Selective acylation of epsilon-amino groups

Abstract

The present invention relates to the acylation of proteins. More particularly, the invention relates to a one-step process for selectively acylating the free epsi-amino group of insulin, insulin analog, or proinsulin in the presence of a free alpha-amino group.

We claim:  
     
       1. A process of selectively acylating proinsulin, insulin, or an insulin analog having one or more free α-amino groups, and a free ε-amino group with a fatty acid, which comprises reacting the ε-amino group with a soluble activated fatty acid ester at pH greater than about 9.0 in a polar solvent. 
     
     
       2. The process of  claim 1  wherein the protein is insulin or an insulin analog. 
     
     
       3. The process of  claim 2  wherein the protein is human insulin. 
     
     
       4. The process of  claim 2  wherein the protein is an insulin analog. 
     
     
       5. The process of  claim 4  wherein the protein is Lys B28 Pro B29 -human insulin. 
     
     
       6. The process of  claim 1  wherein the activated fatty acid ester is a N-hydroxysuccinimide ester of a C 14  to C 18  fatty acid. 
     
     
       7. The process of  claim 2  wherein the activated fatty acid ester is a N-hydroxysuccinimide ester of a C 14  to C 18  fatty acid. 
     
     
       8. The process of  claim 3  wherein the activated fatty acid ester is a N-hydroxysuccinimide ester of a C 14  to C 18  fatty acid. 
     
     
       9. The process of  claim 4  wherein the activated fatty acid ester is a N-hydroxysuccinimide ester of a C 14  to C 18  fatty acid. 
     
     
       10. The process of  claim 5  wherein the activated fatty acid ester is a N-hydroxysuccinimide ester of a C 14  to C 18  fatty acid. 
     
     
       11. The process of  claim 6  wherein the activated fatty acid ester is a N-hydroxysuccinimide ester of palmitic acid. 
     
     
       12. The process of  claim 7  wherein the activated fatty acid ester ms a N-hydroxysuccinimide ester of palmitic acid. 
     
     
       13. The process of  claim 8  wherein the activated fatty acid ester ms a N-hydroxysuccinimide ester of palmitic acid. 
     
     
       14. The process of  claim 9  wherein the activated fatty acid ester is a N-hydroxysuccinimide ester of palmitic acid. 
     
     
       15. The process of  claim 10  wherein the activated fatty acid ester ms a N-hydroxysuccinimide ester of palmitic acid. 
     
     
       16. A process of selectively acylating proinsulin, insulin, or an insulin analog having one or more free α-amino groups, and a free ε-amino group with a fatty acid, which comprises reacting the free ε-amino group with a soluble activated fatty acid ester in a semi-aqueous solvent at a pH from about 9.0 to 12.0. 
     
     
       17. The process of  claim 16 , wherein the protein is human insulin. 
     
     
       18. The process of  claim 17 , wherein the pH is from about 9.5 to about 10.5. 
     
     
       19. The process of  claim 18 , wherein the semi-aqueous solvent is acetonitrile and water. 
     
     
       20. The process of  claim 19 , wherein the solvent is 50% acetonitrile. 
     
     
       21. The process of  claim 20 , wherein the fatty acid ester is N-succinimidyl palmitate. 
     
     
       22. A one step process of acylating proinsulin, insulin or an insulin analog having one or more free α- amino groups and a free ε - amino group comprising selectively introducing an acyl group so that it is covalently bonded to the ε - amino group at a pH greater than about  9 . 0  in an aqueous solvent or a semi - aqueous solvent wherein the ratio of mono - acylated ε - amino insulin, proinsulin or insulin analog to mono - acylated α - amino insulin, proinsulin or insulin analog is greater than about  5 .   
     
     
       23. The one step process of  claim 22  wherein an acyl group is selectively covalently bonded to the ε- amino group of human insulin.   
     
     
       24. The one step process of  claim 22  wherein an acyl group is selectively covalently bonded to the ε- amino group of an insulin analog.   
     
     
       25. The one step process of  claim 24  wherein an acyl group is selectively covalently bonded to the ε- amino group of Lys   B28   Pro   B29 - human insulin.   
     
     
       26. The one step process of  claim 22  wherein the solvent is a semi- aqueous solvent.   
     
     
       27. The one step process of  claim 26  wherein the semi- aqueous solvent has a pH of from about  9 . 0  to  12 . 0 .   
     
     
       28. The one step process of  claim 27  wherein the semi- aqueous solvent has a pH of from about  9 . 5  to about  10 . 5 .   
     
     
       29. The one step process of  claim 28  wherein the semi- aqueous solvent is acetonitrile and water.   
     
     
       30. The one step process of  claim 29  wherein the semi- aqueous solvent is  50   %  acetonitrile.   
     
     
       31. The one step process of  claim 22  wherein the selectively introduced acyl group is a fatty acyl group. 
     
     
       32. The one step process of  claim 22  wherein the ε- amino group is reacted with a soluble activated fatty acid ester.   
     
     
       33. The one step process of  claim 22  wherein the ratio of mono- acylated ε - amino insulin, proinsulin or insulin analog to mono - acylated α - amino insulin, proinsulin or insulin analog is greater than about  10 .   
     
     
       34. The one step process of  claim 22  wherein the ratio of mono- acylated ε - amino insulin, proinsulin or insulin analog to mono - acylated α - amino insulin, proinsulin or insulin analog is greater than about  50 .   
     
     
       35. A one step process of acylating proinsulin, insulin or an insulin analog having one or more free α- amino groups and a free ε - amino group comprising selectively introducing an acyl group so that it is covalently bonded to the ε - amino group in a non - aqueous organic solvent in the presence of a base with a basicity equivalent to a pK   a    greater than or equal to  10 . 75  in water wherein the ratio of mono - acylated ε - amino insulin, proinsulin or insulin analog to mono - acylated α - amino insulin, proinsulin or insulin analog is greater than about  5 .   
     
     
       36. The one step process of  claim 35  wherein an acyl group is selectively covalently bonded to the ε- amino group of human insulin.   
     
     
       37. The one step process of  claim 35  wherein an acyl group is selectively covalently bonded to the ε- amino group of an insulin analog.   
     
     
       38. The one step process of  claim 37  wherein an acyl group is selectively covalently bonded to the ε- amino group of Lys   B28   Pro   B29 - human insulin.   
     
     
       39. The one step process of  claim 35  wherein the selectively introduced acyl group is a fatty acyl group. 
     
     
       40. The one step process of  claim 35  wherein the ε- amino group is reacted with a soluble activated fatty acid ester.   
     
     
       41. The one step process of  claim 35  wherein the ratio of mono- acylated ε - amino insulin, proinsulin or insulin analog to mono - acylated α - amino insulin, proinsulin or insulin analog is greater than about  10 .   
     
     
       42. The one step process of  claim 35  wherein the ratio of mono- acylated ε - amino insulin, proinsulin or insulin analog to mono - acylated α - amino insulin, proinsulin or insulin analog is greater than about  50 .   
     
     
       43. A process of acylating proinsulin, insulin or an insulin analog having one or more free α- amino groups and a free ε - amino group comprising selectively introducing an acyl group so that it is covalently bonded to the ε - amino group without the use of amino - protecting groups at a pH greater than about  9 . 0  in an aqueous solvent or a semi - aqueous solvent wherein the ratio of mono - acylated ε - amino insulin, proinsulin or insulin analog to mono - acylated α - amino insulin, proinsulin or insulin analog is greater than about  5 .   
     
     
       44. The process of  claim 43  wherein an acyl group is selectively covalently bonded to the ε- amino group of human insulin.   
     
     
       45. The process of  claim 43  wherein an acyl group is selectively covalently bonded to the ε- amino group of an insulin analog.   
     
     
       46. The process of  claim 45  wherein an acyl group is selectively covalently bonded to the ε- amino group of Lys   B28   Pro   B29 - human insulin.   
     
     
       47. The process of  claim 43  wherein the solvent is a semi- aqueous solvent.   
     
     
       48. The process of  claim 47  wherein the semi- aqueous solvent has a pH of from about  9 . 0  to  12 . 0 .   
     
     
       49. The process of  claim 48  wherein the semi- aqueous solvent has a pH of from about  9 . 5  to about  10 . 5 .   
     
     
       50. The process of  claim 49  wherein the semi- aqueous solvent is acetonitrile and water.   
     
     
       51. The process of  claim 50  wherein the semi- aqueous solvent is  50   %  acetonitrile.   
     
     
       52. The process of  claim 43  wherein the selectively introduced acyl group is a fatty acyl group. 
     
     
       53. The process of  claim 43  wherein the ε- amino group is reacted with a soluble activated fatty acid ester.   
     
     
       54. The process of  claim 43  wherein the ratio of mono- acylated ε - amino insulin, proinsulin or insulin analog to mono - acylated α - amino insulin, proinsulin or insulin analog is greater than about  10 .   
     
     
       55. The process of  claim 43  wherein the ratio of mono- acylated ε - amino insulin, proinsulin or insulin analog to mono - acylated α - amino insulin, proinsulin or insulin analog is greater than about  50 .   
     
     
       56. A process of acylating proinsulin, insulin or an insulin analog having one or more free α- amino groups and a free ε - amino group comprising selectively introducing an acyl group so that it is covalently bonded to the ε - amino group without the use of amino - protecting groups in a non - aqueous organic solvent in the presence of a base with a basicity equivalent to a pK   a    greater than or equal to  10 . 75  in water wherein the ratio of mono - acylated ε - amino insulin, proinsulin or insulin analog to mono - acylated α - amino insulin, proinsulin or insulin analog is greater than about  5 .   
     
     
       57. The process of  claim 56  wherein an acyl group is selectively covalently bonded to the ε- amino group of human insulin.   
     
     
       58. The process of  claim 56  wherein an acyl group is selectively covalently bonded to the ε- amino group of an insulin analog.   
     
     
       59. The process of  claim 58  wherein an acyl group is selectively covalently bonded to the ε- amino group of Lys   B28   Pro   B29 - human insulin.   
     
     
       60. The process of  claim 56  wherein the selectively introduced acyl group is a fatty acyl group. 
     
     
       61. The process of  claim 56  wherein the ε- amino group is reacted with a soluble activated fatty acid ester.   
     
     
       62. The one step process of  claim 56  wherein the ratio of mono- acylated ε - amino insulin, proinsulin or insulin analog to mono - acylated α - amino insulin, proinsulin or insulin analog is greater than about  10 .   
     
     
       63. The one step process of  claim 56  wherein the ratio of mono- acylated ε - amino insulin, proinsulin or insulin analog to mono - acylated α - amino insulin, proinsulin or insulin analog is greater than about  50 .