UNEQUIVOCAL LOCATION OF SITES OF N-OXIDATION USING NATURAL-ABUNDANCE LONG-RANGE H-1, N-15 GHNMQC 2-DIMENSIONAL NMR

A method is described for unequivocally establishing the site of N-oxi dation which relies on the relatively large downfield shift of aliphat ic nitrogen resonances following oxidation. The technique described is based on N-15 at natural abundance and does not require labelling. N- 15 chemical shifts are established on the basis of long-range H-1-N-15 heteronuclear coupling pathways detected via two-dimensional NMR usin g the GHNMQC pulse sequence. The oxidized nitrogen in the two piperazi ne-containing systems studied was shifted downfield by approximately 68 ppm; the opposite, non-oxidized nitrogen in the piperazine ring was shifted upfield by an average of -6 ppm (N-15 chemical shifts are ref erenced to liquid ammonia with a chemical shift of 0 ppm). N-oxidation perturbations in N-15 chemical shifts were first parameterized using an oxazolidinone antibiotic (eperezolid) and its N-oxide as a model sy stem. After the N-15 Shift due to N-oxidation was determined, the meth od was used to establish unequivocally the site of N-oxidation in PNU- 101387, a piperazine-containing anxiolytic agent. Conventional spectro scopic methods were equivocal and could not reliably establish the sit e of N-oxidation. (C) 1998 John Wiley & Sons, Ltd.