Synthesis of macromolecular systems via lipase catalyzed biocatalytic reactions: applications and future perspectives.

Enzymes, being remarkable catalysts, are capable of accepting a wide range of complex molecules as substrates and catalyze a variety of reactions with a high degree of chemo-, stereo- and regioselectivity in most of the reactions. Biocatalysis can be used in both simple and complex chemical transformations without the need for tedious protection and deprotection chemistry that is very common in traditional organic synthesis. This current review highlights the applicability of one class of biocatalysts viz."lipases" in synthetic transformations, the resolution of pharmaceutically important small molecules including polyphenols, amides, nucleosides and their precursors, the development of macromolecular systems (and their applications as drug/gene carriers), flame retardants, polymeric antioxidants and nanocrystalline solar cells, etc.

Novozym 435-catalyzed syntheses of polyesters and polyamides of medicinal and industrial relevance.

The adverse impact of chemical and biochemical waste on the environment and human health poses a serious challenge in today's World. The best way to address these challenges is to reduce the waste by developing more efficient processes and technologies, based on the principles of "green chemistry". Some of these synthetic approaches involving the chemoenzymatic synthetic methodologies are discussed herein. These lead to the formation of unique nanomaterials with diverse applications, such as drugs/gene delivery systems, flame retardant materials, conducting polymers, controlled release systems, diagnostic agents, and polymeric electrolytes for nanocrystalline solar cells.

Design, synthesis and evaluation of novel PEGylated curcumin analogs as potent Nrf2 activators in human bronchial epithelial cells.

Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) is a central transcription factor that regulates the anti-oxidant defense system and is considered as a modifier for several inflammatory diseases. Thus, activation of Nrf2 provides pivotal therapeutic target for developing therapy against these diseases. Herein, a chemo-enzymatic methodology is designed and developed to make PEGylated curcumins as water soluble drug candidates with enhanced aqueous solubility and bioavailability. For this, curcumin was judiciously converted to diester (1) using ethyl α-bromoacetate and potassium carbonate. The diester 1 in subsequent step was copolymerized with poly(ethylene glycol) using Candida antarctica lipase [CAL-B, Novozym 435] under solventless condition. C. antarctica selectively does trans-esterification and only catalyses reaction of the primary hydroxyls of poly(ethylene glycol). It does not affect the secondary enolic hydroxyls of curcumin, thus leaving behind the active group unaltered. A luciferase based reporter gene assay was used for primary screening for identifying a novel Nrf2 activator. Most of the PEGylated curcumin analogs strongly activate Nrf2 several folds higher than the free curcumin but copolymer 3a was identified as the most potent Nrf2 activator. Copolymer 3a induces Nrf2-driven NQO1 expression in a concentration dependent manner. Furthermore, a plausible mechanism for quantitative structure-activity relationship is also discussed.

Design, synthesis and anti-inflammatory evaluation of PEGylated 4-methyl and 4,8-dimethylcoumarins.

Aberrant interaction between the leukocyte and the endothelial cell (EC) resulting from the deregulated expression of cell adhesion molecules (CAMs) on the endothelium results in uncontrolled inflammation leading to various inflammatory disorders. The existing drugs used to modulate the cytokine-induced expression of cell molecules have severe side effects. Therefore, there is an unmet therapeutic need to develop potent and safe drugs to treat inflammatory disorders. In the present study, novel PEGylated and non-PEGylated 4-methyl and 4,8-dimethylcoumarin derivatives were designed, synthesized and, evaluated for ICAM-1 inhibitory activity. The PEGylated coumarins were synthesized in two different ways. In the first approach, diesters of 4-methyl and 4,8-dimethylcoumarin were co-polymerized, separately with poly(ethylene glycol) using Candida antarctica lipase under solventless conditions. In the other approach, 4-methyl and 4,8-dimethylcoumarins were suitably converted to their bromo analogues and were tethered to already synthesized PEGylated polymers. Synthesized derivatives were evaluated for anti-inflammatory activities with respect to their ability to inhibit the TNF-alpha induced ICAM-1 (intercellular cell adhesion molecule-1) on human endothelial cells. It was found that PEGylated 4-methyl and 4,8-dimethylcoumarin derivatives were more effective than their non-PEGylated analogues to inhibit ICAM-1 expression. The present study opens new vista for PEGylated non-steroidal anti-inflammatory compounds and their further investigations.

Specificities of acetoxy derivatives of coumarins, biscoumarins, chromones, flavones, isoflavones and xanthones for acetoxy drug: protein transacetylase.

The earlier work carried out in our laboratory led to the identification of a novel rat liver microsomal enzyme termed as acetoxy drug: protein transacetylase (TAase), catalyzing the transfer of acetyl group from polyphenolic acetates (PA) to functional proteins. In this paper, we have reported the comparison of the specificities of acetoxy derivatives of coumarins, biscoumarins, chromones, flavones, isoflavones and xanthones with special reference to the phenyl moiety/bulky group on the pyran ring of PA. The results clearly indicated that compounds having phenyl moieties, when used as the substrates, resulted in a significant reduction of TAase catalyzed activity. The alteration in TAase catalyzed activation of NADPH cytochrome c reductase and inhibition of benzene-induced micronuclei in bone marrow cells by PA were in tune with their specificities to TAase.

Microwave-assisted synthesis of antimicrobial dihydropyridines and tetrahydropyrimidin-2-ones: novel compounds against aspergillosis.

Ten 4-aryl-1,4-dihydropyridine and three 4-aryl-1,2,3,4-tetrahydropyrimidin-2-one derivatives have been synthesized and examined for their activity against pathogenic strains of Aspergillus fumigatus and Candida albicans. Although none of the three compounds belonging to pyrimidin-2-one series showed any activity against two pathogens, two of the compounds of the dihydropyridine series, that is, diethyl 4-(4-methoxyphenyl)-2,6-dimethyl-1,4-dihydropyridin-3,5-dicarboxylate and dimethyl 4-(4-methoxyphenyl)-2,6-dimethyl-1,4-dihydropyridin-3,5-dicarboxylate, exhibited significant activity against A. fumigatus in disc diffusion, microbroth dilution and percent spore germination inhibition assays. The most active diethyl dihydropyridine derivative exhibited a MIC value of 2.92 microg/disc in disc diffusion and 15.62 microg/ml in microbroth dilution assays. The MIC(90) value of the most active compound by percent germination inhibition assay was found to be 15.62 microg/ml. The diethyl dicarboxylate derivative of dihydropyridine also exhibited appreciable activity against C. albicans. The in vitro toxicity of the most active diethyl dihydropyridine derivative was evaluated using haemolytic assay, in which the compound was found to be non-toxic to human erythrocytes even at a concentration of 625 microg/ml. The standard drug amphotericin B exhibited 100% lysis of erythrocytes at a concentration almost 16 times less than the safer concentration of the most active dihydropyridine derivative.

Nanosphere-mediated delivery of vitamin E increases its efficacy against oxidative stress resulting from exposure to amyloid beta.

Oxidative stress is an early hallmark of affected neurons in Alzheimer's disease (AD). The antioxidant vitamin E provided limited neuroprotection in AD, which may have derived from its lipophilic nature and resultant inability to quench cytosolic reactive oxygen species (ROS), including those generated from antecedent membrane oxidative damage. We examined herein whether or not encapsulation into polyethylene glycol (PEG)-based nanospheres, which can enter the cytosol, improved the efficacy of vitamin E against amyloid-beta(Abeta)-induced ROS. Unexcapsulated vitamin E prevented Abeta-induced ROS in cultured SH-SY-5Y human neuroblastoma cells only if present prior to, or applied simultaneously with, Abeta treatment. By contrast, encapsulated vitamin E was equally effective if administered 1 hr after Abeta exposure. These findings suggest suggests that nanosphere-mediated delivery methods may be a useful adjunct for antioxidant therapy in AD.

Mechanism of biochemical action of substituted 4-methylcoumarins. Part 11: Comparison of the specificities of acetoxy derivatives of 4-methylcoumarin and 4-phenylcoumarin to acetoxycoumarins: protein transacetylase.

Our earlier observations led to the identification of a microsomal enzyme termed as acetoxy drug: protein transacetylase (TAase) catalyzing the transfer of acetyl groups from acetylated polyphenols to the receptor proteins. TAase was conveniently assayed by the irreversible inhibition of cytosolic glutathione S-transferase (GST) by the model acetoxycoumarin, 7,8-diacetoxy-4-methylcoumarin (1). The specificities of the acetoxy group on the benzenoid ring and position of the pyran carbonyl group of the coumarin with respect to oxygen heteroatom for the catalytic activity of TAase were also reported earlier. In this communication, we have demonstrated that the acetoxy coumarins and acetoxy dihydrocoumarins having a methyl group instead of a phenyl ring at the C-4, when used as the substrates, resulted in enhancement of TAase activity, while the saturation of double bond at C-3 and C-4 position had no effect on TAase activity. A comparison of the optimized structures of 1 and 7,8-diacetoxy-4-phenylcoumarin (2) suggested that the observed influence may be due to out of plane configuration of the phenyl ring at C-4. Further, the TAase-catalyzed activation of NADPH cytochrome c reductase and inhibition of aflatoxin B1 (AFB1)-DNA binding by acetoxy 4-phenylcoumarins and dihydrocoumarins were significantly lower as compared to those caused by acetoxy 4-methylcoumarins.

Influence of EDA-pi interactions in drug encapsulation using nanospheres.

We have evaluated the influence of aromatic and hydrophobic interactions on the strength and selectivity of encapsulation using polymeric nanospheres.

Supramolecular assemblies based on copolymers of PEG600 and functionalized aromatic diesters for drug delivery applications.

A chemoenzymatic approach has been developed to synthesize poly(ethylene glycol)-based amphiphilic copolymers under mild reaction conditions that self-assemble in aqueous media to form polymeric nanomicelles in the range of 20-50 nm. The supramolecular organization of polymeric nanomicelles was studied by 1H NMR longitudinal relaxation time (T1) and light scattering techniques (static and dynamic). Interestingly, the enzyme novozyme-435 plays an important role in controlling the polymerization and distribution of polymer chains, which is critical for the formation of nanomicelles with unimodal distributions. The methodology developed is highly flexible as it allows the introduction of various functionalities in the polymeric nanomicelles. These self-organized nanomicelles are highly efficient drug delivery vehicles for hydrophobic and partially hydrophilic drugs, both transdermally and orally, as they have the ability to encapsulate guest molecules during self-organization. In vivo studies by encapsulating anti-inflammatory agents (aspirin and naproxen) in these polymeric nanomicelles and by applying topically resulted in significant reduction in inflammation. The % reduction in inflammation using polymeric nanomicelles containing aspirin and naproxen was 62 and 64%, respectively.

Acetoxy drug: protein transacetylase of buffalo liver-characterization and mass spectrometry of the acetylated protein product.

The purification and characterization of the buffalo liver microsomal transacetylase (TAase) catalyzing the transfer of acetyl groups from a model acetoxy drug: 7,8-diacetoxy-4-methylcoumarin (DAMC) to GST3-3 has been described here. The enzyme was routinely assayed using DAMC and cytosolic GST as the substrates and was partially purified from microsomes of the buffalo liver. The enzyme was found to have approximate molecular of weight 65 kDa. The action of TAase and DAMC on liver cytosolic GST resulted in the formation of monoacetoxymonohydroxy-4-methylcoumarin (MAMHC) and 7,8-dihydroxy-4-methylcoumarin (DHMC), although the former was the major metabolite. The buffalo liver microsomal TAase exhibited hyperbolic kinetics and yielded K(m) (1667 microM) and V(max) (192 units) when the concentration of DAMC was varied keeping the concentration of GST constant. After having characterized the nature of the substrates and a product of the TAase-catalyzed reaction, we set out to identify the acetylated protein which is another product of the reaction. GST3-3 was used as a model protein substrate for the action of TAase using DAMC as the acetyl donor. The subunit of control and modified GST3-3 were separated by SDS-polyacrylamide gel electrophoresis (PAGE) and digested with trypsin. The tryptic peptides were extracted from the gel pieces and analyzed by matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOFMS). The data search for calibrated and labeled mass peaks of peptides was performed on the Matrix Science Server using the search engine Mascot. The peptide maps so obtained covered 97% of the GST3-3 sequence. On comparison of MALDI peptide maps of modified and control GST, seven new peaks were recognized corresponding to the potentially acetylated peptides in peptide map. The mass value of each of them was 42 Da higher than the theoretical mass of a non-modified GST3-3 tryptic peptide, strongly suggesting acetylation. By examining the fragmentation patterns and by comparing experimental and predicted values for MS/MS daughter ions, the identity of the seven acetylated GST tryptic peptides could be confirmed by the application of LC/MS/MS. In the modified GST, N-terminal proline and six lysines (Lys(51), Lys(82), Lys(123), Lsy(181), Lys(191) and Lys(210)) were found to be acetylated. The structure of acetylated GST revealed that the lysines that underwent acetylation were peripheral in positions.

"Green"-enzymatic synthesis of pegylated phenolic macromer and polymer.

Environmentally benign synthesis of novel pegylated polyphenolics, by combining the extraordinary selectivities of a lipase and an oxidase to develop polymeric electrolytes for applications in dye sensitised solar cells.

Synthesis, characterization and in vitro anti-invasive activity screening of polyphenolic and heterocyclic compounds.

Invasion is the hallmark of malignant tumors, and is responsible for the bad prognosis of the untreated cancer patients. The search for anti-invasive treatments led us to screen compounds of different classes for their effect in an assay for invasion. Thirty-nine new compounds synthesized in the present study along with 56 already reported compounds belonging mainly to the classes of lactones, pyrazoles, isoxazoles, coumarins, desoxybenzoins, aromatic ketones, chalcones, chromans, isoflavanones have been tested against organotypic confronting cultures of invasive human MCF-7/6 mammary carcinoma cells with embryonic chick heart fragments in vitro. Three of them (a pyrazole derivative, an isoxazolylcoumarin and a prenylated desoxybenzoin) inhibited invasion at concentrations as low as 1 microM; instead of occupying and replacing the heart tissue within 8 days, the MCF-7/6 cells grew around the heart fragments and left it intact, when treated with these compounds. At the anti-invasive concentration of 1 microM, the three compounds did not affect the growth of the MCF-7/6 cells, as shown in the sulforhodamine B assay. Aggregate formation on agar was not stimulated by any of the three anti-invasive compounds, making an effect on the E-cadherin/catenin complex improbable. This is an invasion suppressor that can be activated in MCF-7/6 cells by a number of other molecules. Our data indicate that some polyphenolic and heterocyclic compounds are anti-invasive without being cytotoxic for the cancer cells.

Mechanism of biochemical action of substituted 4-methylbenzopyran-2-ones. Part 10: identification of inhibitors for the liver microsomal acetoxycoumarin: protein transacetylase.

The quantitative structure-activity relationship (QSAR) studies conducted by us earlier revealed the cardinal role of the pyran ring carbonyl group in the acetoxy polyphenolic compounds for the acetoxy polyphenol:protein transacetylase (TAase) activity. Hence, an attempt was made to examine whether such substrate analogues of benzopyran acetates which lack in the pyran ring carbonyl group, such as 7-acetoxy-2,3-dihydro-2,2-dimethylbenzopyran (BPA), cetachin pentaacetate (CPA) and hematoxylin pentaacetate (HPA) could inhibit the 7,8-diacetoxy-4-methylcoumarin (DAMC):protein (glutathione-S-transferase) transacetylase activity. These compounds were indeed found to remarkably inhibit the TAase activity in a concentration dependent manner and exerted their inhibitory action very rapidly. Further BPA, CPA and HPA were found to abolish the TAase mediated activation of NADPH cytochrome C reductase as well as the inhibition of liver microsome catalyzed aflatoxin B(1) (AFB(1))-DNA binding by DAMC very effectively. These results strongly suggest that the acetoxybenzopyrans merit as potent inhibitors of TAase.

Synthetic and novel biocatalytic resolution studies on (+/-)-5/6/7-acetoxy-4-aryl-3,4-dihydrocoumarins.

Eleven (+/-)-5/6/7-acetoxy-4-aryl-3,4-dihydrocoumarins have been synthesised in two steps starting from the coupling of cinnamic acid/substituted cinnamic acid with appropriate phenols, followed by acetylation in 50-83% overall yields. All hydroxy- and acetoxycoumarins were unambiguously identified on the basis of their spectral data. Candida antarctica lipase-catalysed deacetylation of these racemic acetoxydihydrocoumarins in dioxane occurred with moderate enantioselectivity. This is one of the rare examples of resolution using phenolic ester moiety as a remote handle for chiral recognition by a lipase.

Enzymatic synthesis of multi-component copolymers and their structural characterization.

The use of enzymes in synthetic applications has increased dramatically in the recent years and the field of polymer science is part of this trend. Synthesis of a variety of polymers using lipase catalyzed (Candida antarctica) polymerization reactions has led to a variety of new materials with interesting properties in our laboratories. This paper describes the synthesis of multi-component polyesters and mixed polymers having polyester and polyamide linkages under solvent-less conditions using Candida antarctica lipase B. The effect of a third component, i.e. a series of 1,omega-alkanediols (1,4-butanediol, 1,6-hexanediol, 1,8-octanediol, 1,10-decanediol, 1,12-dodecanediol, 1,14-tetradecanediol and 1,16-hexadecanediol) on the copolymerization reaction of dimethyl 5-hydroxyisophthalate with poly(ethylene glycol 600) has been studied and the mechanism for the incorporation of the third component is proposed. We have also studied the effect of different functional groups during terpolymerization reaction of dimethyl 5-hydroxyisophthalate with poly(ethylene glycol) by adding a third component having different functionalities (1,6-hexanediol, 1,6-hexanediamine or 1,6-hexanedithiol) and compared the effect of hydroxyl, amine and thiol groups on the polymerization reactions.

Establishment of the enzymatic protein acetylation independent of acetyl CoA: recombinant glutathione S-transferase 3-3 is acetylated by a novel membrane-bound transacetylase using 7,8-diacetoxy-4-methyl coumarin as the acetyl donor.

The current knowledge on biological protein acetylation is confined to acetyl CoA-dependent acetylation of protein catalyzed by specific acetyl transferases and the non-enzymatic acetylation of protein by acetylated xenobiotics such as aspirin. We have discovered a membrane-bound enzyme catalyzing the transfer of acetyl groups from the acetyl donor 7,8-diacetoxy-4-methyl coumarin (DAMC) to glutathione S-transferase 3-3 (GST3-3), termed DAMC:protein transacetylase (TAase). The purified enzyme was incubated with recombinant GST3-3 subunit and DAMC, the modified protein was isolated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in gel digested with trypsin and the tryptic digest was analyzed by mass spectrometry. The N-terminus and six lysines, Lys-51, -82, -124, -181, -191 and -210, were found to be acetylated. The acetylation of GST3-3 described above was not observed in the absence of either DAMC or TAase. These results clearly establish the phenomenon of protein acetylation independent of acetyl CoA catalyzed by a hitherto unknown enzyme (TAase) utilizing a certain xenobiotic acetate (DAMC) as the active acetyl donor.