Carnitine (1, 3-hydroxy-4-trimethylammoniobutyrate) is essential as a donor and acceptor of acyl groups in cellular metabolism. The major solution conformation of carnitine about C3-C4 contains the gauche relationship between the trimethylammonium and hydroxy groups, while two of the three low-energy, staggered conformations about C2-C3 are significantly populated. For studies of carnitine's protein binding sites, we designed conformationally defined cyclohexyl carnitine analogs (2-hydroxy-3-trimethylammoniocyclohexanecarboxylate) which all contain the favored carnitine conformation about the C3-C4 bond. Of the four possible diastereomers for these analogs, we synthesized three (2-4) that contain different carnitine conformations about C2-C3. Diastereomers 2 and 3 were prepared via the major diastereomeric products resulting from reduction of ethyl 5-chloro-3-nitrosalicylate (7). Compound 4, which could not be obtained in practical quantities by the above method, was prepared stereoselectively via opening of the epoxide of 3-(benzyloxycarbonylamino) cyclohexene (23) with Et2AlCN. Compounds 2-4 were not substrates for pigeon breast carnitine acetyltransferase but were weak, competitive inhibitors with Kivalues from 2.9 to 4.1 mM. In contrast, compounds 2 and 4 were not inhibitors of neonatal rat cardiac myocyte CPT- 2, while compound 3 was a modest competitive inhibitor (Ki; 5.3 mM). These results suggest differences between the carnitine binding sites of CAT and CPT-2. © 1994, American Chemical Society. All rights reserved.