Method for solid phase peptide synthesis

Abstract

A novel method for synthesizing a Hirulog peptide is devised.

Claims

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 ##STR00008## with the proviso that .[.then.]. .Iadd.when .Iaddend.A.[., where including.]. .Iadd.includes .Iaddend.a residue X2, .Iadd.A .Iaddend.is .[.always.]. linked via O to said handle or linker, and wherein R is a solid phase, and wherein R.sub.1.[.,.]. .Iadd.and .Iaddend.R.sub.2.[., R.sub.3.]. are.[.,.]. independently, H, 4-(C.sub.1-C.sub.4 alkyl) or 4-(C.sub.1-C.sub.4 alkyl) or 4-(C.sub.1-C.sub.4 alkoxy) or 4-(C.sub.1-C.sub.4 alkoxy), and .[.may be.]. .Iadd.are .Iaddend.the same or different with the proviso that only one of R.sub.1, R.sub.2 .[.may.]. .Iadd.can .Iaddend.be H, and wherein R.sub.2 may optionally be 2-Cl .[.with the proviso that then.]. .Iadd.when .Iaddend.R.sub.1 is H, b) or of the formula III ##STR00009## with the proviso that .[.then.]. .Iadd.when .Iaddend.A.[., where including.]. .Iadd.includes .Iaddend.a residue X2, .Iadd.A .Iaddend.is linked via O to said handle or linker.[.,.]. .Iadd.and .Iaddend.R .[.being defined as above.]. .Iadd.is a solid phase.Iaddend., c) or of the formula IV ##STR00010## wherein R is defined as above and R.sub.1, R.sub.2, R.sub.3 are, independently, H, C.sub.1-C.sub.4 alkyl or C.sub.1-C.sub.4 alkoxy, and .[.may be.]. .Iadd.are .Iaddend.the same or different with the .[.provisio.]. .Iadd.proviso .Iaddend.that only one of R.sub.1, R.sub.2 .[.may.]. .Iadd.can .Iaddend.be H, and wherein L is A(L=A) or wherein L is of formula V ##STR00011## 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.]. .Iadd.wherein .Iaddend.W is of formula II as defined or is of formula VI, ##STR00012## .[.the above definitions for radicals R, R.sub.1 and R.sup..sub.2 applying.]..

3. The peptide-resin conjugate of claim 2, .[.characterized in that.]. .Iadd.wherein .Iaddend.W is of formula VII, ##STR00013## .[.the above definitions for radicals R.sub.1 and R.sub.2 applying and.]. .Iadd.wherein .Iaddend.R.sub.1, R.sup..sub.2 are, independently, H, methyl or methoxy with the .[.provisio.]. .Iadd.proviso .Iaddend.that only one of R.sub.1, R.sub.2 .[.may.]. .Iadd.can .Iaddend.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.]. .Iadd.wherein .Iaddend.X2 is not Trp, Cys or Arg.

6. The peptide-resin conjugate according to claim 1, .[.characterized in that.]. .Iadd.wherein .Iaddend.X1 comprises 0 to 50 amino acid residues.

7. The peptide-resin conjugate according to claim 1, .[.characterized in that.]. .Iadd.wherein .Iaddend.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.]. .Iadd.wherein .Iaddend.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.sub.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.]. .Iadd.wherein .Iaddend.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.]. .Iadd.wherein .Iaddend.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.]. .Iadd.wherein .Iaddend.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.]. .Iadd.wherein .Iaddend.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.]. .Iadd.wherein .Iaddend.R9 is tertiary-butyl.

15. The peptide-resin conjugate according to claim 1, .[.characterized in that.]. .Iadd.wherein .Iaddend.the -Arg(R2)-Pro- which is the thrombin cleavage site, is -Arg[psiCH.sub.2NH]Pro-.

.Iadd.16. A Hirulog peptide synthesized using the peptide-resin conjugate according to claim 1..Iaddend.

.Iadd.17. The Hirulog peptide of claim 16, wherein the Hirulog peptide comprises bivalirudin..Iaddend.

.Iadd.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.sub.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 ##STR00014## 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.sub.1 and R.sub.2 are independently, H, 4-(C.sub.1-C.sub.4 alkyl) or 4-(C.sub.1-C.sub.4 alkyl) or 4-(C.sub.1-C.sub.4 alkoxy) or 4-(C.sub.1-C.sub.4 alkoxy), and are the same or different with the proviso that only one of R.sub.1, R.sub.2 can be H, and wherein R.sub.2 may optionally be 2-Cl when R.sub.1 is H, b) or of the formula III ##STR00015## 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 ##STR00016## wherein R is defined as above and R.sub.1, R.sub.2, R.sub.3 are, independently, H, C.sub.1-C.sub.4 alkyl or C.sub.1-C.sub.4 alkoxy, and are the same or different with the proviso that only one of R.sub.1, R.sub.2 can be H, and wherein L is A(L=A) or wherein L is of formula V ##STR00017## and wherein W allows of cleaving the peptide moiety under weakly acidic conditions of 0.1% to 30% trifluoroacetic acid..Iaddend.

.Iadd.19. The process according to claim 18, further comprising deprotecting the protected peptide to provide a deprotected peptide..Iaddend.

.Iadd.20. The process according to claim 19, wherein the deprotecting is conducted concomitant with cleaving..Iaddend.

.Iadd.21. The process according to claim 19, wherein the deprotecting is conducted after cleaving..Iaddend.

.Iadd.22. The process according to claim 19, wherein the deprotecting is conducted with a composition comprising a strong acid..Iaddend.

.Iadd.23. The process according to claim 19, wherein the deprotecting is conducted with a composition comprising trifluoroacetic acid..Iaddend.

.Iadd.24. The process according to claim 22, wherein the composition further comprises a scavenger..Iaddend.

.Iadd.25. The process according to claim 24, wherein the scavenger comprises thioanisole, phenol, trialkylsilane, or a combination thereof..Iaddend.

.Iadd.26. The process according to claim 18, wherein the cleaving is conducted with a composition comprising a weak acid..Iaddend.

.Iadd.27. The process according to claim 18, wherein the cleaving is conducted with a composition comprising trifluoroacetic acid..Iaddend.

.Iadd.28. The process according to claim 19, further comprising precipitating the deprotected peptide..Iaddend.

.Iadd.29. The process according to claim 19, further comprising contacting the deprotected peptide with methyl-tertbutyl-ether..Iaddend.

.Iadd.30. The process according to claim 18, wherein W is of formula II..Iaddend.

.Iadd.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..Iaddend.

.Iadd.32. The process according to claim 18, wherein R9 is tertiary-butyl..Iaddend.

.Iadd.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..Iaddend.

.Iadd.34. The process according to claim 18, wherein R3 is trityl- and that R4, R5, R6, R7 and R8 are tertiary-butyl..Iaddend.

.Iadd.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 ##STR00018## 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.sub.1 and R.sub.2 are independently, H, 4-(C.sub.1-C.sub.4 alkyl) or 4-(C.sub.1-C.sub.4 alkyl) or 4-(C.sub.1-C.sub.4 alkoxy) or 4-(C.sub.1-C.sub.4 alkoxy), and are the same or different with the proviso that only one of R.sub.1, R.sub.2 can be H, and wherein R.sub.2 may optionally be 2-Cl when R.sub.1 is H, b) or of the formula III ##STR00019## 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 ##STR00020## wherein R is defined as above and R.sub.1, R.sub.2, R.sub.3 are, independently, H, C.sub.1-C.sub.4 alkyl or C.sub.1-C.sub.4 alkoxy, and are the same or different with the proviso that only one of R.sub.1, R.sub.2 can be H, and wherein L is A(L=A) or wherein L is of formula V ##STR00021## and wherein W allows of cleaving the peptide moiety under weakly acidic conditions of 0.1% to 30% trifluoroacetic acid..Iaddend.

.Iadd.36. The process according to claim 35, wherein the deprotecting is conducted concomitant with cleaving..Iaddend.

.Iadd.37. The process according to claim 35, wherein the deprotecting is conducted after the cleaving..Iaddend.

.Iadd.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..Iaddend.

.Iadd.39. The process according to claim 35, wherein X2 is not Trp, Cys or Arg..Iaddend.

.Iadd.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)..Iaddend.

.Iadd.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.sub.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..Iaddend.

.Iadd.42. The process according to claim 35, wherein the Hirulog peptide is Bivalirudin..Iaddend.

Description

EXPERIMENTS

1. Synthesis of Boc-D-Phe-Pro-Arg(Pbf)-Pro-Gly-Gly-Gly-Gly-Asn(Trt)-Gly-Asp(tBu)-Phe-Glu(tBu)-Glu(tBu)-Ile-Pro-Glu(tBu)-Glu(tBu)-Tyr(tBu)-Leu-O-2-CTC (SEQ ID NO: 16) (Protected protected Hirulog-8, Described in EP489 070, Carboxyterminally Conjugated in Ester Linkage to 2-CTC Resin)

(1) All reagents were sourced from EMD Biosciences (Madison, Wis./U.S.A.; Novabiochem-brand). Polystyrene-based 2-C1Trt (CTC) resin (Cbl Patras, Greece), preloaded with Fmoc-Leu-OH, was of 100-200 mesh as regards the base polymer and of 60-200 mesh as regards the preloaded, final CTC resin product. Loading density was about 0.60 mmol/g Individual amino acids were sourced as either Fmoc amino acids or, in case of D-Phe, as readily Boc-protected Boc-D-Phe. Couplings were carried out with TCTU in dichloromethane/N-methylpyrrolidone (NMP), in the presence of Hnig-Base (disopropyl-ethyl-amine, DIEA). Usually, 1.5 eq. of the Fmoc or Boc protected amino acid were used, except for coupling of Fmoc-Arg(Pbf), where 2.5 eq. were used. Similarly, the standard coupling reaction time of 60 min. (at 30 C.) was extended to 90 min. in case of Fmoc-Arg (Pbf). In process control of coupling efficiency was effected by means of the Kaiser test or Chloranil tests.

(2) Fmoc deprotection was carried out with 3-4 cycles of 20% piperidine in NMP at 30 C., with suitable rinsing with NMP in between.

2. Synthesis of Boc-D-Phe-Pro-Arg(Pbf)-Pro-Gly-Gly-Gly-Gly-Asn(Trt)-Gly-Asp(tBu)-Phe-Glu(tBu)-Glu(tBu)-Ile-Pro-Glu(tBu)-Glu(tBu)-Tyr(tBu)-Leu-OH (SEQ ID NO: 17)

(3) Cleavage from 48.3 g resin (about 100 ml swollen resin) as generated in experiment 1 above was achieved with 3 cycles of 15 min. each at 15 C., 2% (w/w) TFA, 1% (w/w) triethylsilane (TES) in dichloromethane. The reaction was stirred by nitrogen bubbling; the colour of the reaction changed from cycle to cycle from yellow/orange to brownish. After each cycle, cleavage reaction was directly quenched by pouring the whole reaction broth into dilute pyridin (pyridine/ethanol 1:9 (v/v)). Resin was then removed by filtration with a frit and subjected to the next cycle. All filtrates were pooled, concentrated to an orange semi-liquid under vacuo (RotaVap), washed with DCM, resuspended in 400 ml double distilled water, stirred at room temperature, filtrated, washed with water and dried. Yield was 28.8 g of a slightly yellow powder of analytical quality (90% pure). Product was analyzed by HPLC and LC-MS.

3. Global Deprotection, Synthesis of NH2-D-Phe-Pro-Arg-Pro-Gly-Gly-Gly-Gly-Asn-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Tyr-Leu-OH (SEQ ID NO: 15)

(4) Global deprotection was carried out in DCM diluted with cleavage cockatail (CC), DCM: CC=1:10 (v/v). CC was made up of TFA/thioanisole/phenol/water/TES in the mixing ratio (% w/w): 89:2.5:2.5:5.0:1.0.1 g of dry product from experiment 2 was dissolved in 10 ml DCM diluted as said above with CC and stirred for 5 hours at room temperature. The product was then recovered by addition of 50 ml methyl-tertbutyl-ether (MTBE, Fluka Chemie, Buchs/Switzerland), cooling the reaction down to 0 C. in a water bath for 30 min. under stirring and filtrating off the salt precipitate that has formed in the whiletime. The filter cake is rinsed with MTBE several times which is then dried at room temperature, yielding 0.8 g of a crude product of about 55% purity as determined by HPLC. The total yield jointly over steps 2 and 3 is about 55%.

4. Comparative Cleavage Experiments and LC-MS Analytics for Synthesis of Hirulog-8 or its C-Terminal Tetrapeptide Fragment Either on Wang Resin or on CTC Resin

(5) Using HPLC LC-MS analytics, it could be shown that upon cleavage from resin and global deprotection at strongly acidic conditions, 1-10% of the peptide product proved alkylated in case of Wang resin, whereas no such modification could be observed upon cleavage from CTC resin. MS analysis allowed of mapping that modification to the tyrosyl residue. Synthetic procedure as described above.