- Structure and monomer/dimer equilibrium for the guanylyl cyclase domain of the optogenetics protein RhoGC
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RhoGC is a fusion protein from the aquatic fungus Blastocladiella emersonii, combining a type I rhodopsin domain with a guanylyl cyclase domain. It has generated excitement as an optogenetics tool for the manipulation of cyclic nucleotide signaling pathways. To investigate the regulation of the cyclase activity, we isolated the guanylyl cyclase domain from Escherichia coli with (GCwCCRho) and without (GCRho) the coiledcoil linker. Both constructs were constitutively active but were monomeric as determined by size-exclusion chromatography and analytical ultracentrifugation, whereas other class III nucleotidyl cyclases are functional dimers. We also observed that crystals of GCRho have only a monomer in an asymmetric unit. Dimers formed when crystals were grown in the presence of the non-cyclizable substrate analog 2′,3′-dideoxyguanosine- 5′-triphosphate, MnCl2, and tartrate, but their quaternary structure did not conform to the canonical pairing expected for class III enzymes. Moreover, the structure contained a disulfide bond formed with an active-site Cys residue required for activity. We consider it unlikely that the disulfide would form under intracellular reducing conditions, raising the possibility that this unusual dimer might have a biologically relevant role in the regulation of full-length RhoGC. Although we did not observe it with direct methods, a functional dimer was identified as the active state by following the dependence of activity on total enzymeconcentration. The low affinity observed for GCRhomonomers is unusual for this enzyme class and suggests that dimer formation may contribute to light activation of the full-length protein.
- Kumar, Ramasamy P.,Morehouse, Benjamin R.,Fofana, Josiane,Trieu, Melissa M.,Zhou, Daniel H.,Lorenz, Molly O.,Oprian, Daniel D.
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Read Online
- Photochemical Properties of New Photolabile cAMP Derivatives in a Physiological Saline Solution
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Three new photolabile esters of cAMP (2-anthraquinonyl)methyl (1a), (7-methoxycoumarinyl)methyl (2a), and 2-naphthylmethyl (3a), have been developed.The stability and photochemical properties of these derivatives were compared to the previously reported ones in a physiological saline solution (1percent DMSO in Ringer's solution, pH 7.4).We found that 2a had satisfactory stability (t1/2 > 1000 h) in the dark and was photolyzed to release the parent cAMP on 340 nm irradiation (Φapp = 0.10, Ε340 = 6730) more efficiently than previously reported caged cAMPs.A biological test using the melanophores of the medaka (Oryzias latipes) revealed that 2a penetrated into the melanophores, inactive before irradiation and activated to release cAMP upon irradiation.We have developed a new caged cAMP which can be used in the investigation of biological responses regulated by intracellular cAMP concentrations using living cells.
- Furuta, Toshiaki,Torigai, Hiromi,Sugimoto, Masazumi,Iwamura, Michiko
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Read Online
- Method for synthesizing cyclic adenosine monophosphate
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The invention discloses a method for synthesizing cyclic adenosine monophosphate, belonging to the field of synthesis of nucleosides in organic chemistry. The method comprises the following reaction steps: with adenylic acid as a raw material, performing one-step cyclization under the action of acetic anhydride so as to obtain acetylated cyclic adenosine monophosphate; and then performing alkalinehydrolysis so as to obtain cyclic adenosine monophosphate. The method only needs two steps of reaction in the whole process, is simple to operate, avoids usage of considerable solvents that are usedin traditional synthesis processes, has been verified in a kg-grade scale and has industrial application prospects.
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Page/Page column 4-8
(2019/01/24)
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- ADENOSINE ANALOG AND ITS USE IN REGULATING THE CIRCADIAN CLOCK
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Provided are a kind of nucleoside analogue compounds, and compositions comprising these compounds and pentostatin, their use for modulating circadian rhythm, preferably, for shifting circadian phase, and methods for modulating circadian rhythm, preferably, for shifting circadian phase via these compounds or the compositions.
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Paragraph 0166; 0171; 0172; 0208
(2018/08/12)
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- Spectral evolution of a photochemical protecting group for orthogonal two-color uncaging with visible light
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Caged compounds are molecules rendered functionally inert by derivatization with a photochemical protecting group. We describe the design logic behind the development of a diethylaminocoumarin (DEAC) caging chromophore, DEAC450, that absorbs blue light strongly (ε450 = 43,000 M-1 cm-1) and violet light 11-fold more weakly. The absorption minimum is in the wavelength range (340-360 nm) that is traditionally used for photolysis of many widely used nitroaromatic caged compounds (e.g., 4-carboxymethoxy-5,7- dinitroindolinyl(CDNI)-GABA). We used this chromophore to synthesize DEAC450-caged cAMP and found this probe was very stable toward aqueous hydrolysis in the electronic ground state but was photolyzed with a quantum efficiency of 0.78. When DEAC450-cAMP and CDNI-GABA where co-applied to striatal cholinergic interneurons, the caged compounds were photolyzed in an chromatically orthogonal manner using blue and violet light so as to modulate the neuronal firing rate in a bidirectional way.
- Olson, Jeremy P.,Banghart, Matthew R.,Sabatini, Bernardo L.,Ellis-Davies, Graham C. R.
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supporting information
p. 15948 - 15954
(2013/11/06)
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- Factors influencing the operational stability of NADPH-dependent alcohol dehydrogenase and an NADH-dependent variant thereof in gas/solid reactors
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The continuous enzymatic gas/solid bio-reactor serves both for the production of volatile fine chemicals and flavors on an industrial scale and for thermodynamically controlled investigation of substrate and water effects on enzyme preparations for research purposes. Here, we comparatively investigated the molecular effects on the operational stability of NADPH-dependent Lactobacillus brevis alcohol dehydrogenase and an NADH-dependent variant thereof, LbADH G37D, in the gas/solid bioreactor. The reference reaction is the reduction of acetophenone to (R)-1-phenylethanol with concomitant oxidation of 2-propanol to acetone for the purpose of regeneration of the redox cofactor. It could be clearly shown that not the thermostability of the cofactor, but the thermostability of the proteins in the solid dry state govern the order of magnitude of the operational stability of both purified enzymes in the gas/solid reactor at low thermodynamic activity of water and substrate. However, at higher thermodynamic activity the operational stability in the gas/solid reactor is overlaid by stabilizing and destabilizing effects of the substrates that require further investigation. We demonstrated first evidence that the substrate affinity of the two variants in the gas/solid reactor is similar to the affinity in aqueous medium. We could also show that partial unfolding of the proteins with subsequent aggregation are the factors governing protein thermo-in-stability both in the dissolved and in the dry state. Thus, stability investigations of enzymes in the dry state are suggested to predict their basal level of operational stability in gas/solid reactions.
- Kulishova, Liliya,Dimoula, Kerasina,Jordan, Max,Wirtz, Astrid,Hofmann, Diana,Santiago-Schuebel, Beatrix,Fitter, Joerg,Pohl, Martina,Spiess, Antje C.
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experimental part
p. 271 - 283
(2011/02/22)
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- Cytidylyl and uridylyl cyclase activity of bacillus anthracis edema factor and bordetella pertussis CyaA
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Cyclic adenosine 3′,5′-monophosphate (cAMP) and cyclic guanosine 3′,5′-monophosphate (cGMP) are second messengers for numerous mammalian cell functions. The natural occurrence and synthesis of a third cyclic nucleotide (cNMP), cyclic cytidine 3′,5′-monophosphate (cCMP), is a matter of controversy, and almost nothing is known about cyclic uridine 3′,5′-monophosphate (cUMP). Bacillus anthracis and Bordetella pertussis secrete the adenylyl cyclase (AC) toxins edema factor (EF) and CyaA, respectively, weakening immune responses and facilitating bacterial proliferation. A cell-permeable cCMP analogue inhibits human neutrophil superoxide production. Here, we report that EF and CyaA also possess cytidylyl cyclase (CC) and uridylyl cyclase (UC) activity. CC and UC activity was determined by a radiometric assay, using [α-32P]CTP and [α-32P]UTP as substrates, respectively, and by a high-performance liquid chromatography method. The identity of cNMPs was confirmed by mass spectrometry. On the basis of available crystal structures, we developed a model illustrating conversion of CTP to cCMP by bacterial toxins. In conclusion, we have shown both EF and CyaA have a rather broad substrate specificity and exhibit cytidylyl and uridylyl cyclase activity. Both cCMP and cUMP may contribute to toxin actions.
- Goettle, Martin,Dove, Stefan,Kees, Frieder,Schlossmann, Jens,Geduhn, Jens,Koenig, Burkhard,Shen, Yuequan,Tang, Wei-Jen,Kaever, Volkhard,Seifert, Roland
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experimental part
p. 5494 - 5503
(2011/04/16)
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- Coumarinylmethyl esters for ultrafast release of high concentrations of cyclic nucleotides upon one- and two-photon photolysis
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(Chemical Equation Presented) Shedding light: Efficient activation of cyclic nucleoside monophosphates (cNMPs) can be achieved upon one- and two-photon flash photolysis of novel photolabile coumarinylmethyl esters of cAMP and cGMP (A = adenosine, G = guanosine) as well as their 8-bromosubstituted derivatives. The phototriggers show high solubility in water and permit space- and time-resolved investigations of the molecular mechanisms of cyclic nucleotide dependent processes.
- Hagen, Volker,Dekowski, Brigitte,Nache, Vasilica,Schmidt, Reinhard,Geissler, Daniel,Lorenz, Dorothea,Eichhorst, Jenny,Keller, Sandra,Kaneko, Hiroshi,Benndorf, Klaus,Wiesner, Burkhard
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p. 7887 - 7891
(2007/10/03)
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- Hydrolytic reactions of diadenosine 5′,5′-triphosphate
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The hydrolysis of diadenosine 5′,5′-triphosphate to AMP and ADP has been studied over a wide pH-range. Under acidic conditions the reaction shows a first-order dependence on the hydronium ion concentration. Below pH 3 the rate-increase begins to level off. From pH 6 to 9 the hydrolysis is slow and pH-independent. Base-catalysed hydrolysis is observed in NaOH-solutions. Under alkaline conditions an intramolecular nucleophilic attack on the phosphate producing 3′,5′-cAMP is also observed, but it is slower than the intermolecular reaction. Depurination of the adenosine moieties competes with the hydrolysis both under acidic and alkaline conditions, but the mechanisms are different. The temperature-dependence of the hydrolysis of Ap3A and the depurination of adenosine moieties were studied under acidic conditions, and the activation parameters of the reactions were calculated. The results of the work reflect the fact that the negatively charged polyphosphate group is very resistant towards nucleophilic attack. An efficient catalysis is only observed under acidic conditions, where the phosphate group becomes protonated. General acids or bases did not catalyse the hydrolysis. Furthermore, hydroxide ion catalysed cleavage is only observed at high base concentrations and other negatively charged nucleophiles did not attack the phosphate groups of diadenosine polyphosphates.
- Mikkola, Satu
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p. 770 - 776
(2007/10/03)
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- Thermodynamics of the hydrolysis reactions of adenosine 3′,5′-(cyclic)phosphate(aq) and phosphoenolpyruvate(aq); the standard molar formation properties of 3′,5′-(cyclic)phosphate(aq) and phosphoenolpyruvate(aq)
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Molar calorimetric enthalpy changes ΔrHm(cal) have been measured for the biochemical reactions {cAMP(aq) + H2O(l) = AMP(aq)} and {PEP(aq) + H2O(l) = pyruvate(aq) + phosphate(aq)}. The reactions were catalyzed, respectively, by phosphodiesterase 3prime;,5prime;-cyclic nucleotide and by alkaline phosphatase. The results were analyzed by using a chemical equilibrium model to obtain values of standard molar enthalpies of reaction ΔrHmo for the respective reference reactions {cAMp-(aq) + H2O(l) = HAMP-(aq)} and {PEP3-(aq) + H2O(l) = pyruvate-(aq) + HPO42-(aq)}. Literature values of the apparent equilibrium constants K′ for the reactions {ATP(aq) = cAMP(aq) + pyrophosphate(aq)K {ATP(aq) + pyruvate(aq) = ADP(aq) + PEP(aq)}, and {ATP(aq) + pyruvate(aq) + phosphate(aq) = AMP(aq) + PEP(aq) + pyrophosphate(aq)} were also analyzed by using the chemical equilibrium model. These calculations yielded values of the equilibrium constants K and standard molar Gibbs free energy changes ΔrGmo for ionic reference reactions that correspond to the overall biochemical reactions. Combination of the standard molar reaction property values (K, ΔrH mo, and ΔrGmo) with the standard molar formation properties of the AMP, ADP, ATP, pyrophosphate, and pyruvate species led to values of the standard molar enthalpy ΔfHmo, and Gibbs free energy of formation ΔfGmo and the standard partial molar entropy Smo of the cAMP and PEP species. The thermochemical network appears to be reasonably well reinforced and thus lends some confidence to the accuracy of the calculated property values of the variety of species involved in the several reactions considered herein. Published by Elsevier Ltd.
- Goldberg, Robert N.,Tewari, Yadu B.
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p. 1809 - 1830
(2007/10/03)
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- Deactivation behavior and excited-state properties of (coumarin-4-yl)methyl derivatives. 2. Photocleavage of selected (coumarin-4-yl)methyl-caged adenosine cyclic 3′,5′-monophosphates with fluorescence enhancement
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A series of axial and equatorial diastereomers of (coumarin-4-yl)methyl-caged adenosine cyclic 3′,5′-monophosphates (cAMPs), 1-6, having methoxy, dialkylamino, or no substituent in the 6- and/or 7-positions, and their corresponding 4-(hydroxymethyl)coumarin photoproducts 7-12 have been synthesized. The photochemical and UV/vis spectroscopical properties (absorption and fluorescence) of 1-6 and 7-12 have been examined in methanol/aqueous HEPES buffer solution. Donor substitution in the 6-position causes a strong bathochromic shift of the long-wavelength absorption band, whereas substitution in the 7-position leads only to a weak red shift. The photochemical cleavage of the caged cAMPs was investigated, and the photoproducts were analyzed. Photochemical quantum yields, fluorescence quantum yields, and lifetimes of the excited singlet states were determined. The highest values of photochemical quantum yields (photo-SN1 mechanism) were obtained with caged cAMPs having a donor substituent in the 7-position of the coumarin moiety, caused by electronic stabilization of the intermediately formed coumarinylmethyl cation. With donor substitution in the 6-position, the resulting moderate electronic stabilization of the coumarinylmethyl cation is overcompensated by the strong bathochromic shift, reducing the energy gap between the excited-state S1 and the corresponding coumarinylmethyl cation. The rate constant for the ester cleavage and liberation of cAMP is about 109 s-1, estimated for the axial isomer of 6 by analysis of the fluorescence increase of the alcohol 12 formed upon laser pulse photolysis.
- Eckardt, Torsten,Hagen, Volker,Schade, Bjoern,Schmidt, Reinhardt,Schweitzer, Claude,Bendig, Juergen
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p. 703 - 710
(2007/10/03)
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- Highly efficient and ultrafast phototriggers for cAMP and cGMP by using long-wavelength uv/vis-activation
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Caged cyclic nucleotides exhibiting remarkable advantageous properties have been developed through the use of novel photolabile coumarinylmethyl protecting groups (see scheme). They serve as excellent intracellular sources of cAMP and cGMP and allow the study of spatial- and time-dependent aspects of cyclic nucleotide signaling.
- Hagen, Volker,Bendig, Juergen,Frings, Stephan,Eckardt, Torsten,Helm, Siegrun,Reuter, Dirk,Kaupp, U. Benjamin
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p. 1046 - 1048
(2007/10/03)
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- Deactivation behavior and excited-state properties of (coumarin-4- yl)methyl derivatives. 1. Photocleavage of (7-methoxycoumarin-4-yl)methyl- caged acids with fluorescence enhancement
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The photochemistry of the (7-methoxycoumarin-4-yl)methyl (MCM) carboxylates 3a-d, the mesylate 4, and the phosphates 5a-e has been examined under near physiological conditions in acetonitrile or methanol/aqueous HEPES buffer solution, respectively. Analysis of photoproducts as well as measurements of photochemical quantum yields, fluorescence quantum yields, and lifetimes for the excited singlet state verified the similar photochemical and photophysical behavior of all the esters studied here. 4- (Hydroxymethyl)-7-methoxycoumarin (2) and the corresponding free acids were obtained as major products upon irradiation. The rates of deactivation of the excited MCM derivatives 3a-5e were found to be dependent on the leaving group ability of the anion concerned as well as on the solvent polarity. The polarity dependence and the exclusive formation of 18O-labeled 2 during irradiation of 5a in 18O-labeled water indicate that photocleavage of the excited singlet state of the MCM caged compounds 3a-5e proceeds via a photo S(N)1 mechanism (solvent-assisted photoheterolysis).
- Schade, Bjoern,Hagen, Volker,Schmidt, Reinhard,Herbrich, Ralph,Krause, Eberhard,Eckardt, Torsten,Bendig, Juergen
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p. 9109 - 9117
(2007/10/03)
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- Photochemistry of phosphate esters: α-keto phosphates as a photoprotecting group for caged phosphate
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Irradiation of two families of α-keto phosphates yielded rearrangement products and deprotected phosphates as the major products. For both sets of reactants, the triplet excited state of the ketone reacted with quantum efficiencies that ranged from 0.10 to 0.38. Desyl phosphates yielded 2-phenylbenzo[6]furan independent of the nature of the solvent whereas phosphate esters of α-hydroxy-p-methoxyacetophenone rearranged to esters of p-methoxyphenylacetic acid. In all cases, the phosphate group with the remaining ligands intact was released in nearly quantitative yield. The desyl group was further developed as a cage ligand for cAMP. Upon photolysis, the desyl caged ester of cAMP (13) quantitatively released the nucleotide with a quantum efficiency of 0.33 ± 0.01 and a unimolecular rate constant of 7.1 × 108s-1. Additional synthetic, product, and mechanistic studies are reported for the two series of α-keto phosphates.
- Givens, Richard S.,Athey, Phillip S.,Matuszewski, Bogdan,Kueper III, L. William,Xue, Jie-You,Fister, Thomas
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p. 6001 - 6012
(2007/10/02)
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- A CONVENIENT SYNTHESIS OF ADENOSINE 3',5'-CYCLIC PHOSPHATE (cAMP) BENZYL AND METHYL TRIESTERS
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The benzyl amd methyl triesters of cAMP were synthesized from cAMP tri-n-butylammonium salt by alkylation with alkyl halides in the presence of Na2CO3 in dimethylacetamide (DMA).
- Kataoka, Shigehiro,Uchida, Riichiro,Yamaji, Nobuyuki
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p. 1351 - 1356
(2007/10/02)
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- Studies on the Synthesis of Compounds Related to Adenosine 3',5'-Cyclic phosphate. IV. The Synthesis of 2-Sulfo- and 2-Carboxy-adenosine 3',5'-Cyclic Phosphate
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The reactions of 2-mercapto-1,N6-etheno c-AMP (c-AMP: adenosine 3',5'-cyclic phosphate) (V) and 2-mercapto c-AMP (III) with hydrogen peroxide afforded 2-sulfo-1,N6-etheno c-AMP (VIII) and 2-sulfo c-AMP (IX), respectively.Deblocking of the etheno residue of VIII afforded IX.Potassium cyanide reacted with 2-bromo-1,N6-etheno c-AMP (VI) in dimethylformamide (DMF) at room temperature to yield 2-cyano-1,N6-etheno c-AMP (X), which was isolated by Dowex 50-X8 (H+) column chromatography.Compound (X) was hydrolyzed to 2-carbamoyl-1,N6-etheno c-AMP (XI), then to 2-carboxy-1,N6-etheno c-AMP (XII) by aqueous sodium hydroxyde.Compound XII was converted to 1,N6-etheno c-AMP (IV) by heating in dimethylsulfoxide (DMSO).Deblocking of the etheno residue of XI and XII afforded 2-carbamoyl c-AMP (XIII) and 2-carboxy c-AMP (XIV), respectively.Compound XIII was hydrolyzed by aqueous sodium hydroxyde to yield XIV.Keywords- cyclic AMP derivative; etheno group; N-bromosuccinimide; mass spectrum; UV; liquid chromatography
- Yamaji, Nobuyuki,Tahara, Kyoko,Kato, Motohiko
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p. 115 - 119
(2007/10/02)
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