Enantioselective synthesis of tetrafluorinated glucose and galactose

Polyfluorinated carbohydrates have emerged as interesting probes to investigate "polar hydrophobicity" effect(s) in protein-carbohydrate interactions. A convenient enantioselective synthesis of tetrafluorinated analogues of two of the most important monosaccharides, D-glucose and D-galactose, is reported, as well as our first results regarding the glycosylation of these sugar analogues.     

Investigation of enzyme activity by SERRS using poly-functionalised benzotriazole derivatives as enzyme substrates

New methods of measuring biologically relevant concentrations of enzymes are necessary to allow greater understanding of biological systems. We have previously shown that aryl azo benzotriazolyl alkyl esters can act as enzyme substrates, with the progress of the reaction being monitored using SERRS (see Nat. Biotechnol., 2004, 22, 1133, ref. ). This is a wholly novel analytical application of SERRS, and the low detection levels of the technique allow for an ultra-sensitive enzyme assay. Masked enzyme substrates are used that are invisible to SERRS until enzymatic hydrolysis. Turnover of the substrate by the enzyme leads to the release of the surface-seeking dye necessary for SERRS, and intense signals are produced. Here we report an improved synthesis of 2H-benzotriazolyl alkyl esters via nucleophilic substitution of a chloromethyl ester by benzotriazolyl azo dyes, giving up to a ten-fold increase on previously reported yields. Introduction of electron-withdrawing groups to the benzotriazole ring allows control over the SERRS properties of the compounds. This is of great significance in expanding the synthetic flexibility and subsequently the fundamental use of these compounds as ultra-sensitive and selective reporters of enzyme activity.     

Ferricenium salts as true substrates of glucose oxidase

The steady-state kinetics of D-glucose oxidation by ferricenium dyes RFc⁺PF₆ ^- (R = H, Me, Et, n-Bu, MeCH₂CMe₂, and Cl) and 1,1′-Et₂Fc+PF₆-catalyzed by glucose oxidase from Aspergillus niger was investigated as a function of RFc⁺ and D-glucose concentrations at pH 6.7, 25°C in the presence of 2% (v/v) Triton X-100. The enzymatic bleaching is characterized by large steady-state portions on the kinetic curves for all ferricenium ions studied. The reaction follows the MichaelisMenten kinetics demonstrating a high affinity of RFc⁺ toward the active site of reduced glucose oxidase (GO). The reaction rate is weakly sensitive to the nature of RFc⁺, and the apparent V_m(app) values decrease only twofold on going from the most to the least reactive salt in the series (l,l′-Et₂Fc⁺ and CIFc⁺, respectively), although their observed redox potentials differ by 160 mV. Remarkably, the reactivity of RFc⁺ does not increase with increasing their oxidative power. The apparent Michaelis constants K_m(app) are also weakly sensitive to the nature of RFc⁺. The profiles for the steady-state rate vs [HFc⁺] and [D-glucose] were rationalized in terms of the “ping-pong” mechanism typical of the catalysis by GO. Ferrocenecarboxylic acid (FcCOOH) appeared to be a competitive inhibitor of GO with the inhibition constant of (3 ±1) x 10-³M. The pH profile for the ferricenium fading is bell-shaped with the optimum around 7.5. A simple routine for a rapid in situ preparation of the ferricenium dye, which is ready for spectrophotometric assaying of the GO activity, is presented. The apparent V_m(app) and K_m(app) values for this substrate are similar to those for HFc⁺PF₆.     

Amperometric enzyme electrodes: Part II. Conducting salts as electrode materials for the oxidation of glucose oxidase

Results are reported for the direct oxidation of the enzyme glucose oxidase on 7 different one-dimensional conducting donor acceptor salts. Experiments conducted with the enzyme in bulk solution are shown to be in good agreement with theory. Three salts, made of the cations tetrathiafulvalinium (TTF⁺), N-methylphenazinium or quinolinium with the anion tetracyanoquinodimethanide (TCNQ^?) had the lowest background currents and were used to make membrane sensors for glucose. Analysis of the variation of current with glucose concentration identified the rate limiting processes as transport of gluycose through the membrane and electrochemical kinetics under unsaturated and saturated conditions respectively. The electrochemical rate constants for these three materials were all greater than 10^?2 cm s^?1. TTF⁺TCNQ^? is the material of choice and linear calibration plots were obtained for glucose concentrations between 50 μmol dm^?3 and 10 mmol dm^?3.     

New approaches to side-chain fluorinated bioimidazoles: 4-alkynylimidazoles as substrates for fluorination

4-Alkynylimidazoles have been prepared and their behavior as substrates for “FBr” addition (NBS+Et₃N·3HF) have been studied. Facile Markownikov addition to ethylnylimidazole 2 gave fluorobromoolefin 9a, a potential synthon for the 2-imidazolyl-2-fluoro-1-ethenyl moiety. Reaction of the lithium salt of 2 with diethyl oxalate produced imidazolylpropynoic ester 3b. However, because of the deactivating effect of the ester functionality, all attempts to carry out addition of “FBr” to 3b met with failure. Reduction of the ester gave the hydroxymethyl-substituted acetylene 4. Addition of “FBr” to this substrate and reductive removal of bromine from the product produced fluoroolefins 12, precursors to E- and Z-β-fluorourocanic acids. The same fluoroolefins can be used as intermediates in the synthesis of β-fluorohistidinols.     

Cu(II)-nitroxyl radicals as catalytic galactose oxidase mimics

Results from Hammett correlation studies and primary kinetic isotope effects for the CuCl-TEMPO catalysed aerobic benzyl alcohol oxidations are inconsistent with an oxoammonium based mechanism. We postulate a copper-mediated dehydrogenation mechanism, in which TEMPO regenerates the active Cu(II)-species. This mechanism is analogous to that observed for Galactose Oxidase and mimics thereof.     

Colorimetric quantification of galactose using a nanostructured multi-catalyst system entrapping galactose oxidase and magnetic nanoparticles as peroxidase mimetics

A colorimetric method for quantification of galactose, which utilizes a nanostructured multi-catalyst system consisting of Fe(3)O(4) magnetic nanoparticles (MNPs) and galactose oxidase (Gal Ox) simultaneously entrapped in large pore sized mesocellular silica, is described. Gal Ox, immobilized in a silica matrix, promotes reaction of galactose to generate H(2)O(2) that subsequently activates MNPs in silica mesopores to convert a colorimetric substrate into a colored product. By using this colorimetric method, galactose can be specifically detected. Along with excellent reusability via application of simple magnetic capturing, enhanced operational stability was achieved by employing a cross-linked enzyme aggregate (CLEA) method for Gal Ox immobilization. This protocol leads to effective prevention of enzyme leaching from the pores of mesocellular silica. The analytical utility of the new colorimetric biosensor was demonstrated by its use in diagnosing galactosemia, a genetic metabolic disorder characterized by the inability to utilize galactose, through analysis of clinical dried blood spot specimens. A microscale well-plate format was employed that possesses a multiplexing capability. The multi-catalyst system entrapping Gal Ox and MNPs represents a new approach for rapid, convenient, and cost-effective quantification of galactose in human blood and it holds promise as an alternative method for galactosemia diagnosis, replacing the laborious procedures that are currently in use.     

Oxidatively robust monophenolate-copper(II) complexes as potential models of galactose oxidase

Cupric complexes of a novel phenanthroline-phenolate ligand have strongly distorted coordination geometries and electrochemical properties conducive to modeling the spectroscopy and reactivity of the enzyme galactose oxidase.     

Superacids as catalysts of the oxidation of inorganic substrates

The oxidation of sulfur dioxide, nitrogen dioxide, carbon monoxide, etc., in anhydrous trifluoroacetic acid (TFA) at room temperature was observed. The oxidative ability of anhydrous TFA in reactions with inorganic substances was shown to be related to molecular oxygen dissolved in it. The dissolved molecular oxygen endowed TFA with the properties of a superacid with pK _a ? ?12. Mechanisms of active oxygen reactions with inorganic substrates were suggested.     

Cholesterol oxidation by microencapsulated cholesterol oxidase

Cholesterol oxidase fromBrevibacterium was microencapsulated using a phase-separation technique. The stability of the enzyme during storage and the activity of the microencapsulated enzyme were determined and compared with the soluble enzyme. The effects of various parameters during microencapsulation on enzyme activity are discussed.     

N-Salicylideneaminoacidato copper(II) complexes as galactose oxidase model compounds

[CuL·B]^q model systems, where L²⁻ is the tridentate Schiff base ligand formed by the condensation of salicylaldehyde with alanine, B is imidazole, q=−1, 0 and +1, are optimized at B3LYP/6-31G* level of theory. Their electronic structure is described in terms of Mulliken population analysis and reactivity indices of Fukui. The total energy of [CuL·B]^q species increases with the electron removal. The reactivity indices suitable for the alcohol (sugar) adducts formation (CuO_sugar and O_phenoxylH_sugar interactions) are in the neutral molecule as well as in the singlet cation. Despite the similar trends in Cu–O_phenoxyl bonding and significant O_phenoxyl spin density in triplet cation, the catalytic mechanism of sugars oxidation proposed for the galactose oxidase cannot be used in our system because the [CuL·B]⁺ formation is energetically unfavorable. The imidazole nitrogen deprotonation is more probable than of the alanine ternary carbon atom.     

Kinetics of cholesterol oxidation by cholesterol oxidase

Kinetic studies of cholesterol oxidation catalyzed by soluble cholesterol oxidase fromBrevibacterium were conducted. The optimum temperature and pH were found to be 40–45°C and 7.0, respectively. A plot of initial reaction rate versus cholesterol concentration is sigmoidal in shape. Analysis of the data suggests that the reaction follows a concerted model and not a stepwise model.     

Bioelectrochemical response of the polyaniline galactose oxidase electrode

Galactose oxidase has been immobilized in a polyaniline film. The response current of the galactose oxidase electrode is a function of the applied potential and increases as the pH increases from 5.61 to 7.25. The optimum pH of the immobilized galactose oxidase is 7.25. The activation energy of the enzyme-catalysed reaction is 41.8 kJ mol⁻¹. The response current of the enzyme electrode shows good reproducibility at temperatures below the optimum temperature of 30.4°C and increases as the galactose concentration increases from 0.2 to 6 mmol dm⁻³. Thus the polyaniline galactose oxidase electrode can be used to determine galactose concentration.     

Glycoprotein labeling using engineered variants of galactose oxidase obtained by directed evolution

A directed evolution approach has been used for the generation of variants of galactose oxidase (GOase) that can selectively oxidize glycans on glycoproteins. The aldehyde function introduced on the glycans D-mannose (Man) and D-N-acetyl glucosamine (GlcNAc) by the enzyme variants could then be used to label the glycoproteins and also whole cells that display mannosides on their surface.     

Catalytic effects of galactose oxidase on micelle-forming galactolipids

Catalytic effects of galactose oxidase on the oxidation of beta-D-galactose-carrying lipids with an oligo-ethylene glycol spacer (number of ethylene glycol units (n)=1, 2, 3, 6, 9, 13, and 20) were examined. The affinity of galactose oxidase for the galactose residue in the amphiphile (estimated by the inverse of the Michaelis constant, K(m)) was much higher than those for free D-galactose and small beta-D-galactopyranosides, and dependent on the length of the ethylene glycol spacer. That is, both below and above the critical micellar concentration, the 1/K(m) values decreased with an increase in the n value. The effectiveness of the enzyme, which can be estimated by the k(cat)/K(m) value, showed the same tendency as the 1/K(m) value. These results could be attributed to the role of the nonpolar environment around the galactose residue in the binding by the enzyme. A significant enhancement of the enzymatic oxidation of galactose residue on the liposome surface was also observed.     

TEMPO-derivatives as catalysts in the oxidation of primary alcohol groups in carbohydrates

Primary hydroxyl groups in aqueous starch, pullulan and methyl α- -glucopyranoside were oxidised to the corresponding carboxylic acid functionalities by TEMPO-(4-X)-derivatives using sodium hypochlorite as the primary oxidant. All the combinations of substrates and nitroxyl radicals resulted in stoichiometric conversions, and the selectivity for oxidation of primary hydroxyls was high. Some depolymerisation occurred throughout the oxidation, especially when 4-acetoxy and 4-mesyl-TEMPO were used. The pH window of the activity of the inexpensive 4-acetamido-TEMPO was found to be substantially lower from that of the other tested TEMPO-derivatives; thus allowing milder reaction conditions. At pH 8, the rate of oxidation was ca. two times higher when 4-acetamido-TEMPO was used compared to the other catalysts.     

Suicide substrates: mechanism-based enzyme inactivators

This review article defines suicide substrates as a class of irreversible inactivators of specific target enzymes where the target enzyme participates in its own destruction by catalytic unmasking of a latent functional group at some stage in the catalytic cycle of the enzyme. Criteria for evaluation of suicide substrates are presented. Then, examples of the various types of latent functional groups and their likely enzymic routes of activation are presented for both natural and synthetic compounds. These examples include gabaculine, secobarbital, allopurinol, clavulanate and penicillin sulfones, tranylcypramine, rhizobitoxin, fluoroalanine, 5-fluorodeoxyuridylate and mitomycin.