CA2024855C - Process and intermediates for producing glucagon - Google Patents

Abstract

Disclosed herein is an improved process for preparing the nonacosapeptide glucagon. The process comprises the serial coupling of two highly purified fragments, glucagon(15-22) and glucagon(7-14) fragments, to a modified glucagon(23-29) fragment having a benzhydrylamine type resin attached to the .omega.-carboxyl of Asp at position 28.

Description

2~24~

Process Intermediates for Producinq Glucaqon Field of the Invention This invention relates to an improved process for preparing human glucagon (hereinafter referred to as "glucagon"), and to intermediates used in the process.

Backqround of the Invention Glucagon, a peptide hormone composed of 29 amino acids, is produced in the alpha cells of islets of Langerhans in the pancreas of a mammal, including man. The hormone regulates glycogenolysis and gluconeogenesis. It can act as a hyperglycemic agent, increasing blood glucose concentration by activating hepatic glycogenolysis. Hence, glucagon is indicated for the treatment of insulin-induced hypoglycemia;
see "Martindale, The Extra Pharmacopocia", 29th ed., J.E.F. Reynolds, Ed., The Pharmaceutical Press, London, UK, 1989, p. 1574.

Glucagon has the following structure:

H-His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr-OH

The preparation of glucagon by chemical synthesis has been r~ported on several occasions.
The first successful synthesis was accomplished by E. WUnsch and G. Wendlberger, Chem. Ber., 101, 3659 (1968); see also E. WUnsch and G. Wendlberger, US patent 3,642,763, issued February 15, 1972. The latter investigators used 2~)2~

the classical method of peptide synthesis involving the condensation of fragments in solution (i.e. the solution phase peptide synthesis). In 1975, the Protein Synthesis Group of the Shanghai Institute of Biochemistry reported the preparation of glucagon by a combination of solution phase and solid phase methods of synthesis; see Scientia Sinica, 18, 745 (1975) and Chem. Abstr., 86, 90223c (1977).
A solid phase synthesis was first reported by S.
Mojsov and R.B. Merrifield, Biochemistry, 20, 2950 (1981). Other reports of the synthesis of glucagon include:
J.E. Shields and E.L. Smithwick, US patent 3,887,538, issued June 3, 1975;
M. Fujino et al., Chem. Pharm. Bull., 26, 539 (1978);
B.F. Lundt et al., Res. Discl., 181, 246 (1979), see also Chem. Abstr., 91, 57515e (1979);
M. S. Verlander et al., US patent 4,351,762, issued September 28, 1982;
S. Mojsov and R.B. Merrifield, Eur. J.
Biochem., 145, 601 (1984); and J.P. Tam and R.B. Merrifield, US patent 4,507,230, issued March 26, 1985.

Although some of the aforementioned processes can produce glucagon in a reasonable yield and purity, a facile and reliable synthesis for producing glucagon on a commercial scale is needed.

The present process can be accomplished simply and rapidly to produce large quantities of glucagon. By the particular choice of starting materials, intermediates and reaction conditions, the present process produces glucagon ~ ~4~ 5 5 3 effectively, free of significant racemization and troublesome byproducts, and with a purity of greater than 98%. The process is distinguished from the aforementioned prior art processes by the serial coupling of two key fragments to a glucagon(23-29) fragment attached to a solid support via the side chain carboxyl of Asp at position 28.

Sllmm~ry of the Invention The process of this invention for preparing glucagon comprises:
(a)coupling a glucagon(15-22) fragment of formula 1 X-Asp(W1)-Ser(W2)-Arg(W3)-Arg(W3)-Ala-Gln-Asp(W1)-Phe-OH

wherein X is an ~-amino protective group, W1 is a protective group for the ~-carboxyl of Asp, w2 is a protective group for the hydroxyl of Ser and W3 is a protective group for the guanidino group of Arg, to a glucagon(23-29)-resin of formula 2 H-Val-Gln-Trp(W4)-Leu-Met(O)-Asp(Q)-Thr(W5)-O-Y
?
wherein W4 is a protective group for the aromatic nitrogen atom of Trp, W5 is protective group for the hydroxyl of Thr, Q is a benzhydrylamine type resin and Y is a carboxyl protective group, to obtain a glucagon(15-29)-resin of formula 3 X-Asp(W1)-Ser(W2)-Arg(W3)-Arg(W3)-Ala-Gln-Asp(W1)-Phe-Val-Gln-Trp(W4)-Leu-Met(O)-Asp(Q)-Thr(W5)-O-Y 3 ~,,,v~

1 ~

20~2 ~5~3 wherein Q, W1 to W5 inclusive, X and Y are as defined above, (b)selectively removing the a-amino protective group of the glucagon~15-29)-resin to obtain the corresponding glucagon(15-29)-resin of formula 3 wherein X is hydrogen, (c)coupling a glucagon(7-14) fragment of formula X-Thr (W5) -Ser(W2)-Asp(W1)-Tyr(W6)-Ser(W2)-Lys (W7) -Tyr(W )-Leu-OH 4 wherein Wl, W2, W5 and X are as defined herein, W
is a protective group for the hydroxyl of Tyr and W7 iS a protective group for the ~-amino group of Lys, with the last-named glucagon(15-29)-resin to obtain a glucagont7-29)-resin of formula 5 X-Thr(W )-Ser(W)-Asp(W1)-Tyr(W6)-Ser(W2)-Lys (W7) Tyr(W )-Leu-Asp(W1)-Ser(W2)-Arg (W3) -Arg (W3) -Ala-Gln-Asp(W)-Phe-Val-Gln-Trp(W)-Leu-Met(O)-Asp(Q)-Thr (W5 ) -O-Y 5 wherein Q, W1 to W7 inclusive, X and Y are as defined above, (d) stepwise coupling the required a-amino protected amino acids to the glucagon(7-29)-resin to obtain a glucagon(1-29)-resin of formula X-His(W )-Ser(W2)-Gln-Gly-Thr (W5) -Phe-Thr(W )-Ser(W )-Asp(W1)-Tyr(W6)-Ser(W2)-Lys (W7) -Tyr(W6)-Leu-Asp(W )-Ser(W )-Arg~W )-Arg(W )-Ala-Gln-Asp(W )-Phe-Val-Gln-Trp(W )-Leu-Met(O)-Asp(Q)-Thr(W )-O-Y 6 wherein Q, W1 to W7 inclusive, X and Y are as defined above and w8 is a protective group for the imidazole ring of His, 2 0 ~ 5 (e)selectively removing the a-amino protective group of the glucagon(1-29)-resin to obtain the corresponding glucagon(1-29)-resin of formula 6 wherein X is hydrogen, and (f)deprotecting the last-named glucagon(1-29)-resin to obtain glucagon.

Also included within the scope of this invention are the glucagon(15-22) fragment of formula 1, the glucagon(23-29)-resin of formula 2, the glucagon(15-29)-resin of formula 3 wherein X is hydrogen or an a-amino protective group, the glucagon(7-14) fragment of formula 4, the glucagon(7-29)-resin of formula 5 and the glucagon(1-29)-resin of formula 6 wherein X is hydrogen or an a-amino protective group.

Details of the Invention The term "residue" with reference to an amino acid means a radical derived from the corresponding a-amino acid by eliminating the hydroxyl of the carboxy group and one hydrogen of the a-amino group. The term "amino acid residue" can include radicals derived from side chain protected amino acids.

In general, the abbreviations used herein for designating the amino acids and the protective groups are based on recommendations of the IUPAC-IUB Commission on Biochemical Nomenclature, see Biochemistry, 11, 1726-1732 (1972). For instance, His, Trp, Gln, Ala, Gly, Arg, Asp, Phe, Ser, Leu, Asn, Thr, Lys, Val, Met, Met(O) and Tyr represent the "residues" of L-histidine, L-tryptohane, L-glutamine, L-alanine, glycine, L-arginine, L-aspartic acid, L-phenylalanine, L-20~ ~3~

serine, L-leucine, L-asparagine, L-threonine, L-lysine, L-valine, L-methionine, L-methionine sulfoxide and L-tyrosine, respectively.

The term "photosensitive spacer" or "photolabile spacer", designated by the symbol "S", as used herein in connection with the preparation of the glucagon(15-22) and glucagon(7-14) fragments described hereinafter, when incorporated into a peptide-resin system, links the first amino acid building block to the resin by orthogonal covalent bonds; the unit or spacer being further characterized in that the bond between the spacer and the first amino acid residue can be cleaved by photolysis to afford the peptide (or the first amino acid residue) with a C-terminal carboxyl. For examples of such spacers, see D.H. Rich and S.K. Gurwara, Canadian patent 1,108,348, issued September 1, 1981; J.P.
Tam et al., J. Amer. Chem. Soc., 102, 6117 (1980); F.S. Tjoeng and G.A. Heavner, J. Org.
Chem., 48, 355 (1983); and J. Gauthier, Canadian patent application, SN 547,394, filed September 21, 1987. When utilized herein, the spacer is first attached to the resin to give the solid support of formula J-S-P wherein J is bromo, chloro or iodo, and S is a photosensitive spacer and P is a resin. Preferred spacers are represented by the formulae -CH(CH3)CO ~ OCH2CO- and -CH(CH3)CO ~ CH2CO- .

when the resin is one of the benzhydrylamine type, and -CH(CH3)CO- when the resin is one of the styrene divinylbenzene type.

2024~5~
. .

The term "benzhydrylamine type resin", as used herein, means a benzhydrylamine resin of the type commonly employed in solid phase peptide synthesis ~SPPS). Such resins include benzhydrylamine resin (BHA) and 4-methylbenzhydrylamine resin.

Turning to the process of this invention, one feature is the protection of labile side chain groups of the various amino acid residues with suitable protective groups to prevent a chemical reaction from occurring at those sites until after the completion of the stepwise coupling to produce the glucagon(1-29)-resin of formula 6.

Preferred protective groups for amino acids with labile side chain groups are tosyl (Tos) for His and Arg, benzyl (Bzl) for Ser and Thr, cyclohexyloxy (OChl), benzyloxy (OBzl) or 2,6-dichlorobenzyloxy (O-Cl2Bzl) for Asp, 2,6-dichlorobenzyl for Tyr, 2-chlorobenzyloxycarbonyl (ClZ) for Lys and formyl (For) for Trp. In a broader sense, the oxygen of methionine sulfoxide is a protective group for Met.

Another common feature is the protection of the a-amino group of an amino acid while the free carboxyl group of that reactant is coupled with the free a-amino group of the second reactant;
the a-amino protective group being one that can be selectively removed to allow the subsequent coupling step to take place at the amino group from which the protective group is removed. A
preferred a-amino protective group, which is represented herein for the disclosed process by the symbol "X", is t-butyloxycarbonyl (Boc).

2~3~5 A preferred carboxyl protective group is benzyl.

Turning now to the starting materials for the process. Two of the starting materials, i.e. the glucagon(15-22) fragment of formula 1 and the glucagon(7-14) fragment of formula 4, are prepared readily in a high state of purity from corresponding protected peptide-resins from which the glucagon fragments are cleaved by photolysis.
This manner of generation of these individual peptide fragments enables one to purify important intermediates products before further coupling, thus decreasing the chances of carrying undesirable impurities through to the final product.

More specifically, a convenient and practical photochemical resin for the preparation of glucagon fragments of formulae 1 and 4 is obtained by modifying commercially benzyhydrylamine (BHA) resin or 4-methyl-benzhydrylamine resin by attaching a photo-sensitive spacer thereto. Preferred spacers have been noted previously. A practical and efficient photochemical resin for the present purpose is 4-(2-chloropropionyl)phenoxyacetyl BHA resin.

To initate the preparation of the fragments of formula 1 and 4, a first amino acid is coupled to the photolabile resin. The preparation of the amino acid-resin is exemplified as follows: An a-amino protected amino acid, e.g. Na-Boc-Phe-OH or Na-Boc-Leu-OH, is coupled to a solid support of formula J-S-P wherein J is bromo, chloro or iodo and S and P are as defined herein, e.g. 4-(2-chloropropionyl)phenoxyacetyl BHA-resin, in the 20~8~

presence of potassium fluoride or cesium chloride to give the corresponding solid support having an a-amino protected amino acid linked thereto.
Thereafter, the a-amino protective group of the latter resin derivative is removed to give the desired amino acid-resin with a free amino group.

The preceding amino acid-resin, in which the amino acid residue is either Phe or Leu, is utilized to prepare respectively a protected peptide resin in which the peptide portion corresponds to that of the desired fragments of formulae 1 and 4. This transformation of the amino acid resin is accomplished by stepwise coupling thereto in the desired order the appropriate a-amino protected amino acids. (For a recent review of solid phase synthesis, see J.M. Stewart and J.D. Young, "Solid Phase Peptide Synthesis", 2nd ed, Pierce Chemical Company, Rockford, Illinois, USA, 1984.) More explicitly, the coupling of the amino acid residues is achieved by using dicyclohexylcarbodiimide ~optionally adding 1-hydroxybenzotriazole, 3-hydroxy-4-oxo-3,4-dihydro-1,2,3-benzotriazine or N-hydroxysuccinimide) as the coupling agent, or by employing the "mixed anhydride" activated form of the a-amino protected acids. Another useful coupling agent is benzotriazol-1-yloxytri-(dimethylamino)phosphonium hexafluorophosphate (BOP), described by B. Castro et al., Tetrahedron Letters, 14, 1219 (1975). Each a-amino protected amino acid or protected fragment is introduced into the reaction system in a relatively slight excess (1.5 to 2 molar equivalents). The success of the coupling reaction at each stage is monitored by the ninhydrin reaction as described by E. Kaiser et al., Anal. Bioch., 34, 595 20î48~

(1970). Removal of the a-amino protective group completes the coupling cycle. In the instance where the a-amino protective group is a t-butyloxycarbonyl, trifluoroacetic acid in methylene chloride is used to remove the protective group.

The cleavage of the protected peptide-resins so obtained to give the desired fragments of formulae 1 and 4 is achieved by photolysis. The photolysis is accomplished by dissolving or suspending the protected peptide-resin in a photolytically stable liquid medium; for example, dioxane, dimethylformamide, methanol, ethanol or N-methylpyrrolidine; purging the solution or suspension of the peptide-resin with argon or nitrogen to remove any dissolved oxygen; and then irradiating the suspension or solution with photolytically effective ultraviolet light. In practice, irradiation at a wavelength of 350 nm has been found to be very effective. In this manner, the glucagon(15-22) fragment of formula 1 and the glucagon(7-14) fragment of formula 4 are obtained with a high degree of purity.

The glucagon(23-29)-resin of formula 2 is another requisite starting material for the present process. This peptide-resin serves as the solid support system for the process. In contrast to previously reported solid phase syntheses of glucagon which use a C-terminus attached resin, the present process uses as a starting material this particular glucagon(23-29)-resin in which a benzhydrylamine-type resin is attached to the side chain carboxyl of Asp at position 28. The use of this glucagon(23-29)-resin with its different point of attachment of 2 0 ~

the resin provides the present process with some very practical advantages. More particularly, it has been realized that with the use of the peptide-resin of formula 2, the subsequent couplings of fragments and amino-acid residues according to the present process are completed rapidly and effectively with as little as 1.5 molar equivalents of activated ester or symmetrical anhydride (no double coupling required). In practice, the glucagon(23-29) resin, prepared by SPPS from N-(9-fluorenylmethyl-oxycarbonyl)-Asp(BHA resin)-Thr(Bzl)-OBzl, has been found to be a good choice of starting material.

With reference to the process, the two fragments of formula 1 and 4 are coupled successively and in proper order with the glucagon(23-29)-resin of formula 2, via the glucagon(15-29)-resin of formula 3, to yield the glucagon(7-29)-resin of formula 5. The above noted coupling techniques are employed effectively and smoothly.

Thereafter, the glucagon(7-29)-resin of formula 5 is coupled stepwise, and in the order of the amino acid sequence of glucagon, with the remaining amino acid residues, using the coupling conditions described hereinbefore, to yield the glucagon(1-29)-resin of formula 6 in which X is hydrogen.

Subsequent deprotection of the latter peptide resin, followed by standard purification techniques, yields pure glucagon. The deprotection can be achieved with hydrogen fluoride, preferably in the presence of p-cresol 2~24~5~

and p-thiocresol in dimethyl sulfide, which simultaneously removes the side chain protective groups and cleaves the peptide residue from the resin. An efficient method of deprotection (using HF) of the latter peptide resin is the low high procedure described by S. Mojsov and R.B.
Merrifield, Eur. J. Biochem., 145, 601 (1984).

The following examples illustrate further this invention. Abbreviations used in the examples include Boc: t-butyloxycarbonyli But:t-butyl;
CH2Cl2: methylene chloride; DCC: N,N/-dicyclo-hexylcarbodiimide; DIEA: diisopropylethylamine;
DMF: dimethylformamide; Et2O:diethyl ether;
EtOAc: ethyl acetate; EtOH: ethanol; Fmoc: 9-fluorenylmethyloxycarbonyl; HOBT: 1-hydroxybenzotriazole; MeOH: methanol and TFA:
trifluoroacetic acid. Solution percentages are calculated on a volume/volume basis unless stated otherwise. Temperatures refer to the centigrade scale. The following terms are trademarks: Pyrex and Vydac.

Example 1 Preparation of4-(2-chloropropionyl)phenoxyacetyl BHA-resin 4-(2-Chloropropionyl)phenoxyacetic acid (8.35 g, 34.5 mmoles) and HOBT (4.66 g, 34.5 mmoles) were dissolved separately in DMF
(2 x 40 ml). The two solutions were mixed and the resulting mixture was cooled at 0 ~ for 20 min. A solution of DCC in CH2Cl2 (27.5 ml, 1.256 mmoles/ml) was added to the solution. The ~ mixture of activated acid was stirred for 30 min.at 0 ~. The free base of benzhydrylamine copoly (styrene-1% divinylbenzene) resin (200-400 mesh, 202~5 50.0 g, amine content = 0.46 mmole/g) was generated with DIEA in CH2Cl2. The resulting resin was stirred in CH2C12 (900 ml). The above noted mixture of activated acid was added in one portion to the stirred resin. The resulting mixture was stirred for 20 h at room temperature.
The resin was collected by filtration, washed with DMF (3X), MeOH (3X), CH2Cl2 (3X), EtOH (3X) and finally dried to constant weight in a vacuum oven to yield 54.3 g of resin. The Kaiser test, E. Kaiser et al., Anal. Biochem., 34, 595 (1970), was negative indicating no starting material.

Example 2 BOC-AMINO-ACID RESINS:

a) Preparation of 4-[2-(Boc-phenylalanyl)-propionyl]phenoxyacetyl BHA-resin 4-[2-Chloropropionyl)phenoxyacetyl]-BHA resin of Example 1 (80.0 g, amine content: 0.70 mmole/g of resin) was stirred in dry DMF (1600 ml) for 10 min to allow the resin to swelli then potassium fluoride (29.23 g) and Boc-Phe-OH (59.4 g) were added in portions to the mixture. The reaction mixture was stirred for 24 h at 60 ~ and then filtered. The collected resin was washed three times each with DMF, H2O, dioxane, MeOH, CH2C12 and EtOH. The resin was dried to constant weight in a vacuum oven to afford free-flowing granules (90.0 g).

Titration with the picric acid indicated an amino content of 0.47 mmoles/g for phenylglycine.

202485~

b) P r e P a r a t i o n o f 4 - [ 2 - ( B o c -leucyl)propionyl]phenoxyacetyl BHA-resin The title compound was obtained by following the procedure of example 2a and replacing Boc-Phe-OH with Boc-Leu-OH.

Example 3 PROTECTED FRAGMENTS:
a) Preparation of protected qlucaqon(15-22) fraqment:

The Boc-amino acid resin of Example 2a (15.0 g, amine content: 0.47 mmole/g) was used to form the glucagon(15-22) fragment attached to the resin by a modification of the solid phase techniques of R.B. Merrifield, J. Amer. Chem.
Soc., 85, 2149 (1963). The selected Boc-amino acids were added to the growing peptidyl-resin chain by the DCC-HOBT activated acid method which comprised adding DCC (2 equiv.) in CH2C12 to a cold solution of HOBT (2 equiv.) and the selected Boc-amino acid (2 equiv.) in DMF, stirring the mixture at 0 ~ for 30 min., and adding the mixture to a suspension of the growing peptidyl resin in CH2C12. The coupling protocol consisted of i) deprotection with 45% TFA in CH2Cl2 (twice for 5 min, once for 25 min) (ii) neutralization with 5% DIEA in CH2C12 (twice for 3 min) and (iii) coupling by the DCC-HOBT or activated ester method. Intermediate washes were done successively with CH2C12, 50% isopropanol in CH2Cl2, isopropanol and CH2Cl2. The coupling reactions were monitored by the Kaiser test supra, and the fluorescamine test, see A.M. Felix and M.H. Jimenez, Anal. Biochem., 52, 377 (1973).
The time required to complete the coupling 2024~
. .

reaction ranged from 4 to 24 h. The final product was washed with DMF, CH2C12, isopropanol, CH2Cl2 and EtOH, and then dried under vacuum to give 25 g (86% yield) of the peptidylresin 4-[2-(Boc-Asp(OChl)-Ser(Bzl)-Arg(Tos)-Arg(Tos)-Ala-Gln-Asp(OChl)-Phe)propionyl]phenoxyacetyl BHA-resin.

The latter peptidylresin was subjected to photolysis as follows: The peptidylresin (25g) was suspended in a mixture of DMF (7.5 l) and EtOH (3.9 l) in a Pyrex vessel. The suspension was purged with argon. While being subjected to a continuous stream of argon, the suspension was stirred and irradiated at 0 ~ at a wavelength of 350 nm for 70 h. The suspension was filtered.
The filtrate was concentrated to dryness under reduced pressure at room temperature. The residual oil was triturated with anhydrous Et2O
to give a white solid. The solid was collected, washed with anhydrous Et2O and dried under vacuum over P2O5 to give 10 g of the corresponding protected N-terminal, free C-terminal carboxyl segment, Boc-Asp(Chl)-Ser(Bzl)-Arg(Tos)-Arg(Tos)-Ala-Gln-Asp(OChl)-Phe-OH (100% yield, 98.7% pure by HPLC).

b) Preparation of protected qlucaqon(7-14) fraqment The peptidylresin, 4-[2-(Boc-Thr(Bzl)-Ser(Bzl)-Asp(OChl)-Tyr(Cl2Bzl)-Ser(Bzl)-Lys(ClZ)-Tyr(Cl2Bzl)-Leu)propionyl]phenoxyacetyl BHA-resin (28.3 g), was obtained in a 95% yield in the same manner as described for the previous peptidylresin using the Boc-amino acid resin of Example 2b (15g, amine content: 0.49 mmole/g).

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Subsequent photolysis of the instant peptidyl resin (28.3 g) in the same manner afforded 11.0 g of the corresponding protected N-terminal, free C-terminal carboxyl segment, Boc-Thr(Bzl)-Ser(Bzl)-Asp(OChl)-Tyr(Cl2Bzl)-Ser(Bzl)-Lys(ClZ)-Tyr(Cl2Bzl)-Leu-OH (82% yield, 97.5% pure by HPLC).

c) Preparation of protected qlucaqon(28-29) fraqment i) H-Thr(OBzl)-OBzl: A mixture of H-Thr-OH
(90.5 g), p-toluenesulfonic acid monohydrate (188 g), benzyl alcohol (760 ml) and toluene (1520 ml) was heated at reflux temperature (with azeotropic distillation of H2O) for 19 h. The reaction mixture was cooled and diluted with EtOAc (1000 ml). Aqueous Na2CO3 (0.5 N) was added until the pH of the mixture was 9. The phases were separated. The organic phase was washed with fresh aqueous Na2CO3 and water and dried (MgSO4). The addition of oxalic acid (92 g) in MeOH (200 ml) to the organic phase gave a precipitate. After the mixture had been stored at 4 for 24 h, the precipitate was collected and washed with hexane and EtOH.
Recrystallization of the precipitate from EtOH
gave H-Thr(Bzl)-OBzl as a hemioxalate salt (40 g, mp 163-164 ~).

ii) Fmoc-Asp(OBu )-Thr(Bzl)-OBzl: DDC (16.09 g) in EtOAc (100 ml) was added to a suspension of Fmoc-Asp(OBut)-OH (32.1 g) and N-hydroxysuccinimide (9.35 g) in EtOAc at O . The mixture was stirred at 0 ~ for 1.5 h and then at 20-22 ~ for 3.5 h. H-Thr(Bzl)-OBzl (obtained from 33.4 g, of its corresponding hemioxalate salt) in EtOAc 20~48~S

(100 ml) was added dropwise to the mixture at 0 ~. The resultant mixture was stirred 18 h at room temperature (20 - 22 ~) and then filtered.
The filtrate was concentrated to dryness. The oily residue (80 g) was placed on a silica gel column (25 cm x 9 cm, 650 g of SiOz). The column was eluted at 2 psi with EtOH-hexane (1:3).
Fraction size was 400 ml. Fractions 6 to 23 were pooled and concentrated. Addition of hexane to the concentrate precipitated the title compound (41.2 g, mp 83-85 ~).

iii) Fmoc-Asp-Thr(Bzl)-OBzl: Fmoc-Asp(Bu )-Thr(Bzl)-OBzl (200 mg) was added to 25% TFA in CH2Cl2 (10 ml) at 0~. The stirred mixture was allowed to come to room temperature over a period of 0.5 h. The mixture was evaporated to dryness.
the solid was washed with hexane and dried to give the title compound (150 mg, mp 90-95 ~).

Example 4 Preparation of Glucaqon(1-29)-resin By following the procedure of Example 3, the title compound was prepared. Namely, Fmoc-Asp-Thr(Bzl)-OBzl of Example 3c (1.6 g) was coupled with BHA resin (2.0 g) by the DCC-HOBT method.
The resulting peptide-resin was subjected to deprotection (removal of Fmoc) in 45% TFA in CH2Cl2; followed by coupling the resulting H-Asp-(BHA resin)-Thr(Bzl)-OBzl with the appropriate Boc-amino acids by the DCC-HOBT method. The resulting glucagon(23-29)-resin, i.e. H-Val-Gln-Trp(For)-Leu-Met(O)-Asp(BHA resin)-Thr(Bzl)-OBzl, was coupled serially (DCC-HOBT method with the protected glucagon(15-22) fragment of Example 3a and the protected glucagon(7-14) fragment of 2024~

Example 3b. Thereafter, the resulting protected glucagon(7-29)-resin was coupled stepwise with the appropriate Boc-amino acids [DCC-HOBT method, except for Gly (Boc-Gly-OH) and His (Boc-His(Tos)-OH) which were coupled by the DCC
symmetrical anhydride method].

The symmetrical anhydride method comprised adding DCC (2 equiv.) in CH2Cl2 to the selected Boc-amino acid (4 equiv.) in CH2Cl2 at 0 stirring the mixture at 0 ~ for 30 min, filtering the mixture and adding the filtrate to a suspension of the peptidyl resin in CH2Cl2.
Accordingly, the protected glucagon(1-29)-resin, i.e. H-His(Tos)-Ser(Bzl)-Gln-Gly-Thr(Bzl)-Phe-Thr(Bzl)-Ser(Bzl)-Asp(OChl)-Tyr(Cl2Bzl)-Ser(Bzl)-Lys (ClZ) -Tyr (Cl2Bzl)-Leu-Asp (OChl) -Ser (Bzl) -Arg(Tos)-Arg(Tos)-Ala-Gln-Asp(OChl)-Phe-Val-Gln-Trp(For)-Leu-Met(O)-Asp(BHA resin)-Thr(Bzl)-OBzl (7.10 g, 84% yield), was obtained.

Example 5 Preparation of Glucaqon The preceding protected glucagon(1-29)-resin (16.3 g) was mixed with p-cresol (6.0 g) and p-thiocresol (2.0 g) in dimethylsulfide (56 ml).
The mixture was placed under nitrogen and cooled to -70 ~. Anhydrous hydrogen fluoride (20 ml) was distilled into the mixture. The mixture was stirred at 0 ~ for 2 h under nitrogen, and then concentrated under reduced pressure at 0 ~. The residue was washed with EtOAc and dried. The resulting solid was mixed with p-cresol (12 g).
Anhydrous hydrogen fluoride (120 ml) was distilled into the mixture at -70 ~. Thereafter, the mixture was stirred at 0 ~ for 45 min. The 2~ 5 ~
mixture was concentrated to dryness under reduced pressure at 0~. The residue was washed with EtOAc and then extracted with 5% aqueous acetic acid. The extract was lyophized to give s 2.91g (59% yield) of the crude product.

The crude product (1.4 g) was purified by reversed-phase chromatography on a Pharmacia*
octadecasilyl-silica column (2.5 x 35 cm, C-18, Vydac*, 10~ particle size) using a gradient of 0.06% TFA in H2O and 0.06% TFA in H2O: -acetonitrile (1:1). The fractions comprising the major peptide peak (W detection at 254 nm) were pooled and freeze dried to give 0.49 g lS (26% overall yield starting from the BHA resin) of the title compound with a purity of greater than 98% as indicated by analytical HPLC. The hyperglycemic potency of the glucagon so obtained was evaluated in rabbits according to procedure described in the British Pharmacopocia Appendix XIV, A-142 (1980). The glucagon prepared by the present process was found to be equipotent to the glucagon standard.

* Trademark .

Claims (4)

1. A glucagon (23-29)-resin of the formula:

H-Val-Gln-Trp(W4)-Leu-Met(O)-Asp(Q)-Thr(W5)-O-Y

wherein W4 is a protective group for the aromatic nitrogen atom of Trp, W5 is a protective group for the hydroxyl of Thr, Q is a benzhydrylamine type resin and Y is a carboxyl protective group.

2. A glucagon (23-29)-resin of claim 1 wherein W4 is formyl, W5 is benzyl, Q is a benzhydrylamine type resin and Y is benzyl.

3. A glucagon (23-29)-resin of claim 1 or 2 wherein Q is a benzhydrylamine resin or a

4-methylbenzhydrylamine resin.