Peptide C alpha-amides, methods for preparing same and uses thereof as precursors of peptide C alpha-thioesters for protein synthesis

Abstract

The subject matter of the present invention is peptide C.sup.-amides which are precursors of peptide C.sup.-thioesters, characterized in that they comprise the radical of general formula (I) in which X, R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5, R.sub.6, R.sub.7, R.sub.8, R.sub.9, n and A are as defined in Claim 1. The subject matter of the present invention is also the use of these peptide C.sup.-amides for obtaining peptide C.sup.-thioesters. The subject matter of the present invention is also the use of these peptide C.sup.-amides for obtaining peptides or proteins, in particular of therapeutic interest, by direct use as a crypto-thioester partner in NCL reactions. ##STR00001##

Claims

1. The peptide C.sup.-amide having the general formula (II): ##STR00064## in which: Peptide1 represents the R.sub.18-(Xaa).sub.k- group in which: k is an integer ranging from 1to 100, Xaa represents, independently of one another, an amino acid residue originating from an amino acid of formula HXaa-OH, and when k is greater than or equal to 2, each of said Xaa is connected to its neighboring Xaa via a peptide bond, R.sub.18 is a hydrogen atom or a substituent of the N-terminal end included in the Xaa residue, one of R.sub.2, R.sub.3, R.sub.4 or R.sub.5 represents the radical BC-D in which: D represents a hydrogen atom or a solid support suitable for solid phase peptide synthesis (SPPS), C is absent or represents an arm that can be used for SPPS, B represents: ##STR00065## in which E is absent or represents the -(Xaa).sub.i- group in which: i represents an integer ranging from 1 to 20, each Xaa represents, independently of one another, an amino acid residue, and when i is greater than or equal to 2, each of said Xaa is connected to its neighboring Xaa via a peptide bond, the others of said R.sub.2, R.sub.3, R.sub.4 or R.sub.5 which do not represent BC-D, then represent, independently of one another, a hydrogen atom, or an alkyl radical having from 1 to 5 carbon atoms, or a phenyl radical (C.sub.6H.sub.5), on the condition that at most two of said radicals R.sub.2, R.sub.3, R.sub.4 or R.sub.5 represent at the same time a phenyl, A represents an aryl or heteroaryl radical chosen from the group comprising the radical of formula: ##STR00066## in which * indicates the point of attachment of A in the compound of formula (II), R.sub.10 represents an alkyl radical having from 1 to 10 carbon atoms, R.sub.11 and R.sub.12 represent, independently of one another, a hydrogen atom, a halogen atom chosen from the group comprising Cl, Br, I and F, a CN radical, an NO.sub.2 radical, a CF.sub.3 radical, a phenyl radical (C.sub.6H.sub.5), a CONH.sub.2 radical, an R.sub.10 radical, an OR.sub.10 radical, an SR.sub.10 radical, an N(R.sub.10).sub.2 radical, a COOR.sub.10 radical, a CONHR.sub.10 radical or a CON(R.sub.10).sub.2 radical, R.sub.10 being as previously defined, the radical of formula: ##STR00067## in which * indicates the point of attachment of A in the compound of formula (II), at least one of X.sub.1, X.sub.2, X.sub.3, X.sub.4 and X.sub.5 represents a nitrogen (N) atom, a COH radical or a CSH radical, the others of said X.sub.1, X.sub.2, X.sub.3, X.sub.4 or X.sub.5 which do not represent N, COH or CSH, then representing, independently of one another, a CR.sub.11 radical with R.sub.11 as previously defined, the radical of formula: ##STR00068## in which * indicates the point of attachment of A in the compound of formula (II), at least one of X.sub.1, X.sub.2, X.sub.3 and Y.sub.4 represents a nitrogen (N) atom, a COH radical or a CSH radical, the others of said X.sub.1, X.sub.2 or X.sub.3 which do not represent N, COH or CSH, then representing, independently of one another, a CR.sub.11 radical with R.sub.11 as previously defined, Y.sub.1, Y.sub.2, Y.sub.3 and Y.sub.4 (on condition that Y.sub.4 does not represent COH or CSH) represent, independently of one another, depending on whether they are linked via a single or double bond, a nitrogen (N) atom or an NR.sub.10 group with R.sub.10 as previously defined, a CH or CH.sub.2 group, a CR.sub.11 or CHR.sub.11 or CR.sub.11R.sub.12 group with R.sub.11 and R.sub.12 as previously defined, a carbonyl (CO), an oxygen (O) atom or a sulfur (S) atom, with the condition that at most two of said Y.sub.1, Y.sub.2, Y.sub.3 and Y.sub.4 represent at the same time an oxygen atom or a sulfur atom, one of said Y.sub.1, Y.sub.2, Y.sub.3 or Y.sub.4 possibly being absent, so as to form a 5-membered ring, thereby promoting an intramolecular rearrangement of said peptide C.sup.-amide (II) leading to the following peptide thioester (II-1): ##STR00069##

2. The peptide C.sup.-amide as claimed in claim 1, wherein C represents an arm that can be used for Fmoc SPPS.

3. The peptide C.sup.-amide as claimed in claim 2, wherein C represents an acid-labile arm chosen from the group comprising a Rink (4-[(2,4-dimethoxyphenyl)methyl]phenoxyacetyl) arm, a Wang (4-alkoxybenzyl) arm, a Sieber (xanthen-3-yloxyalkyl) arm, a PAL (4-2,5-dimethoxyalkoxybenzyl) arm, a 2-chlorotrityl arm, a PAM (phenylacetamidomethyl) arm, a SASRIN (2-methoxy-4-alkoxybenzyl) arm or an MBHA (4-methyl)benzhydryl arm.

4. The peptide C.sup.-amide as claimed in claim 1, wherein D represents a solid support suitable for Fmoc SPPS and is chosen from the group comprising a polyacrylamide resin, a polystyrene resin or a polystyrene/polyethylene glycol (PEG) mixed resin.

5. The peptide C.sup.-amide as claimed in claim 1, wherein the radical A has the formula: ##STR00070## in which X.sub.1 represents COH, and each of X.sub.2, X.sub.3, X.sub.4 and X.sub.5 is as defined in claim 1 and * indicates the point of attachment of A in the compound of formula (II).

6. The peptide C.sup.60 -amide as claimed in claim 5, wherein each of X.sub.2, X.sub.3 or X.sub.5 represents CH and X.sub.4 represents CR.sub.11 with R.sub.11 as defined in claim 1.

7. The peptide C.sup.-amide as claimed in claim 1, wherein n is an integer equal to 0, and the radical A has the formula: ##STR00071## in which X.sub.1 represents a nitrogen (N) atom and each of said X.sub.2, X.sub.3, X.sub.4 and X.sub.5 is as defined in claim 1.

8. The peptide C.sup.-amide as claimed in claim 7, wherein each of X.sub.2, X.sub.3 or X.sub.5 represents CH and X.sub.4 represents CH or CR.sub.11 with R.sub.11 representing OCH.sub.3 or N(CH.sub.3).sub.2.

9. The peptide C.sup.-amide as claimed in claim 1, wherein n is an integer equal to 0, and the radical A has the formula: ##STR00072## in which X.sub.1 or Y.sub.4 represents COH or N, the other said X.sub.1 or Y.sub.4 which does not represent COH or N, and also each of said X.sub.2, X.sub.3, Y.sub.1, Y.sub.2 and Y.sub.3 are as defined in claim 1.

10. The peptide C.sup.-amide as claimed in claim 1, wherein n is an integer equal to 0, and the radical A has the formula: ##STR00073## in which X.sub.1 or X.sub.2 represents COH or N, the other of said X.sub.1 or X.sub.2 which does not represent COH or N and also each of said X.sub.3, Y.sub.1, Y.sub.2, Y.sub.3 and Y.sub.4 are as defined in claim 1.

11. The peptide C.sup.-amide as claimed claim 1, wherein: each of said R.sub.3, R.sub.4, or R.sub.5 represents H, R.sub.2 represents BC-D in which B represents: ##STR00074## and C, D, and E are as defined in claim 1.

12. A process for preparing a peptide C.sup.-amide of formula (II) as defined in claim 1 by Fmoc SPPS, comprising a step of elongation of the compound (Ia) ##STR00075## wherein X represents a sulfur or selenium atom; R.sub.1 represents a hydrogen atom or a protective group for the sulfur or for the selenium which is compatible with conditions of elongation by Fmoc SPPS; one of R.sub.2, R.sub.3, R.sub.4 or R.sub.5 represents the radical BC-D in which: D represents a hydrogen atom or a solid support suitable for SPPS, C is absent or represents an arm that can be used for SPPS, B represents a divalent radical comprising a heteroatom, the others of said R.sub.2, R.sub.3, R.sub.4 or R.sub.5 which do not represent BC-D, then represent, independently of one another, a hydrogen atom, or an alkyl radical having from 1to 5carbon atoms, or a phenyl radical (C.sub.6H.sub.5 ), on the condition that at most two of said radicals R.sub.2, R.sub.3, R.sub.4 or R.sub.5-represent at the same time a phenyl, R.sub.20 represents a hydrogen or R.sub.14, R.sub.14 represents R.sub.13 and R.sub.13 represents a protective group for the amine function, or R.sub.14 represents a protected aminoacyl residue of formula R.sub.13-Xaa-with Xaa representing an amino acid residue originating from an amino acid of formula HXaa-OH, A represents an aryl or heteroaryl radical chosen from the radical of formula: ##STR00076## in which * indicates the point of attachment of A in the compound of formula (Ia), R.sub.10 represents an alkyl radical having from 1 to 10 carbon atoms, R.sub.11 and R.sub.12 represent, independently of one another, a hydrogen atom, a halogen atom chosen from Cl, Br, I and F, a CN radical, an NO.sub.2 radical, a CF.sub.3 radical, a phenyl radical (C.sub.6 H.sub.5 ), a CONH.sub.2 radical, an R.sub.10 radical, an OR.sub.10 radical, an SR.sub.10 radical, an N(R.sub.10 ) radical, a COOR.sub.10 radical, a CONHR.sub.10 radical or a CON(R.sub.10 ).sub.2 radical, with R.sub.10 as previously defined, the radical of formula: ##STR00077## in which * indicates the point of attachment of A in the compound of formula (la), at least one of X.sub.1, X.sub.2, X.sub.3, X.sub.4 and X.sub.5 represents a nitrogen (N) atom, a COH radical or a CSH radical, the others of said X.sub.1, X.sub.2, X.sub.3, X.sub.4 or X.sub.5 which do not represent N, COH or CSH, then representing, independently of one another, a CR.sub.11 radical with R.sub.11 as previously defined, the radical of formula: ##STR00078## in which * indicates the point of attachment of A in the compound of formula (Ia), at least one of X.sub.1, X.sub.2, X.sub.3 and Y.sub.4 represents a nitrogen (N) atom, a COH Radical or a CSH radical, the others of said X.sub.1, X.sub.2, and X.sub.3 which do not represent N, COH or CSH, then representing, independently of one another, a CR.sub.11 radical with R.sub.11 as previously defined, Y.sub.1, Y.sub.2, Y.sub.3 and Y.sub.4 (on condition that Y.sub.4 does not represent COH or CSH) represent, independently of one another, depending on whether they are linked via a single or double bond, a nitrogen (N) atom or an NR.sub.10 group with R.sub.10 as previously defined, a CH or CH.sub.2 group, a CR.sub.11 or CHR.sub.11 or CR.sub.11R.sup.12 group with R.sub.11 and R.sub.12 as previously defined, a carbonyl (CO), an oxygen (O) atom or a sulfur (S) atom, with the condition that at most two of said Y.sub.1, Y.sub.2, Y.sub.3 and Y.sub.4 represent at the same time an oxygen atom or a sulfur atom, one of said Y.sub.1, Y.sub.2, Y.sub.3 or Y.sub.4 possibly being absent, so as to form a 5-membered ring said elongation step making it possible to add Peptide1 as defined in claim 1.

13. A process for preparing a peptide C.sup.-thioester of general formula (IX):
Peptide1-SR(IX) in which: R represents a radical originating from a thiol of formula RSH, and is an alkyl, aryl, arylalkyl, cycloalkyl, heterocycloalkyl or heteroaryl group, it being possible for each of said groups to also comprise one or more conventional substituents chosen from halogen, carboxyl, sulfonate, ammonium, alcohol, ether, alkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, alkoxy, haloalkyl, arylalkyl, heteroarylalkyl and arylheterocycloalkyl Peptide1 is as defined in claim 1, said process comprising a reaction for thioesterification between the peptide C.sup.-amide of formula (II) as defined in claim 1, in which R.sub.1 is equal to H, and a thiol of formula RSH, in order to obtain the peptide C.sup.-thioester of formula (IX) as defined above, and the compound of formula (Ib): ##STR00079## in which X, m, R.sub.2, R.sub.3, R.sub.4, R.sub.5, R.sub.6, R.sub.7, n, R.sub.8, R.sub.9 and A are as defined in claim 1.

14. A process for preparing a peptide of general formula (X):
Peptide1-Yaa-Peptide2-R.sub.15(X) in which: Peptide1 is as defined in claim 1, Yaa is an amino acid residue originating from an amino acid of formula HYaa-OH chosen from the group comprising a cysteine, a homocysteine, a -mercaptovaline, a -mercaptoleucine, a -mercaptoisoleucine, a -mercaptophenylalanine, a-mercaptoproline, a -mercaptoproline, a -mercaptovaline, a -mercaptoisoleucine, a -mercaptoleucine, a -mercaptolysine, a -mercaptoproline, or an amino acid substituted on its nitrogen atom N.sup.with a group containing a - or -aminothiol function, said group being chosen from the group comprising: ##STR00080## Peptide2=(Xaa).sub.I, with I=an integer ranging from 1 to 60, Xaa represents, independently of one another, an amino acid residue originating from an amino acid of formula HXaa-OH, and when I is greater than or equal to 2, each of said Xaa is connected to its neighbor Xaa via a peptide bond, R.sub.15 represents OH, NH.sub.2 or a radical of general formula (I) in which X represents a sulfur atom and R.sub.1 is a protective group for sulfur which is stable with respect to a treatment with TFA and stable under NCL conditions, wherein: a peptide C.sup.-amide of general formula (II) as defined in claim 1, in which R.sub.1=H or a group which is labile under NCL native chemical ligation conditions, is reacted, by means of an NCL reaction, with a peptide possessing an N-terminal - or -aminothiol residue of general formula (XI):
HYaa-Peptide2-R.sub.15(XI) in which Yaa, R.sub.15 and Peptide2 are as defined above, in order to obtain the peptide of general formula (X) as defined above and the compound of formula (Ia) ##STR00081## wherein X represents a sulfur or selenium atom; R.sub.1 represents a hydrogen atom or a protective group for the sulfur or for the selenium which is compatible with conditions of elongation by Fmoc SPPS; one of R.sub.2,R.sub.3, R.sub.4 or R.sub.5 represents the radical BC-D in which: D represents a hydrogen atom or a solid support suitable for SPPS, C is absent or represents an arm that can be used for SPPS, B represents a divalent radical comprising a heteroatom, the others of said R.sub.2, R.sub.3, R.sub.4 or R.sub.5 which do not represent BC-D, then represent, independently of one another, a hydrogen atom, or an alkyl radical having from 1 to 5 carbon atoms or a phenyl radical (C.sub.6H.sub.5), on the condition that at most two of said radicals R.sub.2, R.sub.3, R.sub.4 or R.sub.5-represent at the same time a phenyl, R.sub.20 represents a hydrogen or R.sub.14, R.sub.14 represents R.sub.13 and R.sub.13 represents a protective group for the amine function, or R.sub.14 represents a protected aminoacyl residue of formula R.sub.13-Xaa-with Xaa representing an amino acid residue originating from an amino acid of formula HXaa-OH, A represents an aryl or heteroaryl radical chosen from the radical of formula: ##STR00082## in which * indicates the point of attachment of A in the compound of formula (Ia),3 R.sub.10 represents an alkyl radical having from 1 to 10 carbon atoms, R.sub.11 and R.sub.12 represent, independently of one another, a hydrogen atom, a halogen atom chosen from Cl, Br, I and F, a CN radical, an NO.sub.2 radical, a CF.sub.3 radical, a phenyl radical (C.sub.6H.sub.5), a CONH.sub.2 radical, an R.sub.10 radical, an OR.sub.10 radical, an SR.sub.10 radical, an N(R.sub.10).sub.2 radical, a COOR.sub.10 radical, a CONHR.sub.10 radical or a CON(R.sub.10).sub.2 radical, with R.sub.10 as previously defined, the radical of formula: ##STR00083## in which * indicates the point of attachment of A in the compound of formula (la), at least one of X.sub.1, X.sub.2, X.sub.3, X.sub.4 and X.sub.5 represents a nitrogen (N) atom, a COH radical or a CSH radical, the others of said X.sub.1, X.sub.2, X.sub.3, X.sub.4 or X.sub.5 which do not represent N, COH or CSH, then representing, independently of one another, a CR.sub.11 radical with R.sub.11 as previously defined, the radical of formula: ##STR00084## in which * indicates the point of attachment of A in the compound of formula (Ia), at least one of X.sub.1, X.sub.2, X.sub.3 and Y.sub.4 represents a nitrogen N atom, a COH radical or a CSH radical, the others of said X.sub.1, X.sub.2 and X.sub.3 which do not represent N, COH or CSH, then representing, independently of one another, a CR.sub.11 radical with R.sub.11 as previously defined, Y.sub.1, Y.sub.2, Y.sub.3 and Y.sub.4 (on condition that Y.sub.4 does not represent COH or CSH) represent, independently of one another, depending on whether they are linked via a single or double bond, a nitrogen (N) atom or an NR.sub.10 group with R.sub.10 as previously defined, a CH or CH.sub.2 group, a CR.sub.11 or CHR.sub.11 or CR.sub.11R.sub.12 group with R.sub.11 and R.sub.12 as previously defined, a carbonyl (CO), an oxygen (O) atom or a sulfur (S) atom, with the condition that at most two of said Y.sub.1, Y.sub.2, Y.sub.3 and Y.sub.4 represent at the same time an oxygen atom or a sulfur atom, one of said Y.sub.1, Y.sub.2, Y.sub.3 or Y.sub.4 possibly being absent, so as to form a 5-membered ring.

Description

(1) The invention will be understood more clearly in the light of the nonlimiting and purely illustrative examples which follow. FIGS. 6 to 26 make it possible to illustrate said examples below.

(2) FIGS. 1 to 5, relating to the prior art, have been commented upon in the introduction of the present application.

(3) FIG. 1 represents the steps of solid phase peptide synthesis (SPPS).

(4) FIG. 2 represents the protective groups (Boc and Fmoc) for amines used in SPPS.

(5) FIG. 3 represents an NCL native chemical ligation reaction between a peptide C.sup.-thioster (compound (a)) and a peptide bearing an N-terminal cysteine (compound (b)).

(6) FIG. 4 represents the use of peptide C.sup.-N-alkyl(or aryl)-N-(-mercaptoalkyl)-amides (e) for: the synthesis of peptide C.sup.-thioesters (a) (by acyl transfer from N to S) (FIGS. 4-1 and 4-2), direct use in NCL (FIG. 4-3).

(7) FIG. 5-1 represents a scheme of the principle of the synthesis of peptide C.sup.--mercapto-amides of type (e), detailing the difficult step of N-acylation of the secondary amine.

(8) FIG. 5-2 is a comparison between the acylation of a secondary amine bearing a 2-hydroxybenzyl group (j) and that of a secondary amine bearing a 2-methoxybenzyl group (m), and illustrates more particularly the principle of the assistance, by a 2-hydroxybenzyl group, of the acylation of a secondary amine.

(9) FIG. 6 is a synthesis scheme illustrating the preparation of six compounds of the invention corresponding to general formula (Ia) (compounds 3a, 3b, 3c, 3a, 3b and 3c).

(10) FIG. 7 is a synthesis scheme illustrating the preparation of a peptide C.sup.-amide of general formula (II) (compounds 5 and 5) from a compound of general formula (Ia) (compound 3a).

(11) FIG. 8 represents the chromatogram of an HPLC/MS analysis of the peptide 2 represented in FIG. 6.

(12) FIGS. 9, 10 and 11 represent the chromatograms of respective HPLC/MS analyses of the peptides 3a, 3b and 3c represented in FIG. 6.

(13) FIGS. 12 and 13 represent the chromatograms of respective HPLC/MS analyses of the peptides 4a and 4b represented in FIG. 7.

(14) FIG. 14 represents the chromatogram of an HPLC/MS analysis of the peptide 5 represented in FIG. 7.

(15) FIG. 15 represents the use of a peptide C.sup.-amide of general formula (II) (compound 5) for obtaining a peptide C.sup.-thioester (compound 6).

(16) FIG. 16 represents the use of a peptide C.sup.-amide of general formula (II) (compound 5) in an NCL reaction for obtaining a model peptide (compound 9) (reactivity of crypto-thioester type).

(17) FIG. 17 is a synthesis scheme illustrating the preparation of three compounds of the invention corresponding to general formula (Ia) (compounds 12, 13 and 13).

(18) FIG. 18 represents the chromatogram of an HPLC/MS analysis of compound 13 represented in FIG. 17.

(19) FIG. 19 is a synthesis scheme illustrating the preparation of a peptide C.sup.er-amide of general formula (II) (compounds 14 and 14) from a compound of general formula (Ia) (compound 12).

(20) FIG. 20 represents the chromatogram of an HPLC/MS analysis of the peptides 14 and 14 represented in FIG. 19.

(21) FIG. 21 is a synthesis scheme illustrating the preparation of a peptide C.sup.er-amide of general formula (II) (compounds 15 and 15) from a compound of general formula (Ia) (compound 13).

(22) FIG. 22 represents the chromatogram of an HPLC/MS analysis of the peptide 15 represented in FIG. 21.

(23) FIG. 23 represents the use of the peptides 14/14 in an NCL native chemical ligation reaction so as to give a peptide 17, followed by a one-pot oxidative folding in order to obtain a peptide 19.

(24) FIG. 24 represents the chromatogram of an HPLC/MS analysis of the peptide 16 represented in FIG. 23.

(25) FIG. 25 represents the chromatogram of an HPLC/MS analysis of the peptide 17 represented in FIG. 23.

(26) FIG. 26 represents the chromatogram of an HPLC/MS analysis of the peptide 19 represented in FIG. 23.

EXAMPLES

(27) In examples 1 and 2 described below, in the radical of formula (I) or in the compound (Ia): X represents a sulfur atom, R.sub.1 represents a hydrogen atom or a trityl (Trt) group, R.sub.2 represents a group BC-D in which: B represents CO-Leu-Tyr-Arg-Ala-Gly-O, C is absent and D represents a hydrogen atom, or B represents CO-Leu-Tyr(t-Bu)-Arg(Pbf)-Ala-Gly-O, C represents a Wang arm (CH.sub.2C.sub.6H.sub.4OCH.sub.2) and D represents a solid support such as a resin of polystyrene type, R.sub.3, R.sub.4 and R.sub.5 each represent a hydrogen atom, m is equal to n which is equal to 0, A representing an aryl group of general formula:

(28) ##STR00038## in which X.sub.1 represents COH, each of X.sub.2, X.sub.3 and X.sub.5 represents CH and X.sub.4 represents CH, COCH.sub.3 or CNO.sub.2, and, if it is the compound (Ia), R.sub.20 represents a hydrogen atom.

(29) When D represents a hydrogen atom, this means that the radical (I) is not attached to a solid support. In this case, R.sub.1 represents H and R.sub.2 represents a group BC-D in which B represents CO-Leu-Tyr-Arg-Ala-Gly-O, C is absent and D represents a hydrogen atom.

(30) When D represents a solid support, this means that the radical (I) is attached to said solid support (said solid support being an integral part of the definition of the radical (I)). In this case, R.sub.1 represents trityl (Trt) and R.sub.2 represents a group BC-D in which B represents CO-Leu-Tyr(t-Bu)-Arg(Pbf)-Ala-Gly-O, C represents a Wang arm (CH.sub.2C.sub.6H.sub.4OCH.sub.2) and D represents a solid support such as a resin of polystyrene type.

(31) The products obtained were analyzed by HPLC on a column: nucleosil C18 300 5 m, 4.6250 mm, gradient: 5-50% MeCNH.sub.2O+0.1% TFA over the course of 30 min, flow rate: 1 mlmin, followed by UV detection (=276 nm or 360 nm) and then MALDI-TOF mass spectrometry.

Example 1

Preparation of a Peptide C-Amide of General Formula (II)

1) Synthesis of a Compound of General Formula (Ia)

(32) The synthesis of a compound of general formula (Ia) is illustrated in FIG. 6. Compounds 3a, 3b, 3c and 3a, 3b and 3c correspond to general formula (Ia) in which: for compounds 3a, 3b and 3c, X represents S, R.sub.1 represents Trt, R.sub.3, R.sub.4 and R.sub.5 represent H, m is equal to n which is equal to 0, R.sub.20 represents a hydrogen atom, R.sub.2 represents

(33) ##STR00039##
and A represents respectively:

(34) ##STR00040## for compounds 3a, 3b and 3c, X represents S, R.sub.1 represents H, R.sub.3, R.sub.4 and R.sub.5 represent H, m is equal to n which is equal to 0, R.sub.20 represents a hydrogen atom, R.sub.2 represents CO-Leu-Tyr-Arg-Ala-Gly-OH, and A represents respectively:

(35) ##STR00041##

Synthesis of the Peptidyl Resin 2

(36) The synthesis scheme is illustrated in FIG. 6.

(37) Compound 1 corresponds to the formula H-E-OC-D as defined above in which: E represents Leu-Tyr(t-Bu)-Arg(Pbf)-Ala-Gly, C represents a Wang arm and D represents a resin of polystyrene type.

(38) Compound 2 corresponds to general formula (IVa) as defined above:

(39) ##STR00042##

(40) in which

(41) X represents S, R.sub.1 represents Trt, R.sub.3, R.sub.4, R.sub.5, R.sub.6 and R.sub.7 represent H, m is equal to 0, and R.sub.2 represents:

(42) ##STR00043##

(43) The peptidyl resin 2 of formula:

(44) ##STR00044##

(45) was synthesized under standard conditions (ABI 433 synthesizer, Fastmoc program). An aliquot of resin is treated for 2 h with 87.5:5:5:2.5 CF.sub.3COOHphenolH.sub.2Otri-isopropylsilane so as to detach and deprotect the peptide 2 in order to obtain the peptide 2, which is precipitated by dilution in a 1:1 petroleum ether/Et.sub.2O mixture, washed (Et.sub.2O), dried, and then analyzed by HPLC and mass spectrometry (see FIG. 8).

(46) Peptide 2 (see FIG. 8):

(47) Empirical formula: C.sub.29H.sub.47N.sub.9O.sub.8S;

(48) HPLC: t.sub.R=14.41 min (C18, gradient: 5-50% MeCNH.sub.2O+0.1% TFA over the course of 30 min);

(49) UV detection (=276 nm);

(50) MS (MALDI-TOF): m/z observed=682.0 ([MH].sup.+ calculated for C.sub.29H.sub.47N.sub.9O.sub.8S=682.3).

(51) Reductive Aminations

(52) The synthesis scheme is illustrated in FIG. 6.

(53) 0.1 mmol of the peptidyl resin 2 of formula:

(54) ##STR00045##

(55) is solvated in the mixture dimethylformamide/methanolacetic acid (9:9:2).

(56) 10 equivalents of aldehyde (of general formula VII) as represented in FIG. 6 are then dissolved in 4 ml of dimethylformamide/methanol (1:1) and are added to the peptidyl resin 2. The resulting suspension is stirred for 45 minutes. The resin is then washed with the dimethylformamide/methanol mixture (1:1).

(57) 20 equivalents of NaBH.sub.3CN are dissolved in 4 ml of dimethylformamide/methanolacetic acid (9:9:2) and then added to the resin. The resulting suspension is stirred for 30 minutes. The resin is washed with the dimethylformamide/methanolacetic acid mixture (9:9:2). An aliquot of resin is treated for 2 h with 87.5:5:5:2.5 TFA/PhOH/H.sub.2O/i-Pr.sub.3SiH so as to detach and deprotect the peptide 3a, 3b or 3c in order to obtain respectively the peptide 3a, 3b or 3c, which is precipitated by dilution in a 1:1 petroleum ether/Et.sub.2O mixture, washed (Et.sub.2O), dried and then analyzed by HPLC and mass spectrometry (see FIGS. 9, 10 and 11).

(58) Peptide 3a (FIG. 9):

(59) Empirical formula: C.sub.36H.sub.52N.sub.10O.sub.11S;

(60) HPLC: t.sub.R=20.44 min (C18, gradient: 5-50% MeCNH.sub.2O+0.1% TFA over the course of 30 min);

(61) UV detection (=360 nm);

(62) MS (MALDI-TOF): m/z observed=833.3 ([M1-1].sup.+ calculated for C.sub.36H.sub.52N.sub.10O.sub.11S=833.4).

(63) Peptide 3b (FIG. 10):

(64) Empirical formula: C.sub.36H.sub.53N.sub.9O.sub.9S;

(65) HPLC: t.sub.R=19.17 min (gradient: 5-50% MeCNH.sub.2O+0.1% TFA over the course of 30 min);

(66) UV detection (=276 nm);

(67) MS (MALDI-TOF): m/z obtained=788.3 ([MH].sup.+ calculated for C.sub.36H.sub.53N.sub.9O.sub.9S=788.4).

(68) Peptide 3c (FIG. 11):

(69) Empirical formula: C.sub.37H.sub.55N.sub.9O.sub.10S;

(70) HPLC: t.sub.R=19.59 min (C18, gradient: 5-50% MeCNH.sub.2O+0.1% TFA over the course of 30 min);

(71) UV detection (=276 nm);

(72) MS (MALDI-TOF): m/z obtained=818.4 ([MH].sup.+ calculated for C.sub.37H.sub.55N.sub.9O.sub.105=818.4).

2) Synthesis of a Peptide C-Amide of General Formula (II)

(73) The synthesis of a peptide C.sup.-amide of general formula (II) is illustrated in FIG. 7. Compounds 5 and 5 correspond to general formula (II) in which: for compound 5, X represents S, R.sub.1 represents Trt, R.sub.3, R.sub.4 and R.sub.5 represent H, Peptide1 represents Ac-Tyr(tBu)-Arg(Pbf)-Phe-Gly, m is equal to n which is equal to 0, R.sub.2 represents

(74) ##STR00046##
and A represents

(75) ##STR00047## for compound 5, X represents S, R.sub.1 represents H, R.sub.3, R.sub.4 and R.sub.5 represent H, Peptide1 represents Ac-Tyr-Arg-Phe-Gly-, m is equal to n which is equal to 0, R.sub.2 represents CO-Leu-Tyr-Arg-Ala-Gly-OH, and A represents:

(76) ##STR00048##

(77) Coupling of Fmoc-Glycine onto the Supported N-Alkyl Cysteine 3a

(78) The coupling of Fmoc-glycine onto the peptidyl resin 3a is illustrated in FIG. 7.

(79) The coupling of Fmoc-glycine onto the peptidyl resin 3a is carried out under standard conditions (ABI 433, Fastmoc program). The peptidyl resin 3a is solvated in DMF. 10 equivalents of protected amino acid (Fmoc-Gly-OH as represented in FIG. 7) are then activated with 10 equivalents of HBTU O-benzotriazole-N,N,N,N-tetramethyluronium hexafluorophosphate) in the presence of 10 equivalents of HOBt (1-hydroxybenzotriazole) and 20 equivalents of DIEA (diisopropylethylamine) and then added to the resin, which is then stirred for 30 minutes at ambient temperature. The resin is rinsed with DMF and CH.sub.2Cl.sub.2 and then the Fmoc group is cleaved using piperidine (20% in NMP), three successive treatments of five minutes each. The resin is rinsed with DMF and CH.sub.2Cl.sub.2. An aliquot of resin 4a thus obtained is treated for 2 h with 87.5:5:5:2.5 TFA/PhOH/H.sub.2O/i-Pr.sub.3SiH so as to detach and deprotect the peptide 4a and to obtain the peptide 4a, which is analyzed by HPLC and mass spectrometry (FIG. 12).

(80) Peptide 4a (FIG. 12):

(81) Molecular formula: C.sub.38H.sub.55N.sub.11O.sub.12S;

(82) HPLC: t.sub.R=20.53 min (C18, gradient: 5-50% MeCNH.sub.2O+0.1% TFA over the course of 30 min);

(83) UV detection (=276 nm);

(84) MS (MALDI-TOF): m/z obtained=890.4 ([MH].sup.+ calculated for C.sub.38H.sub.55N.sub.11O.sub.12S=890.4).

(85) Coupling of Fmoc-Alanine onto the Supported N-Alkyl Cysteine 3a

(86) The coupling of Fmoc-alanine onto the peptidyl resin 3a is illustrated in FIG. 7.

(87) The peptidyl resin 3a is solvated in DMF. 10 equivalents of protected Fmoc-alanine (Fmoc-Ala-OH as represented in FIG. 7) are then activated with 10 equivalents of HBTU in the presence of 10 equivalents of HOBt and 20 equivalents of DIEA in DMF and of the peptidyl resin 3a for 30 minutes at ambient temperature. This step is carried out twice. The resin is rinsed with DMF and DCM and then the amine is deprotected with piperidine (20% in NMP), three times for five minutes. The resin is rinsed with DMF and DCM. An aliquot of resin 4b thus obtained is treated for 2 h with 87.5:5:5:2.5 TFA/PhOH/H.sub.2O/i-Pr.sub.3SiH so as to detach and deprotect the peptide 4b and to obtain the peptide 4b, which is analyzed by HPLC and mass spectrometry (FIG. 13).

(88) Peptide 4b (FIG. 13):

(89) Molecular formula: C.sub.39H.sub.57N.sub.11O.sub.12S;

(90) HPLC: t.sub.R=21.50 min (gradient: 5-50% MeCNH.sub.2O+0.1% TFA over the course of 30 min DAD detection);

(91) UV detection (=276 nm);

(92) MS (MALDI-TOF): m/z obtained=904.5 ([MFI].sup.+ calculated for C.sub.39H.sub.57N.sub.11O.sub.12S=904.4).

(93) Elongation of the Peptide 5 Using the Compound 4a Obtained

(94) The elongation of the peptide 5 is represented in FIG. 7.

(95) The continuation of the elongation of the peptide is carried out under standard conditions (ABI 433, Fastmoc program) with the peptidyl resin 4a. An aliquot of resin 5 is treated for 2 h with 87.5:5:5:2.5 TFA/PhOH/H.sub.2O/i-Pr.sub.3SiH so as to obtain the peptide 5 which is precipitated by dilution in a 1:1 petroleum ether/Et.sub.2O mixture, washed (Et.sub.2O), dried and then analyzed by HPLC and mass spectrometry (FIG. 14).

(96) Peptide 5 (FIG. 14):

(97) Molecular formula: C.sub.39H.sub.57N.sub.11O.sub.12S;

(98) HPLC: t.sub.R=21.50 min (gradient: 5-50% MeCNH.sub.2O+0.1% TFA over the course of 30 min DAD detection);

(99) UV detection (=276 nm);

(100) MS (MALDI-TOF): m/z obtained=904.5 ([MH].sup.+ calculated for C.sub.39H.sub.57N.sub.11O.sub.12S=904.4).

Example 2

Use of the Peptide C-Amide of General Formula (II)

(101) 1) in a Thioesterification Reaction in an Acidic Medium

(102) The use of a peptide C.sup.-amide 5 of general formula (II) in a thioesterification reaction is illustrated in FIG. 15.

(103) 2.5 mol of peptide 5 (peptide of general formula (II)) and 100 mol of mercaptophenylacetic acid (namely a thiol of general formula RSH with R representing pHOOCCH.sub.2C.sub.6H.sub.4 as represented in FIG. 15) are dissolved in 1 ml of the mixture H.sub.2OMeCN (7:3)+0.1% TFA. The reaction mixture is stirred for 48 hours at ambient temperature. The progression of the reaction is monitored by HPLC (FIG. 15).

(104) The desired peptide C.sup.-thioester of formula 6 and compound 7 corresponding to general formula (Ia) in which:

(105) X represents S, R.sub.1 represents H, R.sub.3, R.sub.4 and R.sub.5 represent H, R.sub.20 represents H, m is equal to n which is equal to 0, R.sub.2 represents CO-Leu-Tyr-Arg-Ala-Gly-OH, and A represents:

(106) ##STR00049##
are thus obtained.

(107) This compound of formula (Ia) is particularly advantageous owing to its characteristic absorption in the ultraviolet range (max400-450 nm): HPLC analysis with detection in this wavelength range makes it possible to easily detect it and characterize it, which will also allow those skilled in the art to conclude that the peptide C.sup.-thioester of formula (IX) as defined above has indeed been formed.

(108) Peptide C.sup.-thioester 6 (FIG. 15):

(109) ##STR00050##

(110) 2) in an NCL Native Chemical Ligation Reaction

(111) The use of a peptide C.sup.-amide 5 of general formula (II) in a native chemical ligation reaction is illustrated in FIG. 16.

(112) The peptide C.sup.-amide of general formula (II) (compound 5), by virtue of its crypto-thioester properties, can be used directly in an NCL native chemical ligation reaction, without converting it beforehand to the peptide C.sup.-thioester (FIG. 16).

(113) 2.5 mol of crude crypto-thioester peptide 5 and 1.5 equivalents of cysteinyl peptide 8 are dissolved in 1 ml of degassed solution containing 50 mM of mercaptophenylacetic acid, 20 mM of triscarboxyethylphosphine and 200 mM of phosphate buffer, pH=7. The reaction mixture is stirred for 24 h at 37 C. under argon.

(114) A peptide 9 and a compound 7 of general formula (Ia) are thus obtained.

(115) Peptide 9 (FIG. 16): Ac-Tyr-Arg-Phe-Gly-Cys-Leu-Tyr-Arg-Ala-Gly-OH

(116) In examples 3 and 4 described below, in the radical of formula (I) or in the compound (Ia): X represents a sulfur atom,

(117) R.sub.1 represents a hydrogen atom, a trityl (Trt) group, or a tert-butylsulfanyl (StBu) group, R.sub.2 represents a group BC-D in which: B represents CONH, where C is absent and D represents a hydrogen atom, or C represents a Rink arm (CH[2,4-di-MeO-C.sub.6H.sub.4]-C.sub.6H.sub.4OCH.sub.2CONHCH.sub.2) and D represents a solid support such as a resin of ChemMatrix or polystyrene type, R.sub.3, R.sub.4 and R.sub.5 each represent a hydrogen atom, m is equal to n which is equal to 0,

(118) A representing an aryl group of general formula:

(119) ##STR00051## in which X.sub.1 represents COH, each of X.sub.2, X.sub.3 and X.sub.5 represents CH and X.sub.4 represents CNO.sub.2,

(120) and, if it is the compound (Ia), R.sub.20 represents a hydrogen atom.

(121) When D represents a hydrogen atom, this means that the radical (I) is not attached to a solid support. In this case, R.sub.1 represents H or a tert-butylsulfanyl (StBu) group and R.sub.2 represents a group BC-D in which B represents CONH, C is absent and D represents a hydrogen atom.

(122) When D represents a solid support, this means that the radical (I) is attached to said solid support (said solid support being an integral part of the definition of the radical (I)). In this case, R.sub.1 represents a trityl (Trt) group or a tert-butylsulfanyl (StBu) group and R.sub.2 represents a group BC-D in which B represents CONH, C represents a Rink arm (CH[2,4-di-MeO-C.sub.6H.sub.4]-C.sub.6H.sub.4OCH.sub.2CONH) and D represents a solid support such as a resin of ChemMatrix or polystyrene type.

(123) The products obtained were analyzed by HPLC on a column: nucleosil C18 300 5 m, 4.6250 mm, gradient: 5-50% MeCN/H.sub.2O+0.1% TFA over the course of 30 min, flow rate: 1 ml/min, followed by UV detection (=276 nm or 320 nm) and then by MALDI-TOF mass spectrometry.

Example 3

Preparation of Peptide C-Amide of General Formula (II)

1) Synthesis of a Compound of General Formula (Ia)

(124) The synthesis of a compound of general formula (Ia) is illustrated in FIG. 17.

(125) Compounds 12, 13 and 13 correspond to general formula (Ia) in which: for compounds 12 and 13, X represents S, R.sub.3, R.sub.4 and R.sub.5 represent H, m is equal to n which is equal to 0, R.sub.20 represents a hydrogen atom,

(126) R.sub.2 represents

(127) ##STR00052##
A represents:

(128) ##STR00053##

(129) and R.sub.1 represents Trt (12), or StBu (13), for compound 13, X represents S, R.sub.1 represents StBu, R.sub.3, R.sub.4 and R.sub.5 represent H, m is equal to n which is equal to 0, R.sub.20 represents a hydrogen atom, R.sub.2 represents CONH.sub.2, A represents:

(130) ##STR00054##

Synthesis of the Cysteinyl Resins 10 and 11

(131) The synthesis scheme is illustrated in FIG. 17.

(132) Compounds 10 and 11 correspond to general formula (IVa) as defined above:

(133) ##STR00055##

(134) in which

(135) X represents S, R.sub.3, R.sub.4, R.sub.5, R.sub.6 and R.sub.7 represent H, m is equal to 0, R.sub.2 represents

(136) ##STR00056##
and R.sub.1 represents Trt (10) or StBu (11).

(137) The cysteinyl resins 10 and 11 of respective formula:

(138) ##STR00057## were synthesized under standard conditions (ABI 433 synthesizer, Fastmoc program).

(139) Reductive Aminations

(140) The synthesis scheme is illustrated in FIG. 17.

(141) 0.1 mmol of the peptidyl resin 10 or 11 as defined respectively above is solvated in the mixture dimethylformamide/methanol/acetic acid (9:9:2).

(142) 167 mg (10 equivalents) of 2-hydroxy-5-nitrobenzaldehyde dissolved in 4 ml of dimethylformamide/methanol (1:1) are added to the resin. The resulting suspension is stirred for 45 minutes. The resin is then washed with the dimethylformamide/methanol (1:1) mixture.

(143) 126 mg (20 equivalents) of NaBH.sub.3CN dissolved beforehand in 4 ml of dimethylformamide/methanolacetic acid (9:9:2) are added to the resin. The resulting suspension is stirred for 30 minutes. The resin is washed with the dimethylformamide/methanolacetic acid (9:9:2) mixture. An aliquot of the resin 13 is treated for 2 h with 92.5:5:2.5 TFAH.sub.2O/i-Pr.sub.3SiH in order to obtain the compound 13, which is analyzed by HPLC and mass spectrometry after evaporation of the TFA (see FIG. 18).

(144) Compound 13 (FIG. 18):

(145) Empirical formula: C.sub.14H.sub.21N.sub.3O.sub.4S.sub.2;

(146) HPLC: t.sub.R=23.4 min (C18, gradient: 5-50% MeCNH.sub.2O+0.1% TFA over the course of 30 min);

(147) UV detection (=320 nm);

(148) MS (ESI+): m/z observed=360.1 ([MH].sup.+ calculated for C.sub.14H.sub.22N.sub.3O.sub.4S.sub.2=360.1).

2) Synthesis of a Peptide C-Amide of General Formula (II)

(149) The synthesis of a peptide C.sup.-amide of general formula (II) is illustrated in FIG. 19. Compounds 14, 14, 15 and 15 correspond to general formula (II) in which: for compound 14, X represents S, R.sub.1 represents Trt, R.sub.3, R.sub.4 and R.sub.5 represent H, Peptide1 represents H-Ala-Ser(tBu)-Cys(Trt)-Asn(Trt)-Gly-Val-Cys(Trt)-Pro-Phe-Glu(OtBu)-Met-Pro-Pro-Cys(Trt)-Gly-Thr(tBu)-Ser(tBu)-Ala-, m is equal to n which is equal to 0, R.sub.2 represents

(150) ##STR00058##
and A represents:

(151) ##STR00059## for compound 14, X represents S, R.sub.1 represents H, R.sub.3, R.sub.4 and R.sub.5 represent H, Peptide1 represents H-Ala-Ser-Cys-Asn-Gly-Val-Cys-Pro-Phe-Glu-Met-Pro-Pro-Cys-Gly-Thr-Ser-Ala-, m is equal to n which is equal to 0,

(152) R.sub.2 represents CONH.sub.2, and A represents:

(153) ##STR00060## for compound 15, X represents S, R.sub.1 represents StBu, R.sub.3, R.sub.4 and R.sub.5 represent H, Peptide1 represents H-Leu-Tyr(tBu)-Arg(Pbf)-Ala-Gly-, m is equal to n which is equal to 0, R.sub.2 represents

(154) ##STR00061##
and A represents

(155) ##STR00062## for compound 15, X represents S, R.sub.1 represents StBu, R.sub.3, R.sub.4 and R.sub.5 represent H, Peptide1 represents H-Leu-Tyr(tBu)-Arg(Pbf)-Ala-Gly-, m is equal to n which is equal to 0, R.sub.2 represents CONH.sub.2, and A represents:

(156) ##STR00063##

(157) Elongation of the Peptide 14 Using Compound 12

(158) The elongation of the peptide 14 is represented in FIG. 19.

(159) The elongation of the peptide is carried out under standard conditions (ABI 433, Fastmoc program, HCTU as coupling agent) with the resin 12, by carrying out twice in a row the coupling of the first amino acid Fmoc-Ala-OH before deprotecting the Fmoc group and continuing the elongation.

(160) The peptidyl resin 14 obtained is treated for 2 h with 87.5:5:5:2.5 TFA/PhOH/H.sub.2O/i-Pr.sub.3SiH so as to obtain the peptide 14 which is precipitated by dilution in a 1:1 petroleum ether/Et.sub.2O mixture, washed (Et.sub.2O) and then dried under reduced pressure. The precipitate is taken up in demineralized water and lyophilized. It is then analyzed by HPLC and mass spectrometry (FIG. 21). The peptide amide 14 is obtained by mixing with the peptide thioester 14 (the two compounds are in equilibrium in solution).

(161) Peptide 14 (FIG. 20):

(162) Molecular formula: C.sub.85H.sub.126N.sub.23O.sub.31S.sub.5;

(163) HPLC: (gradient: 5-50% MeCNH.sub.2O+0.1% TFA over the course of 30 min UV detection, =276 nm) t.sub.R=25.3 min;

(164) MS (ESI+): m/z obtained=1055.9 ([M+2H].sup.2+).

(165) Peptide 14 (FIG. 20):

(166) Molecular formula: C.sub.85H.sub.126N.sub.23O.sub.31S.sub.5;

(167) HPLC: t.sub.R=23.8 min (gradient: 5-50% MeCNH.sub.2O+0.1% TFA over the course of 30 min;

(168) UV detection (=276 nm);

(169) MS (ESI+): m/z obtained=1055.9 ([M+2H].sup.2+).

(170) Elongation of the Peptide 15 Using Compound 13

(171) The elongation of the peptide 15 is represented in FIG. 21.

(172) The elongation of the peptide is carried out under standard conditions (ABI 433, Fastmoc program, HCTU as coupling agent) with the resin 13, by carrying out twice in a row the coupling of the first amino acid Fmoc-Ala-OH before deprotecting the Fmoc group and continuing the elongation.

(173) The peptidyl resin 15 obtained is treated for 2 h with 97.5:5:5:2.5 TFA/i-Pr.sub.3SiH so as to obtain the peptide 15 which is precipitated by dilution in a 1:1 petroleum ether/Et.sub.2O mixture, washed (Et.sub.2O), dried and then analyzed by HPLC and mass spectrometry (FIG. 21).

(174) Peptide 15 (FIG. 22):

(175) Molecular formula: C.sub.43H.sub.65N.sub.11O.sub.11S.sub.2;

(176) HPLC: t.sub.R=31.0 min (gradient: 5-50% MeCNH.sub.2O+0.1% TFA over the course of 30 min);

(177) UV detection (=320 nm);

(178) MS (MALDI-TOF): m/z obtained=976.4 ([MH].sup.+ calculated for C.sub.43H.sub.65N.sub.11O.sub.11S.sub.2=976.4).

Example 4

Use of the Peptide C-Amide of General Formula (II) in a Native Chemical Ligation (NCL) Reaction

(179) The use of a peptide C.sup.-amide 14 of general formula (II) in a native chemical ligation reaction is illustrated in FIG. 23.

(180) The peptide C.sup.-amide of general formula (II) (compound 14), by virtue of its crypto-thioester properties, can be used directly in an NCL native chemical ligation reaction, without converting it beforehand to the peptide C.sup.-thioester (FIG. 23).

(181) Preparation of the Cysteinyl Peptide 16 Partner

(182) The peptide 16 (HCRCIPVGLIGYCRNPSG-OH) was synthesized under standard conditions (ABI 433 synthesizer, Fastmoc program, coupling agent: HCTU). The resin obtained after automated elongation is treated for 2 h with 87.5:5:5:2.5 TFA/PhOH/H.sub.2O/i-Pr.sub.3SiH so as to detach and deprotect the peptide 16, which is precipitated by dilution in a 1:1 petroleum ether/Et.sub.2O mixture, washed (Et.sub.2O) and then dried. The precipitate is taken up in demineralized water and then lyophilized. It is then analyzed by HPLC and mass spectrometry (FIG. 24).

(183) Peptide 16 (FIG. 24):

(184) Molecular formula: C.sub.81H.sub.135N.sub.25O.sub.22S.sub.3;

(185) HPLC: t.sub.R=24.3 min (gradient: 5-50% MeCNH.sub.2O+0.1% TFA over the course of 30 min);

(186) UV detection (=276 nm);

(187) MS (MALDI-TOF): m/z obtained=1906.9 ([MH]+ calculated for C.sub.81H.sub.136N.sub.25O.sub.22S.sub.3=1906.9).

(188) Native Chemical Ligation Between the Peptides 14/14 and 16

(189) 2.5 mol of crude crypto-thioester peptide 14/14 and 1.5 equivalents of crude cysteinyl peptide 16 are dissolved in 1 ml of a deoxygenated solution containing 25 mM of mercaptophenylacetic acid, 50 mM of triscarboxyethylphosphine and 200 mM of phosphate buffer, pH=7. The reaction mixture is stirred for 3 h at 37 C. under an argon atmosphere. A peptide 17 and a compound 18 of general formula (Ia) are thus obtained. The peptide 17 is purified by semi-preparative HPLC and then lyophilized. It is obtained with a yield of 65%.

(190) Peptide 17 (FIG. 25):

(191) Molecular formula: C.sub.156H.sub.249N.sub.45O.sub.48S.sub.7;

(192) HPLC: t.sub.R=14.6 min (gradient: 30-45% MeCNH.sub.2O+0.1% TFA over the course of 30 min);

(193) UV detection (=276 nm);

(194) MS (MALDI-TOF): m/z obtained=3745.6 ([M+H].sup.+ calculated for C.sub.156H.sub.250N.sub.45O.sub.48S.sub.7=3745.6).

(195) Native Chemical Ligation Between the Peptides 14/14 and 16 Followed by a One-Pot Oxidative Folding

(196) 2.5 mol of crude crypto-thioester peptide 14/14 and 3.75 mol (1.5 equivalents) of crude cysteinyl peptide 16 are dissolved in 1 ml of a deoxygenated solution containing 25 mM of mercaptophenylacetic acid, 50 mM of triscarboxyethylphosphine and 200 mM of phosphate buffer, pH=7. The reaction mixture is stirred for 3 h at 37 C. under an argon atmosphere. The reaction mixture is directly used in a step of oxidative folding of the ligation product 17 (final concentration of 17: 13 M) by dilution in a 1:1 mixture of isopropanol and of a buffer containing 0.2 mM of Tris, pH 8.6, 2 mM of EDTA, 1.82 mM of glutathione (GSH) and 0.78 mM of oxidized glutathione (GSSG), and stirring of the resulting solution for 24 hours at 4 C. The mini-protein 19 comprising three disulfide bridges is purified by semi-preparative HPLC and then lyophilized. 19 is obtained with a yield of 55%.

(197) Peptide 19 (FIG. 26):

(198) Molecular formula: C.sub.156H.sub.243N.sub.45O.sub.48S.sub.7;

(199) HPLC: t.sub.R=28.4 min (gradient: 30-45% MeCNH.sub.2O+0.1% TFA over the course of 30 min);

(200) UV detection (=276 nm);

(201) MS (MALDI-TOF): m/z obtained=3739.5 ([MH]+ calculated for C.sub.156H.sub.244N.sub.45O.sub.48S.sub.7=3739.5).