Enzymatic Methoxycarbonylation of Tyrosol and Hydroxytyrosol.

Tyrosol and hydroxytyrosol are powerful phenolic antioxidants occurring in olive oil and in by-products from olive processing. Due to their high polarity, esterification or other lipophilization is necessary to make them compatible with lipid matrices. Hydroxytyrosol methyl carbonate is a more effective antioxidant than dibutylhydroxytoluene or α-tocopherol and together with tyrosol methyl carbonate exerts interesting pharmacological properties.

An Exploratory Study of the Enzymatic Hydroxycinnamoylation of Sucrose and Its Derivatives.

Phenylpropanoid sucrose esters are a large and important group of natural substances with significant therapeutic potential. This work describes a pilot study of the enzymatic hydroxycinnamoylation of sucrose and its derivatives which was carried out with the aim of obtaining precursors of natural phenylpropanoid sucrose esters, e.g.

Acuminosylation of Tyrosol by a Commercial Diglycosidase.

A commercial glycosidase mixture obtained from (Aromase H2) was found to comprise a specific diglycosidase activity, β-acuminosidase, alongside undetectable levels of β-apiosidase. The enzyme was tested in the transglycosylation of tyrosol using 4-nitrophenyl β-acuminoside as the diglycosyl donor. The reaction was not chemoselective, providing a mixture of Osmanthuside H and its counterpart regioisomer 4-(2-hydroxyethyl)phenyl β-acuminoside in 58% yield.

From Hamamelitannin Synthesis to the Study of Enzymatic Acylations of D-Hamamelose.

The bioactive natural substance, hamamelitannin, was effectively synthesized in two ways. The chemical acylation of 2,3--isopropylidene-α,β-D-hamamelofuranose promoted by BuSnO using 3,4,5-tri--acetylgalloyl chloride, followed by the deprotection provided hamamelitannin in 79%. Pilot enzymatic benzoylation of D-hamamelose using vinyl benzoate (4 equiv.

Synthesis of Tyrosol and Hydroxytyrosol Glycofuranosides and Their Biochemical and Biological Activities in Cell-Free and Cellular Assays.

Tyrosol (T) and hydroxytyrosol (HOT) and their glycosides are promising candidates for applications in functional food products or in complementary therapy. A series of phenylethanoid glycofuranosides (PEGFs) were synthesized to compare some of their biochemical and biological activities with T and HOT. The optimization of glycosylation promoted by environmentally benign basic zinc carbonate was performed to prepare HOT α-L-arabino-, β-D-apio-, and β-D-ribofuranosides.

Transrutinosylation of tyrosol by flower buds of Sophora japonica.

Dried flower buds of Japanese sophora (Sophora japonica) comprising rutinosidase activity were tested in rutinosylation of tyrosol via transglycosylation process from rutin. Optimal conditions for transrutinosylation of tyrosol were 49 mM rutin and 290 mM tyrosol, giving maximum conversion up to 66.4% and 24% yield of isolated and purified rutinoside.

Positional specificity of Flavobacterium johnsoniae acetylxylan esterase and acetyl group migration on xylan main chain.

A new Flavovacterium johnsoniae isolate encodes an enzyme that is essentially identical with a recently discovered novel acetylxylan esterase, capable of liberating 3-O-acetyl group from 4-O-methyl-d-glucuronic acid-substituted xylopyranosyl (Xylp) residues (Razeq et al., 2018). In addition to deesterification of the 2-O-MeGlcA-substituted Xylp residues in acetylglucuronoxylan, the enzyme acts equally well on doubly acetylated Xylp residues from which it liberates only the 3-O-acetyl groups, leaving the 2-O-acetyl groups untouched.

Comparative study of relationship between structure of phenylethanoid glycopyranosides and their activities using cell-free assays and human cells cultured in vitro.

The study focused on protective potential of phytochemicals applicable in prevention and health protection is of great importance. Various structures of these compounds and a wide range of their biological activities have inspired organic chemists to sythesize their effective analogues in order to further increase their efficacy. The aims of our study were (i) to synthesize phenylethanoid glycopyranosides: salidroside (SALI - tyrosol β-d-glucopyranoside), tyrosol β-d-galactopyranoside (TYBGAL), tyrosol α-d-galactopyranoside (TYAGAL), tyrosol α-d-mannopyranoside (TYAMAN), hydroxytyrosol α-d-mannopyranoside (HOTAMA), homosyringyl β-d-glucopyranoside (HSYGLU), hydroxytyrosol β-d-xylopyranoside (HOTXYL) and hydroxysalidroside (HOSALI); (ii) to determine their antioxidant capacities (cell-free approaches); (iii) to evaluate their cytotoxicity (MTT test), protectivity against hydrogen peroxide (HO; comet assay) and effect on the intracellular glutathione level (iGSH; flow cytometry) in experimental system utilizing human hepatoma HepG2 cells.

Reaction mechanism of β-apiosidase from Aspergillus aculeatus.

Apiosidases are glycosidases relevant for aroma development during fermentation of wines and black tea. Reaction mechanism of apiosidase from Aspergillus aculeatus in commercial glycanase Viscozyme L was studied by H NMR technique. Study of hydrolysis of 4-nitrophenyl β-D-apiofuranoside revealed that this reaction proceeds with inversion of hydroxyl group in the anomeric center, which confirms inverting mechanism of the enzyme and its inability to catalyze transapiosylation in syntheses of apiosides.

Salidroside, a Chemopreventive Glycoside, Diminishes Cytotoxic Effect of Cisplatin in Vitro.

Natural products represent the source or the inspiration for the majority of the active ingredients of medicines because of their structural diversity and a wide range of biological effects. Our aims in this study were (i) to synthesize enzymatically salidroside (SAL), the most effective phenylethanoid glycoside in Rhodiola species; (ii) to examine its antioxidant capacity using cell-free assays (reducing power, DPPH radicals scavenging and Fe -chelating assays); (iii) to assess its DNA-protective potential on plasmid DNA (DNA topology assay) and in HepG2 cells (comet assay) damaged by Fe ions and hydrogen peroxide, respectively; and (iv) to investigate the effects of SAL, cisplatin (CDDP) and combined treatments of SAL + CDDP on cell viability (MTT test), level of DNA damage (comet assay), proliferation, cell cycle (flow cytometry) and the expression of signalling molecules associated with cell growth and apoptotic pathways (Western immunoblotting). We found out that SAL manifested low antioxidant and DNA-protective capacity in all assays used.

A selective and mild glycosylation method of natural phenolic alcohols.

Several bioactive natural p-hydroxyphenylalkyl β-D-glucopyranosides, such as vanillyl β-D-glucopyranoside, salidroside and isoconiferin, and their glycosyl analogues were prepared by a simple reaction sequence. The highly efficient synthetic approach was achieved by utilizing acetylated glycosyl bromides as well as aromatic moieties and mild glycosylation promoters. The aglycones, p-O-acetylated arylalkyl alcohols, were prepared by the reduction of the corresponding acetylated aldehydes or acids.

Efficient chemoenzymatic synthesis of 4-nitrophenyl β-d-apiofuranoside and its use in screening of β-d-apiofuranosidases.

4-Nitrophenyl β-d-apiofuranoside as a chromogenic probe for detection of β-d-apiofuranosidase activity was prepared in 61% yield from 2,3-isopropylidene-α,β-d-apiofuranose through a sequence of five reactions. The synthesis involves one regioselective enzymatic step-benzoylation of primary hydroxyl of 2,3-isopropylidene-α,β-d-apiofuranose catalysed by Lipolase 100T and stereoselective β-d-apiofuranosylation of p-nitrophenol using BF3⋅OEt2/Et3N. The product was used for screening of β-d-apiofuranosidase activity in 61 samples of crude commercial enzymes and plant materials.

Effective enzymatic caffeoylation of natural glucopyranosides.

Reaction system was developed for enzymatic caffeoylation of model saccharidic acceptor methyl β-d-glucopyranoside to obtain exclusively methyl 6-O-caffeoyl-β-D-glucopyranoside. Reaction with starting concentration of acceptor 0.2 M provided 73% yield of purified product within 17 days.

Synthesis of 4-nitrophenyl caffeate and its use in assays of caffeoyl esterases.

We have prepared 4-nitrophenyl caffeate by a combination of standard procedures of organic synthesis and enzymatic deacetylation. Based on hydrolysis of 4-nitrophenyl caffeate, a convenient spectrophotometric assay was developed for specific monitoring of caffeoyl esterase. The method is fast and easy to perform, and it requires no expensive equipment.

Donor specificity and regioselectivity in Lipolase mediated acylations of methyl α-D-glucopyranoside by vinyl esters of phenolic acids and their analogues.

Methyl α-D-glucopyranoside as a model acceptor was acylated by several phenolic and non-phenolic vinyl esters using immobilised Lipolase. Donor specificity and regioselectivity of reaction were investigated. Conversion and rate of acylation by structurally varied donors indicates that the synthetic reactivity of Lipolase corresponds to the hydrolytic activity of feruloyl esterase type A.

Action of xylan deacetylating enzymes on monoacetyl derivatives of 4-nitrophenyl glycosides of β-D-xylopyranose and α-L-arabinofuranose.

Measurements of esterase activity by enzyme-coupled assays on monoacetates of 4-nitrophenyl β-D-xylopyranoside and 4-nitrophenyl α-L-arabinofuranoside showed that acetylxylan esterases of families 1, 4 and 5 produced by Trichoderma reesei and Penicillium purpurogenum have a strong preference for deacetylation of position 2 in xylopyranosides. The acetylxylan esterases exhibit only weak activity on acetylated arabinofuranosides, with 2-acetate as the best substrate. Acetyl esterases of family 16 produced by the same two fungi deacetylate in xylopyranosides preferentially positions 3 and 4.

Preparation of regioselectively feruloylated p-nitrophenyl alpha-L-arabinofuranosides and beta-D-xylopyranosides-convenient substrates for study of feruloyl esterase specificity.

p-Nitrophenyl alpha-L-arabinofuranoside and beta-D-xylopyranoside mono-O-ferulates were prepared by 4-O-acetylferuloylation of corresponding enzymatically prepared di-O-acetates followed by deacetylation. An alternative mild acylation catalysed by zinc oxide was tested on xylopyranoside derivatives. The chemoselective methanolysis of the acetyl groups using neutral catalyst dibutyltin oxide at reflux was used as deacetylation method.

The vicinal hydroxyl group is prerequisite for metal activation of Clostridium thermocellum acetylxylan esterase.

Positional specificity of NodB-like domain of a multidomain xylanase U from Clostridium thermocellum (CtAxe) was investigated. Of three monoacetates of 4-nitrophenyl beta-d-xylopyranoside the acetylxylan esterase domain showed a clear preference for the 2-acetate. Moreover, the enzyme was significantly activated by Co(2+).

Substrate and positional specificity of feruloyl esterases for monoferuloylated and monoacetylated 4-nitrophenyl glycosides.

4-Nitrophenyl glycosides of 2-, 3-, and 5-O-(E)-feruloyl- and 2- and 5-O-acetyl-alpha-L-arabinofuranosides and of 2-, 3-, and 4-O-(E)-feruloyl- and 2-, 3- and 4-O-acetyl-beta-D-xylopyranosides, compounds mimicking natural substrates, were used to investigate substrate and positional specificity of type-A, -B, and -C feruloyl esterases. All the feruloyl esterases behave as true feruloyl esterases showing negligible activity on sugar acetates. Type-A enzymes, represented by AnFaeA from Aspergillus niger and FoFaeII from Fusarium oxysporum, are specialized for deferuloylation of primary hydroxyl groups, with a very strong preference for hydrolyzing 5-O-feruloyl-alpha-L-arabinofuranoside.

The acetates of p-nitrophenyl alpha-L-arabinofuranoside--regioselective preparation by action of lipases.

All possible di-O-acetates and mono-O-acetates of p-nitrophenyl alpha-L-arabinofuranoside were prepared by chemoenzymatic way using lipases. The 2,3-di-O-acetate was obtained in 90% yield by deacetylation of the primary acetyl group of per-O-acetylated p-nitrophenyl alpha-L-arabinofuranoside by Candida cylindracea lipase (CCL) or Candida rugosa lipase (LAY). The 2,5- and 3,5-di-O-acetates were obtained by acetylation of p-nitrophenyl alpha-L-arabinofuranoside by Pseudomonas cepacia lipase (LPS-30) in organic solvents.

Enzyme-coupled assay of acetylxylan esterases on monoacetylated 4-nitrophenyl beta-D-xylopyranosides.

Three different monoacetates of 4-nitrophenyl beta-D-xylopyranoside were tested as substrates for beta-xylosidase and for microbial carbohydrate esterases and a series of non-hemicellulolytic esterases. The acetyl group in 2-O-acetyl, 3-O-acetyl, and 4-O-acetyl 4-nitrophenyl beta-D-xylopyranoside makes the glycoside resistant to the action of beta-xylosidase (EC 3.2.

Deoxy and deoxyfluoro analogues of acetylated methyl beta-D-xylopyranoside--substrates for acetylxylan esterases.

Four modified substrates for acetylxylan esterases, 2-deoxy, 3-deoxy, 2-deoxy-2-fluoro, and 3-deoxy-3-fluoro derivatives of di-O-acetylated methyl beta-D-xylopyranoside were synthesized via 2,3-anhydropentopyranoside precursors. Methyl 2,3-anhydro-4-O-benzyl-beta-D-ribopyranoside was transformed into methyl 2,3-anhydro-4-O-benzyl-beta-D-lyxopyranoside in three steps. The epoxide ring opening of 2,3-anhydropentopyranosides was accomplished either by hydride reduction or hydrofluorination.

Lipase-catalysed preparation of acetates of 4-nitrophenyl beta-D-xylopyranoside and their use in kinetic studies of acetyl migration.

Di-O-acetates and mono-O-acetates of 4-nitrophenyl beta-D-xylopyranoside were prepared by use of lipase PS-30. Polarity of organic solvents and reaction time affected the regioselectivity of the di-O-acetylation as well as the yields of monoacetates. The kinetics of acetyl groups migration in these derivatives was studied in aqueous media using HPLC.

An efficient chemoenzymatic route to methyl 4-O-benzyl-2,3-anhydro-beta-D-lyxopyranoside from methyl beta-D-xylopyranoside.

Methyl 4-O-benzyl-2,3-anhydro-beta-D-lyxopyranoside, an intermediate for the preparation of methyl beta-D-xylopyranoside derivatives modified at C-2, was obtained in five steps in 58% yield. The synthetic sequence starts from methyl beta-D-xylopyranoside through two main steps involving regioselective enzymatic acetylation and deacetylation catalyzed by lipase PS.

Purification and properties of a feruloyl esterase involved in lignocellulose degradation by Aureobasidium pullulans.

The lignocellulolytic fungus Aureobasidium pullulans NRRL Y 2311-1 produces feruloyl esterase activity when grown on birchwood xylan. Feruloyl esterase was purified from culture supernatant by ultrafiltration and anion-exchange, hydrophobic interaction, and gel filtration chromatography. The pure enzyme is a monomer with an estimated molecular mass of 210 kDa in both native and denatured forms and has an apparent degree of glycosylation of 48%.