Acyl-enzyme Research Articles

How Clavulanic Acid Inhibits Serine β-Lactamases.

Clavulanic acid is a medicinally important inhibitor of serine β-lactamases (SBLs). We report studies on the mechanisms by which clavulanic acid inhibits representative Ambler class A (TEM-116), C (Escherichia coli AmpC), and D (OXA-10) SBLs using denaturing and non-denaturing mass spectrometry (MS). Similarly to observations with penam sulfones, most of the results support a mechanism involving acyl enzyme complex formation, followed by oxazolidine ring opening without efficient subsequent scaffold fragmentation (at pH 7.5). This observation contrasts with previous MS studies, which identified clavulanic acid scaffold fragmented species as the predominant SBL bound products. In all the SBLs studied here, fragmentation was promoted by acidic conditions, which are commonly used in LC‑MS analyses. Slow fragmentation was, however, observed under neutral conditions with TEM-116 on prolonged reaction with clavulanic acid. Although our results imply clavulanic acid scaffold fragmentation is likely not crucial for SBL inhibition in vivo, development of inhibitors that fragment to give stable covalent complexes is of interest.

Enzymatic acylation of cyanidin-3-O-glucoside with aromatic and aliphatic acid methyl ester: Structure–stability relationships of acylated derivatives

Anthocyanins are water-soluble pigments, but they tend to be unstable in aqueous solutions. Modification of their molecular structure offers a viable approach to alter their intrinsic properties and enhance stability. Aromatic and aliphatic acid methyl esters were used as acyl donors in the enzymatic acylation of cyanidin-3-O-glucoside (C3G), and their analysis was conducted using ultraperformance liquid chromatography–mass spectrometry (UPLC-MS). The highest conversion rate achieved was 96.41 % for cyanidin-3-O-(6″-feruloyl) glucoside. Comparative evaluations of stability revealed that aromatic acyl group–conjugated C3G exhibited superior stability enhancement compared with aliphatic acyl group derivatives. The stability of aliphatic C3G decreased with increasing carbon chain length. The molecular geometries of different anthocyanins were optimized, and energy level calculations using density functional theory (DFT) identified their sites with antioxidant activities. Computational calculations aligned with the in vitro antioxidant assay results. This study provided theoretical support for stabilizing anthocyanins and broadened the application of acylated anthocyanins as food colorants and nutrient supplements.

Substrate Trapping in Polyketide Synthase Thioesterase Domains: Structural Basis for Macrolactone Formation.

Emerging antibiotic resistance requires continual improvement in the arsenal of antimicrobial drugs, especially the critical macrolide antibiotics. Formation of the macrolactone scaffold of these polyketide natural products is catalyzed by a modular polyketide synthase (PKS) thioesterase (TE). The TE accepts a linear polyketide substrate from the termina PKS acyl carrier protein to generate an acyl-enzyme adduct that is resolved by attack of a substrate hydroxyl group to form the macrolactone. Our limited mechanistic understanding of TE selectivity for a substrate nucleophile and/or water has hampered development of TEs as biocatalysts that accommodate a variety of natural and non-natural substrates. To understand how TEs direct the substrate nucleophile for macrolactone formation, acyl-enzyme intermediates were trapped as stable amides by substituting the natural serine OH with an amino group. Incorporation of the unnatural amino acid, 1,3-diaminopropionic acid (DAP), was tested with five PKS TEs. DAP-modified TEs (TE DAP ) from the pikromycin and erythromycin pathways were purified and tested with six full-length polyketide intermediates from three pathways. The erythromycin TE had permissive substrate selectivity, whereas the pikromycin TE was selective for its native hexaketide and heptaketide substrates. In a crystal structure of a native substrate trapped in pikromycin TE DAP , the linear heptaketide was curled in the active site with the nucleophilic hydroxyl group positioned 4 Å from the amide-enzyme linkage. The curled heptaketide displayed remarkable shape complementarity with the TE acyl cavity. The strikingly different shapes of acyl cavities in TEs of known structure, including those reported here for juvenimicin, tylosin and fluvirucin biosynthesis, provide new insights to facilitate TE engineering and optimization.

Capturing acyl-enzyme intermediates with genetically encoded 2,3-diaminopropionic acid for hydrolase substrate identification.

Catalytic mechanism-based, light-activated traps have recently been developed to identify the substrates of cysteine or serine hydrolases. These traps are hydrolase mutants whose catalytic cysteine or serine are replaced with genetically encoded 2,3-diaminopropionic acid (DAP). DAP-containing hydrolases specifically capture the transient thioester- or ester-linked acyl-enzyme intermediates resulting from the first step of the proteolytic reaction as their stable amide analogs. The trapped substrate fragments allow the downstream identification of hydrolase substrates by mass spectrometry and immunoblotting. In this protocol, we provide a detailed step-by-step guide for substrate capture and identification of the peptidase domain of the large tegument protein deneddylase (UL36USP) from human herpesvirus 1, both in mammalian cell lysate and live mammalian cells. Four procedures are included: Procedure 1, DAP-mediated substrate trapping in mammalian cell lysate (~8 d); Procedure 2, DAP-mediated substrate trapping in adherent mammalian cells (~6 d); Procedure 3, DAP-mediated substrate trapping in suspension mammalian cells (~5 d); and Procedure 4, substrate identification and validation (~12-13 d). Basic skills to perform protein expression in bacteria or mammalian cells, affinity enrichment and proteomic analysis are required to implement the protocol. This protocol will be a practical guide for identifying substrates of serine or cysteine hydrolases either in a complex mixture, where genetic manipulation is challenging, or in live cells such as bacteria, yeasts and mammalian cells.

Spatiotemporal and direct capturing global substrates of lysine-modifying enzymes in living cells

Protein-modifying enzymes regulate the dynamics of myriad post-translational modification (PTM) substrates. Precise characterization of enzyme-substrate associations is essential for the molecular basis of cellular function and phenotype. Methods for direct capturing global substrates of protein-modifying enzymes in living cells are with many challenges, and yet largely unexplored. Here, we report a strategy to directly capture substrates of lysine-modifying enzymes via PTM-acceptor residue crosslinking in living cells, enabling global profiling of substrates of PTM-enzymes and validation of PTM-sites in a straightforward manner. By integrating enzymatic PTM-mechanisms, and genetically encoding residue-selective photo-crosslinker into PTM-enzymes, our strategy expands the substrate profiles of both bacterial and mammalian lysine acylation enzymes, including bacterial lysine acylases PatZ, YiaC, LplA, TmcA, and YjaB, as well as mammalian acyltransferases GCN5 and Tip60, leading to discovery of distinct yet functionally important substrates and acylation sites. The concept of direct capturing substrates of PTM-enzymes via residue crosslinking may extend to the other types of amino acid residues beyond lysine, which has the potential to facilitate the investigation of diverse types of PTMs and substrate-enzyme interactive proteomics.

Strategy for the Enzymatic Acylation of the Apple Flavonoid Phloretin Based on Prior α-Glucosylation.

The acylation of flavonoids serves as a means to alter their physicochemical properties, enhance their stability, and improve their bioactivity. Compared with natural flavonoid glycosides, the acylation of nonglycosylated flavonoids presents greater challenges since they contain fewer reactive sites. In this work, we propose an efficient strategy to solve this problem based on a first α-glucosylation step catalyzed by a sucrose phosphorylase, followed by acylation using a lipase. The method was applied to phloretin, a bioactive dihydrochalcone mainly present in apples. Phloretin underwent initial glucosylation at the 4'-OH position, followed by subsequent (and quantitative) acylation with C8, C12, and C16 acyl chains employing an immobilized lipase from Thermomyces lanuginosus. Electrospray ionization-mass spectrometry (ESI-MS) and two-dimensional nuclear magnetic resonance spectroscopy (2D-NMR) confirmed that the acylation took place at 6-OH of glucose. The water solubility of C8 acyl glucoside closely resembled that of aglycone, but for C12 and C16 derivatives, it was approximately 3 times lower. Compared with phloretin, the radical scavenging capacity of the new derivatives slightly decreased with 2,2-diphenyl-1-picrylhydrazyl (DPPH) and was similar to 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS•+). Interestingly, C12 acyl-α-glucoside displayed an enhanced (3-fold) transdermal absorption (using pig skin biopsies) compared to phloretin and its α-glucoside.

Enzymatically acylated naringin with gut modulation potential

BackgroundNaringin is one of the main flavonoids in citrus fruits and byproducts. This flavanone has been shown to be a good antioxidant nutraceutical component, and it also has potential as a gut microbiome modulator, although its applications in final formulations represent a challenge due to its low solubility, both in water and in organic solvents. This work addresses this problem by functionalizing naringin through enzymatic acylation. ResultsThe enzymatic acylation catalyzed by the lipase Novozym® 435 and using acyl donors of different chain lengths, acetate (C2), propionate (C3), and laurate (C12), yielded in conversions of 95% at 24 h and 100% at 48 h, generating a monoacylated product. Both the aqueous and solvent solubility of acylated naringin products were improved while maintaining or even increasing their antioxidant activity. ConclusionsThis acylation process significantly enhanced both the water and solvent solubility of the acylated naringin products while preserving or even enhancing their antioxidant activity. In addition to the gut-modulating properties of flavonoids, acylating them with short- and medium-chain fatty acids could enhance their potential applications in the emerging field of research dedicated to understanding and modulating gut health.How to cite: Gutiérrez-Navarro E, Padilla-de la Rosa JD, Macías A, et al. Enzymatically acylated naringin with gut modulation potential. Electron J Biotechnol 2024; 68. https://doi.org/10.1016/j.ejbt.2023.12.003.

The chemoenzymatic synthesis of glycan-terminated oligo(Leu)x

Introduction: Glycopeptides contain carbohydrate moieties (glycans) covalently attached to the side chain and/or terminal peptide units. Since glycans are present on cell surfaces, these constructs can potentially address a wide array of therapeutic functions. To overcome the deficiencies associated with current synthetic routes to glycopeptides, such as costly processes and toxic reagents, this work aimed to develop versatile environmentally friendly protease-catalyzed peptide synthesis routes to peptides decorated with a glycan at their N-terminus.Methods: “Grafters” were first synthesized that consist of a glycan conjugated directly, or through a spacer, to the amine group of L-Phe-ethyl ester (Phe-OEt). The role of Phe-OEt is to increase the conjugate’s recognition by the protease (papain) catalytic active site. A series of grafters were synthesized with variation of the glycan structure, linkage-chemistry, and presence of an oligo (ethylene glycol) “spacer” of varied length between the glycan and Phe-OEt moiety. High grafter efficiency will result by the successful acceptance of the grafter at the enzymes S1/S2 subsites, formation of an acyl enzyme complex and subsequent conversion to glycan-terminated oligo(Leu)x (x ≥ 1), as opposed to construction of non-glycan N-terminated oligo(Leu)x.Results and discussion: While glycan-Phe-OEt grafters without a spacer between the glycan and Phe-OEt resulted in low grafter efficiency (8.3% ± 2.0%), insertion of a short oligo (ethylene glycol) spacer between the glycan and Phe-OEt moieties (glycan-PEGn-Phe-OEt, n ≥ 3) increased the grafter efficiency by 3-fold–24.5% ± 1.8%. In addition, computational modeling was performed using Rosetta software provided insights on a molecular level of how grafter efficiency is influenced by the PEG spacer length.

Thioesterases as tools for chemoenzymatic synthesis of macrolactones.

Macrocycles are a key functional group that can impart unique properties into molecules. Their synthesis has led to the development of many outstanding chemical methodologies and yet still remains challenging. Thioesterase (TE) domains are frequently responsible for macrocyclization in natural product biosynthesis and provide unique strengths for the enzymatic synthesis of macrocycles. In this feature article, we describe our work to characterize the substrate selectivity of TEs and to use these enzymes as biocatalysts. Our efforts have shown that the linear thioester activated substrates are loaded on TEs with limited substrate selectivity to generate acyl-enzyme intermediates. We show that cyclization of the acyl-enzyme intermediates can be highly selective, with competing hydrolysis of the acyl-enzyme intermediates. The mechanisms controlling TE-mediated macrocyclization versus hydrolysis are a significant unsolved problem in TE biochemistry. The potential of TEs as biocatalysts was demonstrated by using them in the chemoenzymatic total synthesis of macrocyclic depsipeptide natural products. This article highlights the strengths and potential of TEs as biocatalysts as well as their limitations, opening exciting research opportunities including TE engineering to optimize these powerful biocatalysts.

Efficient regioselective enzymatic acylation of troxerutin: difference characterization of in vitro cellular uptake and cytotoxicity.

In this study, we developed a multi-site acylation strategy to improve the lipophilicity and cellular uptake of troxerutin, a natural flavonoid with many health-promoting bioactivities. By clarifying the acylation properties of troxerutin catalyzed by lipases from different sources, a series of troxerutin ester derivatives acylated at different sites was synthesized, including troxerutin dipropyl (TDP), tripropyl (TTP), tetrapropyl (TEP), dibutyl (TDB), monohexyl (TMH), monooctyl (TMO) and monodecyl (TMD) esters. Interestingly, the troxerutin esters acylated at multiple sites with shorter fatty chains (TDP, TTP and TEP) had similar lipophilicity to the mono-acylated esters bearing longer fatty chains (TMH, TMO and TMD, respectively) and meanwhile demonstrated surprisingly lower cytotoxicity than that of the long fatty-chain mono-esters. In particular, the multi-acylated esters with shorter fatty chains showed remarkably higher cellular uptake than the mono-esters with long fatty chains. In vitro gastrointestinal digestion suggested that the multi-acylated esters of troxerutin were more resistant to gastrointestinal degradation than the mono-esters. These results indicated that multi-site acylation with short fatty chains could be an effective alternative to introducing one-site mono-acylation for the modification of troxerutin and other flavonoid compounds.

A light-driven enzymatic enantioselective radical acylation.

Enzymes are recognized as exceptional catalysts for achieving high stereoselectivities1-3, but their ability to control the reactivity and stereoinduction of free radicals lags behind that of chemical catalysts4. Thiamine diphosphate (ThDP)-dependent enzymes5 are well-characterized systems that inspired the development of N-heterocyclic carbenes (NHCs)6-8 but have not yet been proved viable in asymmetric radical transformations. There is a lack of a biocompatible and general radical-generation mechanism, as nature prefers to avoid radicals that may be harmful to biological systems9. Here we repurpose a ThDP-dependent lyase as a stereoselective radical acyl transferase (RAT) through protein engineering and combination with organophotoredox catalysis10. Enzyme-bound ThDP-derived ketyl radicals are selectively generated through single-electron oxidation by a photoexcited organic dye and then cross-coupled with prochiral alkyl radicals with high enantioselectivity. Diverse chiral ketones are prepared from aldehydes and redox-active esters (35 examples, up to 97% enantiomeric excess (e.e.)) by this method. Mechanistic studies reveal that this previously elusive dual-enzyme catalysis/photocatalysis directs radicals with the unique ThDP cofactor and evolvable active site. This work not only expands the repertoire of biocatalysis but also provides a unique strategy for controlling radicals with enzymes, complementing existing chemical tools.

Enzymatic Acylation of Black Rice Anthocyanins and Evaluation of Antioxidant Capacity and Stability of Their Derivatives.

In this study, the structure of the anthocyanin fractions isolated from black rice (Oryza sativa L.) was modified by the enzyme catalysis method using caffeic acid as an acyl donor. At the same time, the effects of the acylation on the lipophilicity, antioxidant activity, and stability of black rice anthocyanins were comprehensively evaluated. The structural analyses of acylated derivatives based on ultraviolet-visible spectroscopy, Fourier-transform infrared spectroscopy, ultra-high-performance liquid chromatography-high-resolution mass spectrometry, and thermogravimetric analysis revealed that caffeic acid was efficiently grafted onto the anthocyanins of black rice through an acylated reaction, while the acylation binding site was on glucoside. When the mass ratios of anthocyanins to caffeic acid were 1:1, the A319/AVis-max value of acylated anthocyanins reached 6.37. Meanwhile, the lipophilicity of acylated derivatives was enhanced. The antioxidant capacity (DPPH and FRAP) and stability (thermal, pH, and light stability) were significantly increased. Overall, the study results provide deeper insights into controlling anthocyanin homeostasis in food processing, broadening the application of colored grain products.

Nanofructosome encapsulated CalB enzyme immobilized silica-coated magnetic nanoparticles for rapid enzymatic hydrolysis and acylation

Nanofructosome encapsulated CalB enzyme immobilized silica-coated magnetic nanoparticles for rapid enzymatic hydrolysis and acylation

PENICILLIN ACYLASE: A RETROSPECTIVE OF STUDYING THE KINETICS AND THERMODYNAMICS OF PRACTICALLY SIGNIFICANT REACTIONS

The review considers the contribution of works carried out at the scientific school of Ilya Vasilievich Berezin to the study of the kinetics and thermodynamics of penicillin acylase-catalyzed reactions. Methods for determining the activity of penicillin acylases, the reversibility of the enzymatic hydrolysis of a number of penicillins, cephalosporins and related compounds, the influence of the β-lactam ring on the thermodynamics of the synthesis of new penicillins and cephalosporins by direct condensation as well as by acyl transfer, the issues of optimizing the conditions for enzymatic acyl transfer and the use of supersaturated reagent solutions are discussed. The role of chromogenic substrates in the study of penicillin acylase, the possibility of using the methods of titration of active sites of the enzyme and the creation of “smart” biocatalysts based on penicillin acylase due to the formation of conjugates with stimulus-sensitive polymers are considered.

Natural protein engineering in the Ω-loop: the role of Y221 in ceftazidime and ceftolozane resistance in Pseudomonas-derived cephalosporinase.

A wide variety of clinically observed single amino acid substitutions in the Ω-loop region have been associated with increased minimum inhibitory concentrations and resistance to ceftazidime (CAZ) and ceftolozane (TOL) in Pseudomonas-derived cephalosporinase and other class C β-lactamases. Herein, we demonstrate the naturally occurring tyrosine to histidine substitution of amino acid 221 (Y221H) in Pseudomonas-derived cephalosporinase (PDC) enables CAZ and TOL hydrolysis, leading to similar kinetic profiles (k cat = 2.3 ± 0.2 µM and 2.6 ± 0.1 µM, respectively). Mass spectrometry of PDC-3 establishes the formation of stable adducts consistent with the formation of an acyl enzyme complex, while spectra of E219K (a well-characterized, CAZ- and TOL-resistant comparator) and Y221H are consistent with more rapid turnover. Thermal denaturation experiments reveal decreased stability of the variants. Importantly, PDC-3, E219K, and Y221H are all inhibited by avibactam and the boronic acid transition state inhibitors (BATSIs) LP06 and S02030 with nanomolar IC50 values and the BATSIs stabilize all three enzymes. Crystal structures of PDC-3 and Y221H as apo enzymes and complexed with LP06 and S02030 (1.35-2.10 Å resolution) demonstrate ligand-induced conformational changes, including a significant shift in the position of the sidechain of residue 221 in Y221H (as predicted by enhanced sampling well-tempered metadynamics simulations) and extensive hydrogen bonding between the enzymes and BATSIs. The shift of residue 221 leads to the expansion of the active site pocket, and molecular docking suggests substrates orientate differently and make different intermolecular interactions in the enlarged active site compared to the wild-type enzyme.

Acyl CoA oxidase: from its expression, structure, folding, and import to its role in human health and disease.

β-oxidation of fatty acids is an important metabolic pathway and is a shared function between mitochondria and peroxisomes in mammalian cells. On the other hand, peroxisomes are the sole site for the degradation of fatty acids in yeast. The first reaction of this pathway is catalyzed by the enzyme acyl CoA oxidase housed in the matrix of peroxisomes. Studies in various model organisms have reported the conserved function of the protein in fatty acid oxidation. The importance of this enzyme is highlighted by the lethal conditions caused in humans due to its altered function. In this review, we discuss various aspects ranging from gene expression, structure, folding, and import of the protein in both yeast and human cells. Further, we highlight recent findings on the role of the protein in human health and aging, and discuss the identified mutations in the protein associated with debilitating conditions in patients.

Coumarins as factor XIIa inhibitors: Potency and selectivity improvements using a fragment-based strategy

Previously, we described weak coumarin inhibitors of factor XIIa, a promising target for artificial surface-induced thrombosis and various inflammatory diseases. In this work, we used fragment-based drug discovery approach to improve our coumarin series. First, we screened about 200 fragments for the S1 pocket. The S1 pocket of trypsin-like serine proteases, such as factor XIIa, is highly conserved and is known to drive a major part of the association energy. From the screening, we selected fragments displaying a micromolar activity and studied their selectivity on other serine proteases. Then, these fragments were merged to our coumarin templates, leading to the generation of nanomolar inhibitors. The mechanism of inhibition was further studied by mass spectrometry demonstrating the covalent binding through the formation of an acyl enzyme complex. The most potent compound was tested in plasma to evaluate its stability and efficacy on coagulation assays. It exhibited a plasmatic half-life of 1.9 h and a good selectivity for the intrinsic coagulation pathway over the extrinsic one.

Enzymatic Modification of Pomace Olive Oil with Natural Antioxidants: Effect on Oxidative Stability.

Enzymatic lipophilization has been proposed as a cost-effective strategy to produce new liposoluble antioxidant compounds. In this study, modified oils rich in structured phenolipids were prepared via one-pot enzymatic acylation of hydroxytyrosol (HTYR), vanillyl alcohol (VA) and homovanillyl alcohol (HVA) with pomace olive oil (POO) in solvent-free conditions using immobilized lipase on biogenic nanoparticles. The effect of temperature (30-70 °C) and enzyme concentration (0.1-1%, w/w) on the efficiency of the bioprocess as well as the reusability of the nanobiocatalyst were thoroughly investigated. The modified oils exhibited increased antioxidant activity compared to the control oil according to DPPH and CUPRAC assays (p < 0.05). The oxidative stability of pomace olive oil was also significantly enhanced after modification, as depicted by the K232 values and TBARS contents under accelerated oxidation at 60 °C (p < 0.05). Moreover, a fortified mayonnaise containing modified oil with HTYR was prepared that was noticeably stable compared to the control mayonnaise at 28 °C for 5 months (p < 0.05). Enzymatically modified oils have great potential for application in the nutraceutical and food industry, encouraging the exploitation of immobilized lipases as effective and green catalytic tools.

Thermally Stable and Reusable Silica and Nano-Fructosome Encapsulated CalB Enzyme Particles for Rapid Enzymatic Hydrolysis and Acylation.

This study reports the preparation of silica-coated and nano-fructosome encapsulated Candida antarctica lipase B particles (CalB@NF@SiO2) and a demonstration of their enzymatic hydrolysis and acylation. CalB@NF@SiO2 particles were prepared as a function of TEOS concentration (3-100 mM). Their mean particle size was 185 nm by TEM. Enzymatic hydrolysis was performed to compare catalytic efficiencies of CalB@NF and CalB@NF@SiO2. The catalytic constants (Km, Vmax, and Kcat) of CalB@NF and CalB@NF@SiO2 were calculated using the Michaelis-Menten equation and Lineweaver-Burk plot. Optimal stability of CalB@NF@SiO2 was found at pH 8 and a temperature of 35 °C. Moreover, CalB@NF@SiO2 particles were reused for seven cycles to evaluate their reusability. In addition, enzymatic synthesis of benzyl benzoate was demonstrated via an acylation reaction with benzoic anhydride. The efficiency of CalB@NF@SiO2 for converting benzoic anhydride to benzyl benzoate by the acylation reaction was 97%, indicating that benzoic anhydride was almost completely converted to benzyl benzoate. Consequently, CalB@NF@SiO2 particles are better than CalB@NF particles for enzymatic synthesis. In addition, they are reusable with high stability at optimal pH and temperature.

Enzymatic acylation of blueberry (Vaccinium ashei) pomace anthocyanins and determination of their cytotoxic properties

To expand the application of acylated blueberry pomace anthocyanins in food and nutraceuticals, their enzymatic acylation reaction and the cytotoxic response were explored. In this study, enzymatic acylation was carried out with cyanidin-3-O-glucoside (C3G) as the main body and Novozyme 435 as the biocatalyst. With a series of improvements, the reaction was carried out under the conditions of lauric acid as the acyl donor, tert-amyl alcohol as the reaction medium, the molar ratio of C3G to lauric acid of 1:10, the reaction time of 24 h and the reaction temperature of 60 °C, and 35.29 % acylation rate was obtained. Moreover, it was found that the lipophilicity, thermostability and photostability of the enzymatic acylated blueberry pomace anthocyanins were improved compared with the untreated blueberry pomace anthocyanins. Cytotoxicity studies showed that acylated blueberry pomace anthocyanins inhibited tumor cells (HeLa, HepG2, and B16 cells) significantly and were less toxic to normal cells (L929 cells) at the maximum concentration (1000 µg/mL) and the longest duration of action (72 h). This study established a method for acylation of blueberry pomace anthocyanins that may be applied to other natural sources of anthocyanins.