AB INITIO AND DFT INVESTIGATION OF THE MECHANISM AND HYDRATION PATTERN OF SIALIDASE AND ITS INHIBITORS KRISHNAN CHANDRASEKARAN NATIONAL UNIVERSITY OF SINGAPORE 2010 AB INITIO AND DFT INVESTIGATION OF THE MECHANISM AND HYDRATION PATTERN OF SIALIDASE AND ITS INHIBITORS KRISHNAN CHANDRASEKARAN M.S. (By Research) Chemistry National University of Singapore A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF CHEMISTRY NATIONAL UNIVERSITY OF SINGAPORE 2010 DEDICATED TO LORD SRI KRISHNA ACKNOWLEDGEMENT S I pay my respectful thanks from the bottom of my heart to Lord Sri Savitri and Lord Sri Gayatri for illuminating, guiding and inspiring my intellect to complete this research study successfully. I wish to pay my sincere thanks to my supervisor Associate Professor Ryan, P.A. Bettens for his patronage, supervision, gentle conduct and guidance to complete this course. I am ever grateful for financial assistance provided by the National University of Singapore, Dept. of chemistry to complete this course and for fulfilling my goal of higher research study in chemistry. I am thankful to my friend Dr. Sanjiv Kumar Yadav, Research Fellow, Department of Physiology, NUS for his constant support and ceaseless encouragement to finish this course. I owe to my dearest and noble friend Mr. B.T.S Ramanujam, Research Scholar, National Chemical Laboratory, Pune, India for his ceaseless impetus to complete this course. I sincerely thank Mr. Karthik Sekar, Graduate Student for his crucial support to complete this work. Also I extend my thanks to my friends Ms. Tan Amelia and Dr. T. Velmurugan for their encouragement to complete this course. I am ever grateful to my beloved parents and my siblings for their profound love, hospitality, prayers for my health and education, faith, ceaseless support and encouragement to complete this research study and for their dedication to send me the overseas. I wish to express my deep sense of indebtedness to Ms. Inthrani Raja Indran, Research Scholar, Department of Physiology, NUS for her cardinal role to complete this research study. K. CHANDRASEKARAN i TABLE OF CONTENTS Chapter General Introduction Chapter Theoretical Methodology 2.1 Introduction 2.2 The Schrodinger Equation 2.2.1 Born Oppenheimer Approximation 2.2.2 The One-Electron Approximation 11 2.2.3 The LCAO approximation 12 2.2.4 Approximate methods used to solve Schrodinger Equation 13 2.2.5 Variation Method 13 2.3 Perturbation Theory 13 2.4 Hartree-Fock Method 14 2.4.1 Roothan-Hall Method 17 2.4.2 Restricted Hartree Fock Method 18 2.5 Electron Correlation 19 2.5.1 Moller-plesset Method 19 2.6 Density Functional Theory (DFT) 20 2.6.1 Local Density Functional Theory (DFT) 23 2.6.2 Gradient Corrected Methods 23 2.6.3 Hybrid DFT Methods 24 2.7 Solvation Models 24 2.7.1 Solvent\solute descriptor models 25 2.7.2 Statistical Model 25 2.7.3 Molecular Simulations 25 2.7.4 Polarizable continuum model 25 ii 2.7.5 Conductor like PCM (CPCM) model 26 2.7.6 Cluster Continuum solvation model 27 2.8 Basis Set 29 2.8.1 Minimal Basis Set 31 2.8.2 Split Valence Basis Set 31 2.8.3 Polarized Basis Set 32 2.8.4 Diffuse Basis Set 32 2.8.5 High Angular Momentum Basis Set 33 Chapter Catalytic Mechanism of Sialildase enzyme 34 3.0 Introduction 34 3.1 Method of Calculation 35 3.2 Results and Discussion 36 3.3 Catalytic Path Step – II 37 3.4 Catalytic Path Step – III 37 3.5 Catalytic Path Step – IV 38 3.6 Catalytic Path Step – V 39 3.7 Catalytic Path Step – VI 40 3.8 Catalytic Path Step – VII 41 3.9 Catalytic Path Step – VIII 42 3.9.1 Conclusion 44 Chapter Explicit cluster continuum investigation of sialyate compounds 45 4.1 Introduction 45 4.2 Method of Calculation 46 4.2.1 Solvation free energy 47 4.2.2 Gibbs Free Energy 48 iii 4.3 Results and Discussion 49 4.3.1Explicit cluster continuum solvation analysis of sialyate-guandino complex 49 4.4 Cluster continuum studies of sialosyl cation complexes 54 4.5 Explicit cluster continuum solvation analysis of sialyate anion 59 4.6 Cluster continuum solvation analysis of sialyl zwitter ion 63 4.9 Conclusion 72 Chapter Cluster continuum solvation analysis of sialidase inhibitors 73 5.1 DANA Complex 73 5.2 Results and Discussion 74 5.2.1 Cluster continuum solvation analysis of DANA-guandino complex 74 5.3 4-Amino-DANA complex 78 5.3.1 Results and discussion 79 5.4 Solvation analysis of 4-guandino-DANA complex 83 5.4.1 Results and Discussion 85 5.5 Tamiflu-guandino complex 89 5.5.1 Results and Discussion 90 5.6 BCX-guandino complex 94 5.6.1 Results and Discussion 95 5.7 Cluster continuum investigation of anionic substrates 99 5.7.1 Solvation analysis of DANA anion 99 5.7.2 Solvation analysis of 4-amino-DANA anion 103 5.7.3 Cluster continuum solvation analysis of 4-guandino-DANA anion 107 5.7.4 Solvation analysis of tamiflu anion 111 5.7.5 Cluster continuum solvation analysis of BCX anion 115 5.8 Conclusion 130 iv Chapter Effect of hydration on the binding affinity of the substrate 131 6.1 Introduction 131 6.2 Method of Calculation 131 6.3 Results and Discussion 131 6.3.1 Sialyate Complex 131 6.4 Binding affinity of DANA as a function of water molecules 133 6.5 Binding analysis of 4-amino-DANA as a function of water molecules 135 6.6 Binding affinity of 4-guandino-DANA 136 6.7 Investigation of binding affinity of tamiflu 138 6.8 Analysis of binding affinity of BCX as a function of water molecules 140 6.8.1 Binding affinity of sialosyl cationic complex 142 6.9 Conclusion 151 Chapter Effect of substituents on the binding affinity of substrates 153 7.1 Introduction 153 7.2 Effect of C4 substituents on the binding affinity of DANA 153 7.2.1 Effect of C4 substituent in solvent phase 156 7.3 Effect of C7 substituents on the binding affinity of DANA 157 7.4 Effect of C7 substituent of DANA in solvent phase 160 7.5 Effect of C7 substituents on the binding affinity of N-DANA 161 7.5.2 Binding affinity of C7 substituent of N-DANA in solvent phase 164 7.6 Effect of C7 substituents on the binding affinity of 4-guandino-DANA 165 7.6.1 Binding of C7 substituents of guandino-DANA in solvent phase 168 7.7 Effect of C12 substituents on the binding affinity of tamiflu 169 7.7.2 Effect of C12 substituents on the binding affinity in solvent phase 173 7.8 Effect of C6 substituents on the binding affinity of tamiflu 174 v 7.9 Conclusion 182 Conclusion 183 References 186 vi LIST OF TABLES Chapter Table I Solvation free energy and hydration energy of sialyate-guandino complex 68 Table II Solvation free energy and water binding energies of sialosyl cationic complex 69 Table III Solvation free energy and water binding energy of sialyate anion 70 Table IV Solvation free energy and hydration energy of sialyl zwitter ion 71 Chapter Table I Solvation free energy and water binding energy of DANA complex 120 Table II Solvation free energy and hydration energy of N-DANA complex 121 Table III Solvation free energy and hydration energy of 4-guandino-DANA complex 122 Table IV Solvation free energy and water binding of energy of tamiflu complex 123 Table V Solvation free energy and water binding energy of BCX complex 124 Table VI Solvation free energy and water binding energy of DANA anion 125 Table VII Solvation free energy and hydration energy of N-DANA anion 126 Table VIII Solvation free energy and hydration energy of 4-guandino-DANA anion 127 Table IX Solvation free energy and water binding energy of tamiflu anion 128 Table X Solvation free energy and water binding energy of BCX anion 129 Chapter Table I Binding energy of sialyate complex 145 Table II Binding affinity of DANA as a function of water molecules 146 Table III Binding energy of N-DANA as a function of water molecules 147 Table IV Binding energy of 4-guandino-DANA as a function of water molecules 148 Table V Binding affinity of tamiflu as a function of water molecules 149 Table VI Binding affinity of BCX as a function of water molecules 150 Table VII Binding affinity of cationic substrate 151 vii TABLE II EFFECT OF C7 SUBSTITUENT ON THE BINDING ENERGY OF DANA (GAS & SOLVENT PHASE) Ligand Binding Energy HF\6-31g (kcal/mol) 105.61 Ligand Binding Energy B3LYP\6-31G(d) (kcal/mol) 105.20 13.14 Solvated Ligand Binding Energy B3LYP\631G(d) (kcal/mol) 10.00 Charges on the oxygen O16 r(C2=C3) (Å) r(C2–O16) (Å) 0.310 -0.789 1.382 1.319 Amino 107.69 109.65 6.10 18.64 -0.803 -0.803 1.319 1.380 Methoxy 106.34 109.51 5.85 12.80 -0.797 -0.797 1.319 1.381 Fluorine 106.34 109.42 6.26 12.99 -0.805 -0.805 1.319 1.380 Methyl 107.86 110.66 6.02 12.87 -0.796 -0.796 1.319 1.382 Chlorine 87.60 96.31 10.48 1.75 -0.786 -0.786 1.490 1.382 Thiol 103.40 108.56 3.60 12.71 -0.795 -0.795 1.319 1.379 CF3 104.69 108.12 1.50 13.61 -0.791 -0.791 1.318 1.381 Guandino 113.68 115.42 11.94 17.57 -0.798 -0.798 1.322 1.322 Substituents Hydroxyl Solvated Ligand Binding Energy HF\6-31g (kcal/mol) Charges on the carbon C2 177 TABLE III EFFECT OF C7 SUBSTITUENTS ON LIGAND BINDING ENERGY OF 4-AMINO-DANA Ligand Binding Energy HF\6-31g (kcal/mol) 107.95 Ligand Binding Energy B3LYP\6-31G(d) (kcal/mol) 112.62 6.33 Solvated Ligand Binding Energy B3LYP\ 6-31G(d) (kcal/mol) 18.03 Charges on the oxygen O16 r(C2=C3) (Å) r(C2–O16) (Å) 0.313 -0.789 1.320 1.382 Amino 108.03 110.44 6.52 12.84 0.336 -0.789 1.322 1.373 Methoxy 105.93 109.25 6.42 13.38 0.313 -0.784 1.320 1.381 Fluorine 105.92 109.35 6.63 13.58 0.316 -0.780 1.320 1.379 Chlorine 104.99 108.15 6.41 13.28 0312 -0.766 1.320 1.380 Methyl 111.02 115.33 3.91 13.16 0.327 -0.782 1.321 1.375 Thiol 107.04 109.67 6.62 13.42 0.334 -0.772 1.320 1.372 Guandino 106.11 109.08 3.42 18.93 0.349 -0.803 1.320 1.374 CF3 104.39 108.08 6.34 44.77 0.31 -0.769 1.32 1.38 Substituent Hydroxyl Solvated Ligand Binding Energy HF\6-31g (kcal/mol) Charges on the carbon C2 178 TABLE IV EFFECT OF C7 SUBSTITUENTS ON LIGAND BINDING ENERGY OF 4-GUANDINO-DANA Ligand Binding Energy HF\6-31g (kcal/mol) 105.42 Ligand Binding Energy B3LYP\6-31G(d) (kcal/mol) 108.95 6.43 Solvated Ligand Binding Energy B3LYP\631G(d) (kcal/mol) 11.45 Amino 107.93 110.62 3.12 8.90 0.350 -0.797 1.322 1.368 Methoxy 107.04 110.02 7.94 18.3 0.330 -0.791 1.323 1.375 Fluorine 104.18 109.2 4.96 13.15 0.329 -0.795 1.322 1.374 Chlorine 105.85 108.15 8.05 14.04 0.320 -0.776 1.321 1.376 Methyl 108.2 111.06 7.97 14.07 0.341 -0.788 1.322 1.370 Thiol 107.45 109.8 8.02 13.7 0.356 -0.788 1.321 1.365 CF3 105.28 108.84 7.64 14.07 0.315 -0.777 1.320 1.376 Substituent Hydroxyl Solvated Ligand Binding Energy HF\6-31g (kcal/mol) Charges on the carbon C2 Charges on the oxygen O16 r(C2=C3) (Å) r(C2–O16) (Å) 0.342 -0.801 1.32 1.376 179 TABLE V EFFECT OF C12 SUBSTITUENT ON LIGAND BINDING ENERGY OF TAMIFLU COMPOUND Ligand Binding Energy B3LYP\6-31G(d) (kcal/mol) Tamiflu Ligand Binding Energy HF\6-31g (kcal/mol) 118.34 Charges on the carbon C4 Charges on the C13 Carbon r(C2=C3) (Å) 3.59 Solvated Ligand Binding Energy B3LYP\631G(d) (kcal/mol) 20.83 122.21 -0.104 -0.107 1.326 Methoxy 117.27 121.56 6.66 14.81 -0.119 -0.1 1.327 Ethoxy 117.34 121.66 6.85 15.07 -0.106 -0.101 1.327 Methyl 119.91 124.03 5.11 13.84 -0.107 -0.112 1.327 Ethyl 119.87 124.12 5.36 14.54 -0.12 -0.112 1.326 Chlorine 110.9 117.72 0.63 12.85 -0.09 -0.093 1.326 Fluorine 116.67 121.21 5.86 14.12 -0.092 -0.093 1.326 Thiol 115.38 119.32 5.38 13.69 -0.104 -0.1 1.326 Guandino 115.67 118.38 11.19 16.66 -0.108 -0.096 1.327 CF3 115.53 120.81 5.18 14.09 -0.091 -0.093 1.324 C12 Substituents of tamiflu Solvated Ligand Binding Energy HF\6-31g (kcal/mol) 180 TABLE VI EFFECT OF SUBSTITUENTS ON TAMIFLU AT C6 POSITION Substituents at the C6 position of tamiflu Ligand binding energy HF\6-31G (kcal/mol) Ligand Binding energy RB3LYP\ 6-31G(d) (kcal/mol) Charge density on carbon C4 atom C4=C13 Bond distance (Å) Dipole Moment in Debyes (D) Tamiflu 118.34 122.21 -0.107 1.326 9.07 Guandino 117.73 121.35 -0.119 1.325 11.61 Methyl 116.82 121.15 -0.092 1.325 10.7 Fluorine 114.55 119.45 -0.12 1.326 11.39 Methyl amine 117.06 121.36 -0.092 1.325 10.61 Thiol 115.07 118.95 -0.091 1.325 10.96 181 7.9 CONCLUSION The investigation of effect of C4 substituent on DANA discloses that the methyl, amino and methoxy group at the C4 position improves the binding affinity of DANA and hence these compounds can be further explored to design the sialidase antiviral drugs. The analysis of C7 substituents on the DANA discloses that the amino, methoxy, and methyl group moderately increases the binding affinity. Especially, guandino at the C7 position enormously increases the binding affinity of DANA. Therefore, 7-guandino-DANA will find major application in the design of sialidase antiviral drugs. The analysis of C7 substituents on the N-DANA shows that the amino and methyl group at the C7 position increases the binding of N-DANA. Hence, methyl and amino derivatives will significantly aid the in the development of sialidase antiviral drugs. The effect of C7 substituent on the 4-guandino-DANA reveals that the amino and methoxy group increases the binding of 4-guandino-DANA and these two derivatives will be used in the design of modern sialidase antiviral drugs. The analysis of C12 substituent on tamiflu signifies that the methyl and ethyl group at the C12 position increases the binding energy. Hence, ethyl and methyl derivatives of tamiflu will provide a potential opportunity to develop potent sialidase inhibitors. Hence, effect of substituents study declares that the substituent with higher binding affinity will be beneficial to develop modern anti influenza drugs. 182 CONCLUSION Ab initio investigation of catalytic path of sialidase enzyme reveals that the catalytic mechanism proceeds through the formation of sialosyl cation intermediate. The structure of cation intermediate will provide an impetus to the development of sialidase inhibitor. The cluster continuum solvation analysis of sialyate compounds reports that the solvation free energy of sialyate-guandino complex and sialyate anion increases as a function of water molecules. Thus it nullifies the specific binding of explicitly water molecules in the functional site of sialyate compounds. The cluster continuum solvation analysis of cationic complex and zwitter ion shows that the solvation free energy of cationic complex and zwitter ion decreases initially and later increases as function of water molecules. Thus it accommodates the specific binding of water molecule in the explicit mono-hydrated and dihydrated structures. Hence, carboxylate oxygen, C2 carbon and C7 hydroxyl group were the best water binding sites for the sialyl cation and zwitterion structures and presence of water molecules in these sites were validated. The explicitly added water molecule decreases the binding energy of the sialyate complex and hence it indicates that explicit hydration is detrimental to the binding affinity. However, it increases the binding energy of sialosyl cationic complex and hence it favors the binding affinity of sialosyl cation complex. The cluster continuum solvation investigation of DANA, 4-amino-DANA and 4-guandinino-DANA reveals that the solvation free energy of the substrate complexes increases as a function of water molecule and thus it invalidates the presence of water molecules in its functional site. This system attains a limiting effect on the solvation free energy at the tetra-hydrated structure. Although these compound structurally differ from sialyate compound, but it follows the solvation free energy pattern same as with the parent sialyate compound. Meanwhile, explicit hydration decreases the binding affinity of all the pyranose ring derivatives such as DANA, 4-amino-DANA and 4-guandino DANA and hence, it is detrimental to the binding affinity of the sialidase inhibitors. The investigation of C4 substituents on DANA found that the amino, methoxy and methyl group were the promising substituents and attains a higher binding affinity than the parent DANA. The 183 guandino and methyl group at the C7 position attains a higher binding affinity than parent DANA. Hence, this substituent at C4 & C7 position of DANA will result in the development of modern anti influenza drugs. The substituent analysis on 4-amino-DANA discloses those methyl and methoxy groups were effective in increasing the binding affinity of 4-aminoDANA and hence, further investigation on this compound will a yield promising result in the development of sialidase inhibitors. The investigation of C7 substituent on the guandino compound reveals that the methyl, methoxy, amino and thiol group attains a higher binding affinity than the parent compound and hence, furthering research based on this template will yield a sialidase inhibitors with a higher binding affinity. The cluster continuum solvation analysis of tamiflu discloses the solvation free energy of tamiflu-guandino complex and its anion increases as a function of water molecule and attains a limiting value at the tetrahydrated structure due to the hydrogen bonding cooperative effects. The tetra and tri-hydrated structure of tamiflu anion attains a low solvation free energy than the parent tamiflu and hence, the presence of water molecules in the functional site is validated for both the explicit hydrated structures. Absence of oxygen in the carbocyclic ring improves the solvation free energy and hence it attains a higher solvation energy than its corresponding pyranose ring derivatives. The explicit hydration linearly decreases the binding affinity of the tamiflu and hence, explicit hydration is detrimental to the binding affinity of tamiflu. Effect of C12 substituents analysis on tamiflu indicates that the methyl and ethyl group favors the binding affinity of tamiflu and hence tamiflu with an alkyl derivative will provide a higher binding affinity inhibitor. However, C6 substituents on the tamiflu were not successful in increasing the binding affinity and hence amino is the best candidate for C6 functional group. The cluster continuum solvation analysis of BCX-guandino complex reveals that the solvation free energy decreases as a function of water molecules initially and later it increases. Thus it validates the presence of water molecules in a di-hydrated structure of BCX. However, the cluster continuum studies of BCX anion divulges that the solvation free energy decreases as a function of water molecules and authenticates the presence of water molecules in a mono-hydrated and tri- 184 hydrated explicit structure. 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Soc. 1997, 119, 681-690. 191 192 [...]... function of water molecules and hence, this finding will facilitate the development of sialidase inhibitors Chapter 7 deals with the effect of substituents on the binding affinity of the sialidase substrates The effect of C4 substituent on the DANA xiii discloses that the methoxy and amino group increases the binding affinity of DANA Similarly the analysis of C7 substituent on the binding affinity of DANA... determine the properties of molecules Thus these methods can, therefore, provide the benefits of some of the more expensive ab initio methods essentially at the cost of Hartree Fock calculations 2.2 THE SCHRöDINGER EQUATION Ab initio molecular orbital theory is mainly employed for predicting the properties of atomic and molecular systems It relies on the fundamental laws of quantum mechanics and uses... susbstituents of DANA 154-155 Figure 7-14, C7 substituents of DANA 158-160 Figure 15-22, C7 substituents of N-DANA 162-164 Figure 23-29, C7 substituents of guandino-DANA 166-168 Figure 30-39, C12 substituents of tamiflu 170-172 Figure 40-44, C6 substituents of tamiflu 174-175 xi SUMMARY This research study addresses the ab initio and DFT investigation of sialidase enzyme mechanism and hydration pattern of its inhibitor. .. continuum analysis delineates the specific binding effect of explicit water molecules in the functional site of the respective sialidase substrates Chapter-3 explains the catalytic path of sialidase enzyme using the ab initio method The mechanistic investigation of sialidase enzyme reveals that the enzyme mechanism proceeds through the carbocation intermediate formation and not by the covalent intermediate... on the binding affinity of the sialidase substrates The investigation of explicit water molecules on the binding affinity of the sialyate complex, DANA-complex, N-DANA complex, 4-guandino-DANA complex and tamiflu complex indicates that the binding affinity of the sialidase substrates decreases as a function of water molecules Hence, it authenticates that the explicit hydration is detrimental to the. .. enzyme catalysis and in the design of sialidase inhibitors Explicit solvation analysis of sialosyl cation provides precise information about the water mediated catalytic path Functional and structural information about sialidase has a significant effect on the discovery of sialidase inhibitors.13 The sialidase is involved in the cleavage of terminal sialic acid from ketosidic linkage of adjacent carbohydrate...Chapter 7 Table I Binding energy of C4 substituents of DANA 176 Table II Binding energy of C7 substituents of DANA 177 Table III Binding energy of C7 substituents of 4-amino-DANA 178 Table IV Binding energy of C7 substituent of 4-guandino-DANA 179 Table V Binding energy of C12 substituents of tamiflu 180 Table VI Binding energy of C6 substituents of tamiflu 181 viii LIST OF FIGURES Chapter... of nuclei and electrons, ψ is the wavefunction known as the Eigen function and E is the energy of the system The Hamiltonian operator24 is a sum of the kinetic and potentially energy of the system Aside from a few smaller systems, the exact solution to the Schrodinger equation is impossible Electronic structure methods are characterized by their various mathematical approximations to its solution The. .. perturbation on the HF problem In the MP scheme, the wave function and the energy are expanded in the power series of the perturbation Perturbation theory offers another method for finding quantum mechanical wave functions Perturbation theory is based upon dividing the Hamiltonian into two parts The first is one for which eigen functions and eigen values of the known parameters The first part and associated... valuable to gain insight into the discrete role of water molecule in folding and hydrogen bonding in the vicinity of sialidase active sites This solvation treatment will predict better water binding sites of ligands and the burial of water molecules in the vicinity of functional sites Besides, it helps to identify and characterize the binding sites where water improves the overall binding Theoretical and . addresses the ab initio and DFT investigation of sialidase enzyme mechanism and hydration pattern of its inhibitor. Effect of substituents at the key functional site of inhibitor is studied and validated. UNIVERSITY OF SINGAPORE 2010 AB INITIO AND DFT INVESTIGATION OF THE MECHANISM AND HYDRATION PATTERN OF SIALIDASE AND ITS INHIBITORS KRISHNAN CHANDRASEKARAN. AB INITIO AND DFT INVESTIGATION OF THE MECHANISM AND HYDRATION PATTERN OF SIALIDASE AND ITS INHIBITORS KRISHNAN CHANDRASEKARAN