Chiral Recognition of Chiral (Hetero)Cyclic Derivatives Probed by Tetraaza Macrocyclic Chiral Solvating Agents via ¹H NMR Spectroscopy

Abstract

In the field of chiral recognition, chiral cyclic organic compounds, especially heterocyclic organic compounds, have attracted little attention and have been rarely studied as chiral substrates by means of ¹H NMR spectroscopy. In this paper, enantiomers of thiohydantoin derivatives, representing typical five-membered N,N-heterocycles, have been synthesized and utilized for assignment of absolute configuration and analysis of enantiomeric excess. All enantiomers have been successfully differentiated with the assistance of novel tetraaza macrocyclic chiral solvating agents (TAMCSAs) by ¹H NMR spectroscopy. Surprisingly, unprecedented nonequivalent chemical shift values (up to 2.052 ppm) of the NH proton of substrates have been observed, a new milestone in the evaluation of enantiomers. To better understand the intermolecular interactions between host and guest, Job plots and theoretical calculations of (S)-G1 and (R)-G1 with TAMCSA 1a were investigated and revealed significant geometric differentiation between the diastereomers. In order to evaluate practical applications of the present systems in analyzing optical purity of chiral substrates, enantiomeric excesses of a typical substrate (G1) with different optical compositions in the presence of a representative TAMCSA (1a) can be accurately calculated based on the integration of the NH proton’s signal peaks. Importantly, this work provides a significant breakthrough in exploring and developing the chiral recognition of chiral heterocyclic organic compounds by ¹H NMR spectroscopy.

Graphical Abstract

Nuclear magnetic resonance (NMR) spectroscopy plays an increasingly important role as a fast and powerful analytical tool in the field of chiral recognition, including the assignment of absolute configuration of chiral molecules and determination of enantiomeric excess of chiral compounds.^1–5 These parameters constitute important and fundamental characterization data in many related research areas, such as in the evaluation of asymmetric synthetic^6,7 and natural products,^8,9 as well as in fields such as food science^10–12 and chiral materials.^13,14 In particular, in chiral pharmaceutical chemistry, different enantiomers of chiral drugs have different biological and pharmaceutical activities, with one producing beneficial outcomes to combat human diseases, whereas the other one may have a detrimental, if not toxic effect to health.^15–17 Over the past decade, NMR spectroscopy, assisted by the use of chiral oriented solvents,^18,19 and especially chiral solvating agents (CSAs) as chiral auxiliaries, has received considerable attention in chiral recognition^20–23 due to several advantages, such as fast and convenient operation, accurate and reliable measurement, low sample consumption, and need for small amounts of deuterated solvent. In addition, multiple field windows are often available for observing split proton signals by ¹H NMR spectroscopy, an ubiquitous technique available in nearly all chemical and related laboratories.^24,25 However, developing highly sensitive, effective, and versatile chiral auxiliaries, especially CSAs, remains a challenging undertaking. In reported work, a few CSAs, such as chiral amines,²⁶ carboxylic acids,²⁷ amides,^28,29 crown ethers,^30,31 calixirenes,³² and others,³³ have been developed and utilized for this purpose with the assistance of ¹H NMR spectroscopy. However, CSAs with outstanding features are still a scarce commodity. Herein, we report tetraaza macrocyclic chiral solvating agents (TAMCSAs) with unique structures containing two amide groups (CONHs), two phenolic hydroxyl groups (PhOHs), and two amino groups (NHs), along with an overall C₂-symmetry and a well-defined cavity, to further explore their chiral discriminating capability for a wide range of chiral substrates by means of ¹H NMR spectroscopy (Figure 1).

Figure 1.

Structures of TAMCSAs 1a–1d.

In our previous work, the potential chiral discriminating capability of a few TAMCSAs has been successfully evaluated by chiral recognition of α-hydroxy acids and N-Ts-α-amino acids with one chiral center,³⁴ dipeptide derivatives with two centers,³⁵ tripeptide derivatives with three centers,³⁶ and chiral ester derivatives.³⁷ These results show that TAMCSAs are highly sensitive, effective, and versatile CSAs.

In reported work, several chiral chain organic compounds have been often used as chiral substrates for the determination of the absolute configuration and evaluation of enantiomeric excess, including chiral amines,^38,39 alcohols,^39–41 amino alcohols,^39,42 carboxylic acids,^43,44 and amino acids or their derivatives.⁴⁵ On the other hand, chiral cyclic organic compounds have been scarcely investigated as chiral substrates by means of ¹H NMR spectroscopy.⁴⁶ Yet, single enantiomers of chiral cyclic organic compounds can have high biological and pharmaceutical activity and can be used as clinical chiral drugs, as well as in synthetic/catalytic applications. For example, the following thiohydantoin derivatives have been used as antiprostate cancer agents (Figure 2a)⁴⁷ and as chiral catalysts for iodoamination of alkenes (Figure 2b).⁴⁸

Figure 2.

Structures of antiprostate cancer agent (a) and chiral alkene iodoamination catalyst (b).

In this paper, enantiomers of chiral thiohydantoin derivatives, as a representative class of five-membered N,N-heterocyclic organic compounds, were prepared and used as chiral substrates for chiral recognition in the presence of TAMCSAs by ¹H NMR spectroscopy.

EXPERIMENTAL SECTION

Synthesis of TAMCSAs 1a–1d.

Chiral diimines 3a–3d (Figure 3a) were prepared from 1,2-(1S,2S)-(+)-cyclohexane-diamine and d-phenylglycine in three steps and 80–85% yields and were used without further purification, according to a literature protocol.⁴⁹ TAMCSAs 1a–1d were synthesized by the intramolecular reductive C–C coupling reaction of 3a–3d at −18–0 °C in a dried DMF dilute suspension under the nitrogen atmosphere^50,51 and were further purified by column chromatography on silica gel to afford enantiopure compounds in 13–25% yields. Meanwhile, chiral compounds 2a–2d (Figure 3b) were obtained as byproducts in 11–15% yields. The detailed synthetic routes and procedures are available in the Supporting Information (page S-3).

Figure 3.

Structures of chiral compounds 2a−2d and 3a−3d.

TAMCSAs 1a–1c and chiral compounds 2a–2c were characterized as new compounds by ¹H NMR, ¹³C NMR, HRMS, and IR methods with the exception of TAMCSA 1d and 2d, which have been previously reported.³⁴ Crystals of TAMCSA 1c suitable for single-crystal X-ray diffraction were obtained through slow evaporation of acetone solution at ambient temperature. Absolute configurations of the newly generated chiral carbon atoms (C15 and C23) of the ArCH*NH groups of TAMCSA 1c were assigned to possess S, S chirality based on X-ray crystallographic analysis (Figure 4).

Figure 4.

X-ray crystal structure of TAMCSA 1c·2·(CH₃)₂CO drawn at 30% probability thermal ellipsoids.

Unfortunately, crystals of TAMCSAs 1a and 1b suitable for X-ray diffraction analysis were not obtained. Therefore, their NOESY spectra were examined for assignment of the absolute configuration of the two newly produced chiral carbon atoms of the ArCH*NH groups. The hydrogen atoms (ArCH*NH) of TAMCSAs 1a and 1b show strong NOESY correlated ¹H NMR signals with the two CH protons (α-H) of the d-phenylglycine moieties. This result suggests that two pairs of closely spaced hydrogen atoms of TAMCSAs 1a and 1b (α-Hs and ArCH*NHs, 3R and 5S, and 8R and 6S) are located on the same side of the macrocyclic framework (Figure 1). Based on these NOESY spectra, the absolute configurations of the newly generated chiral carbon atoms (ArCH*NH) of TAMCSAs 1a and 1b have been assigned as S, S. The structural characterization data and spectra are available in the Supporting Information (pages S-3–S-4 and S-21–S-30).

Synthesis of Enantiomers of Thiohydantion Derivatives G1–G13.

Enantiomers of thiohydantoin derivatives G1–G7 (R¹ = H) were prepared from d- and l-α-amino acids with phenyl isothiocyanate according to the reported synthetic procedure in 50–80% yields.⁵² In order to compare the electronic effects of different substituent groups of the chiral thiohydantoin derivatives on chiral discrimination, the enantiomers of G8–G13 (R¹ = CF₃) were prepared from 3,5-bis(trifluoromethyl)phenyl isothiocyanate with d- and l-α-amino acids, containing the same amino acid moieties (Figure 5).

Figure 5.

Structures of the enantiomers of thiohydantoin derivatives.

Among them, the chemical structures of (S)-G8, (S)-G12, and (R)-G8–13 constitute new optically pure compounds and have been characterized by ¹H NMR, ¹³C NMR, ¹⁹F NMR, HRMS, and IR methods. Detailed synthetic routes and procedures are available in the Supporting Information (pages S-5–S-7 and S-31–S-46).

Chiral Recognition of Enantiomers of Thiohydantion Derivatives G1–G13.

¹H NMR spectra of (±)-G1–G13 were measured in the presence of TAMCSAs 1a–1d in CDCl₃ on a 400 MHz spectrometer based on the optimized chiral discriminating conditions (molar ratio of host and guest = 1:1 and a concentration of 5 mM in most cases).

RESULTS AND DISCUSSION

To explore the discrimination of enantiomers of chiral thiohydantoin derivatives and evaluate the chiral discriminating capability of TAMCSAs 1a–1d, a sample of (±)-G1 with TAMCSA 1a (molar ratio = 1:1) was prepared at a concentration of 5 mM in CDCl₃, and its ¹H NMR spectrum was measured on a 400 MHz spectrometer. The results show that an unprecedented nonequivalent chemical shift value (ΔΔδ = 1.853 ppm) was observed by means of separated ¹H NMR signals of the NH proton of (±)-G1, with a high baseline resolution. Meanwhile, ΔΔδ (0.319 ppm) of the CH proton was also exhibited. Subsequently, a sample of (S)-G1 was added to the aforementioned solution of (±)-G1 with TAMCSA 1a, and its ¹H NMR spectrum was measured under the same conditions. The absolute configuration of enantiomers of (±)-G1 were easily determined by the change of the integration of ¹H NMR signal peak areas of the split protons. Partial expanded spectra and ΔΔδ values of the split protons (NH and CH) of (±)-G1 in the presence of TAMCSA 1a are shown in Figure 6.

Figure 6.

¹H NMR spectrum and expanded spectra of (±)-G1 (a,b), and (S)-G1/(R)-G1 (1.6:1) (c,d) in the presence of TAMCSA 1a in CDCl₃ (400 MHz), [TAMCSA 1a] = 5 mM, “○ (red)” stand for (S)-G1, “● (red)” stands for (R)-G1.

Encouraged by these remarkable chiral discriminating results, we sought to obtain better chiral discriminating effects, including superior baseline resolution and more clearly separated ¹H NMR signals with fewer (if any) overlapping peaks. To this effect, chiral discriminating conditions were screened and optimized by ¹H NMR measurements involving different concentrations and molar ratios of host and guest. Based on these results, a concentration of 5 mM of guest (in most cases) and 1:1 molar ratio of host and guest are more suitable for this study. Their partial ¹H NMR spectra are available in the Supporting Information (page S-7, Figures S2 and S3).

Under optimized chiral discriminating conditions, 51 samples of (±)-G1–13 were prepared in the presence of TAMCSAs 1a–1d in CDCl₃, and their ¹H NMR spectra were measured on a 400 MHz spectrometer, with the exception of the (±)-G1 and TAMCSA 1a combination, which has been measured under the aforementioned optimized conditions [1:1 molar ratio of (±)-G1 and TAMCSA 1a, [(±)-G1] = 5 mM]. The results show that the enantiomers of all thiohydantoin derivatives were clearly differentiated by the separated ¹H NMR signals of the related protons of (±)-G1–13 in the presence of TAMCSAs 1a–1d. Different chemical shifts for multiple types of protons of enantiomers of all of the thiohydantoin derivatives were clearly exhibited. Importantly, several highly significant chiral discriminating outcomes were obtained in the ¹H NMR spectra of (±)-G1–13. Among 52 samples, ΔΔδ values (red) of the NH proton of 12 samples exceed 1.530 ppm. Most astonishingly, the maximum ΔΔδ value of the NH proton of (±)-G8 is as high as 2.052 ppm in the presence of TAMCSA 1c. In addition, ΔΔδ values (green) of the NH proton of 33 samples are between 1.024 and 1.496 ppm. The ΔΔδ values (blue) of the NH proton of seven samples range between 0.582 and 0.942 ppm. To the best of our knowledge, such large nonequivalent chemical shift values (up to 2.052 ppm) have never been previously observed in the presence of CSAs by means of ¹H NMR spectroscopy. Subsequently, samples of (S) or (R)-G1–13 were added to the aforementioned solution of (±)-G1–13 with TAMCSAs 1a–1d, and their ¹H NMR spectra were also measured on a 400 MHz spectrometer under the same conditions. The assignments of enantiomers of (±)-G1–13 were easily determined based on integration changes of the ¹H NMR signal peak areas of the corresponding split protons. The ΔΔδ values and partial spectra of the NH proton, as the most representative case (ΔΔδ ≥ 1.269 ppm, 24 samples), are summarized in Figure 7. ΔΔδ values and partial spectra of other NH protons (ΔΔδ < 1.269 ppm, 28 samples), with the exception of ΔΔδ values of other split protons of chiral thiohydantoin derivatives, are available in the Supporting Information (pages S-8–S-10, Figure 5S, Table S2).

Figure 7.

Nonequivalent chemical shift values (ΔΔδ ≥ 1.269 ppm) and partial spectra of the NH proton of (±)-G1–G4, and (±)-G8–G13 in the presence of TAMCSAs 1a–1d in CDCl₃ (400 MHz) at a concentration of 5 mM, and ^b10 mM; [○ (red)] and [● (red)] stand for (S)- and (R)-enantiomers; ΔΔδ = |Δδ_S − Δδ_R|, Δδ_S = δ_S − δ_free, Δδ_R = δ_R − δ_free. ¹H NMR spectra of all examples in this work were measured on a JEOL spectrometer at 400 MHz (type of NMR probe: broadband gradient autotune).

Moreover, remarkable changes of chemical shifts (Δδ, ppm) of the NH proton of enantiomers of (±)-G1–13 were also observed in the presence of TAMCSAs 1a–1d. Compared with chemical shift values of the NH proton of free (±)-G1–13, chemical shifts of the NH proton of the (S)-enantiomer of the aforementioned guests shifted drastically downfield (by up to 2.025 ppm, Δδ_S = δ_S – δ_free). However, chemical shifts of the NH proton of the (R)-enantiomer moved slightly downfield or upfield (Δδ_R = δ_R – δ_free) in most cases. These results suggest that the diastereomeric complexes of (±)-G1–13 with TAMCSAs 1a–1d exhibit larger geometric variances, presumably due to differential intermolecular interactions between hosts and the two enantiomers of guests. Detailed changes of chemical shift values of the NH proton of (±)-G1–13 in the presence of TAMCSAs 1a–1d are available in the Supporting Information (page S-10, Table S3).

In addition, in order to investigate the temperature effect on enantiodifferentiation, a sample of (±)-G6 with TAMCSA 1a in CDCl₃ was prepared (molar ratio = 1:1, 5 mM), and its ¹H NMR spectra were measured on a 400 MHz spectrometer from 298 to 248 K. The results show that ΔΔδ of the NH proton of (±)-G6 becomes larger, ranging from 1.074 ppm (298 K) to 1.211 ppm (258 and 253 K) as the temperature gradually decreases. However, at 248 K, the ΔΔδ value (1.208 ppm) of the NH proton of (±)-G6 starts to get smaller than that (1.211 ppm) at 253 and 258 K, because samples started precipitating, as the temperature decreased (Figure 8).

Figure 8.

Nonequivalent chemical shift values (ΔΔδ, ppm) and partial spectra of the NH proton of (±)-G6 in the presence of TAMCSA 1a at different temperatures in CDCl₃ (400 MHz). [○ (red)] and [● (red)] stand for (S)- and (R)-G6.

Encouraged by the unprecedented nonequivalent chemical shift values noted above, we were keenly interested in investigating the type of intermolecular interactions between host and guest and deducing a possible mechanism of chiral recognition. First, Job plots of (±)-G1 were obtained in the presence of TAMCSA 1a by ¹H NMR titration experiments.⁵³ A maximum value (X × Δδ_SR = 0.121 ppm, X × Δδ_S = 0.141 ppm, X × Δδ_R = 0.020 ppm) of the NH proton of (±)-G1 was exhibited at a molar fraction of X = 0.5 in the presence of TAMCSA 1a (Figure 9).

Figure 9.

Job plots for complexes of (S)-G1 and (R)-G1 with TAMCSA 1a. Δδ stands for chemical shift change of the NH proton of (R)-G1 [(●) red] and (S)-G1 (●) in the presence of TAMCSA 1a in CDCl₃ at room temperature (400 MHz). X stands for the molar fraction of (±)-G1, (X = [(±)-G1]/[(±)-G1 + TAMCSA 1a]).

This result suggests that a pair of diastereomeric complexes with 1:1 stoichiometry is formed between (±)-G1 and TAMCSA 1a.

To further understand the intermolecular interaction between (±)-G1 and TAMCSA 1a, the geometries of (R)-G1 and (S)-G1 with TAMCSA 1a were optimized by using density functional theory (DFT) at the B3LYP/3–21G* level.⁵⁴ The continuum model (SMD) for chloroform was used in all NMR calculations to simulate the solvent effects. The proposed models show that two hydrogen bonds are formed between (R)-G1 and TAMCSA 1a (NH···(H)OPh, 2.062 Å; PhNCO···HNCO, 1.902 Å) (Figure 10a). However, only one strong hydrogen bond is formed between (S)-G1 and TAMCSA 1a (NH···OCNH) with a bond length of 1.779 Å (Figure 10b).

Figure 10.

Proposed DFT models for the hydrogen bonding interactions between TAMCSA 1a and (R)-G1 (a) and (S)-G1 (b).

The chemical shift values (δ, ppm) of the NH proton of (R)-G1 and (S)-G1 in the presence of TAMCSA 1a and their equivalent chemical shift (Δδ, ppm) are obtained by DFT/SMD calculations. These results are in agreement with the observed chemical shift values and nonequivalent chemical shift values of the same proton (as shown in the Supporting Information, page S-11, Table S4). The Cartesian coordinates and total energies of the complexes of (S)-G1 and (R)-G1 with TAMCSA 1a are obtained by means of B3LYP/3–21G* structural optimization and are available in the Supporting Information (pages S-11–S-21, Tables S5–S9).

Now that the chiral discriminating capability of TAMCSAs 1a–1d has been effectively established by examining the differentiation of chiral thiohydantoin derivatives (±)-G1–13, and practical application in determining enantiomeric purity can be further explored. Subsequently, samples containing (S)-G6 with 10, 20, 30, 50, 70, 85, and 90% ee were prepared in the presence of TAMCSA 1a in CDCl₃ at room temperature, and their ¹H NMR spectra were measured on a 400 MHz NMR spectrometer. Enantiomeric excesses (ee) for all samples were accurately calculated based on the integration of the NH proton of (S)-G6 and (R)-G6, featuring well-separated ¹H NMR signals, and with large enough nonequivalent chemical shifts and good baseline resolution (Figure 11). Excellent linear correlation between the theoretical (X) and observed (Y) ee % values was obtained in the presence of TAMCSA 1a (as shown in Supporting Information page S-8, Figure S4).

Figure 11.

Determination of enantiomeric excesses of G6 with different optical purities, ee (%) = {[(S)-G6 − (R)-G6]/[(S)-G6 + (R)-G6]} × 100%. Overlaid ¹H NMR spectra of the NH proton of (S)-G6 [(○) red] and (R)-G6 [(●) red] in the presence of an equal amount of TAMCSA 1a in CDCl₃. [G6] = 5 mM. (Scans: 8; Data processing: MestReNova software; type of NMR probe: broadband gradient autotune).

CONCLUSIONS

In summary, chiral recognition by means of ¹H NMR spectroscopy has been successfully investigated by virtue of outstanding differentiations of chemical shifts of the split protons of enantiomers of thiohydantoin derivatives (±)-G1–13. Surprisingly large nonequivalent chemical shift values (up to 2.052 ppm) of enantiomers of thiohydantoin derivatives in the presence of TAMCSAs 1a–1d have been established. Practical applications in the analysis of enantiomeric excess (ee) have been achieved by evaluating the integration area of the NH proton of G1 (up to 90% ee) with the assistance of TAMCSA 1a. In addition, the intermolecular interaction between G1 and TAMCSA 1a has been explored by Job plots and theoretical chemical calculations. More importantly, this work provides a new benchmark in the field of chiral recognition by ¹H NMR spectroscopy since unprecedented nonequivalent chemical shifts of chiral thiohydantoin derivatives have been observed in the presence of a family of highly sensitive and effective TAMCSAs. Furthermore, this work also provides a significant breakthrough in exploring and developing the chiral recognition of chiral heterocyclic compounds, especially chiral N,N-heterocyclic organic compounds as potential chiral drugs, by means of ¹H NMR spectroscopy.