1. 1Department of Chemistry, Wayne State University, Detroit, MI, 48202
Gas-Phase Conformations and Energetics of Sodium Cationized 2’-Deoxyguanosine and Guanosine: IRMPD Action Spectroscopy and Theoretical Studies
Yanlong Zhu1, Lucas Hamlow1, Chenchen He1, Xun Bao1, Juehan Gao2, Jos Oomens2, M. T. Rodgers1*
1Department of Chemistry, Wayne State University, Detroit, MI, 48202
2Radboud University Nijmegen, Institute for Molecules and Materials, FELIX Facility, Toernooiveld 7, 6525ED Nijmegen, The Netherlands

2. Introduction
The local structures of DNA and RNA are influenced by protonation, deprotonation and noncovalent binding interactions with metal cations.
Effects of the conformations of DNA and RNA nucleic acids
Neutralize the overall negative charge along deprotonated phosphate backbone
Conformations of phosphate moieties
Nucleobase flipping
Sugar puckering
H-bonding or π-stacking interactions
Stabilize quadruplex structures
dAdo or Ado
dGuo or Guo
dCyd or Cyd
dThd or Thd
X = H or OH
DNA duplexes are mainly stabilized by the hydrogen bonding interaction between bases on the two strands and base stacking within each strand. At low pH, adenine can be protonated to form A+C and A+G base pairs instead of the complimentary AT base pair. The protonation of cytosine leads to C+G base pairs that help stabilize triplex formations.
(The importance of metal cations interacting with DNA was first realized in the 1920s, when studies reported on the need for metal cations to be present in cells to help neutralize the overall negative charge on DNA.)
Under normal physiological conditions, DNA is deprotonated at the phosphate group. The presence of metal cations can neutralize the overall negative charge on DNA. In the late 1960s, binding of Pt(Platinum) to DNA bases has been found to be an effective antitumor agent. This suggests that the metal cation nucleic acid interaction may regulate gene expression and thereby act as drugs. In recent years, a major focus of metal-DNA studies has been identifying the role metal cations play in stabilizing quadruplex structures.
Numerous studies have shown that metals can bind almost anywhere on the DNA molecule. Metal cations are usually found near the negatively charged phosphate groups on the DNA backbone and the next most popular sites are the nucleobases. The proper placement of metal cations on nucleobases may enhance Watson-Crick bonding between complimentary pairs.
Na+
dUrd or Urd
W. Saenger, Principles of Nucleic Acid Structure, Springer, New York, 1988.
Lippert, B. Coordin Chem Rev 2000, 200, 487.

3. IRMPD Spectroscopy Setup
Infrared Multiple Photon Dissociation
(IRMPD) Action Spectroscopy
Free Electron Laser (FEL)
This slide shows the IRMPD experimental setup. The wanted ions of mononucleotides are generated by electrospray ionization from the prepared solutions and accumulated in a hexapole, prior to being injected to the Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometer by a RF octopole ion guide. Ion capturing was affected using electrostatic pulsing of the octopole without causing collisional heating of the ions. In addition, the ions were left to cool to the room temperature in the ICR cell by radiative emission. The ions of interest was mass isolated and then irradiated for 3s by the free electron laser for infrared experiments (FELIX) to induce IR photodissociation.
In an FEL, electrons are accelerated in a linear accelerator (LINAC) to relativistic speed and injected into a periodic magnetic structure called wiggler or undulator. The wiggling motion that the electrons undergo as a consequence of the lorentz force, causes them to emit synchrotron radiation. By capturing the light in an optical cavity or optical resonator which is formed by two mirrors, freshly injected electron bunches can be made to interact with the circulating light pulses, thereby generating stimulated emssion. The wavelength of the stimulated radiation is determined by the FEL resonance condition, which states that the light wave should advance the electron beam by exactly one wavelength over one period of the undulator. The dependence of the resonance wavelength on the magnetic field can be used to conveniently tune the output wavelength of the FEL. The gap between the magnets can be opened and closed, thus varying the magnetic field strength and hence the lasing wavelength. In our study, the FELIX beam energy is typically around 40 MeV, which allows the wavelength to be tuned roughly between 5 and 20 um, corresponding to photon energies of cm-1 respectively.
To generate an IRMPD spectrum, the fragment intensities in all channels are summed and divided by the total ion intensity. This total fragment yield is then plotted as a function of the wavelength, giving the IRMPD spectra.
IRMPD yield = (∑If)/(Ip + ∑If)
1mM dGuo or Guo, NaCl
MeOH:H2O=90%:10%
Polfer, N. C.; Oomens, J.; Suhai, S.; Paizs, B. J Am Chem Soc 2007, 129, 5887.

4. IRMPD Mechanism Absorption of photons by a resonant vibrational mode
Intramolecular Vibrational Redistribution (IVR)
Ei ≥ D0  unimolecular dissociation
IRMPD is a process where the initial absorption of photon takes place at a resonant vibrational mode. Instead of following a coherent ladder climbing mechanism in a single vibrational level, a bath of background vibrational states are anharmonically coupled to the resonant vibrational mode and the energy from the photon are statistically redistributed to these background vibrational states. This process of redistributing energy to the vibrational background states is also known as IVR. Energies from the subsequent photons are redistributed and stored in these background vibrational states through the same fashion and repeat itself many times until enough internal energy is achieved to reach the threshold for dissociation and leads to the fragmentation of molecules.
Polfer, N. C.; Oomens, J. Mass Spectrom Rev 2009, 28, 468.

5. Theoretical Calculations
Simulated Annealing (Hyperchem, Amber force field)
Calculate candidate structures for higher level optimization
Quantum Chemical Calculations (Gaussian)
Optimization and vibrational frequency analyses:
B3LYP/6-31G*
Single point energy calculations
B3LYP/6-311+G(2d,2p)
Frequencies were scaled by a factor of
Calculated vibrational frequencies were broadened using a 20 cm-1 fwhm Gaussian line shape
Geometry optimization and frequency analyses of these species are performed at the B3LYP/6-31G* level of theory, whereas single-point energies are calculated at the B3LYP/6-311+G(2d,2p) level of theory to determine the relative stabilities of these conformations.
Full width at half maximum

6. Conformation of Sodium Cationized Nucleoside
1. Sodium Cation Binding Position
2. Nucleobase Orientation
anti-orientation
Facilitates
Watson-Crick
Base pairing
syn-orientation
Disrupts
Watson-Crick
Base pairing
3. Sugar Configration
C2’-endo
C3’-endo
endo-configuration
C2’ or C3’ atom
on the same side of
the ring as C5’ atom
C5’
C5’
C3’
C2’
C2’
C3’
exo-configuration
C2’ or C3’ atom
on the opposite side of
the ring as C5’ atom
C2’-exo
C3’-exo
C5’
C5’
C3’
C2’
C2’
C3’

7. IRMPD Spectra of Sodium Cationized dGuo and Guo
[dGuo+Na]+
[dGuo+Na]+
[Guo+Na]+
Fragmentation pathways of [dGuo+Na]+ and [Guo+Na]+:
Major: [Nuo+Na]+  [Gua+Na]+ + Sugar
Minor: [Nuo+Na]+  Na+ + Nuo
[Guo+Na]+

8. Ground-State Structures of [dGuo+Na]+ and [Guo+Na]+
[dGuo+Na]+(O6,N7)A
[dGuo+Na]+(O6,N7)A
Na+--- O6,N7
anti, C3’-endo
0.0 kJ/mol
[Guo+Na]+(O6,N7)A
[Guo+Na]+(O6,N7)A
Na+--- O6,N7
anti, C2’-endo
0.0 kJ/mol

9. Sodium Cation Binding to dGuo at O6 and N7
[dGuo+Na]+ IRMPD Spctrum
[dGuo+Na]+
(O6,N7)B
anti, C2’-endo
5.7 kJ/mol
[dGuo+Na]+(O6,N7)B
[dGuo+Na]+
(O6,N7)C
anti, C2’-endo
5.8 kJ/mol
[dGuo+Na]+(O6,N7)C
[dGuo+Na]+(O6,N7)D
[dGuo+Na]+
(O6,N7)D
syn, C2’-endo
14.2 kJ/mol

10. Sodium Cation Binding to Guo at O6 and N7
[Guo+Na]+ IRMPD Spctrum
[Guo+Na]+
(O6,N7)B
syn, C2’-endo
1.4 kJ/mol
[Guo+Na]+(O6,N7)B
[Guo+Na]+
(O6,N7)C
anti, C3’-endo
3.9 kJ/mol
[Guo+Na]+(O6,N7)C
[Guo+Na]+(O6,N7)D
[Guo+Na]+
(O6,N7)D
anti, C2’-endo
7.0 kJ/mol

11. Sodium Cation Binding to dGuo and Guo at N3
[dGuo+Na]+ IRMPD Spectrum
[dGuo+Na]+
(N3,O4′,O5′)A
syn, C2’-exo
53.3 kJ/mol
[dGuo+Na]+(N3,O4′,O5′)A
[Guo+Na]+ IRMPD Spectrum
[Guo+Na]+
(N3,O4’,O5’)A
syn, C2’,C3’-endo
58.0 kJ/mol
[Guo+Na]+(N3,O4’,O5’)A

12. Conclusions
Fragmentation pathways of [dGuo+Na]+ and [Guo+Na]+:
Major: [Nuo+Na]+  [Gua+Na]+ + Sugar
Minor: [Nuo+Na]+  Na+ + Nuo
In both cases, preferential binding position of the sodium cation is O6 and N7 position on guanine.
Nucleobase remains in an anti-orientation.
Sugar puckering of [dGuo+Na]+: C3’-endo
Sugar puckering of [Guo+Na]+: C2’-endo

13. Conclusions VS. VS. [dGuo+Na]+ Na+--- O6,N7 anti, C3’-endo [Guo+Na]+
[dGuo+H]+
H+--- N7
anti, C3’-endo
[Guo+H]+
H+--- N7
anti, C3’-endo
Wu, R. R.; Yang, B.; Berden, G.; Oomens, J.; Rodgers, M. T. J Phys Chem B 2014, 118,

14. Acknowledgements Professor M. T. Rodgers Rodgers Group Members:
Harrison Roy
Ranran Wu
Chenchen He
Lucas Hamlow
FELIX Group
Dr. Cliff Frieler
Thomas Rumble Fellowship
FELIX Facility
National Science Foundation