Patent ID: 8846614
Filing Date: 2014-09-30
Classification: A61K,C07K

Abstract:
1. A process for the production of pramlintide, a disulfide-bridged peptide of formula (1): wherein the Cys-Cys line indicates a cystine disulfide bridge; comprising: (a) coupling Fmoc-Tyr(X)-OH to an acid-labile Rink amide AM resin with a substitution value ranging from 0.15-0.60 mmol/g, in a single coupling reaction, to provide a sidechain-protected resin-bound tyrosine residue, Fmoc-Tyr(X)-Resin; then, (b) removing the N-terminal Fmoc protecting group thereof using piperidine in 1-hydroxybenzotriazole (HOBt) in polar aprotic solvent to provide an N-terminal free amino group; then, (c) assembling each respective Fmoc-protected aminoacid residue sequentially C-terminal to N-terminal under conditions suitable for peptide coupling, to provide sidechain-protected resin-bound peptide of the formula (2RBP): Fmoc-Asn(Y)-Asn(Y)-Phe-Gly-Pro-Ile-Leu-Pro-Pro-Thr(X)-Asn(Y)-Val-Gly-Ser(X)-Asn(Y)-Thr(X)-Tyr(X)-Resin (SEQ ID NO:2); wherein (X) and (Y) signify acid-labile sidechain protecting groups, wherein (X) can be removed under acidic conditions to yield a free OH group and (Y) can be removed under acidic conditions to yield a free NH or SH group; then, (d) removing the N-terminal Fmoc group using piperidine in 1-hydroxybenzotriazole (HOBt) in polar aprotic solvent to provide an N-terminal free amino group; then, (e) coupling Fmoc-Ser(X)-Ser(Ψ wherein each wavy line indicates a point of bonding in the peptide chain, to yield a sidechain-protected resin-bound peptide of Formula (3RBP): Fmoc-Ser(X)-Ser(Ψ (f) further assembling each respective Fmoc-protected aminoacid residue sequentially C-terminal to N-terminal, under conditions suitable for stepwise N-terminal Fmoc removal and peptide coupling, to the sidechain-protected resin-bound peptide of formula (3RBP), to provide a sidechain-protected resin-bound peptide of formula (4RBP), wherein the Ser(Ψ Fmoc-Lys(Boc)-Cys(Y)-Asn(Y)-Thr(X)-Ala-Thr(X)-Cys(Y)-Ala-Thr(X)-Gln(Y)-Arg(Pbf)-Leu-Ala-Asn(Y)-Phe-Leu-Val-His(Y)-Ser(X)-Ser(Ψ with a purity of crude peptide of ≧30%; then, (g) sidechain-deprotecting and cleaving a 37-mer cysteine-reduced peptide of Formula (4) from the resin by contacting with acid, H-Lys-Cys-Asn-Thr-Ala-Thr-Cys-Ala-Thr-Gln-Arg-Leu-Ala-Asn-Phe-Leu-Val-His-Ser-Ser-Asn-Asn-Phe-Gly-Pro-Ile-Leu-Pro-Pro-Thr-Asn-Val-Gly-Ser-Asn-Thr-Tyr-NH 2 (formula (4); SEQ ID NO:5); then, (h) oxidizing the peptide of formula (4) to the disulfide-bridged peptide of Formula (1) by air oxidation, or hydrogen peroxide or copper sulphate or iodine oxidation; and, (i) purifying the peptide of formula (4), or the peptide of formula (1), or optionally both, by RP-HPLC, wherein the peptide of formula (1) has a purity of 98%, or of 99%, or of 99.9% by weight.