Computational Modeling of Varying

Nucleophile Activity on the RNA Cleavage Transition State

Jennifer Andre, Aastha Chokshi, Daniel Greenberg, Jay Karandikar, Steven Kunis, Joshua Loughran, Elena Malloy, David

Mazumder, Dushyant Patel, Jeffrey Wu, Grace Zhang

Advisor: Adam CassanoAssistant: Zack Vogel

RNA Cleavage

• Role of RNA

• RNA Cleavageo Importance & Ubiquityo Change or inhibit gene expression

• Understanding the Mechanismo Potential Applications

Artificial Nucleases Target harmful RNA Cure diseases

DNA RNA Protein

Observing the Nucleophile

The strength of a nucleophile depends upon how negative its charge is.

Transition States

Harris and Cassano, Current Opinion in Chemical Biology, 2008, 12, 626-639

Describing the Transition State

Computational Chemistry

• Life Span of Transition State: 10-13 Seconds

• Allows us to visualize structures that cannot be observedo Distanceso Chargeso Angles

Abu-Awwad, Fakhr, Computational & Biophysical Chemistry, 2007

Electronic Structure Methods

Locke, W., Molecular Orbital Theory, 2006

• Levels of Theory: DFT, Hartree-Fock, MP2, MP4,....

• Basis Set: Set of functions to model molecular orbitals

• Larger basis set = better model, but more computing time

Models

• GaussView

• Simplification

• Structureo Variation of the electronegative portiono Effects on nucleophile

Lönnberg, Org. Biomol. Chem., 2011, 9, 1687–1703

Nucleophile

Optimization

• Finding molecular geometries:o bond lengthso bond angleso charges

• Ground state and transition state

• B3LYP/6-31++G(d,p)

Frequency

• Determines if molecule at minimum or transition state

• Calculating atomic motion within moleculeso Vibrations within bondso Thermochemical data

• B3LYP/6-31++G(d,p)

The Ground State

P-O (Nuc)

P-O (LG)

Charge (ONuc)

Charge (OLG)

-CH3 4.565 1.664 -0.929 -0.72

-CFH2 4.56078 1.66327 -0.9 -0.717

-CF2H 4.53447 1.64745 -0.897 -0.692

-CF3 4.439 1.661 -0.873 -0.71

Transition StatesP-O

(Nuc)P-O(LG)

P-O(Nuc) + P-

O(LG)

-CH3 1.78 2.36 4.14

-CFH2 1.78 2.43 4.21

-CF2H 1.79 2.44 4.23

-CF3 1.78 2.50 4.28

The Phenol Leaving Group

• Structure:o Methyl group by nucleophileo Phenol leaving group

• Different basis seto B3LYP/6-311++G(d,p)

• Test effect of good leaving group on transition state

o Reactant-likeo P-ONuc Longer (2.31Å vs 1.78Å)

o P-OLg Shorter (1.83Å vs 2.36Å)

Energy Calculations

• Calculating Electronic Energy

• Methodso B3LYPo MPWB1K - more accurate

G = (((Ee +ZPVE) + Evib + Erot + Etrans)+ RT) - TS

• Both the ground state and the transition state

∆G = Gtransition state - Gground state

k = kB T h-1 e -∆G/RT

Ribeiro A, Ramos M, Fernandes P. Benchmarking of DFT functionals for the hydrolysis of phosphodiester bonds. J. Chem. Theory Comput. 2010; 6: 2281-2292.Lopez X, Dejaegere A, Leclerc F, York D, Karplus M. Nucleophilic attack on phosphate diesters: a density functional study of in-line reactivity in dianionic, monoanionic, and neutral systems. J. Phys. Chem. 2006; 110(23): 11525-11539.

Energy Results - B3LYP

Ground State Energy (kJ/mol)

Transition State Energy (kJ/mol)

ΔG‡

(kJ/mol)Rate Constant

-CH3 -2298667.1 -2298564.5 102.6 6.46 X 10-6

-CFH2 -2559250.3 -2559143.3 107.0 1.09 X 10-6

-CF2H -2819863.0 -2819743.1 119.9 6.08 X 10-9

-CF3 -3081577.4 -3081460.8 129.9 1.06 X 10-10

Energy Results - MPWB1K

Ground State Energy (kJ/mol)

Transition State Energy (kJ/mol)

ΔG‡

(kJ/mol)Rate Constant

-CH3 -2298016.3 -2297921.9 94.4 1.76 X 10-4

-CFH2 -2558531.8 -2558431.5 100.2 1.65 X 10-5

-CF2H -2819084.0 -2818969.5 114.5 5.37 X 10-8

-CF3 -3081577.4 -3081460.8 116.7 2.18 X 10-8

Energy Results - GraphBrønsted Plot Comparing Acidity of Nucleophile and

Reaction Rate

Conclusions

As pKa decreases (acidity increases):

• free energy of activation increases.

• leaving group bond length increases.

• nucleophile bond length stays roughly the same.

• TS becomes more dissociative.

• nucleophile charge becomes less negative.

As nucleophile charge becomes less negative, ...

• leaving group bond length increases.

Why are our results important?

Showed that RNA cleavage can be manipulated

• Methyl group

• Distances

• Transition states

Therapies

• Ras

• VEGF - Vascular Endothelial Growth Factor

• EGF - Epidermal Growth Factor

Future Investigations

• Changing leaving group

• Changing nucleophile

• Larger basis set

Thank you!Team 1 Project Advisor: Dr. Adam Cassano

Assistant: Zack Vogel

Program Administrators:

Dr. David Miyamoto,

Dr. Steve Surace,

Janet Quinn,

Anna Mae S. Dinio-Bloch

Sponsors,

Without whom NJGSS'12 would not have been possible.

Questions?

Works Cited

Abu-Awwad, Fakhr, Computational & Biophysical Chemistry, 2007

Gaussian 09, Revision A.02, M. J. Frisch, G. W. Trucks, H. B. Schlegel, G. E. Scuseria, M. A. Robb, J. R. Cheeseman, G. Scalmani, V. Barone, B. Mennucci, G. A. Petersson, H. Nakatsuji, M. Caricato, X. Li, H. P. Hratchian, A. F. Izmaylov, J. Bloino, G. Zheng, J. L. Sonnenberg, M. Hada, M. Ehara, K. Toyota, R. Fukuda, J. Hasegawa, M. Ishida, T. Nakajima, Y. Honda, O. Kitao, H. Nakai, T. Vreven, J. A. Montgomery, Jr., J. E. Peralta, F. Ogliaro, M. Bearpark, J. J. Heyd, E. Brothers, K. N. Kudin, V. N. Staroverov, R. Kobayashi, J. Normand, K. Raghavachari, A. Rendell, J. C. Burant, S. S. Iyengar, J. Tomasi, M. Cossi, N. Rega, J. M. Millam, M. Klene, J. E. Knox, J. B. Cross, V. Bakken, C. Adamo, J. Jaramillo, R. Gomperts, R. E. Stratmann, O. Yazyev, A. J. Austin, R. Cammi, C. Pomelli, J. W. Ochterski, R. L. Martin, K. Morokuma, V. G. Zakrzewski, G. A. Voth, P. Salvador, J. J. Dannenberg, S. Dapprich, A. D. Daniels, O. Farkas, J. B. Foresman, J. V. Ortiz, J. Cioslowski, and D. J. Fox, Gaussian, Inc., Wallingford CT, 2009.

Harris and Cassano, Current Opinion in Chemical Biology, 2008, 12, 626-639

Locke, W., Molecular Orbital Theory, 2006

Lönnberg, Org. Biomol. Chem., 2011, 9, 1687–1703

Lopez X, Dejaegere A, Leclerc F, York D, Karplus M. Nucleophilic attack on phosphate diesters: a density functional study of in-line reactivity in dianionic, monoanionic, and neutral systems. J. Phys. Chem. 2006; 110(23): 11525-11539.

Ribeiro A, Ramos M, Fernandes P. Benchmarking of DFT functionals for the hydrolysis of phosphodiester bonds. J. Chem. Theory Comput. 2010; 6: 2281-2292.