Method of preparing peptides comprising a lipophilically modified lysine side chain

Patent metadata

Field	Value
Publication number	US-11021512-B2
Application number	US-201716338213-A
Country	US
Kind code	B2
Filing date	Oct 10, 2017
Priority date	Oct 10, 2016
Publication date	Jun 1, 2021
Grant date	Jun 1, 2021

Abstract

The present invention relates to a method of preparing a lysine side-chain modified peptide by solid phase peptide synthesis.

First claim

The invention claimed is:

1. A method of preparing an isolated exendin-4 derivative peptide comprising a lipophilically modified lysine side chain, comprising the steps of:
(i) assembling the amino acid sequence of said peptide with protected reactive functional groups in the side chains in a step-wise manner using solid phase peptide synthesis (SPPS), wherein the side chain of lysine to be modified is protected by a trityl-based protecting group;
(ii) drying the solid phase resin after the assembling of the amino acid sequence has been completed;
(iii) treating the dried resin several times with a solution of trifluoroacetic acid (TFA) in dichloromethane (DCM) in order to deprotect the lysine side chain to be modified;
(iv) neutralizing the resin;
(v) coupling at least one activated 9-fluorenylmethyloxycarbonyl (Fmoc)-bound linker moiety to the deprotected lysine side chain;
(vi) deprotecting the terminal functional group of the linker coupled to the lysine side chain in step (v);
(vii) coupling an activated lipophilic moiety to the deprotected terminal functional group of the linker of step (vi);
(viii) drying the resin; and
(ix) cleaving the peptide from the resin,
thereby preparing the isolated exendin-4 derivative peptide comprising a lipophilically modified lysine side chain.

2. The method of claim 1 , wherein the trityl-based protecting group is monomethoxytrityl (Mmt) or 4-methyltrityl (Mtt).

3. The method of claim 1 , wherein the activated lipophilic moiety is an activated fatty acid.

4. The method of claim 1 , wherein the exendin-4 derivative has a length of between 30 and 44 amino acids, wherein
(i) the sequence identity to wild-type exendin-4 in the region corresponding to amino acids 1-13 of wild-type exendin is at least 65%, or
(ii) the sequence identity to wild-type exendin-4 in the region corresponding to amino acids 22-39 of wild-type exendin is at least 70%, or
(iii) the lipophilically modified lysine side chain is at position 14 (Lys(14)) with respect to the amino acid positions of wild-type exendin-4.

5. The method of claim 4 , wherein the exendin-4 derivative has a length between 38 and 40 amino acids.

6. The method of claim 1 , wherein, in step (iii), the dried resin is treated at least seven times for at least 5 minutes, with a solution of about 1% (v/v) TFA in DCM.

7. The method of claim 1 , further comprising the steps of:
(viii-a) analyzing the deprotection yield of step (iii) by cleaving a test sample of the resin bound peptide; and
(viii-b) optionally repeating steps (iii) to (viii-a) until the content of cleaved peptide containing the modified lysine side chain is at least 85% compared to peptide without modified lysine side chain.

8. The method of claim 7 , wherein the content of cleaved peptide containing the modified lysine side chain is at least 90% compared to peptide without modified lysine side chain.

9. The method of claim 7 , wherein the content of cleaved peptide containing the modified lysine side chain is at least 95% compared to peptide without modified lysine side chain.

10. The method of claim 1 , wherein the isolated peptide has the amino acid sequence H-dS-Q-G-T-F-T-S-D-L-S-K-Q-K(γE-Palm)-E-S-K-A-A-Q-D-F-I-E-W-L-K-A-G-G-P-S-S-G-A-P-P-P-S-NH 2 (SEQ ID NO.: 1).

11. The method of claim 1 , wherein the cleavage of step (ix) is performed with a cleavage cocktail comprising at least 90% (v/v) trifluoroacetic acid and at least 1% (v/v) ethanedithiol, which is free of thioanisole and ethylmethylsulfide.

12. The method of claim 11 , wherein the cleavage cocktail further comprises a suitable indole compound.

13. The method of claim 12 , wherein the suitable indole compound is 3-methylindole.

14. The method of claim 1 , wherein the cleavage of step (ix) is performed with a cleavage cocktail comprising at least 80% (w/w) trifluoroacetic acid and at least 1% (w/w) 1,2-ethanedithiol, which is free of thioanisole and ethylmethylsulfide.

15. The method of claim 14 , wherein the cleavage cocktail consists of 88.2-98.3% (w/w) trifluoroacetic acid, 1-4.3% (w/w) 1,2-ethanedithiol and 0.7-7.5% (w/w) 3-methylindole.

16. The method of claim 1 , further comprising the steps of:
(x) filtering the peptide solution after cleavage;
(xi) distilling the filtered peptide solution under vacuum;
(xii) adding the residual fraction from the distillation to an antisolvent comprising a dialkyl ether and a heptane;
(xiii) stirring the precipitated solution
(xiv) filtering the precipitated solution;
(xv) washing the precipitate; and
(xvi) drying the wet peptide.

17. The method of claim 16 , wherein in step (xii), the antisolvent is a mixture consisting of diisopropyl ether (DIPE) and n-heptane.

18. The method of claim 1 , comprising the steps of:
(i) assembling the amino acid sequence of a peptide of SEQ ID NO.: 1 with protected reactive functional groups in the side chains in a step-wise manner using solid phase peptide synthesis (SPPS), wherein the side chain of lysine 14 is protected by monomethoxytrityl (Mmt);
(ii) drying the solid phase resin for at least 5 hours at room temperature after the assembling of the amino acid sequence has been completed;
(iii) treating the dried resin nine times for 10 minutes each with a solution of 1% (v/v) trifluoroacetic acid (TFA) in dichloromethane (DCM) in order to deprotect the lysine side chain at position 14;
(iv) neutralizing the resin with a solution of 3% diisopropyl ethylamine (DIPEA) in DCM, until the pH of the solution remains at ≥8;
(v) coupling an activated Fmoc-Glu-OtBu linker moiety to the deprotected lysine side chain under basic conditions;
(vi) cleaving the Fmoc group of the linker coupled to the lysine side chain in step (v) with 20% (v/v) piperidine in DMF;
(vii) coupling activated palmitic acid to the deprotected terminal functional group of the linker of step (vi);
(viii) drying the resin;
(viii-a) analyzing the deprotection yield of step (iii) by cleaving a test sample of the resin bound peptide;
(viii-b) repeating steps (iii) to (viii-a) until the content of cleaved peptide containing the modified lysine side chain is at least 85%; and
(ix) cleaving the peptide from the resin at 25° C. using a cleavage cocktail comprising at least 90% (v/v) trifluoroacetic acid and at least 1% (v/v) ethanedithiol, which is free of thioanisole and ethylmethyl sulfide.

19. The method of claim 1 , comprising the steps of:
(i) assembling the amino acid sequence of a peptide of SEQ ID NO.: 1 with protected reactive functional groups in the side chains in a step-wise manner using solid phase peptide synthesis (SPPS), wherein the side chain of lysine 14 is protected by monomethoxytrityl (Mmt);
(ii) drying the solid phase resin for at least 5 hours at room temperature after the assembling of the amino acid sequence has been completed;
(iii) treating the dried resin nine times for 10 minutes each with a solution of 1% (v/v) trifluoroacetic acid (TFA) in dichloromethane (DCM) in order to deprotect the lysine side chain at position 14;
(iv) neutralizing the resin with a solution of 3% diisopropyl ethylamine (DIPEA) in DCM, until the pH of the solution remains at ≥8;
(v) coupling an activated Fmoc-Glu-OtBu linker moiety to the deprotected lysine side chain under basic conditions;
(vi) cleaving the Fmoc group of the linker coupled to the lysine side chain in step (v) with 20% (v/v) piperidine in DMF;
(vii) coupling activated palmitic acid to the deprotected terminal functional group of the linker of step (vi);
(viii) drying the resin;
(v

Assignees

• Sanofi Sa

Inventors

• Henkel Bernd
• Pleuss Norbert
• Hoerlein Rolf-Ludwig

Classifications

• C07K14/605

  Glucagons · CPC title

• C07K14/57563

  Vasoactive intestinal peptide [VIP]; Related peptides · CPC title

• C07K14/46 Primary

  from vertebrates · CPC title

• C07K1/34

  by filtration, ultrafiltration or reverse osmosis · CPC title

• C07K1/30

  by precipitation · CPC title