Conformational Studies of Carnitine and Acetylcarnitine: Design and Synthesis of Active-Site Probes of Carnitine Acetyltransferase.
Date of Award
Doctor of Philosophy (PhD)
The conformations of carnitine (Cn) and acetylcarnitine (AcCn) have been investigated through a combination of methods. The solid-state structures of their zwitter-ionic forms have been determined by single-crystal X-ray analyses. The crystal structures reveal different backbone conformations for the two molecules. Carnitine exists in an extended conformation (a,g $\sp-$ conformations for the C1-C2-C3-C4 and N1-C4-C3-O3 torsion angles, respectively), while AcCn exhibits a folded conformation ($g\sp-,g\sp-$). The solution conformations of Cn and AcCn have been investigated by high resolution $\sp1$H NMR coupling data. A simple equation has been developed for the prediction of torsion angles from $\sp3 J$ (HCCH) coupling constants. This equation employs experimentally determined substituent constants, which can account for solvent effects. The conformation and populations of conformers of AcCn and Cn in D$\sb2$O have been estimated by use of this new Karplus relationship. The assignment of diasteriotopic protons of Cn and AcCn was made by analogy with the lowest energy conformations as determined by molecular mechanics (MM2) calculations parameterized with atomic charges from ab initio (3-21G) calculations. Populations of conformers, arising from rotation about the C2-C3 and C3-C4 bonds were calculated. Both compounds adopt a highly preferred $g\sp-$ conformation about the N1-C4-C3-O3 torsion angle. In contrast, the C1-C2-C3-C4 torsion angle exhibits substantial rotational freedom between $g\sp-$ and a. In Cn the anti conformer dominates, whereas in AcCn the $g\sp-$ conformer is most prevalent. The relative energetics of extended and folded conformers suggest that binding of either Cn or AcCn to carnitine acetyltransferase (CAT) occurs with the folded form. The syntheses of seventeen conformationally rigid morpholinium analogues of Cn and AcCn are described. These compounds exhibit predictable stereo- and regio-chemistry because of stereospecific ring closures of their acyclic precursors. The single-crystal X-ray structures for seven of the compounds are presented. The synthesis, resolution, and solid-state structure of a sulfonate analogue of Cn (sulfocarnitine) are also described. These compounds have been tested as inhibitors of pigeon CAT and rat-liver carnitine palmitoyltransferase (CPT). The results support the hypothesis of a folded conformation for enzyme-bound Cn or AcCn. The inhibitors also reveal distinctly different modes of Cn recognition by CAT compared to CPT.
Colucci, William Jay, "Conformational Studies of Carnitine and Acetylcarnitine: Design and Synthesis of Active-Site Probes of Carnitine Acetyltransferase." (1987). LSU Historical Dissertations and Theses. 4348.