Anti-inflammatory choline based ionic liquids: Insights into their lipophilicity, solubility and toxicity parametrites

The impact on in vivo efficacy and safety of two novel ionic liquids based on the association of choline with nonsteroidal anti-inflammatory drugs, ketoprofen and naproxen forming IL-APIs, was evaluated. Their lipophilicity, solubility and toxicity were assessed aiming the illustration of the pharmaceutical profile and potential toxic impact. Partition coefficientwas determined usingmicelles of hexadecylphosphocholine and UV–Vis derivative spectroscopy. Additionally, solubility in phosphate buffer pH 7.4 wasmeasured using amodified shake flaskmethod and UV–Vis spectroscopy as detection technique. Ultimately, toxicity was considered resorting to a fully automated cytochrome c oxidase assay based onmicrofluidics. The obtained results demonstrated that the IL-APIs' drug format has the ability to interact with biological membranes and also improves solubility up to 58 times. Moreover, it was evidenced that, although being a nutrient, choline influences the IL-APIs' toxicity. The studied anti-inflammatory IL-APIs exhibited promising properties regarding their incorporation in pharmaceutical formulations

Genetically engineered proteins with two active sites for enhanced biocatalysis and synergistic chemo- and biocatalysis

Enzyme engineering has allowed not only the de novo creation of active sites catalysing known biological reactions with rates close to diffusion limits, but also the generation of abiological sites performing new-to-nature reactions. However, the catalytic advantages of engineering multiple active sites into a single protein scaffold are yet to be established. Here, we report on proteins with two active sites of biological and/or abiological origin, for improved natural and non-natural catalysis. The approach increased the catalytic properties, such as enzyme efficiency, substrate scope, stereoselectivity and optimal temperature window, of an esterase containing two biological sites. Then, one of the active sites was metamorphosed into a metal-complex chemocatalytic site for oxidation and Friedel–Crafts alkylation reactions, facilitating synergistic chemo- and biocatalysis in a single protein. The transformations of 1-naphthyl acetate into 1,4-naphthoquinone (conversion approx. 100%) and vinyl crotonate and benzene into 3-phenylbutyric acid (≥83%; e.e. >99.9%) were achieved in one pot with this artificial multifunctional metalloenzyme.This work was funded by grant ‘INMARE’ from the European Union’s Horizon 2020 (grant agreement no. 634486), grants PCIN-2017-078 (within the Marine Biotechnology ERA-NET), CTQ2016-79138-R, BIO2016-76601-C3-1-R, BIO2016-76601-C3-3-R, BIO2017-85522-R, RTI2018-095166-B-I00 and RTI2018-095090-B-100 from the Ministerio de Economía y Competitividad, the Ministerio de Ciencia, Innovación y Universidades (MCIU), the Agencia Estatal de Investigación (AEI), the Fondo Europeo de Desarrollo Regional (FEDER) and the European Union (EU). P.N.G. and R.B. acknowledge the support of the UK Biotechnology and Biological Sciences Research Council (BBSRC; grant No. BB/M029085/1) and the Centre of Environmental Biotechnology Project and the Supercomputing Wales project, which are partly funded by the European Regional Development Fund (ERDF) through the Welsh Government. The authors gratefully acknowledge the financial support provided by the ERDF. C.C. thanks the Ministerio de Economía y Competitividad and FEDER for a Ph.D. fellowship (Grant BES-2015-073829). J.L.G.-A. thanks the support of the Spanish Ministry of Education, Culture and Sport through the National Program FPU (FPU17/00044). I.C.-R. thanks the Regional Government of Madrid for a fellowship (PEJ_BIO_AI_1201). The authors would like to acknowledge S. Ciordia and M. C. Mena for MALDI-TOF/TOF analysis. We thank the staff of both the European Synchrotron Radiation Facility (ESRF, Grenoble, France), for providing access and technical assistance at beamline ID30A-1/MASSIf-1, and the Synchrotron Radiation Source at Alba (Barcelona, Spain), for assistance at BL13-XALOC beamline. The authors would also like to acknowledge M. J. Vicente and M. A. Pascual at the Servicio Interdepartamental de Investigación (SIDI) of the Autonomous University of Madrid for the ESI-MS analyses

Transforming an esterase into an enantioselective catecholase through bioconjugation of a versatile metal-chelating inhibitor

4 pags., 3 figs.Metal complexes introduced into protein scaffolds can generate versatile biomimetic catalysts endowed with a variety of catalytic properties. Here, we synthesized and covalently bound a bipyridinyl derivative to the active centre of an esterase to generate a biomimetic catalyst that shows catecholase activity and enantioselective catalytic oxidation of (+)-catechin.We acknowledge the financial support of the European Union’s Horizon 2020 (GA 101000327), and the Ministerio de Ciencia e Innovacio´n and Agencia Estatal de Investigacio´n (AEI) (DOI 10.13039/501100011033), and the ‘‘NextGenerationEU/PRTR’’ (PID2020-112758RB-I00, PDC2021-121534-I00, TED2021-130544B-I00, PID2019-105838RB-C31, PID2019-105838RB-C33).Peer reviewe

Surface-active Ionic Liquids for Micellar Extraction of Piperine from Black Pepper

The final publication is available via https://doi.org/10.5560/znb.2013-3196.We present the application of ionic liquid-aqueous micellar solutions as isolation media for the pharmaceutically active ingredient piperine from black pepper. Several surface-active ionic liquids including a biodegradable betaine derivative were used for the extraction of piperine, and a strong correlation between extraction yield and the critical micelle concentration of the respective ionic liquid was found. A scaled strategy for the isolation of piperine was developed that allowed recovery and recycling of the aqueous ionic liquid micellar solution for five runs without any loss in extraction efficiency

Nicotinamide Adenine Dinucleotide-Dependent Redox-Neutral Convergent Cascade for Lactonizations with Type II Flavin-Containing Monooxygenase

A nicotinamide adenine dinucleotide (NADH)-dependent redox-neutral convergent cascade composed of a recently discovered type II flavin-containing monooxygenase (FMO-E) and horse liver alcohol dehydrogenase (HLADH) has been established. Two model reaction cascades were analyzed for the synthesis of g-butyrolactone and chiral bicyclic lactones. In the former cascade, all substrates were converted into one single product g-butyrolactone with high atom efficiency. More than 130 mM g-butyrolactone were obtained when applying 100 mM cyclobutanone and 50 mM 1,4-butanediol in this cascade. In the second cascade where bicyclo[4.2.0] octan-7-one and cis-1,2-cyclohexanedimethanol were coupled, the ketone substrate was converted to the corresponding normal lactone with an ee value of 89-74% (3aS, 7aS) by FMO-E alone and the abnormal lactone with an ee value of > 99% (3aR, 7aS) was formed by both HLADH and FMO-E

Manipulating the stereoselectivity of the thermostable Baeyer-Villiger monooxygenase TmCHMO by directed evolution

Baeyer-Villiger monooxygenases (BVMOs) and evolved mutants have been shown to be excellent biocatalysts in many stereoselective Baeyer-Villiger transformations, but industrial applications are rare which is partly due to the insufficient thermostability of BVMOs under operating conditions. In the present study, the substrate scope of the recently discovered thermally stable BVMO, TmCHMO from Thermocrispum municipale, was studied. This revealed that the wild-type (WT) enzyme catalyzes the oxidation of a variety of structurally different ketones with notable activity and enantioselectivity, including the desymmetrization of 4-methylcyclohexanone (99% ee, S). In order to induce the reversal of enantioselectivity of this reaction as well as the transformations of other substrates, directed evolution based on iterative saturation mutagenesis (ISM) was applied, leading to (R)-selectivity (94% ee) without affecting the thermostability of the biocatalyst