Activities Of Entities Research Articles

1,3,5-Triazine: Recent Development in Synthesis of its Analogs and Biological Profile.

Triazine is an important pharmacophore in the field of research for the development of novel medications due to its presence in numerous powerful physiologically active compounds with significant medical potential, such as anti-tumor, anti-viral, anti-inflammatory, anti-microbial, anti- HIV, anti-leishmanial and others. The easy availability of triazine, high reactivity, simple synthesis of their analog, and their notable broad range of biological activities have garnered chemist interest in designing s-triazine-based drugs. The interest of medicinal chemists has been sparked by the structure-activity relationship of these biologically active entities, leading to the discovery of several promising lead molecules. Its importance for medicinal chemistry research is demonstrated by the remarkable progress made with triazine derivatives in treating a variety of disorders in a very short period. Authors have collated and reviewed the medicinal potential of s-triazine analogous to afford medicinal chemists with a thorough and target-oriented overview of triazine-derived compounds. We hope the present compilation will help people from the industry and research working in the medicinal chemistry area.

Violation of the rights of older adults in Ecuador

The rights, exonerations, and benefits of older persons are violated by not being applied or executed, being contemplated in the Constitution of the Republic of Ecuador. The objectives are to guarantee the full exercise and exercise of the fundamental rights of older persons, which, in addition to being enshrined in the Constitution, are included in international human rights instruments and related laws. It is important to energize the rights and benefits of older adults, through workshops, lectures, forums, reaching out to neighborhood leaders. The Constitution guarantees good living, and this vulnerable group is protected not only by national regulations, but also by international regulations, so that any type of action that violates their rights is eradicated. For this reason, it is intended that topics of studies be exposed, that promote awareness in society, so that they are active entities with knowledge in the legal framework in favor of the elderly, and thus they can truly have a dignified old age and with the full enjoyment and fulfillment of its benefits.

Hybrid/Chimera Drugs - Part 1 - Drug Hybrids Affecting Diseases of the Central Nervous System.

This review, focused on hybrid drugs, is the third in a series of reviews, where the first two reviews dealt with a) dimeric drugs, b) mutual prodrugs - codrugs. The compounds designated as hybrids are comprised of two (and sometimes three) biologically active entities, linked by metabolically stable bridges. In some cases, one of the two components of the hybrids serves as a carrier for the second component, and most frequently, the components elicit their individual biological properties, which are commonly synergistic or complementary. Due to the very large number of publications dealing with hybrid drugs, the present review is restricted to hybrids acting in the central nervous system. Future reviews will cover fields such as antimicrobial, anticancer, and antiviral hybrids, and cardiovascular active hybrids. The selected articles reviewed herein were published between the years 2000-2022 with partial coverage of the year 2023.

Quinoline Synthesis: Nanocatalyzed Green Protocols-An Overview.

Heterocyclic compounds are of great interest in our daily lives. They are widely distributed in nature and are synthesized in laboratories. Heterocycles play an important role in the metabolism of all living cells, including vitamins and coenzyme precursors like thiamine and riboflavin. Furthermore, heterocyclic systems are essential building blocks for creating innovative materials with intriguing electrical, mechanical, and biological properties. Also, more than 85% of all biologically active chemical entities comprise a heterocycle. As a result, heterocycle synthesis piqued researchers' curiosity, and in recent decades, chemists have concentrated more on nitrogen-containing cyclic nuclei in structures. Quinoline and its derivatives exhibit several biological functions, including antimicrobial, anticancer, antimalarial, anti-inflammatory, antihypertensive, and antiasthmatic effects. In addition, over a hundred quinoline-based drugs are available to treat a variety of disorders. Because of its biological importance, researchers developed one-pot synthetic methods employing effective acid/base catalysts (Lewis acids, Brønsted acids, and ionic liquids), reagents, and transition-metal-based catalysts. These methods have some downsides, including longer reaction times, harsher reaction conditions, creation of byproducts, costly catalysts, use of hazardous solvents, an unacceptable economic yield, and catalyst recovery. Researchers' focus has switched to creating environmentally friendly and effective methods for the synthesis of quinoline derivatives as a result of these methodologic shortcomings. Because of its special qualities, the use of nanocatalysts or nanocomposites offers an option for the effective synthesis of quinolines. This review focuses on the published research articles on nanocatalysts to synthesize substituted quinoline derivatives. This review covers all contributions until May 2024, focusing on quinoline ring building and mechanistic issues. With the aid of this review, we anticipate that synthetic chemists will be able to develop more effective methods of synthesizing quinolines.

АНАЛІТИКА ДІЯЛЬНОСТІ ПІДПРИЄМСТВ УКРАЇНИ З ВИРОБНИЦТВА ЦЕМЕНТУ В УМОВАХ СИСТЕМНИХ ВИКЛИКІВ

The article examines the activity of Ukrainian cement production enterprises in conditions of systemic challenges. The steady growth of the cement market in Ukraine in 2021 relative to 2020 has been determined. However, the full-scale war in Ukraine significantly affected the activities of cement manufacturing enterprises. The hostilities on the territory of the country have led to a decrease in the demand for cement due to the suspension of construction projects and infrastructure works, as well as due to instability and economic uncertainty. This was reflected in the decrease in business activity in the cement market in Ukraine. In 2022, the number of active cement production entities decreased by 22.2% compared to 2021. The decrease in the number of business entities in the cement industry of Ukraine led to a decrease in the number of employees, particularly, the number of employees decreased by 21.8% in 2022 compared to 2021. Such trends continued in the reduction of personnel costs of cement production enterprises by 25.1% over the last year under review. It has been proven that the most noticeable negative impact of the war on the development of the cement industry in Ukraine can be traced in the dynamics of the volumes of products sold (goods, services) of enterprises in 2022 compared to 2021: the value of this indicator decreased by UAH 7,499,298.9 thousand. or more than a third. As a result, the financial results of cement production enterprises in 2022 significantly worsened compared to 2021. In general, the level of profitability of the operational activities of cement production enterprises in 2022 significantly decreased compared to the previous three years. The most significant decrease was in profitability of operational activity resulting in a reduction of this indicator to the level of -25.6% in 2022. In general, the system challenges for the development of the cement market in Ukraine derived from war require adequate strategic decisions to support this exceptionally important activity for the post-war recovery of Ukraine. The further research of business responses for the current challenges is a perspective of the further studies in the field.

Features of Accounting under Uncertainty and Risk Conditions

Introduction. The purpose of the activity of each commercial organization is to make a profit, and therefore it is especially important, given the limited labor, material and financial resources, to achieve the planned performance indicators presented in both financial and non-financial assessments. Risks arising in conditions of external and internal uncertainty have an impact on the implementation of the financial and economic activity plan. To ensure the continuity of an economic entity's activities, it is necessary to form a flexible accounting system that accumulates information not only about the facts of economic life that have occurred, but also their characteristics from the perspective of risk-oriented management, in order to reduce the impact of uncertainty conditions on the financial results and property status of the organization. Purpose. Identification of accounting system problems in conditions of risk and uncertainty. Methods. The work uses general scientific research methods - comparison and analysis at each of the presented stages of the study based on an information base, including open data sources, scientific publications of Russian and foreign authors. Results. The key results of the study are 1) the differentiation of the definitions of "risk" and "uncertainty"; 2) the comparison of the chronology of the development of risk management and accounting; 3) the establishment of conceptual problems in accounting under conditions of risk and uncertainty. Conclusions. This study confirms and identifies conceptual problems in accounting in conditions of uncertainty and risk, pointing to the need to develop organizational and methodological foundations based on theoretical principles, which in turn will increase the competitiveness of economic entities of different forms of ownership and types of activity.

New accents of security-oriented management of logistics activity entities in the conditions of digitalization

The paper focuses on management issues in the field of sustainable functioning and development of business entities on the basis of guaranteeing their economic security. The industry specialization of entities specializing in logistics activities is taken into account. New aspects of security-oriented management of logistics entities are defined and taken into account, in particular in the languages of digitalization, which bring to the fore the issues of information and cyber security, digital communications, as well as cashless electronic payments. On this basis, the purpose of the research is to identify new accents of security-oriented management of logistics entities in the conditions of digitalization. The components of the economic security of logistics entities that require priority attention in terms of strengthening during the digital transformation of the economy and society have been determined. The toolkit of safety-oriented management under modern conditions for subjects of logistics activity is indicated. It is shown that under modern conditions, business entities specializing in logistics activities should focus on two mega aspects when forming their own security-oriented management systems. These are: (1) operational challenges caused by critical instability and the consequences of a full-scale war; (2) new opportunities, realities, as well as challenges of global and comprehensive digital transformation of economic relations. It was concluded that the new emphasis of security-oriented management of logistics entities concerns all the leading components of their economic security.

Eco-friendly Synthesis of Indole Conjugated Chromeno[d]Pyrimidines as Anti-cancer Agents and their Molecular Modelling Studies

Introduction: Many medicinally active new chemical entities depend on indole conjugated chromeno[d]pyrimidine derivatives as a building block. The synthesis of 4-(1H-indol-3-yl)-3,4- dihydro-1H-chromeno[4,3-d]pyrimidine-2,5-dione 4 were achieved in the current study by treating 4- hydroxy-2H-chromen-2-one 1, indole aldehydes 2, and urea/thiourea 3 in the presence of L-proline. Methods: By adopting the above protocol, we were able to synthesize eight compounds, i.e. 4-(1Hindol- 3-yl)-3,4-dihydro-1H-chromeno[4,3-d]pyrimidine-2,5-diones (4a-4h), in the presence of Lproline as a catalyst in ethanol as solvent for 2-3 hours at 70-75°C with decent yields of 80-85%, and their structures were ascertained by various spectral techniques. They were further screened for their potentiality to inhibit cancer growth in HepG2 and MDA-MD-231 cells. Results: The scope of the synthesis of biological relevant Indole conjugated Chromeno[ d]Pyrimidines by three-component reaction (MCRs) process was investigated. The most optimised conditions obtained were 0.3 eq of L-proline for 2 hours at 70-75°C which gave the best yield (85%). The few advantages of this newly developed method are excellent yields, no metal catalyst, less toxic solvents, simple workup no chromatographic column purifications. On further screening for their anticancer activities, out of all, the compound 4b displayed noteworthy cytotoxicity with IC50 values of 8.1 and 9.2 μM against HepG2 and MDA-MD-231, respectively. Additionally, in silico studies also supported that compound 4b had favourable binding energy (-7.8 kcal/mol) when compared to the co-crystal ligand (LS5) in inhibiting the human cyclin-dependent kinase 2 (CDK2) protein. Conclusion: In conclusion, we have developed a simple, convenient, and efficient method for the synthesis of structurally diverse indole conjugated chromeno[d]pyrimidine analogues in the presence of L-proline as catalyst in ethanol as solvent with good yields. Further, the in vitro cytotoxic studies against HepG2 and MDA-MD-231 cells demonstrated that the synthesized compounds had good to reasonable activity, except for compound 4d.

Engineering a Ce Promoter into a Three-Dimensional Porous Mo2C@NC Heterostructure for Hydrogen Evolution Electrocatalysis via Weakening the Mo-H Bond Strength.

The pursuit of highly efficient electrocatalysts for the alkaline hydrogen evolution reaction (HER) is of paramount importance for water splitting. However, it is still a formidable task in Mo2C-based materials because of the agglomeration and strong Mo-H binding of Mo2C units. Herein, a novel CeOCl-CeO2/Mo2C heterostructure nesting within a three-dimensional porous nitrogen-doped carbon matrix has been designed and used for catalyzing HER via simultaneous morphology and heterointerface engineering. As expected, the optimal CeOCl-CeO2(0.2)/Mo2C@3DNC exhibits impressive HER activity, with a low overpotential of 156 mV at a current density of 10 mA cm-2 coupled with a slight Tafel slope of 62.20 mV dec-1. Introducing a Ce promoter, that is CeOCl and CeO2, would endow the interface with an internal electric field and electron redistribution between CeOCl-CeO2 and Mo2C induced by the heterogeneous work function difference. Moreover, experimental investigation and density functional calculations confirm that the CeOCl-CeO2/Mo2C heterointerface can downshift the d-band center of the active Mo center, weakening the strength of the Mo-H coupling. This proposed concept, engineering Ce-based promoters into active entities involved in the heterostructure to modulate intermediate adsorption, offers a great opportunity for the design of superior electrocatalysts for energy conversion.

Self-organization of anti-aligning active particles: Waving pattern formation and chaos.

Recently, it has been shown that purely anti-aligning interaction between active particles may induce a finite wavelength instability. The formed patterns display intricate spatiotemporal dynamics, suggesting the presence of chaos. Here, we propose a quasi-one-dimensional simplification of the particle interaction model. This simplified model allows us to deduce amplitude equationsthat describe the collective motion of the active entities. We show that these equationsexhibit chaotic orbits. Furthermore, via direct numerical simulations of the particle's system, we discuss the pertinence of these amplitude equationsapproach for describing the particle's self-coordinated motions.

DIVERSITY OF FINANCING SOURCES OF NON-PROFIT ORGANIZATIONS IN ROMANIA AND THE EU – AN OVERVIEW

Financing is an asset or obstacle to the entity's activity, be it economic or not-forprofit. The impact that funding has on non-profit organizations is closely linked to the specificities arising from service and non-commercial status in society. The main objective of this paper is to present and appreciate diversity in funding non-profit organizations in Romania and the European Union during 2018-2022. In this regard, we are looking at the possibility of funding for NGOs from the European Union. The databases studied are Web of Science – for specialized articles – and Eurostat – for economic data and statistics. For database processing, VOSviewer – for bibliometric analysis – and Excel – for graphs and statistics were used. Making an inventory of the main categories of financial resources available today for Romanian NGOs leads us to conclude that NGOs have access to many funding opportunities. However, specific funding sources are available only to this type of organization

Methanolic extracts of litchi (Litchi chinensis Sonn.): A novel approach of targeting glucose-6-phosphate dehydrogenase for liver cancer therapy

Cancer metabolism has emerged as a potential target for innovative therapeutic approaches in the treatment of cancer. Cancer metabolism has received much attention, particularly in relation to glucose metabolism. It has been observed that human malignancies have high levels of glucose-6-phosphate dehydrogenase (G6PD) activity which is an important enzyme of glucose metabolism. This overactivity is associated with the cell death and angiogenesis, highlighting its potential as a viable target for cancer treatment. This study was conducted to examine the methanolic extracts from the seeds, bark and leaves of litchi (Litchi chinensis Sonn.) in order to discover effective compounds targeting G6PD and potentially active entities against liver cancer. Plant extract screening for the target protein was carried out through enzymatic activity assay. The recombinant plasmid pET-24a-HmG6PD was expressed in E. coli (BL21-DE3) strain, then purified and assessed using metal affinity chromatography with Ni-NTA columns and SDS-PAGE. The cytotoxicity of plant extracts against liver cancer HepG2 cells was assessed using the MTT assay. All three extracts demonstrated significant inhibitory effects (>80% inhibition) against G6PD. They were then subjected to testing at various concentrations, and their IC50 values were subsequently determined. The extracts of litchi (leaf, IC50: 1.199 μg/mL; bark, IC50: 2.350 μg/mL; seeds, IC50: 1.238 μg/mL) displayed significant inhibition of G6PD activity at lower concentrations. Subsequently, the leaf extract of litchi was further assessed for its impact on HepG2 cell lines in a dose-dependent manner and exhibited strong potential as an inhibitor of cancer cell progression. Moreover, the results of acute toxicity study in mice revealed nontoxic effects of litchi leaf extract on hepatocytes. The results imply that Litchi chinensis leaf extract could be considered as a promising candidate for safer drug development in the treatment of liver cancer.

Isolation and Total Synthesis of PM170453, a New Cyclic Depsipeptide Isolated from Lyngbya sp.

In our continuing search for biologically active new chemical entities from marine organisms, we have isolated a new cyclic depsipeptide, PM170453 (1), from a cyanobacterium of the genus Lyngbya sp., collected in the Indo-Pacific Ocean. Structure elucidation of the isolated compound was determined by spectroscopic methods including MS, 1H, 13C and 2D-NMR. To solve the supply problem for 1 and progress pharmaceutical development, the total synthesis of 1 that involves a total of 20 chemical steps in a convergent process was carried out. Its in vitro cytotoxic activity against four human tumor cell lines, as well as the inhibition of the interaction between the programmed cell death protein 1 PD-1 and its ligand PD-L1 were also evaluated.

Asymmetric synthesis of inositol derivatives

Both the enantiomers of racemic 4-O-tosyl-6-O-benzyl-myo-inositol-1,3,5-orthoformate (2) were isolated by preferential crystallization and used to synthesize several enantiomeric myo-inositol derivatives - natural ononitol, natural laminitol, precursors for myo-inositol phosphates and an oxabicyclo [2.2.1] heptane derivative. This work demonstrates the potential of the enantiomeric tosylates D2 and L2 to serve as versatile starting materials for the absolute asymmetric synthesis of inositol derivatives and other natural products from symmetric myo-inositol since no optically active molecular entity was required for the resolution of 2.

QUALITATIVE ANALYSIS ON THE INTERFERENCE AND CAUSALITY BETWEEN ACCOUNTING AND AUDITING FOR ROMANIAN COMPANIES “TOP TRADED” LISTED AT THE BUCHAREST STOCK EXCHANGE

The instability generated by legislative changes, price volatility, difficulties in obtaining and maintaining financial stability are realities that characterize the current economic situation, and the first indications of significant uncertainties regarding the continuity of an entity's activity can be obtained from the financial statements and the audit report, consequently, it becomes essential to assume the responsibilities that administrators and financial auditors have in assessing the adequacy of the going concern principle. The objective of the research is the analysis of how the auditor, by belonging to the Big 4 or Non-Big 4 category, the audit opinion, the number of key audit aspects included in the audit report, confirms the positive or negative implications on the principle of business continuity. The research methodology involved qualitative analysis by manually extracting data from the audit reports issued for the period 2018-2022, for a sample that includes 45 companies "top traded" from Romania listed on the Bucharest Stock Exchange (BSE). Following the analysis, it was found that the assumption of continuity of activity in the preparation of the individual annual financial statements, assumed by the management of the company, is adequate in 78% of the cases for the studied sample.

Copper Oxide Nano Biochar from Spent Coffee Grounds for Phosphate Removal and its Application as an Antibacterially Active Entity

From the viewpoint of both eutrophication and sustainable use of phosphate, the removal and recovery of phosphate from wastewater are important. Adsorption is seen as a viable alternative for effective phosphate removal, even at low concentrations. It is very simple to operate and cheaper. Among the various adsorbents tested, biomass-derived nanomaterials, such as nanobiochar, have shown promising efficiency. However, the use of pristine biochar is often less effective and difficult to recycle. In the present study, copper oxide-modified nanobiochar from spent coffee grounds is presented as an effective phosphate adsorbent. The adsorbent was prepared by the acid digestion of spent coffee grounds, followed by the co-precipitation of copper metal. The developed adsorbent was characterized by BET, FTIR, and XRD. Batch mode adsorption studies were conducted to assess the adsorption efficiency of the developed adsorbent and to investigate the effect of pH, initial concentration, contact time, and adsorbent dose. It was observed that acidic conditions favored the adsorption of phosphate, with maximum adsorption efficiency (93%) at pH 3. The maximum equilibrium phosphate adsorption capacity in this study was 50.2 mg/g at 25 oC, pH 3, a phosphate concentration of 20 mg/L, and an adsorbent dose of 35 mg/mL. The batch experimental data fit the Freundlich isotherm with regression (R2 = 0.991), which signifies that the surface of the adsorbent is heterogeneous. Adsorption kinetic data were best fitted with the pseudo-second-order kinetic model (R2 = 0.996), indicating that the adsorption process was dominated by chemisorption. The copper oxide nanoparticles and Cu/NBC showed relatively higher zone inhibition in gram-positive bacteria than in gram-negative bacteria at similar concentrations. This might be due to the higher activity of the nanoparticle extract on gram-positive bacteria, as most nanoparticle extracts were more active in gram-positive bacteria. This difference may be explained by the difference in the structure of the cell wall in gram-positive bacteria, which consists of a single layer, and in gram-negative bacteria, which has a multi-layered structure and is quite complex. In the majority of test bacteria, Cu/NBC showed better activity. The higher activity of this nanomaterial might be associated with the number of bioactive metabolites and their synergetic activities.

Financial Reporting, Criminal-Juridical Protection, And Corruption in Albania

Purpose: The purpose of this paper is role of financial reporting and the accounting profession in the identification of corruption what is foreseen in the Criminal Code of the Republic of Albania regarding the activity of businesses that violate financial reporting in accordance with accounting standards, how they stand against each other with the aim of a true and fair view of the activities of each entity. Methods: This paper comes as an empirical study of the regulatory framework in Albania in the field of financial reporting and criminal-juridical protection provided in under Criminal Code of the Republic of Albania, as two influential indicators in the wide discussed phenomenon of corruption in our country. The study employs a combination of qualitative and quantitative methodologies. Through empirical and statistical analysis, the actuality in Albania will be understood in the trinomial of financial reporting, criminal-juridical protection, and corruption. At the end, suggestions for the main actors will be presented for the role of financial reporting in the fight against corruption and organized crime, identifying it as a main tool. Results and Discussion: Corruption and organized crime are increasingly finding themselves in many dimensions in the world of globalization and information technology development. Through the "Action plan for fighting corruption and economic crime" (2022), IFAC has provided a framework for how to improve the role of the accounting profession in the fight against corruption, aiming for alignment with the UN SDGs. In this context, Albania has also undertaken its reforms in several areas, such as in the field of financial reporting, justice, etc., focusing on strengthening institutions, increasing public trust, and involving professionals with integrity and an elevated level of professionalism. Implications of the Research: Albania has been working for a long time now to align its legislation with that of the EU, in the framework of EU Integration. Among other areas, there have been a lot of developments toward the harmonization of the legal framework in the field of accounting and audit profession regulation. One of the crucial points in financial information publication is the process of preparation and auditing of financial statements. The legal framework altogether with the reporting standards should aim to encourage the publication of a true and fair view of financial information. It is recommended that the accounting professionals should show caution to prepare financial statements in accordance with national accounting standards, or international financial reporting standards evidencing the true and fair view of the entity's activity. Originality/Value: This paper presents the effect of financial reporting and criminal-juridical protection on the level of corruption in Albania. This goal is achieved by evidencing statistical data on economic entities operating in our country, describing an analysis of criminal-juridical protection provided for in the Criminal Code in force in the Republic of Albania, as well as based on a literature studied to understand how the financial reporting of entities is an important instrument for identifying corruption and what is foreseen in this criminal offense, as a means of preventing corruption and conducting business activity in accordance with legal acts.

Development and characterisation of buccal dispersible film, syrup and effervescent granules of extemporaneous antigout remedy

Homemade medicinal remedies used to treat both acute and chronic ailments for centuries need to be transformed into modern evidence-based dosage forms. Therefore, the present study aimed to develop buccal dispersible film, syrup and effervescent granules of an extemporaneous antigout homemade remedy. The dosage forms were prepared using holistic remedy (HR) as an active entity and excipients masking disagreeable features. The dosage forms and HR were subjected to physicochemical evaluation, and standardised by a reversed-phase HPLC method using chlorogenic acid, caffeic acid, vanillin, and ferulic acid as analytical standards. The standardised dosage forms were investigated for antigout activity using an appropriate model. The dosage forms were found to be physically elegant and phytochemically aligned with HR. The developed dosage forms contained the selected markers and possessed antigout activity. Hence, these standardised and evidence-based dosage forms may have wider acceptance in the market compared to HR.

Secondary Amine Catalysis in Enzyme Design: Broadening Protein Template Diversity through Genetic Code Expansion

AbstractSecondary amines, due to their reactivity, can transform protein templates into catalytically active entities, accelerating the development of artificial enzymes. However, existing methods, predominantly reliant on modified ligands or N‐terminal prolines, impose significant limitations on template selection. In this study, genetic code expansion was used to break this boundary, enabling secondary amines to be incorporated into alternative proteins and positions of choice. Pyrrolysine analogues carrying different secondary amines could be incorporated into superfolder green fluorescent protein (sfGFP), multidrug‐binding LmrR and nucleotide‐binding dihydrofolate reductase (DHFR). Notably, the analogue containing a D‐proline moiety demonstrated both proteolytic stability and catalytic activity, conferring LmrR and DHFR with the desired transfer hydrogenation activity. While the LmrR variants were confined to the biomimetic 1‐benzyl‐1,4‐dihydronicotinamide (BNAH) as the hydride source, the optimal DHFR variant favorably used the pro‐R hydride from NADPH for stereoselective reactions (e.r. up to 92 : 8), highlighting that a switch of protein template could broaden the nucleophile option for catalysis. Owing to the cofactor compatibility, the DHFR‐based secondary amine catalysis could be integrated into an enzymatic recycling scheme. This established method shows substantial potential in enzyme design, applicable from studies on enzyme evolution to the development of new biocatalysts.

Secondary Amine Catalysis in Enzyme Design: Broadening Protein Template Diversity through Genetic Code Expansion.

Secondary amines, due to their reactivity, can transform protein templates into catalytically active entities, accelerating the development of artificial enzymes. However, existing methods, predominantly reliant on modified ligands or N-terminal prolines, impose significant limitations on template selection. In this study, genetic code expansion was used to break this boundary, enabling secondary amines to be incorporated into alternative proteins and positions of choice. Pyrrolysine analogues carrying different secondary amines could be incorporated into superfolder green fluorescent protein (sfGFP), multidrug-binding LmrR and nucleotide-binding dihydrofolate reductase (DHFR). Notably, the analogue containing a D-proline moiety demonstrated both proteolytic stability and catalytic activity, conferring LmrR and DHFR with the desired transfer hydrogenation activity. While the LmrR variants were confined to the biomimetic 1-benzyl-1,4-dihydronicotinamide (BNAH) as the hydride source, the optimal DHFR variant favorably used the pro-R hydride from NADPH for stereoselective reactions (e.r. up to 92 : 8), highlighting that a switch of protein template could broaden the nucleophile option for catalysis. Owing to the cofactor compatibility, the DHFR-based secondary amine catalysis could be integrated into an enzymatic recycling scheme. This established method shows substantial potential in enzyme design, applicable from studies on enzyme evolution to the development of new biocatalysts.