pH-dependence of complexion constants and complex mobility in capillary electrophoresis separations of dipeptide enantiomers

The chiral separation of the LL- and DD-enantiomers of the dipeptides Ala-Tyr, Phe-Phe, and Asp-PheOMe has been investigated at pH 2.5 and pH 3.5 using beta-cyclodextrin (beta-CD), heptakis-(2,6-di-O-methyl)-beta-cyclodextrin, and heptakis-(2,3,6-tri-O-methyl)-beta-cyclodextrin as chiral selectors. According to electrospray mass spectrometry, heptakis-(2,6-di-O-methyl)-beta-cyclodextrin was a mixture of six isomers. Reversal of the enantiomer migration order upon increasing the buffer pH from 2.5 to 3.5 was observed for all peptides with beta-cyclodextrin, for Ala-Tyr and Phe-Phe in the presence of heptakis-(2,3,6-tri-O-methyl)-beta-cyclodextrin, and for Ala-Tyr using heptakis-(2,6-di-O-methyl)-beta-cyclodextrin. The migration behavior could be explained on the basis of the complexation constants and the mobilities of the peptide-cyclodextrin complexes. Both, the binding constants and complex mobilities decreased with increasing pH as the overall-charge of the peptides decreased. While the complexation constants primarily determined the migration order at pH 2.5, complex mobility dominated in most cases at pH 3.5.     

Heptakis(2-O-methyl-3,6-di-O-sulfo)-beta-cyclodextrin: a single isomer, 14-sulfated beta-cyclodextrin for use as a chiral resolving agent in capillary electrophoresis

The first single-isomer, 14-sulfated beta-cyclodextrin, the sodium salt of heptakis(2-O-methyl-3,6-di-O-sulfo)-beta-cyclodextrin (HMdiSu) has been used to separate 24 pharmaceutical weak base enantiomers in pH 2.5 background electrolytes using capillary electrophoresis. For the weakly binding bases, the cationic effective mobilities decreased, approached zero, and then increased again. For the strongly binding bases, the cationic effective mobilities decreased, became anionic at very low concentrations of HMdiSu, passed an anionic mobility maximum, then decreased again as the HMdiSu concentration was increased. Viscosity corrections according to Walden's rule did not eliminate these unexpected effective mobility extrema. The mobility extrema were rationalized by extending the charged resolving agent migration model (CHARM model) to include ionic strength effects.     

Migration order of dipeptide and tripeptide enantiomers in the presence of single isomer and randomly sulfated cyclodextrins as a function of pH

The present study was conducted in order to evaluate the cyclodextrin (CD)-mediated chiral separation of peptide enantiomers as uncharged analytes at pH 5.3 using randomly sulfated beta-cyclodextrin, heptakis-6-sulfato-beta-CD and heptakis-(2,3-diacetyl-6-sulfato)-beta-CD as chiral selectors. Although less effective compared to stronger acidic conditions, the CDs proved to be suitable chiral selectors for the present set of peptides at pH 5.3. The carrier ability of the negatively charged CDs upon reversal of the applied voltage may also be exploited leading to a reversal of the migration order. In addition, reversal of the enantiomer migration order upon increasing the buffer pH from 2.5 to 5.3 was also observed for Ala-Tyr in the presence of randomly sulfated beta-CD, for Ala-Phe, Ala-Tyr, Phe-Phe, Asp-PheNH(2) and Gly-Ala-Phe in the presence of heptakis-6-sulfato-beta-CD, and for Phe-Phe and Ala-Leu in the presence of heptakis-(2,3-diacetyl-6-sulfato)-beta-CD. The migration behavior could be explained on the basis of the complexation constants and the mobilities of the peptide-CD complexes. While a change in the affinity pattern of the CDs upon increasing the pH was observed for some peptides, complex mobility was the primary factor for other peptide-CD combinations affecting the enantiomer migration order at the two pH values studied.     

Enantioseparation of chiral thiobarbiturates using cyclodextrin-modified capillary electrophoresis

The racemates of several chiral thiobarbiturates were separated by using different cyclodextrins in capillary electrophoresis (CE). Six neutral and negatively charged cyclodextrins 1 (CDs) were employed as chiral separators whereof five led to successful separation of enantiomeric thiobarbiturate pairs. The CDs used were the native alpha-CD, beta-CD, gamma-CD, and heptakis-(2,6-di-O-methyl)-beta-cyclodextrin (HDM) as well as heptakis-(2,3-di-O-methyl-6-sulfato)-beta-cyclodextrin (HDMS) and heptakis-(2,3-di-O-acetyl-6-sulfato)-beta-CD (HDAS). Five of the six chiral thiobarbiturates studied could be resolved at a basic pH value of 9.4 and a phosphate buffer concentration of 100 mM in a fused-silica capillary. Structurally related substances showed a similar behavior in separation: 1 and 2 bearing the center of chirality in the side chain at C5 can be best separated using gamma-CD, the N-alkyl-substituted compounds 3 and 4 as well as the N/S-dialkyl-substituted compound 5 could be resolved with HDM. Using the neutral CDs, the migration times were relatively small (< 11 min). 3 and 4 could be also resolved by means of the negatively charged HDMS. In the latter case, the migration time is twice as long as with HDM.     

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.     

Chromatographic evaluation and comparison of three beta-cyclodextrin-based stationary phases by capillary liquid chromatography and pressure-assisted capillary electrochromatography

Enantiomer separations were performed on three beta-cyclodextrin-based chiral stationary phases (CSP) containing the pernaphthylcarbamoylated beta-cyclodextrin (CSP 1), peracetylated beta-cyclodextrin (CSP 2) and permethylated beta-cyclodextrin (CSP 3) as chiral selectors by capillary liquid chromatography and pressure-assisted capillary electrochromatography in this study. Triethylammonium acetate/MeOH or phosphate buffer/MeOH was used as the mobile phase. The experimental factors affecting chiral separations have been examined for each CSP, including pH of the buffers, methanol content and applied voltage. Under optimal separation conditions, a number of racemic compounds were resolved into their enantiomers on three cyclodextrin-based CSP. A comparative study on the performance of three CSP revealed the presence of carbonyl functional groups as well as aromatic rings in the cyclodextrin derivatives, enhanced the interaction between the analytes and CSP, and thus improved enantioselectivity of the CSP.     

Resolution of chiral barbiturates into enantiomers by reversed-phase high-performance liquid chromatography using methylated beta-cyclodextrins

The correlation between the capacity factors of enantiomers of chiral barbiturates and the concentrations of beta-cyclodextrin, heptakis(2,6-di-O-methyl)-beta-cyclodextrin and heptakis(2,3,6-tri-O-methyl)-beta-cyclodextrin dissolved in the mobile phase was studied using LiChrosorb RP-18 as the stationary phase. Owing to the very strong adsorption of permethylated beta-cyclodextrin on the ODS surface a chiral stationary phase is generated dynamically and forms complexes with the solutes; this mechanism has been found to be the only factor responsible for the chiral recognition of the investigated compounds at all applied concentrations. The inclusion of barbiturates in the cavities of permethylated beta-cyclodextrin involves a distinct and entirely new kind of enantioselectivity compared with that observed for beta-cyclodextrin and its dimethyl derivative. Using permethylated beta-cyclodextrin baseline resolutions have been obtained with barbiturates containing a chiral centre in the heterocyclic ring or in the aliphatic side-chain.     

Chiral resolution of atropine, homatropine and eight synthetic tropinyl and piperidinyl esters by capillary zone electrophoresis with cyclodextrin additives

Chiral resolution of atropine, homatropine and eight synthetic tropinyl and piperidinyl esters were studied by capillary zone electrophoresis with cyclodextrin additives. Atropine and eight synthetic derivatives were successfully resolved by heptakis-(2,3,6-tri-O-methyl)-beta-cyclodextrin (TM-beta-CD) at concentrations ranging from 10 to 40 mM. Homatropine was baseline resolved by 10 mM beta-cyclodextrin and hydroxypropyl-beta-cyclodextrin (HP-beta-CD), respectively. The developed method was employed for the determination of atropine enantiomers in human serum.     

Enantioseparation of chiral sulfoxides and sulfinate esters by capillary electrophoresis

Forty-one chiral sulfoxides and sulfinate esters were separated using sulfated beta-cyclodextrin and carboxymethyl beta-cyclodextrin as chiral selectors. Binding constants of some analytes to both chiral selectors were measured in order to examine and help explain the observed migration behavior and enantioselectivity trends. Overall, sulfated beta-cyclodextrin separated a greater number of compounds, and had better separating capabilities than did carboxymethyl beta-cyclodextrin for these analytes. This was true even though all of the analytes showed much stronger binding to carboxymethyl beta-cyclodextrin than to sulfated beta-cyclodextrin. General procedures to optimize the separation, by varying pH, selector concentration, and organic modifier concentration were examined and discussed. Chiral selector concentration had the greatest effect on enantioseparation, with higher concentrations of selector giving better peak-to-peak separations. Organic modifier had an adverse affect on resolution, with increasing amounts giving lower mobility differences. Lastly, pH had only a minimal effect on separation.     

Heptakis(6-amino-6-deoxy)-beta-cyclodextrin as a chiral selector for the separation of anionic analyte enantiomers by capillary electrophoresis

A hepta-substituted beta-cyclodextrin bearing seven amino groups, heptakis(6-amino-6-deoxy)-beta-cyclodextrin (per-6-NH2-beta-CD) was successfully used as a chiral selector for the enantioseparation of different anionic analytes. The running buffer pH and chiral selector concentration were the studied parameters crucial in achieving the maximum possible enantioresolution. Enantiomeric separation of a mixture of seven carboxybenzyl-amino acids was achieved in 24 min. Excellent resolution was obtained for carboxybenzyl-tryptophan (Rs = 11.2).     

Synthesis of phosphoryl-tethered beta-cyclodextrins and their molecular and chiral recognition thermodynamics

Two novel phosphoryl-bridged bis- and tris(beta-cyclodextrin)s of different tether lengths, i.e., bis[m-(N-(6-cyclodextryl)-2-aminoethylaminosulfonyl)phenyl]-m-(chlorosulfonyl)phenylphosphine oxide (5) and tris[m-(N-(6-cyclodextryl)-8-amino-3,6-diazaoctylaminosulfonyl)phenyl]phosphine oxide (6), have been synthesized by reactions of 6-oligo(ethylenediamino)-6-deoxy-beta-cyclodextrins with tris[m-(chlorosulfonyl)phenyl]phosphine oxide. The complex stability constants (K(S)), standard molar enthalpy (Delta H degrees ), and entropy changes (Delta S degrees ) were determined at 25 degrees C for the inclusion complexation of phosphoryl-modified bis- and tris-cyclodextrins (5 and 6, respectively), mono[6-O-(ethoxyhydroxyphosphoryl)]-beta-cyclodextrin (2), mono[6-O-(diethylamino-ethoxyphosphoryl)]-beta-cyclodextrin (3), and mono[6-O-(diphenoxyphosphoryl)]-beta-cyclodextrin (4) with representative alicyclic and N-Cbz-D/L-alanine guests in 0.1 M phosphate buffer solution at pH 7.2 by means of titration microcalorimetry. The thermodynamic parameters obtained indicate that the charge-dipole interaction between the phosphoryl moiety and the negatively charged guests, as well as the conformational difference of modified beta-cyclodextrins in aqueous solution, significantly contribute to the inclusion complexation and the enhanced chiral discrimination. The interactions and binding modes between the hosts and chiral guests were further studied by two-dimensional NMR spectroscopy to elucidate the influence of the structural features of hosts on their increased chiral recognition ability and to establish the correlation between the conformation of the resulting complexes and the thermodynamic parameters obtained.     

Capillary electrophoretic chiral separation of Cinchona alkaloids using a cyclodextrin selector

A new capillary electrophoretic method for the chiral separation of four major Cinchona alkaloids (quinine/quinidine and cinchonine/cinchonidine) was developed using heptakis-(2,6-di-O-methyl)-beta-cyclodextrin as the chiral selector. The inner walls of the separation capillary were modified with a thin polyacrylamide layer, which substantially reduced the electroosmotic flow and improved the chiral resolution and the reproducibility of the migration time of the analytes. Various operation parameters were optimised, including the pH, the capillary temperature, the concentration of the background electrolyte, and the concentration of the chiral selector. Baseline separation of the two diastereomer pairs was achieved in 12 minutes in ammonium acetate background electrolyte pH 5.0 with addition of cyclodextrin in a concentration of 3 mM or higher.     

Combining magnetic resonance spectroscopies, mass spectrometry, and molecular dynamics: investigation of chiral recognition by 2,6-di-O-methyl-beta-cyclodextrin

EPR spectroscopy has been employed to investigate the formation of complexes between heptakis-(2,6-O-dimethyl)-beta-cyclodextrin (DM-beta-CD) and different enantiomeric pairs of chiral nitroxides of general structure PhCH2NO.CH(R)R'. Accurate equilibrium measurements of the concentrations of free and included radicals afforded the binding constant values for these nitroxides. The relationship between the stereochemistry of the DM-beta-CD complexes and the thermodynamics of complexation was elucidated by correlating EPR data with 1H-1H NOE measurements carried out on the complexes between DM-beta-CD and the structurally related amine precursors of nitroxides. NOE data suggested that inclusion of the stereogenic center in the DM-beta-CD cavity occurs only when the R substituent linked to the chiral carbon contains an aromatic ring. For these types of complexes, molecular dynamics simulation indicated that the depth of penetration of the stereogenic center into the cyclodextrin cavity is determined by the nature of the second substituent (R') at the asymmetric carbon and is responsible for the observed chiral selectivity. Analysis of mass spectra showed that, for the presently investigated amines, electrostatic external adducts of CDs with protonated amines are detected by ESI-MS.     

七-(2,6-二-O-甲基)-β-环糊精对1,1'-联萘酚对映体手性识别的电喷雾质谱研究

利用电喷雾质谱研究了β-环糊精、七-(2,6-二-O-甲基)-β-环糊精作为手性识别试剂对1,1'-联萘酚对映体的手性识别效应. 实验结果表明, 在气相中, β-环糊精与七-(2,6-二-O-甲基)-β-环糊精都可以与联萘酚形成非共价复合物. 对形成的复合物的串联质谱研究表明, β-环糊精不能识别联萘酚对映体, 而七-(2,6-二-O-甲基)-β-环糊精对联萘酚对映体有较强的手性识别效应. 进一步研究表明七-(2,6-二-O-甲基)-β-环糊精与联萘酚对映体混合比例以及CID能量对于手性识别并无影响.     

Comparative study on the enantiomer separation of 1,1'-binaphthyl-2,2'diyl hydrogenphosphate and 1,1'-bi-2-naphthol by liquid chromatography and capillary electrophoresis using single and combined chiral selector systems

The chiral recognition ability of single and dual selectors, that were used as additives, have been investigated by HPLC and CE. Native beta- and gamma-cyclodextrins, permethylated beta-cyclodextrin, hydroxypropyl-beta-cyclodextrin, cholic acid and taurodeoxycholic acid sodium salts were applied as chiral selectors, whereas the atropisomers of 1,1'-binaphthyl-2,2'-diyl hydrogenphosphate, and 1,1'-bi-2-naphthol served as model compounds. It was found that all investigated selectors, except for gamma-cyclodextrin, display the same affinity pattern for binaphthyl enantiomers, i.e., binding the S more strongly than the R enantiomer. However, the differences in the phase distribution of chiral selectors led to the opposit elution order of enantiomers: with cyclodextrins, the first eluted is S enantiomer, while R is the first eluted for bile salts. Under the conditions studied, cyclodextrins (except gamma-cyclodextrin), as well as cholic acid sodium salts acting singly, enable the separation of 1,1'-binaphthyl-2,2'-diyl hydrogenphosphate enantiomers both by HPLC and CE methods, while 1,1'-bi-2-naphthol enantiomers were resolved only under CE conditions with permethylated cyclodextrin or bile salts. In both techniques the application of dual systems could improve resolution or make it worse (oreven cancel), depending on the sign of enantioselectivity of particular selectors, their concentrations and localization: mobile or stationary phase. It has been found that the mechanism of separation as well as interactions occurring between two selectors may be followed by using combined HPLC and CE methods. The obtained results proved that, as well as beta-CD, TM-beta-D and gamma-CD also form inclusion complexes with cholic acid sodium salts. The reversal of elution order may be realized by two procedures: changing a single selector, i.e., cyclodextrin on cholic acid sodium salt or vice versa, and by changing the proportion of selectors in the combined bile salt-cyclodextrin system.     

毛细管电泳法手性拆分14种二肽

以咔唑-9-乙基氯甲酸酯(CEOC)作为柱前衍生试剂,采用毛细管电泳对14种二肽进行了手性拆分。以5种二肽为代表,考察了缓冲液种类、浓度、pH值、二元手性选择剂的组合配比等因素对二肽的拆分效果,优化了实验条件。在各自的优化条件下,14种二肽手性拆分的分离度均在3.63以上,最高分离度可达43.14(Gly-Ala)。     

Chiral separation of dansyl amino acids in capillary electrophoresis using mono-(3-methyl-imidazolium)-beta-cyclodextrin chloride as selector

Enantioseparations of fourteen dansyl amino acids were achieved by using a positively-charged single-isomer beta-cyclodextrin, mono-(3-methyl-imidazolium)-beta-cyclodextrin chloride, as a chiral selector. Separation parameters such as buffer pH, selector concentration, separation temperature, and organic modifier were investigated for the enantioseparation in order to achieve the maximum possible resolution. Chiral separation of dansyl amino acids was found to be highly dependent on pH since the degree of protonation of these amino acids can alter the strength of electrostatic interaction and/or inclusion complexation between each enantiomer and chiral selector. In general, the chiral resolution of dansyl amino acids was enhanced at higher pH, which indicates that the carboxylate group on the analytes may interact with the imidazolium group of cationic cyclodextrin. For most analytes, a distinct maximum in enantioresolution was obtained at pH 8.0. Moreover, the chiral separation can be further improved by careful tuning of the separation parameters such as higher selector concentration (e.g. 10 mM), lower temperature, and addition of methanol. Enantioseparation of a standard mixture of these dansyl amino acids was further achieved in a single run within 30 min.     

Enantiomeric separation of glycyl dipeptides by capillary electrophoresis with cyclodextrins as chiral selectors

Uncharged cyclodextrins were tested as chiral selectors for the enantiomeric separation of 13 glycyl dipeptides with capillary electrophoresis. Initial experiments were performed on 10 mmol/L of a cyclodextrin in 0.1 mol/L phosphoric acid -0.088 mol/L triethanolamine. Some of the resolved dipeptides were nonaromatic, which is noteworthy since, to our knowledge, no examples of the separation of small, nonaromatic molecules have been published. Mobility difference plots for Gly-DL-Leu and Gly-DL-Phe with heptakis(2,6-di-O-methyl)-beta-cyclodextrin showed relatively flat profiles in a large concentration range, which is an advantage for the development of robust quantitative analytical methods. The use of a background electrolyte (BGE) solution with pH 3.0 gave irreproducible results for two of the dipeptides, the acidic Gly-DL-Asp and Gly-DL-Glu; this pH is not advisable for the development of robust methods for these two peptides. The need for purer chiral selectors was demonstrated by comparing different batches of heptakis(2,6-di-Omethyl)-beta-cyclodextrin from the same supplier. A BGE consisting of malonic acid and triethanolamine was introduced to give better buffer capacity than the original BGE at pH 3.0.     

Comparing cyclodextrin derivatives as chiral selectors for enantiomeric separation in capillary electrophoresis

A total of 26 different cyclodextrin (CD) derivatives with different functional groups and degrees of substitution were tested against 35 basic pharmaceutical compounds in an effort to investigate their effectiveness as chiral selectors for enantiomeric separation in capillary electrophoresis (CE). Testing was performed under the same conditions using a low pH buffer (25 mM phosphate buffer at pH approximately 2.5). Five CD derivatives, namely, highly sulfated-beta-CD, highly sulfated-beta-CD, hydroxypropyl-beta-CD (degree of substitution approximately 1), heptakis-(2,6-O-dimethyl)-beta-CD, and heptakis(2,3,6-O-trimethyl)-beta-CD were identified to be most effective for enantiomeric separations and have a wide range of enantiomeric selectivity towards the model compounds. Over 90% of the model compounds were enantiomerically resolved with the five identified CD derivatives, at a minimum resolution of 0.5. An additional 20 compounds were also tested to demonstrate the validity of the identified CD derivatives. The five CD derivatives were recommended as the starting chiral selectors in developing enantiomeric separation methods by CE.     

Spectrophotometric and calorimetric titration studies on molecular recognition of camphor and borneol by nucleobase-modified beta-cyclodextrins

A series of modified beta-cyclodextrins with nucleobase substituents, that is, mono(6-ade-6-deoxy)-beta-cyclodextrin (2) and mono(6-ura-6-deoxy)-beta-cyclodextrin (3) as well as mono(6-thy-6-deoxy)-beta-cyclodextrin (4), were selected as molecular receptors to investigate their conformation and inclusion complexation behaviors with some chiral molecules, that is, (+)-camphor, (-)-camphor, (+)-borneol, and (-)-borneol, by spectrophotometric and microcalorimetric titrations in aqueous phosphate buffer solution (pH 7.2) at 298.15 K. Circular dichroism and NMR studies demonstrated that these nucleobase-modified beta-cyclodextrins adopted a co-inclusion mode upon complexation with guest molecules; that is, the originally self-included nucleobase substituents of the host did not move out from the beta-cyclodextrin cavity, but coexisted with guest molecule in the beta-cyclodextrin cavity upon inclusion complexation. Significantly, these nucleobase-modified beta-cyclodextrins efficiently enhanced the molecular binding ability and the chiral recognition ability of native beta-cyclodextrin, displaying enantioselectivity up to 3.7 for (+)-camphor/(-)-camphor pair by 2 and 3.5 for (-)-borneol/(+)-borneol pair by 3. The enhanced molecular/chiral recognition abilities of 2-4 toward (+/-)-camphor were mainly attributed to the increased entropic gains due to the extensive desolvation effects, while the favorable enthalpic gains originating from the good size-fit relationship as well as the hydrogen bond interactions between host and guest result in the enhanced molecular/chiral recognition abilities of 2-4 toward (+/-)-borneol.