Syntheses, Structures, and Enzymatic Evaluations of Conformationally Constrained, Analogue Inhibitors of Carnitine Acetyltransferase: (2R,6R)-, (2S,6S)-, (2R,6S)-, and (2S,6R)-6-(Carboxylatomethyl)-2-(hydroxymethyl)-2,4,4- trimethylmorpholinium

The syntheses and structures of the four stereoisomers of 6-(carboxylatomethyl)-2-(hydroxymethyl)- 2,4,4-trimethylmorpholinium, 1, are described. The key step in the synthetic strategy involves an intramolecular Michael addition reaction. Condensation of nonracemic 3-(methylamino)-2-methylpropane- l,2-diol, 3, with methyl 4-bromo-2-butenoate followed by intramolecular Michael addition gives a mixture of two diastereomers of methyl 2-[4,6-dimethyl-6-(hydroxymethyl)morpholinyl]- acetate, 5. The diastereomeric ratio of the products in this reaction changes from 6:1 to 1:1 with a change in solvent from diethyl ether:methanol (35:1, v:v) to methanol. The structures and absolute configurations of 1 were determined by single crystal X-ray analyses. In crystals and solution, the morpholinium rings adopt a chair conformation with carboxylatomethyl occupying an equatorial position. All four stereoisomers inhibit pigeon breast carnitine acetyltransferase (CAT). Of this series, (2S,6R)-1 binds to CAT most strongly with a K_i of 190 ± 20 μM and an IC₅₀ of 0.42 mM. The enzymatic assays of 1 confirm that CAT recognizes both configurations at C2 and C6 in the analogues. CAT has a different conformation when it binds carnitine or acetylcamitine than when it binds 1. This latter conformation may resemble that when CAT catalyzes acetyl transfer.