Fluorous "racemic" mixture synthesis: polysaccharide-based chiral columns for simultaneous demix and enantioseparation of racemic fluorous tagged compounds

The proof of concept experiments of fluorous "racemic" mixture synthesis (FRMS) is shown using polysaccharide-based chiral stationary phases. The mixture of racemic O-benzoylmandelate derivatives bearing different lengths of fluorous cleavable tags undergoes sequential reactions to provide individual derivatives as well as their enantiomers resolved on polysaccharide-based chiral HPLC columns (DAICEL CHIRALCEL and CHIRALPAK series).     

Spectroscopic rationalization of the separation abilities of decaproline chiral selector in dichloromethane-isopropanol solvent mixture

A chiral column, with decaproline as the chiral selector, has broad chiral selectivity. To understand the separation mechanism of this chiral column, multiple spectroscopic techniques, including optical rotation, electronic circular dichroism, infrared absorption and vibrational circular dichroism, have been used here to study the conformation of the decaproline oligomer in isopropanol(IPA)/dichloromethane(DCM) mixtures. These studies indicate that decaproline oligomer adopts polyproline II conformation in IPA/DCM solvent system (0% IPA approximately 100% IPA). Hydrogen bonding interactions between C=O groups of decaproline and IPA molecules increase as the content of IPA in the solvent mixture increases up to 60% and become less significant from then onwards. These spectroscopic observations are found to have a good correlation with the enantiomeric separation of racemic 2,2,2-trifluoro-1-[10-(2,2,2-trifluoro-1-hydroxy-ethyl-anthracen-9-yl]-ethanol by the decaproline column.     

Enantiomeric Polyketides from the Starfish‐Derived Symbiotic Fungus Penicillium sp. GGF16‐1‐2

One new racemic mixture, penicilliode A ( 1 ) and four pairs of enantiomeric polyketides, penicilliode B and C ( 2 and 3 ) and coniochaetone B and C ( 4 and 5 ), were obtained from the starfish‐derived symbiotic fungus Penicillium sp. GGF16‐1‐2. Interestingly, the strain GGF16‐1‐2 can produce enantiomers. The absolute configuration of 1 was determined by X‐ray diffraction (XRD) analysis, and the absolute configurations of 2 – 4 were determined by the optical rotation (OR) values and electronic circular dichroism (ECD) calculations. Compounds 1 – 5 were firstly isolated from the marine‐derived fungus Penicillium as racemates, and 2 – 5 were separated by HPLC with a chiral stationary phase. All the compounds were evaluated for their antibacterial, cytotoxic and inhibitory activities against PDE4D2.     

The Enantiomeric Separation of Tetrahydrobenzimidazoles Cyclodextrins‐ and Cyclofructans

High performance liquid chromatography (HPLC) and capillary electrophoresis (CE) were used to examine the enantiomeric separation of a series of 17 racemic tetrahydrobenzimidazole analytes. These compounds were prepared as part of a synthetic program directed towards a select group of pyrrole‐imidazole alkaloids. This group of natural products has a unique framework of pyrrole‐ and guanidine‐containing fused rings which can be constructed through the intermediacy of a tetrahydrobenzimidazole scaffold. Several bonded cyclodextrin‐ (both native and derivatized) and derivatized cyclofructan‐based chiral stationary phases were evaluated for their ability to separate these racemates via HPLC. Similarly, several cyclodextrin derivatives and derivatized cyclofructan were evaluated for their ability to separate this set of chiral compounds via CE. Enantiomeric selectivity was observed for the entire set of racemic compounds using HPLC with resolution values up to 3.0. Among the 12 different CSPs, enantiomeric recognition was most frequently observed with the Cyclobond RN and LARIHC CF6‐P, while the Cyclobond DMP yielded the greatest number of baseline separations. Fifteen of the analytes showed enantiomeric recognition in CE with resolution values as high as 5.0 and hydroxypropyl‐γ‐cyclodextrin was the most effective chiral additive. Chirality 25:133–140, 2013. © 2012 Wiley Periodicals, Inc.     

Fundamentals of chirality,resolution, and enantiopure molecule synthesis methods

The chirality of molecules is a concept that explains the interactions in nature. We may observe the same formula but different organizations revolving around the chiral center. Since Pasteur's meticulous observation of sodium ammonium tartrate crystals' structure, scientists have discovered many features of chiral molecules. The number of newly approved single enantiomeric drugs increases every year and takes place in the market. Thus, separation or resolution methods of racemic mixtures are of continued importance in the efficacy of drugs, installation of affordable production processes, and convenient synthetic chemistry practice. This article presents the asymmetric synthesis approaches and the classification of direct resolution methods of chiral molecules.     

Enantiomeric resolution of kielcorin derivatives by HPLC on polysaccharide stationary phases using multimodal elution

Analytical HPLC methods using carbamate chiral stationary phases of polysaccharide derivatives were developed for the enantiomeric resolution of five racemic mixtures of xanthonolignoids: rac-trans-kielcorin C, rac-cis-kielcorin C, rac-trans-kielcorin D, rac-trans-isokielcorin D, and rac-trans-kielcorin E. The separations were evaluated with the stationary phases cellulose tris-3,5-dimethylphenylcarbamate, amylose tris-3,5-dimethylphenylcarbamate, amylose tris-(S)-1-phenylethylcarbamate, and amylose tris-3,5-dimethoxyphenylcarbamate under normal, reversed-phase, and polar organic elution conditions. Chiral recognition of those chiral stationary phases, the influence of mobile phases on the enantiomers separation, and the effects of structural features of the solutes on the chiral discrimination observed are discussed. The best performance was achieved on an amylose tris-3,5-dimethylphenylcarbamate phase. Polar organic conditions gave shorter retention factors and better resolutions and were a valuable alternative to the alcohol-hexane or reversed-phase conditions.     

Chiral recognition ability and solvent versatility of bonded amylose tris(3,5-dimethylphenylcarbamate) chiral stationary phase: enantioselective liquid chromatographic resolution of racemic N-alkylated barbiturates and thalidomide analogs

The chiral recognition ability and solvent versatility of a new chiral stationary phase containing amylose 3,5-dimethylphenylcarabamate immobilized onto silica gel (CHIRALPAK IA) is investigated. Thus, the direct enantioselective separation of a set of racemic N-alkylated barbiturates and 3-alkylated analogs of thalidomide was conducted using different nonstandard solvents as eluent and diluent, respectively in high-performance liquid chromatography (HPLC). The separation, resolution, and elution order of the investigated compounds were compared on both immobilized and coated amylose tris(3,5-dimethylphenylcarbamate) chiral stationary phases (Chiralpak IA and Chiralpak AD, respectively) using a mixture of n-hexane/2-propanol (90:10 v/v) as mobile phase with different flow-rates and fixed UV detection at 254 nm. The effect of the immobilization of the amylose tris(3,5-dimethylphenylcarbamate) chiral stationary phase on silica (Chiralpak IA) on the chiral recognition ability was noted as the bonded phase (Chiralpak IA) was superior in chiral recognition and possesses a higher resolving power in most of the reported cases than the coated one (Chiralpak AD). A few racemates were not or poorly resolved on the immobilized Chiralpak IA or the coated Chiralpak AD when using standard solvents were most efficiently resolved on the immobilized Chiralpak IA upon using nonstandard solvents. Furthermore, the immobilized phase withstands the nonstandard (prohibited) HPLC solvents such as dichloromethane, ethyl acetate, tetrahydrofuran, methyl-tert-butyl ether, and others when used as eluents or as a dissolving agent for the analyte itself. The direct analysis of a real sample extracted from plasma using DCM on Chiralpak IA is also shown.     

Determination of the absolute configuration of secondary alcohols with Horeau's method including enantioselective gas chromatography

The reaction of optically active secondary alcohols with excess of racemic 2-phenylbutyric acid anhydride in pyridine proceeds at different rates to the diastereoisomeric esters (kinetic partial resolution). According to Horeau the (unknown) absolute configuration of the alcohol can be derived from the optical rotation of the remaining excess of 2-phenylbutyric acid in the reaction mixture. Measuring the optical rotation may be very difficult in cases of small absolute rotation values and may be inaccurate due to the necessity to completely remove all chiral impurities. The application of Horeau's method is greatly facilitated by gas chromatographic determination of the enantiomeric ratio of the remaining 2-phenylbutyric acid after methylation using a short capillary column with heptakis(2,6-di-O-methyl-3-O-pentyl)-β-cyclodextrin as a chiral stationary phase. Baseline resolution of the enantiomers is achieved after approximately 10 min of retention time. Due to the high selectivity of capillary gas chromatography the probability of impurities in the mixture to interfere with the measurement of the enantiomeric ratio is extremely low. © 1994 Wiley-Liss, Inc.     

Enantiomeric Crystallization from DL-Aspartic and DL-Glutamic Acids: Implications for Biomolecular Chirality in the Origin of Life

Amino acids in living systems consist almost exclusively of the L-enantiomer. How and when this homochiral characteristic of life came to be has been a matter of intense investigation for many years. Among the hypotheses proposed to explain theappearance of chiral homogeneity, the spontaneous resolution of conglomerates seems one of the most plausible. Racemic solids may crystallize from solution either as racemic compounds(both enantiomeric molecules in the same crystal), or lesscommonly as conglomerates (each enantiomer molecule separate indifferent enantiomeric crystals). Only conglomerates can developa spontaneous resolution (one of the enantiomeric molecule crystallizes preferentially, the other one remains in solution).Most of natural amino acids are racemic compounds at moderatetemperatures. How can we expect a hypothetical spontaneous resolution of these amino acids if they are not conglomerates?In this paper we show how DL-aspartic and DL-glutamic amino acids(racemic compounds), crystallize at ambient conditions as trueconglomerates. The experimental conditions here described,that allows this `anomalous' behaviour, could be also found innatural sedimentary environments. We suggest that these experimental procedures and its natural equivalents, have apotential interest for the investigation of the spontaneous resolution of racemic compounds comprising molecules associatedwith the origin of life.     

Polarimetric detection in high-performance liquid chromatography

Chiroptical detection for HPLC is particularly useful as a selective detection method for chiral molecules, and in enantiomeric purity determination with partial chiral separation or without chiral separation. The recent development of laser-based polarimeters with microdegree sensitivity has increased the applicability of optical rotation detection in HPLC. The detection limit of these instruments is submicrogram on-column for many chiral compounds in analytical HPLC. A variety of applications of the selective detection of optically active molecules are reviewed. The use of polarimetric detection with partial chiral separation is considered, both as an aid to method development and for enantiomeric purity determination. Finally applications to enantiomeric purity determination without chiral separation are reviewed, with the dual use of nonchirally selective and chiroptical detectors to determine the total amount and optical purity of the analyte. Determinations of chiral purity for samples of high enantiomeric excess are described, which with laser-based instrumentation may give accuracies of better than +/- 1% with sample loadings of 50 micrograms on an achiral column. Applications to the study of enantioselective reactions are also considered, with determination of enantiomeric excess in near-racemates to better than +/- 0.1%.     

Homochiral oligopeptides via surface recognition and enantiomeric cross impediment in the polymerization of racemic phenylalanine N-carboxyanhydride crystals suspended in water

As part of our program on the search of possible prebiotic routes for the formation of oligopeptides of homochiral sequence (isotactic) from racemic precursors in aqueous environment, we report the polymerization of racemic crystals of phenylalanine N-carboxyanhydrides, enantioselectively tagged with five deuterium atoms, suspended in water containing various amine initiators. Racemic mixtures of isotactic oligopeptides, comprising up to 25 repeat units of the same handedness, as the dominant component for each length, were observed in a MALDI-TOF mass spectrometry analysis. The racemic mixtures of the peptides could be desymmetrized by initiating the polymerization reaction with water-soluble methyl esters of either enantiopure alpha-amino acids or dipeptides. A three-step mechanism is proposed to account for these results: (i) Surface recognition of the chiral initiator by the chiral sites present at specific faces of the crystal; (ii) Oligopeptide elongation at the polymer/crystal interface; and (iii) Self-assembly of the short isotactic peptides into racemic antiparallel beta-sheets as templates followed by cross-enantiomeric impediment in the growth of enantiomeric chains at the peptide beta-sheet/crystal interface.     

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.     

Chiral HPLC separation and CD spectra of the enantiomers of the alkaloid tacamonine and related compounds.

The HPLC enantiomeric separation of racemic indole alkaloids tacamonine, 17 alpha-hydroxytacamonine, deethyleburnamonine, and vindeburnol was accomplished using Chiralpak AD and Chiralcel OD as chiral stationary phases. Small structural differences affect the enantioselectivity ability of these phases. Single enantiomers of tacamonine and vindeburnol were isolated by semipreparative HPLC and their CD spectra and optical rotations were measured.     

Impact of substituents on the enantioseparation of racemic 2-amidotetralins on polysaccharide stationary phases. I. chiralcel OD

The direct enantiomeric separation of 32 racemic 2-amidotetralins on the commercially available tris-(3,5-dimethylphenylcarbamate) derivative of cellulose, coated on silica gel (Chiralcel OD), is presented. To date, the selection of a column for the chiral separation of a racemic mixture is done empirically. Studying the impact of small changes in the chemical structure of a series of amidotetralins on the separation behavior may help to give an insight in the chiral recognition mechanism. The amidotetralins differed structurally in three of their substituents, which were never directly located on the chiral carbon atom. The enantiomers of 24 out of 32 amidotetralins could be resolved with a resolution >1.5. Hydrogen bonding and π-π interactions are supposed to be the major analyte-chiral stationary phase (CSP) interactions. However, the spatial arrangement of the enantiomers may play an important role too. Increasing the bulkiness of the acyl substituent led to an increase in the resolution (R_S), whereas a more bulky substituent on the aromatic ring resulted in a very low resolution. The introduction of a chlorine atom into the acyl substituent additionally increased the resolving power. Chirality 8:574–578, 1996. © 1997 Wiley-Liss, Inc.     

π-Facial hydrogen bonding in the chiral resolving agent 2,2,2-trifluoro-1-(9-anthryl)-ethanol and its racemic modification

2,2,2-Trifluoro-(9-anthryl)-ethanol (TFAE) has been extensively used, in its pure enantiomeric forms, as a chiral solvating agent in nuclear magnetic resonance spectroscopy (NMR). It has also played an important role in the development of chiral stationary phases in liquid chromatography (LC). X-ray crystallography of the enantiomeric and racemic crystals shows, in both cases, the formation of an intermolecular hydrogen bond between the O? H and the π-face of one of the rings of the anthracene aromatic system.¹ Few examples of such hydrogen bonding have been published previously, and those that have are not as clear cut as in this case. An explanation for the hydrogen bonding is sought using molecular modelling via the PM3 analytically derived molecular electrostatic potentials. Using NMR and dynamic lineshape analysis, the barrier to rotation about the aryl-carbon bond is estimated, indicating the C? CF₃ bond to be perpendicular to the anthracene axis in nonpolar solution. This conformation is identical to the conformation in the crystal. Evidence is also presented to support the formation of intermolecular π-facial hydrogen bonding in TFAE solutions. It is thought that such hydrogen bonding may be implicated in chiral recognition using this compound. © 1994 Wiley-Liss, Inc.     

Synthesis, enantiomeric resolution, and selective C-11 methylation of a highly selective radioligand for imaging the norepinephrine transporter with positron emission tomography

Reboxetine, 2-[alpha-(2-ethoxyphenoxy)benzyl]morpholine, is a highly selective norepinephrine transporter (NET) blocker that has been used for the treatment of depression. Its methyl analogue, 2-[alpha-(2-methoxyphenoxy)benzyl]morpholine (MRB), has been radiolabeled with C-11 for studies of the NET system with positron emission tomography (PET). The normethyl precursor, 2-[alpha-(2-hydroxyphenoxy)benzyl]morpholine (desethylreboxetine), was synthesized in 6% overall yield via a multi-step regio- and stereo-specific synthesis, starting from a mono-O-protected catechol. The resulting racemic mixture of desethylreboxetine was resolved by chiral HPLC to provide the (2S,3S) and (2R,3R) enantiomers in >98% enantiomeric excess. These enantiomers were then used as precursors for radiosynthesis to prepare enantiomerically pure individual 11C-labeled MRB enantiomers for comparative PET studies in baboons. Selective C-11 methylation at the phenolic oxygen with [11C]CH3I was achieved in the presence of excess base. After HPLC purification, racemic ((2S,3S)/(2R,3R)) or enantiomerically pure ((2S,3S) or (2R,3R)) [11C]MRB was obtained in 61-74% decay-corrected radiochemical yields from [11C]CH3I in a synthesis time of 40 min with a radiochemical purity of >96% and a specific activity of 1.7-2.3 Ci/micromol (63-85 GBq/micromol) corrected from the end of bombardment (EOB).     

Some applications of chiral liquid affinity chromatography using bovine serum albumin as a stationary phase

The enantioselectivity excerted by many proteins can be utilized for direct optical resolution in liquid chromatographic processes whereby the protein is used as a stationary phase. Bovine serum albumin (BSA), covalently bound to a suitable support, has been shown to act as a chiral discriminator for a variety of racemic organic compounds in aqueous buffers. Columns packed with BSA-silica can be used for determination of enantiomeric composition in aqueous solvents at very low concentrations by HPLC. This technique opens up new possibilities for the preparative isolation of micrograms amounts of enantiomers and for studies of stereoselectivity and mechanisms in enzymatic and microbial reactions.     

How do reaction conditions affect the enantiopure synthesis of 2‐substituted‐1,4‐benzodioxane derivatives?

Several biologically active compounds structurally include the enantiopure 2‐substituted‐1,4‐benzodioxane scaffold. The straightforward racemization that affects reactions involving most of the common chemical reactives is thus a crucial issue. The developing of a completely stereo‐controlled synthetic route that does not affect the enantiomeric excess is consequently mandatory. It is also important to set up a reliable chiral HPLC method, able to follow the reaction, and to improve the synthetic performances. Here, we report the chiral investigation of two different synthons, we specifically evaluated the synthetic pathways that could be run in order to afford them, avoiding the racemization processes, which could normally occur in basic conditions. In addition, we developed peculiar chiral HPLC methods in order to resolve the enantiomers, define the enantiomeric excess, and fully characterize these compounds.     

Stereochemical studies of hexylitaconic acid,an inhibitor of p53–HDM2 interaction

Hexylitaconic acid (1) is an intriguing natural product possessing a chiral carbon, and both its enantiomers have been found in nature. Enantiomeric pure (+)-(1) and (?)-(1) were successfully prepared by racemic synthesis followed by enantiomeric separation in a chiral HPLC system. Their absolute configurations were clarified by the vibrational circular dichroism technique using their methyl esters 2 and lactones 3. Their inhibitory activities against the interaction of p53–HDM2 were also examined.