Reactivity Analysis of New Multivalent Electrophilic Probes for Affinity Labeling of Carbohydrate Binding Proteins.

We have designed and synthesized six different multivalent electrophiles as carbohydrate affinity labeling probes. Evaluation of the reactivity of the electrophiles against peanut agglutinin (PNA) and Ricinus communis agglutinin (RCA) showed that p- and m-aryl sulfonyl fluoride are effective protein reactive groups that label carbohydrate binding lectins in a ligand-dependent fashion at a nanomolar probe concentration. Analysis of the selectivity of affinity labeling in the presence of excess BSA as a nonspecific protein indicated that m-arylsulfonyl fluoride is a more selective protein-reactive group, albeit with attenuated reactivity. Further analysis showed that the labeling efficiency of the multivalent electrophilic probes can be improved by employing reaction conditions involving 25 °C instead of typically employed 4 °C. Both isomers of arylsulfonyl fluoride groups together represent promising affinity labels for target identification studies that could serve as more efficient alternatives to photoreactive groups.

Crystal structures of peanut lectin in the presence of synthetic ß-N- and ß-S-galactosides disclose evidence for the recognition of different glycomimetic ligands.

Carbohydrate-lectin interactions are involved in important cellular recognition processes, including viral and bacterial infections, inflammation and tumor metastasis. Hence, structural studies of lectin-synthetic glycan complexes are essential for understanding lectin-recognition processes and for the further design of promising chemotherapeutics that interfere with sugar-lectin interactions. Plant lectins are excellent models for the study of the molecular-recognition process. Among them, peanut lectin (PNA) is highly relevant in the field of glycobiology because of its specificity for ß-galactosides, showing high affinity towards the Thomsen-Friedenreich antigen, a well known tumor-associated carbohydrate antigen. Given this specificity, PNA is one of the most frequently used molecular probes for the recognition of tumor cell-surface O-glycans. Thus, it has been extensively used in glycobiology for inhibition studies with a variety of ß-galactoside and ß-lactoside ligands. Here, crystal structures of PNA are reported in complex with six novel synthetic hydrolytically stable ß-N- and ß-S-galactosides. These complexes disclosed key molecular-binding interactions of the different sugars with PNA at the atomic level, revealing the roles of specific water molecules in protein-ligand recognition. Furthermore, binding-affinity studies by isothermal titration calorimetry showed dissociation-constant values in the micromolar range, as well as a positive multivalency effect in terms of affinity in the case of the divalent compounds. Taken together, this work provides a qualitative structural rationale for the upcoming synthesis of optimized glycoclusters designed for the study of lectin-mediated biological processes. The understanding of the recognition of ß-N- and ß-S-galactosides by PNA represents a benchmark in protein-carbohydrate interactions since they are novel synthetic ligands that do not belong to the family of O-linked glycosides.

Protein Sensing Device with Multi-Recognition Ability Composed of Self-Organized Glycopeptide Bundle.

We designed and synthesized amphiphilic glycopeptides with glucose or galactose at the C-terminals. We observed the protein-induced structural changes of the amphiphilic glycopeptide assembly in the lipid bilayer membrane using transmission electron microscopy (TEM) and Fourier transform infrared reflection-absorption spectra (FTIR-RAS) measurements. The glycopeptides re-arranged to form a bundle that acted as an ion channel due to the interaction among the target protein and the terminal sugar groups of the glycopeptides. The bundle in the lipid bilayer membrane was fixed on a gold-deposited quartz crystal microbalance (QCM) electrode by the membrane fusion method. The protein-induced re-arrangement of the terminal sugar groups formed a binding site that acted as a receptor, and the re-binding of the target protein to the binding site induced the closing of the channel. We monitored the detection of target proteins by the changes of the electrochemical properties of the membrane. The response current of the membrane induced by the target protein recognition was expressed by an equivalent circuit consisting of resistors and capacitors when a triangular voltage was applied. We used peanut lectin (PNA) and concanavalin A (ConA) as target proteins. The sensing membrane induced by PNA shows the specific response to PNA, and the ConA-induced membrane responded selectively to ConA. Furthermore, PNA-induced sensing membranes showed relatively low recognition ability for lectin from Ricinus Agglutinin (RCA120) and mushroom lectin (ABA), which have galactose binding sites. The protein-induced self-organization formed the spatial arrangement of the sugar chains specific to the binding site of the target protein. These findings demonstrate the possibility of fabricating a sensing device with multi-recognition ability that can recognize proteins even if the structure is unknown, by the protein-induced self-organization process.

Poly(triazolyl methacrylate) glycopolymers as potential targeted unimolecular nanocarriers.

Synthetic glycopolymers are increasingly investigated as multivalent ligands for a range of biological and biomedical applications. This study indicates that glycopolymers with a fine-tuned balance between hydrophilic sugar pendant units and relatively hydrophobic polymer backbones can act as single-chain targeted nanocarriers for low molecular weight hydrophobic molecules. Non-covalent complexes formed from poly(triazolyl methacrylate) glycopolymers and low molecular weight hydrophobic guest molecules were characterised through a range of analytical techniques - DLS, SLS, TDA, fluorescence spectroscopy, surface tension analysis - and molecular dynamics (MD) modelling simulations provided further information on the macromolecular characteristics of these single chain complexes. Finally, we show that these nanocarriers can be utilised to deliver a hydrophobic guest molecule, Nile red, to both soluble and surface-immobilised concanavalin A (Con A) and peanut agglutinin (PNA) model lectins with high specificity, showing the potential of non-covalent complexation with specific glycopolymers in targeted guest-molecule delivery.

Uniform and Easy-To-Prepare Glycopolymer-Brush Interface for Rapid Protein (Anti-)Adhesion Sensing.

Glycopolymers have emerged as powerful and versatile glycan analogues for the investigation of cellular signal transduction. In this study, a layer of the glycopolymer-brush (GlyB) interface was functionalized on the surface of gold substrates. In order to enhance the capability and accessibility of this transducer interface, a combined protocol of copper(0)-mediated living radical polymerization (Cu(0)-LRP) with subsequent "CuAAC" click reaction was utilized to synthesize a set of novel glycopolymer precursors with a tunable scaffold structure and pyranose ligands. The resulting glycopolymer exhibited a fine-tuned molecular weight with a minor dispersity of 1.27. Through surface plasmon resonance (SPR) analysis, various GlyB interfaces exhibiting different saccharide moieties (glucose, mannose, and galactose) were examined to study their adhesion or antiadhesion potential toward three types of proteins, concanavalin A, bovine serum albumin, and peanut agglutinin (PNA). The strong affinity between poly(galactose) and PNA was further employed to construct a proof-of-concept aggregation-mediated sensing system. This minimal naked-eye sensor that consisted of only two substances, namely, gold nanoparticles and glycopolymers, was characterized and tested for its potential in protein quantification.

Detection of the cancer-associated T antigen using an Arachis hypogaea agglutinin biosensor.

An impedimetric biosensor was developed for the selective detection of the cancer-associated T antigen, using the lectin from Arachis hypogaea (peanut agglutinin, PNA) as the recognition element. The increase in the biosensor's impedance after sample incubation was indicative of lectin recognition and complex formation between PNA and glycoproteins containing T antigen. When using asialofetuin as model glycoprotein, a minimum amount of 100 ng of glycoprotein could be detected, generating an increase in impedance of 7.2%. Albumin did not cause interference in the detection of T-carrying glycoproteins up to a concentration of 0.01 mg ml-1. The biosensor was used to evaluate the T-antigen expression in serum samples and was able to discriminate between control samples (of individuals without cancer) and case samples from patients with diverse types of carcinomas (skin, colon, breast, prostate, stomach, kidney, lung, liver and rectum) in which an increase in the expression of T antigen is well-known. The same samples were analyzed with a Vicia villosa agglutinin biosensor that has specificity for the cancer-associated Tn antigen, to compare the expression of both antigens in the diverse carcinomas. The results were different for both biosensors, confirming that the use of different lectins allows to monitor different antigen expression. Furthermore, combining different lectins, glycosylation profiles for each carcinoma type can be obtained. This work demonstrates the feasibility of employing PNA to selectively recognize the T epitope in glycoproteins and the proposed biosensor could be used for high-throughput, label-free profiling of the cancer-associated T antigen in serum samples.

Collagen microencapsulation recapitulates mesenchymal condensation and potentiates chondrogenesis of human mesenchymal stem cells - A matrix-driven in vitro model of early skeletogenesis.

Mesenchymal condensation is a critical transitional stage that precedes cartilage or bone formation. A microencapsulation technique was previously established to entrap mesenchymal stem cells (MSC) in nanofibrous collagen meshwork. We hypothesize that collagen microencapsulation of MSCs mimics the mesenchymal cell condensation process. Specifically, human MSCs at different concentrations were microencapsulated in collagen for different time points before evaluation for early skeletogenesis markers. A transient upregulation of mesenchymal condensation markers including peanut agglutinin, fibronectin, integrins α5 and αv, an enhanced nuclear localization of SOX9 and binding interactions with COL2A1, and other changes in chondrogenic, hypertropic and osteogenic marker were demonstrated. Collagen microencapsulation upregulated both the chondrogenic and the osteogenic transcription factors and the encapsulated hMSCs hold the potential to differentiate towards both chondrogenic and osteogenic lineages. We also hypothesize that collagen microencapsulation potentiates MSC chondrogenesis. Particularly, chondrogenic differentiation of hMSCs were induced at different time post-encapsulation before evaluation for chondrogenesis outcomes. Sustained SOX9, ACAN and COL2A1 expression were noted and the timing to induce supplement chondro-inductive factors matters. This study reports an extracellular matrix-based in vitro model of mesenchymal condensation, an early stage in skeletogenesis, contributing to rationalizing development-inspired tissue engineering.

Egg autofluorescence and options for detecting peanut agglutinin binding for the identification of Haemonchus contortus eggs in fecal samples.

Quantifying eggs from Haemonchus and other trichostrongyle genera in sheep and goat fecal samples is important for evaluating control and treatment strategies for this family of nematodes with divergent pathologies, capabilities for anthelmintic resistance and environmental susceptibilities. Unfortunately, egg morphology among most of the genera do not differ enough to support the accurate identification of these genera with standard microscopic techniques. Several studies have identified specific lectins which bind selectively to sugars located on the egg surfaces for individual genera among the trichostrongyles. To detect lectins binding to these eggs, they must be directly or indirectly bound to fluorophores, and observed with an epi-fluorescence microscope. The binding of multiple lectins to isolated eggs from a fecal sample can be simultaneously detected if fluorophores are used whose excitation and emission spectra do not overlap, and this would enable the development of a fluorescence-based diagnostic test that identifies multiple trichostrongyle genera within each sample. The present study compared the usefulness of different, commercially available detection systems for use in detecting lectin binding to trichostrongyle eggs. Comparisons were made using the detection of PNA binding to H. contortus eggs with the goal of finding three systems with color spectra that do not overlap. These evaluations included both fluorophores directly conjugated to PNA in a one-step incubation protocol and a two-step incubation protocol involving biotinylated PNA and streptavidin conjugated to different fluorophores. Autofluorescence can affect the efficiency of any fluorescence-based detection system, and significant autofluorescence was observed among the unstained H. contortus eggs with the DAPI-type fluorescence filter, but it was significantly lower with the FITC-type filter and was virtually absent with the rhodamine-type filter. This study demonstrated that all the PNA detection methods tested with H. contortus eggs generated fluorescence intensities (FIs) that were significantly above the autofluorescence generated by the eggs among the three different fluorescence filters. Fluorescence intensities from PNA directly conjugated to either the FITC or rhodamine fluorophores were not different, but the lower autofluoresence in the rhodamine-type filter will enable this fluorophore to be detected more efficiently. Use of biotinylated PNA combined with streptavidin-conjugated to synthetic fluorophores (Alexa Fluor 405, 488 and 546) significantly increased FIs over that of the directly conjugated PNA, but there were no significant differences in FIs among these three biotin-avidin conjugation fluorophores. This biotin-avidin system required two incubation steps. Doubling the concentration of PNA also provided increased FI, at least for the biotin-avidin system. Adding an additional amplification step to the biotin-avidin system involving biotinylated anti-streptavidin followed by the streptavidin-Alexa Fluor complex also provided additional fluorescence.

Methodological improvement of fluorescein isothiocyanate peanut agglutinin (FITC-PNA) acrosomal integrity staining for frozen-thawed Japanese Black bull spermatozoa.

This study aimed to improve the staining of frozen-thawed Japanese Black bull sperm acrosomes with fluorescein isothiocyanate-conjugated peanut agglutinin (FITC-PNA). Spermatozoa were washed, fixed with 1-3% paraformaldehyde (PFA) in suspension for 10, 20, and 30 min, permeabilized with 0-2% Triton X-100 for 5 min, stained with FITC-PNA, and mounted with different antifade agents (0.22 M 1,4-diazabicyclo [2,2,2] octane (DABCO), SlowFade®, and ProLong®) in suspension (In-suspension) or on a smear (On-smear). The spermatozoa were categorized into seven pattern types either immediately or after storage for 24 hr. Experiment 1 showed that 1) the In-suspension method was better than the On-smear method; 2) if spermatozoa were stained using the In-suspension method and examined immediately, the best antifade agent was SlowFade®; 3) if samples were to be stored after staining using the On-smear method, DABCO should be avoided; 4) if spermatozoa were stained using the In-suspension method, storage of the stained samples was not recommended; and 5) if samples were to be stored after staining using the In-suspension method, ProLong® might be the best antifade agent. The results of experiment 2 showed that the concentration of Triton X-100 could be reduced to 0.1 from 1%. The results of experiment 3 showed that the paraformaldehyde concentration used for a 30 min fixation could be reduced from 3 to 2%. It is expected that the improved staining protocol will be useful to determine bull sperm acrosomal integrity.

Supramolecular nanofiber of pyrene-lactose conjugates and its two-photon fluorescence imaging.

A lactose modified pyrene derivative (Py-Lac) was synthesized, with which novel twisted supramolecular nanofibers in diameter about 20 nm were constructed by self-assembly. The nanofibers showed solid-state fluorescence between 400 nm and 650 nm with the maximum emission at 495 nm. Furthermore, its recognition reaction with PNA lectin was investigated by fluorescence spectra and turbidity assays. It is interesting found that the supramolecular assembly as multivalent glycocluster exhibited unique and selectively binding interactions with PNA lectin with the binding constant of 5.74 × 106 M-1. Moreover, compound Py-Lac showed two-photon fluorescence imaging with Hep G2 cells.

Peanut agglutinin specifically binds to a sperm region between the nucleus and mitochondria in tunicates and sea urchins.

Peanut agglutinin (PNA) is an established marker of the mammalian acrosome. However, we observed that PNA specifically binds to a unique intracellular structure alongside the nucleus in ascidian sperm. Here, we characterize the PNA-binding structure in sperm of marine invertebrates. PNA bound to the region between the mitochondrion and nucleus in spermatozoa of ascidians, sea urchins, and an appendicularian. However, PNA-binding substances were not exposed by the calcium ionophore ionomycin in three ascidian species, indicating that it is a distinct structure from the acrosome. Instead, the ascidian PNA-binding region was shed with the mitochondrion from the sperm head via an ionomycin-induced sperm reaction. The ascidian PNA-binding substance appeared to be solubilized with SDS, but not Triton X-100, describing its detergent resistance. Lectins, PHA-L4 , SSA, and MAL-I were detected at an area similar to the PNA-binding region, suggesting that it contains a variety of glycans. The location and some of the components of the PNA-binding region were similar to known endoplasmic reticulum (ER)-derived structures, although the ER marker concanavalin A accumulated at an area adjacent to but not overlapping the PNA-binding region. Therefore, we conclude that ascidian sperm possess a non-acrosomal, Triton-resistant, glycan-rich intracellular structure that may play a general role in reproduction of tunicates and sea urchins given its presence across a wide taxonomic range.

Peanut agglutinin and ß-cyclodextrin functionalized polymer monolith: Microextraction of IgG galactosylation coupled with online MS detection.

A facile online method coupling polymer monolithic microextraction (PMME) with mass spectrometry (MS) was developed for the detection of Immunoglobulin G (IgG) galactosylation glycopeptides. A peanut agglutinin-ß-cyclodextrin (PNA-ß-CD) functionalized poly(hydroxyethyl methylacrylate-ethyleneglycol dimethacrylate) monolith was designed via a click reaction. Thanking to the specificity of PNA-ß-CD for the targets, the material exhibited enhanced enrichment selectivity and extraction efficiency for IgG galactosylation glycopeptides. Under optimal conditions, the developed method gave a linear range of 0.005-5 pmol for IgG glycopeptides with the regression coefficient greater than 0.9990, and the detection limit of IgG galactosylation glycopeptides as low as 0.5 fmol was achieved. The PMME-MS method was applied to IgG galactosylation glycopeptides in real samples including human serum and acute myelogenous leukemia (AML) cell lysate. A series of unique IgG galactosylation glycopeptides were captured by the monolith in the complex samples, indicating satisfactory enrichment ability for IgG galactosylation glycopeptides. The quick and integrated online PMME-MS method exhibited high selectivity for IgG galactosylation, demonstrating its perspectives on the development and broad applications of MS in studying galactosylation proteins regulated biological processes.

Evaluation of a novel fluorescent nanobeacon for targeted imaging of Thomsen-Friedenreich associated colorectal cancer.

The Thomsen-Friedenreich (TF) antigen represents a prognostic biomarker of colorectal carcinoma. Here, using a nanobeacon, the surface of which was fabricated with peanut agglutinin as TF-binding molecules, we demonstrate that the nanobeacon is able to detect TF antigen in frozen and freshly biopsied polyps using fluorescence microscopy. Our results provide important clues about how to detect aberrant colonic tissues in the most timely fashion. Given the versatile application method for this topical nanobeacon, the protocol used in this work is amenable to clinical colonoscopy. Moreover, the prospects of clinical translation of this technology are evident.

Hyaluronate-Peanut Agglutinin Conjugates for Target-Specific Bioimaging of Colon Cancer.

Colon cancer is one of the most common death-related cancers in the world. For treating colon cancer, it is crucial to detect and remove malignant lesions early. Here, we developed hyaluronate (HA)-peanut agglutinin (PNA) conjugates for the bioimaging of colon cancer. The HA-PNA conjugates were successfully synthesized by the coupling reaction between aldehyde-modified HA and the N-terminal amine group of PNA. For diagnostic imaging, rhodamine B (RhoB) was chemically conjugated onto PNA in HA-PNA conjugates. After intraluminal injection of HA-PNA-RhoB conjugates into tumor-bearing mice, small-sized colon cancers could be effectively visualized by ex vivo imaging with an in vivo imaging system (IVIS) and a two-photon microscope. With these results taken together, we could confirm the feasibility of HA-PNA-RhoB conjugates as a bioimaging agent for detecting colon cancers.

Synthesis of ß-galactosylamides as ligands of the peanut lectin. Insights into the recognition process.

The synthesis of mono and divalent ß-galactosylamides linked to a hydroxylated chain having a C2 symmetry axis derived from l-tartaric anhydride is reported. Reference compounds devoid of hydroxyl groups in the linker were also prepared from ß-galactosylamine and succinic anhydride. After functionalization with an alkynyl residue, the resulting building blocks were grafted onto different azide-equipped scaffolds through the copper catalyzed azide-alkyne cycloaddition. Thus, a family of structurally related mono and divalent ß-N-galactopyranosylamides was obtained and fully characterized. The binding affinities of the ligands towards the model lectin PNA were measured by the enzyme-linked lectin assay (ELLA). The IC50 values were significantly higher than that of galactose but the presence of hydroxyl groups in the aglycone chain improved lectin recognition. Docking and molecular dynamics experiments were in accordance with the hypothesis that a hydroxyl group properly disposed in the linker could mimic the Glc O3 in the recognition process. On the other hand, divalent presentation of the ligands led to lectin affinity enhancements.

Host-guest interactions and controllable capture and release of proteins based on cationic perylene bisimides.

A system of controllable capture and release of protein was constructed by multiple, interconnected supramolecular binding modules based on lactose modified mono-cationic perylene bisimide derivatives, cucurbit[8]uril (CB[8]), 1-adamantanamine (ADA) and peanut agglutinin (PNA) lectins.

The Impact of Heteromultivalency in Lectin Recognition and Glycosidase Inhibition: An Integrated Mechanistic Study.

The vision of multivalency as a strategy limited to achieve affinity enhancements between a protein receptor and its putative sugar ligand (glycotope) has proven too simplistic. On the one hand, binding of a glycotope in a dense glycocalix-like construct to a lectin partner has been shown to be sensitive to the presence of a third sugar entity (heterocluster effect). On the other hand, several carbohydrate processing enzymes (glycosidases and glycosyltransferases) have been found to be also responsive to multivalent presentations of binding partners (multivalent enzyme inhibition), a phenomenon first discovered for iminosugar-type inhibitory species (inhitopes) and recently demonstrated for multivalent carbohydrate constructs. By assessing a series of homo- and heteroclusters combining α-d-glucopyranosyl-related glycotopes and inhitopes, it was shown that multivalency and heteromultivalency govern both kinds of events, allowing for activation, deactivation or enhancement of specific recognition phenomena towards a spectrum of lectin and glycosidase partners in a multimodal manner. This unified scenario originates from the ability of (hetero)multivalent architectures to trigger glycosidase binding modes that are reminiscent of those harnessed by lectins, which should be considered when profiling the biological activity of multivalent architectures.

Identification of sperm equatorial segment protein 1 in the acrosome as the primary binding target of peanut agglutinin (PNA) in the mouse testis.

Peanut agglutinin (PNA), a plant lectin protein that recognizes the galactose ß (1 -> 3) N-acetylgalactosamine carbohydrate sequence, has been widely used as a sperm acrosome-specific marker; however, the acrosomal glycoproteins that specifically bind to PNA have yet to be identified. We herein purified and identified PNA-binding glycoproteins in the mouse testis using biotinylated PNA and streptavidin-coupled magnetic beads, and liquid chromatography-tandem mass spectrometry (LC-MS/MS), respectively. In six repeated experiments, sperm equatorial segment protein 1 (SPESP1) was detected most frequently as a PNA-binding glycoprotein, followed by dipeptidase 3, proacrosin-binding protein, and acrosin prepropeptide. The identification of SPEPS1 in the testis lysate and its PNA-bound fraction was verified with lectin and Western blot analyses, and the co-localization of PNA and SPEPS1 in acrosomes was confirmed with lectin- and immunohistochemistry. Since the PNA reactivity of sperm acrosomes was observed not only in normal mice, but also in SPESP1-deficient mice, although at lower levels, PNA was also considered to bind to other candidate glycoproteins. The present study identified SPESP1 in the acrosome as the primary binding target of PNA in the mouse testis. Further defining the specific lectin-glycoprotein relationships in individual cells will enhance the value of lectin histochemistry.

Quantitative approach to lectin-based glycoprofiling of thymic tissues in the control- and the dexamethasone-treated mice.

Dexamethasone (DEX) is the most commonly used synthetic glucocorticoid in treatment of various inflammatory conditions. Here we focused on evaluating the effect of DEX on apoptosis and glycan profile in the mouse thymic tissues. Histological examinations revealed that the DEX treatment cause severe alterations in thymus, such as disruption of thymic capsule, impaired epithelial cell-thymocyte contacts, cellular loss and increased apoptosis. The identification of thymic glycans in the control- and the DEX-treated mice was carried out by using a panel of five plant lectins, Maackia amurensis agglutinin (MAA), peanut agglutinin (PNA), Sambucus nigra agglutinin (SNA), Concanavalin A (ConA) and wheat germ agglutinin (WGA). Lectin histochemistry results showed that glycosylation pattern of thymus changes upon DEX treatment. For further detailed quantitative analyses of the binding intensities for each lectin, histochemical data were scored as high positive (HP), mild positive (MP) and low positive (LP) and differences among signaling densities were investigated. The staining patterns of thymic regions observed with lectin histochemistry suggest that DEX can affect the thymic glycan profile as well as thymocyte apoptosis. These results are consistent with the opinion that not only sialic acid, but also other sugar motifs may be responsible for thymocyte development.

Glycopolymeric gel stabilized N-succinyl chitosan beads for controlled doxorubicin delivery.

Here we report the synthesis and study of N-succinyl chitosan based hydrogel beads, stabilized with glycopolymeric network (NSC/Glc-gel) for application in anticancer drug delivery of doxorubicin (DOX). The bio-recognition of lectins by NSC/Glc-gel bead was also studied by UV-vis spectrophotometry. The beads were characterized using FT-IR, SEM and Thermogravimetric analysis. The extent of DOX loading was proportional to the degree of succinylation and the swelling kinetics of the beads showed pH dependency. The beads exhibited sustained release of DOX over a period of more than 15 days in an acidic pH, mimicking the microenvironment of tumor cells, and even lesser release at physiological pH. Release exponent 'n' derived from Korsmeyer-Peppas model implied that NSC88/Glc-gel (88% succinylation of chitosan) beads followed fickian diffusion controlled release mechanism whereas NSC75/Glc-gel (75% succinylation of chitosan) beads follow zero order release profile. The synthesized beads also displayed specificity to lectin Concanavalin A.