Spectroscopic analysis on structure–affinity relationship in the interactions of different oleanane‐type triterpenoids with bovine serum albumin

Oleanane‐type triterpenoids serve as an important group of plant secondary metabolites with a variety of biological activities and the C‐3 position substitution pattern is a significant structural feature for their biological activities. Three selected oleanane‐type triterpenoids (glycyrrhizin, glycyrrhetinic acid, and carbenoxolone) bearing different substituents (glucuronic acid dimer, hydroxyl, and succinyl groups) at the C‐3 position were studied for their affinities to bind bovine serum albumin (BSA) by steady‐state fluorescence, synchronous, three‐dimensional fluorescence and ultraviolet–visible (UV–vis) absorption spectra. The binding mechanism of the triterpenoids to BSA is due to the formation of the triterpenoids–BSA complex and the binding affinity is strongest for carbenoxolone and ranked in the order carbenoxolone > glycyrrhetinic acid > glycyrrhizin. The thermodynamic parameters calculated at different temperatures showed that triterpenoids binding to BSA primarily depended on hydrophobic interaction and hydrogen bonding. The distance between the bound triterpenoid and BSA was determined on the basis of the Förster's energy transfer theory. Displacement experiments using phenylbutazone and ibuprofen showed the binding site of triterpenoids on BSA at subdomain IIA (Sudlow's site I). The effect of triterpenoids on BSA conformation was analyzed by UV–vis absorption, and synchronous and three‐dimensional fluorescence spectra. These results revealed that the C‐3 position substitution pattern significantly affects the structure–affinity relationships of oleanane‐type triterpenoid binding to BSA and further affects the bioavailability of triterpenoids in the blood circulatory system. Copyright © 2014 John Wiley & Sons, Ltd.     

The Effect of an Amino‐Acid Bridge on Binding Affinity and Cleavage Efficiency of Pyrenyl‐Macrocyclic Polyamine Conjugates toward DNA

A series of pyrenyl‐macrocyclic polyamines 5a – 5c have been prepared and characterized. Their DNA‐cleavage properties were examined under physiological conditions. Without the presence of other additives, the DNA cleavage ability of 5a – 5c showed the order of 5c > 5a > 5b . Absorption and fluorescence experiments showed the binding affinity of 5a – 5c to DNA. The interactions of 5a – 5c with CT‐DNA indicated that the DNA binding ability followed an order according to their cleavage efficiency. All the results indicated that the structures of amino‐acid bridge in the ligands may affect the DNA binding and cleavage ability. The cleavage‐mechanism studies indicated that singlet oxygen and superoxide free radicals were involved in the catalytic DNA cleavage process.     

Synthesis and carbohydrate binding studies of fluorescent α-amidoboronic acids and the corresponding bisboronic acids

Fluorescent boronic acids are very useful for the design and synthesis of carbohydrate sensors. In an earlier communication, we first described the effort of developing water soluble fluorescent α-amidoboronic acids, which change fluorescence upon sugar binding. In this report, we describe a general method of functionalizing such boronic acids and their applications in the preparation of bis-α-amidoboronic acids with significantly enhanced binding for oligosaccharides as compared to their monoboronic acid counterparts. The advantages of good water solubility, easy modification to generate diversity, and modularity in synthesis will make α-amidoboronic acids very useful building blocks for future synthesis of boronic acid-based fluorescent sensors.     

Spectroscopic and molecular docking study on the structure–affinity relationship and mechanism in the interaction of genistein and its derivatives with bovine serum albumin

In this paper, the interaction of genistein (GEN) and its four derivatives (GEN1–4) with bovine serum albumin (BSA) were investigated by ultraviolet–visible absorption spectra, fluorescence, synchronous fluorescence, three‐dimensional fluorescence spectroscopy, circular dichroism and molecular docking techniques. The experimental results showed that the intrinsic fluorescence of BSA was quenched by genisteins and was due to the formation of a genisteins–BSA complex. The quenching constant, binding constants, binding sites, intermolecular distances and thermodynamic properties were calculated at 298 K, 306 K and 310 K. Site marker competitive experiments indicated that the binding site of genisteins to BSA was mainly located in subdomain IIA. The conformational investigation showed that the presence of 0020 genisteins led to changes in the secondary structure of BSA and induced the slight unfolding of protein polypeptides, which confirmed some micro‐environmental and conformational changes of BSA molecules. Furthermore, the binding affinity decreased in the order GEN1 > GEN > GEN4 > GEN3 > GEN2, which revealed that different type and position of substituents of genistein significantly influenced the affinity of compounds to BSA. The number of hydroxyl groups on the ring A was the most important factor because increasing the hydroxyl groups on ring A clearly enhanced the binding affinity. However, trifluoromethylation did not much affect the affinity, alkylation, esterification and difluoromethylation slightly enhanced the binding affinity. The results obtained herein will provide valuable information about the pharmacokinetics at a molecular level and be a useful guideline for the further design of much more suitable genistein derivatives.     

Quantitative analyses of individual sugars in mixture using FRET‐based biosensors

Molecular biosensors were developed and applied to measure individual sugars in biological mixtures such as bacterial culture broths. As the sensing units, four sugar‐binding proteins (SBPs for allose, arabinose, ribose, and glucose) were selected from the Escherichia coli genome and connected to a cyan fluorescent protein and yellow fluorescent protein via dipeptide linkers (CFP‐L‐SBP‐YFP). The putative sensors were randomized in the linker region (L) and then investigated with regard to the intensity of fluorescence resonance energy transfer on the binding of the respective sugars. As a result, four representatives were selected from each library and examined for their specificity using 16 available sugars. The apparent dissociation constants of the allose, arabinose, ribose, and glucose sensors were estimated to be 0.35, 0.36, 0.17, and 0.18 μM. Finally, the sugar sensors were applied to monitor the consumption rate of individual sugars in an E. coli culture broth. The individual sugar profiles exhibited a good correlation with those obtained using an HPLC method, confirming that the biosensors offer a rapid and easy‐to‐use method for monitoring individual sugars in mixed compositions. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012     

Probing the general time scale question of boronic acid binding with sugars in aqueous solution at physiological pH

The boronic acid group is widely used in chemosensor design due to its ability to reversibly bind diol-containing compounds. The thermodynamic properties of the boronic acid-diol binding process have been investigated extensively. However, there are few studies of the kinetic properties of such binding processes. In this report, stopped-flow method was used for the first time to study the kinetic properties of the binding between three model arylboronic acids, 4-, 5-, and 8-isoquinolinylboronic acids, and various sugars. With all the boronic acid-diol pairs examined, reactions were complete within seconds. The k(on) values with various sugars follow the order of D-fructose>D-tagatose>D-mannose>D-glucose. This trend tracks the thermodynamic binding affinities for these sugars and demonstrates that the 'on' rate is the key factor determining the binding constant.     

Concanavalin A‐immobilized magnetic nanoparticles for selective enrichment of glycoproteins and application to glycoproteomics in hepatocelluar carcinoma cell line

Protein glycosylation is one of the most important PTMs in biological organism. Lectins such as concanavalin A (Con A) have been widely applied to N‐glycosylated protein investigation. In this study, we developed Con A‐immobilized magnetic nanoparticles for selective separation of glycoproteins. At first, a facile immobilization of Con A on aminophenylboronic acid‐functionalized magnetic nanoparticles was performed by forming boronic acid‐sugar‐Con A bond in sandwich structure using methyl α‐D ‐mannopyranoside as an intermedium. The selective capture ability of Con A‐modified magnetic nanoparticles for glycoproteins was tested using standard glycoproteins and cell lysate of human hepatocelluar carcinoma cell line 7703. In total 184 glycosylated sites were detected within 172 different glycopeptides corresponding to 101 glycoproteins. Also, the regeneration of the protein‐immobilized nanoparticles can easily be performed taking advantage of the reversible binding mechanism between boronic acid and sugar chain. The experiment results demonstrated that Con A‐modified magnetic nanoparticles by the facile and low‐cost synthesis provided a convenient and efficient enrichment approach for glycoproteins, and are promising candidates for large‐scale glycoproteomic research in complicated biological samples.     

A benzoboroxole‐based affinity ligand for glycoprotein purification at physiological pH

Developing ligands capable of carbohydrate recognition has become increasingly important as the essential roles of glycoproteins and glycolipids in a diverse array of cellular signaling, pathophysiology, and immune response mechanisms are elucidated. Effective ligands for the glycan portion of glycoproteins and glycolipids are needed for pre‐enrichment proteomics strategies, as well as for the purification of individual glycoproteins from complex biological milieu encountered both in biochemistry research and bio‐pharmaceutical development. In this work, we developed a carbohydrate specific affinity ligand for glycoprotein purification using a one‐pot, multi‐component synthesis reaction (Ugi synthesis) and an amine‐functionalized benzoboroxole moiety immobilized on agarose beads. Benzoboroxoles are unique boronic acid derivatives that have recently been found to bind specifically to the cis‐diol groups of carbohydrates at physiological pH, with superior affinity to any other Wulff‐type boronic acid. The solid‐phase affinity ligand developed herein specifically binds the carbohydrate moiety of the glycoprotein glucose oxidase, as well as a fluorescein isothiocyanate‐dextran, as shown through deglycosylation binding studies. Additionally, the ligand is able to purify glucose oxidase from crude Escherichia coli lysate, at physiological pH, equitably to commercially available boronic acid‐functionalized agarose beads that required alkaline pH conditions. Thus, this affinity ligand is a marked improvement on current, commercially available boronic acid‐based glycoprotein enrichment matrices and has the potential to exhibit high individual glycoprotein specificity because of the additional functional groups available for variation on the Ugi scaffold. Copyright © 2015 John Wiley & Sons, Ltd.     

UDP‐sugar pyrophosphorylase is essential for arabinose and xylose recycling,and is required during vegetative and reproductive growth in Arabidopsis

Numerous nucleotide sugars are needed in plants to synthesize cell wall polymers and glycoproteins. The de novo synthesis of nucleotide sugars is of major importance. During growth, however, some polymers are broken down to monosaccharides. Reactivation of these sugars into nucleotide sugars occurs in two steps: first, by a substrate‐specific sugar‐1‐kinase and, second, by UDP‐sugar‐pyrophosphorylase (USP), which has broad substrate specificity. A knock‐out of the USP gene results in non‐fertile pollen. By using various genetic complementation approaches we obtained a strong (>95%) knock‐down line in USP that allowed us to investigate the physiological role of the enzyme during the life cycle. Mutant plants show an arabinose reduction in the cell wall, and accumulate mainly two sugars, arabinose and xylose, in the cytoplasm. The arabinogalactanproteins in usp mutants show no significant reduction in size. USP is also part of the myo‐inositol oxygenation pathway to UDP‐glucuronic acid; however, free glucuronic acid does not accumulate in cells, suggesting alternative conversion pathways of this monosaccharide. The knock‐down plants are mostly sterile because of the improper formation of anthers and pollen sacks.     

Enantiopurity and absolute configuration determination of arene cis‐dihydrodiol metabolites and derivatives using chiral boronic acids

The relative merits of the methods employed to determine enantiomeric excess (ee) values and absolute configurations of chiral arene and alkene cis‐1,2‐diol metabolites, including boronate formation, using racemic or enantiopure (+) and (?)‐2‐(1‐methoxyethyl)phenylboronic acid (MEPBA), are discussed. Further applications of: 1) MEPBA derived boronates of chiral mono‐ and poly‐cyclic arene cis‐dihydrodiol, cyclohex‐2‐en‐1‐one cis‐diol, heteroarene cis/trans‐2,3‐diol, and catechol metabolites in estimating their ee values, and 2) new chiral phenylboronic acids, 2‐[1‐methoxy‐2,2‐dimethylpropyl]phenyl boronic acid (MDPBA) and 2‐[1‐methoxy‐1‐phenylmethyl]phenyl boronic acid (MPPBA) and their advantages over MEPBA, as reagents for stereochemical analysis of arene and alkene cis‐diol metabolites, are presented.     

One‐step synthesis of nitrogen,boron co‐doped fluorescent carbon nanoparticles for glucose detection

Heteroatom‐doped carbon nanoparticles (CNPs) have attracted considerable attention due to an effective improvement in their intrinsic properties. Here, a facile and simple synthesis of nitrogen, boron co‐doped carbon nanoparticles (NB‐CNPs) from a sole precursor, 3‐aminophenylboronic acid, was performed via a one‐step solid‐phase approach. Because of the presence of boronic acid, NB‐CNPs can be used directly as a fluorescent probe for glucose. Based on a boronic acid‐triggered specific reaction, we developed a simple NB‐CNP probe without surface modification for the detection of glucose. When glucose was introduced, the fluorescence of NB‐CNPs was suppressed through a surface‐quenching states mechanism. Obvious fluorescence quenching allowed the highly sensitive determination of glucose with a limit of detection of 1.8 μM. Moreover, the proposed method has been successfully used to detect glucose in urine from people with diabetes, suggesting potential application in sensing glucose.     

Design,synthesis and characterisation of affinity ligands for glycoproteins

The concepts of rational design and solid phase combinatorial chemistry were used to develop affinity adsorbents for glycoproteins. A detailed assessment of protein–carbohydrate interactions was used to identify key residues that determine monosaccharide specificity, which were subsequently exploited as the basis for the synthesis of a library of glycoprotein binding ligands. The ligands were synthesised using solid phase combinatorial chemistry and were assessed for their sugar‐binding ability with the glycoenzymes, glucose oxidase and RNase B. Partial and completely deglycosylated enzymes were used as controls. The triazine‐based ligand, histamine/tryptamine (8/10) was identified as a putative glycoprotein binding ligand, since it displayed particular affinity for glucose oxidase and other mannosylated glycoproteins. Experiments with deglycosylated control proteins, specific eluants and retardation in the presence of competing sugars strongly suggest that the ligand binds the carbohydrate moiety of glucose oxidase rather than the protein itself. Copyright © 1999 John Wiley & Sons, Ltd.     

Expression pattern and ligand‐binding properties of odorant‐binding protein 13 from Monochamus alternatus hope

Odorant‐binding proteins (OBPs) are believed to play an important role in olfactory recognition. In this study, expression pattern and fluorescence binding characteristics of MaltOBP13 from the Japanese pine sawyer beetle, Monochamus alternatus Hope, were investigated via qPCR analysis of MaltOBP13 mRNA level and binding assay of MaltOBP13 and ligands. qPCR monitoring indicated MaltOBP13 mainly expressed in newly emerged males, particularly highly expressed in the last abdominal segment of males, and the expression level was significantly higher in 13‐day‐old mated adults than those of other stages. To further understand the function of the MaltOBP13 protein in odorant reception, the binding affinity of recombinant MaltOBP13 to ligands was tested by fluorescence binding assays with N‐phenyl‐1‐naphthylamine as a fluorescent probe. The results of this assay indicated that MaltOBP13 exhibited a high binding affinity for pine volatiles and binding capacity was higher in acidic conditions than in neutral environment, indicating a possible role in finding host plants.     

Binding of N-dansylgalactosamine to winged-bean tuber lectin: studies by fluorescence quenching titrations

The winged-bean tuber lectin binds to N-dansyl(5-dimethylaminonaphthalene-1-sulphonic acid)galactosamine, leading to a 12.5-fold increase in dansyl fluorescence with a concomitant 25 nm blue-shift in the emission maximum. The enhancement of fluorescence intensity was completely reversed by the addition of methyl alpha-galactopyranoside. The lectin has two binding sites per molecule for this fluorescent sugar and an association constant of 2.59.10(5) M-1 at 25 degrees C. The binding of N-dansylgalactosamine to the lectin shows that it can accommodate a large hydrophobic substituent on the C-2 carbon of D-galactose. Studies with other sugars indicate that a hydrophobic substituent with alpha-conformation at the anomeric position increases the affinity of binding. The C-4 and C-6 hydroxyl groups are also critical for sugar binding to this lectin.     

Conditions with high intracellular glucose inhibit sensing through glucose sensor Snf3 in Saccharomyces cerevisiae

Gene expression in micro‐organisms is regulated according to extracellular conditions and nutrient concentrations. In Saccharomyces cerevisiae, non‐transporting sensors with high sequence similarity to transporters, that is, transporter‐like sensors, have been identified for sugars as well as for amino acids. An alternating‐access model of the function of transporter‐like sensors has been previously suggested based on amino acid sensing, where intracellular ligand inhibits binding of extracellular ligand. Here we studied the effect of intracellular glucose on sensing of extracellular glucose through the transporter‐like sensor Snf3 in yeast. Sensing through Snf3 was determined by measuring degradation of Mth1 protein. High intracellular glucose concentrations were achieved by using yeast strains lacking monohexose transporters which were grown on maltose. The apparent affinity of extracellular glucose to Snf3 was measured for cells grown in non‐fermentative medium or on maltose. The apparent affinity for glucose was lowest when the intracellular glucose concentration was high. The results conform to an alternating‐access model for transporter‐like sensors. J. Cell. Biochem. 110: 920–925, 2010. © 2010 Wiley‐Liss, Inc.     

Design and Application of Highly Responsive Fluorescence Resonance Energy Transfer Biosensors for Detection of Sugar in Living Saccharomyces cerevisiae Cells

A protein sensor with a highly responsive fluorescence resonance energy transfer (FRET) signal for sensing sugars in living Saccharomyces cerevisiae cells was developed by combinatorial engineering of the domain linker and the binding protein moiety. Although FRET sensors based on microbial binding proteins have previously been created for visualizing various sugars in vivo, such sensors are limited due to a weak signal intensity and a narrow dynamic range. In the present study, the length and composition of the linker moiety of a FRET-based sensor consisting of CFP-linker₁-maltose-binding protein-linker₂-YFP were redesigned, which resulted in a 10-fold-higher signal intensity. Molecular modeling of the composite linker moieties, including the connecting peptide and terminal regions of the flanking proteins, suggested that an ordered helical structure was preferable for tighter coupling of the conformational change of the binding proteins to the FRET response. When the binding site residue Trp62 of the maltose-binding protein was diversified by saturation mutagenesis, the Leu mutant exhibited an increased binding constant (82 μM) accompanied by further improvement in the signal intensity. Finally, the maltose sensor with optimized linkers was redesigned to create a sugar sensor with a new specificity and a wide dynamic range. When the optimized maltose sensors were employed as in vivo sensors, highly responsive FRET images were generated from real-time analysis of maltose uptake of Saccharomyces cerevisiae (baker's yeast).     

Oligohis‐tags: mechanisms of binding to Ni2+‐NTA surfaces

Since immobilized metal ion affinity chromatography (IMAC) was first reported, several modifications have been developed. Among them, Ni²⁺ immobilized by chelation with nitrilotriacetic acid (NTA) bound to a solid support has become the most common method for the purification of proteins carrying either a C‐ or N‐terminal histidine (His) tag. Despite its broad application in protein purification, only little is known about the binding properties of the His‐tag, and therefore almost no thermodynamic and kinetic data are available. In this study, we investigated the binding mechanism of His‐tags to Ni²⁺‐NTA. Different series of oligohistidines and mixed oligohistidines/oligoalanines were synthesized using automated solid‐phase peptide synthesis (SPPS). Binding to Ni²⁺‐NTA was analyzed both qualitatively and quantitatively with surface plasmon resonance (SPR) using commercially available NTA sensor chips from Biacore. The hexahistidine tag shows an apparent equilibrium dissociation constant (K_D) of 14 ± 1 nM and thus the highest affinity of the peptides synthesized in this study. Furthermore, we could demonstrate that two His separated by either one or four residues are the preferred binding motifs within hexahis tag. Finally, elongation of these referred motifs decreased affinity, probably due to increased entropy costs upon binding. Copyright © 2009 John Wiley & Sons, Ltd.     

Fluorene‐based boronic acids as fluorescent chemosensor for monosaccharides at physiological pH

Two fluorene‐based boronic acids, 9,9‐dimethyl‐9H‐fluoren‐2‐yl‐2‐boronic acid (1) and 9,9‐dimethyl‐9H‐fluoren‐2,7‐diyl‐2,7‐diboronic acid (2), were synthesized and their sensing abilities for detection of D ‐monosaccharides were investigated by fluorescence at physiological pH. It was found that both boronic acids 1 and 2 have high selectivity and sensitivity for D ‐fructose with stability constant of 47.2 and 412.9, respectively. The sensor 2 showed a linear response toward D ‐fructose in the concentration range from 5 × 10^–5 to 10^–1 mol L^–1 with the detection limit of 2 × 10^–5 mol L^–1. Copyright © 2014 John Wiley & Sons, Ltd.     

Homofermentative production of optically pure <Emphasis Type="SmallCaps">l</Emphasis>-lactic acid from sucrose and mixed sugars by batch fermentation of <Emphasis Type="Italic">Enterococcus faecalis</Emphasis> RKY1

In the present study, lactic acid fermentation was carried out by batch culture of Enterococcus faecalis RKY1 using sucrose and mixed sugars as the major substrate. Maximum lactic acid productivity (5.2 g/L/h) was recorded when 50 and 100 g/L of sucrose were used as a carbon source. Sucrose concentration higher than 150 g/L resulted in the decrease of lactic acid productivity due to inhibition by high substrate concentration, but lactic acid productivity was remained > 3.0 g/L/h until the sucrose used for lactic acid fermentation increased up to 150 g/L. L-Lactic acid content of the total lactic acid produced from sucrose and mixed sugars was higher than 98%. When the fermentation media contained sucrose, the kinetic parameters showing specific rates such as μ, qS, and qP were relatively lower than those of fermentation using glucose as a sole carbon source, which might be due to additional time requirement to induce invertase enzyme for utilization of sucrose. There was no carbon catabolite repression observed when the sugar mixtures containing sucrose, glucose, and/ or fructose were used as a carbon source for lactic acid fermentation by E. faecalis RKY1.     

Study on the synthesis of sulfonamide derivatives and their interaction with bovine serum albumin

Three sulfonamide derivatives (SAD) were first synthesized from p‐hydroxybenzoic acid and sulfonamides (sulfadimidine, sulfamethoxazole and sulfachloropyridazine sodium) and were characterized by elemental analysis, ¹H NMR and MS. The interaction between bovine serum albumin (BSA) and SAD was studied using UV/vis absorption spectroscopy, fluorescence spectroscopy, time‐resolved fluorescence spectroscopy and circular dichroism spectra under imitated physiological conditions. The experimental results indicated that SAD effectively quenched the intrinsic fluorescence of BSA via a static quenching process. The thermodynamic parameters showed that hydrogen bonding and van der Waal's forces were the predominant intermolecular forces between BSA and two SADs [4‐((4‐(N‐(4,6‐dimethylpyrimidin‐2‐yl)sulfamoyl)phenyl)carbamoyl)phenyl acetate and 4‐((4‐(N‐(5‐methylisoxazol‐3‐yl)sulfamoyl)phenyl)carbamoyl)phenyl acetate], but hydrophobic forces played a major role in the binding process of BSA and 4‐((4‐(N‐(6‐chloropyridazin‐3‐yl)sulfamoyl)phenyl) carbamoyl)phenyl acetate. In addition, the effect of SAD on the conformation of BSA was investigated using synchronous fluorescence spectroscopy and circular dichroism spectra. Molecular modeling results showed that SAD was situated in subdomain IIA of BSA. Copyright © 2014 John Wiley & Sons, Ltd.