Monochromophoric Design Strategy for Tetrazine-Based Colorful Bioorthogonal Probes with a Single Fluorescent Core Skeleton

Article abstract of DOI:10.1021/jacs.7b10433

Fluorogenic bioorthogonal probes are ideal for fluorescent imaging in live cell conditions. By taking advantage of the dual functionality of tetrazine (Tz), as a bioorthogonal reaction unit as well as a fluorescence quencher, a fluorophore-Tz conjugate (FL_Tz) has been utilized for fluorescent live cell imaging via inverse electron-demand Diels-Alder (iEDDA) type bioorthogonal reactions. However, most FL_Tz strategies rely on a donor-acceptor-type energy transfer mechanism, which limits red-shifting of probes' emission wavelength without deterioration of the fluorescent turn-on/off ratio. To address this constraint, herein we present a monochromophoric design strategy for making a series of FL_Tzs spanning a broad range of emission colors. For the systematic comparison of design strategies with minimized structural differences, we selected indolizine-based emission-tunable Seoul-Fluor (SF) as a model fluorophore system. As a result, by inducing strong electronic coupling between Tz and π-conjugation systems of an indolizine core, we efficiently quench the fluorescence of SF-tetrazine conjugates (SF_Tzs) and achieved more than 1000-fold enhancement in fluorescence after iEDDA reaction with trans-cyclooctene (TCO). Importantly, we were able to develop a series of colorful SF_Tzs with a similar turn-on/off ratio regardless of their emission wavelength. The applicability as bioorthogonal probes was demonstrated with fluorescence bioimaging of innate microtubule and mitochondria using docetaxel-TCO and triphenylphosphonium-TCO in live cells without washing steps. We believe this study could provide new insight for the reliable and generally applicable molecular design strategy to develop bioorthogonal fluorogenic probes having an excellent turn-on ratio, regardless of their emission wavelength.

Full text of DOI:10.1021/jacs.7b10433

Subscriber access provided by UNIVERSITY OF ADELAIDE LIBRARIES
Article
Mono-chromophoric Design Strategy for Tetrazine-based Colorful
Bioorthogonal Probes with a Single Fluorescent Core Skeleton
Youngjun Lee, Wansang Cho, June Sung, Eunha Kim, and Seung Bum Park
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.7b10433 • Publication Date (Web): 14 Dec 2017
Downloaded from http://pubs.acs.org on December 14, 2017
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a free service to the research community to expedite the
dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts
appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been
fully peer reviewed, but should not be considered the official version of record. They are accessible to all
readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered
to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published
in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just
Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor
changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers
and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors
or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
Journal of the American Chemical Society is published by the American Chemical
Society. 1155 Sixteenth Street N.W., Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 11
Journal of the American Chemical Society
1
2
3
4
5
6
7
8
9
Mono-chromophoric Design Strategy for Tetrazine-based Colorful
Bioorthogonal Probes with a Single Fluorescent Core Skeleton
Youngjun Lee1, Wansang Cho1, June Sung2, Eunha Kim*2, Seung Bum Park*1,3
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
1CRI Center for Chemical Proteomics, Department of Chemistry, Seoul National University, Seoul 08826, Korea
2Department of Molecular Science and Technology, Ajou University, Suwon 16499, Korea
3Department of Biophysics and Chemical Biology, Seoul National University, Seoul 08826, Korea
KEYWORDS bioorthogonal reaction, fluorogenic probe, tetrazine, Seoul-Fluor, live cell imaging
ABSTRACT: Fluorogenic bioorthogonal probe is ideal for fluorescent imaging in live cell condition. By taking advantage of
dual functionality of tetrazine (Tz), bioorthogonal reaction unit as well as fluorescence quencher, fluorophore-Tz conjugate
(FLTz) has been utilized for fluorescent live cell imaging via inverse electron-demand Diels-Alder (iEDDA) type bioorthog-
onal reaction. However, most of FLTz strategies rely on donor-acceptor type energy transfer mechanism which limits red-
shifting of probes’ emission wavelength without deterioration of fluorescent turn-on/off ratio. To address this constraint,
herein we present mono-chromophoric design strategy for making a series of FLTzs spanning a broad range of emission
colors. For the systematic comparison of design strategies with minimized structural differences, we selected indolizine-
based emission-tunable Seoul-Fluor (SF) as a model fluorophore system. As a result, by inducing strong electronical cou-
pling between Tz and π-conjugation systems of indolizine core, we efficiently quench the fluorescence of SF-tetrazine con-
jugates (SFTzs) and achieved more than 1000-fold enhancement in fluorescence after iEDDA reaction with trans-cyclooctene
(TCO). Importantly, we were able to develop a series of colorful SFTzs with similar turn-on/off ratio regardless of their
emission wavelength. The applicability as bioorthogonal probes was demonstrated with fluorescence bioimaging of innate
microtubule and mitochondria using docetaxel-TCO and triphenylphosphonium-TCO in live cells without washing steps.
We believe this study could provide new insight for the reliable and generally applicable molecular design strategy to de-
velop bioorthogonal fluorogenic probes having excellent turn-on ratio, regardless of their emission wavelength.
Fluorescent imaging techniques have revolutionized the
way to understand biological systems in nanoscopic,1 mi-
croscopic,2 and macroscopic levels.3 To date, fluorescent
proteins and immunofluorescence techniques have been
most widely used for imaging target of interest (TOI).4
However, the requirement of genetic engineering, fixation,
cell membrane penetration or other treatment conditions
makes these techniques unsuitable for imaging of innate
proteins under the live cell environment, especially for pri-
mary cells or clinical samples. Alternative method is mak-
ing fluorescent conjugate, which binds specifically to TOI
protein.5 The simplest approach for fluorescent conjugate
is the direct coupling of fluorochromes to ligands of TOI
proteins, but this approach can reduce the binding affinity
of ligand-fluorochrome probes or redirect these probes to
off-target proteins in a non-specific manner.6 Moreover,
extensive washing is required to remove an excess amount
of fluorescent conjugates to get the higher signal-to-noise
ratio. By taking advantage of bioorthogonal chemistry
(highly specific and working in aqueous condition),7 re-
searchers now can develop exquisite ligands for protein bi-
oimaging with a minimal perturbation of original binding
affinity, due to much smaller size of bioorthogonal tags for
the ligand modification compared to that of direct fluoro-
phore ligation.8
Figure 1. (a) Schematic representation of fluorescence turn-
on event of fluorophore-tetrazine conjugate (FLTz), induced
by bioorthogonal cycloaddition reaction with trans-cy-
clooctene (TCO). (b) Bi-chromophore type FLTz quenched by
dipole-dipole energy transfer (left) or electron-exchange en-
ergy transfer (right). (c) Reported trends of change in fluores-
cence on/off ratio of FLTz depending on the emission wave-
lengths of fluorophores. (d) Mono-chromophore type FLTz,
quenched by optically inactive S0–S1 transition (dark-state
quenching).
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 2 of 11
Among various bioorthogonal reactions, recent ad- on SF system, we designed SFTz analogs through Tz modi-
fication at the R1, R2, or R3 position of SF to systematically
compare TBET-, FRET-, or mono-chromophore based
FLTzs. SFTzs have fast reaction kinetics with TCO in aque-
ous condition and turn-on/off ratio up to 1000-fold with
full-visible-color emission ranges. With SFTzs, we success-
fully visualized innate microtubule and mitochondria us-
ing TCO-ligand complexes in live cell conditions without
washing steps. To the best of our knowledge, this is the first
report about FLTz turn-on probes with a single core skele-
ton covering a full range of visible color, for the washing-
free fluorescent live-cell imaging of innate TOI proteins
and intracellular organelles.
1
2
3
4
5
6
7
8
vances in inverse electron-demand Diels-Alder (iEDDA)
reaction received an enormous attention because of its su-
perior kinetics and specificity under the physiological con-
dition.9 Based on dual functionality of tetrazine (Tz),
bioorthogonal reactive group10 as well as fluorescence
quencher,10e,10f fluorophore-Tz conjugate (FLTz) has been at
the center of attention.8b,11 FLTzs can selectively react with
trans-cyclooctene (TCO)-ligand complex via iEDDA reac-
tion as a simultaneous bioorthogonal fluorogenic reaction,
which allows fluorescence imaging of multiple intracellu-
lar proteins8a,12 under the live cell condition without wash-
ing steps (Figure 1a).
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Up to now, several approaches have been pursued to
generate the FLTz having better turn-on efficiency with a
wider range of fluorescent emission wavelength.9 From a
molecular architectural point of view, the earlier version of
FLTz has aliphatic linkers between fluorophore (donor) and
Tz (acceptor) for simple conjugation,11a and the fluores-
cence of FLTz is assumed to be quenched by transferring
fluorophore’s excited energy to Tz quencher via long-range
dipole-dipole interaction, also known as Förster resonance
energy transfer (FRET) (Figure 1b, left).13 A few years later,
a new type of molecular design has emerged via direct con-
junction (without aliphatic linkers) of Tz to the flat organic
fluorophores in an out-of-plane shape, due to the steric
hindrance from adjacent substituents,11b–d which is known
as through-bond energy transfer (TBET) strategy.14 Consid-
ering its close proximity (< 10 Å) between donor (D) and
acceptor (A) along with electronically de-coupled struc-
tures, an excited donor of a TBET pair might transfer its
energy to Tz quencher via short-range electron-exchange
mechanism (Figure 1b, right).15 Although FRET- and TBET-
based strategies were successfully applied to several fluo-
rochromes for the development of fluorogenic FLTzs, how-
ever, a significant decrease in fluorescence turn-on/off ra-
tio at longer emission wavelength is a problem yet to be
solved (Figure 1c). Therefore, there is a high demand in al-
ternative molecular design strategies for FLTz having high
turn-on/off ratio independent from FLTz’s emission wave-
length, to succeed high quality multiplex fluorescent imag-
ing of TOI proteins in live cells.
RESULTS AND DISCUSSION
Initial Design and Synthesis. Many of blue or green
color-emitting FLTzs showed remarkable turn-on/off ratio
by taking advantage of donor-acceptor(D-A) type bi-chro-
mophoric design strategy.11b–d However, orange, red, or far-
red emitting FLTzs suffered drastic deterioration of their
on/off ratio,11a,11c,11f implying the turn-on/off efficiency of D-
A type FLTz pairs is presumably dependent on the energy
level of donor fluorophores.17 In other words, fluorophores
with red-shifted emission wavelengths (>550 nm) have a
lower efficiency of transferring their excited energy to Tz,
causing more severe residual background fluorescence
than that of short-wavelength FLTz, in the case of D-A type
bi-chromophoric system. To overcome this wavelength-
dependent quenching efficiency, we envisioned an alterna-
tive molecular design approach, the mono-chromophoric
strategy, which features strong electronical coupling be-
tween Tz and fluorophores for the development of super-
dark FLTz.
Many of Tz derivatives, even with multiple aromatic
groups, are known as non-fluorescent.18 Once excited, Tz
derivatives should undergo non-radiative energy decay
process due to the lowest lying dark-state at S1 (dark-state
quenching),19 presumably originated from non-radiative
n→π* transition.20 In this context, we hypothesized that
unlike previous energy-transfer quenching in D-A type (bi-
chromophoric) FLTz system, which is sensitive to donor’s
energy level, mono-chromophoric FLTz system would have
inherent n→π* transition stemmed from Tz, and it could
allow highly efficient dark-state quenching independent
from donor’s energy level. After the spontaneous iEDDA
reaction of Tz with TCO, irreversible releases of two nitro-
gen atoms from Tz would cause huge changes in non-
bonding orbitals of mono-chromophoric FLTz system.
Herein, we report the development of SFTz, a new series
of FLTz fluorogenic probes using emission-tunable Seoul-
Fluor (SF) fluorescent core skeleton16 via mono-chromo-
phoric design strategy (Figure 1d). Based on our under-
standing of structure-photophysical property relationship
Figure 2. Molecular structures of Seoul-Fluor (SF) and SF-Tetrazine conjugates (SFTzs)
ACS Paragon Plus Environment

Page 3 of 11
Journal of the American Chemical Society
Subsequently, the lowest lying non-radiative n→π* transi- aliphatic amine, 4-(6-methyl-1,2,4,5-tetrazin-3-yl)benzyla-
1
2
3
4
5
6
7
8
tion might be vanished in this state, which allows fluores-
cence turn-on via optically active π→π* transition origi-
nated from the embedded fluorophores after iEDDA reac-
tion. To prove this hypothesis, we designed and synthe-
sized a series of FLTz derivatives sharing a single core skel-
eton, an emission-tunable indolizine-based Seoul-Fluor, to
minimize other factors from structural differences.
mine, with an aid of HATU and DIPEA. The final SFTz prod-
ucts were thoroughly purified by preparative high-perfor-
mance liquid chromatography before checking their pho-
tophysical properties. More detailed information for syn-
thetic procedure and full characterization of all new com-
pounds is available in supplementary information.
Change in Absorption Property of SFTz01–05 upon TCO
Cycloaddition Reaction. To investigate the effect of mo-
lecular design on the electronical orthogonality between
indolizine and Tz, we checked absorption spectra of SFTzs
before and after the bioorthogonal reaction with (E)-cy-
clooct-4-enol (TCO). Before the reaction with TCO, we ob-
served two different absorption peaks around 400–420 nm
and 500–550 nm from SFTz01, SFTz03, SFTz04 and SFTz05
(Figure 3, grey line). On the contrary, SFTz02 has an absorp-
tion peak around 400–420 nm, but indistinguishable ab-
sorption maxima around 500–550 nm (Figure 3c, grey line).
Considering Tz analogs generally have a distinct weak ab-
sorption peak around 500–550 nm (Figure 3a, grey
line),18b,20 this observation indicates that SFTz02 has
stronger coupled states between SF and Tz than others.
Owing to its full-color tunable fluorescence property
and synthetic versatility, SF scaffold can be an ideal fluor-
ophore system to test our molecular design strategy. For
the systematic comparison of each design strategy of FLTz
with SF scaffold, we utilized three distinct modification
sites, the R1, R2, and R3 positions of SF scaffold. As shown
in Figure 2, SFTz01 and SFTz02 were designed to incorporate
Tz chromophore without aliphatic linkers at the R1 and R2
positions, respectively. For SFTz01, Tz-containing aryl moi-
ety at the R1 position can be geometrically tilted from in-
dolizine π-system,16d leading to de-coupling of π-systems
between indolizine and Tz, which is a hallmark of a TBET-
based FLTz. Special attention was paid toward the molecu-
lar architecture of SFTz02, because the π-conjugation sys-
tems of two chromophores, indolizine and Tz, can be
strongly coupled each other due to the negligible rota-
tional barrier of single-bond between indolizine and Tz.
Thus, we hypothesized SFTz02 might behave as a mono-
chromophoric FLTz. In the case of SFTz03–05, we introduced
Tz to SF scaffold through aliphatic linkers at the R1, R2, and
R3 positions, respectively, as analogs of FRET-based FLTzs.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
More clearly, the electronical orthogonality of SFTz ana-
logs could be confirmed by analyzing absorption changes
after iEDDA reaction with TCO. If Tz and fluorophore have
two independent and electronically de-coupled states, the
cycloaddition reaction of SFTz with TCO will cause inde-
pendent dissipation of absorption peak around 500–550
nm, which is originated from Tz’s absorption, while most
of absorption patterns in other wavelength ranges should
be intact. On the other hand, if Tz and indolizine are
strongly coupled and act as a single chromophore, the
iEDDA reaction with TCO will lead to overall hypso-
chromic shift in absorption spectra due to the reduction of
π-conjugation length. After bioorthogonal reaction with
TCO, SFTz01, SFTz03, SFTz04, and SFTz05 showed marginal
to negligible changes in absorption spectra except clear
disappearance of absorption around 500–550 nm (Figure 3,
inset graphs). As described earlier, FRET-based SFTz03–
SFTz05 were constructed via the conjugation of two chro-
mophores, indolizine and Tz, with aliphatic linkers. There-
fore, electronically de-coupled states between SF and Tz
were expected, which leads to negligible changes in ab-
sorption spectra of SFTz03–SFTz05 after iEDDA reaction. In
the case of TBET-based SFTz01, we proposed that Tz moiety
at the R1 position is geometrically tilted and electronically
de-coupled from π-system of indolizine, which was
strongly supported by marginal changes in absorption
spectrum of SFTz01 around 400 nm (indolizine’s π→π* ab-
sorption band) after iEDDA reaction. These observations
confirmed that SFTz01 and SFTz03–SFTz05 have electroni-
cally de-coupled π-systems between two chromophores.
On the other hand, drastic overall changes of SFTz02 in the
absorption spectra and hypsochromic shift of the absorp-
tion maximum (from 415 to 386 nm) upon TCO treatment
clearly indicate that mono-chromophoric molecular de-
sign of SFTz02 results in strong electronical coupling be-
tween two chromophores, indolizine and Tz. Collectively,
we were convinced that the electronical orthogonality of π-
conjugation system between Tz and indolizine is highly de-
pendent on applied molecular design strategies.
For the synthesis of SFTz01 (Scheme 1), we used palla-
dium-mediated C–H activation of indolizine core and in-
corporated Tz moiety at the R1 position in the presence of
3-(4-iodophenyl)-6-methyl-1,2,4,5-tetrazine and Pd(OAc)2
with AgOAc.16d SFTz02 was prepared via direct conversion
of nitrile group at the R2 position of indolizine moiety into
Tz under the microwave irradiation using NH2NH2, ace-
tonitrile, and Zn(OTf)2, followed by acid-mediated oxida-
tion step with NaNO2.21 In cases of SFTz03, SFTz04, and
SFTz05, carboxyl acid moiety at the R1, R2, or R3 positions of
corresponding SF’s was coupled with Tz moiety containing
Scheme 1. Synthetic Scheme of SFTz01−03
N
N
N
N
N
N
N
N
N
BocHN
I
N
BocHN
O
N
, AgOAc
Pd(OAc)2
O
O
N
DMF, 80 o
C
O
SFTz01
N
BocHN
NH2NH
2, CH3
CN
N
BocHN
µ
wave
O
N
,
Zn(OTf)2
N
O
N
then NaNO
2, 1N HCl
N
N
O
CN
N
SFTz02
O
N
O
H
1) LiOH, r.t
THF/MeOH/H2O (2:1:1)
N
BocHN
N
BocHN
N
O
N
N
N
2) HATU, DIPEA, DMF, r.t
O
N
N
O
H2N
O
N
N
SFTz03
N
N
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 4 of 11
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 3. The changes in absorption spectra for Tz-containing molecules before (grey line) and after (blue line) the reaction with
TCO: (a) 3,6-Dimethyl-1,2,4,5-tetrazine, (b) SFTz01, (c) SFTz02, (d) SFTz03, (e) SFTz04, (f) SFTz05. All spectra were measured in ace-
tonitrile:H2O (1:1 = v:v) mixture at room temperature. Final concentration of dye and TCO was 20 µM and 200 µM, respectively.
Change in Fluorescence Property of SFTz01–05 upon
Cycloaddition Reaction with TCO. Next, we investi-
gated fluorescence changes of SFTz01–05 by comparing the
emission spectra before and after (when reached the max-
imum intensity in fluorescence) reacting with TCO. Inter-
estingly, the strong electronical coupled SFTz02 results in
remarkable enhancement of turn-on/off ratio via complete
dark-state quenching (Figure 4b). As shown in Table 1,
SFTz02 showed completely quenched fluorescence property
(quantum yield: 0.2%), while others showed residual fluo-
rescence (quantum yield: up to 4%) before reaction with
TCO. Consequently, the residual off-state fluorescence of
those FRET- or TBET-type SFTzs lead to the moderate
on/off enhancement (10–70 fold) for SFTz01 and SFTz03–05,
but super-dark quenching of SFTz02 lead to the exceptional
enhancement, more than 1,000-fold, of fluorescence on/off
signal. This result suggests that our mono-chromophoric
design strategy can generate a series of effectively
quenched fluorophores by direct conjugation of Tz to the
π-conjugated core structure of fluorophore, which
prompted us to develop a colorful series of mono-chromo-
phoric SFTzs with ensured quenching efficiency even at the
longer emission wavelength.
Reaction Kinetics. Next, we evaluated the reaction kinet-
ics of SFTz series by tracking the change in fluorescence in-
tensity over the time, after the treatment of TCO. As shown
in Table 1 and Figure S1, all SFTz series showed fast reaction
kinetics (k2 = ~10 M-1s-1) in 1:1 acetonitrile/water condition,
which is comparable with widely used bioorthogonal reac-
tions, such as copper-catalyzed azide-alkyne cycloaddi-
tions (CuAAC, >10 M-1s-1)22 or photo-click cycloadditions
(<60 M-1s-1).22b,23
Rational Expansion of Multi-color SFTzs Based on
Mono-chromophoric Strategy. Based on our knowledge
about emission modularity of SF,16b we designed a series of
SFTz02 analogs to span a wide emission range simply by
changing the electronic nature of substituents at the R1
Figure 4. The changes in emission spectra for SFTz01−SFTz05 before (grey line) and after (blue line) the reaction with TCO: (a)
SFTz01, (b) SFTz02, (c) SFTz03, (d) SFTz04, (e) SFTz05. All spectra were measured in acetonitrile:H2O (1:1 = v:v) mixture at room
temperature. Final concentration of dye and TCO was 20 µM and 200 µM, respectively. Each SFTz was excited at the corresponding
largest excitation maxima of the fluorogenic form.
ACS Paragon Plus Environment

Page 5 of 11
Journal of the American Chemical Society
Table 1. Photophysical Properties for SFTzs Before and Af-
ter the Reaction with trans-cyclooctene (TCO).
reduced HOMO-LUMO energy gap of SF system (Figure 5c
1
2
3
4
5
6
7
8
and Table S1), which is expected to induce a bathochromic
shift of emission wavelength compared to that of SFTz02.
We also observed the similar trends in HOMO-LUMO en-
ergy gaps according to the changes of substituents at the R1
position using 4,5-dihydropyridazine form as SFPy analogs
(Figure S2 and Table S2).
Accordingly, we synthesized SFTz06 (R1 = methoxy; calcu-
lated energy gap = 5.846 eV), SFTz07 (amino; 5.736 eV), and
SFTz08 (diethylamino; 5.722 eV) to generate a multi-color
set of bioorthogonal SFTz probes (Figure 6a). The synthetic
procedure for these analogs was identical to that of SFTz02
(see supplementary information). With these new analogs
in hand, we first evaluated their electronical orthogonality
b
c
d
k2
λex
λem
Fold
a
e
Probe
Φoff
Φon
(M-1s-1)
(on/off)f
(nm) (nm)
SFTz01
SFTz02
SFTz03
SFTz04
SFTz05
a,e
0.011
0.002
0.042
0.012
0.017
21
24
15
15
8.5
416
375
401
375
401
532
484
522
493
530
0.168
0.683
0.583
0.616
0.325
20
> 1,000
14
67
24
9
Φ
off
and Φon denote absolute quantum yield of SFTz and
SFPy form, respectively. bSecond-order rate constant measured
at 20 °C. The largest excitation maxima at the given maximal
emission wavelength. Maximal emission wavelength. Fold
enhancement of fluorescence intensity upon reaction with
TCO. All experimental data were measured in acetoni-
trile:H2O (1:1 = v:v) mixture. Final concentration of SFTz and
TCO was 20 µM and 200 µM, respectively. λem, Φoff, and Φon
were measured at the corresponding λex of each SFTz.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
c
d
f
position of mono-chromophoric SFTz. As shown in Figure
5a, we introduced electron-donating substituents guided
by Hammett constant24 [methoxy (σp = -0.27), amino (-
0.66), and diethylamino group (-0.72)] at the R1 position of
SFPy—the corresponding iEDDA reaction product of SFTz
with TCO having 1,4-dihydropyridazine moiety—to induce
a bathochromic shift of emission wavelength of SFTz-TCO
adducts. The trend in emission wavelength changes of SFPy
analogs was predicted by density functional theory (DFT)
calculation at CAM-B3LYP/6-31G* level.25 It is worth men-
tioning that during the continuous monitoring of fluoro-
genic reaction for SFTz02 with TCO using mass spectros-
copy, we observed a progressive disappearance of mass
peak of SFTz02 until the fluorescence signal reached to
maximum, along with the gradual emergence of a new
mass peak, which is matched with that of two different
types of SFTz-TCO adducts, 1,4- and 4,5-dihydropyridazine
forms. In this study, we mainly considered 1,4-dihydro-
pyridazine isomer as a representative fluorogenic SFPy on
the basis of previous reports.11a,11b,11g As shown in Figure 5b,
electron-donating groups at the R1 position can raise the
energy level of the highest occupied molecular orbital
(HOMO) more than that of the lowest unoccupied molec-
ular orbital (LUMO). Therefore, the substitution of meth-
oxy, amino, and diethylamino groups results in the
Figure 6. (a) Molecular structures of SFTz06 (left), SFTz07
(middle), and SFTz08 (right). (b) The changes in absorption
spectra for SFTz06−08, before (grey) and after (blue) the reac-
tion with TCO. (c) The changes in emission spectra for
SFTz06−08, before (grey) and after (blue) the reaction with
TCO. Final concentration of dye and TCO was 20 µM and 200
µM, respectively. Each SFTz was excited at the corresponding
largest excitation maxima of the fluorogenic form.
Figure 5. (a) A structure of representative fluorescence on-
state SF analog, SFPy, generated from cycloaddition reaction of
SFTz02 with TCO, and Hammett constant values (σp) of H,
OCH3, NH2, and NEt2 functional groups. (b) Calculated
HOMO and LUMO energy values (DFT, CAM-B3LYP/6-31G*)
of SFPy having H, OCH3, NH2, and NEt2 groups at the R1 posi-
tion. (c) The changes in the HOMO-LUMO energy gap of
SFPys with corresponding functional groups at the R1 position.
Figure 7. (a) Normalized emission spectra of SFTz02, SFTz06,
SFTz07, and SFTz08 after the reaction with TCO (SFTzs 20 µM,
TCO 200 µM). (b) Photographic images of each SFTz irradiated
at 365 nm, before and after reacting with TCO.
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 6 of 11
Table 2. Photophysical Properties for Mono-chromo-
phoric SFTzs Before and After the Reaction with TCO.
sulting mono-chromophoric SFTzs, including SFTz02, cover
1
2
3
4
5
6
7
8
blue to orange visible-color emission (Figure 7 and Table
2). The most remarkable achievement of our mono-chro-
mophoric SFTz system is the complete quenching of resid-
ual fluorescence at the off-state, regardless of emission
wavelength (Figure 6c and Table 2). Due to complete dark-
state quenching, fluorescence on/off ratio of all mono-
chromophoric SFTzs showed remarkable triple or quadru-
ple-digit-fold enhancement (600–1000 fold), independent
from their emission wavelength, even in the case of SFTz08
with emission wavelength of >580 nm (Table 2).
b
c
d
k2
λex
λem
Fold
a
e
Probe
Φoff
Φoff
(M-1s-1)
24
(on/off)f
(nm) (nm)
SFTz02
0.002
375
383
390
401
484
505
562
581
0.683
0.485
0.064
0.045
> 1,000
> 1,000
> 600
SFTz06 0.001
SFTz07 0.002
23
22
22
SFTz08 0.001
> 600
9
a,e
Φ
off
and Φon denote absolute quantum yield of SFTz and
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
SFPy form, respectively. bSecond-order rate constant measured
at 20 °C. cThe largest excitation maxima at the given maximal
TD-DFT calculation. To decipher the fluorescence turn-
on mechanism and photophysical property of SFTz02 and
its analogs (SFTz06–08), we conducted time-dependent
(TD)-DFT calculation (CAM-B3LYP/6-31G*)25 with energy
minimized conformers of SFTzs, and its corresponding
iEDDA reaction products having 1,4-dihydropyridazine
form (SFPy). Our major concern was the probability of op-
tical S0–S1 transition at the lowest-lying 1st singlet excited
state, which is known as the most important electronic
state for fluorescence phenomenon according to Kasha’s
rule.26 In this regard, geometry optimization of the 1st ex-
cited state for mono-chromophoric SFTzs and SFPys was
conducted. Then, molecular orbitals and oscillator
strength values (f)27—the optical transition probability—
between S0 and S1 states were investigated using quantum
mechanical calculation. As shown in Figure 8a and Table
S3, the dominant orbitals contributing to S0–S1 transition
of all mono-chromophoric SFTzs were expected to have
d
f
emission wavelength. Maximal emission wavelength. Fold
enhancement of fluorescence intensity upon reaction with
TCO. All experimental data were measured in acetoni-
trile:H2O (1:1 = v:v) mixture. Final concentration of SFTz and
TCO was 20 µM and 200 µM, respectively. λem, Φoff, and Φon
were measured at the corresponding λex of each SFTz.
between indolizine and Tz by checking the changes in ab-
sorption spectra, before and after TCO treatment (Figure
6b). Similar with SFTz02, all three compounds showed dis-
tinct hypsochromic shifts of absorption maxima (425→393,
436→399, and 457→409 nm for SFTz06, SFTz07, and SFTz08,
respectively), which clearly suggested strong electronical
coupling between Tz and indolizine aromatic π-system.
Upon the reaction with TCO, newly synthesized SFTzs
showed decent reaction kinetics (>20 M-1s-1), similar to that
of SFTz02 (Figure S3). Emission maxima of SFTz06 (505 nm),
SFTz07 (562 nm), and SFTz08 (581 nm) were well matched-
with DFT calculation (Figure S4 and Figure S5), and the re-
Figure 8. Molecular orbital distribution, vertical transition energy, and oscillator strength values (f) of SFTz02, SFTz06, SFTz07, and
SFTz08, obtained by TD-DFT calculation (CAM-B3LYP/6-31G*) of corresponding first excited state optimized structures. (a) The
quenched SFTz compounds before the reaction with TCO; (b) The fluorogenic SFPy products after the formation of TCO-adducts.
Mainly contributing orbital of S0−S1 for each compound was illustrated. H and L stand for HOMO and LUMO, respectively.
ACS Paragon Plus Environment

Page 7 of 11
Journal of the American Chemical Society
Table 3. Photophysical Properties for SFTzs Before and Af-
ter the Reaction with TCO.g
1
2
3
4
5
6
7
8
9
b
c
(nm)
401
d
k2
λex
λem
Fold
a
e
Probe
Φoff
Φon
(M-1s-1)
22
(on/off)f
(nm)
581
SFTz08
SFTz09
SFTz10
a,e
0.001
0.005
0.013
0.045
0.016
0.028
> 600
15
418
608
625
30
Figure 9. Molecular structures of SFTz08, SFTz09, and SFTz10
1.2
438
3
for the direct comparison of mono- and bi-chromophoric de-
sign strategies on SFTz. SFTz08, SFTz09, and SFTz10 are designed
as long-emission wavelength analogs for the SFTz02 (mono-
chromophoric SFTz) and SFTz04–05 (bi-chromophoric SFTz),
respectively.
Φ
off
and Φon denote absolute quantum yield of SFTz and
SFPy form, respectively. bSecond-order rate constant measured
at 20 °C. The largest excitation maxima at the given maximal
c
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
d
f
emission wavelength. Maximal emission wavelength. Fold
enhancement of fluorescence intensity upon reaction with
TCO. Experimental data were measured in acetonitrile:H2O
(1:1 = v:v) mixture for SFTz08 and SFTz09, and in acetonitrile for
SFTz10. Final concentration of SFTz and TCO was 20 µM and
200 µM, respectively. λem, Φoff, and Φon were measured at the
corresponding λex of each SFTz.
n→π* type transition.20 Based on orbital distribution of the
lowest 1st excited state of all SFTzs, we confirmed that non-
bonding orbitals from the nitrogen atom-rich Tz signifi-
cantly contribute to n→π* transition within the molecule.
On top of that, the f values for all SFTzs are very close to
zero, suggesting that optical transition between S0 and S1 is
almost forbidden.27 Therefore, low optical transition possi-
bility for S0–S1 transition strongly indicates that mono-
chromophoric SFTzs would lose their excited energy via
non-radiative decay process. Note that, this is a completely
different quenching mechanism from TBET- or FRET-
based systems, whose quenching performance is governed
by energy transfer efficiency from the excited fluorophore
to the electronically de-coupled Tz quencher.
fluorophores becomes “allowed” after iEDDA reaction and
it makes SFPys fluorescent. We also observed almost iden-
tical results in the case of 4,5-dihydropyridazine isomer as
SFPys (Figure S6 and Table S5). In summary, computational
calculation indicates that direct incorporation of Tz to SF
makes optically inactive n→π* transition (low f value) as
major S0–S1 transition for SFTzs, which quenches the fluo-
rescence process of chromophores. Destruction of Tz in
SFTzs, induced by bioorthogonal iEDDA reaction with TCO,
makes optically active π→π* transition dominant (high f
value) for S0–S1 transition in SFPys, which turns on the mol-
ecules’ fluorescence.
After the cycloaddition reaction of SFTz with TCO, re-
moval of two nitrogen atoms as well as extinction of aro-
matic system of Tz might cause a huge transformation of
non-bonding orbitals of SFTz. Therefore, optically inactive
n→π* transition at the lowest lying state is no longer valid
after iEDDA reaction, but π→π* transition becomes the
most important transition between S0 and S1 states in the
all SFPys (Figure 8b and Table S4). Besides, f values for S0–
S1 transition in SFPys (from 0.41 to 0.53) are significantly
higher than that of S0–S1 transition in SFTzs (from 0.0031 to
0.0040). In other words, radiative energy loss of excited
Direct Comparison of Mono- vs Bi-chromophoric
SFTzs with Long Emission Wavelength. Next, we pur-
sued direct comparison of mono- vs bi-chromophoric de-
sign strategy by analyzing turn-on/off ratio at the longer
emission wavelength. For the comparison with SFTz08, the
representative long wavelength compound for mono-chro-
mophoric SFTz, we simply introduced an electron-donating
diethylamino group at the R1 position of SFTz04 and SFTz05,
Figure 10. Fluorogenic bioorthogonal imaging of innate microtubules with SFTz in a fixed-cell condition. HeLa human cervical
carcinoma cells were treated with DMSO (a, c) or Dox-TCO (b, d) for 1 h. TO-PRO-3 Iodide (1 μM) was treated for nucleus staining.
After brief washing with PBS, SFTz02* (a, b) or SFTz08* (c, d) was treated to the cells (Final concentration = 10 µM). Immediately
after addition of the probes, the cell images were observed with fluorescence microscope without washing. Selective microtubule
staining was further confirmed with immunofluorescence using α-tubulin antibody (g–h). Scale bar, 10 µm.
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 8 of 11
to yield SFTz09 and SFTz10, respectively, as representative SFTz02* and SFTz08*, respectively (Figure S9). We first used
1
2
3
4
5
6
7
8
long-wavelength bi-chromophoric SFTzs (Figure 9). It turns
out that after TCO treatment, electronically de-coupled bi-
chromophoric systems, SFTz09–10, showed noticeable de-
preciation of turn-on/off ratio (67→30-fold for SFTz04→
SFTz09 and 25→3-fold for SFTz05→SFTz10) as the fluoro-
phore’s emission coverage was shifted to the longer emis-
sion area (Table 1, Table 3, Figure S7, and Figure S8). On
the contrary, mono-chromophoric design strategy allowed
SFTz08 to preserve compelling turn-on/off efficiency (over
600 fold), comparable to that of SFTz02 (over 1000 fold), at
the long emission area. The slight reduction in turn-on/off
ratio of SFTz08 might be originated from innate low quan-
tum yield of its fluorogenic form. Collectively, this compar-
ison experiment demonstrated the unique advantage of
mono-chromophoric design strategy, which guarantees ex-
cellent fluorescence off/on ratio, independent from the
emission wavelength of fluorophores. By taking advantage
of wavelength-independent quenching mechanism, differ-
ent from conventional energy-transfer type quenching
(FRET or TBET), mono-chromophoric approach could pro-
vide a robust and generally applicable molecular design
strategy for the development of multi-color bioorthogonal
fluorogenic probes with high turn-on ratio.
docetaxel-TCO conjugate (Dox-TCO) for fluorescent bi-
oimaging of microtubule (Figure 10). HeLa human cervical
carcinoma cells were fixed and incubated with Dox-TCO
for 1 h and washed briefly with PBS. Immediately after the
addition of SFTz02* or SFTz08* to the cells, bright cytoplas-
mic fluorescent signal was generated and the intracellular
fluorescent images resemble typical spindle structures of
microtubules (Figure 10b, d). Control experiments con-
firmed that there is a negligible background signal inside
the cells and no background signal outside of the cells even
with longer exposure time (Figure 10a, c). Therefore, we
could achieve a sharp contrast in fluorescent signal be-
tween cytoplasmic and outside of fixed cells without wash-
ing steps to remove excess SFTz probes. Further immuno-
fluorescence with α-tubulin antibody confirmed the spe-
cific staining of microtubules with Dox-TCO/SFTzs (Figure
10e–h).
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Considering the importance of mitochondrial phenotyp-
ing,28 we then explored the use of SFTzs in fluorescence im-
aging of mitochondria in live cells (Figure 11). After 1 h in-
cubation in the presence or absence of triphenylphospho-
nium (TPP)-TCO, HeLa cells were washed and treated with
SFTz02* or SFTz08*. In the absence of TPP-TCO, no specific
staining of mitochondria nor no background signal was ob-
served, even without washing steps (Figure 11a, i). On the
other hand, TPP-TCO/SFTz02* or TPP-TCO/SFTz08* treat-
ment produced crisp fluorescent mitochondrial images
with an exceptional resolution (Figure 11b,e, j). Plot inten-
sity values of pixels along a red line (Figure 11c, d) revealed
a sharp contrast between mitochondrion and other areas,
Bio-Application. Lastly, we applied SFTz02 and SFTz08 for
cellular imaging to demonstrate the usefulness of SFTzs as
multi-color bioorthogonal fluorogenic imaging probes. For
the practical usage of SFTz probes, we changed the R3 posi-
tion of SF system—proven as orthogonal to the fluores-
cence property16c,16e—by introducing the water soluble
moiety such as piperazine and carboxyl group to yield
Figure 11. Fluorogenic bioorthogonal imaging of mitochondria with SFTz02* and SFTz08* in the live cell condition without washing
steps. HeLa human cervical carcinoma cells were treated with DMSO (a, i) or triphenylphosphonium (TPP)-TCO (10 µM, b, c, e,
g, h, j, l) with MitoTracker-Deep Red (f–h, k, l) for 40 min. After brief washing with PBS, SFTz02* (a, b, c, e, g, h) or SFTz08* (i, j, l)
were treated to the cells (Final concentration = 10 µM). Images were immediately observed with fluorescence microscope without
washing. Plot intensity values of pixels along a red line on (c) was analyzed with Image J program (d). ROI, region of interest.
ACS Paragon Plus Environment

Page 9 of 11
Journal of the American Chemical Society
Notes
which validated complete fluorescence quenching of SFTzs
1
2
3
4
5
6
7
8
The authors declare no competing financial interest.
under the physiological culture-media condition (zero in-
tensity was observed in the extracellular area). To confirm
the selectivity of TPP-TCO/SFTz02* and TPP-TCO/SFTz08*
for mitochondria, HeLa cells were co-treated with com-
mercially available mitochondria staining dye, Mito-
Tracker Deep Red, and we observed excellent co-localiza-
tion of the signal between MitoTracker Deep Red and
SFTz02* or SFTz08*. Together, highly efficient and selective
fluorescent staining of intracellular TOI protein and intra-
cellular organelle was possible without multiple washing
steps using SFTzs in live cell condition.
ACKNOWLEDGMENTS
This work was supported by the Creative Research Initiative
Grant (2014R1A3A2030423), Bio & Medical Technology Devel-
opment Program (2012M3A9C4048780) and Basic Research
Program (2009-0093826 and 2016R1C1B2014699) through the
National Research Foundation of Korea (NRF) funded by the
Korean Government (Ministry of Science, ICT & Future Plan-
ning). Y.L. is grateful for his predoctoral fellowship awarded
by the BK21 Plus Program. W.C. is grateful for the NRF-2016-
Fostering Core Leaders of the Future Basic Science Pro-
gram/Global Ph.D. Fellowship Program (2016H1A2A1908141).
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
CONCLUSION
In summary, mono-chromophoric design strategy enables
the development of fluorogenic probe SFTzs, having ex-
traordinary turn-on/off ratio covering a full visible color
range, for the live cell imaging via rapid and catalyst-free
bioorthogonal reaction. Strong electronical coupling be-
tween SF core and Tz chromophore causes dark-state
quenching, which is completely different from conven-
tional FRET- or TBET-type energy transfer mechanism.
Wavelength independent dark-state quenching enables ef-
ficient widening of the emission color range of SFTzs, vali-
dating generality and robustness of this mono-chromo-
phoric strategy. We successfully demonstrate an applica-
bility of SFTzs for bioorthogonal fluorescent imaging of TOI
proteins and subcellular organelles in live cell conditions
without washing steps.
REFERENCES
(1) (a) Fernández-Suárez, M.; Ting, A. Y. Nat. Rev. Mol. Cell Biol.
2008, 9, 929–943. (b) Hell, S. W. Nat. Methods 2009, 6, 24–32.
(2) (a) Lavis, L. D.; Raines, R. T. ACS Chem. Biol. 2008, 3, 142–
155. (b) Lavis, L. D.; Raines, R. T. ACS Chem. Biol. 2014, 9, 855–866.
(c) Dean, K. M.; Palmer, A. E. Nat. Chem. Biol. 2014, 10, 512–523.
(3) (a) Weissleder, R. Nat. Biotechnol. 2001, 19, 316–317. (b)
Ntziachristos, V.; Ripoll, J.; Wang, L. V.; Weissleder, R. Nat. Bio-
technol. 2005, 23, 313–320.
(4) (a) Chudakov, D. M.; Matz, M. V.; Lukyanov, S.; Lukyanov,
K. A. Physiol. Rev. 2010, 90, 1103–1163. (b) Odell, I. D.; Cook, D. J.
Invest. Dermatol. 2013, 133, e4.
(5) (a) van Dam, G. M.; Themelis, G.; Crane, L. M. A.; Harlaar,
N. J.; Pleijhuis, R. G.; Kelder, W.; Sarantopoulos, A.; de Jong, J. S.;
Arts, H. J. G.; van der Zee, A. G. J.; Bart, J.; Low, P. S.; Ntziachristos,
V. Nat. Med. 2011, 17, 1315–1319. (b) Choi, H. S.; Gibbs, S. L.; Lee, J.
H.; Kim, S. H.; Ashitate, Y.; Liu, F.; Hyun, H.; Park, G.; Xie, Y.; Bae,
S.; Henary, M.; Frangioni, J. V. Nat. Biotechnol. 2013, 31, 148–153.
(c) Xue, L.; Karpenko, I. A.; Hiblot, J.; Johnsson, K. Nat. Chem.
Biol. 2015, 11, 917–923. (d) Kim, E.; Yang, K. S.; Kohler, R. H.;
Dubach, J. M.; Mikula, H.; Weissleder, R. Bioconjug. Chem. 2015,
26, 1513–1518. (e) Lee, M. M.; Gao, Z.; Peterson, B. R. Angew. Chem.
Int. Ed. 2017, 56, 6927–6931.
(6) Kim, E.; Yang, K. S.; Giedt, R. J.; Weissleder, R. Chem. Com-
mun. 2014, 50, 4504–4507.
(7) (a) Sletten, E. M.; Bertozzi, C. R. Angew. Chem. Int. Ed.
2009, 48, 6974–6998. (b) Patterson, D. M.; Nazarova, L. A.;
Prescher, J. A. ACS Chem. Biol. 2014, 9, 592–605.
When applying this mono-chromophoric strategy, a dihy-
dropyridazine moiety—product of TCO-Tz reaction—will
directly affect the π-electronic system of the resulting
fluorophores after TCO-FLTz reaction. Considering photo-
physical properties of our mono-chromophoric probes,
this group would act like an EWG. Therefore, our mono-
chromophoric strategy will be a suitable to the fluoro-
phores having specific position, where an EWG is essential
to exhibit fluorescent properties. In this regard, the EWG
position of charge-transfer type fluorophores, such as
NBD, acedan or coumarin derivatives, might be a potential
candidate for a tetrazine incorporation. We believe mono-
chromophoric design strategy will be useful to provide
highly reliable and generally applicable method for the de-
velopment of bioorthogonal fluorogenic probes with excel-
lent turn-on/off ratio in vitro and in vivo.
(8) (a) Reiner, T.; Earley, S.; Turetsky, A.; Weissleder, R. Chem-
BioChem 2010, 11, 2374–2377. (b) Cserép, G. B.; Herner, A.; Kele, P.
Methods Appl. Fluoresc. 2015, 3, 042001.
(9) Oliveira, B. L.; Guo, Z.; Bernardes, G. J. L. Chem. Soc. Rev.
2017, 46, 4895–4950.
(10) (a) Blackman, M, L.; Royzen, M.; Fox, J. M. J. Am. Chem.
Soc. 2008, 130, 13518–13519. (b) Devaraj, N. K.; Weissleder, R.;
Hilderbrand, S. A. Bioconjug. Chem. 2008, 19, 2297–2299. (c) Tay-
lor, M. T.; Blackman, M. L.; Dmitrenko, O.; Fox, J. M. J. Am. Chem.
Soc. 2011, 133, 9646–9649. (d) Knall, A.-C.; Slugovc, C. Chem. Soc.
Rev. 2013, 42, 5131–5142. (e) Selvaraj, R.; Fox, J. M. Curr. Opin. Chem.
Biol. 2013, 17, 753–760. (f) Mayer, S.; Lang, K. Synthesis 2017, 49,
830–848.
(11) (a) Devaraj, N. K.; Hilderbrand, S.; Upadhyay, R.;
Mazitschek, R.; Weissleder, R. Angew. Chem. Int. Ed. 2010, 49,
2869–2872. (b) Carlson, J. C. T.; Meimetis, L. G.; Hilderbrand, S.
A.; Weissleder, R. Angew. Chem. Int. Ed. 2013, 52, 6917–6920. (c)
Wu, H.; Yang, J.; Šečkutė, J.; Devaraj, N. K. Angew. Chem. Int. Ed.
2014, 53, 5805–5809. (d) Meimetis, L. G.; Carlson, J. C. T.; Giedt,
R. J.; Kohler, R. H.; Weissleder, R. Angew. Chem. Int. Ed. 2014, 53,
7531–7534. (e) Knorr, G.; Kozma, E.; Herner, A.; Lemke, E. A.; Kele,
P. Chem. Eur. J. 2016, 22, 8972–8979. (f) Wieczorek, A.; Werther,
P.; Euchner, J.; Wombacher, R. Chem. Sci. 2017, 8, 1506–1510. (g)
ASSOCIATED CONTENT
Supporting Information.
General experimental information, supporting figures, experi-
mental procedure for live cell fluorescence image, 1H and 13C spec-
tra of new compounds, references.
This material is available free of charge via the Internet at
http://pubs.acs.org.”
AUTHOR INFORMATION
Corresponding Author
*ehkim01@ajou.ac.kr
*sbpark@snu.ac.kr
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 10 of 11
Kozma, E.; Estrada Girona, G.; Paci, G.; Lemke, E. A.; Kele, P.
Chem. Commun. 2017, 53, 6696–6699.
1
(12) (a)Budin, G.; Yang, K. S.; Reiner, T.; Weissleder, R. Angew.
Chem. Int. Ed. 2011, 50, 9378–9381. (b) Yang, K. S.; Budin, G.;
Reiner, T.; Vinegoni, C.; Weissleder, R. Angew. Chem. Int. Ed.
2012, 51, 6598–6603.
(13) (a) Förster, T. Ann. Phys. 1948, 2, 55–75. (b) Förster, T. Disc.
Faraday Soc. 1959, 27, 7–17.
2
3
4
5
6
7
8
9
(14) Jiao, G.-S.; Thoresen, L. H.; Burgess, K. J. Am. Chem. Soc.
2003, 125, 14668–14669. (b) Bandichhor, R.; Petrescu, A. D.; Vespa,
A.; Kier, A. B.; Schroeder, F.; Burgess, K. J. Am. Chem. Soc. 2006,
128, 10688–10689.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(15) (a) Dexter, D. L. J. Chem. Phys. 1953, 21, 836–850. (b) Otsuki,
J.; Kanazawa, Y.; Kaito, A.; Islam, D.-M. S.; Araki, Y.; Ito, O. Chem.
Eur. J. 2008, 14, 3776–3784. (c) Hurenkamp. J. H. Tuning Energy
Transfer Between Chromophores Switchable Molecular Photonic
Systems. Ph.D. Thesis, University of Groningen, 2008.
(16) (a)Kim, E.; Koh, M.; Ryu, J.; Park, S. B. J. Am. Chem. Soc.
2008, 130, 12206–12207. (b) Kim, E.; Koh, M.; Lim, B. J.; Park, S. B.
J. Am. Chem. Soc. 2011, 133, 6642–6649. (c) Lee, Y.; Na, S.; Lee, S.;
Jeon, N. L.; Park, S. B. Mol. BioSyst. 2013, 9, 952–956. (d) Choi, E.
J.; Kim, E.; Lee, Y.; Jo, A.; Park, S. B. Angew. Chem. Int. Ed. 2014,
53, 1346–1350. (e) Kim, E.; Lee, Y.; Lee, S.; Park, S. B. Acc. Chem.
Res. 2015, 48, 538–547.
(17) (a) Anslyn, E. V.; Dougherty, D. A. Modern Physical Organic
Chemistry; University Science Books, Sausalito, CA. 2005. (b)
Turro, N. J.; Ramamurthy, V.; Scaiano, J. C. Principles of Molecular
Photochemistry: An Introduction, 1st edition; University Science
Books: Sausalito, CA, 2008.
(18) (a) Gong, Y-H.; Miomandre, F.; Méallet-Renault, R.; Badré,
S.; Galmiche, L.; Tang, J.; Audebert, P.; Clavier, G. Eur. J. Org.
Chem. 2009, 6121–6128. (b) Clavier, G.; Audebert, P. Chem. Rev.
2010, 110, 3299–3314.
(19) Escudero, D. Acc. Chem. Res. 2016, 49, 1816–1824.
(20) Plugge, M.; Alain-Rizzo, V.; Audebert, P.; Brouwer, A. M. J.
Photochem. Photobiol. A Chem. 2012, 234, 12–20.
(21) Yang, J.; Karver, M. R.; Li, W.; Sahu, S.; Devaraj, N. K. An-
gew. Chem. Int. Ed. 2012, 51, 5222–5225.
(22) (a) Presolski, S. I.; Hong, V.; Cho, S.-H.; Finn, M. G. J. Am.
Chem. Soc. 2010, 132, 14570–14576. (b) Lang, K.; Chin, J. W. ACS
Chem. Biol. 2014, 9, 16–20.
(23) (a) Song, W.; Wang, Y.; Qu, J.; Lin, Q. J. Am. Chem. Soc.
2008, 130, 9654–9655. (b) Song, W.; Wang, Y.; Qu, J.; Madden, M.
M.; Lin, Q. Angew. Chem. Int. Ed. 2008, 47, 2832–2835. (c) Yu, Z.;
Pan, Y.; Wang, Z.; Wang, J.; Lin; Q. Angew. Chem. Int. Ed. 2012, 51,
10600–10604.
(24) Hansch, C.; Leo, A.; Taft, R. W. Chem. Rev. 1991, 91, 165–
195.
(25) (a) Adamo, C.; Jacquemin, D. Chem. Soc. Rev. 2013, 42, 845–
856. (b) Liu, X.; Qiao, Q.; Tian, W.; Liu, W.; Chen, J.; Lang, M. J.;
Xu, Z. J. Am. Chem. Soc. 2016, 138, 6960–6963.
(26) (a) Kasha, M. Disc. Faraday Soc. 1950, 9, 14–19. (b) Klán, P.;
Wirz, J. Photochemistry of Organic Compounds: From Concepts to
Practice; Wiley-Blackwell, 2009.
(27) (a) Hilborn, R. C. Am. J. Phys. 1982, 50, 982–986. (b) Lee,
Y.; Jo, A.; Park, S. B. Angew. Chem. Int. Ed. 2015, 54, 15689–15693.
(c) Valeur, B.; Berberan-Santos, M. N. Molecular Fluorescence:
Principles and Applications, Second Edition; Wiley-VCH: Wein-
heim, 2012.
(28) Alirol, E.; Martinou, J. C. Oncogene 2006, 25, 4706–4716.
ACS Paragon Plus Environment

Page 11 of 11
1
Journal of the American Chemical Society
2
3
SYNOPSIS TOC
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
ACS Paragon Plus Environment