Enantiodifferentiation of chiral diols and diphenols via recognition-enabled chromatographic ¹⁹F NMR

Abstract

A novel and efficient method for distinguishing between chiral diols and diphenols has been established through the use of ¹⁹F NMR spectroscopy. Central to this system's efficacy is a chiral amine, strategically modified with a CF₃ group. This amine reacts in-situ with 2-formylphenylboronic acid to create a chiral ¹⁹F-labeled probe. This probe demonstrates discriminatory capabilities by interacting with hydroxy-containing analytes to form boronic esters. These esters produce distinct ¹⁹F NMR signals that vary according to their stereoconfiguration, facilitating accurate chiral differentiation. The method's resolution capacity was demonstrated by successfully identifying 12 distinct chiral analytes (six pairs of enantiomers) in complex mixtures, highlighting its extensive potential in diverse chiral analysis applications.

Graphical abstract

1. Introduction

Chiral compounds, particularly those rich in hydroxy and phenol groups, are crucial in a broad spectrum of natural products, biological substances, and synthetic molecules [1]. Among these, chiral diols and diphenols are particularly valuable as they are used as chiral auxiliaries and catalysts, facilitating the selective production of chiral products in a single configuration [[2], [3], [4], [5]]. This has led to a significant boost in the synthesis of novel chiral diols and diphenols, driven by their unique biological activities and synthetic applications [6,7]. Despite progress in automating reaction conditions, optimizing these enantioselective reactions remains inefficient due to the lack of swift chiral analytical techniques. Chiral high-performance liquid chromatography (HPLC) is the predominant method for assessing enantiomeric purity [8,9]. However, it often requires significant adjustments to the mobile and stationary phases and is time-intensive, rendering it less suitable for high-throughput scenarios. For compounds with low UV absorption, chiral gas chromatography becomes necessary, but this method is even slower and less sensitive. Circular dichroism (CD) spectroscopy offers another option for chiral analysis, but its sensitivity to concentration changes and susceptibility to interference often make it impractical for complex mixtures [[10], [11], [12], [13]]. These limitations underscore the pressing need for developing more rapid and accurate detection methods for chiral diol and diphenol compounds. Advances in this area would markedly improve the production efficiency and effectiveness of chiral compounds in specific configurations, a key factor in fields like pharmaceuticals and organic chemistry.

Nuclear magnetic resonance (NMR) is a rapid and precise analytical technique that provides detailed structural information about compounds at the atomic level. For enantiodifferentiation, which is the process of distinguishing between mirror-image molecules, chiral agents like chiral derivatizing agents (CDAs) and chiral solvating agents (CSAs) are commonly employed [[14], [15], [16], [17], [18], [19]]. These methods often depend on the ¹H NMR signals of the compounds being analyzed, leading to signal overlap in complex mixtures. An alternative NMR strategy for chiral discrimination involves the use of chiral oriented systems [[20], [21], [22]]. The scope of this approach has been broadened by integrating multinuclear NMR, enhancing its applicability [23]. Recently, there's been a resurgence in the use of ¹⁹F NMR-based detection methods, largely due to the 100% natural abundance of ¹⁹F and the rarity of organofluorine compounds in nature [[24], [25], [26], [27], [28], [29]]. By employing ¹⁹F-decorated derivatizing agents, it's possible to distinguish chiral amines, amino acids, and carboxylic acids through their unique ¹⁹F NMR signals [[30], [31], [32], [33], [34]]. This approach enhances the ability to analyze complex mixtures containing multiple components [[29], [30], [31]]. Specifically, when the mixtures include fluorinated compounds as target analytes, it becomes feasible to simultaneously distinguish between various analytes through the use of chiral solvating agents and ¹⁹F NMR [35]. Our research has focused on developing ¹⁹F-labeled probes that can reversibly bind to a variety of analytes [[36], [37], [38], [39], [40]]. By adjusting the chemical exchange rate to slower speeds on the NMR timescale, we've created probes targeting a wide range of analytes. The resulting ¹⁹F NMR signal is typically precise and correlates directly with each component in the sample. The interpretation of these signals is akin to chromatographic analysis, hence this method is often termed "recognition-enabled chromatographic ¹⁹F NMR" [29,41]. It is important to emphasize that the differentiation mechanism employed in our method is fundamentally distinct from that used in chiral liquid chromatography analysis, which is based on physical separation. While this approach has been effective for the enantioanalysis of analytes, such as amines, alcohols, amides, sulfoxides, and N-heterocycles [36,[40], [41], [42]], the enantiodifferentiation of diols and diphenols using existing probes has not been satisfactory. To address this, we introduce a novel system specifically designed for the chiral discrimination of these types of analytes. This system is constructed with a chiral amine with a strategically labeled CF₃ group and 2-formylphenylboronic acids. The key to this system's efficacy lies in the strong binding affinity of the hydroxy-containing analytes to the boronic acids [43]. This interaction results in the formation of diastereoisomeric boronic esters, which exhibit distinctively separated ¹⁹F NMR signals, enabling efficient chiral discrimination. Owing to the system's robust resolving capability, it can simultaneously identify as many as 12 different chiral analytes, marking a significant advancement in the chiral analysis of complex mixtures.

The study of reversible condensation between boronic acids and diols has led to advancements in NMR analysis through the creation of chiral agents. In 2003, Fabio Prati's team developed (S)-(+)-N-acetylphenylglycine boronic acid, for measuring the enantiomeric excess (ee) of 1,2-diols using ¹H NMR [44]. In 2022, Song Ling's group introduced a novel chiral derivatizing agent, derived from chiral 1,2-diphenylethylenediamine, for analyzing alcohol compounds and determining the ee of chiral diols [45]. In 2009, Tony D. James' research introduced a method using a three-component system to determine ee in chiral diols. This method involves forming diastereoisomeric boronic esters, producing distinct ¹⁹F NMR signals for different chiral diols (Fig. 1a) [46]. Building on these foundational contributions, our research aims to develop a more sensitive method for analyzing chiral diols and diphenols. We propose using (S)-2,2,2-trifluoro-1-(4-methoxyphenyl)ethanamine (1), 2-formylphenylboronic acid (2), and chiral diols/diphenols (Fig. 1b). The strategic placement of a trifluoromethyl group near the analyte's chiral center enhances resolving ability. With three chemically equivalent fluorine atoms, our system offers superior sensitivity, potentially accelerating detection speed and facilitating high-throughput analysis.

Fig. 1.

Chiral discrimination of diols using boronic acid derivatives.

2. Experimental section

2.1. Preparation of NMR samples

For analytes A_2-9, dissolve the analyte in acetone-d6 to a concentration of 34 mM. For analytes A₁, A_10-20, use CDCl₃ instead. Prepare 11.4 mM solutions of 1 (23.3 mg in 10 mL solvent) and 34 mM solutions of 2 in acetone-d6 (for the analysis of A_2-9) and CDCl₃ (for the analysis of A₁, A_10-20). Mix 300 μL of the chiral amine, 100 μL of 2, and 100 μL of the analyte in a 3 mL vial, then transfer to an NMR tube for ¹⁹F NMR analysis. For 1,1′-binaphthalene-2,2′-diol (BINOL) and derivatives, add a small amount of powdered molecular sieve to the NMR tube to facilitate analyte-probe association.

2.2. NMR measurements

For Fig. 2c−g, and 3a−t, 4, ¹⁹F NMR spectra were recorded on a Bruker Avance neo 600 NMR spectrometer (565 MHz for ¹⁹F nucleus) equipped with a BBFO probe at 298 K, using a default relaxation delay (D1) of 1 s and a scan number of 64. For all ¹⁹F NMR spectra, proton decoupling was performed.

Fig. 2.

(a) Reaction between 1 (6.8 mM) and 2 (6.8 mM); (b) Formation of boronic acid esters; (c) ¹⁹F NMR chemical shifts of ¹⁹F-labeled amine 1 (6.8 mM); (d) ¹⁹F NMR spectrum of a mixture of 1 (6.8 mM) and 2 (6.8 mM); (e) ¹⁹F NMR spectrum of a mixture of 1 (6.8 mM), 2 (6.8 mM), and 4-A_12S (6.8 mM); (f) ¹⁹F NMR spectrum of a mixture of 1 (6.8 mM), 2 (6.8 mM), and 4-A_12R (6.8 mM); (g) ¹⁹F NMR spectrum of a mixture of 1 (6.8 mM), 2 (6.8 mM), and 4-A₁₂ (6.8 mM).

Fig. 3.

(a, j−t) ¹⁹F NMR spectra of mixtures of 1 (6.8 mM) and 2 (6.8 mM) and diol analytes (ca. 6.8 mM) in CDCl₃; (b–i) ¹⁹F NMR spectra of mixtures of 1 (6.8 mM) and 2 (6.8 mM) and various racemic analytes (ca. 6.8 mM) in acetone-d₆.

Fig. 4.

¹⁹F NMR spectra of mixtures of 1 (6.8 mM) and 2 (6.8 mM) and six pairs of enantiomers (6.8 mM) in CDCl₃. ¹⁹F NMR spectra were recorded on a Bruker Avance II 600 NMR spectrometer (565 MHz for the ¹⁹F nucleus) using a scan number of 64.

3. Results and discussions

Our investigation began with an analysis of the reaction between the CF₃-tagged amine 1 and boronic acid 2. Initially, these two reactants were combined (Fig. 2a), followed by the recording of their ¹⁹F NMR spectrum. As illustrated in Fig. 2, the condensation between 1 and 2 yielded new ¹⁹F NMR signals (Fig. 2d). These signals were attributed to the formation of imine 3 (Fig. 2a). A key observation from our study is the high efficiency of this condensation reaction. Remarkably, a majority of amine 1 is converted into 3 upon treatment with an equimolar amount of boronic acid 2 (Fig. S1). Furthermore, our studies reveal that this condensation reaction is reversible. This was evidenced by the fact that treating 3 with an excess of 1-phenylethan-1-amine results in the release of the ¹⁹F-labeled amine 1 (Fig. S2). Subsequently, we selected 1,1,2-triphenylethane-1,2-diol (A₁₂) as a representative analyte to evaluate the effectiveness of 3 in distinguishing between the enantiomers of chiral diols (Fig. 2b). The introduction of both the R and S forms of diol A₁₂ to 3 resulted in distinctive ¹⁹F NMR signals (Fig. 2e and f). These unique signals are attributed to the formation of diastereoisomeric boronic esters (Fig. 2b). The chemical shifts of these signals varied depending on the stereoconfiguration of the analyte, showcasing the method's sensitivity to stereochemical differences. Remarkably, when we analyzed racemic mixtures of A₁₂, we observed two separate ¹⁹F NMR signals. These signals matched precisely with those obtained from the analysis of the enantiopure forms of A₁₂ conducted independently (Fig. 2g). This consistency further reinforces the viability of this approach for the accurate recognition and enantiodifferentiation of analytes with multiple hydroxy groups. To assess the scope of our method, we expanded our study to include chiral discrimination of various diols and diphenols (Fig. 3 and Fig. S3). Notably, 1,2-diols are recognized for their extensive antibacterial properties and are crucial for chiral synthesis.

We evaluated the discrimination capability using a parameter named "Resolution (R_s)", calculated through equation (1) [47].

$$R_{\mathrm{s}}=\frac{{\delta }_{\mathrm{A}-}{\delta }_{\mathrm{B}}}{W_{\mathrm{h}}(\mathrm{A})+W_{\mathrm{h}}(\mathrm{B})}$$ (1)

This involves calculating the difference in ¹⁹F NMR signal chemical shifts (δ_A and δ_B) and the linewidths at half maximum height of these signals [W_h(A) and W_h(B)]. It is noteworthy that there is an alternative parameter, the quotient E, introduced by Pérez-Trujillo et al. [48] This parameter is designed for evaluating the efficacy of chiral solvating agents and is based on two NMR signals with identical linewidths. However, in cases where the linewidths of the ¹⁹F NMR signals vary between different enantiomers, the use of R_s is more appropriate.

For the series of aliphatic diols (A_1-9), we observed R_s values ranging from 1.5 to 2.6 (Fig. 3a–i). The relatively modest R_s values can be attributed to the similarity of the CH₂-bearing substituents on the chiral carbinol, differing mainly in more distant structural elements. This method showed excellent tolerance to various functional groups, successfully analyzing enantiomers containing ester (A₉), halide (A₇), and ether (C–O–C) linkages (A₈). A notable increase in R_s values was observed for analytes with a phenyl ring in the side chain (A₁₀), suggesting enhanced resolution. When multiple phenyl rings are present (A₁₁, A₁₂), the ¹⁹F NMR signals exhibited a more distinct chemical shift difference, likely due to the shielding effect of the phenyl rings on the nearby CF₃ group. Beyond 1,2-diols, the method effectively discriminated 1,3-diols (A₁₃) and various chiral diphenols (A_15-20). Interestingly, the detection of diphenols was enhanced by incorporating molecular sieves to aid in boronic ester formation, reflecting the lower nucleophilicity of phenols compared to alcohols. BINOL and its derivatives were all effectively resolved, achieving R_s values as high as 133.9. These compounds are pivotal in asymmetric synthesis and chiral separation. Intriguingly, BINOL and its derivatives exhibit an interesting characteristic in the current ¹⁹F NMR-based detection. Specifically, the ¹⁹F NMR signal for the R enantiomer of BINOL appears at a lower field than that of the S enantiomer. This trend, however, reverses in BINOL derivatives that have 3,3′-substituents. This inversion suggests that the substituents near the phenol groups significantly influence the ¹⁹F NMR signals of the boronic ester adducts. It implies that these substituents induce notable changes in the electronic environment around the CF₃ group on the probe, altering the observed NMR signal. This observation underlines the sensitivity of ¹⁹F NMR spectroscopy to changes in the electronic environment, particularly in the context of stereochemistry and substituent effects. In the case of analytes A₂-A₉, using CDCl₃ as the solvent resulted in a partial overlap of the ¹⁹F NMR signals between the newly generated signal and that of imine 3. To enhance chiral discrimination for these analytes, deuterated acetone was employed as an alternative solvent. This adjustment underscores that optimizing the detection medium can significantly improve the resolving capability of the current detection system (Fig. S4). In comparison to the CDA developed by James and coworkers [48], our investigation reveals that for the enantiodifferentiation of aliphatic diols, both methods exhibit comparable performance. Notably, in the case of diphenol analytes, our approach demonstrates enhanced enantiodifferentiation (Table S1). To further understand the chiral discrimination mechanism, we conducted density functional theory (DFT) calculations on the diastereoisomeric boronic esters resulting from reactions between 3 and both A_10R and A_10S (Fig. S5). Additionally, the formation of these boronic esters is corroborated by ¹H NMR spectroscopic analysis of mixtures containing compounds 1, 2, and A₁₀ (Fig. S6). Our findings reveal that in their optimized conformations, the CF₃ group in the boronic esters derived from A_10S is shielded by the adjacent phenyl group (Fig. S5). This aligns with the ¹⁹F NMR spectroscopy results, where the A_10S's signal is more upfield than A_10R's (Fig. 3j). Interestingly, nuclear Overhauser effect spectroscopy (NOESY) experiments did not detect any notable nuclear Overhauser effect (NOE) correlation signals between the protons in the diol and amine segments (Fig. S7b and Fig. S8b). The lack of NOE correlations suggests a flexible C–N bond, potentially leading to rapid interconversion among various conformations. This result, while not definitive, suggests that the diastereoisomers may not have a single, stable prevailing conformation under the experimental conditions.

The approach of using multi-substrate screening has emerged as an effective method for investigating asymmetric catalytic reactions [[49], [50], [51]]. This technique significantly streamlines substrate screening processes, enhancing experimental productivity. Central to this strategy is the ability to identify multiple chiral analytes concurrently, a capability growing in importance. Utilizing the excellent resolving power of our technique, we explored its potential in simultaneously detecting various chiral analytes. In this experiment, we prepared a solution containing six pairs of enantiomeric isomers and recorded its ¹⁹F NMR spectrum. As illustrated in Fig. 4, this resulted in 12 distinct ¹⁹F signals, each accurately corresponding to the individual chiral analytes. This level of performance, when compared with traditional chiral HPLC analysis, demonstrates superior resolution capabilities, signifying a major advancement in the field of chiral analytical techniques. To further validate the effectiveness of our method in assessing the enrichment of chiral samples, we selected 1,1′-bi-2-naphthol (A₁₅) as a representative test analyte. This selection was based on its cost-effectiveness and the ready availability of both enantiomers at high levels of enantiopurity. Due to the steric hindrance associated with A₁₅, its interaction with the in-situ formed CF₃-labeled probe 3 exhibits slight variations. To account for this, we introduced a correction coefficient to counteract the bias stemming from the differing binding affinities (for details, see Fig. S9). This coefficient was derived by analyzing the ratio of the ¹⁹F NMR signals for the R and S enantiomers in a racemic mixture of A₁₅. We then used this correction factor to adjust the integrations of the ¹⁹F NMR signals from samples with enantiomeric enrichment, ensuring accurate ee evaluations. The results, as illustrated in Table 1, show that the ee values determined using our method closely matched the actual enantiomeric composition of the samples. The average discrepancy between the measured and actual ee values was less than 1% (for details, see Fig. S10). Notably, only a minimal quantity of 0.7 mg (6.8 mM) of chiral amine 1 was required for routine enantiomeric analysis, highlighting the method's cost-effectiveness and efficiency.

Table 1.

Evaluation of the ee values.

1,1′-bi-2-naphthol (A15)
actual ee (%)	calculated ee (%)	|calculated ee − actual ee|(%)
0	0	0
10.1	10.6	0.5
23.9	24.0	0.1
36.2	36.0	0.2
40.3	40.3	0
49.1	48.9	0.3
59.8	59.9	0.1
70.1	70.1	0
80.0	79.9	0.1
89.8	89.8	0

4. Conclusion

In summary, our study introduces a novel method for the swift chiral discrimination of a variety of diols and diphenols using ¹⁹F NMR spectroscopy. This approach utilizes an in-situ formed chiral boronic acid, synthesized through the condensation of readily accessible 2-formylphenyl boronic acid with a strategically chosen chiral amine, featuring a CF₃ label. This resultant boronic acid intermediate can interact with various diols and diphenols, resulting in diastereoisomeric boronic esters that exhibit distinct ¹⁹F NMR chemical shifts. This method's resolving power enabled the simultaneous identification of as many as 12 chiral analytes, thus facilitating multicomponent chiral analysis in complex mixtures. Additionally, we successfully demonstrated the method's capability for precise ee assessment. With its operational simplicity, broad scope of applicable analytes, and cost-effectiveness, this method is poised to significantly enhance high-throughput chiral analysis in a variety of scenarios.

CRediT authorship contribution statement

Yilin Zeng: Writing – original draft, review & editing, Methodology, Investigation, Formal analysis, Conceptualization. Wenjing Bao: Writing – review & editing, Methodology, Investigation. Guangxing Gu: Writing – review & editing, Methodology, Investigation, Conceptualization. Yanchuan Zhao: Writing – original draft, review & editing, Supervision, Project administration, Funding acquisition, Conceptualization.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Biographies

Yilin Zeng received her B.S. degree from the School of Nuclear Technology and Chemical Biology of Hubei University of Science and Technology in 2022. She is currently a master student jointly trained by the School of Chemistry and Materials Science of Sichuan Normal University and Key Laboratory of Fluorine and Nitrogen Chemistry and Advanced Materials of the Chinese Academy of Sciences.

Wenjing Bao received the B.S. degrees in College of Chemistry and Molecular Sciences from Wuhan University in the year of 2020. She is currently a Ph.D. student at Key Laboratory of Fluorine and Nitrogen Chemistry and Advanced Materials, Shanghai Institute of Organic Chemistry, University of Chinese Academy of Sciences, Chinese Academy of Science.

Guangxing Gu received his B.S. degrees in School of Chemistry & Chemical Engineering from Henan University of Science and Technology in 2020. He received his master degree in School of Chemistry and Chemical Engineering from Shanghai University of Engineering Science in 2023, He is currently a research assistant at Key Laboratory of Fluorine and Nitrogen Chemistry and Advanced Materials, Shanghai Institute of Organic Chemistry, University of Chinese Academy of Sciences, Chinese Academy of Science.

Yanchuan Zhao received his B.S. degree in chemistry from Jilin University and his Ph.D. degree from the Shanghai Institute of Organic Chemistry (SIOC) in organic chemistry. Following postdoctoral studies in the Department of Chemistry at Massachusetts Institute of Technology (MIT), he started his independent academic career at SIOC in 2017, where he is currently a Research Professor in the Key Laboratory of Fluorine and Nitrogen Chemistry and Advanced Materials of the Chinese Academy of Sciences. His research interests include organofluorine chemistry, NMR-based chemosensing, molecular recognition, and organic porous crystals.

Appendix A. Supplementary data

The following is/are the supplementary data to this article.