Synthesis of 5, 6-Dichloro-Benzimidazole-(2′, 5′)-Nucleotide Trimer

Abstract

The chemical synthesis of the trimeric 5, 6-dichloro-1-β-D-ribofuranosylbenzimidazole-(2′, 5′)-diphosphate using the phosphotriester approach is described.     

Non-Glycosidic Derivatives of 5-Fluorouracil

Abstract

The synthesis and the chemical and biological properties of several non-glycosidic derivatives of 5-fluorouracil are described.     

The Synthesis of 5′-O-Triphosphate-4N-(ω-aminoalkyl)deoxycytidine A Useful Precursor to the Generation of Differently Labeled Triphosphates

Abstract

A chemical synthesis is described for 5′-O-triphosphate-4N-[6-(-γ-aminopropylamidosuccinylamido)-hexyl]deoxycytidine (13), the substrate for synthesis of 5′-O-triphosphate deoxycytidine derivatives (17-19) labeled with biotin, fluorescein and photoreactive azide.     

Some Aspects of the Chemical Synthesis of Oligoribonucleotides

Abstract

The present position regarding the protection of the 2′- and 5′-hydroxy functions in the chemical synthesis of oligoribonucleotides is discussed.     

Indole-3-acetic Acid Synthesis in Tumorous and Nontumorous Species of Nicotiana

The synthesis of indole-3-acetic acid (IAA) in the enzyme extracts of Nicotiana glauca, Nicotiana langsdorffii, their F1 hybrid, their amphidiploid hybrid, and the nontumorous mutant of the hybrid was investigated. Tryptamine, a possible precursor of IAA biosynthesis in Nicotiana tabacum, was not found in the callus tissue of N. glauca, N. langsdorffii, and their F1 hybrid.

In petiole slices, the synthesis of IAA progressively increased during 5 hours of incubation in [¹⁴C]tryptophan. The rate of synthesis was about equal in the hybrid and N. langsdorffii but lower in N. glauca on either a cell or fresh weight basis. It was also found that tryptophan was about 25 times more efficient than tryptamine in promoting synthesis of IAA in petiole slices.

It was found that indoleacetaldehyde oxidase, indoleacetaldehyde reductase, and tryptophan aminotransferase activities were present in all of the species examined; however, tryptophan decarboxylase activity was not found. The tryptophan aminotransferase activity in N. glauca, N. langsdorffii, and the nontumorous mutant required α-ketoglutaric acid and pyridoxal 5-phosphate whereas the addition of pyridoxal 5-phosphate seemed not to increase the enzyme activity in tumor plants.

The tryptophan aminotransferase in the amphidiploid hybrid was partially purified by acetone precipitation. The enzyme activity had a temperature optimum at 49 C and a pH optimum at 8.9. It is suggested that there is an indolepyruvic acid pathway in the synthesis of IAA in the Nicotiana species examined.

     

Synthesis of Terminal Nucleoside Phosphates and Thiophosphates via Phosphoramidite Chemistry

Abstract

Synthetic oligonucleotides for use in biological processes often require a 5′-terminal phosphate function. To study enzymatic reactions this group can be replaced by a thiophosphate function. Most chemical methods for the synthesis of these termini base on phosphotriester chemistry.     

Synthesis of 3′-Azido-3′-Deoxythymidine-Terminated Oligonucleotide

Abstract

A method of completely chemical synthesis of 3′-azido-3′-deoxythymidine-terminated oligonucleotides via 5′-H-phosphonate of AZT is described.     

Nitrile-hydrolyzing enzyme from Meyerozyma guilliermondii and its potential in biosynthesis of 3-hydroxypropionic acid

3-Hydroxypropionic acid (3-HP) is an important platform chemical in organic synthesis. Traditionally, 3-HP was produced by chemical methods and fermentation process. In this work, a novel enzymatic method was developed for green synthesis of 3-HP. A yeast strain harboring nitrile-hydrolyzing enzyme was newly isolated from environmental samples using 3-hydroxypropionitrile (3-HPN) as the sole nitrogen source. It was identified to be Meyerozyma guilliermondii CGMCC12935 by sequencing of the 18S ribosomal DNA and internal transcribed spacer, together with analysis of the morphology characteristics. The catalytic properties of M. guilliermondii CGMCC12935 resting cells were determined, and the optimum activity was achieved at 55 °C and pH 7.5. The enzyme showed broad substrate specificity towards nitriles, especially 3-HPN, aminoacetonitrile and 3-cyanopyridine. The presence of Ag⁺, Pb²⁺ and excess substrate inhibited the enzyme activity, whereas 5% (v/v) ethyl acetate had a positive effect on the enzyme activity. M. guilliermondii CGMCC12935 resting cells by addition of 3% glucose could thoroughly hydrolyze 500 mM 3-HPN into 3-HP within 100 h and the maximal accumulative production of 3-HP reached 216.33 mM, which was over twofolds than the control group with no additional glucose. And this work would lay the foundation for biological production of 3-HP in industry.

     

Etude exploratoire de la spermatogenèse induite par la chaleur chez l'escargot Helix aspersa en hibernation: Rôle du cerveau / An exploratory study on temperature-induced spermatogenesis in the hibernating snail Helix aspersa: the role of the brain

Summary

The male cells in the ovotestis of hibernating snails undergo multiplication when the temperature of the environment is raised from 5°C to 25°C. If the temperature is maintained at 25°C for 4 weeks the process of spermatogenesis is completed but the rate of spermatogenesis (DNA synthesis) starts decreasing from the 3rd week (Table 1; Fig. 1).

Brain ablation in hibernating snails maintained at 25°C causes a significant increase in DNA synthesis exclusively in male cells of the ovotestis. This suggests that the brain exerts an inhibitory influence on spermatogenic multiplication. This influence is effective only during the first and the fourth week of exposure of hibernating snails to 25°C (Fig. 1; Table 1) indicating the existence of an endogenous cyclical control. Spermiogenesis is, however, not affected by brain extirpation (Fig. 4 A,B,C).

When reimplanted in the head haemocoel the brain appears normal histologically (Fig. 5 A,B) and it reestablishes the inhibitory influence on DNA synthesis in the ovotestis only during the first day of temperature-induction. During the 1st, 3rd and 4th week the reimplanted brain, deprived of its neural connections, fails to exert its inhibitory influence suggesting that for this influence to function neural connections to the brain are essential. Surprisingly, the implanted brain seems to inhibit DNA synthesis during the 2nd week of temperature-induction (Table 1).

These experiments show that the brain control temperature-induced spermatogenic multiplication in the ovotestis in snails at the onset of hibernation and this control is exerted by one or more inhibitory factors originating from the brain which may function in concert or independently to produce the neuroendocrine effect. It seems therefore justified to consider spermatogenesis in hibernating snails as being neuroendocrinologically controlled.     

Protein metabolism in senescing wheat leaves : determination of synthesis and degradation rates and their effects on protein loss

Wheat leaves (Triticum aestivum L.) at the moment of their maximum expansion were detached and put in darkness. Their protein, RNA and DNA contents, as well as their rates of protein synthesis and degradation, were measured at different times from 0 to 5 days after detachment. Rates of protein synthesis were measured by incorporation into proteins of large amounts of [³H]leucine. Fractional rates of protein degradation were estimated either from the difference between the rates of synthesis and the net protein change or by the disappearance of radioactivity from proteins previously labeled with [³H]leucine or [¹⁴C]proline.

Protein loss reached a value of 20% during the first 48 hours of the process. RNA loss paralleled that of protein, whereas DNA content proved to be almost constant during the first 3 days and decreased dramatically thereafter.

Measurements of protein synthesis and degradation indicate that, in spite of a slowdown in rate of protein synthesis, an increased rate of protein breakdown is mainly responsible for the observed rapid protein loss.

     

Decarboxylation of Oxalic Acid during Ripening of Banana Fruit (Musa sapientum L.)

ABSTRACT

Japanese apricot, Prunus mume Sieb. et Zucc., biosynthesizes the l-phenylalanine-derived cyanogenic glucosides prunasin and amygdalin. Prunasin has biological properties such as anti-inflammation, but plant extraction and chemical synthesis are impractical. In this study, we identified and characterized UGT85A47 from Japanese apricot. Further, UGT85A47 was utilized for prunasin microbial production. Full-length cDNA encoding UGT85A47 was isolated from Japanese apricot after 5?- and 3?-RACE. Recombinant UGT85A47 stoichiometrically catalyzed UDP-glucose consumption and synthesis of prunasin and UDP from mandelonitrile. Escherichia coli C41(DE3) cells expressing UGT85A47 produced prunasin (0.64 g/L) from racemic mandelonitrile and glucose. In addition, co-expression of genes encoding UDP-glucose biosynthetic enzymes (phosphoglucomutase and UTP-glucose 1-phosphate uridiltransferase) and polyphosphate kinase clearly improved prunasin production up to 2.3 g/L. These results showed that our whole-cell biocatalytic system is significantly more efficient than the existing prunasin production systems, such as chemical synthesis.     

The Effects of 2-5A on Protein Synthesis in Wheat Germ Extracts and Tobacco Protoplasts

Abstract

Nonphosphorylated 2-5A inhibited translation and caused RNA degradation in wheat germ extract, whereas 3-5A had no effect. Protein synthesis inhibition by 2-5A was observed in tobacco protoplasts. 70 kD 2-5A-binding protein was found in potato leaf extracts by chemical crosslinking.     

The Chemical Synthesis of Biochemically Active Oligoribonucleotides Using Dimethylaminomethylene Protected Purine H-Phosphonates

Abstract

Dimethylaminomethylene was applied as the protecting group for the exocyclic amino groups of adenosine and guanosine in the automated chemical synthesis of oligoribonucleotides on a polymer bound support. The dimethyl-aminomethylene protecting group can be removed at room temperature under conditions where the concomitant loss of the 2′-protection group can be excluded. The transformation of 2′-O-(t-butyldimethylsilyl)-5′-O-(4,4′-dimethoxytrityl) protected nucleosides to 3′-H-phosphonates yields synthons, well suited for the automated chemical synthesis of oligoribonucleotides. Using these H-phosphonate monomers, a coupling time of two minute: is sufficient to obtain average coupling yields of more than 98 %. Synthesized RNA is recognized as a substrate in an enzymatic reaction, forms the expected secondary structures and is suitable for NMR structural investigations.     

Synthesis,Specific Intracellular Delivery and Biological Activity of Novel Stabilized (2′-5′)(A)n Analogues

Abstract

5’ and 2’ stabilized (2′-5′)(A)_n analogues were synthesized by chemical modifications of enzymatically polymerized (2′-5′)(A)_n oligomers. They exhibit an increased antiviral activity after micro-injection in HeLa cell cytoplasm in agreement with their augmented metabolic stability. Their specific in vitro delivery to mouse leukemia cells after encapsulation in targetted liposomes leads to a transient inhibition of protein synthesis and an antiviral activity.     

Production of Nucleic Acid-Related Substances by Fermentative Processes

An adenine-requiring mutant (KY7208) of Brevibacterium ammoniagenes ATTC 6872 was found to accumulate an appreciable quantity of IMP and hypoxanthine in the culture liquid.

Crystalline IMP was isolated from culture broth of KY7208 by the use of ion-exchange columns. The preparation obtained was definitely identified as 5′-IMP, based on the results on paperchromatography, UV and IR absorption spectra, and analyses of its hydrolysates.

Growth responses of this mutant were demonstrated to adenine and adenosine, but not to 5′-AMP, 3′-AMP and 5′-AMP.

Over 5 mg of IMP per ml of broth were produced by the organism in natural medium consisting of glucose, yeast extract, urea, high concentrations of phosphate and magnesim salts, and others. The chemical changes showed that hypoxanthine first accumulated in the earlier stage of fermentation, and IMP synthesis then took place with the disappearance of hypoxanthine in the later stage of fermentation.     

Ultrafiltration,a useful method for isolation of intermediates in native chemical ligation exemplified with the total synthesis of Sortase AΔN59

In this paper, ultrafiltration was employed to facilitate the isolation of intermediates in native chemical ligation. Depending on the molecular weight cutoff of the membrane used, molecules with different sizes could be purified, separated, or concentrated by the ultrafiltration process. Total chemical synthesis of the polypeptide chain of the enzyme Sortase A_ΔN59 was used as an example of the application of ultrafiltration in chemical protein synthesis. Sortase A is a ligase that catalyzes transpeptidation reactions between proteins that have C‐terminal LPXTG recognition sequence and Gly5‐ on the peptidoglycan of bacterial cell walls [3]. Ultrafiltration technique facilitated synthesis of Sortase A_ΔN59 and was a promising tool in isolation of intermediates in native chemical ligation. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.     

Ab-inito Quantum Mechanical Calculations of NMR Chemical Shifts in Nucleic Acids Constituents II Conformational Dependence of the lH and 13C Chemical Shifts in the Ribose

Abstract

The magnetic shielding constant of the different ¹³C and ¹³H nuclei of a deoxyribose are calculated for the C2′ endo and C3′ endo puckerings of the furanose ring as a function of the conformation about the C4′C5′ bond. For the carbons the calculated variations are of several ppm, the C3′ endo puckering corresponding in most cases to a larger shielding than the C2′ endo one. For the protons the calculated variations of chemical shifts are all smaller than 1.3 ppm, that is of the order of magnitude of the variation of the geometrical shielding produced on these protons by the other units of a DNA double helix, with a change of the overall structure of the helix. The computations carried out on the deoxyribose ?3′ and 5′ phosphates for several conformations of the phosphate group tend to show that the changes of conformation of the charged group of atoms produce chemical shift variations smaller than the two conformational parameters of the deoxyribose itself. The calculations carried out for a ribose do give the general features of the differences between the carbon and proton spectra of deoxynucleosides and nucleosides.

The comparison of the measured and calculated phosphorylation shifts tend to show that the counterion contributes significantly, for some nuclei of the deoxyribose, to the shifts measured. The calculated magnitude of this polarization effect on carbon shifts suggests a tentative qualitative interpretation of carbon spectra of the ribose part of DNA double helices.     

Metabolism of alpha-Ketoglutarate by Roots of Woody Plants

The uptake and metabolism of α-ketoglutarate-5-¹⁴C by peach, apple, and privet root tissues were studied over various time intervals. As much as 80% of the absorbed ¹⁴C appeared as ¹⁴CO₂ in 320 minutes in peach roots. Apple and privet roots were less effective in this conversion with the bulk of the ¹⁴C found in the organic acid fraction. This indicates differences in organic acid metabolism among species of woody plants.

The ¹⁴C accumulated in malate earlier and in larger quantities than in citrate. Both glutamate and aspartate were labeled in 10 minutes and glutamate was labeled as early as 3 minutes. The labeling pattern does not clearly distinguish between the synthesis of glutamate by glutamic dehydrogenase or by transamination with oxaloacetate.

The rapid metabolism of α-ketoglutarate to glutamate by the 3 species studied indicates the presence of enzyme systems important in amino acid synthesis in the roots of woody plants.

     

Azione Esercitata Da Alcuni Inibitori Respiratori Sull'Inverdimento Di Apici Caulinari Di Piantine Eziolate Di Pisello

Abstract

Action of some respiratory inhibitors on the greening of apices of etiolated pea. — Aim of the research is to study the effect of some of the best kown respiratory inhibitors on the greening of etiolated apices of peas excised from the plant and kept in light in solutions containing saccharose. The inhibitors studied may act on more than one of the reactions leading to the synthesis of the chlorophyll molecule.

The following can be concluded from the present study:

1. A good level of chemical energy seems to be necessary for greening.

2. Terminal iron-oxidases seem to be necessary in this process, while a stimulating effect on greening may be attributed to an inhibition of the terminal copper-oxidases (particularly ascorbic-oxidase); the effect can be explained with the hormonal control of greening by the indoleacetic/ascorbic system.

3. Enzymes of the Krebs cycle regulate both the uptake of the essential metabolites starting from the chlorophyll molecule and the production of chemical energy.

4. Some enzymes of the glycolysis take part in some of the reactions leading to the synthesis of the chlorophyll molecule.     

Enhanced synthesis of poly gamma glutamic acid by increasing the intracellular reactive oxygen species in the Bacillus licheniformis Δ1-pyrroline-5-carboxylate dehydrogenase gene ycgN-deficient strain

Poly gamma glutamic acid (γ-PGA) is an anionic polyamide with numerous applications. Previous studies revealed that L-proline metabolism is implicated in a wide range of cellular processes by increasing intercellular reactive oxygen species (ROS) generation. However, the relationship between L-proline metabolism and γ-PGA synthesis has not yet been analyzed. In this study, our results confirmed that deletion of Δ1-pyrroline-5-carboxylate dehydrogenase gene ycgN in Bacillus licheniformis WX-02 increased γ-PGA yield to 13.91 g L⁻¹, 85.22% higher than that of the wild type (7.51 g L⁻¹). However, deletion of proline dehydrogenase gene ycgM had no effect on γ-PGA synthesis. Furthermore, a 2.92-fold higher P5C content (19.24 μmol gDCW⁻¹) was detected in the ycgN deficient strain WXΔycgN, while the P5C levels of WXΔycgM and the double mutant strain WXΔycgMN showed no difference, compared to WX-02. Moreover, the ROS level of WXΔycgN was increased by 1.18-fold, and addition of n-acetylcysteine (antioxidant) decreased its ROS level, which further reduced γ-PGA synthesis capability of WXΔycgN. Collectively, our results demonstrated that proline catabolism played an important role in maintaining ROS homeostasis, and deletion of ycgN-enhanced P5C accumulation, which induced a transient ROS signal to promote γ-PGA synthesis in B. licheniformis.