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.     

Mediated amperometric biosensors for d-galactose,glycolate and l-amino acids based on a ferrocene-modified carbon paste electrode

Direct-current cyclic voltammetry is used to investigate the suitability of a ferrocene derivative as a mediator with galactose, glycolate and l-amino acid oxidases. The three enzymes coupled catalytically to ferrocene monocarboxylic acid exhibiting homogeneous second-order rate constants in the range 0.4 × 10⁵ to 8.5 × 10⁵ l mol^?1 s^?1. Enzyme electrodes which responded to d-galactose, glycolate or l-amino acids were constructed. The appropriate oxidase was retained behind a dialysis membrane at a carbon paste electrode containing the poorly soluble derivative 1,1′-dimethylferrocene. All the electrodes responded rapidly to millimolar concentrations of their respective substrates producing 95% of the steady-state current response in <2 min. This general method of biosensor construction should be widely applicable to oxidases and other oxidoreductase enzymes.     

Regio- and stereoselective syntheses of l-pentose derivatives from l-arabinose

Novel l-arabinose, l-ribose, 2-deoxy-l-ribose and 2-fluoro-2-deoxy-l-arabinose derivatives were synthesized from readily available l-arabinose. Different synthetic routes to methyl 3,5-di-O-acylated-l-arabino(ribo)furanosides as valued intermediates for the preparation of C-2 functionalized l-pentoses were investigated via regioselective transformations of 1,2-di-O-acetyl-3,5-di-O-pivaloyl-l-arabinofuranose, and selective acylation of methyl l-arabinofuranoside with 4-chlorobenzoyl or pivaloyl chloride. Short three-five-step syntheses of methyl 2-deoxy-α-l-ribofuranoside, its 3,5-di-O-acyl derivatives, valuable precursors for preparation of antiviral 2′-deoxy-l-nucleosides, were accomplished via simple and efficient reduction of methyl 2-O-mesyl-l-arabinofuranoside with L-Selectride or tandem reaction involving a complex hydride 2-deoxygenation/acylation of intermediate 2-deoxysugar. A new synthesis of 2′-deoxy-2′-fluoro-β-l-arabinofuranosyl thymine (l-FMAU) was performed using a mild fluorination of protected l-ribofuranoside and a novel sequence of conversions for the preparation of 2-deoxy-2-fluoro-l-arabinofuranoside derivatives.     

Enzymatic assay with detection by enhanced luminol chemiluminescence in a reversed micellar system: determination of l-amino acids and glucose

A detection system for hydrogen peroxide, i.e., luminol chemiluminescence (CL) in a hexadecyltrimethylammonium bromide (CTAB) reversed micellar system, was coupled to enzyme reactions. The use of CTAB reversed micellar medium allows one to conduct both the oxidase enzymatic and CL detection reactions simultaneously at mild pH (l-amino acid system, pH 8.7; glucose system, pH 8.5) in the absence of any co-oxidant or catalyst. Based on this result, simple and unique determinations of l-amino acids and glucose as substrates were developed. The calibration graph for a representative amino acid, l-phenylalanine, was linear in the concentration range 4.0×10^?6?200×10^?6M with a relative standard deviation of 5.78% (five determinations). The method established for l-phenylalanine was also applicable for the assay of fourteen other l-amino acids. The calibration graph for glucose was linear in the concentration range 5.4×10^??540×10^?6M with a relative standard deviation of 4.27% (eight determinations). This method was compared with a standard spectrophotometric method (hexokinase) and successfully applied to the determination of glucose in human serum.     

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.     

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.     

Enzymatic determination of l-lysine by flow-injection techniques

An enzymatic assay that is highly selective for l-lysine, based on flow-injection techniques combined with spectrophotometric detection, is presented. l-Lysine-α-oxidase (E.C. 1.4.3.14) from Trichoderma viride and horseradish peroxidase were used in a coupled enzyme assay. Peroxide produced in the first reaction was converted by peroxidase with phenol and 4-aminoantipyrine to a quinoneimine dye detectable at 500 nm. An analytical enzyme reactor filled with coimmobilized enzymes was incorporated in the flow-injection system. The assay has a measuring frequency of 30 samples h^?1 and a response time of less than 2 min. To adapt the assay to high concentrations of l-Lysine and to minimize interferences, the injected sample volume was reduced to 2 μ-l, resulting in a linearity range of 1–16 mM l-lysine with a sensitivity of 6–7 mV 1 mmol^?1, a limit of detection (3σ) of 1 mM and a reproducibility of 0.5% (repetitive injection of a 10 mM l-lysine sample). The enzyme cartridge is stable for several months and thousands of measurements.     

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.     

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.     

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.     

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.     

Plant tissue-based membrane biosensor for l-ascorbic acid

Coupling of a slice of the mesocarp of squash (Cucurbita pepo) or cucumber (Cucumis sativus) to a Clark-type oxygen electrode allows 0.02–0.57 mmol l^?1l-ascorbic acid to be determined amperometrically. The method is based on monitoring the decrease in the curretn of oxygen at an applied potential of ?650 mV vs. Ag/AgCl; oxygen is consumed in the analyte oxidation catalyzed by ascorbate oxidase in the plant tissue. One tissue slice serves for 50–80 measurements at 30°C and pH 6. Spare slices can be stored for at least a year in aqueous 50% glycerol without substantial loss of enzyme activity. The biosensor is highly selective towards ascorbic acid with a response time of 70–90 s, the relative standard deviation being about 3%. Satisfactory results were obtained in the analysis of some fruit juices and vitamin tablets.     

Vibrational analysis of l-alanine and deuterated analogs

Raman spectra of the polycrystalline l-alanine analogs CH₃CH(NH⁺₃)COO^?, CH₃CH(ND⁺₃)-COO^?, CD₃CD(NH⁺₃)COO^?, and CD₃CD(ND⁺₃)COO^? have been obtained. A normal coordinate analysis is carried out based on the experimental frequencies of the four isotopic analogs and a 34 parameter valence-type force field defined in terms of local symmetry coordinates. The final refinement, in which five stretching force constants are constrained to fixed values obtained from bond length data, results in an average error of 7 cm^?1 (0.9%) for the observed frequencies of the four isotopically substituted molecules. Band assignments are given in terms of the potential energy distribution for local symmetry coordinates. For non-deuterated l-alanine, the vibrations above 1420 cm^?1 and below 950 cm^?1 may be described as localized group vibrations. By contrast, the eight modes in the middle frequency range, viz. the three skeletal stretching, the COO^? symmetric stretching, one NH⁺₃ rocking, the symmetric CH₃ deformation, and the two methyne CH deformation vibrations, are very strongly coupled to one another. Some decoupling appears to take place in the perdeutero molecule, and all but five modes can be described as localized group vibrations.     

Enantioselective solubilization of DL-amino acids in organic solvents containing N-alkyl-L-proline derivatives and copper(II) ions

Enantioselective solubilization of DL-amino acids has been observed in various organic solvents containing copper(II) complexes of N-alkyl-L-proline, and the enantioselectivity appears to be caused by the formation of diastereomeric mixed-ligand complexes and differences in their stability.     

Development of a disposable miniature l-lysine sensor

A hybrid l-lysine sensor consisting of an immobilized l-lysine decarboxylase and a miniature bacterial CO₂ sensor was fabricated using semiconductor techniques. The bacteria was immobilized in a calcium alginate gel in a miniature oxygen electrode cell together with the electrolyte. The enzyme was immobilized in a bovine serum albumin matrix on a gas-permeable membrane. The cell was formed on a silicon substrate by anisotropic etching and had a two-gold-electrode configuration. The response time of the l-lysine sensor was 1–3 min. The optimum pH was 6.0 and the optimum temperature was 33°C. The response to l-lysine concentration was linear from 25 to 400 μM. Reproducible responses were obtained by adding more than 1 μM pyridoxal-5′-phosphate. The sensor had excellent selectivity for l-lysine and a stable response for more than 25 repetitive operations.     

Synthesis of l,l-puromycin

l,l-Puromycin, a diastereomer of the natural peptidyl nucleoside antibiotic puromycin, has been synthesized from l-xylose in 13 steps.     

Intermolecular/interionic interactions in l-isoleucine-, l-proline-, and l-glutamine-aqueous electrolyte systems

Speed of sound and density values for ternary systems (amino acid + salt + water): l-isoleucine/l-proline/l-glutamine in aqueous solutions of 1.5 M KCl, 1 M KNO₃, and 0.5 M K₂SO₄ have been measured for several concentrations of amino acids at different temperatures (303.15, 308.15, 313.15, 318.15, and 323.15 K). Using speed of sound and density data, the thermodynamic parameters such as isentropic compressibility (κ_s), change in isentropic compressibility (Δκ_s) and relative change (Δκ_s/κ₀) in isentropic compressibility have been computed. The isentropic compressibility values decrease with increase in the amino acid concentration as well as with temperature. The decrease in κ_s values with increase in concentration of l-isoleucine/l-proline/l-glutamine in 1.5 M KCl, 1 M KNO₃, and 0.5 M K₂SO₄ has been ascribed to an increase in the number of incompressible zwitterions in solutions, and the formation of ‘zwitterions-ions’ and ‘zwitterions-water dipole’ entities in solutions. The decrease in κ_s values with increase in temperature has been attributed to the corresponding decrease of κ_relax (a relaxational part of compressibility), which is dominant over the corresponding increase of κ_∞ (an instantaneous part of compressibility). The trends of variation of Δκ_s and Δκ_s/κ₀ with variations in solute concentration and temperature have also been discussed in terms of solute-solute and solute-solvent intermolecular/interionic interactions operative in the systems.     

De novo approach to l-anhydrohexitol nucleosides as building blocks for the synthesis of l-hexitol nucleic acids (l-HNA)

A stereoselective and scalable route to 1,5-anhydrohexitol nucleoside analogues belonging to l-series as building blocks for l-HNA oligonucleotide synthesis has been efficiently tuned. Key to the successful outcome of our approach is the development of a DDQ-mediated domino reaction, which leads to the formation of an unsaturated 1,6-anhydrosugar derivative. Sugar elaborations and base insertion then enable to synthesize six-membered nucleosides.     

Studies towards the synthesis of ertugliflozin from l-Arabinose

A new method for the diastereoselective synthesis of enantiomerically pure ertugliflozin was developed. The crucial step involves an aldol condensation between 1-(4-chloro-3-(4-ethoxybenzyl)phenyl)ethanone and (4R,5R)-5-(((tert-butyldimethylsilyl)oxy)methyl)-2,2-dimethyl-5-((trityloxy)methyl)-1,3-dioxolane-4-carbaldehyde, which was prepared from known 2-C-trityloxymethyl-2,3-O-isopropylidene-l-erythrose (easily accessible in three steps from l-arabinose) by standard reduction/oxidation and protection/deprotection manipulations. Dihydroxylation of the aldol condensation product and further global deprotection led to the formation of the target molecule.     

Studies on indolic mould metabolites. Total synthesis of l-prolyl-2-methyltryptophan anhydride and deoxybrevianamide e

Syntheses of l - prolyl - 2 - methyl - l - tryptophan anhydride, l -prolyl - 2 - methyl - d - tryptophan anhydride, deoxybrevianamide E, and l - prolyl - 2 - (1,1 - dimethylallyl) - d- tryptophan anhydride are described. A route for the conversion of deoxybrevianamide E into brevianamide E has also been examined.