Acylation process for preparation of N-substituted peptide
The present invention relates to a facile acylation process for preparation of N-Substituted peptide and proteins. More specifically, the invention relates to acylating a peptide or a protein with deprotected acylating agent.
1. A process for acylation of a peptide or a protein comprising an acylatable lysine residue with an acylating agent
comprising a lipophilic moiety, an amino acid group comprising an amine reactive imide ester, and at least one protected carboxyl group comprising an alkyl ester that can be deprotected by hydrolysis under acid conditions;
wherein
said peptide or protein is selected from the group consisting of glucagon-like peptide- 1 (GLP-1), glucagon like peptide-2 (GLP-2), insulin, and analogues thereof,
comprising the steps of:
a) deprotection of said acylating agent in a solution comprising a mixture of an acid and a solvent to provide a deprotected acylating agent;
b) equilibrating the deprotected acylating agent of step (a) in a solution at temperature below 25° C. with pH in a basic range;
c) reacting the solution of the equilibrated deprotected acylating agent of step (b) without isolation or purification with a peptide or a protein present in an aqueous solution at basic pH to obtain a N-Substituted peptide or protein; wherein the deprotected equilibrated acylating agent is added to the peptide or protein at a controlled rate to maintain the basic pH; and
d) optionally, quenching the N-Substituted peptide or protein obtained from step (c) by addition of acid or primary amine comprising compound.
2. The process as claimed in claim 1 , wherein the lipophilic moiety is is an alkyl lipophilic moiety.
3. The process as claimed in claim 1 , wherein the the alkyl ester is tert-butyl ester.
4. The process as claimed in claim 1 , wherein the amine reactive imide ester is N-hydroxysuccinimide ester.
5. The process as claimed in claim 1 , wherein the concentration of acylating agent in the deprotecting solution of step (a) is in the range of 0.1-1000 mg/ml.
6. The process as claimed in claim 1 , wherein the acid in mixture of step (a) is selected from a group comprising trifluoroacetic acid (TEA), phosphoric acid, formic acid, sulfuric acid and hydrochloric acid.
7. The process as claimed in claim 1 , wherein the solution for carrying out the deprotection in step (a) comprises 95% TFA and 5% water.
8. The process as claimed in claim 1 , wherein equilibration of the deprotected acylating agent in step (b) is achieved by adjusting the pH to a basic pH in a solution comprising a mixture of an aprotic solvent and a base solution.
9. The process as claimed in claim 8 , wherein the base solution is selected from a group comprising triethylamine (TEA), alkali metal hydroxide and combinations thereof.
10. The process as claimed in claim 8 , wherein the basic pH is in the range of 7 to 12.5.
11. The process as claimed in claim 1 , wherein the glucagon-like peptide-1 analogue (GLP-1) is liraglutide precursor.
12. The process as claimed in claim 1 , wherein the aqueous solution containing the peptide or the protein in step (c) comprises water, base, acid. aprotic solvent, or combinations thereof.
13. The process as claimed in claim 1 , wherein the peptide or protein concentration in the aqueous solution in step (c) is in the range of 1 mg/ml to 30 mg/ml.
14. The process as claimed in claim 1 , wherein the basic pH of the peptide or protein in the aqueous solution in step (c) is in the range of 9 to 12.5.
15. The process as claimed in claim 1 , wherein the reaction in step (c) between the deprotected equilibrated acylating agent of step (b) with the peptide or the protein is carried out at pH in the range of 10 to 12.
16. The process as claimed in claim 1 , wherein the mole ratio of the acylating agent to the peptide or the protein in step (c) ranges from 0.1:1 to 10:1.
17. The process as claimed in claim 1 , wherein the quenching of N-Substituted peptide or protein in step (d) is carried out at pH 8.
18. The process as claimed in claim 1 , wherein the primary amine comprising compound for quenching N-Substituted peptide or protein is Tris(hydroxymethyl)aminomethane.
19. The process as claimed in claim 1 , wherein the protected carboxyl group comprising an alkyl ester is tertiary butyl ester.
20. The process as claimed in claim 1 , wherein the amine reactive imide ester is N-hydroxysuccinimide ester.
21. The process as claimed in claim 5 , wherein the concentration of acylating agent in the deprotecting solution of step (a) is less than 500 mg/ml.
22. The process as claimed in claim 10 , wherein the basic pH is in the range of 9-10.
23. The process as claimed in claim 13 , wherein the peptide or protein concentration in the aqueous solution in step (c) is in the range of 5 mg/ml to 25 mg/ml.
24. The process as claimed in claim 14 , wherein the basic pH of the peptide or protein in the aqueous solution in step (c) is in the range of 11.2 to 11.8.
25. The process as claimed in claim 15 , wherein the pH in the reaction in step (c) between the deprotected equilibrated acylating agent of step (b) with the peptide or the protein is carried out at pH in the range of 10.5 to 11.8.
26. The process as claimed in claim 16 , wherein the mole ratio of the acylating agent to the peptide or the protein is in the range of 1.5:1 to 3:1.
27. A process for acylation of a acylatable peptide or protein comprising a liraglutide precursor having amino acid sequence as set forth in SEQ ID NO. 1, with a protected acylating agent having amino acid group comprising a tertiary butyl ester carboxyl protecting group, a N-hydroxysuccinimide ester reactive ester group and a N-hexadecanoylglutamic acid lipophilic moiety, said acylating agent having the following structure:
comprising the steps of:
a) deprotection of said protected acylating agent in a solution comprising a mixture of an acid comprising trifluoroacetic acid (TFA) and an aqueous solvent to provide a deprotected acylating agent;
b) equilibrating the deprotected acylating agent of step n a solution at temperature below 25° C. with pH in a basic range;
c) reacting the solution of the equilibrated deprotected acylating agent of step (b) without isolation or purification with a peptide or a protein present in an aqueous solution at basic pH to obtain N-substituted peptide or protein; wherein the deprotected equilibrated acylating agent is added to the peptide or protein at a controlled rate to maintain the basic pH; and
d) optionally, quenching the N-Substituted peptide or protein obtained from step (c) by addition of trifluoroacetic acid (TFA) or tris(hydroxymethyl)aminomethane.
28. The acylating agent in claim 1 wherein the lipophilic moiety is a hexadecanoyl glutamic acid, the amine reactive ester is N-hydroxysuccinimide ester and the protected carboxyl group comprising an alkyl ester is tent-butyl ester.