Deacylation Reaction Research Articles

An efficient one-step synthesis of a new series of mutifunctional olefins and direct hydroxymethylation of α-substituted β-keto esters

An efficient one-step procedure for the synthesis of a new series of multifunctional olefins by condensation of α-substituted β-diketones as well as β-phosphonoesters and benzoylated β-ketoesters with formaldehyde, using potassium carbonate in refluxing THF, followed by a deacylation reaction, is herein described. In contrast, the α-substituted β-keto esters, bearing an acyl moiety, only undergo a hydroxymethylation reaction, with no metal catalysts/activating agents, as is usually required for the process, affording the corresponding α-hydroxymethyl β-keto esters in high yields.

Green Enzymatic Synthesis of Geranyl Butyrate: Process Optimization and Mechanistic Insights.

Flavor esters are organic compounds widely used in the food industry to enhance the aroma and taste of products. However, most chemical processes for the production of these flavoring compounds use toxic organic solvents. Some organic solvents derived from petroleum can leave behind residual traces in food products, which may raise concerns about potential health risks and contamination. In this study, we employ Eversa Transform 2.0, a commercial lipase derived from the lipase from Thermomyces lanuginosus, to produce geranyl butyrate in aqueous media. The chemical process was optimized using the Taguchi method, and a conversion of 93% was obtained at the optimal reaction conditions of: 1:5 molar ratio (v/v), 15% biocatalyst load (w/w), at 50 °C, in 6 h. Classic (molecular dynamics) and quantum (density functional theory) simulations unveiled amino acid residues involved in the stabilization of the enzyme-substrate complex. Detailed QM/MM mechanistic studies identified the nucleophilic attack of the deacylation reaction as the rate-limiting step of the entire mechanism, which has a free energy barrier of 14.0 kcal/mol.

The mechanism of ceftazidime and cefiderocol hydrolysis by D179Y variants of KPC carbapenemases is similar and involves the formation of a long-lived covalent intermediate.

Klebsiella pneumoniae carbapenemase (KPC) variants have been described that confer resistance to both ceftazidime-avibactam and cefiderocol. Of these, KPC-33 and KPC-31 are D179Y-containing variants derived from KPC-2 and KPC-3, respectively. To better understand this atypical phenotype, the catalytic mechanism of ceftazidime and cefiderocol hydrolysis by KPC-33 and KPC-31 as well as the ancestral KPC-2 and KPC-3 enzymes was studied. Steady-state kinetics showed that the D179Y substitution in either KPC-2 or KPC-3 is associated with a large decrease in both kcat and KM such that kcat/KM values were largely unchanged for both ceftazidime and cefiderocol substrates. A decrease in both kcat and KM is consistent with a decreased and rate-limiting deacylation step. We explored this hypothesis by performing pre-steady-state kinetics and showed that the acylation step is rate-limiting for KPC-2 and KPC-3 for both ceftazidime and cefiderocol hydrolysis. In contrast, we observed a burst of acyl-enzyme formation followed by a slow steady-state rate for the D179Y variants of KPC-2 and KPC-3 with either ceftazidime or cefiderocol, indicating that deacylation of the covalent intermediate is the rate-limiting step for catalysis. Finally, we show that the low KM value for ceftazidime or cefiderocol hydrolysis of the D179Y variants is not an indication of tight binding affinity for the substrates but rather is a reflection of the deacylation reaction becoming rate-limiting. Thus, the hydrolysis mechanism of ceftazidime and cefiderocol by the D179Y variants is very similar and involves the formation of a long-lived covalent intermediate that is associated with resistance to the drugs.

Optimization and theoretical analysis of lipase-catalyzed enzymatic esterification of glycerol for efficient glycerides synthesis

Eversa® Transform 2.0 is a lipase derived from Thermomyces lanuginosus produced by a genetically modified microorganism. It has a low production cost, high substrate specificity, and high catalytic activity in organic synthesis. The Taguchi method was used to evaluate the best conditions for the enzymatic esterification of glycerol with acetic acid. A yield of 84.8% was obtained, under the optimal conditions (temperature = 40 °C; molar ratio glycerol/acid = 1:1; biocatalyst = 15% w/w; time = 12.5 h). After the statistical analysis, the temperature was found to be the most significant parameter influencing the reaction conversion. A theoretical study was carried out to generate a homology model of the enzyme, based on other natural lipases. Molecular docking, molecular dynamics, and QM/MM simulations were applied to understand the mechanism of esterification and to derive thermodynamic and kinetic data. The nucleophilic attack step was identified as the rate-limiting step for both acylation (13.1 kcal/mol) and deacylation (13.8 and 12.9 kcal/mol) reaction mechanisms. Although the enzyme is capable of esterifying all three alcohol groups of glycerol, the esterification of the primary alcohols is thermodynamically more favorable (5 kcal/mol), especially at higher temperatures, than their secondary counterpart.

New ε-N-thioglutaryl-lysine derivatives as SIRT5 inhibitors: Chemical synthesis, kinetic and crystallographic studies

SIRT5 has been implicated in various physiological processes and human diseases, including cancer. Development of new highly potent, selective SIRT5 inhibitors is still needed to investigate disease-related mechanisms and therapeutic potentials. We here report new ε-N-thioglutaryllysine derivatives, which were designed according to SIRT5-catalysed deacylation reactions. These ε-N-thioglutaryllysine derivatives displayed potent SIRT5 inhibition, of which the potential photo-crosslinking derivative 8 manifested most potent inhibition with an IC50 value of 120 nM to SIRT5, and low inhibition to SIRT1-3 and SIRT6. The enzyme kinetic assays revealed that the ε-N-thioglutaryllysine derivatives inhibit SIRT5 by lysine-substrate competitive manner. Co-crystallographic analyses demonstrated that 8 binds to occupy the lysine-substate binding site by making hydrogen-bonding and electrostatic interactions with SIRT5-specific residues, and is likely positioned to react with NAD+ and form stable thio-intermediates. Compound 8 was observed to have low photo-crosslinking probability to SIRT5, possibly due to inappropriate position of the diazirine group as observed in SIRT5:8 crystal structure. This study provides useful information for developing drug-like inhibitors and cross-linking chemical probes for SIRT5-related studies.

Unveiling the structural features that regulate carbapenem deacylation in KPC-2 through QM/MM and interpretable machine learning.

Resistance to carbapenem β-lactams presents major clinical and economical challenges for the treatment of pathogen infections. The fast hydrolysis of carbapenems by carbapenemase-producing bacterial strains enables the effective deactivation of carbapenem antibiotics. In this study, we aim to unravel the structural features that distinguish the notable deacylation activity of carbapenemases. The deacylation reactions between imipenem (IPM) and the KPC-2 class A serine-based β-lactamases (ASβLs) are modeled with combined quantum mechanical/molecular mechanical (QM/MM) minimum energy pathway (MEP) calculations and interpretable machine-learning (ML) methods. We first applied a dual-level computational protocol to achieve fast sampling of QM/MM MEPs. A tree-based ensemble ML model was employed to learn the MEP activation barriers from the conformational features of the KPC-2/IPM active site. The barrier-predicting model was then unboxed using the Shapley additive explanation (SHAP) importance attribution methods to derive mechanistic insights, which were also verified by additional QM/MM wavefunction analysis. Essentially, we show that potential hydrogen bonding interactions of the general base and the tautomerization states of the carbapenem pyrroline ring could concertedly regulate the activation barrier of KPC-2/IPM deacylation. Nonetheless, we demonstrate the efficacy of interpretable ML to assist the analysis of QM/MM simulation data for robust extraction of human-interpretable mechanistic insights.

800 QM/MM minimum energy pathway conformations for the deacylation reactions of KPC-2/Imipenem

800 QM/MM minimum energy pathway conformations for the deacylation reactions of KPC-2/Imipenem

800 QM/MM minimum energy pathway conformations for the deacylation reactions of KPC-2/Imipenem

800 QM/MM minimum energy pathway conformations for the deacylation reactions of KPC-2/Imipenem

QM/MM Simulations Reveal the Determinants of Carbapenemase Activity in Class A β-Lactamases.

β-lactam antibiotic resistance in Gram-negative bacteria, primarily caused by β-lactamase enzymes that hydrolyze the β-lactam ring, has become a serious clinical problem. Carbapenems were formerly considered “last resort” antibiotics because they escaped breakdown by most β-lactamases, due to slow deacylation of the acyl-enzyme intermediate. However, an increasing number of Gram-negative bacteria now produce β-lactamases with carbapenemase activity: these efficiently hydrolyze the carbapenem β-lactam ring, severely limiting the treatment of some bacterial infections. Here, we use quantum mechanics/molecular mechanics (QM/MM) simulations of the deacylation reactions of acyl-enzyme complexes of eight β-lactamases of class A (the most widely distributed β-lactamase group) with the carbapenem meropenem to investigate differences between those inhibited by carbapenems (TEM-1, SHV-1, BlaC, and CTX-M-16) and those that hydrolyze them (SFC-1, KPC-2, NMC-A, and SME-1). QM/MM molecular dynamics simulations confirm the two enzyme groups to differ in the preferred acyl-enzyme orientation: carbapenem-inhibited enzymes favor hydrogen bonding of the carbapenem hydroxyethyl group to deacylating water (DW). QM/MM simulations of deacylation give activation free energies in good agreement with experimental hydrolysis rates, correctly distinguishing carbapenemases. For the carbapenem-inhibited enzymes, free energies for deacylation are significantly higher than for the carbapenemases, even when the hydroxyethyl group was restrained to prevent interaction with the DW. Analysis of these simulations, and additional simulations of mutant enzymes, shows how factors including the hydroxyethyl orientation, the active site volume, and architecture (conformations of Asn170 and Asn132; organization of the oxyanion hole; and the Cys69-Cys238 disulfide bond) collectively determine catalytic efficiency toward carbapenems.

Graph-learning guided mechanistic insights into imipenem hydrolysis in GES carbapenemases

Pathogen resistance to carbapenem antibiotics compromises effective treatments of superbug infections. One major source of carbapenem resistance is the bacterial production of carbapenemases which effectively hydrolyze carbapenem drugs. In this computational study, the deacylation reaction of imipenem (IPM) by GES-5 carbapenemases (GES) is modeled to unravel the mechanistic factors that facilitate carbapenem resistance. Hybrid quantum mechanical/molecular mechanical (QM/MM) calculations are applied to sample the GES/IPM deacylation barriers on the minimum energy pathways (MEPs). In light of the recent emergence of graph-based deep-learning techniques, we construct graph representations of the GES/IPM active site. An edge-conditioned graph convolutional neural network (ECGCNN) is trained on the acyl-enzyme conformational graphs to learn the underlying correlations between the GES/IPM conformations and the deacylation barriers. A perturbative approach is proposed to interpret the latent representations from the graph-learning (GL) model and extract essential mechanistic understanding with atomistic detail. In general, our study combining QM/MM MEPs calculations and GL models explains mechanistic landscapes underlying the IPM resistance driven by GES carbapenemases. We also demonstrate that GL methods could effectively assist the post-analysis of QM/MM calculations whose data span high dimensionality and large sample-size.

Multiscale Simulations Identify Origins of Differential Carbapenem Hydrolysis by the OXA-48 β-Lactamase.

OXA-48 β-lactamases are frequently encountered in bacterial infections caused by carbapenem-resistant Gram-negative bacteria. Due to the importance of carbapenems in the treatment of healthcare-associated infections and the increasingly wide dissemination of OXA-48-like enzymes on plasmids, these β-lactamases are of high clinical significance. Notably, OXA-48 hydrolyzes imipenem more efficiently than other commonly used carbapenems, such as meropenem. Here, we use extensive multiscale simulations of imipenem and meropenem hydrolysis by OXA-48 to dissect the dynamics and to explore differences in the reactivity of the possible conformational substates of the respective acylenzymes. Quantum mechanics/molecular mechanics (QM/MM) simulations of the deacylation reaction for both substrates demonstrate that deacylation is favored when the 6α-hydroxyethyl group is able to hydrogen bond to the water molecule responsible for deacylation but disfavored by the increasing hydration of either oxygen of the carboxylated Lys73 general base. Differences in free energy barriers calculated from the QM/MM simulations correlate well with the experimentally observed differences in hydrolytic efficiency between meropenem and imipenem. We conclude that the impaired breakdown of meropenem, compared to imipenem, which arises from a subtle change in the hydrogen bonding pattern between the deacylating water molecule and the antibiotic, is most likely induced by the meropenem 1β-methyl group. In addition to increased insights into carbapenem breakdown by OXA β-lactamases, which may aid in future efforts to design antibiotics or inhibitors, our approach exemplifies the combined use of atomistic simulations in determining the possible different enzyme–substrate substates and their influence on enzyme reaction kinetics.

Efficient Construction of 3‐Fluoroalkylated Oxindoles Enabled by Zwitterion Catalysis

Abstract3,3‐Disusbtituted oxindoles are important compounds in organic synthesis, and methods to access this class of compounds have always been sought after. Herein, we have identified a new method where a zwitterion generated using a combination of phosphine and para‐quinone methide could efficiently catalyze the 1,6‐conjugate addition of α‐fluoro‐β‐ketoamides to isatin‐derived para‐quinone methides to access 3‐fluoroalkylated oxindoles. It was further shown that the methodology could be extended to arylaldehyde‐derived p‐QMs. Moreover, one of the conjugate addition products was efficiently transformed into oxoindolinyl fluoroacetamide derivative through a K2CO3 mediated deacylation reaction.

Calcium‐Catalyzed Intramolecular Hydroamination‐Deacylation Reaction of in situ formed β‐Amino Allenes

AbstractWe have developed a simple, One‐Pot, three‐component reaction of tert‐propargyl alcohols, primary amines and acyl ketones to synthesize fully substituted pyrroles and pyridine derivatives in good to excellent yields with large substrate diversity. An eco‐friendly calcium catalyst catalyzes the reaction to form the key intermediate β‐amino allene that undergoes subsequent Thorpe‐Ingold effect assisted hydroamination and aromaticity driven deacylation reaction to yield fully substituted five and six‐membered azacyclic compounds.magnified image

Study on the Degradation of Tetracycline in Wastewater by Micro-nano bubbles Activated Hydrogen Peroxide

Recently, the micro-nano bubble (MB) technology has attracted people's attention due to its special advantages. Here, we carried out the technology of combining MB and hydrogen peroxide (MB/H2O2) to achieve efficient degradation of tetracycline wastewater. The effect of MB/H2O2 technology on the degradation efficiency of tetracycline was deeply analyzed by investigating the reaction time, H2O2 dosage, pH and MB inlet flow. The results showed that the degradation rate of tetracycline hydrochloride by MB/H2O2 technology can reach 92.43%, which is 9.44 times and 3.94 times that of MB and H2O2 alone. Through electron spin resonance (ESR) analysis and free radical quenching experiments, a possible mechanism for MB/H2O2 technology to efficiently degrade TC was proposed. In the MB/H2O2 system, the high temperature and high pressure environment generated when MB ruptures can activate H2O2 to obtain a higher number of active oxygen species. •OH is the main reactive oxygen radical in the process of MB/H2O2 degradation of TC, followed by HO2•/•O2-. In addition, the possible intermediate products of the oxidation TC process were identified by HPLC-MS technology. Under the action of •OH and HO2•/•O2− free radicals, TC molecules undergo demethylation and hydroxylation, ring-opening reactions, isomerization, deethylation, deacylation, deamination and dehydration reactions to generate intermediate products and finally convert them into CO2 and H2O. The development of MB/H2O2 technology can potentially be used to efficiently remove TC substances in the water environment and provide a new method for water purification. Graphical For the first time, the process of combining micro-nano bubbles (MB) with hydrogen peroxide to degrade tetracycline hydrochloride was proposed, and the possible reaction degradation pathways and TC degradation mechanisms in the MB/H2O2 system were discussed.

Understanding the Fatty Acids Removal from Bacterial Lipopolysaccharides via Enzymatically Activated Hydrolysis in Water

Lipopolysaccharides (LPS) belongs to one of the strongest pathogens groups that can stimulate the human immune system. This molecule also known as endotoxin comes from the outer membrane of Gram-negative bacteria cell wall. Gram-negative bacteria shed-out LPS in the form of blebs from its outer membrane particularly after biofilms successfully mature in events where stagnated waters occur. Despite its pathophysiological toxicity mechanism are well- established, little is known about its interaction in water sources that ultimately reach people. LPS molecules are an important pathogen group that triggers a signal within the innate immune system. To occur the LPS agonist role in humans, it is necessary to stimulate the toll-like receptor 4 (TLR4) by using its six acyl chain from its lipid A moiety. The effect of this LPS triggered signal includes an initial strong inflammatory response by the immune system that subsequently is counteracted via the human enzyme acyloxyacyl hydrolase (AOAH). In nature, AOAH detoxifies LPS by removing fatty acids from lipid A component. The main purpose of this study is to mimic this biological response in-vitro by exploring a different enzyme in order to develop a sustainable water treatment technology. In this study we aim to explore a new detoxification mechanism to remove the fatty acids chains from the molecule using an enzymatic de-acylation procedure. The product of the de-acylation reaction had been characterized via gas chromatography (GC), free fatty acid assays (FFA), Fourier-transform infrared spectroscopy (FTIR), among others. Results have demonstrated viability of the reaction that will serve as the groundwork principle to develop further technology for water purification applications.The details for this investigation methods and analytical techniques will be presented.

Mechanistic Basis of OXA-48-like β-Lactamases' Hydrolysis of Carbapenems.

Carbapenem-hydrolyzing class D β-lactamases (CHDLs) are an important source of resistance to these last resort β-lactam antibiotics. OXA-48 is a member of a group of CHDLs named OXA-48-like enzymes. On the basis of sequence similarity, OXA-163 can be classified as an OXA-48-like enzyme, but it has altered substrate specificity. Compared to OXA-48, it shows impaired activity for carbapenems but displays an enhanced hydrolysis of oxyimino-cephalosporins. Here, we address the mechanistic and structural basis for carbapenem hydrolysis by OXA-48-like enzymes. Pre-steady-state kinetic analysis indicates that the rate-limiting step for OXA-48 and OXA-163 hydrolysis of carbapenems is deacylation and that the greatly reduced carbapenemase activity of OXA-163 compared to that of OXA-48 is due entirely to a slower deacylation reaction. Furthermore, our structural data indicate that the positioning of the β5-β6 loop is necessary for carbapenem hydrolysis by OXA-48. A major difference between the OXA-48 and OXA-163 complexes with carbapenems is that the 214-RIEP-217 deletion in OXA-163 creates a large opening in the active site that is absent in the OXA-48/carbapenem structures. We propose that the larger active site results in less constraint on the conformation of the 6α-hydroxyethyl group in the acyl-enzyme. The acyl-enzyme intermediate assumes multiple conformations, most of which are incompatible with rapid deacylation. Consistent with this hypothesis, molecular dynamics simulations indicate that the most stable complex is formed between OXA-48 and imipenem, which correlates with the OXA-48 hydrolysis of imipenem being the fastest observed. Furthermore, the OXA-163 complexes with imipenem and meropenem are the least stable and show significant conformational fluctuations, which correlates with the slow hydrolysis of these substrates.

Control of RNA with quinone methide reversible acylating reagents.

Caging RNA by polyacylation (cloaking) has been developed recently as a simple and rapid method to control the function of RNAs. Previous approaches for chemical reversal of acylation (uncloaking) made use of azide reduction followed by amine cyclization, requiring ∼2-4 h for the completion of cyclization. In new studies aimed at improving reversal rates and yields, we have designed novel acylating reagents that utilize quinone methide (QM) elimination for reversal. The QM de-acylation reactions were tested with two bioorthogonally cleavable motifs, azide and vinyl ether, and their acylation and reversal efficiencies were assessed with NMR and mass spectrometry on model small-molecule substrates as well as on RNAs. Successful reversal both with phosphines and strained alkenes was documented. Among the compounds tested, the azido-QM compound A-3 displayed excellent de-acylation efficiency, with t1/2 for de-acylation of less than an hour using a phosphine trigger. To test its function in RNA caging, A-3 was successfully applied to control EGFP mRNA translation in vitro and in HeLa cells. We expect that this molecular caging strategy can serve as a valuable tool for biological investigation and control of RNAs both in vitro and in cells.

KPC-2 β-lactamase enables carbapenem antibiotic resistance through fast deacylation of the covalent intermediate

Serine active-site β-lactamases hydrolyze β-lactam antibiotics through the formation of a covalent acyl-enzyme intermediate followed by deacylation via an activated water molecule. Carbapenem antibiotics are poorly hydrolyzed by most β-lactamases owing to slow hydrolysis of the acyl-enzyme intermediate. However, the emergence of the KPC-2 carbapenemase has resulted in widespread resistance to these drugs, suggesting it operates more efficiently. Here, we investigated the unusual features of KPC-2 that enable this resistance. We show that KPC-2 has a 20,000-fold increased deacylation rate compared with the common TEM-1 β-lactamase. Furthermore, kinetic analysis of active site alanine mutants indicates that carbapenem hydrolysis is a concerted effort involving multiple residues. Substitution of Asn170 greatly decreases the deacylation rate, but this residue is conserved in both KPC-2 and non-carbapenemase β-lactamases, suggesting it promotes carbapenem hydrolysis only in the context of KPC-2. X-ray structure determination of the N170A enzyme in complex with hydrolyzed imipenem suggests Asn170 may prevent the inactivation of the deacylating water by the 6α-hydroxyethyl substituent of carbapenems. In addition, the Thr235 residue, which interacts with the C3 carboxylate of carbapenems, also contributes strongly to the deacylation reaction. In contrast, mutation of the Arg220 and Thr237 residues decreases the acylation rate and, paradoxically, improves binding affinity for carbapenems. Thus, the role of these residues may be ground state destabilization of the enzyme-substrate complex or, alternatively, to ensure proper alignment of the substrate with key catalytic residues to facilitate acylation. These findings suggest modifications of the carbapenem scaffold to avoid hydrolysis by KPC-2 β-lactamase.

Exploring the Catalytic Reaction of Cysteine Proteases.

Cysteine proteases play a major role in many life processes and are the target of key drugs. The reaction mechanism of these enzymes is a complex process, which involves several steps that are divided into two main groups: acylation and deacylation. In this work, we studied the energy profile for the acylation and a part of the deacylation reaction of three different enzymes, cruzain, papain, and the Q19A-mutated papain with the benzyloxycarbonyl-phenylalanylarginine-4-methylcoumaryl-7-amide (CBZ-FR-AMC) substrate. The calculations were performed using the EVB and PDLD/S-LRA methods. The overall agreement between the calculated and observed results is encouraging and indicates that we captured the correct reaction mechanism. Finally, our finding indicates that the minimum of the reaction profile, between the acylation and deacylation steps, should provide an excellent state for the binding of covalent inhibitors.

Синтез 3-арилизоксазолов и их сульфамидных производных

Great interest in binuclear aromatic systems containing an isoxazole ring and a sulfamide group is due to the effect of two pharmacophore groups at once on the biological activity of these compounds. This article is devoted to the development of a method for the synthesis of 3-arylisoxazole-containing compounds and their sulfamide derivatives from simple and accessible products of organic synthesis. The target products of the developed multistage schemes are derivatives of various bicyclic systems containing an isoxazole ring and a second aromatic ring associated with a sulfofragment. The synthesis of 3-aryl-5-acylaminoisoxazoles was carried out by sequential conversion of methyl esters of aromatic carboxylic acids into the corresponding nitriles by reaction with acetonitrile in the presence of sodium hydride in dioxane. At the next stage, the nitriles reacted with hydroxylamine in an aqueous solution of sodium hydroxide to form the corresponding bicyclic amines, which were then acylated in acetonitrile with acetic acid chloride in the presence of pyridine. The total yields of 3-aryl-5-N-acylaminoisoxazoles were at least 60%. It was found that the sulfonylchlorination of 3-aryl-5-N-acylamino derivatives of isoxazole, depending on the experimental conditions and the structure of the starting substrates, forms both mono- and disulfochlorides. The regioselectivity of the sulfochlorination reaction of the synthesized bicyclic systems was proved by 1H NMR spectroscopy. The influence of various factors on the course of the sulfochlorination reaction of the synthesized bicyclic systems was studied. The dependence of the direction of the electrophilic attack on the structure of the compounds and on the conditions of the experiment was established. As a result of the performed research, both mono and disulfochlorination products were obtained. With an increase in the reaction time, the disulfonylchlorination product accumulates and the deacylation reaction proceeds in parallel. Convincing proof of the structure of all synthesized compounds has been carried out using a complex of modern methods of physical and chemical analysis.