Molecular recognition of sialyl Lewis(x) and related saccharides by two lectins.

The interaction of sialyl Lewis(x), Lewis(x), and alpha-L-Fuc-(1-->3)-beta-D-GlcNAc with isolectin A from Lotus tetragonolobus (LTL-A), and with Aleuria aurantia agglutinin (AAA) was studied using NMR experiments and surface plasmon resonance. Both lectins are specific for fucose residues. From NMR experiments it was concluded that alpha-L-Fuc-(1-->3)-beta-D-GlcNAc and Lewis(x) bound to both lectins, whereas sialyl Lewis(x) only bound to AAA. Increased line broadening of 1H NMR signals of the carbohydrate ligands upon binding to AAA and LTL-A suggested that AAA bound to the ligands more tightly. Further comparison of line widths showed that for both lectins binding strengths decreased from alpha-L-Fuc-(1-->3)-beta-D-GlcNAc to Lewis(x) and were lowest for sialyl Lewis(x). Surface plasmon resonance measurements were then employed to yield accurate dissociation constants. TrNOESY, QUIET-trNOESY, and trROESY experiments delivered bioactive conformations of the carbohydrate ligands, and STD NMR experiments allowed a precise epitope mapping of the carbohydrates bound to the lectins. The bioactive conformation of Lewis(x) bound to LTL-A, or AAA revealed an unusual orientation of the fucose residue, with negative values for both dihedral angles, phi and psi, at the alpha(1-->3)-glycosidic linkage. A similar distortion of the fucose orientation was also observed for sialyl Lewis(x) bound to AAA. From STD NMR experiments it followed that only the L-fucose residues are in intimate contact with the protein. Presumably steric interactions are responsible for locking the sialic acid residue of sialyl Lewis(x) in one out of many orientations that are present in aqueous solution. The sialic acid residue of sialyl Lewis(x) bound to AAA adopts an orientation similar to that in the corresponding sialyl Lewis(x)/E-selectin complex.     

Carbohydrate chips for studying high-throughput carbohydrate-protein interactions

Carbohydrate-protein interactions play important biological roles in living organisms. For the most part, biophysical and biochemical methods have been used for studying these biomolecular interactions. Less attention has been given to the development of high-throughput methods to elucidate recognition events between carbohydrates and proteins. In the current effort to develop a novel high-throughput tool for monitoring carbohydrate-protein interactions, we prepared carbohydrate microarrays by immobilizing maleimide-linked carbohydrates on thiol-derivatized glass slides and carried out lectin binding experiments by using these microarrays. The results showed that carbohydrates with different structural features selectively bound to the corresponding lectins with relative binding affinities that correlated with those obtained from solution-based assays. In addition, binding affinities of lectins to carbohydrates were also quantitatively analyzed by determining IC(50) values of soluble carbohydrates with the carbohydrate microarrays. To fabricate carbohydrate chips that contained more diverse carbohydrate probes, solution-phase parallel and enzymatic glycosylations were performed. Three model disaccharides were in parallel synthesized in solution-phase and used as carbohydrate probes for the fabrication of carbohydrate chips. Three enzymatic glycosylations on glass slides were consecutively performed to generate carbohydrate microarrays that contained the complex oligosaccharide, sialyl Le(x). Overall, these works demonstrated that carbohydrate chips could be efficiently prepared by covalent immobilization of maleimide-linked carbohydrates on the thiol-coated glass slides and applied for the high-throughput analyses of carbohydrate-protein interactions.     

Multivalent Sialosides: A Tool to Explore the Role of Sialic Acids in Biological Processes

Sialic acids (Sias) are fascinating nine‐carbon monosaccharides that are primarily found on the terminus of the oligosaccharide chains of glycoproteins and glycolipids on cell surfaces. These Sias undergo a variety of structural modifications at their hydroxy and amine positions, thereby resulting in structural diversity and, hence, coordinating a variety of biological processes. However, deciphering the structural functions of such interactions is highly challenging, because the monovalent binding of Sias is extremely weak. Over the last decade, several multivalent Sia ligands have been synthesized to modulate their binding affinity with proteins/lectins. In this Minireview, we highlight recent developments in the synthesis of multivalent Sia probes and their potential applications. We will discuss four key multivalent families, that is, polymers, dendrimers, liposomes, and nanoparticles, and will emphasize the major parameters that are essential for the specific interactions of these molecules with proteins in biological systems.     

Surface-grafted poly(acrylic acid) brushes as a precursor layer for biosensing applications: effect of graft density and swellability on the detection efficiency

Carboxyl groups along poly(acrylic acid) (PAA) brushes attached to the surface of a gold-coated substrate served as the precursor moieties for the covalent immobilization of amino-functionalized biotin or bovine serum albumin (BSA) to form a sensing probe for streptavidin (SA) or anti-BSA detection, respectively. Surface-grafted PAA brushes were obtained by acid hydrolysis of poly(tert-butyl acrylate) brushes, formerly prepared by surface-initiated atom transfer radical polymerization of tert-butyl acrylate. As determined by surface plasmon resonance, the PAA brushes immobilized with functionalized biotin or BSA probes not only showed good binding with the designated target analytes but also maintained a high resistance to nonspecific protein adsorption, especially those PAA brushes with a high surface graft density. Although the probe binding capacity can be raised as a function of the graft density of the PAA brushes or the amount of carboxyl groups along the PAA chains, the accessibility of the target analyte to the immobilized probe was limited at the high graft density of the PAA brushes. The effect was far more apparent for the BSA-anti-BSA probe-analyte pair than for the much smaller biotin-SA probe-analyte pair. The impact of the swellability of the PAA brushes, as tailored by the degree of carboxyl group activation, on both the sensing probe immobilization and analyte detection was also addressed. This investigation demonstrated that PAA brushes having a defined graft density have a promising potential as a precursor layer for biosensing applications.     

Chemoenzymatic synthesis and fluorescent visualization of cell-surface selectin-bound sialyl Lewis x derivatives

Sialyl Lewis x (sLe(x)) derivatives conjugated to readily visualized molecular labels are useful chemical probes to study selectin-carbohydrate interactions. Localization of the selectins on the surface of leukocytes and activated endothelial cells can be detected through fluorescence of bound selectin ligands. Herein we present a short chemoenzymatic synthesis of a fluorescently labeled bivalent sLe(x) conjugate. The use of an amino-substituted monovalent sLe(x) to obtain fluorescent- and biotin-labeled sLe(x) derivatives is also described. The cell-staining utility of the fluorescent sLe(x) conjugates is demonstrated for a HUVEC cell line expressing E-selectin and for CHO-K1 cells expressing either L- or E-selectin.     

Rapid, photoactivatable turn-on fluorescent probes based on an intramolecular photoclick reaction

Photoactivatable fluorescent probes are invaluable tools for the study of biological processes with high resolution in space and time. Numerous strategies have been developed in generating photoactivatable fluorescent probes, most of which rely on the photo-"uncaging" and photoisomerization reactions. To broaden photoactivation modalities, here we report a new strategy in which the fluorophore is generated in situ through an intramolecular tetrazole-alkene cycloaddition reaction ("photoclick chemistry"). By conjugating a specific microtubule-binding taxoid core to the tetrazole/alkene prefluorophores, robust photoactivatable fluorescent probes were obtained with fast photoactivation (～1 min) and high fluorescence turn-on ratio (up to 112-fold) in acetonitrile/PBS (1:1). Highly efficient photoactivation of the taxoid-tetrazoles inside the mammalian cells was also observed under a confocal fluorescence microscope when the treated cells were exposed to either a metal halide lamp light passing through a 300/395 filter or a 405 nm laser beam. Furthermore, a spatially controlled fluorescent labeling of microtubules in live CHO cells was demonstrated with a long-wavelength photoactivatable taxoid-tetrazole probe. Because of its modular design and tunability of the photoactivation efficiency and photophysical properties, this intramolecular photoclick reaction based approach should provide a versatile platform for designing photoactivatable fluorescent probes for various biological processes.     

Peptide-modified inverted colloidal crystal scaffolds with bone marrow stromal cells in the treatment for spinal cord injury

Carboxyl groups of surface-tethered poly(acrylic acid) (PAA) brushes should be able to serve as versatile moieties for a wide range of chemical modifications, including an attachment of bioactive species that can act as sensing probes for biosensors. In this research, poly(tert-butyl acrylate) (Pt-BA) brushes were prepared by surface-initiated atom transfer radical polymerization of tert-butyl acrylate. PAA brushes were then obtained after removal of the tert-butyl groups from the Pt-BA brushes by acid hydrolysis. The carboxyl group density of the PAA brushes can be varied as a function of chain length or molecular weight. The reactivity of the carboxyl groups of PAA brushes towards the immobilization of biotin, a frequently used model bioactive probe in biosensing applications, was evaluated. Qualitative determination of streptavidin (SA) binding to the biotin-attached PAA brushes was verified by fluorescence microscopy. The efficiency of the PAA brushes to act as a three dimensional (3D) precursor layer for biosensing applications was further demonstrated using surface plasmon resonance (SPR), where the biotin-attached PAA brushes showed an enhanced signal for the biospecific binding of SA in comparison with a self-assembled monolayer (SAM) of a carboxyl-terminated alkanethiol, used as a model two-dimensional (2D) conventional precursor layer. The PAA brushes showed very low non-specific interactions with two other tested proteins of a similar pI but different sizes. This desirable feature should be highly beneficial for the development of biosensors.     

Nitroxide spin probe study of probe size,hydrogen bonding and polymer matrix rigidity effects on poly(acrylic acid)/poly(ethylene oxide) complexes

An electron spin resonance (ESR) spin probe study was performed on 1 : 1 by weight poly(acrylic acid) (PAA)/poly(ethylene oxide) (PEO) complex over the 100–450 K temperature range with a series of tetramethylpiperidyloxy‐based spin probes. Measurements of the parameters T_5mT, Ta and Td demonstrated the effects of probe size and the strength of hydrogen bonding. The probes in the series Tempone, Tempo, Tempol and Tamine (respectively 4‐oxo‐, unsubstituted, 4‐hydroxy‐ and 4‐amino‐2,2,6,6,‐tetramethylpiperidine ‐1‐oxyl) displayed noticeable increases in the hydrogen‐bonding effect, as indicated by Ta and Td. These increases correlated with increasing hydrogen bond acceptor strength. On the other hand, as the probe size became larger, T_5mT gradually increased due to the free volume decrease. These effects were analyzed using the established theoretical relationship of T_5mT to probe volume expressed by f. Meanwhile, in order to investigate the effect of polymer matrix rigidity, a similar study was performed with a nitroxide spin probe, 2,2,6,6‐tetramethyl‐1‐piperidine‐1‐oxyl (Tempo), on PAA/PEO complexes of different weight compositions. The quantitative fast motion fraction in the composite ESR spectrum was calculated. The influence of changes in the composition of PAA on the molecular mobility was characterized by changes of the spectral parameters and τ_c. The molecular mobility was shown to diminish with increasing content of PAA in PAA/PEO blends duo to the restriction of the polymer matrix rigidity increase. Copyright © 2003 John Wiley & Sons, Ltd.     

Assembly of p-selectin ligands on a polymeric template

High-affinity receptor-ligand interactions frequently involve molecular interactions at two distinct sites. A derivatized polyacrylic-based polymer was synthesized to allow substitution with multiple ligands (e.g., L(1) and L(2)) on the backbone. Two-site P-selectin-ligand interactions were first studied with SiaLe(x) (L(1)) and tyrosine sulfate (L(2)) covalently incorporated onto the flexible polymer. In competition assays, a marked synergistic inhibitory effect was observed when the polymer presented both L(1) and L(2) as opposed to either ligand alone. In a second approach, the SiaLe(X) ligand was reduced in complexity so that L(1) was fixed as Le(x) or Le(a), and alternative L(2) groups (to mimic sialic acid) were investigated. Certain combinations of L(1) and L(2) were better antagonists of P-selectin than SiaLe(x) itself. These approaches offer the potential of facilitating the discovery of novel inhibitors of receptors or enzymes.     

Highly Selective Mitochondrial Targeting by a Ruthenium(II) Peptide Conjugate: Imaging and Photoinduced Damage of Mitochondrial DNA

Mitochondrial DNA (mtDNA) plays a crucial but incompletely understood role in cellular biochemistry and etiology of numerous disease states. Thus, there is an urgent need for targeted probes that can dynamically respond to changes to mtDNA such as copy number in live cells, but it is difficult to permeate the mitochondrial membrane of the living cell. Now, a ruthenium(II) light‐switching probe targeted by peptide vectorization selectively to mitochondrial nucleoids is presented. Evidence for DNA binding by the probe in live cells is derived from confocal fluorescence microscopy, resonance Raman, and luminescence lifetime imaging. While viable under imaging conditions, specific staining of mitochondrial DNA permitted efficient and selective photoinduced toxicity on a cell‐by‐cell basis under higher excitation intensities. This powerful combination of imaging and photocytotoxicity is an important step towards realizing phototheranostic application of such Ru^II probes.     

Selectivity among two lectins: probing the effect of topology, multivalency and flexibility of "clicked" multivalent glycoclusters

The design of multivalent glycoconjugates has been developed over the past decades to obtain high-affinity ligands for lectin receptors. While multivalency frequently increases the affinity of a ligand for its lectin through the so-called "glycoside cluster effect", the binding profiles towards different lectins have been much less investigated. We have designed a series of multivalent galactosylated glycoconjugates and studied their binding properties towards two lectins, from plant and bacterial origins, to determine their potential selectivity. The synthesis was achieved through copper(I)-catalysed azide-alkyne cycloaddition (CuAAC) under microwave activation between propargylated multivalent scaffolds and an azido-functionalised carbohydrate derivative. The interactions of two galactose-binding lectins from Pseudomonas aeruginosa (PA-IL) and Erythrina cristagalli (ECA) with the synthesized glycoclusters were studied by hemagglutination inhibition assays (HIA), surface plasmon resonance (SPR) and isothermal titration microcalorimetry (ITC). The results obtained illustrate the influence of the scaffold's geometry on the affinity towards the lectin and also on the relative potency in comparison with a monovalent galactoside reference probe.     

Efficient solid-phase synthesis of trifunctional probes and their application to the detection of carbohydrate-binding proteins

[structure: see text] An efficient solid-phase synthesis of trifunctional probes containing a photoreactive group, a reporter tag, and a carbohydrate ligand was developed. Labeling studies with these probes demonstrate that specific lectins can be labeled with high sensitivity and selectivity. This technique serves as a powerful tool for the rapid detection and profiling of lectins.     

Computer simulation study of probe-target hybridization in model DNA microarrays: effect of probe surface density and target concentration

We use lattice Monte Carlo simulations to study the thermodynamics of hybridization of single-stranded "target" genes in solution with complementary "probe" DNA molecules immobilized on a microarray surface. The target molecules in our system contain 48 segments and the probes tethered on a hard surface contain 8-24 segments. The segments on the probe and target are distinct, with each segment representing a sequence of nucleotides that interacts exclusively with its unique complementary target segment with a single hybridization energy; all other interactions are zero. We examine how surface density (number of probes per unit surface area) and concentration of target molecules affect the extent of hybridization. For short probe lengths, as the surface density increases, the probability of binding long stretches of target segments increases at low surface density, reaches a maximum at an intermediate surface density, and then decreases at high surface density. Furthermore, as the surface density increases, the target is less likely to bind completely to one probe; instead, it binds simultaneously to multiple probes. At short probe lengths, as the target concentration increases, the fraction of targets binding completely to the probes (specificity) decreases. At long probe lengths, varying the target concentration does not affect the specificity. At all target concentrations as the probe length increases, the fraction of target molecules bound to the probes by at least one segment (sensitivity) increases while the fraction of target molecules completely bound to the probes (specificity) decreases. This work provides general guidelines to maximizing microarray sensitivity and specificity. Our results suggest that the sensitivity and specificity can be maximized by using probes 130-180 nucleotides long at a surface density in the range of 7 x 10(-5)- 3 x 10(-4) probe molecules per nm(2).     

Beyond carbohydrate binding: new directions in plant lectin research

Although for a long time carbohydrate binding property has been used as the defining feature of lectins, studies carried out mostly during the last two decades or so demonstrate that many plant lectins exhibit specific interactions with small molecules that are predominantly hydrophobic in nature. Such interactions, in most cases, appear to be at specific sites that do not interfere with the ability of the lectins to recognise and bind carbohydrates. Further, several of these ligands have binding affinities comparable to those for the binding of specific carbohydrates to the lectins. Given the ability of lectins to specifically recognise the glycocode (carbohydrate code) on different cell surfaces and distinguish between diseased and normal tissues, these additional sites may be viewed as potential drug carrying sites that could be exploited for targeted delivery to sites of choice. Porphyrin-lectin complexes are especially suited for such targeting since porphyrins are already under investigation in photodynamic therapy for cancer. This review will provide an update on the interactions of plant lectins with non-carbohydrate ligands, with particular emphasis on porphyrin ligands. The implications and potential applications of such studies will also be discussed.     

Synthesis of Homo- and Heterofunctionalized Glycoclusters and Binding to Pseudomonas aeruginosa Lectins PA-IL and PA-IIL

Homo- and heterofunctionalized glycoclusters with galactose and/or fucose residues targeting both PA-IL and PA-IIL lectins of Pseudomonas aeruginosa were synthesized using "Click" chemistry and DNA chemistry. Their binding to lectins (separately or in a mixture) was studied using a DNA Directed Immobilization carbohydrate microarray. Homoglycoclusters bind selectively to their lectin while the heteroglycocluster binds simultaneously both lectins with a slight lower affinity.     

Quantitative description and local structures of trivalent metal ions Eu(III) and Cm(III) complexed with polyacrylic acid

The trivalent metal ion (M(III)=Cm, Eu)/polyacrylic acid (PAA) system was studied in the pH range between 3 and 5.5 for a molar PAA-to-metal ratio above 1. The interaction was studied for a wide range of PAA (0.05 mg L(-1)-50 g L(-1)) and metal ion concentrations (2x10(-9)-10(-3) M). This work aimed at 3 goals (i) to determine the stoichiometry of M(III)-PAA complexes, (ii) to determine the number of complexed species and the local environment of the metal ion, and (iii) to quantify the reaction processes. Asymmetric flow-field-flow fractionation (AsFlFFF) coupled to ICP-MS evidenced that size distributions of Eu-PAA complexes and PAA were identical, suggesting that Eu bound to only one PAA chain. Time-resolved laser fluorescence spectroscopy (TRLFS) measurements performed with Eu and Cm showed a continuous shift of the spectra with increasing pH. The environment of complexed metal ions obviously changes with pH. Most probably, spectral variations arose from conformational changes within the M(III)-PAA complex due to pH variation. Complexation data describing the distribution of complexed and free metal ion were measured with Cm by TRLFS. They could be quantitatively described in the whole pH-range studied by considering the existence of only a single complexed species. This indicates that the slight changes in M(III) speciation with pH observed at the molecular level do not significantly affect the intrinsic binding constant. The interaction constant obtained from the modelling must be considered as a mean interaction constant.     

Bifunctional Fluorescent Probe for Sequential Sensing of Thiols and Primary Aliphatic Amines in Distinct Fluorescence Channels

Thiols and primary aliphatic amines (PAA) are ubiquitous and extremely important species in biological systems. They perform significant interplaying roles in complex biological events. A single fluorescent probe differentiating both thiols and PAA can contribute to understanding the intrinsic inter‐relationship of thiols and PAA in biological processes. Herein, we rationally constructed the first fluorescent probe that can respond to thiols and PAA in different fluorescence channels. The probe exhibited a high selectivity and sensitivity to thiols and PAA. In addition, it displayed sequential sensing ability when the thiols and PAA coexisted. The application experiments indicated that the probe can be used for sensing thiols and PAA in human blood serum. Moreover, the fluorescence imaging of endogenous thiols and PAA as well as antihypertensive drugs captopril and amlodipine in living cells were successfully conducted.