Fast, cell-compatible click chemistry with copper-chelating azides for biomolecular labeling

Article abstract of DOI:10.1002/anie.201108181

Bring your own copper: Copper-chelating azides undergo much faster click reactions (CuAAC) than nonchelating azides under a variety of biocompatible conditions. This kinetic enhancement allows site-specific protein labeling to be performed on the surface of living cells with only 10-40μM Cu ^I/Cu^II (see scheme). Detection sensitivity was also increased for CuAAC detection of alkyne-modified proteins and RNA. Copyright

Full text of DOI:10.1002/anie.201108181

Angewandte
Chemie
DOI: 10.1002/anie.201108181
Bioorthogonal Click Chemistry
Fast, Cell-Compatible Click Chemistry with Copper-Chelating Azides
for Biomolecular Labeling**
Chayasith Uttamapinant, Anupong Tangpeerachaikul, Scott Grecian, Scott Clarke,
Upinder Singh, Peter Slade, Kyle R. Gee, and Alice Y. Ting*
The copper-catalyzed azide–alkyne cycloaddition, or
CuAAC, has been used extensively for the conjugation,
immobilization, and purification of biomolecules.[1] Despite
excellent reaction kinetics, high specificity, and bioorthogon-
ality, CuAAC has been used to a far lesser extent in the
cellular context because of toxicity caused by the CuI-
mediated generation of reactive oxygen species (ROS) from
O2.[2] One way to address this problem is to remove the CuI
requirement, by using alkynes activated by ring strain.[3–5]
However, even the fastest of the strained cyclooctynes[6] react
with azides more than tenfold slower than terminal alkynes in
the presence of CuI (kobs ꢀ 1mꢁ1 sꢁ1 for (aza)dibenzocyclo
octyne[6] compared to kobs ꢀ 10–100mꢁ1 sꢁ1 per 10–100 mm CuI/
CuII for CuAAC[7]). A second approach to improve cell
compatibility is to use water-soluble ligands such as tris-
(hydroxypropyltriazolylmethyl)amine (THPTA),[8] bis[(tert-
butyltriazoyl)methyl]-[(2-carboxymethyltriazoyl)methyl]-
amine (BTTAA),[9] or bis(l-histidine)[10] for CuI. These
ligands both accelerate the cycloaddition reaction and act as
sacrificial reductants, helping to protect cells and biomole-
cules from ROS.[8]
Here we explore a third approach to improve the cell
compatibility and performance of CuAAC. In general,
decreasing the copper concentration lowers the toxicity of
CuAAC to cells, but this is accompanied by a large decrease
in reaction kinetics.[9] We reasoned that it might be possible to
compensate for this decrease by using an azide reaction
partner that contains an internal copper-chelating moiety
(Figure 1A), which would raise the effective copper concen-
tration at the reaction site. This concept has been explored for
azide–alkyne reactions in organic solvents, with CuII rather
than CuI species, and at very high copper (10 mm) and
reactant (200–400 mm) concentrations,[11,12] but never before
under conditions relevant to biomolecular labeling. The goal
of our study was to examine the effect of substrate chelation
assistance on CuAAC kinetics and biocompatibility.
The rate-determining step of CuAAC is postulated to be
the formation of the metallacycle from the CuI acetylide and
the organic azide.[15] We decided to test whether an organic
azide containing an internal CuI ligand could accelerate
formation of the metallacycle and hence the overall rate of
the CuAAC reaction. We prepared two azides with proximal
pyridine nitrogen atoms to chelate the CuI ion (picolyl azides
2 and 4), as well as their nonchelating carbocyclic analogues,
1 and 3 (Figure 2).
CuAAC reaction timecourses were measured using 7-
ethynylcoumarin, a fluorogenic alkyne whose quantum yield
(QY) increases from 1% to 25% upon reaction with azides[4]
(Figure 2A). Assays were first performed with 10 mm CuSO4
in the absence of CuI ligands. Reaction timecourses are shown
in Figure S1 (see Supporting Information) and values for
percent conversion into product after 10 and 30 minutes are
given in Figure 2B. Whereas the conventional azides 1 and 3
give no detectable product after 30 minutes under these
conditions, the picolyl azides 2 and 4 give 81% and 38%
product yields, respectively, after 30 minutes. We examined
a few other picolyl azide derivatives as well. The methyl ester
5 gives results similar to the acid 4. Substitution of the
aromatic ring with an electron-donating methoxy group
(azide 6) further accelerates the CuAAC reaction, while an
electron-withdrawing chloride substituent (azide 7) reacts
slower than the other picolyl azide derivatives. These
observations are consistent with a mechanism in which rate
acceleration is caused by coordination of the pyridyl nitrogen
atom to CuI or CuI acetylide, since an electron-donating group
will increase the electron density on this nitrogen atom,
improving coordination.
We further investigated picolyl azide 4, because it is the
synthetic precursor of the ligase substrate and fluorophore
conjugates, described later in this work. We repeated the
CuAAC reaction, but this time at three different copper
concentrations (10, 40, and 100 mm), either in the absence or
the presence of CuI ligand THPTA (4 molar equivalents
relative to copper). Figure 2C shows the timecourses of these
six reactions, as well as control reactions using the non-
chelating analogue of 4, azide 3.
[*] C. Uttamapinant, A. Tangpeerachaikul, Prof. A. Y. Ting
Department of Chemistry, Massachusetts Institute of Technology
77 Massachusetts Avenue, Room 18-496, Cambridge, MA 02139
(USA)
E-mail: ating@mit.edu
Dr. S. Grecian, Dr. S. Clarke, Dr. U. Singh, Dr. P. Slade, Dr. K. R. Gee
Life Technologies, Eugene, OR 97402 (USA)
[**] We thank Carolyn Kwa, Daniel Liu, and Ken Loh for assistance with
neuron culture, Jennifer Yao for LplA enzymes, and Peng Zou for
critical reading of the manuscript. Prof. M. G. Finn (Scripps)
provided the initial batch of THPTA ligand. Funding was provided by
the NIH (R01 GM072670), the Dreyfus Foundation, and the
American Chemical Society. C.U. was supported by the C.P. Chu and
Y. Lai summer graduate fellowship (MIT).
As has previously been observed, the addition of THPTA
increases the CuAAC reaction rate. For the conventional
azide 3, product is undetectable after 30 minutes in the
absence of THPTA (consistent with Figure 2B), whereas the
reactions at 100 and 40 mm copper proceed to completion
Supporting information for this article (figures and experimental
details) is available on the WWW under http://dx.doi.org/10.1002/
anie.201108181.
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picolyl azide 4 at all three Cu
concentrations in the absence
of THPTA are at least as high
as the reaction rates of con-
ventional azide 3 in the pres-
ence of THPTA.
Based on the dramatic
effects observed in vitro, we
tested the utility of copper-
chelating azides to fluores-
cently label proteins in a cel-
lular setting. To target the
picolyl azide moiety to spe-
cific cellular proteins, we
turned to our PRIME (probe
incorporation mediated by
enzymes[17]) protein-labeling
method. A panel of E. coli
lipoic acid ligase (LplA)
mutants was prepared, each
with a mutation at the gate-
keeper residue Trp37.[17–19]
We synthesized
a
picolyl
azide derivative that matches
the substrate requirements
for LplA, i.e., with a carbox-
ylic acid joined by four meth-
ylene groups to the picolyl
azide moiety (picolyl azide 8;
structure in Figure 1B; syn-
thesis in Figure S2 of the
Figure 1. Chelation-assisted CuI-catalyzed click reaction for site-specific and metabolic labeling of biomole-
cules. A) Generic reaction scheme for CuI-catalyzed, picolyl azide-alkyne cycloaddition (chelation-assisted
CuAAC). B) Site-specific probe targeting to cell-surface proteins by PRIME and chelation-assisted CuAAC. An
engineered PRIME ligase (LplA Trp37!Val) first ligates a picolyl azide derivative (picolyl azide 8) onto LplA
acceptor peptide (LAP, blue), which is genetically fused to a protein of interest (POI). Picolyl azide-modified
proteins are then derivatized with a terminal alkyne-probe conjugate (red circle), by live cell compatible
chelation-assisted CuAAC. BTTAA[9] and THPTA[2] are CuI tris-triazole ligands. C) Labeling of newly
synthesized RNAs (top) and proteins (bottom) in cells using alkynyl metabolites and chelation-assisted
CuAAC. EU is a uridine surrogate[13] and Hpg is a methionine surrogate.[14] Alkyne-labeled RNAs and
proteins are derivatized after cell fixation with picolyl azide-fluorophore conjugates (red circle).
Supporting
Information).
In vitro screening by HPLC
of six LplA mutants (W37G,
A, V, I, L, S) showed that the
valine mutant (W37VLplA) was
most efficient at recognizing
picolyl azide 8 and catalyzing
its covalent, ATP-dependent
ligation to the 13 amino acid
recognition
sequence
of
within 30 minutes when THPTA is added. As expected,
lowering the copper concentration lowers the reaction rate.
Dramatic rate enhancements are seen for all six con-
ditions when azide 3 is substituted by the chelation-competent
azide 4 (Figure 2C). Without THPTA, reactions proceed to
completion within 30 minutes for the two higher Cu concen-
trations (100 and 40 mm), in striking contrast to azide 3. When
THPTA is added, azide 4 reacts to completion within
5 minutes at all three copper concentrations. In other words,
the use of chelating azide 4 far offsets the decrease in the
CuAAC reaction rate caused by lowering the Cu concen-
tration. The effect is so strong that the reaction rate of
chelating azide 4 at the lowest Cu concentration (10 mm)
exceeds the reaction rate of the nonchelating azide 3 at the
highest Cu concentration (100 mm). It is also noteworthy that
the use of picolyl azide 4 instead of the conventional azide 3
can more than offset the effect of omitting the accelerating
ligand THPTA. Figure 2C shows that the reaction rates with
LplA, namely LAP (LplA acceptor peptide; Supporting
Information Figure S3).[20]
To test LplA-catalyzed picolyl azide targeting on cells, we
prepared human embryonic kidney (HEK) cells expressing
LAP-tagged cyan fluorescent protein (CFP) targeted to the
cell surface. Picolyl azide 8, W37VLplA, and ATP were added
for 20 minutes to these transfected cells. LAP-conjugated
picolyl azide was then detected by CuAAC with Alexa
Fluor 647-alkyne. Figure 3 and Figure S4 show that labeling
was easily detectable and specific to transfected cells.
To quantitatively examine the contribution of chelation
assistance to labeling efficiency in the context of cell-surface
fluorescence experiments, we repeated the labeling under
several more conditions. We varied the copper concentration
(10, 40, and 100 mm; same as in Figure 2C). We added either
THPTA ligand used in our in vitro experiments (Figure 2) or
BTTAA ligand, which was recently reported by Wu and co-
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Figure 3. Comparison of protein labeling signals on live cells using PRIME
and CuAAC, with and without chelating azide. Two-step, site-specific
protein labeling was performed as depicted in Figure 1B on HEK cells
expressing LAP-tagged cyan fluorescent protein fused to the transmem-
brane domain of the PDGF receptor (LAP-CFP-TM). In the first step, either
W37VLplA was used to target picolyl azide 8 to LAP, or wild-type LplA was
used to target nonchelating 8-azidooctanoic acid. In the second step,
CuAAC was performed for 5 min with Alexa Fluor 647-alkyne and CuSO4
(10, 40, or 100 mm), with either THPTA or BTTAA ligand (in fivefold excess
over CuSO4). Live cells were imaged immediately and representative
images are shown in Figure S4. To quantify labeling signals, the mean
Alexa Fluor 647 and mean CFP intensities were calculated for >90 cells for
each condition, ratioed and averaged. Error bars, ꢂstandard error of the
mean (s.e.m.).
ison shown in Figure S4 (cell images) and Figure 3 (quanti-
tation of these cell images).
Several trends are apparent from Figure 3. First, for the
conventional azide, 8-azidooctanoic acid, decreasing the
copper concentration reduces the cell-labeling signal, as
expected. Second, BTTAA does indeed give higher signals
than THPTA, but not as much as previously reported,[9] and
not at the lowest copper concentration of 10 mm. Third,
replacement of 8-azidooctanoic acid on LAP with the
chelation-competent picolyl azide 8 boosts cell signal at all
Cu concentrations 4- to 38-fold. When differences in picolyl
azide versus alkyl azide enzymatic ligation efficiencies are
taken into account, the difference is 2.7- to 25-fold (Support-
ing Information, Figure S5). The signal enhancements are
greatest at the higher Cu concentrations of 40 and 100 mm.
Similar to the in vitro data shown in Figure 2C, the signal
enhancement caused by picolyl azide more than offsets the
decrease in the CuAAC rate caused by lowering the Cu
concentration. For example, the signal with picolyl azide at
10 mm Cu (+ THPTA) is still 1.6-fold (corrected value) greater
than the signal with alkyl azide at 100 mm Cu (+ THPTA).
Comparisons in the presence of BTTAA ligand show that
picolyl azide at 40 mm Cu gives a 3.9-fold (corrected value)
greater signal than alkyl azide at 100 mm Cu. This experiment
also shows that the rate enhancement caused by picolyl azide
(compared to the nonchelating alkyl azide) is much greater
than the rate enhancement due to switching from a previous-
generation ligand (THPTA) to the newest-generation ligand
(BTTAA). Overall, the best cell-labeling results are obtained
using picolyl azide 8 in combination with BTTAA ligand and
either 40 or 100 mm CuSO4.
Figure 2. In vitro analysis of CuAAC rates with chelating azides.
A) Fluorogenic click reaction with 7-ethynylcoumarin[16] used to mea-
sure CuAAC reaction progress. B) Various chelating azide structures
tested and their CuAAC reaction yields after 10 min and 30 min.
Reactions were run with 10 mm CuSO4 and no CuI ligand. Complete
reaction traces are shown in Figure S1 of the Supporting Information.
C) Kinetic comparison of chelating azide 4 and its nonchelating benzyl
counterpart 3 at different copper concentrations. CuAAC product was
detected using the assay in (A). Solid lines indicate reactions in which
the CuI ligand THPTA was added in fourfold molar excess over CuSO4.
Measurements were performed in triplicate. Error bars, ꢂstandard
deviation (s.d.)
workers to be superior to THPTA and to give the fastest and
most cell-compatible CuAAC labeling to date.[9,21]
As a control, we compared LAP-expressing cells labeled
with LplA and 8-azidooctanoic acid, an alkyl azide that is
incapable of chelation assistance.[22] Since this enzymatic
ligation may have different kinetics than picolyl azide 8
ligation catalyzed by W37VLplA, we compared their labeling
yields on cells after 20 minutes (Supporting Information,
Figure S5). Although picolyl azide 8 ligation is faster, the
difference in yield after 20 min of labeling is at most 1.5-fold
over that of 8-azidooctanoic acid ligation. This correction
factor can therefore be applied to the multivariate compar-
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Using these optimized labeling conditions, we tested the
site specificity of cell-surface protein labeling using LplA and
CuAAC. In Figure 4, HEK cells expressing the transmem-
brane construct LAP-neurexin-1b (LAP is extracellular) were
For maximal versatility, we also developed a similar but
reverse-order site-specific protein labeling scheme based on
LplA ligation of an alkyne, followed by CuAAC derivatiza-
tion with picoyl azide-fluorophore conjugates (Supporting
Information, Figure S6). The alkyne substrate for LplA is 10-
undecynoic acid, and it is best ligated by W37VLplA. Figure S7
compares the original labeling scheme to this reverse-order
scheme. Picolyl azide ligation, followed by CuAAC with
fluorophore-alkyne, gives an approximately 2.4-fold greater
signal on average than alkyne ligation followed by CuAAC
with fluorophore-picolyl azide. This may reflect higher
efficiency for the enzymatic ligation of picolyl azide 8
compared to 10-undecynoic acid.
Since strain-promoted cycloaddition is frequently used as
an alternative to CuAAC in the cellular context, including by
us,[22] we compared it to our newly optimized CuAAC, in
terms of both labeling signal and cell toxicity. Figure S8A in
the Supporting Information shows that picolyl azide ligation
to LAP on cells, followed by CuAAC at 50 mm CuSO4 gives far
greater signal than alkyl azide ligation to LAP followed by
addition of dibenzocyclooctyne.[23] We also compared the
toxicity of these labeling conditions using the CellTiter-Glo
assay, which measures cellular ATP levels. Figure S8B shows
that both our CuAAC and strain-promoted cycloaddition
conditions are equally nontoxic.
A demanding test of the biocompatibility of our new
CuAAC is to perform it on neuron cultures. These delicate
cells show morphological changes in the presence of even low
concentrations of toxic substances. We transfected five-day-
old hippocampal rat neuron cultures with plasmids for
Homer1b-GFP (a postsynaptic marker) and LAP-neuro-
ligin-1 (a postsynaptic transmembrane adhesion protein
with an extracellular LAP tag). On day 11, picolyl azide 8
was ligated to LAP for 20 minutes, then CuAAC was
performed with THPTA and either 50 mm or 300 mm CuSO4.
Figure 4B shows that both conditions produce specific label-
ing, with Alexa Fluor 647 on neuroligin-1 colocalized with
Homer1b-GFP marker. However, the high copper concen-
tration causes focal swelling in the neurons (white arrows),
which is a sign of toxicity. The use of chelation-assisted
CuAAC allows the concentration of Cu to be decreased to
levels minimally toxic to neurons, with little sacrifice to the
signal intensity.
A faster and more biocompatible CuAAC labeling
protocol also benefits the detection of metabolically labeled
proteins and RNA. To demonstrate this, we used either
conventional CuAAC or chelation-assisted CuAAC to image
cellular RNAs and proteins metabolically labeled with 5-
ethynyluridine (EU)[13] or l-homopropargylglycine (Hpg),[14]
respectively (Figure 1C and Figure 5). Detection of these
alkynes on fixed cells was accomplished with a 1.8–2.7-fold
improvement in average signal intensity with Alexa
Fluor 647-picolyl azide compared to Alexa Fluor 647-alkyl
azide.
Figure 4. Site-specific labeling of cell-surface proteins with an engi-
neered picolyl azide ligase and chelation-assisted CuAAC. A) Labeling
of LAP-neurexin-1b on the surface of live HEK cells. First, picolyl azide
8 was ligated to LAP using 10 mm W37VLplA and 1 mm ATP for 20 min.
Second, the cell media was replaced with 20 mm Alexa Fluor 647-
alkyne, 40 mm CuSO4, and 200 mm BTTAA for 5 min. Negative controls
are shown with ATP omitted, wild-type LplA in place of W37VLplA, and
a Lys!Ala mutation in LAP. Histone2B-YFP was used as a transfection
marker. DIC=differential interference contrast image, AF 647=Alexa
Fluor 647. B) Labeling of LAP-neuroligin-1 on the surface of living
hippocampal rat neurons. 11 day-old neuron cultures expressing LAP-
neuroligin-1 and Homer1b-GFP were labeled with picolyl azide 8 as
in (A), followed by Alexa Fluor 647-alkyne. Cells were imaged live after
brief rinsing. CuAAC labeling was performed at the two copper
concentrations indicated, in the presence of 5:1 THPTA/Cu molar ratio
and 50 mm 4-hydroxy-2,2,6,6-tetramethylpiperidine (Tempol). Alexa
Fluor 647 images in the second column correspond to the boxed
regions 1 and 2, shown at higher zoom. White arrows denote regions
of focal swelling when 300 mm CuSO4 is used. All scale bars, 10 mm.
GFP=green fluorescent protein.
labeled first with W37VLplA and picolyl azide 8, then with
Alexa Fluor 647-alkyne in the presence of 40 mm CuSO4 and
200 mm BTTAA ligand. Transfected cells (expressing a nuclear
yellow fluorescent protein (YFP) marker) were strongly
labeled with a ring of Alexa Fluor 647 fluorescence, whereas
neighboring untransfected cells were unlabeled. Negative
controls with ATP omitted, wild-type LplA in place of
W37VLplA, or an alanine mutation in LAP did not result in
Alexa Fluor 647 labeling. The use of the picolyl azide ligase in
combination with chelation-assisted CuAAC thus seems
clearly advantageous, dramatically increasing the signal with-
out sacrificing specificity.
In summary, the use of copper-chelating azides dramat-
ically accelerates the CuAAC reaction under conditions
relevant to biomolecular labeling. We see this advance as
complementary to advances in ligand design, which have also
led to CuAAC rate acceleration and reduced cell toxicity.[2,9]
4
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Keywords: click chemistry · fluorescent probes ·
.
lipoic acid ligase · metabolic labeling · protein engineering
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Received: November 21, 2011
Revised: February 13, 2012
Published online: && &&, &&&&
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Communications
Bioorthogonal Click Chemistry
C. Uttamapinant, A. Tangpeerachaikul,
S. Grecian, S. Clarke, U. Singh, P. Slade,
K. R. Gee, A. Y. Ting*
&&&& — &&&&
Fast, Cell-Compatible Click Chemistry
with Copper-Chelating Azides for
Biomolecular Labeling
Bring your own copper: Copper-chelating
azides undergo much faster click reac-
tions (CuAAC) than nonchelating azides
under a variety of biocompatible condi-
tions. This kinetic enhancement allows
site-specific protein labeling to be per-
formed on the surface of living cells with
only 10–40 mm CuI/CuII (see scheme).
Detection sensitivity was also increased
for CuAAC detection of alkyne-modified
proteins and RNA.
6
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Angew. Chem. Int. Ed. 2012, 51, 1 – 6
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