Fluorometric quantification of total d-gluconate by a flow-injection system using an immobilized-enzyme reactor

A method for the quantification of total d-gluconate by flow-injection analysis was developed using an immobilized-enzyme reactor and fluorescence detection. d-Gluconate was quantified using co-immobilized gluconate kinase (GK) and 6-phosphogluconate dehydrogenase (PGDH) reactor. d-Gluconate was phosphorylated to 6-phospho-d-gluconate by GK in the presence of ATP, and then the 6-phospho-d-gluconate produced was oxidized by PGDH with NADP⁺. The NADPH produced by the GK-PGDH reactor was monitored fluorometrically at 455 nm (excitation at 340 nm). A linear relationship between the responses and concentrations of d-gluconate was obtained in the ranges of 1.0 × 10⁻⁶-1.6 × 10⁻⁴ M. The relative standard deviation for 10 successive injections was 0.57% at the 0.1 mM level. This analytical method was applied to the quantification of d-gluconate in honeys, vinegars and noble rot wines, and the results showed good agreement with those obtained using the conventional F-kit method.     

Fluorometric quantification of total d-gluconate by a flow-injection system using an immobilized-enzyme reactor

A method for the quantification of total d-gluconate by flow-injection analysis was developed using an immobilized-enzyme reactor and fluorescence detection. d-Gluconate was quantified using co-immobilized gluconate kinase (GK) and 6-phosphogluconate dehydrogenase (PGDH) reactor. d-Gluconate was phosphorylated to 6-phospho-d-gluconate by GK in the presence of ATP, and then the 6-phospho-d-gluconate produced was oxidized by PGDH with NADP⁺. The NADPH produced by the GK-PGDH reactor was monitored fluorometrically at 455 nm (excitation at 340 nm). A linear relationship between the responses and concentrations of d-gluconate was obtained in the ranges of 1.0 × 10⁻⁶–1.6 × 10⁻⁴ M. The relative standard deviation for 10 successive injections was 0.57% at the 0.1 mM level. This analytical method was applied to the quantification of d-gluconate in honeys, vinegars and noble rot wines, and the results showed good agreement with those obtained using the conventional F-kit method.     

Determination of L- and D-enantiomers of methotrexate using amperometric biosensors

In order to determine the enantiopurity of methotrexate (Mtx), seven biosensors were proposed for the assay of l-Mtx and three biosensors for the assay of d-Mtx. The biosensors were designed using physical and chemical immobilization of glutamate oxidase and/or l-amino acid oxidase (l-AAOD) and/or horseradish peroxidase (HRP) for the assay of l-methotherexate, and d-amino acid oxidase (d-AAOD) and HRP for the assay of d-Mtx. Electrode characteristics were obtained and compared for the different carbon paste based biosensors. The linear concentration ranges for the proposed biosensors were in the ranges of fmol l⁻¹ to pmol l⁻¹, magnitude order with limits of detection in the fmol l⁻¹ to nmol l⁻¹ concentration range. All biosensors were successful for the determination of the enantiopurity of Mtx as raw material, and in its pharmaceutical formulations (tablets and injections).     

Lipase-catalyzed synthesis of biodegradable copolymer containing malic acid units in solvent-free system

This work is to explore a new route to synthesize functional polyesters bearing pendant hydroxyl groups. The approach is via biocatalyzed direct polycondensation. l-Malic acid, adipic acid and 1,8-octanediol were used as comonomers and lipase Novozym 435 as a biocatalyst. ¹H NMR studies on the structure of the products indicated that Novozym 435 was strictly selective for esterification of l-malic acid carboxyl groups while leaving the hydroxyl groups unchanged. The influences of the monomer feeding ratio, reaction temperature, and reaction time on the molecular weight of the products were investigated. By varying l-malic acid feed ratio in the total monomers from 0 to 20 mol%, the molecular weight (M_W) of the product changed from 9.5 kilo Dalton (kD) to 4.7 kD while reaction was held at 70 °C for 48 h. The maximum M_W could reach 7.4 kD at 80 °C when varying temperature between 70 and 90 °C if l-malic acid is 20 mol% and reaction time is 48 h. At 75 °C the M_W increased from 5.2 kD to 6.6 kD when reaction time was elongated from 48 h to 72 h. However, little change in M_W was observed at 80 and 85 °C when the reaction time was above 48 h. Thermal property of the copolyesters was studied by differential scanning calorimetry (DSC). Increasing the l-malic acid content in copolyesters resulted in melting temperature depression.     

Sequential fluorometric quantification of γ-aminobutyrate and l-glutamate using a single line flow-injection system with immobilized-enzyme reactors

A single line flow-injection system with immobilized-enzyme reactors is proposed for the sequential quantification of γ-aminobutyrate (GABA) and l-glutamate. A co-immobilized l-glutamate oxidase and catalase reactor and an immobilized GABase reactor were introduced into the flow line in series. Sample and reagent were injected into the flow line using an open sandwich method. GABA was selectively detected by GABase when α-ketoglutarate at a high concentration and NADP⁺ were injected as the reagents with a sample. When GABA at a high concentration and NADP⁺ were injected as the reagents with a sample, l-glutamate only was determined by the series of enzymatic reactions. NADPH produced by the immobilized-enzyme reactors was monitored fluorometrically at 455 nm (excitation at 340 nm). Linear relationships between the responses and concentrations of GABA or l-glutamate were observed in the ranges of 5.0 × 10⁻⁶-5.0 × 10⁻⁴ M and 1.0 × 10⁻⁵-5.0 × 10⁻⁴ M, respectively. The relative standard deviations for ten successive injections were less than 2% at the 0.5 mM level. This analytical method was applied to the sequential quantification of GABA and l-glutamate that were produced and consumed, respectively, by lactic acid bacteria, and the results showed good agreement with those obtained using liquid chromatography.     

Construction of a new firefly bioluminescence system using l-luciferin as substrate

l-Luciferin can be converted into d-luciferin with an enzyme/co-factor system consisting of firefly luciferase, an esterase, ATP, Mg²⁺, and coenzyme A. By this means, a new firefly bioluminescence system can be constructed that uses l-luciferin as the substrate.     

On-line microdialysis assay of l-lactate and pyruvate in vitro and in vivo by a flow-injection system with a dual enzyme electrode

A flow-injection biosensor system with an on-line microdialysis sampling is proposed for the simultaneous assay of l-lactate and pyruvate in serum and rat brain. The dialysate collected in the sample loop by perfusing Ringer’s solution through the microdialysis probe is automatically injected into the flow-injection line with a dual enzyme electrode arranged in parallel for the flow direction. The dual enzyme electrode is constructed by hybridizing a poly(1,2-diaminobenzene) film to two sensing parts, which respond selectively to l-lactate and pyruvate, respectively, without any cross-reactivity. Both the sensing parts respond linearly to the concentrations of both analytes between 0.01 and 5 mM, without any interference from oxidizable species and low-molecular weight proteins present in the dialysate. The proposed flow-injection analysis (FIA) method can be successfully applied to the simultaneous in vitro and in vivo assays of both analytes in serum and rat brain, respectively. The system can be automatically processed at an analytical speed of 19 dialysates h⁻¹ over a period of 5 h.     

A convenient synthesis of l-ribose from d-fructose

An efficient method for the stereoselective synthesis of l-ribose was accomplished starting from commercially inexpensive d-fructose. The intermediates in the process can serve as versatile precursors for the preparation of l-nucleoside analogues.     

Selection of synthetic receptors capable of sensing the difference in binding of d-Ala-d-Ala or d-Ala-d-Lac ligands by screening of a combinatorial CTV-based library

Screening of a combinatorial CTV-based artificial, synthetic receptor library 1 {1-13, 1-13, 1-13} for binding of a variety d-Ala-d-Ala and d-Ala-d-Lac containing ligands (6-11) was carried out in phosphate buffer (0.1 N, pH=7.0). After screening and Edman sequencing, synthetic receptors were found containing amino acid sequences, which are either characteristic for binding dye labeled d-Ala-d-Ala or d-Ala-d-Lac containing ligands. For example, receptors capable of binding d-Ala-d-Ala containing ligands 6, 7, 9 and 11 contained—almost in all cases—at least one basic amino acid residue—predominantly Lys—in their arms. This was really a striking difference with the arms of the receptors capable of binding d-Ala-d-Lac containing ligands 8 and 10, which usually contained a significant number of polar amino acids (Gln and Ser), especially in ligand 8, but hardly any basic amino acids. Use of different (fluorescent) dye labels showed that the label has a profound, albeit not decisive, influence on the binding by the receptor. A hit from the screening of the CTV-library with FITC-peptidoglycan (6) was selected for resynthesis and validation.     

High-molar-mass hyaluronan degradation by Weissberger's system: Pro- and anti-oxidative effects of some thiol compounds

Pro- and anti-oxidative effects of two thiol compounds, d-penicillamine and reduced l-glutathione, on the kinetics of degradation of high-molar-mass hyaluronan samples were monitored via rotational viscometry. The degradation was induced under aerobic condition by the so-called Weissberger′s system [ascorbate plus Cu(II)]. Electron paramagnetic resonance spectroscopy was used to confirm the presence of free radicals generated during the biopolymer degradation. Infrared spectroscopy and non-isothermal chemiluminometry were used to characterize the biopolymers after processing.     

Resolution of racemic Vigabatrin using tartaric acid

An efficient and simple resolution methodology for the preparation of (S)- and (R)-Vigabatrin has been developed. In addition, a method of preparation for the novel compounds Vigabatrin-l-tartarate and Vigabatrin-d-tartarate is also described. The title compounds have been synthesized via resolution of Vigabatrin using commercially available l-(+)- and d-(?)-tartaric acids respectively.     

Effects of molecular weight and small amounts of d-lactide units on hydrolytic degradation of poly(l-lactic acid)s

Films of poly(l-lactic acid) (PLLA) with different number-average molecular weights (M_n) and d-lactide unit contents (X_d) were made amorphous and the effects of molecular weight and small amounts of d-lactide units on the hydrolytic degradation behavior in phosphate-buffered solution at 37 °C of PLLA were investigated. The degraded films were investigated using gravimetry, gel permeation chromatography, polarimetry, differential scanning calorimetry, X-ray diffractometry, and tensile testing. To exclude the effects of crystallinity on the hydrolytic degradation, the films were made amorphous by melt-quenching. The incorporation of small amounts of d-lactide units drastically enhanced the hydrolytic degradation of PLLA. In the period of 0-32 weeks, the hydrolytic degradation rate constant (k) of PLLA films increased with increasing X_d, while the k values did not depend on M_n. This means that the effects of X_d on the hydrolytic degradation rate of the films are higher than those of M_n. In contrast, in the period of 32-60 weeks neither X_d nor M_n was a crucial parameter to determine k values, probably because in addition to these parameters the differences in the amount of catalytic oligomers accumulated in films and crystallinity affect the hydrolytic degradation behavior of the films. The initially amorphous PLLA films remained amorphous even after the hydrolytic degradation for 60 weeks.     

Flow-injection fluorometric quantification of pyruvate using co-immobilized pyruvate decarboxylase and aldehyde dehydrogenase reactor: Application to measurement of acetate, citrate and l-lactate

A flow-injection system for the quantification of pyruvate based on the coupled reactions of pyruvate decarboxylase (PDC) and aldehyde dehydrogenase (AlDH) was conceived and optimized. A co-immobilized PDC and AlDH reactor was introduced into the flow line. Sample and reagent (NAD⁺) were injected into the flow line by an open sandwich method and the increase of NADH produced by the immobilized-enzyme reactor was monitored fluorometrically at 455 nm (excitation at 340 nm). Linear relationships between the responses and concentrations of pyruvate were observed in the ranges of 2.0 × 10⁻⁵ to 1.5 × 10⁻³ M at the flow rate of 1.0 ml min⁻¹ and 5.0 × 10⁻⁶ to 1.0 × 10⁻³ M at the flow rate of 0.5 ml min⁻¹. The relative standard deviation for 10 successive injections was 0.95% at the 1.0 mM level. This FIA system for pyruvate was applied to the measurement of acetate, citrate and l-lactate.     

One-pot inversion of d-mannono-1,4-lactone for the practical synthesis of l-ribose

l-Ribose was synthesized in a concise manner from d-mannono-1,4-lactone using one-pot inversion conditions. Treatment of d-mannono-1,4-lactone with piperidine, followed by mesylation-induced S_N2-type O-alkylation, afforded the desired one-pot inversion in an optimum yield, and the following straightforward transformations provided l-ribose in good yields.     

Determination of l-ascorbic acid in wines by direct injection liquid chromatography using a polymeric column

A rapid method for the identification and quantification of l-ascorbic acid in wines by direct injection liquid chromatography equipped with a UV detection was developed. The levels of ascorbic acid were determined using a polymeric PLRP-S 100 A (5 μm) column (150 mm × 4.6 mm) with a mobile water/trifluoroacetic acid (99/1, v/v) phase. The method is rapid (less than 5 min) and sensitive (LOQ of 5 mg L⁻¹). The calibration curve of ascorbic acid was linear (r = 0.999) over a concentration range between 1 and 200 mg L⁻¹. Repeatability was less than 2.5% and the recovery over 95%.     

Flow-injection chemiluminescence determination of ascorbic acid by use of the cerium(IV)-Rhodamine B system

A highly sensitive flow-injection (FI) method with chemiluminescence (CL) detection is used for the determination of l-ascorbic acid. The method is based on the CL reaction of Rhodamine B with cerium(IV) in sulfuric acid media. l-Ascorbic acid is suggested to be a catalyst utilized in the energy-transferred excitation process. The proposed procedure allows quantitation of l-ascorbic acid in the range 3.8×10⁻¹³ to 1.0×10⁻¹⁰ mol l⁻¹ with a correlation coefficient of 0.9998 (n=5) and relative standard deviation (R.S.D.) of 0.92% (n=11) at 1.0×10⁻¹¹ mol l⁻¹. The detection limit (3×blank) was 1.0×10⁻¹³ mol l⁻¹. The method is successfully used to determine l-ascorbic acid in fresh vegetables. The possible mechanism of the chemiluminescence in the system is discussed.     

A d,l-proline catalyzed diastereoselective trimolecular condensation: an approach to the one-pot synthesis of perhydrofuro[3,2-b]pyran-5-ones

d,l-Proline was found to catalyze efficiently the one-pot trimolecular condensation of indoles, a sugar hydroxyaldehyde, and Meldrum’s acid followed by intramolecular cyclization with evolution of carbon dioxide and elimination of acetone to afford 7-(1H-3-indolyl)-2,3-dimethoxyperhydrofuro[3,2-b]pyran-5-ones. The reaction proceeded cleanly at ambient temperature to afford the products in good yields with high diastereoselectivity.     

Swelling and hydrolytic degradation of poly(d,l-lactic acid) in aqueous solutions

Low molecular weight poly(lactic acid) was synthesized by direct polycondensation of lactic acid. The oligomers were characterized by viscometry, light scattering, and gel permeation chromatography (GPC). The swelling behaviour of tablets made of the above polymer immersed in buffer solutions at 37 °C was studied. In the same experiments, the hydrolytic stability of d,l-PLA was assessed by measuring the weight loss after drying the tablets. In order to inhibit any degradation due to bacteria, formaldehyde was added in the solution as biostatic factor. The effect of an incorporated drug on the swelling behaviour of d,l-PLA tablets was also considered. It was found that the incorporation of drug in d,l-PLA tablets increases their swelling index, probably due to the creation of additional porosity in the specimens or other interaction between drug and polymeric matrix.     

d-Poly(lactide) and LHRH decapeptide stereointeractions investigated by vibrational spectroscopy

The interactions between poly(d-lactic acid) (PDLA) and l-configured leuprolide (LHRH) to form heterocomplexes were investigated by a combination of infrared (IR), Raman and near infrared spectroscopy (NIR). It was found that an α crystal with 10₃ helical conformation of PDLA is formed in the PDLA/LHRH heterocomplexes with various LHRH loadings, whereas the secondary structures of LHRH in these heterocompelexes are greatly affected by the blend ratio of LHRH and PDLA. Based on the analysis of IR, Raman and NIR spectra of the various heterocomplexes, it is suggested that stereoselective Van der Waals interactions, consisting of interwined α helices of PDLA and LHRH, is responsible for the driving force of PDLA and LHRH stereocomplexation. To clarify the solid structure and the potential interaction in these heterocomplexes with various LHRH loadings, temperature-dependent IR spectral measurements were also employed. On the basis of the results presented, a model for the PDLA and LHRH stereointeraction process was proposed.     

New building blocks for peptide and depsipeptide synthesis: hexafluoroacetone protected l-homoisoserine and d,l-homoisocysteine derivatives

Efficient syntheses of l-homoisoserine and d,l-homoisocysteine derivatives starting from l-malic and d,l-thiomalic acid using hexafluoroacetone as protecting and activating agent are described. The new compounds are interesting building blocks for the preparation of non-natural peptides and depsipeptides as well as for the construction of new GABA derivatives.