1. Infrared spectroscopy of hydrogen-bonded clusters of protonated histidine Department of Chemistry, School of Science, Kitasato University, Japan Makoto Kondo, Yasutoshi Kasahara, Haruki Ishikawa

2. Histidine (His) One of the essential amino acids Imidazole ring in the side chain Involved in active sites in many enzyme proteins - Flexible conformation plays a critical role in biological processes. - Conformation and intermolecular interaction of His are very important in its reactivity and functions.

3. K. Oda, Acta. Cryst., B28, 639 (1972). A. B. Ahmed, Spectrochimica Acta Part A, 79, 554 (2011). J. J. Madden, Acta. Cryst., B28, 2377 (1972). His zwHis HCl His 2HCl Conformation of His in crystal Conformation of His depends on protonation and hydrogen-bond structure in the crystal. What happens in the gas-phase? His zw H-bond Intra-molecular: 1 Inter-molecular: 7 In crystalIn gas-phase

4. Questions: - Which protonated form is stable in the hydrogen- bonded cluster ? - Is there any relation between the protonation and H-bond sites ? Protonated His (HisH + )  E 0 = 0.0 kcal/mol  E 0 = 0.87 kcal/mol IR photodissociation (PD) spectroscopy of HisH + -(MeOH) n = 1, 2 ImH + formNH 3 + form

5. IR PD spectroscopy Clusters are generated by ESI method. Sample: MeOH solution of His hydrochloride monohydrate. Concentration:0.5 mM Infusion rate:5  ℓ /min High voltage:2 ~ 3 kV The clusters generated were mass-selected the first quadrupole mass filter, and then irradiated with infrared light. The fragment ions detected by the second mass analyzer. IR Q-Mass#1Q-Mass#2

6. IR spectrum of HisH + -(MeOH) 1 Isomer B Free (NH) His 3490 Free (OH) MeOH 3666 Isomer A Two isomers exist ! 3169 H-bonded (NH) His 3562 Free (OH) COOH

7. IR spectrum of HisH + -(MeOH) 1  E 0 = 0.0 kcal/mol  E 0 = 0.12 kcal/mol Isomer A Isomer B M05-2/6-31++G(d, p) ImH + NH 3 +

8. Protonation vs. H-bond Each protonation form has its own favorite H-bond position. B.E. = 15.4 kcal/mol 14.2 kcal/mol 12.5 kcal/mol 16.0 kcal/mol ImH + form NH 3 + form

9. IR spectrum of HisH + -(MeOH) 2 Free (OH) MeOH 3673  E 0 = 0.0 kcal/mol Isomer A  E 0 = 0.11 kcal/mol Isomer B ImH + NH 3 + 3498 Free (NH) His

10. The structures of HisH + -(MeOH) n (n = 1, 2) were determined by IR PD spectroscopy and the DFT calculation. Q. Which protonated form is stable in the hydrogen-bonded cluster ? A. ImH + form is the most stable for n = 1 and 2. But, the energy difference is reduced by the solvation. Q. Is there any relation between the protonation and H-bond sites ? A. Yes. Each protonation form has its own favorite H-bond position. Summary

12. H-bonding position of MeOH ImH + formNH 3 + form B.E. = 11.1 kcal/mol 15.0 kcal/mol

13. The generations of HisH + -(MeOH) 1, 2 were confirmed. Mass spectrum of His

14. Obs.Ref. Ref. ） Dugourd et al. PCCP, (2014) 16, 1257 UV spectrum of HisH + Short-lived excite state is confirmed !

15. Simulated spectra of HisH + -(MeOH) 2