Deviations from Beer's law in electronic absorption and circular dichroism: Detection for enantiomeric excess analysis

The electronic absorption (UV) to circular dichroism (CD) signal ratio can be used for enantiomeric excess (ee) analysis within linear range. However, CD detection often requires a high sample concentration where deviations from Beer's law may occur. Individual enantiomers of four chiral compounds were separated from commercial racemates by semipreparative high‐performance liquid chromatography (HPLC) with chiral columns. They were used to trace possible deviations in both UV and CD detection on achiral HPLC with a photodiode array detector and a CD detector. The CD/UV ratios for samples with the same ee value decreased by up to 7.8 to 52% when the injection volume increased, indicating that the linear standard curve of ee versus CD/UV is only valid within a narrow range. To extend the sample amount to a wider range, a data‐processing method was developed based on two second‐order polynomial functions, which were constructed to fit the relationship between the intensities of the UV and CD signals for two enantiomers. Moreover, a more simplified method based on a third‐order polynomial function was established to calculate the ee values. The variations between the predicted and experimental ee values were within ±0.08 for both methods. To our knowledge, this is the first study that the deviations from Beer's law are considered in both UV and CD detection for ee analysis.     

Generation of molecular chiral asymmetry through stirred crystallization

In our earlier work we established that stirred crystallization of achiral compounds that crystallize in enantiomeric forms result in spontaneous chiral symmetry breaking. The asymmetry thus spontaneously generated is confined to the solid state. In this article, we present a case in which the crystal enantiomeric excess (CEE) can be converted to molecular enantiomeric excess (EE) through a solid state reaction which relates the enantiomeric form of the crystal to the enantiomeric form of the product. Such a process not only provides a means of detecting the CEE generated in stirred crystallization but it is also a means through which chiral asymmetry generated spontaneously is "propagated" to generate chiral compounds with enantiomeric excess.     

On-column deracemization of an atropisomeric biphenyl by quinine-based stationary phase and determination of rotational energy barrier by enantioselective stopped-flow HPLC and CEC.

The reversible enantiomerization of axially chiral 2'-dodecyloxy-6-nitrobiphenyl-2-carboxylic acid was studied in the presence of a brush type chiral stationary phase based on O-(tert-butylcarbamoyl) quinine as chiral selector unit by stopped-flow high-performance liquid chromatography (sfHPLC) and capillary electrochromatography (sfCEC). After initial separation of the enantiomers in the first section of the column, the flow was stopped and the resolved species allowed to enantiomerize on-column. From this conversion, which could be determined from the enantiomeric ratios at different enantiomerization times, kinetic rate constants were calculated. By sfHPLC at a constant temperature of 15 degrees C, kinetic rate constants in the presence of the CSP were found to be 4.1 x 10(-5) s(-1) and 2.2 x 10(-5) s(-1) for the (-) and (+)-enantiomers, respectively, corresponding to half-lives of 279 and 530 min. Thus, apparent activation energies of enantiomerization were calculated to be 93.0 and 94.6 kJ mol(-1) for the (-) and (+)-enantiomers. On the macroscopic level, the apparent difference of rotational energy barriers and kinetic rate constants for both enantiomers is reflected as deracemization. For example, starting from a racemic mixture, an enantiomeric excess (ee) of 14% was seen in the stopped-flow HPLC experiment described after an enantiomerization time of 220 min at 15 degrees C, and a maximal ee of 17% can be approximated after infinite enantiomerization time. There is good agreement between HPLC and CEC results as well as their experimental errors, confirming that the new sfCEC technique may be a valuable and convenient tool to study interconversion processes.     

Time-resolved fluorescent chirality sensing and imaging of malate in aqueous solution

Chiral discrimination of malates in aqueous solutions at near-neutral pH is achieved through fluorescence measurement and imaging using the europium-tetracycline complex (EuTc) as a fluorescent probe. The method is based on the significantly different fluorescence properties of the ternary complexes (Eu-Tc-malate) formed between EuTc and the enantiomeric malates. The enantiomeric excess (ee) of chiral malates can be quantified by both steady-state and time-resolved fluorescence, using either a conventional fluorescence microplate reader or fluorescence imaging. It offers a facile and sensitive method for high-throughput chiral discrimination.     

A simple method to determine the enantiomeric ratio in enantioselective biocatalysis

The enantiomeric ratio (E) is commonly used to characterize the enantioselectivity in enzyme-catalyzed kinetic resolution. In this paper this parameter is directly derived from the enantiomeric excess of substrate and product. This is formally more correct than using Chen's equation after calculating the degree of conversion from both ee values using the relation of Sih and Wu. New expressions and useful graphs have been generated for reversible and irreversible uni-uni reactions. The theoretical predictions have been verified experimentally for various reactions. Values for E and the thermodynamic equilibrium constant,K_EQ, were obtained for a ( -dehalogenase-catalyzed dehalogenation, a hydrolysis reaction by porcine pancreatic lipase, and for C. Cylindracea lipase-catalyzed esterification and transesterification. In view of the current developments in the field of chiral analysis, this method is an easily available tool in the quantitative treatment of enzyme-catalyzed resolution of enantiomers.     

Parallel SFC/MS-MUX screening to assess enantiomeric purity

Enantiomeric excess (ee) was evaluated for two internally synthesized compound libraries using a high-throughput automated, intelligent four-channel parallel supercritical fluid chromatography/mass spectrometry system equipped with a multiplexed ion source interface (SFC/MS-MUX). The two libraries contained compounds spanning a wide range of enantiomeric ratios with structurally diverse chemical scaffolds and stereogenic centers. The system analyzed each sample simultaneously against four chiral columns using up to six organic modifiers. Enhancements to our previously published parallel supercritical fluid chromatography/mass spectrometry system were implemented to address the challenges associated with automated trace enantiomer identification and quantitation. A reversal of enantiomer elution order was observed for several samples across multiple CSPs and modifiers. The relationship between elution order and % ee accuracy is presented for compounds exhibiting high, middle and low % ee values. Despite incidences in which the minor enantiomer eluted prior to the major enantiomer with less than baseline resolution, the overall % ee was in agreement with separations in which full baseline resolution was achieved. The methods presented here demonstrate the value and utility of high-throughput ee determinations to support drug discovery and development programs.     

Asymmetric autocatalysis and its application to chiral discrimination

Chiral pyrimidyl, quinolyl, and pyridyl alkanols act as asymmetric autocatalysts with significant amplification of enantiomeric excess (ee) in the enantioselective addition of diisopropylzinc to pyrimidine-5-, quinoline-3-, and pyridine-3-carbaldehydes, respectively. 2-Alkynyl-5-pyrimidyl alkanol with as low as 0.6% ee automultiplies during the consecutive asymmetric autocatalysis with increasing ee to as high as >99.5%. Asymmetric autocatalysis is applied to chiral discrimination of organic compounds. In the presence of methyl mandelate or 2-butanol with very low ee's (0.05-0.1%) as chiral initiators, the reaction between pyrimidine-5-carbaldehyde and diisopropylzinc affords pyrimidyl alkanol with higher ee's with the correlated absolute configurations to those of the chiral initiators. Chirality of amino acids (such as leucine) and helicenes with very low ee's are also discriminated by asymmetric autocatalysis, affording pyrimidyl alkanol with very high ee's. Asymmetric autocatalysis also discriminates the chirality of primary alcohols-alpha-d, monosubstituted [2.2]paracyclophanes and octahedral cobalt complex with achiral ligands of which the chirality is due to the topology of coordination of the achiral ligand. Even the chirality of inorganic crystals such as quartz and sodium chlorate is discriminated by asymmetric autocatalysis of pyrimidyl alkanol. Thus, asymmetric autocatalysis provides a unique method for the discrimination of chiral compounds and crystals.     

Organocatalytic synthesis of 5-hydroxyisoxazolidine catalyzed by camphor sulfonyl hydrazines through aza-Michael addition/cyclization

A series of chiral 5-hydroxy isoxazolidines has been successfully synthesized through camphor sulfonyl hydrazine-catalyzed asymmetric aza-Michael addition reaction between N,O-protected hydroxyamines and enals. Moderate yields with moderate to good enantioselectivities (up to 96% enantiomeric excess [ee]) were achieved. It provides an alternative asymmetric approach to preparing isoxazolidine derivatives.     

Strain engineering for stereoselective bioreduction of dicarbonyl compounds by yeast reductases

Pure chiral molecules are needed in the pharmaceutical and chemical industry as intermediates for the production of drugs or fine chemicals. Microorganisms represent an attractive alternative to chemical synthesis since they have the potential to generate single stereoisomers in high enantiomeric excess (ee). The baker's yeast Saccharomyces cerevisiae can notably reduce dicarbonyl compounds (in particular alpha- and beta-diketones and keto esters) to chiral alcohols with high ee. However, products are formed at a low rate. Moreover, large amounts of co-substrate are required for the regeneration of NADPH that is the preferred co-factor in almost all the known dicarbonyl reductions. Traditionally, better ee, reduction rate and product titre have been achieved via process engineering. The advent of recombinant DNA technology provides an alternative strategy to improve productivity and yield by strain engineering. This review discusses two aspects of strain engineering: (i) the generation of strains with higher reductase activity towards dicarbonyl compounds and (ii) the optimisation of co-substrate utilisation for NADPH cofactor regeneration.     

Resolution of (±)‐β‐methylphenylethylamine by a novel chiral stationary phase for Pirkle‐type column chromatography

In this study, a new Pirkle‐type chiral column stationary phase for resolution of β‐methylphenylethyl amine was described by using activated Sepharose 4B as a matrix, L ‐tyrosine as a spacer arm, and an aromatic amine derivative of L ‐glutamic acid as a ligand. The binding capacities of the stationary phase were determined at different pH values (pH = 6, 7, and 8) using buffer solutions as mobile phase, and enantiomeric excess (ee) was determined by HPLC equipped with chiral column. The ee was found to be 47%. Chirality, 2010. © 2009 Wiley‐Liss, Inc.     

Expeditious synthesis and chromatographic resolution of (+)- and (-)-trans-1-benzylcyclohexan-1,2-diamine hydrochlorides

The hitherto unknown (-)- and (+)-1-benzylcyclohexan-1,2-diamine hydrochlorides 4a. HCl and 4b. HCl were synthesized by means of diastereoselective alpha-iminoamine rearrangement with subsequent imine reduction and hydrogenolysis. The relative trans-configuration as well as the absolute (1S,2R) and (1R,2S) configurations of 4a and 4b, respectively, were elucidated on the basis of an X-ray analysis of 3b. HCl. The enantiomeric excess (ee) values of the title compounds (>99%) were determined by chiral HPLC on a Chirex (D) Penicillamine column.     

Lipases and whole cell biotransformations of 2-hydroxy-2-(ethoxyphenylphosphinyl)acetic acid and its ester

A wide spectrum of commercially available lipases and microbial whole cells catalysts were tested for biotransformations of 2-hydroxy-2-(ethoxyphenylphosphinyl)acetic acid 1 and its butyryl ester. The best results were achieved for biocatalytic hydrolysis of ester: 2-butyryloxy-2-(ethoxyphenylphosphinyl)acetic acid 2 performed by lipase from Candida cylindracea, what gave optically active products with 85% enantiomeric excess, 50% conversion degree and enantioselectivity 32.9 for one pair of enantiomers. Also enzymatic systems of Penicillium minioluteum and Fusarium oxysporum were able to hydrolyze tested compound with high enantiomeric excess (68–93% ee), enantioselectivity (44 for one pair of enantiomers) and conversion degree about 50–55%. Enzymatic acylation of hydroxyphosphinate was successful in case when porcine pancreas lipase was used. After 4 days of biotransformation the conversion reaches 45% but the enantiomeric enrichment of the isomers mixture do not exceed 43%. Obtained chiral compounds are valuable derivatizing agents for spectroscopic (NMR) evaluation of enantiomeric excess for particular compounds (e.g. amino acids).     

Enzymatic and chromatographic chiral discrimination of racemic (thio)glycidyl esters: Part I. A chemoenzymatic approach to both enantiomers of thioglycidyl esters

Both hitherto unknown (+)-(R)- and (?)-(S)-thioglycidyl esters, (R)-( 2 ) and (S)-( 2 ), have been synthesized with different high enantiomeric excesses (ee) by two routes from the corresponding rac-glycidyl esters rac-( 1 ). The first includes a porcine pancreatic lipase (PPL)-mediated kinetic resolution of these esters followed by sulfuration with practically complete inversion to the (+)-(R)-enantiomer (+)-(R)-( 2 ) (36–86% ee). (?)-(S)-Thioglycidyl esters (?)-(S)-( 2 ) are obtained by the reverse reaction sequence (43–80% ee). In the latter case the hydrolysis rate is lower than that of analogous glycidyl esters. Moreover, the dependence of enantiomeric excess on the size of the acyl-group is of the opposite tendency. Therefore, in both cases suitable selection of the acid residue gives rise to maximum enantioselectivity. The irreversible lipase-catalyzed acylation of rac-glycidol and rac-thioglycidol, however, was found to be a less suitable alternative. The enantiomeric excess of recovered homochiral esters was determined by chiral chromatography using modified cellulose stationary phases (OB, OD). © 1993 Wiley-Liss, Inc.     

Highly Efficient Asymmetric Additions of Diethylzinc to Aldehydes Triply Activated by Chiral Phosphoramide‐Zn(II) Complexes Derived From Cinchona Alkaloids

New chiral phosphoramide ligands derived from cinchona alkaloids were developed, which react with diethylzinc to form chiral phosphoramide‐Zn(II) complexes containing two Lewis bases and one Lewis acid. These trifunctional complexes can serve as highly efficient chiral catalysts for triple activation of enantioselective addition reactions of diethylzinc with aldehydes to give desired alcohol products with excellent yields and enantiomeric excess (ee) values up to 99%. Chirality 25:561‐566, 2013. © 2013 Wiley Periodicals, Inc.     

Insect pest control agents: Novel chiral butanoate esters (juvenogens)

During the investigation of ester derivatives (juvenogens, biochemically activated insect hormonogenic compounds) of biologically active alcohols with potential application in insect pest control, a need for availability of all existing stereoisomers of ethyl N-{2-[4-(2-butanoyloxycyclohexyl)methyl]phenoxy}ethyl carbamate occurred. They were synthesized from their chiral precursors, the corresponding stereoisomers of 2-(4-methoxybenzyl)cyclohexyl butanoate, by removing their protecting group (methyl), and by subsequent condensation of the aromatic hydroxyl moiety with ethyl N-(2-bromoethyl) carbamate. The requested enantiomers of 2-(4-methoxybenzyl)cyclohexyl butanoate were obtained by a Candida antarctica lipase-mediated transesterification and chiral resolution of the respective racemic cis- and trans-isomers of 2-(4-methoxybenzyl)cyclohexanol either directly or after a subsequent chemical esterification of the chiral precursor. In this synthesis, two convenient butanoic acid activating esters, vinyl butanoate and 2,2,2-trifluoroethyl butanoate, were employed, and the chiral precursors in the synthesis of the target molecules were obtained in 41-48% yields (i.e., 82-96% conversion), and with enantiomeric purity ee=96-98%, respectively. The enantiomeric purity of the products was determined by chiral HPLC analysis, and their absolute configuration was assigned on the basis of analyzing the (1)H and (19)F NMR spectra of their diastereoisomeric Mosher acid (3,3,3-trifluoromethyl-2-methoxy-2-phenylpropanoic acid) esters.     

Facile entry to (-)-(R)- and (+)-(S)-mexiletine

The title compounds, 1a and 1b, have been synthesized in a three-step sequence starting from (-)-(S) and (+)-(R)-propylene oxide, respectively, in acceptable overall yields. The enantiomeric excess values for 1a and 1b were 96% and 93% respectively, as assessed by HPLC analysis on a chiral stationary phase of the corresponding N-acetyl derivatives. The synthetic route herein presented may represent a facile entry to highly enriched mexiletine enantiomers, alternative to those previously reported in the literature.     

Utilization of deep‐sea microbial esterase PHE21 to generate chiral sec‐butyl acetate through kinetic resolutions

We previously identified and characterized 1 novel deep‐sea microbial esterase PHE21 and used PHE21 as a green biocatalyst to generate chiral ethyl (S)‐3‐hydroxybutyrate, 1 key chiral chemical, with high enantiomeric excess and yield through kinetic resolution. Herein, we further explored the potential of esterase PHE21 in the enantioselective preparation of secondary butanol, which was hard to be resolved by lipases/esterases. Despite the fact that chiral secondary butanols and their ester derivatives were hard to prepare, esterase PHE21 was used as a green biocatalyst in the generation of (S)‐sec‐butyl acetate through hydrolytic reactions and the enantiomeric excess, and the conversion of (S)‐sec‐butyl acetate reached 98% and 52%, respectively, after process optimization. Esterase PHE21 was also used to generate (R)‐sec‐butyl acetate through asymmetric transesterification reactions, and the enantiomeric excess and conversion of (R)‐sec‐butyl acetate reached 64% and 43%, respectively, after process optimization. Deep‐sea microbial esterase PHE21 was characterized to be a useful biocatalyst in the kinetic resolution of secondary butanol and other valuable chiral secondary alcohols.     

Photobiocatalyzed asymmetric reduction of ketones using Chlorella sp. MK201

Aromatic ketones were reduced using suspension culture of Chlorella sp. MK201 under fluorescent light illumination producing the corresponding chiral alcohols in high yields with excellent enantiomeric excess (ee). For example, 2′,3′,4′,5′,6′-pentafluoroacetophenone at 0.25?mg/ml was converted to the corresponding (S)-alcohol in 80?% yield with?>99?% ee by 1?mg dry wt of Chlorella/ml in 12?h illumination (2,000 lux).     

Chiral symmetry-breaking transition in growth front of crystal phase of 1,1'-binaphthyl in its supercooled melt

Although the theory of spontaneous chiral symmetry-breaking in open systems was proposed some time ago, experimental realization of this phenomenon has not been achieved. In this article, we note that the crystal growth front of 1,1'-binaphthyl shows many of the characteristics of an open system in which chiral symmetry-breaking has occurred. We studied the temperature profiles of the crystallizing surface and obtained X-ray diffraction data of the crystals grown from the melt under controlled conditions. The data show that, under appropriate conditions, the observed bimodal probability distribution of enantiomeric excess (ee) with maxima approximately 60% is due exclusively to chiral crystals and not due to racemic crystals of 1,1'-binaphthyl that can also form at large supercooling. The mass independence of the ee shows that the growing front maintains a constant ee, which is a clear signature of open systems in steady state. Chirality 16:131-136, 2004.     

Enantiomeric impurities in chiral catalysts,auxiliaries, and synthons used in enantioselective syntheses. Part 5

The enantiomeric excess of chiral starting materials is one of the important factors determining the enantiopurity of products in asymmetric synthesis. Fifty‐one commercially available chiral reagents used as building blocks, catalysts, and auxiliaries in various enantioselective syntheses were assayed for their enantiomeric purity. The test results were classified within five impurities level (ie, <0.01%, 0.01%‐0.1%, 0.1%‐1%, 1%‐10%, >10%). Previously from 1998 to 2013, several reports have been published on the enantiomeric composition of more than 300 chiral reagents. This series of papers is necessitated by the fact that new reagents are forthcoming and that the enantiomeric purity of the same reagent can vary from batch to batch and/or from supplier to supplier. This report presents chiral liquid chromatography (LC) and gas chromatography (GC) methods to separate enantiomers of chiral compounds and evaluate their enantiomeric purities. The accurate and efficient LC analysis was done using newly introduced superficially porous particle (SPP 2.7 μm) based chiral stationary phases (TeicoShell, VancoShell, LarihcShell‐P, and NicoShell).