Oligomerization of 3′-amino-3′-deoxyguanosine-5′-phosphorimidazolidate on a d(CpCpCpCpC) template

Summary 3-Amino-3-deoxyguanosine-5-phosphorimidazolidate (ImpGnh ₂) oligomerizes more rapidly and regiospecifically than related nucleotide derivatives on a d(CpCpCpCpC) template. The greater nucleophilicity of the amino group leads to efficient oligomerization even when the structure of the double-helical complex formed by the template and the substrate is not optimal for reaction. The use of amine-containing analogues should permit us to develop models of potentially prebiotic polymerization reactions that cannot be studied easily using natural nucleotides.     

Synthesis of 3′-N-Substituted 3′-Amino-3′-Deoxythymidine Derivatives

Abstract

A series of 3′-N-substituted 3′-amino-3′-deoxythymidine derivatives with alkyl, alkenyl and alkylaryl substituents was synthesized by two methods. The first method involved the reaction of 1-(2,3-dideoxy-3-0-mesyl-5-0-trityl-β-D-threo-pentofuranosyl)thymine with an appropriate amine. In the second method, 3′-amino-5′-0-trityl-3′-deoxy-thymidine served as a synthetic precursor which was reacted with an appropiate aldehyde or ketone followed by sodium borohydride reduction. An improved synthesis of 3′-amino-3′-deoxythymidine from 3′ -azido-5′-0-trityl-3′-deoxythymidine using sodium borohydride was also described.     

Effect of guanosine 3′:5′-monophosphate on the hydroosmotic activity of adenosine 3′:5′-monophosphate in toad urinary bladder

Guanosine 3′:5′-monophosphate has a slight hydroosmotic effect on toad urinary bladder. Furthermore, this nucleotide strongly inhibits the responses to 3′:5′-adenosine monophosphate and oxytocin. The response to an increase in medium tonicity is not modified by the guanosine nucleotide. A role for guanosine 3′:5′-monophosphate in the regulation of water permeability in toad urinary bladder is proposed.     

Synthesis of 3′-Amino-3′-deoxyguanosine 5′-Triphosphate

Abstract

Phosphorylation of 2′-0-acetyl-3′-trifluoroacetamido-3′-deoxy-N²-palmitoylguanosine with N-morpholino-O, O-bis(1-benzotriazolyl)phos-phate gives a 5′-phosphotriester. Removal of the benzotriazolyl group and addition of pyrophosphoric acid gave, after deblocking all protecting groups, GTP(3′NH₂).     

Effects of Exogenous Adenosine 5′-triphosphate 3′-diphosphate on Growth and Sporulation of Bacillus subtilis

Exogenous adenosine 5′-triphosphate 3′-diphosphate (pppApp) had interesting effects on the cell cycle of B. subtilis IFO 3027. The growth rate was reduced by the addition of 1 mm pppApp, and the vegetative cell form was significantly changed. Moreover, the sporulation frequency was increased by 100 times or more as compared with the culture without pppApp. The sporulation process seemed to be stimulated around t₀. pppGpp and ppGpp also showed the same effects as pppApp. Among these effects, depression in growth rate was restored by Mg²⁺ and Ca²⁺, and stimulation of sporulation was inhibited by Mg²⁺, Ca²⁺ and certain carbon sources, such as glucose and glycerol. On the other hand, casamino acids or monovalent cations showed no influence on the pppApp effects. pppApp was not incorporated into cells in experiments with radioactive pppApp.     

An effect of insulin on adipose-tissue adenosine 3′:5′-cyclic monophosphate phosphodiesterase

1. 3':5'-Cyclic nucleotide phosphodiesterase activity was measured in homogenates prepared from epididymal fat-pads and isolated fat-cells incubated in the absence and presence of insulin. 2. Homogenates of insulin-treated tissues showed an increase in phosphodiesterase activity compared with controls. No effect of insulin was observed when the hormone was added directly to homogenates. 3. There was kinetic evidence for the presence of two 3':5'-cyclic nucleotide phosphodiesterases in adipose tissue. Insulin raised the maximal velocity of the low-K(m) enzyme and lowered the K(m) of the higher-K(m) enzyme. 4. It is suggested that the effect of insulin on adipose tissue phosphodiesterase accounts for the ability of this hormone to lower cyclic-AMP concentration in the tissue.     

V-cGAPs: attenuators of 3′3′-cGAMP signaling

Cyclic GMP-AMPs (cGAMPs) are new members of the cyclic dinucleotide family of second messenger signaling molecules identified in both bacteria and mammalian cells. A recent study by Gao et al. published in Cell Research has identified and characterized three 3′3′-cGAMP-specific phosphodiesterases (termed as V-cGAP1/2/3) in V. cholerae, thereby providing mechanistic insights into the function of these enzymes that degrade cGAMPs.Despite their indispensable roles in the composition of DNA and RNA, as well as serving as energy sources, nucleotides are also well known as crucial signaling molecules in all domains of life. Cyclic dinucleotides (CDNs) represent an important and growing family of second messengers, which have been previously recognized as key modulators governing a variety of cellular activities in bacteria, and more recently, in mammalian cells. c-di-GMP and c-di-AMP, the first two members of the CDN family, have been implicated in central bacterial processes, and likely act as universal bacterial secondary messengers^1,2. The latest addition to the bacterial CDN family is 3′3′-cGAMP, a hybrid molecule that is synthesized from ATP and GTP by DncV (a cyclase from V. cholerae) and shown to promote intestinal colonization of V. cholerae by downregulating chemotaxis³. Predicted homologs of DncV are present in many other bacterial species³, indicating that 3′3′-cGAMP may also regulate a wide range of cellular functions, similar to c-di-GMP and c-di-AMP. The research on CDNs as second messengers reached new heights following the recent identification of 2′3′-cGAMP, a noncanonical CDN in mammalian cells containing mixed 2′,5′ (at GpA step) and 3′,5′ (at ApG step) linkages, which is synthesized by cGAMP synthase (cGAS) in response to the presence of DNA in the cytosol^4,5,6. A remarkable set of new discoveries have revealed that all the CDNs described above are able to bind and activate STING, the central adaptor in the cytosolic DNA sensing pathway, thereby promoting the innate immune response in mammalian cells by inducing the expression of Type I interferon (IFN)^7,8,9.Given their critical roles in a variety of important cellular processes, the cellular levels of CDNs have to be tightly controlled by the coordinated action of counteracting cyclases and degradation enzymes. To date, several phosphodiesterases (PDEs) have been found to hydrolyze c-di-GMP (EAL or HD-GYP domain-containing enzymes)¹ and c-di-AMP (DHH-DHHA or HD domain-containing enzymes)^2,10 (Figure 1). In addition, recent research reported that ENPP1 (ecto-nucleotide pyrophosphatase/phosphodiesterase) is the dominant 2′3′-cGAMP hydrolyzing enzyme in mammalian cells¹¹ (Figure 1). A new study by Gao et al.¹² has now identified the first three 3′3′-cGAMP-specific PDEs in V. cholerae and provided detailed insights into their enzymatic mechanisms.Open in a separate windowFigure 1Schematic representation of degradation enzymes identified for different cyclic dinucleotides and the related hydrolysis products. The various protein domains are highlighted by different shapes and colors. Note that the newly identified V-cGAPs belong to the HD-GYP domain-containing PDEs.There are a total of 36 potential PDE genes (containing EAL, HD-GYP or DHH domains) in the V. cholerae genome. To search for 3′3′-cGAMP-specific PDE(s), Gao et al.¹² established an efficient and sensitive eukaryotic screening system by taking advantage of the ability of 3′3′-cGAMP to activate STING and induce type I IFN expression in mammalian cells. By overexpressing the 3′3′-cGAMP synthetase DncV together with the 36 potential PDEs in 293 cells, the authors could monitor IFN-β promoter activation to identify the PDE(s) that could degrade 3′3′-cGAMP. To exclude false-positives, Gao et al. further purified the PDEs that potentially target 3′3′-cGAMP based on the initial screening, and incubated these enzymes with chemically synthesized 3′3′-cGAMP. The treated 3′3′-cGAMP molecules were further assayed by either adding to PFO-permeabilized THP-1 cells to examine IRF3 phosphorylation levels or through loading on HPLC to monitor the generation of new products. As a result of the screening and validation, the authors successfully identified three HD-GYP domain-containing proteins that could degrade 3′3′-cGAMP, named VCA0681, VCA0210 and VCA0931 (designated as V-cGAP1, 2 and 3, respectively).To determine the substrate specificity of V-cGAPs, different cGAMP linkage isomers (3′3′-, 3′2′-, 2′3′-, and 2′2′-cGAMPs) were incubated with the purified V-cGAPs. The results of both IRF3 phosphorylation in THP-1 cells and HPLC assays clearly indicated that V-cGAPs only degrade 3′3′-cGAMP, but not other cGAMP linkage isomers. The 3′3′-cGAMP PDE activity of V-cGAPs was further confirmed by dosage- and time-dependent enzymatic assays. By using mutant proteins, the authors also confirmed that both the HD and GYP motifs within V-cGAPs are critical for PDE activity.Combining detailed HPLC analysis, mass spectrometry and enzymatic treatment, Gao et al. definitively established that 3′3′-cGAMP is first hydrolyzed by all three V-cGAPs to generate linear 5′-pApG, which is further hydrolyzed into 5′-ApG only by V-cGAP1. These results show that V-cGAP2 and V-cGAP3 have only PDE activity, while V-cGAP1 has both PDE and 5′-nucleotidase activities. The authors also found that V-cGAP1 has a much higher activity for linearization of 3′3′-cGAMP to 5′-pApG than V-cGAP2 and 3, with the later two V-cGAPs exhibiting similar kinetics of degradation.The cellular level of 3′3′-cGAMP has to be tightly regulated by a combination of counteracting synthesis and degradation enzymes. Since the expression level of DncV was found to be inducible by outside signals to enhance intestinal colonization and infectivity, it is very likely that the expression level of V-cGAPs will also be regulated by 3′3′-cGAMP production. Indeed, the authors proved that V-cGAP expression is greatly and readily enhanced after arabinose-induced DncV expression in a ΔdncV mutant V. cholerae strain, at both mRNA (by qRT-PCR) and protein (by immunoblot analysis) levels. To confirm the in vivo function of V-cGAPs, the authors performed both “chemotactic” and “infant mouse colonization competition” assays by using V-cGAP1/2/3 single-, double-, or triple-deletion V. cholerae strains. All the in vivo data clearly established that V-cGAPs counteract DncV function and exert a crucial role in regulating bacterial infectivity.The large amount of insightful data presented by Gao et al. has elucidated detailed information regarding the identification and characterization of 3′3′-cGAMP-specific phosphodiesterases, thereby providing valuable insights into our understanding of the regulatory mechanisms of cGAMP signaling in bacteria. Clearly, further structural work will be necessary to understand the intermolecular interactions between 3′3′-cGAMP and V-cGAPs, and provide insights into the mechanism by which V-cGAPs preferentially attack the phosphodiester bond at the GpA step.     

Studies on the hydrolysis of 3-methyl-2′-deoxycytidine in aqueous solution A synthesis of 3-methyl-2′-deoxyuridine

The hydrolysis of 3-methyl-2′-deoxycytidine in aqueous solution has been investigated. Varying proportions of 3-methylcytosine, 3-methyluracil and 3-methyl-2′-deoxyuridine are formed depending upon conditions of pH and temperature. All three hydrolytic products are formed at pH 6.8 and 90°C. At pH 2, depyrimidination of 3-methylcytosine occurs as the only hydrolysis product. When the pH is increased to 12, 3-methyl-2′-deoxycytidine on heating at 90°C is completely deaminated to 3-methyl-2′-deoxyuridine with few side products formed. This reaction serves as the basis for a convenient synthesis of 3-methyl-2′-deoxyuridine. The 300 MHz spectra of 3-methyl-2′-deoxycytidine and 3-methyl-2′-deoxyuridine indicate that the sugar ring in these compounds is predominantly in ²E conformation.     

Evaluation of Different Types of End-Capping Modifications on the Stability of Oligonucleotides Toward 3′- and 5′-Exonucleases

Abstract

Synthetic oligonucleotides are increasingly used because of their potential activity as regulators of gene expression. One of their major drawbacks is instability toward nucleases, in particular exonucleases. In this article, we studied some terminal modifications that can enhance exonuclease resistance, such as end-capping with alkylic chains (1,3-propanediol and 1,6-hexanediol), and with a modified nucleotide (2′,3′ -secouridine). These compounds were compared with the parent (natural) oligodeoxynucleotide and with different analogs containing a progressive number of phosphorothioate linkages. The resistance toward SVPDE and CSPDE (a 3′ - and a 5′ -exonuclease) was assessed, in vitro, by two independent techniques, UV and HPLC. Our results showed that the stability of all the modified oligonucleotides was at least 12 times that of the parent compound.     

Synthesis and Evaluation of Antiviral Activity of 3′-C-Cyano-3′-Deoxynucleosides

Abstract

A series of 3′-C-cyano-3′-deoxy and 3′-C-cyano-2′,3′-dideoxy-nucleoside analogues of thymidine, uridine, cytidine and adenosine have been prepared. Their antiviral activity was assessed in various assay systems and while none of the compounas proved specifically active against human immunodeficiency virus, some compounds had marked activity against other viruses.     

Antiviral Activities of β-Enantiomers of 3′-Substituted-3′-deoxythymidine Analogs

Abstract

Several β-L-3′-substituted-3′-deoxythymidine were stereospecifically synthesized. None of these analogs inhibited HIV-1 nor HBV replication in vitro suggesting that these β-L-pyrimidine derivatives may not be efficiently phosphorylated inside the cells.     

Influence of theophylline on concentrations of cyclic 3′,5′-adenosine monophosphate and cyclic 3′,5′-guanosine monophosphate of rat brain

The influence of theophylline (2.5–100 mg/kg p.o.) on cyclic 3,5-adenosine monophosphate (cAMP) and cyclic 3,5-guanosine monophosphate (cGMP) in brain of Sprague-Dawley rats (0.5–3.0 hr after administration of theophylline) was investigated. It was found that theophylline increases cAMP and cGMP levels when administered in a dose of 25 mg/kg or higher. A significant decrease of cGMP level was observed after administration of 10 mg/kg. The results of this study suggest that the influence of theophylline on cyclic nucleotide levels of rat brain is the result of two factors: (a) inhibitory properties of theophylline on cAMP and cGMP phosphodiesterases and (b) competition of theophylline with adenosine.     

Effects of adenosine 3′ : 5′-monophosphate and guanosine 3′ : 5′-monophosphate on calcium uptake and phosphorylation in membrane fractions of vascular smooth muscle

The effects of adenosine 3′ : 5′-monophosphate (cyclic AMP), guanosine 3′ : 5′-monophosphate (cyclic GMP) and exogenous protein kinase on Ca uptake and membrane phosphorylation were studied in subcellular fractions of vascular smooth muscle from rabbit aorta. Two functionally distinct fractions were separated on a continuous sucrose gradient: a light fraction enriched in endoplasmic reticulum (fraction E) and a heavier fraction containing mainly plasma membranes (fraction P).While cyclic AMP and cyclic GMP had no effect on Ca uptake in the absence of oxalate, both cyclic nucleotides inhibited the rate of oxalate-activated Ca uptake when used at concentrations higher than 10^?5 M. The addition of bovine heart protein kinase to either fraction produced an increase in the rate of oxalate-activated Ca uptake which was further augmented by cyclic AMP. Cyclic GMP caused smaller stimulations of protein kinase-catalyzed Ca uptake than cyclic AMP.Mg-dependent phosphorylation, attributable to endogenous protein kinase(s), was inhibited in fraction E by low concentrations (10^?8 M) of both cyclic AMP and cyclic GMP. In fraction P, an inhibition by cyclic AMP occurred also at a concentration of 10^?8 M, while with cyclic AMP a concentration of 10^?5 M was required for a similar inhibition. Bovine heart protein kinase stimulated the phosphorylation of the membrane fractions much more than Ca uptake. In fraction E, in the presence of bovine protein kinase, both cyclic AMP and cyclic GMP stimulated phosphorylation up to 200%. Under these conditions, no stimulation was observed in fraction P.These results are compatible with the hypothesis that in vascular smooth muscle soluble rather than particulate protein kinases are involved in the regulation of intracellular Ca concentration.     

Stability of plant mRNAs depends on the length of the 3′-untranslated region

Eukaryotic mRNAs that prematurely terminate translation are recognized and degraded by nonsense mediated decay (NMD). This degradation pathway is well studied in animal and yeast cells. The data available imply that NMD also takes place in plants. However, the molecular mechanism of recognition and degradation of plant RNAs containing premature terminator codon (PTC) is not known. Here we report that in plant cells this mechanism involves the recognition of the sizes of the 3'-untranslated regions (3'UTR). Plant 3'UTRs longer than 300 nucleotides induce mRNA instability. Contrary to mammalian and yeast cells, this destabilization does not depend on the presence of any specific sequences downstream of the terminator codon. Unlike nuclear-produced mRNAs, plant virus vector long 3'UTR-containing RNAs, which are synthesized directly in the cytoplasm, are stable and translated efficiently. This shows that RNAs produced in the cytoplasm by viral RNA-dependent RNA polymerase are able to avoid the proposed mechanism.     

Effect of dibutyryl cyclic adenosine 3′5′-monophosphate on mitotic activity in the thyroid of hypophysectomized rats

Summary Effect of dibutyryl cyclic adenosine 35-monophosphate (dbcAMP) on mitotic activity in the thyroid of hypophysectomized rats has been examined. It has been demonstrated that dbcAMP stimulates the incidence of mitoses in the thyroid follicular cells. It is therefore suggested that cAMP may be a mediator of the proliferogenic effect of TSH on the thyroid in vivo. Cyclic AMP could also release some unidentified growth-promoting factors for the thyroid. A direct stimulating effect of dbcAMP on the proliferation of the thyroid follicular cells is assumed to be possible as well.     

The effect of the 3′ → 5′ exonuclease of T7 DNA polymerase on frameshifts and deletions

Both spontaneous frameshift mutation and deletion mutation were measured in a T7 phage deficient in the 3′ → 5′ exonuclease of T7 DNA polymerase. It was found that the absence of this exonuclease caused a marked increase in the revision of both plus one and minus one mutations. The exonuclease deficiency caused essentially no effect on the frequency of deletion between 10-bp direct repeats even when the segment between the direct repeats contained a 25-bp palindrome.     

New Phosphonoformic Acid Derivatives of 3′-Azido-3′-Deoxythymidine

New 5-alkyl ethoxy- and aminocarbonylphosphonates of 3-azido-3-deoxythymidine (AZT) were synthesized, and their antiviral properties in HIV-1-infected cell cultures and stability to chemical hydrolysis were studied. The AZT 5-aminocarbonylphosphonates were shown to be significantly more stable in phosphate buffer (pH 7.2) than the corresponding ethoxycarbonylphosphonates. The therapeutic (selectivity) index of some of the compounds exceeded that of the parent AZT due to their higher antiviral activity.     

Synthesis of Analogues of 3′-Deoxypsicothymidine

Abstract

Preparation of 3′-deoxypsicothymidines bearing a tether group at O1′ is described. Selective protection of the primary hydroxy functions of the starting nucleoside is briefly discussed.