Boosting the Metathesis Activity of Molybdenum Oxo Alkylidenes by Tuning the Anionic Ligand σ Donation.

The catalytic performances of molecular and silica-supported molybdenum oxo alkylidene species bearing anionic O ligands [ORF9, OTPP, OHMT - where ORF9 = OC(CF3)3, OTPP = 2,3,5,6-tetraphenylphenoxy, OHMT = hexamethylterphenoxy] with different σ-donation abilities and sizes are evaluated in the metathesis of both internal and terminal olefins. Here, we show that the presence of the anionic nonafluoro-tert-butoxy X ligand in Mo(O){═CH-4-(MeO)C6H4}(THF)2{X}2 (1; X = ORF9) significantly increases the catalytic performances in the metathesis of both terminal and internal olefins. Its silica-supported equivalent displays slightly lower activity, albeit with improved stability. In sharp contrast, the molecular complexes with large aryloxy anionic X ligands show little activity, whereas the activity of the corresponding silica-supported systems is greatly improved, illustrating that surface siloxy groups are significantly smaller anionic ligands. Of all of the systems, compound 1 stands out because of its unique high activity for both terminal and internal olefins. Density functional theory modeling indicates that the ORF9 ligand is ideal in this series because of its weak σ-donating ability, avoiding overstabilization of the metallacyclobutane intermediates while keeping low barriers for [2 + 2] cycloaddition and turnstile isomerization.

Uncovering selective and active Ga surface sites in gallia-alumina mixed-oxide propane dehydrogenation catalysts by dynamic nuclear polarization surface enhanced NMR spectroscopy.

Gallia-alumina (Ga,Al)2O3(x : y) spinel-type solid solution nanoparticle catalysts for propane dehydrogenation (PDH) were prepared with four nominal Ga : Al atomic ratios (1 : 6, 1 : 3, 3 : 1, 1 : 0) using a colloidal synthesis approach. The structure, coordination environment and distribution of Ga and Al sites in these materials were investigated by X-ray diffraction, X-ray absorption spectroscopy (Ga K-edge) as well as 27Al and 71Ga solid state nuclear magnetic resonance. The surface acidity (Lewis or Brønsted) was probed using infrared spectroscopy with pyridine and 2,6-dimethylpyridine probe molecules, complemented by element-specific insights (Ga or Al) from dynamic nuclear polarization surface enhanced cross-polarization magic angle spinning 15N{27Al} and 15N{71Ga} J coupling mediated heteronuclear multiple quantum correlation NMR experiments using 15N-labelled pyridine as a probe molecule. The latter approach provides unique insights into the nature and relative strength of the surface acid sites as it allows to distinguish contributions from Al and Ga sites to the overall surface acidity of mixed (Ga,Al)2O3 oxides. Notably, we demonstrate that (Ga,Al)2O3 catalysts with a high Al content show a greater relative abundance of four-coordinated Ga sites and a greater relative fraction of weak/medium Ga-based surface Lewis acid sites, which correlates with superior propene selectivity, Ga-based activity, and stability in PDH (due to lower coking). In contrast, (Ga,Al)2O3 catalysts with a lower Al content feature a higher fraction of six-coordinated Ga sites, as well as more abundant Ga-based strong surface Lewis acid sites, which deactivate through coking. Overall, the results show that the relative abundance and strength of Ga-based surface Lewis acid sites can be tuned by optimizing the bulk Ga : Al atomic ratio, thus providing an effective measure for a rational control of the catalyst performance.

N-Heterocyclic Carbene Coordination to Surface Copper Sites in Selective Semihydrogenation Catalysts from Solid-State NMR Spectroscopy.

Supported metal nanoparticles are a very large class of heterogeneous catalysts. While detailed structure-activity relationships require a molecular-level description of the interactions between the metal surfaces and ligands/substrates, this description is rarely accessible. Thus, most insights are derived from models based on single crystals. With the goal to understand alkyne semihydrogenation catalysts based on Cu functionalized with N-heterocyclic carbene (NHC), we cross this gap by investigating NHC-stabilized molecular complexes, supported single sites and nanoparticles by solid-state NMR combined with computations. We show that in silica-supported Cu single sites, Cu retains the coordination geometry observed in molecular compounds, while, for supported Cu nanoparticles, which are active and selective for the semihydrogenation of alkynes, NHC binding is favored at Cu adatoms atop of copper surface, thus paralleling conclusions of surface science studies on single crystals.

A Formulation Protocol with Pyridine to Enable Dynamic Nuclear Polarization Surface-Enhanced NMR Spectroscopy on Reactive Surface Sites: Case Study with Olefin Polymerization and Metathesis Catalysts.

Dynamic nuclear polarization surface-enhanced NMR spectroscopy (DNP-SENS) has emerged as a powerful characterization tool in material chemistry and heterogeneous catalysis by dramatically increasing, by up to 2 orders of magnitude, the NMR signals associated with surface sites. DNP-SENS mostly relies on using exogenous polarizing agents (PAs), typically dinitroxyl radicals, to boost the NMR signals. However, the PAs may interact with the surface or even react with surface sites, thus leading to loss or quenching of DNP enhancements. Herein, we describe the development of a DNP-SENS formulation that allows broadening the application of DNP-SENS to samples containing highly reactive surface sites, namely a Ziegler-Natta propylene polymerization catalyst, a sulfated zirconia-supported metallocene, and a silica-supported cationic Mo alkylidene. The protocol consists of adsorbing pyridine prior to the DNP formulation (TEKPol/TCE). The addition of pyridine not only preserves the PAs and thereby restores the DNP enhancement but also allows probing Lewis/Brønsted acid surface sites that are often present on these catalysts.

Proton-Detected Multidimensional Solid-State NMR Enables Precise Characterization of Vanadium Surface Species at Natural Abundance.

Heterogeneous catalysts fulfill vital roles in industrial processes; however, the nature of the catalytic surfaces, typically either containing a low abundance of active sites or being amorphous in nature, leads to difficulties when attempting to study the structure of the active sites. In this work, we show how making use of fast MAS ssNMR allows one to efficiently detect well-resolved 1H-detected spectra of heterogeneous catalysts. This approach was applied to study the structure of surface species resulting from the grafting of VO(OiPr)3 onto a partially dehydroxylated silica using the surface organometallic chemistry approach. The use of 1H sensitivity enabled detection of various hetero- and homonuclear correlation spectra in order to study the structure of this system and to resolve the structure of the grafted vanadium complex. More specifically, VO(OiPr)3 grafts through both protonolysis and opening of siloxane bridges to generate a bis-grafted species.

Well-Defined Silica-Supported Tungsten(IV)-Oxo Complex: Olefin Metathesis Activity, Initiation, and Role of Brønsted Acid Sites.

Despite the importance of the heterogeneous tungsten-oxo-based olefin metathesis catalyst (WO3/SiO2) in industry, understanding of its initiation mechanism is still very limited. It has been proposed that reduced W(IV)-oxo surface species act as precatalysts. In order to understand the reactivity and initiation mechanism of surface W(IV)-oxo species, we synthesized a well-defined silica-supported W(IV)-oxo species, (≡SiO)WO(OtBuF6)(py)3 (F6@SiO2-700; OtBuF6 = OC(CH3)(CF3)2; py = pyridine), via surface organometallic chemistry (SOMC). F6@SiO2-700 was shown to be highly active in olefin metathesis upon removal of pyridine ligands through the addition of tris(pentafluorophenyl)borane (B(C6F5)3) or thermal treatment under high vacuum. The metathesis activity toward olefins with and without allylic C-H groups, namely ß-methylstyrene and styrene, respectively, was investigated. In the case of styrene, we demonstrated the role of surface OH groups in initiating metathesis activity. We proposed that the presence of strong Brønsted acidic OH sites, which likely arises from the presence of adjacent W sites in the catalyst as revealed by 15N-labeled pyridine adsorption, can assist styrene metathesis. In contrast, initiation of olefins with allylic C-H groups (e.g., ß-methylstyrene) is independent of the surface OH density and likely involves an allylic C-H activation mechanism, like the molecular W(IV)-oxo species. This study indicates that initiation mechanisms depend on the olefinic substrates and reveals the synergistic effect of Brønsted acidic surface sites and reduced W(IV) sites in the initiation of olefin metathesis.

Silica-Supported Molybdenum Oxo Alkylidenes: Bridging the Gap between Internal and Terminal Olefin Metathesis.

Grafting a molybdenum oxo alkylidene on silica (partially dehydroxylated at 700 °C) affords the first example of a well-defined silica-supported Mo oxo alkylidene, which is an analogue of the putative active sites in heterogeneous Mo-based metathesis catalysts. In contrast to its tungsten analogue, which shows poor activity towards terminal olefins because of the formation of a stable off-cycle metallacyclobutane intermediate, the Mo catalyst shows high metathesis activity for both terminal and internal olefins that is consistent with the lower stability of Mo metallacyclobutane intermediates. This Mo oxo metathesis catalyst also outperforms its corresponding neutral silica-supported Mo and W imido analogues.

Studying assembly of the BAM complex in native membranes by cellular solid-state NMR spectroscopy.

Significant progress has been made in obtaining structural insight into the assembly of the ß-barrel assembly machinery complex (BAM). These crystallography and electron microscopy studies used detergent as a membrane mimetic and revealed structural variations in the central domain, BamA, as well as in the lipoprotein BamC. We have used cellular solid-state NMR spectroscopy to examine the entire BamABCDE complex in native outer membranes and obtained data on the BamCDE subcomplex in outer membranes, in addition to synthetic bilayers. To reduce spectral crowding, we utilized proton-detected experiments and employed amino-acid specific isotope-labelling in (13C, 13C) correlation experiments. Taken together, the results provide insight into the overall fold and assembly of the BAM complex in native membranes, in particular regarding the structural flexibility of BamC in the absence of the core unit BamA.

Formation of the ß-barrel assembly machinery complex in lipid bilayers as seen by solid-state NMR.

The ß-barrel assembly machinery (BAM) is a pentameric complex (BamA-E), which catalyzes the essential process of ß-barrel protein insertion into the outer membrane of E. coli. Thus far, a detailed understanding of the insertion mechanism has been elusive but recent results suggest that local protein motion, in addition to the surrounding membrane environment, may be of critical relevance. We have devised a high-sensitivity solid-state NMR approach to directly probe protein motion and the structural changes associated with BAM complex assembly in lipid bilayers. Our results reveal how essential BamA domains, such as the interface formed by the polypeptide transport associated domains P4 and P5 become stabilized after complex formation and suggest that BamA ß-barrel opening and P5 reorientation is directly related to complex formation in membranes. Both the lateral gate, as well as P5, exhibit local dynamics, a property that could play an integral role in substrate recognition and insertion.

Diastereoisomers of l-proline-linked trityl-nitroxide biradicals: synthesis and effect of chiral configurations on exchange interactions.

The exchange (J) interaction of organic biradicals is a crucial factor controlling their physiochemical properties and potential applications and can be modulated by changing the nature of the linker. In the present work, we for the first time demonstrate the effect of chiral configurations of radical parts on the J values of trityl-nitroxide (TN) biradicals. Four diastereoisomers (TNT1, TNT2, TNL1 and TNL2) of TN biradicals were synthesized and purified by the conjugation of a racemic (R/S) nitroxide with the racemic (M/P) trityl radical vial-proline. The absolute configurations of these diastereoisomers were assigned by comparing experimental and calculated electronic circular dichroism (ECD) spectra as (M, S, S) for TNT1, (P, S, S) for TNT2, (M, S, R) for TNL1 and (P, S, R) for TNL2. Electron paramagnetic resonance (EPR) results showed that the configuration of the nitroxide part instead of the trityl part is dominant in controlling the exchange interactions and the order of the J values at room temperature is TNT1 (252 G) > TNT2 (127 G) ≫ TNL2 (33 G) > TNL1 (14 G). Moreover, the J values of TNL1/TNL2 with the S configuration in the nitroxide part vary with temperature and the polarity of solvents due to their flexible linker, whereas the J values of TNT1/TNT2 are almost insensitive to these two factors due to the rigidity of their linkers. The distinct exchange interactions between TNT1,2 and TNL1,2 in the frozen state led to strongly different high-field dynamic nuclear polarization (DNP) enhancements with ε = 7 for TNT1,2 and 40 for TNL1,2 under 800 MHz DNP conditions.

Rapid Prediction of Multi-dimensional NMR Data Sets Using FANDAS.

Solid-state NMR (ssNMR) can provide structural information at the most detailed level and, at the same time, is applicable in highly heterogeneous and complex molecular environments. In the last few years, ssNMR has made significant progress in uncovering structure and dynamics of proteins in their native cellular environments [1-4]. Additionally, ssNMR has proven to be useful in studying large biomolecular complexes as well as membrane proteins at the atomic level [5]. In such studies, innovative labeling schemes have become a powerful approach to tackle spectral crowding. In fact, selecting the appropriate isotope-labeling schemes and a careful choice of the ssNMR experiments to be conducted are critical for applications of ssNMR in complex biomolecular systems. Previously, we have introduced a software tool called FANDAS (Fast Analysis of multidimensional NMR DAta Sets) that supports such investigations from the early stages of sample preparation to the final data analysis [6]. Here, we present a new version of FANDAS, called FANDAS 2.0, with improved user interface and extended labeling scheme options allowing the user to rapidly predict and analyze ssNMR data sets for a given protein-based application. It provides flexible options for advanced users to customize the program for tailored applications. In addition, the list of ssNMR experiments that can be predicted now includes proton (1H) detected pulse sequences. FANDAS 2.0, written in Python, is freely available through a user-friendly web interface at http://milou.science.uu.nl/services/FANDAS .

Supramolecular Organization and Functional Implications of K+ †Channel Clusters in Membranes.

The segregation of cellular surfaces in heterogeneous patches is considered to be a common motif in bacteria and eukaryotes that is underpinned by the observation of clustering and cooperative gating of signaling membrane proteins such as receptors or channels. Such processes could represent an important cellular strategy to shape signaling activity. Hence, structural knowledge of the arrangement of channels or receptors in supramolecular assemblies represents a crucial step towards a better understanding of signaling across membranes. We herein report on the supramolecular organization of clusters of the K+ channel KcsA in bacterial membranes, which was analyzed by a combination of DNP-enhanced solid-state NMR experiments and MD simulations. We used solid-state NMR spectroscopy to determine the channel-channel interface and to demonstrate the strong correlation between channel function and clustering, which suggests a yet unknown mechanism of communication between K+ channels.

A DNP-supported solid-state NMR study of carbon species in fluid catalytic cracking catalysts.

A combination of solid-state NMR techniques supported by EPR and SEM-EDX experiments was used to localize different carbon species (coke) in commercial fluid catalytic cracking catalysts. Aliphatic coke species formed during the catalytic process and aromatic coke species deposited directly from the feedstock respond differently to dynamic nuclear polarization signal enhancement in integral and crushed FCC particles, indicating that aromatic species are mostly concentrated on the outside of the catalyst particles, whereas aliphatic species are also located on the inside of the FCC particles. The comparison of solid-state NMR data with and without the DNP radical at low and ambient temperature suggests the proximity between aromatic carbon deposits and metals (mostly iron) on the catalyst surface. These findings potentially indicate that coke and iron deposit together, or that iron has a role in the formation of aromatic coke.

A Two-Tailed Phosphopeptide Crystallizes to Form a Lamellar Structure.

The crystal structure of a designed phospholipid-inspired amphiphilic phosphopeptide at 0.8 Šresolution is presented. The phosphorylated ß-hairpin peptide crystallizes to form a lamellar structure that is stabilized by intra- and intermolecular hydrogen bonding, including an extended ß-sheet structure, as well as aromatic interactions. This first reported crystal structure of a two-tailed peptidic bilayer reveals similarities in thickness to a typical phospholipid bilayer. However, water molecules interact with the phosphopeptide in the hydrophilic region of the lattice. Additionally, solid-state NMR was used to demonstrate correlation between the crystal structure and supramolecular nanostructures. The phosphopeptide was shown to self-assemble into semi-elliptical nanosheets, and solid-state NMR provides insight into the self-assembly mechanisms. This work brings a new dimension to the structural study of biomimetic amphiphilic peptides with determination of molecular organization at the atomic level.

EGFR Dynamics Change during Activation in Native Membranes as Revealed by NMR.

The epidermal growth factor receptor (EGFR) represents one of the most common target proteins in anti-cancer therapy. To directly examine the structural and dynamical properties of EGFR activation by the epidermal growth factor (EGF) in native membranes, we have developed a solid-state nuclear magnetic resonance (ssNMR)-based approach supported by dynamic nuclear polarization (DNP). In contrast to previous crystallographic results, our experiments show that the ligand-free state of the extracellular domain (ECD) is highly dynamic, while the intracellular kinase domain (KD) is rigid. Ligand binding restricts the overall and local motion of EGFR domains, including the ECD and the C-terminal region. We propose that the reduction in conformational entropy of the ECD by ligand binding favors the cooperative binding required for receptor dimerization, causing allosteric activation of the intracellular tyrosine kinase.

1 H-Detected Solid-State NMR Studies of Water-Inaccessible Proteins In†Vitro and In†Situ.

1 H detection can significantly improve solid-state NMR spectral sensitivity and thereby allows studying more complex proteins. However, the common prerequisite for 1 H detection is the introduction of exchangeable protons in otherwise deuterated proteins, which has thus far significantly hampered studies of partly water-inaccessible proteins, such as membrane proteins. Herein, we present an approach that enables high-resolution 1 H-detected solid-state NMR (ssNMR) studies of water-inaccessible proteins, and that even works in highly complex environments such as cellular surfaces. In particular, the method was applied to study the K+ channel KcsA in liposomes and in situ in native bacterial cell membranes. We used our data for a dynamic analysis, and we show that the selectivity filter, which is responsible for ion conduction and highly conserved in K+ channels, undergoes pronounced molecular motion. We expect this approach to open new avenues for biomolecular ssNMR.

High resolution observed in 800 MHz DNP spectra of extremely rigid type III secretion needles.

The cryogenic temperatures at which dynamic nuclear polarization (DNP) solid-state NMR experiments need to be carried out cause line-broadening, an effect that is especially detrimental for crowded protein spectra. By increasing the magnetic field strength from 600 to 800 MHz, the resolution of DNP spectra of type III secretion needles (T3SS) could be improved by 22 %, indicating that inhomogeneous broadening is not the dominant effect that limits the resolution of T3SS needles under DNP conditions. The outstanding spectral resolution of this system under DNP conditions can be attributed to its low overall flexibility.

An Efficient Labelling Approach to Harness Backbone and Side-Chain Protons in (1) H-Detected Solid-State NMR Spectroscopy.

(1) H-detection can greatly improve spectral sensitivity in biological solid-state NMR (ssNMR), thus allowing the study of larger and more complex proteins. However, the general requirement to perdeuterate proteins critically curtails the potential of (1) H-detection by the loss of aliphatic side-chain protons, which are important probes for protein structure and function. Introduced herein is a labelling scheme for (1) H-detected ssNMR, and it gives high quality spectra for both side-chain and backbone protons, and allows quantitative assignments and aids in probing interresidual contacts. Excellent (1) H resolution in membrane proteins is obtained, the topology and dynamics of an ion channel were studied. This labelling scheme will open new avenues for the study of challenging proteins by ssNMR.

Insight into the Supramolecular Architecture of Intact Diatom Biosilica from DNP-Supported Solid-State NMR Spectroscopy.

Diatom biosilica is an inorganic/organic hybrid with interesting properties. The molecular architecture of the organic material at the atomic and nanometer scale has so far remained unknown, in particular for intact biosilica. A DNP-supported ssNMR approach assisted by microscopy, MS, and MD simulations was applied to study the structural organization of intact biosilica. For the first time, the secondary structure elements of tightly biosilica-associated native proteins in diatom biosilica were characterized in situ. Our data suggest that these proteins are rich in a limited set of amino acids and adopt a mixture of random-coil and ß-strand conformations. Furthermore, biosilica-associated long-chain polyamines and carbohydrates were characterized, thereby leading to a model for the supramolecular organization of intact biosilica.

Efficient Dynamic Nuclear Polarization at 800 MHz/527 GHz with Trityl-Nitroxide Biradicals.

Cross-effect (CE) dynamic nuclear polarization (DNP) is a rapidly developing technique that enhances the signal intensities in magic-angle spinning (MAS) NMR spectra. We report CE DNP experiments at 211, 600, and 800 MHz using a new series of biradical polarizing agents referred to as TEMTriPols, in which a nitroxide (TEMPO) and a trityl radical are chemically tethered. The TEMTriPol molecule with the optimal performance yields a record (1) H NMR signal enhancement of 65 at 800 MHz at a concentration of 10 mM in a glycerol/water solvent matrix. The CE DNP enhancement for the TEMTriPol biradicals does not decrease as the magnetic field is increased in the manner usually observed for bis-nitroxides. Instead, the relatively strong exchange interaction between the trityl and nitroxide moieties determines the magnetic field at which the optimum enhancement is observed.