CHAPTER 8 The Chemical Reactions of DNA Damage and Degradation KENT S. GATES Department of Chemistry, University of Missouri, Columbia, MO 8.1. Introduction and Historical Perspective 334 8.1.1. The Importance of DNA Damage 334 8.1.2. Chemical Agents that Damage DNA and the Chemistry of DNA Degradation 334 8.2. Reactions of Electrophiles with DNA 335 8.2.1. Nucleophilic Sites in DNA and the Factors that Determine the Sites at Which Electrophiles React with DNA 335 8.2.2. The Sequence Context of a DNA Base Affects its Nucleophilicity 336 8.2.3. The Nature of the Reactive Intermediate Involved in the Alkylation Reaction Infl uences Atom Site Selectivity 336 8.2.4. Noncovalent Association of the Alkylating Agent can Direct Reaction to Selected Atoms and Selected Sequences in DNA 337 8.3. Possible Fates of Alkylated DNA 337 8.3.1. Alkylation at Some Sites in DNA Yields Stable Lesions 337 8.3.2. Alkylation at Some Sites in DNA Yields Unstable (Labile) Lesions 338 8.3.3. Deglycosylation Reactions of Alkylated Bases 338 8.3.4. Ring-Opening Reactions of Alkylated Bases 339 8.3.5. Alkylation of Adenine and Cytosine Residues can Accelerate Deamination 341 8.3.6. Reactions Where the DNA Base Serves as a Leaving Group: Thiolysis, Hydrolysis, Elimination, and Reversible DNA Alkylation 341 8.4. Selected Examples of DNA-Alkylating Agents 344 8.4.1. Episulfonium Ions 344 8.4.2. Aziridinium Ions 347 8.4.3. Alkenylbenzenes: DNA Alkylation by Resonance-Stabilized Carbocations 349 8.5. Reaction of Radical Intermediates with DNA 351 8.5.1. General Considerations Regarding Hydrogen Atom Abstraction from the 2Ј-Deoxyribose Sugar 351 8.5.2. Strand Cleavage Stemming from Abstraction of the C1Ј-Hydrogen Atom 351 Reviews of Reactive Intermediate Chemistry. Edited by Matthew S. Platz, Robert A. Moss, Maitland Jones, Jr. Copyright © 2007 John Wiley & Sons, Inc. 333 334 THE CHEMICAL REACTIONS OF DNA DAMAGE AND DEGRADATION 8.5.3. Strand Cleavage Initiated by Abstraction of the 4Ј-Hydrogen Atom 353 8.5.4. Oxidative RNA Damage 355 8.5.5. General Considerations Regarding Reactions of Radicals with the DNA Bases 355 8.5.6. Hydrogen Atom Abstraction from the C5-Methyl Group of Thymine 356 8.5.7. Addition of Radicals to C5 of Thymine Residues 358 8.5.8. Addition of Radicals to C6 of Thymine Residues 359 8.5.9. Addition of Radicals to Guanine Residues 359 8.5.10. Damage Amplifi cation: Reaction of DNA Radical Intermediates with Proximal DNA Bases and Sugars 360 8.6. Examples of DNA-Damaging Radicals 362 8.6.1. γ-Radiolysis 362 8.6.2. Tirapazamine 362 8.6.3. Photolytic Generation of Hydroxyl Radical 366 Ϫ 8.6.4. Agents that Reduce Molecular Oxygen to Superoxide Radical (O2• ) 366 8.7. Conclusion and Outlook 367 Suggested Reading 368 References 368 8.1. INTRODUCTION AND HISTORICAL PERSPECTIVE 8.1.1. The Importance of DNA Damage The sequence of heterocyclic bases in double-helical deoxyribonucleic acid (DNA) forms the genetic code that serves as the blueprint for all cellular operations.1,2 Accu- rate readout of genes during transcription of cellular DNA is required for production of functional proteins.2 In addition, faithful replication of DNA must take place dur- ing cell division to yield daughter cells containing exact copies of the genetic code.2,3 With this said, it is not surprising that chemical modifi cation of cellular DNA can have profound biological consequences including induction of DNA repair proteins, inhibition of cell growth (cell cycle arrest), or cell death via either necrotic or apop- totic mechanisms.4–7 Several cellular systems repair damaged DNA.8–11 Nonetheless, in attempts to replicate damaged DNA, polymerases may introduce errors into the genetic code (mutagenesis).12–19 Accordingly, the study of agents that damage DNA is of both practical and fundamental importance to diverse fi elds including medici- nal chemistry, carcinogenesis, toxicology, and biotechnology. 8.1.2. Chemical Agents that Damage DNA and the Chemistry of DNA Degradation There is rather a small number of functional groups or structural motifs that have the ability to carry out effi cient modifi cation of cellular DNA.20–24 These molecules are of chemical and biological interest because they successfully execute a diffi cult balancing act—they possess suffi cient reactivity to make and break covalent bonds REACTIONS OF ELECTROPHILES WITH DNA 335 DNA–Nu: + E+ DNA–Nu+–E • + R• R DNA base DNA–H++R• DNA• R–H Scheme 8.1 within DNA, yet possess suffi cient stability to survive in the aqueous environment of the cell. Almost all of the cellular DNA damage reactions carried out by drugs, toxins, and mutagens fall into just two general categories: (1) the reaction of a DNA nucleophile with an electrophile or (2) the reaction of a DNA pi bond or C–H bond with a radical (Scheme 8.1). In the following sections of this chapter, we will exam- ine the degradation of DNA that is initiated by its reaction with electrophiles and radicals. In addition, we will consider examples that illustrate chemical strategies by which organic molecules can deliver these highly reactive intermediates to DNA un- der physiological conditions. It should be noted that many of the reaction mechanisms shown in this review are schematic in nature. For example, protonation, deproton- ation, and proton transfer steps may not be explicitly depicted or may not distinguish specifi c acid–base catalysis from general acid–base catalysis. In some cases, arrows indicating electron movement are used to direct the reader’s attention to sites where the “action” is occurring rather than to illustrate a complete reaction mechanism. 8.2. REACTIONS OF ELECTROPHILES WITH DNA 8.2.1 Nucleophilic Sites in DNA and the Factors that Determine the Sites at Which Electrophiles React with DNA Virtually, all of the heteroatoms in DNA (Fig. 8.1) have the potential to act as nucleophiles in reaction with electrophiles.25,26 As one might expect, access to some sites is limited in double-stranded DNA relative to single-stranded DNA.25,26 Interestingly, however, reac- tions are not completely precluded even at locations on Watson–Crick hydrogen bond- ing surfaces of the bases that reside near the helical axis of the duplex. The factors that determine the atom site selectivity for a given DNA-alkylating agent are complex.25–29 A recent detailed study of alkylation by diazonium ions led to the conclusion that atom site selectivities seen in duplex DNA do not refl ect intrinsic nucleophilicities of the hetero- atoms in the nucleobases.29 Rather, placement of the nucleobases into the environment of the double helix substantially alters the nucleophilicity of base heteroatoms. Factors that alter the nucleophilicities of various heteroatoms in the DNA bases, when placed within the context of double helix, include proximity of the polyanionic sugar-phosphate backbone, lower dielectric constants in the DNA grooves relative to bulk water, and in- teraction of the inherent dipoles of the nucleobases with the electrostatic environment of the double helix (e.g., charges of the backbone and neighboring bases).29 336 THE CHEMICAL REACTIONS OF DNA DAMAGE AND DEGRADATION 5′ HO C5′ B HO B O ′ O C4 C1′ C3′ C2′ O O OH –O –O P P O O O B O B O O 3′ HO HO OH DNA Backbone RNA Backbone B=DNA Base Major Groove Major Groove CH3 H O N H H 5 H 5 O N 3 3 7 6 NN 7 6 NN N H 1 R N H 1 R N 1N 8 1 O 8 O 9 9 H N N 2 N N 2 N 3 R 3 R H A-T G-C Minor Groove Minor Groove R=DNA Backbone Figure 8.1. See color insert. 8.2.2. The Sequence Context of a DNA Base Affects its Nucleophilicity The sequence context in which a given DNA base resides can also affect its nucleophilicity. For example, many studies have documented the fact that fl anking bases have a marked effect on the reaction of the N7-position of guanine residues with positively charged electrophiles such as aziridinium ions and diazaonium ions.30–34 The observed sequence specifi city for DNA alkylation by these agents often correlates well with the calculated sequence-dependent variations in the mo- lecular electrostatic potential at the N7-position of the guanine alkylation site.30–35 For example, for many agents, alkylation at 5Ј-GGG sequences is markedly favored over that at 5Ј-CGC sequences (where the G is alkylated). 8.2.3. The Nature of the Reactive Intermediate Involved in the Alkylation Reaction Infl uences Atom Site Selectivity The favored sites for alkylation of duplex DNA vary depending upon the nature of the alkylating agent. For example, in the alkylation of double-stranded DNA by POSSIBLE FATES OF ALKYLATED DNA 337 dimethylsulfate, the preferred sites of attack follow the order: N7G ϾϾ N3A ϾϾ N1AϳN3AϳN3G ϾϾ O6G.25,26 On the contrary, when methyldiazonium ion is the alkylating agent, the preferred sites of attack are: phosphate oxygen Ͼ N7G Ͼ O2T Ͼ O6G Ͼ N3A.25,26 Such trends have often been rationalized by noting that hard, SN1-type alkylating agents such as methyldiazonium ion display increased reactivity with hard oxygen nucleophiles in DNA.25–28 This analysis is of some practical value in predicting and rationalizing alkylation site preferences; however, in some cases the traditional classifi cations of alkylating agents as “SN1 or SN2” may be inaccurate and, therefore, confusing. For example, primary diazonium ions that have typically 28 been classifi ed as SN1-type alkylating agents, in fact, are known to react strictly 36,37 via bimolecular SN2 mechanisms.