1. Combining the Power of IRMPD with Ion-Molecule Reactions: The Structure and Reactivity of Radical Ions of Cysteine and its Derivatives M. Lesslie 1, J. Lawler 1, G. Berden 2, J. Oomens 2, J.K.-C. Lau 3,4, K.W.M. Sui 3,4, A. C. Hopkinson 4, V. Steinmetz 5, P. Maitre 5, V. Ryzhov 1 1 Northern Illinois University, Dekalb, IL (USA). 2 FELIX Radboud University, Nijmegen (NL). 3 University of Windsor, Ontario (CA). 4 York University, Ontario (CA). 5 Université Paris Sud, Orsay (FR).

2. Free Radicals & Oxidative Damage

3. Importance of Biological Sulfur Radicals Fragmentation, cross-linking, disulfide bond cleavage… Antioxidants Key role at active sites (Ribonucleotide reductases) Reversible radical storage α-carbon radicals (mostly glycine) J. Stubbe and P. Riggs-Gelasco, Trends Biochem. Sci. 1998, 23, 438-443.

4. Cysteine & Homocysteine Alkali Adducts Radical Rearrangement Biological Perspective How do alkali metal ions affect radical reactivity and structure? H. Lodish, A. Berk, S. L. Zipursky, P. Matsudaira, D. Baltimore and J. Darnell, 2000. X ?

5. Techniques Sulfur Radical Formation Solution phase: Cys(SH) + R’ONO ⇌ Cys(SNO) + R’OH Gas phase: [Cys(SNO)] +  [Cys(S )] + + NO Ion-Molecule Reactions (IMR) Differences in reactivity suggest differences in structure Regiospecific IMRs: S highly reactive / α-C not reactive Gas-phase infrared spectroscopy Infrared multiple photon dissociation (IRMPD) [M+Hcy] + FELIX (FTICR) & [M+Cys] + CLIO (QIT) Theoretical calculations DFT B3LYP/6-311++G(d,p) Structural elucidation / barriers to rearrangement CID

6. Infrared Multiple Photon Dissociation 6 N. C. Polfer, Chemical Society Reviews, 2011, 40, 2211-2221. Action spectroscopy: Dissociation indicates absorption Monitor fragmentation yield vs. wavelength *CLIO: ESI-QIT

7. Ion-Molecule Reactions Modified Bruker Esquire 3000 ESI-QIT

8. Protonated Cys Radical Cation I. Captodatively stabilized α- carbon radical II. Sulfur radical (initially formed) Rearrangement (II  I) barrier: ~160 kJ mol -1 B3LYP/6-311++G(d,p) level Relative energies in kJ mol -1 Sinha, R.; Maitre, P.; Piccirillo, S.; Chiavarino, B.; Crestoni, M.; Fornarini, S., Phys Chem Chem Phys 2010, 12,9794-9800. J. Zhao, K. W. M. Siu and A. C. Hopkinson, Phys. Chem. Chem. Phys. 2008, 10, 281-288. X

9. Probing Cys Radical Alkali Adducts with IMRs Disulfide bond transferRadical Recombination Reactivity of all [M+Cys] + suggests sulfur-based radical – no rearrangement

10. [Li+Cys] + [Na+Cys] + [K+Cys] + [H+Cys] + S-rad 3 αC-rad 1 Exp S-rad 1 S-rad 2 S-rad 3 36.0  Li + S-rad 2 27.6  Li +  αC-rad 1 -34.3 S-rad 1 0.0  Li +  IRMPD of Cys Metal Adducts [Li+Cys] + *CLIO Facility B3LYP/6-311++G(d,p) level, FWHM = 30 cm -1 Relative energies in kJ mol -1

11. S-rad 3 αC-rad 1 Exp S-rad 1 S-rad 2 15.1  Na + S-rad 3 35.6  Na +  αC-rad 1 -38.9 S-rad 1 0.0  Na + S-rad 3 αC-rad 1 Exp S-rad 1 S-rad 2 K+K+  S-rad 3 27.6 K+K+  αC-rad 1 -48.1 K+K+  S-rad 2 7.1 K+K+  S-rad 1 0.0 [K+Cys] + [Na+Cys] + Alkali metal adducts of cysteine radicals are tridentate sulfur-based radicals. B3LYP/6-311++G(d,p) level, FWHM = 30 cm -1 Relative energies in kJ mol -1

12. Reactivity Analysis [X+Cys(S )] + + NO  [X+Cys(SNO)] + X+X+ Rate Constant (cm 3 molecules -1 s -1 ) % of Collision Rate X +… S Distance (Å) H+H+ 8.0 x 10 -11 12 2.35 Li + 1.4 x 10 -10 22 2.55 Na + 2.2 x 10 -10 34 2.92 K+K+ 3.5 x 10 -10 56 3.39 K+K+   Na +  Li +  2.35 Å 2.92 Å3.39 Å 2.55 Å Increasing Reactivity Increasing X +… S Distance

13. Homocysteine Radical Cation Rearrangement barrier (S  α-C ): ~130 kJ mol -1 S. Osburn, T. Burgie, G. Berden, J. Oomens, R. A. O’Hair and V. Ryzhov, J. Phys. Chem. A 2012, 117, 1144-1150.   [H+Hcy(S )] + does not rearrange Difference in reactivity attributed to N-H … S bond length Reactivity with dimethyl disulfide *FELIX Facility

14. IMRs of [M+Hcy] + Highly Reactive Minimal or No Reactivity [M+Hcy] + + CH 3 SSCH 3 [M+Hcy] + + NO [M+Hcy] + lack of reactivity suggests migration to the α-carbon.

15. IRMPD of Hcy Radical Metal Adducts [Li+Hcy] + [Na+Hcy] + [K+Hcy] + [H+Hcy] + S-rad αC-rad 1 αC-rad 2 Exp Li +  S-rad 0.0 Li +  αC-rad 1 -14.6 Li +  αC-rad 2 -39.7 [Li+Hcy] + *FELIX Facility B3LYP/6-311++G(d,p) level, FWHM = 30 cm -1 Relative energies in kJ mol -1

16. [K+Hcy] + [Na+Hcy] + αC-rad 1 αC-rad 2 Exp S-rad Na +  S-rad 0.0 Na +  αC-rad 1 -36.0 Na +  αC-rad 2 -43.1 S-rad αC-rad 1 αC-rad 2 Exp K+K+  S-rad 0.0 K+K+  αC-rad 1 -46.9 K+K+  αC-rad 2 -51.9 Metal adducts of homocysteine radicals are bidentate α-carbon radicals. B3LYP/6-311++G(d,p) level, FWHM = 30 cm -1 Relative energies in kJ mol -1

17. [ M+Hcy ] + Isomerization Relative energies in kJ mol -1 B3LYP/6-311++G(d,p) level a S. Osburn, T. Burgie, G. Berden, J. Oomens, R. A. O’Hair and V. Ryzhov, J. Phys. Chem. A 2012, 117, 1144-1150. M+M+ Critical TS aH+aH+ 131.8 Li + 98.7 Na + 90.0 K+K+ 81.2 Alkali metal ions decrease TS energy to accessible level

18. Summary [M+Cys] + are sulfur-based tridentate species [M+Hcy] + rearrange to captodatively stabilized α-carbon structures Alkali metal adducts appear to lower rearrangement barrier Regiospecific IMRs provide quick insight on radical location IRMPD & DFT calculations confirm specific molecular structure  Li +  X

19. Acknowledgements FELIX: Geil Berden, Jos Oomens CLIO: Vincent Steinmetz, Philippe Maitre Calculations: Justin Kai-Chi Lau, A.C. Hopkinson, K.W.M. Siu Department of Chemistry and Biochemistry, NIU: Victor Ryzhov John Lawler, Jarrod Ragusin

21. B3LYP/6-311++G(d,p) level, scaling factor = 0.976, FWHM = 30 cm -1 Relative enthalpies in kcal mol -1 21 1657 1605 1476 1384 1294 1122 Vibrational motion of [Li + Hcys]  + vibrational mode at 1657 cm -1 Li + 

22. B3LYP/6-311++G(d,p) level, scaling factor = 0.976, FWHM = 30 cm -1 Relative enthalpies in kcal mol -1 22 1657 1605 1476 1384 1294 1122 Vibrational motion of [Li + Hcys]  + vibrational mode at 1605 cm -1 Li + 

23. B3LYP/6-311++G(d,p) level, scaling factor = 0.976, FWHM = 30 cm -1 Relative enthalpies in kcal mol -1 23 1657 1605 1476 1384 1294 1122 Vibrational motion of [Li + Hcys]  + vibrational mode at 1476 cm -1 Li + 

24. B3LYP/6-311++G(d,p) level, scaling factor = 0.976, FWHM = 30 cm -1 Relative enthalpies in kcal mol -1 24 1657 1605 1476 1384 1294 1122 Vibrational motion of [Li + Hcys]  + vibrational mode at 1384 cm -1 Li + 

25. B3LYP/6-311++G(d,p) level, scaling factor = 0.976, FWHM = 30 cm -1 Relative enthalpies in kcal mol -1 25 1657 1605 1476 1384 1294 1122 Vibrational motion of [Li + Hcys]  + vibrational mode at 1294 cm -1 Li + 

26. B3LYP/6-311++G(d,p) level, scaling factor = 0.976, FWHM = 30 cm -1 Relative enthalpies in kcal mol -1 26 1657 1605 1476 1384 1294 1122 Vibrational motion of [Li + Hcys]  + vibrational mode at 1122 cm -1 Li + 