Effect of Additives on the Selectivity and Reactivity of Enzymes.

Enzymes have been widely used as efficient, eco-friendly, and biodegradable catalysts in organic chemistry due to their mild reaction conditions and high selectivity and efficiency. In recent years, the catalytic promiscuity of many enzymes in unnatural reactions has been revealed and studied by chemists and biochemists, which has expanded the application potential of enzymes. To enhance the selectivity and activity of enzymes in their natural or promiscuous reactions, many methods have been recommended, such as protein engineering, process engineering, and media engineering. Among them, the additive approach is very attractive because of its simplicity to use and high efficiency. In this paper, we will review the recent developments about the applications of additives to improve the catalytic performances of enzymes in their natural and promiscuous reactions. These additives include water, organic bases, water mimics, cosolvents, crown ethers, salts, surfactants, and some particular molecular additives.

Enzymatic enantioselective aldol reactions of isatin derivatives with cyclic ketones under solvent-free conditions.

Nuclease p1 from Penicillium citrinum was observed to directly catalyze the asymmetric aldol reactions between isatin derivatives and cyclic ketones with high isolated yields (up to 95%) and moderate to good stereoselectivity (dr up to >99/1, ee up to 82%). A series of reaction conditions were investigated in detail, and the addition of deionized water had a big influence upon the enzyme activity. This case of biocatalytic promiscuity not only widens the applicability of nuclease p1 to new chemical transformation in organic synthesis, but also provides a potentially valuable method to construct pharmaceutically active compounds in medicinal chemistry.

Unexpected three-component domino synthesis of pyridin-2-ones catalyzed by promiscuous acylase in non-aqueous solvent.

The Acylase "Amano" (AA)-catalyzed synthesis of valuable pyridin-2-ones via domino Knoevenagel condensation-Michael addition-intramolecular cyclization-oxidization reaction between aldehyde, cyanoacetamide and ethyl acetoacetate or cyclohexyl acetoacetate was developed in the sense of a one-pot strategy. Various aliphatic, aromatic and hetero-aromatic pyridin-2-ones could also be produced in the reaction. The mechanism was illustrated according to the controlled reaction, pyridin-2-one was formed via the oxidization by oxygen at the final step. This simple and efficient enzymatic domino reaction not only widens its application of AA to organic synthesis, but is also an attractive way for the synthesis of heterocyclic compounds.

Reagentless biosensor based on layer-by-layer assembly of functional multiwall carbon nanotubes and enzyme-mediator biocomposite.

A simple and controllable layer-by-layer (LBL) assembly method was proposed for the construction of reagentless biosensors based on electrostatic interaction between functional multiwall carbon nanotubes (MWNTs) and enzyme-mediator biocomposites. The carboxylated MWNTs were wrapped with polycations poly(allylamine hydrochloride) (PAH) and the resulting PAH-MWNTs were well dispersed and positively charged. As a water-soluble dye methylene blue (MB) could mix well with horseradish peroxidase (HRP) to form a biocompatible and negatively-charged HRP-MB biocomposite. A (PAH-MWNTs/HRP-MB)(n) bionanomultilayer was then prepared by electrostatic LBL assembly of PAH-MWNTs and HRP-MB on a polyelectrolyte precursor film-modified Au electrode. Due to the excellent biocompatibility of HRP-MB biocomposite and the uniform LBL assembly, the immobilized HRP could retain its natural bioactivity and MB could efficiently shuttle electrons between HRP and the electrode. The incorporation of MWNTs in the bionanomultilayer enhanced the surface coverage concentration of the electroactive enzyme and increased the catalytic current response of the electrode. The proposed biosensor displayed a fast response (2 s) to hydrogen peroxide with a low detection limit of 2.0×10⁻7 mol/L (S/N=3). This work provided a versatile platform in the further development of reagentless biosensors.

Integrating In Silico and In vitro approaches to dissect the stereoselectivity of Bacillus subtilis lipase A toward ketoprofen vinyl ester.

The asymmetric catalysis, as the character of enzyme, attracts increasing attention from the scientific and industrial communities. In this study, the Bacillus subtilis lipase A, as a model enzyme, is studied systematically to dissect its stereoselectivity toward (rac)-ketoprofen vinyl ester using a combination scheme of molecular docking and quantum mechanical/molecular mechanical (QM/MM) analysis. In this procedure, the rational orientation of the two enantiomers of ketoprofen vinyl ester is obtained with the AutoDock performing, and then, the steric contacts between the enzyme and substrate in the docking outputs are examined visually at the atomic level with a small-probe technique. Subsequently, the binding energies of the enzyme-substrate complexes are calculated using an ONIOM (Our own N-layered Integrated Molecular Orbital + Molecular mechanics)-based QM/MM protocol. The results obtained from the theoretical studies show that the B. subtilis lipase A prefer to hydrolyze the (R )-ketoprofen vinyl ester when compared to its (S )-enantiomer, with a relatively high E (stereoselectivity) value of 31.28 charactering its enantioselectivity. Furthermore, to verify the conclusions from the computational analysis, the B. subtilis lipase A gene is cloned to overexpress the recombinant B. subtilis lipase A, and its stereoselectivity was determined. Satisfactorily, the experimental results are in well agreement with the theoretical predictions because the (R )-ketoprofen vinyl ester is found as the preferring enantiomer of the B. subtilis lipase A, with experimentally measured E value of 36.7. We therefore expect that this in silico-in vitro hybrid approach can provide a new and effective avenue to predict the catalytic activity of and to investigate the molecular mechanism of enzyme-mediated asymmetric catalysis and help in understanding the enzymatic process and in rational enzyme design.

A Combination of Computational and Experimental Approaches to Investigate the Binding Behavior of B.sub Lipase A Mutants with Substrate pNPP.

The formation of so-called enzyme-substrate complex is the key step for a successful enzyme-catalysis reaction. Enzymes use substrate-binding energy both to promote ground-state association and to stabilize the reaction transition state selectively. Some residues besides the catalytic triads play important roles toward the substrate binding process. In this study, we employed ONIOM methodology and docking to explore the influence of individual amino acids of Bacillus subtilis (B.sub) lipase A on the hydrolysis reaction, with the aim to guide mutagenesis experiments on the basis of computational framework. Subsequently, the B.sub lipase A is modified experimentally with different non-polar residues at the position 12, which is spatially adjacent to the active site, by using site-directed mutagenesis. We obtain a good correlation model between the computationally predicted binding energies and the experimental measured affinities, with a correlation coefficient r=0.78. It is largely unexplored that the combination of docking and quantum mechanical/molecular mechanical (QM/MM) analyses is used in conjunction with experimental procedure to investigate the enzyme catalysis process. We therefore expect that this work could provide a new pathway for exploring the molecular mechanism of enzyme-substrate recognition and interaction.

Multidrug nanoparticles based on novel random copolymer containing cytarabine and fluorodeoxyuridine.

Novel multidrug nanoparticles were self-assembled from the random copolymer containing cytarabine and fluorodeoxyuridine. The multidrug copolymer carrying 28.7wt.% of cytarabine and 29.1wt.% of fluorodeoxyuridine was prepared by radical polymerization combined with enzymatic selective transesterification. Homopolymers of the two drugs were also synthesized by the same method. And the polymers were characterized by FTIR, (1)H NMR, and gel permeation chromatography (GPC). Self-assembly of the multidrug copolymer was verified by UV-vis and fluorescence spectroscopy. The morphology of nanoparticles formed from the copolymer was investigated by transmission electron microscopy (TEM) and dynamic light scattering (DLS), which indicated that the nanoparticles were regular spheres with a diameter of 133+/-28nm. In vitro drug release studies illustrated that the two synergistic anticancer agents could be simultaneously released from the multidrug nanoparticles.

Hydrolase-catalyzed fast Henry reaction of nitroalkanes and aldehydes in organic media.

Nitroalkanes underwent fast additions to a variety of structurally diverse aldehydes under the catalysis of d-aminoacylase in DMSO. The influences of reaction conditions including solvents, temperature, enzyme concentration and molar ratio of substrates were systematically investigated. Seventeen products were obtained in short time with moderate to high yields. It is the first report on hydrolase-catalyzed fast Henry reaction in organic solvent.

Enzymatic synthesis of amoxicillin via a one-pot enzymatic hydrolysis and condensation cascade process in the presence of organic co-solvents.

A cascade reaction combining the enzymatic hydrolysis of Penicillin G potassium salt (PGK) with the kinetically controlled enzymatic coupling of in situ formed 6-aminopenicillanic acid (6-APA) with p-hydroxyphenylglycine methyl ester (D-HPGM) to give amoxicillin as the final product by using a single enzyme has been demonstrated successfully. Ethylene glycol (EG) was employed as a component of reaction buffer to improve the synthesis yield. Reaction parameters, including different cosolvents, EG content, the loading of immobilized penicillin G acylase (IPA), and reaction temperature and time were studied to evaluate their effects on the reaction. The best result of 55.2% yield was obtained from the reaction which was carried out in the mixed media containing 40% sodium dihydrogen phosphate buffer (apparent pH 6.0) and 60% EG (v/v), with the initial concentration 150 mM and 450 mM of PGK and D-HPGM, respectively, catalyzed by 50 IU/mL IPA at 25 degrees C for 10 h. The IPA could be recycled for nine batches without obviously losing of catalytic activity. The important strategy will have potential application in the beta-lactam antibiotics industry due to the advantages of saving the effort of isolating 6-APA, reducing usual enzymatic steps and the industrial cost of amoxicillin synthesis.

Facile synthesis of novel mutual derivatives of nucleosides and pyrimidines by regioselectively chemo-enzymatic protocol.

We established a facile regioselectively chemo-enzymatic synthesis procedure for the preparation of mutual derivatives of nucleosides and pyrimidines by sequential Markovnikov addition and acylation. Firstly, pyrimidine derivatives containing vinyl ester group were synthesized from pyrimidines and divinyl esters through Markovnikov addition catalyzed by K(2)CO(3) in DMSO at 80 degrees C, and the yields were ranged from 50% to 87%. Then regioselective acylation of ribavirin and cytarabine with pyrimidine vinyl ester was catalyzed by CAL-B (immobilized lipase from Candida antarctica) in anhydrous acetone. Reaction conditions of enzymatic acylation including enzyme resource and solvents were optimized. A series of mutual derivatives of nucleosides and pyrimidines were synthesized successfully and characterized with NMR, IR, and HRMS. This chemo-enzymatic protocol involving sequential Markovnikov addition and acylation provided a novel way of synthesizing complicated functional compounds regioselectively which was hard to be achieved either by chemical or by enzymatic methods.

Thermal treatment of galactose-branched polyelectrolyte microcapsules to improve drug delivery with reserved targetability.

A novel multilayered drug delivery system by LbL assembly of galactosylated polyelectrolyte, which is possible to have the potential in hepatic targeting by the presence of galactose residues at the microcapsule's surface, is designed. Thermal treatment was performed on the capsules and a dramatic thermal shrinkage up to 60% decrease of capsule diameter above 50 degrees C was observed. This thermal behavior was then used to manipulate drug loading capacity and release rate. Heating after drug loading could seal the capsule shell, enhancing the loading capacity and reducing the release rate significantly. Excellent affinity between galactose-binding lectin and heated galactose-containing microcapsules were observed, indicating a stable targeting potential even after high temperature elevating up to 90 degrees C.

Hepatic-targeting microcapsules construction by self-assembly of bioactive galactose-branched polyelectrolyte for controlled drug release system.

We describe the construction of hepatic-targeting microcapsules by self-assembly of chemo-enzymatic synthesized poly(vinyl galactose ester-co-methacryloxyethyl trimethylammonium chloride) (PGEDMC) containing galactose branches, which can be specifically recognized by membrane bound galactose receptors (ASGPR), for acyclovir (ACV) controlled release system. Alternate deposition of PGEDMC and poly(sodium 4-styrenesulfonate) (PSS) was carried out on ACV microcrystals. It was revealed that the drug release rate decreases with the increase of coated layer number and a microcapsule-drying treatment would enhance the sustained release effect probably because of a multilayer shrink and tightness during the process. The complete release of ACV yielded a hollow PGEDMC/PSS multilayered network with favorable integrity and nano-thickness by TEM and SEM. The potential targetability of the system was proved in vitro by PNA lectin recognition. Lectin hardly adsorbed on the film where the outmost layer was a polyanion or a polycation without galactose component. Whilst the galactose-containing layer (PGEDMC) was the outmost layer, a significant lectin combination was observed. This technique could provide a promising way to encapsulate and deliver various target substances in biological and pharmaceutical applications.

Promiscuous zinc-dependent acylase-mediated carbon-carbon bond formation in organic media.

A zinc-dependent acylase, D-aminoacylase from Escherichia. Coli, displays a promiscuous activity to catalyze the carbon-carbon bond formation reaction of 1,3-dicarbonyl compounds to methyl vinyl ketone in organic media.

N-methylimidazole significantly improves lipase-catalysed acylation of ribavirin.

N-methylimidazole, a molecular solvent, but also, in cationic form, a component of 1-alkyl-3-methylimidazolium ([C(n)MIM]+) ionic liquids, showed promise as an additive in accelerating remarkably transesterification catalyzed by lipase acrylic resin from Candida antarctica (CAL-B).

Bioactive galactose-branched polyelectrolyte multilayers and microcapsules: self-assembly, characterization, and biospecific lectin adsorption.

We describe the fabrication of multilayers and microcapsules with biologically designed targeting activity using chemoenzymatic synthesized carbohydrate-branched polyelectrolytes. A novel cationic d-galactose-branched copolymer [poly(vinyl galactose ester-co-methacryloxyethyl trimethylammonium chloride), PGEDMC] is alternated with poly(styrene sulfonate) (PSS) to form thin multifilms by the layer-by-layer (LbL) technique on such different solid surfaces as quartz slides, poly(ethylene terephthalate) (PET) films, silicon wafers, and polystyrene (PS) microparticles. The experimental protocols were first optimized on flat, smooth silica substrates using UV-vis, contact angle, and atomic force microscopy (AFM) measurements. The film properties of PGEDMC/PSS multilayers are modified by varying polyelectrolyte concentration, ionic strength, and counteranion types. Hollow capsules were formed after the removal of colloidal templates; transmission (TEM) and scanning (SEM) electron microscopy were used to verify the LbL process integrity. PGEDMC/PSS planar films and capsules carrying beta-galactose as recognition signals have specific recognition abilities with peanut agglutinin (PNA) lectin rather than concanavalin A (Con A) lectin observed by fluorescence spectroscopy.

Basic ionic liquid as catalysis and reaction medium: a novel and green protocol for the Markovnikov addition of N-heterocycles to vinyl esters, using a task-specific ionic liquid, [bmIm]OH.

A basic ionic liquid, 1-methyl-3-butylimidazolium hydroxide ([bmIm]OH), has been introduced as a catalyst and reaction medium for the Markovnikov addition of N-heterocycles to vinyl esters under mild conditions. The evidence for the role of this basic ionic liquid [bmIm]OH in promoting the Markovnikov addition has been given. On the basis of the evidence, a mechanism was postulated.

Markedly enhancing lipase-catalyzed synthesis of nucleoside drugs' ester by using a mixture system containing organic solvents and ionic liquid.

Eightfold higher yields and three times faster reaction rates were achieved by means of using a mixture solvent system composed of 90% acetone and 10% [BMIM]BF4 in the lipase-catalyzed regioselective synthesis of polymerizable ester of nucleoside drugs.

Controllable synthesis of polymerizable ester and amide prodrugs of acyclovir by enzyme in organic solvent.

A facile control of the acylation position at the primary hydroxyl and amino of acyclovir, respectively, was achieved and five polymerizable acyclovir prodrugs were synthesized. Various reaction conditions were studied in detail. Thus, lipase acrylic resin from Candida antarctica (CAL-B) in pyridine or acetone showed high chemo-selectivity toward the primary hydroxyl of acyclovir. However, lipase PS 'Amano' (PS) in DMSO selectively acylated the amino group. The selectivity of PS could be adjusted by changing reaction solvents. The acyclovir vinyl derivatives obtained would be important monomers used for the preparation of macromolecular nucleoside drugs.

Chemo-enzymatic synthesis of raffinose-branched polyelectrolytes and self-assembly application in microcapsules.

A novel biocompatible polyelectrolyte poly(vinyl raffinose-co-acrylic acid) (PRCA) containing a raffinose branch was prepared via redox polymerization using Fe(2+)/K(2)S(2)O(8)/H(2)O(2) starting from enzymatically-synthesized monomer: 1-O-vinyldecanedioyl raffinose. Copolymers with different monomer feed ratios were prepared and characterized with IR, NMR, and GPC. PRCA can be alternated with polycation to form microcapsules on a crystals template by electrostatic layer-by-layer technique. The multilayers of PRCA/poly(methacryloyloxyethyl dimethylbenzyl ammonium chloride) (PMBA) on quartz slides and PRCA/poly(dimethyldiallyl ammonium chloride) (PDDA) on acyclovir crystals template were fabricated and characterized with UV-Vis spectra, the microelectrophoretic measurement, and TEM. Hollow capsules can be formed after the removal of acyclovir crystals template in a buffer solution. The nano-capsule-carrying galactose residue is a potential targeting drug-controlled delivery systems.

Hydrolase-catalyzed Michael addition of imidazoles to acrylic monomers in organic medium.

Hydrolase-catalyzed Michael addition of imidazole derivatives to acrylic monomers in organic medium was described. A serial of N-substituted imidazole derivatives were successfully synthesized in moderate yields by the catalysis of hydrolases in organic medium. Nine commercially available hydrolases from different sources were screened and all of them were found to be able to catalyze this type of addition reaction. The reaction yields depended on the solvent properties and the solvents with higher log P value supported this enzyme-catalyzed reaction to give higher conversion. Influence of the structure of the Michael acceptor and donor on the enzymatic Michael addition was also investigated. The acceptor with shorter alcohol chain afforded a higher yield. A more rapid conversion was observed when the donor had an electron-withdrawing group. This hydrolase-catalyzed Michael addition reaction has widened the applicability of biocatalysts in organic and bioorganic synthesis.