A chiral trimethyl lock based on the vicinal disubstituent effect: prolonged release of camptothecin into cancer cells.

Synthesis and in vitro testing of a prodrug designed for the controlled delivery of the anticancer drug camptothecin within pancreatic cancer cells are reported. Our study reveals a non-conventional pharmacokinetic release characterized by an exponential pattern before reaching the half-life (t1/2) and a linear pattern thereafter. The release mechanism was triggered either by hydrolytic enzymes and/or by the acid microenvironment of cancer cells.

Applications of biocatalytic CC bond reductions in the synthesis of flavours and fragrances.

Industrial biotechnology and biocatalysis can provide very effective synthetic tools to increase the sustainability of the production of fine chemicals, especially flavour and fragrance (F&F) ingredients, the market demand of which has been constantly increasing in the last years. One of the most important transformations in F&F chemistry is the reduction of CC bonds, typically carried out with metal-catalysed hydrogenations or hydride-based reagents. Its biocatalytic counterpart is a competitive alternative, showcasing a range of advantages such as excellent chemo-, regio- and stereoselectivity, ease of implementation, mild reaction conditions and modest environmental impact. In the present review, the application of biocatalysed alkene reductions (from microbial fermentations with wild-type strains to engineered isolated ene-reductase enzymes) to synthetic processes useful for the F&F industry will be described, highlighting not only the exquisite stereoselectivity achieved, but also the overall improvement when chirality is not involved. Multi-enzymatic cascades involving CC bioreductions are also examined, which allow much greater chemical complexity to be built in one-pot biocatalytic systems.

Combination of transjugular intrahepatic portosystemic shunt and antegrade through-the-TIPS coil embolization for bleeding mixed-type ectopic ileal varices.

A 61-year-old man with alcoholic cirrhosis and a history of severe cholecystitis leading to secondary thrombosis of the recanalized paraumbilical vein was admitted to our hospital for recurrent gastrointestinal bleeding and severe anemia. Capsule endoscopy and CT angiography detected profuse bleeding in the proximal ileum from ectopic ileal varices. Hepatic venous-portal gradient (HVPG) measurement was consistent with severe portal hypertension. Persistent bleeding despite transjugular intrahepatic portosystemic shunt (TIPS) placement required a combined approach with antegrade through-the-TIPS coil embolization of the ileal varices.

Towards compact laser-driven accelerators: exploring the potential of advanced double-layer targets.

The interest in compact, cost-effective, and versatile accelerators is increasing for many applications of great societal relevance, ranging from nuclear medicine to agriculture, pollution control, and cultural heritage conservation. For instance, Particle Induced X-ray Emission (PIXE) is a non-destructive material characterization technique applied to environmental analysis that requires MeV-energy ions. In this context, superintense laser-driven ion sources represent a promising alternative to conventional accelerators. In particular, the optimization of the laser-target coupling by acting on target properties results in an enhancement of ion current and energy with reduced requirements on the laser system. Among the advanced target concepts that have been explored, one appealing option is given by double-layer targets (DLTs), where a very low-density layer, which acts as an enhanced laser absorber, is grown to a thin solid foil. Here we present some of the most recent results concerning the production with deposition techniques of advanced DLTs for laser-driven particle acceleration. We assess the potential of these targets for laser-driven ion acceleration with particle-in-cell simulations, as well as their application to PIXE analysis of aerosol samples with Monte Carlo simulations. Our investigation reports that MeV protons, accelerated with a ∼20 TW compact laser and optimized DLTs, can allow performing PIXE with comparable performances to conventional sources. We conclude that compact DLT-based laser-driven accelerators can be relevant for environmental monitoring.

Stereoselective synthesis of whisky lactone isomers catalyzed by bacteria in the genus Rhodococcus.

Whisky lactone is a naturally occurring fragrance compound in oak wood and is widely used as a sensory additive in food products. However, safe and efficient methods for the production of its individual enantiomers for applications in the food industry are lacking. The aim of this study was to develop an efficient and highly stereoselective process for the synthesis of individual enantiomeric forms of whisky lactones. The proposed three-step method involves (1) column chromatography separation of a diastereoisomeric mixture of whisky lactone, (2) chemical reduction of cis-and trans-whisky lactones to corresponding syn-and anti-diols, and (3) microbial oxidation of racemic diols to individual enantiomers of whisky lactone. Among various bacteria in the genera Dietzia, Gordonia, Micrococcus, Rhodococcus, and Streptomyces, R. erythropolis DSM44534 and R. erythropolis PCM2150 effectively oxidized anti-and syn-3-methyl-octane-1,4-diols (1a-b) to corresponding enantiomerically pure cis-and trans-whisky lactones, indicating high alcohol dehydrogenase activity. Bio-oxidation catalyzed by whole cells of these strains yielded enantiomerically pure isomers of trans-(+)-(4S,5R) (2a), trans-(-)-(4R,5S) (2b), and cis-(+)-(4R,5R) (2d) whisky lactones. The optical density of bacterial cultures and the impact of the use of acetone powders as catalysts on the course of the reaction were also evaluated. Finally, the application of R. erythropolis DSM44534 in the form of an acetone powder generated the enantiomerically enriched cis-(-)-(4S,5S)-isomer (2c) from the corresponding syn-diol (1b). The newly developed method provides an improved approach for the synthesis of chiral whisky lactones.

Dependence of 1H-NMR T 1 relaxation time of trimethylglycine betaine deep eutectic solvents on the molar composition and on the presence of water.

1H-NMR spin lattice relaxation times (T 1), measured by inversion recovery technique, allowed to establish the stoichiometric coefficient (ratio between the H-bond acceptor and H-bond donor) of a series of trimethylglycine betaine/diol based deep eutectic solvents (DESs); ethylene glycol, triethylene glycol and 1,3-propandiol were selected as H-bond donors. The maximum amount of water tolerated by the DES, before its complete hydration, was determined as well. Finally, the method was validated comparing the eutectic composition of the betaine/glycol system with that determined by means of differential scanning calorimetry analysis; the stoichiometric coefficients were identical.

Thermal stabilization of diverse biologics using reversible hydrogels.

Improving the thermal stability of biologics, including vaccines, is critical to reduce the economic costs and health risks associated with the cold chain. Here, we designed a versatile, safe, and easy-to-use reversible PEG-based hydrogel platform formed via dynamic covalent boronic ester cross-linking for the encapsulation, stabilization, and on-demand release of biologics. Using these reversible hydrogels, we thermally stabilized a wide range of biologics up to 65°C, including model enzymes, heat-sensitive clinical diagnostic enzymes (DNA gyrase and topoisomerase I), protein-based vaccines (H5N1 hemagglutinin), and whole viruses (adenovirus type 5). Our data support a generalized protection mechanism for the thermal stabilization of diverse biologics using direct encapsulation in reversible hydrogels. Furthermore, preliminary toxicology data suggest that the components of our hydrogel are safe for in vivo use. Our reversible hydrogel platform offers a simple material solution to mitigate the costs and risks associated with reliance on a continuous cold chain for biologic transport and storage.

Silk Vista Baby flow diverter stent for ruptured intracranial aneurysms: a retrospective observational study.

PURPOSE: Flow diversion changed the approach to complex intracranial aneurysms, leading to a widespread use and a rapid technological evolution. Indeed, indications continued to expand, including ruptured intracranial aneurysms in selected cases. Recently, new devices have been designed specifically to target smaller vessels. Therefore, we conducted a multicenter study to evaluate clinical outcome, complications, and occlusion rate of patients with ruptured aneurysms treated with new generation low profile Silk Vista Baby (SVB) flow diverter stent (FD). METHODS: We performed a retrospective observational study on consecutive patients who underwent treatment with SVB for ruptured aneurysms at 12 Italian centers. Primary end point was favorable clinical outcome rate, defined as modified ranking score (mRS) of 0-2 at the 3 months. Secondary outcomes were complication rate, aneurysm re-rupture, and complete aneurysm occlusion at last radiological follow-up. RESULTS: Twenty-five patients were included; at 3 months' follow-up, 19 patients (79.1%) had favorable clinical outcome (mRS 0-2). Three patients (12.5%) died during follow-up. In-stent thrombosis occurred in two cases (8.3%), managed with glycoprotein IIb/IIIA and intra-stent angioplasty, without clinical consequences. In 18 (85.7%) patients, complete occlusion at 3 months was demonstrated. No rebleeding occurred during follow-up. Presentation with unfavorable World Federation of Neurosurgical Societies grading system (WFNS) and posterior circulation location were both significantly correlated with unfavorable clinical outcome (p = 0.005 and p = 0.02). CONCLUSIONS: Our data suggests that low profile FD treatment of ruptured intracranial aneurysms located distally of the circle of Willis is feasible. New generation low profile FD may represent an alternative option in carefully selected cases.

Chemoenzymatic Synthesis of the Most Pleasant Stereoisomer of Jessemal.

We describe the asymmetric synthesis of the most pleasant enantiomer of Jessemal fragrance. The key steps are (i) the one-pot reduction of an α-chloro-tetrasubstituted cyclohexenone to give the chlorohydrin, catalyzed by two stereoselective redox enzymes (an ene-reductase and an alcohol dehydrogenase); (ii) the regioselective epoxide ring-opening with organocuprate or organolithium nucleophiles. Density functional theory calculations together with the Curtin-Hammett principle allowed the rationalization of the regioselectivity.

"A Study in Yellow": Investigations in the Stereoselectivity of Ene-Reductases.

Ene-reductases from the Old Yellow Enzyme (OYE) superfamily are a well-known and efficient biocatalytic alternative for the asymmetric reduction of C=C bonds. Considering the broad variety of substituents that can be tolerated, and the excellent stereoselectivities achieved, it is apparent why these enzymes are so appealing for preparative and industrial applications. Different classes of C=C bonds activated by at least one electron-withdrawing group have been shown to be accepted by these versatile biocatalysts in the last decades, affording a vast range of chiral intermediates employed in the synthesis of pharmaceuticals, agrochemicals, flavours, fragrances and fine chemicals. In order to access both enantiomers of reduced products, stereodivergent pairs of OYEs are desirable, but their natural occurrence is limited. The detailed knowledge of the stereochemical course of the reaction can uncover alternative strategies to orient the selectivity via mutagenesis, evolution, and substrate engineering. An overview of the ongoing studies on OYE-mediated bioreductions will be provided, with particular focus on stereochemical investigations by deuterium labelling.

Environment Controls Biomolecule Release from Dynamic Covalent Hydrogels.

Moldable hydrogels composed of dynamic covalent bonds are attractive biomaterials for controlled release, as the dynamic exchange of bonds in these networks enables minimally invasive application via injection. Despite the growing interest in the biomedical application of dynamic covalent hydrogels, there is a lack of fundamental understanding as to how the network design and local environment control the release of biomolecules from these materials. In this work, we fabricated boronic-ester-based dynamic covalent hydrogels for the encapsulation and in vitro release of a model biologic (ß-galactosidase). We systematically investigated the role of network properties and of the external environment (temperature and presence of competitive binders) on release from these dynamic covalent hydrogels. We observed that surface erosion (and associated mass loss) governed biomolecule release. In addition, we developed a statistical model of surface erosion based on the binding equilibria in a boundary layer that described the rates of release. In total, our results will guide the design of dynamic covalent hydrogels as biomaterials for drug delivery applications.

Synthesis of Polycyclic Fused Indoline Scaffolds through a Substrate-Guided Reactivity Switch.

Zn(II)-catalyzed divergent synthesis of functionalized polycyclic indolines through formal [3 + 2] and [4 + 2] cycloadditions of indoles with 1,2-diaza-1,3-dienes (DDs) is reported. The nature and type of substituents of substrates are found to act as a chemical switch to trigger two distinct reaction pathways and to obtain two different types of products upon the influence of the same catalyst. The mechanism of both [4 + 2] and [3 + 2] cycloadditions was investigated and fully rationalized by density functional theory (DFT) calculations.

Conversion of Oleic Acid into Azelaic and Pelargonic Acid by a Chemo-Enzymatic Route.

A chemo-enzymatic approach for the conversion of oleic acid into azelaic and pelargonic acid is herein described. It represents a sustainable alternative to ozonolysis, currently employed at the industrial scale to perform the reaction. Azelaic acid is produced in high chemical purity in 44% isolation yield after three steps, avoiding column chromatography purifications. In the first step, the lipase-mediated generation of peroleic acid in the presence of 35% H2O2 is employed for the self-epoxidation of the unsaturated acid to the corresponding oxirane derivative. This intermediate is submitted to in situ acid-catalyzed opening, to afford 9,10-dihydroxystearic acid, which readily crystallizes from the reaction medium. The chemical oxidation of the diol derivative, using atmospheric oxygen as a stoichiometric oxidant with catalytic quantities of Fe(NO3)3∙9∙H2O, (2,2,6,6-tetramethylpiperidin-1-yl)oxyl (TEMPO), and NaCl, affords 9,10-dioxostearic acid which is cleaved by the action of 35% H2O2 in mild conditions, without requiring any catalyst, to give pelargonic and azelaic acid.

Biliary Leakage After Hepatobiliary and Pancreatic Surgery: A Classification System to Guide the Proper Percutaneous Treatment.

PURPOSE: To investigate the effectiveness of percutaneous approaches to treat bile leak and to propose an anatomical classification of biliary fistula to guide the most appropriate percutaneous approach. MATERIALS AND METHODS: Fifty-six patients with bile leakage after hepatobiliary surgery were included. Based on preoperative images and postoperative fistulogram images, three categories of bile leakage were defined. Every category was treated with non-surgical approaches (internal-external percutaneous drainage, percutaneous/endoscopic biliodigestive anastomosis with rendez-vous technique and biliodigestive percutaneous anastomosis with totally radiologic rendez-vous). RESULTS: In 44/56 (78%) patients, anatomical conformation was "direct communication" (bile ducts upstream from the leak present a direct communication with downstream ducts) and their treatment was conventional percutaneous drainage. In 5/56 (9%), anatomical conformation was "indirect communication" (bile ducts upstream from the leak communicate with downstream ducts through a bile collection) and treatment was percutaneous/endoscopic rendez-vous technique. In 7/56 (12%), anatomical conformation was "no communication" (ducts upstream from the leak are completely excluded from ducts downstream) and treatment was totally radiologic rendez-vous. In 54/56 (96%) during the follow-up, cholangiography revealed complete resolution of the leak without residual stenosis and drains were removed. Complications occurred in 12/56 (21%). Procedure-related mortality was 0%. Ten patients, after > 6 months from resolution of their fistula and drain removal, died due to cancer recurrence. Currently, 44/56 patients (77%) at long-term follow-up (> 12 months) are alive, without bile leak. CONCLUSION: Our classification helps to choose the most proper percutaneous approach in all kinds of bile leakage, even in severe cases; these are safe techniques with a high success rate.

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.

Exploiting the vicinal disubstituent effect on the diastereoselective synthesis of γ and δ lactones.

Trifluoroacetic acid catalysed lactonization of vicinal disubstituted γ-hydroxyesters was investigated in different solvents. The reaction kinetics, monitored by NMR spectroscopy, showed that: (i) the vic-disubstituent effect is stereoselective since the anti diastereoisomer ring closes substantially more rapidly than the syn isomer ring; (ii) the anti-vic effect is much stronger than the classical Thorpe-Ingold effect (known also as the gem-disubstituent effect), instead the syn diastereoisomers have rate constants comparable to that of the gem-disubstituted ester; (iii) the vic-effect can be enhanced by increasing the steric hindrance of one of the two substituents or carrying out the reaction in non-polar solvents. DFT computations of energy barriers (ΔG‡) were in good agreement with the experimental data. The distortion/interaction-activation strain model together with the Winstein-Holness kinetic scheme gave more insights into the origin of the vic-effect. An application of this effect consists of the diastereomeric resolution of disubstituted γ and δ lactones, among which are the naturally occurring Nicotiana t. lactone, the whisky and cognac oak lactones, and the Aerangis lactone. Both cis and trans diastereoisomers of these lactones were isolated in good yield and with high diastereomeric excess (de >92%). The selectivities of the diastereomeric resolution process, determined by NMR spectroscopy, are reported as well.

One-Pot Multi-Enzymatic Synthesis of the Four Stereoisomers of 4-Methylheptan-3-ol.

The use of pheromones in the integrated pest management of insects is currently considered a sustainable and environmentally benign alternative to hazardous insecticides. 4-Methylheptan-3-ol is an interesting example of an insect pheromone, because its stereoisomers are active towards different species. All four possible stereoisomers of this compound were prepared from 4-methylhept-4-en-3-one by a one-pot procedure in which the two stereogenic centres were created during two sequential reductions catalysed by an ene-reductase (ER) and an alcohol dehydrogenase (ADH), respectively.

Exploitation of a Multienzymatic Stereoselective Cascade Process in the Synthesis of 2-Methyl-3-Substituted Tetrahydrofuran Precursors.

Enantiopure 2-methyl-3-substituted tetrahydrofurans are key precursors of several biologically active products (drugs, flavors, and agrochemicals). Thus, a stereocontrolled and efficient methodology for the obtainment of these synthons is highly desirable. We exploited a two-step multienzymatic stereoselective cascade reduction of α-bromo-α,ß-unsaturated ketones to give the corresponding bromohydrins in good yields, with high ee and de values. The cascade process is catalyzed by an ene-reductase and an alcohol dehydrogenase. Further manipulations of these bromohydrins, by two diastereodivergent routes, allowed the preparation of the tetrahydrofuran synthons. One route is based on a lipase catalyzed cleavage of the protecting group. The second route is characterized by a camphor sulfonic acid mediated isomerization of a ß-hydroxyepoxide to give the tetrahydrofuran-2-ol. Finally, the synthesis of the most odorous and pleasant stereoisomer of the roasted meat aroma, i.e., (2S,3R)-2-methyl-3-thioacetate tetrahydrofuran, is reported as well.

Stereoselective synthesis of hernandulcin, peroxylippidulcine A, lippidulcines A, B and C and taste evaluation.

The first stereoselective synthesis of lippidulcines A, B and C has been accomplished starting from (+)-hernandulcin, which has been prepared on a multigram scale. The previously assigned absolute configurations have been confirmed. The key steps of this synthesis are based on a modified version of the Kornblum-DeLaMare rearrangement, and on a highly regioselective and stereoselective ketone reduction with the MeCBS reagent. The taste evaluations indicate that none of these sesquiterpenes are sweet, instead the lippidulcine A is a cooling agent with a mint after taste.

A Rapid and High-Throughput Assay for the Estimation of Conversions of Ene-Reductase-Catalysed Reactions.

A fast and sensitive colorimetric assay (FRED, fast and reliable ene-reductases detection) that allows the estimation of levels of conversion of ene-reductase (ER)-catalysed reactions has been developed. The activated olefin is reduced by ER at the expense of NAD(P)H cofactor, whose regeneration is carried out in situ by the glucose/glucose dehydrogenase system. Subsequently, the consumption of the co-substrate glucose is determined colorimetrically by a multienzymatic system. The FRED assay offers a wide range of possible applications, from enzyme fingerprinting and kinetic analysis, to primary screening of enzyme libraries and optimisation of ERs' performances under different reaction conditions.