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. 2019 Jun 1:479:41-47.
doi: 10.1016/j.carres.2019.05.002. Epub 2019 May 16.

A substrate tagging and two-step enzymatic reaction strategy for large-scale synthesis of 2,7-anhydro-sialic acid

Wanqing Li  1 , Tamashree Ghosh  1 , Yuanyuan Bai  1 , Abhishek Santra  1 , An Xiao  1 , Xi Chen  2
Affiliations

A substrate tagging and two-step enzymatic reaction strategy for large-scale synthesis of 2,7-anhydro-sialic acid

Wanqing Li et al. Carbohydr Res. .

Abstract

A sialyltransferase acceptor tagging and two-step enzymatic reaction strategy has been developed for multigram-scale chemoenzymatic synthesis of 2,7-anhydro-N-acetylneuraminic acid (2,7-anhydro-Neu5Ac), a compound that can serve as a sole carbon source for the growth of Ruminococcus gnavus, a common human gut commensal. Different approaches of introducing hydrophobic UV-active tags to lactose as well-suited sialyltransferase acceptors have been explored and a simple two-step high-yield chemical synthetic procedure has been identified. The UV-active hydrophobic tag facilitates monitoring reaction progress and allows facile product purification by C18-cartridges. A two-step enzyme-catalyzed reaction procedure has been established to combine with C18 cartridge-based purification process for high-yield production of the desired product in multigram scales with the recycled use of chromophore-tagged lactoside starting material and sialoside intermediate. This study demonstrated an environmentally friendly highly-efficient synthetic and purification strategy for the production of 2,7-anhydro-Neu5Ac to explore its potential functions.

Keywords: 2,7-Anhydro-N-Acetylneuraminic acid; Chemoenzymatic synthesis; Sialic acid; Sialidase; Sialyltransferase; Substrate tagging.

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Figures

Fig. 1
Fig. 1
The structure of 2,7-anhydro-Neu5Ac (1).
Scheme 1
Scheme 1
Synthesis of LacβProNHCbz (3) and LacβProNHFmoc (4) from LacβProN3 (2).
Scheme 2
Scheme 2
Two-step enzymatic reaction cycle for the production of 2,7-anhydro-Neu5Ac from Neu5Ac. A) Step A, OP2E α2–3-sialylation system containing NmCSS and PmST1_M144D for the formation of Neu5Acα2–3LacβProNHCbz (5) or Neu5Acα2–3LacβNHCbz (7) from LacβProNHCbz (3) or LacβNHCbz (6) in the presence of Neu5Ac and CTP; and B) Step B, SpNanB-catalyzed formation of 2,7-anhydro-Neu5Ac (1) from Neu5Acα2–3LacβProNHCbz (5) or Neu5Acα2–3LacβNHCbz (7) produced in Step A.
Scheme 3
Scheme 3
Multigram (5 g)-scale protection group-free two-step synthesis of LacβNHCbz (6) from lactose.
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