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1.
J Am Chem Soc ; 145(18): 10268-10274, 2023 May 10.
Artigo em Inglês | MEDLINE | ID: mdl-37104685

RESUMO

Dynamic nuclear polarization (DNP) is a hyperpolarization method that is widely used for increasing the sensitivity of nuclear magnetic resonance (NMR) experiments. DNP is efficient in solid-state and liquid-state NMR, but its implementation in the intermediate state, namely, viscous media, is still less explored. Here, we show that a 1H DNP enhancement of over 50 can be obtained in viscous liquids at a magnetic field of 9.4 T and a temperature of 315 K. This was accomplished by using narrow-line polarizing agents in glycerol, both the water-soluble α,γ-bisdiphenylen-ß-phenylallyl (BDPA) and triarylmethyl radicals, and a microwave/RF double-resonance probehead. We observed DNP enhancements with a field profile indicative of the solid effect and investigated the influence of microwave power, temperature, and concentration on the 1H NMR results. To demonstrate potential applications of this new DNP approach for chemistry and biology, we show hyperpolarized 1H NMR spectra of tripeptides, triglycine, and glypromate, in glycerol-d8.

2.
Angew Chem Int Ed Engl ; 62(24): e202216610, 2023 06 12.
Artigo em Inglês | MEDLINE | ID: mdl-37009775

RESUMO

Here we uncover collagen, the main structural protein of all connective tissues, as a redox-active material. We identify dihydroxyphenylalanine (DOPA) residues, post-translational oxidation products of tyrosine residues, to be common in collagen derived from different connective tissues. We observe that these DOPA residues endow collagen with substantial radical scavenging capacity. When reducing radicals, DOPA residues work as redox relay: they convert to the quinone and generate hydrogen peroxide. In this dual function, DOPA outcompetes its amino acid precursors and ascorbic acid. Our results establish DOPA residues as redox-active side chains of collagens, probably protecting connective tissues against radicals formed under mechanical stress and/or inflammation.


Assuntos
Di-Hidroxifenilalanina , Tirosina , Di-Hidroxifenilalanina/química , Tirosina/química , Colágeno/química , Oxirredução , Aminoácidos/metabolismo
3.
J Am Chem Soc ; 144(3): 1164-1168, 2022 01 26.
Artigo em Inglês | MEDLINE | ID: mdl-35029974

RESUMO

Dynamic nuclear polarization (DNP) is a powerful method to enhance NMR sensitivity. Much progress has been achieved recently to optimize DNP performance at high magnetic fields in solid-state samples, mostly by utilizing the solid or the cross effect. In liquids, only the Overhauser mechanism is active, which exhibits a DNP field profile matching the EPR line shape of the radical, distinguishable from other DNP mechanisms. Here, we observe DNP enhancements with a field profile indicative of the solid effect and thermal mixing at ∼320 K and a magnetic field of 9.4 T in the fluid phase of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) lipid bilayers doped with the radical BDPA (1,3-bis(diphenylene)-2-phenylallyl). This interesting observation might open up new perspectives for DNP applications in macromolecular systems at ambient temperatures.

4.
Angew Chem Int Ed Engl ; 58(5): 1402-1406, 2019 01 28.
Artigo em Inglês | MEDLINE | ID: mdl-30485626

RESUMO

Nuclear magnetic resonance (NMR) techniques play an essential role in natural science and medicine. In spite of the tremendous utility associated with the small energies detected, the most severe limitation is the low signal-to-noise ratio. Dynamic nuclear polarization (DNP), a technique based on transfer of polarization from electron to nuclear spins, has emerged as a tool to enhance sensitivity of NMR. However, the approach in liquids still faces several challenges. Herein we report the observation of room-temperature, liquid DNP 13 C signal enhancements in organic small molecules as high as 600 at 9.4 Tesla and 800 at 1.2 Tesla. A mechanistic investigation of the 13 C-DNP field dependence shows that DNP efficiency is raised by proper choice of the polarizing agent (paramagnetic center) and by halogen atoms as mediators of scalar hyperfine interaction. Observation of sizable DNP of 13 CH2 and 13 CH3 groups in organic molecules at 9.4 T opens perspective for a broader application of this method.

5.
Magn Reson Med ; 75(3): 985-96, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-25884985

RESUMO

PURPOSE: Overhauser dynamic nuclear polarization (DNP) allows the production of liquid hyperpolarized substrate inside the MRI magnet bore as well as its administration in continuous flow mode to acquire MR images with enhanced signal-to-noise ratio. We implemented inversion recovery preparation in order to improve contrast-to-noise ratio and to quantify the overall imaging performance of Overhauser DNP-enhanced MRI. METHOD: The negative enhancement created by DNP in combination with inversion recovery (IR) preparation allows canceling selectively the signal originated from Boltzmann magnetization and visualizing only hyperpolarized fluid. The theoretical model describing gain of MR image intensity produced by steady-state continuous flow DNP hyperpolarized magnetization was established and proved experimentally. RESULTS: A precise quantification of signal originated purely from DNP hyperpolarization was achieved. A temperature effect on longitudinal relaxation had to be taken into account to fit experimental results with numerical prediction. CONCLUSION: Using properly adjusted IR preparation, the complete zeroing of thermal background magnetization was achieved, providing an essential increase of contrast-to-noise ratio of DNP-hyperpolarized water images. To quantify and optimize the steady-state conditions for MRI with continuous flow DNP, an approach similar to that incorporating transient-state thermal magnetization equilibrium in spoiled fast field echo imaging sequences can be used.


Assuntos
Processamento de Imagem Assistida por Computador/métodos , Imageamento por Ressonância Magnética/métodos , Processamento de Sinais Assistido por Computador , Animais , Aorta/fisiologia , Desenho de Equipamento , Espectroscopia de Ressonância Magnética , Camundongos , Modelos Teóricos , Imagens de Fantasmas , Água
6.
J Am Chem Soc ; 136(44): 15533-6, 2014 Nov 05.
Artigo em Inglês | MEDLINE | ID: mdl-25333422

RESUMO

Dynamic nuclear polarization (DNP)-enhanced solid-state NMR spectroscopy has been shown to hold great potential for functional studies of membrane proteins at low temperatures due to its great sensitivity improvement. There are, however, numerous applications for which experiments at ambient temperature are desirable and which would also benefit from DNP signal enhancement. Here, we demonstrate as a proof of concept that a significant signal increase for lipid bilayers under room-temperature conditions can be achieved by utilizing the Overhauser effect. Experiments were carried out on aligned bilayers at 400 MHz/263 GHz using a stripline structure combined with a Fabry-Perot microwave resonator. A signal enhancement of protons of up to -10 was observed. Our results demonstrate that Overhauser DNP at high field provides efficient polarization transfer within insoluble samples, which is driven by fast local molecular fluctuations. Furthermore, our experimental setup offers an attractive option for DNP-enhanced solid-state NMR on ordered membranes and provides a general perspective toward DNP at ambient temperatures.


Assuntos
Bicamadas Lipídicas/química , Espectroscopia de Ressonância Magnética/métodos , Temperatura
7.
Phys Chem Chem Phys ; 16(35): 18781-7, 2014 Sep 21.
Artigo em Inglês | MEDLINE | ID: mdl-25078259

RESUMO

Here we show how fast dynamics between radicals and solvent molecules in liquid solutions can be detected by comparison of coupling factors determined by nuclear magnetic relaxation dispersion (NMRD) measurements and dynamic nuclear polarization (DNP) enhancement measurements at high magnetic field (9.2 T). This is important for a theoretical understanding of the Overhauser DNP mechanism at high magnetic fields and thus for optimization of the DNP agent/target system for high resolution liquid state NMR applications. Mixtures of the solution of TEMPOL radicals in water, toluene, acetone and DMSO have been investigated. The results are compared to the classical hard-sphere model and molecular dynamic simulations. Our results clearly indicate that fast sub-ps dynamics, which are not related to classical rotational or translational motion of the molecules, significantly contribute to the Overhauser DNP mechanism at high magnetic fields.

8.
J Am Chem Soc ; 135(51): 19275-81, 2013 Dec 26.
Artigo em Inglês | MEDLINE | ID: mdl-24279469

RESUMO

Dynamic nuclear polarization (DNP) enhances the sensitivity of solid-state NMR (SSNMR) spectroscopy by orders of magnitude and, therefore, opens possibilities for novel applications from biology to materials science. This multitude of opportunities implicates a need for high-performance polarizing agents, which integrate specific physical and chemical features tailored for various applications. Here, we demonstrate that for the biradical bTbK in complex with captisol (CAP), a ß-cyclodextrin derivative, host-guest assembling offers a new and easily accessible approach for the development of new polarizing agents. In contrast to bTbK, the CAP-bTbK complex is water-soluble and shows significantly improved DNP performance compared to the commonly used DNP agent TOTAPOL. Furthermore, NMR and EPR data reveal improved electron and nuclear spin relaxation properties for bTbK within the host molecule. The numerous possibilities to functionalize host molecules will permit designing novel radical complexes targeting diverse applications.

9.
Phys Chem Chem Phys ; 15(16): 6049-56, 2013 Apr 28.
Artigo em Inglês | MEDLINE | ID: mdl-23493879

RESUMO

We have performed liquid state ("Overhauser") Dynamic Nuclear Polarization (DNP) experiments at high magnetic field (9.2 T, corresponding to 260 GHz EPR and 400 MHz (1)H-NMR resonance frequency) on aqueous solutions of (14)N-TEMPOL nitroxide radicals. Integrated signal enhancements exceeding -80 were observed for the water protons at microwave superheated temperatures (160 °C) and still -14 at ambient temperatures (45 °C) relevant to biological applications. Different contributions contributing to the DNP enhancement such as saturation factor, leakage factor and sample temperature under microwave irradiation could be determined independently for a high spin concentration of 1 M, allowing the calculation of the coupling factors as a function of temperature and a quantitative comparison of this parameter with values derived from field dependent relaxation measurements or predictions from MD simulation.


Assuntos
Óxidos N-Cíclicos/química , Água/química , Campos Magnéticos , Micro-Ondas , Simulação de Dinâmica Molecular , Nitrogênio/química , Óxidos de Nitrogênio/química , Marcadores de Spin , Temperatura
10.
J Magn Reson ; 356: 107564, 2023 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-37852111

RESUMO

Pulse electron double resonance (PELDOR), also called double electron-electron resonance (DEER), is a technique capable of measuring the strength of electron spin dipolar interactions, revealing spin-spin distance distributions in ordered and disordered solid materials. Previous work has shown that PELDOR signals acquire an out-of-phase component under conditions of high electron spin polarization, such as at low temperatures and high fields. In this paper, we show theoretically and experimentally that the size and sign of this effect depends on the macroscopic shape of the sample and its orientation in the external magnetic field. This effect is caused by dipolar interactions between distant spins and provides new insights into the fundamental physics of PELDOR.

11.
J Phys Chem Lett ; 14(31): 7059-7064, 2023 Aug 10.
Artigo em Inglês | MEDLINE | ID: mdl-37526333

RESUMO

Dynamic nuclear polarization (DNP) is routinely used as a method for increasing the sensitivity to nuclear magnetic resonance (NMR). Recently, high-field solid-effect DNP in viscous liquids on 1H nuclei was demonstrated using narrow-line polarizing agents. Here we expand the applicability of DNP in viscous media to 13C nuclei. To hyperpolarize 13C nuclei, we combined solid-effect 1H DNP with a subsequent transfer of the 1H polarization to 13C via insensitive nuclei enhanced by polarization transfer (INEPT). We demonstrate this approach using a triarylmethyl radical as a polarizing agent and glycerol-13C3 as an analyte. We achieved 13C enhancement factors of up to 45 at a magnetic field of 9.4 T and room temperature.

12.
J Magn Reson ; 337: 107185, 2022 04.
Artigo em Inglês | MEDLINE | ID: mdl-35276481

RESUMO

In DNP experiments, NMR signal intensity is increased by transferring the much larger electron spin polarization to nuclear spins via microwave irradiation. Here we describe the design and performance of a probehead that makes it possible to perform Overhauser DNP experiments at 1H and 13C in liquid samples with a volume of up to 100 nl. We demonstrate on a 13C-labeled sodium pyruvate sample in water that proton decoupling under DNP conditions is possible with this new triple-resonance DNP probehead. In addition, the heat dissipation from the sample has been greatly improved with our new probe design. This makes it possible to keep liquid samples at a constant temperature under irradiation with a high-frequency 263 GHz microwave gyrotron with a few watts of output power. This improved performance opens up the possibility to disentangle the role of sample temperature and applied microwave power for DNP efficiency in liquids and to obtain a quantitative determination of EPR saturation by observing the suppression of paramagnetic shift as a function of microwave power.


Assuntos
Micro-Ondas , Prótons , Espectroscopia de Ressonância Magnética , Temperatura , Água/química
13.
Magn Reson (Gott) ; 3(1): 101-110, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-37905182

RESUMO

Double electron-electron resonance (DEER) spectroscopy measures the distribution of distances between two electron spins in the nanometer range, often on doubly spin-labeled proteins, via the modulation of a refocused spin echo by the dipolar interaction between the spins. DEER is commonly conducted under conditions where the polarization of the spins is small. Here, we examine the DEER signal under conditions of high spin polarization, thermally obtainable at low temperatures and high magnetic fields, and show that the signal acquires a polarization-dependent out-of-phase component both for the intramolecular and intermolecular contributions. For the latter, this corresponds to a phase shift of the spin echo that is linear in the pump pulse position. We derive a compact analytical form of this phase shift and show experimental measurements using monoradical and biradical nitroxides at several fields and temperatures. The effect highlights a novel aspect of the fundamental spin physics underlying DEER spectroscopy.

14.
J Am Chem Soc ; 133(23): 9052-62, 2011 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-21548577

RESUMO

PpoA is a fungal dioxygenase that produces hydroxylated fatty acids involved in the regulation of the life cycle and secondary metabolism of Aspergillus nidulans . It was recently proposed that this novel enzyme employs two different heme domains to catalyze two separate reactions: within a heme peroxidase domain, linoleic acid is oxidized to (8R)-hydroperoxyoctadecadienoic acid [(8R)-HPODE]; in the second reaction step (8R)-HPODE is isomerized within a P450 heme thiolate domain to 5,8-dihydroxyoctadecadienoic acid. In the present study, pulsed EPR methods were applied to find spectroscopic evidence for the reaction mechanism, thought to involve paramagnetic intermediates. We observe EPR resonances of two distinct heme centers with g-values typical for Fe(III) S = (5)/(2) high-spin (HS) and Fe(III) S = (1)/(2) low-spin (LS) hemes. (14)N ENDOR spectroscopy on the S = (5)/(2) signal reveals resonances consistent with an axial histidine ligation. Reaction of PpoA with the substrate leads to the formation of an amino acid radical on the early millisecond time scale concomitant to a substantial reduction of the S = (5)/(2) heme signal. High-frequency EPR (95- and 180-GHz) unambiguously identifies the new radical as a tyrosyl, based on g-values and hyperfine couplings from spectral simulations. The radical displays enhanced T(1)-spin-lattice relaxation due to the proximity of the heme centers. Further, EPR distance measurements revealed that the radical is distributed among the monomeric subunits of the tetrameric enzyme at a distance of approximately 5 nm. The identification of three active paramagnetic centers involved in the reaction of PpoA supports the previously proposed reaction mechanism based on radical chemistry.


Assuntos
Biocatálise , Sistema Enzimático do Citocromo P-450/química , Sistema Enzimático do Citocromo P-450/metabolismo , Ácidos Graxos/metabolismo , Oxigênio/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Sistema Enzimático do Citocromo P-450/genética , Espectroscopia de Ressonância de Spin Eletrônica , Radicais Livres/química , Heme/química , Heme/metabolismo , Histidina/química , Mutação , Proteínas Recombinantes de Fusão/genética , Tirosina/química
15.
J Am Chem Soc ; 133(34): 13375-9, 2011 Aug 31.
Artigo em Inglês | MEDLINE | ID: mdl-21702503

RESUMO

Pulsed Electron-Electron Double Resonance (PELDOR) on double-stranded DNA (ds-DNA) was used to investigate the conformational flexibility of helical DNA. Stretching, twisting, and bending flexibility of ds-DNA was determined by incorporation of two rigid nitroxide spin labels into a series of 20 base pair (bp) DNA duplexes. Orientation-selective PELDOR experiments performed at both X-band (9 GHz/0.3 T) and G-band (180 GHz/6.4 T) with spin label distances in the range of 2-4 nm allowed us to differentiate between different simple models of DNA dynamics existing in the literature. All of our experimental results are in full agreement with a dynamic model for ds-DNA molecules, where stretching of the molecule leads to a slightly reduced radius of the helix induced by a cooperative twist-stretch coupling.


Assuntos
DNA/química , Modelos Moleculares , Conformação de Ácido Nucleico , Pirenos/química , Análise Espectral
16.
Nat Commun ; 12(1): 6880, 2021 11 25.
Artigo em Inglês | MEDLINE | ID: mdl-34824218

RESUMO

Nuclear magnetic resonance (NMR) spectroscopy is a powerful and popular technique for probing the molecular structures, dynamics and chemical properties. However the conventional NMR spectroscopy is bottlenecked by its low sensitivity. Dynamic nuclear polarization (DNP) boosts NMR sensitivity by orders of magnitude and resolves this limitation. In liquid-state this revolutionizing technique has been restricted to a few specific non-biological model molecules in organic solvents. Here we show that the carbon polarization in small biological molecules, including carbohydrates and amino acids, can be enhanced sizably by in situ Overhauser DNP (ODNP) in water at room temperature and at high magnetic field. An observed connection between ODNP 13C enhancement factor and paramagnetic 13C NMR shift has led to the exploration of biologically relevant heterocyclic compound indole. The QM/MM MD simulation underscores the dynamics of intermolecular hydrogen bonds as the driving force for the scalar ODNP in a long-living radical-substrate complex. Our work reconciles results obtained by DNP spectroscopy, paramagnetic NMR and computational chemistry and provides new mechanistic insights into the high-field scalar ODNP.


Assuntos
Ressonância Magnética Nuclear Biomolecular/métodos , Água/química , Isótopos de Carbono/química , Teoria da Densidade Funcional , Ligação de Hidrogênio , Campos Magnéticos , Simulação de Dinâmica Molecular , Temperatura
17.
Phys Chem Chem Phys ; 12(22): 5786-90, 2010 Jun 14.
Artigo em Inglês | MEDLINE | ID: mdl-20461255

RESUMO

Dynamic nuclear polarization (DNP) at high magnetic fields (9.2 T, 400 MHz (1)H NMR frequency) requires high microwave power sources to achieve saturation of the EPR transitions. Here we describe the first high-field liquid-state DNP results using a high-power gyrotron microwave source (20 W at 260 GHz). A DNP enhancement of -29 on water protons was obtained for an aqueous solution of Fremy's Salt; in comparison the previous highest value was -10 using a solid-state microwave power source (maximum power 45 mW). The increased enhancements are partly due to larger microwave saturation and elevated sample temperature. These experimentally observed DNP enhancements, which by far exceed the predicted values extrapolated from low-field DNP experiments, demonstrate experimentally that DNP is possible in the liquid state also at high magnetic fields.


Assuntos
Espectroscopia de Ressonância Magnética/instrumentação , Espectroscopia de Ressonância Magnética/métodos , Micro-Ondas , Simulação de Dinâmica Molecular , Compostos Nitrosos/química
18.
Magn Reson (Gott) ; 1(2): 275-284, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-37904825

RESUMO

A continuous flow dynamic nuclear polarization (DNP) employing the Overhauser effect at ambient temperatures can be used among other methods to increase sensitivity of magnetic resonance imaging (MRI). The hyperpolarized state of water protons can be achieved by flowing aqueous liquid through a microwave resonator placed directly in the bore of a 1.5 T MRI magnet. Here we describe a new open Fabry-Pérot resonator as DNP polarizer, which exhibits a larger microwave exposure volume for the flowing liquid in comparison with a cylindrical TE013 microwave cavity. The Fabry-Pérot resonator geometry was designed using quasi-optical theory and simulated by CST software. Performance of the new polarizer was tested by MRI DNP experiments on a TEMPOL aqueous solution using a blood-vessel phantom. The Fabry-Pérot resonator revealed a 2-fold larger DNP enhancement with a 4-fold increased flow rate compared to the cylindrical microwave resonator. This increased yield of hyperpolarized liquid allows MRI applications on larger target objects.

19.
Nat Commun ; 11(1): 2315, 2020 05 08.
Artigo em Inglês | MEDLINE | ID: mdl-32385229

RESUMO

As established nearly a century ago, mechanoradicals originate from homolytic bond scission in polymers. The existence, nature and biological relevance of mechanoradicals in proteins, instead, are unknown. We here show that mechanical stress on collagen produces radicals and subsequently reactive oxygen species, essential biological signaling molecules. Electron-paramagnetic resonance (EPR) spectroscopy of stretched rat tail tendon, atomistic molecular dynamics simulations and quantum-chemical calculations show that the radicals form by bond scission in the direct vicinity of crosslinks in collagen. Radicals migrate to adjacent clusters of aromatic residues and stabilize on oxidized tyrosyl radicals, giving rise to a distinct EPR spectrum consistent with a stable dihydroxyphenylalanine (DOPA) radical. The protein mechanoradicals, as a yet undiscovered source of oxidative stress, finally convert into hydrogen peroxide. Our study suggests collagen I to have evolved as a radical sponge against mechano-oxidative damage and proposes a mechanism for exercise-induced oxidative stress and redox-mediated pathophysiological processes.


Assuntos
Colágeno/química , Tendões/química , Animais , Materiais Biocompatíveis/química , Biopolímeros/química , Di-Hidroxifenilalanina/química , Espectroscopia de Ressonância de Spin Eletrônica , Radicais Livres/química , Oxirredução , Estresse Oxidativo , Ratos , Espécies Reativas de Oxigênio/química
20.
J Magn Reson ; 264: 68-77, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26920832

RESUMO

Dynamic nuclear polarization (DNP) at high magnetic fields has recently become one of the major research areas in magnetic resonance spectroscopy and imaging. Whereas much work has been devoted to experiments where the polarization transfer from the electron spin to the nuclear spin is performed in the solid state, only a few examples exist of experiments where the polarization transfer is performed in the liquid state. Here we describe such experiments at a magnetic field of 9.2 T, corresponding to a nuclear Larmor frequency of 400 MHz for proton spins and an excitation frequency of 263 GHz for the electron spins. The technical requirements to perform such experiments are discussed in the context of the double resonance structures that we have implemented. The experimental steps that allowed access to the enhancement factors for proton spins of several organic solvents with nitroxide radicals as polarizing agents are described. A computational scheme for calculating the coupling factors from molecular dynamics (MD) simulations is outlined and used to highlight the limitations of the classical models based on translational and rotational motion that are typically employed to quantify the observed coupling factors. The ability of MD simulations to predict enhancements for a variety of radicals and solvent molecules at any magnetic field strength should prove useful in optimizing experimental conditions for DNP in the liquid state.

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