Genetically encoded tetrazine amino acid directs rapid site-specific in vivo bioorthogonal ligation with trans-cyclooctenes

Article abstract of DOI:10.1021/ja2109745

Bioorthogonal ligation methods with improved reaction rates and less obtrusive components are needed for site-specifically labeling proteins without catalysts. Currently no general method exists for in vivo site-specific labeling of proteins that combines fast reaction rate with stable, nontoxic, and chemoselective reagents. To overcome these limitations, we have developed a tetrazine-containing amino acid, 1, that is stable inside living cells. We have site-specifically genetically encoded this unique amino acid in response to an amber codon allowing a single 1 to be placed at any location in a protein. We have demonstrated that protein containing 1 can be ligated to a conformationally strained trans-cyclooctene in vitro and in vivo with reaction rates significantly faster than most commonly used labeling methods.

Full text of DOI:10.1021/ja2109745

Communication
pubs.acs.org/JACS
Genetically Encoded Tetrazine Amino Acid Directs Rapid Site-Specific
in Vivo Bioorthogonal Ligation with trans-Cyclooctenes
Jason L. Seitchik,† Jennifer C. Peeler,† Michael T. Taylor,‡ Melissa L. Blackman,‡ Timothy W. Rhoads,§
Richard B. Cooley,§ Christian Refakis,† Joseph M. Fox,‡ and Ryan A. Mehl*,§
†Department of Chemistry, Franklin & Marshall College, Lancaster, Pennsylvania 17604-3003, United States
‡Brown Laboratory, Department of Chemistry and Biochemistry, University of Delaware, Newark, Delaware 19716, United States
§Department of Biochemistry and Biophysics, Oregon State University, Corvallis, Oregon 97331, United States
S
* Supporting Information
A bioorthogonal ligation reaction on proteins that is going to
ABSTRACT: Bioorthogonal ligation methods with im-
proved reaction rates and less obtrusive components are
needed for site-specifically labeling proteins without
catalysts. Currently no general method exists for in vivo
site-specific labeling of proteins that combines fast reaction
rate with stable, nontoxic, and chemoselective reagents. To
overcome these limitations, we have developed a tetrazine-
containing amino acid, 1, that is stable inside living cells.
We have site-specifically genetically encoded this unique
amino acid in response to an amber codon allowing a
single 1 to be placed at any location in a protein. We have
demonstrated that protein containing 1 can be ligated to a
conformationally strained trans-cyclooctene in vitro and in
vivo with reaction rates significantly faster than most
commonly used labeling methods.
advance in vivo labeling will need to (i) have components that
are unreactive to cell culturing conditions, (ii) be rapid and
quantitative, (iii) be encoded genetically for any site on the
protein, and (iv) result in a stable linkage facilitating labeling
with diverse probes. In order to satisfy these labeling criteria
and overcome current limitations, we set out to develop a
genetically encodable functionality based on the tetrazine−
alkene coupling reaction.
Inverse electron-demand Diels−Alder reactions of s-
tetrazines are venerable reactions in organic synthesis.9
Recently, we introduced the bioorthogonal reaction between
tetrazines and trans-cyclooctene derivatives,10 a method of
rapid bioconjugation which has been applied by a number of
groups.11 The foundation of this bioorthogonal reaction is a
photochemical, flow-chemistry method that provided the first
general and scalable route to functionalized trans-cyclo-
octenes.12 These strained alkenes have allowed for tetrazines
with reduced reactivities to be used that are more stable toward
nucleophiles (i.e., water and thiols) but still yield fast coupling
reactions (coupling rates with k2 > 1000 M−1 s−1).10,11,13
To develop an unnatural amino acid that could partner in the
tetrazine ligation, one of the reactive partners needs to be
attached to an amino acid, while the other needs to be attached
to a useful label. The trans-cyclooctene functionality is stable
under biological conditions and is relatively easy to
functionalize.11a,b,d,f Accordingly, we set out to develop a
tetrazine containing amino acid that is stable to cell culturing
conditions but still sufficiently reactive with strained alkenes.
The tetrazines that have been studied in conjugations with
trans-cyclooctene, such as amido-substituted 3,6-di(2-pyridyl)-
s-tetrazines and monoaryl-s-tetrazines, are stable in biological
milieu but display slow background reactivity that would be
incompatible with cellular growth conditions.11c,13,14 Thus, we
aimed to design a tetrazine derivative that would be stable in
cells, 4-(6-methyl-s-tetrazin-3-yl)aminophenylalanine (1). Add-
ing electron-donating methyl and secondary amine substituents
to the 3 and 6 positions of the tetrazine should improve its
stability against nucleophilic attack. The tetrazine-UAA 1 was
synthesized in five steps from commercially available materials
(see Supporting Information (SI) for details).
nderstanding protein networks and structural changes is
U
key to revealing the secrets of biology. Currently available
in vitro bioorthogonal ligation methods for site-specifically
labeling proteins are limited by the reaction rates or bulk of
reactive components. Also, no general method exists for in vivo
site-specific labeling of proteins with a catalyst-free reaction or
reactants that are stable, nontoxic and chemoselective.
A wide variety of functionalities have been genetically
incorporated for the site-specific labeling of proteins, but
these methods are not effective in vivo because they have
modest coupling rates.1 A robust but slow labeling reaction
involves azides with unstrained alkynes and Cu+ catalyst (k2 =
10−2−10−4 M−1 s−1).2 Strained, fluorinated alkyne-containing
amino acids can be used without Cu+ but still have modest
coupling rates (k2 = 1 M−1 s−1).1a,c,3 Genetically encoded
cyanobenzathiazole condensations and photo-click reactions
have been developed with rate constants reaching 10 M−1 s−1
but have side reactions in vivo or need short wavelengths of
light.4 A wide variety of reactions have also been developed but
show varying levels of side reactions in vivo (using cyclooctynes,
aldehydes, phosphine labeling reagents).5 Alternative genetic
incorporation methods such as self-labeling tag sequences6 and
fluorescent protein fusions7 are nontoxic and can exhibit fast
coupling rates, but the components are often bulky and can
rarely be moved to multiple locations within the labeled protein
without compromising the function of the system of interest.8
Received: November 22, 2011
Published: January 26, 2012
© 2012 American Chemical Society
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While the electron donating 3-amino substituent of 1 makes
this tetrazine stable and soluble in biological conditions, it also
decreases the rate of Diels−Alder reactions. Accordingly, we
chose to combine this amino acid with derivatives of compound
2, a strained trans-cyclooctene (s-TCO) that we recently
developed.13 With tetrazines, compound 2 displays relative
rates that are significantly faster than our original system based
on 5-hydroxy-trans-cyclooctene.10
Using 1 mM 1, 124 mg of GFP-1 was purified per liter of
medium, while GFP-wt yielded 220 mg/L under similar
conditions (no GFP is produced in the absence of 1). To
further demonstrate that 1 can be incorporated into
recombinant proteins using pDule-mtaF, we compared the
masses of GFP-1 to GFP-wt using ESI-Q-Tof mass analysis.
The native GFP-wt has the expected mass of 27 827 1 Da,
and GFP-1 exhibits the expected mass increase to 27 971
1
We set out to evolve the Methanococcus jannaschii (Mj)
tyrosyltRNA synthetase (RS)/tRNACUA pair since it has
previously been used to incorporated UAAs of similar structure
in response to an amber codon.15 To evolve the orthogonal
MjTyrRS/tRNACUA pair capable of incorporating 1 in
Escherichia coli, we used a library of the synthetase (RS) gene
that was randomized for the codons corresponding to six active-
site residues within 7 Å of the bound tyrosine.15 We performed
two rounds of alternating positive and negative selection on this
library (see SI for details). Plasmids from surviving clones were
transformed into cells with a plasmid containing a GFP gene
interrupted with an amber codon.6g,16 We assessed 96 colonies
for UAA-dependent expression of GFP. The top 20 performing
clones showed >300 mg/L of GFP-1 expression in the presence
of 1 and no detectable GFP fluorescence over background in
the absence of 1 (SI, Sup. Figure 2). Sequencing these 20
clones revealed one unique RS sequence contain the mutations
(Y32E, L65A, A107E, F108P, Q109S, D158G, L162G).
To further characterize the incorporation of 1 into proteins
in response to the amber codon, the selected RS was cloned
into a pDule vector that contains one copy of Mj tRNACUA to
create pDule-mtaF.6g,16,17 Expression of GFP gene interrupted
by an amber codon at site 150 in the presence of pDule-mtaF
was efficient and dependent on the presence of 1 (Figure 1B).
Da, verifying that 1 is incorporated at single site (SI, Sup.
Figure 2). The site of 1 incorporation was confirmed by
analysis of the tandem mass spectrometry (MS/MS)
fragmentation series of the relevant tryptic peptide (Figure
1C).18 Overall, the results of protein expression with affinity
purification, SDS-PAGE, MS, and MS/MS analysis demon-
strate the high fidelity and efficient incorporation of 1 at a
genetically programmed site in GFP using pDule-mtaF. Since
expressions contained 1 for 40 h in media at 37 °C, this also
confirms that stability of 1 to cell growth and protein
purification conditions.
The conjugated π systems of tetrazines cause them to absorb
visible light between 510 and 550 nm, enabling them to quench
many attached fluorescent probes.5g This quenching has proven
useful because once the tetrazine undergoes cycloaddition with
alkenes the conjugated system is removed and the coupled
product ceases to quench nearby fluorophores. When 1 is
encoded at site 150 in GFP, ∼12 Å from the protein
chromophore, the protein fluorescence is quenched 11-fold
(Figure 2A,B). Incubating GFP-1 (3 μM) with 39 μM 2 shows
Figure 2. Characterization of tetrazine transcyclooctene reaction on
GFP. (A) Quenched GFP-1 is “turned on” by coupling with 2, forming
fluorescence GFP-3. (B) Excitation at 488 nm produces low
flourescene for GFP-1, while the reaction forming GFP-3 produces
full fluorescence for GFP. (C) ESI-Q-Tof MS analysis of product from
GFP-1 with 2 demonstrates specific and quantitative labeling of GFP-
1. (D) MALDI MS analysis of GFP-1 reaction with diacetyl-
fluorescein-labeled sTCO.
Figure 1. Genetic incorporation of 4-(6-methyl-s-tetrazin-3-yl)-
aminophenylalanine (1) into proteins. (A) Amino acid 1 reacts with
sTCO (2) and then loses nitrogen gas to form the stable conjugate 3.
(B) The evolved MjRS/tRNACUA pair in pDule-mtaF allows for site-
specific incorporation of 1 in response to an amber codon. Lane 2
shows expression levels of GFP-wt from pBad-GFP-His6. Production
of GFP-1 from pBad-GFP-150TAG-His6 is dependent on 1 in the
growth media: lane 3, without 1 present; lane 4, with 1 mM 1 present.
Protein was purified by Co2+ affinity chromatography, separated by
SDS-PAGE and stained with Coomassie. (C) MS/MS fragmentation
of tryptic peptides derived from GFP-1 samples demonstrates the
efficient high-fidelity incorporation of a single 1 UAA in response to an
amber stop codon using the mtaF synthetase. The spectra confirm 1
incorporation at codon 150. The fragmentation sites are illustrated
above the spectrum.
a complete return of fluorescence in <30 s, indicating GFP-3
was formed. ESI-Q-Tof of the desalted reaction mixture
confirmed the quantitative conversion of GFP-1 (expected
27 971 Da; observed 27 971 1 Da) into GFP-3 (expected 28
094 Da; observed 28093 1 Da (SI, Sup. Figure 2).
It has been shown that tetrazine−alkene reaction rates are
solvent dependent and are accelerated in polar solvents.10 In
order to determine this rate constant for this reaction at a
specific site in a protein the increase in GFP-3 fluorescence was
followed in a PBS buffer at pH 7, 22 °C. The kinetics of the
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reaction were performed under pseudo-first-order conditions as
verified by a single exponential fit for product formation
([GFP] was 0.10 μM and [2] ranged from 27.0 to 6.8 μM).
The in vitro second-order rate constant for GFP-1 with 2 is 880
10 M−1 s−1 (Figure 3A). This verifies that this site-specific
In vivo site-specific reaction rate constants have yet to be
determined for bioorthogonal reactions. To determine the rate
constant for this reaction inside living cells, E. coli expressing
GFP-1 was pelleted and resuspended in PBS buffer. In vivo
kinetic studies were performed similarly to the in vitro studies
and the five pseudo-first-order rate constants were used to
determine the bimolecular rate constant. The in vivo second-
order rate constant for GFP-1 with 2 is 330
20 M−1 s−1
(Figure 3B). Since identical conditions were used for in vitro
and in vivo kinetic measurements, the 60% rate constant
reduction is most likely a result of the in vivo cellular
environment or hampered uptake of 2. Limiting cellular uptake
of 2 would result in non-exponential kinetics for the formation
of GFP-3, which was not observed (Figure 3B). Also, the rate
constant for the overall process measured in cells is comparable
to the in vitro rate constants indicating the uptake step cannot
be significantly slower than the reaction step. Molecular
crowding or electrostatic changes induced by the internal
cellular environment could account for the rate change. The in
vivo coupling reaction was also verified in rich media and on
live cells. No toxicity was evident as judged by comparing cell
viability of cells harboring the sTCO reagent to those that did
not (SI, Sup. Figures 5 and 6). While the in vivo reaction is
slower than the in vitro, the coupling rate is still sufficient to
label proteins in their native environment.
To verify that this labeling strategy is general, 2 was
conjugated to a diacetyl-fluorescein dye, forming 4 (SI, Sup.
Scheme 2). The fluorescently labeled trans-cyclooctene 4 was
then reacted with GFP-1, similarly to reactions with 2.
Quantitative labeling of GFP-1 would result in GFP-5 mass
of 28 653 Da (Figure 2D), but in aqueous conditions
deacetylated products would be expected. While the
fluorescein-labeled protein ionized poorly via ESI-MS,
MALDI MS was used to confirm labeling (expected 28 653
Da; observed 28 635 100 Da). This confirms that protein-
containing 1 is reactive with labeled strained trans-cyclooctenes.
In conclusion, we report a general method for the site-
specific incorporation of a novel, stable tetrazine containing
amino acid, 1, into recombinant proteins in E. coli. We
demonstrated that protein containing 1 can be quantitatively
labeled with strained trans-cyclooctenes and that the resulting
ligation product is stable to in vivo cellular conditions. We
determined the in vitro and in vivo rate constants for this
bioorthogonal coupling to be significantly faster than currently
used site-specific labeling reactions.1a,2−4 The speed of this
catalyst-free reaction and the stability of reacting components
and ligation product will make it an advantageous tool for
studying complex protein networks and conformational
changes in vivo and in vitro. Our future efforts will focus on
developing this reaction for use in labeling proteins in live yeast
and mammalian cells and in engineering protein interactions.
Figure 3. Rate constant determination for in vitro and in vivo reaction
of GFP-1 and 2. (A) In vitro kinetics resulted in rate k = 880 10 M−1
s−1. (B) In vivo kinetics resulted in k = 330 20 M−1 s−1. For both
experiments unimolecular rate constants were calculated by fitting the
rate of GFP-3 formation to a single exponential at five different
concentrations of 2. Inset is bimolecular rate constant determination
using unimolecular kobs = k[2].
tetrazine−strained cyclooctene reaction is orders of magnitude
faster than established site-specific labeling methods.
Thus far, tetrazine−alkene coupling reactions on engineered
proteins have only been characterized in vitro and on cell
surfaces. While it is possible to genetically encode ketone, azide
and alkyne functionality site-specifically on amino acids, little
labeling has been done on these engineered proteins in vivo
with these amino acids. To determine if this bioorthogonal
reaction is rapid and quantitative in vivo, E. coli cells containing
expressed GFP-1 were incubated with 0.1 mM 2 in PBS buffer
at room temperature. As expected, the fluorescence returned in
<1 min, indicating that GFP-3 had been formed. The cells were
cooled and washed twice with PBS at 0 °C to remove unreacted
2. ESI-Q-Tof MS of affinity purified his-tag protein resulted in a
molecular mass matching only GFP-3 (Figure 2C). This verifies
that 2 can be taken up by E. coli cells, the reaction is facile, and
the conjugated product is stable in vivo. This demonstrates the
first site-specific in vivo reaction for this type of bioorthogonal
ligation.
ASSOCIATED CONTENT
■
S
* Supporting Information
Experimental and analytical details; complete ref 6e. This
material is available free of charge via the Internet at http://
pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
ryan.mehl@oregonstate.edu
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Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by F&M Hackman and Eyler funds,
NSF-MCB-0448297, Research Corporation (CC6364), NSF-
DMR-09-69301, HHMI undergraduate science program, and
P20 RR017716 from the COBRE Program of the NCRR.
Spectra were obtained with instrumentation supported by
NIEHS #P30ES000210, NSF CRIF:MU grants CHE 0840401
and CHE-0541775. We thank Dr. Scott Brewer for assistance
with kinetics measurements and data analysis.
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