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 HIV integrase by standard Fmoc-polyamide chemistry was unsuccessful (Trt-protected Cys) or difficult (Acm-protected Cys); after 10-12 couplings, deprotection became hindered and a major portion (30%-50%) of the growing peptide chains became inaccessible to reagents. The synthesis was repeated using N-(2-hydroxy-4-methoxybenzyl) (Hmb) backbone protection at two strategic positions in the sequence, and Acm protection of Cys. The synthesis was trouble-free and progressed smoothly to completion. Electrospray mass spectrometric analysis at each step of the cleavage and purification showed that Hmb groups become O-acetylated during treatment of the peptide with acetic anhydride in the presence of tertiary base. The acetylated Hmb was refractory to cleavage with TFA/scavenger mixtures but could be readily removed following deacetylation with piperidine. The target peptide was purified to > 98% purity and an overall yield of 15%.
Packman
L C
LC
Cambridge Centre for Molecular Recognition, University of Cambridge, UK.
Quibell
M
M
Johnson
T
T
D000595:Amino Acid Sequence; D000840:Anisoles / Q000737:chemistry*; D004254:DNA Nucleotidyltransferases / Q000138:chemical synthesis*; D006678:HIV / Q000201:enzymology*; D019426:Integrases; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000138:chemical synthesis*
D000840:Anisoles; D010446:Peptide Fragments; D004254:DNA Nucleotidyltransferases; D019426:Integrases