Method for solid phase peptide synthesis

Abstract

A novel method for synthesizing a Hirulog peptide is devised.

The invention claimed is: 
     
       1. A peptide-resin conjugate A-W, wherein A=P-X1-Asp(R4)-Phe-Glu(R5)-Glu(R6)-Ile-Pro-Glu(R7)-Glu(R8)-Tyr(R9)-X2 (SEQ ID NO:3), wherein X1 is a peptidyl moiety of 0 to 200 amino acids, X1 optionally comprising protection groups on individual amino acid side chains, wherein R9 is an amino side chain protection group and wherein X2 is a single amino acid residue linked to the solid phase via —O— and optionally being side chain or C-terminally protected, and wherein P is H or is a protection group selected from the group consisting of Boc, Fmoc, Dde, Nps, Alloc, Z, and R4, R5, R6, R7 and R8 are amino acid side chain protection groups, and
 wherein W is a solid phase composite comprising a resin handle or linker 
 a) of the formula II 
 
       
         
           
           
               
               
           
         
         with the proviso that then when A, where including includes a residue X2, A is always linked via —O— to said handle or linker, 
         and wherein R′″ is a solid phase, and wherein R″ 1 , and R″ 2 , R″ 3  are, independently, H, 4-(C 1 -C 4  alkyl) or 4′-(C 1 -C 4  alkyl) or 4-(C 1 -C 4  alkoxy) or 4′-(C 1 -C 4  alkoxy), and may be are the same or different with the proviso that only one of R″ 1 , R″ 2  may can be H, and wherein R″ 2  may optionally be 2-Cl with the proviso that then when R″ 1  is H, 
         b) or of the formula III 
       
       
         
           
           
               
               
           
         
         with the proviso that then when A, where including includes a residue X2, A is linked via —O— to said handle or linker, and R′″ being defined as above is a solid phase, 
         c) or of the formula IV 
       
       
         
           
           
               
               
           
         
         wherein R′″ is defined as above and R″ 1 , R″ 2 , R″ 3  are, independently, H, C 1 -C 4  alkyl or C 1 -C 4  alkoxy, and may be are the same or different with the provisio proviso that only one of R″ 1 , R″ 2  may can be H, and 
         wherein L is A(L=A) or wherein L is of formula V 
       
       
         
           
           
               
               
           
         
         and wherein W allows of cleaving the peptide moiety under weakly acidic conditions of 0.1% to 30% trifluoroacetic acid. 
       
     
     
       2. The peptide-resin conjugate of  claim 1 , characterized in that wherein W is of formula II as defined or is of formula VI, 
       
         
           
           
               
               
           
         
         the above definitions for radicals R′″, R″ 1  and R ″   2  applying. 
       
     
     
       3. The peptide-resin conjugate of  claim 2 , characterized in that wherein W is of formula VII, 
       
         
           
           
               
               
           
         
         the above definitions for radicals R″ 1  and R″ 2  applying and wherein R″ 1 , R ″   2  are, independently, H, methyl or methoxy with the provisio proviso that only one of R″ 1 , R″ 2  may can be H, and that, where A including a residue X2 is linked via —N— to said handle or linker of formula VII, independently are methyl or methoxy, preferably are methoxy. 
       
     
     
       4. The peptide-resin conjugate of  claim 1 , wherein the handle or linker of formula II is selected from the group consisting of 2-chloro-trityl, 4-methoxy-trityl, 4,4′-dimethoxytrityl and 4-methyltrityl. 
     
     
       5. The peptide-resin conjugate according to  claim 1 , characterized in that wherein X2 is not Trp, Cys or Arg. 
     
     
       6. The peptide-resin conjugate according to  claim 1 , characterized in that wherein X1 comprises 0 to 50 amino acid residues. 
     
     
       7. The peptide-resin conjugate according to  claim 1 , characterized in that wherein A=P-X1-Asp(R4)-Phe-Glu(R5)-Glu(R6)-Ile-Pro-Glu(R7)-Glu(R8)-Tyr(R9)-Leu-O (SEQ ID NO:2). 
     
     
       8. The peptide-resin conjugate of  claim 1 , characterized in that wherein R9 is tertiary-butyl. 
     
     
       9. The peptide-resin conjugate according to  claim 1 , wherein A=Boc-D-Phe-Pro-Arg(R2)-Pro-Gly-Gly-Gly-Gly-Asn(R3)-Gly-Asp(R4)-Phe-Glu(R5)-Glu(R6)-Ile-Pro-Glu(R7)-Glu(R8)-Tyr(R9)-Leu-O— or A=Fmoc-D-Phe-Pro-Arg(R2)-Pro-Gly-Gly-Gly-Gly-Asn(R3)-Gly-Asp(R4)-Phe-Glu(R5)-Glu(R6)-Ile-Pro-Glu(R7)-Glu(R8)-Tyr(R9)-Leu-O— or A=NH 2 -D-Phe-Pro-Arg(R2)-Pro-Gly-Gly-Gly-Gly-Asn(R3)-Gly-Asp(R4)-Phe-Glu(R5)-Glu(R6)-Ile-Pro-Glu(R7)-Glu(R8)-Tyr(R9)-Leu-O— and wherein R2, R3, R4, R5, R6, R7, R8, R9 are amino side chain protection groups and wherein R1 is an insoluble solid phase. 
     
     
       10. The peptide-resin conjugate according to  claim 1 , characterized in that wherein the solid phase is polymeric and has a mesh size of less than 700 (US Bureau of Standards). 
     
     
       11. The peptide-resin conjugate according to  claim 9 , characterized in that wherein R2 is pentamethyldihydrobenzofuranyl, adamantyloxy-carbonyl or isobornyloxycarbonyl, R9 is tert-butyl or a derivative thereof and that R3 to R8 are acid-labile protection groups. 
     
     
       12. The peptide-resin conjugate according to  claim 9 , characterized in that wherein R2 is Pbf and that R4 to R9 are acid-labile protection groups that require at least 50% trifluoroacetic acid for removal. 
     
     
       13. The peptide-resin conjugate according to  claim 12 , characterized in that wherein R3 is trityl- and that R4, R5, R6, R7 and R8 are tertiary-butyl. 
     
     
       14. The peptide-resin conjugate according to  claim 13 , characterized in that wherein R9 is tertiary-butyl. 
     
     
       15. The peptide-resin conjugate according to  claim 1 , characterized in that wherein the -Arg(R2)-Pro- which is the thrombin cleavage site, is -Arg[psiCH 2 NH]Pro-. 
     
     
       16. A Hirulog peptide synthesized using the peptide-resin conjugate according to claim 1. 
     
     
       17. The Hirulog peptide of claim 16, wherein the Hirulog peptide comprises bivalirudin. 
     
     
       18. A process of using a peptide resin conjugate A-W for the synthesis of Bivalirudin, the process comprising
 cleaving a protected peptide from the peptide resin conjugate A-W,   wherein
 A=Boc-D-Phe-Pro-Arg(R2)-Pro-Gly-Gly-Gly-Gly-Asn(R3)-Gly-Asp(R4)-Phe-Glu(R5)-Glu(R6)-Ile-Pro-Glu(R7)-Glu(R8)-Tyr(R9)-Leu-O— or 
 A=Fmoc-D-Phe-Pro-Arg(R2)-Pro-Gly-Gly-Gly-Gly-Asn(R3)-Gly-Asp(R4)-Phe-Glu(R5)-Glu(R6)-Ile-Pro-Glu(R7)-Glu(R8)-Tyr(R9)-Leu-O— or 
 A=NH 2 -D-Phe-Pro-Arg(R2)-Pro-Gly-Gly-Gly-Gly-Asn(R3)-Gly-Asp(R4)-Phe-Glu(R5)-Glu(R6)-Ile-Pro-Glu(R7)-Glu(R8)-Tyr(R9)-Leu-O— 
 wherein R2, R3, R4, R5, R6, R7, R8, R9 are amino side chain protection groups, and 
   wherein W is a solid phase composite comprising a resin handle or linker
 a) of the formula II 
   
       
         
           
           
               
               
           
         
         
           
             with the proviso that when A includes a residue X2, A is linked via —O— to said handle or linker, 
             wherein R′″ is a solid phase, and wherein R″ 1  and R″ 2  are independently, H, 4-(C 1 -C 4  alkyl) or 4′-(C 1 -C 4  alkyl) or 4-(C 1 -C 4  alkoxy) or 4′-(C 1 -C 4  alkoxy), and are the same or different with the proviso that only one of R″ 1 , R″ 2  can be H, and wherein R″ 2  may optionally be 2-Cl when R″ 1  is H, 
           
           b) or of the formula III 
         
       
       
         
           
           
               
               
           
         
         
           
             with the proviso that when A includes a residue X2, A is linked via —O— to said handle or linker and R′″ is a solid phase, 
           
           c) or of the formula IV 
         
       
       
         
           
           
               
               
           
         
         
           wherein R′″ is defined as above and R″ 1 , R″ 2 , R″ 3  are, independently, H, C 1 -C 4  alkyl or C 1 -C 4  alkoxy, and are the same or different with the proviso that only one of R″ 1 , R″ 2  can be H, and
 wherein L is A(L=A) or wherein L is of formula V 
 
         
       
       
         
           
           
               
               
           
         
         and wherein W allows of cleaving the peptide moiety under weakly acidic conditions of 0.1% to 30% trifluoroacetic acid. 
       
     
     
       19. The process according to claim 18, further comprising deprotecting the protected peptide to provide a deprotected peptide. 
     
     
       20. The process according to claim 19, wherein the deprotecting is conducted concomitant with cleaving. 
     
     
       21. The process according to claim 19, wherein the deprotecting is conducted after cleaving. 
     
     
       22. The process according to claim 19, wherein the deprotecting is conducted with a composition comprising a strong acid. 
     
     
       23. The process according to claim 19, wherein the deprotecting is conducted with a composition comprising trifluoroacetic acid. 
     
     
       24. The process according to claim 22, wherein the composition further comprises a scavenger. 
     
     
       25. The process according to claim 24, wherein the scavenger comprises thioanisole, phenol, trialkylsilane, or a combination thereof. 
     
     
       26. The process according to claim 18, wherein the cleaving is conducted with a composition comprising a weak acid. 
     
     
       27. The process according to claim 18, wherein the cleaving is conducted with a composition comprising trifluoroacetic acid. 
     
     
       28. The process according to claim 19, further comprising precipitating the deprotected peptide. 
     
     
       29. The process according to claim 19, further comprising contacting the deprotected peptide with methyl-tertbutyl-ether. 
     
     
       30. The process according to claim 18, wherein W is of formula II. 
     
     
       31. The process according to claim 30, wherein formula II is selected from the group consisting of 2-chloro-trityl, 4-methoxy-trityl, 4,4′-dimethoxytrityl and 4-methyltrityl. 
     
     
       32. The process according to claim 18, wherein R9 is tertiary-butyl. 
     
     
       33. The process according to claim 18, wherein R2 is selected from the group consisting of pentamethyldihydrobenzofuranyl, adamantyloxy-carbonyl, isobornyl-oxy-carbonyl, pentamethylenchromanesulfonyl, 4-methoxy-2,3,6-trimethylbenzenesulfonyl and its 4-tert.butyl-2,3,5,6-tetramethyl homologue or Boc. 
     
     
       34. The process according to claim 18, wherein R3 is trityl- and that R4, R5, R6, R7 and R8 are tertiary-butyl. 
     
     
       35. A process of using a peptide resin conjugate A-W for the synthesis of a Hirulog peptide, the process comprising
 cleaving a protected peptide from the peptide resin conjugate A-W, and   deprotecting the protected peptide,   wherein A=P-X1-Asp(R4)-Phe-Glu(R5)-Glu(R6)-Ile-Pro-Glu(R7)-Glu(R8)-Tyr(R9)-X2 (SEQ ID NO:3), wherein X1 is a peptidyl moiety of 0 to 200 amino acids, X1 optionally comprising protection groups on individual amino acid side chains, wherein R9 is an amino side chain protection group and wherein X2 is a single amino acid residue linked to the solid phase via —O— and optionally being side chain or C-terminally protected, and wherein P is H or is a protection group selected from the group consisting of Boc, Fmoc, Dde, Nps, Alloc, Z, and R4, R5, R6, R7 and R8 are amino acid side chain protection groups, and   wherein W is a solid phase composite comprising a resin handle or linker
 a) of the formula II 
   
       
         
           
           
               
               
           
         
         
           
             with the proviso that when A includes a residue X2, A is linked via —O— to said handle or linker, 
             wherein R′″ is a solid phase, and wherein R″ 1  and R″ 2  are independently, H, 4-(C 1 -C 4  alkyl) or 4′-(C 1 -C 4  alkyl) or 4-(C 1 -C 4  alkoxy) or 4′-(C 1 -C 4  alkoxy), and are the same or different with the proviso that only one of R″ 1 , R″ 2  can be H, and wherein R″ 2  may optionally be 2-Cl when R″ 1  is H, 
           
           b) or of the formula III 
         
       
       
         
           
           
               
               
           
         
         
           
             with the proviso that when A includes a residue X2, A is linked via —O— to said handle or linker and R′″ is a solid phase, 
           
           c) or of the formula IV 
         
       
       
         
           
           
               
               
           
         
         
           
             wherein R′″ is defined as above and R″ 1 , R″ 2 , R″ 3  are, independently, H, C 1 -C 4  alkyl or C 1 -C 4  alkoxy, and are the same or different with the proviso that only one of R″ 1 , R″ 2  can be H, and 
             wherein L is A(L=A) or wherein L is of formula V 
           
         
       
       
         
           
           
               
               
           
         
         and wherein W allows of cleaving the peptide moiety under weakly acidic conditions of 0.1% to 30% trifluoroacetic acid. 
       
     
     
       36. The process according to claim 35, wherein the deprotecting is conducted concomitant with cleaving. 
     
     
       37. The process according to claim 35, wherein the deprotecting is conducted after the cleaving. 
     
     
       38. The process according to claim 35, wherein W is of formula II and is selected from the group consisting of 2-chloro-trityl, 4-methoxy-trityl, 4,4′-dimethoxytrityl and 4-methyltrityl. 
     
     
       39. The process according to claim 35, wherein X2 is not Trp, Cys or Arg. 
     
     
       40. The process according to claim 35 wherein A=P-X1-Asp(R4)-Phe-Glu(R5)-Glu(R6)-Ile-Pro-Glu(R7)-Glu(R8)-Tyr(R9)-Leu-O (SEQ ID NO:2). 
     
     
       41. The process according to claim 35, wherein A=Boc-D-Phe-Pro-Arg(R2)-Pro-Gly-Gly-Gly-Gly-Asn(R3)-Gly-Asp(R4)-Phe-Glu(R5)-Glu(R6)-Ile-Pro-Glu(R7)-Glu(R8)-Tyr(R9)-Leu-O— or A=Fmoc-D-Phe-Pro-Arg(R2)-Pro-Gly-Gly-Gly-Gly-Asn(R3)-Gly-Asp(R4)-Phe-Glu(R5)-Glu(R6)-Ile-Pro-Glu(R7)-Glu(R8)-Tyr(R9)-Leu-O— or A=NH 2 -D-Phe-Pro-Arg(R2)-Pro-Gly-Gly-Gly-Gly-Asn(R3)-Gly-Asp(R4)-Phe-Glu(R5)-Glu(R6)-Ile-Pro-Glu(R7)-Glu(R8)-Tyr(R9)-Leu-O— and wherein R2, R3, R4, R5, R6, R7, R8, R9 are amino side chain protection groups. 
     
     
       42. The process according to claim 35, wherein the Hirulog peptide is Bivalirudin.