Abstract
AbstractVarious deuterated propanes and butanes have been synthesized to serve as mechanistic probes to explore the C-H hydroxylation occurring at the hydrophobic pocket within the particulate methane monooxygenase (pMMO) from M. capsulatus (Bath). The enantiomeric excess (ee) of the oxidation products for [1,1,1-2H3]butane and [1,1,1,4,4,4-2H6]butane was determined to be 94% and 86%, respectively, in favor of the formation of the 2R-alcohol. According to our previous experiment, in the case of [2,2-2H2]butane, the insertion of the CH3CD2- end was more favorable than the CH3CH2- end due to the tightness of the hydrophobic pocket. The van der Waal radius of the C-D bond is about 0.01 A shorter than that of C-H bond. Therefore, the different ee value between [1,1,1-2H3]butane and [1,1,1,4,4,4-2H6]butane may simply reflect the higher steric hinderance of 4-CH3- end of [1,1,1-2H3]butane adjacent to the edge of the hydrophobic pocket. This steric effect may control the stereochemistry in order to bias the hydroxylation toward the R-alcohol at the 4-CD3- end. From GC-MS analysis of m/z 141 and 144 ratio of the correponding trifluoroacetic ester derivatives of [1,1,1-2H3]butan-2-ol products we measured, we obtained kCH3-/kCD3-= 0.73(0.01), indicating that the insertion is in preference for the CD3CH2- end relative to CH3CH2- end. In the case of [2-2H1]propane, we measured a modest kinetic isotope effect (KIE) of hydrogen vs. deuterium of 5.79(0.04), in full agreement with KIE value previously determined for cryptically chiral ethane and d.l-chiral butane (5.5). These data provide further support for the concerted oxene insertion instead of radical rebound mechanism in the hydroxylation reaction of propane molecule mediated by pMMO. Finally, we conducted experiments on the oxidation of the trans-2-butene by pMMO. The enantiomeric excess of the products trans-2,3-epoxybutane was determined to be 10.2% in favor of (2S,3S)-products. The epoxidation of vinylic carbon mediated by pMMO does not give rise to the same high chiral selectivity in comparison with the hydroxylation of the aliphatic compound. This result indicates that the sp3 carbon center encounters a stronger stereoelectronic orientation relative to the sp2 carbon center at the active site.