1. Ultrafast Spectroscopic and ab initio Computational Investigations on Solvation Dynamics of Neutral and Deprotonated Tyrosine 70 th ISMS TI03 Champaign, IL E-mail: tfujiwa@bgsu.edutfujiwa@bgsu.edu Center for Photochemical Sciences Bowling Green State University National Research Council of Canada Marek Z. Zgierski Takashige Fujiwara

2. Aromatic Amino Acids of Importance WYF Macromolecular luminescence of natural proteins is largely contributed by: The macromolecular luminescence is perturbed by micro-environment: ‣ Chromophore presence in a polypeptide chain ‣ Structural influence of the protein W Y F TryptophanTyrosine Phenylalanine The prime interest: to use luminescence as a probe for the study of structural properties of proteins. Photophysical/photochemical mechanisms of Tyr in ultrafast time scale is known little, as opposed to Trp.

3. Time-Resolved Fluorescence Upconversion Setup

4. Data Analyses in Upconversion Signals ‣ Wavelength Corrections (λ flu –1 = λ obs –1 – λ gate –1 ) ‣ Spectral Response Correction (Monochromator) ‣ Group Velocity Distribution (GVD) Correction ‣ Light/Raman Scatter Removal (Reconstruction) ‣ Fluorescence Decays for Specific Wavelengths RamanLaser Tyrosine in water (GVD-corrected) Water (GVD-corrected) Raman 308 nm Laser 280 nm ~300 fs (Gauss-2D) Raw Spectral profiles Water Raman Light Scatter

5. Temporal Profiles of Tyrosine in Water Temporal profiles (Raw Data)

6. Re-constructed TRFS of Neutral Tyrosine in Water wavelength / nm time / ps fluorescence intensity slow ~ 3.45 ns (TCSPC) fast ~ 7–10 ps 1mM @280nm

7. Spectral Solvent Dependence of Tyrosine Steady-state spectra in various organic solvents — n-hexane, dioxane, methanol, DMSO, and water — EmissionAbsorption ‣ Solvent polarity (dielectric constants, Ɛ r ) ‣ Hydrogen bondings ‣ Conformational isomers (Rotamers) ‣ Excimer formations (phenol+Hex or Diox) ‣ Solvent polarity (dielectric constants, Ɛ r ) ‣ Hydrogen bondings ‣ Conformational isomers (Rotamers) ‣ Excimer formations (phenol+Hex or Diox)

8. Re-constructed TRFS of Tyrosine in Methanol Single-exponential decay: 4.0 ns from TCSPC 1mM tyr/MeOH Exc@280 nm ~ 4.0 ns (TCSPC)

9. Fluorescence Decay Profiles of Tyrosine in DMSO 310 nm Tyr/DMSO, exc@280 nm 1.6 ns TCSPC, Exc@290 nm Res. ~700 ps (fwhm) ~10 ns

10. Re-constructed TRFS of Tryptophan in Water 345 nm 1mM Trp/H 2 O Exc@280 nm

11. Aqueous Tyrosine in Various pH Micro-environments Absorption Emission pK a =9.3 pK a =2.2 pK a =10.1 de-protonated tyrosine intramolecular ion pair doubly de- protonated tyrosinate zwitteriondianionanion pH (basicity) at pH=7

12. Excitation Wavelength Dependence of Fluorescence Spectra IIIIII τ slow ~4.3 ns τ slow ~2.6 ns τ fast <100 ps A mixture of neutral, deprotonated, and doubly-deprotonated tyrosines abs Tyrosine in pH 12 buffer solution TCSPC, Exc@290 nm Res. ~700 ps (fwhm)

13. Time-Resolved Fluorescence Decays Exc@320 nm Tyrosine in pH 12 solution Exc@290 nm Tyrosine in pH 9.5 solution ~2.6 ns

14. Time-Resolved Fluorescence Spectra of Aqueous Tyrosine at pH 12 Tyr (pH12) Exc@300 nm ~23 ps 2–4 ns wavelength / nm time / ps Fluorescence Intensity

15. Electron Transfer in Tyrosyl Radical e–e– high-pH range + e–e– H+H+ ++ mid-pH range Proton Coupled Electron Transfer (CPET) Tyrosyl Radical Decay Mechanism e–e– H+H+ D.R. Weinberg, C.J. Gagliardi, J.F. Hull, C.F. Murphy, C.A. Kent, B.C. Westlake, A. Paul, D.H. Ess, D.G. McCafferty, and T.J. Meyer, Chem. Rev. 112, 4016 (2012). D. V. Bent and E. Hayon, JACS, 97, 2599 (1975). Phenolic pK a : pK a (S 0 ) ~ 10.3 pK a (S 1 ) ~ 4.2 Increasing acidity upon excitation Proton Coupled ET reactionET reaction

16. Transient Absorption Spectra of Deprotonated Tyrosine Adopted from C.V. Pagba, S.-H. Chi, J. Perry, and B.A. Barry, J. Phys. Chem. B. 119, 2726 (2015). 18 ps (1000 ps) S n ←S 1 anion S n ←S 1 zwitterion tyr 14 ps (910 ps) 16 ps (910 ps) 54 ps e aq – (720 nm) S n ←S 1 dianion tyr 19 ps (580 ps) 23 ps (500 ps)

17. Optimized Structures of Tyrosine and Tyrosinate Tyrosine, S 0 (MP2/cc-pVDZ) Zwitterion, S 0 (DFT/cc-pVDZ, H 2 O + PCM) Anion, S 1 CC2/cc-pVDZ Anion, S 1 CC2/cc-pVDZ Dianion, S 1 CC2/cc-pVDZ

18. Computed Excited-State Absorption and Emission Comparison of Steady-State Spectra with Computed Oscillator Strengths TDDFT/cc-pVDZ with SCRF PCM (water, Onsager) Non-equilibrated

19. Summary We studied photophysical properties of tyrosine in various micro- environments using femtosecond fluorescence up-conversion and TDDFT and CC2 computations. Aqueous tyrosine does not show significant ultrafast solvation in the earlier time, as opposed to tryptophan in water. The observed fluorescence decays are consistent with the formerly reported transient absorption. The excited-state dynamics on a single- and doubly-deprotonated tyrosine under various pH solutions were investigated. In high basicity, tyrosine shows different absorption/emission spectra, and a total spectrum consists of a combination of these individual spectra that depend on the pH of the solution. Ohio Supercomputer Center (OSC) Acknowledgments Support: