Enantioseparation Using Cellulose Tris(3,5-dimethylphenylcarbamate) as Chiral Stationary Phase for HPLC: Influence of Molecular Weight of Cellulose.

The cellulose oligomers with different degrees of polymerization (DP), 7, 11, 18, 24, 26, 40 and 52, were prepared by hydrolysis of microcrystalline cellulose with phosphoric acid. These oligomers including the starting microcrystalline cellulose (DP 124) were converted to tris(3,5-dimethylphenylcarbamate) (CDMPC) derivatives by the reaction with an excess of 3,5-dimethylphenyl isocyanate to be used as the chiral stationary phase (CSP) in high-performance liquid chromatography (HPLC). The structures of the CDMPC derivatives were investigated by infrared spectroscopy (IR), ¹H-NMR, circular dichroism (CD) and size exclusion chromatography (SEC), and the DPs of the derivatives estimated by SEC agreed with those estimated by ¹H-NMR. After coating the derivatives on silica gel, their chiral recognition abilities were evaluated using eight racemates under a normal phase condition with a hexane-2-propanol (99/1) mixture as an eluent. The chiral recognition abilities of 7- and 11-mers, particularly the former, were lower than those of the higher oligomers from DP 18 to 52, which had rather similar abilities to that of 124-mer, although the abilities depended on the racemates. DP 18 seems to be sufficient for CDMPC to exhibit chiral recognition similar to that of the CDMPC with larger DPs.

Immobilized-type chiral packing materials for HPLC based on polysaccharide derivatives.

The polysaccharide-based chiral packing materials (CPMs) for high-performance liquid chromatography (HPLC) have been recognized as the most powerful ones for the analyzing and preparative separating of the chiral compounds. These CPMs have been conventionally prepared by coating polysaccharide derivatives on a silica gel support. This means that the solvents, which swell or dissolve the derivatives on the silica gel and reduce the performance of the chiral columns, do not allow to be applied as components of the eluents. Therefore, the polysaccharide-based CPMs can be used with a rather limited number of eluents. In order to enhance the versatility of the eluent selection for more practical and economical chromatographic enantioseparations, the polysaccharide derivatives must be immobilized onto the silica gel. This review summarizes our latest studies on the development of the immobilized-type CPMs via the radical copolymerization and the polycondensation of the polysaccharide derivatives bearing small amounts of vinyl groups and alkoxysilyl groups, respectively.

Immobilization of polysaccharide derivatives onto silica gel Facile synthesis of chiral packing materials by means of intermolecular polycondensation of triethoxysilyl groups.

The 3,5-dimethylphenylcarbamates of cellulose and amylose bearing a small amount of 3-(triethoxysilyl)propyl residues were synthesized by a simple process and efficiently immobilized onto a silica gel support by intermolecular polycondensation of the triethoxysilyl groups. The obtained chiral packing materials (CPMs) were evaluated by high-performance liquid chromatography. The polysaccharide derivatives containing about 1-2% of the 3-(triethoxysilyl)propyl residue were efficiently immobilized with a high chiral recognition. The immobilized CPMs could be used with the eluents containing chloroform and tetrahydrofuran, which cannot be used with the conventional coated-type CPMs. By using these eluents, the chiral recognitions for many racemates could be improved.

Influence of vinyl monomers and temperature on immobilization of cellulose 3,5-dimethylphenylcarbamate onto silica gel as chiral stationary phases for high-performance liquid chromatography.

Cellulose 3,5-dimethylphenylcarbamates bearing a low content of a vinyl group at the 6-position on the glucose units were synthesized by a previously developed regioselective method and chemically immobilized onto a vinylized silica gel as chiral stationary phases (CSPs) for high-performance liquid chromatography (HPLC). The immobilization of the derivatives was performed through a radical polymerization reaction with AIBN as the initiator in the presence of toluene. The effects of vinyl monomers, such as isoprene, 2,3-dimethyl-1,3-butadiene (DMBD), ethylene glycol dimethacrylate (EDMA) and 1,5-hexadiene, on the immobilization and enantioselectivities of the derivatives were investigated. The effect of the temperature used for the radical polymerization reaction on the immobilization was also examined. In addition, the direct comparison of the chiral recognition abilities of the laboratory-made and commercially available columns was discussed.

High-performance liquid chromatographic enantioseparations on capillary columns containing monolithic silica modified with amylose tris(3,5-dimethylphenylcarbamate).

Monolithic capillary columns containing native silica were modified by in situ coating with amylose tris(3,5-dimethylphenylcarbamate) and applied for enantioseparations in capillary liquid chromatography. Capillary columns were examined for 10 standard racemic compounds in order to compare the performance of monolithic silica columns with the common, 4.6mm I.D. high-performance liquid chromatographic columns packed with particulate silica. The effects of polysaccharide coating and of the linear velocity of the mobile phase on peak performance were studied. Enantioseparations with an analysis time below 1min were achieved for some chiral analytes.

Cellulose dimethylphenylcarbamate-bonded carbon-clad zirconia for chiral separation in high performance liquid chromatography.

Porous zirconia particles are very robust material and have received considerable attention as a stationary phase support for HPLC. We prepared cellulose dimethylphenylcarbamate-bonded carbon-clad zirconia (CDMPCCZ) as a chiral stationary phase (CSP) for separation of enantiomers of a set of 14 racemic compounds in normal phase (NP) and reversed-phase (RP) liquid chromatography. Retention and enantioselectivity on CDMPCCZ were compared to those on CDMPC-coated zirconia (CDMPCZ) to see how the change in immobilization method of the chiral selector affects the retention and chiral selectivity. In NPLC, retention was longer and the number of resolved racemates was smaller on CDMPCCZ than on CDMPCZ. However, chiral selectivity factors for some resolved racemates were better on CDMPCCZ than on CDMPCZ. The longer retention on CDMPCCZ is likely due to strong, non-chiral discriminating interactions with the carbon layer on CDMPCZ. In RPLC only two racemates were resolved on CDMPCCZ, but retention times were shorter than, and resolutions were comparable to, those in NPLC, indicating a potential for improving chromatographic performance of the CDMPCCZ column in RPLC with optimized column preparation and separation conditions.

High-performance liquid chromatographic enantioseparation using chitin carbamate derivatives as chiral stationary phases.

Chitin carbamate derivatives including 4-substituted and 3,5-disubstituted phenylcarbamates, 1-phenylethylcarbamates, and cycloalkylcarbamates were synthesized and coated on macroporous silica gel to evaluate their chiral recognition abilities as chiral stationary phases (CSPs) for high-performance liquid chromatography (HPLC). Among the derivatives, the 3,5-dimethylphenyl, 4-chlorophenyl, and 4-trifluoromethylphenylcarbamates showed relatively high-chiral recognition abilities when a hexane-2-propanol mixture was used as the eluent. The CSPs based on the chitin 3,5-dimethylphenyl and 3,5-dichlorophenylcarbamates could be stably used in the presence of chloroform and ethyl acetate as a component of the eluents, and a few racemates were more sufficiently resolved by the addition of a small amount of chloroform in the mobile phase. Some racemates were more efficiently resolved under the reversed phase condition.

High-performance liquid chromatographic enantioseparations on monolithic silica columns containing a covalently attached 3,5-dimethylphenylcarbamate derivative of cellulose.

Covalent immobilization of 3,5-dimethylphenylcarbamate derivative of cellulose was performed in situ onto native silica monoliths cladded in a 50 mm x 4.6 mm polyether ether ketone high-performance liquid chromatographic (HPLC) column. The covalent attachment of cellulose derivative in the range of 16-19% (w/w) was performed via an epoxide moiety. The column obtained by this technique combines the high enantiomer-resolving ability of the polysaccharide derivative with favourable dynamic properties of monolithic HPLC columns. The covalent attachment of the cellulose derivative enables this column to be used in combination with the mobile phases which are incompatible with coated-type polysaccharide columns due to solubility of chiral selector in some organic solvents.

Comparative enantioseparation of selected chiral drugs on four different polysaccharide-type chiral stationary phases using polar organic mobile phases.

The enantiomers of randomly selected chiral drugs and drug analogs of various structural and pharmacological groups were resolved on four different polysaccharide-type chiral stationary phases (CSP) using pure methanol and acetonitrile as mobile phases. Polysaccharide phenylester type CSP, Chiralcel-OJ although resolving the enantiomers of some chiral drugs was less universal in the combination with methanol and acetonitrile as mobile phases. Among polysaccharide phenylcarabamates amylose tris(3,5-dimethylphenylcarbamate) (Chiralpak-AD) was superior over the corresponding cellulose derivative, cellulose tris(3,5-dimethylphenylcarbamate) (Chiralcel-OD). However, another derivative of cellulose, namely, cellulose tris(3,5-dichlorophenylcarbamate) (CDCPC) exhibited higher chiral recognition ability compared to Chiralpak-AD material. This study confirms previous findings about the applicability of polysaccharide type CSPs in so called polar organic mode as well as shows high potential of CDCPC as a practically useful CSP for High performance liquid chromatography (HPLC) enantioseparations.

Comparative study on the application of capillary liquid chromatography and capillary electrochromatography for investigation of enantiomeric purity of the contraceptive drug levonorgestrel.

In the last few years, it has been shown that capillary electrochromatography (CEC) is a promising technique for enantioseparations. However, to date almost no studies are published on a critical comparison of CEC and its pressure-driven counterpart, capillary liquid chromatography (CLC) for real samples. In this study, the goal was to compare CLC and CEC for the determination of the enantiomeric purity of the contraceptive drug levonorgestrel and its pharmaceutical formulation. The study prevailed that not all potential advantages of CEC over CLC can easily be transformed in a real gain of detection limit of the enantiomeric impurity. However, certain advantages of CEC over CLC have been unambiguously shown.

Enantioseparation using ortho- or meta-substituted phenylcarbamates of amylose as chiral stationary phases for high-performance liquid chromatography.

Six ortho- and six meta-substituted phenylcarbamate derivatives of amylose were prepared and their chiral recognition abilities were evaluated as chiral stationary phases (CSPs) for high-performance liquid chromatography (HPLC). The substitution at the meta-position on the aromatic ring was more preferable than that at the ortho-position to obtain CSPs with a high chiral recognition ability, and the introduction of either an electron-withdrawing or electron-donating substituent can improve the chiral resolving power of the meta-substituted phenylcarbamates of amylose. The chiral recognition ability of the amylose phenylcarbamates and elution order of the enantiomers were significantly dependent on the position, nature and number of the substituents on the phenyl group. Correlations between the chiral recognition ability and the N-H frequencies in the IR spectra and the chemical shifts of the N-H protons in the (1)H NMR spectra of the carbamate moieties of the amylose derivatives were discussed. The structures of the amylose derivatives were also investigated by circular dichroism spectroscopy.

Organic-inorganic hybrid materials for efficient enantioseparation using cellulose 3,5-dimethylphenylcarbamate and tetraethyl orthosilicate.

The hybrid bead-type chiral packing material (CPM) for preparative enantioseparation has been prepared from the cellulose 3,5-dimethylphenylcarbamate containing a small number of 3-(triethoxysilyl)propyl groups in the presence of tetraethyl orthosilicate, by a sol-gel reaction in an aqueous surfactant solution. The obtained hybrid bead-type CPM was packed into a column and evaluated by high-performance liquid chromatography. When compared with the commercially available Chiralpak IB, which is prepared by the immobilization of cellulose 3,5-dimethylphenylcarbamate on silica gel, the hybrid bead-type CPM was shown to exhibit a similar chiral recognition and possess a higher loading capacity.

Enantioseparation using urea- and imide-bearing chitosan phenylcarbamate derivatives as chiral stationary phases for high-performance liquid chromatography.

Completely deacetylated chitosan was prepared by the treatment of commercial chitosan with 50% aqueous NaOH, and then derivatized into several new chitosan phenylcarbamate derivatives having a urea and an imide moiety at the 2-position of the glucosamine ring by the reaction with isocyanate and phthalic anhydride/isocyanate, respectively. The chitosan derivatives were coated on macroporous silica gel and evaluated as chiral stationary phases (CSPs) for high-performance liquid chromatography. The chiral recognition ability of the chitosan derivative was improved using the completely deacetylated chitosan. Among the novel chitosan derivatives, the 3,5-dimethyl-, 3,5-dichloro-, and 3,4-dichlorophenylcarbamate derivatives were found to possess relatively high chiral resolution abilities. The CSPs based on the chitosan phenylcarbamate-urea and -imide derivatives were stable in the presence of chloroform and ethyl acetate as a component of the eluents, and some racemates were better resolved by such eluents. The dichlorophenylcarbamate-imide derivatives showed a high chiral recognition for metal acetylacetonate complexes. The enantiomerization of Al(acac)3 was performed on the chitosan 3,5-dichlorophenylcarbamate-imide derivative CSP and the resulting chromatogram showed a 26% (+)-isomer enrichment.

Preparation and chiral recognition ability of crosslinked beads of polysaccharide derivatives.

The spherical beads consisting of cellulose 3,5-dimethylphenylcarbamate with partial hydroxyl groups were prepared to be used as chiral packing materials (CPMs) for HPLC. The beads were obtained without using macroporous silica gel, which is usually used as the support of the CPMs based on the polysaccharide derivatives. After the crosslinking in the bead with diisocyanates, such as 4,4'-diphenylmethane diisocyanate (MDI), 4,4'-dibenzyl diisocyanate (DBDI), tolylene-2,4-diisocyanate (TDI), and m-xylylene diisocyanate (XDI), the obtained beads were packed into an HPLC column. As the content of the hydroxyl groups of the cellulose derivatives decreased, the obtained CPM exhibited a higher chiral recognition ability. The beads possessed a higher loading capacity than the CPM prepared by coating the cellulose derivative on silica gel. The crosslinked beads could be used with the eluent containing chloroform. The amylose derivative beads were also prepared as a CPM for chiral HPLC.

Immobilized polysaccharide derivatives: chiral packing materials for efficient HPLC resolution.

Polysaccharide-based chiral packing materials (CPMs) for high-performance liquid chromatography have frequently been used not only to determine the enantiomeric excess of chiral compounds but also to preparatively resolve a wide range of racemates. However, these CPMs can be used with only a limited number of solvents as mobile phases because some organic solvents, such as tetrahydrofuran, chloroform, and so on, dissolve or swell the polysaccharide derivatives coated on a support, e.g., silica gel, and destroy their packed columns. The limitation of mobile phase selection is sometimes a serious problem for the efficient analytical and preparative resolution of enantiomers. This defect can be resolved by the immobilization of the polysaccharide derivatives onto silica gel. Efficient immobilizations have been attained through the radical copolymerization of the polysaccharide derivatives bearing small amounts of polymerizable residues and also through the polycondensation of the polysaccharide derivatives containing a few percent of 3-(triethoxysilyl)propyl residue.

One-pot synthesis of polysaccharide 3,5-dimethylphenylcarbamates having a random vinyl group for immobilization on silica gel as chiral stationary phases.

Different kinds of vinyl groups were randomly introduced onto the glucose units of cellulose 3,5-dimethylphenylcarbamates by a one-pot method using the bifunctional reagents dec-1-ene-10-isocyanate, 2-isocyanatoethyl methacrylate, and methacryloyl chloride. The chiral recognition properties of the prepared derivatives were then evaluated by coating and immobilizing them on silica gel as HPLC packing material. Immobilization was carried out by radical copolymerization with a vinyl comonomer, 1,5-hexadiene, in toluene at 80 degrees C. The effects of the structures and content of the vinyl groups on the immobilization and on enantioseparations were investigated. This one-pot method was also extended to the synthesis of amylose 3,5-dimethylphenylcarbamates having a random vinyl group. Comparisons of the chiral resolution powers of our laboratory-made packing materials and the newly commercialized Chiralpak IA with immobilized amylose 3,5-dimethylphenylcarbamate and Chiralpak IB with immobilized cellulose 3,5-dimethylphenylcarbamate are discussed.

Enantioseparation using alkoxyphenylcarbamates of cellulose and amylose as chiral stationary phase for high-performance liquid chromatography.

Cellulose and amylose phenylcarbamates having one or two alkoxy groups on a phenyl ring were synthesized, and their chiral recognition abilities as chiral stationary phases for HPLC were evaluated. Compared to the 4-methoxyphenylcarbamates of cellulose and amylose, which are known to show a poor chiral recognition, the 3-methoxyphenylcarbamates exhibited much higher chiral recognitions. For cellulose derivatives, as the bulkiness of the 3-alkoxy group increased, the chiral recognition ability increased. On the other hand, for the amylose derivatives, a clear relation between the chiral recognition and the bulkiness of the alkoxy group was not observed, and the 3-methoxy, ethoxy, and isopropoxyphenylcarbamates showed relatively high chiral recognitions. The introduction of two methoxy groups to the meta-positions decreased the chiral recognition ability. In order to discuss the relationship between the structure and chiral recognition ability of the alkoxyphenylcarbamates, their molecular models were constructed.

High-performance liquid chromatographic enantioseparations on capillary columns containing crosslinked polysaccharide phenylcarbamate derivatives attached to monolithic silica.

Monolithic capillary columns containing native silica gel were covalently modified with 3,5-disubstituted phenylcarbamate derivatives of cellulose and amylose and applied for enantioseparations in capillary LC. The method previously used for covalent immobilization of polysaccharide phenylcarbamate derivatives onto the surface of microparticulate silica gel was successfully adapted for in situ modification of monolithic fused-silica capillary columns. The effects of the nature of polysaccharide and the substituents, as well as of multiple covalent immobilization of polysaccharide derivative on chromatographic performance of capillary columns were studied. The capillary columns obtained using this technique are stable in all solvents commonly used in LC and exhibit promising enantiomer resolving ability.

Pore formation of thermostable direct hemolysin secreted from Vibrio parahaemolyticus in lipid bilayers.

Vibrio parahaemolyticus secretes thermostable direct hemolysin (TDH), a major virulence factor. Earlier studies report that TDH is a pore-forming toxin. However, the characteristics of pores formed by TDH in the lipid bilayer, which is permeable to small ions, remain to be elucidated. Ion channel-like activities were observed in lipid bilayers containing TDH. Three types of conductance were identified. All the channels displayed relatively low ion selectivity, and similar ion permeability. The Cl- channel inhibitors, DIDS, glybenclamide, and NPPB, did not affect the channel activity of pores formed by TDH. R7, a mutant toxin of TDH, also forms pores with channel-like activity in lipid bilayers. The ion permeability of these channels is similar to that of TDH. R7 binds cultured cells and liposomes to a lower extent, compared to TDH. R7 does not display significant hemolytic activity and cell cytotoxicity, possibly owing to the difficulty of insertion into lipid membranes. Once R7 is assembled within lipid membranes, it may assume the same structure as TDH. The authors propose that the single glycine at position 62, substituted with serine in the R7 mutant toxin, plays an important role in TDH insertion into the lipid bilayer.