The synthesis and biological activity of thiolcarnitine and its thiolesters

Abstract

Acetyl-D,L-thiolcarnitine was synthesized by the acid-catalyzed addition of thiolacetic acid to 4trimethylammonio-2-butenoic acid. AcetyI-D,L-thiolcarnitinc was the precursor of D,L-thiolcarnitine. which was preparcd by base hydrolysis. -I-hiolcarnitine significantly enriched in thc L-isomer was prepared from acetyI-D,L-thiolcarnitine using carnitine acetyltransferase as the resolving agent. The C2, C~ and C,, carnitine thiolesters were obtained by acylating thiolcarnitine with the corresponding N-hydroxysuccinimide esters. As a substrate for carnitine acetyltransferase, acetylthiolcarnitine gave the same kinetic constants as did acetylcarnitine; on the other hand, thiolcarnitine and carnitine gave the same K,o but the V ..... with thiolcarnitine was less than 5% of the value obtained with carnitine. With thiolcarnitine and acetyhhiolcarnitine as reactants, the measured K,q, at 30 C and pH 7.0, for the reacti~)n catalyzed by carnitine acetyhransferase (see below) was 4.6_+. I. acetylthiolcarnitine+ CoA --acetyI-CoA + thiolcarnitinc Lipids 18: 382-386, }983.A published abstract ( 1 ), appearing in 1970, stated that a long-chain carnitine thiolester was biologically active as an analog of the corresponding carnitine ester. Thiolcarnitine (i.e., 4-trimethylammonio-3-mercaptobutanoate) attracted our attention because its availability would allow us to synthesize affinity labels and photoaffinity labels for carnitine and carnitine ester binding sites by derivatizing the thiol group, just as we have done with CoA (2-4). We therefore developed a synthesis for thiolcarnitine and its thiolesters and evaluated their biological activity rather extensively. As we were completing these experiments, Ferri et al. (5) published a note on the mitochondrial handling of D,L-thiolcarnitine and acetyI-D,L-thiolcarnitine, thereby establishing that thiolcarnitine is indeed biologically active as a substitute for carnitine.This communication reports a synthetic route to thiolcarnitine and its thioIesters that is relatively simple, uses reagents and equipment that are commonly found in biochemical laboratories and yields products in amounts suitable for most biochemical studies. A procedure for enriching the L-isomer content of thiolcarnitine (to 77-86%) was also developed. A synthesis of thiolcarnitine has not been published previously. Preliminary reports of our experiments have appeared (6,7).

S‐(p‐Azidophenacyl)thiocarnitine specifically binds to the γ‐butyrobetaine‐binding protein of Agrobacterium sp. and can be used as a photoaffinity label

“…was synthesized as described [l]. PAP-TC was synthesized by coupling thiocarnitine [3] with the photolabile ligand p-azidophenacylbromide [2]. Purification was performed by preparative TLC (cellulose-coated plate without fluorescent indicator) and detection by exposing the plate to 12 vapour.…”

“…Purification was performed by preparative TLC (cellulose-coated plate without fluorescent indicator) and detection by exposing the plate to 12 vapour. Synthesis of ["%Z]PAP-TC was carried out with DL-[14C]carnitine hydrochloride (lOpCi/~mol) as starting material and essentially as described [2,3], with the exception that H2S04 was omitted in the first step of the reaction. This modification greatly increased the yield of the synthesis.…”

Photoaffinity labelling of carnitine acetyltransferase with S-(p-azidophenacyl)thiocarnitine

“…synthesized by the method of Ziegler et al (1967), DL-acetylthiocarnitine was synthesized by the method of Duhr et al (1983), and acetyl-CoA was prepared by the method of Simon & Shemin (1953).…”

“…Aquasol-2 universal scintillation cocktail was purchased from New England Nuclear. Methods PAP-TC was prepared by coupling DL-thiocarnitine (Duhr et al, 1983) with the photolabile ligand p-azidophenacyl bromide (Hixson & Hixson, 1975). [We are now aware that a synthesis of DL-thiocarnitine was first described in a patent awarded to Sigma-Tau Industrie Farmaceutiche Riunite (1982).]…”

“…(KBr disc), 2160 cm-' (-N3); 'H n.m.r. (2H20) (see Duhr et al, 1983), 8 2.92 (2H), 3.30 (9H), 3.62 (1H), 3.71 (2H), 4.28 (2H), 7.18 (aromatic, doublet, 2H) and 8.05 p.p.m. (aromatic, doublet, 2H); t.l.c.…”