Photolysis of hydroxocobalamin in the presence of plasmid DNA (pBR322) results in DNA cleavage. Temporal control of hydroxyl radical production and DNA strand scission by hydroxocobalamin was demonstrated using a 2-deoxyribose assay and a plasmid relaxation assay, respectively. The light-driven hydroxocobalamin-mediated catalytic formation of hydroxyl radicals was demonstrated using radical scavenging studies of DNA cleavage and via recycling of a hydroxocobalamin-resin several times without loss of efficacy

Lawrence, David S.

Shell, Thomas A.

PubMed

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A New Trick (Hydroxyl Radical Generation) For An Old Vitamin(B12)Thomas A. Shell and David S. LawrenceDepartment of Chemistry, Division of Medicinal Chemistry and Natural Products, Department ofPharmacology University of North Carolina, Chapel Hill, NC 27599AbstractPhotolysis of hydroxocobalamin in the presence of plasmid DNA (pBR322) results in DNAcleavage. Temporal control of hydroxyl radical production and DNA strand scission byhydroxocobalamin was demonstrated using a 2-deoxyribose assay and a plasmid relaxation assay,respectively. The light-driven hydroxocobalamin-mediated catalytic formation of hydroxylradicals was demonstrated using radical scavenging studies of DNA cleavage and via recycling ofa hydroxocobalamin-resin several times without loss of efficacy.Hydroxyl radicals (·OH) have been used to assess DNA structure1 and to elucidate DNA-binding sites for proteins2 and small molecules.3 An example of the Fenton reaction, inwhich Fe(II)·EDTA reduces H2O2, is the most commonly used method to generate ·OH.However, for intracellular purposes, a limitation of this method is the inability to preciselycontrol when the reaction starts and stops. A Fenton-like reaction utilizing a caged Cu(II)ion that can be released by photolysis of the ligand cage has been reported.4 This strategyfurnishes control over the initiation of the Fenton reaction, albeit not the termination step.Several light-mediated “Fenton reagents” have been developed.5 In contrast to the Fentonreaction, the majority of these compounds have the disadvantage of stoichiometric radicalproduction. TiO2 does catalytically produce ·OH upon exposure to light,6 however sinceTiO2 induces intracellular oxidative damage in the absence of light,7 it cannot be used in alight-dependent fashion in cell-based studies. By contrast, synchrotron X-ray8 and γ9irradiation can be used to precisely initiate and terminate ·OH production. Although thesemethods are advantageous for their ability to provide time resolved information, they are notwidely available and they cannot be used to direct oxidative damage to specific intracellularor molecular sites.Cobalamins, a class of compounds of which Vitamin B12 is a member, could be useful forgenerating radical species under cellular conditions. For example, photolysis of the cobalt-carbon bond in methylcobalamin (MeCbl; 1 where X = Me) generates cob(II)alamin (B12R)and methyl radical (·CH3) (Scheme 1A).10 Based on this precedent, we wondered whetherhydroxocobalamin (B12a where X = OH) could undergo an analogous reaction to generate·OH and B12R (Scheme 1B). In the presence of oxygen, the latter species is rapidly oxidizedto regenerate B12a.10 We therefore investigated the possibility that the combination of lightand B12a could be used to oxidatively damage biomolecules via the generation of ·OH.tshell@email.unc.edu and lawrencd@email.unc.edu.Supporting Information Available: Figures S1-S22 and procedures are available free of charge via the internet at http://pubs.acs.org.NIH Public AccessAuthor ManuscriptJ Am Chem Soc. Author manuscript; available in PMC 2012 February 23.Published in final edited form as:J Am Chem Soc. 2011 February 23; 133(7): 2148–2150. doi:10.1021/ja111585c.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author ManuscriptInitial studies employed a plasmid relaxation assay to observe the ·OH-mediated conversionof circular, supercoiled DNA (Form I) to relaxed, circular DNA (Form II). A Pyrex filter(>300 nm) was used to photolyze B12a in the presence of pBR322 DNA. Analysis byagarose gel electrophoresis revealed a band corresponding to Form II DNA, but only fromthe light-exposed reaction mixtures containing B12a (Fig. S1). By contrast, light in theabsence of B12a or B12a in the absence of light, does not result in significant DNA damage.DNA cleavage by B12a does not occur in the absence of O2, an observation consistent with aradical-mediated mechanism of DNA damage (Fig. S3).11 In addition, the ·OH scavengerssorbitol12 and sodium benzoate,13 efficiently prevent DNA cleavage (Fig. S5), therebysupporting the notion that damage is mediated by ·OH. The light-dependence of DNAcleavage was assessed by photolyzing B12a for various time periods followed by incubationin the dark (Fig. S8). Short photolysis periods followed by dark incubation resulted insignificantly less strand scission than photolyzing for the same total time period,demonstrating that the species formed by the photolysis of B12a does not continue to damageDNA without continued exposure to light.We employed an established ·OH assay to assess light-mediated production of ·OH.14Deoxyribose is attacked by ·OH, generating malondialdehyde that, upon heating withthiobarbituric acid (TBA), produces a chromophore (532 nm).15 Unfortunately, we foundthat the presence of B12a during the heating step interferes with the assay. Therefore, B12awas immobilized on an aminebearing resin (2) using 1,1′-carbonyldi-(1,2,4-triazole) (Fig.S10). Deoxyribose was incubated with the B12a-resin under photolytic and non-photolyticconditions and the resin was subsequently removed prior to the heating step. The light-driven nature of B12a-resin-mediated ·OH production is evident by the time-dependentproduction of the TBA-malondialdehyde chromophore in the presence (Fig. 1, averageabsorbance increase per period: 0.104), but not in the absence (average absorbance increaseper period: 0.003), of light.Photolysis of the Co-methyl bond in MeCbl generates Co(II)-cobalamin and ·CH3 (Scheme1A). The former, upon oxidation, produces B12a,10 which should generate DNA damaging·OH under photolytic conditions (Scheme 1B). However, ·CH3 is also capable of causingDNA strand scission.16 We assessed the relative contribution of these two radical species toDNA damage using sorbitol and sodium benzoate (·OH traps) and 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO; a carbon-centered radical trap17). TEMPO does nothave an appreciable effect on the ability of MeCbl to damage DNA, but sorbitol and sodiumbenzoate almost completely protect DNA from damage (Fig. S11). Therefore, the initialstoichiometric production of ·CH3 is small compared to that of the B12a-mediated generationof ·OH.The notion that B12a catalytically produces ·OH is supported by radical trapping studies ofDNA damage by re-using the B12a-resin 2 several times without loss in its capacity todamage deoxyribose (Fig. S13). Furthermore, a B12a concentration less than the pBR322concentration was used to demonstrate catalytic production of strand scission events. Basedon the number of observable strand scission events, B12a induced a minimum of 20damaging events per molecule during 90 min of light exposure (Fig. 2); an underestimatesince plasmid relaxation assays do not visualize all DNA nicks. The amount of DNAdamage per molecule increased with longer exposure to light. A band corresponding tolinear DNA (form III) resulting from nonrandom double-strand cleavage was observed(Figs. S15-16),18 indicative of two radicals originating near each other in time and in space.A B12a-spermine conjugate 3 was synthesized (Fig. S17) and proved useful for directingdamage to DNA in an environment with a high concentration of a hydroxyl radical trap.Shell and Lawrence Page 2J Am Chem Soc. Author manuscript; available in PMC 2012 February 23.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author ManuscriptConjugate 3 forms a DNA complex with an apparent binding constant (Kapp = 1.5 × 108 ±3.4 × 106 M-1) similar to the reported value for spermine (5.9 × 108 M-1).19 The B12a-spermine conjugate induces DNA damage more effectively than B12a (Fig. S20). Moreimportantly, 10000 equiv. of sorbitol failed to significantly inhibit DNA damage induced by3, whereas only 100 equiv. of sorbitol significantly inhibited strand scission due to B12a(Fig. S22), suggesting that 3 generates ·OH near the DNA 3 it is bound to.Light-dependent ·OH production by B12a enjoys several characteristics that make itpotentially useful as a generator of ·OH for biological studies. Light is increasinglyemployed in modern cell biology to control intracellular processes via cell-embeddedphotosensitive bioactive reagents.20 The combination of light and light-sensitive reagentsprovides the investigator with exquisite temporal control over cellular biochemistry. Spatialcontrol of oxidative damage is afforded by the addition of a binding moiety to B12a becausethe highly reactive ·OH is generated proximal to the target biomolecule. In addition, light-induced radical generation with B12a is catalytic, whereas radical generation by othersystems is stoichiometric. Future research will focus on conjugating recognition moieties toB12a to direct oxidative damage to specific macromolecules and organelles.Supplementary MaterialRefer to Web version on PubMed Central for supplementary material.AcknowledgmentsWe thank the NIH (CA079954).References1. Tullius, TD. Radiation Research. Moriarty, M., editor. Vol. 2. Allen Press; Lawrence, KS: 2000. p.3332. a Jain SS, Tullius TD. Nat. Protoc. 2008; 3:1092. [PubMed: 18546600] b Frazee RW, Taylor JA,Tullius TD. J. Mol. Biol. 2002; 323:665. [PubMed: 12419257] c Zaychikov E, Schickor P,Denissova L, Heumann H. Methods Mol. Biol. 2001; 148:49. [PubMed: 11357607]3. Trauger JW, Dervan PB. Methods Enzymol. 2001; 340:450. [PubMed: 11494863]4. Ciesienski KL, Haas KL, Dickens MG, Tesema YT, Franz KJ. J. Am. Chem. Soc. 2008; 130:12246.[PubMed: 18714999]5. a Haruna S, Kuroi R, Kajiwara K, Hashimoto R, Matsugo S, Tokumaru S, Kojo S. Bioorg. Med.Chem. Lett. 2002; 12:675. [PubMed: 11844698] b Adam W, Cadet J, Dall'Acqua F, Epe B,Ramaiah D, Saha Moeller CR. Angew. Chem., Intl. Ed. Eng. 1995; 34:107. c Matsugo S, KawanishiS, Yamamoto K, Sugiyama H, Matsuura T, Saito I. Angew. Chem., Int. Ed. Engl. 1991; 30:1351. dAveline BM, Matsugo S, Redmond RW. J. Am. Chem. Soc. 1997; 119:11785. e Tobin D,Arvanitidis M, Bisby RH. Biochem. Biophys. Res. Commun. 2002; 299:155. [PubMed: 12435402]f Kitano H, Maeda Y, Furukawa K, Yamamoto T, Izumida R, Matsugo S. Photochem. Photobiol.1995; 62:809. [PubMed: 8570717] g Guptasarma P, Balasubramanian D, Matsugo S, Saito I.Biochemistry. 1992; 31:4296. [PubMed: 1567875] h Takeuchi T, Matsugo S, Morimoto K.Carcinogenesis. 1997; 18:2051. [PubMed: 9395201]6. a Chang C-Y, Hsieh Y-H, Hsieh L-L, Yao K-S, Cheng T-C. J. Hazard. Mat. 2009; 166:897. bMatthews RH. J. Chem. Soc. Chem Comm. 1983:177.7. Trouiller B, Reliene R, Westbrook A, Solaimani P, Schiestl RH. Cancer Res. 2009; 69:8784.[PubMed: 19887611] Gurr J-R, Wang ASS, Chen C-H, Jan K-Y. Toxicology. 2005; 213:66.[PubMed: 15970370]8. a Adilakshmi T, Soper SFC, Woodson SA. Methods Enzymol. 2009; 468:239 - 58. [PubMed:20946773] b Brenowitz M, M. R, C. Dhavan G, Takamoto K. 2002; 12:648–53.9. Ottinger LM, Tullius TD. J. Am. Chem. Soc. 2000; 122:5901.Shell and Lawrence Page 3J Am Chem Soc. Author manuscript; available in PMC 2012 February 23.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author Manuscript10. a Taylor R, Smucker L, Hanna ML, Gill J. Arch. Biochem. Biophys. 1973; 156:521. [PubMed:4718782] b Schrauzer GN, Lee L-P, Sibert JW. J. Am. Chem. Soc. 1970; 92:2997. [PubMed:5446952]11. Shell TA, Mohler DL. Curr. Org. Chem. 2007; 11:1525.12. Morelli R, Russo-Volpe S, Bruno N, Scalzo RL. J. Agric. Food Chem. 2003; 51:7418. [PubMed:14640593]13. Beauchamp C, Fridovich I. J. Biol. Chem. 1970; 245:4641. [PubMed: 5466067]14. Halliwell B, Gutteridge JMC, Aruoma OI. Anal. Biochem. 1987; 165:215. [PubMed: 3120621]15. a Gutteridge JMC. FEBS Lett. 1981; 128:343. [PubMed: 7262325] b Cheeseman KH, Beavis A,Esterbauer H. Biochem. J. 1988; 252:64. c Gutteridge JMC, Halliwell B. Biochem. J. 1988;253:932. [PubMed: 2845941]16. a Mohler DL, Downs JR, Hurley-Predecki AL, Sallman JR, Gannett PM, Shi X. J. Org. Chem.2005; 70:909. b Mohler DL, Dain DR, Kerekes AD, Nadler WR, Scott TL. Bioorg. Med. Chem.Lett. 1998; 8:871. [PubMed: 9871557]17. Connolly TJ, Baldoví MV, Mohtat N, Scaiano JC. Tetrahedron Lett. 1996; 37:4919.18. Povirk LF, Wübker W, Köhnlein W, Hutchinson F. Nucleic Acids Res. 1977; 4:3573. [PubMed:73164]19. Kapp PD. Biochemistry. 1984; 23:4821. [PubMed: 6238622]20. Lee HM, Larson DR, Lawrence DS. ACS Chem. Biol. 2009; 19:409. [PubMed: 19298086]Shell and Lawrence Page 4J Am Chem Soc. Author manuscript; available in PMC 2012 February 23.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author ManuscriptScheme 1.(A) Photolysis of MeCbl to furnish CH3· and (B) proposed light-dependent generation of·OH by B12a.Shell and Lawrence Page 5J Am Chem Soc. Author manuscript; available in PMC 2012 February 23.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author ManuscriptFigure 1.Light-driven ·OH production via photolysis of the B12a-resin conjugate 2. The conjugate 2was suspended in a deoxyribose solution (50 mM in pH 7.4 PBS). The suspension wassequentially photolyzed (60 min total), stored in the dark (100 min total), and photolyzed(60 min total), with aliquots removed and analyzed at various time points.Shell and Lawrence Page 6J Am Chem Soc. Author manuscript; available in PMC 2012 February 23.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author ManuscriptFigure 2.Light-mediated DNA (30 μM/bp in pH 7.4 PBS) by hydroxocobalamin (300 pM). Sampleswere irradiated with Pyrex-filtered light from a mercury arc lamp.Shell and Lawrence Page 7J Am Chem Soc. Author manuscript; available in PMC 2012 February 23.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author Manuscript

A New Trick (Hydroxyl Radical Generation) for an Old Vitamin (B 12 )

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A New Trick (Hydroxyl Radical Generation) for an Old Vitamin (B 12 )

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