Pulsed Dynamic Nuclear Polarization with Trityl Radicals

Abstract

Continuous-wave (CW) dynamic nuclear polarization (DNP) is now established as a method of choice to enhance the sensitivity in a variety of NMR experiments. Nevertheless, there remains a need for the development of more efficient methods to transfer polarization from electrons to nuclei. Of particular interest are pulsed DNP methods because they enable a rapid and efficient polarization transfer that, in contrast with CW DNP methods, is not attenuated at high magnetic fields. Here we report nuclear spin orientation via electron spin-locking (NOVEL) experiments using the polarizing agent trityl OX063 in glycerol/water at a temperature of 80 K and a magnetic field of 0.34 T. ¹H NMR signal enhancements up to 430 are observed, and the buildup of the local polarization occurs in a few hundred nanoseconds. Thus, NOVEL can efficiently dynamically polarize ¹H atoms in a system that is of general interest to the solid-state DNP NMR community. This is a first, important step toward the general application of pulsed DNP at higher fields.

Graphical Abstract

In dynamic nuclear polarization (DNP), electron spin polarization is transferred to nuclei via microwave irradiation at or near the electron Larmor frequency. DNP thereby enhances the nuclear spin polarization and can be used to increase the signal intensities in nuclear magnetic resonance (NMR) experiments. This requires the introduction of unpaired electrons into the NMR sample in the form of polarizing agents. When DNP and NMR experiments are performed at the same magnetic field and temperature, a maximum signal enhancement of γ_e/γ_1H = 658 can be achieved for protons, where γ_e and γ_1H are the gyromagnetic ratios of electrons and protons, respectively.

DNP is currently successfully utilized in a variety of NMR experimental protocols.^1–5 In magic-angle spinning (MAS) NMR, samples are dynamically polarized in situ, typically at temperatures of 80–100 K.^6–11 In contrast, in dissolution DNP, a static sample is polarized in a frozen, glassy matrix and subsequently melted with hot solvent such that solution NMR or MRI can be performed.^12–14 The mechanisms of DNP typically employed in these experiments are the cross-effect (CE)^15–18 and the solid-effect (SE).^19–23 Both of these mechanisms rely on state mixing from electron–nucleus interactions (as well as electron–electron interactions in case of the CE) in the laboratory frame and require continuous-wave (CW) microwave irradiation at high power to generate enhanced polarization of the nuclear spin manifold. Unfortunately, at higher magnetic fields the state mixing in the laboratory frame decreases due to the increasing strength of the electron and nuclear Zeeman terms, and the DNP efficiency attenuates as ~1/B₀ for the CE and ~1/B₀² for the SE. In MAS NMR experiments at higher fields (≥600 MHz/395 GHz) DNP enhancements are currently well below the theoretical maximum.^24–26 In dissolution DNP the detrimental field dependence is partially circumvented by performing DNP at 95 GHz (3.4 T or 144 MHz), followed by sample shuttling to higher field for the NMR experiment; however, as a consequence, polarization buildup is done at very low temperature, typically ~1.2 K, and the ¹³C buildup times are very long, on the order of a few hours.^27,28

While the detrimental field dependence is inherent to the CW DNP mechanisms SE and CE, it is possible to use other DNP mechanisms where the efficiency does not depend on the magnetic field. In particular, one can adapt approaches originally developed to detect low-γ nuclei in time-domain NMR experiments such as INEPT in solution²⁹ and cross-polarization in solids.^30,31 In these methods, energy level degeneracy, and thereby strong state mixing, is created in the rotating frame by the application of microwave and RF pulses. The Hamiltonian in the rotating frame contains no Zeeman terms and therefore the state mixing is not decreased at high magnetic fields. Moreover, there is the additional benefit that, compared with high-power CW microwave radiation, generating high-power microwave pulses is technically less challenging.

To date, several forms of pulsed DNP have been proposed. These include DNP in the nuclear rotating frame,^32,33 the dressed state solid effect (DSSE),^34,35 polarization of nuclear spins enhanced by ENDOR (PONSEE),^36,37 and nuclear spin orientation via electron spin locking (NOVEL).^38–40 In this last scheme, which is based on the method of cross-polarization, polarization is efficiently transferred from electrons to nuclei using a rotating frame/lab frame Hartmann–Hahn matching condition ω_1S = ω_0I, where ω_1S = γ_eB₁ is the electron Rabi frequency and ω_0I = γ_nB₀ is the nuclear Larmor frequency.

The initial NOVEL experiments reported at ≤0.34 T or 15 MHz were performed on a single crystal of silicon doped with boron acceptors⁴⁰ and later in diamond.⁴¹ In both cases the enhancements were small and the buildup of polarization was limited by the slow spin diffusion in the ²⁹Si or ¹³C spin reservoirs; however, the low Larmor frequencies of these two spin species facilitated satisfying the NOVEL matching condition. Very recently NOVEL was employed to polarize bulk ¹H, and because of improvements in instrumentation and sample preparation the enhancements were larger. Enhancements of 163 and 323 were observed in a single crystal of benzophenone doped with diphenylnitroxide and in partially deuterated polystyrene doped with BDPA, respectively.⁴² In single crystals of naphthalene doped with pentacene,^43–47 p-terphenyl doped with pentacene,⁴⁸ and fluorene doped with acridine,^49,50 NOVEL has been used in combination with the integrated solid effect (ISE) to transfer polarization from photoexcited triplet states to ¹H.⁵¹ Because of the non-Boltzmann polarization of these photoexcited triplet states, enhancements >γ_e/γ_1H were obtained in these experiments.

Here we report NOVEL experiments using the polarizing agent trityl OX063 in a glycerol/water matrix at a temperature of 80 K and a magnetic field of 0.34 T. This highly stable radical is widely used in MAS DNP, dissolution DNP, and in vivo oxygen/EPR imaging.^12,52 The glycerol/water matrix facilitates application of NOVEL to biological structures by sample doping with trityl, similar to doping with biradicals in current CE MAS DNP.^7,53–56 The ¹H NMR signal enhancements we obtain are large, up to 430, and the buildup of bulk ¹H hyperpolarization is rapid, <8 s.

A description of the polarization transfer in NOVEL from a single unpaired electron (S = 1/2) to a single proton (I = 1/2) requires the following Hamiltonian in the rotating frame⁵⁷

$$H=\mathrm{\Delta }{\widehat{S}}_z-{\omega }_{01\mathrm{H}}{\widehat{I}}_z+A_{zx}{\widehat{S}}_z{\widehat{I}}_x+A_{zy}{\widehat{S}}_z{\widehat{I}}_y+A_{zz}{\widehat{S}}_z{\widehat{I}}_z+{\omega }_{1\mathrm{S}}{\widehat{S}}_x$$ (1)

where Δ = ω_0S − ω_μw with ω_0S the electron Larmor frequency and ω_μw the microwave frequency. The other terms are the nuclear Zeeman term, the secular and pseudosecular hyperfine interaction with a nearby proton, and the microwave irradiation, respectively. An approximate energy level degeneracy is created when the electrons are spin-locked by on-resonance microwave irradiation (Δ = 0) with a field strength of ω_01H. Strong state mixing then arises from the pseudosecular terms of the proton hyperfine interaction (i.e., the dipolar interaction), which is on the order of a megahertz for trityl OX063.^58,59

Figure 1 shows 15 MHz ¹H NMR spectra of a 50 μL sample of d₈-glycerol/D₂O/H₂O 60:30:10 v/v/v doped with 10.5 mM trityl OX063 acquired with the NOVEL sequence “on” and “off”. (See the Experimental Methods section for a detailed description of the experimental protocol.) Acquisition of the off-signal with a sufficiently high signal-to-noise ratio requires ~12 h, whereas acquisition of the on-signal consumes ~15 min. The enhancement, ε = (I_on/I_off) − 1, with the NOVEL sequence on is 380 (±50). The phase of the on-signal is opposite to the phase of the off-signal, which is due to the choice of the phase of the electron spin locking pulse relative to the 90° flip pulse (see Scheme 1).⁴² The on-spectrum shows a central peak of a fwhm (full width at half-maximum) of ~15 kHz, which is typical for protons in this solid matrix,⁶⁰ and two shoulders, which are offset with respect to the central peak by roughly 50 kHz. Possibly these shoulders arise from protons that interact with the unpaired electron on trityl.

Figure 1.

¹H NMR signal enhancement by NOVEL DNP on a sample of d₈-glycerol/D₂O/H₂O (60/30/10 v/v/v) doped with 10.5 mM trityl OX063. The off-signal is an average of 4096 acquisitions, and the on-signal is an average of 256 acquisitions. Before each ¹H NMR acquisition, polarization is built up by repeating the electron spin lock at the NOVEL condition at a 1 kHz rate for 8 s. See also the Experimental Methods section.

Scheme 1.

NOVEL Pulse Sequence

Figure 2 shows the enhancement of the ¹H NMR signal as a function of the length of the locking pulse (mixing time). The transfer of polarization from the electron to protons is nearly complete after ~300 ns. After the initial rise the enhancement drops slightly and transient oscillations are present in the curve, reflecting the coherent nature of the polarization transfer. Similar oscillations have been observed in ¹H–¹³C cross-polarization experiments in a ferrocene single crystal⁶¹ as well as in NOVEL experiments on single crystals.^49,50 For our system the oscillations are expected to be attenuated by the powder averaging and because electron–nuclear dipolar interactions occur with many different protons located both on the trityl molecule and in the solvent.^58,59

Figure 2.

¹H NMR signal enhancement by NOVEL DNP as a function of the length of the electron spin-locking pulse (mixing time). Each ¹H NMR spectrum is an average of 128 acquisitions. Before each ¹H NMR acquisition, polarization is built up by repeating the electron spin lock at the NOVEL condition at a 1 kHz rate for 2 s.

Figure 3 shows the enhancement of the ¹H NMR signal as a function of the applied microwave field strength, γ_eB₁ (in MHz), and we consequently refer to this figure as a microwave field profile. The enhancement reaches a maximum around 15 MHz. Thus, the polarization transfer is most efficient at the NOVEL condition γ_eB₁ = γ_nB₀. The fwhm of the microwave field profile is 6 MHz, which roughly corresponds to the fwhm of the central peak in the echo-detected EPR spectrum of trityl OX063, which is 7 MHz, shown in Figure 4. Above γ_eB₁ ≈ 20 MHz a significant enhancement of about~ 15% of the maximum remains. This is presumably due to higher order effects.⁴⁷

Figure 3.

Microwave field profile. ¹H NMR signal enhancement by NOVEL DNP as a function of the applied microwave field strength. The line connecting the dots is a guide to the eye. Each ¹H NMR spectrum is an average of 128 acquisitions. Before each ¹H NMR acquisition, polarization is built up by repeating the electron spin lock at the NOVEL condition at a 1 kHz rate for 2 s.

Figure 4.

Zeeman field profile. Field dependence of the ¹H NMR signal enhanced by NOVEL DNP (black dots, black line to guide the eye) together with the echo-detected EPR spectrum (red line) of a 10.5 mM trityl OX063 sample. The echo-detected EPR spectrum was recorded at a microwave frequency of 9.7837 GHz. The central peak occurs at 348.98 mT, which corresponds to a ¹H Larmor frequency of 14.859 MHz. To acquire the profile, we performed NOVEL experiments at this magnetic field, while the microwave frequency was swept. To compare the field profile to the echo-detected EPR spectrum we transformed the microwave frequency values to magnetic field values.

Figure 4 shows the NOVEL enhancement Zeeman field profile together with the echo-detected EPR spectrum of the same sample. The central peak in the field profile is wider than the central peak in the EPR spectrum: fwhm = 20 MHz as opposed to 7 MHz (0.1 mT = 2.8 MHz). Thus, NOVEL occurs even when the microwaves are off resonance (Δ ≠ 0). In this case the nutation frequency of the electrons is given by ${\omega }_{\text{eff}}={({\mathrm{\Delta }}^2+{\omega }_{1\mathrm{S}}^2)}^{1/2}$, where ω_1S ≈ 2π × 15 MHz. The small contribution of Δ makes the NOVEL matching condition relatively broad. Remarkably, when going further off-resonance, both above and below the central peak, the enhancement does not decay to zero but remains ~10% of the maximum enhancement on resonance. (Note that around 348.35 mT the phase of the enhanced ¹H NMR signal is inverted.) Also, two side peaks are observed, one positive around 349.9 mT and one negative around 348.0 mT. We suspect that in these far-off-resonance regions second-order terms give rise to a small transfer of polarization.

The echo-detected EPR spectrum of trityl OX063 in Figure 4 also exhibits two sidebands, separated roughly 15 MHz from the central peak. In EPR spectra of low concentration trityl samples (≤0.2 mM) “spin-flip” lines, which are due to forbidden hyperfine transitions, are observed at these field positions;⁶² however, the intensity of these spin-flip lines is much smaller than the intensity of the sidebands in our spectrum. This might be related to the high trityl concentration in our DNP samples, 10.5 mM for the sample in Figure 4. Recently trityl OX063 has been shown to aggregate in aqueous solutions at concentrations >1 mM.⁶³ We performed NOVEL experiments with various concentrations of trityl and found that the enhancements increase roughly up to 10 mM. At higher concentrations the echo-detected EPR spectra are strongly distorted, presumably due to aggregation effects, and enhancements decrease.

The number of electrons in our sample is much smaller than the number of protons to be polarized. Thus, polarization of bulk protons requires nuclear spin diffusion.⁶⁴ The buildup of this hyperpolarization takes much longer than the initial polarization transfer from electron to nearby proton.⁶⁵ We measured this buildup time, T_B, on a sample that contained 6.4 mM to be 7.6 s. Note that this is much shorter than the nuclear T₁, which we found to be 26 s on the same sample.

To “pump” the proton polarization as efficiently as possible, we should maximize the repetition rate of the lock pulse sequence during bulk-polarization buildup. This rate is currently limited by the duty cycle of the TWT amplifier, which is 1%, and the electronic longitudinal relaxation time, T_1e, of trityl. On a sample containing 6.4 mM trityl OX063 we measured T_1e via inversion recovery to be 2.1 ms. For this same sample a lock sequence repetition time of 1 ms was found to yield the optimal enhancement.

In NMR a flip-back pulse of phase −x after a spin-lock period can be used to bring the magnetization back along z, either for prolonged storage or to allow a shorter recycle delay between acquisitions.⁶⁶ In our NOVEL experiments a flip-back pulse immediately after the electron spin lock shortens the optimal shot-repetition time for the lock pulses to 0.5 ms, and, moreover, it increases the final enhancement by 15%.

What makes trityl OX063 such an efficient polarizing agent for NOVEL? (i) The EPR spectrum of trityl in frozen solution is very narrow. In our sample the central line has an fwhm of 7 MHz. This allows efficient excitation of all electrons in the sample with a 16 ns 90° pulse (corresponding to a bandwidth of 63 MHz). (ii) Trityl OX063 and its relatives were designed to minimize the isotropic hyperfine interaction with protons. At the same time there are many protons close enough to the unpaired electron to allow a strong dipolar interaction, that is, protons on the –CH₂– groups and in the solvent.^58,59 All trityl radicals contribute efficiently to DNP at the NOVEL condition γ_eB₁ = γ_nB₀. (iii) Trityl OX063 has a very small g-anisotropy and a relatively long T_1e,⁶⁷ which allows efficient spin-locking. On the other hand, the electronic T_1e is not too long to prohibit fast repetition of the locking sequence.

In conclusion, our experiments show that NOVEL can very efficiently polarize protons in a system that is of general interest to the solid-state DNP NMR community. This is a first, important step toward the general application of pulsed DNP at higher fields. Future work would include the development of high-frequency microwave sources that are able to generate the high-power pulses necessary to perform electron spin locking at higher fields.^68,69 At W band (95 GHz/3.4 T/144 MHz), amplifiers capable of producing kilowatt microwave pulses are already commercially available, and with a specialized setup NOVEL DNP at this frequency seems within reach. In addition one could think of alternative pulsed DNP methods, for which trityl OX063 in a frozen glycerol/water matrix is a good model system.

EXPERIMENTAL METHODS

NMR/EPR/DNP experiments at 15 MHz/9.8 GHz were performed on a Bruker ElexSys E580 X-band EPR spectrometer using an EN 4118X-MD4 (ENDOR) probe containing a dielectric microwave resonator. Microwave pulses were amplified using a 1 kW TWT amplifier 117X (Applied Systems Engineering, Fort Worth, TX). The ENDOR RF coil was tuned with an external tuning-matching circuit. NMR signals were detected with an iSpin-NMR System (SpinCore Technologies) with RF pulses amplified using a 1000 W LPI-10-21001 linear pulse amplifier (ENI, Rochester, NY). The sample temperature was kept at 80 K using a CF 935 flow cryostat with liquid nitrogen as a cryogen and an ITC 503S temperature controller (Oxford Instruments).

Solid-state ¹H NMR signals were acquired using a solid echo pulse sequence (π/2)_X − τ − (π/2)_Y − t − echo – with 90° pulses of 2.5 μs and t = 20 μs, with the phases of the pulses cycled through the conventional eight steps. Proton–proton dipole couplings lead to a short nuclear T₂ in our sample. Each acquisition consisted of 128 points and was performed with a detection bandwidth of 1 MHz. The signal was zero filled up to 1024 points prior to Fourier transformation.

Echo-detected EPR spectra were obtained using a Hahn echo pulse sequence (π/2)_X − t − (π)_X − t − echo – with 90° pulses of 16 ns and t = 500 ns using a two-step phase cycle. At each field position 100 acquisitions were performed with a repetition rate of 1 kHz. To record the echo intensity we set the integration window to cover the entire echo. Rise and fall time of the microwave pulses is ~2 ns.

NOVEL experiments were performed at the magnetic field that gave the strongest EPR echo intensity. The NOVEL pulse sequence is shown in Scheme 1. A presaturation sequence consisting of 16 120° pulses 10 ms apart is used to remove previously existing nuclear polarization. Subsequently, polarization is built up by running the NOVEL sequence for several seconds with a repetition rate of 1 kHz. The microwave cavity is maximally overcoupled, and the microwave power attenuation was set to 8 dB to generate a γ_eB₁ field of 15.6 MHz (corresponding to a 90° pulse of 16 ns) to fulfill the NOVEL condition. The length of the lock pulse was generally set to 500 ns. After hyperpolarization buildup an NMR experiment is performed. NMR signal enhancements by NOVEL were determined by comparing the spectral intensities of the on and off signals.

Samples for DNP experiments contained ~10 mM trityl OX063 in d₈-glycerol/D₂O/H₂O 60/30/10 v/v/v (a.k.a. “DNP juice”) with 1 M urea.⁷⁰ Exact trityl concentrations were determined by UV–vis absorption spectroscopy. Thin-wall precision quartz EPR sample tubes with an OD of 4 mm were used (Wilmad-LabGlass), which were filled to a sample volume of 50 μL.