Oxazolone versus macrocycle structures for Leu-enkephalin b2-b4: insights from infrared multiple-photon dissociation spectroscopy and gas-phase hydrogen/deuterium exchange.

The collision-induced dissociation (CID) products b(2)-b(4) from Leu-enkephalin are examined with infrared multiple-photon dissociation (IR-MPD) spectroscopy and gas-phase hydrogen/deuterium exchange (HDX). Infrared spectroscopy reveals that b(2) exclusively adopts oxazolone structures, protonated at the N-terminus and at the oxazolone ring N, based on the presence and absence of diagnostic infrared vibrations. This is correlated with the presence of a single HDX rate. For the larger b(3) and b(4), the IR-MPD measurements display diagnostic bands compatible with a mixture of oxazolone and macrocycle structures. This result is supported by HDX experiments, which show a bimodal distribution in the HDX spectra and two distinct rates in the HDX kinetic fitting. The kinetic fitting of the HDX data is employed to derive the relative abundances of macrocycle and oxazolone structures for b(3) and b(4), using a procedure recently implemented by our group for a series of oligoglycine b fragments (Chen et al. J. Am. Chem. Soc.2009, 131(51), 18272-18282. doi: 10.1021/ja9030837). In analogy to that study, the results suggest that the relative abundance of the macrocycle structure increases as a function of b fragment size, going from 0% for b(2) to approximately 6% for b(3), and culminating in 31% for b(4). Nonetheless, there are also surprising differences between both studies, both in the exchange kinetics and the propensity in forming macrocycle structures. This indicates that the chemistry of "head-to-tail" cyclization depends on subtle differences in the sequence as well as the size of the b fragment.