Chemoenzymatic Synthesis Of HNK-1-Containing O-Mannose Glycans

The HNK-1 epitope(human natural killer-1 epitope)is a unique trisaccharide structure(HSO3-3GLcA?1-3Gal?1-4GlcNAc)containing a terminal 3-O-sulfated glucuronic acid(GlcA)?1-3-linked to N-acetyllactosamine(LacNAc,LN),which connected to the terminal of glycolipids as well as N-and O-glycans.It is mainly expressed in the nervous system,especially in brain and peripheral nervous tissues and plays important biological rolesHNK-1 glycans have many physiological effects in brain tissue and peripheral nervous tissues which perform different functions at different stages of the development of nervous system.This neural specific epitope plays crucial roles in synaptic plasticity,spatial memory formation,motor nerves regeneration,and peripheral nerve repair.HNK-1 is also the target of autoimmune antibodies in patients with Guillain-Barre syndrome and other neuropathies due to its high autoimmunogenicity.Its derivatives and mimetics have shown great potential in antigen-specific treatment for antimyelin-associated glycoprotein(anti-MAG)neuropathy.Besides,HNK-1 glycans can promote synaptic myelin regeneration and reconstruction which can promote functional recovery for injured spinal cordHNK-1 epitope was initially discovered on O-mannosylated glycoproteins in brain tissues in 1997.Despite their abundance,studies on mammalian O-mannosylation have been mainly focused on ?-dystroglycan(oc-DG).According to the initial connecting type,the O-mannose glycans are mainly classified into four core types:Core M0,Core M1,Core M2 and Core M3.To date,more than 20 O-mannosyl glycans have been identified,including 5 O-mannosyl glycans containing HNK-1 epitope which later characterized as a unique component of Core Ml and Core M2 O-mannosyl glycans.O-mannosyl glycans account for over 30%of total O-glycans in brain tissues.It is therefore not surprising that the HNK-1 epitope on branched O-mannosyl glycans plays important roles in brain development and remyelination.However,at present,only the first two steps of glycosylation are known in the O-mannose biosynthetic pathway,and the related enzymes for further extensions have not been confirmed.Besides,because of the structural complexity,it is very difficult to isolate and identify from the organisms,and more potential O-mannose glycans remain to be discovered and confirmed in the future.This makes it difficult to reveal their structure and activity relationship between O-mannose and related proteins at the molecular level.The first task to solve this problem is to build an abundant O-mannosyl glycan library.Due to the structural complexity of the HNK-1 epitope,the synthesis of HNK-1-containing O-mannose glycans is very challenging.Until now,the synthesis of HNK-1 epitope is just based on the simple trisaccharide structure and its derivatives,there are no reports about the synthesis of HNK-1-containing O-mannose glycans.There are some challenges in the synthesis of them:the stereoselectivity and regioselectivity of the glucuronide glycosidic bond,the site-specific sulfation of glucuronic acid,the connection of HNK-1 trisaccharide with the core structure of O-mannose,and the extension of the carbohydrate chains.Therefore,the chemical synthesis of O-mannose glycans involved multiple steps,complicated protective group operation,resulting in low overall yield.Besides,due to the difficulty of recombinant expression of sulfotransferase,the current enzymatic synthesis can only obtain nonsulfated HNK-1 structure or its analogue.So far,a practical synthetic approach for the synthesis of O-mannose containing the HNK-1 epitope is still missing.In this thesis,given the synthetic challenges of HNK-1 epitope-containing O-mannose glycans,we have developed a highly efficient chemoenzymatic synthesis strategy.15 O-mannose glycans containing sulfated HNK-1 epitope and 15 O-mannose glycans containing nonsulfated HNK-1 epitope were systematically and efficiently synthesized for the first time at preparative scales.This thesis mainly contains the following parts:(1)A HNK-1 lactone trisaccharide donor ?-32 and three mannosyl building block receptors ?-33??-39??-42 were rationally designed,and the Core Ml intermediate ?-1 and Core M2 intermediates ?-2 and ?-9 of HNK-1 epitope O-mannose were efficiently synthesized through chemoenzymatic approaches.(2)Four enzymatic modules were successfully developed,including ?1-4-galactosylation,?1-3-glucuronylation,?1-3-fucosylation and ?2-3-sialylation systems(3)Two core structures III-2 and III-9 were synthesized and used as key intermediates for the synthesis of HNK-1 epitope-containing O-mannose Core M2 glycans.Three enzymatic modules were sequentially applied to and introduce galactose,fucose,sialic acid residues for chain elongation for the synthesis of fourteen Core M2 HNK-1 epitope O-mannose ?-3—?-15.(4)Starting from three core structures ?-44—?-46,fifteen corresponding nonsulfated HNK-1 O-mannose ?-16—?-30 were systematically synthesized using a diversity-oriented enzymatic modular assembly strategy.The innovations of the research including the following parts(1)In this research,a highly efficient chemoenzymatic synthesis strategy was developed.Fifteen highly challenging sulfated HNK-1 epitope-containing O-mannose glycans and fifteen nonsulfated HNK-1-containing O-mannose glycans were efficiently and systematically synthesized for the first time(2)The systematic and efficient synthesis of O-mannose glycans containing HNK-1 epitope provides important standards for the further study of the biological functions of O-mannose glycans,revealing the function and mechanism of related carbohydrates in brain tissues at the molecular level,and providing new methods of diagnosis and treatment of related neurological diseases(3)The chemoenzymatic strategy developed in this paper is also suitable for the synthesis of other complex glycoconjugates containing HNK-1 epitope,such as the synthesis of related N-glycans.