Fluorescent sensing of triphosphate nucleotides via anthracene derivatives

A nucleotide is composed of a nucleobase, a five-carbon sugar, and phosphate groups. Recognition of these three sites can provide useful information for the development of selective fluorescent receptors for a specific nucleotide. In this paper, anthracene derivatives with two imidazolium groups at the 1,8- and 9,10-positions, quaternary ammonium groups, or the boronic acid group were examined for the recognition of nucleotides, such as ATP, GTP, CTP, TTP, UTP, ADP, and AMP, via fluorescence changes. The anthracene group provides the interaction between the bases of the nucleotides. The imidazolium and quaternary ammonium groups induce hydrogen bonding interactions with the phosphate groups of the nucleotides. The boronic acid group can interact with the ribose of the nucleotides.     

A supramolecular ON-OFF-ON fluorescence assay for selective recognition of GTP

With the objective of developing small molecule based receptors for nucleosides and nucleotides, interactions of a cyclic donor-acceptor conjugate 1 with adenosine, AMP, ADP, CTP, UTP, ITP, ATP, and GTP have been investigated by absorption, steady-state, and time-resolved fluorescence, cyclic voltammetry (CV), NMR, and fluorescence indicator displacement techniques. Titration of 1 with the fluorescent indicator, 8-hydroxy-1,3,6-pyrene trisulfonate (HPTS), resulted in nearly complete fluorescence quenching of HPTS, along with 25% hypochromicity in its absorption spectrum. Benesi-Hildebrand analysis gave a 1:1 stoichiometry for the complex between the receptor 1 and HPTS with an association constant (Kass) of 4.66 x 104 M-1 in buffer. The driving force for such a complexation was evaluated to be the synergistic effects of pi-stacking and electrostatic interactions inside the cavity as confirmed by the effect of ionic strength, temperature, and the negative results obtained with the model compound 2. Titration of the nonfluorescent complex [1.HPTS] with various nucleosides and nucleotides resulted in revival of fluorescence of the indicator, HPTS. It was observed that GTP induces maximum displacement of HPTS from the complex [1.HPTS] with an overall fluorescence enhancement of ca. 150-fold. The addition of adenosine, AMP, ADP, CTP, and UTP showed negligible changes, whereas ca. 45- and 50-fold enhancement was observed with ATP and ITP, respectively. The competitive displacement of the indicator by various analytes is found to be in the order GTP (buffer) approximately GTP (biofluid) > ITP approximately ATP > UTP > CTP approximately ADP approximately AMP approximately Ade. By virtue of having a better pi-electron cloud, GTP undergoes effective electronic, pi-stacking, and electrostatic interactions inside the cavity and forms a stable complex with the receptor 1. The uniqueness of this assay is that it differentiates GTP from ATP and other nucleotides and signals the event through a visual "turn on" fluorescence mechanism in buffer as well as in biological fluids.     

Capillary electrophoresis apparatus equipped with a bioluminescence detector using a batch- or flow-type detection cell

We developed a capillary electrophoresis (CE) apparatus equipped with a batch- or flow-type bioluminescence (BL) detection cell. Firefly luciferin-luciferase BL reaction was used to analyze samples of nucleotides, such as ATP, dATP, ADP, GTP, UTP, CTP, ITP, and TTP. In the CE apparatus with the batch-type cell, ATP was detected at concentrations of 5-100 microM, while the other nucleotides were not detected at concentrations less than 500 microM. The electropherogram of ATP included two BL peaks; the latter peak showed peculiar broadening, which continued up to ca. 2.5 h. In the CE apparatus with the flow-type cell, ATP, dATP, and ADP were detected with single peaks with detection limits of 1, 75, and 100 microM, respectively. The other nucleotides, GTP, UTP, CTP, ITP, and TTP, were not detected at concentrations less than 0.5 mM. A mixture of 10 microM ATP and 100 microM dATP was examined using the CE apparatus with the flow-type BL detection cell. ATP and dATP were separated using running buffer at pH 10 containing 1 mM phenylboronic acid. The interaction between ATP and phenylboronic acid delayed the migration time of ATP.     

A cholic acid-based fluorescent chemosenor for the detection of ATP

A novel ditopic cholic acid-based fluorescent chemosensor for ATP, 1a, was designed and synthesized. Its interactions with phosphates, AMP, ADP, ATP, CTP, GTP, and TTP have been investigated. When ATP was added to a 1:1 aqueous CH3CN solution of the sensor at pH 7.4, a significant decrease in fluorescence of 1a was observed, whereas other guest molecules showed a much smaller effect. The complex between 1a and ATP was confirmed through combined UV, 1H, 13C and 31P NMR spectroscopic methods. The uniqueness of the new sensor is that it binds with ATP 33-124 times more selectively than other nucleotides, as evidenced from the respective binding constants. 1a is a highly sensitive sensing probe; as little as 30 nM ATP can cause 15% fluorescence quenching of the sensor.     

Borate-nucleotide complex formation depends on charge and phosphorylation state

Flow injection analysis with electrospray ionization mass spectrometry was used to investigate borate-nucleotide complex formation. Solutions containing 100 microM nucleotide and 500 microM boric acid in water-acetonitrile-triethylamine (50:50:0.2, v/v/v; pH 10.3) showed that borate complexation with nicotinamide nucleotides was significantly influenced by the charge on the nicotinamide group and the number of phosphate groups on the adenine ribose. Borate binding decreased in the order of NAD(+), NADH, NADP(+) and NADPH. To investigate the relationship between complex formation and phosphorylation, association constants (K(A)) of borate-adenine (AMP, ADP, ATP), -guanine (GMP, GDP, GTP), -cytidine (CMP, CDP, CTP) and -uridine (UMP, UDP, UTP) complexes were compared. The results showed that the number of nucleotide phosphate groups was inversely proportional to the relative abundance of the borate complexes, with the K(A) of borate-nucleotide complex decreasing in the order mono-, di- and tri-phosphates (AMP approximately GMP approximately CMP approximately UMP > ADP approximately GDP approximately CDP approximately UDP > GTP > ATP approximately CTP approximately UTP). At pH 7.4, using ammonium bicarbonate buffer, only borate-NAD(+) complex was observed. This indicates that the borate-NAD(+) complex may be the most physiologically relevant of those studied.     

Sensitive determination of nucleotides and polynucleotides based on the fluorescence quenching of the Tb3+-Tiron complex

 A sensitive method using fluorescence quenching for the determination of nucleotides (ATP, ADP, AMP, CTP, UTP) and polynucleotides[poly(A), poly(I), poly(U)] is proposed. It is based on the ability of nucleotides and polynucleotides to inhibit the formation of a strongly fluorescent complex of Tb³⁺ ion with Tiron. The possibilities of spectrofluorimetric measurements of these systems were studied under optimal conditions (pH 6.9 in hexamethylene tetramine-HCl buffer, 1.2×10^-6 mol/L of Tb³⁺, 4.0×10^-6 mol/L of Tiron, λ_ex=317 nm, λ_em=546 nm). The results showed that the Tb³⁺-Tiron complex could be used as a fluorescence test for the phosphate moieties of nucleotides and polynucleotides. The detection limits are 0.3, 1.2, 3.7, 0.2, 0.3, 1.1, 0.6 and 0.9 ng/mL for ATP, ADP, AMP, CTP, UTP, poly(A), poly(I), and poly(U), respectively. The relative standard deviations (6 replicates) are within 4.0% in the middle of the linear range. The fluorescence quenching mechanism of these systems is also discussed. Received: 16 July 1996 / Revised: 13 November 1996 / Accepted: 13 November 1996     

über Struktur und Aktivität der den H2O2-Zerfall katalysierenden Cu2+-Komplexe VII

It has been stated in a preceding paper [3] that only parts of a ligand coordinated to a metal ion can be oxidized by H₂O₂ (= peroxidative activity). Considering the reversal of this statement to be true, it is shown by means of the peroxidative activity of the Cu²⁺-complexes of ATP, ITP, CTP, UTP, and TTP that in these complexes the heteroaromatic groups contribute to the coordination of Cu²⁺ ion. By analogy with the Cu²⁺-ATP-complex, where a macrocyclic phosphate-metal-adenine chelate is formed [4], and based on his experimental results, the author considers the existence of such a macrocyclic chelate in the copper complexes of ITP, GTP, CTP, UTP, and TTP as established. The coordination sites of the heteroaromatic groups in these complexes are discussed.     

基于咪唑的荧光传感器对磷酸二氢根离子的高选择性识别

设计并合成了基于咪唑基团的高选择性的荧光传感器, 分别利用荧光和紫外-可见光谱研究了其对阴离子的识别. 结果显示, 该类荧光传感器只在H₂PO₄^-离子存在下发生显著的荧光猝灭现象, 并且产生一个新的荧光发射峰, 因此可用于乙腈溶液中H₂PO₄^-的快速有效检测.     

Separation of nucleoside mono-, di- and triphosphates by capillary electrophoresis via cadmium complexation

Summary The CE separation of twelve nucleotides (5′-mono-, di-, triphosphates of adenosine, guanosine, cytidine and uridine) was improved by adding cadmium ion to the ammonium citrate/citric acid buffer (pH 5, ionic strength 100 mM). Cadmium ion acts as a complexing agent for some nucleotides (ATP, CTP, GTP, UTP, GDP). In order to accelerate the separation, the electroosmotic flow was reversed by flushing the fused-silica capillary with 0.2 % aqueous solution of the polycationic surfactant hexadimethrine bromide. A good separation of the twelve nucleotides studied was then achieved on a dynamically coated capillary in less than 5 min by using an ammonium citrate/citric acid buffer (pH 5, ionic strength 100 mM) to which 2 mM cadmium ion has been added. High peak efficiencies were obtained (210 000 theoretical plates) and the resolution between two adjacent peaks was always greater than 1.5.     

Delayed luminescence analysis (DLA) of purine and pyrimidine ribose and deoxyribose nucleotide triphosphates in picomole quantities

A discovery is reported of a new system that enables one to quantitate the amounts of separated nucleotide triphosphates in picomole quantities. This system of delayed luminescence analysis (DLA) is sensitive to both purine and pyrimidine ribose and deoxyribose nucleotide triphosphates. A crude luciferin-luciferase (substrate-enzyme) preparation from firefly lanterns, in the presence of nucleotide triphosphate, is utilized to generate light that is detected by a liquid scintillation counter with the coincidence of the photomultiplier tubes turned off. Light is produced in a delayed fashion, the maximum emission being dependent on the type of nucleotide. Purine nucleotides (GTP, ITP, dATP, dGTP) give maximal light emission at approximately 2 mins; with the pyrimidine nucleotides the time required for maximal light emission was 5 min for UTP, dUTP, and TTP, 10 min for CTP, and 12 min for dCTP. A linear relationship on a log-log plot of light emission vs. concentration of nucleotide is demonstrated with ITP, dATP, UTP, and CTP.     

Hydrophilic interaction chromatography of nucleotides and their pathway intermediates on titania

Nucleotides and their pathway intermediates play important roles in all living species. They are essential cellular components in energy transfer, metabolic regulatory processes and biosynthesis. Titania (TiO(2)) has strong Lewis acid sites which have an affinity for the strongly electronegative phosphonate group of nucleotides. Herein a bare titania column (150 mm x 4.6 mm I.D., 3 microm) with UV detection at 254 nm was used for the separation of a set of nucleotides (AMP, ADP, ATP, UMP, UDP, UTP, GMP, GDP, GTP, CMP and CTP) and their intermediates (NAD, NADH, UDP-Glu and UDP-GluNAc). Addition of phosphate to the eluent suppresses the ligand-exchange interactions with the titania surface such that hydrophilic interaction chromatography (HILIC) separations may be performed. Increasing the %ACN resulted in increasing retention and efficiency (up to 13,000, 9500 and 4500 plates/m for AMP, ADP and ATP, respectively). The effects of pH, buffer concentration and other eluent anions (fluoride and acetate) were also studied. Fifteen nucleotides and their intermediates were separated in 26 min (R(minimum)>1.3) using an one-step gradient.     

ATP detection using a label-free DNA aptamer and a cationic tetrahedralfluorene

A simple and sensitive method for ATP detection using a label-free DNA aptamer as the recognition element and ethidium bromide (EB) as the signal reporter is reported. The ATP-binding aptamer undergoes a conformational switch from the aptamer duplex to the aptamer/target complex upon target binding, which induces the fluorescence change of intercalated EB emission. Good selectivity between ATP and CTP, GTP or UTP has been demonstrated, which is due to the specific recognition between the ATP aptamer and ATP. Using EB alone as a signal reporter, the ATP detection limit was estimated to be approximately 0.2 mM. When a light harvesting cationic tetrahedralfluorene was used as an energy donor to sensitize the intercalated EB emission, a 10-fold increase in detection limit and a 2-fold increase in detection selectivity was demonstrated. The sensitivity and selectivity of the tetrahedralfluorene sensitized assay is comparable to or better than most fluorescent ATP assays with multiple labels.     

In vitro nonenzymatic glycation of guanosine 5′-triphosphate by dihydroxyacetone phosphate

Dihydroxyacetone phosphate (DHAP) is a glycolytic intermediate that has been found to be significantly elevated in the erythrocytes of diabetic patients and patients with triosephosphate isomerase deficiency. DHAP spontaneously breaks down to methylglyoxal, a potent glycating agent that reacts with proteins and nucleic acids in vivo to form advanced glycation endproducts (AGEs). Like methylglyoxal, DHAP itself is also a glycating metabolite, capable of condensing with proteins and altering their structure or function. The objective of this investigation was to evaluate the susceptibility of nucleotides to nonenzymatic attack by DHAP, and to determine the factors influencing the rate and extent of nucleotide glycation by this sugar. Of the four nucleotide triphosphates (ATP, CTP, GTP and UTP) that were studied, only GTP was reactive, forming a wide range of UV and fluorescent products with DHAP. Increases in temperature and nucleotide concentration enhanced the rate and extent of GTP glycation by DHAP and promoted the heterogeneity of AGEs. Capillary electrophoresis, HPLC, and mass spectrometry allowed for a thorough analysis of the glycated products and demonstrated that the reaction of DHAP with GTP occurred via the classical Amadori pathway.     

Using a deoxyribozyme ligase and rolling circle amplification to detect a non-nucleic acid analyte, ATP

An allosteric ribozyme (aptazyme) has been used to transduce the binding of a small organic analyte (ATP) into the ligation of a circular template for rolling circle amplification (RCA). An ATP-activated deoxyribozyme ligase was immobilized on a glass slide and, upon addition of ATP, catalyzed the ligation of a circular padlock probe. The ligated products could be directly amplified and visualized via RCA. The coupled reaction exhibited could detect as little as 1 muM of ATP and could discriminate against structurally similar nucleotides such as GTP, CTP, and UTP. Cooperative ATP activation of the deoxyribozyme was faithfully mimicked by RCA, yielding an amplified "switch" that was responsive to ATP concentration.     

Preparation of a mixture of nucleoside triphosphates suitable for use in synthesis of nucleotide phosphate sugars from ribonucleic acid using nuclease P1, a mixture of nucleoside monophosphokinases and acetate kinase

This paper (1) describes the enzymatic synthesis of a mixture of adenosine, guanosine, cytidine, and uridine triphosphates (ATP, GTP, CTP, and UTP) from ribonucleic acid (RNA). RNA was hydrolyzed by nuclease P1 to a mixture of 5'-nucleoside monophosphates. This mixture was converted to the nucleoside triphosphates using a mixture of nucleoside monophosphate kinases and acetate kinase, with acetyl phosphate as the ultimate phosphoryl donor. The nucleoside monophosphokinases were extracted from brewer's yeast in a four-step procedure. The specific activity of the yeast enzyme preparation after gel permeation chromatography was sufficiently high that the yeast kinases could be immobilized in volumes that were practical for laboratory scale syntheses. Conversions from NMP to NTP in a mixture containing 0.34 mol of total nucleoside phosphates were: ATP, 90%; GTP, 90%; CTP, 60%; and UTP, 40%.     

Nucleotide recognition by protonated aminoglycosides

Interactions of protonated forms of kanamycin A with nucleotides and several simple phosphate anions have been studied by potentiometric and NMR titrations. The affinity of kanamycin A to anions is comparable to that observed with other aliphatic polyammonium receptors of similar charge, but it discriminates triphosphate nucleotides with different nucleobases with binding constants following the order GTP?CTP ≈ ATP. Kanamycin A also binds the respective uncharged nucleosides with the same selectivity. Binding of ATP is exothermic with a negative entropic contribution in contrast to what is expected for simple ion pairing. Other tested aminoglycosides, amikacin and streptomycin, bind ATP less efficiently than kanamycin A. Models of structures of kanamycin A complexes with ATP and GTP obtained by molecular mechanics (OPLS-2005) calculations based on ¹H and ³¹P NMR data confirm the possibility of nucleotide discrimination by simultaneous ion pairing of terminal nucleotide phosphate groups with ammonium sites of rings B and C and hydrogen bonding of the nucleobase at the ring A of the aminoglycoside.     

Unique X-ray sheet structure of 1,8-bis(imidazolium) anthracene and its application as a fluorescent probe for DNA and DNase

A new imidazolium anthracene derivative 1 was synthesized, and its unique X-ray crystal structure was examined. In aqueous solutions, probe 1 exhibited a selective fluorescent quenching effect only with DNA among various anions including the nucleotides investigated. This probe was further applied to monitor the activity of DNase.     

A naphthalimide–calixarene as a two-faced and highly selective fluorescent chemosensor for Cu or F

A new naphthalimide–calix[4]arene was synthesized as a two-faced and highly selective fluorescent chemosensor for Cu²⁺ or F⁻. This chemosensor displayed a selective fluorescence quenching effect only with Cu²⁺ among the various metal ions. On the other hand, among the various anions, the title chemosensor displayed a selective fluorescence quenching effect only with F⁻. The binding mode with F⁻ was further investigated using fluorescence changes and ¹H NMR experiments.     

Ion-pair reversed phase HPLC determination of nucleotides,nucleosides and nucleobases — Application to nucleotide metabolism in hepatocytes

Chromatographia - Three groups of metabolites were analyzed in extracts of rat hepatocytes by an HPLC method: (i) nucleotides (ATP, ADP, AMP, GTP, GDP, UTP, UDP, IMP, UMP), (ii) nucleosides and...     

Smectic Mesophases from Dihydroxy Derivatives of Quaternary Alkylammonium Salts

Long alkyl-chain quaternary ammonium salts bearing two hydroxyethyl groups at the polar head group form smectic A mesophases at high temperatures and ordered smectic T mesophases of tetragonal symmetry at lower temperatures. The latter phase resembles a supermolecular grid (figure); it is formed by a hydrogen-bonding network between the hydroxy groups themselves on the one hand, and between the hydroxy groups and the bromide anions on the other, which is perfectly congruous with the tetragonal packing of the quaternary ammonium groups.