University of Groningen
Synthesis of Health-Promoting Carbohydrates
Verkhnyatskaya, Stella
DOI:
10.33612/diss.158661500
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Publication date: 2021
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Verkhnyatskaya, S. (2021). Synthesis of Health-Promoting Carbohydrates. University of Groningen. https://doi.org/10.33612/diss.158661500
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1.1
Introduction
Glycans are the most diverse class of biopolymers and this diversity is crucial for many cellular processes, including cell-to-cell recognition.1,2 The diversity of glycans
originates from their structure. They are built from monosaccharide blocks that are linked to each other in either 1,2-trans- or 1,2-cis-fashion (Scheme 1), and furthermore, each monosaccharide has several hydroxyl groups and this leads to the possibility to create linear or branched oligo- or polymer structures. When only one hydroxyl connects to the next sugar a linear polymer is created, whilst branched structures can be created with additional carbohydrates attached to the same core building block. Impressively, the human glycome (the entirety of carbohydrates both free and bound) is constructed from only ten monosaccharide building blocks giving rise to a tremendous number of glycans.3 For instance, one class of compounds of the human
glycome, human milk oligosaccharides (HMOs) that are present in human milk, are constructed from five different monosaccharides (D-glucose, D-galactose, N-acetyl-D -glucosamine, L-fucose, and N-acetylneuraminic acid) and there are more than 200 structures identified.4 These molecules have an important role in infant health
development, specifically, they have nutritional value for beneficial bacteria, prevent pathogenic bacteria to adhere to epithelial cells, and serve as immune modulators. 4
Chapter 1
14
Chapter 1
Scheme 1. Illustration of 1,2-trans and 1,2-cis glycosidic linkages. The numbering starts from the anomeric position 1. 1,2-trans or 1,2-cis refers to the relative orientation of the substituents at positions C-1 and C-2.
In comparison to the human glycome, the bacterial glycome is significantly larger and is based on more than 25 abundant monosaccharides in combination with a variety of genera- or strain-specific monosaccharides.5-7 Because the bacterial cell envelope is
covered with a layer of polysaccharides that contain unique structural features, that are different from the human glycome,5,7 those cell envelope structures are recognized by
the immune system allowing for the differentiation between pathogenic and beneficial bacteria. Therefore, researchers are investigating specific structural features of the cell envelope of pathogenic bacteria to potentially produce vaccines.8 When it comes to
beneficial bacteria, knowing which structural feature of the glycan leads to a health-promoting effect can give a possibility to supply these molecules artificially to mimic that effect.
To investigate which structural components are responsible for specific health effects, pure samples are a must. However, isolating glycans from natural sources is often complicated by the presence of several cell components and other glycans, which also hampers a thorough characterization. Therefore synthetic chemistry is employed to produce pure samples for characterization and biological evaluation.
1.2
The glycosylation reaction
To chemically synthesize a part of a glycan (oligosaccharide) a carefully designed synthetic plan is necessary. Two types of molecules are necessary to build a glycosidic bond: a donor, containing an activatable leaving group at the anomeric center, and an acceptor (the nucleophile) (Scheme 2). Several choices have to be made, for instance, the choice of protecting group at the anomeric position, that can be activated selectively at will, and which protecting groups will be used at the other positions on the carbohydrate structure, as they affect the reactivity of both donor and acceptor and can influence the stereochemical outcome. Often a synthetic strategy includes an orthogonal protecting group to allow the decoration or elongation, and a protecting group that can direct the formation of one type of linkages, either 1,2-trans or 1,2-cis linkages.
There are two main participants in a glycosylation reaction: donor 1 and acceptor 2, which is generally another carbohydrate molecule (Scheme 2). When a donor undergoes activation, several reactive species may be produced: covalent species such as triflate 3, and oxacarbenium 4. The oxacarbenium ion species gives the reaction a
more SN1-like character, while covalent species can be substituted in an SN2-fashion.
15 depends on many factors such as stability of the reactive species, temperature, the concentration of reactants, and the reactivity of the acceptor.9 One way to gain control
over stereoselectivity is to introduce an ester as a protecting group at the C-2 position, in which case the coupling reaction may occur via dioxolenium species 5, giving trans-linked product 7. However, no general method for the construction of cis-linkages has been developed to date.10,11
Scheme 2. General glycosylation mechanism; a new glycosidic bond is shown in red (R – protecting group, G – protecting group at C-2, either benzyl-like or acetyl-like; LG – leaving group)
1.3
Thesis Outline
The aim of the work described in this thesis was to design and synthesize oligosaccharides with potentially health-promoting beneficial effects for infant health development. In Chapter 2 non-digestible carbohydrates are reviewed, including galacto- (GOS) and fructooligosaccharides (FOS), pectins, and exopolysaccharides among others, that can mimic functions of HMOs and serve as prebiotics, i.e. improve the growth of beneficial bacteria. Inspired to mimic the function of HMOs, a fucosylated cyclodextrin was designed and the projects on this topic are described in Part 1. The synthesis of fucosylated β-cyclodextrin proceeded with high stereo- and regioselectivity yielding di-α-fucosylated β-cyclodextrin (DFβ-CD), which is described in Chapter 3. Intriguingly, the described fucosylation was highly stereo- and regioselective even though an excess of a donor was used per hydroxyl/monosaccharide residue. To understand the origin of the selectivity observed in this coupling, molecular dynamics simulations were performed, revealing an unexpected SNi-type mechanism, and various interactions between the benzyl groups
of the donor molecule and the cyclodextrin barrel (Chapter 4). The di-α-fucosylated β-cyclodextrin (DFβ-CD) was prepared with a biological function in mind. In Chapter 5 its digestibility and effect on bacterial adhesion were studied. Impressively, DFβ-CD was resistant to digestive and microbial enzymes in vitro and demonstrated anti-adhesive properties against enterotoxigenic Escherichia coli strain O78:H11.
Part 2 of this thesis is dedicated to the efforts towards the synthesis of the repeating unit of the exopolysaccharide (EPS) of Bifidobacterium adolescentis (probiotic bacterium that may give beneficial health effects to the host). This EPS contains 6-deoxy-L-talose (6dTal) residues,12 which is a rare monosaccharide that has all
16
Chapter 1
hydroxyl groups in the cis-orientation. This EPS contains 6dTal residues that are linked via (1→3)-cis- and (1→2)-trans-bonds, and the trans-linked residues contain a glucose residue at the O-3 position. Chapter 6 is focused on the synthesis of the trans-linked part of the repeating unit. Two strategies were evaluated: generation of orthogonally protected 6dTal building blocks, where hydroxyl groups can be liberated at will; and a regioselective glycosylation strategy in which the glucose was introduced on a 6-deoxytalose diol-acceptor. The second part of the molecule contains only cis-linkages, and a thorough evaluation of different methods to construct cis-taloside linkages is described in Chapter 7. Chapter 7 covers the reactivity and selectivity of 6-deoxytalose building blocks in an attempt to construct cis-glycosidic bonds with primary and secondary acceptors. After optimizing the conditions for cis-glycosylation for 6-deoxytalose, the total synthesis of the full nonasaccharide repeating unit was successfully accomplished, as reported in Chapter 8.
1.4
References
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4. Bode, L. Glycobiology 2012, 22, 1147-1162.
5. Adibekian, A.; Stallforth, P.; Hecht, M.; Werz, D. B.; Gagneux, P.; Seeberger, P. H. Chem. Sci. 2011, 2, 337-344.
6. Toukach, P. V.; Egorova, K. S. Nucleic Acids Res. 2016, 44, D1229-D1236.
7. Herget, S.; Toukach, P. V.; Ranzinger, R.; Hull, W. E.; Knirel, Y. A.; von der Lieth, C. BMC Struct. Biol. 2008, 8, 35.
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12. Nagaoka, M.; Muto, M.; Yokokura, T.; Mutai, M. J. Biochem. 1988, 103, 618-621.
