Metabolism of Pyridine Coenzymes in Microorganisms

NADP was enzymatically synthesized from NAD and p-nitrophenyl phosphate or nucleoside monophosphate with the enzyme preparation of Proteus mirabilis (IFO 3849). In this phosphotransferring reaction, ATP did not serve as phosphoryl donor.

In addition to NADP, an unidentified substance (Compound I) showing fluorescence with methyl ethyl ketone and having no coenzyme activity to glutamic dehydrogenase was synthesized. The yield of NADP was usually below 30 per cent of Compound I.

NADP was isolated from the reaction mixture and its coenzyme activity to some dehydrogenases was demonstrated.

A new derivative of NAD (Compound I) synthesized from NAD and p-nitrophenyl phosphate by the enzyme preparation of Proteus mirabilis (IFO 3849), was isolated from the reaction mixture.

After degradation of this compound with snake venom nucleotide pyrophosphatase, Compound III was obtained. 5′-NMN was phosphorylated to Compound IV by the same enzyme preparation of P. mirabilis. By the determination of chemical constituents and the degradation with phosphomonoesterases, Compounds III and IV were identified as nicotinamide riboside 2′(3′),5′-diphosphate, and Compound I was identified as NADP analog which was formed by phosphorylation at the 2′ or 3′ position of the nicotinamide ribose moiety, not at the 2′ position of adenosine moiety of NAD.     

The chloroplast chlL gene of the green alga Chlorella vulgaris C-27 contains a self-splicing group I intron

The chlL gene product is involved in the light-independent synthesis of chlorophyll in photosynthetic bacteria, green algae and non-flowering plants. The chloroplast genome of Chlorella vulgaris strain C-27 contains the first example of a split chlL gene, which is interrupted by a 951?bp group I intron in the coding region. In vitro synthesized pre-mRNA containing the entire intron and parts of the flanking exon sequences is able to efficiently self-splice in vitro in the presence of a divalent and a monovalent cation and GTP, to yield the ligated exons and other splicing intermediates characteristic of self-splicing group I introns. The 5′ and 3′ splice sites were confirmed by cDNA sequencing and the products of the splicing reaction were characterized by primer extension analysis. The absence of a significant ORF in the long P9 region (522?nt), separating the catalytic core from the 3′ splice site, makes this intron different from the other known examples of group I introns. Guanosine-mediated attack at the 3′ splice site and the presence of G-exchange reaction sites internal to the intron are some other properties demonstrated for the first time by an intron of a protein-coding plastid gene.     

Androsterone Derivatives Substituted at Position 16: Chemical Synthesis,Inhibition of Type 3 17β-hydroxysteroid Dehydrogenase,Binding Affinity for Steroid Receptors and Proliferative/antiproliferative Activity on Shionogi (AR +) Cells

A series of androsterone (ADT) derivatives substituted at position 16 were efficiently synthesized in short reaction sequences; the ether analogues were also synthesized in the case of the methyl and allyl derivatives. The aim of this study was to develop inhibitors of the steroidogenic enzyme type 3 17 β -hydroxysteroid dehydrogenase and then evaluate their ability to inhibit this activity in transfected HEK-293 cells. For each compound we measured the percentage of inhibition of the transformation of 4-androstene-3,17-dione, the natural substrate of this steroidogenic enzyme, into the active androgen testosterone. The synthesized compounds proved to be weak inhibitors of this enzyme, but interestingly, these ADT derivatives do not bind to androgen, estrogen, glucocorticoid, and progestin receptors, suggesting no unsuitable receptor-mediated effects. One exception, 16 α -(3′-bromopropyl)-5 α -androstane-3 α,17 β -diol (8), the only compound bearing a hydroxy group at position 17 β instead of a ketone, showed a strong binding affinity for the androgen receptor (70% at 1 μM) and also exhibited an antiproliferative activity on Shionogi (AR +) cells (86% at 1 μM), which was comparable to that of hydroxyflutamide, a pure antiandrogen (100% at 1 μM).     

Synthesis and Anti‐HIV Activity of 4′‐Cyano‐2′,3′‐didehydro‐3′‐deoxythymidine

A new anti‐HIV agent 4′‐cyano‐2′,3′‐didehydro‐3′‐deoxythymidine (9) was synthesized by allylic substitution of the 3′,4′‐unsaturated nucleoside 14, having a leaving group at the 2′‐position, with cyanotrimethylsilane in the presence of SnCl₄. Evaluation of the anti‐HIV activity of 9 showed that this compound is much less potent than the recently reported 2′,3′‐didehydro‐3′‐deoxy‐4′‐(ethynyl)thymidine (1).     

Effect of Imino Group of a Linker Arm at the C5 Position of a Pyrimidine Nucleoside on the Thermal Stabilities of DNA/DNA and DNA/RNA Duplexes

The modified ODN's bearing C5‐substituted 2′‐deoxyuridine derivative were synthesized by a post‐synthetic modification with an unsymmetrical triamine. The effect of the C5‐substituent on the duplex formation with complementary DNA or RNA differed with the position of an imino group in the linker‐arms.     

Synthesis of 2-(2′-Hydroxy-2′, 2′-diphenylethyl)-8-hydroxy-quinoline and Its Biological Activity

2-(2′-Hydroxy-2′,2′-diphenylethyl)-8-hydroxyquinoline was prepared via Grignard reaction involving the activated methyl group in position 2. This compound inhibited the action of the phenol oxidase prepared from prepupae of housefly. In a dipping test of the final instar larvae of housefly, it showed some inhibitory effects on the metamorphosis.     

Index

Anticancer role of oxindole compounds is well documented. Here, we synthesized new derivatives of 3-hydroxy-2-oxindole functionalized at position 3 (1a–f) which are expected to have antiproliferative activity in cancer cells. Human prostate cancer cell line (DU145) was treated with the synthesized derivatives at 40-μM concentration for 24, 48, and 72 h. Compounds 1-ethyl-3-hydroxy-1,1′,3,3′-tetrahydro-2H,2′H-3,3′-biindole-2,2′-dione (1d), 5-bromo-1-ethyl-3-hydroxy-1,1′,3,3′-2H,2′H-3,3′-biindole-2,2′-dione (1e), and 5-chloro-1-ethyl-3-hydroxy-1,1′,3,3′-tetrahydro-2H,2′H-3,3′-biindole-2,2′-dione (1f) were found to significantly reduce DU145 cell viability at 48 and 72 h whereas no significant changes were observed up to 24 h. The compounds 1e and 1f showed the most cytotoxicity effect and had a similar antiproliferative activity on DU145 cell line. They have halogen and ethyl substitutions at positions 5 and 1, respectively. The IC₅₀ of compound 1e for DU145 and A375 cells at 48 h was determined. The apoptotic effects and cell cycle progression of compound 1e at 1/2 × IC₅₀ (55 μM) concentration in DU145 cells were investigated by nuclei staining, comet assay, flow cytometry, and scanning electron microscopy (SEM). The results obtained showed that this compound increased the percentage of tail DNA, increased the occurrence of the sub-G1 phase, and induced G2M arrest and apoptosis in DU145 cells after exposure for 48 h to a 55-μM concentration. The SEM images revealed cell contraction at 24 h, cell condensation, plasma membrane blebbing, and formation of apoptotic bodies at 48 and 72 h. These observations suggest that the antiproliferative activity of compound 1e may be to induce apoptosis in DU145 cells.     

Catalysis by molecular iodine: a rapid synthesis of 1,8-dioxo-octahydroxanthenes and their evaluation as potential anticancer agents

Molecular iodine facilitated the reaction of 5,5-dimethyl-1,3-cyclohexanedione with aromatic aldehydes in iso-propanol affording a variety of 1,8-dioxo-octahydroxanthenes in high yields. Most of the compounds synthesized showed good anti-proliferative properties in vitro against three cancer cell lines and 9-(2-hydroxyphenyl)-3,3,6,6-tetramethyl-3,4,5,6,7,9-hexahydro-1H-xanthene-1,8(2H)-dione possessing a 2-hydroxy phenyl group at C-9 position was found to be promising. Further structure elaboration of the same compound and the crystal structure analysis and hydrogen bonding patterns of another compound that is, 9-(4-methoxyphenyl)-3,3,6,6-tetramethyl-3,4,5,6,7,9-hexahydro-1H-xanthene-1,8(2H)-dione prepared by using this methodology is presented.     

Enzymatic incorporation and fluorescent labelling of cyclooctyne-modified deoxyuridine triphosphates in DNA

The amino group of 5-aminopropargyl-2′-deoxyuridine-5′-triphosphate was labelled with dibenzocyclooctyne (DIBO) and two derivatives of bicyclo [6.1.0] non-4-yne (BCN) with short and long linkers to produce three different cycloalkyne-modified deoxyuridine triphosphates. BCN was successfully incorporated into DNA at multiple sites by enzyme-mediated primer extension and the polymerase chain reaction (PCR). Efficient fluorescent labelling of the BCN-DNA and DIBO-DNA with Cy3-azide was demonstrated.     

2′-C-Cyano-2′-deoxy-1-β-D-arabinofuranosylcytosine (CNDAC): A Mechanism-Based DNA-Strand-Breaking Antitumor Nucleoside1

Abstract

The antitumor mechanism of action of 2′-C-cyano-2′-deoxy-1-β-d-arabinofuranosylcytosine (CNDAC) has been examined. CNDAC was designed as a potentially DNA-self-strand-breaking nucleoside. It had potent antitumor effects against various solid tumors in vitro as well as in vivo. Using a chain-extension method with Vent (exo^?) DNA polymerase and a short primer/template system, we found that 5′-triphosphate of CNDAC (CNDACTP) was incorporated into the primer at a site opposite a guanine residue in the template. After further chain-extension reaction of the primer containing CNDAC at the 3′-terminus, chain elongation was not observed. Therefore, CNDACTP appeared to act as a chain-terminator. Analyses of the structure of the 3′-terminus in the primer revealed 2′-C-cyano-2′,3′-didehydro-2′,3′-dideoxycytidine (ddCNC) together with CNDAC and 2′-C-cyano-2′-deoxy-1-β-d-ribofuranosylcytosine (CNDC). The existence of ddCNC in the 3′-end of the primer would be due to the self-strand-break by the nucleotide incorporated next to CNDAC. We also found that CNDAC was epimerized to CNDC in near-neutral to alkaline media. Therefore, CNDC found in the primer was epimerized after incorporation of CNDACTP into the primer. We also described the metabolism of CNDAC.     

Specific Deprotonation Reactions of the Pyrimidine Radical Cation Resulting from the Menadione Mediated Photosensitization of 2′-Deoxycytidine

Menadione(2-methyl-1, 4-naphthoquinone) was shown to sensitize 2′-deoxycytidine to near ultraviolet light according to two main mechanisms. Reaction of a water molecule with the initially photo-induced pyrimidine radical cation and subsequent addition of molecular oxygen leads to the preponderant formation of the four cis and trans diastereoisomers of 5,6-dihydroxy-5,6-dihydro-2′-deoxyuridine. Pyrimidine ring opening and rearrangement products are also generated through the intermediate 6-hydroxy-5,6-dihydro-2′-deoxyurid-5-yl radical. The competitive deprotonation reaction of the radical cation is likely to involve two sites. Loss of an amino group proton is the likely initial event to explain the formation of 2′-deoxyuridine which is resistant to further photooxidation. The second deprotonation reaction involves the osidic carbon C(1′). The resulting radical will further react with oxygen leading to the release of free cytosine with concomitant formation of 2-deoxy-D-ribono-1,4-lactone. This reaction which is not prevented by hydroxyl radical scavengers constitutes to our knowledge the first example of a pyrimidine radical which is able to initiate selective intramolecular reaction at position 1 within the sugar moiety.     

Labeled Nucleoside Triphosphates with Reversibly Terminating Aminoalkoxyl Groups

Nucleoside triphosphates having a 3′-ONH₂ blocking group have been prepared with and without fluorescent tags on their nucleobases. DNA polymerases were identified that accepted these, adding a single nucleotide to the 3′-end of a primer in a template-directed extension reaction that then stops. Nitrite chemistry was developed to cleave the 3′-ONH₂ group under mild conditions to allow continued primer extension. Extension-cleavage-extension cycles in solution were demonstrated with untagged nucleotides and mixtures of tagged and untagged nucleotides. Multiple extension-cleavage-extension cycles were demonstrated on an Intelligent Bio-Systems Sequencer, showing the potential of the 3′-ONH₂ blocking group in “next generation sequencing.”     

Detection of bovine leukocyte adhesion deficiency by nonisotopic ligase chain reaction

A nonisotopic ligase chain reaction (LCR) assay was developed to detect the mutation (D128G; Shuster et al. (1992) PNAS 89, 9225-9) for bovine leukocyte adhesion deficiency (BLAD). Two sets of diagonally opposed discriminating LCR primers that differentiate the normal and BLAD allele were designed so that the 3′ end of each primer overlapped the D128G mutation. These discriminating primers were synthesized with a 5′ biotin and could be captured using streptavidin-coated microtitre wells. A common set of primers that abut these discriminating primers were also synthesized and 3′-tailed with digoxigenin-ddUTP. Captured LCR products were then detected using antidigoxigenin antibodies coupled to alkaline phosphatase. The assay readout was a chemiluminescent signal generated by the hydrolysis of Lumi-Phos TM 530 and the entire assay including DNA isolation can be completed within 8 h.     

Synthesis and Properties of DNA Dumbbells Containing Chemically Active Substituted Pyrophosphate Internucleotide Groups

Abstract

DNA dumbbells with substituted pyrophosphate groups at a definite position of the sugar-phosphate backbone were synthesized by condensation of terminal 5′-phosphomonester- and 3′-methylphosphodiester groups in nicked dumbbells. N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide was used as a condensing agent. An efficient method for producing extended oligonucleotides carrying an O-methyl-substitued 3′-phosphate group was developed. Properties of the modified DNA-dumbbells were investigated. The substituted pyrophosphate group in the DNA dumbbells was efficiently cleaved under the action of N-methylimidazole or ethylendiamine aqueous solutions at pH 8.0.     

Changes of 5＇ Terminal Nucleotides of PCR Primers Causing Variable T-A Cloning Efficiency

T-A cloning takes advantage of the unpaired adenosyl residue added to the 3＇ terminus of amplified DNAs by Taq and other thermostable DNA polymerase and uses a Ilnearlzed plasmld vector with a protruding 3＇ thymldylate residue at each of Its 3＇ termini to clone polymerase chain reaction （PCR）-derived DNA fragments. It Is a simple, reliable, and efficient Ilgatlon-dependent cloning method for PCR products, but the drawback of variable cloning efficiency occurs during application. In the present work, the relationship between variable T-A cloning efficiency and the different 5＇ end nucleotlde base of primers used In PCR amplification was studied. The results showed that different cloning efficiency was obtained with different primer pairs containing A, T, C and G at the 5＇ terminus respectively. The data shows that when the 5＇ end base of primer pair was adenosyl, more white colonies could be obtained In cloning the corresponding PCR product In comparison with other bases. And the least white colonies were formed when using the primer pair with 5＇ cytldylate end. The gluanylate end primers resulted In almost the same cloning efficiency In the white colonies amount as the thymldylate end primer did, and this efficiency was much lower than that of adenosyl end primers. This presumably is a consequence of variability In 3＇dA addition to PCR products mediated by Taq polymerase. Our results offer instructions for primer design for researchers who choose T-A cloning to clone PCR products.     

The strand transfer oligonucleotide inhibitors of HIV-integrase

Retroviral integrase participates in two catalytic reactions, which require interactions with the two ends of the viral DNA in the 3′processing reaction, and with a targeted host DNA in the strand transfer reaction. The 3′-hydroxyl group of 2′-deoxyadenosine resulting from the specific removing of GT dinucleotide from the viral DNA in the processing reaction provides the attachment site for the host DNA in a transesterification reaction. We synthesized oligonucleotides (ONs) of various lengths that mimic the processed HIV-1 U5 terminus of the proviral long terminal repeat (LTR) and are ended by 2′-deoxyadenosine containing a 3′-O-phosphonomethyl group. The duplex stability of phosphonomethyl ONs was increased by covalent linkage of the modified strand with its complementary strand by a triethylene glycol loop (TEG). Modified ONs containing up to 10 bases inhibited in vitro the strand transfer reaction catalyzed by HIV-1 integrase at nanomolar concentrations.     

Synthesis of 3′-Fluoro-3′-Deoxyribonucleosides; Anti-HIV-1 and Cytostatic Properties

Abstract

It has generally proven difficult to synthesize ribonucleosides with sugar modifications at the 3′ position. We now present a practical route for the synthesis of ribonucleosides with a 3′ fluorine substituent. Nucleosides with the xylo configuration were prepared by sugar-base condensation. Tritylation of the unprotected nucleosides gave a mixture of 2′,5′ and 3′,5′ bistritylated nucleosides which were difficult to characterize. Therefore the necessary precursors were synthesized in a step-wise fashion, starting with selective deprotection of the 2′-acyl group, followed by tritylation. This gave the 2′,5′-tritylated xylonucleosides in good yield. Reaction with diethylaminosulfur trifluoride and deprotection with 80 % acetic acid provided the 3′-fluoro-3′-deoxyribonucleosides 1, 2 and 4. The cytidine derivative was synthesized from 1 by reaction with trifluoromethanesulfonic anhydride followed by ammonia. Treatment of 4 with adenosine deaminase yielded 5.     

Mechanism of primer synthesis by the herpes simplex virus 1 helicase-primase

We utilized templates of defined sequence to investigate the mechanism of primer synthesis by herpes simplex virus 1 helicase-primase. Under steady-state conditions, the rate of primer synthesis and the size distribution of products remained constant with time, suggesting that the rate-limiting step(s) of primer synthesis occur(s) during primer initiation (at or before the formation of the pppNpN dinucleotide). Consistent with this idea, increasing the concentration of NTPs required for dinucleotide synthesis increased the rate of primer synthesis, whereas increasing the concentration of NTPs not involved in dinucleotide synthesis inhibited primer synthesis. Due to these effects on primer initiation, varying the NTP concentration could affect start site selection on templates containing multiple G-pyr-pyr initiation sites. Increasing the NTP concentration also increased the processivity of primase. However, even at very high concentrations of NTPs, elongation of the dinucleotide into longer products remained relatively inefficient. Primase did not readily elongate preexisting primers under conditions where free template was present in large excess of enzyme. However, if template concentrations were lowered such that primase synthesized primers on all or most of the template present in the reaction, then primase would elongate previously synthesized primers.     

Chemoenzymatic Synthesis of 3′-Deoxy-3′-(4-Substituted-Triazol-1-YL)-5-Methyluridine

An efficient protocol has been developed for the synthesis of a small library of 3′-deoxy-3′-(4-substituted-triazol-1-yl)-5-methyluridine using Cu(I)-catalyzed Huisgen–Sharpless–Meldal 1,3-dipolar cycloaddition reaction of 3′-azido-3′-deoxy-5-methyluridine with different alkynes under optimized condition in an overall yields of 76%–92%. Here, the azido precursor compound, i.e., 3′-azido-3′-deoxy-5-methyluridine was chemoenzymatically synthesized from D-xylose in good yield. Some of the alkynes used in cycloaddition reaction were synthesized by the reaction of hydroxycoumarins or naphthols with propargyl bromide in acetone using K₂CO₃in excellent yields. All synthesized compounds were unambiguously identified on the basis of their spectral (IR, ¹H-, ¹³C NMR spectra, and high-resolution mass spectra) data analysis.