Massive investments are made in utilization of biomass for fuel and chemicals. Still, a range of processes need to be optimized. To this end, characterization of enzymes involved in degradation of the plant cell wall is important. However, the plant components of interest are highly complex structures, which complicates their isolation from biological sources. Therefore, chemical synthesis stands as the method of choice. The project presents routes to synthesize key fragments found in the plant cell wall.
The plant cell wall is a complex amalgam of polysaccharides and proteins, embedded in a highly hydrated matrix. While cellulose and hemicellulose are the most abundant polysaccharides, the project focuses on one group of non-cellulosic polysaccharides, namely pectin. Pectic polymers play crucial roles in signaling and in defense responses against herbivores and pathogens.
Rhamnogalacturonan II (RG-II) is a highly conserved cell wall pectic oligosaccharide. In industry, RG-II is mainly obtained from red wine after extractions and purification in several steps. In recent years, RG-II has attracted considerable interest due to potential desirable effects in human nutrition and health.
RG-II is composed of six structurally different oligosaccharides referred to as side chains A-F, which are attached to a linear homogalacturonan (HG) backbone. In the project, a pentasaccharide portion of the RG-II polysaccharide was synthesized. The synthesis commenced with commercially available D-galactose pentaacetate from which four discrete galactose building blocks were prepared. In this way, the synthesis of a HG backbone with the possibility to install branching was successfully accomplished by iterative glycosylations of these monosaccharide building blocks. To this end, a 3-deoxy-D-manno-2- octulosonic acid (Kdo) residue derived from side chain C was specifically targeted.
Furthermore, the optimal conditions for each glycosylation step was established. These achievements allowed for the assembly of tetra-galactoside with good to excellent yields (68 to 85%) in the glycosylation steps and high selectivity for the desired α-(1→4)-linked products. The target oligo-galacturonate was finally obtained after chemoselective deprotection of the 6-positions, followed by oxidation of the corresponding primary alcohol and introduction of the benzyl esters.
Moreover, the synthesis of Kdo on large scale was accomplished via a base-catalyzed aldol condensation between D-arabinose and oxaloacetic acid. In turn, this result enabled the development of a new shorter and higheryielding protocol to prepare a benzyl ester fluoride donor from the ammonium salt of Kdo. Finally, this donor was coupled to the HG backbone affording the desired pentasaccharide with high α-selectivity.
In conclusion, the developed protocol will allow for the introduction of other side-chains derived from RG-II, and could potentially be expanded to prepare a longer backbone chain via the iterative coupling of versatile monosaccharide building blocks. All the substrates that will be accessible after the final deprotection steps will prove to be useful for pectic enzyme studies.
Illustration:
Section of a wall in growing plant cells.