Synthesis of thymosin α1 by fragment condensation using tert.-butyl side chain protection

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Abstract
A novel synthesis of thymosin α1 by classical methods using seven tert. -butyl side chain protected fragments is described. Optimum conditions were found for the final DCC/HOBt coupling of the two key intermediates; decapeptide and octadecapeptide. Thymosin α1 was purified by two stages of preparative HPLC (partial purification with C8 and final purification with C18 reverse phase silica gel) to give a 30% overall yield for the final four stages of synthesis (including catalytic hydrogenation of octadecapeptide, coupling, deprotection and purification). The product was shown to be homogeneous by thin-layer and paper high voltage electrophoresis, isoelectric focusing analysis, thin-layer chromatography and high performance liquid chromatography. Amino acid analysis, optical rotation, 1 H-n.m.r. spectroscopy, FAB mass spectroscopy and peptide mapping after tryptic digestion confirmed the structure of thymosin α1. Three minor stereoisomer contaminants were isolated by HPLC and characterized as [D-Lys14]-thymosin α1, [D-Lys17]-thymosin α1 and [D-Ala3]-thymosin α1 resulting from racemization at Lys14, Lys17 and Ala3 during the coupling of the fragments. A final contaminant, isolated by HPLC, was characterized as Nα-isobutyloxycarbonyl-thymosin α1 (15–28), which results from “wrong way opening” of an activated mixed anhydride.

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