Assessment and acceleration of binding energy calculations for protein–ligand complexes by the fragment molecular orbital method

In the field of drug discovery, it is important to accurately predict the binding affinities between target proteins and drug applicant molecules. Many of the computational methods available for evaluating binding affinities have adopted molecular mechanics‐based force fields, although they cannot fully describe protein–ligand interactions. A noteworthy computational method in development involves large‐scale electronic structure calculations. Fragment molecular orbital (FMO) method, which is one of such large‐scale calculation techniques, is applied in this study for calculating the binding energies between proteins and ligands. By testing the effects of specific FMO calculation conditions (including fragmentation size, basis sets, electron correlation, exchange‐correlation functionals, and solvation effects) on the binding energies of the FK506‐binding protein and 10 ligand complex molecule, we have found that the standard FMO calculation condition, FMO2‐MP2/6‐31G(d), is suitable for evaluating the protein–ligand interactions. The correlation coefficient between the binding energies calculated with this FMO calculation condition and experimental values is determined to be R = 0.77. Based on these results, we also propose a practical scheme for predicting binding affinities by combining the FMO method with the quantitative structure–activity relationship (QSAR) model. The results of this combined method can be directly compared with experimental binding affinities. The FMO and QSAR combined scheme shows a higher correlation with experimental data (R = 0.91). Furthermore, we propose an acceleration scheme for the binding energy calculations using a multilayer FMO method focusing on the protein–ligand interaction distance. Our acceleration scheme, which uses FMO2‐HF/STO‐3G:MP2/6‐31G(d) at R_int = 7.0 Å, reduces computational costs, while maintaining accuracy in the evaluation of binding energy. © 2015 Wiley Periodicals, Inc.     

Template‐Assembled Synthetic G‐Quadruplex (TASQ): A Useful System for Investigating the Interactions of Ligands with Constrained Quadruplex Topologies

A new biomolecular device for investigating the interactions of ligands with constrained DNA quadruplex topologies, using surface plasmon resonance (SPR), is reported. Biomolecular systems containing an intermolecular‐like G‐quadruplex motif 1 (parallel G‐quadruplex conformation), an intramolecular G‐quadruplex 2 , and a duplex DNA 3 have been designed and developed. The method is based on the concept of template‐assembled synthetic G‐quadruplex (TASQ), whereby quadruplex DNA structures are assembled on a template that allows precise control of the parallel G‐quadruplex conformation. Various known G‐quadruplex ligands have been used to investigate the affinities of ligands for intermolecular 1 and intramolecular 2 DNA quadruplexes. As anticipated, ligands displaying a π‐stacking binding mode showed a higher binding affinity for intermolecular‐like G‐quadruplexes 1 , whereas ligands with other binding modes (groove and/or loop binding) showed no significant difference in their binding affinities for the two quadruplexes 1 or 2 . In addition, the present method has also provided information about the selectivity of ligands for G‐quadruplex DNA over the duplex DNA. A numerical parameter, termed the G‐quadruplex binding mode index (G4‐BMI), has been introduced to express the difference in the affinities of ligands for intermolecular G‐quadruplex 1 against intramolecular G‐quadruplex 2 . The G‐quadruplex binding mode index (G4‐BMI) of a ligand is defined as follows: G4‐BMI=K_D^intra/K_D^inter, where K_D^intra is the dissociation constant for intramolecular G‐quadruplex 2 and K_D^inter is the dissociation constant for intermolecular G‐quadruplex 1 . In summary, the present work has demonstrated that the use of parallel‐constrained quadruplex topology provides more precise information about the binding modes of ligands.     

Mechanistic Insight into Nanomolar Binding of Multivalent Neoglycopeptides to Wheat Germ Agglutinin

Multivalent carbohydrate–protein interactions are frequently involved in essential biological recognition processes. Accordingly, multivalency is often also exploited for the design of high‐affinity lectin ligands aimed at the inhibition of such processes. In a previous study (D. Schwefel et al., J. Am. Chem. Soc. 2010 , 132, 8704–8719) we identified a tetravalent cyclopeptide‐based ligand with nanomolar affinity to the model lectin wheat germ agglutinin (WGA). To unravel the structural features of this ligand required for high‐affinity binding to WGA, we synthesized a series of cyclic and linear neoglycopeptides that differ in their conformational freedom as well as the number of GlcNAc residues. Combined evidence from isothermal titration calorimetry (ITC), enzyme‐linked lectin assays (ELLA), and dynamic light scattering (DLS) revealed different binding modes of tetra‐ and divalent ligands and that conformational preorganization of the ligands by cyclization is not a prerequisite for achieving high binding affinities. The high affinities of the tetravalent ligands rather stem from their ability to form crosslinks between several WGA molecules. The results illustrate that binding affinities and mechanisms are strongly dependent on the used multivalent system which offers opportunities to tune and control binding processes.     

Thermodynamic Parameters for the Association of Fluorinated Benzenesulfonamides with Bovine Carbonic Anhydrase II

This paper describes a calorimetric study of the association of a series of seven fluorinated benzenesulfonamide ligands (C₆H_nF_5?nSO₂NH₂) with bovine carbonic anhydrase II (BCA). Quantitative structure–activity relationships between the free energy, enthalpy, and entropy of binding and pK_a and log P of the ligands allowed the evaluation of the thermodynamic parameters in terms of the two independent effects of fluorination on the ligand: its electrostatic potential and its hydrophobicity. The parameters were partitioned to the three different structural interactions between the ligand and BCA: the Zn^II cofactor–sulfonamide bond (≈65 % of the free energy of binding), the hydrogen bonds between the ligand and BCA (≈10 %), and the contacts between the phenyl ring of the ligand and BCA (≈25 %). Calorimetry revealed that all of the ligands studied bind in a 1:1 stoichiometry with BCA; this result was confirmed by ¹⁹F NMR spectroscopy and X‐ray crystallography (for complexes with human carbonic anhydrase II).     

Regioselective Hydroxylation of C12–C15 Fatty Acids with Fluorinated Substituents by Cytochrome P450 BM3

We demonstrate herein that wild‐type cytochrome P450 BM3 can recognize non‐natural substrates, such as fluorinated C₁₂–C₁₅ chain‐length fatty acids, and show better catalysis for their efficient conversion. Although the binding affinities for fluorinated substrates in the P450 BM3 pocket are marginally lower than those for non‐fluorinated substrates, spin‐shift measurements suggest that fluoro substituents at the ω‐position can facilitate rearrangement of the dynamic structure of the bulk‐water network within the hydrophobic pocket through a micro desolvation process to expel the water ligand of the heme iron that is present in the resting state. A lowering of the Michaelis–Menten constant (K_m), however, indicates that fluorinated fatty acids are indeed better substrates compared with their non‐fluorinated counterparts. An enhancement of the turnover frequencies (k_cat) for electron transfer from NADPH to the heme iron and for C? H bond oxidation by compound I (Cpd I) to yield the product suggests that the activation energies associated with going from the enzyme–substrate (ES state) to the corresponding transition state (ES^≠ state) are significantly lowered for both steps in the case of the fluorinated substrates. Delicate control of the regioselectivity by the fluorinated terminal methyl groups of the C₁₂–C₁₅ fatty acids has been noted. Despite the fact that residues Arg47/Tyr51/Ser72 exert significant control over the hydroxylation of the subterminal carbon atoms toward the hydrocarbon tail, the fluorine substituent(s) at the ω‐position affects the regioselective hydroxylation. For substrate hydroxylation, we have found that fluorinated lauric acids probably give a better structural fit for the heme pocket than fluorinated pentadecanoic acid, even though pentadecanoic acid is by far the best substrate among the reported fatty acids. Interestingly, 12‐fluorododecanoic acid, with only one fluorine atom at the terminal methyl group, exhibits a comparable turnover frequency to that of pentadecanoic acid. Thus, fluorination of the terminal methyl group introduces additional interactions of the substrate within the hydrophobic pocket, which influence the electron transfers for both dioxygen activation and the controlled oxidation of aliphatics mediated by high‐valent oxoferryl species.     

Application of plug–plug technique to ACE experiments for discovery of peptides binding to a larger target protein: A model study of calmodulin‐binding fragments selected from a digested mixture of reduced BSA

To discover peptide ligands that bind to a target protein with a higher molecular mass, a concise screening methodology has been established, by applying a “plug–plug” technique to ACE experiments. Exploratory experiments using three mixed peptides, mastoparan‐X, β‐endorphin, and oxytocin, as candidates for calmodulin‐binding ligands, revealed that the technique not only reduces the consumption of the protein sample, but also increases the flexibility of the experimental conditions, by allowing the use of MS detection in the ACE experiments. With the plug–plug technique, the ACE–MS screening methodology successfully selected calmodulin‐binding peptides from a random library with diverse constituents, such as protease digests of BSA. Three peptides with K_d values between 8–147 μM for calmodulin were obtained from a Glu‐C endoprotease digest of reduced BSA, although the digest showed more than 70 peaks in its ACE–MS electropherogram. The method established here will be quite useful for the screening of peptide ligands, which have only low affinities due to their flexible chain structures but could potentially provide primary information for designing inhibitors against the target protein.     

Two Distinct Thermal Stabilities of DNA and Enzymatic Activities of DNase I in a Multistep Assembly with Carbazole Ligands: Different Binding Characteristics for Duplex and Quadruplex DNA

A partially hydrophobic carbazole ligand ((Im⁺)₂Cz: 2,2′‐(9‐ethyl‐9 H‐carbazole‐3,6‐diyl)bis(ethyne‐2,1‐diyl)bis(1,3‐dimethyl‐1 H‐imidazol‐3‐ium)) adopts two different binding states (binding states I and II) in its interactions with calf‐thymus (ct‐) DNA. Two distinct binding states were identified by biphasic UV/Vis and circular dichroism (CD) spectral changes during the titration of DNA into the carbazole ligand. At low concentrations of ct‐DNA, (Im⁺)₂Cz binds to nearly every part of ct‐DNA (binding state I). By contrast, an increased concentration of ct‐DNA results in a switch in the DNA‐binding state, so that the ligands are bound per five DNA base pairs. Similarly, a monocationic carbazole ligand (Im⁺Cz: 2‐((6‐bromo‐9‐ethyl‐9 H‐carbazol‐3‐yl)ethynyl)‐1,3‐dimethyl‐1 H‐imidazol‐3‐ium) also shows biphasic UV/Vis spectral changes during the titration of ct‐DNA into Im⁺Cz, which suggests two different binding states of the Im⁺Cz ligand with ct‐DNA. The stepwise equilibrium of the ligand–DNA‐complex formation is capable of switching the thermal stability of ct‐DNA, as well as the enzymatic activity of deoxyribonuclease (DNase I). In binding state I, the (Im⁺)₂Cz ligands interact with nearly every base pair in ct‐DNA and stabilize the double‐helix structure, which results in a larger increase in the melting temperature of the ct‐DNA than that observed with binding state II. On the other hand, the (Im⁺)₂Cz ligand significantly reduces the enzymatic activity of DNase I in binding state I, although the enzymatic activity is recovered once the binding state of the ligand–DNA complex is changed to binding state II. The (Im⁺)₂Cz ligand was also employed as a binder for G‐quadruplex DNA. In contrast to the stepwise complex formation between (Im⁺)₂Cz and ct‐DNA, (Im⁺)₂Cz shows a monotonous UV/Vis spectral response during the titration of G‐quadruplex DNA into (Im⁺)₂Cz, which suggests a single binding state for (Im⁺)₂Cz with G‐quadruplex DNA.     

Exploring Weak Ligand–Protein Interactions by Long‐Lived NMR States: Improved Contrast in Fragment‐Based Drug Screening

Ligands that have an affinity for protein targets can be screened very effectively by exploiting favorable properties of long‐lived states (LLS) in NMR spectroscopy. In this work, we describe the use of LLS for competitive binding experiments to measure accurate dissociation constants of fragments that bind weakly to the ATP binding site of the N‐terminal ATPase domain of heat shock protein 90 (Hsp90), a therapeutic target for cancer treatment. The LLS approach allows one to characterize ligands with an exceptionally wide range of affinities, since it can be used for ligand concentrations [L] that are several orders of magnitude smaller than the dissociation constants K_D. This property makes the LLS method particularly attractive for the initial steps of fragment‐based drug screening, where small molecular fragments that bind weakly to a target protein must be identified, which is a difficult task for many other biophysical methods.     

Synthetic dsDNA‐Binding Peptides Using Natural Compounds as Model

We have developed a series of short DNA‐binding peptides containing newly synthesized, unnatural as well as natural amino acid building blocks. By a combinatorial‐library approach, oligopeptides were developed with moderate dsDNA‐binding affinities. Two strategies were used to further enhance the binding affinity of the lead peptides: Ac‐Arg‐Ual‐Sar‐Chi‐Chi‐Chi‐Arg‐NH₂ and Ac‐Arg‐Cbg‐Cha‐Chi‐Chi‐Tal‐Arg‐NH₂. Site‐selective amino acid substitutions increased the binding affinities up to 2 × 10^?5 M . Further enhancement of the binding affinities could be achieved by coupling of an acridine intercalating unit, using linker arms of different length and flexibility. With the introduction of a new lysine‐based acridine unit, different types of oligopeptide–acridine conjugates were designed using known dsDNA‐binding ligands as model compounds. The binding capacities of these new oligopeptide–acridine conjugates have been investigated by a fluorescent intercalator (ethidium bromide) displacement (FID) assay. With the synthesis of the dipeptide–acridine conjugates, binding affinities in the low micromolar range were obtained (6.4 × 10^?6 M ), which is similar to the binding strength of the well‐known DNA binder Hoechst 33258.     

Ligand–Receptor Binding Affinities from Saturation Transfer Difference (STD) NMR Spectroscopy: The Binding Isotherm of STD Initial Growth Rates

The direct evaluation of dissociation constants (K_D) from the variation of saturation transfer difference (STD) NMR spectroscopy values with the receptor–ligand ratio is not feasible due to the complex dependence of STD intensities on the spectral properties of the observed signals. Indirect evaluation, by competition experiments, allows the determination of K_D, as long as a ligand of known affinity is available for the protein under study. Herein, we present a novel protocol based on STD NMR spectroscopy for the direct measurements of receptor–ligand dissociation constants (K_D) from single‐ligand titration experiments. The influence of several experimental factors on STD values has been studied in detail, confirming the marked impact on standard determinations of protein–ligand affinities by STD NMR spectroscopy. These factors, namely, STD saturation time, ligand residence time in the complex, and the intensity of the signal, affect the accumulation of saturation in the free ligand by processes closely related to fast protein–ligand rebinding and longitudinal relaxation of the ligand signals. The proposed method avoids the dependence of the magnitudes of ligand STD signals at a given saturation time on spurious factors by constructing the binding isotherms using the initial growth rates of the STD amplification factors, in a similar way to the use of NOE growing rates to estimate cross relaxation rates for distance evaluations. Herein, it is demonstrated that the effects of these factors are cancelled out by analyzing the protein–ligand association curve using STD values at the limit of zero saturation time, when virtually no ligand rebinding or relaxation takes place. The approach is validated for two well‐studied protein–ligand systems: the binding of the saccharides GlcNAc and GlcNAcβ1,4GlcNAc (chitobiose) to the wheat germ agglutinin (WGA) lectin, and the interaction of the amino acid L ‐tryptophan to bovine serum albumin (BSA). In all cases, the experimental K_D measured under different experimental conditions converged to the thermodynamic values. The proposed protocol allows accurate determinations of protein–ligand dissociation constants, extending the applicability of the STD NMR spectroscopy for affinity measurements, which is of particular relevance for those proteins for which a ligand of known affinity is not available.     

Fluorinated Carbohydrates as Lectin Ligands: Dissecting Glycan–Cyanovirin Interactions by Using 19F NMR Spectroscopy

NMR spectroscopy and isothermal titration calorimetry (ITC) are powerful methods to investigate ligand–protein interactions. Here, we present a versatile and sensitive fluorine NMR spectroscopic approach that exploits the ¹⁹F nucleus of ¹⁹F‐labeled carbohydrates as a sensor to study glycan binding to lectins. Our approach is illustrated with the 11 kDa Cyanovirin‐N, a mannose binding anti‐HIV lectin. Two fluoro‐deoxy sugar derivatives, methyl 2‐deoxy‐2‐fluoro‐α‐D ‐mannopyranosyl‐(1→2)‐α‐D ‐mannopyranoside and methyl 2‐deoxy‐2‐fluoro‐α‐D ‐mannopyranosyl‐(1→2)‐α‐D ‐mannopyranosyl‐(1→2)‐α‐D ‐mannopyranoside were utilized. Binding was studied by ¹⁹F NMR spectroscopy of the ligand and ¹H–¹⁵N HSQC NMR spectroscopy of the protein. The NMR data agree well with those obtained from the equivalent reciprocal and direct ITC titrations. Our study shows that the strategic design of fluorinated ligands and fluorine NMR spectroscopy for ligand screening holds great promise for easy and fast identification of glycan binding, as well as for their use in reporting structural and/or electronic perturbations that ensue upon interaction with a cognate lectin.     

Targeting programmed cell death 4 (PDCD4) with biogenic compounds in ARDS by Gaussian process‐based QSAR virtual screening

The programmed cell death 4 (PDCD4) has recently been recognized as a new and attractive target of acute respiratory distress syndrome. Here, we attempted to discover new and potent PDCD4 mediator ligands from biogenic compounds using a synthetic strategy of statistical virtual screening and experimental affinity assay. In the procedure, a Gaussian process‐based quantitative structure‐activity relationship regression predictor was developed and validated statistically based on a curated panel of structure‐based protein‐ligand affinity data. The predictor was integrated with pharmacokinetics analysis, chemical redundancy reduction, and flexible molecular docking to perform high‐throughput virtual screening against a distinct library of chemically diverse, drug‐like biogenic compounds. Consequently, 6 hits with top scores were selected, and their binding affinities to the recumbent protein of human PDCD4 were identified, 3 out of which were determined to have high or moderate affinity with K_d at micromolar level. Structural analysis of protein‐ligand complexes revealed that hydrophobic interactions and van der Waals contacts are the primary chemical forces to stabilize the complex architecture of PDCD4 with these mediator ligands, while few hydrogen bonds, salt bridges, and/or π‐π stacking at the complex interfaces confer selectivity and specificity for the protein‐ligand recognition. It is suggested that the statistical Gaussian process‐based quantitative structure‐activity relationship screening strategy can be successfully applied to rational discovery of biologically active compounds. The newly identified molecular entities targeting PDCD4 are considered as promising lead scaffolds to develop novel chemical therapeutics for acute respiratory distress syndrome.     

Two silver(I) complexes with bis(benzimidazole)‐2‐oxopropane ligands: Syntheses,crystal structures and DNA binding studies

Two Ag(I) complexes, [Ag₂(bobb)₂]⋅(NO₃)₂ ( 1 ) and [Ag₂(crotonate)₂(aobb)]_n ( 2 ) (bobb =1,3‐bis(1‐benzylbenzimidazol‐2‐yl)‐2‐oxapropane; aobb =1,3‐bis(1‐allylbenzimidazol‐2‐yl)‐2‐oxopropane), have been synthesized and characterized using elemental analysis, electrical conductivities, infrared and UV–visible spectral measurements and single‐crystal X‐ray diffraction. Complex 1 is binuclear and three‐coordinated by two N atoms from two bobb ligands, while complex 2 is a unique metal organic compound with diamond‐like multinuclear Ag centers with each Ag bridged by two aobb ligands and two crotonate ions to form one‐dimensional single polymer chain structures and extended into two‐dimensional frameworks through π–π and intermolecular C─H⋅⋅⋅O hydrogen bonds. The adjacent Ag(I) centers are bridged by allyl from aobb which is not only a σ‐bonding ligand, but also a π‐acid ligand. The DNA binding modes of complexes 1 and 2 were investigated using electronic absorption titration, fluorescence spectra and viscosity measurements. The results suggest that the two complexes bind to DNA via an intercalative mode, and their binding affinity for DNA follows the order 2  >  1 . This is due to the chelating effects which can enhance the planar functionality of the metal complexes.     

Small Cause,Great Impact: Modification of the Guanidine Group in the RGD Motif Controls Integrin Subtype Selectivity

Due to its unique role as a hydrogen‐bond donor and its positive charge, the guanidine group is an important pharmacophoric group and often used in synthetic ligands. The chemical modification of the guanidine group is often considered to destroy its function. Herein, we show that the N‐methylation, N‐alkylation, or N‐acylation of the guanidine group can be used to modify the receptor subtype specificity of the integrin ligand cilengitide. Using the αvβ6/α5β1‐biselective ligand c(isoDGRkphg) and the αvβ6‐specific ligand c(FRGDLAFp(NMe)K(Ac) as examples, we show that the binding affinities of the ligands can be fine‐tuned by this method to enhance the selectivity for αvβ6. Furthermore, we describe a new strategy for the functionalization of integrin ligands. By introducing longer N‐alkylguanidine and N‐acylguanidine groups, we are able to simultaneously identify a hitherto unknown anchoring point and enhance the subtype selectivity of the ligand.     

Compressed images for affinity prediction (CIFAP): a study on predicting binding affinities for checkpoint kinase 1 protein inhibitors

Analyses of known protein–ligand interactions play an important role in designing novel and efficient drugs, contributing to drug discovery and development. Recently, machine learning methods have proven useful in the design of novel drugs, which utilize intelligent techniques to predict the outcome of unknown protein–ligand interactions by learning from the physical and geometrical properties of known protein–ligand interactions. The aim of this study is to work through a specific example of a novel computational method, namely compressed images for affinity prediction (CIFAP), in which binding affinities for structurally related ligands in complexes with human checkpoint kinase 1 (CHK1) are predicted. The CIFAP algorithm presented here relates published pIC ₅₀ values of 57 compounds, derived from a thienopyridine pharmacophore, in complexes with CHK1 to their two‐dimensional (2D) electrostatic potential images compressed in orthogonal dimensions. Patterns obtained from the 2D images are then used as inputs in regression and learning algorithms such as support vector regression (SVR) and adaptive neuro‐fuzzy inference system (ANFIS) methods to validate the experimental pIC ₅₀ values. This study revealed that the 2D image pixels in the vicinity of bound ligand surfaces provide more relevant information to make correlations with the empirical pIC ₅₀ values. As compared with ANFIS, SVR gave rise to the lowest root mean square errors and the greatest correlations, suggesting that SVR could be a plausible choice of machine learning methods in predicting binding affinities by CIFAP. Copyright © 2013 John Wiley & Sons, Ltd.     

A Stable Monomeric SiO2 Complex with Donor–Acceptor Ligands

Isolation of a monomeric SiO₂ compound 3 as a stable donor–acceptor complex with two different ligands —a σ‐donating ligand (pyridine, dimethylaminopyridine, N ‐heterocyclic carbene) and a donor–acceptor ligand (iminophosphorane)—is presented. The SiO₂ complex 3 is soluble in ordinary organic solvents and is stable at room temperature in solution and in the solid state. Of particular interest, 3 remains reactive and can be used as a stable and soluble unimolecular SiO₂ reagent.     

Electronic Olfactory Sensor Based on A. mellifera Odorant‐Binding Protein 14 on a Reduced Graphene Oxide Field‐Effect Transistor

An olfactory biosensor based on a reduced graphene oxide (rGO) field‐effect transistor (FET), functionalized by the odorant‐binding protein 14 (OBP14) from the honey bee (Apis mellifera) has been designed for the in situ and real‐time monitoring of a broad spectrum of odorants in aqueous solutions known to be attractants for bees. The electrical measurements of the binding of all tested odorants are shown to follow the Langmuir model for ligand–receptor interactions. The results demonstrate that OBP14 is able to bind odorants even after immobilization on rGO and can discriminate between ligands binding within a range of dissociation constants from K_d=4 μM to K_d=3.3 mM . The strongest ligands, such as homovanillic acid, eugenol, and methyl vanillate all contain a hydroxy group which is apparently important for the strong interaction with the protein.     

Ethylene polymerization by the new chromium catalysts based on amino‐pyrrolide ligands

A series of amino‐pyrrolide ligands ( 1–4a ) and their derivatives amino‐thiophene ligand ( 5a ), amino‐indole ligand ( 6a ) were prepared. Chromium catalysts, which were generated in situ by mixing the ligands with CrCl₃(thf)₃ in toluene, were tested for ethylene polymerization. The preliminary screening results revealed that the tridentate amino‐pyrrolide ligands containing soft pendant donor, 3a, 4a /CrCl₃(thf)₃ systems displayed high catalytic activities towards ethylene polymerization in the presence of modified methyaluminoxane. The electronic and steric factors attached to the ligand backbone significantly affected both the catalyst activity and the polymer molecular weight. Complex 4b was obtained by the reaction of CrCl₃(thf)₃ with one equivalent of the lithium salts of 4a , which was the most efficient ligand among the tested ones. The effect of polymerization parameters such as cocatalyst concentration, ethylene pressure, reaction temperature, and time on polymerization behavior were investigated in detail. The resulting polymer obtained by 4b display wax‐like and possess linear structure, low molecular weight, and unimodal distribution. © 2008 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 47: 713–721, 2009     

Unwinding DNA and RNA with Synthetic Complexes: On the Way to Artificial Helicases

Synthetic helicases can be designed on the basis of ligands that bind more strongly to single‐stranded nucleic acids than to double‐stranded nucleic acids. This can be achieved with ligands containing phenyl groups, which intercalate into single strands, but due to their small size not into double strands. Moreover, two phenyl rings are combined with a distance that allows bis‐intercalation with only single strands and not double strands. In this respect, such ligands also mimic single‐strand binding (SSB) proteins. Exploration with more than 23 ligands, mostly newly synthesised, shows that the distance between the phenyl rings and between those and the linker influence the DNA unwinding efficiency, which can reach a melting point decrease of almost ΔT_m=50 °C at much lower concentrations than that with any other known artificial helicases. Conformational pre‐organisation of the ligand plays a decisive role in optimal efficiency. Substituents at the phenyl rings have a large effect, and increase, for example, in the order of H<F<Cl<Br, which illustrates the strong role of dispersive interactions in intercalation. Studies with homopolymers revealed significant selectivity: for example, with a ligand concentration of 40 μM at 35 °C, only GC double strands melt (ΔT_m=48 °C), whereas the AT strand remains untouched, and with poly(rA)–poly(rU) as an RNA model one observes unfolding at 29 °C with a concentration of only 30 μM .     

Clustering of Escherichia coli type-1 fimbrial adhesins by using multimeric heptyl α-D-mannoside probes with a carbohydrate core

Heptyl α‐D ‐mannoside (HM) is a strong inhibitor of the FimH lectin that mediates the initial adhesion of the uropathogenic Escherichia coli (E. coli) to the bladder cells. We designed a set of multivalent HM ligands based on carbohydrate cores with structural valencies that range from 1 to 7. The chemical strategy used to construct the regular hydrophilic structures consisted of the repetition of a critical glucoside fragment. A primary amino group was grafted at the sugar reducing end to couple the multimers to a fluorescent label. A one‐pot synthetic approach was developed to tether the ligands and the fluorescein isothiocyanate (FITC) probe to the scaffold simultaneously. Isothermal calorimetry with the monomeric FimH lectin revealed nanomolar affinities and saturation of all structurally available binding sites on the multivalent HM ligands. Direct titrations domain showed almost strict correlation of enthalpy–entropy compensation with increasing valency of the ligand, whereas reverse titration calorimetry demonstrated negative cooperativity between the first and the second binding site of the divalent heptyl mannoside. A multivalency effect was nevertheless observed by inhibiting the haemagglutination of type‐1 piliated UTI89 E. coli, with a titer as low as 60 nM for the heptavalent HM ligand. An FITC‐labeled HM trimer showed capture and cross‐linking of living bacteria in solution, a phenomenon not previously described with low‐valency ligands.