Enantiodifferentiation of multifunctional tertiary alcohols by NMR spectroscopy with a Whelk-O type chiral solvating agent

A Whelk-O type chiral solvating agent (CSA) allows NMR enantioresolution of multifunctional tertiary alcohols. Crystallographic analysis of the CSA revealed two conformers, which can be expected to coexist in solution. One conformation affords a chiral cleft that can engage a single enantiomer in simultaneous hydrogen bonding, π–π stacking, and CH/π interactions. This chiral recognition process yields nonequivalent NMR signals for the enantiomers of eighteen substrates even when substoichiometric amounts of the CSA are used.     

Quantitative 1H NMR method for the routine spectroscopic determination of enantiomeric purity of active pharmaceutical ingredients fenfluramine, sertraline, and paroxetine

Determining the enantiomeric purity of chiral therapeutic agents is important in the development of active pharmaceutical ingredients (API). A strategy for determining the enantiomeric purity of three APIs was developed using nuclear magnetic resonance (NMR) and the chiral solvating agent (CSA) 1,1-bi-2-naphthyl (1). While chiral chromatography is widely used to evaluate enantiomeric purity, it can sometimes suffer from tedious sample preparation obviating rapid measurements that are sometimes needed during the manufacture of such agents. The techniques described herein provide comparable enantiomeric purity results with those obtained with traditional chiral HPLC and other published methods for these compounds. Chiral analysis of standard samples of methylbenzylamine enantiomeric mixtures using 1 were found to be quantitative to approximately 1% minor enantiomer. Enantiomeric purity determination by NMR utilizing chiral solvating agents do not require special instrumental techniques, chemical derivatization or standards and is therefore ideally suited for rapid routine analysis. As a result, the technique demonstrated is commonly used in our laboratory as a complementary or alternative method to chiral HPLC or optical rotation measurements for routine determination of enantiomeric purity.     

手性溶剂-NMR法测定2,4-滴丙酸对映体纯度的定量分析

研究了手性溶剂法测定2,4-滴丙酸的^1H、^13C谱,在满足NMR准确定量所要求的分离度和信噪比的条件下,能准确测定手性化合物的对映体纯度。比较了以对映体百分含量（R％）和对映体过量（ee）表示手性农药的对映体纯度的差别,发现以对映体百分含量代替对映体过量来表示手性农药的对映体纯度更为准确。     

Chiral recognition of peptide enantiomers by cinchona alkaloid derived chiral selectors: mechanistic investigations by liquid chromatography, NMR spectroscopy, and molecular modeling

The chiral recognition mechanism of a cinchona alkaloid based chiral selector for N-protected peptide enantiomers was investigated. A chiral stationary phase derived from this selector was employed for liquid chromatographic enantiomer separations. It showed exceptionally high enantiomer discrimination for the (all-R)- and (all-S)-enantiomers of dialanine (alpha = 20), while a pronounced loss of chiral recognition occurred upon the insertion of an additional alanine residue into the peptide backbone. This reduction of enantioselectivity was investigated in great detail by NMR spectroscopy of complexes of the chiral selector and the analyte enantiomers accompanied by molecular modeling studies. Investigation of intramolecular NOEs provided the conformational states of the free and complexed forms of the selector. The analysis of complexation-induced shifts yielded information on intermolecular interactions and allowed us to propose binding models, which were further supported by the observation of intermolecular NOEs, indicating the relative arrangements of selector and analytes. Stochastic molecular dynamics simulations were able to reproduce the chromatographic retention orders and energy differences, as well as the intermolecular NOEs. The computational data were used to evaluate the intermolecular forces responsible for analyte binding. In addition, the relative contributions of the fragments of the chiral selector to the enantioselective binding event were assessed. A spatial arrangement of the chiral selector and the analyte allowing the primary ionic interaction as well as hydrogen bonding and pi-pi-stacking to take place simultaneously was found to be essential to obtain very high enantioselectivities.     

Signal separation and determination of the enantiomeric purity of primary amines with (-)-myrtenal--a 13C NMR study

The applicability of (-)-myrtenal as a chiral derivatizing agent in combination with (13)C NMR spectroscopy was investigated. (13)C NMR was found to be a valuable tool for the identification and enantiomer differentiation of primary amines including beta-amino alcohols and vicinal diamines. The enantiomeric excess could be determined via automated deconvolution and integration, and was found to be in good accordance with the expected values even in the cases, when enantiomer differentiation was not possible in (1)H NMR spectra.     

Determination of the enantiomeric purity of epinephrine by HPLC with circular dichroism detection

Several hundred drug substances approved by the U.S. Food and Drug Administration are chiral molecules. For the enantiomeric purity assessment, current practice is to develop separation techniques using chiral columns or mobile phase modifiers to separate enantiomers before detection. An alternative approach is to use currently accepted HPLC assay methods and use chiral-specific detectors to confirm whether the correct enantiomer is present. In this paper, adding a circular dichroism (CD) detector to an achiral HPLC method from the US Pharmacopeia (USP) is shown to be amenable for the determination of the enantiomeric purity of epinephrine, a substance used to treat anaphylaxis. This HPLC-UV-CD approach was able to detect the inactive D-(+) enantiomer at 1% of the total epinephrine composition. The linearity, accuracy, and precision of HPLC-UV-CD were evaluated and compared to analyses using a chiral HPLC method. Additionally, an epinephrine drug product was analyzed for assay (concentration) and enantiomeric purity. The results from achiral and chiral methods were identical within the experimental error. Overall, achiral chromatography performed using a USP method with CD detection may serve as a general means of determining chiral drug enantiomer purity and avoids the need for the development of additional chiral-specific methods for each individual drug.     

Kinetic method for the simultaneous chiral analysis of different amino acids in mixtures

The kinetic method has been extended to enantiomeric excess (ee) determinations on amino acids present in mixtures. Singly charged trimeric clusters [Cu(II)(ref*)(2)(A(m)) - H](+) are readily generated by electrospraying solutions containing Cu(II), a chiral reference ligand (ref*), and the amino acids (analytes A(m), m = 1-3). A trimeric cluster ion for each amino acid is individually mass-selected and then collisionally activated to cause dissociation by competitive loss of either the reference ligand or the analyte. For each analyte in the mixture, as shown from separate experiments, the logarithm of the ratio of the fragment abundances for the complex containing one enantiomer of this analyte expressed relative to that for the fragments of the corresponding complex containing the other enantiomer is linearly related to the enantiomeric composition of the amino acid. Formation and dissociation of each trimeric complex ion are shown to occur independently of the presence of other analytes. Chiral selectivity appears to be an intrinsic property and the chiral selectivity R(chiral(m)) measured from the mixture of analytes is equal to R(chiral) measured for the pure analyte. The sensitive nature of the methodology and the linear relationship between the logarithm of the fragment ion abundance ratio and the optical purity, characteristic of the kinetic method, allow the determination of chiral impurities of less than 2% ee in individual compounds present in mixtures by simply recording the ratios of fragment ion abundances in a tandem mass spectrum.     

Extension of chromatographically derived chiral recognition systems to chiral recognition and enantiomer analysis by electrospray ionization mass spectrometry

Chiral recognition by positive ion electrospray ionization (ESI) mass spectrometry is demonstrated through the adaptation of chromatographically derived chiral recognition systems. Solutions of soluble analogues of chiral selectors used in Pirkle-type chiral stationary phases, when mixed with a chiral analyte, whose enantiomers are known to be resolved on the analogous chiral stationary phase, are shown to afford selector–analyte complexes in the mass spectrum. Pseudo-enantiomeric chiral selectors, where each pseudo-enantiomer has a different mass and a higher affinity for the opposite analyte enantiomer of its pseudo-antipode, were prepared. When mixed with a chiral analyte, solutions of these pseudo-enantiomeric selectors afford selector–analyte complexes in the ESI-mass spectrum where the relative intensities of the selector–analyte complexes are dependent on the enantiomeric composition of the analyte. Additionally, the sense of the observed chiral recognition is in agreement with the sense of chiral recognition observed chromatographically.     

The effects of aromatic substituents on the chromatographic enantioseparation of diarylmethyl esters with the whelk-O1 chiral stationary phase

Members of a series of diarylmethanols, diarylmethyl pivalates, and diarylmethyl acetates (analyte sets 1-26) were enantioresolved with the (S,S)-Whelk-O1 chiral stationary phase (CSP). An analogue of the (S,S)-Whelk-O1 selector was combined with enantioenriched samples of the various diarylmethyl pivalates and thereby used as a chiral solvating agent (CSA) for high field 1H NMR studies. The absolute configurations of a number of chiral diarylmethyl pivalates were assigned using this approach, and hydrolysis of the pivalates allowed assignment of the absolute configurations of the corresponding diarylmethanols. Chromatographic, 1H NMR, and X-ray evidence are given in support of a chiral recognition model for the enantioresolution of diarylmethyl esters on this CSP.     

Determination of enantiomeric compositions of DOPA by tandem mass spectrometry using the kinetic method with fixed ligands

A fixed ligand (FL) version of the kinetic method was applied to rapid, simple, and accurate chiral analysis of DOPA, which is an important drug used for treatment of Parkinson's disease. Singly charged clusters containing the transition metal ion Cu(II), pyridyl ligands which serve as a fixed ligand, some amino acid as a reference, and the analyte DOPA were generated by electrospray ionization. The cluster ion of interest was mass-selected, and the kinetics of its competitive unimolecular dissociations was investigated in an ion trap mass spectrometer. The chiral selectivity (R(chiral)), the ratio of the two fragment ion abundances when the cluster contains one pure enantiomer of the analyte expressed relative to that for the other enantiomer, varies with fixed ligands, references, and transition metals. Chiral discrimination was optimized in 1,10-phenanthroline as a FL, L-Phe and L-Pro as a reference, and Cu(II) as a central metal ion. Quantitative determinations of the enantiomeric composition of DOPA were achieved using two-point calibration curves. The linear relationship between the logarithm of the fragment ion abundance ratio (ln R) and enantiomeric compositions (ee%) of the DOPA allows the determination of the chiral purity of enantiomeric mixtures.     

核磁共振技术测定联萘酚的对映体纯度

手性联萘酚((±)-BINOL)制备是国内高校常开设的一个实验,其产品的对映体纯度测试是实验的重要一环。以2-甲酰基苯硼酸和(S)-(?)-1-苯乙胺的混合物作为手性试剂与联萘酚发生Bull-James Assembly反应,以此设计了核磁共振氢谱(¹H NMR)测定联萘酚对映体纯度的实验。实验结果表明,通过反应生成的对映体混合物的核磁信号,能准确地计算出(±)-BINOL的对映体纯度。以选定的苯环氢核信号计算出的(R)-BINOL含量与理论ee值有着良好的线性关系(R²=0.9999)。此实验方法能够快速完成大量学生样品的测量,同时大大减少实验废液的产生量。学生通过此实验可对核磁共振技术有更进一步的了解。     

Evaluation of the interaction between sitagliptin and cyclodextrin derivatives by capillary electrophoresis and nuclear magnetic resonance spectroscopy

An aqueous capillary electrophoretic method was developed for chiral analysis of the novel anti-diabetic drug, sitagliptin. The acid-base profiling of the analyte was carried out using both capillary electrophoresis and nuclear magnetic resonance pH titrations. The apparent complex stability and chiral separation properties were investigated with 30 different cyclodextrins under acidic conditions. The effect of concentration and pH of the BGE, temperature of the capillary, and the type and concentration of the chiral selector on the enantiomer resolution were thoroughly investigated. The effects of dual cyclodextrin systems on separation were also extensively studied. Complete separation of racemic sitagliptin with good resolution (R(S)=2.24) was achieved within a short time (15 min) with optimized parameters (10°C, pH=4.4, 40 mM phosphate buffer) of a sulfobutylether-β-cyclodextrin (averaged degree of substitution ~4) and native β-cyclodextrin dual system. The averaged stoichiometry of the inclusion complex was determined using the Job plot method with both (1)H and (19)F NMR experiments and resulted in a 1:1 complex. The structure of the inclusion complex was elucidated using 2-D ROESY NMR experiments.     

Chiral capillary electrophoretic analysis of the enantiomeric purity of a pharmaceutical compound using sulfated beta-cyclodextrin

A practical chiral capillary electrophoresis method using randomly sulfated beta-cyclodextrin was developed for the quantitative determination of the chiral purity of a pharmaceutical compound. A systematic method development approach was conducted by modifying selected parameters such as the concentration of the chiral selectors, buffer pH, organic modifiers, buffer concentrations and type, temperature and applied voltage. The results of the investigation permitted an improved understanding of the separation mechanism. Two facile strategies for the reversal of the enantiomer elution order are also described. The optimized method was validated in terms of variability of the chiral selector, linearity, sensitivity, accuracy, recovery, ruggedness, and precision.     

Enantiomeric discrimination by double quantum excited selective refocusing (DQ-SERF) experiment

The differences in chemical shift anisotropies, dipolar couplings, and quadrupolar couplings of two enantiomers in the chiral liquid crystalline media are employed to visualize enantiomers. In spite of the fact that proton has high magnetic moment and is abundantly present in all the chiral molecules, 1H NMR is not exploited to its full potential because of severe overlap of unresolved transitions arising from long- and short-distance couplings. Furthermore, the two spectra from R and S enantiomers result in doubling of the number of observable transitions. The present study demonstrates the application of the selectively excited homonuclear double quantum (DQ) coherence correlated to its single quantum coherence of an isolated methyl group in a chiral molecule. The DQ dimension retains only the passive couplings within the protons of the methyl group while the long-distance passive couplings are refocused, removing the overlap of central transitions, and each enantiomer displays a doublet instead of a triplet unlike in regular selective refocusing experiment. The doublet separation being different for each enantiomer results in their discrimination. The cross section taken along the single quantum dimension pertaining to each transition in the DQ dimension provides the one-dimensional spectra for each individual enantiomer with the complete removal of the overlapped transitions from the other enantiomer. The experiment is robust, the pulse sequence is easy to implement, and the methodology has been demonstrated on different chiral molecules.     

Separation of Enantiomers of Drugs by Capillary Electrophoresis with Permethyl‐gamma‐Cyclodextrin as Chiral Solvating Agent

High‐throughput screening is a promising new approach in analytical chemistry. Within the framework of an extended screening program (The German‐Chinese Drug Screening Program), the enantioseparation of 86 drugs was investigated by capillary zone electrophoresis in the presence of the chiral solvating agent (CSA) octakis‐(2,3,6‐tri‐O‐methyl)‐γ‐cyclodextrin (TM‐γ‐CD). By this means, 15 drugs could be separated into enantiomeric pairs. Approximate measures for the degree of interaction (migration retardation factor, R_m) and for the degree of enantiomer recognition (migration separation factors, α_m) revealed intriguing patterns that were compared with those found for native γ‐cyclodextrin (γ‐CD). Although there is a distinct influence of the analyte structure on the electrophoretic data, interpretation remains difficult. Most remarkably, permethylation of γ‐CD leads neither to a higher affinity nor to better chiral recognition, in contrast to the findings with α‐CD.     

Conformational arm-wrestling: battles for stereochemical control in benzamides bearing matched and mismatched chiral 2- and 6-substituents

The orientation of a tertiary amide group adjacent to an aromatic ring may be governed by the stereochemistry of an adjacent chiral substituent. With a chiral substituent in both ortho positions, matched/mismatched pairs of isomers result. Evidence for matched stereochemistry is provided by the clean NMR spectra of single conformers, while mismatching gives poor or unexpected selectivities in the formation of chiral substituents, or mixtures of amide conformers. Attempts to use the match-mismatch effect to select for racemic pairs of enantiomeric substituents, and hence develop a "racemate-sequestering" reagent, are described, along with the use of "matching" to scavenge a single enantiomer of a diamine from material of incomplete enantiomeric purity.     

Elucidation of the chiral recognition mechanism of cinchona alkaloid carbamate-type receptors for 3,5-dinitrobenzoyl amino acids

A cinchona alkaloid having extraordinary chiral discriminatory powers (alpha = 32.6 for dinitrobenzoyl leucine) is developed as a chiral stationary phase (CSP) for chromatography. An explanation of how chiral discrimination takes place is presented. Using a soluble analogue of the CSP, we found that NMR spectrometry indicates that 1:1 complexes exist for both optical isomers interacting with the CSP, that the free base form of the CSP exists in an open/closed ratio of 35/65 but that the protonated, bound-state form is exclusively in the anti-open conformation, and that significant intermolecular NOEs exist for the more stable diastereomeric complex but not for the less stable complex. Stochastic molecular dynamics simulations were carried out in solvents of low and high dielectric. The chromatographic retention orders and free energy differences of analyte binding to CSP were reproduced computationally as were the observed intra- and intermolecular NOEs. Data from the simulation were used to evaluate the intermolecular forces responsible for analyte binding as well as to discern fragments of the CSP doing most of the work of holding the complexes together. The enantiodifferentiating forces and the parts of the CSP most responsible for chiral discrimination are described. Moments of distributions of key dihedral angles and distances between centroids were used to assess the relative rigidity of the competing diastereomeric complexes. Simultaneous multiple-contact ion-pairing, hydrogen bonding, and pi-stacking are possible for the longer retained enantiomer only. An X-ray crystallographic study of the more stable complex confirms the conclusions derived from chromatography, NMR spectroscopy, and molecular modeling.     

Anomalous NMR behavior of meso compounds with remote stereogenic centers on addition of chiral shift reagent or chiral solvating agent

A problem has arisen in using chiral shift reagents (CSR) and chiral solvating agents (CSA) to determine meso and racemic forms of diastereoisomers in which the stereogenic centers of the molecules are separated by achiral spacers. It is found that NMR signals of both meso and racemic forms of diastereoisomers may exhibit doubling on addition of CSR/CSA, which means that unequivocal assignments cannot be made without characterizing the effects for separate meso and racemic forms; this is particularly important for additions of CSR/CSA at relatively low concentrations, which always result in the splitting of some NMR signals of diastereoisomers. The phenomenon is demonstrated in the (31)P NMR spectra of meso and racemic forms of three spermine-bridged gem-disubstituted cyclotriphosphazatrienes, 1a-c, and compared with analogous achiral molecules, the per-substituted spermine-bridged cyclotriphosphazatrienes 2a-d. As expected, only one set of (31)P NMR signals was observed for the achiral compounds 2a-d, even on addition of CSA. Two sets of (31)P NMR ABX multiplets corresponding to meso and racemic diastereoisomers were observed for compounds 1a-c; on addition of CSA, the signals of at least one of the multiplets for each compound separated into more than the expected groups of three lines with an intensity distribution of 2:1:1. To understand this phenomenon, the meso and racemic forms of 1a and 1b and the meso form of 1c have been separated and characterized by X-ray crystallography. On addition of CSA to the racemic forms of 1a and 1b, the (31)P NMR spectrum shows the expected doubling of signals, but, unexpectedly, the same is observed for each of the meso forms of 1a-c. Analogous results using both CSA and CSR have been obtained for the meso and racemic forms of the diastereoisomeric piperazine-bridged macrocyclic-phosphazene compound, 3, whereas no effect was observed for the two meso forms of the doubly bridged macrocyclic-phosphazene compound 4. The phenomenon of doubling of the (31)P NMR signals of the meso form of singly bridged cyclotriphosphazatrienes, 1a-c and 3, is explained by consideration of the equilibrium in solution of independent complexation of a chiral ligand with molecules that have two chiral cyclophosphazene moieties separated by an achiral spacer group. The results show that the stereogenicity of such diastereoisomeric molecules in solution cannot be characterized unequivocally by NMR measurements on addition of either CSR or CSA.     

Enantiomer discrimination by mass spectrometry: noncovalent interactions of an N-derivatized dipeptide with various cinchona alkaloid derivatives and comparison with enantioselective liquid-phase separations

The enantiomer discrimination properties of cinchona alkaloid derived chiral selectors (CSs) towards a dipeptide analyte are examined by electrospray ionization mass spectrometry. The complexes formed between the CSs and the analyte enantiomers owing to various noncovalent interactions are analyzed and the magnitudes of enantiomer discrimination are determined from the complexes mass spectrometric intensities. The influence of different structural features of the CSs on enantioselectivity is discussed. The enantiomer discrimination results obtained by mass spectrometry are compared with those from related liquid chromatography enantiomer separations. A certain coherence between the chromatographic and mass spectrometric enantioselectivities could be established and the enantiomer discrimination patterns, i.e., the relative binding strengths, were identical for the two techniques. Thus, the use of mass spectrometry as a screening tool in the development of new CSs for chromatographic applications seems feasible.     

Determination of the enantiomeric purity of nucleoside analogs related to d4T and acyclovir,new potential antiviral agents,using liquid chromatography on cellulose chiral stationary phases

We reported a method of determination of enantiomeric purity of the new potential antiviral agents by direct analytical HPLC. Those agents are nucleoside analogs, having one chiral center. They are synthesized as a single enantiomer (R or S) by an asymmetric pathway. The chiral stationary phases chosen are silica-based cellulose tris-3,5-dimethylphenylcarbamate (Chiralcel OD-H), or tris-methylbenzoate (Chiralcel OJ). Resolution was achieved using normal-phase chromatography with a mobile phase consisting of n-hexane-alcohol (ethanol or 2-propanol) in various percentages. Furthermore the effects of structural features on retention, selectivity and resolution, as well as on the elution order were thoroughly studied. Differences in the lipophilicity of the compounds were also examined.