ROLES OF ELECTROSPRAY MASS-SPECTROMETRY, COUNTERION DISTRIBUTION MONITORING AND N-(2-HYDROXY-4-METHOXYBENZYL) BACKBONE PROTECTION IN PEPTIDE-SYNTHESIS

The synthesis of a 36-residue fragment of HN integrase by standard Fmo c-polyamide chemistry was unsuccessful (Trt-protected Cys) or difficul t (Acm-pro tected Cys); after 10-12 couplings, deprotection became hin dered and a major portion (30%-50%) of the growing peptide chains beca me inaccessible to reagents. The synthesis was repented using N-(2-hyd roxy-4-methoxybenzyl) (Hmb) backbone protection at two strategic posit ions in the sequence, and Acm protection of Cys. The synthesis was tro uble-free and progressed smoothly to completion. Electrospray mass spe ctrometric analysis at each step of the cleavage and purification show ed that Hmb groups become O-acetylated during treatment of the peptide with acetic anhydride in the presence of tertiary base. The acetylate d Hmb was refractory to cleavage with TFA/scavenger mixtures bur could be readily removed following deacetylation with piperidine. The targe t peptide was purified to >98% purity and an overall yield of 15%.