Determination of cysteine concentration by fluorescence increase: reaction of cysteine with a fluorogenic aldehyde

Spectroscopic detection of small organic compounds using sensors designed either to monitor covalent-bond formation or to detect in non-covalent fashion has attracted increased interest recently.1–4 Although fluorescent and visual detection methods for free amino acids,1 amines,2 and thiols3 have been reported, selective detection of cysteine by fluorescence or by color change has only been reported by the Strongin group.5 Development of simpler methods for the determination of cysteine concentration is of current interest.5,6 One key for useful cysteine detection is discrimination of cysteine from other amino acids, amines, and thiols. The Strongin group used a xanthene dye possessing aldehydes that afforded thiazolidine by the reaction with cysteine under alkaline conditions, resulting in a decrease in fluorescence and a color change.5 Their results stimulated us to examine the utility of our recently developed fluorogenic aldehyde7 for the selective detection of cysteine. Our fluorogenic aldehydes can be used to monitor the progress of aldehyde transformations through increased fluorescence.7 Monitoring of an increase in fluorescence from a low level of fluorescence is typically more reliable than monitoring reduction in fluorescence intensity of a highly fluorescent sample when changes in fluorescence are small.7 Here we report a fluorescence growth method for cysteine detection employing our fluorogenic aldehyde 1 at neutral pH (Scheme 1).8,9 Aldehyde 1 was mixed with a series of amino acids, glutathione (reduced, g-L-glutamyl-L-cysteinylglycine), and glucose in aqueous buffer (pH 7) and the fluorescence (lex 250 nm, lem 380 nm) was monitored (Figs. 1 and 2). The reaction with cysteine resulted in an increase in fluorescence, while mixture with other amino acids or with glucose did not change the fluorescence; the reaction with glutathione showed only a small increase in fluorescence (Fig. 1a). When the fluorescence intensity was measured at 30 min, only the reaction with cysteine provided values meaningfully higher than the blank (without amino acid) (Fig. 1b). These results indicate that this fluorescence assay selectively detects cysteine and discriminates the aminothiol of cysteine from other simple thiols. Other amino acids including methionine, serine, lysine, proline, and histidine, as well as glutathione were all inactive in this assay, indicating that formation of thiazolidine 2 is key for the selective detection of cysteine. Although iminium or hemiaminal formation from aldehydes and amines (including amino acids) and monothiohemiacetal formation from aldehydes and thiols have been used for the detection of amines and thiols, these reactions were reversible and higher concentrations of analytes were typically required for the detection.9 Our results indicated that the level of formation of hemiaminal or monothiohemiacetal was far below the formation of thiazolidine 2. The time course of the fluorescence assay is shown in Fig. 2a. At 30 min, less than 0.5 mM of 2 was formed in the reaction with cysteine, determined based on the fluorescence intensity of 2 in the same buffer. The fluorescence of the reaction with cysteine was still increasing at 200 min. The fluorescence spectra of the reaction mixtures at 200 min are shown in Fig. 2b. Under these conditions complete conversion of 1 to 2 was not necessary to detect cysteine. In many fluorescence-based detection methods where decreases in fluorescence were monitored, it was often difficult to detect the small decrease in fluorescence at the initial stages of the reactions because of the intense fluorescence of the fluorescent reactant. Since our detection method monitored increase in fluorescence,