Esterification of borate with NAD+and NADH as studied by electrospray ionization mass spectrometry and11B NMR spectroscopy

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Abstract
This paper describes for the first time the direct measurement of boric acid (B(OH)3) and borate (B(OH)4−) adduction to NAD+ and NADH by electrospray ionization mass spectrometry (ESI-MS) and 11B NMR spectroscopy. The analysis demonstrates that borate binds to both cis-2,3-ribose diols on NAD+ forming borate monoesters (1 : 1 addition), borate diesters (1 : 2 addition) and diborate esters (2 : 1 addition), whereas, only borate monoesters were formed with NADH. MS in the negative ion mode showed borate was bound to a cis-2,3-ribose diol and not to the hydroxyl groups on the phosphate backbone of NAD+, and MS/MS showed that the 1 : 1 addition monoester contained borate bound to the adenosine ribose. Boron shifts of borate monoesters and diesters with NAD+ were observed at 7.80 and 12.56 ppm at pH 7.0 to 9.0. The esterifications of borate with NAD+ and NADH were pH dependent with maximum formation occurring under alkaline conditions with significant formation occurring at pH 7.0. Using ESI-MS, the limit of detection was 50 µM for NAD+ and boric acid (1 : 1) to detect NAD+–borate monoester at pH 7.0. These results suggest esterification of borate with nicotinamide nucleotides could be of biological significance. Copyright © 2003 John Wiley & Sons, Ltd.