Development Of Highly Efficient Strategies For The Synthesis Of O-mannose Glycans And Mannopentaose

Cell surface glycans with diverse structures play pivotal roles in many important physiological and pathogenic processes.Owing to its structural complexity, it is almost impossible to obtain structurally defined carbohydrates inappreciable amounts from natural sources. Thus, the limited sources of glycans samples set a bottleneck to structural and functional studies of bioactive glycans. Fortunately, there has been a great effort invested in establishing chemical and chemoenzymatic approaches for the synthesis of oligosaccharides, which provides powerful access to obtaining glycans and glycoconjugates for biological investigations.This thesisis mainly composed of two parts:the chemoenzymatic synthesis of six Core M1 O-mannose glycans and the one-pot chemical synthesis of a unsymmetrical 3,6-branched mannopentaose (Man5).Two highly efficient strategies were developed for the synthesis of these two type of glyans at preparative scale which can serve as invaluble probes for their biological studies.1 Chemoenzymatic synthesis of Core Ml O-mannose glycansRecent years, more and more effort has been devoted to the study of the "uncommon" O-mannose glycans due to their diverse structures and essential bioactivities. O-mannose glycans on α-dystroglycan (α-DG) directly link with the extracellular matrix and play pivotal roles in the structure and function of the dystrophin-glycoprotein complex (DGC). Defects in O-mannosylation of α-DG resulting from the abnormal changes of biosynthetic pathway of O-mannose glycans can cause several congenital muscular dystrophies (CMDs). Moreover, O-mannose glycans are also closely associated with tumor metastasis and certain arenaviruses infection of host cells. However, neither the biosynthetic pathway nor the precise function of this diverse glycan library have not been fully understood yet. The first step to solve these problems is to acquire sufficient amount of structurally well-defined O-mannose glycan probes to be used in carbohydrate microarray to gain molecular insight into these processes.Nevertheless, due to the challenges of stereoselective formation of the sialic acid and fucose linkages in O-mannose glycans and also their high structural diversity, the synthetic studies of O-mannose glycans are still far behind their biological investigations:(1) The previous protocols mainly focused on certain single structures and only two O-mannose glycan structures have been synthesized (one Core M1 tetrasaccharide and one Core M3 trisaccharide). (2) Most of the developed chemical strategies were time consuming and labor intensive with poor stereoselectivity and low yield. The enzymatic approaches on the other hand are generally limited by the low efficiency, low substrate tolerability, and low expression level of the enzymes used. (3) More complex structures such as tetra-or penta-saccharide containing sLNGc, Lewis x (Lex) and sialyl Lewis x (sLex) have not been prepared in any form. Therefore, there is an urgent need to develop a diversity-oriented synthesis strategy for the synthesis of O-mannose glycans covering as many distinct types of core structures as possible.To solve the above problems, we developed a diversity-oriented chemoenzymatic synthetic strategy for the synthesis of a-dystroglycan (a-DG) core Ml O-mannose glycans using a sequential three-step one-pot multienzyme (OPME) glycosylation process. The research was carried out from the following aspects:(1) Chemical preparation of Core Ml O-mannose disaccharyl serineA flexible chemical approach was developed based on diacetone and t-butyldimethyl silyl (TBDMS) protecting group strategy and two efficient steps of glycosylation with high stereoselectivity for the efficient preparation of Core M1 O-mannose disaccharyl serine.(2) Enzymatic enlongation of Core M1 O-mannose glycansThree bacterial "one-pot multienzyme" (OPME) glycosylation systems developed for β1-4-galactosylation, α2-3-sialylation and α1-3-fucosylation, respectively, were employed to the biomimetic enlogation of Core M1 O-mannose glycans from the chemically synthesized Core M1 O-mannose disaccharyl serine as the initial substrate. Besides, the sequences of the enzymatic reactions were appropriately adjusted to achieve the diverse preparation of Core M1 O-mannose glycans.As results, six Core M1 O-mannose glycans were achieved as their Fmoc-Serine derivatives in preparative scales for the first time with a more than 70% yield per enzymatic step. Besides, three Core M1 O-mannosyl tetra-and penta-saccharides containing sLNGc, Lewis x (Lex) and sialyl Lewis x (sLex) moieties were synthesized for the first time.2 Chemical synthesis of unsymmetrical 3,6-branched mannopentaose (Man5)Man5 is the common core structure of oligomannose and the hybrid type of N-glycans, and it is also the most abundant structure of the glycans on gp120 which is the envelope glycoprotein of HIV-1. There is convincing evidence that Man5 domainon gp120 is the recognition site of a broadly potent neutralizing human monoclonal antibody,2G12. Man5 is also the primary target for a HIV envelope binding lectin, cyanovirin-N (C VN). Therefore, large amount of pure Man5 is needed for the construction of HIV vaccine based on clustered Man5 and for the investigations of interactions between gp120 oligomannose and their receptors.However, most of the previous synthetic protocols mainly focused on linear or branched symmetrical sequences and there are still no efficient approaches for the assembly of branched unsymmetrical oligomannoses.To solve the above problems, an expeditious three-component, one-pot protocol based on regioselective glycosylation of a light protected acceptor was developed and the research was carried out from the following aspects:(1) Three initial mannose building blocks were efficiently constructed.(2) The conditions of two regioselective glycosylation were optimized by stepwise protocol with the three building blocks and mannopentaose was obtained in 38% yield.(3) A one-pot strategy was conducted employing the same three building blocks giving the unsymmetrical 3,6-branched mannopentaose in a yield of 61%, which was much higher than that of the stepwise synthesis,38%.3 Conclusions and noveltiesIn summary, we developed two powerful strategies for the synthesis of two types of bioactive oligosaccharides, especially for the synthesis of α-DG O-mannose glycans. The efficiency and flexibility of the strategies can also be employed for the synthesis of other oligosaccharides with similar structures, which will accelerate the biological investigations of the oligosaccharides and help lay the basis for the diagnosis and treatment of certain diseases. The main novelties were listed as follows:(1) A flexible approach for efficient preparation of Core M1 O-mannose disaccharyl serine was developed based on a diacetone and TBDMS protecting group strategy and two steps of efficient glycosylation with high stereoselectivity.(2) A diversity-oriented strategy for the chemoenzymatic synthesis of Core M1 O-mannose glycans based on bacterial sequential "one-pot multienzyme" (OPME) glycosylation process was developed for the first time.(3) Efficient preparation of six Core M1 O-mannose glycans in preparative scales was achieved for the first time.(4) Core M1 O-mannose tetra-and penta-saccharides containing sLNGc, Lex and sLex moieties were prepared for the first time.(5) A one-pot chemical synthetic strategy was developed for the first time for the efficient synthesis of unsymmetrical 3,6-branched mannopentaose (Man5).(6) Twenty-four new carbohydrate building blocks was synthesized for the first time and among which three oligosaccharides with completely novel backbones were obtained for the first time.