FR2655348A1 - Reaction and solubilising medium for peptides and process for the synthesis of peptides using this medium - Google Patents

Abstract

The present invention relates to a reaction and solubilising medium for the synthesis of peptides. This reaction and solubilising medium for peptide synthesis and/or purification contains a diluent A chosen from the group of water-immiscible solvents and a phenol B. Application to the synthesis of peptides which are used, where appropriate, for the synthesis of medicinal products, vaccines, and food or plant-protection products.

Description

The present invention relates to a reaction medium and solubilizer for the synthesis of peptides. It also relates to a peptide synthesis process in this same medium.

There are many methods of peptide synthesis in the liquid phase. They all have common basic points which consist in - optionally protecting the function of the side chain of an amino acid by a cleavable protecting group at the end of peptide synthesis; protecting the amine function (N a) of said amino acid with a cleavable protective group after the condensation of the amino acid; activating the carboxylic acid function of said protected amino acid; and then condensing it with an amino acid or a peptide whose C terminal function is protected and whose amine function is free.

the peptide is obtained by total deprotection of the protecting groups after the condensation of all the amino acids.

The condensation reaction can be carried out both in the homogeneous liquid phase and in the heterogeneous phase (for example Merrifield synthesis).

In general, the synthesis of peptides requires the protection of the carboxylic function of the C-terminal amino acid in the form of esters.

In the case of peptide syntheses in homogeneous phase, the ester will be chosen from
methyl, benzyl and tert-butyl esters,
the polymer esters soluble in organic solvents
for example the polyether esters of MUTTER M. and Al
Justus Liebigs Annalen der Chemie 1975 pp. 901. 915.

or better, among the esters claimed in the patent RP deposited by
the Applicant (No. FR 89 06700 on GPC esters).

In the case of peptide syntheses in heterogeneous phase,
the ester may in particular be an ester of insoluble polymers
in organic solvents
The following polymers may be mentioned in a non-exhaustive manner: the styrene divinylbenzene copolymer introduced by RB MERRIFIELD (JACS 1963. 85, pp. 2149, 2154) polydimethylacrylamide-co-Boc-Ala-N'-acroyl-hexylethylenediamine E. ATHERTON and RC SHEPPARD (JACS 97 1975 pp. 6584.6585) * polystyrene grafted on Kel-F (TREGEAR and Al 1966 US Patent
3,700,609 and Chem.Abot. 71 p 508241 (1969) * cellulose
The heterogeneous phase peptide syntheses can be carried out either in a stirred reactor, in a column reactor, or by any other technique (for example membrane use).
The yield and purity of the final product is highly dependent on the yield of each step, partly because of the geometric increase in losses, and partly because of the problems of separating the desired product from the by-products. . This problem is amplified during the progression of synthesis as the number of amino acids increases.

In addition, in the specific case where the condensation is carried out in a homogeneous liquid medium, all these methods have the same disadvantage their volume productivity is low because of the poor solubility of the amino acids or the intermediate protected peptides.

This disadvantage is pointed out by many authors such as M. NARITA, K. ISHIKAWA, J. Y. CHEN and Y. KIM, Int. J. Peptide Protein Res., 24, 580 (1984).

E. GROSS and J. MEIENHOFER, The Peptides; Analysis, Synthesis, Biology,
Academic Press, 1, 45, (1979).

M. MUTTER and E. BAYER, The Peptides; Analysis, Synthesis, Biology,
Academic Press, 2, 288 (1980).

 FUHRHOP and PENZCIN, Organic Synthesis, Verlag Chemie, 4.1.2. Peptides, 219 (1984).

a) The productivity per unit volume is particularly very low when water-immiscible solvents such as chloroform or ethyl acetate are used. By way of example, the nitro 4 benzyl ester of N- (2-nitro-phenylsulfenyl) -α-benzyl-phenylalanyl-N-nitro-L-arginine is synthesized at a rate of 0.016 mol per liter in chloroform: E. Wünsch , Methoden der Organischem Chemie, XV-2, von Peptiden Synthesis, Georg Thieme Verlag, 108 (1974).

For its part, the methyl ester of benzyloxycarbonyl-L-prolyl-L-tyrosine was prepared at a concentration of 0.050 mol per liter in ethyl acetate.
M. BODANSKY, A. BODANSKY, The Practice of Peptide Synthesis, Springer-Verlag, 140, 1984.

Water-miscible solvents have specific disadvantages and are hardly more advantageous.

Indeed, while they sometimes make it possible to synthesize the peptides in a more concentrated medium, they prohibit the direct purification by washing with water, which is necessary to remove the reagents introduced in excess and the products of the coupling reaction. .

As a result, the water-miscible solvents are usually distilled and then replaced with water-immiscible solvents in order to carry out the washes. We therefore return to the previous problem because the purification step requires very large solvent volumes, while the treatment of the reaction mass has been made more complex by the introduction of additional distillation and redissolution operations.

Thus, the dipeptide Z-Cys (S-BZl) -Tyr OEt prepared in THF (2 mole / liter) was transferred into ethyl acetate (0.1 mole / liter) before being washed with water. 'water.

M. BODANSKY, The Practice of Peptide Synthesis, Springer Verlag, 129, 1984.

The protected dipeptide Z-Lys (Z) -Gly-OEt synthesized in acetonitrile (0.13 mol / liter) was purified after replacement of the initial solvent with ethyl acetate (0.05 mol / liter, Ibid 150, 1984) while
Z-Ala-Tyr 0CH3 was prepared in dimethylformamide (0.3 mole / liter) before being purified by washing with water in solution in ethyl acetate (0.1 mol / liter) (Ibid 148, 1984).

The use of these water-miscible solvents, which in general are polar, can lead to additional disadvantages in terms of industrial hygiene (DMSO, HMPT), as well as in terms of chemical selectivity.

In general, polar solvents promote the racemization of the N-protected activated amino acid, according to the development of
DS KEMP, Peptides, Analysis, Synthesis, Biology; 1, 354-355, 1979.

Thus obtaining concentrated solutions of peptide intermediates in water-immiscible organic solvents compatible with the current manufacturing standards (Good Manufacturing Practices) remains an unresolved problem.

This is why one of the aims of the present invention is to provide a reaction medium which increases the yield of peptide condensations.

Another object of the present invention is to find a reaction medium which makes it possible to facilitate the syntheses and purifications of peptides, in particular peptides of 2 to 50 amino acids in general, and in particular from 3 to 20 amino acids, or amino-peptides. acids.

Finally, another aim is to provide a reaction medium allowing a peptide synthesis in a homogeneous medium at high concentrations (for example at least equal to 0.1 in general at 0.2 M.

These and other purposes which will appear later, are achieved by a reaction medium and solubilizing for synthesis and / or peptide purification which comprises a diluent A selected from the group of water immiscible solvents and a phenol B .

The amount of phenol B is preferably at least 1/200 of diluent A.

There is no strict upper limit. Thus, the use of phenol B (pure product or mixture) without diluent A, is part of the invention insofar as the following conditions are met - The melting point of phenol B is at most equal to 50 ° C ( in the present description the zeros are not significant figures unless otherwise specified) - The phenol B may be separated from the reaction product by distillation or reduced pressure: - The phenol B is not miscible with the water in all proportions - Phenol B does not form a stable emulsion capable of causing the elimination of co-products by washing with water.

The halogeno phenols advantageously di-, and preferably mono chloro, as well as the light alkyl phenols respond, for example, to these constraints.

However, it is preferable that there is a diluent A and a phenol B.

Advantageously, the mass ratio between said phenol B and said diluent
A is between 1/200 and 1, preferably between 1/20 and 1/2.

The above values, expressed in mass ratio, are well suited for phenol itself and for phenols whose molecular mass is not of an order of magnitude different from that of phenol itself (C 6 H 5 OH).

For phenols of high molecular weight, the molar concentration per liter is at least 10-2, preferably between 0.1 and 2, preferably between 0.5 and 1.5 M.

When diluents A and phenols B are not miscible in any proportion, the upper limit of the phenol (s) content is the lower of the two limits: that mentioned above and the solubility limit.

By the expression phenol B, it is necessary to include phenols or mixtures of phenols.

The diluents A are sufficiently polar organic solvents to dissolve at least 1%, preferably at least 2% by weight of the phenol (C 6 H 6 OH) and are sufficiently hydrophobic to be immiscible with water in any proportion.

It is preferable that the water can dissolve at most 10% of diluent A advantageously at most 1% by weight and this even in the presence of phenol B as a third solvent.

The diluents A can be mixtures, including petroleum fractions. Naturally, under the operating conditions, the diluents A must be inert with respect to the phenols and peptide synthesis reagents used.

Preferred families of diluents are selected from the group consisting of aromatic derivatives, ethers, esters and halogenated solvents.

As paradigms of members of these families, halogenated aliphatic derivatives include dichloromethane, 1,2-dichloroethane, 1,1,1-trichloroethane, aromatic derivatives, toluene and halogenated aromatic derivatives. chlorobenzene, such as esters of ethyl acetate and isopropyl acetate, as ethers, tert-butyl methyl ether and anisole.

For reasons of industrial economy, it is preferable that the diluent
A is distillable at atmospheric pressure or under primary or secondary vacuum.

In general, the phenol B is chosen from the group of compounds corresponding to the following formula I.

(R1) n - Ar - O - H (I) in which
Ar represents a monocyclic, polycyclic, heterocyclic or non-aromatic aromatic radical
- The substituents R1, similar or different, represent
a halogen unit, preferably fluorine, chlorine, bromine
a group -Z-R2, where Z can be
- a simple connection;
an oxygen atom; where R2 represents a hydrogen atom, an optionally hydroxylated or mono or poly halogenated alkyl or aryl radical of at most 8 carbon atoms, n represents the number of substituents and is equal to O or to an integer at most equal to number of substitutable positions on aromatic rings and their mixtures.

Alkyl groups (as defined in the dictionary of Chemistry
Duval Presses Scientifiques Internationale PARIS VIe 1959) can in particular be linear or branched aliphatic residues of at most 6 carbon atoms or arylaliphatic residues.

The number of substitutable positions is easily determinable using simple rules known to those skilled in the art.

For example, when Ar = phenyl n <5
Ar = pyridyl S 4
Ar = naphthyl <7
Ar = quinolyl 4 6
Advantageously, the phenol B has a number of carbon atoms of at most 30 carbon atoms, preferably at most 20 carbon atoms.

It is desirable that the vicinal positions of the phenol function are unsubstituted or occupied by non-bulky groups.

Are considered bulky radicals connected to said vicinal positions by a tertiary or secondary carbon.

Mono cyclic compounds are those which give the best compromise between efficiency and cost; those with 6 knuckles (pyridinyl or phenyl nucleus) are preferred.

It is also desirable that Z-R 2 can not be more than three times, preferably more than 2 times, hydroxyl.

Advantageously, in formula I, the radicals R are chosen from the group consisting of methyl, ethyl, propyl and butyl radicals, trifluoromethyl and pentafluoroethyl radicals, methoxy, ethoxy, propyloxy and butyloxy-phenyl radicals. hydroxy phenyl, and Ar OH, - phenyloxy, hydroxyphenyloxy, - fluorine, chlorine and bromine radicals.

In order not to overload the phenol molecule, it is desirable that in the formula I n be at most equal to 5.

Among the phenols giving the best results, mention should be made of: - optionally monosubstituted hydroxy-pyridines, - optionally monosubstituted hydroxy quinolines, - monohalo phenols (preferably mono chloro), - polyhalo phenols, (preferably polyfluoro), phenols mono- or disubstituted by alkyl radicals of C1 to C4, alkoxy of C1 to C4, perfluoroalkyl of C1 to C2 and 2,2,2 trifluoroethyl, - diphenols, - phenol in the narrow sense, - naphthols optionally mono or di substituted.

The media according to the present invention make it possible to dissolve an optionally protected amino acid at a concentration of at least 0.05 M.

The amino acid can be C or N protected; in the latter case, the acid group can be activated on the acid function, by the protected side acid functions, the amino acids can be natural or synthetic.

The medium according to the present invention also makes it possible to dissolve a protected peptide.

The medium according to the present invention also makes it possible to dissolve unprotected peptides (HCl, Pro-Gly-NH2, HCl, His-Trp, 2HCL, Arg-Pro) and to synthesize.

Since the medium according to the present invention solubilizes both amino acids, peptides and blocking or activating reagents, it makes it possible to carry out "in situ" blockages, or even the activation of amino acids.

The peptide may be C or N protected; in the latter case, the acid group can be activated on the acid function.

Of course, the functions of the amino acids, other than the acid or amine functions targeted by the synthesis, can also be protected if necessary according to the usual techniques in the field.

The medium of the present invention makes it possible to significantly increase the solubility of the peptides in the organic phase.

It allows, in general, to change the order of magnitude of the solubility in the favorable direction, taking as a basis the solubility of the peptide in organic diluent A in the absence of phenolic additive.

The medium makes it possible to carry out the different steps of the peptide synthesis under conditions allowing a good volume yield.

In particular, it increases the productivity of the condensation steps.

The present invention also relates to a process for the synthesis of peptides possibly protected in a liquid medium, starting from an initial peptide or amino acid, optionally protected on its acid function and in that an amino acid is added. activated on the acid function and protected on the amine function, in which the reagents are dissolved in the medium according to the invention.

In the same way, the present invention also aims at a process for the synthesis of peptides which may be protected in a liquid medium, starting with an initial amino acid or peptide, solubilized according to the invention and that another peptide is condensed. having its activated C terminal acid function and its protected N-terminal amine function, wherein the reagents are dissolved in a medium according to the invention.

For example, the amine function is protected by a carbamate group or by reaction with a betadicarbonyl compound.

Advantageously, the acid function of the N-protected reagents is activated by an organic or inorganic acid chloride, by an alkyl chloroformate, by a carbodiimide or by an ester activated by their carbonyl diimidazole, or by an imidazole acyl.

After condensation of an amino-acid or a protected-N-peptide with the initial peptide or amino acid, the amine-N-terminal function is released by any method, and advantageously by hydrogenolysis or by acidic or basic solvolysis. or by photolysis.

The amino acid or C-terminal peptide protected (preferably by esterification or amidation) initial is usually introduced into the reaction medium, then the amino acid or peptide activated on its acid function and protected on its function amine is added, the order of addition of the reagents being indifferent, after reaction co-products and excess reagents are removed from the organic phase by washing with acidic aqueous solutions, basic or neutral, then the amine function is released and finally a new amino acid or a new peptide activated on its acid function and protected on its amine function, is introduced.

Surprisingly, the medium potentiates the reactivity of the peptides with the amino acids and significantly improves the kinetics of the condensation.

An important advantage of the process is that it removes excess reagents and condensation co-products from the concentrated organic phase by simply washing with water, facilitated by the anti-emulsifying effect of the phenols, a contam effect during of the study that led to the present invention. The excess reagents are, for example, amino acids or activated and N-protected peptides, or C-protected amino acids or peptides, or catalysts for accelerating coupling such as hydroxy benzotriazole, imidazole,
N-hydroxysuccinimide, etc .....

The medium according to the present invention also makes it possible to increase the productivity of the cleavage steps of the protecting groups. It also allows the cleavage of the acid-sensitive groups, for example t-butyloxycarbonyl, by means of common reagents such as trifluoroacetic acid or hydrochloric acid, as well as the cleavage of the hydrogenolysable groups, for example benzyloxycarbonyl, benzyl ethers and esters or the nitro group protecting the guanidino function of the arginine side chain.

Said medium allows the cleavage of basolabile groups such as sulfofluorenyl-methyl-oxycarbonyl by common reagents such as diethylamine or piperidine. It also allows the cleavage of photolysable protecting groups. It also allows the cutting of protective groups by electrochemical reduction.

An important advantage of the process is, in the homogeneous phase, that it allows the catalysts to be removed from the cleavage steps by simple operations: the acidic or basic catalysts can be removed by aqueous washing while the hydrogenolysis catalysts such as the palladium absorbed on charcoal can be removed by filtration; the peptide or peptide intermediate remaining in concentrated organic solution.

Another advantage of the solubilization process is that it is compatible with all the operations necessary for the synthesis of the peptides and that it allows a direct sequence between the different stages of condensation, cleavage of protecting groups and purification by washing with water. water without changing the solvent, and optionally without isolation of the peptide.

It thus allows the repetitive synthesis of peptides.

Due to the repetitiveness of the synthesis, the method of the present invention can be advantageously implemented in an automated form.

The method is particularly useful for the synthesis of poorly soluble peptides in organic solvents such as peptides containing glycyl, arginyl, glutaminyl, asparaginyl, serinyl, threonyl and prolyl residues;
With regard to the parameters not covered by the present description, the process according to the present invention is carried out under customary conditions.

The peptides resulting from the synthesis may be used optionally for the synthesis of drugs, vaccines, agri-food or phytosanitary products.

The present invention is illustrated by the following nonlimiting examples.

Protocol of solubilization tests
The solubilities of the peptide intermediate were determined as follows, at a temperature of 25 ° C.

An exactly weighed amount of the peptide intermediate is placed in a Pyrex tube. The solvent or mixture of solvents to be tested is then introduced using a precision pipette so as to obtain a mass ratio of peptide / volume of solvent equal to 8g / 100cm3.

The tube closed with a screw cap is then placed on a HEIDOLPH brand agitator, model TOP-MIX, vibrating at its maximum speed for 60 seconds.

In the case where the solid is completely dissolved, the solubility is noted> 8g / 100cm3, otherwise the volume of solvent is doubled and the process is repeated.

At the end of 4 successive dilutions without total dissolution, the measurements are taken up on a smaller quantity of solid.

When the solubility in a given solvent was found to be less than 0.05 g / 100 cc, it was further verified that heating at 40 C and additional stirring for 4 minutes did not change the result.

Notes: the composition of the solvent / additive mixtures is weighted.

SOLUBILIZATION OF DIFFERENT PEPTIDES
EXAMPLE 1 Hydrochloride of the methyl ester of L-serinyl-L-tyrosine

(HCl, Ser-Tyr-OCH3)
RESULTS
Solubility in dichloromethane: S <0.05g / 100cm3
Solubility in the dichloromethane / phenol mixture; 4/1: 8>s> 4g / 100cm3
EXAMPLE 2 Trifluoroacetate of the methyl ester of L-tryptophanyl-L-serinyl-L-tyrosine (CF 3 COOH, Trp-Ser-Tyr-OCH 3):
The solubilities measured as in Example 1 are the following in dichloromethane: s <0.05 g / 100 cm3.

 in the dichloromethane / phenol mixture; 4/1: 8> s> 4g / 100cm3.

Example 3 Methyl Ester of the
L-L-tryptophanyl- serinyl-L-tyrosine
(Trp-Ser-Tyr-OCH3):
Solubilities measured as in Example 1 in dichloromethane: <0.05 g / 100 cm 3 in the dichloromethane / phenol mixture; 4/1: s> 8g / 100cm3. dichloromethane / t-butyl-4-phenol; 4/1: s> 8g / 100cm3. dichloromethane / dimethoxy-2,6-phenol; 4/1: s> 8g / 100cm3. dichlormethane / 2,6-dimethylphenol; 4/1: s> 8g / 100cm3. Dichloromethane / 2,6-dichlorophenol; 4/1: s> 8g / 100cm3. dichloromethane / hydroxy-2-pyridine; 5/1: s> 8g / 100cm3 dichloromethane / 2-methoxyphenol; 4/1: s> 16g / 100 cm3. dichloromethane / trifluoromethyl-3-phenol; 4/1: s> 8g / 100cm3. dichloromethane / 2,3,4,5,6-pentafluoro phenol; 4/1: s> 8g / 100cm3 ethyl acetate: s <0.05g / 100cm3. ethyl acetate / phenol; 9/1: s = 8g: 100cm3
Example 4: L-Histidinyl-L-Tryptophan Hydrochloride
(HCl, His, Trp)
Solubilities measured as in Example 1.

dichloromethane: s <0.05 g / 100 cm3. dichloromethane / phenol; 4/1: s = 2 g / 100cm3
Example 5: N-Benzyloxycarbonyl-L-pyroglutanyl-L-histidine (Zp Glu-His)
Solubilities measured as in Example 1. dichloromethane: s <0.05 g / 100 cm3. dichloromethane / phenol; 4/1: 8>s> 4g / 100cm3. dichloromethane / methoxy-4-phenol; 4/1: 2>s> lg / 100cm3. dichloromethane / hydroxy-2-pyridine; 5/1: s = 0.3g / 100cm3. dichloromethane / trifluoromethyl-3-phenol; 4/1: 8>s> 4g / lOOcm3. chloro-2-phenol: 8>s> 4g / 100cm3. pentafluorophenol / dichloromethane; 1/4: 8>s> 4g / 100cm3
Example 6: L-arginyl-L-proline dihydrochloride
(2 HCl, Arg-Pro)
Solubilities measured as in Example 1. dichloromethane: s <0.05 g / 100 cm3. dichloromethane / phenol; 4/1: s = 2 g / 100cm3. dichloromethane / chloro-2-phenol; 1/1: 2>s> lg / lOOcm3
Example 7: L-Leucyl-L-Arginyl-L-Prolyl-Glycinamide Hydrochloride
(HCl, Leu-Arg-Pro-Gly-NH2)
Solubilities measured as in Example 1. dichloromethane: s <0.05g / 100cm. dichloromethane / methoxy-4-phenyl 4/1: s = 4g / 10Oc3. dichloromethane / phenol; 4/1: s = 4g / 100cm3. dichloromethane / trifluoromethyl-3-phenol; 4/1: s = 4g / 100cm3. dichloromethane / chloro-2-phenol; 2/1: 2>s> lg / lOOcm3. dichloromethane / 2,3,4,5,6-pentafluoro-phenol; 4/1: 4>s> 2 g / 100 cm 3 chloro-2-phenol: 4>s> 2 g / 100 cm 3. dichloromethane / tert-butyl-4-phenol; 4/1: 1>s> 0.5g / 100cm3. dichloromethane / hydroxy-2-pyridine; 5/1: s = 1 g / 100cm3
By way of comparison, dimethylformamide, an additive which does not form part of the invention, was tested. dichloromethane / dimethylformamide; 4/1: s <0.5g / 100cm3
Example 8: Hydrochloride of L-prolyl-glycinamide
(HCl, Pro-Gly-NH2).

Solubilities measured as in Example 1. dichloromethane: s <0.05 g / 100 cm 3 dichloromethane / phenol; 4/1: s> 16g / 100cm3. dichloromethane / methoxy-4-phenol; 4/1: 8>s> 4g / 100cm3. dichloromethane / chloro-2-phenol; 2/1: 2>s> lg / 100cm3. dichloromethane / hydroxy-2-pyridine; 5/1: 4>s> 2g / 100cm3
By way of comparison, dimethylformamide, an additive which does not form part of the invention, has been tried. dichloromethane / dimethylformamide; 4/1: s <0.5g / lOOcm3
Example 9: N - # - Trifluoroacetyl-L-Lysyl-L-Proline
(Lys (TFA) - Pro)
Solubilities measured as in Example 1. dichloromethane: s <0.05 g / 100 cm3. ethyl acetate: s <0.05 g / 100 cm3 dichloromethane / phenol; 4/1: s> 20 g / 100cm3. dichloromethane / tert-butyl-4-phenol; 4/1: s> 8 g / 100cm3. dichloromethane / dimethoxy-2,6-phenol; 4/1: s> 8 g / 100cm3. dichloromethane / dimethyl-2,6-phenol; 4/1: s = 8 g / lOOcm3. dichloromethane / hydroxy-2-pyridine; 5/1: s> 8 g / 100cm3. dichloromethane / 2,6-dichlorophenol; 4/1: s> 8 g / 100cm3. dichloromethane / methoxy-2-phenol; 4/1: s> 8 g / 100cm3. dichloromethane / trifluoromethyl-3-phenol; 4/1: s> 8 g / 100cm3. dichloromethane / chloro-2-phenol; 4/1: s> 8 g / lOOcm3. dichloromethane / 2,3,4,5,6-pentafluoro-phenol; 4/1: s> 8 g / 100 cc ethyl acetate / phenol; 9/1: s = 8 g / 100cm3
Example 10: Methyl ester of N-tertiobutyloxycarbonylpentaglycine
(Boc-Gly-Gly-Gly-Gly-Gly-OCH3): (Boc glyg OCH3)
The solubility in dichloromethane was measured by HPLC, with external calibration, on a saturated solution of Boc-Gly5-OCH3.

The other solubilities were measured as in Example 1.

dichloromethane: s = 0.33 g / 100 cm3 dichloromethane / phenol; 20/1: s = 0.5 g / lOOcm3. dichlormethane / phenol; 4/1: s = 4 g / 100cm3. anisole: s <0.05 g / 100cm3. anisole / phenol; 4/1: s = 4 g / 100cm3. chloro-2-phenol: s> 8 g / 100 cm3. dichloromethane / chloro-2-phenol; 4/1: 2>s> 1 g / 100cm3. dichloromethane / methoxy-4-phenol; 4/1: 4>s> 2 g / 100cm3
EXAMPLE 11 Benzyl Ester of N-tertiobutyloxycarbonyl pentaglycine
(Boc-Gly-Gly-Gly-Gly-Gly-O-CH2-Ph)
The solubility in dichloromethane was measured by HPLC by external calibration on the supernatant of a saturated solution; the other solubilities were measured as in Example 1. dichloromethane: s = 0.07 g / 100 cm3. dichloromethane: phenol; 9/1: s> 5 g / lOOcm3
By way of comparison, additives not forming part of the invention have been tested. dichloromethane / trifluoroethanol; 9/1: s <2 g / 100cm3 * dichloromethane / octanol-2; 9/1: s <2 g / 100cm3 * dichioromethane / pivalic acid; 9/1: s <2 g / 100cm3 *. dichloromethane / dimethylformamide; 9/1: s <2 g / lOOcm3 * dichloromethane saturated with LiCl: s <2 g / 100cm3 * * Concentrations lower than 2g / 100cm3 have not been tested.

Example 12: Methyl ester of N-benzyloxycarbonyl-B acid
benzyl-L-aspartyl-amino-1-cyclopropane-carboxylic acid
(Z-Asp- (OBzl) - Acc-OCH3)
Solubilities measured as in Example 1. methyl and tert. butyl: s <0.5 g / 100 cm3 methyl ether and tert. butyl / tert.butyl-4-phenol; 6/1
s> 1.2 g / 100cm3. chlorobenzene: s <1.5 g / 100 cm3. chlorobenzene / tert.butyl-4-phenol; 4/1: s> 20 g / 100 cc anisole: s> 2.5 g / 100 cm3. anisole / tert.butyl-4-phenol; 4/1: s> 25 g / lOOcm3. ethyl acetate: s = 2.5 g / 100 cm3. ethyl acetate / 2-hydroxypyridine (saturated solution)
10>s> 5g / 100cm3
SYNTHESIS OF PEPTIDES
Example 13 Propyl ester of L-aspartyl-amino-1-cyclopropane carboxylic acid
(Asp-Acc-Opr).

Note: this dipeptide ester is a sweetener having a sweetening power approximately 200 times higher than that of sucrose according to C. MAPELLI,
Mr. GARY NEWTON, CE RINGOLD and CH STAMMER, Int. J.Peptide Protein Res.

30, 498-510 (1987).

Retape 1: Condensation at the concentration of 0.66 mole / liter of
CH2 CL2 (320 g).

In a 100 cm 3 Pyrex tri-neck reactor cooled by a water bath at 17 ° C. and inerted with a stream of dry nitrogen, the solids are successively charged onto a solid anisole (37.5 cm 3) root. following
Benzyl-aspartic acid N-benzyloxycarbonyl-B (25 mmol),
N-ethyl-N '- (3-dimethylamino-propyl) carbodiimide hydrochloride (30 mmol) and 1-hydroxypyridine (42.5 mmol).

The suspension fluidizes rapidly and leads to a solution to which are added in turn after 15 minutes
Propyl amino-1-cyclopropane carboxylate (30 mmol) and tert-butyl-4-phenol (5 g).

After stirring overnight, the reaction mixture is washed at 30 ° C. with 25 cm 3 of 2N hydrochloric acid and then 3 times with 13 cm 3 of an aqueous solution consisting of 10 cm 3 of demineralized water and 3 cm 3 of saturated brine. NaCl. The combined aqueous phases are counter-extracted with 2.5 cm3 of anisole.

Step 2: Hydrogenolysis of the protective groups chained with the step of
condensation.

The organic phase (40 cm 3 is transferred to a 200 cm 3 tri-neck pyrex reactor containing 100 cm 3 of distilled water and 2.8 g of 3% palladium on carbon, previously inert under nitrogen.

The mixture brought to 55 ° C. is placed at ambient pressure under a stream of hydrogen for 4 hours.

Retape 3: Isolation of the peptide
The reaction mixture is filtered on Millipore 5A, the catalyst is washed on the filter with 10 cm3 of water at 35 ° C twice.

The organic phase is decanted and discarded.

The aqueous phase is washed with 30 cm3 of ethyl acetate 3 times and then concentrated by vacuum distillation to a residual mass of 60 g.

A crystallization occurs spontaneously.

The suspension, added with 240 cm3 of acetone is filtered on fritted glass of porosity n 4.

The solid washed with acetone and dried to constant weight weighs 4.41 g.

The filtrate concentrated to dryness is dissolved in 11 cm3 of water.

The addition of acetone (66 cm3) makes it possible to isolate a second batch of solid weighing 1.52 g after drying under vacuum.

Results
The propyl ester of L-aspartyl-amino-1-cycloprane-carboxylic acid monohydrate, thus obtained with a yield of 86/0, is a white solid melting at 179 ° C.

Its 1H NMR spectrum recorded at 360 Mhz in solution in DMSO is compliant.

Elemental analysis: C 11, H 18, N 2, 05, 1H 2 O
Calculated: C, 47.83; H, 7.3; N, 10.14; 34.76 Found: C 47.69 H 7.1 N 10.01; O 34.81
Water content (Karl Fischer): 6.48%. Potentiometric dosing
Amine function: 102%
Acidic function: 98%
Example 14
By way of comparison, a test according to Example 13 in which either the addition of 4-tert-butylphenol or the addition of 2-hydroxy-pyridine is omitted rapidly leads to an inactive reaction mixture.

PURIFICATION OF PEPTIDES
BY EXTRACTION LIOUIDE-LIOUIDE
Example 15: Benzyl ester of glycyl-glycyl-L-phenylalanyl-L-leucine
(Gly-Gly-Phe-Leu-OBzl)
In order to purify this tetrapeptide, by washing with water at the end of its synthesis, the partition of this peptide between dichloromethane and water was studied as follows.

A solution of Gly-Gly-Phe-Leu-OBzl hydrochloride (0.9 mmol) in dichloromethane (12 cm3) is stirred vigorously in the presence of an aqueous solution of KHCO 3 (2 N, 3 cm 3). This mixture provides an emulsion that remains stable for several days. A comparative test was carried out in the presence of phenol
Example 16
A partition test carried out according to Example 15 after the addition of 3.9 g of phenol to the organic solution gives, after identical stirring, two clear liquid phases, the decantation of which is complete in less than 15 minutes.

HPLC analysis shows that the aqueous phase contains only 0.4% of the engaged tetrapeptide.

Example 17 Benzyl Ester of the
N-butyloxycarbonyl-L-phenylalanyl-L-valine
(Boc-Phe-Val-OBzl).

In a 50 cm3 pyrex glass reactor are successively introduced at room temperature the 2-hydroxy-pyridinic ester of N-tert.butyloxycarbonyl-L-phenylaline (3.6 mmol, 1.23 g)
Hydrochloride of the benzyl ester of Valine (3 mmol, 0.73 g)
The dichloromethane (15 cm3) and then stirring the N-methyl morpholine (3 mmol).

After 68 hours at room temperature, the reaction mixture is washed successively with 5 cm 3 of dilute sulfuric acid (95 μ pH 2), with 5 cm 3 of water and then with 5 cm 3 of KHCO 3. 2N in solution in water.

A stable emulsion is formed.

On the contrary, when a dichloromethane / phenol (9/1) mixture is used as the reaction solvent, all the washes proceed without difficulty and the settling is rapid.

EXAMPLE 18
Subject: Study of the influence of phenol during peptide coupling.

Coupling studied:
Boc-Phe-O-Py + HCl, Val-OBzl + NMM
CH2 Cl2 (with or without phenol)
Boc-Phe-Val OBzl + HCl, NMM
Operating mode
The coupling was carried out by bringing into contact in methylene chloride at reflux, on the one hand, 1.2 equivalents of N 2 -hydropyridine ester of N-butyloxy L phenylalanine (Boc-Phe-OPy) and ester hydrochloride. benzyl valine (Val OBzl) an equivalent in the presence of N methyl morpholine (NMM).

The disappearance of Val OBzl and the appearance of said peptide were measured. Correlation is perfect to indeterminations of measurements.

The results are collated in the following table, in which the peptide yield is shown and, which is much more significant of the activation and kinetics problems, the residual OBz1 Val.

Two types of tests were carried out in a perfectly homogeneous medium, on the one hand with phenol C6 H50H in methylene chloride (10% by weight of phenol), and on the other hand without phenol.

The results show a highly significant kinetic difference, since 90% of said peptide is obtained in 2 hours in methylene chloride alone, and in 20 minutes in the 10% by weight mixture of methylene chloride phenol.

RESULTS: In the table is specified the coupling efficiency as well as
the percentage of Val OBzl over time.

<Tb>

TIME <SEP> 10 <SEP> min <SEP> 30 <SEP> min <SEP> 1 <SEP> hour <SEP> 2 <SEP> h <SEP> 30
<tb> Yield <SEP> without <SEP>#OH<SEP><SEP> 56 <SEP>% <SEP> 80 <SEP>% <SEP> 86 <SEP>% <SEP> 95 <SEP>%
<tb>
<tb><SEP> Coupling <SEP> with <SEP>#OH<SEP><SEP> 78 <SEP>% <SEP> 93 <SEP>% <SEP> 96 <SEP>% <SEP> 99 <SEP> %
<tb> Val <SEP> OBzl <SEP> without <SEP>#OH<SEP><SEP> 44 <SEP>% <SEP> 20 <SEP>% <SEP> 14 <SEP>% <SEP> 5 <SEP >%
<tb> Residual
<tb> with <SEP>#OH<SEP><SEP> 22 <SEP>% <SEP> 7 <SEP>% <SEP> 4 <SEP>% <SEP> 1 <SEP>%
<Tb>

Claims (25)

1) Reaction and solubilizing medium for the synthesis and / or peptide purification characterized in that it advantageously comprises a diluent A selected from the group of water immiscible solvents and a phenol B. 2) Medium according to claim 1 characterized in that the mass ratio between said phenol B and said diluent A is between 1/200 and 1 preferably between 1/20 and 1/2. 3) Medium according to one of claims 1 and 2, characterized in that said diluent is selected from the group consisting of aromatics, ethers, esters and halogenated solvents. 4) Medium according to one of claims 1 to 3 characterized in that said phenol is selected from the group of compounds corresponding to the following general formula I.  an oxygen atom; where R2 represents a hydrogen atom, an optionally hydroxylated or mono or poly halogenated alkyl or aryl radical of at most 8 carbon atoms, n represents the number of substituents and is equal to O or to an integer at most equal to number of substitutable positions on aromatic rings and their mixtures 5) Medium according to one of claims 1 to 4 characterized in that said phenol B has a number of carbon atoms of at most 30 carbon atoms, preferably at most 20 carbon atoms.  - a simple connection;  a group -Z-R2, where Z can be  a halogen unit, preferably fluorine, chlorine, bromine  - The substituents R1, similar or different, represent  Ar represents a monocyclic, polycyclic, heterocyclic or non-aromatic aromatic radical (R1) n - Ar - O - H (I) in which 6) Medium according to one of claims 1 to 5 characterized in that the vicinal positions of the phenol function are unsubstituted or occupied by non-bulky groups. 7) Medium according to one of claims 4 to 6 characterized in that the -Ar radical is a monocyclic aromatic preferably having 6 knuckles. 8) Medium according to one of claims 4 to 7, characterized in that in formula I, the radicals R are chosen from the group consisting of - methyl, ethyl, propyl, butyl radicals, - trifluoromethyl and pentafluoromethyl radicals, the methoxy, ethoxy, propyloxy, butyloxy, phenyl, hydroxy phenyl and Ar OH radicals; the phenyloxy, hydroxyphenyloxy, fluorine, chlorine and bromine radicals. 9) Medium according to one of claims 4 to 8 characterized in that in the formula I n is at most equal to 5. 10) Medium according to one of claims 1 to 6, 8 and 9 characterized in that said phenol A is selected from the group consisting of - optionally monosubstituted hydroxy-pyridines, - hydroxy quinolines optionally monosubstituted, - monohalo phenols ( preferably mono chloro), poly (halo) phenols (preferably polyfluoro), phenols mono or di substituted with C1 to C4 alkyl radicals, C1 to C4 alkoxy, perfluoroalkyl C1 to C2 and 2, 2,2, trifluoroethyl, - diphenols, - phenol in the narrow sense, - optionally mono or di substituted naphtholes. 11) Medium according to one of claims 1 to 10 characterized in that it further comprises a protected amino acid. 12) Medium according to claim 11 characterized in that said amino acid is protected C. 13) Medium according to claim 11 characterized in that said amino acid is N protected. 14) Medium according to claim 12 characterized in that said amino acid is activated on the acid function. 15) Medium according to one of claims 1 to 10 characterized in that it further comprises a protected peptide. 16) A medium according to claim 15 characterized in that said peptide is C protected. 17) A medium according to claim 15 characterized in that said peptide is N protected. 18) A medium according to claim 16 characterized in that said peptide is activated on the acid function. 19) A method for synthesizing peptides optionally protected in a liquid medium, starting from an initial peptide or amino acid, protected on its acid function and in that an activated amino acid is added to the acid function and protected on the amine function, characterized in that the reagents are dissolved in the medium according to one of claims 1 to 10. 20) A method for synthesizing peptides, optionally protected in a liquid medium, starting from an initial amino acid or peptide, solubilized according to claim 1 and condensing another peptide having its activated C terminal acid function and its function protected N-terminal amine, characterized in that the reagents are dissolved in a medium according to one of claims 1 to 10. 21) A method according to any one of claims 19 and 20 characterized in that the amine function is protected by a carbamate group or by reaction with a betadicarbonyl compound. 22) Process according to any one of claims 19 to 21, characterized in that the C-terminal acid function of the initial peptide or amino acid is preferably protected by esterification or amidation. 23) Process according to any one of claims 19 to 21, characterized in that the acid function of the N-protected reagents is activated by an organic or inorganic acid chloride, an alkyl chloroformate, a carbodiimide or by an activated ester or an imidazole acyl. 24) Process according to any one of claims 19 to 21, characterized in that after condensation of an amino acid or a peptide-N protected with the peptide or initial amino acid, the amine-N-function. The terminal is liberated by hydrogenolysis or by acidolysis or by basolysis. 25) A method according to any one of claims 19 to 24 characterized in that 1 amino acid or C-terminal peptide protected initial is introduced into the reaction medium, then the amino acid or peptide activated on its acid function and protected on its amine function is added, the order of addition of the reagents being indifferent. After reaction, the co-products and the excess reagents are removed from the organic phase by washing with acidic, basic or neutral aqueous solutions, then the amine function is released and finally a new amino acid or a new activated peptide on its function acid and protected on its amine function, is introduced.