Vicinal Hydroxyl Research Articles

Carbohydrate isomer resolution via multi-site derivatization cyclic ion mobility-mass spectrometry.

Oligosaccharides serve many roles in extant life and may have had a significant role in prebiotic chemistry on the early Earth. In both these contexts, the structural and isomeric diversity among carbohydrates presents analytical challenges necessitating improved separations. Here, we showcase a chemical derivatization approach, where 3-carboxy-5-nitrophenylboronic acid (3C5NBA) is used to label vicinal hydroxyl groups, amplifying the structural difference between isomers. We explore the applicability of state-of-the-art ion mobility - mass spectrometry (IM-MS) instrumentation in the analysis of derivatized carbohydrates. In particular we focus on the resolving power required for IM separation of derivatized isomers. A recently developed cyclic ion mobility (cIM) mass spectrometer (MS) was chosen for this study as it allows for multi-pass IM separations, with variable resolving power (Rp). Three passes around the cIM (Rp ∼ 120) enabled separation of all possible pairs of four monosaccharide standards, and all but two pairs of eight disaccharide standards. Combining cIM methodology with tandem mass spectrometry (MS/MS) experiments allowed for the major products of each of the 3C5NBA carbohydrate derivatization reactions to be resolved and unequivocally identified.

The Structural Signs of Sweetness in Artificial Sweeteners: A Rotational Study of Sorbitol and Dulcitol

A gas-phase study on the artificial sweeteners sorbitol and dulcitol has been carried out for the first time by using a combination of chirped-pulse Fourier-transform microwave (CP-FTMW) spectroscopy and laser ablation (LA). The isolation conditions provided by the supersonic expansion reveal the intrinsic conformational structures of these sweeteners. The three and five observed conformers for sorbitol and dulcitol, respectively, are stabilized by networks of cooperative intramolecular hydrogen bonds between vicinal hydroxyl groups in clockwise or counterclockwise arrangements. Suitable places in the structure of seven out of eight conformers identified for both polyalcohols meet the requirements of the glucophore proposed by Shallenberger and Acree's molecular theory of sweet taste. Present results provide the first linkage between sweetness and structure in sugar alcohols.

Catechins and other flavonoids as protectors against undesired modifications of biomolecules

Undesired modifications of biomolecules (proteins, lipids and nucleic acids), mainly oxidation by reactive oxygen species, underlay many diseases and apparently aging. Compounds counteracting these modifications can therefore be candidates for potential drugs and aging-modifying agents. Naturally occurring compounds are especially interesting in this respect since knowledge of their properties may be a basis for dietary recommendations and functional food production. The aim of our studies was to compare the antioxidant properties and efficiency of a range of flavonoids and other natural antioxidants in protection against protein modifications and lipid peroxidation. We compared the ABTS*-scavenging activity capacity of a range of flavonoids and selected other antioxidants, their iron binding capacity estimated by the ferrozine competition test and strong iron binding evaluated by a modified deoxyribose degradation test, their potency to prevent peroxynitrite reactions in three different test systems, based on fluorescein bleaching, tyrosine nitration and serum albumin thiol oxidation as well as oxidation of dihydrorhodamine and fluorescein, and protein tyrosine chlorination by hypochlorous acid. The sequence of protective capacity of the antioxidants was different in various systems. Correlation analysis revealed that the hydroxyl group at the R04 position of the flavonoids contributes significantly to prevention of fluorescein bleaching, R3-OH is important for prevention of thiol oxidation while R5-OH and R03-OH are significant in prevention of tyrosine nitration. The total number of hydroxyl groups correlated with the ability of flavonoids to prevent oxidation reactions and the presence of vicinal hydroxyl groups correlated with flavonoid reactivity in all systems used [1]. Flavonoids were also active in preventing nitration of intracellular proteins. Flavonoids and other antioxidants were also compared as protective agents against glycation of serum albumin induced by various sugars (glucose, fructose and ribose) [2] and reactive aldehydes (glyoxal and methylglyoxal) [3] and blood plasma lipoproteins against lipid peroxidation induced by various agents (AAPH, peroxynitrite and hypochlorous acids). Catechins showed the highest ABTS*-scavenging capacity, the highest stoichiometry of Fe3+ reduction in the FRAP assay and belonged to the most efficient compounds in protection SIN-1 induced oxidation of dihydrorhodamine 123, AAPH-induced fluorescein bleaching and hypochlorite-induced fluorescein bleaching. (+)-Catechin and (-)-epicatechin were the most effective compounds in protection against AAPH-induced erythrocyte hemolysis while (-)-epicatechin gallate, (-)-epigallocatechin gallate and (-)-epigallocatechin protected at lowest concentrations against hypochlorite-induced hemolysis. The results demonstrate high potency of flavonoids in protection against undesired modifications of biomolecules, even at micromolar concentrations.

Synthesis and plant growth modulation of tris (2-hydroxyethyl)ammonium boron-containing compounds

To develop boron deficiency treatment composite preparations for significant agricultural crops tris(2-hydroxyethyl)ammonium complexes containing boron and lower dicarboxylic acid (C2-C4) anions were synthesized and characterized. It was shown, that formation and stabilization of complexes containing a greater number of carbon atoms or intermolecular ?- conjugation (e.g. maleic acid) is related to space and electrostatical hurdles, respectively. According to NMR spectroscopy, in case of tartaric acid complex vicinal hydroxyl bounding with boron was found. The preexisted boratrane azeotropic water distillation synthetic method was modernized (videlicet optimal solvent mixture and raw materials ratio were chosen). Various triethanolamine and boric acid reaction mediums, i.e. nonpolar (toluene), polar aprotic (dimethylsulphoxide), protic (isopropanol, 2-butanol) solvents and their mixtures, were tested. In the issue optimal synthetic method, utilizing isopropanol/2-butanol mixture in ratio 3 to 1, was elaborated. In comparison to standard azeotropic water-isopropanol distillation the yield of the process was exceeded to 12.08% (from 82.70% to 94.78%) and low impurity concentrations in product was committed. Besides alternative laboratory solvent-free boratrane synthetic method was developed and optimal rinsing fluid composition was found. During agricultural experiments substance effectiveness in germination power and germinability of beet seeds and productivity of sugar beet was studied. Boratrane was found to be slightly effective for seed germinability stimulation. Boratrane-containing composition (i.e. boratrane + tris(2-hydroxyethyl)ammonium o-cresoxyacetate + 1-chloromethylsilatrane) was shown to have the best results in apical root length, average root-crop and average plant weigth increasing in comparison with the control.

Solvent-enabled control of reactivity for liquid-phase reactions of biomass-derived compounds

The use of organic solvents in biomass conversion reactions can lead to high rates and improved selectivities. Here, we elucidate the effects of organic solvent mixtures with water on the kinetics of acid-catalysed dehydration reactions of relevance to biomass conversion. Based on results from reaction kinetics studies, combined with classical and ab initio molecular dynamics simulations, we show that the rates of acid-catalysed reactions in the liquid phase can be enhanced by altering the extents of solvation of the initial and transition states of these catalytic processes. The extent of these effects increases as the number of vicinal hydroxyl or oxygen-containing groups in the reactant increases, moving from an alcohol (butanol), to a diol (1,2-propanediol), to a carbohydrate (fructose). We demonstrate that the understanding of these solvation effects can be employed to optimize the rate and selectivity for production of the biomass platform molecule hydroxymethylfurfural from fructose.

Tethering Dual Hydroxyls into Mesoporous Poly(ionic liquid)s for Chemical Fixation of CO2 at Ambient Conditions: A Combined Experimental and Theoretical Study

Vicinal dual hydroxyl functional mesoporous poly(ionic liquid)s with large surface area and high ionic liquid (IL) content were synthesized through the copolymerization of epoxy-containing IL monomers and divinylbenzene, followed by ring opening in water. They acted as recyclable heterogeneous organocatalysts in the cycloaddition of a series of epoxides with CO2 under mild conditions (down to ambient conditions). The catalyst can be lightly recovered and reused with stable activity. The remarkable performance was attributable to the abundant mesoporosity and the synergistic effect of vicinal dual hydroxyls as hydrogen-bonding donors and halogen anions as nucleophiles. Density functional theory calculation, comprising the structural optimization, the energetic profile, and the charge and energy decomposition analysis by the combination of the extended transition state energy decomposition scheme with the natural orbitals for chemical valence approach, was performed to afford insight, indicating that the vi...

Multifunctional self-assembling hydrogel from guar gum

Facile and “green” techniques for hydrogel fabrication are in high demand to satisfy a wide range of practical applications. To the best of our knowledge, this report presents the first description of an efficient, one-flask method for preparing a guar gum (GG)-based self-assembling hydrogel, which takes advantage of the formation of cyclic acetals between the vicinal hydroxyl groups and carbonyl groups, as introduced via the regioselective oxidation of GG using sodium periodate (NaIO4). The final product was achieved without the addition of any crosslinking agents. The self-assembling hydrogel was rapidly formed under ambient conditions, and demonstrated remarkable self-healing performance, as well as thermally responsive properties associated with sol-gel transitions. Furthermore, the GG hydrogel had the ability to remove copper ions (Cu2+) from the surrounding aqueous solution. This removal can be accounted for due to the large number of active sites (i.e., hydroxyl groups) within its porous three-dimensional structure. The adsorption equilibrium was achieved after 5h, and the amount of adsorbed Cu2+ was 944mg/g GG when an initial concentration of 25mg/mL CuSO4 in solution was used. The adsorption isotherm data correlated well with the Freundlich model (R2>0.99), suggesting that the Cu2+ was removed through multilayered and complex adsorption mechanisms.

I6 Natural product-based protein amyloid inhibitors

Toxic protein amyloid formation has been implicated in more than a dozen protein misfolding diseases such as Alzheimer’s disease, Parkinson’s disease, and type 2 diabetes. Currently there are no effective disease-modifying drugs available. One common therapeutic strategy is to discover and develop protein amyloid inhibitors that can slow down, prevent, or remodel toxic amyloids. Natural products are a major class of amyloid inhibitors, and several dozens of natural product-based amyloid inhibitors have been identified and characterized in recent years. Using amyloidogenic proteins amylin, amyloid beta-peptide, and tau as model systems, we screened libraries of natural compounds used in Complementary and Alternative Medicine. We identified multiple potent inhibitors, including rosmarinic acid and baicalein (200 nM and 1 μM respectively in apparent IC 50 ) [1] . These compounds disaggregate amyloidogenic protein fibrils, significantly reduce protein amyloid-induced cytotoxicity and toxic amyloid oligomers in circulating sera and/or cerebrospinal fluid from diseased animal models. Dissecting the functional groups of these compounds, we demonstrated, for the first time to our knowledge, that the vicinal hydroxyl groups of the catechol groups played key functional roles in amyloid inhibition in more than two dozen catechol-containing compounds, including many plant- and fruit-derived flavonoids and other phenolic compounds [2] . Compounds with multiple catechol groups, such as rosmarinic acid, exhibited additive effects. We provided further mass spectrometric evidence that incubating several of these catechol-containing inhibitors with amyloidogenic proteins leads to covalent adducts consistent with Schiff base conjugation mechanism to interfere with toxic amyloid formation. Consistent with forming covalent adducts via the mechanism of quinone intermediates, we demonstrated that many catechol-containing natural products significantly increase amyloid inhibition effects under autooxidation or oxidizing conditions. Lastly, I will discuss our approach to enhance certain natural product solubility/bioavailability by engineering natural compound-incorporating nanoparticles [3] . Acknowledgement This work is in part supported by Virginia Tech new faculty start-up funds, Commonwealth Health Research Board (CHRB), Alzheimer’s and Related Diseases Research Award Fund (ARDRAF) from Virginia Center on Aging, Diabetes Action Research and Education Foundation (DAREF), and Virginia Tech Center for Drug Discovery (VTCDD).

Structure-activity relationships for flavone interactions with amyloid β reveal a novel anti-aggregatory and neuroprotective effect of 2′,3′,4′-trihydroxyflavone (2-D08)

Naturally-occurring flavonoids have well documented anti-aggregatory and neuroprotective properties against the hallmark toxic protein in Alzheimer’s disease, amyloid β (Aβ). However the extensive diversity of flavonoids has limited the insight into the precise structure-activity relationships that confer such bioactive properties against the Aβ protein. In the present study we have characterised the Aβ binding properties, anti-aggregatory and neuroprotective effects of a discreet set of flavones, including the recently described novel protein sumoylation inhibitor 2′,3′,4′-trihydroxyflavone (2-D08). Quercetin, transilitin, jaceosidin, nobiletin and 2-D08 were incubated with human Aβ1–42 for 48h in vitro and effects on Aβ fibrillisation kinetics and morphology measured using Thioflavin T (ThT) and electron microscopy respectively, in addition to effects on neuronal PC12 cell viability. Of the flavones studied, only quercetin, transilitin and 2-D08 significantly inhibited Aβ1–42 aggregation and toxicity in PC12 cells. Of those, 2-D08 was the most effective inhibitor. The strong anti-amyloid activity of 2-D08 indicates that extensive hydroxylation in the B ring is the most important determinant of activity against β amyloid within the flavone scaffold. The lack of efficacy of jaceosidin and nobiletin indicate that extension of B ring hydroxylation with methoxyl groups result in an incremental loss of anti-fibrillar and neuroprotective activity, highlighting the constraint to vicinal hydroxyl groups in the B ring for effective inhibition of aggregation. These findings reveal further structural insights into anti-amyloid bioactivity of flavonoids in addition to a novel and efficacious anti-aggregatory and neuroprotective effect of the semi-synthetic flavone and sumoylation inhibitor 2′,3′,4′-trihydroxyflavone (2-D08). Such modified flavones may facilitate drug development targeting multiple pathways in neurodegenerative disease.

Hyperbranched-polyol-tethered poly (amic acid) electrospun nanofiber membrane with ultrahigh adsorption capacity for boron removal

The development of efficient adsorbents with high sorption capacity remains as a challenge for the removal of micropollutants occurred globally in water resources. In this work, poly (amic acid) (PAA) electrospun nanofiber membranes grafted with hyperbranched polyols were synthesized and used for boron removal. The PAA nanofiber was reacted with hyperbranched polyethylenimine (HPEI) and further with glycidol to introduce the vicinal hydroxyl groups. The chemical composition and surface characteristics of the obtained PAA-g-PG membranes were evaluated by FESEM, FTIR, XPS and water contact angles (WCA) measurements. The boron adsorption thermodynamics and kinetics were investigated systematically. The results showed that the PAA nanofiber spun from concentration of 15% had uniform morphology and narrow diameter distribution. The PAA-g-PG nanofiber membrane had a maximum boron uptake of 5.68mmol/g and could adsorb 0.82mmol/g boron from a 5mg/L solution in 15min. Both the high surface area of nanofibers and the hyperbranched structure should contribute to the high boron uptake and high adsorption rate. The nanofiber membrane obeyed the Langmuir adsorption model and the pseudo-first-order kinetic model. The regeneration efficiency of the nanofiber membrane remained 93.9% after 10 cycled uses, indicating good regenerability of the membrane.

Polymercaptan-based polyurethane foams

Rigid thiourethane foams were prepared from vegetable oil-based polythiols (polymercaptans) and compared with soy- and castor oil-based polyurethane reference foams. Three types of polymercaptans were tested, soybean oil-based one with thiol groups only, epoxidized soybean oil-based with vicinal thiol and hydroxyl groups and castor oil-based with hydroxyl and thiol groups separated with several methylene units. Physical, mechanical, and thermal characteristics of polyol- and thiol based foams were similar to petrochemical foams used as heat insulation materials in construction, appliances, etc.

Amylin Amyloid Inhibition by Flavonoid Baicalein: Key Roles of Its Vicinal Dihydroxyl Groups of the Catechol Moiety.

Amyloid formation of the 37-residue amylin is involved in the pathogenesis of type 2 diabetes and, potentially, diabetes-induced neurological deficits. Numerous flavonoids exhibit inhibitory effects against amylin amyloidosis, but the mechanisms of inhibition remain unclear. Screening a library of natural compounds uncovered a potent lead compound, the flavone baicalein. Baicalein inhibits amylin amyloid formation and reduces amylin-induced cytotoxicity. Analogue analyses demonstrated, for the first time, key roles of the vicinal hydroxyl groups on the A-ring. We provided mass spectrometric evidence that incubating baicalein and amylin leads to their conjugation, consistent with a Schiff base mechanism.

Improvement of enantioselectivity of the B-type halohydrin hydrogen-halide-lyase from Corynebacterium sp. N-1074

Halohydrin hydrogen-halide-lyase (H-Lyase) is a bacterial enzyme involved in the degradation of halohydrins. This enzyme catalyzes the intramolecular nucleophilic displacement of a halogen by a vicinal hydroxyl group in halohydrins, producing the corresponding epoxides. The H-Lyases have been classified into A, B and C subtypes based on amino acid sequence similarities. These enzymes have attracted much attention as industrial catalysts in the synthesis of chiral chemicals from prochiral halohydrins. In the present study, we constructed mutants of B-type H-Lyase from Corynebacterium sp. N-1074 (HheB) displaying higher enantioselectivity by structure-based site-directed mutagenesis and random mutagenesis. A triple mutant of HheB exhibited 98.5% enantioselectivity, the highest ever reported, toward (R)-4-chloro-3-hydroxy-butyronitrile production, with the yield reaching approximately two-fold that of the wild-type enzyme. We discuss the structural basis of the high enantioselectivity and productivity of the mutant by comparing the crystal structures of the mutant HheB and the wild-type enzyme in complex with or without the substrate analogue.

Insights into furanose solution conformations: beyond the two-state model.

A two-state model is commonly used for interpreting ring conformations of furanoses based on NMR scalar (3) J-coupling constants, with the ring populating relatively narrow distributions in the North and the South of the pseudorotation itinerary. The validity of this simple approach has been questioned, and is examined here in detail employing molecular dynamics (MD) simulations with a new GLYCAM force field parameter set for furanoses. Theoretical (3) J-coupling constants derived from unrestrained MD simulations with the new furanose-specific parameters agreed with the experimental coupling constants to within 1Hz on average. The results confirm that a two state model is a reasonable description for the ring conformation in the majority of methyl furanosides. However, in the case of methyl α-D-arabinofuranoside the ring populates a continuum of states from North to South via the eastern side of the pseudorotational itinerary. Two key properties are responsible for these differences. Firstly, East and West regions in β- and α-anomers, respectively, are destabilized by the absence of the anomeric effect. And, secondly, East or West conformations can be further destabilized by repulsive interactions among vicinal hydroxyl groups and ring oxygen atoms when the vicinal hydroxyl groups are in syn-configurations (such as in ribose and lyxose) more so than when in anti (arabinose, xylose).

Early diagnosis of fungal infections using piezomicrogravimetric and electric chemosensors based on polymers molecularly imprinted with d-arabitol

An elevated concentration of d-arabitol in urine, especially compared to that of l-arabitol or creatinine, is indicative of a fungal infection. For that purpose, we devised, fabricated, and tested chemical sensors determining d-arabitol. These chemosensors comprised the quartz crystal resonator (QCR) or extended-gate field-effect transistor (EG-FET) transducers integrated with molecularly imprinted polymer (MIP) film recognition units. To this end, we successfully applied a covalent approach to molecular imprinting, which involved formation of weak reversible covalent bonds between vicinal hydroxyl groups of arabitol and boronic acid substituents of the bithiophene functional monomer used. The MIP films were synthesized and simultaneously deposited on gold electrodes of quartz crystal resonators (Au-QCRs) or Au-glass slides by oxidative potentiodynamic electropolymerization. With the QCR and EG-FET chemosensors, the d-arabitol concentration was determined under flow-injection analysis and stagnant-solution binding conditions, respectively. Selectivity with respect to common interferences, and l-arabitol in particular, of the devised chemosensors was superior. Limits of detection and linear dynamic concentration ranges of the QCR and EG-FET chemosensors were 0.15mM and 0.15 to 1.25mM as well as 0.12mM and 0.12 to 1.00mM, respectively, being lower than the d-arabitol concentrations in urine of patients with invasive candidiasis (>220μM). Therefore, the devised chemosensors are suitable for early diagnosis of fungal infections caused by Candida sp. yeasts.

Singlet-Oxygen Generation in Alkaline Periodate Solution.

A nonphotochemical generation of singlet oxygen ((1)O2) using potassium periodate (KIO4) in alkaline condition (pH > 8) was investigated for selective oxidation of aqueous organic pollutants. The generation of (1)O2 was initiated by the spontaneous reaction between IO4(-) and hydroxyl ions, along with a stoichiometric conversion of IO4(-) to iodate (IO3(-)). The reactivity of in-situ-generated (1)O2 was monitored by using furfuryl alcohol (FFA) as a model substrate. The formation of (1)O2 in the KIO4/KOH system was experimentally confirmed using electron spin resonance (ESR) measurements in corroboration with quenching studies using azide as a selective (1)O2 scavenger. The reaction in the KIO4/KOH solution in both oxic and anoxic conditions initiated the generation of superoxide ion as a precursor of the singlet oxygen (confirmed by using superoxide scavengers), and the presence of molecular oxygen was not required as a precursor of (1)O2. Although hydrogen peroxide had no direct influence on the FFA oxidation process, the presence of natural organic matter, such as humic and fulvic acids, enhanced the oxidation efficiency. Using the oxidation of simple organic diols as model compounds, the enhanced (1)O2 formation is attributed to periodate-mediated oxidation of vicinal hydroxyl groups present in humic and fulvic constituent moieties. The efficient and simple generation of (1)O2 using the KIO4/KOH system without any light irradiation can be employed for the selective oxidation of aqueous organic compounds under neutral and near-alkaline conditions.

Targeted acylation for all the hydroxyls of (+)-catechin and evaluation of their individual contribution to radical scavenging activity

The reactivity profile of all the hydroxyl groups in (+)-catechin towards acylation and their respective contribution to radical scavenging activity were systematically explored in this work. Selective acylation of the hydroxyls on different rings was carried out employing either a basic or acidic activation strategy. Monoacylation of B ring was achieved effectively with the aid of dimethyltin dichloride. Monoacylation of A ring was accomplished by sequential protection and deprotection of B and C rings. Based on specific acylation of all the individual hydroxyls of (+)-catechins, the structure radical scavenging activity relationship of each hydroxyl of (+)-catechin was established. It was demonstrated that the vicinal phenolic hydroxyls on B ring played the most important role in the ABTS radical scavenging activity and those on A and C rings made a much smaller contribution. This study has laid solid groundwork for further modification of the catechins and improvement of their properties.

Surface peeling of cellulose nanocrystals resulting from periodate oxidation and reductive amination with water-soluble polymers

Cellulose nanocrystals (CNCs) were first submitted to periodate oxidation for increasing reaction times and subsequently to reductive amination with water-soluble amine-terminated polymers. The morphology and chemical properties of the samples were characterized by imaging (TEM), spectroscopic (FTIR and 13C solid-state NMR), and scattering techniques (DLS and WAXS). The data revealed that the periodate oxidation of CNCs resulted in an increase in the degree of oxidation (DO) with reaction time and that most of the created carbonyls were converted into hemiacetals as a result of their interaction with vicinal hydroxyl groups. Like in the case of cellulose microfibrils, CNCs were oxidized from the surface to the core of the particles. For low reaction times and concomitant low DOs, the morphology and crystallinity of the CNCs were mildly altered. A prolonged oxidation resulted in the separation of the originally laterally assembled crystallites and the data are consistent with the presence of dangling chains resulting from the cleavage of glucosyl units at the surface of the thin elongated cellulose nanoparticles. A comparison with literature data further showed that the oxidation reaction was not primarily governed by the degree of crystallinity. Remarkably, after reductive amination with water-soluble polymer chains, thin unoxidized crystalline cellulose nanoparticles were recovered. This situation seems to result from the surface peeling of the CNCs due to reaction of surface-oxidized dangling chains with water-soluble polymers and their subsequent solubilization.

Crystal structures of halohydrin hydrogen-halide-lyases from Corynebacterium sp. N-1074.

Halohydrin hydrogen-halide-lyase (H-Lyase) is a bacterial enzyme that is involved in the degradation of halohydrins. This enzyme catalyzes the intramolecular nucleophilic displacement of a halogen by a vicinal hydroxyl group in halohydrins to produce the corresponding epoxides. The epoxide products are subsequently hydrolyzed by an epoxide hydrolase, yielding the corresponding 1, 2-diol. Until now, six different H-Lyases have been studied. These H-Lyases are grouped into three subtypes (A, B, and C) based on amino acid sequence similarities and exhibit different enantioselectivity. Corynebacterium sp. strain N-1074 has two different isozymes of H-Lyase, HheA (A-type) and HheB (B-type). We have determined their crystal structures to elucidate the differences in enantioselectivity among them. All three groups share a similar structure, including catalytic sites. The lack of enantioselectivity of HheA seems to be due to the relatively wide size of the substrate tunnel compared to that of other H-Lyases. Among the B-type H-Lyases, HheB shows relatively high enantioselectivity compared to that of HheBGP1 . This difference seems to be due to amino acid replacements at the active site tunnel. The binding mode of 1, 3-dicyano-2-propanol at the catalytic site in the crystal structure of the HheB-DiCN complex suggests that the product should be (R)-epichlorohydrin, which agrees with the enantioselectivity of HheB. Comparison with the structure of HheC provides a clue for the difference in their enantioselectivity.

Functionality of surface-coupled oxidised glycosaminoglycans towards fibroblast adhesion

Glycosaminoglycans are able to bind many growth factors and adhesive proteins, which affect cell activities such as adhesion, migration, growth and differentiation. Chondroitin sulphate, hyaluronan, sulphated hyaluronan and heparin were oxidised here (aldehyde glycosaminoglycans) to generate aldehydes on vicinal hydroxyl groups of the uronic monomers of glycosaminoglycans for subsequent direct covalent binding to amino-terminated model substrata. The properties of modified surfaces were monitored by water contact angle, zeta potential, ellipsometry measurements and atomic force microscopy showing successful immobilisation of aldehyde glycosaminoglycans. Wetting properties and zeta potentials were related to sulphate content of aldehyde glycosaminoglycans with aldehyde heparin as most wettable and negative surface and aldehyde hyaluronan as the least. The thickness of surface layers measured by ellipsometry indicated a predominant side-on immobilisation of all aldehyde glycosaminoglycans. Atomic force microscopy studies showed that immobilisation of aldehyde hyaluronan lead to a rather smooth surface coating while immobilisation of sulphated aldehyde glycosaminoglycans was characterised by a globular appearance of surfaces with higher roughness. The experiments with human fibroblast studying adhesion under serum-free conditions were carried out to learn about bioactivity of aldehyde glycosaminoglycans. It was observed that the increase in sulphation degree of aldehyde glycosaminoglycans was accompanied by increased adhesion and spreading of cells with stronger expression of focal adhesions and cytoskeletal structures. By contrast, cell adhesion and spreading were lower on aldehyde hyaluronan. Immunofluorescence staining of cells in contact with aldehyde hyaluronan revealed a stronger expression of CD44, which can represent an alternative route of cell adhesion. The results show that oxidised glycosaminoglycans can be successfully applied for the development of bioactive surface coatings. The created biomimetic microenvironment may be useful to engineer surfaces of implants and scaffolds for tissue regeneration.