Formation of 8-<i>S</i>-L-Cysteinyladenosine from 8-Bromoadenosine and Cysteine

Abstract

When 8-bromoadenosine was incubated with cysteine at pH 7.2 and 37°C, an exclusive product was generated. This product was identified as a cysteine substitution derivative of adenosine at the 8 position, 8-S-Lcysteinyladenosine. The reaction accelerated as pH increased from mildly acidic to basic conditions. The isolated cysteine adduct of adenosine decreased with a half-life of 15 h at pH 7.2 and 37°C. Similar results were obtained for the incubation of 8-bromo-2′-deoxyadenosine and 8-bromoadenosine 3′,5′-cyclic monophosphate with cysteine. These results suggest that 8-bromoadenine in nucleotides, RNA, and DNA can react with thiols, resulting in adducts under physiological conditions. Key words 8-bromoadenosine; cysteine; cysteinyladenosine; 8-bromo-2′-deoxyadenosine; 8-bromo-cAMP HOBr can react with nucleic acid bases. HOBr reacted with thymidine, resulting in thymidine glycol and its phosphate derivative in phosphate buffer. 9) 2′-Deoxyguanosine (dGuo) and 2′-deoxycytidine (dCyd) are major targets in the reaction of HOBr. In in vitro studies, 8-bromo-2′-deoxyguanosine (8-BrdGuo) and 5-bromo-2′-deoxycytidine (5-Br-Cyd) were generated from dGuo and dCyd by reactions with reagent HOBr, an EPO/H 2 O 2 /Br − system, and other oxidant/Br − systems. 10-13)Recently, 8-Br-dGuo was detected in urine from healthy volunteers. 14) In diabetic patients, the urinary 8-Br-dGuo level was found to be 8-fold higher than that in healthy volunteers. This implies that nucleoside bromination occurs naturally in healthy humans and that inflammatory diseases greatly increase its level. For adenine (Ade) in nucleoside, the reactivity with HOBr is low. However, it has been reported that 8-bromoadenine (8-Br-Ade) is the major purine oxidation product generated in double-stranded DNA by either reagent HOBr or an EPO/H 2 O 2 /Br − system. 15)In our previous paper, we reported that the incubation of 8-bromoguanosine (8-Br-Guo) with L-cysteine (Cys) under physiological conditions (pH 7.4 and 37°C) generated 8-S-Lcysteinylguanosine (Cys-Guo) and guanosine (Guo). 16) In the present study, we investigated the reaction of 8-bromoadenosine (8-Br-Ado), 8-bromo-2′-deoxyadenosine (8-BrdAdo), and 8-bromoadenosine 3′,5′-cyclic monophosphate (8-Br-cAMP) with Cys, and report here the identification of the product and its comparison with the reaction of 8-Br-Guo with Cys. ResultsA solution of 100 µM 8-Br-Ado and 50 mM Cys was incubated in 100 mM potassium phosphate buffer at pH 7.2 and at a temperature of 37°C for 4 h. When the reaction mixture was analyzed by reversed phase (RP) HPLC, an unknown product peak showing a UV spectrum with λ max =279 nm was eluted at the retention time of 4.4 min in the chromatogram with a small peak of adenosine (Ado) (Fig. 1). Ado was confirmed by coincidence of the RP-HPLC retention time and UV and MS spectra of an authentic Ado. The unknown product was collected and subjected to spectrometric measurements. The product showed an electrospray ionization time of flight (ESI-TOF) MS spectrum with m/z=298 and 385 in the nega...