Synthetic Applications Research Articles

Microbial Synthetic Epigenetic Tools Design and Applications.

Microbial synthetic epigenetics offers significant opportunities for the design of synthetic biology tools by leveraging reversible gene control mechanisms without altering DNA sequences. However, limited understanding and a lack of technologies for thorough analysis of the mechanisms behind epigenetic modifications have hampered their utilization in biotechnological applications. In this review, we explore advancements in developing epigenetic-based synthetic gene regulatory tools at both transcriptional and post-transcriptional levels. Furthermore, we examine strategies developed to construct epigenetic-based circuits that provide controllable and stable gene regulation, aiming to boost the performance of microbial chassis cells. Finally, we discuss the current challenges and perspectives in the development of synthetic epigenetic tools.

Isolable Spirocyclic Silylone: π-Delocalized Spiro[3.3]heptasila-2,6-diylidone.

Strained cyclic tetrylones are important synthons due to various synthetic applications. Connecting two cyclic tetrylone rings through a single shared quaternary group 14 element atom to form a spirocyclic molecule has been unexplored both theoretically and experimentally. The formation of a spirocyclic motif has been a synthetic challenge. In contrast, the reaction of amidinato disilicon(I) 1, (Me3P)2SiCl4, and KC8 afforded π-delocalized spiro[3.3]heptasila-2,6-diylidone2 and tetrasilacyclobutadiene byproducts 3 and 4. Compound 2 is the smallest spirocyclic tetrylone derivative, which is composed of a σ-type lone pair and delocalized π bond in each all-silicon spirocyclic ring. The electronic property is supported by its coordination with a W(CO)5 moiety.

Genetically encoded Boolean logic operators to sense and integrate phenylpropanoid metabolite levels in plants.

Synthetic biology has the potential to revolutionize biotechnology, public health, and agriculture. Recent studies have shown the enormous potential of plants as chassis for synthetic biology applications. However, tools to precisely manipulate metabolic pathways for bioproduction in plants are still needed. We used bacterial allosteric transcription factors (aTFs) that control gene expression in a ligand-specific manner and tested their ability to repress semi-synthetic promoters in plants. We also tested the modulation of their repression activity in response to specific plant metabolites, especially phenylpropanoid-related molecules. Using these aTFs, we also designed synthetic genetic circuits capable of computing Boolean logic operations. Three aTFs, CouR, FapR, and TtgR, achieved c. 95% repression of their respective target promoters. For TtgR, a sixfold de-repression could be triggered by inducing its ligand accumulation, showing its use as biosensor. Moreover, we designed synthetic genetic circuits that use AND, NAND, IMPLY, and NIMPLY Boolean logic operations and integrate metabolite levels as input to the circuit. We showed that biosensors can be implemented in plants to detect phenylpropanoid-related metabolites and activate a genetic circuit that follows a predefined logic, demonstrating their potential as tools for exerting control over plant metabolic pathways and facilitating the bioproduction of natural products.

Tillage rotation and biostimulants can compensate for reduced synthetic agrochemical application in a dryland cropping system

Tillage rotation and biostimulants can compensate for reduced synthetic agrochemical application in a dryland cropping system

Ortho-Alkynyl Benzyl Alcohols as C6 Synthons in Regioselective Construction of Polysubstituted Naphthalenes.

A straightforward methodology for the assembly of polysubstituted naphthalenes from ortho-alkynyl benzyl alcohols, enabled by using catalytic amounts of Tf2O, has been developed. This transformation not only features transition-metal free and without using other bases and additives but also provides a new synthetic application for ortho-alkynyl benzyl alcohols, i.e., as C6 synthons for the construction of PAHs.

The elimination of two restriction enzyme genes allows for electroporation-based transformation and CRISPR-Cas9-based base editing in the non-competent Gram-negative bacterium Acinetobacter sp. Tol 5

ABSTRACT Environmental isolates are promising candidates for new chassis of synthetic biology because of their inherent capabilities, which include efficiently converting a wide range of substrates into valuable products and resilience to environmental stresses; however, many remain genetically intractable and unamenable to established genetic tools tailored for model bacteria. Acinetobacter sp. Tol 5, an environmentally isolated Gram-negative bacterium, possesses intriguing properties for use in synthetic biology applications. Despite the previous development of genetic tools for the engineering of strain Tol 5, its genetic manipulation has been hindered by low transformation efficiency via electroporation, rendering the process laborious and time-consuming. This study demonstrated the genetic refinement of the Tol 5 strain, achieving efficient transformation via electroporation. We deleted two genes encoding type I and type III restriction enzymes. The resulting mutant strain not only exhibited marked efficiency of electrotransformation but also proved receptive to both in vitro and in vivo DNA assembly technologies, thereby facilitating the construction of recombinant DNA without reliance on intermediate Escherichia coli constructs. In addition, we successfully adapted a CRISPR-Cas9-based base-editing platform developed for other Acinetobacter species. Our findings provide genetic modification strategies that allow for the domestication of environmentally isolated bacteria, streamlining their utilization in synthetic biology applications. IMPORTANCE Recent synthetic biology has sought diverse bacterial chassis from environmental sources to circumvent the limitations of laboratory Escherichia coli strains for industrial and environmental applications. One of the critical barriers in cell engineering of bacterial chassis is their inherent resistance to recombinant DNA, propagated either in vitro or within E. coli cells. Environmental bacteria have evolved defense mechanisms against foreign DNA as a response to the constant threat of phage infection. The ubiquity of phages in natural settings accounts for the genetic intractability of environmental isolates. The significance of our research is in demonstrating genetic modification strategies for the cell engineering of such genetically intractable bacteria. This research marks a pivotal step in the domestication of environmentally isolated bacteria, promising candidates for emerging synthetic biology chassis. Our work thus significantly contributes to advancing their applications across industrial, environmental, and biomedical fields.

Understanding the stability of a plastic-degrading Rieske iron oxidoreductase system.

Rieske oxygenases (ROs) are a diverse metalloenzyme class with growing potential in bioconversion and synthetic applications. We postulated that ROs are nonetheless underutilized because they are unstable. Terephthalate dioxygenase (TPADO PDB ID 7Q05)is a structurally characterized heterohexameric α3β3 RO that, with its cognate reductase (TPARED), catalyzes the first intracellular step of bacterial polyethylene terephthalate plastic bioconversion. Here, we showed that the heterologously expressed TPADO/TPARED system exhibits only ~300 total turnovers at its optimal pH and temperature. We investigated the thermal stability of the system and the unfolding pathway of TPADO through a combination of biochemical and biophysical approaches. The system's activity is thermally limited by a melting temperature (Tm) of 39.9°C for the monomeric TPARED, while the independent Tm of TPADO is 50.8°C. Differential scanning calorimetry revealed a two-step thermal decomposition pathway for TPADO with Tm values of 47.6 and 58.0°C (ΔH = 210 and 509 kcal mol-1, respectively) for each step. Temperature-dependent small-angle x-ray scattering and dynamic light scattering both detected heat-induced dissociation of TPADO subunits at 53.8°C, followed by higher-temperature loss of tertiary structure that coincided with protein aggregation. The computed enthalpies of dissociation for the monomer interfaces were most congruent with a decomposition pathway initiated by β-β interface dissociation, a pattern predicted to be widespread in ROs. As a strategy for enhancing TPADO stability, we propose prioritizing the re-engineering of the β subunit interfaces, with subsequent targeted improvements of the subunits.

Coumarin—Synthetic Methodologies, Pharmacology, and Application as Natural Fluorophore

Coumarin—Synthetic Methodologies, Pharmacology, and Application as Natural Fluorophore

Inhibitors application time and pasture canopy capture regulate gaseous losses of urine-N

AbstractTechnologies have been developed for the in-situ treatment of urine patches deposited by grazing livestock to mitigate nitrogen (N) losses using N transformation inhibitors. For this mitigation to be effective, close contact between the applied inhibitors and the N in the urine patch is required (similar to N-fertilisers coated with inhibitors). This research aimed to determine the proportions of urine-N that mixed with inhibitor at or exceeding the threshold concentration (inhibitor concentration at which the nitrification rate is reduced by at least 40%) when inhibitors were applied to simulated urine patches at 4, 24 and 48 h after synthetic urine application. Three commonly used nitrification inhibitors (NIs) [dicyandiamide (DCD), 3,4-dimethylpyrazole phosphate (DMPP), and 2-chloro-6-(trichloromethyl) pyridine (nitrapyrin)] were applied at 40 mL of inhibitor per urine-patch at two different concentrations. The field studies were undertaken in two dairy-grazed pasture soils with contrasting drainage. Large proportions of applied NIs (38%–59% DCD, 27%–58% DMPP, and 31%–58% nitrapyrin) were retained in the pasture canopy. In most cases, the inhibitor threshold concentration was present only within the top 0–20 mm of the soil, with only 16%–40% of the urine-N present. In some cases, the proportions of urine-N intercepted was 12%–15% higher when inhibitors were applied 4 h after urine application compared to delayed application of 24 and 48 h after urine application. Our results revealed that a substantial proportion of N in the urine-patch remained out of the reach of the inhibitor solution. This is possibly due to the small volume (40 mL per 2 L urine patch, 1:50) of the inhibitors applied, with up to 59% of inhibitor solution retained in the pasture canopy. The time delays (4 to 48 h) between the urine deposition and the inhibitor application could have also contributed to this poor physical mixing between inhibitor and urine. Increasing the volume of water applied with the inhibitor and assessing the effect of rainfall/irrigation on increasing urine-N and inhibitor mixing warrants further consideration.

Trihaloisocyanuric Acids: Useful Reagents for Halogenation Reactions and Heterocyclic Scaffold Construction

Abstract: Trihaloisocyanuric acids [1,3,5-trihalo-1,3,5-triazine-2,4,6-(1H,3H,5H)-triones] are commercially available or easily prepared solids. They are highly reactive, stable, easily handled, and have an excellent atom economy, transferring up to three halogen atoms to organic substrates. In these regards, the present review summarizes their synthetic applications as safe and convenient reagents. Therefore, electrophilic halogenation reactions of alkenes, alkynes, arenes, heteroarenes, carbonyl compounds, and heteroatoms, as well as radical halogenation involving saturated substrates and in situ halogenated intermediates for Appel-type reactions are presented and discussed. Remarkably, applications of trihaloisocyanuric acids in processes for the construction of heteroarene scaffolds based on electrophilic halo- and oxidative cyclization, multicomponent reactions, and telescopic reactions are also given.

The accelerated tensor Kaczmarz algorithm with adaptive parameters for solving tensor systems

Solving tensor systems is a common task in scientific computing and artificial intelligence. In this paper, we propose a tensor randomized average Kaczmarz method with adaptive parameters that exponentially converges to the unique least Frobenius norm solution of a given consistent tensor system under the t-product structure. In order to accelerate convergence, a tensor average Kaczmarz method based on stochastic heavy ball momentum technique (tAKSHBM) is proposed. The tAKSHBM method utilizes iterative information to update parameters instead of relying on prior information, addressing the problem in the adaptive learning of parameters. Additionally, the tAKSHBM method based on Fourier transform is proposed, which can be effectively implemented in a distributed environment. It is proven that the iteration sequences generated by all the proposed methods are convergent for given consistent tensor systems. Finally, we conduct experiments on both synthetic data and practical applications to support our theoretical results and demonstrate the effectiveness of the proposed algorithms.

Ruthenium(II)-Catalyzed Oxidative Annulation of Imidazo[1,5-a]quinolin-2-iums Salts and Internal Alkynes via C-H Bond Activation.

Ruthenium(II)-catalyzed synthesis of π-conjugated fused imidazo[1,5-a]quinolin-2-ium derivatives have been achieved via C-H activation of quinoline-functionalized NHC (NHC=N-heterocyclic carbene) and oxidative coupling with internal alkynes. The reaction occurred with high efficiency, broad substrate scope, tolerates a wide range of functional groups and utilized into a gram-scale. Synthetic applications of the coupled product have been exemplified in the late-stage derivatization of various highly functionalized scaffolds. Moreover, most of the annulated products exhibit intense fluorescence and have potential applications in optoelectronic devices. Mechanistic studies have provided insights into the spectroscopic characterization of key five-membered ruthenacycle intermediate and Ru(0) sandwich species. Based on several control experiments, deuterium-kinetic isotope effect, and thermodynamic activation parameters the mechanistic finding demonstrated that fused imidazo-[1,5-a]quinolin-2-ium C(2)-H bond cleavage is the rate-determining step and ruling out the possibility of reductive elimination for controlling the rate of reaction.

Functional and structural characterization of an IclR family transcription factor for the development of dicarboxylic acid biosensors.

Prokaryotic transcription factors (TFs) regulate gene expression in response to small molecules, thus representing promising candidates as versatile small molecule-detecting biosensors valuable for synthetic biology applications. The engineering of such biosensors requires thorough in vitro and in vivo characterization of TF ligand response as well as detailed molecular structure information. In this work, we functionally and structurally characterize the Pca regulon regulatory protein (PcaR) transcription factor belonging to the IclR transcription factor family. Here, we present in vitro functional analysis of the ligand profile of PcaR and the construction of genetic circuits for the characterization of PcaR as an in vivo biosensor in the model eukaryote Saccharomyces cerevisiae. We report the crystal structures of PcaR in the apo state and in complex with one of its ligands, succinate, which suggests the mechanism of dicarboxylic acid recognition by this transcription factor. This work contributes key structural and functional insights enabling the engineering of PcaR for dicarboxylic acid biosensors, in addition to providing more insights into the IclR family of regulators.

Joint Inversion of Gravity and Magnetic Data based on the Modified Structural Similarity Index for the Structural Consistency Constraint

Joint inversion is a crucial approach to mitigate the non-uniqueness problem in geophysical inversion. Nevertheless, existing joint inversion methods fall short of meeting the stringent requirements of high-precision exploration, necessitating the development of new techniques. In this paper, we introduce the Structural Similarity Index (SSIM) as a novel structural consistency constraint for the joint inversion of gravity and magnetic data. Compared with the results of cross-gradient inversion, our method demonstrates outstanding performance and stability. SSIM inversion not only introduces a new class of joint inversion with structural constraints but also enhances the consistency of inversion results in the distribution of physical attribute values. The structural constraints of SSIM inversion are more comprehensive and robust, significantly improving the reliability of the inversion. Both synthetic and real data applications demonstrate that the proposed method can effectively handle both synthetic and real data, yielding outstanding results.

Synthesis and Utilization of 1H-Indazole N-Oxides in the Production of C3-Functionalized 1H-Indazoles

The medicinal importance, natural rarity, and challenges associated with the synthesis of C3-functionalized 1H-indazoles have propelled the development of novel and practical 1H-indazole N-oxides for the production of diverse arrays of C3-functionalized 1H-indazoles. Utilizing 1H-indazole N-oxides has demonstrated remarkable efficacy in selectively introducing diverse functional groups, including amino (–NHAr), chloro (–Cl), hydroxy (–OH), sulfonyl (–SO2Ar), aromatic (–Ar), olefin, alkyl, and N-formyl (–NRCHO) groups, to indazole pharmacophore molecules. This review aims to offer a concise overview of synthetic approaches and practical applications of 1H-indazole N-oxides, including recent studies conducted by the authors. Transformative reactions involving 1H-indazole N-oxides not only offer strategies for synthesizing C3-functionalized 1H-indazoles but also hold significant potential in medicinal chemistry.

Potential Landslide Identification in Baihetan Reservoir Area Based on C-/L-Band Synthetic Aperture Radar Data and Applicability Analysis

The Baihetan reservoir region is characterized by complex geomorphology, significant altitude differences, and rugged terrain. Geological hazards in such areas are often characterized by high concealment, wide distribution, and difficulty in field investigation. Traditional identification techniques are unable to detect and monitor geological hazards on a large scale with high efficiency and accuracy. In recent decades, interferometric synthetic aperture radar (InSAR) techniques, such as small baseline subset InSAR (SBAS-InSAR), have been widely applied to landslide identification. However, due to factors such as vegetation and the degree of landslide deformation, single-band synthetic aperture radar (SAR) still has certain limitations in detecting landslides. In this study, SBAS-InSAR was conducted based on ALOS-2 and Sentinel-1 ascending-descending images covering the Baihetan reservoir region. Deformation identification results were utilized to conduct a statistical analysis of the SAR detection performance and landslide characteristics, and the effect of vegetation on the detection effectiveness of different SAR bands was discussed. The study revealed that when surface vegetation coverage reaches a high degree, the percentage of areas with coverage greater than 0.6 is greater than 95%, the SAR coherence is mainly affected by vegetation thickness; the comparison of the difference change in the average coherence of the C/L bands among the four vegetation types shows that the ratio of the average coherence of the L-bands to the C-bands increases by a factor of three with the increase in thickness and the transition from crops to shrubs and trees. The results showed that the L-band has better detectability than the C-band in alpine-canyon terrain with vegetation coverage and complex vegetation composition. However, considering the high temporal resolution and accessibility of Sentinel-1 SAR data, it is still the main data choice for wide-area identification of landslides in the reservoir area, while other satellite-borne SAR data with different wavelengths and resolutions, such as ALOS, can be used to assist in the identification and monitoring of landslide hazards with significant magnitude of deformations and dense vegetation coverage. Therefore, the combined utilization of multi-band SAR data has the potential to enhance the dependability of landslide identification and monitoring, resulting in more accurate detection results.

Switchable chemoselective aryne reactions between nucleophiles and pericyclic reaction partners using either 3-methoxybenzyne or 3-silylbenzyne.

Arynes are known to serve as highly reactive benzene-based synthons, which have gained numerous successes in preparing functionalized arenes. Due to the superb electrophilic nature of these fleeting species, however, it is challenging to modulate the designated aryne transformation chemoselectively, when substrates possess multiple competing reaction sites. Here, we showcase our effort to manipulate chemoselective control between two major types of aryne transformations using either 3-methoxybenzyne or 3-silylbenzyne, where nucleophilic addition-triggered reactions and non-polar pericyclic reactions could be differentiated. This orthogonal chemoselective protocol is found to be applicable between various nucleophiles, i.e., imidazole, N-tosylated/N-alkyl aniline, phenol, and alcohol, and an array of pericyclic reaction partners, i.e., furan, cyclopentadiene, pyrrole, cycloheptatrienone, and cyclohexene. Beyond arylation reactions, C-N bond insertion, Truce-Smiles rearrangement, and nucleophilic annulation are appropriate reaction modes as well. Moreover, this chemoselective protocol can find potential synthetic application.

In(OTf)3 promoted sonochemical approach to 3-(2-chloropyrimidin-4-yl)indoles: Their in silico and in vitro evaluation against SIRT1

We report exploring the 3-(2-chloropyrimidin-4-yl)indole framework for the identification of new inhibitors of SIRT1. A library of compounds based on this framework was synthesized via an ultrasound assisted heteroarylation approach. The methodology involved In(OTf)3 mediated CC bond forming reaction between 2,4-dichloropyrimidine and indoles to give the desired products in good to acceptable yields. The regioselectivity of this approach appeared to be a key feature as the methodology involved sonochemical replacement of the (i) C-3 hydrogen of indole and (ii) C-4 chloro group of the pyrimidine ring at the same time. Its synthetic application was demonstrated by converting one of the synthesized compounds into the naturally occurring alkaloid Meridianin G. The in silico docking of 3-(2-chloropyrimidin-4-yl)indoles prepared showed promising interactions with SIRT1 in addition to probable selectivity towards this protein over SIRT2. A majority of compounds participated in H-bond interaction with GLN345 residue mainly through their indole “NH” moiety. However, higher binding affinities were observed for compounds possessing a sulfonamide moiety at C-5 position of the indole ring. Correlating the findings of in silico studies a number of synthesized compounds showed inhibition of SIRT1 in vitro. With the inhibition > 70 % the compound 3e, 3o and 3p were identified as initial hit molecules when 3p emerged as most promising among them. The SAR (Structure-Activity-Relationship) analysis also suggested that a sulfonamide moiety at C-5 position was beneficial for the high SIRT1 inhibition whereas a free NH moiety of the indole ring was essential for activity. Based on in silico and in vitro studies including ADME predictions the compound 3p appeared as a key agent for further pharmacological evaluation.

Redox Relay-Induced C-S Radical Cross-Coupling Strategy: Application in Nontraditional Site-Selective Thiocyanation of Quinoxalinones.

Oxidative cross-coupling is a powerful strategy to form C-heteroatom bonds. However, oxidative cross-coupling for constructing C-S bond is still a challenge due to sulfur overoxidation and poisoning transition-metal catalysts. Now, electrochemical redox relay using sulfur radicals formed in situ from inorganic sulfur source offers a solution to this problem. Herein, electrochemical redox relay-induced C-S radical cross-coupling of quinoxalinones and ammonium thiocyanate with bromine anion as mediator is presented. The electrochemical redox relay comprised initially the formation of sulfur radical via indirect electrochemical oxidation, simultaneous electrochemical reduction of the imine bond, electro-oxidation-triggered radical coupling involving dearomatization-rearomatization, and the reformation of the imine bond through anodic oxidation. Applying this strategy, various quinoxalinones bearing multifarious electron-deficient/-rich substituents at different positions were well compatible with moderate to excellent yields and good steric hindrance compatibility under constant current conditions in an undivided cell without transition-metal catalysts and additional redox reagents. Synthetic applications of this methodology were demonstrated through gram-scale preparation and follow-up transformation. Notably, such a unique strategy may offer new opportunities for the development of new quinoxalinone-core leads.

Generation and Application of Aryl Radicals Under Photoinduced Conditions.

Photoinduced aryl radical generation is a powerful strategy in organic synthesis that facilitates the formation of diverse carbon-carbon and carbon-heteroatom bonds. The synthetic applications of photoinduced aryl radical formation in the synthesis of complex organic compounds, including natural products, physiologically significant molecules, and functional materials, have received immense attention. An overview of current developments in photoinduced aryl radical production methods and their uses in organic synthesis is given in this article. A generalized idea of how to choose the reagents and approach for the generation of aryl radicals is described, along with photoinduced techniques and associated mechanistic insights. Overall, this article offers a critical assessment of the mechanistic results as well as the selection of reaction parameters for specific reagents in the context of radical cascades, cross-coupling reactions, aryl radical functionalization, and selective C-H functionalization of aryl substrates.