Engineering an unnatural Nα‐anchored disulfide into BPTI by total chemical synthesis: structural and functional consequences

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Abstract
A disulfide-engineered analogue of bovine pancreatic trypsin inhibitor (BPTI), ((Nα-(CH2)2S-)Gly38)BPTI, has been prepared using a thioester-mediated auxiliary functional group chemical ligation of a Nα-ethanethiol-containing peptide segment with a peptide-αCOSR segment. In this study, Nα-(ethanethiol)Gly38 replaces the native Cys38, providing the sulfhydryl group required for ligation and folding. Comparisons between ((Nα-(CH2)2SH)Gly38)BPTI, synthetic native BPTI and reference BPTI purchased from Sigma were made using mass spectroscopy, enzyme inhibitor association constant determination (Ka) and 1H-nuclear magnetic resonance total correlated spectroscopy (1H-NMR TOCSY) measurements. The Ka value for ((Nα-(CH2)2SH)Gly38)BPTI was approximately 20-fold lower than synthetic and reference BPTI, which was attributed to perturbations in the binding loop of the protein (near Cys14). This hypothesis was confirmed by two-dimensional (2D) 1H-NMR TOCSY experiments. The data reported here demonstrate that total chemical synthesis by auxiliary functional group chemical ligation is a practical method for the synthesis of a novel class of biologically active protein analogues containing additional functional groups linked to the protein backbone