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The Journal of Lipid Research, Vol. 39, 1118-1126, May 1998
Copyright © 1998 by Lipid Research, Inc.

Paper on Methodology

Synthesis of an unsaturated fatty acid analogue (18-(4'-azido-2'-hydroxybenzoylamino)-oleic acid) and its interaction with lysophosphatidylcholine: acyl-CoA-O-acyltransferase

Lars Gehringa, Dirk Haaseb, Kai Habbena, Claus Kerkhoffa, Hartmut H. Meyerb, and Volkhard Kaevera
a Institut für Molekularpharmakologie, Medizinische Hochschule Hannover, D-30623 Hannover, Germany
b Institut für Organische Chemie, Universität Hannover, D-30167 Hannover, Germany

Correspondence to: Volkhard Kaever.

Acylation/deacylation reactions represent a basic requirement of triglyceride as well as phospholipid metabolism, and maintenance of membrane lipid composition. In order to examine enzymes participating in these pathways, we synthesized 18-(4'-azido-2'-hydroxybenzoylamino)-oleic acid, an iodinable photoaffinity analogue of oleic acid as a new tool for analyzing enzymes, especially those binding unsaturated fatty acids or acyl-CoAs. For the synthesis of {omega}-amino-oleic acid, coupling two bifunctional C9-components was used. The described synthesis scheme is also suited for the specific generation of other fatty acid analogues with distinct positions of the double bond. The functionality of 18-(4'-azido-2'-hydroxybenzoylamino)-oleic acid was investigated with the enzyme lysophosphatidylcholine:acyl-CoA-O-acyltransferase (LAT) [EC 2.3.1.23], an enzyme that shows high specificity towards (poly)unsaturated fatty acyl-CoAs. It could be shown that the photolabel, esterified with coenzyme A, acts in the dark as a reversible inhibitor of the enzyme activity, but photolysis of the label results in irreversible inactivation of LAT. This inactivation could be prevented by addition of the native substrate arachidonyl-CoA during photolysis. Several proteins could be specifically visualized using the iodinated analogue.

The data indicate that this new photoaffinity label may have application to identify and characterize lipid biosynthetic enzymes using unsaturated fatty acids as well as acyl-CoA binding proteins and the active site of these proteins.—Gehring, L., D. Haase, K. Habben, C. Kerkhoff, H. H. Meyer, and V. Kaever. Synthesis of an unsaturated fatty acid analogue (18-(4'-azido-2'-hydroxybenzoylamino)-oleic acid) and its interaction with lysophosphatidylcholine:acyl-CoA-O-acyltransferase. J. Lipid Res. 1998. 39: 1118–1126.

Supplementary key words: 18-(4'-azido-2'-hydroxybenzoylamino)-oleoyl-CoA, {omega}-amino - oleic acid, photoaffinity label, acyl-CoA analogue


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