Sialosides chemoenzymatic synthesis

Figure 2 One-pot three-enzyme chemoenzymatic synthesis of sialosides containing sialic acid modifications. In this strategy, mannose or ManNAc derivatives are chemically or enzymatically synthesized. These compounds are then used by a recombinant coli K-12 sialic acid aldolase to obtain sialic acids and their derivatives followed by an N. meningitidis CMP-sialic acid synthetase for the formation of CMP-sialic acids. From which, sialic acids can be transferred to lactose, LacNAc, galactose, GalNAc, or their derivatives by a multifunctional P. muitocida sialyltransferase (PmSTl) or a P, damseia a2,6-sialyltransferase (Pd2,6ST) to form a2,3- or a2,6-linked sialosides in one pot without the isolation of intermediates.

Highly efficient chemoenzymatic synthesis of naturally occurring and non-natural a-2,6-linked sialosides a P. damsela a-2,6-sialyltransferase with extremely flexible donor-substrate 26. 

Scheme 3. One-pot three-enzyme chemoenzymatic synthesis of sialosides containing natural...

Table 2. One-Pot Three-Enzyme Chemoenzymatic Synthesis of a2 -Linked Sialosides Containing Naturally Occurring and Non-natural Sialic Acids... 

Scheme 6. One-pot three-enzyme chemoenzymatic synthesis of pNP-tagged o2,3- and a2,6-linked sialoside libraries containing a list of naturally occurring sialic acidforms.

Varki, A., and Chen, X. (2008) Combinatorial chemoenzymatic synthesis and high-throughput screening of sialosides. ACS Chem. Biol., 3, 567-576. 

Yu, H., Huang, S., Chokhawala, H., Sun, M., Zheng, H., and Chen, X., Highly efficient chemoenzymatic synthesis of naturally occurring and non-natural a-2,6-linked sialosides A P. damsela a-2,6-sialyltransferase with extremely flexible donor-substrate specificity. Angew. Chem. Int. Ed. 2006,45 (24), 3938-3944. 

Yu H, Chokhawala HA, Huang S, Chen X. One-pot three-enzyme chemoenzymatic approach to the synthesis of sialosides containing natural and non-natural functionalities. Nature Protocols 2006 1 2485-2492. 

Aldolases, CMP-Sia synthetases, and/or sialyltransferases have been combined in a number of one-pot, chemoenzymatic syntheses of naturally occurring sialosides (e.g., see Ichikawa et /. ). The use of permissive enzymes broadens the scope of these one-pot methods and has facilitated the synthesis of a number of unnatural sialosides. The Chen group reported a Pasteurella multocida sialyltransferase that can function as an a2-3 sialyltransferase, o 2-6 sialyltransferase, o 2-3 sialidase, or a2-3 rr j-sialidase, depending on the pH of the reaction mixture. Using this enzyme in a one-pot reaction in combination with E. coli aldolase and... 

Once chemoenzymatically produced, these unnatural sialosides have demonstrated utility in a variety of biological applications. A sialoside consisting of 9-0-acetylated sialyl Tn antigen (sTn) was produced by one-pot synthesis and conjugated to human serum albumin to be used to evaluate the role of acetylation in carbohydrate-protein interactions. Para-nitrophenol-tagged sialosides can be used in a colorimetric assay to evaluate sialidase specificity. Biotin-tagged sialosides have been immobilized on a streptavidin-coated surface to study protein-carbohydrate interactions with surface plasmon resonance techniques. Size-defined... 

In sialoside synthesis, the use of enzymes can dramatically reduce the multiple protection-deprotection steps and provide great stereo- and regio-selectivity. Many enzymes accept uimatural substrates. Genetic engineering can further alter their stability, broaden their substrate specificity, and increase their specific activity [73]. Therefore, chemoenzymatic approaches using enzymes in combination with classical carbohydrate chemistry opened up a new venue, avoiding many of the problems encountered in traditional chemical synthesis. 

The total synthesis of sialosides by using the chemoenzymatic approach is as follows [74]. Sialic acid itself can be synthesized from ManNAc, mannose, or their derivatives by sialic acid aldolase enzyme through aldol condensation reaction. If ManNAc is chemically or enzymatically modified at C2, C4—C6 positions, sialic acid has structural modifications at C5, C7-C9 positions, respectively. The sialic acids are subsequently activated by a CMP-siahc acid synthetase to form a CMP-sialic acid, which is the donor used by sialyltransferases. Because CMP-sialic acid is tmstable, the CMP-Neu5Ac synthetase is valuable for the preparative enzymatic synthesis of sialosides. In the last steps, the CMP-sialic acid is transferred to galactose or GalNAc terminated glycosides by sialyltransferases to form structurally defined sialosides. Examples are that Chen and co-workers have recently developed a one-pot multienzyme system for the efficient synthesis of a-sialosides (Table 2) [12,76,79]. In this system, recombinant E. coli K-12 sialic acid aldolase catalyzed the synthesis of sialic acid precursors for... 

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