Insulinotropic peptide synthesis using solid and solution phase combination techniques

• US 20080004429A1
• Filed: 06/21/2007
• Published: 01/03/2008
• Est. Priority Date: 06/23/2006
• Status: Abandoned Application

• Current Assignee: Roche Palo Alto LLC (Roche Holding AG)
• Original Assignee: Roche Palo Alto LLC (Roche Holding AG)
• Inventors: Roberts, Christopher, Chen, Lin, Han, Yeun-Kwei
• Application Number:
  US11/821,017
• Publication Number:
  US 20080004429A1
• Time in Patent Office: Days
• US Class Current: 530/330
• CPC Class Codes: A61K 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• CPC Class Codes: C07K 14/605 Glucagons

Abstract

The present invention relates to the preparation of insulinotropic peptides that are synthesized using a solid and solution phase (“hybrid”) approach. Generally, the approach includes synthesizing three different peptide intermediate fragments using solid phase chemistry. Solution phase chemistry is then used to add additional amino acid material to one of the fragments. The fragments are then coupled together in the solid solution phase. The use of a pseudoproline in one of the fragments eases solid phase synthesis of that fragment and also eases subsequent solution phase coupling of this fragment to other fragments. The present invention is very useful for forming insulinotropic peptides such as GLP-1(7-36) and its natural and non-natural counterparts.

Claims

1. A method of making an insulinotropic peptide, comprising the steps of:

a) preparing a peptide fragment including the amino acid sequence HX⁸EX¹⁰ (SEQ.

ID NO.

     6) wherein X⁸ and X¹⁰ are each residues of an achiral amino acid, or said fragment is a counterpart thereof including the X⁸ and X¹⁰ residues, each of H, E, X⁸ and X¹⁰ optionally including side chain protection; and

b) incorporating the peptide fragment into an insulinotropic peptide.

2. The method of claim 1, wherein X8 is an amino acid residue corresponding to methyl alanine.

3. The method of claim 1, wherein X¹⁰ is an amino acid residue corresponding to glycine.

4. The method of claim 1, wherein the insulinotropic peptide includes the amino acid sequence (SEQ. ID No. 5)

HX⁸EX¹⁰TFTSDVSSYLEGQAAKEFIAWLVKX³⁵R

and counterparts thereof, wherein each of the symbols X at positions, 8, 10, and 35 independently denotes an achiral, optionally sterically hindered amino acid residue;

and wherein one or more of the amino acid residues optionally includes side chain protection.

5. The method of claim 4, wherein at least one of X⁸ and X³⁵ is a residue of Aib.

6. The method of claim 4, wherein X¹⁰ is a residue of glycine.

7. The method of claim 1, wherein the insulinotropic peptide includes the amino acid sequence HX⁸EX¹⁰TFTSDVX^17-18YLEGQAAKEFIAWLVKX³⁵R (SEQ ID NO. 13) and counterparts thereof, wherein the symbols X⁸, X¹⁰, and X³⁵ each independently denotes an achiral amino acid residue, and X^17-18 is a residue of a pseudoproline, said amino acid residues optionally including side chain protection.

8. The method of claim 7, wherein X^17-18 has the formula

9. A peptide fragment or counterpart thereof having the amino acid sequence HX⁸EX¹⁰ (SEQ ID NO. 6), wherein X⁸ and X¹⁰ are each residues of an achiral amino acid, each of H, E, X⁸ and X¹⁰ optionally including side chain protection.

10. The peptide fragment of claim 9, wherein X⁸ is a residue of Aib and X¹⁰ is a residue of glycine.

11. A method of making an insulinotropic peptide, comprising the steps of:

a) preparing a peptide fragment or a counterpart thereof including the amino acid sequence TFTSDVX^17-18YLEG (SEQ. ID No.

     8) wherein the residue denoted by the symbol X^17-18 is a dipeptide residue of a pseudoproline; and

b) incorporating the peptide fragment into an insulinotropic peptide.

12. The method of claim 11, wherein X^17-18 has the formula

13. The method of claim 12, wherein Φ

represents a residue of Ser optionally including side chain protection.

14. The method of claim 12, wherein R² is —

CH—

.

15. The method of claim 12, wherein R¹ is

16. The method of claim 15 wherein each of R³ and R⁴ is methyl.

17. The method of claim 12, wherein the insulinotropic peptide includes the amino acid sequence HX⁸EX¹⁰TFTSDVX^17-18YLEGQAAKEFIAWLVKX³⁵R (SEQ ID NO. 13) and counterparts thereof, wherein the symbols X⁸, X¹⁰, and X³⁵ each independently denotes an achiral amino acid residue, and X^17-18 is a residue of a pseudoproline, said amino acid residues of the sequence optionally including side chain protection.

18. The method of claim 12, wherein the insulinotropic peptide includes the amino acid sequence (SEQ. ID No. 5)

HX⁸EX¹⁰TFTSDVSSYLEGQAAKEFIAWLVKX³⁵R

and counterparts thereof, wherein each of the symbols X at positions, 8, 10, and 35 independently denotes an achiral, optionally sterically hindered amino acid residue;

and wherein one or more of the amino acid residues optionally includes side chain protection.

19. A peptide or a counterpart thereof including the amino acid sequence TFTSDVX^17-18YLEG (SEQ. ID NO. 8) wherein the residue denoted by the symbol X^17-18 is a dipeptide residue of a pseudoproline;

said amino acid residues optionally including side chain protection.

20. A method of making an insulinotropic peptide, comprising the steps of:

a) preparing a peptide fragment or counterpart thereof including the amino acid sequence QAAKEFIAWLVKX³⁵ (SEQ ID NO.

     9), wherein X³⁵ is a residue of an achiral amino acid, said residues of the sequence optionally including side chain protection; and

b) incorporating the peptide fragment into an insulinotropic peptide.

21. The method of claim 20, wherein X³⁵ is an amino acid residue of methylalanine.

22. The method of claim 20, wherein the peptide fragment includes the amino acid sequence QAAKEFIAWLVKX³⁵R (SEQ ID NO. 12).

23. The method of claim 22, wherein the R does not include side chain protection.

24. The method of claim 20, wherein the insulinotropic peptide includes the amino acid sequence HX⁸EX¹⁰TFTSDVX^17-18YLEGQAAKEFIAWLVKX³⁵R (SEQ ID NO. 13) and counterparts thereof, wherein the symbols X⁸, X¹⁰, and X³⁵ each independently denotes an achiral amino acid residue, said amino acid residues of the sequence optionally including side chain protection;

and wherein X^17-18 is a reside of a pseudoproline.

25. The method of claim 20, wherein the insulinotropic peptide includes the amino acid sequence (SEQ. ID No. 5)

HX⁸EX¹⁰TFTSDVSSYLEGQAAKEFLAWLVKX³⁵R

and counterparts thereof, wherein each of the symbols X at positions, 8, 10, and 35 independently denotes an achiral, optionally sterically hindered amino acid residue;

and wherein one or more of the amino acid residues optionally includes side chain protection.

26. A method of making an insulinotropic peptide, comprising the steps of:

a) providing a first peptide fragment including the amino acid sequence HX⁸EX¹⁰ (SEQ ID NO.

     6), wherein X⁸ and X¹⁰ are each residues of an achiral amino acid, each of H and E optionally including side chain protection;

b) providing a second peptide fragment including the amino acid sequence TFTSDVX^17-18YLEG (SEQ ID NO.

     8) wherein the residue denoted by the symbol X^17-18 is a dipeptide residue of a pseudoproline, said amino acid residues of the sequence optionally including side chain protection;

c) coupling the first fragment to the second fragment to provide a third peptide fragment including the amino acid sequence HX⁸EX¹⁰TFTSDVX^17-18YLEG (SEQ ID NO.

     11), said amino acid residues of the sequence optionally including side chain protection;

d) providing a fourth peptide fragment including the amino acid sequence QAAKEFIAWLVKX³⁵ (SEQ ID NO.

     9), wherein X³⁵ is a residue of an achiral amino acid, said amino acid residues of the sequence optionally including side chain protection;

e) coupling the fourth peptide fragment to arginine in order to provide a fifth peptide fragment including the amino acid sequence QAAKEFIAWLVKX³⁵R (SEQ ID NO.

     12), said residues of the sequence optionally including side chain protection; and

f) coupling the fifth fragment to the third fragment in order to provide an insulinotropic peptide including the amino acid sequence HX⁸EX¹⁰TFTSDVX^17-18YLEGQAAKEFIAWLVKX³⁵R (SEQ ID NO.

     13), said residues of the sequence optionally including side chain protection.

27. A method of making an insulinotropic peptide, comprising the steps of:

a) providing a first peptide fragment including the amino acid sequence HX⁸EX¹⁰ (SEQ ID NO.

     6), wherein X⁸ and X¹⁰ are each residues of an achiral amino acid, each of H, E, X⁸ and X¹⁰ optionally including side chain protection;

b) providing a second peptide fragment including the amino acid sequence TFTSDVX^17-18YLEG (SEQ ID NO.

     8) wherein the residue denoted by the symbol X^17-18 is a dipeptide residue of a pseudoproline, said amino acid residues of the sequence optionally including side chain protection;

c) coupling the first fragment to the second fragment to provide a third peptide fragment including the amino acid sequence HX⁸EX¹⁰ TFTSDVX^17-18YLEG (SEQ ID NO.

     11), said amino acid residues of the sequence optionally including side chain protection;

d) providing a fourth peptide fragment including the amino acid sequence QAAKEFIAWLVKX³⁵ (SEQ ID NO.

     9), wherein X³⁵ is a residue of an achiral amino acid, said amino acid residues of the sequence optionally including side chain protection;

e) coupling the fourth peptide fragment to arginine in order to provide a fifth peptide fragment including the amino acid sequence QAAKEFIAWLVKX³⁵R (SEQ ID NO.

     12), said residues of the sequence optionally including side chain protection; and

f) coupling the fifth fragment to the third fragment in order to provide an insulinotropic peptide of the formula (SEQ. ID No.

     5) HX⁸EX¹⁰TFTSDVSSYLEGQAAKEFIAWLVKX³⁵R and counterparts thereof, wherein each of the symbols X at positions, 8, 10, and 35 independently denotes an achiral, optionally sterically hindered amino acid residue; and

wherein one or more of the amino acid residues optionally includes side chain protection.