Trisindoline synthesis and anticancer activity

Expression of a Rhodococcus-derived oxygenase gene in Escherichia coli yielded indigo metabolites with cytotoxic activity against cancer cells. Bioactivity-guided fractionation of these indigo metabolites led to the isolation of trisindoline as the agent responsible for the observed in vitro cytotoxic activity against cancer cells. While the cytotoxicity of etoposide, a common anticancer drug, was dramatically decreased in multidrug-resistant (MDR) cancer cells compared with treatment of parental cells, trisindoline was found to have similar cytotoxicity effects on both parental and MDR cell lines. In addition, the cytotoxic effects of trisindoline were resistant to P-glycoprotein overexpression, one of the most common mechanisms of drug resistance in cancer cells, supporting its use to kill MDR cancer cells.     

Cytotoxic properties of immunoconjugates containing melittin-like peptide 101 against prostate cancer: in vitro and in vivo studies

Background: Monoclonal antibodies (MAbs) can target therapy to tumours while minimising normal tissue exposure. Efficacy of immunoconjugates containing peptide 101, designed around the first 22 amino acids of bee venom, melittin, to maintain the amphipathic helix, to enhance water solubility, and to increase hemolytic activity, was assessed in nude mice bearing subcutaneous human prostate cancer xenografts. Methods: Mouse MAbs, J591 and BLCA-38, which recognise human prostate cancer cells, were cross-linked to peptide 101 using SPDP. Tumour-bearing mice were used to compare biodistributions of radiolabeled immunoconjugates and MAb, or received multiple sequential injections of immunoconjugates. Therapeutic efficacy was assessed by delay in tumour growth and increased mouse survival. Results: Radiolabeled immunoconjugates and antibodies showed similar xenograft tropism. Systemic or intratumoural injection of immunoconjugates inhibited tumour growth in mice relative to carrier alone, unconjugated antibody and nonspecific antibody-peptide conjugates and improved survival for treated mice. Conclusions: Immunoconjugates deliver beneficial effects; further peptide modifications may increase cytotoxicity.     

Enzymatic synthesis of water-soluble derivatives of salicylic acid in organic media

A novel enzymatic method for preparing water-soluble derivatives of salicylic acid catalyzed by immobilized lipase was described. This study is the first to describe the enzymatic transesterification of methyl salicylate in organic solvents with different hydroxyl donors. The acyl-transfer between methyl salicylate and sorbitol was best supported by solvents of log P values –0.33 to 1.4. With Candida antarctica lipase in tert-amyl alcohol, a sorbitol conversion yield of 98% can be obtained by transesterification with sorbitol and methyl salicylate in one step.     

In vitro and in vivo inhibition of plant polyamine oxidase activity by polyamine analogues

Polyamine oxidase from Avena sativa L. cv. Cristal seedlings was purified to homogeneity using a simple four-step purification protocol including an infiltration washing technique. The enzyme had a high affinity for spermidine and spermine (K_m ∼ 5.5 and 1.2 μM, respectively), and also oxidized norspermidine (K_m ∼ 64.0 μM). Natural and synthetic diamines, cyclohexylamine, the putrescine analogue 1-aminooxy-3-aminopropane, and several polyamine analogues had inhibitory effects on polyamine oxidase activity and none were substrates. No inhibitory effect was observed on spermidine oxidation when the reaction product 1,3-diaminopropane was added. By contrast, 1-aminooxy-3-aminopropane showed mixed inhibition kinetics and a K_i value of 0.113 mM. In addition, in vitro enzymatic activity assays showed that the oligoamine [3,8,13,18,23,28,33,38,43,48-deca-aza-(trans-25)-pentacontene], the tetramine 1,14-bis-[ethylamino]-5,10-diazatetradecane, and the pentamine 1,19-bis-[ethylamino]-5,10,15-triazanonadecane, displayed potent competitive inhibitory activities against polyamine oxidase with K_i values of 5.8, 110.0 and 7.6 nM, respectively, where cyclohexylamine was a weak competitive inhibitor with a K_i value of 0.5 mM. These analogues did not inhibit mycelial growth of the fungus Sclerotinia sclerotiorum (Lib.) De Bary and the bacterium Pseudomonas viridiflava (Burkholder) Dowson in vitro. On the contrary, with concentrations similar to those used for polyamine analogues, guazatine (a well-known fungicide and at the same time, a polyamine oxidase inhibitor) inhibited (∼85%) S. sclerotiorum mycelial growth on Czapek-Dox medium.Finally, the analogue 1,19-bis-ethylamino-5,10,15-triazanonadecane inhibited polyamine oxidase activity observed in segments of maize leaves in vivo. The results obtained provide insights into research on the influence of polyamine oxidase activity on plant biotic and abiotic stresses.     

Lipase-catalysed synthesis of olvanil in organic solvents

The release rate of vanillylamine from its hydrochloride salt was the limiting step in the lipase-catalysed synthesis of olvanil, a capsaicin analogue amide, in organic solvents. When the tertiary amine base concentration (N,N-diisopropylethylamine) was increased from 20 mM to 360 mM, the initial rate of amide synthesis increased proportionally. At a 12 molar excess of N,N-diisopropylethylamine and 30 min of preincubation, both the initial rate and total conversion were the same as those with free vanillylamine (80% conversion in 20 h). This result was independent of the organic solvent used. It is also shown that N,N-diisopropylethylamine does not enhance lipase activity.     

Enzymatic conversion of sunflower oil to biodiesel in a solvent-free system: Process optimization and the immobilized system stability

The feasibility of using the commercial immobilized lipase from Candida antarctica (Novozyme 435) to synthesize biodiesel from sunflower oil in a solvent-free system has been proved. Using methanol as an acyl acceptor and the response surface methodology as an optimization technique, the optimal conditions for the transesterification has been found to be: 45 ^oC, 3% of enzyme based on oil weight, 3:1 methanol to oil molar ratio and with no added water in the system. Under these conditions, >99% of oil conversion to fatty acid methyl ester (FAME) has been achieved after 50 h of reaction, but the activity of the immobilized lipase decreased markedly over the course of repeated runs. In order to improve the enzyme stability, several alternative acyl acceptors have been tested for biodiesel production under solvent-free conditions. The use of methyl acetate seems to be of great interest, resulting in high FAME yield (95.65%) and increasing the half-life of the immobilized lipase by about 20.1 times as compared to methanol. The reaction has also been verified in the industrially feasible reaction system including both a batch stirred tank reactor and a packed bed reactor. Although satisfactory performance in the batch stirred tank reactor has been achieved, the kinetics in a packed bed reactor system seems to have a slightly better profile (93.6 ± 3.75% FAME yield after 8–10 h), corresponding to the volumetric productivity of 48.5 g/(dm³ h). The packed bed reactor has operated for up to 72 h with almost no loss in productivity, implying that the proposed process and the immobilized system could provide a promising solution for the biodiesel synthesis at the industrial scale.     

A rational approach to the regioselective deacetylation of 2′,3′,5′-tri-O-acetyluridine by Novozym 435 catalysed alcoholysis

To give a rational explanation for the behaviour of 2′,3′,5′-tri-O-acetyluridine (TAU) catalysed alcoholysis using Novozym 435, the commercial biocatalyst with immobilized Candida antarctica lipase B (CALB), a set of experiments analyzing the role of the alcohol/substrate (A/S) molar ratio, alcohol/biocatalyst (A/B) and substrate/biocatalyst (S/B) mass ratios were carried out. At a A/S = 120 and a S/B = 6.16, 2′,3′-di-O-acetyluridine (DAU) was obtained in 92% at 22 h. The observed trend towards the exclusive formation of DAU at very high alcohol amounts can be explained on the basis of the change of substrate orientation from normal to inverse. The simple molecular modelling analysis supports that key O/H atoms from TAU and the resulting intermediates display the adequate distances to generate productive binding only when the inverse coordination of TAU is present through the 5′-moiety of TAU, at high ethanol concentrations. At these conditions a possible allosteric-like effect of ethanol, combined with water in an H-network in the catalytic triad and in its neighbourhood, could explain the high selectivity towards the production of DAU at selected conditions.     

Continuous synthesis of alkyl ferulate by immobilized Candida antarctica lipase at high temperature

1-Pentyl, 1-hexyl and 1-heptyl ferulates were continuously synthesized at 60–90°C using a reactor system in which a column packed with ferulic acid powders and another column packed with immobilized Candida antarctica lipase particles were connected in series. Conversions greater than 0.9 were achieved for the synthesis of the 1-hexyl and 1-heptyl ferulates at 90°C. The system could be stably operated for the 1-heptyl ferulate synthesis at 90°C for at least two weeks.     

Development of yeast cells displaying <Emphasis Type="Italic">Candida antarctica</Emphasis> lipase B and their application to ester synthesis reaction

We isolated the lipase B from Candida antarctica CBS 6678 (CALB CBS6678) and successfully constructed CALB-displaying yeast whole-cell biocatalysts using the Flo1p short (FS) anchor system. For the display of CALB on a yeast cell surface, the newly isolated CALB CBS6678 exhibited higher hydrolytic and ester synthesis activities than the well-known CALB, which is registered in GenBank (Z30645). A protease accessibility assay using papain as a protease showed that a large part of CALB, approximately 75%, was localized on an easily accessible part of the yeast cell surface. A comparison of the lipase hydrolytic activities of yeast whole cells displaying only mature CALB (CALB) and those displaying mature CALB with a Pro region (ProCALB) revealed that mature CALB is preferable for yeast cell surface display using the Flo1p anchor system. Lyophilized yeast whole cells displaying CALB were applied to an ester synthesis reaction at 60°C using adipic acid and n-butanol as substrates. The amount of dibutyl adipate (DBA) produced increased with the reaction time until 144 h. This indicated that CALB displayed on the yeast cell surface retained activity under the reaction conditions.     

Microwave-assisted synthesis of new benzimidazole derivatives with lipase inhibition activity

Abstract

A practical protocol has been used for the synthesis of benzimidazoles. The reaction of iminoester hydrochlorides of phenylacetic with 4,5-dichloro-1,2-phenylenediamine under microwave irradiation leads to the benzimidazole derivatives with good yields and in short reaction times. After the synthesis of benzimidazoles, we synthesized ester and hydrazide derivatives under microwave irradiation with good yields. All compounds were evaluated with regard to pancreatic lipase activity and 3b, 3c, 5a and 6a showed lipase inhibition at various concentrations.     

Rational design of enantioselective enzymes requires considerations of entropy

Entropy was shown to play an equally important role as enthalpy for how enantioselectivity changes when redesigning an enzyme. By studying the temperature dependence of the enantiomeric ratio E of an enantioselective enzyme, its differential activation enthalpy (Delta(R-S)DeltaH(++)) and entropy (Delta(R-S)DeltaS(++)) components can be determined. This was done for the resolution of 3-methyl-2-butanol catalyzed by Candida antarctica lipase B and five variants with one or two point mutations. Delta(R-S)DeltaS(++) was in all cases equally significant as Delta(R-S)DeltaH(++) to E. One variant, T103G, displayed an increase in E, the others a decrease. The altered enantioselectivities of the variants were all related to simultaneous changes in Delta(R-S)DeltaH(++) and Delta(R-S)DeltaS(++). Although the changes in Delta(R-S)DeltaH(++) and Delta(R-S)DeltaS(++) were of a compensatory nature the compensation was not perfect, thereby allowing modifications of E. Both the W104H and the T103G variants displayed larger Delta(R-S)DeltaH(++) than wild type but exhibited a decrease or increase, respectively, in E due to their different relative increase in Delta(R-S)DeltaS(++).     

Chemo-enzymatic synthesis of the glycosylated alpha-mating factor of Saccharomyces cerevisiae and analysis of its biological activity

The effect of glycosylation on a bioactive peptide was studied using yeast Saccharomyces cerevisiae alpha-mating factor, which is composed of 13 amino acids. In this study, we prepared glycosylated alpha-mating factor by chemo-enzymatic synthesis. At first, N-acetylglucosaminyl alpha-mating factor (Trp-His-Trp-Leu-Gln(GlcNAc)-Leu-Lys-Pro-Gly-Gln-Pro-Met-Tyr) was chemically synthesized by the solid-phase method. Then, using the transglycosylation activity of Mucor hiemalis endo-beta-N-acetylglucosaminidase, we synthesized glycosylated alpha-mating factor with a glutamine-linked sialo complex type oligosaccharide. The biological activity of alpha-mating factor derivatives was examined by means of a growth arrest assay using secreted-protease-defective a cells of S. cerevisiae. The results showed that the bioactivity of glycosylated alpha-mating factor was lower than that of native alpha-mating factor. However, when sialic acid was removed from the complex type sugar chain of glycosylated alpha-mating factor, its bioactivity was recovered. Glycosylated alpha-mating factor exhibited higher resistance against proteolysis than native alpha-mating factor. It was found that the bioactivity of N-acetylglucosaminyl alpha-mating factor was higher than that of alpha-mating factor. Circular dichroism studies indicated that a slight change in the structure of alpha-mating factor may influence its activity.     

Synthesis of resveratrol analogues, and evaluation of their cytotoxic and xanthine oxidase inhibitory activities

Thirteen resveratrol (=5-[(E)-2-(4-hydroxyphenyl)ethenyl]benzene-1,3-diol) analogues with a CHO group have been prepared by partial synthesis from resveratrol. The synthesized compounds have been evaluated for their cytotoxic activity against a human nasopharyngeal epidermoid tumor cell line KB, as well as for their xanthine oxidase inhibitory activity. Compounds 2, 3, and 6a showed the most significant cytotoxic activities against the cell line KB, and compound 2 also exhibited strong xanthine oxidase inhibitory activity.     

The mechanism of solvent effect on the positional selectivity of Candida antarctica lipase B during 1,3-diolein synthesis by esterification

We investigated the influence of solvent on the positional selectivity of Novozym 435 which was the immobilized Candida antarctica lipase B (CALB) during the esterification of oleic acid with glycerol for 1,3-diolein preparation previously. Herein, molecular modeling was used to elucidate the underlying mechanism of the solvent effect on the positional selectivity of the enzyme. The results showed that the binding energy of sn-1 hydroxyl of glycerol molecular with CALB became higher, and the binding energy of sn-2 hydroxyl of glycerol molecular with CALB became lower along with the increase of the solvent log P. It was demonstrated that, increasing log P of the solvent, the enzyme selectivity to sn-1 hydroxyl of glycerol molecular grew weaker, and the selectivity to sn-2 hydroxyl of glycerol molecular grew stronger.     

Effect of additives on the esterification activity of immobilized Candida antarctica lipase

In this work, the stabilizing effect of bovine serum albumin (BSA), peptone (PEP), and polyethylene glycol (PEG) during immobilization of Candida antarctica lipase on activated carbon was investigated. The influence of enzyme concentration and type of additive, added during the immobilization procedure, was studied using a 2² factorial central composite design. The goal was to maximize the synthetic activity of butyl butyrate, using butyric acid and butanol as substrate in n-heptane. An increase of 31–58% in the esterification activity was obtained when enzyme concentration on the supernatant was enhanced from 86.50 U m L⁻¹ to 226.80 U mL⁻¹. An enhancement in esterification activity of 38–68.95% was observed, depending on the initial enzyme concentration, when PEP was used instead of BSA. No significant increase in the esterification activity was observed when PEP was replaced by PEG. However, thermal stability tests at 50 °C showed that PEG had a higher stabilizing effect.     

Solvent-free synthesis of glyceryl ferulate using a commercial microbial lipase

A process was optimized for the enzymatic synthesis of glyceryl ferulate with a yield of up to 96% using a vacuum-rotary evaporation strategy under following conditions: 15 mmol glycerol, 1.5 mmol ethyl ferulate, 170 mg Candida antarctica lipase, at 60°C for 10 h and under a vacuum of 10 mm Hg. The immobilized lipase can be used 10 times.     

Modelling and experimental studies on lipase-catalyzed isoamyl acetate synthesis in a microreactor

A lipase-catalyzed synthesis of isoamyl acetate was studied in a continuously operated pressure-driven microreactor. The esterification of isoamyl alcohol and acetic acid occurred at the interface between n-hexane and an aqueous phase with dissolved lipase B from Candida antarctica. By adjusting flow rates of both phases, a parallel laminar flow with liquid–liquid boundary in the middle of the microchannel could be reestablished and a separation of phases was achieved at the y-shaped exit of the microreactor. Since product remained in the organic phase, this also enabled its continuous separation from the aqueous phase with the enzyme. A three-dimensional mathematical model was developed, considering the velocity profile developed for steady-state conditions between two immiscible fluids. The model contained convection, diffusion, and enzyme reaction terms, where esterification rate was described with a Ping-Pong Bi-Bi mechanism and inhibition by both substrates. Experimental data, which were in good agreement with model simulations, have demonstrated 35% conversion at residence time 36.5 s at 45 °C and at 0.5 M acetic acid and isoamyl alcohol inlet concentrations, which is much faster as in any literature reported so far. According to model simulations, obtained by non-equidistant finite differences numerical solutions of complex non-linear equations system, further microreactor design and process optimization are feasible.     

Chemo-enzymatic synthesis of the carbohydrate antigen N-glycolylneuraminic acid from glucose

N-Glycolylneuraminic acid (Neu5Gc) is a non-human sialic acid, which may play a significant role in human pathologies, such as cancer and vascular disease. Further studies into the role of Neu5Gc in human disease are hindered by limited sources of this carbohydrate. Using a chemo-enzymatic approach, Neu5Gc was accessed in six steps from glucose. The synthesis allows access to gram-scale quantities quickly and economically and produces Neu5Gc in superior quality to commercial sources. Finally, we demonstrate that the synthesized Neu5Gc can be incorporated into the cell glycocalyx of human cells, which do not naturally synthesize this sugar. The synthesis produces Neu5Gc suitable for in vitro or in vivo use.     

Regio- and enantio-selective acetylation of 3,3,3-trifluoro-2-arylpropane-1,2-diols using Candida antarctica lipase

Of nine commercially available lipases, lipase SP 435 from Candida antarctica, showed moderate enantioselectivity (E=17) for acetylation of racemic 3,3,3-trifluoro-2-phenylpropane-1,2-diol, 2, with vinyl acetate in diisopropyl ether (S selectivity). The other eight had low selectivities, with E values below 10. The selectivity and reactivity of SP 435 for 2 was markedly improved in dichloroethane (E=41). Moreover, SP 435 had moderate to high selectivity for the related compounds 3,3,3-trifluoro-2-(1-naphthyl)-propane-1,2-diol, 4, (E=20), 3,3,3-trifluoro-2-(indol-3-yl)propane-1,2-diol, 6, (E=80), and 3,3,3-trifluoro-2-(pyrrol-2-yl)-propane-1,2-diol, 8, (E=17).     

Design,synthesis, and evaluation of the antiproliferative activity of hydantoin-derived antiandrogen-genistein conjugates

Androgen receptor (AR) signaling is vital to the viability of all forms of prostate cancer (PCa). With the goal of investigating the effect of simultaneous inhibition and depletion of AR on viability of PCa cells, we designed, synthesized and characterized the bioactivities of bifunctional agents which incorporate the independent cancer killing properties of an antiandrogen and genistein, and the AR downregulation effect of genistein within a single molecular template. We observed that a representative conjugate, 9b, is much more cytotoxic to both LNCaP and DU145 cells relative to the antiandrogen and genistein building blocks as single agents or their combination. Moreover, conjugate 9b more effectively down regulates cellular AR protein levels relative to genistein and induces S phase cell cycle arrest. The promising bioactivities of these conjugates warrant further investigation.