Mild hydrolysis of chemically stable valerolactams by a biocatalytic ATP-dependent system fueled by metaphosphate.

Medium-sized 5- and 6-membered ring lactams are molecules with remarkable stability, in contrast to smaller ß-lactams. As monomers, they grant access to nylon-4 and nylon-5, which are alternative polyamides to widespread caprolactam-based nylon-6. Chemical hydrolysis of monocyclic γ- and δ-lactams to the corresponding amino acids requires harsh reaction conditions and up to now, no mild (enzymatic) protocol has been reported. Herein, the biocatalytic potential of a pair of heterologously expressed bacterial ATP-dependent oxoprolinases - OplA and OplB - was exploited. Strong activity in the presence of excess of ATP was monitored on δ-valerolactam and derivatives thereof, while trace activity was detected on γ-butyrolactam. An ATP recycling system based on cheap Graham's salt (sodium metaphosphate) and a polyphosphate kinase allowed the use of catalytic amounts of ATP, leading to up to full conversion of 10 mM δ-valerolactam at 30 °C in aqueous medium. Further improvements were obtained by co-expressing OplA and OplB using the pETDuet1 vector, a strategy which enhanced the soluble expression yield and the protein stability. Finally, a range of phosphodonors was investigated in place of ATP. With acetyl phosphate and carbamoyl phosphate, turnover numbers up to 176 were reached, providing hints on a possible mechanism, which was studied by 31P-NMR.

Enzymatic Oxidative Cascade for Oxofunctionalization of Fatty Acids in One-Pot.

The biocatalytic conversion of fatty acids to α-ketoacids was accomplished by the action of two enzymes combined in a simultaneous one-pot two-step cascade. In the first step, P450 monooxygenase from Sphingomonas paucimobilis used hydrogen peroxide in the so-called peroxygenase mode for the regio- and enantioselective formation of α-hydroxyacids. In the next step, these hydroxyacid intermediates were further oxidized to the corresponding α-ketoacids by an α-hydroxyacid oxidase from Aerococcus viridans at the expense of molecular oxygen, thereby regenerating hydrogen peroxide used in the first step. Overall, the cascade was designed to employ catalytic quantities of hydrogen peroxide and proceeded at room temperature in dilute aqueous H2O2 solutions (≤0.01%). This setup could be applied to the conversion of a range of fatty acids (C6:0 to C10:0) and was scaled up to allow the production of 2-oxooctanoic acid in 91% isolated yield.

PQQ-dependent Dehydrogenase Enables One-pot Bi-enzymatic Enantio-convergent Biocatalytic Amination of Racemic sec-Allylic Alcohols.

The asymmetric amination of secondary racemic allylic alcohols bears several challenges like the reactivity of the bi-functional substrate/product as well as of the α,ß-unsaturated ketone intermediate in an oxidation-reductive amination sequence. Heading for a biocatalytic amination cascade with a minimal number of enzymes, an oxidation step was implemented relying on a single PQQ-dependent dehydrogenase with low enantioselectivity. This enzyme allowed the oxidation of both enantiomers at the expense of iron(III) as oxidant. The stereoselective amination of the α,ß-unsaturated ketone intermediate was achieved with transaminases using 1-phenylethylamine as formal reducing agent as well as nitrogen source. Choosing an appropriate transaminase, either the (R)- or (S)-enantiomer was obtained in optically pure form (>98 % ee). The enantio-convergent amination of the racemic allylic alcohols to one single allylic amine enantiomer was achieved in one pot in a sequential cascade.

Biotransformation of progesterone and testosterone enanthate by Circinella muscae.

In this study, the biotransformation of progesterone (1) and testosterone enanthate (5) using the whole cells of Circinella muscae was investigated for the first time. Microbial transformation of 1 with C. muscae afforded three known metabolites including 9α-hydroxyprogesterone (2), 14α-hydroxyprogesterone (3) and 6ß,14α dihydroxyprogesterone (4) after 6 days of incubation at 26 °C. The biotransformation of 5 with C. muscae yielded a new metabolite; 8ß,14α-dihydroxytestosterone (8), in addition to two known metabolites; 6ß-hydroxytestosterone (6), and 9α-hydroxytestosterone (7). The structure of the metabolites were established on the basis of spectroscopic data.

Biocatalytic Enantioselective Oxidation of Sec-Allylic Alcohols with Flavin-Dependent Oxidases.

The oxidation of allylic alcohols is challenging to perform in a chemo- as well as stereo-selective fashion at the expense of molecular oxygen using conventional chemical protocols. Here, we report the identification of a library of flavin-dependent oxidases including variants of the berberine bridge enzyme (BBE) analogue from Arabidopsis thaliana (AtBBE15) and the 5-(hydroxymethyl)furfural oxidase (HMFO) and its variants (V465T, V465S, V465T/W466H and V367R/W466F) for the enantioselective oxidation of sec-allylic alcohols. While primary and benzylic alcohols as well as certain sugars are well known to be transformed by flavin-dependent oxidases, sec-allylic alcohols have not been studied yet except in a single report. The model substrates investigated were oxidized enantioselectively in a kinetic resolution with an E-value of up to >200. For instance HMFO V465S/T oxidized the (S)-enantiomer of (E)-oct-3-en-2-ol (1 a) and (E)-4-phenylbut-3-en-2-ol with E>200 giving the remaining (R)-alcohol with ee>99% at 50% conversion. The enantioselectivity could be decreased if required by medium engineering by the addition of cosolvents (e. g. dimethyl sulfoxide).

Extending Designed Linear Biocatalytic Cascades for Organic Synthesis.

Artificial cascade reactions involving biocatalysts have demonstrated a tremendous potential during the recent years. This review just focuses on selected examples of the last year and putting them into context to a previously published suggestion for classification. Subdividing the cascades according to the number of catalysts in the linear sequence, and classifying whether the steps are performed simultaneous or in a sequential fashion as well as whether the reaction sequence is performed in vitro or in vivo allows to organise the concepts. The last year showed, that combinations of in vivo as well as in vitro are possible. Incompatible reaction steps may be run in a sequential fashion or by compartmentalisation of the incompatible steps either by using special reactors (membrane), polymersomes or flow techniques.

Enantioselective biocatalytic formal α-amination of hexanoic acid to l-norleucine.

A three-step one-pot biocatalytic cascade was designed for the enantioselective formal α-amination of hexanoic acid to l-norleucine. Regioselective hydroxylation by P450CLA peroxygenase to 2-hydroxyhexanoic acid was followed by oxidation to the ketoacid by two stereocomplementary dehydrogenases. Combination with final stereoselective reductive amination by amino acid dehydrogenase furnished l-norleucine in >97% ee.

A novel approach for bioconjugation of Rhizomucor miehei lipase (RML) onto amine-functionalized supports; Application for enantioselective resolution of rac-ibuprofen.

Since the amine groups are highly reactive toward various functional moieties, the formation of covalent bonds between a biomolecule and an insoluble amine-functionalized support is the most frequently used technique in immobilization protocols. A new approach based on the Ugi four-component reaction was used for immobilization of Rhizomucor miehei lipase (RML) as a model enzyme on amine-functionalized silica and silica nanoparticles (SBA-15). For this, the amine-modified supports were prepared and the structural properties of the functionalized supports, prior to and after functionalization were characterized by using IR, SEM, TGA, DTA, TEM. Immobilization of RML on the aminated carriers was performed under extremely mild conditions (25 °C, pH 7). The results revealed very rapid immobilization of 150 and 200 mg of RML on 1 g of silica-NH2 and SBA-NH2, respectively, producing 95-100% of immobilization yield. The specific activity and optimum pH of the immobilized preparations and the effect of temperature and co-solvents on their stabilities as well as the reusability of the derivatives were tested. The immobilized preparations were also used as enantioselective catalyst in kinetic resolution of racemic ibuprofen. Among them, Silica-RML showed the best selectivity with 92.2% enantiomeric excess (ee) and E-value of 33.9.

Biocatalytic Oxidative Cascade for the Conversion of Fatty Acids into α-Ketoacids via Internal H2 O2 Recycling.

The functionalization of bio-based chemicals is essential to allow valorization of natural carbon sources. An atom-efficient biocatalytic oxidative cascade was developed for the conversion of saturated fatty acids to α-ketoacids. Employment of P450 monooxygenase in the peroxygenase mode for regioselective α-hydroxylation of fatty acids combined with enantioselective oxidation by α-hydroxyacid oxidase(s) resulted in internal recycling of the oxidant H2 O2 , thus minimizing degradation of ketoacid product and maximizing biocatalyst lifetime. The O2 -dependent cascade relies on catalytic amounts of H2 O2 and releases water as sole by-product. Octanoic acid was converted under mild conditions in aqueous buffer to 2-oxooctanoic acid in a simultaneous one-pot two-step cascade in up to >99 % conversion without accumulation of hydroxyacid intermediate. Scale-up allowed isolation of final product in 91 % yield and the cascade was applied to fatty acids of various chain lengths (C6:0 to C10:0).

Enantioselective Oxidative Aerobic Dealkylation of N-Ethyl Benzylisoquinolines by Employing the Berberine Bridge Enzyme.

N-Dealkylation methods are well described for organic chemistry and the reaction is known in nature and drug metabolism; however, to our knowledge, enantioselective N-dealkylation has not been yet reported. In this study, exclusively the (S)-enantiomers of racemic N-ethyl tertiary amines (1-benzyl-N-ethyl-1,2,3,4-tetrahydroisoquinolines) were dealkylated to give the corresponding secondary (S)-amines in an enantioselective fashion at the expense of molecular oxygen. The reaction is catalyzed by the berberine bridge enzyme, which is known for CC bond formation. The dealkylation was demonstrated on a 100 mg scale and gave optically pure dealkylated products (ee>99 %).

C-20 Ketone reduction of hydrocortisone by Fusarium solani and Aspergillus ochraceus.

The biotransformation of hydrocortisone (1) by Fusarium solani and Aspergillus ochraceus was investigated for the first time. After 10 days at 30 °C, just one metabolite was produced by both fungi: 11ß, 17α, 20ß, 21-tetrahydroxypregn-4-en-3-one (2) established on the basis of spectroscopic data. The reaction was reduction of the 20-carbonyl group. Time course study determined by HPLC showed 60 and 45 % yield for the metabolite by F. solani and A. ochraceus, respectively.

A new abietane diterpenoid from Salvia xanthocheila Boiss.

From the chloroform extract of aerial part of Salvia xanthocheila Boiss. one new abietane diterpenoid, xantoquinone (5α,6α-dimethoxy-7,11,14-trioxoabieta-8,12-diene) and one known oleanene triterpenoid namely 1ß,3ß-dihydroxy-12-oleanene were isolated. The structure of the new terpenoid was elucidated by comprehensive spectroscopic analysis including electron ionisation-mass spectra, (1)H NMR, (13)C NMR, distortionless enchancement by polarisation transfer, H,H correlation spectroscopy, heteronuclear multiple-quantum coherence and heteronuclear multiple-bond correlation.

A new triterpene from Salvia xanthocheila Boiss.

From the chloroform extract of the aerial parts of Salvia xanthocheila, one new triterpene, together with two known diterpenes, two known flavonoids and a phytosterol was isolated. On the basis of comprehensive spectroscopic analyses, including electron ionisation mass spectra, ¹H-NMR, ¹³C-NMR, 1-D nuclear Overhauser effect, distortionless enchancement by polarisation transfer, H,H correlation spectroscopy, heteronuclear multiple quantum coherence, rotating frame Overhauser enhancement spectroscopy spectra and comparison with spectral data of known compounds, the structure of new compounds was established as 1ß,3ß-dihydroxy-olean-9(11),12-dienyl (1). The five known compounds (2-6) were 7α-acetoxyroyleanone, taxodione, salvigenin, apigenin-7,4'-dimethyl ether and ß-sitosterol, respectively. These known structures are isolated from the aerial parts of S. xanthocheila for the first time.

Essential oil from aerial parts of of Betonica grandiflora Willd. from Iran.

The chemical composition of the essential oil obtained from the aerial parts of Betonica grandiflora Willd., growing wild in Iran, was analysed by gas chromatography-mass spectrometry for the first time. Overall, 40 volatile components were identified on the basis of their mass spectra characteristics and retention indices in which ledol (21.8%), myrtenyl acetate (21.7%), eudesm-7(11)-en-4-ol (6.5%), trans-caryophyllene (5.5%), α-bisabolol (4.9%) and isolongifolol (4.5%) were the major constituents. Oxygenated sesquiterpenes, monoterpene hydrocarbons and sesquiterpene hydrocarbons were the main groups of compounds with 45.6%, 23.1% and 18.4%, respectively. The oil was moderately active against Bacillus subtilis and Staphylococcus aureus and inactive against Enterococcus faecalis and Klebsiella pneumoniae.