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.

Solid-State Preparation and Characterization of 2-Hydroxypropylcyclodextrins-Iodine Complexes as Stable Iodophors.

The use of iodine as antiseptic poses some issues related to its low water solubility and high volatility. Stable solid iodine-containing formulations are highly advisable and currently limited to the povidone-iodine complex. In this study, complexes of molecular iodine with 2-hydroxypropyl α-, ß- and γ-cyclodextrins were considered water-soluble iodophors and prepared in a solid state by using three different methods (liquid-assisted grinding, co-evaporation and sealed heating). The obtained solids were evaluated for their iodine content and stability over time in different conditions using a fully validated UV method. The assessment of the actual formation of an inclusion complex in a solid state was carried out by thermal analysis, and the presence of iodine was further confirmed by SEM/EDX and XPS analyses. High levels of iodine content (8.3-10.8%) were obtained with all the tested cyclodextrins, and some influence was exerted by the employed preparation method. Potential use as solid iodophors can be envisaged for these iodine complexes, among which those with 2-hydroxypropyl-α-cyclodextrin were found the most stable, regardless of the preparation technique. The three prepared cyclodextrin-iodine complexes proved effective as bactericides against S. epidermidis.

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.

Spectroscopic investigation on 1,2-substituted ferrocenes with only planar chirality: How chiroptical data are related to absolute configuration and to substituents.

Single enantiomers of three 1,2-substituted ferrocene derivatives, i.e. 1-methoxymethyl-2-hydroxymethylferrocene (1), 1-formyl-2-hydroxymethylferrocene (2) and 1-iodo-2-hydroxymethylferrocene (3), sharing the common hydroxymethyl substituent and the presence of planar chirality only, were investigated for their spectroscopic (IR and UV) and chiroptical (VCD and ECD) properties. Both enantiomers of 1 were obtained for the first time in optically pure form by lipase-catalyzed kinetic resolution of the corresponding racemate (±)-1 and separately converted into formyl derivatives (+)-2 and (-)-2. The experimental spectroscopic and chiroptical data were compared with DFT calculated spectra and excellent correspondence was found for all compounds, allowing one to confirm the previously assigned absolute configurations. The common features in the VCD spectra of a doublet between 940 and 965 cm-1 and the short-wavelength (about 200 nm) doublet and the longest wavelength band in the ECD spectra were analyzed to test whether they may be taken as markers of the absolute configuration (AC). The predominance of conformers with intramolecular hydrogen bond for the first two investigated compounds is predicted by conformational analysis and also confirmed by NMR.

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.

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.

Effect of cyclodextrin additives on azithromycin in aqueous solution and insight into the stabilization mechanism by sulfobutyl ether-ß-cyclodextrin.

The stability of azithromycin in buffered aqueous solution at pH 6.7 was investigated in the presence of different cyclodextrin (CD) additives by HPLC monitoring of the drug concentration over time. In the presence of γ-CDs, either in native or derivatized form, the long-term stability of azithromycin was sensibly decreased with respect to the reference sample without any additives, whereas the opposite effect was observed with all the three tested ß-CDs. The most effective stabilization of the drug was obtained by using sulfobutyl ether-ß-cyclodextrin, which allowed a concentration of azithromycin in solution at 99% up to 6 months at room temperature. The positive action of sulfobutyl ether-ß-cyclodextrin was mainly exerted through the suppression of a degradation pathway leading to the opening of lactone ring of azithromycin. The formation of dynamic inclusion complexes in solution was ruled out by NMR data and stabilization of azithromycin by the amphiphilic sulfobutyl ether-ß-cyclodextrin through surfactant-like effects was proposed on the basis of the strict similarity, either in the degradation profiles and in the NMR data, with a solution of the drug in the presence of sodium hexylsulphonate as surfactant.

Ferrocenes with simple chiral substituents: an in-depth theoretical and experimental VCD and ECD study.

Circular dichroism spectra in the IR range (VCD = vibrational circular dichroism) and in the UV range (ECD = electronic circular dichroism) have been recorded for both enantiomers of simple mono-substituted ferrocenes containing chiral pendants: 1-acetoxyethylferrocene, 1, 1-methoxyethylferrocene, 2, and 1-hydroxyethylferrocene, 3; the related disubstituted 1,1'-bis(1-hydroxyethyl)ferrocene, 4, was also considered. These two types of spectra, with the support of DFT calculations, concur to unequivocally confirm the absolute configuration for 1-4. In particular, our computational results point out the clear advantage of using an anharmonic oscillator model for the interpretation of VCD spectra of chiral ferrocenes. Interesting conformational properties are either confirmed or established by the technique, like the eclipsed conformation of the two cyclopentadienyl rings and an intra-molecular interaction involving the OH for 3. For 4, NMR, VCD and IR spectra are compatible with dimer formation and in this case a distorted conformation is predicted. Of utmost importance for the absolute configuration assignment in mono-substituted ferrocenes, we were able to identify a diagnostic VCD band at 950 cm-1 and a (low intensity) ECD band that clearly indicate the absolute configuration of the whole series.

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.

Enantioseparation of aldols by high-performance liquid chromatography on polysaccharide-based chiral stationary phases that bear chlorinated substituents.

Two families of aldols, obtained from the condensation of aromatic aldehydes with cyclohexanone or acetone (ten examples in each group), were analyzed by high-performance liquid chromatography in normal phase elution mode on three polysaccharide-based chiral stationary phases of the Lux series, namely, Lux Cellulose-2, Lux Cellulose-4 and Lux Amylose-2, which share the common feature of chlorinated substituents in the chiral selectors. Following simple optimization steps, the enantioseparation of all aldols derived from cyclohexanone was achieved and the highest values of separation factor (α, 1.32 < α < 2.20) and resolution (Rs , 4.5 < Rs <17.2) were observed on Lux Cellulose-2, with the only exception of the 4-nitro-substituted derivative that was better resolved on Lux Cellulose-4. On the contrary, Lux Amylose-2 was the best choice for aldols derived from acetone and only specific analytes in this group were resolved on the cellulose-based supports. A variable-temperature study of selected compounds allowed us to determine thermodynamic parameters of the enantioseparation process, which was enthalpy-controlled in all the cases except one.

Synthesis and characterization of some chiral metal-salen complexes bearing a ferrocenophane substituent.

The C(2)-symmetrical "salen" ligand (+)-9 bearing two [5]ferrocenophane substituents has been prepared in five steps starting from readily available diacetylferrocene, p-hydroxybenzaldehyde and (R,R)-N,N'-diphenylethylenediamine. Reaction of (+)-9 with Mn(OAc)(3), Co(OAc)(2) ZnEt(2) or UO(2)(OAc)(2) gave the corresponding metal-complexes which were characterised by spectroscopic methods.

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.

Preparation of a chiral ferrocenyl derivative containing a biphenyl unit via nickel-mediated homocoupling.

Ferrocenyl derivative 2, which possesses a biphenyl unit, was prepared by nickel(0)-mediated homocoupling of a chiral ferrocenyloxazoline. Compound 2 exists as an equilibrium mixture of two diastereomers, in a ratio dependent on temperature and solvent, due to the partially hindered rotation around biaryl bond. Upon complexation with Cu(I) or Zn(II), complete fixation of the stereogenic axis was obtained and a single stable atropoisomer was formed, whose absolute configuration was assigned on the basis of its NOESY spectrum. The 2-Cu(I) complex was used as catalyst in the cyclopropanation of styrene.