Investigation of Bis(α-Phenylethylamido)Methylphosphonates by NMR Spectroscopy

Abstract

The influence of the relative location of chiral carbon and phosphorus atoms and the position of the aryl-group on diastereomeric anisochronity in non-racemic mixtures of enantiomers has been examined. The optical isomers of methanephosphonic acid bis(α-phenylethylamide) (I), which contain pro-chiral phosphorus atom, were synthesized by the reaction of methanephosphonic acid dichloride(II) with (+)- or (-) -α-phenylethylamine (III). The extent of chemical shift nonequivalence in ³¹P and ¹H NMR spectra of non-racemic mixtures of RR- and SS-I was negligible. It can be explained by the absence of aryl-amino group, favouring intermolecular association, effect of SCADA¹, and the long distance between the chiral center and pro-chiral phosphorus. Phosphonylation of non-racemic mixtures of (+)- and (-)-III by II proceeds stereospecifically giving mixture of RR-, SS-I, and two meso-compounds analyzed by ³¹P ¹H NMR.     

PI by NMR: Probing CH–π Interactions in Protein–Ligand Complexes by NMR Spectroscopy

While CH–π interactions with target proteins are crucial determinants for the affinity of arguably every drug molecule, no method exists to directly measure the strength of individual CH–π interactions in drug–protein complexes. Herein, we present a fast and reliable methodology called PI (π interactions) by NMR, which can differentiate the strength of protein–ligand CH–π interactions in solution. By combining selective amino-acid side-chain labeling with ¹H-¹³C NMR, we are able to identify specific protein protons of side-chains engaged in CH–π interactions with aromatic ring systems of a ligand, based solely on ¹H chemical-shift values of the interacting protein aromatic ring protons. The information encoded in the chemical shifts induced by such interactions serves as a proxy for the strength of each individual CH–π interaction. PI by NMR changes the paradigm by which chemists can optimize the potency of drug candidates: direct determination of individual π interactions rather than averaged measures of all interactions.     

Fluorinated PLGA Nanoparticles for Enhanced Drug Encapsulation and 19F NMR Detection

In the continuous search for multimodal systems with combined diagnostic and therapeutic functions, several efforts have been made to develop multifunctional drug delivery systems. In this work, through a covalent approach, a new class of fluorinated poly(lactic-co-glycolic acid) co-polymers (F-PLGA) were designed that contain an increasing number of magnetically equivalent fluorine atoms. In particular, two novel compounds, F₃-PLGA and F₉-PLGA, were synthesized and their chemical structure and thermal stability were analyzed by solution NMR, DSC, and TGA. The obtained F-PLGA compounds were proven to form in aqueous solution colloidal stable nanoparticles (NPs) displaying a strong ¹⁹F NMR signal. The fluorinated NPs also showed an enhanced ability to load hydrophobic drugs containing fluorine atoms compared to analogous pristine PLGA NPs. Preliminary in vitro studies showed high cell viability and the NP ability to intracellularly deliver and release a functioning drug.     

Investigation of Geometrical and Conformational Isomers of Cobalt(II) Bis(diphenylacetato)-bis(γ-picolinate) by Dynamic NMR Spectroscopy and Molecular Mechanics

Cobalt(II) bis(diphenylacetato)-bis(-picolinate) is synthesized and its geometrical and conformational isomers are investigated using dynamic NMR and molecular mechanics methods. The complex in the solution is shown to exist as cis and trans isomers, which are detected at 263 K in the course of a slow intermolecular ligand exchange. At T < 223 K, the intramolecular rotation of the phenyl radicals about the ^–O₂C–CH(Ph)₂ and ^–O₂CCH(Ph)–Ph bonds slows, which results in the formation of different conformers. The energies of the retarded intermolecular exchange and of the retarded rotation of the phenyl radicals were calculated from ¹H NMR and molecular mechanics data.     

Determination of the Chemical Structure of Poly-β (-)-pinene by NMR Spectroscopy

This paper is dedicated to the memory of our friend and colleague Annalaura Segre.

The chemical structure of a series of β (-)-pinene polymers (PBP) obtained by radiation-induced polymerization, free radical initiation, cationic polymerization over a Friedel-Craft catalyst and by coordinative polymerization over a Ziegler-Natta catalyst has been fully elucidated by ¹H and ¹³C-NMR spectroscopy. 2D NMR techniques have been applied in order to assign all the NMR resonances to the structures of the PBP investigated. The NMR spectra show that the most regular PBP structure is obtained by radiation-induced polymerization followed by the free radical initiated polymerization. The most defective structure has been observed in the case of PBP prepared by cationic mechanism over a Friedel-Crafts catalyst. The discussion accounts for different types of defects and cross-links present in the PBPs investigated whose fundamental structure is based on the p-menthene repeating unit.

NMR self-diffusion measurements have been performed to evaluate the molecular weight of all the PBP investigated. The highest molecular weight (2600 Dalton) was found in the case of PBP prepared by Ziegler-Natta catalyst, while the lowest molecular weight was found in the case of PBP prepared by radiation-induced polymerization (about 1000 Dalton).     

Unleashing the Potential of 17O NMR Spectroscopy Using Mechanochemistry

¹⁷O NMR spectroscopy has been the subject of vivid interest in recent years, because there is increasing evidence that it can provide unique insight into the structure and reactivity of many molecules and materials. However, due to the very poor natural abundance of oxygen-17, ¹⁷O labeling is generally a prerequisite. This is a real obstacle for most research groups, because of the high costs and/or strong experimental constraints of the most frequently used ¹⁷O-labeling schemes. Here, we show for the first time that mechanosynthesis offers unique opportunities for enriching in ¹⁷O a variety of organic and inorganic precursors of synthetic interest. The protocols are fast, user-friendly, and low-cost, which makes them highly attractive for a broad research community, and their suitability for ¹⁷O solid-state NMR applications is demonstrated.     

Real-Time Analysis of Folding upon Binding of a Disordered Protein by Using Dissolution DNP NMR Spectroscopy

The kinase inhibitory domain of the cell cycle regulatory protein p27^Kip1 (p27) was nuclear spin hyperpolarized using dissolution dynamic nuclear polarization (D-DNP). While intrinsically disordered in isolation, p27 adopts secondary structural motifs, including an α-helical structure, upon binding to cyclin-dependent kinase 2 (Cdk2)/cyclin A. The sensitivity gains obtained with hyperpolarization enable the real-time observation of ¹³C NMR signals during p27 folding upon binding to Cdk2/cyclin A on a time scale of several seconds. Time-dependent intensity changes are dependent on the extent of folding and binding, as manifested in differential spin relaxation. The analysis of signal decay rates suggests the existence of a partially folded p27 intermediate during the timescale of the D-DNP NMR experiment.     

The 3F Library: Fluorinated Fsp3-Rich Fragments for Expeditious 19F NMR Based Screening

Fragment-based drug discovery (FBDD) is a popular method in academia and the pharmaceutical industry for the discovery of early lead candidates. Despite its wide-spread use, the approach still suffers from laborious screening workflows and a limited diversity in the fragments applied. Presented here is the design, synthesis, and biological evaluation of the first fragment library specifically tailored to tackle both these challenges. The 3F library of 115 fluorinated, Fsp³-rich fragments is shape diverse and natural-product-like with desirable physicochemical properties. The library is perfectly suited for rapid and efficient screening by NMR spectroscopy in a two-stage workflow of ¹⁹F NMR and subsequent ¹H NMR methods. Hits against four diverse protein targets are widely distributed among the fragment scaffolds in the 3F library and a 67 % validation rate was achieved using secondary assays. This collection is the first synthetic fragment library tailor-made for ¹⁹F NMR screening and the results demonstrate that the approach should find broad application in the FBDD community.     

Stereoselective Synthesis of Fluorinated Galactopyranosides as Potential Molecular Probes for Galactophilic Proteins: Assessment of Monofluorogalactoside–LecA Interactions

The replacement of hydroxyl groups by fluorine atoms on hexopyranoside scaffolds may allow access to invaluable tools for studying various biochemical processes. As part of ongoing activities toward the preparation of fluorinated carbohydrates, a systematic investigation involving the synthesis and biological evaluation of a series of mono- and polyfluorinated galactopyranosides is described. Various monofluorogalactopyranosides, a trifluorinated, and a tetrafluorinated galactopyranoside have been prepared using a Chiron approach. Given the scarcity of these compounds in the literature, in addition to their synthesis, their biological profiles were evaluated. Firstly, the fluorinated compounds were investigated as antiproliferative agents using normal human and mouse cells in comparison with cancerous cells. Most of the fluorinated compounds showed no antiproliferative activity. Secondly, these carbohydrate probes were used as potential inhibitors of galactophilic lectins. The first transverse relaxation-optimized spectroscopy (TROSY) NMR experiments were performed on these interactions, examining chemical shift perturbations of the backbone resonances of LecA, a virulence factor from Pseudomonas aeruginosa. Moreover, taking advantage of the fluorine atom, the ¹⁹F NMR resonances of the monofluorogalactopyranosides were directly monitored in the presence and absence of LecA to assess ligand binding. Lastly, these results were corroborated with the binding potencies of the monofluorinated galactopyranoside derivatives by isothermal titration calorimetry experiments. Analogues with fluorine atoms at C-3 and C-4 showed weaker affinities with LecA as compared to those with the fluorine atom at C-2 or C-6. This research has focused on the chemical synthesis of “drug-like” low-molecular-weight inhibitors that circumvent drawbacks typically associated with natural oligosaccharides.     

Aromatic 19F–13C TROSY—[19F, 13C]-Pyrimidine Labeling for NMR Spectroscopy of RNA

We present the access to [5-¹⁹F, 5-¹³C]-uridine and -cytidine phosphoramidites for the production of site-specifically modified RNAs up to 65 nucleotides (nts). The amidites were used to introduce [5-¹⁹F, 5-¹³C]-pyrimidine labels into five RNAs—the 30 nt human immunodeficiency virus trans activation response (HIV TAR) 2 RNA, the 61 nt human hepatitis B virus ϵ (hHBV ϵ) RNA, the 49 nt SAM VI riboswitch aptamer domain from B. angulatum, the 29 nt apical stem loop of the pre-microRNA (miRNA) 21 and the 59 nt full length pre-miRNA 21. The main stimulus to introduce the aromatic ¹⁹F–¹³C-spin topology into RNA comes from a work of Boeszoermenyi et al., in which the dipole-dipole interaction and the chemical shift anisotropy relaxation mechanisms cancel each other leading to advantageous TROSY properties shown for aromatic protein sidechains. This aromatic ¹³C–¹⁹F labeling scheme is now transferred to RNA. We provide a protocol for the resonance assignment by solid phase synthesis based on diluted [5-¹⁹F, 5-¹³C]/[5-¹⁹F] pyrimidine labeling. For the 61 nt hHBV ϵ we find a beneficial ¹⁹F–¹³C TROSY enhancement, which should be even more pronounced in larger RNAs and will facilitate the NMR studies of larger RNAs. The [¹⁹F, ¹³C]-labeling of the SAM VI aptamer domain and the pre-miRNA 21 further opens the possibility to use the biorthogonal stable isotope reporter nuclei in in vivo NMR to observe ligand binding and microRNA processing in a biological relevant setting.     

Exploring the Structural Details of Cu(I) Binding to α-Synuclein by NMR Spectroscopy

The aggregation of α-synuclein (AS) is selectively enhanced by copper in vitro, and the interaction is proposed to play a potential role in vivo. In this work, we report the structural, residue-specific characterization of Cu(I) binding to AS and demonstrate that the protein is able to bind Cu(I) with relatively high affinity in a coordination environment that involves the participation of Met1 and Met5 residues. This knowledge is a key to understanding the structural-aggregation basis of the copper-catalyzed oxidation of AS.     

The Investigation of Oxygen Absorption over LaOF by Means of Raman Spectroscopy

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Structure of a Protein–RNA Complex by Solid-State NMR Spectroscopy

Solid-state NMR (ssNMR) is applicable to high molecular-weight (MW) protein assemblies in a non-amorphous precipitate. The technique yields atomic resolution structural information on both soluble and insoluble particles without limitations of MW or requirement of crystals. Herein, we propose and demonstrate an approach that yields the structure of protein–RNA complexes (RNP) solely from ssNMR data. Instead of using low-sensitivity magnetization transfer steps between heteronuclei of the protein and the RNA, we measure paramagnetic relaxation enhancement effects elicited on the RNA by a paramagnetic tag coupled to the protein. We demonstrate that this data, together with chemical-shift-perturbation data, yields an accurate structure of an RNP complex, starting from the bound structures of its components. The possibility of characterizing protein–RNA interactions by ssNMR may enable applications to large RNP complexes, whose structures are not accessible by other methods.     

Hydrolysis of Tetraglycine by a Zr(IV)-Substituted Wells–Dawson Polyoxotungstate Studied by Diffusion Ordered NMR Spectroscopy

The use of diffusion ordered NMR spectroscopy (DOSY) for the analysis of complex reaction mixtures involving polyoxometalates (POMs) was demonstrated for the hydrolysis of the peptide tetraglycine by the K₁₅H[Zr(α₂-P₂W₁₇O₆₁)₂]·25H₂O Wells–Dawson type cluster. ¹H DOSY NMR studies have shown that severe signal overlap observed in the one-dimensional ¹H NMR spectrum of reaction mixtures containing a POM and peptides could be overcome by the two-dimensional character of a DOSY NMR measurement. A clear distinction between the ¹H NMR signals of the products formed during the hydrolysis of 5.0 mM of tetraglycine catalyzed by 1.0 mM of K₁₅H[Zr(α₂-P₂W₁₇O₆₁)₂]·25H₂O was observed based on the extra dimension containing information about diffusion coefficients that distinguishes a typical DOSY measurement from conventionally used 1D ¹H NMR. The spectrum clearly shows the presence of 5 species with diffusion coefficients of 3.71 × 10^?10 m²/s (3.91; 3.84; 3.82 and 3.62 ppm), 4.39 × 10^?10 m²/s (3.87; 3.76 and 3.61 ppm), 5.26 × 10^?10 m²/s (3.67 and 3.63 ppm), and 7.46 × 10^?10 m²/s (3.37 ppm) that are assigned to the non-hydrolyzed tetraglycine, the hydrolysis intermediate products triglycine and glycylglycine, and the end product of hydrolysis glycine, respectively. In addition, a signal assigned to cyclic glycylglycine, with a diffusion coefficient practically identical to the diffusion coefficient of glycylglycine was observed at 3.86 ppm. In addition, ¹H and ³¹P NMR spectroscopy were further used to study the binding of tetraglycine to K₁₅H[Zr(α₂-P₂W₁₇O₆₁)₂]·25H₂O and the solution speciation of K₁₅H[Zr(α₂-P₂W₁₇O₆₁)₂]·25H₂O.     

Sensitive Morphological Characterization of Oriented High-Density Lipoprotein Nanoparticles Using 31P NMR Spectroscopy

The biological function of high-density lipoprotein (HDL) nanoparticles, the so-called good cholesterol that is associated with a low risk of heart disease, depends on their composition, morphology, and size. The morphology of HDL particles composed of apolipoproteins, lipids and cholesterol is routinely visualised by transmission electron microscopy (TEM), but higher-resolution tools are needed to observe more subtle structural differences between particles of different composition. Here, reconstituted HDL formulations are oriented on glass substrates and solid-state ³¹P NMR spectroscopy is shown to be highly sensitive to the surface curvature of the lipid headgroups. The spectra report potentially functionally important differences in the morphology of different HDL preparations that are not detected by TEM. This method provides new morphological insights into HDL comprising a naturally occurring apolipoprotein A-I mutant, which may be linked to its atheroprotective properties, and holds promise as a future research tool in the clinical analysis of plasma HDL.     

A (31) P NMR Investigation of the CoPi Water-Oxidation Catalyst

Beneath the sheets: (31) P?NMR data suggests that phosphates are liberated freely in the interlayer of a cobalt-hydroxide water-oxidation catalyst. The cobalt-hydroxide sheets are separated by an interlayer region with water, counterions and phosphate, which help to shuttle protons as the layer develops charge.     

Investigation of nanodispersion in polystyrene–montmorillonite nanocomposites by solid-state NMR

Nanocomposites result from combinations of materials with vastly different properties in the nanometer scale. These materials exhibit many unique properties such as improved thermal stability, reduced flammability, and improved mechanical properties. Many of the properties associated with polymer–clay nanocomposites are a function of the extent of exfoliation of the individual clay sheets or the quality of the nanodispersion. This work demonstrates that solid-state NMR can be used to characterize, quantitatively, the nanodispersion of variously modified montmorillonite (MMT) clays in polystyrene (PS) matrices. The direct influence of the paramagnetic Fe³⁺, embedded in the aluminosilicate layers of MMT, on polymer protons within about 1 nm from the clay surfaces creates relaxation sources, which, via spin diffusion, significantly shorten the overall proton longitudinal relaxation time (T). Deoxygenated samples were used to avoid the particularly strong contribution to the T of PS from paramagnetic molecular oxygen. We used T as an indicator of the nanodispersion of the clay in PS. This approach correlated reasonably well with X-ray diffraction and transmission electron microscopy (TEM) data. A model for interpreting the saturation-recovery data is proposed such that two parameters relating to the dispersion can be extracted. The first parameter, f, is the fraction of the potentially available clay surface that has been transformed into polymer–clay interfaces. The second parameter, ϵ, is a relative measure of the homogeneity of the dispersion of these actual polymer–clay interfaces. Finally, a quick assay of T is reported for samples equilibrated with atmospheric oxygen. Included are these samples as well as 28 PS/MMT nanocomposite samples prepared by extrusion. These measurements are related to the development of high-throughput characterization techniques. This approach gives qualitative indications about dispersion; however, the more time-consuming analysis, of a few deoxygenated samples from this latter set, offers significantly greater insight into the clay dispersion. A second, probably superior, rapid-analysis method, applicable to oxygen-containing samples, is also demonstrated that should yield a reasonable estimate of the f parameter. Thus, for PS/MMT nanocomposites, one has the choice of a less complete NMR assay of dispersion that is significantly faster than TEM analysis, versus a slower and more complete NMR analysis with sample times comparable to TEM, information rivaling that of TEM, and a substantial advantage that this is a bulk characterization method. © 2003 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys 41: 3188–3213, 2003     

Investigation of the Interaction between Isoflavonoids and Bovine Serum Albumin by Fluorescence Spectroscopy

The interactions of bovine serum albumin （BSA） with three structurally related isoflavonoids, genistein, puerarin and daidzein, were studied under physiological conditions by fluorescence spectroscopic technique. The quenching mechanism of these compounds with BSA was suggested as static quenching and the binding constants were determined at different temperatures based on the fluorescence quenching results. The transfer efficiency of energy and distance between the acceptor and BSA were investigated on the basis of the mechanism of the Forster energy transference. According to the thermodynamic parameters it has been suggested that the acting force be mainly hydrophobic force. The comparison of binding potency of the three isoflavonoids to BSA showed that the substitution by 5-OH and 8-Glc could enhance the binding affinity. All these obtained in the work can make us better understand the mode of the action and pharmacological activities of the isoflavonoids.     

Detection of picramic acid and picramate in henné products by NMR Spectroscopy

Painting and body art are increasing their utilisation as well as their cultural impact, since piercing and tattoos are expanding social phenomena, involving many young people. However, the utilised materials often enter the market with insufficient control and several cases of skin damages are reported. Safety of the utilised products must be ensured by adequate quality controls which must be easily made, rapid, low cost, clear and persuasive. The method here reported, regards the analysis on the possible presence of picramic acid in the ethyl acetate extracts of commercial henné powders by NMR Spectroscopy. In the proton spectrum, three sets of peaks could be detected, corresponding to the three classes of makers resonances: hennosides, typical markers of Lawsonia inermis, the henné plant; picramic acid or picramate; fatty acids. In particular, the set of signals corresponding to hennosides can be used as markers of the utilisation of the correct raw material of henné.