Binding studies of adhesion/growth-regulatory galectins with glycoconjugates monitored by surface plasmon resonance and NMR spectroscopy.

Functionalized fluorescent glycans have the potential to act as tools to detect and analyze protein-carbohydrate interactions. We present here a facile strategy for immobilization of functionalized lactose as a model disaccharide. Bioactivity was tested with three members of the adhesion/growth-regulatory galectins family in different types of assay, i.e. matrix in surface plasmon resonance (SPR), free ligand in solution by STD/trNOESY and docking measurements. In all cases, the activity of the disaccharide was maintained. The attachment of this new fluorescent glycoconjugate to the surface results in a well-defined interface, enabling desired orientational flexibility and enhanced access of binding partners. The results indicate that this new glycoconjugate exhibits binding affinity to galectin-1, 3 and CG-16. Kinetic analysis of the interaction between these galectins and immobilized glycoconjugate by SPR yielded a K(D) of 1.01 mM for galectin-1, 83.5 microM for galectin-3 and 0.28 mM for CG-16. No major contacts to the aglyconic part were detected, which might compromise the specificity of the binding process with other headgroups. Thus, testing these proteins offers the potential for medical applications to detect these endogenous effectors or further derivatives and characterize their carbohydrate specificity.

Catalytic conjugate additions of geminal bis(sulfone)s: expanding the chemistry of sulfones as simple alkyl anion equivalents.

The value of cyclic gem-bis(sulfone) 4 as a simple alkyl nucleophile equivalent in catalytic C-C bond-forming reactions is demonstrated. The 1,4-type nucleophilic additions of bis(sulfone) 4 to alpha,beta-unsaturated ketones take place by assistance of catalytic guanidine base. On the other hand, pyrrolidines are able to catalyze the conjugate addition of 4 to both enones and enals, likely by means of iminium ion activation. Upon exploration of the best chiral pyrrolidine catalyst, it has been found that the addition of 4 to enals catalyzed by diphenylprolinol silyl ether 10 proceeds with very high enantioselectivity (beta-aryl-substituted enals >95% ee; beta-alkyl substituted enals up to 94% ee; ee = enantiomeric excess). Further reductive desulfonation of adducts gives rise to the corresponding beta-methyl aldehydes, as well as the derived alcohols, acetals, and methyl esters after simple (Mg, MeOH) well-established protocols. Application of the procedure to the synthesis of biologically relevant phenethyl building blocks is shown. Most interestingly, alpha-alkylation of initially obtained bis(sulfone) adducts can be done even with less reactive alkylating reagents, such as long linear-chain or branched-chain alkyl halides. Accordingly, upon the desulfonation process, a general, experimentally simple and highly enantioselective access to beta-branched aldehydes, alcohols, or esters is possible. Further exploration of the method includes the use of chiral alpha,beta-unsaturated aldehydes derived from citronellal as the Michael acceptor partners. In these instances, the sense of the conjugate addition of 4 is controlled by the chirality of the pyrrolidine catalyst, thus allowing for a stereochemically predictable access to 1,3-dimethyl arrays, such as those present in deoxygenated polyketide-type natural products. The intramolecular variation of this technology by using doubly unsaturated aldehyde-ester 22 illustrated the site selectivity of the procedure and its potential for tandem processes leading to highly substituted polycyclic systems, such as 24.

Glycan tagging to produce bioactive ligands for a surface plasmon resonance study via immobilization on different surfaces.

Suitable glycan derivatives will find immediate application in the study of their interactions. Here, we present an efficient synthetic strategy to introduce a fluorescent tag functionalized with an amino group into a model disaccharide structure (lactose). This strategy led to the maintenance of bioactivity, checked by the study of the interaction of this bioconjugate with a plant lectin (mistletoe lectin 1) by NMR spectroscopy, computational docking, and surface plasmon resonance (SPR). To demonstrate the versatility of this approach, we immobilized the new glycan derivatives on different surfaces, and a comparative analysis is presented and can be successfully used for biomimetic carbohydrate-protein interaction studies on the SPR biochip.

Assessing carbohydrate-carbohydrate interactions by NMR spectroscopy: the trisaccharide epitope from the marine sponge Microciona prolifera.

WEAK RECOGNITION PROCESSES: Weak calcium-mediated carbohydrate-carbohydrate interactions have been detected by DOSY and TRNOESY NMR methods by employing a gold glyconanoparticle as a multivalent system. In addition, 3D models of trisaccharide-Ca(II)-trisaccharide complexes based on results from molecular dynamics simulations are proposed. Diffusion-ordered NMR spectroscopy (DOSY-NMR) and TR-NOESY-NMR experiments are used to detect ligand binding to macromolecular receptors. These techniques have been applied to detect weak carbohydrate-carbohydrate self-recognition in solution, making use of sugar-decorated gold nanoparticles as the "macromolecule" and the same carbohydrate as the ligand. Changes in the diffusion coefficient of the free carbohydrate in the presence of the glyconanoparticle (only with Ca(II) ions in the sample solution), as well as changes in the sign of the sugar NOE peaks--positive for the free sugar (in the presence or absence of Ca(II)) and negative for the sugar only in the simultaneous presence of the glyconanoparticle and Ca(II) ions--have been taken as proof of weak Ca(II)-mediated carbohydrate-carbohydrate interactions in solution. Although different methods such as SPR, TEM, and AFM have been used in the past to detect carbohydrate-carbohydrate interactions with the aid of gold nanoparticles and gold selfassembled monolayers, they are restricted to high-affinity ranges. The methods used in this study allow expansion of the number of techniques to tackle this relevant biological problem, also for approaching ligand-receptor interactions below the high-affinity range. Additionally, 3D models of trisaccharide-Ca(II)-trisaccharide complexes based on results from molecular dynamics simulations are proposed.

Highly acylated (acetylated and/or p-coumaroylated) native lignins from diverse herbaceous plants.

The structure of lignins isolated from the herbaceous plants sisal ( Agave sisalana), kenaf ( Hibiscus cannabinus), abaca ( Musa textilis) and curaua ( Ananas erectifolius) has been studied upon spectroscopic (2D-NMR) and chemical degradative (derivatization followed by reductive cleavage) methods. The analyses demonstrate that the structure of the lignins from these plants is highly remarkable, being extensively acylated at the gamma-carbon of the lignin side chain (up to 80% acylation) with acetate and/or p-coumarate groups and preferentially over syringyl units. Whereas the lignins from sisal and kenaf are gamma-acylated exclusively with acetate groups, the lignins from abaca and curaua are esterified with acetate and p-coumarate groups. The structures of all these highly acylated lignins are characterized by a very high syringyl/guaiacyl ratio, a large predominance of beta- O-4' linkages (up to 94% of all linkages), and a strikingly low proportion of traditional beta-beta' linkages, which indeed are completely absent in the lignins from abaca and curaua. The occurrence of beta-beta' homocoupling and cross-coupling products of sinapyl acetate in the lignins from sisal and kenaf indicates that sinapyl alcohol is acetylated at the monomer stage and that, therefore, sinapyl acetate should be considered as a real monolignol involved in the lignification reactions.

"Click" saccharide/beta-lactam hybrids for lectin inhibition.

Hybrid glycopeptide beta-lactam mimetics designed to bind lectins or carbohydrate recognition domains in selectins have been prepared according to a "shape-modulating linker" design. This approach was implemented using the azide-alkyne "click" cycloaddition reaction, and as shown by NMR/MD experiments, binding of the resulting mimetics to Ulex Europaeus Lectin-1 (UEL-1) occurred after a "bent-to-extended" conformational change around a partially rotatable triazolylmethylene moiety.

Versatile strategy for the synthesis of biotin-labelled glycans, their immobilization to establish a bioactive surface and interaction studies with a lectin on a biochip.

The emerging role of glycans as versatile biochemical signals in diverse aspects of cellular sociology calls for establishment of sensitive methods to monitor carbohydrate recognition by receptors such as lectins. Most of these techniques involve the immobilization of one of the binding partners on a surface, e.g. atomic force microscopy, glycan array and Surface Plasmon Resonance (SPR), hereby simulating cell surface presentation. Here, we report the synthesis of fluorescent glycoconjugates, with a functionalization strategy which avoids the frequently occurring ring opening at the reducing end for further immobilization on a surface or derivatization with biotin. In order to improve the versatility of these derivatized glycans for biological studies, a new approach for the synthesis of biotinylated and fluorescent glycans has also been realized. Finally, to illustrate their usefulness the neoglycoconjugates were immobilized on different surfaces, and the interaction analysis with a model lectin, the toxin from mistletoe, proved them to act as potent ligands, underscoring the merit of the presented synthetic approach.

NMR studies on the conformation of oligomannosides and their interaction with banana lectin.

The conformational and dynamic behaviour of three mannose containing oligosaccharides, a tetrasaccharide with alpha1-->2, and alpha1-->3, and a penta and a heptasaccharide with alpha1-->2, alpha1-->3, and alpha1-->6 linkages has been evaluated by molecular mechanics and dynamics simulations and NMR spectroscopical methods. It is found that they display a fair amount of conformational freedom, with one major and one minor conformation per glycosidic linkage. The evaluation of their recognition by banana lectin has also been performed by STD NMR methods and a preliminary view of their putative interaction mode has been carried out by means of docking procedures.

Use of the 1H nuclear magnetic resonance spectra signals from polyphenols and acids for chemometric characterization of cider apple juices.

The low field region (5.8-9.0 ppm) corresponding to aromatic protons and the region 1.8-3.0 ppm of the (1)H NMR spectra were used for characterization and chemometric differentiation of 52 apple juices obtained from six cider apple varieties. The data set consisted of 14 integrated areas corresponding to resonances from acids and phenolic compounds. Multivariate procedures based on hierarchical cluster and discriminant analysis were performed on selected signals of the spectra to determine whether it was possible to distinguish the different juices. Cluster analysis was able to satisfactorily classify the six apple varieties. Discriminant analysis, by means of stepwise procedure for variables selection and leave-one-out for cross-validation, was applied to 40 samples from the year 2001, obtaining recognition and prediction abilities of 100%. The most discriminant variables corresponded to poliphenols, (-)-epicatechin, phloridzin-phloretin, and p-coumaric, chlorogenic, and malic acids. The classification model was applied to 12 samples from apples harvested in the years 2002 and 2003, and the prediction ability was 91.7%.