In this study, we developed an efficient
fluorous tagging strategy
for site-selective enzymatic fucosylation/sialylation in which appending
a readily removable auxiliary tag (SF
17
) to the reducing end of an acceptor provided the regioselectivity
control of a given wild-type fucosyltransferase/sialyltransferase.
By combining this strategy with the sequential one-pot enzymatic system
of HMO production, various fucosylated para-HMOs,
such as LNFP I, LNFP II, LNFP III, LNFP V, LNDFH I, LNDFH II, TF-LNT,
F-pLNH I, F-pLNH IV, DF-pLNH I isomer, DF-pLNH II, TF-pLNH I, TF-pLNH II, F-pLNnH, F-pLNnH I, DF-pLNnH, TF-pLNnH, and TetraF-pLNO, were synthesized. Furthermore,
the SF
17
-tagged acceptors improved
the reaction rates of the enzymatic glycan chain extension. Terminal
α2,6-sialylation on SF
17
-tagged pLNnH was directly achieved using wild-type
α2,6-sialyltransferase from Photobacterium damsela (Pd26ST). Moreover, enzyme kinetics revealed that the catalytic
efficiency (k
cat/K
M) of α1,4-fucosylation of Bf13FT (α1,3/4-fucosyltransferase
from Bacteroides fragilis) on SF
17
-tagged LNFP V acceptors could
be enhanced by 55 times compared with tagging with an azidohexyl aglycone.
We developed an efficient and reliable strategy for generating complex
and diverse natural HMO libraries for functional gut microbiome studies.
This SF
17
-assisted glycosylation
strategy provides a practical approach to harness the extreme substrate
flexibility offered by readily available bacterial glycosyltransferases,
which enables the site-selective fucosylation/sialylation on para-HMOs.
