Capillary electrophoretic chiral resolution of vicinal diols by complexation with borate and cyclodextrin: Comparative studies on different cyclodextrin derivatives

Capillary electrophoretic enantioseparation of compounds containing vicinal diol groups have been investigated using different β-cyclodextrin (CD) derivatives and borate as a background electrolyte. Both native β-CD and several β-CD derivatives are examined. Chiral recognition is attributed to both enantioselective inclusion of the analyte into the chiral cavity of the CD and complexation with borate. The influence of concentration of the chiral selector, pH, and organic modifiers on the resolution was studied. Four diols were baseline separated. Chirality 9:153–156, 1997. © 1997 Wiley-Liss, Inc.     

Separation of cefoperazone enantiomers using beta-cyclodextrin as chiral additive by capillary zone electrophoresis

A capillary electrophoresis method was developed to separate the enantiomers of cefoperazone. Different cyclodextrins, including alpha-cyclodextrin (alpha-CD), beta-cyclodextrin (beta-CD), gamma-cyclodextrin (gamma-CD), 2-hydroxypropyl-beta-cyclodextrin (HP-beta-CD), and methyl-beta-cyclodextrin (Me-beta-CD), were tested as chiral additives in the running buffer. The effect of various parameters on enantioseparation such as concentration of NaH(2)PO(4), buffer pH, and CD concentration was also studied. The cefoperazone enantiomers were baseline separated under conditions of 0.04 mmol/L beta-CD, 75 mmol/L NaH(2)PO(4) buffer at pH 4.0. A fused silica capillary (40 cm effective length x 75 microm ID) was used. The applied voltage and capillary temperature were 20 kV and 25 degrees C, respectively. Under these conditions, linear calibration curves were obtained in the 5-500 microg/ml range using UV detection at 280 nm. The limit of detection for both isomers was 0.1 microg/ml. The method was used for the analysis of different pharmaceutical preparations (dose) and biological samples containing cefoperazone.     

Determination of enantiomeric excess for 2,3-dihydroxy-3-phenylpropionate compounds by capillary electrophoresis using hydroxypropyl-beta-cyclodextrin as chiral selector

A new capillary zone electrophoresis (CZE) method was developed to separate three chiral 2,3-dihydroxy-3-phenylpropionate enantiomers using neutral hydroxypropyl-beta-CD (HP-beta-CD) as chiral selector and borate as background electrolyte. The results showed that HP-beta-CD exhibited good enantioselectivity and high resolution was achieved under the optimum condition of pH 10.3, 200 mM borate buffer containing 6% methanol and 50 mM HP-beta-CD at 15 kV and 20 degrees C within 16 min. The precision of the method was <0.9% for migration time and 4.5% for corrected peak area. In addition, the developed method was successfully applied to the determination of enantiomeric excess (ee) of synthetic 2,3-dihydroxy-3-phenylpropionate samples. With this method, low as 0.2% impurity of the undesirable enantiomer in the presence of high amount of target enantiomer was determined. The results demonstrated that the proposed CZE method is a simple and useful technique and is applicable to ee assay of 2,3-dihydroxy-3-phenylpropionate enantiomers.     

Epoxide hydrolase-mediated enantioconvergent bioconversions to prepare chiral epoxides and alcohols

A number of epoxide hydrolase (EH)-mediated bioconversions have been developed to prepare single enantiomeric product from racemic substrates with a yield greater than 50%. Enantioconvergent hydrolysis using single or two EHs possessing complementary enantio- and regio-selectivity, EH-based chemoenzymatic reactions, and EH-triggered cascade-reactions have been developed for the preparation of chiral epoxides, epoxyalcohols, tetrahydrofuran derivatives and vicinal diols. All these bioconversions are based on stereochemical flexibilities of various EHs and can be used in total synthesis of biologically active compounds without the formation of unwanted enantiomers.     

Capillary electrophoresis of phosphorylated amino acids with fluorescence detection

A rapid and sensitive capillary electrophoresis (CE) method coupled with fluorescence detection was developed for identification of protein phosphorylation by determination of phosphoamino acids. Naphthalene-2,3-dicarboxaldehyde (NDA), a fluorescence derivatization reagent, was used to label protein hydrolysate. The optimal derivatization reaction was performed with 3.5mM NDA, 40 mM NaCN and 20mM borate buffer (pH 10.0) for 15 min. The baseline separation of three phosphorylated amino acids could be obtained in less than 180 s with good repeatability by using 30 mM borate (pH 9.2) containing 2.0mM beta-cyclodextrin (beta-CD) as the running buffer. The detection limits for phosphothreonine, phosphotyrosine and phosphoserine were 7.0 x 10(-9)M, 5.6 x 10(-9)M and 7.2 x 10(-9)M, respectively (S/N=3). Also, the interference from other protein amino acids with large molar excess over that of phosphoamino acids was studied. With beta-casein as the analysis protein, this method was successfully validated.     

Production of chiral epoxides: Epoxide hydrolase-catalyzed enantioselective hydrolysis

Chiral epoxides are highly valuable intermediates, used for the synthesis of pharmaceutical drugs and agrochemicals. They have broad scope of market demand because of their applications. A major challenge in modern organic chemistry is to generate such compounds in high yields, with high stereo- and regio-selectivities. Epoxide hydrolases (EH) are promising biocatalysts for the preparation of chiral epoxides and vicinal diols. They exhibit high enantioselectivity for their substrates, and can be effectively used in the resolution of racemic epoxides through enantioselective hydrolysis. The selective hydrolysis of a racemic epoxide can produce both the corresponding diols and the unreacted epoxides and vicinal diol has prompted researchers to explore their use in the synthesis of epoxides and diols with high ee values.     

Stereocontrolled synthesis of 1-acetylen-2,3-di-o-benzyl-tetrahydrofurans, 1,4-anhydro-arabinitol,and alpha,beta-dihydroxy-gamma-alkyl-butyrolactones

This article describes a concise and efficient synthesis of 1-acetylen-2,3-di-O-benzyl-tetrahydrofurans from tartaric acid esters using as the key step the stereocontrolled cyclization of Co(2)(CO)(6)-complexed propargylic diols. This molecule led to enantiomerically pure 1,4-anhydro-arabinitol and alpha,beta-dihydroxy-gamma-alkyl-butyrolactones. In the latter case, the critical methylene oxidation at the oxygen vicinal position was performed by RuO(4).     

A simple, inexpensive, high pressure liquid chromatographic method for separating cytokinins in plant extracts

Separation of a mixture of the main cytokinins occurring naturally in plant tissues was achieved by high pressure liquid chromatography using insoluble polyvinylpyrrolidone as the solid support. The separation of each cytokinin was first assessed over a range of salt and l-butanol concentrations and pH using a mixture of borate buffer and l-butanol as the mobile phase to determine the conditions necessary for optimum resolution. A discrete separation of zeatin, N-6-(Delta-2-isopentenyl)adenine, their related ribonucleosides, and kinetin was achieved using a simple isocratic elution with 0.025 m borate buffer at pH 6.8 and 4% (v/v) l-butanol. A number of cytokinin-active compounds were detected in cabbage extracts by the Amaranthus betacyanin bioassay using this separation technique.     

Yb(fod)3 in the spectroscopic determination of the configuration of chiral diols: a survey of the lanthanide diketonate method

A modification of the diketonate method for the determination of the absolute configuration of chiral vicinal diols, first proposed by Nakanishi, is taken under object and tested with a set of aliphatic and aromatic substrates; Yb(fod)(3) (fod = 6,6,7,7,8,8,8-heptafluoro-2,2-dimethyl-3,5-octanedionate) is employed as the chromophoric species. The replacement of dpm (dpm = dipivalomethanate) with fod ensures the formation of strong and stable signals in the UV region, whose signs are related to the configuration of the chelating molecule. The choice of ytterbium as the central cation introduces two major advantages: the electronic f-f transition of the rare-earth supplies a second spectral window in the near-IR, which is sensitive to the chemical environment around the atom. This allowed us to ascertain the number of different species present in solution and the observation of an induced CD also for strongly UV-absorbing substrates. The pseudocontact shifts induced in the (1)H-NMR spectrum by the unpaired electron of the lanthanide ion provide valuable structural information on the adducts.     

Development of a validated capillary electrophoresis method for enantiomeric purity control and quality control of levocetirizine in a pharmaceutical formulation

A chiral capillary electrophoresis method has been developed for the quantification of 0.1% of the enantiomeric impurity (dextrocetirizine) in levocetirizine and determination of both in pharmaceuticals using sulfated-β-cyclodextrins (CDs) as chiral selector. Several parameters affecting the separation were studied such as the type and concentration of chiral selectors, buffer composition and pH, organic modifier, mixtures of two CDs in a dual system, voltage, and temperature. The optimal separation conditions were obtained using a 50 mM tetraborate buffer (pH 8.2) containing 1% (w/v) sulfated-β-CDs on a fused-silica capillary. Under these conditions, the resolution of two enantiomers was higher than 3. To validate the method, the stability of the solutions, robustness (two level half fraction factorial design for 5 factors using 19 experiments [2(n-1)+3]), precision, linearity (dextrocetirizine 0.25-2.5 μg/ml, R(2) = 0.9994, y = 0.0375x + 0.0008; levocetirizine 15-100 μg/ml, R(2) = 0.9996, y = 0.0213x + 0.0339), limit of detection (0.075 μg/ml, 0.03% m/m), limit of quantification (0.25 μg/ml, 0.1% m/m), accuracy (dextrocetirizine 84-109%, levocetirizine 97.3-103.1%), filter effect, and different CD batches were examined. The validated method was further applied to bulk drug and tablets of levocetirizine.     

Enantioseparation of amfepramone (rac-diethylpropion): preparative separation of the enantiomers and enantioselective analysis

The enantiomers of the anorectic drug amfepramone [rac-diethylpropion, rac-2-(diethylamino)-1-phenyl-1-propanone; rac-DEP] were separated in the preparative scale by crystallization. With enantiopure di-O-benzoyltartaric acid as salt-forming chiral selector, diastereoisomeric salts of DEP enantiomers with a final purity of more than 97.5% were obtained. Analytical liquid chromatographic and electrophoretic methods for the control of the enantiomeric purity and the stoichiometry of the salts were developed. The enantioseparation of rac-DEP by capillary electrophoresis (CE) using hydroxypropyl-beta-cyclodextrin (HP-beta-CD) as chiral discriminator and phosphate buffer (pH 3.3) as run buffer led to good separations. HPLC methods were developed using polysaccharide chiral stationary phases (CSP). The separation of the two enantiomers and the two main degradation products (1-phenyl-1,2-propanedione and propiophenone), known from solid and liquid pharmaceutical preparations, was attained in one run on the silica-based CSP cellulose tris(3,5-dimethylphenylcarbamate) (Chiralcel OD). The conditions which might affect the enantioselectivity and the quality of the enantiomeric separation were investigated for Chiralcel OD and the related CSP amylose tris(3,5-dimethylphenylcarbamate) (Chiralpak AD). Both CSPs showed very similar chromatographic properties. The separation factors could be influenced significantly by varying the polar organic modifier added to the mobile phase.     

Detection of d-Serine in rat brain by capillary electrophoresis with laser induced fluorescence detection

A capillary zone electrophoresis method with laser induced fluorescence detection for the chiral separation of highly fluorescent enantiomeric derivatives of d/l-Serine from 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-d/l-Serine) was developed and optimized. Enantiomeric separation of NBD-d/l-Serine was accomplished by using 40 mM hydroxypropyl-beta-cyclodextrin (HP-beta-CD) contained in 100 mM borate buffer, pH 10.0. A 70 cm (effective length of 50 cm) uncoated fused-silica capillary at a voltage of 15 kV was used for the separation. The optimized electrophoretic conditions were subsequently applied to the analysis of d-Serine in rat brain, and satisfactory analytical results with respect to accuracy were obtained. This assay showed acceptable precision, with linearity in the d-Serine concentration range of 0.2-20.0 microM. The limit of detection for d-Serine was 3.0 x 10(-7)M.     

Inactivation of soman by beta-cyclodextrin

Soman (pinacolyl methylphosphonofluoridate), a mixture of four stereoisomers, is inactivated appreciably in Tris buffer, pH 7.40, mu = 0.155 at 25 degrees C by beta-cyclodextrin (cycloheptaamylose, beta-CD). Under these conditions, the dissociation constant Kd of the 1:1 complex formed by beta-CD and soman and the rate constant k2 for the phosphonylation of beta-CD by soman are (0.53 +/- 0.05)mM and (5.9 +/- 0.6) X 10(-2) min-1 respectively. It results that the inactivation of soman by the mono-anion of beta-CD is about 2,600 times faster than the hydrolysis of soman by the hydroxide ion. The inactivation of both P(-) isomers of soman by beta-CD proceeds apparently at the same rate but both P(+) isomers react more slowly. Thus the interaction is stereospecific. The inactivation of soman by beta-CD appears to be as fast in human plasma in vitro as in Tris buffer.     

Solid phase synthesis and biological evaluation of enantiomerically pure wasp toxin analogues PhTX-343 and PhTX-12

PhTX-343 and PhTX-12, analogues of the natural polyamine wasp toxin PhTX-433, were synthesised in 40-60% yields as pure enantiomers using solid phase synthesis techniques. Capillary electrophoresis procedures were developed for chiral separation and determination of enantiomeric purity (ee) of the enantiomers of PhTX-343 and PhTX-12. The methods were optimised with respect to chiral selector, buffer pH, and temperature around the capillary. Thus, rac-PhTX-343 was resolved using a separation buffer containing 30 mM heptakis-(2, 6-di-O-methyl)-beta-cyclodextrin in 50 mM 6-aminocarproic acid (pH 4. 0) at 15 degrees C. rac-PhTX-12 was not resolvable in this system, but could be resolved using a separation buffer containing 10% w/v of dextrin 10, a linear maltodextrin, in 50 mM 6-aminocaproic acid (pH 4.0) at 15 degrees C. Using these methods, the optical purity of the synthetic enantiomers was determined to be ee > 99%. The enantiomers were also characterised by chiroptical methods. The antagonist potency of the enantiomers was tested on nicotinic acetylcholine receptors (human muscle-type nAChR) expressed in TE671 cells, ionotropic glutamate receptors in Xenopus laevis oocytes (expressing recombinant GluR1flop receptors), and locust muscle ionotropic glutamate receptors sensitive to quisqualate (qGluR). The potencies of each pair of enantiomers were similar (eudismic ratio close to 1).     

Determination of the dissociation constants of ropinirole and some impurities and their quantification using capillary zone electrophoresis

Ropinirole, 4-[2-(dipropylamino)ethyl]-1,3-dihydro-2H-indol-2-one, is a potent anti-Parkinson’s disease drug developed by SmithKline Beecham Pharmaceuticals. Capillary zone electrophoresis (CZE) was used for the determination of the dissociation constants of ropinirole and five structurally related impurities, potentially formed during its synthesis and for separation and quantification of these substances. The dissociation constants obtained from the CZE measurements were confirmed by UV spectrophotometry for some of the test compounds, obtaining a good agreement between the values. Careful optimization of the running buffer composition permitted base-line resolution of the six compounds in a borate buffer containing acetonitrile and magnesium sulfate (a 100 mM borate buffer containing 30 mM MgSO₄ and 20 vol.% of acetonitrile). It was shown that CZE can determine the level of these impurities, down to a level of 0.05% of the main component within 15 min.     

High-performance capillary electrophoresis of glycoproteins: the use of modifiers of electroosmotic flow for analysis of microheterogeneity.

High-performance capillary electrophoresis (HPCE) is rapidly gaining acceptance as an analytical tool for the study of biological macromolecules. In the present study, the utility of HPCE for separation of glycoproteins is highlighted using a pure ovalbumin preparation. Ovalbumin, the 43-kDa glycoprotein of avian egg white, is known to be heterogeneous in nature with at least nine different carbohydrate structures having been identified on the single Asn residue. HPCE separation in an 87-cm capillary containing borate buffer and 1 mM putrescine resolves five major protein peaks in less than 30 min under nondenaturing conditions. This effect appears to be specific to glycoproteins since analysis of the nonglycosylated protein carbonic anhydrase under the same conditions showed no enhanced separation. The sodium borate buffer is proposed to play a key role in the separation by preferentially complexing with the diols of specific carbohydrate moieties on ovalbumin. Addition of putrescine enhances resolution by slowing bulk flow through the capillary and allowing electrophoretic separation of what is deduced to be closely related glycoforms of ovalbumin. Dephosphorylation of the ovalbumin with either calf intestinal alkaline phosphatase or potato acid phosphatase results in a shift of all peaks to a more rapid migration time and is consistent with a loss of negative charge. This suggests that all major ovalbumin isoforms are phosphorylated to the same degree and that heterology among ovalbumin isoforms resides solely in the carbohydrate structure. The enhanced resolution obtained with the employment of longer capillaries and modifiers of endo-osmotic flow was not restricted to ovalbumin since partial resolution of pepsin isoforms was observed under the same conditions.     

Synthesis of epoxide and vicinal diol regioisomers from docosahexaenoate methyl esters

Docosahexaenoic acid (22:6(n-3)) was recently shown to be metabolized by liver microsomes to vicinal diol regioisomers. To identify the diols, and to compare their biological actions with those of epoxide precursors, we developed a chemical method to synthesize microgram to milligram amounts of epoxides and corresponding diols. In brief, methylated docosahexaenoate was reacted for 15 min with 0.1 eq m-chloroperoxybenzoic acid. After normal- and reverse-phase high performance liquid chromatography, six products were isolated. The underivatized or hydrogenated products were characterized and identified using capillary gas-liquid chromatography and mass spectrometry. The products were identified as 19,20-, 16,17-, 13,14-, 10,11-, 7,8-, and 4,5-epoxy-docosapentaenoate. Per incubation, the total epoxide yield from 22:6(n-3) was 8.6%. By reincubating unused substrate 10-20 times (cycling), the total epoxide could be increased to 55-70%. As found for epoxides of arachidonic and eicosapentaenoic acids, the yield of individual regioisomers increased as the distance between the targeted double bond and carbomethoxy group increased. Each epoxide regioisomer was hydrolyzed to its corresponding vicinal diol. The gas-liquid chromatographic retention times and mass spectra of the diol products were found to match those of metabolites produced by cytochrome P-450 monooxygenases.     

Enantiomerization of 3-carbethoxy-l,4-benzodiazepin-2-one: combined chiral HPLC and spectroscopic study.

Recently developed chiral HPLC columns CHIRIS AD1 and CHIRIS AD2 have been demonstrated to separate racemic, configurationally unstable ethyl-7-chloro-2-oxo-5-phenyl-2,3-dihydro-1H-1,4-benzodiazepine-3-carboxylate (1) and its 3-methyl congener 2; fast on-column enantiomerization of configurationally unstable 1 was observed, however. Addition of 0.1% of AcOH to the eluting mixture inhibits enantiomerization, whereas the same percentage of Et(3)N completely precludes enantioseparation, suggesting base-catalysis by free beta-aminoethyl groups, present in low percentage in chiral stationary phase (CSP). When both CSPs were prepared under conditions of nonexhaustive acylation by N-DNB-alpha-aminoacids, no separation of 1 was observed. The rate of enantiomerization on CHIRIS AD2 was determined at 25 degrees C, the mechanism is discussed, and experimental results correlated with calculated relative stabilities of the tautomers la-c. Absolute (3S) configuration of (+) enantiomers of 1 and 2 was determined by comparison of their eluation profile to that of (+/-)-3 and (3S)-(+)-3, taking into account relative (psia or psie) configuration of the prevailing conformer in solution.     

Enantiomeric separation of chiral ruthenium(II) complexes using capillary electrophoresis

Capillary zone electrophoresis (CZE) and micellar capillary electrophoresis (MCE) were applied for the enantiomeric separation of nine mononuclear tris(diimine)ruthenium(II) complexes as well as the separation of all stereoisomers of a dinuclear tris(diimine)ruthenium(II) complex. Nine cyclodextrin (CD) based chiral selectors were examined as run buffer additives to evaluate their effectiveness in the enantiomeric separation of tris(diimine)ruthenium(II) complexes. Seven showed enantioselectivity. Sulfated gamma-cyclodextrin (SGC), with four baseline and three partial separations, was found to be the most useful chiral selector. In CZE mode, the derivatized gamma-CDs were more effective than beta-CDs while sulfated CDs work better than carboxymethyl CDs. In MCE mode, hydroxypropyl beta-CD separated the greatest number of tris(diimine) ruthenium(II) complexes. The effects of chiral selector concentration, run buffer pH and concentration, the concentration ratio between chiral selector and other factors were investigated.     

Structure–function relationships of epoxide hydrolases and their potential use in biocatalysis

Background

Chiral epoxides and diols are important synthons for manufacturing fine chemicals and pharmaceuticals. The epoxide hydrolases (EC 3.3.2.-) catalyze the hydrolytic ring opening of epoxides producing the corresponding vicinal diol. Several isoenzymes display catalytic properties that position them as promising biocatalytic tools for the generation of enantiopure epoxides and diols.

Scope of review

This review focuses on the present data on enzyme structure and function in connection to biocatalytic applications. Available data on biocatalysis employed for purposes of stereospecific ring opening, to produce chiral vicinal diols, and kinetic resolution regimes, to achieve enantiopure epoxides, are discussed and related to results gained from structure–activity studies on the enzyme catalysts. More recent examples of the concept of directed evolution of enzyme function are also presented.

Major conclusions

The present understanding of structure–activity relationships in epoxide hydrolases regarding chemical catalysis is strong. With the ongoing research, a more detailed view of the factors that influence substrate specificities and stereospecificities is expected to arise. The already present use of epoxide hydrolases in synthetic applications is expected to expand as new enzymes are being isolated and characterized. Refined methodologies for directed evolution of desired catalytic and physicochemical properties may further boost the development of novel and useful biocatalysts.

General significance

The catalytic power of enzymes provides new possibilities for efficient, specific and sustainable technologies to be developed for production of useful chemicals.