Bioorganic & Medicinal Chemistry Letters 23 (2013) 3864–3867

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Bioorganic & Medicinal Chemistry Letters

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Formation of 8-S-L-cysteinylguanosine from 8-bromoguanosineand cysteine q

0960-894X/$ - see front matter � 2013 The Authors. Published by Elsevier Ltd. All rights reserved.http://dx.doi.org/10.1016/j.bmcl.2013.04.084

q This is an open-access article distributed under the terms of the CreativeCommons Attribution-NonCommercial-No Derivative Works License, which per-mits non-commercial use, distribution, and reproduction in any medium, providedthe original author and source are credited.⇑ Corresponding author. Tel.: +81 86 271 8346; fax: +81 86 271 8320.

E-mail address: tsuzuki@shujitsu.ac.jp (T. Suzuki).

Toshinori Suzuki ⇑, Aya Kosaka, Michiyo InukaiSchool of Pharmacy, Shujitsu University, Okayama 703-8516, Japan

a r t i c l e i n f o

Article history:Received 9 March 2013Revised 23 April 2013Accepted 26 April 2013Available online 9 May 2013

Keywords:8-BromoguanosineCysteine8-S-L-cysteinylguanosineGuanosine

a b s t r a c t

When 8-bromoguanosine was incubated with cysteine at pH 7.4 and 37 �C, a previously unidentifiedproduct was formed as a major product in addition to guanosine. The product was identified as a cysteinesubstitution derivative of guanosine at the 8 position, 8-S-L-cysteinylguanosine. The reaction was accel-erated under mildly basic conditions. The cysteine adduct of guanosine was fairly stable and decomposedwith a half-life of 193 h at pH 7.4 and 37 �C. Similar results were observed for incubation of 8-bromo-20-deoxyguanosine with cysteine. The results suggest that 8-bromoguanine in nucleosides, nucleotides,RNA, and DNA can react with thiols resulting in stable adducts.

� 2013 The Authors. Published by Elsevier Ltd. All rights reserved.

Eosinophil peroxidase (EPO) plays an important role in defensemechanisms against parasites mediated by the oxidation of Br�.1

EPO generates a reactive species hypobromous acid (HOBr) fromH2O2 and Br�. An EPO/H2O2/Br� system in the presence of a plasmaconcentration of Cl� can react with nucleosides to formbrominated nucleosides including 8-bromo-20-deoxyguanosine(8-Br-dGuo).2–4 Meanwhile, myeloperoxidase (MPO), an enzymesecreted from neutrophil and monocytic cells, generates hypochlo-rous acid (HOCl) from H2O2 and Cl�.5,6 The formed HOCl is also ofcentral importance in host defense mechanisms. MPO can generatechlorinated nucleosides including 8-chloro-20-deoxyguanosine (8-Cl-dGuo).7 It has been reported that an MPO/H2O2/Cl� system inthe presence of a plasma concentration of Br� also generates bro-minated nucleosides.8 HOCl formed in the MPO system would re-act with Br�, generating a brominating reagent, HOBr. An in vitroDNA replication study showed that human DNA polymerasesincorporated 20-deoxyguanosine-50-monophosphate (dGMP), 20-deoxyadenosine-50-monophosphate (dAMP), and 20-deoxythymi-dine-50-monophosphate (dTMP) in addition to one-base deletionopposite to an 8-Br-dGuo residue in an oligodeoxynucleotide, sug-gesting that 8-Br-dGuo in DNA is a mutagenic lesion.9 Recently,8-Br-dGuo was detected in urine from healthy volunteers with asimilar concentration of 8-Cl-dGuo, while the concentrations are

one order of magnitude lower than that of 8-oxo-7,8-dihydroxy-20-deoxyguanosine (8-oxo-dGuo).10 In diabetic patients, urinary8-Br-dGuo and 8-Cl-dGuo levels were eightfold higher than thelevels in healthy volunteers, whereas there was a small increasein 8-oxo-dGuo. This implies that 8-Br-dGuo exists in healthy hu-mans and that inflammatory diseases greatly increase its level.Thus, the importance of 8-Br-dGuo is becoming clear. However,there is little information available about the reaction of 8-Br-dGuowith intracellular molecules. In the present study, we investigatedthe reaction of 8-bromoguanosine (8-Br-Guo) with L-cysteine (Cys)and report identification of the products.

A solution of 100 lM 8-Br-Guo (Santa Cruz Biotechnology, TX,USA) and 50 mM Cys (Sigma, MO, USA) was incubated in100 mM potassium phosphate buffer at pH 7.4 and 37 �C for 48 hin the dark. When the reaction mixture was analyzed by reversedphase (RP) HPLC, two product peaks appeared in the chromato-gram (Fig. 1). The products were collected and subjected to spec-trometric measurements. The product eluted at the retentiontime of 6.5 min showed a UV spectrum with kmax = 252 nm andan ESI-TOF/MS spectrum with m/z = 282 in the negative mode.The product was identified as guanosine (Guo) by coincidence ofthe RP-HPLC retention time and UV and MS spectra of an authenticGuo. The product eluted at the retention time of 7.7 min showed aUV spectrum with kmax = 272 nm (Fig. 1, inset). An ESI-TOF/MSspectrum showed m/z = 401 and 314 in the negative mode(Fig. 2). High-resolution ESI-TOF/MS (negative) of the molecularion showed m/z = 401.088576, which agreed with the theoreticalmolecular mass for C13H17N6O7S composition within 1 ppm. 1H-NMR showed no aromatic proton signal and three aliphatic protonsignals in addition to six aliphatic ribose proton signals. 13C-NMR

3020100

Time (min)

400300200Wavelength (nm)

Guo

Cys-Guo

8-Br-Guo

Cys

Figure 1. RP-HPLC chromatogram of a reaction mixture of 8-Br-Guo with Cysdetected at 260 nm. The inset is the on-line UV spectrum of Cys-Guo. A solution of100 lM 8-Br-Guo and 50 mM Cys was incubated in 100 mM potassium phosphatebuffer at pH 7.4 and 37 �C for 48 h in the dark. The HPLC system consisted ofShimadzu LC-10ADvp pumps and an SCL-10Avp system controller. On-line UVspectra were obtained with a Shimadzu SPD-M10Avp UV–vis photodiode-arraydetector. For the RP-HPLC, an Inertsil ODS-3 octadecylsilane column of4.6 � 250 mm and particle size 5 lm (GL Science, Tokyo) was used. The eluentwas 20 mM ammonium acetate (pH 7.0) containing 10% methanol. The columntemperature was 40 �C and the flow rate was 1 mL/min.

100

50

0

600500400300200

401

314

Figure 2. Negative ion electrospray ionization time of flight mass spectrometry(ESI-TOF/MS, MicroTOF, Bruker) spectrum of 8-S-L-cysteinylguanosine (Cys-Guo).

100

80

60

40

20

0967248240

Time (h)

A B100

80

60

40

20

01007550250

Cys (mM)

Figure 4. (A) The time course of the concentration changes in 8-Br-Guo (circle),Cys-Guo (square), and Guo (triangle) when a solution of 100 lM 8-Br-Guo and50 mM Cys was incubated in 100 mM potassium phosphate buffer at pH 7.4 and37 �C for 0–96 h in the dark. (B) The Cys dose dependence of the concentrationchanges in 8-Br-Guo (circle), Cys-Guo (square), and Guo (triangle) when a solutionof 100 lM 8-Br-Guo and 0–100 mM Cys was incubated in 100 mM potassiumphosphate buffer at pH 7.4 and 37 �C for 48 h in the dark. The concentration wasdetermined by RP-HPLC. Means ± S.D. (n = 3) are presented.

100

80

60

40

20

011109876543

pH

Figure 5. The pH dependence of the concentration changes in 8-Br-Guo (circle),Cys-Guo (square), and Guo (triangle) when a solution of 100 lM 8-Br-Guo and50 mM Cys was incubated in 100 mM potassium phosphate buffer at pH 3–11 and37 �C for 48 h in the dark. The concentration was determined by RP-HPLC.Means ± S.D. (n = 3) are presented.

100

80

60

T. Suzuki et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3864–3867 3865

showed five aromatic carbon signals and seven aliphatic carbonsignals with a carboxyl carbon signal (171.0 ppm). Combiningthese data, the product was identified as a Cys-substituted deriva-tive of Guo at the 8 position, (Cys-Guo).11 The structures of theproducts are shown in Figure 3. Concentrations of the productswere 15.6 ± 0.2 lM for Cys-Guo and 8.9 ± 0.2 lM for Guo with74.3 ± 0.6 lM of unreacted 8-Br-Guo.12 As a control, a similarexperiment was conducted for glycine (Gly). Incubation of a solu-tion of 100 lM 8-Br-Guo with 50 mM Gly at pH 7.4 and 37 �C forup to 120 h showed no product and no consumption of 8-Br-Guo.

Figure 4A shows the time-dependent changes in concentrationsof Cys-Guo, Guo, and 8-Br-Guo, when 100 lM 8-Br-Guo and50 mM Cys were incubated in 100 mM potassium phosphate bufferat pH 7.4 and 37 �C for 0–96 h. The concentrations of Cys-Guo andGuo increased with increasing incubation time. Cys-Guo formedwith a higher concentration than Guo. Figure 4B shows the Cysdose-dependence of concentrations of the products and 8-Br-Guo, when 100 lM 8-Br-Guo and 0–100 mM Cys were incubatedin 100 mM potassium phosphate buffer at pH 7.4 and 37 �C for

NH

N

NO

NH2NBr

R8-Br-Guo

Cys NH

N

NO

NH2N

H2NS

HO O

R

+ NH

N

NO

NH2N

RCys-Guo Guo

Figure 3. Reaction of 8-Br-Guo with Cys. R denotes ribose.

48 h. The concentrations of the products increased with increasingCys dose.

Figure 5 shows the pH dependence of the concentrations of theproducts and 8-Br-Guo. Consumption of 8-Br-Guo increased withincreasing pH value of the solution from pH 7 to pH 8. The concen-tration of Cys-Guo increased with increasing pH from 7 to 8. How-ever, the concentration of Guo gradually decreased as the pHincreased.

Figure 6 shows the stability of Cys-Guo. 100 lM Cys-Guo wasincubated in 100 mM potassium phosphate buffer of pH 3.0, 7.4,

40

20

0967248240

Time (h)

Figure 6. The time course of the concentration changes in Cys-Guo at pH 3.0(circle), pH 7.4 (triangle), and pH 8.5 (square) when a solution of isolated 100 lMCys-Guo was incubated in 100 mM potassium phosphate buffer at pH 3.0, 7.4, or 8.5and 37 �C for up to 96 h in the dark. The concentration was determined by RP-HPLC.Means ± S.D. (n = 3) are presented.

NH

N

NO

NH2NBr

R

+Cys-SH NH

N

HN

O

NH2N

S

R

NH

N

NO

NH2N

R

Cys NH

N

NO

NH2NS

R

CysBr

−Br−, −H+

Cys-SH

8-Br-Guo Cys-Guo

Guo

Scheme 1. Proposed reaction pathways for the reaction of 8-Br-Guo with Cys.

3866 T. Suzuki et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3864–3867

and 8.5 at 37 �C for up to 96 h. At pH 3.0, the concentration of Cys-Guo did not change. At pH 7.4, Cys-Guo decreased time-depen-dently with a half-life of 193 h. Although several products were de-tected in the RP-HPLC chromatogram, Guo did not form. At pH 8.5,the half-life was 244 h. As a control, 8-Br-Guo was incubated underthe same conditions, but no decomposition was observed.

To obtain information about the reaction of an 8-bromoguaninemoiety in deoxyribonucleoside with Cys, similar experiments wereconducted for 8-bromo-20-deoxyguanosine (8-Br-dGuo). When asolution of 100 lM 8-Br-dGuo (Santa Cruz Biotechnology) and50 mM Cys was incubated in 100 mM potassium phosphate bufferat pH 7.4 and 37 �C in the dark, 8-S-L-cysteinyl-20-deoxyguanosine(Cys-dGuo)13 and dGuo were generated. At an incubation time of48 h, the concentrations of the products were 20.1 ± 1.3 lM forCys-dGuo and 9.5 ± 0.7 lM for dGuo with 66.2 ± 2.0 lM of unre-acted 8-Br-dGuo.14

To obtain information about the reaction mechanism generat-ing Guo, two experiments were conducted. 100 lM Cys-Guo wasincubated with 50 mM Cys in 100 mM potassium phosphate buffer(pH 7.4) at 37 �C for up to 72 h and the reaction mixture was ana-lyzed by RP-HPLC. The concentration of Cys-Guo decreased, butGuo did not form. It suggests that Cys does not catalyze the forma-tion of Guo from Cys-Guo. 100 lM 8-Br-Guo was incubated with50 mM NaBH4, a reducing agent, in 100 mM potassium phosphatebuffer (pH 7.4) at room temperature for 1 h. However, the concen-tration of 8-Br-Guo did not decrease and Guo was not detected inthe RP-HPLC chromatogram.

It has been reported that 5-bromo-20-deoxyuridine (5-Br-dUrd)reacts with Cys generating a Cys adduct, 5-S-L-cysteinyl-20-deoxy-uridine (Cys-dUrd), and a debromination product, 20-deoxyuridine(dUrd).15 The dUrd formation in the 5-Br-dUrd/Cys reaction wasmaximal at pH 8, and the Cys-dUrd formation was most favorableat pH 9. The reaction was very slow below pH 5 and above pH 10.The pH profile for the product yields is quite different from that ofthe present 8-Br-Guo/Cys reaction, suggesting that a different reac-tion mechanism exists. In the reaction of 8-Br-Guo with Cys, theyield of Cys-Guo increased with increasing pH with a sigmoidalprofile (Fig. 4). The pKa of Cys is 8.3 for the thiol group.16 At mildlyacidic pH, a simple nucleophilic attack of the thiol group (–SH) ofCys to C8 of 8-Br-Guo and subsequent elimination of Br� and H+

would generate Cys-Guo. The thiolate (–S�) of Cys formed at mildlybasic pH attacks the C8 atom more efficiently. In contrast, the yieldof Guo gradually decreased with increasing pH. For the formationof Guo, the pathway via Cys-Guo was ruled out. Meanwhile, Guowas not generated from 8-Br-Guo by NaBH4. The reaction mecha-nism for the formation of Guo from 8-Br-Guo by Cys is unclear.The possible reaction pathways are summarized in Scheme 1.

Cys-Guo was relatively stable (Fig. 5). If Cys adducts at guanineC8 were formed in RNA and DNA, they would remain for a longperiod. Although the serum concentration of Cys is low (34 lM),the intracellular level of glutathione (GSH, pKa = 8.8) in mamma-lian cells is in the millimolar range (0.5–10 mM).17,18 GSH may alsoreact with 8-bromoguanine, generating stable products as well asfree Cys at guanine C8 in RNA and DNA. These thiol adducts atC8 of guanine can exert an influence on RNA and DNA functionsdifferent from that of 8-bromoguanine. Recently, a new signaltransduction mechanism termed protein S-guanylation was re-vealed.19 8-Nitroguanosine-30,50-cyclic monophosphate (8-NO2-cGMP) reacts with cysteine thiols of a protein Keap1, generatingcGMP adduct of the protein. The formation of S-guanylation regu-lated the redox-sensor signaling protein. The results of the presentstudy suggest that 8-bromoguanosine-30,50-cyclic monophosphate(8-Br-cGMP), either generated by endogenously-formed HOBrfrom guanosine-30,50-cyclic monophosphate (cGMP) or addedexogenously as a reagent, may also react partially with cysteinethiols of proteins resulting in cGMP adducts as well as 8-NO2-cGMP. The reaction of 8-bromoguanine nucleosides with Cys maybe slower than that of 8-nitroguanine nucleosides with Cys. How-ever, 8-bromoguanine nucleosides are relatively stable, whereas 8-nitroguanine nucleosides are labile for N-glycosidic bond, resultingin depurination, with half-lives of 3 min for 8-nitro-20-deoxygua-nosine and 5 h for 8-nitroguanosine at pH 7 and 37 �C.20,21 Thus,8-bromoguanine would stay in nucleosides and nucleic acids fora long period and can react with thiols resulting in adducts.

The present results show that relatively stable Cys adducts canform from 8-bromoguanine nucleosides under neutral conditions.It suggests that these adducts may have some importance in eluci-dating the influence of 8-bromoguanine in nucleosides, nucleo-tides, RNA and DNA in genotoxicity and signal transduction.

References and notes

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10. Asahi, T.; Kondo, H.; Masuda, M.; Nishino, H.; Aratani, Y.; Naito, Y.; Yoshikawa,T.; Hisaka, S.; Kato, Y.; Osawa, T. J. Biol. Chem. 2010, 285, 9282.

11. Spectrometric data of 8-S-L-cysteinylguanosine (Cys-Guo). 1H NMR (500 MHz,DMSO-d6): d (ppm/TMS) 6.92 (s, 2H, NH2), 5.73 (d, J = 6.3 Hz, 1H, H-10), 4.89(dd, 1H, H-20), 4.13 (dd, 1H, H-30), 3.85 (ddd, J = 4.6 Hz, 1H, H-40), 3.71 (ddd, 1H,H-b), 3.67 (ddd, 1H, H-50 or 500), 3.59 (ddd, 1H, H-a), 3.53 (ddd, 1H, H-50 or 500),3.29 (ddd, 1H, H-b). 13C NMR (125 MHz, DMSO-d6): d (ppm/TMS) 171.0(COOH), 156.1 (C-6), 153.5 (C-2), 152.5 (C-4), 142.8 (C-8), 116.8 (C-5), 88.2 (C-10), 85.6 (C-40), 70.6 (C-20), 70.5 (C-30), 62.0 (C-50), 53.9 (C-a), 35.3 (C-b). ESI-TOF/MS (negative ion): m/z 401 [M�H]�, 314 [M�H� Cys]�. HR-ESI-TOF/MS(negative ion): m/z 401.088576 [M�H]� (calculated for C13H17N6O7S,401.088491). UV: kmax 272 nm (pH 7.0).

12. The concentrations of the products were evaluated from integrated peak areason RP-HPLC chromatograms detected at 260 nm and the molecular extinctioncoefficients at 260 nm (e260 nm). The e260 nm values of the authentic sampleswere determined from integration of the H30 proton signal of NMR and theHPLC peak area detected at 260 nm relative to those of Guo (e260

nm = 11,500 M�1 cm�1) in the mixed solution. The estimated e260 nm valueswere 16,000 M�1 cm�1 for 8-Br-Guo and 15,000 M�1 cm�1 for Cys-Guo.

13. Spectrometric data of 8-S-L-cysteinyl-20-deoxyguanosine (Cys-dGuo). 1H NMR(500 MHz, DMSO-d6): d (ppm/TMS) 6.78 (s, 2H, NH2), 6.16 (dd, J = 6.9 and8.0 Hz, 1H, H-10), 4.36 (dd, J = 2.9 Hz, 1H, H-30), 3.78 (ddd, 1H, H-40), 3.68 (ddd,1H, H-b), 3.62 (ddd, 1H, H-50 or 500), 3.53 (dd, 1H, H-a), 3.50 (ddd, 1H, H-50 or500), 3.26 (ddd, 1H, H-b), 3.26 (ddd, 1H, H-20 or 200), 2.04 (ddd, 1H, H-20 or 200).

13C NMR (125 MHz, DMSO-d6): d (ppm/TMS) 170.0 (COOH), 155.8 (C-6), 153.3(C-2), 153.3 (C-4), 142.8 (C-8), 116.9 (C-5), 87.6 (C-40), 83.5 (C-10), 71.0 (C-30),62.0 (C-50), 53.9 (C-a), 36.7 (C-20), 35.3 (C-b). ESI-TOF/MS (negative ion): m/z385 [M�H]�. HR-ESI-TOF/MS (negative ion): m/z 385.092824 [M�H]�

(calculated for C13H17N6O6S 385.093577). UV: kmax 272 nm (pH 7.0).14. The concentrations of the products were evaluated from integrated peak areas

on RP-HPLC chromatograms detected at 260 nm and the molecular extinctioncoefficients at 260 nm (e260 nm). The e260 nm values of the authentic sampleswere determined from integration of the H30 proton signal of NMR and theHPLC peak area detected at 260 nm relative to those of dGuo (e260

nm = 11,800 M�1 cm�1) in the mixed solution. The estimated e260 nm valueswere 16,100 M�1 cm�1 for 8-Br-dGuo and 15,400 M�1 cm�1 for Cys-dGuo.

15. Wataya, Y.; Negishi, K.; Hayatsu, H. Biochemistry 1973, 12, 3992.16. Wang, K.; Wen, Z.; Zhang, W.; Xian, M.; Cheng, J. P.; Wang, P. G. Bioorg. Med.

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