Chemical synthesis and biological activity of novel brominated 7-deazaadenosine-3',5'-cyclic monophosphate derivatives.

Synthetic derivatives of cyclic adenosine monophosphate, such as halogenated or other more hydrophobic analogs, are widely used compounds, to investigate diverse signal transduction pathways of eukaryotic cells. This inspired us to develop cyclic nucleotides, which exhibit chemical structures composed of brominated 7-deazaadenines and the phosphorylated ribosugar. The synthesized 8-bromo- and 7-bromo-7-deazaadenosine-3',5'-cyclic monophosphates rank among the most potent activators of cyclic nucleotide-regulated ion channels as well as cAMP-dependent protein kinase. Moreover, these substances bind tightly to exchange proteins directly activated by cAMP.

Synthesis and evaluation of c-di-4'-thioAMP as an artificial ligand for c-di-AMP riboswitch.

Cyclic-di-adenosine monophosphate (c-di-AMP) is a bacterial second messenger that binds to an RNA receptor called riboswitch and regulates its downstream genes involving cell wall metabolism, ion transport, and spore germination. Therefore, the c-di-AMP riboswitch can be a novel target of antibiotics. In this study, we synthesized c-di-4'-thioAMP (1), which possesses a sulfur atom instead of an oxygen atom in the furanose ring, as a candidate of a bioisoster for natural c-di-AMP. The resulting 1 bound to the c-di-AMP riboswitch with a micromolar affinity (34.8µM), and the phosphodiesterase resistance of 1 was >12-times higher than that of c-di-AMP. Thus, 1 can be considered to be a stable ligand against a c-di-AMP riboswitch.

Mechanisms of ATP to cAMP Conversion Catalyzed by the Mammalian Adenylyl Cyclase: A Role of Magnesium Coordination Shells and Proton Wires.

We report a mechanism of adenosine triphosphate (ATP) to cyclic adenosine monophosphate (cAMP) conversion by the mammalian type V adenylyl cyclase revealed in molecular dynamics (MD) and quantum mechanics/molecular mechanics (QM/MM) simulations. We characterize a set of computationally derived enzyme-substrate (ES) structures showing an important role of coordination shells of magnesium ions in the solvent accessible active site. In the lowest energy ES conformation, the coordination shell of MgA2+ does not include the Oδ1 atom of the conserved Asp440 residue. Starting from this conformation, a one-step reaction mechanism is characterized that includes proton transfer from the ribose O3'H3' group in ATP to Asp440 via a shuttling water molecule concerted with PA-O3A bond cleavage and O3'-PA bond formation. The energy profile of this route is consistent with the observed reaction kinetics. The computed energy profiles initiated from higher energy ES complexes are characterized by larger energy expenses to complete the reaction. Consistent with experimental data, we show that the Asp440Ala mutant of the enzyme should exhibit a reduced but retained activity. All considered reaction pathways include proton wires from the O3'H3' group via shuttling water molecules.

Fluorescent modification of adenosine 3',5'-monophosphate: spectroscopic properties and activity in enzyme systems.

The synthesis of a highly fluorescent analog of adenosine 3',5'-monophosphate, namely, 1,N(6)-ethenoadenosine 3',5'-monophosphate, has provided a powerful probe for systems involving adenosine 3',5'-monophosphate. The potential utility of this analog is indicated by its long fluorescent lifetime, detectability at low concentration, and relatively long wavelength of excitation (300 nanometers). In protein kinase systems it is a highly acceptable substitute for adenosine 3',5'-monophosphate.

Open-closed switching of synthetic tubular pores.

While encouraging progress has been made on switchable nanopores to mimic biological channels and pores, it remains a great challenge to realize long tubular pores with a dynamic open-closed motion. Here we report µm-long, dynamic tubular pores that undergo rapid switching between open and closed states in response to a thermal signal in water. The tubular walls consist of laterally associated primary fibrils stacked from disc-shaped molecules in which the discs readily tilt by means of thermally regulated dehydration of the oligoether chains placed on the wall surfaces. Notably, this pore switching mediates a controlled water-pumping catalytic action for the dehydrative cyclization of adenosine monophosphate to produce metabolically active cyclic adenosine monophosphate. We believe that our work may allow the creation of a variety of dynamic pore structures with complex functions arising from open-closed motion.

Synthesis, structure, and reactivity of adenosine cyclic 3',5'-phosphate benzyl triesters.

A series of triesters of adenosine cyclic 3',5'-phosphate was synthesized by treatment of the free acid with various diazoalkanes (R=H, CH3, C6H5,0-NO2C6H4, p-NO2C6H4, p-CH3C6H4). The resulting diastereomeric mixtures were separated into their axial and equatorial components. Hydrolysis of the compounds was examined as well as photolysis of the photolabile o-nitrobenzyl ester. All compounds were then tested for their ability to activate the cAMP-dependent protein kinase and for their ability to serve as a substrate for the cAMP phosphodiesterase showing almost no effect on either enzyme. In a biological assay the benzyl triesters were able to penetrate into C 6 rat glioma cells and to induce the typical morphological alteration of the cell shape known for high cellular levels of cAMP. It was concluded that the benzyl triesters of cAMP are useful derivatives which can be efficiently and specifically converted to the parent nucleotide. Benzyl derivatives of biologically active phosphodiesters may provide a useful tool for study in biology and pharmacology.

Synthesis of some 1, 8- and 2, 8-disubstituted derivatives of adenosine cyclic 3', 5'-phosphate and their interaction with some enzymes of cAMP metabolism.

1, 8-Disubstituted derivatives of adenosine cyclic 3', 5'-phosphate (cAMP) were synthesized by N-oxidation or N-methylation of previously reported 8-substituted cAMP derivatives to yield 8-bromoadenosine cyclic 3', 5'-phosphate 1-oxide and 8-(benzylthio)-1-methyladenosine cyclic 3', 5'-phosphate. Substituents were introduced into the 8 position of 2-methyladenosine cyclic 3', 5'-phosphate and 2-butyladenosine cyclic 3', 5'-phosphate by bromination, followed by treatment with sodium benzylmercaptide, sodium p-chlorothiophenolate, or, in the former case, sodium azide. Each of the 1,8- and 2,8-disubstituted derivatives of cAMP was tested as activators of cAMP-dependent protein kinase and as substrates for the inhibitors of cyclic nucleotide phosphodiesterases. Depending on the substitutions, examples were found where the disubstituted derivatives were either more active, equally as active or less active than the monosubstituted parent compounds as protein kinase activators. For the compounds reported, 8-substitution completely or substantially eliminated the ability of 1- or 2-substituted derivatives of cAMP to serve as substrates for phosphodiesterase and diminished the ability of these latter derivatives to inhibit cAMP hydrolysis.

Synthesis and enzymatic and inotropic activity of some new 8-substituted and 6,8-disubstituted derivatives of adenosine cyclic 3',5'-monophosphate.

The synthesis of certain new 8-(arythio)- and 8-(alkylthio)-cAMP derivatives and N6-alkyl- and N6-dialkyl-8-(arylthio) and -8-(alkylthio) derivatives of cAMP is reported. On the basis of activation of protein kinase, several N6-alkyl-8-(benzylthio)-cAMP derivatives were selected for evaluation as inotropic agents using cat papillary muscle in vitro. Activity in these studies resulted in the selection of several analogues for in vivo studies in the anesthetized dogs. The best inotropic agent selected on the basis of in vivo studies was N6-butyl-8-(benzylthio)-cAMP (26), which exhibited an increase in blood-flow rate of 85% with no increase in heart rate. A large-scale synthesis of 26 from cAMP is reported via N1-alkylation, followed by a Dimroth rearrangement, reduction, bromination, and nucleophilic displacement via benzyl mercaptan. The N6-alkyl-8-substituted-cAMP derivatives represent a new class of potent inotropic agents. The direct mechanism of action of 26 suggests the possible utility of this cyclic nucleotide to treat clinical myocardial infarction by rapid intravenous infusion.

Transport of cyclic AMP and synthetic analogs in the perfused rat liver.

The purpose of the present work was to investigate the transport of cyclic AMP (cAMP) and analogs in the rat liver. The experimental system was the isolated once-through perfused liver. Transport was measured by employing the multiple-indicator dilution technique. The single-pass recovery of tracer [(32)P]cAMP was equal to 94.4 +/- 1. 4%; no significant extracellular transformation of cAMP occurred during a single passage. The unidirectional influx rates of dibutyryl-cAMP were a saturable function of its concentration, with K(m) = 72.75 +/- 9.24 microM and V(max) = 0.464 +/- 0.026 micromol min(-1) (mL cellular space)(-1). The unidirectional influx rates of cAMP were much lower than those of dibutyryl-cAMP and were a linear function of the concentration (up to 100 microM). The transfer coefficient for influx (k(in)) was equal to 0.860 +/- 0.058 mL min(-1) (mL extracellular space)(-1). cAMP inhibited the influx of dibutyryl-cAMP; the IC(50) was 0.83 mM. The following series of increasing unidirectional influx rates was found: cAMP < monobutyryl-cAMP approximately 2-aza-epsilon-cAMP < rp-cAMPS approximately sp-cAMPS < 8-Br-cAMP approximately dibutyryl-cGMP approximately 8-Cl-cAMP < O-dibutyryl-cAMP. There was no precise correlation between the rates of influx of the various cyclic nucleotides and their lipophilicity. It was concluded that the penetration of cAMP and its analogs into the liver cells was a facilitated process. Lipophilicity was not the only factor determining the rate of transport. The transformation of dibutyryl-cAMP was limited by both transport and activity of the intracellular enzymic systems. The intracellular transformation of exogenous cAMP, however, was limited by the transport process.

Lanthanide ions for the first non-enzymatic formation of adenosine 3',5'-cyclic monophosphate from adenosine triphosphate under physiological conditions.

Adenosine 3',5'-cyclic monophosphate (cAMP) is formed from adenosine triphosphate at pH 8 and 50 degrees C by use of lanthanide ions. Pr3+ and La3+ are the most active. The cAMP formation is more efficient at higher pH, where the mixture is made homogeneous by the addition of beta-cyclodextrin. The potential functioning of lanthanide ions as the catalytic center of an artificial adenylate cyclase is indicated.

Novel 3',5'-cyclic nucleotide analogue: adenosine 3',5'-cyclic boranomonophosphate.

A general procedure for the first synthesis of a 3',5'-cyclic boranomonophosphate was established. Specifically, adenosine 3',5'-cyclic boranomonophosphosphate (cyclic AMPB, 4c), a P-borane (BH(3)) analogue of adenosine 3',5'-cyclic monophosphate (cAMP), was synthesized via a phosphite approach in good yield. The method is also applicable for syntheses of natural cAMP and its phosphorothioate analogue. The two diasteromers of cyclic AMPB 4c were separated, and their chemical structures were established via spectroscopic methods.

Synthesis, photochemistry and application of (7-methoxycoumarin-4-yl)methyl-caged 8-bromoadenosine cyclic 3',5'-monophosphate and 8-bromoguanosine cyclic 3',5'-monophosphate photolyzed in the nanosecond time region.

New caged derivatives of hydrolysis-resistant 8-bromoadenosine cyclic 3',5'-monophosphate (8-Br-cAMP) and 8-bromoguanosine cyclic 3',5'-monophosphate (8-Br-cGMP) are described. The compounds are the axial and equatorial isomers of the (7-methoxycoumarin-4-yl)methyl (MCM) esters of cyclic nucleotides. Synthesis is accomplished by treatment of 4-bromomethyl-7-methoxycoumarin with the tetra-n-butylammonium salts of the 8-bromo-substituted cyclic nucleotides or with the free acids of 8-Br-cAMP and 8-Br-cGMP in the presence of silver(I) oxide. MCM-caged 8-Br-cAMP and MCM-caged 8-Br-cGMP liberate 8-Br-cAMP and 8-Br-cGMP during irradiation with ultraviolet light within a few nanoseconds. They show favorable absorption properties and quantum yields and are resistant to hydrolysis in aqueous buffer solutions. The moderate fluorescence properties of the caged compounds in comparison with the strongly fluorescent 4-hydroxymethyl-7-methoxycoumarin (MCM-OH) photoproduct allow the indirect estimation of the amount of photolytically released cyclic nucleotides in aqueous buffer solutions using fluorescence measurements. Their usefulness for physiological studies has been examined in a mammalian cell line expressing the cyclic nucleotide-gated ion channel of bovine olfactory sensory neurons using the patch-clamp technique and confocal laser scanning microscopy. The caged compounds serve as efficient and rapid intracellular sources of 8-Br-cAMP and 8-Br-cGMP. However, at least in HEK 293 cells, fluorescence signals cannot be used to monitor the photolysis of MCM-caged 8-Br-cAMP and 8-Br-cGMP, due to quenching of the fluorescence of MCM-OH.

[A facile synthesis of adenosine 3', 5'-cyclic methylphosphonate and methylphosphonothioate].

A facile synthesis of adenosine 3', 5'-cyclic methylphosphonate (1) and 3', 5'-cyclic methylphosphonothioate (2) was accomplished by treatment of 2'-protected adenosine with O,O-bis (1-benzotriazolyl) methylphosphonate or O,O-bis (1-benzotriazolyl) methylphosphonothioate under mild conditions, respectively. The ability of the compounds to inhibit DNA synthesis in mice hepatoma was tested. A pair of diasteromers of 2'-protected (1) was separated and the configuration at the phosphorus atom was identified.

Azide-specific labeling of biomolecules by Staudinger-Bertozzi ligation phosphine derivatives of fluorescent probes suitable for single-molecule fluorescence spectroscopy.

We describe the synthesis of phosphine derivatives of three fluorescent probes that have a brightness and photostability suitable for single-molecule fluorescence spectroscopy and microscopy: Alexa488, Cy3B, and Alexa647. In addition, we describe procedures for use of these reagents in azide-specific, bioorthogonal labeling through Staudinger-Bertozzi ligation, as well as procedures for the quantitation of labeling specificity and labeling efficiency. The reagents and procedures of this report enable chemoselective, site-selective labeling of azide-containing biomolecules for single-molecule fluorescence spectroscopy and microscopy.