Cyclic Nucleoside Research Articles

Effect of Mutations at the Monomer-Monomer Interface of cAMP Receptor Protein on Specific DNA Binding

To determine the thermodynamic role of binding of an operon to cAMP receptor protein (CRP) in the activation of transcription, isothermal titration calorimetry measurements were performed on the binding of three 40-base pair DNA sequences to the cyclic nucleoside complexes of CRP and its mutants at 296 K. The three 40-base pair sequences consisted of a consensus DNA (conDNA) duplex derived from the CRP-binding site sequences of the operons activated by CRP and two DNA sequences based on the CRP-binding site sequences of the lac operon (lacDNA) and of the gal operon (galDNA). The mutants of CRP consisted of a T127L mutant, a S128A mutant, and a mutant containing both mutations (CRP*) which not only alter the transcriptional activity of the CRP complexes but also are involved in the monomer-monomer interfacial interactions of the CRP dimer. The binding reactions of the DNA duplexes to the fully cNMP-ligated CRP-mutant complexes were endothermic with binding constants as high as 6.6 +/- 1.1 x 10(6) M-1 (conDNA.CRP(cAMP)2). ConDNA binding to the unligated T127L and CRP* mutants was observed as well as conDNA and lacDNA binding to CRP with cAMP bound to only one monomer. The reduction of the binding constants with increase in KCl concentration indicated the formation of two ion pairs for the cAMP-ligated CRP and S128A complexes and four ion pairs for the cAMP-ligated T127L and CRP* complexes. Reduction of the DNA binding constants upon substitution of D2O for H2O in the buffer, the large heat capacity changes, and the enthalpy-entropy compensation exhibited by the binding reactions indicate the importance of dehydration in the binding reaction. Small angle neutron scattering measurements on the lacDNA.CRP(cAMP)2 complex in D2O/H2O mixtures show that the DNA is bent around the cAMP-ligated protein in solution.

Constrained analogues of 2′-nor cyclic nucleoside monophosphates

The synthesis of constrained analogues of 2′-nor-cyclic nucleosides monophosphates containing a thiomethylene tether was readily accomplished from the oxathiolane intermediate 4. The uracil, cytosine, and 5-bromocytosine spirophosphate analogues 22, 16, and 25 were inhibitory to HCMV replication in Flow 2002 cells.

Synthesis and antiviral evaluation of 4′-hydroxymethyl-2′, 3′-dideoxy-3′-thianucleosides and their cyclic monophosphates

Novel 4′-hydroxymethyl-2′,3′-dideoxy-3′-thianucleosides and their cyclic monophosphates have been synthesized and evaluated for anti-HIV activity. This is the first report of the use of the phosphorodiamidite methodology to prepare cyclic nucleoside monophosphates.

Effect of cAMP Binding Site Mutations on the Interaction of cAMP Receptor Protein with Cyclic Nucleoside Monophosphate Ligands and DNA

Although cAMP binding to wild type cAMP receptor protein (CRP) induces specific DNA binding and activates transcription, cyclic nucleoside monophosphate (cNMP) binding to the CRP mutant Ser128 --> Ala does not, whereas the double CRP mutant Thr127 --> Leu/Ser128 --> Ala activates transcription even in the absence of cNMP. Isothermal titration calorimetry measurements on the cNMP binding reactions to the S128A and T127L/S128A mutants show that the reactions are mainly entropically driven as is cAMP binding to CRP. In contrast to cAMP binding to CRP, the binding reactions are noncooperative and exothermic with binding enthalpies (DeltaHb) ranging from -23.4 +/- 0.9 kJ mol-1 for cAMP binding to S128A at 39 degrees C to -4.1 +/- 0.6 kJ mol-1 for cAMP binding to T127L/S128A at 24 degrees C and exhibit enthalpy-entropy compensation. To account for the inactivity of the S128A mutant, in vitro and in vivo DNA binding experiments were performed on the cAMP-ligated S128A mutant. The cAMP-ligated S128A mutant binds to the consensus DNA binding site with approximately the same affinity as that of cAMP-ligated CRP but forms a different type of complex, which may account for loss of transcriptional activity by the mutant. Energy minimization computations on the cAMP-ligated S128A mutant show that amino acid conformational differences between S128A and CRP occur at Ser179, Glu181, and Thr182 in the center of the DNA binding site, implying that these conformational changes may account for the difference in DNA binding.

Synthesis of Methylene-Bridged Analogues of Nicotinamide Riboside, Nicotinamide Mononucleotide and Nicotinamide Adenine Dinucleotide

Abstract 5-O-tert-Butyldimethylsilyl-1,2-O-isopropylidene-3(R)-(nicotinamid-2-ylmethyl)-α-D-ribofuranose (11a) and −3(R)-(nicotinamid-6-ylmethyl)-α-D-ribofuranose (11b) were prepared by condensation of 5-O-tert-butyldimethylsilyl-1,2-O-isopropylidene-α-D-erythro-3-pentulofuranose (10) with lithiated (LDA) 2-methylnicotinamide and 6-methylnicotinamide, respectively, and then deprotected to give 1,2-O-isopropylidene-3-(R)-(nicotinamid-2-ylmethyl)-α-D-ribofuranose(12a) and 1,2-O-isopropylidene-3(R)-(nicotinamid-6-ylmethyl)-α-D-ribofuranose (12b). Benzoylation as well as phosphorylation of compounds 12 afforded the corresponding 5-O-benzoate (13b) and 5-O-monophosphates (14a and 14b). Treatment of 13b with CF3COOH/H2O caused 1,2-de-O-isopropylidenation with simultaneous cyclization to the corresponding methylene-bridged cyclic nucleoside - 3′,6-methylene-1-(5-O-benzoyl-β-D-ribofuranose)-3-carboxamidopyridinium trifluoro-acetate (8b) - restricted to the “anti” conformation. In a similar manner compounds 14a an...

Synthesis and antiviral evaluation of quinazoline, thieno‐[2,3‐d]pyrimidine, and lumazine analogues of 3′‐fluoro‐3′‐deoxythymidine (FLT)

Abstract2,4(1H,3H)‐Quinazolinediones 3a–c, lumazine (3d) and thieno[2,3‐d]pyrimidine‐2,4(1H,3H)‐dione (8) were silylated and condensed with methyl 2,3‐dideoxy‐3‐fluoro‐5‐O‐(4‐phenylbenzoyl)‐β‐D‐erythro‐pentofuranoside (2) by using trimethylsilyl triflate as a catalyst to afford the corresponding cyclic nucleosides 4 and 9 and acyclic nucleosides 5 and 10. Removal of the protecting group from the glycon moiety was achieved in good yields by treatment with sodium methoxide in methanol at room temperature. The new FLT analogues were devoid of activity against HIV‐1 and HSV‐1.

Type I benzophenone-mediated nucleophilic reaction of 5'-amino-2',5'-dideoxyguanosine. A model system for the investigation of photosensitized formation of DNA-protein cross-links.

5'-Amino-2',5'-dideoxyguanosine has been synthesized in order to investigate the intramolecular reactivity of an amino group toward the guanine radical produced by type I photosensitization mechanism. Benzophenone-mediated photosensitization of 5'-amino-2',5'-dideoxyguanosine in aerated aqueous solution results in the formation of a predominant cyclic nucleoside together with an unstable nucleoside precursor. The two modified nucleosides have been isolated by reverse phase high performance liquid chromatography and characterized by spectroscopic measurements including 13C and 1H NMR, fast atom bombardment mass spectroscopy, and UV absorption. The stable photoproduct has been identified as 9-oxa-2,4-diazabicyclo[4.2.1]non-2-en-7-ol, 3-amino- (1R-exo), whereas its precursor has been assigned as acetic acid, [(7-hydroxy-9-oxa-2,4-diazabicyclo[4.2.1]non-2-en-3-yl)amino]oxo- (1R-exo). A reaction mechanism, involving nucleophilic addition of the sugar amino group to guanine radical intermediates, is proposed to explain the formation of the two photoproducts.

Synthesis of cyclic and acyclic nucleoside analogues having a thiophene or dihydrothiophene ring fused to the d side of an uracil

AbstractSynthesis of cyclic and acyclic nucleosides was achieved by alkylation of various new aglycones following the Vorbrüggen and Niedballa's procedure [6]. The analytical results allowed us to conclude that the alkylation occured on N1 site and led to the β anomeric form for the cyclic nucleosides.

Synthesis of (1R,3R,5S)-1-Amino-3-(hydroxymethyl)bicyclo[3.1.0]hexane as a Precursor for the Synthesis of Carbocyclic Nucleosides

The stereocontrolled synthesis has been achieved of a 1,5-methano-1- amino-5-(hydroxymethyl)-cyclopentane, a potential component for carbo- cyclic nucleosides. Stereocontrol was manifest by converting (R)-2-(( benzyloxy)ethyl)oxirane specifically to (2S,3S)-2-amino-2,3-methanoadi- pate through a series of lactones. This aminocyclopropanecarboxylate was then cyclized to the corresponding cyclopentanone ester. Reduction of the ketone, elimination, and hydrogenation of the double bond led primarily to the cyclopentane with the amino and ester groups trans (9/1). Enolization followed by an ammonium chloride quench then inver- ted this to a mixture in which the cis isomer was dominant (4/1). Simple functional group manipulation then gave the target (1R,3R,5S)-1-amino- 3-(hydroxymethyl)bicyclo[3.1.0]hexane

Purified trichosanthin (GLQ223®) exacerbation of indirect HIV-associated neurotoxicity in vitro

A formulated preparation of trichosanthin (GLQ223, Pharmaceutical Development Group, Genelabs Inc., Redwood City, California, USA) has been shown to selectively inhibit HIV replication in vitro in lymphocytes and macrophages. In view of recent anecdotal reports of central nervous system (CNS) complications associated with trichosanthin use in some HIV-infected patients, we evaluated any potential drug effects leading to neurotoxicity using a human brain cell aggregate model. Brain cell aggregate cultures were incubated with dilutions of purified trichosanthin alone (trichosanthin), supernatants of HIV-infected macrophage cultures (S-HIV), supernatants of uninfected macrophage cultures (S-U), supernatants of purified trichosanthin-treated uninfected macrophage cultures (S-trichosanthin), or supernatants of purified trichosanthin-treated HIV-infected macrophage cultures (S-HIV-trichosanthin). Treatment with purified trichosanthin alone at up to 2 micrograms/ml, with S-U or with S-trichosanthin, produced no morphological signs of toxicity to brain cell aggregate cultures. S-trichosanthin treatment at 2 micrograms/ml did not result in a significant change in cyclic nucleoside phosphorylase (CNP) activity. Treatment of the brain aggregates with S-HIV and S-HIV-trichosanthin did, however, result in morphological alteration of the brain aggregates, with S-HIV-trichosanthin-treated brain aggregates showing the most severe damage. Although purified trichosanthin did not appear to be directly toxic to human brain aggregate cultures, trichosanthin treatment of infected macrophages may have increased the morphological alterations caused by supernatants of HIV-infected macrophages. These experimental observations may explain anecdotal reports of adverse CNS reactions in association with trichosanthin treatment of HIV-infected patients and emphasize the neurotoxic potential of any therapy targeted at HIV-infected macrophages.

Differentiation of Anomeric & Positional Isomers of Acyclic & Cyclic Nucleosides by FAB Tandem Mass Spectrometry & NMR

Abstract We have been interested in developing routine methods, using standard 1H and 13C NMR spectroscopy of assigning anomeric configuration for both acyclic and cyclic nucleosides of the types (1) & (2) respectively. In the latter class of compounds ve have also sought to determine the site of glycosidation (viz N1 or N3 with respect to the imidazole ring). We have extended this study using FAB tandem mass spectrometry1 to identify stereochemical differences.

ChemInform Abstract: Nucleosides. Part 25. Synthesis of Alicyclic and Cyclic Nucleosides of 4(5H)‐Oxopyrazolo(3,4‐d)pyrimidine and 4‐Aminopyrazolo(3,4‐d)pyrimidine and Their Antileishmanial Activity.

AbstractThe title compounds (V), (VIIb), (IX) and (XIII)‐(XVI), which represent nonclassical nucleosides of the xanthine oxidase inhibitor allopurinol (I) and 4‐aminopyrazolo(3,4‐d)pyrimidine, are prepared in order to evaluate their antileishmanial activity.

NMR studies of a bacterial cell culture medium (LB Broth): cyclic nucleosides in yeast extracts

The composition of LB broth (tryptone, yeast extract and NaCl) was investigated by 1H, 31P-NMR spectroscopy, FPLC and gel electrophoresis. An enexpected finding was the high level of 2′,3′-cyclic nucleotides, detected by characteristic 31P-NMR resonances in the region 20–21 ppm, originating from the yeast component. 31P-NMR resonances for cyclic nucleotides were observed during the autolysis of Saccharomyces cerevisiae cells, and in model reactions of RNase with RNA.

NMR studies of a bacterial cell culture medium (LB Broth): cyclic nucleosides in yeast extracts

The composition of LB broth (tryptone, yeast extract and NaCl) was investigated by 1H,31P-NMR spectroscopy, FPLC and gel electrophoresis. An unexpected finding was the high level of 2'3'-cyclic nucleotides, detected by characteristic 31P-NMR resonances in the region 20-21 ppm, originating from the yeast component. 31P-NMR resonances for cyclic nucleotides were observed during the autolysis of Saccharomyces cerevisiae cells, and in model reactions of RNase with RNA.

Cyclic nucleotide dynamics in the rat hypothalamus during osmotic stimulation: in vivo and in vitro studies

The molecular mechanisms which regulate expression of vasopressin (AVP)- and oxytocin (OT)-encoding genes are unknown. We have investigated the regulatory role of one class of second messenger, the cyclic nucleosides, by examining levels of both adenosine 3′,5′-monophosphate (cAMP) and guanosine 3′,5′-monophosphate (cGMP) in hypothalamic nuclei of rats during osmotic stimulation. In vivo studies, in which rats were given 2% saline to drink for different periods (salt loading), demonstrated elevated levels of cAMP in the supraoptic nucleus (SON) after 2 days. Raised levels were also evident at 3 and 7 days. A similar (less marked) pattern was observed in the paraventricular nucleus (PVN) but not in the suprachiasmatic nucleus (SCN). cGMP was present at much lower levels than cAMP and did not exhibit parallel dynamics during salt loading; however, significant changes in cGMP levels were found in the SON and PVN. In vitro studies, in which explant cultures of punched hypothalamic nuclei were challenged with hypertonic media, demonstrated that increasing medium osmolality from 290 to 310 mOsm/kg doubled the level of cAMP in the SON but did not change levels in the PVN or SCN. A greater stimulus, 325 mOsm/kg, caused a 4-fold increase in SON cAMP, and small cAMP responses in the PVN and SCN. Marked cGMP responses were also observed in the SON following stimulation at 310 and 325 mOsm/kg, smaller responses being found in the PVN and SCN. These results are consistent with previous demonstrations of SON neuron osmosensitivity. The present study has provided evidence to support the hypothesis that cyclic nucleotides mediate osmotic effects on hypothalamic gene expression, and describes an in vitro model system whereby this hypothesis may be directly tested.

Calculations of the conformational properties of acyclonucleosides: Part 2. Comparison of active and nonactive compounds

Two related molecules from the acyclic nucleoside family 9-(1,3-dihydroxy-2-propoxymethyl) guanine (DHPG) and 9-(1,5-dihydro-4-hydroxymethyl-3-oxapentyl-2- [R])guanine (2′, 3′- secoG) have been compared by means of force field conformational analysis. They are respectively active and nonactive analogs of the antivirial compound 9-((2-hydroxyethoxy)methyl)guanine (ACG). As in the case of ACG many local minima are found for both molecules, indicating their great flexibility. For all three molecules conformations similar to those occurring in cyclic nucleosides have energies from 3 to 7 kcal mol −1 above the most stable minima.

Measurement of cytoplasmic free Ca2+ and cyclic purine nucleoside monophosphates in rat hepatocytes during receptor-mediated endocytosis of transferrin and asialoglycoproteins

Measurement of cytoplasmic free Ca2+ and cyclic purine nucleoside monophosphates in rat hepatocytes during receptor-mediated endocytosis of transferrin and asialoglycoproteins

Inhibition of erythrocyte plasma membrane NADH dehydrogenase by nucleotides and uncouplers

Erythrocyte ghost NADH dehydrogenase is inhibited in a competitive fashion by ATP and ADP whereas other nucleoside di- and triphosphates, cyclic nucleosides, as well as non-phosphorylating ATP analogs are relatively ineffective. In addition, this enzyme, measured with ferricyanide as electron acceptor, is inhibited by uncouplers of oxidative phosphorylation (proton-conducting reagents), the inhibition being competitive in character (i.e., the uncouplers were without influence upon maximum velocity). The effectiveness of the uncouplers was in the order of their hydrophobic character with the presence of the alkyl side chain rendering nonyl-dinitrophenol much more active than 2,6-dinitrophenol itself. Hydrophobic compounds that are not protonophores (e.g., eosin, proflavin or valinomycin) were not inhibitory. Whereas adenine nucleotides probably inhibit NADH oxidation competitively through structural similarity with the substrate, it appears unlikely that uncouplers compete at the NADH site directly. Rather, the apparently-competitive inhibition in the latter case may reflect competition for proton transfer to an acceptor residing in a hydrophobic region of the enzyme complex.

Structural modifications at the 2'— and 3'— positions of some pyrimidine nucleosides as determinants of their interaction with the mouse erythrocyte nucleoside transporter

Modifications in the sugar moiety of pyrimidine nucleosides may affect their ability to function as permeants of the mouse erythrocyte nucleoside transporter. In this investigation, a number of synthetic uracil and thymine nucleosides which differ from the physiological nucleosides, uridine, deoxyuridine and thymidine, through structural changes at the 2′- and 3′-positions were studied. Interaction of the analogs with the transporter has been assessed in terms of their affinities for an external site on the transporter as well as their abilities to effect trans-acceleration of thymidine efflux. 1-(β- D-Arabinofuranosyl) uracil (araU) and 1-(β- D-arabinofuranosyl)thymine (araT) were comparable to thymidine as permeants while nucleosides in which the 3′-hydroxyl was replaced with hydrogen or a halogen had a decreased affinity for the transporter. 3′-Fluoro-3′-deoxy-araU weakly accelerated thymidine efflux while its ribo-isomer and the other 3′-halogeno-3′deoxy-arabino analogs as well as dideoxythymidine inhibited efflux. The absence of 2′- and 3′-carbons in acyclothymidine and acyclo-uridine strongly decreased the affinities of these nucleosides for the transporter; efflux of thymidine was not accelerated in the presence of these compounds. The conformationally constrained cyclic nucleoside 2,2′-anhydro-araU had a very low affinity for the transporter, and influx of the radiolabeled compound could not be demonstrated. The results suggest that modification at the 3′-position, loss of a portion of the sugar ring, and lack of conformational flexibility are factors which decrease the abilities of some pyrimidine nucleosides to function as permeants. It is suggested that combined effects of substituents which play a role in determining nucleoside conformation should be considered in assessing structural requirements for permeants of the transporter.

Protein kinase and specific phosphate acceptor proteins associated with tupaia herpesvirus.

A protein kinase activity was found to be associated with tree shrew (tupaia) herpesvirus. The protein kinase was characterized with respect to its requirements for enzymatic activity. A divalent cation such as Mg2+ or Mn2+ was necessary as well as ATP as the phosphate donor. Distinct tupaia herpesvirus polypeptides (molecular weights of 100,000 [100K], 82K, and 53K) were found to be phosphate acceptor proteins when 5 mM Mg2+ was used. At a higher Mg2+ concentration (20 mM), additional viral proteins (220K, 71K, 31K, and 20K) were phosphorylated. The viral phosphoproteins were analyzed by chemical and enzymatic hydrolyses. The predominant sites of phosphorylation were the beta-OH groups of the serine and threonine residues of these tupaia herpesvirus proteins. Kinase activity was not stimulated by cyclic nucleoside monophosphates. Endogenously added proteins did not enhance protein kinase activity. Protein kinase activity was inhibited by 5'-p-fluorosulfonylbenzoyladenosine.