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.

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.

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.

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.

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.

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.

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.

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.

Spontaneous Keto-Enol Tautomerization in the Crystal Lattice Visualized with the Help of Water Encapsulated in Hydrophilic Reservoirs.

Keto-enol tautomerism in the solid phase is a process that is particularly difficult to follow. In this work we demonstrate how it can be done by introducing deuterium into the crystal lattice of organic compounds which tend to form hydrates. In our studies we explored H-D exchange in the crystals stored in contact with deuterium oxide vapors. Employing barbituric acid (BA) and (+)-catechin (CAT) as model samples and by using advanced solid-state NMR spectroscopy and mass spectrometry, we revealed that not only OH and NH protons of these chemicals undergo exchange to deuterium in a crystal lattice, but also usually immobile protons, that is, (Ar)CH (in CAT) and CH2 (in BA) are exchanged as a result of keto-enol tautomerism.

Correction: Analysis of local molecular motions of aromatic sidechains in proteins by 2D and 3D fast MAS NMR spectroscopy and quantum mechanical calculations.

Correction for 'Analysis of local molecular motions of aromatic sidechains in proteins by 2D and 3D fast MAS NMR spectroscopy and quantum mechanical calculations' by Piotr Paluch et al., Phys. Chem. Chem. Phys., 2015, 17, 28789-28801.

1H-31P CPVC NMR method under Very Fast Magic Angle Spinning for analysis of dipolar interactions and dynamics processes in the crystalline phosphonium tetrafluoroborate salts.

We present an NMR methodology which can be used to study the dynamical processes occurring in organophosphorus compounds that belong to the group of the organic ionic plastic crystals (OIPCs). As model samples we employed two phosphonium tetrafluoroborate salts; (t-Bu)3PH+BF4- (1) and (Me)3PH+BF4- (2). Both samples possess in their structures direct H-P bonds, and both undergo complex thermal processes in the solid state, forming below the melting point three or four phases, respectively. 1H-31P CPVC (Cross-Polarization Variable Contact) measurements were performed under Very Fast Magic Angle Spinning with speed equal to 50 or 60 kHz, in order (i) to establish the hydrogen-phosphorus dipolar couplings, and (ii) to correlate the dipolar splitting values with molecular motions of the cation. Our project is divided into three sections. In the first part we present DSC studies of (1) and (2), to verify whether these samples fulfill the requirements that define them as OIPC. The second part is dedicated to a discussion of the theoretical aspects of 1H-31P CPVC and especially an analysis of the influence of different parameters, e.g. CSA31P, H-H mismatch, rf-inhomogeneity, dipolar truncation, and the type of dynamics through the motionally averaged <ηD> asymmetry value on the NMR response. The third part shows experimental 1H-31P CPVC data and applicability of these measurements to study H-P distances and dynamics. The complex molecular motion for sample (2), including rotation and diffusion, versus temperature is then postulated on the bases of the changes of H-P dipolar splitting.

Application of 1H and 27Al magic angle spinning solid state NMR at 60kHz for studies of Au and Au-Ni catalysts supported on boehmite/alumina.

In this work for the first time we show the power of solid state NMR spectroscopy in structural analysis of alumina and catalysts supported on the alumina surface employing very fast (60kHz) magic angle spinning (MAS) technique. In the methodological part we demonstrate that under such MAS condition, cross-polarization (CP) from proton to aluminum is an efficient process when a very weak 27Al RF field is applied. The mechanism of CP transfer and the Hartmann-Hahn (H-H) matching conditions were tested for 27Al RF fields equal to 3.3 and 8.3kHz. It has been found that double quantum (DQ) CP/MAS is the best choice for H-H set with RF =3.3kHz. It has been also proved that the quality of 1H-27Al CP/MAS spectra strongly depends on 27Al carrier offset. Applied to γ-alumina, this method revealed that 1H-27Al CP/MAS at 60kHz is extremely useful for mapping the distribution of hydroxyl groups on the surface. Indeed, the AlV sites, which are not easily detected with Single Pulse Experiment (SPE), are clearly seen when 1H-27Al CP/MAS is applied. Utilizing 2D 1H-27Al CP/MAS HETCOR experiment it was possible to assign the proton positions and to correlate them with aluminum centers. Studies of mono- (Au) and bi- (Au-Ni) metallic catalysts supported on boehmite/alumina carrier employing 1D and 2D HETCOR experiments clearly show that distributions of hydroxyl groups for both systems are dramatically different.

Imaging the spatial distribution of radiofrequency field, sample and temperature in MAS NMR rotor.

We investigate using nutation experiments the spatial distribution of radiofrequency (rf) field, sample, temperature and cross-polarization transfer efficiency in 1.3 mm rotor. First, two-dimensional (2D) 1H nutation experiments on silicone thin cylinders in the presence of B0 field gradient generated by shim coils are used to image the spatial distribution of rf field inside the rotor. These experiments show that the rf field is asymmetrical with respect to the center of the rotor. Moreover, they show the large inhomogeneity that still remains across the sample when using spacers, and that even in this case, the rf-field close to the drive cap is decreased to ca. only 20% of its maximum value. Such 2D nutation experiment in the presence of B0 field gradient are also employed to demonstrate the migration of adamantane sample from the center of the rotor to its ends during Magic-Angle Spinning (MAS). Furthermore, 2D 1H nutation experiments on nickelocene exhibiting temperature-dependent isotropic chemical shift provides insights into the temperature distribution inside rotor. Finally three-dimensional (3D) 1H → 13C Cross-Polarization under MAS (CPMAS) nutation experiment indicates that only nuclei subject to the largest rf field contribute to the CPMAS transfer, when using rf field of constant amplitude on both channels. Such high selectivity allows the determination of accurate dipolar coupling constants in the Cross-Polarization with Variable Contact (CP-VC) experiment under fast MAS, at the expense of low sensitivity. Conversely when using ramped-amplitude on the 1H channel during the CPMAS transfer, nuclei subject to smaller rf field contributes to the transfer, which increases the sensitivity of CPMAS experiment but does not allow an accurate determination of dipolar coupling constants using CP-VC experiment.

Analysis of local molecular motions of aromatic sidechains in proteins by 2D and 3D fast MAS NMR spectroscopy and quantum mechanical calculations.

We report a new multidimensional magic angle spinning NMR methodology, which provides an accurate and detailed probe of molecular motions occurring on timescales of nano- to microseconds, in sidechains of proteins. The approach is based on a 3D CPVC-RFDR correlation experiment recorded under fast MAS conditions (ν(R) = 62 kHz), where (13)C-(1)H CPVC dipolar lineshapes are recorded in a chemical shift resolved manner. The power of the technique is demonstrated in model tripeptide Tyr-(d)Ala-Phe and two nanocrystalline proteins, GB1 and LC8. We demonstrate that, through numerical simulations of dipolar lineshapes of aromatic sidechains, their detailed dynamic profile, i.e., the motional modes, is obtained. In GB1 and LC8 the results unequivocally indicate that a number of aromatic residues are dynamic, and using quantum mechanical calculations, we correlate the molecular motions of aromatic groups to their local environment in the crystal lattice. The approach presented here is general and can be readily extended to other biological systems.

Fine refinement of solid state structure of racemic form of phospho-tyrosine employing NMR Crystallography approach.

We present step by step facets important in NMR Crystallography strategy employing O-phospho-dl-tyrosine as model sample. The significance of three major techniques being components of this approach: solid state NMR (SS NMR), X-ray diffraction of powdered sample (PXRD) and theoretical calculations (Gauge Invariant Projector Augmented Wave; GIPAW) is discussed. Each experimental technique provides different set of structural constraints. From the PXRD measurement the size of the unit cell, space group and roughly refined molecular structure are established. SS NMR provides information about content of crystallographic asymmetric unit, local geometry, molecular motion in the crystal lattice and hydrogen bonding pattern. GIPAW calculations are employed for validation of quality of elucidation and fine refinement of structure. Crystal and molecular structure of O-phospho-dl-tyrosine solved by NMR Crystallography is deposited at Cambridge Crystallographic Data Center under number CCDC 1005924.

Insights into the tautomerism in meso-substituted corroles: a variable-temperature 1H, 13C, 15N, and 19F†NMR spectroscopy study.

Tris(pentafluorophenyl)corrole and its (15)N-enriched isotopomer were studied in [D8]toluene solution by 1D and 2D variable-temperature NMR techniques to establish the mechanisms of tautomerization of the NH protons inside the interior of the corrole macrocycle. Three such rate processes could be identified of which two modulate the spectral line shapes at temperatures above 205 K and the third is NMR-inaccessible as it is very fast. The latter involves the proton engaged in an unsymmetrical proton sponge unit formed by two pyrrole nitrogen atoms. Temperature and concentration dependences of the two remaining processes were determined. One of them is purely intramolecular and the other is intermolecular at low temperatures, with growing contribution of an intramolecular mechanism at elevated temperatures. The proposed microscopic mechanisms of all these processes are semi-quantitatively confirmed by quantum chemical calculations using density functional theory.

Ibuprofen in mesopores of Mobil Crystalline Material 41 (MCM-41): a deeper understanding.

In this work, we compared two methods (incipient wetness and melting) for the encapsulation of ibuprofen in the pores of Mobil Crystalline Material 41 (MCM-41) through NMR (nuclear magnetic resonance) spectroscopy. (1)H NMR spectra were recorded under very fast MAS (sample spinning 60 kHz) conditions in both 1D and 2D mode (NOESY sequence). We also performed (13)C cross-polarization magic angle spinning (CP/MAS) experiments, (13)C single pulse experiments (SPE), and (1)H-(13)C HSQC HR/MAS (heteronuclear single quantum coherence high resolution) HR/MAS correlations. Evaluation of the encapsulation methods included an analysis of the filling factor of the drug into the pores. The stability of Ibu/MCM in an environment of ethanol or water vapor was tested. Our study showed that melting a mixture of Ibu and MCM is a much more efficient method of confining the drug in the pores compared to incipient wetness. The optimal experiments for the former method achieved a filling factor of approximately 60%. We concluded that the major limitation to the applicability of the incipient wetness method (filling factor ca. 20%) is the high affinity of solvent (typically ethanol) for MCM-41. We found that even ethanol vapor can remove Ibu from the pores. When a sample of Ibu/MCM was stored for a few hours in a closed vessel with ethanol vapor, Ibu was transported from the pores to the outer walls of MCM. We observed a similar phenomenon with water vapor, although this process is slower compared to the analogous procedure using ethanol. Our study clearly demonstrates that existing methods used to encapsulate drugs in mesoporous silica nanoparticles (MSNs) require reevaluation.

The structure of cyclolinopeptide A in chloroform refined by RDC measurements.

Three-dimensional structures of molecules traditionally assigned from nuclear Overhauser effects and vicinal coupling constants are recently complemented by measurements of residual dipolar couplings. Residual dipolar couplings measured in a stretched poly(dimethylsiloxane) gel were used to determine the structure of cyclolinopeptide A in chloroform solution at -50 °C. After structure refinement, conformational details of main cluster were discussed in relation to crystal and nuclear Overhauser effect derived structures.

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.