Endor Determined Structure and Conformation of Spin-Labeled Methyl L-Phenylalanate in Frozen Solutions

Abstract

The conformation of L-phenylalanine and methyl L-phenylalanate acylated at the amino nitrogen position with the nitroxyl spin-label 2,2,5,5-tetramethyl- l-oxypyrroline-3-carboxylic acid has been determined by ENDOR spectroscopy and computer based molecular modeling. ENDOR spectra of spin-labeled L- phenylalanine and the corresponding methyl ester of L-phenylalanine and of their 8-fluoro and £-fluoro derivatives in perdeuterated methanol or chloroform/toluene exhibited well resolved resonance absorptions from protons and fluorines of the amino acid moiety. From the analysis of the dependence of the ENDOR spectra on Hq, we have identified principal hyperfine coupling (hfc) components of each class of nuclei. Under the point-dipole approximation, the dipolar hfc components yielded estimates of electron- nucleus separations with less than 5% uncertainties based on ENDOR line widths. ENDOR determined distance constraints were then used to assign molecular conformations through torsion angle search calculations. These calculations were carried out for a molecular model constructed from X-ray defined molecular fragments. The resulting structure was close to the ideal g- rotamer (%1 ca. -75°) in methanol. In toluene/chloroform the value of Xi was ca. -100°. With the fluorine substituent in the ortho position of the phenyl side chain, two conformations were detected corresponding to values of +92° and -73° for %2 due to the rotation around the cβ-cY bond. The conformation of spin-labeled methyl L-phenylalanate in methanol differed only slightly from that in toluene/chloroform and was closely similar to the structure of spin-labeled L-phenylalanine. (Supported by GM 21900 and AA 06374).