The degradation of ribonucleic acid in the cotyledons of Pisum arvense

1. A ribonuclease has been partially purified from the cotyledons of germinating seed of Pisum arvense. 2. The enzyme degrades ribopolynucleotides to adenosine 3'-phosphate, guanosine 3'-phosphate and the cyclic nucleotides cytidine 2',3'-phosphate and uridine 2',3'-phosphate; no resistant ;core' remains. 3. The activity of RNA-degrading enzymes in the cotyledons increases to a maximum during the first 5 days of germination, passes through a minimum around the eighth day, and thereafter increases again. 4. Ion-exchange chromatography of methanol-soluble extracts of cotyledons revealed the presence, amongst other components, of the 2'-, 3'- and 5'-phosphates of cytidine and uridine, the 3'- and 5'-phosphates of adenosine, and guanosine 5'-phosphate. 5. Seed soaked in a solution containing [(32)P]orthophosphate gave a methanol-soluble fraction containing labelled nucleoside 5'-phosphates, but nucleoside 2'- and 3'-phosphates were not labelled. 6. It is believed that the nucleoside 2'- and 3'-phosphates arise by the action of ribonuclease on cotyledon RNA.     

The modified nucleosides of tRNAs. II. Synthesis of 2''-O-methylcytidylyl (3''-5'') cytidine.

The synthesis of 2'-O-methylcytidylyl (3'-5')cytidine by the triester method using as protecting groups, 2,2,2-trichloroethyl for phosphate hydroxyl group, p-chlorophenyoxyacetyl for 5-hydroxyl group, methoxymethylidene for 2',3'-cis-diol system, and benzoyl for the exo-amino group of cytidine is presented. The obtained product was characterised by UV, electrophoresis, chromatography and an enzymatic digestion.     

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)     

Kinetic studies on degradation of nucleotides catalyzed by phosphodiesterase-phosphomonoesterase from Fusarium moniliforme

Degradation of the 2'-phosphates, 3'-phosphates, 5'-phosphates, 2':3'-cyclic phosphates, 3':5'-cyclic phosphates, and 5'-(p-nitrophenylphosphates) of adenosine, guanosine, cytidine, and uridine catalyzed by Fusarium phosphodiesterase-phosphomonoesterase was followed by means of high performance liquid chromatography. All the nucleotides were susceptible to the enzyme to a greater or lesser degree, and the kinetic constants, Km and kcat, were determined at pH 5.3 and 37 degrees C. These constants were affected by both the nucleoside moiety and the position of the phosphate. Judged from kcat/Km, the 3'-phosphates, 2':3'-cyclic phosphates, and 5'-(p-nitrophenylphosphates) were good substrates, whereas the 2'-phosphates, 5'-phosphates, and 3':5'-cyclic phosphates were poor substrates except for adenosine 2'-phosphate, adenosine 5'-phosphate, and cytidine 5'-phosphate, which were hydrolyzed relatively easily. Among the phosphodiesters, the 2':3'-cyclic phosphates of adenosine, guanosine, and cytidine; and the 3':5'-cyclic phosphates of adenosine and cytidine were degraded into nucleoside and inorganic phosphate without release of intermediary phosphomonoester into the medium. Other phosphodiesters were degraded stepwise releasing definite intermediates.     

Synthesis and use of 3'-(azidoiodosalicyl) derivatives of 2', 5'-dideoxyadenosine as photoaffinity ligands for adenylyl cyclase

3'-[(4-Azidosalicyl)glycyl]-2',5'-dideoxyadenosine (1), 3'- [(4-azidosalicyl)-gamma-aminobutyryl]-2',5'-dideoxyadenosine (2), and the (125)I-labeled mono- and diiodinated analogs of 1 were synthesized and tested as photoaffinity probes for adenylyl cyclases. Kinetics for inhibition of purified type I enzyme by 1 was noncompetitive with respect to Mn(*)5'-ATP in the absence of light, implying a P-site mechanism of inhibition. In a UV-dependent manner both 1 and 2 and the iodinated derivative of 1 irreversibly inactivated membrane-bound and purified forms of recombinant type I bovine adenylyl cyclase expressed in ovarian cells of either the fall armyworm (Sf9) or Trichoplasia ni (High Five). Irreversible inactivation was independent of 5'-ATP and was prevented by 2', 5'-dideoxyadenosine. Adenylyl cyclase, whether purified from bovine brain or in membranes from High Five cells expressing type I enzyme, when subjected to UV irradiation in the presence of (125)I-labeled 1 resulted in radioactive incorporation into protein migrating at approximately 116 kDa. The cross-linking of 1 and its iodinated derivative with adenylyl cyclase suggests potential for such compounds to be useful in structural studies of adenylyl cyclases or of other proteins for which adenine nucleosides are substrates or allosteric regulators.     

2',5'-Oligoadenylate trimer core and the cordycepin analog augment the tumoricidal activity of human natural killer cells

Interferon (IFN) augments the lytic activity of natural killer (NK) cells, inhibits the transformation of human peripheral blood lymphocytes (PBL) by Epstein Barr virus (EBV), and induces a 2',5'-oligoadenylate (2',5'-An) synthetase. Exogenous 2',5'-An by itself can inhibit the transformation of human PBL by EBV. The present studies report that 2',5'-An and its cordycepin analog also augmented the tumoricidal activity of human NK cells. Incubation of nylon wool-passed PBL for 1 to 2 hr with the 5'-dephosphorylated core trimer of 2',5'-An boosted natural killing of tumor target cells modestly, but consistently. The cordycepin analog (3'-deoxyadenylate) also augmented NK activity. The optimal concentration both of 2',5'-A3 core and of 2',5'-3'dA3 core was 50 microM, and the optimal time for this effect was 2 hr of treatment. Kinetic analysis revealed that 2',5'-A3 core increased the lytic rate of NK cells by about one-third. This increase was due to an even greater increase (about 50%) in the lytic activity of individual NK cells, coupled with a slight decrease in the number of actual NK effector cells. In contrast, 3',5'-A3 core did not increase NK activity even at 300 microM, at which point it was toxic. In addition, to rule out a pro-drug effect as the basis for the boosting of NK activity by 2',5'-A3 core and by 2',5'-3'dA3 core, the effect of adenosine and cordycepin monomers on NK activity was tested. Neither adenosine nor cordycepin, tested at 150 microM (three times the optimal concentration of the trimer cores), boosted NK activity. The addition of 2'-deoxycoformycin (2 microM) had no effect on the actions of adenosine and cordycepin monomers. The data presented here demonstrate that 2',5'-A3 core and its analog 2',5'-3'dA3 core have another IFN-like action, augmentation of NK activity, in addition to inhibiting EBV-induced transformation.     

Acceptor activity of 4-N-acetylcytidine in the synthesis of (3'-5')-internucleotide bond catalyzed by pancreatic nuclease]

Cytidine and 4-N-acetylcytidine were compared as phosphate acceptors in dinucleoside monophosphate synthesis catalyzed by pancreatic ribonuclease with uridine-2',3'-cyclophosphate and cytidine-2',3'-cyclo phosphate as phosphate donors. Because of low solubility of 4-N-acetylcytidine in water, the synthesis was carried out in aqueus-organic media. The results obtained indicate that acetylation of the exoaminogroup of cytidine decreases its acceptor activity. For the first time uridilyl-(3'-5')-4-N-acetylcytidine and cytidilyl-(3'-5')-4-N-acetylcytidine are prepared enzymatically by pancreatic ribonuclease.     

Mouse neuroblastoma adenylate cyclase. Adenosine and adenosine analogues as potent effectors of adenylate cyclase activity.

1. Intact mouse neuroblastoma NS20 cells, in the presence of cyclic adenosine 3':5'-monophosphate (cAMP) phosphodiesterase inhibitor, responded to adenosine (200 muM) and 2-chloroadenosine (200 muM) with a 20-fold increase in intracellular cAMP levels. AMP (200 muM) additions caused only a 3.5-fold cAMP level elevation. ATP, ADP, guanosine, cytidine, uridine, and guanine, all at 200 muM, had no effect on the cAMP level of these cells. 2. Homogenate NS20 adenylate cyclase activity was increased 2.5- to 4-fold by addition of 200 muM adenosine, 2-chloroadenosine, 2-hydroxyadenosine, or 8-methylaminoadenosine. Prostaglandin E1 additions (1.4 muM) produced about an 8-fold stimulation of homogenate cyclase activity. The Km of homogenate cyclase activation by adenosine and 2-chloroadenosine was 67.6 and 6.7 muM, respectively. Addition of 7-deazaadenosine, tolazoline, yohimbine, guanosine, cytosine, guanine, 2-deoxy-AMP, and adenine 9-beta-D-xylopyranoside, all at 200 muM were found to be without effect on homogenate NS20 adenylate cyclase. Two classes of inhibitors of homogenate NS20 adenylate cyclase activity were observed. One class, which included AMP, adenine, and theophylline, blocked 2-chloroadenosine but not prostaglandin E1 stimulation of cyclase. Theophylline was shown to be a competitive inhibitor of 2-chloroadenosine, with a Ki of 35 muM. The second class of inhibitors, which included 2'- and 5'-deoxyadenosine, inhibited unstimulated, 2-chloroadenosine and prostaglandin E1-stimulated homogenate cyclase activity to about the same degree. 3. Activation of NS20 homogenate adenylate cyclase by adenosine appears to be noncooperative. 4. The inhibitory action of putative "purinergic" neurotransmitters is postulated to be due to their effects on adenylate cyclase activity.     

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.     

Structural requirements of (2'-5') oligoadenylate for protein synthesis inhibition in human fibroblasts.

The structural requirements of (2'-5')-oligoadenylic acid (pppA(2'p5'A)x, X greater than or equal to 1 or (2'-5'An) for inhibition of protein synthesis in cells were examined with a modified calcium-coprecipitation technique, using a series of trinucleotide analogs (pppA2'p5'A2'p5'N, N=rC, rG, rU, T, dC, dG, dA). In this system both the degree and the duration of the inhibition of protein synthesis were dependent on the added concentration of (2'-5')A3. Of all the heterotrimers, only the deoxy A derivative was active as an inhibitor of protein synthesis, while the other members of the analog series were found to have no inhibitory effects. In competition experiments between (2'-5')A3 and the non-active analogs, three heterotrimers were shown to reduce the activity of (2'-5')A3 in protein inhibition. In contrast, the dephosphorylated (2'-5')A3 had no inhibitory effect and was not effective in blocking (2'-5')A3. These results indicate that the 5'-terminal triphosphate is important for binding of (2'-5')A3 to the site of (2'-5')An action and the adenine base at the 2'-terminus is important for activating the machinery responsible for protein synthesis inhibition in the cells, most likely the (2'-5')An-activated nuclease.     

Synthesis of branched ribonucleotides

A synthetic strategy for branched ribonucleotides, which have recently been discovered, was described. A fully protected adenosine unit (5) having the tris (4,5-dichlorophthalimido)trityl (CPTr), bis(anilino)phosphoryl (BAP), and bis(phenylthio)phosphoryl (BPTP) groups as the 5'-, 2'-, and 3'-hydroxyl protecting groups, respectively, was synthesized from adenosine by a five-step reaction involving a new method for the 2'-O-phosphorylation by the use of hexaethylphosphorous triamide. The selective deprotection of appropriate protecting groups from 5 followed by stepwise condensation with two different ribonucleoside derivatives (7 and 10) gave a protected branched ribonucleotide (11) via a 3'-phosphorylated 2'-5' dinucleotide (8). Deprotection of 11 and 8 gave a branched trinucleotide (12) and 3'-phosphorylated dinucleotide (13).     

Crystal structures of the complexes of trichosanthin with four substrate analogs and catalytic mechanism of RNA N-glycosidase

Four substrate analogs-nicotinamide adenine dinucleotide, adenylyl (3', 5') guanosine, guanylyl (3',5') adenosine, and adenosine 2', 5'-diphosphate-have been used to prepare the complexes with trichosanthin (TCS), a type I ribosome-inactivating protein that possesses the activity of N-glycosidase. The crystal structures of the complexes have been determined and refined at high resolution. The refined structures show that the N-glycosidic bonds of all the four substrate analogues are hydrolyzed and a common structure is shared by the four complexes, in which only adenine, the product of the enzymatic reaction, is bound in the active center. The structure is compared with those of native trichosanthin and a previously reported trichosanthin-NADPH complex in which the N-glycosidic bond is uncleaved. The structural comparison shows that the conformation of Tyr70 obviously differs from those in the latter two structures, i.e., the side chain of Tyr70 is rotated along its Cbeta-Cgamma bond by approximately 70 degrees. The water molecule found to be preassociated with the N-glycosidic bond in the TCS-NADPH complex structure and proposed to be the water candidate responsible for hydrolyzing the N-glycosidic bond disappears in the trichosanthin-product complex structure. Based on the comparison of the three structures representing the different stages of the enzymatic reaction, the catalytic mechanism of RNA N-glycosidase has been further elucidated. Proteins 2000;39:37-46.     

Interaction of 2',3'-di-O-nitro-5'-(N-ethylcarboxamido)adenosine with adenosine receptors in intestinal smooth muscle

The adenosine derivative, 2'3'-di-O-nitro-(5'-N-ethylcarboxamido)adenosine (DINECA), caused relaxation in several isolated smooth muscle preparations including guinea pig taenia caeci, beef coronary arteries, and rabbit small intestine. In rabbit small intestine the response profile of DINECA action differed from that of established adenosine receptor agonists and, in contrast with the latter, its relaxant effect was only partially reversed by the antagonist 8-p-sulfophenyltheophylline. Concentration-response curves to 5'-(N-ethylcarboxamido)adenosine (NECA), but not those to DINECA, were significantly shifted to the right by 100 microM of 8-sulfophenyltheophylline. Tissues exposed previously to DINECA became refractory to adenosine, an effect not observed with tissues exposed to NECA, suggesting that DINECA became bound to adenosine receptors. Adenylate cyclase from neuroblastoma cells, containing Ra-type adenosine receptors, was stimulated by 2-chloroadenosine and NECA but not by DINECA. The results suggest that most of the smooth muscle relaxant actions of DINECA are not due to interaction with adenosine receptors but are probably due to its function as a nitrate. However, DINECA appears to interact with adenosine receptors, causing long lasting inhibition of adenosine action in rabbit intestine. Such actions may contribute to the overall response to DINECA application in vivo, although lowering of blood pressure due to the high reactivity of the vasculature to nitrates may be the initial and major effect.     

Elevated levels of (2''-5'')oligoadenylic acid polymerase activity in growth-arrested human lymphoblastoid Namalva cells.

(2'-5')Oligoadenylic acid [(2'-5')An] polymerase activity was measured in extracts of human lymphoblastoid cells of the Namalva line cultured under different conditions. Exponentially growing cells had a relatively low polymerase activity level, whereas cells grown to limit density showed elevated levels. When fresh medium was added to growth-arrested cells, (2'-5')An polymerase activity decreased concomitantly with the initiation of active deoxyribonucleic acid synthesis. An increase in polymerase activity level was also observed after exponentially growing cells were transferred from medium containing 20% serum to fresh medium containing 0.2% serum. These cells diminished deoxyribonucleic acid synthesis and remained quiescent until 20% serum was again added. Polymerase activity level decreased as the cells entered into S phase. The addition of the inhibitor of deoxyribonucleic acid synthesis, hydroxyurea, to exponentially growing cells did not increase polymerase level, indicating that cells blocked in S phase and at the G1-S boundary maintained the basal level of this enzyme. Degradation of labeled (2'-5')An was measured in extracts of Namalva cells cultured under different conditions, but no significant differences among degradative activities were observed. Since (2'-5')An polymerase activity is one of the enzymatic activities induced by interferon, we measured interferon titers in Namalva cell medium. Less than 1 reference unit per ml was detected in cells grown under different conditions. Moreover, the increase in (2'-5')An polymerase activity level in cells transferred from 20 to 0.2% serum was not prevented by including anti-lymphoblastoid interferon antibody in the medium. These results suggest that the activity level of (2'-5')An polymerase is regulated in Namalva cells on the basis of the growth status of the cells and that this regulatory mechanism is apparently not activated by interferon.     

3'-Deoxy-3'-fluoro analogs of core trimers of 2-5A: effect on lytic activity of human NK-lymphocytes]

The effect of core trimers, (2'-5')-analogues of oligoadenylic acid containing 9-(3-deoxy-3-fluoro-beta-D-xylofuranosyl)adenine (AF) and 3'-deoxy-3'-fluoroadenosine (AF) in various positions of the oligomer chain, on the lytic activity of human natural killer cells (NK cells) was studied in three different ways. The cellular cytotoxicity was determined using a highly sensitive nonradioactive approach employing a chelate europium-diethylenetriamino-pentaacetic acid complex (Eu-DTPA). It was shown that all fluorodeoxyanalogues enhance the lytic activity of intact NK lymphocytes, which follows from the lysis rate constant k2. At the same time, the substitution of either the central adenosine fragment or (to a greater extent) the 5'-terminal residue of (2'-5')A3 with AF causes a decrease in the number of active NK cells, which, unlike the case of the natural core trimer, leads to a loss of the capacity to increase the activity of NK. By contrast, isomeric ribo-analogues. (2'-5')(AF)A2 and (2'-5')A(AF)A, and trimers with the 2'(3')-terminal nucleotide substituted by AF or AF increased the activity of NK cells with an effectiveness close to or higher than the natural trimer (2'-5')A3. Inasmuch as isomeric xylo- and ribo-3'-deoxy-3'-fluoroanalogues of (2'-5')A3 are stereochemically modified oligomers, the data unambiguously suggest that the spatial structure of these trimers affects the increase in the lytic activity of NK cells.     

Covalent labeling of adenylyl cyclase cytosolic domains with gamma-methylimidazole-2',5'-dideoxy-[gamma-(32)P]3'-ATP and the mechanism for P-site-mediated inhibition

A truncated first cytosolic domain of type V adenylyl cyclase (VC(1)) and a truncated second cytosolic domain of type II adenylyl cyclase (IIC(2)) were used alone and in the readily reversible complex (VC(1).IIC(2)) to evaluate interactions with each other and with reversible and irreversible P-site ligands. Enzyme activity was used to assess formation and dissolution of VC(1).IIC(2). The data suggest that binding of 2',5'-dideoxy-3'-ATP to VC(1) and IIC(2) prevented formation of VC(1).IIC(2) and that 2',5'-dideoxy-3'-ATP dissociation occurred slowly. To enable configuration specific cross-linking to the catalytic site, 2',5'-dideoxyadenosine 3'-[gamma-(1-methylimidazole)-triphosphate] (gamma-MetIm-2', 5'-dd-3'-ATP) and 2',5'-dd-adenosine 3'-(gamma-azidoanilido)-triphosphate (gamma-azidoanilido-2', 5'-dd-3'-ATP) were synthesized, the former also as its gamma-(32)P-labeled analog. gamma-Azidoanilido-2',5'-dd-3'-ATP exhibited an inhibitory potency comparable with that of 2', 5'-dd-3'-ATP. gamma-MetIm-2',5'-dd-[gamma-(32)P]3'-ATP labeled the individual VC(1) and IIC(2) domains comparably and covalently to approximately 20% within 1 h. Formation of VC(1).IIC(2) resulted in reduced labeling of VC(1) but enhanced labeling of IIC(2). The data imply that formation of the catalytically active VC(1).IIC(2) complex affects the interaction of each domain with the 2', 5'-dd-3'-ATP, the binding of which also affects the interaction between the two cytosolic domains, leading to a pseudo-irreversible inhibition.     

Differentiation of a human monocyte-like cell line by (2′–5′) oligoisoadenylate

Treatment of a human monocyte-like cell line (U-937) by (2'-5')ApApA, the 5' dephosphorylated product of (2'-5')oligo-isoadenylate [oligo(A)] synthetase, an interferon-induced enzyme, was able to induce differentiation, mimicking the effect of interferon treatment. Treatment of U-937 cells with (2'-5')ApApA resulted in morphologic changes, new (monocyte-associated) membrane antigen expression, and acquisition of the capacity to mediate antibody-dependent cellular cytotoxicity (ADCC). (2'-5')ApA and (3'-5')ApApA were without effect. A myeloid cell line (HL-60) which differentiates in response to other agents, but not to alpha-interferon, was not able to differentiate in response to (2'-5')ApApA, despite the ability of interferon to induce (2'-5')oligo (A) synthetase.     

Double-stranded RNA causes synthesis of 2',5'-oligo(A) and degradation of messenger RNA in interferon-treated cells

Interferon-treated HeLa cells were incubated with [3H]uridine to label mRNA and were then exposed to the double-stranded RNA poly(inosinic acid).poly(cytidylic acid) (In.Cn). The incubation with In.Cn greatly enhanced the decay of mRNA. When the cells were incubated in this way in the presence of cycloheximide, which blocks ribosome movement along mRNA, extensive polysome degradation was detected in interferon-treated cells. Products of degradation of mRNA were recovered from monosomes which were presumably formed as a result of endonucleolytic breaks of mRNA. This endonucleolytic activity was correlated with the formation of 2',5'-oligo(A) by an enzyme induced by interferon and activated by double-stranded RNA; the 2',5'-oligo(A) was previously shown to activate an endonuclease in cell extracts. The 2',5'-oligo(A) levels in cells were measured by a competition-binding assay. Details of the procedure used are described, including synthesis of highly radioactive (2'-5')pppA3[32P]cytidine 3',5'-diphosphate, separation of 2',5'-oligo(A) binding from degrading activities, and specificity of the assay.     

Effects of 2'-O-(trifluoromethyl)adenosine on oligodeoxynucleotide hybridization and nuclease stability.

The synthesis and properties of oligodeoxynucleotides (ODNs) containing 2'- O -(trifluoromethyl)adenosine (2) are described. 2'- O -(Trifluoromethyl)adenosine (2) or N 6-(benzoyl)-2'- O -(trifluoromethyl)adenosine (6) was obtained in 22 or 32% yield by treating 2'- O -[(methylthio)thiocarbonyl]-3',5'- O -(1,1,3, 3-tetraisopropyldisiloxane-1,3-diyl)(TIPDS)adenosine (4) or N 6, N 6-(dibenzoyl)-2'- O -[(methylthio)thiocarbonyl]-3',5'- O -(TIPDS)-adenosine (5), respectively, with pyridinium poly-(hydrogen fluoride) in the presence of 1,3-dibromo-5,5-dimethylhydantoin. Nucleoside 2 was incorporated into DNA hexadecamers. ODNs that contained 2 reduced the thermal stability of duplexes with their complementary DNAs but increased the thermal stability of duplexes with their complementary RNAs. Furthermore, ODNs containing 2 were slightly more resistant to snake venom phosphodiesterase than an unmodified ODN.     

Introduction of alkynyl chains on C-8 of adenosine led to very selective antagonists of the A(3) adenosine receptor.

Some 8-alkynyladenosines were synthesized and evaluated for their adenosine receptor activity, utilizing radioligand binding studies (A(1), A(2A), A(3)) or adenylyl cyclase activity assays (A(2B)). Furthermore, the maximal induction of guanosine 5'-(gamma-thio)triphosphate ([35S]GTPgammaS) binding to G proteins and the inhibition of NECA-stimulated binding, in membranes of CHO cells which express the human A(3) receptor, were used to determine the intrinsic activity of these nucleosides at the A(3) adenosine receptor. The results showed that these new adenosine derivatives are very selective ligands for the A(3) receptor subtype and behave as adenosine antagonists, since they do not stimulate basal [35S]GTPgammaS binding, but inhibit NECA-stimulated binding. This is the first report that adenosine derivatives, with unmodified ribose moiety, are adenosine receptor antagonists.