Formation of 8-S-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-L-cysteinyladenosine. 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.

Eosinophil peroxidase (EPO), an enzyme secreted from eosinophil granulocytes, generates the reactive species hypobromous acid (HOBr) from H₂O₂ and Br⁻.^1,2) The human plasma concentration of Br⁻ is 39–84 µM, compared with a concentration of 100 mM for Cl⁻.³⁾ EPO uses the plasma concentration of Br⁻ selectively in the presence of 100 mM Cl⁻, resulting in HOBr. The HOBr thus formed plays an important role in the body’s defense against parasites. Meanwhile, myeloperoxidase (MPO), an enzyme secreted from neutrophil and monocytic cells, generates hypochlorous acid (HOCl) from H₂O₂ and Cl⁻.^4–6) The HOCl thus formed is also of central importance in host defense mechanisms. Since HOCl can react with Br⁻ to generate HOBr, a portion of HOCl formed by the MPO system would react with a plasma concentration Br⁻, converting it to HOBr.^7,8)

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-Br-dGuo) and 5-bromo-2′-deoxycytidine (5-Br-Cyd) were generated from dGuo and dCyd by reactions with reagent HOBr, an EPO/H₂O₂/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/H₂O₂/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-L-cysteinylguanosine (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-Br-dAdo), 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.

Results

A 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 negative mode (Fig. 2A). High-resolution ESI-TOF/MS (negative) of the molecular ion showed m/z=385.094002, which agreed with the theoretical molecular mass for C₁₃H₁₇N₆O₆S within 2 ppm. ¹H-NMR in D₂O showed an aromatic proton signal and three aliphatic proton signals in addition to six ribose proton signals. ¹³C-NMR showed five aromatic carbon signals and seven aliphatic carbon signals with a carboxyl carbon signal (174.8 ppm). Combining these data, the product was identified as a Cys-substituted derivative of Ado at the 8 position, 8-S-L-cysteinyladenosine (Cys-Ado) (Fig. 2B). Concentrations of these products were 22.5±0.3 µM for Cys-Ado and 0.7±0.0 µM for Ado with 74.0±1.1 µM of unreacted 8-Br-Ado. As a control, a similar experiment was conducted for glycine (Gly). Incubation of a solution of 100 µM 8-Br-Ado with 50 mM Gly at pH 7.2 at 37 °C for up to 24 h showed no product and no consumption of 8-Br-Ado.

Fig. 1. RP-HPLC Chromatogram of a Reaction Mixture of 8-Br-Ado with Cys Detected at 260 nm

A solution of 100 µM 8-Br-Guo and 50 mM Cys was incubated in 100 mM potassium phosphate buffer at pH 7.2 and 37°C for 4 h. The HPLC system consisted of LC-10ADvp pumps and an SPD-M10Avp UV-Vis photodiode-array detector (Shimadzu, Kyoto, Japan). For the RP-HPLC, an Inertsil ODS-3 octadecylsilane column of 4.6×250 mm and a particle size of 5 µm (GL Sciences, Tokyo) was used. The eluent was 20 mM ammonium acetate (pH 7.0) containing 25% methanol. The column temperature was 40°C and the flow rate was 1 mL/min.

Fig. 2. (A) Negative Ion Electrospray Ionization Time of Flight Mass Spectrometry (ESI-TOF/MS, MicroTOF, Bruker) Spectrum of 8-S-L-Cysteinyladenosine (Cys-Ado); (B) Structure of Cys-Ado

Figure 3A shows time-dependent changes in the concentrations of Cys-Ado, Ado, and 8-Br-Ado, when 100 µM 8-Br-Ado and 50 mM Cys were incubated in 100 mM potassium phosphate buffer at pH 7.2 at 37°C for 0–4 h. The concentrations of Cys-Ado and Ado increased with increased incubation time. 8-Br-Ado was converted to Cys-Ado almost exclusively. Figure 3B shows the Cys dose-dependence of concentrations of the products and 8-Br-Ado, when 100 µM 8-Br-Ado and 0–100 mM Cys were incubated in 100 mM potassium phosphate buffer at pH 7.2 at 37°C for 4 h. The concentrations of the products increased with increasing Cys dose. Figure 3C shows the pH dependence of the concentrations of the products and 8-Br-Ado. Consumption of 8-Br-Ado increased with increasing pH value of the solution. The concentration of Cys-Ado increased with increasing pH, whereas the concentration of Ado remained low throughout the pH range examined. Figure 3D shows the stability of Cys-Ado. One hundred micromolar Cys-Ado purified by RP-HPLC was incubated in 100 mM potassium phosphate buffer of pH 7.2 at 37°C for up to 72 h. Cys-Ado decreased time-dependently with a half-life of 15.1 h. Although products from Cys-Ado were detected in the RP-HPLC chromatogram, Ado did not form. A major product showed an ESI-TOF/MS spectrum with m/z=769 in the negative mode. High-resolution ESI-TOF/MS (negative) of the molecular ion showed m/z=769.178688, which agreed with the theoretical molecular mass for C₂₆H₃₃N₁₂O₁₂S₂ (m/z=769.178780) within 1 ppm. The major product is presumed to be a dimer of Cys-Ado, minus two hydrogen atoms [(Cys-Ado)₂–2H]. Further studies are needed to clarify the structure of this major product and its reaction mechanism.

Fig. 3. (A) The Time–Course of the Concentration Changed in 8-Br-Ado (Circle), Cys-Ado (Triangle), and Ado (Square) When a Solution of 100 µM 8-Br-Ado and 50 mM Cys Was Incubated in 100 mM Potassium Phosphate Buffer at pH 7.2 and 37°C for 0–4 h; (B) The Cys Dose-Dependence of the Concentration Changed in 8-Br-Ado (Circle), Cys-Ado (Triangle), and Ado (Square) When a Solution of 100 µM 8-Br-Ado and 0–100 mM Cys Was Incubated in 100 mM Potassium Phosphate Buffer at pH 7.2 and 37°C for 4 h; (C) The pH Dependence of the Concentration Changed in 8-Br-Ado (Circle), Cys-Ado (Triangle), and Ado (Square) When a Solution of 100 µM 8-Br-Ado and 50 mM Cys Was Incubated in 100 mM Potassium Phosphate Buffer at pH 5.2–10.2 and 37°C for 4 h; (D) The Time–Course of the Concentration Changed in Cys-Ado at pH 7.2 and 37°C When a Solution of Isolated 100 µM Cys-Ado Was Incubated in 100 mM Potassium Phosphate Buffer for Up to 72 h

The half-life was calculated from the first-order rate constant obtained using the nonlinear least-squares fitting algorithm in Igor Pro (Wavemetrics). All the reaction mixtures were analyzed by RP-HPLC. Means±S.D. (n=3) are presented.

To obtain information about the reaction of an 8-bromoadenine moiety in deoxyribonucleoside with Cys, similar experiments were conducted for 8-bromo-2′-deoxyadenosine (8-Br-dAdo). A solution of 100 µM 8-Br-dAdo and 50 mM Cys was incubated in 100 mM potassium phosphate buffer of pH 7.2 at 37°C for 4 h. When the reaction mixture was analyzed by RP-HPLC, a product peak showing a UV spectrum with λ_max=279 nm was eluted with a small peak of dAdo. From MS and NMR data, the product was identified as a Cys-substituted derivative of dAdo at the 8 position, 8-S-L-cysteinyl-2′-deoxyadenosine (Cys-dAdo). Concentrations of these products were 25.1±0.3 µM for Cys-dAdo and 0.6±0.1 µM for dAdo with 70.3±1.1 µM of unreacted 8-Br-dAdo. The half-life of Cys-dAdo at pH 7.2 and at 37°C was 19.0 h.

To obtain information about the reaction of an 8-bromoadenine moiety in nucleotide with Cys, similar experiments were conducted for 8-bromoadenosine 3′,5′-cyclic monophosphate (8-Br-cAMP). A solution of 100 µM 8-Br-cAMP and 50 mM Cys was incubated in 100 mM potassium phosphate buffer of pH 7.2 at 37°C for 4 h. When the reaction mixture was analyzed by RP-HPLC, a product peak showing a UV spectrum with λ_max=279 nm was eluted with a small peak of cAMP. From MS and NMR data, the product was identified as a Cys-substituted derivative of cAMP at the 8 position, 8-S-L-cysteinyladenosine 3′,5′-cyclic monophosphate (Cys-cAMP). Concentrations of the products were 20.0±0.1 µM for Cys-cAMP and 1.2±0.0 µM for cAMP with 76.6±0.2 µM of unreacted 8-Br-cAMP. The half-life of Cys-cAMP at pH 7.2 and at 37°C was 12.9 h.

Discussion

Similar to the reaction of 8-Br-Guo with Cys, the major product of the reaction of 8-Br-Ado with Cys was a cysteine compound substituted at position 8 at the purine base. The reaction of 8-Br-Ado with Cys, however, proceeds faster than that of 8-Br-Guo. Whereas 48 h was required for ca. 25% consumption of 8-Br-Guo in the reaction of 100 µM 8-Br-Guo with 50 mM Cys at pH 7.4 and 37°C,¹⁵⁾ 8-Br-Ado was ca. 25% consumed within a reaction time of 4 h in the reaction of 100 µM 8-Br-Ado with 50 mM Cys at pH 7.2 and 37°C (Fig. 3A). Although Guo was formed with more than half the amount of the resulting Cys-Guo product in the reaction of 8-Br-Guo with Cys, the amount of Ado formed was low in the reaction of 8-Br-Ado with Cys. 8-Br-Ado was converted to Cys-Ado almost exclusively. Similar results were obtained for the reaction of 8-Br-dAdo and 8-Br-cAMP with Cys. The reaction formula is shown in Chart 1. The yield of Cys-Ado increased with increasing pH from mildly acidic to basic conditions (Fig. 3C). The pK_a of Cys is 8.3 for the thiol group.¹⁷⁾ The thiolate (–S⁻) of Cys formed by deprotonation of the thiol group (–SH) would attack the C8 atom of 8-Br-Ado, generating Cys-Ado. Whereas Cys-Guo was fairly stable, with a half-life of 193 h at pH 7.4 and 37°C, Cys-Ado was less stable, with a half-life of only 15.1 h at pH 7.2 and 37°C, resulting in products other than Ado. Similar half-lives were obtained for Cys-dAdo and Cys-cAMP. The Cys adducts formed in RNA and DNA may remain for a certain period of time.

Chart 1

Reaction of 8-Br-Ade in 8-Br-Ado, 8-Br-dAdo, and 8-Br-cAMP with Cys. R denotes either ribose, 2-deoxyribose or ribose 3,5-cyclic phosphate.

Regarding mutations caused by the bromination of DNA bases, primer extension reactions catalyzed by human DNA polymerases on DNA templates including 8-Br-dGuo, 5-Br-dCyd, and 8-Br-dAdo, has been reported.¹⁸⁾ An 8-Br-dGuo-modified template incorporated incorrect bases, whereas 5-Br-dCyd- and 8-Br-dAdo-modified templates incorporated only correct bases. This result implies that 8-Br-dGuo is the only mutagenic lesion, not 5-Br-dCyd and 8-Br-dAdo. In the present study, 8-Br-dAdo reacted with Cys resulting in an adduct. If 8-Br-dAdo in DNA can react with various thiols including glutathione and proteins close to DNA, and resulting in the formation of adducts, mutagenic events may be initiated.

cAMP is a ubiquitous second chemical messenger that couples extracellular signals to intracellular responses in all cell types.¹⁹⁾ Since cAMP cannot pass through the plasma membrane, 8-Br-cAMP, a membrane permeable cAMP analog, is used as a standard tool for investigations of biochemical and physiological signal transduction pathways.²⁰⁾ 8-Br-cAMP shows excellent activation potentials for protein kinase A, as well as relatively long-acting effects due to its resistance to cAMP phosphodiesterase. Recently, a new signal transduction mechanism termed protein S-guanylation was revealed.²¹⁾ 8-Nitroguanosine 3′,5′-cyclic monophosphate (8-NO₂-cGMP) reacts with cysteine thiols of protein Keap1, generating a cGMP adduct of the protein. The formation of S-guanylation regulated the redox-sensor signaling protein. The results of the present study suggest that 8-Br-cAMP, either generated by endogenously-formed HOBr from cAMP or added exogenously as a reagent, may also react partially with the cysteine thiols of proteins resulting in cAMP adducts (S-adenylation) as well as 8-NO₂-cGMP. Since the half-life of Cys-cAMP at pH 7.2 and 37°C was 12.9 h, it may be sufficiently stable as a signal adduct.

The present results show that 8-Br-Ade nucleoside and nucleotide can react with Cys more rapidly than the reaction of 8-Br-Gua nucleoside, resulting in Cys adducts with a certain degree of stability under neutral conditions. These adducts may have some importance in elucidating the influence of 8-Br-Ade in nucleosides, nucleotides, RNA and DNA in terms of genotoxicity and signal transduction.

Experimental

Materials

8-Br-Ado and Cys were purchased from Sigma-Aldrich (MO, U.S.A.). 8-Br-dAdo was obtained from Alfa Aesar (Lancashire, U.K.) and 8-Br-cAMP from Nacalai Tesque (Kyoto, Japan). Water was purified with a Millipore Milli-Q deionizer.

HPLC and MS Conditions

The HPLC system consisted of LC-10ADvp pumps and an SPD-M10Avp UV-Vis photodiode-array detector (Shimadzu, Kyoto, Japan). For the RP-HPLC, an Inertsil ODS-3 octadecylsilane column of 4.6×250 mm and a particle size of 5 µm (GL Sciences, Tokyo) was used. The eluent was 20 mM ammonium acetate (pH 7.0) containing 25% methanol. The column temperature was 40°C and the flow rate was 1 mL/min. The RP-HPLC chromatogram was detected at 260 nm. ESI-TOF/MS measurements were performed on a MicroTOF spectrometer (Bruker, Bremen, Germany) in negative mode. The sample isolated by RP-HPLC was directly infused into the MS system by a syringe pump without a column.

Spectrometric Data

Spectrometric Data of Cys-Ado

ESI-TOF/MS (negative mode): m/z 298 and 385. High resolution (HR)-ESI-TOF/MS (negative mode): m/z 385.094002 Obsd, (Calcd for C₁₃H₁₇N₆O₆S 385.093577). UV: λ_max=279 nm (pH 7.0). ¹H-NMR (500 MHz, D₂O): δ (ppm/TSP-d₄) 8.04 (s, 1H, H-2), 6.00 (d, 1H, H-1′), 4.94 (dd, 1H, H-2′), 4.45 (dd, 1H, H-3′), 4.31 (m, 1H, H-4′), 4.26 (dd, 1H, H-α), 4.03 (dd, 1H, H-β), 3.90 (ABX, 2H, H-5′ and 5″), 3.69 (dd, 1H, H-β). ¹³C-NMR (125 MHz, D₂O): δ (ppm/TSP-d₄) 174.8 (COOH), 156.9, 154.3 (C-2), 152.8, 152.1, 122.3, 92.0 (C-1′), 89.6 (C-4′), 75.5 (C-2′), 73.8 (C-3′), 64.9 (C-5′), 57.4 (C-α), 36.2 (C-β).

Spectrometric Data of Cys-dAdo

ESI-TOF/MS (negative mode): m/z 282 and 369. HR-ESI-TOF/MS (negative mode): m/z 369.098991 Obsd, (Calcd for C₁₃H₁₇N₆O₅S 369.098662). UV: λ_max=279 nm (pH 7.0). ¹H-NMR (500 MHz, D₂O): δ (ppm/TSP-d₄) 8.04 (s, 1H, H-2), 6.39 (dd, 1H, H-1′), 4.67 (m, 1H, H-3′), 4.27 (dd, 1H, H-α), 4.18 (m, 1H, H-4′), 3.02 (dd, 1H, H-β), 3.89 (ABX, 2H, H-5′ and 5″), 3.70 (dd, 1H, H-β), 2.94 (m, 1H, H-2′ or 2″), 2.41 (m, 1H, H-2′ or 2″). ¹³C-NMR (125 MHz, D₂O): δ (ppm/TSP-d₄) 174.8 (COOH), 156.7, 154.0 (C-2), 152.9, 151.9, 122.2, 90.6 (C-4′), 88.4 (C-1′), 74.6 (C-3′), 65.0 (C-5′), 57.4 (C-α), 40.9 (C-2′), 36.1 (C-β).

Spectrometric Data of Cys-cAMP

ESI-TOF/MS (negative mode): m/z 360 and 447. HR-ESI-TOF/MS (negative mode): m/z 447.049477 Obsd, (Calcd for C₁₃H₁₆N₆O₈PS 447.049342). UV: λ_max=279 nm (pH 7.0). ¹H-NMR (500 MHz, D₂O): δ (ppm/TSP-d₄) 8.15 (s, 1H, H-2), 6.05 (s, 1H, H-1′), 5.14 (dd, 1H, H-3′), 4.86 (d, 1H, H-2′), 4.46 (ABX, 2H, H-5′ and 5″), 4.26 (m, 1H, H-4′), 4.24 (dd, 1H, H-α), 4.05 (dd, 1H, H-β), 3.64 (dd, 1H, H-β). ¹³C-NMR (125 MHz, D₂O): δ (ppm/TSP-d₄) 174.8 (COOH), 156.4, 154.6 (C-2), 153.4, 151.5, 121.9, 95.3 (C-1′), 79.8 (C-3′), 75.0 (C-4′), 74.4 (C-2′), 69.9 (C-5′), 57.2 (C-α), 36.2 (C-β).

Quantitative Procedures

The concentrations of the products were evaluated according to integrated peak areas on RP-HPLC chromatograms detected at 260 nm and by the molecular extinction coefficients at 260 nm (ε_260 nm). The ε_260 nm value of 16000 M⁻¹ cm⁻¹ was used for 8-Br-Ado, 8-Br-dAdo, and 8-Br-cAMP. The ε_260 nm values of Cys-Ado, Cys-dAdo, and Cys-cAMP were determined from the integration of proton signals of NMR and the HPLC peak area detected at 260 nm relative to those of Ado or dAdo (ε_260 nm=14900 M⁻¹ cm⁻¹) in the mixed solution. The estimated ε_260 nm values were 9900 M⁻¹ cm⁻¹ for Cys-Ado, 9600 M⁻¹ cm⁻¹ for Cys-dAdo, and 9900 M⁻¹ cm⁻¹ for Cys-cAMP.

Conflict of Interest

The authors declare no conflict of interest.

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