Enzymatic biosynthesis of novel 2-(2-phenylethyl)chromone glycosides catalyzed by UDP-glycosyltransferase UGT71BD1.

2-(2-Phenylethyl)chromones (PECs) are the main bioactive components of agarwood which showed diverse pharmaceutical activities. Glycosylation is a useful structural modification method to improve compounds' druggability. However, PEC glycosides were rarely reported in nature which largely limited their further medicinal investigations and applications. In this study, the enzymatic glycosylation of four naturally separated PECs 1-4 was achieved using a promiscuous glycosyltransferase UGT71BD1 identified from Cistanche tubulosa. It could accept UDP-Glucose, UDP-N-acetylglucosamine and UDP-xylose as sugar donors and conduct the corresponding O-glycosylation of 1-4 with high conversion efficiencies. Three O-glucosylated products 1a (5-hydroxy-2-(2-phenylethyl)chromone 8-O-ß-D-glucopyranoside), 2a (8-chloro-2-(2-phenylethyl)chromone 6-O-ß-D-glucopyranoside) and 3a (2-(2-phenylethyl)chromone 6-O-ß-D-glucopyranoside) were prepared and structurally elucidated as novel PEC glucosides based on NMR spectroscopic analyses. Subsequent pharmaceutical evaluation found that 1a showed remarkably improved cytotoxicity against HL-60 cells, whose cell inhibition rate was 19 times higher than that of its aglycon 1. The IC50 value of 1a was further determined to be 13.96 ± 1.10 µM, implying its potential as a promising antitumor-leading candidate. To improve the production of 1, docking, simulation and site-directed mutagenesis were performed. The important role of P15 in the glucosylation of PECs was discovered. Besides, a mutant K288A with a two-fold increased yield for 1a production was also afforded. This research reported the enzymatic glycosylation of PECs for the first time, and also provide an eco-friendly pathway for the alternative production of PEC glycosides for leading compounds discovery.

Improved preparative enzymatic glycosylation of vancomycin aglycon and analogues.

Modifications to the enzymatic glycosylation of vancomycin and its residue 4 thioamide analogue are detailed that significantly reduce the enzyme loading and amount of glycosyl donor needed for each glycosylation reaction, provide a streamlined synthesis and replacement for the synthetic UDP-vancosamine glycosyl donor to improve both access and storage stability, and permit a single-pot, two-step conversion of the aglycons to the fully glycosylated synthetic glycopeptides now conducted at higher concentrations. The improvements are exemplified with the two-step, one-pot glycosylation of [Ψ[C(=S)NH]Tpg4]vancomycin aglycon (92%) conducted on a 400 mg scale (2 mg to 1 g scales) and vancomycin aglycon itself (5 mg scale, 84%).

The first report of enzymatic transglycosylation catalyzed by family GH84 N-acetyl-ß-d-glucosaminidase using a sugar oxazoline derivative as a glycosyl donor.

O-Glycosylated N-acetyl-ß-d-glucosamine-selective N-acetyl-ß-d-glucosaminidase (O-GlcNAcase), belonging to glycoside hydrolase family 84 (GH84), is known as a retaining glycosidase with the possibility of enzymatic transglycosylation. However, no enzymatic transglycosylation catalyzed by GH84 O-GlcNAcase has been reported. Here, enzymatic transglycosylation catalyzed by GH84 O-GlcNAcase was first reported. The enzymatic transglycosylation catalyzed by the GH84 O-GlcNAcase from Bacteroides thetaiotaomicron (BtGH84 O-GlcNAcase) was attained using 1,2-oxazoline derivative of N-acetyl-d-glucosamine (GlcNAc oxazoline) as a glycosyl donor substrate. The ß-linked N-acetyl-d-glucosamine (GlcNAc) derivative was enzymatically synthesized using N-(2-hydroxyethyl)acrylamide as an acceptor substrate. Interestingly, the ß1,6-linked disaccharide derivative of GlcNAc was also obtained in the case of using the GlcNAc derivative with a triazole-linked acrylamide group as an acceptor substrate. Additionally, a one-pot chemo-enzymatic transglycosylation starting from unprotected GlcNAc through GlcNAc oxazoline successfully showed through the combination with the direct synthesis of GlcNAc oxazoline in water and the enzymatic transglycosylation.

The mechanism of in vitro non-enzymatic glycosylation inhibition by Tartary buckwheat's rutin and quercetin.

Tartary buckwheat is rich in rutin, quercetin, and other flavonoids, which exert prominent effects by inhibiting non-enzymatic glycosylation. In this study, an in vitro non-enzymatic glycosylation model was established, and the inhibitory effects of rutin and quercetin on the early, middle, and late products of non-enzymatic glycosylation were determined. Furthermore, their effects on the formation of advanced glycation end products (AGEs) and on protein functional groups and secondary structure were analyzed. These findings provided a theoretical basis for further investigation of the mechanism via which Tartary buckwheat's rutin and quercetin inhibited non-enzymatic glycosylation. The results showed that rutin and quercetin inhibited the formation of fructosamine, dicarbonyl compounds, and fluorescent AGE in a concentration-dependent manner. Rutin and quercetin exhibited antioxidant activity and could reduce the formation of protein oxidation products. The highest clearance rates for DPPH and ABTS+ were 62.74 % and 71.14 %, respectively.

Composition, anti-LDL oxidation, and non-enzymatic glycosylation inhibitory activities of the flavonoids from Mesembryanthemum crystallinum.

This study identified the constituents of purified flavonoid (PEF) isolated from Mesembryanthemum crystallinum and examined their inhibitory effects on low-density lipoprotein (LDL) oxidation and non-enzymatic glycosylation. More than 30 kinds of flavonoid compounds were identified in M. crystallinum, including tangeretin, nobiletin, farrerol, protocatechuic aldehyde, diosmin, and rutin. Moreover, tangeretin corresponds to approximately 51% of the total identified flavonoids. PEF had a low IC50 value for 1,1-diphenyl-2-picrylhydrazyl radicals (DPPH·), hydroxyl radical (·OH), and superoxide anion free radical (O 2 - · ) scavenging. They were found to effectively delay and inhibit the production of conjugated diene (CD) and thiobarbituric acid reactive substance (TBARS) during LDL oxidation. Meanwhile, scanning electron microscopy (SEM) of the LDL oxidation incubation system with PEF showed a smooth and dense surface, with no obvious cavitation phenomenon. Furthermore, PEF effectively inhibited the production of LDL glycosylation products and showed a strong inhibitory effect in the latter stage. The electrophoresis of advanced glycosylation end products (AGEs) further confirmed that PEF can effectively prevent the cross-linking between glucose and proteins, protecting LDL from glycosylation-induced damage.

Enzymatic Synthesis of Resveratrol α-Glucoside by Amylosucrase of Deinococcus geothermalis.

Glycosylation of resveratrol was carried out by using the amylosucrase of Deinococcus geothermalis, and the glycosylated products were tested for their solubility, chemical stability, and biological activities. We synthesized and identified these two major glycosylated products as resveratrol-4'-O-α-glucoside and resveratrol-3-O-α-glucoside by nuclear magnetic resonance analysis with a ratio of 5:1. The water solubilities of the two resveratrol-α-glucoside isomers (α-piceid isomers) were approximately 3.6 and 13.5 times higher than that of ß-piceid and resveratrol, respectively, and they were also highly stable in buffered solutions. The antioxidant activity of the α-piceid isomers, examined by radical scavenging capability, showed it to be initially lower than that of resveratrol, but as time passed, the α-piceid isomers' activity reached a level similar to that of resveratrol. The α-piceid isomers also showed better inhibitory activity against tyrosinase and melanin synthesis in B16F10 melanoma cells than ß-piceid. The cellular uptake of the α-piceid isomers, which was assessed by ultra-performance liquid chromatography (UPLC) analysis of the cell-free extracts of B16F10 melanoma cells, demonstrated that the glycosylated form of resveratrol was gradually converted to resveratrol inside the cells. These results indicate that the enzymatic glycosylation of resveratrol could be a useful method for enhancing the bioavailability of resveratrol.

Utilization of Maillard reaction in moist-dry-heating system to enhance physicochemical and antioxidative properties of dried whole longan fruit.

This research aimed to enhance the physicochemical and antioxidant properties of dried whole longan fruit using Maillard reaction or non-enzymatic glycosylation (glycation) in a moist-dry-heating system at 60 °C with approximately 75% relative humidity for 5-50 days. During Maillard reaction, the browning index (BI) of the fruits increased significantly while lightless, redness and yellowness decreased. Interestingly, the rare sugars especially D-psicose and D-allose gradually increased by 2-3 folds when compared to the initial Maillard reaction. The development of D-mannose was additionally established through the glycation. The degree of glycation increased with the decrease of free amino acid, suggesting that conjugation of sugar with amino acids was involved. SDS-PAGE confirmed that the high molecular weight (HMW) of conjugated sugar-amino acid was the Maillard reaction product. The antioxidative properties including DPPH and ABTS radical scavenging activities, also ferric reducing antioxidant power (FRAP) were also increased as Maillard reaction progressed, which showed the activities in the range of 43.2-94.1 mg GAE/100 g dry basis, 0.23-3.09 g TE/100 g dry basis, and 0.35-5.95 g FeSO4/100 g dry basis, respectively. This study demonstrated a practical approach of Maillard reaction for the development of dried longan fruit with high antioxidative properties.

Anion exchange resins in phosphate form as versatile carriers for the reactions catalyzed by nucleoside phosphorylases.

In the present work, we suggested anion exchange resins in the phosphate form as a source of phosphate, one of the substrates of the phosphorolysis of uridine, thymidine, and 1-(ß-ᴅ-arabinofuranosyl)uracil (Ara-U) catalyzed by recombinant E. coli uridine (UP) and thymidine (TP) phosphorylases. α-ᴅ-Pentofuranose-1-phosphates (PF-1Pis) obtained by phosphorolysis were used in the enzymatic synthesis of nucleosides. It was found that phosphorolysis of uridine, thymidine, and Ara-U in the presence of Dowex® 1X8 (phosphate; Dowex-nPi) proceeded smoothly in the presence of magnesium cations in water at 20-50 °C for 54-96 h giving rise to quantitative formation of the corresponding pyrimidine bases and PF-1Pis. The resulting PF-1Pis can be used in three routes: (1) preparation of barium salts of PF-1Pis, (2) synthesis of nucleosides by reacting the crude PF-1Pi with an heterocyclic base, and (3) synthesis of nucleosides by reacting the ionically bound PF-1Pi to the resin with an heterocyclic base. These three approaches were tested in the synthesis of nelarabine, kinetin riboside, and cladribine with good to excellent yields (52-93%).

Leloir glycosyltransferases of natural product C-glycosylation: structure, mechanism and specificity.

A prominent attribute of chemical structure in microbial and plant natural products is aromatic C-glycosylation. In plants, various flavonoid natural products have a ß-C-d-glucosyl moiety attached to their core structure. Natural product C-glycosides have attracted significant attention for their own unique bioactivity as well as for representing non-hydrolysable analogs of the canonical O-glycosides. The biosynthesis of natural product C-glycosides is accomplished by sugar nucleotide-dependent (Leloir) glycosyltransferases. Here, we provide an overview on the C-glycosyltransferases of microbial, plant and insect origin that have been biochemically characterized. Despite sharing basic evolutionary relationships, as evidenced by their common membership to glycosyltransferase family GT-1 and conserved GT-B structural fold, the known C-glycosyltransferases are diverse in the structural features that govern their reactivity, selectivity and specificity. Bifunctional glycosyltransferases can form C- and O-glycosides dependent on the structure of the aglycon acceptor. Recent crystal structures of plant C-glycosyltransferases and di-C-glycosyltransferases complement earlier structural studies of bacterial enzymes and provide important molecular insight into the enzymatic discrimination between C- and O-glycosylation. Studies of enzyme structure and mechanism converge on the view of a single displacement (SN2)-like mechanism of enzymatic C-glycosyl transfer, largely analogous to O-glycosyl transfer. The distinction between reactions at the O- or C-acceptor atom is achieved through the precise positioning of the acceptor relative to the donor substrate in the binding pocket. Nonetheless, C-glycosyltransferases may differ in the catalytic strategy applied to induce nucleophilic reactivity at the acceptor carbon. Evidence from the mutagenesis of C-glycosyltransferases may become useful in engineering these enzymes for tailored reactivity.

Antioxidant and antiglycation activities of traditional plants and identification of bioactive compounds from extracts of Hordeum vulgare by LC-MS and GC-MS.

Glycation has been involved in Schiff base reaction lead to hyperglycemia at cellular level. The current study aimed to identify the bioactive compounds from selected folkloric plants for their antiglycation and antioxidant potential. Methanol extracts demonstrated the highest activities, therefore, it was further fractionated using n-hexane, dichloromethane, ethyl acetate, and methanol solvents to isolate the nonpolar compounds from the Hordeum vulgare. Moreover, n-hexane and dichloromethane fractions of H. vulgare demonstrated the best antioxidant (61.58% and 62.89%) and antiglycation activities (72.52% and 61.52%) at 2 mg/ml, respectively. Analytical techniques of LC-MS and GC-MS were employed for identification of bioactive compounds; Biochanin A in dichloromethane (DCM) and Vitamin E in n-hexane fractions. There was a strong correlation between antioxidant and antiglycation activities (r = 0.97 and r = 0.96) of DCM & n-hexane fractions of H. vulgare. Findings of this study established the role of Biochanin A and Vit E from H. vulgare as potent antiglycation agents. PRACTICAL APPLICATIONS: The results of this study confirmed the potential role of Black Barley has involved in the inhibition of protein glycation, which can be the potential treatment to reduce the complications of Diabetic Patients. The Black Barley has a rich source of identified compounds Biochanin A and Vitamin E. We can use this plant as a staple food in curing the severity of diabetes. The other practical approach is to use this plant as an ingredient of different food products. The extraction of identified bioactive compounds from the plant will be a good and cheap source of the treatment.

Chemoenzymatic synthesis of carboxylate-terminated maltooligosaccharides and their use for cross-linking of chitin.

In this paper, we report chemoenzymatic synthesis of maltooligosaccharides having carboxylate groups at both ends (carboxylate-terminated maltooligosaccharides, GlcA-Glcn-GlcCOONa). The products were further used as cross-linker for water-soluble chitin (WSCh) to obtain network chitins. The synthesis of GlcA-Glcn-GlcCOONa was achieved by thermostable phosphorylase-catalyzed enzymatic α-glucuronylation using α-d-glucuronic acid 1-phosphate with a carboxylated maltooligosaccharide, which was prepared by chemical oxidation at the reducing end of maltoheptaose with sodium hypoiodite. The structures of GlcA-Glcn-GlcCOONa were evaluated by 1H NMR and MALDI-TOF mass spectra. The obtained GlcA-Glcn-GlcCOONa were used as cross-linker for WSCh by condensation in the presence of condensing agent. The reaction mixtures totally turned into hydrogel form in most cases. Morphologies of lyophilized samples (cryogels) from the hydrogels were evaluated by SEM measurement. The hydrogels could be converted into films by pressing. Furthermore, mechanical properties of the hydrogels and films were investigated by compression and tensile tests, respectively.

Evaluation of Immunoreactivity of Pea (Pisum sativum) Albumins in BALB/c and C57BL/6 Mice.

Green pea (Pisum sativum) is a component of European cuisine; however, an estimated 0.8% of Europeans suffer from allergies to pea proteins. We examined the immunoreactive potential of pea albumins (PA) in BALB/c and C57BL/6 mice. Mice were orally gavaged with PA or glycated pea albumins (G-PA) for 10 consecutive days, in combination with an adjuvant. Both PA and G-PA increased PA-specific serum antibody titers to about 212 for anti-PA IgG, ∼27 for anti-PA IgA, and ∼27.8 for anti-PA IgA in fecal extracts (p < 0.001). On day 42 postexposure, the antibodies titers decreased and were greater in BALB/c compared to C57BL/6 mice (p < 0.05). Distribution of CD4+ and CD8+ T cells in lymphoid tissues presented strain-specific differences. PA was found to induce lymphocyte proliferation; however, G-PA did not. Both PA and G-PA changed CD4+ and CD8+ T cells percentages in some lymphoid tissues; however, this did not impact cytokines production by splenocyte cultures evidenced by the stimulation of Th1, Th2, and Th17 cells. The observed immunomodulatory properties of PA and G-PA and lack of a sign of allergic reaction render them suitable for supplements in personalized diets, but further research is needed to precisely understand this activity.

Aqueous One-pot Synthesis of Glycopolymers by Glycosidase-catalyzed Glycomonomer Synthesis Using 4,6-Dimetoxy Triazinyl Glycoside Followed by Radical Polymerization.

Glycopolymers have attracted increased attention as functional polymeric materials, and simple methods for synthesizing glycopolymers remain needed. This paper reports the aqueous one-pot and chemoenzymatic synthesis of four types of glycopolymers via two reactions: the ß-galactosidase-catalyzed glycomonomer synthesis using 4,6-dimetoxy triazinyl ß-D-galactopyranoside and hydroxy group-containing (meth)acrylamide and (meth)acrylate derivatives as the activated glycosyl donor substrate and as the glycomonomer precursors, respectively, followed by radical copolymerization of the resulting glycomonomer and excess glycomonomer precursor without isolating the glycomonomers. The resulting glycopolymers bearing galactose moieties exhibited specific and strong interactions with the lectin peanut agglutinin as glycoclusters.

Liquid chromatography quadrupole-Orbitrap mass spectrometry for the simultaneous analysis of advanced glycation end products and protein-derived cross-links in food and biological matrices.

Advanced glycation end products (AGEs) and protein cross-links have been extensively investigated in both food and biomedical fields over the past years. Although there are a few chromatographic and immunological methods for the analysis of selected AGEs, there is no method available for comprehensive simultaneous analysis of major AGEs found in processed foods and biological samples. In the present study, we have reported a validated UHPLC-MS/MS method for simultaneous identification and quantification of 15 different AGEs, furosine (an indicator of Amadori products), 2 protein-derived cross-links (lanthionine and lysinoalanine) and 2 amino acids (Lys and Arg). The analytes were separated on a reversed phase C-18 column and quantified accurately based on the isotope dilution method, where 9 stable isotope-labelled internal standards were used to quantify 20 different analytes using an Orbitrap mass analyzer. The method showed acceptable linearity, accuracy and precision. The LOD and LOQ values in plasma were in the range of 0.30-19.02 and 0.87-57.06 ng/mL, respectively. The recovery values at the three spiked levels were in the range of 71-110%, with some exceptions. The intraday and interday precision were in the range of 1.5-13.2%, however, quantification of N-ɛ-(carboxymethyl)lysine accompanied slightly higher interday precision (30.7%). The applicability of the method was successfully assessed by analyzing AGEs and protein cross-links in six different complex matrices including Ultra-High Temperature (UHT) processed milk, roasted chicken breast meat, roasted chicken skin, roasted pork liver, bovine plasma and perfusion liquid.

Effects of piceatannol on the structure and activities of bovine serum albumin: A multi-spectral and molecular modeling studies.

Piceatannol (PIC) displays a wide spectrum of biological activities, such as antioxidation, antibacterial activity and anti-inflammation, but the biochemical and molecular mechanism is not fully understood. In this study, the interaction of PIC with bovine serum albumin (BSA) was studied by fluorescence spectroscopy, ultraviolet-visible absorption spectroscopy, circular dichroism spectroscopy and molecular simulation. The effects of PIC on BSA non-enzymatic glycosylation, fibrillation, thermal stability, and structure information were also studied. The results showed that the formation of PIC-BSA complex by mainly hydrogen-bonding forces resulted in the conformational changes of protein. PIC inhibited the formation of ß-sheets structures of BSA. BSA still maintained the esterase-like good activity in the presence of PIC. In addition, PIC significantly reduced the degree of BSA glycosylation. These results provided a basis for the molecular interaction between PIC and protein, and suggested the potential effect of PIC in preventing the progression of diabetes mellitus.

Protective effects of carnosic acid against aging in a premature cellular senescence model and in a D-galactose induced mouse model / 药学学报

This study aimed to investigate the effect and possible mechanism of carnosic acid (CA) on delaying aging. The effects of CA on senescence-related β-galactosidase (SA-β-Gal) activity and expressions of p53, p21 and p16 were evaluated by an oxidative challenge induced premature 2BS cell senescence model. Meanwhile, the animal experiment was approved by the Ethics Committee of Zhejiang Hospital. Male C57 BL/6J mice were injected with 100 mg·kg-1·d-1 D-galactose (D-gal) for 8 weeks to establish an aging model in vivo, and CA at 5 and 10 mg·kg-1·d-1 were given ig administration at the same time. Morris water maze test was used to test the spatial memory ability. Then the serum and tissue samples were collected for the detections of malondialdehyde (MDA), total superoxide dismutase (T-SOD), interleukin-6 (IL-6), tumor necrosis factor α (TNFα) and advanced glycation end products (AGEs) as well as the protein expression of p53, p21 and p16 in hippocampus of brain. The results showed that H2O2 induced increment of SA-β-Gal activity (95%) was prevented by CA treatment (35%) and the enhanced protein expressions of p53, p21 and p16 in H2O2 exposed 2BS cells were alleviated by CA treatment, suggesting a potent protective role of CA against premature senescence induced by oxidative challenge. For in vivo study, D-gal induced declined spatial memory ability was partly reversed by CA administration. Besides, the serum and cerebral levels of MDA, IL-6, TNFα and AGEs were attenuated by CA treatment when compared to those in model mice. And the protein expressions of p53, p21 and p16 in mice hippocampus were suppressed by CA in D-gal treated mice. Taken together, our results showed that CA protects premature senescence induced by oxidative stress and D-gal, which is related to its antioxidative, antiinflammatory roles and inhibition on non-enzymatic glycosylation.

Deep Eutectic Solvents as New Reaction Media to Produce Alkyl-Glycosides Using Alpha-Amylase from Thermotoga maritima.

Deep Eutectic Solvents (DES) were investigated as new reaction media for the synthesis of alkyl glycosides catalyzed by the thermostable α-amylase from Thermotoga maritima Amy A. The enzyme was almost completely deactivated when assayed in a series of pure DES, but as cosolvents, DES containing alcohols, sugars, and amides as hydrogen-bond donors (HBD) performed best. A choline chloride:urea based DES was further characterized for the alcoholysis reaction using methanol as a nucleophile. As a cosolvent, this DES increased the hydrolytic and alcoholytic activity of the enzyme at low methanol concentrations, even when both activities drastically dropped when methanol concentration was increased. To explain this phenomenon, variable-temperature, circular dichroism characterization of the protein was conducted, finding that above 60 °C, Amy A underwent large conformational changes not observed in aqueous medium. Thus, 60 °C was set as the temperature limit to carry out alcoholysis reactions. Higher DES contents at this temperature had a detrimental but differential effect on hydrolysis and alcoholysis reactions, thus increasing the alcoholyisis/hydrolysis ratio. To the best of our knowledge, this is the first report on the effect of DES and temperature on an enzyme in which structural studies made it possible to establish the temperature limit for a thermostable enzyme in DES.

Biosynthesis of Novel Shikonin Glucosides by Enzymatic Glycosylation.

Shikonin, a natural naphthoquinone, has attracted much attention due to its various biological activities. Two shikonin glucosides, shikonin-1',8-di-O-ß-D-glucopyranoside (1) and shikonin-1'-O-ß-D-glucopyranoside (2), were biosynthesized through in vitro enzymatic glycosylation and their structures were elucidated using spectroscopic techniques. The water-solubility and stability of compounds 1 and 2 were significantly higher than those of the parent compound. Furthermore, compound 2 showed moderate cytotoxicity against six cancer cell lines, with IC50 values ranging from 36.10 to 67.47 µM. This research indicated that in vitro enzymatic glycosylation of shikonin is an effective strategy to improve it water solubility and chemical stability.

Enzymatic synthesis of avermectin B1a glycosides for the effective prevention of the pine wood nematode Bursaphelenchus xylophilus.

Avermectin produced by Streptomyces avermitilis is an anti-nematodal agent against the pine wood nematode Bursaphelenchus xylophilus. However, its potential usage is limited by its poor water solubility. For this reason, continuous efforts are underway to produce new derivatives that are more water soluble. Here, the enzymatic glycosylation of avermectin was catalyzed by uridine diphosphate (UDP)-glycosyltransferase from Bacillus licheniformis with various UDP sugars. As a result, the following four avermectin B1a glycosides were produced: avermectin B1a 4″-ß-D-glucoside, avermectin B1a 4″-ß-D-galactoside, avermectin B1a 4″-ß-L-fucoside, and avermectin B1a 4″-ß-2-deoxy-D-glucoside. The avermectin B1a glycosides were structurally analyzed based on HR-ESI MS and 1D and 2D nuclear magnetic resonance spectra, and the anti-nematodal effect of avermectin B1a 4″-ß-D-glucoside was found to exhibit the highest activity (IC50 = 0.23 µM), which was approximately 32 times greater than that of avermectin B1a (IC50 = 7.30 µM), followed by avermectin B1a 4″-ß-2-deoxy-D-glucoside (IC50 = 0.69 µM), avermectin B1a 4″-ß-L-fucoside (IC50 = 0.89 µM), and avermectin B1a 4″-ß-D-galactoside (IC50 = 1.07 µM). These results show that glycosylation of avermectin B1a effectively enhances its in vitro anti-nematodal activity and that avermectin glycosides can be further applied for treating infestations of the pine wood nematode B. xylophilus.