Precipitation-free high-affinity multivalent binding by inline lectin ligands.

Multivalent ligand-protein interactions are a key concept in biology mediating, for example, signalling and adhesion. Multivalent ligands often have tremendously increased binding affinities. However, they also can cause crosslinking of receptor molecules leading to precipitation of ligand-receptor complexes. Plaque formation due to precipitation is a known characteristic of numerous fatal diseases limiting a potential medical application of multivalent ligands with a precipitating binding mode. Here, we present a new design of high-potency multivalent ligands featuring an inline arrangement of ligand epitopes with exceptionally high binding affinities in the low nanomolar range. At the same time, we show with a multi-methodological approach that precipitation of the receptor is prevented. We distinguish distinct binding modes of the ligands, in particular we elucidate a unique chelating binding mode, where four receptor binding sites are simultaneously bridged by one multivalent ligand molecule. The new design concept of inline multivalent ligands, which we established for the well-investigated model lectin wheat germ agglutinin, has great potential for the development of high-potency multivalent inhibitors as future therapeutics.

Optimising broadband pulses for DEER depends on concentration and distance range of interest.

EPR distance determination in the nanometre region has become an important tool for studying the structure and interaction of macromolecules. Arbitrary waveform generators (AWGs), which have recently become commercially available for EPR spectrometers, have the potential to increase the sensitivity of the most common technique, double electron-electron resonance (DEER, also called PELDOR), as they allow the generation of broadband pulses. There are several families of broadband pulses, which are different in general pulse shape and the parameters that define them. Here, we compare the most common broadband pulses. When broadband pulses lead to a larger modulation depth, they also increase the background decay of the DEER trace. Depending on the dipolar evolution time, this can significantly increase the noise level towards the end of the form factor and limit the potential increase in the modulation-to-noise ratio (MNR). We found asymmetric hyperbolic secant (HS{1,6}) pulses to perform best for short DEER traces, leading to a MNR improvement of up to 86 % compared to rectangular pulses. For longer traces we found symmetric hyperbolic secant (HS{1,1}) pulses to perform best; however, the increase compared to rectangular pulses goes down to 43 %.

Conformationally Unambiguous Spin Label for Exploring the Binding Site Topology of Multivalent Systems.

Multivalent carbohydrate-lectin interactions are a key concept in biological processes mediating, for example, signaling and adhesion. Binding affinities of multivalent ligands often increase by orders of magnitude compared to a monovalent binding situation. Thus, the design of multivalent ligands as potent inhibitors is a highly active field of research, where knowledge about the binding site topology is crucial. Here, we report a general strategy for precise distance measurements between the binding sites of multivalent target proteins using monovalent ligands. We designed and synthesized Monovalent, conformationally Unambiguously Spin-labeled LIgands (MUeSLI). Distances between the binding sites of the multivalent model lectin wheat germ agglutinin in complex with a GlcNAc-derived MUeSLI were determined using pulsed electron paramagnetic resonance spectroscopy. This approach is an efficient method for exploring multivalent systems with monovalent ligands, and it is readily transferable to other target proteins, allowing the targeted design of multivalent ligands without structural information available.

Fucose-Functionalized Precision Glycomacromolecules Targeting Human Norovirus Capsid Protein.

Norovirus infection is the major cause of nonbacterial gastroenteritis in humans and has been the subject of numerous studies investigating the virus's biophysical properties and biochemical function with the aim of deriving novel and highly potent entry inhibitors to prevent infection. Recently, it has been shown that the protruding P domain dimer (P-dimer) of a GII.10 Norovirus strain exhibits two new binding sites for l-fucose in addition to the canonical binding sites. Thus, these sites provide a novel target for the design of multivalent fucose ligands as entry inhibitors of norovirus infections. In this current study, a first generation of multivalent fucose-functionalized glycomacromolecules was synthesized and applied as model structures to investigate the potential targeting of fucose binding sites in human norovirus P-dimer. Following previously established solid phase polymer synthesis, eight precision glycomacromolecules varying in number and position of fucose ligands along an oligo(amidoamine) backbone were obtained and then used in a series of binding studies applying native MS, NMR, and X-ray crystallography. We observed only one fucose per glycomacromolecule binding to one P-dimer resulting in similar binding affinities for all fucose-functionalized glycomacromolecules, which based on our current findings we attribute to the overall size of macromolecular ligands and possibly to steric hindrance.

Redox-Active Tetraruthenium Macrocycles Built from 1,4-Divinylphenylene-Bridged Diruthenium Complexes.

Metallamacrocylic tetraruthenium complexes were generated by treatment of 1,4-divinylphenylene-bridged diruthenium complexes with functionalized 1,3-benzene dicarboxylic acids and characterized by HR ESI-MS and multinuclear NMR spectroscopy. Every divinylphenylene diruthenium subunit is oxidized in two consecutive one-electron steps with half-wave potential splittings in the range of 250 to 330 mV. Additional, smaller redox-splittings between the +/2+ and 0/+ and the 3+/4+ and 2+/3+ redox processes, corresponding to the first and the second oxidations of every divinylphenylene diruthenium entity, are due to electrostatic effects. The lack of electronic coupling through bond or through space is explained by the nodal properties of the relevant molecular orbitals and the lateral side-by-side arrangement of the divinylphenylene linkers. The polyelectrochromic behavior of the divinylphenylene diruthenium precursors is retained and even amplified in these metallamacrocyclic structures. EPR studies down to T=4 K indicate that the dications 1-H(2+) and 1-OBu(2+) are paramagnetic. The dications and the tetracation of macrocycle 3-H display intense (dications) or weak (3-H(4+) ) EPR signals. Quantum chemical calculations indicate that the four most stable conformers of the macrocycles are largely devoid of strain. Bond parameters, energies as well as charge and spin density distributions of model macrocycle 5-H(Me) were calculated for the different charge and spin states.