Superior Fluorogen-Activating Protein Probes Based on 3-Indole-Malachite Green

Article abstract of DOI:10.1021/acs.orglett.7b02055

A series of novel fluorogenic dyes based on 3-indole-Malachite Green, MGs 5-7, have been developed that are dark in solution but highly fluorescent when bound to the cognate reporter, fluorogen-activating protein (FAP). Significantly, the new MGs 5-7 prob

Full text of DOI:10.1021/acs.orglett.7b02055

Letter
pubs.acs.org/OrgLett
Superior Fluorogen-Activating Protein Probes Based on 3‑Indole−
Malachite Green
Qingyang Zhang,†,‡,∥ Qinghua Wang,†,‡,∥ Ying Sun, Limin Zuo, Verena Fetz, and Hai-Yu Hu*
‡
§
⊥
,†,‡
†
‡
State Key Laboratory of Bioactive Substances and Function of Natural Medicine and Beijing Key Laboratory of Active Substances
Discovery and Drugability Evaluation, Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical
College, Beijing 100050, China
§
Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100050,
China
⊥
̈
Department of Chemical Biology, Helmholtz-Zentrum fur Infektionsforschung (HZI), Inhoffenstrasse 7, 38124 Braunschweig,
Germany
*
S Supporting Information
ABSTRACT: A series of novel fluorogenic dyes based on 3-indole−
Malachite Green, MGs 5−7, have been developed that are dark in
solution but highly fluorescent when bound to the cognate reporter,
fluorogen-activating protein (FAP). Significantly, the new MGs 5−7
probes are superior to the traditional MG 1 with high fluorescent
efficiency and low toxicity to cells while maintaining the large
“
(
pseudo-Stokes” shifts (Δλ = λ − λ ) and the malachite green
ex
em
MG)-like fluorescence OFF−ON switching mechanism in both live
mammalian cells and bacterial cells when bound to FAP.
luorescence imaging is a powerful approach for noninvasive
F
and real-time visualization and tracking of biomolecules and
biological processes in living cells and animals. The develop-
1
ment of convenient and high-efficiency fluorophores is essential
2
for the rapidly developing imaging technology. Toward this end,
numbers of fluorophores have been developed, including
3
4
5
6
7
naphthalimide, styryl, coumarin, BODIP,Y and cyanine.
However, to achieve a high signal-to-noise ratio for visualization
and quantification, extensive and time-consuming washing of
cells to remove free fluorophore is required, rendering the
continuous monitoring of biological processes virtually impos-
Figure 1. (a) Framework of malachite green (MG). (b) Absorbance
spectrum of MG 1 in PBS.
8
sible. This has led to a push in the development of
fluoromodule-based reporter/probe systems that feature ro-
weak fluorescence when excited by the Y band, only the X band
was used as an excitation band for fluorescence imaging studies
(λex 630 nm/λem 650 nm) and exhibited small Stokes shifts (Δλ =
20 nm). A small Stokes shift can cause self-quenching and
measurement error by excitation light and scattered light. To
address this issue, great effects have been dedicated, and three
bustly activatable probes that could be optimized for real-time
9
fluorescence imaging.
Fluorogen-activating proteins (FAPs) are a class of
fluoromodules that bind a nonfluorescent small molecule in a
10
noncovalent fashion and exhibit fluorescence. FAP technology
was first introduced in 2008 and has been successfully used to
series of Fo
̈
rster resonance energy transfer (FRET)-based MG
11
fluorogens with large pseudo-Stokes shifts have been devel-
monitor receptor trafficking in live cells. MG (Figure 1a) is one
such fluorogen whose fluorescence is strongly quenched by
conformational flexibility in solution. FAP/MGs display two
excitation (absorbance) bands, a main band (X) contributed by
13
oped. However, some problems are encountered with them
because they contain complicated structures, require multistep
reactions, and give low yields, and FRET strategies do not
14
12
entirely eliminate the self-quenching and inner-filter effects.
rings A and B and a minor band (Y) contributed by ring C. For
instance, the most commonly used cell-permeable FAP
fluorogen, FAP/MG 1 (see Figure 3), exhibits two excitation
bands, the minor Y band with maximum excitation at 470 nm and
the main X band with maximum excitation at 630 nm (Figure
Fluorescent labels that have a large Stokes shift, low toxicity,
and are bright and cell permeable are preferred for biological
15
imaging. Herein, we demonstrate that a series of novel MG
1b). Both of the excitation Y and X bands give an emission
Received: July 5, 2017
maximum at 650 nm. However, since FAP/MG 1 displayed very
©
XXXX American Chemical Society
A
DOI: 10.1021/acs.orglett.7b02055
Org. Lett. XXXX, XXX, XXX−XXX

Organic Letters
Letter
analogues with rational modifications of the Y band contributor
ring C show improved quantum yield, large “pseudo-Stokes”
shift (Δλ = λ − λ = 115 nm), and lower cell toxicity,
improving the application of FAP/MGs in live-cell fluorescence
microscopy. To our knowledge, use of a single MG fluorogen to
achieve a large wavelength difference between maximum
excitation and emission peaks by internal charge transfer
Theoretical calculations suggested that increasing the charge
transfer and enhancing the π-conjugation of the bottom half
could improve the fluorescence efficiency of MGs. Therefore, we
hypothesized modulation of the photophysical properties of MG
via fusion of the aromatic rings to the ring C phenyl unit.
Six new MG derivatives with naphthalene (MG 2), quinolone
(MG 3), or an indole group (MGs 4−7) and, for comparison,
control MG fluorogen MG 1 (Figure 3) were synthesized in high
ex
em
(
ICT) mechanism has not been reported.
A large Stokes shift is desirable in fluorescent labeling
applications of dyes, as it reduces self-quenching effects and
16
interference from an excitation source. Exploration of multi-
fluorophores with energy donor−acceptor architectures to
achieve large pseudo-Stokes shifts of MG fluorogens has been
1
7
reported. We intend to explore an alternative approach to
increase the “pseudo-Stokes” shift in MGs by stabilizing the
excited state through structural relaxation. ICT has been utilized
with the aim of achieving a large wavelength difference between
the maximum excitation and emission peaks in common organic
1
8
systems, while this strategy has not been adopted in MGs and
their analogues.
Quantum chemical calculations have been proven to be an
important tool for investigation of the relationships between
structures and spectral properties of the organic molecules and
for the interpretation of experimental results. Lately, time-
dependent density functional theory (TD-DFT) appears to be
one of the most successful methods in terms of the balance
between accuracy and speed, and it has been widely used in
electronic transition energy predictions. To gain insight into
the relationship between molecular structure and photophysical
properties, TD-DFT calculations were performed on MG 1 using
B3LYP/TZP (basis set) in ADF 2016. Figure 2 shows the
Figure 3. Structures of malachite green (MG) synthesized.
yields (see the synthetic procedures in the SI for details).We first
explored the effects of the aromatic substitution on the
fluorogen’s absorption spectrum, FAP_dH6.2 binding ability,
and fluorescent spectrum in PBS. The photophysical properties
of fluorogen MGs 2−7 along with control fluorogen MG 1 are
summarized in Table 1 and Figures S3 and 4.
19
As expected, the presence of a conjugated polycyclic aromatic
moiety on ring C could impact on the absorption of X or/and Y
band. However, the fluorescence quantum yields of MGs 2−4 are
much lower than that of MG 1 after binding with FAP_dH6.2
and showed no fluorescence or only weak fluorescence. The
fluorescence quantum yields of MGs 5−7 are comparable to MG
1
and showed a good fluorescence turn-on response to
FAP_dH6.2. Moreover, MGs 5−7 retained the remarkable
selectivity of FAP over various tested human and mice proteins in
vitro (Figure S5). Crystallography of the FAP/MG complex
reveals that MG binds FAP as left-handed propeller conformers
with somewhat variable rotation angles along the C2 axis (Figure
12,20
S1).
TD-DFT calculations showed that ring C of MG 1 and
MGs 5−7 were left-handed propellers, while MGs 2−4 were
right-handed propellers (Figure S2). We inferred that only left-
handed propeller MG fluorogen could become fluorescent when
binding with FAP. Figure 4a shows the UV/vis spectra of MG 1
and 3-indole conjugated fluorogens MGs 5−7. Compared with
MG 1, MGs 5−7 were found to exhibit efficient ICT emission (Y
band), resulting in large “pseudo-Stokes” shifts along with
significantly enhanced emission intensity. Concentration−effect
relationships between MGs and FAP_dH6.2 were observed
Figure 2. Frontier orbital of MG 1 and its electron transition from
HOMOs to LUMO estimated by TD-DFT calculations to produce X
and Y bands.
(
Figure S6−9). When excited at a long wavelength band (X
electron transition of MG 1 from the highest occupied molecular
orbitals (HOMOs) to the lowest unoccupied molecular orbital
band), maximum fluorescence intensities showed an unambig-
uous enhancement from MGs 5−7 compared to MG 1 (2213,
2007, 2384 vs 1283).
(
LUMO). The HOMO and LUMO densities of MG 1 are mainly
located on the top half of the fluorogen, whereas HOMO−1 and
HOMO−2 densities are mainly localized on ring C on the down
half of MG 1. Theoretical calculations showed the ICT transition
in MG 1 appears at the excitation Y band attributed to HOMO−
More interestingly, fluorescence intensities of MG 5, MG 6,
and MG 7 were 3.8, 2.6, and 4.1 times higher compared with MG
1 at short Y band excitation, respectively (Table 1 and Figure S4).
In general, the electron-donating substituent on the 3-indole
group could increase the fluorescence intensity. The “pseudo-
Stokes” shifts of MG 5 (115 nm), MG 6 (125 nm), and MG 7
(115 nm) were shorter than MG 1 (180 nm); nevertheless, they
2
to LUMO transition; meanwhile, both HOMO → LUMO and
HOMO−1 → LUMO transitions contribute to the excitation of
12
X band, which are in good agreement with the literature.
B
DOI: 10.1021/acs.orglett.7b02055
Org. Lett. XXXX, XXX, XXX−XXX

Organic Letters
Letter
g
Table 1. Photophysical Properties of Fluorogen MGs 1−7
a
b
c
ε × 10 (M cm−1
3
−1
d
e
fluorogen
MG 1
λab (nm)
λex (nm)
λem (nm)
)
FI
ΦF
K (pM)
D
460
470
630
510
630
650
650
665
665
ND
670
630
630
635
635
630
630
13.0
30.7
9.8
278
1283
77
0.154
0.296
ND
1.533
6
15
505
20
MG 2
0.813
6
31.6
38.4
35.6
25.2
31.6
24.8
30.8
25.2
398
0.105
ND
f
MG 3
MG 4
MG 5
640
625
515
ND
ND
ND
625
515
590
510
600
515
575
192
0.068
0.215
0.153
0.224
0.196
0.140
0.112
2.061
1.487
1055
2213
716
5
85
510
85
515
75
MG 6
MG 7
2.346
1.721
5
2007
1147
2384
5
32.0
a
b
c
d
e
λ = absorption maximum. λ = excitation maximum. λ = emission maximum. FI = fluorescence intensity. Φ = fluorescence quantum yield.
ab
ex
em
F
f
g
ND= not detectable. Solvent conditions: 0.01 M PBS containing 1.0 % DMSO and 2.0 μM FAP_dH6.2, pH 7.4.
Figure 4. (a) Absorption spectra of MG 1 and MGs 5−7. (b)
Fluorescence spectra of MG 5, MG 5/FAP, MG 5/FAP + 1.0 equiv of
MG 1, and MG 5/FAP + 4.0 equiv of MG 1.
Figure 5. Confocal microscopic images of HEK293A_GFP_ dH6.2
cells incubated with MG 1, MG 5, and MG 5 for 15 min and then 1.0
equiv of MG 1 for 15 min, respectively. Blue fluorescence signals from
Hoechst 33258 stain; green from GFP (λex = 488 ± 20 nm, λem = 500 ±
5 nm); red from MG/FAP (λex = 514 ± 20 nm, λem = 630 ± 25 nm);
merge from bright-field and blue, green, red fluorescence signals. Scale
bar = 20 μm.
are sufficient for avoiding the overlap of excitation peak and
emission peak. MG 5 was later used in the cellular studies.
MG 5 is fluorescence-off in solution, but intensely fluorescent,
with the excitation and emission maxima at 515 and 630 nm,
respectively, upon binding to FAP_dH6.2. However,
FAP_dH6.2/MG 1 shows no fluorescence when excited at 515
nm, while the binding abilities of MG 1 and MG 5 to FAP_dH6.2
are comparable. We hypothesized that the MG 5/FAP_dH6.2/
MG 1 system could exhibit an OFF−ON−OFF fluorescence
intensity change in the fluorescence spectra. Figure 4b and Figure
S10 show the fluorescence spectral changes of MG 5 before and
after addition of FAP_dH6.2 and the changes during the titration
of MG 1. Accordingly, the fluorescence brightness of MG 5
increased by 817-fold upon binding with FAP_dH6.2 and then
exhibited 90% fluorescence decrease after addition of 4.0 equiv of
MG 1. The fluorescence modulation of MG 5 with FAP_dH6.2
and MG 1 clearly demonstrates an OFF−ON−OFF switch as
expected.
2
that MG 5 with large “pseudo-Stokes” shifts allowed for no-wash
imaging of FAP inside mammalian cells and bacterial cells with
reasonable incubation time. Similar to the PBS solution, the MG
5/FAP/MG 1 system could exhibit an OFF−ON−OFF
fluorescence switch. The fluorescent OFF mode of
FAP_dH6.2 expressed cells could be switched ON in the
presence of MG 5 and then exhibited ∼50% fluorescence
decrease after addition of 1.0 equiv of MG 1.
To localize FAP and further verify the selectivity of MG 5 to
FAP in cells, HEK293A cells transiently expressing FAP_GFP
fusion protein were generated by transfection of the plasmid
pcDNA3.1 encoding FAP_GFP and incubated with MG 5.
Confocal images of the GFP channel present the location of the
FAP_GFP protein in the cell. MG 5/FAP signal confocal
microscopy analysis reveals a high level of colocalization of the
GFP signal with the MG 5/FAP signal, which indicates the
remarkable selectivity of MG 5 to FAP. Z-Stack confocal imaging
studies showed a clear intracellular distribution of both of the
GFP and MG 5/FAP signals, including the nucleus in living cells
(Figure S14 and S15). Moreover, it is noteworthy that MG 5 was
found to be much less toxic than MG 1 in three tested human cell
lines (for details, see the SI, Table S1).
Inspired by the large “pseudo-Stokes” shifts results and an
OFF−ON−OFF switch for FAP_dH6.2 imaging in PBS
solution, we investigated the applicability of MG 5 for live cell
imaging. In this study, FAP_dH6.2 expressing mammalian cells
HEK293A and bacterial cells E. coli were generated and used,
2
1
respectively. Typically, the HEK293A_dH6.2 cells and E.
coli_dH6.2 bacterial strains were separately incubated with 2 μM
of MG 5 or MG 1 for 15 min at 37 °C and subsequently subjected
to confocal microscopy for fluorescence imaging (Figure 5). As
shown in Figures S12−S14, strong fluorescence emission was
observed after incubation of MG 5 with FAP_dH6.2 expressing
mammalian and bacterial cells, whereas similar cellular
incubation with MG 1 led to no obvious fluorescence when a
In summary, we have rationally designed and synthesized a
series of novel 3-indole−Malachite Green-based FAP fluorogens,
MGs 5−7. The important features of this class of FAP fluorogens
514 nm laser excitation was applied. These results demonstrated
C
DOI: 10.1021/acs.orglett.7b02055
Org. Lett. XXXX, XXX, XXX−XXX

Organic Letters
Letter
are the efficient internal charge transfer resulting significant
fluorescence enhancements, remarkable large “pseudo-Stokes”
shifts, low toxicity to cells, as well as very fast onset in response to
FAP in both live mammalian cells and bacterial cells. They have
the potential to be an alternative to FRET-based MG fluorogens
with large pseudo-Stokes shifts in multiplexing applications with
FAP imaging.
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ASSOCIATED CONTENT
Supporting Information
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The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.orglett.7b02055.
2
Synthetic procedures and spectral data for all new
compounds, UV−vis, FI spectral studies, cell cytotoxic
studies, and confocal imaging studies (PDF)
1
(
Zakel, K. L.; Adler, S.; Fitzpatrick, J. A.; Woolford, C. A.; Yan, Q.;
Vasilev, K. V.; Berget, P. B.; Bruchez, M. P.; Jarvik, J. W.; Waggoner, A.
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AUTHOR INFORMATION
Corresponding Author
■
(c) Zhang, M.; Chakraborty, S. K.; Sampath, P.; Rojas, J. J.; Hou, W.;
*E-mail: haiyu.hu@imm.ac.cn.
Saurabh, S.; Thorne, S. H.; Bruchez, M. P.; Waggoner, A. S. J. Clin. Invest.
2015, 125, 3915−3927.
ORCID
(12) Szent-Gyorgyi, C.; Stanfield, R. L.; Andreko, S.; Dempsey, A.;
Hai-Yu Hu: 0000-0002-4760-6865
Author Contributions
∥Q.Z. and Q.W. contributed equally.
Ahmed, M.; Capek, S.; Waggoner, A. S.; Wilson, I. A.; Bruchez, M. P. J.
Mol. Biol. 2013, 425, 4595−4613.
(13) (a) Naganbabu, M.; Perkins, L. A.; Wang, Y.; Kurish, J.; Schmidt,
B. F.; Bruchez, M. P. Bioconjugate Chem. 2016, 27, 1525−1531.
Notes
(b) Szent-Gyorgyi, C.; Schmidt, B. F.; Fitzpatrick, J. A.; Bruchez, M. P. J.
The authors declare no competing financial interest.
Am. Chem. Soc. 2010, 132, 11103−11109. (c) Yushchenko, D. A.;
Zhang, M.; Yan, Q.; Waggoner, A. S.; Bruchez, M. P. ChemBioChem
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(
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14) Fan, J.; Hu, M.; Zhan, P.; Peng, X. Chem. Soc. Rev. 2013, 42, 29−
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15) (a) Chang, M. C.; Chantzis, A.; Jacquemin, D.; Otten, E. Dalton.
ACKNOWLEDGMENTS
■
We thank Prof. Mark Bro
̈
nstrup (Helmholtz Centre for Infection
Research) for providing the FAP_dH6.2 plasmid and for
valuable discussions. Financial support from the Sino-German
research project GZ 1271, PUMC Youth Fund (3332016056),
and Beijing Nova Program (Z16111000490000) is acknowl-
edged.
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Parvez, M.; McDonald, R. Angew. Chem., Int. Ed. 2011, 50, 12214−
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