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FRET‐Based Direct and Continuous Monitoring of Human Fucosyltransferases Activity: An Efficient synthesis of Versatile GDP‐ L ‐Fucose Derivatives from Abundant d‐ Galactose
Author(s) -
Maeda Takahiro,
Nishimura ShinIchiro
Publication year - 2008
Publication title -
chemistry – a european journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.687
H-Index - 242
eISSN - 1521-3765
pISSN - 0947-6539
DOI - 10.1002/chem.200700760
Subject(s) - fucose , galactose , chemistry , förster resonance energy transfer , biochemistry , fluorescence , physics , quantum mechanics
We have developed a facile and versatile protocol for the continuous monitoring of human fucosyltransferases activity by using fluorescence energy resonance transfer (FRET), and have explored the feasibility of its use in an inhibitor screening assay. A convenient sugar nucleotide with a fluorogenic probe, 6‐deoxy‐6‐ N ‐(2‐naphalene‐2‐yl‐acetamide)‐β‐ L ‐galactopyranos‐1‐yl‐guanosine 5′‐diphosphate disodium salt ( 1 ), was efficiently synthesized from naturally abundant D ‐galactopyranose via a key intermediate, 6‐azide‐1,2,3,4‐tetra‐ O ‐benzoyl‐6‐deoxy‐β‐ L ‐galactopyranose ( 10 ). It was demonstrated that the combined use of the glycosyl donor 1 and a dansylated acceptor substrate, sialyl‐α2,3‐LacNAc derivative ( 2 ) allowed us to carry out highly sensitive, direct, and continuous in vitro monitoring of the generation of sialyl Lewis X (SLe x ), which is catalyzed by human α‐1,3‐fucosyltransferase VI (FUT‐VI). A kinetic analysis revealed that compound 1 was an excellent donor substrate ( K M =0.94 μ M and V max =0.14 μ M min −1 ) for detecting human FUT‐VI activity. To the best of our knowledge, this synthetic fluorogenic probe is the most sensitive and selective donor substrate for FUT‐VI among all of the known GDP‐Fuc analogues, including the parent GDP‐Fuc. When a dansylated asparagine‐linked glycopeptide 20 , which is derived from egg yolk was employed as an alternate acceptor substrate, a FRET‐based assay with compound 1 could be used to directly monitor the α1,6‐fucosylation at the reducing terminal GlcNAc residue by human FUT‐VIII ( K M =175 μ M and V max =0.06 μ M / min); this indicates that the present method might become a general protocol for the characterization of various mammalian fucosyltransferases in the presence of designated fluorogenic acceptor substrates. The present protocol revealed that compound 23 , which was obtained by a 1,3‐dipolar cycloaddition between the disodium salt 16 and 1‐ethynyl‐naphthalene exhibits highly potent inhibitory effects against the FUT‐VI‐mediated sialyl Lewis X synthesis (IC 50 =5.4 μ M ).