Mechanism Of Acylation Research Articles

Influence of Wobbling Tryptophan and Mutations on PET Degradation Explored by QM/MM Free Energy Calculations.

Plastic-degrading enzymes, particularly poly(ethylene terephthalate) (PET) hydrolases, have garnered significant attention in recent years as potential eco-friendly solutions for recycling plastic waste. However, understanding of their PET-degrading activity and influencing factors remains incomplete, impeding the development of uniform approaches for enhancing PET hydrolases for industrial applications. A key aspect of PET hydrolase engineering is optimizing the PET-hydrolysis reaction by lowering the associated free energy barrier. However, inconsistent findings have complicated these efforts. Therefore, our goal is to elucidate various aspects of enzymatic PET degradation by means of quantum mechanics/molecular mechanics (QM/MM) reaction simulations and analysis, focusing on the initial reaction step, acylation, in two thermophilic PET hydrolases, LCC and PES-H1, along with their highly active variants, LCCIG and PES-H1FY. Our findings highlight the impact of semiempirical QM methods on proton transfer energies, affecting the distinction between a two-step reaction involving a metastable tetrahedral intermediate and a one-step reaction. Moreover, we uncovered a concerted conformational change involving the orientation of the PET benzene ring, altering its interaction with the side-chain of the "wobbling" tryptophan from T-stacking to parallel π-π interactions, a phenomenon overlooked in prior research. Our study thus enhances the understanding of the acylation mechanism of PET hydrolases, in particular by characterizing it for the first time for the promising PES-H1FY using QM/MM simulations. It also provides insights into selecting a suitable QM method and a reaction coordinate, valuable for future studies on PET degradation processes.

The Evolution of the Acylation Mechanism in β-Lactamase and Rapid Protein Dynamics.

β-Lactamases are a class of well-studied enzymes that are known to have existed since billions of years ago, starting as a defense mechanism to stave off competitors and are now enzymes responsible for antibiotic resistance. Using ancestral sequence reconstruction, it is possible to study the crystal structure of a laboratory resurrected 2-3 billion year-old β-lactamase. Comparing the ancestral enzyme to its modern counterpart, a TEM-1 β-lactamase, the structural changes are minor, and it is probable that dynamic effects play an important role in the evolution of function. We used molecular dynamics simulations and employed transition path sampling methods to identify the presence of rate-enhancing dynamics at the femtosecond level in both systems, found that these fast motions are more efficiently coordinated in the modern enzyme, and examined how specific dynamics can pinpoint evolutionary effects that are essential for improving enzymatic catalysis.

Direct Synthesis of C4-Acyl Indoles via C-H Acylation.

The direct synthesis of C4-acyl indoles deprived of C2 and C3 substituents has proven to be challenging, with scarce efficient synthetic routes being reported. Herein, we disclose a highly site-selective palladium-catalyzed C-H acylation for the construction of C4-acyl indoles via a Catellani-Lautens cyclization strategy. In addition, we systematically studied the ortho C-H acylation mechanism of iodoaniline through density functional theory (DFT) calculations and combined experimental results to elucidate the principle of high chemoselectivity brought by triazine benzoate as an acylation reagent.

Front Cover: Expanding the Substrate Scope of Acyltransferase LovD9 for the Biosynthesis of Statin Analogues (Chem. Eur. J. 42/2023)

Vinyl and p-nitrophenyl esters have emerged as alternative substrates for the LovD9 acylation mechanism, showing product yields as high as those obtained by α-dimethylbutyryl-S-methyl-3-mercaptopropionate (DMB-SMMP), the thioester for which LovD9 was evolved. More information can be found in the Research Article by G. Jiménez-Osés, F. López-Gallego and co-workers (DOI: 10.1002/chem.202300911).

Coenzyme A binding sites induce proximal acylation across protein families

Lysine Nɛ-acylations, such as acetylation or succinylation, are post-translational modifications that regulate protein function. In mitochondria, lysine acylation is predominantly non-enzymatic, and only a specific subset of the proteome is acylated. Coenzyme A (CoA) can act as an acyl group carrier via a thioester bond, but what controls the acylation of mitochondrial lysines remains poorly understood. Using published datasets, here we found that proteins with a CoA-binding site are more likely to be acetylated, succinylated, and glutarylated. Using computational modeling, we show that lysine residues near the CoA-binding pocket are highly acylated compared to those farther away. We hypothesized that acyl-CoA binding enhances acylation of nearby lysine residues. To test this hypothesis, we co-incubated enoyl-CoA hydratase short chain 1 (ECHS1), a CoA-binding mitochondrial protein, with succinyl-CoA and CoA. Using mass spectrometry, we found that succinyl-CoA induced widespread lysine succinylation and that CoA competitively inhibited ECHS1 succinylation. CoA-induced inhibition at a particular lysine site correlated inversely with the distance between that lysine and the CoA-binding pocket. Our study indicated that CoA acts as a competitive inhibitor of ECHS1 succinylation by binding to the CoA-binding pocket. Together, this suggests that proximal acylation at CoA-binding sites is a primary mechanism for lysine acylation in the mitochondria.

Transition metal-free regioselective direct acylation of quinazolines for the synthesis of acylquinazoline derivatives.

An efficient, mild method for direct regioselective acylation of quinazolines under metal-free conditions was developed with bis(trifluoroacetoxy)iodobenzene and trimethylsilyl azide at ambient temperature. The acylation reaction of quinazolines with aldehydes gave the corresponding acyl quinazolines in ethyl acetate with good to excellent yields and excellent functional group tolerance and site selectivity. In addition, the mechanism of the direct acylation of quinazolinone was investigated through HPLC-HRMS (high pressure liquid chromatography-high resolution mass spectrometry) and EPR (electron paramagnetic resonance) strategies.

Mechanism of Friedel-Crafts Acylation Using Metal Triflate in Deep Eutectic Solvents: An Experimental and Computational Study.

In this paper, we develop a method for Friedel-Crafts acylation using metal triflate in deep eutectic solvents. Various metal triflates were tested and provided good to excellent yields of corresponding ketone products. The density functional theory calculation revealed the metal effects on the formation of active intermediate acylium triflate as well as the acidic condition. The metal triflate in the deep eutectic solvent can be recovered and reused with a little loss in the catalytic activity.

CATALYSIS OF TRIVALENT PHOSPHORUS COMPOUNDS OF THE REACTIONS OF SUBSTITUTED BENZOIC ACIDS WITH ANILINE

The catalytic preparation of substituted benzanilides by the reaction of substituted benzoic acids with aniline is an important model process that has been intensively deve­lo­ped recently, in the field of the «green chemistry» concept, direct catalytic amidation, and its study is an urgent scientific and practical task.
 Within the framework of solving this problem, the catalysis of the acylation of aniline by substituted benzoic acids with trivalent phosphorus compounds P(III) was studied. It was established that P(III) in the amount of only 2% mol. from substituted benzoic acid effectively catalyze this reaction, which proceeds in boiling low-polar solvents with intensive water distillation in an air atmosphere, which leads to obtaining anilides of substituted benzoic acids with a yield close to quantitative. The new catalysts, phosphorus trichloride and tribromide, phosphorous acid, successfully complement the catalytic system tetrabutoxytitanate/polybutoxytitanate, previously used for these purposes, allowing to obtain anilides of amino­benzoic acids with greater selectivity, as well as anilides of oxybenzoic acids.
 The study of the kinetic regularities of the reaction catalyzed by phosphorus trichloride showed a weak effect of substituents: |ρ| ≤ 0.61. Hammett dependences are represented by curves with a maximum, straight line segments are characteristic only for certain groups of substituents, both for meta- and para-substituted and for orthosubstituted benzoic acids.
 One of the possible mechanisms of acylation is the formation in situ in the first mi­nutes of the reaction of aniline phosphite, which can act as an oxygen-nucleophilic catalyst, and, upon interaction with substituted benzoic acid, forms the corresponding benzoyl phosphite, which is then attacked by a free aniline molecule to obtain substituted benzanilide.
 The ortho effect for P(III) catalysis is absent or very weak, in the case of aminobenzoic acids. At the same time, anthranilic acid can form a phosphite with phosphorous acid, which directly participates in catalysis.

Acylation of Anthocyanins and Their Applications in the Food Industry: Mechanisms and Recent Research Advances.

Anthocyanins are extensively used as natural non-toxic compounds in the food industry due to their unique biological properties. However, the instability of anthocyanins greatly affects their industrial application. Studies related to acylated anthocyanins with higher stability and increased solubility in organic solvents have shown that the acylation of anthocyanins can improve the stability and fat solubility of anthocyanins. However, relevant developments in research regarding the mechanisms of acylation and applications of acylated anthocyanins are scarcely reviewed. This review aims to provide an overview of the mechanisms of acylation and the applications of acylated anthocyanins in the food industry. In the review, acylation methods, including biosynthesis, semi-biosynthesis, and chemical and enzymatic acylation, are elaborated, physicochemical properties and biological activities of acylated anthocyanins are highlighted, and their application as colourants, functionalizing agents, intelligent indicators, and novel packaging materials in the food industry are summarized. The limitations encountered in the preparation of acylated anthocyanins and future prospects, their applications are also presented. Acylated anthocyanins present potential alternatives to anthocyanins in the food industry due to their functions and advantages as compared with non-acylated analogues. It is hoped that this review will offer further information on the effective synthesis and encourage commercialization of acylated anthocyanins in the food industry.

Mechanisms and inhibition of Porcupine-mediated Wnt acylation.

Wnt signalling is essential for regulation of embryonic development and adult tissue homeostasis1-3, and aberrant Wnt signalling is frequently associated with cancers4. Wnt signalling requires palmitoleoylation on a hairpin 2 motif by the endoplasmic reticulum-resident membrane-bound O-acyltransferase Porcupine5-7 (PORCN). This modification is indispensable for Wnt binding to its receptor Frizzled, which triggers signalling8,9. Here we report four cryo-electron microscopy structures of human PORCN: the complex with the palmitoleoyl-coenzyme A (palmitoleoyl-CoA) substrate; the complex with the PORCN inhibitor LGK974, an anti-cancer drug currently in clinical trials10; the complex with LGK974 and WNT3A hairpin 2 (WNT3Ap); and the complex with a synthetic palmitoleoylated WNT3Ap analogue. The structures reveal that hairpin 2 of WNT3A, which is well conserved in all Wnt ligands, inserts into PORCN from the lumenal side, and the palmitoleoyl-CoA accesses the enzyme from the cytosolic side. The catalytic histidine triggers the transfer of the unsaturated palmitoleoyl group to the target serine on the Wnt hairpin 2, facilitated by the proximity of the two substrates. The inhibitor-bound structure shows that LGK974 occupies the palmitoleoyl-CoA binding site to prevent the reaction. Thus, this work provides a mechanism for Wnt acylation and advances the development of PORCN inhibitors for cancer treatment.

Biochemical Mechanisms of Sirtuin-Directed Protein Acylation in Hepatic Pathologies of Mitochondrial Dysfunction.

Mitochondrial protein acetylation is associated with a host of diseases including cancer, Alzheimer’s, and metabolic syndrome. Deciphering the mechanisms regarding how protein acetylation contributes to disease pathologies remains difficult due to the complex diversity of pathways targeted by lysine acetylation. Specifically, protein acetylation is thought to direct feedback from metabolism, whereby nutritional status influences mitochondrial pathways including beta-oxidation, the citric acid cycle, and the electron transport chain. Acetylation provides a crucial connection between hepatic metabolism and mitochondrial function. Dysregulation of protein acetylation throughout the cell can alter mitochondrial function and is associated with numerous liver diseases, including non-alcoholic and alcoholic fatty liver disease, steatohepatitis, and hepatocellular carcinoma. This review introduces biochemical mechanisms of protein acetylation in the regulation of mitochondrial function and hepatic diseases and offers a viewpoint on the potential for targeted therapies.

Insights into acylation mechanisms: co-expression of serine carboxypeptidase-like acyltransferases and their non-catalytic companion paralogs.

SUMMARYSerine carboxypeptidase‐like acyltransferases (SCPL‐ATs) play a vital role in the diversification of plant metabolites. Galloylated flavan‐3‐ols highly accumulate in tea (Camellia sinensis), grape (Vitis vinifera), and persimmon (Diospyros kaki). To date, the biosynthetic mechanism of these compounds remains unknown. Herein, we report that two SCPL‐AT paralogs are involved in galloylation of flavan‐3‐ols: CsSCPL4, which contains the conserved catalytic triad S‐D‐H, and CsSCPL5, which has the alternative triad T‐D‐Y. Integrated data from transgenic plants, recombinant enzymes, and gene mutations showed that CsSCPL4 is a catalytic acyltransferase, while CsSCPL5 is a non‐catalytic companion paralog (NCCP). Co‐expression of CsSCPL4 and CsSCPL5 is likely responsible for the galloylation. Furthermore, pull‐down and co‐immunoprecipitation assays showed that CsSCPL4 and CsSCPL5 interact, increasing protein stability and promoting post‐translational processing. Moreover, phylogenetic analyses revealed that their homologs co‐exist in galloylated flavan‐3‐ol‐ or hydrolyzable tannin‐rich plant species. Enzymatic assays further revealed the necessity of co‐expression of those homologs for acyltransferase activity. Evolution analysis revealed that the mutations of the CsSCPL5 catalytic residues may have taken place about 10 million years ago. These findings show that the co‐expression of SCPL‐ATs and their NCCPs contributes to the acylation of flavan‐3‐ols in the plant kingdom.

Chemical Biology Tools for Protein Lysine Acylation.

Lysine acylation plays pivotal roles in cell physiology, including DNA transcription and repair, signal transduction, immune defense, metabolism, and many other key cellular processes. Molecular mechanisms of dysregulated lysine acylation are closely involved in the pathophysiological progress of many human diseases, most notably cancers. In recent years, chemical biology tools have become instrumental in studying the function of post-translational modifications (PTMs), identifying new "writers", "erasers" and "readers", and in targeted therapies. Here, we describe key developments in chemical biology approaches that have advanced the study of lysine acylation and its regulatory proteins (2016-2021). We further discuss the discovery of ligands (inhibitors and PROTACs) that are capable of targeting regulators of lysine acylation. Next, we discuss some current challenges of these chemical biology probes and suggest how chemists and biologists can utilize chemical probes with more discriminating capacity. Finally, we suggest some critical considerations in future studies of PTMs from our perspective.

Chemical Biology Tools for Protein Lysine Acylation

AbstractLysine acylation plays pivotal roles in cell physiology, including DNA transcription and repair, signal transduction, immune defense, metabolism, and many other key cellular processes. Molecular mechanisms of dysregulated lysine acylation are closely involved in the pathophysiological progress of many human diseases, most notably cancers. In recent years, chemical biology tools have become instrumental in studying the function of post‐translational modifications (PTMs), identifying new “writers”, “erasers” and “readers”, and in targeted therapies. Here, we describe key developments in chemical biology approaches that have advanced the study of lysine acylation and its regulatory proteins (2016–2021). We further discuss the discovery of ligands (inhibitors and PROTACs) that are capable of targeting regulators of lysine acylation. Next, we discuss some current challenges of these chemical biology probes and suggest how chemists and biologists can utilize chemical probes with more discriminating capacity. Finally, we suggest some critical considerations in future studies of PTMs from our perspective.

A DFT study on the mechanism and regioselectivity of NHC-catalyzed double acylation of aromatic 1,2-diketones with α,β-unsaturated ketones

The possible mechanisms and the origin of regioselectivity of the N-heterocyclic carbene (NHC)-catalyzed double acylation reaction of aromatic 1,2-diketones with α,β-unsaturated ketones have been theoretically studied using density functional theory.

Acylation of m-cresol with acetic acid supported by in-situ ester formation on H-ZSM-5 zeolites

Friedel-Crafts acylation of phenolic compounds with carboxylic acids catalyzed by Brønsted-acid zeolites is an interesting reaction that could find application in the production of fuels and chemicals from renewable feedstocks. The mechanism of direct acylation of m-cresol with acetic acid over protonic zeolites and the role that the confining environment may have on reaction rate and product selectivity are topics of current interest. Here, we evaluate this reaction at varying conditions over H-ZSM-5 catalysts with different acid site densities. By decoupling the effect of parasitic phenomena – diffusion and catalyst deactivation – we have been able to evaluate the true kinetics of this reaction. We have found that, in the absence of a solvent, the reaction over H-ZSM-5 zeolites under commonly studied conditions (Si/Al = 40) at 250 °C is often influenced by the diffusion of reactants to active sites. By further decreasing active site density and operating under diffusion-free conditions, the true activation barrier for acetophenone (2-hydroxyl-4-methylacetophenone) formation obtained on H-ZSM-5 under surface-saturation conditions is relatively low (41 kJ/mol). This value is significantly lower than the barrier associated with the dehydration of the carboxylic acid to form surface acyl species when not assisted by the formation of intermediate ester species. Based on reaction kinetic analysis, it is proposed that the formation of acylium ion from adsorbed m-tolyl acetate is the rate-limiting step. However, feeding the intermediate ester – i.e., m-tolyl acetate – to act as acylating agent, results in reduced rates compared to those in which the acylating agent is acetic acid. This result reveals that diffusion of m-tolyl acetate is slow and that the kinetically relevant ester is formed within the confines of the zeolite pores. By co-feeding water with m-tolyl acetate, we reveal that the bulky ester is hydrolyzed to form smaller molecules that diffuse faster inside zeolite pores and subsequently enhance the reaction rate. Vapor phase acylation of m-cresol with acetic acid over low acid site density H-Beta zeolite confirms the role of the active intermediate ester in accelerating the rate of acylated product formation. Catalyst deactivation during reaction increases with the partial pressure of acetic acid, even under the zero-order kinetic regime, during which the partial pressure of produced gaseous ketene remains constant. This result would indicate that this usual coke precursor is not the main cause of deactivation, but rather bulkier compounds formed by subsequent esterification of acetic acid with the acylated product (e.g., 2-hydroxyl-4-methylacetophenone).

Substrate and product complexes reveal mechanisms of Hedgehog acylation by HHAT.

Hedgehog proteins govern crucial developmental steps in animals and drive certain human cancers. Before they can function as signaling molecules, Hedgehog precursor proteins must undergo amino-terminal palmitoylation by Hedgehog acyltransferase (HHAT). We present cryo-electron microscopy structures of human HHAT in complex with its palmitoyl-coenzyme A substrate and of a product complex with a palmitoylated Hedgehog peptide at resolutions of 2.7 and 3.2 angstroms, respectively. The structures reveal how HHAT overcomes the challenges of bringing together substrates that have different physiochemical properties from opposite sides of the endoplasmic reticulum membrane within a membrane-embedded active site for catalysis. These principles are relevant to related enzymes that catalyze the acylation of Wnt and of the appetite-stimulating hormone ghrelin. The structural and mechanistic insights may advance the development of inhibitors for cancer.

Visible‐Light‐Induced Decarboxylative Acylation of Pyridine N‐Oxides with α‐Oxocarboxylic Acids Using Fluorescein Dimethylammonium as a Photocatalyst

AbstractHerein, the development of a visible‐light‐induced catalytic system to achieve the decarboxylative acylation of pyridine N‐oxides with α‐oxocarboxylic acids, at room temperature and using the organic dye fluorescein dimethylammonium as a new type of photocatalyst, is reported. A series of 2‐arylacylpyridine N‐oxides were selectively synthesized in moderate to good yields by controlling the polarity of the reaction solvent. The developed strategy was successfully applied in the synthesis of an important intermediate of the drug, acrivastine, on a gram scale. Notably, this is the first time that fluorescein dimethylammonium has been used to catalyze the Minisci‐type C−H decarboxylative acylation reaction. The mechanism of decarboxylative acylation was studied by capturing adducts of acyl radicals and 1,1‐diphenylethylene to confirm a radical mechanism. The disclosed catalytic system provides a green synthetic strategy for decarboxylative acylation without the use of additional oxidants or metal catalysts.magnified image

Mechanistic insight on the inhibition of D, D-carboxypeptidase from Mycobacterium tuberculosis by β-lactam antibiotics: an ONIOM acylation study

Mycobacterium tuberculosis cell wall is intricate and impermeable to many agents. A D, D-carboxypeptidase (DacB1) is one of the enzymes involved in the biosynthesis of cell wall peptidoglycan and catalyzes the terminal D-alanine cleavage from pentapeptide precursors. Catalytic activity and mechanism by which DacB1 functions is poorly understood. Herein, we investigated the acylation mechanism of DacB1 by β-lactams using a 6-membered ring transition state model that involves a catalytic water molecule in the reaction pathway. The full transition states (TS) optimization plus frequency were achieved using the ONIOM (B3LYP/6-31 + G(d): AMBER) method. Subsequently, the activation free energies were computed via single-point calculations on fully optimized structures using B3LYP/6-311++(d,p): AMBER and M06-2X/6-311++(d,p): AMBER with an electronic embedding scheme. The 6-membered ring transition state is an effective model to examine the inactivation of DacB1 via acylation by β-lactams antibiotics (imipenem, meropenem, and faropenem) in the presence of the catalytic water. The ΔG# values obtained suggest that the nucleophilic attack on the carbonyl carbon is the rate-limiting step with 13.62, 19.60 and 30.29 kcal mol−1 for Imi–DacB1, Mero–DacB1 and Faro–DacB1, respectively. The electrostatic potential (ESP) and natural bond orbital (NBO) analysis provided significant electronic details of the electron-rich region and charge delocalization, respectively, based on the concerted 6-membered ring transition state. The stabilization energies of charge transfer within the catalytic reaction pathway concurred with the obtained activation free energies. The outcomes of this study provide important molecular insight into the inactivation of D, D-carboxypeptidase by β-lactams. Communicated by Ramaswamy H. Sarma

Kinetics of 2-Methylfuran Acylation with Fatty Acid Anhydrides for Biorenewable Surfactants

Friedel-Crafts acylation using long-chain fatty acid derivatives and biomass-derived furans is the key reaction to produce alkyl furan ketones, an important precursor for biorenewable oleo-furan surfactants. In this work, the steady-state acylation kinetics and reaction mechanism were investigated using a model system of 2-methylfuran and n-octanoic anhydride in a fixed-bed tubular reactor using Al-MCM-41, a mesoporous aluminosilicate. An apparent activation energy (15.5 +or- 1.4 kcal mol- 1) was obtained for the formation of the acylated product, 2-octanoyl-5-methylfuran (2O5MF), for a temperature range of 348-408 K. The apparent reaction rate orders were ~0.6 and ~0.5 in the 2-methylfuran and anhydride concentrations, respectively, while near-zero apparent rate orders were measured in the product concentrations, indicating negligible product inhibition. An Eley-Rideal catalytic acylation mechanism was proposed to explain the experimentally observed apparent rate orders.