Atomic-resolution chemical characterization of (2x)72-kDa tryptophan synthase via four- and five-dimensional 1H-detected solid-state NMR.

NMR chemical shifts provide detailed information on the chemical properties of molecules, thereby complementing structural data from techniques like X-ray crystallography and electron microscopy. Detailed analysis of protein NMR data, however, often hinges on comprehensive, site-specific assignment of backbone resonances, which becomes a bottleneck for molecular weights beyond 40 to 45 kDa. Here, we show that assignments for the (2x)72-kDa protein tryptophan synthase (665 amino acids per asymmetric unit) can be achieved via higher-dimensional, proton-detected, solid-state NMR using a single, 1-mg, uniformly labeled, microcrystalline sample. This framework grants access to atom-specific characterization of chemical properties and relaxation for the backbone and side chains, including those residues important for the catalytic turnover. Combined with first-principles calculations, the chemical shifts in the ß-subunit active site suggest a connection between active-site chemistry, the electrostatic environment, and catalytically important dynamics of the portal to the ß-subunit from solution.

Carbon-13 chemical shift tensor measurements for nitrogen-dense compounds.

This paper reports the principal values of the 13 C chemical shift tensors for five nitrogen-dense compounds (i.e., cytosine, uracil, imidazole, guanidine hydrochloride, and aminoguanidine hydrochloride). Although these are all fundamentally important compounds, the majority do not have 13 C chemical shift tensors reported in the literature. The chemical shift tensors are obtained from 1 H→13 C cross-polarization magic-angle spinning (CP/MAS) experiments that were conducted at a high field of 18.8 T to suppress the effects of 14 N-13 C residual dipolar coupling. Quantum chemical calculations using density functional theory are used to obtain the 13 C magnetic shielding tensors for these compounds. The best agreement with experiment arises from calculations using the hybrid functional PBE0 or the double-hybrid functional PBE0-DH, along with the triple-zeta basis sets TZ2P or pc-3, respectively, and intermolecular effects modeled using large clusters of molecules with electrostatic embedding through the COSMO approach. These measurements are part of an ongoing effort to expand the catalog of accurate 13 C chemical shift tensor measurements, with the aim of creating a database that may be useful for benchmarking the accuracy of quantum chemical calculations, developing nuclear magnetic resonance (NMR) crystallography protocols, or aiding in applications involving machine learning or data mining. This work was conducted at the National High Magnetic Field Laboratory as part of a 2-week school for introducing undergraduate students to practical laboratory experience that will prepare them for scientific careers or postgraduate studies.

Zwitterionic or Not? Fast and Reliable Structure Determination by Combining Crystal Structure Prediction and Solid-State NMR.

When it comes to crystal structure determination, computational approaches such as Crystal Structure Prediction (CSP) have gained more and more attention since they offer some insight on how atoms and molecules are packed in the solid state, starting from only very basic information without diffraction data. Furthermore, it is well known that the coupling of CSP with solid-state NMR (SSNMR) greatly enhances the performance and the accuracy of the predictive method, leading to the so-called CSP-NMR crystallography (CSP-NMRX). In this paper, we present the successful application of CSP-NMRX to determine the crystal structure of three structural isomers of pyridine dicarboxylic acid, namely quinolinic, dipicolinic and dinicotinic acids, which can be in a zwitterionic form, or not, in the solid state. In a first step, mono- and bidimensional SSNMR spectra, i.e., 1H Magic-Angle Spinning (MAS), 13C and 15N Cross Polarisation Magic-Angle Spinning (CPMAS), 1H Double Quantum (DQ) MAS, 1H-13C HETeronuclear CORrelation (HETCOR), were used to determine the correct molecular structure (i.e., zwitterionic or not) and the local molecular arrangement; at the end, the RMSEs between experimental and computed 1H and 13C chemical shifts allowed the selection of the correct predicted structure for each system. Interestingly, while quinolinic and dipicolinic acids are zwitterionic and non-zwitterionic, respectively, in the solid state, dinicotinic acid exhibits in its crystal structure a "zwitterionic-non-zwitterionic continuum state" in which the proton is shared between the carboxylic moiety and the pyridinic nitrogen. Very refined SSNMR experiments were carried out, i.e., 14N-1H Phase-Modulated (PM) pulse and Rotational-Echo Saturation-Pulse Double-Resonance (RESPDOR), to provide an accurate N-H distance value confirming the hybrid nature of the molecule. The CSP-NMRX method showed a remarkable match between the selected structures and the experimental ones. The correct molecular input provided by SSNMR reduced the number of CSP calculations to be performed, leading to different predicted structures, while RMSEs provided an independent parameter with respect to the computed energy for the selection of the best candidate.

Solid-State NMR-Driven Crystal Structure Prediction of Molecular Crystals: The Case of Mebendazole.

Among all possible NMR crystallography approaches for crystal-structure determination, crystal structure prediction - NMR crystallography (CSP-NMRX) has recently turned out to be a powerful method. In the latter, the original procedure exploited solid-state NMR (SSNMR) information during the final steps of the prediction. In particular, it used the comparison of computed and experimental chemical shifts for the selection of the correct crystal packing. Still, the prediction procedure, generally carried out with DFT methods, may require important computational resources and be quite time-consuming, especially if there are no available constraints to use at the initial stage. Herein, the successful application of this combined prediction method, which exploits NMR information also in the input step to reduce the search space of the predictive algorithm, is presented. Herein, this method was applied on mebendazole, which is characterized by desmotropism. The use of SSNMR data as constraints for the selection of the right tautomer and the determination of the number of independent molecules in the unit cell led to a considerably faster process, reducing the number of calculations to be performed. In this way, the crystal packing was successfully predicted for the three known phases of mebendazole. To evaluate the quality of the predicted structures, these were compared to the experimental ones. The crystal structure of phase B of mebendazole, in particular, was determined de novo by powder diffraction and is presented for the first time in this paper.

"Crystal memory" Affects the Properties of Peptide Hydrogels - The Case of the Cyclic Tyr-Tyr dipeptide.

In this work a relationship between the crystal form and morphology and rheological properties of peptide-based hydrogels is examined. We show, that under favorable circumstances a correlation between a starting solid material and a self-assembly processes in solution can exist, leading to different properties of a resulting soft matter. This observation, together with an in-depth analysis of the influence of stereochemistry of self-assembled (ll) and (dl) Tyr-Tyr cyclic dipeptides (cYY) on the observed relationship between gelation and crystallization allowed us to gain a deeper understanding of the peptide hydrogelation processes at a molecular level, using liquid state NMR, rheological studies and scanning electron microscopy. In the course of our studies, several crystal forms of (ll)-cYY has been discovered and described in details using single crystal X-ray diffraction, as well as advanced solid state NMR, X-ray diffraction of powders, thermal analysis, FTIR, circular dichroism and crystal structure prediction (CSP) calculations. Subsequently, we found that while (ll)-cYY easily assembles into hydrogels with different properties depending on the starting solid form, (dl)-cYY always precipitated as one crystal form in the tested conditions. Molecular-level justification for this observation is given.

Combining heteronuclear correlation NMR with spin-diffusion to detect relayed Cl-H-H and N-H-H proximities in molecular solids.

Analysis of short-to-intermediate range intermolecular interactions offers a great way of characterizing the solid-state organization of small molecules and materials. This can be achieved by two-dimensional (2D) homo- and heteronuclear correlation NMR spectroscopy, for example, by carrying out experiments at high magnetic fields in conjunction with fast magic-angle spinning (MAS) techniques. But, detecting 2D peaks for heteronuclear dipolar coupled spin pairs separated by greater than 3 Å is not always straightforward, particularly when low-gamma quadrupolar nuclei are involved. Here, we present a 2D correlation NMR experiment that combines the advantages of heteronuclear-multiple quantum coherence (HMQC) and proton-based spin-diffusion (SD) pulse sequences using radio-frequency-driven-recouping (RFDR) to probe inter and intramolecular 1H-X (X = 14N, 35Cl) interactions. This experiment can be used to acquire 2D 1H{X}-HMQC filtered 1H-1H correlation as well as 2D 1H-X HMQC spectra. Powder forms of dopamine·HCl and l-histidine·HCl·H2O are characterized at high fields (21.1 T and 18.8 T) with fast MAS (60 kHz) using the 2D HMQC-SD-RFDR approach. Solid-state NMR results are complemented with NMR crystallography analyses using the gauge-including projector augmented wave (GIPAW) approach. For histidine·HCl·H2O, 2D peaks associated with 14N-1H-1H and 35Cl-1H-1H distances of up to 4.4 and 3.9 Å have been detected. This is further corroborated by the observation of 2D peaks corresponding to 14N-1H-1H and 35Cl-1H-1H distances of up to 4.2 and 3.7 Å in dopamine·HCl, indicating the suitability of the HMQC-SD-RFDR experiments for detecting medium-range proximities in molecular solids.

Narrowing down the conformational space with solid-state NMR in crystal structure prediction of linezolid cocrystals.

Many solids crystallize as microcrystalline powders, thus precluding the application of single crystal X-Ray diffraction in structural elucidation. In such cases, a joint use of high-resolution solid-state NMR and crystal structure prediction (CSP) calculations can be successful. However, for molecules showing significant conformational freedom, the CSP-NMR protocol can meet serious obstacles, including ambiguities in NMR signal assignment and too wide conformational search space to be covered by computational methods in reasonable time. Here, we demonstrate a possible way of avoiding these obstacles and making as much use of the two methods as possible in difficult circumstances. In a simple case, our experiments led to crystal structure elucidation of a cocrystal of linezolid (LIN), a wide-range antibiotic, with 2,3-dihydroxybenzoic acid, while a significantly more challenging case of a cocrystal of LIN with 2,4-dihydroxybenzoic acid led to the identification of the most probable conformations of LIN inside the crystal. Having four rotatable bonds, some of which can assume many discreet values, LIN molecule poses a challenge in establishing its conformation in a solid phase. In our work, a set of 27 conformations were used in CSP calculations to yield model crystal structures to be examined against experimental solid-state NMR data, leading to a reliable identification of the most probable molecular arrangements.

Complete resonance assignment of a pharmaceutical drug at natural isotopic abundance from DNP-Enhanced solid-state NMR.

Solid-state dynamic nuclear polarization enhanced magic angle spinning (DNP-MAS) NMR measurements coupled with density functional theory (DFT) calculations enable the full resonance assignment of a complex pharmaceutical drug molecule without the need for isotopic enrichment. DNP dramatically enhances the NMR signals, thereby making possible previously intractable two-dimensional correlation NMR spectra at natural abundance. Using inputs from DFT calculations, herein we describe a significant improvement to the structure elucidation process for complex organic molecules. Further, we demonstrate that a series of two-dimensional correlation experiments, including 15N-13C TEDOR, 13C-13C INADEQUATE/SARCOSY, 19F-13C HETCOR, and 1H-13C HETCOR, can be obtained at natural isotopic abundance within reasonable experiment times, thus enabling a complete resonance assignment of sitagliptin, a pharmaceutical used for the treatment of type 2 diabetes.

NMR Crystallography Reveals Carbonate Induced Al-Ordering in ZnAl Layered Double Hydroxide.

Layered double hydroxides (LDHs) serve a score of applications in catalysis, drug delivery, and environmental remediation. Smarter crystallography, combining X-ray diffraction and NMR spectroscopy revealed how interplay between carbonate and pH determines the LDH structure and Al ordering in ZnAl LDH. Carbonate intercalated ZnAl LDHs were synthesized at different pH (pH 8.5, pH 10.0, pH 12.5) with a Zn/Al ratio of 2, without subsequent hydrothermal treatment to avoid extensive recrystallisation. In ideal configuration, all Al cations should be part of the LDH and be coordinated with 6 Zn atoms, but NMR revealed two different Al local environments were present in all samples in a ratio dependent on synthesis pH. NMR-crystallography, integrating NMR spectroscopy and X-ray diffraction, succeeded to identify them as Al residing in the highly ordered crystalline phase, next to Al in disordered material. With increasing synthesis pH, crystallinity increased, and the side phase fraction decreased. Using 1 H-13 C, 13 C-27 Al HETCOR NMR in combination with 27 Al MQMAS, 27 Al-DQ-SQ measurements and Rietveld refinement on high-resolution PXRD data, the extreme anion exchange selectivity of these LDHs for CO3 2- over HCO3 - was linked to strict Al and CO3 2- ordering in the crystalline LDH. Even upon equilibration of the LDH in pure NaHCO3 solutions, only CO3 2- was adsorbed by the LDH. This reveals the structure directing role of bivalent cations such as CO3 2- during crystallization of [M2+ 4 M3+ 2 (OH)2 ]2+ [A2- ]1 ⋅yH2 O LDH phases.

Crystal Engineering of Supramolecular 1,4-Benzene Bisamides by Side-Chain Modification - Towards Tuneable Anisotropic Morphologies and Surfaces.

Benzene bisamides are promising building blocks for supramolecular nano-objects. Their functionality depends on morphology and surface properties. However, a direct link between surface properties and molecular structure itself is missing for this material class. Here, we investigate this interplay for two series of 1,4-benzene bisamides with symmetric and asymmetric peripheral substitution. We elucidated the crystal structures, determined the nano-object morphologies and derived the wetting behaviour of the preferentially exposed surfaces. The crystal structures were solved by combining single-crystal and powder X-ray diffraction, solid-state NMR spectroscopy and computational modelling. Bulky side groups, here t-butyl groups, serve as a structure-directing motif into a packing pattern, which favours the formation of thin platelets. The use of slim peripheral groups on both sides, in our case linear perfluorinated, alkyl chains, self-assemble the benzene bisamides into a second packing pattern which leads to ribbon-like nano-objects. For both packing types, the preferentially exposed surfaces consist of the ends of the peripheral groups. Asymmetric substitution with bulky and slim groups leads to an ordered alternating arrangement of the groups exposed to the surface. This allows the hydrophobicity of the surfaces to be gradually altered. We thus identified two leitmotifs for molecular packings of benzene bisamides providing the missing link between the molecular structure, the anisotropic morphologies and adjustable surface properties of the supramolecular nano-objects.

A toolbox for improving the workflow of NMR crystallography.

NMR crystallography is a powerful tool with applications in structural characterization and crystal structure verification, to name two. However, applying this tool presents several challenges, especially for industrial users, in terms of consistency, workflow, time consumption, and the requirement for a high level of understanding of experimental solid-state NMR and GIPAW-DFT calculations. Here, we have developed a series of fully parameterized scripts for use in Materials Studio and TopSpin, based on the .magres file format, with a focus on organic molecules (e.g. pharmaceuticals), improving efficiency, robustness, and workflow. We separate these tools into three major categories: performing the DFT calculations, extracting & visualizing the results, and crystallographic modelling. These scripts will rapidly submit fully parameterized CASTEP jobs, extract data from the calculations, assist in visualizing the results, and expedite the process of structural modelling. Accompanied with these tools is a description on their functionality, documentation on how to get started and use the scripts, and links to video tutorials for guiding new users. Through the use of these tools, we hope to facilitate NMR crystallography and to harmonize the process across users.

17 O solid-state NMR at ultrahigh magnetic field of 35.2 T: Resolution of inequivalent oxygen sites in different phases of MOF MIL-53(Al).

MIL-53(Al) is a member of the most extensively studied metal-organic framework (MOF) families owing to its "flexible" framework and superior stability. 17 O solid-state NMR (SSNMR) spectroscopy is an ideal site-specific characterization tool as it probes local oxygen environments. Because oxygen local structure is often altered during phase change, 17 O SSNMR can be used to follow phase transitions. However, 17 O is a challenging nucleus to study via SSNMR due to its low sensitivity and resolution arising from the very low natural abundance of 17 O isotope and its quadrupolar nature. In this work, we describe that by using 17 O isotopic enrichment and performing 17 O SSNMR experiments at an ultrahigh magnetic field of 35.2 T, all chemically and crystallographically inequivalent oxygen sites in two representative MIL-53(Al) (as-made and water adsorbed) phases can be completely resolved. The number of signals in each phase is consistent with that predicted from the space group refined from powder X-ray diffraction data. The 17 O 1D magic-angle spinning (MAS) and 2D triple-quantum MAS (3QMAS) spectra at 35.2 T furnish fine information about the host-guest interactions and the structural changes associated with phase transition. The ability to completely resolve multiple chemically and crystallographically inequivalent oxygen sites in MOFs at very high magnetic field, as illustrated in this work, significantly enhances the potential for using the NMR crystallography approach to determine crystal structures of new MOFs and verify the structures of existing MOFs obtained from refining powder X-ray diffraction data.

Structure Solution of Nano-Crystalline Small Molecules Using MicroED and Solid-State NMR Dipolar-Based Experiments.

Three-dimensional electron diffraction crystallography (microED) can solve structures of sub-micrometer crystals, which are too small for single crystal X-ray crystallography. However, R factors for the microED-based structures are generally high because of dynamic scattering. That means R factor may not be reliable provided that kinetic analysis is used. Consequently, there remains ambiguity to locate hydrogens and to assign nuclei with close atomic numbers, like carbon, nitrogen, and oxygen. Herein, we employed microED and ssNMR dipolar-based experiments together with spin dynamics numerical simulations. The NMR dipolar-based experiments were 1H-14N phase-modulated rotational-echo saturation-pulse double-resonance (PM-S-RESPDOR) and 1H-1H selective recoupling of proton (SERP) experiments. The former examined the dephasing effect of a specific 1H resonance under multiple 1H-14N dipolar couplings. The latter examined the selective polarization transfer between a 1H-1H pair. The structure was solved by microED and then validated by evaluating the agreement between experimental and calculated dipolar-based NMR results. As the measurements were performed on 1H and 14N, the method can be employed for natural abundance samples. Furthermore, the whole validation procedure was conducted at 293 K unlike widely used chemical shift calculation at 0 K using the GIPAW method. This combined method was demonstrated on monoclinic l-histidine.

Fast Acquisition of Proton-Detected HETCOR Solid-State NMR Spectra of Quadrupolar Nuclei and Rapid Measurement of NH Bond Lengths by Frequency Selective HMQC and RESPDOR Pulse Sequences.

Fast magic-angle spinning (MAS), frequency selective (FS) heteronuclear multiple quantum coherence (HMQC) experiments which function in an analogous manner to solution SOFAST HMQC NMR experiments, are demonstrated. Fast MAS enables efficient FS excitation of 1 H solid-state NMR signals. Selective excitation and observation preserves 1 H magnetization, leading to a significant shortening of the optimal inter-scan delay. Dipolar and scalar 1 H{14 N} FS HMQC solid-state NMR experiments routinely provide 4- to 9-fold reductions in experiment times as compared to conventional 1 H{14 N} HMQC solid-state NMR experiments. 1 H{14 N} FS resonance-echo saturation-pulse double-resonance (RESPDOR) allowed dipolar dephasing curves to be obtained in minutes, enabling the rapid determination of NH dipolar coupling constants and internuclear distances. 1 H{14 N} FS RESPDOR was used to assign multicomponent active pharmaceutical ingredients (APIs) as salts or cocrystals. FS HMQC also provided enhanced sensitivity for 1 H{17 O} and 1 H{35 Cl} HMQC experiments on 17 O-labeled Fmoc-alanine and histidine hydrochloride monohydrate, respectively. FS HMQC and FS RESPDOR experiments will provide access to valuable structural constraints from materials that are challenging to study due to unfavorable relaxation times or dilution of the nuclei of interest.

Centerband-Only Detection of Exchange NMR with Natural-Abundance Correction Reveals an Expanded Unit Cell in Phenylalanine Crystals.

The NMR pulse sequence CODEX (centerband-only detection of exchange) is a widely used method to report on the number of magnetically inequivalent spins that exchange magnetization via spin diffusion. For crystals, this rules out certain symmetries, and the rate of equilibration is sensitive to distances. Here we show that for 13 C CODEX, consideration of natural abundance spins is necessary for crystals of high complexity, demonstrated here with the amino acid phenylalanine. The NMR data rule out the C2 space group that was originally reported for phenylalanine, and are only consistent with a larger unit cell containing eight magnetically inequivalent molecules. Such an expanded cell was recently described based on single crystal data. The large unit cell dictates the use of long spin diffusion times of more than 200 seconds, in order to equilibrate over the entire unit cell volume of 1622 Å3 .

Ultrafast 1H MAS NMR Crystallography for Natural Abundance Pharmaceutical Compounds.

Magic angle spinning (MAS) NMR is a powerful method for the study of pharmaceutical compounds, and probes with spinning frequencies above 100 kHz enable an atomic-resolution analysis of sub-micromole quantities of fully protonated solids. Here, we present an ultrafast NMR crystallography approach for structural characterization of organic solids at MAS frequencies of 100-111 kHz. We assess the efficiency of 1H-detected experiments in the solid state and demonstrate the utility of 2D and 3D homo- and heteronuclear correlation spectra for resonance assignments. These experiments are demonstrated for an amino acid, U-13C,15N-histidine, and also for the significantly larger, natural product Posaconazole, an antifungal compound investigated at natural abundance. Our results illustrate the power for characterizing organic molecules, enabled by exploiting the increased 1H resolution and sensitivity at MAS frequencies above 100 kHz.

A GIPAW versus GIAO-ZORA-SO study of 13C and 15N CPMAS NMR chemical shifts of aromatic and heterocyclic bromo derivatives.

Theoretical simulation of NMR parameters in compounds bearing heavy atoms generally requires the application of relativistic corrections. We report herein the theoretical characterization of 13C and 15N CPMAS NMR of known bromo-derivative crystals by using both the GIPAW and the combined GIAO-ZORA-SO approximation methods. Several statistical analyses were performed to compare both approaches, with non-relativistic GIPAW method being more useful to predict the 13C and 15N chemical shifts. The problem of applying GIPAW to crystal structures showing static or dynamic crystalline disorder of the special class resulting in half-protons will be discussed in detail.

5-amino-2-methylpyridinium hydrogen fumarate: An XRD and NMR crystallography analysis.

Single-crystal X-ray diffraction structures of the 5-amino-2-methylpyridinium hydrogen fumarate salt have been solved at 150 and 300 K (CCDC 1952142 and 1952143). A base-acid-base-acid ring is formed through pyridinium-carboxylate and amine-carboxylate hydrogen bonds that hold together chains formed from hydrogen-bonded hydrogen fumarate ions. 1 H and 13 C chemical shifts as well as 14 N shifts that additionally depend on the quadrupolar interaction are determined by experimental magic angle spinning (MAS) solid-state nuclear magnetic resonance (NMR) and gauge-including projector-augmented wave (GIPAW) calculation. Two-dimensional homonuclear 1 H-1 H double-quantum (DQ) MAS and heteronuclear 1 H-13 C and 14 N-1 H spectra are presented. Only small differences of up to 0.1 and 0.6 ppm for 1 H and 13 C are observed between GIPAW calculations starting with the two structures solved at 150 and 300 K (after geometry optimisation of atomic positions, but not unit cell parameters). A comparison of GIPAW-calculated 1 H chemical shifts for isolated molecules and the full crystal structures is indicative of hydrogen bonding strength.

Identifying aspirin polymorphs from combined DFT-based crystal structure prediction and solid-state NMR.

A combined experimental and computational approach was used to distinguish between different polymorphs of the pharmaceutical drug aspirin. This method involves the use of ab initio random structure searching (AIRSS), a density functional theory (DFT)-based crystal structure prediction method for the high-accuracy prediction of polymorphic structures, with DFT calculations of nuclear magnetic resonance (NMR) parameters and solid-state NMR experiments at natural abundance. AIRSS was used to predict the crystal structures of form-I and form-II of aspirin. The root-mean-square deviation between experimental and calculated 1 H chemical shifts was used to identify form-I as the polymorph present in the experimental sample, the selection being successful despite the large similarities between the molecular environments in the crystals of the two polymorphs.

Polymorphic Forms of Valinomycin Investigated by NMR Crystallography.

A dodecadepsipeptide valinomycin (VLM) has been most recently reported to be a potential anti-coronavirus drug that could be efficiently produced on a large scale. It is thus of importance to study solid-phase forms of VLM in order to be able to ensure its polymorphic purity in drug formulations. The previously available solid-state NMR (SSNMR) data are combined with the plane-wave DFT computations in the NMR crystallography framework. Structural/spectroscopical predictions (the PBE functional/GIPAW method) are obtained to characterize four polymorphs of VLM. Interactions which confer a conformational stability to VLM molecules in these crystalline forms are described in detail. The way how various structural factors affect the values of SSNMR parameters is thoroughly analyzed, and several SSNMR markers of the respective VLM polymorphs are identified. The markers are connected to hydrogen bonding effects upon the corresponding (13C/15N/1H) isotropic chemical shifts of (CO, Namid, Hamid, Hα) VLM backbone nuclei. These results are expected to be crucial for polymorph control of VLM and in probing its interactions in dosage forms.