MAGNETIC RESONANCE IN CHEMISTRY Magn. Reson. Chem. 2005; 43: 902–909 Published online 22 August 2005 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/mrc.1652 Quantitative determination of SH groups using 19 F NMR spectroscopy and disulfide of 2,3,5,6-tetrafluoro-4-mercaptobenzoic acid Dmitrii I. Potapenko, 1,2 Elena G. Bagryanskaya, 1 Igor A. Grigoriev, 3 Aleksander M. Maksimov, 3 Vladimir A. Reznikov, 3 Vyacheslav E. Platonov, 3 Thomas L. Clanton 5 and Valery V. Khramtsov 4,5∗ 1 International Tomography Center, Novosibirsk 630090, Russia 2 Novosibirsk State University, Novosibirsk 630090, Russia 3 Institute of Organic Chemistry, Novosibirsk 630090, Russia 4 Institute of Chemical Kinetics and Combustion, Novosibirsk 630090, Russia 5 Dorothy M. Davis Heart and Lung Research Institute, The Ohio State University, Columbus, OH 43210, USA Received 23 March 2005; Revised 23 June 2005; Accepted 27 June 2005 A new method of measurement of thiol concentration by 19 F NMR spectroscopy is developed. The method is based on the detection of products of the exchange reaction of thiols with a newly synthesized fluorinated disulfide, 2,3,5,6-tetrafluoro-4-mercaptobenzoic acid (BSSB). A significant broadening of the 19 F NMR signal of BSSB in the presence of thiols was observed and attributed to the exchange reaction between the parent disulfide and 2,3,5,6-tetrafluoro-4-mercaptobenzoic acid. The rate constant for this reaction was found to be equal to .63 ± 11/ × 10 3 M −1 s −1 at pH 7.0. The method was applied for the measurement of concentration of glutathione and albumin in rat blood. Copyright  2005 John Wiley & Sons, Ltd. KEYWORDS: NMR; thiols; thiol measurements; 19 F; thiol-disulfide exchange; glutathione; fluorinated disulfides; benzoic acid INTRODUCTION The role of thiols in metabolic processes and in support- ing intracellular and extracellular homeostasis in vivo is widely appreciated. Among low-molecular-weight thiols, glutathione, GSH, and cysteine are the most significant. Particularly GSH, presenting in various cells in mM con- centration range, is considered to be the most important player in defining cellular ‘redox state’. 1 The role of labile thiol groups in supporting the structure and function of proteins is also well documented. The main protein com- ponents of blood, hemoglobin, and albumin are important examples of macromolecular thiols, having in their structure two and one SH groups easily available for modification, correspondingly. 2–5 L Correspondence to: Valery V. Khramtsov, Dorothy M. Davis Heart & Lung Research Institute, 201 HLRI, 473 W12th Ave, The Ohio State University, Columbus, OH 43210, USA. E-mail: khramtsov-1@medctr.osu.edu Contract/grant sponsor: RFBR; Contract/grant numbers: 05-04-48632, 05-04-48483. Contract/grant sponsor: NIH; Contract/grant number: K01 EB03519, HL 53333. Contract/grant sponsor: INTAS; Contract/grant number: 03-55-1740. Contract/grant sponsor: Ministry of Education; Contract/grant number: RF A03-2.11-822. Currently existing methods of SH group registra- tion (including photometric measurements using Ellman’s reagent 6 ) require optically transparent samples or prior iso- lation of SH-containing compounds 7–9 and, therefore, cannot be applied in vivo. Several years ago we proposed an elec- tron paramagnetic resonance (EPR) spectroscopy approach for thiol detection, which has better potential for in vivo use. 10 – 12 The method is based on thiol–disulfide exchange reactions of stable biradical disulfides with thiols, which result in formation of monoradical products followed by significant changes in EPR spectrum. The possibility of work- ing in nontransparent media and the high sensitivity of EPR are the most important advantages of this noninvasive technique. However biological reduction of the probes in EPR-silent products significantly limits the applications of the method. The development of fluorine-containing disul- fides and application of the 19 F NMR technique for thiol detection overcomes the latter disadvantage of the EPR approach. Moreover, the NMR approach could enable imag- ing of SH-containing compounds in living organisms. In the present paper a fluorine-containing disulfide of 4-mercapto- 2,3,5,6-tetrafluorobenzoic acid (BSSB) was synthesized. The mechanism of the reaction of BSSB with a number of thi- ols of biological importance was studied and applications of BSSB to measure SH compounds in rat blood is demon- strated. Copyright  2005 John Wiley & Sons, Ltd.