15N and 13C{14N} NMR investigation of the major nitrogen-containing segment in an aquatic fulvic acid: evidence for a hydantoin derivative.

A nitrogen-rich segment in a fulvic acid (FA) from Pony Lake, a coastal pond in Antarctica, was investigated by (15)N and (13)C{(14)N} solid-state NMR techniques. As reported previously, the (13)C{(14)N} spectrum of C bonded to N exhibits a peak at 157 ppm that is assigned to an sp(2)-hybridized carbon bonded to at least two nitrogen atoms. This segment contains 48% of all N in the sample. (15)N NMR shows distinct signals, 20 ppm upfield and downfield from the typical peptide resonance; dipolar dephasing confirmed that they are due to protonated N. The well-resolved downfield peak, which accounts for 1/4 of the spectral area, cannot be assigned to aromatic heterocycles, such as purines, because the fraction of aromatic C bonded to N in this sample is very small. Analysis of (15)N chemical-shift trends and (15)N NMR of model compounds, such as arginine and its derivatives, excludes assignment to a guanidinium ion or to substituted guanidino groups. Similarly, ureido groups, -NH-CO-NH-, that are not bonded to a second C = O do not match the observed (15)N peaks in the FA, since both N resonate upfield from the peptide resonance. On the other hand, all chemical shifts are matched within the observed range by the -C(alkyl)-NH-CO-NH-CO-C structure found in two nonaromatic heterocycles, hydantoin and dihydrouracil. The five-membered hydantoin ring, which is found in the purine metabolite allantoin, provides a better match than the six-membered dihydrouracil ring. Regular uracil or thymine fails to produce adequate agreement with observed chemical shifts.