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3D Double-Quantum/Double-Quantum Exchange Spectroscopy of Protons under 100 kHz Magic Angle Spinning Published as part of The Journal of Physical Chemistry virtual special issue “Recent Advances in Connecting Structure, Dynamics, and Function of Biomolecules by NMR”. Rongchun Zhang,†,∥,⊥ Nghia Tuan Duong,‡,∥ Yusuke Nishiyama,*,‡,§ and Ayyalusamy Ramamoorthy*,† †

Biophysics and Department of Chemistry, University of Michigan, Ann Arbor, Michigan 48109-1055, United States RIKEN CLST-JEOL Collaboration Center, RIKEN, Yokohama, Kanagawa 230-0045, Japan § JEOL Resonance Inc., Musashino, Akishima, Tokyo 196-8558, Japan ‡

S Supporting Information *

ABSTRACT: Solid-state 1H NMR spectroscopy has attracted much attention in the recent years due to the remarkable spectral resolution improvement by ultrafast magic-angle-spinning (MAS) as well as due to the sensitivity enhancement rendered by proton detection. Although these developments have enabled the investigation of a variety of challenging chemical and biological solids, the proton spectral resolution is still poor for many rigid solid systems owing to the presence of conformational heterogeneity and the unsuppressed residual proton−proton dipolar couplings even with the use of the highest currently feasible sample spinning speed of ∼130 kHz. Although a further increase in the spinning speed of the sample could be beneficial to some extent, there is a need for alternate approaches to enhance the spectral resolution. Herein, by fully utilizing the benefits of double-quantum (DQ) coherences, we propose a single radio frequency channel proton-based 3D pulse sequence that correlates double-quantum (DQ), DQ, and single-quantum (SQ) chemical shifts of protons. In addition to the two-spin homonuclear proximity information, the proposed 3D DQ/DQ/SQ experiment also enables the extraction of three-spin and four-spin proximities, which could be beneficial for revealing the dipolar coupled proton network in the solid state. Besides, the 2D DQ/DQ spectrum sliced at different isotropic SQ chemical shift values of the 3D DQ/DQ/SQ spectrum will also facilitate the identification of DQ correlation peaks and improve the spectral resolution, as it only provides the local homonuclear correlation information associated with the specific protons selected by the SQ chemical shift frequency. The 3D pulse sequence and its efficiency are demonstrated experimentally on small molecular compounds in the solid state. We expect that this approach would create avenues for further developments by suitably combining the benefits of partial deuteration of samples, selective excitation/decoupling pulses, heteronuclear spins for spectral editing, and nonuniform sampling.

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resolution as well as the sensitivity.24−33 However, for rigid crystalline solids, the proton spectral resolution is still limited by the residual higher order terms of 1H−1H dipolar couplings as well as due to the conformational heterogeneity and the anisotropic bulk magnetic susceptibility broadening in the sample, which are unlikely to be completely suppressed by higher spinning speeds. Some aspects of this limitation can be overcome by deuteration to dilute the highly abundant protons, particularly for the biological samples,34−37 and also by using multidimensional or selective-excitation experiments for sitespecific selection or filtering of signals to enhance spectral

INTRODUCTION

There is considerable current interest in the development of novel NMR techniques that could provide atomic insights into the molecular structures and dynamics for numerous nonsoluble and noncrystallizable systems, such as amyloid fibrils,1−7 membrane proteins,8−15 and the supramolecular complex materials like bone.16,17 Solid-state NMR spectroscopy has been well utilized for probing structures in a length scale ranging from an angstrom to several hundred nanometers and dynamics in a time scale from picoseconds to seconds in a variety of proteins and materials.18−23 Particularly, due to the rapid advance and applications of ultrafast magic-angle-spinning (MAS) probe technology in the recent years, proton-detected solid-state NMR techniques have attracted much attention, mostly due to the significant improvement in spectral © 2017 American Chemical Society

Received: April 12, 2017 Revised: May 23, 2017 Published: May 24, 2017 5944

DOI: 10.1021/acs.jpcb.7b03480 J. Phys. Chem. B 2017, 121, 5944−5952

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The Journal of Physical Chemistry B resolution.38−45 Although the use of such sophisticated techniques at ultrafast MAS conditions brings out the treasure hidden within the mighty spin systems in the sample, unfortunately, the signal sensitivity is still greatly compromised by the limited sample volume due to the extremely small rotor size (∼0.7 to ∼1.3 mm in diameter). As a result, applications of multidimensional solid-state NMR experiments are still limited for samples where less abundant, low-γ nuclei (such as 13C and/or 15N) are involved.46−50 Therefore, single-channel proton-based experiments are useful under ultrafast MAS due to the high natural abundance and high sensitivity of protons. Such proton-only based solid-state NMR experiments will create avenues for high-throughput studies on scarcely available samples and therefore would enable the effective utilization of a plethora of solid-state NMR techniques in many different areas. Indeed, the increasing number of studies on the design of novel materials including graphene, polymorphic pharmaceuticals, MOFs and other nanomaterials that cannot be studied at atomic resolution by other means also create the need for highthroughput solid-state NMR experiments. Recent studies reported in the literature have successfully introduced proton-only multidimensional solid-state NMR experiments. 2D correlation of double-quantum (DQ)/singlequantum (SQ) and SQ/SQ chemical shift interactions of protons under ultrafast MAS conditions are particularly valuable to probe two-spin proximity in addition to the enhanced spectral resolution offered by the DQ coherence.51−60 The recently reported 2D chemical shift anisotropy/chemical shift (CSA/CS) correlation of protons allow for the accurate measurement of CSA tensors of protons that are highly valuable for probing site-specific chemical environment and of course for the use of CSA tensors in NMR based dynamics studies.61−65 Our previous studies have also reported proton-based multidimensional experiments demonstrating additional possibilities and applications. A 3D 1H SQ/DQ/ SQ chemical shift correlation experiment has been shown to offer insights into the three-spin proximity and the molecular arrangement that the regular 2D DQ/SQ experiment fails to offer.42 Another recently demonstrated 3D 1H DQ/CSA/SQ experiment has been demonstrated to be useful in the extraction of CSA tensors of protons whose signals are otherwise overlapped in the regular 2D CSA/CS experiment.38 Although Spiess and co-workers have proposed 13C based DQ/DQ MAS exchange NMR experiment for probing slow molecular motions by exploiting the spinning sidebands of 1D 13 C spectrum at different mixing periods,66 it has not been explored to probe proton homonuclear proximity mostly due to low spectral resolution and extremely fast spin diffusion under slow spinning speed. Continuing on our development of multidimensional techniques to study solids under ultrafast MAS conditions, here we propose a 3D 1H DQ/DQ/SQ chemical shift correlation experiment that correlates the DQ signals among different proton spin pairs. Such an experiment not only offers the two-spin proximity information but also provides insights into three-spin and four-spin proximities, which is beneficial for understanding the proton dipolar coupling network in the molecular system under investigation. In addition, the 2D DQ/DQ spectrum sliced at the isotropic SQ chemical shift frequency position of the 3D DQ/DQ/SQ spectrum is shown to contain the local homonuclear correlation information related to the specific proton only, and thus simplifies the identification of DQ peaks and further improves the spectral resolution. Experimental results obtained from

powder samples of small molecular compounds including a dipeptide are also reported in this study.

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EXPERIMENTAL SECTION Samples. 15N3-L-histidine·HCl·H2O powder sample was purchased from Acros Organics (Morris Plains, NJ) and used without any further purification. A powder sample of Nacetyl-15N-L-valyl-15N-L-leucine (NAVL) was prepared as explained elsewhere.67 About 0.8 mg of 15N3-L-histidine·HCl· H2O packed in a 1 mm rotor and 0.3 mg of NAVL packed in a 0.75 mm rotor were used to carry out the experiments presented in this study. Solid-State NMR Spectroscopy. All NMR experiments were performed on a 600 MHz ECZ600R solid-state NMR spectrometer equipped with a 1.0 mm or 0.75 mm doubleresonance ultrafast MAS probe (JEOL Resonance Inc.). Although the samples used in this study were labeled with 15 N isotope, unlabeled samples are sufficient for the proposed 3D experiment; 1H−15N dipolar couplings are fully suppressed by the fast MAS and there is no need to perform any heteronuclear decoupling for the experiments reported in this study. Other experimental details are given in the figure captions or in the main text below. 3D DQ/DQ/SQ Pulse Sequence. The 3D pulse sequence used in this study is shown in Figure 1. Dipolar recoupling

Figure 1. 3D 1H DQ/DQ/SQ solid-state MAS NMR experiment. Radio-frequency pulse sequence for the 3D 1H DQ/DQ/SQ chemical shift correlation experiment (top) and the coherence selection pathway (bottom). Rotor-synchronized DQ recoupling schemes were applied to excite and reconvert the DQ coherences, whereas fpRFDR with XY414 phase cycling was used for homonuclear magnetization exchange.52,53 A Z-filter time (tZF) of ∼1 ms was applied before the final 90° pulse to eliminate residual transverse magnetization after the conversion of DQ to ZQ coherences. The following phase cyclings were used in acquiring the spectra reported in this study: ϕ1 = 0, ϕ2 = 0123, ϕ3 = 0, ϕ4 = 0000111122223333, ϕ5 = 0, ϕrec = 0202202002022020.

schemes of back-to-back (BABA)-XY1651,68 were employed for DQ excitation and reconversion, whereas a finite-pulse radiofrequency-driven dipolar recoupling (fpRFDR)52,53,69,70 sequence was applied for the mixing of zero-quantum proton magnetization after the first DQ reconversion. A recently reported phase cycling scheme, XY414, that renders efficient recoupling of 1H−1H dipolar couplings and is tolerant to experimental imperfections and chemical shift anisotropy, was used for the 180° pulses in the fp-RFDR sequence.53 The increments in the t1 and t2 periods are synchronized to the rotor period so that spinning sidebands are folded onto the center bands in these indirect dimensions. A Z-filter (tZF ∼ 1 ms) was employed before the final 90° read pulse to suppress the residual transverse magnetization after the second DQ reconversion period. A four-step phase cycling was used to 5945


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The Journal of Physical Chemistry B obtain the DQ coherence in both t1 and t2 periods, resulting in a total of 16 steps phase cycling. States-TPPI method71 was used for the signal acquisition of both t1 and t2 periods. Experiments on a powder sample of 15N3-L-histidine·HCl· H2O were performed under 70 kHz MAS with a 90° pulse length of 1 μs and a recycle delay of 6.0 s. The recycle delay was optimized to balance the signal-to-noise ratio and the total duration of the 3D experiment. The 3D spectra of 15N3-Lhistidine·HCl·H2O were obtained from 16 t1 and 16 t2 increments and by coadding 16 scans for each increment. The DQ excitation/reconversion time was optimized by performing regular 1D DQ filtered proton experiments with t1 = 0, and it was set as 114.3 μs (equal to eight rotor periods). Experiments on NAVL sample were performed under 100 kHz MAS with a 90° pulse length of 0.6 μs and a recycle delay of 2.0 s. The 3D spectrum of NAVL was obtained from 32 t1 and 32 t2 increments and coadding 16 scans for each increment. The DQ excitation/reconversion time was set as 80 μs (equal to eight rotor periods). fpRFDR was also applied during the recycle delay to accelerate the overall T1 relaxation of protons.72

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RESULTS AND DISCUSSION With the expansion to the second dimension, the 2D spectrum generally provides a higher spectral resolution than the 1D spectrum. Furthermore, due to the doubled spectral width of the DQ dimension, better separations of peaks are achieved. Therefore, by further expanding the 2D experiment into a 3D experiment and correlating DQ/DQ chemical shifts, the 3D DQ/DQ/SQ chemical shift correlation experiment will be able to provide a much higher resolution spectrum and enable probing multispin proximity. The feasibility and performance of the above-mentioned 3D sequence is demonstrated on the powder samples of L-histidine·HCl·H2O and NAVL as explained below. 3D DQ/DQ/SQ Spectrum of L-Histidine·Hcl·H2O at 70 kHz MAS. After initial optimization of the parameters in the pulse sequence and other experimental conditions, the 3D experiment was performed on a powder sample of L-histidine· HCl·H2O at 70 kHz MAS and the resultant spectra are given in Figure 2 −4. As expected, the DQ2/SQ (i.e., F2/F3) spectrum shown in Figure 2b is similar to the conventional 2D DQ/SQ chemical shift correlation spectrum of protons, where a spin pair close to each other generates a DQ signal within a proper DQ excitation time, and has the same DQ chemical shift (the sum of the isotropic chemical shift of the two spins) along the DQ dimension. From the 2D DQ/SQ chemical shift correlation spectrum sliced out of the 3D spectrum, we are able to probe the two-spin correlation dependent on the DQ excitation/reconversion time. It is worth noting that the 2D DQ1/SQ (F1/F3) spectrum (Figure 2c) extracted from the 3D spectrum has more cross peaks when compared to the 2D DQ2/SQ (F2/F3) spectrum (Figure 2b), although all the DQ signals were excited and reconverted with the same excitation and reconversion times. This is because of the protonmagnetization exchange during the fpRFDR mixing time and DQ excitation and reconversion periods. For example, there is a cross peak (indicated by the blue solid line in Figure 2c) between the NHb SQ chemical shift (δNHb) and DQ chemical shift (δH1+δH2) of H1 and H2. In the first DQ excitation/ reconversion period (periods ab and cd), the dipolar coupling recoupled by BABA-XY16 sequence induced the DQ signal between H1 and H2. The DQ free precession (in the period bc) gives a peak at the DQ chemical shift of (δH1 + δH2) along

Figure 2. 2D spectra extracted from the 3D DQ/DQ/SQ spectrum. 2D skyline projections extracted from the 3D DQ/DQ/SQ experiment on L-histidine·HCl·H2O with a fpRFDR mixing time of 0.457 ms. (a) Schematic molecular structure of L-histidine·HCl·H2O. (b) 2D DQ2/SQ (F2/F3) projection. (c) 2D DQ1/SQ (F1/F3) projection. (d) 2D DQ1/DQ2 (F1/F2) projection. The solid blue horizontal line in Figure 2c indicates the correlation between NHb (or NHa) isotropic chemical shift (δNHb or δNHa) and the DQ chemical shift of H2 and H1 (δH1 + δH2).

the DQ1 dimension. In fact, there are two mechanisms for magnetization transfer between NHb and H2 (or H1) as explained below. In the first mechanism, during the fp-RFDR mixing time (period de), there will be magnetization exchange among the protons, such as NHb and H2 (or H1). In the second mechanism, the H2 (or -H1) magnetization involving NHb-H2 or (-H1) DQ coherence in the second DQ excitation period can also transfer the magnetization originated from H2 (or H1) to NHb as discussed below in detail for DQ1/DQ2 slices along the SQ axis. This mechanism can be active with a combination of spin diffusion to another nuclei X and NHb-X DQ coherence formation. Therefore, there is a cross peak with a chemical shift of δNHb along the SQ dimension and a DQ chemical shift of (δH1+δH2) along the DQ1 dimension in the 2D DQ1/SQ spectrum. Similarly, the magnetization exchange between NHa and H2 (or H1) will also induce a DQ/SQ cross peak with a chemical shift of δNHa along the SQ dimension and a DQ chemical shift of (δH1 + δH2) along the DQ1 dimension in the 2D DQ1/SQ spectrum. In terms of this aspect, with a comparison to the DQ2/SQ (F2/F3) spectrum, the “additional” cross peak in DQ1/SQ (F1/F3) spectrum could be used to indicate the proximity between a spin pair and the third spin, where the proximity between the third spin and one of the spins of the pair could be probed by fpRFDR mixing or DQ magnetization transfer. Figure 2d shows the DQ1/DQ2 correlation spectrum obtained from the skyline projection of the 3D DQ/DQ/SQ correlation spectrum with a fp-RFDR mixing time of 0.457 ms (equal to 32 rotor periods). As is shown above, a peak at the DQ dimension indicates a spin pair close to each other. Herein, the cross peak in the DQ1/DQ2 spectrum indicates the proximity between two spin pairs, where 5946


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Figure 3. 2D DQ1/DQ2 spectra extracted from the 3D spectrum. 2D DQ1/DQ2 (F1/F2) slices at the isotropic chemical shift frequency of H1 (a), NH3+ (b), and H3/H4 (c) along the SQ dimension in the 3D DQ/DQ/SQ spectrum with a fpRFDR mixing time of 0.457 ms. The DQ1/DQ2 cross peaks of (a) (δH3/H4−H3/H4, δH1−NHb), (b) (δNHa−NHb, δH3N+−H1/H3N+/H2), and (c) (δH1−NHb, δH3/H4−H3/H4) are indicated by the red circle.

Figure 4. 2D DQ1/DQ2 spectra extracted from the 3D DQ/DQ/SQ spectrum. 2D DQ1/DQ2 (F1/F2) skyline projections extracted from the 3D DQ/DQ/SQ spectrum of L-histidine·HCl·H2O with (a) a mixing time of 0.457 ms without fpRFDR, a fpRFDR mixing time of (b) 0.457 ms and (c) 2.286 ms. The DQ1/DQ2 cross peaks between two spin pairs, (H1−NHb, H1−NHa), are indicated by the magenta circles in Figure 4a,b.

peaks appear at ω1 = ω2 on the diagonal line with a slope of 1, and the cross peaks appear at (ω1, ω2), indicating the presence of correlation between different proton spins. The 1H DQ2/SQ correlation spectrum shown in Figure 2b can be displayed in this representation by performing the shearing transformation, where the SQ/SQ representation directly provides intuitive SQ/SQ correlation peaks as shown in Figure S1A. However, it becomes more complicated if the DQ frequency is coupled to multiple SQ resonances as shown in Figure 2c. In fact, fpRFDR and DQ mixing between the DQ1 and DQ2 dimensions introduce additional cross peaks in the DQ1/SQ spectrum (Figure 2c) compared to the DQ2/SQ spectrum. For example, the DQ1 frequency originating from NHa and NHb (δDQ1 ∼ 30 ppm) couples to not only the SQ resonances at δSQ = 12.6 ppm (NHa) and 17.4 ppm (NHb) but also the resonances at δSQ = 9.4 ppm (H1), 8.5 ppm (H3N+), and 7.6 ppm (H2). From such a DQ/SQ representation, it is clear that the DQ1/SQ correlation peaks originate from magnetization transfer from NHa or NHb to H1, H3N+, and H2. However, after the application of shearing transformation as shown in Figure S1B, such specific correlation information is lost. Therefore, we use the conventional DQ/SQ representation throughout the manuscript for the display of DQ/SQ correlation spectra which retains the benefits offered by the DQ dimension in comparison to the SQ dimension. The DQ1/DQ2 slices at the isotropic chemical shift of H1 and NH3+ only contain the DQ/DQ cross peaks related to the proton H1 or NH3+, as indicated in Figure 3a,b, respectively. Indeed, in Figure 3a, all the peaks in the DQ2 dimension include H1 in a pair, because SQ magnetization is directly

each spin pair is indicated by their values along the DQ1 or DQ2 dimension. In fact, the cross peaks are from the magnetization exchange during the fpRFDR mixing time and DQ mixing. For example, if there is DQ/DQ cross peak between AB and BC spin pairs, then it means that the three spins, A, B, and C, are close to each other depending on the fpRFDR mixing time and DQ recoupling time. However, if spin B consists of two unresolved sites (B1 and B2 with the same chemical shift frequency), then the correlation between AB and BC spin pairs observed in the DQ1/DQ2 spectrum could indicate the four-spin correlation; for example, between AB1 and B2C, etc. In the meantime, if two spin pairs AB and CD both induce DQ signals, and A (or B) has magnetization exchange with C (or D) during the fpRFDR mixing, then there will be a cross peak between AB and CD in the DQ1/DQ2 spectrum. This would enable the DQ1/DQ2 spectrum to probe the three-spin and four-spin proximities. However, although the DQ dimension has a doubled spectral width compared to the SQ dimension, the peaks are still severely overlapped along the DQ dimension. Herein, by taking the DQ1/DQ2 slices at specific SQ chemical shift frequency, the 2D DQ1/DQ2 spectral slices are simpler for interpretation as they only provide information associated with the selected protons as shown in Figure 3 for protons H1, NH3+ and H3/H4. Before further discussing the spectra obtained from the 3D pulse sequence, we briefly discuss the ways to present 2D DQ/ SQ spectra, because the DQ/SQ spectrum is often transformed to a SQ/SQ representation by using a shearing transformation to overcome the spectral folding problem and for an easy interpretation.56 In the SQ/SQ representation, autocorrelation 5947


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Figure 5. High-resolution 1H/1H correlation spectra of NAVL under 100 kHz MAS. (a) Chemical structure of NAVL. (b) 2D DQ/SQ chemical shift correlation spectrum obtained from the regular 2D DQ/SQ experiment with BABA-XY16 for DQ excitation/reconversion. (c) 2D DQ2/SQ (F2/F3), (d) 2D DQ1/SQ (F1/F3), and (e) 2D DQ1/DQ2 (F1/F2) skyline projection extracted from the 3D DQ/DQ/SQ spectrum that was obtained with a fpRFDR mixing time of 3.2 ms. (f) 2D DQ1/DQ2 skyline projection extracted from the 3D DQ/DQ/SQ spectrum that was obtained with a mixing time of 0.5 ms but without applying fpRFDR.

time as short as 0.457 ms without fp-RFDR pulses, the spin diffusion among protons could be reasonably ignored. Herein, the cross peaks in Figure 4a should be resulting from magnetization exchange during the DQ recoupling periods. For example, after the first DQ reconversion period, spin B has partial magnetization originated from spin A through the A-B DQ correlation. In the second DQ excitation/reconversion period, spin B will induce DQ coherence with spin C and thus gives the DQ signals. As a result, there will be a cross peak at (δA + δB, δC + δB) in the DQ1/DQ2 spectrum. Therefore, without any fpRFDR mixing, the cross peaks actually contain only the three-spin proximity information. On the contrary, in the presence of fpRFDR mixing, the four-spin correlation could also be observed in the DQ1/DQ2 slices, such as the cross peak at (δ H3/H4−H3/H4 , δ H 1 −NH b ) in Figure 3a, (δ NH a −NH b ,

transformed from DQ coherence in the DQ2 dimension. However, some of the peaks in the DQ1 dimension do not include H1, indicating four-spin proximity through magnetization transfer as discussed below. For example, if we look at the H1 related correlations in Figure 3a, the DQ cross peak at (δH1−NHb, δH1−NHa) actually indicates the three-spin proximity, between the H1−NHa spin pair and NHb. Though H1−NHb and H 1−NHa proximities are determined by the DQ coherences in the DQ1 and DQ2 dimensions, respectively, the NHa−NHb proximity can be deduced from the DQ1/DQ2 cross peak through fpRFDR mixing or DQ recoupling. The cross peak arising from DQ recoupling is demonstrated in Figure 4a where multiple cross peaks in the DQ1/DQ2 spectrum are observed without fpRFDR mixing. For a mixing 5948


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Figure 6. 2D spectral slices extracted from 3D DQ/DQ/SQ spectrum of NAVL. 2D DQ1/DQ2 (F1/F2) slices taken at the isotropic chemical shift frequencies of H2 (a), H3 (b), and H4 (c) along the SQ dimension in the 3D DQ/DQ/SQ spectrum of NAVL with a fpRFDR mixing time of 3.2 ms.

δH3N+−H1/H3N+/H2) in Figure 3b, and (δH1−NHb, δH3/H4−H3/H4) in Figure 3c, where the spin pair indicated by the DQ1 chemical shift does not include any proton of the pair indicated by the DQ2 chemical shift, as highlighted by the red circles in Figure 3. These four-spin correlations never appear in the absence of fpRFDR mixing (Figure 4a). Hence, the DQ1/DQ2 spectra obtained at different isotropic SQ chemical shifts could provide multiple three-spin and four-spin proximity information. The DQ/DQ cross peak at (δH1−NHb, δH1−NHa) is present in both DQ1/DQ2 spectra that were acquired with and without fpRFDR pulses, as shown in Figure 4a,b, respectively. This cross peak arises from two types of contributions: one is the magnetization transfer between these three protons during the fpRFDR mixing time, and the other is due to the magnetization exchange during the DQ recoupling periods. The cross peak at (δH1−NHb, δH1−NHa) acquired without a fpRFDR mixing is relatively weak as compared to those appearing in spectra acquired with a fpRFDR mixing. This means that contribution from fpRFDR is the major source of magnetization transfer, as compared to DQ recoupling. Because the DQ/DQ cross peaks in the DQ1 and DQ2 dimensions were largely correlated by fpRFDR mixing, it is expected that with increasing the fpRFDR mixing time, the cross peak intensity will be enhanced, as shown in Figure 4b and 4c, where the mixing time was 0.457 and 2.286 ms, respectively. Therefore, by changing fp-RFDR mixing time, it is possible to estimate the distance between two different spin pairs, and thus to probe four-spin proximity. 3D DQ/DQ/SQ Spectrum of NAVL at 100 kHz MAS. To further demonstrate the feasibility of the 3D experiment presented in this study, we extended the demonstration of 3D experiments on a dipeptide powder sample of NAVL under 100 kHz MAS. The chemical structure of NAVL is shown in Figure 5a. The high-resolution 1H spectra of NAVL under ultrafast MAS up to 90 kHz are reported elsewhere.42 Here, 100 kHz MAS spinning gives a better resolution, in particular on the aliphatic regions (H6 and H7) around 1 ppm, as shown on the projections along the SQ dimension in Figure 5b−d. The regular 2D DQ/SQ chemical shift correlation spectrum is given in Figure 5b, which provides the two-spin proximity information with a DQ excitation/reconversion time of 80 μs. It is clearly shown that the DQ2/SQ (F2/F3, Figure 5c) spectrum, extracted from the 3D DQ/DQ/SQ spectrum, is basically the same as the regular 2D DQ/SQ spectrum except for the peak intensities. On the contrary, the DQ1/SQ (F1/F3,

Figure 5d) spectrum gives more cross peaks in comparison to the DQ2/SQ (F2/F3) spectrum. For example, the cross peak at (10.8 ppm, 13.1 ppm) (as indicated by the red solid circle in Figure 5d) arises from the magnetization exchange between H1 and H2 or H5 during the fpRFDR mixing time and/or the second DQ excitation period, whereas the DQ coherence between H2 and H5 are observed in the DQ1 dimension. It is also worth noting that there are also some cross peaks missing in the DQ1/SQ (F1/F3) spectrum in comparison to the regular 2D DQ/SQ or the 2D DQ2/SQ (F2/F3) spectrum, such as the DQ peaks between H1 and H3 (or H4). That is because the DQ conversion efficiencies are too low for the magnetization of H1 and H3 (or H4) to survive after two DQ filtering periods and/or spin diffusion to the other protons by the fpRFDR mixing. The DQ1/DQ2 (F1/F2) skyline projection extracted from the 3D DQ/DQ/SQ spectrum with a fpRFDR mixing time of 3.2 ms is shown in Figure 5e, where the DQ/DQ cross peaks indicate the proximity of different spin pairs. However, the overlapped DQ peaks could hinder the unambiguous assignments, in particular when the SQ chemical shifts are close to each other. For better interpretation, DQ1/ DQ2 slice along the isotropic SQ chemical shift is required and is shown in the following. It is worth noting that with a comparison to Figure 5e, some cross peaks (indicated by the cyan circles) are missing in the DQ1/DQ2 spectrum shown in Figure 5f when there is no fpRFDR mixing. That is because the cross peaks in the DQ1/DQ2 spectrum without fpRFDR mixing only contain the three-spin proximity information as discussed above, where the two spin pairs share one common proton. Moreover, the intensities of such cross peaks are generally below the detectable level due to the limited magnetization exchange through the DQ recoupling periods. The DQ1/DQ2 spectra sliced at the isotropic SQ chemical shift frequencies of H2, H3, and H4 show only the spin proximity information related to the corresponding proton DQ frequency in the DQ2 dimension, as shown in Figure 6a−c, respectively. Interestingly, we observed a four-spin correlation related to H6/H7 protons as indicated by the cross peak at (δ6/7−6/7,, δ2−1), (δ6/7−6/7,, δ3−1), (δ6/7−6/7,, δ4−1) in the DQ1/ DQ2 spectra sliced at the chemical shift of H2, H3, and H4, respectively. In fact, within a DQ excitation/reconversion time of 80 μs, the DQ coherences between proton 1 and proton 2, proton 3, proton 4 were all induced as well as the autocorrelation among the methyl groups (H6 and H7), which were also well demonstrated in the regular 2D DQ/SQ 5949


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nevertheless bound to be an intellectual exploitation of the very rich network of dipolar coupled protons.

spectrum (Figure 5b) and also in the 2D DQ2/SQ projections from the 3D spectrum (Figure 5c). As a 3.2 ms fpRFDR mixing would result in a widespread magnetization exchange among all protons as demonstrated in our previous study,42 it is not surprising that we observed all the four-spin proximity DQ/DQ cross peaks mentioned above. It is also worth noting that the intensity of the cross peak (δ6/7−6/7, δ3−1) in Figure 6b is weaker than that of the cross peak observed at (δ6/7−6/7, δ4−1) in Figure 6c, because the H4−H1 pair is closer to the H6/7-H6/7 pair than the H3−H1 pair and thus induces stronger DQ correlation signals. However, due to the large line width for a fully protonated sample, DQ peaks along DQ2 dimension are still overlapped. Therefore, by reduction of the fpRFDR mixing time or DQ excitation time by replacing the recoupling sequences other than BABA-XY16, the magnetization is mainly transferred among nearest protons and thus will further simplify the spectra as also clearly shown in Figure 4.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jpcb.7b03480. Shearing transformation on related DQ/SQ spectra (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*Y. Nishiyama. E-mail: [email protected]. *A. Ramamoorthy. E-mail: [email protected]. ORCID

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Rongchun Zhang: 0000-0002-2480-2652 Nghia Tuan Duong: 0000-0001-5761-3666 Ayyalusamy Ramamoorthy: 0000-0003-1964-1900

CONCLUSION Multiple quantum coherences73−75 have been well utilized in both solution and solid-state NMR studies, and a plethora of novel techniques have been reported. In this study, we proposed a single-channel proton-based 3D DQ/DQ/SQ chemical shift correlation experiment to probe multispin proximities under ultrafast MAS, which has been demonstrated to enhance the proton spectral resolution and the signal sensitivity. Besides the DQ2/SQ (F2/F3) spectrum, which provides the two-spin proximity information like the regular 2D DQ/SQ spectrum, the DQ1/SQ (F1/F3) and DQ1/DQ2 (F1/ F2) spectra extracted from the 3D spectrum are both able to provide three-spin and four-spin spatial proximity information. The two-spin proximity is determined by the DQ recoupling time, whereas the three-spin and four-spin proximities are codetermined by the DQ recoupling time as well as the fpRFDR mixing time. If the two-spin pairs share a common spin, the DQ1/DQ2 (F1/F2) spectrum will provide three-spin proximity information; otherwise, it will offer the four-spin proximity information, i.e., the proximity between two different spin pairs. When the DQ1/DQ2 (F1/F2) slice is taken at a specific SQ chemical shift, the spectral contents are simplified as it only gives the local information related to the specific selected protons and thus will facilitate the DQ resonance assignments. Overall, we believe that the elegant 1H 3D DQ/ DQ/SQ pulse sequence under ultrafast MAS will be beneficial for probing the proximity among different proton spins and thus enable a high-throughput investigation of structure and dynamics of various materials at high resolution, needless to mention that the proposed 3D experiment will benefit from the use of faster magic angle spinning, higher external magnetic fields, and partial deuteration of samples. A straightforward application of the use of double quantum coherence in multidimensional experiments is the measurement of chemical shift tensors of protons as recently demonstrated with the use of a 3D NMR experiment38 which will be bound to happen as higher magnetic fields are introduced for studies on solids.40,76,77 Further developments in the pulse sequence utilizing selective excitation, constant time evolution, and nonuniform sampling would further expand the utility of the proposed 3D experiment. Though an extension of this study to explore the involvement of higher order coherences of protons, and/or the higher order terms of 1H−1H dipolar couplings, may sound like a pure academic interest at present, it would

Present Address ⊥

State Key Laboratory of Medicinal Chemical Biology, Nankai University, Tianjin, 300071, P. R. China.

Author Contributions ∥

These authors have made equal contributions to this study.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was supported by funds from National Institutes of Health (GM084018 A.R.). The authors thank JEOL RESONANCE for the support and interest in our ultrafast MAS research.

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REFERENCES

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