A method of the rapid preparation of adenosine 5'-gamma-[32P] triphosphate by chemical synthesis.

A new chemical method for the synthesis of adenosine 5'-gamma-[32P] triphosphate has been developed based on the reaction of adenosine 5'-diphosphate with ethyl chloroformate. The resulting active mixed anhydride was able to react with [32P]-triethylammonium orthophosphate to give gamma-[32P]ATP.     

A Method of the Rapid Preparation of Adenosine 5′-γ-[32P] Triphosphate by Chemical Synthesis

A new chemical method for the synthesis of adenosine 5′-γ-[³²p] triphosphate has been developed based on the reaction of adenosine 5′- diphosphate with ethyl chloro-formate. The resulting active mixed anhydride was able to react with [³²p]-triethylammonium orthophosphate to give γ-[³²p]atp.     

Formation of stable anhydrides from CoA transferase and hydroxamic acids.

Acetohydroxamic acid reacts with the enzyme-CoA form of succinyl-CoA:3-ketoacid coenzyme A transferase to give an inactive product with a rate constant of 860 M-1 min-1 at pH 8.1, 25 degrees C. The reaction is reversible in the presence of coenzyme A and has an equilibrium constant of 0.040. The product is an anhydride that is an analog of the intermediate that has been postulated in the normal catalytic pathway; it is inactive because coenzyme A does not react with the acyl group of the hydroxamic acid. The equilibrium constant for formation of the anhydride from the thil ester of enzyme and methyl 3-mercaptopropionate is 75 times larger than the equilibrium constant of 2.2 for the formation of N,O-diacetylhydroxylamine from acetohydroxamic acid and acetyl-CoA. This shows that the enzyme stabilizes the anhydride at the active site by at least -2.6 kcal mol-1. Succinomonohydroxamic acid reacts with enzyme-CoA as both a substrate and an inactivator, with relative rate constants of 25:1. The inactivation is irreversible, indicating that the enzyme provides a larger stabilization of at least -5.9 kcal mol-1 for the anhydride of an analog of the specific substrate, succinate. The results are consistent with the hypothesis that the enzyme stabilizes an anhydride that is formed at the active site during turnover of normal substrates through a stepwise reaction mechanism.     

Antibacterial Activity of 2′-Esters of Erythromycin

The effect of esterification at the 2'-position of desosamine on the antibacterial activity of erythromycin was investigated by determining the bacteriostatic and bactericidal activities of erythromycin and a number of its 2'-esters on S. aureus and relating these activities to the hydrolysis rates of the esters. These studies, together with comparison of the inhibition of protein synthesis in a cell-free system isolated from S. aureus, lead to the conclusion that 2'-esters of erythromycin are inactive until hydrolyzed. Loss of activity appears to result from inability of erythromycin esters to bind to bacterial ribosomes and thus inhibit synthesis of protein.     

Tin and thallium reagents for transfer of the 1,2-di-tert-butylcyclopentadienyl ligand to transition metals

New tin and thallium reagents capable of transferring the 1,2-di-tert-butylcyclopentadienyl moiety are easily prepared and utilized in furthering the transition metal organometallic chemistry of this intersting ligand. Lithium 1,2-di-tert-butylcyclopentadienide (1) reacts cleanly and selectively with SnClMe₃ to give 2,3-di-tert-butyl-5-trimethylstannyl-1,3-cyclopentadiene (2), which in turn reacts with Re(CO)₅Br to form the half-sandwich complex [Re(η⁵-C₅H₃(1,2-Bu^t)₂)(CO)₃] (3). The reaction between thallium ethoxide and 1,5-ditert-butyl-1,3-cyclopentadiene in hexane affords the excellent cyclopentadienyl transfer reagent, thallium 1,2-di-tert-butylcyclopentadienide (4). The thallium salt reacts with [Ru(COD)Cl₂]_n to give the sandwich complex [Ru(η⁵-C₅H₃(1,2-Bu₂^t)₂)] (5).     

Kinetic studies on the diaqua form of cis-platin and various nucleobases

Kinetic studies on cis-[Pt(NH3)2(OH2)2]2+ and various nucleobases show that this ion reacts more quickly with guanosine than with adenosine, cytidine, and thymidine, and that a monophosphoric acid unit considerably enhances the rate of reaction of guanosine; the kinetic preference of 5'-GMP over 5'-AMP may point to a greater thermodynamic selectivity.     

Triaryl(Oxy)Phosphine Dibromide- A Convenient Reagent for the Preparation of S-Arylthioinosines and N 6, 5′-Disubstituted Adenosine Derivatives from Inosine

Abstract

Inosine isopropylidene 1 reacts with triphenylphosphine/phosphite dibromide and thiophenol to give 5′-bromo-S-phenylthioinosine 5 which is a versatile precursor for 5′,N ⁶-disubstituted adenosine derivatives.     

Specificity of S-adenosylmethionine synthetase for ATP analogues mono- and disubstituted in bridging positions of the polyphosphate chain

The entire family of ATP analogues that are either mono- or disubstituted with imido and methylene bridges in the polyphosphate chain of ATP have been investigated as substrates and inhibitors of S-adenosylmethionine synthetase (ATP:L-methionine S-adenosyltransferase). The disubstituted analogues adenosine 5'-(alpha,beta:beta,gamma-diimidotriphosphate) (AMPNPNP) and adenosine 5'-(alpha,beta:alpha,beta'-diimidotriphosphate) [AMP(NP)2] have been synthesized for the first time, and a new route to adenosine 5'-(alpha,beta:beta,gamma-dimethylenetriphosphate) (AMPCPCP) has been developed. S-Adenosylmethionine synthetase catalyzes a two-step reaction: the intact polyphosphate chain is displaced from ATP, yielding AdoMet and tripolyphosphate, followed normally, but not obligatorily, by the hydrolysis of the tripolyphosphate to pyrophosphate and orthophosphate. Uniformly, the imido mono- or disubstituted derivatives are both better substrates and better inhibitors than their methylene counterparts. AMPNPNP reacts rapidly to give a single equivalent of product per active site, but subsequent turnovers are at least 1000-fold slower, enabling it to be used to quantify enzyme active site concentrations. In contrast, AMPCPCP is not detectably a substrate (less than 10(-5)% of ATP). AMP(NP)2, a branched isomer of linear AMPNPNP, was not a substrate but was a linear competitive inhibitor, greater than 100 fold more potent than ADP, indicating a reasonable degree of bulk tolerance at the alpha-phosphoryl group binding site.(ABSTRACT TRUNCATED AT 250 WORDS)     

Synthesis of (Z)-(2,3-bis-hydroxymethyl)methylenecyclopropane analogues of purine nucleosides

Synthesis of (Z)-(2,3-bis-hydroxymethyl)methylenecyclopropane analogues of nucleosides adenosine 10a, 10b, 10c and 17 is described. Epimerization of Feist's acid (11) using acetic anhydride gave cyclic anhydride 12 which was reduced in situ to give diol 13. Acetylation (compound 14) followed by addition of bromine led to dibromo derivative 15. Alkylation-elimination of adenine with 15 afforded, after deacetylation, analogue 10a. Similar treatment of 2-amino-6-chloropurine and 2,6-diaminopurine led to diacetates 16 and 18. Deprotection then gave compounds 17 and 10c. Hydrolysis of 17 furnished guanine analogue 10b. Compounds 10a, 10b or 10c were inactive against HCMV, HSV-1, HSV-2, EBV VZV and HBV. Analogues 10a and 10b were also assayed for anti-HIV activity. Compound 10a was effective in HIV-1/MT-2 culture with EC50/CC50 33/> 100 microM but 10b was inactive. Analogue 10a was not a substrate for adenosine deaminase.     

Aminoacyl-tRNA Analogues; Synthesis,Purification and Properties of 3′-Anthraniloyl Oligoribonucleotides

Abstract

Reaction of isatoic anhydride with adenosine, adenosine 5′-phosphate, oligoribonucleotides or with the E. coli tRNA^Val led to attachment of an anthraniloyl residue at 2′-or 3′-OH groups of 3′-terminal ribose residue. No protection of the S'-hydroxyl group or internal 2′-hydroxyl groups is required for this specific reaction. Anthraniloyl-tRNA which is an analogue of aminoacyl-tRNA forms a ternary complex with EF-Tu*GTP. The anthraniloyl-residue is used as a fluorescent reporter group to monitor interactions with proteins.

     

Characterization of the interaction of ophiobolin A and calmodulin

1. The fungal toxin ophiobolin A reacts with the epsilon-amino group of lysine to give a conjugated enamine produce with lambda max at 272 nm and a molar extinction of 19,200 per M/cm. 2. Bovine brain calmodulin reacts with ophiobolin A to give a lambda max at 272 nm. 3. One mol of calmodulin reacts with two moles of ophiobolin A. Reaction of 1 mol of ophiobolin A inactivates 1 mol of calmodulin. 4. Ophiobolin A-treated calmodulin is resistant to tryptic cleavage at lysine 77. 5. Ophiobolin A also inhibits Dictyostelium calmodulin which has glutamine instead of lysine at residue 77.     

Crystal structures of T. b. rhodesiense adenosine kinase complexed with inhibitor and activator: implications for catalysis and hyperactivation

Background

The essential purine salvage pathway of Trypanosoma brucei bears interesting catalytic enzymes for chemotherapeutic intervention of Human African Trypanosomiasis. Unlike mammalian cells, trypanosomes lack de novo purine synthesis and completely rely on salvage from their hosts. One of the key enzymes is adenosine kinase which catalyzes the phosphorylation of ingested adenosine to form adenosine monophosphate (AMP) utilizing adenosine triphosphate (ATP) as the preferred phosphoryl donor.

Methods and Findings

Here, we present the first structures of Trypanosoma brucei rhodesiense adenosine kinase (TbrAK): the structure of TbrAK in complex with the bisubstrate inhibitor P¹,P⁵-di(adenosine-5′)-pentaphosphate (AP5A) at 1.55 Å, and TbrAK complexed with the recently discovered activator 4-[5-(4-phenoxyphenyl)-2H-pyrazol-3-yl]morpholine (compound 1) at 2.8 Å resolution.

Conclusions

The structural details and their comparison give new insights into substrate and activator binding to TbrAK at the molecular level. Further structure-activity relationship analyses of a series of derivatives of compound 1 support the observed binding mode of the activator and provide a possible mechanism of action with respect to their activating effect towards TbrAK.     

An active-site-directed adenosine triphosphate analogue binds to the beta-subunits of factor F1 mitochondrial adenosine triphosphatase with its triphosphate moiety.

The reaction of the mixed anhydride of [3H]ATP and mesitylenecarboxylic acid and soluble mitochondrial adenosine triphosphatase is accompanied by the covalent binding of one molecule of the inhibitor to a molecule of the enzyme and results in the inhibition of adenosine triphosphatase activity by more than 90%. The electrophoresis of adenosine triphosphatase modified by reaction with the mixed anhydride of [3H]ATP and mesitylenecarboxylic acid in polyacrylamide gel in the presence of sodium dodecyl sulphate showed that the inhibitor is bound to the beta-subunit of the enzyme. The results suggest that ATP may also bind to the beta-subunit of the adenosine triphosphatase with its triphosphate moiety.     

Yeast DNA 3'-repair diesterase is the major cellular apurinic/apyrimidinic endonuclease: substrate specificity and kinetics

DNA strand breaks with damaged 3' termini are potentially toxic lesions caused by free radicals. The purified yeast diesterase that removes small nucleotide fragments from such 3' termini in oxidized DNA has been further characterized with respect to its substrate specificity. In addition to the 3'-phosphoglycolaldehyde esters used to monitor the activity during purification, the enzyme efficiently hydrolyzed a variety of other 3'-esters in DNA. These included 3'-phosphates, 3'-(2,3-didehydro-2,3-dideoxyribose phosphates), and the 3'-blocking damages formed in vivo in Escherichia coli by H2O2 or in vitro by DNA treatment with bleomycin. This same transition metal-dependent enzyme also constitutes the major yeast endonuclease for apurinic/apyrimidinic sites in DNA, hydrolyzing these damages to yield normal 3'-hydroxyl nucleotides and 5'-phosphoryl base-free sugar termini (a Type II apurinic/apyrimidinic endonuclease). Yeast 3'-phosphoglycolaldehyde diesterase therefore appears to be involved in two distinct pathways of DNA repair: initiation of the repair of oxidative strand breaks in DNA and the restoration of sites of base loss caused by many types of DNA-damaging agents.     

Synthesis and properties of diastereoisomers of adenosine 5'-(O-1-thiotriphosphate) and adenosine 5'-(O-2-thiotriphosphate).

The chemical synthesis of adenosine 5'-(O-1-thiotriphosphate) (ATPalphaS) and adenosine 5'-(O-2-thiotriphosphate) (ATPbetaS) is described. Both exist as a pair of diastereomers, A and B. The isomers of ATPalphaS can be distinguished on the basis of their different reaction rates with myokinase as well as nucleoside diphosphate kinase. With both enzymes, isomer A reacts fast whereas isomer B reacts considerably more slowly. Phosphorylation of a mixture of isomers of ADPalphaS with pyruvate or acetate kinase yields ATPalphaS, isomer A, whereas the phosphoryl transfer with creatine or arginine kinase yields isomer B. The isomers of ATPbetaS differ in their reactivity with myosin. Isomer A is readily hydrolyzed, whereas isomer B is not. However, isomer B reacts faster with nucleoside diphosphate kinase and ADP than isomer A. Phosphoryl transfer with pyruvate kinase onto ADPbetaS yields ATPbetaS, isomer A, with acetate kinase, isomer B.     

Synthesis and reactions of a nucleoside derivative of phosphoric sulfonic anhydride. Studies related to the mechanisms of coupling reactions in the chemical synthesis of oligodeoxyribonucleotides by phosphotriester procedures

The synthesis of a model compound, diphenylphosphoric toluene-p-sulfonic anhydride, an arylsubstituted phosphoric sulfonic mixed anhydride, is described. Using the same procedure a thymidyl substituted derivative was prepared. The phosphoric sulfonic anhydride is the presumed intermediate in oligonucleotide coupling reactions involving phosphodiester activation by arenesulfonyl derivatives. This mixed anhydride reacts with a variety of nucleophiles. It can be converted to phophotriester derivatives in the presence of simple alcohols. Phosphotriester formation using the 5'-hydroxyl of a thymidine derivative requires additionally a catalyst such as N-methylimidazole. The reactive intermediate produced upon the addition of N-methylimidazole to the phosphoric sulfonic anhydride has been observed spectroscopically using 31P-NMR.     

Oxidation Chemistry of Dopamine: Possible Insights into the Age-Dependent Loss of Dopaminergic Nigrostriatal Neurons

The oxidation chemistry of the chemical neurotransmitter dopamine (DA) has been investigated using electrochemical approaches. At physiological pH in aqueous solution DA is initially oxidized at a pyrolytic graphite electrode in a reversible 2e, 2H⁺ reaction to give DA-o-quinone (1). Deprotonation and intramolecular cyclization of 1 yields 5,6-dihydroxyin-doline (3) which is rapidly further oxidized (2e, 2H⁺) to 6-hydroxyindoline-6-one (4; dopaminochrome). Compound 4 then rearranges to 5,6-dihydroxyindole (5) which for the first time has been unequivocally identified as an oxidation product of DA. When preconcentrated on a preparative reversed-phase HPLC column 5 undergoes an unusual nonoxidative dimerization reaction to give 2-(5 ,6-dihydroxyindoline-3-yl)-5,6-dihydroxyindole (7). This 2,3′-linked indole-indoline dimer is extremely toxic when centrally administered to laboratory mice and evokes complex behavioral responses including convulsions, tremor, and hyperactivity. DA is known to undergo autoxidation in the cytoplasm of dopaminergic substantia nigra neurons to give neuromelanin polymer. Based on the results obtained in this study it is speculated that 5, a necessary intermediate in the latter reaction, is adsorbed on neuromelanin and reacts to form 7 which along with DA-o-quinone (1) and dopaminochrome (4) might be endotoxins that contribute to the age-dependent degeneration of dopaminergic SN neurons. In the presence of L-cysteine DA-o-quinone (1) reacts to give 5-S-cysteinyldopamine (8) not 6-S-cysteinyldopamine as has been suggested by other investigators.     

Why do asthmatic subjects respond so strongly to inhaled adenosine?

Bronchospasm induced by adenosine is blocked by representatives of all the major classes of drugs used in the treatment of asthma. Understanding the mechanism of this bronchospasm may help understand the way these drugs work. Clinical studies have suggested involvement of neural pathways, mast-like cells and mediators such as histamine, serotonin and lipoxygenase products. There is a strong link between responsiveness to adenosine and eosinophilia. In different animal models A1, A2b and A3 adenosine receptor subclasses have all been implicated in inducing bronchospasm. whilst occupation of the A2a receptor generally has no, or the opposite effect. At least two different mechanisms, both involving neural pathways, exist. One, involving the adenosine A1 receptor, functions in mast cell depleted animals; the other requires interaction with a population of mast-like cells activated over A2b or A3 receptors. Not only histamine but also serotonin and lipoxygenase products released from the mast-like cells are potential mediators. In animal models good reactivity to adenosine receptor agonists is generally only found when the animals are first sensitized and exposed to allergen in ways likely to induce an allergic inflammation. An exception is the BDE rat, which reacts to adenosine receptor agonists such as APNEA or NECA even without allergen exposure. This rat strain does however show evidence of spontaneous eosinophilic inflammation in the lung even without immunization. As mast cells both release adenosine and respond to adenosine, adenosine provides a non-specific method of amplifying specific signals resulting from IgE/antigen interaction. This mechanism may not only have a pathological significance in asthma; it may be part of a normal bodily defense response that in asthmatic subjects is inappropriately activated.     

A sensitive radioimmunoassay for adenosine in biological samples

A simple, sensitive, specific, and reproducible radioimmunoassay for the measurement of adenosine in biological materials has been developed. Adenosine antibody was obtained by immunizing rabbits with an immunogen prepared by conjugating 2′,3′-disuccinyladenosine to human serum albumin. By succinylating adenosine in samples at the 2′- and 3′-O positions with a premixed reagent consisting of succinic anhydride, triethylamine, and dioxane, the assay became sensitive enough to detect less than picomole amounts of adenosine in minute quantities of tissues. The corss-reactivity of structurally related compounds with the antibody was mostly negligible except for 2′-deoxyadenosine, whose usual concentration was very low. The use of this method made it possible to measure adenosine without any prior purification procedure. The immunoreactive materials in various biological samples disappeared during incubation of the samples with adenosine deaminase.     

Properties of enzyme fraction A from Chlorella and copurification of 3' (2'), 5'-biphosphonucleoside 3' (2')-phosphohydrolase, adenosine 5'phosphosulfate sulfohydrolase and adenosine-5'-phosphosulfate cyclase activities.

Enzyme fraction A from Chlorella which catalyzes the formation of adenosine 5'-phosphosulfate from adenosine 3'-phosphate 5'-phosphosulfate is further characterized. Fraction A is found to contain an Mg2+ -activated and Ca2+ -inhibited 3' (2')-nucleotidase specific for 3' (2'), 5'-biphosphonucleosides. This activity has been named 3' (2), 5'-biphosphonucleoside 3' (2')-phosphohydrolase. The A fraction is also found to contain an activity which catalyzes the formation of adenosine 3':5'-monophosphate (cyclic AMP) from adenosine 5'-phosphosulfate (adenosine 5'-phosphosulfate cyclase). Under the same conditions of assay, 5'-ATP and 5'-ADP are not substrated for cyclic AMP formation. Unlike the 3' (2'), 5'-biphosphonucleoside 3' (2')-phosphohydrolase activity, the adenosine 5'-phosphosulfate cyclase activity does not require Mg2+, requires NH+4 or Na+, and is not inhibited by Ca2+. The A fraction also contains an adenosine 5'-phospho sulfate sulfohydrolase activity which forms 5'-AMP and sulfate. The three activities remain together during purification and acrylamide gel electrophoresis of the purified preparation yields a pattern where only one protein band has all three activities. The phosphohydrolase can be separated from the other two activities by affinity chromatography on agarose-hexyl-adenosine 3'n5'-bisphosphate yielding a phosphohydrolase preparation showing a single band on gel electrophoresis. The adenosine 5'-phosphosulfate cyclase may provide an alternate route of cyclic AMP formation from sulfate via ATP sulfurylase, but its regulatory significance in Chlorella, if any, remains to be demonstrated. In sulfate reduction, the phosphohydrolase may serve to provide a readily utilized pool of adenosine 5'-phosphosulfate as needed by the adenosine 5'-phosphosulfate sulfotransferase. The cyclase and sulfohydrolase activities would be regarded as side reactions incidental to this pathway, but may be of importance in other metabolic and regulatory reactions.