Metal ions as novel polarizing agents for dynamic nuclear polarization enhanced NMR spectroscopy

High-spin complexes of Gd(III) and Mn(II) were introduced as polarizing agents (PAs) for solid-state dynamic nuclear polarization (DNP) in 2011. This dissertation was undertaken in 2013, with the intention of exploring these PAs further. Major goals of this work were to understand their DNP mechanism(s) and explore their application in biomolecular research. This cumulative thesis details the methods, advantages, and practical implications of using high-spin PAs for MAS DNP. Data from electron paramagnetic resonance (EPR) and NMR spectroscopy are discussed for a complete understanding of DNP mechanisms. Out of the two main mechanisms − solid effect (SE) and cross effect (CE − active under experimental conditions of solid-state DNP, commonly used nitroxide PAs evoke CE owing to their broad EPR spectra. On the other hand, DNP mechanisms evoked by high-spin metal ions seem non-trivial due to additional features (originating from spin-orbit coupling or zero field splitting) in their EPR spectra. The features of the EPR signal generally influence the shape of enhancement profiles. Therefore, the metal ion with a simpler EPR signal i.e., Gd(III) , is chosen as the starting point for the investigation of DNP mechanisms. Varying concentrations (2, 10, 20 mM) of a water-soluble and stable complex Gd-DOTA was dissolved as the PA in a glycerol-water solution of 13C,15N - urea. Field profiles of DNP enhancement on each nuclear type (1H, 13C, and 15N) establishes SE as the active DNP mechanism at the smallest PA concentration (2 mM). This confirms the theoretical predictions that narrow line width of the Gd(III) EPR signal arising from the central transition (CT, ms = -1/2 +1/2) allows for resolved SE DNP. However, that is no longer the case at higher PA concentrations of 10 and 20 mM. At higher Gd(III) concentrations, the CE mechanism contributes significantly and varies with nuclear Larmor frequency (ωn) of the concerned nuclei. The enhancement maxima shifts towards the EPR resonance as the contribution from CE increases. This shift is evident in the field profiles of 15N and 13C, whereas that of 1H is least influenced. This observation can be explained by combining theoretical estimates with the experimental data; the CE is evoked by increased dipolar coupling (Dee) – a prerequisite for CE – between neighboring Gd(III) spins as the statistical inter-spin distance shortens at elevated concentrations. This finding is important because the knowledge of active DNP mechanisms is essential for accurate interpretation of results from DNP experiments. From the experiments on Gd-DOTA it becomes clear that concentration, inter-spin distances, and hence induced Dee are intertwined. In order to explicitly address the influence of inter-spin distances on DNP mechanisms we started a collaboration with the group of Adelheid Godt (Bielefeld). In this collaborative project, bis-complexes of the type Gd(III)-spacer-Gd(III) with variable spacer lengths were investigated. These PAs provided an excellent model system where the influence of only inter-spin distances can be determined for a fixed Gd(III) concentration. A small PA concentration of 4 mM is used to ensure absence of significant inter-molecular dipolar interactions. A mono-Gd complex of similar geometry and chemistry is taken as a reference for SE DNP. The mono-Gd complex yields enhancements arising from SE as expected from negligible inter-molecular Dee. The contribution of CE increases as the inter-spin distances between Gd(III) ions become shorter going from 3.4 nm 2.1 nm 1.4 nm 1.2 nm due to corresponding increase in Dee. The extent of CE on ωn follows the same trend as for Gd-DOTA. Highest CE contribution is observed on nuclei with the smallest ωn 15N because smaller ωn approaches the width of the EPR signal, this is an additional requirement for CE DNP. The field position for maximum DNP enhancement corresponding to Gd-DOTA, is used for DNP experiments on Ubiquitin with an attached Gd-tag as PA. The success of DNP on this sample illustrates the possibility of site-directed DNP with metal ions tags as PAs. As a perspective Gd-tags can be used to examine change in conformation of a protein that would give higher enhancements due to CE if two Gd(III) labeled domains are closer in space. In a separate project, Mn(II) (s=5/2) bound to the divalent site of a hammerhead ribozyme was used as a PA which resulted in the first demonstration of intra-complex DNP using an intrinsically bound metal ion PA

Effect of cyanobacterial exopolysaccharides on salt stress alleviation and seed germination

Abstract: Effect of exopolysaccharides (EPS) produced by a consortium of cyanobacteria on germination of three crops wheat, maize and rice was studied at different salt concentrations. Production of EPS was found to be stimulated by salts, which in turn had a significant Na + removal capability from aqueous solution. Seed germination, vigor index and mobilization efficiency in all the three crops remarkably improved when cyanobacterial EPS was applied. While germination improved significantly by 13 to 30%, mobilization efficiency increased marginally by 1.03 to 1.1 times and vigor index increased by 1.15 to 2.4 times in these crops in response to EPS under non-saline conditions. Salinity had an inhibitory effect on seed germination of all the species showing 18 to 54% reduction. However, in the presence of EPS, the salt induced inhibition diminished to 13 to 18%. Inhibitory effect of salt on chlorophyll concentration, vigor index and mobilization efficiency of the seedlings was much less in these crops in the presence of EPS, indicating the latter's role in salt stress alleviation

Bis-Gadolinium Complexes for Solid Effect and Cross Effect Dynamic Nuclear Polarization

Kaushik M, Qi M, Godt A, Corzilius B. Bis-Gadolinium Complexes for Solid Effect and Cross Effect Dynamic Nuclear Polarization. Angewandte Chemie International Edition. 2017;56(15):4295–4299

Dynamic nuclear polarization for sensitivity enhancement in modern solid-state NMR

The field of dynamic nuclear polarization has undergone tremendous developments and diversification since its inception more than 6 decades ago. In this review we provide an in-depth overview of the relevant topics involved in DNP-enhanced MAS NMR spectroscopy. This includes the theoretical description of DNP mechanisms as well as of the polarization transfer pathways that can lead to a uniform or selective spreading of polarization between nuclear spins. Furthermore, we cover historical and state-of-the art aspects of dedicated instrumentation, polarizing agents, and optimization techniques for efficient MAS DNP. Finally, we present an extensive overview on applications in the fields of structural biology and materials science, which underlines that MAS DNP has moved far beyond the proof-of-concept stage and has become an important tool for research in these fields

Dynamic nuclear polarization of nucleic acid with endogenously bound manganese.

We report the direct dynamic nuclear polarization (DNP) of (13)C nuclei of a uniformly [(13)C,(15)N]-labeled, paramagnetic full-length hammerhead ribozyme (HHRz) complex with Mn(2+) where the enhanced polarization is fully provided by the endogenously bound metal ion and no exogenous polarizing agent is added. A (13)C enhancement factor of ε = 8 was observed by intra-complex DNP at 9.4 T. In contrast, "conventional" indirect and direct DNP experiments were performed using AMUPol as polarizing agent where we obtained a (1)H enhancement factor of ε ≈ 250. Comparison with the diamagnetic (Mg(2+)) HHRz complex shows that the presence of Mn(2+) only marginally influences the (DNP-enhanced) NMR properties of the RNA. Furthermore two-dimensional correlation spectra ((15)N-(13)C and (13)C-(13)C) reveal structural inhomogeneity in the frozen, amorphous state indicating the coexistence of several conformational states. These demonstrations of intra-complex DNP using an endogenous metal ion as well as DNP-enhanced MAS NMR of RNA in general yield important information for the development of new methods in structural biology

Trehalose matrices for high temperature dynamic nuclear polarization enhanced solid state NMR

International audienceTrehalose matrix in combination with a tailor designed dinitroxide radical represents a promising DNP medium for high-sensitivity solid-state NMR of biological substrates at elevated temperatures where conventional water-based formulations melt down

Gd(III) and Mn(II) complexes for dynamic nuclear polarization : small molecular chelate polarizing agents and applications with site-directed spin labeling of proteins

We investigate complexes of two paramagnetic metal ions Gd3+ and Mn2+ to serve as polarizing agents for solid-state dynamic nuclear polarization (DNP) of 1H, 13C, and 15N at magnetic fields of 5, 9.4, and 14.1 T. Both ions are half-integer high-spin systems with a zero-field splitting and therefore exhibit a broadening of the mS = −1/2 ↔ +1/2 central transition which scales inversely with the external field strength. We investigate experimentally the influence of the chelator molecule, strong hyperfine coupling to the metal nucleus, and deuteration of the bulk matrix on DNP properties. At small Gd-DOTA concentrations the narrow central transition allows us to polarize nuclei with small gyromagnetic ratio such as 13C and even 15N via the solid effect. We demonstrate that enhancements observed are limited by the available microwave power and that large enhancement factors of >100 (for 1H) and on the order of 1000 (for 13C) can be achieved in the saturation limit even at 80 K. At larger Gd(III) concentrations (≥10 mM) where dipolar couplings between two neighboring Gd3+ complexes become substantial a transition towards cross effect as dominating DNP mechanism is observed. Furthermore, the slow spin-diffusion between 13C and 15N, respectively, allows for temporally resolved observation of enhanced polarization spreading from nuclei close to the paramagnetic ion towards nuclei further removed. Subsequently, we present preliminary DNP experiments on ubiquitin by site-directed spin-labeling with Gd3+ chelator tags. The results hold promise towards applications of such paramagnetically labeled proteins for DNP applications in biophysical chemistry and/or structural biology

Atomic-Scale Structure and its Impact on Chemical Properties of Aluminum Oxide Layers Prepared by Atomic Layer Deposition on Silica

We report the atomic-scale structure of alumina layers obtained by atomic layer deposition (ALD) of trimethylaluminium onto partially dehydroxylated silica. Such a detailed insight into the atomic structure of the species formed with increasing Al content was gained using a variety of one- and two-dimensional solid-state nuclear magnetic resonance (NMR) experiments involving 27Al, 1H and 29Si nuclei. 15N dynamic nuclear polarization surface-enhanced NMR spectroscopy (15N DNP SENS) and infrared spectroscopy using 15N-labelled pyridine as a probe molecule reveal that aluminium oxide layers on amorphous silica contain both strong Bronsted and strong Lewis acid sites, whereby the relative abundance and nature of these sites, and therefore the acidity of the surface, evolve with increasing thickness of the alumina film. <br /

Atomic-Scale Structure and Its Impact on Chemical Properties of Aluminum Oxide Layers Prepared by Atomic Layer Deposition on Silica

International audienc

Atomic-Scale Structure and Its Impact on Chemical Properties of Aluminum Oxide Layers Prepared by Atomic Layer Deposition on Silica

Alumina and aluminosilicates, prepared under various synthesis conditions, play a central role in heterogeneous catalysis with a broad range of industrial applications. We report herein the atomic-scale structure of alumina layers obtained by atomic layer deposition (ALD) of trimethylaluminum onto partially dehydroxylated silica. Such a detailed insight into the atomic structure of the species formed with increasing Al content was gained using a variety of one- and two-dimensional solid-state nuclear magnetic resonance (NMR) experiments involving 27Al, 1H, and 29Si nuclei. Multicomponent fittings of the 1D and 2D experimental data sets allowed us to show that at 3.4 wt % of deposited Al, a submonolayer containing [4]Al(3Si), [4]Al(4Si), and [5]Al(2Si) species forms on the silica surface, with most of these sites carrying OH groups. The films obtained after additional ALD cycles (depositing 9.2 or 15.4 wt % Al) feature characteristics of an amorphous alumina phase with a high concentration of [5]Al species and abundant OH groups. The most probable species at the interface between silica and alumina are [4]Al(2Si), [4]Al(3Si), and [5]Al(2Si). 15N dynamic nuclear polarization surface-enhanced NMR spectroscopy (15N DNP SENS) and infrared spectroscopy using 15N-labeled pyridine as a probe molecule reveal that aluminum oxide layers on amorphous silica contain both strong Brønsted and strong Lewis acid sites, whereby the relative abundance and nature of these sites, and therefore the acidity of the surface, evolve with increasing thickness of the alumina films (controlled by the number of ALD cycles). This study provides the first in-depth atomic-scale description of (sub-)nanometer-scale aluminum oxide films prepared by ALD as a function of their growth on a partially dehydroxylated silica support, opening the way to molecular-level understanding of the catalytic activity of such heterogeneous catalysts with tailored acidity.ISSN:0897-475