n-carbamoyl-d-p-hydroxyphenylglycine production using immobilized d-hydantoinase from recombinant E. coli

An immobilized d-hydantoinase was characterized and employed to produce n-carbamoyl-d-p-hydroxyphenylglycine (CpHPG) in a repeated batch process. The V_max and K_m of the immobilized d-hydantoinase at 50°C were 6.28 mm min⁻¹ g⁻¹ biocatalyst and 71.6 mm, respectively. The product CpHPG did not inhibit the activity of d-hydantoinase. Optimal reaction temperature was 60°C. A decrease in activity of immobilized d-hydantoinase due to thermal inactivation could be described as first-order decay; the deactivation energy was 23.97Kcal mol⁻¹. Under process conditions (50°C, 10% w/v substrate, and pH 8.5), the half-life of the immobilized d-hydantoinase was eight batches. The attrition of immobilized d-hydantoinase particles with a large amount of insoluble substrate particles during stirring resulted in fine biocatalyst particles. In addition to the thermal inactivation, the loss of fine biocatalyst particles during the recovery step contributed to the low operational stability.     

A facile synthesis of 5-thio-l-fucose and 5-thio-d-arabinose from d-arabinose

5-Thio-l-fucopyranose tetraacetate was synthesized in 11 steps from or d-arabinose diethyl dithioacetal by one-carbon elongation at C-5. Highly diastereo-selective addition of MeLi in ether to a derivative was achieved to give the corresponding 6-deoxy-β-d-altrofuranose isomer in good yield. A sulfur atom was introduced at C-5 of 6-deoxy-d-altrofuranose derivatives via substitution of a 5-tosylate with KSAc in HMPA with inversion of configuration, giving 5-thio-l-fucopyranose. A derivative was also prepared from 6-deoxy-β-d-altrofuranose derivatives. 5-Thio-d-arabinopyranose tetraacetate, the 5-demethyl analog of 5-thio-l-fucose, was also synthesized from in 5 steps. 5-Thio-d-arabinose showed weak inhibitory activity against α-l-fucosidase from bovine kidney (K_{i = 0.77 mM}).     

A microplate assay for d-xylose/ d-glucose isomerase

A modification of the resorcinol method of Kulka¹ for the determination of ketoses is described. Though being a stopped enzyme test, it is much more sensitive than the carbazol method and by applying microtiter plates and measuring with an ELISA reader, a large number of tests can be performed within a short time, thereby facilitating initial velocity studies.

The test is linear up to a concentration of 2.5 m -xylulose even in the presence of 10 m -xylose and 2 m -fructose in the presence of 10 m -glucose. The sensitivity is 25 μ for xylulose and 38 μ for fructose. The test method is insensitive to perturbations of substances frequently used in isolation procedures such as ammonium sulfate, Triton X-100, PEG 6000, sodium dodecyl sulfate, and ethanol in moderate concentrations.     

Hypotensive effects of [d-Tyr,d-Thr]neuropeptide Y(27–36)

An analogue of the 10 C-terminal amino acids of neuropeptide Y (NPY) containing taining three d-isomeric substitutions (27–36-d) has been synthesized and its cardiovascular activity studied in Sprague-Dawley (SD) and spontaneously hypertensive (SHR) rats. Intravenous administration of 1000 nmol/kg 27–36-d decreases MAP in SHR (−59.9 ± 5.0 mmHg) and SD rats (−44.4 ± 4.7 mmHg). The hypotension produced by 1000 nmol/kg 27–36-d diminished by 71.2% following pretreatment with the histamine receptor antagonist diphenhydramine, although histamine depletion with compound does not significantly alter this hypotension. These data suggest that NPY(27–36)-d produces a profound and sustained hypotension in two strains of rat which is partially attributable to activity at histamine receptors.     

Determination of [d-Ala, d-Leu]enkephalin and the metabolites containing C-terminal d-leucine by high-performance liquid chromatography

A high-performance liquid chromatographic procedure has been developed for the determination of [d-Ala², d-Leu⁵]enkephalin (DADLE) and the fragments containing d-leucine in rat blood. The procedure was applied to the determination of blood levels of [³H-d-Leu⁵]DADLE and the C-terminal fragments after intravenous administration of [³H-d-Leu⁵]DADLE to a rat. Unlabelled DADLE and the C-terminal fragments were spiked as carriers to rat blood samples and the blood samples were extracted with 1% trifluoroacetic acid in methanol. The recoveries from rat blood were quantitative for all compounds. DADLE and the C-terminal four fragments were well separated on a reversed-phase column with gradient elution using a mobile phase composed of 0.14% HClO₄ and acetonitrile.     

A comparative study of the O-3 reactivity of isomeric N-dimethylmaleoyl-protected d-glucosamine and d-allosamine acceptors

Four isomeric N-dimethylmaleoyl 4,6-O-benzylidene-protected d-hexosamine acceptors (2, 3, 4, and 5) with all possible configurations at C-1 and C-3 (e.g., derived from d-glucosamine and d-allosamine) were prepared, and the assessment of their O-3 relative reactivity through competition experiments using the known per-O-acetylated d-galactopyranosyl trichloroacetimidate donor (15) was then carried out. The reactivities are in the order 4 ? 2 > 5 > 3. The analysis of the NMR spectra of 2–5 at different temperature and modeling experiments carried out on analogs of 2–5 (DFT) and on the acceptors themselves (MM) are coincident, and have helped to establish the stability of the different hydrogen bonds, and of the conformers which carry them. The whole results suggest that the electronic effects (hydrogen bonds) are required to explain the observed trend, in spite of the axial conformation of the most reactive hydroxyl group. The steric effects appear only when hydrogen bonds are weak.     

A fluorescence-based assay for measuring N-acetyl-1-d-myo-inosityl-2-amino-2-deoxy-α-d-glucopyranoside deacetylase activity

Here we report a new fluorescence-based assay for measuring MshB (N-acetyl-1-d-myo-inosityl-2-amino-2-deoxy-α-d-glucopyranoside deacetylase) activity. The current assay for measuring MshB activity requires the fluorescent labeling of reaction mixtures and subsequent analysis using high-performance liquid chromatography (HPLC), resulting in a significant amount of processing time per sample. Here we describe a more rapid fluorescnce-based assay for the measurement of MshB activity that does not require HPLC analysis and can be carried out in multiwell plates. This fluorescamine (FSA)-based assay was used to determine the steady-state parameters for the deacetylation of N-acetyl-glucosamine (GlcNAc) by MshB, and the results from these experiments support the hypothesis that the inositol moiety primarily contributes to the affinity of GlcNAc–Ins (N-acetyl-1-d-myo-inosityl-2-amino-2-deoxy-α-d-glucopyranoside) for MshB. The rapid nature of this assay will aid efforts toward a more detailed biochemical characterization of MshB. Furthermore, because this assay relies on the formation of a primary amine, it could be adapted to measure the activity of mycothiol-S-conjugate amidase, a metal-dependent amidase that is a potential drug target involved in the mycothiol detoxification pathway.     

A facile synthesis of d-ribo-C20-phytosphingosine and its C2 epimer from d-ribose

A facile synthetic route to d-ribo-C₂₀-phytosphingosine 31 and its C2 epimer 32 is described. The Overman rearrangement of allylic trichloroacetimidates derived from the known ribose derivative 7 has been used as the key step. The subsequent functional group interconversions in rearranged products 14 and 15 followed by Wittig olefination, Pd/C-mediated reduction and the removal of protecting groups successfully constructed the final molecules.     

Synthesis of 6-deoxy-5-thio-d-glucose

Three routes were investigated for the conversion of d-glucose into the title compound. In the first approach, reduction of the 5,6-thürane ring of 5,6-dideoxy-5,6-epithio-1,2-O-isopropylidene α-d-glucofuranose (17) as well as that of its 3-O-allyl derivative (13) with lithium aluminium hydride was investigated; 17 afforded the corresponding 6-deoxy derivative besides di-, tri-, and poly-mers, whereas only polymers were formed from 13. In the second approach, the oxirane ring of was reduced by sodium borohydride and the resulting 6-deoxy derivative was converted into the 5-thiobenzoate; the corresponding hex-4-enofuranose was formed as a byproduct. In the third approach partial mesylation of methyl 5-thio-α-d-glucopyranoside was attempted, but the 6-mesylate 27 could be isolated only in modest yield (28%) together with rearranged 2,5-thioanhydromannofuranoside derivatives. The mechanism of this rearrangement is discussed in detail. The 6-mesylate 27 was converted via the 6-iodo derivative into the title compound.     

One-pot enzymatic production of 2-acetamido-2-deoxy-d-galactose (GalNAc) from 2-acetamido-2-deoxy-d-glucose (GlcNAc)

2-Acetamido-2-deoxy-d-galactose (GalNAc) is a common monosaccharide found in biologically functional sugar chains, but its availability is often limited due to the lack of abundant natural sources. In order to produce GalNAc from abundantly available sugars, 2-acetamido-2-deoxy-d-glucose (GlcNAc) was converted to GalNAc by a one-pot reaction using three enzymes involved in the galacto-N-biose/lacto-N-biose I pathway of bifidobacteria. Starting the reaction with 600 mM GlcNAc, 170 mM GalNAc was produced at equilibrium in the presence of catalytic amounts of ATP and UDP-Glc under optimized conditions. GalNAc was separated from GlcNAc using water-eluting cation-exchange chromatography with a commonly available cation-exchange resin.     

Free l-amino acids and d-aspartate content in the nervous system of Cephalopoda. A comparative study

We have determined the content of free l-amino acids and d-aspartate in the nervous tissue of three representative cephalopods: Sepia officinalis, Octopus vulgaris, and Loligo vulgaris, and the optic lobes of adult and embryo Sepia officinalis. Taurine is the most abundant amino acid in the cephalopod nervous tissue. Its content amounts to more than 50% of the total free amino acids. The other most concentrated amino acids are Glu, Ala, Asp, and GABA. High concentrations of d-aspartate were found in the nervous tissue of all cephalopods examined (7–12 μmol/g wet tissue) which represents 50–80% of the total aspartate (d + l), depending on the animal. Among the various regions of the brain of Octopus vulgaris, d-aspartate was found to be evenly distributed in the various regions of the brain. In nerve tissue of Sepia officinalis, there is no significant difference in the pattern of free l-amino acids, in particular of the d-aspartate concentration, between adults and embryos, except for GABA, Gly, His and Thr. This suggests that d-aspartate in nerve tissue of the Cephalopoda is of endogenous origin and not a product of accumulation from exogenous sources. From a comparative study of the content of d-aspartate in the nervous tissue of different animals, we found that protostomia contain a significantly higher amount than deuterostomia. Thus, d-aspartate could be a criterion to distinguish the protostomia phyla from the deuterostomia phyla.     

Glycosylation studies on conformationally restricted 3,5-O-(di-tert-butylsilylene)-d-galactofuranosyl trichloroacetimidate donors for 1,2-cis α-d-galactofuranosylation

Conformationally restricted 3,5-O-di-tert-butylsilylene-d-galactofuranosyl trichloroacetimidate donors were synthesized from allyl α-d-galactofuranoside for the construction of 1,2-cis α-d-galactofuranosyl linkages. Glycosylation reactions were performed with several acceptors, including d-galactono-1,4-lactone, d-rhamnopyranosyl, and d-mannopyranosyl derivatives. The influence of the temperature and the reaction solvents was evaluated, as well as the 6-O-substitution pattern of the donor. The higher α-selectivities were obtained at −78 °C in diethyl ether as solvent. 6-O-Acetyl substitution on constrained donor increased the α-selectivity compared to the 6-O-benzyl substitution. Almost no selectivities were observed in the non-participating solvent CH₂Cl₂. In contrast, ethereal solvents enhanced the α-selectivity suggesting a participating effect in the reaction intermediate.     

The glutathione dependence of inorganic sulfate formation from l- or d-cysteine in isolated rat hepatocytes

The GSH dependence of the metabolic pathways involved in the conversion of cysteine to sulfate in intact cells has been investigated. It was found that hepatocyte-catalysed sulfate formation from added

Full-size image

-cysteine did not occur if hepatocyte GSH was depleted beforehand, but was restored when GSH levels recovered. Furthermore, sulfate formation did not recover in GSH-depleted hepatocytes if GSH synthesis was prevented with buthionine sulfoximine. Thiosulfate formation was, however, markedly enhanced in GSH-depleted hepatocytes. These results suggest that thiosulfate is an intermediate in the formation of inorganic sulfate from

Full-size image

-cysteine and that GSH was required for the conversion of thiosulfate to inorganic sulfate. Much less sulfate was formed if the cysteine was replaced with cysteinesulfinate. Furthermore, sulfate formation from

Full-size image

-cysteine was markedly inhibited by the addition of the transaminase inhibitor

Full-size image

-cycloserine or the γ-cystathionase inhibitor

Full-size image

-propargylglycine. The major routes of sulfate formation from

Full-size image

-cysteine therefore seems to involve pathways that do not involve

Full-size image

-cysteinesulfinate. Similar amounts of sulfate were formed from

Full-size image

-cysteine as

Full-size image

-cysteine. Thiosulfate instead of sulfate was also formed in GSH-depleted hepatocytes. However, sulfate formation from

Full-size image

-cysteine differed from

Full-size image

-cysteine in that it was inhibited by the

Full-size image

-aminoacid oxidase inhibitor sodium benzoate and was not affected by transaminase or γ-cystathionase inhibitors. These results suggest that thiosulfate is an intermediate in sulfate formation from

Full-size image

-cysteine and involves the oxidation of

Full-size image

-cysteine by

Full-size image

-amino acid oxidase to form β-mercaptopyruvate.     

Evidence of benzilic rearrangement during the electrochemical oxidation of d-glucose to d-glucaric acid

During the course of the 2,2,6,6-tetramethyl-1-piperidinyloxy free radical-catalyzed electrochemical oxidation of d-glucose to d-glucaric acid a new side-product was observed. This compound was isolated and identified as a tricarboxylic acid of unique structure, which was named maribersonic acid. Its structure was proven by different experiments coupled with several analytical methods, and its appearance during the electrochemical oxidation of d-glucose was rationalized through a thorough study.     

Broadband dielectric spectroscopy and calorimetric investigations of d-lyxose

Using broadband dielectric spectroscopy, we have studied different types of relaxation processes, namely, primary (α), secondary (β), and another sub-T_g process called γ-process, in the supercooled state of d-lyxose, over a wide frequency (10^-2–) and temperature range (120–340 K). In addition, the same sample was analyzed by differential scanning calorimeter. The temperature dependence of the relaxation times as well as the dielectric strength of different processes has been critically examined. It has been observed that the slower secondary relaxation (designated as β-) process shifts to lower frequencies with increasing applied pressure, but not the faster one. This pressure dependence indicates that the observed slower secondary relaxation (β-) is Johari–Goldstein relaxation process and faster one (γ-process) is probably the rotation of hydroxymethyl (–CH₂OH) side group attached to the sugar ring, that is, of intramolecular origin.     

Pharmacokinetic, metabolic, and antidiarrheal properties of (d and l) heptapeptides of sorbin in rodent

The C-terminal heptapeptide-amide (C7-sorbin) is the minimal biologically active fragment of sorbin inducing an increase in intestinal hydroelectrolytic absorption. An analogue (D7-sorbin), characterized by the replacement of the ultimate C-terminal amino acid l-alanine-amide by d-alanine-amide, was synthetized. For pharmacokinetic studies, D7-sorbin and C7-sorbin were tritium labeled. After IV injection, clearances were 10.6 and 30.2 ml⁻¹ for D7-sorbin and C7-sorbin, respectively, and MRT were 34 and 18 min. After SC administration, C_max attained 0.41% and 0.12% of the dose/ml, respectively. The IP route showed a 45-min delay before C_max and a 100% bioavailability for both peptides. D7-sorbin was principally excreted in urine, as shown by balance study, and in part in intact form, as controlled by mass spectrometry. D7-sorbin induced a significant decrease of the VIP-induced ileal secretion, previously observed with C7-sorbin. The change of l-Ala to d-Ala increased the stability of the synthetic C-terminal peptide of sorbin whereas its biological activity, bioavailability, and route of elimination were unchanged.     

Modulation of UV-light-induced skin inflammation by d-alpha-tocopherol and l-ascorbic acid: a clinical study using solar simulated radiation

Objective: In this clinical trial we studied whether oral supplementation with d-alpha-tocopherol (α-Toc), l-ascorbic acid (Asc), or α-Toc combined with Asc influenced the solar simulated radiation (SSR) induced skin inflammation in healthy volunteers. Methods: We investigated the following groups in a prospective, randomized and placebo controlled study: Group (1) α-Toc 2 g / day, group (2) Asc 3 g / day, group (3) α-Toc 2 g / day combined with Asc 3 g / day, and group (4) placebo. Before and 50 days after supplementation we analyzed α-Toc and Asc concentrations in keratinocytes. The dose response curve of UV erythema was determined by reflectance spectrophotometry and the minimal erythema dose (MED) by visual grading before and after supplementation. Results: 50 days after supplementation α-Toc keratinocyte levels were increased in groups (1) and (3), Asc concentrations were elevated in groups (2) and (3), and the a/γ-Toc ratio increased in groups (1) and (3). The dose response curve of UVR induced erythema showed a significant flattening and the MED increased from 103 ± 29 mJ/cm² (before supplementation) to 183 ± 35 mJ/cm² (after supplementation) in group (3), while there were no significant changes in groups (1) and (2) after vitamin supplementation. Conclusion: α-Toc and Asc act synergistically in suppression of the sunburn reaction.     

Visible wavelength spectrophotometric assays of l-aspartate and d-aspartate using hyperthermophilic enzyme systems

Methods with which to simply and rapidly assay l-aspartate (l-Asp) and d-aspartate (d-Asp) would be highly useful for physiological research and for nutritional and clinical analyses. Levels of l- and d-Asp in food and cell extracts are currently determined using high-performance liquid chromatography. However, this method is time-consuming and expensive. Here we describe a simple and specific method for using an l-aspartate dehydrogenase (l-AspDH) system to colorimetrically assay l-Asp and a system of three hyperthermophilic enzymes—aspartate racemase (AspR), l-AspDH, and l-aspartate oxidase (l-AO)—to assay d-Asp. In the former, the reaction rate of nicotinamide adenine dinucleotide (NAD⁺)-dependent l-AspDH was measured based on increases in the absorbance at 438 nm, reflecting formation of formazan from water-soluble tetrazolium-1 (WST-1), using 1-methoxy-5-methylphenazinum methyl sulfate (mPMS) as a redox mediator. In the latter, d-Asp was measured after first removing l-Asp in the sample solution with l-AO. The remaining d-Asp was then changed to l-Asp using racemase, and the newly formed l-Asp was assayed calorimetrically using NAD⁺-dependent aspartate dehydrogenase as described above. This method enables simple and rapid spectrophotometric determination of 1 to 100 μM l- and d-Asp in the assay systems. In addition, methods were applicable to the l- and d-Asp determinations in some living cells and foods.