Dynamic Nuclear Polarization with Vanadium(IV) Metal Centers

Abstract

•Harnessed V4+ metal centers as polarizing agents for dynamic nuclear polarization•Determined V4+ spin-diffusion barrier size using V4+-to-1H polarization transfer•Traced polarization pathways using V4+-1H spin “rulers”•Direct polarization of 1H nuclei located 12.6 Å from the V4+ center Dynamic nuclear polarization (DNP) offers a strategy to amplify the inherently insensitive nuclear magnetic resonance (NMR) signal by orders of magnitude, dramatically enhancing the NMR studies of molecules and materials. To date, most DNP studies have relied on exogenous polarizing agents (PAs) with narrow electron paramagnetic resonance (EPR) lines. Expanding the catalog of PAs to paramagnetic centers at the core of active sites in enzymes and functional materials will offer the opportunity to illuminate the local structure around these metal centers during chemical transformations. This study is the first demonstration of DNP using V4+ centers with broad EPR lines as PAs to enhance and detect the NMR signal of 1H nuclei at discrete distances from the V4+ center. These results pave the way toward endogenous DNP studies of active molecules and materials in which paramagnetic metals play key functional roles. Dynamic nuclear polarization (DNP) harnesses the large polarization of electron spins to dramatically increase nuclear magnetic resonance (NMR) sensitivity. This study expands the scope of DNP beyond its traditional focus on hyperpolarizing the solvent network using exogenous polarizing agents (PAs). We introduce 1H DNP with endogenous V4+ centers positioned in a set of vanadyl complexes with tunable V4+-1H distances. We traced the polarization transfer from V4+ to 1H spins, specifically differentiating between direct V4+-1Hs polarization transfer and the 1H spin-diffusion-mediated bulk solvent 1H polarization buildup and illuminated the effect of the V4+-1H distance on these processes. These results deepen our understanding of polarization pathways and expand the catalog of PAs to broad-line transition metals. This study establishes crucial first steps toward employing strategically positioned endogenous paramagnetic metal centers for DNP and the conceptual framework of hyperfine DNP spectroscopy that merges both spatial and chemical diagnosis of target nuclear spins. Dynamic nuclear polarization (DNP) harnesses the large polarization of electron spins to dramatically increase nuclear magnetic resonance (NMR) sensitivity. This study expands the scope of DNP beyond its traditional focus on hyperpolarizing the solvent network using exogenous polarizing agents (PAs). We introduce 1H DNP with endogenous V4+ centers positioned in a set of vanadyl complexes with tunable V4+-1H distances. We traced the polarization transfer from V4+ to 1H spins, specifically differentiating between direct V4+-1Hs polarization transfer and the 1H spin-diffusion-mediated bulk solvent 1H polarization buildup and illuminated the effect of the V4+-1H distance on these processes. These results deepen our understanding of polarization pathways and expand the catalog of PAs to broad-line transition metals. This study establishes crucial first steps toward employing strategically positioned endogenous paramagnetic metal centers for DNP and the conceptual framework of hyperfine DNP spectroscopy that merges both spatial and chemical diagnosis of target nuclear spins. Nuclear magnetic resonance (NMR) spectroscopy, a widely used tool for elucidating fundamental chemical, structural, and dynamical information in molecules and materials, is inherently limited by the poor polarization of nuclear spins. Dynamic nuclear polarization (DNP) is the most broadly applicable hyperpolarization method for enhancing the NMR signal by orders of magnitudes, relying on polarization transfer from highly polarized electron spins (e) to the surrounding nuclear spins (n). In a typical DNP experiment, a source of unpaired electron spins—known as a polarizing agent (PA)— is mixed with the sample in a 1H-rich glassing matrix. Microwave (μw) irradiation near the electron paramagnetic resonance (EPR) frequency of the PA can drive polarization transfer from the electron to the surrounding 1H nuclear spins. Current state-of-the-art DNP methodologies have already transformed the scope of NMR in fields from structural biology to materials science.1Lilly Thankamony A.S. Wittmann J.J. Kaushik M. Corzilius B. Dynamic nuclear polarization for sensitivity enhancement in modern solid-state NMR.Prog. Nucl. Magn. Reson. Spectrosc. 2017; 102–103: 120-195Crossref PubMed Scopus (237) Google Scholar To date, nitrogen-centered nitroxide or carbon-centered trityl radicals are used nearly exclusively as PAs, owing to their stability, solubility, molecular geometry, relatively long electron spin relaxation time, and an electron spin g factor near 2.0, matching that of a free electron.1Lilly Thankamony A.S. Wittmann J.J. Kaushik M. Corzilius B. Dynamic nuclear polarization for sensitivity enhancement in modern solid-state NMR.Prog. Nucl. Magn. Reson. Spectrosc. 2017; 102–103: 120-195Crossref PubMed Scopus (237) Google Scholar However, these PAs are exogenously introduced and do not serve as a polarization source to report on specific locales around paramagnetic active sites in molecules and functional materials. A huge opportunity exists in the use of paramagnetic transition metal centers, intrinsic to the system of interest, as PAs. To date, highly electronically symmetric paramagnetic metals, such as Gd3+, Mn2+, and Cr3+, with narrow central EPR transition bands have been used as exogenous PAs for high-field (> 5 T) DNP.2Corzilius B. Smith A.A. Barnes A.B. Luchinat C. Bertini I. Griffin R.G. High-field dynamic nuclear polarization with high-spin transition metal ions.J. Am. Chem. Soc. 2011; 133: 5648-5651Crossref PubMed Scopus (94) Google Scholar, 3Kaushik M. Bahrenberg T. Can T.V. Caporini M.A. Silvers R. Heiliger J. Smith A.A. Schwalbe H. Griffin R.G. Corzilius B. Gd(iii) and Mn(ii) complexes for dynamic nuclear polarization: small molecular chelate polarizing agents and applications with site-directed spin labeling of proteins.Phys. Chem. Chem. Phys. 2016; 18: 27205-27218Crossref PubMed Google Scholar, 4Corzilius B. Michaelis V.K. Penzel S.A. Ravera E. Smith A.A. Luchinat C. Griffin R.G. Dynamic nuclear polarization of (1)H, (13)C, and (59)Co in a tris(ethylenediamine)cobalt(III) crystalline lattice doped with Cr(III).J. Am. Chem. Soc. 2014; 136: 11716-11727Crossref PubMed Scopus (45) Google Scholar, 5Corzilius B. eMagRes Harris R.K. Wasylishen R.L. Paramagnetic metal ions for dynamic nuclear polarization. Wiley & Sons, 2018Google Scholar Corzillius and coworkers employed endogenous paramagnetic metal centers, such as Mn2+, of a hammerhead ribozyme complex to enhance the 13C-NMR signal, and Leskes and coworkers used Mn2+ and Fe3+ ions to hyperpolarize 7/6Li and 17O in battery materials.6Wenk P. Kaushik M. Richter D. Vogel M. Suess B. Corzilius B. Dynamic nuclear polarization of nucleic acid with endogenously bound manganese.J. Biomol. NMR. 2015; 63: 97-109Crossref PubMed Scopus (41) Google Scholar, 7Wolf T. Kumar S. Singh H. Chakrabarty T. Aussenac F. Frenkel A.I. Major D.T. Leskes M. Endogenous dynamic nuclear polarization for natural Abundance 17O and lithium NMR in the bulk of inorganic solids.J. Am. Chem. Soc. 2019; 141: 451-462Crossref PubMed Scopus (44) Google Scholar, 8Harchol A. Reuveni G. Ri V. Thomas B. Carmieli R. Herber R.H. Kim C. Leskes M. Endogenous dynamic nuclear polarization for sensitivity enhancement in solid-state NMR of electrode materials.J Phys Chem C Nanomater Interfaces. 2020; 124: 7082-7090Crossref PubMed Scopus (15) Google Scholar However, many transition metal ions, such as Ni+, Cu2+, Ni3+, and V4+, that are widely present and central to the function of battery materials, catalytic compounds, and metalloenzymes, are considered inaccessible to DNP due to their wide EPR lines and g values that are significantly shifted from 2.0. Expanding DNP capabilities to utilize these metal centers as PAs would provide a significant step toward DNP-enhanced NMR studies with endogenous paramagnetic metal centers. Attaining local chemical and structural information with DNP-enhanced NMR studies using endogenous paramagnetic transition metals necessitates a rigorous understanding of their polarization pathways. Fundamentally, bulk polarization of nuclear spins by DNP comprises two stages: first, polarization transfer from the PAs to discrete nuclear spins by PA-nuclear spin hyperfine interactions; second, polarization transfer by nuclear spin diffusion, in which polarized nuclear spins exchange energy with nearby, unpolarized spins to propagate polarization to bulk nuclei. These processes, and the detected NMR spectrum, are influenced by paramagnetic effects, such as paramagnetic relaxation enhancement (PRE)9Bloembergen N. On the interaction of nuclear spins in a crystalline lattice.Physica. 1949; 15: 386-426Crossref Scopus (713) Google Scholar, 10Blumberg W.E. Nuclear spin-lattice relaxation caused by paramagnetic impurities.Phys. 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These paramagnetic effects give rise to the “spin-diffusion barrier”15Khutsishvili G. Spin diffusion and magnetic relaxation of nuclei.Sov. Phys. JETP. 1962; 15: 909-913Google Scholar that defines how effectively the nuclear spins can transfer the polarization from near the paramagnetic center outward to other nuclear spins through nuclear spin diffusion after getting hyperpolarized. A number of studies have explored the concept of the spin-diffusion barrier around lanthanides, transition-metal centers, and organic radicals.16Ramanathan C. Dynamic nuclear polarization and spin diffusion in nonconducting solids.Appl. Magn. Reson. 2008; 34: 409-421Crossref Scopus (60) Google Scholar, 17Canarie E.R. Jahn S.M. Stoll S. Quantitative structure-based prediction of electron spin decoherence in organic radicals.J. Phys. Chem. Lett. 2020; 11: 3396-3400Crossref PubMed Scopus (18) Google Scholar, 18Tan K.O. Mardini M. Yang C. Ardenkjær-Larsen J.H. Griffin R.G. Three-spin solid effect and the spin diffusion barrier in amorphous solids.Sci. Adv. 2019; 5eaax2743Crossref PubMed Scopus (20) Google Scholar The exact size of the spin-diffusion barrier is a critical parameter in determining the DNP polarization pathway and buildup rate, as it determines the location of the nearest nuclear spins that serve as a conduit for nuclear spin diffusion to remote nuclei. Hence, the rate of nuclear spin diffusion depends on the closest paramagnetic metal-nuclear spin distance for nuclei located beyond the spin-diffusion barrier that should give rise to a gradient of nuclear spin-diffusion rates. Many DNP models rely on knowledge of the spin-diffusion barrier and the polarization transfer rate,19Pinon A.C. Spin diffusion in dynamic nuclear polarization nuclear magnetic resonance. EPFL, 2018https://infoscience.epfl.ch/record/256344?ln=enGoogle Scholar, 20Smith A.A. Corzilius B. Barnes A.B. Maly T. Griffin R.G. Solid effect dynamic nuclear polarization and polarization pathways.J. Chem. Phys. 2012; 136015101Crossref PubMed Scopus (85) Google Scholar, 21Wittmann J.J. Eckardt M. Harneit W. Corzilius B. Electron-driven spin diffusion supports crossing the diffusion barrier in MAS DNP.Phys. Chem. Chem. Phys. 2018; 20: 11418-11429Crossref PubMed Google Scholar which can be aided by experimentally validated knowledge of the effect of the electron-nuclear spin distance on the DNP buildup rates. These rates determine the spatial propagation of nuclear hyperpolarization and modulate the sensitivity enhancement by DNP. In this study, we demonstrate the viability of V4+ ions as PAs to enhance the 1H-NMR signal of localized protons around the transition metal center at 6.9 T. We designed a series of vanadyl complexes with deliberately installed 1H-containing trimethylene groups at varying distances from the V4+ center on an otherwise nuclear-spin-free ligand backbone.22Graham M.J. Yu C.J. Krzyaniak M.D. Wasielewski M.R. Freedman D.E. Synthetic approach to determine the effect of nuclear spin distance on electronic spin decoherence.J. Am. Chem. Soc. 2017; 139: 3196-3201Crossref PubMed Scopus (43) Google Scholar These transition metal-nuclear spin “rulers” allow a systematic study of the effectiveness of the 1Hs to conduct the spin-diffusion process as a function of their distance to the V4+ centers. We present the first demonstration of DNP using a wide-line transition metal by broadband irradiation of the V4+ EPR transitions. A versatile and unique (to date) DNP NMR instrument powered by a tunable-frequency (193–201 GHz) solid-state microwave source with arbitrary waveform generation (AWG) enabled these experiments. DNP-enhanced 1H-NMR spectra and polarization buildup curves quantified the radius of the spin-diffusion barrier to be between 4.0–6.6 Å and revealed that spin-diffusion-mediated bulk 1H polarization buildup directly depends on the position of the trimethylene 1H nuclei relative to the barrier. Crucially, we demonstrate direct polarization transfer to, and NMR detection of, 1H nuclei located 12.6 Å away from the V4+ center via V4+-1H hyperfine interaction. This work comprises the first systematic study of the effect of the spin-diffusion barrier around a paramagnetic metal center on polarization transfer and DNP buildup rates and paves the way toward elucidating structural and chemical information around paramagnetic active sites and cofactors. To distinguish DNP of local, select nuclear spins from DNP of bulk nuclei in order to achieve global NMR sensitivity enhancement, we dub this novel category of experiments hyperfine DNP spectroscopy. The V4+-1H rulers, i.e., the vanadyl complexes with controlled average V4+-1H distances (RV-H), are shown in Figure 1A, with RV-H = 4.0, 6.6, 9.3, and 12.6 Å for complexes 1–4, respectively. The complexes with chemical formulas (Ph4P)2[VO(C3H6S2)2] (1), (Ph4P)2[VO(C5H6S4)2] (2), (Ph4P)2[VO(C7H6S6)2] (3), and (Ph4P)2[VO(C9H6S8)2] (4) have tetraphenylphosphonium cations and were dissolved in 99.5% deuterated dimethylformamide (DMF). The field-swept echo-detected EPR spectra of the vanadyl complexes were recorded at a μw frequency (ωμw) of 240 GHz by sweeping the field from 8.4 to 9 T at 5 K (see Figure S1). The principle components of the g factor and hyperfine coupling (A) between paramagnetic V4+ ([Ar]3d1, S = 1⁄2) and 100% abundant 51V isotope (I = 7⁄2) were extracted by fitting these echo-detected EPR spectra using EasySpin.23Stoll S. Schweiger A. EasySpin, a comprehensive software package for spectral simulation and analysis in EPR.J. Magn. Reson. 2006; 178: 42-55Crossref PubMed Scopus (3750) Google Scholar Figure 1B depicts the EPR line shapes simulated at 6.9 T based on the fitted EPR parameters. The g and A tensor values found for all vanadyl complexes are well resolved at the high field and frequency employed in this study (Table S1 and S2) and are in agreement with previously reported values for the same complexes determined by X-band CW EPR analysis.22Graham M.J. Yu C.J. Krzyaniak M.D. Wasielewski M.R. Freedman D.E. Synthetic approach to determine the effect of nuclear spin distance on electronic spin decoherence.J. Am. Chem. Soc. 2017; 139: 3196-3201Crossref PubMed Scopus (43) Google Scholar The EPR lines of the vanadyl complexes span more than 3 GHz, which are significantly broader compared to the nitroxide-based radicals that span 0.6–1 GHz at 6.9 T and 4 K. The EPR spectrum is inhomogenously broadened as a result of the g-anisotropy of V4+, as corroborated by data fitting in EasySpin (Figure S1; Table S1). While transition metal centers can have EPR line broadening spanning 100s of GHz,24Barra A.-L. Gräslund A. Andersson K.K. The use of very high frequency EPR (VHF-EPR) in studies of radicals and metal sites in proteins and small inorganic models.in: Grinberg O.Y. Berliner L.J. Very High Frequency (VHF) ESR/EPR. Springer, 2004: 145-163Crossref Google Scholar only narrow-line radicals (line width < 800 MHz) have been utilized in the current state-of-the-art DNP experiments due to the limited μw frequency range in commercial instruments. In this context, paramagnetic metals with EPR line widths exceeding 1 GHz are categorized as wide-line PA for DNP. In the previous EPR study of these complexes, the size of the spin-diffusion barrier was reported to be between 4.0 and 6.6 Å. Here, we explore the viability of V4+ centers as PA for DNP, determine the size of spin-diffusion barrier under DNP conditions (at high field of 6.9 T) using NMR detection, and study its influence on the DNP process. To determine whether the V4+ ions are viable polarization agents, we investigated the DNP frequency profiles for 1–4. The DNP frequency profiles of these broad-line V4+ centers were recorded using the EPR-NMR pulse sequence, in which the μw irradiation frequency was varied over a 3 GHz span (Figure 2A). The NMR signal enhancement factors were determined by calculating the ratio ε = (SON − SOFF)/SOFF, where SON and SOFF are NMR signal intensities under μw-on and μw-off conditions at equal buildup times, respectively. Figure 2B shows the DNP frequency profiles recorded with chirped μw pulse trains. The DNP frequency profiles across all complexes were broad and asymmetric with maximum positive and negative enhancement positions separated by ∼ 1 GHz. These complexes exhibited larger negative enhancements at around 192.4 GHz compared to positive enhancements at around 191.5 GHz. The same general features for the DNP profiles were observed with monochromatic CW irradiation with the exception of lower overall enhancement values (εCW-DNP, Figure S2). The DNP frequency profiles of the complexes provide key insight into the mechanism of polarization transfer in these systems. The DNP profiles for all complexes display a sharper intensity slope at the higher frequency end and a longer tail toward low frequencies, reflecting the broad and asymmetric dispersion of the EPR spectrum caused by inhomogeneous broadening (Figure 1B). This indicates that the underlying mechanism is the differential solid effect (SE).25Wenckebach W.T. The solid effect.Appl. Magn. Reson. 2008; 34: 227-235Crossref Scopus (45) Google Scholar In conventional SE, electron-nuclear dipolar interactions permit the forbidden electron-nuclear double quantum and zero quantum excitations that lead to the characteristic positive and negative enhancements separated by twice the nuclear Larmor frequency. Differential SE gives rise to the superposition of such SE profiles whose center frequencies span the inhomogeneously broadened EPR line. In such cases, the shape (width and symmetry) of DNP frequency profiles is dominated by the EPR line shape, which in the current case resulted in an asymmetric and broad DNP frequency profile. The basic feature of such DNP profiles could be replicated by numerical simulations in which the difference of the positive and negative enhancements, according to SE DNP for each frequency point, was calculated from the respective EPR signal intensity to compute the net DNP enhancement and the DNP profile reconstructed for each complex (see Figure S3). DNP-enhanced 1H-NMR spectra measured at the maximum positive enhancement frequency (ωμw = 191.30, 191.50, 191.35, and 191.40 GHz for 1–4, respectively) yielded εChirp-DNP ∼ 1, 19, 12, and 9 for 1–4 using chirped μw pulses (Figure 3, solid line). CW μw irradiation yielded enhancements of εCW-DNP ∼ 0.5, 1.7, 1.7, and 1.8 for 1–4, respectively (Figure 3, dashed-lines). Corresponding 1H-NMR spectra collected at the maximum negative enhancement with chirped μw pulses revealed similar enhancement values (Figure S4). The higher enhancement observed in sample 2 is attributed to the high solubility of the complex in the DMF solvent (77.2 mM),26We hypothesize that the solubility of complex 2 in DMF is particularly high due to the optimal balance between the non-polar nature of the organic ligands and the overall charge of the complex. With the longer linkers in complex 3 and 4, the nonpolar nature of those ligands are dominant and inhibit solubility in DMF. With complex 1, the ligand is more ionic in nature, and combined with the 2– charge of the complex may render the complex highly ionic, hence reducing its solubility in DMF. Taken together, we propose that the nonpolar nature of the ligands on complex 2 balance the ionic nature of the complex, thereby enhancing its solubility in DMF.Google Scholar resulting in a relatively large number of V4+ metal centers in the sample. Crucially, these results demonstrate, for the first time, 1H polarization enhancement with a wide-line EPR transition in V4+ paramagnetic ions using broadband microwave pulses. The significant DNP performance boost obtained by transitioning from CW monochromatic to broadband chirp train μw irradiation has recently been reported in organic biradicals.27Kaminker I. Han S. Amplification of dynamic nuclear polarization at 200 GHz by arbitrary pulse shaping of the electron spin saturation profile.J. Phys. Chem. Lett. 2018; 9: 3110-3115Crossref PubMed Scopus (14) Google Scholar Broadband pulse trains are crucial for DNP to access a greater population of wide-line transition metal centers that constructively participate in polarization enhancement. The inhomogeneous broadened EPR lines due to large g-anisotropy in these metals spread out the electron spin density across a wide frequency range. As a result, monochromatic CW microwave irradiation would only engage a small population of the V4+ centers in DNP that, hence, results in small NMR signal enhancements. Thus, by using shaped μw pulses to fully saturate the broad EPR transition, we could successfully access V4+ spin centers that generate significant polarization enhancement. The gain factor was εChirp-DNP/εCW-DNP > 4 for all samples 1–4, reaching up to ∼10 for 2 (with εChirp-DNP = −33 and εCW-DNP = −3.5 in the region of negative enhancement). By implementing broadband chirp μw pulses, we demonstrate 1H polarization with V4+ paramagnetic metal centers traditionally believed to be inaccessible for DNP. To realize DNP with endogenous paramagnetic metal centers, we need to understand the polarization transfer mechanism and pathways. Specifically, we need to know the polarization characteristics of protons relative to their proximity to the polarization agent. Because differential SE DNP is fundamentally based on electron-nuclear dipolar interactions, complexes 1–4 present a unique opportunity to systematically investigate polarization pathways with modular V4+-1H dipolar interactions. Essential to this investigation is identifying the distinct nuclear spins participating in the polarization process. Based on our sample preparation, the enhanced 1H-NMR signal observed can originate from three different sources: the trimethylene moieties on the vanadyl complex, the tetraphenylphosphonium (PPh4+) counterion, and the 0.5% protons in the DMF-d7 solvent. To begin our investigation, we turned to the DNP-enhanced NMR spectra of 1–4 (Figure 3), which show a common narrow signal across the complexes. However, the 1H-NMR spectral line shapes of 4 clearly indicate the presence of two spectral components with different line widths. In order to distinguish between the two signals in 4 and identify the common narrow signal across 1–3, the inter-pulse delay (τ) was varied between 30–200 μs (see Figure S5) to observe whether the broad signal in 4 can be selectively suppressed with increasing τ. The 1H spectra of 4 recorded with τ = 50 μs (cyan, solid line) and τ = 200 μs (magenta, dashed line) are shown in Figure 4A. With a 200-μs inter-pulse delay, we observe only a single narrow spectral component. This indicates that the 1H nuclear spins constituting the broad spectral component have significantly shorter transverse relaxation times (T2n) relative to those of the narrow spectral component. A deconvolution of the 1H-NMR acquired with τ = 50 μs revealed that the narrow peak is centered at −5 ppm and the broad peak shifted upfield to −14 ppm (Figure S7). The combination of a shorter T2n, a broad line shape, and an upfield-shifted peak position suggests that these nuclear spins experience greater paramagnetic effects (PRE and PCS) than those of the narrow signal. This is further reinforced by solution-state 1H-NMR spectra of 4, wherein the peaks of the complex protons (centered at 2.22 ppm) were broader compared to that of the solvent 1Hs (counterion protons at 7.77 ppm and the DMF protons at 7.91, 2.80, and 2.64 ppm) as shown in Figure S6. The upfield shift at low temperatures has also been reported in previous work on S = ½ vanadium complexes.28Morse D.B. Hendrickson D.N. Rauchfuss T.B. Wilson S.R. Highly oxidizing organometallics: physicochemical characterization of (methylcyclopentadienyl)vanadium(IV) trichloride and related vanadium(III) and titanium(III) derivatives.Organometallics. 1988; 7: 496-502Crossref Scopus (20) Google Scholar Thus, we assigned the broad-signal component with shorter T2n to the protons covalently attached to the complex, referred to as “complex protons,” and we assigned the narrow signal with longer T2n to “solvent protons” that include protons on the counter ions and the DMF solvent. To discount the possibility of the counterion protons being too close to the V4+ centers leading to the broad signal, we performed additional pulsed hyperfine EPR experiments (known as electron spin echo envelope modulation [ESEEM]) to detect V4+-31P hyperfine couplings (ESEEM is sensitive to V4+-31P distances in the 3–7 Å range) between V4+ of the complexes and 31P of the PPh4 ions (data not shown). However, no modulations were observed, indicating that the counterions are not in close proximity to V4+. This observation is consistent with the size of the solvation shell being sufficiently large for the tetraphenylphosphonium ion, such that the ions are separated far enough from the vanadyl complex. Moreover, the broad signal was absent in complexes 1–3, which further confirms that only the protons on the complex give rise to this signal. The absence of the broad components in samples 1–3 is a result of paramagnetic quenching due to strong PRE at shorter RV-H compared to 4. In contrast, the complex 1Hs of sample 4 are clearly visible as a broad spectral component. With the chemical identity of the protons that constitute the observed NMR signals confirmed, we then proceeded to investigate the 1H polarization buildup times of the different nuclei. The DNP buildup times of the 1H-NMR signal of complexes 2–4 were recorded, as shown in Figure 4B, including those of the two 1H signals of 4. Note that the DNP buildup of complex 1 is not shown, as there was no DNP enhancement observed with this sample (Figure 3). To test whether the total sample concentration affected the buildup curve, two concentrations of sample 2, 77.2 mM (at saturation) and 13 mM (comparable with the other three complexes) were measured. All DNP buildup curves were fitted to a stretched exponential:I=I0[1−e−(tTDNP)n]where I0 is the NMR signal intensity at DNP saturation, TDNP is the time constant for polarization buildup, and n is the stretch factor. The value of n (≤ 1) provides key information about the nature of the polarization process. We expect that polarization buildup dominated by 1H nuclear spin diffusion to be a mono-exponential process, resulting in n ∼ 1. Should other processes, such as V4+-1H hyperfine coupling interactions, contribute to polarization buildup, we expect a multi-exponential process that lowers n toward a value of 0.5.29Tse D. Hartmann S.R. Nuclear spin-lattice relaxation via paramagnetic centers without spin diffusion.Phys. Rev. Lett. 1968; 21: 511-514Crossref Scopus (157) Google Scholar The TDNP, n, and I0 values obtained from the fits are given in Table 1.Table 1Fitted Parameters for the Proton Buildup Curves in 2–4 for Chirped DNP ExperimentsComplex#TDNP (s)NI04 (complex)18.2 ± 1.50.60 ± 0.031.003 ± 0.0204 (solvent)47.7 ± 6.50.79 ± 0.061.021 ± 0.049388.0 ± 4.00.78 ± 0.011.176 ± 0.0182142.7 ± 28.30.80 ± 0.091.053 ± 0.0722 (77 mM)120.4 ± 9.00.93 ± 0.021.253 ± 0.048 Open table in a new tab Between the two types of 1H nuclei, the complex protons on 4 have the shortest TDNP and smallest n values relative to the solvent protons in 2–4. These nuclei exhibit a stretching parameter (n = 0.6) that is very close to 0.5, which we, therefore, attribute to polarization by V4+-1H hyperfine interactions and not nuclear spin diffusion. Polarization via hyperfine interactions involves the direct transfer of polarization through the double and zero quantum transitions. This single-step process is expected to be faster than polarization by the stochastic, multi-step nuclear-spin-diffusion processes. The