Search for: All records

Nuclear spin relaxation mechanisms are often difficult to isolate and identify, especially in molecules with internal flexibility. Here we combine experimental work with computation in order to determine the major mechanisms responsible for 31 P spin–lattice and singlet order (SO) relaxation in pyrophosphate, a physiologically relevant molecule. Using field-shuttling relaxation measurements (from 2 μT to 9.4 T) and rates calculated from molecular dynamics (MD) trajectories, we identified chemical shift anisotropy (CSA) and spin–rotation as the major mechanisms, with minor contributions from intra- and intermolecular coupling. The significant spin–rotation interaction is a consequence of the relatively rapid rotation of the –PO 3 2− entities around the bridging P–O bonds, and is treated by a combination of MD simulations and quantum chemistry calculations. Spin–lattice relaxation was predicted well without adjustable parameters, and for SO relaxation one parameter was extracted from the comparison between experiment and computation (a correlation coefficient between the rotational motion of the groups). 

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. 

31 P NMR spectroscopy and the study of nuclear spin singlet relaxation phenomena are of interest in particular due to the importance of phosphorus-containing compounds in physiology. We report the generation and measurement of relaxation of 31 P singlet order in a chemically equivalent but magnetically inequivalent case. Nuclear magnetic resonance singlet state lifetimes of 31 P pairs have heretofore not been reported. Couplings between 1 H and 31 P nuclei lead to magnetic inequivalence and serve as a mechanism of singlet state population conversion within this molecule. We show that in this molecule singlet relaxation occurs at a rate significantly faster than spin–lattice relaxation, and that anticorrelated chemical shift anisotropy can account for this observation. Calculations of this mechanism, with the help of molecular dynamics simulations and ab initio calculations, provide excellent agreement with the experimental findings. This study could provide guidance for the study of 31 P singlets within other compounds, including biomolecules. 

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.