The chemistry of the bifunctional alkylating agent phosphoramide musta
rd and model phosphoramides was probed by multinuclear NMR spectroscop
y as a function of pH. Between pH 1 and 11, both the P-31 and N-15 res
onances for phosphoramide mustard displayed a single monobasic titrati
on curve with a pK(a) of 4.9. The protonation below pH 4.9 correlates
with the loss in reactivity of the mustard. The O-17 NMR spectrum of O
-17-enriched phosphoramide mustard shows little change with pH. The da
ta on the mustard was compared to N-15 and P-31 NMR data on N-15-enric
hed phosphoramidic acid, phosphorodiamidic acid, and phosphoric triami
de. Contrary to the conclusions of previous studies, our combined P-31
, N-15, and O-17 NMR results are more consistent with N-protonation of
phosphoramide mustard rather than an O-protonation. Theoretical calcu
lations on the phosphoramidic acid, phosphorodiamidic acid, and phosph
oric triamide show O-protonation to be more stable in the gas phase. F
or the latter two compounds, the calculations suggest that N-protonati
on may be the most stable protonated form in the aqueous phase. These
findings influence our understanding of the structure-activity relatio
nships of phosphoramide mustards.