Chiral Solid Solutions for the NMRAnalysis of Enantiomers: A Potential New Approach to Chiral Analysis

Differences between the solid-state C CP-MAS NMR spectra of holemic samples of the two enantiomers of 2,2-dihydroxy-1,1binaphthyl (binol) were not sufficiently emphatic to reliably distinguish them, though they are readily distinguishable from the spectrum of the bimate of the compound crystallized from an equimatic sample. Inducing an additional chiral environment by cocondensation with sucrose as a chiral selector (CS) provided a method to yield differential spectra for the two enantiomers and thus effect enantiodifferentiation by way of solid-state NMR using weak interactions from a CS.


Introduction
One of the most important and challenging determinations in chemistry is the state of chirality ("enantiodifferentiation, " i.e., the identi�cation of an enantiomer, the quanti�cation of enantiomeric composition, and/or the determination of absolute con�guration [1,2]), ever more so with the increasingly stringent requirements placed upon the pharmaceutical industry for enantiomerically pure drugs.As part of our investigations [3] into chiral processes involving weak interactions, in particular the ESDAC [3][4][5][6][7] (enantiomer self-disproportionation on achiral chromatography) phenomenon stemming from our original observations [4,8] of 9-hydroxy cineole (1) and its related metabolites (Figure 1) such as the dihydroxylated compounds 2,9-(2) and 2,10dihydroxy-1,8-cineole (3) [9], we were greatly interested in new methods of enantiodifferentiation. 9-Hydroxy cineole (1) is an interesting compound because it readily undergoes SDE (self-disproportionation of enantiomers) [10], for example, by ESDAC [4] and also by sublimation [8], since it, like 2 and 3, is obtained as a scalemate from its natural source.A scalemate is a sample containing an unequal mixture of enantiomers-the preferred terminology and descriptors set out recently [11,12] with respect to chiral systems are used herein.us, not only were we interested in absolute con�guration determination but also quanti�cation of the enantiomeric composition (ec), and for which we had to take care not to elicit perturbation of the ec from its original state.
Fortunately, however, many methods are available to accomplish enantiodifferentiation and even within NMR there are a number of broad approaches [13][14][15][16][17][18][19] including chemical derivatization, chiral solvating agents (CSAs), and chiral complexation.In addition, chiral cavitands, either under isotropic conditions [17,[20][21][22][23][24] or otherwise [25], have also been explored with respect to evaluation of the ec and/or identi�cation of the predominant enantiomer.e most formidable advances within NMR of recent times in this context have involved the application of chiral liquid crystals (CLCs) [26][27][28][29][30][31][32][33][34][35][36][37][38]-originating from the pioneering work [39,40] of Snyder et al.-for determination of the ec or even for absolute con�guration determination.Such are the dramatic differences in favorable cases that even (�uoro)hydrocarbon enantiomers [31][32][33] and enantiomers chiral by virtue of isotopic substitution [36] have been successfully analyzed using this methodology.e great success of CLCs lies in the strong response by various order-dependent parameters to the small differences between the enantiomers in the chiral environment based on their orientation to the �eld; though differential orientation of enantiomers by other, �eldindependent media is also possible [41,42].However, despite all efforts, a general solution [13,19,27,28,43] to the central questions has not been found.A general solution is not only F 1: e structures of compounds 1-3.e recently described [11] notation for indicating relative stereochemistry as an extension to the Natta projection convention proposed by Giulio Natta  is used and for which the "2" near to the C-2 and C-8 atoms indicates that the number of stereoisomers present in the sample or under consideration is two.
desirable in its own right but also to limit the shortcomings of the various present methodologies [13-15, 28, 41, 42] which include technical and interpretative challenges.All of the previous work, however, suggests that if a general solution is to be found, then the solution will likely involve a compact state and/or aligned media.In this work, the aim is to examine a new approach to chiral analysis in the solid state by use of a chiral selector (CS), namely, sucrose, to effect the enantiodifferentiation of 2,2 � -dihydroxy-1,1 � -binaphthyl (4, binol) by means of 13 C CP-MAS NMR (cross-polarizationmagic angle spinning NMR).

Results and Discussion
One intriguing notion is whether enantiomers are able to display observable spectroscopic differences under isotropic conditions (in addition to parity violation effects [44]): in dilute solution (considering the typical concentrations used for solution-state NMR), the presumption is that the analyte molecules are well separated and effectively prevented from intimate interaction with each other by solvation.is is obviously not the case in the solid state but, thus far at least, there are no reports of such observations.Applying the same rationalization for an isolated pair of chiral molecules (considering them equivalent in energy) to a chiral molecule in a matrix of identical molecules, then the presumption would be that this system is equivalent to its mirror-image molecule also surrounded by identical molecules.Distinctions between holemates (i.e., enantiopure samples) and their equimates (i.e., samples consisting of 1 : 1 ratios of the two enantiomers) have been reported in nonisotropic phases [13,18,[45][46][47], and distinctions have even been made on the order-dependent parameters for cases where the holmate and the bimate (i.e., the "racemate") exhibit identical isotropic chemical shis and anisotropic values [48,49].ese differences can even permit quantitation of the ec for scalemates in favorable cases in either of the aforementioned situations.However, these observations are attributed to the different space groups that these particular enantiomers and their corresponding equimates belong to [50,51] in the former case, or in the latter case, due to the differing molecular symmetries and packings for the molecules in the holematic and bimatic states [48,49].ese results have been suitably reviewed [50,51].Given the lack of differential observations between the spectra of two enantiomers in the con�nes of the solid state leads either to the conclusion that there is no discernible effect or that the same net effect is resultant for both enantiomers-and also the equimate in cases where the space groups are the same or the compound behaves as a homomate (conglomerate) or unimate (pseudoracemate).Alternatively, perturbation of the chiral environment in the solid state by the introduction of a CS could also lead to enantiodifferentiation. is approach does not seem to have been considered previously, though NMR examination of the enclathration in the solid phase of an enantiomer by a chiral medium has been reported [52] and reviewed [51].us, whilst noncovalent interactions for the creation of a chiral environment have been useful for the analysis of enantiomers [13][14][15][16][17][18][19]28], for example, CSAs have been in use for some time now and have been subsequently followed by CLCs [51], the next logical step is chiral solid solutions (CSSs).Of note, in a weak chiral environment (induced by itself nonetheless), 1 readily displays chiralstate dependent differential behavior and this led us to the premise that perhaps not just in solution-state NMR, but also in solid-state NMR similar phenomena could be observed based on the analogous phenomena of SIDA (self-induced diastereomeric anisochronism) [3,53] in the former method.Herein, enantiodifferentiation by an extremely weak external CS, namely, sucrose, as the provider of a chiral environment, is examined.
To explore the aforementioned premise, binol (4) was examined by solid-state 13 C CP-MAS NMR.e two enantiomers of binol (R-4 and S -4, see Figure 2) each crystallizes in the P3 2 space group whilst the bimate (i.e., a "racemic compound, " bim-4) crystallizes in the Iba2 space group [54].Four samples consisting of holemic samples of each of the two enantiomers (R-4 and S-4), an equimate sample as a mechanical mixture of the two (R-4 + S-4) prepared by the addition and thorough solid-phase mixing of equal amounts of R-4 and S-4 using a vortex mixer for 10-15 minutes, and an equimate sample prepared as the bimate (bim-4) by crystallization of an equimatic sample from chloroform were examined.Although solid-phase admixture of the enantiomers R-4 and S-4 has been reported [54] to rapidly yield the bimate, morphing was not apparent in this study upon mixing and the bimate was only formed aer dissolution and removal of the solvent by evaporation.e results are portrayed in Figure 3 where the spectrum, as expected, of the bimate (bim-4) is clearly distinct from the spectra of the two enantiomers R-4 and S-4 and the mechanical mixture of the two (R-4 + S-4).However, although there are slight discernible differences between each of the enantiomers and the mechanical mixture in the �gure shown, for example, the intensity of the pairs of signals at ca. 112 and 154 ppm and the signal at ca. 134 ppm, these apparent distinctions are blurred by the run-to-run inconsistencies of the acquired spectra and thus cannot by unequivocally attributed to the inherent differences between the two enantiomers R-4 and S-4 and the mechanical mixture R-4 + S-4.To con�rm that the differences were not due to sample inconsistencies, the samples were also examined by solution-state NMR, whereby the spectra of R-4 and S-4 were found to be, to all intents and purposes, identical by 1 H and 13 C NMR.
To perturb the intrinsic chiral environment and thereby create discernible differences, an enhanced chiral environment was induced in the samples by the admixture of a CS, and for this purpose sucrose was used.Physical intimacy between the hydrophobic and hydrophilic compounds was ensured by the use of �nely powdered samples� in the case of sucrose, consumer-grade crystals were ground in a mortar and pestle prior to admixture whilst the binol (4) was used as received.Following weighing and admixture, thorough dispersion and intimate contact between the crystals was ensured by several minutes of vortex mixing.Several samples were again prepared: the two enantiomers (R-4 and S-4, each ca. 1 : 1 with the CS and two further samples of R-4 in a 1.19 : 1 ratio and a 0.55 : 1 ratio), the equimate as a mechanical mixture (R-4 + S-4, ca. 1 : 1 with the CS), and the bimate (bim-4, ca. 1 : 1 with the CS).Again, although in particular comparisons discernible differences were apparent in the 13 C solid-state NMR, the run-to-run inconsistencies of the acquired spectra again prevented any �rm conviction of a distinction between either of the enantiomers (R-4 and S-4) and the mechanical mixture of the two (R-4 + S-4).Indeed, for all directly comparable spectra, reliable distinctions between the samples consisting solely of 4 (R-4, S-4, R-4 + S-4, and bim-4) and the admixture of each sample with sucrose were not forthcoming.Needless to say, variation of the enantiomer to sugar ratio failed to realize any signi�cant perturbation.
To accentuate the perturbations of the chiral environment, each of the binol (4) admixtures with sucrose was taken up in DMSO and the resulting solutions then reduced to dryness under high vacuum.ese cocondensed samples were then subsequently again examined by 13 C CP-MAS NMR.For the  enantiomer (R-4) with sucrose, new distinctions were not apparent above that of the run-to-run inconsistencies.However, most intriguingly, upon close examination of the S-enantiomer (S-4) with sucrose, the spectrum of the dried sample was distinct, but this was only evident aer the course of several weeks (Figure 4).e main distinctions were for the pairs of signals at ca. 112 ppm and ca.154 ppm wherein both pairs were substantially increased in intensity, similarly also for the signal at 134 ppm.e two peaks at 127 ppm were altered such that a reversal of signal intensity relative between the two occurred.e freshly dried sample also showed these distinctions, but the perturbations were only very slight and were intermediate between the physically mixed-only sample of S-enantiomer (S-4) with sucrose and the cocondensed aged sample.It is of considerable note that the differences displayed by the aged S-enantiomer (S-4) with sucrose sample appear to exceed the run-to-run inconsistencies and that these distinctions are thus attributable to the induced chiral environment provided by the CS.In principle, therefore, this method could provide a means for enantiomer identi�cation or even determination of ec with appropriate data processing (e.g., deconvolution of signals).It must be noted though that cocondensation will form the bimate if the R-enantiomer (R-4) is present, so cocondensation is limited to homomates and unimates and not applicable to systems that display heteromatic behavior such as bimates-as is the case here.Moreover, this time-dependent change in the spectra was not apparent for the physically mixed-only sample of the Senantiomer (S-4) with sucrose.Also, clearly distinct changes were not evident over the same course of time for any of the R-enantiomer (R-4) with sucrose samples.And �nally, differences in the spectra of the CS sucrose were not in evidence.
It should be noted that although CP is not conducive to quantitative studies, in this work, we are not comparing atoms in different molecules nor are we comparing different atoms within the one molecule, but rather the same atoms under different chiral conditions.is has been shown to be a valid assumption [46].us, by maintaining constant CP conditions across the samples, valid comparisons were ensured.

Conclusions
us, this as yet unexplored avenue of examination could potentially yield a general and easy-to-implement solution for chiral analysis.e obvious appeals of this approach are its simplicity, the ease of sample recovery (in this particular case, only liquid-liquid extraction), the circumvention of speci�c interactions, and the dispensing of any special technique for sample preparation or spectral interpretation.us, whilst the power of CLCs at present is unparalleled, the technical and interpretative challenges limit the chance of a general solution being easily found.With further work it is hoped that development of this approach could be advanced to the stage where enantiomeric identi�cation and the quanti�cation of enantiomeric excess can be accomplished, not only in speci�c cases, but as a more general solution.

Experimental
13 C CP-MAS NMR spectra were acquired without lock at ambient temperature in a �eld of 9.4 T (100 MHz) using 4 mm ZrO 2 rotors spun at 9 kHz with a contact time of 16 ms and postacquisition delay of 300 s.A long contact time was utilized to facilitate observation of the quaternary carbons via long-range couplings; thus, a compromise value was applied between the one-bond and multiple-bond couplings with this compromise value being determined in practice from a short series of measurements using optimized conditions to locate an appropriately sufficient value.e 90 ∘ pulse for 1 H in the irradiation channel (3.90 s) was found by acquisition of a series of 13 C CP-MAS NMR spectra on a standard adamantane sample and locating the optimal value at a set power level.is same power level was used for the Hartman-Hahn condition where CP was effected by continuous wave irradiation of both the 1 H and 13 C nuclei.e power level for the Hartman-Hahn condition on 13 C was found by varying the power level applied to the 13 C nuclei for a standard adamantane sample at a set contact time (5 ms) and locating the optimal value for maximal signal response.Power levels amounted to −3.5 and 0.32 dB for the 1 H and 13 C nuclei, respectively, utilizing 100 and 300 Watt ampli�ers, respectively.Typically, a spectrum was acquired for 12 h consisting of 146 scans. 1 k data points were collected for each FID (acquisition time = 6.4 ms) which were processed by zero-�lling to 2 k and applying a line broadening of 100 Hz.Selected samples were also rerun to gauge run-to-run aberrations, either sequentially for the same sample insertion, at different sessions for the same sample, or by repacking the sample rotor with the same material.For comparative purposes, spectra were vertically scaled and shied horizontally.Solution-state spectra were acquired as previously described [55,56].

F 4 :
e 13 C solid-state NMR spectra of the S-enantiomer of binol (S-4): an aged sample aer cocondensation with sucrose from DSMO solution (trace a), a freshly dried sample aer cocondensation (trace b), a physically mixed-only sample of S-enantiomer (S-4) and sucrose, ca. 1 : 1 (trace c), and the pure S-enantiomer (S-4) (trace d).