Unequivocal location of sites ofN-oxidation using natural abundance long-range1H,15N GHNMQC two-dimensional NMR

Abstract

A method is described for unequivocally establishing the site of N-oxidation which relies on the relatively large downfield shift of aliphatic nitrogen resonances following oxidation. The technique described is based on 15N at natural abundance and does not require labelling. 15N chemical shifts are established on the basis of long-range 1H–15N heteronuclear coupling pathways detected via two-dimensional NMR using the GHNMQC pulse sequence. The oxidized nitrogen in the two piperazine-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 (15N chemical shifts are referenced to liquid ammonia with a chemical shift of 0 ppm). N-oxidation perturbations in 15N chemical shifts were first parameterized using an oxazolidinone antibiotic (eperezolid) and its N-oxide as a model system. After the 15N shift due to N-oxidation was determined, the method was used to establish unequivocally the site of N-oxidation in PNU-101387, a piperazine-containing anxiolytic agent. Conventional spectroscopic methods were equivocal and could not reliably establish the site of N-oxidation. © 1998 John Wiley & Sons, Ltd.