Synthesis of hyaluronic acid oligosaccharides and exploration of a fluorous-assisted approach
Graphical abstract
Introduction
Hyaluronic acid is a negatively charged linear polysaccharide that belongs to the glycosaminoglycan family. It is constituted by the repetition of disaccharide units of d-glucuronic acid (GlcA)-β(1→3)-N-acetyl-d-glucosamine (GlcNAc)-β(1→4). Hyaluronic acid is the only glycosaminoglycan that does not contain sulfate groups. Despite its simple chemical structure, hyaluronic acid is involved in a wide range of biological processes, such as cell-migration, recognition and tumor invasion.1 The specific biological activities of hyaluronic acid strongly depend on the length and molecular weight of the carbohydrate chain.2 For example, high molecular weight polymers are anti-angiogenic while smaller oligosaccharide fragments induce angiogenesis. In this context, well-defined synthetic oligosaccharides are useful to elucidate the role of hyaluronic acid chains in nature. In particular, synthetic oligosaccharides, with defined sequence and length, are required to study, at the molecular level, the interactions between hyaluronic acid and certain protein receptors that trigger the biological functions of this natural product. These studies can potentially lead to the interference and control of a particular biological process.
Several approaches have been reported for the synthesis of hyaluronic acid oligosaccharides.3, 4, 5 Two main strategies have been employed for the preparation of these molecules.6 In the pre-glycosylation oxidation strategy, glucuronic acid building blocks are directly used in coupling reactions.7, 8, 9, 10, 11, 12, 13, 14 The low reactivity of these units can afford low to moderate yields in the glycosylation reactions.15, 16, 17 For this reason, an alternative post-glycosylation oxidation approach has been also considered. In this second method, glucose moieties are used for the assembly of the oligosaccharide chain.6, 18, 19, 20, 21, 22, 23 Then, glucose units are oxidized to the corresponding glucuronic acids to give the final hyaluronic acid molecules. The oxidation of highly elaborated intermediates is, however, a complex and challenging synthetic step.
Recently, several methods have been reported to accelerate the preparation of hyaluronic acid oligomers, facilitating the purification of synthetic intermediates. Huang and Huang6 developed a pre-activation based iterative one-pot strategy in which anomeric reactivity adjustment and intermediate oligosaccharide purifications are not required. Using a fully automated solid-phase approach, Codée, van der Marel and co-workers24, 25 described the synthesis of hyaluronic acid fragments up to the pentadecamer level.
Here, we describe the total synthesis of one tri- and one tetrasaccharide that correspond to the structure of hyaluronic acid, using a stepwise strategy. Glucuronic acid building blocks were directly employed for the glycosylation reactions. Additionally, we have explored the use of a fluorous tag to facilitate the preparation of this type of compounds.
Section snippets
Results and discussion
For the preparation of hyaluronic acid oligosaccharides, we have followed the retrosynthetic analysis shown in Scheme 1. We envisaged that final molecules, such as tetrasaccharide 1, could be obtained from the corresponding fully protected precursors, such as 2, by a three-step sequence of deprotection reactions (basic hydrolysis, selective N-acetylation, and hydrogenolysis). The oligosaccharide backbone was assembled by linking monosaccharide building blocks 3, 4, and 5. The levulinoyl (Lev)
Conclusion
We have described a strategy for the preparation of hyaluronic acid oligosaccharides. Suitably protected GlcA and glucosamine monosaccharide building blocks were used for the assembly of the hyaluronic acid chain. Participating groups at position 2 of these units ensured the selective formation of the desired β glycosidic bonds. A sequence of deprotection reactions efficiently afforded the final tri- and tetrasaccharide fragments. An alternative fluorous-assisted approach was also explored.
General procedures
Thin layer chromatography (TLC) analyses were performed on silica gel 60 F254 precoated on aluminum plates (Merck) and the compounds were detected by staining with sulfuric acid/ethanol (1:9), with cerium (IV) sulfate (10 g)/phosphomolybdic acid (13 g)/sulfuric acid (60 mL) solution in water (1 L), or with anisaldehyde solution [anisaldehyde (25 mL) with sulfuric acid (25 mL), ethanol (450 mL) and acetic acid (1 mL)], followed by heating at over 200 °C. Column chromatography was carried out on silica
Acknowledgments
We thank the Spanish Ministry of Economy and Competitiveness (Grants CTQ2009-07168 and CTQ2012-32605) and the European Union (FEDER support) for financial support.
References (36)
- et al.
J. Biol. Chem.
(2003) - et al.
Eur. J. Cell Biol.
(2006) - et al.
Tetrahedron
(2004) - et al.
Carbohydr. Res.
(1996) - et al.
Tetrahedron Lett.
(2007) - et al.
Carbohydr. Res.
(2010) - et al.
Carbohydr. Res.
(2011) - et al.
Tetrahedron Lett.
(1993) - et al.
Carbohydr. Res.
(1994) - et al.
Tetrahedron: Asymmetry
(1994)
Carbohydr. Res.
Carbohydr. Res.
Adv. Carbohydr. Chem. Biochem.
Tetrahedron
J. Org. Chem.
Adv. Synth. Catal.
Chem.-Eur. J.
J. Org. Chem.
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