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
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:
Infrared Spectroscopy Ion structure characterization of metal-ion complexes by inspecting the IR spectrum. IRMPD to produce “action spectrum” “Fingerprint” region ( cm -1 ) Characteristic modes ( cm -1 )
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)
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
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) 5 Hz, ~50 mJ/pulse Good IRMPD spectroscopy on many species bound by less than 2.5 eV
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
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 +
Expectations from Models Amide oxygen O a always binds Terminal OH never binds O t, N t, Ring fairly competitive O t somewhat favored
The Dipeptides Terminal CO Amide CO Amide NH Terminal OH Ring
Initial Impressions Similarity of spectra suggests common binding mode Major peaks correspond well to characteristic normal modes
Good Fits
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
+ ¯ + ¯ M + Trp Zwitterion M + Trp Charge-solvated Zwitterions?
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)
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?
CS ZW Ba +2 Trp Spectrum Success! The zwitterion is favored
ZwitterionCharge-solvated Dipeptide Complexes Are Not Zwitterions
Amide O a Is Always Bound An example: Na + AlaPhe Best amide-unbound conformer Only amide-bound structures give good fits and good stabilities
Terminal OH is never bound An example: Na + AlaPhe OH bound structures have low stability and poor fit in the CO stretch region
Endo Exo costs ~35 kJ/mol shifts OH bending frequency by cm -1 Terminal OH Never Binds, is always Endo
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 +
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