Synthesis and application of RG-II oligosaccharides

Carbohydrates are the predominant component of biomass on earth as they are an integral part of all living species, including plants, animals, and bacteria. Carbohydrates have important structural and functional roles in biology but remain, in contrast to the other two main classes of biopolymers, oligonucleotides and proteins, poorly understood. Plants produce an enormous diversity of carbohydrates that fulfill essential roles during the life cycle of the plant. Plants contain many unique carbohydrates that are not found in animals but are still mostly conserved across the plant kingdom. The vast majority of plant carbohydrates are part of the cell wall. Plant carbohydrates are major nutritional resources in food and feed and receive an enormous interest as sources of renewable materials and for the production of fuels and chemicals. A prerequisite to enhance the economic viability of plant biomass as a renewable resource is a detailed knowledge of the carbohydrate structures in the cell wall and how they are constructed by the plant. However, access to pure carbohydrates for structural and biological studies is difficult due to their high molecular complexity. The structurally most complex carbohydrate in nature is rhamnogalacturonan-II (RG-II), a highly conserved pectic polysaccharide, containing twelve different types of monosaccharides that are connected through 20 different linkages. The complex structure of RG-II poses enormous challenges for organic chemists, including the synthesis of rare and acidic monosaccharides, a highly congested structure, and a high number of 1,2 -cis-glycosidic bonds. We will develop new synthetic strategies to access oligosaccharide fragments of the RG-II structure, which will be printed on glass slides in a robot assisted manner to investigate carbohydrate-protein interactions. The availability of such glycan arrays with highly complex RG-II fragments will for the first time enable the systematic elucidation of enzymes (glycosyltransferases) involved in RG-II biosynthesis and the characterization of molecular tools (antibodies), aiming at detecting and monitoring specific RG-II epitopes within plant cell walls. The generated knowledge will facilitate future developments towards improved biomass digestibility, material strength of plant-derived products, and the shelf life of fruits and vegetables.

Plant cell walls are one of nature's most impressive engineering achievements. They give plants their strength and flexibility, and they are also a major renewable resource for textiles, paper, building materials, and bio-based fuels. A particularly important, but very rare, component of these walls is rhamnogalacturonan-II (RG-II) - a remarkably complex sugar polymer that is essential for normal plant growth and development. RG-II is part of the pectin matrix in the cell wall and acts like a molecular "cross-brace." It forms boron-dependent cross-links that help create a three-dimensional network, contributing to cell wall stability, mechanical strength, and controlled porosity. Despite its crucial role and conserved structure across land plants, RG-II is extremely difficult to study because it contains many unusual sugar units and linkages, and occurs only in very small amounts in nature. This has severely limited research progress and the development of tools to understand and exploit plant cell wall architecture. Our project set out to overcome this obstacle by using advanced organic chemistry to build RG-II fragments in the laboratory. The goal was to create pure, well-defined pieces of RG-II that can be used as molecular tools to study how this polymer is made, how it functions, and how it can be detected in different plant tissues. During the project, we achieved two major breakthroughs: 1) We succeeded in the total synthesis of RG-II side chain A, the portion of RG-II that carries the apiose residue involved in borate cross-linking. 2) We prepared a pentasaccharide fragment of RG-II side chain B containing the rare sugars apiose and aceric acid, which do not occur in most common carbohydrates and must be built from scratch. These synthetic RG-II fragments are now being used to investigate its 3D-structure and to create glycan arrays - microscopic "sugar chips" on which many different carbohydrate structures are printed. By screening these arrays with antibodies that recognize pectins, and with candidate biosynthetic enzymes, we can begin to identify RG-II-specific antibodies and the glycosyltransferases that build RG-II in the plant's Golgi apparatus. Better tools to visualize and understand RG-II will help scientists unravel how plant cell walls are assembled and maintained, and how their properties can be tuned.