Triphosphate Anion Research Articles

Transport of Nucleoside Triphosphates into Cells by Artificial Molecular Transporters.

Chemically modified nucleoside triphosphates (NTPs) are widely exploited as unnatural metabolites in chemical biology and medicinal chemistry. Because anionic NTPs do not permeate cell membranes, their corresponding neutral precursors are employed in cell-based assays. These precursors become active metabolites after enzymatic conversion, which often proceeds insufficiently. Here we show that metabolically-active NTPs can be directly transported into eukaryotic cells and bacteria by the action of designed synthetic nucleoside triphosphate transporters (SNTTs). The transporter is composed of a receptor, which forms a non-covalent complex with a triphosphate anion, and a cell-penetrating agent, which translocates the complex across the plasma membrane. NTP is then released from the complex in the intracellular milieu and accumulates in nuclei and nucleoli in high concentration. The transport of NTPs proceeds rapidly (seconds to minutes) and selectively even in the presence of other organic anions. We demonstrate that this operationally simple and efficient means of transport of fluorescently labelled NTPs into cells can be used for metabolic labeling of DNA in live cells.

Di- and Triphosphate Recognition and Sensing with Mono- and Dinuclear Fluorescent Zinc(II) Complexes: Clues for the Design of Selective Chemosensors for Anions in Aqueous Media.

The synthesis of a new ligand (L1) containing two 1,4,7-triazacyclononane ([9]aneN3 ) moieties linked by a 4,5-dimethylenacridine unit is reported. The binding and fluorescence sensing properties toward Cu2+ , Zn2+ , Cd2+ , and Pb2+ of L1 and receptor L2, composed of two [9]aneN3 macrocycles bridged by a 6,6''-dimethylen-2,2':6',2''-terpyridine unit, have been studied by coupling potentiometric, UV/Vis absorption, and emission measurements in aqueous media. Both receptors can selectively detect Zn2+ thanks to fluorescence emission enhancement upon metal binding. The analysis of the binding and sensing properties of the Zn2+ complexes toward inorganic anions revealed that the dinuclear Zn2+ complex of L1 selectively binds and senses the triphosphate anion (TP), whereas the mononuclear Zn2+ complex of L2 displays selective recognition of diphosphate (DP). Binding of TP or DP induces emission quenching of the Zn2+ complexes with L1 and L2, respectively. These results are exploited to discuss the role played by pH, number of coordinated metal cations, and binding ability of the bridging units in metal and/or anion coordination and sensing.

From Flexible to Constrained Tris(tetraamine) Ligands: Synthesis, Acid–Base Properties, and Structural Effect on the Coordination Process with Nucleotides

AbstractThis work presents the synthesis and characterization of L2 and L3, which formed with the previously described ligand L1 a family of three tris(tetraamine) ligands based on a mesitylenyl spacer. The trimeric ligands differ from one to the other by the nature of their tetraamine moieties: triethylenetetraamine (L1), cyclen (L2), and constrained cyclen (L3). The impact of the rigidification of the structure from L1 to L3 on the acid–base properties is investigated, and the behavior of the ligands towards triphosphate (TriP), adenosine monophosphate (AMP), adenosine diphosphate (ADP), and adenosine triphosphate (ATP) anion receptors is explored by potentiometric investigations. To characterize and understand the driving forces implicated in the supramolecular assemblies, results are supported by 1H and 31P NMR measurements performed over a wide pH range. ATP appears as the most complexed anion and particular attention is given to comparison with its inorganic triphosphate analogue to highlight the contribution of π‐stacking interactions. Results unambiguously show the various coordination scheme induced by the structure of the ligands.

Sensing of adenosine-5′-triphosphate anion in aqueous solutions and mitochondria by a fluorescent 3-hydroxyflavone dye

The current work demonstrates the formation of complexes between the tetraanion adenosine-5′-triphosphate (ATP) and the flavone derivative 3-hydroxy-4′-(dimethylamino)flavone (FME). Two kinds of complexes are evidenced. The higher affinity ATP–FME complex corresponds to a stacking of the two aromatic molecules and leads to a strong hypochromicity of the absorption spectrum of the dye. The lower affinity (ATP) 2–FME complex results in a strong increase of the fluorescence intensity (∼20-fold), due mainly to the appearance of the anionic form of FME, as shown by the important red shift (60 nm) of both excitation and emission spectra. Molecular modeling indicates that this anionic form results from the deprotonation induced by the influence of the tetra-charged triphosphate group of the ATP molecules. Using its strong enhancement of fluorescence intensity in the presence of ATP, the dye was used successfully to monitor the succinate-induced production of endogenous ATP in mitochondria. As a consequence, FME can be considered as a starting point to design efficient ATP sensors.

A potentiometric and NMR investigation of triphosphate recognition by linear and bismacrocyclic octaamines possessing a 1,4-xylenyl linker

The host–guest binding interaction between two symmetrical octaazaligands containing a para-xylenyl spacer and triphosphate anion is reported. The ligands studied here are the para-xylenylbiscyclam (AMD3100) and its linear analogue the para-xylenylbis-1,4,8,11-tetraazaundecane, obtained by linking the two corresponding cyclic or open-chain tetraamines. The purpose of this study is to evaluate the effect of these two different architectures on the factors governing the recognition of the triphosphate anion. The results clearly indicate that compared to its biscyclam analogue, the linear octaamine presents a better triphosphate recognition in the whole p[H] range. On the one hand, the capacity of the octaamine to form easily polyprotonated species constitutes the driving force favouring the formation of ternary species, and, on the other hand, the adaptability of this receptor in organizing hydrogen bonds represents also a benefit for the binding of triphosphate substrate. The polyprotonated ligand is sufficiently flexible to wrap itself round the anion in a way to achieve an optimal fit for the best host–guest recognition.

Complexation of the triphosphate anion: tuning the structure of cyclen based macrotricycles with 1,3-dimethylbenzene and 2,6-dimethylpyridine linkers. A potentiometric and NMR study

The host-guest interaction between orthophosphate, pyrophosphate and triphosphate anions and three cyclen-based macrotricyclic ligands was investigated by potentiometric measurements and NMR spectroscopy. The ligands differ from one another by the nature of their spacers, which are 1,3-dimethylbenzene (TMC), 2,6-dimethylpyridine (TPyC) or a combination of the two (TMPyC). In aqueous solution, each ligand gave protonated species that further formed ternary complexes after binding with anions; these complexes were analyzed as a result of hydrogen bond formation and coulombic attraction between the organic host and the inorganic guest. The equilibrium constants found for all the detected species are reported and the selectivity, illustrated with species distribution diagrams, is discussed. The results unambiguously showed that the ligand possessing a single supplementary anchoring site (the pyridinyl spacer) exhibited the greatest affinity for the phosphate species in a large p[H] range.

Synthesis and Structure Characterization of Piperazine-1,4-diium Triphosphate

[C 4 N 2 H 12 ] 2 HP 3 O 10 · H 2 O, a new hydrate organic cation monohydrogeno-triphosphate, has been synthesized and characterized by X-ray diffraction, IR spectroscopy, mass angle spinning (MAS), NMR, and thermal analysis. The title compound crystallizes in a monoclinic unit cell with a = 11.072(5), b = 12.324(2), c = 13.122(5) Å, β = 93.85(5)°, Z = 4, V = 1787(1) Å 3 , and a noncentrosymmetric space group P2 1 (no. 4). Crystal structure is determined and refined to R = 0.057 using 4262 independent reflections. The atomic arrangement can be described as a typical layers organization. Layers built by HP 3 O 10 4− anions and water molecules are parallel to the (a, b) planes. Between these layers the piperazinium cations, which form hydrogen bonds with oxygen atoms of the triphosphate anion, are located.

Cluster Phase Reactions: Alkylation of Triphosphate and DNA Anions with Alkylammonium Cations

Electrospray ionization is used to create what are demonstrated to be highly reactive, metastable clusters of alkylammonium cations and triphosphate dianions. Collision-induced activation of the clusters leads to either S_N2 or E2 reactions, resulting in the formation of an alkyltriphosphate or triphosphate anion, respectively. The series of tetraalkylammonium ions from tetramethylammonium to tetraheptylammonium is studied. The S_N2 pathway is favored over the E2 pathway. The replacement of a proton in the triphosphate dianion with a sodium cation in these clusters leads to enhanced yields of the E2 product. PM5 calculations of the reaction energetics correlate well with the observed product distributions. The doubly deprotonated DNA oligomer CCGG can also be alkylated by CID of its cluster with a tetraalkylammonium cation. The cluster phase reactions described in this study serve to illustrate a general scheme which we propose for the rational design of ionic clusters where reactivity is ensured by inhibiting direct dissociation. The experimental methodology implied by the cluster design criteria expands the utility of small molecular clusters for structure−reactivity studies and directed chemical synthesis.

Conformation-dependent intermolecular interaction energies of the triphosphate anion with divalent metal cations. Application to the ATP-binding site of a binuclear bacterial enzyme. A parallel quantum chemical and polarizable molecular mechanics investigation.

We have explored the conformation-dependent interaction energy of the triphosphate moiety, a key constituent of ATP and GTP, with a closed-shell divalent cation, Zn2+, used as a probe. This was done using the SIBFA polarizable molecular mechanics procedure. We have resorted to a previously developed approach in which triphosphate is built out from its elementary constitutive fragments, and the intramolecular, interfragment, interaction energies are computed simultaneously with their intermolecular interactions with the divalent cation. This approach has enabled reproduction of the values of the intermolecular interaction energies from ab initio quantum-chemistry with relative errors <3%. It was extended to the complex of a nonhydrolyzable analog of ATP with the active site of a bacterial enzyme having two Mg2+ cations as cofactors. We obtained following energy-minimization a very close overlap of the ATP analog over its position from X-ray crystallography. For models of the ATP analog-enzyme complex encompassing up to 169 atoms, the values of the SIBFA interaction energies were found to match their DFT counterparts with relative errors of <2%.

Conformational analysis of three pyrophosphate model species: Diphosphate, methyl diphosphate, and triphosphate

Whereas theoretical investigations of the energetic origin of hydrolyzing a pyrophosphate linkage abound, few studies have focused on the energetics of the rotation of this linkage. This less-studied property of the pyrophosphate linkage was investigated here by use of ab initio calculations to characterize the conformational space of three model species of pyrophosphate anions: diphosphate (P2O), methyl diphosphate (CH3P2O), and triphosphate (P3O). By carefully selecting conformationally distinct rotational isomers of the three model compounds, their potential surfaces were thoroughly explored. In addition to showing that a terminal phosphate group is indeed very flexible in accordance with the general perception of free rotation, a number of intriguing features of this linkage emerged from the ab initio calculations, which include an influential sp3-hybridized CHO intramolecular hydrogen bond in methyl diphosphate, and a highly restricted rotational space pertaining to the central pyrophosphate linkage of the triphosphate anion. These ab initio findings were then evaluated by, and proved insightful in, follow-up examinations of experimentally determined complex structures of proteins and their dinucleotide or adenine and guanine triphosphate ligands. © 1999 John Wiley & Sons, Inc. J Comput Chem 20: 1702–1715, 1999

Synthetic Polyphosphonates, Polyphosphates, and Phosphonocarboxylates as Allosteric Effectors of Hemoglobin

Abstract Synthetic phosphonocarboxylates, diphosphonates, polyphosphates, and some triphosphonic acids were studied as effectors in the binding of dioxygen to human hemoglobin. Ethane-1,2-diphosphonic acid, and triphosphate anion were strong effectors.

Calculation of the vibrational spectrum of the triphosphate anion

A large amount of experimental material on the vibrational spectra of phosphates with different degrees of condensation has now been accumulated. In the interpretation of these spectra empirical assignment of the vibrational frequencies based on symmetry considerations and an assumption regarding the characteristic nature of the vibrations of different groups of atoms is widely used. Calculations of the vibrational spectra, performed for a small number of compounds (see, e.g., [1-6], in general confirm the results of empirical assignment and hence confirms the assumption regarding the characteristic nature of a number of stretching vibrations in different phosphates. However in [7] on the basis of the calculation of the vibrational spectrum of the triphosphate anion P30~ a conclusion was reached that only some of the frequencies can be assigned to vibrations of specific groups of atoms (the terminal PO~, middle P02, and bridge POP, groups), but the majority of normal modes are mainly mixed. This, however, contradicts the results of the calculation performed for the P30~7 anion in [6]. Taking into account the existence of limitations and assumptions in [6, 7], whose effect on the interpretation of the spectra will be discussed below, we recalculated the vibrational spectrum of triphosphate anion to resolve the existing contradiction.

Crystal growth of sodium triphosphate hexahydrate from aqueous solutions

The growth rate (R) of the various crystallographic faces of a single sodium triphosphate hexahydrate crystal, Na5P3O10·6H2O, is measured as a function of the supersaturation in solutions prepared from both anhydrous phases: sodium triphosphate Phase I (Na5P3O10-I) and sodium triphosphate Phase II (Na5P3O10-II). It is very striking that at the same degree of supersaturation the linear growth rate of the (100) faces and the (001) faces of the hexahydrate in a solution made of Phase I is much higher than in a solution of Phase II. Since it has been shown that the structure of the hexahydrate crystals is the same in both cases there must be a difference in structure of the solute in the solution. Particularly the difference in the structure of the P3O10 group, and in the sodium co-ordination between the crystal structures of Phase I and Phase II, is determinative of the number and shape of the ions in the supersaturated solutions. In view of this we assume that the rupture of the bond between the sodium cation and triphosphate anion is the rate-determining step for crystal growth of hexahydrate.