1. Chem 6440/7440 Computational Studies of the Oxidation of Guanine Barbara H. Munk Computational Chemistry 6440/7440

2. Chem 6440/7440 Overview  Background  Research Plan  Results to date  Next Steps  Summary

3. Chem 6440/7440 Background nOxidation of nucleobases and nucleotides followed by strand scission of the DNA/RNA is a major pathway in mutagenesis, carcinogenesis, aging and cell death Burrows, C.J.; Muller, J.G.; Oxidative Nucleobase Modifications Leading to Strand Scission; Chem. Rev. 1998, 98, 1109-1151.

4. Chem 6440/7440 Background Burrows, C.J.; Muller, J.G.; Oxidative Nucleobase Modifications Leading to Strand Scission; Chem. Rev. 1998, 98, 1109-1151.

5. Chem 6440/7440 Background nGuanine has a lower redox potential than other nucleobases and chemical oxidation of this base is observed experimentally nOxidants include reactive oxygen species, ionizing radiation, and transition metal complexes nReactive oxygen species include: HO·, RO·, ROO·, and O 2 · Baik, M.H.; Silverman, J.S.; Yang, I.V.; Ropp, P.A.; Szalai, V.A.; Yang, W.; and Thorp, W.H.; Using Density Functional Theory to Design DNA Base Analogues with Low Oxidation Potentials; J. Phys. Chem. B.; 2001, 105, 6437-6444

6. Chem 6440/7440 Background Nucleobase and Nucleoside Numbering Schemes Burrows, C.J.; Muller, J.G.; Oxidative Nucleobase Modifications Leading to Strand Scission; Chem. Rev. 1998, 98, 1109-1151.

7. Chem 6440/7440 Background Oxidation of guanine can occur at three sites

8. Chem 6440/7440 Background nProducts formed by attack at C-4 and C-5 revert to guanine nOxidation at C-8 leads to two forms of DNA damage

9. Chem 6440/7440 Background

10. Chem 6440/7440 Research Plan nUse a minimally substituted guanine structure nEvaluate oxidation at C-4, C-5 and C-8 positions with ·OH, ·OCH 3, and ·OOH

11. Chem 6440/7440 Research Plan nCalculate the enthalpy and free energy of reaction, and forward and reverse barrier heights using Gaussian (Development Version) on Linux operating system nElectron correlation important  Use Density Functional Theory – B3LYP nBasis set – 6-31G(d)  Works well for organic molecules  Polarization functions give molecular flexibility Prat, F.; Houk, K.N.; Foote, C.S.; Effect of Guanine Stacking on the Oxidation of 8-Oxoguanine in B-DNA. J. Am. Chem. Soc. 1998, 120, 845-846. Sugiyama, H.; Saito, I.; Theoretical Studies of GG-Specific Photocleavage of DNA via Electron Transfer: Significant Loweering of Ionization potential and 5’ Localization of HOMO of Stacked GG Bases in B-Form DNA. J. Am. Chem. Soc. 1996, 118, 7063-7068.

12. Chem 6440/7440 Results to Date

13. Chem 6440/7440 Reactions with ·OH Enthalpy of Reaction (Kcal/mol) Barrier Height Forward (Kcal/mol) Barrier Height Reverse (Kcal/mol) Free Energy of Reaction (Kcal/mol) Barrier Height Forward (Kcal/mol) Barrier Height Reverse (Kcal/mol) 4-hydroxyguanine-18.26-1.6216.64-8.198.2816.47 5-hydroxyguanine-13.67-4.339.34-3.985.419.37 8-hydroxyguanine-33.06**-23.61** * To be determined

14. Chem 6440/7440 Guanine 4-Hydroxyguanine radical 4-Hydroxyguanine transition state

15. Chem 6440/7440 Guanine 5-Hydroxyguanine radical 5-Hydroxyguanine transition state

16. Chem 6440/7440 Guanine8-hydroxyguanine radical

17. Chem 6440/7440 Reactions with ·OCH 3 Enthalpy of Reaction (Kcal/mol) Barrier Height Forward (Kcal/mol) Barrier Height Reverse (Kcal/mol) Free Energy of Reaction (Kcal/mol) Barrier Height Forward (Kcal/mol) Barrier Height Reverse (Kcal/mol) 4-methoxyguanine-2.8610.3713.238.7621.5612.80 5-methoxyguanine0.626.886.2612.2218.636.41 8-methoxyguanine-18.981.0920.07-7.3412.1319.47

18. Chem 6440/7440 Guanine 4-Methoxyguanine radical 4-Methoxyguanine transition state

19. Chem 6440/7440 Guanine 5-Methoxyguanine radical 5-Methoxyguanine transition state

20. Chem 6440/7440 Guanine 8-Methoxyguanine transition state 8-Methoxyguanine radical

21. Chem 6440/7440 Reactions with ·OOH Enthalpy of Reaction (Kcal/mol) Barrier Height Forward (Kcal/mol) Barrier Height Reverse (Kcal/mol) Free Energy of Reaction (Kcal/mol) Barrier Height Forward (Kcal/mol) Barrier Height Reverse (Kcal/mol) 4-hydroperoxy guanine complex 11.4613.291.8323.0325.302.27 4-epoxyguanine15.95**18.44** * To be determined

22. Chem 6440/7440 Guanine 4-Hydroperoxy guanine complex 4-Epoxyguanine

23. Chem 6440/7440 Reactions with ·OOH Enthalpy of Reaction (Kcal/mol) Barrier Height Forward (Kcal/mol) Barrier Height Reverse (Kcal/mol) Free Energy of Reaction (Kcal/mol) Barrier Height Forward (Kcal/mol) Barrier Height Reverse (Kcal/mol) 5-hydroperoxy guanine complex 13.94**25.60** 5-epoxyguanine15.95**18.44** * To be determined

24. Chem 6440/7440 Guanine5-Epoxyguanine 5-Hydroperoxy guanine complex

25. Chem 6440/7440 Reactions with ·OOH Enthalpy of Reaction (Kcal/mol) Barrier Height Forward (Kcal/mol) Barrier Height Reverse (Kcal/mol) Free Energy of Reaction (Kcal/mol) Barrier Height Forward (Kcal/mol) Barrier Height Reverse (Kcal/mol) 8-hydroperoxy guanine complex -5.94**5.80 8-oxo-guanine**** 2,6-diamino-5- formamido-4- hydroxy pyrimidine 0.71**0.49** * To be determined

26. Chem 6440/7440 Guanine 2,6-Diamino-5- formamido-4- hydroxy pyrimidine 8-Hydroperoxy guanine complex

27. Chem 6440/7440 Next Steps nIdentify transition states for  8-hydroxyguanine  4, 5, and 8 hydroperoxyguanine  4 and 5 epoxyguanine  8-oxo-guanine  2, 6-diamino-5-formamido-4-hydroxy pyrimidine (FAPy-G)

28. Chem 6440/7440 Summary nOxidation by ·OH and ·OCH3 at the C-8 position appears to be thermodynamically more favorable than oxidation at C-4 and C-5 nOxidation by ·OOH appears to be a multistep process nOxidation at the C-4 and C-5 positions may proceed through an epoxide intermediate

29. Chem 6440/7440 Acknowledgements  Dr. H.B. Schlegel  Schlegel Group  Dr. Smriti Anand  Dr. Hrant Hratchian  Jie Li  Stan Smith  Funding  Dept. of Chemistry, WSU  NSF  Gaussian Inc.  Computer Time  NCSA  WSU- C&IT

30. Chem 6440/7440 Reactions Generating ·OH

31. Chem 6440/7440 Supplemental Material nAlkoxyl radicals RO· can be generated via radical ring opening of epoxides with a nickel catalyst or via hydroperoxides nROO· are generated in vivo, as lipid hydroperoxides are produced as a consequence of cellular exposure to oxidative stress