A NIR-emitting cyanine with large Stokes shifts for live cell imaging: Large impact of the phenol group on emission

Article abstract of DOI:10.1039/c9cc06831g

There are a limited number of near-infrared (NIR) emitting (λ_em = 700-900 nm) molecular probes for imaging applications. A NIR-emitting probe that exhibits emission at ～800 nm with a large Stokes shift was synthesized and found to exhibit excellent selectivity towards mitochondria for live-cell imaging. The photophysical properties were attributed to an excited cyanine structure via intramolecular charge transfer (ICT) involving a phenol group.

Full text of DOI:10.1039/c9cc06831g

Showcasing research from Professor Yi Pang’s laboratory,
Department of Chemistry, University of Akron, Akron,
Ohio, USA.
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Volume 55 Number 88 14 November 2019 Pages 13187–13316
A NIR-emitting cyanine with large Stokes shifts for live cell
imaging: large impact of the phenol group on emission
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There are a limited number of near-infrared (NIR) emitting
molecular probes available for imaging applications. A
fluorescent cyanine with NIR emission (λem ≈ 800 nm)
and large Stokes shift was found to exhibit excellent
selectivity towards mitochondria for live-cell imaging.
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A NIR-emitting cyanine with large Stokes shifts for
live cell imaging: large impact of the phenol
group on emission†
Cite this: Chem. Commun., 2019,
5, 13223
5
Received 2nd September 2019,
Accepted 26th September 2019
Dipendra Dahal,
Michael Konopka
Lucas McDonald, Sabita Pokhrel, Sailaja Paruchuri,
and Yi Pang
*
DOI: 10.1039/c9cc06831g
rsc.li/chemcomm
1
6,17
There are a limited number of near-infrared (NIR) emitting (kem
=
and cyanine as fluorophores.
However, these fluorophores
700–900 nm) molecular probes for imaging applications. A NIR-emitting have small Stokes shifts (Dl E 10–60 nm), which prevent the
probe that exhibits emission at B800 nm with a large Stokes shift optimum collection of fluorescence signals and hamper their
1
8–20
was synthesized and found to exhibit excellent selectivity towards broad applications in bioimaging.
Therefore, developing
mitochondria for live-cell imaging. The photophysical properties fluorophores with large Stokes shifts is desirable, in order to
were attributed to an excited ‘‘cyanine structure’’ via intramolecular realize the full potential of sensitive fluorescence imaging. In
charge transfer (ICT) involving a phenol group.
addition, NIR-emitting fluorescent probes with significant Stokes
shifts are also useful for single excitation multicolor live-cell
21
Visualization of specific organelles and organs by fluorescent imaging, which plays an important role in biological studies.
agents has been the center of recent research because of their
high sensitivity, low cost, and high specificity. The applications (Fig. 1), which gives NIR emission with very large Stokes shifts
depend on the probe’s ability to target the specific organelles (or (Dl E 234 nm).
Recently, our group synthesized NIR emitting probe 1
1–3
2
2,23
The molecular structure of 1 integrates an
organs) without perturbation of their morphology and physiology. ‘‘excited state intramolecular proton transfer (ESIPT)’’ group
For example, mitochondria are double layer membrane-bound with a hemicyanine unit via sharing a meta-phenylene bridge.
organelles which exist in eukaryotic cells and are responsible for Interestingly, probe 1 exhibits excellent selectivity toward intra-
a
the generation of the entire cellular energy in an animal cell in the cellular lysosomes, due to the low pK (= 5.72) of its phenolic
4–7
form of ATP. The function of mitochondria is dependent on the
electrochemical gradient potential (Dc ) across mitochondrial
m
membranes or electrochemical proton motive force (Dp), which is
a combination of mitochondrial membrane potential and mito-
chondrial pH gradient generated by a respiratory electron trans-
4,7–9
port chain (ETC).
Damage and dysfunction of mitochondria
are associated with various diseases including aging, cancer,
neurodegenerative diseases, Alzheimer’s disease, Huntington
7,10–12
disease, diabetes, and Parkinson’s disease.
Most of the fluorescent probes absorb and emit in the visible
region, and their application is limited by high phototoxicity and
1
3
interference from background fluorescence.
NIR-emitting
1
4
fluorescent probes are useful for bioimaging applications, as
they suffer less auto-fluorescence from the biological samples
1
5
and can achieve deeper tissue penetration. Current NIR-
emitting probes include BODIPY, benzofuran, rhodamine,
Department of Chemistry, The University of Akron, Akron, OH 44325, USA.
E-mail: yp5@uakron.edu
†
Electronic supplementary information (ESI) available: Synthesis and character-
1
ization of 2 and 3; H NMR and mass spectral data; and additional UV-vis
Fig. 1 Chemical structures of probes 1–3 and some commercial mito-
absorption and fluorescence data. See DOI: 10.1039/c9cc06831g
chondria staining dyes.
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proton and its ability to give a large fluorescence turn on in an
À
+
22
acidic environment (ArO + H - ArOH). It should be noted
that the effective chromophores in 1 can be approximated by
hemicyanine and ESIPT fragments, due to p-conjugation inter-
2
4
ruption by a meta-phenylene bridge. In an effort to further
examine the structural impact on the fluorescence properties,
herein, we illustrate the synthesis of 2, in which the effective
chromophore is nearly the same as that in 1. In probe 2, the
phenolic proton can dissociate but does not undergo any
intramolecular proton transfer reaction. Interestingly, probe 2
exhibits NIR emission (lem E 800 nm) with a very large Stokes
shift (e.g. Dl E 161 nm), in sharp contrast to conventional Scheme 1 The proposed transformation of excited 2 to its keto form 4,
and their equilibrium via proton dissociation.
cyanine dyes (Dl E 10–60 nm). Although the phenolic proton
a
of 2 is acidic (pK E 4), probe 2 exhibits remarkable selectivity
toward mitochondria when used to stain biological cells.
The desired compound 2 was synthesized by the reaction of
2
2
-hydroxy-5-methylisophthalaldehyde (dialdehyde) with 1-ethyl-
-methylbenzothiazolium salt in 75% yield (see ESI† for detail)
and was characterized by NMR and mass spectrometry. The
1
H NMR spectrum of 2 gave sharp doublet signals at 8.0 and
8.4 ppm, which were attributed to newly formed vinyl bonds (Fig. 2).
The high-resolution mass spectrum revealed m/z 483.1119 that was
the deprotonated state of 2 without counter ions (Fig. S4, ESI†); calcd
for 483.1553. Compound 3 (m/z 454.1500, ESI,† Fig. S7; calcd for
454.1526) was also synthesized to aid the spectral study.
The UV-vis absorption spectra of 2 revealed the absorption
peaks at labs E 395 and a broad shoulder at 456 nm, similar to
that observed for 1 due to the presence of identical effective
2
2
Fig. 3 UV-vis absorption (broken line) and emission spectra (solid line) of
chromophores in 1 and 2. However, a new absorption peak at
2
2 2
and 3 in CH Cl . The excitation wavelengths were 687 nm for 2 and
l
l
max E 717 nm was observed for 2. The absorption bands at
abs E 395 and 456 nm were attributed to the enol form (or acid
3
95 nm for 3.
form) 2a, while the band at 717 nm was attributed to its base
form 2b (Scheme 1). Since only one set of resonance signals (e.g. substituents, as the NIR emission band was not observed
1
for H
b
and H
c
) was observed in the H NMR spectrum, for the model compound 3 (labs = 395 nm and lem = 456 nm;
compound 2 was assumed to be present predominantly in its Fig. S12 and S13, ESI†).
enol form 2a in a polar aprotic solvent.
Examination of 2 in different solvents revealed that its NIR
Interestingly, the emission of 2 showed lem E 784 nm (Fig. 3 absorption peak was affected by solvent polarity (labs = 728 nm
and Table 1). The results indicated a strong synergistic inter- in DMSO, but 589 nm in aqueous solution) (Table 1 and Fig. S8,
action between the two hemicyanine substituents in 2, since ESI†). However, the emission wavelength of 2 was less affected
the lem value of 2 was significantly red-shifted from that of 1 (lem = 770–804 nm) (Fig. S9, ESI†). DFT calculations with the
2
2
(l
em E 681 nm). The observed spectral red-shift was appar- TD-SCF method revealed that the keto tautomer of 2 in CH
2 2
Cl
ently dependent on the presence of the –OH group between the showed absorption (labs = 714 nm) and emission (lem = 780 nm)
(
(
Fig. S10, ESI†), which closely matched the experimental values
abs = 717 nm and lem = 784 nm).
l
Table 1 Photophysical properties of compounds 2 and 3 in different
solvents
Compound 2
Compound 3
Solvents
l
abs
l
em
(Ffl
)
l
abs
l
em
(Ffl)
DCM
DMF
DMSO
EtOH
MeCN
392, 456, 717
388, 489, 728
388, 728
385, 654
382, 702
382, 633
382, 589
784
803
803
798
795
794
770
0.11
0.04
0.04
0.03
0.04
0.02
0.01
395
383
387
384
382
383
380
456
470
467
460
460
460
460
0.004
0.002
0.002
0.002
0.002
0.002
0.002
1
Fig. 2 H NMR spectrum of 2 in DMSO-d
6
, showing the resonance signals MeOH
of aromatic protons (the alkyl region is omitted for clarity).
Water
1
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Fig. 5 Fluorescence spectra of 2 (10 mM) in different pH buffers in water
(
excitation at 448 nm). The inset shows the UV-absorption of the corres-
ponding solution.
Fig. 4 Fluorescence spectra of 2 at room temperature and low tempera-
ture, with lex of 450 nm (dotted lines) and 650 nm (solid lines).
It should be notable that there was a significant difference
between the optical properties of 1 and 2. Under alkaline
The observed NIR emission from 2 could be attributed to the conditions (e.g., pH = 9–11), the resulting phenoxide from 1 was
22
formation of its keto form 4 (Scheme 1). Traditionally, one non-fluorescent as ESIPT was no longer operative. However, the
would assume that 4b is the species responsible for the NIR phenoxide anion of 2 was still fluorescent, although the fluorescence
emission. An interesting question is where the protonated keto intensity was higher at acidic pH (Fig. 5). Since the phenolic proton
form 4a would give emission. In order to shed some light on of 2 was quite acidic, the formation of the phenoxide anion at
their emissive properties, we decided to freeze the dilute physiological pH allowed the excitation of 2 at a longer wavelength,
solution of 2 by liquid nitrogen, which would eliminate the thereby raising the prospect for bioimaging applications. Evaluation
dynamic impact of chemical equilibrium between 2a and 2b. of 2 also revealed that the probe was insensitive to different anions
À
Thus, a solution of 2 (in EtOH) in a quartz tube was cooled by such as acetate (ACO ), adenosine monophosphate (AMP), adeno-
immersing it into liquid nitrogen in a quartz Dewar, and the sine diphosphate (ADP), adenosine triphosphate (ATP), pyropho-
2
À
2À
fluorescence emission spectra were acquired by exciting the sphate (PPi), biphosphate (HPO ), thiosulfate (S O ), perchlorate
4
2
3
À À
À
sample at 450 nm (Fig. S14, ESI†) and 650 nm (Fig. S15, ESI†) (ClO
at different temperatures. At low temperature (at À189 1C), and some biothiols like cysteine (Cys), glutathione (GSH), and
emission of 2 became more intense and blue-shifted to lem bovine serum albumin (BSA) (Fig. S19a and S20a, ESI†). Considering
00 nm (Fig. 4), as the probe molecules were frozen in a rigid the high abundance of glutathione in cells, compound 2 was treated
4 3 2 2
), bisulfite (HSO ), fluoride (F ), hydrogen peroxide (H O )
E
7
solvent matrix (EtOH m.p. = À112 1C). However, when the enol with very high concentration of GSH (up to 10 mM), the optical
form 2a was excited selectively at 450 nm, the emission spectra properties of compound 2 were not affected by the use of 1 mM
revealed only one peak that had similar lem when the sample (50 equivalents) of GSH. However, absorbance was blue shifted
was excited at 650 nm (lex for keto form 2b). The result indicated (Fig. S19b, ESI†) and emission intensity decreased (Fig. S20b,
that the enol form 2a also gave NIR emission.
ESI†) with the addition of 5 mM (250 equivalents) concentration
The pH response of 2 was examined by acquiring the optical of GSH.
spectra in different pH buffer solutions in water. In strongly
The fluorescence properties of 2 encouraged us to seek their
acidic buffer pH = 1–3, 2 exhibited only one absorption band at potential applications for bioimaging. Cell viability studies by
385 nm (Fig. 5 and Fig. S17, ESI†), indicating that the equili- performing MTT assay showed the half-maximal cell inhibitory
brium was completely shifted to 2a (Scheme 1). As pH increased, concentration (IC50) of 24.72 mM for 2 (Fig. S21, ESI†), indicating its
an additional absorption peak was observed at B593 nm, which low toxicity. When using 2 to stain normal human lung fibroblast
could be attributed to the deprotonation of the phenolic proton. (NHLF) cells, confocal microscopy imaging (Fig. 6B and Fig. S22,
a
The pK of 2 was determined to be 4.0 by the Boltzmann fitting ESI†) displayed fluorescence signals in a non-uniform pattern,
(Fig. S18, ESI†), showing that the phenolic proton was quite indicating that the dye might bind to intracellular organelles
acidic as a consequence of two electron-withdrawing benzo- selectively. Co-staining of 2 with MitoTracker Green FM showed
thiazolium groups. Interestingly, the fluorescence of 2 revealed good colocalization (Fig. 6C). The results confirmed that 2 exhibited
only one major emission peak at lem E 788 nm in the entire pH excellent selectivity to the mitochondria, with a Pearson’s correlation
range examined. When pH = 1–3, probe 2 was expected to be in coefficient 0.79 (Fig. 6C and F). The results indicated that 2 was
the enol form 2a, as revealed from its UV-vis spectra. Upon selective toward mitochondrial organelles, despite the fact that
excitation, 2a could undergo the intramolecular charge transfer the probe would give higher fluorescence in an acidic environ-
(
ICT), which led to its keto tautomer 4a (Scheme 1). A notable ment (pK
feature was that the keto form 4a was only formed in the excited Probe 2 could be considered as a ‘‘functional mitochondrial
state, giving the fluorescence at lem E 788 nm with a very large probe’’ which is accumulated into mitochondria due to the
a
= 4.01, and Fig. 5).
Stokes shift.
m
potential gradient (Dc ) of the mitochondrial matrix, as suggested
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We acknowledge support from NIH (Grant No. 1R15GM126438-
0
1A1) and partial support from Coleman Endowment from The
University of Akron. We thank Savannah Snyder for acquiring mass
spectrometric data.
Conflicts of interest
There are no conflicts to declare.
References
1
T. Nagaya, Y. A. Nakamura, P. L. Choyke and H. Kobayashi, Front.
Oncol., 2017, 7, 314.
2
M. Grossi, M. Morgunova, S. Cheung, D. Scholz, E. Conroy,
M. Terrile, A. Panarella, J. C. Simpson, W. M. Gallagher and
D. F. O’Shea, Nat. Commun., 2016, 7, 10855.
Fig. 6 Normal human lung fibroblast (NHLF) cells co-stained with 2 (1 mM)
and MitoTracker green (200 nM), viewed in green (A) and NIR channel (B),
merged A and B (C) and bright field (D). (E) The plot of relative intensity vs.
distance for probe 2 and MitoTracker Green for the region in the white line
in (A) and (B), and (F) correlation plot between MitoTracker Green and 2.
Excitation/emission lex/lem = 488/525 nm for MitoTracker Green, and
3 A. C. Sedgwick, L. Wu, H.-H. Han, S. D. Bull, X.-P. He, T. D. James,
J. L. Sessler, B. Z. Tang, H. Tian and J. Yoon, Chem. Soc. Rev., 2018,
47, 8842–8880.
4
N. Zhao, S. Chen, Y. Hong and B. Z. Tang, Chem. Commun., 2015, 51,
3599–13602.
S. Gandre-Babbe and A. M. van der Bliek, Mol. Biol. Cell, 2008, 19,
402–2412.
1
5
6
40/(680–735) nm for 2.
2
6
7
J. R. Friedman and J. Nunnari, Nature, 2014, 505, 335.
Z. Xu and L. Xu, Chem. Commun., 2016, 52, 1094–1119.
by using 2 in fixed cells (Fig. S24, ESI†). Compound 2 was also
treated in different cell lines of human oligodendrocytes
8 E. Gottlieb, S. M. Armour, M. H. Harris and C. B. Thompson, Cell
Death Differ., 2003, 10, 709.
9
S. W. Perry, J. P. Norman, J. Barbieri, E. B. Brown and H. A. Gelbard,
Biotechniques, 2011, 50, 98–115.
(MO.3.13 cells, Fig. S23, ESI†), the colocalization of 2 with
MitoTracker Green demonstrated that there was proper overlapping 10 Y. Liu, J. Zhou, L. Wang, X. Hu, X. Liu, M. Liu, Z. Cao, D. Shangguan
and W. Tan, J. Am. Chem. Soc., 2016, 138, 12368–12374.
1 B. Liu, M. Shah, G. Zhang, Q. Liu and Y. Pang, ACS Appl. Mater.
Interfaces, 2014, 6, 21638–21644.
between MitoTracker Green and compound 2 with a Pearson’s
correlation coefficient of 0.67 which indicated that probe 2 was
1
selective to mitochondrial organelles in the live oligodendrocyte 12 L. Long, M. Huang, N. Wang, Y. Wu, K. Wang, A. Gong, Z. Zhang and
J. L. Sessler, J. Am. Chem. Soc., 2018, 140, 1870–1875.
3 K. Singh, A. M. Rotaru and A. A. Beharry, ACS Chem. Biol., 2018, 13,
cells. It should be noted that fluorescence imaging was acquired
with a 680–735 nm emission filter (due to the availability), and
1
1785–1798.
brighter imaging could be observed if the emission filter was near 14 S. Wang, Y. Fan, D. Li, C. Sun, Z. Lei, L. Lu, T. Wang and F. Zhang,
Nat. Commun., 2019, 10, 1058.
5 E. I. Altıno ˇg lu, T. J. Russin, J. M. Kaiser, B. M. Barth, P. C. Eklund,
M. Kester and J. H. Adair, ACS Nano, 2008, 2, 2075–2084.
the peak fluorescence at B788 nm. The results pointed that 2 could
be a useful molecular probe for bioimaging.
1
In summary, NIR (lem E 800 nm) emitting probe 2 was 16 S. Zhang, T. Wu, J. Fan, Z. Li, N. Jiang, J. Wang, B. Dou, S. Sun,
F. Song and X. Peng, Org. Biomol. Chem., 2013, 11, 555–558.
7 X. Zhang, Q. Sun, Z. Huang, L. Huang and Y. Xiao, J. Mater.
Chem. B, 2019, 7, 2749–2758.
synthesized, which showed large Stokes shifts via the ICT
mechanism. Probe 2 possessed an acidic phenol group with
1
pK
a
= 4.01, which allowed the formation of the keto-form to 18 W. Hu, H. Ma, B. Hou, H. Zhao, Y. Ji, R. Jiang, X. Hu, X. Lu, L. Zhang,
Y. Tang, Q. Fan and W. Huang, ACS Appl. Mater. Interfaces, 2016, 8,
facilitate ICT interaction in the excited state. This extended the
conjugation, thereby shifting the absorption and emission to
the NIR region. The acidic phenol group in the meta-phenylene
bridge plays a vital role in the optical properties of 2, as
compound 3 without the phenol group gave weak emission at
a much shorter wavelength (lem E 470). Probe 2 with double
positive charges was assumed to play a decisive role in the
observed mitochondrial selectivity, due to Coulombic attraction
toward the negative potential gradient of the mitochondrial
matrix. Low cytotoxicity of 2 and its attractive NIR emission
suggest that the probe could have promising application in live-
cell imaging.
1
2039–12047.
1
2
2
9 B. Wang, X. Cui, Z. Zhang, X. Chai, H. Ding, Q. Wu, Z. Guo and
T. Wang, Org. Biomol. Chem., 2016, 14, 6720–6728.
0 W. Sun, S. Guo, C. Hu, J. Fan and X. Peng, Chem. Rev., 2016, 116,
7768–7817.
1 K. D. Piatkevich, B. P. English, V. N. Malashkevich, H. Xiao,
S. C. Almo, R. H. Singer and V. V. Verkhusha, Chem. Biol., 2014,
2
2 D. Dahal, L. McDonald, X. Bi, C. Abeywickrama, F. Gombedza,
M. Konopka, S. Paruchuri and Y. Pang, Chem. Commun., 2017, 53,
3697–3700.
3 Y. Guo, D. Dahal, Z. Kuang, X. Wang, H. Song, Q. Guo, Y. Pang and
A. Xia, AIP Adv., 2019, 9, 015229.
4 Q. Chu, Y. Pang, L. Ding and F. E. Karasz, Macromolecules, 2003, 36,
3848–3853.
1, 1402–1414.
2
2
2
1
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