Preparation of molecularly imprinted polymers for warfarin and coumachlor by multi-step swelling and polymerization method and their imprinting effects.

Monodisperse molecularly imprinted polymers (MIPs) for warfarin (WF) and coumachlor (CC), MIPWF and MIPCC, respectively, were prepared using 4-vinylpyridine (4-VPY) as a functional monomer and ethylene glycole dimethacrylate (EDMA) as a crosslinker by multi-step swelling and polymerization. Six kinds of MIPWF, MIPWF1 - MIPWF6, were prepared varying the concentrations of WF and 4-VPY, while maintaining the EDMA concentration constant, and their retention and molecular recognition properties were evaluated using a mixture of sodium phosphate buffer and acetonitrile as a mobile phase in LC. In addition to shape recognition, hydrogen bonding, ionic and hydrophobic interactions could affect the retention and molecular recognition of WF on MIPWF, and ionic interactions seem to govern the retention and molecular recognition of WF above mobile phase pH 6 associated with higher molar ratio of 4-VPY to EDMA. Furthermore, MIPCC was prepared under the same conditions with MIPWF6, which gave the highest imprinting factor for WF. WF could be recognized more strongly on MIPCC than MIPWF6, and the imprinting factors of WF on MIPWF6 and MIPCC, respectively, are 2.68 and 5.03 using 20mM sodium phosphate buffer - acetonitrile (30/70, v/v)(final pH 6.1) as the mobile phase. This result indicates that the use of CC as a template molecule instead of WF could be useful for getting a higher imprinting factor for WF and for avoiding the leakage problem in the assay of WF in LC.

Molecularly imprinted polymer for glutathione by modified precipitation polymerization and its application to determination of glutathione in supplements.

Molecularly imprinted polymers (MIP) particles for glutathione (GSH) with a narrow particle size distribution were prepared by modified precipitation polymerization using methacrylic acid as a functional monomer, divinylbenzene as a crosslinker and water as a co-solvent. The particle diameters of the MIP and non-imprinted polymer (NIP) prepared under the optimum conditions were 3.81±0.95 (average±standard deviation) and 3.39±1.22µm, respectively. The retention and molecular-recognition properties of the prepared MIP were evaluated using a mixture of acetonitrile and water as a mobile phase in hydrophilic interaction chromatography. With an increase of acetonitrile content, the retention factor of GSH was increased on the MIP. In addition to shape recognition, hydrophilic interactions seem to work for the recognition of GSH on the MIP. The MIP had a specific molecular-recognition ability for GSH, while glutathione disulfide, l-Glu, l-Cys, Gly-Gly and l-Cys-Gly could not be retained or recognized on the MIP. The effect of column temperature revealed that the separation of GSH on the MIP was entropically driven. Binding experiments and Scatchard analyses revealed that one binding sites were formed on both the MIP and NIP, while the MIP gave higher affinity and capacity for GSH than the NIP. Furthermore, the MIP was successfully applied for determination of GSH in the supplements.

Separation of enantiomers on chiral stationary phase based on cellulase: Effect of preparation method and silica particle diameters on chiral recognition ability.

Cellulase (Cel) was immobilized onto aminopropyl-silica gels via its amino and carboxy groups, respectively, using N,N'-disuccinimidyl carbonate, and 1-ethyl-3-(3'-dimethylaminopropyl)carbodimide and N-hydroxysulfosuccinimide. They were termed N-Cel and C-Cel, respectively. Despite their smaller retention factors on a C-Cel column, the enantioseparation factors and resolution of ß-blockers, propranolol, alprenolol, oxprenolol and pindolol, were similar with N- and C-Cel columns. In addition, C-Cel was prepared using aminopropyl-silica gels, whose nominal particle diameters were 5 and 3, and 2.1µm, respectively. A C-Cel column prepared with 2.1-µm aminopropyl-silica gels gave the highest enantioselectivity and column efficiency among three C-Cel columns. Furthermore, the influence of N,N-dimethyl-n-octylamine (DMOA) or cellobiose concentrations on the retentivity and enantioselectivity for ß-blockers on a C-Cel column was investigated. The results indicate that single-site competition of ß-blockers with DMOA or cellobiose on the catalytic binding site of Cel and the further bindings at the secondary site in a non-competitive fashion could occur. Furthermore, the enantioselective bindings of ß-blockers could occur at the catalytic biding cite of Cel and at the secondary binding site.

Preparation of molecularly imprinted polymers for strychnine by precipitation polymerization and multistep swelling and polymerization and their application for the selective extraction of strychnine from nux-vomica extract powder.

Monodisperse molecularly imprinted polymers for strychnine were prepared by precipitation polymerization and multistep swelling and polymerization, respectively. In precipitation polymerization, methacrylic acid and divinylbenzene were used as a functional monomer and crosslinker, respectively, while in multistep swelling and polymerization, methacrylic acid and ethylene glycol dimethacrylate were used as a functional monomer and crosslinker, respectively. The retention and molecular recognition properties of the molecularly imprinted polymers prepared by both methods for strychnine were evaluated using a mixture of sodium phosphate buffer and acetonitrile as a mobile phase by liquid chromatography. In addition to shape recognition, ionic and hydrophobic interactions could affect the retention of strychnine in low acetonitrile content. Furthermore, molecularly imprinted polymers prepared by both methods could selectively recognize strychnine among solutes tested. The retention factors and imprinting factors of strychnine on the molecularly imprinted polymer prepared by precipitation polymerization were 220 and 58, respectively, using 20 mM sodium phosphate buffer (pH 6.0)/acetonitrile (50:50, v/v) as a mobile phase, and those on the molecularly imprinted polymer prepared by multistep swelling and polymerization were 73 and 4.5. These results indicate that precipitation polymerization is suitable for the preparation of a molecularly imprinted polymer for strychnine. Furthermore, the molecularly imprinted polymer could be successfully applied for selective extraction of strychnine in nux-vomica extract powder.

Molecularly imprinted polymer for caffeic acid by precipitation polymerization and its application to extraction of caffeic acid and chlorogenic acid from Eucommia ulmodies leaves.

Molecularly imprinted polymers (MIPs) for caffeic acid (CA) were prepared using 4-vinylpyridine and methacrylamide (MAM) as functional monomers, divinylbenzene as a crosslinker and acetonitrile-toluene (3:1, v/v) as a porogen by precipitation polymerization. The use of MAM as the co-monomer resulted in the formation of microsphere MIPs and non-imprinted polymers (NIPs) with ca. 3- and 5-µm particle diameters, respectively. Binding experiments and Scatchard analyses revealed that the binding capacity and affinity of the MIP to CA are higher than those of the NIP. The retention and molecular-recognition properties of the prepared MIPs were evaluated using water-acetonitrile and sodium phosphate buffer-acetonitrile as mobile phases in hydrophilic interaction chromatography (HILIC) and reversed-phase chromatography, respectively. In HILIC mode, the MIP showed higher molecular-recognition ability for CA than in reversed-phase mode. In addition to shape recognition, hydrophilic interactions seem to work for the recognition of CA on the MIP in HILIC mode, while hydrogen bonding and hydrophobic interactions seem to work for the recognition of CA in reversed-phase mode. The MIP had a specific molecular-recognition ability for CA in HILIC mode, while other structurally related compounds, such as chlorogenic acid （CGA), gallic acid, protocatechuic acid and vanillic acid, could not be recognized by the MIP. Furthermore, the MIP was successfully applied for extraction of CA and CGA in the leaves of Eucommia ulmodies in HILIC mode.

Molecularly imprinted polymer for chlorogenic acid by modified precipitation polymerization and its application to extraction of chlorogenic acid from Eucommia ulmodies leaves.

Molecularly imprinted polymers (MIPs) for chlorogenic acid (CGA) were prepared by modified precipitation polymerization using methacrylic acid as a functional monomer, divinylbenzene as a crosslinker and methanol or dimethylsulfoxide as a co-solvent. The prepared MIPs were microspheres with a narrow particle size distribution. Binding experiments and Scatchard analyses revealed that two classes of binding sites, high and low affinity sites, were formed on the MIP. The retention and molecular-recognition properties of the prepared MIP were evaluated using a mixture of water and acetonitrile as a mobile phase in hydrophilic interaction chromatography. With an increase of acetonitrile content, the retention factor of CGA was increased on the MIP. In addition to shape recognition, hydrophilic interactions seem to work for the recognition of CGA on the MIP. The MIP had a specific molecular-recognition ability for CGA, while other related compounds, such as caffeic acid, gallic acid, protocatechuic acid and vanillic acid, could not be recognized by the MIP. Furthermore, the MIP for CGA was successfully applied for extraction of CGA in the leaves of Eucommia ulmodies.

Separation of enantiomers on chiral stationary phase based on chicken α1-acid glycoprotein: effect of silica particle diameters on column performance.

The effects of silica particle diameters on performances of chicken α1-acid glycoprotein (c-AGP)-immobilized silica particle columns were investigated. c-AGP was immobilized onto aminopropyl silica particles, whose nominal particle diameters were 5, 3 and 2.1 µm, activated with N,N'-disuccinimidyl carbonate. The retention factor (k), enantioseparation factor (α), resolution (Rs) and height equivalent to a theoretical plate (H) of solutes on three c-AGP columns were evaluated using a mixture of phosphate buffer and organic modifier as a mobile phase in LC. There were not so much differences in their k and α values among three c-AGP columns, while their Rs values were in the order of 2.1 µm>3 µm>5 µm silica particles and their H values were in the reversed order. Since three c-AGP columns gave almost the same enantioseparation factors for solutes, their highest Rs and lowest H values on a c-AGP-immobilized column prepared with 2.1-µm silica particles came from its highest column efficiency among there c-AGP columns. These results suggest that 2.1-µm silica particles could be useful for the preparation of c-AGP- or protein-based CSPs.

Monodisperse, molecularly imprinted polymers for creatinine by modified precipitation polymerization and their applications to creatinine assays for human serum and urine.

Molecularly imprinted polymers (MIPs) for creatinine were prepared by modified precipitation polymerization using methacrylic acid as a functional monomer and divinylbenzene as a crosslinker. The prepared MIPs were monodispersed with a narrow particle size distribution. Binding experiments and Scatchard analyses revealed that two classes of binding sites, high- and low-affinity sites, were formed on the MIPs. The retention and molecular-recognition properties of the MIPs were evaluated by hydrophilic interaction chromatography using a mixture of ammonium acetate buffer and acetonitrile as a mobile phase. With an increase of acetonitrile content, the retention factor of creatinine was increased on the MIP. In addition to shape recognition, hydrophilic interactions seemed to enhance the recognition of creatinine on the MIP. The MIPs' molecular-recognition ability was specific for creatinine; the structurally related compounds such as hydantoin, 1-methylhydantoin, 2-pyrrolidone, N-hydroxysuccinimide and creatine were not recognized. Furthermore, the creatinine concentrations in human serum and urine were successfully determined by direct injection of the deproteinized serum and diluted urine samples onto the MIP.

Preparation of monodisperse curcumin-imprinted polymer by precipitation polymerization and its application for the extraction of curcuminoids from Curcuma longa L.

A monodisperse molecularly imprinted polymer (MIP) for curcumin was first prepared by precipitation polymerization using methacrylamide (MAM) and 4-vinylpyridine as functional co-monomers, divinylbenzene as a crosslinker, and a mixture of acetonitrile and toluene as a porogen. The use of MAM as the co-monomer resulted in the formation of a monodisperse MIP and non-imprinted polymer (NIP). MIP and NIP, respectively, were monodispersed with a narrow particle size distribution (3.3 ± 0.09 and 3.5 ± 0.10 µm). In addition to shape recognition, hydrophobic and hydrogen-bonding interactions affected the retention and molecular-recognition of curcumin on the MIP. The MIP for curcumin could extract curcuminoids (curcumin, demethoxycurcumin, and bisdemethoxycurcumin) in Curcuma longa L.

Interaction of cepharanthine with immobilized heat shock protein 90α (Hsp90α) and screening of Hsp90α inhibitors.

Heat shock protein 90α (Hsp90α) immobilized on aminopropyl silica gels was prepared via the N- or C-terminal, which was termed Hsp90α-NT or Hsp90α-CT, respectively. Binding interactions of biscoclaurine alkaloids (cepharanthine (CEP), berbamine (BBM), isotetrandrine (ITD), and cycleanine (CCN)) with Hsp90α were examined using the Hsp90α-NT or -CT columns by frontal and zonal chromatography studies. The dissociation constants of CEP, BBM, ITD, and CCN to Hsp90α-NT were estimated to be 5.3, 18.6, 46.3, and 159 µM, respectively, by frontal chromatography techniques. Similar results were obtained with the Hsp90α-CT column. These data suggest that these biscoclaurine alkaloids interact with the middle domain of Hsp90α. This was confirmed by demonstrating that CEP competed with endothelial nitric oxide synthase at the middle domain of Hsp90α, where it was shown to have a dissociation constant of 15 nM. Furthermore, the Hsp90α-NT column was applied for preliminary screening of natural Hsp90α inhibitors by zonal chromatography studies.

Preparation of magnetic molecularly imprinted polymers for bisphenol A and its analogues and their application to the assay of bisphenol A in river water.

Magnetic molecularly imprinted polymers (M-MIPs) for bisphenol A (BPA) and its structural analogues have been prepared by a multi-step swelling and polymerization method using uniformly-sized magnetic particles as a shape template. Binding experiments and Scatchard analyses revealed that two classes of binding sites were formed on M-MIP(BPA). The retention and molecular-recognition properties of M-MIPs for BPA and its structural analogues were evaluated using a mixture of phosphate buffer and acetonitrile or a mixture of water and acetonitrile as a mobile phase by LC. M-MIPs for BPA and [²H16]BPA (BPA-d16), M-MIP(BPA) and M-MIP(BPA-d16), showed almost the same imprinting factors for BPA, while M-MIP for bisphenol B (2,2-bis(4-hydroxyphenyl)butane, BPB), M-MIP(BPB), gave the moderate imprinting factor for BPA. Furthermore, M-MIP(BPB) was applied for the selective extraction and determination of BPA in environmental water samples. The concentration of BPA in river water samples was determined to be 68 ng/L.

Preparation of molecularly imprinted polymers for organophosphates and their application to the recognition of organophosphorus compounds and phosphopeptides.

Monodisperse molecularly imprinted polymers (MIPs) for diphenyl phosphate (DPP) and 1-naphthyl phosphate (1-NapP) have been prepared by a multi-step swelling and polymerization method using 4-vinylpyridine as a functional monomer, glycerol dimethacrylate as a crosslinker and cyclohexanol or 1-hexanol as a porogen. The retention and molecular-recognition properties of these MIPs for organophosphorus compounds were evaluated by HPLC using a mixture of phosphate buffer and acetonitrile as an eluent. In addition to shape recognition, hydrogen bonding and hydrophobic interactions could play an important role in the retention and molecular recognition of DPP and 1-NapP. Furthermore, the MIPs were applied to the separation of adenosine and adenosine phosphates (AMP, ADP and ATP). These phosphates were retained on the MIPs according to the number of phosphate groups in the molecule and were well separated from one another. Hydrogen bonding and hydrophobic interactions seemed to affect the retention and recognition of adenosine phosphates in low acetonitrile content, while hydrophilic interactions affected these properties in high acetonitrile content. Finally, the MIPs were applied to the trapping of phosphopeptides. The MIPs non-selectively trapped phosphopeptides, which have phosphorylated tyrosine, serine or threonine in the sequences, and successfully trapped four phosphopeptides in tryptic digests of bovine α-casein.

Monodispersed molecularly imprinted polymer for creatinine by modified precipitation polymerization.

A monodispersed molecularly imprinted polymer (MIP) for creatinine was prepared by modified precipitation polymerization. The retention and molecular-recognition properties of the prepared MIP were evaluated by the hydrophilic interaction chromatography mode using a mixture of ammonium acetate buffer and acetonitrile as a mobile phase in liquid chromatography. The MIP had a specific recognition ability for creatinine, while other structurally related compounds, such as hydantoin, 1-methylhydantoin, 2-pyrrolidone, N-hydroxysuccinimide and creatine, could not be recognized on the MIP. In addition to shape recognition, hydrophilic interactions could work for the recognition of creatinine on the MIP.

Simultaneous determination of non-steroidal anti-inflammatory drugs in river water samples by liquid chromatography-tandem mass spectrometry using molecularly imprinted polymers as a pretreatment column.

A restricted access media-molecularly imprinted polymer (RAM-MIP) for flufenamic acid has been developed for the simultaneous determination of non-steroidal anti-inflammatory drugs (NSAIDs) in river water samples. The RAM-MIP was prepared using 4-vinylpyridine and ethylene glycol dimethacrylate as a functional monomer and cross-linker, respectively, by a multi-step swelling and polymerization method followed by a surface modification technique. The RAM-MIP for flufenamic acid showed excellent molecular recognition abilities for flufenamic acid and mefenamic acid, and moderate molecular recognition abilities for indomethacin, etodolac and ketoprofen. The simultaneous determination of NSAIDs (mefenamic acid, indomethacin, etodolac and ketoprofen) in river water samples was carried out by LC-MS/MS using the RAM-MIP for flufenamic acid as a pretreatment column. The concentrations of mefenamic acid, indomethacin and etodolac in river water samples were determined to be 0.4, 0.7 and 0.3ng/L, respectively, while ketoprofen was below the limit of quantitation.

Molecularly imprinted polymers for simultaneous determination of antiepileptics in river water samples by liquid chromatography-tandem mass spectrometry.

A restricted access media-molecularly imprinted polymer (RAM-MIP) for cyclobarbital has been developed for selective extraction of antiepileptics in river water samples. The RAM-MIP was prepared using 4-vinylpyridine and ethylene glycol dimethacrylate as a functional monomer and cross-linker, respectively, by a multi-step swelling and polymerization method followed by a surface modification technique. The RAM-MIP for cyclobarbital showed molecular recognition abilities for phenobarbital, amobarbital and phenytoin as well as cyclobarbital. Thus, selective analysis of antiepileptics in river water samples was attained with RAM-MIP extraction followed by column-switching liquid chromatography-tandem mass spectrometry. The concentrations of phenobarbital and phenytoin in river water samples were about 15 and 4 ng/L, respectively, while that of amobarbital was below the limit of quantitation.

Multiple ligand-binding properties of the lipocalin member chicken alpha1-acid glycoprotein studied by circular dichroism and electronic absorption spectroscopy: the essential role of the conserved tryptophan residue.

Multiple ligand-binding properties of the 30-kDa chicken alpha(1)-acid glycoprotein (cAGP), a member of the lipocalin protein family, were investigated for the first time by using circular dichroism (CD) and UV/Vis absorption spectroscopy methods. By measuring induced CD (ICD) spectra, high-affinity binding (K(a) approximately 10(5)-10(6) M(-1)) of several drugs, dyes and natural compounds to cAGP was demonstrated including antimalarial agents (quinacrine, primaquine), phenotiazines (chlorpromazine, methylene blue), propranolol, non-steroidal antiinflammatory drugs (ketoprofen, diclofenac), tamoxifen, diazepam, tacrine, dicoumarol, cationic dyes (auramine O, thioflavine T, ethidium bromide), benzo[a]pyrene, L-thyroxine, bile pigments (bilirubin, biliverdin), alkaloids (piperine, aristolochic acid), saturated and unsaturated fatty acids. Analysis of the extrinsic CD spectra with the study of the covalently modified protein and CD displacement experiments revealed that a single Trp26 residue of cAGP conserved in the whole lipocalin family is part of the binding site, and it is essentially involved in the ligand-binding process via pi-pi stacking interaction resulting in the appearance of strong induced CD bands due to the non-degenerate intermolecular exciton coupling between the pi-pi* transitions of the stacked indole ring-ligand chromophore. The finding that cAGP is able to accommodate a broad spectrum of ligands belonging to different chemical classes suggests that its core beta-barrel cavity is unusually wide containing overlapping sub-sites. Significance of these new data in understanding of the ligand-binding properties of other lipocalins, especially that of human AGP, and potential practical applications are briefly discussed. Overall, cAGP serves as a simple, ultimate model to extend our knowledge on ligand-binding properties of lipocalins and to study the role of tryptophan residues in molecular recognition processes.

Investigation of chiral recognition mechanism on chicken alpha(1)-acid glycoprotein using separation system.

Chicken alpha1-acid glycoprotein (alpha1-AGP) consists of 183 amino acid residues and has only one Trp residue at the 26 position. In this study, the Trp26 residue was modified with 2-nitrophenylsulfenyl chloride and chiral separation of neutral, acidic and basic compounds was examined on chicken alpha1-AGP and Trp-modified chicken alpha1-AGP columns. Chiral separation of propranolol, alprenolol and oxprenolol was lost on the Trp-modified chicken alpha1-AGP column, while chlorpheniramine, ketoprofen and benzoin were still enantioseparated on the Trp-modified chicken alpha1-AGP column despite of lower enantioselectivity than that on the chicken alpha1-AGP column. These results suggest that the Trp26 residue could be responsible for chiral recognition of these compounds. Competition studies using N,N-dimethyl-n-octylamine (DMOA) as a competitor indicated that propranolol, alprenolol and oxprenolol competed with DMOA on a single binding site near the Trp26 region and that further bindings of chlorpheniramine, ketoprofen and benzoin occurred at the secondary binding site in a non-competitive fashion with DMOA.

Identification of disulfide bonds and site-specific glycosylation in chicken alpha1-acid glycoprotein by matrix-assisted laser desorption ionization time-of-flight mass spectrometry.

Recently, we reported the amino acid sequence of chicken alpha1-acid glycoprotein (chicken alpha1-AGP) [Biochem. Biophys. Res. Commun. 295 (2002) 587]. In this study, we located the disulfide bonds and site-specific glycosylation in chicken alpha1-AGP using tryptic digests of carbamidomethylated chicken alpha1-AGP, carbamidomethylated completely deglycosylated chicken alpha1-AGP (cd-alpha1-AGP), and nonreduced denatured cd-alpha1-AGP by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Based on the detection of peptides mlz 3037.4 (amino acid sequences 69-76 plus 161-183) and 3453.3 (amino acid sequences 69-80 plus 161-183), the two disulfide bonds of chicken alpha1-AGP were determined to be located at Cys 6-Cys 146 and Cys 73-Cys 163. The results also showed that Asn 16, 70, 77, and 87 were fully glycosylated and that Asn 62 was partially glycosylated.

Separation of basic drug enantiomers by capillary electrophoresis using chicken alpha1-acid glycoprotein: insight into chiral recognition mechanism.

Recombinant chicken alpha(1)-acid glycoprotein (alpha(1)-AGP) was prepared by the Escherichia coli expression system and completely deglycosylated alpha(1)-AGP (cd-alpha(1)-AGP) was obtained by treatments of native alpha(1)-AGP with a mixture of endoglycosidase and N-glycosidase. The average molecular masses of chicken alpha(1)-AGP, cd-alpha(1)-AGP and recombinant alpha(1)-AGP were estimated to be about 29 200, 21 700 and 20 700, respectively, by matrix-assisted laser desorption-time of flight-mass spectrometry. We compared the chiral recognition ability of chicken alpha(1)-AGP, cd-alpha(1)-AGP and recombinant alpha(1)-AGP using them as chiral selectors in capillary electrophoresis. The chicken alpha(1)-AGP showed higher resolution for eperisone, pindolol and tolperisone than cd-alpha(1)-AGP or recombinant alpha(1)-AGP. Recombinant alpha(1)-AGP still showed chiral recognition for three basic drugs tested. By addition of propranolol as a competitor in the separation solution in CE, no enantioseparations of three basic drugs were observed with chicken alpha(1)-AGP, cd-alpha(1)-AGP or recombinant alpha(1)-AGP. These results reveal that the protein domain of the chicken alpha(1)-AGP is responsible for the chiral recognition ability, and that the chiral recognition site(s) for basic drugs exists on the protein domain.

Protein domain of chicken alpha(1)-acid glycoprotein is responsible for chiral recognition.

Ovoglycoprotein from chicken egg whites (OGCHI) has been used as a chiral selector to separate drug enantiomers. However, neither the amino acid sequence of OGCHI nor the responsible part for the chiral recognition (protein domain or sugar moiety) has yet to be determined. First, we isolated a cDNA clone encoding OGCHI, and clarified the amino acid sequence of OGCHI, which consists of 203 amino acids including a predictable signal peptide of 20 amino acids. The mature OGCHI shows 31-32% identities to rabbit and human alpha(1)-acid glycoproteins (alpha(1)-AGPs). Thus, OGCHI should be the chicken alpha(1)-AGP. Second, the recombinant chicken alpha(1)-AGP was prepared by the Escherichia coli expression system, and its chiral recognition ability was confirmed by capillary electrophoresis. Since proteins expressed in E. coli are not modified by any sugar moieties, this result shows that the protein domain of the chicken alpha(1)-AGP is responsible for the chiral recognition.