1. Robert C. Dunbar Case Western Reserve University Nick C. Polfer, Jos Oomens FOM-Institute for Plasma Physics Structure Investigation of Cation-Pi Complexes of Alkali Metal Ions with Aromatic Dipeptides by IRMPD Spectroscopy $$$ FOM, NHMFL, NSF OSU Molecular Spectroscopy Symposium

2. Overview Previous IR spectroscopy of M + (Amino Acid) Move toward Polypeptides: Look at M + (Dipeptide) M + = Na +, K + Dipeptide = AlaPhe, PheAla  Amide linkage  Possible cation - pi interaction with Phe Features of these systems:

3. Infrared Spectroscopy Ion structure characterization of metal-ion complexes by inspecting the IR spectrum.  IRMPD to produce “action spectrum”  “Fingerprint” region (400-1500 cm -1 )  Characteristic modes (1000-1800 cm -1 )

4. Action Spectroscopy and IRMPD Infrared Multiple Photon Dissociation IR photon typically 0.1 eV Dissociation energy typically 3 eV Many photons delivered sequentially by an intense, short laser pulse (IRMPD) M + AlaPhe M + + AlaPhe (or other fragments)

5. Light Source The Free Electron Laser gives  Convenient sweep across the chemically informative IR spectrum  High intensity and energy per pulse  Tight collimation of beam

6. Free Electron Laser -- FELIX  Free Electron Laser for Infared EXperiments  FOM Institute for Plasma Physics, Netherlands  IR light source for spectroscopy  Tunable from 4.5 to >35 microns (2200 to <285 cm-1)  (Continuous sweep over range of factor of 3)  Macropulses @ 5 Hz, ~50 mJ/pulse  Good IRMPD spectroscopy on many species bound by less than  2.5 eV

8. Experimental Protocol  Electrosprayed M + L accumulates, cools in hexapole  Transfer M + L to ICR, radiative cooling  FTICR eject sequence isolates M + L in cell  ~10 FELIX macropulses pass through ion cloud  FTICR detect sequence measures relative abundance of M + L ions and photofragment ions Duration 500 msec 1 sec 100 msec 2 sec 100 msec

9. Peptide Model Binding Sites DFT / mpw1pw91 (kJ/mol) N t 61 O a 125 K+K+ Small-molecule model calculations OH 31 O t 84 R 69 N t 96 O a 165 OH 55 O t 118 R 99 Na +

10. Expectations from Models  Amide oxygen O a always binds  Terminal OH never binds  O t, N t, Ring fairly competitive  O t somewhat favored

11. The Dipeptides Terminal CO Amide CO Amide NH Terminal OH Ring

12. Initial Impressions  Similarity of spectra suggests common binding mode  Major peaks correspond well to characteristic normal modes

13. Good Fits

14. Definite Conclusions (Stabilities and Spectra)  No Zwitterions  Amide O always bound  OH never bound  Free OH always endo Analyzing the Structures Put together  Spectra  Calculated stabilities of conformers  Calculated IR spectra of conformers

15. + ¯ +  ¯ M + Trp Zwitterion M + Trp Charge-solvated Zwitterions?

16. Charge-Solvated Example: K + Phenylalanine Experimental spectrum (charge-solvated) This demonstrates the characteristic peaks: CS: 1150 and 1725 cm -1 ZW: 1675 and 1400 cm -1 Charge solvated Zwitterion See Polfer, Paisz, Snoek, Compagnon, Suhai, Meijer, Von Helden, Oomens, JACS 127, 8571 (2005)

17. Zwitterion Example: A Doubly-Charged Metal Ion Gas-phase zwitterions in gas-phase amino acid complexes are unusual (Arg, Pro) A striking new result M +2 Ions: See if the higher electrostatic forces can stabilize the salt bridge and favor the zwitterion Success in forming BaTrp +2 by electrospray. Is it a zwitterion?

18. CS ZW Ba +2 Trp Spectrum Success! The zwitterion is favored

19. ZwitterionCharge-solvated Dipeptide Complexes Are Not Zwitterions

20. Amide O a Is Always Bound An example: Na + AlaPhe Best amide-unbound conformer Only amide-bound structures give good fits and good stabilities

21. Terminal OH is never bound An example: Na + AlaPhe OH bound structures have low stability and poor fit in the CO stretch region

22. Endo Exo  costs ~35 kJ/mol  shifts OH bending frequency by 50-100 cm -1 Terminal OH Never Binds, is always Endo

23.  O t or N t binding? Two More Difficult Questions  Ring binding?  Binding to the O terminus or the N terminus is very similar in spectrum and stability. Possible mixtures.  Good evidence for ring binding of Na +  Ring bound is similar to free ring for K +

24. Cytochrome C vs K + AlaPhe K + AlaPhe Cyt C Amide II NH Amide I CO COOH OH Oomens et al, PCCP, 2005 The future: Maybe bigger and bigger biomolecules? Wavenumber cm -1