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.

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.

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.

Late-stage oxyfunctionalization of terpenoid scaffolds has been recognized as a powerful tool in the synthesis of complex molecules such as natural products to achieve their efficient diversification. Selective C−H oxidation of such hydrocarbon scaffolds remains challenging for chemical catalysts because of their insufficient regio-and stereoselectivity. To achieve this goal, cytochrome P450 monooxygenases are often used as biocatalysts. Here, we demonstrate the successful P450catalyzed chemo-, regio-, and stereoselective oxidation of the tobacco cembranoid β-cembrenediol. This 14-membered macrocycle possesses a broad range of biological activities including antitumor-promoting and neuroprotective effects and carries seven potential sites for allylic hydroxylation as well as three epoxidation sites. On the basis of first-sphere active site mutagenesis, we generated in a few rounds a P450 BM3 minimal library and screened for β-cembrenediol oxidation activity. Several P450 BM3 variants were evolved, enabling the regioselective hydroxylation of the neighboring positions C-9 (100% regioselectivity and a diastereomeric ratio of 89:11 in the case of the F87A/I263L mutant) and C-10 (97% regioselectivity and a diastereomeric ratio of 74:26 in the case of the L75A/V78A/F87G mutant) of β-cembrenediol.

The role of liquid confinement on the asymmetric Rh catalysis was studied using the 1,2-addition of phenylboroxine (2)t oN-tosylimine 1 in the presence of [RhCl(C 2 H 4 ) 2 ] 2 and chiral dienel igandsa sb enchmark reaction. To get access to Rh complexes of differentpolarity,enantiomerically pure C 2 -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.Amicroemulsion containing the equal amounts of H 2 O/KOH and toluene/reactants was formulated using the hydrophilic sugar surfactant n-octyl b-d-glucopyranoside( C 8 G 1 )t om ediate the miscibility between the nonpolarr eactants and KOH, neededt oa ctivate the Rh-diene complex. Prominent features of this organized reaction medium are its temperature insensitivity as well as the presence of water and toluenerich compartments with ad omain size of 55 confirmed by small-angle X-ray scattering (SAXS). Althoughb icyclooctadiene ligands 4a,b,e performed equally well under homogeneous and microemulsion conditions, ligands 4c,d gave a different chemoselectivity.F or norbornadienes 5, 6,h owever, microemulsions markedly improved conversion and enantioselectivity as well as reaction rate, as was confirmed by kinetic studies using ligand 5b.Scheme1.EnvisionedRh-catalyzed asymmetric 1,2-additions in the presence of novel chiral bicyclo[3.3.0]octadiene and norbornadiene basedl igands 4-6 (ME:microemulsion). Scheme2.Synthesis of novel chiral bicyclo[3.3.0.]octadiene ligands 4.For details see the SupportingInformation.