Studies on the Oxidation of Aromatic Amines Catalyzed by Trametes versicolor Laccase.

The bio-oxidation of a series of aromatic amines catalyzed by T. versicolor laccase has been investigated exploiting either commercially available nitrogenous substrates [(E)-4-vinyl aniline and diphenyl amine] or ad hoc synthetized ones [(E)-4-styrylaniline, (E)-4-(prop-1-en-1-yl)aniline and (E)-4-(((4-methoxyphenyl)imino)methyl)phenol]. At variance to their phenolic equivalents, the investigated aromatic amines were not converted into the expected cyclic dimeric structures under T. versicolor catalysis. The formation of complex oligomeric/polymeric or decomposition by-products was mainly observed, with the exception of the isolation of two interesting but unexpected chemical skeletons. Specifically, the biooxidation of diphenylamine resulted in an oxygenated quinone-like product, while, to our surprise, in the presence of T. versicolor laccase (E)-4-vinyl aniline was converted into a 1,2-substited cyclobutane ring. To the best of our knowledge, this is the first example of an enzymatically triggered [2 + 2] olefin cycloaddition. Possible reaction mechanisms to explain the formation of these products are also reported.

Stereoselective Biocatalyzed Reductions of Ginger Active Components Recovered from Industrial Wastes.

Ginger is among the most widespread and widely consumed traditional medicinal plants around the world. Its beneficial effects, which comprise e. g. anticancer and anti-inflammatory activities as well as gastrointestinal regulatory effects, are generally attributed to a family of non-volatile compounds characterized by an arylalkyl long-chained alcohol, diol, or ketone moiety. In this work, ginger active components have been successfully recovered from industrial waste biomass of fermented ginger. Moreover, their recovery has been combined with the first systematic study of the stereoselective reduction of gingerol-like compounds by isolated alcohol dehydrogenases (ADHs), obtaining the enantioenriched sec-alcohol derivatives via a sustainable biocatalytic path in up to >99 % conversions and >99 % enantiomeric/diastereomeric excesses.

Functional Characterization and Synthetic Application of Is2-SDR, a Novel Thermostable and Promiscuous Ketoreductase from a Hot Spring Metagenome.

In a metagenome mining-based search of novel thermostable hydroxysteroid dehydrogenases (HSDHs), enzymes that are able to selectively oxidize/reduce steroidal compounds, a novel short-chain dehydrogenase/reductase (SDR), named Is2-SDR, was recently discovered. This enzyme, found in an Icelandic hot spring metagenome, shared a high sequence similarity with HSDHs, but, unexpectedly, showed no activity in the oxidation of the tested steroid substrates, e.g., cholic acid. Despite that, Is2-SDR proved to be a very active and versatile ketoreductase, being able to regio- and stereoselectively reduce a diversified panel of carbonylic substrates, including bulky ketones, α- and ß-ketoesters, and α-diketones of pharmaceutical relevance. Further investigations showed that Is2-SDR was indeed active in the regio- and stereoselective reduction of oxidized steroid derivatives, and this outcome was rationalized by docking analysis in the active site model. Moreover, Is2-SDR showed remarkable thermostability, with an apparent melting temperature (TM) around 75 °C, as determined by circular dichroism analysis, and no significant decrease in catalytic activity, even after 5 h at 80 °C. A broad tolerance to both water-miscible and water-immiscible organic solvents was demonstrated as well, thus, confirming the potential of this new biocatalyst for its synthetic application.

Studies on the Catalytic Promiscuity of Limonene Epoxide Hydrolases in the Non-hydrolytic Ring Opening of 1,2-Epoxides.

The non-hydrolytic ring opening of 1,2-epoxides in the presence of limonene epoxide hydrolases (LEHs) and different nucleophiles has been investigated. Lyophilized, wild-type LEHs were tested in selected water-saturated organic solvents in the presence of cyclohexene oxide as substrate and different alcohols, thiols and primary amines as nucleophiles. Although the LEHs retained an appreciable catalytic activity under different reaction conditions, formation of the desired 1,2-substituted cyclohexanols was not observed. Alternatively, LEH variants incapable of performing the hydrolytic reaction were generated by site-directed mutagenesis and tested in aqueous media in the presence of different water-soluble nucleophiles and cyclohexene oxide. Under defined reaction conditions, an acceleration of up to about threefold of the spontaneous reaction rate was observed in the presence of sodium azide and potassium thiocyanate as nucleophiles.

Chemo-enzymatic synthesis of (E)-2,3-diaryl-5-styryl-trans-2,3-dihydrobenzofuran-based scaffolds and their in vitro and in silico evaluation as a novel sub-family of potential allosteric modulators of the 90 kDa heat shock protein (Hsp90).

Herein we propose a facile, versatile and selective chemo-enzymatic synthesis of substituted (E)-2,3-diaryl-5-styryl-trans-2,3-dihydrobenzofurans based on the exploitation of the laccase-mediated oxidative (homo)coupling of (E)-4-styrylphenols. Thanks to this novel synthetic strategy, a library of benzofuran-based potential allosteric activators of the Heat shock protein 90 (Hsp90) was easily prepared. Moreover, considering their structural analogies to previously reported allosteric modulators, the sixteen new compounds synthesized in this work were tested in vitro for their potential stimulatory action on the ATPase activity of the molecular chaperone Hsp90. Combining experimental and computational results, we propose a mechanism of action for these compounds, and expand the structure-activity relationship (SAR) information available for benzofuran-based Hsp90 activators.

Correction: Self-assembled 4-(1,2-diphenylbut-1-en-1-yl)aniline based nanoparticles: podophyllotoxin and aloin as building blocks.

Correction for 'Self-assembled 4-(1,2-diphenylbut-1-en-1-yl)aniline based nanoparticles: podophyllotoxin and aloin as building blocks' by Gaia Fumagalli, et al., Org. Biomol. Chem., 2017, DOI: 10.1039/c6ob02591a.

Self-assembled 4-(1,2-diphenylbut-1-en-1-yl)aniline based nanoparticles: podophyllotoxin and aloin as building blocks.

The ability of 4-(1,2-diphenylbut-1-en-1-yl)aniline as a self-assembly inducer is reported. The conjugation of this moiety with aloin or podophyllotoxin resulted in spherical nanoparticles that were characterized by Dynamic Light Scattering (DLS), Transmission Electron Microscopy (TEM) and NanoSight technology. A preliminary biological evaluation on two cancer cell lines is reported.

Novel thermostable amine transferases from hot spring metagenomes.

Hot spring metagenomes, prepared from samples collected at temperatures ranging from 55 to 95 °C, were submitted to an in silico screening aimed at the identification of novel amine transaminases (ATAs), valuable biocatalysts for the preparation of optically pure amines. Three novel (S)-selective ATAs, namely Is3-TA, It6-TA, and B3-TA, were discovered in the metagenome of samples collected from hot springs in Iceland and in Italy, cloned from the corresponding metagenomic DNAs and overexpressed in recombinant form in E. coli. Functional characterization of the novel ATAs demonstrated that they all possess a thermophilic character and are capable of performing amine transfer reactions using a broad range of donor and acceptor substrates, thus suggesting a good potential for practical synthetic applications. In particular, the enzyme B3-TA revealed to be exceptionally thermostable, retaining 85% of activity after 5 days of incubation at 80 °C and more than 40% after 2 weeks under the same condition. These results, which were in agreement with the estimation of an apparent melting temperature around 88 °C, make B3-TA, to the best of our knowledge, the most thermostable natural ATA described to date. This biocatalyst showed also a good tolerance toward different water-miscible and water-immiscible organic solvents. A detailed inspection of the homology-based structural model of B3-TA showed that the overall active site architecture of mesophilic (S)-selective ATAs was mainly conserved in this hyperthermophilic homolog. Additionally, a subfamily of B3-TA-like transaminases, mostly uncharacterized and all from thermophilic microorganisms, was identified and analyzed in terms of phylogenetic relationships and sequence conservation.

Structural and biochemical insights into 7ß-hydroxysteroid dehydrogenase stereoselectivity.

Hydroxysteroid dehydrogenases are of great interest as biocatalysts for transformations involving steroid substrates. They feature a high degree of stereo- and regio-selectivity, acting on a defined atom with a specific configuration of the steroid nucleus. The crystal structure of 7ß-hydroxysteroid dehydrogenase from Collinsella aerofaciens reveals a loop gating active-site accessibility, the bases of the specificity for NADP(+) , and the general architecture of the steroid binding site. Comparison with 7α-hydroxysteroid dehydrogenase provides a rationale for the opposite stereoselectivity. The presence of a C-terminal extension reshapes the substrate site of the ß-selective enzyme, possibly leading to an inverted orientation of the bound substrate. Proteins 2016; 84:859-865. © 2016 Wiley Periodicals, Inc.

Regioselective alcoholysis of silychristin acetates catalyzed by lipases.

A panel of lipases was screened for the selective acetylation and alcoholysis of silychristin and silychristin peracetate, respectively. Acetylation at primary alcoholic group (C-22) of silychristin was accomplished by lipase PS (Pseudomonas cepacia) immobilized on diatomite using vinyl acetate as an acetyl donor, whereas selective deacetylation of 22-O-acetyl silychristin was accomplished by Novozym 435 in methyl tert-butyl ether/ n-butanol. Both of these reactions occurred without diastereomeric discrimination of silychristin A and B. Both of these enzymes were found to be capable to regioselective deacetylation of hexaacetyl silychristin to afford penta-, tetra- and tri-acetyl derivatives, which could be obtained as pure synthons for further selective modifications of the parent molecule.

Dicarboxylic esters: Useful tools for the biocatalyzed synthesis of hybrid compounds and polymers.

Dicarboxylic acids and their derivatives (esters and anhydrides) have been used as acylating agents in lipase-catalyzed reactions in organic solvents. The synthetic outcomes have been dimeric or hybrid derivatives of bioactive natural compounds as well as functionalized polyesters.

The quest for new mild and selective modifications of natural structures: laccase-catalysed oxidation of ergot alkaloids leads to unexpected stereoselective C-4 hydroxylation.

Laccase-catalysed oxidation of ergot alkaloids in the absence of chemical mediators allowed the unexpected isolation of the mono-hydroxylated derivatives of compounds 2-7. Structure determination by NMR techniques clearly indicated that hydroxylation took place at the C-4 benzylic position. Quite notably, the proposed protocol allowed, for the first time, functionalisation at the C-4 position of the ergoline skeleton. Depending on the absence or on the presence of a C-10 α-methoxy substituent, hydroxylation was either stereoselective (furnishing C-4α OH derivatives) or gave rise to a C-4α/C-4ß OH mixture in a 2:1 ratio, respectively.

In search of sustainable chemical processes: cloning, recombinant expression, and functional characterization of the 7α- and 7ß-hydroxysteroid dehydrogenases from Clostridium absonum.

Nicotinamide adenine dinucleotide phosphate-dependent 7α-hydroxysteroid dehydrogenase (7α-HSDH) and 7ß-hydroxysteroid dehydrogenases (7ß-HSDH) from Clostridium absonum catalyze the epimerization of primary bile acids through 7-keto bile acid intermediates and may be suitable as biocatalysts for the synthesis of bile acids derivatives of pharmacological interest. C. absonum 7α-HSDH has been purified to homogeneity and the N-terminal sequence has been determined by Edman sequencing. After PCR amplifications of a gene fragment with degenerate primers, cloning of the complete gene (786 nt) has been achieved by sequencing of C. absonum genomic DNA. The sequence coding for the 7ß-HSDH (783 nt) has been obtained by sequencing of the genomic DNA region flanking the 5' termini of 7α-HSDH gene, the two genes being contiguous and presumably part of the same operon. After insertion in suitable expression vectors, both HSDHs have been successfully produced in recombinant form in Escherichia coli, purified by affinity chromatography and submitted to kinetic analysis for determination of Michaelis constants (K (m)) and specificity constants (k (cat)/K (m)) in the presence of various bile acids derivatives. Both enzymes showed a very strong substrate inhibition with all the tested substrates. The lowest K (S) values were observed with chenodeoxycholic acid and 12-ketochenodeoxycholic acid as substrates in the case of 7α-HSDH, whereas ursocholic acid was the most effective inhibitor of 7ß-HSDH activity.

Exploiting enzymatic regioselectivity: a facile methodology for the synthesis of polyhydroxylated hybrid compounds.

Polyhydroxylated hybrid molecules have been synthesized using a protocol based on the regioselective acylation of the target compounds with activated dicarboxylic acids catalyzed by Novozym-435. The procedure implies that the mixed ester derivatives prepared and isolated from the first esterification step act as acylating agents in the second esterification step.

Enzymatic kinetic resolution of silybin diastereoisomers.

In nature, the flavonolignan silybin (1) occurs as a mixture of two diastereomers, silybin A and silybin B, which in a number of biological assays exhibit different activities. A library of hydrolases (lipases, esterases, and proteases) was tested for separating the silybin A and B diastereomers by selective transesterification or by stereoselective alcoholysis of 23-O-acetylsilybin (2). Novozym 435 proved to be the most suitable enzyme for the preparative production of both optically pure silybins A and B by enzymatic discrimination. Gram amounts of the optically pure substances can be produced within one week, and the new method is robust and readily scalable to tens of grams.

Discovery and Characterization of a Novel Thermostable ß-Amino Acid Transaminase from a Meiothermus Strain Isolated in an Icelandic Hot Spring.

A Meiothermus strain capable of using ß-phenylalanine for growth is isolated by culture enrichment of samples collected in hot environments and the genome is sequenced showing the presence of 22 putative transaminase (TA) sequences. On the basis of phylogenetic and sequence analysis, a TA termed Ms-TA2 is selected for further studies. The enzyme is successfully produced in Escherichia coli Rosetta(DE3) cells, with 70 mg of pure protein obtained from 1 L culture after purification by affinity chromatography. Ms-TA2 shows high activity toward (S)-ß-phenylalanine and other (S)-ß-amino acids, as well as a preference for α-ketoglutarate and aromatic aldehydes as amino acceptors. Moreover, Ms-TA2 is shown to be a thermostable enzyme by maintaining about 60% of the starting activity after 3 h incubation at 50 °C and showing a melting temperature of about 73 °C. Finally, a homology-based structural model of Ms-TA2 is built and key active site interactions for substrate and cofactor binding are analyzed.

Dispersed phantom scatterer technique reveals subtle differences in substrate recognition by phospholipase D inactive mutants.

An elastic light-scattering-based method that makes use of phantom nanoparticles as a substrate organizer (see illustration) allowed the quantitative evaluation of the molecular recognition events between a series of inactivated mutants of phospholipase D and lysophosphatidylcholine. The results highlight the remarkable effects on binding capability caused by single amino acid substitutions.

Stereoselectivity Switch in the Reduction of α-Alkyl-ß-Arylenones by Structure-Guided Designed Variants of the Ene Reductase OYE1.

Ene reductases from the Old Yellow Enzyme (OYE) family are industrially interesting enzymes for the biocatalytic asymmetric reduction of alkenes. To access both enantiomers of the target reduced products, stereocomplementary pairs of OYE enzymes are necessary, but their natural occurrence is quite limited. A library of wild type ene reductases from different sources was screened in the stereoselective reduction of a set of representative α-alkyl-ß-arylenones to investigate the naturally available biodiversity. As far as the bioreduction of the ethyl ketone derivatives concerns, the results confirmed the distinctiveness of the OYE3 enzyme in affording the reduced product in the (S) configuration, while all the other tested ene reductases from the Old Yellow Enzymes family showed the same stereoselectivity toward the formation of corresponding (R) enantiomer. A possible determinant role of the "hot spot" residue in position 296 for the stereoselectivity control of these reactions was confirmed by the replacement of Phe296 of OYE1 with Ser as found in OYE3. Further investigations showed that the same stereoselectivity switch in OYE1 could be achieved also by the replacement of Trp116 with Ala and Val, these experimental results being rationalized by structural and docking studies. Moreover, an additive effect on the stereoselectivity of OYE1 was observed when coupling the selected mutations in position 296 and 116, thus providing two extremely enantioselective variants of OYE1 (W116A-F296S, W116V-F296S) showing the opposite stereoselectivity of the wild type enzyme. Lastly, the effects of the mutations on the bioreduction of carvone enantiomers were investigated as well.

Self-assembling Releasable Thiocolchicine-Diphenylbutenylaniline Conjugates.

The design and the synthesis of new self-assembling conjugates is reported. The target compounds are characterized by the presence of a self-immolative linker that secures a controlled release induced by lipase cleavage. 4-(1,2-Diphenylbut-1-en-1-yl)aniline is used as a self-assembling inducer and amino-thiocolchicine as prototype of drug. The release of thiocolchicine derivative has been demonstrated in vitro in the presence of porcine pancreatic lipase and Celite-supported lipase. The formation of nanoparticles is confirmed by dynamic light scattering, atomic force microscopy, and fluorescence microscopy. The antiproliferative activity has been proved on two human cancer cell lines.