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Urease is an enzyme containing a dinuclear nickel active center responsible for the hydrolysis of urea into carbon dioxide and ammonia. Interestingly, inorganic models of urease are unable to mimic its mechanism despite their similarities to the enzyme active site. The reason behind the discrepancy in urea decomposition mechanisms between inorganic models and urease is still unknown. To evaluate this factor, we synthesized two bis-nickel complexes, [Ni2L(OAc)] (1) and [Ni2L(Cl)(Et3N)2] (2), based on the Trost bis-Pro-Phenol ligand (L) and encompassing different ligand labilities with coordination geometries similar to the active site of jack bean urease. Both mimetic complexes produced ammonia from urea, (1) and (2), were ten- and four-fold slower than urease, respectively. The presence and importance of several reaction intermediates were evaluated both experimentally and theoretically, indicating the aquo intermediate as a key intermediate, coordinating urea in an outer-sphere manner. Both complexes produced isocyanate, revealing an activated water molecule acting as a base. In addition, the reaction with different substrates indicated the biomimetic complexes were able to hydrolyze isocyanate. Thus, our results indicate that the formation of an outer-sphere complex in the urease analogues might be the reason urease performs a different mechanism.

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Novel functionalized indolines were synthesized from 2-(((N-aryl)amino)methyl)acrylates and formamides under ultrasonic irradiation for the first time. Aiming to develop a straightforward and easy-to-implement methodology for the synthesis of indolines, an instrumentation setup was designed, including ultrasound (US) equipment (Ultrasonic Horn; tip diameter of 12.7 mm, 20 kHz, maximum power of 400 W), an open reaction flask, and an inexpensive and green catalyst (1 mol%; FeSO4·7H2O; CAS: 7782-63-0) without the need for anhydrous conditions. The use of the sono-Fenton process in the presence of formamides and 2-(((N-aryl)amino)methyl)acrylates afforded a broad range of functionalized indolines within 60 s in high yields. Several experimental parameters of the ultrasound-assisted reaction were evaluated, such as amplitude (40-80%), sonication time (15-60 s), and pulsed ultrasonic irradiation. A 60 s silent reaction did not produce the desired indoline. The optimized conditions for US-mediated reactions allowed the production of functionalized indolines in high isolated yields (up to 99%, 60 s reaction, pulse ration 1 s:1 s, US amplitude 60 %).

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An ultrafast (10 s) methodology to construct novel highly functionalized 2-quinolinones from N-(o-ethynylaryl)acrylamides (1,7-enynes) is described for the first time. Microwave irradiation enabled the ultrafast synthesis of 2-quinolinone-fused γ-lactones from Fenton's reagents in formamide. After six key consecutive reactions, including a diastereoselective step, 2-quinolinone-fused γ-lactones were obtained in good overall yield (up to 46%; 10 s).

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Coixspirolactams, spiro[oxindole-γ-lactones], are found in adlay seeds and exhibit anticancer activity. A novel synthetic methodology was developed to enable an easy access to (±)-coixspirolactam A and a large number of new coixspirolactams in excellent overall yields. The exquisite exploitation of formamide reactivity was essential for the construction of oxindole and lactone scaffolds.

RESUMO

Several enzymatic reactions of heteroatom-containing compounds have been explored as unnatural substrates. Considerable advances related to the search for efficient enzymatic systems able to support a broader substrate scope with high catalytic performance are described in the literature. These reports include mainly native and mutated enzymes and whole cells biocatalysis. Herein, we describe the historical background along with the progress of biocatalyzed reactions involving the heteroatom(S, Se, B, P and Si) from hetero-organic substrates.

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The conversion of carbon dioxide into important industrial feedstock is a subject of growing interest in modern society. A possible way to achieve this goal is by carrying out the CO2/methanol cascade reaction, allowing the recycle of CO2 using either chemical catalysts or enzymes. Efficient and selective reactions can be performed by enzymes; however, due to their low stability, immobilization protocols are required to improve their performance. The cascade reaction to reduce carbon dioxide into methanol has been explored by the authors, using, sequentially, alcohol dehydrogenase (ADH), formaldehyde dehydrogenase (FalDH), and formate dehydrogenase (FDH), powered by NAD+/NADH and glutamate dehydrogenase (GDH) as the co-enzyme regenerating system. All the enzymes have been immobilized on functionalized magnetite nanoparticles, and their reactions investigated separately in order to establish the best performance conditions. Although the stepwise scheme led to only 2.3% yield of methanol per NADH; in a batch system under CO2 pressure, the combination of the four immobilized enzymes increased the methanol yield by 64 fold. The studies indicated a successful regeneration of NADH in situ, envisaging a real possibility of using immobilized enzymes to perform the cascade CO2-methanol reaction.

RESUMO

A novel electroactive macromonomer based on poly(l-lactic acid) (PLLA) with (3,4-ethylenedioxythiophene) (EDOT) functional end groups, was prepared by a traditional approach of organometallic polymerization with stannous octanoate [Sn(oct2)] and enzymatic polymerization using immobilized Candida antarctica Lipase B (CAL-B) and Amano lipase Pseudomonas cepacia(PS-IM), as catalysts. In the synthetic strategy, (2,3-dihydrothieno[3,4-b] dioxin-2-yl)methanol (EDOT-OH) was used to initiate the ring opening polymerization of lactide to yield PLLA with EDOT end group. All macromonomers (EDOT-PLLA) were characterized by 1H and 13C RMN, MALDI-TOF, GPC and EDX. Moreover, ICP-OES analysis showed the presence of Sn traces in the material synthesized by the traditional approach, but that pathway led to macromonomers with higher molecular weight while the enzymatic route led to completely metal-free macromonomers with medium and lower molecular weights. Also, electrochemical and chemical polymerization of EDOT-PLLA were tested showing that it is possible to prepare degradable conducting polymers based on poly(3,4-ethylenedioxythiphene) (PEDOT). The biocatalytic synthesis is a very promising and environmental friendly pathway for the preparation of biodegradable materials for short time applications.

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ABSTRACT The conversion of carbon dioxide into important industrial feedstock is a subject of growing interest in modern society. A possible way to achieve this goal is by carrying out the CO2/methanol cascade reaction, allowing the recycle of CO2 using either chemical catalysts or enzymes. Efficient and selective reactions can be performed by enzymes; however, due to their low stability, immobilization protocols are required to improve their performance. The cascade reaction to reduce carbon dioxide into methanol has been explored by the authors, using, sequentially, alcohol dehydrogenase (ADH), formaldehyde dehydrogenase (FalDH), and formate dehydrogenase (FDH), powered by NAD+/NADH and glutamate dehydrogenase (GDH) as the co-enzyme regenerating system. All the enzymes have been immobilized on functionalized magnetite nanoparticles, and their reactions investigated separately in order to establish the best performance conditions. Although the stepwise scheme led to only 2.3% yield of methanol per NADH; in a batch system under CO2 pressure, the combination of the four immobilized enzymes increased the methanol yield by 64 fold. The studies indicated a successful regeneration of NADH in situ, envisaging a real possibility of using immobilized enzymes to perform the cascade CO2-methanol reaction.

RESUMO

ABSTRACT Several enzymatic reactions of heteroatom-containing compounds have been explored as unnatural substrates. Considerable advances related to the search for efficient enzymatic systems able to support a broader substrate scope with high catalytic performance are described in the literature. These reports include mainly native and mutated enzymes and whole cells biocatalysis. Herein, we describe the historical background along with the progress of biocatalyzed reactions involving the heteroatom(S, Se, B, P and Si) from hetero-organic substrates.

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A novel and highly efficient synthetic approach to pyrroloindolines has been developed. The process is based on tandem radical addition/cyclization with inexpensive iron catalyst. This method tolerates a wide range of N-methyl-N-arylacrylamides as well carbamoyl radicals, providing access to a variety of functionalized 3,3-disubstituted oxindoles, key intermediates for many bioactive pyrroloindolines such as (±)-esermethole, (±)-deoxyeseroline, and (±)-physovenol methyl ether.

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Nanorattles, comprised of a nanosphere inside a nanoshell, were employed as the next generation of plasmonic catalysts for oxidations promoted by activated O2 . After investigating how the presence of a nanosphere inside a nanoshell affected the electric-field enhancements in the nanorattle relative to a nanoshell and a nanosphere, the SPR-mediated oxidation of p-aminothiophenol (PATP) functionalized at their surface was investigated to benchmark how these different electric-field intensities affected the performances of Au@AgAu nanorattles, AgAu nanoshells and Au nanoparticles having similar sizes. The high performance of the nanorattles enabled the visible-light driven synthesis of azobenzene from aniline under ambient conditions. As the nanorattles allow the formation of electromagnetic hot spots without relying on the uncontrolled aggregation of nanostructures, it enables their application as catalysts in liquid phase under mild conditions using visible light as the main energy input.

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The importance of chiral compounds in all fields of technology and life sciences is shown. Small chiral molecules are mainly used as building blocks in the synthesis of more complex and functionalized compounds. Nature creates and imposes stereoselectivity by means of enzymes, which are highly efficient biocatalysts. The use of whole cells as a biocatalyst source is a promising strategy for avoiding some drawbacks associated with the use of pure enzymes, especially their high cost. The use of free cells is also challenging, since cell lysis can also occur under the reaction conditions. However, cell immobilization has been employed to increase the catalytic potential of enzymes by extending their lifetimes in organic solvents and non-natural environments. Besides, immobilized cells maintain their biocatalytic performance for several reaction cycles. Considering the above-mentioned arguments, several authors have synthesized different classes of chiral compounds such as alcohols, amines, carboxylic acids, amides, sulfides and lactones by means of immobilized cells. Our aim was to discuss the main aspects of the production of chiral compounds using immobilized cells as a source of biocatalysts, except under fermentation conditions.

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Freshwater contamination usually comes from runoff water or direct wastewater discharges to the environment. This paper presents a case study which reveals the impact of these types of contamination on the sediment bacterial population. A small stretch of Lerma River Basin, heavily impacted by industrial activities and urban wastewater release, was studied. Due to industrial inputs, the sediments are characterized by strong hydrocarbon concentrations, ranging from 2 935 to 28 430µg·kg(-1) of total polyaromatic hydrocarbons (PAHs). These sediments are also impacted by heavy metals (e.g., 9.6µg·kg(-1) of Cd and 246µg·kg(-1) of Cu, about 8 times the maximum recommended values for environmental samples) and polychlorinated biphenyls (ranging from 54 to 123µg·kg(-1) of total PCBs). The bacterial diversity on 6 sediment samples, taken from upstream to downstream of the main industrial and urban contamination sources, was assessed through TRFLP. Even though the high PAH concentrations are hazardous to aquatic life, they are not the only factor driving bacterial community composition in this ecosystem. Urban discharges, leading to hypoxia and low pH, also strongly influenced bacterial community structure. The bacterial bioprospection of these samples, using PAH as unique carbon source, yielded 8 hydrocarbonoclastic strains. By sequencing the 16S rDNA gene, these were identified as similar to Mycobacterium goodii, Pseudomonas aeruginosa, Pseudomonas lundensis or Aeromonas veronii. These strains showed high capacity to degrade naphthalene (between 92 and 100% at 200mg·L(-1)), pyrene (up to 72% at 100mg·L(-1)) and/or fluoranthene (52% at 50mg·L(-1)) as their only carbon source on in vitro experiments. These hydrocarbonoclastic bacteria were detected even in the samples upstream of the city of Salamanca, suggesting chronical contamination, already in place longer before. Such microorganisms are clearly potential candidates for hydrocarbon degradation in the treatment of oil discharges.

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The hypervalent selenium- and tellurium-containing compounds (halo-organoselenuranes and halo-organotelluranes) were treated with amino acids to evaluate their reactivity and chemoselectivity by (1)H, (13)C, (77)Se and (125)Te NMR spectroscopy. The study of forced thermal stability was performed and analyzed by NMR. The organotelluranes remained stable at temperatures around 60 °C but in the case of organoselenuranes, there was formation of new products at 37 °C as a result of halogen loss. (77)Se and (125)Te NMR spectroscopy has proved to be a very efficient and fast technique to evidence the high selectivity of organochalcogenanes against l-amino acids, specific to l-cysteine.

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A series of hypervalent selenium- and tellurium-containing compounds (organoselenuranes and organotelluranes) was evaluated aiming novel inhibitors of a threonine protease, namely the 20S proteasome (20S PT). In vitro assays demonstrated high inhibitory potency and specificity of these compounds toward the ß2 catalytic site of the 20S PT. Organotelluranes were identified as more potent inhibitors than organoselenuranes since their IC50 ranged from 3.5 to 16 µM while for organoselenuranes the IC50 ranged from 16 to 35 µM indicating great potential to be explored in 20S proteasome inhibition. Cellular assays with those compounds were employed to verify the cytotoxicity and ability to inhibit 20S proteasome in cell. These assays demonstrated that organoselenuranes are capable of maintaining their selectivity in cell while the organotelluranes became inactive under cellular conditions. Stability studies of the organochalcogenanes were performed by (77)Se and (125)Te NMR analysis. It was observed that organotelluranes are stable under enzymatic assay conditions and, organoselenuranes, the structures responsible for inhibitory activity are cyclized organoselenuranes.

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We employed thiol-funcionalized AgAu nanoshells (AgAu NSs) as supports for the covalent attachment of lipases (BCL, Burkholderia cepacia lipase; PPL, pancreatic porcine lipase). Specifically, we were interested in investigating the effect of the nature/size of the spacer in AgAu NSs-functionalized organic thiols over the covalent attachment of lipases. The catalytic performance of AgAu-lipase systems was measured in the kinetic resolution of (R,S)-1-(phenyl)ethanol via a transesterification reaction. In comparison to free BCL, the lipase attached to AgAu NSs using a small spacer such as cysteamine or mercaptoacetic acid, with the largest spacer mercaptoundecanoic acid, had the fastest conversion rate. The recycling potential for BCL was investigated. After three reaction cycles, the enzyme activity was kept at around 90% of the initial value. The results described herein show that the size of the spacer plays an important role in optimizing lipase activities in metallic nanoshells as solid supports.

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Lipase-catalyzed kinetic resolution of aryltrimethylsilyl chiral alcohols through a transesterification reaction was studied. The optimal conditions found for the kinetic resolution of m- and p-aryltrimethylsilyl chiral alcohols, led to excellent results, high conversions (c = 50%), high enantiomeric ratios (E > 200) and enantiomeric excesses for the remaining (S)-alcohol and (R)-acetylated product (>99%). However, kinetic resolution of o-aryltrimethylsilyl chiral alcohols did not occur under the same conditions applied to the other isomers.

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The enzymatic kinetic resolution of tert-butyl 2-(1-hydroxyethyl) phenylcarbamate via lipase-catalyzed transesterification reaction was studied. We investigated several reaction conditions and the carbamate was resolved by Candida antarctica lipase B (CAL-B), leading to the optically pure (R)- and (S)-enantiomers. The enzymatic process showed excellent enantioselectivity (E > 200). (R)- and (S)-tert-butyl 2-(1-hydroxyethyl)phenylcarbamate were easily transformed into the corresponding (R)- and (S)-1-(2-aminophenyl)ethanols.

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Several microorganisms were isolated from soil/sediment samples of Antarctic Peninsula. The enrichment technique using (RS)-1-(phenyl)ethanol as a carbon source allowed us to isolate 232 psychrophile/psychrotroph microorganisms. We also evaluated the enzyme activity (oxidoreductases) for enantioselective oxidation reactions, by using derivatives of (RS)-1-(phenyl)ethanol as substrates. Among the studied microorganisms, 15 psychrophile/psychrotroph strains contain oxidoreductases that catalyze the (S)-enantiomer oxidation from racemic alcohols to their corresponding ketones. Among the identified microorganisms, Flavobacterium sp. and Arthrobacter sp. showed excellent enzymatic activity. These new bacteria strains were selected for optimization study, in which the (RS)-1-(4-methyl-phenyl)ethanol oxidation was evaluated in several reaction conditions. From these studies, it was observed that Flavobacterium sp. has an excellent enzymatic activity at 10 °C and Arthrobacter sp. at 15 and 25 °C. We have also determined the growth curves of these bacteria, and both strains showed optimum growth at 25 °C, indicating that these bacteria are psychrotroph.

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A new series of organotelluranes were synthesized and investigated, and the structure-activity relationships in cysteine proteases inhibition were determined. It was possible to identify the relevance of structural components linked to the reactivity of these compounds as inhibitors. For example, dibromo-organotelluranes showed to be more reactive than dichloro-organotelluranes towards cysteine cathepsins V and S. Besides, no remarkable enantio-selectivity was verified. In general the achiral organotelluranes were more reactive than the chiral congeners against cysteine cathepsins V and S. A reactivity order for organochalcogenanes and cysteine cathepsins was proposed after the comparison of the inhibitory potencies of organotelluranes with the related organoselenanes.

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