US2008033147A1PendingUtilityA1

Method For Producing Acylated Peptides

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Assignee: NOVO NORDISK ASPriority: Sep 25, 2002Filed: Aug 22, 2007Published: Feb 7, 2008
Est. expirySep 25, 2022(expired)· nominal 20-yr term from priority
Inventors:Dorte Lunoe DunweberInge Holm JensenLouis B. Hansen
G01N 33/68C07K 14/57563C07K 1/1077C07K 1/006G01N 33/6842C07K 14/605C07K 14/62
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Claims

Abstract

The present invention provides a method for acylating one or more amino groups of a peptide where the acylation reaction is to be performed in an aqueous mixture containing less than 10% w/w aprotic polar solvent.

Claims

exact text as granted — not AI-modified
1 . A method for producing an N-acylated peptide, said method comprising: 
 a) reacting a peptide having at least one free amino group with an acylating agent of the general formula I                        wherein 
 n is 0-8;  
 R 1  is COOR 4 ;  
 R 2  is a lipophilic moiety;  
 R 3  together with the carboxyl group to which R 3  is attached designate a reactive ester or a reactive N-hydroxy imide ester; and  
 R 4  is selected from hydrogen, C 1-12 -alkyl and benzyl,  
   under basic conditions in an aqueous mixture containing less than 10% w/w aprotic polar solvent; and      b) if R 4  in the acylating agent of step a) is not hydrogen, saponifying the acylated peptide ester group (COOR 4 ) under basic conditions;    in order to produce said N-acylated peptide.    
   
   
       2 . The method according to  claim 1 , wherein said reaction in step a) takes place in an aqueous mixture containing less than 8% w/w aprotic polar solvent.  
   
   
       3 . The method according to  claim 1 , wherein said reaction in step a) takes place in an aqueous mixture containing less than 5% w/w aprotic polar solvent.  
   
   
       4 . The method according to  claim 1 , wherein said reaction in step a) takes place in an aqueous mixture containing less than 3% w/w aprotic polar solvent.  
   
   
       5 . The method according to  claim 1 , wherein the acylating agent is added to the reaction mixture in step a) as a solid.  
   
   
       6 . The method according to  claim 1 , wherein said reaction in step a) takes place in the presence of an aprotic polar solvent.  
   
   
       7 . The method according to  claim 6 , wherein said aprotic polar solvent is selected from the group consisting of N-methyl-2-pyrrolidone, tetrahydrofurane and dimethylsulfoxide.  
   
   
       8 . The method according to  claim 6 , wherein all of the aprotic solvent is added to the reaction mixture as a solvent for the acylating agent.  
   
   
       9 . The method according to  claim 6 , wherein the acylating agent is added to the reaction mixture as a solution which is stabilized by adding an acid.  
   
   
       10 . The method according to  claim 9 , wherein said acid is added to the aprotic polar solvent in a concentration from 0.01% w/w to 1% w/w.  
   
   
       11 . The method according to  claim 9 , wherein said acid is added to the aprotic polar solvent in a concentration from 0.05% w/w to 0.5% w/w.  
   
   
       12 . The method according to  claim 9 , wherein said acid is selected from the group consisting of sulphuric acid, methanesulphonic acid and trifluoroacetic acid.  
   
   
       13 . The method according to  claim 1 , wherein the reaction in step a) takes place in the absence of an aprotic polar solvent.  
   
   
       14 . The method according to  claim 1 , wherein R 4  is hydrogen.  
   
   
       15 . The method according to  claim 1 , wherein R 4  is selected from C 1-8 -alkyl and benzyl.  
   
   
       16 . The method according to  claim 1 , wherein R 3  together with the carboxyl group to which R 3  is attached designate a reactive N-hydroxy imide ester.  
   
   
       17 . The method according to  claim 1 , wherein the acylated peptide ester is saponified in step b) at a pH value in the range of 10-14.  
   
   
       18 . The method according to  claim 1 , wherein the acylated peptide ester is saponified in step b) at pH range from 9-13.  
   
   
       19 . The method according to  claim 1 , wherein pH of the reaction mixture in step a) is from pH 9 to pH 13.  
   
   
       20 . The method according to  claim 1 , wherein pH of the reaction mixture in step a) is from pH 10 to pH 12.  
   
   
       21 . The method according to  claim 1 , wherein pH of the reaction mixture in step a) is from pH 11.0 to pH 11.5.  
   
   
       22 . The method according to  claim 1 , wherein the temperature of the reaction mixture in step a) is in the range of 0-50° C.  
   
   
       23 . The method according to  claim 1 , wherein the temperature of the reaction mixture in step a) is in the range from 5-40° C.  
   
   
       24 . The method according to  claim 1 , wherein the temperature of the reaction mixture in step a) is in the range from 10-30° C.  
   
   
       25 . The method according to  claim 1 , wherein R 2  is selected from C 3-39 -alkyl, C 3-39 -alkenyl, C 3-39 -alkadienyl and steroidal residues.  
   
   
       26 . The method according to  claim 25 , wherein R 2 —C(═O)— is selected from the group consisting of lithocholoyl and hexadecanoyl.  
   
   
       27 . The method according to  claim 1 , wherein said peptide used as starting material for step a) has a peptide purity of at least 80 as determined by RP-HPLC.  
   
   
       28 . The method according to  claim 1 , wherein said peptide used as starting material for step a) has a peptide purity of at least 90% as determined by RP-HPLC.  
   
   
       29 . The method according to  claim 1 , wherein said peptide used as starting material for step a) has a peptide purity of at least 93% as determined by RP-HPLC.  
   
   
       30 . The method according to  claim 1 , wherein said peptide used as starting material for step a) has a peptide purity of at least 95% as determined by RP-HPLC.  
   
   
       31 . The method according to  claim 1 , wherein said peptide used as starting material for step a) has a peptide purity of at least 97% as determined by RP-HPLC.  
   
   
       32 . The method according to  claim 1 , wherein said peptide is selected from the group consisting of GLP-1, exendin-4, GLP-2, glucagon, insulin, analogues thereof and derivatives of any of the foregoing.  
   
   
       33 . The method according to  claim 1 , wherein said peptide is a GLP-1 agonist.  
   
   
       34 . The method according to  claim 1 , wherein said peptide is selected from the group consisting of exendin-3, exendin-4, Arg 34 -GLP-1(7-37), Gly 8 -GLP-1(7-36)-amide, Gly 8 -GLP-1(7-37), Val 8 -GLP-1(7-36)-amide, Val 8 -GLP-1(7-37), Val 8 Asp 22 -GLP-1(7-36)-amide, Val 8 Asp 22 -GLP-1(7-37), Val 8 Glu 22 -GLP-1(7-36)-amide, Val 8 Glu 22 -GLP-1(7-37), Val 8 Lys 22 -GLP-1(7-36)-amide, Val 8 Lys 22 -GLP-1(7-37), Val 8 Arg 22 -GLP-1(7-36)-amide, Val 8 Arg 22 -GLP-1(7-37), Val 8 His 22 -GLP-1(7-36)-amide, Val 8 His 22 -GLP-1(7-37), des(B30) human insulin and analogues thereof.  
   
   
       35 . The method according to  claim 1 , wherein said peptide is selected from HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPSKKKKKK-NH2 (ZP-10) and analogues thereof.  
   
   
       36 . The method according to  claim 1 , wherein the reaction mixture in step a) comprises a buffer which is suitable for maintaining a substantially constant pH during the reaction.  
   
   
       37 . The method according to  claim 1 , wherein said peptide is not insulin or an analogue thereof.

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