Solid-state NMR backbone chemical shift assignments of α-synuclein amyloid fibrils at fast MAS regime.

The α-synuclein (α-syn) amyloid fibrils are involved in various neurogenerative diseases. Solid-state NMR (ssNMR) has been showed as a powerful tool to study α-syn aggregates. Here, we report the 1H, 13C and 15N back-bone chemical shifts of a new α-syn polymorph obtained using proton-detected ssNMR spectroscopy under fast (95 kHz) magic-angle spinning conditions. The manual chemical shift assignments were cross-validated using FLYA algorithm. The secondary structural elements of α-syn fibrils were calculated using 13C chemical shift differences and TALOS software.

Influence of heterochirality on the structure, dynamics, biological properties of cyclic(PFPF) tetrapeptides obtained by solvent-free ball mill mechanosynthesis.

Cyclic tetrapeptides c(Pro-Phe-Pro-Phe) obtained by the mechanosynthetic method using a ball mill were isolated in a pure stereochemical form as a homochiral system (all L-amino acids, sample A) and as a heterochiral system with D configuration at one of the stereogenic centers of Phe (sample B). The structure and stereochemistry of both samples were determined by X-ray diffraction studies of single crystals. In DMSO and acetonitrile, sample A exists as an equimolar mixture of two conformers, while only one is monitored for sample B. The conformational space and energetic preferences for possible conformers were calculated using DFT methods. The distinctly different conformational flexibility of the two samples was experimentally proven by Variable Temperature (VT) and 2D EXSY NMR measurements. Both samples were docked to histone deacetylase HDAC8. Cytotoxic studies proved that none of the tested cyclic peptide is toxic.

Solvent-Free Mechanosynthesis of Oligopeptides by Coupling Peptide Segments of Different Lengths - Elucidating the Role of Cesium Carbonate in Ball Mill Processes.

We report an idea for the synthesis of oligopeptides using a solvent-free ball milling approach. Our concept is inspired by block play, in which it is possible to construct different objects using segments (blocks) of different sizes and lengths. We prove that by having a library of short peptides and employing the ball mill mechanosynthesis (BMMS) method, peptides can be easily coupled to form different oligopeptides with the desired functional and biological properties. Optimizing the BMMS process we found that the best yields we obtained when TBTU and cesium carbonate were used as reagents. The role of Cs2CO3 in the coupling mechanism was followed on each stage of synthesis by 1H, 13C and 133Cs NMR employing Magic Angle Spinning (MAS) techniques. It was found that cesium carbonate acts not only as a base but is also responsible for the activation of substrates and intermediates. The unique information about the BMMS mechanism is based on the analysis of 2D NMR data. The power of BMMS is proved by the example of different peptide combinations, 2+2, 3+2, 4+2, 5+2 and 4+4. The tetra-, penta-, hexa-, hepta- and octapeptides obtained under this project were fully characterized by MS and NMR techniques.

Arsenic(III) oxide intercalate with ammonium chloride: crystal structure revision and thermal characterization.

According to the crystal structure determination by Edstrand & Blomqvist [Ark. Kemi (1955), 8, 245-256], intercalate NH4Cl·As2O3·0.5H2O ({\bf Y_{{NH}_{4}Cl}}) is not isostructural with compound KCl·As2O3·0.5H2O. This is very unlikely because both NH4Br·2As2O3 and KBr·2As2O3 as well as NH4I·2As2O3 and KI·2As2O3 are isostructural. Hence, intercalate {\bf Y_{{NH}_{4}Cl}} has been studied using single-crystal X-ray diffraction in addition to attenuated total reflection Fourier transform infrared (ATR-FTIR) and 15N solid-state magic-angle spinning nuclear magnetic resonance (ssNMR) spectroscopies. These techniques indicate that revising the previous crystal structure model is necessary. Compound {\bf Y_{{NH}_{4}Cl}} crystallizes in space group P6/mmm with unit-cell parameters a = 5.25420 (10) Šand c = 12.6308 (3) Šand is isostructural with KCl·As2O3·0.5H2O. The presence of two symmetry-independent ammonium cations in the structure has been unequivocally confirmed using 15N ssNMR spectroscopy. The 15N ssNMR spectrum of intercalate {\bf Y_{{NH}_{4}Cl}} has been compared with analogous spectra of NH4Br·2As2O3 and NH4I·2As2O3 which allowed for a probable assignment of signals to ammonium cations occupying particular sites in the crystal structures. Thermogravimetry, differential scanning calorimetry and variable-temperature ATR-FTIR spectra have revealed that intercalate {\bf Y_{{NH}_{4}Cl}} is dehydrated between 320 and 475 K. Upon cooling or standing in moist air water is re-absorbed. Dehydration leads to significant shortening of the c unit-cell parameter as revealed by powder X-ray diffraction [c = 12.1552 (7) Šat 293 K]. Compound {\bf Y_{{NH}_{4}Cl}} decomposes on prolonged heating above 490 K to arsenic(III) oxide and ammonium chloride.

New salts of teriflunomide (TFM) - Single crystal X-ray and solid state NMR investigation.

New salts of teriflunomide TFM (drug approved for Multiple Sclerosis treatment) with inorganic counterions: lithium (TFM_Li), sodium (TFM_Na), potassium (TFM_K), rubidium (TFM_Rb), caesium (TFM_Cs) and ammonium (TFM_NH4) were prepared and investigated employing solid state NMR Spectroscopy, Powder X-ray Diffraction PXRD and Single Crystal X-ray Diffraction (SC XRD). Crystal and molecular structures of three salts: TFM_Na (CCDC: 2173257), TFM_Cs (CCDC: 2165288) and TFM_NH4 (CCDC: 2165281) were determined and deposited. Compared to the native TFM, for all crystalline salt structures, a conformational change of the teriflunomide molecule involving about 180-degree rotation of the end group, forming an intramolecular hydrogen bond N-H⋯O is observed. By applying a complementary multi-technique approach, employing 1D and 2D solid state MAS NMR techniques, single and powder X-ray diffraction measurements, as well as the DFT-based GIPAW calculations of NMR chemical shifts for TFM_Na and TFM_Cs allowed to propose structural features of TFM_Li for which it was not possible to obtain adequate material for single crystal X-Ray measurement.

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.

1H-Detected Biomolecular NMR under Fast Magic-Angle Spinning.

Since the first pioneering studies on small deuterated peptides dating more than 20 years ago, 1H detection has evolved into the most efficient approach for investigation of biomolecular structure, dynamics, and interactions by solid-state NMR. The development of faster and faster magic-angle spinning (MAS) rates (up to 150 kHz today) at ultrahigh magnetic fields has triggered a real revolution in the field. This new spinning regime reduces the 1H-1H dipolar couplings, so that a direct detection of 1H signals, for long impossible without proton dilution, has become possible at high resolution. The switch from the traditional MAS NMR approaches with 13C and 15N detection to 1H boosts the signal by more than an order of magnitude, accelerating the site-specific analysis and opening the way to more complex immobilized biological systems of higher molecular weight and available in limited amounts. This paper reviews the concepts underlying this recent leap forward in sensitivity and resolution, presents a detailed description of the experimental aspects of acquisition of multidimensional correlation spectra with fast MAS, and summarizes the most successful strategies for the assignment of the resonances and for the elucidation of protein structure and conformational dynamics. It finally outlines the many examples where 1H-detected MAS NMR has contributed to the detailed characterization of a variety of crystalline and noncrystalline biomolecular targets involved in biological processes ranging from catalysis through drug binding, viral infectivity, amyloid fibril formation, to transport across lipid membranes.

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.

Hadamard acquisition of 13 C-13 C 2-D correlation NMR spectra.

We show that a multiselective excitation with Hadamard encoding is a powerful tool for 2-D acquisition of 13 C─13 C homonuclear correlations. This method is not designed to improve the sensitivity, but rather to reduce the experiment time, provided there is sufficient sensitivity. Therefore, it allows fast acquisition of such 2-D spectra in labeled molecules. The technique has been demonstrated using a U─13 C─15 N histidine hydrochloride monohydrate sample allowing each point of the build-up curves of the 13 C─13 C cross-peaks to be recorded within 4 min 35 s, which is very difficult with conventional methods. Using the U─13 C─15 N f-MLF sample, we have demonstrated that the method can be applied to molecules with 14 13 C resonances with a minimum frequency separation of 240 Hz.

Crystal structures of two furazidin polymorphs revealed by a joint effort of crystal structure prediction and NMR crystallography.

This work presents the crystal structure determination of two elusive polymorphs of furazidin, an antibacterial agent, employing a combination of crystal structure prediction (CSP) calculations and an NMR crystallography approach. Two previously uncharacterized neat crystal forms, one of which has two symmetry-independent molecules (form I), whereas the other one is a Z' = 1 polymorph (form II), crystallize in P21/c and P1 space groups, respectively, and both are built by different conformers, displaying different intermolecular interactions. It is demonstrated that the usage of either CSP or NMR crystallography alone is insufficient to successfully elucidate the above-mentioned crystal structures, especially in the case of the Z' = 2 polymorph. In addition, cases of serendipitous agreement in terms of 1H or 13C NMR data obtained for the CSP-generated crystal structures different from the ones observed in the laboratory (false-positive matches) are analyzed and described. While for the majority of analyzed crystal structures the obtained agreement with the NMR experiment is indicative of some structural features in common with the experimental structure, the mentioned serendipity observed in exceptional cases points to the necessity of caution when using an NMR crystallography approach in crystal structure determination.

Automated Backbone NMR Resonance Assignment of Large Proteins Using Redundant Linking from a Single Simultaneous Acquisition.

Thanks to magic-angle spinning (MAS) probes with frequencies of 60-100 kHz, the benefit of high-sensitivity 1H detection can now be broadly realized in biomolecular solid-state NMR for the analysis of microcrystalline, sedimented, or lipid-embedded preparations. Nonetheless, performing the assignment of all resonances remains a rate-limiting step in protein structural studies, and even the latest optimized protocols fail to perform this step when the protein size exceeds ∼20 kDa. Here, we leverage the benefits of fast (100 kHz) MAS and high (800 MHz) magnetic fields to design an approach that lifts this limitation. Through the creation, conservation, and acquisition of independent magnetization pathways within a single triple-resonance MAS NMR experiment, a single self-consistent data set can be acquired, providing enhanced sensitivity, reduced vulnerability to machine or sample instabilities, and highly redundant linking that supports fully automated peak picking and resonance assignment. The method, dubbed RAVASSA (redundant assignment via a single simultaneous acquisition), is demonstrated with the assignment of the largest protein to date in the solid state, the 42.5 kDa maltose binding protein, using a single fully protonated microcrystalline sample and 1 week of spectrometer time.

The influence of the stereochemistry and C-end chemical modification of dermorphin derivatives on the peptide-phospholipid interactions.

In this work the conformation of dermorphin, Tyr-D-Ala-Phe-Gly-Tyr-Pro-Ser-NH2, an opioid peptide and its analogues with different stereochemistry of alanine and different C-terminus is studied in aqueous and membrane environments. Using two-dimensional NMR techniques we demonstrate that in D2O/H2O peptides with D-alanine have extended conformation, while for the L-isomers more compact conformation is preferred. The analysis of ROESY HR MAS spectra of the peptides interacting with the DMPC bilayer indicates that both stereoisomers have still more extended conformation compared to aqueous phase, as shown by much weaker intermolecular interactions. The influence of Ala residue stereochemistry is also reflected in the interactions of the studied peptides with model membranes, as shown by the 31P NMR static spectra, in which the shapes of the phosphorus NMR signals originating from D-isomers correspond to spherically shaped vesicles in the presence of external magnetic field, in comparison to a more elongated ones observed for L-isomers, while TEM photographs shows that upon addition of D-isomers larger lipid vesicles are formed, in contrast to smaller ones for L-isomers. The location of aromatic fragments of dermorphins in the membrane is determined based on static 2H NMR and 1H1H RFDR MAS experiments. All aromatic rings were found to be inserted in the hydrophobic part of the bilayer, with the exception of the Tyr5 rings of D-Ala dermorphins. The influence of the C-terminal modification was found to be almost imperceptible.

Modification of bacterial nanocellulose properties through mutation of motility related genes in Komagataeibacter hansenii ATCC 53582.

Bacterial nanocellulose (BNC) produced by Komagataeibacter hansenii has received significant attention due to its unique supernetwork structure and properties. It is nevertheless necessary to modify bacterial nanocellulose to achieve materials with desired properties and thus with broader areas of application. The aim here was to influence the 3D structure of BNC by genetic modification of the cellulose producing K. hansenii strain ATCC 53582. Two genes encoding proteins with homology to the MotA and MotB proteins, which participate in motility and energy transfer, were selected for our studies. A disruption mutant of one or both genes and their respective complementation mutants were created. The phenotype analysis of the disruption mutants showed a reduction in motility, which resulted in higher compaction of nanocellulose fibers and improvement in their mechanical properties. The data strongly suggest that these genes play an important role in the formation of BNC membrane by Komagataeibacter species.

Evaluation of excitation schemes for indirect detection of 14N via solid-state HMQC NMR experiments.

It has previously been shown that 14N NMR spectra can be reliably obtained through indirect detection via HMQC experiments. This method exploits the transfer of coherence between single-(SQ) or double-quantum (DQ) 14N coherences, and SQ coherences of a suitable spin-1/2 'spy' nucleus, e.g., 1H. It must be noted that SQ-SQ methods require a carefully optimized setup to minimize the broadening related to the first-order quadrupole interaction (i.e., an extremely well-adjusted magic angle and a highly stable spinning speed), whereas DQ-SQ ones do not. In this work, the efficiencies of four 14N excitation schemes (DANTE, XiX, Hard Pulse (HP), and Selective Long Pulse (SLP)) are compared using J-HMQC based numerical simulations and either SQ-SQ or DQ-SQ 1H-{14N} D-HMQC experiments on l-histidine HCl and N-acetyl-l-valine at 18.8 T and 62.5 kHz MAS. The results demonstrate that both DANTE and SLP provide a more efficient 14N excitation profile than XiX and HP. Furthermore, it is shown that the SLP scheme: (i) is efficient over a large range of quadrupole interaction, (ii) is highly robust to offset and rf-pulse length and amplitude, and (iii) is very simple to set up. These factors make SLP ideally suited to widespread, non-specialist use in solid-state NMR analyses of nitrogen-containing materials.

Analysis of HMQC experiments applied to a spin ½ nucleus subject to very large CSA.

The acquisition of solid-state NMR spectra of "heavy" spin I = 1/2 nuclei, such as 119Sn, 195Pt, 199Hg or 207Pb can often prove challenging due to the presence of large chemical shift anisotropy (CSA), which can cause significant broadening of spectral lines. However, previous publications have shown that well-resolved spectra can be obtained via inverse 1H detection using HMQC experiments in combination with fast magic angle spinning. In this work, the efficiencies of different 195Pt excitation schemes are analyzed using SIMPSON numerical simulations and experiments performed on cis- and transplatin samples. These schemes include: hard pulses (HP), selective long pulses (SLP) and rotor-synchronized DANTE trains of pulses. The results show that for spectra of species with very large CSA, HP is little efficient, but that both DANTE and SLP provide efficient excitation profiles over a wide range of CSA values. In particular, it is revealed that the SLP scheme is highly robust to offset, pulse amplitude and length, and is simple to set up. These factors make SLP ideally suited to widespread use by "non-experts" for carrying out analyses of materials containing "heavy" spin I = 1/2 nuclei that are subject to very large CSAs. Finally, the existence of an "intermediate" excitation regime, with an rf-field strength in between those of HP and SLP, which is effective for large CSA, is demonstrated. It must be noted that in some samples, multiple sites may exist with very different CSAs. This is the case for 195Pt species with either square-planar or octahedral structures, with large or small CSA, respectively. These two types of CSAs can only be excited simultaneously with DANTE trains, which scale up the effective rf-field. Another way to obtain all the information is to perform two different experiments: one with SLP and the second with HP to excite the sites with moderate/large and small/moderate CSAs, respectively. These two complementary experiments, recorded with two different spinning speeds, can also be used to discriminate the center-band resonances from the spinning sidebands.

Understanding the formation of apremilast cocrystals.

Apremilast (APR), an anti-psoriatic agent, easily forms isostructural cocrystals and solvates with aromatic entities, often disobeying at the same time Kitaigorodsky's rule as to the saturation of possible hydrogen-bonding sites. In this paper the reasons for this peculiar behavior are investigated, employing a joint experimental and theoretical approach. This includes the design of cocrystals with coformers having a high propensity towards the formation of both aromatic-aromatic and hydrogen-bonding interactions, determination of their structure, using solid-state NMR spectroscopy and X-ray crystallography, as well as calculations of stabilization energies of formation of the obtained cocrystals, followed by crystal structure prediction calculations and solubility measurements. The findings indicate that the stabilization energies of cocrystal formation are positive in all cases, which results from strain in the APR conformation in these crystal forms. On the other hand, solubility measurements show that the Gibbs free energy of formation of the apremilast:picolinamide cocrystal is negative, suggesting that the formation of the studied cocrystals is entropy driven. This entropic stabilization is associated with the disorder observed in almost all known cocrystals and solvates of APR.

Simple and Robust Study of Backbone Dynamics of Crystalline Proteins Employing 1H-15N Dipolar Coupling Dispersion.

We report a new solid-state multidimensional NMR approach based on the cross-polarization with variable-contact pulse sequence [ Paluch , P. ; Pawlak , T. ; Amoureux , J.-P. ; Potrzebowski , M. J. J. Magn. Reson. 233 , 2013 , 56 ], with 1H inverse detection and very fast magic angle spinning (νR = 60 kHz), dedicated to the measurement of local molecular motions of 1H-15N vectors. The introduced three-dimensional experiments, 1H-15N-1H and hCA(N)H, are particularly useful for the study of molecular dynamics of proteins and other complex structures. The applicability and power of this methodology have been revealed by employing as a model sample the GB-1 small protein doped with Na2CuEDTA. The results clearly prove that the dispersion of 1H-15N dipolar coupling constants well correlates with higher order structure of the protein. Our approach complements the conventional studies and offers a fast and reasonably simple method.

(Ar-CO-C[triple bond, length as m-dash]C)(PEt3)Au and (Ar-C[triple bond, length as m-dash]C)(PEt3)Au complexes bearing pyrenyl and ferrocenyl groups: synthesis, structure, and luminescence properties.

Two types of (acetylide)(triethylphosphine)gold(i) complexes ArCOC[triple bond, length as m-dash]CAuPEt3 (1a and 1b) and ArC[triple bond, length as m-dash]CAuPEt3 (2a and 2b) bearing Ar = pyren-1-yl or ferrocenyl group were synthesized and the effect of a carbonyl moiety on the structure, propensity to ligand scrambling in solution and luminescence properties were investigated. We found that the complexes bearing acetylenic ketone-derived ligands underwent ligand scrambling in solution to afford mixtures of ArCOC[triple bond, length as m-dash]CAuPEt3 and [(ArCOC[triple bond, length as m-dash]C)2Au]-[Au(PEt3)2]+. The latter complexes were isolated and their structures were confirmed by single crystal X-ray diffraction studies. The aurophilic interaction of AuAu in these complexes resulted in the formation of wire-like structures.

In Depth Analysis of Chiroptical Properties of Enones Derived from Abietic Acid.

With the use of inexpensive commercially available abietic acid, a whole series of abietane enones were prepared in high yields. The structures of all the products obtained were determined by comprehensive spectroscopic analysis with particular emphasis on the use of advanced NMR techniques, comparison with previously reported data and, where possible, by single crystal X-ray diffraction. However, in cases where X-ray crystallography was not applicable or compounds tested were unstable, a final stereochemical assignment could be inferred only by electronic circular dichroism (ECD) supported by vibrational circular dichroism to increase credibility. To reveal the relationship between structure and chiroptical properties, we used combined experimental and theoretical analysis of geometries, structural parameters, and chiroptical properties of all enones synthesized. A thorough analysis of their conformational flexibility by examining the effect of solvent and temperature on the ECD spectra was also used to achieve desired objectives. As a result, the impact of substituents adjacent to the enone chromophore on the conformation was determined by demonstrating that even slight changes in the position of hydroxyl and isopropyl groups attached to carbon C13 may substantially affect ECD curves' pattern, leading in some cases to Cotton effects sign reversal.

New synthetic pathway leading to oxospirochlorins.

In this work we propose a completely new approach for the synthesis of spirochlorin derivatives based on the use of an imino-keto intermediate formed in situ from 2-amino-5,10,15,20-tetraphenylporphyrins and inverse electron demand Diels-Alder (iEDDA) cycloaddition with 3,6-di-2-pyridyl-1,2,4,5-tetrazine. The mechanism of reaction was analyzed employing theoretical methods by comparing the difference in energy of Frontier Molecular Orbitals (FMO) for appropriate reagents. Ground-state molecular electrostatic (ESP) potential maps were employed as additional tools allowing explanation of the reactivity of substrates. The new class of spirochlorin compounds was fully characterized by means of mass spectrometry, IR, liquid and solid state NMR and X-ray crystallography. Correlation between molecular structure and optical properties for the obtained title compounds is discussed.