Photo-induced and Rapid Labeling of Tetrazine-Bearing Proteins via Cyclopropenone-Caged Bicyclononynes

Article abstract of DOI:10.1002/anie.201908209

Inverse electron-demand Diels–Alder cycloadditions (iEDDAC) between tetrazines and strained alkenes/alkynes have emerged as essential tools for studying and manipulating biomolecules. A light-triggered version of iEDDAC (photo-iEDDAC) is presented that confers spatio-temporal control to bioorthogonal labeling in vitro and in cellulo. A cyclopropenone-caged dibenzoannulated bicyclo[6.1.0]nonyne probe (photo-DMBO) was designed that is unreactive towards tetrazines before light-activation, but engages in iEDDAC after irradiation at 365 nm. Aminoacyl tRNA synthetase/tRNA pairs were discovered for efficient site-specific incorporation of tetrazine-containing amino acids into proteins in living cells. In situ light activation of photo-DMBO conjugates allows labeling of tetrazine-modified proteins in living E. coli. This allows proteins in living cells to be modified in a spatio-temporally controlled manner and may be extended to photo-induced and site-specific protein labeling in animals.

Full text of DOI:10.1002/anie.201908209

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International Edition: DOI: 10.1002/anie.201908209
German Edition: DOI: 10.1002/ange.201908209
Bioorthogonal Reactions
Hot Paper
Photo-induced and Rapid Labeling of Tetrazine-Bearing Proteins via
Cyclopropenone-Caged Bicyclononynes
Susanne V. Mayer+, Anton Murnauer+, Marie-Kristin von Wrisberg, Marie-Lena Jokisch, and
Kathrin Lang*
Abstract: Inverse electron-demand Diels–Alder cycloaddi-
tions (iEDDAC) between tetrazines and strained alkenes/
alkynes have emerged as essential tools for studying and
manipulating biomolecules. A light-triggered version of iED-
DAC (photo-iEDDAC) is presented that confers spatio-
temporal control to bioorthogonal labeling in vitro and
in cellulo. A cyclopropenone-caged dibenzoannulated bicyclo-
[6.1.0]nonyne probe (photo-DMBO) was designed that is
unreactive towards tetrazines before light-activation, but
engages in iEDDAC after irradiation at 365 nm. Aminoacyl
tRNA synthetase/tRNA pairs were discovered for efficient site-
specific incorporation of tetrazine-containing amino acids into
proteins in living cells. In situ light activation of photo-DMBO
conjugates allows labeling of tetrazine-modified proteins in
living E. coli. This allows proteins in living cells to be modified
in a spatio-temporally controlled manner and may be extended
to photo-induced and site-specific protein labeling in animals.
labeling and manipulating proteins in living systems.[1a,3a]
Highly specific and orthogonal Pyrrolysyl-tRNA synthetase
(PylRS)/tRNACUA pairs have allowed the site-specific incor-
poration of UAAs bearing strained alkene/alkyne moieties,
such as norbornene (Nor),[4] 1,3-disubstituted cyclopropene
(Cp),[5] bicyclo[6.1.0]nonyne (BCN),[6] and trans-cyclooctene
(TCO)[6a,7] into proteins in E. coli and mammalian cells and
their subsequent chemoselective labeling with tetrazine
conjugates.[8] iEDDAC reactions have found application for
imaging of cell-surface and intracellular proteins, for labeling
and identifying proteomes in E. coli, mammalian cells, and
multicellular organisms[5] as well as for selectively inhibiting
a specific target protein within living cells.[9] Furthermore,
TCO-bearing UAAs have enabled tetrazine-triggered protein
decaging and activation in living cells.[10] Likewise, tetrazine-
bearing amino acids have been incorporated into proteins in
E. coli using engineered variants of the Methanocaldococcus
jannaschii (Mj) Tyrosyl-tRNA synthetase (TyrRS)/tRNACUA
pair, and tetrazine-modified proteins have been labeled with
strained TCO fluorophores in ultra-rapid iEDDAC (second
order rate constants up to 105 mÀ1 sÀ1).[11]
Recent advancements in bioorthogonal chemistries have
shown a growing interest in using external stimuli to induce
bioorthogonal reactivity. In particular, photo-inducible reac-
tions have emerged as a method to exert control over when
and where bioorthogonal partners react with each other.[12]
Key advances include light-triggered tetrazole-alkene photo-
click chemistry,[13] visible-light induced [4+2] cycloaddition
between 9,10-phenanthrenequinone and vinyl ethers[14] as
well as photo-induced activation of cyclopropenone-caged
cyclooctynes for strain-promoted azide–alkyne cycloadditions
(photo-SPAAC).[15] Importantly, also versions of photo-in-
duced iEDDAC have been reported, such as light/enzyme-
triggered redox-activation of dihydrotetrazines to tetra-
zines[16] and the modular caging of cyclopropenes.[17] As the
former approach is so far limited to dihydrotetrazines that are
stable to spontaneous air oxidation, and photo-induced
iEDDAC with described caged cyclopropenes show very slow
reaction rates (k2 ꢀ 10À2–10À4 mÀ1 sÀ1), there is however
a pressing need for novel, photo-inducible iEDDAC reactions
with fast kinetics.
Introduction
Bioorthogonal chemistries offer a versatile platform to
probe and control biomolecules.[1] Progress over the last two
decades in the formulation of more selective and faster
bioorthogonal reagents has led to far-reaching applications in
biology, medicine, and materials.[2] An especially exciting
class of bioorthogonal reactions is represented by inverse
electron-demand Diels–Alder cycloadditions (iEDDAC) be-
tween s-tetrazines and alkene or alkyne dienophiles.[3] In
combination with site-specific incorporation of unnatural
amino acids (UAAs) into proteins via genetic code expansion,
iEDDAC reactions have emerged as indispensable tools for
[*] S. V. Mayer,[+] A. Murnauer,[+] M. K. von Wrisberg, M. L. Jokisch,
Prof. Dr. K. Lang
Center for Integrated Protein Science Munich (CIPSM), Department
of Chemistry, Group of Synthetic Biochemistry, Technical University
of Munich, Institute for Advanced Study
Lichtenbergstr. 4, 85748 Garching (Germany)
E-mail: kathrin.lang@tum.de
[+] These authors contributed equally to this work.
Supporting information and the ORCID identification number(s) for
the author(s) of this article can be found under:
https://doi.org/10.1002/anie.201908209.
Herein we report a rapid light-triggered tetrazine ligation
by developing a photo-activatable BCN-based probe that
confers spatial and temporal control to labeling of tetrazine-
bearing proteins in living cells. Inspired by innovative work
from Popik and co-workers on photo-activated cyclooc-
tynes,[18] we designed and synthesized a cyclopropenone-
caged dibenzoannulated BCN derivative (photo-DMBO)
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KGaA. This is an open access article under the terms of the Creative
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work is properly cited, the use is non-commercial and no modifica-
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Scheme 1. Photochemical decarbonylation of a cyclopropenone-caged
BCN-probe (photo-DMBO) activates reactivity towards tetrazine-bear-
ing proteins, conferring spatial and temporal control to protein label-
ing.
that is quantitatively decarbonylated to the corresponding
alkyne (DMBO) by short UV irradiation at 365 nm. While
photo-DMBO is inert towards tetrazines, the decaged version
reacts rapidly and selectively with tetrazine-bearing proteins
via iEDDAC (Scheme 1).
Figure 1. Site-specific incorporation of tetrazine-bearing amino acids
into proteins in bacteria and selective protein labeling via iEDDAC with
BCN- (3) and TCO- (4) fluorophore conjugates. a) Structures of TetK
(1) and mTetK (2). b) Incorporation of 1 and 2 into sfGFP-150TAG-
His6. left: Coomassie stained SDS-PAGE; middle: a-His6 WB, right:
Coomassie stained SDS-PAGE of purified sfGFP mutants. c) ESI-MS-
characterization of purified sfGFP-N150TetK-His6 and sfGFP-
N150mTetK-His6. d) SDS-PAGE fluorescence imaging and ESI-MS
confirm selective and quantitative labeling of sfGFP-mTetK with BCN-
TAMRA e) Selective labeling of mTetK bearing sfGFP in E. coli cell
lysate with fluorophore conjugates 3 and 4. Structures of 3 and 4 are
shown in the Supporting Information, Figure S2. Cbb: Coomassie
brilliant blue, Fl: Fluorescence.
To use this approach for rapid, photo-induced protein
labeling in living cells, we have synthesized novel tetrazine-
bearing amino acids and found a highly efficient PylRS
mutant for their site-specific incorporation into proteins in
E. coli. Tetrazine-modified proteins react with dibenzoannu-
lated BCN with second-order rate constants of about
50mÀ1 sÀ1, putting them among the fastest bioorthogonal
photo-induced reactions. Light-triggered iEDDAC employ-
ing photo-DMBO and our novel tetrazine-amino acids works
selectively and rapidly (completion within a few minutes at
low mm concentrations) on purified proteins, in complex
mixtures as well as on living E. coli. The strong dependence
on light allows for both sequential dual modification of
proteins as well as for protein labeling with spatio-temporal
control in living systems.
modified amino acids at hand, we set out to site-specifically
incorporate 1 and 2 into proteins in cellulo. A large variety of
structurally diverse UAAs have been genetically encoded in
response to an amber codon introduced into a gene of interest
by employing PylRS/tRNACUA pairs from Methanosarcina
species.[1a,21] None of the so far developed PylRS variants
accepts however lysine derivatives with bulky polar side
chains such as 1 and 2. Guided by structural analyses of the C-
terminal catalytic center of wt PylRS and its mutants,[22] we
screened a panel of > 50 different M. barkeri PylRS mutants
for their ability to direct the selective and site-specific
incorporation of 1 and 2. Gratifyingly, Mb PylRS mutant
(Y271G and C313V, Mb numbering, dubbed TetRS) that has
not been described so far led to the efficient incorporation of
both 1 and 2 into C-terminally His-tagged superfolder green
fluorescent protein (sfGFP) containing a premature amber
codon. SDS-PAGE and a-His6 western blot (WB) analysis
confirmed very good incorporation efficiencies for both 1 and
2 and full-length sfGFP-1-His6 and sfGFP-2-His6 were
isolated in good yields (Figure 1b, ca. 80 mgLÀ1 of culture).
Similarly, myoglobin (Myo) and ubiquitin (Ub) bearing site-
specifically introduced amber codons produced good yields of
protein in presence, but not absence of 1 and 2. The selective
and site-specific incorporation of tetrazine-bearing amino
Results and Discussion
Genetic Encoding and Rapid Labeling of Methyl-tetrazine Amino
Acids
To guarantee efficient and quantitative protein labeling in
living cells we aimed at genetically encoding flexible UAAs
bearing small and physiologically stable tetrazine moieties. As
methyl-substituted tetrazines have been identified as the most
stable derivatives with an optimal stability to reactivity
balance,[11b, 19] we designed lysine-based methyl-substituted
tetrazine amino acids TetK(1) and mTetK(2) (Figure 1a). The
amino acids were synthesized by reacting the corresponding
nitriles (3-hydroxyproprionitrile for 1 and 3-(hydroxyme-
thyl)benzonitrile for 2) with acetonitrile in the presence of
hydrazine and zinc- or nickel-triflate to form the respective
dihydrotetrazine alcohols, which were oxidized and coupled
to the e-amino group of lysine to yield 1 and 2 (Supporting
Information).[20] With efficient access to flexible tetrazine-
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acids was further confirmed by electrospray ionization mass
spectrometry (ESI-MS) of purified proteins (Figure 1c;
Supporting Information, Figure S1).
decarbonylation to form DMBO by irradiation above 350 nm.
Synthesis of photo-9 (Figure 2a) started with Sonogashira
cross-coupling of 3-iodoanisol and 3-ethynylanisol to give the
symmetrical acetylene 5. Partial hydrogenation of 5 using
Lindlarꢀs catalyst generated Z-alkene 6, which was subjected
to cyclopropanation with ethyl diazoacetate to yield 7. The
ester group in 7 was reduced to the corresponding hydroxyl
group and protected by acetylation (8) before subsequent
Friedel–Crafts alkylation with tetrachlorocyclopropene in the
presence of AlCl3 to yield photo-9 after in situ hydrolysis of
the corresponding dichlorocyclopropene intermediate (Fig-
ure 2a, Supporting Information).[24] Despite its considerable
ring strain, photo-9 is stable in aqueous buffers at physio-
logical pH over several days (Supporting Information, Fig-
ure S8a). Upon 5 min of irradiation at 365 nm, photo-9
decarbonylates quantitatively to form DMBO compound 9
(Figure 2b). Photo-decaging is effective in different organic
solvents (MeOH, DMSO) and in aqueous buffers. To test
reactivity of photo-9 towards methyl-tetrazine amino acid 2,
we incubated a 100 mm solution of 2 with a five-fold excess of
photo-9. Only upon UV irradiation of the mixture (5 min at
365 nm), amino acid 2 reacted quantitatively to the cyclo-
addition compound 10, while in the absence of UV irradiation
no formation of product 10 could be observed even after
prolonged incubation (Figure 2c), confirming the exquisite
photo-inducibility of the cycloaddition reaction.
As expected, purified proteins that bear site-specifically
incorporated methyl-tetrazines reacted readily and quantita-
tively with BCN- (3) or TCO-modified (4) fluorophores as
shown by SDS-PAGE based fluorescence imaging and ESI-
MS analysis (Figure 1d; Supporting Information, Figures S2,
S3). Labeling of proteins containing 1 or 2 was also specific
towards the E. coli proteome as shown by selective labeling of
over-expressed intracellular and cell-surface proteins bearing
tetrazine amino acids with fluorophores 3 or 4 via iEDDAC
(Figure 1e; Supporting Information, Figures S4–S7).
Development of a Rapid, Photo-induced iEDDAC
With tetrazine-modified proteins at hand, we set out to
develop photochemically triggered iEDDAC (photo-iED-
DAC) that confers temporal and spatial control to the
labeling of target proteins.[12a] To achieve this goal we
explored the photochemical generation of reactive BCN-
probes. Inspired by work from Popik, who described cyclo-
propenone-caged dibenzocyclooctynes for photo-SPAAC,[18]
we aimed at designing a cyclopropenone-caged version of
BCN that displays a tetrazine-reactive triple bond upon
photochemical decarbonylation (Scheme 1). As cycloprope-
none compounds without aryl substituents are known to
absorb and thereby photo-decage at wavelengths below
250 nm,[23] we decided to synthesize a cyclopropenone-caged
dibenzoannulated BCN probe (cyclopropenone-caged
Rapid, Light-Induced Protein Labeling via photo-iEDDAC
We next set out to test if tetrazine-modified proteins could
be labeled in a photo-induced and selective fashion with
functionalized photo-DMBO compounds. By decorating
di(methoxybenzo)bicyclo[6.1.0]nonyne,
dubbed
photo-
DMBO, Scheme 1), which should be amenable to photo-
Figure 2. Establishing photo-iEDDAC reactivity. a) Synthesis of photo-9. Conditions: a) 1.2 equiv 3-ethynylanisole, 3 equiv DIPEA, 0.1 equiv CuI,
0.05 equiv Pd(PPh3)4, in THF, reflux, overnight, (71%); b) 20% w/w Lindlar catalyst under H2 atmosphere, in hexane, 2 h, rt, (77%); c) 2.5 equiv
ethyl diazoacetate, 0.06 equiv CuSO4, in toluene, 758C, overnight, (18%); d) 2 equiv LiAlH4, in Et2O, 08C–rt, 2 h, (68%); e) 2.6 equiv Ac2O,
0.05 equiv DMAP, 4.9 equiv NEt3, in DCM, 08C–rt, 2 h, (70%); f) 1 equiv tetrachlorocyclopropene, 3 equiv AlCl3, in DCM, À208C–rt, 4 h, aqueous
workup (55%). b) Short UV irradiation (5 min, 365 nm) converts photo-9 quantitatively to 9. c) Photo-9 reacts with amino acid 2 only when
irradiated at 365 nm for 5 min to form iEDDAC product 10.
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photo-DMBO with a PEG-linker and a primary amino group
bearing BocK at position 150 instead of mTetK did not result
for further functionalization with fluorophores we generated
water-soluble photo-11 (Figure 3a) that is quantitatively
decarbonylated to 11 by irradiation at 365 nm for 5 min
(Figure S8b). Incubation of a 10 mm solution of sfGFP-2-His6
with a 25-fold excess of 11 led to quantitative sfGFP-labeling
within 5 min (Supporting Information, Figure S9). Similarly
an irradiated solution (365 nm, 5 min) of sfGFP-2-His6 and
photo-11 yielded quantitative sfGFP-modification within
short incubation times (Figure 3b; Supporting Information,
Figure S9). Importantly, incubation in the absence of light did
not yield any cycloaddition product. Furthermore, sfGFP
in any labeling, confirming the selectivity of photo-iEDDAC
(Supporting Information, Figure S9). Also, TetK-modified
proteins (sfGFP-1-His6) reacted selectively upon light-acti-
vation with photo-11, albeit much more slowly, and quanti-
tative protein labeling with 25-fold excess of 11 was only
achieved after several hours of incubation (Supporting
Information, Figure S10). To check how DMBO compounds
performed in SPAAC reactions, we incubated an azide-
bearing sfGFP[25] with photo-11. Also this labeling took 2–
3 hours to go to completion, highlighting the superior
reactivity of DMBO in iEDDAC versus SPAAC (Supporting
Information, Figure S11). Furthermore, azide-modified ami-
no acids are not completely stable towards reduction to the
corresponding amine in the cytosol of E. coli and therefore
our stable methyl-tetrazine amino acids make for more
reliable reporters in live E. coli. To benchmark photo-DMBO
reactivity against previously reported cyclopropenone-caged
cycloalkynes,[18] we synthesized a cyclopropenone-caged di-
benzocyclooctyne (photo-12) that contains no extra ring
strain on the dibenzoannulated cyclooctyne[26] and incubated
it after light irradiation with sfGFP-2-His6 (Supporting
Information, Figure S12). No cycloaddition product was
observed even after overnight incubation, though decarbon-
ylation to 12 had proceeded cleanly as proven by LC-MS,
highlighting that the extra ring strain in DMBO is needed for
reaction with methyl-tetrazines.[2c,27] Incubation of photo-12
with azide-bearing sfGFP under UV-irradiation resulted in
approximately 60% SPAAC product upon overnight incuba-
tion, demonstrating that the extra strain in DMBO results
also in higher reactivity towards azides (Supporting Informa-
tion, Figures S11, S13). Interestingly, also a recently reported
dibenzoannulated and cyclopropenone-caged silicon-contain-
ing cycloheptyne (photo-13)[28] failed in reacting with tetra-
zine- or azide-modified proteins under identical conditions
(Supporting Information, Figures S14, S15).
Encouraged by the short incubation times needed for
quantitative iEDDAC labeling between DMBO compounds
and sfGFP-2-His6, we set out to determine on-protein rate
constants in a more quantitative way. As previously observed
and exploited for determination of on-protein kinetics in vitro
and in cellulo, site-specific incorporation of tetrazine amino
acids in vicinity to the sfGFP chromophore (N150) lead to
sfGFP-fluorescence quenching that is restored after iEDDAC
of the tetrazine moiety with a strained trans-cyclooctene
label.[11] Similarly, also genetic encoding of flexible mTetK
into sfGFP-N150TAG led to sfGFP chromophore quenching
and fluorescence recuperated after reaction with 11 (Fig-
ure 3c).
Figure 3. Characterization of photo-iEDDAC on tetrazine-bearing pro-
teins. a) Structures of water-soluble photo-DMBO conjugates. Both
photo-11 and the Cy5-conjugate photo-14 are decarbonylated by short
irradiation at 365 nm to quantitatively form 11 and 14, respectively.
b) Quantitative and rapid labeling of sfGFP-N150mTetK with photo-11
analyzed by LC-MS. c) Excitation at 488 nm produces quenched
fluorescence for sfGFP-N150mTetK, which is restored after reaction
with 11. d) Determination of second-order rate constant k2 of sfGFP-
N150mTetK and 11. Inset shows fluorescence increase (508 nm) of
sfGFP-N150mTetK upon addition of a 20-fold excess of 11 over time.
e) Rapid protein labeling with fluorophore-conjugate 14. f) Selective
and light-induced labeling of mTetK-bearing sfGFP with photo-14
towards the E. coli proteome.
By following the exponential increase over time in sfGFP-
fluorescence, we were able to quantify second order on-
protein rate constants under pseudo first-order conditions.
We determined a rate constant of about 50mÀ1 sÀ1 (Figure 3d;
Supporting Information, Figure S16), which is nearly twice as
À
fast as iEDDAC between mTetK and BCN OH and 50 or
1000 times faster than the corresponding reactions with 1,3-
disubstituted cyclopropene or norbornenol (Supporting In-
formation, Figures S17–S19), alkene reporters that are rou-
tinely used for biomolecule labeling.[4–5,29] Reaction between
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sfGFP-2-His6 and 11 is also significantly faster than reaction
between 11 and a sfGFP mutant bearing a previously
described phenylalanine based tetrazine amino acid
(TetF),[11b] which is presumably due to higher flexibility and
on-protein accessibility of mTetK versus TetF (Supporting
Information, Figure S20). The fast kinetic rate constants
enable photo-triggered labeling of mTetK-modified proteins
with photo-11 within minutes at low mm or nm concentrations
and put photo-iEDDAC reactions between DMBO and
tetrazines among the fastest photo-induced bioorthogonal
reactions, several orders of magnitude faster than recently
described approaches with caged cyclopropenes (k2 ꢀ 10À2–
10À4 mÀ1 sÀ1),[17] and considerably faster than most photo-
SPAAC reactions with strained dibenzocyclooctynes (k2
ꢀ 10À1–10À2 mÀ1 sÀ1),[30] and on par with the recently described
and so far fastest photo-SPAAC reaction employing photo-
oxa-dibenzocylooctyne (k2 ꢀ 40mÀ1 sÀ1).[31]
Photo-iEDDAC Confers Spatio-temporal Control to Protein
Labeling on Living Cells
Figure 4. Photo-iEDDAC labeling on living cells. a) In gel fluorescence
imaging and fluorescence microscopy show efficient and light-induced
labeling of a cell-surface protein in living E. coli. b) Structure of
bifunctional linker photo-16, displaying a BCN and a photo-DMBO
moiety. c) The DMBO moiety in photo-16 becomes only available upon
UV-irradiation and thereby allows sequential labeling of live E. coli with
temporal and spatial control.
To create fluorescent photo-DMBO probes, the primary
amino group of photo-11 was coupled to succinimidyl esters
of Cy5 and tetramethylrhodamine (TAMRA) dyes, creating
photo-14 (Figure 3a; Supporting Information, Figure S21)
and photo-15 (Supporting Information, Figure S22). Photo-
DMBO fluorophores react rapidly, specifically and quantita-
tively with recombinant proteins bearing site-specifically
incorporated mTetK upon irradiation at 365 nm, as confirmed
by LC-MS and SDS-PAGE-based fluorescence imaging (Fig-
ure 3e; Supporting Information, Figures S21, S22). Light-
induced labeling with photo-14 and photo-15 is also distinct
towards cellular proteins of the E. coli proteome, as shown by
specific and selective labeling of mTetK-bearing proteins in
E. coli lysates with minimal background. Importantly, effi-
cient labeling is reliant on irradiation at 365 nm (Figure 3 f).
tetrazine ligation prior to UV irradiation and cells are
efficiently labeled with a methyl-tetrazine–fluorophore con-
jugate (Tet-Cy5) only after light activation (Figure 4c;
Supporting Information, Figure S24), conferring mutual or-
thogonality to iEDDA cycloadditions with tetrazines and
providing in principle control over protein-labeling both in
time and space.[32a,33]
Encouraged by specific modification of mTetK-bearing Conclusion
proteins in cell lysates we set out to use photo-iEDDAC for
temporally controlled labeling of living E. coli. We over-
expressed outer membrane protein OmpC-Y232mTetK on
the surface of E. coli cells, washed cells and treated them with
photo-14 either in the presence or absence of UV light.
Labeling was specific for mTetK-bearing cells that were
irradiated at 365 nm (Figure 4a; Supporting Information,
Figure S23). As photo-DMBO reactivity is strictly controlled
by light and remains dormant until activated by UV
irradiation, photo-iEDDAC can be used in one-pot reactions
with iEDDAC for sequential dual labeling of proteins.[32] To
show this mutual orthogonality, we constructed a heterobi-
functional crosslinker, which unifies two moieties displaying
comparable iEDDAC reactivities, namely BCN and DMBO,
in one molecule for sequential one-pot labeling (Figure 4b).
Photo-16 displays both BCN- and photo-DMBO reactivity
and permits controlled assembly of different methyl-tetrazine
tagged conjugates. E. coli cells expressing mTetK-bearing
OmpC on their cell surface were treated with photo-16,
allowing covalent attachment of photo-16 via its BCN moiety.
The photo-DMBO functionality in photo-16 is refractory to
We have reported a novel light-induced iEDDAC be-
tween tetrazines and a cyclopropenone-caged dibenzoannu-
lated BCN probe (photo-DMBO). We have demonstrated the
efficient site-specific incorporation of methyl-tetrazine modi-
fied amino acids 1 and 2 into proteins in E. coli and their
efficient, specific, and light-triggered labeling with photo-
DMBO fluorophore conjugates. Photo-iEDDAC proceeds
with on-protein rate-constants of about 50mÀ1 sÀ1, on par with
the fastest bioorthogonal photo-induced reactions, enabling
protein labeling within minutes at low mm concentrations.
These reactions are 2–4 orders of magnitude faster than
iEDDAC using cyclopropene or norbornene as dienophiles
and nearly twice as fast as cycloadditions between BCN and
metabolically stable methyl-tetrazines. While we have dem-
onstrated the advantages of our approach in vitro and in
living E. coli, the ability to incorporate UAAs in mammalian
cells and C. elegans,[34] zebrafish,[35] and D. melanogaster[36]
using engineered PylRS/tRNACUA pairs suggests that it may
be possible to extend the photo-induced labeling approach
described here to site-specifically label proteins in animals in
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holt, J. M. Verkade, M. Wiessler, C. Schultz, J. C. van Hest, F. L.
a photo-induced fashion, which might be especially attractive
in combination with multi-photon activation of photo-DMBO
compounds.[37] Exploitation of photo-iEDDAC and genet-
ically encodable tetrazine reporters to study receptor activa-
tion is focus of current studies in our lab. We furthermore
expect that other fields such as the fabrication of micro-
arrays,[38] biosensors, and the preparation of multifunctional
material may benefit from this photo-triggered and rapid
iEDDAC reaction.
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Acknowledgements
This work was supported by the Excellence Initiative CIPSM
and the DFG through the following programs: SFB1035,
GRK1721 and SPP1623 (to K.L). K.L. is a Mçssbauer
Professor at TUM-IAS and as such acknowledges funding
by the Excellence Initiative and the EU Marie Curie
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Conflict of interest
The authors declare no conflict of interest.
Keywords: bioorthogonal reactions ·photo-induced labeling ·
protein labeling ·tetrazine ·unnatural amino acids
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