Renewable Resources for Enantiodiscrimination: Chiral Solvating Agents for NMR Spectroscopy from Isomannide and Isosorbide

A new family of chiral selectors was synthesized in a single synthetic step with yields up to 84% starting from isomannide and isosorbide. Mono- or disubstituted carbamate derivatives were obtained by reacting the isohexides with electron-donating arylisocyanate (3,5-dimethylphenyl- or 3,5-dimethoxyphenyl-) and electron-withdrawing arylisocyanate (3,5-bis(trifluoromethyl)phenyl-) groups to test opposite electronic effects on enantiodifferentiation. Deeper chiral pockets and derivatives with more acidic protons were obtained by derivatization with 1-naphthylisocyanate and p-toluenesulfonylisocyanate, respectively. All compounds were tested as chiral solvating agents (CSAs) in 1H NMR experiments with rac-N-3,5-dinitrobenzoylphenylglycine methyl ester in order to determine the influence of different structural features on the enantiodiscrimination capabilities. Some selected compounds were tested with other racemic analytes, still leading to enantiodiscrimination. The enantiodiscrimination conditions were then optimized for the best CSA/analyte couple. Finally, a 2D- and 1D-NMR study was performed employing the best performing CSA with the two enantiomers of the selected analyte, aiming to determine the enantiodiscrimination mechanism, the stoichiometry of interaction, and the complexation constant.


■ INTRODUCTION
Chirality plays a crucial role in medicinal, biological, and synthetic chemistry. Since most of the active pharmaceutical ingredients (APIs) are optically active molecules, there is a growing need for simple, fast, easy, and robust methods for determining the purity of scalemic mixtures. To this aim, the main analytical methods of interest are chiral chromatography (chiral gas chromatography 1 or chiral high-performance liquid chromatography 2,3 ), chiral electrophoresis, 4,5 and chiral spectroscopies. 6 Among these latter, NMR spectroscopy, a reliable routine technique, has received much attention. 7,8 The main strategies to determine the enantiomeric composition via NMR are the use of chiral derivatizing agents (CDAs), chiral solvating agents (CSAs), chiral lanthanide shift reagents (CLSRs), or chiral liquid crystals (CLCs). 9−12 In particular, CSAs are interesting compounds: they are simply added to an analyte solution without the need for timeconsuming derivatization steps, and the enantiomeric composition of the chiral compound can be directly determined from a 1 H NMR spectrum.
Enantiodifferentation relies on secondary interactions, such as ion pairing, π−π, Coulombic and hydrogen bond interactions: diastereomeric adducts are formed in situ, by means of a fast (on the NMR time-scale) complexation equilibrium, and hence, the final measured spectrum is a timeaverage of the bound and unbound form of the substrate. Therefore, if the two enantiomers of the analyte are characterized by different association constants, an additional differentiation of the chemical shift could occur, even though thermodynamic differentiation is not necessary to observe enantiodiscrimination in NMR. Furthermore, given that no covalent derivatization is employed, in principle analytes can be recovered at the end of the analysis. This aspect is of primary importance when difficult-to-access costly compounds are analyzed. 12 A common strategy for the synthesis of new CSAs relies on the use of simple and easy to functionalize chiral platforms. 7,13−22 These compounds are easily derivatized, and their enantiodiscrimination properties are modulated by the introduction of suitable functional groups. Rigid structures have proven to be suitable in enhancing the selectivity toward particular analytes, 18,21 while more flexible structures have been employed to enhance CSA versatility. Usually, the observed chemical-shift differentiation derives from the anisotropy of aromatic groups present in the structure of chiral agents. In fact a tweezer-like bis-thiourea derivative, which possesses the above-discussed structural features, has been successfully used as a CSA. 13 Starting from these considerations, we reasoned that a fast and easy way to obtain new CSAs could be represented by the use of isohexides. Isohexides, namely (3R,3aR,6R,6aR)hexahydrofuro [3,2-b]furan-3,6-diol and (3R,3aR,6S,6aR)hexahydrofuro [3,2-b]furan-3,6-diol respectively known as isomannide 1 and isosorbide 2 (Figure 1), are byproducts of the starch industry, arising from dehydration of D-mannitol and D-sorbitol. 23 These commercially available starting materials provide an easy and inexpensive access to optically pure functionalized compounds. Indeed, through a simple derivatization of the two hydroxyl groups, the characteristic chiral cavity of their scaffold ( Figure 1) can be functionalized, thus leading to new compounds, whose properties depend not only on the nature of the introduced moieties but also on the different stereochemistry of the native hydroxyl groups. Due to these characteristics, isohexides were successfully employed as starting materials in the preparation of chiral ligands, 24 organocatalysts, 25 and chiral ionic liquids. 26,27 In particular, starting from isomannide, bidentate ligands 24,28−30 and ionic molecular tweezers 27,31 were obtained by virtue of the endo arrangement of the hydroxyl groups, which allows the appended units to be sufficiently close to each other. However, the interaction of two appended moieties was even observed in some isosorbide derivatives, due to their particular spatial arrangement. 31 In our previous works, we demonstrated that some isohexide derivatives could be successfully employed as CSAs in NMR studies. 32,33 These positive preliminary results prompted us to expand the scope, synthesizing new isohexide derivatives to be employed in NMR enantiodiscrimination studies ( Figure 2). In particular, derivatization of the hydroxyl groups as arylcarbamates was chosen to obtain new chiral shift agents that could establish multiple intermolecular interactions, such as π−π interactions through the aromatic groups and dipole−dipole interactions and/or hydrogen bond interactions through the carbamoyl group. To study the influence of different parameters on the enantiorecognition process, such as isohexide stereochemistry and nature, number, and position of the derivatizing moieties on the chiral scaffold, a family of mono-and dicarbamates was easily synthesized from parent isomannide and isosorbide. Arylcarbamoyloxy derivatives, containing respectively electron-donating groups (3,5-dimethyl-, 3a−7a, or 3,5-dimethoxy-, 3d−7d) or electron-withdrawing groups (3,5-bis(trifluoromethyl)-, 3c−7c) were selected to test the influence of opposite electronic effects on enantiodifferentiation. 1-Naphthylcarbamoyloxy derivatives 3b−7b were synthesized to obtain a deeper chiral pocket, and p-toluenesulfonylcarbamoyloxy derivatives 3e−7e were endowed with more acidic protons ( Figure 2). All products were fully characterized (Figures S1−S55, Supporting Information), and their enantiodiscrimination ability was studied by 1 H NMR spectroscopy.
■ RESULTS AND DISCUSSION Synthesis of Compounds 3−7. The synthesis of compounds 3−7 was performed following a general protocol: isomannide 1 or isosorbide 2 was reacted with an arylisocyanate 8 employing a catalytic amount of dimethylamino-  The Journal of Organic Chemistry pubs.acs.org/joc Article pyridine (DMAP) in dry tetrahydrofuran as the solvent (Scheme 1). In order to obtain mono-or disubstituted derivatives, a different stoichiometry was employed. For the synthesis of compounds 4 and 7, an excess of aryl-isocyanate was added, while to enhance the selectivity toward the monoderivatization a 3-fold excess of the reacting isohexide 1 or 2 was used. In both cases, DMAP was employed as the catalyst, except for compounds 3e, 5e, and 6e. While monoderivatization of symmetric isomannide 1 led to one product, reaction of isosorbide 2 afforded a mixture of the two possible isomers 5 and 6 in an ∼1:1 ratio. The nonselective monoderivatization of isosorbide was not an issue, since we were interested in both isomers; a selective derivatization could be easily obtained exploiting an initial selective protection as reported in the literature. 32,34 In all cases, pure compounds were obtained in good yields after chromatographic purification, with the only exception of 4a−d, 7a−c that crystallized from the crude and/or were recrystallized after a simple workup. Enantiodiscrimination Tests. Compounds 3−7 were tested for their enantiodiscriminating properties toward selected racemic substrates ( Figure 3) in 1 H NMR experiments. In the initial part of the work we focused on the enantiodiscrimination of amino acid derivatives. Initially, rac-N-3,5-dinitrobenzoylphenylglycine methyl ester (3,9) was used to test the different chiral auxiliaries, as the 3,5-DNB aromatic moiety can establish π−π interactions with the CSAs, leading to an enhancement in enantiodifferentiation. Furthermore, as already reported, 13 the introduction of a 3,5-DNB moiety allows for having some diagnostic signals to study enantiodiscrimination phenomena, since its protons resonate in a spectral region free from CSA signals ( Figure S62, Supporting Information).
Enantiodiscrimination tests were performed by adding 1 equiv of CSA (3-7) to a 30 mM solution of 9 in CDCl 3 as the solvent. Splitting of selected NMR signals was employed as a measure of enantiodiscrimination magnitude (Table 1).
Regarding the signal shift, a common trend could be observed for all the CSAs, showing a low-frequency shift for the p-DNB (up to 0.098 ppm) proton and a high-frequency shift for the −NH proton of 9 (up to 0.252 ppm) (Figures S63−64, S66−67, and S69, Supporting Information). This evidence suggests an interaction between the aromatic groups of CSAs and the electron-poor phenyl ring of 9, with the p-DNB proton in the shielding cone of CSAs. It is also possible to assume −NH as one of the major sites of intermolecular interaction, as suggested by the high shifts always observed.
To assess if the higher nonequivalence observed for 7c derives from a cooperative action of the two aromatic groups, 1:2 mixtures of 9 and monoderivatives 5c and 6c (Table 2, entries 2 and 4) were analyzed. Doubling the concentration of monoderivative 5c did not give any enantiodiscrimination (entry 2). Conversely, better enantiodifferentiation could be observed for 6c, but the nonequivalence values were lower than those obtained with compound 7c (compare entries 4−5). These results suggest the cooperative effect of the two substituents in 7c (Table 2).
It is to note that the nonequivalences measured for protons of 9 in the presence of 7c were comparable 13,35,36 or even higher 37−39 than those reported in the literature for the same analyte.
Compound 7c was then tested with other racemic analytes (10−12). Nonequivalences were detected only for proton signals of 10, while 2,2,2-trifluoro-1-(9-anthryl)ethanol 11 or α-methylbenzylamine 12 was not discriminated (Figures S72− S77, Supporting Information). However, ΔΔδ values measured for the proton signals of derivative 10, the electron-rich analogue of 9, were lower than those recorded for the protons of 9 (Figure 4 and Figure S73, Supporting Information), so confirming that the π−π interactions between electronically complementary aromatic rings play a minor if not negligible role in the enantiorecognition process.
Derivatives 3e−7e, possessing a more acidic carbamoyl proton, were tested with amine 12 (Table 3). For this substrate, the signals of the proton of the stereocenter and the methyl protons were chosen as diagnostic, since they resonate in sufficiently free spectral regions. In particular, nonequivalence of the signals of the methyl protons was always observed, while the signal of the methine proton was split only employing compounds 4e, 6e, 7e (entries 5−10, Table 3). In all cases, a strong interaction between CSAs and the amine can be inferred on the basis of the large shift of the signals ( Figure  S78−S82, Supporting Information). Compound 4e was the best CSA for this substrate, leading to good nonequivalences for diagnostic signals. The use of a higher amount of CSAs did not lead to significantly better results, as shown in Table 3. Optimization of enantiodiscrimination tests was performed, aimed at determining the best conditions for the enantiorecognition study, on the best CSA-analyte couple (7c and 9). To this aim 1:1 mixtures of 7c and 9 at higher concentrations (45 and 60 mM) and 5 mM solutions of 9 containing different amounts of 7c were analyzed (Table 4, Figure S83−S84 Supporting Information).
The nonequivalence undergoes a significant increase in the range 5 mM to 30 mM, whereas only small changes are observed further increasing the concentration up to 60 mM (Table 4, Figure S84, Supporting Information).
Interestingly, for a 5 mM substrate concentration, in the presence of 6 equiv of CSA a 4-fold increase of enantiomer differentiation was obtained (Table 4, Figure S83, Supporting Information). The best results were obtained working with a 60 mM or a 45 mM equimolar solution of 9 and 7c (Table 4, entry 3 and entry 2). In this case, the signal of −NH proton could be clearly detected, making these two conditions both suitable for enantiodifferantion mechanism studies ( Figure  S84, Supporting Information). The choice fell on 45 mM solutions to avoid the risk of observing precipitation of the analytes over time and to use a slightly lower amount of the prepared CSA.
Finally, additional experiments have been carried out to compare the enantiomeric ratio (er) in 7c/9 mixtures (nominal enantiomeric ratio (R)-9/(S)-9 79:21 and 98.5:1.5), on the basis of NMR spectroscopy and chiral chromatography determinations. The results from the two techniques were in very good agreement (Figures S88−S90).
NMR Characterization of CSA 7c. In order to analyze the enantiodiscrimination mechanism between CSA 7c and compound 9, complete characterization of CSA 7c was needed. To this aim, 1D and 2D NMR experiments were performed, in CDCl 3 at 45 mM concentration. Discussion of homo-and heterocorrelations detected in COSY, ROESY, and HSQC spectra is reported in the Supporting Information.  Characterization data are collected in Table S1, Supporting Information and reported in Figure 5.
Considering the conformation of CSA 7c in solution, the two 3,5-bis(trifluoromethyl)phenyl moieties bound to the rigid core did not show any particular conformational prevalence: as an example, the magnitude of ROE effects given by the NH-7 proton at the frequencies of H 5 , H 1 , H 6a , and H 4 were comparable ( Figure S86b, Supporting Information), indicating that the 3,5-bis(trifluoromethyl)phenyl moiety bound to the C 5 site, named Ar-5, is freely rotating around the C 5 −O bond. Analogously, the NH-10 proton gave dipolar interactions with protons H 1 , H 2 , and H 3 ( Figure S86b, Supporting Information). These last effects were once again comparable in magnitudes; therefore, the 3,5-bis(trifluoromethyl)phenyl moiety bound to the C 2 carbon, named Ar-2, is itself freely rotating around the C 2 −O bond.
Compound (R)-9 showed a strong preference for the interaction at the 3,5-bis(trifluoromethyl)phenyl moiety Ar-2. In fact, a remarkably high complexation shift of +0.444 ppm was measured for the proton NH-10, as well as very high complexation shifts were measured for the protons H 1 and H 6 (Table 5, Figure 6). Interestingly, negligible complexation shifts were measured for the protons H 11 /H 8 of the 3,5bis(trifluoromethyl)phenyl group (Table 5). Therefore, the intermolecular adduct is mainly stabilized by a strong network of hydrogen-bond interactions involving the hydrogen-bond donor group NH-10 of the CSA and, reasonably, the electronacceptor oxygen atoms of its rigid isosorbide skeleton. Every proton of (R)-9 showed relevant complexation shifts in the presence of the CSA, with higher values for its NH proton ( Table 6). Even if a strong preference for the interaction at the NH-10 moiety of Ar-2 can be assessed, the interaction must also involve moiety Ar-5, although to a minor extent, as witnessed by the complexation shifts measured for NH-7 (+0.123 ppm), H 6a (Δδ = −0.135 ppm), and H 5 (Δδ = −0.069 ppm). The very high shift value of −0.260 ppm measured for H 6 probably comes from the contribution of the interaction both at the NH-10 and NH-7, which likely entails closeness of the aromatic moiety of (R)-9 to the isosorbide skeleton causing shielding of proton H 6 .
The interaction of CSA 7c with (S)-9 involves once again both the NH-7 and NH-10 moieties, with a slight preference for NH-7 (Table 5). Only NH proton of (S)-9 showed  ΔΔδ = |Δδ R − Δδ S | where Δδ R = δ R mixture − δ free and Δδ S = δ S mixture − δ free , being δ R mixture and δ S mixture the chemical shifts of the two enantiomers in the presence of the CSA. b Para-proton of the 3,5-DNB moiety. c Ortho-protons of the 3,5-DNB moiety. d Methine proton of the stereocenter. e Signal not detected due to superimposition with the resonance of aromatic protons.
The Journal of Organic Chemistry pubs.acs.org/joc Article significant complexation shift (Table 6). Therefore, it can be concluded that the acidic NH group of moiety Ar-5 is mainly involved in nonselective interactions with both enantiomers, whereas the enantiodiscrimination mainly originates from the strong preference of NH-10 for (R)-9. The above conclusions were supported also by the nature of proximity constraints arising from intermolecular ROE effects detected in the two mixtures. In particular, relevant inter-ROEs were found between the ortho protons of (R)-9 and proton H 1 , H 6a , NH-7, and NH-10 ( Figure 7b). The ROE effect at H 11 was more intense than it was at H 8 , confirming that the 3,5dinitrophenyl group of the substrate mainly lies on the rigid skeleton of the CSA with a preference for the interaction at NH-10 of Ar-2.
Analogous but weaker and less selective intermolecular ROE effects were detected in the mixture (S)-9/7c, indicating a minor preference for the interaction at NH-10 of Ar-2 ( Figure  7a).

■ CONCLUSIONS
A new family of chiral solvating agents (CSAs) 3−7 was easily synthesized starting from isomannide and isosorbide. Following the same protocol, by a single synthetic step new monoand disubstituted carbamates were easily obtained and purified. Different phenyl isocyanates were selected to test different aspects, such as the influence of opposite electronic effects, the elongation of the arms of the chiral clamp, and the influence of the acidity of carbamic −NH on the enantiodiscrimination.
All the prepared CSAs were tested employing rac-N-3,5dinitrobenzoylphenylglycine methyl ester 9 as a representative analyte. The results clearly showed that the chiral structure of isohexides is well suitable for building-up chiral auxiliaries that can be successfully employed in enantiodiscrimination   The Journal of Organic Chemistry pubs.acs.org/joc Article processes. The large portfolio of derivatives allowed us to study the influence of different parameters, such as stereochemistry and degree of derivatization of the central chiral scaffold as well as structural and electronic properties of the derivatizing agent, on the enantiodiscriminating capabilities. The cooperative action of two derivatizing moieties, the interaction with the NH groups, the minor role played by π−π interaction between electronically complementary aromatic rings emerged as peculiar characteristics of the enantiorecognition and the best nonequivalences were obtained with derivative 7c containing two electron-poor 3,5-bis(trifluoromethyl)phenylcarbamoyloxy groups.
The study of the enantiodiscrimination mechanism allowed us to establish that (R)-9 showed a stronger interaction with CSA 7c than its enantiomer, with a strong preference for the interaction with one of the 3,5-bis(trifluoromethyl)phenyl moieties of the CSA and with the intermolecular adduct being mainly stabilized by a strong network of hydrogen bonds interactions. In particular, enantiodiscrimination mainly originated from the NH-10 preference for (R)-9.
■ EXPERIMENTAL SECTION Materials and General Methods. All the reactions involving sensitive compounds were carried out under dry Ar, in flame-dried glassware. If not noted otherwise, reactants and reagents were commercially available and used as received from TCI-Chemicals and Sigma-Aldrich. TLC analyses were carried out with Merk 60 F254 plates (0.2 mm). 1 H NMR spectra were recorded in Chloroform-d, Acetone-d 6 , Methanol-d 4 , and DMSO-d 6 on a JEOL ECZ400S or JEOL ECZ500R spectrometer. The following abbreviations are used: s = singlet, bd = broad singlet, d = doublet, dd = double doublet, ddd = double double doublet, dt = double triplet, t = triplet, td = triple doublet, tdd = triple double doublet, q = quartet, h = heptet m = multiplet. 13 C NMR spectra were recorded at 101 MHz on a JEOL ECZ400S or at 126 MHz on a JEOL ECZ500R spectrometer. 19 F spectra were recorded at 471 MHz on a JEOL ECZ500R spectrometer. 1 H and 13 C NMR chemical shifts (ppm) are referred to TMS as the external standard. Melting points were measured on a Reichert Thermovar Type 300429 Microscope. Optical rotations were measured in 1 dm cells at the sodium D line, using an Anton Paar MCP 300 Polarimeter. NMR characterization of compound 7c and the study of the interaction mechanism were performed on an INOVA600 spectrometer operating at 600 MHz for 1 H nuclei. The samples were analyzed in CDCl 3 solution; the temperature was controlled (25°C). For all the 2D NMR spectra the spectral width used was the minimum required in both dimensions. The gCOSY (gradient COrrelation SpectroscopY) map was recorded by using a relaxation delay of 1 s, 128 increments of 8 transients, each with 2K points. The 2D-ROESY (Rotating-frame Overhauser Enhancement SpectroscopY) maps were recorded by using a relaxation time of 3 s and a mixing time of 0.5 s; 128 increments of 16 transients of 2Kpoints each were collected. The 1D-ROESY spectra were recorded using a selective inversion pulse with 1024 transients, a relaxation delay of 1 s, and a mixing time of 0.5 s. The gHSQC (gradient Heteronuclear Single Quantum Coherence) map was recorded with a relaxation time of 1.2 s, 128 increments with 32 transients, each of 2K-points. Elemental analyses were obtained using an Elementar Vario MICRO cube equipment. Isomannide (1), isosorbide (2), aryl isocyanates (8), rac-propylene oxide, 3,5-dinitrobenzoyl chloride, 3,5-dimethoxybenzoyl chloride, and diazabiciclo [5,4,0]undec-7-en (DBU) were purchased from Merck and used as received.