Towards the Design of Carnitine Acyltransferase Inhibitors: Modeling the Conformational Behavior of (R)-Carnitine, (R)-Acetylcarnitine, Morpholinium rings, and 2-Oxo-1,3,6-dioxazaphosphacinium rings.

Víctor Manuel Rosas-García

Dissertation submitted to the Faculty of
Virginia Polytechnic Institute and State University
in partial fulfillment of the requirements for the degree of

Doctor of Philosophy
in
Chemistry

APPROVED:
Richard D. Gandour
Mark Anderson
David Bevan
Harry Gibson
Thomas Hudliçky
James Tanko

Blacksburg, Virginia

KEYWORDS: carnitine , acylcarnitine , modeling of morpholinium rings , phosphonate , modeling of medium-sized rings , conformational analysis

Copyright 1997, Víctor M. Rosas-García


Abstract

Full grid-search semiempirical calculations (AM1 and AM1/COSMO) on zwitterionic acetylcarnitine and carnitine, cationic acetylcholine and choline, and 3-acetoxypropanoate and 3-hydroxypropanoate in the gas phase and solution were performed. The calculated {delta}H°hydr for hydrolyses of acetylcarnitine to carnitine and of acetylcholine to choline show reasonable agreement with the experimental values in unbuffered solution (acetylcarnitine: -4.63 kcal/mol calc. vs. -7.43 kcal/mol exp.; acetylcholine: -3.20 kcal/mol calc. vs. -3.06 kcal/mol exp.) The results suggest that a change in the conformational populations of acetylcarnitine-carnitine upon hydrolysis maintains a nearly constant polarity, which keeps the work of desolvation of the products to a minimum. Acetylcholine-choline and acetoxypropanoate-hydroxypropanoate present a much higher work of desolvation, therefore yielding a lower free enthalpy of hydrolysis. Ab initio calculations at the RHF/6-31G* level for the carnitines and the cholines, and RHF/6-31+G for the propanoates, were done to calibrate the quality of the AM1 results for both the gas phase and in solution. The calculations in the gas phase involved full optimization of the AM1-optimized structures at the RHF/6-31G* level and RHF/6-31+G level, and single points at the MP2//RHF/6-31G* and MP2//RHF/6-31+G level to estimate correlation effects. The ab initio calculations in solution were single points on the AM1-optimized geometries and used the Tomasi solvent model. The ab initio results confirmed the qualitative reliability of the semiempirical results.

The conformational behavior of several 4,4-dimethylmorpholinium rings and 4,4-dimethyl-2-oxo-1,3,6-dioxazaphosphacinium rings was examined by molecular mechanics (AMBER* and AMBER*-GB/SA). The contrast between the behavior of these heterocycles and that of the parent saturated hydrocarbon systems formed a picture of the conformational behavior of these six- and eight-membered heterocycles. Influences of factors such as shortened bond lengths, varied bond angles, presence or absence of lone pairs and substituents, and dipolar alignment are described. Morpholinium rings show increased stabilization of the twist-boat with 1,1,3,3-digem substitution, as compared to the parent cyclohexane systems. In the gas phase, the lowest chair/twist-boat energy gap is found in 2-(hydroxymethyl)-2,4,4-trimethylmorpholinium at 1.14 kcal/mol. The gap in the congruent hydrocarbon system is 5.23 kcal/mol. Differential solvation destabilizes the lowest energy twist-boat found in the gas phase, increasing the energy gap to 2.62 kcal/mol. The lowest chair/twist-boat energy gap in GB/SA water amounts to 1.45 kcal/mol, stabilized by solvation from an initial 2.13 kcal/mol in the gas phase.

In the dioxazaphosphacinium rings, the preferred conformation in the gas phase is the boat-chair (BC) and the populations are conformationally heterogeneous. As substituents approach a 1,1,3,3-digem pattern, the twist-chair (TC) and twist-boat (TB) conformers are stabilized. Solvation favors boat-boat (BB) conformers, with the substituents exerting influence on the conformational preference only to stabilize the TB in two instances ( [cis-substituted ring] and [disubstituted ring]). Solvation reduces the heterogeneity of the conformational populations.

Modeling of phosphonate moieties required development of molecular mechanics parameters for dimethyl methylphosphonate. Dimethyl methylphosphonate conformations were calculated at the RHF/6-31+G* level. Charges were calculated by the CHelpG scheme. The results were used to generate AMBER* parameters for modeling of alkylphosphonates in the gas phase and in solution. Comparison of the results of our AMBER* parameters against three other common force fields (MM2*, MMX and UFF) showed that AMBER* reproduced better the ab initio results when comparing absolute deviations in bond lengths, bond angles and torsion angles. The modified AMBER* reproduced better than the other three force fields several X-ray geometries of alkylphosphonates.

Part of this work was supported by NIH Grant GM 42016


Table of Contents

Title pageTowards the Design of Carnitine Acyltransferase Inhibitors: Modeling the Conformational Behavior of (R)-Carnitine, (R)-Acetylcarnitine, Morpholinium rings, and 2-Oxo-1,3,6-dioxazaphosphacinium rings.
Dedication
Acknowledgments
1 Introduction.
1.1.Purpose of study.
2 Historical.
2.1.Introduction.
2.2.Acyltransferase inhibitors.
2.2.1.Competitive reversible inhibitors.
2.2.2.Competitive irreversible inhibitors.
2.2.3.Noncompetitive reversible inhibitors.
2.2.4.Noncompetitive irreversible inhibitors.
3 Acetylcarnitine hydrolysis.
3.1.Review of literature.
3.1.1.Acylcarnitines in cellular metabolism.
3.1.2.Thermodynamics of acetylcarnitine hydrolysis.
3.1.2.1.High-energy compounds.
3.1.2.2.Effects of solvation on thermodynamic quantities.
3.1.2.3.Relationship between conformational preference and thermodynamics.
3.2.Results and Discussion.
3.2.1.Conformational analyses in gas phase.
3.2.1.1.Acetylcarnitine vs. carnitine.
3.2.1.2.Acetylcholine vs. choline.
3.2.1.3.Acetoxypropanoate vs. hydroxypropanoate.
3.2.1.4.Carnitines vs. cholines.
3.2.1.5.Carnitines vs. propanoates.
3.2.2.Ab initio results in the gas phase.
3.2.3.Comparison of AM1 vs. ab initio.
3.2.4.Conformational analyses including the effect of solvent.
3.2.4.1.Acetylcarnitine vs. carnitine
3.2.4.2.Acetylcholine vs. choline.
3.2.4.3.Acetoxypropanoate vs. hydroxypropanoate.
3.2.4.4.Carnitines vs. cholines.
3.2.4.5.Carnitines vs. propanoates.
3.2.5.Ab initio results including solvent.
3.2.6.Thermochemical calculations from semiempirical results.
3.2.7.Modeling of tetrahedral intermediate: preliminary results.
4 Effects of heteroatoms on conformational stability.
4.1.General effects of introduction of heteroatoms.
4.2.The anomeric effect.
4.3.The gauche effect.
4.4.Conformational analysis of heterocycles.
4.4.1.Six-membered rings.
4.4.2.Eight-membered rings.
4.5.Conformational analyses of morpholinium rings.
4.5.1.Modeling in the gas phase.
4.5.1.1.Cyclohexanes.
4.5.1.2.Morpholinium rings.
4.5.2.Modeling in GB/SA water.
4.6.Conformational analyses of 2-oxo-1,3,6-dioxazaphosphacinium rings.
4.6.1.Modeling in the gas phase.
4.6.1.1.Cyclooctanes.
4.6.1.2.Dioxazaphosphacinium rings.
4.6.2.Modeling in GB/SA water.
5 Modeling of phosphonates.
5.1.Modeling of phosphorus species.
5.1.1.Ab initio methods.
5.1.2.Semiempirical methods.
5.1.3.Molecular mechanics methods.
5.2.Development of AMBER* parameters for phosphonates.
5.2.1.Calculations on dimethyl methylphosphonate.
5.2.2.Comparison vs. crystal structures.
6 Methods.
6.1.General methods and software.
6.1.1.Molecular mechanics.
6.1.2.Semiempirical calculations.
6.1.3.Ab initio calculations.
6.2.Acetylcarnitine calculations.
6.2.1.Conformational search strategy by molecular mechanics.
6.2.2.Conformational populations.
6.2.3.Semiempirical calculations on carnitines, cholines and propanoates.
6.2.4.Ab initio calculations on carnitines, cholines and propanoates.
6.3.Conformational search in six-membered rings.
6.4.Conformational search and analyses in eight-membered rings.
6.5.AMBER* parametrization of phosphonates.
6.6.Least-squares fit of phosphonate rings to tetrahedral intermediate.
6.7.Hardware.
7 Conclusions.
7.1.Summary
7.2.Future work.
Bibliography
A Naming and numbering scheme for cyclooctane conformers.
A.1.Boat-chair family.
A.2.Chair-chair family.
A.3.Boat-boat family.
B Rules for naming hetero-substituted cyclooctanes.
C Software standards used.
D Chemical files included.
E README: Notes on the creation of a dissertation in ETD-ML.
E.1.ETD-ML source encoding.
E.2.Virtual Reality Modeling Language (VRML).
E.3.Chemical Markup Language (CML).
E.4.Babel, PDB, Chime.
E.5.Reformatting the molecules.
E.6.Footnotes.
E.7.Notes on MOL notation substitution.
E.8.README.TXT: Files in this ETD.
Vita

List of Tables

3.1.Comparison of AM1 vs. 6-31G* calculations on carnitines and cholines in the gas phase.
3.2.Comparison of AM1 vs 6-31+G calculations on propanoates in the gas phase.
3.3.Comparison of AM1 vs. 6-31G* calculations on carnitines and cholines in water.
3.4.Comparison of AM1 vs. 6-31+G calculations on propanoates in water.
3.5.Comparison of {delta}Hhydr (kcal/mol) calculated at {epsilon}=78.3 vs. calorimetric values.
3.6.Expectation values for Dipoles ({mu}) and Enthalpies of Solvation ({delta}H°solv)
3.7.RMS deviation after least-squares fitting of global minimum of rings to global minimum of intermediate in GB/SA water.
3.8.RMS deviation after least-squares fitting of second most populated conformer of ring to global minimum of intermediate in GB/SA water.
4.1.Comparison of relative energies in the gas phase (kcal/mol) of cyclohexane conformations.
4.2.Comparison of morpholinium vs. dimethylcyclohexane in the gas phase.
4.3.Comparison of energies of trimethylmorpholinium vs. trimethylcyclohexane in the gas phase (kcal/mol).
4.4.Comparison of energies of tetramethylcyclohexane vs. tetramethylmorpholinium in the gas phase (kcal/mol).
4.5.Comparison of energies of ethyltrimethylcyclohexane vs. (hydroxymethyl)trimethylmorpholinium in the gas phase (kcal/mol).
4.6.Conformational and solvation energies (kcal/mol) of dimethylmorpholinium in the gas phase and GB/SA water.
4.7.Conformational and solvation energies (kcal/mol) of trimethylmorpholinium in the gas phase and GB/SA water.
4.8.Conformational and solvation energies (kcal/mol) of tetramethylmorpholinium in the gas phase and GB/SA water.
4.9.Conformational and solvation energies (kcal/mol) of (hydroxymethyl)trimethylmorpholinium in the gas phase and GB/SA water.
4.10.Conformers of cyclooctane within 3.0 kcal/mol of global minima in the gas phase.
4.11.Conformers of tetramethylcyclooctane within 3.0 kcal/mol of global minima in the gas phase.
4.12.Conformers of pentamethylcyclooctane within 3.0 kcal/mol of global minima in the gas phase.
4.13.Conformers of hexamethylcyclooctane within 3.0 kcal/mol of global minima in the gas phase.
4.14.Conformers of dioxazaphosphacinium within 3.0 kcal/mol of global minima in the gas phase.
4.15.Conformers of cis-methyl dioxazaphosphacinium within 3.0 kcal/mol of global minimum in the gas phase.
4.16.Conformers of trans-methyl dioxazaphosphacinium within 3.0 kcal/mol of global minimum in the gas phase.
4.17.Conformers of dimethyl dioxazaphosphacinium within 3.0 kcal/mol of global minimum in the gas phase.
4.18.Conformers of dioxazaphosphacinium within 3.0 kcal/mol of global minimum in water.
4.19.Conformers of cis-methyl dioxazaphosphacinium within 3.0 kcal/mol of global minimum in water.
4.20.Conformers of trans-methyl dioxazaphosphacinium within 3.0 kcal/mol of global minimum in water.
4.21.Conformers of dimethyl dioxazaphosphacinium within 3.0 kcal/mol of global minimum in water.
5.1.Predicted bond lengths and angles of dimethyl methylphosphonate.
5.2.RMS fit of molecular mechanics to ab initio geometries.
5.3.Relative conformational energies (kcal/mol)
5.4.Comparison of Molecular Mechanics results vs. X-ray.
5.5.RMS values of MM vs. X-ray including all heavy atoms.
5.6.RMS values of MM vs. X-ray including phosphonate heavy atoms only.

List of Figures

1.1.4,4-dimethylmorpholinium ring.
1.2.2,4,4,-trimethyl-2-oxo-1,3,6-dioxazaphosphacinium ring.
2.1.Tetrahedral intermediate for Carnitine-CoA acyl transfer.
2.2.Aminocarnitine.
2.3.Hemiacylcarnitinium moiety.
2.4.3-hydroxy-5,5-dimethylhexanoic acid.
2.5.3-amino-5,5-dimethylhexanoic acid.
2.6.4-hexadecyl-2,4,4-trimethyl-2-oxo-1,3,6-dioxazaphosphacinium bromide.
2.7.6-(Carboxylatomethyl)-2-(hydroxymethyl)-2,4,4-trimethylmorpholinium.
2.8.Anderson's phosphonate.
2.9.Anderson's phosphate.
3.1.Comparison of free energies of hydrolysis between acetylcarnitine and acetylcholine.
3.2.``Folded'' conformer of carnitine.
3.3.``Extended'' conformer of carnitine.
3.4.Hydrolyses of extended and folded conformers.
3.5.Labels for torsion angles used in acetylcarnitine.
3.6.Changes in population and dipole moments upon hydrolysis.
3.7.Differences in the energies of solvation for acetylcarnitine, acetylcholine and 3-acetoxypropanoate.
3.8.Global minimum for (R,S) tetrahedral intermediate in GB/SA water.
3.9.3D Model of the R,S tetrahedral intermediate in GB/SA water.
3.10.Global minimum for (R,R) tetrahedral intermediate in GB/SA water.
3.11.3D Model of the R,R tetrahedral intermediate in GB/SA water.
3.12.Atom pairs used in the least squares fit between the phosphonate rings and the tetrahedral intermediate.
4.1.Orbital and hyperconjugation description of the anomeric effect.
4.2. Crown (D4d)
4.3.Chair-chair, CC (C2v)
4.4.Twist-chair-chair, TCC (D2)
4.5.Twist-chair, TC (C2v)
4.6.Chair, C (C2h)
4.7.Boat-chair, BC (Cs)
4.8.Twist-boat-chair, TBC (C2)
4.9.Boat-boat, BB (D2d)
4.10.Twist-boat, TB (S4)
4.11.Boat, B (D2d)
4.12.Structure of explosive HMX.
4.13.2-(Hydroxymethyl)-2,4,4-trimethylmorpholinium studied by Savle et al.
4.14.Labeling of substituent positions in cyclohexane.
5.1.Fit of AMBER* phosphonate parameters for P-C bond stretch to 6-31+G* curve.
5.2.Fit of AMBER* phosphonate parameters for P-O bond stretch to 6-31+G* curve.
5.3.Fit of AMBER* phosphonate parameters for O-P-O bond angle to 6-31+G* curve.
5.4.Fit of AMBER* phosphonate parameters for C-P=O bond angle to 6-31+G* curve.
5.5.Fit of AMBER* phosphonate parameters for H-C-P=O torsion angle to 6-31+G* curve.
5.6.Fit of AMBER* phosphonate parameters for C-O-P-O torsion angle to 6-31+G* curve.
5.7.Conformers of dimethyl methylphosphonate.
5.8.Comparison of molecular mechanics C-O-P=O torsion profiles vs. ab initio.
5.9.First crystal structure used for comparison (ZAYSIB).
5.10.Additional crystal structures used in the comparison.
A.1.``Left-oriented'' and ``right-oriented'' BCs
A.2.Labels for the orientations of substituents in BC
A.3.``Left-oriented'' and ``right-oriented'' TBCs
A.4.Labels for the orientations of substituents in TBC
A.5.CC numbering and labeling scheme.
A.6.Crown.
A.7.TCC numbering and labeling scheme.
A.8.TC numbering and labeling scheme.
A.9.C numbering and labeling scheme.
A.10.B numbering and labeling scheme.
A.11.BB numbering and labeling scheme.
A.12.TB numbering and labeling scheme.
B.1.Example conformer.

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