Communications
DOI: 10.1002/anie.200705805
Bioorthogonal Chemistry
A Photoinducible 1,3-Dipolar Cycloaddition Reaction for Rapid,
Selective Modification of Tetrazole-Containing Proteins**
Wenjiao Song, Yizhong Wang, Jun Qu, Michael M. Madden, and Qing Lin*
Bioorthogonal chemistry provides an exciting new strategy to
visualize protein expression, track protein localization, mea-
sure protein activity, and identify protein interaction partners
in living systems.[1] Two steps are typically involved in this
approach:1) the incorporation of a bioorthogonal group into
a protein through either a biochemical pathway or semisyn-
Scheme 1. Photoactivated 1,3-dipolar cycloaddition reaction between a
2,5-diaryl tetrazole and a substituted alkene dipolarophile.
thesis; 2) a site-specific reaction between the protein that
carries the bioorthogonal group and a cognate small-molecule
probe. Although a plethora of methods have been developed
to address the first step, such as non-sense suppression
mutagenesis,[2] expressed protein ligation,[3] metabolic engi-
neering,[4] and tagging-via-substrate,[5] only a small number of
bioorthogonal reactions are known for the second step. These
site-specific reactions include the acid-catalyzed nucleophilic
addition of hydrazine to a ketone or aldehyde,[6] Staudinger
ligation,[7] CuI-catalyzed azide–alkyne 1,3-dipolar cycloaddi-
tion (click chemistry),[8] strain-promoted azide–alkyne 1,3-
dipolar cycloaddition,[9] and the oxidative coupling of ani-
line.[10] To fully realize the potential of bioorthogonal
chemistry in probing protein function, there is an urgent
need for the discovery of additional bioorthogonal reactions
with robust reaction attributes. Herein, we report a bioor-
thogonal, photoinducible 1,3-dipolar cycloaddition reaction
that allows rapid and highly selective modification of proteins
carrying a diaryl tetrazole group in biological media.
Forty years ago, Huisgen and co-workers reported a
photoactivated 1,3-dipolar cycloaddition reaction between
2,5-diphenyltetrazole and methyl crotonate.[11] A concerted
reaction mechanism was proposed, whereby the diaryl
tetrazole undergoes a facile cycloreversion reaction upon
photoirradiation to release N2 and generate in situ a nitrile
imine dipole, which cyclizes spontaneously with an alkene
dipolarophile to afford a pyrazoline cycloadduct (Scheme 1).
The photolysis of diaryl tetrazoles was found to be extremely
efficient upon UV irradiation at 290 nm, with quantum yields
in the range 0.5–0.9.[12] Despite its robust mechanism, this
photoactivated reaction has seen very few applications in the
past four decades.[13]
In our initial studies, we identified an extremely mild
photoactivation procedure in the use of a hand-held UV lamp
from UVP (UVM-57, 302 nm, 115 V, 0.16 amps). Under these
mild conditions, the solvent compatibility, functional-group
tolerance, regioselectivity, and yield of the photoactivated 1,3-
dipolar cycloaddition reaction were excellent.[14] We then
examined the reaction kinetics by incubating a tetrazole
peptide with acrylamide in phosphate-buffered saline (PBS)
at pH 7.5 under UV light (302 nm; see Figure S1 in the
Supporting Information). We found that the photolysis of the
tetrazole peptide to generate the nitrile imine intermediate
was extremely rapid, with a first-order rate constant k1 =
0.14 sÀ1; the subsequent cycloaddition with the dipolarophile
acrylamide proceeded very efficiently, with a second-order
rate constant k2 = 11.0mÀ1 sÀ1.[15] In the absence of a dipolar-
ophile, however, slow formation of the nitrile imine–H2O
adduct was observed (see Figure S2 in the Supporting
Information). Moreover, the pyrazoline cycloadducts
showed strong fluorescence with variable emissions in the
region of 487–538 nm, depending on the structure of the
dipolarophile (see Figure S3 in the Supporting Information),
and a high fluorescence quantum yield (F = 0.29).
To examine the bioorthogonality of this reaction for
residue-specific protein modification, we introduced the
diphenyltetrazole moiety into the lysozyme by acylating the
surface lysine residues of the lysozyme with the water-soluble
tetrazole succinimide 1 (Figure 1a). A mixture of tetrazole-
modified lysozymes (44% monoacylated, 5% bisacylated)
was obtained, together with the unmodified lysozyme (51%).
After the removal of small molecules by size-exclusion
chromatography, the mixture was incubated with acrylamide
(50 equiv) and irradiated with UV light (302 nm) for 2 min
(Figure 1a). The pyrazoline product was analyzed along with
the unmodified lysozyme and the tetrazole-modified lyso-
zyme by LC–ESIMS. The expected intact masses of the
lysozyme, the tetrazole-modified lysozyme (Lyso-Tet), and
the pyrazoline product (Lyso-Pyr) were observed (Figure 1b–
d). The conversion of Lyso-Tet into Lyso-Pyr was estimated to
be 90% on the basis of LC–MS analysis (see Figure S5 in the
Supporting Information). No obvious side products, such as
[*] W. Song, Y. Wang, M. M. Madden, Prof. Dr. Q. Lin
Department of Chemistry, State University of NewYork at Buffalo
Buffalo, NY 14260 (USA)
Fax: (+1)716-645-6963
E-mail: qinglin@buffalo.edu
Dr. J. Qu
Department of Pharmaceutical Sciences
State University of NewYork at Buffalo
Buffalo, NY 14260 (USA)
[**] Q.L. is grateful to the Petroleum Research Fund, University at
Buffalo, and NewYork State Center of Excellence in Bioinformatics
and Life Sciences for financial support.
Supporting information for this article is available on the WWW
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ꢀ 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2008, 47, 2832 –2835