Report

Teaser, summary, work performed and final results

Periodic Reporting for period 2 - GLYCO-TOOLS (Bio-Inspired Tools for Glycoscience)

Teaser

Cell surface carbohydrates play key roles in cell recognition mechanisms. O-glycosylation is a ubiquitous post-translational modification that is highly dynamic and responsive to cellular stimuli through the action of cycling enzymes. Expression of specific O-glycans is linked...

Summary

Cell surface carbohydrates play key roles in cell recognition mechanisms. O-glycosylation is a ubiquitous post-translational modification that is highly dynamic and responsive to cellular stimuli through the action of cycling enzymes. Expression of specific O-glycans is linked to changes in gene expression in, for example, inflammatory bowel disease, cystic fibrosis and several types of cancer.
Protein-carbohydrate interactions typically exhibit high specificity and weak affinities toward their carbohydrate ligand. This low affinity is compensated in nature by the architecture of the protein, the host presenting the carbohydrate ligands in a multivalent manner or as clusters on the cell or mucosal surface. This effect is known as the multivalency or “cluster–glycoside effect” and has been well documented for lectin–carbohydrate interactions as increasing ligand affinity and selectivity. The fundamental understanding of these glycosylation patterns at molecular and functional levels will allow mechanisms associated with bacterial-host interactions, bowel disease and several cancers to be defined, which will facilitate the identification of effective treatments and diagnostics for these conditions in due course.
This is a multidisciplinary project involving synthetic organic and inorganic chemistry, enzymology and glycobiology. The proposal centres on the development of expedient synthetic and chemo-enzymatic methodologies for the preparation of novel multivalent O-glycan probes that will be used in the screening of O-glycosylation-linked interactions in health and in disease. These studies will help us understand the parameters controlling the combinatorial diversity of O-glycans and the implications of such diversity on receptor binding and subsequent intracellular signalling, which in turn will lead us to the development of new glycan-based diagnostic tools and therapeutics.

This research programme is comprised of three main objectives:

A. Development of expedient and catalytic methods for oligosaccharide assembly, which will pave the way to the automation of oligosaccharide synthesis.
B. Chemical and enzymatic synthesis of O-glycan targets. Harnessing of the glyco-synthetic machinery for the combinatorial synthesis of O-glycan probes.
C. Preparation of novel glyco-nanoparticles for the development of glycan-based targeted drug delivery systems (Smart Glyco-Nanomaterials).

Work performed

During the first 18 months of the project, work towards WP1, WP2 and WP3 has been carried out in parallel to address some of the key proposed aims of the project.
Specifically:

WP1/WP2: We have developed a bifunctional organocatalyst for the stereoselective glycosylation of 2-nitrogalactals to access mucin type O-glycans and is now reported in Org. Lett. 2016, 18, 4222.

WP1: An organocatalytic method, using cooperative Brønsted acid-type organocatalysis has been developed for the stereoselective synthesis of deoxyglycosides is now published in J. Org. Chem. 2017, 82, 407.

WP2: A novel class of imidazolium-based labels have been developed to harness the glycosynthetic machinery of human milk and we have demonstrated the technology on the chemo-enzymatic synthesis of biologically relevant O-glycans. The work has now been published in Org. Biomol. Chem. 2017, DOI: 10.1039/C7OB00550D.

WP3: A series of mucin type O-glycans have been conjugated onto fluorescent QDs and the novel probes evaluated against a panel of cancer cell lines. Initial experiments suggest different internalization profiles based on the type of glycan on the QD. A manuscript is currently in preparation.

WP3: A new class of sp3 nanocrystalline carbon dots have been developed as non-toxic fluorescent platforms for intracellular delivery. The work has now been pulished in Nanoscale 2016 , 8, 18630

Final results

Access to structurally defined oligosaccharide libraries is key for the advancement of glycobiology research and also for carbohydrate-based drug discovery. Therefore, the development of general and efficient methodologies for the synthesis of oligosaccharide tools which are not restricted to specialized labs, has the potential to transform the field.

During the first period of this programme, we have devoted efforts towards addressing the major hurdles in oligosaccharide synthesis. The ability to perform O-glycosylation reactions in a catalytic and stereoselective manner is one of the main remaining challenges in carbohydrate chemistry. To address this, our team has developed a series of novel and more efficient catalytic methods for the synthesis of an important class of O-glycosides (namely 2- and 2,6-deoxyglycosides). Using our novel methodologies, a series of biologically important glycosides, which are otherwise not available, have been prepared and will be evaluated in biological studies in due course. Another of the main problems for the automation of oligosaccharide synthesis is the requirement for purification after each step, which is normally accomplished by chromatographic methods. To address this, our team had developed the Ionic Catch and Release Oligosaccharide Synthesis methodology (ICROS) that relies on the use of solution phase ionic-liquid-based purification probes (ITags) which are compatible with chemical and enzymatic transformations. We have taken this techonology a step further and demonstrated that ITag-labeled glycan probes could be used to harness the natural biosynthetic machinery present in human breast-milk to access biologically important oligosaccharides. We showed that we can circumvent lengthy and costly chemical syntheses, as well as the need to isolate a specific enzymes, which can be challenging to express and applicable only to small-scale production. (http://blogs.rsc.org/ob/2017/05/24/synthesis-of-challenging-oligosaccharides-by-harnessing-glycosyltranferase-activity-directly-from-human-breast-milk/).

Another aspect of the programme is aimed at the development and application of carbohydrate-coated fluorescent nanoparticles (NPs) as a powerful tool to screen for carbohydrate-mediated interactions and exploit those for therapeutic applications. We had previously shown that glycan-coated fluorescent CdSe/ZnS quantum dot (QD) could be used to enable control of nanodot uptake and intracellular localization in cancer and in immortalised corneal epithelial cells and that the inherent fluorescent of the nanoparticle was ideal for live cell imaging. However, although we showed that glycan density mitigates the inherent toxicity of heavy metals such CdSe, these QDs remain less than ideal for in vivo applications. In order to broaden the scope of functional nanomaterials for biomedical purposes, we have now develop a one-pot, three-minute, gram-scale synthesis of water-soluble, and fluorescent carbon dots (FCDs) from simple and cheap sugar starting materials. We have successfully demonstrate the utility of lactose functionalized FCDots as non-toxic fluorescent intracellular delivery vehicles.

Website & more info

More info: http://galanresearch.com.