NMR spectroscopy is an important technique for the study of biological fluids and intact cells (1-8), Compared with other analytical methods, it is nondestructive and noninvasive, and it allows delicately balanced chemical and cellular processes to be observed directly at the molecular level. 13C, 15N, 31P, and !H NMR spectroscopies have all been used to study biological fluids and/or intact cells.Because of the inherent low NMR sensitivities and low natural abundances of 13C and 15N, isotopically enriched compounds generally are used in 13C and l5N NMR studies. The advantage is a relatively simple spectrum that consists of resonances from the enriched compounds and their metabolites superimposed on much weaker background resonances. However, isotopically enriched compounds are required, and, in the case of intact cell studies, they must be incorporated into the cells. Nevertheless, 13C and 15N NMR with isotopically enriched compounds are widely used and important methods for the study of metabolic processes in intact cells, 31P, which has a natural abundance of 100% and a high inherent NMR sensitivity, is also widely used. Because there are few phosphorus-containing compounds at detectable levels, 31P NMR spectra of biological fluids and intact cells generally are relatively simple. For the same reason, however, 31P NMR can be used to study only a limited number of compounds.the analog-to-digital converter. This spectrum and all the other spectra presented In this article were measured with a Varlan VXR-500S spectrometer operated in the unlocked mode.
The alkylating agent isophosphoramide mustard (IPM) spontaneously forms a relatively stable aziridine derivative which can be directly observed using NMR spectroscopy. The protonations of IMP and its aziridine were probed using 1H, 31P, 15N, and 17O NMR spectroscopy. The positions of the 31P, 15N, and 17O resonances of IPM between pH 2 and 10 each exhibit a single monobasic titration curve with the same pKa of 4.31 +/- 0.02. On the basis of a comparison with other compounds and our earlier work with phosphoramide mustard, the NMR results for IPM indicate that protonation occurs at nitrogen and not oxygen. Over this same pH range, each of the 1H, 31P, and 15N resonances of IPM-aziridine also show a single monobasic titration with a pKa of 5.30 +/- 0.09. The magnitude of the change in chemical shifts suggests that the protonation of the IPM-aziridine occurs at the ring nitrogen. Theoretical gas-phase calculations of PM, IPM, and IPM-aziridine suggest O-protonation to be more likely; however, aqueous phase calculations predict the N-protonated forms to be most stable. Furthermore, for PM and IPM-aziridine, which contain nonequivalent nitrogens, the theoretical calculations and experimental data both agree as to which nitrogen undergoes protonation. These results suggest that the IMP-aziridine remains unprotonated under physiological conditions and may, in part, explain the lower alkylating activity of IPM as compared to PM.
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