Chemo-enzymatic epoxidation of oleic acid and methyl oleate in solvent-free medium

Chemo-enzymatic epoxidation of oleic acid (OA) and its methyl ester has been performed using hydrogen peroxide and immobilized lipase from Candida antarctica (Novozym® 435). The purpose of the study was to characterize the reaction under solvent-free conditions. The reaction temperature had a significant impact on epoxidation of OA. At lower temperatures, the substrate conversion was hindered by the formation of solid epoxystearic acid product. Nearly 90% conversion of OA to the epoxide product was obtained after 6?h at 50°C. Longer reaction times at 40°C and above resulted in by-product formation and eventually lowered the product yield. In contrast, the reaction with methyl oleate (MO) was less influenced by temperature. Almost complete epoxidation was achieved at 40–60°C; the higher the temperature the shorter was the reaction time. The main epoxidation product obtained was epoxystearic acid methyl ester (EME), and the remaining was epoxystearic acid (EA) formed by the hydrolytic action of the lipase. Recycling of the lipase for epoxidation of MO at 50°C indicated that the immobilized enzyme was prone to activity loss.     

Oxidation of isoeugenol by Nocardia iowensis

Isoeugenol is a starting material for both the synthetic and biotechnological production of vanillin and vanillic acid. Nocardia iowensis DSM 45197 (formerly Nocardia species NRRL 5646) resting cells catalyze the conversion of isoeugenol to vanillic acid, vanillin, vanillyl alcohol and guaiacol. The present study used a variety of chemical, microbial and enzymatic approaches to probe the pathways used by N. iowensis in the oxidation of isoeugenol to these products. Of three possible pathways considered, initial side-chain olefin epoxidation, epoxide hydrolysis to a vicinal diol, and diol cleavage to vanillin and subsequently further oxidation to vanillic acid appears as the most likely route. Isoeugenol was not oxidized to ferulic acid, a well-known microbial transformation precursor for vanillin and vanillic acid. ¹⁸O-Labeled oxygen (one atom) and water (two oxygen atoms) were incorporated into vanillic acid during the whole-cell biotransformation reaction with isoeugenol indicating the likely involvement of oxygenase and hydrolase systems in the bioconversion reaction. Vanillin was converted to singly labeled vanillic acid in the presence of H₂¹⁸O suggesting the presence of an aldehyde oxidase. Cell extracts achieved the conversion of isoeugenol to vanillic acid and vanillin without cofactors. Partial fractionation of two enzyme activities supported the presence of isoeugenol monooxygenase and vanillin oxidase activities in N. iowensis.     

Biosynthesis of ursolic acid derivatives by microbial metabolism of ursolic acid with Nocardia sp. strains—Proposal of new biosynthetic pathways

Our studies of the microbial-metabolism of triterpenoid ursolic acid by various Nocardia sp. strains, have led to the proposal of two novel pathways to produce triterpenoid derivatives. Nocardia sp. NRRL 5646, Nocardia sp. 44822 and Nocardia sp. 44000 generated the following ursolic acid derivatives: ursolic acid methyl ester, ursonic acid, ursonic acid methyl ester, 3-oxoursa-1,12-dien-28-oic acid and 3-oxoursa-1,12-dien-28-oic acid methyl ester. Nocardia sp. 45077 synthesized ursonic acid and 3-oxoursa-1,12-dien-28-oic acid while Nocardia sp. 46002 produced only ursonic acid and Nocardia sp. 43069 showed no metabolism at all. The conversion of ursolic acid by Nocardia sp. NRRL 5646 was independent of the medium used for the fermentation. An increase in temperature from 28 °C to 36 °C doubled the reaction rate of the biotransformation. The analysis of ursane metabolites was done by HPLC, while their structures were established using HPLC–APCI_pos-MS/MS and HPLC–NMR spectroscopy. The pseudo molecular ion peaks were determined by HPLC–APCI_pos-MS and used to measure their molecular weight. The product ion spectra of the metabolites showed the characteristic fragments of Δ¹²-oleanes and Δ¹²-ursanes indicating that a substitution in ring A or B was responsible for the decrease in molecular weight.Based on these results, two new biosynthetic pathways are proposed. These new pathways can presumably be used as strategic routes for the biotechnological production of triterpenoid derivatives. It is assumed that a 3β-hydroxysteroid dehydrogenase and a 3-ketosteroid-Δ¹-dehydrogenase are involved in the transformation of the steroid.     

Caffeoylquinic Acids: Separation Method,Antiradical Properties and Cytotoxicity

Twelve chlorogenic acid derivatives and two flavones were isolated from Moquiniastrum floribundum (Asteraceae, other name: Gochnatia floribunda). Compounds were evaluated in relation to their cytotoxicity and antiradical properties. Cytotoxicity was not observed for compounds, however, chlorogenic acid derivatives showed antiradical activity and were more active than the Trolox standard. Quinic acid esterified with caffeoyl group at C‐4 position showed higher antiradical activity compared to acylation at C‐3 or C‐5 positions. Additional caffeoyl groups esterified in quinic acid increase the antiradical activity observed for 4‐caffeoylquinic acid. Excepted to 3,4‐dicaffeoylquinic acid methyl ester, methyl ester derivatives show higher capacity of trapping radicals than their respective acids. Consequently, the presence of caffeoyl group at C‐4 position of quinic acid is suggested as fundamental to obtain the highest antiradical activity.     

Easy Access to Enantiomerically Pure Nonproteinogenic Amino Acids

The protease from Bacillus licheniformis (alcalase) shows a remarkable broad substrate tolerance and high enantioselectivity against nonproteinogenic racemic amino acid derivatives. N‐acetyl protected amino acid esters of mono‐, di‐ or tri‐substituted phenyl alanines and even tert.‐leucine were hydrolyzed with high enantioselectivity. The obtained mixtures of (S)‐N‐acetyl amino acid and (R)‐N‐acetyl amino acid ester can easily be separated. The R‐ or S‐amino acids were obtained by acidic cleavage of the optically pure derivatives or the (R)‐ester was racemized by treatment with potassium t‐butylate.     

Synthesis,Anticonvulsant Activity and Metabolism of 4‐chlor‐3‐methylphenoxyethylamine Derivatives of Trans‐2‐aminocyclohexan‐1‐ol

In this study, we report the synthesis, spectral characterization, antiepileptic activity and biotransformation of three new, chiral, N‐aminoalkyl derivatives of trans – 2 aminocyclohexan‐1‐ol: 1 (R enantiomer), 2 (S enantiomer) and 3 (racemate). Antiepileptic activity of the titled compounds was studied using MES and scMet. Moreover, in this study, the biotransformation of 1 , 2 and 3 in microbial model (Cunninghamella), liver microsomal assay as well as in silico studies (MetaSite) was evaluated. Studies have indicated that 1 , 2 and 3 have good antiepileptic activity in vivo, comparable to valproate. Biotransformation assays showed that the most probable metabolite (indicated in every tested assays) was M1 . The microbial model as well as in silico study showed no difference in biotransformation between tested enantiomers. However, in a rat liver microsomal study compound 1 and 2 (R and S enantiomer) had different main metabolite – M2 for 1 and M1 for 2 . MS/MS fragmentation allowed us to predict the structures of obtained metabolites, which were in agreement with 1°alcohol ( M1 ) and carboxylic acid ( M2 ). Our research has shown that microbial model, microsomal assay, and computational methods can be included as useful and reliable tools in early ADME‐Tox assays in the process of developing new drug candidates. Chirality 27:163–169, 2015. © 2014 Wiley Periodicals, Inc.     

The immunotropic activity of lupane and oleanane 2,3-<Emphasis Type="Italic">seco</Emphasis>-triterpenoids

The immunotropic effect of the 2,3-seco derivatives of allobetulon, betulonic acid, and the methyl ester of betulonic acid were studied. It was found that the highest activity is shown by compounds with an oleanane fragment. The presence of a free C28-carboxyl group enhances the activity of lupane 2,3-seco derivatives. A significant contribution to the development of the immune response is introduced by a functional group at the C3 atom in the A ring—the C3-carboxy derivatives intensify the processes of antibody production and alter the number and ratio of leukocyte forms. It is shown that 2,3-seco-1-cyano-19β,28-epoxy-18α-olean-3-oic acid stimulates humoral immunity with a positive influence on hematopoiesis.     

Application of (S)‐TBMB carboxylic acid‐based on‐line HPLC‐exciton CD analysis to acyclic vicinal alcohols and amino alcohols

Applications of the on‐line HPLC‐exciton CD analysis using (S)‐2‐tert‐butyl‐2‐methyl‐1,3‐benzodioxole‐4‐carboxylic acid [(S)‐TBMBC‐OH] that can simultaneously determine the enantiomeric compositions and the absolute configuration of cyclohexane‐1,2‐diols and diamines as well as acyclic vicinal diols and amino alcohols were studied. Di‐O‐ or di‐N,O‐(S)‐TBMBC derivatives of acyclic terminal vicinal diols, 2‐hydroxy‐1‐amines, and nonterminal vicinal diols gave symmetrical exciton CD spectra between enantiomers, indicating their absolute configurations. However, Di‐N,O‐(S)‐TBMBC derivatives of 2‐amino‐1‐ols did not always give symmetrical exciton CD spectra between enantiomers, but their 2‐phthalimido‐1‐O‐(S)‐TBMBC derivatives gave symmetrical exciton CD spectra, indicating their absolute configurations. All these (S)‐TBMBC derivatives were separated by normal‐phase HPLC and unequivocally determined by the on‐line HPLC‐exciton CD analysis without recourse to reference samples. Chirality 11:149–159, 1999. © 1999 Wiley‐Liss, Inc.     

Kinetic resolution of N‐acetyl‐DL‐alanine methyl ester using immobilized Escherichia coli cells bearing recombinant esterase from Bacillus cereus

D‐alanine is widely used in medicine, food, additives, cosmetics, and other consumer items. Esterase derived from Bacillus cereus WZZ001 exhibits high hydrolytic activity and stereoselectivity. In this study, we expressed the esterase gene in Escherichia coli BL21 (DE3). We analyzed the biocatalytic resolution of N‐acetyl‐DL‐alanine methyl ester by immobilized whole E. coli BL21 (DE3) cells, which were prepared through embedding and cross‐linking. We analyzed biocatalytic resolution under the optimal conditions of pH of 7.0, temperature of 40°C and substrate concentration of at 700 mM with an enantiomeric excess of 99.99% and e.e._p of 99.50%. The immobilized recombinant B. cereus esterase E. coli BL21 (DE3) cells exhibited excellent reusability and retained 86.04% of their initial activity after 15 cycles of repeated reactions. The immobilized cells are efficient and stable biocatalysts for the preparation of N‐acetyl‐D‐alanine methyl esters.     

Multi‐steps green process for synthesis of six‐membered functional cyclic carbonate from trimethylolpropane by lipase catalyzed methacrylation and carbonation,and thermal cyclization

A highly functionalized six‐membered cyclic carbonate, methacrylated trimethylolpropane (TMP) cyclic carbonate, which can be used as a potential monomer for bisphenol‐free polycarbonates and isocyanate‐free polyurethanes, was synthesized by two steps transesterifications catalyzed by immobilized Candida antarctica lipase B, Novozym^®435 (N435) followed by thermal cyclization. TMP was functionalized as 70 to 80% selectivity of mono‐methacrylate with 70% conversion was achieved, and the reaction rate was evaluated using various acyl donors such as methacrylic acid, methacrylate‐methyl ester, ‐ethyl ester, and ‐vinyl ester. As a new observation, the fastest rate obtained was for the transesterfication reaction using methacrylate methyl ester. Byproducts resulted from leaving groups were adsorbed on the molecular sieves (4Å) to minimize the effect of leaving group on the equilibrium. The difference of reaction rate was explained by molecular dynamic simulations on interactions between carbonyl oxygen and amino acid residues (Thr 40 and Glu 157) in the active site of lipase. Our docking studies revealed that as acyl donor, methyl ester was preferred for the initial conformation of the first tetrahederal intermediate with hydrogen bonding interactions. TMP‐monomethacrylate (TMP‐mMA) cyclic carbonate was obtained in 63% yield (74.1% calculated in 85% conversion) from the lipase‐catalyzed carbonation reaction of TMP‐mMA with dimethylcarbonate, and followed by thermal cyclization of the monocarbonate at 90°C. From the multiple reactions demonstrated in gram scale, TMP‐mMA cyclic carbonate was obtained as a green process without using chlorinated solvent and reagent. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 32:83–88, 2016     

Production of a pharmaceutical intermediate via biohydroxylation using whole cells of Rhodococcus rubropertinctus N82

Rhodococcus rubropertinctus N82 possesses unique regiospecific hydroxylation activity in biotransformation of compounds. In this study, the ability of whole cells of the strain R. rubropertinctus N82 in biotransformation was studied. The hydroxylation activity resulted in transforming 6,7-dihydro-4H-thieno[3,2-c]-pyridine-5-carboxylic acid tert-butyl ester (LS1) into 2-hydroxy-6,7-dihydro-4H-thieno[3,2-c]-pyridine-5-carboxylic acid tert-butyl ester (LP1), a pharmaceutical intermediate. By optimizing conditions for the hydroxylating biotransformation using whole cells of R. rubropertinctus N82 as biocatalyst, 3.3?mM LP1 was successfully produced from 4?mM LS1 with a molar yield of 83%. Thus, effective method was newly developed to produce LP1, which is a synthetic intermediate of a platelet inhibitor active pharmaceutical ingredient drug, prasugrel.     

Changes in Renal Function and Oxidative Status Associated with the Hypotensive Effects of Oleanolic Acid and Related Synthetic Derivatives in Experimental Animals

Purpose

The triterpene oleanolic acid (OA) is known to possess antihypertensive actions. In the present study we to compared the effects of the triterpene on mean arterial blood pressure (MAP) and kidney function following acute administration in normotensive animals with those of its related oleanane synthetic derivatives (brominated oleanolic acid, Br-OA and oleanolic acid methyl ester, Me-OA). We also used experimental models of hypertension to further explore the effects of sub-chronic oral OA treatment and evaluated influences on oxidative status.

Methods

OA was extracted from dried flower buds of Syzygium aromaticum using a previously validated protocol in our laboratory. Me-OA and Br-OA were synthesized according to a method described. Rats were supplemented with lithium chloride (12 mmol L^-1) prior to experimentation in order to raise plasma lithium to allow measurements of lithium clearance and fractional excretion (FE_Li) as indices of proximal tubular Na⁺ handling. Anaesthetized animals were continuously infused via the right jugular with 0.077M NaCl. MAP was measured via a cannula inserted in the carotid artery, and urine was collected through a cannula inserted in the bladder. After a 3.5 h equilibration, MAP, urine flow, electrolyte excretion rates were determined for 4 h of 1 h control, 1.5 h treatment and 1.5 h recovery periods. OA, Me-OA and Br-OA were added to the infusate during the treatment period. We evaluated sub-chronic effects on MAP and kidney function in normotensive Wistar rats and in two animal models of hypertension, spontaneously hypertensive rats (SHR) and Dahl salt-sensitive (DSS) rats, during 9-week administration of OA (p.o.). Tissue oxidative status was examined in these animals at the end of the study. Increasing evidence suggests that and renal function disturbances and oxidative stress play major roles in the pathogenesis of hypertension.

Results

Acute infusion OA and oleanane derivatives displayed qualitatively similar effects in decreasing MAP and increasing urinary Na⁺ outputs. The drugs increased the FE_Na and FE_Li without influencing GFR indicating that at least part of the overall natriuretic effect involved proximal tubular Na⁺ reabsorption. Sub-chronic OA administration (p.o.) also elicited hypotensive responses in Wistar, DSS and SHR rats. The MAP lowering effect was more marked in hypertensive animals and were positively correlated with increased urinary Na⁺ excretion. Compared with respective control rats, OA treatment reduced malondialdehyde (MDA, a marker of lipid peroxidation) and increased activities of antioxidant enzymes; superoxide dismutase and glutathione peroxidase in hepatic, cardiac and renal tissues.

Conclusions

OA and oleanane derivatives have similar effects on MAP, kidney function and oxidative stress. The amelioration of oxidative stress and blood pressure lowering effects by OA are more marked in hypertensive animals and correlated with an increased urinary Na⁺ output.

Novelty of the Work

The results of this study are novel in that they show 1) a correlation between blood pressure reduction and increased urinary Na⁺ excretion by OA, 2) a more marked MAP reduction in hypertensive animals and 3) a drug-induced decrease in proximal tubule Na⁺ reabsorption. The results may also be clinically relevant because OA is effective via oral administration.     

Characterization of mycosporine‐serine‐glycine methyl ester,a major mycosporine‐like amino acid from dinoflagellates: a mass spectrometry study

Several unknown mycosporine‐like amino acids (MAAs) have been previously isolated from some cultured species of toxic dinoflagellates of the Alexandrium genus (Dinophyceae). One of them, originally called M‐333, was tentatively identified as a shinorine methyl ester, but the precise nature of this compound is still unknown. Using a high‐resolution reversed‐phase liquid chromatography mass spectrometry analyses (HPLC/MS), we found that natural populations of the red tide dinoflagellate Prorocentrum micans Ehrenberg showed a net dominance of M‐333 together with lesser amounts of other MAAs. We also documented the isolation and characterization of this MAA from natural dinoflagellate populations and from Alexandrium tamarense (Lebour) Balech cultures. Using a comparative fragmentation study in electrospray mass spectrometry between deuterated and non‐deuterated M‐333 compounds and synthesized mono and dimethyl esters of shinorine, this novel compound was characterized as mycosporine‐serine‐glycine methyl ester, a structure confirmed by nuclear magnetic resonance. These isobaric compounds can be differentiated by their fragmentation patterns in MS³ experiments because the extension and the specific site of the methylation changed the fragmentation pathway.     

Investigation of the stereoselective in vitro metabolism of the chiral antiasthmatic/antiallergic drug flezelastine by high-performance liquid chromatography and capillary zone electrophoresis

An achiral HPLC method using a silica gel column as well as two independent chiral analytical methods by HPLC and capillary zone electrophoresis (CZE) were developed in order to investigate the in vitro metabolism of the racemic antiasthmatic/antiallergic drug flezelastine. The chiral HPLC analysis was performed on a Chiralpak AD column, which also allowed the simultaneous separation of the N-dephenethyl metabolite. The chiral separation by CZE was achieved by the addition of β-cyclodextrin to the run buffer. The stereoselectivity of the in vitro biotransformation of flezelastine was investigated using liver homogenates of different species. Depending on the species, diverse stereoselective aspects were demonstrated. The determination of the enantiomeric ratios of flezelastine and of N-dephenethylflezelastine after incubations of racemic flezelastine with liver microsomes revealed that porcine liver microsomes showed the greatest enantioselectivity of the biotransformation. (−)-Flezelastine was preferentially metabolized. After incubations with bovine liver microsomes the enantiomer of N-dephenethylflezelastine formed from (+)-flezelastine dominated. Incubations of the pure enantiomers of flezelastine with induced rat liver microsomes resulted in the stereoselective formation of a hitherto unknown metabolite, which was only detected in samples of (+)-flezelastine. Initial structure elucidation of the compound indicated that the new     

Isomerization of octadecapentaenoic acid (18:5n‐3) in algal lipid samples under derivatization for GC and GC‐MS analysis

During gas chromatography (GC) analysis of fatty acid (FA) composition of the dinoflagellate Gymnodinium kowalevskii, we found unex‐pectedly low and irreproducible content of all‐cis‐3,6,9,12,15‐octadecapentaenoic acid (18:5n‐3), which is an important chemotaxonomic marker of several classes of microalgae. We compared chromatographic behavior of 18:5n‐3 methyl ester and other GC derivatives obtained using different conventional methods of derivatization. The use of methods based on saponification or base‐catalyzed transesterification resulted in a mixture of double‐bond positional isomers of 18:5. On a SUPELCOWAX 10 column, the equivalent chain length (ECL) value for authentic 18:5n‐3 methyl ester was 20.22, whereas the main component after base‐catalyzed methylation had ECL 20.88. Attempts to prepare N‐acyl pyrrolidides or 4,4‐dimethyloxazoline (DMOX) derivatives of 18:5n‐3 also gave inadequate results. These derivatives also showed a main peak corresponding to isomerized 18:5. Mass spectra for both DMOX and pyrrolidide derivatives of this compound showed the base peak at m/z 139, probably corresponding to 2,6,9,12,15‐18:5 acid. Of all methods tested for methylation, only derivatization with 5% HCl or 1% sulphuric acid in methanol gave satisfactory results. Therefore, GC or GC‐mass spectrometry analyses of algal lipids containing 18:5n‐3 may be inaccurate when base‐catalyzed methods of FA derivatization are applied. The best and simplest way to avoid incorrect GC results is to use standard acid‐catalyzed methylation.     

Oxidative metabolism by Thalassiosira weissflogii (Bacillariophyceae) of a diol-ester of okadaic acid, the diarrhetic shellfish poisoning

Previous investigations into the comparative toxicity of the diarrhetic shellfish poisoning (DSP) toxins to Thalassiosira weissflogii (Grun.) Fryxell et Hasle found that this diatom oxidatively metabolized okadaic acid diol‐ester (OA diol‐ester) to a more water‐soluble product. This oxidative transformation of OA diol‐ester by the diatom is significant for two reasons. First, it is known that dinophysistoxin‐4 (DTX‐4), the primary DSP toxin produced by the dinoflagellate Exuviaella lima (Ehr.) Butschli, will be hydrolyzed to the diol‐ester following cell rupture (e.g. ingestion by a predator). Second, it implies that the ester, an uncharged, lipophilic intermediate, can easily enter cells and therefore may play an important role in the uptake and transfer of DSP toxins through the food web. It has been suggested that the water soluble DTX‐4 may also be the form in which DSP toxins are excreted from the producing cell. Therefore, the stability of DTX‐4 was examined when incubated either in fresh seawater medium into which washed cells of E. lima were introduced or in seawater medium conditioned by E. lima cells. Rapid hydrolysis of DTX‐4 to the diol‐ester took place in both cases. Thus, regardless of the route by which DTX‐4 is liberated from the cell, either by cell disruption or excretion, the diol‐ester will be the dominant form of the toxin to challenge associated organisms. To examine the metabolism of OA diol‐ester by T. weissflogii in more detail, serial cultures of the diatom were challenged with OA diol‐ester at a concentration of 2.0 μg·mL^?1. The metabolism and fate of the diol‐ester in both cellular and medium fractions were monitored over 3 days using liquid chromatography with either ultraviolet (LC‐UV) or mass spectrometric (LC‐MS) detection. During the course of the experiment, all of the diol‐ester was metabolized. LC‐MS analysis revealed the presence of multiple oxidative products of OA diol‐ester in the medium fraction, including a carboxylic acid derivative. The major metabolites were isolated in sufficient quantity to permit structural elucidation by NMR and MS. All the metabolites identified resulted from oxidation of the diol‐ester side chain with the primary sites of attack at the terminal, subterminal, and unsaturated carbons. OA was found in both cellular and medium fractions, and its production was directly correlated with the metabolism of the diol‐ester. The relative partitioning of both OA diol‐ester and its oxidation products between cells and medium supports the contention that OA diol‐ester can readily enter cells, be metabolized, and then excreted in more water‐soluble forms.     

Optical resolution of two isomeric naphthylalanines by immobilized enyzmes

Optical resolution of β-(1-naphthyl)alanine and β-(2-naphthyl)alanine have been efficiently carried out through enzymatic hydrolysis of their methyl ester and/or N-acetyl ester derivatives by immobilized enzymes. Difficulties related to the lipophilic character of these amino acids were overcome by using emulsions of n-butyl acetate–water as reaction medium. The use of an automatic recirculating apparatus allowed reproducible and repetitive use of the immobilized biocatalysts.     

Cytotoxic Constituents and Mechanism from Peganum harmala

Peganum harmala L. is a traditional Chinese and Uygur medicine used to treat cancer. Bioactivity‐guided fractionation was applied to determine the cytotoxic constituents from P. harmala. A novel triterpenoid and a phenolic glycoside were isolated and identified, as well as seven known compounds. The novel metabolites were elucidated to be 3α‐acetoxy‐27‐hydroxyolean‐12‐en‐28‐oic acid methyl ester ( 1 , OA) and N‐acetyl‐9‐syringinoside ( 9 ). Some compounds exhibited potent cytotoxicity against human tumor cells. Among them, OA showed the highest cytotoxicity against human lung cancer cells A549 with an IC₅₀ value of 8.03 ± 0.81 μm . OA had a potent anti‐NSCLC cell activity by interfering with the epidermal growth factor receptor (EGFR) activation and its downstream signaling, and could exert an antiproliferative effect by inactivation of EGFR‐driven antiapoptotic pathway followed by the release of mitochondrial cytochrome c, which might prove to be a promising leading compound for the development of an anti‐lung cancer drug.     

Chemo-enzymatic epoxidation of oleic acid and methyl oleate in solvent-free medium

Chemo-enzymatic epoxidation of oleic acid (OA) and its methyl ester has been performed using hydrogen peroxide and immobilized lipase from Candida antarctica (Novozym® 435). The purpose of the study was to characterize the reaction under solvent-free conditions. The reaction temperature had a significant impact on epoxidation of OA. At lower temperatures, the substrate conversion was hindered by the formation of solid epoxystearic acid product. Nearly 90% conversion of OA to the epoxide product was obtained after 6 h at 50°C. Longer reaction times at 40°C and above resulted in by-product formation and eventually lowered the product yield. In contrast, the reaction with methyl oleate (MO) was less influenced by temperature. Almost complete epoxidation was achieved at 40-60°C; the higher the temperature the shorter was the reaction time. The main epoxidation product obtained was epoxystearic acid methyl ester (EME), and the remaining was epoxystearic acid (EA) formed by the hydrolytic action of the lipase. Recycling of the lipase for epoxidation of MO at 50°C indicated that the immobilized enzyme was prone to activity loss.     

Enantioselective enzymatic hydrolysis of racemic drugs by encapsulation in sol–gel magnetic sporopollenin

Candida rugosa lipase was encapsulated within a sol–gel procedure and improved considerably by fluoride-catalyzed hydrolysis of mixtures of octyltriethoxysilane and tetraethoxysilane in the presence of magnetic sporopollenin. The catalytic properties of the immobilized lipases were evaluated into model reactions, i.e., the hydrolysis of p-nitrophenylpalmitate (p-NPP), and the enantioselective hydrolysis of racemic naproxen methyl ester, mandelic acid methyl ester or 2-phenoxypropionic acid methyl ester that were studied in aqueous buffer solution/isooctane reaction system. The encapsulated magnetic sporopollenin (Spo-M-E) was found to give 319 U/g of support with 342% activity yield. It has been observed that the percent activity yields and enantioselectivity of the magnetic sporopollenin encapsulated lipase were higher than that of the encapsulated lipase without support. The substrate specificity of the encapsulated lipase revealed more efficient hydrolysis of the racemic naproxen methyl ester and 2-phenoxypropionic acid methyl ester than racemic mandelic acid methyl ester. It was observed that excellent enantioselectivity (E > 400) was obtained for encapsulated lipase with magnetic sporopollenin with an ee value of S-Naproxen and R-2 phenoxypropionic acid about 98%.