Quantitative 1H-1H Distances in Protonated Solids by Frequency

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Spectroscopy and Photochemistry; General Theory

Quantitative 1H-1H Distances in Protonated Solids by Frequency Selective Recoupling at Fast Magic Angle Spinning NMR Nghia Tuan Duong, Sreejith Raran-Kurussi, Yusuke Nishiyama, and Vipin Agarwal J. Phys. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acs.jpclett.8b02189 • Publication Date (Web): 24 Sep 2018 Downloaded from http://pubs.acs.org on September 26, 2018

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The Journal of Physical Chemistry Letters

Quantitative 1H-1H Distances in Protonated Solids by Frequency Selective Recoupling at Fast Magic Angle Spinning NMR

Nghia Tuan Duong$, Sreejith Raran-Kurussi, Yusuke Nishiyama$#@ and Vipin Agarwal@ $

RIKEN-JEOL Collaboration Centre, RIKEN, Yokohama, Kanagawa 230-0045, Japan #

JEOL RESONANCE Inc., Musashino, Akishima, Tokyo 196-8558, Japan

TIFR Centre for Interdisciplinary Sciences, Tata Institute of Fundamental Research Hyderabad, Sy. No. 36/P, Gopanpally, Ranga Reddy District, Hyderabad 500 107, India

1 ACS Paragon Plus Environment

The Journal of Physical Chemistry Letters 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ABSTRACT: Nuclear magnetic resonance (NMR) spectroscopy of protons in protonated solids is challenging. Fast magic angle spinning (MAS) and homonuclear decoupling schemes, in conjunction, with high magnetic fields have improved the proton resolution. However, experiments to quantitatively measure 1H-1H distances still remain elusive due to the dense proton-proton dipolar coupling network. A novel MAS solid-state NMR pulse sequence is proposed to selectively recouple and measure inter-proton distances in protonated samples. The phase-modulated sequence combined with a judicious choice of transmitter frequency is used to measure quantitative 1H-1H distances on the order of 3Å in L-histidine.HCl.H2O, despite the presence of other strongly coupled protons. This method provides a major boost to NMR crystallography approaches for structures determination of pharmaceutical molecules by directly measuring 1H-1H distances. The band-selective nature of the sequence also enables observing selective 1H-1H correlations (e.g. HN-HN/HN-Hα/ΗΝ-ΗMehtyl) in peptides and proteins, which should serve as useful restraints in structure determination.

TOC GRAPHICS

KEYWORDS: selective recoupling, proton-proton distances, solid-state NMR, protonated samples, fast MAS


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X-ray or neutron diffractions methods have revolutionized our understanding of molecular structures and their organization in crystal lattices. However, for number of samples lacking long-range order, diffraction data is not easily available. Nuclear Magnetic Resonance (NMR) crystallography has emerged as the primary method to decipher the structures of molecules in the disordered physical state.1,2 NMR crystallography combines inputs from solid-state NMR, powder diffraction, molecular modeling and DFT calculation.3 This whole process of structural characterization via NMR crystallography can be simplified or improved if quantitative 1H-1H distances were accessible. However, the measurement of 1H-1H dipolar couplings is challenging due to the presence of a dense network of dipolar-coupled spins even in the fast MAS regime. Traditionally, selectively labelled samples have been used to measure homonuclear

13

C-13C

dipolar couplings.4 Presently, these rare nuclear couplings can be measured in natural abundant sample by employing dynamic nuclear polarization.5,6 Protons have ~100% natural abundance so similar artificial spares labelling methods is not feasible for estimation of dipolar coupling. Frequency selective recoupling has been used to measure to measure both heteronuclear and homonuclear dipolar couplings in uniformly labelled samples, e.g. Frequency-selective rotational echo-double resonance (fs-REDOR) is routinely used to measure weak heteronuclear dipolar couplings.7,8 The frequency selection circumvents the problems of dipolar truncation and permits measurement of weak couplings in the presence of stronger couplings.9 The rotational resonance and rotational resonance in the tilted frame experiments were amongst the earliest recoupling experiments used to measure selective distances between homonuclear spin pairs.10,11 A second-approach aims at simultaneous recoupling of the chemical shift and the dipolar Hamiltonian that in turn makes the zero/double quantum (ZQ/DQ) dipolar Hamiltonian time-dependent in the chemical-shift interaction frame except at particular choice of the transmitter frequency.12-14 Examples of this approach are a) SEASHORE (shift-evolution-



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assisted selective homonuclear recoupling) and b) COMICS (cosine-modulated recoupling with chemical shift introduction) sequence.12-14 Another approach for selective recoupling was based on the idea of creating an effective Ising Hamiltonian in uniformly

13

C-labeled samples. Khaneja

et al. employed triple oscillating field technique (TOFU)15 while Levitt and co-workers proposed the truncated dipolar recoupling (TDR) method that combines symmetry sequences with selective pulses to create the Ising Hamiltonian.16 Amongst these only the TDR approach has been used to measure 1H-1H dipolar coupling for the –CH2 moiety of glycine.17 Otherwise, proton spin-diffusion (PSD) curves recoded via two-dimensional

1

H-1H or CHHC/NHHC

experiments are used to encode 1H-1H distances.18-21 However, extraction of 1H-1H distances from PSD curves remains challenging.1,21-24 Recently, we introduced the band-selective spectral spin diffusion sequence to selectively recouple protons using weak rf fields.25 This approach also yields only qualitative 1H-1H distances. In this study, we propose a novel first-order DQ recoupling pulse sequence to measure 1H-1H distances in protonated solids. The sequence is termed as SElective Recoupling of Protons (SERP). As a proof of concept, several 1H-1H distances are measured in L-histidine.HCl.H2O. We also show that SERP can be used as a proton band-selective recoupling sequence in U[13C,15N]-MLF/ubiquitin

and

provide

both

qualitative

and

quantitative

restraints

in

macromolecules. The SERP sequence involves application of rotor-synchronized phase-modulated pulses (Figure 1a). The phase modulation is implemented in a manner similar to the phase modulated Lee-Goldburg (PMLG) decoupling except that the total change of phase, φ, during the sequence is different from 207.8° necessary to satisfy the Lee-Goldburg condition.26,27 The ten-pulse P0 block followed by a P180 block (a 180o phase shift compared to the P0 block) ensures that the quantization axis of the chemical shift Hamiltonian is parallel to the Z-axis after every P0P180 block.28 A simulation comparing the evolution of one-spin (with chemical shift-offset) during the


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P0 and the P0P180 block is shown in Figure 1d. The recoupling takes places during the 2n (n is an integer number) cycles of the P0P180 block. The m cycles of P0P180P90P270 block is introduced to ensure a constant time (CT) mixing sequence by keeping n+m constant. The CT version compensates for magnetization loss due to relaxation or high-order coherent effects. The 1D/2D implementation of the sequence is depicted in figure 1b).

Figure 1 Pulse sequence a) for SERP recoupling, φk represent the phase of the kth pulse (b) for 1D/2D

13

C/15N filtered experiment with 1H-1H mixing. The filled rectangles denote π/2 pulses.

The shaped pulse on the

13

C/15N channel is a 180°-Q3 pulse used to select only one of the

resonance.29 c) chemical structure of histidine. d) Simulation depicting evolution of single spin zmagnetization during P0 and P0P180 blocks. e) Simulated polarization transfer efficiency (H5 H7) as a function of time for the ring protons of histidine during SERP mixing. SERP recoupling requires optimization of four parameters namely the MAS frequency ( ν r ), phase (φ), the cycle time ( τ c =P0+P180) or the modulation frequency ( ν m = 1 / τ c ) and the rf amplitude ( ν1H ). These parameters were optimized (Figure 2a-f) through numerical simulation (SIMPSON30,31) to observe selective polarization transfer between the H5 H7 ring protons of 5 ACS Paragon Plus Environment


histidine despite the presence of the multiple strong couplings (Figure 1c). Figure 1e) shows a four-spin simulation for optimized parameters (discussed below) of the SERP recoupling sequence. The negative and selective H5H7 polarization transfer indicates that SERP is a first-order DQ selective homonuclear recoupling sequence. In the simulations, ν r was fixed while φ, ν m and ν 1H were independently varied while observing polarization transfer from I zH 5 to I zH 7 , I zH 6 and I zH 8 . Figure 2a-c) shows the transfer efficiency as a function of φ and ν m , with ν1H =100 kHz. When φ < 350° or ν m = ν r , non-selective polarization transfer is observed from H5 to other protons. However, at ν m = 0.5ν r (dashed black line) and φ ~450°-780°, only selective H5 H7 transfer is observed. The analogous experimental plots (Figure 2g-i) show comparable regions of selective ( ν m = 0.5ν r and φ ~450°-780°) and nonselective transfer to simulations. A zoom up of the region enclosed by the white boxes depicting selective transfer is provided in the SI (Figure S1). At ν m = 0.5ν r , φ and ν1H were independently varied (Figure 2d-f). Selective H5 H7 polarization transfer is observed when φ ~450°- 780° and rf field ~75-140 kHz (highlighted by crosses in Figure 2d). Different ν1H only changes the scaling factor of the recoupled dipolar Hamiltonian. At these conditions, the polarization transfer from H5 to H6 and H8 proton is truncated despite the stronger dipolar couplings. The experimental plots (Figure 2j-l) are in reasonable agreement with simulations except for broader regions of polarization transfer due to rf inhomogeneity and finite linewidth of proton resonances. Additional simulations of SERP recoupling (Figure S2, S3) as a function of the number of spins and MAS frequency reconfirm SERP being a first-order DQ recoupling sequence and that polarization transfer occurring exclusively between the two spins equidistant from the transmitter-offset frequency.


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Figure 2: a-f) Numerical simulations and g-l) experimental demonstration of selective polarization transfer from H5H7 during SERP recoupling. Transfer efficiency is plotted as a 7 ACS Paragon Plus Environment


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function φ versus ν m at ν1H = 100 kHz: a-c) simulated and g-i) experimental. As a function of φ versus ν1H at ν m = 34.04kHz : d-f) simulated and j-l) experimental. The transmitter frequency was set to the mean chemical shift of H5 and H7. The details of the simulated spin system and experimental parameters are provided in the SI. The SERP sequence can be classified as a C221 sequence with P0 as the basic element in the framework of the symmetry-based sequences. The sequence recouples the Iz (µ=0) term of the isotropic chemical shift, chemical shift anisotropy (CSA) and the heteronuclear dipolar Hamiltonian and the µ=0, 2 (ZQ and DQ, respectively) term of the homonuclear dipolar Hamiltonians. The effect of the heteronuclear dipolar couplings is minimized by heteronuclear decoupling during SERP mixing while the proton CSAs are too small to cause appreciable change to magnetization trajectories. Therefore, the only relevant terms are the isotropic chemical shift and the homonuclear dipolar Hamiltonians. Thus, for a two-spin system the effective Hamiltonian in the rf interaction frame can be written as:

Hˆ = ω d 2 I1+ I 2+ + ω d* 2 I1− I 2− + ω d 0 I1+ I 2− + ω d* 0 I1− I 2+ + ω 1( 0) I1z + ω 2( 0 ) I 2 z

Eq. 1

or

Hˆ = ω d 2 I1+ I 2+ + ω d* 2 I1− I 2− + ω d 0 I1+ I 2− + ω d* 0 I1− I 2+ + Ξ ( I1z − I 2 z ) + Σ ( I1z + I 2z )

Eq. 2

where ω d 2 and ω d 0 represent the dipolar coupling constants in DQ and ZQ sub-space,

(

)

(

)

respectively. ω n(0 ) is the isotropic chemical shift terms, Ξ = ω 1(0) − ω 2(0) 2 and Σ = ω 1(0) + ω 2(0) 2 . The Hamiltonian in the chemical shift interaction frame can be represented by: Eq. 3 In general, Eq.3 is time-dependent and is averaged out. However, for a spin pair, when either the sum (Σ) or the difference (Ξ) of the isotropic chemical shifts is zero, Eq.3 becomes timeindependent and recouples either the DQ or the ZQ dipolar Hamiltonian, respectively. In a


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multiple-spin system, DQ recoupling condition can be selectively satisfied for a spin-pair by placing the transmitter frequency at the center of the two resonances (Σ = 0). The ZQ recoupling condition is automatically satisified when the isotropic chemical shift of the two spins is identical .i.e., for overlapped peaks ( (Ξ) = 0) and is independent of the position of the carrier frequency. Figure 3a-c) demonstrate the experimental efficacy of frequency selective 1H-1H recoupling between different proton pairs in U-[13C and/or

15

N] histidine sample. Figure 3a shows selective

polarization transfer from H5 to a group of H6, H8 and NH3+ protons indicating the band selective nature of SERP while Figure 3b shows selective H5 ↔ H7 transfer between resolved spins. Even for overlapped peaks (H2/H3) selective transfer is observed e.g. H2/H3 H5 (Figure 3 c). The 1H projections along the dotted lines are plotted below Figure 3a-c). Figure 3de) shows the NMR signal amplitude for selective polarization transfer from H5/H7 to H6/H8 as a function of recoupling duration. The first dipolar oscillation is visible while at longer recoupling time oscillations are damped. The thick red and black bands represent the confidence interval of fitting and correspond to distance ranges given in Table 1. The insets in Figure 3d-e) depict the root mean square error between experiment and simulated data as a function of dipolar coupling. We find that the buildup curves of SERP recoupling can actually distinguish a difference of 0.1-0.2 Å in distance between the H7-H6 and H7-H8 in histidine (Figure 3e). The distances estimated from SERP experiments appear to be about 0.1-0.2Å smaller compared to similar distances from X-ray (CSD entry: HISTCM01) and neutron diffraction (CSD entry: HISTCM12) structures (Table 1).32,33 This could arises due to distribution of chemical shifts resulting from anisotropic bulk magnetic susceptibility broadening.34 When a distribution of chemical shift is considered, the transfer of polarization is faster by about 2-6τr (Figure S4) while for fitting of data in Figure 3d-e) a unique chemical shifts is considered during simulation; thus, accounting for observed shorter distances. Figure 3f) shows the polarization transfer curves from H5H7 and vice versa. Here, unlike build-up curves in Figure 3d-e), the magnetization



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continues to grow at longer mixing time. In small molecules, intermolecular and intramolecular distances between the same spin pairs can be comparable and will result in a cumulative response. This will always be an unavoidable situation in small molecules while determining medium to long 1H-1H distances. In case of histidine, multiple H5-H7 distances are present (Figure 3i, shows intramolecular and intermolecular H5-H7 spin pairs in histidine at a distance of 4.1Å, 3.2Å, 5.7Å and 6.1Å, respectively). The unequal population of these spin-pairs results in (0)

the peculiar transfer profile (Figure S5). For overlapped peaks, (i.e. ω 1

= ω 2(0) ), the ZQ

recoupling condition (Eq. 3) is always satisfied and spin dynamics is more complex. However, even in this situation it is possible to selectively transfer magnetization from one of the overlapped protons to another proton in the molecule. For example H2/H3 are overlapped and selective polarization transfer from H3H5 (Figure 3g) is observed by selecting the C3 resonance in the

13

C dimension. Although the build-up curve does not show a clear oscillation,

the first maximum is indicative of H3-H5 distance of approximately 2.3-2.6Å. H3 belongs to the CH2 moiety and therefore, has two intramolecular and one-intermolecular H3-H5 pairs between 2.53-4.20Å. The ZQ build up between overlapped H2-H3 (Figure 3h and Figure S6) were measured by incrementing the P0P180P90P270 block to ensure only ZQ transfer. The simulations for H5-H7 and H2-H3 were performed using structural inputs in order to understand the observed buildup profiles and not to extract distances.


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Figure 3 a-c) Demonstration of 2D frequency selective 1H-1H recoupling in U-[13C and/or

15

N] L-

Histidine. a) H5 H6/NH3+/H8, b) H5 and H7 c) H2/H3 H5, all experimental parameters are provided in the SI. Positive contours are depicted in green while negative contours are depicted in brown. In each case, the proton transmitter was placed at the mean frequency of the two protons to be selectively recoupled. d-f) represent the experimental (circles) and simulated (solid lines/colored regions) polarization build up between selective pair of spins as a function of recoupling time. The thick red and black bands represent the confidence interval of the fitted 11 ACS Paragon Plus Environment


distances in d) and e). d) H5 H6/H8, e) H7 H6/H8 f) H5↔H7, g) H3H5, h) ZQ transfer between H2 H3 i) different distances mapped on the crystal structure of histidine.

Table 1 Inter proton distances from different spectroscopic methods

Distance

SERP (Å)

X-ray (Å)

Neutron (Å)

H7-H6

2.26-2.43

2.38

2.46

H7-H8

2.31-2.48

2.55

2.57

H5-H6

2.44-2.63

2.47

2.53

H5-H8

2.80-3.08

3.37

3.24

As discussed above frequency selective recouping have been proposed previously in carbons to measure distances up to a few angstrom.12-14 The relatively large chemical shift dispersion in carbons combined with weaker dipolar couplings result in very narrow recoupling bandwidth (typically less than ~300Hz). For longer distances, the recoupled bandwidth can be smaller than the linewidths.12-14 In contrast, the dipolar couplings amongst protons are larger and the chemical shift dispersion is smaller than carbons. Under our experimental conditions, we numerically observe that the ϕ

480o

and ϕ

680o

conditions have a scaling factor of approximately

0.22 and 0.11, respectively. In reality, the scaling factor is complex and depends on several factors such as the net flip angle, number of pulses that define the total flip angle during P0/P180, the Fourier coefficient of the recoupled Hamiltonians in the interaction frame, the rf amplitude which indirectly manifests itself by changing the magnitude of the Fourier coefficient of the recoupled Hamiltonian and φ. A discussion on the effective field direction and corresponding scaling factor for the two conditions is provided in the SI. The observed scaling factors correspond to a recoupling bandwidth of 0.5-2kHz for 1H-1H distances on the order of 4Å. The smaller scaling factor for the ϕ

680o

condition results in narrower bandwidth than the


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ϕ 480 condition as observed through simulations (Figure 4 a-c) and experiments (Figure 4d-f). o

The offset of 0, -1.0 and -1.5kHz (Figure 4a-f) corresponds to the chemical shift centers between the H5-H7, H5-H6 and H5-H8 pairs of protons in histidine. The 680° condition results in higher transfer efficiencies compared to the 480° condition both in experiments and simulations (Figure 4a-f and Figure S2). In contrast to simulations (Figure 4a-c), the experimental bandwidth (Figure 4d-f) of recoupling is broader most likely due to the finite linewidths and lower experimental transfer efficiency compared to simulation. The SERP recoupling is also useful to probe selective HN-HN, HN-Hα and HN-Hmethyl spatial correlations in larger macromolecules such as proteins. Figure 4g) shows selective HN-HN (longer than 4Å) for U-[13C,15N] for MLF and Figure 4h) shows selective HN-Hα and HN-HMethyl contacts in U-[13C,15N] Ubiquitin, respectively. The selective correlations observed via SERP recoupling are analogous to those previously accessible only in perdeuterated proteins with protonation at selective sites.35 Such structurally selective contacts will serve as useful structural restraints in structure determination of protonated solids. As a real application, we also attempted to measure the HNA46 to HαA46/F45 distance in ubiquitin (Figure S7). The initial results on the measurement of 1H-1H distances in large molecules are very promising. SERP can be used in both in a quantitative and qualitative sense by recording spectra at one mixing time. The incorporation of very rarely used quantitative distance restraints and vector angle restraints to the qualitative distance restraints are known to significantly improve the quality of solid-state NMR structures.36,37



Figure 4 a-c) simulated and d-f) experimental peak intensities of Hn (n=6,7,8) as a function of the 1H transmitter frequency during H5Hn SERP recoupling at ϕ of 480° and 680° for a duration of 1.8ms and 3.0ms, respectively. (a, d) H7, (b, e) H6, (c, f) H8. Selective g) HN-HN, and h) HN-Hα (black) / HN-Hmehtyl (red) SERP correlation spectra for MLF and Ubiquitin, respectively. The proton offsets were set to 8.8ppm (MLF), 6.85 (black line) and 4.85ppm (red line) for Ubiquitin. The positive (negative) contours are depicted in green (brown, black, red).


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To conclude, we propose a novel frequency selective dipolar recoupling sequence to measure quantitative 1H-1H distances in protonated solids. Distances measured via SERP should complement the data obtained from spin diffusion measurements and assist in NMR crystallography approaches to structures of small molecules. For 1H-1H distances on the order of ~ 3Å, dipolar oscillations are easily observed. For measurement of longer distances dipolar oscillations are damped but the buildup rate can help distinguish protons based on distances. The damping in smaller molecules can have several possible origins, such as a cumulative response from intra and intermolecular distances; the labile protons can have a distribution of distances, relaxation processes, ZQ recoupling in case of protons with overlapped chemical shifts and inhomogeneous broadening of peaks. The band-selective nature of SERP recoupling should be particularly useful in identifying both quantitative and qualitative distance restraints in protonated macromolecules. We envisage 1H-1H dipolar couplings should also serve as useful probes in characterizing dynamics.

ASSOCIATED CONTENT

Supporting Information. Numerical simulations; sample preparation details and the NMR experimental parameters are included. The Supporting Information is available free of charge on the ACS Publications. AUTHOR INFORMATION Corresponding Authors: Email: [email protected], [email protected] ACKNOWLEDGMENT: VA would like to acknowledge Japanese Society for Promotion of Sciences fellowship ID no: S17066 and financial support from Department of Science and Technology, Government of



India, via grant number: ECR/2017/001450. We also acknowledge Prof Matthias Ernst, Prof P.K. Madhu and Dr Kaustubh Mote for useful discussions. We declare no competing financial interests. References (1) (2) (3) (4)

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Distance Measurements by Solid-State NMR. J. Chem. Phys. 2008, 128 (1), 015103– 015109. Marin-Montesinos, I.; Mollica, G.; Carravetta, M.; Gansmüller, A.; Pileio, G.; Bechmann, M.; Sebald, A.; Levitt, M. H. Truncated Dipolar Recoupling in Solid-State Nuclear Magnetic Resonance. Chem. Phys. Lett. 2006, 432 (4-6), 572–578. Mollica, G.; Madhu, P. K.; Ziarelli, F.; Thévand, A.; Thureau, P.; Viel, S. Towards Measurement of Homonuclear Dipolar Couplings in 1H Solid-State NMR: Recoupling with a Rotor-Synchronized Decoupling Scheme. Phys. Chem. Chem. Phys. 2012, 14 (13), 4359–7. Caravatti, P.; Neuenschwander, P.; Ernst, R. R. Characterization of Heterogeneous Polymer Blends by Two-Dimensional Proton Spin Diffusion Spectroscopy. Macromolecules 1985, 18 (1), 119–122. Lange, A.; Seidel, K.; Verdier, L.; Luca, S.; Baldus, M. Analysis of Proton−Proton Transfer Dynamics in Rotating Solids and Their Use for 3D Structure Determination. J. Am. Chem. Soc. 2003, 125 (41), 12640–12648. Lange, A.; Luca, S.; Baldus, M. Structural Constraints From Proton-Mediated Rare-Spin Correlation Spectroscopy in Rotating Solids †. J. Am. Chem. Soc. 2002, 124 (33), 9704–9705. Eléna, B.; Pintacuda, G.; Mifsud, N.; Emsley, L. Molecular Structure Determination in Powders by NMR Crystallography From Proton Spin Diffusion. J. Am. Chem. Soc. 2006, 128 (29), 9555–9560. Suter, D.; Ernst, R. Spin Diffusion in Resolved Solid-State NMR Spectra. Phys. Rev., B Condens. Matter 1985, 32 (9), 5608–5627. Kubo, A.; Mcdowell, C. A. Spectral Spin Diffusion in Polycrystalline Solids Under MagicAngle Spinning. J Chem Soc, Faraday Trans 1988, 84 (11), 3713–3730. Veshtort, M.; Griffin, R. G. SPINEVOLUTION: a Powerful Tool for the Simulation of Solid and Liquid State NMR Experiments. J. Magn. Reson. 2006, 178 (2), 248–282. Jain, M. G.; Lalli, D.; Stanek, J.; Gowda, C.; Prakash, S.; Schwarzer, T. S.; Schubeis, T.; Castiglione, K.; Andreas, L. B.; Madhu, P. K.; et al. Selective 1H-1H Distance Restraints in Fully Protonated Proteins by Very Fast Magic-Angle Spinning Solid-State NMR. J. Phys. Chem. Lett. 2017, 8 (11), 2399–2405. Vinogradov, E.; Madhu, P. K.; Vega, S. High-Resolution Proton Solid-State NMR Spectroscopy by Phase-Modulated Lee–Goldburg Experiment. Chem. Phys. Lett. 1999, 314 (5-6), 443–450. Mote, K. R.; Agarwal, V.; Madhu, P. K. Five Decades of Homonuclear Dipolar Decoupling in Solid-State NMR: Status and Outlook. Prog. Nucl. Magn. Reson. Spectrosc 2016. Leskes, M.; Madhu, P. K.; Vega, S. A Broad-Banded Z-Rotation Windowed PhaseModulated Lee–Goldburg Pulse Sequence for 1H Spectroscopy in Solid-State NMR. Chem. Phys. Lett. 2007, 447 (4-6), 370–374. Emsley, L.; Bodenhausen, G. Gaussian Pulse Cascades: New Analytical Functions for Rectangular Selective Inversion and in-Phase Excitation in NMR. Chem. Phys. Lett. 1990, 165 (6), 469–476. Bak, M.; Rasmussen, J. T.; Nielsen, N. C. SIMPSON: a General Simulation Program for Solid-State NMR Spectroscopy. J. Magn. Reson. 2000, 147 (2), 296–330. Tošner, Z.; Andersen, R.; Stevensson, B.; Edén, M.; Nielsen, N. C.; Vosegaard, T. Computer-Intensive Simulation of Solid-State NMR Experiments Using SIMPSON. J. Magn. Reson. 2014, 246, 79–93. Oda, K.; Koyama, H. A Refinement of the Crystal Structure of Histidine Hydrochloride Monohydrate. Acta Crystallogr. B 1972, 28 (2), 639–642. Fuess, H.; Hohlwein, D.; Mason, S. A. Neutron Diffraction Study of L-Histidine 17 ACS Paragon Plus Environment


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Hydrochloride Monohydrate. Acta Crystallogr. B 1977, 33 (3), 654–659. VanderHart, D. L.; Earl, W. L.; Garroway, A. N. Resolution in 13C NMR of Organic Solids Using High-Power Proton Decoupling and Magic-Angle Sample Spinning. J. Magn. Reson. (1969) 1981, 44 (2), 361–401. Linser, R.; Bardiaux, B.; Higman, V.; Fink, U.; Reif, B. Structure Calculation From Unambiguous Long-Range Amide and Methyl 1H−1H Distance Restraints for a Microcrystalline Protein with MAS Solid-State NMR Spectroscopy. J. Am. Chem. Soc. 2011, 133 (15), 5905–5912. Rienstra, C. M.; Tucker-Kellogg, L.; Jaroniec, C. P.; Hohwy, M.; Reif, B.; McMahon, M. T.; Tidor, B.; Lozano-Pérez, T.; Griffin, R. G. De Novo Determination of Peptide Structure with Solid-State Magic-Angle Spinning NMR Spectroscopy. Proc. Natl. Acad. Sci. U.S.A. 2002, 99 (16), 10260–10265. Franks, W. T.; Wylie, B. J.; Schmidt, H. L. F.; Nieuwkoop, A. J.; Mayrhofer, R.-M.; Shah, G. J.; Graesser, D. T.; Rienstra, C. M. Dipole Tensor-Based Atomic-Resolution Structure Determination of a Nanocrystalline Protein by Solid-State NMR. Proc. Natl. Acad. Sci. U.S.A. 2008, 105 (12), 4621–4626.


Page 18 of 22

a)

1

H

τr CP

* 15N/13C

tp

φ1 φ2 φ3 φ4 φ5 φ6 φ7 φ8 φ9 φ10

*

CP

][

2n

m

= P0 P180 P0 P180 P90 P270

mixing

P0=

b)

[

1H-1H

het. dec.

]

e)

φk1....φk5=φ*((k-1)+0.5)/5 φk6....φk10=φ*((10-k)+0.5)/5+π

x y-x-y

CP

1H-1H

mixing

t2

c)

*

t1

CP

WALTZ

ACS Paragon Plus Environment

Intensity

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


0.9 0.7 0.5 0.3 0.1 -0.1 -0.3

d) Intensity

Page 19 of 22

φ = 480o H5 H7

H6 H8

0 1 2 3 4 5 6 τ (ms) mix

1.0 0.8 0.6 0.4 0.2 0

1 2 τ

P0 P0-P180 3 4 5 6

mix

(ms)


-0.1

200

-0.2

30 1000

40

ν

50

m

(kHz)

-0.2

1000

φ (degree)

X

600

X

0.3 0.2 0.1 0

X

200

-0.1

200 30

0.4

400

0

60

d)

800

400

-0.1

40

50

ν

m

(kHz)

0.4 0.3 0.2

600

0.1

400

0 -0.1

200

75

100 125

ν 1 (kHz)

25

g)

φ (degree)

800

-0.1

200

-0.2

1000

40

50

ν m (kHz)

60

X X

400

0.2 0.1 0

X

200

0.3

-0.1 -0.2

0

25

50

ν

1

H

75 100 125

(kHz)

-0.1

200

-0.2

40

50

ν

m

(kHz)

60

f)

800

0.4 0.3 0.2

600

0.1

400

0 -0.1

200

ν 1 (kHz)

25

ν r = 68.04 kHz

-0.1

200

-0.2

30

40

50

ν m (kHz)

60

k)

800

0.2 0.1 0

200

-0.1 -0.2

0

25

50

ν

1

H

75 100 125

(kHz)


75

100 125

ν 1 (kHz)

i)

0

400

-0.1

200

-0.2

0

30

40

50

ν m (kHz)

60

l)

800

70

0.4 0.3

600

0.2 0.1

400

0

200 0

0.2 0.1

600

1000 0.3

400

H

800

70

0.4

600

0

0.2

0

400

50

1000

0.1

600

0

-0.2

100 125

h)

1000 0.4

600

75

800

70

j)

800

0

0.2

0

400

30

H

1000

0.1

600

0

50

Experiments: Source spin H5,

1000

0

-0.2

φ (degree)

H

400

1000

φ (degree)

50

0.1

30

-0.2

25

600

60

e)

800

φ (degree)

0

0.1

0.2

φ (degree)

400

600

c)

800

φ (degree)

0.1

0.2

H8

φ (degree)

600

b)

800

φ (degree)

φ (degree)

0.2

H6

φ (degree)

a)

800

Simulations: Source spin H5, νr = 68.04 kHz

H7

φ (degree)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 20 of 22

-0.1 -0.2

0

25

50

ν

1

H

75 100 125

(kHz)

H5

H7 H6

H6

H5

H5

H8

H5

NH3

NH3 22.0

18.0

14.0

10.0

6.0

δ2 -1H (ppm)

H5

c)

H7

H5

30 H5

20.0

50

NH3 16.0

H3

H3

40

H7

H5

24.0

2.0

H7

12.0

8.0

δ2 -1H (ppm)

H5

H2

15

20

H2

10

δ2 -1H (ppm)

0

5

H6

0.11

0.9

0.7

0.5

0.3

0.1

0.1-

0.3-

0.5-

0.7-

H7

b)

δ1 -13C

H7

0.9-

200.0

δ1 -15N (ppm) 190.0 180.0 170.0

a)

0.5

d)

0.5

H7 (Exp.) H5 (Exp.)

0.3

H5 ! H5 !

0

0.5

1

0.2

2

4

D

6 HH

0.1

H6 H5(Experiment) H6 (Exp.) H8 H5(Experiment) H8 (Exp.)

1.5

2

Time (ms)

2.5

6 3 0

8 10 12

(kHz)

0

3

g)

0.3

9

6

0.5

h)

1.0

1

1.5

2

Time (ms)

H2

2.5

H6

0.4

0.2

0

1

2

3

Time (ms)

4

5

H5

H8 3.25Å H5 5.70Å H7 3.19Å H7 0Å H6 4.1

48

H8 Å 57

0.5

1.0

1.5

2.0

Time (ms)

2.5

3.0

0.0

H7 2.

Å

0.2

0.0 0.0

0

3

H2 (Exp.) H3(Exp.) H2 (Sim.) H3 (Sim.)

Intensity

Intensity

H5 (Exp.) H7 (Exp.)

0.1

i)

0.6

H3 H3

10 12

H3

0.8 0.4

8

D DHH (kHz) (kHz) HH

H7 ! H6H6 (Experiment) H7 (Exp.) H7H7 ! H8H8 (Experiment) (Exp.)

0

3.2 Å (sim) 4.1 Å (sim) 5.7 Å (sim)

0.2

3Å

0.1

12

2.5

0.2

Intensity

Intensity

18 15 12 9 6 3 0

%RMSD residuals

Intensity

H5 H7

0.4

0.3

0.6

f)

0.5

0.4

0.4

0

e)

% residuals RMSD

0.6

2.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


δ1 -15N (ppm) 190.0 180.0 170.0

Page 21 of 22

0

1

2

3

4

Time (ms)


5

6

6


a)

Intensity

Simulations

0

H7

b)

H6

0

-0.1

-0.1

-0.2

-0.2

-0.2

-0.3

-0.3

-0.3

-0.4

-0.4

φ =480

-0.5 -1.5

-1 -0.5

0

0.5

offset (kHz)

d)

-0.6

1

-1 -0.5

0.5

offset (kHz)

e)

H7

0

-0.05

-0.05

-0.05

-0.1

-0.1

-0.1

-0.15

-0.15

-0.15

g)

F3

110

120

L2

F3-OH

M1

130 14

12 1H

10

8

6

-1.5

4

2

Chemical Shifts (ppm)

-1 -0.5

0

0.5

offset (kHz)

100.0

offset (kHz)

-0.3

1

0.5

-0.3

1

1

H8

φ =480 φ =680 -1.5

-1 -0.5

0

0.5

1

offset (kHz)

Hα

h)

Hmethyl

110.0

0.5

0

offset (kHz)

120.0

0

Hα F45/A46

HN A46

130.0

-1 -0.5

-1 -0.5

-0.25

φ =680

15N


100

-1.5

Missing Aliphatic protons

-0.3

-0.25

φ =680

-1.5

-0.2

φ =480


-0.25

F3(Aromatic)

Intensity

0

-0.2

φ =680

f)

H6

0

φ =480

φ =480

-0.6

1

0

-0.2

H8

-0.5

φ =680 -1.5

c)

-0.4

φ =480

-0.5

φ =680

-0.6

Experiments

0

-0.1

15N

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 22 of 22

11.0

9.0


7.0

1H

5.0

MPD peak

3.0

1.0


-1.0

Quantitative 1H-1H Distances in Protonated Solids by Frequency

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