Water (1)H relaxation rate measurements of (15)N-(2)H-TEMPONE solutions at temperatures ranging from 298 to 328 K have been performed as a function of magnetic field from 0.00023 to 9.4 T, corresponding to (1)H Larmor frequencies of 0.01 to 400 MHz. The relaxation profiles were analyzed according to the full theory for dipolar and contact relaxation, and used to estimate the coupling factor responsible for observed solution DNP effects. The experimental DNP enhancement at (1)H Larmor frequency of 15 MHz obtained by saturating one of the lines of the (15)N doublet is only ca. 20% lower than the limiting value predicted from the relaxation data, indicating that the experimental DNP setup is nearly optimal, the residual discrepancy arising from incomplete saturation of the other line.

Dynamic nuclear polarization is emerging as a potential tool to increase the sensitivity of NMR aiming at the detection of macromolecules in liquid solution. One possibility for such an experimental design is to perform the polarization step between electrons and nuclei at low magnetic fields and then transfer the sample to a higher field for NMR detection. In this case, an independent optimization of the polarizer and detection set ups is required. In the present paper we describe the optimization of a polarizer set up at 15 MHz (1)H NMR/9.7 GHz EPR frequencies based on commercial hardware. The sample consists of the nitroxide radical TEMPONE-D,(15)N in water, for which the dimensions were systematically decreased to fit the homogeneous B(1) region of a dielectric ENDOR resonator. With an available B(1) microwave field up to 13 G we observe a maximum DNP enhancement of -170 at room temperature by irradiating on either one of the EPR lines. The DNP enhancement was saturated at all polarizer concentrations. Pulsed ELDOR experiments revealed that the saturation level of the two hyperfine lines is such that the DNP enhancements are well consistent with the coupling factors derived from NMRD data. By raising the polarizing field and frequencies 10-fold, i.e. to 140 MHz (1)H/94 GHz EPR, we reach an enhancement of -43 at microwave field strengths (B(1) approximately 5 G). The results are discussed in view of an application for a DNP spectrometer.

We propose the use of the pulse electron double resonance (ELDOR) method to determine the effective saturation factor of nitroxide radicals for dynamic nuclear polarization (DNP) experiments in liquids. The obtained values for the nitroxide radical 15 N at different concentrations are rationalized in terms of spin relaxation and are shown to fulfil the Overhauser theory.Dynamic nuclear polarization (DNP) in aqueous solution at ambient conditions is a major topic of current efforts to enhance the sensitivity of high resolution NMR and magnetic resonance imaging. [1][2][3] In the liquid state, DNP is governed by the Overhauser mechanism, 4,5 in which polarization is transferred from paramagnetic centers to coupled nuclear spins by microwave pumping of the EPR line. The enhancements e depend on four factors, i.e. the ratio of the gyromagnetic constants of the electron spin g s and the target nucleus g I , the coupling factor x, the leakage factor f and the effective saturation factor s eff of the EPR line: 6In recent studies, nitroxide radicals have been favoured as polarizing agents for DNP since they are soluble in water, well compatible with biological systems, non-toxic and have been found to account for large DNP enhancements at magnetic fields up to 9 T. 7,8 However, the determination of the saturation factor for this class of polarizers has emerged as one of the major difficulties in rationalizing the observed DNP enhancements in terms of the Overhauser equation (1). [8][9][10][11][12] Due to the strong hyperfine coupling between the electron and the nitrogen nucleus, which splits the EPR spectrum into two ( 15 N) or three ( 14 N) separate lines, the saturation factor, defined as s eff = (S B À hS z i)/S B with S B the Boltzmann polarization of the electron spin, cannot be simply extracted from the saturation behaviour of the pumped line according to the Bloch equation, as formerly suggested for trityl radicals. 13 Moreover, the expectation value of the electron spin polarization hS z i depends on the population of all energy levels involved. In recent years, this issue has been debated in the literature and several theoretical models were proposed to calculate the saturation factor for nitroxides 9-11 but to date no direct experimental approach has been provided. In this paper, we show that the effective saturation factor in Overhauser DNP can be determined by a pulsed electron double resonance (ELDOR) experiment, 14 which measures the intensity of a hyperfine line when pumping a coupled hyperfine line. This approach is demonstrated with a 15 N containing nitroxide because this gives the maximum enhancement for DNP and is relevant for future DNP applications. However, it is fully applicable to radicals incorporating a 14 N nucleus as well. Our experiments were performed at X-band (9.7 GHz electron Larmor frequency) in a commercial dielectric EPR/ENDOR resonator (Bruker EN4118X-MD4), as used for DNP, 12 that is overcoupled to permit irradiation at two frequencies with Dn up to 100 MHz. The saturation factors...

A DNP set-up is described where a liquid sample is hyperpolarized by the electron-nucleus Overhauser effect in a field of 0.34 T and transferred to a field of 14.09 T for NMR detection. In contrast to a previous set-up, using two dedicated magnets for polarization and detection, a dedicated ferroshim system was inserted into the bore of a 14.09 T shielded cryomagnet to provide a homogeneous low-field region in the stray field above the magnetic center. After polarization in the low-field the sample is transferred to the high-field magnetic center within 40 ms by a pneumatic shuttle system. In our set-up a standard high-resolution inverse (1)H/(13)C selective probe was used for NMR detection and a homebuilt EPR cavity, operating in the TM(110) mode was used for polarisation. First experimental data are presented. We observed a maximum proton Overhauser enhancement of up to epsilon(HF) = -3.7 in the high-field position for a 5 mM 4-Oxo-TEMPO-D,(15)N (TEMPONE)/H(2)O sample. While this reproduces the DNP enhancement observed also in the old set-up, with the new set-up we observe enhancement on larger molecules that were impossible to enhance in the old set-up. Therefore, we can demonstrate for the first time Overhauser enhanced high resolution proton spectra of glucose and 2,2-dimethyl-2-silapentane-5-sulfonic acid sodium salt (DSS) in D(2)O, where the high resolution spectrum was acquired in the high-field position after polarizing the sample in the low-field.

Dynamic nuclear polarization (DNP) permits increasing the NMR signal of nuclei by pumping the electronic spin transitions of paramagnetic centers nearby. This method is emerging as a powerful tool to increase the inherent sensitivity of NMR in structural biology aiming at detection of macromolecules. In aqueous solution, additional technical issues associated with the penetration of microwaves in water and heating effects aggravate the performance of the experiment. To examine the feasibility of low-field (9.7 GHz/0.35 T) DNP in high resolution NMR, we have constructed the prototype of a two-field shuttle DNP spectrometer that polarizes nuclei at 9.7 GHz/0.35 T and detects the NMR spectrum at 14 T. We report our first (1)H and (13)C DNP enhancements with this spectrometer. Effective enhancements up to 15 were observed for small molecules at (1)H 600 MHz/14 T as compared to the Boltzmann signal. The results provide a proof of principle for the feasibility of a shuttle DNP experiment and open up perspectives for the application potential of this method in solution NMR.