Reaction and dissolving medium for peptides and synthesis...

Details Reaction and dissolving medium for peptides and synthesis... Reaction and dissolving medium for peptides and synthesis...

1998-06-12

2002-01-29

Low, Christopher S. F. (Department: 1653)

Chemistry: natural resins or derivatives; peptides or proteins;

Peptides of 3 to 100 amino acid residues

Synthesis of peptides

C530S338000

Reexamination Certificate

active

06342582

ABSTRACT:

BACKGROUND OF THE INVENTION
The present invention relates to a reaction and dissolving medium for the synthesis of peptides. It also relates to a method for the synthesis of peptides in said medium.
A large number of methods exist for the synthesis of peptides in liquid phase. They all have fundamental common features such as:
if necessary, protecting the function of the side chain of a peptide by a protective group cleavable at the end of the peptide synthesis;
protecting the amine function (&agr;N) of a peptide by a protective group cleavable after the condensation reaction;
activating the carboxylic acid function of a protected peptide; and
then subjecting the peptide to a condensation reaction with a peptide in which the C-terminal function is protected and in which the amine function is free.
The peptide is obtained by total deprotection from protective groups after the condensation reaction of all of the amino acids.
The condensation reaction can be carried out either in homogeneous liquid phase or in heterogeneous phase (for example, the 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 synthesis in homogeneous phase, the ester will be chosen from:
the methyl, -benzyl and tert-butyl esters;
the ester of polymers soluble in organic solvents,
for example the ester of polyethers described by M. Mutter et al., Justus Liebigs Annalen der Chemie 1975 pp. 901-915, hereby incorporated by reference;
or preferably from the esters disclosed in the French Patent Application No. FR 89 06700, which corresponds to U.S. Ser. No. 592,028, filed on Oct. 2, 1990, which discloses GPC esters and is hereby incorporated by reference.
In the case of peptide syntheses in the heterogeneous phase, the ester will be, in particular, an ester of polymers insoluble in organic solvents. The following polymers may be mentioned, without this list being limiting:
the styrene-divinylbenzene copolymer introduced by R. B. Merrifield (J.A.C.S. 1963, 85 p. 2149, 2154) hereby incorporated by reference;
co-Boc-&bgr;-Ala-N′-acroylyl-hexamethylenediamine polydimethylacrylamide described by E. Atherton and R. C. Sheppard (J.A.C.S. 97 1975 p. 6584, 6585), hereby incorporated by reference;
polystyrene grafted on Kel-F (Tregear et al. 1966, U.S. Pat. No. 3,700,609 and Chem. Abot. 71 p. 508241 (1969)), both of which are hereby incorporated by reference; and cellulose.
Peptide syntheses in heterogeneous phase may be carried out in a stirred reactor, in a column reactor or by any other technique (for example use of a membrane).
The yield and the purity of the final product to a large extent depend on the yield from each step, on the one hand because of the geometric growth in the losses and on the other hand because of the problems in separating the desired product from the by-products. This problem is amplified as the synthesis progresses when the number of amino acids increases.
Moreover, in the specific case where the condensation reaction is carried out in a homogeneous liquid medium, all of these methods have a common disadvantage: their productivity per unit volume is low because of the poor solubility of the amino acids or of the intermediate protected peptides.
This disadvantage is indicated by numerous authors, such as:
M. Narita, K. Ishikawa, J. Y. Chen and Y. Kin, 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).
The productivity-per unit volume is in particular very low when water-immiscible solvents such as chloroform or ethyl acetate are used. By way of example, the 4-nitrobenzyl ester of &agr;-N-(2-nitrophenylsulfenyl)-&ohgr;-N-benzylphenylalanyl-nitro-L-arginine is synthesized in an amount of 0.016 mol per liter in chloroform: E. Wünsch, Methoden der Organischem Chemie {Methods in Organic Chemistry}, XV-2, Synthese von Peptiden {Peptide Synthesis}, Georg Thieme Verlag, 108 (1974).
For its part, the methyl ester of benzyoxycarbonyl-L-prolyl-L-tyrosine has been prepared in a concentration of 0.050 mol per liter in ethyl acetate: M. Bodansky and A. Bodansky, The Practice of Peptide Synthesis, Springer-Verlag, 140, 1984.
The water-miscible solvents have specific disadvantages and are hardly more advantageous. In fact, although they sometimes allow the synthesis of peptides to be carried out in a more concentrated medium, they prohibit their direct purification by washing with water, which is necessary to remove the reagents introduced in excess and the products of the coupling reaction.
Consequently, the water-miscible solvents are usually distilled and then replaced by water-immiscible solvents in order to proceed with washing. This therefore comes back to the previous problem because the purification step requires very large volumes of solvent, while the treatment of the reaction mass has been made more complex by the introduction of supplementary distillation and redissolving operations.
Thus, the dipeptide Z-Cys(S-BZl)-Tyr OEt prepared in THF (2 mole/liter) has been transferred to ethyl acetate (0.1 mol/liter) before being washed with 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) has been purified after replacing the initial solvent by ethyl acetate (0.05 mol/liter) (Ibid. 150, 1984), while Z-Ala-Tyr-OCH
3
has been prepared in dimethylformamide (0.3 mol/liter) before being purified by washing with water, in a solution of ethyl acetate (0.1 mol/liter) (Ibid. 148, 1984).
The use of these water-miscible solvents, which generally are polar, can lead to additional disadvantages with respect to industrial hygiene (DMSO, HMPT), and with respect to the chemical selectivity. In general, the polar solvents in fact promote the racemization of the N-protected activated amino acid, according to the implementation by D. S. Kemp, Peptides, Analysis, Synthesis, Biology; 1, 354-355, 1979.
Thus, the production of concentrated solutions of peptide intermediates in organic solvents which are immiscible with water and compatible with current production standards (Good Manufacturing practice) remains an unresolved problem.
It is for this reason that one of the aims of the present invention is to provide a reaction medium which increases the yield from peptide condensation reactions. Another aim of the present invention is to provide a reaction medium which makes it possible to facilitate the syntheses and the purifications of peptides, in particular the peptides having 2 to 50 amino acids in general, and in particular 3 to 20 amino acids.
Finally, another aim is to provide a reaction medium allowing a peptide synthesis in homogeneous medium at high concentrations (for example at least about 0.1 and in general about 0.2 M, depending upon the number of amino acids in the peptide—shorter chain length peptides can of course be synthesized at higher molar concentrations than can longer chain length peptides).
These aims and others, which will become apparent from the text which follows, are achieved by means of a reaction and dissolving medium for peptide synthesis and/or purification which comprises a diluent A chosen from at least one of a group of water-immiscible diluents A and at least one phenol B.
In the present application, the term “phenol B” is defined to mean any hydroxyaromatic compound, including mixtures of hydroxyaromatic compounds. The term “water-immiscible diluent A” is defined to mean a diluent that is immiscible in water in some proportion. In other words, when the diluent is added to water, two phases should be formed.
The amount of phenol B is preferably at least 1/200 of the diluent A. There is no strict upper limit to the amount of phenol B to dilu

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