aspartatic acid pH2.5 / 1 .9 1 ,7 gastric mucosa protease 37°C

Thermolysin from Metal loprotease pH7.5 / 5.2 4,0 Bacillus 37°C

thermoproteolyticus

rokko

Protease P "Amano" Serine protease pH7.5 / 39.8 20,1 6SD from Aspergillus 37°C

melleus (Seaprose)

No enzyme (blank) pH2.5 / <1 <1

37°C

No enzyme (blank) pH7.5 / <1 <1

37°C

3. Reaction of Isonicotinoyl-L-valine ester and 1 ,12-Diaminododecane to N,N-Bis- (Isonicotinoyl-L-valovD-l ,12-diaminododecane

3.1 Comparison of different enzymes

500μηηοΙβ of 1 ,12-Diaminododecane was weighed in 1 .5ml vials and dissolved in 950μΙ acetonitrile solution, containing l OO mole of Isonicotinoyl-L-valine methylester. 1 .Omg of enzyme preparation, dissolved in 50μΙ phosphate buffer pH 7.5 (containing 250mM potassium phosphate and 1 mM calcium chloride) was added and the vials were shaken for 24h at 37°C and 1000rpm. Conversion of Isonicotinoyl-L-valine methylester to the respective mono- and diamide was analysed after 24 hours by HPLC.

3.2 Comparison of different solvents

225 mole of 1 ,12-Diaminododecane and 500μηηοΙβ of Isonicotinoyl-L-valine methylester were weighed in 1 .5ml vials and dissolved in 950μΙ solvent. 5. Omg of Subtilisin Carlsberg from Bacillus licheniformis preparation, dissolved in 50μΙ phosphate buffer pH 7.5 (containing 250mM potassium phosphate and 1 mM calcium chloride) was added and the vials were shaken for 24h at 37°C and l OOOrpm.

Conversion of Isonicotinoyl-L-valine methylester to the respective mono- and diamide was analysed after 24 hours by HPLC.

3.3 Comparison of different amounts of buffer

Substrate mixtures containing 225 mole of 1 ,12-Diaminododecane, 500μηηοΙβ of Isonicotinoyl-L-valine methylester and 950-990μΙ acetonitrile, depending on the volume of buffer were prepared. 5.0mg of Subtilisin Carlsberg from Bacillus licheniformis preparation was weighed in 1 .5ml vials and dissolved in 10-50μΙ phosphate buffer pH 7.5 (containing 250mM potassium phosphate and 1 mM calcium chloride). The substrate mixture was added and the vials were shaken for 24h at 37°C and l OOOrpm.

3.4 Comparison of different additives

Substrate mixtures containing 225μηηοΙβ of 1 ,12-Diaminododecane, 500μηηοΙβ of Isonicotinoyl-L-valine methylester, the desired amount of additive and 950μΙ (with DMSO) or 990μΙ (with Urea) acetonitrile were prepared. 5.0mg of Subtilisin Carlsberg from Bacillus licheniformis preparation was weighed in 1 .5ml vials and dissolved in 10μΙ phosphate buffer pH 7.5 (containing 250mM potassium phosphate and 1 mM calcium chloride). The substrate mixture was added and the vials were shaken for 24h at 37°C and l OOOrpm.

3.5 Comparison of different temperatures using 95% acetonitrile with 5% buffer as solvent

225 mole of 1 ,12-Diaminododecane and 500μηηοΙβ of Isonicotinoyl-L-valine methylester were weighed in 1 .5ml vials and dissolved in 950μΙ acetonitrile. 5.0mg of Subtilisin Carlsberg from Bacillus licheniformis preparation, dissolved in 50μΙ phosphate buffer pH 7.5 (containing 250mM potassium phosphate and 1 mM calcium chloride) was added and the vials were shaken for 24h at different temperatures and l OOOrpm.

3.6 Comparison of different temperatures using 99% acetonitrile with 1 % buffer as solvent

A substrate mixture containing 225 mole of 1 ,12-Diaminododecane, 500μηηοΙβ of Isonicotinoyl-L-valine methylester and 990μΙ acetonitrile was prepared. 5.0mg of Subtilisin Carlsberg from Bacillus licheniformis preparation was weighed in 1 .5ml vials and dissolved in 10μΙ phosphate buffer pH 7.5 (containing 250mM potassium phosphate and 1 mM calcium chloride). The substrate mixture was added and the vials were shaken for 24h at different temperatures and 1000rpm.

3.7 Comparison of different substrate concentrations

Substrate mixtures containing the desired amounts of 1 ,12-Diaminododecane and Isonicotinoyl-L-valine methylester and 950μΙ acetonitrile were prepared. For 1200mM ester and saturated di-amine the mixture was filtrated after stirring 18h at 50°C to remove undissolved di-amine. 5.0mg of Subtilisin Carlsberg from Bacillus

licheniformis preparation, dissolved in 50μΙ phosphate buffer pH 7.5 (containing 250mM potassium phosphate and 1 mM calcium chloride) was added to the substrate mixture and the vials were shaken for 24h at 37°C and 1000rpm.

Conversion to Conversion to

Concentration Concentration of di-amide mono-amide of ester [mM] di-amine [mM] [mM/24h] [mM/24h]

100 50 0.1 2.1

500 225 2.3 60.4

max 389.6

1200 171 .9

(saturated) 9.5 3.8 Comparison of different enzyme concentrations using 99% acetonitrile with 1 % buffer as solvent

A substrate mixture containing 225 mole of 1 ,12-Diaminododecane, 500μηηοΙβ of Isonicotinoyl-L-valine methylester and 990μΙ acetonitrile was prepared. The desired amount of Subtilisin Carlsberg from Bacillus licheniformis preparation was weighed in 1 .5ml vials and 10μΙ phosphate buffer pH 7.5 (containing 250mM potassium phosphate and 1 mM calcium chloride) was added. The substrate mixture was added and the vials were shaken for 24h at 37°C and l OOOrpm.

3.9 Comparison different enzyme concentrations using 95% acetonitrile with 5% buffer as solvent

A substrate mixture containing 225 mole of 1 ,12-Diaminododecane, 500μηηοΙβ of Isonicotinoyl-L-valine methylester and 950μΙ acetonitrile was prepared. The desired amount of Subtilisin Carlsberg from Bacillus licheniformis preparation was weighed in 1 .5ml vials and 50μΙ phosphate buffer pH 7.5 (containing 250mM potassium phosphate and 1 mM calcium chloride) was added. The substrate mixture was added and the vials were shaken for 24h at 37°C and 1000rpm.

3.10 Comparison of different Isonicotinoyl-L-valine esters

Substrate mixtures containing 225 mole of 1 ,12-Diaminododecane, 500μηηοΙβ of the desired Isonicotinoyl-L-valine ester and 990μΙ acetonitrile were prepared. 5.0mg of Subtilisin Carlsberg from Bacillus licheniformis preparation was weighed in 1 .5ml vials and dissolved in 10μΙ phosphate buffer pH 7.5 (containing 250mM potassium phosphate and 1 mM calcium chloride). The substrate mixture was added and the vials were shaken for 24h at 37°C and 1000rpm.

Conversion to Conversion to

di-amide mono-amide

Ester [mM/24h] [mM/24h]

-OtBu 0 2.0

-OMe 5.3 83.7

^*) similar conversion wit hout enzyme

3.1 1 Comparison of different acyl/protection groups using the methylester as the substrate

Substrate mixtures containing 225 mole of 1 ,12-Diaminododecane, 500μηηοΙβ of the desired N-protected/acylated L-valine methylester and 990μΙ acetonitrile were prepared. 5.0mg of Subtilisin Carlsberg from Bacillus licheniformis preparation was weighed in 1 .5ml vials and dissolved in 10μΙ phosphate buffer pH 7.5 (containing 250mM potassium phosphate and 1 mM calcium chloride). The substrate mixture was added and the vials were shaken for 24h at 37°C and 1000rpm.

3.12 Comparison of different acyl/protection groups using the phenylester as the substrate

Substrate mixtures containing 225 mole of 1 ,12-Diaminododecane, 500μηηοΙβ of the desired N-protected/acylated L-valine phenylester and 990μΙ acetonitrile were prepared. 5.0mg of Subtilisin Carlsberg from Bacillus licheniformis preparation was weighed in 1 .5ml vials and dissolved in 10μΙ phosphate buffer pH 7.5 (containing 250mM potassium phosphate and 1 mM calcium chloride). The substrate mixture was added and the vials were shaken for 24h at 37°C and 1000rpm.

Claims

Claims

Process for the manufacture of compounds according to formula I

(I)

comprising reacting a compound of formula II with a compound of formula in a solvent S, at a temperature Θ, in the presence of a protease P, wherein compound II is defined as compound II = T or compound II = Q, with

= T),

and

= Q).

and

compound (III) is defined as

wherein

M is selected from

und II = T; and

R¹ and R² are independently selected from hydrogen atom, Ci-C₄ alkyl, Ci-C₄ hydroxyalkyl, Ci-C₄ thioether, C6-C20 aryl, C7-C20 alkylaryl, C7-C20

alkylhydroxyaryl, C₄-C2o alkyl heteroaryl, Ci-C₄ alkyl-C(O)Y; one of X¹ , X² is nitrogen, the other is carbon;

L is selected from C2-C20 alkyl, C6-C20 aryl, C7-C20 alkylaryl;

X and Y are independently selected from -OH, -NH₂, -NHR³, -NR³R⁴;

R³ and R⁴ are independently selected from C1-C6 alkyl and C₇-C20 alkylaryl; n is selected from 0, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20;

R^A is selected from C2-C20 alkyl, C6-C20 aryl, C7-C20 alkylaryl.

Process according to claim 1 , wherein compound II is selected from compound II = T with

(II = T),

and wherein

L is selected from C2-C20 alkyl, C6-C20 aryl, C7-C20 alkylaryl.

3. Process according to claim 2, wherein L is selected from C2-C20 alkyl.

4. Process according to claims 1 to 3, wherein R¹ is selected from hydrogen atom, Ci-C₄ alkyl, Ci-C₄ thioether, C6-C20 aryl, C7-C20 alkylaryl.

5. Process according to claims 1 to 4, wherein R¹ is selected from Ci-C₄ alkyl.

6. Process according to claims 1 to 5, wherein the solvent S is selected from S1 = mixture of 90% (v/v) to 99.9% (v/v) aprotic organic solvent and 10% (v/v) to 0.1 % (v/v) aqueous buffer at a pH between pH 6.5 and pH 8.5, S2 = mixture of 90% (v/v) to 99.9% (v/v) aprotic organic solvent and 10% (v/v) to 0.1 % (v/v) aqueous buffer at a pH between pH 7 and pH 8, S3 = mixture of 90% (v/v) to 99.9% (v/v) acetonitrile and 10% (v/v) to 0.1 % (v/v) aqueous buffer at a pH between pH 6.5 and pH 8.5, S4 = mixture of 90% (v/v) to 99.9% (v/v) 1 ,4-Dioxane and 10% (v/v) to 0.1 % (v/v) aqueous buffer at a pH between pH 6.5 and pH 8.5, S5 = mixture of 90% (v/v) to 99.9% (v/v) ethylene glycol dimethyl ether and 10% (v/v) to 0.1 % (v/v) aqueous buffer at a pH between pH 6.5 and pH 8.5, S6 = mixture of 90% (v/v) to 99.9% (v/v) terf-Butanol and 10% (v/v) to 0.1 % (v/v) aqueous buffer at a pH between pH 6.5 and pH 8.5, S7 = mixture of 95% (v/v) to 99% (v/v) aprotic organic solvent and 5% (v/v) to 1 % (v/v) aqueous buffer at a pH between pH 6.5 and pH 8.5, S8 = mixture of 95% (v/v) to 99% (v/v) acetonitrile and 5% (v/v) to 1 % (v/v) aqueous buffer at a pH between pH 6.5 and pH 8.5, S9 = mixture of 95% (v/v) to 99% (v/v) 1 ,4-Dioxane and 5% (v/v) to 1 % (v/v) aqueous buffer at a pH between pH 6.5 and pH 8.5, S10 = mixture of 95% (v/v) to 99% (v/v) ethylene glycol dimethyl ether and 5% (v/v) to 1 % (v/v) aqueous buffer at a pH between pH 6.5 and pH 8.5, S1 1 = mixture of 95% (v/v) to 99% (v/v) terf-Butanol and 5% (v/v) to 1 % (v/v) aqueous buffer at a pH between pH 6.5 and pH 8.5, S12 = mixture of 95% (v/v) to 99% (v/v) aprotic organic solvent and 5% (v/v) to 1 % (v/v) aqueous buffer at a pH between pH 7 and pH 8, S13 = mixture of 95% (v/v) to 99% (v/v) acetonitrile and 5% (v/v) to 1 % (v/v) aqueous buffer at a pH between pH 7 and pH 8, S14 = mixture of 95% (v/v) to 99% (v/v) 1 ,4-Dioxane and 5% (v/v) to 1 % (v/v) aqueous buffer at a pH between pH 7 and pH 8, S15 = mixture of 95% (v/v) to 99% (v/v) ethylene glycol dimethyl ether and 5% (v/v) to 1 % (v/v) aqueous buffer at a pH between pH 7 and pH 8, S16 = mixture of 95% (v/v) to 99% (v/v) terf-Butanol and 5% (v/v) to 1 % (v/v) aqueous buffer at a pH between pH 7 and pH 8.

7. Process according to claims 1 to 6, wherein the temperature Θ is selected in the range from θ = (ΘΟΡΤ - 50°C) to θ = (ΘΟΡΤ + 10°C) or in the range from θ = (ΘΟΡΤ - 30°C) to θ = (ΘΟΡΤ + 5°C) or in the range from (ΘΟΡΤ - 10°C) to (ΘΟΡΤ + 5°C) with the proviso that the reaction mixture used for performing the process should be in the liquid state at the temperature Θ selected.

8. Process according to claims 1 to 7, wherein the protease P is a serine

protease or a cysteine protease.

9. Process according to claims 1 to 8, wherein the protease P is a subtilisin- like serine protease.

Process according to claim 1 , wherein compound II is selected from compound II = T with

and wherein

M is selected from

and wherein

L is selected from C2-C20 alkyl, C6-C20 aryl, C7-C20 alkylaryl, and wherein

R^A is selected from C2-C20 alkyl, C6-C20 aryl, C7-C20 alkylaryl, and wherein

R¹ is selected from hydrogen atom, Ci-C₄ alkyl, Ci-C₄ thioether, C6-C20 aryl, C7-C20 alkylaryl,

and wherein

one of X¹ , X² is nitrogen, the other is carbon,

and wherein

the solvent S is S1 = mixture of 90% (v/v) to 99.9% (v/v) aprotic organic solvent and 10% (v/v) to 0.1 % (v/v) aqueous buffer at a pH between pH 6.5 and pH 8.5,

and wherein

the temperature Θ is selected in the range from θ = (ΘΟΡΤ - 50°C) to Θ = (ΘΟΡΤ + 10°C) with the proviso that the reaction mixture used for performing the process should be in the liquid state at the temperature Θ selected,

and wherein

the protease P is a serine protease or a cysteine protease.

11. Process according to claim 1 , wherein compound II is selected from

compound II = T with

(II = T),

and wherein M is selected from

and wherein

L is selected from C2-C20 alkyl, C6-C20 aryl, C7-C20 alkylaryl, and wherein

R^A is selected from C2-C20 alkyl, C6-C20 aryl, C7-C20 alkylaryl, and wherein

R¹ is selected from hydrogen atom, Ci-C₄ alkyl, Ci-C₄ thioether, C6-C20 aryl, C7-C20 alkylaryl,

and wherein

one of X¹ , X² is nitrogen, the other is carbon,

and wherein

the solvent S is S1 = mixture of 90% (v/v) to 99.9% (v/v) aprotic organic solvent and 10% (v/v) to 0.1 % (v/v) aqueous buffer at a pH between pH 6.5 and pH 8.5,

and wherein

the temperature Θ is selected in the range from θ = (ΘΟΡΤ - 50°C) to Θ = (ΘΟΡΤ + 10°C) with the proviso that the reaction mixture used for performing the process should be in the liquid state at the temperature Θ selected,

and wherein

the protease P is a subtilisin-like serine protease.

12. Process according to claim 1 , wherein compound II is selected from

compound II = T with

and wherein

M is selected from

and wherein

L is selected from C2-C20 alkyl,

and wherein

R^A is selected from C2-C20 alkyl, C6-C20 aryl, C7-C20 alkylaryl, and wherein

R¹ is selected from hydrogen atom, Ci-C₄ alkyl, Ci-C₄ thioether, C6-C20 aryl, C7-C20 alkylaryl,

and wherein

X¹ is carbon and X² is nitrogen,

and wherein

the solvent S is selected from S2 = mixture of 90% (v/v) to 99.9% (v/v) aprotic organic solvent and 10% (v/v) to 0.1 % (v/v) aqueous buffer at a pH between pH 7 and pH 8, S3 = mixture of 90% (v/v) to 99.9% (v/v) acetonitrile and 10% (v/v) to 0.1 % (v/v) aqueous buffer at a pH between pH 6.5 and pH 8.5, S4 = mixture of 90% (v/v) to 99.9% (v/v) 1 ,4- Dioxane and 10% (v/v) to 0.1 % (v/v) aqueous buffer at a pH between pH 6.5 and pH 8.5, S5 = mixture of 90% (v/v) to 99.9% (v/v) ethylene glycol dimethyl ether and 10% (v/v) to 0.1 % (v/v) aqueous buffer at a pH between pH 6.5 and pH 8.5, S6 = mixture of 90% (v/v) to 99.9% (v/v) terf-Butanol and 10% (v/v) to 0.1 % (v/v) aqueous buffer at a pH between pH 6.5 and pH 8.5,

and wherein

the temperature Θ is selected in the range from θ = (ΘΟΡΤ - 50°C) to Θ = (ΘΟΡΤ + 10°C) with the proviso that the reaction mixture used for performing the process should be in the liquid state at the temperature Θ selected,

and wherein

the protease P is a serine protease or a cysteine protease.

Process according to claim 1 , wherein compound II is selected from compound II = T with

and wherein

M is selected from

and wherein

L is selected from C2-C20 alkyl,

and wherein

R^A is selected from C2-C20 alkyl, C6-C20 aryl, C7-C20 alkylaryl, and wherein

R¹ is selected from hydrogen atom, Ci-C₄ alkyl, Ci-C₄ thioether, C6-C20 aryl, C7-C20 alkylaryl,

and wherein

X¹ is carbon and X² is nitrogen, and wherein

the solvent S is selected from S2 = mixture of 90% (v/v) to 99.9% (v/v) aprotic organic solvent and 10% (v/v) to 0.1 % (v/v) aqueous buffer at a pH between pH 7 and pH 8, S3 = mixture of 90% (v/v) to 99.9% (v/v) acetonitrile and 10% (v/v) to 0.1 % (v/v) aqueous buffer at a pH between pH 6.5 and pH 8.5, S4 = mixture of 90% (v/v) to 99.9% (v/v) 1 ,4- Dioxane and 10% (v/v) to 0.1 % (v/v) aqueous buffer at a pH between pH 6.5 and pH 8.5, S5 = mixture of 90% (v/v) to 99.9% (v/v) ethylene glycol dimethyl ether and 10% (v/v) to 0.1 % (v/v) aqueous buffer at a pH between pH 6.5 and pH 8.5, S6 = mixture of 90% (v/v) to 99.9% (v/v) terf-Butanol and 10% (v/v) to 0.1 % (v/v) aqueous buffer at a pH between pH 6.5 and pH 8.5,

and wherein

the temperature Θ is selected in the range from θ = (ΘΟΡΤ - 50°C) to Θ = (ΘΟΡΤ + 10°C) with the proviso that the reaction mixture used for performing the process should be in the liquid state at the temperature Θ selected,

and wherein

the protease P is a subtil isin-like serine protease.

14. Process according to claim 1 , wherein compound II is selected from

compound II = Q with

= Q).

and wherein

M is selected from

and wherein

R^A is selected from C2-C20 alkyl, C6-C20 aryl, C7-C20 alkylaryl, and wherein

R¹ and R² are independently selected from hydrogen atom, Ci-C₄ alkyl, Ci-C₄ hydroxyalkyl, Ci-C₄ thioether, C6-C20 aryl, C7-C20 alkylaryl, C7-C20 alkylhydroxyaryl, C₄-C2o alkylheteroaryl,

and wherein one of X¹, X² is nitrogen, the other is carbon,

and wherein

X is selected from -OH, -NH₂, -NHR³, -NR³R⁴;

and wherein

R³ and R⁴ are independently selected from C1-C6 alkyl and C₇-C20 alkylaryl;

and wherein

n is selected from 0, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10;

and wherein

the solvent S is S1 = mixture of 90% (v/v) to 99.9% (v/v) aprotic organic solvent and 10% (v/v) to 0.1 % (v/v) aqueous buffer at a pH between pH 6.5 and pH 8.5,

and wherein

the temperature Θ is selected in the range from θ = (ΘΟΡΤ - 50°C) to Θ = (ΘΟΡΤ + 10°C) with the proviso that the reaction mixture used for performing the process should be in the liquid state at the temperature Θ selected,

and wherein

the protease P is a serine protease or a cysteine protease.

15. Process according to claim 1 , wherein compound II is selected from

compound II = Q with

(II = Q)

and wherein

M is selected from

and wherein

R^A is selected from C2-C20 alkyl, C6-C20 aryl, C7-C20 alkylaryl, and wherein

R¹ and R² are independently selected from hydrogen atom, Ci-C₄ alkyl, Ci-C₄ hydroxyalkyl, Ci-C₄ thioether, C6-C20 aryl, C7-C20 alkylaryl, C7-C20 alkylhydroxyaryl, C₄-C2o alkyl heteroaryl,

and wherein one of X¹, X² is nitrogen, the other is carbon,

and wherein

X is selected from -OH, -NH₂, -NHR³, -NR³R⁴;

and wherein

R³ and R⁴ are independently selected from C1-C6 alkyl and C₇-C20 alkylaryl;

and wherein

n is selected from 0, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10;

and wherein

the solvent S is S1 = mixture of 90% (v/v) to 99.9% (v/v) aprotic organic solvent and 10% (v/v) to 0.1 % (v/v) aqueous buffer at a pH between pH 6.5 and pH 8.5,

and wherein

the temperature Θ is selected in the range from θ = (ΘΟΡΤ - 50°C) to Θ = (ΘΟΡΤ + 10°C) with the proviso that the reaction mixture used for performing the process should be in the liquid state at the temperature Θ selected,

and wherein

the protease P is a subtil isin-like serine protease.

16. Process according to claim 1 , wherein compound II is selected from

compound II = Q with

= 0),

and wherein

M is selected from

and wherein

R^A is selected from C2-C20 alkyl, C6-C20 aryl, C7-C20 alkylaryl, and wherein

R¹ and R² are independently selected from hydrogen atom, Ci-C₄ alkyl, Ci-C₄ hydroxyalkyl, Ci-C₄ thioether, C6-C20 aryl, C7-C20 alkylaryl, C7-C20 alkylhydroxyaryl, C₄-C2o alkyl heteroaryl,

and wherein X¹ is carbon and X² is nitrogen,

and wherein

X is selected from -OH,

and wherein

n is selected from 0, 1 , 2, 3,

and wherein

the solvent S is selected from S2 = mixture of 90% (v/v) to 99.9% (v/v) aprotic organic solvent and 10% (v/v) to 0.1 % (v/v) aqueous buffer at a pH between pH 7 and pH 8, S3 = mixture of 90% (v/v) to 99.9% (v/v) acetonitrile and 10% (v/v) to 0.1 % (v/v) aqueous buffer at a pH between pH 6.5 and pH 8.5, S4 = mixture of 90% (v/v) to 99.9% (v/v) 1 ,4- Dioxane and 10% (v/v) to 0.1 % (v/v) aqueous buffer at a pH between pH 6.5 and pH 8.5, S5 = mixture of 90% (v/v) to 99.9% (v/v) ethylene glycol dimethyl ether and 10% (v/v) to 0.1 % (v/v) aqueous buffer at a pH between pH 6.5 and pH 8.5, S6 = mixture of 90% (v/v) to 99.9% (v/v) terf-Butanol and 10% (v/v) to 0.1 % (v/v) aqueous buffer at a pH between pH 6.5 and pH 8.5,

and wherein

the temperature Θ is selected in the range from θ = (ΘΟΡΤ - 50°C) to Θ = (ΘΟΡΤ + 10°C) with the proviso that the reaction mixture used for performing the process should be in the liquid state at the temperature Θ selected,

and wherein

the protease P is a serine protease or a cysteine protease.

17. Process according to claim 1 , wherein compound II is selected from

compound II = Q with

(II = Q)

and wherein

M is selected from

and wherein

R^A is selected from C2-C20 alkyl, C6-C20 aryl, C7-C20 alkylaryl and wherein

R¹ and R² are independently selected from hydrogen atom, Ci-C₄ alkyl, Ci-C₄ hydroxyalkyl, Ci-C₄ thioether, C6-C20 aryl, C7-C20 alkylaryl, C7-C20 alkylhydroxyaryl, C₄-C2o alkyl heteroaryl,

and wherein

X¹ is carbon and X² is nitrogen,

and wherein

X is selected from -OH,

and wherein

n is selected from 0, 1 , 2, 3,

and wherein

the solvent S is selected from S2 = mixture of 90% (v/v) to 99.9% (v/v) aprotic organic solvent and 10% (v/v) to 0.1 % (v/v) aqueous buffer at a pH between pH 7 and pH 8, S3 = mixture of 90% (v/v) to 99.9% (v/v) acetonitrile and 10% (v/v) to 0.1 % (v/v) aqueous buffer at a pH between pH 6.5 and pH 8.5, S4 = mixture of 90% (v/v) to 99.9% (v/v) 1 ,4- Dioxane and 10% (v/v) to 0.1 % (v/v) aqueous buffer at a pH between pH 6.5 and pH 8.5, S5 = mixture of 90% (v/v) to 99.9% (v/v) ethylene glycol dimethyl ether and 10% (v/v) to 0.1 % (v/v) aqueous buffer at a pH between pH 6.5 and pH 8.5, S6 = mixture of 90% (v/v) to 99.9% (v/v) terf-Butanol and 10% (v/v) to 0.1 % (v/v) aqueous buffer at a pH between pH 6.5 and pH 8.5,

and wherein

the temperature Θ is selected in the range from θ = (ΘΟΡΤ - 50°C) to Θ = (ΘΟΡΤ + 10°C) with the proviso that the reaction mixture used for performing the process should be in the liquid state at the temperature Θ selected,

and wherein

the protease P is a subtil isin-like serine protease.