Measurement of the Dexree of Coupled Isotopic Enrichment of Different Positions in an Antibiotic Peptide by NMR

An experimental strategy for determining the extent to which multiply isotopically labeled fragments are incorporated intact into relatively complicated compounds of interest is presented. The NMR methods employed are based on isotope-filtered one-dimensional spectra and difference HSQC spectra incorporating a spin echo designed to report on the presence of a second NMR active isotope at a coupled site. They supplement existing methods for determining the extent of isotopic incorporation at individual sites to reveal whether two coupled labeled sites in a precursor are incorporated as an intact unit into products. The methods described also circumvent ¹H signal overlap and distinguish between the effects of different nitrogens coupled to individual carbons. The somewhat complicated case of valclavam illustrates the method's utility in measuring the J coupling constants between ¹³C and nearby sites that are only fractionally labeled with ¹⁵N, and measuring the fraction of molecules in which ¹³C is coupled to ¹⁵N, at each of several sites. The ¹⁵N of [2-¹³C, ¹⁵N] -labeled glycine is found to be incorporated into all three N positions of valclavam but most heavily into the N11 position. Specifically, ¹⁵N and ¹³C are incorporated into the N11 and C10 positions together as an ¹⁵N¹³C fragment approximately 8% of the time, whereas ¹⁵N is incorporated largely independently at the other positions,