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Glycosyldisulfides from dynamic combinatorial libraries as O-glycoside mimetics for plant and endogenous lectins: Their reactivities in solid-phase and cell assays and conformational analysis by molecular dynamics simulations
Abstract
Dynamic combinatorial library design exploiting the thiol-disulfide exchange readily affords access to glycosyldisulfides. In order to reveal lectin-binding properties of this type of non-hydrolyzable sugar derivative, libraries originating from a mixture of common building blocks of natural glycans and thiocompounds were tested against three plant agglutinins with specificity to galactose, fucose or N-acetylgalactosamine, respectively, in a solid-phase assay. Extent of lectin binding to matrix-immobilized neoglycoprotein presenting the cognate sugar could be reduced, and evidence for dependence on type of carbohydrate was provided by dynamic deconvolution. Glycosyldisulfides also maintained activity in assays of increased physiological relevance, that is, using native tumor cells and also adding to the test panel an endogenous lectin (galectin-3) involved in tumor spread and cardiac dysfunction. N-Acetylgalactosamine was pinpointed as the most important building block of libraries for the human lectin and the digalactoside as most potent compound acting on the toxic mistletoe agglutinin which is closely related to the biohazard ricin. Because this glycosyldisulfide, which even surpasses lactose in inhibitory capacity, rivals thiodigalactoside as inhibitor, their degrees of intramolecular flexibility were comparatively analyzed by computational calculations. Molecular dynamics runs with explicit consideration of water molecules revealed a conspicuously high degree of potential for shape alterations by the disulfide's three-bond system at the interglycosidic linkage. The presented evidence defines glycosyldisulfides as biologically active ligands for lectins.
... As such, solidphase assays with surface-immobilized (neo)glycoconjugates or glycans and labeled galectins or determination of direct ligand binding in solution using fluorescence polarization, frontal affinity chromatography, and isothermal titration calorimetry showed merit to detect differences in binding properties among galectins (Leffler and Barondes 1986;Lee et al. 1990;Ahmad et al. 2002;Hirabayashi et al. 2002;Sörme et al. 2004;Leppänen et al. 2005). In order to substitute the artificial matrix by a physiological surface, the design of the inhibition assay had been extended to using a human tumor or Chinese hamster ovary cells in vitro (André et al. 2006;Patnaik et al. 2006). This experimental setting with cells, the multivalency of galectins apparent from their activity as hemagglutinins and the versatility of neoglycoconjugates, serving as potent lectin ligands (Rapoport et al. 2007), prompted us to explore the following approach: to combine availability of a panel of labeled neoglycoconjugates, which present different carbohydrate headgroups, with cell surface binding of galectins. ...
... The human galectins originated from recombinant production and were then purified by affinity chromatography on lactosylated Sepharose 4B, prepared by divinyl sulfone activation (Gabius 1990), as a crucial step, analyzed for purity by one-and two-dimensional gel electrophoresis, gel filtration, and nano-electrospray ionization mass spectrometry (Kopitz et al. 2001;André et al. 2004), and checked for binding activity prior to and after biotinylation by hemagglutination, solid-phase assays, and cell binding (André et al. 2006). Proteolytic truncation of full-length galectin-3 used extensive collagenase digestion to yield the C-terminal CRD (Kopitz et al. 2001). ...
... ont testé, sur phase solide et sur des cellules, un système mettant en jeu des sucres portant sur leur position anomère un thiol libre, pouvant former des ponts disulfures et créant ainsi des ligands di-sacchraridiques. [72] Ramström et al. ont recouru à la formation d'hémithioacétal sur un coeur portant des saccharides pour identifier les inhibiteurs de la β-galactosidase. [73] Klebe, Hirsch et coll. ...
La bactérie Pseudomonas aeruginosa a développé une résistance accrue aux antibiotiques. Des alternatives se développent, notamment l’étude d’agents antiadhésifs. Cette stratégie vise à bloquer les protéines à la surface de la bactérie pour empêcher son adhésion sur les cellules hôtes. Au sein du laboratoire, des glycoclusters antiadhésifs ont déjà été synthétisés et ont fait leurs preuves in vivo. L’alliance de la multivalence et de la chimie combinatoire dynamique pourrait nous permettre d’identifier de nouveaux glycoclusters d’intérêt biologique. Des briques moléculaires dithiophénol glycosylées ont été étudiées pour la reconnaissance de lectines bactériennes. Celles-ci ont montré la formation préférentielle de macrocycles de type glycodyn[3]arènes et glycodyn[4]arènes. Des études biologiques sont en cours pour évaluer leur efficacité et leur toxicité in vitro. En parallèle de ces travaux, une nouvelle génération de briques moléculaires a été imaginée. Elle devra répondre à une problématique de double chimie combinatoire dynamique pour permettre la polymérisation des glycodyn[n]arènes en glycodynamères. Une brique dithiophénol incorporant une fonction polymérisable (aldéhyde/hydrazide) sera nécessaire pour atteindre les glycodynamères, ainsi que des bras espaceurs hydrosolubles pour la polymérisation. Ceci permettra de passer de l’échelle de la lectine (glycodyn[n]arènes) à celle de la bactérie (glycodynamères). Deux chimies combinatoires dynamiques orthogonales seront alors mises en jeu sur la base de briques dithiophénol d’une part et aldéhyde/hydrazide d’autre part.
... Ye (2 × 10 8 CFU) were fixed in 3% paraformaldehyde for 2 h at room temperature, washed three times in phosphate-buffered saline (PBS), and stored at −80 • C in PBS containing 15% of glycerol. To determine galectin binding, 2 × 10 7 fixed bacteria were incubated with label free rGal1 as described [59] at a final concentration of 3.3 mM (100 µg/mL) for 1 h at 37 • C. After two washes with PBS/Tween 0.1%, Gal1 binding was detected by incubation with a rabbit anti-human Gal1 antibody for 45 min at 4 • C. Cells were then washed twice in 0.1% PBS-Tween, next, resuspended in 50 µL of PBS with a polyclonal anti-rabbit FITC-conjugate antibody (1/200), and incubated for 30 min on ice. ...
Yersinia enterocolitica (Ye) inserts outer proteins (Yops) into cytoplasm to infect host cells. However, in spite of considerable progress, the mechanisms implicated in this process, including the association of Yops with host proteins, remain unclear. Here, we evaluated the functional role of Galectin-1 (Gal1), an endogenous β-galactoside-binding protein, in modulating Yop interactions with host cells. Our results showed that Gal1 binds to Yops in a carbohydrate-dependent manner. Interestingly, Gal1 binding to Yops protects these virulence factors from trypsin digestion. Given that early control of Ye infection involves activation of macrophages, we evaluated the role of Gal1 and YopP in the modulation of macrophage function. Although Gal1 and YopP did not influence production of superoxide anion and/or TNF by Ye-infected macrophages, they coordinately inhibited nitric oxide (NO) production. Notably, recombinant Gal1 (rGal1) did not rescue NO increase observed in Lgals1−/− macrophages infected with the YopP mutant Ye ∆yopP. Whereas NO induced apoptosis in macrophages, no significant differences in cell death were detected between Gal1-deficient macrophages infected with Ye ∆yopP, and WT macrophages infected with Ye wt. Strikingly, increased NO production was found in WT macrophages treated with MAPK inhibitors and infected with Ye wt. Finally, rGal1 administration did not reverse the protective effect in Peyer Patches (PPs) of Lgals1−/− mice infected with Ye ∆yopP. Our study reveals a cooperative role of YopP and endogenous Gal1 during Ye infection.
... In recent years, numerous extended linkage modes that consist of two (or even more) bridging atoms in the place of a native O-glycosidic bond have been designed (Scheme 1(A2)) [7,8]. The extended, three-bond interglycosidic connections, including S-S [12][13][14][15], Se-S, Se-Se [16], C-S [17,18], C-N [19], N-O [20], and SO 2 -N [21] bonds, provide specific conformational properties, and, in some cases, advantageous binding capabilities to carbohydrate mimetics. ...
Oligosaccharides and glycoconjugates are abundant in all living organisms, taking part in a multitude of biological processes. The application of natural O-glycosides in biological studies and drug development is limited by their sensitivity to enzymatic hydrolysis. This issue made it necessary to design hydrolytically stable carbohydrate mimetics, where sulfur, carbon, or longer interglycosidic connections comprising two or three atoms replace the glycosidic oxygen. However, the formation of the interglycosidic linkages between the sugar residues in high diastereoslectivity poses a major challenge. Here, we report on stereoselective synthesis of carbon-sulfur-bridged disaccharide mimetics by the free radical addition of carbohydrate thiols onto the exo-cyclic double bond of unsaturated sugars. A systematic study on UV-light initiated radical mediated hydrothiolation reactions of enoses bearing an exocyclic double bond at C1, C2, C3, C4, C5, and C6 positions of the pyranosyl ring with various sugar thiols was performed. The effect of temperature and structural variations of the alkenes and thiols on the efficacy and stereoselectivity of the reactions was systematically studied and optimized. The reactions proceeded with high efficacy and, in most cases, with complete diastereoselectivity producing a broad array of disaccharide mimetics coupling through an equatorially oriented methylensulfide bridge.
... Various dynamic reversible reactions have been proposed and applied in protein-directed DCC, which can be divided into three main types. These include addition-elimination reactions at carbonyl groups (such as imine and hydrazone formation) [24,[35][36][37][38][39][40][41][42][43][44][45][46][47][48][49], thiol exchange reactions (such as thiol-disulphide exchange [50][51][52][53][54][55][56][57], thiol-enone reaction [58] and the hemithioacetal reaction) [59,60], and boronate ester formation reaction (Table 3) [25,61,62]. Diselenide exchange and selenenylsulphide exchange reactions have also been shown to react reversibly in water at neutral pH [63,64], although to date there are no examples of inhibitor discovery via protein-directed DCC using these reactions. ...
Protein-directed dynamic combinatorial chemistry is an emerging technique for efficient discovery of novel chemical structures for binding to a target protein. Typically, this method relies on a library of small molecules that react reversibly with each other to generate a combinatorial library. The components in the combinatorial library are at equilibrium with each other under thermodynamic control. When a protein is added to the equilibrium mixture, and if the protein interacts with any components of the combinatorial library, the position of the equilibrium will shift and those components that interact with the protein will be amplified, which can then be identified by a suitable biophysical technique. Such information is useful as a starting point to guide further organic synthesis of novel protein ligands and enzyme inhibitors. This review uses literature examples to discuss the practicalities of applying this method to inhibitor discovery, in particular, the set-up of the combinatorial library, the reversible reactions that may be employed, and the choice of detection methods to screen protein ligands from a mixture of reversibly forming molecules.
... It recognizes non-reducing α-D-mannose structures and for this reason the thioanalogs 1-thio-D-mannose (3) and methyl 6-thio-α-D-mannopyranoside (6) were synthesized. To obtain further structural diversity, the 1-thio analogs of N-acetyl-β-D-glucosamine (4) and N-acetyl-β-D-galactosamine (5) were also synthesized (André, Pei et al. 2006). The syntheses of compounds 3 and 6, exemplified in Scheme 4, were performed in few steps from the suitably derivatised carbohydrates by reaction with thioacetate, and subsequent deprotection. ...
Licentiate Thesis Stockholm 2006 Akademisk avhandling som med tillstånd av Kungliga Tekniska Högskolan i Stockholm framlägges till offentlig granskning för avläggande av licentiatexamen i organisk kemi, tisdagen den 2 maj, kl 10.00 i sal F3, KTH, Lindstedtsvägen 26, Stockholm. Avhandlingen försvaras på engelska.
Galectins, a class of carbohydrate‐binding proteins, play a crucial role in various physiological and disease processes. Therefore, the identification of ligands that efficiently bind these proteins could potentially lead to the development of new therapeutic compounds. In this study, we present a method that involves screening synthetic click glycopeptide libraries to identify lectin‐binding ligands with low micromolar affinity. Our methodology, initially optimized using Concanavalin A, was subsequently applied to identify binders for the therapeutically relevant galectin 1. Binding affinities were assessed using various methods and showed that the selected glycopeptides exhibited enhanced binding potency to the target lectins compared to the starting sugar moieties. This approach offers an alternative means of discovering galectin‐binding ligands as well as other carbohydrate‐binding proteins, which are considered important therapeutic targets.
Lymphocyte activation gene 3 (LAG3) is an inhibitory immune checkpoint receptor that restrains autoimmune and antitumor responses, but its evolutionarily conserved cytoplasmic tail lacks classical inhibitory motifs. Major histocompatibility complex class II (MHC class II) is an established LAG3 ligand, and fibrinogen-like protein 1 (FGL1), lymph node sinusoidal endothelial cell C-type lectin (LSECtin), and Galectin-3 have been proposed as alternative binding partners that play important roles in LAG3 function. Here, we used a fluorescent human T cell reporter system to study the function of LAG3. We found that LAG3 reduced the response to T cell receptor stimulation in the presence of MHC class II molecules to a lesser extent compared with the receptor programmed cell death protein 1. Analysis of deletion mutants demonstrated that the RRFSALE motif in the cytoplasmic tail of LAG3 was necessary and sufficient for LAG3-mediated inhibition. In this system, FGL1, but not LSECtin or Galectin-3, acted as a LAG3 ligand that weakly induced inhibition. LAG3-blocking antibodies attenuated LAG3-mediated inhibition in our reporter cells and enhanced reporter cell activation even in the absence of LAG3 ligands, indicating that they could potentially enhance T cell responses independently of their blocking effect.
Glycosylation of peptides and proteins is a widely employed strategy to mimic important post-translational modifications or to modulate the physicochemical properties of peptides to enhance their delivery. Furthermore, glycosylation via a sulfur atom imparts increased chemical and metabolic stability to the resulting glycoconjugates. Herein, we report a simple and chemoselective procedure to prepare disulfide-linked glycopeptides. Acetate-protected glycosylsulfenyl hydrazines are shown to be highly reactive with the thiol group of cysteine residues within peptides, both in solution and as part of conventional solid-phase peptide synthesis protocols. The efficiency of this glycosylation methodology with unprotected carbohydrates is also demonstrated, which avoids the need for deprotection steps and further extends its utility, with disulfide-linked glycopeptides produced in excellent yields. Given the importance of glycosylated peptides in structural glycobiology, pharmacology, and therapeutics, the methodology outlined provides easy access to disulfide-linked glycopeptides as molecules with multiple biological applications.
A one-pot reaction methodology has been developed for the preparation of mixed S–Se-linked pseudodisaccharides by the treatment of glycosyl halides with a combination sodium sulfide nonahydrate (Na2S⋅9H2O) and carbon disulfide (CS2) in the presence of alkyl and glycosyl diselenides. The reaction condition is fast, odourless, high yielding and suitable for scale-up synthesis. Exclusive formation of anomerically beta-linked products was obtained from anomeric glycosyl bromides.
A significantly fast one step reaction condition has been developed for the synthesis of unsymmetrical anomeric glycosyl disulfide derivatives in excellent yield by the treatment of anomeric glycosyl bromides with a combination of carbon disulfide (CS2) and sodium sulfide nonahydrate (Na2S ⋅ 9H2O) in the presence of symmetrical alkyl, aryl and glycosyl disulfides at room temperature. Exclusive formation of anomerically beta‐disulfides was observed under the reaction conditions. Most of the reactions are high yielding and suitable for scale‐up synthesis. A fast one step reaction condition has been developed for the synthesis of unsymmetrical anomeric glycosyl disulfide derivatives from anomeric glycosyl bromides in excellent yield.
A convenient odourless reaction condition has been developed for the preparation of symmetrical glycosyl disulfide derivatives and aryl glycosyl disulfide derivatives from corresponding glycosyl iodides via in situ formation of glycosyl Bunte salts as thiol surrogates. The reaction has been further extended towards the preparation of glycosyl sulphide derivatives from corresponding Bunte salt in the presence of sodium sulphide. Most of the reactions were high yielding and suitable for scaling up.
Glycosyl selenocyanate derivatives were prepared in water using KSeCN as selenium precursor. Reaction of glycosyl selenocyanate with sugar electrophiles in the presence of hydrazine monohydrate furnished Se-linked pseudodisaccharides.
Chemometrics and Intelligent Laboratory Systems
Available online 3 February 2021, 104266
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QSAR study of unsymmetrical aromatic Disulfides as potent avian SARS-CoV main protease inhibitors using quantum chemical descriptors and statistical methods
Author links open overlay panelSamirChtitaaAssiaBelhassanbMohamedBakhouchcAbdelali IdrissiTaouratidAdnaneAouidatebSalahBelaidieMohammedMoutaabbidaSaidBelaaouadaMohammedBouachrinebfTaharLakhlifib
a
Laboratory of physical chemistry of materials, Faculty of sciences Ben M’Sik, Hassan II University of Casablanca, B.P. 7955, Sidi Othmane, Casablanca, Morocco
b
Molecular Chemistry and Natural Substances Laboratory, Department of chemistry, Faculty of sciences, University Moulay Ismail, Meknes, Morocco
c
Laboratory of Bioorganic Chemistry, Department of Chemistry, Faculty of Sciences, Chouaïb Doukkali University, 24000, El Jadida, Morocco
d
Laboratory of biological chemistry applied to the environment, Department of chemistry, Faculty of sciences, University Moulay Ismail, Meknes, Morocco
e
Laboratory of Molecular Chemistry and Environment, Group of Computational and pharmaceutical Chemistry, University of Biskra, BP145 07000, Biskra, Algeria
f
High School of Technology of Khenifra, Sultane Slimane University, B.P. 591, Khenifra, Morocco
Received 5 October 2020, Revised 9 January 2021, Accepted 30 January 2021, Available online 3 February 2021.
https://doi.org/10.1016/j.chemolab.2021.104266
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Highlights
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Quantum descriptors were calculated using DFT calculation for forty disulfide derivatives as inhibitors of the SARS Coronavirus.
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Hundred linear pentavariate and quadrivariate robust validated models were established
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Models were validated internally as well as externally along with Y-Randomization according to the OECD principles and acceptance criteria of Golbraikh and Tropsha’s
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Anti-SARS-CoV main protease of studied compounds is governed by five descriptors
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Structural requirements for great SARS-CoV inhibitor will be by substituting disulfides with smaller size electron withdrawing groups.
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New compounds with good SARS-CoV inhibitors activities have been designed.
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In silico prediction studies on ADMET pharmacokinetics properties were conducted.
Abstract:
In silico research was executed on forty unsymmetrical aromatic disulfide derivatives as inhibitors of the SARS Coronavirus (SARS-CoV-1). Density functional theory (DFT) calculation with B3LYP functional employing 6-311+G(d,p) basis set was used to calculate quantum chemical descriptors. Topological, physicochemical and thermodynamic parameters were calculated using ChemOffice software. The dataset was divided randomly into training and test sets consisting of 32 and 8 compounds, respectively. In attempt to explore the structural requirements for bioactives molecules with significant anti-SARS-CoV-2 activity, we have built valid and robust statistics models using QSAR approach. Hundred linear pentavariate and quadrivariate models were established by changing training set compounds and further applied in test set to calculate predicted IC50 values of compounds. Both built models were individually validated internally as well as externally along with Y-Randomization according to the OECD principles for the validation of QSAR model and the model acceptance criteria of Golbraikh and Tropsha’s. Model 34 is chosen with higher values of R2, R2test and Q2cv (R2 = 0.838, R2test= 0.735, Q2cv = 0.757).
It is very important to notice that anti-SARS-CoV main protease of these compounds appear to be mainly governed by five descriptors, i.e. highest occupied molecular orbital energy (EHOMO), energy of molecular orbital below HOMO energy (EHOMO-1), Balaban index (BI), bond length between the two sulfur atoms (S1S2) and bond length between sulfur atom and benzene ring (S2Bnz). Here the possible action mechanism of these compounds was analyzed and discussed, in particular, important structural requirements for great SARS-CoV main protease inhibitor will be by substituting disulfides with smaller size electron withdrawing groups. Based on the best proposed QSAR model, some new compounds with higher SARS-CoV inhibitors activities have been designed. Further, in silico prediction studies on ADMET pharmacokinetics properties were conducted
The disulfide bond plays an important role in the formation and stabilisation of higher order structures of peptides and proteins, while in recent years interest in this functional group has been extended to carbohydrate chemistry. Rarely found in nature, glycosyl disulfides have attracted significant attention as glycomimetics, with wide biological applications including lectin binding, as key components of dynamic libraries to study carbohydrate structures, the study of metabolic and enzymatic studies, and even as potential drug molecules. This interest has been accompanied and fuelled by the continuous development of new methods to construct the disulfide bond at the anomeric centre. Glycosyl disulfides have also been exploited as versatile intermediates in carbohydrate synthesis, particularly as glycosyl donors. This review focuses on the importance of the disulfide bond in glycobiology and in chemistry, evaluating the different methods available to synthesise glycosyl disulfides. Furthermore, we review the role of glycosyl disulfides as intermediates and/or glycosyl donors for the synthesis of neoglycoproteins and oligosaccharides, before finally considering examples of how this important class of carbohydrates have made an impact in biological and therapeutic contexts.
Functional pairing between cellular glycoconjugates and tissue lectins like galectins has wide (patho)physiological significance. Their study is facilitated by non-hydrolyzable derivatives of the natural O-glycans, such as Sand Se-glycosides. The latter enable extensive analyses by specific 77 Se NMR spectroscopy, but still remain underexplored. By the example of selenodigalactoside (SeDG) and the human galectins 1 and 3, we evaluate diverse 77 Se NMR detection methods and propose selective 1 H, 77 Se heteronuclear Hartmann-Hahn transfer for efficient use in competitive NMR screening against a selenoglycoside spy ligand. By fluorescence anisotropy, circular dichroism, and isothermal titration calorimetry (ITC) we show that affinity and thermodynamics of SeDG binding by galectins are similar to thiodi-galactoside (TDG) and N-acetyllactosamine (LacNAc), confirming that Se substitution has no major impact. ITC data in D 2 O vs. H 2 O are similar for TDG and LacNAc binding by both galectins, but a solvent effect, indicating solvent rearrangement at the binding site, is hinted for SeDG and clearly observed for LacNAc dimers with extended chain length.
The fundamental importance of protein‐glycan recognition calls for specific and sensitive high‐resolution techniques for their detailed analysis. After introduction of 19F NMR to study the recognition of fluorinated glycans, we present new 77Se NMR methodology for complementary studies of selenoglycans with optimised resolution and sensitivity, where direct NMR detection on 77Se is replaced by its indirect observation in a 2D 1H,77Se HSQMBC spectrum. In contrast to OH/F substitution, O/Se exchange allows to target the glycosidic bond. As an example, selenodigalactoside recognition by three human galectins and a plant toxin is readily indicated by signal attenuation and linebroadening in the 2D 1H,77Se HSQMBC spectrum, where CPMG‐INEPT long‐range transfer ensures maximal detection sensitivity, clean signal phases, and reliable ligand ranking. By monitoring competitive displacement of a selenated spy ligand, the selective 77Se NMR approach may be used to screen also non‐selenated compounds. Finally, 1H,77Se CPMG‐INEPT transfer allows to combine further NMR sensors of molecular interaction with the specificity and resolution of 77Se NMR.
The heteroaromatic fused diazabicyclic “bimane” ring system, discovered four decades ago, is endowed with remarkable chemical and photophysical properties. No carbohydrate derivatives of bimanes have, however, been described thus far. Here we report on the syntheses of a range of bimanes decorated with various glycosyl residues. Mono- and disaccharide residues were attached to syn- or anti-bimane central cores via thio-, disulfido- or selenoglycosidic linkages to obtain novel fluorescent or nonfluorescent glycoconjugates. Cu(I)-catalyzed cycloaddition of glycosyl azides to a bimane diethynyl derivative furnished further bivalent glycoconjugates with sugar residues linked to the central bimane core via 1,2,3-triazole rings. We have determined the crystal and molecular structures of several glycosylated and non-glycosylated bimanes and report fluorescence data for the new compounds.
Dynamic combinatorial chemistry (DCC) has repeatedly proven to be an effective approach to generate directed ligand libraries for macromolecular targets. In the absence of an external stimulus, a dynamic library forms from reversibly reacting building blocks and reaches a stable thermodynamic equilibrium. However, upon addition of a macromolecular host which can bind and stabilize certain components of the library, the equilibrium composition changes and induces an evolution‐like selection and enrichment of high‐affinity ligands. A valuable application of this so‐called target‐directed DCC (tdDCC) is the identification of potent ligands for pharmacologically relevant targets. Over time, the term tdDCC has been applied to describe a number of different experimental set‐ups, leading to some ambiguity concerning its definition. This article systematically classifies known procedures for tdDCC and related approaches, with a special focus on the methods used for analysis and evaluation of experiments.
Straightforward S-S bond formation via the oxidation of S-acetyl group by iodide was reported here. The reaction was further applied in the synthesis of per-O-acetylated glycosyl disulfides. These studies demonstrated great improvement in reaction rate, yield and general convenience in the presence of N-Iodosuccinimide. Furthermore, selectively deacetylated glycosyl thiols were obtained in high yields when these per-O-acetylated glycosyl disulfides were reduced with tris(2-carboxyethyl)-phosphine (TCEP). Our method supplied an efficient way to obtain both per-O-acetylated glycosyl disulfides and per-O-acetylated glycosyl thiols in which the sulfur group was located at any position.
The role of sulfur in glycosidic bonds has been evaluated using quartz crystal microbalance methodology. Synthetic routes towards α1-2- and α1-6-linked dimannosides with S- or O-glycosidic bonds have been developed, and the recognition properties assessed in competition binding assays with the cognate lectin concanavalin A. Mannose-presenting QCM sensors were produced using photoinitiated, nitrene-mediated immobilization methods, and the subsequent binding study was performed in an automated flow-through instrumentation, and correlated with data from isothermal titration calorimetry. The recorded Kd-values corresponded well with reported binding affinities for the O-linked dimannosides with affinities for the α1-2-linked dimannosides in the lower micromolar range. The S-linked analogs showed slightly disparate effects, where the α1-6-linked analog showed weaker affinity than the O-linked dimannoside, as well as positive apparent cooperativity, whereas the α1-2-analog displayed very similar binding compared to the O-linked structure.
With the aim to develop compounds able to target multiple metabolic pathways and, thus, to lower the chances of drug resistance, we investigated the anti-trypanosomal activity and selectivity of a series of symmetric diglycosyl diselenides and disulfides. Of 18 compounds tested the fully acetylated forms of di-β-D-glucopyranosyl and di-β-D-galactopyranosyl diselenides (13 and 15, respectively) displayed strong growth inhibition against the bloodstream stage of African trypanosomes (EC50 0.54 μM for 13 and 1.49 μM for 15) although with rather low selectivity (SI < 10 assayed with murine macrophages). Nonacetylated versions of the same sugar diselenides proved to be, however, much less efficient or completely inactive to suppress trypanosome growth. Significantly, the galactosyl (15), and to a minor extent the glucosyl (13), derivative inhibited glucose catabolism but not its uptake. Both compounds induced redox unbalance in the pathogen. In vitro NMR analysis indicated that diglycosyl diselenides react with glutathione, under physiological conditions, via formation of selenenylsulfide bonds. Our results suggest that non-specific cellular targets as well as actors of the glucose and the redox metabolism of the parasite may be affected. These molecules are therefore promising leads for the development of novel multitarget antitrypanosomal agents.
The emerging significance of recognition of cellular glycans by lectins for diverse aspects of pathophysiology is a strong incentive for considering development of bioactive and non-hydrolyzable glycoside derivatives, for example by introducing S/Se atoms and the disulfide group instead of oxygen into the glycosidic linkage. We report the synthesis of 12 bivalent thio-, disulfido- and selenoglycosides attached to benzene/naphthalene cores. They present galactose, for blocking a plant toxin, or lactose, the canonical ligand of adhesion/growth-regulatory galectins. Modeling reveals unrestrained flexibility and inter-headgroup distances too small to bridge two sites in the same lectin. Inhibitory activity was first detected by solid-phase assays using a surface-presented glycoprotein, with relative activity enhancements per sugar unit relative to free cognate sugar up to nearly 10fold. Inhibitory activity was also seen on lectin binding to surfaces of human carcinoma cells. In order to proceed to characterize this capacity in the tissue context monitoring of lectin binding in the presence of inhibitors was extended to sections of three types of murine organs as models. This procedure proved to be well-suited to determine relative activity levels of the glycocompounds to block binding of the toxin and different human galectins to natural glycoconjugates at different sites in sections. The results on most effective inhibition by two naphthalene-based disulfides and a selenide raise the perspective for broad applicability of the histochemical assay in testing glycoclusters that target biomedically relevant lectins.
Novel strategies for the efficient synthesis of unsymmetrical glycosyl disulfides are reported. Glycosyl disulfides are increasingly important as glycomimetics and molecular probes in glycobiology. Sialosyl disulfides are synthesised directly from the chlorosialoside Neu5Ac2Cl, proceeding via a thiol-disulfide exchange reaction between the sialosyl thiolate and symmetrical disulfides. This methodology was adapted and found to be successfully applicable to the synthesis of unsymmetrical glucosyl disulfides under mild conditions.
We present an implementation of explicit solvent particle mesh Ewald (PME) classical molecular dynamics (MD) within the PMEMD molecular dynamics engine, that forms part of the AMBER v14 MD software package, that makes use of Intel Xeon Phi coprocessors by offloading portions of the PME direct summation and neighbor list build to the coprocessor. We refer to this implementation as pmemd MIC offload and in this paper present the technical details of the algorithm, including basic models for MPI and OpenMP configuration, and analyze the resultant performance. The algorithm provides the best performance improvement for large systems ( 400,000 atoms), achieving a ˜35% performance improvement for satellite tobacco mosaic virus (1,067,095 atoms) when 2 Intel E5-2697 v2 processors (2 x 12 cores, 30M cache, 2.7GHz) are coupled to an Intel Xeon Phi coprocessor (Model 7120P–1.238/1.333 GHz, 61 cores). The implementation utilizes a two-fold decomposition strategy: spatial decomposition using an MPI library and thread-based decomposition using OpenMP. We also present compiler optimization settings that improve the performance on Intel Xeon processors, while retaining simulation accuracy.
A cooxidation of carbohydrate and terpene thiols gives mixtures of disulfides containing 51 - 90% of the unsymmetric product. Membranoprotective and antioxidant properties of obtained unsymmetric and symmetric disulfides were evaluated based on their ability to inhibit the H2O2-induced hemolysis of erythrocytes, as well as the accumulation of secondary products of the peroxy oxidation of lipids and the oxidation of hemoglobin.
The ability of glycosyldiselenides to act as lectin ligands and their selective detection in plasma by 77Se NMR is reported
A wide range of symmetrical and unsymmetrical glycosyl disulfides is synthesized with focus on the use of alpha-glycosyl thiols. Oxidation of alpha-glycosyl thiols with iodine leads to symmetrical alpha,alpha-glycosyl disulfides, while unsymmetrical disulfides are readily synthesized from alpha- and beta-glycosyl thiols under the action of DDQ. Thus, glycosyl disulfides containing at least one alpha-glycosidic linkage are made available.
An easy and mild method has been developed for the synthesis of mixed glycosyl disulfides/selenenylsulfides from glycosyl halides and diaryl/dialkyl dichalcogenides in the presence of benzyltriethylammonium tetrathiomolybdate [(BnEt3N)2MoS4]. The salient feature of this method is the sulfur transfer from [BnEt3N]2MoS4 to form glycosyl disulfides which with excess tetrathiomolybdate further undergo exchange reaction with other dichalcogenides in a one-pot operation.
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We present an implementation of explicit solvent all atom classical molecular dynamics (MD) within the AMBER program package that runs entirely on CUDA-enabled GPUs. First released publicly in April 2010 as part of version 11 of the AMBER MD package and further improved and optimized over the last two years, this implementation supports the three most widely used statistical mechanical ensembles (NVE, NVT, and NPT), uses particle mesh Ewald (PME) for the long-range electrostatics, and runs entirely on CUDA-enabled NVIDIA graphics processing units (GPUs), providing results that are statistically indistinguishable from the traditional CPU version of the software and with performance that exceeds that achievable by the CPU version of AMBER software running on all conventional CPU-based clusters and supercomputers. We briefly discuss three different precision models developed specifically for this work (SPDP, SPFP, and DPDP) and highlight the technical details of the approach as it extends beyond previously reported work [Götz et al., J. Chem. Theory Comput. 2012, DOI: 10.1021/ct200909j; Le Grand et al., Comp. Phys. Comm. 2013, DOI: 10.1016/j.cpc.2012.09.022].We highlight the substantial improvements in performance that are seen over traditional CPU-only machines and provide validation of our implementation and precision models. We also provide evidence supporting our decision to deprecate the previously described fully single precision (SPSP) model from the latest release of the AMBER software package.
Our recent description of pulse sequences for the intramolecular relay of 1H polarization to the 19F reporter of a carbohydrate ligand after saturation transfer from a cognate lectin prompted us to test the applicability of this technique for inhibitor screening. By strategically combining synthetic organic chemistry and cell assays with 19F-NMR-based competition experiments, we document the validity of this approach. Two mannose-specific leguminous agglutinins as receptors, the α-methyl derivative of 2-deoxy-2-fluoro-D-mannopyranoside as sensor and synthetic mannosides selected to represent different inhibitory capacities were used to establish a test panel. Signal amplitudes were found to vary among the two related lectins, and their precipitation by glycodendrimers enabled inherent limits to be set. The obtained experimental basis was then broadened by assaying a galactopyranoside-binding plant toxin, using 6-deoxy-6-fluoro-D-galactopyranose as a sensor molecule. The easy identification of the two anomeric signals by 19F NMR spectroscopy enabled ready detection of the preference of this lectin for the α-anomer and allowed the two individual inhibition profiles to be deduced. These 19F NMR spectroscopic data were in accord with the activities of inhibitors used to protect cells from toxicity. Our results therefore provide an experimental basis for 19F-NMR-based inhibitor screening.
The use of thioglycosides and other glycan derivatives with anomeric sulfur linkages is gaining increasing interest, both in synthesis and in various biological contexts. Herein, we demonstrate the occurrence and circumvention of anomerization during 1-S-glycosylation reactions, and present highly efficient and stereocontrolled syntheses of a series of photoprobe-thiosaccharide conjugates. Mutarotation of glycosyl thiols proved to be the origin of the anomeric mixtures formed, and kinetic effects could be used to circumvent anomerization. The synthesized carbohydrate conjugates were then evaluated by both solution- and solid-phase-based techniques. Both binding results showed that the S-linked glycosides interact with their cognate lectins comparably to the corresponding O-analogs in the present cases, thus demonstrating the reliability of the solid-support platform built upon our photo-initiated carbohydrate immobilization method for probing protein bindings, and showing the potential of combining these two means for studying carbohydrate-protein inter-actions.
Reversible covalent bond formation under thermodynamic control adds reactivity to self-assembled supramolecular systems, and is therefore an ideal tool to assess complexity of chemical and biological systems. Dynamic combinatorial/covalent chemistry (DCC) has been used to read structural information by selectively assembling receptors with the optimum molecular fit around a given template from a mixture of reversibly reacting building blocks. This technique allows access to efficient sensing devices and the generation of new biomolecules, such as small molecule receptor binders for drug discovery, but also larger biomimetic polymers and macromolecules with particular three-dimensional structural architectures. Adding a kinetic factor to a thermodynamically controlled equilibrium results in dynamic resolution and in self-sorting and self-replicating systems, all of which are of major importance in biological systems. Furthermore, the temporary modification of bioactive compounds by reversible combinatorial/covalent derivatisation allows control of their release and facilitates their transport across amphiphilic self-assembled systems such as artificial membranes or cell walls. The goal of this review is to give a conceptual overview of how the impact of DCC on supramolecular assemblies at different levels can allow us to understand, predict and modulate the complexity of biological systems.
The chemical behavior of S-glycopyranosyl-N-monoalkyl dithiocarbamates (DTCs) as masked 1-glycosyl thiols, easily prepared by the nucleophilic displacement of 1-halo sugars with dithiocarbamate salts of primary amines, has been studied and synthetically exploited. This behavior relies on the abstraction of the proton of the carbamate functionality that allows controlled access to thiolate sugar intermediates. The basic character of the DTC salts used as reagents leads to thiolates that evolve in situ to symmetrical diglycosyldisulfides (DGDSs) when long reaction times are allowed. Alternatively, controlled unmasking of the thiolate function can be efficiently attained by treatment with an external base of isolated anomeric glycosyl DTCs, the formation of which is prevalent when using short reaction times. In this manner, a second methodology for the preparation of symmetrical DGDSs and a chemical protocol for the S-glycosylation of any electrophilic substrate are established. The applications of this last strategy for the preparation of thioglycosyl vinyl sulfones, thiodisaccharides, and S-linked homo- and heterodivalent neoglycoconjugates are described as a proof-of-concept of the great potential of the sugar DTCs in any chemical scenario in which the covalent attachment of a thiol sugar is required. The evaluation of the biological functionality of some divalent sulfurated sugar systems is also described.
Fuzzy logic systems can be exploited for defining the degrees of true or false binding between calcium mediated multivalent lactose and peanut agglutinin lectin, which are difficult to define with Boolean logic.
As a novel branch of combinational chemistry, dynamic combinatorial chemistry (DCC) can be viewed as a technique which combines library synthesis and screening in one pot. By addition of molecular target, ligangds, which show binding affinity or strong interaction with the molecular target, can be amplified an young but rapidly growing branch of combinatorial chemistry, has been widely used in organic chemistry, biochemistry, material fields. Ligands in the library can be amplified, since synthesis of the library is screened by a molecular target. Therefore, these structures could be identified easily. Consequently DCC has been widely used in the lead discovery, material chemistry and other fields. On the basis of the principle and method of DCC, this review emphasizes the three factors of DCC, including molecular targets (bio-enzyme, lectin, nucleic acid, organic molecule, inorganic molecule); reaction (disulphide chemistry, ammoniation reduction reaction, hydrazone chemistry, etc.) and analytical method. Meanwhile, limitation, current situation and future development of DCC were also discussed in this paper.
A high-yielding, one-pot methodology for preparing unsymmetrical glycosyl disulfides derived from sugar, alkyl/aryl or cysteine thiols is reported using 1-chlorobenzotriazole (BtCl) as the oxidant. The highlight of the method is the low temperature of coupling (−78°C) as well as the in situ trapping of the sulfenyl intermediate, which ensures that no homodimer of R1SH (R1SSR1) is formed. The coupling efficiency is independent of sugar type, thiol position in the sugar, sugar-protecting groups, and the various products serve to illustrate the rapid synthetic access to a number of model systems in glycobiology.
Methodology for the efficient and facile synthesis of glycosyl disulfides is reported. A one-pot procedure employing mild conditions using diethyl azodicarboxylate is described to synthesise a series of glycosyl disulfides in excellent yields.
A protocol based on saturation transfer difference (STD)-NMR spectroscopy competition experiments is presented as a fast compound-screening method (30min acquisition time per compound) capable of evaluating ligand properties for the lectin site with the intention of selecting compounds for iterative refinement. Glycosides with variations in the aglycon and plant toxin viscumin (VAA) were studied systematically. The best hits were further analyzed via docking simulations to rationalize an increased affinity structurally. The strategy described can readily be extended to other medically relevant lectins.
Molecular recognition: Dynamic combinatorial libraries (DCLs) exploiting multivalency effects and metal coordination have been employed for carbohydrate-protein recognition. The interaction of a three-component DCL based on 2,2'-bipyridine (bipy)-Fe(II) complexes with concanavalin A (ConA; see scheme) results in enhanced binding by multivalent presentation with a bias towards mannose-containing library components.
The ring-opening reaction of sugar 3,4-epoxides by 2,3,4,6-tetra-O-acetyl-1-thio-β-d-galactopyranose () as a nucleophile led to (1 → 3)- and (1 → 4)-thiodisaccharides. High regio- and diastereoselectivities were achieved in the synthesis of the per-O-acetyl derivative of the β-d-Galp-S-(1 → 4)-4-thio-α-d-Glcp-O-iPr (). Analogues of the 4-thiolactoside have been prepared, with the β-d-Galp non-reducing end S-linked to d-Glcp, d-Gulp and d-Idop. A similar regioselective attack of on C-4 of 2-propyl 3,6-di-O-acetyl-3,4-epithio-α-d-galactopyranoside () led to 2-propyl 3,4-dithiolactoside derivative . During this reaction the free 3-SH group of underwent oxidative dimerization or oxidative coupling with the SH function of to give the respective disulfides. Glycosylation of the thiol group of using trichloroacetimidate derivatives of β-d-Galp or β-d-Galf afforded the corresponding branched dithiotrisaccharides. The free compounds were evaluated as inhibitors of the E. coli β-galactoside. The bis(2-propyl 3,4-dithiolactosid-3-yl)-disulfide, obtained from , displayed the strongest inhibitory activity in these series of glycomimetics and proved to be a non-competitive inhibitor (K(i) = 95 μM).
Galectin-3 is expressed and secreted by immune cells and has been implicated in multiple aspects of the inflammatory response. It is a glycan binding protein which can exert its functions within cells or exogenously by binding cell surface ligands, acting as a molecular bridge or activating signalling pathways. In addition, this lectin has been shown to bind to microorganisms. In this study we investigated the interaction between galectin-3 and Neisseria meningitidis, an important extracellular human pathogen, which is a leading cause of septicaemia and meningitis. Immunohistochemical analysis indicated that galectin-3 is expressed during meningococcal disease and colocalizes with bacterial colonies in infected tissues from patients. We show that galectin-3 binds to N. meningitidis and we demonstrate that this interaction requiresfull-length, intact lipopolysaccharide molecules. We found that neither exogenous nor endogenous galectin-3 contributes to phagocytosis of N. meningitidis; instead exogenous galectin-3 increases adhesion to monocytes and macrophages but not epithelial cells. Finally we used galectin-3 deficient (Gal-3(-/-) ) mice to evaluate the contribution of galectin-3 to meningococcal bacteraemia. We found that Gal-3(-/-) mice had significantly lower levels of bacteraemia compared with wild-type mice after challenge with live bacteria, indicating that galectin-3 confers an advantage to N. meningitidis during systemic infection.
Ligand DiscoveryDCC Strategies in Targeting Biological SystemsDynamic Diversity Generation for Biological SystemsApplications of DCC in Biological SystemsConclusions and Future ProspectsAcknowledgmentsReferences
Ras biological activity necessitates membrane anchorage that depends on the Ras farnesyl moiety and is strengthened by Ras/galectin-1 interactions. We identified a hydrophobic pocket in galectin-1, analogous to the Cdc42 geranylgeranyl-binding cavity in RhoGDI, possessing homologous isoprenoid-binding residues, including the critical L11, whose RhoGDI L77 homologue changes dramatically on Cdc42 binding. By substituting L11A, we obtained a dominant interfering galectin-1 that possessed normal carbohydrate-binding capacity but inhibited H-Ras GTP-loading and extracellular signal-regulated kinase activation, dislodged H-Ras(G12V) from the cell membrane, and attenuated H-Ras(G12V) fibroblast transformation and PC12-cell neurite outgrowth. Thus, independently of carbohydrate binding, galectin-1 cooperates with Ras, whereas galectin-1(L11A) inhibits it.
A soluble lectin which agglutinates trypsin-treated rabbit erythrocytes was purified from calf heart using affinity chromatography on asialofetuin-Sepharose. Its molecular weight was determined by gel filtration to be approximately 17,000. On polyacrylamide gel electrophoresis in sodium dodecyl sulfate, the predominant molecular species had a molecular weight of 9,000, suggesting that the lectin is a dimer. Binding studies performed with iodinated lectin revealed that neuraminidase-treated calf erythrocytes contained approximately 5 X 10(6) lectin binding sites per cell. Native calf and rabbit erythrocytes bound the lectin, but human and rat erythrocytes required neuraminidase and trypsin treatment, respectively, for lectin binding to occur. A number of saccharides, glycopeptides, and glycoproteins possess haptene inhibitory activity toward lectin binding to erythrocytes. The most potent of these have either galactose beta leads to galactose beta leads to, galactose beta N-acetylglucosamine beta leads to, or galactose beta leads to N-acetylglucosamine beta leads to sequences at their nonreducing termini. Lactose and galactose beta 1 leads to 3N-acetylgalactosamine are the next best haptenes. Finally, alpha-linked galactose residues and free galactose are very weak haptenes. The presences of a terminal sialic acid residue impairs haptene activity in all instances. Calf heart also contains a membrane-associated lectin which is very similar but not identical with the soluble lectin. A soluble beta-galactoside binding lectin was also isolated from calf lung. It has the same molecular size and subunit structure as the soluble heart lectin and is antigenically identical. In binding studies, the pattern of inhibition by various haptenes was the same for all three lectins.
Carbohydrate binding specificity of the galactose-specific, major lectin of mistletoe extract (ML-1) was studied by an inhibition assay using monosaccharides, monosaccharide derivatives, disaccharides, and compounds containing multiple galactosyl terminals. The results indicate that 1) both alpha- and beta-galactosyl residues are recognized equally well; 2) each of the hydroxyl groups of galactose contributes to varying degrees to the binding process, the 4-OH being the most important and the 6-OH the least important hydroxyl group; 3) disaccharide sequences of Gal beta 2Gal and Gal beta 3Gal have much higher affinity than galactose, whereas affinity of all other Gal-disaccharides is only slightly better than galactose; 4) macromolecular ligands having 10 or more terminal galactosyl residues have 500-fold higher affinity than Gal; and 5) a group on ML-1 with pK alpha of 4.8 appears to be involved in the binding of ligand.
Binding characteristics of human spleen soluble galactoside-binding protein (galaptin) were studied using simple galactosides, galactose-terminated disaccharides, cluster glycosides containing up to 6 terminal lactosyl residues, bovine serum albumin derivatives containing 7 to 40 lactosyl residues, desialylated serum glycoproteins, and glycopeptides derived thereof as inhibitors in a newly developed binding assay. In this assay, aminohexyl lactoside was attached to divinyl sulfone-activated Sepharose, which was then used to bind 125I-galaptin. Similarly derivatized Sepharose containing mannoside served as a control. The assay is sensitive, maintains linearity in the concentration range of 125I-galaptin tested, and has very low nonspecific binding. The following new findings were made. 1) All the alpha-D-galactopyranosides with non-sugar aglycon were better inhibitors than the corresponding beta-D-galactopyranoside. 2) The S-galactosides were better inhibitors than the corresponding O-galactosides, regardless of the anomeric configuration. 3) Many Gal beta 1-4- and Gal beta 1-3-linked disaccharides were tested. Although the galaptin did not appear to recognize N-acetylglucosamine as a monosaccharide, the presence of this sugar penultimate to galactose increased the binding affinity by as much as 500-fold, as was the case for N-acetyllactosamine. Of a particular importance is the presence of an equatorial 3-OH group on this sugar. We synthesized the 3-deoxy derivative of N-acetyllactosamine and found that it had 50-fold lower binding affinity compared to N-acetyllactosamine. 4) The binding sites of this lectin do not seem to be operating in a cooperative fashion, since synthetic lactose-containing divalent ligands with various inter-galactose distances did not increase the binding affinity significantly.
The parameters that affect the interaction of ligands with a fucose-binding lectin from rat liver have been examined. 125I-Fucosyl-bovine serum albumin (Fuc-BSA) containing 50 residues of fucose/molecule was used as the standard ligand. At low initial concentrations of ligand (10 ng/ml) and lectin (140 ng/ml), the reaction reaches equilibrium at pH 7.8, 23 degrees C, within 40 min. The binding of ligands is Ca2+ dependent with half-maximal binding occurring at 54 microM Ca2+; of several metal ions tested, only Sr2+ partially replaced Ca2+. Binding was maximal between pH 7.6 and 8.6, fell slightly up to pH 10, but fell markedly below pH 7. The lectin-ligand complexes dissociated at low pH, on removal of Ca2+, or in the presence of a large excess of competing ligand. The apparent association constant (Ka) for Fuc-BSA was 1.75 X 10(8) M-1. The fucose content of the Fuc-BSA also influenced binding, with little apparent binding below 24 fucose residues/molecule and maximal binding from 40 to 50 fucose residues/molecule. With knowledge of the parameters influencing binding, sensitive reproducible assays for the lectin were developed. The binding specificity of the lectin was examined by measuring the inhibition of 125I-Fuc-BSA binding by neoglycoproteins, monosaccharides, and glycosides or by direct binding of neoglycoproteins. Galactosides and beta-linked fucosides were the best ligands among the neoglycoproteins, with much weaker binding by mannosyl- or N-acetylglucosaminyl-BSA. On the basis of the pattern of inhibition of Fuc-BSA binding by various monosaccharides and glycosides, it is possible to propose the conformations of saccharides that best fit the lectin-binding site. The C1 conformation of N-acetyl-D-galactosamine fits best, although other not obviously related monosaccharides such as L-fucose, L-arabinose, and D-mannose can also assume conformations that permit them to be effective inhibitors. The pattern of binding of neoglycoproteins to the lectin differs from that of other pure hepatic lectins. Thus, the fucose lectin has a high affinity for Fuc-BSA and galactosyl-BSA but a low affinity for N-acetylglucosaminyl-BSA. The galactose lectin binds only galactosyl-BSA and shows little binding with either N-acetylglucosaminyl-BSA or Fuc-BSA. In contrast, the mannose/N-acetylglucosamine lectin binds N-acetylglucosaminyl-BSA and Fuc-BSA but not galactosyl-BSA.
Binding of a series of mammalian glycoconjugates to three soluble rat lung lectins was determined with a quantitative assay. The three lectins, RL-14.5, RL-18, and RL-29, had a similar apparent affinity for lactose and associated with the same critical determinants, which included positions 4 and 6 of Gal and part of Glc. Derivatization at position 3 of Glc in lactose markedly reduced reactivity with the three lectins. For RL-14.5 and RL-29 the determinant extended specifically to the 3-hydroxyl of Glc which must be equatorial. In contrast, the stereochemical requirements for RL-18 were less specific, and Gal beta 1-3GalNAc bound as well as lactose. For RL-29 activity was markedly enhanced by GalNAc alpha 1-3 substitution on Gal, a modification which had little effect with RL-18 and inhibited binding to RL-14.5. Combinations of these residues in larger oligosaccharides and glycopeptides did not substantially enhance binding above that which might be expected from the sum of the constituent beta-galactoside residues. Although these lectins showed overlapping specificities, their binding properties are sufficiently different to suggest selective interactions with naturally occurring mammalian glycoconjugates.
The plasma membrane establishes the interface for the communication of cells with the environment. Thus, surface determinants govern the reactivity and capacity of cells to respond to external signals. Changes in their profile, for example in malignant transformation, and manifestation of cell-type-specific features apparently hold inspiring lessons in store for us on how they are translated into cellular responses. But before turning to the signaling routes the biochemical modes for coding signals warrant a comment, as proteins are often unduly portrayed as the decisive hardware. In contrast, and actually prominent among the biochemical systems to store information, carbohydrate epitopes of cellular glycoconjugates favorably combine high-density coding with strategic positioning, rendering them readily accessible for interactions with adaptor molecules. The interaction with lectins is the ignition key to start glycoconjugate-mediated biosignaling. Several plant lectins, especially due to their mitogenicity, have become a popular type of laboratory tool to elicit cell responses and to analyze biochemical pathways leading from initial binding to measured activity such as enhanced proliferation. With emerging insights into the roles of mammalian (endogenous) lectins and the promising perspective for medical applications, emphasis in this area is shifting from model studies with plant proteins toward work with the physiological effectors. By targeting branchend epitopes of glycan chains two classes of endogenous lectins, i.e. galectins and selectins, are remarkably well suited to establish initial contacts with the cell surface. Indeed, these lectins - in their interplay with certain cognate binding partners - are being defined as potent signal inducers. Consequently, we can take aspects of their activity profiles as incentive to dissect underlying routes of signal transmission with an eye more on principles than on intricate case-specific details. Hence, regulation of cell growth by cascades of mitogen-activated protein kinases (MAPKs), cyclins/cyclin-dependent kinases and inhibitors thereof, of cell survival by the phosphatidylinositol 3′-OH kinase (PI3K)/Akt pathway, the remodeling of the cytoskeleton by integrin-mediated cell adhesion, the implication of p53 in regulating cell fate and details of programmed cell death by the intrinsic and extrinsic routes for induction of apoptosis will be discussed. Moreover, we will look at selectin-induced signaling during leukocyte homing. Explicitly, it is the aim of our review to familiarize glycoscientists, whose main interest is to scrutinize the structural aspects or to develop applications, with basic concepts of cellular signaling triggered by these interactions.
La reaction du melange thioacetate de potassium-acide thioacetique avec l'azido-2 benzylidene-4,6 desoxy-2 O-methyl-1 O-trifluoromesyl-3 gulopyranoside donne l'acetamido-2 O-benzylidene-4,6 didesoxy-2,3 O-methyl-1 hexeno-2 pyranoside; avec le chlorure d'azido-2 desoxy-2 tri-O-acetyl-3,4,6 galactopyranosyle on obtient l'acetamido-2 S-acetyl-1 desoxy-2 tri-O-acetyl thio-1 galactopyranose
Owing to the expression of more than one type of galectin in animal tissues, the delineation of the functions of individual members of this lectin family requires the precise definition of their carbohydrate specificities. Thus, the binding properties of chicken liver galectin (CG-16) to glycoproteins (gps) and Streptococcus pneumoniae type 14 polysaccharide were studied by the biotin/avidin-mediated micro titre-plate lectin-binding assay and by the inhibition of lectin-glycan interactions with sugar ligands. Among 33 glycans tested for lectin binding, CG-16 reacted best with human blood group ABO (H) precursor gps and their equivalent gps, which contain a high density of D-galactopyranose(beta1-4)2-acetamido-2-deoxy-D-glucopyranose [Gal(beta1-4)GlcNAc] and Gal(beta1-3)GlcNAc residues at the nonreducing end, but this lectin reacted weakly or not at all with A-, H-type and sialylated gps. Among the oligosaccharides tested by the inhibition assay, the tri-antennary Gal(beta1-4)GlcNAc (Tri-II) was the best. It was 2.1 x 10(3) nM and 3.0 times more potent than Gal and Gal(beta1-4)GlcNAc (II)/Gal(beta1-3) GlcNAc(beta1-3)Gal(beta1-4)Glc (lacto-N-tetraose) respectively. CG-16 has a preference for the beta -anomer of Gal at the nonreducing end of oligosaccharides with a Gal(beta1-4) linkage > Gal(beta1-3) greater than or equal to Gal(beta1-6). From the results, it can be concluded that the combining site of this agglutinin should be a cavity type, and that a hydrophobic interaction in the vicinity of the binding site for sugar accommodation increases the affinity. The binding site of CG-16 is as large as a tetrasaccharide of the beta -anomer of Gal, and is most complementary to lacto-N-tetraose and Gal(beta1-4)GlcNAc related sequences.
A novel metal-assisted assembly of multivalent carbohydrate ligands is described. A bipyridine-modified N-acetylgalactosamine (bipy-GalNAc) undergoes Fe(II)-induced self-association to form a trimeric GalNAc ligand (Fe11(bipy-GalNAc)3). The synthetic GalNAc cluster strongly binds to Vicia villosa B4 lectin and Glycin Max lectin, which recognizes multiple GalNAc residues. The trimeric GalNAc ligand is formed as a mixture of four diastereomeric isomers: Δ-fac, Λ-fac, Δ-mer, and Λ-mer. These stereoisomers are in a dynamic equilibrium at room temperature. The equilibrium allows the spatial arrangement of the three GalNAc residues to change in order to fit into a multivalent carbohydrate binding site of the lectins. Detailed analysis of the kinetic and thermodynamic data for the isomerization can provide structural information of the carbohydrate binding site of the lectins.
Mixed glycosyl disulfides are not only glycomimetics but also
glycosyl donors that may be readily constructed in either armed
ether-protected or disarmed ester-protected and in soluble or
solid-supported forms from corresponding glycosyl methanethiosulfonates and
used in the glycosylation of a variety of representative acceptors.
An asymmetric disulfide linkage, formed by conjugation of a 5-nitropyridine-2-sulfenyl activated thioglycoside and a protein or pre-assembled peptide sequence, represents a good structural mimic of natural asparagine glycosylation.
Neoglycoproteins were prepared with chemoenzymatically synthesized complex biantennary N-glycan derivatives the nonreducing ends of which bear typical sequences found in glycoproteins. A chemically obtained biantennary heptasaccharide-azide was reduced and acylated with a 6-aminohexanoyl spacer. Elongation of the deprotected heptasaccharide using glycosyltransferases yielded a biantennary nonasaccharide with terminal galactose residues and two undecasaccharides terminating with alpha 2,6- or alpha 2,3-linked sialic acid. The free amino group of the spacer of these oligosaccharides was converted into an isothiocyanate. Its subsequent coupling to bovine serum albumin gave neoglycoproteins with a yield of 2.4-3.6 glycan chains per carrier molecule. This versatile synthetic pathway allows employment of a wide variety of complex-type glycans, which can be introduced to various test systems in vitro and in vivo to evaluate potential biomedical applications. Solid-phase assays with biotinylated sugar receptors revealed discriminatory binding properties of the three neoglycoproteins, especially for the mistletoe lectin. This direct assay system is preferable to the measurement of inhibitory capacities with respect to model ligands. Ligand type- and cell type-dependent quantitative differences in the binding properties of the probes were detected by FACScan analyses with a panel of tumor cell lines and by monitoring of staining in tissue sections for small cell and non-small-cell lung cancer and mesotheliomas. Biodistribution of iodinated neoglycoproteins in mice gave a prolonged presence of the sialylated probes in serum. Relative to the nonasaccharide, the uptake, especially of the iodinated neoglycoprotein with alpha 2,3-sialylated ligand chains, was clearly elevated in mice for kidneys and Ehrlich tumors. On the basis of the documented feasibility of these applications, it is concluded that the further elaboration of glycan chain variants by the described synthetic approach in combination with the given test panel is warranted to evaluate the potential of complex glycan chain-carrying neoglycoproteins for diagnostic and therapeutic purposes.
Dynamic molecular recognition of synthetic carbohydrate ligands by lectins is described. When bipyridine-modified GalNAc is reacted with Fe(II), four diastereomers of the trivalent GalNAc ligand are formed in dynamic equilibrium at room temperature. The equilibrium allows the three GalNAc residues to adjust their spatial orientation on the metal template to fit into the binding pocket of various GalNAc-specific lectins.
The scientific description of lectins, a class of sugar-binding proteins distinct from both sugar-specific enzymes and antibodies, began nearly 100 years ago. The natural ligands of lectins include the sugar moieties of cell glycoproteins, glycolipids, and proteoglycans. Lectins can selectively recognize these potential information-storing or -transferring structural elements and so contribute to regulating biological processes. Plant lectins are now a well-established means of characterizing and localizing glycoconjugate-bound sugar determinants. However, work on endogenous lectins is still confronted by many open questions, whose answers require detailed experiments using a suitable model system. Tumors offer an obvious model, particularly since they allow the possible diagnostic and therapeutic uses of lectins to be explored directly. Tumor lectinology employs a broad range of methods, from the chemical synthesis of the sugar components of neoglycoproteins, and their use to seek out cellular sugar receptors with histochemical and cell-biological techniques, to the biochemical and immunological characterization of the lectins expressed by tumor cells and the use of homology analysis to find further functional domains. The outlook for clinical applications depends on the harmonious integration of these disparate disciplines in tumor lectinology.
Several monomeric and dimeric Lewis glycolipids have been investigated by NMR spectroscopy, and structural aspects were modelled by computer. From the pseudo-C2-symmetric tetrasaccharide unit that forms the recognition domain of the Lewis Y and Lewis b antigens, a totally C2-symmetric tetrasaccharide was designed that contains the structural element common to all Lewis antigens. Finally, a model for the presentation of dimeric Lewis antigens at membrane surfaces was derived. The overall shapes of the dimeric Lewis oligosaccharides are defined by the connectivity of the sugar residues within rigid tri- and tetrasaccharide building blocks.
The conformational behaviour of N,N′-diacetyl-4-thiochitobiose
(1) has been studied using a combination
of NMR spectroscopy (NOE data) and molecular mechanics calculations. Analogies
and differences with the natural compound N,N′-diacetylchitobiose
(2) have been found. Moreover, the
study of its bound conformation to the lectin wheat germ agglutinin has also
been studied using TR-NOE experiments. A process of conformational selection
is observed and only one of the conformers present in aqueous solution for
the free state is bound by the lectin.
The two thio analogues (2 and 3) of TMSEt galabioside [2-(trimethylsilyl)ethyl 4-O-(α-D-galactopyranosyl)-β-D-galactopyranoside, 1], having anomeric sulfur instead of anomeric oxygen atoms, were synthesized and their conformations investigated by NMR and computational (MM 3) methods. A spacer galabioside was covalently coupled to aminated microtiter plates, and binding of a bacterial pilus adhesin (PapG) to the plates was inhibited by the soluble ligands 1, 2 and 3. The ligand 2, which has an intersaccharidic sulfur linkage, was a much less efficient inhibitor than 1, which has the natural oxygen linkage. The inhibitory power of ligand 3 was only slightly less than that of 1. An NMR experiment with 1 and 2, in which hydroxyl-group hydrogens had been partially (50%) substituted by deuterium, demonstrated the presence (in 1) and absence (in 2) of an intramolecular (HO 2′ – HO 6) hydrogen bond. This result indicates that the conformations of 1 and 2 are different and that the difference is sufficient to cause the observed (≈ 30 times) reduction of the saccharide-protein binding strength.
2-Amino C-glycerolipid 1b was synthesized by using the Ramberg−Bäcklund rearrangement as the key step. β-C-Glycerolipid 1b exhibits in vitro antiproliferative effects strikingly similar to those of O-glycoside analogue 1a.
Versatile synthetic procedures make saccharide derivatives with substitution at various sites available. Because of the particular involvement of galactosides in biorecognition processes, with lectins affecting growth regulation and adhesion, lactose has become a popular starting point for systematic structure variation. Since shape and flexibility are key parameters for ligand binding, we studied the conformational behavior of aza/carba-C/O analogues of lactose (compound 1, aza-C; compound 2, carba-O). Computational calculations and NMR spectroscopy revealed conspicuous differences between 1 and 2 in the extents of accessible conformational area and flexibility. The aza-C derivative also revealed clear shifts in population density relative to lactose, attributable to the exo-anomeric effect. The assumption that the derivatives can act as glycomimetics was substantiated by binding studies with a plant lectin. The aza-C analogue 1 maintained a higher degree of flexibility when bound than the O-glycosidically linked carbadisaccharide 2. The experimental detection of exclusive NOEs characteristic of different conformers of bound 1 [i.e., H1′/H4 (syn-Φ), H1′/H3 (anti-Ψ), and H2′/H4 (anti-Φ) NOE cross-peaks] argues in favor of the ligand still harboring a certain degree of flexibility when bound to mistletoe lectin (VAA). In contrast, there is unambiguous evidence for binding of the major exo-anomeric conformer of 2 by VAA, as indicated by the presence of the exclusive NOE of H7eq′/H4. Evidently, structural variations between closely related compounds can translate into clear differences in the lectin-bound states of glycomimetics. (© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2004)
Oligosaccharides, complex carbohydrates that comprise 2–30 monosaccharides linked by glycosidic bonds, are being developed as therapeutic agents for a variety of indications. The large-scale production of oligosaccharides achieved by enzymatic synthesis has encouraged the preclinical and clinical evaluation of these carbohydrates for efficacy in infectious and inflammatory diseases, transplantation, and other conditions. Many of the promising oligosaccharide drugs in development interfere with adhesion events between different cell types, and between antibodies and cells. In addition, complex carbohydrate-based agents are being developed for use in metabolic and cardiovascular diseases, as cancer vaccines, and in drug delivery. Also, diverse efforts to construct combinatorial libraries of complex carbohydrates are opening the door to broader screening approaches.
Asymmetric non-reducing disaccharides containing an interglycosidic disulfide linkage were synthesised under mild conditions through reaction of tetraacetyl-β-d-glucopyranosyl methanethiolsulfonate with O-acetylated 1-thio-aldopyranoses. The preferred conformation around the SS bond is close to that observed in unconstrained disulfides (−90°).
Combinatorial chemistry has contributed significantly to understanding the structure–function relationships of biologically important molecules such as proteins and nucleic acids. However, carbohydrates and carbohydrate conjugates, which have been identified as key modulators of several biological functions have not enjoyed the same measure of success. The complexity and synthetic challenges of carbohydrate conjugates have resulted in a number of conceptual approaches to rapidly access sufficient quantities of these biomolecules. This article summarizes these combinatorial approaches and also highlights fully automated library synthesis of artificial glycopeptides with the goals of understanding their biological roles.
Galectin-1, S-type β-galactosyl-binding lectins present in vertebrate and invertebrate species, are dimeric proteins that participate in cellular adhesion, activation, growth regulation, and apoptosis. Two high-resolution crystal structures of B. arenarum galectin-1 in complex with two related carbohydrates, LacNAc and TDG, show that the topologically equivalent hydroxyl groups in the two disaccharides exhibit identical patterns of interaction with the protein. Groups that are not equivalent between the two sugars present in the second moiety of the disaccharide, interact differently with the protein, but use the same number and quality of interactions.The structures show additional protein-carbohydrate interactions not present in previously reported lectin-lactose complexes. These contacts provide an explanation for the enhanced affinity of galectin-1 for TDG and LacNAc relative to lactose. Galectins are in dimer-monomer equilibrium at physiological protein concentrations, suggesting that this equilibrium may be involved in organ-specific regulation of activity. Comparison of B. arenarum with other galectin-1 structures shows that among different galectins there are significant changes in accessible surface area buried upon dimer formation, providing a rationale for the variations observed in the free-energies of dimerization.The structure of the B. arenarum galectin-1 has a large cleft with a strong negative potential that connects the two binding sites at the surface of the protein. Such a striking characteristic suggests that this cleft is probably involved in interactions of the galectin with other intra or extra-cellular proteins. Proteins 2000;40:378–388. © 2000 Wiley-Liss, Inc.
Glycoconjugates play important roles in biological reactions (for example sialyl Lewisx in homing of leukocytes and mannose-6-phosphate in targeting of lysosomal enzymes) and thus aberration in carbohydrate structures in glyco-conjugates can lead to abnormal biological behaviors. In fact, glycoconjugates expressed on the surfaces of tumor and cancer cells are considerably different from those of the normal cells, at least quantitatively. There are many known carbohydrate-deficient glycoprotein syndromes. As recognition of carbohydrate groups is mostly performed by carbohydrate-binding proteins, aberration in these proteins also results in disease status (for example I-cell disease). Many pathogens use carbohydrates as recognition markers for invasion (examples are influenza virus and cholera toxin). The carbohydrate receptors in various organs can be used for targeting drugs, antibodies and even DNAs. Conjugation of polysaccharides derived from pathogenic micro-organisms with appropriate proteins provides effective vaccines against the micro-organisms.
Protein (lectin)-carbohydrate (cellular glycoconjugate) recognition is operative in biochemical information transfer. Galectins
constitute a family of endogenous galactoside-binding lectins with conserved features in the binding site. The members of
this lectin category are assumed to be involved in cell adhesion and growth regulation. To assess to what extent the different
modes of binding-site presentation and/or carbohydrate fine-specificities will affect aspects of galectin behavior, homodimeric
cross-linking galectin-1 and monomeric chimeric galectin-3, with its collagenase-sensitive stalk linked to the carbohydrate-recognition
domain, were investigated. Cell-surface expression of the two galectins and accessible galectin-binding sites on various tumor
cell lines was ascertained by FACScan analysis. In particular, ligand accessibility for the two galectins differed for the
tested cell line types. Binding of tumor cells to laminin and plasma or placental fibronectin was generally reduced by treatment
of cells or matrix with galectins. Galectin-3 was more efficient than galectin 1 at impairing laminin's potency as matrix.
Cell binding of galectin-1, on the other hand, proved on average more effective for blocking cell association to fibronectins
after its preincubation with cell suspensions. Differences were also apparent in the biodistribution of the galectins, where
an avian homolog of galectin-1 served as the control to distinguish effects of spatial and sugar-binding features. Histopathological
analysis of lymph-node-negative and -positive breast and colorectal carcinomas (n = 180 including 60 metastatic lesions) indicated a correlation of either increased galectin-1 binding and reduced galectin-3
expression or reduced binding of both galectins with the occurrence of lymph node lesions. Together with data on the heparin-binding
lectin, revealing reduced expression to be associated with a positive lymph-node status in the breast cancer group, these
results can be interpreted to reflect cell-type-dependent requirements of galectin ligand presentation during the metastatic
cascade. By introducing mammalian lectins to lectin-histochemical studies, the detection of quantitative differences in glycosylation
brings an understanding of its cell biological significance one step closer.
To study conformational parameters of ligands before and after complex formation with the galactoside-binding agglutinin of Viscum album L. (VAA) in solution, combined computer-assisted random walk molecular mechanics (RAMM) calculations extended by conformational clustering analysis (CCA), molecular dynamics (MD) simulations as well as two-dimensional rotating-frame nuclear Overhauser effect (ROE) and two-dimensional nuclear Overhauser effect (NOE) spectroscopy NMR experiments were employed. Derivatives of the naturally occurring disaccharides Galbeta1-3GlcNAcbeta1-R and Galbeta1-3GalNAcbeta1-R as well as of a synthetic high-affinity binding partner, i.e. the disaccharide Galbeta1-2Galbeta1-R', were chosen as ligands in this study. The disaccharides displayed inherent flexibility in the valley of the global minimum between phi/psi combinations of (40 degrees/60 degrees) and (40 degrees/-60 degrees). Calculations of the de-N-acetylated sugars revealed that presence of this group did not markedly influence the distribution of low-energy conformers in the phi, psi, epsilon plot. Occupation of side minima at phi/psi (180 degrees/0 degrees) or (0 degrees/180 degrees) is either unlikely or low according to the results of MD simulations and RAMM calculations extended by CCA. Notably, these side minima define conformations which are not stable during a MD simulation. Transitions to other minima occur already a few picoseconds after the start of the simulation. NMR experiments of the free-state ligand confirmed the validity of the data sets obtained by the calculations. Following the description of the conformational space in the free-state NMR experiments were performed for these disaccharides complexed with VAA. They yielded two interresidual contacts for Galbeta1-3GlcNAcbeta1-R and Galbeta1-2Galbeta1-R'. The ligand conformations in the complex did not deviate markedly from those of a minimum conformation in the free state. One- and two-dimensional transferred nuclear Overhauser enhancement (TRNOE) experiments at different mixing times excluded the influence of spin-diffusion effects. When the NOE build-up curves in the three studied cases were compared, the residual mobility of the penultimate carbohydrate unit of Galbeta1-3GalNAcbeta1-R was observed to be higher than that of the respective hexopyranose unit of the other two bound ligands. Due to the availability of the conformational parameters of Galbeta1-2Galbeta1-R' in association with a galectin, namely the beta-galactoside-binding protein from chicken liver, it is remarkable to note that this ligand displays different conformations in the binding sites of either the plant or the animal lectin. They correspond to local energy-minimum conformations in the phi,psi, epsilon plot and substantiate differential conformer selection by these two lectins with identical nominal monosaccharide specificity.
To study conformational parameters of ligands before and after complex formation with the galactoside-binding agglutinin of Viscum album L. (VAA) in solution, combined computer-assisted random walk molecular mechanics (RAMM) calculations extended by conformational clustering analysis (CCA), molecular dynamics (MD) simulations as well as two-dimensional rotating-frame nuclear Overhauser effect (ROE) and two-dimensional nuclear Overhauser effect (NOE) spectroscopy NMR experiments were employed. Derivatives of the naturally occurring disaccharides Gal beta 1-3GlcNAc beta 1-R and Gal beta 1-3GalNAc beta 1-R as well as of a synthetic high-affinity binding partner, i.e. the disaccharide Gal beta 1-2Gal beta 1-R', were chosen as ligands in this study. The disaccharides displayed inherent flexibility in the valley of the global minimum between Phi/Psi combinations of (40 degrees/60 degrees) and (40 degrees/-60 degrees). Calculations of the de-N-acetylated sugars revealed that presence of this group did not markedly influence the distribution of low-energy conformers in the Phi, Psi, E plot. Occupation of side minima at Phi Psi(180 degrees/0 degrees) or (0 degrees/180 degrees) is either unlikely or low according to the results of MD simulations and RAMM calculations extended by CCA. Notably, these side minima define conformations which are not stable during a MD simulation. Transitions to other minima occur already a few picoseconds after the start of the simulation. NMR experiments of the foe-state ligand confirmed the validity of the data sets obtained by the calculations. Following the description of the conformational space in the free-state NMR experiments were performed for these disaccharides complexed with VAA. They yielded two interresidual contacts for Gal beta 1-3GlcNAc beta 1-R and Gal beta 1-2Gal beta 1-R'. The ligand conformations in the complex did not deviate markedly from those of a minimum conformation in the foe state. One-and two-dimensional transferred nuclear Overhauser enhancement (TRNOE) experiments at different mixing times excluded the influence of spin-diffusion effects. When the NOE build-up curves in the three studied cases were compared, the residual mobility of the penultimate carbohydrate unit of Gal beta 1-3GalNAc beta 1-R was observed to be higher than that of the respective hexopyranose unit of the other two bound ligands. Due to the availability of the conformational parameters of Gal beta 1-2Gal beta 1-R' in association with a galectin, namely the beta-galactoside-binding protein from chicken liver, it is remarkable to note that this ligand displays different conformations in the binding sites of either the plant or the animal lectin. They correspond to local energy-minimum conformations in the Phi, Psi, E plot and substantiate differential conformer selection by these two lectins with identical nominal monosaccharide specificity.
Mediation of cellular interactions by protein (lectin)-carbohydrate recognition presupposes the expression of respective surface determinants. Due to the importance of cellular contacts between bone marrow stromal cells, recently shown to express cell surface lectins, and tumor or normal progenitor cells for biosignaling and marrow egress, quantitation of cell surface sugar receptor expression by a panel of chemically glycosylated enzymes (tetrameric E. coli beta-galactosidase) for human leukemia/lymphoma cells was initiated. Cells of the new B lymphoblastoid line Croco II that are partially positive for the CD15-specific epitope expressed receptors for various sugar specificities on their surface, fulfilling an indispensable prerequisite for establishment of glycobiological interactions. Binding studies with increasing neoglycoenzyme concentrations up to saturation in four cases disclosed values for apparent affinity constants in the range of 25-200 nM with 0.25-3 x 10(5) bound probes per cell. The presence of receptors for constituents of carbohydrate chains of cellular glycoconjugates was also ascertained biochemically, namely for beta-galactosides, alpha-mannosides, alpha-fucosides and N-acetylgalactosaminides. Expression of this property was modulated by changes in the culture conditions, as revealed by binding studies with cells, derived from growth in medium containing different serum concentrations. These findings indicate that cell surface sugar receptors of tumor cells warrant further attention with respect to recognitive interactions.
Molecular recognition can be mediated by protein (lectin)-carbohydrate interaction, explaining the interest in this topic. Plant lectins and, more recently, chemically glycosylated neoglycoproteins principally allow to map the occurrence of components of this putative recognition system. Labelled endogenous lectins and the lectin-binding ligands can add to the panel of glycohistochemical tools. They may be helpful to derive physiologically valid conclusions in this field for mammalian tissues. Consequently, experiments were prompted to employ the abundant beta-galactoside-specific lectin of human nerves in affinity chromatography and in histochemistry to purify and to localize its specific glycoprotein ligands. In comparison to the beta-galactoside-specific plant lectins from Ricinus communis and Erythrina cristagalli, notable similarities were especially detectable in the respective profiles of the mammalian and the Erythrina lectin. They appear to account for rather indistinguishable staining patterns in fixed tissue sections. Inhibitory controls within affinity chromatography, within solid-phase assays for each fraction of lectin-binding glycoproteins and within histochemistry as well as the demonstration of crossreactivity of the three fractions of lectin-binding glycoproteins with the biotinylated Erythrina lectin in blotting ascertained the specificity of the lectin-glycoprotein interaction. In addition to monitoring the accessible cellular ligand part by the endogenous lectin as probe, the comparison of immunohistochemical and glycohistochemical detection of the lectin in serial sections proved these methods for receptor analysis to be rather equally effective. The observation that the biotinylated lectin-binding glycoproteins are also appropriate ligands in glycohistochemical analysis warrants emphasis.(ABSTRACT TRUNCATED AT 250 WORDS)
Affinity chromatography provides a powerful tool for isolation of carbohydrate-binding proteins. However, the choice of the ligand and spacer has an important impact on effectiveness. The influence of several different ligands on qualitative and quantitative aspects of the purification of two beta-galactoside-specific lectins has been evaluated. Sepharose was modified by coupling four types of neoglycoproteins (galactosylated or lactosylated bovine serum albumin with increasing sugar content) and two naturally occurring asialoglycoproteins at similar densities. Carbohydrate ligands at essentially equal density were made accessible to the lectins by seven commonly used methods. The yield of mistletoe lectin was high when lactosylated neoglycoproteins were used for separation. For these resins the sugar incorporation exceeded 10 sugar groups per protein carrier molecule. The yield was similarly high with the asialoglycoproteins and with lactose; the sugar was coupled to the resin as a p-aminophenyl derivative or by means of divinyl sulfone activation. An epoxy group in linkages of galactose or lactose decreased the binding capacity. A quantitatively similar degree of protein yields was obtained for the beta-galactoside-binding protein of bovine heart, although different proteins were obtained when neoglycoproteins were used as ligand. The nature of the affinity ligand in lectin purification can increase the yield and may also influence the profile of the carbohydrate-binding proteins.
Three entirely different tumor types were investigated biochemically for the presence and characteristics of endogenous carbohydrate-binding proteins in an inbred Brown Norway rat, an outbred Sprague-Dawley rat, and an outbred Han:NMRI mouse. The patterns under investigation included specificities for alpha- and beta-galactosyl, alpha-mannosyl, and alpha-fucosyl moieties, respectively, and specificities for heparin, analyzed by affinity chromatography on resins with immobilized sugars or glycoproteins and polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The patterns were divided into categories according to dependence of the binding activity on the presence of Ca2+ and dependence on extraction conditions. Rhabdomyosarcoma revealed only Ca2+-independent activities, i.e., activities with specificity for beta-galactosides at a molecular weight of 12,000, with specificity for alpha-galactosides at molecular weights of 29,000, 43,000, and 45,000, with specificity for heparin at molecular weights of 13,000 and 16,000, and with specificities for mannose and fucose at molecular weights ranging from 62,000 to 70,000. For the spontaneous mammary adenocarcinoma the pattern was entirely different and more diverse, including species with the Ca2+ requirement. Extracts with the use of 0.2 M NaCl (salt) and 2% Triton X-100 (detergent) from teratoma contained at least nine different carbohydrate-binding proteins. The only similarities between the pattern of endogenous carbohydrate-binding proteins from teratoma and from mammary adenocarcinoma were beta-galactoside-binding proteins, one with a Ca2+ requirement and one without a Ca2+ requirement, and the heparin-binding proteins. These heparin-binding proteins were the only types of carbohydrate-binding proteins common to all three tumor types. The analysis indicates that certain bands represented newly identified proteins capable of binding to galactose-, mannose- or fucose-containing glycoconjugates, respectively. When assayed with rabbit erythrocytes, the different fractions showed agglutination activity. They can thus be termed "endogenous lectins." The use of endogenous lectin patterns as potential diagnostic markers in addition to the corresponding changes in the glycoconjugate composition is proposed.
Soybean agglutinin (SBA) (Glycine max), which is a tetrameric GalNAc/Gal-specific lectin, has recently been reported to form unique, highly organized cross-linked complexes with a series of naturally occurring and synthetic multiantennary carbohydrates with terminal GalNAc or Gal residues [Gupta, D., Bhattacharyya, L., Fant, J., Macaluso, F., Sabesan, S., & Brewer, C. F. (1994) Biochemistry 33, 7495-7504]. In order to elucidate the nature of these complexes, the X-ray crystallographic structure of SBA cross-linked with a biantennary analog of the blood group I carbohydrate antigen is reported. The structure reveals that lattice formation is promoted uniquely by the bridging action of the bivalent pentasaccharide (beta-LacNAc)2Gal-beta-R, where R is -O(CH2)5COOCH3 and the beta-LacNAc moieties are linked to the 2 and 6 positions of the core Gal. The structure of SBA complexed with the synthetic biantennary pentasaccharide has thus been determined by molecular replacement techniques and refined at 2.6 A resolution to an R value of 20.1%. The crystals are hexagonal with a P6(4)22 space group, which differs significantly from that of crystals of the free protein. In the structure, each monomeric asymmetric unit contains a Man9 oligomannose-type chain at Asn 75, with only the first two GlcNAc residues visible. The overall tertiary structure of the SBA subunit is similar to that of other legume lectins as well as certain animal lectins. However, the dimer interface in the SBA tetramer is unusual in that only one complete peptide chain is sterically permitted, thus requiring juxtapositioning of one C-terminal fragmented subunit together with an intact subunit. Association between SBA tetramers involves binding of the terminal Gal residues of the pentasaccharide at identical sites in each monomer, with the sugar cross-linking to a symmetry-related neighbor molecule. The cross-linking pentasaccharide is in a conformation that possesses a pseudo-2-fold axis of symmetry which lies on a crystallographic 2-fold axis of symmetry of the lattice. Hence, the symmetry properties of the bivalent oligosaccharide as well as the lectin are structural determinants of the lattice. The results are discussed in terms of multidimensional carbohydrate-lectin cross-linked complexes, as well as the signal transduction properties of multivalent lectins.
The thermodynamics of the binding of derivatives of galactose and lactose to a 14 kDa beta-galactoside-binding lectin (L-14) from sheep spleen has been studied in 10 nM phosphate/150 mM NaCl/10 mM beta-mercaptoethanol buffer, pH 7.4, and in the temperature range 285-300 K using titration calorimetry. The single-site binding constants of various sugars for the lectin were in the following order: N-acetyl-lactosamine thiodigalactoside > 4-methylumbelliferyl lactoside > lactose > 4-methylumbelliferyl alpha-D-galactoside > methyl-alpha-galactose > methyl-beta-galactose. Reactions were essentially enthalpically driven with the binding enthalpies ranging from -53.8 kJ/mol for thiodigalactoside at 301 K to -2.2 kJ/mol for galactose at 300 K, indicating that hydrogen-bonding and van der Waals interactions provide the major stabilization for these reactions. However, the binding of 4-methylumbelliferyl-alpha-D-galactose displays relatively favourable entropic contributions, indicating the existence of a non-polar site adjacent to the galactose-binding subsite. From the increments in the enthalpies for the binding of lactose, N-acetyl-lactosamine and thiodigalactoside relative to methyl-beta-galactose, the contribution of glucose binding in the subsite adjacent to that for galactose shows that glucose makes a major contribution to the stability of L-14 disaccharide complexes. Observation of enthalpy-entropy compensation for the recognition of saccharides such as lactose by L-14 and the absence of it for monosaccharides such as galactose, together with the lack of appreciable changes in the heat capacity (delta Cp), indicate that reorganization of water plays an important role in these reactions.
Among the limitations to the practical therapeutic oligopeptide are low oral availability, indifferent aqueous solubility, and an astonishing efficient sequestration and biliary elimination by a multi-capacity liver transporter. Given the purposed use of N- and O- linked saccharides as functional appendages of eukaryotic peptides and proteins, a strategy of glycopeptide mimicry was examined for the oligopeptide renin inhibitor, ditekiren. The anticipation was that the saccharide would impart significant aqueous solubility, and might impact beneficially on the remaining two limitations. Execution of this approach was achieved by the removal of the (dimethylethoxy)carbonyl amino terminus of ditekiren, and its substitution by Boc-L-asparagine N-linked mono- and disaccharides. Potent hypotensive activity, as measured by a human renin-infused rat assay, is observed for virtually all of these structures (N-linked beta-pyranose D-N-acetyglucosaminyl, D-glucosaminyl, D-N-acetylgalactosaminyl, D-mannosyl, D-galactosyl, D-maltosyl, D-cellobiosyl, D-chitobiosyl, but not L-fucosyl). The basis for this dramatic improvement (relative to ditekiren in the same assay) is the diversion of the peptide clearance from rapid liver biliary clearance to slower urinary clearance (Fisher, J. F.; Harrison, A. W.; Wilkinson, K. F.; Rush, B. R.; Ruwart, M. J. J. Med. Chem. 1991, 34, 3140). Guided by the human renin-infused rat hypertension assay, an evaluation of the linker-saccharide pairing was made. Loss of hypotensive activity is observed upon substitution of the Boc-L-asn by Boc-D-asn, and by removal of the Boc amino terminus of the glycopeptide. Potent hypotensive activity is preserved by replacement of the Boc-L-asn linker by succinate, malate, tartrate, and adipate linkers. With the longer adipate spacer, attachment of the saccharide to the P-3 phenylalanine--with omission of the P-4 proline--retains activity. These data suggest value to the glycopeptide guise for preserving the in vivo activity, and for the beneficial manipulation of pharmacodynamics, of this renin inhibitory oligopeptide. This strategy may have general applicability.
The 1H and 13C NMR data at neutral pH are presented for methyl 4-thio-beta- and alpha-maltoside (1 and 2) together with methyl 1-thio-alpha-D-glucopyranoside (3) and methyl 4-thio-alpha-D-glucopyranoside (4) as reference compounds. Furthermore, the NMR data at high and low pH are presented for the 4-amino-4-deoxy analogues of methyl alpha-maltoside (5 and 6) and the 5-amino-5-deoxy analogue (8) together with reference compounds methyl 4-amino-4-deoxy-alpha-D-glucopyranoside (7) and 1-deoxynojirimycin (9). The experimental NMR data are assigned by 1- and 2-dimensional spectroscopy at 500 and 600 MHz. The conformational preferences of the maltose analogues 1, 2, 5, 6 and 8 are evaluated by difference NOE experiments, 13C-1H long-range coupling constants, chemical-shift comparison with model compounds and hard-sphere force field calculations for 1 using Monte Carlo simulations. Additionally, the results are compared with extensive experimental NOE data for methyl alpha- and beta-maltoside and the results discussed in light of earlier studies.
Viscumin (the major lectin of mistletoe extract), also known as ML-1, and ricin (RCA II) belong to a group of heterodimeric toxic lectins composed of an A chain, which inhibits protein synthesis, and a B chain, which mediates entry into the cell in a galactose-specific manner. Although most of the binding force for the association of viscumin with galactose-containing ligands is generated by the nonreducing terminal galactose residue, a particular hydroxyl group on the penultimate sugar also appears to participate in the binding, suggesting that viscumin has an extended combining site. In this paper, we give further examples of affinity enhancement by the hydroxyl group situated on the penultimate sugar next to the glycosidic linkage of the terminal galactose. The structure with highest affinity for viscumin thus far discovered is beta-D-Gal-(1-->2)-beta-D-Gal. In contrast to viscumin, ricin does not have this extended binding area, as none of the disaccharides tested exhibited significant affinity enhancement.
As a part of our studies of selectin blockers, we prepared 1-(2-tetradecylhexadecyl)-3'-O-sulfo Le(X) 1 and 1-(2-tetradecylhexadecyl) sLe(X) 2 and examined their inhibitory activities against natural ligand (sLe(X)) binding to E-, P-, and L-selectins. Compounds 1 and 2 were 2 times more potent than the sLe(X) tetrasaccharide toward E-selectin binding and up to 4 times more potent than sLe(X) toward P- and L-selectin binding. Interestingly, compound 1 provided dose-dependent protective effects against an immunoglobulin E-mediated skin reaction in mouse ears. This protective effect was associated with diminished tissue accumulation of neutrophils in the ear (as assessed by myeloperoxidase). These findings indicate that the modification of sLe(X) or 3'-O-sulfo Le(X) with a "branched anchor", a 2-tetradecylhexadecyl group, is useful in the design of a more potent selectin blocker, which has broad inhibitory activities toward all selectins.
VMD is a molecular graphics program designed for the display and analysis of molecular assemblies, in particular biopolymers such as proteins and nucleic acids. VMD can simultaneously display any number of structures using a wide variety of rendering styles and coloring methods. Molecules are displayed as one or more "representations," in which each representation embodies a particular rendering method and coloring scheme for a selected subset of atoms. The atoms displayed in each representation are chosen using an extensive atom selection syntax, which includes Boolean operators and regular expressions. VMD provides a complete graphical user interface for program control, as well as a text interface using the Tcl embeddable parser to allow for complex scripts with variable substitution, control loops, and function calls. Full session logging is supported, which produces a VMD command script for later playback. High-resolution raster images of displayed molecules may be produced by generating input scripts for use by a number of photorealistic image-rendering applications. VMD has also been expressly designed with the ability to animate molecular dynamics (MD) simulation trajectories, imported either from files or from a direct connection to a running MD simulation. VMD is the visualization component of MDScope, a set of tools for interactive problem solving in structural biology, which also includes the parallel MD program NAMD, and the MDCOMM software used to connect the visualization and simulation programs. VMD is written in C++, using an object-oriented design; the program, including source code and extensive documentation, is freely available via anonymous ftp and through the World Wide Web.
Through a computer modeling and simulation technique, we investigated the binding mode of a complex of E-selectin-GSC-150, which is a novel selectin blocker. GSC-150 is the 3'-sulfated Lewis X derivative with a long, branched alkyl chain. Initial attempts to construct a model for E-selectin-GSC-150 complex were performed based on a previously reported model of E-selectin-sialyl Lewis X (sLex) complex [Kogan, T.P.; Revelle, B.M.; Tapp, S.; Scott, D.; Beck, P.J.J. Biol. Chem. 1995, 270, 14047-14055]. In our model, the carbohydrate portion of GSC-150 interacted with the protein in a similar manner as that of sLex reported previously. Interestingly, each of the branched alkyl chains extended on the surface of E-selectin and interacted with two different hydrophobic portions. One of these hydrophobic portions consists of Tyr44, Pro46, and Tyr48. Another portion forms a shallow cavity, and it consists of Ala9, Leu114, and the alkyl moieties of the side chains of Lys111, Lys112, and Lys113. A subsequent 200-ps molecular dynamics simulation in solution revealed that the interactions involved in the sugar portion of the ligand were relatively weak, whereas the hydrophobic interactions involved in the branched alkyl chains were fairly stable in solution. These results suggest that the branched alkyl chain serves as an "anchor" for the tight binding of GSC-150 on the surface of E-Selectin. This is the first attempt to evaluate the dynamics of E-Selectin-ligand interactions in solution, and it sheds light on the nature of ligand recognition by selectins.
Protein and lipid glycosylation is no longer considered as a topic whose appeal is restricted to a limited number of analytical experts perseveringly pursuing the comprehensive cataloguing of structural variants. It is in fact arousing curiosity in various areas of basic and applied bioscience. Well founded by the conspicuous coding potential of the sugar part of cellular glycoconjugates which surpasses the storage capacity of oligonucleotide- or oligopeptide-based code systems, recognition of distinct oligosaccharide ligands by endogenous receptors, i.e. lectins and sugar-binding enzymes or antibodies, is increasingly being discovered to play salient roles in animal physiology. Having inevitably started with a descriptive stage, research on animal lectins has now undubitably reached maturity. Besides listing the current categories for lectin classification and providing presentations of the individual families and their presently delineated physiological significance, this review places special emphasis on tracing common structural and functional themes which appear to reverberate in nominally separated lectin and animal categories as well as lines of research which may come to fruition for medical sciences.
The galactoside-binding lectin from mistletoe (Viscum album L.) is a biological response modifier, eliciting e.g. enhanced secretion of cytokines. This immunological activity warrants the further analysis of its ligand-binding properties with special attention paid to blood group epitopes. To avoid the microheterogeneity and complexity of naturally occurring glycoproteins, chemically strictly defined neoglycoconjugates and a panel of synthetic oligosaccharides were employed in solid-phase assays for direct binding and assessment of the relative inhibitory capacity. Since label incorporation into the lectin, although performed under protective conditions, or surface immobilization by adsorption to plastic may affect its affinity characteristics, the extent of neoglycoconjugate binding in the absence of any interfering substance and in the presence of oligosaccharides was determined comparatively with labeled and with immobilized lectin. In principle, these two factors could be excluded to markedly alter binding features. In addition to lactose, the blood group determinants H and B were strongly reactive. A fucose residue can thus especially be accommodated to the binding site when linked to the non-reducing unit. N-Acetyllactosamine was nearly as potent as an inhibitor as lactose. Lec and the A determinant were notably inferior to the other ABH blood group epitopes. Le(a) and Le(x) and their sialylated derivatives displayed only very weak binding capacity. Among the two natural isomers of sialyllactose, the alpha 2,6-form displayed a higher level of inhibitory capacity than the alpha 2,3-derivative. Isomeric variants of the Thomsen-Friedenreich antigen, too, reduced lectin binding to the lactose-carrying polymer. Their capacities were surpassed by those of the H and the B determinants and a related form of the latter, the P1 epitope. An overlap of specificity with the immunomodulatory human galectin-3 is thus measurable for H/B-like structures. The documented differential reactivity of the mistletoe lectin to blood group oligosaccharides may have a bearing on the responsiveness of blood group-positive cell populations.
Isothermal titration calorimetry (ITC) measurements of the binding 1-beta carbohydrate-substituted galactopyranoside derivatives to galectin-1 from bovine spleen, a dimer with one binding site per subunit, were performed at 283-285 and 298 K. The disaccharides were lactose, methyl beta-lactoside, lactulose, 4-O-beta-D-galactopyranosyl-D-mannopyranoside, 3-O-beta-D-galactopyranosyl-D-arabinose, 2'-O-methyllactose, lacto-N-biose, N-acetyllactosamine, and thiodigalactopyranoside. The site binding enthalpies, DeltaHb, are the same at both temperatures and range from -42.2 +/- 3.3 kJ mol-1 for thiodigalactopyranoside to -24.5 +/- 0.5 kJ mol-1 for lacto-N-biose, and the site binding constants range from 4.86 +/- 0.78 x 10(3) M-1 for methyl beta-lactoside at 297.8 K to 6.54 +/- 0.97 x 10(4) M-1 for N-acetyllactosamine at 281.3 K. The binding reactions are enthalpically driven, exhibit enthalpy-entropy compensation, and, with the exception of N-acetyllactosamine, follow a van't Hoff dependence of the binding constant on temperature. The number of contacts at distances <4.0 A between the disaccharide and galectin was determined from the energy-minimized conformation of the complex derived from the X-ray crystallographic structure of the galectin-N-acetyllactosamine complex determined by Liao et al. [Liao, D. I., Kapadia, G., Ahmed, H., Vasta, G. R., and Herzberg, O. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 1428-1432]. The binding enthalpies calculated from changes in the solvent-accessible surface areas of the galectin binding site upon binding of the disaccharide were in close agreement with the experimental values for lactose, lactulose, lacto-N-biose, and N-acetyllactosamine, all of which exhibit binding enthalpies >-36 kJ mol-1. Differential scanning calorimetry measurements on solutions of galectin and its disaccharide complexes show that the galectin dimer does not dissociate upon denaturation in contrast to the legume lectins. At the denaturation temperature, the galectin in the absence of sugar exists as a tetramer, and the extent of this association is substantially reduced in the presence of a disaccharide.
NOE measurements and molecular mechanics calculations have been performed to study the conformational behaviour of Fuc(alpha 1-3)GlcNAc and its thioglycoside analogue in solution. Experimental data show that, in contrast with the natural O-disaccharide, which is basically monoconformational, the S-analogue shows two conformational families, namely syn and anti.