Solid phase synthesis of h(Gly2)GLP-2

1. A method of preparing a peptide comprising the amino acid sequence His-Gly-Asp-Gly-Ser-Phe-Ser-Asp-Glu-Met-Asn-Thr-Ile-Leu-Asp-Asn-Leu-Ala-Ala-Arg-Asp-Phe-Ile-Asn-Trp-Leu-Ile-Gln-Thr-Lys-Ile-Thr-Asp (SEQ ID NO:1) via a fragment-based assembly, the method comprising the steps of:

(a) providing a first peptide fragment comprising the amino acid sequence X-Ser-Phe-Ser-Asp-Glu-Met-Asn-Thr-Ile-Leu-Asp-Asn-Leu-Ala-Ala-Arg-Asp-Phe-Ile-Asn-Trp-Leu-Ile-Gln-Thr-Lys-Ile-Thr-Asp (SEQ ID NO:2), wherein X is a first protection group and wherein the C-terminal residue of the first peptide fragment is conjugated to a support;

(b) providing a second peptide fragment comprising the amino acid sequence Y-His-Gly-Asp-Gly (SEQ ID NO:3), wherein Y is a second protection group;

(c) removing the first protection group from the first peptide fragment; and

(d) coupling the second peptide fragment to the N-terminally deprotected, support-conjugated first peptide fragment;

wherein aspartamide by-product formation is reduced during preparation of the peptide via the fragment-based assembly in comparison to a peptide comprising the amino acid sequence of SEQ ID NO:1 prepared by a non-fragment based assembly.

2. The method of claim 1 , wherein the first peptide fragment and/or the second peptide fragment are prepared by solid phase peptide synthesis, and wherein optionally, in case the second peptide fragment is prepared by solid phase peptide synthesis, the second peptide fragment is cleaved from the support prior to coupling the second peptide fragment to the N-terminally deprotected first peptide fragment, and/or wherein optionally, in case the first peptide fragment is prepared by solid phase peptide synthesis, the first peptide fragment coupled to the second peptide fragment is cleaved from the support.

3. The method of claim 2 , wherein the first peptide fragment and/or the second peptide fragment are provided by conjugating the C-terminal amino acid residue to a support and sequentially adding appropriately protected amino acids to the N-terminus of the C-terminal, support-conjugated residue(s).

4. The method of claim 3 , wherein amino acids to be sequentially added to the N-terminus of the C-terminal, support-conjugated residue(s) of the first peptide fragment and/or the second peptide fragment are each protected by a protection group selected from the group consisting of Boc and Fmoc.

5. The method of claim 1 , wherein the first protection group is Fmoc.

6. The method of claim 1 , wherein the second protection group is an acid-labile protection group, selected from the group consisting of Boc and benzyloxycarbonyl.

7. The method of claim 1 , wherein the histidine residue of the second peptide fragment is protected at the side chain with a protection group selected from the group consisting of trityl, Boc, Bom and Bum.

8. The method of claim 1 , wherein the aspartic acid residue of the second peptide fragment is protected at the side chain with a tert-butyl ester protection group.

9. The method of claim 1 , comprising:

(a) providing by solid phase synthesis a first peptide fragment comprising the amino acid sequence X-Ser-Phe-Ser-Asp-Glu-Met-Asn-Thr-Ile-Leu-Asp-Asn-Leu-Ala-Ala-Arg-Asp-Phe-Ile-Asn-Trp-Leu-Ile-Gln-Thr-Lys-Ile-Thr-Asp (SEQ ID NO:2) by peptide synthesis, wherein X is a Fmoc protection group and wherein the C-terminal residue of the first peptide fragment is conjugated to a support;

(b) providing by solid phase synthesis a second peptide fragment comprising the amino acid sequence Y-His-Gly-Asp-Gly (SEQ ID NO:3) by peptide synthesis, wherein Y is an acid-labile protection group, selected from the group consisting of Boc and benzyloxycarbonyl, and wherein the C-terminal residue of the second peptide fragment is conjugated to a support;

(c) cleaving the second peptide fragment from the support;

(d) optionally purifying the cleaved second peptide fragment;

(e) removing the Fmoc protection group from the first peptide fragment, by adding a secondary amine selected from the group consisting of piperidine, piperazine, morpholine and dicyclohexylamine;

(f) coupling the second peptide fragment to the support-conjugated first peptide fragment by adding the purified second peptide fragment to the N-terminally deprotected, support-conjugated first peptide fragment;

(g) cleaving the support-conjugated first peptide fragment coupled to the second peptide fragment from the support; and

(h) optionally purifying the cleaved first peptide fragment coupled to the second peptide fragment, wherein optionally the support is a functionalized polymer.

10. The method of claim 9 , wherein the C-terminal amino acid of the first peptide fragment and/or the second peptide fragment is attached to the functionalized polymer by means of a linker.

11. A method of preparing a pharmaceutical composition containing a peptide comprising the amino acid sequence His-Gly-Asp-Gly-Ser-Phe-Ser-Asp-Glu-Met-Asn-Thr-Ile-Leu-Asp-Asn-Leu-Ala-Ala-Arg-Asp-Phe-Ile-Asn-Trp-Leu-Ile-Gln-Thr-Lys-Ile-Thr-Asp (SEQ ID NO:1), the method comprising the steps of:

(a) preparing the peptide according to the method of claim 1 ; and

(b) preparing a pharmaceutical composition containing the peptide prepared in step (a).

12. The method of claim 11 , wherein the pharmaceutical composition further comprises phosphate buffer in an amount sufficient to adjust the pH of the composition to a physiologically tolerable level.

13. The method of claim 11 , wherein the pharmaceutical composition further comprises L-histidine.

14. The method of claim 11 , wherein the pharmaceutical composition further comprises a bulking agent selected from the group consisting of mannitol and sucrose.

15. The method of claim 11 , wherein the pharmaceutical composition is provided as an injectable dosage form.

16. The method of claim 1 , further comprising purifying the second peptide fragment to remove a [beta]-Asp analogue prior to coupling the second peptide fragment to the first peptide fragment.