Department of Chemistry Unravelling the Potential of Sulfur-Substituted DNA Analogues as UVA Photosensitizers MARVIN POLLUM and CARLOS E. CRESPO-HERNÁNDEZ American Society for Photobiology June 15, 2014

Department of Chemistry Photosensitizers Compounds that initiate a chemical reaction following absorption of light Medicinal Uses Treatment of various skin diseases Cancer treatment Alone No Effect Photosensitizer Light + Chemical Change

Photosensitizers Necessary Properties Strong absorption of light Department of Chemistry Department of Chemistry Photosensitizers Necessary Properties Strong absorption of light For medicinal application longer wavelengths desired Figure: Moreira, M.C.; Prado, R.; Campos, Applied Biomedical Engineering. 2011

Photosensitizers Necessary Properties Strong absorption of light Department of Chemistry Department of Chemistry Photosensitizers Necessary Properties Strong absorption of light For medicinal application longer wavelengths desired Light absorption populates reactive excited states Typically lowest energy triplet state because of long lifetime S(*) T1 S0 3O2 1O2 Biomolecule Type I Type II

Thiobases as Photosensitizers Department of Chemistry Department of Chemistry Thiobases as Photosensitizers Sulfur-substituted DNA base analogues Begin with thiothymine Change site of sulfur substitution Change degree of sulfur substitution Impact of sulfur substitutions on: Absorption properties Excited-state dynamics Propose thiothymines as potential photosensitizers?

Steady-State Absorption Department of Chemistry Department of Chemistry Steady-State Absorption UVC UVB UVA Tune absorption over 100 nm

Excited-State Dynamics Department of Chemistry Department of Chemistry Excited-State Dynamics Broadband Transient Absorption Spectroscopy ~200 femtosecond time resolution Pump Laser Pulse Probe Broadband Pulse Delay time I0 I Sample Detector ~320 to 710 nm

Dynamics of 2-thiothymine Department of Chemistry Department of Chemistry Dynamics of 2-thiothymine 0 ps 2.5 ps 2.5 ps 250 ps PBS Buffer, pH 7.4 λex = 316 nm No change in lifetime of experiment (1000 ps) Pollum, M. and Crespo-Hernández, C. E. J. Chem. Phys. 2014, 140, 071101

Dynamics of 2-thiothymine Department of Chemistry Department of Chemistry Dynamics of 2-thiothymine PBS Buffer, pH 7.4 λex = 316 nm Thiobase τISC (ps) Triplet Yield 2tT 0.62 0.9 ± 0.1 4tT 2,4dtT Pollum, M. and Crespo-Hernández, C. E. J. Chem. Phys. 2014, 140, 071101 Taras-Goślińska, K. et al. J. Photochem. Photobio. A. 2014. 89

Dynamics of 4-thiothymine Department of Chemistry Department of Chemistry 0 ps 1.5 ps Dynamics of 4-thiothymine 1.5 ps 80 ps PBS Buffer, pH 7.4 λex = 340 nm No change in lifetime of experiment (1000 ps) Reichardt, C. and Crespo-Hernández, C. E.. J. Phys. Chem. Lett. 2010, 1, 2239-2243

Dynamics of 4-thiothymine Department of Chemistry Department of Chemistry Dynamics of 4-thiothymine PBS Buffer, pH 7.4 λex = 340 nm Thiobase τISC (ps) Triplet Yield 2tT 0.62 0.9 ± 0.1 4tT 0.24 0.85 ± 0.15 2,4dtT Reichardt, C. and Crespo-Hernández, C. E.. J. Phys. Chem. Lett. 2010, 1, 2239-2243

Dynamics of 2,4-dithiothymine Department of Chemistry Department of Chemistry 0 ps 1.5 ps Dynamics of 2,4-dithiothymine 1.5 ps 100 ps PBS Buffer, pH 7.4 λex = 335 nm No change in lifetime of experiment (1000 ps) Pollum, M. and Crespo-Hernández, C. E. (Unpublished). 2014

Dynamics of 2,4-dithiothymine Department of Chemistry Department of Chemistry Dynamics of 2,4-dithiothymine PBS Buffer, pH 7.4 λex = 335 nm Thiobase τISC (ps) Triplet Yield 2tT 0.62 0.9 ± 0.1 4tT 0.24 0.85 ± 0.15 2,4dtT 0.37 N.D. Pollum, M. and Crespo-Hernández, C. E. (Unpublished). 2014

Conclusions Sulfur substitution of thymine results in: Department of Chemistry Department of Chemistry Conclusions Sulfur substitution of thymine results in: Red shift of absorption up to 110 nm Population of reactive triplet excited states Ultrafast High yields In all three derivatives UVC UVB UVA Thiobase τISC (ps) Triplet Yield 2tT 0.62 0.9 ± 0.1 4tT 0.24 0.85 ± 0.15 2,4dtT 0.37 N.D.

Conclusions Propose 4tT and 2,4dtT as effective UVA photosensitizers Department of Chemistry Department of Chemistry Conclusions Propose 4tT and 2,4dtT as effective UVA photosensitizers UVC UVB UVA Thiobase τISC (ps) Triplet Yield 2tT 0.62 0.9 ± 0.1 4tT 0.24 0.85 ± 0.15 2,4dtT 0.37 N.D. Figure: Moreira, M.C.; Prado, R.; Campos, Applied Biomedical Engineering. 2011

Future Work Test 4tT and 2,4dtT as a UVA photosensitizer in cells Department of Chemistry Department of Chemistry Future Work Test 4tT and 2,4dtT as a UVA photosensitizer in cells Use these spectroscopic techniques to screen for other potential photosensitizers

Acknowledgments Dr. Carlos Crespo Group Members Department of Chemistry Department of Chemistry Acknowledgments Dr. Carlos Crespo Group Members National Science Foundation (CHE-1255084)

Department of Chemistry

Experimental Methods Femto- to millisecond time window Department of Chemistry Department of Chemistry Experimental Methods Femto- to millisecond time window Or any combination of these properties

Department of Chemistry UVA Photosensitizers 4-thiothymidine shown to be a good UVA photosensitizer Incorporation into DNA Cancer Cell Targeting Reelfs, O. et. Al. Nucl. Acids Res. 2011, 1. Reelfs, O. Karran, P. Young, A.R. Photochem. Photobio. Sci. 2012, 11, 148.

Type I or Type II Mechanism? Department of Chemistry Department of Chemistry Type I or Type II Mechanism? Singlet Oxygen Yields Collaboration with Steffen Jockusch Direct measurement by 1O2 luminescence Relatively Low Thiobase Singlet Oxygen Yield 4tT 0.17 ± 0.02 2tT N.D. 2,4dtT 0.08 ± 0.01

Singlet oxygen phosphorescence standard SJ_032814 1 Singlet oxygen phosphorescence standard air saturated TRIS buffer (D2O) pH = 7.5 lex = 355 nm lex = 355 nm obs = 1270 nm intensity Phenalenone 1O2= 0.981 normalized intensity l (nm) time (µs) 1R. Schmidt, C. Tanielian, R. Dunsbach, C. Wolf, J. Photochem. Photobiol. A 1994, 79, 11-17.

UVA Photosensitizers Two Modes of Photosensitization Type I Type II Department of Chemistry Department of Chemistry UVA Photosensitizers Two Modes of Photosensitization Type I Direct Reaction Type II Indirect Reaction Singlet Oxygen Generation Typically from the T1 state because of long lifetime S(*) T1 S0 3O2 1O2 Biomolecule Type I Type II

Site and Degree of Substitution Effects Department of Chemistry Department of Chemistry Site and Degree of Substitution Effects Thiobase τISC (ps) τSQ (ps) Triplet Yield 4tT 0.24 84 0.85 ± 0.15 2tT 0.62 49 0.9 ± 0.1 2,4dtT 0.37 35 N.D. Reichardt, C. and Crespo-Hernández, C. E.. J. Phys. Chem. Lett. 2010, 1, 2239-2243 Pollum, M. and Crespo-Hernández, C. E. J. Chem. Phys. 2014, 140, 071101

Background UV Radiation UVC 0% UVB 5% UVA 95% Department of Chemistry Epidermis Dermis UVC 0% UVB 5% UVA 95% Ozone Layer

Background Ultrafast relaxation of the natural bases Department of Chemistry Department of Chemistry Background Ultrafast relaxation of the natural bases Ring puckering at C5 in uracil and thymine

Transient Absorption Spectroscopy (TAS) Department of Chemistry Department of Chemistry Transient Absorption Spectroscopy (TAS) Wavelength Δ A Time 1 S0 S1 Sn T1 Tn Probe Pulse White light ~325 to 710 nm Wavelength Δ A Time 2 ISC Wavelength Δ A Time 3 Absorption spectra of transient species IC Wavelength Δ A Pump Pulse ISC Time 4 Wavelength Δ A Time 5

Transient Absorption Spectroscopy (TAS) Department of Chemistry Department of Chemistry Transient Absorption Spectroscopy (TAS) Wavelength Δ A Wavelength Absorbance Time 1 Wavelength Δ A Time 2 Wavelength Δ A Time 3 Time Absorbance 1 2 3 4 5 Wavelength Δ A Time 4 Wavelength Δ A Time 5

Propose Good UVA Photosensitizers? Department of Chemistry Department of Chemistry Propose Good UVA Photosensitizers? UVC UVB UVA Thiobase τISC (ps) Triplet Yield Singlet Oxygen Yield 4tT 0.21 0.85 ± 0.15 0.17 ± 0.02 2tT 0.69 0.9 ± 0.1 N.D. 2,4dtT 0.52 0.08 ± 0.01

Background Photodynamic Therapy (PDT) Used to treat: Cancer Department of Chemistry Department of Chemistry Background Photodynamic Therapy (PDT) Used to treat: Cancer Variety of skin diseases www.photolitec.org/Tech_PDT

UVC UVB UVA

Sulfur-Substituted Thymine Department of Chemistry Department of Chemistry Sulfur-Substituted Thymine UVC UVB UVA

Dynamics of 2-thiothymine Department of Chemistry Department of Chemistry Dynamics of 2-thiothymine 0 ps 2.5 ps 2.5 ps 250 ps PBS Buffer, pH 7.4 λex = 316 nm No change in lifetime of experiment (1000 ps) Pollum, M. and Crespo-Hernández, C. E. J. Chem. Phys. 2014, 140, 071101

Dynamics of 4-thiothymine Department of Chemistry Department of Chemistry 0 ps 1.5 ps Dynamics of 4-thiothymine 1.5 ps 80 ps PBS Buffer, pH 7.4 λex = 340 nm No change in lifetime of experiment (1000 ps) Reichardt, C. and Crespo-Hernández, C. E.. J. Phys. Chem. Lett. 2010, 1, 2239-2243

Dynamics of 2,4-dithiothymine Department of Chemistry Department of Chemistry 0 ps 1.5 ps Dynamics of 2,4-dithiothymine 1.5 ps 100 ps PBS Buffer, pH 7.4 λex = 335 nm No change in lifetime of experiment (1000 ps) Pollum, M. and Crespo-Hernández, C. E. (Unpublished). 2014

Propose Good UVA Photosensitizers? Department of Chemistry Department of Chemistry Propose Good UVA Photosensitizers? UVC UVB UVA Thiobase τISC (ps) Triplet Yield 4tT 0.24 0.85 ± 0.15

Propose Good UVA Photosensitizers? Department of Chemistry Department of Chemistry Propose Good UVA Photosensitizers? X UVC UVB UVA Thiobase τISC (ps) Triplet Yield 4tT 0.24 0.85 ± 0.15 2tT 0.62 0.9 ± 0.1

Propose Good UVA Photosensitizers? Department of Chemistry Department of Chemistry Propose Good UVA Photosensitizers? UVC UVB UVA Thiobase τISC (ps) Triplet Yield 4tT 0.24 0.85 ± 0.15 2,4dtT 0.37 N.D.

Future Work Test 4tT and 2,4dtT as a UVA photosensitizer in cells Department of Chemistry Department of Chemistry Future Work Test 4tT and 2,4dtT as a UVA photosensitizer in cells Use these spectroscopic techniques to screen for other potential photosensitizers Study the dynamics of thiobases in DNA oligomers