Synthesis and characterization of selenotrisulfide-derivatives of lipoic acid and lipoamide

Abstract

Thiol-containing compounds, such as glutathione and cysteine, react with selenite under specific conditions to form selenotrisulfides. Previous studies have focused on isolation and characterization of intermolecular selenotrisulfides. This study describes the preparation and characterization of intramolecular selenotrisulfide derivatives of lipoic acid and lipoamide. These derivatives, after separation from other reaction products by reverse-phase HPLC, exhibit an absorbance maximum at 288 nm with an extinction coefficient of 1,500 M(-1) small middle dotcm(-1). The selenotrisulfide derivative of lipoic acid was significantly stable at or below pH 8.0 in contrast to several other previously studied selenotrisulfides. Mass spectral analysis of the lipoic acid and lipoamide derivatives confirmed both the expected molecular weights and also the presence of a single atom of selenium as revealed by its isotopic distribution. The selenotrisulfide derivative of lipoic acid was found to serve as an effective substrate for recombinant human thioredoxin reductase as well as native rat thioredoxin reductase in the presence of NADPH. Likewise, the lipoamide derivative was efficiently reduced by NADH-dependent bovine lipoamide dehydrogenase. The significant in vitro stability of these intramolecular selenotrisulfide derivatives of lipoic acid can serve as an important asset in the study of such selenium adducts as model selenium donor compounds for selenophosphate biosynthesis and as rate enhancement effectors in various redox reactions.

Figure 1

Spectral analysis of the reaction of selenite with DHLA. Spectra were recorded after addition of 10 μl of a reaction mixture containing DHLA and selenite in an acidic solution of 50% EtOH (Materials and Methods) to 1 ml of 100% EtOH. Ratios given for each spectrum represent the molar ratio of free sulfhydryl to selenium in the reaction mixture.

Figure 2

Reverse-phase HPLC analysis of the products formed by reaction of selenite with DHLA. Spectrophotometric analysis of reaction products monitored at (A) 215 nm and (B) 288 nm. Arrow in B indicates the peak with absorption maximum at 288 nm, which corresponds to the selenium-containing derivative of DHLA. No compounds with significant absorbance at 288 nm were detected in the elution profile before the application of the MeOH gradient. Chromatographic details are described in Materials and Methods.

Figure 3

Absorption spectra of selenotrisulfide derivatives. (A) Selenium-containing derivative of DHLA. (B) Selenium-containing derivative of DHLN. Spectra were recorded during HPLC analysis using an integrated Diode array spectrophotometer in Hewlett–Packard series 1050 HPLC system.

Figure 4

Proposed structures of selenotrisulfide derivatives. (A) Schematic representation of LASe. (B) Schematic representation of LNSe.

Figure 5

pH stability of LASe. (A) Total area units of the peak representing LASe recorded during HPLC analysis are plotted as a function of the pH of the buffer in which LASe was incubated. (B) Total area units of the peak representing oxidized lipoic acid released from LASe are plotted as a function of the pH of the buffer in which LASe was incubated. The area units given in both plots are the average of at least three independent experiments, and the error bars represent one standard deviation from the mean. For LASe, the absorbance was followed at 288 nm, and for oxidized lipoic acid at 333 nm. Other experimental details are described in Materials and Methods.

Figure 6

Reduction of LASe by recombinant hTrxR and rat liver TrxR. Reaction mixtures (1 ml), containing 0.1 M sodium phosphate buffer (pH 7.0), 1.0 mM, and 300 μM LASe were incubated with recombinant hTrxR [1 μg, 4 units of activity with 5,5′ dithiobis(2-nitrobenzoic acid) as substrate] or native rat TrxR [1 μg, 42 units of activity with 5,5′ dithiobis(2-nitrobenzoic acid) as substrate]. Samples were taken at the indicated time intervals and stabilized for HPLC analysis by reaction with 200 mM iodoacetic acid. The area of the peak representing LASe determined for each time point by HPLC analysis was converted to a percentage of the initial LASe peak area of a sample taken before enzyme addition. The area units given in both plots are the average of at least three independent experiments, and the error bars represent one standard deviation from the mean.

Figure 7

Reduction of LNSe by bovine LD. Reaction mixtures contained 0.1 M sodium phosphate buffer (pH 6.5), 1.0 mM NADPH, 300 μM LNSe, and 33 ng LD. After removal of an initial aliquot before addition of enzyme, samples were taken at 1-min intervals up to 4 min. The area of the peak representing LNSe determined for each time point by HPLC analysis was converted to a percentage of the initial LNSe peak. The area units given are the average of at least three independent experiments, and the error bars represent one standard deviation from the mean.