Natural Compounds against Alzheimer’s Disease: Molecular Recognition of Aβ1–42 Peptide by Salvia sclareoides Extract and its Major Component,Rosmarinic Acid,as Investigated by NMR

Amyloid peptides, Aβ1–40 and Aβ1–42, represent major molecular targets to develop potential drugs and diagnostic tools for Alzheimer’s Disease (AD). In fact, oligomeric and fibrillar aggregates generated by these peptides are amongst the principal components of amyloid plaques found post mortem in patients suffering from AD. Rosmarinic acid has been demonstrated to be effective in preventing the aggregation of amyloid peptides in vitro and to delay the progression of the disease in animal models. Nevertheless, no information is available about its molecular mechanism of action. Herein, we report the NMR characterization of the interaction of Salvia sclareoides extract and that of its major component, rosmarinic acid, with Aβ1–42 peptide, whose oligomers have been described as the most toxic Aβ species in vivo. Our data shed light on the structural determinants of rosmarinic acid–Aβ1–42 oligomers interaction, thus allowing the elucidation of its mechanism of action. They also provide important information for the rational design of new compounds with higher affinity for Aβ peptides to generate new anti‐amyloidogenic molecules and/or molecular tools for the specific targeting of amyloid aggregates in vivo. In addition, we identified methyl caffeate, another natural compound present in different plants and human diet, as a good ligand of Aβ1–42 oligomers, which also shows anti‐amyloidogenic activity. Finally, we demonstrated the possibility to exploit STD‐NMR and trNOESY experiments to screen extracts from natural sources for the presence of Aβ peptide ligands.     

Delineating Binding Modes of Gal/GalNAc and Structural Elements of the Molecular Recognition of Tumor‐Associated Mucin Glycopeptides by the Human Macrophage Galactose‐Type Lectin

The human macrophage galactose‐type lectin (MGL) is a key physiological receptor for the carcinoma‐associated Tn antigen (GalNAc‐α‐1‐O‐Ser/Thr) in mucins. NMR and modeling‐based data on the molecular recognition features of synthetic Tn‐bearing glycopeptides by MGL are presented. Cognate epitopes on the sugar and matching key amino acids involved in the interaction were identified by saturation transfer difference (STD) NMR spectroscopy. Only the amino acids close to the glycosylation site in the peptides are involved in lectin contact. Moreover, control experiments with non‐glycosylated MUC1 peptides unequivocally showed that the sugar residue is essential for MGL binding, as is Ca²⁺. NMR data were complemented with molecular dynamics simulations and Corcema‐ST to establish a 3D view on the molecular recognition process between Gal, GalNAc, and the Tn‐presenting glycopeptides and MGL. Gal and GalNAc have a dual binding mode with opposite trend of the main interaction pattern and the differences in affinity can be explained by additional hydrogen bonds and CH–π contacts involving exclusively the NHAc moiety.     

Structural determination of abutilins A and B,new flavonoids from Abutilon pakistanicum,by 1D and 2D NMR spectroscopy

Two new flavonoids, abutilin A and B, were isolated from the chloroform soluble fraction of Abutilon pakistanicum and their structures assigned from ¹H and ¹³C NMR spectra, DEPT and by 2D COSY, HMQC and HMBC experiments. Ferulic acid (3), (E)‐cinnamic acid (4), 5‐hydroxy‐4′,6,7,8‐tetramethoxyflavone (5), kaempferol (6), luteolin (7) and luteolin 7‐O‐β‐D ‐glucopyranoside (8) have also been reported from this species. Copyright © 2009 John Wiley & Sons, Ltd.     

Separation of nine compounds from Salvia plebeia R.Br. using two‐step high‐speed counter‐current chromatography with different elution modes

Nine compounds were successfully separated from Salvia plebeia R.Br. using two‐step high‐speed counter‐current chromatography with three elution modes. Elution–extrusion counter‐current chromatography was applied in the first step, while classical counter‐current chromatography and recycling counter‐current chromatography were used in the second step. Three solvent systems, n‐hexane/ethyl acetate/ethanol/water (4:6.5:3:7, v/v), methyl tert‐butyl ether/ethyl acetate/n‐butanol/methanol/water (6:4:1:2:8, v/v) and n‐hexane/ethyl acetate/methanol/water (5:5.5:5:5, v/v) were screened and optimized for the two‐step separation. The separation yielded nine compounds, including caffeic acid ( 1 ), 6‐hydroxyluteuolin‐7‐glucoside ( 2 ), 5,7,3′,4′‐tetrahydroxy‐6‐methoxyflavanone‐7‐glucoside ( 3 ), nepitrin ( 4 ), rosmarinic acid ( 5 ), homoplantaginin ( 6 ), nepetin ( 7 ), hispidulin ( 8 ), and 5,6,7,4′‐tertrahydroxyflavone ( 9 ). To the best of our knowledge, 5,7,3′,4′‐tetrahydroxy‐6‐methoxyflavanone‐7‐glucoside and 5,6,7,4′‐tertrahydroxyflavone have been separated from Salvia plebeia R.Br. for the first time. The purities and structures of these compounds were identified by high‐performance liquid chromatography, electrospray ionization mass spectrometry, ¹H and ¹³C NMR spectroscopy. This study demonstrates that high‐speed counter‐current chromatography is a useful and flexible tool for the separation of components from a complex sample.     

Postcolumn determination of polyphenolic antioxidants in Cirsium vulgare (Savi) Ten. extracts

Novel methods for the determination of polyphenolic antioxidants present in extracts from inflorescences of Cirsium vulgare (Savi) Ten. based on ultra‐high performance liquid chromatography with photodiode array and chemiluminescence detection have been developed. Under the optimized conditions of chromatographic separation the analytical characteristic of the method was performed. The proposed method was successfully applied to the determination of ten polyphenols present in inflorescences of Cirsium vulgare . A comparison of the contents of analytes in extracts prepared by using various extraction media (methanol, ethanol, 70% methanol, 70% ethanol, and water) was carried out for the first time. For the postcolumn detection of scavenging activity of polyphenolic antioxidants against reactive oxygen species (H₂O₂, ^•OH, O₂^{• −}) three systems based on chemiluminescence of luminol were used. A review of the current scientific literature shows that this is the first report on the application of luminol‐based postcolumn detection for the on‐line investigation of ^•OH scavenging activity. The main compound determined in extracts from inflorescences of Cirsium vulgare was apigenin 7‐O‐glucuronide, whereas the highest antioxidant activity was observed for chlorogenic acid, luteolin 7‐O‐glucoside, and apigenin.     

UPLC‐QTOF‐MSE‐based diagnostic product ion filtering to unveil unstable C6‐C2 glucoside conjugates in Forsythia suspensa

Forsythia suspensa contains C₆‐C₂ glucoside conjugates (CCGCs) that are chemically unstable, thereby hindering their isolation and purification. In the present study, ultra‐performance liquid chromatography‐quadrupole time‐of‐flight mass spectrometry (UPLC‐QTOF) was utilized to screen and identify unstable CCGCs in the fruits and leaves of F. suspensa without any tedious isolation and purified process based on independent information acquisition (also called MS^E) and individual MS/MS experiments. Diagnostic product ion filtering (DPIF) was further applied to mine unknown analogs in MS^E high energy levels based on characteristic m/z of key substructures. A modified nomenclature for CCGCs is hereby proposed to facilitate discussions. Possible fragmentation pathways of major types of known CCGCs were proposed and used for deducing their structures. A total of 8 potentially new CCGCs were discovered and initially identified. The accuracy of their identification was further verified by structural elucidation of 3 unstable CCGCs isolated from the fruits of F. suspensa using 1D and 2D‐NMR spectroscopy. The established UPLC‐QTOF‐MS^E‐based DPIF technique facilitates the rapid discovery and direct identification of unstable CCGCs in fruits and leaves of F. suspensa .     

Triterpenoid Saponins from Luculia pincia Hook

Two new triterpenoid saponins, cincholic acid-3-O-β-D-xylopyranoside, 28-O-β-D-glucopyranosyl ester (I), quinovic acid28-O-β-D-glucopyranosyl ester (4), and a new phenolic glucoside, 4-[4‘‘-O-( 2‘‘, 3‘‘, 5‘‘, 6‘‘-tetrahydroxy phenyl)-β-D-glucoside]-l-butene (2), along with five known triterpenoid saponins and one phenolic glucoside were isolated from the n-butanol fraction of the stems of Luculia pinciana Hook. Their structures were established by means of spectroscopic methods.     

Guest–host chemistry with dendrimers: Stable polymer assemblies by rational design

A new type of guest has been designed and synthesized for the exo‐type supramolecular functionalization of adamantyl‐urea‐terminated poly(propylene imine) dendrimers. This new type of guest motif features a uriedo methane sulfonic acid moiety that binds very selectively to the surfaces of dendrimers via a combination of noncovalent interactions forming well‐defined complexes. The guest–host properties have been examined for a fifth‐generation adamantyl‐urea‐functionalized poly(propylene imine) dendrimer capable of binding 32 guest molecules and for a model host molecule that can bind only one guest molecule. The guest–host chemistry has been studied with ¹H NMR spectroscopy, nuclear Overhauser enhancement spectroscopy NMR spectroscopy, T₁‐relaxation NMR experiments, and IR spectroscopy. The 1:32 ratio with the dendrimer has been confirmed unambiguously from ¹H NMR spectra of the complex after size exclusion chromatography. Competition experiments with guests bearing a carboxylic acid instead of a sulfonic acid in the binding motif have demonstrated that the sulfonic acid has superior binding strength. Also, the importance of a combination of noncovalent interactions has been shown via competition experiments with a guest lacking the uriedo moiety. © 2004 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 42: 3792–3799, 2004     

Constituents of the Whole Herb of Clinoponium laxiflorum

The isolation and identification of twenty‐two components (including one new compound) from the whole herb of Clinoponium laxiflorum (Hay) Matsum (Labiatae) are described. Their structures were determined on the basis of spectral and chemical transformation. One new compound is methyl rosmarinate. The other twenty‐one compounds include three steroids (α‐spinasterol, α‐spinasteryl‐3‐O‐β‐D‐glucopyranoside, and β‐sitosteryl‐3‐O‐β‐glucopyranoside), three triterpenes (oleanolic acid, ursolic acid, and betulinic acid), nine flavonoids (didymin, apigenin‐7‐O‐β‐glucopyranoside, luteolin‐7‐O‐β‐glucopyranoside, isosakuranetin, narigenin, apigenin, luteolin, narirutin, and hesperidin), three lignolic acids (rosmarinic acid, 3‐(3,4‐dihydroxyphenyl)lactic acid, and caffeic acid), and three phenols (4‐hydroxybenzaldehyde, 3,4‐dihydroxybenzaldehyde, and 3,4‐dihydroxybenzoic acid).     

Investigation of the binding of a carbohydrate-mimetic peptide to its complementary anticarbohydrate antibody by STD-NMR spectroscopy and molecular-dynamics simulations

Saturation transfer difference (STD)‐NMR spectroscopy was used to probe experimentally the bioactive solution conformation of the carbohydrate mimic MDWNMHAA 1 of the O‐polysaccharide of Shigella flexneri Y when bound to its complementary antibody, mAb SYA/J6. Molecular dynamics simulations using the ZymeCAD? Molecular Dynamics platform were also undertaken to give a more accurate picture of the conformational flexibility and the possibilities for bound ligand conformations. The ligand topology, or the dynamic epitope, was mapped with the CORCEMA‐ST (COmplete Relaxation and Conformational Exchange Matrix Analysis of Saturation Transfer) program that calculates a total matrix analysis of relaxation and exchange effects to generate predicted STD‐NMR intensities from simulation. The comparison of these predicted STD enhancements with experimental data was used to select a representative binding mode. A protocol that employed theoretical STD effects calculated at snapshots during the entire course of a molecular dynamics (MD) trajectory of the peptide bound to the Fv portion of the antibody, and not the averaged atomic positions of receptor–ligand complexes, was also examined. In addition, the R factor was calculated on the basis of STD (fit) to avoid T1 bias, and an effective R factor, R_eff, was defined such that if the calculated STD (fit) for proton k was within error of the experimental STD (fit) for proton k, then that calculated STD (fit) for proton k was not included in the calculation of the R factor. This protocol was effective in deriving the antibody‐bound solution conformation of the peptide which also differed from the bound conformation determined by X‐ray crystallography; however, several discrepancies between experimental and calculated STD (fit) values were observed. The bound conformation was therefore further refined with a simulated annealing refinement protocol known as STD‐NMR intensity‐restrained CORCEMA optimization (SICO) to give a more accurate representation of the bound peptide epitope. Further optimization was required in this case, but a satisfactory correlation between experimental and calculated STD values was obtained. Attempts were also made to obtain STD enhancements with a synthetic pentasaccharide hapten, corresponding to the O‐polysaccharide, while bound to the antibody. However, unfavorable kinetics of binding in this system prevented sufficient STD build‐up, which, in turn, hindered a rigorous analysis via full STD build‐up curves.     

Chemical Composition,Antioxidant and Enzyme Inhibitory Activities of Onosma bourgaei and Onosma trachytricha and in Silico Molecular Docking Analysis of Dominant Compounds

The aim of this study was to investigate the chemical composition, antioxidant and enzyme inhibitory activities of methanol (MeOH) extracts from Onosma bourgaei (Boiss.) and O. trachytricha (Boiss.). In addition, the interactions between phytochemicals found in extracts in high amounts and the target enzymes in question were revealed at the molecular scale by performing in silico molecular docking simulations. While the total amount of flavonoid compounds was higher in O. bourgaei, O. trachytricha was richer in phenolics. Chromatographic analysis showed that the major compounds of the extracts were luteolin 7-glucoside, apigenin 7-glucoside and rosmarinic acid. With the exception of the ferrous ion chelating assay, O. trachytricha exhibited higher antioxidant activity than O. bourgaei. O. bourgaei exhibited also slightly higher activity on digestive enzymes. The inhibitory activities of the Onosma species on tyrosinase were almost equal. In addition, the inhibitory activities of the extracts on acetylcholinesterase (AChE) were stronger than the activity on butyrylcholinesterase (BChE). Molecular docking simulations revealed that luteolin 7-glucoside and apigenin 7-glucoside have particularly strong binding affinities against ChEs, tyrosinase, α-amylase and α-glucosidase when compared with co-crystallized inhibitors. Therefore, it was concluded that the compounds in question could act as effective inhibitors on cholinesterases, tyrosinase and digestive enzymes.     

Preparative separation of flavonoids in plant extract of Smilacis Glabrae Roxb. by high performance counter‐current chromatography

Four flavonoids, isoastilbin, astilbin, isoengelitin, and engelitin were isolated and purified simultaneously from Smilacis Glabrae Roxb. for the first time by high performance counter‐current chromatography using a system consisting of n‐hexane–n‐butanol–water (1:2:3, v/v/v). A total of 392.6 mg of astilbin, 71.4 mg of isoastilbin, 47.4 mg of engelitin, and 10.3 mg of isoengelitin were purified from 1.89 g of the ethyl acetate extract of Smilacis Glabrae Roxb. in six runs, each at over 94.51% purity as determined by HPLC. The structures of the four compounds were identified by their retention time, the LC‐ESI‐MSⁿ in the negative ion mode, and confirmed by ¹H‐NMR experiments. The characteristic LC‐ESI‐MS fragmentation patterns of the four compounds were discussed.     

Combination of supercritical fluid extraction with counter‐current chromatography to isolate anthocyanidins from the petals of Chaenomeles sinensis based on mathematical calculations

Supercritical fluid extraction (SFE) coupled with high‐speed counter‐current chromatography (HSCCC) was successfully used for the extraction and on‐line isolation of the anthocyanidins from the petals of Chaenomeles sinensis in two stages. The SFE parameters were optimized by an orthogonal test, and the solvent systems of SFE and HSCCC were calculated and optimized with the help of a multiexponential function model. In the first stage, the lower phase of the solvent system of n‐butanol/tert‐butyl methyl ether/acetonitrile/0.1% aqueous TFA (0.715:1.0:0.134:1.592, v/v/v/v) was used as both the SFE modifier and the HSCCC stationary phase, after extraction, the extractants were pumped into HSCCC column, and then eluted with the corresponding upper phase to isolate the moderately hydrophobic compounds. In the second stage, the upper phase of the solvent system of n‐butanol/ethyl acetate/acetonitrile/0.1% aqueous TFA (1.348:1.0:0.605:2.156, v/v/v/v) was used as both the SFE modifier and the HSCCC stationary phase, followed by elution with the corresponding lower phase to separate the hydrophobic compounds. With the help of two‐stage SFE/HSCCC, six compounds including delphinidin‐3‐O‐glucoside (Dp3G), cyanidin‐3‐O‐glucoside (Cy3G), peonidin‐3‐O‐glucoside (Pn3G), delphinidin (Dp), peonidin (Pn), and malvidin (Mv) were successfully separated within 300 min. The targeted compounds were identified by UV spectrophotometry, MS, and NMR spectroscopy. This research has opened up great prospects for the industrial application of SFE–HSCCC for the automatic extraction and separation of unstable compounds.     

Leptocarposide: a new triterpenoid glycoside from Ludwigia leptocarpa (Onagraceae)

A new triterpenoid bidesmoside (leptocarposide) possessing an acyl group in their glycosidic moiety (1), together with the known luteolin‐8‐C‐glucoside (2) and 1‐O‐β‐d ‐glucopyranosyl‐(2S,3R,8E)‐2‐[(2′R)‐2‐hydroxypalmitoylamino]‐8‐octadecen‐1,3‐diol (3) was isolated from the n‐butanol‐soluble fraction of whole plant of Ludwigia leptocarpa (Nutt) Hara (Onagraceae). Structure of compound 1 has been assigned on the basis of spectroscopic data (¹H and ¹³C NMR, ¹H‐¹H COSY, HSQC, HMBC, and ROESY), mass spectrometry, and by comparison with the literature. This compound was further screened for its potential antioxidant properties by using the radical scavenging assay model 2,2‐diphenyl‐1‐picrylhydrazyl and reveals non‐potent antioxidant activities, while compound 2 shows SC₅₀ of 0,038 mM. Copyright © 2013 John Wiley & Sons, Ltd.     

Efficient method for screening and identification of radical scavengers in the leaves of Olea europaea L

In this article, an efficient method was developed to screen, isolate and identify the major radical scavengers in the leaves of Olea europaea L. by DPPH‐HPLC‐DAD, HSCCC and NMR. The method of DPPH‐HPLC‐DAD was used to screen the major radical scavengers. It was found that three major constituents (A, B, C) in the extract of the leaves of O. europaea L. possessed potential antioxidant activities. In order to identify the chemical structures of those compounds, the HSCCC method with a two‐phase solvent system composed of petroleum ether–ethyl acetate–water at an optimized volume ratio of 6:600:700 (v/v/v) together with column chromatography was developed to isolate and purify the active compounds. Pure compounds A (225 mg), B (10 mg) and C (12 mg) with purities 92.6, 95.1 and 96.4%, respectively, were obtained from the crude sample (500 mg). Their structures were identified as oleuropein (A), luteolin‐7‐O‐glucoside (B) and verbascoside (C) by ¹H‐NMR and ¹³C‐NMR. Copyright © 2010 John Wiley & Sons, Ltd.     

Flavonoid binding to a multi-drug-resistance transporter protein: an STD-NMR study

Flavonoids are well known to inhibit the function of the multi-drug-resistance (mdr) transporter by interacting with their ATP binding domains. The precise orientation of these molecules inside the ATP binding pocket is still unclear. We applied the saturation transfer difference (STD) NMR technique to investigate the binding of the flavonoid luteolin and its 7-O--D-glycopyranoside to the recombinant nucleotide binding domain (NBD2) of mouse-mdr. First, this NMR technique confirmed binding of both ligands to NBD2, as was determined from tryptophan fluorescence-quenching experiments. Further, the results suggest binding of both luteolin and its 7-O--D-glycopyranoside by their polar groups at positions 4, 5, and 3 to the protein.Abbreviations pgp p-Glycoprotein - NBD2 C-Terminal nucleotide binding domain of mouse multi drug resistance protein (mdr)     

Ligand–Receptor Binding Affinities from Saturation Transfer Difference (STD) NMR Spectroscopy: The Binding Isotherm of STD Initial Growth Rates

The direct evaluation of dissociation constants (K_D) from the variation of saturation transfer difference (STD) NMR spectroscopy values with the receptor–ligand ratio is not feasible due to the complex dependence of STD intensities on the spectral properties of the observed signals. Indirect evaluation, by competition experiments, allows the determination of K_D, as long as a ligand of known affinity is available for the protein under study. Herein, we present a novel protocol based on STD NMR spectroscopy for the direct measurements of receptor–ligand dissociation constants (K_D) from single‐ligand titration experiments. The influence of several experimental factors on STD values has been studied in detail, confirming the marked impact on standard determinations of protein–ligand affinities by STD NMR spectroscopy. These factors, namely, STD saturation time, ligand residence time in the complex, and the intensity of the signal, affect the accumulation of saturation in the free ligand by processes closely related to fast protein–ligand rebinding and longitudinal relaxation of the ligand signals. The proposed method avoids the dependence of the magnitudes of ligand STD signals at a given saturation time on spurious factors by constructing the binding isotherms using the initial growth rates of the STD amplification factors, in a similar way to the use of NOE growing rates to estimate cross relaxation rates for distance evaluations. Herein, it is demonstrated that the effects of these factors are cancelled out by analyzing the protein–ligand association curve using STD values at the limit of zero saturation time, when virtually no ligand rebinding or relaxation takes place. The approach is validated for two well‐studied protein–ligand systems: the binding of the saccharides GlcNAc and GlcNAcβ1,4GlcNAc (chitobiose) to the wheat germ agglutinin (WGA) lectin, and the interaction of the amino acid L ‐tryptophan to bovine serum albumin (BSA). In all cases, the experimental K_D measured under different experimental conditions converged to the thermodynamic values. The proposed protocol allows accurate determinations of protein–ligand dissociation constants, extending the applicability of the STD NMR spectroscopy for affinity measurements, which is of particular relevance for those proteins for which a ligand of known affinity is not available.     

Stacking Structure of Confined 1‐Butanol in SBA‐15 Investigated by Solid‐State NMR Spectroscopy

Understanding the complex thermodynamic behavior of confined amphiphilic molecules in biological or mesoporous hosts requires detailed knowledge of the stacking structures. Here, we present detailed solid‐state NMR spectroscopic investigations on 1‐butanol molecules confined in the hydrophilic mesoporous SBA‐15 host. A range of NMR spectroscopic measurements comprising of ¹H spin–lattice (T₁), spin–spin (T₂) relaxation, ¹³C cross‐polarization (CP), and ¹H,¹H two‐dimensional nuclear Overhauser enhancement spectroscopy (¹H,¹H 2D NOESY) with the magic angle spinning (MAS) technique as well as static wide‐line ²H NMR spectra have been used to investigate the dynamics and to observe the stacking structure of confined 1‐butanol in SBA‐15. The results suggest that not only the molecular reorientation but also the exchange motions of confined molecules of 1‐butanol are extremely restricted in the confined space of the SBA‐15 pores. The dynamics of the confined molecules of 1‐butanol imply that the ¹H,¹H 2D NOESY should be an appropriate technique to observe the stacking structure of confined amphiphilc molecules. This study is the first to observe that a significant part of confined 1‐butanol molecules are orientated as tilted bilayered structures on the surface of the host SBA‐15 pores in a time‐average state by solid‐state NMR spectroscopy with the ¹H,¹H 2D NOESY technique.     

Isolation,purification, and identification of the main phenolic compounds from leaves of celery (Apium graveolens L. var. dulce Mill./Pers.)

We developed a simple and meaningful preparative method for the separation and purification of the main phenolic compounds from the leaves of celery (Apium graveolens L. var. dulce Mill./Pers.) and we established an accurate and specific analytical method for the identification of the main phenolic compounds from celery leaves. The crude extract from celery leaves was prefractioned by polyamide resin to enrich the phenolic compounds. They were then purified further by preparative high‐performance liquid chromatography, and seven main phenolic compounds were obtained: including chlorogenic acid, luteolin 7‐O‐β‐d‐ apiofuranosyl(1→2)‐β‐d‐ glucopyranoside, luteolin 7‐O‐β‐d‐ glucopyranoside, apiin, chrysoeriol 7‐O‐β‐d‐ apiofuranosyl(1→2)‐β‐d‐ glucopyranoside, luteolin 7‐O‐[β‐d‐ apiofuranosyl(1→2)‐(6′′‐O‐malonyl)]‐β‐d‐ glucopyranoside, and apigenin 7‐O‐[β‐d‐ apiofuranosyl(1→2)‐(6′′‐O‐malonyl)]‐β‐d‐ glucopyranoside. Their purities were measured by using high‐performance liquid chromatography, and their chemical structures were confirmed using UV spectrophotometry, ultra high performance liquid chromatography with quadrupole time‐of‐flight tandem mass spectrometry, and NMR spectroscopy. Our studies indicate that preparative high‐performance liquid chromatography combined with polyamide resin is a simple and meaningful preparative method for the separation and purification of phenolic compounds from the leaves of celery or other plants, and the use of UV spectrophotometry, ultra high performance liquid chromatography with quadrupole time‐of‐flight tandem mass spectrometry, and NMR spectroscopy is an accurate and specific analytical method for the identification of phenolic compounds.     

Differential Epitope Mapping by STD NMR Spectroscopy To Reveal the Nature of Protein–Ligand Contacts

Saturation transfer difference (STD) NMR spectroscopy is extensively used to obtain epitope maps of ligands binding to protein receptors, thereby revealing structural details of the interaction, which is key to direct lead optimization efforts in drug discovery. However, it does not give information about the nature of the amino acids surrounding the ligand in the binding pocket. Herein, we report the development of the novel method differential epitope mapping by STD NMR (DEEP‐STD NMR) for identifying the type of protein residues contacting the ligand. The method produces differential epitope maps through 1) differential frequency STD NMR and/or 2) differential solvent (D₂O/H₂O) STD NMR experiments. The two approaches provide different complementary information on the binding pocket. We demonstrate that DEEP‐STD NMR can be used to readily obtain pharmacophore information on the protein. Furthermore, if the 3D structure of the protein is known, this information also helps in orienting the ligand in the binding pocket.