ENDOR DETERMINED MOLECULAR GEOMETRIES OF SPIN-LABELED FLUOROANILIDES IN FROZEN SOLUTION
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With use of fluorine and proton electron nuclear double resonance (ENDOR) spectroscopy, we have determined the molecular geometries of randomly oriented 2-fluoro-, 3-fluoro-, and 4-fluoroanilide derivatives of the spin-label 2,2,5,5-tetramethylpyrroline-1-oxyl-3-carboxylic acid in frozen perdeuterated methanol. Under conditions of very low modulation depth (35 kHz) of the radiofrequency field to ensure that ENDOR line widths were not distorted, ENDOR resonance absorptions were observed specific for the protons and fluorine substituents of the anilide ring. The ENDOR shifts of each fluorine and proton substituent were shown to correspond to principal hyperfine coupling (hfc) components and to exhibit axial symmetry. The ENDOR results also demonstrated that for each fluoroanilide isomer there are two conformers of the anilide ring constrained by the nonbonded 1,6-syn periplanar interactions of an ortho proton or fluorine substituent with the carbonyl oxygen. The electron-nucleus separations were calculated from the dipolar hfc contributions according to the point-dipole, strong-field approximation. Assignments of the resonance frequencies of ENDOR features were confirmed on the basis of spectral simulations. To assess the accuracy of this method of deriving structural details from ENDOR spectra of molecules in frozen solutions, we have compared the ENDOR-determined electron-nucleus separations with those predicted through computer-based molecular graphics modeling of X-ray-derived atomic coordinates of the spin-label and fluoroanilide moieties covalently bonded through a planar amide group. With assignment of the effective dipole of the nitroxyl group to the midpoint of the N-O bond, there was less than a 5% discrepancy between the ENDOR-determined and molecular model results for electron-nucleus distances over a 5-11- range. The ENDOR and molecular modeling results demonstrated that the fluorines and protons of the anilide group are essentially coplanar with the pyrrolinyl ring, the maximum deviation of the electron-nucleus vector from the molecular plane of the spin-label being less than 21. This application of ENDOR spectroscopy to nitroxide spin-labels provides a basis for a general method to determine detailed molecular geometries of spin-labeled molecules in frozen solution. 1988, American Chemical Society. All rights reserved.