Amino acids and peptides. XVI. Synthesis of a tetrapeptide sequence (A9-A12) of glucagon.

A synthesis of the tetrapeptide sequence As-Ag of glucagon is described that employs various blocking groups, coupling procedures, and routes. The threonyl-phenylalanyl-threonyl-seryl sequence A5-A8 in the hyperglycemic hormone, glucagon (Foa and Galansino, 1962), has been prepared by three different groups within the past four years (Beyerman and Bontekoe, 1964; Schroder and Gibian, 1962; Wunsch and Wendleberger, 1964). In connection with our earlier work on protected glucagon fragments (Costopanagiotis, Handford, and Weinstein, 1968), there are given here some new approaches to the dipeptides N-benzyloxycarbonyl-L-threonyl-L-phenylalanine methyl ester (I) and N-benzyloxycarbonyl-L-threonyl-L-serine methyl ester (II), as well as the tetrapeptide Nbenzyloxycarbonyl-L-threonyl-L-phenylalanyl-L-threonyl-L-serine methyl ester (III). N-Benzyloxycarbonyl-L-threonine (IV) was reacted with L-phenylalanine methyl ester (V) with the aid of either N,N'-dicyclohexylcarbodiimide (Sheehan and Hess, 1955) or 2-ethyl-5-phenylisoxazolium-3'-sulfonate (Woodward, Olofson, and Mayer, 1961) to afford the dipeptide I. Carbodiimide is favored in this case, as the isoxazolium agent provided an impure, gummy product, which required extensive recrystallization. An attempt to make N-benzyloxycarbonyl-L-threonine 2,4,5-trichlorophenyl ester (VI) yielded an unstable oil. If isolated, this particular compound would have been a valuable addition to the list of 2,4,5-trichlorophenyl esters (Pless and Boissonnas, 1963) that have been widely employed in recent years to facilitate the construction of peptides (Schroder and Lubke, 1964). A similar effort to obtain N-benzyloxycarbonyl-L-threonine ^-nitrophenyl ester (VII) gave only a minor amount of the activated ester (Bodansky and Ondetti, 1966), while the alternative 2,4-dinitrophenyl ester (VIII) is apparently easily available by the same procedure (Rocchi, Marchiori, and Scoffone, 1963). No report has appeared to date on the preparation of N-benzyloxycarbonyl-L-threonine pentachlorophenyl ester (IX). The coupling of compound IV and L-serine methyl ester (X) with either N,N'-dicyclohexyl carbodiimide or 2-ethyl-5-phenylisoxazolium-3'-sulfonate produced dipeptide II. Here the isoxazolium salt gave the superior yield, in contrast with the observations described earlier for dipeptide I. The disparity may be due to a combination of both solvent (dichloromethane vs. acetonitrile) and temperature factors (0° vs. 10°C). Treatment of dipeptide I with aqueous alkali or hydrazine furnished NManuscript received June 1, 1967. THE OHIO JOURNAL OF SCIENCE 68(2): 104, March, 1968. No. 2 AMINO ACIDS AND PEPTIDES 105 benzyloxycarbonyl-L-threonyl-L-phenylalanine (XI) and N-benzyloxycarbonylL-threonyl-L-phenylalanine hydrazide (XII), respectively. An azide coupling between hydrazide XII and L-threonyl-L-serine methyl ester (XIII), modified by the use of /-butyl nitrite with dimethylformamide as the solvent (Honz and Rudlinger, 1961; Schroder, 1964; Mazur and Schlatter, 1964), formed the desired tetrapeptide III. The same reaction, but with the use of sodium nitrite and hydrochloric acid-acetic acid (Greenstein and Winitz, 1961), gave tetrapeptide III in an equivalent amount. Alternatively, an azide coupling between hydrazide XII and 1-hydroxypiperidine (Handford, Jones, Young, and Johnson, 1965) afforded N-benzyloxycarbonyl-L-threonyl-L-serine 1-piperidyl ester (XIV). If the mineral acid was replaced by acetic acid, citric acid, or formic acid, no reaction occurred between hydrazide XII and 1-hydroxypiperidine. The activated ester XIV, on treatment with the amine XIII in the presence of one equivalent of acetic acid, yielded the tetrapeptide III. However, this coupling went only very slowly, and the resultant low yield may well have been caused by appreciable cyclization of the amine XIII to the corresponding diketopiperazine (XV). Finally, treatment of tetrapeptide III with hydrazine afforded N-benzyloxycarbonyl-L-threonylL-phenylalanyl-L-threonyl-L-serine hydrazide (XVI).