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 15N and 13C{14N} solid-state NMR techniques. As reported previously, the 13C{14N} spectrum of C bonded to N exhibits a peak at 157ppm that is assigned to an sp2-hybridized carbon bonded to at least two nitrogen atoms. This segment contains 48% of all N in the sample. 15N NMR shows distinct signals, 20ppm 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 15N chemical-shift trends and 15N NMR of model compounds, such as arginine and its derivatives, excludes assignment to a guanidinium ion or to substituted guanidino groups. Similarly, ureido groups, NHCONH, that are not bonded to a second C=O do not match the observed 15N 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 CalkylNHCONHCOC 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. Copyright (c) 2011 John Wiley & Sons, Ltd.