Dinucleotide Analogues Research Articles

Oligonucleotide Analogues with Integrated Bases and Backbone. Part 13

AbstractThe self‐complementary UA and AU dinucleotide analogues 41–45, 47, 48, and 51–60 were prepared by Sonogashira coupling of 6‐iodouridines with C(5′)‐ethynylated adenosines and of 8‐iodoadenosines with C(5′)‐ethynylated uridines. The dinucleotide analogues associate in CDCl3 solution. The C(6/I)‐unsubstituted AU dimers 51 and 54 prefer an anti‐oriented uracilyl group and form stretched linear duplexes. The UA propargyl alcohols 41 and 43–45 possess a persistent intramolecular O(5′/I)H⋅⋅⋅N(3/I) H‐bond and, thus, a syn‐oriented adeninyl and a gt‐ or tg‐oriented ethynyl moiety; they form corrugated linear duplexes. All other dimers form cyclic duplexes characterized by syn‐oriented nucleobases. The preferred orientation of the ethynyl moiety (the C(4′),C(5′) torsion angle) defines a conformation between gg and one where the ethynyl group eclipses O(4′/I). The UA dimers 42, 47, and 48 form Watson–Crick H‐bonds, the AU dimers 56 and 58–60 H‐bonds of the Watson–Crick‐type, the AU dimers 53 and 55 reverse‐Hoogsteen, and 57 Hoogsteen H‐bonds. The pairing mode depends on the substituent of C(5′/I) (H, OSiiPr3; OH) and on the H‐bonds of HOC(5′/I) in the AU dimers. Association constants were derived from the concentration‐dependent chemical shift for HN(3) of the uracilyl moiety; they vary from 45–104 M−1 for linear duplexes to 197–2307 M−1 for cyclic duplexes. The thermodynamic parameters were determined by van't Hoff analysis of the temperature‐dependence of the (concentration‐dependent) chemical shift for HN(3) of the uracilyl moiety. Neglecting stacking energies, one finds an average energy of 3.5–4.0 kcal/mol per intermolecular H‐bond. Base stacking is evidenced by the temperature‐dependent CD spectra. The crystal structure of 54 shows two antiparallel chains of dimers connected by Watson‐Crick H‐bonds. The chains are bridged by a strong H‐bond between the propargylic OH and OC(4) and by weak reverse A ⋅ A Hoogsteen H‐bonds.

Structural Determinants for N1/N7 Cyclization of Nicotinamide Hypoxanthine 5‘-Dinucleotide (NHD+) Derivatives by ADP-Ribosyl Cyclase from Aplysia californica: Ca2+-Mobilizing Activity of 8-Substituted Cyclic Inosine 5‘-Diphosphoribose Analogues in T-Lymphocytes

A series of nicotinamide hypoxanthine 5'-dinucleotide (NHD+) analogues modified at C-8 (2-5) and 7-deaza-NHD+ were synthesized, and cyclization in the presence of Aplysia ADP-ribosyl cyclase was studied. All 8-substituted NHD+ analogues were converted into their N1-cyclic forms by the enzyme, while in contrast, 7-deaza-NHD+ 17 was hydrolyzed into 7-deazainosine 5'-diphosphoribose (7-deaza-IDPR) 25. Correlations are made showing that the conformation of the NHD+ substrate is the key to successful cyclization. The pharmacological activities of these novel cIDPR derivatives were evaluated in both permeabilized and intact Jurkat T-lymphocytes. The results show that in permeabilized cells both 8-iodo 1g and 8-N3-N1-cIDPR 1d have an activity comparable to that of cADPR, while 8-iodo 1g and 8-phenyl-N1-cIDPR 1c have a small but significant effect in intact cells and can therefore be regarded as membrane-permeant; thus, cIDPR derivatives are emerging as important novel biological tools to study cADPR-mediated Ca2+ release in T-cells.

Specificity of recognition of mRNA 5′ cap by human nuclear cap-binding complex

The heterodimeric nuclear cap-binding complex (CBC) binds to the mono-methylated 5' cap of eukaryotic RNA polymerase II transcripts such as mRNA and U snRNA. The binding is important for nuclear maturation of mRNAs and possibly in the first round of translation and nonsense-mediated decay. It is also essential for nuclear export of U snRNAs in metazoans. We report characterization by fluorescence spectroscopy of the recognition of 5' capped RNA by human CBC. The association constants (K(as)) for 17 mono- and dinucleotide cap analogs as well as for the oligomer m7GpppA(m2') pU(m2')pA(m2') cover the range from 1.8 x 10(6) M(-1) to 2.3 x 10(8) M(-1). Higher affinity for CBC is observed for the dinucleotide compared with mononucleotide analogs, especially for those containing a purine nucleoside next to m7G. The mRNA tetramer associates with CBC as tightly as the dinucleotide analogs. Replacement of Tyr138 by alanine in the CBP20 subunit of CBC reduces the cap affinity except for the mononucleotide analogs, consistent with the crystallographic observation of the second base stacking on this residue. Our spectroscopic studies showed that contrary to the other known cap-binding proteins, the first two nucleotides of a capped-RNA are indispensable for its specific recognition by CBC. Differences in the cap binding of CBC compared with the eukaryotic translation initiation factor 4E (eIF4E) are analyzed and discussed regarding replacement of CBC by eIF4E.

IMP dehydrogenase from the protozoan parasite Toxoplasma gondii.

The opportunistic apicomplexan parasite Toxoplasma gondii damages fetuses in utero and threatens immunocompromised individuals. The toxicity associated with standard antitoxoplasmal therapies, which target the folate pathway, underscores the importance of examining alternative pharmacological strategies. Parasitic protozoa cannot synthesize purines de novo; consequently, targeting purine salvage enzymes is a plausible pharmacological strategy. Several enzymes critical to purine metabolism have been studied in T. gondii, but IMP dehydrogenase (IMPDH), which catalyzes the conversion of IMP to XMP, has yet to be characterized. Thus, we have cloned the gene encoding this enzyme in T. gondii. Northern blot analysis shows that two IMPDH transcripts are present in T. gondii tachyzoites. The larger transcript contains an open reading frame of 1,656 nucleotides whose deduced protein sequence consists of 551 amino acids (TgIMPDH). The shorter transcript is an alternative splice product that generates a 371-amino-acid protein lacking the active-site flap (TgIMPDH-S). When TgIMPDH is expressed as a recombinant protein fused to a FLAG tag, the fusion protein localizes to the parasite cytoplasm. Immunoprecipitation with anti-FLAG was employed to purify recombinant TgIMPDH, which converts IMP to XMP as expected. Mycophenolic acid is an uncompetitive inhibitor relative to NAD+, with a intercept inhibition constant (Kii) of 0.03+/-0.004 microM. Tiazofurin and its seleno analog were not inhibitory to the purified enzyme, but adenine dinucleotide analogs such as TAD and the nonhydrolyzable beta-methylene derivatives of TAD or SAD were inhibitory, with Kii values 13- to 60-fold higher than that of mycophenolic acid.

Cyclic ADP-Ribose Analogues Containing the Methylenebisphosphonate Linkage: Effect of Pyrophosphate Modifications on Ca2+ Release Activity

Analogues of cyclic ADP-ribose (cADPR) incorporating a methylenebisphosphonate linkage in the place of the pyrophosphate have been synthesized from nicotinamide adenine dinucleotide analogues enzymatically using Aplysia californica ADP-ribosyl cyclase. Methylenebisphosphonate cyclic ADP-ribose (cADPR[CH(2)]) and methylenebisphosphonate cyclic 3-deaza-ADP-ribose (3-deaza-cADPR[CH(2)]) showed full agonist activity for release of Ca(2+) ions from sea urchin egg homogenates. The EC(50) for cADPR[CH(2)] was 856 nM and that for 3-deaza-cADPR[CH(2)] was 300 nM, about 15- and 5-fold less potent than cADPR, respectively.

Characterization of oxidized nicotinamide adenine dinucleotide (NAD +) analogues using a high-pressure-liquid-chromatography-based NAD +-glycohydrolase assay and comparison with fluorescence-based measurements

A high-pressure-liquid-chromatography (HPLC)-based technique was developed to assess the oxidized nicotinamide adenine dinucleotide (NAD +)-glycohydrolase activity of the catalytic domain of Pseudomonas exotoxin A containing a hexa-His tag. The assay employs reverse-phase chromatography to separate the substrate (NAD +) and products (adenosine 5′-diphosphate-ribose and nicotinamide) produced over the reaction time course, whereby the peak area of nicotinamide is correlated using a standard curve. This technique was used to determine whether the NAD + analogue, 2′-F-ribo-NAD +, was a competing substrate or a competitive inhibitor for this toxin. This NAD + analogue was hydrolyzed at a rate of 0.2% that of NAD + yet retained the same binding affinity for the toxin as the parent compound. Finally, the rate that a fluorescent NAD + analogue, ε-NAD +, is hydrolyzed by the toxin was also investigated. This analogue was hydrolyzed six times slower than NAD + as determined using HPLC. The rate of hydrolysis of ε-NAD + calculated using the fluorometric version of the assay shows a sixfold increase in reaction rate compared to that determined by HPLC. This HPLC-based assay is adaptable to any affinity-tagged enzyme that possesses NAD +-glycohydrolase activity and offers the advantage of directly measuring the enzyme-catalyzed hydrolytic rate of NAD + and its analogues.

Synthesis, conformational analysis, and biological activity of new analogues of thiazole-4-carboxamide adenine dinucleotide (TAD) as IMP dehydrogenase inhibitors

Thiazole-4-carboxamide adenine dinucleotide (TAD) analogues T-2′-MeAD ( 1) and T-3′-MeAD ( 2) containing, respectively, a methyl group at the ribose 2′- C-, and 3′- C-position of the adenosine moiety, were prepared as potential selective human inosine monophosphate dehydrogenase (IMPDH) type II inhibitors. The synthesis of heterodinucleotides was carried out by CDI-catalyzed coupling reaction of unprotected 2′- C-methyl- or 3′- C-methyl-adenosine 5′-monophosphate with 2′,3′- O-isopropylidene-tiazofurin 5′-monophosphate, and then deisopropylidenation. Biological evaluation of dinucleotides 1 and 2 as inhibitors of recombinant human IMPDH type I and type II resulted in a good activity. Inhibition of both isoenzymes by T-2′-MeAD and T-3′-MeAD was noncompetitive with respect to NAD substrate. Binding of T-3′-MeAD was comparable to that of parent compound TAD, while T-2′-MeAD proved to be a weaker inhibitor. However, no significant difference was found in inhibition of the IMPDH isoenzymes. T-2′-MeAD and T-3′-MeAD were found to inhibit the growth of K562 cells (IC 50 30.7 and 65.0 μM, respectively).

Relationship between Charge-Transfer Interactions, Redox Potentials, and Catalysis for Different Forms of the Flavoprotein Component ofp-Cresol Methylhydroxylase

Thirty-three variants of the flavoprotein component of p-cresol methylhydroxylase that contain noncovalently or covalently bound flavin adenine dinucleotide (FAD) analogues were studied. A very good correlation was found between the efficiency of p-cresol oxidation by these proteins and E(CT), the energy for the maximum wavelength for the charge-transfer band of the complex between the bound flavin and 4-bromophenol, a substrate mimic. The correlation covers a range of k(cat) values that spans over 5 orders of magnitude and values of E(CT) that span 900 mV, and the analysis of the data provided a value of the transfer coefficient, alpha, of 0.31. This study demonstrates clearly that the redox properties of both the bound substrate and the flavin cofactor must be taken into account to explain the relative catalytic efficiencies of the variant flavoproteins.

Oligonucleotide Analogues with a “Nucleobase‐Including Backbone”. Part 10. Design, Synthesis, and Association of Ether‐Linked Dimers.

The dinucleoside analogues 24, 25, 28–30, and 33 associate in CDCl3 solution. Association constants, as determined from the concentration-dependent chemical shift for HN(3) of the uridine moiety and from thermodynamic parameters, range from 265 M−1 (33) to 3220 M−1 (30). The association of 31 in CDCl3 is too strong to be determined (concentration independent δ(HN(3)) of ca. 12.8 ppm) and the fully deprotected dimer 32 proved insufficiently soluble in CDCl3. This observation strongly evidences that structural differentiation of oligonucleotides and their analogues into backbone and nucleobases is not required for pairing. The dinucleotide analogues were prepared by O-alkylation of C(8)-unsubstituted or of C(8)-oxymethylated, partially protected adenosines by the C(6)-mesyloxy- or C(6)-halomethylated uridines 20–22, followed by partial or total deprotection.

Oligonucleotide Analogues with a ‘Nucleobase‐Including Backbone’. Part 10

AbstractThe dinucleoside analogues 24, 25, 28–30, and 33 associate in CDCl3 solution. Association constants, as determined from the concentration‐dependent chemical shift for HN(3) of the uridine moiety and from thermodynamic parameters, range from 265 M−1 (33) to 3220 M−1 (30). The association of 31 in CDCl3 is too strong to be determined (concentration independent δ(HN(3)) of ca. 12.8 ppm) and the fully deprotected dimer 32 proved insufficiently soluble in CDCl3. This observation strongly evidences that structural differentiation of oligonucleotides and their analogues into backbone and nucleobases is not required for pairing. The dinucleotide analogues were prepared by O‐alkylation of C(8)‐unsubstituted or of C(8)‐oxymethylated, partially protected adenosines by the C(6)‐mesyloxy‐ or C(6)‐halomethylated uridines 20–22, followed by partial or total deprotection.

Novel nicotinamide adenine dinucleotide analogues as selective inhibitors of NAD +-dependent enzymes

Three novel dinucleotide analogues of nicotinamide adenine dinucleotide (NAD +) have been synthesised from d-ribonolactone. These compounds incorporate a thiophene moiety in place of nicotinamide and are hydrolytically stable. They have been evaluated as inhibitors of adenosine diphosphate ribosyl cyclase, glutamate dehydrogenase and Sir2 acyltransferase activities. Enzyme specificity and a high level of inhibition was observed for the dehydrogenase.

‘Ethylene‐Bis[phosphonate] Nucleic Acids’: Novel Monomeric Synthons for the Solid‐Phase Synthesis of (PCH2CH2P)‐Bridged Oligonucleotides

An efficient synthesis of modified deoxyribonucleosidic building units, containing the 'ethylene-bisphosphonate nucleic acids' (EBPNAs) 1 or 2, has been developed. By means of solution-phase incorporation of 11, the oxidized form of 2, dinucleotide analogs of type 13 were prepared. The same strategy was used to incorporate 2 into the 5'-terminal position of the 12-mer homopolynucleotide methylphosphonate 14. A preliminary approach for solid-phase incorporation of the nucleosidic building block 1 into a preselected position of an oligonucleotide sequence to give 19 is presented, a reaction performed by means of a coupling strategy involving catalytic oxazaphospholidine-ring cleavage.

Hydrogen Bond Mediated Association of Dinucleotide Analogs

Abstract Several dinucleotide analogs, in which two of the nucleobases adenine, thymine and uracil are connected by a hexadecamethylene linker have been synthesized and studied for their base-base interaction in a chloroform solution. Using 1H NMR spectroscopic techniques intramolecular base pair formation through hydrogen bonding is observed at ambient temperatures but found to strongly depend on the identity of paired bases. Thus, whereas cyclization through intramolecular base-base interactions predominate for adenine–(CH2)16–thymine and adenine–(CH2)16–uracil, no intramolecular adenine-adenine pairing was observed. Upon addition of acetic acid to adenine–(CH2)16–thymine, strong adenine-acetic acid interactions result in the disruption of preformed intramolecular adenine-thymine base pairs.

Tissue-specific and subcellular localization of phototropin determined by immuno-blotting

Phototropin (phot) is a UV/blue- light receptor mediating phototropic reactions of plants as a response to unilateral irradiation. Using an antiserum directed against the N-terminal part of Arabidopsis phot1, we show here cross-reaction with phototropin from Avena sativa, Eruca sativa, Glycine max, Lepidium sativum, Lycopersicon esculentum, Pisum sativum, Sinapis alba, and Zea mays. In all investigated plants, blue light irradiation led to a gel mobility shift of phototropin corresponding to an apparent increase in size of 2-3 kDa. This increase is transient: the apparent size of the phototropin band reverted back to the original size in the dark within 60-90 min. The capacity for in vitro phosphorylation increased to 350% ( A. sativa) and 200% ( L. sativum) at 90 min after a blue light pulse without an increase in the amount of phototropin protein. Starting from coleoptile tips of monocots that contained the highest concentration of phototropin, we found an exponential decrease in basipetal sections of equal size while a linear decrease was determined for dicots in basipetal sections starting from the section below the hypocotyl hook. We confirmed the membrane association of all phototropin in dark-grown seedlings; after a 2-min blue light pulse, however, 20% of phototropin was found in the cytosolic fraction and only 80% in the membrane fraction. Both fractions showed the gel mobility shift indicating light-dependent autophosphorylation. Detergent-free solubilization of phototropin with chaotropic reagents was investigated with etiolated A. sativa seedlings. Up to 95% of phototropin was solubilized with a mixture of sodium bromide and sodium diphosphate, and subsequently subjected to affinity purification using Cibachron Blue 3GA-agarose as a dinucleotide analogue. Immediately after solubilization, soluble phototropin still showed blue-light-dependent autophosphorylation but lost its activity within less than 1 h.

Synthesis and Resolution of Dinucleotide(TpAZT) Phosphoramidates.

AbstractFor Abstract see ChemInform Abstract in Full Text.

Oligonucleosides with a Nucleobase‐Including Backbone; Synthesis and Self‐Association of Novel Dinucleotide Analogues.

ChemInformVolume 34, Issue 31 Natural Products Oligonucleosides with a Nucleobase-Including Backbone; Synthesis and Self-Association of Novel Dinucleotide Analogues. Andrew J. Matthews, Andrew J. Matthews Lab. Org. Chem., ETH-Hoenggerberg, CH-8093 Zuerich, Switz.Search for more papers by this authorPunit K. Bhardwaj, Punit K. Bhardwaj Lab. Org. Chem., ETH-Hoenggerberg, CH-8093 Zuerich, Switz.Search for more papers by this authorAndrea Vasella, Andrea Vasella Lab. Org. Chem., ETH-Hoenggerberg, CH-8093 Zuerich, Switz.Search for more papers by this author Andrew J. Matthews, Andrew J. Matthews Lab. Org. Chem., ETH-Hoenggerberg, CH-8093 Zuerich, Switz.Search for more papers by this authorPunit K. Bhardwaj, Punit K. Bhardwaj Lab. Org. Chem., ETH-Hoenggerberg, CH-8093 Zuerich, Switz.Search for more papers by this authorAndrea Vasella, Andrea Vasella Lab. Org. Chem., ETH-Hoenggerberg, CH-8093 Zuerich, Switz.Search for more papers by this author First published: 25 July 2003 https://doi.org/10.1002/chin.200331229Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat No abstract is available for this article. Volume34, Issue31August 5, 2003 RelatedInformation

Dinucleotide analogues as novel inhibitors of RNA-dependent RNA polymerase of hepatitis C Virus.

Replication of hepatitis C virus (HCV) RNA is catalyzed by the virally encoded RNA-dependent RNA polymerase NS5B. It is believed that the viral polymerase utilizes a de novo or primer-independent mechanism for initiation of RNA synthesis. Our previous work has shown that dinucleotides were efficient initiation molecules for NS5B in vitro (W. Zhong, E. Ferrari, C. A. Lesburg, D. Maag, S. K. Ghosh, C. E. Cameron, J. Y. Lau, and Z. Hong, J. Virol. 74:9134-9143, 2000). In this study, we further demonstrated that dinucleotide analogues could serve as inhibitors of de novo initiation of RNA synthesis directed by HCV NS5B. Both mononucleotide- and dinucleotide-initiated RNA syntheses were affected by dinucleotide analogues. The presence of the 5'-phosphate group in the dinucleotide compounds was required for efficient inhibition of de novo initiation. Optimal inhibitory activity also appeared to be dependent on the base-pairing potential between the compounds and the template terminal bases. Because the initiation process is a rate-limiting step in viral RNA replication, inhibitors that interfere with the initiation process will have advantages in suppressing virus replication. The use of dinucleotide analogues as inhibitor molecules to target viral replication initiation represents a novel approach to antiviral interference.

Oligonucleosides with a nucleobase-including backbone; synthesis and self-association of novel dinucleotide analogues.

The synthesis and self-association of protected oxymethylene-bridged UA analogues are described.

Synthesis and Resolution of Dinucleotide(TpAZT) Phosphoramidates

Dinucleotide (TpAZT) phosphoramidates were synthesized through Atherton-Todd reaction of dinucleoside H-phosphonates and amino acid methyl esters, and their diastereomers (Rp and Sp) were separated by crystallization. It was showed that the cheap methyl esters of natural alanine and phenylalanine could act as new chiral auxiliaries for large-scale synthesis of dinucleotide analogs.

Cytotoxicity and cellular differentiation activity of methylenebis(phosphonate) analogs of tiazofurin and mycophenolic acid adenine dinucleotide in human cancer cell lines

Mycophenolic acid (MPA) is a fungally-derived inhibitor of inosine 5′-monophosphate dehydrogenase (IMPDH). MPA binds IMPDH at the nicotinamide sub-site of the NAD cofactor binding domain leaving the adenosine sub-site empty. In order to improve the binding affinity we synthesized MPA analogs by linking adenosine 5′-methylenebis(phosphonate) with mycophenolic alcohols containing 2-, 4-, and 6-carbon atoms in their aliphatic side chain. Adenine dinucleotide analogs of tiazofurin, selenazofurin and benzamide riboside were synthesized as P 1, P 2-disubstituted pyrophosphates. Cytotoxicity of each analog was examined in human colon adenocarcinoma HT-29 and erythroleukemia K562 cells, and induction of differentiation in K562 cells by these agents was determined. Mycophenolic acid is currently used as an immunosuppressant but its anticancer action is limited by inactivation due to rapid glucuronidation. The new analogs show resistance to metabolism to inactive species and exhibit enhanced cytotoxicity in tumor cell lines, and therefore could be useful as anticancer agents.