Synthesis of thymosin alpha 1 by fragment condensation using tert.-butyl side chain protection.
A novel synthesis of thymosin alpha 1 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 alpha 1 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, 1H-n.m.r. spectroscopy, FAB mass spectroscopy and peptide mapping after tryptic digestion confirmed the structure of thymosin alpha 1. Three minor stereoisomer contaminants were isolated by HPLC and characterized as [D-Lys14]-thymosin alpha 1, [D-Lys17]-thymosin alpha 1 and [D-Ala3]-thymosin alpha 1 resulting from racemization at Lys14, Lys17 and Ala3 during the coupling of the fragments. A final contaminant, isolated by HPLC, was characterized as N alpha-isobutyloxycarbonyl-thymosin alpha 1 (15-28), which results from "wrong way opening" of an activated mixed anhydride.
D000595:Amino Acid Sequence; D007202:Indicators and Reagents; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D009904:Optical Rotation; D010446:Peptide Fragments / Q000138:chemical synthesis; D000077596:Thymalfasin; D013947:Thymosin / Q000031:analogs & derivatives* / Q000138:chemical synthesis
D007202:Indicators and Reagents; D010446:Peptide Fragments; D013947:Thymosin; D000077596:Thymalfasin