Molecular Insight into Long-Wavelength Fluorogenic Dye Design: Hydrogen Bond Induces Activation of a Dormant Acceptor

Article abstract of DOI:10.1002/chem.201504133

The detection of chemical or biological analytes in response to molecular changes relies increasingly on fluorescence methods. Therefore, there is a substantial need for the development of improved fluorogenic dyes. In this study, we demonstrated how an intramolecular hydrogen bond activates a dormant acceptor through a charge induction between phenolic hydrogen and a heteroaryl nitrogen moiety. As a result, a new fluorochrome is produced, and the molecule exhibits a strong fluorescent emission. When the strength of the hydrogen bonding was increased by conformational locking, the obtained dye emitted at longer wavelengths and fluoresced under physiological conditions. The dye was implemented in a turn-ON system responsive to hydrogen peroxide. The molecular insight provided by this study should assist in the design of fluorescent dyes that are suitable for in vitro and in vivo applications.

Full text of DOI:10.1002/chem.201504133

DOI: 10.1002/chem.201504133
Communication
&
Fluorescent Dyes
Molecular Insight into Long-Wavelength Fluorogenic Dye Design:
Hydrogen Bond Induces Activation of a Dormant Acceptor
Einat Kisin-Finfer, Orit Redy-Keisar, Michal Roth, Ronen Ben-Eliyahu, and Doron Shabat*[a]
inflammation model in mice. The modular structure of the
Abstract: The detection of chemical or biological analytes
QCy7 fluorochrome enabled us to develop a library of dye
in response to molecular changes relies increasingly on
compounds that can be used for turn-ON probe design.[14,15]
fluorescence methods. Therefore, there is a substantial
It is tremendously difficult to predict the fluorescence char-
need for the development of improved fluorogenic dyes.
acteristics of a given dye, since there are numerous pathways
In this study, we demonstrated how an intramolecular hy-
for non-radiative decay in which an excited molecule can lose
drogen bond activates a dormant acceptor through
its energy. Non-radiative electron decay can be significantly de-
a charge induction between phenolic hydrogen and a het-
creased by locking the excited molecule in a planar conforma-
eroaryl nitrogen moiety. As a result, a new fluorochrome is
tion. Here we report a new study that provides molecular in-
produced, and the molecule exhibits a strong fluorescent
sight into an intramolecular hydrogen-bond bridge, between
emission. When the strength of the hydrogen bonding
a donor and a dormant acceptor. By conjugation with an addi-
was increased by conformational locking, the obtained
tional acceptor, the hydrogen bonding is harnessed to produce
dye emitted at longer wavelengths and fluoresced under
a long-wavelength fluorogenic dye.
physiological conditions. The dye was implemented in
The general molecular structure of a dye activated through
a turn-ON system responsive to hydrogen peroxide. The
hydrogen bonding is illustrated in Figure 1. The dye is com-
molecular insight provided by this study should assist in
posed of a latent phenol donor conjugated at the para posi-
the design of fluorescent dyes that are suitable for in vitro
tion to an active acceptor moiety and at the ortho position to
and in vivo applications.
a dormant acceptor (structure I). Upon formation of an intra-
molecular hydrogen bond between the donor and the dor-
mant acceptor (II), three functions are achieved: 1) the dor-
The detection of chemical or biological analytes, in response
to molecular changes,[1,2] relies increasingly on fluorescence.[3–6]
Thus, there is a demand for more sensitive, more specific, and
more versatile fluorescent molecules that can be used for in
vitro and in vivo applications.[7–9] Such molecules should have
a high physiological stability, high quantum yields of fluores-
cence, long emission wavelengths, large Stokes shifts, and
mant acceptor gains a positive charge enabling it to act as an
active acceptor, 2) the phenol latent donor gains a negative
charge allowing it to act as an active donor, and 3) the hydro-
gen bridge locks the molecule in a planar conformation. At the
same time, an intramolecular charge transfer (ICT) from the
phenol donor to either one of the two acceptors forms a new
donor–acceptor pair with longer p-electron conjugation (III
and IV). As a result, a long-wavelength-emitting fluorochrome
is formed.
a
good photostability.[10–12] We have recently developed
a novel class of turn-ON cyanine-based probes with a long-
wavelength fluorescence emission.[13] The probes are based on
the fluorochrome QCy7, which is generated upon removal of
a specific trigger moiety by an analyte of interest. Upon trig-
gering, a unique change in the p-electron system leads to gen-
eration of a cyanine dye with strong near-infrared (NIR) fluores-
cence. In a representative example, the probe enabled detec-
tion of endogenous hydrogen peroxide, produced in an acute
To test the proposed hydrogen-bridge formation mode of
action of a dormant acceptor, we prepared dyes 1 and 2
(Figure 2). Dye 1 is composed of the indolium general acceptor
(blue) and 2-pyridine moiety (green); the 2-pyridine is capable
of forming a hydrogen bond with the phenol donor (structure
1’). Dye 2 is an analogue of dye 1 in which the 2-pyridine
moiety is replaced with a 4-pyridine. The hydrogen bond
cannot form in dye 2 and, therefore, dye 2 served as a negative
control for the hydrogen-bridge activation pathway expected
for dye 1.
[a] Dr. E. Kisin-Finfer,+ Dr. O. Redy-Keisar,+ M. Roth, R. Ben-Eliyahu,
Prof. Dr. D. Shabat
School of Chemistry
The synthesis of dyes 1 and 2 was achieved as outlined in
Figure 3. Compound 1a was subjected to Miyaura borylation
using bis(pinacolato)diboron, tricyclohexylphosphine as
a ligand, and bis(dibenzylideneacetone)palladium(0) as a cata-
lyst to obtain the aryl boronic ester 1b. Ester 1b was then cou-
pled with 2-bromopyridine using tetrakis(triphenylphosphine)-
palladium(0) as a catalyst by the Suzuki cross-coupling reaction
to afford compound 1c. In order to prepare compound 2a,
Raymond and Beverly Sackler Faculty of Exact Sciences
Tel Aviv University, Tel Aviv 69978 (Israel)
Fax: (+972)3-640-9293
E-mail: chdoron@post.tau.ac.il
[+] E.K.F. and O.R.K. contributed equally to this work
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/chem.201504133. This contains full experimental
details, characterization data of all new compounds and spectroscopic
assay conditions.
Chem. Eur. J. 2015, 21, 18566 – 18570
18566
ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Communication
The pKa value of 8.0 measured
for dye 1 is not sufficient to
maintain
a
hydrogen bond
under physiological conditions
at pH 7.4. Furthermore, dye 1 ex-
hibited fluorescence emission
with a lmax of 610 nm. This wave-
length is below the optimal NIR
window suitable for in vivo use.
To further increase the strength
of the intramolecular hydrogen
bond and the increase the emis-
sion wavelength, we sought to
design
a molecular structure
with a “carbon bridge” between
the phenol moiety and the
Figure 1. Graphical illustration of a dormant acceptor activated by hydrogen bonding to produce a new long-
wavelength emitting fluorochrome.
pyridine
dormant
acceptor
(Figure 6). We reasoned that this
modification would lock the molecule in a planar conformation
and thereby increase the strength of the hydrogen bond. In
addition, the planarization of the molecule should minimize
non-radiative electron decay of the excited state, thus increas-
ing the fluorescence quantum yield and yielding fluorescence
at longer wavelengths.
Based on the design presented in Figure 6, we chose to pre-
pare dye 3 (Figure 7). The molecular structure of the dye in-
cludes a conjugated carbon bridge between the two aryl rings
and a conjugated indolium acceptor at the ortho position of
the hydroxylphenol. The nitrogen atom of the quinoline
moiety is capable of forming a hydrogen bond with the phe-
nolic hydrogen as illustrated in Figure 7. As described for the
pyridine-based dye, an ICT from the phenol donor to either
one of the two acceptors will form a new donor–acceptor pair
with elongated p-electron conjugation.
Figure 2. 2-Pyridine dormant acceptor (structure 1) and negative control 4-
pyridine (structure 2) used to validate the hydrogen-bridge activation.
the boronic ester 1b was coupled with 4-bromopyridine hy-
drochloride using similar Suzuki cross-coupling conditions. Fi-
nally, the Suzuki cross-coupling products 1c and 2a were con-
densed with indolium propanesulfonate acceptor 1d to afford
dyes 1 and 2, respectively.
The absorbance spectra of the dyes 1 and 2 measured in
acetonitrile were almost identical with maxima at 450 nm
(Figure 4). However, the fluorescence spectra of the two dyes
were entirely different. Dye 2 exhibited a minor fluorescence
emission with a lmax of 520 nm, whereas dye 1 showed
a strong fluorescence emission with a lmax of 610 nm (F=
10.5%). This result provides support for our suggested dor-
mant-acceptor activation concept in which a hydrogen bridge
formed in dye 1 turns on the pyridine dormant acceptor and
thereby produces an active fluorochrome. Such a structure
cannot be formed in control dye 2 and, therefore, no substan-
tial fluorescence was observed.
The synthesis of dye 3 was achieved as shown in Figure 8.
Commercially available 10-hydroxybenzoquinoline was ortho-
formylated using paraformaldehyde and magnesium chloride
to afford aldehyde 3a, which was condensed with indolium ac-
ceptor 1d to afford dye 3.
The absorbance and the fluorescence spectra of dye 3 were
initially measured in acetonitrile (Figure 9). Benzoquinoline dye
3 exhibited a notable redshift in comparison to dye 1. The
maximum absorbance wavelength of dye 3 was 480 nm and
the emission wavelength was 670 nm. In addition, the ob-
served fluorescence intensity was also notably higher than that
of pyridine-based dye 1 (F=15.7%). These changes are attrib-
uted to the extended conjugation and the stronger hydrogen
bond in dye 3 relative to dye 1. The fluorescence emission of
dye 3 is well within the NIR window, making it a candidate for
in vivo imaging applications. However, under aqueous condi-
tions, the dormant-acceptor activation mode of action pro-
posed in Figure 7 will be ON only if the intramolecular hydro-
gen bond is preserved at physiological pH.
To assess whether the hydrogen bridge of dye 1 is formed
under physiological conditions, we measured the pKa of the
phenols in dyes 1 and 2. The pKa values were determined by
monitoring absorbance as a function of pH (Figure 5). The
phenol of dye 1 had a pKa of 8.0, significantly higher than that
of dye 2 (pKa of 6.4). This difference, which could be observed
through changes of color, is attributed to the hydrogen bond
formation with the nitrogen of the 2-pyridine ring of dye 1;
such a hydrogen bond cannot be formed in dye 2.
As predicted, the measured pKa of dye 3 was significantly
higher than that of dye 1, with a value of 11.3 (see the Sup-
porting Information for details). A phenol with this pKa value
should be able to preserve the hydrogen bond also under
Chem. Eur. J. 2015, 21, 18566 – 18570
www.chemeurj.org
18567
ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Communication
physiological conditions. Indeed,
the observed fluorescent emis-
sion of dye 3 at pH 7.4 indicates
that the hydrogen bridge is
formed (Figure 10). Dye 3 also
showed a substantial NIR fluo-
rescence emission in aqueous
buffer at physiological pH.
As illustrated in Figure 7, the
dormant-acceptor
activation
mechanism of dye 3 relies on
the hydrogen bridge formation
between the phenol and the
quinolone nitrogen atom. There-
fore, masking of the phenol by
a protecting group should elimi-
nate the hydrogen bridge and
the corresponding fluorochrome.
This approach is commonly used
to construct turn-ON fluorescent
probes, where the masking is
performed with an analyte-re-
sponding group. To demonstrate
such a probe based on benzo-
quinoline dye 3, we evaluated
the turn-ON behavior of a probe
Figure 3. Chemical synthesis of dyes 1 and 2.
obtained through masking of
the phenol moiety by a benzyl
boronate ester protecting group (Figure 11; 4). This group can
be removed by a reaction with hydrogen peroxide through an
oxidation–elimination sequence. Incubation of hydrogen per-
oxide with probe 4 should result in the formation of intermedi-
ate 4a. The latter should then undergo 1,6-quinone-methide
elimination to release the active form of dye 3.
Figure 4. A) Absorbance and B) fluorescence spectra of dyes 1 and 2 [50 mm]
in acetonitrile.
Figure 6. Molecular structure of dye with pyridine as a dormant acceptor
fused through a carbon bridge.
Figure 7. Activation of bridged quinoline dormant acceptor by hydrogen
Figure 5. Measurement of pKa values for dyes 1 and 2 (lmax 540 nm).
bonding to produce a new long-wavelength emitting fluorochrome.
Chem. Eur. J. 2015, 21, 18566 – 18570
www.chemeurj.org
18568
ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Communication
Figure 8. Chemical synthesis of dye 3.
Figure 12. Chemical synthesis of probe 4.
The fluorescence emission spectra of probe 4 versus that of
dye 3 in phosphate-buffered saline (PBS) at pH 7.4 are present-
ed in Figure 13A. Dye 3 shows strong emission with a maxi-
mum wavelength of 650 nm, whereas probe 4 exhibits a minor
emission in this wavelength range. Incubation of hydrogen
peroxide with probe 4 resulted in a significant increase of fluo-
rescent signal, whereas no increase was observed upon incu-
bation in the absence of the hydrogen peroxide (Figure 13B).
These results demonstrate implementation of a turn-ON, long-
wavelength fluorochrome probe based on the hydrogen-
bridge activation pathway.
Figure 9. A) Absorbance (lex 480 nm) and B) fluorescence (lem 670 nm) spec-
tra of dye 3 [50 mm] in acetonitrile.
Figure 10. A) Absorbance (lex 500 nm) and B) fluorescence spectra (lem
650 nm) of dye 3 [50 mm] in PBS at pH 7.4.
Figure 13. A ) Fluorescence spectra (lex 480 nm) of probe 4 (blue) and dye 3
(red; [50 mm]) in PBS at pH 7.4; B) fluorescence emission (lem 650 nm) of
probe 4 [50 mm] in the presence (red) and in the absence (blue) of hydrogen
peroxide [100 mm].
Intramolecular hydrogen bonding contributes to the photo-
chemical process known as the excited-state intramolecular
proton transfer (ESIPT).[16–18] Although, this phenomenon has
been characterized in studies of fluorogenic dyes[19–21] and
photoacids,[22] there is a lack of information on the influence of
hydrogen bond strength and pKa on the fluorescent properties
(wavelength emission and fluorescence intensity) of fluorogen-
ic dyes. Interestingly, the hydrogen-bonding interaction in the
excited state of a green fluorescence protein (GFP)-like chro-
mophore led to fluorescence quenching due to a rapid internal
conversion or proton/electron transfer.[23] In another related ex-
ample, an intramolecular hydrogen bond produced a xanthene
dye with NIR fluorescence emission.[24]
Figure 11. Mechanism of hydrogen peroxide activated NIR fluorescent turn-
ON based on dye 3.
The synthesis of probe 4 was achieved as illustrated in
Figure 12. The phenol moiety of compound 3a was alkylated
with benzyl iodide 4b to produce compound ether 4c, which
was reacted with indolium 1d to afford probe 4.
In this study, we demonstrated that an intramolecular hydro-
gen bond can activate a dormant acceptor through a charge
induction between phenolic hydrogen and a heteroaryl nitro-
Chem. Eur. J. 2015, 21, 18566 – 18570
www.chemeurj.org
18569
ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Communication
[2] A. Romieu, Org. Biomol. Chem. 2015, 13, 1294–1306.
[3] X. Chen, T. Pradhan, F. Wang, J. S. Kim, J. Yoon, Chem. Rev. 2012, 112,
1910–1956.
[4] Y. Yang, Q. Zhao, W. Feng, F. Li, Chem. Rev. 2013, 113, 192–270.
[5] M. H. Lee, J. S. Kim, J. L. Sessler, Chem. Soc. Rev. 2015, 44, 4185–4191.
[6] V. S. Lin, W. Chen, M. Xian, C. J. Chang, Chem. Soc. Rev. 2015, 44, 4596–
4618.
[7] H. Kobayashi, M. Ogawa, R. Alford, P. L. Choyke, Y. Urano, Chem. Rev.
2010, 110, 2620–2640.
[8] R. Weissleder, C. H. Tung, U. Mahmood, A. Bogdanov, Nat. Biotechnol.
1999, 17, 375–378.
[9] G. Blum, G. von Degenfeld, M. J. Merchant, H. M. Blau, M. Bogyo, Nat.
Chem. Biol. 2007, 3, 668–677.
[10] S. Gnaim, D. Shabat, Acc. Chem. Res. 2014, 47, 2970–2984.
[11] E. Kisin-Finfer, D. Shabat, Bioorg. Med. Chem. 2013, 21, 3602–3608.
[12] O. Redy-Keisar, K. Huth, U. Vogel, B. Lepenies, P. H. Seeberger, R. Haag,
D. Shabat, Org. Biomol. Chem. 2015, 13, 4727–4732.
[13] N. Karton-Lifshin, E. Segal, L. Omer, M. Portnoy, R. Satchi-Fainaro, D.
Shabat, J. Am. Chem. Soc. 2011, 133, 10960–10965.
[14] N. Karton-Lifshin, L. Albertazzi, M. Bendikov, P. S. Baran, D. Shabat, J.
Am. Chem. Soc. 2012, 134, 20412–20420.
[15] O. Redy-Keisar, E. Kisin-Finfer, S. Ferber, R. Satchi-Fainaro, D. Shabat, Nat.
Protoc. 2013, 9, 27–36.
[16] J. Lee, C. H. Kim, T. Joo, J. Phys. Chem. A 2013, 117, 1400–1405.
[17] F. A. Chipem, A. Mishra, G. Krishnamoorthy, Phys. Chem. Chem. Phys.
2012, 14, 8775–8790.
[18] J. Zhao, S. Ji, Y. Chen, H. Guo, P. Yang, Phys. Chem. Chem. Phys. 2012, 14,
8803–8817.
gen moiety. The activated acceptor can then form a new elon-
gated p-electron pattern in conjugation with a donor moiety
from a separate location on the molecule. The intramolecular
hydrogen bond also induces conformational locking of the
conjugated backbone. Such an effect is known to reduce non-
radiative decay of excited electrons. We increased the hydro-
gen bond strength through bridging of the molecular skeleton
in the benzoquinoline. This improved the conjugation and pla-
narization of the dye molecule and, consequently, shifted the
fluorescence to a longer emission wavelength in the NIR
region. Due to the increased strength of the hydrogen bond
(pKa of 11.3) in the benzoquinoline molecule, the resulting dye
strongly fluoresced under physiological conditions (see the
Supporting Information for fluorescence quantum yields). The
dye was effectively implemented in a turn-ON probe by mask-
ing of the phenol moiety with a responsive substrate for hy-
drogen peroxide. We anticipate that the molecular insights
provided by this study will assist in the design of fluorescent
dyes that can be used for in vitro and in vivo applications.
Acknowledgements
[19] Z. Song, R. T. Kwok, E. Zhao, Z. He, Y. Hong, J. W. Lam, B. Liu, B. Z. Tang,
ACS Appl. Mater. Interfaces 2014, 6, 17245–17254.
D.S. thanks the Israel Science Foundation (ISF), the Binational
Science Foundation (BSF), and the German Israeli Foundation
(GIF) for financial support. This work was also partially support-
ed by grants from the Israeli National Nanotechnology Initia-
tive (INNI), Focal Technology Area (FTA) program: Nanomedi-
cine for Personalized Theranostics, and The Leona M. and
Harry B. Helmsley Nanotechnology Research Fund.
[20] R. Hu, J. Feng, D. Hu, S. Wang, S. Li, Y. Li, G. Yang, Angew. Chem. Int. Ed.
2010, 49, 4915–4918; Angew. Chem. 2010, 122, 5035–5038.
[21] J. Piechowska, K. Huttunen, Z. Wrobel, H. Lemmetyinen, N. V. Tkachen-
ko, D. T. Gryko, J. Phys. Chem. A 2012, 116, 9614–9620.
[22] K. Y. Chen, C. C. Hsieh, Y. M. Cheng, C. H. Lai, P. T. Chou, Chem. Commun.
2006, 4395–4397.
[23] G. J. Huang, J. H. Ho, C. Prabhakar, Y. H. Liu, S. M. Peng, J. S. Yang, Org.
Lett. 2012, 14, 5034–5037.
[24] M. Sibrian-Vazquez, J. O. Escobedo, M. Lowry, F. R. Fronczek, R. M.
Strongin, J. Am. Chem. Soc. 2012, 134, 10502–10508.
Keywords: dyes/pigments · fluorescence · hydrogen bonds ·
IR spectroscopy · molecular probes
Received: October 14, 2015
Published online on November 3, 2015
[1] Y. Tang, D. Lee, J. Wang, G. Li, J. Yu, W. Lin, J. Yoon, Chem. Soc. Rev.
2015, 44, 5003–5015.
Chem. Eur. J. 2015, 21, 18566 – 18570
www.chemeurj.org
18570
ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim