A Kinetic Trapping Approach for Facile Access to 3FaxNeu5Ac and a Photo-Cross-Linkable Sialyltransferase Probe

Sialic acid (Neu5Ac) is installed onto glycoconjugates by sialyltransferases (STs) using cytidine monophosphate-Neu5Ac (CMP-β-d-Neu5Ac) as their donor. The only class of cell-active ST inhibitors are those based on a 3FaxNeu5Ac scaffold, which is metabolically converted into CMP-3FaxNeu5Ac within ce...

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Published in:Journal of the American Chemical Society 2024-10, Vol.146 (42), p.28630-28634
Main Authors: Kumawat, Dhanraj, Gray, Taylor E., Garnier, Cole R., Bui, Duong T., Li, Zhixiong, Jame-Chenarboo, Zeinab, Jerasi, Jeremy, Wong, Warren O., Klassen, John S., Capicciotti, Chantelle J., Macauley, Matthew S.
Format: Article
Language:English
Subjects:
alkaline phosphatase
class
cytidine
fluorine
fructose-bisphosphate aldolase
glycoconjugates
glycosyltransferases
ligands
sialic acid
stereochemistry
stereoselectivity
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Summary:Sialic acid (Neu5Ac) is installed onto glycoconjugates by sialyltransferases (STs) using cytidine monophosphate-Neu5Ac (CMP-β-d-Neu5Ac) as their donor. The only class of cell-active ST inhibitors are those based on a 3FaxNeu5Ac scaffold, which is metabolically converted into CMP-3FaxNeu5Ac within cells. It is essential for the fluorine to be axial, yet stereoselective installation of fluorine in this specific orientation is challenging. Sialic acid aldolase can convert 3-fluoro­pyruvate and 2-acetamido-2-deoxy-d-mannopyranose (ManNAc) to 3FNeu5Ac, but stereocontrol of the fluorine in the product has not been possible. We hypothesized that the 3Fax kinetic product of a sialic acid aldolase reaction could be trapped by coupling with CMP-sialic acid synthetase to yield CMP-3FaxNeu5Ac. Here, we report that highly active CMP-sialic acid synthetase and short reaction times produce exclusively CMP-3FaxNeu5Ac. Removal of CMP from CMP-3FaxNeu5Ac under acidic conditions unexpectedly led to 3-fluoro-β-d-Neu5Ac 2-phosphate (3FaxNeu5Ac-2P). Alkaline phosphatase successfully converted 3FaxNeu5Ac-2P to 3FaxNeu5Ac, enabling stereochemically controlled access to 3FaxNeu5Ac, which is effective in lowering the sialoglycan ligands for Siglecs on cells. Moreover, our kinetic trapping approach could be used to access CMP-3FaxNeu5Ac with modifications at the C5, C9, or both positions, which enabled the chemo­enzymatic synthesis of a photo-cross-linkable version of CMP-3FaxNeu5Ac that selectively photo-cross-linked to ST6GAL1 over two other STs.
ISSN:0002-7863
1520-5126
1520-5126
DOI:10.1021/jacs.4c10835