Quantitative analysis of 14N quadrupolar coupling using 1H detected 14N solid-state NMR.

Magic-angle spinning solid-state NMR is increasingly utilized to study the naturally abundant, spin-1 nucleus 14N, providing insights into the structure and dynamics of biological and organic molecules. In particular, the characterisation of 14N sites using indirect detection has proven useful for complex molecules, where the 'spy' nucleus provides enhanced sensitivity and resolution. Here we exploit the sensitivity of proton detection, to indirectly characterise 14N sites using a moderate rf field to generate coherence between the 1H and 14N at moderate and fast-magic-angle spinning frequencies. Efficient numerical simulations have been developed that have allowed us to quantitatively analyse the resulting 14N lineshapes to determine both the size and asymmetry of the quadrupolar interaction. Exploiting only naturally occurring abundant isotopes will aid the analysis of materials with the need to resort to isotope labelling, whilst providing additional insights into the structure and dynamics that the characterisation of the quadrupolar interaction affords.


Introduction
Nitrogen-14 has a natural abundance of 499.6% and a moderate gyromagnetic ratio (g14 N /g1 H B 0.07), making it a potentially attractive nucleus for high-resolution solid-state NMR studies of nitrogen containing compounds. However, 14 N is a spin-1 nucleus with a nuclear quadrupole interaction (NQI) typically on the order of 1-5 MHz, making detection challenging. The NQI is however sensitive to variations in molecular symmetry, geometry and electrostatic environment, which in combination with its high natural abundance and prevalence in organic and biological materials, makes the development of methods to characterize 14 N sites desirable. To this end, a number of methods have been presented in the literature, particularly within the last ten years, that facilitate the determination of NQI parameters of nitrogen sites in a range of organic, biological and pharmaceutical materials.
Despite the size of the NQI and the width of resulting signal, ultra-wideline excitation methods developed by Schurko and co-workers have provided accurate NQI parameters at 14 N sites in a number of organic and pharmaceutical compounds. [1][2][3] The resolution of this method is inherently limited by the width of static 14 N signals, which overlap and hinder interpretation, limiting this technique to compounds with few (currently fewer than 3) unique 14 N sites.
Methods for detection of overtone (Dm = AE2) transitions on static samples, [4][5][6] and more recently under MAS 7-12 and DOR 13 have provided alternative methods for characterizing 14 N sites. Unlike Dm = AE1 14 N signals, overtone signals are not affected by the large (MHz) quadrupole broadening. However, there are sensitivity concerns when exciting this ''forbidden'' transition, which are further exacerbated by the slow nutation and correspondingly low excitation bandwidth of overtone signals, making simultaneous detection of multiple 14 N overtone signals difficult. 8 A third strategy for detection of 14 N is the indirect detection method, pioneered independently by Gan and Bodenhausen's laboratories, [14][15][16][17][18][19][20][21][22][23][24][25] where 14 N sites are detected via their interaction with coupled spin-1/2 'spy' nuclei; typically 1 H or 13 C, using pulse sequences qualitatively similar to HMQC experiments. Transfer of polarisation between the 14 N and the 'spy' nucleus occurs due to high-order cross-terms between the quadrupolar and 14 N'spy' dipolar Hamiltonians, known as the 'residual dipolar splitting' (RDS), as well as a contribution from the J-coupling, in experiments known as J-HMQCs. Alternatively, polarisation transfer can be driven by the recoupling of the 1 H/ 14 N dipolar coupling using rotary resonance (R 3 ), REDOR or symmetry based recoupling sequences, in sequences that have been generally termed D-HMQC experiments. 19,26,27 Similarly, double cross-polarization schemes have also been proposed for indirect detection which function well at higher spinning speeds. 28 These indirect techniques benefit from the sensitivity and resolution that are afforded by the 'spy' nuclei and do not require excitation or detection of the entire first-order broadened 14 N spectrum. Accordingly, they have found application to the study of structure and dynamics of a number of systems. 18,29,30 One of the major obstacles to the more widespread application of these techniques is their relatively poor spy nucleus/ 14 N transfer efficiencies, which usually result in a B90% drop in sensitivity compared to the 1 H spin-echo, and the strong dependency on high 14 N RF amplitudes, 31 this is particularly acute at moderate spinning speeds (o60 kHz). This, in turn, renders the accurate de novo determination of 14 N NQI parameters from these experiments difficult, since precise determination of the quadrupolar coupling constant (C Q ) and asymmetry parameter (Z) requires fitting of the 14 N lineshape, which cannot be achieved without high quality spectra with a high S/N ratio.
In this study we present a method for indirect detection of 14 N signals via 1 H's with enhanced efficiency at moderate to fast MAS frequencies , with transfer efficiencies of up to 27.5%, enabling acquisition of 1 H/ 14 N correlation spectra with improved signal/noise ratios and well-defined lineshapes in the 14 N dimension. This method, which is derived from our earlier work on the detection of 14 N via 13 C, 32 differs from previously published methods of indirect 14 N detection based on the HMQC pulse sequence, in that polarisation transfer occurs not under free evolution or established methods of heteronuclear dipolar recoupling, but rather under relatively long (100s ms) periods of moderate (30-70 kHz) RF irradiation on the 14 N channel (see Fig. 1). As described in our earlier studies, during these periods of irradiation both single and double quantum coherences are generated with efficiencies in excess of 20% of the initial equilibrium magnetization. 32 As per previous indirect detection sequences the indirect 14 N signals in 2D spectra are not affected by the typically large (MHz) first order quadrupolar interaction providing that the t 1 increments are rotor synchronised and the magic-angle accurately set. This results in 14 N signals at a frequency which is the sum of the chemical shift and second order isotropic quadrupolar shift (d iso Q ), given for the general case in ppm by: 29 where I and m are the spin quantum numbers and u 0 is the Larmor frequency in Hz. Eqn (1) simplifies to: In the case of 14 N, when I = 1 and m = +1 or 0. The quadrupolar product, w q , is given by: If the isotropic chemical shift, d iso CS , is known, for example from studies of 15 N spectra, then the 14 N d iso Q contribution to the 14 N shift of a given peak can be determined by subtraction of the 15 N d iso CS from the observed 14 N shift, allowing the determination of w q . Previous studies have shown the experimentally measured w q to be consistent with previously determined or calculated values of C Q and Z for glycine and peptides of AGG and b-DA, 14,16,30 and has been used to investigate H-bonding in pharmaceuticals. 29 However, in order to quantitatively determine C Q and Z precisely from such data, Z must be determined by fitting the 14 N lineshape to simulations.
The simulation of indirectly detected 14 N lineshapes is difficult since the lineshape depends on numerous parameters, including quadrupolar broadening, and the orientation of the quadrupolar tensor with respect to the dipolar and CSA tensors. Therefore, determination of 14 N NQI parameters has not been previously attempted due to (a) poor efficiency of indirect detection methods leading to noisy and ill-defined 14 N lineshapes unsuitable for fitting, and (b) computationally expensive nature of simulating these experiments. In this work, we address both of these issues and present a highly efficient 1 H/ 14 N correlation experiment, and fast and accurate simulations which in combination allow the de novo determination of 14 N NQI parameters from organic solids with a variety of 14 N sites with various NQI parameters. These fast and accurate numerical simulations of each 14 N site allows the fitting of simulations to experimental lineshapes and the simultaneous determination of 14 N NQI parameters at multiple sites from a 2D data set.

Materials
Samples of histidine hydrochloride monohydrate (HisÁHClÁH 2 O), L-histidine (L-His), N-acetyl-valine (NAV) and valine were purchased from Sigma-Aldrich (UK) and used without further purification. and 3D-14 N/ 1 H/ 1 H correlation (B) spectra reported in this study. The length of the excitation (t exc ) and reconversion pulses (t rec ) and the t 1 increments are chosen to ensure that the sequence is rotor synchronised, p/2 and p pulses are highlighted in black and grey respectively. For the 3D experiment, proton mixing during t m was conducted using a RFDR pulse train, pulses have been omitted for clarity. The pulse sequence for indirect detection of 14 N via 1 H used in this work is shown in Fig. 1. Pulse lengths for 1 H pulses were p/2 = 1.55 ms, p = 3.10 ms on both samples, 14 N pulses for excitation and reconversion were applied at 62.5 kHz for 575 ms, and 30 kHz for 390 ms for L-His and HisÁHClÁH 2 O, respectively. The L-His spectrum was recorded with 112 scans per t 1 increment, with 160 complex increments and a recycle delay of 2 s and the HisÁHClÁH 2 O spectrum was recorded with 64 scans and 254 t 1 increments with a recycle delay of 2 s. Both were recorded using States-TPPI. 33 Both datasets were zero-filled to 2048 Â 2048 points and 2D Fourier transformed without a window function.
Experiments on NAV were performed on a Bruker Avance II spectrometer at 20 T (Larmor frequencies of 850 MHz and 61.4 MHz for 1 H and 14 N) using a JEOL 1 mm double resonance MAS probe. The sample was spun with a MAS frequency of 78 kHz. Spectra were acquired using the pulse sequence in Fig. 1. 1 H pulses were p/2 = 1.1 ms, p = 2.2 ms and 14 N excitation and reconversion pulses were applied at 72 kHz for 370 ms. 128 complex t 1 increments were recorded with 136 scans per increment, in a phase sensitive manner, using States-TPPI. 33 The recycle delay was 3 s. Data was zero-filled to 2048 points in each dimension before 2D Fourier transform. 3D 14 N resolved 1 H/ 1 H correlation spectra were acquired the pulse scheme shown in Fig. 1B. Following 14 N evolution, protons were allowed to evolve under the proton chemical shift prior a period of RFDR proton/proton mixing 34 prior to detection. Data was acquired with States-TPPI 33 in both indirect dimensions with 16 and 32 increments in the indirect 14 N and 1 H dimensions respectively. Data was processed in nmrPipe 35 and visualised in Analysis 2.4.0, modified to include 14 N parameters. 36 In all spectra, the 14 N dimension is referenced to the 14 N resonance of NH 4 Cl at 39.3 ppm, ensuring that the 14 N shifts can be compared directly with values on the scale typically used for referencing 15 N in biological solids. 37

Numerical simulations
All NMR simulations were performed with the Spinach library versions 1.6 and 2.2. 38 Each nitrogen site simulated was approximated as a pair of nuclei comprising the 14 N nucleus and the closest bound proton. Simulations included dipolar coupling, CSA and NQI parameters for both spins that were initially obtained from CASTEP calculations 39 or literature values. These initial calculated and literature values are shown in Table 1. Relative orientations of 14 N NQI and 14 N and 1 H CSA tensors were also obtained from the literature, and are also given in Table 1, defined with respect to the PAS of the 1 H-14 N dipolar tensor. Relaxation was neglected in the simulations, and an apodization function applied to simulated FIDs before Fourier transform that reflected the effective T 2 of the experimental 14 N signal. An input script for the experiment described in this paper is available in the latest release of the Spinach library. For simulations on sites in L-His and HisÁHClÁH 2 O, powder averaging was performed over 770 pairs of a and b angle orientations calculated using the Lebedev method. A Floquet rank of 45 was sufficient for convergence for all sites in L-His and HisÁHClÁH 2 O except the N d site of HisÁHClÁH 2 O, where a rank of 55 was required due to the increased C Q magnitude. Simulations of NAV used 1454 a and b angle orientations and a Floquet rank of 55. To fit the experimentally determined lineshapes the simulated 14 N signals, C Q and Z were systematically varied in steps of 50 kHz and 0.1 respectively and the RMSD between experiment and simulation calculated.

CASTEP calculations
To calculate the NQI we used the CASTEP density function theory (DFT) package [39][40][41][42] which uses the gauge including projector augmented wave (GIPAW) algorithm. We used the Perdew-Burke-Ernzerhof (PBE) generalised gradient approximation (GGA) with ultrasoft pseudopotentials and 0.1k points Å 3 . We tested the value of the NQI by converging against the planewave energy cut-off tolerance.

Results and discussion
Histidine hydrochloride monohydrate and L-histidine In order to assess the effects of alternate hydrogen bonding patterns and changes in local geometry on 14 N NQI parameters Table 1 Parameters describing the spin interactions and their relative geometries found in the compounds used in this study

Sample
Site at a number of different nitrogen sites, and how these changes affect 14 N lineshapes and peak positions, we initially investigated samples of L-His and HisÁHClÁH 2 O. These two salts of histidine exhibit different protonation states (see Fig. 2) and H-bonding networks, resulting in a variation in local electronic structure with the associated variation in 14 N NQI parameters (Table 1). Fig. 2A 51 Using a D-HMQC sequence, with R 3 recoupling, transfer efficiencies of 5-6% have been reported for the NH 3 + site in glycine at 20 kHz MAS. 16 The experiment performed here was optimised (in terms of 14 N offset and pulse length) such that efficiency was maximally distributed over the two heterocyclic 14 N sites, and as such, 14 N pulse widths and amplitudes are possible that increase the efficiency at any of the three sites, at the expense of signal intensity at the other sites. This results in a spectrum with a quality and signal/noise ratio such that subtle spectral features such as the shoulders on the peaks of N e and N d are observed which can aid in the characterisation of the quadrupolar interaction. Fig. 2B Table 2.
The d iso Q can be used to estimate the magnitude of the C Q and Z at each site, using eqn (1)-(3). A contour plot of values of d iso Q at 14.1 T, calculated from eqn (1), as a function of the magnitude of the C Q and Z are shown in Fig. S1 (ESI †). As expected from eqn (2), C Q and Z are correlated, however at this field the d iso Q shows only a limited dependence on the asymmetry parameter Z and thus C Q can be readily constrained to a range of B200 kHz irrespective of the asymmetry parameter. In   possible values of Z. The C Q of the NH 3 + site in both compounds can be constrained to a smaller range of C Q magnitudes, since w q and, therefore, 14 N d iso Q is less dependent on Z when the C Q is smaller. The agreement of the 14 N d iso Q determined from indirect detection methods in this paper, as compared to those calculated from literature values for 14 N chemical shift and NQI tensors in the histidine compounds studied here is demonstrated in Table 2. In general, the 14 N d iso Q observed in this work is smaller than that expected from literature values obtained by other techniques, or CASTEP calculated values. This discrepancy is most severe at the amine sites of both histidine molecules, with the HisÁHClÁH 2 O experimental NH 3 + 14 N d iso Q being 14.1 ppm lower than the calculated value, and that site in L-His was found to be 9 ppm lower than the literature/calculated value. At the remaining heterocyclic sites, the N e site in HisÁHClÁH 2 O was found to deviate most from the 14 N d iso Q value calculated from literature NQI parameter values, at 5.3 ppm. These deviations, being most severe for the highly mobile primary amine sites in histidine compounds, may be due to comparisons being made with NQI parameters determined by different techniques at cryogenic temperatures rather than room temperature, freezing out motions that could scale the 14 N NQI in our experiments, or comparisons with CASTEP calculations that do not take into account molecular motions.

Quantitative analysis of histidine spectra
Having recorded indirect 14 N detected spectra with very high efficiencies, the quality of data was high enough to observe subtle spectral features in the 14 N lineshapes, for example, the shoulders on the sides of N e and N d peaks in the HisÁHClÁH 2 O spectrum. As it is not possible to accurately determine C Q and Z from the quadrupolar product alone, a more accurate analysis was undertaken fitting the 14 N lineshapes in the indirect dimension. Numerical simulations were fitted to the experimentally determined lineshapes at each 14 N site in the two histidine compounds, by systematically searching a C Q and Z parameter space spanning C Q = AE0.25 MHz and Z = AE0.25 of the published or calculated values. Plots of the root mean squared deviation (RMSD) between simulated and experimental spectra for each of the 14 N sites in the histidine samples studied is shown in Fig. 3. The fits of these lineshapes were conducted with the quadrupolar and chemical shielding tensors oriented with respect to the 1 H/ 14 N dipolar coupling according the values given in Table 1. Simulations indicate that these parameters do indeed contribute to the overall 1 H/ 14 N lineshape, however for compounds with a small C Q these effects are negligible with the linewidths that are experimentally obtainable.
There is a strip of relatively good agreement (RMSD o B0.3) through the RMSD plot for each 14 N site where the simulated 14 N d iso Q is consistent with that observed experimentally. A single black contour line through each plot marks the experimentally observed 14 N d iso Q , which are also plotted on the contour plot in Fig. S1 (ESI †), and tabulated in Table 2. This line generally coincides with the area of lowest RMSD in each plot. This shows that the largest contribution to the quality of the simulations fit is determined from the 14 N d iso Q , which is to be expected since it determines the position of the peak. In the RMSD plot for the N d of HisÁHClÁH 2 O, Fig. 3C 52 Additionally, visual comparison of the simulated lineshape with the experimental lineshape ( Fig. 2A, yellow traces) shows a good agreement. This plot demonstrates that using a simulation fitting approach, one can define Z to a range of BAE0.1, which allows the C Q to be determined with a precision of tens of kHz, which is at least an order of magnitude more precise than one can achieve considering only the 14 N d iso Q . In the RMSD plots for the N e sites of both HisÁHClÁH 2 O (Fig. 3B) and L-His (Fig. 3E), the fit of the lineshape with respect to Z is somewhat less robust. In the case of the N e site in However, the fit is not as good as that of the N e site in the hydrochloride salt; in L-His the RMSD minima are broader, and the RMSD is not as low (minimum RMSD = 0.24). This is probably due to there being less well-defined features in the experimental lineshape at this site (as shown in Fig. 2B) compared to the N e in HisÁHClÁH 2 O (Fig. 2A). However, the N e sites are expected to have similar chemical environments and hydrogen bond geometries, and therefore similar 14 N spectral parameters in terms of their 14 N EFG and CSA tensors which give rise to the features observed in the 14 N lineshapes, and one of the RMSD minima (at C Q = 1.35 MHz, Z = 0.95-1.00) is consistent with expected values.
The NH 3 + sites in both HisÁHClÁH 2 O and L-His show a poor fit of the simulated lineshape with respect to Z, with the minimum RMSD encompassing almost the whole range of Z values simulated, and not falling below RMSD = 0.3. This means it is not possible to use these plots to constrain the C Q at either site more precisely than was achieved by considering simply the 14 N d iso Q . In the case of the HisÁHClÁH 2 O the rather poorly defined minima reflects the featureless nature of the peak without any discontinuities, shoulders or splittings, making a distinctive fit difficult. This may be due to the fast 1 H T 2 * decoherence at this site leading to a broadening of the signal and the loss of any distinctive features, indeed simulated spectra exhibited well defined splittings and shoulders before the application of the apodization to match the experimentally observed decay. We attribute this rapid relaxation to the molecular motions present at the site, [53][54][55] which, in the case of NH 3 + groups have previously been shown to influence the SQ 14 N lineshape. 18 At the NH 3 + site in L-His, a well-defined splitting was observed in the 14 N lineshape. However, the fit of the simulated spectra to this lineshape was the least satisfactory of all those investigated here, with a minimum RMSD = 0.35. As the experimental and simulated slices (Fig. 2B, red traces) show, the experimental spectrum observed was significantly broader, with more features, than the best fit simulated spectrum. The reason for this may be that at the NH 3 + , the 14 N is coupled to three separate protons, which cannot be resolved in the 1 H spectrum, and so their contributions to the 14 N lineshape are superimposed in the 14 N dimension. The simulated lineshape only takes into account an approximation of the spin system with 14 N coupled to a single amine proton. This would mean that the simulation only accounted for part of the experimental lineshape, and that the remaining features were due to coupling to the other protons at the NH 3 + . Similar effects could also arise through the presence of conformational disorder in the sample or the presence of multiple polymorphs.
The differences observed between the fitted NH 3 + lineshape and those experimentally obtained may also reflect the limitations of our current simulations which ignore the effects of relaxation and motional processes. Although such effects could be incorporated into the calculations, these simulations are already computationally demanding and, practically, close to the limit of what we can achieve in a reasonable timeframe (several weeks of supercomputer time) when fitting a grid of parameters to experimental data. In contrast; fits of the heterocyclic 14 N sites exhibit a better fit with the experimental data, with the C Q and Z in good agreement with published or calculated values. This is particularly clear for the N d site where the larger C Q is larger.

N-Acetyl-valine spectrum
Further proton detected experiments were performed on NAV (see Fig. 4), which exhibits a larger NQI than the sites studied in the histidine samples, with a C Q of 3.21 MHz and a Z = 0.32, 48 and as such mirrors the properties of 14 N sites within the peptide bonds of proteins and peptides. 56 Fig. 4A 48 The efficiency of the two-way 14 N transfer is 9% with respect to the 1 H spin echo signal under the same conditions. This is lower than the efficiencies of the proton detected experiment generally observed on the lower magnitude C Q sites in the histidine compounds. This is a trend observed in all indirect 14 N detection experiments and can be attributed to the increased magnitude of the 14 N C Q in NAV, which is likely to mean that the pulses applied on the 14 N channel excite the far broader 14 N spectrum to a lesser extent.

Quantitative analysis of N-acetyl-valine spectra
A quantitative analysis of the NAV was conducted in a similar manner to that utilized for the histidine compounds. A plot of the 14 N d iso Q , calculated from eqn (2), as a function of C Q and Z at 20.0 T, with the experimentally measured NAV 14 N d iso Q marked, is shown in Fig. S2 (ESI †). From this plot, one can read off the upper and lower limits placed on the C Q at the 14 N site in NAV by the 14 N d iso Q and determine the range of C Q magnitudes consistent with the 14 N d iso Q as 2.83-3.26 MHz, for all values of Z. This range of 430 kHz is far larger than the range of B200 kHz that could be obtained for the moderate C Q (1.0-1.5 MHz) sites found in histidines at 14.1 T. This is since when the C Q is large, the effects of Z on the 14 N d iso Q will be larger, as evident from eqn (2), since Z and C Q are correlated, and the contribution of Z is scaled by the C Q magnitude. Fig. 4B shows a contour plot of the RSMD of simulated spectra with respect to the experimental 14 N lineshape in NAV as a function of the C Q and Z used in the simulations. A comparison of the best-fit 14 N simulated lineshape with the experimental lineshape is seen in the projections in Fig. 4. The quality of the fit is high, with a minimum RMSD = 1.5, however the RMSD minimum is relatively broad; covering a region of C Q magnitude of 75 kHz (C Q = 3.1 MHz-C Q = 3.175 MHz), and range of Z values of 0.32 (0-0.32). The range of values covered by this RMSD minimum slightly underestimates the literature values of C Q = 3.21 MHz, Z = 0.32. 48 However, the black contour line which indicates the 14 N d iso Q calculated from the measured 14 N shift at the centre of gravity of the 14 N lineshape is also underestimated by the best RMSD fit, and in fact agrees very well with the literature values. The lack of accuracy with which the simulations fit the data suggest that there are features of the experimental 14 N lineshape that are not accounted for in the simulations. The reason for this is unclear. Simulations to ascertain if the relative orientations of the quadrupolar, dipolar and chemical shift anisotropy influenced the lineshape were conducted (see Fig. S3 and S4, ESI †). Simulations in the absence of the chemical shielding anisotropy (CSA) (Fig. S3, ESI †) show that the proton lineshape is strongly correlated to the b Euler angle describing the orientation of the quadrupolar tensor with respect to the 1 H/ 14 N dipolar coupling tensor, whilst the influence of the corresponding a Euler angle is limited to the lineshape. Similarly, introduction of the CSA into the simulations (Fig. S4, ESI †) resulted in minor changes to the lineshape. Although the effects of tensor orientation are pronounced when the linewidths are small, many of these factors are masked with the currently attainable experimental resolution, which together with the computationally demanding nature of the simulations and large parameter space precludes a more rigorous statistical fitting of the lineshape. Alternatively, it is plausible that the signal is broadened or shifted by higher-order terms (42) in the quadrupolar Hamiltonian that are not accounted for in the simulations and not removed by MAS. For sites with lower (o1.5 MHz) quadrupolar couplings, it was verified that these terms did not affect the simulated lineshapes. However, this was not verified for the case where the quadrupolar coupling was large (B3 MHz) and high order terms are expected to be more significant, as running the simulations with the full Hamiltonian was found to be prohibitively time consuming. Finally, it may be that the 14 N spectrum of this site is broadened by high mobility of the amide nitrogen, although we acknowledge that this is not reflected in the size of the quadrupolar interaction.

Multidimensional 14 N correlation spectroscopy
The quadrupolar interaction provides a sensitive reporter of secondary structure within proteins, with variations of up to 200 kHz reported between alpha-helical and beta-sheet structures, corresponding to variation in d iso Q of B130 ppm in the proton detected spectra reported here. Despite the large variation in d iso Q reported for proteins, for larger biomolecules its application will remain challenging due to the limited chemical shift dispersion in the amide protons. Although further enhancements in resolution and sensitivity can be realised through the application of faster MAS and higher 14 N rf fields, 26,32 for larger biomolecules it is clear that higher-dimensionality spectra will be required to aid resolution and assignment. In Fig. 1B we describe a 3D experiments which allows the acquisition of a 14 N resolved 1 H/ 1 H correlation experiment. Analogous in many ways to the 15 N filtered 1 H/ 1 H NOESY experiment frequently employed in the liquid state, the 1 H/ 1 H correlations are now resolved according to the d iso Q of the adjacent 14 N site. The efficiency of the experiment ensures that such data sets can be acquired in as little as 2 days, with the overall duration limited by the phase cycling employed (Fig. 5).

Conclusions
We have demonstrated the application of a novel method of indirect detection of 14 N via 1 H under moderate and fast MAS frequencies, applied to two forms of histidine and NAV. Spectra with up to 27.5% efficiency can be recorded using this method, resulting in a large increase in S/N and spectral quality, over previous methods. The data presented have been fitted to simulated lineshapes to extract NQI parameters for nitrogen sites in different chemical environments with increased precision. The 1 H detected 14 N experiments described here are not as limited in the number of sites that can studied as previous methods such as piecewise detection, indeed the variation observed in C Q served to enhance the resolution. Furthermore, exploitation of 1 H detection facilitates its application to unlabelled biomolecules, providing a viable route to study the structure and dynamics complex natural organic and biomolecular systems.

Conflicts of interest
There are no conflicts to declare.