Tokiko Murashima

Separation of enantiomers on a pepsin-bonded column

Abstract Commercial crystalline pepsin from porcine stomach mucosa was characterized by high-performance capillary electrophoresis and matrix-assisted laser desorption ionization time-of-flight mass spectrometry. These results suggest that the average molecular mass of the pepsin is about 34 900, and that it contains heterogeneity due to the autodigestion. The pepsin was covalently bound to porous aminopropylsilica materials using an N,N′-disuccinimidyl carbonate reaction. By using a mixture of phosphate buffer and organic modifier as an eluent, basic and uncharged enantiomers were resolved, while no resolution of acidic enantiomers was observed.

Separation of enantiomers on a lysozyme-bonded silica column

Abstract A lysozyme-bonded silica column has been developed for direct separation of enantiomers. Lysozyme was covalently bound to porous silica materials using an N,N′-disuccinimidyl carbonate reaction. By using a mixture of phosphate buffer and organic modifier as an eluent, basic and unchanged enantiomers were resolved, while no resolution of acidic enantiomers was observed.

Retentive and enantioselective properties of ovomucoid-bonded silica columns. Influence of protein purity and isolation method

Abstract The influence of protein purity and isolation method on the chiral resolution of racemates on ovomucoid-bonded materials has been investigated. Chicken ovomucoid (OMCHI) and turkey ovomucoid (OMTKY) were obtained from commercial sources, and were isolated by precipitation with acetone from their corresponding egg whites. Further, crude OMCHI protein was fractionated by cation-exchange chromatography into two fractions, pure OMCHI and an unknown protein fraction. The OMCHI, fractionated OMCHI and OMTKY proteins were characterized by N-terminal sequencing and trypsin-inhibitory activity. Their purity was checked by reversed-phase chromatography. The results reveal that the commercial OMCHI, and the isolated OMCHI and OMTKY are crude preparations and that an unknown protein fraction is present in each crude ovomucoid protein. The OMCHI, fractionated OMCHI and OMTKY proteins were bound to aminopropyl-silica gels activated by N, N′-disuccinimidyl carbonate to test the retentive and enantioselective properties of those materials. The result suggests that the unknown protein fraction could be responsible for chiral recognition ability of the OMCHI and OMTKY columns.

Retention and enantioselective properties of ovomucoid-bonded silica columns Influence of physical properties of base materials and spacer length

Abstract The influence of the physical properties of base silica materials and spacer length on chiral separation of enantiomers on ovomucoid (OVM)-bonded materials was investigated. With regard to the pore size of the base silica materials, the 300-A materials gave higher enantioselectivity, than the 120-A materials, despite the smaller amounts of bonded OVM proteins. However, higher resolution was obtained with the latter materials. With regard to the spacer length, aminopropyl (AP)-, aminobutyl-, N-(4-aminobutyl)-3-aminopropyl- and N-(6-aminohexyl)-3-aminopropyl-silica gels were activated by N,N′-di-succinimidyl carbonate (DSC) and the proteins were bound. The first two materials showed excellent chiral resolution properties for the solutes tested, and the AP materials gave higher enantioselectivity and resolution. On the other hand, only oxprenolol enantiomers were slightly resolved on the last two materials. Also, AP-silica gels activated by DSC were compared with glycerylpropyl (GP)-silica gels activated by 1,1′-carbonyldiimidazole. The former materials gave higher resolution for acidic and basic solutes despite the lower enantioselectivity, whereas for the unchanged hexobarbital the latter materials gave higher enantioselectivity and almost equal resolution. The above results reveal that the 120-A base silica gels are more suitable than the 300- A base silica gels for obtaining larger amounts of bonded OVM proteins and that a less hydrophobic spacer such as an AP group and a hydrophilic spacer such as a GP group are suitable.

Structure determination of a kaempferol 3-rhamnosyldiglucoside from Impatiens balsamina.

Abstract The chemical structure of a kaempferol 3-rhamnosyldiglucoside, isolated from white petals of Impatiens balsamina was unambiguously determined to be kaempferol-3- O -[2″- O -α- l -rhamnopyranosyl-3″ O -β- d -glucopyrano-syl]-P-D-glucopyranoside based on the spectroscopic techniques.

Retention and enantioselective properties of racemic compounds on modified ovomucoid columns: II. Reaction with glyceraldehyde, formaldehyde and glutaric anhydride

Abstract Modified ovomucoid (OVM) columns were prepared by reaction with glyceraldehyde, formaldehyde and glutaric anhydride. The retention and enantioselective properties of racemic compounds on these modified OVM columns were compared with those on an unmodified OVM column. The retentions of racemic compounds on the modified OVM columns were lower than or approximately equal to those on the unmodified OVM column (except for basic compounds on the OVM column reacted with glutaric anhydride). The modified OVM columns gave lower or approximately equal enantioselectivities than the unmodified OVM column for acidic and unchanged compounds, whereas the modified OVM columns gave higher enantioselectivity for basic compounds. These differences may be mainly attributed to changes in protein conformation, especially changes in chiral recognition site(s) as a result of modification. The results reveal that modification of OVM proteins may be effective for the chiral separation of basic compounds on an OVM column.

The specific inhibition of crystal growth of monohydrogen potassium L-tartrate by d-catechin

Crystal growth of monohydrogen potassium L-tartrate in an ethanolic aqueous solution was specifically inhibited by d-catechin, but not by either its epimeric isomer at C3, l-epicatechin or by gallic acid and caffeic acid. 3D-Structure similarity search of d-catechin with two molecules of the tartrate and docking study of d-catechin with the crystal model of the tartrate suggested that d-catechin mimics a structure consisting of the two tartrate molecules in the inhibition. Differences in the conformation of the catechol moieties of d-catechin and l-epicatechin may explain the distinct inhibitory effects of the epimeric isomers.