2022 Volume 70 Issue 7 Pages 458-468
A lot of chiral stationary phases (CSPs) have been introduced for the purpose of analytical and preparative separations of enantiomers. CSPs based on proteins and glycoproteins have unique properties among those CSPs. This review article deals with the preparation of CSPs based on proteins and glycoproteins, their chiral recognition properties and mechanisms, focusing on the CSPs investigated in our group. The dealt proteins and glycoproteins are including bovine serum albumin, human serum albumin, lysozyme, pepsin, human α1-acid glycoprotein (AGP), chicken ovomucoid and chicken ovoglycoprotein (named chicken AGP).
In biological processes, the behaviors of enantiomers are totally different in the reaction with enzymes and the binding to receptors and other molecules.1,2) Because enzymes and receptors, which are chiral molecules, can differentiate enantiomers.1,2) Therefore, pharmacokinetics/pharmacodynamics and/or pharmacological activity/toxicity of enantiomers in the natural worlds, especially in humans, are different.1,2) Recently, attention has been paid to the determination of chiral pollutants, pharmaceuticals, agrochemicals and so on, in environmental samples and biota.3–5) Chiral stationary phases (CSPs) have played a key role in analytical and preparative separations of enantiomers.1,6,7) Those include Pirkle-type, polysaccharide derivatives, native and derivatized cyclodextrins, glycopeptides, crown ethers, proteins/glycoproteins, ligand-exchange type, chiral polymers and molecularly imprinted polymers.1,6,7) Recently, nanoparticles and nanomaterials modified with chiral selectors were used in capillary electrochromatography (CEC) mode to improve the chiral resolution ability by immobilizing large amounts of chiral selectors.8–10) Chiral metal organic frameworks (MOFs) and covalent organic frameworks were newly introduced as CSPs.11–14)
In the early 1950s, the bindings of the enantiomers of an anionic azo-dye to bovine serum albumin (BSA)15) or human serum albumin (HSA)16) were examined. Binding of the (−)-form to BSA with an affinity of about 2-fold higher than that of the (+)-form was observed, while the difference in the binding to HSA was almost insignificant. Furthermore, it was reported that (−)-Trp was bound to serum albumin (bovine mercaptoalbumin or HSA) with a higher affinity than the (+)-form, which had almost no binding to serum albumins.17) (−)-Trp was bound to serum albumin predominantly at one site in a highly stereospecific manner: the indole ring, carboxy group and α-hydrogen of (−)-Trp interacted with the a protein. Especially, a close fit to the protein at the α-hydrogen of (−)-Trp was important.17) These studies clearly indicated that serum albumins could differentiate enantiomers of some ligands, and that they could be used as chiral selectors for the enantioseparations. In 1973, BSA-succinoylaminoethyl-agarose beads were used for the separation of Trp enantiomers. On the BSA-agarose beads, (+)- and (−)-Trp were clearly resolved, and the (+)-enantiomer eluted first. This result was consistent with the bindings of Trp enantiomers to BSA in the solution described above. This was the first report on the use of CSPs based on a protein for the enantioseparation purposes in LC.18)
A wide range of compounds, especially pharmaceuticals or agrochemicals, can be enantioseparated on CSPs based on proteins and glycoproteins. However, in general they are unstable against heat, acids, organic modifiers and so on because those induce the reversible or irreversible changes in their conformation. Proteins so far employed have included BSA,19) HSA,20) trypsin,21) α-chymotrypsin,22) lysozyme,23) pepsin,24) fatty acid-binding protein,25) penicillin G-acylase,26) streptavidin27) and lipase B,28) while glycoproteins have included human α1-acid glycoprotein (AGP, hAGP),29) chicken ovomucoid (OMCHI),30) chicken ovoglycoprotein (OGCHI)31) (named chicken AGP (cAGP)32)), avidin,33) riboflavin binding protein,34) ovotransferrin (conalbumin),35) cellobiohydrolase,36) glucoamylase,37) antibody (immunoglobulin G),38) α3β4-nicotinic acetylcholine receptor39) and human liver organic cation transporter.40) Physical properties of those proteins and glycoproteins used as the CSPs are shown in Table 1.
| Protein | Molecular mass (Da) | Carbohydrate content (%) | Isoelectric point | Origin |
|---|---|---|---|---|
| Protein | ||||
| Bovine serum albumin (BSA) | 66000 | —a) | 4.7 | Bovine serum |
| Human serum albumin (HSA) | 66000 | — | 4.7 | Human serum |
| Trypsin | 23000 | — | 10.1–10.8 | Pancreas |
| α-Chymotrypsin | 25000 | — | 8.1–8.6 | Pancreas |
| Lysozyme | 14300 | — | 10.5 | Egg white |
| Pepsin | 34600 | — | <1 | Porcine stomach |
| Fatty acid binding protein | 14000 | — | 9.0 | Chicken liver |
| Penicillin G-acylase | 85000 | — | 8.1 | Bacterium |
| Streptavidin | 53000 | — | 5 | Bacterium |
| Lipase B | 33000 | 6.0 | Fungus | |
| Glycoprotein | ||||
| Human α1-acid glycoprotein (AGP, hAGP) | 41000–43000 | 45 | 2.7–3.8 | Human serum |
| 33000 | 35 | 2.7–3.8 | ||
| Ovomucoid (OMCHI) | 28000 | 30 | 4.1 | Egg white |
| Ovoglycoprotein (OGCHI) (chicken α1-acid glycoprotein (cAGP)) | 30000 | 25 | 4.1 | Egg white |
| Avidin | 66000 | 7 | 10 | Egg white |
| Riboflavin binding protein | 32000–36000 | 14 | 4 | Egg white |
| Ovotransferrin (conalbumin) | 77000 | 2.6 | 6.1 | Egg white |
| Cellulase | Fungus | |||
| Cellobiohydrolase I | 65000 | 6 | 3.9 | |
| Cellobiohydrolase II | 53000 | 5.9 | ||
| Cellobiohydrolase 58 | 60000 | 3.8 | ||
| Glucoamylase | Fungus | |||
| Glucoamylase G1 | 94000 | 30–35 | ||
| Glucoamylase G2 | 85000 | |||
| Antibody (immunoglobulin G) | 150000 | 2–3 | 6–8 | Vertebrate |
| α3β4-Nicotinic acetylcholine receptor | 309000 | Cell line | ||
| Human liver organic cation transporter | 61000 | Cell line |
a) No sugar moieties.
Those proteins and glycoproteins were adsorbed or immobilized onto solid supports as chiral selectors in LC. They were also used as chiral selectors in CEC after being adsorbed, immobilized and encapsulated onto the capillary, or the immobilized proteins and glycoproteins being packed into the capillary.
A lot of book chapters41–44) and review articles45–50) dealing with proteins and glycoproteins as chiral selectors in LC or CEC have been published. This review article deals with the preparation of CSPs based on proteins and glycoproteins, their chiral recognition properties and mechanisms, focusing on the CSPs investigated in our group.
Silicas and polymers have been utilized as the solid supports for the preparation of CSPs based on proteins and glycoproteins to attain the high-performance separations.47) The former supports were used as particles, capillaries or monoliths,46,47) while the latter supports have been used as particles or monoliths.47)
Two methods, physical absorption and covalent immobilization of proteins and glycoproteins, were mainly employed for the preparation of CSPs.46,47) Onto bare or anion-exchange silicas, the proteins and glycoproteins were physically adsorbed. To avoid the elution of the proteins and glycoproteins, they were crosslinked with glutaraldehyde on the silicas.46,47) Covalent immobilization of the proteins and glycoproteins onto functionalized supports was conducted via their amino, carboxy and sulfhydryl groups.43) In addition, glycoproteins were immobilized onto hydrazide-functionalized supports via their carbohydrates.51)
Proteins and glycoproteins were covalently immobilized onto glycidyl (epoxy)-, dihydroxypropyl (diol)- and aminopropyl (amino)-supports via their amino groups. As shown in Chart 1, proteins and glycoproteins were reacted with epoxy-supports, yielding the secondary amine.52) As shown in Chart 2, after activation of diol-supports with 1,1′-carbonyldiimidazole (CDI), proteins and glycoproteins were reacted, yielding the carbamate bond.20) Activation of diol-supports with N,N′-disuccinimidylcarbonate (DSC) resulted in the same CSPs with those activated with CDI. Reaction of proteins and glycoproteins with diol-supports activated by tresyl chloride (CF3CH2SO2Cl) yielded the secondary amine as shown in Chart 1. Furthermore, diol-supports were oxidized to the corresponding aldehyde-supports by sodium periodate. Proteins and glycoproteins were immobilized onto the aldehyde-supports via Shiff base formation, followed by reductive amination with sodium cyanoborohydride as shown in Chart 3.
Protein and glycoprotein were immobilized onto amino-supports activated by DSC, yielding the urea bond (Chart 4). N,N′-Disuccinimidylsuberate (DSS) as well as DSC was used for the activation of amino-supports.53) Furthermore, proteins and glycoproteins were immobilized to amino-supports using glutaraldehyde as a crosslinker, yielding the Schiff base followed by reductive amination46) (Chart 3).
Protein and glycoproteins via their carboxy groups were immobilized onto amino-supports by 1-ethyl-3-(3′-dimethylaminopropyl)carbodimide (EDC) and N-hydroxysulfosuccinimide (HSSI)54) (Chart 5), yielding the amide bond.
Moreover, proteins and glycoproteins via their sulfhydryl groups were immobilized onto amino-supports activated by succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) (Chart 6) or succinimidyl iodoacetate (SIA).55)
Covalent immobilization of glycoproteins onto hydrazide-functionalized supports was attained as shown in Chart 7.51)
As shown in Table 1, a lot of CSPs based on proteins and glycoproteins have been introduced. We will focus on CSPs investigated in our groups as mentioned in Introduction.
3.1. Proteins3.1.1. BSAThough it was considered that BSA had 582 amino-acid sequences, Tyr156 was lacking in the previous sequence.56) BSA consists of 583 amino-acid sequences with a molecular mass of 66 kDa. BSA was first immobilized onto agarose and used for the enantioseparation of Trp: (+)- and (−)-Trp were enantioseparated using the CSPs based on BSA.18) BSA was immobilized onto silicas by Allenmark et al.19) Though silicas were mainly used as the base materials, polymers also were used for the immobilization of BSA in LC.57)
It was reported that CSPs based on BSA-fragments, immobilized onto silica particles, had lower capacity and enantioselectivity than the CSPs based on intact BSA.58,59) Next, CSPs based on the N-terminal half of BSA-fragments, which were mainly cleaved between Asp307 and Phe308 by pepsin and whose molecular masses were approx. 35000 Da, were isolated.60,61) The intact BSA and BSA-fragments were immobilized onto amino-silicas activated by DSC. The immobilized amounts of the BSA-fragments were 2.2–2.7 times higher than those of the intact BSA. Enatioseparations of 2-arylpropionic acid derivatives, benzodiazepines, warfarin and benzoin were attained with CSPs based on the BSA-fragments in LC. CSPs based on the BSA-fragments gave higher enantioselectivity for lorazepam and benzoin because of the higher immobilized amounts, and lower enantioselectivity for other compounds, compared with CSPs based on intact BSA.61)
In CEC, capillaries filled with silica particles consisting of BSA crosslinked with glutaraldehyde have been prepared for the resolution of Trp enantiomers.62) A CEC column was prepared with physically adsorbed BSA on strong anion exchange packings.63) BSA was immobilized onto the silica capillary wall64) after etching the capillary wall with sodium hydroxide. The silica capillary was reacted with 3-glycidoxypropyltrimethoxysilane followed by hydrolysis with hydrochloric acid. The obtained diol-silicas were activated with tresyl chloride, and then BSA was immobilized. Enantiomers of dinitrophenyl amino-acids and 3-hydroxy-1,4-benzodiazepines were separated. BSA was immobilized onto aldehyde- or amino-silica monoliths via Shiff base formation followed by reduction with sodium cyanoborohydride and used for separation of Trp enantiomers in CEC.65) Furthermore, BSA was immobilized onto a carboxylated multi-walled carbon nanotube (cMWCNT) using water-soluble carbodiimide. The aminopropyl fused-silica capillary was crosslinked with cMWCNT-BSA using glutaraldehyde. cMWCNTs would have a significant effect to enhance the phase ratio of a CEC column and to improve the enantioselectivity in CEC.66) BSA was adsorbed in a capillary wall using polydopamine (PDA) as the adhesion layer and surface functionalization agent, and a MOF as supporting platform and biomacromolecule immobilization carrier, respectively.67)
A carboxylated single-walled carbon nanotube (cSWCNT) was cut into short pipes by oxidation with mineral acid. BSA was immobilized to the cSWCNT using EDC and HSSI (Chart 5). Enantioseparation of Trp was attained utilizing CSPs based on cSWCNT-BSA, adsorbed onto microchannels in microchip CEC.68) Graphene oxide (GO)-Fe3O4 nanocomposites were synthesized by a chemical precipitation method. BSA was adsorbed onto to the obtained nanocomposites, and then packed into the polydimethylsiloxane (PDMS) chip. The magnetic GO-Fe3O4-BSA nanocomposites were used for enantioseparations of Trp, Thr and propranolol in CEC with applying an external magnetic field.69) Polydopamine–GO (PDA-GO) was coated on the PDMS chip. Furthermore, BSA was adsorbed onto the PDA-GO layer. The PDA-GO-BSA nanocomposites showed the efficient separation of enantiomers of amino-acids (Trp and Thr) and dipeptide in CEC.70)
BSA was permeated into the phospholipid bilayer membrane, and then a phospholipid-BSA was coated onto the capillary walls.71) A first coating layer in the capillary was 1-(4-iodobutyl)-1,4-dimethylpiperazin-1-ium iodide, which effectively suppressed the electroosmotic flow and stabilized the phospholipid-BSA. Furthermore, BSA was encapsulated into silica monoliths using a sol-gel method by three steps: 1) formation of sol particles during hydrolysis and condensation, 2) addition of BSA into the sol, and 3) trapping BSA in the growing silicate network.72) The prepared monoliths were used for enantioseparation of Trp in CEC.
3.1.2. HSAHSA consists of 585 amino-acid sequences with a molecular mass of 66 kDa. Domenici et al. first immobilized HSA onto diol-silica particles20) and used in LC. Recently, HSA was immobilized onto polymer monoliths, copolymers of glycidyl methacrylate (GMA) and ethylene glycol dimethacrylate (EDMA), GMA-co-EDMA, by several methods (epoxy, CDI, DSS and Shiff base methods) as described in Section 2.73) The Schiff base method (Chart 3) gave the highest resolution in enantioseparations of warfarin and Trp.74) Furthermore, HSA-immobilized silica monoliths gave the highest retention and higher or comparable resolution and efficiency compared with HSA-immobilized silica particles and GMA-co-EDMA monoliths.74) CSPs based on HSA immobilized via its sulfhydryl groups by the SMCC (Chart 6) and SIA methods55) gave better enantioselectivity and stability than those prepared by the Schiff base method.
CSPs based on the N-terminal half of HSA-fragments, whose molecular mass are about 35000 Da, were prepared.75) The HSA-fragments were immobilized onto amino-silicas activated by DSC. CSPs based on the HSA-fragments had lower enantioselectivity than CSPs based on intact HSA for enantiomers of warfarin, 2-arylpropionic acid derivatives and benzodiazepines. It was reported that the primary binding sites of warfarin were on an N-terminal half of HSA (Site I), and those of 2-arylpropionic acid derivatives and benzodiazepines were on the other half of HSA (Site II).75) However, it was found that the (R)- and (S)-warfarin adopted very similar conformations in a co-crystal structure of each enantiomer with HSA and had many of the same specific contacts with amino-acid residues at the binding site. It resulted in the lack of stereospecificity of the HSA-warfarin interaction.76) Therefore, it is questionable whether the primary binding sites of HSA might be the same as the enantioselective binding sites on HSA.
In CEC, HSA-immobilized LC silica packing materials were packed into fused-silica capillaries.77) HSA was coated onto the capillary by physical adsorption.78) Enantioseparation of warfarin showing strong bindings to HSA was attained by using the coated capillary, but no enantioseparation of Trp showing weaker bindings to HSA. This is due to the low effective protein concentration on the capillary wall. GO-modified silica monoliths were prepared using three types of amine spacers for GO immobilization, including ammonia, ethanediamine (EDA) and polyethyleneimine.79) GO-EDA-HSA-based silica monoliths exhibited the best chiral recognition ability for enantiomers of Phe, Trp, warfarin, ibuprofen, azelastine, salbutamol, nefopam, propranolol and chlortrimeton among the prepared HSA-monoliths in CEC. Furthermore, HSA and cellulase were co-immobilized on a poly(GMA-co-EDMA) monolith. CSPs based on the mixed selectors exhibited a broader range of enantioselectivity than the single selector in CEC.80)
3.1.3. Lysozyme and PepsinHaginaka et al.23,24) developed CSPs based on lysozyme and pepsin, respectively. Basic (chlorpheniramine, alprenolol and tolperisone) and uncharged (benzoin and lorazepam) enantiomers were resolved in LC on lysozyme-based CSPs.23) On pepsin-based CSPs, basic (homochlorcyclizine, verapamil and alprenolol) and uncharged (oxazepam) enantiomers were also separated.24) However, no resolution of acidic enantiomers was observed on CSPs based on either lysozyme or pepsin. CSPs based on pepsin could be extensively applied to separations of basic enantiomers in CEC mode as described below.
In CEC using CSPs based on lysozyme, the fused-silica capillary walls were coated with a multilayer assembly of exfoliated γ-zyrconium phosphate and lysozyme, and Trp enantiomers were separated.81) After the disulfide bonds of lysozyme chain were reduced by tris (2-carboxyethyl)phosphine, the resultant partially unfolded monomer aggregated to form an amyloid-like 2D nanofilm.82) The aminopropyl-fused silica capillary was coated with the lysozyme-nanofilms. The obtained capillary column was used for the separations of basic enantiomers.82) Furthermore, the capillary walls were coated with a phospholipid coating with lysozyme83) as the chiral selector permeated into the phospholipid bilayer membrane as described in section 3.1.1. for BSA. Enatioseparation of Trp was attained with the phospholipid-lysozyme membrane.
In CEC using CSPs based on pepsin, silica monoliths functionalized with 3-aminopropyl trimethoxysilane were reacted with glutaraldehyde and pepsin. The resulting Schiff bases and residual aldehyde groups were reduced to the secondary amines and hydroxy groups, respectively, using sodium cyanoborohydride84) and used for the enantioseparation of nefopam hydrochloride. The cSWCNT was incorporated into poly(GMA-co-EDMA) monoliths and reacted with pepsin via conversion of epoxide groups to amine groups using glutaraldehyde as the crosslinker.85) The resulting Schiff base was reduced to the secondary amine. The pepsin-modified poly(GMA-co-EDMA) monoliths with cSWCNT exhibited much higher enantioselectivity than those without cSWCNT for basic drugs such as nefopam, clenbuterol, amlodipine and propranolol. Furthermore, GO-EDA-pepsin-based silica monoliths79) were prepared with the same method with GO-EDA-HSA-based silica monoliths. Furthermore, instead of cSWCNT and GO, amino-modified mesoporous silica nanoparticles were incorporated into poly(GMA-co-EDMA) monoliths.86) In this case, it was supposed that pepsin could be immobilized to amino-modified mesoporous silica nanoparticles and polymer monoliths. Similarly, amino-modified mesoporous silica nanoparticles contributed to excellent enantioselectivity for basic drugs (azelastine, salmeterol, clenbuterol, propranolol, nefopam, ritodrine and so on). A zeolitic imidazolate framework-8 (ZIF-8) via layer-by-layer self-assembly was grown on poly(GMA-co-EDMA) monoliths.87) Pepsin was covalently immobilized onto the surface of the amino-modified ZIF-8 through the Schiff base method. Separations of hydroxychloroquine, chloroquine, hydroxyzine, nefopam, clenbuterol and amlodipine enantiomers were attained with higher resolution values compared with the monoliths without ZIF-8. Similarly, zeolitic imidazolate framework-4, 5-imidazoledicarboxylic acid was used to immobilize pepsin in poly(GMA-co-EDMA) monoliths.88) Furthermore, gold nanoparticles (AuNP) via Au-S bond were incorporated onto poly(GMA-co-EDMA) monoliths. Pepsin was immobilized to the surface of the carboxylated AuNP.89) The pepsin-immobilized monoliths loaded AuNP showed excellent resolution for basic enantiomers.
3.2. Glycoproteins3.2.1. Human α1-Acid Glycoprotein (AGP, hAGP)AGP (orosomucoid (ORM)) consists of 183 amino-acid residues46) with an average molecular mass of approx. 33 kDa, measured by matrix-assisted laser-desorption ionization time-of-flight mass spectrometry.90) Besides the high heterogeneity of glycans, two variants are encoded by different genes. The F1*S variant (ORM1) is by the alleles of the same gene, while the A variant (ORM2) is by a different gene.91)
CSPs based on AGP were introduced by Hermansson.29) Silica particles, silica monoliths and GMA-co-EDMA monoliths were used as the support and their retentive and enantioselective properties were compared.51) Due to the higher surface area of the silica monolith, a more protein per unit volume was immobilized compared to a silica particle or GMA-co-EDMA monolith. The AGP-immobilized-silica monoliths gave higher retention, and better resolution and efficiency than the AGP-immobilized-silica particles or -GMA-co-EDMA monoliths.51)
A wide range of enantiomers were separated using AGP-based CSPs in LC.46,92) The optimization procedure for AGP-based CSPs was proposed by changing mobile phase pH, type and concentration of organic modifier and so on with the premise that the method can be used in LC/MS.93)
AGP-immobilized LC silica particle32) were packed into fused-silica capillaries and used in CEC mode.
3.2.2. OMCHIOMCHI is a glycoprotein from chicken and has a trypsin inhibitory activity.94) CSPs based on OMCHI were introduced by Miwa et al.30) OMCHI was immobilized onto aminopropyl-silica particles30) or amino-polymer particles,95) both of which were activated by DSC. CSPs based on OMCHI were utilized for the enantioseparations of pharmaceuticals in formulations and drugs in biological fluids in LC.46) The OMCHI column (commercially available as the OVM column) provided better long-term stability for repetitive injections compared to the commercially available AGP column.96) Two columns were also compared: the OMCHI column tended to separate large molecules better than the AGP column.97)
In CEC, OMCHI was encapsulated in silica monoliths and used for enantioseparation of benzoin, eperisone and chlorpheniramine.72)
3.2.3. cAGPHaginaka et al.31) isolated a protein from crude OMCHI preparations and named OGCHI as reported by Ketterer.98) Crude OMCHI preparations included 10% of OGCHI.31) The pI values of OMCHI and OGCHI are the same, as shown in Table 1. Therefore, OGCHI could be contaminated in OMCHI preparations. OGCHI was immobilized onto aminopropyl-silica particles activated with DSC. The CSPs based on OGCHI gave excellent chiral recognition ability, while CSPs based on the pure OMCHI showed no chiral recognition ability. Furthermore, OGCHI was preferentially immobilized onto amino-silicas activated by DSC in the presence of OMCHI.99) CSPs based on ovoglycoprotein from Japanese quail (OGJPQ) were also prepared.100) The CSPs based on OGCHI gave better chiral resolution for basic compounds but less chiral resolution for acidic compounds than the CSPs based on OGJPQ.
OGCHI was immobilized onto aminopropyl-silica particles by reaction with its amino and carboxy group, as shown in Charts 4 and 5, respectively.101) Figures 1A and 1B show separations of 2-phenylpropionic acid and naproxen on the CSPs based on OGCHI, immobilized via its amino and carboxy group, respectively. When the amino group of OGCHI was utilized for immobilization onto functionalized silicas, acidic ligands could not be enantioseparated. These results suggest that electrostatic interactions between an amino group of OGCHI and a carboxy group of a ligand work for the chiral recognition.
Reproduced with permission from Fig. 5 in ref. 101.
Recently, the amino-acid sequence of OGCHI was determined by isolation of a cDNA clone encoding OGCHI.32) OGCHI showed 31–32% identities with rabbit and human AGPs. Thus, OGCHI were named chicken AGP (cAGP), a member of the lipocalin family.
Aminopropyl-silica particles, whose nominal particle diameters were 5, 3 and 2.1 µm, were activated with DSC, and then cAGP was immobilized onto them.102) There were not so much differences in the retention and enantioseparation factors among three CSPs based on cAGP, while their resolution values were in the order of 2.1 > 3 > 5 µm of silica particle diameters and their height equivalent of a theoretical plate values were in the reversed order. These results indicate that silica particles of 2.1-µm diameters are suitable for the preparation of CSPs based on cAGP among silica particles evaluated.
Chiral recognition mechanisms of hAGP, turkey ovomucoid third domain (OMTKY3) and cAGP will be discussed based on chromatographic behaviors, NMR measurements, and molecular modeling and ligand docking studies. It is considered that the chiral recognition site(s) of those glycoproteins are located on the protein domain.
4.1. Human α1-Acid Glycoprotein (AGP, hAGP)It was supposed that the ligand-binding to hAGP occurred at a single binding site at the protein domain, or more than one binding site.103) Non-covalent interactions such as the hydrophobic, electrostatic and hydrogen bonding interactions could work in separations of enantiomers on CSPs based on AGP.92,93) Native hAGP consists of about 70% of F1*S variant (ORM1) and 30% of A variant (ORM2). ORM1 has the ligand-binding sites named lobs I, II and III, while ORM2 has only lobs I and II.104) Figure 2 shows crystal structures of ORM1 (left) and ORM2 (middle) of hAGP, and a constructed model structure of cAGP (right).105) The lobes I–III of hAGP engaged in the ligand-bindings.104)
Lobes I, II and III are highlighted in green, orange and cyan, respectively. The amino acids, which consist of feasible ligand-binding sites for cAGP, are colored in magenta. For interpretation of the references to color in this figure legend, the reader is referred to the Web version of the original article. Reproduced with permission from Fig. 7 in ref. 105.
Enantioselective bindings of coumarin derivatives such as warfarin, acenocoumarol and phenprocoumon to ORM 1 and ORM2 of hAGP were investigated. All examined compounds bound stronger to ORM1 than to ORM2.91) ORM1 and native hAGP preferred the binding of (S)-enantiomers of warfarin and acenocoumarol. Furthermore, a new homology model of hAGP was built and the models of ORM1 and ORM2 suggested that the binding cavity for ORM1, including W122, was the same with ORM2, and that ORM2 formed a smaller, and more hydrophobic cavity compared to ORM1.91) The docking results of (R)- and (S)-acenocoumarol to both variants indicated that the binding to ORM1 was more favorable than ORM2, but that energy differences between (R)- and (S)-enantiomers were not significant, showing a slight preference for (S)-enantiomers in the both variants.91)
Circular dichroism (CD) methods were utilized for evaluation of ligand-binding properties of hAGP.106) The induced CD spectra of drug–hAGP complexes were observed with different classes of drugs. Additional CD experiments, using recombinant hAGP mutants, indicated that the W25A could not induce extrinsic CD signal with any ligands, but that no changes occurred in the ligand-binding ability of W122A. These results suggest that W25 was essentially involved in the ligand-bindings of hAGP by π–π stacking mechanism.106)
4.2. OMTKY3The chiral recognition mechanism of a ligand on OMTKY3 was investigated by using NMR measurements, and molecular modeling and ligand docking.107) As shown in Fig. 3, there are chiral and achiral binding sites on OMTKY3: in the former, hydrophobic, electrostatic and hydrogen bonding interactions work for the chiral recognition of the ligand, while in the latter, only hydrophobic interactions work for the achiral recognition. Figure 4 shows the chiral binding model for (R)- and (S)-U-80413, one of 2-arylpropionic acid derivatives, on OMTKY3.107) The carboxy groups of (R)- and (S)-U-80413 engage in electrostatic interactions with the positive charge on Arg21. The carbonyl groups of (R)- and (S)-U-80413 share hydrogen bonds with NH3+ group of Lys34. The distinguishing difference between the enantiomers is the proximity of the phenyl group of (R)-U-80413 and Phe53. These mean that the (R)-enantiomer has three-point interactions with OMTKY3, while (S)-enantiomer has two-point interactions. The elution order of U-80413, which was (S)/(R), on CSPs based on OMTKY3 was consistent with the docking study.
The white curved graphic represents the protein backbone. Selected protein side chains are also shown in white. Ligands are labeled according to their R or S chirality and numbered according to their position among the 100 lowest energy minimized binding orientations. Reproduced with permission from Fig. 18 in ref. 107.
Reproduced with permission from Fig. 19 in ref. 107.
cAGP has only one W residue at the 26 position in 183 amino-acid sequences.29) The W26 was modified with 2-nitrophenylsulfenyl chloride and enantioseparations of some compounds were examined on CSPs based on native cAGP and W26-modified cAGP.108) Chiral separations of β-blockers such as alprenolol, oxprenolol and propranolol were completely lost on the CSPs based on W26-modified cAGP, while benzoin, ketoprofen and chlorpheniramine enantiomers were still separated despite lower enantioseparation factors than those on CSPs based on native cAGP. These results suggest that the W26 could be involved in chiral recognition of these compounds. Competition studies using N,N-dimethyl-n-octylamine as a competitor also supported the above results.108) Furthermore, CD methods were used for ligand-binding properties of cAGP. The extrinsic CD spectra with the W26-modified cAGP and CD displacement experiments revealed that a single W26 residue of cAGP conserved in the whole lipocalin family is part of the binding site and is essentially involved in the ligand-binding process via π–π stacking interactions between the indole ring of W26 and the ligand.109)
Recently, the model structure of cAGP was constructed using ORM2 of hAGP.105) Subsequently, benzoin, chlorpheniramine and propranolol enantiomers were docked onto a certain cavity of the constructed model structure of cAGP.105) Both enantiomers of benzoin were docked onto a similar position of cAGP (Fig. 5A). It was the case that both enantiomers of chlorpheniramine were docked onto the almost the same position with benzoin enantiomers (Fig. 5B). However, propranolol enantiomers slightly shifted to the small cliff near H25 and W26 as shown in Fig. 5C. For chlorpheniramine, an amino group of (R)-chlorpheniramine could interact with a carboxy group of E168 at a distance of 2.6 Å as shown in Fig. 6A, while a carboxy group of D161 is close to an amino group of (S)-chlorpheniramine at a distance of 3.0 Å as shown in Fig. 6B. In addition, as shown in Fig. 6B, Y47 was located nearby (S)-chlorpheniramine with a distance of 3.9 Å. Furthermore, a carbonyl group of the main chain between R128 and T129 is close to the chlorine atom of (S)-chlorpheniramine with a distance of 3.6 Å. These results indicate that (S)-chlorpheniramine interacts with cAGP more tightly than (R)-chlorpheniramine. Furthermore, (R)- and (S)-propranolol were docked to the constructed model structure of cAGP105) (Fig. 7). Interestingly, the docked propranolol was slightly shifted toward H25 and W26 compared with the dockings of bezoin and chlorpheniramine enantiomers. The amino group of (R)-propranolol interacted with a carbonyl group of the main chain between R128 and T129 at a distance of 2.7 Å, while the hydroxy group of (S)-propranolol interacted with the same carbonyl group at a distance of 2.8 Å. Y47 was also involved in the binding of (R)-propranolol with a distance of 4.7 Å. These results suggest that the (R)-propranolol binding to cAGP preferred to the (S)-propranolol binding. The elution orders of benzoin, chlorpheniramine and propranolol enantiomers on CSPs based on cAGP in LC were consistent with the docking results on the model structure of cAGP.
The constructed model structure of cAGP is illustrated as surface mode and the docked compounds are shown as ball-and-stick models, respectively. Reproduced with permission from Fig. 2 in ref. 105.
The docked ligands are represented in either green or blue for carbon atoms. Besides, nitrogen and oxygen atoms are colored in blue and red in each. The remarked amino-acid residues are labeled and shown in gray for carbon atoms, and feasible interactions are depicted as dotted lines. For interpretation of the references to color in this figure legend, the reader is referred to the Web version of the original article. Reproduced with permission from Fig. 4 in ref. 105.
The docked lignds are represented as in Fig. 6. For interpretation of the references to color in this figure legend, the reader is referred to the Web version of the original article. Reproduced with permission from Fig. 5 in ref. 105.
The ligand-binding sites of ORM1 and ORM2 of hAGP, and cAGP are completely different as shown in Fig. 2.105) As described in Section 4.1, a constructed model of hAGP could not explain chiral bindings of acenocoumarol on lobes I–III on ORM1 or lobs I–II on ORM2.104) On the other hand, CD methods showed that W25 was essentially involved in the ligand-bindings of hAGP.109) Taking into account CD results on hAGP and cAGP, and ligand-bindings of cAGP, chiral binding sites could be existing near W25 on hAGP and W26 on cAGP.
CSPs based on proteins and glycoproteins have been prepared and used for the enantioseparations of various compounds in LC and CEC. The supports used were mainly silicas and polymers, which are used as a particle, capillary or monolith. Proteins so far used were BSA and HSA, trypsin, α-chymotrypsin, lysozyme, pepsin, fatty acid-binding protein, penicillin G-acylase, streptavidin and lipase, while glycoproteins so far used were AGP (hAGP), OGCHI (now named cAGP), avidin, riboflavin binding protein, ovotransferrin, cellobiohydrolases, glucoamylase, antibody, α3β4-nicotinic acetylcholine receptor and human liver organic cation transporter.
CPSs based on proteins and glycoproteins can be utilized for the separations of not only enantiomers but also diastereomers, including pharmaceuticals and agrochemicals. In the future, a new protein or glycoprotein that has excellent chiral recognition properties can be found and introduced as CSPs. By genetic technology it is possible to prepare a protein that is stable and has excellent chiral recognition abilities. Furthermore, the domain or fragment of a protein, which has excellent chiral recognition abilities, will be prepared also by genetic technology.
I would like to thank my mentors and collaborators, especially the late Prof. Emeritus Toyozo Uno, Prof. Emeritus Terumichi Nakagawa, Prof. Thomas Pinkerton, Prof. Irving Wainer, Prof. Yutaka Sadakane, Prof. Ferenc Zsila, Prof. Qiang Fu, Dr. Hiroo Wada, Prof. Hisami Matsunaga, Prof. Taku Yamashita, Ms. Yuki Okazaki, Ms. Chino Kagawa, Ms. Naoko Kanasugi, Ms. Yukiko Miyano, Ms. Tokiko Murashima, Ms. Yuki Saizen, Ms. Chikako Seyama and Ms. Hisako Takehira.
The author declares no conflict of interest.
This review of the author’s work was written by the author upon receiving the 2018 Pharmaceutical Society of Japan Award for Divisional Scientific Contribution.
