Kira A. Armacost

236 Pages·2013·7.03 MB·English

by  Kira Armacost

Exploring the Claisen Rearrangement and Aldol Reaction “on Water” and in
Catalytic Antibodies using QM/MM Simulations and the Examination of Ligand
Induced Conformational Changes in Alkanesulfonate Monooxygenase using
Molecular Dynamics

by

Kira A. Armacost

A dissertation submitted to the Graduate Faculty of
Auburn University
in partial fulfillment of the
requirements for the Degree of
Doctor of Philosophy

Auburn, Alabama
May 5, 2013

Copyright 2013 by Kira Allyce Armacost

Approved by

Orlando Acevedo, Chair, Associate Professor of Chemistry
J. Vincent Ortiz, Ruth W. Molette Professor and Chairman
Konrad Patkowski, Assistant Professor of Chemistry
Michael Squillacote, Associate Professor of Chemistry
Abstract

The “on water” environment, defined by the absence of water solubility of the
reactants has been reported to provide increased rate accelerations, yields, and specificity
for several types of organic reaction classes compared to organic solvents. The aromatic
Claisen rearrangements of allyl p-R-phenyl ethers (R = CH , Br, and OCH ) and allyl
3 3
naphthyl ether, and another class of reaction, the aldol reaction have been investigated to
determine their on water enhancements using QM/MM Monte Carlo calculations and
free-energy perturbation theory. The aldol reaction was further studied in catalytic
antibody 33F12 to determine the mechanistic properties and its comparison with the aldol
reaction in condensed phase simulations. In another study, molecular dynamics
calculations were performed on monooygenase systems to identify the protein
fluctuations with various bound ligands (reduced flavin mononucleotide (FMNH ), a
2
C4a-peroxyflavin intermediate, alkanesulfonate, FMNH -alkanesulfonate, and C4a-
2
peroxyflavin intemediate-alkanesulfonate) and an apo (free of ligand) structure. Salt
bridge formations have been shown to participate in dynamic conformational changes for
the ligand bound structures between Glu20-Arg297 and Asp111-Arg297. Also, Arg226
was confirmed to stabilize the peroxy group of the c4a-peroxyflavin ligand, and possibly
help contributing to the sulfite release.

ii
Acknowledgements

My sincere and utmost appreciation is given to my advisor, Dr. Orlando Acevedo,
for all the guidance he has given me through my graduate school career. I am extremely
thankful for all the mentoring and support I have received from him. Next, I’d like to
thank Dr. Vincent Ortiz, Dr. Konrad Patkowski, Dr. Michael Squillacote and Dr. Paul
Cobine for serving as my committee members and for the support I’ve gotten in my
dissertation process. To my past and present research lab mates, Samba, Billy, Caley,
Symon, Nicole and Bin, I’d like to thank each one of you for helping with everything I’ve
needed and being a great support system. Last, but definitely not least, I’d like to
especially thank my dad, mom and brother who have dealt with all of my calls home and
extreme levels of stress. Without my family, I wouldn’t have gotten to where I am today.

iii
Table of Contents

Abstract ............................................................................................................................... ii
Acknowledgements ............................................................................................................ iii
List of Tables ..................................................................................................................... vi
List of Schemes ................................................................................................................. vii
List of Figures .................................................................................................................... ix
Chapter 1: Introduction ......................................................................................................1
1.1 Chemical Reactions in Aqueous Solutions ................................................................1
1.2 “On Water” Reactions ................................................................................................2
1.3 The Claisen Rearrangement .......................................................................................5
1.4 The Aldol Reaction ....................................................................................................9
1.5 Catalytic Aldolase Antibody ....................................................................................15
1.6 Alkanesulfonate Monooxygenase ............................................................................20
Chapter 2: Methods ..........................................................................................................28
2.1 QM/MM ...................................................................................................................28
2.1.1 PDDG/PM3 ...................................................................................................33
2.1.2 Polarized Continuum Model (PCM) .............................................................37
2.1.3 OPLS .............................................................................................................39
2.1.4 Free Energy Perturbation ..............................................................................40
2.1.5 Monte Carlo ..................................................................................................43
2.1.6 Periodic Boundary Conditions ......................................................................44
2.2 Molecular Dynamics ................................................................................................45
2.2.1 Integration Algorithms ..................................................................................48
iv
2.2.2 SHAKE Algorithm ........................................................................................52
2.2.3 Amber Biomolecular Package ......................................................................53
2.3 Principal Component Analysis .................................................................................57
Chapter 3: Claisen Rearrangement: Insight into Solvent Effects and “on Water”
Reactivity from QM/MM Simulations...............................................................................65
3.1 Introduction ..............................................................................................................66
3.2 Methods ....................................................................................................................67
3.3 Structures ..................................................................................................................70
3.4 Energetics .................................................................................................................74
3.5 Solvent Effects on the Rate of Reaction ...................................................................77
3.6 Solvent Polarization .................................................................................................78
3.7 Substituent Effects on Energetics .............................................................................79
3.8 Solute-Solvent Energy Pair Distributions ................................................................82
3.9 Radial Distribution Functions ..................................................................................89
3.10 Hydrophobic Effects ..............................................................................................91
3.11 Continuum Solvent Models ....................................................................................92
3.12 Conclusions ................................................................................................................95
Chapter 4: Understanding the Aldol Reaction from Catalytic Antibodies to “On Water”
Organocatalysts using QM/MM Calculations ...................................................................97
4.1 Introduction ..............................................................................................................98
4.2 Methods ..................................................................................................................102
4.2.1 Setup and Processing for Antibody 33F12 .................................................102
4.2.2 QM/MM Method ........................................................................................103
4.2.3 Monte Carlo Simulation Protocol ...............................................................105
4.2.4 Polynomial Quadrature Method ..................................................................106
4.3 Results and Discussion ...........................................................................................106
v
4.3.1 Aldol Reaction in Antibody 33F12 ............................................................106
4.4 Future Work ...........................................................................................................112
4.4.1 Aldol reaction with an Organocatalyst in the Gas Phase ............................112
4.4.2 Aldol Reaction with an Organocatalyst in Solution ...................................112
4.5 Conclusion ..............................................................................................................116
Chapter 5: Flavin-Induced Active Site Conformational Changes in Alkanesulfonate
Monooxygenase Predicted from Molecular Dynamic Simulations .................................118
5.1 Introduction ............................................................................................................119
5.2 Methods ..................................................................................................................121
5.2.1 Enzyme Setup .............................................................................................121
5.2.2 MD Simulation Protocol .............................................................................122
5.2.3 Principal Component Analysis ...................................................................123
5.2.4 Analysis.......................................................................................................124
5.3 Results and Discussion ...........................................................................................124
5.3.1 Cluster Analysis ..........................................................................................124
5.3.2 Role of Arg297 ...........................................................................................127
5.3.3 Salt Bridge Formations Between Glu20-Arg297 and Asp111-Arg297 ......129
5.3.4 Octanesulfonate and its Role in SsuD .........................................................133
5.3.4 Conformational Fluctuations Occurring in SsuD .......................................136
5.4 Conclusion ..............................................................................................................145
References .......................................................................................................................147
Appendix I .................................................................................................................... I – 1
Appendix II .................................................................................................................. II – 1
vi
List of Tables

Table 1.1 Percent Yields of Different Solvents for the Claisen Rearrangement of
Allyl Naphthyl Ether ..................................................................................7
Table 3.1 Computed Bond Lengths (Å) for the Allyl p-Tolyl Ether Claisen
Rearrangement Transition Structures at 25°C at 1 at ...............................74
Table 3.2 Absolute Free Energy of Activation, G‡ (kcal/mol), at 25 C for the
Claisen Rearrangement of Allyl p-Tolyl Ether and the G‡ Relative to
“In Water” using QM/MM .......................................................................76
Table 3.3 Absolute Free Energy of Activation, G‡ (kcal/mol), at 25 C for the
Claisen Rearrangement of Allyl p-R-Phenyl Ethers and the G‡
Relative to R = CH using QM/MM simulations .....................................80
3
Table 3.4 Computed Free Energy of Activation, G‡ (kcal/mol), at 25 C for the
Claisen Rearrangement of Allyl Naphthyl Ether and the G‡ Relative to
“In Water ..................................................................................................81
Table 3.5 Solute-Solvent Energy Pair Distributions for the Claisen Rearrangement
of Allyl p-R-Phenyl Ethers and Allyl Naphthyl Ether for the Reactant
(GS) and Transition Structure (TS) “in water” and “on water” integrated
to -4.0 kcal/mol (and -3.5 kcal/mol in parenthesis ...................................86
Table 3.6 Charges for the Claisen Rearrangement Transition Structure (and
Reactant in Parenthesis) of Allyl p-R-Phenyl Ether ................................86
Table 3.7 Number of Water Molecules Interacting with the Reacting Oxygen of
Allyl p-R-Phenyl Ethers and Allyl Naphthyl Ether for the Claisen
Rearrangement Reactant (GS) and Transition Structure (TS) “in water”
and “on water” from Radial Distribution Functions ................................91
Table 3.8 Gas-Phase Activation Barriers, G‡ (kcal/mol), at 25 C for the Claisen
Rearrangement of Allyl p-R-Phenyl Ethers (R = OCH , CH , and Br) ..93
3 3
Table 3.9 Free Energy of Activation, G‡ (kcal/mol), at 25 C for the Claisen
Rearrangement of Allyl p-R-Phenyl Ethers in Solution using B3LYP/6-
311+G(2d,p)/PCM ...................................................................................94
Table 5.1 Dominant clusters for SsuD unbound and with various bound ligands.
The RMSds are the average values over the range the cluster occurs ...125
Table 5.2 RMSF for interactions listed (average RMSF of residues) ...................144
Table 5.3 Average difference in fluctuations of interactions with comparison of
each bound structure (b) to the unbound (apo) structure (a). The Arg297-
Flavin and Arg226-Flavin (b) differences are compared to FMNH (a)
2
and Arg226-OCS (b) is compared to OCS (a) ......................................144

vii
List of Figures

Figure 1.1 The reaction of quadricyclane and dimethyl azodicarboxylate .................4
Figure 1.2 Conversion of chorsimate to prephenate ....................................................6
Figure 1.3 Enamine mechanism of acetone and 4-nitrobenzaldehyde ......................10
Figure 1.4 Hydrophobic organocatalyst with gem-diphenyl group ..........................11
Figure 1.5 Transition state models of the enamine mechanism (A: Zimmerman-
Traxler model and B: DFT models) .........................................................12
Figure 1.6 Role of water in the reaction of acetone and benzaldehyde with
organocatalyst ..........................................................................................13
Figure 1.7 Transition state models of benzaldehyde and acetone with catalyst ........13
Figure 1.8 Mechanism of class-I aldolase antibodies (Enz=enzyme, B=base) ........16
Figure 1.9 Mechanism for trapping Lys residue in active site by antibodies (Ab)
33F12 and 38C2 .......................................................................................17
Figure 1.10 DFT calculations yielding the transition structures .................................18
Figure 1.11 Desulfonation mechanism of alkanesulfonate monooxygenase ..............21
Figure 1.12 A: Flavin mononucleotide (FMN) and B: Flavin Adenoside Dinucleotide
(FAD) ......................................................................................................21
Figure 1.13 Flavin-dependent monooxygenase systems .............................................22
Figure 1.14 SsuD enzyme. (a) View perpendicular to β-barrel. (b) View along β-
barrel axis .................................................................................................23
Figure 1.15 The kinetic mechanism for the reduction of flavin by SsuE ....................23
Figure 1.16 Mechanism for formation of C4a-(hydro)peroxyflavin and C4a-peroxy
flavin intermediates ..................................................................................24
Figure 1.17 Catalytic mechanisms of C4a-(hydro)peroxyflavin (A) and C4a-
peroxyflavin (B) ......................................................................................27
Figure 2.1 The QM region represents the bond making/bond breaking portion,
where the MM represents the remainder of the system ...........................29
Figure 2.2 Lennard-Jones Potential graphical representation ...................................32
Figure 2.3 Description of the link atom for ethane, where two methyl fragments are
generated and link atom (L) replaces the q-m bond ................................33
Figure 2.4 Thermodynamic cycle for molecules A and B in two different solvent .42
Figure 2.5 Representation of double wide sampling ................................................43
Figure 2.6 Periodic boundary conditions in two dimensions ....................................45
Figure 2.7 Atomic positions in a 3-dimensional space ..............................................47
Figure 2.8 The forms of the Verlet algorithm. (a) Original. (b) Leap frog method. (c)
Velocity Verlet algorithm ........................................................................51
Figure 2.9 Flow chart for the AMBER program .......................................................54
Figure 2.10 Scree plot (circle represents “elbow” region of plot) ..............................53

viii
Figure 3.1 Free energy map (kcal/mol) computed for the “on water” Claisen
rearrangement of allyl p-tolyl ether using the reaction coordinates R
OC
and R . Illustrated structure is the transition structure from the
CC
QM/MM/MC simulations ........................................................................71
Figure 3.2 Illustration of the “on water” allyl p-tolyl ether transition structure from
the QM/MM/MC Claisen rearrangement calculations .............................72
Figure 3.3 Solute-solvent energy pair distributions for the Claisen rearrangement of
allyl p-tolyl ether in p-chlorophenol, transition structure (solid black) and
reactant (dashed black), and in n-decylamine, transition structure (solid
blue) and reactant (dashed blue), at 25 C. The ordinate records the
number of solvent molecules that interact with the solutes and their
interaction energy on the abscissa ............................................................83
Figure 3.4 Solute-solvent energy pair distributions for the Claisen rearrangement of
allyl p-R-phenyl ethers: “in water” transition structure (solid red), “in
water” reactant (dashed red), “on water” transition structure (solid black),
and “on water” reactant (dashed black) at 25 C .....................................87
Figure 3.5 Computed O(ether)H(water) radial distribution function for the Claisen
rearrangement reactions of allyl p-R-phenyl ethers: “in water” transition
structure (solid red), “in water” reactant (dashed red), “on water”
transition structure (solid black), and “on water” reactant (dashed black)
at 25 C .....................................................................................................88
Figure 4.1 Illustration of reduced antibody 33F12 consisting of 135 residues,
benzaldehyde, TyrL36, the enamine formed between acetone and
LysH93, and a 22 Å water cap ...............................................................108
Figure 4.2 Free-energy profile (kcal/mol) for the aldol reaction between
benzaldehyde, the enamine formed between acetone and LysH93, and
TyrL36 in antibody 33F12. Maximum free-energy values are truncated to
70 kcal/mol for clarity ............................................................................110
Figure 4.3 A close-up of the aldol reaction transition state between the LysH93-
enamine and benzaldehyde in the active site of antibody 33F12 with 2
nearby water molecules retained. Average distances over the final 25
million configurations given in angstroms .............................................112
Figure 4.4 re- face transition state geometry (a) is C-C bond formation transition
structure, and (b) is the proton transfer transition structure ...................113
Figure 4.5 si- face transition state geometry (a) is C-C bond formation transition
structure, and (b) is the proton transfer transition structure ...................114
Figure 4.6 Illustration of the organocatalyst-enamine aldol reaction with
benzaldehyde on water from the QM/MM MC/FEP simulations ..........115
Figure 4.7 Overlay of the organocatalyst-enamine transition states with the
favorable re-face (beige) and unfavorable si-face benzaldehyde (blue)
conformations .........................................................................................116
Figure 5.1 RMSd for WT SsuD unbound and bound with various substrates, with
respect to the initial structure .................................................................126
Figure 5.2 Cluster structure 9 from the FMNH bound WT SsuD system
2
highlighting the interacting distance between the phosphorous of PO and
4
NH of Arg297 .......................................................................................127
2
ix
Figure 5.3 Distance calculations between the phosphorus of the flavin phosphate
group with NH of Arg297 for all WT SsuD structures containing a flavin
2
group ......................................................................................................128
Figure 5.4 Salt bridge between Glu20 and Arg297 for WT SsuD with no bound
substrate occurring at 30ns .....................................................................130
Figure 5.5 Hydrogen bonds formed between Glu20 and Arg297 for all simulations
(No occupancy above 0.1% for the apo structure) ................................130
Figure 5.6 Salt bridge between Asp111 and Arg297 in WT SsuD with docked
FMNH . Illustrated structure given at 11.8ns .......................................131
2
Figure 5.7 Hydrogen bonds formed between Asp111 and Arg297 for all simulations
(No occupancy above 0.1% for the FMNO- structure) ..........................131
Figure 5.8 Distance calculations showing a salt bridge between Glu20 and Arg297
occurring over a large portion of the FMNO- simulation and electrostatic
interactions between the peroxy group of FMNO- and NH of Arg226,
2
represented by the illustrated cluster structure for the enzyme ..............132
Figure 5.9 Distance calculations showing a salt bridge between Glu20 and Arg297
occurring over a large portion of the FMNO--OCS simulation and
electrostatic interactions between the peroxy group of FMNO--OCS and
NH of Arg226, as shown in both the distance calculation and the
2
illustrated cluster structure for the enzyme ............................................133
Figure 5.10 All hydrogen bond interactions occupying above 3% of the simulation
time for the FMNH in the FMNH bound and FMNH -OCS bound SsuD
2 2 2
systems ...................................................................................................134
Figure 5.11 Distance calculations for WT SsuD containing octanesulfonate as a
substrate for the sulfure of OCS and NH of Arg226 with the dominant
2
cluster structures for the OCS and FMNH -OCS simulation illustrated
2
................................................................................................................135
Figure 5.12 Distance calculation of the peroxy group of the flavin group with
octanesulfonate for the FMNO--OCS SsuD simulation .........................136
Figure 5.13 Eigenvalue scree plot indicating the total percent variance for each
principal component. Numbers next to points represent the sum of the
total variance at the given PC ................................................................137
Figure 5.14 Root mean square fluctuations of FMNH (solid black) with the apo
2
structure (solid red). The secondary structure is located with blue and
orange bars (alpha helices are in orange and beta sheets are in blue). PC
represents the first three dominant principal components......................139
Figure 5.15 Root mean square fluctuations of FMNO- (solid black) compared to the
apo structure (solid red). The secondary structure is located with blue and
orange bars (alpha helices are in orange and beta sheets are in blue). PC
represents the first three dominant principal components......................140
Figure 5.16 Root mean square fluctuations of OCS (solid black) compared to the apo
structure (solid red). The secondary structure is located........................141
Figure 5.17 Root mean square fluctuations of FMNH -OCS (solid black) compared to
2
the apo structure (solid red). The secondary structure is located with blue
and orange bars (alpha helices are in orange and beta sheets are in blue).
PC represents the first three dominant principal components ................142
x