Systematic Evaluation of Bioorthogonal Reactions in Live Cells with Clickable HaloTag Ligands: Implications for Intracellular Imaging

Article abstract of DOI:10.1021/jacs.5b06847

Bioorthogonal reactions, including the strain-promoted azide-alkyne cycloaddition (SPAAC) and inverse electron demand Diels-Alder (iEDDA) reactions, have become increasingly popular for live-cell imaging applications. However, the stability and reactivity

Full text of DOI:10.1021/jacs.5b06847

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Article
Systematic Evaluation of Bioorthogonal Reactions in Live Cells with
Clickable HaloTag Ligands: Implications for Intracellular Imaging
Heather E. Murrey, Joshua Capps Judkins, Christopher W. am Ende, T. Eric Ballard, Yinzhi Fang, Keith
A Riccardi, Li Di, Edward Guilmette, Joel W Schwartz, Joseph Michael Fox, and Douglas S. Johnson
J. Am. Chem. Soc., Just Accepted Manuscript • Publication Date (Web): 13 Aug 2015
Downloaded from http://pubs.acs.org on August 13, 2015
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Systematic Evaluation of Bioorthogonal Reactions in Live
Cells with Clickable HaloTag Ligands: Implications for Intra-
cellular Imaging
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Heather E. Murrey,*,†,§ Joshua C. Judkins,† Christopher W. am Ende,† T. Eric Ballard,†, ‡ Yinzhi Fang,ǁ Keith
Riccardi,‡ Li Di,‡ Edward R. Guilmette,§ Joel W. Schwartz,§ Joseph M. Fox,ǁ and Douglas S. Johnson*,†
†Worldwide Medicinal Chemistry, Pfizer Worldwide Research and Development, Cambridge, Massachusetts 02139, United States
‡Pharmacokinetics, Dynamics and Metabolism, Pfizer Worldwide Research and Development, Groton, Connecticut, 06340, Unitꢀ
ed States
§Neuroscience and Pain Research Unit, Pfizer Worldwide Research and Development, Cambridge, Massachusetts 02139, United
States
ǁBrown Laboratories, Department of Chemistry and Biochemistry, University of Delaware, Newark, Delaware 19716, United
States
KEYWORDS: click chemistry; bioorthogonal reactions; clickable HaloTag; fluorophores; intracellular imaging; SPAAC; iEDDA
ABSTRACT: Bioorthogonal reactions, including the strainꢀpromoted azideꢀalkyne cycloaddition (SPAAC) and inverse electron deꢀ
mand DielsꢀAlder (iEDDA) reactions, have become increasingly popular for live cell imaging applications. However, the stability and
reactivity of reagents has never been systematically explored in the context of a living cell. Here we report a universal, organelleꢀ
targetable system based on HaloTag protein technology for directly comparing biorthogonal reagent reactivity, specificity, and stability
using clickable HaloTag ligands in various subcellular compartments. This system enabled a detailed comparison of the bioorthogonal
reactions in live cells and informed the selection of optimal reagents and conditions for live cell imaging studies. We found that the
reaction of sTCO with monosubstituted tetrazines is the fastest reaction in cells, however both reagents have stability issues. To address
this, we introduced a new variant of sTCO, AgꢀsTCO, which has much improved stability and can be used directly in cells for rapid
biorthogonal reactions with tetrazines. Utilization of Ag complexes of conformationally strained transꢀcyclooctenes should greatly exꢀ
pand their usefulness especially when paired with less reactive, more stable tetrazines.
cellular biomolecules.4,5 These reactions typically follow secondꢀ
order kinetics and have reaction rate constants ranging from 10ꢀ4
Mꢀ1sꢀ1 to 106 Mꢀ1sꢀ1;5,31,32 however, these rate constants are typiꢀ
cally measured in vitro and are not necessarily reflective of rates in
complex biological systems. While many reactions have been
evaluated in vitro or at the cell surface, relatively few bioorthogoꢀ
nal reactions have been carried out in an intracellular context. In
addition, with more than 20 unique reactions,5 selecting the apꢀ
propriate chemistry for a given application can be daunting. We
envisioned developing a system that would enable the unbiased
investigation of bioorthogonal reaction progression in the comꢀ
plex intracellular biological mileu of living cells, where issues
such as reagent permeability, selectivity, and crossꢀreactivity
could be explored.
Recently, methods have been developed to label proteins with
smallꢀmolecules using genetically encoded fusion proteins with
the covalent selfꢀlabeling O6ꢀalkyl guanineꢀDNA alkyltransferase
(AGT) known as SNAPꢀtag33,34 and an engineered haloalkane
dehalogenase known as HaloTag.35 These proteins covalently
react with small molecule substrates and allow selective labeling
within living systems. AGT fusion proteins can be covalently
Introduction
Probing the biological functions of proteins and small moleꢀ
cules is essential to our mechanistic understanding of cell bioloꢀ
gy. Recent developments in bioorthogonal chemistry1ꢀ5 have
provided a means to chemically tag biomolecules in live cells
without significantly perturbing their native functions. These
bioorthogonal chemical tags provide unique handles that, after
ligation with specific reporters, enable visualization and/or idenꢀ
tification of biological targets. Bioorthogonal tags have been
introduced into proteins in a number of ways: reaction with
clickable covalent inhibitors,6,7 ligandꢀdirected affinityꢀbased
labeling with tosyl8 and other reactive groups,9,10 photoaffinity
labeling with clickable photoprobes,11,12 ligation using enzymes
(such as lipoic acid ligase,13ꢀ15 protein farnesyltransferase16 or
sortase17), metabolic labeling18 (clickable glycans,19,20 amino acꢀ
ids,21 lipids,22,23, acetylꢀCoA24 and SꢀadenosylꢀLꢀmethionine anaꢀ
logs25), and genetic encoding of unnatural amino acids.26,27 Theꢀ
se techniques are finding great utility in drug discovery efforts
for target identification, targetꢀengagement, and target validation
directly in living systems.28ꢀ30
With the widespread use of bioorthogonal labeling strategies,
there is increasing effort to develop novel reactions for targeting
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Scheme 1. Overall scheme for organelleꢀtargetable HaloTagꢀbased evaluation of strain promoted azideꢀalkyne cycloaddition (SPAAC, top) and
inverse electron demand DielsꢀAlder (iEDDA, bottom) biorthogonal chemistry with analysis by fluorescence microscopy and inꢀgel fluoresꢀ
cence.
labeled with O6ꢀbenzylguanine derivatives and HaloTag fusion
proteins covalently bind chloroalkane ligands. One advantage of
these genetic fusion techniques is that they can be designed to
target selected organelles in live cells.36,37 There are now a numꢀ
ber of recombinant methods that can be used to incorporate
bioorthogonal groups into proteins, either via direct encoding of
unnatural amino acids,27,38ꢀ40 or through fusion of an acceptor
peptide for ligaseꢀmediated attachment of a bioorthogonal
tag.14,15 Several of these methods have been applied to liveꢀcell
imaging.14,41ꢀ43 However, these techniques are not yet able to
accept the full spectrum of bioorthogonal tagging groups, many
of which are sterically demanding and hydrophobic.
demand DielsꢀAlder (iEDDA) reaction of strained cycloalkenes
and cyclooctynes with tetrazines.31,58,59 We used bioorthogonal
fluorophore reporters to investigate reaction rates, reagent speciꢀ
ficity, cellular availability, stability, and optimal conditions for
intracellular labeling in living mammalian cells using inꢀgel fluoꢀ
rescence and fluorescence microscopy (Scheme 1). This system
provides a novel, liveꢀcell platform for the unbiased investigation
of bioorthogonal chemistry in different subcellular organelles
and will be a useful tool to characterize novel reactions and
fluorophores in the context of a living cell.
Results and Discussion
Herein, we report the development and validation of an orgaꢀ
nelleꢀtargetable model system that can be used to systematically
evaluate bioorthogonal reactions in live cells. We adapted the
HaloTag protein labeling technology35,44 by synthesizing chloroꢀ
alkane derivatives incorporating various bioorthogonal groups
and covalently displaying these groups on HaloTagꢀfusion proꢀ
teins. Previously, this technology has been used to label Haloꢀ
Tagꢀfusion proteins in live cells with chloroalkaneꢀlinked fluoꢀ
rescent molecules,45ꢀ50 photosensitizing chromophores,51 photoꢀ
reactive kinase inhibitors52,53 and hydrophobic tags.54,55 Haloꢀ
Tagꢀprotein fusions were selected to localize the HaloTag proꢀ
tein, and hence the bioorthogonal reaction, to different subcelluꢀ
lar compartments, including the nucleus, cytosol, plasma memꢀ
brane and endoplasmic reticulum (ER). Because HaloTag labelꢀ
ing is broadly general, it can be used to incorporate a wide range
of bioorthogonal reaction partners thereby enabling the comparꢀ
ative study of reaction efficiency and selectivity. Moreover, the
same HaloTag constructs can be used to directly incorporate
fluorophores, thereby providing a fluorescence readout for quanꢀ
tifying the efficiency of bioorthogonal methods of attaching the
same fluorophores. Overall, this system enables a quantitative
comparison between different bioorthogonal ligations, as well as
the participating chemical groups, in the complex microenviꢀ
ronments within the confines of a living cell, where true
bioorthogonality can be evaluated.
Design and Synthesis of Clickable HaloTag Ligands and
Bioorthogonal Fluorophores. We developed a model system
based on HaloTag protein technology to examine bioorthogonal
reactions inside living cells and optimize conditions for liveꢀcell
fluorescence imaging applications (Scheme 1). A suite of
bioorthogonal HaloTag ligands were synthesized (Figure 1A) to
investigate two prominent bioorthogonal reactions that have
become increasingly popular for liveꢀcell labeling: SPAAC and
iEDDA (see Supporting Information (SI) for synthesis). For
SPAAC, several cyclooctynes with enhanced reaction kinetics
due to increased strain energy have been introduced, including
the dibenzocyclooctynes DIBO,60 DBCO61 (also known as
DIBAC) and BARAC,62 and the bicyclononyne BCN.63 We
chose to prepare HaloTag ligands based on the commercially
available cyclooctynes DBCO 1 and BCN 2 (Figure 1A) and
investigate the SPAAC reaction with fluorophoreꢀazides (i.e., 15,
19 and 23, Figure 1BꢀD). In addition, we examined the reverse
biorthogonal group pairing, by preparing chloroalkaneꢀazide
HaloTag ligand 3 to tag the protein with an azide, followed by
reaction with DBCOꢀ and BCNꢀlinked fluorescent reporters 16
and 17, respectively. For iEDDA, we prepared HaloTags ligands
incorporating seven different dienophiles that cover a broad
range of reactivity, including the parent transꢀcyclooctene 4,64 the
conformationally strained transꢀcyclooctenes known as sTCO (5
and 6)65 and dTCO 7,32 as well as derivatives of norbornene (8)66
and cyclopropene (9)67,68 (Figure 1A). The iEDDA reaction was
examined using fluorophoreꢀtetrazines (i.e., 13, 14, 20-22, Figure
1BꢀD) as the reaction partner. We also examined the reaction of
tetrazineꢀchloroalkane 10 and methylꢀtetrazineꢀchloroalkane
To validate this system, we investigated two prominent
bioorthogonal chemistries that are amenable to live cell labeling,
the strainꢀpromoted azideꢀalkyne cycloaddition (SPAAC) beꢀ
tween cyclooctynes and azides56,57 and the inverse electronꢀ
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O
O
Cl
1
2
3
A
R
N
O
B
H
N
O
N
N
N
N
N
4
5
6
7
8
9
R
H
CO2
N
R=H, 5-TAMRA-Tz 13
R=Me, 5-TAMRA-Tz-Me 14
O
N3
H
6
O
5
N3
DBCO 1
BCN 2
Azide 3
HN
R
O
5-TAMRA-azide 15
H
10
11
12
13
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H
O
H
O
H
H
O
H
H
O
H
H
H
N
O
N
O
2
H
O
TCO 4
sTCO 5
Ag-sTCO 6
5/6-TAMRA-DBCO 16
O
O
H
H
O
H
O
N
O
H
O
H
O
dTCO 7
Nbor 8
Cprop 9
5-TAMRA-BCN 17
H
O
N
N
O
N
O
H
O
N
H
H
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CO2
N
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N
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R
R=H, Tz 10
R=Me, Me-Tz 11
TAMRA 12
C
33
D
34
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36
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N
O
O
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F
F
R=
R'=
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NH
R
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OR'
O
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N3
O
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Dipivaloyl-fluorescein-azide 19
R=
R=
BODIPY-FL-Tz 22
O
N
N
N
N
HN
R
R=
R'=
or H
O
O
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BODIPY-FL-azide 23
N3
20
Fluorescein-diacetate-Tz 20
Fluorescein-Tz 21
Figure 1. Structures of bioorthogonal (A) HaloTag chloroalkane ligands and (B) TAMRAꢀ, (C) Fluoresceinꢀ, and (D) BODIPYꢀfluorophores.
1166,69ꢀ71 with the BCNꢀ and TCOꢀlinked fluorophores (17 and 18,
respectively) to study the reverse pairing where the tetrazine was
the protein tag and the strained alkyne and alkene was incorpoꢀ
rated in the reporter molecule. We chose to investigate the reacꢀ
tions with clickable versions of three fluorophores, carboxytetꢀ
tein expression and reaction colocalization by fluorescence miꢀ
croscopy. When expressed in mammalian cells, the organelleꢀ
targeting sequence directs expression of the HaloTag protein
inside the lumen of the organelle of interest. Localized HaloTag
fusion protein can then be covalently labeled with various clickꢀ
able HaloTag ligands (i.e., 1ꢀ11) to tag the protein followed by
reaction with the cognate bioorthogonal fluorophore reporters
(Scheme 1). Expression and localization of fusion proteins tarꢀ
geting the nucleus, endoplasmic reticulum (ER), cytosol, and
plasma membrane (Table 1) were confirmed by liveꢀcell fluoresꢀ
cence microscopy (Figure S1) prior to analysis of the bioorthogꢀ
onal reactions.
ramethylrhodamine
(TAMRA),
boron
dipyrromethene
(BODIPY), and fluorescein that have been used extensively for
liveꢀcell imaging (Figure 1BꢀD).
Organelle-Targeted HaloTag Fusion Proteins. HaloTag
fusion proteins were designed to contain three components: 1)
an Nꢀterminal HaloTag protein to react with bioorthogonal Halꢀ
oTag ligands, 2) an organelleꢀtargeting protein sequence, and 3) a
fluorescent protein (GFP or mCherry) for confirmation of proꢀ
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Table 1. OrganelleꢀTargeted HaloTag Constructs Table 2. Live cell reaction rate data and efficiency for SPAAC
1
2
3
4
5
6
7
8
reactions.
HaloTag Construct
Organelle
EC50
HaloTag TAMRA-
t1/2
Emax
(%)b
Halo-H2B-GFP
Halo-H2B-mCherry
Nucleus
Ligand
DBCO (1)
BCN (2)
azide
(µM)a
(min)b
Halo-mCherry
Nucleus/Cytosol
Cytosol
15
0.4
4.2
94
67
Halo-GAP43-GFP
Halo-GAP43-mCherry
Halo-KDEL
15
10.7
40.1
Endoplasmic Reticulum
aFrom dose response of TAMRAꢀazide for 2 h
Halo-KDEL-GFP
Halo-KDEL-mCherry
Halo-mCherry-PDGFR
9
bFrom time course with 25 µM TAMRAꢀazide
Extracellular Plasma Membrane
10
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60
SPAAC Reactions in Live HeLa Cells for Strained Alkyne
Tags with Azide Reporters. For SPAAC ligations, HeLa cells
expressing HaloꢀH2BꢀGFP were labeled with strained alkyne
ligands DBCO 1 or BCN 2, followed by reaction with a dose
response of TAMRAꢀazide 15 for 2h (Figure 2AꢀB). Reactions
were immediately quenched with 500 µM 2ꢀ(2ꢀ(2ꢀ(2ꢀ
azidoethoxy)ethoxy)ethoxy)ethanꢀ1ꢀamine (azideꢀamine 24, Figꢀ
ure S3A) and processed as described in the Materials and Methꢀ
ods section to determine the EC50 for each reaction. Repreꢀ
sentative gels for the SPAAC reaction of TAMRAꢀazide 15 with
the DBCO 1ꢀ and BCN 2ꢀHaloꢀH2BꢀGFP conjugates are shown
in Figure S4A and Figure 2A, respectively and the EC50 curves
from quantitation of 3ꢀ6 replicates for each reaction are shown
in Figure 2B. The reactions were further investigated using a
timecourse with 25 µM 15 to determine the t1/2 and Emax values
(Figure 2CꢀD, Table 2). Representative gels from the timecourse
reaction of TAMRAꢀazide 15 with DBCO 1ꢀ (Figure S4B) and
BCN 2-HaloꢀH2BꢀGFP conjugates (Figure 2C) are shown.
Quantification of the inꢀgel fluorescence and graph of the oneꢀ
phase association curves from 3ꢀ6 replicates are reported in Figure
2D. Interestingly, we found significant differences between
EC50, t1/2, and Emax values for DBCO 1 compared with BCN 2
when reacted with 15 (Table 2). The reaction between 1 and 15
was ~10ꢀfold faster and had a 20ꢀfold lower EC50. Consistent
with our observations for DBCO in these liveꢀcell studies, rate
enhancements up to 400ꢀfold over in vitro reaction rates have
been reported for more lipophilic cyclooctynes in biological enꢀ
vironments.56,74,75 In addition, we observed a significant differꢀ
ence in the saturating levels (Emax) for SPAAC ligations in the
context of a living cell. Here, 1 proceeded with >90% efficiency
(Emax = 94%), whereas 2 had an average Emax of 67% (Table 2).
These findings demonstrate the utility of our model system to
directly compare bioorthogonal reaction parameters in live cells.
Furthermore, the reaction parameters calculated herein are a
reflection of multiple variables, including the reaction kinetics, as
well as the permeability and diffusion of the reactants. Thereꢀ
fore, the differences in rates we observe cannot easily be predictꢀ
ed from in vitro models of reaction kinetics. Taken together, our
studies suggest that the combination of DBCO with TAMRAꢀ
azide is an efficient SPAAC reaction for liveꢀcell nuclear labeling
in mammalian cells.
Efficiency of Clickable HaloTag Ligand Conjugation to
HaloTag Fusion Proteins. We initially investigated the
SPAAC and iEDDA reactions in the nucleus of live HeLa cells
using HaloꢀH2BꢀGFP, which has high protein expression levels
and excellent nuclear localization. Our initial investigations used
TAMRA as the fluorophore reporter as it exhibits low backꢀ
ground fluorescence for live cell imaging and has a relatively
uniform distribution within HeLa cells.72,73 The capacity of Halꢀ
oTag ligands 1-11 to label HaloꢀH2BꢀGFP in HeLa cells was first
examined using a competitive pulseꢀchase assay with fluorescent
TAMRA HaloTag ligand 12. HaloꢀH2BꢀGFP expressing HeLa
cells were labeled with HaloTag ligands 1-11, and chased with
TAMRA ligand 12. The ability of nonꢀfluorescent ligands 1-11 to
inhibit incorporation of 12 was determined by inꢀgel fluoresꢀ
cence (Figure S2). While we did not observe complete saturation
of HaloTag protein under these conditions, the incorporation of
clickable HaloTag ligands 1-11 was similar (~70ꢀ79% incorporaꢀ
tion), enabling a semiꢀquantitative comparison of subsequent
SPAAC and iEDDA reactions.
Comparison of Bioorthogonal Reactions in Live HeLa
Cells. The SPAAC and iEDDA ligations were evaluated in cells
by reacting the clickable HaloTag protein conjugates of 1ꢀ11 with
the corresponding clickable TAMRAꢀbased fluorescent reporters
13ꢀ18. The TAMRAꢀlabeled proteins were detected by inꢀgel
fluorescence to quantify the click chemistry reaction product as
well as any nonspecific protein labeling. Total HaloTag protein
was measured by western blot to control for protein loading. To
account for data fluctuations, variability between experiments,
and the dynamic nature of a live cell system, we included
TAMRA chloroalkane ligand 12 as a positive control to deterꢀ
mine the total amount of HaloTag protein that could be fluoresꢀ
cently labeled per experiment (See Materials and Methods for
detailed procedures). We first analyzed a dose response for each
reaction to determine the halfꢀmaximal effective concentration
(EC50), defined as the dose halfway between the baseline and
saturating level for each reaction. The saturating level for each
reaction was based on the plateau from normalized curve fit data
in which we observed no further significant increase in fluoresꢀ
cence intensity between the clickable HaloTag conjugate and
bioorthogonal TAMRA reporter. An optimized dose was then
selected for timecourse studies to determine the reaction halfꢀlife
(t1/2), defined as the timepoint halfway between the baseline and
saturating level for the indicated dose. In addition, we calculated
the efficiency for each reaction (Emax) from timecourse data,
which is defined as the saturating level (plateau from curve fit
data) for each reaction when normalized to the TAMRA Haloꢀ
Tag ligand 12 control. The reaction rate data reflect both the
HaloTag labeling and the biorthogonal reaction. As the HaloTag
labeling efficiency was consistent from experiment to experiment
(Figure S2), differences in these values mostly reflect differences
between the bioorthogonal reactions.
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Figure 2. Analysis of SPAAC reactions of DBCO 1ꢀ and BCN 2ꢀHaloTag conjugates with TAMRAꢀazide 15 in the nucleus of live HeLa cells
expressing HaloꢀH2BꢀGFP. (A) Cells expressing HaloꢀH2BꢀGFP were treated with 10 µM chloroalkane ligand BCN 2 for 30 min to generate
the BCN 2ꢀHaloꢀH2BꢀGFP conjugate followed by reaction with 50 nMꢀ250 µM TAMRAꢀazide 15 for 2 h at 37 °C and analyzed by inꢀgel fluoꢀ
rescence (top panel) and western blot of total HaloTag protein (bottom panel). As a control, HaloꢀH2BꢀGFP was treated with TAMRA ligand
12 to determine the maximum fluorescent labeling per experiment (lane 1). (B) Doseꢀresponse curves from densitometry analysis of gels from
the SPAAC reaction of TAMRAꢀazide 15 with DBCO 1ꢀHalo (orange) and BCN 2ꢀHalo (green) conjugates. (C) Representative gel of SPAAC
timecourse experiments with BCN 2ꢀHalo and 25 µM TAMRAꢀazide 15 analyzed by inꢀgel fluorescence (top panel) and western blot of total
HaloTag protein (bottom panel). (D) Oneꢀphase association curves from timecourse densitometry analysis of SPAAC reactions between DBCO
1ꢀHalo (orange) and BCN 2ꢀHalo (green) reacting with 25 µM TAMRA–azide 15 for 1ꢀ240 min at 37 °C. Curves were generated in GraphPad
Prism from fluorescence intensity measurements of n = 3 – 6 independent replicates as described in the experimental section, and reported as
the mean ± SEM.
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Table 3. Live cell reaction rate data and efficiency for iEDDA
reactions.
concentrations (vide infra, Figure S6, S8 and S13), we used lower
concentrations of the fluorophoreꢀtetrazine for the iEDDA dose
response and timecourse studies. The strained alkyne BCN 2,
which has recently been reported to undergo iEDDA
reactions,39,76,77 and DBCO 1 were also investigated in these studꢀ
ies. HaloꢀH2BꢀGFP incorporating the TCO 4, BCN 2, DBCO
1, NBor 8 and Cprop 9 HaloTag ligands were initially reacted
with a dose response of TAMRAꢀTz 13 for 1 h, and the reacꢀ
EC50
(µM)a
2.1
HaloTag
Ligand
t1/2
Emax
TAMRA-
Tz (13)
(min)b (%)b
BCN (2)
TCO (4)
16.9
13.8
51
47
Tz (13)
1.4
tions were quenched with 100 µM (4ꢀ(1,2,4,5ꢀtetrazinꢀ3ꢀ
yl)phenyl)methanamine (Tzꢀamine 27, Figure S3B) and proꢀ
cessed as described. Figure 3A contains a representative gel for
the reaction between TCO 4ꢀHaloꢀH2BꢀGFP conjugate and
TAMRAꢀTz 13, and a representative gel for the reaction of the
BCN 2ꢀHaloꢀH2BꢀGFP conjugate with 13 is included in Figure
S5A. Under these conditions, TCO 4 had a slightly lower EC50
and the reaction proceeded to a greater extent compared to BCN
2 (Figure 3B and Table 3). In contrast, DBCO 1 did not react
with TAMRAꢀTz 13 (Figure S6). This is consistent with previꢀ
ous reports,77,78 and is likely due to increased sterics present in
the tricyclic dibenzocyclooctyne ring system. We next investiꢀ
gated the reaction timecourse of BCN 2 and TCO 4 with 2 µM
TAMRAꢀTz 13 to determine the t1/2 and Emax values. Repreꢀ
sentative gels for the timecourse reaction of TAMRAꢀTz 13 with
TCO 4 or BCN 2 are shown in Figure 3C and Figure S5B, reꢀ
spectively. Here, we observed much faster reaction rates than
the SPAAC ligations, consistent with the faster in vitro kinetics
reported for iEDDA ligations. Interestingly, BCN 2 was found
to undergo an iEDDA reaction with TAMRAꢀTz 13 almost as
fast as TCO 4 in the nucleus of live mammalian cells, and both
BCN (2)
TCO (4)
Tz-Me (14)
Tz-Me (14)
n.d.
n.d.
44.8
46.4
31
27
sTCO (5)
Tz (13)
Tz (13)
Tz (13)
0.1
0.036
0.05
2.2
1.4
5.2
67
86
77
Ag-sTCO
(6)
dTCO (7)
aFrom dose response of TAMRAꢀTz 13 for 1 h
bFrom time course with 2 ꢁM 13 or 14
n.d. = not determined
iEDDA Reactions in Live HeLa Cells for Strained Alkene
and Alkyne Tags with Tetrazine Reporters. We next evaluꢀ
ated the iEDDA reaction of tetrazines with four prominent
dienophiles covering a broad range of reactivity. We calculated
the EC50, t1/2, and Emax values as described above and in the
Materials and Methods section. Due to faster kinetics for iEDꢀ
DA reactions compared to SPAAC reactions, and the propensity
of monosubstituted tetrazines for nonspecific labeling at high
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Figure 3. Analysis of iEDDA reactions of BCN 2ꢀ and TCO 4ꢀHaloTag conjugates with TAMRAꢀTz 13 in the nucleus of live HeLa cells exꢀ
pressing HaloꢀH2BꢀGFP. (A) Cells were treated with 10 µM chloroalkane ligand TCO 4 for 30 minutes to generate the TCO 4ꢀHaloꢀH2Bꢀ
GFP conjugate followed by reaction with 1 nMꢀ20 µM TAMRAꢀTz 13 for 1 h at 37 °C and analyzed by inꢀgel fluorescence (top panel) and
western blot of total HaloTag protein (bottom panel). As a control, HaloꢀH2BꢀGFP was treated with TAMRAꢀligand 12 to determine the maxꢀ
imum fluorescence labeling per experiment (lane 1). (B) Doseꢀresponse curves from densitometry analysis of gels from the iEDDA reaction of
TAMRAꢀTz 13 with BCN 2ꢀHalo (black) and TCO 4ꢀHalo (blue) conjugates. (C) Representative gel of iEDDA timecourse reaction between 2
µM TAMRAꢀTz 13 and TCO 4ꢀHalo conjugate analyzed by inꢀgel fluorescence (top panel) and western blot of total HaloTag protein (bottom
panel). (D) Oneꢀphase association curves from timecourse densitometry analysis of iEDDA reactions between BCN 2ꢀHalo (black) or TCO 4ꢀ
Halo (blue) with 2 µM TAMRAꢀTz 13 for 0.5 – 120 min at 37 °C. Curves were generated in GraphPad Prism from fluorescence intensity
measurements of n = 3 – 6 independent replicates as described in the experimental section, and reported as the mean ± SEM.
reactions proceeded with similar efficiencies (Figure 3D and
Table 3).
that 8 and 9 are either not stable in mammalian cells or not reacꢀ
tive under the liveꢀcell labeling conditions implemented here.
Various tetrazines have been synthesized for liveꢀcell labeling,
and 3ꢀmethylꢀ6ꢀaryltetrazine derivatives have been used for liveꢀ
cell imaging applications due to their improved stability.70,71
Therefore, we were curious to examine how this tetrazine variant
behaved in our model system. To this end, we determined the
halfꢀlives and efficiency for the BCN 2ꢀHalo and TCO 4ꢀHalo
conjugates with TAMRAꢀTzꢀMe 14 (Figure 1B). Consistent with
published in vitro kinetic data,82,83 14 reacts about 3 times more
slowly than TAMRAꢀTz 13 under identical parameters (Figure S9
and Table 3). In addition, reaction with TAMRAꢀTzꢀMe 14 was
also less efficient, reacting with only ~25ꢀ30% efficiency when
compared to our positive TAMRA control within the 4 h
timecourse.
As DBCO was found to rapidly undergo the SPAAC reaction
with TAMRAꢀazide 15, we were curious to determine how the
rate of this reaction compared with iEDDA reactions of 2 and 4
with 13. To provide a direct comparison between reaction rates,
we determined the halfꢀlife and efficiency of this reaction under
identical conditions as the iEDDA reactions. Here, we observed
a halfꢀlife for this SPAAC reaction of ~15 min, which was alꢀ
most as fast in live cells as the iEDDA reactions of 4 and 2 with
13 (Figure S7). This surprising rate enhancement for SPAAC
reactions of DBCO underscores the importance of measuring
relative reaction rates within a cellular context, and not relying
solely on in vitro rate data.
Under the same conditions used in Figure 3A, we did not obꢀ
serve significant reaction with either the NBor 8 or Cprop 9
HaloTag conjugates (data not shown), even though the HaloTag
ligands were effectively conjugated to the HaloTag protein (Figꢀ
ure S2). To more accurately assess the reactivity of NBor 8 and
Cprop 9 HaloTag ligands in our cellular system, which display
sluggish in vitro reaction rates compared to other dienoꢀ
philes,66,68,79ꢀ81 we evaluated higher concentrations of TAMRAꢀ
Tz 13 over a longer 4 h reaction period. Even under these modiꢀ
fied conditions, we were only able to detect ~10ꢀ20% reaction
(Figure S8). Furthermore, we observed significant background
protein labeling by 13 at these high concentrations, and backꢀ
Overall, these studies clearly demonstrate significant differꢀ
ences in labeling rates and stability of reactants when placed in
the context of a living cell, and should help provide guidance
when choosing the reaction of choice and labeling conditions for
a particular application.
SPAAC and iEDDA Ligations of Azide and Tetrazine
Tags with Strained Alkyne and Alkene Reporters. Our liveꢀ
cell model system provided interesting observations regarding
SPAAC and iEDDA reactions when either the strained alkyne or
alkene was introduced into the cell as a tag prior to reaction with
the appropriate azide or tetrazine reporter. We also investigated
the reverse situation where an azide or tetrazine was introduced
ground labeling dominated at 100 µM. These studies suggest
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Figure 4. Stability of conformationally strained transꢀcyclooctene derivatives. (A) H NMR spectra (600 MHz, CD3OD) showing degradation
1
and some transꢀcis isomerization of fresh sTCO 5 (bottom) and following a 3ꢀday incubation neat at 30 °C (top). (B) H NMR spectra (600
MHz, CD3OD) showing no degradation or transꢀcis isomerization of fresh AgꢀsTCO 6 (bottom) and following a 3 day incubation neat at 30 °C
(top). (C) Stability of TCOꢀderivatized HaloTag ligands 4-7 following formation of HaloꢀH2BꢀGFP conjugates inside the nucleus over 24 h.
HeLa cells expressing HaloꢀH2BꢀGFP were labeled with 10 µM chloroalkane ligands 4ꢀ7 for 30 min at 37 °C. Unbound ligand was washed out
of the cells for 1 h, and the t=0 timepoint was labeled with a saturating dose of 2 µM TAMRAꢀTz 13 for 1 h and analyzed by inꢀgel fluoresꢀ
cence. All other timepoints were labeled with TAMRAꢀTz 13 after the indicated incubation times in media. For each timepoint, we labeled
HaloꢀH2BꢀGFP or HaloꢀKDEL with TAMRA ligand 12 as a positive control to account for HaloTag conjugate protein turnover during the
course of the experiment. Graphs represent fluorescence intensity measurements from 3 replicates, and are reported as the mean ± SEM. (D)
Stability of TCO 4-7 HaloꢀKDEL conjugates inside the lumen of the ER over 24 h. HeLa cells expressing HaloꢀKDEL were labeled, collected,
and analyzed as in (C).
into the cell as a tag followed by reaction with the appropriate
strained alkyne or alkene reporter. For the SPAAC reactions,
HaloꢀH2BꢀGFP incorporating azide HaloTag ligand 3 was reactꢀ
ed with a timecourse of 25 µM TAMRAꢀDBCO 16 or TAMRAꢀ
BCN 17 (Figure S10). Here, we observed an interesting disconꢀ
nect where the azide tag /cycloalkyne reporter combination did
not perform as well and generally had decreased reaction effiꢀ
ciency relative to the analogous cycoalkyne tag/azide reporter
systems. To better understand this disconnect, we determined
the passive permeability of the clickable reporters 13ꢀ18 in lowꢀ
efflux MDCKII cells (Table S1).84 We found that TAMRAꢀazide
15 had moderate permeability, while TAMRAꢀBCN 16 and
TAMRAꢀDBCO 17 had very low permeability. We also investiꢀ
gated the stability of 13ꢀ18 in HeLa cells and found that
TAMRAꢀazide 15 was completely stable over a 24 h period,
while TAMRAꢀBCN 17 exhibited considerable instability with
only 6% remaining after 24 h (Table S2). Therefore the reduced
efficiency of the azide tag /cycloalkyne reporter combination can
be explained, in part, by the poorer permeability and stability of
the cycloalkyne reporters which leads to lower effective intracelꢀ
lular concentrations of these reagents. In addition, we observed
a significant amount of background protein labeling using 25 µM
16 and 17 (Figure S12E and F) that was not observed with
TAMRAꢀazide 15 (Figure S12A). This is most likely due to nonꢀ
specific labeling of cysteine thiol biomolecules which has been
shown to be a limitation of cyclooctynes including BCN in cell
lysates and inside live cells.85 These observations have implicaꢀ
tions for probe design and argue for preferential placement of
cycloalkyne bioorthogonal tags directly on the probe or biologiꢀ
cal component to be labeled rather than the reporter group.
For iEDDA reactions, the efficiency of reaction between the
HaloTag conjugate of Tz 10 and 2 µM TAMRAꢀTCO 18 deꢀ
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Figure 5. Analysis of iEDDA reactions of sTCO 5ꢀ, AgꢀsTCO 6ꢀ, and dTCO 7ꢀHaloTag conjugates with TAMRAꢀTz 13 in the nucleus of live
HeLa cells expressing HaloꢀH2BꢀGFP. (A) Doseꢀresponse curves for the iEDDA reaction of TAMRAꢀTz 13 with the HaloTag conjugates of
sTCO 5 (red), AgꢀsTCO 6 (blue), and dTCO 7 (black) chloroalkane ligands. (B) One phase association plot of the iEDDA reaction timecourse
between 2 µM TAMRAꢀTz 13 and the HaloTag conjugates of chloroalkane ligands 5ꢀ7. Curve fits represent full 2 h timecourse, and are shown
here up to 60 min to highlight differences in the early part of the curves. See Figure S13 for representative gels of each reaction. Data is reꢀ
ported as the mean ± SEM for 3 replicates.
creased by ~50% (Figure S11) compared to the corresponding
reaction where TCO 4 was incorporated as the tag and TAMRAꢀ
Tz 13 was used as the reporter (Figure 3). Monoꢀsubstituted
tetrazines82 have been reported to have limited stability in serum
upon prolonged exposure and this could account for part of the
reduced reaction efficiency. In addition, TAMRAꢀTz 13 displays
nonꢀspecific labeling at high concentrations (Figure S8 and
S12D) so it is likely that a component of the instability is due to
covalent modification of biomolecules within the cell. We also
found that TAMRAꢀTCO 18 was considerably more stable than
TAMRAꢀBCN 17 in HeLa cells (Table S2). Consistent with the
improved stability, very little background labeling was observed
for TAMRAꢀTCO 18 under the conditions of our experiments
(Figure S12B). Even though TCO has been shown to undergo
partial isomerization to the cisꢀisomer within the cell,86 this isoꢀ
mer is inert and does not cause nonspecific labeling of backꢀ
ground proteins. This isomerization may slightly lower the Emax
for the iEDDA reaction between 10 and 18, but it is not a limitaꢀ
tion because the selectivity is not affected.
Interestingly, the TzꢀMe 11 HaloTag conjugate retained high
efficiency when incubated with TAMRAꢀTCO 18 (Figure S11).
In addition, TAMRAꢀTzꢀMe 14 was more stable in HeLa cells
(Table S2) and displayed much less background labeling (Fig S9
and S12C) compared to TAMRAꢀTz 13 (Figure S8 and S12D).
These observations are consistent with prior reports that 3,6ꢀ
disubstituted tetrazines are more stable than monoꢀsubstituted
tetrazines,64,82,83,87 and suggest that disubstituted tetrazines may
be useful as protein tagging agents for live cell bioorthogonal
labeling in mammalian cells particularly when paired with more
reactive TCO reporters, as is the case in bacteria.32,38
far proved unsuccessful.14,39 Recent reports have shown that
these conformationally strained derivatives can undergo isomeriꢀ
zation to the unreactive cis isomer in the presence of high thiol
concentrations, presumbably via a free radical mediated pathꢀ
way.32,65 Using our model system, we investigated both the stabilꢀ
ity and reactivity of these two highly reactive TCO derivatives to
assess their utility as tags for bioorthogonal labeling in mammaliꢀ
an cells.
We first examined the stability of sTCO when heated neat at
o
30 C for 3 days and observed almost complete decomposition
with a trace of isomerization to the cis isomer (Figure 4A). We
sought to find a way to stabilize the sTCO reagent to permit
prolonged storage. The preparation of sTCO involves phoꢀ
toisomerization of the cisꢀcyclooctene in a flow reactor where the
transꢀisomer is isolated through selective complexation with Agꢀ
NO3ꢀimpregnated silica gel.65,88 The sTCO is then liberated by
stirring the silica with NH4OH. We hypothesized that preparing
the sTCO reagent as the silver complex would provide long term
stability for storage, yet could be applied directly to cell culture
which contains high concentrations of NaCl that could release
the reactive sTCO in situ. First we prepared the AgꢀsTCO Halꢀ
oTag ligand 6 (Figure 1A) and demonstrated that it was stable
when heated at 30 °C for 3 days (Figure 4B), unlike the parent
sTCO ligand 5 (Figure 4A). In addition, we synthesized a Haloꢀ
Tag reagent derived from the newly reported dTCO32 (Figure
1A, ligand 7), which has increased stability compared to sTCO
while displaying similar reactivity.
We next examined the relative stability of the TCO series of
HaloTag ligands (compounds 4ꢀ7) following conjugation to Haꢀ
loꢀ H2BꢀGFP in the nucleus or HaloꢀKDEL in the endoplasmic
reticulum (ER) (Figure 4CꢀD). HeLa cells were transfected with
either HaloꢀH2BꢀGFP or HaloꢀKDEL and HaloTag ligands 4ꢀ7
were added to make the corresponding clickable HaloTag conjuꢀ
gates and excess unbound ligand was washed out of the cells.
TAMRAꢀTz 13 was added at the indicated time points over 24 h
to evaluate how much of the TCOꢀHalo conjugate was stable
and could undergo the iEDDA reaction. Results were quantified
by inꢀgel fluorescence and Western blotting. The reaction of
Investigation of Conformationally Strained TCO-
Derivatives. Recently, two conformationally strained transꢀ
cyclooctene derivatives have been described that display faster
reactivity relative to simpler TCO derivatives. These groups
include the strained transꢀcyclooctene sTCO65, which displays the
fastest reactivity, and the cisꢀdioxolaneꢀfused transꢀcyclooctene
dTCO, which displays better stability and solubility.32 To date,
their use in live cells has been limited to applications as reporter
molecules,32,38 and efforts to genetically encode or to use ligases
to selectively introduce sTCO or dTCO into proteins have thus
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Figure 6. Confocal images of the SPAAC and iEDDA reactions of clickable BCN 2ꢀ, DBCO 1ꢀ, and TCO 4ꢀHaloTag conjugates with clickaꢀ
ble TAMRA fluorophores in live cells expressing HaloꢀH2BꢀGFP. HaloꢀH2BꢀGFP was labeled with 10 µM BCN 2 (A), DBCO 1 (B), or TCO 4
(C) chloroalkane ligands for 30 minutes, followed by reaction with a dose response of TAMRAꢀazide 15 (SPAAC, AꢀB) or TAMRAꢀTz 13
(iEDDA, C) for 30 min at 37 °C and imaged live after a 2 h washout period. Total nuclei were visualized with Hoescht 33342 and are shown in
blue (column 1 on the left). Cells expressing HaloꢀH2BꢀGFP are shown in green (column 2). SPAACꢀlabeled nuclei are shown in red (column
3). Overlays were created between green and red channels to demonstrate colocalization (yellow) between transfection and SPAAC chemistry
(column 4). All images were collected under identical parameters on the same day. Scale bar = 20 µm.
TCO 4ꢀHaloꢀH2BꢀGFP with TAMRAꢀTz 13 progressed to the
same extent over 24 h indicating that TCO 4 was stable in the
nucleus under these conditions. In contrast, less TAMRAꢀTz 13
reacted over the 24 h period with the conformationally strained
TCO derivatives presumably due to isomerization to the unreacꢀ
tive cis isomer with prolonged incubation. AgꢀsTCO 6 and
sTCO 5 had the lowest stability, leading to significant loss of
reactivity after 24 h, while dTCO 7 had intermediate stability.
However, in the ER, all the transꢀcyclooctenes appeared stable
for up to 24 h (Figure 4D), suggesting that differences in subcelꢀ
lular microenvironments can affect reagent stability. The ER
provides a highly oxidizing environment relative to the rest of
the cell89 and further study will be needed to determine if that
contributes to the enhanced stability of transꢀcyclooctenes in the
ER relative to the nucleus. Nonetheless, our data suggests that
conformationally strained TCOs are useful as tagging molecules
in the ER, where some enzymeꢀmediated protein labeling reacꢀ
tions are not compatible.90 Importantly, sTCO 5 and AgꢀsTCO 6
behaved almost identically demonstrating that the Ag is effecꢀ
tively decomplexed when 6 is added to cells.
Reaction rates were then examined between TAMRAꢀTz 13
and the more reactive dienophiles 5ꢀ7 in the nucleus to deterꢀ
mine how these values compare to TCO 4 under the same reacꢀ
tion conditions. Consistent with the increased reactivity obꢀ
served from in vitro kinetics data,32,65 we observed over 14ꢀfold
lower EC50 values for the sTCO 5, AgꢀsTCO 6 and dTCO 7
HaloꢀH2BꢀGFP conjugates when compared to TCO 4, with the
lowest EC50 ~ 36 nM for AgꢀsTCO 6 (Figure 5A, Table 3). Repꢀ
resentative gels for each reaction are shown in Figure S13A.
Furthermore, a comparison of reaction halfꢀlife values demonꢀ
strates that AgꢀsTCO 6 (halfꢀlife ~ 1.4 min) and sTCO 5 (halfꢀ
life ~ 2.2 min) react the fastest, followed by dTCO 6, and finally
TCO 4 (Figure 5B and Table 3). Representative gels for
timecourse reactions are shown in Figure S13B. In addition, the
reactions with conformationally strained TCO derivatives 5ꢀ7
have the highest efficiencies with Emax values in the 67ꢀ86%
range compared to 47% for TCO 4. Overall, these data demonꢀ
strate that the sTCO reagents (5 and 6) have the fastest kinetics
and highest efficiency for live cell labeling, enabling reaction
saturation within minutes following addition of the tetrazine
reporter. Furthermore, consistent with the above stability data,
sTCO was clearly liberated from the silver complex 6 when addꢀ
ed to the cell culture since AgꢀsTCO 6 performed identically or
slightly better than the parent sTCO 5. In addition, AgꢀsTCO 6
stock solutions remained stable up to 8 months when stored at ꢀ
20
pound, required frequent preparation and/or resynthesis of the
ligand when stored at ꢀ20 C. We also tested sTCO 5 and Agꢀ
°C, whereas sTCO 5 stock solutions, and even neat comꢀ
°
sTCO 6 in a standard MTT cell viability assay in HeLa cells and
found no cell toxicity over a 24 h period (Figure S14). Given
that AgꢀsTCO has greatly improved stability for storage, we feel
this reagent will expand the utility of sTCO iEDDA chemistry in
cells.
Characterization of SPAAC and iEDDA Reactions in Dif-
ferent Organelles. We next investigated the performance of
the SPAAC and iEDDA reactions in the endoplasmic reticulum
and cytosol, where unique subcellular microenvironments can
affect the stability (Figure 4CꢀD) and, potentially, reactivity of
the bioorthogonal groups investigated here. We chose the fastꢀ
est dienophiles, AgꢀsTCO 6 and dTCO 7, to evaluate the iEDꢀ
DA reaction. For SPAAC chemistry, we examined both the
DBCO 1 and BCN 2 chloroalkane ligands. We restricted the
bioorthogonal reactions to the lumen of the ER using HaloTagꢀ
KDEL. For cytosolic localization, we designed a HaloTagꢀ
GAP43ꢀGFP construct which is present throughout the cytosol,
and excluded from the nucleus (Figure S1C). HeLa cells were
transfected with either construct and treated under identical
conditions used for evaluation of the nuclear construct, Haloꢀ
H2BꢀGFP. Overall, both the SPAAC and iEDDA reactions
proceeded with similar reaction rates in both the cytosol and ER
(Figure S15 and Table 4). The reaction of AgꢀsTCO 6 and TCO
7 with TAMRAꢀTz 13 mirrored what was seen in the nucleus
(Figure S15CꢀD and Table 4).
As expected, AgꢀsTCO 6 was slightly faster than dTCO 7, and
both reactions proceeded with similar efficiencies (Table 4).
Furthermore, the SPAAC reaction between TAMRAꢀazide 15
and DBCO 1 was approximately 5ꢀfold faster and proceeded
withgreater efficiency than the reaction with BCN 2 (Figure
S15AꢀB and Table 4). These data suggest that both SPAAC and
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iEDDA ligations are compatible with different subcellular comꢀ
partments.
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Figure 7. Confocal images of the SPAAC and iEDDA reactions between clickable HaloTag conjugates and clickable fluorescein derivatives in
different subcellular organelles. (A) Confocal images of nuclei expressing HaloꢀH2BꢀmCherry labeled with BCN 2 followed by SPAAC reacꢀ
tion with 50 µM FlꢀdiPivꢀazide 19 for 1 h under live (top panels) and MeOH fixed (bottom panels) conditions. Total nuclei were visualized
with Hoescht 33342 and are shown in blue (column 1 on left). Cells expressing mCherry are shown in red (column 2). Click chemistry is
shown in green (column 3). Overlays were created between red, green, and blue channels to demonstrate colocalization (yellow) between transꢀ
fection and bioorthogonal chemistry (column 4). (B) Confocal images of the ER expressing HaloꢀKDELꢀmCherry labeled with BCN 2 folꢀ
lowed by SPAAC with 50 µM FlꢀdiPivꢀazide 19 for 1 h under live (top panels) and MeOH fixed (bottom panels) conditions. (C) Confocal imꢀ
ages of nuclei expressing HaloꢀH2BꢀmCherry labeled with TCO 4 followed by iEDDA reaction with 1 µM FlꢀdiAcꢀTz 20 for 1 h. (D) Confocal
images of the ER expressing HaloꢀKDELꢀmCherry labeled with TCO 4 followed by iEDDA reaction with 1 µM FlꢀdiAcꢀTz 20 for 1 h. (E)
Confocal images of the cytoplasm/nucleus expressing HaloꢀmCherry labeled with TCO 4 followed by iEDDA reaction with 1 µM FlꢀdiAcꢀTz
20 for 1 h. (F) Confocal images of cell surface expressing HaloꢀmCherryꢀPDGFR labeled with TCO 4 followed by iEDDA reaction with cellꢀ
impermeable FlꢀTz 21. All images were acquired after a 2 h washout period. Scalebar = 20 µm.
Evaluation of SPAAC and iEDDA Reactions with
Bioorthogonal TAMRA Fluorophores for Intracellular Im-
aging in the Nucleus. With the systematic evaluation of the
bioorthogonal reactions in hand, we were in a position to evalu
ate the SPAAC and iEDDA reactions using our clickable reaꢀ
gents for the purpose of intracellular imaging in live cells. For
the SPAAC reaction, HeLa cells expressing HaloꢀH2BꢀGFP
were treated with either DBCO ligand 1 or BCN ligand 2, folꢀ
lowed by reaction with a dose response of TAMRAꢀazide 15.
Cells were imaged live after a 2 h washout period. Representaꢀ
tive images for the reaction of BCN 2ꢀHaloꢀH2BꢀGFP (Figure
6A) and DBCO 1ꢀHaloꢀH2BꢀGFP (Figure 6B) conjugates with
TAMRAꢀazide 15 are shown. Overall, we observed differences
in the intensity of nuclear labeling consistent with our reaction
rate data in live cells (Table 2). The rate enhancement observed
with the HaloTag conjugate of DBCO 1 enabled significant reacꢀ
tion to be visualized with as little as 0.5 µM TAMRAꢀazide 15
and the reaction becomes saturated with optimal signalꢀtoꢀnoise
levels with only 2 µM TAMRAꢀazide 15 (Figure 6B). In conꢀ
trast, 10 µM TAMRAꢀazide 15 is required to visualize the reacꢀ
tion with BCN 2, and 25 µM 15 is necessary to achieve adequate
signalꢀtoꢀnoise levels (Figure 6A). Background fluorescence
becomes evident with 25 µM TAMRAꢀazide 15, and dominates
at 100 µM 15 (Figure 6A, bottom panel). These data suggest that
25 µM is an upper limit to the amount of TAMRAꢀazide 15 that
can be successfully used for these live cell imaging studies.
For iEDDA reactions, HeLa cells expressing HaloꢀH2BꢀGFP
Table 4. Live cell reaction rate data in different organelles.
HaloTag
Ligand
t1/2
Emax
TAMRA- Organelle (min) (%)
Cytosol
ER
20.1a
25.2a
5.3a
70
66
86
78
BCN (2)
Azide 15
Azide 15
Cytosol
ER
DBCO (1)
6.6a
Cytosol
ER
2.1b
1.2b
2.7b
82
68
75
Ag-sTCO (6)
dTCO (7)
Tz 13
Tz 13
Cytosol
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ER
4.7b
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1
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aFrom time course with 25 µM TAMRAꢀazide
bFrom time course with 2 µM TAMRAꢀTz
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9
were treated with chloroalkane TCO ligand 4 followed by reacꢀ
tion with a dose response of TAMRAꢀTz 13 (Figure 6C) for 30
min, and imaged live after a 2 h washout. For iEDDA reaction
with 13, we observed a direct correlation between our inꢀgel
fluorescence analysis and imaging, with significant signal obꢀ
served using 0.5 µM TAMRAꢀTz 13 and optimal signalꢀtoꢀnoise
levels using 2 µM TAMRAꢀTz 13. Background labeling domiꢀ
lower membrane permeability. Fixing the cells in MeOH comꢀ
pletely removed background, and revealed selective labeling in
both the nucleus (Figure 7A, bottom panel) and ER (Figure 7B,
bottom panel). BODIPYꢀFLꢀazide 23 displayed a different patꢀ
tern of nonꢀspecific accumulation, with high concentrations of
unreacted fluorophore that appeared to accumulate in organelles
(Figure S17AꢀB, top panels). This background was not completeꢀ
ly removed after fixation (Figure S17AꢀB, bottom panels), sugꢀ
gesting that BODIPYꢀFLꢀazide 23 may not be as useful for inꢀ
tracellular imaging.
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nated when 10 µM TAMRAꢀTz 13 was used (Figure 6C, bottom
panel), suggesting lower concentrations (≤2 µM) should be used
for live cell imaging studies in mammalian cells. Here, we also
examined the reverse bioorthogonal group pairing where Tz
ligand 10 or TzꢀMe ligand 11 was incorporated into HaloꢀH2Bꢀ
GFP as the tag, and TAMRAꢀTCO 18 served as the fluorescent
reporter (Figure S16). We observed labeling with a low dose of
TAMRAꢀTCO (0.1 µM), but the labeling did not increase in a
dose proportionate fashion when up to 25 µM TAMRAꢀTCO 18
was used (Figure S16A). This data suggests that the monosubstiꢀ
tuted Tz 10 is not stable in the cells and a proportion decomposꢀ
es before reacting with TAMRAꢀTCO 18. In contrast, the less
reactive MeꢀTz 11 HaloTag conjugate behaved differently. No
reaction with MeꢀTz 11 was observed when 0.1 µM TAMRAꢀ
TCO 18 was used, however significant labeling was seen with 2
µM 18 and the reaction appeared to reach saturation when 10
µM 18 was used with good colocalization (Figure S16B). This
data is consistent with the results from the inꢀgel fluorescence
analysis where the reaction of the TzꢀMe 11 HaloTag conjugate
retained high efficiency when incubated with TAMRAꢀTCO 18,
while the reaction of the Tz 10 conjugate was less efficient (Figꢀ
ure S11). It is also worthwhile to note that very little background
fluorescent labeling or nonꢀspecific fluorophore accumulation
was observed with TAMRAꢀTCO 18, even up to 25 µM (Figure
S16). This is also consistent with the low background observed
based on inꢀgel fluorescence data (Figure S12B). These observaꢀ
tions highlight how TCOꢀbased reporters not only provide exꢀ
cellent reactivity, but also label tetrazineꢀtagged proteins with
high selectivity.
Evaluation of SPAAC and iEDDA Reactions with
Bioorthogonal Fluorescein and BODIPY-FL Fluorophores
for Intracellular Imaging in Different Organelles. We next
explored the SPAAC and iEDDA ligations in different orgaꢀ
nelles using two green fluorophores amenable to intracellular
labeling: BODIPYꢀFL and Fluorescein (Fl). HeLa cells were
transfected with the intracellular organelleꢀtargeting constructs
for HaloTag expression in the nucleus (HaloꢀH2BꢀmCherry),
and ER (HaloꢀKDELꢀmCherry). For the SPAAC ligations, Halꢀ
oTag protein was labeled with BCN ligand 2 followed by reacꢀ
tion with the green cell permeable fluorophores, fluoresceinꢀ
dipivalateꢀazide (FlꢀdiPivꢀazide) 19 (Figure 7) and BODIPYꢀFLꢀ
azide 23 (Figure S17). Here, we observed significant background
fluorescence for both 19 and 23, even after a 2 h washout. Flꢀ
diPivꢀazide 19 signal appeared to be present diffusely throughout
the cytosol and nucleus of live cells (Figure 7AꢀB, top panels).
This background accumulation is likely due to the high concenꢀ
tration of fluorophore needed to effectively promote the SPAAC
reactions, resulting in trapping of negatively charged fluorescein
once the pivaloyl groups have been removed by endogenous
esterases inside the cell. This limits passive diffusion of unreactꢀ
ed fluorophore out of the cell during the washout stage due to
We also evaluated iEDDA reactions in the nucleus (Haloꢀ
H2BꢀmCherry), ER (HaloꢀKDELꢀmCherry), cytosol (Haloꢀ
mCherry), and at the extracellular face of the plasma membrane
(HaloꢀmCherryꢀPDGFR). HeLa cells expressing the intracellular
constructs were labeled with TCO HaloTag ligand 4, followed by
reaction with 1 µM fluoresceinꢀdiacetateꢀTz (FlꢀdiAcꢀTz) 20
(Figure 7CꢀE) or BODIPYꢀFLꢀTz 22 (Figure S17CꢀF) for 30
min. Tetrazines have been reported to quench the fluorescence
of some fluorophores, resulting in turnꢀon fluorescence upon
reaction with transꢀcyclooctenes.91 Therefore, we initially atꢀ
tempted to image subcellular compartments without a washout
step prior to imaging in live cells using these “turnꢀon” tetrazineꢀ
linked fluoresceinꢀ and BODIPYꢀFL fluorophores. However,
the background fluorescence we observed for intracellular reacꢀ
tion was too high to distinguish specific labeling (data not
shown). This is in part due to stability issues with the tetrazine
which can lead to fluorescent impurities (i.e., see Table S2 for
stability of TAMRAꢀTz 13 in HeLa cells).70,92 Fluorophoreꢀ
tetrazines with >1000ꢀfold fluorescence increases have recently
been developed, and these may provide better tools for intracelꢀ
lular iEDDA imaging in live cells with no washout period.70,71
Following a 2 h washout period, we were able to observe selecꢀ
tive labeling with no visible background for both FlꢀdiAcꢀTz 20
and BODIPYꢀFLꢀTz 22 in the nucleus (Figure 7C and S17C),
ER (Figure 7D and S17D), and cytosol (Figure 7E and S17E).
In addition, we demonstrated selective extracellular labeling with
cellꢀimpermeable fluoresceinꢀTz (FlꢀTz) 21 (Figure 7F), whereas
cellꢀpermeable BODIPYꢀFLꢀTz 22 labeled both intracellular
(present in the secretory pathway) and extracellular Haloꢀ
mCherryꢀPDGFR (Figure S17F), demonstrating that iEDDA
reactions are successful for both intracellular and extracellular
labeling. Of note, FlꢀTz 21 is amenable to selective extracellular
labeling, even when the bioorthogonal reaction partner is present
intracellularly.
Based on these studies, the cellꢀpermeable iEDDA fluoroꢀ
phoreꢀtetrazines at the concentrations tested here exhibit excelꢀ
lent properties for live cell intracellular imaging experiments in
all the organelles tested, whereas the high concentrations of
fluorophoreꢀazides required for efficient SPAAC labeling are not
ideal for live cell imaging and cells need to be fixed to remove
background fluorescence.
Rapid Intracellular Imaging with Conformationally
Strained trans-Cyclooctenes using the iEDDA Reaction.
Finally, we evaluated the fastest iEDDA reactions observed in
this work, for rapid labeling in the nucleus at low fluorophore
concentrations. Here, we analyzed a dose response of TAMRAꢀ
Tz 13 after 5 min of incubation with the AgꢀsTCO 6ꢀHaloꢀH2Bꢀ
GFP and dTCO 7ꢀHaloꢀH2BꢀGFP conjugates compared to the
TCO 4ꢀHaloꢀH2BꢀGFP conjugate. Reactions were quenched
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Journal of the American Chemical Society
for 5 min with 100 µM Tzꢀamine (Figure S3), and imaged after a
Page 12 of 27
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1 h washout period. Here, we observed some signal for both
AgꢀsTCO 6ꢀHalo (Figure 8A) and dTCO 7ꢀHalo (Figure 8B)
conjugates at 100 nM after 5 min (top row), with significant laꢀ
beling and excellent signalꢀtoꢀnoise levels at 1 µM (third row).
Furthermore, labeling intensity increased with increasing fluoroꢀ
phore concentration, and appeared to saturate with 1ꢀ5 µM
TAMRAꢀTz 13 within the 5 min incubtion (Figure 8A and 8B).
In contrast, we needed ~10ꢀfold more fluorophore to see any
signal with TCO 4ꢀHalo (Figure 8C), further highlighting the
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Figure 8. Rapid intracellular imaging of iEDDA reactions in live HeLa cells. HaloꢀH2BꢀGFP expressing HeLa cells were labeled with Haloꢀ
Tag chloroalkane ligands TCO 4, dTCO 7, or AgꢀsTCO 6 followed by reaction with a dose response of TAMRAꢀTz 13 for 5 min at 37 °C.
Reactions were quenched with Tzꢀamine 27 followed by a 1 h washout prior to imaging. Cells were imaged live under identical parameters and
representative images are shown for AgꢀsTCO 6ꢀHalo (A), dTCO 7ꢀHalo (B), and TCO 4ꢀHalo (C). Total nuclei were visualized with Hoescht
33342 and are shown in blue (column 1 on left). Cells expressing HaloꢀH2BꢀGFP are shown in green (column 2). Click chemistry is shown in
red (column 3). Overlays were created between red and green channels to demonstrate colocalization (yellow) between transfection and
bioorthogonal chemistry (column 4). Scale bar = 20 ꢁm.
superiority of the AgꢀsTCO and dTCO reagents over TCO for
rapid intracellular live cell imaging.
known. Our studies clearly demonstrate that iEDDA ligations
are the fastest reactions in multiple cellular compartments of live
cells, enabling reaction completion within minutes when selectꢀ
ing the fastest dienophiles, sTCO and dTCO. Previous attempts
to tag proteins with sTCO or dTCO using genetically encoded
amino acids or lipoic acid ligase have been unsuccessful.14,31
Here, we demonstrate that protein conjugates can be made in
living mammalian cells using Halotag ligands of both sTCO (5,
6) and dTCO (7). The intracellular stability of sTCO and dTCO
was organelle dependent, with significant loss of reactivity in the
cell nucleus after 24 hours, but high stability in the ER over 24
hours. Thus, while conformationally strained transꢀcyclooctenes
display remarkable kinetics, their utility as tagging molecules is
somewhat tempered by their organelleꢀdependent stability. The
impact is somewhat alleviated because the major side reaction is
isomerization to the unreactive cisꢀisomer which appears to be
“silent” in the cells. In contrast, cyclooctynes can undergo covaꢀ
lent modification with cysteine and other thiolꢀcontaining bioꢀ
molecules which can lead to issues associated with nonspecific
background labeling. Another advantage of iEDDA reactions
with transꢀcyclooctenes is that efficient labeling can be achieved
using low concentrations of fluorophoreꢀtetrazines, thereby reꢀ
ducing the chance for nonꢀspecific fluorophore accumulation
and labeling in live cells, an issue which is present with the high
fluorophore concentrations required for SPAAC reactions or
iEDDA reactions with less reactive dienophiles.
Conclusions. Overall, our HaloTag model system provides a
unique and unbiased method to systematically evaluate
bioorthogonal labeling strategies directly inside living mammaliꢀ
an cells, as well as in different subcellular organelles. Using this
approach, we were able to rapidly assess various biorthogonal
groups for fast, efficient, and selective SPAAC and iEDDA ligaꢀ
tions for live cell imaging applications. While the SPAAC reacꢀ
tions were slower overall in live cells, we were still able to identiꢀ
fy conditions for intracellular live cell imaging studies using
TAMRA fluorophores for both BCN and DBCO. However, in
many cases the high fluorophore concentrations required for live
cell labeling studies limits the fluorophore selection due to backꢀ
ground fluorescence accumulation. Unexpectedly, we discovered
that the SPAAC reaction of DBCO 1 when conjugated to Haloꢀ
Tag proteins was nearly as rapid as the iEDDA reaction with
slower dienophiles, TCO and BCN, in different subcellular
structures of live cells. This rate acceleration allowed the use of
lower concentrations of the fluorophore reporter and suggests
that this group may also have utility for intracellular imaging
applications where DBCO is used as a tag. When DBCO was
linked to the reporter, we observed extensive background labelꢀ
ing at the high concentrations required for the SPAAC reaction,
limiting its use in these instances.
For the first time, proteins have been tagged in living mammaꢀ
lian cells with sTCO and dTCO, which when reacted with teꢀ
trazines constitutes the fastest bioorthogonal coupling reaction
The modest shelfꢀlife of sTCO has also limited its usefulness.
We observed degradation when sTCO was stored neat or as a
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Journal of the American Chemical Society
DMSO stock solution. In addition, freezeꢀthawing of stock #V660ꢀ20) at the restriction sites BglII/PstI. A (SerꢀGlyꢀGlyꢀ
1
2
3
4
5
6
7
8
solutions led to decomposition/isomerization, and we found it
was best to prepare singleꢀuse frozen aliquots to prevent freezeꢀ
thaw decomposition. We addressed the shelfꢀlife issue by makꢀ
ing a stable AgꢀsTCO complex and demonstrated that it can be
used directly in cells for reaction with tetrazineꢀlinked fluoroꢀ
phores. This silver complex has been stored as a stock solution
Gly)9 bridge was synthesized between Halo and mCherry.
HeLa Cell Culture and Transfection. HeLa cells were
maintained in growth media containing Dulbecco’s modified
eagle medium (DMEM, Life Technologies) supplemented with
10% (v:v) heat inactivated FBS (Life Technologies), 100
units/mL penicillin and streptomycin (Life Technologies), and
10 mM Hepes (Life Technologies) in a humidified incubator set
in ethanol for 8 months at ꢀ20
°C without any observable loss of
reactivity.
To date, most cellꢀlabeling studies involving iEDDA have
focused on use of tetrazines as reporter molecules with strained
alkenes and alkynes as the tags. In a ‘reversed’ approach, our
data shows that methylꢀtetrazine tag 14 could be used as the
protein tag in labeling studies with TAMRAꢀTCO 18 as the reꢀ
porter. Moving forward, we expect there will be considerable
advantages to utilizing stable tetrazine tags with the more reacꢀ
tive conformationally strained sTCO or dTCO based reporters.
We are presently preparing AgꢀsTCO and AgꢀdTCO fluorescent
reporters for this purpose and results will be disclosed in due
course. Further expansion of the suite of stable tetrazines with
reduced size and good solubility is also an important goal to
enhance their utility as tagging molecules.
The selection of an appropriate bioorthogonal labeling strateꢀ
gy for a given application is a difficult task, requiring multiple
considerations to minimally perturb the system of interest. We
hope these investigations will help provide guidance for selecting
the appropriate reaction and reagents for various live cell imagꢀ
ing applications. We believe it can also serve as a platform to
evaluate new bioorthogonal reactions5,93,94 in live cells where one
could easily prepare HaloTag ligands of newly developed
bioorthogonal groups to evaluate the chemistry in live cells and
within the various intracellular organelles. The development of
improved fluorophores is also an exciting area of research.50,95
This platform is ideally suited to help evaluate and optimize new
fluorophores including fluorogenic fluorophores for noꢀwash
live cell imaging71,92,96 as well as fluorophores for superꢀ
resolution imaging.41,97,98
at 37 °C/5% CO2. HeLa cells were transfected with a 3:1 ratio
9
of lipofectamine 2000 (Life Technologies):DNA according to
the manufacturer’s protocol in antibioticꢀfree DMEM/10% FBS
at 80ꢀ90% confluence. Cells were incubated for 3 h at 37
°C/5% CO2 and media was changed into growth media for 16ꢀ
20 h prior to HaloTag labeling experiments.
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Preparation of Clickable HaloTag Ligand and Fluoro-
phore Stock Solutions. HaloTag Ligands 1-11 were prepared as
10 mM stocks (1000X) in anhydrous DMSO within 1ꢀweek of
NMR confirmation of ligand structure. Stocks were frozen as
singleꢀuse aliquots at ꢀ20 °C, and kept for up to 6 months due to
stability issues. sTCO ligand 5 and cyclopropene ligand 9 were
especially unstable, and required constant monitoring and resynꢀ
thesis. AgꢀsTCO ligand 6 was prepared as a 10 mM stock in
EtOH, and maintained stability over at least 8 months when
stored at ꢀ20 °C. Fluorophore tetrazine derivatives were preꢀ
pared as 10 mM stocks in anhydrous DMSO, and serially diluted
to 1000X working stocks. Solutions were frozen at ꢀ20 °C and
remained stable up to 3 freezeꢀthaw cycles. Strained alkyne and
azide fluorophores were prepared as 100 mM stock solutions in
anhydrous DMSO, and frozen as 1000X working stocks at ꢀ20
°C. Aliquots were used up to 3 times before disposal without
any loss of reactivity. TCOꢀTAMRA fluorophore was prepared
as a 10 mM stock in anhydrous DMSO, aliquoted, and stored at
ꢀ20 °C as singleꢀuse aliquots.
Labeling of HaloTag fusion proteins with Bioorthogonal
Ligands and Evaluation of SPAAC and iEDDA Reactions
in Live Cells. HeLa cells expressing HaloTag constructs were
treated in 6ꢀwell dishes with 1 mL of 10 µM HaloTag ligands 1-
12 in growth media for 0.5 h at 37 °C/5% CO2. Samples labeled
with 12 served as a positive control to determine the maximum
amount of HaloTag protein labeling per experiment. Cells were
washed three times in DPBS and incubated in 2 mL new media
for 1 h with one media change to remove unbound HaloTag
ligands. For SPAAC reactions, HeLa cells were labeled with
chloroalkane ligands 1-3 followed by either a dose response of 50
nMꢀ250 µM fluorophores 15-17 for 2 h, or a timecourse of 25
Experimental Section
Materials and Methods. Synthesis of chloroalkane HaloTag
ligands 1ꢀ11 and bioorthogonal fluorophores are described in the
Supporting Information. All reagents were purchased from
SigmaꢀAldrich unless otherwise noted.
Molecular Cloning. Nꢀterminal HaloTag ORF fusion proꢀ
tein plasmids in pFN21A mammalian expression vector were
purchased for targeted HaloTag expression in the lumen of the
endoplasmic reticulum (human KDELR3; Promega FHC05579),
nucleus (human HIST1H2BK; Promega FHC05625), and cytoꢀ
sol (human GAP43;
Promega FHC02950).
KDELR3,
µM fluorophore for 30 s ꢀ 4 h in growth media. Reactions were
immediately quenched by washing cells two times in 500 µM
azideꢀamine 24, DBCOꢀamine 26 (Click Chemistry Tools), or
BCNꢀamine 25 in PBS (SPAAC Quench buffer). For iEDDA,
HeLa cells labeled with chloroalkane ligands 2, and 4-11 were
treated with either a dose response from 1 nMꢀ20 µM fluoroꢀ
phores 13, 14, 17, and 18 for 1 h, or a timecourse of 2 µM fluorꢀ
ophore for 10 sꢀ2 h in growth media. Cells were quenched by
washing two times in 100 µM Tetrazineꢀamine 27 (Click Chemisꢀ
try Tools) or TCOꢀamine 28 in PBS (iEDDA Quench buffer).
Cells were scraped in 1 mL quench buffer, spun at 2000 x g for 3
min, the buffer was aspirated and cell pellets were immediately
frozen on dry ice.
HIST1H2BK, and GAP43 plasmids were cut with PmeI/NotI,
and ligated to a NotI/SmaI fragment of either EGFP (pEGFPꢀ
N1, Clontech) or mCherry (pmCherryꢀN1, Clontech) to create
fluorescent protein tags on each construct.
The Cꢀterminal HaloTag ORF pHTN mammalian expression
vector was purchased from Promega (G772A). A cytoꢀ
solic/nuclear expressed HaloꢀmCherry fusion construct was
generated by ligating an NheI/EcoRV Halo fragment into
pmCherryꢀN1 (Clontech) mammalian expression vector, cut
with NheI/PspOMI (blunt filled). For extracellular HaloTag
protein expression (HaloꢀmCherryꢀPDGFR), a construct was
generated by ligating a synthetic, stop codon eliminated Haloꢀ
mCherry fusion to the transmembrane domain of the PDGFR in
the pDisplay vector (Invitrogen/LifeTechnologies catalog
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HeLa Cell Lysis, SDS-PAGE, In-Gel Fluorescence, and
Western Blotting. For SPAAC, cell pellets were lysed by soniꢀ
labeled HaloTag protein degradation during the course of the
experiment.
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cation in 100 µL 1% SDS/SPAAC quench buffer. For iEDDA,
Evaluation of SPAAC and iEDDA Reactions for
Live/Fixed Cell Imaging. HeLa cells were plated on polyꢀ
lysine coated glassꢀbottom dishes (MatTek, P35GCꢀ1.5ꢀ14C),
transfected, and treated as described for inꢀgel fluorescence with
the following modifications. Following fluorophore incubation
in culture media, cells were washed 3 x 1 mL PBS with the apꢀ
propriate quench reagent, and quenched for an additional 5 min
in media. Cells were washed 3 x 1 mL media to remove quench
reagent and incubated in cell culture media for 1ꢀ2 h prior to
imaging. Cells were washed one time in phenol redꢀfree DMEM
(Life Technologies)/10% FBS, and media was replaced with
phenolꢀred free DMEM/10% FBS supplemented with 10
µg/mL Hoescht 33342 (Life Technologies, H3570) for nuclear
labeling 5 min prior to imaging. Cells were fixed in iceꢀcold
MeOH for 10 min, washed 3 x 1 mL PBS and incubated in 1 mL
PBS overnight at 4 °C. PBS was aspirated and 100 µL
VECTASHIELD (Vector Laboratories) cell mounting media
containing DAPI (Vector Laboratories, Hꢀ1200) was added prior
to imaging.
cell pellets were lysed by sonication in 100 µL 1% SDS/iEDDA
quench buffer. Protein concentrations were determined with a
BCA protein assay (ThermoꢀFisher) and cell lysates were norꢀ
malized by protein concentration. Samples were prepared in 1X
LDS sample buffer (Life Technologies), with 10 µg protein loadꢀ
ed per well, and separated by SDSꢀPAGE on NuPage 4ꢀ12%
BisꢀTris gels in MES running buffer (Life Technologies).
TAMRAꢀfluorescence was analyzed on a Typhoon variable
mode imager (GE Healthsciences) using a TAMRA filter. Gels
were then transferred to nitrocellulose using iBLOT (Life Techꢀ
nologies), blocked in Odyssey blocking buffer (LiCor) for 1 h at
RT, and incubated in antiꢀHaloTag pAb (Promega, G9281) at
1:2000 overnight in TBST. Membranes were washed 3 times in
TBST, and incubated in antiꢀrabbit IRDye 800CW (LiCor) at
1:10,000 in TBST for 1 h at RT. Membranes were washed three
times in TBST and imaged on the Odyssey Infrared Imager
(LiCor).
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Data Quantification. Fluorescence intensity measurements
were quantified in ImageJ 1.45 (NIH) for both TAMRA fluoresꢀ
cence and total HaloTag protein expression. Inꢀgel fluorescence
(TAMRA) signal first was normalized to total HaloTag protein
expression signal (Western blot). The positive TAMRAꢀcontrol
(ligand 12) was set at 100% for each experiment. SPAAC and
iEDDA data were normalized to this value, and reported as a
percent of control. Data from 3ꢀ6 independent replicates were
quantified and plotted as the log[dose] vs. response for generaꢀ
tion of EC50 values. Curves were fit using a four parameter doseꢀ
response curve in GraphPad Prism version 6.03 for Windows,
Fluorescence Microscopy. Live and fixed cells were imaged
on a Zeiss AxioObserver.Z1 with a Yokagawa CSUꢀX1M 5000
spinning disk system using a Zeiss PlanApochromatic 40x/1.3 or
63x/1.4 oil immersion objectives. The imaging system was
maintained in a 37 °C heated incubation chamber along with
humidified stageꢀtop incubation components set at 37 °C/ 5%
CO2 for live cell imaging. Excitation of Hoescht and DAPI was
carried out with a 405 nm laser and emission spectra were colꢀ
lected between 440ꢀ480 nM.
Excitation of fluorescein,
BODIPYꢀFL, and eGFP was carried out using a 488 nm laser
and emission spectra were collected between 520ꢀ550 nm.
TAMRA and mCherry were excited with a 561 nm laser and
emission spectra were collected between 620ꢀ670 nm. Images
were acquired using a Photometrics Evolve 512 Delta camera
using the appropriate filter conditions for the indicated fluoroꢀ
phores with the ZEN Blue 2012 v. 8.1 software (Carl Zeiss Miꢀ
croscopy).
GraphPad
Software,
La
Jolla
California
USA,
www.graphpad.com. For timecourse experiments, data was
plotted as the percent of control vs. time and fit with pseudoꢀ
first order association kinetics where Y=Y0 + (PlateauꢀY0)*(1ꢀ
exp(ꢀK*x)) where the Plateau = Emax and K = rate constant exꢀ
pressed as the reciprocal of x in units. Y0 was set as a constant =
0. All data is reported as the mean ± SEM from 3ꢀ6 independꢀ
ent replicates.
Supporting Information Available: Supporting figures and
synthetic methods. This material is available free of charge via
the Internet at http://pubs.acs.org.
Pulse-Chase Analysis of HaloTag Ligand Incorporation
into HaloTag Protein. HeLa cells expressing HaloꢀH2BꢀGFP
were treated with 10 µM HaloTag ligands 1-12 for 30 min in
growth media at 37 °C/5% CO2, washed three times with PBS,
and incubated for 1 h in growth media with one media change to
remove unbound HaloTag ligand. Samples were treated with 1
µM HaloTag ligand 12 for 10 minutes at 37 °C/5% CO2, washed
three times with PBS and processed as described after lysis in
100 µL 1% SDS with sonication.
ACKNOWLEDGMENT
This work was supported by NIH Grant R01EB014354 from
NIBIB. Spectra were obtained with instrumentation supported by
NIH
P20GM104316,
P30GM110758, S10RR026962ꢀ01,
S10OD016267ꢀ01 and NSF grants CHE 0840401 and CHEꢀ
1229234.
Analysis of the Stability of TCO Derivatives in the Nu-
cleus and ER of Live HeLa Cells. HeLa cells transfected with
HaloꢀH2BꢀGFP (nucleus) or HaloꢀKDEL (ER) were analyzed
for dienophile stability. Cells treated with 10 µM HaloTag ligꢀ
ands 4ꢀ7 and 12 were incubated in DMEM/10% FBS for 0.5 h
followed by a 1 h washout period prior to reaction with
TAMRAꢀtetrazine 13 at 37 °C/5% CO2. HeLa cells were incuꢀ
bated with a saturating dose of 2 µM 5ꢀTAMRAꢀtetrazine 13 for
1 h at 37 °C/5% CO2 at the indicated timepoints. Cells were
washed with PBS, scraped in 1 mL PBS, pelleted at 2000 x g for
3 min, and processed as described previously for inꢀgel fluoresꢀ
cence. Each timepoint contained 12 as a control to account for
We would like to thank Kay Ahn and Jemy Gutierrez for valuaꢀ
ble discussions, Katherine Hales for assistance with confocal
microscopy, and Shannon Cawley and Brendon Kapinos for LCꢀ
MS sample analysis.
The authors declare no competing financial interests.
AUTHOR INFORMATION
Corresponding Authors
*doug.johnson@pfizer.com
hmurrey@ITS.JNJ.com
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176x137mm (300 x 300 DPI)
ACS Paragon Plus Environment

Page 23 of 27
Journal of the American Chemical Society
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174x59mm (300 x 300 DPI)
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 24 of 27
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176x62mm (300 x 300 DPI)
ACS Paragon Plus Environment

Page 25 of 27
Journal of the American Chemical Society
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174x96mm (300 x 300 DPI)
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 26 of 27
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175x61mm (300 x 300 DPI)
ACS Paragon Plus Environment

Page 27 of 27
Journal of the American Chemical Society
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84x46mm (300 x 300 DPI)
ACS Paragon Plus Environment