Method for preparing peptides by assembling multiple peptide fragments

Patent metadata

Field	Value
Publication number	US-10072041-B2
Application number	US-201213985369-A
Country	US
Kind code	B2
Filing date	Feb 15, 2012
Priority date	Feb 16, 2011
Publication date	Sep 11, 2018
Grant date	Sep 11, 2018

Abstract

Method for preparing a peptide assembly of n fragments and n−1 amino acids bearing a thiol function, represented by the formula:

A 1 -C 1 -A 2 -C 2 -A 3 - . . . -C i−1 -A i - . . . -C n−1 -A n   (I)

in which A 1 , A 2 , A 3 , . . . A i . . . , A n are peptide fragments, C 1 , C 2 , C 3 . . . C i−1 . . . C n−1 are amino acid residues bearing a thiol function, n is comprised between 3 and 50, and i is 2 to n, in which a peptide-thioester is prepared of formula: A 1 -SR (II) in which A 1 is a peptide fragment and SR is an alkyl thioester residue, R being alkyl optionally substituted, starting from a bis(2-sulphanylethyl)amino peptide.

First claim

The invention claimed is:

1. A method for preparing a peptide assembly of n fragments and n−1 amino acids bearing a thiol moiety, represented by the formula:
A 1 -C 1 -A 2 -C 2 -A 3 - . . . -C i−1 -A i - . . . -C n−1 -A n   (I)

in which A 1 , A 2 , A 3 , . . . A i . . . , A n are peptide fragments,
C 1 , C 2 , C 3 . . . C i−1 . . . C n−1 are amino acid residues bearing a thiol moiety,
n is comprised between 3 and 50, and
i is any integer comprised between 2 and n, the method comprising the steps of
treating a bis(2-sulphanylethyl)amino peptide: A 1 -SEAoff in which SEAoff is a cyclic bis(2-sulphanylethyl)amino group

with a thiol R—SH, in the presence of a cyclic disulphide reducing agent to prepare a peptide-thioester of formula:
A 1 -SR  (II)

in which A 1 is a peptide fragment and SR is an alkyl thioester residue, R being able to be an optionally substituted alkyl radical,
preparing a peptide fragment of formula (IV) by condensing a peptide fragment of structure:
H—C 1 (SR′)-A 2 -SEAoff  (III)

in which C 1 , A 2 and SEAoff are defined as above and (SR′) represents a disulphide residue on the thiol of the amino acid C 1 , with the peptide thioester of formula (II) in the presence of an aromatic thiol ArSH, to obtain the peptide fragment of structure:
A 1 -C 1 -A 2 -SEAoff  (IV)

in which A 1 , C 1 , A 2 and SEAoff are as defined previously, preparing a peptide thioester of formula:
A 1 -C 1 -A 2 -SR  (II′)

in which A 1 , C 1 , A 2 and R are as defined previously, by treating the peptide fragment of formula (IV) with a thiol R—SH, in the presence of a cyclic disulphide reducing agent,
preparing a peptide fragment of formula (IV′) by condensing a peptide fragment of structure:
H—C 2 (SR′)-A 3 -SEAoff  (III′)

in which SEAoff and R′ and C 2 and A 3 are defined as above, with the peptide thioester of formula (II′) in the presence of an aromatic thiol (ArSH), in order to produce a peptide fragment of structure:
A 1 -C 1 -A 2 -C 2 -A 3 -SEAoff  (IV′)

in which A 1 , C 1 , A 2 , C 2 , A 3 and SEAoff are as defined previously,
and reiterating these 2 operations up to n−2 times, in order to obtain a peptide fragment of structure:
A 1 -C 1 -A 2 -C 2 -A 3 - . . . -C i−1 -A i - . . . -C n−2 -A n−1 -SEAoff  (IV m )

in which A 1 , A 2 , A 3 , . . . A i . . . , A n−1 , C 1 , C 2 , C 3 . . . C i−1 . . . C n−2 and SEAoff are as defined previously, followed by
implementing a native ligation reaction of this obtained peptide fragment (IV m ), with a peptide of formula:
H—C n−1 -A n (V n )

in which C n−1 and A n are as defined previously, in the presence of a cyclic disulphide reducing agent, in order to produce the multiple peptide assembly of general formula (I) wherein assembly proceeds from the peptide N-terminal end to the C-terminal end, and wherein a peptide assembly of n fragments is prepared in a “one pot” reaction in the absence of a step of isolating intermediates.

2. A method according to claim 1 , wherein a peptide assembly of 3 or 4 fragments is prepared, of structures A 1 -C 1 -A 2 -C 2 -A 3 or A 1 -C 1 -A 2 -C 2 -A 3 -C 3 -A 4 .

3. A method according to claim 1 , wherein C 1 , C 2 , C 3 . . . C i−1 . . . C n−1 represent Cys residues.

4. A method according to claim 1 , wherein one or more of the peptide fragments A 2 . . . A i . . . A n−1 can bear one or more modified amino acids.

5. A method according to claim 1 , wherein the condensing of the peptide-thioester with a peptide fragment of structure (III) is carried out in the presence of 4-mercaptophenylacetic acid.

6. A method for manufacturing a pharmaceutical composition wherein the method comprises:
manufacturing a polypeptide of formula (I) according to claim 1 , and
preparing a pharmaceutical composition comprising a polypeptide or peptide thus prepared, in the pure state or in combination with one or more compatible and pharmaceutically acceptable adjuvants.

7. A method for manufacturing a diagnostic device wherein the method comprises:
manufacturing a polypeptide of formula (I) according to claim 1 , and
preparing a formulation or forming of this polypeptide in a form suitable for use for a diagnostic device.

8. A method according to claim 1 , wherein the cyclic disulphide reducing agent is tris(2-carboxyethyl)phosphine.

9. A method according to claim 1 , wherein n is comprised between 3 and 20.

10. A method according to claim 1 , wherein n is comprised between 3 and 10.

Assignees

• Melnyk Oleg
• Ollivier Nathalie
• Mhidia Reda
• Dheur Julien
• Centre Nat Rech Scient
• Univ De Lille 1 Sciences Et Technologies

Inventors

• Melnyk Oleg
• Ollivier Nathalie
• Mhidia Reda
• Dheur Julien

Classifications

• C07K1/026 Primary

  by fragment condensation in solution · CPC title

• C07K1/003

  by transforming the C-terminal amino acid to amides · CPC title