Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
  • C07K1/04—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length on carriers
  • C07K1/042—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length on carriers characterised by the nature of the carrier
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
  • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
  • C07K5/08—Tripeptides
  • C07K5/0802—Tripeptides with the first amino acid being neutral
  • C07K5/0812—Tripeptides with the first amino acid being neutral and aromatic or cycloaliphatic

Definitions

• the invention relates to the field of organic chemistry, and more particularly to the synthesis of peptides or proteins from amino acids.
• the invention relates to a process for the synthesis of peptides in vitro, in the liquid phase, which does not use a solid support or insoluble resin.
• This process is based on the use of a new family of anchoring molecules, namely derivatives of polyolefins or oligomers of polyolefins or polyalkenes, which, linked to an amino acid or an amino acid derivative, exhibit good solubility in nonpolar solvents, but have low solubility in water.
• This process makes it possible to obtain peptides which are purer and / or easier to purify than the known processes on solid support or on liquid support. It is easy to automate.
• All amino acids have at least two reactive chemical functions: the amine function (Na center) and the carboxylic acid function (C-terminal). Certain amino acids also have side chains capable of reacting with the Na or C-terminal centers.
• the key to a peptide synthesis strategy lies in the choice of protective groups (also called protection groups) by which the reactive centers are protected during certain stages of the process.
• protection groups also called protection groups
• each addition of an amino acid requires a cycle of steps: protection - activation / coupling - deprotection.
• the protective groups are cleaved to generate the target peptide.
• the common basic cycle protection - activation / coupling - deprotection is invariable but only the protective group used. More precisely, such a cycle comprises several stages:
• the target peptide is obtained by total deprotection of the protective groups.
• a first, a second and a third amino acid (here called AA1, AA2 and AA3) are supplied.
• the amine function (Na) of the amino acid AA2 is temporarily protected by a tert-butoxycarbonyl group (commonly called “Boc”).
• Boc tert-butoxycarbonyl group
• Then activates the carboxylic acid function (C-terminal) of said protected amino acid which is condensed with the free amine function (Na) (ie unprotected) of a methyl ester of amino acid AA1, which generates a dipeptide.
• the amino function (Na) of the amino acid AA2 is deprotected (ie the protective group Boc is cleaved with an acid, such as trifluoroacetic acid) from the dipeptide formed.
• the amino function (Na) of the amino acid AA3 is protected.
• the carboxylic acid function (C-terminal) of the amino acid AA3 which is coupled with the amine function (Na) of the amino acid AA2 (of the deprotected dipeptide) is activated to generate the protected tripeptide.
• a tripeptide is obtained. This process includes a minimum of five reaction steps, two of which are (activation / coupling - deprotection) for each additional amino acid added to the peptide.
• the derivative obtained is coupled via the activation of its carboxylic acid (C-terminal), to the amine function (Na) of the amino acid AA1 anchored on the resin, which generates an N-protected dipeptide and anchored on the resin. .
• the Fmoc group of the dipeptide is cleaved (on AA2).
• the protection of the amino function (Na) of the amino acid AA3 by an Fmoc group is followed by activation of its carboxylic acid (C-terminal) then, we proceed to the coupling of the species obtained with the anchored dipeptide and having the free amine function, thus generating an N-protected tripeptide, which remains fixed on the solid support by the acid function (C-terminal) of its amino acid AA1.
• This process includes a minimum of seven reaction steps, two of which (activation / coupling - deprotection) for each additional amino acid added to the peptide.
• the carboxylic acid (C-terminal) function of the starting amino acid is protected in the form of a methyl ester and the following amino acids are successively condensed after the protection of their amine function (Na) by a benzyloxycarbonyl group and the activation of their carboxylic acid function (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 yield. By way of example, there may be mentioned the synthesis of ACTH with an overall yield of approximately 7%, described by Schwyzer and Sieber (Helv. Chim. Acta 1966, 49, 134-158).
• the amino acid or the protein can be linked to a protective group called a solubilizer, such as phenylazobenzyl sulfonylethyloxy (OPSE), described in EP 0 017 536 (CM Industries).
• OPSE phenylazobenzyl sulfonylethyloxy
• this protective group allows the solubilization of the amino acid or of the peptide synthesized in N, N-dimethylformamide.
• the purification is done by precipitation and filtration of the solids, which gives rise to operational difficulties.
• EP 2 612 845 A1 and US 2014/0296483 (Ajinmoto Co., Inc.) have described new protective groups (or anchoring molecules) allowing the solubilization of the amino acid and the peptide.
• the purification is done either by simple washing with water, or by precipitation and filtration. Thanks to these protective groups very lipophilic of the carboxylic acid function, bivalirudin, an anticoagulant consisting of 20 amino acid residues was prepared with an overall yield of 73% and a purity of 84% (see D. Takahashi et al., Angew. Chem. Int. Ed., 2017, 56, 7803-7807).
• its limit namely the number of amino acid residues capable of being anchored, remains unknown.
• the financial cost and the environmental cost of these anchor molecules constitute a handicap.
• Solid phase peptide synthesis has been described by Merrifield (J. Am. Chem. Soc., 1963, 85, 2149-2154). It consists in fixing the carboxylic acid (C-terminal) function of the first amino acid or of the peptide on an insoluble resin (support). Consequently, the reagents are used in excess in order to ensure the total conversion of the activation / coupling steps. Purifications are done by simple filtration and washing of the resin. Although this technique is automated and simpler, many drawbacks exist such as the cost and loss of the reagents used in excess, and the lack of homogeneity of the peptides synthesized because it is almost impossible to obtain homogeneous peptides: it is said that the system is degenerate. In addition, the purification in Preparative high performance liquid chromatography of these heterogeneous peptides is expensive because it consumes a lot of solvents and is not environmentally acceptable.
• a yield of less than 100% implies not only the loss of reagents, but also the formation of side products which may be difficult to separate from the target peptide; insofar as one wishes to have peptides as pure as possible to clearly characterize their biological effects, this means that either one accepts the additional cost that the purification presents, or one accepts the presence of impurities likely to generate a risk error in appreciation of the biological effects observed.
• the problem which the present invention seeks to solve is to present a method of synthesis of purer peptides or proteins, with higher yield, more ecological, less expensive and which is automated.
• This process should be suitable for at least all natural amino acids and preferably for a broad spectrum of non-natural amino acids.
• This method should not involve expensive or difficult-to-synthesize anchor molecules or supports.
• the present invention proposes to solve the difficulties left in the prior art by the use of polyolefins or polyolefin oligomers or polyalkenes for the production of high purity peptides or proteins in the liquid phase.
• polyolefins and in particular of polyisobutenes (PIB) derivatives, as anchor molecules or liquid support, allows the solubilization of amino acids and the synthesis of peptides in organic solution (solvents halogenated and non-halogenated), while facilitating their purification by simple extraction or washing, in this case with water or a water / ethanol or water / acetonitrile mixture, or by simple filtration.
• some of these anchoring molecules in particular certain polyisobutenes (PIB) derivatives
• PIB polyisobutenes
• the object of the invention is therefore a process for the synthesis of peptides or proteins by successively elongating the second end (Na) of a peptide chain whose first end is fixed, by means of its carboxylic acid function ( C-terminal) or of its amine function (Na), on an anchoring molecule soluble in an apolar solvent, characterized in that said anchoring molecule comprises a polyolefin chain with at least 10 units of monomers, and preferably between 15 and 50 units.
• it is functionalized at one of its ends, to allow the anchoring of the first amino acid.
• said anchoring molecule is a polyolefin.
• said anchoring molecule comprises only a single polyolefin chain; this polyolefin chain can thus be obtained by a simple polymerization reaction (of isobutene).
• Said anchoring molecule can comprise, in each of its units, alkyl groups which may or may not be identical, which are preferably selected from the group formed by methyl and ethyl.
• Said polyolefin chain advantageously has a weight-average molecular mass of between 600 and 20,000, and preferably between 700 and 15,000.
• Said polyolefin chain can comprise a number of carbon-carbon bonds unsaturated not exceeding 5%, and preferably not exceeding 3%.
• it is a polyisobutene chain.
• said anchoring molecule comprises a polyolefin chain (or is a polyolefin chain) which is terminated by a group selected from the group formed by:
• X is selected from the group formed by: -OH, -NH2, -SH;
• ⁇ Z is O or absent
• ⁇ X 1 is selected from the group formed by: -OH, - NH2, -SH, -NH-NH2, -CXRR 1 , -C 6 H 3 R '(CRX)
• X is selected from the group formed by -OH, - NH2, -SH, and R is selected from the group formed by -H, Aryl, Heteroaryl, and R 'is selected from the group formed by -H, -Alkyl, -O-Alkyl, -Aryl, - O-Aryl, Heteroaryl, -O-Heteroaryl,
• This group then represents the functionalization of the polyolefin chain.
• Said first end of said peptide chain is a first unit of amino acid AA1; it is on this first amino acid AA1 that the anchoring molecule is linked, either on its carboxylic acid function (C-terminal), or on its amine function (Na).
• Said peptide chain is formed of n amino acid units; its second end is another amino acid unit AAn.
• the peptide chain is lengthened by successive elongation, and during each of these elongation steps another unit of amino acid AA (n + 1) is added to said second end.
• the amine function (Na) of the amino acids used in the process according to the invention can be protected by a Boc or Fmoc group, or by any other suitable protective groups.
• Natural and / or non-natural and / or synthetic amino acids can be used in said peptide chain.
• the method according to the invention comprises at least one step in which said peptide chain is attached to said anchor molecule and is separated from the reaction medium by extraction in an apolar solvent.
• the process according to the invention makes it possible to obtain very pure peptides or proteins, which are cleaved from their anchoring molecule after the last stage of elongation of the peptide chain, to be used according to their destination, for example as active ingredient for preclinical or clinical trials.
• amino acid natural amino acids and non-natural amino acids.
• Natural amino acids include the L-form of the amino acids that can be found in naturally occurring proteins, that is: 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).
• the “unnatural” amino acids include form D 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. They also include unnatural amino acids, such as:
• Aib 2-aminoisobutyric acid
• Nal2 2-naphthylalanine
• protected amino acid is used here also to designate temporarily protected amino acids, as described above, in particular; for example the amine function (Na) can be protected by an Fmoc, Boc, benzyl group or any other suitable protective group.
• polyolefins or more precisely polyolefin oligomers (polyolefins also being called polyalkenes), and their derivatives as anchoring molecules or protective group, whether of the carboxylic acid function ( C-terminal) of the amino acid or of the peptide, or of the amine function (Na) of the amino acid or of the peptide, or of the side chain of said amino acid or of peptide (in the form of ester, amide, ether bonds , thioether or any other function) in the liquid phase.
• Polyolefin molecules include a chain of carbon atoms linked by single bonds.
• polymers can comprise branches consisting of identical or different, but preferably identical, alkyl groups.
• polymers are used with a number of monomer units of at least 10 and preferably between 15 and 50.
• Homopolymers are preferred, but copolymers can also be used, and in the latter case the number of bonds unsaturated in the chain of carbon atoms advantageously does not exceed 5%, and preferably does not exceed 3%.
• PIB polyisobutenes
• 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.
• these anchoring molecules are linked to the carboxylic acid function of an amino acid (C-terminal) or to the amine function (Na) by a covalent bond of amide, ester, benzyl, allyl type or all other functions.
• This supposes that the anchor molecules are engaged in a suitably functionalized form, which is called in the present description "derivatives of PIB", knowing that this term also includes here the derivatives of anchor molecules which are not derivatives of polyisobutene, but which are derivatives of other polyolefins according to the definition given above.
• This functionalization of the anchor molecule is generally a terminal functionalization, preferably at one of the ends of the chain of carbon atoms; it will be described below.
• polyolefin oligomers used as anchor molecule are typically characterized by a mass average molecular weight, but it is also possible to use "pure" oligomers which contain identical molecules of a given chain length.
• This reaction between the PIB derivative and the amino acid leads to a product characterized in that when the PIB derivative is linked to an amino acid or an amino acid derivative, possibly having a protected side chain, a molecule with low solubility in water ( ⁇ 30 mg / ml).
• the process for the synthesis of peptides, possibly protected, in the liquid phase (solution) according to the invention is characterized in that an amino acid or a peptide is solubilized in an organic medium by a PIB derivative linked to the carboxylic acid function (C-terminal) or to the amine function (Na) of the amino acid or of the peptide.
• the PIB derivative acts as an anchor molecule or liquid support for the amino acid or peptide which is synthesized by successive attachment of amino acids to the last amino acid attached to this anchored molecule.
• the anchor molecule also serves as a protective group during the synthesis of the peptide during successive iterations.
• the amino acid or optionally protected peptide anchored on a PIB molecule is characterized in that the carboxylic acid function (C-terminal) or the amine function (Na) of said amino acid or peptide is linked by a covalent bond of the type ester, amide, benzyl, allyl or any other chemical function to a lipophilic PIB derivative, giving a very low solubility in water ( ⁇ 30 mg / ml).
• said anchor molecule which is preferably a derivative of PIB, acts as a liquid support for the synthesis of peptides or proteins.
• This derivation of the amino acid (Na-protected or not) or the peptide (Na-protected or not) with the PIB derivative significantly increases the solubility of said amino acid or of said peptide in non-polar organic liquid phase. More specifically, these amino acids and these peptides become soluble in organic solvents, such as halogenated solvents (methylene chloride, chloroform), ethyl acetate, tetrahydrofuran, cyclohexane, hexane (s) or aromatic solvents such as benzene or toluene.
• organic solvents such as halogenated solvents (methylene chloride, chloroform), ethyl acetate, tetrahydrofuran, cyclohexane, hexane (s) or aromatic solvents such as benzene or toluene.
• the amino acids and peptides attached to a PIB derivative have a high partition coefficient for the organic phase during an extraction / decantation in the presence of water or a water / ethanol or water / acetonitrile mixture. , thus allowing their simple and rapid purification.
• the present invention also provides a process for the synthesis of peptides (protected or not), in the liquid phase, characterized in that one starts from a amino acid or peptide in solution (or an amino acid derivative or peptide in solution), which will be linked to one of the anchoring molecules as defined above, via the function carboxylic acid (C-terminal) or of the amino function (Na) of the amino acid derivative or of the starting peptide, and that the following amino acids or peptides are added or condensed, which are protected on their function amine (Na) and optionally on their side chain, after activation of their carboxylic acid function (C-terminal).
• the activation of the carboxylic acid function (C-terminal) of the Na-protected amino acid or of the peptide can be carried out by all known techniques of synthesis via the formation of an anhydride by means of various reagents such as: carbodiimide , acid chloride, alkyl chloroformate, or any other technique for activating an Na-protected amino acid.
• the process for the synthesis of peptides according to the invention is also characterized in that the amino acids or the peptides to be condensed are then added. These amino acids or peptides being activated on their acid function and protected on their amine (Na) function, and protected if necessary also on their side chain.
• Reaction scheme 3 shows the first step in coupling the acid function of the first amino acid (AA1), in this case phenylalanine (Phe), to a PIB derivative functionalized with a phenol.
• AA1 first amino acid
• Phe phenylalanine
• the amino acid AA1 is engaged in the protected state by an Fmoc group.
• DCM dichloromethane
• EDC 1- ethyl-3- (3-dimethylaminopropyl) carbodiimide
• HOBt hydroxy-benzotriazole
• DMAP 4-dimethyl-aminopyridine.
• Reaction diagram 4 shows the variant in which AA1 is engaged in the protected state by a Boc group.
• the protection group (Fmoc or Boc) is cleaved to release the amine function (Na) from AA1.
• This so-called deprotection step is illustrated in reaction diagrams 5 for the Fmoc case and 6 for the Boc case. It leads to a molecule which we call here PIB-AA1.
• ACN means acetonitrile
• HNEÎ2 means diethylamine
• TFA means trifluoroacetic acid
• the first step is repeated by adding to the PIB-AA1 molecule, the second amino acid (here called AA2), in this case glycine (Gly) in the protected state (Boc or Fmoc); the acid (C-terminal) function of AA2 (whose Na function is protected) binds to the N-terminal function of PIB-AA1.
• the Na function of PIB-AA1 -AA2 is deprotected, generating the dipeptide PIB-AA1 -AA2 anchored on its support.
• a fifth step the first step is repeated, adding to the molecule PIB-AA1 -AA2, the third amino acid (here called AA3), in this case tyrosine protected on its side chain by a tert-butyl group, to the 'protected state (Boc or Fmoc); the acid function (C-terminal) of AA3 (whose Na function is protected) binds to the Na function of PIB-AA1 - AA2.
• the Na function of PIB-AA1 -AA2-AA3 is deprotected, and the anchored tripeptide shown in reaction scheme 7 is obtained.
• this process makes it possible, by successive iterations, to add successive amino acids to the amino acid and then to the peptide attached to the PIB derivative, thereby obtaining a peptide having the desired sequence.
• the peptide being attached to the liquid support, it can be separated at any time, and in particular after the last iteration, from all polar products by extraction in an apolar solvent. At the end of this elongation sequence, the peptide can be detached from the support polymer; thus the peptide loses its solubility in an apolar phase, and it can be separated from the support polymer, for use in accordance with its destination.
• reaction scheme 8 For the tripeptide of reaction scheme 7.
• TFA trifluoroacetic acid
• TIPS triisopropylsilane
• water can be used.
• Figure 9 shows a number of PIB derivatives with their functionalization which are suitable as a liquid carrier for carrying out the present invention.
• X is a group selected from the group formed by: OH, NH 2 , NH-NH2, SH;
• ⁇ R is a group selected from the group formed by: H, aryl, hetero-aryl;
• R ’ is a group selected from the group formed by: H, alkyl, O-alkyl, hetero-aryl, O- (hetero-aryl);
• R is a group selected from the group formed by: H, alkyl, O-alkyl, hetero-aryl, O- (hetero-aryl),
• Y is a group selected from the group formed by: O, CH2CH2.
• n is an integer which is typically greater than 10, and advantageously between 15 and 50.
• X can be a free amine, a hydrazine, an alcohol, a thiol or a phenol.
• Certain PIB derivatives which can be used in the context of the present invention are commercially available as ligands for homogeneous catalysis.
• the preferred anchor molecules namely polyisobutene derivatives, can be prepared from biobased isobutene.
• the concept of biobased content is defined in ISO 16620-1: 2015 “Plastics - Biobased content - part 1: General principles”, in particular by a definition of the terms “biobased synthetic polymer”, “biobased synthetic polymer content”, “Bio-based carbon content” and “bio-based mass content”, as well as in ISO 16620-2: 2015 “Plastics - Bio-based content - part 2: Determination of bio-based carbon content” and ISO 16620-3: 2015 “ Plastics - Biobased content - part 3: Determination of the biobased synthetic polymer content ”, for the methods of determination and quantification of the biobased character.
• the anchoring molecules have a content of biobased carbon greater than 90%, preferably greater than 93%, and even more preferably greater than 95%.
• 20 example -X, -Z-CehhX 1 or -CR ” CH-CHX as defined above), is between 600 and 20,000, and preferably between 700 and 15,000. Beyond a molecular mass around 20,000 these molecules have too high a viscosity, which would risk limiting their solubility in the solvents (halogenated or not) used for the activation / coupling step.
• Certain PIB derivatives which can be used in the context of the present invention are commercially available as ligands for homogeneous catalysis.
• the preferred anchor molecules namely polyisobutene derivatives, can be prepared from biobased isobutene.
• the concept of biobased content is defined in ISO 16620-1: 2015 “Plastics - Biobased content - part 1: General principles”, in particular by a definition of the terms “biobased synthetic polymer”, “biobased synthetic polymer content”, “Bio-based carbon content” and “bio-based mass content”, as well as in ISO 16620-2: 2015 “Plastics - Bio-based content - part 2: Determination of bio-based carbon content” and ISO 16620-3: 2015 “ Plastics - Biobased content - part 3: Determination of the biobased synthetic polymer content ”, for the methods of determination and quantification of the biobased character.
• the anchoring molecules have a content of biobased carbon greater than 90%, preferably greater than 93%, and even more preferably greater than 95%.
• the process according to the invention has many advantages.
• a first advantage is that it allows peptide production in the liquid phase, where the peptide (Na-protected or not) linked to the anchoring molecule, defined above, remains in organic solution.
• a second advantage is that it makes it possible to obtain peptides of high purity by a simple washing with water or a water / ethanol or water / acetonitrile mixture, or by filtration, thus generating the elimination of the sub- products (salts, acids or any other molecular species occurring for example during the deprotection of the amine function) which are not linked to the derivative of polyolefins or oligomers of polyolefins or polyalkenes and of reagents in excess of the organic phase.
• Organic solvents such as cyclohexane, heptane (s), hexane (s) which have flash points ⁇ 15 ° C, are suitable for solubilizing derivatives of polyolefins or oligomers of polyolefins or polyalkenes during the extraction or washing.
• the method according to the invention therefore makes it possible to avoid all the purification steps necessary in the prior art methods.
• a third advantage, which is particularly important, is that the process according to the invention makes it possible to synthesize peptides or even proteins, by adjusting the length of the derivative of polyolefins or oligomers of polyolefins or polyalkenes, by making them more lipophilic.
• Another advantage is the possibility of checking the purity of the peptide being synthesized, at any time, by taking an aliquot followed by an analysis by the various techniques known to those skilled in the art (such as spectrometry of mass, high performance liquid chromatography, nuclear magnetic resonance of the proton or carbon-13).
• the preferred anchor molecules namely polyisobutene derivatives, can be prepared from biobased isobutene, as explained above.
• the possibility of automating the process according to the invention and the possibility of recycling the anchoring molecules (polyolefins 22 or oligomers of polyolefins or polyalkenes).
• the peptide is deprotected from its protective groups and finally from the anchoring molecule by one of the reactions usually used in peptide synthesis (such as hydrolysis , saponification, hydrogenolysis), which releases the anchor molecule.
• the anchor molecule can be recycled. Thanks to their high purity, the peptides or proteins produced by this process can be used as pharmaceutical products (drugs and vaccines), cosmetic products, phytosanitary products or food products, or to access any of these products.
• Examples 1 and 2 illustrate two variants of the coupling reaction of the first amino acid (AA1) of the target peptide (engaged in the reaction in the Na-protected form by Fmoc or Boc) with a liquid support (in this case a derivative of GDP).
• Examples 3 and 4 illustrate two variants of the deprotection of the following amino acid (AA2), engaged in the reaction in the Na-protected form by Fmoc or Boc (here called, respectively, "derivative of Fmoc” or " derived from Boc ”).
• Example 5 illustrates the release of the peptide from the anchor molecule, in accordance with reaction scheme 8 above.
• chlorinated solvents namely DCM.
• solvents of lower harmfulness such as: tetrahydrofuran, 2-methyltetrahydrofuran, ethylene carbonate, pure or as a mixture.
• the N-protected amino acid (Fmoc-AA-OH or Boc-AA-OH) (1, 3 mmol) was dissolved in DCM (5 mL) with magnetic stirring and under a nitrogen atmosphere, then cooled to 0 ° C in an ice bath.
• the PIB derivative (free amine, alcohol, thiol or phenol) (1 mmol) dissolved in DCM (5 mL), 4-dimethylaminopyridine (DMAP) (0.3 mmol).
• DMAP 4-dimethylaminopyridine
• the tert-butylcarbamate derivative (1 mmol) was dissolved in the in DCM (5 mL) with magnetic stirring, and was cooled to 0 ° C in an ice bath.
• a DCM / TFA mixture (1/1) 35 ml was added, then the reaction medium was stirred at room temperature for 1 h.
• the solvents were evaporated under reduced pressure. Cyclohexane was added to the residue which was then washed three times with water or with a water / ethanol or water / acetonitrile mixture, then with a saturated aqueous solution of sodium hydrogencarbonate, and with a saturated aqueous solution of chloride. sodium.
• the organic phase was dried over Na2SC> 4, filtered, and the solvent was evaporated under reduced pressure.

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