Selective functionalization of a genetically encoded alkene-containing protein via photoclick chemistry in bacterial cells

Article abstract of DOI:10.1021/ja803598e

We report a tetrazole-based, photoclick chemistry that can be employed to selectively functionalize an alkene genetically encoded in a protein inside E. coli cells. The reaction involved the treatment of E. coli cells with cell-permeable tetrazoles followed by a brief photo irradiation at 302 nm (4 min) and an overnight incubation at 4 °C. This in vivo alkene functionalization procedure was simple, straightforward, and nontoxic to E. coli cells. Additionally, fluorescent adducts were formed, facilitating the monitoring of the reaction in vivo. This reaction should offer a new tool for the study of alkene-containing proteins in living systems. Copyright

Full text of DOI:10.1021/ja803598e

Published on Web 07/02/2008
Selective Functionalization of a Genetically Encoded Alkene-Containing
Protein via “Photoclick Chemistry” in Bacterial Cells
Wenjiao Song,† Yizhong Wang,† Jun Qu,‡ and Qing Lin*,†
Departments of Chemistry and Pharmaceutical Sciences, State UniVersity of New York at Buffalo,
Buffalo, New York 14260
Received May 14, 2008; E-mail: qinglin@buffalo.edu
Selective functionalization of proteins via bioorthogonal chemistry
provides a powerful strategy to study protein function in living
systems.1 A major bottleneck in this approach has been the dearth of
unique, bioorthogonal reactions that can be employed to functionalize
chemical reporters incorporated site-selectively into proteins. As a
result, only a few chemical reporters such as aldehyde,2 ketone,3 azide,4
and acetylene5 have been used successfully in living systems. The
functionalization of these reporters relies essentially on three bioor-
thogonal reactions: (1) nucleophilic addition to carbonyl by hydrazine
and hydroxylamine; (2) azide-acetylene cycloaddition either catalyzed
by CuI (“click chemistry”)6 or accelerated by ring strain;7 and (3)
Figure 1. Scheme for selective functionalization of Z-domain protein encoding
Staudinger ligation.4 To expand structural diversity of chemical
O-allyl-tyrosine via a photoclick chemistry.
reporters for multiplexed applications in living systems,8 there is a
strong demand for new enabling bioorthogonal reactions.
Alkene is a versatile functional group in organic transformation for
which several selective, water-compatible chemistries have been
developed, including olefin metathesis,9 Diels-Alder reaction,10 and
1,3-dipolar cycloaddition reactions.11 A number of alkene-containing
amino acids have been incorporated site-specifically into proteins,
including homoallylglycine,12 O-allyl-tyrosine,13 and dehydroalanine,14
because of their unique reactivity. To our knowledge, however, alkene
functionalization using cell-permeable reagents in living cells has not
been reported. Herein, we report the first selective functionalization
of O-allyl-tyrosine genetically encoded in a Z-domain protein using a
photoactivated, nitrile imine mediated 1,3-dipolar cycloaddition reaction
(“photoclick chemistry”) in Escherichia coli (Figure 1). While we have
recently reported the selective labeling of tetrazole-containing proteins
in biological media using this chemistry,15 the work described here
represents the first time that this photoclick chemistry is applied to
living cells and in a reverse manner involving an unactivated alkene.
As a first step to establish the utility of photoclick chemistry in
selectively functionalizing a genetically encoded alkene, we incubated
a panel of diaryltetrazole compounds with a mutant Z-domain
genetically encoding O-allyl-tyrosine at residue 7 position (alkene-
Z).13 In parallel, we also performed the same incubations with the
wild-type Z-domain protein (wt-Z) as a control. The mixtures were
photoirradiated with a 302-nm hand-held UV lamp for 10 min, and
then the products were analyzed by sodium dodecyl sulfate-polyacry-
Figure 2. Screening for diaryltetrazoles that selectively react with O-allyl-
lamide gel electrophoresis (SDS-PAGE). Since the photoclick chem-
tyrosine-containing Z-domain (m) over the wild-type Z-domain (w) by in-gel
fluorescence analysis: (a) structures of diaryltetrazoles; (b) SDS-PAGE gel
istry produces fluorescent pyrazoline cycloadducts,15 we performed
in-gel fluorescence analyses to follow the product formation (Figure
2). Among 12 diaryltetrazoles analyzed, 3 showed selective product
formation only with alkene-Z, but not with wt-Z (1, 2, 7 in Figure 2),
suggesting that both neutral (e.g., H in tetrazole 1) and electron-
withdrawing substituents (e.g., F in tetrazoles 2 and 7) on the N-aryl
ring can give rise to selective reactivity toward O-allyl-tyrosine. The
cycloadduct formation involving tetrazole 1 was further confirmed by
unambiguous identification of the pyrazoline mass (+292.13 Da) in
fluorescence images of the pyrazoline cycloadducts under 365-nm UV
illumination (top panels) and by Coomassie blue staining (bottom panels).
Reaction conditions: 250 µM tetrazole, 15 µM Z-domain in PBS buffer, pH
7.5, 302-nm photoirradiation for 10 min.
both trypsin- and V8-digested samples in the nanoLC-tandem mass
spectrometry analyses.16
To determine the rate of cycloaddition, we performed a kinetic study
of a model reaction between tetrazole 1 and allyl phenyl ether.17 The
photolysis of tetrazole 1 to generate the nitrile imine was found to be
very rapid, and essentially complete within 2 min. The subsequent
cycloaddition reaction, however, was much slower with an apparent
† Department of Chemistry.
‡ Department of Pharmaceutical Sciences.
9
9654 J. AM. CHEM. SOC. 2008, 130, 9654–9655
10.1021/ja803598e CCC: $40.75
2008 American Chemical Society

C O M M U N I C A T I O N S
Figure 4. SDS-PAGE analysis of E. coli cell lysates expressing either alkene-Z
or wt-Z after treatment with tetrazole 1: left panel, Coomassie blue staining;
right panel, fluorescence in-gel imaging (λex ) 365 nm). The experiment was
repeated three times independently and similar results were obtained.
the tetrazole reactivity and apply this chemistry to alkene functional-
ization in mammalian cells are currently underway.
Figure 3. Selective functionalization of alkene-Z by tetrazole 1 in E. coli
cells: CFP channel (top row) and DIC channel (bottom row) images of bacterial
cells expressing either alkene-Z (a,c) or wt-Z (b,d) proteins after treatment with
100 µM tetrazole 1. An 100× oil immersion lens was used.
Acknowledgment. We gratefully acknowledge the Petroleum
Research Fund (PRF No. 45503-G4), SUNY Buffalo, and New York
State Center of Excellence in Bioinformatics and Life Sciences for
financial support. We thank Prof. Peter Schultz at Scripps Research
Institute for providing the pLEIZ, pLEIZ-7TAG, pBK-ALRS plasmids
used in this study.
second-order rate constant of 0.00202 ( 0.00007 M-1 s-1, based on
the HPLC analysis.16 By comparison, the cycloaddition reaction
between tetrazole 1 and acrylamide showed an apparent second-order
rate constant of 0.15 M-1 s-1, 75-fold faster than that of allyl phenyl
ether,16 presumably due to the lower LUMO energy of acrylamide,
and thus better dipole HOMO-dipolarophile LUMO overlap.18
To examine whether this photoclick chemistry can be used to
functionalize alkene-containing proteins in E. coli, BL21(DE3) cells
expressing either wt-Z or O-allyl-tyrosine containing Z-domain proteins
were suspended in the PBS buffer containing 5% glycerol and 100
µM tetrazole 1. After incubation at 37 °C for 30 min, the cell
suspensions were irradiated with a hand-held 302-nm UV lamp for 4
min. The bacterial cells were then incubated at 4 °C overnight to allow
the cycloaddition reaction to proceed to completion. Because spec-
troscopic properties16 of the pyrazoline match closely to that of cyan
fluorescent protein (CFP), we were able to use a fluorescent microscope
equipped with the CFP filter set (ex 438/24 nm, em 483/32 nm) to
monitor the cycloaddition reaction in vivo. In the CFP channel, only
E. coli cells expressing alkene-Z showed strong fluorescence while
cells expressing wt-Z did not (Figure 3a,b), indicating that the
cycloaddition reaction is selective toward the alkene functional group.19
In the DIC channel, the cell density for the alkene-Z expressing cells
was lower than that of the wt-Z expressing cells (Figure 3c,d), which
can be attributed to significantly slower bacterial growth rate in the
GMML medium (the culture medium used for the genetic incorporation
of O-allyl-tyrosine). There was no apparent treatment-induced toxicity
observed, including potential photo damages, presumably due to short
irradiation time (4 min).
To verify that the fluorescence observed in E. coli arose from the
photoclick chemistry, the treated bacterial cells were lysed and the
lysates were subjected to in-gel fluorescence analysis. Only one
fluorescent band with the size matching that of Z-domain was observed
in the alkene-Z lysate, but not in the wt-Z lysate (Figure 4), indicating
that indeed the fluorescence was due to selective formation of the
fluorescent pyrazoline-Z adduct.
In summary, we have demonstrated that a tetrazole-based, photoclick
chemistry can be employed to selectively functionalize an alkene
genetically encoded in a protein inside E. coli cells. The reaction
procedure was simple, straightforward, and nontoxic to E. coli cells
with the only required external reagents to be tetrazoles and photons.
Additionally, fluorescent cycloadducts were formed, which enabled a
facile monitoring of the reaction in vivo. The efforts to further optimize
Supporting Information Available: Experimental procedures and
characterization data for all compounds. This material is available free of
charge via the Internet at http://pubs.acs.org.
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