Neutron diffraction structure of (2R,3R)-L-(-)-[2-D]carnitine tetrachloroaurate, [(CH3)3N-CH2-CHOH-CHD-COOH]+[AuCl4]-:: Determination of the absolute stereochemistry of the crotonobetaine-to-carnitine transformation catalyzed by L-carnitine dehydratase from Escherichia coli

Single-crystal neutron diffraction has been used to determine the stereochemical course of the hydration of trans-crotonobetaine to L-(-)-carnitine by the enzyme L-carnitine dehydratase. Firstly, an X-ray analysis of the undeuterated carnitinium salt [(CH3)(3)N-CH2-CH(OH)-CH2-COOH](+)[AuCl4](-) confirmed that the absolute configuration at the C-3 position of L-(-)-carnitine (the CHOH group) is indeed R. This was accomplished using the gold atom as an anomalously-scattering source. Then stereospecifically deuterated L-(-)-[2-D] carnitine was prepared by the hydration of trans-crotonobetaine in D2O using purified L-carnitine dehydratase from Escherichia coli. The subsequent neutron analysis of deuterated [(CH3)(3)N-CH2-CH(OH)-CH2-COOH](+)[AuCl4](-) revealed that the CHD group at position C-2 also had the absolute R configuration, thus establishing that the addition of D2O across the C=C double bond of trans-crotonobetaine proceeds by a stereospecific syn pathway. In Contrast, all other hydration-dehydration reactions examined thus far show that, when the proton added or abstracted is bonded to a carbon atom that is adjacent to a carboxylate group, the absolute stereochemistry of the resulting transformation is anti. Crystallographic details for [(CH3)(3)N-CH2-CH(OH)-CHD-COOH](+)[AuCl4](-) are as follows: space group P2(1)2(1)2(1) (orthorhombic), a = 10.855(2), b = 11.678(3), c = 22.776(6) Angstrom; Z = 8; final agreement factor for the neutron analysis at 15 K: R(F-0) = 0.097 based on 1140 reflections with I > 3 sigma(I).