Benzyloxycarbonylprolylprolinal, a transition-state analogue for prolyl oligopeptidase, forms a tetrahedral adduct with catalytic serine, not a reactive cysteine.

N-Benzyloxycarbonyl-l-prolyl-l-[1-13C]prolinal was synthesized starting with reduction of l-[1-13C]Pro to l-[1-13C]prolinol, followed by coupling with N-benzyloxycarbonyl-l-Pro to N-benzyloxycarbonyl-l-Pro-l-[1-13C]prolinol (Z-Pro-[1-13C]prolinol), and finally oxidation of the alcohol to the aldehyde with dimethyl sulphoxide. While the 13C NMR chemical shift of the aldehyde carbon is 202 p.p.m., that of the aldehyde hydrate is between 91.6 and 91.8 p.p.m., that of the dithiothreitol adduct is between 74.8 and 75.0 p. p.m., and that in the presence of the serine protease prolyl oligopeptidase is at 92.3 p.p.m.. The linewidth of the latter is 114 Hz, roughly consistent with the molecular mass of 80 kDa reported for the enzyme. Inverse detection experiments gave a 1H resonance at 5.29 p.p.m. with a linewidth of 80 Hz, also consistent with the expected chemical shift and linewidth for a hemiacetal bound to such a large enzyme, while the free hydrate gave resonances at 5.18 and 5. 25 p.p.m., with very much narrower linewidths. It is concluded that Z-Pro-prolinal, a putative transition-state analogue for prolyl oligopeptidase, forms a tetrahedral complex with the enzyme at its catalytic serine, rather than at a neighbouring cysteine that was found to be highly reactive according to chemical modification studies.

Solution structure of vasoactive intestinal polypeptide (11-28)-NH2, a fragment with analgesic properties.

An 18-residue-long fragment of vasoactive intestinal polypeptide [VIP(11-28)-NH2] that is known to be analgesic was synthesized by solid-phase t-Boc methodology on a 4-methylbenzhydrylamine resin. Circular dichroism spectroscopy gave evidence that the peptid acquires about 60% helical structure in 50/50 methanol/phosphate buffer, pH 6.0, and 65% (+/-5%) helicity in 80/20 methanol/phosphate buffer pH 7.0, A 2.0 mM solution of VIP (11-28) NH2 in 80% methanol, 20% phosphate buffer pH 7.0 was subjected to 2-dimensional nuclear magnetic resonance (NMR) studies The NMR results suggested formation of an extended helical structure extending from residue 11 to 27 essentially the same region found to be helical in a VIP(1-28)-NH2 and log. This finding suggests that the sequence required for analgesia assumes a helical structure at the receptor.