Enantioenriched Boron C,N-Chelates via Chirality Transfer.

Molecules stereogenic only at tetrahedral boron atoms show great promise for applications, for example as chiroptical materials, but are scarcely investigated due to their synthetic challenge. Hence, this study reports a two-step synthesis of enantioenriched boron C,N-chelates. First, the diastereoselective complexation of alkyl/aryl borinates with chiral aminoalcohols furnished boron stereogenic heterocycles in up to 86 % yield and d.r. >98 : 2. Treatment of these O,N-complexes with chelate nucleophiles was surmised to transfer the stereoinformation via the ate-complex into the C,N-products. This chirality transfer succeeded by substitution of the O,N-chelates with lithiated phenyl pyridine to give boron stereogenic C,N-chelates in up to 84 % yield and e.r. up to 97 : 3. The chiral aminoalcohol ligands could be recovered after isolation of the C,N-chelates. The chirality transfer tolerated alkyl, alkynyl and (hetero-)aryl moieties at boron and could be further extended by post-modification: transformations such as catalytic hydrogenations or sequential deprotonation/electrophilic trapping were feasible while maintaining the stereochemical integrity of the C,N-chelates. Structural aspects of the boron chelates were studied by variable temperature NMR and X-ray diffraction.

Chasing Self-Assembly of Thioether-Substituted Flavylium Salts in Solution and Bulk State.

Two series of flavylium triflates carrying alkoxy side chains in the A-ring (benzo unit of chromylium salt) and thioethers in the B ring (phenyl unit) (On -Fla-Sm ) as well as thioethers at both A and B ring (Sn -Fla-Sm ) were synthesized in order to understand the effect of thioether functionalization on their self-assembly and electronic properties. Concentration-dependent and diffusion ordered (DOSY) NMR experiments of O1 -iV-Fla-S3 indicate the formation of columnar H-aggregates in solution with antiparallel intracolumnar stacking of the AC unit (chromylium) of the flavylium triflate, in agreement with the solid state structure of O1 -V-Fla-S1 . Thioether substitution on the B ring changes the linear optical properties in solution, whereas it has no effect on the A ring. According to differential scanning calorimetry, polarizing optical microscopy and X-ray diffraction bulk self-assembly of these ionic liquid crystals (ILCs) depends on the total number of side chains, yielding SmA and LamCol phases for ILCs with 2-3 chains and Colro , Colh phases for ILCs with 3-6 chains. Thus, we demonstrated that thioethers are a useful design tool for ILCs with tailored properties.

Modulating chitin synthesis in marine algae with iminosugars obtained by SmI2 and FeCl3-mediated diastereoselective carbonyl ene reaction.

Strategies for synthesizing polyhydroxylated piperidines such as iminosugars have received broad attention. These substances are known to interact with carbohydrate related enzymes, glycosidases and glycosyltransferases, to which also the large enzyme families of chitin synthases and cellulose synthases belong. Many chemical and biological aspects of chitin synthases remain unexplored due to the fact that modulating substances are hardly available or expensive. Starting from enantiopure D- and L-amino acids, a series of iminosugars was prepared by a Lewis acid-catalyzed cyclization of amino acid-derived unsaturated aldehydes as key step. Therefore, different Lewis acids were tested. For samarium diiodide we observed a superior stereoselectivity in comparison to iron(III) chloride and methylaluminium dichloride. To increase water solubility for testing and measurement of enzyme activity, the cyclization products were further functionalized. We established a novel biological chitin synthesis test system which allows quantitative investigation of chitin synthesis in the chitin fiber producing diatom algae Thalassiosira in vivo under the light microscope. None of the compounds displayed cytotoxicity, but two of the four iminosugars increased the length of the chitin fibers produced. This is a strong indicator that these compounds mimic carbohydrates responsible for restarting chitin polymerization.

Synthesis of Highly Functionalized Hydrindanes via Sequential Organocatalytic Michael/Mukaiyama Aldol Addition and Telescoped Hydrozirconation/Cross-Coupling as Key Steps: En Route to the AB System of Clifednamides.

The AB ring systems of the clifednamide family, polycyclic tetramate macrolactames (PoTeMs), were prepared by a new, convergent approach employing an intramolecular Diels-Alder (IMDA) reaction. Key steps comprise an organocatalytic Michael addition (>90% enantiomeric excess (ee)), a Mukaiyama aldol reaction for the convergent installation of a diene moiety, and a telescoped hydrozirconation/cross-coupling grafting an enone. The following IMDA furnished a highly functionalized hydrindane (diastereomeric ratio (dr) = 91:1) with the same configuration as the clifednamide scaffold. Advantages of this route are only one required protecting group, 13% overall yield over 9 steps (reduced from previously 17 steps/1.3% overall), and the potential access to the key intermediates in the clifednamide biosynthesis.

Asymmetric Catalysis in Liquid Confinement: Probing the Performance of Novel Chiral Rhodium-Diene Complexes in Microemulsions and Conventional Solvents.

The role of liquid confinement on the asymmetric Rh catalysis was studied using the 1,2-addition of phenylboroxine (2) to N-tosylimine 1 in the presence of [RhCl(C2 H4 )2 ]2 and chiral diene ligands as benchmark reaction. To get access to Rh complexes of different polarity, enantiomerically pure C2 -symmetric p-substituted 3,6-diphenylbicyclo[3.3.0]octadienes 4 and diastereomerically enriched unsymmetric norbornadienes 5 and 6 carrying either the Evans or the SuperQuat auxiliary were synthesized. A microemulsion containing the equal amounts of H2 O/KOH and toluene/reactants was formulated using the hydrophilic sugar surfactant n-octyl ß-d-glucopyranoside (C8 G1 ) to mediate the miscibility between the nonpolar reactants and KOH, needed to activate the Rh-diene complex. Prominent features of this organized reaction medium are its temperature insensitivity as well as the presence of water and toluene-rich compartments with a domain size of 55 Šconfirmed by small-angle X-ray scattering (SAXS). Although bicyclooctadiene ligands 4 a,b,e performed equally well under homogeneous and microemulsion conditions, ligands 4 c,d gave a different chemoselectivity. For norbornadienes 5, 6, however, microemulsions markedly improved conversion and enantioselectivity as well as reaction rate, as was confirmed by kinetic studies using ligand 5 b.

Chemoenzymatic Route to Oxyfunctionalized Cembranoids Facilitated by Substrate and Protein Engineering.

Cembranoids constitute a large family of 14-membered oxygenated macrocyclic diterpenoids with potential as therapeutic agents. Selective late-stage oxidations of cembranoid scaffolds remain a challenge for chemical catalysts but can be accomplished by enzymes. Here, a new chemoenzymatic route to oxyfunctionalized 14-membered macrocycles including cembranoids is described. This route combines a metal-catalyzed ring-closing metathesis with a subsequent P450 BM3-catalyzed hydroxylation and delivers cembranoid-like analogues. Systematic substrate probing with a set of synthetic 14-membered macrocycles revealed that the regioselectivity of a P450 BM3-based biocatalyst increased with increasing ring rigidity as well as size and polarity of the exocyclic substituents. Enzyme regioselectivity could further be improved by first-sphere active site mutagenesis. The V78A/F87A variant catalyzed hydroxylation of cembranoid-ol (9S/R)-3 d with 90 % regioselectivity for C5 position. Extensive NMR analysis of Mosher esters and single crystal X-ray structure determination revealed a remarkable diastereoselectivity of this P450 BM3 mutant depending on substrate stereochemistry, which led exclusively to the syn-cembranoid-diols (5S,9S)-4 and (5R,9R)-4.

The strength of the template effect attracting nucleotides to naked DNA.

The transmission of genetic information relies on Watson-Crick base pairing between nucleoside phosphates and template bases in template-primer complexes. Enzyme-free primer extension is the purest form of the transmission process, without any chaperon-like effect of polymerases. This simple form of copying of sequences is intimately linked to the origin of life and provides new opportunities for reading genetic information. Here, we report the dissociation constants for complexes between (deoxy)nucleotides and template-primer complexes, as determined by nuclear magnetic resonance and the inhibitory effect of unactivated nucleotides on enzyme-free primer extension. Depending on the sequence context, Kd's range from 280 mM for thymidine monophosphate binding to a terminal adenine of a hairpin to 2 mM for a deoxyguanosine monophosphate binding in the interior of a sequence with a neighboring strand. Combined with rate constants for the chemical step of extension and hydrolytic inactivation, our quantitative theory explains why some enzyme-free copying reactions are incomplete while others are not. For example, for GMP binding to ribonucleic acid, inhibition is a significant factor in low-yielding reactions, whereas for amino-terminal DNA hydrolysis of monomers is critical. Our results thus provide a quantitative basis for enzyme-free copying.

High-fidelity recognition of RNA: solution structure of a DNA:RNA hybrid duplex with a molecular cap.

Binding RNA targets, such as microRNAs, with high fidelity is challenging, particularly when the nucleobases to be bound are located at the terminus of the duplex between probe and target. Recently, a peptidyl chain terminating in a quinolone, called ogOA, was shown to act as a cap that enhances affinity and fidelity for RNAs, stabilizing duplexes with Watson-Crick pairing at their termini. Here we report the three-dimensional structure of an intramolecular complex between a DNA strand featuring the ogOA cap and an RNA segment, solved by NMR and restrained torsion angle molecular dynamics. The quinolone stacks on the terminal base pair of the hybrid duplex, positioned by the peptidyl chain, whose prolinol residue induces a sharp bend between the 5' terminus of the DNA chain and the glycine linked to the oxolinic acid residue. The structure explains why canonical base pairing is favored over hard-to-suppress mismatched base combinations, such as T:G and A:A, and helps to design improved high-fidelity probes for RNA.

Entropy is key to the formation of pentacyclic terpenoids by enzyme-catalyzed polycyclization.

Polycyclizations constitute a cornerstone of chemistry and biology. Multicyclic scaffolds are generated by terpene cyclase enzymes in nature through a carbocationic polycyclization cascade of a prefolded polyisoprene backbone, for which electrostatic stabilization of transient carbocationic species is believed to drive catalysis. Computational studies and site-directed mutagenesis were used to assess the contribution of entropy to the polycyclization cascade catalyzed by the triterpene cyclase from A. acidocaldarius. Our results show that entropy contributes significantly to the rate enhancement through the release of water molecules through specific channels. A single rational point mutation that results in the disruption of one of these water channels decreased the entropic contribution to catalysis by 60 kcal mol(-1) . This work demonstrates that entropy is the key to enzyme-catalyzed polycyclizations, which are highly relevant in biology since 90 % of all natural products contain a cyclic subunit.

Isolation of ZnO-binding 12-mer peptides and determination of their binding epitopes by NMR spectroscopy.

Inorganic-binding peptides are in the focus of research fields such as materials science, nanotechnology, and biotechnology. Applications concern surface functionalization by the specific coupling to inorganic target substrates, the binding of soluble molecules for sensing applications, or biomineralization approaches for the controlled formation of inorganic materials. The specific molecular recognition of inorganic surfaces by peptides is of major importance for such applications. Zinc oxide (ZnO) is an important semiconductor material which is applied in various devices. In this study the molecular fundamentals for a ZnO-binding epitope was determined. 12-mer peptides, which specifically bind to the zinc- or/and the oxygen-terminated sides of single-crystalline ZnO (0001) and (000-1) substrates, were selected from a random peptide library using the phage display technique. For two ZnO-binding peptides the mandatory amino acid residues, which are of crucial importance for the specific binding were determined with a label-free nuclear magnetic resonance (NMR) approach. NMR spectroscopy allows the identification of pH dependent interaction sites on the atomic level of 12-mer peptides and ZnO nanoparticles. Here, ionic and polar interaction forces were determined. For the oxygen-terminated side the consensus peptide-binding sequence (HSXXH) was predicted in silico and confirmed by the NMR approach.

15N relaxation NMR studies of prolyl oligopeptidase, an 80 kDa enzyme, reveal a pre-existing equilibrium between different conformational states.

Open and closed: The characterization of protein mobility is crucial for the understanding of biological functions. We have applied NMR spectroscopy to study the conformational dynamics of the 80 kDa enzyme prolyl oligopeptidase (POP). Our results revealed that POP is highly dynamic and that inhibition of catalytic activity shifts this conformational equilibrium towards a less dynamic state.

Influence of N-alkyl substituents and counterions on the structural and mesomorphic properties of guanidinium salts: experiment and quantum chemical calculations.

A series of N-4-(4'-alkoxybiphenyl)-N',N',N",N"-tetramethylguanidinium salts was synthesized with varying alkoxy chain lengths and additional N-alkyl substituents, each with a number of different counterions. X-ray crystal-structure analyses of 1b I, 1b PF(6), 2a I, and 4a I reveal bilayer structures in the solid state and, for the 1b and 1b PF(6) salts, a hydrogen-bond-type connectivity between the guanidinium N-H group and the anion is found. For the N-alkyl homologues 2a I and 4a I the anion is still oriented close to the head group, although at a larger distance. Ion pairs are present also in solution, as demonstrated by (1)H NMR: the N-H chemical shift shows a good linear correlation with the radius, and hence the hardness, of the anion. The intramolecular conformational flexibility of 1b I, 2b I, 3b I, and 4b I was studied by temperature-dependent (1)H NMR spectroscopy and discrete activation barriers were determined for rotations about each of the three C-N partial double bonds of the guanidinium core. The relative heights of the individual barriers change between the N-H and the N-alkylguanidinium salts. A fourth barrier is observed for the rotation about the N-biphenyl bond. DFT calculations of charge densities show that the positive charge resides primarily on the central carbon atom. Rotational barriers were calculated for N'-substituted 2-amino-1,3-dimethylimidazolidinium cations as models, and are in qualitatively good agreement with the NMR data. Mesomorphic properties were studied by differential-scanning calorimetry, polarizing optical microscopy, and X-ray diffraction (WAXS/SAXS). All liquid-crystalline guanidinium salts exhibit smectic A mesophases. Clearing temperatures show a linear correlation with the anionic radius. Substitution of the N-H group with methyl, ethyl, or propyl results in decreasing mesophase widths and a concomitant shrinkage of the layer spacings.

Interaction of Structurally Diverse Phenolic Compounds with Porcine Pancreatic α-Amylase.

A blood glucose level lowering effect is postulated for polyphenols (PPs), which is in part attributed to the inhibition of α-amylase. To estimate structure-effect relationships, chlorogenic acid (CA), phlorizin (PHL), epigallocatechin gallate (EGCG), epicatechin (EC), and malvidin-3-glucoside (Mlv-3-glc) were used as inhibitors in an enzyme assay, on the basis of the conversion of GalG2CNP by α-amylase. The detection of CNP was performed by UV/vis spectroscopy. The data reveal that the inhibitor strength decreases as follows: EGCG > Mlv-3-glc > EC > PHL ∼ CA. Detection of the substrate conversion by isothermal titration calorimetry supports these results. All PPs showed mixed inhibition, except for CA and EGCG wherein the competitive proportion was predominant. Investigations by saturation transfer difference NMR revealed interaction of PPs with α-amylase prevalently based on interactions with the aromatic or conjugated system. A correlation between the extent of the conjugated system and the IC50 of the PP could be found.

Baicalin, a prodrug able to reach the CNS, is a prolyl oligopeptidase inhibitor.

Prolyl oligopeptidase is a cytosolic serine peptidase that hydrolyzes proline-containing peptides at the carboxy terminus of proline residues. It has been associated with schizophrenia, bipolar affective disorder, and related neuropsychiatric disorders and therefore may have important clinical implications. In a previous work, we used (19)F NMR to search for new prolyl oligopeptidase inhibitors from a library of traditional Chinese medicine plant extracts, and identified several extracts as powerful inhibitors of this peptidase. Here, the flavonoid baicalin was isolated as the active component of an extract of Scutellaria baicalensis roots having prolyl oligopeptidase inhibitory activity. Baicalin inhibited prolyl oligopeptidase in a dose-dependent manner. Inhibition experiments using baicalin analogs showed that the sugar moiety was not necessary for activity. The IC(50)s of baicalin and its aglycone derivative baicalein were rather similar, showing that the sugar moiety was not involved in the interaction of baicalin with POP. These results were confirmed by saturation transfer difference NMR experiments. To further understand the absorption and transport mechanisms of baicalin and baicalein, we evaluated their transport in vitro through the gastrointestinal tract and the blood-brain barrier using a Parallel Artificial Membrane Permeability Assay. The molecule which potentially crosses both barriers was identified as baicalein, the aglycone moiety of baicalin. Our results show that baicalin is a new prodrug able to inhibit prolyl oligopeptidase. As baicalin is a natural compound with a long history of safe administration to humans, it is a highly attractive base from which to develop new treatments for schizophrenia, bipolar affective disorder, and related neuropsychiatric diseases.

Peptide NMHRYPNQ of the cellular prion protein (PrP(C)) inhibits aggregation and is a potential key for understanding prion-prion interactions.

Pathogenesis of transmissible spongiform encephalopathies is correlated with a conversion of the normal cellular form of the prion protein (PrP(C)) into the abnormal isoform (scrapie form of PrP). Contact of the normal PrP with its abnormal isoform, the scrapie form of PrP, induces the transformation. Knowledge of molecules that inhibit such contacts leads to an understanding of the mechanism of the aggregation, and these molecules may serve as leads for drugs against transmissible spongiform encephalopathies. Therefore, we screened a synthetic octapeptide library of the globular domain of the human PrP(C) for binding affinity to PrP(C). Two fragments with binding affinity, 149YYRENMHR156 and 153NMHRYPNQ160, were identified with K(d) values of 21 and 25 microM, respectively. A 10-fold excess of peptide 153NMHRYPNQ160 inhibits aggregation of the PrP by 99%. NMR and mass spectrometry showed that the binding region of the peptide 153NMHRYPNQ160 is located at helix 3 of the PrP.

A new side opening on prolyl oligopeptidase revealed by electron microscopy.

Prolyl oligopeptidase (POP) has gained importance as a target for the treatment of neuropsychiatric diseases and cognitive disturbances. Therefore, a variety of strategies are currently used to identify POP inhibitors. Here we performed electron microscopy (EM) studies of human POP. Our data reveal for the first time the presence of a new side opening in POP that was not observed in any of the crystallographic structures described to date. Finally, molecular dynamics, the relevant normal modes that contribute to the fluctuation of the catalytic triad residues and the algorithm CAVERN also support the existence of a new large side opening on POP.

Direct observation of ligand binding to membrane proteins in living cells by a saturation transfer double difference (STDD) NMR spectroscopy method shows a significantly higher affinity of integrin alpha(IIb)beta3 in native platelets than in liposomes.

About 30% of the proteins in mammalian systems are membrane bound or integrated (e.g., GPCRs). It is inherently difficult to investigate receptor-ligand interactions on a molecular level in their natural membrane environment. Here, we present a new method based on saturation transfer difference (STD) NMR to characterize at an atomic level binding interactions of cell surface proteins in living cells. Implemented as a double difference technique, STD NMR allows the direct observation of binding events and the definition of the binding epitopes of ligands. The binding of the pentapeptide cyclo(RGDfV) to the surface glycoprotein integrin alpha(IIb)beta3 of intact human blood platelets can be detected by saturation transfer double difference (STDD) NMR in less than an hour. A 5-fold higher STD response reflects a significantly higher affinity of integrin alpha(IIb)beta3 in native platelets than in liposomes, which demonstrates the importance of studying membrane proteins in their natural environment. Also, the binding mode of cyclo(RGDfV) in the arginine glycine region is slightly different when interacting with native integrin in platelets compared to integrin reintegrated into liposomes.