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

• Published in: Journal of mass spectrometry. 2003-06, Vol.38 (6), p.632-640
• Main Authors: Kim, Danny H., Marbois, Beth N., Faull, Kym F., Eckhert, Curtis D.
• Format: Article
• Language: English
• Subjects: 11B NMR spectroscopy; borate adduction; borate complexes; Borates - chemistry; Carbohydrates. Nucleosides and nucleotides; Chemistry; electrospray ionization mass spectrometry; Esterification; Exact sciences and technology; Guanosine Diphosphate - analysis; Guanosine Diphosphate - chemistry; Hydrogen-Ion Concentration; Molecular Structure; NAD; NAD - chemistry; Nuclear Magnetic Resonance, Biomolecular; Nucleosides, nucleotides and oligonucleotides; Organic chemistry; Preparations and properties; Spectrometry, Mass, Electrospray Ionization
• ISSN: 1076-5174; 1096-9888
• DOI: 10.1002/jms.476

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.