Substrate Urea Research Articles

Organic Synthesis Away from Equilibrium: Contrathermodynamic Transformations Enabled by Excited-State Electron Transfer.

ConspectusChemists have long been inspired by biological photosynthesis, wherein a series of excited-state electron transfer (ET) events facilitate the conversion of low energy starting materials such as H2O and CO2 into higher energy products in the form of carbohydrates and O2. While this model for utilizing light-driven charge transfer to drive catalytic reactions thermodynamically "uphill" has been extensively adapted for small molecule activation, molecular machines, photoswitches, and solar fuel chemistry, its application in organic synthesis has been less systematically developed. However, the potential benefits of these approaches are significant, both in enabling transformations that cannot be readily achieved using conventional thermal chemistry and in accessing distinct selectivity regimes that are uniquely enabled by excited-state mechanisms. In this Account, we present work from our group that highlights the ability of visible light photoredox catalysis to drive useful organic transformations away from their equilibrium positions, addressing a number of long-standing synthetic challenges.We first discuss how excited-state ET enabled the first general methods for the catalytic anti-Markovnikov hydroamination of unactivated alkenes with alkyl amines. In these reactions, an excited-state iridium(III) photocatalyst reversibly oxidizes secondary amine substrates to their corresponding aminium radical cations (ARCs). These electrophilic N-centered radicals can then react with olefins to furnish valuable tertiary amine products with complete anti-Markovnikov regioselectivity. Notably, some of these products are less thermodynamically stable than their corresponding amine and alkene starting materials. We next present a strategy for light-driven C-C bond cleavage within various aliphatic alcohols mediated by homolytic activation of alcohol O-H bonds by excited-state proton-coupled electron transfer (PCET). The resulting alkoxy radical intermediates then undergo C-C β-scission to ultimately provide isomeric linear carbonyl products that are often higher in energy than their cyclic alcohol precursors. Applications of this chemistry for the light-driven depolymerization of lignin biomass, commercial phenoxy resin, hydroxylated polyolefin derivatives, and thermoset polymers are presented as well. We then describe a method for the contrathermodynamic positional isomerization of highly substituted olefins by means of cooperative photoredox and chromium(II) catalysis. In this work, generation of an allylchromium(III) species that can undergo highly regioselective in situ protodemetalation enables access to a less substituted and thermodynamically less stable positional isomer. Product selectivity in this reaction is determined by the large differential in oxidation potentials between differently substituted olefin isomers. Lastly, we discuss a light-driven deracemization reaction developed in collaboration with the Miller group, wherein a racemic urea substrate undergoes spontaneous optical enrichment upon visible light irradiation in the presence of an iridium(III) chromophore, a chiral Brønsted base, and a chiral peptide thiol. Excellent levels of enantioselectivity are achieved via sequential and synergistic proton transfer (PT) and H atom transfer (HAT) steps. Taken together, these examples highlight the ability of excited-state ET events to enable access to nonequilibrium product distributions across a wide range of catalytic, redox-neutral transformations in which photons are the only stoichiometric reagents.

Artificial cells with viscoadaptive behavior based on hydrogel-loaded giant unilamellar vesicles.

Viscoadaptation is an essential process in natural cells, where supramolecular interactions between cytosolic components drive adaptation of the cellular mechanical features to regulate metabolic function. This important relationship between mechanical properties and function has until now been underexplored in artificial cell research. Here, we have created an artificial cell platform that exploits internal supramolecular interactions to display viscoadaptive behavior. As supramolecular material to mimic the cytosolic component of these artificial cells, we employed a pH-switchable hydrogelator based on poly(ethylene glycol) coupled to ureido-pyrimidinone units. The hydrogelator was membranized in its sol state in giant unilamellar lipid vesicles to include a cell-membrane mimetic component. The resulting hydrogelator-loaded giant unilamellar vesicles (designated as HL-GUVs) displayed reversible pH-switchable sol-gel behavior through multiple cycles. Furthermore, incorporation of the regulatory enzyme urease enabled us to increase the cytosolic pH upon conversion of its substrate urea. The system was able to switch between a high viscosity (at neutral pH) and a low viscosity (at basic pH) state upon addition of substrate. Finally, viscoadaptation was achieved via the incorporation of a second enzyme of which the activity was governed by the viscosity of the artificial cell. This work represents a new approach to install functional self-regulation in artificial cells, and opens new possibilities for the creation of complex artificial cells that mimic the structural and functional interplay found in biological systems.

Exploring Urease Inhibition by Coumarin Derivatives through in silico and in vitro Methods

In this report, we designed and synthesized ten N-(R-phenyl)-3-carboxamide-coumarin derivatives (2a-2j), exploring the coumarin nucleus, and an R-phenyl group as a structural scaffold and a peptide bond as a linker between them. The structure-activity relationships were investigated with different R-substituents (H, Br, NO2, Cl) in ortho, meta, or para positions from the phenyl group. Coumarins were obtained in good yields (72-95%), and in vitro screening against Canavalia ensiformis urease showed potential inhibitory percentages ranging from 42 to 65%. Half-maximal inhibitory concentration (IC50) values were determined for the best compounds 2b (R = 2-Br) and 2d (R = 4-Br). After conducting molecular docking and molecular dynamics on urease from C. ensiformis and Helicobacter pylori, potential binding modes for the most effective compounds 2b and 2d showed that these derivatives are able to interact with the crucial residue Cys592, thereby blocking the access of the urea substrate to the active site. According to density functional theory (DFT) calculations, 4-Br substitution on H. pylori urease was necessary for interacting with catalytic amino acids on the active site.

Enzymatically-induced dynamic assemblies from surface functional stomatocyte nanoreactors.

Collective behavior has become a recent topic of investigation in systems chemistry. In pursuing this phenomenon, we present polymersome stomatocytes loaded with the enzyme urease, which show basic stigmergy-based communication and are capable of signal production, reception, and response by clustering with surface complementary binding partners. The collective behavior is transient and based on the widely known pH-sensitive non-covalent interactions between nitrilotriacetic acid (NTA) and histidine (His) moieties attached to the surface of urease-loaded and empty stomacytes, respectively. Upon the addition of the substrate urea, the urease stomatocytes are able to increase the environmental pH, allowing the NTA units to interact with the surface histidines on the complementary species, triggering the formation of transient clusters. The stomatocytes display a maximum clustering interaction at a pH between 6.3 and 7.3, and interparticle repulsive behavior outside this range. This leads to oscillating behavior, as the aggregates disassemble when the pH increases due to high local urease activity. After bulk pH conditions are restored, clustering can take place again. Within the detectable region of dynamic light scattering, individual stomatocytes can aggregate to agglomerates with 10 times their volume. Understanding and designing population behavior of active colloids can facilitate the execution of cooperative tasks, which are not feasible for individual colloids.

Urea-Functionalized Fe4 L6 Cages for Supramolecular Gold Catalyst Encapsulation to Control Substrate Activation Modes.

The excellent catalytic performances of enzymes in terms of activity and selectivity are an inspiration for synthetic chemists and this has resulted in the development of synthetic containers for supramolecular catalysis. In such containers the local environment and pre-organization of catalysts and substrates leads to control of the activity and selectivity of the catalyst. Herein we report a supramolecular strategy to encapsulate single catalysts in a urea-functionalized Fe4 L6 cage, which can co-encapsulate a functionalized urea substrate through hydrogen bonding. Distinguished selectivity is obtained, imposed by the cage as site isolation only allows catalysis through π activation of the substrate and as a result the selectivity is independent of catalyst concentration. The encapsulated catalyst is more active than the free analogue, an effect that can be ascribed to transitionstate stabilization rather than substrate pre-organization, as revealed by the MM kinetic data. The simple strategy reported here is expected to be of general use in many reactions, for which the catalyst can be functionalized with a sulfonate group required for encapsulation.

Urea‐Functionalized Fe4L6 Cages for Supramolecular Gold Catalyst Encapsulation to Control Substrate Activation Modes

AbstractThe excellent catalytic performances of enzymes in terms of activity and selectivity are an inspiration for synthetic chemists and this has resulted in the development of synthetic containers for supramolecular catalysis. In such containers the local environment and pre‐organization of catalysts and substrates leads to control of the activity and selectivity of the catalyst. Herein we report a supramolecular strategy to encapsulate single catalysts in a urea‐functionalized Fe4L6 cage, which can co‐encapsulate a functionalized urea substrate through hydrogen bonding. Distinguished selectivity is obtained, imposed by the cage as site isolation only allows catalysis through π activation of the substrate and as a result the selectivity is independent of catalyst concentration. The encapsulated catalyst is more active than the free analogue, an effect that can be ascribed to transitionstate stabilization rather than substrate pre‐organization, as revealed by the MM kinetic data. The simple strategy reported here is expected to be of general use in many reactions, for which the catalyst can be functionalized with a sulfonate group required for encapsulation.

Abundance, diversity and physiological preferences of comammox Nitrospira in urban groundwater

Complete ammonia oxidizer (comammox Nitrospira), catalyze complete nitrification process in a single organism, are frequently detected in groundwater ecosystem. However, the ecological niches and environmental driving factors of comammox Nitrospira in urban groundwater are largely unknown. Here we investigated the communities of ammonia oxidizers in urban groundwater located in Shanghai city, China. Quantitative analysis demonstrated the dominance of comammox Nitrospira over classical ammonia oxidizers (ammonia-oxidizing archaea and bacteria, AOA and AOB). Phylogenetic analysis showed clades B and A2 comprise the majority of comammox Nitrospira groups. Temperature was one of the most vital factors affecting comammox Nitrospira community. Furthermore, clade A comammox Nitrospira can be enriched by urea substrate, which was in line with the ability of utilizing urea by the pure clade A comammox culture Nitrospira inopinata. In addition, we observed that relatively low temperature (<20 °C) and high copper levels (>0.04 mg L−1) can stimulate the growth of comammox Nitrospira. Overall, this study revealed the presence, diversity and physiological preferences of comammox Nitrospira in urban groundwater nitrification, shedding insights on the ecological roles of comammox Nitrospira in subsurface environment.

Effect of regulating urease activity on the properties of bio-CaCO3 precipitated on recycled aggregates

The high porosity of attached mortar on recycled aggregates leads to high water absorption and low strength, which hinders its application efficiency in concrete production. Microbial induced carbonate precipitation is a promising approach for enhancing recycled aggregates. The difficulty at present is that the low bonding between biogenic CaCO3 and aggregates leads to low strengthening efficiency. The cohesion among CaCO3 and its bonding with aggregates have been found to be closely influenced by the precipitation rate. While in the ureolytic process, urease activity is the key to control the precipitation process. In this study, the precipitation process was controlled by regulating the release rate of urease activity and its effect on the mechanical properties of the deposited CaCO3 was studied. The results showed that controlling the initiation time of induced bacterial urease and the concentration of precipitated precursors (calcium and urea) could effectively slow down the release rate of initial urease activity, thus regulating the precipitation rate. It was found that the bacteria cultivated without substrate urea can slowly release urease activity, and the urea decomposed process can be sustained until 14 d, and the best cohesion of biogenic CaCO3 could be obtained at 0.5 M of calcium and urea. The aggregates treated by the bacteria cultivated with urea showed a mass loss of 1.88% and water absorption reduction of 8.9%. While the ones treated by the bacteria cultivated without urea showed a mass loss of 1.70% and water absorption reduction of 29.83%, and the ones treated by the pellets centrifuged by the bacteria cultivated without urea showed a mass loss of 1.44% and water absorption reduction of 22.46%. It proved that the method of bacterial cultivation without urea can efficiently enhance the bonding strength between biogenic CaCO3 and aggregates, thus further improving the properties of aggregates, which was of great significance to improve the application efficiency of recycled aggregate in concrete.

An Improved Method for Determining Urease Activity from Electrical Conductivity Measurements.

We present an improved approach to evaluating the activity of urease from electrical conductivity (EC) measurements. In this approach, chemical equilibrium modeling via PHREEQC is used in conjunction with empirical equations for computing EC to develop a function that relates the increase in EC during urea hydrolysis in a closed reactor to the concentration of ammonium species present (and concentration of urea remaining) in the reaction solution. By applying this function to data from continuous measurement of EC during urea hydrolysis, we obtain a profile of the concentration of the urea substrate with time, which is then used to determine the urease activity. The activity of commercially available urease extracted from jack beans was determined using this method and compared well to the activity determined using Nessler's reagent, a commonly used colorimetric assay. This EC-based method is inexpensive and can be used for accurate determination of urease activity for a variety of applications.

The Isolation, Immobilization, and Characterization of Urease from The Seeds of Winged Bean (Psophocarpus tetragonolobus (L.) DC.

Urease has been utilized in the field of health and industry. Urease is commonly used in the form of free enzyme, so that the utilization is limited. Urease efficiency can be improved using immobilization enzyme. This research aimed to do the urease isolation, immobilization, and characterization from the winged bean seeds. This research was started by determining the amino-acid content of winged bean seeds using the Liquid chromatography-mass spectrometry (LCMS). The winged bean seeds were germinated and extracted. The obtained crude extract’s activity was determined using Nessler reagent and measured using UV-Vis spectrophotometer with the wavelength of 500 nm. The urease of winged bean seeds was immobilized using the alginate matrix. The optimization of urease-immobilized beads could be made through the variations of natrium alginate concentration and beads formation periods in solution CaCl2. Characterization free and immobilized urease were made using the variations of urea substrate concentration, pH, temperature, and also the repeated utilization of immobilized urease. Winged bean seeds are rich with essential amino acid, such as leucine, isoleucine, histidine, phenylalanine, and valine. The urease obtained from the winged bean seeds had the optimum activity in the germination period of 8 days. The urease immobilization showed the optimum condition in the natrium alginate concentration of 5% (w/v) and beads formation period in solution CaCl2 for 60 minutes. The characterization results of free urease and immobilization had the optimum condition at the urea substrate of 0.2 M, and pH 7. Free urease had the optimum temperature of 35 oC, while the immobilized urease had the optimum temperature of 40 oC. The immobilized urease had the utilization stability up to 5 times with the relative activity of 48%. The EDX analysis results showed that the alginate did not contain N, while alginate urease beads contained N as much as 12%.

Identification and expression of bifunctional acid urea-degrading enzyme/urethanase from Enterobacter sp. R-SYB082 and its application in degradation of ethyl carbamate in Chinese rice wine (Huangjiu).

Ethyl carbamate (EC) is a potential carcinogen existing in fermented foods such as Chinese rice wine (Huangjiu). Since urea is an important precursor of EC, the degradation of urea could be an effective way to reduce EC in foods. In this study, an Enterobacter sp. R-SYB082 with acid urea degradation characteristics was obtained through microbial screening. Further research isolated a new acid urea-degrading enzyme from R-SYB082 strain - ureidoglycolate amidohydrolase (UAH) - which could degrade EC directly. The cloning and expression of UAH in Escherichia coli BL21 (DE3) suggested that the activity of urea-degrading enzyme reached 3560 U L-1 , while urethanase activity reached 2883 U L-1 in the optimal fermentation condition. The enzyme had the dual ability of degrading substrate urea and product EC. The removal rate of EC in Chinese rice wine could reach 90.7%. This study provided a new method for the integrated control of EC in Chinese rice wine and other fermented foods. © 2022 Society of Chemical Industry.

Dihydropyrimidones: A ligands urease recognition study and mechanistic insight through in vitro and in silico approach

Scaffold varied dihydropyrimidone derivatives 1–20 were evaluated for their selective urease inhibitory kinetics potential. Compounds 1, 2, 3, 4, 5, 6, and 12 were found to be the most promising urease inhibitors and showed the inhibition (Ki values) within the range of 9.9 ± 0.5 to 18.3 ± 0.4 µM. Lineweaver–Burk plot, Dixon plot and their secondary replots confirm that all these molecules have followed competitive mode of inhibition. Docking arrangements (MOE) revealed that all the ligands bind in the active site and therefore compete with substrate urea. Molecular docking studies of all compounds have confirmed the binding interactions of various ligands with the amino acid residues as well as Ni atoms of active site. Furthermore, these compounds 1–20 were also tested for their cytotoxicity against human neutrophils and plants and were found to be non-toxic.

One-step immunoassay without washing steps for influenza A virus detection using ISFET

A one-step immunoassay for influenza A virus detection was developed using two different microbeads and a filter-inserted bottle. Two bead types with diameters of 15 (capture bead) and 3 (detection bead) μm were prepared to specifically detect influenza A virus. Anti-influenza A virus antibodies were coated on both bead types, whereas urease was immobilized only on the detection bead. An influenza A-positive sample could form a sandwich complex with the capture and detection beads; this complex would not pass through the filter, which had a controlled pore size. As the detection bead was used at a limiting concentration, it would be prevented from crossing the filter; thus, it would further react with the substrate urea and consequently increase the pH. An influenza A-negative sample would fail to form the sandwich complex in the presence of the capture and detection beads. Accordingly, the detection bead would pass through the filter into the urea buffer and increase the pH. The pH change in the urease reaction could be quantitatively measured by an indicator such as phenol red or using ion-selective field-effect transistor (ISFET). This one-step immunoassay was used for the detection of influenza A virus in real samples. The receiver operating characteristic (ROC) plot analysis showed an area under the curve (AUC) value of 0.931; the sensitivity and specificity of the assay was 80% and 90%, respectively, at a cutoff value of 0.9986. These results demonstrate that the one-step immunoassay could increase the sensitivity of influenza A virus detection in real samples.

Single-particle analysis of urea amidolyase reveals its molecular mechanism.

Urea amidolyase (UA), a bifunctional enzyme that is widely distributed in bacteria, fungi, algae, and plants, plays a pivotal role in the recycling of nitrogen in the biosphere. Its substrate urea is ultimately converted to ammonium, via successive catalysis at the C-terminal urea carboxylase (UC) domain and followed by the N-terminal allophanate hydrolyse (AH) domain. Although our previous studies have shown that Kluyveromyces lactis UA (KlUA) functions efficiently as a homodimer, the architecture of the full-length enzyme remains unresolved. Thus how the biotin carboxyl carrier protein (BCCP) domain is transferred within the UC domain remains unclear. Here we report the structures of full-length KlUA in its homodimer form in three different functional states by negatively-stained single-particle electron microscopy. We report here that the ADP-bound structure with or without urea shows two possible locations of BCCP with preferred asymmetry, and that when BCCP is attached to the carboxyl transferase domain of one monomer, it is attached to the biotin carboxylase domain in the second domain. Based on this observation, we propose a BCCP-swinging model for biotin-dependent carboxylation mechanism of this enzyme.

Isotopic evidences of the preferential coordination between 12CO2 and urease enzyme

The active Ni(II) centre of metalloenzyme urease forms a coordination with substrate urea prior to the hydrolysis of urea. The present study provided the direct experimental evidences of isotope preferential coordination between Ni(II) metal centre of urease enzyme and substrate urea, which eventually alters the product yield. Furthermore, in-vitro experiments revealed that the specific 12CO2 isotopic species bridges between heavier isotope of substrate urea [13CO(NH2)2] and Ni(II) centre of urease for more effective coordination, which ultimately enhances the yield of the reaction. Finally, the in-vitro observations have been validated under in-vivo physiological conditions exploiting the urease activity of the gastric pathogen Helicobacter pylori present in human stomach. Hence, the present study paves the way for better understanding of the isotope-selective catalytic activity of urease enzyme and exemplifies the link between 12CO2 and urease enzyme.

Study on Removal of Heavy Metal Ni Pollution by Microbial Mineralization Consolidation

In this study, a strain of Pichia pastoris strain N was screened from the soil around the local electroplating plant. This carbonate mineralization bacteria produced urease during the growth and metabolism process, decomposing the substrate urea and producing ammonia ions. And carbonate ions, mineralized to consolidate the heavy metal Ni in the soil. The basic physiological and biochemical properties, growth curve, urease activity and influencing factors of strain N and mineralization analysis experiments were analyzed. The conclusions of this experiment can provide a theoretical basis for the removal of heavy metals from soil in the field and can improve the application value of this technology.

Facile Electrochemical Intramolecular Amination of Urea-Tethered Terminal Alkenes for the Synthesis of Cyclic Ureas.

Facile intramolecular amination of unactivated alkenes has been achieved by using electricity as a catalyst that helps to generate an intermediate and accelerates formation of cyclic ureas in high yields. Using this method, no metal catalysts were used. During electrolysis, a nitrogen radical was formed at the urea substrate that cyclised with the alkene and generated a terminal carbon radical which further formed a bond with the 2,2,6,6‐tetramethylpiperidine‐N‐oxyl radical (TEMPO). This method of electrolysis not only gives cyclic ureas but also functionalises terminal unactivated alkenes. This method can be considered to be environmentally friendly given that it avoids the issues of toxicity or complicated metal ligands and could therefore be potentially employed in green chemistry.

Anti-Helicobacterpylori effectiveness and targeted delivery performance of amoxicillin-UCCs-2/TPP nanoparticles based on ureido-modified chitosan derivative

The amoxicillin-UCCs-2/TPP nanoparticles constructed with ureido-modified chitosan derivative UCCs-2 and sodium tripolyphosphate (TPP) played an important role to deliver drug to achieve more efficacious and specific eradication of Helicobacterpylori (H. pylori) in vitro. In this study, the anti-H. pylori effectiveness in vivo and uptake mechanism was investigated in details, including the effect of temperature, pH values and the addition of competitive substrate urea on uptake. Compared with unmodified nanoparticles, a more efficacious and specific anti-H. pylori activities were obtained in vivo by using this biological chitosan derivative UCCs-2. Histological staining and immunological analysis verified that the amoxicillin-UCCs-2/TPP nanoparticles could diminish the proinflammatory cytokines levels and alleviate the inflammatory damages caused by H. pylori infection. The uredio-modified nanoparticles also have favorable gastric retention property, which is beneficial for the oral drug delivery to targeted eradicate H. pylori infection in stomach. These findings suggest that this targeted drug delivery system may serve for specific treatment of H. pylori infection both in vitro and in vivo, which can also be used as promising nanocarriers for other therapeutic reagents to target H. pylori.

Enzyme-induced biomineralization of cupric subcarbonate for ultrasensitive colorimetric immunosensing of carcinoembryonic antigen

Herein we combine the enzyme-nanoprobe induced biomineralization of cupric subcarbonate with a sensitive copper chromogenic reaction to develop an ultrasensitive colorimetric immunosensing method. The nanoprobe was prepared through the surface assembly of signal antibody and high-content urease on the nanocarrier of gold nanoparticle (Au NP). Upon sandwich immunoreaction at a magnetic bead (MB)-based immunosensing platform, the analyte of carcinoembryonic antigen and the Au NP/urease nanoprobe were quantitatively captured onto the MB surface successively to form an immunocomplex. The enzyme reaction of the nanoprobe resulted in the catalyzed hydrolysis of the urea substrate to produce large amounts of OH− and HCO3−. These ions were further combined with added Cu2+ to form the biomineralization of cupric subcarbonate rapidly on the immunocomplex. Based on the acid-release of Cu2+ from the biomineralized products for the chromogenic reaction, a red complex was obtained and employed for the ultrasensitive colorimetric signal transduction. Under the optimum conditions, a linear range over five orders of magnitude was obtained with a low detection limit of 0.45 pg/mL. In addition, this method features low cost, rapid and convenient operation as well as satisfactory reproducibility, stability and accuracy. It thus owns great potentials for practical applications.

Optimal control of nutrition restricted dynamics model of Microalgae biomass growth model

The biomass of the microalgae is very potential to be proposed as an alternative renewable energy resources because it could be extracted into lipid. Afterward, the lipid could be processed to get the biodiesel or bioethanol. The extraction of the biomass on lipid synthesis process is very important to be studied because the process just gives some amount of lipid. A mathematical model of restricted microalgae biomass growth just gives 1/3 proportion of lipid with respect to the biomass in the synthesis process. An optimal control is designed to raise the ratio between the number of lipid formation and the microalgae biomass to be used in synthesis process. The minimum/ Pontryagin maximum principle is used to get the optimal lipid production. The simulation shows that the optimal lipid formation could be reach by simultaneously controlling the carbon dioxide, in the respiration and photosynthesis the process, and intake nutrition rates of liquid waste and urea substrate. The production of controlled microalgae lipid could be increase 6.5 times comparing to the uncontrolled one.