Solid phase peptide synthesis via side chain attachment
AssigneeChemical & Biopharmaceutical Laboratories of Patras S.A.
InventorKleomenis K. Barlos
Life of this patent
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Abstract
The present application discloses peptides and peptaibols of high purity may be obtained by solid phase peptide synthesis using as the starting resin hydroxy amino acids, hydroxy amino acid amides, hydroxy amino alcohols or small peptides containing hydroxy amino acids attached to polymers through their side chain.
Claims24
A peptide resin conjugate of the formula E-D-2-Nal-Cys(A)-Tyr(C)-D-Trp(F)-Lys(E)-Val-Cys(A)-Thr(Resin)-NH2 wherein:
- D-designates the chirality of the amino acid that follows as a D-amino acid;
- each A is independently a thiol protecting group selected from Trt, Mmt, Acm, and StBu;
- C is a hydroxy protecting group selected from 2-chlorotrityl (Clt), Trt, and tBu;
- F is H or Boc;
- E is an amino protecting group selected from Mtt, Mmt, Trt, and Boc;
- and
- Resin is H or an acid labile resin suitable for solid-phase peptide synthesis.
A peptide of the formula E-Phe-Val-Asn(A)-Gln(A)-His(A)-Leu-Cys(B)-Gly-Ser(C)-His(A)-Leu-Val-Glu(C)-Ala-Leu-Tyr(C)-Leu-Val-Cys(A)-Gly-Glu(C)-Arg(D)-Gly-Phe-Phe-Tyr(C)-Thr(C)-Pro-Lys(E)-Thr(Resin)-O-C wherein:
- A is H or a carboxamido or imidazol protecting group selected from Trt, Mtt, and Mmt;
- B is a Cys protecting group selected from Mmt, Trt, Acm, and StBu;
- C is a hydroxy, carboxyl or phenoxy protecting group selected from tBu, Trt, and 2-chlorotrityl (Clt);
- D is a guanidine protecting group selected from Pbf and Pmc;
- E is H or an amino protecting group selected from Fmoc, Boc, Trt, Nps, Mtt, Mmt, and 2-chlorotrityl (Clt);
- and
- Resin is H or an acid labile resin suitable for solid-phase peptide synthesis.
A peptide resin conjugate of the formula E-Ser(C)-His(A)-Leu-Val-Glu(C)-Ala-Leu-Tyr(C)-Leu-Val-Cys(B)-Gly-Glu(C)-Arg(D)-Gly-Phe-Phe-Tyr(C)-Thr(C)-Pro-Lys(E)-Thr(Resin)-O- wherein:
- A is a carboxamido or imidazol protecting group selected from Trt, Mtt, and Mmt;
- B is a Cys protecting group selected from Mmt, Trt, Acm, and StBu;
- each C is independently a hydroxy, carboxyl or phenoxy protecting group selected from tBu, Trt, and 2-chlorotrityl (Clt);
- D is H or a guanidine protecting group selected from Pbf and Pmc;
- E is H or an amino protecting group selected from Fmoc, Boc, Trt, Nps, Mtt, Mmt, and 2-chlorotrityl (Clt);
- and
- Resin is H or an acid labile resin suitable for solid-phase peptide synthesis.
A peptide resin conjugate of the formula E-Cys(B)-Ser(C)-Asn(A)-Leu-Ser(C)-Thr(C)-Cys(B)-Val-Leu-Gly-Lys(E)-Leu-Ser(C)-Gln(A)-Glu(C)-Leu-His(A)-Lys(E)-Leu-Gln(A)-Thr(C)-Tyr(C)-Pro-Arg(D)-Thr(C)-Asn(A)-Thr(C)-Gly-Ser(C)- Gly-Thr(Resin)-Pro-NH2, wherein:
- each A is independently a carboxamido or imidazol protecting group selected from Trt, Mtt, and Mmt;
- each B is independently a Cys protecting group selected from Mmt, Trt, Acm, and StBu;
- each C is independently a hydroxyl, carboxy, or phenoxy protecting group selected from of tBu, Trt, and 2-chlorotrityl (Clt);
- D is a guanidine protecting group selected from Pbf and Pmc;
- each E is independently H or an amino protecting group selected from Fmoc, Boc, Trt, Nps, Mtt, Mmt, and 2-chlorotrityl (Clt);
- and
- Resin is H or an acid labile resin suitable for solid-phase peptide synthesis.
A process for the preparation of salmon calcitonin the process comprising:
- condensing the partially protected 11-32 peptide resin conjugate fragment of salmon calcitonin of the formula H-Lys(E)-Leu-Ser(C)-Gln(A)-Glu(C)-Leu-His(A)-Lys(E)-Leu-Gln(A)-Thr(C)-Tyr(C)-Pro-Arg(D)-Thr(C)-Asn(A)-Thr(C)-Gly-Ser(C)-Gly-Thr(Resin)-Pro-NH2 with a 1-10 partially protected fragment of salmon calcitonin of the formula E-Cys(B)-Ser(C)-Asn(A)-Leu-Ser(C)-Thr(C)-Cys(B)-Val-Leu-Gly-OH;
- wherein
- each A is independently a carboxamido or imidazol protecting group selected from Trt, Mtt, and Mmt;
- each B is independently a Cys protecting group selected from Mmt, Trt, Acm, and StBu;
- each C is independently a hydroxyl, carboxy or phenoxy protecting group selected from tBu, Trt, and 2-chlorotrityl (Clt);
- D is H or a guanidine protecting group selected from Pbf and Pmc;
- each E is independently H or an amino protecting group selected from Fmoc, Boc, Trt, Nps, Mtt, Mmt, and 2-chlorotrityl (Clt);
- and
- Resin is H or an acid labile resin suitable for solid-phase peptide synthesis.
A partially protected peptide resin conjugate of the formula E-His(A)-Gly-Glu(C)-Gly-Thr(C)-Phe-Thr(C)-Ser(C)-Asp(C)-X-Lys(E)-Gln(A)-Met-Glu(C)-Glu(C)-Glu(C)-Ala-Val-Arg(D)-Leu-Phe-Ile-Glu(C)-Trp(F)-Leu-Lys(E)-Asn(A)-Gly- Gly-Pro-Ser(C)-Ser(C)-Gly-Ala-Pro-Pro-Pro-Ser(Resin)-NH2 wherein:
- X is Leu-Ser(tBu) or Leu-ψSer;
- each A is independently H or a carboxamido or imidazol protecting group selected from Trt, Mtt, and Mmt;
- each C is independently a hydroxy, carboxyl, or phenoxy protecting group selected from tBu, Trt, and 2-chlorotrityl (Clt);
- D is H or a guanidine protecting group elected from Pbf and Pmc;
- each E is independently H or an amino protecting group selected from Fmoc, Boc, Trt, Nps, Mtt, Mmt, and 2-chlorotrityl (Clt);
- F is H or Boc;
- and
- Resin is an acid labile resin suitable for solid-phase peptide synthesis.
A partially protected peptide resin conjugate Y-Glu(C)-Glu(C)-Ala-Val-Arg(D)-Leu-Phe-Ile-Glu(C)-Trp(F)-Leu-Lys(E)-Asn(A)-Gly-Gly-Pro-Ser(C)-Ser(C)-Gly-Ala-Pro-Pro-Pro-Ser(Resin)-NH2 that is the exenatide fragments 12-39, 13-39, 14-39, wherein:
- Y is H, E-Glu(C), E-Met-Glu(C), E-Gln(A)-Met-Glu(C) or E-Lys(E′)-Gln(A)-Met-Glu(C);
- wherein:
- each A is independently H or a carboxamido protecting group selected from Trt, Mtt, and Mmt;
- E and E′ are each independently H or an amino protecting group selected from Fmoc, Boc, Trt, Nps, Mtt, Mmt, and 2-chlorotrityl (Clt);
- F is H or Boc;
- each C is independently a hydroxy or carboxyl protecting group selected from tBu, Trt, and 2-chlorotrityl (Clt);
- D is H or a guanidine protecting group selected from Pbf and Pmc;
- and
- Resin is H or an acid labile resin suitable for solid-phase peptide synthesis.
A partially protected peptide that is E-His(A)-Gly-Glu(C)-Gly-Thr(C)-Phe-Thr(C)-Ser(C)-Asp(C)-X-Y in particular sequences corresponding to the exenatide fragments 1-11, 1-13, 1-14 and 1-15, wherein:
- X is Leu-Ser(tBu) or Leu-ψSer;
- E is an amino protecting group selected from Fmoc, Boc, Trt, Nps, Mtt, Mmt, and 2-chlorotrityl (Clt);
- A is H, a carboxamido or imidazol protecting group selected from Trt, Mtt, and Mmt;
- each C is independently a hydroxy or carboxyl protecting group selected from tBu, Trt, and 2-chlorotrityl (Clt);
- and
- Y is OZ, Lys(E)-Gln(A)-OZ, Lys(E)-Gln(A)-Met-OZ, Lys(E)-Gln(A)-Met-Glu(C)-OZ, wherein each Z is independently H or a group which activate electrophilically carboxyl groups selected from Bt, Su, Pfp, Tcp, and Pnp.
A partially protected peptide resin conjugate of the formula E-Lys(E)-Cys(B)-Asn(A)-Thr(C)-Y-Cys(B)-Y-Gln(A)-Arg(D)-Leu-Ala-Asn(A)-Phe-Leu-Val-His(A)-X-Asn(A)-Asn(A)-Phe-Gly-Pro-Ile-Leu-Pro-Pro-Thr(C)-Asn(A)-Val-Gly- Ser(C)-Asn(A)-Thr(C)-Tyr(Resin)-NH2 wherein X=Ser(tBu)-Ser(tBu) or Ser(tBu)-ψSer, wherein:
- Y is Ala-Thr(tBu) or Ala-ψThr;
- each A is independently H, a carboxamido or imidazol protecting group selected from Trt, Mtt, and Mmt;
- each B is independently a Cys protecting group selected from Mmt, Trt, Acm, and StBu;
- each C is independently hydroxyl, carboxy or phenoxy protecting group selected from tBu, Trt, and 2-chlorotrityl (Clt);
- D is H or a guanidine protecting group selected from Pbf and Pmc;
- E is H or an amino protecting group selected from Fmoc, Boc, Trt, Nps, Mtt, Mmt, and 2-chlorotrityl (Clt);
- and
- Resin is H or an acid labile resin suitable for solid-phase peptide synthesis.
A protected or partially protected peptide resin conjugate with the sequence Z-Asn(A)-Phe-Leu-Val-His(A)-X-Asn(A)-Asn(A)-Phe-Gly-Pro-Ile-Leu-Pro-Pro-Thr(C)-Asn(A)-Val-Gly-Ser(C)-Asn(A)-Thr(C)-Tyr(Resin)-NH2 in particular the protected or partially protected 10-38, 11-38, 12-38 and 14-38 partially protected pramlintide fragments wherein:
- Z is H or E-Gln(A)-Arg(D)-Leu-Ala, E-Arg(D)-Leu-Ala, E-Leu-Ala or E-Ala;
- X is Ser(tBu)-Ser(tBu) or Ser(tBu)-ψSer;
- A is H or a carboxamido or imidazol protecting group selected from Trt, Mtt, and Mmt;
- C is a hydroxyl, carboxy or phenoxy protecting group selected from tBu, Trt, and 2-chlorotrityl (Clt);
- D is H or a guanidine protecting group selected from Pbf and Pmc;
- E is H or an amino protecting group selected from Fmoc, Boc, Trt, Nps, Mtt, Mmt, and 2-chlorotrityl (Clt);
- and
- Resin is H or an acid labile resin suitable for solid-phase peptide synthesis.
A process for the preparation of partially protected and oxidized pramlintide fragments of formulae I, II, III, or IV:
- wherein:
- each Y is independently Ala-Thr(tBu) or Ala-ψThr;
- Z is H or a group selected from Bt, Su, Pfp, Tcp, and Pnp;
- each A is independently H or a carboxamido or imidazol protecting group selected from Trt, Mtt, and Mmt;
- each C is independently a hydroxy, carboxyl or phenoxy protecting group selected from tBu, Trt, or 2-chlorotrityl (Clt);
- each D is independently H or a guanidine protecting group selected from Pbf and Pmc;
- each E is independently H or an amino protecting group selected from Fmoc, Boc, Trt, Nps, Mtt, Mmt, or 2-chlorotrityl (Clt);
- said process comprising:
- assembling protected peptides on an acid sensitive resin, such as a 2-CTC-resin;
- cleaving the protected peptide from the resin;
- and
- oxidizing the resin-bound peptides by contacting the resin-bound peptides with mild acid, such as dilute trifluoroacetic acid in an organic solvent, such as dichloromethane, wherein the solvent comprises a 2-200 molar excess of iodine over the fragment.
A process for the preparation of bivalirudin comprising:
- condensing a bivalirudin peptide resin conjugate fragment of the formula:
- E-X-Tyr(Resin)-Leu-O-C;
- wherein:
- X is an 11-18 partially protected bivalirudin fragment;
- E is H or an amino protecting group selected from Fmoc, Boc, Trt, Nps, Mtt, Mmt, and 2-chlorotrityl (Clt);
- C is a hydroxyl carboxyl protecting group selected from tBu, Trt, and 2-chlorotrityl (Clt);
- and
- Resin is H or an acid labile resin suitable for solid-phase peptide synthesis;
- in solution or on solid phase with a bivalirudin peptide resin conjugate fragment of the formula:
- E-D-Phe-Y-OZ;
- wherein:
- Y is a 2-10 partially protected bivalirudin fragment;
- Z is H or a group selected from Bt, Su, Pfp, Tcp, and Pnp;
- and
- D-designates the chirality of the amino acid that follows as being a D-amino acid;
- to give:
- E-D-Phe-Y-X-Tyr(Resin)-Leu-O-C;
- wherein the method further comprises:
- deprotecting, or cleaving from the resin and deprotecting to provide bivalirudin;
- of purifying the bivalirudin by chromatography;
- and
- lyophilizing the bivalirudin to provide bivalirudin in >99% purity.
A protected or partially protected peptide resin conjugate with the formula E-Ser(C)-Tyr(C)-Ser(C)-Met-Glu(C)-His(A)-Phe-Arg(D)-Trp(F)-Gly-Lys(E)-Pro-Val-Gly-Lys(E)-Lys(E)-Arg(D)-Arg(D)-Pro-Val-Lys(E)-Val-Tyr(Resin)-Pro-O-C, wherein:
- A is H or a carboxamido or imidazol protecting group selected from Trt, Mtt, and Mmt;
- each C is independently a hydroxy, carboxyl or phenoxy protecting group selected from tBu, Trt, and 2-chlorotrityl (Clt);
- each D is independently H or a guanidine protecting group selected from Pbf and Pmc;
- each E is independently H or an amino protecting group selected from Fmoc, Boc, Trt, Nps, Mtt, Mmt, and 2-chlorotrityl (Clt);
- F is H or Boc and
- Resin is H or an acid labile resin suitable for solid-phase peptide synthesis.
A protected or partially protected fragment with the formula E-X-Tyr(Resin)-Pro-O-C wherein:
- X is a ACTH(1-24) sequence;
- E is H or an amino protecting group selected from Fmoc, Boc, Trt, Nps, Mtt, Mmt, and 2-chlorotrityl (Clt);
- C is a hydroxy, carboxyl or phenoxy protecting group selected from tBu, Trt, and 2-chlorotrityl (Clt);
- and
- Resin is H or an acid labile resin suitable for solid-phase peptide synthesis.
Dependencies are inferred from the claim text.