Studies in Peptide Synthesis

The preparation of polypeptide and protein analogues by conventional or fragment condensation synthesis is often a complex and time-consuming procedure. The combination of protected natural peptide fragments, isolated by enzymatic cleavage, and synthetic peptides could give these analogues more rapidly and conveniently than before. A semisynthetic approach is therefore an alternative approach for effective introduction of amino acid substitutions in enzymes and proteins. Insulin offers an excellent opportunity in which this approach can be investigated as tryptic cleavage removes the B-chain C-terminal octapeptide to give des-octapeptide insulin. This thesis reports the synthesis of protected B-chain C-terminal peptides that could be coupled to a similarly protected des-octapeptide insulin. Complete deprotection would then yield a semisynthetic insulin.
The intended use of acid labile groups such as t-butoxycarbonyl and benzhydryl esters for the final protected fragments imposed severe restrictions on planning and synthesising the protected fragments. The syntheses were therefore devised such that fully protected peptides could be prepared by conventional strategy and then converted to a much milder protected form suitable for coupling.
The 4-picolyl ester method [R. Gamble, R. Garner and G. T. Young, Nature, Lond. 217, 247 (1967)] was used to synthesise the fully protected natural octapeptide (1) using the t-butoxycarbonyl group for α-amino protection, except for the ultimate glycine residue which was incorporated as its benzyloxycarbonyl derivative, piperidino-oxycarbonyl for ε-amino lysine protection and benzyl ethers for threonine and tyrosine hydroxyl group protection. The use of these groups was such that by a series of deprotection-reprotection steps the mildly protected derivative (2) could be prepared and this would be in a form ready for coupling. The synthetic route for the preparation of (1) is outlined in Figure 1. The combination of N, N'-dicyclohexylcarbodi-imide and 1-hydroxybenzotriazole for coupling reactions was employed throughout the synthesis due to its advantages with regard to speed, simplicity of use and absence of racemisation [W. König and R. Geiger, Chem.Ber. 103, 2034 (1970)].
The piperidino-oxycarbonyl group had been proposed for α- and ε-protection by Stevenson [D. Stevenson and G, T. Young, J.Chem.Soc. (C), 2389 (1969)] and was introduced by either piperidino succinimido carbonate or piperidino 2,4,5-trichlorophenyl carbonate. Nash [P. P. Nash, "Synthetic Studies Related to Peptides from Bee Venom". D.Phil. Thesis, Oxford 1973] found the latter preferable and obtained α-benzyloxycarbonyl-ε-piperidino-oxycarbonyl-L-Iysine which was converted to α-t-butoxycarbonyl-ε-piperidino-oxycarbonyl-L-lysine by treatment with hydrogen bromide in acetic acid and the t-butoxycarbonyl azide. This method proved unsatisfactory because of low yields and impurities thought to be L-lysine and ε-benzyloxycarbonyl-L-lysine arising at each stage. A new synthetic route was proposed as in Figure 2. Treatment of ε-benzyloxycarbonyl-L-lysine with t-butoxycarbonyl azide gave α-t-butoxycarbonyl-ε-benzyloxycarbonyl-L-lysine in 91% yield. Hydrogenation in 80% aqueous ethanol gave α-t-butoxycarbonyl-L-lysine in 83.2% yield which was reacted with piperidino 2,4,5-trichlorophenyl carbonate to give α-t-butoxycarbonyl-ε-piperidino-oxycarbonyl-L-lysine, which was isolated as its dicyclohexylammonium salt in 68% yield.
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