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.     

Inhibition of chloramphenicol binding to Escherichia coli 70S ribosomes by 2'(3')-O-aminoacyl-dinucleoside phosphates derived from the aminoacyl-tRNA acceptor terminus.

The effect of 2' and 3'-O-aminoacyl-dinucleoside phosphates cytidylyl(3'-5')-2'(3')-O-L-phenyl-alanyladenosine (I), cytidylyl(3'-5')-3'-deoxy-2'-O-L-phenylalanyladenosine (IIa), cytidylyl(3'-5')-2'-deoxy-3'-O-L-phenylalanyladenosine (IIIa), cytidylyl(3'-5')-3'-deoxy-2'-O-glycyladenosine (IIb), cytidylyl(3'-5')-2'-deoxy-3'-O-glycyladenosine (IIIb), cytidylyl(3'-5')-3'-deoxy-2'-O-L-leucyladenosine (IIc), cytidylyl(3'-5')-2'-deoxy-3'-O-L-leucyladenosine (IIIc), cytidylyl(3'-5')-3'-O-L-phenylalanyladenosine (IIId) as analogs of the 2'(3')-aminoacyl-tRNA termini, on chloramphenicol binding to 70S Excherichia coli ribosomes was investigated. The association constants (Kb) of the investigated compounds were determined by the equilibrium dialysis method. Based on the constancy of Kb over the range of inhibitor concentration, it was determined that the binding site of the 2' isomers IIa-IIc overlaps with the chloramphenicol site, whereas the variability of Kb for the 3' isomers IIIb, IIIc and especially IIIa seems to indicate that they do not achieve a complete fit. The consistently higher values of the Kb values for the 3' isomers IIIa-IIIc relative to that of the 2' isomers IIa-IIc also indicate a stabilization of the binding of the former due to a specific interaction between its amino acid portion and a ribosomal site.     

Synthesis and separation of diastereomers of uridine 2',3'-cyclic boranophosphate

The first boron-containing 2',3'-cyclic phosphate-modified analogue, uridine 2',3'-cyclic boranophosphate (2',3'-cyclic-UMPB), was synthesized. 5'-O-Protected uridine was cyclophosphorylated by diphenyl H-phosphonate to yield uridine 2',3'-cyclic H-phosphonate, which upon silylation followed by boronation and subsequent acid treatment gave 2',3'-cyclic-UMPB in high yield. The two diastereomers of 2',3'-cyclic-UMPB were separated by HPLC. An alternative method for synthesis of uridine 2',3'-cyclic phosphorothioate (2',3'-cyclic-UMPS) via H-phosphonate was also described.     

Guanosine tetraphyosphate and its analogues. Chemical synthesis of guanosine 3',5'-dipyrophosphate, deoxyguanosine 3',5'-dipyrophosphate, guanosine 2',5'-bis(methylenediphosphonate), and guanosine 3',5'-bis(methylenediphosphonate).

A new procedure for the synthesis of the pyrophosphate bond has been employed in the preparation of nucleoside dipyrophosphates from nucleoside 3',5'-diphosphates. The method makes use of a powerful phosphorylating agent generated in a mixture of cyanoethyl phosphate, dicyclohexylcarbodiimide, and mesitylenesulfonyl chloride in order to avoid possible intramolecular reactions between the two phosphate groups on the sugar ring. That such reactions can readily occur was shown by the facile cyclization of deoxyguanosine 3',5'-diphosphate to P1,P2-deoxyguanosine 3',5'-cyclic pyrophosphate in the presence of dicyclohexylcarbodiimide alone. The phosphorylation reagent was initially tested in the conversion of deoxyguanosine 3',5'-diphosphate to the corresponding 3',5'-dipyrophosphate and was then used to phosphorylate 2'-O-(alpha-methoxyethyl)guanosine 3',5'-diphosphate, which had been prepared from 2'-O-(alpha-methoxyethyl)guanosine. In the latter case, the addition of the two beta phosphate groups was accomplished in 40% yield. Removal of the methoxyethyl group from the phosphorylated product gave guanosine 3',5'-dipyrophosphate, which was shown to be identical with guanosine tetraphosphate prepared enzymatically from a mixture of GDP and ATP. A modification of published procedures was also necessary to effect the synthesis of guanosine bis(methylenediphosphonate). Guanosine was treated with methylenediphosphonic acid and dicyclohexylcarbodiimide in the absence of added base. The product consisted of a mixture of guanosine 2',5' - and 3',5'-bis(methylenediphosphonate), which was resolved by anion-exchange chromatography. The 2',5' and 3',5' isomers are interconvertible at low pH, with the ultimate formation of an equilibrium mixture having a composition ratio of 2:3. The predominant constituent of this mixture has been unequivocally identified as the 3',5' isomer by synthesis from 2'-O-tetrahydropyranylguanosine.     

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.     

Specificities of rabbit anti-(2',5')oligoadenylate antibodies towards phosphorothioate and seco analogs of oligoadenylates.

Rabbit antibodies to 2',5'-linked triadenylate were prepared by immunization with (2',5')A3 conjugated via the 2'3'-levulinic group, (2'5')A3-Lev, to BSA. New radioimmunoassay for (2',5')oligoadenylates was developed using 125I thyrosine labeled derivative of (2',5')A3-Lev. Reactivity of antibodies with phosphorothioate and seco analogs of oligoadenylates was studied. It was found that (i) stereospecific substitution of the diastereotopic oxygens with sulfur in the internucleotide phosphodiester linkages changes the immunoreactivity of such analogs; (ii) the seco analogs of oligoadenylates display in some cases a rather high reactivity.     

Expression of the 2',3'-cAMP-adenosine pathway in astrocytes and microglia

Many organs express the extracellular 3',5'-cAMP-adenosine pathway (conversion of extracellular 3',5'-cAMP to 5'-AMP and 5'-AMP to adenosine). Some organs release 2',3'-cAMP (isomer of 3',5'-cAMP) and convert extracellular 2',3'-cAMP to 2'- and 3'-AMP and convert these AMPs to adenosine (extracellular 2',3'-cAMP-adenosine pathway). As astrocytes and microglia are important participants in the response to brain injury and adenosine is an endogenous neuroprotectant, we investigated whether these extracellular cAMP-adenosine pathways exist in these cell types. 2',3'-, 3',5'-cAMP, 5'-, 3'-, and 2'-AMP were incubated with mouse primary astrocytes or primary microglia for 1 h and purine metabolites were measured in the medium by mass spectrometry. There was little evidence of a 3',5'-cAMP-adenosine pathway in either astrocytes or microglia. In contrast, both cell types converted 2',3'-cAMP to 2'- and 3'-AMP (with 2'-AMP being the predominant product). Although both cell types converted 2'- and 3'-AMP to adenosine, microglia were five- and sevenfold, respectively, more efficient than astrocytes in this regard. Inhibitor studies indicated that the conversion of 2',3'-cAMP to 2'-AMP was mediated by a different ecto-enzyme than that involved in the metabolism of 2',3'-cAMP to 3'-AMP and that although CD73 mediates the conversion of 5'-AMP to adenosine, an alternative ecto-enzyme metabolizes 2'- or 3'-AMP to adenosine.     

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.     

3' processing of human pre-U2 small nuclear RNA: a base-pairing interaction between the 3' extension of the precursor and an internal region.

The spliceosomal small nuclear RNAs U1, U2, U4, and U5 are transcribed by RNA polymerase II as precursors with extensions at their 3' ends. The 3' processing of these pre-snRNAs is not understood in detail. Two pathways of pre-U2 RNA 3' processing in vitro were revealed in the present investigation by using a series of human wild-type and mutant pre-U2 RNAs. Substrates with wild-type 3' ends were initially shortened by three or four nucleotides (which is the first step in vivo), and the correct mature 3' end was then rapidly generated. In contrast, certain mutant pre-U2 RNAs displayed an aberrant 3' processing pathway typified by the persistence of intermediates representing cleavage at each internucleoside bond in the precursor 3' extension. Comparison of the wild-type and mutant pre-U2 RNAs revealed a potential base-pairing interaction between nucleotides in the precursor 3' extension and a sequence located between the Sm domain and stem-loop III of U2 RNA. Substrate processing competition experiments using a highly purified pre-U2 RNA 3' processing activity suggested that only RNAs capable of this base-pairing interaction had high affinity for the pre-U2 RNA 3' processing enzyme. The importance of this postulated base-pairing interaction between the precursor 3' extension and the internal region between the Sm domain and stem-loop III was confirmed by the results obtained with a compensatory substitution that restores the base pairing, which displayed the normal 3' processing reaction. These results implicate a precursor-specific base-paired structure involving sequences on both sides of the mature cleavage site in the 3' processing of human U2 RNA.     

Synthesis and studies of 3'-C-trifluoromethyl nucleoside analogues bearing adenine or cytosine as the base

3'-Deoxy-3'-C-CF3, 2',3'-dideoxy-3'-C-CF3 and 2',3'-unsaturated-3'-C-CF3 nucleoside derivatives of adenosine and cytidine have been synthesized. All these derivatives were prepared by glycosylation of adenine and uracil with a suitable peracylated 3-trifluoromethyl sugar precursor. The resulting protected nucleosides were subject to appropriate chemical modifications to afford the target nucleoside derivatives. Additionally, the chemical stability in acidic and neutral media of the 2',3'-dideoxy-3'-C-CF3 and 2',3'-unsaturated-3'-C-CF3 nucleoside derivatives of adenosine was compared to that of their parent nucleosides 2',3'-dideoxyadenosine (ddA) and 2',3'-dideoxy-2',3'-didehydroadenosine (d(4)A). Our results confirm that addition of a trifluoromethyl group at C-3' on such nucleoside derivatives appears to confer increased chemical stability toward acid-catalyzed cleavage of the glycosidic bond comparatively to their parent counterparts. When evaluated for their antiviral activity in cell culture experiments, two compounds, namely, 2',3'-dideoxy-3'-C-CF3-adenosine and 2',3'-dideoxy-2',3'-didehydro-3'-C-CF3-cytidine exhibited moderate anti-HBV activity with EC50 values of 10 and 5 microM, respectively.     

Mitochondrial gene transfer in pieces: fission of the ribosomal protein gene rpl2 and partial or complete gene transfer to the nucleus.

Mitochondrial genes are usually conserved in size in angiosperms. A notable exception is the rpl2 gene, which is considerably shorter in the eudicot Arabidopsis than in the monocot rice. Here, we show that a severely truncated mitochondrial rpl2 gene (termed 5' rpl2) was created by the formation of a premature stop codon early in eudicot evolution. This 5' rpl2 gene was subsequently lost many times from the mitochondrial DNAs of 179 core eudicots surveyed by Southern hybridization. The sequence corresponding to the 3' end of rice rpl2 (termed 3' rpl2) has been lost much more pervasively among the mitochondrial DNAs of core eudicots than has 5' rpl2. Furthermore, where still present in these mitochondrial genomes, 3' rpl2 always appears to be a pseudogene, and there is no evidence that 3' rpl2 was ever a functional mitochondrial gene. An intact and expressed 3' rpl2 gene was discovered in the nucleus of five diverse eudicots (tomato, cotton, Arabidopsis, soybean, and Medicago). In the first three of these species, 5' rpl2 is still present in the mitochondrion, unlike the two legumes, where both parts of rpl2 are present in the nucleus as separate genes. The full-length rpl2 gene has been transferred intact to the nucleus in maize. We propose that the 3' end of rpl2 was functionally transferred to the nucleus early in eudicot evolution, and that this event then permitted the nonsense mutation that gave rise to the mitochondrial 5' rpl2 gene. Once 5' rpl2 was established as a stand-alone mitochondrial gene, it was then lost, and was probably transferred to the nucleus many times. This complex history of gene fission and gene transfer has created four distinct types of rpl2 structures or compartmentalizations in angiosperms: (1) intact rpl2 gene in the mitochondrion, (2) intact gene in the nucleus, (3) split gene, 5' in the mitochondrion and 3' in the nucleus, and (4) split gene, both parts in the nucleus.     

Effect of cyclic nucleotides and isopropylnoradrenaline on oxygen tension and absorption]

Isopropylnoradrenaline (ISO), 3',5'-AMP and dibutyryl-3',5'-AMP decreased the oxygen tension (pO2) in the liver and the spleen and increased the body oxygen consumption (VO2). Time dynamics of these two effects was closely correlated for ISO and 3',5'-AMP. An increase of heat output was not accompanied by any significant changes in the respiration coefficient. Pempidine and dihydroergotamine failed to prevent 3',5'-AMP effects; inderal somewhat decreased these effects. Apparently, the catecholamine influence upon pO2 was a result of the VO2 increase through 3'5'-AMP effects are largely direct, but they include the in vivo and beta-receptor component; 2',3'-AMP decreased pO2 and VO2.     

Repressible extracellular phosphodiesterases showing cyclic 2'',3''- and cyclic 3'',5''-nucleotide phosphodiesterase activities in Neurospora crassa.

Two molecular species of repressible extracellular phosphodiesterases showing cyclic 2',3'- and cyclic 3',5'-nucleotide phosphodiesterase activities were detected in mycelial culture media of wild-type Neurospora crassa and purified. The two molecular species were found to be monomeric and polymeric forms of an enzyme constituted of identical subunits having molecular weights of 50,000. This enzyme had the same electrophoretic mobility as repressible acid phosphatase. The enzyme designated repressible cyclic phosphodiesterase showed pH optima of 3.2 to 4.0 with a cyclic 3',5'-AMP substrate and 5.0 to 5.6 with a cyclic 2',3'-AMP substrate. Repressible cyclic phosphodiesterase was activated by MnCl2 and CoCl2 with cyclic 2',3'-AMP as substrate and was slightly activated by MnCl2 with cyclic 3',5'-AMP. The enzyme hydrolyzed cyclic 3',5'- and cyclic 2',3'-nucleotides, in addition to bis-rho-nitrophenyl phosphate, but not certain 5' -and 3'-nucleotides. 3'-GMP and 3'-CMP were hydrolyzed less efficiently. Mutant strains A1 (nuc-1) and B1 (nuc-2), which cannot utilize RNA or DNA as a sole source of phosphorus, were unable to produce repressible cyclic phosphodiesterase. The wild type (74A) and a heterocaryon between strains A1 and B1 produced the enzyme and showed growth on orthophosphate-free media containing cyclic 2',3'-AMP or cyclic 3',5'-AMP, whereas both mutants showed little or no growth on these media.     

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.     

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.     

Interplay between AAUAAA and the trans-splice site in processing of a Caenorhabditis elegans operon pre-mRNA

About half of Caenorhabditis elegans genes have a 1-2 bp mismatch to the canonical AAUAAA hexamer that signals 3' end formation. One rare variant, AGUAAA, is found at the 3' end of the mai-1 gene, the first gene in an operon also containing gpd-2 and gpd-3. When we expressed this operon under heat shock control, 3' end formation dependent on the AGUAAA was very inefficient, but could be rescued by a single bp change to create a perfect AAUAAA. When AGUAAA was present, most 3' ends formed at a different site, 100 bp farther downstream, right at the gpd-2 trans-splice site. Surprisingly, 3' end formation at this site did not require any observable match to the AAUAAA consensus. It is possible that 3' end formation at this site occurs by a novel mechanism--trans-splicing-dependent cleavage--as deletion of the trans-splice site prevented 3' end formation here. Changing the AGUAAA to AAUAAA also influenced the trans-splicing process: with AGUAAA, most of the gpd-2 product was trans-spliced to SL1, rather than SL2, which is normally used at downstream operon trans-splice sites. However, with AAUAAA, SL2 trans-splicing of gpd-2 was increased. Our results imply that (1) the AAUAAA consensus controls 3' end formation frequency in C. elegans; (2) the AAUAAA is important in determining SL2 trans-splicing events more than 100 bp downstream; and (3) in some circumstances, 3' end formation may occur by a trans-splicing-dependent mechanism.     

Effect of 2'-5' phosphodiesters on DNA transesterification by vaccinia topoisomerase

Vaccinia topoisomerase forms a covalent DNA-(3'-phosphotyrosyl)-enzyme intermediate at a pentapyrimidine target site 5'-CCCTTp downward arrow in duplex DNA. By introducing single 2'-5' phosphodiesters in lieu of a standard 3'-5' phosphodiester linkage, we illuminate the contributions of phosphodiester connectivity to DNA transesterification. We find that the DNA cleavage reaction was slowed by more than six orders of magnitude when a 2'-5' linkage was present at the scissile phosphodiester (CCCTT(2')p downward arrow(5')A). Thus, vaccinia topoisomerase is unable to form a DNA-(2'-phosphotyrosyl)-enzyme intermediate. We hypothesize that the altered geometry of the 2'-5' phosphodiester limits the ability of the tyrosine nucleophile to attain a requisite, presumably apical orientation with respect to the 5'-OH leaving group. A 2'-5' phosphodiester located to the 3' side of the cleavage site (CCCTTp downward arrowN(2')p(5')N) reduced the rate of transesterification by a factor of 500. In contrast, 2'-5' phosphodiesters at four other sites in the scissile strand (TpCGCCCTpT downward arrowATpTpC) and five positions in the nonscissile strand (3'-GGGpApApTpApA) had no effect on transesterification rate. The DNAs containing 2'-5' phosphodiesters were protected from digestion by exonuclease III. We found that exonuclease III was consistently arrested at positions 1 and 2 nucleotides prior to the encounter of its active site with the modified 2'-5' phosphodiester and that the 2'-5' linkage itself was poorly hydrolyzed by exonuclease III.     

Properties of a cyclic 3'5'-nucleotide phosphodiesterase from Vigna mungo

Cyclic AMP phosphodiesterase (PDE) partially purified from roots of Vigna mungo exhibited optimum activity at pH 5.5 to 6.0 and maximum enzyme activity at 50 degrees C. Levels of PDE activity in roots remained relatively constant from the first to the eleventh day after germination; on the twelfth day there was a 400% increase in PDE activity. The enzyme was stable for at least 48 hours at 28 degrees C, retaining 92% of its original activity. Plant growth hormones including gibberellic acid, indoleacetic acid and kinetin at 1.0 and 10.0 microM concentrations did not have any significant effect on enzyme activity. Nucleotides tested including cyclic 2'3' AMP, cyclic 2'3' GMP completely abolished enzyme activity at 1.0mM while cyclic 3'5' GMP, cyclic 3'5' GMP, 2'deoxy 5' ATP, 2'deoxy 5'GTP and 5'ADP were also inhibitory to the enzyme. The enzyme was stimulated by Mg2+, Fe2+ and NH4+ while Cu2+ and Fe3+ were inhibitory. Theophylline, caffeine, phosphate, pyrophosphate and EDTA were inhibitory to the enzyme.     

Metabolism and anti-human immunodeficiency virus-1 activity of 2-halo-2',3'-dideoxyadenosine derivatives

Both 2',3'-dideoxyadenosine and 2',3'-dideoxyinosine have been shown (Mitsuya, H., and Broder, S. (1987) Nature 325, 773-778) to have in vitro activity against the human immunodeficiency virus-1 (HIV). However, these dideoxynucleosides may be catabolized by human T cells, even when adenosine deaminase is inhibited by deoxycoformycin. To overcome this problem, we have synthesized the 2-fluoro-, 2-chloro-, and 2-bromo-derivatives of 2',3'-dideoxyadenosine. The metabolism and anti-HIV activity of the 2-halo-2',3'-dideoxyadenosine derivatives and of 2',3'-dideoxyadenosine were compared. The 2-halo-2',3'-dideoxyadenosine derivatives were not deaminated significantly by cultured CEM T lymphoblasts. Experiments with 2-chloro-2',3'-dideoxyadenosine showed that the T cells converted the dideoxynucleoside to the 5'-monophosphate, 5'-diphosphate, and 5'-triphosphate metabolites. At concentrations lower than those producing cytotoxicity in uninfected cells (3-10 microM), the 2-halo-2',3-dideoxyadenosine derivatives inhibited the cytopathic effects of HIV toward MT-2 T lymphoblasts, and retarded viral replication in CEM T lymphoblasts. Experiments with a deoxycytidine kinase-deficient mutant CEM T cell line showed that this enzyme was necessary for the phosphorylation and anti-HIV activity of the 2-chloro-2',3'-dideoxyadenosine. In contrast, 2',3'-dideoxyadenosine was phosphorylated by the deoxycytidine kinase-deficient mutant and retained anti-HIV activity in this cell line. Thus, the 2-halo derivatives of 2',3'-dideoxyadenosine, in contrast to 2',3'-dideoxyadenosine itself, are not catabolized by T cells. Their anti-HIV and anti-proliferative activities are manifest only in cells expressing deoxycytidine kinase. The in vivo implications of these results for anti-HIV chemotherapy are discussed.