Preparation of pyridine-N-glucuronides of tobacco-specific nitrosamines

Nicotine and cotinine are metabolized to pyridine-N-glucuronides in humans. This suggests that the analogous metabolites of the carcinogenic nicotine- related nitrosamines N'-nitrosonornicotine (NNN), 4-(methylnitrosamino)-1-( 3-pyridyl)-1-butanone (NNK), and 4-(methylnitrosamino)-1-(3-pyridyl)-1-buta nol (NNAL) should also be formed in people exposed to these compounds via t obacco products. We describe the synthesis of the appropriate pyridine-N-gl ucuronides: pyridyl-N-beta -D-glucopyranuronosyl-N'-nitrosonornicotinium in ner salt (NNN-N-Gluc, 8), 4-(methylnitrosamino)-1-(3-pyridyl-N-beta -D-gluc opyranuronosyl)-1-butanonium inner salt (NNK-N-Gluc, 9), and 4-(methylnitro samino)-1-(3-pyridyl-N-beta -D-glucopyranuronosyl)-1-butanolonium inner sal t (NNAL-N-Gluc, 10). The starting material, methyl 2,3,4-tri-O-acetyl-1-bro mo-1-deoxy-alpha -D-glucopyranuronate (1), is prepared in two steps from gl ucuronolactone. Reactions of 1 with racemic NNN (2), NNK (3), or racemic NN AL (4) are carried out with no solvent and the crude products are deprotect ed by treatment with base, giving the desired N-glucuronides 8-10 in 5-7% o verall yield after HPLC purification. The N-glucuronides were characterized by H-1 NMR, including COSY and NOESY spectra, and by MS and MS/MS. NNN-N-G luc exists as a 52:48 ratio of (E)- and (Z)-rotamers, which were partially separated by HPLC. This ratio was surprisingly similar to the (E):(Z) ratio for NNN itself suggesting hydrogen bonding of the (Z)-nitroso oxygen atom to the 2"-hydroxyl group of the glucuronide moiety. Partial HPLC separation s of the (E)- and (Z)-rotamers of NNK-N-Gluc and the (E)- and (Z)-rotamers as well as the (R)- and (S)-diastereomers of NNAL-N-Gluc were also achieved . The standards prepared in this study as well as the HPLC conditions devel oped for their separation will be important for analysis of these compounds in human urine.