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Determination of absolute configuration via vibrational circular dichroism
Vol. 1, No. 3 2004
Drug Discovery Today: Technologies Editors-in-Chief Kelvin Lam – Pfizer, Inc., USA Henk Timmerman – Vrije Universiteit, The Netherlands DRUG DISCOVERY
TODAY
TECHNOLOGIES
Lead optimization
Determination of absolute configuration via vibrational circular dichroism Tom Kuppens1, Patrick Bultinck1,*, Wilfried Langenaeker2 1 2
Department of Inorganic and Physical Chemistry, Ghent University, Krijgslaan 281 (S3), B-9000 Gent, Belgium Janssen Pharmaceutica N.V., PAS Specification Management, Lammerdries 55, 2250 Olen, Belgium
Vibrational circular dichroism (VCD) provides a growing and promising technology for the determination of the absolute configuration of molecules in solution, including drug molecules. The practical application of VCD spectroscopy consists of the experimental determination and comparison to quantum chemically calculated data. The key features of the VCD technology are presented and an example of an application of the technique is discussed. Introduction Knowledge of the absolute configuration of drug-like molecules is of prime importance because it is well known that different stereoisomers can have very different biological effects. A well-known case where two enantiomers have very different effects is the thalidomide molecule. This drug caused a large number of birth defects when administered to pregnant women as antiemetic. The cause for this tragedy was found in the fact that only one enantiomer had the desired effect whereas the other was teratogenic. As chirality is a feature of many pharmaceutical molecules, techniques to identify the stereochemistry of a molecule are of prime importance in drug discovery. In the present review, the vibrational circular dichroism (VCD) technique is described as a powerful method. An overview is presented *Corresponding author: (P. Bultinck) [email protected] URL: http://www.quantum.ugent.be 1740-6749/$ ß 2004 Elsevier Ltd. All rights reserved.
DOI: 10.1016/j.ddtec.2004.11.004
Section Editors: Paul Lewi, Frits Daeyaert – Center for Molecular Design, Janssen Pharmaceutica N.V., Vosselaar, Belgium Vibrational circular dichroism (VCD) spectroscopy of chiral molecules enables the determination of the absolute stereochemical configuration of chiral molecules by comparing experimental and ab initio calculated spectra. Issues are the assignment of the vibrational spectra and the level and type (DFT/HF) of theoretical treatment used. Kuppens et al. describe the practical and theoretical considerations of the method. The Ghent Quantum Chemistry Group of Professor Bultinck conducts research on the development of a computational methodology for the calculation of VCD spectra, ab initio studies on chelates and macrocyclic complexes, and conformational analysis of polypeptides. Dr Langenaeker has been using VCD for the determination of the absolute configuration of drug candidates at Janssen Pharmaceutica N.V.
of both the experimental VCD measurement as well as the application of quantum chemistry in structure and stereochemistry elucidation, together with an example application in a pharmaceutical context.
Experimental VCD measurement Circular dichroism (CD) is a general term referring to the differential absorption of right and left circularly polarized light. The differential absorbance DA at every frequency is given by:
DA ¼ AL AR
(1)
where AL and AR represent the absorbance for left and right circularly polarized light, respectively. VCD is the measurement of this differential absorbance in the infrared region. www.drugdiscoverytoday.com
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Glossary Born–Oppenheimer approximation: because the mass of the atomic nuclei is far greater than the mass of the electrons orbiting it, the positions of the nuclei are considered to be constant under this approximation. The motion of the electrons can therefore be considered decoupled from the motion of the nuclei, which leads to the elimination of several terms from the Schro¨ dinger equation. Fourier transformed infrared (FT-IR): instead of recording the amount of energy absorbed when the frequency of the infrared (IR) light is varied (conventional or dispersive IR spectroscopy), the IR light is guided through an interferometer. After passing the sample the measured signal is an interferogram. Performing a mathematical Fourier transform on this signal results in a spectrum identical to that from dispersive infrared spectroscopy. Using FT-IR, one has a better signal-to-noise ratio compared to a dispersive instrument. The acquisition time is also much lower. Lock-in amplifier: lock-in amplifiers are used to measure the amplitude and phase of signals buried in noise. They achieve this by acting as a narrow bandpass filter, which removes much of the unwanted noise while allowing through the signal, which is to be measured. The frequency of the signal to be measured and hence the passband region of the filter is set by a reference signal, which has to be supplied to the lock-in amplifier along with the unknown signal. The reference signal must be at the same frequency as the modulation of the signal to be measured. Photoelastic modulator (PEM): instrument used for modulating or varying, at a fixed frequency, the polarization of a beam of light. The PEM contains a photoelastic material that changes its optical properties under pressure. By modulating the pressure on the material with a piezoelectric control, linearly polarized light is converted to circularly polarized.
The key reason for the use of VCD for the determination of absolute configuration lies in the fact that different enantiomers (+ and ) exhibit opposite differential absorbance:
DAþ ¼ DA
(2)
Measuring VCD with a specially equipped FOURIER TRANSFORMED INFRARED (FT-IR) (see Glossary) spectrometer over large spectral regions is becoming more common practice. CD in vibrational transitions, however, is a weak phenomenon,
with magnitudes of typically around 105 absorbance units. The first solution-phase VCD measurements date back to the mid 1970s [1,2] using a dispersive IR spectrometer. At the time, the experiments yielded weak and noisy spectra. Subsequent developments in the photoelastic modulators (PEM) and the development of FT-IR VCD have greatly improved the applicability and quality of VCD, and is making VCD spectroscopy a more routine technique. Today, virtually all VCD spectrometers are equipped with an FT-IR spectrometer. Because of the small magnitude of VCD it requires sophisticated modulation techniques for its observation. The central component in this modulation is the PEM. A periodic stress applied to an isotropic crystal causes the modulation of the polarization of the IR beam between left and right circular polarized light (CPL). A diagram of a FTVCD spectrometer is given in Fig. 1. The randomly polarized modulated beam that exits the FTIR spectrometer first passes through an optical filter, to assure a better signal to noise ratio. Then the beam is polarized by a linear polarizer. The linear polarized light is then alternatively switched at the PEM frequency between left and right CPL. The beam is then transmitted through the sample and is focused on an IR detector. The detected signal is divided into two channels. The first channel, which contains the normal single channel beam spectrum, is low pass filtered. The second channel contains the VCD signal and is high pass filtered and demodulated at the PEM frequency using a lockin amplifier. Commercial setups of FT-IR VCD spectrometers are provided by Bruker Optics [3,4] (http://www.brukeroptics.com), Bomem-BioTools [5,6] (http://www.btools.com), Nicolet/ Thermo (http://www.thermo.com), Jasco [7] (http:// www.jascoinc.com) and Bio-Rad [8,9] (http://www.bio-rad.com).
Figure 1. Diagram of a Fourier transformed vibrational circular dichroism spectrometer setup. The randomly polarized modulated beam that exits the FT-IR spectrometer passes through an optical filter. Then the beam is polarized by a linear polarizer. The linear polarized light is then alternatively switched at the PEM frequency between left and right CPL. The beam is then transmitted through the sample and is focused on an IR detector. The detected signal is divided into two channels. The first channel, which contains the normal single channel beam spectrum, is low pass filtered. The second channel contains the VCD signal and is high pass filtered and demodulated at the PEM frequency using a lock-in amplifier.
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Theory Optical activity manifests itself as a different absorption of left and right CPL, i.e. DA = AL AR or under Beer’s law condition De = eL eR. In a non-absorbing solvent, and for a dilute solution of a chiral molecule [10], the molar absorptivity and differential molar absorptivity can be written as
P eðyÞ ¼ 1:09 1038 y P Dj fj ðyj Þ 38 DeðyÞ ¼ 4:35 10 y Rj fj ðyj Þ
(3)
Dj and Rj in Eq. (3) are the dipole and rotational strengths of the excitation of state g to j at frequency yj, respectively. Dj and Rj are in esu2 cm2. fj is the normalized Lorentzian bandshape. Dj and Rj are given by
Dj ¼ jhgjmel jjij2 Rj ¼ Im ½hgjmel jji hjjmmag jgi
(4)
mel and mmag in Eq. (4) are the electric dipole and magnetic dipole moment operators, respectively [11]. In the harmonic approximation and working with normal coordinates Qi [12], the dipole strength for a fundamental excitation of mode a, is proportional to
@mel 2 @Q
Da01
a
(5)
0
The rotational strength for a fundamental transition [12] of mode a is given by
@mel @mmag @P @Q
Ra01
a
0
a
0
(6)
Pa in Eq. (6) is the conjugated moment for normal mode a. The second term of Eq. (6) requires a non-Born–Oppenheimer approximation, because of the correlation between the nuclear and electronic velocities. Algorithms for the calculation of the required dipole and rotor strengths and thus VCD spectra have been established [13], and VCD data can now be calculated in several popular quantum chemical software packages.
Computational methodology For VCD to be applicable in the determination of the stereochemistry of chiral compounds in solution, the acquired VCD spectra should be interpretable. The interpretation of VCD spectra, however, is not straightforward and requires an algorithm that relates both the structure and spectra. These spectra can be simulated by quantum chemical methods; comparison between the predicted and experimental spectrum makes the identification of the stereoisomer present in solution possible. The calculation of VCD spectra via quantum chemical methods requires several steps. First, a stereoisomer is chosen for which all calculations will be performed. The VCD data for the enantiomer of this stereoisomer are then simply the mirror
image. In the second step, a conformational analysis is conducted for the chosen stereoisomer with the purpose of locating the minimum energy conformations of the molecule. Once the geometry of these conformations is optimized on the required theoretical level, the VCD properties can be calculated. A theoretical VCD spectrum is then obtained by combining all conformations, and in the ultimate step the theoretical VCD spectrum is compared to the experimental one, thereby allowing the assignment of the absolute configuration of the molecule. The aim of the conformational analysis is to find all possible conformations of the molecule in the chosen absolute configuration. Knowledge of the different minimum energy conformations is crucial in structure determination by VCD. Experimental IR and VCD spectra are averaged spectra over all conformations present in solution. In case of a rigid molecule, a single conformer might be sufficient to explain experimental data. If the molecule is flexible, however, equilibrium between the different conformations has to be taken in to account. Different conformational search methods can be used on various levels of theory. An extensive discussion on conformational searches for medicinal molecules can be found in Sadowski et al. [14]. In the next step, the geometry of the minima is optimized at the desired quantum chemical level, followed by the calculation of the vibrational frequencies, IR and VCD intensities. Nowadays, most VCD calculations are carried out using the Density Functional Theory (DFT). DFT methods require a so-called density functional. For VCD calculations, most often hybrid functionals like B3LYP and B3PW91 [15,16] are used because these have been found to give better agreement with experiment compared to Hartree–Fock calculations [17]. Another choice that has to be made is the basis set to be used for the development of the molecular orbitals. Generally speaking, bigger basis sets give better descriptions of the molecule. The drawback, however, is that an increase of the basis set results in a serious increase of the required computer time. An economical compromise between accuracy and computational efficiency is the medium-size 6-31G* basis set [4]. Going to larger basis sets, the cc-pVTZ basis set gives excellent agreement with experiment [4]. Calculations can be carried out with commercially available software such as Gaussian03 (Revision B05, Gaussian, Inc., Pittsburgh, PA, http://www.gaussian.com). Herein Hessians matrices, atomic polar tensors (APT) and atomic axial tensors (AAT) can be calculated using gauge-including/invariant atomic orbitals (GIAOs) allowing the calculation of the VCD properties [18–20]. For each minimum energy conformation the dipole and rotational strengths are calculated. The results are single conformer line spectra, which can be broadened using a Lorentzian band-shape. Because each minimum contributes to the total spectrum in a Boltzmann weighted manner, the www.drugdiscoverytoday.com
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isomer used in the quantum chemical study. Naturally, if the molecule contains several chiral centers, it is necessary to consider many different stereoisomers.
IR and VCD spectra can be simulated as
Dtot ðyÞ ¼ Rtot ðyÞ ¼
X A X
FA DA ðyÞ FA RA ðyÞ
(7)
A
FA in Eq. (7) is weight factor expressing the contribution of each minimum energy conformation to the total spectrum. The weight factor for conformation A is usually taken as the Boltzmann population of this conformation. The DA(y) and RA(y) are the single conformational spectra. Once the total spectra are calculated, one can proceed to the comparison with the experimentally acquired spectra. If a good agreement between experimental and theoretical VCD spectra is obtained, one can conclude that the stereoisomer chosen for the quantum chemical study is also the one in the experimental sample. If a mirror image relationship exists with the experimental spectrum, one can conclude that the experimental sample contains the enantiomer of the stereo-
Application example The application of VCD spectroscopy, especially in a drug discovery environment, has profited greatly from the availability of commercial VCD apparatuses. As described above, the use of VCD for the determination of the absolute configuration of a (drug) molecule, requires quantum chemical calculations. The availability of a stable and sufficiently fast algorithm for the quantum chemical calculation of VCD spectra in widely available software packages, together with the increasing speed of computers and performant computer clusters has further boosted the capacities of VCD. Concurrent with these evolutions, VCD is no longer limited in applicability to relatively small and rigid molecules, but will become a method of increasing popularity for molecules of increasing size and flexibility.
Figure 2. Methyl mandelate and its IR and VCD spectra. (a) Schematic representation of methyl mandelate. (b) Experimental IR spectrum of methyl mandelate (gray). Simulated IR spectrum on B3LYP/6-31G* (green) and cc-pVTZ (blue) level. Experimental intensities are in absorbance units, theoretical intensities in 1040 esu2 cm2 and frequencies in cm1. Normal modes are numbered. (c) Experimental VCD spectrum of R() (red) and S(+) (green) methyl mandelate. Simulated VCD spectrum S(+) methyl mandelate on B3LYP/6-31G* (blue) and cc-pVTZ (brown) level. Experimental intensities are in 105 absorbance units, theoretical intensities in 1044 esu2 cm2 and frequencies in cm1. Normal modes are numbered.
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To illustrate the use of the VCD technology, methyl mandelate (Fig. 2a) is presented as an example, illustrating the different steps involved. Methyl mandelate is the methyl ester of mandalic acid, and the (R)- and (S)-isomers are chiral derivatizing agents for nuclear magnetic resonance (NMR) determination of enantiomeric purity of a-deuterated carboxylic acids, alcohols and amines [21]. VCD spectra for both R()- and S(+)-enantiomers, were measured on a Bruker IFS 66/S FT-IR spectrometer coupled to a PMA37 module. The VCD and IR spectra were recorded at a resolution of 4 cm1. For methyl mandelate the IR spectrum was measured using a Bruker Vector 22 FT-IR spectrometer. In all applications of VCD spectroscopy, the possibility to obtain spectra for both enantiomers greatly increases the quality of the spectra owing to the possibility to reduce several artifacts like baseline shifting [22]. A key feature of the VCD technology is the possibility to record spectra directly in solution phase, thereby opening the way for determination of absolute configurations for molecules without needing to obtain crystals. All calculations on methyl mandelate were done for the molecule in the S configuration. The conformational search strategy was followed, involving a combination of stochastic searching and systematic searching. This resulted in six unique minima. In the next step, the geometry of all minimum energy conformations was optimized using DFT calculations with the B3LYP functional and the 6-31G* and ccpVTZ basis sets, followed by the calculation of the absorption frequencies and dipole and rotor strengths. The line spectra are then broadened via a Lorentzian shape and theoretical spectra are obtained through Boltzmann averaging over all conformations. In Fig. 2b, the simulated and experimental IR spectra are given. The simulated IR spectra (with a Lorentzian broadening of 4 cm1) for both basis sets agree very well with the
experimental spectrum, with band positions and relative intensities in good agreement with experiment. The VCD spectra are depicted in Fig. 2c. One can see that the experimental VCD spectra have mirror image symmetry. Deviations on this symmetry are owing to small concentration differences and baseline effects. Based only on the agreement between theoretical and experimental spectrum, it can be seen that the S(+) spectrum agrees very well with the calculated S spectrum. However, one has to be careful with these practices, because of the nature of the simulated spectra. The harmonic approximation, resulting in higher calculated frequencies (which can partially be corrected with a scaling factor, depending on level of theory) and the fact that computational spectra do not incorporate solvent or intermolecular effects, one has to be absolutely sure that peaks corresponding to the same normal mode are compared. To do that, the peaks are identified based on the agreement between the simulated and experimental IR spectra, supplemented with VCD data. This way of working provides a more objective and robust assignment method, but is labor-intensive. As can be seen in Figs 2b and 2c, the normal modes have been assigned and, based on the agreement between the different modes, the assignment can be made.
Conclusions Compared to other available techniques, VCD offers a new and powerful approach to the determination of chiral molecules in solution phase. X-ray diffraction (XRD) [23,24], enzymatic resolution [25], NMR [26,27], optical rotation [28], electronic circular dichroism (ECD) [29,30] and stereoselective synthesis are techniques that were proven to be utilizable, but not always practicable (Table 1). VCD, by contrast, can be utilized on a wide range of molecules in
Table 1. Comparison summary table Technology 1 Name of specific type Vibrational circular of technology dichroism (VCD)
Technology 2
Technology 3
Technology 4
Technology 5
X-ray diffraction (XRD)
Nuclear magnetic resonance (NMR)
Optical rotation (OR)
Electronic circular dichroism (ECD)
Relatively easy to measure
Applicable to molecules in solution
Pros
Very reliable Well known Applicable in solution on a wide range of Food and Drug Administration molecules (FDA) standard
Cons
Measurements are not straightforward
Only a few bands Higher level Need for good Need for chiral shift are available reagents, chiral additives calculations needed quality single compared to VCD or derivatization crystals Higher level Only one property calculations needed can be compared in compared to VCD contrast to VCD (comparison of all bands in the spectrum) Sign depends on solvent used
References
See Related articles
[23,24]
[26,27]
[28,31]
[29,30] www.drugdiscoverytoday.com
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Related articles Stephens, P.J. (2004) Vibrational circular dichroism spectroscopy: a new tool for the stereochemical characterization of chiral molecules. In Computational medicinal chemistry for drugs discovery (Bultinck, P. et al., eds), pp. 699–725, Marcel Dekker, Inc Polavarapu, P.L. (1994) New spectroscopic tool – absolute-configuration determination of pharmaceutical compounds by vibrational circulardichroism. Spectroscopy 9, 48–55 Nafie, L.A. (1996) Vibrational optical activity. Appl. Spectrosc. 50, A14– A26 Keiderling, T.A. (1990) Vibrational circular dichroism. Comparison of techniques and practical considerations. In Practical Fourier Transform Infrared Spectroscopy. Industrial and Laboratory Chemical Analysis (Ferraro, J.R. et al., eds), pp. 203–284, Academic Press Freedman, T.B. et al. (2003) Determination of the absolute configuration and solution conformation of gossypol by vibrational circular dichroism. Chirality 15, 196–200
solution. The increase in speed and power of computers, the recent advances in computation algorithms and the performance of DFT functionals will only extend the range of molecules that can be operated by VCD. Furthermore, dedicated VCD instruments are now widely available, making VCD spectroscopy a more routine technique. VCD can also be used in other structure and conformationrelated applications on molecules of pharmaceutical interest, i.e. determination of the enantiomeric purity of a sample relative to a known standard. Also determination of the solution conformations of small or larger biological molecules can be probed by VCD. Despite the progress during the last decades, additional work is needed in several areas. In the experimental field, the problem of baseline effects in VCD spectroscopy and relatively long sampling times have to be tackled. Comparison between experimental and theoretical spectra is time consuming and labor-intensive. Algorithms that can handle this comparison and ultimately assign the AC are developed (unpublished material). Also, there is need for improved modeling of the effects of solvent and intermolecular interaction on the observed spectra.
Acknowledgement The authors want to thank Wouter Herrebout (Antwerp University) for scientific and technical assistance.
Outstanding issues Incorporation of solvents in the calculation of the IR and VCD spectra should give better agreement with experiment. Ab initio calculation of anharmonic vibrational circular dichroism intensities as a replacement for the harmonic approximation. Low temperature measurements should give less populated higher energy conformations. This should reduce the computational load, because fewer conformations have to be taken into account to simulate spectra.
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References 1 Holzwart, G. et al. (1974) Infrared circular-dichroism of carbon-hydrogen and carbon–deuterium stretching modes – observations. J. Am. Chem. Soc. 96, 251–252 2 Nafie, L.A. et al. (1976) Vibrational circular-dichroism. J. Am. Chem. Soc. 98, 2715–2723 3 Urbanova, M. et al. (2000) Measurements of concentration dependence and enantiomeric purity of terpene solutions as a test of a new commercial VCD spectrometer. Chirality 12, 199–203 4 Kuppens, T. et al. (2003) Determination of the stereochemistry of 3hydroxymethyl-2,3-dihydro-[1,4]dioxino[2,3-b]pyridine by vibrational circular dichroism and the effect of DFT integration grids. J. Phys. Chem. A 107, 542–553 5 Bose, P.K. et al. (1999) Acetate groups as probes of the stereochemistry of carbohydrates: a vibrational circular dichroism study. Carbohydr. Res. 322, 135–141 6 Stephens, P.J. (2004) Vibrational circular dichroism spectroscopy: a new tool for the stereochemical characterization of chiral molecules. In Computational Medicinal Chemistry for Drugs Discovery (Bultinck, P. ed.), pp. 699–725, Marcel Dekker, Inc 7 Miyazawa, M. et al. (1996) Vibrational circular dichroism of prolinecontaining oligopeptides. Appl. Spectrosc. 50, 644–648 8 Hilario, J. et al. (2001) Polarization modulation Fourier transform infrared spectroscopy with digital signal processing: comparison of vibrational circular dichroism methods. Appl. Spectrosc. 55, 1435–1447 9 Wang, B.L. et al. (1995) Observations on the measurement of vibrational circular-dichroism with rapid-scan and step-scan FT-IR techniques. Appl. Spectrosc. 49, 1347–1355 10 Schellman, J.A. (1975) Circular-dichroism and optical-rotation. Chem. Rev. 75, 323–331 11 Rauk, A. (1991) Vibrational circular dichroism Intensities: ab initio calculations. In New Developments in Molecular Chirality (Mezey, P.G. ed.), pp. 57–92, Kluwer Academic Publishers 12 Freedman, T.B. et al. (2000) Vibrational transition current density in (2S,3S)-oxirane-d(2): visualizing electronic and nuclear contributions to IR absorption and vibrational circular dichroism intensities. J. Mol. Struct. 550, 123–134 13 Stephens, P.J. et al. (1994) Ab initio calculation of vibrational absorption and circular-dichroism spectra using density-functional force-fields. J. Phys. Chem. 98, 11623–11627 14 Sadowski, J. et al. (2004) 3D structure generation and conformational searching. In Computational Medicinal Chemistry for Drugs Discovery (Bultinck, P. ed.), pp. 151–212, Marcel Dekker, Inc 15 Becke, A.D. (1993) Density-functional thermochemistry. 3. The role of exact exchange. J. Chem. Phys. 98, 5648–5652 16 Lee, C.T. et al. (1988) Development of the colle-salvetti correlation-energy formula into a functional of the electron-density. Phys. Rev. B – Condens. Matter 37, 785–789 17 Stephens, P.J. et al. (2000) Determination of the structure of chiral molecules using ab initio vibrational circular dichroism spectroscopy. Chirality 12, 172–179 18 Cheeseman, J.R. et al. (1996) Ab initio calculation of atomic axial tensors and vibrational rotational strengths using density functional theory. Chem. Phys. Lett. 252, 211–220 19 Stephens, P.J. (1985) Theory of vibrational circular-dichroism. J. Phys. Chem. 89, 748–752 20 Stephens, P.J. (1987) Gauge dependence of vibrational magnetic dipole transition moments and rotational strengths. J. Phys. Chem. 91, 1712– 1715 21 Parker, D. (1983) H-1 and H-2 nuclear magnetic-resonance determination of the enantiomeric purity and absolute-configuration of alpha-deuteriated primary carboxylic acids, alcohols, and amines. J. Chem. Soc. – Perkin Trans. 2, 83–88 22 Freedman, T.B. et al. (2003) Determination of the absolute configuration and solution conformation of gossypol by vibrational circular dichroism. Chirality 15, 196–200 23 Flack, H.D. et al. (1999) Absolute structure and absolute configuration. Acta Crystallogr. Sect. A 55, 908–915
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24 Flack, H.D. et al. (2000) Reporting and evaluating absolute-structure and absolute-configuration determinations. J. Appl. Crystallogr. 33, 1143–1148 25 McIninch, J.K. et al. (1998) Determination of the absolute configuration of alpha-hydroxyglycine derivatives by enzymatic conversion and chiral high-performance liquid chromatography. J. Chromatogr. A 828, 191–198 26 Hoye, T.R. et al. (2000) An NMR method for determination of configuration of beta-substituted carboxylic acids. Tetrahedron Lett. 41, 2289–2293 27 Hoye, T.R. et al. (1998) An NMR strategy for determination of configuration of remote stereogenic centers: 3-methylcarboxylic acids. J. Am. Chem. Soc. 120, 4638–4643
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28 Polavarapu, P.L. et al. (2003) Intrinsic rotation and molecular structure. Chirality 15, S143–S149 29 Uray, G. et al. (2001) Absolute configuration in 4-alkyl- and 4-aryl-3,4dihydro-2(1H)-pyrimidones: a combined theoretical and experimental investigation. J. Org. Chem. 66, 6685–6694 30 Pescitelli, G. et al. (2003) Theoretical analysis of the porphyrin–porphyrin exciton interaction in circular dichroism spectra of dimeric tetraarylporphyrins. J. Am. Chem. Soc. 125, 7613–7628 31 Stephens, P.J. et al. (2001) Calculation of optical rotation using density functional theory. J. Phys. Chem. A 105, 5356–5371
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Drug Discovery Today: Technologies Editors-in-Chief Kelvin Lam – Pfizer, Inc., USA Henk Timmerman – Vrije Universiteit, The Netherlands DRUG DISCOVERY
TODAY
TECHNOLOGIES
Lead optimization
Determination of absolute configuration via vibrational circular dichroism Tom Kuppens1, Patrick Bultinck1,*, Wilfried Langenaeker2 1 2
Department of Inorganic and Physical Chemistry, Ghent University, Krijgslaan 281 (S3), B-9000 Gent, Belgium Janssen Pharmaceutica N.V., PAS Specification Management, Lammerdries 55, 2250 Olen, Belgium
Vibrational circular dichroism (VCD) provides a growing and promising technology for the determination of the absolute configuration of molecules in solution, including drug molecules. The practical application of VCD spectroscopy consists of the experimental determination and comparison to quantum chemically calculated data. The key features of the VCD technology are presented and an example of an application of the technique is discussed. Introduction Knowledge of the absolute configuration of drug-like molecules is of prime importance because it is well known that different stereoisomers can have very different biological effects. A well-known case where two enantiomers have very different effects is the thalidomide molecule. This drug caused a large number of birth defects when administered to pregnant women as antiemetic. The cause for this tragedy was found in the fact that only one enantiomer had the desired effect whereas the other was teratogenic. As chirality is a feature of many pharmaceutical molecules, techniques to identify the stereochemistry of a molecule are of prime importance in drug discovery. In the present review, the vibrational circular dichroism (VCD) technique is described as a powerful method. An overview is presented *Corresponding author: (P. Bultinck) [email protected] URL: http://www.quantum.ugent.be 1740-6749/$ ß 2004 Elsevier Ltd. All rights reserved.
DOI: 10.1016/j.ddtec.2004.11.004
Section Editors: Paul Lewi, Frits Daeyaert – Center for Molecular Design, Janssen Pharmaceutica N.V., Vosselaar, Belgium Vibrational circular dichroism (VCD) spectroscopy of chiral molecules enables the determination of the absolute stereochemical configuration of chiral molecules by comparing experimental and ab initio calculated spectra. Issues are the assignment of the vibrational spectra and the level and type (DFT/HF) of theoretical treatment used. Kuppens et al. describe the practical and theoretical considerations of the method. The Ghent Quantum Chemistry Group of Professor Bultinck conducts research on the development of a computational methodology for the calculation of VCD spectra, ab initio studies on chelates and macrocyclic complexes, and conformational analysis of polypeptides. Dr Langenaeker has been using VCD for the determination of the absolute configuration of drug candidates at Janssen Pharmaceutica N.V.
of both the experimental VCD measurement as well as the application of quantum chemistry in structure and stereochemistry elucidation, together with an example application in a pharmaceutical context.
Experimental VCD measurement Circular dichroism (CD) is a general term referring to the differential absorption of right and left circularly polarized light. The differential absorbance DA at every frequency is given by:
DA ¼ AL AR
(1)
where AL and AR represent the absorbance for left and right circularly polarized light, respectively. VCD is the measurement of this differential absorbance in the infrared region. www.drugdiscoverytoday.com
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Glossary Born–Oppenheimer approximation: because the mass of the atomic nuclei is far greater than the mass of the electrons orbiting it, the positions of the nuclei are considered to be constant under this approximation. The motion of the electrons can therefore be considered decoupled from the motion of the nuclei, which leads to the elimination of several terms from the Schro¨ dinger equation. Fourier transformed infrared (FT-IR): instead of recording the amount of energy absorbed when the frequency of the infrared (IR) light is varied (conventional or dispersive IR spectroscopy), the IR light is guided through an interferometer. After passing the sample the measured signal is an interferogram. Performing a mathematical Fourier transform on this signal results in a spectrum identical to that from dispersive infrared spectroscopy. Using FT-IR, one has a better signal-to-noise ratio compared to a dispersive instrument. The acquisition time is also much lower. Lock-in amplifier: lock-in amplifiers are used to measure the amplitude and phase of signals buried in noise. They achieve this by acting as a narrow bandpass filter, which removes much of the unwanted noise while allowing through the signal, which is to be measured. The frequency of the signal to be measured and hence the passband region of the filter is set by a reference signal, which has to be supplied to the lock-in amplifier along with the unknown signal. The reference signal must be at the same frequency as the modulation of the signal to be measured. Photoelastic modulator (PEM): instrument used for modulating or varying, at a fixed frequency, the polarization of a beam of light. The PEM contains a photoelastic material that changes its optical properties under pressure. By modulating the pressure on the material with a piezoelectric control, linearly polarized light is converted to circularly polarized.
The key reason for the use of VCD for the determination of absolute configuration lies in the fact that different enantiomers (+ and ) exhibit opposite differential absorbance:
DAþ ¼ DA
(2)
Measuring VCD with a specially equipped FOURIER TRANSFORMED INFRARED (FT-IR) (see Glossary) spectrometer over large spectral regions is becoming more common practice. CD in vibrational transitions, however, is a weak phenomenon,
with magnitudes of typically around 105 absorbance units. The first solution-phase VCD measurements date back to the mid 1970s [1,2] using a dispersive IR spectrometer. At the time, the experiments yielded weak and noisy spectra. Subsequent developments in the photoelastic modulators (PEM) and the development of FT-IR VCD have greatly improved the applicability and quality of VCD, and is making VCD spectroscopy a more routine technique. Today, virtually all VCD spectrometers are equipped with an FT-IR spectrometer. Because of the small magnitude of VCD it requires sophisticated modulation techniques for its observation. The central component in this modulation is the PEM. A periodic stress applied to an isotropic crystal causes the modulation of the polarization of the IR beam between left and right circular polarized light (CPL). A diagram of a FTVCD spectrometer is given in Fig. 1. The randomly polarized modulated beam that exits the FTIR spectrometer first passes through an optical filter, to assure a better signal to noise ratio. Then the beam is polarized by a linear polarizer. The linear polarized light is then alternatively switched at the PEM frequency between left and right CPL. The beam is then transmitted through the sample and is focused on an IR detector. The detected signal is divided into two channels. The first channel, which contains the normal single channel beam spectrum, is low pass filtered. The second channel contains the VCD signal and is high pass filtered and demodulated at the PEM frequency using a lockin amplifier. Commercial setups of FT-IR VCD spectrometers are provided by Bruker Optics [3,4] (http://www.brukeroptics.com), Bomem-BioTools [5,6] (http://www.btools.com), Nicolet/ Thermo (http://www.thermo.com), Jasco [7] (http:// www.jascoinc.com) and Bio-Rad [8,9] (http://www.bio-rad.com).
Figure 1. Diagram of a Fourier transformed vibrational circular dichroism spectrometer setup. The randomly polarized modulated beam that exits the FT-IR spectrometer passes through an optical filter. Then the beam is polarized by a linear polarizer. The linear polarized light is then alternatively switched at the PEM frequency between left and right CPL. The beam is then transmitted through the sample and is focused on an IR detector. The detected signal is divided into two channels. The first channel, which contains the normal single channel beam spectrum, is low pass filtered. The second channel contains the VCD signal and is high pass filtered and demodulated at the PEM frequency using a lock-in amplifier.
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Theory Optical activity manifests itself as a different absorption of left and right CPL, i.e. DA = AL AR or under Beer’s law condition De = eL eR. In a non-absorbing solvent, and for a dilute solution of a chiral molecule [10], the molar absorptivity and differential molar absorptivity can be written as
P eðyÞ ¼ 1:09 1038 y P Dj fj ðyj Þ 38 DeðyÞ ¼ 4:35 10 y Rj fj ðyj Þ
(3)
Dj and Rj in Eq. (3) are the dipole and rotational strengths of the excitation of state g to j at frequency yj, respectively. Dj and Rj are in esu2 cm2. fj is the normalized Lorentzian bandshape. Dj and Rj are given by
Dj ¼ jhgjmel jjij2 Rj ¼ Im ½hgjmel jji hjjmmag jgi
(4)
mel and mmag in Eq. (4) are the electric dipole and magnetic dipole moment operators, respectively [11]. In the harmonic approximation and working with normal coordinates Qi [12], the dipole strength for a fundamental excitation of mode a, is proportional to
@mel 2 @Q
Da01
a
(5)
0
The rotational strength for a fundamental transition [12] of mode a is given by
@mel @mmag @P @Q
Ra01
a
0
a
0
(6)
Pa in Eq. (6) is the conjugated moment for normal mode a. The second term of Eq. (6) requires a non-Born–Oppenheimer approximation, because of the correlation between the nuclear and electronic velocities. Algorithms for the calculation of the required dipole and rotor strengths and thus VCD spectra have been established [13], and VCD data can now be calculated in several popular quantum chemical software packages.
Computational methodology For VCD to be applicable in the determination of the stereochemistry of chiral compounds in solution, the acquired VCD spectra should be interpretable. The interpretation of VCD spectra, however, is not straightforward and requires an algorithm that relates both the structure and spectra. These spectra can be simulated by quantum chemical methods; comparison between the predicted and experimental spectrum makes the identification of the stereoisomer present in solution possible. The calculation of VCD spectra via quantum chemical methods requires several steps. First, a stereoisomer is chosen for which all calculations will be performed. The VCD data for the enantiomer of this stereoisomer are then simply the mirror
image. In the second step, a conformational analysis is conducted for the chosen stereoisomer with the purpose of locating the minimum energy conformations of the molecule. Once the geometry of these conformations is optimized on the required theoretical level, the VCD properties can be calculated. A theoretical VCD spectrum is then obtained by combining all conformations, and in the ultimate step the theoretical VCD spectrum is compared to the experimental one, thereby allowing the assignment of the absolute configuration of the molecule. The aim of the conformational analysis is to find all possible conformations of the molecule in the chosen absolute configuration. Knowledge of the different minimum energy conformations is crucial in structure determination by VCD. Experimental IR and VCD spectra are averaged spectra over all conformations present in solution. In case of a rigid molecule, a single conformer might be sufficient to explain experimental data. If the molecule is flexible, however, equilibrium between the different conformations has to be taken in to account. Different conformational search methods can be used on various levels of theory. An extensive discussion on conformational searches for medicinal molecules can be found in Sadowski et al. [14]. In the next step, the geometry of the minima is optimized at the desired quantum chemical level, followed by the calculation of the vibrational frequencies, IR and VCD intensities. Nowadays, most VCD calculations are carried out using the Density Functional Theory (DFT). DFT methods require a so-called density functional. For VCD calculations, most often hybrid functionals like B3LYP and B3PW91 [15,16] are used because these have been found to give better agreement with experiment compared to Hartree–Fock calculations [17]. Another choice that has to be made is the basis set to be used for the development of the molecular orbitals. Generally speaking, bigger basis sets give better descriptions of the molecule. The drawback, however, is that an increase of the basis set results in a serious increase of the required computer time. An economical compromise between accuracy and computational efficiency is the medium-size 6-31G* basis set [4]. Going to larger basis sets, the cc-pVTZ basis set gives excellent agreement with experiment [4]. Calculations can be carried out with commercially available software such as Gaussian03 (Revision B05, Gaussian, Inc., Pittsburgh, PA, http://www.gaussian.com). Herein Hessians matrices, atomic polar tensors (APT) and atomic axial tensors (AAT) can be calculated using gauge-including/invariant atomic orbitals (GIAOs) allowing the calculation of the VCD properties [18–20]. For each minimum energy conformation the dipole and rotational strengths are calculated. The results are single conformer line spectra, which can be broadened using a Lorentzian band-shape. Because each minimum contributes to the total spectrum in a Boltzmann weighted manner, the www.drugdiscoverytoday.com
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isomer used in the quantum chemical study. Naturally, if the molecule contains several chiral centers, it is necessary to consider many different stereoisomers.
IR and VCD spectra can be simulated as
Dtot ðyÞ ¼ Rtot ðyÞ ¼
X A X
FA DA ðyÞ FA RA ðyÞ
(7)
A
FA in Eq. (7) is weight factor expressing the contribution of each minimum energy conformation to the total spectrum. The weight factor for conformation A is usually taken as the Boltzmann population of this conformation. The DA(y) and RA(y) are the single conformational spectra. Once the total spectra are calculated, one can proceed to the comparison with the experimentally acquired spectra. If a good agreement between experimental and theoretical VCD spectra is obtained, one can conclude that the stereoisomer chosen for the quantum chemical study is also the one in the experimental sample. If a mirror image relationship exists with the experimental spectrum, one can conclude that the experimental sample contains the enantiomer of the stereo-
Application example The application of VCD spectroscopy, especially in a drug discovery environment, has profited greatly from the availability of commercial VCD apparatuses. As described above, the use of VCD for the determination of the absolute configuration of a (drug) molecule, requires quantum chemical calculations. The availability of a stable and sufficiently fast algorithm for the quantum chemical calculation of VCD spectra in widely available software packages, together with the increasing speed of computers and performant computer clusters has further boosted the capacities of VCD. Concurrent with these evolutions, VCD is no longer limited in applicability to relatively small and rigid molecules, but will become a method of increasing popularity for molecules of increasing size and flexibility.
Figure 2. Methyl mandelate and its IR and VCD spectra. (a) Schematic representation of methyl mandelate. (b) Experimental IR spectrum of methyl mandelate (gray). Simulated IR spectrum on B3LYP/6-31G* (green) and cc-pVTZ (blue) level. Experimental intensities are in absorbance units, theoretical intensities in 1040 esu2 cm2 and frequencies in cm1. Normal modes are numbered. (c) Experimental VCD spectrum of R() (red) and S(+) (green) methyl mandelate. Simulated VCD spectrum S(+) methyl mandelate on B3LYP/6-31G* (blue) and cc-pVTZ (brown) level. Experimental intensities are in 105 absorbance units, theoretical intensities in 1044 esu2 cm2 and frequencies in cm1. Normal modes are numbered.
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To illustrate the use of the VCD technology, methyl mandelate (Fig. 2a) is presented as an example, illustrating the different steps involved. Methyl mandelate is the methyl ester of mandalic acid, and the (R)- and (S)-isomers are chiral derivatizing agents for nuclear magnetic resonance (NMR) determination of enantiomeric purity of a-deuterated carboxylic acids, alcohols and amines [21]. VCD spectra for both R()- and S(+)-enantiomers, were measured on a Bruker IFS 66/S FT-IR spectrometer coupled to a PMA37 module. The VCD and IR spectra were recorded at a resolution of 4 cm1. For methyl mandelate the IR spectrum was measured using a Bruker Vector 22 FT-IR spectrometer. In all applications of VCD spectroscopy, the possibility to obtain spectra for both enantiomers greatly increases the quality of the spectra owing to the possibility to reduce several artifacts like baseline shifting [22]. A key feature of the VCD technology is the possibility to record spectra directly in solution phase, thereby opening the way for determination of absolute configurations for molecules without needing to obtain crystals. All calculations on methyl mandelate were done for the molecule in the S configuration. The conformational search strategy was followed, involving a combination of stochastic searching and systematic searching. This resulted in six unique minima. In the next step, the geometry of all minimum energy conformations was optimized using DFT calculations with the B3LYP functional and the 6-31G* and ccpVTZ basis sets, followed by the calculation of the absorption frequencies and dipole and rotor strengths. The line spectra are then broadened via a Lorentzian shape and theoretical spectra are obtained through Boltzmann averaging over all conformations. In Fig. 2b, the simulated and experimental IR spectra are given. The simulated IR spectra (with a Lorentzian broadening of 4 cm1) for both basis sets agree very well with the
experimental spectrum, with band positions and relative intensities in good agreement with experiment. The VCD spectra are depicted in Fig. 2c. One can see that the experimental VCD spectra have mirror image symmetry. Deviations on this symmetry are owing to small concentration differences and baseline effects. Based only on the agreement between theoretical and experimental spectrum, it can be seen that the S(+) spectrum agrees very well with the calculated S spectrum. However, one has to be careful with these practices, because of the nature of the simulated spectra. The harmonic approximation, resulting in higher calculated frequencies (which can partially be corrected with a scaling factor, depending on level of theory) and the fact that computational spectra do not incorporate solvent or intermolecular effects, one has to be absolutely sure that peaks corresponding to the same normal mode are compared. To do that, the peaks are identified based on the agreement between the simulated and experimental IR spectra, supplemented with VCD data. This way of working provides a more objective and robust assignment method, but is labor-intensive. As can be seen in Figs 2b and 2c, the normal modes have been assigned and, based on the agreement between the different modes, the assignment can be made.
Conclusions Compared to other available techniques, VCD offers a new and powerful approach to the determination of chiral molecules in solution phase. X-ray diffraction (XRD) [23,24], enzymatic resolution [25], NMR [26,27], optical rotation [28], electronic circular dichroism (ECD) [29,30] and stereoselective synthesis are techniques that were proven to be utilizable, but not always practicable (Table 1). VCD, by contrast, can be utilized on a wide range of molecules in
Table 1. Comparison summary table Technology 1 Name of specific type Vibrational circular of technology dichroism (VCD)
Technology 2
Technology 3
Technology 4
Technology 5
X-ray diffraction (XRD)
Nuclear magnetic resonance (NMR)
Optical rotation (OR)
Electronic circular dichroism (ECD)
Relatively easy to measure
Applicable to molecules in solution
Pros
Very reliable Well known Applicable in solution on a wide range of Food and Drug Administration molecules (FDA) standard
Cons
Measurements are not straightforward
Only a few bands Higher level Need for good Need for chiral shift are available reagents, chiral additives calculations needed quality single compared to VCD or derivatization crystals Higher level Only one property calculations needed can be compared in compared to VCD contrast to VCD (comparison of all bands in the spectrum) Sign depends on solvent used
References
See Related articles
[23,24]
[26,27]
[28,31]
[29,30] www.drugdiscoverytoday.com
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Related articles Stephens, P.J. (2004) Vibrational circular dichroism spectroscopy: a new tool for the stereochemical characterization of chiral molecules. In Computational medicinal chemistry for drugs discovery (Bultinck, P. et al., eds), pp. 699–725, Marcel Dekker, Inc Polavarapu, P.L. (1994) New spectroscopic tool – absolute-configuration determination of pharmaceutical compounds by vibrational circulardichroism. Spectroscopy 9, 48–55 Nafie, L.A. (1996) Vibrational optical activity. Appl. Spectrosc. 50, A14– A26 Keiderling, T.A. (1990) Vibrational circular dichroism. Comparison of techniques and practical considerations. In Practical Fourier Transform Infrared Spectroscopy. Industrial and Laboratory Chemical Analysis (Ferraro, J.R. et al., eds), pp. 203–284, Academic Press Freedman, T.B. et al. (2003) Determination of the absolute configuration and solution conformation of gossypol by vibrational circular dichroism. Chirality 15, 196–200
solution. The increase in speed and power of computers, the recent advances in computation algorithms and the performance of DFT functionals will only extend the range of molecules that can be operated by VCD. Furthermore, dedicated VCD instruments are now widely available, making VCD spectroscopy a more routine technique. VCD can also be used in other structure and conformationrelated applications on molecules of pharmaceutical interest, i.e. determination of the enantiomeric purity of a sample relative to a known standard. Also determination of the solution conformations of small or larger biological molecules can be probed by VCD. Despite the progress during the last decades, additional work is needed in several areas. In the experimental field, the problem of baseline effects in VCD spectroscopy and relatively long sampling times have to be tackled. Comparison between experimental and theoretical spectra is time consuming and labor-intensive. Algorithms that can handle this comparison and ultimately assign the AC are developed (unpublished material). Also, there is need for improved modeling of the effects of solvent and intermolecular interaction on the observed spectra.
Acknowledgement The authors want to thank Wouter Herrebout (Antwerp University) for scientific and technical assistance.
Outstanding issues Incorporation of solvents in the calculation of the IR and VCD spectra should give better agreement with experiment. Ab initio calculation of anharmonic vibrational circular dichroism intensities as a replacement for the harmonic approximation. Low temperature measurements should give less populated higher energy conformations. This should reduce the computational load, because fewer conformations have to be taken into account to simulate spectra.
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