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.

The aqueous buffered suspensions of some vegetable flours were tested in the reaction with 1,2-epoxide deriving from (4R)- or (4S)-limonene as well as with the standard substrate methyl oleate epoxide and epoxide hydrolase activity was mainly detected in the flours from soybean, mung bean and red mung bean. A simple protocol was applied for obtaining a raw freeze-dried enzymatic preparation from flours, which maintained most of the activity of the fresh suspension and offered advantages in easy handling, reproducibility and stability over the time. Preferential hydrolysis of one of the diastereoisomers present in the starting (4R)- or (4S)-limonene oxide (cis/trans mixture) was observed and the reaction converged toward the formation of a predominant diol, resulting from the attack on the most (C1)- or less (C2)-substituted carbon of the oxirane ring depending on the cis/trans stereochemistry of the substrate. Biocatalyzed hydrolysis of (S)-limonene oxide proceeded at higher reaction rate compared to the (R)-isomer and with superior stereoconvergence toward a single diol product. The kinetic separation of the slow-reacting diastereoisomers of (R)- and (S)-limonene oxides was optimized by using the enzymatic preparations from red mung bean and soybean, respectively, and trans-(4R)-limonene oxide and cis-(4S)-limonene oxide were obtained in good yield and stereochemical purity.

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.

The biocatalytic epoxidation of ethanolamides of ?-3 fatty acids EPA and DHA, regarded as biologically active ?-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.

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.

In this review the recent reports of biocatalytic reactions applied to the desymmetrization of meso-compounds or symmetric prochiral molecules are summarized. The survey of literature from 2015 up to date reveals that lipases are still the most used enzymes for this goal, due to their large substrate tolerance, stability in different reaction conditions and commercial availability. However, a growing interest is focused on the use of other purified enzymes or microbial whole cells to expand the portfolio of exploitable reactions and the molecular diversity of substrates to be transformed. Biocatalyzed desymmetrization is nowadays recognized as a reliable and efficient approach for the preparation of pharmaceuticals or natural bioactive compounds and many processes have been scaled up for multigram preparative purposes, also in continuous-flow conditions.

The stability of the anti-inflammatory drug nepafenac was investigated in aqueous solutions containing hydroxypropyl-beta-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 a-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. (C) 2020 Elsevier B.V. All rights reserved.

Oxylipins are oxygenated bioactive natural products deriving from the oxidation of polyunsaturated fatty acids (PUFAs) that have attracted increasing interest has for their potent anti-inflammatory activity as well as cancer preventing agents [1]. Considerable synthetic efforts have been devoted to the development of selective processes for the production of single isomers of hydroxy- or epoxy- fatty acids, but in most cases multistep sequences are necessary to control the functionalization of a specific double bond of the polyenic molecules. Lipoxygenases (LOX) are (non-heme) iron-containing enzymes which catalyze the insertion of oxygen into polyunsaturated fatty acids, whereas plant peroxygenases (PXG) contain the heme prosthetic group and use alkyl hydroperoxides to form an oxyferryl specie that is catalytically active in epoxidation of the PUFAs. Since lipoxygenases and peroxygenases are present in a wide variety of plants, particularly legumes and cereals, soybean flour extracts and enzyme-enriched microsomal fractions of oat flour extracts have been proposed as economical alternative to the purified enzymes [2-3]. In spite of their potential in organic synthesis, however, in most cases the reactions with soybean flour and oat flour have been carried out on analytical scale and the products identified by MS-analysis. We decided to investigate the feasibility of preparative reactions in the presence of inexpensive extracts of soybean flour or oat flour using eicosapentaenoic acid (EPA) as test substrate. In this context, we found that the freeze-drying of extracts was advantageous in terms of activity and reproducibility, with a checked stability up to 6 months. The use of lyophilized flour extracts allowed the regio- and steroselective functionalization of EPA and the optimization of reaction and purification conditions led to good yields of isolated products, whose structures were established by 1D- and 2D-NMR experiments. [1] Moro, K; Nagahashi, M; Ramanathan, R; Takabe, K; Wakai T. World J Clin Cases 2016, 4, 155-164. [2] Tu, H.-A. T.; Dobson, E. P.; Henderson, L. C.; Barrow, C. J.; Adcock, J. L. New Biotechnology 2018, 41, 23-33. [3] Piazza, GJ; Foglia, TA ; A Nuñez JAOCS 2001, 78, 589-592.

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.

Lipase-catalyzed benzoylation of amines was shown to be feasible, in some cases with high enantioselectivity, and the best results were obtained using immobilized lipase from Candida antarctica (Novozym 435) and methyl benzoate as acyl donor in the presence of molecular sieves. The procedure was optimized for the resolution of (+/-)-1-methyl-3-phenylpropylamine, a key intermediate in the synthesis of antihypertensive drug labetalol, and the enantiopure (R)-benzamide was then converted into the pharmacologically active isomers of the drug. In comparison with the reported synthesis of chiral isomers of labetalol, this chemoenzymatic route offers the advantage in the lack of any chiral stoichiometric auxiliary.

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. (C) 2018 Elsevier B.V. All rights reserved.

Among some derivatives of milnacipran, a last-generation antidepressive drug with useful application in the treatment of fibromyalgia, N-isobutyramide (±)-4 was identified as a conglomerate-forming compound and characterized using a binary melting diagram and X-ray analysis. Exploiting the conglomerate nature of (±)-4, its enantiomeric resolution by preferential crystallization was shown to be feasible and allowed us to obtain both enantiomers in satisfactory yield and excellent optical purity over a few cycles of entrainment.

Enzymatic catalysis is a valuable approach used in organic synthesis to achieve chemical transformations in mild reaction conditions and with a high degree of selectivity. The use of enzymes, especially lipase, in organic solvent is an advantageous way to obtain single stereoisomers of a drug and biotransformations are today accepted as a powerful methodology for the industrial preparation of chiral pharmaceuticals. Enantiomerically pure molecules containing amino functions are important synthons for the preparation of drugs. Primary and secondary amines, where the amino group is directly located on a stereogenic carbon, have been obtained in good yields and optical purity by lipases-catalyzed kinetic resolution of the racemates through a transamination reaction in the presence of carboxylic esters as acyl donors.1 However, aminomethyl compounds are challenging substrates for their high reactivity and distance from the chiral center. Milnacipran, Z-(±)-2-(aminomethyl)-N,N-diethyl-1-phenyilcyiclopropane, is an active antidepressant drug belonging to the class of inhibitors of the reuptake of serotonin and has recently attracted interest for its painkiller effects in the treatment of fibromyalgia.2 Milnacipran is currently marketed in many countries, but not yet in Italy, in racemic form, however, recent pharmacokinetic studies on single enantiomers showed greater activity for (1S, 2R)-levomilnacipran. The aim of this study is the development of a simpler and more economical strategy alternative to the reported an enantioselective synthesis. The kinetic resolution of racemic milnacipran in the presence of lipase was then investigated and optimized by means of a careful choice of the reaction conditions (lipase source and form, temperature, solvent and acyl donor nature). The amide product and the unreacted substrate were obtained in satisfactory chemical yields and enantiomeric purities.3 During this study we identified one of the amides obtained from the enzyme-catalyzed resolution as a conglomerate, whose nature was confirmed by the physical properties (melting points, solubility and X-ray diffraction). The properties of this specific amide of milnacipran were then exploited for its spontaneous resolution by conglomerate crystallization through a sequence of crystallization steps. Starting from a substrate with quite low enantiomeric excess, crystals of both enantiomers were obtained in enantiopure form.

Le lipasi sono tra gli enzimi impiegati in biocatalisi quelli più utilizzati in sintesi organica asimmetrica, sia per l'ottenimento di molecole con elevata purezza enantiomerica a partire da substrati simmetrici (forme meso) o racemici, sia per la regioprotezione di molecole contenenti più gruppi funzionali della stessa natura chimica. Il largo impiego è giustificato dalla loro versatilità nel riconoscimento molecolare (bassa specificità per il substrato), dal fatto che non necessitano di co-fattori per compiere la loro attività catalitica, dalla stabilità in solventi diversi da quello acquoso e soprattutto dalla elevata regio-, chemio-, stereo- e enantiotoposelettività con cui espletano il riconoscimento molecolare. Da molti anni è nota la capacità delle lipasi di riconoscere stereoselettivamente isomeri contenenti centri stereogenici, recentemente è stata dimostrata la loro abilità nel riconoscere selettivamente anche molecole aventi chiralità planare e assiale. Nell'ambito di questa presentazione saranno illustrati alcuni risultati significativi per comprendere come la biocatalisi, e in particolare l'uso di lipasi in reazioni di transacilazione in solvente organico, possa essere di utilità nell'isolamento di enantioforme otticamente attive dotate di chiralità anche complessa, difficilmente ottenibili con i classici metodi di sintesi o di risoluzione cinetica classica. Come esempi di riconoscimento biocatalitico della chiralità centrale sono riportati la risoluzione di un farmaco chirale commercializzato in forma racemica e un caso in cui sono stati separati i singoli isomeri ottici da una miscela complessa di diastereoisomeri contenente sia il racemato che la corrispondente forma meso. Per chiarire invece le potenzialità dell'enzima nei confronti della risoluzione e/o desimmetrizzazione della chiralità assiale sono illustrati alcuni esempi in cui atropisomeri bifenilici, bipiridinici e fenantrolinici sono stati investigati come substrati. Nella maggior parte dei casi riportati i prodotti delle reazioni sono stati ottenuti per la prima volta in forma otticamente attiva pertanto è stato necessario utilizzare tecniche di cromatografia chirale e metodi spettroscopici (dicroismo circolare accoppiato con spettrometria NMR) come supporto per la determinazione della purezza enantiomerica e per l'assegnazione della configurazione assoluta.

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.

A biocatalysed procedure for the kinetic resolution of milnacipran, (±)-1 was developed and optimized by careful choice of the reaction parameters. The reaction of (±)-1 with methyl iso-butyrate as acyl donor in the presence of Novozyme 435 in tert-butyl methyl ether proceeded with moderate enantioselectivity giving the more pharmacologically active enantiomer of milnacipran (-)-1 as unreacted substrate and the corresponding amide (-)-4 both in optically enriched form. When the enzymatic reaction was prolonged up to 65% substrate conversion enantiopure levomilnacipran (-)-1 (98% ee) was directly recovered from the reaction mixture by simple extraction workup. © 2014 Published by Elsevier B.V.

Il progetto è rivolto alla sintesi chimica orientata verso la diversità molecolare di piccole collezioni di composti con potenziale attività biologica e farmacologica attraverso l'utilizzo di metodologie e strategie sintetiche tradizionali ed innovative, entrambe condotte secondo i principi della chimica sostenibile. Le strutture finali dei composti sono riconducibili a due grandi famiglie di composti naturali: i Curcuminoidi e gli Anfidinolidi, entrambi di estremo interesse nel campo degli antitumorali ed in particolari ai tumori della pelle (melanoma e carcinoma della pelle). E' di fondamentale importanza la capacità di prevedere le caratteristiche strutturali e funzionali delle nuove molecole capaci di svolgere le funzioni biologiche richieste che interessano principalmente nuovi lead farmacologici per agenti tumorali (melanoma e carcinoma della pelle) e di individuare e caratterizzare più profili molecolari di sistemi biologici verso i quali le molecole manifestano attività completamente differenti (es. fragranze).

Using a highly stereoselective enzymatic procedure based on the irreversible transesterification of alcoholic functions in organic solvent, both enantiomers of zingerol and dehydrozingerol . O-methyl derivatives . 1 and . 2 were obtained in high optical purity. The biocatalytic method was then extended to the corresponding biphenyl derivatives . 5-8 for which an one-pot resolution/desymmetrization was carried out on the whole racemic/. meso mixtures to give single stereoisomers with very high enantiomeric and diastereoisomeric excesses. The comparison of kinetic and enantioselective behavior of the lipase AK from . Pseudomonas fluorescens in the transesterification of monomer (±)-. 1 and the related (±)-. 5 and meso-. 6 biphenyl dimers was also made.

New chiral bipyridyl alcohols and the corresponding N,N-dioxide derivatives were obtained with high enantiomeric excess by a chemo-enzymatic procedure. Metalation of commercial 6,6'-dimethyl-2,2'-bipyridine followed by reaction with acetaldehyde furnished an inseparable 1:1 mixture of dl- and meso- 6,6'-bis(2-hydroxypropyl)-2,2'-bipyridine ((±)-7a and meso-7b) that was subjected to lipasecatalysed enantioselective esterification with vinyl acetate in organic solvent. Immobilised Mucor Miehei lipase (Lipozyme ® IM) displayed high stereoselectivity and unreacted alcohol (S,S)-7a, monoacetyl derivative (R,S)-6 and diacetate (R,R)-9 were obtained in high optical purities as the result of the simultaneous resolution of the racemate and desymmetrization of meso isomer. After separation, the chiral compounds were converted into the corresponding N,N-dioxide derivatives with additional axial chirality stereoselectively induced during the N,N-oxidation and some considerations about their stereochemistry and atropisomeric stability were deduced by circular dichroism spectroscopy.

Optically active C2-symmetric 2,2'-bipyridines and their related N,N'-dioxides bearing stereogenic carbons in the side chains have recently attracted great interest in organic and coordination chemistry.1 The access to such derivatives in optically active form has been mostly achieved by "chiral pool" approach or asymmetric synthesis, but a valuable alternative route could rely on kinetic resolution of racemic bipyridine frameworks. In this context, lipase catalysed acylation could provide useful tool for the preparation of enantiopure bipyridine alcohols, as an extension of well-known biocatalysed resolution of phenyl substituted alkanols. However, few data are available on resolution or desymmetrization processes applied to racemic or meso- bipyridine having secondary hydroxy groups in side chain. As part of our continuous interest in the study of lipase behaviour concerning the stereo-recognition of bipyridines and phenanthroline derivatives with central and/or axial chirality,2 we have focused our attention on the lipase catalysed kinetic resolution of racemic C2-symmetric 6,6'-bis(2-hydroxypropyl)-2,2'-bipyridine 2, obtained as an inseparable 1:1 mixture of dl- and meso-forms by metallation and treatment with acetaldehyde of commercially available 3,3'-dimethyl-2,2'-bipyridine. The esterification of the whole stereoisomeric mixture with vinyl acetate as acyl donor in presence of immobilised Mucor Miehei lipase (Lipozyme IM) has resulted in the selective transformation of the alcoholic functions on the stereogenic carbon having R configuration. This selective chiral recognition gave the simultaneous resolution of (±)-2 and the desymmetrization of meso isomer, allowing the recovery of the diacetate (R,R)-4, monoacetyl derivative (R,S)-3 and unreacted alcohol (S,S)-2a, in high optical purities. The absolute configuration was confirmed by NMR spectroscopy. The enantiopure products obtained were then converted into the corresponding N,N'-dioxide derivatives, whose stereochemistry and axial stability were investigated by dynamic circular dichroism spectroscopy.