Crystal structure determination of the β-cyclodextrin-p-aminobenzoic acid inclusion complex from powder X-ray diffraction data

The 1:1 inclusion complex of β-cyclodextrin and p-aminobenzoic acid was prepared and characterized by TG-DTA. The crystal structure of the complex was solved directly from powder X-ray diffraction data using the direct space approach and refined using Rietveld refinement techniques. The complex crystallizes in monoclinic P2₁ space group, with unit cell parameters a = 20.7890 ?, b = 10.2084 ?, c = 15.1091 ?, β = 110.825°, V = 2997 ?³. The amino group is located at the wide side of the β-cyclodextrin cavity, forming hydrogen bonds with β-cyclodextrin, and the carboxyl group is located at the narrow side. The crystallographic data obtained from powder diffraction data were compared with the single crystallographic data, and the result shows that solving crystal structure of cyclodextrins inclusion complexes of such complexity is accessible to powder diffractionists to some extent.     

Characterisation and properties of the inclusion complex of 24-epibrassinolide with β-cyclodextrin

This paper reports the first study of an inclusion complex of abrassinosteroid with -cyclodextrin. The formation of inclusion complexesbetween 24-epibrassinolide and -cyclodextrin was confirmed by theirphysicochemical properties and the compounds were analysed by differentialscanning calorimetry, powder X-ray diffraction, nuclear magnetic resonancespectrometry and scanning electron microscopy. Theoretical calculations usingthe MM+ HyperChem force field showed a preference for inclusion of thesidechain of the epibrassinolide molecule into the -cyclodextrin cavity toform a 1:1 inclusion complex, although complexes involving inclusion ofthe steroidal nucleus also possess a favourable interaction energy. Rice laminainclination assay, employing IAC-103 and IAC-104 cultivars, showed an improvedactivity for the epibrassinolide-cyclodextrin complex compared to theepibrassinolide itself. The results suggest that brassinosteroid complexationwith cyclodextrins may enhance the biological activity of these plant growthregulators.     

Study on the interaction between the inclusion complex of hematoxylin with β-cyclodextrin and DNA

Ultraviolet-visible (UV-vis) spectra, fluorescence spectra, electrochemistry, and the thermodynamic method were used to discuss the interaction mode between the inclusion complex of hematoxylin with β-cyclodextrin and herring sperm DNA. On the condition of physiological pH, the result showed that hematoxylin and β-cyclodextrin formed an inclusion complex with binding ratio n(hematoxylin):n(β-cyclodextrin) = 1:1. The interaction mode between β-cyclodextrin-hematoxylin and DNA was a mixed binding, which contained intercalation and electrostatic mode. The binding ratio between β-cyclodextrin-hematoxylin and DNA was n(β-cyclodextrin -hematoxylin):n(DNA) = 2:1, binding constant was K(?)(298.15K) = 5.29 × 10? L·mol?1, and entropy worked as driven force in this action.     

Molecular simulation of β-cyclodextrin inclusion complex with 2-phenylethyl alcohol

The structures of β-cyclodextrin inclusion complexes with 2-phenylethyl alcohol in vacuum and aqueous solution have been investigated by using molecular dynamics simulation. The inclusion structures and the physicochemical stability of the complexes were also analysed, discussed and validated by ultraviolet spectrums and thermodynamic properties. The results of molecular dynamics simulation indicate that the A-type β-cyclodextrin inclusion complex with 2-phenylethyl alcohol in both vacuum and aqueous solution have better physical stability, and its chemical stability also has obvious promotion than that of free one. Therefore, the β-cyclodextrin can be used to control and regulate the release of the 2-phenylethyl in food.     

Characterization of the mRNAs for α-, β- and γ-actin

The mRNAs for α-, β- and γ-actin have been characterized with respect to molecular weight and poly(A) content. Polyacrylamide gel electrophoresis under denaturing conditions shows that the mRNA for α-actin (muscle-specific actin) is approximately 4.6 × 10⁵ daltons in size, and that the mRNAs for β- and γ-actin (nonmuscle actins) are much larger, approximately 6.6 × 10⁵ daltons in size. We therefore calculate that the noncoding regions of the β- and γ-actin mRNAs contain about 800 nucleotides. This is in marked contrast to the noncoding regions of α-actin mRNA which contain only about 180 nucleotides. During electrophoresis in high-resolution nondenaturing gels, the β-actin mRNA migrates slightly slower than the γ-actin mRNA. This indicates either that β-actin mRNA is about 100 nucleotides longer than γ-actin mRNA, or that these mRNAs differ in secondary structure. Fractionation of actin mRNA on the basis of poly(A) content shows that a substantial portion of the β-actin mRNA, but very little of the α- or γ-actin mRNAs, fails to bind to oligo(dT)-cellulose. Much of this poly(A)-deficient β-actin mRNA, however, does bind to poly(U)-Sepharose, a substrate with higher affinity for short poly(A) sequences. This indicates that many of these β-actin mRNA molecules are polyadenylated, but that they have unusually short poly(A) tails. The finding that β- and γ-actins are translated from mRNAs of different electrophoretic mobility and different poly(A) content strongly suggests that these two closely related proteins are products of different genes.     

Crystal structure of β-barrel assembly machinery BamCD protein complex

The β-barrel assembly machinery (BAM) complex of Escherichia coli is a multiprotein machine that catalyzes the essential process of assembling outer membrane proteins. The BAM complex consists of five proteins: one membrane protein, BamA, and four lipoproteins, BamB, BamC, BamD, and BamE. Here, we report the first crystal structure of a Bam lipoprotein complex: the essential lipoprotein BamD in complex with the N-terminal half of BamC (BamC(UN) (Asp(28)-Ala(217)), a 73-residue-long unstructured region followed by the N-terminal domain). The BamCD complex is stabilized predominantly by various hydrogen bonds and salt bridges formed between BamD and the N-terminal unstructured region of BamC. Sequence and molecular surface analyses revealed that many of the conserved residues in both proteins are found at the BamC-BamD interface. A series of truncation mutagenesis and analytical gel filtration chromatography experiments confirmed that the unstructured region of BamC is essential for stabilizing the BamCD complex structure. The unstructured N terminus of BamC interacts with the proposed substrate-binding pocket of BamD, suggesting that this region of BamC may play a regulatory role in outer membrane protein biogenesis.     

Crystal structure of Escherichia coli BamB, a lipoprotein component of the β-barrel assembly machinery complex

In Gram-negative bacteria, the BAM (β-barrel assembly machinery) complex catalyzes the essential process of assembling outer membrane proteins. The BAM complex in Escherichia coli consists of five proteins: one β-barrel membrane protein, BamA, and four lipoproteins, BamB, BamC, BamD, and BamE. Despite their role in outer membrane protein biogenesis, there is currently a lack of functional and structural information on the lipoprotein components of the BAM complex. Here, we report the first crystal structure of BamB, the largest and most functionally characterized lipoprotein component of the BAM complex. The crystal structure shows that BamB has an eight-bladed β-propeller structure, with four β-strands making up each blade. Mapping onto the structure the residues previously shown to be important for BamA interaction reveals that these residues, despite being far apart in the amino acid sequence, are localized to form a continuous solvent-exposed surface on one side of the β-propeller. Found on the same side of the β-propeller is a cluster of residues conserved among BamB homologs. Interestingly, our structural comparison study suggests that other proteins with a BamB-like fold often participate in protein or ligand binding, and that the binding interface on these proteins is located on the surface that is topologically equivalent to where the conserved residues and the residues that are important for BamA interaction are found on BamB. Our structural and bioinformatic analyses, together with previous biochemical data, provide clues to where the BamA and possibly a substrate interaction interface may be located on BamB.     

The enhancement of the oral bioavailability of γ-tocotrienol in mice by γ-cyclodextrin inclusion

Cyclodextrin (CD) is widely used in the pharmaceutical and nutritional fields to form an inclusion complex with lipophilic compounds for the improvement of their aqueous solubility, stability and diffusibility under physiological conditions. In this study, we investigated the effect of the γ-tocotrienol (γT3) inclusion complex with CD on its oral bioavailability. Five-week-old C57BL6 mice were fed a vitamin E-free diet for 28 days, followed by the oral administration of 2.79 mg of γT3-rich fraction (TRF) extracted from rice bran or the equivalent dose (14.5 mg) of a CD inclusion complex with TRF (TRF/CD). The levels of γT3 in sequentially collected plasma were determined by LC-MS/MS. The pharmacokinetic study revealed that the plasma concentrations of γT3 were increased and peaked at 6 or 3 h after the oral administration of TRF or TRF/CD, respectively (C_max values of 7.9±3.3 or 11.4±4.5 μM, respectively). The area under the curve of plasma γT3 concentration also showed a 1.4-fold increase in the group administered with TRF/CD compared with the TRF-only group. Furthermore, the mice that had received the TRF/CD tended to reduce the endotoxin shock induced by injection with lethal amounts of Escherichia coli lipopolysaccharide, compared with the mice that had received TRF alone. Taken together, our results suggest that the CD inclusion improved γT3 bioavailability, resulting in the enhancement of γT3 physiological activity, which would be a useful approach for the nutrition delivery system.     

Development of inclusion complex of brinzolamide with hydroxypropyl-β-cyclodextrin

Glaucoma is an accumulative optic neuropathy resulted from increasing intraocular pressure. Brinzolamide (BRZ) is a kind of carbonic anhydrase inhibitors for glaucoma treatment. In this study, brinzolamide-hydroxypropyl-β-cyclodextrin (BRZ-HP-β-CD) inclusion complex was prepared by solvent evaporation method to improve the solubility of BRZ and enhance the therapeutic effect of BRZ. The formation of the inclusion complex was confirmed by Fourier transform infrared spectroscopy, differential scanning calorimeter and nuclear magnetic resonance spectroscopy. The solubility of BRZ increased about 10-fold after the formation of the BRZ-HP-β-CD inclusion complex. The in vitro corneal accumulative permeability of the inclusion complex increased 2.91-fold compared to the commercial available formulation (AZOPT^®). In addition, BRZ-HP-β-CD inclusion complex (0.5% BRZ) had an equivalent efficiency of lowering intraocular pressure with AZOPT^® (1% BRZ) in vivo. These results identified the BRZ-HP-β-CD inclusion complex might have a promising future as a novel formulation of BRZ for glaucoma treatment.     

Investigation of inclusion complex of cilnidipine with hydroxypropyl-β-cyclodextrin

The objective of this study was to improve the water-solubility and photostability of cilnidipine by complexing it with hydroxypropyl-β-cyclodextrin (HP-β-CD or HP-beta-CD). The interactions of cilnidipine and HP-β-CD were characterized by ultra violet-visible (UV/VIS) spectroscopy, differential scanning calorimetry (DSC), powder X-ray diffraction (PXRD), Fourier transformation-infrared (FT-IR) spectroscopy and (1)H nuclear magnetic resonance ((1)H NMR) spectroscopy to verify the formation of cilnidipine-HP-β-CD complex inclusion. Moreover, the binding sites in the HP-β-CD structure were also tracked through (1)H NMR spectroscopy analysis. All the characterization information proved the formation of cilnidipine-HP-β-CD inclusion complex, and the results demonstrated the superiority of the inclusion complex in dissolution rates and photostability; in addition, the apparent solubility of cilnidipine was increased more than 10,000-fold in the presence of HP-β-CD. The stability constant (1:1) was found to be 50,116M(-1), suggesting a high tendency of the drug to enter the HP-β-CD cavity. These results identified the cilnidipine-HP-β-CD inclusion complex as an effective new approach to design a novel formulation for pharmaceutical application.     

Spectroscopic characterization of the inclusion complexes of luteolin with native and derivatized β-cyclodextrin

The inclusion complexes of Luteolin (LU) with cyclodextrins (CDs) including β-cyclodextrin (βCD), hydroxypropyl-β-cyclodextrin (HPβCD) and dimethyl-β-cyclodextrin (DMβCD), Scheme 1, have been investigated using the method of steady-state fluorescence. The stoichiometric ratio of the three complexes was found to be 1:1 and the stability constants (K) were estimated from spectrofluorometric titrations, as well as the thermodynamic parameters. Maximum inclusion ability was obtained in the case of HPβCD followed by DMβCD and βCD. Moreover, ¹H NMR and 2D NMR were carried out, revealing that LU has different form of inclusion which is in agreement with molecular modeling studies. These models confirm that when LU–βCD and LU–DMβCD complexes are formed, the B-ring is oriented toward the primary rim; however, for LU–HPβCD complex this ring is oriented toward the secondary rim. The ESR results showed that the antioxidant activity of luteolin was the order LU–HPβCD > LU–DMβCD > LU–βCD > LU, hence the LU-complexes behave are better antioxidants than luteolin free.     

Selective degradation of β- and γ-cyclodextrin by co-digestion using a CGTase fromBacillus ohbensis and α-glucosidase for efficient α-cyclodextrin recovery

A simple and specific recovery method for α-cyclodextrin (α-CD) was developed by employing co-digestion of CD reaction mixtures with CGTase fromBacillus ohbensis and α-glucosidase. The combination of CGTase fromB. ohbensis and α-glucosidase, such as α-amylase, β-amylase, or glucoamylase was examined for the selective degradation of β-and γ-CD in the CD reaction mixture formed by CGTase fromB. macerans. The co-digestion of the CD mixture with Taka-amylase and the CGTase resulted in α-CD and maltodextrins, the combination with β-amylase resulted in α-CD and maltose, and that with glucoamylase resulted in α-CD and glucose. The conditions of selective degradation of β- and γ-CD by co-digestion with the CGTase and glucoamylase were optimized as follows: the incubation pH, 5.5; incubation temperature, 50°C; CGTase concentration, 15 u/g of substrate; glucoamylase, 10 u/g of substrate; substrate concentration, 10% (w/v); the incubation time was fixed for 18 hr from the stand point of operation convenience. Most part of the content was presented in poster session at the 7th International Cyclodextrin Symposium, Tokyo, April 1994.     

Molecular weights of wheat γ2-, β6-, α7-, α8- and α9-gliadins

The molecular weights of wheat γ₂-, β6-, α7-, α8- and α9-gliadins were calculated with the aid of a computer technique from sedimentation equilibrium data obtained in an ultracentrifuge equipped with photoelectric scanner. The dissociative solvents, all at pH 3.1 by addition of HCl, included 3 M urea, 0.15 M KCl; 8 M urea, 0.15 M KCl and 6 M guanidine-HCl. The minimum molecular weights for γ₂-, α7- and α9-gliadins, obtained in 6 M guanidine-HCl, were 34 600, 30 400 and 30 900, respectively. The β6- and α8-gliadins gave minimum molecular weights of 33 000 and 36 900, respectively, in 3 M urea, 0.15 M KCl.     

Crystal structures of rare disaccharides, α-D-glucopyranosyl β-D-psicofuranoside, and α-D-galactopyranosyl β-D-psicofuranoside

The crystal structures of α-D-glucopyranosyl β-D-psicofuranoside and α-D-galactopyranosyl β-D-psicofuranoside were determined by a single-crystal X-ray diffraction analysis, refined to R(1)=0.0307 and 0.0438, respectively. Both disaccharides have a similar molecular structure, in which psicofuranose rings adopt an intermediate form between (4)E and (4)T(3). Unique molecular packing of the disaccharides was found in crystals, with the molecules forming a layered structure stacked along the y-axis.     

Molecular dynamics study of an α-cyclodextrin-phosphatidylinositol inclusion complex

Cyclodextrins are cyclic oligosaccharides known for their ability to include substrate molecules in their hydrophobic cavity. Moreover, cyclodextrins show a hemolytic activity when mm concentrations are added to blood. This hemolysis is commonly interpreted as a massive dissociation of phospholipids from the cell membrane due to the formation of complexes with the cyclodextrins. In the literature, a complexation between -cyclodextrin ( CD) and phosphatidylinositol (PI) specific to the inositol headgroup has been proposed. But the need for the detailed interaction mechanism between the two molecules motivated the present work based on molecular dynamics simulations. Investigation of long range electrostatic interactions shows that a mutual approach of the molecules is only possible when the primary hydroxyl side of CD faces the inositol headgroup of PI. This orientation is also the most favourable from adiabatic- and free-energy profiles calculated along a reaction coordinate that leads to an inclusion of PI into a CD. For free energy simulations, partial hydration of the model has been used. A study of glycosidic bond dihedral angles in CD shows an increase in dihedral fluctuations before complexation and a dihedral freezing once the complex is formed.     

“Induced-fit”-type complex formation of the model enzyme α-cyclodextrin

On the basis of X-ray and neutron data for several α-cyclodextrin·substrate complexes it is shown that basically two different structures for α-cyclodextrin exist, one “tense”, the other “relaxed”. An “induced-fit”-like mechanism for α-cyclodextrin complex formation is proposed.     

Computational investigation on the host–guest inclusion process of norfloxacin into β-cyclodextrin

A theoretical ¹H NMR spectroscopy and thermodynamic analysis of the host–guest inclusion process involving the norfloxacin (NFX) into β-cyclodextrin (β-CD) was carried out. DFT structure and stabilization energies were obtained in both gas and aqueous phases. We could establish that the complex formation is enthalpy driven, and the hydrogen bonds established between NFX and β-CD play a major role in the complex stabilization. Besides, a theoretical ¹H NMR analysis has shown to be a supplementary proceeding to predict appropriately the inclusion mode of norfloxacin molecule into the β-CD. In this work, a theoretical study of the NFX@β-CD complex is reported for the first time, seeking a deep understanding of topology and thermodynamics of the inclusion complex formation.

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Graphical Abstract Topology, thermodynamic and ¹H NMR analysis of NFX@β-CD host-guest complexes

     

Anion inhibition profiles of α-, β- and γ-carbonic anhydrases from the pathogenic bacterium Vibrio cholerae

Among the numerous metalloenzymes known to date, carbonic anhydrase (CA, EC 4.2.1.1) was the first zinc containing one, being discovered decades ago. CA is a hydro-lyase, which catalyzes the following hydration–dehydration reaction: CO₂ + H₂O ⇋ HCO₃⁻ + H⁺. Several CA classes are presently known, including the α-, β-, γ-, δ-, ζ- and η-CAs. In prokaryotes, the existence of genes encoding CAs from at least three classes (α-, β- and γ-class) suggests that these enzymes play a key role in the physiology of these organisms. In many bacteria CAs are essential for the life cycle of microbes and their inhibition leads to growth impairment or growth defects of the pathogen. CAs thus started to be investigated in detail in bacteria, fungi and protozoa with the aim to identify antiinfectives with a novel mechanism of action. Here, we investigated the catalytic activity, biochemical properties and anion inhibition profiles of the three CAs from the bacterial pathogen Vibrio cholera, VchCA, VchCAβ and VchCAγ. The three enzymes are efficient catalysts for CO₂ hydration, with k_cat values ranging between (3.4 − 8.23) × 10⁵ s⁻¹ and k_cat/K_M of (4.1 − 7.0) × 10⁷ M⁻¹ s⁻¹. A set of inorganic anions and small molecules was investigated for inhibition of these enzymes. The most potent VchCAγ inhibitors were N,N-diethyldithiocarbamate, sulfamate, sulfamide, phenylboronic acid and phenylarsonic acid, with K_I values ranging between 44 and 91 μM.