Benzoyl and hydroxybenzoyl esters of coenzyme A. Purification and nuclear magnetic resonance characterization; conformation in solution.

Abstract Benzoyl-CoA and o-, m-, and p-hydroxybenzoyl-CoA were synthesized by reacting the benzoyl chloride (formed from the respective benzoic acid and oxalyl chloride) with reduced CoA. The products were purified by chromatography (Sephadex LH 20, ethanol-water, 40:60 (v/v) and treatment with Chelex. Purity was confirmed in several ways: (a) a single ultraviolet-absorbing spot with a unique Rf value was observed for each of the CoA esters in each of several thin layer chromatographic solvent systems; (b) elemental stoichiometry (C, N, P, S, Li, H) agreed with prediction; (c) the values of ultraviolet extinction coefficients (emm) obtained by dry weight, enzymic release of thiol, and comparison of additive spectra of CoA and the respective benzoate with esterolysis spectra of the benzoylCoA all agreed (see accompanying article (18)); (d) the proton magnetic resonance (PMR) spectra were consistent with the structures of the benzoyl-CoAs and contained no extraneous signals. All of the signals in the PMR spectra of CoA, of acetyl-CoA and of the benzoyl-CoAs were assigned to specific moieties of the respective molecules. A downfield displacement of the CH2-S signal relative to its position in the spectrum of reduced CoA is characteristic of substitution on the sulfur atom, e.g. oxidized CoA, acetyl-CoA, benzoyl-CoA. All other signals of the CoA moiety in spectra of reduced CoA and acetyl-CoA under various conditions have identical chemical shifts. In contrast, in spectra of each of the benzoyl-CoAs in D2O, the (C-1')-H, (C-2)-H and (C-8)-H signals of the adenosyl moiety and the signals of the respective benzoyl protons are displaced upfield with respect to their positions in spectra of equimolar admixtures of reduced CoA with the respective benzoate (all other signals (except CH2-S) are essentially unchanged). These differences in chemical shift (Δδ) are independent of benzoyl-CoA concentration. The Δδ values are decreased by increased temperature and there is no difference between the spectrum of benzoyl-CoA in 70 % methanol-d4 (30 % D2O) and that of the CoA-benzoate admixture in D2O. These PMR data, and concentration, temperature, and solvent data suggested a specific intramolecular complex involving a ring stacking interaction between the adenosyl and benzoyl moieties with the charged and polar groups of the phosphopantetheine moiety facing the exterior, and the methyl and methylene groups forming a hydrophobic interior of the folded molecule. A Corey-Pauling-Koltun molecular model of the folded benzoyl-CoA molecule was constructed which allowed direct measurement of the distances of the three adenosyl protons from the center of the benzene ring. The values of Δδ predicted from these distances (due to the benzene ring current effect) correlated well with those observed. The values of ΔG = -0.7 Cal per mole, ΔH = -8.9 Cal per mole, and ΔS = -27 e.u. calculated from the temperature-dependence data are consistent with the proposed model of the intramolecular complex, involving more than one type of weak interaction. It is noted that approximately 65 % of the molecules of benzoyl-CoA exist in the folded conformation near physiological temperature.