Reconstructing the Discontinuous and Conformational β1/β3‐Loop Binding Site on hFSH/hCG by Using Highly Constrained Multicyclic Peptides

Making peptide‐based molecules that mimic functional interaction sites on proteins remains a challenge in biomedical sciences. Here, we present a robust technology for the covalent assembly of highly constrained and discontinuous binding site mimics, the potential of which is exemplified for structurally complex binding sites on the “Cys‐knot” proteins hFSH and hCG. Peptidic structures were assembled by Ar(CH₂Br)₂‐promoted peptide cyclizations, combined with oxime ligation and disulfide formation. The technology allows unprotected side chain groups and is applicable to peptides of different lengths and nature. A tetracyclic FSH mimic was constructed, showing >600‐fold improved binding compared to linear or monocyclic controls. Binding of a tricyclic hCG mimic to anti‐hCG mAb 8G5 was identical to hCG itself (IC₅₀=260 vs. 470 pM ), whereas this mimic displayed an IC₅₀ value of 149 nM for mAb 3468, an hCG‐neutralizing antibody with undetectable binding to either linear or monocyclic controls.     

Folding and Unfolding of Two Mixed α/β Peptides

We present a molecular dynamics simulation study of two peptides containing α‐ and β‐amino acid residues. According to experiment, the two peptides differ in the dominant fold when solvated in methanol: one shows a helical fold, the other a β hairpin. The simulations at 300 and 340 K were done by starting from a NMR spectroscopic model structure and from an extended (denatured) structure. The typical structural features of the two peptides are reproduced and a folding/unfolding equilibrium is observed on the nanosecond timescale at 300 K. Analysis of proton–proton NOE distance bounds and backbone ³J coupling constants gives results consistent with the experimental data. We conclude that our simulations are complementary to the experiments by providing detailed information on the conformational distributions.     

β‐Aminopeptidase‐Catalyzed Biotransformations of β2‐Dipeptides: Kinetic Resolution and Enzymatic Coupling

We have previously shown that the β‐aminopeptidases BapA from Sphingosinicella xenopeptidilytica and DmpA from Ochrobactrum anthropi can catalyze reactions with non‐natural β³‐peptides and β³‐amino acid amides. Here we report that these exceptional enzymes are also able to utilize synthetic dipeptides with N‐terminal β²‐amino acid residues as substrates under aqueous conditions. The suitability of a β²‐peptide as a substrate for BapA or DmpA was strongly dependent on the size of the C_α substituent of the N‐terminal β²‐amino acid. BapA was shown to convert a diastereomeric mixture of the β²‐peptide H‐β²hPhe‐β²hAla‐OH, but did not act on diastereomerically pure β²,β³‐dipeptides containing an N‐terminal β²‐homoalanine. In contrast, DmpA was only active with the latter dipeptides as substrates. BapA‐catalyzed transformation of the diastereomeric mixture of H‐β²hPhe‐β²hAla‐OH proceeded along two highly S‐enantioselective reaction routes, one leading to substrate hydrolysis and the other to the synthesis of coupling products. The synthetic route predominated even at neutral pH. A rise in pH of three log units shifted the synthesis‐to‐hydrolysis ratio (v_S/v_H) further towards peptide formation. Because the equilibrium of the reaction lies on the side of hydrolysis, prolonged incubation resulted in the cleavage of all peptides that carried an N‐terminal β‐amino acid of S configuration. After completion of the enzymatic reaction, only the S enantiomer of β²‐homophenylalanine was detected (ee>99 % for H‐(S)‐β²‐hPhe‐OH, E>500); this confirmed the high enantioselectivity of the reaction. Our findings suggest interesting new applications of the enzymes BapA and DmpA for the production of enantiopure β²‐amino acids and the enantioselective coupling of N‐terminal β²‐amino acids to peptides.     

Photocrosslinking of RNA and PhotoMet‐Containing Amphiphilic α‐Helical Peptides

Discovering RNA–protein interactions : A library of photoMet‐containing peptides was synthesized by using an Arg‐ and Leu‐rich α‐helical amphiphilic peptide. Irradiation of mixtures of these peptides and Rev‐responsive element (RRE) hairpin RNA promoted formation of covalent adducts. Analysis of one adduct showed that U26 in the bulged stem is responsible for covalent bond formation with the carbene intermediate. This strategy can provide important structural information about RNA–peptide interactions.

     

Short Antimicrobial Lipo‐α/γ‐AA Hybrid Peptides

The last two decades have seen the rise of antimicrobial peptides (AMPs) to combat emerging antibiotic resistance. Herein we report the solid‐phase synthesis of short lipidated α/γ‐AA hybrid peptides. This family of lipo‐chimeric peptidomimetics displays potent and broad‐spectrum antimicrobial activity against a range of multi‐drug resistant Gram‐positive and Gram‐negative bacteria. These lipo‐α/γ‐AA hybrid peptides also demonstrate high biological specificity, with no hemolytic activity towards red blood cells. Fluorescence microscopy suggests that these lipo‐α/γ‐AA chimeric peptides can mimic the mode of action of AMPs and kill bacterial pathogens via membrane disintegration. As the composition of these chimeric peptides is simple, therapeutic development could be economically feasible and amenable for a variety of antimicrobial applications.     

Radiation-induced copolymerization of α,β,β-trifluoroacrylonitrile with α-olefin

Homopolymerization and copolymerization of α,β,β-trifluoroacrylonitrile (FAN) with γ-olefins were carried out in bulk by γ-ray irradiation at 25°C. FAN gives very small quantities of brown and greasy low molecular weight polymer. Cyano groups in FAN polymer were found to be readily hydrolyzed to acid amide groups in the atmosphere. FAN was found to copolymerize with ethylene, propylene, and isobutylene via a radical mechanism to form equimolar copolymers in a wide range of monomer compositions. The polymerization rate increases linearly with FAN fraction in the monomer mixture. These copolymers are also hydrolyzed in the atmosphere, and the hydrolysis proceeds with more difficulty for the copolymer with higher α-olefin. The reactivity ratios r₁ (FAN) and r₂ (α-olefin) were determined to be 0.01 and 0.12 for the FAN/ethylene copolymerization and 0.01 and 0.07 for the FAN/propylene copolymerization. These results confirm that an alternating copolymerization takes place in the FAN/α-olefin system.     

Cell‐Permeable β‐Peptide Inhibitors of p53/hDM2 Complexation

Look at what the cat(ionic motif) dragged in! We report a general strategy to increase the cell permeability of β³‐peptides. Introduction of a minimal cationic motif within the folded structure of a high‐affinity β³‐peptide ligand for hDM2 led to molecules with high 3₁₄‐helical structure, high hDM2 affinity and sufficient cell permeability to upregulate p53‐dependent genes in live mammalian cells. Minimally cationic β³‐peptides represent the critical first step towards a class of protease‐resistant peptidomimetics that might modulate intracellular biological pathways.

     

Efficient Photochemical Synthesis of Peptide‐α‐Phenylthioesters

Low yields and substantial epimerization of peptide‐α‐thioesters often compromise the overall efficiency of native chemical ligation (NCL). Peptide arylthioesters are more reactive than peptide alkylthioesters in NCL, but are also more difficult to handle due to their propensity to hydrolyze, and are therefore often generated in situ. However, pre‐prepared peptide arylthioesters are required for some NCL applications. Here we present a 7‐nitroindoline‐based photochemical method that generates protected peptide phenylthioesters under neutral reaction conditions via their activated esters from photoreactive peptide precursors in high isolated yields, and with low levels of epimerization. This method is fully compatible with Fmoc‐strategy solid‐phase peptide synthesis. Global deprotection with trifluoroacetic acid furnishes peptide phenylthioesters for NCL. Photoreactive peptide precursors can also be converted into their hydrazides in two steps by this method.     

Synthesis of α, α′, β-trideuterovinyl acetate

Reaction conditions for the synthesis of α, α′, β-trideuterovinyl acetate by the continuous vapour phase catalytic reaction of deuteroacetylene and acetic acid-d (CH₃COOD) are described.     

Crystal Structures of BapA Complexes with β‐Lactam‐Derived Inhibitors Illustrate Substrate Specificity and Enantioselectivity of β‐Aminopeptidases

β‐Aminopeptidases have exclusive biocatalytic potential because they react with peptides composed of β‐amino acids, which serve as building blocks for the design of non‐natural peptidomimetics. We have identified the β‐lactam antibiotic ampicillin and the ampicillin‐derived penicilloic acid as novel inhibitors of the β‐aminopeptidase BapA from Sphingosinicella xenopeptidilytica (K_i values of 0.69 and 0.74 mM , respectively). We report high‐resolution crystal structures of BapA in noncovalent complexes with these inhibitors and with the serine protease inhibitor 4‐(2‐aminoethyl)benzenesulfonyl fluoride. All three inhibitors showed similar binding characteristics; the aromatic moiety extended into a hydrophobic binding pocket of the active site, and the free amino group formed a salt bridge with Glu133 of BapA. The exact position of the inhibitors and structural details of the ligand binding pocket illustrate the specificity and the enantioselectivity of BapA‐catalyzed reactions with β‐peptide substrates.     

Acetylcholine Promotes Binding of α‐Conotoxin MII at α3β2 Nicotinic Acetylcholine Receptors

α‐Conotoxin MII (α‐CTxMII) is a 16‐residue peptide with the sequence GCCSNPVCHLEHSNLC, containing Cys2–Cys8 and Cys3–Cys16 disulfide bonds. This peptide, isolated from the venom of the marine cone snail Conus magus, is a potent and selective antagonist of neuronal nicotinic acetylcholine receptors (nAChRs). To evaluate the impact of channel–ligand interactions on ligand‐binding affinity, homology models of the heteropentameric α₃β₂‐nAChR were constructed. The models were created in MODELLER with the aid of experimentally characterized structures of the Torpedo marmorata‐nAChR (Tm‐nAChR, PDB ID: 2BG9) and the Aplysia californica‐acetylcholine binding protein (Ac‐AChBP, PDB ID: 2BR8) as templates for the α₃‐ and β₂‐subunit isoforms derived from rat neuronal nAChR primary amino acid sequences. Molecular docking calculations were performed with AutoDock to evaluate interactions of the heteropentameric nAChR homology models with the ligands acetylcholine (ACh) and α‐CTxMII. The nAChR homology models described here bind ACh with binding energies commensurate with those of previously reported systems, and identify critical interactions that facilitate both ACh and α‐CTxMII ligand binding. The docking calculations revealed an increased binding affinity of the α₃β₂‐nAChR for α‐CTxMII with ACh bound to the receptor, and this was confirmed through two‐electrode voltage clamp experiments on oocytes from Xenopus laevis. These findings provide insights into the inhibition and mechanism of electrostatically driven antagonist properties of the α‐CTxMIIs on nAChRs.     

Accommodation of impurities in α-Al2O3, α-Cr2O3 and α-Fe2O3

Atomic scale computer simulation was used to predict the mechanisms and energies associated with the accommodation of aliovalent and isovalent dopants in three host oxides with the corundum structure. Here we consider a much more extensive range of dopant ions than has previously been the case. This enables a rigorous comparison of calculated mechanism energetics. From this we predict that divalent ions are charge compensated by oxygen vacancies and tetravalent ions by cation vacancies over the full range of dopant radii. When defect associations are included in the model these conclusions remain valid. At equilibrium, defects resulting from extrinsic dopant solution dominate intrinsic processes, except for the largest dopant cations. Solution reaction energies increase markedly with increasing dopant radius. The behaviour of cluster binding energies is more complex.     

β-Fur-2-yl-α-halogenacrylonitrile. I. Darstellung von β-Fur-2-yl-ß-aminoacrylonitrilen und β-Fur-2-yl-α-aminoacrylonitrilen

β-Fur-2-yl-α-halogenacrylonitriles. I. Preparation of β-Fur-2-yl-ß-aminoacrylonitriles and β-Fur-2-yl-α-aminoacrylonitriles β-Fur-2-yl-α-halogenacrylonitriles 1 react with secondary amines to yield β-fur-2-yl-ß-aminonitriles 2 and β-fur-2-yl-α-aminoacrylonitriles 3 . The ¹ H-n.m.r. spectra of the E/Z isomers are discussed.     

Reactions of α-Arylhydrazono-β-oxo-β-phenyl-propionitriles

ω-Cyanoacetophenone was coupled with a variety of aromatic diazonium chlorides to give the corresponding arylazo derivatives 1 a – g . Analysis of the IR and UV spectra of these compounds indicated that they exist mainly as the corresponding arylhydrazones. Compounds 1 a – d, f, g reacted with diazomethane to yield the N-methyl-arylhydrazones 2 a – f . 3-Amino-4-arylazo-5-phenylpyrazoles 5 a – g and 4-arylazo-5-imino-1,3-diphenyl-2-pyrazolines 6 a – g were obtained by the interaction of 1 a – g with hydrazine hydrate and with phenylhydrazine, respectively.     

Asymmetric Epoxidation of α,β‐Unsaturated Aldehydes in Aqueous Media Catalyzed by Resin‐Supported Peptide‐ Containing Unnatural Amino Acids

The enantio‐ and diastereoselective epoxidation of α,β‐unsaturated aldehydes in aqueous media was realized using a resin‐supported peptide catalyst. Introducing the hydrophobic and bulky unnatural amino acid 3‐(1‐pyrenyl)alanine into the peptide sequence was effective for enhancing the reaction rate and enantioselectivity.     

Synthesis of γ‐Fluoro‐α, β‐unsaturated Carboxylic Esters from Saturated α‐Fluoro Aldehydes

γ‐Fluoro‐α, β‐unsaturated carboxylic esters 7a, 7b and 7d and 4‐fluoro‐4‐phenylbut‐3‐enoic ester ( 8 ) are obtained by two alternative pathways from 2‐fluoro aldehydes 5a—d , either by Horner—Wadsworth—Emmons reaction or by Wittig reaction. The aldehydes 5a—d are prepared by Swern oxidation of the corresponding fluorohydrins 4a—d . These are available from α‐olefins by bromofluorination, bromineby‐acetate replacement and subsequent hydrolysis.