Sensitive and selective fluorescence detection of guanosine nucleotides by nanoparticles conjugated with a naphthyridine receptor

Novel fluorescent nanosensors, based on a naphthyridine receptor, have been developed for the detection of guanosine nucleotides, and both their sensitivity and selectivity to various nucleotides were evaluated. The nanosensors were constructed from polystyrene nanoparticles functionalized by (N-(7-((3-aminophenyl)ethynyl)-1,8-naphthyridin-2-yl)acetamide) via carbodiimide ester activation. We show that this naphthyridine nanosensor binds guanosine nucleotides preferentially over adenine, cytosine, and thymidine nucleotides. Upon interaction with nucleotides, the fluorescence of the nanosensor is gradually quenched yielding Stern–Volmer constants in the range of 2.1 to 35.9 mM⁻¹. For all the studied quenchers, limits of detection (LOD) and tolerance levels for the nanosensors were also determined. The lowest (3σ) LOD was found for guanosine 3’,5’-cyclic monophosphate (cGMP) and it was as low as 150 ng/ml. In addition, we demonstrated that the spatial arrangement of bound analytes on the nanosensors’ surfaces is what is responsible for their selectivity to different guanosine nucleotides. We found a correlation between the changes of the fluorescence signal and the number of phosphate groups of a nucleotide. Results of molecular modeling and ζ-potential measurements confirm that the arrangement of analytes on the surface provides for the selectivity of the nanosensors. These fluorescent nanosensors have the potential to be applied in multi-analyte, array-based detection platforms, as well as in multiplexed microfluidic systems.     

Minimization and molecular dynamics studies of guanosine and Z-DNA modified by N-2-acetylaminofluorene

The modeling program AMBER 3.0 was used to study the conformations adopted by the C8-substituted guanosine adduct of the carcinogen N-2-acetylaminofluorene (AAF), called dGuo-AAF. This conformational study was extended to the hexamer d(CGCGCG)₂ in the Z form, modified by AAF at guanine G4 with the carcinogen situated at the helix exterior in two different orientations (named ZAAF-5′ and ZAAF-3′). Considering the importance of electrostatic interactions in case of charged molecules like nucleic acids, minimization and molecular dynamics (MD) were performed using different electrostatic parameters (dielectric functions and scale factors). For that purpose, a subroutine allowing the use of a sigmoidal distance-dependent dielectric function ε_cal has been added to the program. For dGuo-AAF, the results show a great importance of the starting conformation and of the force field parameters on the minimization and MD behaviors. For the AAF-modified Z-DNA, the results display a pronouned dependence on the choice of the set of electrostatic parameters as well. With the distance-dependent dielectric function ε = r, the force field parameters favor the formation of intramolecular H bonds, which can lead to important distortions of Z-DNA. The use of ε = 4r or ε = ε_cal attenuates effectively such a tendency, except in full MD simulations. The dielectric function ε_cal is computing intense, but often similar results are obtained with ε = 4r, especially with the use of the BELLY option. AAF appears to prefer the ZAAF-5′ conformation, i.e., with the fluorene ring stacking on the sugar phosphate backbone of the following 5′ C-G bases. Such a conformation is further stabilized by an intramolecular hydrogen bond between the amino group of the 3′ cytosine and the carbonyl group of AAF. The fluorene ring, although mobile around its long axis, does not flip-flop between its two main orientations, ZAAF-5′ and ZAAF-3′. The presence of the AAF does not prevent the Z_I to Z_II transitions. Whatever the electrostatic parameters, the presence or absence of AAF, the cytosine sugars stay in the C₂′-endo pucker domain. In unmodified Z-DNA, the guanine sugars stay in the C₃′-endo region, except for the terminal guanines which transit to the C₂′endo region. In modified Z-DNA, the sugar of the modified guanine behaves as a terminal guanine sugar.     

Luminescence Properties and Analytical Figures of Merits of Benzo(a)Pyrene Guanosine Adduct Adsorbed on α- β- and γ-Cyclodextrin/NaCl,and Trehalose/NaCl Solid Matrices

Abstract

Several cyclodextrin/NaCl and trehalose/NaCl mixtures were investigated as solid matrices for obtaining room-temperature luminescence from a benzo(a)pyrene (B(a)P) guanosine adduct. Room-temperature fluorescence (RTF) and room-temperature phosphorescence (RTP) intensities from the B(a)P-guanosine adduct were compared for different solid matrices. These results showed that 25% trehalose/NaCl, 1% α-cyclodextrin/NaCl, and 1% γ-cyclodextrin/NaCl solid matrices yielded strong fluorescence signals and moderately strong phosphorescence signals at room temperature from the B(a)P-guanosine adduct. In addition, the luminescence properties of pyrene, guanosine, guanosine 3′ -monophosphate free acid and guanosine 3′-monophosphate sodium salt on 1% α-, β-, and γ- cyclodextrin/NaCl solid matrices were obtained.     

Kinetics and mechanism of the N7-hydroxyalkylation of guanosine

The kinetics of the N₇-hydroxyalkylation of guanosine by the equally substituted epoxide, trans-2,3-epoxybutane, and the unequally substituted epoxides, 3-chloro-1,2-epoxypropane, and 1,2-epoxypropane in glacial acetic acid, have been measured by a spectrophotomeric method over the range 20-40°. Activation parameters have been determined. Comparative rates calculated from the ratios of second-order rate constants indicate that 1,2-epoxypropane reacts with guanosine about three times faster than does trans-2,3-epoxybutane and about two times faster than 3-chloro-1,2-epoxypropane. These results coupled with the results of a previous report on the structural analysis of the products from these reactions are consistent with a “push-pull” mechanism in which N₇ of guanosine reacts preferentially at the least substituted carbon of the epoxide with simultaneous transfer of a proton from acetic acid to the oxygen of the epoxide. The lower reactivities of trans-2,3-epoxybutane and 3-chloro-1,2-epoxypropane in comparison to that of 1,2-epoxypropane are discussed in terms of steric factors and electronic factors which determine the stability of the requisite transition state for a “push-pull” mechanism model.     

The state of the guanosine nucleotide allosterically affects the interfaces of tubulin in protofilament

The dynamics of microtubules is essential for many microtubule-dependent cellular functions such as the mitosis. It has been recognized for a long time that GTP hydrolysis in αβ-tubulin polymers plays a critical role in this dynamics. However, the effects of the changes in the nature of the guanosine nucleotide at the E-site in β-tubulin on microtubule structure and stability are still not well understood. In the present work, we performed all-atom molecular dynamics simulations of a αβα-tubulin heterotrimer harboring a guanosine nucleotide in three different states at the E-site: GTP, GDP-Pi and GDP. We found that changes in the nucleotide state is associated with significant conformational variations at the α-tubulin N- and β-tubulin M-loops which impact the interactions between tubulin protofilaments. The results also show that GTP hydrolysis reduces αβ-tubulin interdimer contacts in favor of intradimer interface. From an atomistic point view, we propose a role for α-tubulin glutamate residue 254 in catalytic magnesium coordination and identified a water molecule in the nucleotide binding pocket which is most probably required for nucleotide hydrolysis. Finally, the results are discussed with reference to the role of taxol in microtubule stability and the recent tubulin-sT2R crystal structures.     

Fluorescence quenching of TMR by guanosine in oligonucleotides

Nucleotide-specific fluorescence quenching in fluorescently labeled DNA has many applications in biotechnology. We have studied the inter- and intra-molecular quenching of tetramethylrhodamine (TMR) by nucleotides to better understand their quenching mechanism and influencing factors. In agreement with previous work, dGMP can effectively quench TMR, while the quenching of TMR by other nucleotides is negligible. The Stern-Volmer plot between TMR and dGMP delivers a bimolecular quenching constant of K _s = 52.3 M⁻¹. The fluorescence of TMR in labeled oligonucleotides decreases efficiently through photoinduced electron transfer by guanosine. The quenching rate constant between TMR and guanosine was measured using fluorescence correlation spectroscopy (FCS). In addition, our data show that the steric hindrance by bases around guanosine has significant effect on the G-quenching. The availability of these data should be useful in designing fluorescent oligonucleotides and understanding the G-quenching process.     

Reactive intermediates in laser photolysis of guanosine

The photochemical reactions of aqueous solution guanosine with acetone as photosensitizer have been investigated by laser flash photolysis. From detailed kinetic analysis, the reaction mechanism has been derived: dehydrogenated guanosine radical G(-H)* and triplet guanosine ³G* were produced via electron transfer and triplet-triplet energy transfer respectively. The half-life time of ³G* has been determined to be 7.4 μs, and the quenching rate constant of Mn²⁺ on it was obtained to be 1.9 × 10⁸ M^-1 s^-1. Based on pH titration, the pKa value of ³G* was also obtained to be 8.7.     

Molecular Recognition of Acetylaminofluorene-and Aminofluorene-modified Guanosine

Abstract

New receptors based upon a carboxamidonaphthyridine unit derivatized with the polyaromatic segments [4-(1-pyrenyl)butyroylamino] and [3-(9-anthracenyl)propanoylamino] have been synthesized and studied by ¹H NMR titration in their binding interaction to the guanosine-C8 adducts from the carcinogens 2-acetylaminofluorene and 2-aminofluorene. The high binding energy, in comparison with that of the non-derivatized analogues, is rationalized in terms of a bi-site interaction (hydrogen bonding and aromatic stacking). Although many factors may contribute to the strength of the host-guest complexes described herein, it appears that in some cases π-π interaction may induce a concomitant bending in the hydrogen bond system.     

Quantum dot nanocrystals having guanosine imprinted nanoshell for DNA recognition

Molecular imprinted polymers (MIPs) as a recognition element for sensors are increasingly of interest and MIP nanoparticles have started to appear in the literature. In this study, we have proposed a novel thiol ligand-capping method with polymerizable methacryloylamido-cysteine (MAC) attached to CdS quantum dots (QDs), reminiscent of a self-assembled monolayer and have reconstructed surface shell by synthetic host polymers based on molecular imprinting method for DNA recognition. In this method, methacryloylamidohistidine-platinium (MAH-Pt(II)) is used as a new metal-chelating monomer via metal coordination-chelation interactions and guanosine templates of DNA. Nanoshell sensors with guanosine templates give a cavity that is selective for guanosine and its analogues. The guanosine can simultaneously chelate to Pt(II) metal ion and fit into the shape-selective cavity. Thus, the interaction between Pt(II) ion and free coordination spheres has an effect on the binding ability of the CdS QD nanosensor. The binding affinity of the guanosine imprinted nanocrystals has investigated by using the Langmuir and Scatchard methods, and experiments have shown the shape-selective cavity formation with O6 and N7 of a guanosine nucleotide (K(a) = 4.841x10(6) mol L(-1)) and a free guanine base (K(a) = 0.894x10(6) mol L(-1)). Additionally, the guanosine template of the nanocrystals is more favored for single stranded DNA compared to double stranded DNA.     

Conformational studies on the four stereoisomers of the novel anticholinergic 4-(dimethylamino)-2-phenyl-2-(2-pyridyl)pentanamide

Summary To interpret differences in the anticholinergic activity among the four steroisomers of 4-(dimethylamino)-2-phenyl-2-(2-pyridyl)pentanamide (1–4), we performed conformational studies using the semiempirical molecular orbital method. The structures of the global minimum-energy conformations obtained for 1–4, however, could not explain the different activities, particularly in terms of distances between the essential pharmacophores. We thus implemented superimposition studies, using the energetically stable conformations of the most active stereoisomer, 1(2S,4R), as a template. The energy penalties for a conformation change of the less active stereoisomers 2–4 from their global minimum-energy structure to a new conformation, fitting onto the global minimum-energy conformation of 1, appear to account for the differences in the pharmacological potency better than using the other conformations of 1 as a template. We thus presume that the global minimum-energy conformation of 1 is closely related to the bioactive conformation for these anticholinergics, and also that the pharmacological potency is linked to how readily these substances can change their conformations to fit the muscarinic receptor.     

Crystal structure and sequential dehydration transitions of disodium guanosine 5′‐monophosphate tetrahydrate

Disodium guanosine 5′‐monophosphate was reported previously to crystallize as both the tetrahydrate and the heptahydrate. We herein report a determination of the molecular and crystal structures of the title tetrahydrated salt, 2Na⁺·C₁₀H₁₂N₅O₈P²⁻·4H₂O. It was found that the structure differs markedly from that of the heptahydrate, but greatly resembles that of disodium deoxyguanosine 5′‐monophosphate tetrahydrate. The C2′—O2′H moiety of ribose is surrounded by hydrophilic moieties and is disordered over two sites. The sugar puckering mode is O4′‐endo‐C1′‐exo at both sites and the conformation around the C4′—C5′ bond is gauche–trans. Powder X‐ray diffraction and thermal analyses revealed that the temperature‐controlled transition from the tetrahydrate to the anhydride proceeded through three intermediate phases between 40 and 60 °C at 0% relative humidity. Large induction periods were observed.     

Synthesis of Guanosine 5′-(β-L-Fucopyranosyl)-Diphosphate Revisited

ABSTRACT

It will be demonstrated that a successful synthesis of β-L-fucopyranose-1-phosphate (2), a key intermediate in the preparation of guanosine 5′-(β-L-fucopyranose)-diphosphate (1), strongly depends on the nature of the acyl protecting groups for the non-anomeric hydroxyl functions. Thus, the perbenzoylated, instead of peracetylated, α-L-fucopyranosyl trichloroacetimidate (11) or the corresponding ethyl β-thiofucopyranoside proved to be a convenient starting compound for the preparation of 2. Further, condensation of N,N′-dicyclohexyl-4-morpholinecarboxamidinium guanosine 5′-morpholidophosphate with excess 2 gave the title compound without concomitant formation of bisguanosine-5′-diphosphate (16).     

Synthesis,crystal structure,and antimicrobial activity of a series of cobalt(III) Schiff base complexes

Abstract

A series of six new mononuclear Schiff base complexes, 1–6 of cobalt(III) of the general formula, [CoLX] or its adduct with methanol, is reported. The pentadentate Schiff base ligand (H₂L) was obtained by the condensation of N-(3-aminopropyl)-N-methylpropane-1,3-diamine with 1-(2-hydroxyphenyl)ethanone (H₂L¹) or 1-(2-hydroxyphenyl)propan-1-one (H₂L²). X stands for the pseudohalides, N₃^–, N(CN)₂^? , and NCS^–. The complexes have been synthesized by the reaction of equimolar amounts of cobalt(II) nitrate with H₂L¹ or H₂L² in the presence of the respective pseudohalide in methanol medium. All the complexes have been characterized by microanalytical, spectroscopic, single crystal XRD (except 3), and other physicochemical studies. Structural studies reveal that the central Co(III) ion in 1, 2, 4, 5, and 6 adopts a distorted octahedral geometry with a CoN₄O₂ chromophore. Weak intermolecular H-bonding and/or π-interactions are operative in these complexes to bind the molecular units. The antimicrobial activity of all the complexes and their constituent Schiff bases has been tested against some common bacteria and fungi.     

Spectral studies of the interaction between sanguinarine and guanosine

The interaction between sanguinarine and guanosine was investigated by using UV-vis and fluorescence spectra at pH 7.2. The binding of sanguinarine to guanosine was substantiated by the hypochromism and bathochromism in the absorption spectra and the emission quenching in fluorescence spectra. The fluorescence lifetime results, the varieties of the fluorophore absorption spectra and the decrease of the binding constant with the increasing temperature all indicate that the fluorescence quenching is static. The ratio and constant of the binding cytidine to sanguinarine are 2 and 6.44 × 10^7, respectively. The result shows that the binding of sanguinarine to guanosine is not only exothermic but also entropy-driven with △H=-8.53kJ/mol, AS = 0.12 kJ/（molK）, and AG =-44.57 kJ/mol at 298.15 K.     

Nucleotide Platination Induced by Visible Light

A two-step photoreaction is induced by irradiation of the Pt^IV complex trans,cis-[Pt(OCOCH₃)₂I₂(en)] with visible light in the presence of guanosine 5′-monophosphate (5′-GMP; see scheme): A photoinduced ligand exchange followed by photoreduction gives the bis-GMP adduct of [Pt(en)]²⁺. Although the dihydroxodiiodo complex also undergoes a photoinduced ligand exchange, no reaction with the nucleotide was observed.     

A quantum-mechanical study of the interaction of glyoxal with guanine

A quantum molecular study by the SCFab initio method of the interaction of glyoxal with guanine provides for the formation of a stable covalent adduct in which the glyoxal fragment forms a complementary cyclic ring attached to the imino N₁ and amino N₂ atoms of guanine with the concomitant migration of the N-bonded H atoms to the oxygens of glyoxal. The reaction should proceed in two steps. The most plausible mechanism involves as the first step the interaction of a carbonyl group of glyoxal with the amino group of guanine followed by a similar interaction at the imino group of guanine, rather than the reverse order of interactions. The respective energy barriers are 49.7 and 63.9 kcal/mole. The intermediate product is also more stable when the adduct occurs first at N₂:30.7 kcal/mole versus 17.9 kcal/mole for the adduct at N₁.     

Expeditious Synthesis of a Trisubstrate Analogue for α(1→3)Fucosyltransferase

Abstract

The synthesis of cyclohexyl 2-acetamido-2-deoxy-3-O-{2-O-[2-(guanosine 5′-O-phosphate)ethyl]-α-L-fucopyranosyl}-β-D-glucopyranoside (1), a potential inhibitor of α(1→3)fucosyltransferases, is described. Target compound 1 was assembled via fucosylation of cyclohexyl 2-acetamido-2-deoxy-4,6-O-isopropylidene-β-D-glucopyranoside (6) with ethyl 2-O-[2-(benzoylhydroxy)ethyl]-3,4-O-isopropylidene-1-thio-β-L-fucopyranoside (5) followed by debenzoylation, subsequent condensation of the resulting compound with 3′,4′ -di-O-benzoyl-5′ -O-(2-cyanoethyl-N,N-diisopropylphosphoramidite)-2-N-diphenylacetylguanosine (10) and deprotection.     

Studies on DNA binding behavior of biologically active Cu(II) complexes of Schiff bases containing acyl pyrazolones and 2-ethanolamine

Pyrazolone derivatives (Z)-4-((2-hydroxyethylimino)(p-tolyl)methyl)-3-methyl-1-phenyl-1H-pyrazol-5(4H)-one [PMP-EA] (1), (Z)-1-(3-chlorophenyl)-4-((2-hydroxyethylimino)(p-tolyl)methyl)-3-methyl-1H-pyrazol-5(4H)-one [MCPMP-EA] (2), and (Z)-4-((2-hydroxyethylimino)(p-tolyl)methyl)-3-methyl-1-p-tolyl-1H-pyrazol-5(4H)-one [PTPMP-EA] (3) have been synthesized and characterized. The molecular geometry of 2 has been determined by single-crystal X-ray study. These ligands exist in amine-one tautomeric form in the solid state. Three copper(II) complexes, [Cu(PMP-EA)(H₂O)₂] (4), [Cu(MCPMP-EA)(H₂O)₂] (5), and [Cu(PTPMP-EA)(H₂O)₂] (6), respectively, have been synthesized using these ligands and characterized by microanalytical data, molar conductivity, IR, UV–Visible, FAB-Mass, magnetic measurement, TG-DTA studies, and ESR spectral studies; Cu(II) is five-coordinated with [ML(H₂O)₂] composition. The interaction of the complexes with CT-DNA (calfthymus) was investigated using different methods. The results suggest that the copper complexes bind to DNA via intercalation and can quench the fluorescence intensity of EB bound to DNA.     

Hydrogen-bonded network and enforced supramolecular cavities in molecular crystals: An orthogonal aromatic-triad strategy. Guest binding,molecular recognition,and molecular alignment properties of a bisresorcinol derivative of anthracene in the crystalline state

Abstract

Crystallization of an orthogonal resorcinol-anthracene-resorcinol compound 1a (host) from an ester solvent such as alkyl benzoate (guest) affords a 1:2 host-guest adduct 1a·2(ester). An essential aspect of the crystal structures of ethyl, propyl, and isobutyl benzoate adducts (space group, P2₁/n) and also that of methyl benzoate adduct (C2/c) is an extensive hydrogen-bonded network of host 1a, leading to a molecular sheet composed of hydrogen-bonded polyresorcinol chains and anthracene columns. This network generates well-defined, cyclophane-like supramolecular cavities, which incorporate two alkyl benzoate molecules in a highly selective manner via a combination of essential host-guest hydrogen-bonding and what may be called the cavity-packing effect. The selectivity factor between methyl benzoate (the lowest-affinity guest) and isobutyl benzoate (the highest-affinity guest) is 1:70 under competitive conditions. The actual geometry of the cavity is somehow dependent on and hence induced-fit adjustable to the guest structures by manipulating the intramolecular (anthracene-resorcinol dihedral angle) and intermolecular conformation (tilt angle between two hydrogen-bonded resorcinol rings) of compound 1a as well as the sheet-to-sheet distance. The adducts 1a·2(guest) can also be obtained by solid-state guest-exchange or guest-binding, respectively, using a preformed adduct or guest-free apohost dipped in an appropriate guest solvent. The methyl benzoate adducts obtained in these ways exhibit the same X-ray powder diffraction pattern as the genuine single-crystal obtained by direct crystallization of host 1a from methyl benzoate. Thus, even internal supramolecular cavities maintained by the hydrogen-bonded network are readily accessible to molecules in bulk solution. In addition, they undergo an induced-fit adjustment to a guest molecule newly added by the guest-exchange or the guest-binding process, during which the crystallinity is maintained. The potential use of symmetrical and divergent multiple hydrogen-bonding sites with an orthogonal aromatic spacer (orthogonal aromatic-triad strategy) is discussed in terms of a tool to construct a new class of porous organic crystals that show novel molecular recognition, crystalline-state guest-binding, and crystalline-phase molecular alignment properties.