Determination of Absolute Configuration of Chiral Molecules

focal point review

YANAN HE,* BO WANG, AND RINA K. DUKOR BIOTOOLS,INC., 17546 BEELINE HIGHWAY,JUPITER,FLORIDA 33458 LAURENCE A. NAFIE DEPARTMENT OF CHEMISTRY, 1-014CST, SYRACUSE UNIVERSITY, SYRACUSE,NEW YORK 13244

Determination of Absolute Conﬁguration of Chiral Molecules Using Vibrational Optical Activity: A Review

Determination of the absolute handedness, single crystals of the pure chiral sample of VCD, with the recent implementation of known as absolute configuration (AC), of chiral molecules as required for X-ray analysis. By ROA subroutines in commercial quantum molecules is an important step in any field comparing the sign and intensity of the chemistry software, ROA will in the future related to chirality, especially in the pharma- measured VOA spectrum with the correspond- complement VCD for AC determination. In this ceutical industry. Vibrational optical activity ing ab initio density functional theory (DFT) review, the basic principles of the application of calculated VOA spectrum of a chosen configu- (VOA) has become a powerful tool for the VCD to the determination of absolute configuration, one can unambiguously assign the AC of determination of the AC of chiral molecules in ration in chiral molecules are described. The a chiral molecule. Comparing measured VOA the solution state after nearly forty years of steps required for VCD spectral measurement spectra with calculated VOA spectra of all the evolution. VOA offers a novel alternative, or and calculation are outlined, followed by brief conformers can also provide solution-state descriptions of recently published papers re- supplement, to X-ray crystallography, permit- conformational populations. VOA consists of porting the determination of AC in small ting AC determinations on neat liquid, oil, and infrared vibrational circular dichroism (VCD) organic, pharmaceutical, and natural product solution samples without the need to grow and vibrational Raman optical activity (ROA). molecules. Currently, VCD is used routinely by research- ers in a variety of backgrounds, including Index Headings: Absolute configuration; Vibra- Received 12 April 2011; accepted 26 April tional optical activity; VOA; Vibrational circu- 2011. molecular chirality, asymmetric synthesis, chi- * Author to whom correspondence should be ral catalysis, drug screening, pharmacology, lar dichroism; VCD; Raman optical activity; sent. E-mail: [email protected]. and natural products. Although the application ROA; Chiral pharmaceutical molecules; Natu- DOI: 10.1366/11-06321 of ROA in AC determination lags behind that ral product molecules.

0003-7028/11/6507-0699$2.00/0 Volume 65, Number 7, 2011 Ó 2011 Society for Applied Spectroscopy APPLIED SPECTROSCOPY 699 focal point review

INTRODUCTION the same but their VCD or ROA spectra cades of development since its discovery 6–9 ibrational optical activity are equal in intensity but opposite in in the 1970s, vibrational optical (VOA)1–5 is a spectroscopic sign (mirror images of each other), as activity (VOA) has evolved into a well-established technique used routine- V measure of the differential re- shown in Fig. 1 for (1R)-(þ)- and (1S)- ly for the AC determination of small sponse of a chiral molecule to left versus (-)-camphor. The VCD spectrum of a organic molecules including natural right circularly polarized radiation dur- chiral molecule can be calculated using 10,11 an ab initio density functional theory products and pharmaceutical com- ing a vibrational transition. VOA con- pounds.5,12 VOA has also been success- (DFT) method. The absolute configura- sists of infrared vibrational circular fully applied for the determination of tion (AC) of a chiral molecule can be dichroism, known as VCD, and vibra- chiral purity (or %EE) of a changing tional Raman optical activity, known as determined by comparing the measured enantiomeric mixture,13,14 monitoring ROA. The infrared (IR) or Raman VCD or ROA spectrum with the calcu- reactions that involve chiral mole- spectrum of a pair of enantiomers is lated spectrum. After nearly four de- cules,15 and characterization of biomolecules such as peptides,16,17 pro- teins,16,17 carbohydrates,18,19 nucleic ac- ids,20,21 andevenviruses.22,23 AC determination has become increasingly important for the pharmaceutical industry because currently more than two thirds of the drugs on the global market are chiral drugs serving myriad thera- peutic areas, such as anxiety, indiges- tion, heartburn, arthritis, AIDS, cancer, allergies, etc. In 2010 nine out of ten top-selling blockbuster drugs, including Lipitor and Plavix, were chiral drugs. With the recent rapid development of biotherapeutic pharmaceuticals, chiral drugs will play an even more important role in saving the lives of patients because all bio-drugs are chiral. Since the FDA recommended the use of stereochemically pure drugs in 1992,24 single enantiomer drugs have become the standard in pharmaceutical companies when working with compounds featuring asymmetric centers. Shortening timelines for chiral drug discovery and development usually depends on the efficiency of asymmetric synthesis, enantiomeric purification, and AC determination.12 Techniques that are prevalent in both academia and the pharmaceutical industry for the determination of the AC of chiral molecules include X-ray crystallography, Mosher’s method (NMR), the chiral liquid crystal NMR technique, and VCD.25 X-ray crystallography is still considered the most reliable technique, but it requires a single crystal and typically at least one heavy atom.26 Mosher’s NMR method requires derivatization and is used primarily on FIG. 1. Observed VCD (upper frame) and IR (lower frame) spectra of (þ)- and (-)- alcohols and amines.26 The chiral- camphor in CCl4 (0.8 M); 98 lm path length cell with BaF2 windows; 20 min collection for both enantiomers; instrument optimized at 1400 cm-1 with 4 cm-1 liquid-crystal NMR method generally resolution. Solvent-subtracted IR and enantiomer-subtracted VCD spectra are requiresalargersamplesize(40to50 shown. milligrams) and complex experimental

700 Volume 65, Number 7, 2011 FIG. 2. Left panel: observed and calculated ROA (upper frame) and Raman (lower frame) spectra of (-)-(S)-(a)-pinene (neat liquid, 10 min accumulation, 7 cm-1 resolution, 532 nm at 200 mW). Right panel: observed and calculated VCD (upper frame) and IR (lower frame) spectra of (-)-(S)-(a)-pinene (neat liquid, 25 lm path length, 20 min scan, 4 cm-1 resolution, 1400 cm-1 setting for PEM). procedures.27 The VOA technique com- ization of biomolecules.29 ROA has publications using ROA for AC deter- plements the X-ray and NMR methods also been used for the determination mination have appeared. well because AC determination by of AC,30,31 but its application currently VOA does not require a single crystal, lags behind that of VCD. Recently DEFINITIONS OF does not entail molecular derivatization Gaussian Inc. (Wallingford, CT) imple- VIBRATIONAL CIRCULAR or intermolecular interactions, and is mented ROA calculations in the Gauss- DICHROISM AND RAMAN suitable for samples in liquid, solid, and ian09program,32 which should OPTICAL ACTIVITY gas phases. Dedicated Fourier trans- facilitate AC determinations by ROA. Vibrational circular dichroism has form (FT) VCD instrumentation was Figure 2 shows a comparison of the AC only one form, deﬁned as the difference commercialized ﬁrst by BioTools with determination of (-)-(S)-(a)-pinene by in the absorbance A m¯ of a chiral 28 ð Þ ABB Bomem in 1997, and since then VCD versus by ROA. It is clear that the molecule for left circularly polarized many pharmaceutical companies and AC of (a)-pinene can be determined (LCP) versus right circularly polarized academic groups have been using VCD equally well by either VCD or ROA. (RCP) infrared radiation during vibra- as a routine method for the AC In this Focal Point Review the tional excitation:1 determination of chiral molecules in theoretical background, instrumentation, the solution state. More than an esti- and methodology for AC determination DAðm¯Þ = ALðm¯Þ-ARðm¯Þð1Þ mated 3000 ACs have been determined by VOA are described, and some by VCD in the past few years, and the examples of recent applications of AC Here the decadic absorbance for unpo- number is increasing rapidly every determination by VCD are highlighted. larized absorption is given by year. ROA instrumentation was com- ROA is included in the introduction, but Aðm¯Þ = ½A ðm¯ÞþA ðm¯Þ=2 mercialized by BioTools in 2003 and the main focus of this review is on AC L R has been used mainly for the character- determination by VCD because very few =-log10½Iðm¯Þ=I0ðm¯Þ ð2Þ

APPLIED SPECTROSCOPY 701 focal point review

FIG. 3. Optical diagram of a ChiralIR-2XTM instrument from BioTools, Inc. for the measurement of VCD spectra.

where Iðm¯Þ and I0ðm¯Þ are the transmis- Incident circular polarization ROA METHODOLOGY sion intensities with and without the (ICP-ROA) is the original form of 6 Method of Absolute Conﬁguration sample, respectively, and where m¯ is the ROA, first measured in 1973 and Determination. 7 For VCD, the AC wavenumber frequency of the radiation. confirmed in 1975, in which the determination of a chiral molecule is VCD is also defined in terms of the difference in Raman intensity for RCP made by comparing the experimental IR molar absorptivity as versus LCP incident radiation states is and VCD spectra of the unknown measured while using fixed non-ellipti- Deðm¯Þ = e ðm¯Þ-e ðm¯Þð3Þ sample with those of the corresponding L R cal (unpolarized or linearly polarized) calculated IR and VCD spectra of the where the molar absorptivity of a sample polarization state a for the scattered molecule using a chosen AC. If the sign of path length b and concentration C is radiation. Scattered circular polarization and relative intensity of the observed ROA (SCP-ROA), first measured in bands in the VCD spectrum of the eðm¯Þ = Aðm¯Þ=bC ð4Þ 1988,33 is the difference in Raman sample are the same as that of the intensity for RCP versus LCP scattered calculated spectrum, the AC of the In contrast to VCD, ROA has four radiation while using fixed non-elliptical sample is the same as the AC chosen circular polarization (CP) forms. These incident polarization state a. SCP-ROA for the calculation. If the bands of the are given by the following deﬁnitions:1 is the form of ROA used in commercial observed VCD spectra are the opposite sign of those calculated, the sample has ROA instruments. Two new forms of ICP-ROA: DI ðm¯Þ = IRðm¯Þ-ILðm¯Þð5Þ the opposite AC of that used in the a a a ROA, predicted in 198934 and measured calculation. a a a a few years later,34,35 are called dual SCP-ROA: DI ðm¯Þ = IRðm¯Þ-ILðm¯Þð6Þ Vibrational Optical Activity In- circular polarization in-phase and out- R L strumentation. As with infrared spec- DCPI-ROA: DIIðm¯Þ = IR ðm¯Þ-ILðm¯Þð7Þ of-phase ROA (DCPI-ROA and DCPII- trometers, there are two types of VCD ROA), which, as seen in Eqs. 7 and 8, instruments, dispersive and Fourier and are differences in Raman intensity using transform.36 Before the advent of the both incident and scattered circular 37 DCP -ROA: DI ðm¯Þ = IRðm¯Þ-ILðm¯Þ FT-VCD spectrometer in 1979, all II II L R polarization states in the same measure- VCD instruments used a monochroma- ð8Þ ments. tor that scanned through a certain

702 Volume 65, Number 7, 2011 FIG. 4. Optical diagram of a ChiralRAMANTM instrument from BioTools, Inc. for the measurement of backscattered SCP- ROA spectra. spectral range. Dispersive VCDs are still taken place in the past decade and were used for biological samples in the mid- incorporated into the ChiralIR-2X FT- IR region, taking advantage of a strong- VCD spectrometer from BioTools, Inc. er source and a narrower spectral These include the use of dual-PEM for range.16,17 Shortly after BioTools intro- baseline stability and artifact suppres- duced the first commercial FT-VCD sion,39 dual source for better signal-to- instrument to the market in 1997, noise ratio,40 introduction of digital Bruker, Jasco, and Thermo-Electron also time-sampling to eliminate external offered VCD accessories for their FT-IR lock-ins and electronic filters, and spec- spectrometers. Despite the different tral range extension to 10 000 cm-1.41,42 designs for VCD spectrometers, all of An optical diagram of a Dual-PEM them use a photo-elastic modulator ChiralIR-2X TM VCD instrument (Bio- (PEM) placed in front of the sample in Tools, Inc.) is shown in Fig. 3. an FT-IR spectrometer to modulate the Raman optical activity instrumenta- IR beam between left- and right-circular tion originated with the ICP form of 28,38 polarizations at high frequency. A ROA, in which the incident light is FIG. 5. Structure of (-)-a-fluoro cy- number of instrumental advances have modulated between right- and left-circu- clohexanone 1.

APPLIED SPECTROSCOPY 703 focal point review

FIG. 6. (Top) Upper panel: optimized geometries, relative energies, and dipole moments for conformers of (2S,2R)-1. Lower panel: comparison of observed IR (lower frames) and VCD (upper frames) spectra of (-)-1 with (a) calculated spectra for (2S,5R)-conﬁguration conformers 5Ia-1, 5Ia-2, 5Ia-3, and 5Ia-4 and with ( b) composite of calculated spectra for (2S,5R)- conﬁguration conformers: 20% 5Ia-1, 20% 5Ia-2, 20% 5Ia-3, and 40% 5Ia-4. IR and VCD bands unique to the higher-energy conformation 5Ia-4 are indicated by þ.98 Adapted from Ref. 98 with permission of John Wiley and Sons. larly polarized states and the scattered Werner Hug is commercially available into different optical paths and are light is linearly polarized or unpolarized. from BioTools.44,45 This ROA spec- displayed on the upper and lower halves Instrumentation for this form of ROA is trometer depolarizes the incident light of a multi-channel charge-coupled de- not currently commercially available,43 completely while the scattered RCP and vice (CCD) detector. The ROA spec- but an SCP-ROA spectrometer Chiral- LCP radiation are measured simulta- trum is obtained by subtracting the LCP RAMANTM based on the design of neously by separating their intensities intensity from the RCP intensity. An

704 Volume 65, Number 7, 2011 FIG. 7. Structures of (4S,7R)-(-) and (4S,7S)-(þ)-4-isopropylidene-7-methyl- 4,5,6,7-tetrahydro-2(1)H-indazoles. optical diagram of a ChiralRAMAN- 2XTM ROA spectrometer is shown in Fig. 4. Vibrational Circular Dichroism and Infrared Measurement. To measure VCD and IR spectra of a sample, one must ﬁrst choose a suitable solvent that has a good spectral window in the IR region of interest. The sample is then dissolved in the solvent and placed in an IR sample cell, which usually has BaF2 or CaF2 windows. An IR spectrum is collected to optimize the concentration

FIG. 9. Optimized conformers, relative energies, and Boltzmann populations for pyrazole diastereomer 2a.62 Reproduced from Ref. 62 with permission of Elsevier.

or path length. In order to have a good desired IR absorbance range. The VCD signal-to-noise ratio for the measured baseline can be corrected by measuring VCD spectrum, the absorbance of the IR and then subtracting the corresponding bands should be in the range of 0.2 to VCD spectrum of one of the following, 0.8 absorbance. If the tested IR intensi- depending on which is available: the ties do not fall into this range, then either enantiomer of the sample (the best), the FIG. 8. 1H and 2H tautomers of pyr- the concentration or the path length racemic mixture of the sample and its azoles 2a and 2b. needs to be adjusted to obtain the enantiomer (second best), or the solvent.

APPLIED SPECTROSCOPY 705 focal point review

FIG. 10. Comparison of VCD (upper frame) and IR (lower FIG. 11. Comparison of the observed IR and VCD spectra for frame) observed spectra for (-)-diastereomer (right axes) (-)-diastereomer (right axes) with the calculated spectra (left with calculated spectra for diastereomer 2a conformers A axes, spectra offset for clarity) for 60% 2a-A þ 40% 2a-B and and B (left axes).62 Reproduced from Ref. 62 with permis- with the Boltzmann population weighted sum of calculated sion from Elsevier. spectra for all eight conformers of 2a.62 Reproduced from Ref. 62 with permission of Elsevier.

The IR baseline can be corrected by and Cheeseman, a decade ago to DFT most recent releases include analytical measuring and then subtracting the IR theory with gauge-invariant atomic or- subroutines for the calculation of all absorbance spectrum of the solvent from bitals (GIAOs), and this approach has needed ROA tensor derivatives, thereby that of the sample. become the standard method for the eliminating the time consuming ﬁnite Vibrational Circular Dichroism theoretical calculation of VCD spec- difference derivative steps that restricted and Raman Optical Activity Calcula- tra.49,50 The latest release of Gaussian the application of ROA to only the tions. Software for VOA Calculations. 0932 also contains software for calculat- smallest of chiral molecules. Now cal- Gaussian, Inc. has pioneered the com- ing ROA intensities. Other programs are culations of ROA can be carried out for mercial availability of software for available that contain VOA software, molecules comparable in size to those calculating VCD spectra from quantum such as Dalton or CADPAC, but these highlighted in this review, albeit at some mechanical ab initio or density function- are less user-friendly or less tested than additional computational time for the al theory programs since Gaussian 94. commercially available programs. The following reasons. The calculation of These programs employ the magnetic Amsterdam Density Functional (ADF) ROA spectra is more extensive and ﬁeld perturbation (MFP) theory of VCD program package recently announced complex relative to that for VCD spectra intensities conceived and implemented commercially available software for because of the higher-order tensor by Stephens46,47 and derived indepen- calculating VCD intensities. derivatives needed to form the observ- dently by Buckingham and Galwas.48 Software for ROA calculations is able ROA invariants, of which there are The MFP approach was extended by available in readily usable form from two for ROA in the usual far-from- Stephens, in collaboration with Frisch Gaussian in versions of 03 and 09. The resonance approximation, and the added

706 Volume 65, Number 7, 2011 whereas the scalar dot product of li and mi can be either positive or negative depending on whether these vectors are pointing roughly in the same or opposite directions. The conversion from a set of dipole or rotational strengths to a full IR or VCD spectrum is given by the expressions m X eðmÞ = -39 DifiðmÞð10Þ 9:184 3 10 i

m X DeðmÞ = RifiðmÞð11Þ FIG. 12. Structures of M- and P-enantiomer of nonamethoxy cyclotriveratry- 2:296 3 10-39 lene 3. i where the line-shape function is typical- sophistication in basis sets needed that development of faster computing power, ly a normalized Lorentzian function with must include diffuse functions. Once more and more VCD calculations have a half width at half-maximum ci for each measured and calculated ROA spectra been carried out with other functionals vibrational mode i and is given by are acquired for a given molecule, the and larger basis sets.11 c =p method of AC determination outlined Simulation of VCD and IR Spectra. f ðmÞ = i ð12Þ i 2 2 below is essentially the same for ROA The output of quantum mechanical ðmi-mÞ þ ci as it is for VCD. Estimates for the calculations of IR absorption and VCD Z additional computational time needed is given by a list of the dipole strength ‘ for an ROA calculation, given the results Di for IR intensities and the rotational fiðmÞ dm = 1 ð13Þ 0 of a VCD calculation that include the strength Ri for VCD intensities for each determination of the contributing con- normal vibrational mode i: Comparison of the individual conformer formers, their equilibrium geometry, and IR and VCD spectra with the observed 2 vibrational force fields, are in the range Di = jlij Ri = Imðli miÞð9Þ spectra often leads to the identification of two to five times longer depending on of the one or two most important the particular choice of basis sets and where li is a vector representing the conformers, usually those with the functionals. electric dipole transition moment of the lowest calculated energies. The calcu- Conformational Search. The first molecule and mi is the corresponding lated normal mode frequencies are step for VCD/ROA calculation is to magnetic dipole transition moment.1–6 typically scaled by a factor in the range obtain all the possible conformations The absolute square of a vector, the of 0.97 to 0.98 to compensate for the using molecular mechanics or semi- square of its length, given in the fact that the calculated frequencies are empirical methods with commercial expression for Di is always positive, based on a harmonic force field whereas programs such as the Spartan program from Wavefunction Inc., Hyperchem from Hypercube, Inc., MacroModel from Schrodinger, Inc., or PC Model from Serena Software. Geometry Optimization and VCD and IR Calculations. The lower-energy conformers (within approximately 5 kcal/mol from the lowest-energy conformer) generated from the conformational search are submitted to Gaussian 0932 or another program for DFT calculations (or other types) of the geometry optimization and for calculations of the force field, vibrational modes, and VCD and IR intensities. The typical functional and basis set for DFT calculations of VCD is B3LYP/ 6-31G(d), which has been found to be a FIG. 13. Structure and isotopic labeling of the P-helical parent Q1A and the good balance between accuracy and cost isotopomers Q2A.74 Reproduced from Ref. 74 with permission of John Wiley and of computing time. Recently with the Sons.

APPLIED SPECTROSCOPY 707 focal point review

reduce intensities and change the loca- tion of spectral peaks. Once the IR spectrum is assigned, the same numbers can be used to correlate the measured to the calculated VCD spectra. At this stage, it is now possible to determine whether the AC chosen for the VCD calculation is the same or the opposite of the AC of the sample used for the VCD measurement. If the correspondence is not yet clear, further theoretical analysis is needed to consider a variety of factors such as solvent effects, intermolecular interactions such as dimerization, the use of improved basis sets, or alternative hybrid density functionals. The most serious possible problem, aside from dimerization, is missing an important conformer in the conformational analysis. This latter pos- sibility, and the fact that the VCD from individual conformers can be significantly different from one another due to the extreme sensitivity of VCD to molecular conformation, reinforces the importance of a careful conformational analysis at the outset of the theoretical calculations. A corollary to this state- ment is that every assignment of AC by VCD includes a determination of the solution-state conformation or solution- state population of the principal conformers of the chiral molecule in question. This additional information is not available from a determination of AC using X-ray crystallography. If desired, dipole and rotational strength values for the measured IR and VCD spectra can be obtained and compared to the calculated dipole and FIG. 14. IR (lower frame) and VCD (upper frame) observed for Q1A (right axes) rotational strengths. This is accom- 74 compared to the calculation for the P-enantiomer shown in Fig. 12 (left axes). plished by first fitting the IR spectrum Reproduced from Ref. 74 with permission of John Wiley and Sons. with a set of Lorentzian line shapes such that each IR band ei(m) is associated with the observed frequencies arise from an spectra begins with an assignment of the a Lorentzian band of a given center anharmonic force field. In addition, measured IR spectrum. If the calculated frequency mi and spectral width. The area under the band is related to the solvent effects lead to further frequency normal modes are numbered from the 1 differences, and sometimes mode-order lowest to the highest frequencies, corre- dipole strength by the expression Z differences. In some programs solvent sponding numbers can be assigned to ‘ -39 eiðmÞ effects can be included in the geometry the measured IR spectrum based on the Di = 9:184 3 10 dðmÞ 0 m optimization and IR and VCD calcula- frequencies and relative intensities of the Z tions. bands. This process is more straightfor- 9:184 3 10-39 After the IR and VCD spectra of the ward for the IR spectrum than for the ffi eiðmÞ dm ð14Þ mi band contributing conformers have been cal- VCD spectrum since all the bands are culated, they are weighted by their positive in the IR absorbance spectrum This is just a mathematical inversion of fractional Boltzmann population and and there is no intensity cancellation like Eq. 10 with support from Eqs. 12 and 13 summed to produce the final calculated there can be in the VCD spectrum where and the assumption that the frequency of IR and VCD spectra. Analysis of these juxtaposed bands of opposite sign can normal mode i is approximately constant

708 Volume 65, Number 7, 2011 FIG. 15. IR (lower frame) and VCD (upper frame) observed FIG. 16. Comparison of difference spectra of IR (lower for Q2A compared to the calculation for the P-enantiomer frame) and VCD (upper frame) observed for (Q1A minus with 13C at three ring methylenes (13C-3) shown in Fig. Q2A) compared to calculations of the P-enantiomers 13.74 Reproduced from Ref. 74 with permission of John (parent minus 13C-3 isotopomer) in the conformation shown Wiley and Sons. in Fig. 13.74 Reproduced from Ref. 74 with permission of John Wiley and Sons. over the range of integration of the band. sured in the sample. If the opposite of an assignment of AC by VCD band Using the same Lorentzian band posi- enantiomer was calculated with a perfect correlation require (1) assigning mea- tions and widths determined for the IR fit, the points in the plot would lie along sured VCD bands to calculated VCD spectrum, the VCD is fit and the a line of slope -1. From such a plot the bands, (2) fitting the selected measured rotational strengths can be extracted in quality of the fit of the measured to the VCD bands to determine their area and the same way using calculated VCD data can be ascertained individual rotational strengths, and (3) Z ‘ visually. This method of AC quality plotting the calculated versus measured De ðmÞ 2 R = 2:296 3 10-39 i dm assessment has been advocated by rotational strengths and/or calculating R i 51 0 m Stephens. From such rotational coefficients as described above. Steps Z -39 strength correlation plots, statistical (1) and (2) are time consuming and 2:296 3 10 2 ffi DeiðmÞ dm measures such as R coefficient of require user judgments. An alternative mi band determination value can be obtained method, in which a quantity called the ð15Þ for both slope equal to þ1 and -1to enantiomeric similarity index (ESI) is aid in the level of confidence associated defined, has been developed to assess Given a set of experimental and calcu- with an assignment of the absolute numerically the degree of similarity lated rotational strengths, a plot of configuration. The use of statistical between a measured and a calculated calculated versus measured rotational measures of comparison of measured VCD spectrum without spectral assign- strengths can be constructed such that and calculated rotational strengths of ment or band area determination.53 The a perfect fit corresponds to all points selected major VCD bands has been ESI is a spectral similarity measure that lying along a line of slope þ1 if the same reported by Minick.52 correlates two sets of spectra with bands enantiomer was calculated as was mea- The methods of assessing the quality at shifted locations. An algorithm using

APPLIED SPECTROSCOPY 709 focal point review

ESI to evaluate the quality of fit between crystallography. In fact, VCD has been the observed and the calculated VCD used to uncover errors in the assignment and IR spectra was developed by Bio- of AC by other methods, including X- Tools in collaboration with Bultinck’s ray crystallography.52,54 group53 and is commercially available as a software program called Compare- DETERMINATION OF VOA. ABSOLUTE CONFIGURATION Even though there is currently no OF SMALL ORGANIC universally accepted standard for assess- MOLECULES ing the statistical confidence in the Over the past several years there has assignment of AC using VCD, it can been a rapid growth in the use of VCD be said that after literally hundreds of to determine the AC of small chiral such assignments, there are no cases in FIG. 17. Structure of perdeuterio- organic molecules. A large variety of phenyl-phenyl-sulfoxide. which a conflict exists between a structural types have been examined, thorough VCD analysis leading to a including epoxides and cyclopro- prediction of AC with the determination panes,55–58 bicyclic and tricyclic struc- of AC by other methods, such as X-ray tures,59–67 chiral structures with no

FIG. 18. Left panel: comparison of the B3LYP/TZ2P and B3PW91/TZ2P IR spectra of 4 to the experimental IR spectrum. The assignment of the experimental spectrum is based on the B3LYP/TZ2P spectrum. Right panel: comparison of the B3LYP/ TZ2P and B3PW91/TZ2P VCD spectra of (R)-1 and (S)-1 to the experimental VCD spectrum of (þ)-4. The assignment of the experimental spectrum is based on the B3LYP/TZ2P spectrum of (S)-1. The calculated IR and VCD spectra have Lorentzian band shapes (c = 4.0 cm-1).88 Adapted from Ref. 88 with permission of Elsevier.

710 Volume 65, Number 7, 2011 Tetra-substituted a-Fluoro Cyclo- data. The absolute configuration of 1 hexanones. Compound 1 (shown in Fig. and its analogs were unambiguously 5) is one of the chiral tetra-substituted a- determined. The comparison of the fluoro cyclohexanones that are asym- experimental spectra with the calculated metric catalysts for oxone epoxidation of spectra is shown in Fig. 6. trans olefins.98 The AC of the epoxida- (4S,7R)-(-) and (4S,7S)-(þ)-4-Iso- tion products can be predicted by the propylidene-7-methyl-4,5,6,7-tetrahy- AC of the catalyst used.98 Ketone 1 and dro-2(1)H-indazoles. Chiral pyrazoles its analogs were synthesized as race- are interesting precursors for the hydro- mates, which were then resolved and the tris(pyrazolyl)borate ligands, which can relative configuration of each of them be used in asymmetric catalysis.62 Such was determined by the NMR method. ligands also have potential use as The (-)-enantiomer of ketone 1 was building blocks for molecular motors.62 dissolved in CDCl3 (0.21 M) and placed In this collaboration between the groups in a 0.1 mm path length cell with BaF2 of Crassous and Nafie, diastereomeric windows. IR and VCD spectra were pyrazoles 2a and 2b (shown in Fig. 7) 0 FIG. 19. Structure of the 2R, 2 S, 3R, measured at 4 cm-1 resolution and 5 h were synthesized, their ACs were deter- 0 0 0 3 R enantiomer of 2,2 -dinitro-2,2 -bia- collection time for both sample and mined, and their tautomeric equilibrium ziridine. solvent, with the instrument optimized was studied by VCD spectroscopy for at 1400 cm-1. All the IR and VCD the first time. Diastereomers 2a and 2b chiral center (atropisomer or molecules spectra were baseline corrected by were prepared from dihydrocarvone by a with axial or helical chirality),68–83 subtracting solvent spectra from those Claisen condensation and then separated sulfur or phosphorus containing of the sample. The absolute configura- by the chiral high-performance liquid compounds,77,84–88 flexible mole- tions of 1 and its analogs were deter- chromatography (HPLC) method. The cules,56,89–91 and other structures that mined by VCD measurements combined VCD and IR of 2a and 2b were -1 do not fit into any of the above with DFT calculation of VCD using measured in CDCl3 at 4 cm resolution categories.92–97 The following examples Gaussian 9899 with B3LYP functional with 9 h collection time for both sample highlight the versatility of VCD as a and 6-31G(d) basis set. The conforma- and solvent, with the instrument opti- powerful tool for the AC determinations tional analysis of 1 and its analogs mized at 1400 cm-1. Each of the of different types of chiral organic yielded the conformation populations diastereomers 2a and 2b has two structures. that give the best fit to the experimental tautomeric forms, shown in Fig. 8. The

FIG. 20. Structures of the three most populated conformers of 5: –udH (63%), –uuC (17%), and –udG (13%) as obtained on the basis of DFT calculations at the B3LYP/6-31þG** level. The population factors are summed over all possible 56 conformations of the CO2Et groups (H atoms are not reported). Reproduced from Ref. 56 with permission of John Wiley and Sons.

APPLIED SPECTROSCOPY 711 focal point review

tion.31,101–108 This collaboration between the groups of Nafie, Luz, and Zimmerman investigated the use of isotopic difference VCD spectra as an aid in the AC determination of chiral molecules for the first time.74 The enantiomers and the racemates of the parent non-isotopic substituted nonamethoxy cyclotriveratrylene (Fig. 12) and the 13C-substituted isotopomer (Fig. 13) were dissolved in CDCl3 and their VCD and IR spectra were measured at 4 cm-1 resolution with 5 h collection, and the instrument optimized at 1400 cm-1. VCD calculations were carried out at the DFT level with Gaussian 03 using a B3LYP functional and a 6-31G(d) basis set. The AC of the parent nonamethoxy cyclotriveratrylene and its 13C-labeled isotopomer were determined by comparing the observed VCD and IR spectra with the calculated spectra (shown in Figs. 14 and 15). The isotopic difference VCD and IR spectra were calculated (Fig. 16), and the good fit between the observed and the calculated isotopic difference spectra confirmed the AC assignments previously obtained. Given the quality of fit for Q1A and Q2A, it is of interest to see whether the difference in the measured and the calculated IR and VCD spectra FIG. 21. Comparison of the experimental data (lower traces) of 5 (black) and its for these two molecules also compare enantiomer (gray) with the Boltzmann weighted average calculated spectra (upper traces) of the 2R, 20S, 3R, 3M0R enantiomer (calculated frequencies are well. The difference spectra shown in scaled by a factor of 0.975).56 Reproduced from Ref. 56 with permission of John Fig. 15 demonstrated that VCD differ- Wiley and Sons. ence spectra for both calculated and measured spectra can be used as a tool to assess the quality of fit of the calculated conformational search and VCD calcu- 2a and 2b could be determined by spectra. Moreover, since only those lations at the DFT level with Gaussian comparing the experimental with the parts of the IR and VCD spectra affected 03100 using B3LYP functional and calculated Boltzmann population by the isotopic substitution contribute to 6-31G(d) basis set revealed that for each weighted sum VCD spectra of the eight the IR and VCD difference spectra, of 2a and 2b there are four lowest- lowest-energy conformers (Fig. 11). some features in the original that do energy conformers dominant in solution. This detailed VCD study enabled the not agree as well may cancel, leaving a For both 2a and 2b two of the four calculation of the percentage of tauto- smaller subset of IR and VCD features lowest-energy conformers are 1H tautomers 1H and 2H (Fig. 8). The averaged for comparison. This is the first example mers and the other two are 2H tauto- calculated VCD over all tautomers and of a comparison of measured and mers. The optimized conformations, all conformers closely reproduced the calculated VCD difference spectra, and relative energies, and Boltzmann popu- experimental VCD spectrum. The com- this approach could be useful in the lations of 2a are shown in Fig. 9. parison between experiment and theory future for AC assignments. Comparing the observed VCD and IR determined precisely the proportions of Perdeuteriophenyl-phenyl-sulfox- spectra with the calculated spectra for each isomer (both tautomers and con- ide. Enantiopure sulfoxides are valuable the two dominant conformers (Fig. 10) formers). chiral starting material and important allowed the identification of experimen- 13C-Labeled Nonamethoxy Cyclo- chiral auxiliaries in organic synthesis.88 tal VCD features that arise from each triveratrylene. The sensitivity of VOA In the past, the AC of optically pure dominant conformer. The respective to the masses of atomic nuclei has been sulfoxides has been determined by the (4S,7R)-(-)- and (4S,7S)-(þ)-absolute reported for molecules that are chiral empirical rule of Mislow et al.109 configurations of pyrazole diastereomers only by virtue of isotopic substitu- However, a recent non-empirical analy-

712 Volume 65, Number 7, 2011 FIG. 22. Structure of (R)-(-)-malathion. sis of a series of ECD spectra of chiral alkyl aryl sulfoxides has found the rule to be wrong for these molecules.110 In this study, the groups of Stephens and Drabowicz synthesized and determined the AC of an isotopically chiral sulfoxide 4 (Fig. 17) using VCD. The geometry optimization and the VCD and IR spectra were calculated using DFT with B3LYP/TZ2P and B3PW91/ TZ2P functional and basis sets. Only one low-energy conformer dominates the solution population and the B3LYP/TZ2P calculated lowest energy conformer is in good agreement with the FIG. 24. Comparison of the measured VCD (upper frame) and IR (lower frame) X-ray determined structure. Based on spectra of (-)-malathion (thin line: CCl4, 0.11 M, BaF2,72lm path length) with the this study and previous work, the predicted (population weighted) spectra of eight energetically preferred confor- B3LYP/TZ2P equilibrium geometry mations for (R)-malathion (bold line: B3LYP/6-31G(d)).112 Reproduced from Ref. agrees best with X-ray data and the 112 with permission of John Wiley and Sons. B3LYP/TZ2P simulated VCD and IR spectra are more accurate than the B3PW91/TZ2P or 6-31G(d) simulated and calculated spectra is not very was determined by VCD and further spectra (shown in Fig. 18). Usually for favorable using the B3LYP functional supported by comparison of ECD mea- sulfoxide containing molecules, the and 6-31G(d) basis set due to a lower surements with TD-DFT calculations of agreement between the experimental accuracy simulation of the frequency of ECD intensities. Due to the ﬂexibility of the S=O stretching mode. In this study, the sample, approximately 300 possible larger basis sets were tested with two conformers were analyzed, but the DFT different functionals and it was found calculation revealed that only a few that B3LYP/TZ2P is a better functional conformers are populated at room tem- and basis set combination for calculating perature (Fig. 20). Comparing the Boltz- the geometry and VCD of sulfoxide mann weighted average calculated containing molecules. (B3LYP/6-31þG**) spectra of the 2R, 2,20-Dinitro-2,20-biaziridine. The 20S, 3R, 30R enantiomers with the 0 0 molecule 2,2 -dinitro-2,2 -biaziridine 5 spectra measured in CDCl3, the AC of possesses many useful functional the two enantiomers of 5 were deter- groups, such as an aziridine ring and mined unambiguously using VCD (Fig. nitro and carboxylic groups, that can 21). The comparison of the measured serve as versatile building blocks for the ECD spectra with the TD-DFT calculat- synthesis of a variety of new compounds ed ECD spectra reinforced the AC of interest for asymmetric synthesis of assignment obtained by VCD. The pharmacologically active substrates.56 In ECD study also indicated that for the FIG. 23. Optimized-geometry of the most stable conformer (B3LYP/ a recent study, a racemic mixture of 5 ECD simulation the Coulomb-attenuated 6-31G(d)) of (R)-malathione.112 Repro- (Fig. 19) was synthesized and the two CAM-B3LYP functional gives better duced from Ref. 112 with permission enantiomers were separated by chiral results than the B3LYP functional, of John Wiley and Sons. HPLC. The AC of the two enantiomers especially for N-containing molecules.

APPLIED SPECTROSCOPY 713 focal point review

FIG. 25. Structure of GT-2331.

This study demonstrated that VCD is suitable not only for rigid molecules but also for flexible molecules. The key step in AC determination of flexible mole- FIG. 26. Upper frame: experimental VCD spectrum of 7 (red) compared to VCD cules using VCD is the conformational spectrum calculated for the mixture of H-bonded tautomers (black). Lower frame: search. With a good strategy and careful experimental VCD spectrum of the enantiomer of 7 (blue) versus VCD spectrum survey, it is no longer a challenging task calculated for the mixture of H-bonded tautomers (black). Asterisks indicate to find all the low-energy conformers in possible small spectral artifacts in the experimental VCD spectrum.52 Repro- a flexible molecule. duced from Ref. 52 with permission of John Wiley and Sons.

DETERMINATION OF 112 ABSOLUTE CONFIGURATION for trade and human health purposes. calculation (B3LYP/6-31G(d)) resulted OF PHARMACEUTICAL Enantiomers of chiral pesticides work in eight conformers within 1 kcal/mol MOLECULES differently in biological systems, and from the lowest-energy conformer and analytical methods to determine the account for greater than 75% of the The number of AC determinations by optical purity, stereoselective bioactivi- calculated population distribution. The VCD has increased dramatically in the ty, and environmental behavior of chiral predicted most stable conformation of pharmaceutical industry in the past few pesticides has been developed.112 Sev- (R)-malathion is shown in Fig. 23. The years into the range of several hundreds eral chiral pesticides, including malathi- measured VCD and IR spectra of (-)- of structures per year. However, for on, are liquid at room temperature, and malathion (CCl4, 0.11 M, BaF2,72lm proprietary reasons fewer than twenty therefore, the absolute configuration of path length) are in good agreement with AC determinations of pharmaceutical these pesticides cannot be determined by the population weighted VCD and IR compounds via VCD method have been X-ray crystallography in the usual spectra of the eight energetically pre- recently reported.52,111–122 The follow- manner. In this study, Izumi and co- ferred conformations for (R)-malathion ing examples demonstrate that VCD has workers determined the AC and solution (Fig. 24). The absolute configuration of become a useful tool in assisting the conformation of malathion using VCD (-)-malathion was therefore assigned as study of structure–activity relationships spectroscopy and a conformational code (R)-(-)-malathion from the VCD anal- (SAR) and/or structure–property rela- that was recently developed by his ysis and corresponds to the reference tionships (SPR) for chiral pharmaceuti- group.112,123 This code can be used in assignment. The use of a fragment- cal compounds. AC analysis for a flexible molecule conformational search with the confor- (R)-(1)-Malathion. Malathion 6 (Fig. without a full conformational search mational code highlights the essential 22) is one of the most widely used using molecular mechanics (MM) calcu- conformational features of the molecular pesticides for suppression of harmful lations. Instead, DFT calculations were fragments and affords insight into the insects, such as the mosquito, and has a carried out for various fragments of (R)- conformational distribution in a solution flexible liquid-state conformation at malathion, namely ethyl propionate, (R)- state for a flexible molecule such as (-)- room temperature.112 Related to this ethyl 2-(methylthio) propanoate, (R)- malathion. This methodology therefore use, the need for pesticide tests for the diethyl 2-(methylthio) succinate, and allows the selection time of the predom- determination of maximum residue lev- O,O,S-trimethyl phosphorodithioate. inant conformations of large and flexible els (MRLs) in food have been addressed The conformational search and DFT chiral molecules to be shortened and the

714 Volume 65, Number 7, 2011 ment has characteristic conformational molecule has also been found to be distribution patterns. A database of the highly dependent on absolute stereo- conformational distribution patterns of chemistry, making the reliable assign- numerous common fragments and an ment of this property a necessity. Two associated conformational analysis previous X-ray diffraction studies on wouldbeusefulforthedesignof this same molecule have assigned ACs bioactive compounds in the future. This that are opposite to each other, leaving example once again demonstrates the its three-dimensional structure uncertain. usefulness of VCD for the AC determi- In view of this, its AC was reinvestigat- nation of conformationally flexible mol- ed using VCD. Results from this study ecules. provided independent assignment of this Histamine H3 Receptor Antagonist important molecule as the (1S,2S)-enan- GT-2331. GT-2331, 7 (Fig. 25), is one tiomer. FIG. 27. Structure of (R)-1-[(4-cyano- of the most potent members of a class of A racemic mixture of 7 was synthe- phenyl)(3-bromo-4-hydroxyphenyl)- sized and the racemate was separated methyl]-1H-[1,2,4]triazole. chiral drug substances used to regulate the synthesis and release of histamine by into purified enantiomers by preparative the histamine H3 receptor and is an supercritical fluid chromatography. accuracy of the determination of abso- important biomarker for pharmaceutical VCD and IR spectra were acquired in -1 lute configuration is significantly im- companies conducting research in this the mid-IR region (2000–800 cm )ata -1 proved. Most large molecules consist of field.52 In addition to its overall struc- resolution of 4 cm . The (1S,2S) the chemical fragments, and each frag- tural features, the bioactivity of this configuration was chosen for calculations and two tautomeric forms N1–H and N3–H were built. Global conformational searches at the molecular mechanics level followed by DFT calculation using the B3LYP functional with a 6-311G(d,p) basis set predicted one dominant conformer (81% population) for the N1–H tautomer and six dominant conformers 97% (total population) for

FIG. 29. ECD spectra. Dark blue trace: experimental spectrum of 8a. Light blue trace: experimental spectrum of compound 8b. Black trace: calculated spectrum for R conﬁguration (average over eight conformers with calculated FIG. 28. VCD spectra. Bottom trace: experimental spectrum of 8a (0.256 M population). The spectra were record- -4 CDCl3). Middle trace: calculated spectrum for R conﬁguration (weighted average ed on 10 M solutions in the range taking into account populations obtained by calculated Gibbs free energies). 190–240 nm and on 10-3 M solutions 116 Upper trace: experimental spectrum of 8b (0.23 M in CDCl3). Calculated in the range 240–340 nm. Adapted frequencies have been shifted by 30 cm-1.116 Adapted from Ref. 116 with from Ref. 116 with permission of permission of American Chemical Society. American Chemical Society.

APPLIED SPECTROSCOPY 715 focal point review

the N3–H tautomer. The combination of literature reported. The simulated IR and all the conformers of N1–H and N3–H VCD spectra predicted for a 65:35 resulted in the lowest-energy N1–H mixture of N1–H and N3–H tautomers conformer being 90–95% populated. are in good agreement with the experi- However, when comparing the Boltz- mental spectra (Fig. 26). This study mann weighted IR and VCD spectra indicated that for molecules that have with the experimental data, the agree- strong intermolecular interactions, the ment is not satisfactory, indicating that Boltzmann distribution based on gas- the simulated spectra based on gas- phase free energies may not be accurate. phase Boltzmann distribution of the When dealing with this type of molecule conformers are not accurate for this one should carry out a very careful molecule in solution state. Comparing population analysis to determine the the experimental IR with the calculated more accurate populations of each IR of each conformer suggested that the conformer. N3–H tautomer was underestimated 1-[(4-Cyanophenyl)(3-bromo-4-hy- using gas-phase free energies. The mole droxyphenyl)methyl]-1H-[1,2,4]tri- fractions of N1–H and N3–H were azole. 1-[(4-Cyanophenyl)(3-bromo-4- estimated empirically using the relative hydroxyphenyl)methyl]-1H-[1,2,4]tri- intensities of bands at 1595 cm-1 and azole 8 is the synthetic precursor for 1- 1575 cm-1. Based on those intensities [(4-cyanophenyl)(3-bromo-4-sulfamoyl- the mole fractions of N1–H and N3–H oxyphenyl)methyl]-1H-[1,2,4]triazole, FIG. 30. Structure of (1R, 5S) azabi- were estimated to be 0.65 and 0.35, which was found to be the most potent cyclo[3,2,1]octane derivative 9. which is in good agreement with the of the achiral and racemic aromatase

FIG. 31. Left panel: observed VCD (upper frame) and IR (lower frame) spectra of 9 compared with those of the calculated spectra of the eight lowest-energy conformers of (1R, 5S). Right panel: observed VCD (upper frame) and IR (lower frame) spectra of 9 compared with those of the calculated Boltzmann averaged spectra of the eight lowest-energy conformers of (1R, 5S).

716 Volume 65, Number 7, 2011 semi-preparative chiral HPLC. After an state of excessive retention of water.120 extensive effort to obtain crystals suit- The SAR study revealed that the (1S, able for X-ray analysis failed, the AC of 5R) stereoisomers had significantly each enantiomer 8a and 8b were deter- higher affinity for the vascular and renal minedbyVCDandreinforcedby receptors than their enantiomers.120 In electronic circular dichroism (ECD). some cases the eutomer was greater than The experimental VCD spectra of 8a 1000-fold more active than the corre- and 8b and the calculated Boltzmann sponding distomer in the vascular-re- averaged VCD (B3LYP/6-31G*) for the ceptor assay.120 Therefore, AC deter- R configuration of 8 are shown in Fig. mination in a timely manner played an 28. The ECD measurement in conjunc- important role in understanding the SAR tion with TDDFT (B3LYP/6-31G*) and/or SPR of these analogs. The calculations reinforced the AC assign- enantiomers of each compound were ment obtained from VCD study (Fig. synthesizedandseparatedbychiral 29). supercritical fluid chromatography. The Azabicyclo[3,2,1]octane Deriva- VCD and IR spectra of both enantiomers

FIG. 32. Structures of chromanes tives. A series of biaryl amides contain- were measured in CDCl3 (0.8 M) using 10-1 and 10-2. ing an azabicyclooctane amine head- a 100 lm path length cell with BaF2 piece were synthesized and evaluated as windows; 8 h collection for both enan- mixed arginine vasopressin (AVP) re- tiomers; instrument optimized at 1400 inhibitors for the treatment of hormone- ceptor antagonists that are being inves- cm-1 with 4 cm-1 resolution. The (1R, dependent breast cancer in postmeno- tigated for use in the treatment of 5S) conﬁguration was built and the pausal women.116 The two enantiomers hyponatremia, hypertension, congestive conformational search was carried out 8a (Fig. 27) and 8b were separated by heart failure, liver cirrhosis, and any using HyperChem (Hypercube, Inc.).

FIG. 33. Calculated VCD spectra, optimized structures, and relative energies of the four lowest-energy conformers of (R)- 10-1.142 Reproduced from Ref. 142 with permission of Elsevier.

APPLIED SPECTROSCOPY 717 focal point review

FIG. 35. Structure of salvileucalin A (11a) and salvileucalin B (11b).

analogs of compound 9 were determined by comparing the observed VCD of each unknown compound with that of compound 9. This study provided a useful example for the determination of AC for analogs used in SAR and/or SPR studies in drug discovery.

DETERMINATION OF ABSOLUTE CONFIGURATION OF NATURAL PRODUCT MOLECULES Within the past four years more than thirty papers have been published on AC determination of natural products. Most of these natural product molecules feature more than one chiral center and their relative stereochemis- tries were determined by X-ray or NMR methods. The majority of these molecules were investigated by the groups of Stephens11,124–128 and Jo- 129–139 FIG. 34. Comparison of the VCD and IR spectra of the measured (þ)-10-1 with the seph-Nathan, but additional calculated VCD and IR spectra of the Boltzmann average of the four lowest- groups in natural-product research have energy conformers of the corresponding (R)-10-1. The comparison establishes begun to take advantage of VCD for the absolute configuration of this molecule as (þ)-(R)-10-1.142 Reproduced from AC determinations.140–149 Nafie pub- Ref. 142 with permission of Elsevier. lished an extensive review on the AC determination of natural products by 10 The resulting conformers were calculat- the overlay of the calculated spectra of VCD in 2008, and as a result only a ed for geometry optimization and IR and the eight low-energy conformers (the few more recent examples are described in this section. VCD intensities at DFT level using left panel of Fig. 31 for compound 9 Chromanes. Two chromanes, 10-1 Gaussian03100 with B3LYP functional (structure shown in Fig. 30)) demon- and 10-2 (Fig. 32), were isolated from and 6-31G(d) basis set. The Gaussian strated the sensitivity of VCD to the Peperomia obtusifolia as racemic mix- calculation resulted in eight low-energy conformational changes. The AC of tures and resolved using chiral conformers that have energies within 1 each enantiomer was determined by HPLC.142 The initial stereochemical kcal/mol from the lowest-energy con- comparing the measured VCD and IR determination was based on an empirical 1 former. These conformers differ by the spectra with those of the calculated rule that the sign of the Lb band of the orientation of the carbonyl and the Boltzmann averaged spectra of the (1R, ECD can predict the AC of the dihy- aromatic groups. The comparison of 5S) configuration (the right panel of Fig. dropyrane ring. Experimentally, the the observed VCD and IR spectra with 31 for compound 9). The ACs of other chromane 10-1 was dissolved in CDCl3

718 Volume 65, Number 7, 2011 former, four conformers representing 75% of the Boltzmann distribution were selected to construct the calculated IR and VCD spectra (Fig. 33). Comparison of the observed spectra with the DFT calculations revealed that the absolute configuration of the chromane 10-1 is (R) (Fig. 34), opposite to the previous assignment based on the observed sign 1 of the Lb ECD band and the empirical rule. ECD calculations based on time- dependent DFT (TDDFT) (B3LYP/ 6-311þþG(2d,2p)//B3LYP/6-31G(d) showed that both P- and M-helicity of the heterocyclic ring could result in the 1 same sign for the ECD Lb band, which results in the failure of the empirical rule in this case. The quality of the fit of the observed VCD/IR spectra with those of the calculated spectra was evaluated by the CompareVOA algorithm,53 and a high confidence level (enantiomeric similarity index (ESI) = 76.6 for chromane 10-1) further confirmed the correctness of the new AC assignment by VCD. Salvileucalin B. Salvileucalin B 11b

FIG. 36. Comparison of the measured VCD (upper frame) and IR (lower frame) (Fig. 35) is the first natural product spectra with the predicted (population weighted) spectra of salvileucalin A, 11a, having a caged carbon framework that with (thin line) the measured VCD spectrum; (thick line) the calculated exhibits high cytotoxicity against human (population weighted) VCD spectrum; (thin line) the measured IR spectrum; and A549 and HT-29 cells.143 In this study (thick line) the calculated (population weighed) IR spectrum.143 Adapted from Ref. Salvileucalin B and its genetically 143 with permission of American Chemical Society. related analog Salvileucalin A were isolated from Salvia leucantha (Mexican bush sage), their structure skeletons were elucidated by extensive NMR in a BaF2 cell (100 lm path length) and stable conformer. The geometry optimi- studies, and their relative configurations the VCD spectra were measured. A zation and IR and VCD intensities of were determined by X-ray crystallogra- conformational search using MMþ and these conformers were calculated using phy.143 The absolute configurations of MMFF force fields in Hyperchem (Hy- Gaussian 0932 at the DFT level with the Salvileucalin A and B were determined percube, Gainsville, FL) and Spartan B3LYP functional and 6-31G(d) basis by VCD spectroscopy in combination (Wavefunction, Irvine, CA) resulted in set. Among the ten low-energy con- with DFT calculations carried out at the 53 conformers with relative energy less formers with relative energy within 1.4 level of B3LYP functional and 6-31G(d) than 3 kcal/mol above that of the most kcal/mol from the lowest-energy con- basis set. The comparison of the observed VCD and IR spectra of Salvileu- calin A with the calculated spectra is shown in Fig. 36. For molecules that have multiple chiral centers in a fused ring system, such as Salvileucalin A and B, the best strategy for AC determination is to first use other methods such as NMR or X-ray to establish the relative stereochemistry, then use VCD to determine the AC. This will significantly reduce the time spent on the calculation of different possible configurations. Isoepitaondiol. The structure of iso- FIG. 37. The original and X-ray/NMR reassigned structures of isoepitaondiol. epitaondiol 12a (Fig. 37), a meroditer-

APPLIED SPECTROSCOPY 719 focal point review

and therefore is more straightforward, efficient, and convenient in practice. The other advantage of VOA is that along with the determination of AC, the solution-state conformational populations via conformational analysis are obtained by determining the most important conformers included in the VOA calculation or by comparing the experimental VOA spectra with the calculated spectra of each conformer. Although many groups have used VCD, ECD, and/or optical rotatory dispersion (ORD) together for AC determination, the VCD method is much easier and more reliable because vibrational transitions have much narrower bandwidths than electronic transitions and VCD spectra are much better resolved than ECD spectra. Additionally vibrational rotational strengths only depend on the wavefunction of the ground electronic state, whereas electronic rotational strengths depend on wavefunctions of both ground and excited electronic states, and the calculation of the ground electronic state wavefunction is more FIG. 38. Experimental and Boltzmann weighted calculated DFT B3LYP/DGTZVP accurate than that of any of the excited VCD spectra of isoepitaondiol diacetate (12b).138 Adapted from Ref. 138 with electronic states. permission of American Chemical Society. The recent examples of AC determination by VCD described here demonstrate that VCD has been well adopted in penoid isolated from Stypopodium fla- a molecule bearing several chiral centers a variety of research areas including the belliforme was reassigned as the diace- on a fused ring system using the basic study of chirality, asymmetric tate to be 12b (Fig. 37).138 The relative methods of NMR, X-ray, and VCD. synthesis or catalysis, drug screening, configuration was determined by X-ray CONCLUSIONS pharmacology, and natural products. crystallography and extensive NMR With the development of the Compare- studies. The absolute configuration was Vibrational optical activity, and espe- VOA algorithm53 for evaluating the determined by VCD in comparison to cially VCD, has evolved to become a quality of fit between the observed and DFT calculations with a B3LYP/ powerful tool for the determination of the calculated spectra, reliable statistical DGDZVP basis set. The IR and VCD the AC of chiral molecules in the measures for AC determination by VCD spectra were measured at a resolution of solution state. Further advancements in will further enhance the growth in the -1 4cm . Conformational searches were VOA instrumentation now allow more application of this methodology. Al- carried out using a Monte Carlo guided accurate VOA measurements with min- though it is not yet widely recognized, protocol using the X-ray coordinates of imal artifacts that might interfere with the availability of ROA instrumentation 12b as the input data. The 13 conform- the interpretation of the experimental and quantum chemistry software make ers obtained from the MMFF molecular data. In addition, the continued devel- possible the use of ROA as a closely mechanics searches were calculated at opment and upgrade of the computing related and complementary tool for DFT level for geometry optimization power and VOA software, together with determination of AC in the solution using a B3LYP hybrid functional and the availability of higher level func- state that is only marginally less effec- the DGDZVP basis set. The two lowest tionals and basis sets, make the predic- tive than its popular cousin VCD. In the energy conformers were Boltzmann tion of VOA spectra even more accurate future, it is likely that both VCD and weighted to simulate the calculated IR and reliable for the unambiguous deter- ROA will be used together in concert or and VCD spectra. The absolute config- mination of AC in chiral organic separately according to ease of sampling uration was unambiguously determined molecules. Compared to other more to determine AC in chiral molecules to a by comparing the calculated with the traditional techniques such as X-ray level of confidence that is even higher observed VCD spectra (Fig. 38). This is and NMR, the VOA method does not than that currently enjoyed today by another example of AC determination of require a single crystal or derivatization VCD alone.

720 Volume 65, Number 7, 2011 ACKNOWLEDGMENTS 19. T. Taniguchi, I. Tone, and K. Monde, 39. L. A. Nafie, Appl. Spectrosc. 54, 1634 Chirality 20, 446 (2008). (2000). The authors would like to thank Drs. Jim 20. L. Ashton, A. Hobro, G. L. Conn, M. Rouhi, 40. L. A. Nafie, H. Buijs, A. Rilling, X. Cao, and Cheeseman and Michael Frisch from Gaussian and and E. W. Blanch, J. Mol. Struct. 883–884, R. K. Dukor, Appl. Spectrosc. 58, 647 Dr. Honggang Li from BioTools for providing 187 (2008). (2004). calculated and experimental ROA spectra. We also 21. Y. Blagoi, G. Gladchenko, L. A. Nafie, T. B. 41. X. Cao, R. D. Shah, R. K. Dukor, C. Guo, T. thank Dr. Elke Debbie and Professor Patrick Freedman, V. Sorokin, V. Valeev, and Y. He, B. Freedman, and L. A. Nafie, Appl. Bultinck from Ghent University in Belgium for Biopolymers 78, 275 (2005). Spectrosc. 58, 1057 (2004). helpful discussion and the development of the 22. L. D. Barron, E. W. Blanch, I. H. McColl, C. 42. C. Guo, R. D. Shah, R. K. 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