Recent developments on creation of artificial metalloenzymes

Organic synthesis using biocatalysts has been developed over many years and is still a prominent area of research. In this context, various hybrid biocatalysts composed of a synthetic metal complex catalyst and a protein scaffold (i.e. “artificial metalloenzymes”) have been constructed. One of the most recent research areas in biocatalysts-mediated synthesis is CC bond/cleavage, the most important type of reaction in organic chemistry. Some of the artificial enzymes were applied to in-cell reactions as well as in vitro systems. The effects of the structural fluctuation in biomacromolecules on their functions have also been realized. This review article includes recent research examples of artificial metalloenzymes used to CC bond formation/cleavage. As a perspective, we also focus on how we apply protein dynamics factor for the creation of new generation artificial metalloenzymes.     

Method for optical resolution of racemic homochlorcyclizine and comparison of optical isomers in antihistamine activity and pharmacokinetics.

A method was developed for semi-preparative scale enantioseparation of racemic homochlorcyclizine (HCZ) by high performance liquid chromatography (HPLC) on Chiralcel OD column. The best resolution was achieved using an eluent composed of n-hexane plus 0.2 M isopropylamine. By this method, about 5.0 mg of racemic HCZ could be resolved completely in one run. The optical purity of the enantiomers were both greater than 99.9%. The studies of antihistamine activity on guinea pig ileum demonstrated that l-HCZ is significantly more potent than d- and racemic HCZ. The pharmacokinetics of d- and l-HCZ after oral administration to rats also differed. The successful resolution of racemic HCZ permits comparison of the pharmacokinetics and antihistamine activity of the enantiomers.     

Effect of lipid composition on phospholipase A2-catalyzed membrane leakage in immobilized liposomes: sensitization for polychlorinated biphenyls detection with antibody affinity column tandem with fluorescent liposome column

Phospholipase A(2) (PLA(2))-catalyzed membrane leakage can be detected by immobilized liposomes containing a self-quenching fluorescent dye, calcein, on an open column using off-line analysis with a fluorescent spectrophotometer. The calcein release was found to be affected by the pH value, incubation time, and liposome compositions. The fluorescent signal from the negatively charged liposomes hydrolyzed by PLA(2) was 5 times higher than that from neutral liposomes. We utilized this enzymatic reaction to amplify signal to detect polychlorinated biphenyls (PCBs). To achieve this goal, we conjugated an analogue of PCB, 3,4-dichloroaniline, to PLA(2). The competitive immunoreaction between the 3,4-dichloroaniline-PLA(2) conjugate and PCB samples on the anti-PCB antibody column caused the release of the bound PLA(2) conjugates in proportion to the PCB concentration. The released PLA(2) conjugates was then passed through the tandem fluorescent liposome column causing release of fluorescent dye from the liposomes. Therefore, the signal of immunocompetitive assay was amplified on the fluorescent liposome column. The tandem column system achieves a high sensitivity by detecting the PCB concentration as low as 0.5 ng/mL in less than 20 min. It has great potential in detecting other pollutants, and has been used for sensitive immunoassays.     

2,2′-Isopropylidenebis[(4S,5R)-4,5-di(2-naphthyl)-2-oxazoline] ligand for asymmetric cyclization-carbonylation of meso-2-alkyl-2-propargylcyclohexane-1,3-diols

The oxidative cyclization-carbonylation of meso-2-alkyl-2-propargylcyclohexane-1,3-diols mediated by Pd(II) with chiral bisoxazoline (box ligand) afforded bicyclic-β-alkoxyacrylates. Based on a ligand screening, 2,2′-isopropylidenebis[(4S,5R)-4,5-di(2-naphthyl)-2-oxazoline] ligand has been developed. The products with a chiral quaternary carbon were obtained in 71-100% yields with 85-95% ee.     

Dioxo-molybdenum(VI) and mono-oxo-molybdenum(IV) complexes supported by new aliphatic dithiolene ligands: new models with weakened Mo=O bond characters for the arsenite oxidase active site

The cis-dioxo-molybdenum(VI) complexes, [MoO2(L(H))2]2- (1b), [MoO2(L(S))(2)]2- (2b), and [MoO2(L(O))2]2- (3b) (L(H) = cyclohexene-1,2-dithiolate, L(S) = 2,3-dihydro-2H-thiopyran-4,5-dithiolate, and L(O) = 2,3-dihydro-2H-pyran-4,5-dithiolate), with new aliphatic dithiolene ligands were prepared and investigated by infrared (IR) and UV-vis spectroscopic and electrochemical methods. The mono-oxo-molybdenum(IV) complexes, [MoO(L(H))2]2- (1a), [MoO(L(S))2]2- (2a), and [MoO(L(O))2]2- (3a), were further characterized by X-ray crystal structural determinations. The IR and resonance Raman spectroscopic studies suggested that these cis-dioxo molybdenum(VI) complexes (1b-3b) had weaker Mo=O bonds than the common Mo(VI)O2 complexes. Complexes 1b-3b also exhibited strong absorption bands in the visible regions assigned as charge-transfer bands from the dithiolene ligands to the cis-MoO2 cores. Because the oxygen atoms of the cis-Mo(VI)O2 cores are relatively nucleophilic, these complexes were unstable in protic solvents and protonation might occur to produce Mo(VI)O(OH), as observed with the oxidized state of arsenite oxidase.     

Peroxotungstate immobilized on ionic liquid-modified silica as a heterogeneous epoxidation catalyst with hydrogen peroxide

Peroxotungstate immobilized on ionic liquid-modified SiO2 is capable of heterogeneously epoxidizing a wide range of olefins with the maintenance of the catalytic activity of homogeneous analogue. The epoxidation was immediately stopped by the removal of the catalyst, and no tungsten species could be found in the filtrate after the removal of the catalyst. These results can rule out any contribution to the observed catalysis from the tungsten species that leached into the reaction solution, and the observed catalysis is truly heterogeneous in nature. Furthermore, the catalyst was reusable without the loss of the catalytic performance.     

Isolation of heptadepsin, a novel bacterial cyclic depsipeptide that inhibits lipopolysaccharide activity

Lipopolysaccharide (LPS) is considered to cause various inflammatory reactions. We searched among microbial secondary metabolites for compounds that could inhibit LPS-stimulated adhesion between human umbilical vein endothelial cells (HUVEC) and human myelocytic cell line HL-60 cells. In the course of our screening, we isolated a novel cyclic depsipeptide, which we named heptadepsin, from the whole culture broth of Paenibacillus sp. The addition of heptadepsin prior to LPS stimulation decreased HL-60 cell-HUVEC adhesion without showing any cytotoxicity. It also inhibited the cellular adhesion induced by lipid A, the active component of LPS, but it did not inhibit TNF-alpha or IL-1beta-induced cell adhesion. The result of surface plasmon resonance (SPR) analysis revealed that heptadepsin interacted with lipid A directly. Thus, heptadepsin, a novel naturally occurring cyclic heptadepsipeptide, was shown to inactivate LPS by direct interaction with LPS.     

Kinetically controlled Ag+-coordinated chiral supramolecular polymerization accompanying a helical inversion

We report kinetically controlled chiral supramolecular polymerization based on ligand–metal complex with a 3 : 2 (L : Ag⁺) stoichiometry accompanying a helical inversion in water. A new family of bipyridine-based ligands (d-L1, l-L1, d-L2, and d-L3) possessing hydrazine and d- or l-alanine moieties at the alkyl chain groups has been designed and synthesized. Interestingly, upon addition of AgNO₃ (0.5–1.3 equiv.) to the d-L1 solution, it generated the aggregate I composed of the d-L1AgNO₃ complex (d-L1 : Ag⁺ = 1 : 1) as the kinetic product with a spherical structure. Then, aggregate I (nanoparticle) was transformed into the aggregate II (supramolecular polymer) based on the (d-L1)₃Ag₂(NO₃)₂ complex as the thermodynamic product with a fiber structure, which led to the helical inversion from the left-handed (M-type) to the right-handed (P-type) helicity accompanying CD amplification. In contrast, the spherical aggregate I (nanoparticle) composed of the d-L1AgNO₃ complex with the left-handed (M-type) helicity formed in the presence of 2.0 equiv. of AgNO₃ and was not additionally changed, which indicated that it was the thermodynamic product. The chiral supramolecular polymer based on (d-L1)₃Ag₂(NO₃)₂ was produced via a nucleation–elongation mechanism with a cooperative pathway. In thermodynamic study, the standard ΔG° and ΔH_e values for the aggregates I and II were calculated using the van''t Hoff plot. The enhanced ΔG° value of the aggregate II compared to that of the formation of aggregate I confirms that aggregate II was thermodynamically more stable. In the kinetic study, the influence of concentration of AgNO₃ confirmed the initial formation of the aggregate I (nanoparticle), which then evolved to the aggregate II (supramolecular polymer). Thus, the concentration of the (d-L1)₃Ag₂(NO₃)₂ complex in the initial state plays a critical role in generating aggregate II (supramolecular polymer). In particular, NO₃⁻ acts as a critical linker and accelerator in the transformation from the aggregate I to the aggregate II. This is the first example of a system for a kinetically controlled chiral supramolecular polymer that is formed via multiple steps with coordination structural change.

The nanoparticles were transformed into the supramolecular polymer as the thermodynamic product, involving a helical inversion from left-handed to right-handed helicity.     

Profiling analysis of oligosaccharides in antibody pharmaceuticals by capillary electrophoresis

Carbohydrate chains in glycoprotein pharmaceuticals have important roles for the expression of their biological activities. Therefore, development of an assessment method for the carbohydrate chains is an important parameter for quality control of glycoprotein pharmaceuticals such as newly developed therapeutic antibodies. In this report, we applied capillary electrophoresis with laser-induced fluorescence detection to the analysis of carbohydrate chains after releasing with glycoamidase followed by derivatization with 3-aminobenzoic acid. We found that four major oligosaccharides present in antibody pharmaceuticals were successfully separated with good resolution. The present method showed good precision in both migration times and relative peak areas, and gave comparable accuracy with that using a derivatization method with 8-aminopyrene-1,3,6-trisulfonate.     

Enantioface-selective palladium-catalyzed silaboration of allenes via double asymmetric induction

Enantioenriched beta-borylallylsilanes were synthesized by palladium-catalyzed enantioface-selective addition of the silicon-boron bond to terminal allenes using a palladium catalyst possessing a chiral monodentate phosphine ligand. Use of a silylborane bearing a chiral auxiliary on the boron atom was beneficial to gain enantioface selectivities as high as 96% de.