Esterification of borate with NAD + and NADH as studied by electrospray ionization mass spectrometry and 11 B NMR spectroscopy

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 11 B 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 µ M 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.