Enlarged analogues of uniconazole,new azole containing inhibitors of ABA 8′-hydroxylase CYP707A

We enlarged the uniconazole (UNI) molecule to find a specific inhibitor of abscisic acid (ABA) 8′-hydroxylase, and synthesized various UNI derivatives that were substituted with hydrophilic and hydrophobic groups at the 4-chlorine of the phenyl group of UNI using click chemistry. Considering its potency in ABA 8′-hydroxylase inhibition, its small effect on seedling growth, and its ease of application, UT4, the UNI derivative containing the C₄ alkyltriazole, was the best candidate for a highly selective inhibitor of ABA 8′-hydroxylase.     

Cloning, Functional Identification and Sequence Analysis of Flavonoid 3′-hydroxylase and Flavonoid 3′,5′-hydroxylase cDNAs Reveals Independent Evolution of Flavonoid 3′,5′-hydroxylase in the Asteraceae Family

Flavonoids are ubiquitous secondary plant metabolites which function as protectants against UV light and pathogens and are involved in the attraction of pollinators as well as seed and fruit dispersers. The hydroxylation pattern of the B-ring of flavonoids is determined by the activity of two members of the vast and versatile cytochrome P450 protein (P450) family, the flavonoid 3′-hydroxylase (F3′H) and flavonoid 3′,5′-hydroxylase (F3′5′H). Phylogenetic analysis of known sequences of F3′H and F3′5′H indicated that F3′5′H was recruited from F3′H before the divergence of angiosperms and gymnosperms. Seven cDNAs were isolated from species of the Asteraceae family, all of which were predicted to code for F3′Hs based on their sequences. The recombinant proteins of four of the heterologously in yeast expressed cDNAs exhibited the expected F3′H activity but surprisingly, three recombinant proteins showed F3′5′H activity. Phylogenetic analyses indicated the independent evolution of an Asteraceae-specific F3′5′H. Furthermore, sequence analysis of these unusual F3′5′H cDNAs revealed an elevated rate of nonsynonymous substitutions as typically found for duplicated genes acquiring new functions. Since F3′5′H is necessary for the synthesis of 3′,4′,5′-hydroxylated delphinidin-derivatives, which normally provide the basis for purple to blue flower colours, the evolution of an Asteraceae-specific F3′5′H probably reflects the adaptive value of efficient attraction of insect pollinators.     

Allele-specific marker development and selection efficiencies for both flavonoid 3′-hydroxylase and flavonoid 3′,5′-hydroxylase genes in soybean subgenus soja

Color is one of the phenotypic markers mostly used to study soybean (Glycine max L. Merr.) genetic, molecular and biochemical processes. Two P450-dependent mono-oxygenases, flavonoid 3′-hydroxylase (F3′H; EC1.14.3.21) and flavonoid 3′,5′-hydroxylase (F3′5′H, EC1.14.13.88), both catalyzing the hydroxylation of the B-ring in flavonoids, play an important role in coloration. Previous studies showed that the T locus was a gene encoding F3′H and the W1 locus co-segregated with a gene encoding F3′5′H in soybean. These two genetic loci have identified to control seed coat, flower and pubescence colors. However, the allelic distributions of both F3′H and F3′5′H genes in soybean were unknown. In this study, three novel alleles were identified (two of four alleles for GmF3′H and one of three alleles for GmF3′5′H). A set of gene-tagged markers was developed and verified based on the sequence diversity of all seven alleles. Furthermore, the markers were used to analyze soybean accessions including 170 cultivated soybeans (G. max) from a mini core collection and 102 wild soybeans (G. soja). For both F3′H and F3′5′H, the marker selection efficiencies for pubescence color and flower color were determined. The results showed that one GmF3′H allele explained 92.2 % of the variation in tawny and two gmf3′h alleles explained 63.8 % of the variation in gray pubescence colors. In addition, two GmF3′5′H alleles and one gmF3′5′h allele explained 94.0 % of the variation in purple and 75.3 % in white flowers, respectively. By the combination of the two loci, seed coat color was determined. In total, 90.9 % of accessions possessing both the gmf3′h-b and gmf3′5′h alleles had yellow seed coats. Therefore, seed coat colors are controlled by more than two loci.     

Caspase-8 and its inhibitors in RCCs in vivo: the prominent role of ARC

Activation of the initiator-caspase, caspase-8 is under tight control of multiple antiapoptotic regulators including ARC, cFlip_S, cFlip_L and PED/PEA-15. Since there is little data regarding the expression of caspase-8 and its antiapoptotic regulators in human tumours in vivo, we analysed their expression in renal cell carcinomas (RCCs) to identify which of these genes might be crucial for the well known impaired apoptosis and—as a result—resistance towards chemotherapy and ionizing radiation of RCCs. Caspase-8, cFlip_S, cFlip_L and PED/PEA-15 mRNA expression was significantly increased only in early stages of RCCs compared to non-neoplastic renal tissue. In contrast, ARC mRNA expression was significantly increased in RCCs of all stages without differences between the tumour stages and grades. Importantly, the relative mRNA expression ratio between ARC and caspase-8 was significantly increased during carcinogenesis and tumour progression. In contrast, the relative mRNA expression ratio between cFlip_S, cFlip_L or PED/PEA-15 and caspase-8 remained constant during all tumour stages. In conclusion, our analysis revealed that ARC is the only caspase-8 inhibiting regulator being constantly overexpressed in RCCs. Furthermore, the balance between antiapoptotic ARC and proapoptotic caspase-8 is the only one to be disturbed during carcinogenesis and tumour progression of RCCs. This inhibition of Caspase-8 might therefore be one example for the multiple antiapoptotic functions of ARC in RCCs possibly contributing to the marked resistance of RCCs towards radio- and chemotherapy and reflects a shift of gene expression towards a more antiapoptotic context in RCCs. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Salicylate–urea-based soluble epoxide hydrolase inhibitors with high metabolic and chemical stabilities

We investigated N-adamantyl-N′-phenyl urea derivatives as simple sEH inhibitors. Salicylate ester derivatives have high inhibitory activities against human sEH, while the free benzoic acids are less active. The methyl salicylate derivative is a potent sEH inhibitor, which also has high metabolic and chemical stabilities; suggesting that such inhibitors are potential lead molecule for bioactive compounds acting in vivo.     

Design,synthesis, and anticancer activity of C8-substituted-4′-thionucleosides as potential HSP90 inhibitors

A series of C8-substituted-4′-thioadenosine analogs 3a–3g, 15, and 17 and their truncated derivatives 4a–4j, 23–25, and 27 have been successfully synthesized from d-ribose and d-mannose, respectively, employing Pummerer type or Vorbrüggen condensation reactions and the functionalization at the C8-position of nucleobase via Stille coupling or nucleophilic aromatic substitution reactions as key steps. All the synthesized compounds were assayed for their HSP90 inhibitory activity, but they were found to be inactive up to 100 μM. However, the 8-iodo derivatives 15, 17, and 27 exhibited potent anticancer activity, indicating that different mechanism of action might be involved in their biological activity.     

Synthesis and evaluation of 5′-modified thymidines and 5-hydroxymethyl-2′-deoxyuridines as Mycobacterium tuberculosis thymidylate kinase inhibitors

We report the synthesis of 5′-modified thymidines (16, 18, 21, 23) and 5,5′-bis-substituted 2′-deoxyuridine analogues (30, 47) as inhibitors of thymidine monophosphate kinase of Mycobacterium tuberculosis (TMPKmt). These analogues were evaluated for their capacity to inhibit TMPKmt and solely two 5′-modified thymidines were found to possess moderate inhibitory activity. In addition, a feasibility study of protecting groups for the 5-CH₂OH moiety of 2′-deoxyuridines is described that enables to introduce the desired 5′-modification.     

Normal coordinate analysis of 5′-dGMP and its deuterated derivatives

By calculations based on the Wilson GF-method and using a valence force field, the vibration modes of 5-dGMP have been assigned. Good agreement was obtained between the calculated and experimental results corresponding to the Raman and infrared spectra of 5-dGMP. The calculations can also predict the displacement of infrared bands observed upon selective deuteration on C8 and simultaneously on the C8, N1 and N2 atoms of the guanine ring. In order to preserve the harmonic approximation of the potential field, the redundancy between the internal coordinates is entirely removed using a matrix-product diagonalization procedure. In this treatment the local symmetry of different constituents of the molecule is taken into consideration, thus avoiding extensive linear combinations of internal coordinates.The extension of these calculations to the guanosine-moiety involved in the double helix structures of DNAs allowed us to reproduce a certain number of the characteristic guanine vibration modes altered by the BZ transitions of poly(dG-dC)·poly(dG-dC) and d(CG)₃·d(CG)₃.     

Semi-rational chemical modification of endoinulinase by pyridoxal 5′-phosphate and ascorbic acid

It is important to improve the quality of the enzyme inulinase used in industrial applications without allowing the treatment to have any adverse effects on enzyme activity. We achieved preferential chemical modification of the non-catalytic domain of endoinulinase (EC 3.2.1.7) to enhance the thermostability of the enzyme. We used pyridoxal 5′-phosphate (PLP) to modify the more accessible lysine residues at the surface of endoinulinase and then performed a necessary step of reduction with ascorbate. Endoinulinase was incubated in the presence of PLP at various concentrations; this step was followed by reduction of the resulting Schiff base and dialysis. The effects of different PLP concentrations and incubation times on enzyme modification were evaluated. Enzyme deactivation was observed immediately after treatment, even at low PLP concentrations, while reactivation was observed for samples treated with low PLP concentrations after a period of time. Structural analysis revealed that the α-helix content increased from 13.60% to 17.60% after applying the modification strategy; consequently, enzyme stabilization was achieved. The melting temperature (T_m) of the modified enzyme increased from 64.1 °C to 72.2 °C, and a comparative study of thermal stability at 25 °C, 45 °C, and 50 °C for 150 min confirmed that the enzyme was stabilized because of increase in its half-life (t_1/2) after PLP modification/ascorbate reduction. The modification process was optimized to achieve the optimum mole ratio for the PLP/endoinulinase (1.37). Excess moles of the modifier are thought to be responsible for enzyme deactivation through unwanted/nonspecific and noncovalent interactions, and the optimization ensured that there was no excess modifier after the desired covalent reaction was complete.     

Synthesis of 8-Bromo- and 8-Azido-2′-deoxyadenosine-5′-O-(1-thiotriphosphate)

Abstract

Treatment of 3′-O-methoxyacetylated 8-bromo-2′-deoxyadenosine (5), with a twofold excess of salicyl phosphorochloridite (6), and subsequent reaction with bis(tri-n-butylammonium) pyrophosphate and oxidation with sulfur followed by removal of the protecting group gives predominantly 8-bromo-2′-deoxyadenosine-5′-O-(1-thiotriphosphate) (7), and minor amounts of the corresponding brominated monothiophosphate. Alternatively, the photoreactive dATP analog 8-azido-2′-deoxyadenosine-5′-O-(1-thiotriphosphate) (11), is obtained by phosphorylation of unprotected 8-azido-2′-deoxyadenosine (9) with a 1.8 molar equivalent excess of thiophosphoryl chloride and bis(tri-n-butylammonium) pyrophosphate. A protection of the nucleobase 6-amino group is not required. The photoaffinity labeling reagent 11, was characterized by ³¹P-NMR and ion-spray mass spectroscopy and its photolysis upon long wavelength UV irradiation was studied. Both α-thioderivatives of 2′-deoxyadenosine triphosphates can be incorporated into plasmid DNA by T7 DNA polymerase. Thus, they can be used for interference studies of protein binding and for cross-linking with amino acids in protein-nucleic acid-complexes.     

2′–5′ oligoadenylate synthetase and its relationship to HLA and genetic markers of insulin-dependent diabetes mellitus

Cytokines and their related enzyme pathways may play a part in the development of insulin-dependent diabetes mellitus (IDDM). We have therefore studied the activity of the enzyme 2′–5′ oligoadenylate synthetase (which is induced by both interferon and the tumour necrosis factors) in circulating mononuclear cells from 40 subjects with IDDM and 32 healthy control subjects. There was no difference in mean basal enzyme activity between the two groups. A polymorphism of the 2′–5′ oligoadenylate synthetase gene, not previously described, was found using the restriction enzymeBam HI. There was no association of 2′–5′ oligoadenylate synthetase genotypes with IDDM, but there was a significant correlation between basal 2′–5′ oligoadenylate synthetase activity and 2′–5′ oligoadenylate synthetase genotypes. Significantly higher mean basal levels of 2′–5′ oligoadenylate synthetase activity were associated with HLA-DQA 4.6 phenotype (determined using the restriction enzymeTaq 1 and a DQA probe) and HLA-DR3 (determined serologically), whereas significantly lower mean levels of enzyme activity were associated with HLA-DQA 5.5 and HLA-DR7, in both IDDM and control subjects. An analysis of variance confirmed that these associations were independent 2′–5′ oligoadenylate synthetase genotype. Likewise, a significantly higher mean level of enzyme activity was associated with the heterozygous 1/3 insulin-related genotype in the IDDM subjects only. This study therefore suggests that the possession of certainHLA haplotypes might be associated with differing levels of basal 2′–5′ oligoadenylate synthetase activity.     

Synthesis and antiviral activities of 3-deaza-3-fluoroaristeromycin and its 5′ analogues

The naturally occurring adenine based carbocyclic nucleosides aristeromycin and neplanocin A and their 3-deaza analogues have found a prominent place in the search for diverse antiviral activity agent scaffolds because of their ability to inhibit S-adenosylhomocysteine (AdoHcy) hydrolase. Following the lead of these compounds, their 3-deaza-3-fluoroaristeromycin analogues have been synthesized and their effect on S-adenosylhomocysteine hydrolase and RNA and DNA viruses determined.     

Uptake, Movement, Activity, and Persistence of an Abscisic Acid Analog (8′ Acetylene ABA Methyl Ester) in Marigold and Tomato

The abscisic acid (ABA) analog 8′ acetylene ABA methyl ester (PBI 429) was evaluated for its potential to alter the growth and moisture use of bedding plants during nursery production. Treating seedlings with the ABA analog as a root-dip slowed moisture use and growth of tomato seedlings under greenhouse conditions. In marigolds, comparable ABA analog treatments had no effect on growth and limited effects on plant moisture use. To determine whether these differences in response to treatment with the ABA analog were associated with differences in absorption of the analog and/or its persistence, the ABA analog was applied either as a foliar spray or root-dip, and the resulting concentrations of the ABA analog were monitored over a 10-day interval in both the roots and the leaves. In both crops, the ABA analog was detected in both leaf and root tissues irrespective of the mode of application, suggesting systemic movement of the analog. Tissue concentrations of the ABA analog were consistently lower in the foliar treatment than in the root-dip. The uptake and the retention of the ABA analog over time was similar in leaves of the two test crops, but less of the ABA analog was absorbed and retained in the roots of marigold plants than in the tomatoes. This suggests that the observed differences in responses of these two plant species to application of ABA analogs may be related to differences in retention or accumulation of ABA in the roots rather than to differences in the total amount of ABA analog absorbed or its movement and retention in the plant system. Levels of endogenous ABA were not significantly altered by application of the ABA analog.     

A chemical synthesis of UDP-LacNAc and its regioisomer for finding ‘oligosaccharide transferases’

A chemical synthesis of uridine 5’-diphospho-N-acetyllactosamine (Galβ(1→4)GlcNAc-UDP; UDP-LacNAc) and Galβ(1→3)GlcNAc-UDP is described. Coupling of the disaccharide imidate derivatives with dibenzylphosphate gave the corresponding 1-phosphates, which were condensed with UMP-imidazolate to give the target UDP-oligosaccharides after purification by anion exchange HPLC and gel filtration column chromatography. Using this methodology a variety of oligosaccharide nucleotide analogues can be synthesized. These UDP-oligosaccharides may be useful for finding so-called `oligosaccharide transferases’, the glycosyltransferases which transfer the oligosaccharide moiety onto glycosyl acceptors.     

5′-Haloacetamido-5′-deoxythymidines: novel inhibitors of thymidylate synthase

5′-Bromoacetamido-5′-deoxythymidine (BAT), 5′-iodoacetamido-5′-deoxythymidine (IAT), 5′-chloroacetamido-5′-deoxythymidine (CAT) and [¹⁴C]BAT were synthesized and their interactions with thymidylate synthase purified from L1210 cells were invesatigated. The inhibitory effects of these compounds on thymidylate synthase were in the order BAT > IAT > CAT, which is in agreement with their cytotoxic effects in L1210 cells. In the presence of substrate during preincubation, the concentration required for 50% inhibition of the enzyme activity by these inhibitors was 4–8 fold higher than it was in the absence of dUMP. The I₅₀ values for BAT were 1·10⁻⁵ M and 1.2·10⁻⁶ M in the presence and absence, respectively, of dUMP during preincubation. These results were in agreement with the observed inhibition of thynmidylate synthase by BAT in intact L1210 cells. A Lineweaver-Burk plot revealed that BAT behaved as a competitive inhibitor. The K_m for the enzyme was 9.2 μM, and the K_i determined for competitive inhibition by BAT was 5.4 μM. Formation of a tight, irreversible compledx is referred from the finding that BAT-inactivation of thymidylate synthase was not reversible on prolonged dialysis and that the enzyme-BAT complex was nondissociable by gel filtration through a Sephadex G-25 column or by TSK-125 column chromatography. Incubation of thymidylate synthase with BAT resulted in time-dependent, irreversible loss of enzyme activity by first-order kinetics. The rate constant for inactivation was 0.4 min⁻¹, and the steady-state constant of inactivation, K_i, was estimated to be 6.6 μM. The 5′-haloacetamido-5′-deoxythymidines provide specific inhibitors of thymidylate synthase that may also serve as reagents for studying the enzyme mechanism.