EP3962922A1 - Method for producing peptides or proteins or peptidomimetics - Google Patents

Abstract

A process for synthesizing peptides or proteins or peptidomimetics by successive elongation, with units, of the second end (primary or secondary amine function, hydroxyl function or thiol function) of a peptide or protein or peptidomimetic chain, characterized in that: said units are selected from the group made up of: α, β or γ-amino acids, α, β or γ-hydroxy acids and α, β or γ-mercapto acids (natural or unnatural or synthetic), the molecules having at least two functional groups; - the first end of said peptide or protein or peptidomimetic is bonded by a covalent bond to an anchoring molecule that is soluble in organic solvents such as halogenated solvents (methylene chloride, chloroform), ethyl acetate, tetrahydrofuran, 2-methyltetrahydrofuran, isooctane, cyclohexane, hexane(s), methylcyclohexane or methyl tert-butyl ether, or aromatic solvents such as benzene or toluene, or any other suitable solvent.

Description

METHOD OF PRODUCTION OF PEPTIDES OR PROTEINS OR

PEPTIDOMIMETICS

Technical field of the invention

The present invention relates to the chemistry of peptides or proteins or peptidomimetics, and more particularly their syntheses by chemical means from bifunctional molecules, and in particular from a, b or g-amino acids and / or a, b or g- acids. hydroxylated and / or a, b or g-mercapto acids.

More specifically, the invention relates to a process for producing peptides or proteins or peptidomimetics in solution. This process does not use conventional protective groups such as te / f-butoxycarbonyl (Boc) or fluorenylmethoxycarbonyl (Fmoc) on the amine function of the a, b, or g-amino acid. Likewise, it is not necessary to use protective groups on the hydroxyl function of a, b, or g-hydroxyl acid, or on the thiol function of a, b, or g-mercapto acid.

This process is based on the use of a, b, or g-amino acids or a, b, or g-hydroxy acids or a, b, or g-mercapto acids activated in the form, respectively, of 2,2 - bis (trifluoromethyl) -1, 3-oxazolidin-5-one or 2,2-bis (trifluoromethyl) -1, 3-oxazinan-6-one, or 2,2-bis (trifluoromethyl) -1, 3-oxazepan -7-one or 2,2-bis (trifluoromethyl) -1, 3-dioxolan-4-one, or 2,2-bis (trifluoromethyl) -1, 3-dioxan-4-one, or 2,2-bis (trifluoromethyl) -1, 3-dioxepan-4-one, or 2,2-bis (trifluoromethyl) -1, 3-oxathiolan-5-one, or 2,2-bis (trifluoromethyl) -1, 3- oxathian- 6-one, or 2,2-bis (trifluoromethyl) -1, 3-oxathiépan-7-one or their derivatives and from a family of anchoring molecules, namely derivatives of polyolefins or oligomers of polyolefins or polyalkenes. The anchor molecule is linked to a molecule having at least two electrophilic and / or nucleophilic functional groups, and in particular to a first acid a, b, or g-amino or acid a, b, or g-hydroxylated or a, b , or g-mercapto acid, which will then be the subject of successive elongation / iteration steps to lead to peptides or proteins or peptidomimetics.

This process makes it possible to obtain peptides or proteins or peptidomimetics in a more efficient way (that is to say with a reduced number of steps), faster, purer or easier to purify than the methods on solid support or in current solution. It is easy to automate.

State of the art

The remarkable growth in the development of therapeutic peptides or proteins or peptidomimetics over the last decade has led to a large number of approvals of new molecules on the market (see the publications of J. Med. Chem., 2018, 22, 1382 -1414, Bioorg. Med. Chem., 2018, 26, 2700-2707); thus, therapy based on peptides or proteins or peptidomimetics has become one of the fastest growing segments in the pharmaceutical industry. Cancers, metabolic diseases and diseases of the central nervous system are the main therapeutic areas accelerating the demand for new peptides or proteins or therapeutic peptidomimetics.

However, a number of bottlenecks prevent the widespread adoption of peptides, or proteins or peptidomimetics in therapy. Mention may be made, for example, of their short metabolic half-life and their hydrophilic nature. Another key factor, and by far the most important, retarding the growth of therapeutic applications of peptides or proteins or peptidomimetics is their mode of production.

The very first liquid-phase peptide synthesis was carried out over a century ago by E. Fisher and E. Fourneau (Ber. Dtsch. Chem. Ges. 1901, 34, 2868-2879). Since then, many chemists have made improvements; this is the case with Bodansky and du Vigneaud (J. Am. Chem. Soc., 1959, 51, 5688-5691), Beyerman et al. (Rec. Trav. Chim., Netherlands 1973, 92, 481-492), of R. K. Sharma and R. Jain (Synlett 2007, 603-606), of Nodal et al. (Nature Chemistry 2017, 9, 571-577), by Liu et al. (Org. Lett., 2018, 20, 612-615) and Muramatsu et al. (ACS Catal., 2018, 8, 2181-2187).

The most frequently used peptide or protein or peptidomimetic synthesis routes involve temporary protection of the amine (Na) function of amino acids. Today, the main protective groups used are the te / f-butoxycarbonyl group, this approach is commonly called the “Boc” strategy, and the fluorenylmethoxycarbonyl group, this approach is commonly called the “Fmoc” strategy. These two peptide synthesis routes are known to those skilled in the art (see Section 7-5 of the “Biochemistry” manual by D. Voet and J .G. Voet, 2 ^nd edition, Brussels 2005). In practice, the amino acids are supplied in the protected state on the amine function (Na) by the Fmoc or Boc group, and are involved directly in the activation / coupling reactions.

Amino acids can be used in liquid phase or on a solid support; in the latter case, the amino acid protected on the amine function (Na) is fixed on a resin insoluble in organic solvents, this is the synthesis of Merrifield (J. Am. Chem. Soc., 1963, 85, 2149- 2154). It is a well-controlled process, which however has some drawbacks such as: the cost of reagents used in excess and the lack of homogeneity of the peptides synthesized. The system is said to be degenerate, which generates additional costs of purifications by preparative high performance liquid chromatography.

In Liquid Phase Peptide Synthesis (SPPL), all reactions take place in homogeneous solution. This methodology has been described by Bodansky and du Vigneaud (J. Am. Chem. Soc., 1959, 51, 5688-5691). The carboxylic acid function (C-terminal) of the starting amino acid is protected in the form of a methyl ester, and the following amino acids are condensed successively after the protection of their amine function (Na) by a carboxybenzyl group (abbreviated Cbz) then the activation of their acid function carboxylic (C-terminal) by a nitrophenyl ester. All synthetic intermediates are purified by precipitation or washing with water (extraction). This peptide synthesis methodology is long, tedious and generates peptides with low yields. By way of example, mention may be made of the synthesis of ACTH with an overall yield of about 7%, described by Schwyzer and Sieber (Helv. Chim. Acta 1966, 49, 134-158).

A modification of this methodology has been reported by Beyerman et al. (Rec. Trav. Chim. Netherlands 1973, 92, 481-492). It consists of protecting the carboxylic acid function (C-terminal) of an amino acid or of a peptide in the form of a benzyl ester and of carrying out the coupling reaction (or condensation) in the presence of an excess of Na-protected amino acid anhydride, with a view to improving yields. Finally, although the yields of the coupling reactions are increased, there is a loss of solubility of the peptide in the organic phase when the latter reaches about five amino acids.

Other strategies allowing the solubilization of amino acids, in order to facilitate peptide synthesis, have been developed. Mention may be made of the work of Narita (Bull. Chem. Soc. Jap., 1978, 51, 1477-1480), the work of Bayer and Mutter (Nature 1972, 237, 512-513) on polyethylene glycol as a solubilization adjuvant, and patents EP 0 017 536 (Sanofi) and EP 2 612 845 A1 and US 2014/0296483 (Ajinomoto Co., Inc.) on solubilizing anchor molecules.

Peptide synthesis strategies are also known which make use of bifunctional groups, that is to say groups which are capable simultaneously of activating the carboxylic acid function (C-terminal) and of protecting the amine function (Na). amino acid, forming highly reactive intermediate ring structures. This is the case with N-carboxyanhydrides (abbreviated NCAs) (see Ber. Dtsch. Chem. Ges., 1906, 39, 857-861; Ber. Dtsch. Chem. Ges., 1907, 40, 3235-3249; Ber. . Dtsch. Chem. Ges., 1908, 41, 1721-172; J. Am. Chem. Soc., 1957, 79, 2153-2159; J. Am. Chem. Soc., 1947, 69, 1551-1552) . Reactive intermediates have been synthesized from amino acids and dichlorodimethylsilane derivatives (see SH van Leeuwen et al., Tetrahedron Letters 2002, 43, 9203-9207 and patent WO 00/37484 A1). Amino acid derivatives activated by boron trifluoride etherate have been invented for the synthesis of peptides (see SH van Leeuwen et al., Tetrahedron Letters 2005, 46, 653- 656). The activation of amino acids in the presence of hexafluoroacetone has also been described (see Chem. Ztg., 1990, 114, 249-251 and J. Spengler et al., Chem. Rev., 2006, 106, 4728- 4746) .

All these methods of synthesizing peptides or proteins or peptidomimetics, in solution or on a solid support, have at least one or more disadvantages among the following: the use of protective groups, the use of reagents in excess, the possibility of racemization, the low solubility of the peptides during synthesis in organic solvents, the limitation of the size of the peptide, costly and polluting purifications, complex experimental protocols or the possibility of polymerization. Overall, the examination of the abundant literature available in the field of peptide synthesis shows that difficulties persist to produce peptides or proteins or peptidomimetics of high purity, with good yields, at low cost and with a lower ecological footprint.

The problem that the present application proposes to solve is the design of a new methodology for synthesizing peptides or proteins or peptidomimetics making it possible to remove the locks linked to their access or production left in the prior art.

Object of the invention

According to the invention, the problem is solved by a method of synthesizing peptides or proteins or peptidomimetics in liquid phase which comprises the combination of two essential characteristics which are detailed below.

The first object of the present invention is a process for the synthesis of peptides or proteins or peptidomimetics by successive elongation of the second end of a molecule of type Q ^a - E - Q ^b , where Q ^a and Q ^b can be identical or different. and represent an electrophilic and / or nucleophilic function, and E represents a spacer. Said second end may in particular be a primary or secondary amine, a hydroxyl or a thiol, of an a, b, g or d-amino acid or a, b, g or d-hydroxyl acid or a, b, g or d -mercapto acid or peptide or protein or peptidomimetic, characterized in that said units are selected from the group formed by: acids a, b, g or d- amino or acids a, b, g or d-hydroxylated or a, b , g or d-mercapto acids (natural or unnatural or synthetic). In addition, the first end of said Q ^a - E - Q ^b type molecule (for example of said a, b, g or d-amino acid or a, b, g or d-hydroxy acid or a, b, g or d-mercapto acid) or of said peptide or protein or peptidomimetic is attached to an anchoring molecule soluble in organic solvents such as halogenated solvents (methylene chloride, chloroform), ethyl acetate, tetrahydrofuran, 2 -methyletetrahydrofuran, isooctane, cyclohexane, hexane (s), methylcyclohexane, methyl tert-butyl ether or aromatic solvents such as benzene or toluene or any other suitable solvent.

The first essential feature is the use of a family of specific anchor molecules. According to the invention, the anchoring molecules are polyolefins or oligomers of polyolefins or polyalkenes. The method according to the invention provides access to high purity peptides or proteins or peptidomimetics (natural or unnatural or synthetic). This process generates savings of steps and atoms due to the absence of the use of protective groups (on the amine, hydroxyl or thiol functions of the main chain) and of coupling agents and therefore, financial savings. In the end, the process is more respectful of the environment.

The second essential feature is the use of bifunctional molecules of type Q ^a - E - Q ^b in which the groups Q ^a and Q ^b may be the same or different, and are selected from the electrophilic groups and / or the nucleophilic groups, and E represents a spacer. Advantageously, Q ^a and Q ^b are selected from the group formed by chemical functions such as: alcohols, aldehydes, primary amines, secondary amines, azides, ethynils, halogens, thiols, vinyls, and / or the spacer E is selected from the group formed by structural units such as: aromatic, heteroaromatic, saturated alkyl chains (branched or not), unsaturated alkyl chains (branched or not), glycols (and preferably polyethylene glycol).

In the advantageous case where one uses as bifunctional molecule of type Q ^a - E - Q ^b , an acid a, b or g-amino or an acid a, b or g-hydroxylated or an a, b or g-mercapto acid, these compounds are used in their activated forms, namely, 2,2- bis (trifluoromethyl) -1, 3-oxazolidin-5-one or 2,2-bis (trifluoromethyl) -1, 3-oxazinan-6 -one or 2,2-bis (trifluoromethyl) -1, 3-oxazepan-7-one or 2,2-bis (trifluoromethyl) -1, 3-dioxolan-4- ones or 2,2-bis (trifluoromethyl) - 1, 3-dioxan-4-one or 2,2-bis (trifluoromethyl) -1, 3-dioxepan-4-one or 2,2-bis (trifluoromethyl) -1, 3-oxathiolan-5-one or 2, 2-bis (trifluoromethyl) -1, 3-oxathian-6-one or 2,2-bis (trifluoromethyl) -1, 3-oxathiepan-7-one or their derivatives.

Figure 1 shows the structure of these activated forms. The latter are prepared from a, b or g-amino acids (this expression meaning here: a-amino acids, b-amino acids or g-amino acids), or a, b or g-hydroxyl acids (this expression meaning here: a-hydroxy acids, b-hydroxy acids or g-hydroxy acids), or a, b or g-mercapto acids (this expression meaning here: a-mercapto acids, b-mercapto acids or g-mercapto acids) corresponding. [Chem 1]

X = NH, N-alkyl, N-aryl S

R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ^{6 AIR} y ^{| e or Ar} y ^{| e}

Diagram n ° 1: Structures of activated shapes

To date, it has never been demonstrated the possibility of easily preparing in solution, peptides consisting of more than four different amino acids or not, using derivatives of activated amino acids in the form of 2,2-bis (trifluoromethyl) -1, 3-oxazolidin-5-one.

It is precisely the object of the invention which proposes the use of activated acids in the form of 2,2-bis (trifluoromethyl) -1, 3-oxazolidin-5-one or 2,2-bis (trifluoromethyl) -1, 3- oxazinan-6-one or 2,2-bis (trifluoromethyl) -1, 3-oxazepan-7-one or 2,2-bis (trifluoromethyl) - 1, 3-dioxolan-4-ones or 2 , 2-bis (trifluoromethyl) -1, 3-dioxan-4-one or 2,2- bis (trifluoromethyl) -1, 3-dioxepan-4-one or 2,2-bis (trifluoromethyl) -1, 3- oxathiolan-5-one or 2,2-bis (trifluoromethyl) -1, 3-oxathian-6-one or 2,2-bis (trifluoromethyl) -1, 3-oxathiepan-7-one or their derivatives in the presence of ' an anchoring molecule allowing the production of peptides or proteins or peptidomimetics of high purity, in liquid phase (or solution).

The method for synthesizing peptides or proteins or peptidomimetics according to the invention proceeds by successive elongation of the second end (primary or secondary amine, hydroxyl or thiol) of a peptide or protein or peptidomimetic chain whose first end is attached to a molecule anchor soluble in an organic solvent. Said anchor molecule comprises a polyolefin or polyolefin or polyalkene oligomer chain having at least 10 monomer units, and preferably between 15 and 350 monomer units.

In an advantageous embodiment, said polyolefin chain is a polyisobutene (PIB) chain. In particular, said anchoring molecule can be a polyolefin. The polyolefin chain can be functionalized at at least one of its ends. Alternatively, the polyolefin or polyolefin or polyalkene oligomer chain can comprise a number of unsaturated carbon-carbon bonds not exceeding 5%, and preferably not exceeding 3%, and / or the anchor molecule can exhibit a number of unsaturated carbon-carbon bonds. mass average molecular weight between 600 and 20,000, and preferably between 700 and 15,000.

In a particular embodiment, said anchoring molecule comprises a polyolefin chain (or is a polyolefin chain) terminated by at least one group selected from the group formed by: a function -X ^a , where X ^a is selected from the group group formed by: -OH, -IMH2, - NHR ^a (R ^a = alkyl or aryl), -SH;

o a function -Y-C6H4X ^b , where

^■ Y is O, S, CH2 or absent,

^■ X ^b is selected from the group formed by: -OH, -IMH2, -NHR ^a , -SH, - CX ^a R ^a R ^b , -C ₆ H ₃ R ^c (CR ^a X ^a ),

where: R ^b is selected from the group consisting of -H, -Aryl, - Heteroaryl, -Alkyl, and

R ^c is selected from the group consisting of -H, -Alkyl, -O-Alkyl, -Aryl, -O-Aryl, -Heteroaryl, -O-Heteroaryl;

o a ^function -CR = CH-CHX ^a or -CR a function ^of H-CH = CH-CHX ^a, wherein X ^a has the meaning defined above, and R ^d is methyl or ethyl.

In particular, X ^a can be a primary or secondary amine function, an alcohol, a thiol or a phenol.

In an advantageous embodiment, the mass average molecular mass of the anchor molecules, apart from the terminal functionalization (for example -X ^a , -Z-CeH4X ^b or -CR ^d = CH-CHX ^a as defined above) , is between 600 and 20,000, and preferably between 700 and 15,000. Above a mass average molecular mass of approximately 20,000, these molecules may have a viscosity that is too high, which would risk limiting their viscosity. solubility in organic solvents used for the coupling / elongation or iteration step.

Certain GDP derivatives used in the context of the present invention are commercially available as ligands for homogeneous catalysis. By way of example, one can use 2-methyl-3- [polyisobutyl (12)] propanol (mass average molecular weight 757, including terminal functionalization) or 4- [polyisobutyl (18)] phenol (mass weight average molecular weight 1104, including terminal functionalization) which are distributed, respectively, under the references 06-1037 and 06-1048 by the company Strem Chemicals. These two molecules are derivatives of polyisobutenes whose chain is terminated, respectively, by a group -CH2-C (CH3) (H) -CH2-OH (ie isopropanol) and by a group -CH2-C (CH3) 2-C6H4-OH (ie phenol).

According to one characteristic of the invention, the use of an anchoring molecule soluble in an organic solvent as described above (and more particularly the use of polyolefins), is capable also of acting as a liquid support or a group. protector of the carboxylic acid function (C-terminal) or of any other chemical function (side chain (s)) of an a, b or g-amino acid or a, b or g-hydroxylated acid or a, b or g-mercapto acid, or any other molecule having at least two functional groups. It also allows the solubilization of the anchored peptides or proteins or peptidomimetics and their syntheses in organic solution (halogenated and / or non-halogenated solvents).

According to another characteristic of the invention, the use of an anchoring molecule soluble in an organic solvent as described above (and more particularly the use of polyolefins), and insoluble in certain polar solvents (such as 'water and / or ethanol and / or acetonitrile), facilitates the purification of a, b or g-amino acids or a, b or g-hydroxy acids or a, b or g-mercapto acids or peptides or proteins or peptidomimetics anchored by simple extraction (washing) or simple filtration on silica. Thus, a simple extraction or a simple filtration makes it possible to obtain the anchored peptides or proteins or peptidomimetics with high chemical purity.

According to yet another characteristic of the invention, commercially available anchoring molecules are used, or anchoring molecules which can be synthesized simply and directly from commercially available precursors, in particular certain polyisobutenes derivatives ( GDP).

According to yet another characteristic of the invention, the a, b or g-amino acids or a, b or g-hydroxylated acids or a, b or g-mercapto acids are reacted in their activated forms, respectively of, 2, 2-bis (trifluoromethyl) -1, 3-oxazolidin-5-one or 2,2- bis (trifluoromethyl) -1, 3-dioxolan-4-one or 2,2-bis (trifluoromethyl) -1, 3-oxathiolan -5-one or (2,2-bis (trifluoromethyl) -1, 3-oxazinan-6-one, 2,2-bis (trifluoroméhyl) -1, 3-dioxan-4-one, 2,2- bis ( trifluoromethyl) -1, 3-oxathian-6-one, 2,2-bis (trifluoromethyl) -1, 3-oxazepan-7-one, 2,2- bis (trifluoromethyl) -1, 3-dioxepan-4-one , 2,2-bis (trifluoromethyl) -1, 3-oxathiépan-7-one and their derivatives, in the presence of the anchor molecule in an appropriate solvent (or a mixture of solvents), at a temperature between -20 ° C and 150 ° C. In one embodiment, the reaction is carried out in any inert liquid solvent (or mixture) capable of to dissolve the reagents. Applicable solvents include, but are not limited to halogenated and non-halogenated hydrocarbons. The preferred solvents are tetrahydrofuran, ethyl acetate, 2-methyltetrahydrofuran, propylene carbonate, or any other solvent or mixture of solvents capable of dissolving these two chemical species.

According to yet another characteristic of the invention, the reactions between the GDP derivatives and the activated a, b or g-amino acids or activated a, b or g-hydroxylated acids or activated a, b or g-mercapto acids take place in batch chemistry (especially in a flask or cistern), but preferably, they are carried out in flow chemistry (also called continuous flow chemistry).

According to yet another characteristic of the invention, the a, b or g-amino acids or a, b or g-hydroxylated acids or a, b or g-mercapto acids having side chains incompatible with the conditions of the reaction of anchoring / elongation or iteration, can be temporarily masked by an appropriate protective group. It can in particular be chosen from the group formed by:

te / f-butoxycarbonyle (abbreviated Boc),

fluorenylmethoxycarbonyl (abbreviated Fmoc),

benzyl (abbreviated Bzl),

trityl (abbreviated Trt),

carboxybenzyl (abbreviated Cbz),

2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl (abbreviated Pbf),

4-methoxy-2,3,6-trimethylbenzenesulphonyl (abbreviated Mtr).

It is also possible to use any other protective group compatible with the present process.

According to yet another characteristic of the invention, said anchoring molecule reacts with a first acid a, b, or g-amino or acid a, b, or g-hydroxylated or a, b, or y-mercapto acid activated ( here abbreviated AAA1), resulting in a covalent bond between the acid a, b, or g-amino or acid a, b, or g-hydroxylated or a, b, or g-mercapto acid and the anchor molecule.

According to yet another characteristic of the invention, said peptide or protein or peptidomimetic chain is formed of n units of a, b or g-amino acids or a, b or g-hydroxyl acids or a, b or g-mercapto acids ; its second end is another unit of a, b or g-amino acid or a, b or g-hydroxyl acid or a, b or g-mercapto acid, here abbreviated AAAn. During the course of the process, the peptide or protein or peptidomimetic chain lengthens by elongation or successive iteration, and during each of these steps another unit of a, b or g-amino acid or a, b or g-hydroxylated acid or a, b or g-mercapto acid is added, here abbreviated AAA (n + 1), to said second end (primary or secondary amine, free alcohol or thiol). This reaction sequence is shown in Reaction Scheme # 2 below.

[Chem 2]

eptide

Z = NH, N-alkyl, N-aryl, O, S

R ¹ , R ^{2, 3} , R ^{4 =} H and / or alkyl and or aryl

n = 0.1

Reaction scheme No. 2: General process for obtaining a peptide According to yet another characteristic of the invention, it is possible to use in said peptide or protein or peptidomimetic chain, a, b or g-amino acids and / or acids. a, b or g-hydroxylated and / or a, b or g-mercapto natural and / or unnatural and / or synthetic acids. According to yet another characteristic of the invention, it is possible to use in said peptidomimetic chain one or more units of molecules of type Q ^a - E - Q ^b , having at least two functional groups, which are identical or different, and which are selected from electrophilic groups and / or nucleophilic groups, and which are separated by a spacer unit E. The Q ^a and / or Q ^b groups may or may not be end groups. The spacer E can be an entity selected from the group formed by:

- aliphatic chains (branched or not and unsaturated or not);

- aryls or heteroaryls (substituted or not).

Advantageously, the molecules of the Q ^a - E - Q ^{b type} carry a terminal function selected from the group formed by the primary amine function, the secondary amine function, the hydroxyl function or the thiol function.

It can easily be seen that said a, b or g-amino acids, said a, b or g-hydroxylated acids and said a, b or g-mercapto acids represent particular cases of bifunctional molecules of the type Q ^a - E - Q ^b . It is the same for said d-amino acids, said d-hydroxy acids and said d-mercapto acids, which however are not necessarily involved in the reaction in their activated form, like other bifunctional molecules which are not a, b or g-amino acids, acids a, b or g-hydroxylated or a, b or g-mercapto acids.

The bifunctional molecule Q ^a - E - Q ^b can have a molecular structure, selected in particular from epoxides, aziridines, thiiranes. Thus, according to the invention, peptidomimetics comprising an epoxy-succinate group, like the E-64 peptide, or even azirido peptides, like Miraziridine, can be prepared according to the invention.

Some examples are given here for bifunctional molecules of type Q ^a - E - Q ^b which can be used in the context of the present invention: sarcosine, 2- (1-aminoethyl) -1, 3-oxazole- acid. 4-carboxylic and (2R, 3R, 4R) -3-hydroxy-2,4,6-trimethyl-heptanoic acid.

Said bifunctional molecule Q ^a - E - Q ^b can in particular be an amino acid according to the definition which is given below. It can also be a peptide, for example a dipeptide, a tripeptide, a tetrapeptide, a pentapeptide, a hexapeptide, a heptapeptide, an octapeptide, a nonapeptide, a decapeptide, or an even longer peptide.

These bifunctional molecules can be introduced into the peptide or protein or peptidomimetic chain by known chemical reactions. They do not bear a protective group on a terminal function selected from the group formed by the primary amine function, the secondary amine function, the hydroxyl function or the thiol function. For example, if said bifunctional molecule is an amino acid or a peptide, it does not bear N-terminal protection; it is possible to protect its side chains or side functions, which are not modified during the elongation of the peptide.

As described above, said a, b or g-amino acids, said a, b or g-hydroxy acids and said a, b or g-mercapto acids are preferentially used in their activated forms.

The units derived from bifunctional molecules which are not selected from among the a, b or g-amino acids, the a, b or g-hydroxyl acids and the a, b or g-mercapto acids, can advantageously be attached to the end C-terminal of said peptidomimetic, or on the terminal end (in particular by functionalization of the primary or secondary amine, hydroxyl or thiol function), or else on the side chain (of at least an a, b or g-amino acid or a, b or g-hydroxylated acid or a, b or g-mercapto acid), or between two units selected from among the a, b or g-amino acids, the a acids, b or g-hydroxy and a, b or g-ercapto acids.

According to an advantageous embodiment, the number of units resulting from bifunctional molecules which are not selected from among the a, b or g-amino acids, the a, b or g-hydroxyl acids and the a, b or g-mercapto acids does not exceed 50% by number, and preferably does not exceed 25% by number.

According to yet another characteristic of the invention, at least one step in which said peptide or protein or peptidomimetic chain is attached to said anchor molecule and is purified from the reaction medium by extraction in an organic solvent (such as cyclohexane, l heptane (s) or any other suitable solvent) immiscible with water (or a water / ethanol mixture or a water / acetonitrile mixture) or by filtration on silica.

According to yet another characteristic of the invention, it makes it possible to obtain peptides or proteins or peptidometics of high purity, after deprotection of the side chains (if necessary), then detachment of their anchoring molecule after the last iteration step. , to be used according to their destination, for example as an active principle for preclinical trials, clinical care or any other applications.

According to yet another feature of the invention, the anchor molecules can be reused (recycled) in the process according to the invention.

A second object of the present invention is a molecule which can be obtained by the process according to the invention. Said molecule comprises an a, b or g-amino acid or a, b or g-hydroxy acid or a, b or g-mercapto acid or peptide or protein or peptidomimetic attached to an anchor molecule.

Description

1. Definitions

In the context of the present invention, by “amino acid” we mean: natural amino acids and unnatural or synthetic amino acids. "Natural" amino acids include the L form of so-called standard proteinogenic amino acids which can be found in proteins of natural origin, ie: alanine (Ala), arginine (Arg), asparagine (Asn) , aspartic acid (Asp), cysteine (Cys), glutamine (Gin), glutamic acid (Glu), glycine (Gly), histidine (His), isoleucine (Ile), leucine (Leu), lysine (Lys), methionine (Met), phenylalanine (Phe), proline (Pro), serine (Ser), threonine (Thr), tryptophan (Trp), tyrosine (Tyr) and valine (Val). They also include other proteinogenic amino acids, and in particular pyrrolysine and selenocysteine.

"Unnatural" amino acids include the D-form of the natural amino acids defined above, the homo forms of certain natural amino acids (such as: arginine, lysine, phenylalanine and serine), and the nor forms of leucine and valine.

"Unnatural" amino acids also include all synthetic amino acids. They also include unnatural amino acids, such as:

Abu = 2-aminobutyric acid CH ₃ -CH ₂ -CH (COOH) (NH ₂ );

iPr = isopropyl-lysine (CH ₃ ) 2C-NH- (CH ₂ ) 4-CH (COOH) (NH ₂ );

Aib = 2-aminoisobutyric acid;

F-trp = N-formyl-tryptophan;

Orn = ornithine;

Nal (2 ’) = 2-naphthylalanine.

This list is obviously not exhaustive.

It is also possible to use unsaturated a and b amino acids, natural or not.

In the context of the present invention, the term "activated amino acid" is used herein to denote activated a, b, or g-amino acids respectively in the form of 2,2-bis (trifluoromethyl) -1, 3-oxazolidin -5-one or (2,2-bis (trifluoromethyl) -1, 3-oxazinan-6-one or 2,2-bis (trifluoromethyl) -1, 3-oxazepan-7-one, and their derivatives, with the possibility or not of a protective group on the side chain.

The present invention is also applicable to the synthesis of peptidomimetics. The precursors used for this synthesis are defined as follows:

The term "a, b or g-hydroxyl acid" is used herein according to the rules of terminology of IUPAC, known to those skilled in the art. Examples are compounds such as lactic acid, malic acid, tartaric acid, salicylic acid or g-hydroxybutyric acid, which are found in nature. In the context of the present invention, it is also possible to use all the “unnatural” a, b or g-hydroxylated acids which also include all the synthetic a, b or g-hydroxylated acids.

The term "activated a, b or g-hydroxy acid" denotes all natural and / or unnatural and / or synthetic a, b or g-hydroxy acids, which have been activated respectively in the form of 2,2-bis ( trifluoromethyl) -1, 3-dioxolan-4-one or 2,2-bis (trifluoromethyl) -1, 3- dioxan-4-one or 2,2-bis (trifluoromethyl) -1, 3-dioxepan-4-one , and their derivatives, with the possibility or not of a protective group on the side chain. The term “a, b or g-mercapto acid” is used here according to the rules of terminology of IUPAC, known to those skilled in the art. Examples are compounds such as thioglycolic acid, 3-mercaptopropionic acid, mercaptobutanoic acid. In the context of the present invention, it is also possible to use all the “unnatural” a, b or g-mercapto acids which also include all the synthetic a, b or g-mercapto acids. The term "activated a, b or g-mercapto acid" denotes all compounds resulting from the activation of a, b or g-mercapto acids (natural, unnatural or synthetic) in the form of 2,2-bis ( trifluoromethyl) -1, 3-oxathiolan-5-one or 2,2- bis (trifluoromethyl) -1, 3-oxathian-6-one or 2,2-bis (trifluoromethyl) -1, 3-oxathiepan-7-one , and their derivatives, with the possibility or not of a protective group on the side chain.

Those skilled in the art know that in this context, the designation a, b, g and d refers to the position of the carbon substituted by the amine (primary or secondary) or hydroxyl or thiol function with respect to the carbon of the carboxylic acid function. (C-terminal).

The term "peptidomimetic" is used according to the state of the art as a functional term for a molecule capable of mimicking or blocking a peptide with respect to its interaction with a specific receptor. In particular, a peptidomimetic can comprise units which are not amino acids.

The abbreviations “DMF”, “DMSO” and “THF”, well known to chemists, denote, respectively, dimethylformamide, dimethylsulfoxide and tetrahydrofuran.

2. Detailed description

A first essential characteristic of the process according to the invention is the use of a, b or g-amino acids or a, b or g-hydroxylated acids or a, b or g-mercapto acids activated in the form of 2, respectively, 2-bis (trifluoromethyl) -1, 3-oxazolidin-5-one or (2,2-bis (trifluoro-methyl) -1, 3-oxazinan-6-one or 2,2-bis (trifluoromethyl) -1, 3-oxazepan-7-one or 2,2-bis (tri-fluoromethyl) -1, 3-dioxolan-4-one or 2,2-bis (trifluoromethyl) -1, 3-dioxan-4-one or 2, 2-bis (tri-fluoromethyl) -1, 3-dioxepan-4-one or 2,2-bis (trifluoromethyl) -1, 3- oxathiolan-5-one or 2,2-bis (trifluoromethyl) -1, 3 -oxathian-6-one or 2,2-bis (trifluoromethyl) - 1, 3-oxathiépan-7-one and their derivatives, in the presence of an anchoring molecule soluble in an organic solvent. organic »any inert liquid solvent (or mixture) capable of dissolving (hot and / or cold) the reactants Applicable solvents include, but are not limited to, halogenated or non-halogenated hydrocarbons.

A, b or g-amino acids or a, b or g-hydroxylated acids or a, b or g-mercapto acids activated in the form of 2,2-bis (trifluoromethyl) -1, 3-oxazolidin-5- respectively one or (2,2-bis (trifluoromethyl) -1, 3-oxazinan-6-one or 2,2-bis (trifluoromethyl) -1, 3-oxazepan-7- one or 2,2-bis (trifluoromethyl) -1, 3-dioxolan-4-one or 2,2-bis (trifluoromethyl) -1, 3-dioxan-4-one or 2,2-bis (trifluoromethyl) -1 , 3-dioxepan-4-one or 2,2-bis (trifluoromethyl) -1, 3- oxathiolan-5-one or 2,2-bis (trifluoromethyl) -1, 3-oxathian-6-one or 2,2 -bis (trifluoromethyl) - 1, 3-oxa-thiepan-7-one and their derivatives, are prepared according to known methods, from a, b or g-amino acids or a, b or g-hydroxyl acids or a, b or g-mercapto acids, natural or unnatural (having a side chain (protected or not)) and hexafluoroacetone. Reaction scheme n ° 3 represents the activation of the different acids, it is a reaction known as such:

[Chem 3]

X = NH, N-alkyl, N-aryl, O, S,

₁ ,,. _{c K} = A ky e or Ary e

R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁶

Reaction scheme no. 3: Activation of a, b or g-amino acids or a, b or g-hydroxyl acids or a, b or g-mercapto acids. According to one characteristic of the invention, which will be described in greater detail below, the anchoring molecules (or protective groups or solubilizing molecules) are polyolefins or more precisely oligomers of polyolefins (polyolefins also being called polyalkenes) and their derivatives, that is to say they are functionalized.

According to another characteristic of the invention, this method of synthesizing peptides or proteins or peptidomimetics (protected or not on their side chains), in the liquid phase, is characterized in that one starts from an anchoring molecule and an a, b or g-amino acid or a, b or g-hydroxyl acid or a, b or g-mercapto acid activated respectively in the form of 2,2-bis (trifluoromethyl) -1, 3-oxazolidin- 5-one or (2,2- bis (trifluoromethyl) -1, 3-oxazinan-6-one or 2,2-bis (trifluoromethyl) -1, 3-oxazepan-7-one or

2.2-bis (trifluoromethyl) -1, 3-dioxolan-4-one or 2,2-bis (trifluoromethyl) -1, 3-dioxan-4-one or

2.2-bis (trifluoromethyl) -1, 3-dioxepan-4-one or 2,2-bis (trifluoromethyl) -1, 3-oxathiolan-5-one or 2,2-bis (trifluoro-methyl) -1, 3 -oxathian-6-one or 2,2-bis (trifluoromethyl) -1, 3-oxathiepan-7-one or their derivatives. A covalent bond is then formed between these two molecular species. The elongation / iteration step consists in adding or condensing the following a, b or g-amino acids or a, b or g-hydroxylated acids or a, b or g-mercapto acids, which are possibly protected on their side chain (in the form of ester, ether, thioester, thioether or any other functions chemicals compatible with this process). Thus, the anchoring molecule plays a role of protective group of the carboxylic acid function (C-terminal) of the first acid a, b or g-amino or acid a, b or g-hydroxylated or a, b or g-mercapto acid.

According to another characteristic of the invention, this method of peptide or protein or peptidomimetic synthesis can be carried out using a fragment of a suitably protected peptide or protein or peptidomimetic and an a, b or g-amino acid or a, b or g acid. -hydroxylated or a, b or g-mercapto acid or peptide or protein or peptidomimetic, anchored on a GDP molecule, allowing after coupling to obtain a longer peptide or protein or peptidomimetic.

According to another characteristic of the invention, this method of peptide or protein or peptidomimetic synthesis can be carried out using molecules Q ^a - E - Q ^b having at least two functional groups Q ^a and Q ^b , which are identical or different, and which are selected from the electrophilic and / or nucleophilic chemical functions. Examples of such structures are styrene oxide, aminothiophenoles or 1-azido-4- (bromomethyl) benzene. These molecules can be directly attached to the anchor molecule or introduced during synthesis on the amine function (primary or secondary) or hydroxyl or thiol, a, b or g-amino acids or a, b or g-hydroxylated acids or a, b or g-mercapto acids or peptides or anchored proteins or peptidomimetics.

The process according to the invention can be carried out in any inert liquid solvent (or mixture) capable of dissolving the reactants (halogenated or not), at a temperature typically between about -20 ° C and about 150 ° C, in a reactor ( in batch or in flow).

According to another characteristic of the invention the a, b or g-amino acid or a, b or y-hydroxylated acid or a, b or g-mercapto acid or peptide or protein or peptidomimetic anchored on a GDP molecule is characterized in that the terminal function of said a, b or y-amino acid or a, b or g-hydroxylated acid or a, b or y-mercapto acid or peptide or protein or peptidomimetic or any other molecule having at least two functional groups is linked by a covalent bond (ester, ether, amide, thioester or any other chemical functions), thus giving a very low solubility in water (<30 mg / ml). It is in this sense that the GDP derivative acts as a liquid carrier or solubilizing molecule for the synthesis of peptides or proteins or peptidomimetics.

By way of illustration, Reaction Scheme No. 4 shows the reaction of an activated amino acid in the form of 2,2-bis (trifluoromethyl) -1, 3-oxazolidin-5-one with a derivative of polyisobutene (abbreviated PIB) which is terminated by a phenol function. In this case, the α-amino acid is L-phenylalanine (Phe).

[Chem 4]

Reaction scheme n ° 4: Anchoring to the liquid support

Thus, the first α-amino acid of the future peptide is attached to the anchor molecule via an ester-type covalent bond.

Reaction diagram 5 shows the elongation or iteration step, i.e. the attachment of a second unit of amino acid, to the first amino acid attached to the anchor molecule. In this case, that second α-amino acid is L-tryptophan (Trp).

[Chem 5]

Reaction scheme n ° 5: Elongation

It is easily seen that this process allows, by successive iterations, to add units of a, b or g-amino acids or a, b or g-hydroxylated acids or a, b or g-mercapto acids on the last acid a, b or g-qhiίhέ or a, b or g-hydroxylated acid or a, b or g-mercapto acid or peptide or protein or peptidomimetic fixed on the derivative of PIB, during synthesis, to obtain a peptide or protein or peptidomimetic having the desired sequence. The peptide or protein or peptidomimetic being chemically linked to the anchor molecule, it can be separated at any time, and in particular after the last iteration step, of all polar products by extraction in an organic solvent such as hexane (s) or cyclohexane and water or in a water / ethanol or water / acetonitrile mixture. At the end of this iteration sequence, and optionally after the deprotection of the side chains, the peptide or protein or peptidomimetic can be detached from the anchor molecule; thus the peptide or the protein or the peptidomimetic loses its solubility in an apolar solvent, and it can be separated from the anchor molecule, for use according to its purpose.

According to another characteristic of the invention, the derivation (or anchoring) of an a, b or y-amino acid or a, b or g-hydroxylated acid or a, b or y-mercapto acid or peptide or protein or peptidomimetic (protected or not on its side chain (s)) with a derivative of PIB in fact causes a significant increase in the solubility of said a, b or y-amino acid or a, b acid or g-hydroxylated or a, b or y-mercapto acid or peptide or protein or peptidomimetic anchored in organic liquid phase. More specifically, these a, b or y-amino acids or a, b or g-hydroxy acids or a, b or y-mercapto acids or peptides or proteins or peptidomimetics anchored on a derivative of PIB become soluble in organic solvents, such as than halogenated solvents (methylene chloride, chloroform), ethyl acetate, tetrahydrofuran, 2-methyletetrahydrofuran, isooctane, cyclohexane, hexane (s), methylcyclohexane, methyl tert-butyl ether propylene carbonate or aromatic solvents such as benzene or toluene or any other suitable solvent. Thus, a, b or y-amino acids or a, b or g-hydroxylated acids or a, b or y-mercapto acids or peptides or proteins or peptidomimetics anchored on a GDP derivative have a high partition coefficient for the phase organic during extraction / decantation, thus allowing simple and rapid purification. At the same time, their solubility in solvents such as water or a mixture of water / ethanol or water / acetonitrile is very low.

According to another characteristic of the invention, the reaction between a derivative of PIB and the first acid a, b or y-amino or acid a, b or g-hydroxylated or a, b or y-mercapto acid activated, optionally having a side chain protected or not (ester, amide, thioester or any other chemical functions), leads to a product whose solubility in water is low (<30 mg / ml).

Thus, when an a, b or y-amino acid or a, b or g-hydroxylated acid or a, b or y-mercapto acid or peptide or protein or peptidomimetic is attached to a derivative of PIB, the latter acts as a liquid support (or anchoring molecule or solubilizing molecule) because the product of this reaction becomes soluble in organic solvents but remains insoluble in solvents such as water or a water / ethanol or water / acetonitrile mixture; this allows its separation from the reaction mixture by phase separation. Reaction Scheme No. 6 shows an example of the step of detaching an octapeptide having the sequence Phe-Tpr-Cys (Bzl) -Trp-Cys (Bzl) -Trp-Trp-Cys (Bzl) -NH2, from anchor molecule (derivative of PIB terminated by a phenol function), on which the peptide is anchored by the carboxylic acid function (C-terminal) of L-phenylalanine. The side chains of L-cysteine residues are protected by a benzyl group (Bzl). The free peptide is insoluble in apolar solvents (ie cyclohexane, hexane (s)), which allows it to be easily separated from the anchor molecule. The anchor molecule can be recovered for reuse in the process.

[Chem 6]

Reaction scheme 6: Detachment of an octapeptide from the anchor molecule The peptide precipitates in solvents such as: ethyl ether, cyclohexane or any other suitable solvent. It can then be used according to its intended purpose.

We now describe a second essential characteristic of the process for preparing peptides or proteins or peptidomimetics according to the invention, namely the use of anchoring molecules that are soluble in certain organic solvents such as: ethyl acetate, tetrahydrofuran, 2-methyletetrahydrofuran, isooctane, cyclohexane, hexane (s), methylcyclohexane, methyl tert-butyl ether or halogenated solvents.

Advantageously, the process according to the invention uses polyolefins or more precisely oligomers of polyolefins (polyolefins also being called polyalkenes), and their derivatives as anchoring molecules or liquid support or protective group, whether for the carboxylic acid function ( C-terminal) of the a, p or y-amino acid or a, b or g-hydroxy acid or a, b or y-mercapto acid or peptide or protein or peptidomimetic, or of the side chain of said a, b acid or y- amino or a, b or g-hydroxylated acid or a, b or y-mercapto acid or peptide or protein or peptidomimetic (in the form of ester, amide, ether, thioester, thioether or any other suitable chemical functions) in liquid phase. Polyolefin molecules consist of a chain of carbon atoms linked together by single bonds. They can include branches made up of alkyl groups identical or different, but preferably identical. Preferably, the polymers consist of a number of monomer units of at least 10 and preferably between 15 and 350. Homopolymers are preferred, but copolymers (saturated or not) can be used. In the case of unsaturated polymers or copolymers, the number of unsaturated bonds in the chain of carbon atoms advantageously does not exceed 5%, and preferably does not exceed 3%.

Preferably, they are derivatives of polyisobutenes (PIB), a class of polymers known since the 1930s of the last century, but derivatives of polypropylenes can also be used.

These anchoring molecules are preferably used in the process according to the invention in the form of functionalized derivatives, as will be explained in greater detail below. Scheme # 7 shows a number of PIB derivatives with their functionalizations which are suitable as a liquid carrier for carrying out the present invention.

[Chem 7]

Diagram n ° 7: General structures of anchoring molecules In these formulas:

• X ^e is a group selected from the group formed by -OH, -NH2, -NHR ^a (R ^a = alkyl or aryl), -SH;

· Ar is an aromatic or heteroaromatic group, substituted or not;

• A is either absent or a group selected from the group formed by: CH2, CH2-CH2, S;

• R ^f is a group selected from the group formed by H, aryl, hetero-aryl, alkyl, O-alkyl, O-aryl, O-hetero-aryl;

· R ^q is a group selected from the group consisting of H, alkyl, O-alkyl, aryl, hetero-aryl, O-aryl, O-hetero-aryl;

• the number n is an integer which is typically greater than 10, and advantageously between 15 and 350.

The number m is either 0 or 1. In particular, the group X ^e can be a function in a primary or secondary amine, an alcohol, a thiol or a phenol.

According to the invention, these anchoring molecules are linked to the carboxylic acid function of an a, b or g-amino acid or a, b or g-hydroxylated acid or a, b or g-mercapto acid (C-terminal ) or all chemical functions of a molecule having at least two functional groups via a covalent bond. This assumes that the anchor molecules are engaged in a suitably functionalized form, which is referred to herein as "GDP derivatives". This term also encompasses here the derivatives of anchoring molecules which are not derivatives of polyisobutene, but which are derivatives of other polyolefins according to the definition which is given above; it includes in particular the derivatives of polyolefin oligomers. This functionalization of the anchor molecule is in general a terminal functionalization, preferably at one of the ends of the chain of carbon atoms; it is described below. According to the invention, the a, b or g-amino acids or a, b or g-hydroxy acids or a, b or g-mercapto acids or peptides or proteins or peptidomimetics can be functionalized on their side chains with derivatives of PIB , in the form of ester, ether, thioether, thioester or any other chemical functions compatible with the present process. This amounts to giving the PIB derivatives a role of protective group (s) of the side chain (s) of said a, b or g-amino acids or a, b or g-hydroxylated or a acids, b or g-mercapto acids or peptides or proteins or peptidomimetics. The chains of polyolefins or oligomers of polyolefins or polyalkenes used as anchoring molecules are typically characterized by an average molecular mass by mass, but it is also possible to use chains of polyolefins or oligomers of polyolefins or polyalkenes called "homogeneous" which comprise identical molecules of a given chain length.

More precisely, the process for the synthesis of peptides or proteins or peptidomimetics, optionally protected on their side chains, in liquid phase (solution) according to the invention, is characterized by the fact that an acid a, b or y- is solubilized. amino or acid a, b or g-hydroxylated or a, b or g-mercapto acid or peptide or protein or peptidomimetic in organic medium by a derivative of PIB linked to the carboxylic acid function of the acid a, b or g-amino or a, b or g-hydroxylated acid or a, b or g-mercapto acid or peptide or protein or peptidomimetic or any other molecule having at least two functional groups. The PIB derivative acts as an anchoring molecule (also referred to here as a “liquid carrier” or “solubilizing molecule”) of the a, b or y-amino acid or a, b or g-hydroxyl acid or a, b or y-mercapto acid or peptide or protein or peptidomimetic or any other molecule having at least two functional groups. Said peptide or protein or peptidomimetic attached to this anchoring molecule is synthesized by successive attachment of a, b or y-amino acids or a, b or g-hydroxyl acids or a, b or y-mercapto acids or any other molecules having at least two functional groups on the last a, b or y-amino acid or a, b or g-hydroxy acid or a, b or y-mercapto acid or any other molecule having at least two functional groups. Thus, the anchor molecule also serves as a protective group for the carboxylic acid function (C-terminal) during the synthesis of the peptide or protein or peptidomimetic during successive iterations.

The carboxylic acid function (C-terminal) of said a, b or y-amino acid or a, b or y-hydroxylated acid or a, b or y-mercapto acid or peptide or protein or peptidomimetic, optionally protected on its chain (s) (s) lateral (s), is linked by a covalent bond of ester, amide, thioester, or any other covalent chemical bond type, to a derivative of lipophilic PIB, giving a very low solubility in water (<30 mg / ml). It is in this sense that the GDP derivative acts as a liquid carrier or solubilizing molecule for the synthesis of peptides or proteins or peptidomimetics.

This derivative of the a, b or y-amino acid or a, b or g-hydroxylated acid or a, b or y-mercapto acid or peptide or protein or peptidomimetic (protected or not on its side chains) with a derivative of PIB significantly increases the solubility of said a, b or y-amino acid or a, b or g-hydroxy acid or a, b or y-mercapto acid or anchored peptide or protein or peptidomimetic, in organic liquid phase. More specifically, these a, b or g-amino acids or a, b or g-hydroxy acids or a, b or g-mercapto acids or peptides or proteins or peptidomimetics anchored on the derivative of PIB become soluble in organic solvents such as : halogenated solvents (methylene chloride, chloroform), ethyl acetate, tetrahydrofuran, 2-methyletetrahydrofuran, isooctane, cyclohexane, hexane (s), methylcyclohexane, methyl tert-butyl ether or aromatic solvents such as benzene or toluene or any other suitable solvent. Consequently, a, b or g-amino acids or a, b or g-hydroxy acids or a, b or g-mercapto acids or peptides or proteins or peptidomimetics attached to a derivative of PIB have a high partition coefficient for the organic phase during extraction / decantation in the presence of cyclohexane or hexane (s) and water or a water / ethanol mixture or else water / acetonitrile, thus allowing their simple and rapid purification.

In one embodiment of the method for synthesizing peptides or proteins or peptidomimetics (protected or not on their side chains), in the liquid phase according to the invention, the starting point is a molecule having at least two functional groups or an acid a, b, or g-amino or a, b, or g-hydroxy acid or a, b, or g-mercapto acid activated respectively in the form of 2,2-bis (trifluoromethyl) -1, 3-oxazolidin-5 -one or (2,2- bis (trifluoromethyl) -1, 3-oxazinan-6-one or 2,2-bis (trifluoromethyl) -1, 3-oxazepan-7-one or

2.2-bis (trifluoromethyl) -1, 3-dioxolan-4-one or 2,2-bis (trifluoromethyl) -1, 3-dioxan-4-one or

2.2-bis (trifluoromethyl) -1, 3-dioxepan-4-one or 2,2-bis (trifluoromethyl) -1, 3-oxathiolan-5-one or 2,2-bis (trifluoromethyl) -1, 3-oxathian -6-one or 2,2-bis (trifluoro-methyl) -1, 3-oxathiepan-7-one or their derivatives, which will be linked to one of the anchoring molecules as defined above, via of a covalent bond, and which one adds or condenses, by successive iterations, the molecules having at least two functional groups or the acids a, b or y-amino or acids a, b or g-hydroxylated or a, b or g-mercapto acids activated and optionally protected on their side chains.

The method for synthesizing peptide or protein or peptidomimetic according to the invention can be carried out using a fragment of peptide or protein or peptidomimetic suitably protected on its side chains and a peptide or protein or peptidomimetic sequence anchored on a GDP molecule allowing, afterwards coupling, to obtain a longer peptide or protein or peptidomimetic.

The method for synthesizing a peptide or protein or peptidomimetic according to the invention can be carried out using molecules having at least two groups functional, namely a group Q ^a and Q ^b group, which may be identical or different and which are selected from the electrophilic groups and / or nucleophilic groups. By way of example, in a first embodiment Q ^a can be an electrophilic group, and Q ^b can be a nucleophilic group, or alternatively, in a second embodiment, Q ^a can be a first electrophilic group and Q ^b a second electrophilic group, or alternatively, in a third embodiment, Q ^a can be a first nucleophilic group and Q ^b a second nucleophilic group; in variants, Q ^a and Q ^b can also denote the same electrophilic group, or even the same nucleophilic group. These molecules having at least two functional groups can be directly anchored on the anchor molecule or introduced during synthesis on the amine (primary or secondary) or hydroxyl or thiol function, of a, b or g-amino acids or a acids , p or y-hydroxy or a, b or y-mercapto acids or anchored peptides or proteins or peptidomimetics.

Advantageously, a slight stoichiometric excess of the a, b or y-amino acid or a, b or g-hydroxylated acid or a, b or y-mercapto acid activated, during each anchoring step, is used. elongation and / or iteration.

These bifunctional molecules can be introduced into the peptide or protein or peptidomimetic chain by known chemical reactions. If necessary, they can be protected or masked (on its nucleophilic function or any other chemical functions requiring it, by means of known reactions) and activated by known techniques.

The units derived from bifunctional molecules which are not selected from among a, b or y-amino acids or a, b or g-hydroxy acids or a, b or y-mercapto acids can advantageously be attached to the C-terminus of said peptide or protein or peptidomimetic, or on the N, O or S-terminal end (in particular by functionalization of the amine, hydroxyl or thiol function), or else on the side chain, or alternatively between two units selected from the acids a , b or y-amino or a, b or g-hydroxy acids or a, b or y-mercapto acids.

According to an advantageous embodiment, the number of units derived from bifunctional molecules which are not selected from among a, b or y-amino acids or a, b or g-hydroxylated acids or a, b or y-mercapto acids does not not exceed 50% in number of units, and preferably does not exceed 25% in number of units, and even more preferably does not exceed 10% in number of units. By way of example, semaglutide, a peptidomimetic comprising on a side chain an α-aminobutyric acid unit, can be prepared using the process according to the invention.

A list of molecules of the a, b, g or d-amino acid type is given here which can be used as units within the framework of the process according to the invention, in addition to the amino acids already mentioned above: the acid d -amino levulinic acid, g-aminobutyric acid, b-aminobutyric acid (also known by the acronym BABA), b-alanine, b-lysine, b-aminoisobutyric acid, bN-Methylamino-L- alanine, (2S, 3S, 8S, 9S) -3-amino-9-methoxy-2,6,8-trimethyl-10-phenyldeca-4,6-dienoic acid (also known as ADDA), G (2R) -2- (methylamino) butanedioic acid (also known as NMDA) and 4-amino-3-hydroxybutanoic acid.

A list of molecules of the a, b, g or d-hydroxylated acid type is given here which can be used as units in the context of the process according to the invention: 4-hydroxybutanoic acid, 2- (hydroxymethyl) acid -3-methylbutanoic and (2R, 3R, 4R) - 3-hyd roxy-2, 4, 6-tri m éthy I heptanoic acid.

A list of molecules of type a, b, y or d-mercapto acid is given here which can be used as units in the context of the process according to the invention: 4-sulfanylbutanoic acid, 2-cyclopropyl-3 acid -sulfanylpropanoic acid, 2-cyclobutyl-3- sulfanylpropanoic acid and 2- (2-sulfanylphenyl) butanoic acid

The method according to the invention has many advantages.

A first advantage is that it allows the production of peptides or proteins or peptidomimetics (protected or not on their side chains) linked to the anchor molecule in the organic liquid phase.

A second advantage is that it makes it possible to obtain peptides or proteins or peptidomimetics (protected or not on their side chains) anchored of high purity by a simple washing (extraction) in an apolar organic solvent and with water or a water / ethanol or even water / acetonitrile mixture or by filtration, thus causing the elimination of by-products (salts, acids or any other molecular species) which are not linked to the derivative of polyolefins or oligomers of polyolefins or polyalkenes such as excess reagents. Organic solvents such as cyclohexane, heptane, hexane (s) which have flash points <15 ° C, are suitable for solubilizing the derivatives of polyolefins or of oligomers of polyolefins or polyalkenes during extraction or processing. washing. The process according to the invention therefore makes it possible to limit the purification steps which are necessary in the processes of the state of the art. A third advantage, which is particularly important, is that the process according to the invention makes it possible to synthesize peptides or proteins or peptidomimetics, by adjusting the length of the derivative of polyolefins or oligomers of polyolefins or polyalkenes, that is to say by making them more lipophilic.

A fourth advantage is the possibility of controlling the purity of the peptide or protein or peptidomimetic during synthesis, at any time, by taking an aliquot followed by analysis by the various techniques known to those skilled in the art (such as than mass spectrometry, high performance liquid chromatography, proton or carbon-13 nuclear magnetic resonance).

A fifth advantage lies in the fact that it is not necessary to use a protective group for the amine function (primary or secondary) or hydroxyl or thiol, respectively, a, b or g-amino acids or a acids, b or g-hydroxy or a, b or g-mercapto acids which generally costs two steps (protection and deprotection). More generally, the process according to the invention allows an optimal economy of atoms because it does not involve either protective groups for the amine (primary or secondary) or hydroxyl or thiol function of the corresponding acids, or coupling agents. . This economy of atoms and of process steps according to the invention generates, in industrial reality, financial savings while reducing the generation of waste, which is a favorable environmental factor unlike current methods.

A sixth advantage of the invention lies in the fact that the activation of the carboxylic acid function (C-terminal) is concomitant with the protection of the amine (primary or secondary) or hydroxyl or thiol function, therefore reducing the number of steps.

A seventh particularly interesting advantage of the invention lies in the production of high purity peptides or proteins or peptidomimetics after the cleavage of the protective groups of the side chains, then of the anchor molecule. This avoids purifying the peptide or protein or peptidomimetic synthesized. As a result, additional savings are generated over known methods. This further limits the environmental impact of the production of peptides or proteins or peptidomimetics.

An eighth advantage of the invention lies in the possibility of accessing peptides or proteins or peptidomimetics of larger sizes, either by modulating the size of the liquid carrier or by introducing it on one or more side chains of a, b or g-amino acids or a, b or g-hydroxylated acids or a, b or g-mercapto acids activated.

Other advantages are the possibility of automating the process according to the invention and the possibility of recycling the extraction solvents and the anchoring molecules (polyolefins or oligomers of polyolefins or polyalkenes). Indeed, when the series of iterations to obtain the sequence of the target peptide or protein or peptidomimetic is completed, the latter is deprotected of its protective groups of the side chains and finally, of the anchor molecule by one of the reactions usually used in peptide synthesis, such as hydrolysis, saponification, hydrogenolysis or any other reaction compatible with the present process.

Thanks to their high purity, the peptides or proteins or peptidomimetics produced by this process can be used as pharmaceutical products (drugs and vaccines), cosmetics, phytosanitary products, agrifood products or as intermediates to synthesize such products.

Example

An octapeptide was prepared using the method according to the invention.

In a first step the following amino acids were activated: activated L-phenylalanine (referred to herein as AAA1), activated L-tryptophan (referred to herein as AAA2) and activated L-cysteine (protected by a benzyl (Bzl) protecting group) ( referred to herein as AAA3). This reaction corresponds to Reaction Scheme # 3 above.

To a solution of the amino acid (10 mmol) in NN-dimethylformamide (5 ml), at room temperature, equipped with a dry ice gas condenser and a bubbler, was condensed hexafluoroacetone in excess (> 2 equivalents). After sixteen hours of stirring at room temperature, the reaction mixture was concentrated to dryness, and the residue was lyophilized. The crude product obtained was dissolved in dichloromethane, filtered and then the solvent was removed under reduced pressure and lyophilized (three times). The 2,2-bis (trifluoromethyl) -1, 3-oxazolidin-5-ones or activated amino acids were obtained as an oil or a solid with yields of between 80-95%. Their formulas are given below in figure 8.

[Chem 8] Figure 8: Structures of activated amino acids

In a second step, an activated amino acid (activated L-phenylalanine referred to herein as AAA1) is coupled to the anchor molecule, in this case a derivative of PIB. This reaction corresponds to Reaction Scheme # 4 above.

A solution of the PIB derivative (31.1 mg, 0.028 mmol) and the activated amino acid (here AAA1) (9.8 mg, 0.031 mmol) in a tetrahydrofuran / hexafluoroisopropanol mixture (2 ml) was heated to 50 ° C for 2 hours then cooled to room temperature. A saturated aqueous solution of sodium hydrogencarbonate (2 ml) was added to the reaction medium and the mixture was stirred at room temperature for thirty minutes. The reaction medium was extracted three times with cyclohexane, washed with brine, dried over sodium sulfate, filtered and concentrated under reduced pressure to yield the H ₂ N-Phe-PIB derivative.

In a third step, the peptide is extended by attaching another activated amino acid (here AAA2). This reaction corresponds to Reaction Scheme # 5 above.

A solution of the hhN-Phe-PIB derivative (1 equivalent) and the following activated amino acid (AAA2) (1, 1 equivalent) in tetrahydrofuran / hexafluoroisopropanol (2 mL, 9/1) was heated to 50 ° C for 2 hours then cooled to room temperature. The reaction mixture was treated as before, to yield the corresponding anchored dipeptide (hhN-Trp-Phe-PIB), and so on. In this case, the iteration was repeated with the activated amino acid AAA3 under the same conditions to obtain the corresponding anchored tripeptide (H2N-Cys (Bzl) -Trp-Phe-PIB).

Other iterations were then carried out until an anchored octapeptide of the sequence H2N-Cys (Bzl) -T rp-T rp-Cys (Bzl) -T rp-Cys (Bzl) -T rp-Phe-PI B.

In a final step, the peptide is unhooked from the anchor molecule. This reaction corresponds to Reaction Scheme # 6 above.

Has a solution of the anchored octapeptide (H2N-Cys (Bzl) -Trp-Trp-Cys (Bzl) -Trp-Cys (Bzl) -Trp- Phe-PIB) (8 mg) in a tetrahydrofuran / water mixture (8 : 2) (2 mL), at room temperature room, was added lithium hydroxide solution (1 M, 2 ml). The reaction mixture was stirred at room temperature for 16 hours. The reaction medium was diluted with a dioxane / HCl solution and the precipitate was washed with cyclohexane.

Claims

1. Process for the synthesis of peptides or proteins or peptidomimetics by successive elongation of the second end, which has a primary or secondary amine function, a hydroxyl function or a thiol function, of a peptide or protein or peptidomimetic chain by units, characterized in that :

said units are selected from the group formed by molecules of type Q ^a - E - Q ^b , where Q ^a and Q ^b can be the same or different, and are selected from electrophilic groups and nucleophilic groups, and E represents a spacer ;

the first end of said peptide or protein or peptidomimetic is attached by a covalent bond to an anchoring molecule soluble in organic solvents such as halogenated solvents (preferably methylene chloride or chloroform), ethyl acetate, tetrahydrofuran, 2-methyletetrahydrofuran, isooctane, cyclohexane, hexane (s), methylcyclohexane, methyl tert-butyl ether or aromatic solvents such as benzene or toluene;

said process does not involve groups protecting the primary amine or secondary amine or hydroxyl or thiol function.

2. Method according to claim 1, characterized in that the groups Q ^a and Q ^b are selected from the group formed by: alcohols, aldehydes, primary amines, secondary amines, azides, ethyniles, halogens, thiols, vinyls, and / or in that the spacer E is selected from the group formed by aromatics, heteroaromatics, saturated alkyl chains (branched or not), unsaturated alkyl chains branched or not), glycols (and preferably polyethylene glycol).

3. Method according to claim 1 or 2, characterized in that said units Q ^a - E - Q ^b Q ^a -EQ ^b are selected from the group formed by: natural or unnatural a, b, g or d-amino acids natural or synthetic, natural or unnatural or synthetic a, b, y or d-hydroxy acids, natural or unnatural or synthetic a, b, y or d-mercapto acids.

4. Method according to claim 3, characterized in that said units of a, b or y-amino acids or a, b or g-hydroxylated acids or a, b or y-mercapto acids are used in an activated form. .

5. Method according to claim 3 or 4, characterized in that said units of a, b or y-amino acids or a, b or g-hydroxy acids or a, b or y-mercapto acids are implemented respectively under the form of 2,2-bis (trifluoromethyl) -1, 3-oxazolidin-5-one or (2,2-bis (trifluoromethyl) -1, 3-oxazinan-6-one or 2,2- bis (trifluoromethyl) -1, 3-oxazepan-7-one or 2,2-bis (trifluoromethyl) -1, 3-dioxolan-4-one or 2,2-bis (trifluoromethyl) -1, 3-dioxan-4 -one or 2,2-bis (trifluoromethyl) -1, 3-dioxepan-4-one or 2,2-bis (trifluoromethyl) -1, 3-oxathiolan-5-one or 2,2-bis (trifluoromethyl) - 1, 3-oxathian-6-one or 2,2-bis (trifluoromethyl) -1, 3-oxathiepan-7-one or their derivatives.

6. Method according to any one of claims 1 to 5, characterized in that said anchoring molecule comprises a polyolefin chain or is a polyolefin chain or a polyolefin or polyalkene oligomer, with at least 10 units of monomers, and of preferably between 15 and 350 monomer units and is preferably a polyisobutene chain.

7. The method of claim 6, characterized in that said polyolefin or polyolefin or polyalkene oligomer chain is functionalized at at least one of its ends.

8. Method according to any one of claims 6 or 7, characterized in that said polyolefin or polyolefin or polyalkene oligomer chain comprises a number of unsaturated carbon-carbon bonds not exceeding 5%, and preferably not exceeding 3%. .

9. Method according to any one of claims 6 to 8, characterized in that said anchor molecule has a mass average molecular weight of between 600 and 20,000, and preferably between 700 and 15,000.

10. Method according to any one of claims 1 to 9, characterized in that said anchoring molecule comprises a polyolefin chain (or is a polyolefin chain) or polyolefin or polyalkene oligomer terminated by at least one group selected from the group. trained by :

o a function -X ^a , where X ^a is selected from the group formed by: -OH, - NH ₂ , -NHR ^a (R ^a = alkyl or aryl), -SH;

o a function -Y-C6H4X ^b , where

^■ Y is O, S, CH ₂ OR absent,

^■ X ^b is selected from the group formed by: -OH, -NH ₂ , -NHR ^a , - SH, -CX ^a R ^a R ^b , -C ₆ H ₃ R ^c (CR ^a X ^a ),

where R ^b is selected from the group formed by -H, -Aryle,

-Heteroaryl, -Alkyl, and R ^c is selected from the group formed by -H, -Alkyl, -O-Alkyl, -Aryl, -O-Aryl, -Heteroaryl, -O-Heteroaryl, o a function -CR ^d = CH-CHX ^a or a -CR ^d function H-CH = CH-CHX ^a , where X ^a is defined as above, and R ^d is methyl or ethyl.

11. Method according to any one of claims 1 to 10, characterized in that:

said first end of said peptide or protein or peptidomimetic chain is a first unit of a, b or g-amino acid or a, b or g-hydroxyl acid or a, b or g-mercapto acid, said peptide or protein or peptidomimetic chain is formed of n units of a, b or g-amino acid and / or a, b or g-hydroxylated acid and / or a, b or g-mercapto acid, and

the second end of said peptide chain is another unit of a, b or g-amino acid or a, b or g-hydroxy acid or a, b or g-mercapto acid.

12. Method according to any one of claims 1 to 11, characterized in that during said elongation another unit of a, b or g-amino acid or a, b or g-hydroxylated acid or a, b is added. or g-mercapto acid at said second end from their respective activated forms.

13. Method according to any one of claims 1 to 12, characterized in that said peptide or said protein or said peptidomimetic is obtained by condensation of a peptide or protein or peptidomimetic anchored on an anchor molecule and of a fragment of peptide or protein or peptidomimetic suitably protected on its side chains.

14. Method according to any one of claims 1 to 13, comprising at least one step in which said peptide or protein or peptidomimetic chain attached to said anchor molecule is separated from the reaction medium by extraction in an organic solvent (such as: cyclohexane, heptane, hexane (s)) by extraction or washing with water or a water / ethanol or water / acetonitrile mixture or by simple filtration.

15. A method according to any one of claims 1 to 14, comprising a step in which said peptide or said protein or said peptidomimetic is detached from said anchor molecule.

16. A molecule obtainable by the process according to any one of claims 1 to 14.