Communications
DOI: 10.1002/anie.201008178
In Vivo Protein Labeling
Genetically Encoded Copper-Free Click Chemistry**
Tilman Plass, Sigrid Milles, Christine Koehler, Carsten Schultz,* and Edward A. Lemke*
The ability to visualize biomolecules within living specimen
by engineered fluorescence tags has become a major tool in
modern biotechnology and cell biology. Encoding fusion
proteins with comparatively large fluorescent proteins (FPs)
as originally developed by the Chalfie and Tsien groups is
currently the most widely applied technique.[1] As synthetic
dyes typically offer better photophysical properties than FPs,
alternative strategies have been developed based on genet-
ically encoding unique tags such as Halo and SNAP tags,
which offer high specificity but are still fairly large.[2] Small
tags like multi-histidine[3] or multi-cysteine motifs[4] may be
used to recognize smaller fluorophores, but within the cellular
environment they frequently suffer from poor specificity as
their basic recognition element is built from native amino acid
side chains. Such drawbacks may be overcome by utilizing
bioorthogonal chemistry that relies on coupling exogenous
moieties of non-biological origin under mild physiological
conditions. A powerful chemistry that fulfils these require-
ments is the Huisgen type (3+2) cycloaddition between azides
and alkynes (a form of click chemistry[5]). By utilizing
supplementation-based incorporation techniques and click
reactions Beatty et al. coupled azide derivatized dyes to
Escherichia coli expressing proteins bearing linear alkynes.[6]
However, this azide–alkyne cycloaddition required copper(I)
as a catalyst (CuAAC), which strongly reduces biocompati-
bility (but see Ref. [7]). This limitation has been overcome by
Bertozzi and co-workers, who showed that the “click”
reaction readily proceeds when utilizing ring-strained alkynes
as a substrate[8] and since then this strain-promoted azide–
alkyne cycloaddition (SPAAC) has found increasing applica-
tions in labeling, for example, carbohydrates,[9] nucleotides,[10]
and lipids.[11] Further expanding the potential of this
approach, Ting and co-workers engineered a lipolic acid
ligase which ligates a small genetically encoded recognition
peptide to a cylcooctyne-containing substrate. In a second
step the incorporated cyclooctyne moiety then functioned as a
specific site for labeling in cells.[12]
The direct genetic encoding of fluorescent unnatural
amino acids (UAAs) has overcome many drawbacks of
previous approaches by offering exquisite specificity, freedom
of placement within the target protein, and minimal, if any,
structural change. This approach was first achieved by
Summerer et al. who evolved a leucyl tRNA/synthetase
(tRNA/RS) pair from E. coli to genetically encode the
UAA dansylalanine into Saccharomyces cerevisiae.[13] In
response to the amber stop codon TAG, dansylalanine was
readily incorporated into proteins by the host translational
machinery. This approach has since been used to genetically
encode several small dyes,[14] but owing to the need to evolve
new tRNA/RS pairs and potential size limitations imposed by
the translational machinery, larger dyes with enhanced
photophysical properties have not yet been encoded.
Targeted incorporation of UAAs should in principle make
it possible to genetically incorporate strained alkyne and
azide functional groups. In fact, a variety of azides have been
genetically encoded by the use of, for example, engineered
tyrosyl Methanococcus jannaschii tRNATyr/RS, leucyl E. coli
tRNALeu/RS, and pyrrolysine Methanosarcina bakeri/mazei
tRNApyl/pylRS[15,16] pairs (Tyr= tyrosine, Leu = leucyl, pyl =
pyrrolysyl). However, genetically encoding the functionality
necessary for metal-free click ligations, that is, the strained
alkyne, has not been achieved to date, owing to the large size
of the cyclic side-chain moiety. Direct encoding of strained
alkynes offers numerous advantages over encoding the azide
functionality. Many commercial compounds, such as fluores-
cent dyes or fatty acids, are only available as the azide
derivatives and thus not compatible with a copper-free click
reaction with the available genetically encoded UAAs.
Another major advantage of directly encoding strained
alkynes is that they can react with fluorogenic azides, which
are dyes whose fluorescence is dramatically quenched in the
azide form and increases strongly after a successful click
reaction (Scheme 1).[17] This fluorogenic approach was
applied, for example, to specifically label lipids within living
mammalian cells with a coumarin azide derivative, which
offered high contrast due to a lack of background fluores-
cence.[11] The benefit of fluorogenic labels has also been
highlighted in recent work of the Weissleder group who
utilized the ability of tetrazines to quench fluorescence and to
react with strained dienophiles in a Diels–Alder cycloaddi-
tion.[18]
[*] T. Plass, S. Milles, C. Koehler, Priv.-Doz. Dr. C. Schultz,
Dr. E. A. Lemke
Structural and Computational Biology Unit and Cell Biology and
Biophysics Unit, EMBL
Meyerhofstrasse 1, 69117 Heidelberg (Germany)
Fax: (+49)6221-397-536
E-mail: schultz@embl.de
[**] We are grateful for the help of V. Vandelinder, A. Neal, and C. Besir in
the preparation of this work. We thank P. G. Schultz for materials.
This study was technically supported by the EMBL-ALMF, -PEPCF,
and -PCF core facilities. T.P. is a VCI and S.M. a BIF fellow. E.A.L.
acknowledges funding by the Emmy Noether program and C.S.
from the Transregio83 of the DFG.
To overcome the current limitations of engineering
copper-free click chemistry into living cells, we aimed to
genetically encode strained alkynes into E. coli. We reasoned
that the natural amber suppressor pyrrolysine tRNApyl/pylRS
pair from M. mazei might be a suitable starting point because
the amino acid substrate binding pocket is partially exposed,
Supporting information for this article (Detailed experimental
methods and compound synthesis is available on the WWW under
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ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2011, 50, 3878 –3881