Solvent-free dynamic nuclear polarization enhancements in organically modified mesoporous silica.

High-field dynamic nuclear polarization is a powerful tool for the structural characterization of species on the surface of porous materials or nanoparticles. For these studies the main source of polarization are radical-containing solutions which are added by post-synthesis impregnation of the sample. Although this strategy is very efficient for a wide variety of materials, the presence of the solvent may influence the chemistry of functional species of interest. Here we address the development of a comprehensive strategy for solvent-free DNP enhanced NMR characterization of functional (target) species on the surface of mesoporous silica (SBA-15). The strategy includes the partial functionalization of the silica surface with Carboxy-Proxyl nitroxide radicals and target Fmoc-Glycine functional groups. As a proof of principle, we have observed for the first time DNP signal enhancements, using the solvent-free approach, for 13C{1H} CPMAS signals corresponding to organic functionalities on the silica surface. DNP enhancements of up to 3.4 were observed for 13C{1H} CPMAS, corresponding to an experimental time save of about 12 times. This observation opens the possibility for the DNP-NMR study of surface functional groups without the need of a solvent, allowing, for example, the characterization of catalytic reactions occurring on the surface of mesoporous systems of interest. For 29Si with direct polarization NMR, up to 8-fold DNP enhancements were obtained. This 29Si signal enhancement is considerably higher than the obtained with similar approaches reported in literature. Finally, from DNP enhancement profiles we conclude that cross-effect is probably the dominant polarization transfer mechanism.

2H NMR study on temperature-dependent water dynamics in amino-acid functionalized silica nanopores.

We prepare various amino-acid functionalized silica pores with diameters of ∼6 nm and study the temperature-dependent reorientation dynamics of water in these confinements. Specifically, we link basic Lys, neutral Ala, and acidic Glu to the inner surfaces and combine 2H nuclear magnetic resonance spin-lattice relaxation and line shape analyses to disentangle the rotational motions of the surfaces groups and the crystalline and liquid water fractions coexisting below partial freezing. Unlike the crystalline phase, the liquid phase shows reorientation dynamics, which strongly depends on the chemistry of the inner surfaces. The water reorientation is slowest for the Lys functionalization, followed by Ala and Glu and, finally, the native silica pores. In total, the rotational correlation times of water at the different surfaces vary by about two orders of magnitude, where this span is largely independent of the temperature in the range ∼200-250 K.

Static field gradient NMR studies of water diffusion in mesoporous silica.

NMR diffusometry is used to ascertain the pore-size dependent water diffusion in MCM-41 and SBA-15 silica over broad temperature ranges. Detailed analysis of 1H and 2H NMR stimulated-echo decays reveals that fast water motion through voids between different silica particles impairs such studies in the general case. However, water diffusion inside single pores is probed in the present approach, which applies high static field gradients to enhance the spatial resolution of the experiment and uses excess water in combination with subzero temperatures to embed the silica particles in an ice matrix and, thus, to suppress interparticle water motion. It is found that the diffusion of confined water slows down by almost two orders of magnitude when the pore diameter is reduced from 5.4 nm to 2.1 nm at weak cooling. In the narrower silica pores, the temperature dependence of the self-diffusion coefficient of water is well described by an Arrhenius law with an activation energy of Ea = 0.40 eV. The Arrhenius behavior extends over a broad temperature range of at least 207-270 K, providing evidence against a fragile-to-strong crossover in response to a proposed liquid-liquid phase transition near 225 K. In the wider silica pores, partial crystallization results in a discontinuous temperature dependence. Explicitly, the diffusion coefficients drop when cooling through the pore-size dependent melting temperatures Tm of confined water. This finding can be rationalized by the fact that water can explore the whole pore volumes above Tm, but is restricted to narrow interfacial layers sandwiched between silica walls and ice crystallites below this temperature. Comparing our findings for water diffusion with previous results for water reorientation, we find significantly different temperature dependencies, indicating that the Stokes-Einstein-Debye relation is not obeyed.

Structural Insights into Peptides Bound to the Surface of Silica Nanopores.

The structure and surface functionalization of biologically relevant silica-based hybrid materials was investigated by 2D solid-state NMR techniques combined with dynamic nuclear polarization (DNP). This approach was applied to a model system of mesoporous silica, which was modified through in-pore grafting of small peptides by solid-phase peptide synthesis (SPPS). To prove the covalent binding of the peptides on the surface, DNP-enhanced solid-state NMR was used for the detection of 15 N NMR signals in natural abundance. DNP-enhanced heterocorrelation experiments with frequency switched Lee-Goldburg homonuclear proton decoupling (1 H-13 C and 1 H-15 N CP MAS FSLG HETCOR) were performed to verify the primary structure and configuration of the synthesized peptides. 1 H FSLG spectra and 1 H-29 Si FSLG HETCOR correlation spectra were recorded to investigate the orientation of the amino acid residues with respect to the silica surface. The combination of these NMR techniques provides detailed insights into the structure of amino acid functionalized hybrid compounds and allows for the understanding for each synthesis step during the in-pore SPPS.

Ultrafast Single-Scan 2D†NMR Spectroscopic Detection of a PHIP-Hyperpolarized Protease Inhibitor.

Two-dimensional NMR spectroscopy is one of the most important spectroscopic tools for the investigation of biological macromolecules. However, due to the low sensitivity of NMR spectroscopy, it takes usually from several minutes to many hours to record such spectra. Here, the possibility of detecting a bioactive derivative of the sunflower trypsin inhibitor-1 (SFTI-1), a tetradecapeptide, by combining parahydrogen-induced polarization (PHIP) and ultrafast 2D NMR spectroscopy is shown. The PHIP activity of the inhibitor was achieved by labeling with O-propargyl-l-tyrosine. In 1D PHIP experiments a signal enhancement of a factor of approximately 1200 compared to standard NMR was found. This enhancement permits measurement of 2D NMR correlation spectra of low-concentrated SFTI-1 in less than 10 seconds, employing ultrafast single-scan 2D NMR detection. As experimental examples PHIP-assisted ultrafast single-scan TOCSY spectra of SFTI-1 are shown.

Efficient Building Blocks for Solid-Phase Peptide Synthesis of Spin Labeled Peptides for Electron Paramagnetic Resonance and Dynamic Nuclear Polarization Applications.

Specific spin labeling allows the site-selective investigation of biomolecules by EPR and DNP enhanced NMR spectroscopy. A novel spin labeling strategy for commercially available Fmoc-amino acids is developed. In this approach, the PROXYL spin label is covalently attached to the hydroxyl side chain of three amino acids hydroxyproline (Hyp), serine (Ser) and tyrosine (Tyr) by a simple three-step synthesis route. The obtained PROXYL containing building-blocks are N-terminally protected by the Fmoc-protection group, which makes them applicable for the use in solid-phase peptide synthesis (SPPS). This approach allows the insertion of the spin label at any desired position during SPPS, which makes it more versatile than the widely used post synthetic spin labeling strategies. For the final building-blocks, the radical activity is proven by EPR. DNP enhanced solid-state NMR experiments employing these building-blocks in a TCE solution show enhancement factors of up to 26 for 1 H and 13 C (1 H→13 C cross-polarization). To proof the viability of the presented building-blocks for insertion of the spin label during SPPS the penta-peptide Acetyl-Gly-Ser(PROXYL)-Gly-Gly-Gly was synthesized employing the spin labeled Ser building-block. This peptide could successfully be isolated and the spin label activity proved by EPR and DNP NMR measurements, showing enhancement factors of 12.1±0.1 for 1 H and 13.9±0.5 for 13 C (direct polarization).

Biofunctionalization of Nano Channels by Direct In-Pore Solid-Phase Peptide Synthesis.

Diatom biosilica are highly complex inorganic/organic hybrid materials. To get deeper insights on their structure at a molecular level, model systems that mimic the complex natural compounds were synthesized and characterized. A simple and efficient peptide immobilization strategy was developed, which uses a well-ordered porous silica material as a support and commercially available Fmoc-amino acids, similar to the known solid-phase peptide synthesis. As an example, Fmoc-glycine and Fmoc-phenylalanine are immobilized on the silica support. The success of functionalization was investigated by 13 C CP MAS and 29 Si CP MAS solid-state NMR. Thermogravimetric analysis (TGA) and elemental analysis (EA) were performed to quantify the functionalization. Changes of the specific surface area, pore volume, and pore diameters in all modification steps were studied by Brunauer-Emmett-Teller based nitrogen adsorption-desorption measurements (BET). The combination of the analytical methods provided high grafting densities of 2.1±0.2 molecules/nm2 on the surface. Furthermore, they allowed for monitoring chemical changes on the pore surface and changes of the pore properties of the material during the different functionalization steps. This universal approach is suitable for the selective synthesis of pores with tunable surface-peptide functionalization, with applications to the synthesis of a big variety of silica-peptide model systems, which in the future may lead to a deeper understanding of complex biological systems.

A disintegrin derivative as a case study for PHIP labeling of disulfide bridged biomolecules.

A specific labeling strategy for bioactive molecules is presented for eptifibatide (integrilin) an antiplatelet aggregation inhibitor, which derives from the disintegrin protein barbourin in the venom of certain rattlesnakes. By specifically labeling the disulfide bridge this molecule becomes accessible for the nuclear spin hyperpolarization method of parahydrogen induced polarization (PHIP). The PHIP-label was synthesized and inserted into the disulfide bridge of eptifibatide via reduction of the peptide and insertion by a double Michael addition under physiological conditions. This procedure is universally applicable for disulfide-containing biomolecules and preserves their tertiary structure with a minimum of change. HPLC and MS spectra prove the successful insertion of the label. 1H-PHIP-NMR experiments yield a factor of over 1000 as lower limit for the enhancement factor. These results demonstrate the high potential of the labeling strategy for the introduction of site selective PHIP-labels into biomolecules' disulfide bonds.

A novel strategy for site selective spin-labeling to investigate bioactive entities by DNP and EPR spectroscopy.

A novel specific spin-labeling strategy for bioactive molecules is presented for eptifibatide (integrilin) an antiplatelet aggregation inhibitor, which derives from the venom of certain rattlesnakes. By specifically labeling the disulfide bridge this molecule becomes accessible for analytical techniques such as Electron Paramagnetic Resonance (EPR) and solid state Dynamic Nuclear Polarization (DNP). The necessary spin-label was synthesized and inserted into the disulfide bridge of eptifibatide via reductive followed by insertion by a double Michael addition under physiological conditions. This procedure is universally applicable for disulfide containing biomolecules and is expected to preserve their tertiary structure with minimal change due to the small size of the label and restoring of the previous disulfide connection. HPLC and MS analysis show the successful introduction of the spin label and EPR spectroscopy confirms its activity. DNP-enhanced solid state NMR experiments show signal enhancement factors of up to 19 in 13C CP MAS experiments which corresponds to time saving factors of up to 361. This clearly shows the high potential of our new spin labeling strategy for the introduction of site selective radical spin labels into biomolecules and biosolids without compromising its conformational integrity for structural investigations employing solid-state DNP or advanced EPR techniques.

Dirhodium Coordination Polymers for Asymmetric Cyclopropanation of Diazooxindoles with Olefins: Synthesis and Spectroscopic Analysis.

A facile approach is reported for the preparation of dirhodium coordination polymers [Rh2 (L1)2 ]n (Rh2 -L1) and [Rh2 (L2)2 ]n (Rh2 -L2; L1=N,N'-(pyromellitoyl)-bis-L-phenylalanine diacid anion, L2=bis-N,N'-(L-phenylalanyl) naphthalene-1,4,5,8-tetracarboxylate diimide) from chiral dicarboxylic acids by ligand exchange. Multiple techniques including FTIR, XPS, and 1 H→13 C CP MAS NMR spectroscopy reveal the formation of the coordination polymers. 19 F MAS NMR was utilized to investigate the remaining TFA groups in the obtained coordination polymers, and demonstrated near-quantitative ligand exchange. DR-UV-vis and XPS confirm the oxidation state of the Rh center and that the Rh-single bond in the dirhodium node is maintained in the synthesis of Rh2 -L1 and Rh2 -L2. Both coordination polymers exhibit excellent catalytic performance in the asymmetric cyclopropanation reaction between styrene and diazooxindole. The catalysts can be easily recycled and reused without significant reduction in their catalytic efficiency.

Structure-based optimization of oxadiazole-based GSK-3 inhibitors.

Inhibition of glycogen synthase kinase-3 (GSK-3) induces neuroprotective effects, e.g. decreases ß-amyloid production and reduces tau hyperphosphorylation, which are both associated with Alzheimer's disease (AD). The two isoforms of GSK-3 in mammalians are GSK-3α and ß, which share 98% homology in their catalytic domains. We investigated GSK-3 inhibitors based on 2 different scaffolds in order to elucidate the demands of the ATP-binding pocket [1]. Particularly, the oxadiazole scaffold provided potent and selective GSK-3 inhibitors. For example, the most potent inhibitor of the present series, the acetamide 26d, is characterized by an IC50 of 2 nM for GSK-3α and 17 nM for GSK-3ß. In addition, the benzodioxane 8g showed up to 27-fold selectivity for GSK-3α over GSK-3ß, with an IC50 of 35 nM for GSK-3α. Two GSK-3 inhibitors were further profiled for efficacy and toxicity in the wild-type (wt) zebrafish embryo assay to evaluate simultaneously permeability and safety.