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A new experimental approach has been developed to study the distribution of local electrostatic potential around specific protons in biologically important molecules. The approach is the development of a method denoted as "spin label/spin probe," which was proposed by one of us (. Mol. Biol. 6:498-507). The proposed method is based upon the quantitative measurement of the contribution of differently charged nitroxide probes to the spin lattice relaxation rate (1/T1) of protons in the molecule of interest, followed by calculation of local electrostatic potential using the classical Debye equation. In parallel, the theoretical calculation of potential distribution with the use of the MacSpartan Plus 1.0 program has been performed. Application of the method to solutions of simple organic molecules (aliphatic and aromatic alcohols, aliphatic carboxylates (propionate anion), and protonated ethyl amine and imidazole) allowed us to estimate the effective potential around the molecules under investigation. These were found to be in good agreement with theoretically expected values. This technique was then applied to zwitterionic amino acids bearing neutral and charged side chains (glycine, lysine, histidine, and aspartic acid). The reliability of the general approach is proved by the data presented in this paper. Application of this new methodology can afford insight into the biochemical significance of electrostatic effects in biological systems.

The conformational preferences of N-methyl derivatives of the dopamine reuptake blocker threo-methylphenidate [Ritalin] and the p-methyl analogue were determined in the solid state and in solution and that of the erythro isomer in solution. The solid-state structures of (±)-threo-N-methyl-α-phenyl-2-piperidineacetic acid methyl ester hydrochloride [(±)-threo-N-methyl-methylphenidate hydrochloride] (2) and (±)-threo-N,p-dimethyl-α-phenyl-2-piperidineacetic acid methyl ester hydrochloride (5) were determined by single crystal X-ray diffraction analysis. (±)-2 underwent spontaneous resolution to give crystalline chiral plates containing two independent molecules in the asymmetric ring, and at each site there is a disorder involving the N-methylpiperidinyl ring methylene and methyl carbon atoms with a 0.710(7):0.290(7) ratio of occupancy factors. The two (2RS,3RS,4SR) major disordered molecules have similar structures consisting of a chair conformation for the piperidine ring with axial N-methyl and CH(Ph)COOMe groups. The two (2RS,3RS,4RS) minor molecules in the disorder also have similar structures and differ from the major ones by epimerization at nitrogen and inversion of the piperidine ring to afford an axial N-methyl group, and an equatorial CH(Ph)COOMe group. (±)-5 gave crystalline plates also containing diaxially disposed piperidinyl-ring substituents. Dissolution in D2O of either 2 or its erythro-epimer (3) each gives a 5:4 ratio of two species in which the major species exhibits an axial N-methyl group and an equatorial CH(Ph)COOMe group while the minor species has a diequatorial arrangement for both substituents. Both of the axial N-methyl threo or erythro major species in D2O are overwhelmingly conformationally biased in favor of an antiperiplanar H(2)···H(3) disposition and one piperidine ring invertomer. Dissolution of the threo or erythro epimers in CD2Cl2 gives the same axial N-methyl/equatorial CH(Ph)COOMe and diequatorially disposed species but now in a reversed ratio [respectively 3:20 for threo and 4:5 for erythro].

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