Abstract
Nanoelectrospray ionization mass spectrometry (nanoESI‐MS) and computer simulation were applied to the characterization of non‐covalent interactions of [Leu^5^]‐enkephalin (LE) and its optical isomers, [D‐Tyr^1^, Leu^5^]‐enkephalin (Y‐LE), [D‐Phe^4^, Leu^5^]‐enkephalin (F‐LE) and [D‐Tyr^1^, D‐Phe^4^, Leu^5^]‐enkephalin (YF‐LE). The dimer formation tendencies of the optical isomers of LE were evaluated by nanoESI‐MS using quadruply deuterated LE (H~2~N‐Tyr‐(2,2‐d~2~)Gly‐(2,2‐d~2~)Gly‐Phe‐Leu‐COOH, d~4~‐LE) as an internal standard. The relative interaction strengths of the optical isomers of LE were estimated to be Y‐LE < F‐LE < LE < YF‐LE. Geometry optimization calculations were performed for interactions in vacuo and in water using a semi‐empirical SCF method (PM3). The initial coordinate of the dimer structure of LE was taken from that obtained from single‐crystalline x‐ray diffraction analysis. Estimates of the interaction strengths of the dimer complexes were based on the heats of formation of a dimer complex (H~d~) and the corresponding monomers (H~m~) using the equation Δ__H__ = H~d~ − 2__H__~m~. The values of Δ__H__ obtained from the calculations for interactions in water decreased in the order Y‐LE > F‐LE > LE > YF‐LE. Since the smaller values of Δ__H__ correspond to stronger interactions between peptides, the results from computer simulations were qualitatively consistent with those obtained from the nanoESI experiments. The possibility of cross‐checking these independent techniques was demonstrated using medium‐sized molecules of biological importance. The agreement of the results from the two techniques suggested that nanoESI experiments, at least qualitatively, reflected the relative interaction strengths of non‐covalently bound enkephalins in aqueous solution. Copyright © 2001 John Wiley & Sons, Ltd.