Fluorescence Quenching Effects of Tetrazines and Their Diels–Alder Products: Mechanistic Insight Toward Fluorogenic Efficiency

Article abstract of DOI:10.1002/ange.202008757

Inverse electron demand Diels–Alder reactions between s-tetrazines and strained dienophiles have numerous applications in fluorescent labeling of biomolecules. Herein, we investigate the effect of the dienophile on the fluorescence enhancement obtained upon reaction with a tetrazine-quenched fluorophore and study the possible mechanisms of fluorescence quenching by both the tetrazine and its reaction products. The dihydropyridazine obtained from reaction with a strained cyclooctene shows a residual fluorescence quenching effect, greater than that exerted by the pyridazine arising from reaction with the analogous alkyne. Linear and ultrabroadband two-dimensional electronic spectroscopy experiments reveal that resonance energy transfer is the mechanism responsible for the fluorescence quenching effect of tetrazines, whereas a mechanism involving more intimate electronic coupling, likely photoinduced electron transfer, is responsible for the quenching effect of the dihydropyridazine. These studies uncover parameters that can be tuned to maximize fluorogenic efficiency in bioconjugation reactions and reveal that strained alkynes are better reaction partners for achieving maximum contrast ratio.

Full text of DOI:10.1002/ange.202008757

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Titel: Fluorescence Quenching Effects of Tetrazines and Their
Diels-Alder Products: Mechanistic Insight Toward Fluorogenic
Efficiency
Autoren: Brismar Pinto-Pacheco, William Paul Carbery, Sameer Khan,
Daniel B Turner, and Daniela Buccella
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Zitierweise: Angew. Chem. Int. Ed. 10.1002/anie.202008757
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10.1002/ange.202008757
Angewandte Chemie
RESEARCH ARTICLE
Fluorescence Quenching Effects of Tetrazines and Their Diels-
Alder Products: Mechanistic Insight Toward Fluorogenic
Efficiency
Brismar Pinto-Pacheco,[a] William P. Carbery,[a] Sameer Khan,[a] Daniel B. Turner*[a][b] and Daniela
Buccella*[a]
[a]
Dr. B. Pinto-Pacheco, Dr. W. P. Carbery, S. Khan, Prof. Dr. D. B. Turner, Prof. Dr. D. Buccella
Department of Chemistry
New York University
100 Washington Square East, New York, NY 10003, USA.
E-mail: dbuccella@nyu.edu (D.B.)
[b]
Current address: Micron School of Materials Science and Engineering
Boise State University
Boise, ID 83725, USA
E-mail: danielturner926@boisestate.edu (D.B.T.)
Supporting information for this article is given via a link at the end of the document.
Abstract: Inverse electron demand Diels–Alder (iedDA) reactions
between s-tetrazines and strained dienophiles have numerous
applications in chemical biology and materials science. Tetrazines
most often quench the emission of pendant fluorophores. This effect,
however, is diminished upon iedDA, leading to the formation of
emissive products with important applications in fluorescence
labelling of biomolecules. Herein we investigate the effect of the
dienophile on the fluorescence enhancement obtained upon reaction
fluorogenic reaction, given the right choice of reaction partners.[9-
22]
This feature can be particularly useful for in situ fluorescence
labeling of cellular species to enable their visualization and
tracking by various microscopy techniques. The fluorogenic
character stems from the strong fluorescence quenching effect
that tetrazines can exert on tethered fluorophores, which leads
to net fluorescence enhancement once the tetrazine core is
broken.
with
mechanisms of fluorescence quenching by both the tetrazine and its
reaction products through combination of linear and
a
tetrazine-quenched fluorophore and study the possible
Significant efforts have been devoted to the optimization of
reaction partners to increase the rates of iedDA reactions for
applications in bioconjugation chemistry.[23] The fluorogenic
properties of these transformations, however, have received
comparably less attention, despite much room for improvement
for applications in which high contrast is important and for
techniques like super-resolution imaging. Important work in this
area has placed an emphasis on maximizing the fluorescence
quenching efficiency of the tetrazine as a means to decrease the
brightness of the tetrazine-functionalized reactant, which can
result in impressive fluorescence enhancement (turn-on ratio)
upon cycloaddition. In this context, recent advances in the
design of fluorogenic iedDA substrates based on quenching by
through-bond energy transfer (TBET) to tetrazine moieties have
resulted in hundred-fold emission enhancements upon
a
ultrabroadband two-dimensional electronic spectroscopy (2D ES)
experiments. The dihydropyridazine product obtained from reaction
of the tetrazine with a strained cyclooctene shows a residual
fluorescence quenching effect, greater than that exerted by the
pyridazine arising from reaction of the tetrazine with the analogous
alkyne. Ultrabroadband 2D ES measurements reveal that resonance
energy transfer is the mechanism responsible for the fluorescence
quenching effect of tetrazines, whereas a mechanism involving more
intimate electronic coupling, most likely photoinduced electron
transfer, is responsible for the quenching effect of the
dihydropyridazine. These studies uncover parameters that can be
tuned to maximize fluorogenic efficiency in bioconjugation reactions
and they reveal that strained alkenes, though optimal for fast kinetics,
are not the best reaction partners for achieving maximum contrast
ratio in fluorescence labeling.
Introduction
Inverse electron demand Diels-Alder (iedDA) reactions between
s-tetrazines and strained dienophiles[1-3] (Figure 1) have become
increasingly widespread, with applications in areas ranging from
chemical biology[4-6] to materials science.[7-8] With remarkably
fast reaction rates, innocuous byproduct formation, and no
catalyst requirement, these transformations offer an excellent
alternative to azide-alkyne cycloadditions and other ‘click’
reactions, especially for bioconjugation reactions performed in
cellulo.[5] Beyond the remarkable kinetics, an attractive feature of
iedDA reactions involving tetrazines is the possibility of obtaining
Figure 1. Inverse electron demand Diels-Alder reaction between s-tetrazines
and common strained dienophiles. With the right choice of reaction partners,
the reaction can be rendered fluorogenic. The mechanism of fluorescence
quenching by tetrazines, however, remains speculative.
fluorescent products from non-emissive precursors, i.e.
a
1
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10.1002/ange.202008757
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RESEARCH ARTICLE
reaction.[24-31] Yet the mechanism responsible for the general
quenching effect of tetrazines on other, commonly used, non-
conjugated fluorophores remains speculative, and the influence
of the dienophile on the photophysical properties of the iedDA
reaction products has been ignored.
exo-bicyclo[6.1.0]non-4-yn-9-yl-methanol (BCN)[34] to enable
direct comparison of the iedDA products.[35] These fused ring
systems had been specifically designed to maximize the rate of
reaction with tetrazines and are commonly used in ligation
applications. Other strained alkenes such as cyclopropenes and
their derivatives also have shown favorable kinetics in iedDA
reactions with tetrazines, including examples of fluorogenic
bioconjugation reactions.[14] As these dienophiles become more
broadly used, their effect on the fluorogenic efficiency of the
reaction with tetrazines ought to be investigated systematically.
The nature of the substituents on the cyclopropene, however,
can lead to a wide variety of products[36] that extend beyond the
scope of this initial study.
A range of fluorophores covering the visible spectrum
(Figure 2) was selected for the quenching studies. The emission
profiles of the fluorophores show various degrees of overlap with
the absorption profile of the tetrazine Q1 (Figure 2A and Table
1), enabling the assessment of both Resonance Energy Transfer
(RET) and non-RET mechanisms. The cycloaddition products,
Q2 and Q3, do not absorb significantly in this region of the
spectrum. We sought to work with fluorophores that would have
a single species in solution (e.g., a single protonation state) at
neutral pH in order to simplify the interpretation of photophysical
studies. The coumarin amide derivative, F2, was prepared from
Coumarin 343 by simple amide coupling procedure with
carbonyldiimidazole (CDI) and ethanolamine (Scheme S2). The
orange-emitting p-methoxystyryl-substituted BODIPY, F4, was
synthesized from Knoevenagel condensation of 1,3,5,7-
tetramethyl BODIPY[37] and p-hydroxybenzaldehyde, followed by
alkylation of the phenol with dimethylsulfate, as detailed in the
Supporting Information (Scheme S3). The product fluorophore
shows a main absorption maximum at 568 nm (ε568 = 8.5×104 M-
1cm-1) and emission maximum at 578 nm (Figure S1), with a
fluorescence quantum yield Φ = 0.56 in MeCN/PBS. The small
Stokes shift is common of BODIPY fluorophores, and the
properties of this new derivative are similar to those of related p-
hydroxystyryl BODIPY dyes, but without the pH dependence of
the fluorescence emission that arises from a phenol moiety.[38]
Other fluorophores used in this study were obtained from
commercial sources (F1, F5) or synthesized by reported
procedures (F3[39]).
Results of steady-state quenching experiments of the
various fluorophore-quencher combinations are shown in Figure
3, with Stern–Volmer constants summarized in Table 2.
Solutions of dihydropyridazine Q2 and pyridazine Q3 quenchers
were prepared in situ by reaction of the dialkyltetrazine with
three equivalents of the corresponding dienophile. Complete
conversion to products was confirmed by the disappearance of
the characteristic tetrazine absorption band in the visible
spectrum (λabs = 526 nm), as well as by LC-MS analysis. Control
quenching experiments without tetrazine were performed to rule
out possible quenching effects of the excess dienophile present
in the mixtures. No fluorescence quenching was observed with
either BCN or TCO alone. The reaction of TCO with tetrazine
and all the fluorescence quenching experiments with the product
dihydropyridazine Q2 were performed under anaerobic
conditions; nevertheless, we saw no evidence of oxidation to the
pyridazine upon exposure to air in the timescale of our
experiments.
In the course of our studies, we have encountered
fluorogenic systems in which the fluorescence quantum yield of
the iedDA product varies significantly with the choice of
dienophile.[32] Furthermore, we and others[25] have found the
luminescence quantum yields of the dihydropyridazine-
functionalized products to be lower than those of the parent,
non-functionalized fluorophores. This difference indicates the
presence of non-radiative deactivation pathways that are still in
operation in the ‘turned-on’ products. In this work, we study the
fluorescence quenching effect of tetrazines and the products
generated in iedDA reactions with various dienophiles,
discussing the implications on the design of fluorogenic
transformations. We provide evidence for a resonance energy
transfer process between a tetrazine and spectrally overlapping
fluorophores, and for a more intimate quenching mechanism
involving
strong
electronic
coupling
between
the
dihydropyridazine product and the fluorophore, likely electron
transfer. These results are significant because they shed light on
critical processes that determine the fluorogenic efficiency and
the scope of fluorescence labeling reactions based on tetrazine
cycloadditions. Furthermore, they offer a unique demonstration
of ultrabroadband two-dimensional electronic spectroscopy (2D
ES) applied to the study of fluorescence quenching pathways.
Results and Discussion
Intermolecular fluorescence quenching by tetrazines,
pyridazines and dihydropyridazines: linear steady state and
time-resolved studies
To investigate the effect of the dienophile on the fluorogenic
efficiency of iedDA reactions, we began by studying the
bimolecular quenching of various fluorophores with increasing
concentrations of 3,6-bis(2-hydroxyethyl)-s-tetrazine, Q1, or of
its reaction products with either a strained alkene or alkyne (Q2
and Q3, respectively, see Figure 2). Although aryl-substituted
tetrazines are used in bioconjugation reactions more often than
the alkyl-substituted counterparts due to their favorable reaction
kinetics and ease of synthesis,[4, 7] we used the alkyl derivatives
because they obviate potential influence of the substituents and
thereby isolate the quenching effect of the tetrazine moiety.
Furthermore, we found Q1, and especially its cycloaddition
reaction products, to have better solubility than some of the
simple aryl tetrazines used in bioconjugation reactions,
particularly in aqueous solvent mixtures at the high
concentrations required for typical intermolecular quenching
experiments (results not shown). Still, because of limitations in
solubility at such high concentrations, all quenching studies
reported herein were conducted in
acetonitrile/aqueous PBS buffer.
a
2:1 mixture of
The dialkyl tetrazine used in this study was synthesized from
3-hydroxypropionitrile and hydrazine hydrate with nickel triflate
as catalyst (Scheme S1), in a protocol optimized from the
method described by Devaraj and coworkes.[25] As dienophiles,
Concentrations of quenchers ranging from micromolar to low
millimolar were employed in order to reach levels of quenching
we
selected
exo-(E)-bicyclo[6.1.0]non-4-en-9-yl-methanol
(hereafter trans-cyclooctene, TCO)[33] and the alkyne analogue
2
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10.1002/ange.202008757
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RESEARCH ARTICLE
Figure 2. Fluorophores (A) and quenchers (B) selected for this study. Panel in the upper right shows the photoluminescence spectrum of each fluorophore (solid
lines, excited at their respective absorption maxima, listed in Table 1) and their overlap with the absorption spectrum of 3,6-bis(2-hydroxyethyl)-s-tetrazine
(dashed line, shaded) in a 2:1 MeCN/PBS buffer mixture. The products of iedDA reaction, Q2 and Q3, with alkene and alkyne TCO and BCN, respectively, do not
show significant absorption in the same spectral window.
Table 1. Photophysical and electrochemical properties of fluorophores used in this study.[a]
Absorption
maximum λabs
(nm)
Emission
maximum λem
(nm)
J[b]
Förster
E(A/A-)
(V vs. SCE)
E(A+/A)
(V vs. SCE)
Compound
Φ
E00 (eV)
(×1013 M-1cm-
1nm4)
radius[c] (Å)
F1
F2
F3
F4
F5
420
441
480
487
512
578
626
0.671(8)
0.64(2)
0.98(2)
0.560(4)
0.54(3)
1.271
1.495
2.204
0.281
0.011
21.6
22.0
25.2
16.3
9.48
-1.381[e]
-1.652[e]
-1.004[e]
-1.015[e]
-0.18[f,g]
1.003[f]
0.890[f]
1.184[e]
0.865[e]
1.280[e,g]
2.76
2.67
2.45
2.17
2.03
504
568d
590
[a] Optical properties determined in a 2:1 MeCN/PBS buffer mixture at 25 °C. Electrochemical parameters determined in MeCN with 0.1 M Bu4NPF6 as supporting
electrolyte. Values in parentheses reflect the uncertainty of the last significant figure. Errors correspond to the standard deviation from triplicate measurements. [b]
Overlap integral with the fluorophore as donor and Q1 as energy acceptor. [c] Determined using the dynamic isotropic limit (2/3) for the orientation factor κ2. [d]
Extinction coefficient ε568= 85000 ± 2000 M-1cm-1. [e] Either cathodic or anodic peak potential (Ep) for an irreversible peak. [f] Half-wave potential (E1/2) for a
reversible peak. [g] Values from reference [40]
.
of the same order of magnitude as those observed for tetrazine-
appended fluorophores (intramolecular quenching, vide infra),
for which the effective molarity of the quencher is much higher.
Comparison of steady-state with analogous time-resolved
measurements (Figures S2, S3 and Table S1) revealed that, in
some cases, the effect of the tetrazine arises from a combination
of dynamic (collisional) and static (involving the formation of
non-emissive complexes in the ground state) quenching. We did
not deconvolute the contributions of the two types of process
because most of the available data were collected at relatively
low concentrations of quenchers for which static quenching is
less prevalent and difficult to quantify accurately. We only report
herein the apparent Stern–Volmer constants extracted from
steady-state measurements. These values suffice to establish,
at least semi-quantitatively, the relative quenching power of the
tetrazine and its various reaction products.
As reflected by the Stern–Volmer constants (Table 2), the
quenching effect of the tetrazine is much greater than that of its
reaction products with various dienophiles, a difference that
supports the application of the iedDA reaction for fluorogenic
purposes.
Intramolecular
fluorescence
quenching
by
tetrazines—with a typical absorption band in the 500–550 nm
range—has been shown to be highly dependent on the emission
wavelength of the linked fluorophores, prompting the proposal of
41]
a RET-based quenching mechanism.[9,
The lack of solvent
dependence on the quenching of geometrically constrained
bichromophoric tetrazine-BODIPY systems lent further credence
to the proposed RET mechanism over an alternative
3
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RESEARCH ARTICLE
Figure 3. Stern–Volmer plots for fluorophores F1-F5 treated with a dialkyltetrazine (Q1), and its reaction products with a strained alkene (Q2) or alkyne (Q3).
Quenching experiments were conducted in a 2:1 MeCN/PBS buffer mixture at 25 °C.
fluorescence emission of tetrazine-functionalized fluorophores
Table 2. Stern–Volmer constants obtained from steady-state fluorescence
will not be fully restored and the maximum possible contrast will
not be achieved upon reaction with TCO (vide infra). Upon close
inspection of the absorption spectrum of the quenchers, a very
weak absorption band at 442 nm (ε442 = 5.8 M-1cm-1) can be
observed for Q2 (Figure S4), thus some contribution from RET
to the quenching by this dihydropyridazine cannot be ruled out
entirely for the blue-green emitters. The extent of quenching,
however, is greater with the orange-red emitting fluorophores,
which show very little spectral overlap with this absorption
feature. An alternative mechanism is thus most likely involved.
Tetrazines, pyridazines, and dihydropyridazines are
electrochemically active and could potentially participate in
electron transfer reactions that provide efficient non-radiative
pathways for decay of excited fluorophores. In the context of
bioorthogonal chemistry, the redox properties of tetrazines—
which enable their photocatalytic or electrochemical generation
quenching experiments.
Q1
Q2
Q3
KSV (M-1)
440 ± 30
680 ± 70
KSV (M-1)
13.9 ± 0.8
20.6 ± 0.1
36 ± 2
KSV (M-1)
10 ± 12
6 ± 2
F1
F2
F3
F4
F5
1200 ± 100
360 ± 20
92 ± 9
6 ± 1
19 ± 1
8.7 ± 0.1
1.7 ± 0.9
37 ± 7
photoinduced electron transfer (PeT) mechanism.[42] Our results
seemed consistent with this notion, with the maximum
fluorescence quenching effect observed for the BODIPY FL
fluorophore, which exhibits the greatest spectral overlap and
longest calculated Förster radius (25.2 Å) with the
dialkyltetrazine as energy acceptor (λabs = 526 nm, ε526 = 546 ± 1
M-1cm-1). Cresyl Violet—showing almost negligible spectral
overlap with the tetrazine—was chosen as a control to support
this mechanistic hypothesis. To our surprise, however, this
fluorophore was subject to significant quenching by the tetrazine
as well. A detailed study of this system, reported elsewhere,[40]
ruled out RET and indicated that an excited state proton transfer
mechanism operates in this particular case.
from
reversible
oxidation
of
the
corresponding
dihydrotetrazines—have been exploited to develop methods for
controlled activation of bioconjugation reactions at surfaces[43]
and in biomaterials.[44] To evaluate the plausibility of
fluorescence quenching by a PeT mechanism, we investigated
the electrochemical properties of various fluorophores and
quenchers used in this study (Figures S5-S11). The main
features of the fluorophores are summarized in Table 1. The
cyclic voltammogram (CV) of the dialkyltetrazine in acetonitrile
with Bu4NPF6 as supporting electrolyte displays a reversible
reduction peak at E1/2 = –0.874 V vs SCE that does not change
in shape and intensity after multiple scans. This wave likely
corresponds to the reversible formation of a radical anion in the
non-aqueous solvent;[42],[45] further reduction to the dianion is not
observed in our scan window. A single irreversible oxidation (Ep,c
For all fluorophores investigated, the extent of fluorescence
quenching exerted by the pyridazine Q3 is almost negligible,
whereas that of the dihydropyridazine Q2 is sizeable. These
results indicate that, in the context of fluorogenic labeling,
4
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10.1002/ange.202008757
Angewandte Chemie
RESEARCH ARTICLE
=
2.045
V
vs SCE) is observed. From these results,
spectroscopy, three types of signal contribute to the spectrum.
Positive (red) ground state bleach (bleach) and stimulated
emission (SE) signals arise from the depletion of the ground
state (S0 → S1) by the pump pulse and coherent emission of the
first excited state (S1 → S0) to the ground state, respectively.
Negative (blue) excited-state absorption (ESA) signal indicates a
transition from the first excited state to some higher-lying excited
state (S1 → Sn), respectively. The bleach and SE signals are
comparable to absorption and fluorescence, respectively, while
the entire 2D electronic spectrum can be reduced in
dimensionality to the equivalent of a TA spectrum by summing
along the excitation axis. The inset below the 2D spectrum is the
TA spectrum for visual comparison, demonstrating that 2D ES
can resolve multiple spectral features that lead to single peaks in
TA spectroscopy. Finally, the absorption spectra of
methoxystyryl BODIPY F4 and tetrazine Q1 are placed on the
right inset as additional reference spectra.
photoinduced oxidation of the tetrazine is predicted to be
thermodynamically unfavorable for all fluorophores (Table
S2),[46] whereas the photoinduced reduction is favorable for all
fluorophores except F5 and thus must be considered still as a
feasible quenching mechanism alternative to RET.
The CV of the dihydropyridazine Q2 in acetonitrile (Figure
S11) shows irreversible reduction and oxidation peaks at Ep,c = –
0.88
photoinduced reduction and oxidation of the quencher are
thermodynamically favorable with fluorophores F1 through F4,
though the photoinduced reduction is more favorable for the
coumarins and methoxystyryl BODIPY and the photoinduced
oxidation is more favorable for BODIPY FL, F3. For cresyl violet,
F5, only the photoinduced oxidation appears thermodynamically
favorable. There is no direct correlation between the redox
potentials and the Stern–Volmer constants extracted from
bimolecular fluorescence quenching experiments, either from
the steady state or time-resolved measurements. Such
correlation, however, may be expected in series of structurally
related compounds for which the reorganization energy and
electronic coupling terms are similar. This is not the case for the
compounds studied here, motivating the use of other
spectroscopic methods to study the energy transfer mechanisms
that apply.
V and Ep,a = 0.73 V vs. SCE, respectively. Both
Analysis of the 2D electronic spectra after addition of the
quenchers enables the evaluation of possible fluorescence
quenching mechanisms, including exciton formation, PeT, RET,
Ultrafast two-dimensional electronic spectroscopy (2D ES)
studies
Pump-probe optical spectroscopy techniques yield valuable
information about energy transfer mechanisms by selectively
exciting an optical transition and probing the time-resolved
dynamics of the resultant excited state. The tetrazine and its
TCO products, however, are inefficient fluorescence quenchers,
and mixtures of fluorophores with tetrazine Q1 and
dihydropyridazine Q2 returned only transient absorption (TA)
spectra that were only subtly different, at best. We therefore
utilized an extension of TA spectroscopy, ultrabroadband two-
dimensional electronic spectroscopy (2D ES), as a means of
resolving weak spectral changes associated with different
fluorescence quenching mechanism. The 2D ES technique has
a
number of advantages over conventional pump-probe
spectroscopies. For instance, congested one-dimensional
spectra, or samples with multiple chromophores, are readily
resolved along the excitation axis in 2D electronic spectra. The
technique also avoids an inconvenient trade-off in ultrafast
transient absorption, where shorter laser pulses with better time
resolution necessitate more excitation bandwidth and decrease
excitation specificity. Many of these same advantages informed
the development of 2D NMR techniques, and the spectral
interpretation of 2D ES proves similar as well.[47-48] Diagonal
peaks in 2D ES correspond to individual electronic transitions
while off-diagonal cross peaks arise from coupled transitions.
Figure 4. (A) Normalized 2D electronic spectrum of methoxystyryl BODIPY F4
at a waiting time of 100 ps. Bleach and stimulated emission features appear
as red, positive (+) signal, while excited-state absorption features appear as
blue, negative (-), signal. Regions marked with dashed violet boxes are areas
of mechanistic interest where signatures of RET or excitonic coupling would
be expected to appear in the presence of tetrazine Q1. Insets to the top, right
and bottom include 1D spectra for ease of comparison. (B) Simplified
schematic representation of the signature spectral features associated with
various possible energy transfer mechanisms between a fluorophore (F,
orange) and a quencher (Q, green). The RET box illustrates the region where
changes in the spectrum due to RET are likely to occur. In FRET between two
fluorophores, the region would likely include the growth of a stimulated
emission band. In experiments with a non-emissive acceptor, like those
described herein, there would be a loss of stimulated emission from the
fluorescent donor and no new peak.
The location and evolution of these cross peaks differ based on
49]
the underlying energy transfer mechanism.[40,
Their
examination thus allows one to rule out certain possible
fluorescence quenching mechanisms while supporting others.
The 2D ES electronic spectrum of methoxystyryl BODIPY F4
in 2:1 MeCN:PBS at a waiting time of 100 ps is presented in
Figure 4 (expanded and explained in detail in Figure S14). This
fluorophore was chosen for further study due to the favorable
overlap of the compound’s absorption and fluorescence with the
excitation bandwidth of the laser (500 – 770 nm). Like in TA
5
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10.1002/ange.202008757
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Figure 5. Salient 2D electronic spectra for mechanistic study of methoxystyryl BODIPY, F4, treated with tetrazine Q1 or dihydropyridazine, Q2. Horizontal and
vertical dashed lines indicate prominent excitation and probe wavelengths in the 2D spectra.
and excited-state proton transfer. Each mechanism has unique
2D ES signatures (Figure 4B), and the latter mechanism was
proposed for the fluorescence quenching of cresyl violet by
tetrazine Q1 using a similar analysis.[40] In the case at hand, the
BODIPY fluorophore lacks exchangeable protons and therefore
only excitonic, PeT, and RET mechanisms need to be
considered.
In studies of light-harvesting proteins and nanoparticle
assemblies, excitonic coupling leads to cross peaks that appear
immediately at common absorption wavelengths, with
subsequent energy transfer between chromophores altering the
intensity of those cross peaks. Photochemical transformations
such as PeT do not lead to the appearance of spectral features
with the same immediacy. Nevertheless, new cross peaks at
common excitation wavelengths but in a new emission region
indicate the alteration of the donor and acceptor electronic
states as would be expected for PeT. This new emission feature
is associated with the donor–acceptor complex, which can
electronic spectrum that defines the range of possible excitation
energies needed to result in acceptor emissions. In the specific
case studied here, the acceptor does not emit appreciable light,
thus the energy transfer will likely be indicated by a loss of SE
signal of the donor in the RET box.
The 800 ps 2D electronic spectra of methoxystyryl BODIPY
F4 with and without tetrazine Q1 along with the 5 ps spectra with
and without dihydropyridazine Q2 are presented in Figure 5.
Additional 2D spectra at waiting times of 200 fs, 1 ps, 10 ps, and
100 ps are presented in Figures S15 and S16 in the SI. When
methoxystyryl BODIPY is exposed to 45 mM (500 equivalents)
of tetrazine Q1, a comparison of the treated and untreated 2D
electronic spectra reveals a reduction in the SE signal in the
former for all waiting times. This reduction in SE signal is
localized in the high excitation energy region of the 2D spectrum,
close to the absorption of tetrazine Q1 at 525 nm.[52] It should be
noted that intermolecular quenching, even at the high
concentrations of quencher employed here is not 100% efficient,
therefore significant emission signal of the unquenched
fluorophore is expected to be present in all of the 2D electronic
spectra taken. Furthermore, because neither Q1 nor Q2 are
emissive, this residual emission from F4 will be the primary
contributor to the 2D spectra.
undergo
a number of transformations over the course of
picoseconds, including back–electron transfer and diffusion.[50-51]
The weaker electronic coupling underpinning RET mechanisms
yield spectra with a new peak at donor excitation and acceptor
emission. This results in a “RET box”, i.e. the area of a 2D
6
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10.1002/ange.202008757
Angewandte Chemie
RESEARCH ARTICLE
The absence of cross peaks at the common frequencies of
methoxystyryl BODIPY and tetrazine Q1 eliminates excitonic
coupling as a possible mechanism, and the lack of rapidly
appearing emission features suggests PeT is a poor fit. In
particular, the lack of change in the ESA region of the
methoxystyryl BODIPY—where new peaks originating from
exciplex or donor-acceptor complex formation would appear—
suggests a different mechanism. The area of reduced SE signal
in the early time 2D electronic spectra corresponds well to the
area indicated by the RET box. Two new ESA features and a
third, red-shifted SE peak appear by 800 ps in the area of
spectral overlap between F4 and Q1. Both the location and time-
scale of the changes[53] suggest that fluorescence quenching by
Q1 proceeds via a RET mechanism. A comparably smaller loss
of ESA signal at 525 nm along the excitation line of F4 may
indicate the bleaching of Q1 due to RET. Under more
conventional FRET conditions this bleaching would fall within the
RET box; the high energy absorption of Q1 relative to F4,
however, results in this alternative placement serving as
secondary evidence suggesting a RET mechanism. The long
time scale and relatively small quenching efficiency is expected
given the relatively small spectral overlap between the two
chromophores and their concentrations in solution.
Intramolecular fluorescence quenching and fluorogenic
iedDA reaction
To assess the impact of the above results on the efficiency of
fluorogenic iedDA reactions, we linked covalently the
dialkyltetrazine moiety to the BODIPY FL fluorophore F3 and
measured the increase in fluorescence emission upon reaction
with either dienophile, TCO or BCN (Figure 6). BODIPY FL was
chosen for this experiment because it exhibited the greatest
quenching by the tetrazine and potential turn-on upon
cycloaddition in our intermolecular studies. As a result, this
fluorophore offered a large dynamic range to evaluate the
differences in fluorogenic efficiency arising from the two classes
of dienophiles studied here. It should be noted, however, that
differences in fluorogenic efficiency upon reaction with different
dienophiles, leading to pyridazine and dihydropyridazine
products, have been observed also with tetrazine-functionalized
fluorescein and rhodamine fluorophores widely employed in
biolabeling applications.[25]
Treatment of BODIPY FL-Tz with excess alkyne led to a 75-
fold fluorescence enhancement, whereas reaction with the
alkene only afforded a ~24-fold enhancement. The latter value is
comparable to those reported for fluorogenic reactions in similar
systems.[9, 14] Oxidation of the dihydropyridazine product to the
corresponding pyridazine by treatment with 2,3-dichloro-5,6-
dicyanobenzoquinone (DDQ) led to further enhancement of the
The changes in the 2D spectrum of F4 in the presence of
dihydropyridazine Q2 proceed much more quickly than in the
tetrazine Q1 case and occur in an entirely different region of the
2D spectrum. Specifically, the ESA features at emission
wavelengths between 550 nm and 600 nm disappear by 5 ps but
recover partially by 100 ps, with nearly full recovery by 800 ps
(Figure S16). The depletion of the ESA features is apparent by
200 fs in the 2D spectra, but close inspection of the transient
absorption traces preserved from the phasing process, while
noisy, indicate that the treated and untreated methoxystyryl
BODIPY are still identical within the noise limit at 100 fs.
As in the case with the tetrazine Q1, 2D ES can rule out
many of the possible mechanisms of energy transfer in the
dihydropyridazine Q2 system. Fluorescence quenching through
RET is unlikely in this case based on the absence of the
required spectral overlap between the possible donor and
acceptor molecules (Q2 has an absorption maximum of 341 nm
with a shoulder at 430 nm). The rapid disappearance of the
BODIPY ESA features by 200 fs suggests a mechanism with
stronger electronic coupling than present in a RET mechanism,
but the early time transient absorption and the reappearance of
ESA signal at 100 ps conflicts with a mechanism based on
excitonic coupling. These observations leave PeT as the most
likely mechanism for fluorescence quenching by the
dihydropyridazine Q2. The relatively low efficiency of
fluorescence quenching by Q2 means that the features of the
transient species, particularly the BODIPY radical anion, may be
covered entirely by the unquenched F4 signal. Unfortunately,
without direct access to the donor-acceptor complex it is
impossible to compare the rate of this transient disappearance
with the rate of ground state recovery and determine whether
the process proceeds through an electron transfer event or
through Dexter-type electron exchange. Nevertheless, the time
scale of the disappearance and recovery of the ESA features—
ultrafast disappearance by 200 fs followed by reappearance
over the course of several hundred picoseconds—is consistent
with the rates of electron transfer and back electron transfer to
the ground state measured by pump-probe spectroscopies in
other systems.[54]
Figure 6. Fluorogenic reaction of tetrazine-functionalized BODIPY with
strained dienophiles (only one isomer of the dihydropyridazine product shown).
The reaction with the alkyne affords full recovery of the fluorescence whereas
that with the alkene does not, due to PeT between the fluorophore and the
dihydropyridazine. Fluorescence emission profiles collected after reaction
completion, λex= 502 nm. Quantum yields determined in 2:1 MeCN/PBS buffer
mixture using fluorescein in 0.1 M NaOH as standard (Reference [55]).
7
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10.1002/ange.202008757
Angewandte Chemie
RESEARCH ARTICLE
fluorescence, but still lower than that observed with the product
of the direct reaction with BCN. This small difference in
fluorescence emission appears to originate from a quenching
effect of the DDQ in the former, as revealed by a control
experiment in which an independently prepared sample of the
pyridazine product P2 was treated with excess of the quinone
(Figure S17).
those obtained with the strained trans-cyclooctenes.[25] It is
important to note that, under oxidizing conditions, the
dihydropyridazine can be converted to a pyridazine post ligation,
leading to further fluorescence enhancement. Nevertheless, in
systems in which the oxidation is not facile or is not easily
controlled, the fluorogenic efficiency of reactions with alkenes
might be limited or vary over time or sample, thus affecting
quantitative analysis.
Comparison of the quantum yields of the products of the
fluorogenic reactions (Figure 6 and Table S3) with that of the
parent, non-functionalized BODIPY FL, reveals full restoration of
the fluorescence emission of BODIPY FL-Tz upon reaction with
BCN. These results are consistent with the trends observed in
the bimolecular quenching experiments, in which the quenching
effect of dihydropyridazine Q2 was consistently greater than that
of pyridazine Q3, and the latter was almost negligible in most
cases. They also suggest that bimolecular quenching
experiments may be valuable for simple evaluation of reaction
partners in the process of optimizing the fluorogenic efficiency of
a transformation. Moreover, quantum yields determined in 2:1
MeCN/PBS buffer differ very little from those determined in
purely aqueous solvent (Table S3), suggesting that the extent of
fluorescence quenching is similar in both and that the
conclusions of our studies are relevant to the interpretation of
systems operating in typical bioconjugation conditions.
The CV of BODIPY FL-Tz in acetonitrile (Figure S12) shows
two reduction waves, one at E1/2 = –0.896 V and a second,
irreversible one at Ep,c = –1.075 V vs SCE. An irreversible
oxidation peak was observed at 1.197 V vs. SCE. These
features match those of the tetrazine and BODIPY FL measured
independently, thus confirming there is no direct electronic
communication between the two chromophores. Significantly,
the quantum yield of P1 increases to Φ=0.42 ± 0.02 in 1,2-
dichloroethane (ε= 10.36), consistent with a decrease in the rate
of photoinduced electron transfer in the solvent of lower
dielectric constant.
Two-dimensional electronic spectroscopy reveals the
fluorescence quenching mechanism of a dialkyltetrazine to be
mainly RET, though PeT is thermodynamically feasible in most
cases investigated here.
With new photochemical mechanistic insight, the
development of fluorogenic reactions with tetrazines can be
approached in a more rational and systematic fashion. One
obvious avenue includes the optimization of various parameters
affecting RET efficiency in order to decrease the fluorescence
emission of the tetrazine-functionalized fluorophore in its ‘off’
state. Yet consideration of the electrochemical properties of the
fluorophores and click reaction products in the design process
may enable the tuning of the ‘on’ state too. Specifically,
decreasing the rate of PeT with the dihydropyridazine product
can result in greater overall fluorescence enhancement upon
bioconjugation. Finally, smart use of the electrochemical
properties of the tetrazines themselves may also help in the
design of better fluorogenic reactions. For example, careful
matching of the fluorophore and tetrazine redox potentials may
enable the engineering of fluorogenic transformations based
entirely on the modulation of PeT rates rather than RET
quenching, thus enabling the use of fluorophores with low
energy emission—which lie beyond the range optimal for RET—
and expanding the scope of these click reactions in the context
of bioimaging applications.
Acknowledgements
Conclusion
This work was supported by the National Science Foundation
under grant CHE-1555116 to D.B and CHE-1552235 to D.B.T.
D.B.T. also acknowledges support from the Alfred P. Sloan
Foundation. W.P.C. acknowledges support from the Margaret
Strauss Kramer Fellowship, and B.P.-P. thanks the NSF for a
Graduate Research Fellowship under Grant No. DGE1342536.
The authors thank Prof. Nicholas Geacintov and Dr. Alexander
Durandin for access to equipment and assistance with
fluorescence lifetime experiments. They also thank María De
Abreu and Dr. Guangqian Zhang for help in the early stages of
these studies.
Inverse electron demand Diels–Alder (iedDA) reactions between
tetrazines and strained dienophiles have become increasingly
used in bioconjugation chemistry, and their fluorogenic character
makes them particularly valuable for labeling and tracking
biomolecules in cellulo. Important efforts have been devoted to
tailoring reaction partners for improved kinetics, stability, and
biocompatibility. In particular, recent design of strained
dienophiles—beyond those developed for other click reactions—
has led to reaction rates unparalleled by any other
bioconjugation reaction to date.
Speed, however, comes at the expense of fluorogenic
efficiency. Though strained alkenes offer the fastest kinetics in
reactions with tetrazines, our study demonstrates that the
product dihydropyridazine can participate in photoinduced
electron transfer processes that impact the fluorescence
efficiency of pendant fluorophores. The quenching effect of the
pyridazine obtained from the strained alkynes, on the other hand,
is almost negligible with all the fluorophores included in our
study. These observations make strained alkynes a better
choice of dienophile for fluorescence labeling applications that
demand high contrast and for super-resolution microscopy.
Other dienophiles such as norbornene and cyclopropenes must
be evaluated further in this context, though the currently
available data shows fluorogenic enhancements comparable to
Keywords: Tetrazine • FRET • photoinduced electron transfer •
2D electronic spectroscopy • quenching mechanism
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9
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Angewandte Chemie
RESEARCH ARTICLE
Entry for the Table of Contents
An ultrafast look at fluorogenic tetrazines. Two-dimensional electronic spectroscopy and
electrochemical studies provide mechanistic insight on the fluorescence quenching effects of tetrazines
and their Diels-Alder cycloaddition products. The studies uncover the influence of the dienophile on the
fluorogenic efficiency of reactions with tetrazine-decorated fluorophores and reveal options for
maximizing contrast in biolabeling applications.
10
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