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.     

Flavonoids and Their Glycosides as Anti‐amyloidogenic Compounds: Aβ1–42 Interaction Studies to Gain New Insights into Their Potential for Alzheimer's Disease Prevention and Therapy

Combining NMR spectroscopy, transmission electron microscopy, biochemical and in vitro toxicity assays, we characterized the effect of flavonoid glycosylation, a chemical modification found very frequently in nature, on their ability to recognize and bind Aβ1–42 oligomers, preventing their aggregation and their neurotoxicity. Our data allow the elucidation of their structure–activity relationships, showing that glycosylation has a modest impact on flavonoid affinity for Aβ oligomers but, at the same time, increases both solubility and chemical stability, thus promoting their beneficial properties against Alzheimer's disease (AD). As flavonoids and their glycosides are widely available in natural foods, our results provide important information for the evaluation of the role of a flavonoid‐rich diet for the prevention of AD. In addition, the structural data collected can be exploited for the rational design of more potent Aβ oligomer inhibitors, useful for the development of new therapies against AD.     

A Disulfide Bridge Allows for Site‐Selective Binding in Liver Bile Acid Binding Protein Thereby Stabilising the Orientation of Key Amino Acid Side Chains

The presence of a disulfide bridge in liver bile acid binding protein (L‐BABP/S‐S) allows for site‐selective binding of two bile acids, glycochenodeoxycholic (GCDA) and glycocholic acid (GCA), differing only in the presence of a hydroxyl group. The protein form devoid of the disulfide bridge (L‐BABP) binds both bile salts without discriminating ability. We investigate the determinants of the molecular recognition process in the formation of the heterotypic L‐BABP/S‐S complex with GCA and GCDA located in the superficial and inner protein sites, respectively. The comparison of the NMR spectroscopy structure of heterotypic holo L‐BABP/S‐S, the first reported for this protein family, with that of the homotypic L‐BABP complex demonstrates that the introduction of a S–S link between adjacent strands changes the conformation of three key residues, which function as hot‐spot mediators of molecular discrimination. The favoured χ₁ rotameric states (t, g⁺ and g^? for E99, Q100 and E109 residues, respectively) allow the onset of an extended intramolecular hydrogen‐bond network and the consequent stabilisation of the side‐chain orientation of a buried histidine, which is capable of anchoring a specific ligand.     

α‐O‐Linked Glycopeptide Mimetics: Synthesis,Conformation Analysis,and Interactions with Viscumin,a Galactoside‐Binding Model Lectin

Efficient cycloaddition of a silylidene‐protected galactal with a suitable heterodiene yielded the basis for a facile diastereoselective route to a glycopeptide‐mimetic scaffold. Its carbohydrate part was further extended by β1–3‐linked galactosylation. The pyranose rings retain their ⁴C₁ chair conformation, as shown by molecular modeling and NMR spectroscopy, and the typical exo‐anomeric geometry was observed for the disaccharide. The expected bioactivity was ascertained by saturation‐transfer‐difference NMR spectroscopy by using the galactoside‐specific plant toxin viscumin as a model lectin. The experimental part was complemented by molecular docking. The described synthetic route and the strategic combination of computational and experimental techniques to reveal conformational properties and bioactivity establish the prepared α‐O‐linked glycopeptide mimetics as promising candidates for further exploitation of this scaffold to give O‐glycans for lectin blocking and vaccination.     

Natural Compounds against Neurodegenerative Diseases: Molecular Characterization of the Interaction of Catechins from Green Tea with Aβ1–42, PrP106–126, and Ataxin‐3 Oligomers

By combining NMR spectroscopy, transmission electron microscopy, and circular dichroism we have identified the structural determinants involved in the interaction of green tea catechins with Aβ1–42, PrP106–126, and ataxin‐3 oligomers. The data allow the elucidation of their mechanism of action, showing that the flavan‐3‐ol unit of catechins is essential for interaction. At the same time, the gallate moiety, when present, seems to increase the affinity for the target proteins. These results provide important information for the rational design of new compounds with anti‐amyloidogenic activity and/or molecular tools for the specific targeting of amyloid aggregates in vivo.     

Serine versus Threonine Glycosylation with α‐O‐GalNAc: Unexpected Selectivity in Their Molecular Recognition with Lectins

The molecular recognition of several glycopeptides bearing Tn antigen (α‐O‐GalNAc‐Ser or α‐O‐GalNAc‐Thr) in their structure by three lectins with affinity for this determinant has been analysed. The work yields remarkable results in terms of epitope recognition, showing that the underlying amino acid of Tn (serine or threonine) plays a key role in the molecular recognition. In fact, while Soybean agglutinin and Vicia villosa agglutinin lectins prefer Tn‐threonine, Helix pomatia agglutinin shows a higher affinity for the glycopeptides carrying Tn‐serine. The different conformational behaviour of the two Tn biological entities, the residues of the studied glycopeptides in the close proximity to the Tn antigen and the topology of the binding site of the lectins are at the origin of these differences.     

Cucurbit[7]uril: A High‐Affinity Host for Encapsulation of Amino Saccharides and Supramolecular Stabilization of Their α‐Anomers in Water

Cucurbit[7]uril (CB[7]), an uncharged and water‐soluble macrocyclic host, binds protonated amino saccharides (D ‐glucosamine, D ‐galactosamine, D ‐mannosamine and 6‐amino‐6‐deoxy‐D ‐glucose) with excellent affinity (K_a=10³ to 10⁴ M ^?1). The host–guest complexation was confirmed by NMR spectroscopy, isothermal titration calorimetry (ITC), and MALDI‐TOF mass spectral analyses. NMR analyses revealed that the amino saccharides, except D ‐mannosamine, are bound as α‐anomers within the CB[7] cavity. ITC analyses reveal that CB[7] has excellent affinity for binding amino saccharides in water. The maximum affinity was observed for D ‐galactosamine hydrochloride (K_a=1.6×10⁴ M ^?1). Such a strong affinity for any saccharide in water using a synthetic receptor is unprecedented, as is the supramolecular stabilization of an α‐anomer by the host.     

Mimicking Chitin: Chemical Synthesis,Conformational Analysis,and Molecular Recognition of the β(1→3) N‐Acetylchitopentaose Analogue

Mimicking Nature by using synthetic molecules that resemble natural products may open avenues to key knowledge that is difficult to access by using substances from natural sources. In this context, a novel N‐acetylchitooligosaccharide analogue, β‐1,3‐N‐acetamido‐gluco‐pentasaccharide, has been designed and synthesized by using aminoglucose as the starting material. A phthalic group has been employed as the protecting group of the amine moiety, whereas a thioalkyl was used as the leaving group on the reducing end. The conformational properties of this new molecule have been explored and compared to those of the its chito analogue, with the β‐1,3 linkages, by a combined NMR spectroscopic/molecular modeling approach. Furthermore, the study of its molecular recognition properties towards two proteins, a lectin (wheat germ agglutinin) and one enzyme (a chitinase) have also been performed by using NMR spectroscopy and docking protocols. There are subtle differences in the conformational behavior of the mimetic versus the natural chitooligosaccharide, whereas this mimetic is still recognized by these two proteins and can act as a moderate inhibitor of chitin hydrolysis.     

A Molecular Paddlewheel with a Sliding Organometallic Latch: Syntheses,X‐Ray Crystal Structures and Dynamic Behaviour of [Cr(CO)3{η6‐2‐(9‐triptycyl)indene}], and of [M(CO)3{η5‐2‐(9‐triptycyl)indenyl}] (M=Mn,Re)

Metal clamping in operation! Deprotonation of [η⁶‐2‐(9‐triptycyl)indene]tricarbonylchromium induces a haptotropic shift of the organometallic fragment from the six‐membered to the five‐membered ring, as in a→b . In the anion, rotation of the molecular paddlewheel is blocked by the bulky tripod. X‐ray crystal structures provide pictures of the system in both its “ON” and “OFF” states.

     

Regio‐ and stereospecific assembly of dispiro[indoline‐3,3′‐pyrrolizine‐1′,5′′‐thiazolidines] from simple precursors using a one‐pot procedure: synthesis,spectroscopic and structural characterization,and a proposed mechanism of formation

The synthesis and characterization of three new dispiro[indoline‐3,3′‐pyrrolizine‐1′,5′′‐thiazolidine] compounds are reported, together with the crystal structures of two of them. (3RS,1′SR,2′SR,7a′SR)‐2′‐(4‐Chlorophenyl)‐1‐hexyl‐2′′‐sulfanylidene‐5′,6′,7′,7a′‐tetrahydro‐2′H‐dispiro[indoline‐3,3′‐pyrrolizine‐1′,5′′‐thiazolidine]‐2,4′′‐dione, C₂₈H₃₀ClN₃O₂S₂, (I), (3RS,1′SR,2′SR,7a′SR)‐2′‐(4‐chlorophenyl)‐1‐benzyl‐5‐methyl‐2′′‐sulfanylidene‐5′,6′,7′,7a′‐tetrahydro‐2′H‐dispiro[indoline‐3,3′‐pyrrolizine‐1′,5′′‐thiazolidine]‐2,4′′‐dione, C₃₀H₂₆ClN₃O₂S₂, (II), and (3RS,1′SR,2′SR,7a′SR)‐2′‐(4‐chlorophenyl)‐5‐fluoro‐2′′‐sulfanylidene‐5′,6′,7′,7a′‐tetrahydro‐2′H‐dispiro[indoline‐3,3′‐pyrrolizine‐1′,5′′‐thiazolidine]‐2,4′′‐dione, C₂₂H₁₇ClFN₃O₂S₂, (III), were each isolated as a single regioisomer using a one‐pot reaction involving l ‐proline, a substituted isatin and (Z)‐5‐(4‐chlorobenzylidene)‐2‐sulfanylidenethiazolidin‐4‐one [5‐(4‐chlorobenzylidene)rhodanine]. The compositions of (I)–(III) were established by elemental analysis, complemented by high‐resolution mass spectrometry in the case of (I); their constitutions, including the definition of the regiochemistry, were established using NMR spectroscopy, and the relative configurations at the four stereogenic centres were established using single‐crystal X‐ray structure analysis. A possible reaction mechanism for the formation of (I)–(III) is proposed, based on the detailed stereochemistry. The molecules of (I) are linked into simple chains by a single N—H…N hydrogen bond, those of (II) are linked into a chain of rings by a combination of N—H…O and C—H…S=C hydrogen bonds, and those of (III) are linked into sheets by a combination of N—H…N and N—H…S=C hydrogen bonds.     

Characterization of Doubly Ionic Hydrogen Bonds in Protic Ionic Liquids by NMR Deuteron Quadrupole Coupling Constants: Differences to H‐bonds in Amides,Peptides, and Proteins

We present the first deuteron quadrupole coupling constants (DQCCs) for selected protic ionic liquids (PILs) measured by solid‐state NMR spectroscopy. The experimental data are supported by dispersion‐corrected density functional theory (DFT‐D3) calculations and molecular dynamics (MD) simulations. The DQCCs of the N−D bond in the triethylammonium cations are the lowest reported for deuterons in PILs, indicating strong hydrogen bonds between ions. The NMR coupling parameters are compared to those in amides, peptides, and proteins. The DQCCs show characteristic behavior with increasing interaction strength of the counterion and variation of the H‐bond motifs. We report the similar presence of the quadrupolar splitting pattern and the narrow liquid line in the NMR spectra over large temperature ranges, indicating the heterogeneous nature of PILs.     

Synthesis of Electron‐Deficient Corona[5]arenes and Their Selective Complexation with Dihydrogen Phosphate: Cooperative Effects of Anion–π Interactions

Reported here are the syntheses, conformational structures, electrochemical properties, and noncovalent anion binding of corona[5]arenes. A (3+2) fragment coupling reaction proceeded efficiently under mild reaction conditions to produce a number of novel heteroatom‐ and methylene‐bridged corona[3]arene[2]tetrazine macrocycles. Selective oxidation of the sulfur atom between two phenylene rings afforded sulfoxide‐ and sulfone‐linked corona[5]arenes in good yields. All corona[5]arenes synthesized adopted similar 1,2,4‐alternate conformational structures, forming pentagonal cavities. The cavity sizes and the electronic properties such as redox potentials, were measured with CV and DPV, and were influenced by the different bridging units. As electron‐deficient macrocycles, the acquired corona[3]arene[2]tetrazines served as highly selective hosts, forming complexes with the hydrogen‐bonded dimer of dihydrogen phosphate through cooperative anion–π interactions.     

The β‐Agostic Structure in (C5Me5)2Sc(CH2CH3): Solid‐State NMR Studies of (C5Me5)2Sc−R (R=Me,Ph, Et)

Multinuclear solid‐state NMR studies of Cp*₂Sc?R (Cp*=pentamethylcyclopentadienyl; R=Me, Ph, Et) and DFT calculations show that the Sc?Et complex contains a β‐CH agostic interaction. The static central transition ⁴⁵Sc NMR spectra show that the quadrupolar coupling constants (C_q) follow the trend of Ph≈Me>Et, indicating that the Sc?R bond is different in Cp*₂Sc?Et compared to the methyl and phenyl complexes. Analysis of the chemical shift tensor (CST) shows that the deshielding experienced by Cβ in Sc?CH₂CH₃ is related to coupling between the filled σ_C‐C orbital and the vacant orbital.     

Supramolecular Complexation in Biological Media: NMR Study on Inclusion of an Anionic Tetraarylporphyrin into a Per‐O‐Methylated β‐Cyclodextrin Cavity in Serum,Blood, and Urine

The supramolecular complexation of 5,10,15,20‐tetrakis(4‐sulfonatophenyl)porphyrin (TPPS) with heptakis(2,3,6‐tri‐O‐methyl)‐β‐cyclodextrin (TMCD) has been known to be highly specific in aqueous media. In this study, we have used NMR spectroscopy to reveal that this supramolecular system also works even in biologically crowded media such as serum, blood, and urine. A ¹³C‐labeled heptakis(2,3,6‐tri‐O‐methyl‐¹³C)‐β‐cyclodextrin (¹³C‐TMCD) was synthesized and studied using one‐dimensional (1D) HMQC spectroscopy in serum and blood. The 1D HMQC spectrum of ¹³C‐TMCD showed clear signals due to the 2‐, 3‐, and 6‐O¹³CH₃ groups, whose chemical shifts changed upon addition of TPPS due to quantitative formation of the ¹³C‐TMCD/TPPS=2/1 inclusion complex in such biological media. The ¹H NMR signals of non‐isotope‐labeled TPPS included by ¹³C‐TMCD were detected using the ¹³C‐filtered ROESY technique. A pharmacokinetic study of ¹³C‐TMCD and its complex with TPPS was carried out in mice using the 1D HMQC method. The results indicated that (1) 1D HMQC is an effective technique for monitoring the inclusion phenomena of ¹³C‐labeled cyclodextrin in biological media and (2) the intermolecular interaction between ¹³C‐TMCD and TPPS is highly selective even in contaminated media like blood, serum, and urine.     

Proton/Hydrogen‐Transfer Coordinate of 2,5‐Dihydroxybenzoic Acid Investigated in a Supersonic Beam: Combined IR/UV Spectroscopy in the S0, S1, and D0 States

As a model system for intramolecular proton/hydrogen‐transfer coordinates, the structure of 2,5‐dihydroxybenzoic acid is investigated for the ground, first electronically excited and also the ionic state. Combined IR/UV spectroscopy in molecular‐beam experiments is applied and the experimental results are interpreted by the application of DFT and CASPT2 methods. No proton or hydrogen transfer is observed, but evidence is given for a hydrogen dislocation of the intramolecular hydrogen bond in the S₁ state and to lesser extent in the D₀ state. To obtain direct information on the proton/hydrogen‐transfer coordinate, IR spectra are recorded both in the region of the OH and especially the CO stretching vibrations by also applying two new variants of combined IR/UV spectroscopy for the S₁ and D₀ states. The CO groups are directly involved in the hydrogen bond and, in contrast to the hydrogen‐bonded OH groups, the CO stretching frequencies can be observed in all electronic states.     

Ionic complexes of 1,1′‐dimethyltitanocene(IV) dichloride with simple α‐amino acids: synthesis,structural characterisation and investigation on hydrolytic stability in aqueous solution

Five cationic complexes of the general formula [Cp′₂Ti(A)₂]²⁺ [Cl^?]₂ [Cp′ = η⁵‐(CH₃)C₅H₄ and A = glycine, 1 ; 2‐methylalanine, 2 ; N‐methylglycine, 3 ; L ‐alanine, 4 ; and D ‐alanine 5 ] were prepared by the reaction of Cp′₂TiCl₂ and the appropriate α‐amino acid in 1:2 molar ratio from methanol–water solution in high yield. Air‐stable crystalline solids, highly soluble in water, were characterized by means of elemental analysis, IR, Raman, ¹H, ¹³C and ¹⁴N NMR spectroscopy. The structure of compound 3 was determined by single crystal X‐ray crystallography: orthorhombic Pbca No. 61, a = 9.5310(3), b = 18.2980(5), c = 26.6350(5) Å, V = 4654 ų, Z = 8. Hydrolytic stability of all compounds in D₂O was investigated using ¹H NMR spectroscopy within the pD interval of 2.9–6.5. All compounds slowly decomposed during 24 h at pD = 2.94, forming a mixture of hydrolytic products [Cp′₂Ti(A)(D₂O)]²⁺, [Cp′₂Ti(D₂O)₂]²⁺ and respective α‐amino acids. By elevating pD to 4.0 and up to 6.5, a yellowish precipitate was formed, which indicates decomposition of the complexes. These compounds were characterized using elemental analyses, IR and Raman spectroscopy and attributed to oligomeric and/or polymeric structures described empirically by the formula Ti(Cp′)_xO_y(OH)_z (x = 0.65; y = 0.3, z = 1.9). Copyright © 2005 John Wiley & Sons, Ltd.