Process for the synthesis of a peptide having a Trp residue

1. A process for making a peptide of the formula (I),

ρGlu-His-Trp-Ser-Tyr-D-Trp-Leu-Arg-Pro-Gly-NH 2 ,  (I)

wherein said process comprises:

(a) temporarily protecting the indole-NH group on the side chain of any tryptophan residue with a side chain protecting group;

(b) temporarily protecting the side chain of the histidine residue with a side chain protecting group;

(c) attaching a cesium salt of Boc-Gly onto a chloromethylated polystyrene resin to yield Boc-Gly resin;

(d) deblocking Boc-Gly-resin by mixing the Boc-Gly-resin with a mixture of a Boc-deblocking organic acid to yield an organic acid salt of H 2 N-Gly-resin;

(e) neutralizing the organic acid salt of H 2 N-Gly-resin with a base to yield H 2 N-Gly-resin;

(f) successively coupling H 2 N-Gly-resin with a Boc-α-amine protected amino acid, said protecting amino acid corresponding to ρGlu-His-Trp-Ser-Tyr-D-Trp-Leu-Arg-Pro-Gly-NH 2 in the order shown from the C-terminal to the N-terminal, wherein said coupling comprises:

(i) reacting H 2 N-Gly-resin with the Boc-α-amine protected amino acid in the presence of a peptide coupling agent to yield a Boc-blocked coupled product;

(ii) deblocking the Boc group from the Boc-blocked coupled product by mixing the Boc-blocked coupled product with a Boc-deblocking organic acid to yield a coupled product;

(iii) reacting the coupled product with a next Boc-α-amine protected amino acid in the presence of a peptide coupling agent to yield a Boc-blocked coupled product;

(iv) deblocking the Boc group from the Boc-blocked coupled product by mixing the Boc-blocked coupled product with a Boc-deblocking organic acid to yield a next coupled product;

(v) repeating steps (d)(iii) and (d)(iv) until the N-terminal amino acid of the peptide of formula (I) has been coupled and the Boc group of the N-terminal amino acid has been deblocked to yield a protected completed peptide-resin;

(vi) removing said side chain protecting group on the histidine residue;

(g) deprotecting the protected indole-NH group on the side chains of the tryptophan residues and cleaving the completed peptide-resin to yield ρGlu-His-Trp-Ser-Tyr-D-Trp-Leu-Arg-Pro-Gly-NH 2 , wherein said deprotecting and cleaving comprises:

(i) making a solution of the protected completed peptide-resin in an alcohol/inert polar aprotic solvent mixture;

(ii) cooling the solution to about 0° C. to about 10° C.;

(iii) bubbling ammonia gas slowly and maintaining cooling until the solution is well saturated with ammonia to yield a saturated solution;

(iv) mixing the saturated solution without cooling for about 15–36 hours until <95% of free methyl ester is converted to the corresponding amide; and

(h) isolating ρGlu-His-Trp-Ser-Tyr-D-Trp-Leu-Arg-Pro-Gly-NH 2 from said solution.

2. A process of claim 1 wherein the indole-NH groups on the side chains of the tryptophan residues are temporarily protected with a formyl group and the side chain of the histidine residue is temporarily protected with a tosyl group.

3. A process according to claim 2 , wherein the following Boc-α-amine amino acids are coupled successively from the C-terminal to the N-terminal, N-α-Boc-L-proline, N-α-Boc-L-arginineHCl, N-α-Boc-L-leucine, N-α-Boc-D-tryptophan (N′-indole-formyl), N-α-Boc-L-tyrosine, N-α-Boc-L-serine hydrate, N-α-Boc-tryptophan(N′-indole-formyl) and N-α-Boc-L-histidine(tosyl)-cyclohexylamine salt to yield a coupled product and further wherein pyroglutamic acid is coupled to the N-terminal of said coupled product.

4. A process according to claim 3 wherein said pyroglutamic acid is temporarily protected with a Boc protecting group.

5. A process according to claim 1 , wherein said indole-NH group of the side chains of any tryptophan residue is protected with an acyl group.

6. A process according to claim 5 , wherein said side chain protecting group is a formyl group.

7. A process according to claim 6 , wherein the formyl protecting groups of the side chains of the tryptophan residues are removed by treatment with a solution comprising a) ammonia, a primary amine, or a secondary amine and b) an alcohol.

8. A process according to claim 7 , wherein said solution comprises ammonia and methanol.

9. A process according to claim 8 , wherein 1-hydroxybenzotriazole (HOBT) is used when coupling N-α-Boc-D-tryptophan(N′-indole-formyl), N-α-Boc-L-tyrosine, N-α-Boc-L-serine hydrate and N-α-Boc-tryptophan (N′-indole-formyl).

10. A process according to claim 5 , wherein said acyl group is removed by treatment with a solution comprising a) ammonia, a primary amine, or a secondary amine and b) an alcohol.

11. A process of claim 1 , wherein said side chain protecting group on the histidine residue is a tosyl group.

12. A process according to claim 11 , wherein the tosyl protecting group of the side chain of the histidine residue is removed by treatment with a solution comprising 1-Hydroxybenzotriazole (HOBT) in dimethylformamide (DMF).