The biochemical mechanism for the formation of the C–P–C bond sequence found in l-phosphinothricin, a natural product with antibiotic and herbicidal activity, remains unclear. To obtain further insight into the catalytic mechanism of PhpK, the P-methyltransferase responsible for the formation of the second C–P bond in l-phosphinothricin, we utilized a combination of stable isotopes and two-dimensional nuclear magnetic resonance spectroscopy. Exploiting the newly emerged Bruker QCI probe (Bruker Corp.), we specifically designed and ran a 13C-31P multiple quantum 1H-13C-31P (HCP) experiment in 1H-31P two-dimensional mode directly on a PhpK-catalyzed reaction mixture using 13CH3-labeled methylcobalamin as the methyl group donor. This method is particularly advantageous because minimal sample purification is needed to maximize product visualization. The observed 3:1:1:3 multiplet specifically and unequivocally illustrates direct bond formation between 13CH3 and 31P. Related nuclear magnetic resonance experiments based upon these principles may be designed for the study of enzymatic and/or synthetic chemical reaction mechanisms. Copyright © 2015 John Wiley & Sons, Ltd.
One-dimensional (1D) quantitative NMR (qNMR) is a useful tool for concentration determination due to its experimental simplicity and the direct proportionality of the integrated signal area to the number of nuclei spin. For complex mixtures, however, signal overlapping often in one-dimensional quantitative 1H NMR (1D 1H qNMR) spectrum limits the accurate quantification of individual compound. Here, we introduced employing joint 1D qNMR methods of different nuclei, such as 1H and 31P (or/and 19F), to quantify multiple compounds in a complex mixture using a single universal concentration reference. When the concentration ratio of several compounds containing different elements in a complex mixture is of interest, the result calculated from measured intensities from 1D qNMR of different nuclei is independent of the gravimetric error from the reference. In this case, the common reference also serves as a ‘quantitative bridge’ among these 1D qNMR of different nuclei. Quantitative analysis of choline, phosphocholine, and glycerophosphocholine mixture is given as an example using trimethylphosphine oxide ((CH3)3P(O)) as concentration reference. Compounds containing multiple elements, such as tetramethylammonium hexafluorophosphate (N+(CH3)4PF6−), are proposed as the common concentration reference for 1H, 13C, 15N, 31P, and 19F qNMR for the quantitative analysis of complex mixture containing these different elements. We anticipate that the proposed joint 1D qNMR approach using a universal concentration reference will be a valuable alternative for simultaneous quantification of multiple compounds in a complex mixture due to its accuracy and single and simple sample preparation. Copyright © 2014 John Wiley & Sons, Ltd.
