Achieving High Affinity towards a Bacterial Lectin through Multivalent Topological Isomers of Calix[4]arene Glycoconjugates

A family of seven topologically isomeric calix[4]arene glycoconjugates was prepared through the synthesis of a series of alkyne‐derivatised calix[4]arene precursors that are suitable for the attachment of sugar moieties by microwave‐assisted copper(I)‐catalysed azide–alkyne cycloaddition (CuAAC). The glycoconjugates thus synthesised comprised one mono‐functionalised derivative, two 1,2‐ or 1,3‐divalent regioisomers, one trivalent and three tetravalent topoisomers in the cone, partial cone or 1,3‐alternate conformations. The designed glycoconjugates were evaluated as ligands for the galactose‐binding lectin PA‐IL from the opportunistic bacterium Pseudomonas aeruginosa, a major causative agent of lung infections in cystic fibrosis patients. Binding affinities were determined by isothermal titration calorimetry (ITC), and the interaction with the lectin was shown to be strongly dependant on both the valence and the topology. Whereas the trivalent conjugate displayed enhanced affinity when compared to a monosaccharide model, the tetravalent conjugates are to‐date the highest‐affinity ligands measured by ITC. The topologies presenting carbohydrates on both faces of calixarene are the most potent ones with dissociation constants of approximately 200 nM . Molecular modelling suggests that such a multivalent molecule can efficiently chelate two of the binding sites of the tetrameric lectin; this explains the 800‐fold increase of affinity achieved by the tetravalent molecule. Surface plasmon resonance (SPR) experiments confirmed that this glycoconjugate is the strongest inhibitor for binding of PA‐IL to galactosylated surfaces for potential applications as an anti‐adhesive agent.     

Substrate‐induced formation of a recognition structure in a four‐polypeptide–lipid monolayer system

Poly(γ‐methyl L ‐glutamate)s with Ser, His, Asp, and Glu residues at the amino terminal as the serine protease catalytic site were prepared. The number‐average degree of polymerization of the polypeptides was 51. A dipalmitoylphosphatidylcholine monolayer containing the polypeptides was formed at the air–water interface and was transferred onto gold‐deposited glass plates. The binding of N‐acetyltyrosine ethyl ester, a typical substrate of the serine protease, to the monolayer was characterized by surface plasmon resonance measurements. The four‐polypeptide–lipid monolayer system conditioned on an aqueous solution containing the substrate N‐acetyltyrosine ethyl ester exhibited Langmuir‐type binding of the substrate. Its binding constant of 6.1 × 10⁴ M⁻¹ was about 20 times larger than that observed for a monolayer prepared on pure water. The behavior may have arisen from a substrate‐induced rearrangement of the four kinds of polypeptides in the monolayer, forming a substrate‐binding structure similar to that found in serine protease. © 2000 John Wiley & Sons, Inc. J Polym Sci A: Polym Chem 38: 2186–2191, 2000     

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.     

Protein Dimerization on a Phosphonated Calix[6]arene Disc

Complex formation between cationic cytochrome c and the water‐soluble, poly‐anionic p ‐phosphonatocalix[6]arene (pclx₆) was investigated. A crystal structure (at 1.8 Å resolution) revealed a remarkable dimeric disc of pclx₆ that acts like glue to mediate a symmetric (C ₂) protein dimer. The calixarene disc has a diameter of about 1.5 nm and masks about 360 Ų of protein surface. The key protein–calixarene contacts occur via two linchpin lysines, with additional contacts provided by a small hydrophobic patch. The protein–calixarene supramolecular assemblies were observed in solution by size‐exclusion chromatography with multi‐angle light scattering and NMR spectroscopy. Using isothermal titration calorimetry and NMR data, an apparent K _d in the low micromolar range was determined for the charge‐rich protein–calixarene complex. In contrast to p ‐sulfonatocalix[4]arene, the larger pclx₆ has a single, well‐defined binding site that mediates the assembly of cytochrome c in solution.     

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.     

Sensing of Double‐Stranded DNA/RNA Secondary Structures by Water Soluble Homochiral Perylene Bisimide Dyes

A broad series of homochiral perylene bisimide (PBI) dyes were synthesized that are appended with amino acids and cationic side chains at the imide positions. Self‐assembly behavior of these ionic PBIs has been studied in aqueous media by UV/Vis spectroscopy, revealing formation of excitonically coupled H‐type aggregates. The interactions of these ionic PBIs with different ds‐DNA and ds‐RNA have been explored by thermal denaturation, fluorimetric titration and circular dichroism (CD) experiments. These PBIs strongly stabilized ds‐DNA/RNA against thermal denaturation as revealed by high melting temperatures of the formed PBI/polynucleotide complexes. Fluorimetric titrations showed that these PBIs bind to ds‐DNA/RNA with high binding constants depending on the number of the positive charges in the side chains. Thus, spermine‐containing PBIs with six positive charges each showed higher binding constants (logK_s=9.2–9.8) than their dioxa analogues (logK_s=6.5–7.9) having two positive charges each. Induced circular dichroism (ICD) of PBI assemblies created within DNA/RNA grooves was observed. These ICD profiles are strongly dependent on the steric demand of the chiral substituents of the amino acid units and the secondary structure of the DNA or RNA. The observed ICD effects can be explained by non‐covalent binding of excitonically coupled PBI dimer aggregates into the minor groove of DNA and major groove of RNA which is further supported by molecular modeling studies.     

Formation and Structure of Fluorescent Silver Nanoclusters at Interfacial Binding Sites Facilitating Oligomerization of DNA Hairpins

Fluorescent, DNA‐stabilized silver nanoclusters (DNA‐AgNCs) are applied in a range of applications within nanoscience and nanotechnology. However, their diverse optical properties, mechanism of formation, and aspects of their composition remain unexplored, making the rational design of nanocluster probes challenging. Herein, a synthetic procedure is described for obtaining a high yield of emissive DNA‐AgNCs with a C‐loop hairpin DNA sequence, with subsequent purification by size‐exclusion chromatography (SEC). Through a combination of optical spectroscopy, gel electrophoresis, inductively coupled plasma mass spectrometry (ICP‐MS), and small‐angle X‐ray scattering (SAXS) in conjunction with the systematic study of various DNA sequences, the low‐resolution structure and mechanism of the formation of AgNCs were investigated. Data indicate that fluorescent DNA‐AgNCs self‐assemble by a head‐to‐head binding of two DNA hairpins, bridged by a silver nanocluster, resulting in the modelling of a dimeric structure harboring an Ag₁₂ cluster.     

Adsorption of Propene on Neutral Gold Clusters in the Gas Phase

The adsorption of propene on neutral gold clusters is investigated in a collision cell under a few collision conditions. The adsorption reaction is studied by pressure‐dependent kinetic measurements and delayed unimolecular dissociation of the excited Au_n?propene complexes. The cluster size (n=9–25) and temperature (T=90–300 K) dependence of the propene adsorption is analyzed. Strong size dependences of the absorption reaction are observed; a larger propene adsorption probability was found for gold clusters composed of an even number of atoms. Propene binding energies are estimated by comparison of the temperature‐dependent unimolecular dissociation rates with rates obtained by using statistical RRKM modeling. The Au_n–propene binding energies decrease non‐monotonously with cluster size and are in the range of 1.2–0.85 eV for n=9–25. Finally, the bonding of C₃H₆ on Au_n is qualitatively described and similarities with the absorption of CO molecules on gold clusters are discussed.     

UVA and UVB‐Induced 8‐Methoxypsoralen Photoadducts and a Novel Method for their Detection by Surface‐Enhanced Laser Desorption Ionization Time‐of‐Flight Mass Spectrometry (SELDI‐TOF MS)

UVA‐activated psoralens are used to treat hyperproliferative skin conditions due to their ability to form DNA photoadducts, which impair cellular processes and may lead to cell death. Although UVA (320–400 nm) is more commonly used clinically, studies have shown that UVB (280–320 nm) activation of psoralen can also be effective. However, there has been no characterization of UVB‐induced adduct formation in DNA alone. As psoralen derivatives have a greater extinction coefficient in the UVB region (11 800 cm^?1 M^?1 at 300 nm) compared with the UVA region (2016 cm^?1 M^?1 at 365 nm), a greater extent of adduct formation is expected. SELDI‐TOF, a proteomic technique that combines chromatography with mass spectrometry, was used to detect photoadduct formation in an alternating A–T oligonucleotide. 8‐Methoxypsoralen (8‐MOP) and DNA solutions were irradiated with either UVA or UVB. An adduct peak was obtained with SELDI‐TOF. For UVB‐activated 8‐MOP, the extent of adducts was three times greater than for UVA. HPLC ESI‐MS analysis showed that UVB irradiation yielded high levels of 3,4‐monoadducts (78% of total adducts). UVA was more effective than UVB at conversion of 4′,5′‐monoadducts to crosslinks (17% vs 4%, respectively). This report presents a method for comparing DNA binding efficiencies of interstrand crosslink inducing agents.     

Quantifying the Effect of Surface Ligands on Dendron–DNA Interactions: Insights into Multivalency through a Combined Experimental and Theoretical Approach

We report the synthesis, DNA binding ability and preliminary gene delivery profiles of dendrons with different amine surface groups, 1,3‐diaminopropane (DAP), N,N‐di‐(3‐aminopropyl)‐N‐(methyl)amine (DAPMA) and spermine (SPM). By using a combination of ethidium bromide displacement, gel electrophoresis and transfection assays, it is shown that the dendrons with SPM groups are the most effective DNA binders, while the DAPMA‐functionalised dendrons were the most effective systems for gene delivery (although the gene delivery profiles were still modest). In order to provide deeper insight into the experimental data, we performed a molecular dynamics simulation of the interactions between the dendrons and DNA. The results of these simulations demonstrated that, in general terms, the enthalpic contribution to binding was roughly proportional to the dendron surface charge, but that dendrons with DAP (and DAPMA) surface amines had significant entropic costs of binding to DNA. In the case of DAP, this is a consequence of the fact that the entire dendron structure has to be organised in order for each individual monoamine charge to make effective contact with DNA. For SPM, however, each surface ligand is already a multivalent triamine, therefore, each individual charge has a much lower entropic cost of binding. For DAPMA, we observed that strong binding of the hindered tertiary amine to the DNA double helix led to ligand back‐folding and significant geometric distortion of DNA. Although this weakens the overall binding, we suggest that this distortion might be an explanation for the experimentally observed enhanced gene delivery, in which DNA compaction is an important step. Overall, this paper demonstrates how structure–activity relationships can be developed for multivalent dendritic ligands and provides insights into the thermodynamics of multivalent interactions.     

Computational study of metathesis degradation of rubber, 1. Distribution of cyclic oligomers via intramolecular metathesis degradation of cis‐polybutadiene

The results of the molecular modelling of the intramolecular metathesis degradation of cis‐polybutadiene (cis‐PB) into cyclic oligomers at PM3 level of theory showed that the chain‐ring equilibrium is completely shifted towards the all‐trans cyclic isomers. The formation of cyclic products, containing from three to six butadiene units, from larger rings is thermodynamically favoured with cyclic butadiene tetramers and pentamers being the main products. These results are in reasonable agreement with most of the available experimental data. The discrepancy observed in some cases between found and calculated ring distributions for the intramolecular metathesis degradation of cis‐PB suggests that the reaction is kinetically controlled under certain conditions.     

Dimeric Calixarenes: A New Family of Major‐Groove Binders

A new class of potent DNA binding agents is presented. Dimeric calix[4]arenes with cationic groups at their upper rims and flexible alkyl bridges can be synthesized from triply acyl‐protected calix[4]arene tetramines in relatively short synthetic sequences (3–5 steps). The compounds attach themselves to double‐stranded nucleic acids in a noncovalent fashion, with micro‐ to nanomolar affinities. Guanidinium headgroups with their extended hydrogen‐bonding “fingers” are more powerful than ammonium groups, and the benzylamine series is superior to the anilinium series (see below). The new ligands easily distinguish between RNA and various DNA types, and produce characteristic changes in UV/Vis, fluorescence, CD, as well as NMR spectra. Especially extended oligonucleotides of more than 100 base pairs are bound with affinities increasing from RNA (10 μM K_d)<AT‐rich (1 μM )<GC‐rich DNA double strands (100–10 nM ). Ethidium bromide displacement studies confirm this order. CE₅₀ values are remarkably low (1–4 μM ), and are more than 300 times lower than that of spermine, which is a typical backbone binder. Stoichiometries are rather high (one calixarene dimer per two BP), suggesting a potential aggregation of bound ligands inside the major groove. Most UV/Vis melting curves display an inverted shape, and start from drastically enhanced absorption intensities for the DNA complexes. DAPI displacement studies prove that up to one equivalent of calixarene dimer can be accommodated in the dye‐loaded DNA. RNA complexation by calixarene dimers is accompanied by a drastic CD spectral transition from the typical A‐form to a perfect B‐signature, providing further experimental evidence for major‐groove binding. The orientation of the ligands can be deduced from NMR titrations and is reproduced in Monte‐Carlo simulations on 1:1 complexes in water.     

The Role of Formamidine Groups in Dextran Based Nonviral Vectors for Gene Delivery on Their Physicochemical and Biological Characteristics

Dextran‐formamidine esters (dextran‐N‐[(dimethylamino)methylene]‐β‐alanine ester) with different degrees of substitution (0.45–0.92) are synthesized in an one‐pot reaction. Dextran (Mw 60 000 g mol^?1) is allowed to react with unprotected beta‐alanine and iminium chloride and investigated regarding the potential as gene delivery system for the transfer of plasmid DNA. With degrees of substitution ≥ 0.63 improved DNA binding with formation of enzymatically stable complexes of about 130–160 nm with negative surface charges are obtained. These physicochemical characteristics correlated with increasing transfection rates in CHO‐K1 cells determined by a luciferase reporter gene assay in dependency of the number of formamidine residues, N/P ratios and amount of DNA. The role of the number of formamidine groups is also highlighted by in vitro cyto‐ and hemotoxicity tests under the chosen conditions. These results indicate that dextran‐formamidine esters are a very promising material for the safe and efficient gene delivery.     

Cooling,Spectroscopy and Non‐Sticking of trans‐Stilbene and Nile Red

We create and study trans‐Stilbene and Nile Red in a cryogenic (7 K) cell with a low density helium buffer gas. No molecule–helium cluster formation is observed, indicating limited atom–molecule sticking in this system. We place an upper limit of 5 % on the population of clustered He–trans‐Stilbene, consistent with a measured He–molecule collisional residence time of less than 1 μs. With its very low energy torsional modes, trans‐Stilbene is less rigid than any molecule previously buffer‐gas‐cooled into the Kelvin regime. We also report cooling and gas phase visible spectroscopy of Nile Red, a much larger molecule. Our data suggest that buffer gas cooling will be feasible for a variety of small biological molecules.     

Silver Ions in Non‐canonical DNA Base Pairs: Metal‐Mediated Mismatch Stabilization of 2′‐Deoxyadenosine and 7‐Deazapurine Derivatives with 2′‐Deoxycytidine and 2′‐Deoxyguanosine

Novel silver‐mediated dA?dC, dA*?dC, and dA*?dG base pairs were formed in a natural DNA double helix environment (dA* denotes 7‐deaza‐dA, 7‐deaza‐7‐iodo‐dA, and 7‐cyclopropyl‐7‐deaza‐dA). 7‐Deazapurine nucleosides enforce silver ion binding and direct metal‐mediated base pair formation to their Watson–Crick face. New phosphoramidites were prepared from 7‐deaza‐dA, 7‐deaza‐7‐iodo‐dA, and 7‐cyclopropyl‐7‐deaza‐dA, which contain labile isobutyryl protecting groups. Solid‐phase synthesis furnished oligonucleotides that contain mismatches in near central positions. Increased thermal stabilities (higher T_m values) were observed for oligonucleotide duplexes with non‐canonical dA*?dC and dA?dC pairs in the presence of silver ions. The stability of the silver‐mediated base pairs was pH dependent. Silver ion binding was not observed for the dA?dG mismatch but took place when mismatches were formed between 7‐deazaadenine and guanine. The specific binding of silver ions was confirmed by stoichiometric UV titration experiments, which proved that one silver ion is captured by one mismatch. The stability increase of canonical DNA mismatches might have an impact on cellular DNA repair.     

Metalloporphyrin–polyelectrolyte assemblies in aqueous solution: Influence of the metal center and the polyelectrolyte architecture

This study investigates the influence of different metal centers in porphyrins and of different polyelectrolyte architectures on the formation of supramolecular metalloporphyrin–polyelectrolyte assemblies in aqueous solution via electrostatic self‐assembly. Metal‐analogues of the tetravalent anionic meso‐tetrakis(4‐sulfonatophenyl)‐porphyrin (with Zn²⁺, Co²⁺, Ni²⁺, Mn³⁺, and Fe³⁺) are combined with the cationic dendrimer of generation 4 or with the linear polydiallyldimethyl‐ammoniumchloride. Dynamic light scattering and atomic force microscopy reveal that the different molecular geometries of the metalloporphyrins resulting from axial ligands determine the size and the stability of the aggregates. A thermodynamic study elucidates the importance of the polymer architecture in controlling the size of the assembly and the role of the metal center. © 2017 Wiley Periodicals, Inc. J. Polym. Sci., Part B: Polym. Phys. 2018 , 56, 484–500     

Polarized light‐induced photoisomerization in glassy poly(methyl methacrylate) and local relaxation processes of the polymer matrix

Photoisomerization kinetics of trans 4‐methoxystilbene induced by irradiation with linearly polarized light was investigated in the glassy state of poly(methyl methacrylate)(PMMA). The reaction was strongly selected by linearly polarized light as revealed by a large dichroic absorption generated upon irradiation. The optical anisotropy increases with increasing irradiation time and passes through a maximum before approaching an equilibrium value, which strongly depends on the experimental temperatures. From the decay of the total absorbance of the trans‐isomer observed upon irradiation with 313 nm UV light, it was found that the rates of the photoisomerization trans → cis were not significantly changed within the temperature range of the experiments. On the other hand, the reorientational relaxation times of the stilbene, which were directly measured by the annealing‐after‐irradiation techniques, depend strongly on temperature. These relaxation data were compared to the previous results obtained with dopants of the size larger than the stilbene and were discussed in conjunction with the local relaxation processes of the PMMA matrix. © 2000 John Wiley & Sons, Inc. J Polym Sci B: Polym Phys 38: 682–690, 2000     

Computational Study of the Interactions between Benzene and Crystalline Ice Ih: Ground and Excited States

Ground‐state geometries of benzene on crystalline ice cluster model surfaces (I_h) are investigated. It is found that the binding energies of benzene‐bound ice complexes are sensitive to the dangling features of the binding sites. We used time‐dependent DFT to study the UV spectroscopy of benzene, ice clusters, and benzene–ice complexes, by employing the M06‐2X functional. It is observed that the size of the ice cluster and the dangling features have minor effects on the UV spectral characteristics. Benzene‐mediated electronic excitations of water towards longer wavelengths (above 170 nm) are noted in benzene‐bound ice clusters, where the cross‐section of photon absorption by water is negligible, in good agreement with recent experimental results (Thrower et al., J. Vac. Sci. Technol. A, 2008, 26 , 919–924). The intensities of peaks associated with water excitations in benzene–ice complexes are found to be higher than in isolated ice clusters. The π→π* electronic transition of benzene in benzene–ice complexes undergoes a small redshift compared with the isolated benzene molecule, and this holds for all benzene‐bound ice complexes.     

Fracture toughness of nylon‐6 blends with maleated rubbers

The fracture toughness of blends of nylon‐6 with maleated ethylene–propylene rubber and maleated styrene/hydrogenated butadiene/styrene triblock copolymer was investigated with a single‐edge‐notched three‐point‐bending instrumented Dynatup test. The blends for which the rubber particle size was less than 0.7 μm fractured in a ductile manner over the whole range of ligament lengths, whereas the blends with particles larger than 0.7 μm showed a ductile‐to‐brittle transition with the ligament length. In this regime, ductile fracture was observed for specimens with short ligaments, whereas brittle fracture was seen for those with long ligaments. The ductile fracture behavior was analyzed with the essential‐work‐of‐fracture model, whereas linear elastic fracture mechanics techniques were used to analyze the brittle fracture behavior. The fact that the ductile fracture energy was larger for the blends with the styrene/hydrogenated butadiene/styrene triblock copolymer than for those with ethylene–propylene rubber was due to the larger dissipative energy density of the blends based on the styrene/hydrogenated butadiene/styrene triblock copolymer. Both the critical strain energy release rate (G_IC) and the plane‐strain critical stress intensity factor (K_IC) increased as the rubber particle size decreased for both blend systems. The G_IC and K_IC parameters had similar values, regardless of the rubber type, when the rubber particle size was fixed. The transition ligament length was near the size criterion for plane‐strain conditions for both blend systems. © 2004 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys 42: 1739–1758, 2004     

Coarse‐grained molecular dynamics simulations of protein–ligand binding

Coarse‐grained molecular dynamics (CGMD) simulations with the MARTINI force field were performed to reproduce the protein–ligand binding processes. We chose two protein–ligand systems, the levansucrase–sugar (glucose or sucrose), and LinB–1,2‐dichloroethane systems, as target systems that differ in terms of the size and shape of the ligand‐binding pocket and the physicochemical properties of the pocket and the ligand. Spatial distributions of the Coarse‐grained (CG) ligand molecules revealed potential ligand‐binding sites on the protein surfaces other than the real ligand‐binding sites. The ligands bound most strongly to the real ligand‐binding sites. The binding and unbinding rate constants obtained from the CGMD simulation of the levansucrase–sucrose system were approximately 10 times greater than the experimental values; this is mainly due to faster diffusion of the CG ligand in the CG water model. We could obtain dissociation constants close to the experimental values for both systems. Analysis of the ligand fluxes demonstrated that the CG ligand molecules entered the ligand‐binding pockets through specific pathways. The ligands tended to move through grooves on the protein surface. Thus, the CGMD simulations produced reasonable results for the two different systems overall and are useful for studying the protein–ligand binding processes. © 2014 Wiley Periodicals, Inc.