Chemically modified graphite electrode with immobilized enzyme as a potentiometric sensor for some l-amino acids

A chemically modified electrode with immobilized enzyme was constructed by covalent attachment of l-amino acid oxidase (E.C. 1.4.3.2) to a graphite rod via chemical modification of the electrode surface. Logarithmic response with concentration of selected l-amino acids was observed in the 10^-2–10^-5 M range. The electrodes displayed slopes of 24–29 mV/decade over the tested concentration range for l-phenylalanine, l-methionine, and l-leucine. The electrode slope degraded by 33% after 78 days under the defined storage conditions. Interaction of hydrogen peroxide with surface groups generated during cyanuric chloride modification appears to be the major contributor to the potentiometric response. Cations change the electrode potential but have essentially no effect on the electrode slope. A plausible model describing the mechanism of response is presented.     

Bioluminescence flow system for determination of branched-chain l-amino acids in serum and urine

A continuous-flow bioluminescence method for determinations of branched-chain l-amino acids in serum and urine is described. Serum can be analyzed directly after simple filtration. Response is linear for 20-2000 pmol in the biological matrix. Leucine dehydrogenase is immobilized onto a nylon coil separated from the reactor coil containing bacterial bioluminescence enzymes. The stability of the immobilized is high (lifetime > two months) and more than 900 samples can be analyzed with the use of a few mg of enzymes. The results obtained agree well with those obtained by ion-exchange chromatography (amino acid analyzer).     

Enzymatic determination of cholesterol, l-amino acids and linoleic acid with a novel redox indicator system

A sensitive kinetic fluorimetric system is proposed for the determination of hydrogen peroxide produced by enzymatic oxidation of cholesterol, l-amino acids and linoleic acid. 2-Hydroxynaphthaldehyde thiosemicarbazone (HNTS) is oxidized by hydrogen peroxide or hydroperoxides in an ammoniacal medium in a Mn(II)-catalyzed reaction to give a fluorescent product (λ_ex = 390 nm, λ_em = 450 nm). The lowest concentration of hydrogen peroxide determined is 50 pmol. Cholesterol was determined in egg yolk, cod liver oil and horse serum. The ranges of concentration for substrates were 0.33–3.74 μM cholesterol; 0.3–10 μM, 0.6–15 μM and 0.75–10 μM, for l-leucine, l-phenyalanine and l- serine, respectively; and 15–150 μM linoleic acid.     

The first chemical conversion of l-proline to l-glutamic acid

The ruthenium tetroxide oxidation of N-acyl-l-proline esters gave the corresponding l-pyroglutamic acid derivatives in good yields with no appreciable racemization, which led to the first chemical conversion of l-proline to l-glutamic acid.     

Bound coefactor/dual enzyme electrode system for l-alanine

A dual enzyme-bound coenzyme electrode system for quantifying l-alanine is described. Commercially available dextran-bound NAD was incorporated into an l-alanine dehydrogenase (E.C. 1.4.1.1)/l-lactate dehydrogenase (E.C. 1.1.1.27) enzyme system and held at the surface of a potentiometric ammonia gas sensor. Using this system, l-alanine calibration curves with a slope of 45 mV/decade and 10^?5 M detection limit were obtained with a sensor lifetime of at least 10 days. This system is potentially useful for the clinical determination of l-alanine in serum.     

A reliable l-lactate electrode with a new membrane for enzyme immobilization for amperometric assay of lactate

Lactate oxidase from Pediococcus species can be rapidly and simply immobilized on a commercially available pre-activated membrane fixed to an amperometric probe detecting hydrogen peroxide, to provide a very sensitive practical l-lactate sensor. At 25°C, in a phosphate buffer pH 7.1, the detection limit is 1.25 × 10^?7 M, calibration is linear between 2.5 × 10^?7 and 2.5 × 10^?4 M, the response time is <2 min, and the probe can be used for hundreds of assays over several weeks. With a microprocessor-based instrument including the same type of electrode, sample injections can be made at 90-s intervals, the response being displayed after only 30 s. High selectivity is achieved because the differential measurement system continuously subtracts currents at the chosen potential arising from the presence of electroactive species. Samples (20 μl) from sera and dairy products were successfully tested without pretreatment; a relative standard deviation between 1 and 3% was routinely obtained. Correlation of the data with data obtained by the conventional spectrophotometric method was excellent.     

From d-xylose to a d-glycero-l-altrose derivative

A key intermediate corresponding to a rare sugar framework has been synthesized, starting from d-xylose, an inexpensive carbohydrate. This approach gave access to new elaborated sugar moieties for structure–activity relationships in the KRN research.     

A flow-injection method for the amperometric determination of l-lactate with immobilized enzymes and a chemically modified electrode

A sensitive flow-injection system for l-lactate is described. Lactate dehydrogenase, LDH, and glutamic-pyruvic transaminase, GPT, were co-immobilized on a gluteraldehyde-activated porous silica support and used in a packed-bed enzyme reactor. l-lactate is oxidized to pyruvate in the presence of NAD⁺, an equivalent amount of NADH being produced. The equilibrium of the reaction is unfavourable, but by co-immobilizing LDH with GPT and adding l-glutamate, the pyruvate reacts and enough force is obtained to drive the oxidation of l-lactate totally to the product side. The NADH formed is then detected electrocatalytically at an electrode chemically modified with Meldola Blue, operated at 0 mV vs. Ag/AgCl. The system responds linearly to injected samples (25 μl) of l-lactate in the concentration range 10 μM–1.5 mM. The maximum sample throughput is 30 h^?1. The LDH/GPT reactor was stable for four weeks when used daily at optimal pH 8.8.     

Synthesis and proposed conformations of l-prolyl-l-leucyl-β-alanine alkylamides

The synthesis of H-Pro-Leu-β-Ala-NH₂, H-Pro-Leu-β-Ala-NHCH₃ and H-Pro-Leu-β-Ala-N(CH₃)₂ is described. On the basis of IR and ¹H NMR spectral data, a 7-membered ring including the NH of β-alanine with the CO of proline should be assigned for the H-Pro-Leu-β-Ala-N(CH₃)₂. Consequently, the plausible conformations for H-Pro-Leu-β-Ala-NH₂ and H-Pro-Leu-β-Ala-NHCH₃ derive from the formation of an 11-membered ring, between the trans amide proton and the CO of Pro, or from the formation of an 8-membered ring, between this carboxamide proton and the CO of Leu, plus the aforementioned 7-membered ring.     

Mediated amperometric biosensor for hypoxanthine based on a hydroxymethylferrocene-modified carbon paste electrode

An amperometric enzyme electrode for the determination of hypoxanthine in fish meat is described. The hypoxanthine sensor was prepared from xanthine oxidase immobilized by covalent binding to cellulose triacetate and a carbon paste electrode containing hydroxymethylferrocene. The xanthine oxidase membrane was retained behind a dialysis membrane at a carbon paste electrode. The sensor showed a current response to hypoxanthine due to the bioelectrocatalytic oxidation of hypoxanthine, in which hydroxymethyiferrocene served as an electron-transfer mediator. The limit of detection is 6 × 10^?7 M, the relative standard deviation is 2.8% (n=28) and the response is linear up to 7 × 10^?4 M. The sensor responded rapidly to a low hypoxanthine concentration (7 × 10^?4 M), the steady-state current response being achieved in less than 1 min, and was stable for more than 30 days at 5 ° C. Results for tuna samples showed good agreement with the value determined by the conventional method.     

Stereoselective synthesis of 3-glycosyl-5-methoxycarbonyl-isoxazolidines from d-galactose and d-glucose

Regio- and stereoselective cycloaddition of methyl acrylate to C-glycosyl nitrones derived from d-galactose and d-glucose, giving 5-methoxycarbonyl-3-(pentoglycos-5-yl or pentitol-1-yl)isoxazolidines, is reported. Transformation of one of them into a 4-hydroxy-2-(pentoglycos-5-yl)pyrrolidine derivative, potentially useful in a route to polyhydroxy-perhydroazaazulenes, was achieved.     

Ortho-substituted aryl monoboronic acids have improved selectivity for d-glucose relative to d-fructose and l-lactate

Ortho-substituted aryl monoboronic acids have been found to have improved selectivity for d-glucose compared to d-fructose and l-lactate. These findings are supported by computational studies on the B3LYP/6-31G(d) level using Gaussian. This finding is of interest for development of boronate based d-glucose sensors.     

Electrochemical enzymatic determinations of ethanol and l-lactic acid with a carbon paste electrode modified chemically with nicotinamide adenine dinucleotide

The oxidized form of nicotinamide adenine dinucleotide (NAD⁺) is chemically immobilized at the surface of a carbon paste electrode containing n-octaldehyde. The NAD⁺ is converted to NADH by oxidation of ethanol and l-lactic acid catalyzed by their respective dehydrogenases, and the NADH formed is oxidized electrochemically to the original NAD⁺, thus giving a well defined linear-sweep voltammetric peak. The peak area is linearly related to the amount of ethanol or l-lactic acid in the range 0.05–2 × 10^-9 mol.     

Improved synthesis of d,l-fluorocitric acid

We propose a decagram synthesis of commercially unavailable fluorocitric acid. The synthesis begins with benzyl fluoroacetate, which is converted to dibenzyl 2-fluoro-3-oxosuccinate, followed by condensation with malonic acid or its monobenzyl ester and subsequent hydrogenolysis.     

High-performance liquid chromatographic detection of L- and D-amino acids by use of immobilized enzyme electrodes as detectors

Two enzyme electrodes based on immobilized L- and D-amino acid oxidases give specific responses to L- and D-amino acids, respectively. They are used as amperometric detectors for high-performance liquid chromatography, by splitting the flow after elution from the column and detecting D-isomers in one line, L-isomers in the other. The detection limit is about 2 pmol for some amino acids (methionine, tyrosine, leucine, and phenylalanine). The procedure is useful for the specific detection of L- and D-amino acids without complicated pretreatment. The electrodes retain most of their original activities after repetitive use for one month.     

Synthesis of d-gluco-, l-ido-, d-galacto-, and l-altro-configured glycaro-1,5-lactams from tartaric acid

The d-gluco-, l-ido-, d-galacto-, and l-altro-configured glycaro-1,5-lactams 1-4 were prepared from the known tartaric anhydride 5 via the aldehyde 6. These lactams are known (1) or potential (2-4) inhibitors of β-d-glucuronidases and α-l-iduronidases. Olefination of 6 to the (E)- and (Z)-alkenes 7 or 8, followed by reagent or substrate controlled dihydroxylation, lactonization, azidation, reduction, and deprotection led in 10 steps and in overall yields of 11-20% to the title lactams.     

Synthesis of chiroptical properties of some N-(3-methyl-2-quinoxaloyl) l-amino acids and their dioxides

A number of N-(3-methyl-2-quinoxaloyl) l-α-amino acids and esters, and their 1,4-dioxides have been prepared. The quinoxaline derivatives of aliphatic and aromaticl-α-amino acids exhibits enantiomorphic CD spectra in ethanol as well as in ethanolic KOH. However, the corresponding quinoxaline-1,4-dioxide derivatives of the l-α-aliphatic and l-α-aromatic amino acids show, in organic solvents, similar CD spectra. This behaviour is attributed to differences in conformational equilibria in both the quinoxaline and the quinoxaline-1,4-dioxide series NMR and mass spectra of these compounds are discussed.     

Amperometric simultaneous sensing system for d-glucose and l-lactate based on enzyme-modified bilayer electrodes

An amperometric biosensor system which uses screen-printed electrodes to simultaneously detect d-glucose and l-lactate has been developed and applied for simple and rapid determination of d-glucose and l-lactate levels in lactic fermenting beverages. The system was constructed from three-dimensionally layered electrodes. Taking into consideration the effects of easily oxidized substances contained in the samples, ferricyanide ions, which are electrochemically oxidized at a lower voltage, were chosen as a mediator. A linear relationship between steady-state current and concentration was found over a range of 1-100 mM (d-glucose) and 1-50 mM (l-lactate); the variation coefficients were 1.43% (n = 10) and 3.50% (n = 10) for the d-glucose and l-lactate sensors, respectively. When applied to lactic fermenting beverages, there was good agreement between the results obtained by the proposed sensing system and those obtained by the HPLC method. Using the proposed method, assays were completed within 5 min.     

Synthesis of hybrids of d-glucose and d-galactose with 1-deoxynojirimycin analogues using ring-closing metathesis

Four hybrids of azasugars with d-glucose and d-galactose have been synthesized from 3-nitro-2,3-unsaturated-O-glycosides. All the hybrid molecules showed moderate activity against β-galactosidase, the one derived from d-glucose and 1,4-dideoxygulonojirimycin 18, and 26, which is a hybrid of d-glucose and 1,4-dideoxymannohomonojrimycin, showed selectivity toward α-glucosidase and β-galactosidase, respectively.