skip to main content


Title: Phosphates form spectroscopically dark state assemblies in common aqueous solutions
Phosphates and polyphosphates play ubiquitous roles in biology as integral structural components of cell membranes and bone, or as vehicles of energy storage via adenosine triphosphate and phosphocreatine. The solution phase space of phosphate species appears more complex than previously known. We present nuclear magnetic resonance (NMR) and cryogenic transmission electron microscopy (cryo-TEM) experiments that suggest phosphate species including orthophosphates, pyrophosphates, and adenosine phosphates associate into dynamic assemblies in dilute solutions that are spectroscopically “dark.” Cryo-TEM provides visual evidence of the formation of spherical assemblies tens of nanometers in size, while NMR indicates that a majority population of phosphates remain as unassociated ions in exchange with spectroscopically invisible assemblies. The formation of these assemblies is reversibly and entropically driven by the partial dehydration of phosphate groups, as verified by diffusion-ordered spectroscopy (DOSY), indicating a thermodynamic state of assembly held together by multivalent interactions between the phosphates. Molecular dynamics simulations further corroborate that orthophosphates readily cluster in aqueous solutions. This study presents the surprising discovery that phosphate-containing molecules, ubiquitously present in the biological milieu, can readily form dynamic assemblies under a wide range of commonly used solution conditions, highlighting a hitherto unreported property of phosphate’s native state in biological solutions.  more » « less
Award ID(s):
2108205 2004217
NSF-PAR ID:
10430630
Author(s) / Creator(s):
; ; ; ; ; ; ; ; ;
Date Published:
Journal Name:
Proceedings of the National Academy of Sciences
Volume:
120
Issue:
1
ISSN:
0027-8424
Format(s):
Medium: X
Sponsoring Org:
National Science Foundation
More Like this
  1. Abstract

    Phosphate is an essential anion in the human body, comprising approximately 1% of the total body weight, and playing a vital role in metabolism, cell membranes, and bone formation. We have recently provided spectroscopic, microscopic, and computational evidence indicating that phosphates can aggregate much more readily in solution than previously thought. This prior work provided indirect evidence through the observation of unusual P NMR relaxation and line‐broadening effects with increasing temperature. Here, we show that, under conditions of slow exchange and selective RF saturation, additional features become visible in chemical exchange saturation transfer (CEST) experiments, which appear to be related to the previously reported phosphate clustering. In particular, CEST shows pronounced dips several ppm upfield of the main phosphate resonance at low temperatures, while direct P spectroscopy does not produce any signals in that range. We study the pH dependence of these new spectroscopic features and present exchange and spectroscopic parameters based on fitting the CEST data. These findings could be of importance in the investigation of phosphate dynamics, especially in the biological milieu.

     
    more » « less
  2. Abstract

    Supramolecular macrocyclic forces have been used to trap phytate,myo‐inositol‐1,2,3,4,5,6‐hexakisphosphate, a key bioanion with multiple roles in metabolic processes. Due to the complex chemistry of six multivalent phosphates surrounding the small, cyclic inositol framework, crystallographic information of simple phytate salts has been elusive. This report represents a combined crystallographic, theoretical, and solution binding investigation of a supramolecular macrocyclic complex of phytate. Together, the results provide significant insight to phytate's intramolecular and intermolecular interactions at the microenvironment level. The macrocycle‐phytate aggregates consist of phytate anionic pairs, each partly sandwiched by two 24‐membered, amide/amine‐based cationic macrocycles. The phytate ion pairs hold the tetrameric macrocyclic array together by six strong intermolecular hydrogen bonds. Both phytates crystallize in 1a5e phosphate conformations (one axial (P2) and five equatorial phosphates). Solution NMR binding studies in 1 : 1 DMSO‐d6 : D2O indicate 2 : 1 macrocycle:phytate associations, suggesting that the sandwich‐like nature of the complex holds together in solution. DFT studies indicate the likely occurrence of dynamic intramolecular interchange of phosphate protons, as well as important roles for the axial (P2) phosphate in both intramolecular and intermolecular hydrogen bonding interactions.

     
    more » « less
  3. Three carboxamidequinoline ligands were synthesized and their complexes with Eu 3+ were used for recognition and detection of organic/inorganic phosphates in water. The signal transduction process is based on an “On–Off–On” switch in the fluorescence signal utilizing changes in the intramolecular charge transfer (ICT). The fluorescence emission of ligands is quenched upon exposure to the Eu 3+ (Off signal). Following the addition of the phosphate analytes the ligand–Eu 3+ complex disassembles, which results in the regeneration of the original emission of the ligand (On signal). In general, the Eu 3+ complexes show higher affinity towards adenosine 5′-triphosphate (ATP) and lower affinity to other phosphates, namely adenosine 5′-diphosphate (ADP), adenosine 5′-monophosphate (AMP), pyrophosphate (H 2 P 2 O 7 2− , PPi), and dihydrogenphosphate (H 2 PO 4 − , Pi). 
    more » « less
  4. null (Ed.)
    To treat impairments in hard tissues or overcome pathological calcification in soft tissues, a detailed understanding of mineralization pathways of calcium phosphate materials is needed. Here, we report a detailed mechanistic study of hydroxyapatite (HA) mineralization pathways in an artificial saliva solution via in situ liquid cell transmission electron microscopy (TEM). It is found that the mineralization of HA starts by forming ion-rich and ion-poor solutions in the saliva solution, followed by coexistence of the classical and nonclassical nucleation processes. For the nonclassical path, amorphous calcium phosphate (ACP) functions as the substrate for HA nucleation on the ACP surface, while the classical path features direct HA nucleation from the solution. The growth of HA crystals on the surface of ACP is accompanied by the ACP dissolution process. The discoveries reported in this work are important to understand the physiological and pathological formation of HA minerals, as well as to engineer the biomineralization process for bone healing and hard tissue repairs. 
    more » « less
  5. Based on Coulomb's Law alone, electrostatic repulsion between two anions is expected to prevent their dimerization. Contrary to that idea, this Tutorial Review will present evidence showing that anion–anion dimers of protic hydroxyanions can form readily, and describe conditions that facilitate their formation. From X-ray crystal structures, we learn that hydroxyanions dimerize and oligomerize by overcoming long-range electrostatic opposition. Common examples are hydroxyanions of phosphate, sulfate, and carbonate, often in partnership with charged and neutral receptors. Short-range hydrogen bonds between anionic donors and acceptors are defined as anti-electrostatic hydrogen bonds (AEHBs) with insight from theoretical studies. While anion dimers are difficult to identify unequivocally in solution, these solution dimers have recently been definitively identified. The development of the supramolecular chemistry of anion–anion dimers has led to applications in hierarchical assemblies, such as supramolecular polymers and hydrogen bonded organic frameworks. 
    more » « less