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Understanding and Controlling Glycosylation Reactions

Periodic Reporting for period 2 - GLYCONTROL (Understanding and Controlling Glycosylation Reactions)

Reporting period: 2018-11-01 to 2020-04-30

Carbohydrates are all around us, they are present in all kingdoms of life and they play important roles in a many biological processes and are relevant to many -if nor nearly all- diseases, including cancer and infectious diseases. To study the role of carbohydrate sin these processes well-defined carbohydrate molecules (oligosaccharides) are required. because isolation of these materials from natural sources is often impossible, chemists are developing methods to make these molecules in the laboratory by organic chemical synthesis. Although great progress has been made in the field, the assembly of complex oligosaccharides continues to pose a big challenge because the central reaction used to stitch carbohydrates together, the glycosylation reaction, remains ill understood. This project is directed at unraveling the mechanistic details behind this crucial reaction and it does so by taking an experimental and computational approach. It surveys the influence of the reactivity of both reaction apartness that are united in the reaction, the so-called donor and acceptor glycosides and tries to match the reactivity of these by external reagents. Using the insight gained in the reaction, complex biologically relevant oligosaccharides will be assembled. Glycans of pathogenic worms (schistosomes) will be synthesised and used for diagnostic purposes. And so-called galactosamine galactans will be synthesised to use these in studies towards the biosynthesis of these molecules, which occur in pathogenic bacteria (such as Pseudomomans aeruginosa) and pathogenic fungi (Aspergillus fumigatus).

also see: https://www.eye-openers.nl/en/meetthecrew/jeroen-codee/
"We have developed a system to systematically determine the reactivity of acceptor glycosides in the glycosylation reaction. This has provide key insight in how the reactivity of the acceptor influences the outcome of the glycosylation reaction. (publication #1 and #6)
The method is being extended to probe a wide variety of acceptors and investigate the reasons behind the reactivity differences. Wit the insight gained, the mechanism of various glycosylation reactions are being probed.

We have developed synthetic methodology that allows one to stereoselectively construct 1,2-cis glucosidic linkages using reactivity modulators that match the reactivity of the donor glycoside to the reactivity of the acceptor glycoside. (publication #2 and #7)
The knowledge gained here is being used to assemble different biologically relevant oligosaccharides.

We have developed a computational method to map the stability of oxocarbenium ions, important reactive intermediates i the glycosylation reaction, as a function of their overall shape. This has allowed us to rationalise the sometimes striking stereoselectivity observed in addition reactions to these ions. (Publication #5).
The developed method is being used to explore different oxocarbenium ions as well as dixolenium ions (Hansen et al. Characterization of Glycosyl Dioxolenium Ions and Their Role in Glycosylation Reactions, ChemRxiv 2019).

We have developed synthetic chemistry (not yet published), which allows the efficient construction of 1,2-cis galactose/galactosamine linkages, which has been used in the assembly of a broad panel of galactosaminogalactans. The generated structures have bene used to unravel the mode of action of biosynthesis enzymes, that degrade these polysaccharides. (Publications #3 and #4)
The chemistry is being used to expand the library of structures, generate new biosynthesis probes and achieve the synthesis of new synthetic targets.

We have developed methodology that has allowed the assembly of Schiostosome derived glycans (not published yet). This has been used to assemble a small set of glycans that have been used to screen sera for the presence of antibodies against these structures, as this is a strong indicator for infection (not published yet)."
The results obtained so far have gone beyond the start-of-the-art as there was no effective methodology available to probe the influence of the reactivity of the acceptor glycosides on the mechanism and outcome of glycosylation reactions. The tool developed will be used in the remainder of the project (and beyond) to investigate a much wider panel of acceptors to delineate structure-reactivty-stereoselectivuty principles.

Th use of reactivity modulators has been presented in the context of a novel protecting group strategy. This approach is conceptually novel and has gone beyond strategies know before as it reliably allows one to match the reactivity of both coupling partners (the donor and acceptor glycosides), bearing uniform protecting groups. The strategy will be adopted for the assembly of other biologically relevant glycans within the project and beyond.

The computational method developed has gone significantly beyond the state-of-the-art as no effective method was available to probe the stability of oxocarbenium ions as a functional of all conformations (shapes) they can adopt. This method is now being used to probe many other oxocarbenium ions and dioxolenium ions (Hansen et al. Characterization of Glycosyl Dioxolenium Ions and Their Role in Glycosylation Reactions, ChemRxiv 2019). It will be used to dissect stereoelectonbic effects for various functional groups and illuminate how these effects shape the outcome of glycosylation reactions.

The methodology developed for the assembly of galactosaminogalactans will be used to generate a larger library of structures. These will serve as probes for other biosynthesis enzymes. attachment of the molecules to carrier porteins will provide model vaccines that can be used to elicit antibodies. These may be used as diagnostic tools, research tools and as therapeutic agents. If successful the GAG-conjugates can be developed into prophylactic vaccines. The synthesis chemistry that has been developed will be translated to other glycans to expand the scope of the methodology and generate other biologically relevant glycans.

The synthesic methodology that has been at the basis of the assembly of the schistosome glycans, can be extended to generate even more complex schistosome glycans and other biologically relevant oligosaccharides. initial biological results have revealed two glycans that can be used in a diagnostic setting to reliably report on schistosome infections. These glycans will be used to screen a larger cohort of patients (sera) to further show applicability of the material.
A computational method reveals the conformational behaviour of glycosyl oxocarbenium ions