Carotenoids as antioxidants to prevent photooxidation

Carotenoids act as antioxidants in photooxidation by quenching singlet oxygen or triplet sensitizer. The antioxidant activities of β‐carotene, lutein and lycopene during photooxidation were investigated by following the formation of methyl linoleate isomeric hydroperoxides. The hydroperoxide formation and the isomeric distribution were determined using high‐performance liquid chromatography and post‐column detection with diphenyl‐1‐pyrenyl phosphine (DPPP). DPPP reacts with both the conjugated and nonconjugated hydroperoxides formed in photooxidation to give fluorescing DPPP‐oxides. Photooxidation with or without added β‐carotene, lutein and lycopene (10, 20 and 40 ppm) were carried out at +3 °C under 2000 lx. All the studied carotenoids were potential antioxidants during photooxidation and their antioxidant activities were concentration‐dependent. There were no significant differences in the antioxidant activities of lycopene and β‐carotene at a concentration of 10 ppm. At a concentration of 20 ppm β‐carotene was a better antioxidant than lycopene or lutein, and at a concentration of 40 ppm lycopene exerted a better antioxidant activity than β‐carotene or lutein. There were no significant differences between lycopene and lutein at a concentration of 20 ppm. The results also showed that carotenoids had no effect on the distribution of isomeric hydroperoxides indicating that the antioxidant mechanism of carotenoids during photooxidation does not involve hydrogen donation. All carotenoids were consumed during photooxidation. At a higher concentration (40 ppm) lycopene was more stable than the other tested carotenoids. That contributed most likely to it having a better antioxidant activity than β‐carotene and lutein.     

Antioxidant activity of extracts from six different Sudanese plant materials

Methanolic extracts obtained by manual solvent extraction (MSE) and accelerated solvent extraction (ASE) of different Sudanese plant materials (Sclerocarya birrea leaves, Salvadora persica bark and leaves, Combretum hartmannianum leaves, Guiera senegalensis leaves and roots) were investigated for their antioxidant activity. There was no significant difference between the two extraction methods (p < 0.01) regarding the total amount of phenolic compounds expressed as gallic acid equivalents (GAE) (52.6–166.7 mg GAE/g total extractable compounds for MSE and 53.1–169.3 mg GAE/g for ASE). In comparison to a control without extract, the extracts were remarkably effective in the β‐carotene bleaching method, whereas the effectiveness was half or less in comparison to Trolox as standard antioxidant. Also using the 1,1‐diphenyl‐2‐picrylhydrazyl (DPPH) method antioxidant activity could be shown in comparison to a control, however, the extracts were less effective than Trolox. No significant difference was found between the two extraction methods. The increase of the peroxide value of sunflower oil during storage at 70°C was markedly lower after addition of the extracts in comparison to the control, but in the Rancimat test (120°C) the extracts showed only a small stabilization factor (F = 0.9–1.4) especially in comparison to Trolox (F = 5.8).     

Effects of Phosphatidylcholine on Interaction of α-Tocopherol and β-Carotene in Photosensitized Oxidation of Emulsions

The interaction between α‐tocopherol (500 mg/kg) and β‐carotene (10 mg/kg) during chlorophyll‐photosensitized oxidation of a sunflower oil emulsion was studied in the presence or absence of phosphatidylcholine (PC, 250 mg/kg) by determining peroxide (POV) and conjugated dienoic acid (CDA) values. Chlorophyll, α‐tocopherol, β‐carotene, and PC contents in the emulsion were also monitored. α‐Tocopherol and β‐carotene individually and interactively decreased the POV and CDA values of oil in the emulsion by singlet oxygen quenching. PC decreased the POV and CDA values of oil, however, the values of the emulsion with added α‐tocopherol, β‐carotene, and PC were not significantly different from those of the emulsion with added α‐tocopherol and β‐carotene without PC. Contents of α‐tocopherol did not change during 24‐h oxidation, whereas co‐present PC significantly caused α‐tocopherol and chlorophyll degradation. β‐Carotene and PC contents significantly decreased to 45.5 and 51.3 %, respectively, after 24 h, and α‐tocopherol protected β‐carotene from degradation. The results suggest that PC had no net effects on the interactive antioxidant activity of α‐tocopherol and β‐carotene during chlorophyll‐photosensitized oxidation of the emulsion through free radical generation, chlorophyll degradation, and lessening the potency of α‐tocopherol as a singlet oxygen quencher.     

In‐vitro study on the efficacy of tannin fractions of edible nuts as antioxidants

Tannin fractions were isolated from crude acetonic extracts of defatted walnut, hazelnut and almond kernels using Sephadex LH‐20 column chromatography. The obtained material was characterized by content of total phenolics and electrophoretic separations using capillary zone electrophoresis (CZE). The antioxidant activities of the tannin fractions were analyzed by several methods: DPPH and ABTS assays, photochemiluminescence (PCL) method, as well as in two lipid model systems: emulsion with β‐carotene‐linoleic acid and L ‐α‐lecithin liposomes. The contents of total phenolics in the tannin fractions of walnuts, hazelnuts and almonds were 550, 329 and 83 mg catechin eq/g, respectively. The electrophoretic profiles of hazelnut and almond tannin fractions were similar, in contrast to the walnut profile. All analyzed fractions exhibited strong antioxidant properties. The antioxidant capacity of lipid‐soluble (ACL) compounds determined by PCL method was the highest for the fraction isolated from walnuts – 7.35 mmol Trolox eq/g. The DPPH radical and the ABTS radical cation were scavenged by the walnut tannin fraction with a higher efficacy than by the two other fractions. EC₅₀ values of the DPPH method were 1.8 times higher for the hazelnut fraction and 2.3 times higher for the almond fraction when compared to the walnut tannins. In turn, the total antioxidant activity values were 8.17, 2.82 and 1.98 mmol Trolox eq/g for the walnut, hazelnut and almond fractions, respectively. On the other hand, in both lipid models applied, lower antioxidant activity of walnut tannins than of hazelnut tannins was noted. The antioxidant effect of almond tannins was weaker or similar than that of walnut tannins in the β‐carotene‐linoleic acid emulsion and the L ‐α‐lecithin liposomal system, respectively.     

Optimization of the production of β‐carotene from molasses by Blakeslea trispora: a statistical approach

The effect of pretreatment of molasses, nitrogen sources, natural oils, fatty acids, antioxidant, precursors, and mixtures of the above substances on β‐carotene production by Blakeslea trispora in shake flask culture was investigated. Also, a central composite design was employed to determine the maximum β‐carotene concentration at optimum values for the process variables (linoleic acid, kerosene, antioxidant). The highest concentration of the carotenoid pigment was obtained in molasses solution treated with invertase. Corn steep liquor and yeast extract at concentrations of 5.0% and 0.5% (w/v), respectively, increased slightly the concentration of β‐carotene, while the natural oils, fatty acids, and precursors (except kerosene) did not improve the production of pigment when they were added separately to the medium. On the other hand, the mixture of linoleic acid, kerosene and antioxidant increased significantly the concentration of β‐carotene. The fit of the model was found to be good. Linoleic acid, kerosene and antioxidant had a strong linear effect on β‐carotene concentration. The concentration of β‐carotene was significantly affected by linoleic acid–antioxidant and kerosene–antioxidant interactions as well as by the negative quadratic effects of these variables. The interaction between linoleic acid–kerosene had no significant linear effect. Maximum β‐carotene concentration (790.0 mg dm^?3) was obtained in culture grown in molasses solution supplemented with linoleic acid (30.74 g dm^?3), kerosene (27.79 g dm^?3) and antioxidant (10.22 g dm^?3). © 2002 Society of Chemical Industry     

Physical State of β‐Carotene at High Concentrations in a Solid Triglyceride Matrix

The highly hydrophobic β‐carotene is often distributed or dissolved in triglycerides to enhance either nutritional or coloring effects. This study aims at elucidating the physical state of β‐carotene that at high concentrations are mixed into a solid high‐melting tri‐glyceride matrix by dissolution at high temperatures (165 °C) in the melted triglyceride. Extensive isomerization of β‐carotene is observed by HPLC after melting crystalline all‐trans β‐carotene and in the solid mixtures of β‐carotene and fully hydrogenated sunflower oil. Crystalline triglyceride is found in the mixed samples by XRPD analysis whereas no signs of crystalline lattice structures of β‐carotene are detected. DSC thermograms show only the melting and recrystallization events of triglycerides, which are affected by the presence of β‐carotene. Severe line broadening is observed in the ¹³C CP/MAS NMR spectra of the β‐carotene‐triglyceride mixtures when compared to crystalline β‐carotene, demonstrating the lack of long‐range order of the carotene. Altogether, the results demonstrate that β‐carotene is present as an amorphous mixture of trans‐ and cis‐isomers dispersed into a structure of crystalline triglyceride in the solid carotene‐triglyceride mixtures. Practical applications: The amorphous structure of trans‐ and cis‐isomers in solid formulations of β‐carotene‐triglyceride mixtures will strongly affect their functional properties related to nutrition and color as food ingredients.     

Synthesis and characterization of novel biocidal cyclodextrin inclusion complexes grafted onto polyamide‐6 fabric by a redox method

The synthesis of immobilized β‐cyclodextrin derivatives onto polyamide‐6 fabric is presented. These novel fabrics were prepared by graft‐copolymerization of glycidyl methacrylate (GMA) onto polyamide 6 fabric, using a chemical redox system K₂S₂O₈/CuSO₄·5H₂O, followed by reaction of β‐cyclodextrins (CD) or monochlorotriazinyl (MCT β‐CD) with the GMA epoxy group. Some biocidal guests were complexed into CD cavity including p‐hydroxy benzoic acid, AgNO₃–ethanolamine mixture, iodine, N,N‐diethyltoluamide (DETA), citronella, jasmine, and sweet basil. Characterization of the novel fabrics was done by Fourier transform infrared spectroscopy (IR), electron scanning microscopy (SEM), and thermo gravimetric analysis (TGA). The biocidal activity of the grafted fabrics was tested against five strains of microorganisms. © 2005 Wiley Periodicals, Inc. J Appl Polym Sci 99: 2586–2593, 2006     

Seed Oils of Five Black Tartary Buckwheat Cultivars with Biochemical Characterization and Antioxidant Properties

Black tartary buckwheat oils (BTBOs) were extracted from five major industrial tartary buckwheat cultivars grown under similar agronomical activities and environmental conditions. These oils were characterized for the bioactive compounds containing fatty acids, β‐carotene, lutein, α‐, β‐, δ‐ and γ‐tocopherol, and for their antioxidant properties. The total tocopherol contents that were obtained ranged from 704.66 to 1156.19 mg/kg, with γ‐tocopherol (588.98–977.91 mg/kg) as the main component. The concentration of lutein ranged from 253.14 to 429.63 mg/kg, which was almost ten times higher than that of β‐carotenoid (46.71–69.2 mg/kg), indicating that black tartary buckwheat seed oils were a good source of lutein. The predominant fatty acids were unsaturated oleic acid (C18:1) (35.27–40.61 %) and linoleic acid (C18:2) (38.25–42.90 %). Excellent values of 2,2‐diphenyl‐1‐picrylhydrazyl radical (DPPH), diammonium salt (ABTS) radical scavenging activities were obtained and the highest oxygen radical absorbance capacity (ORAC) value of 13.89 mmol Trolox equiv/g oil was detected in the variety of Chuanqiao No. 1, which was clearly separated by principal component analysis (PCA) on the basis of the highest content of tocopherols and carotenoids. Moreover, the correlation analysis showed that tocopherols and carotenoids were the major contributors to the antioxidant activities of the BTBOs. This study demonstrates that lipophilic extraction in the tartary buckwheat seed contains many interesting bioactive compounds, which are beneficial for human health.     

Carotenoid pattern in Blakeslea trispora grown on oil‐enriched substrates with regard to triacylglycerol species accumulation

The carotenoid pattern in Blakeslea trispora grown on oil‐enriched substrates was investigated with regard to triacylglycerol (TAG) species accumulation, to assess the interrelationship between these two processes. Analysis of individual carotenoids and TAG was carried out by HPLC. β‐Carotene production was at the expense of lycopene and γ‐carotene formation in cells grown on crude olive pomace oil (COPO) and crude soybean oil (CSO) at two levels of addition (10.0 and 30.0 g/L culture medium). A shift to γ‐carotene synthesis was observed at increased oil level. Cellular lipids produced at the low COPO or CSO levels contained more unsaturated TAG compared with those obtained on glucose as the sole carbon source. With regard to the typical soybean or olive oil TAG profile, cellular TAG had profiles dependent on the type and the amount of the co‐substrates used. In the presence of CSO, the cellular TAG profile was similar to that of the respective oil for both levels of addition. A notable desaturase activity was observed only in the presence of low COPO addition. The present study can serve as a basis for a better understanding of TAG accumulation with regard to β‐carotene production in oil‐enriched substrates.     

Multi‐Enzymatic Synthesis of Optically Pure β‐Hydroxy α‐Amino Acids

A novel enzymatic production system of optically pure β‐hydroxy α‐amino acids was developed. Two enzymes were used for the system: an N‐succinyl L ‐amino acid β‐hydroxylase (SadA) belonging to the iron(II)/α‐ketoglutarate‐dependent dioxygenase superfamily and an N‐succinyl L ‐amino acid desuccinylase (LasA). The genes encoding the two enzymes are part of a gene set responsible for the biosynthesis of peptidyl compounds found in the Burkholderia ambifaria AMMD genome. SadA stereoselectively hydroxylated several N‐succinyl aliphatic L ‐amino acids and produced N‐succinyl β‐hydroxy L ‐amino acids, such as N‐succinyl‐L ‐β‐hydroxyvaline, N‐succinyl‐L ‐threonine, (2S,3R)‐N‐succinyl‐L ‐β‐hydroxyisoleucine, and N‐succinyl‐L ‐threo‐β‐hydroxyleucine. LasA catalyzed the desuccinylation of various N‐succinyl‐L ‐amino acids. Surprisingly, LasA is the first amide bond‐forming enzyme belonging to the amidohydrolase superfamily, and has succinylation activity towards the amino group of L ‐leucine. By combining SadA and LasA in a preparative scale production using N‐succinyl‐L ‐leucine as substrate, 2.3 mmol of L ‐threo‐β‐hydroxyleucine were successfully produced with 93% conversion and over 99% of diastereomeric excess. Consequently, the new production system described in this study has advantages in optical purity and reaction efficiency for application in the mass production of several β‐hydroxy α‐amino acids.

     

Synthesis and characterization of grafting β‐cyclodextrin with chitosan

Two new adsorbents [β‐cyclodextrin–chitosan (β‐CD–CTS) and β‐cyclodextrin‐6–chitosan (β‐CD‐6‐CTS)] were synthesized by the reaction of β‐cyclodextrin (β‐CD) with epoxy‐activated chitosan (CTS) and the sulfonation of the C‐6 hydroxyl group of β‐cyclodextrin with CTS, respectively. Their structures were confirmed by IR spectral analysis and X‐ray diffraction analysis, and their apparent amount of grafting was determined by ultraviolet spectroscopy. The adsorption properties of β‐CD‐CTS and β‐CD‐6‐CTS for p‐dihydroxybenzene were studied. The experimental results showed that the two new adsorbents exerted adsorption on the carefully chosen target. The highest saturated capacity of p‐dihydroxybenzene of β‐CD‐CTS and β‐CD‐6‐CTS were 51.68 and 46.41 mg/g, respectively. © 2004 Wiley Periodicals, Inc. J Appl Polym Sci 94: 860–864, 2004     

Factors affecting droplet size of sodium caseinate‐stabilized O/W emulsions containing β‐carotene

This work was initiated to prepare an oil‐in‐water (O/W) emulsion containing β‐carotene by microfluidization. The β‐carotene was dissolved in triolein and microfluidized with an aqueous phase containing sodium caseinate (SC) as the emulsifier. Microfluization at 140 MPa resulted in O/W emulsions with a mean droplet diameter of ca. 120 nm, which was further confirmed by transmission electron microscopy analysis. The influences of SC concentration and microfluidization parameters on the droplet size of the emulsions were studied. The results showed that the mean droplet diameter decreased significantly (p <0.05) from 310 to 93 nm with the increase in SC concentration from 0.1 to 2 wt‐%. However, a further increase in SC concentration did not much change the droplet diameter, although the polydispersity of the emulsions was slightly improved. The droplet diameter of the emulsions was found to decrease from 200 to 120 nm with increasing microfluidization pressure, with narrower droplet size distribution. The storage study showed that the emulsions were physically stable for about 2 weeks at 4 °C in the dark. The results provide a better understanding of the performance of SC in stabilizing the O/W emulsions.     

Supercritical antisolvent micronisation of synthetic all‐trans‐β‐carotene with tetrahydrofuran as solvent and carbon dioxide as antisolvent

BACKGROUND: Supercritical antisolvent (SAS) micronisation of synthetic trans ‐β‐carotene was studied using tetrahydrofuran (THF) as solvent and supercritical carbon dioxide (CO₂) as antisolvent, with the objective of increasing its bioavailability and facilitating its dispersion in oil and emulsion formulations as a result of its smaller particle size. The micronised powder was analysed by scanning electron microscopy and high‐performance liquid chromatography. Micronisation experiments were performed in order to evaluate the effects of temperature (308.15–333.15 K), pressure (6.5–13 MPa) and concentration of the liquid solution (6–9 g L^?1). The effect of the supercritical CO₂/THF flow ratio in the range between 4 and 44 (on a mass basis) was also analysed. Determinations of equilibrium concentrations of β‐carotene in the CO₂/THF mixture were also performed. RESULTS: The particle size obtained ranged from 1 to 500 µm, with mean particle diameters around 100 µm. Three types of morphology were found in the precipitated powder: crystalline with superficial pores and leaf‐like appearance; crystalline with regular shapes and blade‐like edges; and crystalline without superficial pores and leaf‐like apearance. The Peng–Robinson equation of state was used to calculate the density of the CO₂/THF binary mixture, and the solubility of β‐carotene in this mixture was correlated with its density. CONCLUSION: The use of the SAS technique to micronise β‐carotene proved to be efficient, and the absence of degradation in the micronised powder allows the industrial application of this technique. Copyright © 2008 Society of Chemical Industry     

Effects of culture temperature on the production of bioactive polysaccharides by Agaricus blazei in batch cultures

Effects of culture temperature ranging from 20 to 34 °C on cell growth, polysaccharide biosynthesis and the bioactivity of polysaccharides of Agaricus blazei were evaluated via eight batch cultures in steps of 2 °C in a stirred tank bioreactor. Results indicated that the optimal temperature for the biomass was 28 °C with a cell yield of 780 mg g^?1, while that for polysaccharide formation was 30 °C with a product yield of 230 mg g^?1. Both the β‐glucan content and average molecular weight of the polysaccharides from different temperature‐controlled cultures were closely correlated with their tumour necrosis factor‐α (TNF‐α) release capability on macrophage cells. The polysaccharides from the low temperature range (20–24 °C) not only had higher relative content of β‐glucan and average molecular weight but also exhibited higher bioactivity compared with those from the high temperature range (30–34 °C). The optimal temperature for the production of bioactive polysaccharides of A. blazei was 24 °C, at which their relatively high molecular weight, β‐glucan content and TNF‐α release capability on macrophage cells were 1650 kDa, 188 mg g^?1 and 1560 pg per 5 × 10⁴ cells respectively. Copyright © 2007 Society of Chemical Industry     

Effects of Temperature and Heating Time on the Formation of Four Toxic α,β‐Unsaturated‐4‐Hydroxyaldehydes in Vegetable Oils

The formation of four α,β‐unsaturated hydroxyaldehydes, 4‐hydroxy‐2‐trans‐hexenal (HHE), 4‐hydroxy‐2‐trans‐octenal (HOE), 4‐hydroxy‐2‐trans‐nonenal (HNE), and 4‐hydroxy‐2‐trans‐decenal (HDE), was detected in commercial corn, soybean, peanut, and canola oils heated for 1, 3, and 5 hours at 145, 165, and 185 °C. These four toxic aldehydes were investigated using high‐performance liquid chromatography (HPLC). These oils were selected based upon different degrees of unsaturations, especially their linoleic and linolenic acid concentrations. To select the appropriate conditions of temperatures and heating times, preliminary experiments were conducted using the thiobarbituric acid assay, which detects the formation of secondary‐oxidation products such as aldehydes and related carbonyl compounds. After various heat treatments, the formation of HHE, HOE, HNE, and HDE was detected as 2,4‐dinitrophenyl hydrazine derivatives using HPLC. In general, HHE, HOE, HNE, and HDE formation increased in all four oils with higher temperatures, longer heating times, and higher concentrations of linoleic and linolenic acids in the oils. The formation of HNE in the oils was mostly much higher than the other three 4‐hydroxyaldehyde isomers under the same conditions.     

Characterization of the effects of β‐carotene on the thermal oxidation of triacylglycerols using HPLC‐ESI‐MS

RP HPLC method coupled to ESI‐MS was used for the analysis and characterization of the oxidation of model triacylglycerols (TAGs) in presence of β‐carotene. β‐Carotene was added to the TAGs and oxidized in the Rancimat at 110°C. The samples were separated isocratically using a mixture of isopropanol with methanol and a Phenomenex C18 column. β‐Carotene degradation was measured using high performance TLC. We found that β‐carotene plays an important role during the thermal degradation of high oleic acid model TAGs. Half of the β‐carotene was degraded before 3 h of thermal treatment. β‐Carotene significantly increases the peroxide value of the TAGs after the third hour, suggesting a pro‐oxidant action. However, different TAGs show different activity toward thermal treatment and β‐carotene. The LLL was found to be less stable, OLL and OLO were stable till 10 and 12 h respectively, while POO, OOO, and OSO were the stable TAGs till 14 h. In TAGs, replacing linoleic acid by oleic acid, the stability of the corresponding TAG was found to increase by 2 h. A new class of oxidized TAGs was reported for the first time, together with previously reported species. The proposed mechanism of formation and identification of the newly identified species have been explained. Among the oxidized species of TAGs, mono‐hydroperoxides, bis‐hydroperoxides, epoxy‐epidioxides, and epoxides were the major compounds identified.     

Highly Enantioselective Phase‐Transfer Catalytic α‐Alkylation of α‐tert‐Butoxycarbonyllactams: Construction of β‐Quaternary Chiral Pyrrolidine and Piperidine Systems

A new enantioselective α‐alkylation of α‐tert‐butoxycarbonyllactams for the construction of β‐quaternary chiral pyrrolidine and piperidine core systems is reported. α‐Alkylations of N‐methyl‐α‐tert‐butoxycarbonylbutyrolactam and N‐diphenylmethyl‐α‐tert‐butoxycarbonylvalerolactam under phase‐transfer catalytic conditions (solid potassium hydroxide, toluene, −40 °C) in the presence of (S,S)‐3,4,5‐trifluorophenyl‐3,3′,5,5′‐tetrahydro‐2,6‐bis(3,4,5‐trifluorophenyl)‐4,4′‐spirobi[4H‐dinaphth[2,1‐c:1′,2′‐e]azepinium] bromide [(S,S)‐NAS Br] (5 mol%) afforded the corresponding α‐alkyl‐α‐tert‐butoxycarbonyllactams in very high chemical (up to 99%) and optical yields (up to 98% ee). Our new catalytic systems provide attractive synthetic methods for pyrrolidine‐ and piperidine‐based alkaloids and chiral intermediates with β‐quaternary carbon centers.     

Chemoenzymatic Preparation of Enantiopure Homoadamantyl β‐Amino Acid and β‐Lactam Derivatives

Racemic cis‐10‐azatetracyclo[7.2.0.1^2,6.1^4,8]tridecan‐11‐one was prepared from homoadamant‐4‐ene by chlorosulfonyl isocyanate addition. The transformation of the β‐lactam to the corresponding β‐amino ester followed by Candida antarctica lipase A‐catalyzed enantioselective (E>>200) N‐acylation with 2,2,2‐trifluoroethyl butanoate afforded methyl (1R,4R,5S,8S)‐5‐aminotricyclo[4.3.1.1^3,8]undecane‐4‐carboxylate and the (1S,4S,5R,8R)‐butanamide with>99% ee at 50% conversion. Alternatively, transformation of the β‐lactam to the corresponding N‐hydroxymethyl‐β‐lactam and the following Pseudomonas cepacia (currently Burkholderia cepacia) lipase‐catalyzed enantioseletive O‐acylation provided the (1S,4S,6R,9R)‐alcohol (ee=87%) and the corresponding (1R,4R,6S,9S)‐butanoate (ee>99%). In the latter method, competition for the enzyme between the (1R,4R,6S,9S)‐butanoate, 2,2,2‐trifluoroethyl butanoate and the hydrolysis product, butanoic acid, tended to stop the reaction at about 45% conversion and finally gave racemization in the (1S,4S,6R,9R)‐alcohol with time.     

A concise synthesis of single‐enantiomer β‐lactams and β‐amino acids using Rhodococcus globerulus

BACKGROUND: Pharmaceutical companies continue to evaluate β‐amino acids and β‐lactams in a range of drug candidates. The development of a highly efficient and selective bioresolution of cyclic β‐lactam substrates could yield enantiopure lactams and β‐amino acids with medicinal potential. The aim of this work was to discover and develop a biocatalyst capable of selectively hydrolysing β‐lactam substrates. RESULTS: Screening of our in‐house culture collection led to the discovery of a microorganism, Rhodococcus globerulus (NCIMB 41042) with β‐lactamase activity. Whole‐cell bioresolutions of the β‐lactams 1–4 were successfully carried out and in all cases enantiomeric excesses of the residual lactam and amino acid product were found to be greater than 98%. For one example, the bioresolution was optimised to operate at 60 g L^?1 substrate concentration with a 20% wt/wt cell paste loading. CONCLUSION: A microorganism, Rhodococcus globerulus (NCIMB 41042), capable of selectively hydrolysing a range of cyclic β‐lactams, has been discovered. A scalable whole‐cell bioresolution process has been developed, leading to the synthesis of multigram quantities of enantiomerically pure β‐lactams and β‐amino acids. Copyright © 2007 Society of Chemical Industry     

Molecular Basis for the Enantioselective Ring Opening of β‐Lactams Catalyzed by Candida antarctica Lipase B

Lipase B from Candida antarctica (CAL‐B) catalyzes the slow, but highly enantioselective (E>200), ring‐opening alcoholysis of two bicyclic and two 4‐aryl‐substituted β‐lactams. Surprisingly, the rate of the reaction varies with the nature of the alcohols and was fastest with either enantiomer of 2‐octanol. A 0.5‐g scale reaction with 2‐octanol as the nucleophile in diisopropyl ether at 60 °C yielded the unreacted β‐lactam in 39–46% yield (maximum yield is 50%) with ≥96% ee. The product β‐amino acid esters reacted further by polymerization (not isolated or characterized) or by hydrolysis due to small amounts of water in the reaction mixture yielding β‐amino acids (7–11% yield, ≥96% ee). The favored enantiomer of all four β‐lactams had similar 3‐D orientation of substituents, as did most previously reported β‐lactams and β‐lactones in similar ring‐opening reactions. Computer modeling of the ring opening of 4‐phenylazetidin‐2‐one suggests that the reaction proceeds via an unusual substrate‐assisted transition state, where the substrate alcohol bridges between the catalytic histidine and the nitrogen of the β‐lactam. Computer modeling also suggested that the molecular basis for the high enantioselectivity is a severe steric clash between Ile189 in CAL‐B and the phenyl substituent on the slow‐reacting enantiomer of the β‐lactam.