Sulfonium alkylation followed by 'click' chemistry for facile surface modification of proteins and tobacco mosaic virus

Article abstract of DOI:10.1016/j.tetlet.2008.11.107

Alkylsulfonium salts (ASS) have been shown to act as powerful alkylating agents. However, few studies have addressed the application of sulfonium salts to the modification of biomolecules such as nucleic acids and proteins. Since these large biomolecules play important roles in biological processes, a convenient and fast method for their modification is greatly needed. In this work, for the first time, we used a tandem method of sulfonium alkylation and click chemistry (CuAAC) for modification of biomolecules. Fluorescent labeling of proteins and tobacco mosaic virus were successfully performed after simple incubation of biomolecules with sulfonium salts followed by azido-containing compound at room temperature. This facile bioconjugation assay based on ASS-CuAAC reactions should be useful in protein chemistry and bionanoscience.

Full text of DOI:10.1016/j.tetlet.2008.11.107

Tetrahedron Letters 50 (2009) 759–762
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Tetrahedron Letters
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Sulfonium alkylation followed by ‘click’ chemistry for facile surface
modification of proteins and tobacco mosaic virus
*
Long Yi, Jie Shi, Shuang Gao, Shibo Li, Congwei Niu, Zhen Xi
State Key Laboratory of Elemento-Organic Chemistry and Department of Chemical Biology, Nankai University, Tianjin 300071, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Alkylsulfonium salts (ASS) have been shown to act as powerful alkylating agents. However, few studies
have addressed the application of sulfonium salts to the modification of biomolecules such as nucleic
acids and proteins. Since these large biomolecules play important roles in biological processes, a conve-
nient and fast method for their modification is greatly needed. In this work, for the first time, we used a
tandem method of sulfonium alkylation and click chemistry (CuAAC) for modification of biomolecules.
Fluorescent labeling of proteins and tobacco mosaic virus were successfully performed after simple incu-
bation of biomolecules with sulfonium salts followed by azido-containing compound at room tempera-
ture. This facile bioconjugation assay based on ASS-CuAAC reactions should be useful in protein
chemistry and bionanoscience.
Received 28 October 2008
Revised 21 November 2008
Accepted 26 November 2008
Available online 30 November 2008
Keywords:
Alkylsulfonium
Click chemistry
Protein
Ó 2008 Elsevier Ltd. All rights reserved.
Virus
Bioconjugation assay
The transfer of an intact methyl group from a donor to a suit-
able acceptor is a vitally important biological process. The uni-
versal donor of intact methyl group is AdoMet,1 which is an
important cofactor for enzymes that methylate a diverse array
of nucleophile acceptors within the cell (i.e., DNA, RNA, proteins,
biogenic amines, etc.). In order to have a chemical basis for
understanding the alkyl transferation, a series of methylsulfoni-
um salts as methylating agents have been investigated.2 More-
over, since higher alklysulfonium salts have recently become
readily available,3 increasing studies of their alkylating ability
were undertaken. It is now clear that under mild conditions em-
ployed, alkylsulfonium salts (ASS) indeed act as powerful alkylat-
ing agents.4,5 However, few studies have addressed the
application of sulfonium salts to the modification of biomole-
cules such as nucleic acids and proteins. Since these large bio-
functional groups with high yield. In addition, the resulting
1,2,3-triazoles are stable at aqueous conditions and are bio-
compatible. Recently, click chemistry has prevailed with appli-
cations in organic synthesis, drug discovery, materials science,
and biotechnology, which is proved to be an ideal tool for
incorporating functionalities onto desired scaffolds.7–13 The
present study intends to develop
a tandem ASS-CuAAC for
labeling and modification of biomolecules. After labeling alkyne
groups onto protein surface through sulfonium alkylation, click
chemistry was used to incorporate fluorescent moieties to
these biomolecules.
Modification of proteins is often accomplished by using var-
ious alkylating agents. Moreover, plant virus such as tobacco
mosaic virus (TMV) contains coat proteins, which in principle
can be modified via these alkylating agents. A sulfonium salt
1 as an alkyne-containing alkylating agent was designed and
synthesized (Scheme 1). Compound 1 has potential ability to re-
act with nucleophiles to selectively transfer alkyl group, which
could be employed for surface modification of proteins and
virus.
molecules play important roles in biological processes,
a
convenient and fast method for their modification is greatly
needed. Therefore, we decided to investigate the chemical mod-
ification of biomolecules through alkylation with alkylsufonium
salts.
On the other hand, the Cu(I)-catalyzed azide-alkyne cycload-
dition (CuAAC),6 often referred to as ‘click’ chemistry, is very
chemoselective, only occurring between alkynyl and azido
Bovine serium albumin (BSA) contains many nucleophilic res-
idue such as amino, imidazole, thiol, and carboxylate groups on
its surface. When we combined 1 at room temperature with BSA
in phosphate buffer (pH 8.0), we observed the lowered solubility
of the protein. After incubation for 24 h and then treated with
azide-contained fluorescent compound 2 (Fig. 1A), the samples
* Corresponding author. Tel./fax: +86 22 23504782.
E-mail address: zhenxi@nankai.edu.cn (Z. Xi).
0040-4039/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2008.11.107

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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.

L. Yi et al. / Tetrahedron Letters 50 (2009) 759–762
761
Figure 2. (A) Bioconjugation of BSA and TMV to give fluorescent labeling products. (B) Time-dependent emission spectra of CuAAC reaction between alkylated-modified BSA
with 3. For CuAAC reactions, 3 and the alkyne-modified BSA were mixed in 2 mL Tris-HCl buffer (0.1 M, pH 7.5). After the addition of CuSO4 and ascorbate, emission spectra of
the reaction solution excited at 400 nm were monitored; slit width of both excitation and emission are 5 nm. Dot and dash line in (B) represents BSA treated with ASS or
CuAAC only. (C) Compare the emission spectra of TMV and TMV-1 at the same concentration.
Acknowledgments
This work was supported by the National Key Project for Basic
Research of China (2003CB114403), National Natural Science Foun-
dation of China (20272029, 20572053, 20421202, 20432010), and
Ministry of Education of China (104189).
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.tetlet.2008.11.107.
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