2' Derivatives of guanosine and inosine cyclic 3',5'-phosphates. Synthesis, enzymic activity, and the effect of 8-substituents.

A series of representative derivatives of guanosine cyclic 3',5'-phosphate (cGMP) and inosine cyclic 3',5'-phosphate (cIMP) which contained modifications in either the 2' position or the 8 and 2' positions were synthesized. Three types of derivatives were investigated: (1) derivatives in which the 2' position has been altered to produce a 2'-deoxynucleoside cyclic 3',5'-phosphate or a 9-beta-D-arabinofuranosylpurine cyclic 3',5'-phosphate; (2) 2'-omicron-acyl derivatives; and (3) doubly modified derivatives containing a 2' modification [as in (1) and (2)] and an 8-substitution. 2'-Deoxyinosine cyclic 3',5'-phosphate and 9-beta-D-arabinofuranosylhypoxanthine cyclic 3',5'-phosphate were obtained by HNO2 deamination of 2'-deoxyadenosine cyclic 3',5'-phosphate and 9-beta-D-arabinofuranosyladenine cyclic 3',5'-phosphate (ara-cAMP), respectively. Treatment of 8-bromo-2'-omicron-(p-toluenesulfonyl) adenosine cyclic 3',5'-phosphate with NaSH yielded the intermediate 8,2'-anhydro-9-beta-D-arabinofuranosyl-8-mercaptoadenine cyclic 3',5-phosphate, which was converted directly to 2'-deoxyadenosine cyclic 3',5'-phosphate (dcAMP) by treatment with Raney nickel. 8-Bromo-2'-omicron-(p-toluenesulfonyl) guanosine cyclic 3',5'-phosphate was converted to 8,2'-anhydro-9-beta-D-arabinofuranosyl-8-mercaptoguanine cyclic 3',5'-phosphate, and the latter was desulfurized with Raney nickel to give 2-deoxyguanosine cyclic 3',5'-phosphate. Ara-cAMP, 9-beta-D-arabinofuranosylguanine cyclic 3',5'-phosphate, and 9-beta-D-arabinofuranosyl-8-mercaptoguanine cyclic 3',5'-phosphate have been previously reported (Mian et al. (1974), J. Med. Chem. 17, 259). 8-Bromo-2'-omicron-acetylinosine cyclic 3',5'-phosphate and 8-[(p-chlorophenyl)thio]-2'-omicron-acetylinosine cyclic 3',5'-phosphate were produced by acylation of 8-bromoinosine cyclic 3',5'-phosphate and 8-[(p-chlorophenyl)thio]inosine cyclic 3',5'-phosphate, respectively; while 8-bromo-2'-omicron-butyrylguanosine cyclic 3',5'-phosphate was synthesized by bromination of 2'-omicron-butyrylguanosine cyclic 3',5'-phosphate.     

Poly(A) polymerase from Escherichia coli adenylylates the 3'-hydroxyl residue of nucleosides, nucleoside 5'-phosphates and nucleoside(5')oligophospho(5')nucleosides (NpnN).

The capacity of Escherichia coli poly(A) polymerase to adenylylate the 3'-OH residue of a variety of nucleosides, nucleoside 5'-phosphates and dinucleotides of the type nucleoside(5')oligophospho(5')nucleoside is described here for the first time. Using micromolar concentrations of [alpha-32P]ATP, the following nucleosides/nucleotides were found to be substrates of the reaction: guanosine, AMP, CMP, GMP, IMP, GDP, CTP, dGTP, GTP, XTP, adenosine(5')diphospho(5')adenosine (Ap2A), adenosine (5')triphospho(5')adenosine (Ap3A), adenosine(5')tetraphospho(5')adenosine (Ap4A), adenosine(5')pentaphospho(5')adenosine (Ap5A), guanosine(5')diphospho(5') guanosine (Gp2G), guanosine(5')triphospho(5')guanosine (Gp3G), guanosine(5')tetraphospho(5')guanosine (Gp4G), and guanosine(5')pentaphospho(5')guanosine (Gp5G). The synthesized products were analysed by TLC or HPLC and characterized by their UV spectra, and by treatment with alkaline phosphatase and snake venom phosphodiesterase. The presence of 1 mM GMP inhibited competitively the polyadenylylation of tRNA. We hypothesize that the type of methods used to measure polyadenylation of RNA is the reason why this novel property of E. coli poly(A) polymerase has not been observed previously.     

Degradation by the 2',5'-phosphodiesterase activity of mouse cells requires the presence of a ribo hydroxyl group in the penultimate position of the oligonucleotide substrate

A series of 9-beta-D-xylofuranosyladenine (xyloA or xyloadenosine) substituted analogs of 2-5A core trimer and tetramer were examined for their ability to be degraded by the 2',5'-phosphodiesterase activity of cytoplasmic extracts of mouse L cells. Two distinct groups of xyloA-substituted analogs could be readily discriminated. The first group contained xyloadenosine at the 2'-termini and included A2'p5'A2'p5'(xyloA) and A2'p5'A2'p5'A2'p5'(xyloA). These oligomers behaved as did their parent oligoadenylates in that they were equally sensitive to degradation by the 2',5'-phosphodiesterase activity. The second group of oligonucleotides bore a xyloadenosine residue in the penultimate nucleotide residues of the oligomers and included A2'p5'(xyloA)2'p5'(xyloA), (xyloA)2'p5'(xyloA)2'p5'(xyloA), A2'p5'A2'p5'(xyloA)2'p5'(xyloA) and (xyloA)2'p5' (xyloA)2'p5'(xyloA)2'p5'(xyloA). This group was quite resistant to 2',5'-phosphodiesterase activity. In all, the findings demonstrate that the ribo configuration 3'-hydroxyl group in the penultimate nucleotide of the oligonucleotide substrate is a prerequisite for the 2',5'-phosphodiesterase activity.     

Heterogeneous nuclear RNA promotes synthesis of (2'',5'')oligoadenylate and is cleaved by the (2'',5'')oligoadenylate-activated endoribonuclease.

Heterogeneous nuclear RNA contains double-stranded regions that are not found in mRNA and that may serve as recognition elements for processing enzymes. The double-stranded regions of heterogeneous nuclear RNA prepared from HeLa cells promoted the synthesis of (2',5')oligoadenylate [(2',5')oligo(A) or (2'5')An] when incubated with (2',5')An polymerase. This enzyme is present in elevated levels in interferon-treated cells, and labeled heterogeneous nuclear RNA incubated with extracts of these cells is preferentially cleaved, since mRNA included in the same incubations is not appreciably degraded. The cleavage of heterogenous nuclear RNA is caused by the synthesis of (2'5')An and by a "localized" activation of the (2',5')An-dependent endonuclease, since it was enhanced by ATP, the substrate of the (2',5')An polymerase, and inhibited by 2'-dATP and ethidium bromide. Both of these compounds suppress the synthesis of (2',5')An, the first by competitive inhibition and the latter by intercalating into double-stranded RNA. The possible role of double-stranded regions and of the (2',5')An polymerase-endonuclease system in the processing of heterogeneous nuclear RNA is discussed.     

Interaction of muscle glycogen phosphorylase b with riboflavin, its tetraacetyl derivative and their analogs

The interaction of rabbit skeletal muscle glycogen phosphorylase b with riboflavin, 2',3',4',5'-tetraacetylriboflavin and their analogues, containing different substituents in the positions 6, 8 and 8 alpha, has been studied. Dissociation constant for the complex of the enzyme and riboflavin was determined to be 12.5 microM (pH 6.8; 20 degrees C) by sedimentation velocity method. Riboflavin and its analogues have been found to inhibit glycogen phosphorylase b. The inhibitor half-saturation concentration values increase in the following order: riboflavin (18 microM), 8-methoxy(nor)rifoblavin (23 microM), 8 alpha-bromo-2',3',4',5'-tetraacetylriboflavin (40 microM), 6-bromoriboflavin (40 microM), 8 alpha-hydroxyriboflavin (60 microM), 8-hydroxy(nor)riboflavin (90 microM), 8 alpha-(gamma-carboxypropylamino-2',3',4',5'-tetraacetylriboflav in (90 microM), 8 alpha-[p-(5-ethyl-1,3,4-thiodiazol-2-ylsulfamido)phenylamino ]- 2',3',4',5'-tetraacetylriboflavin (100 microM), 8 alpha-(L-methionyno)-2',3',4',5'-tetraacetylriboflavin (120 microM), 8 alpha-[p-(thiazol-2-ylsulfamido)phenylamino]- 2',3',4',5'-tetraacetylriboflavin (140 microM), 8 alpha-(p-sulfamidophenylamino)-2',3',4',5'-tetraacetylriboflavi n (180 microM), 8 alpha-(p-carboxyphenylamino)-2',3',4',5'-tetraacetylriboflavin+ ++ (210 microM), 2',3',4',5'-tetraacetylriboflavin (250 microM), 8 alpha-(L-homoserino)-2',3',4',5'-tetraacetylriboflavin (340 microM), 8 alpha-(L-glutamo)-2',3',4',5'-tetraacetylriboflavin (360 microM). The existence of glycogen phosphorylase b complexes with riboflavin and its analogues has been proved by methods of absolute and difference spectrophotometry.     

2''5'' oligoadenylate synthetase, an interferon induced enzyme: direct assay methods for the products, 2''5'' oligoadenylates and 2''5'' co-oligonucleotides.

The interferon induced enzyme 2'5' oligoadenylate synthetase produces 2'5' pppA(pA)n the first discovered natural nucleotide with a 2'5' linkage. We describe a direct assay of this enzyme based on separation by thin layer chromatography (TLC) of the substrate ATP and the products 2'5' pppA(pA)n (n larger than or equal to 1). This technique presents obvious advantages compared to the currently used methods. Moreover the enzyme uses other nucleotides as substrates forming co-oligonucleotides 2'5 pppA(pA)n pN (N = U,G,C,dA,dG,dT and dC). Additional procedures are described using different developing solvent systems for the separation of the core-2'5' oligonucleotides (2'5' A(pA)npN) containing AMP-residues entirely and those with another nucleotide at the 2' end.     

A conformational study of adenylyl-(3'',5'')-adenosine and adenylyl-(2'',5'')-adenosine in aqueous solution by carbon-13 magnetic resonance spectroscopy.

The solution conformation of adenylyl-(3',5')-adenosine and adenylyl-(2',5')-adenosine in both the stacked and unstacked states was studied by carbon-13 magnetic resonance spectroscopy. Large chemical shift differences between the base carbons in the dimers and those in the corresponding monomers are attributed in part to the influence of base-base interaction. Carbon-phosphorus couplings across three bonds revealed the preferred populations for certain backbone rotamers, demonstrating that significant changes in conformation about the "c(3')-O and C(5')-O bonds do not occur in the temperature or salt-induced unstacking of adenylyl-(3',5')-adenosine. However, rotations about the C(2')-O and C(5')-O bonds occur in the temperature-mediated unstacking of adenylyl-(2',5')-adenosine.     

Structure of 2',5'-linked tetraribonucleotide loops: a novel RNA motif

We report on the three dimensional structure of an RNA hairpin containing a 2',5'-linked tetraribonucleotide loop, namely, 5'-rGGAC(UUCG)GUCC-3' (where UUCG = U(2'p5')U(2'p5')C(2'p5')G(2'p5')). We show that the 2',5'-linked RNA loop adopts a conformation that is quite different from that previously observed for the native 3',5'-linked RNA loop. The 2',5'-RNA loop is stabilized by (a) U:G wobble base pairing, with both bases in the anti conformation, (b) extensive base stacking, and (c) sugar-base contacts, all of which contribute to the extra stability of this hairpin structure.     

Hydrolysis of some mRNA 5'-cap analogs catalyzed by the human Fhit protein--and lupin ApppA hydrolases.

Hydrolysis of the following four cap analogs: m7G(5')ppp(5')A, m7G(5')ppp(5')m6A, m7G(5')ppp(5')m2'OG and m7G(5')ppp(5')2'dG catalyzed by homogeneous human Fhit protein and yellow lupin Ap3A hydrolase has been investigated. The hydrolysis products were identified by HPLC analysis and the K(m) and Vmax values calculated based on the data obtained by the fluorimetric method.     

2',5'-Phosphodiesterase activity depends upon the presence of a 3'-hydroxyl moiety in the penultimate position of the oligonucleotide substrate

3'-Deoxyadenosine (3'dA, cordycepin)-substituted analogs of 2-5A core 5'-monophosphate (p5'A2'p5'A2'p5'A) were examined for their sensitivity toward degradation by the 2'-phosphodiesterase activity in cytoplasmic extracts of mouse L cells. The analogs, p5'(3'dA)-2'p5'A2'p5'A, p5'(3'dA)2'p5'A2'p5'(3'dA) and p5'A2'p5'A2'p5'(3'dA) were degraded at a rate comparable to p5'A2'p5'A2'p5'A itself. On the other hand, under the assay conditions examined p5'A2'p5'(3'dA)2'p5'A, like p5'(3'dA)2'p5'(3'dA)2'p5'(3'dA), was completely resistant to degradation. The data imply that sensitivity to the 2',5'-phosphodiesterase activity of mouse L cells requires the presence of 3'-hydroxyl moiety in the penultimate nucleotide.     

Inhibition of protein synthesis by the cordycepin analog of (2'-5')ppp(Ap)nA, (2'-5')ppp(3'dAp)n3'dA, in intact mammalian cells

The introduction of the cordycepin analog of (2'-5')An, (2'-5')ppp(3'dAp)n3'dA [referred to as (2'-5')p33'dAn], into mouse L929 cells and cultured human fibroblasts resulted in a dose-dependent inhibition of protein synthesis which was comparable to the inhibition observed by (2'-5')ppp(Ap)nA [referred to as (2'-5')p3An]. The inhibition of protein synthesis by (2'-5')p33'dAn was much more persistent than that of the naturally occurring (2'-5')p3An following prolonged incubation of cells. Furthermore, the (2'-5')p3An was cytotoxic to mammalian cells in culture, whereas the (2'-5')p33'dAn was not.     

Pyrano chalcones and a flavone from Neoraputia magnifica and their Trypanosoma cruzi glycosomal glyceraldehyde-3-phosphate dehydrogenase-inhibitory activities

The fruits of Neoraptua magnifica var. magnifica afforded three new flavonoids: 2'-hydroxy-4,4',-dimethoxy-5',6'-(2',2'-dimethylpyrano)chalcone, 2'-hydroxy-3,4,4'-trimethoxy-5',6'-(2',2'-dimethylpyrano)chalcone, and 3',4'-methylenedioxy-5,7-dimethoxyflavone which were identified on the basis of spectroscopic methods. The known flavonoids 2'-hydroxy-3,4,4',5-tetramethoxy-5',6'-(2',2'-dimethylpyrano)chalcone, 2'-hydroxy-3,4,4',5,6'-pentamethoxychalcone, 3',4'-methylenedioxy-5,6,7-trimethoxyflavone, 3',4'-methylenedioxy-5',5,6,7-tetramethoxyflavone, 3',4',5',5,7-pentamethoxyflavanone and 3',4',5'5,7-pentamethoxyflavone were also identified. The latter flavone was the most active as glyceraldehyde-3-phosphate dehydrogenase-inhibitor.     

Conformational properties of 2',5' linked Rp- and Sp-phosphorothioate oligoadenylates studied by circular dichroism and NMR

2',5'-Linked oligoadenylates (2-5A) are involved in the antiviral action of interferon. The 2-5A binds and activates 2-5A dependent RNase (RNase L), which degrades viral mRNA, resulting in the inhibition of protein synthesis. 2',5'-Linked phosphorothioate oligoadenylates with an Rp configuration bind to and activate the RNase L. On the other hand, 2',5' phosphorothioate oligoadenylate with an Sp configuration weakly binds to the RNase L and is devoid of the RNase L activation ability. Comparative circular dichroism (CD) and NMR studies are carried out to characterize the difference in properties between the two configurations of the 2',5' phosphorothioate oligoadenylates. 2',5' Rp-Phosphorothioate oligoadenylates showed CD spectra similar to those of the corresponding native 2',5' oligoadenylates, while the 2',5' Sp-phosphorothioate oligoadenylates exhibited a weaker CD band compared to the former two, indicating the weaker base-stacking interaction of the 2',5' Sp-phosphorothioate oligoadenylates. The temperature-dependent change in the CD revealed that 2',5' phosphorothioate oligoadenylates showed larger DeltaH(0) and DeltaS(0) values for the thermal transition of the conformation than the corresponding native 2',5' oligoadenylates. The NMR spectral assignment was accomplished by several NMR measuring techniques. The 2'-H of the ribose ring linked to the 2',5' Sp-phosphorothioate showed a higher field chemical shift of the proton NMR than that linked to the corresponding 2',5' Rp-phosphorothioate. 2',5' Rp- and Sp-phosphorothioate oligoadenylates possess a sugar conformation similar to that of the corresponding native 2',5' oligoadenylates.     

Synthesis and biological activity of tubercidin analogues of ppp5'A2'p(5'A2'p)n5'A

A series of tubercidin (7-deazaadenosine) analogues of 2-5A of the general formula p5'(c7A)2'p[5'(c7A)-2'p]n5'(c7A) (n = 0-5) were prepared by lead ion catalyzed polymerization of the 5'-phosphoroimidazolidate of tubercidin. Through the corresponding imidazolidates, these oligonucleotide 5'-monophosphates were converted to the 5'-triphosphates. All reported structures were corroborated by enzyme digestion and 1H or 31P nuclear magnetic resonance. When evaluated for its ability to bind to the 2-5 A-dependent endonuclease of mouse L cells, the tubercidin analogue of trimeric 2-5A, namely, ppp5'(c7A)2'p5'(c7A)2'p5'(c7A), and the corresponding tetramer were bound as effectively as 2-5A itself; nonetheless, it and the corresponding tetramer, ppp5'-(c7A)2'p5'(c7A)2'p5'(c7A)2'p5'(c7A), failed to stimulate the 2-5A-dependent endonuclease as judged by its inability to inhibit translation in extracts of mouse L cells programmed with encephalomyocarditis virus RNA and to give rise to ribosomal RNA cleavage in the same cell system under conditions where 2-5A showed activity at 10(-9) M. The trimer, ppp5'(c7A)2'p5'(c7A)2'p5'(c7A), was an antagonist of 2-5A action in the L cell extract. In the lysed rabbit reticulocyte system, both the trimeric and tetrameric tubercidin 2-5A analogues were bound to the 2-5A-dependent endonuclease as well as 2-5A, but in this case, the tetramer triphosphate, ppp5'(c7A)2'p5'(c7A)2'p5'(c7A)2'p5'(c7A), was just as potent an inhibitor of translation as 2-5A tetramer triphosphate. Moreover, this inhibition was prevented by the established 2-5A antagonist p5'A2'p5'A2'p5'A.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mechanisms of degradation of 2′-5′ oligoadenylates

We have studied the mechanisms of breakdown of 2'-5' oligoadenylates. We monitored the time-courses of degradation of ppp(A2'p5')nA (dimer to tetramer) and of 5'OH-(A2'p5')nA (dimer to pentamer) in unfractionated L1210 cell extract. The 5' triphosphorylated 2'-5' oligoadenylates are converted by a phosphatase activity. However, 2'-5' oligoadenylates are degraded mainly by phosphodiesterase activity which splits the 2'-5' phosphodiester bond sequentially at the 2' end to yield 5' AMP and one-unit-shorter oligomers. The nonlinear least-squares curve-fitting program CONSAM was used to fit these kinetics and to determine the degradation rate constant of each oligomer. Trimers and tetramers, whether 5' triphosphorylated or not, are degraded at the same rate, whereas 5' triphosphorylated dimer is rapidly hydrolyzed and 5'-OH dimer is the most stable oligomer. The interaction between degradation enzymes and the substrate strongly depends on the presence of a 5' phosphate group in the vicinity of the phosphodiester bond to be hydrolyzed; indeed, when this 5' phosphate group is present, as in pp/pA2'p5'A/or A2'/p5'A2'p5'A/, affinity is high and maximal velocity is low. Such a degradation pattern can control the concentration of 2'-5' oligoadenylates active on RNAse L either by limiting their synthesis (5' triphosphorylated dimer is the primer necessary for the formation of longer oligomers) and/or by converting them into inhibitory (e.g., monophosphorylated trimer) or inactive (e.g., nonphosphorylated oligomers) molecules.     

5'-modified agonist and antagonist (2'-5')(A)n analogues: synthesis and biological activity

Two 5'-modified (2'-5')(A)4 oligomers with an increased resistance to phosphatase degradation were synthesized and evaluated for their ability to develop an antiviral response when introduced into intact cells by microinjection or by chemical conjugation to poly(L-lysine). The enzymatic synthesis of 5'-gamma-phosphorothioate and beta,gamma-difluoromethylene (2'-5')(A)4 from adenosine 5'-O-(3-thiotriphosphate) and adenosine beta,gamma-difluoromethylenetriphosphate by (2'-5')-oligoadenylate synthetase is described. The isolation and characterization of these (2'-5')(A)4 analogues were achieved by high-performance liquid chromatography. The structures of 5'-modified tetramers were corroborated by enzyme digestion. These two 5'-modified tetramers compete as efficiently as natural (2'-5')(A)4 for the binding of a radiolabeled (2'-5')(A)4 probe to ribonuclease (RNase) L. Nevertheless, at the opposite to 5'-gamma-phosphorothioate (2'-5')(A)4, beta,gamma-difluoromethylene (2'-5')(A)4 failed to induce an antiviral response after microinjection in HeLa cells. In addition, it behaves as an antagonist of RNase L as demonstrated by its ability to inhibit the antiviral properties of 5'-gamma-phosphorothioate (2'-5')(A)4 when both are microinjected in HeLa cells. The increased metabolic stability of 5'-gamma-phosphorothioate (2'-5')(A)4 as compared to that of (2'-5')(A)4 was first demonstrated in cell-free extracts and then confirmed in intact cells after introduction in the form of a conjugate to poly(L-lysine). Indeed, 5'-gamma-phosphorothioate (2'-5')(A)4-poly(L-lysine) conjugate induces protein synthesis inhibition and characteristic ribosomal RNA cleavages for longer times than unmodified (2'-5')(A)4-poly(L-lysine) in the same cell system.(ABSTRACT TRUNCATED AT 250 WORDS)     

Lsm proteins bind and stabilize RNAs containing 5' poly(A) tracts

Many orthopoxvirus messenger RNAs have an unusual nontemplated poly(A) tract of 5 to 40 residues at the 5' end. The precise function of this feature is unknown. Here we show that 5' poly(A) tracts are able to repress RNA decay by inhibiting 3'-to-5' exonucleases as well as decapping of RNA substrates. UV cross-linking analysis demonstrated that the Lsm complex associates with the 5' poly(A) tract. Furthermore, recombinant Lsm1-7 complex specifically binds 5' poly(A) tracts 10 to 21 nucleotides in length, consistent with the length of 5' poly(A) required for stabilization. Knockdown of Lsm1 abrogates RNA stabilization by the 5' poly(A) tract. We propose that the Lsm complex simultaneously binds the 3' and 5' ends of these unusual messenger RNAs and thereby prevents 3'-to-5' decay. The implications of this phenomenon for cellular mRNA decay are discussed.