Anasztázia Hetényi

Synthesis and transformations of enantiomeric 1,2-disubstituted monoterpene derivatives

Abstract Regio- and stereospecific addition of chlorosulfonyl isocyanate to (+)- and (−)-α-pinene 1 resulted in enantiomerically pure β-lactams 2 , which were converted to enantiomeric β-amino esters 3 and 1,3-amino alcohols 4 and 6 with ee >99%. The resulting 1,3-difunctional compounds 3 , 4 and 6 were transformed to fused saturated 1,3-heterocycles such as tetrahydro-1,3-oxazines 7 and 9 , 2,4-pyrimidinedione 11 and 2-thioxopyrimidin-4-one 13 enantiomers.

Controlled in situ preparation of Aβ(1–42) oligomers from the isopeptide “iso-Aβ(1–42)”, physicochemical and biological characterization

Beta-amyloid (A beta) peptides play a crucial role in the pathology of the neurodegeneration in Alzheimers disease (AD). Biological experiments (both in vitro and animal model studies of AD) require synthetic A beta peptides of standard quality, aggregation grade, neurotoxicity and water solubility. The synthesis of A beta peptides has been difficult, owing to their hydrophobic character, poor solubility and high tendency for aggregation. Recently an isopeptide precursor (iso-A beta(1-42)) was synthesized by Fmoc-chemistry and transformed at neutral pH to A beta(1-42) by O-->N acyl migration in a short period of time. We prepared the same precursor peptide using Boc-chemistry and studied the transformation to A beta(1-42) by acyl migration. The peptide conformation and aggregation processes were studied by several methods (circular dichroism, atomic force and transmission electron microscopy, dynamic light scattering). The biological activity of the synthetic A beta(1-42) was measured by ex vivo (long-term potentiation studies in rat hippocampal slices) and in vivo experiments (spatial learning of rats). It was proven that O-->N acyl migration of the precursor isopeptide results in a water soluble oligomeric mixture of neurotoxic A beta(1-42). These oligomers are formed in situ just before the biological experiments and their aggregation grade could be standardized.

A Foldamer-Dendrimer Conjugate Neutralizes Synaptotoxic β-Amyloid Oligomers

Background and Aims Unnatural self-organizing biomimetic polymers (foldamers) emerged as promising materials for biomolecule recognition and inhibition. Our goal was to construct multivalent foldamer-dendrimer conjugates which wrap the synaptotoxic β-amyloid (Aβ) oligomers with high affinity through their helical foldamer tentacles. Oligomeric Aβ species play pivotal role in Alzheimers disease, therefore recognition and direct inhibition of this undruggable target is a great current challenge. Methods and Results Short helical β-peptide foldamers with designed secondary structures and side chain chemistry patterns were applied as potential recognition segments and their binding to the target was tested with NMR methods (saturation transfer difference and transferred-nuclear Overhauser effect). Helices exhibiting binding in the µM region were coupled to a tetravalent G0-PAMAM dendrimer. In vitro biophysical (isothermal titration calorimetry, dynamic light scattering, transmission electron microscopy and size-exclusion chromatography) and biochemical tests (ELISA and dot blot) indicated the tight binding between the foldamer conjugates and the Aβ oligomers. Moreover, a selective low nM interaction with the low molecular weight fraction of the Aβ oligomers was found. Ex vivo electrophysiological experiments revealed that the new material rescues the long-term potentiation from the toxic Aβ oligomers in mouse hippocampal slices at submicromolar concentration. Conclusions The combination of the foldamer methodology, the fragment-based approach and the multivalent design offers a pathway to unnatural protein mimetics that are capable of specific molecular recognition, and has already resulted in an inhibitor for an extremely difficult target.

Stereoselective synthesis of spiro and condensed pyrazolines of steroidal α,β-unsaturated ketones and nitrilimines by 1,3-dipolar cycloaddition

Effective syntheses of endo- and exocyclic alpha,beta-unsaturated ketones as CC dipolarophiles were carried out in the 13alpha-estrone series. The 1,3-dipolar cycloadditions of 15,16alpha,beta-unsaturated ketones of 13alpha-estrone 3-methyl and 3-benzyl ether with nitrilimines stereoselectively furnished two regioisomers of new condensed pyrazolines in a ratio of 2:1. The main product was the isomer obtained by the attack of the N-terminus of the 1,3-dipole on the carbon atom beta to the carbonyl group of the dipolarophile. The nitrilimine cycloadditions to the 16-methylene-17-ketones of 13alpha-estrone 3-methyl and 3-benzyl ether stereo- and regioselectively furnished spiropyrazolines. The attack of the N-terminus of the dipole occurred on the alpha-carbon of the alpha,beta-unsaturated ketones. The reactions were performed under both homogeneous and heterogeneous conditions. Silver acetate as a base proved more effective than its triethylamine counterpart. Changes in regio- and stereoselectivities were not observed on variation of the conditions of the cycloaddition reactions. The structures of the new products were determined by NMR (one- and two-dimensional) and MALDI TOF MS techniques, with C(70) fullerenes as matrix in the latter case.

Interactions of the carrier ligands of antidiabetic metal complexes with human serum albumin: a combined spectroscopic and separation approach with molecular modeling studies.

The specific binding of carrier ligands of antidiabetic vanadium(IV) and zinc(II) complexes into drug binding pockets of human serum albumin (HSA) has been investigated via displacement reactions of site markers such as warfarin and dansylglycine by different spectroscopic (fluorescence, circular dichroism, NMR) and separation methods (capillary zone electrophoresis, ultrafiltration-UV). Conditional stability constants of the ligands were calculated for the binding at sites I and II of HSA. Binding site I was found to be the primary binding site for 2,6-pyridine dicarboxylic acid (dipic) and picolinic acid (pic), and site II for 6-methylpicolinic acid (6-Mepic) and maltol, although dipic, 6-Mepic and pic displace both site markers at differing extents. The experimental data is complemented by protein-ligand docking calculations for dipic and 6-Mepic which support the observations.

In vitro Anti-diabetic Activity and Chemical Characterization of an Apolar Fraction of Morus alba Leaf Water Extract

The tea from the white mulberry (Morus alba L.) leaf is a worldwide known traditional medicine of type II diabetes. Here, we report the investigation of the dichloromethane‐soluble fraction obtained in a 0.24% m/m yield from the hot water extract of mulberry leaves. A significant, dose‐dependent activity was found by means of the 24‐h glucose consumption of fully differentiated adipocytes both in the absence and presence of insulin. The fraction was characterized by HPLC‐DAD, GC‐MS and GC‐FID. The main constituent (40.3% by means of GC‐FID) was isolated and identified as loliolide by EIMS, HRESIMS and NMR spectroscopy. In the active fraction benzyl alcohol, ethyl benzoate, t‐cinnamic acid, p‐hydroxyacetophenone, t‐coniferyl alcohol and synapil alcohol were also identified by GC‐MS and quantified by GC‐FID (0.7, 1.3, 1.5, 2.9, 7.5 and 2.6%, respectively). Copyright

Solvent‐Enhanced Diastereo‐ and Regioselectivity in the PdII‐Catalyzed Synthesis of Six‐ and Eight‐Membered Heterocycles via cis‐Aminopalladation

The Pd(II)-catalyzed intramolecular oxidative cyclization of tosyl-protected cis- and trans-N-allyl-2-aminocyclohexanecarboxamides was examined, and efficient syntheses of cyclohexane-fused pyrimidin-4-ones and 1,5-diazocin-6-ones were developed. In the course of the research, a marked solvent effect was observed on both the regio- and diastereoselectivity. Additionally, a novel Pd(II)-mediated domino oxidation, oxidative amination reaction was discovered. Our experimental and theoretical findings suggest that the reactions proceed via a cis-aminopalladation mechanism.

Induced Folding of Protein‐Sized Foldameric β‐Sandwich Models with Core β‐Amino Acid Residues

The mimicry of protein-sized β-sheet structures with unnatural peptidic sequences (foldamers) is a considerable challenge. In this work, the de novo designed betabellin-14 β-sheet has been used as a template, and α→β residue mutations were carried out in the hydrophobic core (positions 12 and 19). β-Residues with diverse structural properties were utilized: Homologous β(3) -amino acids, (1R,2S)-2-aminocyclopentanecarboxylic acid (ACPC), (1R,2S)-2-aminocyclohexanecarboxylic acid (ACHC), (1R,2S)-2-aminocyclohex-3-enecarboxylic acid (ACEC), and (1S,2S,3R,5S)-2-amino-6,6-dimethylbicyclo[3.1.1]heptane-3-carboxylic acid (ABHC). Six α/β-peptidic chains were constructed in both monomeric and disulfide-linked dimeric forms. Structural studies based on circular dichroism spectroscopy, the analysis of NMR chemical shifts, and molecular dynamics simulations revealed that dimerization induced β-sheet formation in the 64-residue foldameric systems. Core replacement with (1R,2S)-ACHC was found to be unique among the β-amino acid building blocks studied because it was simultaneously able to maintain the interstrand hydrogen-bonding network and to fit sterically into the hydrophobic interior of the β-sandwich. The novel β-sandwich model containing 25 % unnatural building blocks afforded protein-like thermal denaturation behavior.

Ligand-Induced Flocculation of Neurotoxic Fibrillar Aβ(1-42) by Noncovalent Crosslinking

Aggregation of the amyloid‐β (Aβ) peptides has a pivotal role in Alzheimer’s disease (AD). Small molecules and short peptides/peptidomimetics can exert their full protective effects against Aβ within a short time‐frame, but the exact mechanism of action is unclear. Time‐dependent NMR spectroscopic binding and replacement experiments were carried out for peptide LPFFD and thioflavine T (ThT) on neurotoxic fibrillar Aβ(1–42), which revealed transient binding behavior for both compounds, and complex time‐dependent features in the replacement experiments. The results of particle size measurements through the use of diffuse light‐scattering and transmission electron microscopy support the conclusions that the studied ligands induced interfibrillar association on a short timescale, which explains the NMR spectroscopic binding and replacement results. ζ‐Potential measurements revealed a slightly increased electrostatic stability of the Aβ fibrils upon ligand binding; this suggests that the interfibrillar assembly is driven by specific noncovalent cross‐linking interactions. A specific surface and mobility decrease due to the ligand‐induced flocculation of the Aβ fibrils can explain the neuroprotective effects.

Galectin-1–Asialofetuin Interaction Is Inhibited by Peptides Containing the Tyr-Xxx-Tyr Motif Acting on the Glycoprotein

Galectin‐1 (Gal‐1), a ubiquitous β‐galactoside‐binding protein expressed by various normal and pathological tissues, has been implicated in cancer and autoimmune/inflammatory diseases in consequence of its regulatory role in adhesion, cell viability, proliferation, and angiogenesis. The functions of Gal‐1 depend on its affinity for β‐galactoside‐containing glycoconjugates; accordingly, the inhibition of sugar binding blocks its functions, hence promising potential therapeutic tools. The Tyr‐Xxx‐Tyr peptide motifs have been reported to be glycomimetic sequences, mainly on the basis of their inhibitory effect on the Gal‐1–asialofetuin (ASF) interaction. However, the results regarding the efficacy of the Tyr‐Xxx‐Tyr motif as a glycomimetic inhibitor are still controversial. The present STD and trNOE NMR experiments reveal that the Tyr‐Xxx‐Tyr peptides studied do not bind to Gal‐1, whereas their binding to ASF is clearly detected. 15N,1H HSQC titrations with 15N‐labeled Gal‐1 confirm the absence of any peptide–Gal‐1 interaction. These data indicate that the Tyr‐Xxx‐Tyr peptides tested in this work are not glycomimetics as they interact with ASF via an unrevealed molecular linkage.