Fast MAS Research Articles

Efficient DNP at high fields and fast MAS with antenna-sensitized dinitroxides.

Dynamic Nuclear Polarization (DNP) can significantly enhance the sensitivity of solid-state NMR. In DNP, microwave irradiation induces polarization transfer from unpaired electron spins to 1H nuclear spins via hyperfine couplings and spin-diffusion. The structure of the polarizing agents that host the electron spins is key for DNP efficiency. Currently, only a handful of structures perform well at very high magnetic fields (≥18.8 T), and enhancements are significantly lower than those obtained at lower fields. Here, we introduce a new series of water-soluble nitroxide biradicals with a scaffold augmented by dihydroxypropyl antenna chains that perform significantly better than previous dinitroxides at 18.8 T. The new radical M-TinyPol(OH)4 yields enhancement factors of ∼220 at 18.8 T and 60 kHz MAS, which is a nearly factor 2 larger than for the previous best performing dinitroxides. The performance is understood through 2H ESEEM measurements to probe solvent accessibility, supported by Molecular Dynamics simulations, and by experiments on deuterated samples. We find that the deuterated glycerol molecules in the matrix are located mainly in the second solvation shell of the NO bond, limiting access for protonated water molecules, and restricting spin diffusion pathways. This provides a rational understanding of why the dihydroxypropyl chains present in the best-performing structures are essential to deliver the polarization to the bulk solution.

5D solid-state NMR spectroscopy for facilitated resonance assignment.

1H-detected solid-state NMR spectroscopy has been becoming increasingly popular for the characterization of protein structure, dynamics, and function. Recently, we showed that higher-dimensionality solid-state NMR spectroscopy can aid resonance assignments in large micro-crystalline protein targets to combat ambiguity (Klein et al., Proc. Natl. Acad. Sci. U.S.A. 2022). However, assignments represent both, a time-limiting factor and one of the major practical disadvantages within solid-state NMR studies compared to other structural-biology techniques from a very general perspective. Here, we show that 5D solid-state NMR spectroscopy is not only justified for high-molecular-weight targets but will also be a realistic and practicable method to streamline resonance assignment in small to medium-sized protein targets, which such methodology might not have been expected to be of advantage for. Using a combination of non-uniform sampling and the signal separating algorithm for spectral reconstruction on a deuterated and proton back-exchanged micro-crystalline protein at fast magic-angle spinning, direct amide-to-amide correlations in five dimensions are obtained with competitive sensitivity compatible with common hardware and measurement time commitments. The self-sufficient backbone walks enable efficient assignment with very high confidence and can be combined with higher-dimensionality sidechain-to-backbone correlations from protonated preparations into minimal sets of experiments to be acquired for simultaneous backbone and sidechain assignment. The strategies present themselves as potent alternatives for efficient assignment compared to the traditional assignment approaches in 3D, avoiding user misassignments derived from ambiguity or loss of overview and facilitating automation. This will ease future access to NMR-based characterization for the typical solid-state NMR targets at fast MAS.

Determination of the mutual orientation between proton CSA tensors mediated through band-selective 1H–1H recoupling under fast MAS

The mutual orientation of nuclear spin interaction tensors provides critical information on the conformation and arrangement of molecules in chemicals, materials, and biological systems at an atomic level. Proton is a ubiquitous and important element in a variety of substances, and its NMR is highly sensitive due to their virtually 100% natural abundance and large gyromagnetic ratio. Nevertheless, the measurement of mutual orientation between the 1H CSA tensors has remained largely untouched in the past due to strong 1H–1H homonuclear interactions in a dense network of protons. In this study, we have developed a proton-detected 3D 1H CSA/1H CSA/1H CS correlation method that utilizes three techniques to manage homonuclear interactions, namely fast magic-angle spinning, windowless C-symmetry-based CSA recoupling (windowless-ROCSA), and a band-selective 1H–1H polarization transfer. The asymmetric 1H CSA/1H CSA correlated powder patterns produced by the C-symmetry-based methods are highly sensitive to the sign and asymmetry parameter of the 1H CSA, and the Euler angle β as compared to the symmetric pattern obtained by the existing γ-encoded R-symmetry-based CSA/CSA correlation methods and allows a larger spectral area for data fitting. These features are beneficial for determining the mutual orientation between the nuclear spin interaction tensors with improved accuracy.

Internuclear distance measurements between 1H and 14N in multi-component rigid solids at fast MAS

1H-14N internuclear distances are readily and accurately measured using the symmetry-based phase-modulated resonance-echo saturation-pulse double-resonance (PM-S-RESPDOR) method in rigid solids. The fraction curve, (S0 – S’)/S0, is represented by a single variable of a 1H-14N heteronuclear dipolar coupling, where S0 and S’ are the PM-S-RESPDOR signal intensity with and without 14N PM saturation pulse, respectively. Analytical equation of the fraction curve easily provides 1H-14N couplings. This treatment is only applicable when NH proton resonance is well separated from the other proton peaks. With the limited 1H resolution even at fast MAS > 60 kHz, unfortunately, this condition is not necessarily satisfied especially in multi-component systems which often appear in pharmaceutical applications. To overcome this problem, T-HMQC filtering is applied to suppress the 1H signals other than NH proton prior to the PM-S-RESPDOR experiments. The method is well demonstrated on two components acetaminophen-oxalic acid (APAP-OXA) systems. Further analysis of orientation dependence of T-HMQC and PM-S-RESPDOR shows that the analytical equation can be safely applied in the analysis of T-HMQC filtered PM-S-RESPDOR experiments.

Assignment of aromatic side-chain spins and characterization of their distance restraints at fast MAS

The assignment of aromatic side-chain spins has always been more challenging than assigning backbone and aliphatic spins. Selective labeling combined with mutagenesis has been the approach for assigning aromatic spins. This manuscript reports a method for assigning aromatic spins in a fully protonated protein by connecting them to the backbone atoms using a low-power TOBSY sequence. The pulse sequence employs residual polarization and sequential acquisitions techniques to record HN- and HC-detected spectra in a single experiment. The unambiguous assignment of aromatic spins also enables the characterization of 1H–1H distance restraints involving aromatic spins. Broadband (RFDR) and selective (BASS-SD) recoupling sequences were used to generate HN-ΗC, HC-HN and HC-HC restraints involving the side-chain proton spins of aromatic residues. This approach has been demonstrated on a fully protonated U-[13C,15N] labeled GB1 sample at 95–100 kHz MAS.

Rapid Quantification of Pharmaceuticals via 1H Solid-State NMR Spectroscopy.

The physicochemical properties of active pharmaceutical ingredients (APIs) can depend on their solid-state forms. Therefore, characterization of API forms is crucial for upholding the performance of pharmaceutical products. Solid-state nuclear magnetic resonance (SSNMR) spectroscopy is a powerful technique for API quantification due to its selectivity. However, quantitative SSNMR experiments can be time consuming, sometimes requiring days to perform. Sensitivity can be considerably improved using 1H SSNMR spectroscopy. Nonetheless, quantification via 1H can be a challenging task due to low spectral resolution. Here, we offer a novel 1H SSNMR method for rapid API quantification, termed CRAMPS-MAR. The technique is based on combined rotation and multiple-pulse spectroscopy (CRAMPS) and mixture analysis using references (MAR). CRAMPS-MAR can provide high 1H spectral resolution with standard equipment, and data analysis can be accomplished with ease, even for structurally complex APIs. Using several API species as model systems, we show that CRAMPS-MAR can provide a lower quantitation limit than standard approaches such as fast MAS with peak integration. Furthermore, CRAMPS-MAR was found to be robust for cases that are inapproachable by conventional ultra-fast (i.e., 100 kHz) MAS methods even when state-of-the-art SSNMR equipment was employed. Our results demonstrate CRAMPS-MAR as an alternative quantification technique that can generate new opportunities for analytical research.

NMR Assignment of Methyl Groups in Immobilized Proteins Using Multiple-Bond 13C Homonuclear Transfers, Proton Detection, and Very Fast MAS.

In nuclear magnetic resonance spectroscopy of proteins, methyl protons play a particular role as extremely sensitive reporters on dynamics, allosteric effects, and protein–protein interactions, accessible even in high-molecular-weight systems approaching 1 MDa. The notorious issue of their chemical shift assignment is addressed here by a joint use of solid-state 1H-detected methods at very fast (nearly 100 kHz) magic-angle spinning, partial deuteration, and high-magnetic fields. The suitability of a series of RF schemes is evaluated for the efficient coherence transfer across entire 13C side chains of methyl-containing residues, which is key for establishing connection between methyl and backbone 1H resonances. The performance of ten methods for recoupling of either isotropic 13C–13C scalar or anisotropic dipolar interactions (five variants of TOBSY, FLOPSY, DIPSI, WALTZ, RFDR, and DREAM) is evaluated experimentally at two state-of-the-art magic-angle spinning (55 and 94.5 kHz) and static magnetic field conditions (18.8 and 23.5 T). Model isotopically labeled compounds (alanine and Met-Leu-Phe tripeptide) and ILV-methyl and amide-selectively protonated, and otherwise deuterated chicken α-spectrin SH3 protein are used as convenient reference systems. Spin dynamics simulations in SIMPSON are performed to determine optimal parameters of these RF schemes, up to recently experimentally attained spinning frequencies (200 kHz) and B 0 field strengths (28.2 T). The concept of linearization of 13C side chain by appropriate isotope labeling is revisited and showed to significantly increase sensitivity of methyl-to-backbone correlations. A resolution enhancement provided by 4D spectroscopy with non-uniform (sparse) sampling is demonstrated to remove ambiguities in simultaneous resonance assignment of methyl proton and carbon chemical shifts.

Supramolecular Synthon Identification in Azelaic Acid – Isonicotinamide

Supramolecular synthons are the smallest structural units that contain the information inherent in building large molecular assemblies. Here, we apply solid-state NMR methods for the identification of the synthon in a cocrystal of Azelaic acid [(CH2)7(COOH)2]– Isonicotinamide [C6H6N2O]. It is observed that the spin-lattice relaxation time of protons in the cocrystal is long, implying that the choice of experiments, particularly those that involve low-sensitivity nuclei, could be time consuming. It is noticed that the two dimensional 1H double quantum (DQ) – 1H single quantum (SQ) correlation experiment at very fast MAS (60 kHz) is able to provide information on proton-proton proximities that leads to an understanding of the synthon structure. Experiments such as the CP-Reverse CP and 1H–13C HETCOR have been used to obtain spectral assignment which was additionally confirmed by first-principle GIPAW NMR chemical shift calculations. Present study indicates the basic unit to be a heterosynthon in Azelaic acid (AZA) – Isonicotinamide (INIC) cocrystal formed by two different modes of intermolecular connectivity, viz. acid–pyridine and acid–amide hydrogen bonding interactions. The presence of a CH…O type bond involving a hydrogen of INIC is also indicated from the observation of a larger chemical shift of the corresponding proton. The experimental protocol presented in this paper is expected to be a useful tool for the estimation of the basic structural arrangement of the synthons in a rapid fashion.

Solid-State NMR: Methods for Biological Solids.

In the last two decades, solid-state nuclear magnetic resonance (ssNMR) spectroscopy has transformed from a spectroscopic technique investigating small molecules and industrial polymers to a potent tool decrypting structure and underlying dynamics of complex biological systems, such as membrane proteins, fibrils, and assemblies, in near-physiological environments and temperatures. This transformation can be ascribed to improvements in hardware design, sample preparation, pulsed methods, isotope labeling strategies, resolution, and sensitivity. The fundamental engagement between nuclear spins and radio-frequency pulses in the presence of a strong static magnetic field is identical between solution and ssNMR, but the experimental procedures vastly differ because of the absence of molecular tumbling in solids. This review discusses routinely employed state-of-the-art static and MAS pulsed NMR methods relevant for biological samples with rotational correlation times exceeding 100's of nanoseconds. Recent developments in signal filtering approaches, proton methodologies, and multiple acquisition techniques to boost sensitivity and speed up data acquisition at fast MAS are also discussed. Several examples of protein structures (globular, membrane, fibrils, and assemblies) solved with ssNMR spectroscopy have been considered. We also discuss integrated approaches to structurally characterize challenging biological systems and some newly emanating subdisciplines in ssNMR spectroscopy.

Separating an overlapped 1H peak and identifying its 1H-1H correlations with the use of single-channel 1H solid-state NMR at fast MAS

Fast magic-angle spinning (≥60 ​kHz) technique has enabled the acquisition of high-resolution 1H NMR spectra of solid materials. However, the spectral interpretation is still difficult because the 1H peaks are overlapped due to the narrow chemical shift range and broad linewidths. An additional 13C or 14N or 1H dimension possibly addresses the issues of overlapped proton resonances, but it leads to the elongated experimental time. Herein, we introduce a single-channel 1H experiment to separate the overlapped 1H peak and identify its spatially proximal 1H–1H correlations. This sequence combines selective excitation, selective 1H–1H polarization transfer by selective recoupling of protons (SERP), and broadband 1H recoupling by back-to-back (BABA) recoupling sequences. The concept for 1H separation is based on (i) the selective excitation of a well-resolved 1H peak and (ii) the selective dipolar polarization transfer from this isolated 1H peak to one of the 1H peaks in the overlapped/poor resolution region by SERP and (iii) the detection of 1H–1H correlations from these two 1H peaks to other neighboring 1Hs by BABA. We demonstrated the applicability of this approach to identify overlapped peaks on two molecules, β-L-aspartyl-l-alanine and Pioglitazone.HCl. The sequence allows the clear observation of 1H–1H correlations from an overlapped 1H peak without an additional heteronuclear dimension and ensures efficient polarization transfers that leads to twelve fold reduction in experimental time compared to 14N edited experiments. The limitation and the conditions of applicability for this approach are discussed in detail.

Detection of remote proton-nitrogen correlations by 1H-detected 14N overtone solid-state NMR at fast MAS.

Detecting proton and nitrogen correlations in solid-state nuclear magnetic resonance (NMR) is important for the structural determination of biological and chemical systems. Recent advances in proton detection-based approaches under fast magic-angle spinning have facilitated the detection of 1H-14N correlations by solid-state NMR. However, observing remote 1H-14N correlations by these approaches is still a challenge, especially for 14N sites having large quadrupolar couplings. To address this issue, we introduce the 1H-14N overtone continuous wave rotational-echo saturation-pulse double-resonance (1H-14N OT CW-RESPDOR) sequence. Unlike regular 2D correlation experiments where the indirect dimension is recorded in the time domain, the 1H-14N OT CW-RESPDOR experiment is directly observed in the frequency domain. A set of 1H-14N OT CW-RESPDOR filtered 1H spectra is recorded at varying 14N OT frequencies. Thanks to the selective nature of the 14N OT pulse, the filtered 1H spectra appear only if the 14N OT frequency hits the positions of the 14N OT central band or one of the spinning sidebands. This set of filtered 1H spectra represents a 2D 1H-14N OT correlation map. We have also investigated the optimizable parameters for CW-RESPDOR and figured out that these parameters are not strictly needed for our working magnetic field of 14.1 T. Hence, the experiment is easy to set up and requires almost no optimization. We have demonstrated the experimental feasibility of 1H-14N OT CW-RESPDOR on monoclinic L-histidine and L-alanyl L-alanine. The remote 1H-14N correlations have been efficiently detected, no matter how large the 14N quadrupolar interaction is, and agree with the crystal structures. In addition, based on the remote 1H-14N correlations from the non-protonated 14N site of L-histidine, we can unambiguously distinguish the orthorhombic and monoclinic forms.

Accelerating the acquisition of high-resolution quadrupolar MQ/ST-HETCOR 2D spectra under fast MAS via 1H detection and through-space population transfers

Recently, we established an experimental setup protocol to perform the population transfer from half-integer quadrupolar spin to 1H nuclei under fast MAS in the context of MQ-HETCOR experiments. In this article, we further develop the high-resolution 2D HETCOR methods by ST-based approaches, making use of the sensitivity advantage of STMAS over its MQMAS counterpart. In a similar manner to the previous work, which utilized CP and RINEPT for the population transfer, we also demonstrate the experimental setup protocol for PRESTO. Using {23Na}-1H and {27Al}-1H spin systems of powder samples, we compare a series of MQ/ST-HETCOR 2D spectra to discuss the pros and cons of the distinct MQ/ST-based approaches for spin 3/2 and 5/2 nuclei, respectively. We also incorporate two experimental tricks to reduce the experimental time of such long 2D experiments, the Optimized Rotor-Synchronization (ORS) and the Non-Uniform Sampling (NUS), in the context of high-resolution spectra of half-integer quadrupolar spin nuclei.

A comparison of through-space population transfers from half-integer spin quadrupolar nuclei to 1H using MQ-HETCOR and MQ-SPAM-HETCOR under fast MAS

In this article, we compare the various schemes of magnetization transfer from half-integer quadrupolar spins to 1H nuclei and we establish an efficient protocol to perform these transfers under MQMAS high-resolution with the MQ-HETCOR and MQ-SPAM-HETCOR experiments under fast MAS. The MQMAS efficiencies are analyzed with SIMPSON simulations, and the CPMAS and RINEPT magnetization transfers are compared at 62.5 kHz MAS using {23Na}-1H and {27Al}-1H MQ-HETCOR and MQ-SPAM-HETCOR experiments performed on NaH2PO4, Na2HPO4, Na citrate dihydrate and ipa-AlPO-14 powder samples. We discuss the pros and cons of these approaches, aiming to record 2D spectra of the best possible quality under fast MAS. We also incorporate some experimental approaches to reduce the total experiment time of such long 2D experiments.

Determination of the chemical shift tensor anisotropy and asymmetry of strongly dipolar coupled protons under fast MAS

Orientationally-dependent interactions such as dipolar coupling, quadrupolar coupling, and chemical shift anisotropy (CSA) contain a wealth of spatial information that can be used to elucidate molecular conformations and dynamics. To determine the sign of the chemical shift tensor anisotropy parameter (δaniso), both the |m| ​= ​1 and |m| ​= ​2 components of the CSA need to be symmetry allowed, while the recoupling of the |m| ​= ​1 term is accompanied with the reintroduction of homonuclear dipolar coupling components. Therefore, previously suggested sequences which solely recouple the |m| ​= ​2 term cannot determine the sign a 1H's δaniso in a densely-coupled network. In this study, we demonstrate the CSA recoupling of strongly dipolar coupled 1H spins using the Cnn1(9003601805400360180900) sequence. This pulse scheme recouples both the |m| ​= ​1 and |m| ​= ​2 CSA terms but the scaling factors for the homonuclear dipolar coupling terms are zeroed. Consequently, the sequence is sensitive to the sign of δaniso but is not influenced by homonuclear dipolar interactions.

Efficient symmetry-based γ-encoded DQ recoupling sequences for suppression of t1-noise in solid-state NMR spectroscopy at fast MAS

Solid-state NMR spectroscopy has played a significant role in elucidating the structure and dynamics of materials and biological solids at a molecular level for decades. In particular, the 1H double-quantum/single-quantum (DQ/SQ) chemical shift correlation experiment is widely used for probing the proximity of protons, rendering it a powerful tool for elucidating the hydrogen-bonding interactions and molecular packing of various complex molecular systems. Two factors, namely, the DQ filtering efficiency and t1-noise, dictate the quality of the 2D 1H DQ/SQ spectra. Experimentally different recoupling sequences show varied DQ filtering efficiencies and t1-noise. Herein, after a systematic search of symmetry-based DQ recoupling sequences, we report that the symmetry-based γ-encoded RNnν sequences show superior performance to other DQ recoupling sequences, which not only have a higher DQ recoupling efficiency but can also significantly reduce t1-noise. The origin of t1-noise is further discussed in detail via extensive numerical simulations. We envisage that such γ-encoded RNnν sequences are superior candidates for DQ recoupling in proton-based solid-state NMR spectroscopy due to its capability of efficiently exciting DQ coherences and suppressing t1-noise.

Proton-detected solid-state NMR spectroscopy of spin-1/2 nuclei with large chemical shift anisotropy

Constant-time (CT) dipolar heteronuclear multiple quantum coherence (D-HMQC) has previously been demonstrated as a method for proton detection of high-resolution wideline NMR spectra of spin-1/2 nuclei with large chemical shift anisotropy (CSA). However, 1H transverse relaxation and t1-noise often reduce the sensitivity of D-HMQC experiments, preventing the theoretical gains in sensitivity provided by 1H detection from being realized. Here we demonstrate a series of improved pulse sequences for 1H detection of spin-1/2 nuclei under fast MAS, with 195Pt SSNMR experiments on cisplatin as an example. First, a t1-incrementation protocol for D-HMQC dubbed Arbitrary Indirect Dwell (AID) is demonstrated. AID allows the use of arbitrary, rotor asynchronous t1-increments, but removes the constant time period from CT D-HMQC, resulting in improved sensitivity by reducing transverse relaxation losses. Next, we show that short high-power adiabatic pulses (SHAPs), which efficiently invert broad MAS sideband manifolds, can be effectively incorporated into 1H detected symmetry-based resonance echo double resonance (S-REDOR) and t1-noise eliminated (TONE) D-HMQC experiments. The S-REDOR experiments with SHAPs provide approximately double the dipolar dephasing, as compared to experiments with rectangular inversion pulses. We lastly show that sensitivity and resolution can be further enhanced with the use of swept excitation pulses as well as adiabatic magic angle turning (aMAT).

Effective Hamiltonian and 1H-14N cross polarization/double cross polarization at fast MAS

In this work we investigate in detail the underlying spin-dynamics associated with 1H-14N double CP experiments under fast MAS, recently demonstrated by Carnevale et al. We employ matrix logarithm and Floquet theory to compute numerically the effective Hamiltonian associated to the time-dependent problem. Certain common features related to construction of effective Hamiltonians by both approaches are discussed. The main observations related to 1H-14N CPMAS/double CP transfer are: (a) various spin terms of the effective Hamiltonian strongly depend on the crystallite orientation; (b) significant CP transfer occurs only when the magnitudes of the effective1H and 14N RF strengths are comparable, and simultaneously all pure 14N terms in the effective Hamiltonian are small, except for the longitudinal and the RF terms; (c) the sign of 14N CPMAS signal follows the sign of 14N effective RF strength; (d) sign of the double CP signal is largely independent of crystallite orientation. We predict and verify matching conditions employing multiples of the spinning frequency or involving different 14N RF strengths. We provide an analytical proof for (d). The proof also provides an estimate for the ratio of 1H-14N and 14N-1H transfer amplitudes which is further substantiated through simulations. In addition, we find that double CP signals include contributions from several single-quantum coherences present after the first CP process. The uneven contribution from different coherences leads to a reversal of signal at very short contact times, a feature noted experimentally by Carnevale et al. The connection between CPMAS transfer and efficient spin-lock is discussed and illustrated. The factors affecting second-order quadrupolar lineshapes in double CP experiment are examined. With a linear ramp of 1H RF amplitude we have observed that significant CP transfer occurs for more crystallite orientations resulting in improved sensitivity.

DiSupLo - New extremely easy and efficient method for loading of active pharmaceutical ingredients into the pores of MCM-41 mesoporous silica particles

Abstract DiSupLo (Diffusion Supported Loading) is acronym defining the method of loading of APIs into the pores of mesoporous silica particles (MSP). In this approach the starting material, homogenized physical mixture of two or more components (APIs/MSP) is placed in the closed vessel containing the ethanol. In the first stage of the process, there is no direct contact between solid mixture and liquid solvent, the only one is between the diffusing vapors of ethanol and solid matter. In the second stage, the vapors penetrate the solid state mixture in the whole volume, locally condense and dissolve the API. On this stage API dissolved in the minimal volume of solvent is transported to the pores of MSP. The applicability of method is proved employing selected APIs - Ibuprofen, Flurbiprofen, Ketoprofen, their mixtures and MCM-41 as MSP drug carrier. DiSupLo is quantitative process with high filling factor reaching value much higher compared to classical “wet methods”. Advances Solid State NMR techniques with sample spinning 60 kHz (1H Very Fast MAS, 2D 1H–1H RFDR MAS, 2D 1H–13C invHETCOR), 1H–13C HSQC High Resolution (HR) MAS as well adsorption-desorption, PXRD, DSC and FTIR were employed for analysis of loading and interactions of API's inside the pores.

Can proton-proton recoupling in fully protonated solids provide quantitative, selective and efficient polarization transfer?

Dipolar recoupling sequences have been used to probe spatial proximity of nuclear spins and were traditionally designed to probe rare spins such as 13C and/or 15N nuclei. The multi-spin dipolar-coupling network of the rare spins is weak due to smaller couplings and large chemical shift dispersion. Therefore, the recoupling approaches were tailored to design offset compensated or broadband sequences. In contrast, protons have a substantially stronger dipolar-coupling network and much narrower chemical shift range. Broadband recoupling sequences such as radio-frequency driven recoupling (RFDR), back-to-back (BABA), and lab frame proton-proton spin diffusion have been routinely used to characterize the structures of protein/macromolecules and small molecules. Recently selective 1H-1H recoupling sequences have been proposed that combine chemical shift offset of the resolved proton spectrum (at fast MAS) with first- and second-order dipolar recoupling Hamiltonians to obtain quantitative and qualitative proton distances, respectively. Herein, we evaluate the performances of broadband and selective proton recoupling sequences such as finite pulse RFDR (fp-RFDR), band-selective spectral spin diffusion (BASS-SD), second-order cross-polarization (SOCP), and selective recoupling of proton (SERP) in terms of the selectivity and efficiency of 1H-1H polarization transfers in a dense network of proton spins and explore the possibility of measuring 1H-1H distances. We use theoretical considerations, numerical simulations, and experiments to support the distinct advantages and disadvantages of each recoupling sequence. Experiments were performed on L-histidine.HCl.H2O at a MAS frequency of 71.43 kHz. This study rationalizes the proper selection of 1H-1H recoupling sequences when working with fully protonated solids.

Sensitivity enhancement in 2D Double Cross Polarization experiments under fast MAS by recycling unused protons

We demonstrate sensitivity enhancement via recycling of proton magnetization in 2D Double Cross Polarization (Double CP) experiments performed on fully protonated and uniformly labeled (13C, 15N) samples at a magic angle spinning rate of 60 kHz. Unused proton magnetization is preserved during t1 evolution either by locking it with CW irradiation or by employing rotor-synchronized pi pulses. A flip-back pulse together with a modified second CP block preserves unused proton magnetization resulting in enhanced sensitivity. We have achieved sensitivity enhancements of 15–20% and 25–28% in 1H–13C and 1H–15N 2D Double CP experiments respectively. At shorter recycle delays (∼0.25T1), relative sensitivity enhancements of 40–45% and 55% were obtained in 1H–13C and 1H–15N 2D Double CP experiments respectively. An analysis of the sensitivity enhancements and theoretical estimation of lineshapes in indirect dimension in the presence of proton recycling is provided.