Liquid phase synthesis of peptides and peptide derivatives

Details Liquid phase synthesis of peptides and peptide derivatives Liquid phase synthesis of peptides and peptide derivatives

1994-05-25

1996-05-14

Russel, Jeffrey E.

Chemistry: natural resins or derivatives; peptides or proteins;

Peptides of 3 to 100 amino acid residues

4 to 5 amino acid residues in defined sequence

530341, C07K 102, C07K 110, C07K 5103

Patent

active

055168914

DESCRIPTION:

BRIEF SUMMARY
BACKGROUND OF THE INVENTION

Classical liquid phase peptide synthesis involves the well-known problems of many manipulations and considerable loss of material during the isolation and purification of intermediates.
Some of these problems have been circumvented by using continuous liquid phase techniques. Furthermore, numerous advances made in solid phase peptide synthesis have led to progress in liquid phase synthetic methods. For example, the protecting groups developed in solid phase synthesis are also useful in liquid phase synthesis.
The two major routes in liquid phase synthesis are (1) use of the t-butyloxycarbonyl protecting group ("Boc") to block the non-side chain amino functionality, and (2) use of the 9-fluoroenyl methoxycarbonyl protecting group ("Fmoc") to block the same functionality. For examples, see Thierry, J. et al. J. Med. Chem. 33:2122 (1990) and Hoeg-Jensen, T. et al. Tetrahedron Letters 32:6387 (1991).
No continuous liquid phase peptide synthetic methods using Fmoc as the protecting group and a substituted carbodiimide as the coupling agent have hitherto been reported.


SUMMARY OF THE INVENTION

The present invention relates to a continuous liquid phase synthetic method for preparing a peptide containing 2-10 amino acid residues with or without modification at the C-terminus or an N-acyl derivative thereof, which method comprises the steps of:
(a) coupling a first amino acid or a derivative thereof to a second amino acid via an amide bond in an organic solvent containing a substituted carbodiimide, with the first amino acid or derivative thereof having both its non-side chain carboxyl functionality and its side-chain functionalities, if any and necessary, blocked by base-stable groups and a second amino acid with its non-side chain amino functionality blocked by Fmoc and its side chain functionalities, if any and necessary, blocked by base-stable groups;
(b) detaching the Fmoc with ammonia, a primary amine, or a secondary amine;
(c) coupling an additional amino acid to the Fmoc-detached coupled product via an amide bond in an organic solvent containing a substituted carbodiimide, with the additional amino acid having its non-side chain amino functionality blocked by Fmoc and its side chain functionalities, if any and necessary, blocked by basestable groups;
(d) detaching the Fmoc with ammonia, a primary amine, or a secondary amine;
(e) repeating steps (c) and (d) until the length of the peptide or the N-acyl derivative is achieved;
(f) acylating the non-side chain amino group of the Fmoc-detached coupled product from the previous step; and
(g) detaching the base-stable groups;
In the above-described method, the first amino acid or derivative thereof corresponds to a first residue (i.e., the C-terminal residue) in the peptide or its N-acyl derivative, the second amino acid corresponds to a second residue adjacent to the first residue in the peptide or its N-acyl derivative, and the additional amino acid corresponds to a third residue adjacent to the second residue in the peptide or its N-acyl derivative. (In other word, consecutive amide coupling is performed in the C terminus to N terminus direction with one amino acid linked to another based on the sequence of the peptide or its N-acyl derivative to be synthesized.) Furthermore, when the peptide or the N-acyl derivative contains two amino acid residues, steps (c) through (e) are omitted. In addition, when the peptide, rather than the N-acyl derivative, is to be synthesized, step (f) is omitted.
Note that the carboxyl and amino groups which participate in the formation of an amide bond are called non-side chain functionalities. On the other hand, any functional groups which are not involved in formation of such a bond are called side chain functionalities.
A peptide without modification at the C-terminus refers to a peptide with a carboxylic group at its C-terminus (i.e., .varies.-COOH of the C-terminal residue in most naturally-occurring peptides). A peptide with modification at the C-terminus, on the other hand, refers to a peptid

REFERENCES:
patent: 3121707 (1964-02-01), Anderson et al.
patent: 3769271 (1973-10-01), Southard
patent: 4250086 (1981-02-01), Heavner
patent: 4407794 (1983-10-01), Roques et al.
patent: 4581168 (1986-04-01), Diaz et al.
patent: 4658016 (1987-04-01), Konig et al.
patent: 4755591 (1988-07-01), Konig et al.
patent: 5004781 (1991-04-01), Rink
patent: 5202418 (1993-04-01), Lebl et al.
Carpino et al., "The 9-Fluorenylmethoxycarbonyl Amino-Protecting Group", J. Org. Chem., 37:3404-3409, 1972.
Wang et al., "One-Pot Liquid-Phase Synthesis of DSIP and 5-Phe-DSIP Using Fluoren-9-yl-methoxycarbonyl-Protected Amino Acid Pentafluorophenyl Esters", Synthesis, 9:845-846, 1990.
Gross et al, The Peptides: Analysis, Synthesis, Biology, published 1981 by Academic Press (New York), pp. 22-24, 225.
Jensen, et al., "A New Method for Rapid Solution Synthesis of Shorter Peptides by use of PyBOP", Tetrahedron Letters, vol. 32, No. 44, pp. 6387-6390, 1991.
Lenfant, et al., "Enhancement of the Adherence of Hematopoietic Stem Cells to Mouse Bone Marrow-derived Stromal Cell Line MS-1-T by a Tetrapeptide Acetyl-N-Ser-Asp-Lys-Pro", Exp. Hematol. 17:898-902, 1989.
E. Atherton et al., "The Peptides", vol. 9, pp. 32-39, 1987.
Gregg B. Fields et al., "Solid phase peptide synthesis utilizing 9-fluorenylmethoxycarbonyl amino acids", Aug. 23, 1989, Int. J. Peptide Res. 35, pp. 161, 181-183, 186, 187.
Derek Hudson, "Methodological Implications of Simultancous Solid-Phase Peptide Synthesis. 1. Comparison of Different Coupling Procedures", American Chemical Society, 1988, pp. 618-623.
Thierry et al., "Synthesis and Activity of NAcSerAspLysPro Analogues on Cellular Interactions between T-Cell and Erythrocytes in Rosette Formation", American Chemical Society, 1990, pp. 2123-2127.

Affiliated with

Also associated with

Rating

Liquid phase synthesis of peptides and peptide derivatives does not yet have a rating. At this time, there are no reviews or comments for this patent.

If you have personal experience with Liquid phase synthesis of peptides and peptide derivatives, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Liquid phase synthesis of peptides and peptide derivatives will most certainly appreciate the feedback.

Rate now

Comments { 0 }

Profile ID: LFUS-PAI-O-1896755