GR/M67230
Molecular Basis of Enantiomeric Discrimination
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The Molecular Basis of Enantiomeric Discrimination: Predictive
Optimisation of Chiral HPLC
Final Report
1
Summary
This proposal was for a combined computer simulation and experimental study to develop
new tools for designing and optimising chiral HPLC. Of particular interest was to show that it
is now feasible to perform realistic molecular simulations of HPLC, and that such simulations
can aid in the design and optimisation of HPLC columns. In particular, it was proposed to
carry out a combined simulation / experimental investigation into a possible correlation
between circular dichroism spectra on mixed solutions and chiral HPLC columns based on
the same components to determine whether such a correlation could be relied upon to
optimise the conditions for chiral HPLC. The major findings of this project have been:
(i)
to show that molecular simulation can be used to good effect in understanding
the interactions that drive chiral discrimination in an HPLC column;
(ii)
to develop a new method of calculating ∆∆G values (and hence selectivities)
for chiral HPLC based on atomistic simulations and using explicit solvent; this
method provides a good balance between accuracy and ease of
implementation;
(iii) to show that the correlation between circular dichroism spectra and HPLC
resolving power is also found with cellulose stationary phases, and to verify
from simulations that there is a common molecular mechanism to underpin
this linkage;
(iv)
to show that chiral discrimination in flexible molecules (i.e. compounds that
can exist in a number of different conformations under operating conditions)
must be understood in terms of an ensemble of different dimer geometries, and
involves a subtle balance of discriminations in favour of both enantiomers;
(v)
to verify that solvent structure can have a major effect on chiral
discrimination; in particular, tetrahydrofuran shows both important packing
effects arising from its cyclic shape and hydrogen-bond formation, and the
balance of these two effects can determine which of the favourable analytestationary phase geometries is found most often under the specified operating
conditions.
2
Background/Context
Chiral chromatography is an increasingly important tool in both academic and industrial
research, particularly for the pharmaceutical industry. It is now one of the most efficient and
reliable methods for determining enantiomeric purity and for obtaining enantiomerically pure
samples of new drugs. Yet the technique has been severely hampered by an inability to
predict optimum conditions, or even suitable phases, to effect a given separation. The range
of chiral phases available is both limited and expensive, and finding a suitable match of chiral
phase and chromatographic conditions to a given application is not always possible. These
problems are a serious limitation for chiral HPLC, and indicate an urgent need to develop
better methods for tailoring HPLC to specific applications.
The problem of rational design also occurs when developing drugs and materials, and
in these applications molecular simulation (MS) has become a great benefit over the past
decade; MS is now a standard research tool for designing both drugs and materials, as well as
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for understanding their modes of action. While problems in designing and optimising HPLC
systems are often more complex than those encountered in drug and materials design—due to
the diversity of microscopic structure found at the solid/liquid interface in HPLC—the
revolution in computer hardware and simulation methods over recent years has made it
possible to model very complex systems with an accuracy and realism that was inconceivable
five years ago. This is well illustrated by computational studies of complex heterogeneous
systems, including solid/liquid interfaces,1 chiral discrimination in solute-solvent structuring,2
and adsorption at disordered surfaces.3 Thus one of the aims of this study has been to show
how MS methods can also be used to aid in the design and optimisation of HPLC for chiral
separations.
3
Key Advances and Supporting Methodology
AIMS AND OBJECTIVES
As outlined in the original report, the central aim of this project was to show that molecular
simulation methods can now make a substantial contribution towards understanding the chiral
HPLC at a molecular level. In particular, it was intended to show that realistic simulations of
HPLC are feasible, and that they can aid substantially in understanding the molecular basis
for chiral discrimination with chiral stationary phases. In particular, it was planned to use
molecular simulations to investigate a possible correlation between solution-phase circular
dichroism spectroscopy and enantiomeric separation in chiral HPLC.
On the whole, these objectives have been realised within the project. New methods
for calculating ∆∆G values (and the associated resolution factor, α) have been developed,4,5
and shown to provide a feasible route to estimating enantiomeric resolving powers from
atomistic simulations that incorporate explicit solvent effects.6 Further, the simulations have
allowed a detailed investigation of the molecular basis for chiral discrimination, and have
highlighted the importance of considering an ensemble of chiral interactions in understanding
the net resolving power from stationary phases with conformation flexibility built in.5 Finally,
it has been possible in at least one case study (resolution of chiral metallo-helicates on
cellulose), to show a mechanistic link between the solution phase CD and the chiral HPLC
separation.7,8 In the process, it has been possible to identify some of the characteristics of
when such a link is likely to exist, and therefore to begin to understand how to generalise CD
as a tool for optimising chiral HPLC.
DETAILED RESULTS
Free Energy Methods
One of the aims of this project was to show how molecular simulation could be used to
calculate partition coefficients for HPLC separations. In particular, we were interested in
direct calculation of the ∆∆G for the equilibrium
(R-analyte…stationary phase) + S-analyte ! R-analyte + (stationary phase…S-analyte)
since this is directly related to the resolving power of the column. Existing methods for
calculating free energy differences from simulations currently fall into two categories: (i)
accurate but very time consuming methods, such as thermodynamic integration, and (ii)
computationally cheap but very approximate methods. For systems such as that studied in this
project, which involve complex and flexible compounds, neither of these extremes is
particularly useful.
One of the major achievements of this project has been to develop a new free energy
method that gives a good compromise between realistic sampling of the set conformations
responsible for chiral recognition between flexible molecules, but at a manageable
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Molecular Basis of Enantiomeric Discrimination
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computational cost.4,5,6The method is based on identifying a set of dominant conformations
for both species involved in dimer formation (for HPLC these would be the stationary phase
active constituent and the analyte), locating favourable adsorption geometries for the
resulting dimer, and then estimating the contribution of each dimer conformation to the
equilibrium free energy from short molecular dynamics simulations of each dimer. To
improve efficiency, the conformational and dimer binding stages are combined with a cluster
analysis so that groups of similar conformations can be represented by a single lead
conformation. The resulting set of dimer conformations can be referred to as the ensemble of
dominant conformations (EDC). A very important advantage of this method is that it is
possible to include explicit solvent into the molecular dynamics calculations without
requiring prohibitive computational resources, so that solvent contributions to the free energy
can be included explicitly.
We note that our developments have paralleled similar methodological developments
for drug-protein interactions,9 although for the drug-protein interactions these have only
considered implicit solvent models. The method has been tested with linked dimeric
carbohydrates (LDCs) as the active constituent of the stationary phase, and D- or L-benzoin
as the chiral analyte, and has been shown to be viable and to provide considerable insight into
the mechanism of chiral recognition.4,6
Chiral Recognition
An important theme within the proposal was to develop a molecular understanding of the
process of chiral recognition within HPLC — without such a molecular understanding it is
not possible to exploit rational design methods for optimising HPLC. While there has been
many studies of rigid stationary phases, such as those derived from cyclodextrins, there has
been essentially no consideration in the literature of chiral recognition in flexible molecules.
Within this project we have exploited the concept of the EDC (described above) to
gain a molecular understanding of chiral recognition between the LDC-based stationary
phase and benzoin. Using the methods described above, an EDC was derived for each of
R-benzoin and S-benzoin interacting with the LDC. These ensembles were then
supplemented by replacing the benzoin in a stable benzoin-LDC dimer with its enantiomer
and then optimising its geometry. In this way it was possible to generate an equivalent set of
dimer geometries that contained the dominant binding modes for each enantiomer of benzoin
ELDC / kcal mol
-1
20
10
0
0
4
8
12
LDC conformation
Figure 1: chiral discrimination in the interaction of a linked dimeric carbohydrate (LDC)
and benzoin. The left plot indicates how the free energy of binding each benzoin enantiomer
varies with the LDC conformation. The right hand plot indicates the energy of the LDC
conformation, and hence determines the probability of finding that conformation. The net
discrimination arises as a balance of these two effects.
16
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Molecular Basis of Enantiomeric Discrimination
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on the LDC and from which their relative contribution to the net chiral discrimination could
be studied. The results (see figure 1) showed that the chiral discrimination arises as a subtle
ensemble average, with some LDC conformers favouring the R-benzoin and others the
S-benzoin; the net discrimination was an energy-weighted average of the conformer
discriminations. Further, it was not possible to identify a 3-point interaction that correlated
with the resolving power of the different enantiomers. We conclude that, for flexible
molecules, chiral discrimination must be understood as a net consequence of many subtle and
counteracting effects.
Solvent Influence
The simulations used for the chiral recognition and free energy calculations have also
allowed a detailed investigation of the solvent influence on the chiral recognition. All our
simulations used tetrahydrofuran (THF) as a model solvent, since this was used in the
original experimental HPLC studies using and LDC stationary phase.10 The results have
shown very clearly that THF can have a pronounced influence on the chiral recognition, with
the strength of the discrimination identified in Figure 1 being strongly influenced by the
nature of the interactions between the LDC-benzoin dimer and individual THF molecules. In
particular, the balance between the efficiency of packing the THF rings around the LDCbenzoin dimer and hydrogen bonding to the etheric oxygen in THF led to solvent effects that
could sometimes dominate the net chiral recognition. Such effects have been found before,2,11
but their importance is not widely recognised. In particular, some of the LDC-benzoin dimers
with the highest energy in vacuum ended up having the lowest energy in THF, and therefore
ended up dominating the chiral discrimination in the presence of solvent. It is likely that THF
is an unusual solvent in this regard, in that it gives rise to both hydrogen bonding and strong
packing effects, but the results do illustrate that the choice of solvent could be exploited more
strongly to optimise chiral HPLC.
H2N
NH2
O
O
N
N
EtOH
room temp
2h
N
N
Octahedral M
N
N
Figure 2: metallo-helicates synthesised at Warwick. These
adopt a helical structure that binds preferentially to the major
groove in DNA
Correlation between circular dichroism and chiral HPLC
Due to personnel changes (specifically, the departure of K. Worsfold from AJC’s lab just
before the start of the grant, and the resignation of S. Canon during the project), the emphasis
in the experimental programme was changed from studying LDC stationary phases to using
other materials. In particular, cellulose was found to be a promising stationary phase for
separating some members of a novel class of metallo-supramolecular helicates that have
recently been synthesised at Warwick (see Figure 2).12 These helicates were shown to have a
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Molecular Basis of Enantiomeric Discrimination
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cylindrical architecture of similar dimensions to the protein binding units that target the major
groove of DNA, and induced remarkable structural effects — wrapping up double-stranded
DNA in an intra-molecular fashion. Such structural effects are unprecedented with synthetic
DNA binders, and so marked out these metallo-helicates as important analytes on which to
develop our molecular understanding of chiral HPLC.
Parent compound in 1 M glucose
Me 4 Me derivative in 1 M glucose
I
n
d
u
c
e
d
C
D
/
m
d
e
g
1
0.5
0
-0.5
-1
300
400
500
Wavelength / nm
600
700
Figure 3: Resolving with sugars. Left: separation of racemic parent compound
(Figure 2) into two enantiomers on. Right: induced CD signal for two metallo
helicates in 1 M glucose solution. The compound resolved by cellulose (a glucose
polymer) has a spectrum that follows the actual CD spectrum of the resolved
compounds. The one not resolved by the cellulose has only an unstructured
dispersion induced CD band at long wavelengths.
While it was clear that the helicate was chiral, all existing literature methods for
resolving the enantiomer failed. Inspired by the sugar-based stationary phases described in
our original proposal, but having lost the personnel to synthesise the required amount of the
LDC, we tried cellulose as a stationary phase with an aqueous salt solution as the mobile
phase and then achieved the resolution shown in Figure 3 with paper. Packing columns with
cellulose had an even better effect with the parent compound. A preliminary communication
of this work was published recently7 and a full paper is in preparation8 Subsequent work has
enabled the use of highly organic mobile phases, which facilitates later purification of the
compounds. We have also been attempting to resolve a range of derivatives of the parent
compound, but success has been mixed. Molecular modelling studies were carried out on the
interaction between the helicates and glucose. This revealed up to three sites on the helicate
that generated chiral recognition by the glucose, the strongest of which was in the region of
the imine bond. Substituting this bond with a methyl group rendered the surface of the
helicate in this region effectively achiral so that the discrimination was switched off. X-ray
crystallography and circular dichroism analyses have been used to confirm that the modelling
predictions of the identity of the first eluted enantiomer are in fact correct. Of particular
interest is the fact that the circular dichroism spectrum for the helicate in glucose solution
shows a significant interaction with the glucose if and only if the helicate is subsequently
resolved on paper or within a packed cellulose HPLC column (Figure 3). It was shown that in
this case, the modelling and experimental studies all indicate that the interactions that lead to
chiral discrimination between glucose and the metallo-helicates is determined by local
glucose-helicate interactions, and as such the solution phase circular dichroism experiments
will provide a good indication of how to optimise the chiral separation.
Work using the first batch of resolved helicates has also been published.13
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Structure of the Stationary Phase
Due to the personnel changes identified above, and also due to the need to develop a better
methodology for calculating free energies of binding, the work on characterising the
molecular structure of the LDC stationary phase has not proceeded as far as envisaged within
the original proposal. Never-the-less, it has been possible to generate a 3-dimensional
molecular map of the LDC/graphite stationary phase. The graphite does induce some changes
in the relative energy of the LDC conformations, and so can be expected to lead to some
variation in activity relative to the solution phase behaviour, but in general this was found to
be a modest effect. More generally, one may expect that where the analyte / active constituent
interaction is localised, then there will be a strong correlation between solution phase and
HPLC discriminations. This work is currently being written up for publication.14
4
Dissemination Activities and Further Research
The results of this work have been already been disseminated through presentation at a
number of international conferences. In addition, four publications have already appeared or
are in press. A further three are now being written. The Journals for these publications have
been carefully selected to maximise dissemination in the relevant communities. In particular,
the Journal of Molecular Graphics and Modelling (JMGM) is the modelling journal with the
highest impact factor, and is the natural forum for pharmaceutical modelling. Since chiral
HPLC is an important tool in the pharmaceutical industry, JMGM has been targeted as a
prime journal for this study. The work has also been publicised through the investigators
personal contacts with a number of chemical companies, including Thermohypersil, Glaxo
Smith Kleine and Astra Zeneca.
Research is still in progress to better characterise the link between the solution phase
circular dichroism spectrum and the resolving power of a related HPLC column. This work is
still at a pre-commercial stage but is rapidly developing to the point where industrial funding
to continue the work might be expected.
References
1
2
3
4
5
6
7
8
9
10
11
12
13
14
P.M. Rodger, T.R. Forester, W. Smith, Fl. Phase. Eq., 116, 326–332 (1996)
J. Fidler, P.M. Rodger, A. Rodger, J. Am. Chem. Soc., 116, 7266–7273 (1994)
T.J. Carver, M.G.B. Drew and P.M. Rodger, J. Chem. Soc., Faraday Trans., 92, 5029–5034 (1996)
R. Lukac, A.J. Clark, M.A. San Miguel, A. Rodger, P.M. Rodger, J. Mol. Liq., in press
R. Lukac, A.J. Clark, S. Khalid, A. Rodger, A. Snedden, P.M. Rodger, J. Mol. Liq. 2002, 98-99, 411
R. Lukac, A.J. Clark, M.A. San Miguel, A. Rodger, P.M. Rodger, J. Am. Chem. Soc., in preparation
M.J. Hannon, I. Meistermann, C.J. Isaac, C. Blomme, J.R. Aldrich-Wilson, A. Rodger, Chem. Comm. 2001,
1078
A. Rodger, M.J. Hannon, C. Blomme, I. Meistermann, J. Peberdy, V. Rüdeiker, S. Khalid, P.M. Rodger, J.
Am. Chem. Soc, in preparation
K.L. Mardis, R. Luo, M.K. Gilson, J. Molec. Biol., 2001, 309, 507-517; K. Mardis, R. Luo, L. David, M.
Potter, A. Glemza, G. Payne, M.K. Gilson, J. Bio. Struct. Dyn., 2000, 89-94; R. Luo, M.K. Gilson, J. Am.
Chem. Soc., 2000, 122, 2934-2937
K. Worsfold, PhD. thesis, University of Warwick, 2000
C. Coulombeau, A. Rassat., Bull. Soc., Chim. France, 1966, 12, 3752
M.J. Hannon, V. Moreno, M.J. Prieto, E. Molderheim, E. Sletten, I. Meistermann, C.J. Isaac, K.J. Sanders
and A. Rodger, Angew. Chem., Intl. Ed. Engl., 2001, 40, 879
I. Meistermann, A. Rodger, V. Moreno, M.J. Prieto, E., Molderheim, E. Sletten, S. Khalid, P.M. Rodger, J.
Peberdy, C.J. Isaac, M.J. Hannon, Proc. Nat. Acad. Sci., 2002, 99, 5069
R. Lukac, A.J. Clark, M.A. San Miguel, A. Rodger, P.M. Rodger, J. Mol. Graph. Mod., in preparation