STUDIES IN C-TERMINAL SEQUENCING - NEW REAGENTS FOR THE SYNTHESIS OF PEPTIDYLTHIOHYDANTOINS

In previous studies aimed at the sequencing of peptides and proteins f rom the carboxy terminus, we have derivatized the C-terminus to a thio hydantoin using acetic anhydride and trimethylsilylisothiocyanate (TMS -ITC) and subsequently hydrolyzed it to form a shortened peptide capab le of further degradation and an amino acid thiohydantoin which can be identified by reverse-phase HPLC. Current limitations to this chemist ry include an inability to derivatize proline and low yields with aspa ragine and aspartic acid residues (Bailey et al., 1992). In an attempt to solve some of these problems, we have investigated the use of reag ents other than acetic anhydride for the activation of the C-terminal carboxylic acid. These include 2-fluoro-1-methylpyridinium tosylate, 2 -chloro-1-methylpyridinium iodide, and acetyl chloride. Addition of TM S-ITC to peptides activated by the 2-halo-pyridinium salts formed the expected peptidylthiohydantoin, but in addition formed a peptide chemi cally modified at the C-terminus which was blocked to C-terminal seque nce analysis. This derivative was not obtained when either acetic anhy dride or acetyl chloride was used for activation. Formation of this de rivative was found to require the presence of an isothiocyanate reagen t in addition to the halo-pyridinium salt. Sodium thiocyanate, TMS-ITC , and a new reagent for thiohydantoin synthesis, tributyltinisothiocya nate (TBSn-ITC), were all found to be capable of forming this analogue . Structural elucidation of the C-terminally modified amino acid revea led it to be a 2-imino-pyridinium analogue. Formation of this C-termin ally blocked peptide could be minimized by the use of the 2-chloro-pyr idinium reagent, rather than the 2-fluoro reagent, and by performing t he reaction at a temperature of 50-degrees-C or lower. The 2-halo-pyri dinium reagents offer potential advantages over the use of acetic anhy dride for activation of the C-terminal carboxylic acid. These include: milder reaction conditions, faster reaction times, and the ability to sequence through C-terminal aspartic acid. The TBSn-ITC reagent was f ound to be comparable to TMS-ITC for formation of peptidylthiohydantoi ns.