Simple Substrates Research Articles

Synthesis of Chiral, Fused Tricyclic Molecules by Sequential Metal‐Catalyzed Reactions of Simple Substrates

AbstractA set of complex tricyclic compounds containing several functional groups and multiple stereogenic centers has been prepared in only two steps with excellent diastereo‐ and enantioselectivities. The synthesis takes advantage of the highly versatile and enantioselective Pd‐catalyzed allylic substitution of simple cyclic allylic acetates or carbonates to form chiral 1,6‐, 1,7‐ and 1,8‐enynes, which are then diastereoselectively transformed to the corresponding cyclopentenone‐ and cyclobutene‐based tricyclic compounds.magnified image

Development of an innovative flexible paper-based methanol fuel cell (PB-DMFC) sensing platform – Application to sarcosine detection

This work describes for the first time a paper-based direct methanol fuel cell platform (PB-DMFC) that functions as an energy source and biosensor, assembled on a simple paper substrate for point-of-care (POC) applications, targeting sarcosine as proof-of-concept.Specifically, a methanol fuel cell strip was developed from a square of Whatman paper, acting as substrate. The paper strip was treated with an impermeable agent (paraffin solution) and supported all fuel cell device components, including the electrolyte (Nafion®), anode electrode (carbon black Pt/Ru), cathode electrode (carbon black Pt), and current collectors (silver edges). All the described components formed a flexible single layer that operated in a completely passive mode by adding few microliters of a methanol solution on the anode side and by using atmospheric oxygen on the cathode side. The obtained platform had a stable electrical signal with an average OCV value of 0.45–0.55 V and a maximum power density of 20–50 µW/cm2, depending on the methanol concentration used (0.5 M–2 M). A sensing layer was built in situ on the anode electrode by electropolymerization of a solution of 3,4-ethylenedioxythiophene (EDOT) and pyrrole (Py) as monomers. The obtained PB-DMFC/biosensor was calibrated at room temperature in buffer and healthy human urine and showed linear responses from 1.0 × 10−7 to 1.0 × 10−3 M with a detection limit of 6.6 × 10−8 M. Selectivity studies evidenced signals changing within 1–10%, both in positive and negative directions. Results evidenced good reproducibility.Overall, the obtained results demonstrate a self-sufficient biosensor for the detection of sarcosine consisting of an innovative paper-based methanol fuel cell strip. This concept can open new horizons for massification of biosensors even in places with energy shortage.

Porous Au/AAO: A simple and feasible SERS substrate for dynamic monitoring and mechanism analysis of DNA oxidation

Herein, a simple strategy was proposed to acquire an orderly and controllable periodic structure. Firstly, the optimal parameters of porous anodic aluminum oxide (AAO) were screened out by FDTD solutions. Combined with magnetron sputtering of gold nano-film, the Au/AAO SERS substrate was thus fabricated. The enhancement factor (EF) was calculated to be 4.87 × 106, and SERS spectra with good reproducibility were also acquired. The oxidization process of plasmid DNA was thus monitored under the conditions of low detection power and short integration time. Fenton's reagent was used as oxidizing reagent, and Na2SO4 was applied for the internal standard in SERS analysis. The changes of I657/I998, I723/I998, I1587/I998, I1648/I998 were studied over time. DNA oxidation product 8-OHdG was also detected in this process. Then the relationship between the relative peak intensity ratios and the base structure change in DNA was established. The conclusions of SERS spectral analysis were verified by agarosegelelectrophoresis, DNA oxidative damage gel detection and colorimetric detection of 8-OHdG. The mechanism analysis of DNA oxidation process was thus studied. The simple and feasible detection strategy proposed could extend the application of SERS technology in the process monitoring of photosensitive or other unstable biological samples.

Enantioselective Biocascade Catalysis with a Single Multifunctional Enzyme.

Asymmetric catalytic cascade processes offer direct access to complex chiral molecules from simple substrates and in a single step. In biocatalysis, cascades are generally designed by combining multiple enzymes, each catalyzing individual steps of a sequence. Herein, we report a different strategy for biocascades based on a single multifunctional enzyme that can promote multiple stereoselective steps of a domino process by mastering distinct catalytic mechanisms of substrate activation in a sequential way. Specifically, we have used an engineered 4-oxalocrotonate tautomerase (4-OT) enzyme with the ability to form both enamines and iminium ions and combine their mechanisms of catalysis in a complex sequence. This approach allowed us to activate aldehydes and enals toward the synthesis of enantiopure cyclohexene carbaldehydes. The multifunctional 4-OT enzymes could promote both a two-component reaction and a triple cascade characterized by different mechanisms and activation sequences.

Cultivation of previously uncultured microorganisms with a continuous-flow down-flow hanging sponge (DHS) bioreactor, using a syntrophic archaeon culture obtained from deep marine sediment as a case study.

In microbiology, cultivation is a central approach for uncovering novel physiology, ecology, and evolution of microorganisms, but conventional methods have left many microorganisms found in nature uncultured. To overcome the limitations of traditional methods and culture indigenous microorganisms, we applied a two-stage approach: enrichment/activation of indigenous organisms by using a continuous-flow down-flow hanging sponge bioreactor and subsequent selective batch cultivation. Here, we provide a protocol for this bioreactor-mediated technique using activation of deep marine sediment microorganisms and downstream isolation of a syntrophic co-culture containing an archaeon closely related to the eukaryote ancestor (Candidatus Promethearchaeum syntrophicum strain MK-D1) as an example. Both stages can easily be tailored to target other environments and organisms by modifying the inoculum, feed solution/gases, attachment material and/or cultivation media. We anaerobically incubate polyurethane sponges inoculated with deep-sea methane seep sediment in a reactor at 10 °C and feed anaerobic artificial seawater medium and methane. Once phylogenetically diverse and metabolically active microorganisms are adapted to synthetic conditions in the reactor, we transition to growing community samples in glass tubes with the above medium, simple substrates and selective compounds (e.g., antibiotics). To accommodate for the slow growth anticipated for target organisms, primary cultures can be incubated for ≥6-12 months and analyzed for community composition even when no cell turbidity is observed. One casamino acid- and antibiotic-amended culture prepared in this way led to the enrichment of uncultured archaea. Through successive transfer in vitro combined with molecular growth monitoring, we successfully obtained the target archaeon with its partner methanogen as a pure syntrophic co-culture.

Enantioselective Biocascade Catalysis with a Single Multifunctional Enzyme

AbstractAsymmetric catalytic cascade processes offer direct access to complex chiral molecules from simple substrates and in a single step. In biocatalysis, cascades are generally designed by combining multiple enzymes, each catalyzing individual steps of a sequence. Herein, we report a different strategy for biocascades based on a single multifunctional enzyme that can promote multiple stereoselective steps of a domino process by mastering distinct catalytic mechanisms of substrate activation in a sequential way. Specifically, we have used an engineered 4‐oxalocrotonate tautomerase (4‐OT) enzyme with the ability to form both enamines and iminium ions and combine their mechanisms of catalysis in a complex sequence. This approach allowed us to activate aldehydes and enals toward the synthesis of enantiopure cyclohexene carbaldehydes. The multifunctional 4‐OT enzymes could promote both a two‐component reaction and a triple cascade characterized by different mechanisms and activation sequences.

A targeted metabolomics method for extra- and intracellular metabolite quantification covering the complete monolignol and lignan synthesis pathway

Microbial synthesis of monolignols and lignans from simple substrates is a promising alternative to plant extraction. Bottlenecks and byproduct formation during heterologous production require targeted metabolomics tools for pathway optimization.In contrast to available fractional methods, we established a comprehensive targeted metabolomics method. It enables the quantification of 17 extra- and intracellular metabolites of the monolignol and lignan pathway, ranging from amino acids to pluviatolide. Several cell disruption methods were compared. Hot water extraction was best suited regarding monolignol and lignan stability as well as extraction efficacy. The method was applied to compare enzymes for alleviating bottlenecks during heterologous monolignol and lignan production in E. coli. Variants of tyrosine ammonia-lyase had a considerable influence on titers of subsequent metabolites. The choice of multicopper oxidase greatly affected the accumulation of lignans. Metabolite titers were monitored during batch fermentation of either monolignol or lignan-producing recombinant E. coli strains, demonstrating the dynamic accumulation of metabolites.The new method enables efficient time-resolved targeted metabolomics of monolignol- and lignan-producing E. coli. It facilitates bottleneck identification and byproduct quantification, making it a valuable tool for further pathway engineering studies. This method will benefit the bioprocess development of biotransformation or fermentation approaches for microbial lignan production.

EVALUATION OF SUBSTRATES IN THE EMERGENCE AND INITIAL GROWTH OF Cryptostegia madagascariensis Bojer ex Decne (PERIPLOCOIDEAE, APOCYNACEAE)


 madagascariensis is an invasive species highly adapted to the semiarid region, being widely disseminated through water courses by the wind and fostered by man, particularly for its popularity as an ornamental plant, due to its permanent foliage and intense lilac flowering. The objective of this work was to evaluate the germination and initial development of the gay widow in different substrates. To this end, an Entirely Matched Design (DIC) trial was set up, with 12 treatments, which consisted of the types of substrates (sand; soil; goat manure; bagana; coconut dust; sand + manure; sand + coconut dust; sand + bagana; soil + manure; soil + coconut dust; soil + bagana and soil + manure + bagana) in 4 repetitions of 16 seeds. Styrofoam trays with 128 cells were used, containing the respective substrates, with one seed per cell being sown. At 20 days after sowing, the final evaluation was carried out, measuring: percentage of emergence, plant height; number of leaves; stem diameter; root length; shoot dry weight; root dry weight; Dickson's quality score. With the results, it was observed that the substrates bagana and coconut powder provided better results for germination. In the simple substrates bagana and coconut dust and in the mixtures sand + goat manure, sand + coconut dust and soil + coconut dust, the most expressive values ​​were observed for all the variables studied.

Understanding and Circumventing the Requirement for Native Thioester Substrates for α-Oxoamine Synthase Reactions.

Many enzyme classes require thioester electrophiles such as acyl-carrier proteins and acyl-coenzyme A substrates. For in vitro applications, these substrates can render these chemical transformations impractical. To address this challenge, we have investigated the mechanism of coenzyme A in gating catalysis of one α-oxoamine synthase, SxtA AOS. Through investigating the reactivity of SxtA AOS and corresponding enzyme variants against a panel of substrates and coenzyme A mimics, we determined that activity is gated through the binding of the pantetheine arm and a phosphate group that hydrogen bonds to residue Lys154 that is predicted by an AlphaFold2 model to be located in a tunnel leading to the active site. To provide an economical solution for preparative-scale reactions, in situ transthioesterification was used with pantetheine and simple thioester substrate precursors, resulting in productive reactions. These findings outline a strategy for employing ACP- and CoA-dependent enzymes that are inaccessible through other means without the need for cost-prohibitive coenzyme A or carrier protein-activated substrates.

Rhodium-catalyzed synthesis of 1-silabenzonorbornenes via 1,4-rhodium migration

An efficient synthesis of 1-silabenzonorbornenes was developed from a simple substrate combination of alkynyl(2-alkylallyl)silanes and arylboron reagents under rhodium catalysis through the use of 1,4-rhodium migration. Three carbon–carbon bonds were newly formed during the catalytic process and the reaction pathway was probed by a deuterium labeling experiment as well.

Global protein dynamics as communication sensors in peptide synthetase domains.

Biological activity is governed by the timely redistribution of molecular interactions, and static structural snapshots often appear insufficient to provide the molecular determinants that choreograph communication. This conundrum applies to multidomain enzymatic systems called nonribosomal peptide synthetases (NRPSs), which assemble simple substrates into complex metabolites, where a dynamic domain organization challenges rational design to produce new pharmaceuticals. Using a nuclear magnetic resonance (NMR) atomic-level readout of biochemical transformations, we demonstrate that global structural fluctuations help promote substrate-dependent communication and allosteric responses, and impeding these global dynamics by a point-site mutation hampers allostery and molecular recognition. Our results establish global structural dynamics as sensors of molecular events that can remodel domain interactions, and they provide new perspectives on mechanisms of allostery, protein communication, and NRPS synthesis.

Substrate complexity affects the prevalence and interconnections of antibiotic, metal and biocide resistance genes, integron-integrase genes, human pathogens and virulence factors in anaerobic digestion

Anaerobic digestion (AD) is widely used to treat livestock manure that harbors diverse pollutants (resistance genes (ARGs), metal/biocide resistance genes (MBRGs), integron-integrase genes, human pathogens and pathogen virulence factors (VFs)). However, the interplays of these pollutants and the effects of substrate complexity on pollutants in AD are elusive. This study investigated the dynamics of these pollutants and bacterial communities during AD of swine manure, by metatranscriptomic sequencing and amplicon sequencing of 16 S rRNA and 16 S rRNA gene. The pollutant profiles and bacterial communities differed across AD processes, nevertheless with consistent dominance of ARGs of multi-drugs, tetracycline, aminoglycoside and rifamycin, MBRGs of multi-biocides, multi-metals, copper and arsenic, the integron-integrase gene intI1, potential pathogens of Escherichia coli, Streptococcus gallolyticus and Clostridium perfringens, VFs involved in pathogen adherence, and bacterial phyla of Firmicutes, Bacteroidetes and Proteobacteria. Reduced substrate complexity (replacing a part of swine manure, a complex substrate, with a simple substrate, apple waste or fructose) decreased the prevalence and stochastic turnover of ARGs and MBRGs. Network analyses revealed decreased interplays among pollutants under reduced substrate complexity. Our findings provide a mechanical understanding of diverse pollutants dynamics during AD, and reveal the importance of substrate complexity in controlling prevalence and interplays of pollutants.

Ruthenium-Catalyzed Geminal Hydroborative Cyclization of Enynes.

Disclosed here is the first geminal (gem-) hydroborative cyclization of enynes. Different from known hydroborative cyclizations, this process adds hydrogen and boron to the same position, leading to a new reaction mode. With [Cp*RuCl]4 as catalyst, a range of gem-hydroborated bicyclic products bearing a cyclopropane unit could be rapidly assembled from simple enyne substrates. Control experiments and density functional theory (DFT) calculations provided important insights into the reaction mechanism. Notably, two major competing pathways may operate with substrate-dependence. 1,6-Enynes favor initial oxidative cyclometalation to form a ruthenacyclopentene intermediate prior to engaging hydroborane, while other enynes (e.g., 1,7-enynes) that lack strong propensity toward cyclization prefer initial alkyne gem-(H,B)-addition to form an α-boryl ruthenium carbene followed by intramolecular olefin cyclopropanation. This process also represents the first ruthenium-catalyzed enyne hydroborative cyclization.

Ruthenium‐Catalyzed Geminal Hydroborative Cyclization of Enynes

AbstractDisclosed here is the first geminal (gem‐)hydroborative cyclization of enynes. Different from known hydroborative cyclizations, this process adds hydrogen and boron to the same position, leading to a new reaction mode. With [Cp*RuCl]4as catalyst, a range ofgem‐hydroborated bicyclic products bearing a cyclopropane unit could be rapidly assembled from simple enyne substrates. Control experiments and density functional theory (DFT) calculations provided important insights into the reaction mechanism. Notably, two major competing pathways may operate with substrate‐dependence. 1,6‐Enynes favor initial oxidative cyclometalation to form a ruthenacyclopentene intermediate prior to engaging hydroborane, while other enynes (e.g., 1,7‐enynes) that lack strong propensity toward cyclization prefer initial alkynegem‐(H,B)‐addition to form an α‐boryl ruthenium carbene followed by intramolecular olefin cyclopropanation. This process also represents the first ruthenium‐catalyzed enyne hydroborative cyclization.

Bioproduction of eriodictyol by Escherichia coli engineered co-culture

Eriodictyol (ED) is a flavonoid in the flavanones subclass. It is abundantly present in a wide range of medicinal plants, citrus fruits, and vegetables. In addition, ED owns numerous importantly medicinal bioactivities such as inhibition of proliferation, metastasis and induction of apoptosis in glioma cells or inhibition of glioblastoma migration, and invasion. This study described the heterologous production of ED by E. coli based co-culture engineering system from the simple carbon substrate D-glucose. Two E. coli strains were engineered and functioned as constitutive components of biological system. Specifically, the first strain (upstream module) contained genes for synthesis of p-coumaric acid (pCA) from D-glucose. And, the second strain (downstream module) consisted of genes for the synthesis of ED from pCA. The highest yield in ED production was achieved 51.5 ± 0.4mg/L using stepwise optimal culture conditions, while monoculture was achieved 21.3 ± 0.2mg/L only. In conclusion, co-culture was the most efficient alternative approach for the synthesis of ED and other natural products.

Comparative Experimental and Computational Studies of Hydroxyl and Sulfate Radical-Mediated Degradation of Simple and Complex Organic Substrates.

Persulfate (PS)-based advanced oxidation processes (AOPs) have been promoted as alternatives to H2O2-based AOPs. To gauge the potential of this technology, the PS/Fe(II) and Fenton (H2O2/Fe(II)) processes were comparatively evaluated using formate as a simple target compound and nanofiltration concentrate from a municipal wastewater treatment plant as a complex suite of contaminants with the aid of kinetic modeling. In terms of the short-term rate and extent of mineralization of formate and the nanofiltration concentrate, PS/Fe(II) is less effective due to slow Fe(II)/Fe(III) cycling attributable to the scavenging of superoxide by PS. However, in the concentrate treatment, PS/Fe(II) provided a sustained removal of total organic carbon (TOC), with ∼81% removed after 7 days with SO4•- consistently produced via homolysis of the long-life PS. In comparison, H2O2/Fe(II) exhibited limited TOC removal over ∼57% after 10 h due to the futile consumption of H2O2 by HO•. PS/Fe(II) also offers better performance at transforming humic-like moieties to more biodegradable compounds as a result of chlorine radicals formed by the reaction of SO4•- with the matrix constituents present in the concentrate. The application of PS/Fe(II) is, however, subject to the limitations of slow oxidation of organic contaminants, release of sulfate, and formation of chlorinated byproducts.

Optimizing volatile fatty acid production from municipal sludge: Linking microbial activity with reactor performance

Literature on optimum levels and combinations of bioreactor operating parameters to maximize volatile fatty acids (VFAs) via anaerobic digestion (AD) is still scant and contradictory. This study investigated the interactive impact of digester sludge retention time (SRT), temperature, and pH on optimizing VFAs production and linking reactor performance to acidogenic activities. VFAs production from municipal sludge was maximized in semi-continuous flow acid fermenters (AFs). Batch fermentation tests were utilized for the specific acidogenesis activity assays (SAdA) with a simpler substrate (glucose and acetic acid) and inocula from AFs. Fermenters operated at high pH of 8, low SRT of 2 days, and varying temperatures of 55 and 45 °C had the highest chemical oxygen demand (COD) solubilization and acidification yields. Similar fermenters had the highest VFAs fold increase (1.7–2.2) over the substrate, largest acetic acid contribution to total VFAs (52–58%), and maximum SAdA extent (368–461 mL H2/g VSS inoculum). Maximum hydrogen production rate indirectly represented substrate utilization rate, growth of the acidogens, and hence the specific activities of the acidogenic bacteria in the AFs. These results suggested that batch acidogenic assays could provide quantitative information regarding the presence and activities of cultures in continuously-fed AD utilizing more complex substrates, e.g. municipal sludge. A multiple linear regression model was derived successfully predicting VFAs fold increase of AFs with an R-squared value of 0.9999. The constructed contour plots predicted higher VFAs at relatively high fermenter pH (7.5–8.0), lower SRT (2–2.2 days), and lower temperatures (45–48 °C).

Iridium(III)-Catalyzed Asymmetric Site-Selective Carbene C-H Insertion during Late-Stage Transformation.

C-H functionalization has recently received considerable attention because C-H functionalization during the late-stage transformation is a strong and useful tool for the modification of the bioactive compounds and the creation of new active molecules. Although a carbene transfer reaction can directly convert a C-H bond to the desired C-C bond in a stereoselective manner, its application in late-stage material transformation is limited. Here, we observed that the iridium-salen complex 6 exhibited efficient catalysis in asymmetric carbene C-H insertion reactions. Under optimized conditions, benzylic, allylic, and propargylic C-H bonds were converted to desired C-C bonds in an excellent stereoselective manner. Excellent regioselectivity was demonstrated in the reaction using not only simple substrate but also natural products, bearing multiple reaction sites. Moreover, based on the mechanistic studies, the iridium-catalyzed unique C-H insertion reaction involved rate-determining asynchronous concerted processes.

Construction of Zn-heptapeptide bionanozymes with intrinsic hydrolase-like activity for degradation of di(2-ethylhexyl) phthalate

Nanozyme with intrinsic enzyme-like activity has emerged as favorite artificial catalyst during recent years. However, current nanozymes are mainly limited to inorganic-derived nanomaterials, while biomolecule-sourced nanozyme (bionanozyme) are rarely reported. Herein, inspired by the basic structure of natural hydrolase family, we constructed 3 oligopeptide-based bionanozymes with intrinsic hydrolase-like activity by implementing zinc induced self-assembly of histidine-rich heptapeptides. Under mild condition, divalent zinc (Zn2+) impelled the spontaneous assembly of short peptides (i.e. Ac-IHIHIQI-CONH2, Ac-IHIHIYI-CONH2, and Ac-IHVHLQI-CONH2), forming hydrolase-mimicking bionanozymes with β-sheet secondary conformation and nanofibrous architecture. As expected, the resultant bionanozymes were able to hydrolyze a serious of p-nitrophenyl esters, including not only the simple substrate with short side-chain (p-NPA), but also more complicated ones (p-NPB, p-NPH, p-NPO, and p-NPS). Moreover, the self-assembled Zn-heptapeptide bionanozymes were also proven to be capable of degrading di(2-ethylhexyl) phthalate (DEHP), a typical plasticizer, showing great potential for environmental remediation. Based on this study, we aim to provide theoretical references and exemplify a specific case for directing the construction and application of bionanozyme.

Modeling of biohydrogen production using generalized multi-scale kinetic model: Impacts of fermentation conditions

This paper presents a new multi-scale kinetic model built upon the multi-stage growth Hypothesis for predicting biohydrogen production. The proposed model represents the significant factors affecting biohydrogen production using a sum of first-order kinetic terms with varying dynamics from slow to fast one. The current work investigates 52 case studies of biohydrogen production that show the double first-order kinetic model provides the best modeling fitness (R2 > 0.99). This result suggests two prevalent pathways or microbial groups with distinct dynamics (i.e., fast and slow modes) in biohydrogen production. An increase in temperature (30 °C–43 °C) or substrate concentration (10 g/L to 40 g/L) and the use of simple substrates or mixed cultures can increase the fast-mode dominance up to 100% contribution. Model analysis suggests that the fast mode corresponds to the butyrate production pathway, the growth-associated hydrogen-producing activity, the easily-biodegradable substrates, or the quick hydrogen-producing groups.