High-resolution NMR of hydrogen in organic solids by DNP enhanced natural abundance deuterium spectroscopy

Journal of Magnetic Resonance 259 (2015) 192–198

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High-resolution NMR of hydrogen in organic solids by DNP enhanced natural abundance deuterium spectroscopy Aaron J. Rossini a,b, Judith Schlagnitweit b, Anne Lesage b, Lyndon Emsley a,b,⇑ a b

Institut des Sciences et Ingénierie Chimiques, Ecole Polytechnique Fédérale de Lausanne (EPFL), CH-1015 Lausanne, Switzerland Institut des Sciences Analytiques, Centre de RMN à très hauts champs (CNRS/ENS-Lyon, UCB-Lyon1), Université de Lyon, 69100 Villeurbanne, France

a r t i c l e

i n f o

Article history: Received 5 July 2015 Revised 21 August 2015 Available online 8 September 2015 Keywords: NMR spectroscopy Deuterium Hyper-polarization

a b s t r a c t We demonstrate that high field (9.4 T) dynamic nuclear polarization (DNP) at cryogenic (100 K) sample temperatures enables the rapid acquisition of natural abundance 1H–2H cross-polarization magic angle spinning (CPMAS) solid-state NMR spectra of organic solids. Spectra were obtained by impregnating substrates with a solution of the stable DNP polarizing agent TEKPol in tetrachloroethane. Tetrachloroethane is a non-solvent for the solids, and the unmodified substrates are then polarized through spin diffusion. High quality natural abundance 2H CPMAS spectra of histidine hydrochloride monohydrate, glycylglycine and theophylline were acquired in less than 2 h, providing direct access to hydrogen chemical shifts and quadrupolar couplings. The spectral resolution of the 2H solid-state NMR spectra is comparable to that of 1 H spectra obtained with state of the art homonuclear decoupling techniques. Ó 2015 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

1. Introduction Deuterium solid-state NMR is widely employed to measure hydrogen chemical shifts and probe dynamics by measurement of 2H electric field gradient (EFG) tensors [1–9]. However, since 2 H has very low natural isotopic abundance (0.0115%) and it is an I = 1 quadrupolar nucleus, 2H solid-state NMR is normally performed on isotopically labeled samples. This restricts the widespread use of 2H solid-state NMR. It was also recognized early on that magic angle spinning (MAS) deuterium solid-state NMR could be employed to obtain high-resolution measurement of hydrogen chemical shifts [1–9]. Currently, the most common way to measure hydrogen chemical shifts in solids in the absence of isotopic enrichment/dilution is to measure MAS 1H spectra. However, because of the network of dipolar couplings between abundant 1 H spins, resolution is limited, even with the most advanced dipolar decoupling schemes [10–14] and even at the fastest MAS frequencies available today [13,15,16]. The ability to routinely perform 2H solid-state NMR at natural abundance would be of tremendous benefit. A number of research groups have demonstrated that direct excitation natural abundance 2H solid-state spectra may be acquired from mobile deuterium nuclei (e.g., of methyl groups) ⇑ Corresponding author at: Institut des Sciences et Ingénierie Chimiques, Ecole Polytechnique Fédérale de Lausanne (EPFL), CH-1015 Lausanne, Switzerland. E-mail address: [email protected] (L. Emsley).

with acquisition times of several hours [8,17–21]. Dynamics improves sensitivity by reducing 2H longitudinal relaxation times and partially averaging the quadrupolar interaction. Reduced quadrupolar couplings result in higher sensitivity since the 2H MAS NMR signals are focused into fewer sidebands. For these reasons, acquisition of natural abundance 2H solid-state NMR spectra of rigid solids is much more challenging. Takegoshi and co-workers showed by combining cross-polarization [22] (CP) from 1H to 2H [2,23–26], MAS, high magnetic fields and with co-addition of the spinning sidebands to the isotropic peaks in the NMR spectra, that natural abundance 2H solid-state NMR spectra of the rigid organic solid dimedone could be obtained [27]. Nevertheless, 13 h of signal averaging were required to obtain a reasonable signal to noise ratio (SNR) [27]. Thus, while promising, this approach is still not broadly applied. To reduce the lengthy signal averaging times, the sensitivity of 2H NMR experiments must be substantially increased. It is now possible to enhance the sensitivity of high-field solidstate NMR experiments by several orders of magnitude with dynamic nuclear polarization (DNP) [28–31] where the polarization of unpaired electrons is transferred to NMR active nuclei. Usually the unpaired electrons are introduced by doping the sample with stable exogenous radical polarizing agents [32–34]. High-field DNP enhanced solid-state NMR has been applied to biomolecules dissolved/suspended in solution, to biomolecules associated with membranes or cellular materials [35–42], to inorganic materials and surfaces [43–54] and to organic solids [55–62].

http://dx.doi.org/10.1016/j.jmr.2015.08.020 1090-7807/Ó 2015 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

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For DNP experiments on surfaces and inorganic materials the samples are easily prepared with an incipient wetness impregnation procedure [43,48]. Impregnation introduces radical solution onto the surface or inside the porous volume of many materials, bringing the radicals into proximity to the target nuclei to be polarized and simultaneously providing a suitable medium to propagate polarization from the radical to the target. In the case of nonporous nano or micro-particulate organic solids impregnation can also be used to prepare samples for spin diffusion relayed DNP experiments [56]. In spin diffusion relayed DNP experiments, samples are impregnated with a minimal volume of radical solution, where the impregnating medium is chosen so that the solid to be studied is insoluble, to coat the surface of the particles, leaving the (crystalline or amorphous) solids intact. During DNP enhanced 1 H polarization builds up at the surface of the particles, and 1H–1H spin diffusion relays the polarization into the interior of the material [55,56]. This can be used to polarize non-porous insoluble micro-particulate organic solids [56], and is now being rapidly taken up [54–56,61,62]. Maly et al. have previously applied direct 2H DNP to deuterated analytes dissolved in glass forming solutions, however this approach is not applicable to crystalline organic solids with natural 2 H abundance [63]. Here we apply 1H spin diffusion relayed DNP to polarize protons throughout organic solids [55,56], followed by 1 H–2H CP. This can provide signal enhancements large enough to overcome the 0.0115% natural abundance of 2H. We show that for the organic solids histidine hydrochloride monohydrate (hist), glycylglycine (gly) and theophylline (theo, Scheme 1) this enables the acquisition of natural abundance 2H CPMAS spectra in about an hour. The resolution of the 2H solid-state NMR spectra are comparable to that obtained in 1H spectra with state of the art homonuclear decoupling, enabling the high resolution measurement of hydrogen chemical shifts and 2H EFG tensor parameters. 2. Experimental methods DNP experiments were performed on a 9.4 T 263 GHz/400 MHz Bruker Avance III solid-state DNP NMR spectrometer [64] with a

Scheme 1. Molecular structures of histidine hydrochloride monohydrate (hist), glycylglycine (gly) and theophylline (theo). hist and gly are drawn as zwitterions to reflect the proton positions in the solid state.

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triple resonance 3.2 mm HXY probe configured for 1H–13C–2H experiments. 2H pulse calibrations and 1H–2H CPMAS experiments were optimized with DNP on 12.5 mM AMUPol DSMO-d6/D2O/H2O 60/36/6. The magic angle was adjusted with the 79Br signal of KBr at low temperature. The powdered organic solids were ground by hand for several minutes in an agate mortar and pestle to reduce particle sizes and then impregnated [43] with a minimal volume of a solution of TEKPol in TCE (Table S1) [65]. 1H–2H CPMAS experiments used CP contact times between 9 and 14 ms and a constant 2 H spin lock rf field of 80 kHz. The amplitude of the 1H spin lock was ramped [66] from 46 kHz to 66 kHz. 100 kHz SPINAL-64 1 H heteronuclear decoupling was applied during acquisition [67]. DNP enhanced 2D 1H–13C HETCOR spectra were acquired with mr = 12.5 kHz and 100 kHz eDUMBO1–22 homonuclear decoupling [12] during t1 (Fig. S4). Room temperature ‘‘three pulse” 1H–1H spin diffusion experiments on hist (Fig. S5) were performed with 10.0 kHz MAS on a 2.5 mm HX probe on a Bruker Avance III 11.7 T spectrometer with 100 kHz LG4 decoupling [14,68] with a = 55° during t1. 2H solid-state NMR spectra were simulated with the Solid Lineshape Analysis module (v. 2.2.4) in the Bruker Topspin v3.2 software.

3. Results and discussion In order to enable DNP experiments on the micro-crystalline solids they were all finely ground by hand in a mortar and pestle to reduce the particle size, then impregnated with 14–16 mM solutions of TEKPol [65] in tetrachloroethane (TCE). This impregnating solution provides high DNP enhancements [69] and all the solids studied here are insoluble in TCE. DNP enhanced proton polarization builds up rapidly at the surface of the particles and is then relayed into the interiors of the particles by 1H–1H spin diffusion [55,56]. Note that TCE provides high DNP enhancements without requiring partial deuteration, which should ideally be avoided to eliminate intense solvent 2H signals (Though solvent signals could be suppressed by using spin-echo detection.) [70]. As has been discussed previously, the relayed-DNP enhancements depend strongly on the T1 of the substrate at 100 K [55,56,61]. For demonstration purposes here, we have thus chosen two molecules with long T1, and one compound with a shorter T1. At 105 K the proton T1 of both hist and gly are on the order of several hundred seconds. Very high DNP enhancements can be obtained with spin diffusion relayed DNP due to the long proton T1 relaxation times [55,56]. For hist, the 1H DNP enhancement (eH) of the protons of TCE at the surface of the particles was around 232. The 1H DNP enhancement inside the particles was determined from 1H–13C CPMAS spectra (Fig. S1). The eC CP measured for hist was 144 (Fig. S1). eH for the protons of TCE impregnating powdered gly was 340 and eC CP of 128 was measured for the protons within the gly particles (Fig. S1). The very high eH value likely occurs due to the favorable dielectric properties of powdered gly [71]. Both hist and gly possess relatively long proton T1’s, and polarization (recycle) delays between 60 s and 100 s yield optimal sensitivity. On the other hand theo has rotating methyl groups which result in a proton T1 of only 22 s at 105 K. An eH of 250 was measured for the protons of TCE and eC CP of only 17 was measured for theo as a consequence of its shorter 1H T1 (Fig. S1). This eC CP is similar and slightly higher than value of 12 obtained by Viel and co-workers for theo impregnated with a 16 mM TEKPol TCE solution [72]. Despite the reduced DNP enhancements obtained for theo, high sensitivity is obtained because relatively short optimal polarization delays of 12–30 s can be used. In summary, substantial gains in sensitivity are obtained with spin diffusion relayed DNP for organic solids with both long and

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short T1. This is in line with previous observations where, for example, good sensitivity can be achieved even with compounds having T1 around 100 K as short as 15 s [56]. In the following we exploit this to acquire natural abundance 2H solid-state NMR spectra. Fig. 1 shows the DNP enhanced natural abundance 1H–2H CPMAS solid-state NMR spectra of powdered crystalline samples of hist, gly and theo acquired at a MAS rate (mr) of 5 kHz. Since the MAS rate is much less than the magnitude of the 2H quadrupolar interaction, the spectra are split into spinning sidebands spanning ca. 250 kHz. The isotropic region of the spectra has good resolution and most of the expected resonances are clearly resolved (vide infra). In all cases the 2H NMR signal arising from TCE is weak, likely because the 2H signal of frozen glassy TCE is inhomogeneously broadened, and more affected by the paramagnetic polarization agents. Importantly, Fig. 1 illustrates that high SNR 2H CPMAS spectra can be acquired in short times with DNP (Table 1). The natural abundance 2H CPMAS spectra of hist, gly and theo were acquired in 2.1 h, 1.1 h and 2.3 h, respectively. The SNR of the most and least intense isotropic peaks in the spectrum of hist (which showed the lowest overall sensitivity) were above 20 and 4, respectively. The SNR can be further increased by numerically post-processing the spectra and co-adding each of the spinning sidebands to the isotropic region (Fig. 1) [27]. With this simple procedure the SNR of the most and least intense isotropic peaks increases to about 56 and 12, respectively for hist. Much higher SNR and sensitivity was obtained for theo and gly, because the 2H signals are dispersed over fewer peaks. Note that stable sample spinning (here ±2 Hz) is required to avoid broadening of the peaks by the sideband addition procedure.

Natural abundance 2H CPMAS spectra can be rapidly acquired because of the combined gain in sensitivity obtained from the combination of DNP, CP and reduced sample temperatures. CP from 1H theoretically provides a maximum gain of a factor of c1H/c2H = 6.5. Takegoshi and co-workers observed CP signal gains close to the theoretical maximum for experiments on partially 2H labeled rigid solids [27]. Therefore, we expect that a similar gain in sensitivity is obtained here with CP. In agreement with previous 1H–2H CPMAS experiments on organic solids [21,24,27] we also observe that relatively long CP contact times P9 ms are required to obtain best CP efficiency (Fig. S2). The MAS rate also affects sensitivity. Here we use moderate MAS rates where 1H–2H CP is generally more efficient [27], but we do not see much variation in sensitivity for hist as a function of mr between 5 and 10 kHz (Fig. S3). Faster MAS frequencies provide higher sensitivity by concentrating the 2H NMR signals into fewer spinning sidebands, but slower MAS can also be compensated for by the sideband co-addition procedure. The DNP enhancement for TCE at the surface of the samples is also highest at moderate MAS, around 6 kHz [64,65]. Fastest spin diffusion rates (D) are obtained at reduced MAS rates, leading to faster signal build-up rates and higher DNP enhancements [55]. The exact overall gain in sensitivity provided by 105 K DNP enhanced solid-state NMR as compared to conventional room temperature solid-state NMR experiments depends upon a number of factors such as 1H and 2H relaxation times, CP dynamics, linewidths, paramagnetic quenching, thermal polarization and the improved performance of NMR probes at low T [47,56,57]. While CP likely provides a substantial gain in the magnitude of the 2H NMR signal per scan, the proton T1’s at the low temperatures used for the DNP experiments are relatively long compared to room

Fig. 1. 9.4 T DNP enhanced 105 K natural abundance 1H–2H CPMAS solid-state NMR spectra of (A and B) histidine hydrochloride monohydrate (hist), (C and D) glycylglycine (gly) and (E and F) theophylline (theo) with mr = 5 kHz. CP contact times of 9 ms, 10 ms and 14 ms were used hist, gly, and theo, respectively. Full views of the spinning sideband manifold (left) and expanded views of the isotropic peak region (right). Isotropic spectra with improved signal to noise (upper red traces) were obtained by co-adding each spinning sideband to the isotropic peaks. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Table 1 DNP enhancements, repetition delays, number of scans and SNR of the Highest/Lowest intensity peaks in the DNP enhanced 2H CPMAS spectra. Solid

eC CP

Polarization delay (s)

Scans

SNR ratio of most/least intense isotropic peak

SNR ratios of most/least intense peak width sideband co-addition

Hist gly theo

144 128 17

80 80 12

96 48 680

21/4 24/14 26/4

56/12 87/56 146/19

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temperature proton and deuterium T1’s. However, we usually observe that any losses in sensitivity arising from increased relaxation times are offset by an increase in the NMR signal at 105 K and improved performance of the NMR probe [47,56]. The overall gain in sensitivity obtained with DNP solid-state NMR for impregnated samples is actually often roughly equivalent to the measured DNP enhancement [47,56]. In summary, the combination of CP, DNP and low sample temperature provides a sensitivity gain of about two orders of magnitude for the crystalline organic solids studied here. This enables the rapid acquisition of natural abundance 2H solid-state NMR spectra. We now compare the resolution obtained for 2H to that for state of the art 1H homonuclear dipolar decoupling techniques. The isotropic 2H NMR spectra (obtained with sideband co-addition to improve SNR) of hist and theo are overlaid onto homonuclear decoupled 1H NMR spectra. Note that the peak positions in the 1 H and 2H NMR spectra are not coincident due to the quadrupole induced shift of the 2H peaks (vide infra). For all 2H experiments care was taken to accurately set the magic angle to maximize resolution of the spectra [27,73]. (Note that it is challenging to accurately adjust the angle when the probe is cooled to 105 K as the mechanism can sometimes be frozen into position.) Highresolution dipolar-decoupled 1H MAS NMR spectra of hist and theo were obtained from projections of 105 K DNP enhanced 1 H–13C dipolar HETCOR spectra with 12.5 kHz MAS (Fig. S4) using eDUMBO1–22 homonuclear decoupling [12]. For additional comparison, a high-resolution room temperature 1H–1H spin diffusion correlation spectrum of hist with state of the art LG4 homonuclear decoupling was also acquired [14,68] (Fig. S5). Note that for the room temperature 1H homonuclear decoupling experiments the sample was also restricted to the center region of the rotor to reduce rf inhomogeneity. Fig. 2A and C shows that the resolution of the 105 K DNP enhanced 2H CPMAS spectra is similar or superior to that of the 1 H spectra obtained with eDUMBO1–22 decoupling under identical experimental conditions. Peak widths at half-height (Fig. S6, Table 2) for theo and hist indicate that the 2H CPMAS spectra might provide a slight improvement in resolution compared to the 1H spectra. For theo, all of the peaks give a smaller D1/2(2H) (in ppm) than the corresponding D1/2(1H) with an average 18% improvement in resolution in the 2H spectrum. For hist an average reduction of 4% was observed for the 2H linewidths. However, the resolution of the 105 K 2H CPMAS spectrum is found to be about 30% worse than that observed in a 1H NMR spectrum at 298 K acquired with LG4 homonuclear decoupling (Fig. 2B). The LG4 1H MAS NMR spectrum likely gives better resolution due to the higher sample temperature that leads to dynamics that may reduce inhomogeneous broadening and 1 H–1H dipolar couplings. The 2H solid-state NMR spectra thus provide high resolution without the need for homo-nuclear decoupling sequences, enabling the measurement of hydrogen chemical shifts and sitespecific measurements of 2H electric field gradient (EFG) tensors. However, measurement of hydrogen chemical shifts requires correction of the observed 2H peak positions for the quadrupole induced shift. For example, due to the quadrupole induced shift, the positions of the 2H peaks of hist are between +0.2 and +0.6 ppm higher the peak positions observed in the corresponding homonuclear decoupled 1H NMR spectra (Fig. 2). However, by fitting the spinning sideband manifolds for each peak (Fig. S7, shown for hist) it is possible determine the EFG tensor parameters CQ and gQ. Importantly, simulation of the sideband manifold also provides the true isotropic 2H chemical shifts, which are corrected for the quadrupole induced shift. In this way the isotropic hydrogen chemical shifts can be measured with 2H NMR. Comparison of the hydrogen chemical shifts determined by 2H solid-state NMR

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Fig. 2. Comparison of 2H solid-state NMR spectra (red) and homonuclear decoupled 1 H solid-state NMR spectra (black) for hist (A and B) and theo (C). (A and C) 100 kHz eDUMBO1–22 homonuclear decoupling at 105 K, (B) 100 kHz LG4 decoupling at 298 K. 1H–2H CPMAS spectra were obtained by co-adding all spinning sidebands to the isotropic peaks. The eDUMBO1–22 decoupled 1H spectra were obtained from projections of 1H–13C HETCOR spectra (Fig. S4). The LG4 1H MAS spectrum was taken from a projection of a ‘‘three pulse” 2D 1H–1H correlation spectrum (Fig. S5). Further details are given in the SI. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

and those measured with 1H homonuclear decoupling experiments (Table 2) shows excellent agreement with differences less than 0.2 ppm for both hist and theo.

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Table 2 Comparison of isotropic hydrogen chemical shifts and resolution of peaks measured in DNP enhanced 1H and 2H solid-state NMR experiments.a Decoupling method Hydrogen atom

b

Histidine
HCl
H2O a b c, d d3 e, d f2 g, h Theophylline a b c3, d3

300 K LG4 1

diso( H) (ppm)

DNP enhanced 2H CPMAS

105 K eDUMBO1–22 1

c

D1/2( H) (ppm)

1

diso( H) (ppm)

1

c

D1/2( H) (ppm)

diso(2H) (ppm)

D1/2(2H) (ppm)c

17.4 0.49 12.8 0.72 9.3 0.46 8.6 0.61 7.8 0.43 5.4 0.52 3.0 0.54 Average [D1/2(2H)/D1/2(1H)] = 1.31f

17.4 0.72 12.6 0.73 9.1 0.92 8.0–9.1d 2.81 7.6 1.13 5.65 0.54 2.8 0.86 Average [D1/2(2H)/D1/2(1H)] = 0.96f

17.3 12.6 9.2 8.5d 7.4 –e 2.8

0.81 0.70 0.76 0.92 0.86 1.31 0.69

– – –

15.2 1.19 7.6 1.49 3.2 1.11 Average [D1/2(2H)/D1/2(1H)] = 0.82

15.0 7.6 3.4

1.09 1.15 0.85

– – –

a 1 H chemical shifts and linewidths were measured from a 105 K DNP enhanced 2D 1H–13C HETCOR spectrum acquired with eDUMBO1–22 homonuclear decoupling applied in t1. 2H chemical shifts were obtained from simulations of the wideline DNP enhanced 1H–2H CPMAS spectrum. The simulations take into account quadrupole induced shifts that shift the 2H peak more positive than the true value of the isotropic chemical shift. b Structures with atom labels are shown in Figs. S4 and 2 of the main text. c The full width at half height (linewidth) of the peaks was determined by fitting the isotropic regions of the spectra. d It is difficult to measure the 1H and 2H chemical shifts for the ammonium hydrogen atoms at 105 K. The 1H and 2H NMR signals from the ammonium group are likely broadened due to intermediate timescale rotation of the ammonium group at low temperature. A broad 1H peak covering a range of 8.0–9.1 ppm correlates with 13C peaks of atoms 5 and 6 (with shifts of 53.3 ppm and 25.8 ppm) in the 1H–13C HETCOR spectrum acquired with a 0.25 ms contact time. In the 2H spectrum, the peak overlap makes the precise measurement of the shift difficult and the peaks are probably similarly broad. We have assigned a shift of 8.5 ppm to the ammonium protons based upon the fitting of the 2H spectrum. e The 2H signal arising from the water hydrogen atoms is very weak in the 1H–2H CPMAS spectrum. It is only visible in the spectrum formed from co-addition of the spinning sidebands to the isotropic peak. The water signal is likely weak due to unfavorable CP characteristics and/or exchange broadening. f For histidine only the resolved peaks in the 1H HETCOR projection and 2H CPMAS NMR spectrum (bolded values) were used to calculate the ratio of linewidths due to uncertainty in measuring the linewidths for overlapping peaks.

4. Conclusions

References

In conclusion, spin diffusion relayed impregnation DNP enables substantial enhancements of proton polarization in organic solids, and this can be exploited to rapidly acquire natural abundance 1 H–2H CPMAS solid-state NMR spectra, as demonstrated here for the model organic solids hist, gly and theo. DNP enhanced natural abundance 1H–2H CPMAS is thus a straightforward method for high-resolution measurements of hydrogen chemical shifts and 2 H EFG tensor parameters. The method opens up many perspectives since the resolution is already comparable to state of the art 1H methods, and the 2H spectra presented here can almost certainly be improved upon. For example further gains in resolution for 2H could be realized by obtaining spectra at higher magnetic fields, where second order quadrupole broadening will be further reduced [27].

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Acknowledgments Prof. P. Tordo, Dr. O. Ouari and Dr. G. Casano (Aix-Marseille Université) are thanked for supplying TEKPol. Financial support was from EQUIPEX contract ANR-10-EQPX-47-01, ERC Advanced Grant No. 320860 and the Ecole Polytechnique Fédérale de Lausanne. JS acknowledges support from a Schrödinger Fellowship J 3377-N28 from the Austrian Science Fund.

Appendix A. Supplementary material Additional experimental details, optimization of 1H–2H CPMAS experiments, fits of 2H NMR spectra and comparisons between 1 H and 2H MAS NMR spectra. Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jmr.2015.08.020.

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