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L. Yi et al. / Tetrahedron Letters 50 (2009) 759–762
Scheme 1. Synthesis and structure of a sulfonium salt used in this study.
reaction in real-time. We found that sulfonium alkylation of
proteins can be finished in 4 h by the treatment with ASS in
NaHCO3–Na2CO3 buffer (pH 9.0). The alkyne-labeled BSA was
dialyzed with Tris–HCl buffer (0.1 M, pH 7.5) to remove 1,
and then used for CuAAC reaction. As shown in Figure 2B, fluo-
rescent labeling of BSA was accomplished in 7 h by the
appearance of
a maximum fluorescence emission band at
approximately 480 nm, characteristic to the emission band of
coumarin. Control experiments without either 1 or 3 gave no
such band. These results showed successful selective fluores-
cent labeling of BSA.
Encouraged by the results in proteins, the bioconjugate
reactions were also used to functionalize virus. A TMV virus
contains a single stranded RNA of 6395 nucleotides encapsu-
lated in a helical virion by 2130 coat protein (TMV CP) mono-
mers, showing about 300 nm length.17 TMV particles have
recently shown great potential application in bionano-
science.18–20 As shown in Figure 2A, TMV was first treated
by ASS in NaHCO3–Na2CO3 buffer (pH 9.0) for 4 h. It is noted
that the modified TMV can easily be precipitated during the
reaction. Bioconjugate reactions for TMV were performed with
careful shaking. After centrifugation and washed by Tris–HCl
buffer (0.1 M, pH 7.5) for several times, the alkyne-labeled
virus was used for CuAAC reaction. After 7 h, the TMV-1 was
isolated by centrifugation and washed by buffer.21 As shown
in Figure 2C, the TMV is non-fluorescent, while the TMV-1 at
the same concentration has strong emission at center about
480 nm (Fig. 2C), showing the successful fluorescent labeling
on TMV surface.
The modified TMV was further analyzed by denaturing SDS
gel electrophoresis and transmission electron microscopy
(TEM) (Fig. 3). It is clear that fluorescent dye was attached
to viral protein (Fig. 3, lanes 4 and 5), which is consistent
with result obtained from emission spectra. The SDS gel elec-
trophoresis of modified TMV coat protein (TMV CP) gave three
major bands; all bands showed dye labeling in Figure 3B.
TEM showed that TMV particles were not destroyed by the
ASS-CuAAC reactions (Fig. 3C), implying that the reaction con-
ditions are mild and biofriendly.
Figure 1. (A) Schematic depiction of the generation of fluorescent labeling BSA by
tandem ASS-CuAAC reactions; BSA represents as space-filling model.16 (B) Lanes 1
and 2, BSA with or without DMSO; lanes 3 and 4, BSA + Cu(II)/ascorbate + ligand
N1,N1-diisopropylethane-1,2-diamine; lane 5, BSA + 2; lanes 6 and 7, BSA + Cu(II)/
ascorbate; lanes 8–11, BSA treated with ASS-CuAAC reactions; lanes 8 and 10, the
reaction precipitation; lanes 9 and 11, the reaction solution; lanes 10 and 11,
reaction without the ligand. (C) The same gel of (B) under ultraviolet illumination.
See Supplementary data for detailed experimental process.
were analyzed by denaturing gel electrophoresis, as shown in
Figure 1B and C.14 Compared with the gel results under ultravi-
olet illumination and Coomassie blue staining, at least three
points can be concluded: (I) the fluorescent labeling of proteins
can only be obtained after sulfonium alkylation followed by click
(ASS-CuAAC) reactions; (II) the dye-labeled proteins have de-
creased solubility in aqueous buffer; (III) the click reaction seems
to proceed smoothly even without the ligand used in normal
In summary, for the first time, we developed a tandem meth-
od of sulfonium alkylation and click chemistry for modification
of biomolecules. Fluorescent labeling of proteins and virus were
successfully performed after simple incubation of biomolecules
with sulfonium salts followed by azido-containing compound
at room temperature. We believe that the mild and biocompati-
ble conditions of ASS-CuAAC reactions used here can efficiently
click reaction. The ASS-CuAAC reaction should be
method for biolabeling.
a general
In order to optimize the protocol of ASS-CuAAC reactions,
nonfluorescent 3 was employed as the azido carrying agent
(Fig. 2A). Compound 3 is nonfluorescent because of a photo-in-
duced electron transfer (PET) effect from azido group to cou-
marin group.15 When click reaction occurs between azido and
alkynyl groups, ‘turn-on’ fluorescence could be observed for
the coumarin moiety, which could be used to monitor the
bioconjugate
a wide range of compounds and/or functional
groups to the surface of proteins and virus. This facile bioconju-
gate assay should be useful in protein chemistry and
bionanoscience.