10.1002/anie.202009408
Angewandte Chemie International Edition
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mannose type N-glycan, was labeled with 5g and AgNTf2 in a
PB buffer. Fluorescent SDS-PAGE (Fig. 5C) and MS data (Fig.
5F) validated the successful Cy5 labeling. Furthermore, we
chose a therapeutic IgG antibody, trastuzumab23 (also known as
herceptin) which is glycosylated as well, to perform the reaction
with 5e (Fig. S17) or 5f (Fig. 5B) catalyzed by AgNTf2. The MS
and fluorescent data confirmed the robust conjugation on the
antibody. Notably, the protein conjugation by this approach can
be carried out under quite mild conditions using the DBCO
concentration as low as 167 uM and the AgNTf2 concentration as
low as 16.7 uM.
Additionally, we conducted the reaction on a commercial
bacteriophage, Ph.D.12 phage display peptide library (New
England Biolabs), for Cy5 labeling using 5g (100 uM) and
AgNTf2 (10 uM). As shown in Fig. 5E, fluorescent gel of the
phage proteins implicated the Cy5 tag was successfully attached.
In order to investigate the effect of labeling conditions on phage
amplification, we determined the phage titer after the reaction. It
can be seen from Fig. S20 that the labeled bacteriophage
exhibited good tranduction and titer as similar as the original
phage, which proved the labeling process by this method did not
kill the phage.
Figure 6. Site-specific conjugation of trastuzumab via an Fc binding peptide carrying the caged acylation tag DBCO
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This switched click reaction of DBCOs exhibits a unique feature
that acylation reactivity of DBCOs is caged in absence of silver
catalysts. Therefore, harnessing caging/decaging process on
DBCOs, we successfully achieved site-specific modification of a
commercial antibody drug trastuzumab using a DBCO-tagged
Fc-binding peptide (16) as shown in Fig. 6. The cyclic peptide
was previously reported24 to interact with Fc domain near the
K249. We incubated DBCO-peptide 16 with trastuzumab in a pH
7.4 buffer, allowing the peptide to approach the Fc domain when
DBCO was caged. Then, in a decaging condition with AgNTf2,
the peptide was selectively conjugated on K249 almost
quantitatively. The resulted site-specific conjugate 17 was fully
characterized by MS of full-length IgG, reduced IgG, and peptide
mapping analysis (Fig. S23-S25).
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In conclusion, we have observed an unexpected rearrangement
of DBCOs under TFA treatment and optimized the condition
using silver catalysts. Ag-catalyzed rearrangement of DBCOs
not only deprives its click reactivity of SPAAC, but also renders a
switched acylation reaction with amine substrates selectively.
This method brings new ideas in manipulating click reactivity of
DBCOs, and has shown practical applications in modification
and
fluorescent
labeling
on
peptides/glycopeptides,
robust
proteins/glycoproteins, and bacteriophages via
a
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Acknowledgements
This work was supported by the National Natural Science
Foundation of China (NNSFC, No. 21877116), National Science
& Technology Major Project “Key New Drug Creation and
Manufacturing Program” of China (No. 2018ZX09711002-006,
2019ZX09721001).
24. Yamada, K.; Shikida, N.; Shimbo, K.; Ito, Y.; Khedri, Z.; Matsuda,
Y.; Mendelsohn, B.A. Angew. Chem. Int. Ed. 2019, 58, 5592.
Keywords: click chemistry • DBCO • switched click reaction •
caged acylation reagent • silver catalyst
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