Single-isomer sulfated alpha-cyclodextrins for capillary electrophoresis. Part 2. Hexakis(6-O-sulfo)-alpha-cyclodextrin: synthesis, analytical characterization, and initial screening tests

The second member of the family of single-isomer sulfated alpha-cyclodextrins, the sodium salt of hexakis(6-O-sulfo)-alpha-cyclodextrin (HxS), has been synthesized, analytically characterized, and used as the resolving agent for the capillary electrophoretic separation of the enantiomers of nonionic, weak-acid and weak-base analytes present in our initial screening kit. HxS interacted less strongly with many of the analytes tested than the larger-ring analogs, heptakis(6-O-sulfo)-beta-cyclodextrin (HS) and octakis(6-O-sulfo)-gamma-cyclodextrin (OS). For some of the analytes, the separation selectivities obtained with HxS were complementary to those observed with hexakis(2,3-di-O-acetyl-6-O-sulfo)-alpha-cyclodextrin (HxDAS), HS, and OS. For all analytes, the effective mobilities and separation selectivities as a function of the background electrolyte concentration of HxS followed the trends that were found for HxDAS, HS, and OS.     

Synthesis of mono-6-deoxy-6-N,N,N',N',N'-pentamethylethylenediammonio-cyclomaltoheptaose, a single-isomer, monosubstituted, permanently dicationic beta-CD and its use for enantiomer separations by CE

The dichloride salt of mono-6-deoxy-6-N,N,N',N',N'-pentamethylethylenediammonio-cyclomaltoheptaose (PEMEDA-BCD), the first single-isomer, monosubstituted, permanently dicationic beta-CD has been synthesized, analytically characterized, and used for the capillary electrophoretic separation of the enantiomers of a group of analytes in acidic and basic BGEs. When the concentration of PEMEDA-BCD was changed in the BGEs, the resulting effective mobilities of the analytes and the respective separation selectivities followed the predictions of the ionic strength-corrected charged resolving agent migration model. Good separation selectivities and favorable normalized EOF mobilities allowed for the rapid, efficient separation of the enantiomers of anionic, weak acid and nonionic analytes in the low- and/or high-pH BGEs.     

Experimental verification of a predicted, hitherto unseen separation selectivity pattern in the nonaqueous capillary electrophoretic separation of weak base enantiomers by octakis (2,3-diacetyl-6-sulfato)-gamma-cyclodextrin

The capillary electrophoretic separation of cationic enantiomers with single-isomer multivalent anionic resolving agents was reexamined with the help of the charged resolving agent migration model. Three general model parameters were identified that influence the shape of the separation selectivity and enantiomer mobility difference curves: parameter b, the binding selectivity (K(RCD)/K(SCD)), parameter s, the size selectivity (mu0(RCD)/mu0(SCD)), and parameter a, the complexation-induced alteration of the analyte's mobility (mu0(RCD)/mu0). In addition to the previously observed discontinuity in separation selectivity that occurs as mu(eff) of the less mobile enantiomer changes from cationic to anionic, a new feature, a separation selectivity maximum was predicted to occur in the resolving agent concentration range where both enantiomers migrate cationically provided that (i) K(RCD)/K(SCD) <1 and mu0(RCD)/mu0(SCD) >1 and (K(RCD)mu0(RCD))/(K(SCD)mu0(SCD)) > 1, or (ii) K(RCD)/K(SCD) >1 and mu0(RCD)/mu0(SCD) <1 and (K(RCD)mu0(RCD))/(K(SCD)mu0(SCD)) <1. This hitherto unseen separation selectivity pattern was experimentally verified during the nonaqueous capillary electrophoretic separation of the enantiomers of four weak base analytes in acidic methanol background electrolytes with octakis(2,3-diacetyl-6-sulfato)-gamma-cyclodextrin (ODAS-gammaCD) as resolving agent.     

Single-isomer sulfated alpha-cyclodextrins for capillary electrophoresis: hexakis(2,3-di-O-methyl-6-O-sulfo)-alpha-cyclodextrin, synthesis, analytical characterization, and initial screening tests

The third, concluding member of the family of single-isomer, fully sulfated alpha-cyclodextrins, the sodium salt of hexakis(2,3-di-O-methyl-6-O-sulfo)-alpha-cyclodextrin (HxDMS), has been synthesized on the kilogram scale, completing the nine-member array of the single-isomer, 6-O-sulfo CDs now available. HxDMS was tested for the capillary electrophoretic (CE) resolution of the enantiomers of nonelectrolyte, weak acid and weak base analytes contained in our CD screening kit. HxDMS complexed differently with many of the analytes tested than either its larger-ring analogs, heptakis(2,3-di-O-methyl-6-O-sulfo)-beta-CD (HDMS) and octakis(2,3-di-O-methyl-6-O-sulfo)-gamma-CD (ODMS) or its same-ring, but differently substituted analogs, hexakis(6-O-sulfo)-alpha-CD (HxS) and hexakis(2,3-di-O-acetyl-6-O-sulfo)-alpha-CD (HxDAS). For all analytes, the effective mobilities and separation selectivities as a function of the background electrolyte concentration of HxDMS followed the trends that were found for the other single-isomer, 6-O-sulfo CDs.     

Synthesis of heptakis(2-O-methyl-3-O-acetyl-6-O-sulfo)-cyclomaltoheptaose, a single-isomer, sulfated beta-cyclodextrin carrying nonidentical substituents at all the C2, C3, and C6 positions and its use for the capillary electrophoretic separation of enantiomers in acidic aqueous and methanolic background electrolytes

The sodium salt of heptakis(2-O-methyl-3-O-acetyl-6-O-sulfo)cyclomaltoheptaose (HMAS), the first single-isomer, sulfated beta-cyclodextrin carrying nonidentical substituents at all of the C2, C3, and C6 positions, has been synthesized, analytically characterized, and used for the capillary electrophoretic separation of the enantiomers of a group of 24 weak base pharmaceuticals in acidic aqueous and acidic methanolic background electrolytes. HMAS interacted more strongly with most of the analytes studied than heptakis(2,3-di-O-methyl-6-O-sulfo)cyclomaltoheptaose, but less strongly than heptakis(2,3-di-O-acetyl-6-O-sulfo)cyclomaltoheptaose, the respective analogs with identical substituents at the C2 and C3 positions. The good separation selectivities and favorable normalized electroosmotic flow mobilities allowed for rapid, efficient separation of the enantiomers of 19 of the 24 weak base analytes in the aqueous and methanolic background electrolytes. The trends in the effective mobilities and separation selectivities as a function of the HMAS concentration closely followed the predictions of the ionic strength-corrected charged resolving agent migration model.     

Synthesis of a single-isomer sulfated beta-cyclodextrin carrying nonidentical substituents at all of the C2, C3, and C6 positions and its use for the electrophoretic separation of enantiomers in acidic aqueous and methanolic background electrolytes. Part 2: Heptakis(2-O-methyl-6-O-sulfo) cyclomaltoheptaose

The sodium salt of heptakis(2-O-methyl-6-O-sulfo)cyclomaltoheptaose (HMS), the second single-isomer, sulfated beta-CD carrying nonidentical substituents at all of the C2, C3, and C6 positions, has been synthesized, analytically characterized, and used for the capillary electrophoretic separation of the enantiomers of a group of 23 weak base analytes in acidic aqueous and methanolic BGEs. HMS interacted strongly with only about half of the analytes studied. The good separation selectivities and favorable normalized EOF mobilities allowed for rapid, efficient separation of the enantiomers of 19 of the 23 weak base analytes in the aqueous BGEs, often with separation selectivity values complimentary to those obtained with other single-isomer sulfated CDs. HMS did not prove to be as good a resolving agent in acidic methanolic BGEs as its counterpart, heptakis(2-O-methyl-3-O-acetyl-6-O-sulfo)cyclomaltoheptaose.     

A family of single-isomer,sulfated gamma-cyclodextrin chiral resolving agents for capillary electrophoresis: octa(6-O-sulfo)-gamma-cyclodextrin

The second member of the single-isomer, sulfated gamma-cyclodextrin family, the sodium salt of octa(6-O-sulfo)-gamma-cyclodextrin (OS) has been synthesized, characterized and used to separate the enantiomers of nonelectrolyte, acidic, basic, and ampholytic analytes by capillary electrophoresis in acidic aqueous background electrolytes. The anionic effective mobilities of the nonelectrolyte and anionic analytes increased with increasing concentration of OS. The effective mobilities of strongly complexing cationic analytes became anionic with very low OS concentrations and passed local anionic effective mobility maxima as the OS concentration, and along with it, the ionic strength, of the background electrolyte increased. The effective mobilities of the weakly binding cationic analytes became only slightly anionic at high OS concentration values and did not show the local anionic effective mobility maxima. For nonelectrolyte analytes, separation selectivities decreased with increasing OS concentration. For cationic analytes, separation selectivities were highest where the effective mobilities of the less mobile enantiomers approached zero. OS proved to be a broadly applicable chiral resolving agent.     

Nonaqueous capillary electrophoretic separation of basic enantiomers using heptakis(2,3-dimethyl-6-sulfato)-beta-cyclodextrin.

The enantiomers of 40 basic analytes, mostly pharmaceuticals, were separated by nonaqueous capillary electrophoresis in acidic methanol background electrolytes using the sodium salt of heptakis(2,3-dimethyl-6-sulfato)-beta-cyclodextrin (HDMS-beta-CD). The effective mobilities, separation selectivities, and peak resolution values were determined as a function of the HDMS-beta-CD concentration in the 0-40 mM range and were found to follow the theoretical predictions of the charged resolving agent migration model (CHARM model). Fast, efficient enantiomer separations were achieved for a large number of both very hydrophobic and hydrophilic weak bases.     

Synthesis, analytical characterization and initial capillary electrophoretic use in an acidic background electrolyte of a new, single-isomer chiral resolving agent: heptakis(2-O-sulfo-3-O-methyl-6-O-acetyl)-β-cyclodextrin

The sodium salt of heptakis(2-O-sulfo-3-O-methyl-6-O-acetyl)cyclomaltoheptaose (HAMS), the first single-isomer sulfated β-CD that carries the sulfo group exclusively at the C2 position, has been synthesized. The purity of each synthetic intermediate and of the final product was determined by hydrophilic interaction (HILIC) and reversed-phase HPLC. The structural identity of each intermediate and of the final product was verified by 1-D and 2-D NMR spectroscopy and MALDI-TOF MS. HAMS was used for the capillary electrophoretic separation of the enantiomers of a set of non-ionic and weak base analytes in pH 2.5 background electrolytes. Rapid separations with satisfactory peak resolution values were obtained for most enantiomers using low concentrations of HAMS. The effective mobilities and separation selectivities were dependent on the concentration of HAMS according to the predictions of the charged resolving agent migration model. The separation selectivities observed with HAMS, heptakis(2-O-methyl-3-O-acetyl-6-O-sulfo)cyclomaltoheptaose and heptakis(2-O-methyl-3,6-di-O-sulfo)cyclomaltoheptaose were different for some of the analytes studied in detail.     

Experimental verification of a predicted, previously unseen separation selectivity pattern in the capillary electrophoretic separation of noncharged enantiomers by octakis(2,3-diacetyl-6-sulfato)-gamma-cyclodextrin

The capillary electrophoretic separation of noncharged enantiomers with single-isomer anionic resolving agents is reexamined here with the help of the charged resolving agent migration model. Two general model parameters have been identified that influence the effective mobility, separation selectivity and mobility difference curves of the enantiomers: parameter b, called binding selectivity (K(RCD)/K(SCD)), and parameter s, called size selectivity (mu(o)RCD/mu(o)SCD). Analysis of the model in terms of these parameters indicates that in addition to the known, previously observed separation selectivity vs. resolving agent concentration patterns, a new pattern, increasing separation selectivity with increasing resolving agent concentration, is also possible provided that (i) K(RCD)/K(SCD)<1 and mu(o)RCD/mu(o)SCD>1 and (K(RCD)mu(o)RCD)/(K(SCD)mu(o)SCD)>1, or (ii) K(RCD)/ K(SCD)>1 and mu(o)SCD/mu(o)SCD<1 and (K(RCD)mu(o)RCD)/(K(SCD)mu(o)SCD)<1. This hitherto unseen separation selectivity pattern was experimentally verified during the capillary electrophoretic separation of the enantiomers of O-isopropyl p-nitrophenyl methylphosphonate with the single-isomer octakis(2,3-diacetyl-6-sulfato)-gamma-cyclodextrin as resolving agent.     

Determination of enantiomeric impurity of linezolid by capillary electrophoresis using heptakis-(2,3-diacetyl-6-sulfato)-beta-cyclodextrin

A method for the enantioseparation of linezolid, the first compound of a truly new class of antibiotics-the oxazolidinones, was developed. The elaborated method of linezolid enantiomers separation was successfully performed using an anionic single-isomer cyclodextrin-heptakis-(2,3-diacetyl-6-sulfato)-beta-cyclodextrin (HDAS-beta-CD) as a resolving agent with the help of the charged resolving agent migration model (CHARM model). The best results were obtained with 27.5mM HDAS-beta-CD dissolved in 50mM borate buffer, pH 9.0, 15 degrees C, normal polarity. The facile strategies for the reversal of the enantiomers elution order are also described. Afterwards, the optimized method was validated in terms of sensitivity, linearity, accuracy and precision.     

Use of single-isomer, multiply charge chiral resolving agents for the continuous, preparative-scale electrophoretic separation of enantiomers based on the principle of equal-but-opposite analyte mobilities

A novel approach to continuous, preparative-scale electrophoretic enantiomer separations has been developed based on the observation that stable, equal-but-opposite effective mobilities can be created for the enantiomers of a single-charged analyte by complexing them with a single-isomer, multiply charged resolving agent, provided that the charge of the resolving agent is opposite in sign to that of the uncomplexed analyte enantiomers. When such an analyte-resolving agent system is fed into a continuous, free-flow electrophoretic apparatus, stable, steady-state operating conditions can be established which permit the continuous feeding of the racemic analyte and the collection of pure enantiomers at the opposite sides of the feed stream. This concept is demonstrated via the separation of the enantiomers of terbutaline using heptakis-6-sulfato beta-cyclodextrin as resolving agent, affording production rates as high as 2.8 mg/h in the general-purpose, continuous free-flow electrophoretic system, the Octopus.     

The role of achiral sorbent matrix in chiral recognition of amino acid enantiomers in ligand-exchange chromatography

Summary As an alternative to the known three-point interaction model describing recognition of optical isomers by a chiral resolving agent, a new concept has been developed stating that two interaction points between the resolving agent and the enantiomers are also sufficient for achieving chiral recognition of the latter, provided that the diastereomeric adducts formed by the resolving agent with the enantiomers additionally interact with a non-chiral chromatographic sorbent. This concept is based on the results of ligand-exchange chromatography of -amino acid enantiomers with copper(II) complexes of chiral bifunctional ligands as the resolving agents in chromatographic systems.     

Separation selectivity patterns in the capillary electrophoretic separation of anionic enantiomers by octakis-6-sulfato-gamma-cyclodextrin

The capillary electrophoretic separation of anionic enantiomers with multiply-charged, single-isomer, anionic resolving agents was reexamined with the help of the charged resolving agent migration model. Three general model parameters were identified that influence the shape of the separation selectivity and enantiomer mobility difference curves: parameter b, the binding selectivity (K(RCD)/K(SCD)), parameter s, the size selectivity (micro0RCD/micro0SCD), and parameter a, the complexation-induced alteration of the analyte's mobility (micro0SCD/micro0). Function analysis of the model indicates that there are six unique separation selectivity vs. resolving agent concentration patterns: in two of the patterns, separation selectivity asymptotically increases to the limiting value set by parameter b; in two other patterns, separation selectivity passes a local maximum and asymptotically decreases to the limiting value set by parameter b; and in the last two patterns, separation selectivity passes a local maximum, decreases to unity, then, after reversal of the intrinsic migration order, asymptotically increases to the limiting value set by parameter b. Though the patterns with asymptotically increasing selectivities were observed in earlier work, this paper reports the first experimental verification of the existence of the local selectivity maximum during the capillary electrophoretic separation of the enantiomers of several weak acids in high pH background electrolytes with octakis-6-sulfato-gamma-cyclodextrin as the resolving agent.     

Chiral Resolution of Benzophenone Imine Derivatives of Amino Acid Esters on Chiral Stationary Phases

Abstract

A convenient way of resolving the enantiomers of amino acid esters after their derivatization using benzophenone imine is described. The enantiomers of benzophenone Schiff base derivatives of various amino acid ethyl esters are readily separated on three commercially available chiral stationary phases (CSPs). Among them, CSPs 2 and 3 afford generally the base-line enantioresolution for the analytes studied. From understanding of chromatographic results, a plausible chiral recognition mechanism is discussed.     

Efficient resolution of 2,2'-dihydroxy-1,1'-binaphthyl by inclusion complexation with chiral N-(3-chloro-2-hydroxypropyl)-N,N,N-trimethylammonium chloride

The complete resolution of 2,2[prime or minute]-dihydroxy-1,1[prime or minute]-binaphthyl into its enantiomers by inclusion complexation with a commercially available derivative of choline, is reported. The two enantiomers are recovered in >99% ee from the inclusion complexes by simple dissolution in a diethyl ether-water medium and the resolving agent can be recycled.     

Capillary electrophoretic separation of the enantiomers of organophosphates with a phosphorus stereogenic center using the sodium salt of octakis(2,3-diacetyl-6-sulfo)-gamma-cyclodextrin as resolving agent

The sodium salt of the single-isomer, chiral resolving agent, octakis(2,3-diacetyl-6-sulfo)-gamma-cyclodextrin (ODAS-gammaCD) has been used for the capillary electrophoretic separation of the enantiomers of alkylarylphosphates which carry a phosphorus-based stereogenic center. The effective mobilities and separation selectivities were measured at different ODAS-gammaCD and methanol concentrations to find the conditions under which the minor enantiomers could be adequately quantitated in samples obtained by chemical resolution of the racemic mixtures. This work extends the utility of ODAS-gammaCD to a hitherto unexplored field, the capillary electrophoretic separation of the enantiomers of organophosphorus compounds.     

A resolution of the monodentate P*-chiral phosphine PButC6H4Br-4 and its NMR-deduced absolute configuration

Both enantiomers of the monodentate phosphine PBu^tC₆H₄Br-4 were obtained in an enantiopure state by chromatographic separation of their diastereomeric adducts with a new homochiral ortho-palladated resolving agent derived from α-tert-Bu-substituted tertiary benzylamine. The conformation of the palladacycle and the absolute configuration of the phosphine were determined using ¹H NMR spectroscopy (including NOE technique) and confirmed by an X-ray diffraction study of both diastereomeric complexes. The enantiomers of the resolved phosphine were displaced from the individual diastereomers of the palladium(II) complexes with recovery of the starting resolving agent, and trapped in the form of binuclear coordination complexes of palladium(II).     

Stereoisomer quantification of the beta-blocker drugs atenolol, metoprolol, and propranolol in wastewaters by chiral high-performance liquid chromatography-tandem mass spectrometry

A chiral liquid chromatography-tandem mass spectrometry (HPLC-MS-MS) method was developed and validated for measuring individual enantiomers of three beta-blocker drugs (atenolol, metoprolol, and propranolol) in wastewater treatment plant (WWTP) influents and effluents. Mean recoveries of the pharmaceuticals ranged from 67 to 106%, and the limits of detection of the analytes were 2-17 ng/L in wastewater effluents. The method was demonstrated by measuring, for the first time, the stereoisomer composition of target analytes in raw and treated wastewaters of two Canadian WWTPs. In these trials, racemic amounts of the three drugs were observed in influent of one wastewater treatment plant, but nonracemic amounts were observed in another. Effluents of the two plants contained nonracemic amounts of the drugs. These results indicate that biologically-mediated stereoselective processes that differ among WWTPs had occurred to eliminate individual enantiomers of the target analytes.     

Capillary electrophoretic separation of enantiomers in a high-pH background electrolyte by means of the single-isomer chiral resolving agent octa(6-O-sulfo)-gamma-cyclodextrin

The new, alkali-stable, single-isomer, sulfated gamma-cyclodextrin, the sodium salt of octa(6-O-sulfo)-gamma-cyclodextrin (OS) was used for the first time to separate the enantiomers of non-ionic, acidic, basic and ampholytic analytes by capillary electrophoresis in high-pH aqueous background electrolytes. The effective mobilities and separation selectivities were found to follow trends similar to those observed earlier in acidic aqueous background electrolytes. OS proved to be a broadly applicable chiral resolving agent and afforded adequate peak resolution values with short separation times for a number of non-ionic, weak acid, weak base and ampholytic analytes.