Efficient Screening of Target-Specific Selected Compounds in Mixtures by 19F NMR Binding Assay with Predicted 19F NMR Chemical Shifts

Ligand-based ¹⁹F NMR screening is a highly effective and well-established hit-finding approach. The high sensitivity to protein binding makes it particularly suitable for fragment screening. Different criteria can be considered for generating fluorinated fragment libraries. One common strategy is to assemble a large, diverse, well-designed and characterized fragment library which is screened in mixtures, generated based on experimental ¹⁹F NMR chemical shifts. Here, we introduce a complementary knowledge-based ¹⁹F NMR screening approach, named ¹⁹Focused screening, enabling the efficient screening of putative active molecules selected by computational hit finding methodologies, in mixtures assembled and on-the-fly deconvoluted based on predicted ¹⁹F NMR chemical shifts. In this study, we developed a novel approach, named LEFshift, for ¹⁹F NMR chemical shift prediction using rooted topological fluorine torsion fingerprints in combination with a random forest machine learning method. A demonstration of this approach to a real test case is reported.     

Stereochemical analysis of acrylic copolymers with large polar side groups by NMR spectroscopy

NMR spectra give abundant information at the microstructural level. When pseudoasymmetric carbon atoms are present in the monomeric repeating units, it is possible to obtain valuable information about the relative stereochemical configuration of the side substituents of the pseudoasymmetric center. This is the best way to know the tacticity of a polymer and the cotacticity parameters of copolymers, as well as the average distribution of comonomeric units along the chains, according to the polymerization mechanism. In this work, we present the microstructural and stereochemical characterization of acrylic copolymers prepared by free radical copolymerization of 2-hydroxyethyl methacrylate with a methacrylic ester or methacrylamide derivative, bearing polar side groups of biomedical interest. These copolymer systems were analyzed by ¹H-NMR spectroscopy to determine the average molar composition, and the corresponding reactivity ratios, and by ¹³C-NMR to study the microstructural and stereochemical configuration of comonomer sequences in terms of triads and pentads. Analyses account for the mechanism of polymerization, which fits first order Markov statistics for the addition of comonomers to free radical growing ends. The stereochemical distribution is described by a Bernoullian trial.     

Design and preparation of polyphenyl distance markers for solid-state 19F NMR

With ¹³C-labeled samples, it is possible to measure internuclear distances up to 7 Å by solid-state NMR, thus providing a powerful tool for probing ligand—receptor interactions. However, limitations in measurable distances and appreciable natural abundant ¹³C background signals present problems in solid-state ¹³C NMR. In order to overcome these disadvantages, a set of reference compounds with known F—F distances, namely, quinolinol, p-biphenyl, and p-terphenyl-bearing trifluoromethyl and trifluoromethylthio groups, have been synthesized. The preparation of these reference compounds and means for diluting these references in solid-state NMR are described.     

13C-19F去耦在含氟有机化合物结构鉴定中的应用

对合氟有机化合物开展13C-19F去耦实验,分析去耦效果,结合13C-19F去耦实验归属含氟有机化合物13C NMR化学位移.     

Impedance spectroscopy analysis of Pb5Al3F19

The ac conductivity of Pb₅Al₃F₁₉ between 293 and 743 K has been analyzed within a combined complex impedance, modulus and permittivity formalism. The antiferroelectric phase IV to ferroelastic phase III, and the phase III to paraelastic phase II, transitions have been characterized; the latter is shown to be accompanied by higher F⁻ ion mobility above T=368±5 K. A new dielectric anomaly observed iso-chronously in ε′ (f,T) at 670 K corresponds to an expected, but hitherto unobserved, transition from phase II to the paraelectric prototype phase I of Pb₅Al₃F₁₉.     

NMR spectroscopy as basis for characterization of Pluronic® F108 and its derivatives

The hydroxyl end groups of Pluronic®F108 {a triblock copolymer surfactant of poly(ethylene glycol) and poly(propylene glycol) [PEG‐PPG‐PEG]} were modified into primary amine, sulfonic acid, and quaternary ammonium equivalents for use in affinity chromatography. NMR was used for monitoring the efficacy of modifications on intermediaries and final products. The primary amine equivalents were prepared via conversion of the hydroxyl groups to a tosylate, its displacement with an azide, followed by reduction to the primary amine. The sulfonic acid equivalents were prepared via hydroxyl group tosylation, the displacement of tosylate with thiol, and its oxidation to sulfonic acid. The conversion to trimethyl ammonium was achieved via hydroxyl group tosylation, tosylate displacement by halide, and halide displacement with trimethylamine. © 2000 John Wiley & Sons, Inc. J Appl Polym Sci 78: 109–117, 2000     

4-fluorophenylglycine as a label for 19F NMR structure analysis of membrane-associated peptides

The non-natural amino acid 4-fluorophenylglycine (4F-Phg) was incorporated into several representative membrane-associated peptides for dual purpose. The (19)F-substituted ring is directly attached to the peptide backbone, so it not only provides a well-defined label for highly sensitive (19)F NMR studies but, in addition, the D and L enantiomers of the stiff side chain may serve as reporter groups on the transient peptide conformation during the biological function. Besides peptide synthesis, which is accompanied by racemisation of 4F-Phg, we also describe separation of the epimers by HPLC and removal of trifluoroacetic acid. As a first example, 18 different analogues of the fusogenic peptide "B18" were prepared and tested for induction of vesicle fusion; the results confirmed that hydrophobic sites tolerated 4F-Phg labelling. Similar fusion activities within each pair of epimers suggest that the peptide is less structured in the fusogenic transition state than in the helical ground state. In a second example, five doubly labelled analogues of the antimicrobial peptide gramicidin S were compared by using bacterial growth inhibition assays. This cyclic beta-sheet peptide could accommodate both L and D substituents on its hydrophobic face. As a third example, we tested six analogues of the antimicrobial peptide PGLa. The presence of d-4F-Phg reduced the biological activity of the peptide by interfering with its amphiphilic alpha-helical fold. Finally, to illustrate the numerous uses of l-4F-Phg in (19)F NMR spectroscopy, we characterised the interaction of labelled PGLa with uncharged and negatively charged membranes. Observing the signal of the free peptide in an aqueous suspension of unilamellar vesicles, we found a linear saturation behaviour that was dominated by electrostatic attraction of the cationic PGLa. Once the peptide is bound to the membrane, however, solid-state (19)F NMR spectroscopy of macroscopically oriented samples revealed that the charge density has virtually no further influence on the structure, alignment or mobility of the peptide.     

Ionic mobility and structure of glasses based on indium and bismuth fluorides from data of IR and19F NMR spectroscopy

The InF₃-BaF₂-BiF₃ glass-forming system is investigated by the IR and¹⁹F NMR spectroscopic techniques. The influence of bismuth trifluoride on the dynamic behavior of fluorine ions and the structure of glasses in this system is demonstrated.     

Design and Generation of Highly Diverse Fluorinated Fragment Libraries and their Efficient Screening with Improved 19F NMR Methodology

Fragment screening performed with ¹⁹F NMR spectroscopy is becoming increasingly popular in drug discovery projects. With this approach, libraries of fluorinated fragments are first screened using the direct‐mode format of the assay. The choice of fluorinated motifs present in the library is fundamental in order to ensure a large coverage of chemical space and local environment of fluorine (LEF). Mono‐ and poly‐fluorinated fragments to be included in the libraries for screening are selected from both in‐house and commercial collections, and those that are ad hoc designed and synthesized. Additional fluorinated motifs to be included in the libraries derive from the fragmentation of compounds in development and launched on the market, and compounds contained in other databases (such as Integrity, PDB and ChEMBL). Complex mixtures of highly diverse fluorine motifs can be rapidly screened and deconvoluted in the same NMR tube with a novel on the fly combined procedure for the identification of the active molecule(s). Issues and problems encountered in the design, generation and screening of diverse fragment libraries of fluorinated compounds with ¹⁹F NMR spectroscopy are analyzed and technical solutions are provided to overcome them. The versatile screening methodology described here can be efficiently applied in laboratories with limited NMR setup and could potentially lead to the increasing role of ¹⁹F NMR in the hit identification and lead optimization phases of drug discovery projects.     

Simultaneous 19F NMR Screening of Prolyl Oligopeptidase and Dipeptidyl Peptidase IV Inhibitors

Prolyl oligopeptidase (POP) and dipeptidyl peptidase IV (DPP IV) are serine proteases that belong to the same family of enzymes. These peptidases are relevant because of their association with the pathophysiology of serious illnesses, such as type 2 diabetes (DPP IV), and those related to cognitive disorders (POP). Several NMR‐based screening methods are being used to find and validate new hit scaffolds. In particular, ¹⁹F NMR‐based screening methods have proven to be powerful tools for the discovery and development of new inhibitors. Here we present an accurate and reliable ¹⁹F NMR‐based simultaneous assay that is used to screen for new selective POP and DPP IV inhibitors in compound mixtures. This activity assay consists of the simultaneous performance of POP and DPP‐IV ¹⁹F NMR activity assays in the presence of their fluorine‐containing substrates. Furthermore, the assays were conducted in the presence of 0.01 % v/v of Triton X‐100, which is a detergent that disrupts micelle formation, thereby preventing unspecific aggregate‐based inhibition. Finally, this ¹⁹F NMR methodology was applied to screen for ligands in plant extracts. Our results indicate that this method allows the simultaneous and accurate identification of selective POP and DPP IV inhibitors in these compound mixtures.     

Magnetic field simulations in support of interdiffusion quantification with NMR

Modelling the mixing process of multicomponent liquids is still an open problem. In this work the possibility to use nuclear magnetic resonance (NMR) to measure concentration profiles during interdiffusion of a binary system is discussed. NMR is a non-invasive technique that can be used to measure diffusivities of liquids over the complete viscosity range even for optically non-transparent samples. This work discusses the difficulties that appear when measuring concentration profiles of a binary system. During the mixing process of two different liquids, the concentration gradient leads to a time-dependent distribution of magnetic susceptibility within the sample. NMR experiments as well as numerical simulations are used to show the magnetic field distortions introduced by the susceptibility of pure liquids as well as their mixture in the sample. If the field inhomogeneity is comparable to the chemical shift difference, the broadening of the NMR spectrum leads to important overlapping of the NMR lines. The lack of knowledge about the line shape, influenced by the field inhomogeneity, introduces errors in the concentration profile. Finally, a numerical method based on the simulation of the magnetic field distribution generated by a given concentration gradient is proposed to increase the accuracy of the concentration measurements.     

Solid state 19F NMR study of crystal transformation in PVDF and its nanocomposites

The polymorphism of poly(vinylidene fluoride) (PVDF) and its nanocomposites was studied by means of solid state nuclear magnetic resonance spectroscopy. ¹³C cross polarization magic angle spinning (¹³C CP MAS) NMR spectra were recorded using simultaneous high‐power decoupling on both the proton and fluorine channels. Both ¹H → ¹³C and ¹⁹F → ¹³C CP experiments were conducted, giving identical results apart from intensity variations due to the CP efficiency. Two main resonances for the CF₂ and the CH₂ groups were observed for both neat PVDF (PVDF‐C0) and the nanocomposite containing 2 wt% clay (PVDF‐C2) samples. ¹⁹F CP MAS spectra were obtained from long proton spin‐lock experiments with a shorter contact time. The results showed two strong resonances at ?84 and ?98 ppm with equal intensities, representing the α‐form crystalline structure of PVDF. It was shown that the clay induces the crystallization of PVDF in β‐form. Our earlier investigations using thermal analysis and X‐ray scattering methods also showed crystal transformation of PVDF in its clay nanocomposites. POLYM. ENG. SCI. 46:1684–1690, 2006. © 2006 Society of Plastics Engineers     

Raman spectroscopy, 19F and 31P MAS-NMR of a series of fluorochloroapatites

We report the Raman spectra of a series of fluorochloroapatites Ca₅(PO₄)₃F_1?xCl_x, where x = 0.0, 0.1, 0.2, 0.4, 0.5, 0.6, 0.8, 0.9 and 1.0 (i.e. from pure fluorapatite to chlorapatite). The series did not appear to exhibit any immiscibility, however peak broadening and additional Raman and NMR spectral features on chlorine addition could be interpreted as reduction in symmetry and ordering in the ion channels. All Raman bands showed significant broadening with chlorine substitution, indicating disordering of the crystal lattice or ordering into fluorine and chlorine rich environments. There was also a general trend of the Raman bands to shift to lower wavenumber linearly with chlorine substitution with the ν₁ phosphate band decreasing from 966 cm^?1 for x = 0.0 to 961 cm^?1 for x = 1.0. The full width half maximum (FWHM) of this band increases linearly with chlorine addition from 5 cm^?1 for x = 0.0 to 10 cm^?1 for x = 1.0. The shift in a component in the ν₃ phosphate band with chlorine addition at around 1035 cm^?1 agrees with data in the literature on chlorine containing geological apatites. An additional component was seen at around 586 cm^?1 in the ν₄ phosphate region for chlorapatite and the area of this band decreased linearly to zero as fluorine replaced chlorine. This could be due to E_g symmetry phonons splitting into A_g and B_g modes due to a symmetry change such as hexagonal to monoclinic with chlorine addition. ¹⁹F magic angle spinning nuclear magnetic resonance (MAS-NMR) spectra showed a shift to lower ppm values with chlorine addition and a broadening of resonances, consistent with the Raman data. The ³¹P spectra developed additional shoulders (at around 2 and 4 ppm) with chlorine addition with the main peak position (3.3 ppm for x = 0) decreasing for compositions moving away from x = 0.5 indicating maximum phosphorous nuclear shielding occurs at an approximate F:Cl ratio of 1:1. This discontinuity is a possible indication of a structural transition at x = 0.5 related to local short scale phosphate order ? disorder or a change in crystal symmetry.     

Identification by 19F NMR of traditional Chinese medicinal plants possessing prolyl oligopeptidase inhibitory activity

Prolyl oligopeptidase is a cytosolic serine peptidase that hydrolyzes proline-containing peptides at the carboxy termini of the proline residues. This peptidase has been associated with schizophrenia, bipolar affective disorder, and related neuropsychiatric disorders and might therefore have important clinical implications. Traditional Chinese medicinal (TCM) plants provide a rich source of unexplored compounds for strategies to find novel POP inhibitors, but the traditional methodologies used to identify POP inhibitors could have some limitations when working with natural products: interference with the colorimetric or fluorimetric detection methods commonly used to screen for POP inhibitors can result in the generation of false positives or false negatives. Since NMR screening is less prone to such interference, we decided to explore the use of 19F NMR to screen for POP inhibitors. We synthesized a new 19F-labeled POP substrate--Z-Gly-Pro-Phe-4(CF3)-NH2--and used it to search for new POP inhibitors in TCM plant extracts. We identified several plants with high POP-inhibitory activity and show here that the combination of 19F NMR and TCM plant extracts is a useful tool for identifying new POP inhibitors.     

Detection and quantification of branching in polyacrylates by size-exclusion chromatography (SEC) and melt-state C NMR spectroscopy

Chain branching has been investigated in a homologous series of poly(n-alkyl acrylates) (methyl, ethyl, n-butyl, n-hexyl) obtained by radical polymerization. The total amount of chain branching was quantified using melt-state ¹³C nuclear magnetic resonance (NMR) spectroscopy. It gave access to low degrees of branching in both soluble and insoluble polyacrylates, homopolymers and copolymers. The lowest degree of branching was found for the ethyl member of the series with quantification by conventional solution-state NMR found to take a prohibitively long time. The method proposed here is compared to the ones published previously, and previous literature results are critically reviewed.The presence of long-chain branching (LCB) was selectively detected using multiple-detection size-exclusion chromatography (SEC), with LCB being found for all soluble homopolymers but the poly(n-butyl acrylate). This finding was confirmed by close examination of the Mark-Houwink parameters for the various polyacrylates studied in this work or those previously published.     

Removal of sodium acetate in poly(vinyl alcohol) and its quantification by 1H NMR spectroscopy

A major impurity in poly (vinyl alcohol) (PVA) is sodium acetate which remains after its preparation by a base catalyzed hydrolysis of poly(vinyl acetate), and the amount of sodium acetate in commercial PVA samples may reach several percentages. To establish an optimal condition for the removal of sodium acetate, several washing parameters such as washing period, solvent polarity, and temperature were investigated in this study. Nuclear magnetic resonance (NMR) spectroscopy was successfully applied to determine the residual amounts of sodium acetate in the purified poly(vinyl alcohol). The relative integral value for the methyl peak of sodium acetate in PVA was converted to a relative mass value and finally to the sodium acetate content contained in PVA. The results showed that over 95% of sodium acetate in PVA was removed by a washing of PVA with distilled water within 2 h. When methanol was used as a washing solvent, a higher temperature than room temperature was required for an effective removal of sodium acetate. © 2007 Wiley Periodicals, Inc. J Appl Polym Sci, 2008