Expanding the Use of Peroxygenase from Oat Flour in Organic Synthesis: Enantioselective Oxidation of Sulfides.

Biocatalyzed oxidations are an important target in sustainable synthesis since chemical oxidations often require harsh conditions and metal-based catalysts. A raw peroxygenase-containing enzymatic preparation from oat flour was tested as a biocatalyst for the enantioselective oxidation of sulfides to sulfoxides and the variations of some reaction parameters were evaluated. Under optimal conditions, thioanisole was fully converted into the corresponding (R)-sulfoxide with high optical purity (80% ee) and the same stereopreference was maintained in the oxidation of some other sulfides. Changes in the substituent on the sulfur atom affected the selectivity of the enzyme and the best results were obtained with phenyl methoxymethyl sulfide, which gave the corresponding sulfoxide in 92% ee as exclusive product. The over-oxidation of sulfides to sulfones was instead detected in all the other cases and preferential oxidation of the (S)-enantiomer of the sulfoxide intermediate was observed, albeit with low selectivity. Carrying out the oxidation of thioanisole up to the 29% formation of sulfone led to enhancement of the sulfoxide optical purity (89% ee). The activity in sulfoxidation reactions, in addition to that reported in the epoxidation of different substrates, makes this plant peroxygenase a promising and useful tool in organic synthesis.

Stereoselective Promiscuous Reactions Catalyzed by Lipases.

The ability of lipases to display activity beyond their physiological reactions, so-called "catalytic promiscuity", has gained increasing interest in the last two decades as an important tool for expanding the application of these enzymes in organic synthesis. Some lipases have been shown to be effective in catalyzing a variety of C-C bond formation reactions and most of the investigations have been directed to the optimization of the products yield through a careful tuning of the experimental parameters. Despite the fact that new stereogenic carbons are formed in many of the tested reactions, the target products have been often obtained in racemic form and examples of an efficient asymmetric induction by the used lipases are quite limited. The aim of this review, mainly focused on those lipase-catalyzed promiscuous reactions in which optically active products have been obtained, is to offer a current state of art together with a perspective in this field of asymmetric synthesis.

Stereospecific Epoxidation of Limonene Catalyzed by Peroxygenase from Oat Seeds.

Limonene is one of the most abundant naturally occurring cyclic monoterpenes and has recently emerged as a sustainable alternative to petroleum-based solvents as well as a chemical platform for the production of value-added compounds. The biocatalytic epoxidation of both enantiomers of limonene was carried out in the presence of a peroxygenase-containing preparation from oat (Avena sativa) flour. Different reaction profiles were observed depending on the starting enantiomer of limonene, but in both cases the 1,2-monoepoxide was obtained as the main product with excellent diastereoselectivity. Trans-1,2-monoepoxide and cis-1,2-monoepoxide were isolated from the reaction of (R)-limonene and (S)-limonene, respectively, and the reactions were scaled-up to 0.17 M substrate concentration. The process is valuable for operational simplicity, lack of toxic metal catalysts, and cost-effectiveness of the enzymatic source. Pure stereoisomers of 1,2-monoepoxides of limonene constitute a useful starting material for biorenewable polymers, but can be also converted into other chiral derivatives by epoxide ring opening with nucleophiles. As a proof of concept, a tandem protocol for the preparation of enantiopure (1S,2S,4R)-1,2-diol from (R)-limonene and (1R,2R,4S)-1,2-diol from (S)-limonene was developed.

Biocatalytic regio- and stereoselective access to ω-3 endocannabinoid epoxides with peroxygenase from oat flour.

The biocatalytic epoxidation of ethanolamides of ω-3 fatty acids EPA and DHA, regarded as biologically active ω-3 endocannabinoids, in the presence of a peroxygenase-containing preparation from oat flour was investigated. Good regio- and steroselectivity toward the formation of the epoxide on the terminal double bond in the chain was observed with both these fatty acid derivatives and chiral monoepoxides 1 or 2 in 74% optical purity and 51-53% yields were isolated and spectroscopically characterized. The use of acetone as cosolvent in the reaction medium allowed to increase the concentration of starting substrates up to 40 mM and to further improve the selectivity in the epoxidation of DHA-EA. Due to the easy availability of the enzymatic preparation, the method offers a valuable strategy for the access to oxyfunctionalized derivatives of fatty acids.

Covid-19 and gender: lower rate but same mortality of severe disease in women-an observational study.

BACKGROUND: Gender-related factors might affect vulnerability to Covid-19. The aim of this study was to describe the role of gender on clinical features and 28-day mortality in Covid-19 patients. METHODS: Observational study of Covid-19 patients hospitalized in Bergamo, Italy, during the first three weeks of the outbreak. Medical records, clinical, radiological and laboratory findings upon admission and treatment have been collected. Primary outcome was 28-day mortality since hospitalization. RESULTS: 431 consecutive adult patients were admitted. Female patients were 119 (27.6%) with a mean age of 67.0 ± 14.5 years (vs 67.8 ± 12.5 for males, p = 0.54). Previous history of myocardial infarction, vasculopathy and former smoking habits were more common for males. At the time of admission PaO2/FiO2 was similar between men and women (228 [IQR, 134-273] vs 238 mmHg [150-281], p = 0.28). Continuous Positive Airway Pressure (CPAP) assistance was needed in the first 24 h more frequently in male patients (25.7% vs 13.0%; p = 0.006). Overall 28-day mortality was 26.1% in women and 38.1% in men (p = 0.018). Gender did not result an independent predictor of death once the parameters related to disease severity at presentation were included in the multivariable analysis (p = 0.898). Accordingly, the Kaplan-Meier survival analysis in female and male patients requiring CPAP or non-invasive ventilation in the first 24 h did not find a significant difference (p = 0.687). CONCLUSION: Hospitalized women are less likely to die from Covid-19; however, once severe disease occurs, the risk of dying is similar to men. Further studies are needed to better investigate the role of gender in clinical course and outcome of Covid-19.

Degradation profile of nepafenac in aqueous solution and structural characterization of a novel degradation product.

The stability of the anti-inflammatory drug nepafenac was investigated in aqueous solutions containing hydroxypropyl-ß-cyclodextrin at three different values of pH and degradation products were identified. (2-Amino-3-benzoyl)-oxoacetic acid, previously not reported as nepafenac-related impurity, was isolated and structurally characterized by NMR and ESI-MS analyses. It was also shown that the formation of this α-ketoacid from nepafenac in alkaline water/organic cosolvent solution occurs through an aerobic oxidation of the key intermediate 7-benzoyl-1,3-dihydro-indol-2-one, which in some extent is protected from oxidation in the presence of the cyclodextrin additive.

Lyophilized extracts from vegetable flours as valuable alternatives to purified oxygenases for the synthesis of oxylipins.

In this work, the whole aqueous extracts of soybean flour and oat flour have been used as valuable alternatives to purified oxygenase enzymes for the preparation of oxylipins derived from (5Z,8Z,11Z,14Z,17Z)-eicosapentaenoic acid (EPA). The lipoxygenase activity in the aqueous extracts of soybean (Glycine max. L.) flour was monitored with linoleic acid as substrate and compared with the commercially available purified enzyme (LOX-1). Oat flour extracts (Avena sativa L.) were evaluated for their peroxygenase activity by comparing different enzyme preparations in the epoxidation of methyl oleate. It was found that lyophilization of the aqueous extracts from these vegetable flours offers advantages in terms of enzyme stability, reproducibility and applicability to preparative organic synthesis. The lyophilized enzyme preparations were tested for the oxyfunctionalization of EPA and the formed products were isolated in satisfactory yields. In the presence of lyophilized extract from soybean, EPA gave 15S-hydroxy-(5Z,8Z,11Z,13E,17Z)-eicosapentaenoic acid in enantiopure form as exclusive product. Peroxygenase from oat flour was less selective and catalyzed the formation of different epoxides of EPA. However, the biocatalyzed epoxidation of EPA under controlled conditions allowed to obtain optically active (17R,18S)-epoxy-(5Z,8Z,11Z,14Z)-eicosatetraenoic acid (65% ee) as the main monoepoxide, among the five possible ones.

pH-Dependent stability of azithromycin in aqueous solution and structure identification of two new degradation products.

The degradation profile of azithromycin in buffered solutions was investigated using HPLC and found to be pH dependent in the range of 6.0-7.2. Desosaminylazitromycin, derived from hydrolytic loss of cladinose of the parent molecule, was the major degradation product at pH 6.0 but its amount progressively decreased moving toward pH 7.2. Two additional unreported degradation products were also observed and their structures were fully elucidated by MS- and NMR-spectroscopy to be associated with opening of the macrocyclic lactone ring.

Enantiomerically pure phosphonated carbocyclic 2'-oxa-3'-azanucleosides: synthesis and biological evaluation.

Starting from enantiomeric pure 1-[(3S,5R)- and 1-[(3R,5S)-3-(hydroxymethyl)-2-methylisoxazolidin-5-yl]-5-methylpyrimidine-2,4(1H,3H)-diones (-)7a and (+)7b, obtained by lipase-catalyzed resolution, pure diethyl{[(3S,5R)-2-methyl-5-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)isoxazolidin-3-yl]methyl}phosphonate (-)12a and diethyl{[(3R,5S)-2-methyl-5-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)isoxazolidin-3-yl]methyl}phosphonate (+)12b have been synthesized. The obtained compounds showed no cytotoxic activity versus the U937 cell line in comparison with AZT, and were poorly able to inhibit HIV infection in vitro.

Chiral HPLC analysis of milnacipran and its FMOC-derivative on cellulose-based stationary phases.

The HPLC enantioseparation of the last generation antidepressive drug milnacipran (+/-)-1 was investigated on different cellulose-based chiral stationary phases (CSPs). On carbamate-type columns, Chiralcel OD and OD-H (+/-)-1 could be separated with alpha value about 1.20 but the resolution was quite low because of the tailing of the peaks. Direct determination of (+/-)-1 with high selectivity and resolution was obtained on Chiralcel OJ in normal phase mode elution. Precolumn derivatization of milnacipran with Fmoc-Cl gave compound (+/-)-2 which was enantioseparated on all the investigated CSPs and allowed enhanced UV or fluorimetric detection. The Chiralpak IB, that could be considered the immobilized version of Chiralcel OD-H, was found completely ineffective in the chiral recognition of (+/-)-1 and moderately efficient in the separation of (+/-)-2.

Comparative HPLC enantioseparation of ferrocenylalcohols on two cellulose-based chiral stationary phases.

The direct HPLC enantiomeric separation of several ferrocenylalcohols on the commercially available Chiralcel OD and Chiralcel OJ columns has been evaluated in normal-phase mode. Almost all the compounds were resolved on one or both chiral stationary phases (CSPs) with separation factor (alpha) ranging from 1.06 to 2.88 while the resolution (R(s)) varied from 0.63 to 12.70 In the separation of the alpha-ferrocenylalcohols 1a-e and the phenyl analogues 2a-e, which were all resolved except 1c, a similar trend in the retention behavior for the two series of alcohols was evidenced and the selectivity was roughly complementary on the two investigated CSP. For three ferrocenylacohols, chosen as model compounds, the influence of the mobile phase composition and temperature on the enantioseparation were investigated and additional information on the chiral recognition mechanism were deduced from the chromatographic behavior of their acetylderivatives.

Chloroperoxidase from Caldariomyces fumago is active in the presence of an ionic liquid as co-solvent.

Chloroperoxidase from Caldariomyces fumago catalyses the oxidation of 1,2-dihydronaphthalene to (1R,2R)-(+)-dihydroxytetrahydronaphthalene in homogenous citrate buffer/ionic liquid mixtures, using t-butyl hydroperoxide as O2 donor. It tolerates up to 30 (v/v) 1,3-dimethylimidazolium methylsulfate or 1-butyl-3-methylimidazolium methylsulfate. The enzyme activity in these ionic liquid co-solvent systems is retained for 24 h, but it falls to 3 h using non-ionic organic solvents such as t-BuOH or acetone.