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Understanding and Controlling Glycosylation Reactions

Periodic Reporting for period 4 - GLYCONTROL (Understanding and Controlling Glycosylation Reactions)

Reporting period: 2021-11-01 to 2022-10-31

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 carbohydrates in 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. In this reaction, two forms of the product can form (referred to as the alpha- and beta-products) of which only is desired. 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 partners that are united in the reaction, the so-called “donor” and “acceptor” glycosides and tries to match the reactivity of these by external reagents to achieve high yielding and selective reactions. The gathered knowledge will be applied in syntheses of complex glycans that can be used to answer biological questions and enable the development of new diagnostic tools and innovative vaccines. The synthetic molecules can be used to discover new carbohydrate processing enzymes and open up new avenues to develop antibiotics.
We have developed a system to systematically determine the reactivity of acceptor glycosides in the glycosylation reaction. This has provided key insight in how the reactivity of the acceptor influences the outcome of the glycosylation reaction. We have used this methodology to, for the first time, systematically investigate how the different groups on carbohydrate building blocks influence their reactivity and with that the outcome of the glycosylation reaction. To understand the stability and reactivity of reactive intermediates formed during the reactions, we have developed computational methods that systematically map the effect the ring substituents have on the shape and stability of these species. Our computational work has been combined with state-of-the-art spectroscopy techniques to measure these intermediates.
To modulate the reactivity of the glycosylation reactions and match the reactivity of the two coupling partners (the “donor” and “acceptor”) we have introduced different reactivity modulators and we have shown how to employ these to achieve highly stereoselective glycosylations.
Using the mechanistic insight gained by our computational and experimental investigations, we have been able to develop highly effective glycosylation reactions that have been used to effectively assemble complex biologically relevant oligosaccharides. Glycans of pathogenic worms (schistosomes) have been synthesized and used for diagnostic purposes. With the synthesized glycans we have been able to detect antibodies directed at the glycans of the worms and we have been able to correlate these to recent infections. These preliminary results show that the glycans can be used in future diagnostic tools to help eradicate these impactful infections. So-called galactosaminogalactans have been synthesized and used in studies to unravel the biosynthesis of these molecules, which occur in pathogenic bacteria (such as Pseudomonas aeruginosa) and pathogenic fungi (Aspergillus fumigatus). Using our synthetic glycans the mode of action of several biosynthesis enzymes has been unraveled and this paves the way to interfere with this machinery opening up new avenues for the development of antibiotics. In addition, we have been able through detailed structural studies to discover a new type of molecular interaction that stabilizes the overall structure of the oligosaccharides. We have been able to generate well-defined fragments of polysaccharides of Staphylococcus aureus, a “super bug” that has acquired resistance to almost all available antibiotics. The synthesized structures can be used in innovative synthetic vaccines to combat this successful bacterium. The stereoselective glycosylation methodology that we developed has also allowed us to generate synthetically challenging glycomimetics (“sugar lookalikes”) that can be used to discover and inhibit carbohydrate processing enzymes.

The results of these studies have been published in various publications, that have been published in high tier journals, reaching a large and diverse audience. The results have been presented in many lectures, given at conferences and at universities and research institutes.
Where glycosylation reactions are generally optimized in a time- and labor consuming trial-and-error manner our results have provided handles for rational optimization. The structure-reactivity-stereoselectivity principles that we uncovered will serve as a guide to enable and improve many glycosylation reactions in the future. This will expedite the assembly of glycans and make these highly-sought after commodities available to for further biological and medical research.
The computational methods we have developed have been used to understand the reactivity of “families of compounds” as opposed to studies that focus on a single substrate. This has allowed us to understand how all functional groups present on the carbohydrate rings influence the reactivity of these as stand-alone entities but also in the context of the substituents present. The insight gained has gone far beyond the state-of-the-art as no tools were available to systematically dissect the stereo-electronic effects of these groups.
With our developed methodology we have been able to generate biologically relevant glycans that have never been synthesized before and that therefore have never been available for biological follow-up studies. We have thus been able, by use of the library our synthetic glycans, to unravel the mode of action of different biosynthesis enzymes, the activity of which was previously ill-understood. This will open up possibilities to interfere with these enzymes and in doing so develop new antibiotics.
The insight gained into the glycosylation reaction has made it possible to generate complex bacterial glycans, previously beyond reach, that are now available for the development of synthetic vaccines.