Asymmetric and reduced xanthene fluorophores: Synthesis, photochemical properties, and application to activatable fluorescent probes for detection of nitroreductase

Article abstract of DOI:10.3390/molecules24173206

Xanthene fluorophores, including fluorescein, rhodol, and rhodamines, are representative classes of fluorescent probes that have been applied in the detection and visualization of biomolecules. Turn on activatable fluorescent probes, that can be turned on

Full text of DOI:10.3390/molecules24173206

molecules
Article
Asymmetric and Reduced Xanthene Fluorophores:
Synthesis, Photochemical Properties, and Application
to Activatable Fluorescent Probes for Detection
of Nitroreductase
Kunal N. More 1,† , Tae-Hwan Lim 1,†, Julie Kang 1, Hwayoung Yun 2 , Sung-Tae Yee 1 and
Dong-Jo Chang 1,
*
1
College of Pharmacy and Research Institute of Life and Pharmaceutical Sciences, Sunchon National
University, Suncheon 57922, Korea
College of Pharmacy, Pusan National University, Busan 46241, Korea
Correspondence: djchang@scnu.ac.kr; Tel.: +82-61-750-3765
These two authors contributed equally to this work.
2
*
†
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Academic Editor: David Díez
Received: 21 August 2019; Accepted: 2 September 2019; Published: 3 September 2019
Abstract: Xanthene fluorophores, including fluorescein, rhodol, and rhodamines, are representative
classes of fluorescent probes that have been applied in the detection and visualization of biomolecules.
“Turn on” activatable fluorescent probes, that can be turned on in response to enzymatic reactions,
have been developed and prepared to reduce the high background signal of “always-on” fluorescent
probes. However, the development of activity-based fluorescent probes for biological applications,
using simple xanthene dyes, is hampered by their inefficient synthetic methods and the difficulty
of chemical modifications. We have, thus, developed a highly efficient, versatile synthetic route to
developing chemically more stable reduced xanthene fluorophores, based on fluorescein, rhodol,
and rhodamine via continuous Pd-catalyzed cross-coupling. Their fluorescent nature was evaluated
by monitoring fluorescence with variation in the concentration, pH, and solvent. As an application
to activatable fluorescent probe, nitroreductase (NTR)-responsive fluorescent probes were also
developed using the reduced xanthene fluorophores, and their fluorogenic properties were evaluated.
Keywords: fluorescence; xanthene fluorophore; reduced rhodafluor; activatable fluorescent
probe; nitroreductase
1. Introduction
The xanthene scaffold-based fluorophores, including fluorescein, rhodamine, and their hybrid
structure (rhodol), are among the most commonly used fluorophores, which have been widely applied
as fluorescent probes to detect various biological and cellular processes [1,2]. Rhodol and rhodamine
fluorophores in the form of activity-based fluorescent probes have attracted much interest, due to
their high quantum yields in aqueous solutions, over a broad pH range and better photostability,
as compared to fluorescein in detecting various cellular phenomena [3]. Previously, fluorescent probes
carrying macromolecules or small-molecule ligands for targeted delivery have been developed and
used in biomedical imaging. These fluorescent probes are disadvantageous, due to their “always-on”
nature for imaging which lead to poor target-to-background ratios, that result from non-specific binding.
Activatable “turn-on” fluorescent probes were assisted in overcoming this drawback, as they emit
fluorescence only under specific conditions, such as binding to the target protein [1,4,5]. Our research
group has attempted the development of nitroreductase-responsive fluorescent probes for hypoxia
imaging, based on xanthene fluorophores, such as fluorescein and rhodol fluorophores, which bear a
Molecules 2019, 24, 3206; doi:10.3390/molecules24173206
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carboxylate group [6]. However, the selective functionalization of the terminal -OH or -NH2 group
on the xanthene ring via alkylation or amide coupling is difficult, because carboxylate is a good
leaving group (Figure 1A). Alkylation or amide coupling of typical xanthene fluorophores mainly
produce an undesired product via a reaction of the carboxylate group in its open form, and leads
to low yield of the desired product. We envisioned that alkylation or amide coupling could be
achieved at the desired position by shifting the equilibrium toward closed form (Figure 1B). Reduced
xanthene fluorophores have a benzyl alkoxy group, which is a poorer leaving group, compared to the
carboxylate group in typical xanthene fluorophores. Koide’s group first reported a reduced fluorescein
derivative, containing a benzyl alcohol, instead of benzoic acid, producing Pittsburgh green, and it
was capable of detecting palladium and mercury [7,8]. Urano’s group also reported various reduced
xanthene fluorophores, based on rhodol and rhodamine containing benzyl alcohol (HMDER), benzyl
thiol, and benzyl amine instead of the benzoic acid, and designed a novel activatable fluorescent
probe, using HMDER for in vitro and in vivo imaging of β-galactosidase [9–12]. Subsequently, a few
activatable fluorescent probes, based on reduced rhodafluors for biomedical imaging or sensing ozone,
have been reported [13–22].
Figure 1. Structural analysis of typical and reduced xanthene fluorophores. (
A) Reactivity of fluorescein
and rhodol derivatives in alkylation and amide coupling reactions; and (B) equilibrium between closed
and open forms affecting fluorescence.
Asymmetric and reduced xanthene fluorophores based on fluorescein can be synthesized
from commercially available fluorescein via the reduction and subsequent oxidation of the
carboxylate group of fluorescein (Figure 2A), whereas reduced rhodol and rhodamine fluorophores
can be prepared by a convergent synthesis of the xanthene moiety via the reaction of
2-(4-(dialkylamino)-2-hydroxybenzoyl)benzoic acid with resorcinol or 4-aminophenol, followed by
reduction and subsequent oxidation (Figure 2B,C) [9–11,14,23]. However, the current synthetic methods
have limitations in effectively generating diverse asymmetric and reduced xanthene fluorophores. Not
only different types of fluorophores, such as fluorescein, rhodol, and rhodamine, but also the same type
of fluorophores with different substituents should be individually synthesized. Thus, we have designed
a diversity-oriented strategy, that is based on continuous Pd-catalyzed cross-coupling reactions, using

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fluorescein as the starting material for the efficient synthesis of a series of asymmetric and reduced
xanthene fluorophores (Figure 2D).
Figure 2. Strategies for synthesizing asymmetric and reduced xanthene fluorophores.
Herein, we describe the synthesis and photochemical properties of a series of asymmetric
and reduced xanthene fluorophores with fluorescein, rhodol, and rhodamine scaffolds, that enable
efficient asymmetric N-functionalization or O-functionalization. Nitroreductase (NTR)-responsive
fluorescent probes were developed as activatable fluorescent probes using the various reduced xanthene
fluorophores, which can be applied to the design of new pro-drugs for therapeutics and imaging agents
targeting hypoxia [6,23].
2. Results and Discussion
2.1. Chemistry
We started the synthesis of a series of asymmetric and reduced xanthene fluorophores from
commercially available fluorescein, in order to produce fluorophores with synthetic advantage in O-
and N-functionalization of the xanthene ring, and can be applied as an activatable fluorescent probes.
The asymmetric and reduced fluorescein derivative (3) for O-functionalization was first prepared
from fluorescein via dimethylation, LiAlH4 reduction, and subsequent oxidation with p-chloranil
(Scheme 1) [10]. We attempted to extend our synthetic strategy to various reduced rhodafluors,
including rhodol and rhodamine fluorophores, starting from 3. Asymmetric and reduced rhodol

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derivatives with a monoalkylamino (n-propylamino,
7), dialkylamino (diethylamino,
8), or free amino
(-NH2, 12) group were synthesized from 3 via triflation and subsequent Pd-catalyzed cross-coupling
reaction, which was developed by Tao Peng et al. [24–27].
Scheme 1. Synthesis of reduced fluorescein, rhodol, and rhodamine fluorophores. Reagents and
conditions: i) CH3I or methoxymethyl (MOM)-Cl, K2CO3. DMF, rt,
THF, 0 ◦C; iii) p-chloranil, MeOH, rt,
= 79% over 2 steps, = 88% over 2 steps; iv) Tf2O, pyridine,
CH2Cl2, 0 ◦C, to rt,
= 86%, = 86%; v) amine or imine, Pd catalyst (Pd(OAc)2, Pd2(dba)3 CHCl3
or Pd(PPh3)4), ligand (BINAP or Xantphos), Cs2CO3, toluene, 105 ◦C,
= 20%, = 41 %, = further
1= 99%, 2 = 89%; ii) LiAlH4,
3
4
5
6
·
7
8
9
reaction without purification, 10 = 100%, 11 = 47%; vi) aq. 1 N HCl, THF, rt, 12 = 55% over 2 steps;
vii) TFA, CH2Cl2, rt, 13 = 55%, 14 = 84%; viii) N-phenyl-bis-(trifluoromethanesulfonimide), K2CO3,
CH3CN, 0 ◦C, to rt, 15 = 69%, 16 = 57%; ix) benzophenone imine, Pd(OAc)2, Cs2CO3, BINAP, toluene,
105 ◦C; x) aq. 1 N HCl, THF, rt, 17 = 44% over 2 steps, 18 = 30% over 2 steps.
The cross-coupling reaction, that has been used to prepare the reduced rhodol fluorophores was
optimized by the reaction of triflate
catalysts (Pd(PPh3)4, Pd(OAc)2, and Pd2(dba)3
and base (Cs2CO3 and t-BuONa, data not shown). The use of Pd(OAc)2 (10 mol%), BINAP (16 mol%),
and Cs2CO3 (3 eq.) in toluene at 105 ◦C led to the formation of reduced rhodol derivative
bearing
a diethylamino group in moderate yield (41%). Under the same condition for the synthesis of
cross-coupling reactions of with n-propylamine and benzophenone imine produced reduced rhodol
5
with diethylamine, in the presence of conventional palladium
·CHCl3), ligands (BINAP, Johnphos, and Xantphos),
8
8,
5
7, with an n-propylamino group in 20% yield and fluorophore 9, which was further hydrolyzed, using
1 N HCl in THF to give reduced rhodol derivative 12 with an -NH2 group (55% yield over 2 steps).
The reactions for reduced rhodol 7 and 8 required an excess of n-propylamine (20 eq.) and diethylamine
(10 eq.), due to their low boiling points, which resulted in relatively low yields.
Next, we attempted to synthesize reduced rhodol 13 and 14 with a -OH group, which are
key intermediates for the reduced rhodamine 17 and 18 with an -NH2 group. We designed a
synthetic route, using intermediate 4, bearing a methoxymethyl (MOM)-protected hydroxyl group,
instead of the methoxy group (Scheme 1). Triflate
except that the first methylation was replaced by MOM protection using MOMCl. Based on the
cross-coupling reaction of , Pd(OAc)2 and BINAP were used to synthesize the O-MOM protected
rhodol fluorophores, containing an ethylamino (10) and diethylamino (11) group, but the reactions were
unsuccessful. Thus, the Pd-catalyzed cross-coupling reaction of for reduced rhodols (10 and 11) was
optimized using a variety of Pd catalysts (Pd(OAc)2, Pd(PPh3)4, Pd(dppf)Cl2, and Pd2(dba)3 CHCl3)
6 was synthesized in the same manner as 5,
5
6
·

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and ligands (BINAP, Xantphos, and Johnphos) (data not shown). Thus, 10 was synthesized in
good yield (quant.) under the reaction conditions with Pd2(dba)3
·
CHCl3 (10 mol%), Xantphos
◦
(15 mol%), and Cs2CO3 (3 eq.) in toluene at 105 C, while 11 was prepared in a moderate yield
(47%) by using Pd(PPh3)4 (10 mol%), BINAP (15 mol%) and Cs2CO3 (3 eq.). O-MOM protected
rhodol derivatives (10 and 11) were treated with trifluoroacetic acid to give rhodol fluorophores 13
and 14 bearing a phenolic -OH group for O-functionalization. Subsequently, we tried to synthesize
reduced rhodamine fluorophores for N-functionalization. Triflation of rhodol derivative 13, using
triflic anhydride, gave the desired triflate 15 in very low yield. However, triflation by phenyl triflimide
[N-phenyl-bis(trifluoromethanesulfonimide)] and K2CO3 in acetonitrile afforded the desired rhodol
triflates 15 and 16 in moderate yields (69%, and 57%, respectively). We then performed Pd-catalyzed
cross-coupling reactions of 15 and 16 to prepare reduced rhodamine fluorophores possessing an -NH2
group for N-functionalization. The cross-coupling reaction of 15 and 16 with benzophenone imine,
using Pd(OAc)2, BINAP, and Cs2CO3 in toluene, followed by acidic hydrolysis produced the desired
rhodamine fluorophores 17 and 18 in 44%, and 30% yields, respectively. Previously, various attempts
were made for the synthesis of reduced xanthene fluorophores, which resulted in the synthesis of various
fluorescein, rhodol, and rhodamine fluorophores from more than one synthetic scheme [9–11,14,23].
The continuous Pd-catalyzed cross-coupling reactions enabled the rapid synthesis of novel series of
reduced fluorescein, rhodol, and rhodamine fluorophores in highly efficient and concise synthetic
scheme. The library of chemically-stable reduced fluorescein, rhodols, and rhodamines could be
constructed with this synthetic strategy in moderate- to high-yields. It can be applied to synthesize
various reduced xanthene-based fluorophores with N-functionalization or O-functionalization, which
are useful to the design and discovery of novel activity-based fluorescent probes.
Next, we investigated the reactivity of the reduced xanthene fluorophores in comparison with
typical xanthene fluorophores for the O-alkylation and amide coupling reactions. We performed
O-alkylation of reduced fluorescein
bromide, under basic conditions (Scheme 2). Both methylation and benzylation of
3
and typical fluorescein 22, using methyl iodide and benzyl
produced the
3
desired O-alkylated product in high yields (86%, and 98%, respectively), whereas 22 only gave
undesired ester products, which could not be used as activatable fluorescent probes. But the amide
coupling reactions of two types of rhodol fluorophores (reduced rhodol 12 and typical rhodol 23) were
different from the O-alkylation reactions of 3 and 22. In the amide coupling reactions, both 12 and 23
afforded the desired amide products (21, and 26, respectively). However, under the reaction conditions,
using HOBt and iPrNEt2 in DMF, EDC-coupling of 12 exhibited a six-fold higher yield (50%) than 23
(8%). From the results of alkylation and amide coupling of the two types of xanthene fluorophores,
we concluded that reduced xanthene fluorophores are more beneficial for phenolic O-alkylation and
terminal N-amide coupling, compared to typical xanthene fluorophores.
Scheme 2. Reactivity of reduced fluorescein and typical fluorescein fluorophores on alkylation and
amide coupling reactions. Reagents and conditions: i) CH3I, K2CO3, DMF, rt, 86% for 19, 87% for 24; ii)
benzyl bromide, DBU, acetone, rt, 98% for 20, 71% for 25; iii) EDC, HOBt, iPrNEt2, DMF, rt, 50% for 21
,
8% for 26.

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We further attempted to synthesize activatable fluorescent probes based on the reduced xanthene
fluorophores for sensing nitroreductase, an enzyme that catalyzes the reduction of a nitro group to
an amine via a hydroxyl amine in the presence of NADH and a representative biomarker of hypoxic
cells including solid tumors [28
,29] (Scheme 3). We envisioned that the reduced fluorescein (3), rhodol
(12 and 14), and rhodamine (18) fluorophores will be potential candidates for activatable fluorescent
probes, because they have a free -OH or NH2 group and show strong fluorescence at physiological pH
(pH~7). The only exception is 12, which shows weak fluorescence emission at pH~7, but we prepared
an activatable fluorescent probe, based on 12 because the concentration of fluorophores released from
activatable fluorescent probes can be calculated by the concentration-dependent calibration curve of
the corresponding fluorophore. O-Alkylated NTR-responsive fluorescent probes (27 and 28), based on
reduced fluorescein
according to a previously reported method [
2
and rhodol 14 containing an -OH group were synthesized using Ag2O in toluene,
]. Reduced rhodol and rhodamine-based NTR-responsive
6
fluorescent probes (29 and 30), bearing a carbamate linker, were prepared from reduced xanthene
fluorophores 12 and 18 containing an -NH2 group, respectively, by using 4-nitrobenzyl chloroformate
and iPrNEt2.
Scheme 3. Synthesis of NTR-responsive fluorescent probes with p-nitrobenzyl group. Reagents and
conditions: i) 4-nitrobenzyl bromide, Ag2O, toluene, reflux, 27 = 54%, 28 = 48%; ii) 4-nitrobenzyl
chloroformate, iPrNEt2, CH2Cl2, rt, 29 = 86%, 30 = 47%.
2.2. Photochemical Properties and NTR Reaction
The photochemical properties of the asymmetric and reduced xanthene fluorophores, including
quantum yield, concentration-dependent fluorescence emission, stability, and solvent effect,
were evaluated. The quantum yields of newly synthesized fluorophores were calculated in comparison
with the reference standard, fluorescein (0.1 N NaOH,
Φr = 0.85; Table 1). Most of reduced xanthene
fluorophores exhibited significant quantum yields proving their promising fluorogenic nature. Reduced
fluorophores with a -OH showed high quantum yield compared to fluorophore with an -NH2. Among
all fluorophores, rhodol (13) and rhodamine (17) bearing monoethylamine showed highest quantum
yields of 0.824, and 0.399 respectively, showing their strong fluorogenic nature.
Table 1. Quantum Yields of Synthesized Reduced Xanthene Fluorophores.
Comp.
3
7
8
12
13
14
17
18
λabs
456
518
0.044
473
535
0.010
490
554
467
519
0.013
472
532
510
553
490
537
0.399
504
556
λem
a
0.042 a
0.004 b
0.824 b
0.141 b
Φ
s
a = Quantum yields are measured in water by relative method in comparison with fluorescein (0.1 N NaOH,
Φr = 0.85), b = reported quantum yields [9,10,14].
Next, the fluorescent emission at the maximal absorption wavelength (
and reduced xanthene fluorophores in PBS (phosphate buffered saline; 10 mM, pH 7.4) was evaluated
in a concentration-dependent manner (Figure 3). The reduced fluorescein ( ), rhodol (13 and 14),
λmax) for the asymmetric
3

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and rhodamine (17 and 18), containing a -OH or -NH2 group, showed very strong fluorescence emission
at physiological pH~7.4, whereas reduced rhodol and 8, without the -OH or -NH2 group, showed
relatively weak fluorescence emission at pH~7.4. Reduced rhodol with a -OCH3 and -NEt2 groups
7
8
exhibited extremely low fluorescence and quantum yield, implying that an acidic hydrogen on the
terminal oxygen or nitrogen atom on the xanthene ring is essential for sufficiently intense fluorescence
emission in this series of fluorophores. The rhodol 12 showed exceptionally low fluorescent emission
(about 2000 RFU at 5
µM) in the series, even though it contains an -NH2 group. Of all the reduced
xanthene fluorophoresm containing the novel rhodol (
7, 8
and 12) and rhodamine (17) derivatives,
the rhodamine 17 containing -NHEt and -NH2 groups showed the strongest fluorescence emission
(about 80,000 RFU at 2 µM; Figure 3G).
Figure 3. Concentration-dependent fluorescence spectra of asymmetric and reduced xanthene
fluorophores in PBS (10 mM, pH 7.4).
= 473/535 nm); ( ) rhodol
= 472/532 nm); (
(
A
) fluorescein
3
(
λ
/
λ
= 456/518 nm); (
B
) rhodol
= 467/519
7
ex em
(
λ
/
λ
C
8
(λ
/
λ
= 490/554 nm); (
D
) rhodol 12
(λ /λ
ex em
ex em
ex em
nm); (
E
) rhodol 13 ) rhodol 14
= 490/537 nm); (H) rhodamine 18 (λ /λ = 504/556 nm). *Fluorescence of rhodol (13) and
ex em
(λ
/
λ
F
(
λ
/λ
= 510/553 nm); (G) rhodamine 17
ex em
ex em
(
λ
/
λ
ex em
rhodamine (17) derivatives with monoethylamino group was estimated in the range 0.5 to 2
fluorescence at 5 µM is over threshold.
µM because
However, the pH-dependent fluorescence spectra showed a different trend from physiological pH
(Figure 4). Reduced rhodol fluorophores (13 and 14) showed strong fluorescence in the pH range of 5-11,
indicating that it is feasible to apply them to activatable fluorescent probes for use under physiological
conditions, as demonstrated previously [11,12]. On the other hand, the reduced fluorescein (3) and
rhodamine (17 and 18) fluorophores exhibited, not only strong fluorescence at physiological pH,
but also significantly enhanced fluorescence emission upon lowering the pH. Interestingly, reduced
rhodol 7 and 12, containing a -OCH3 at the end and mono-substituted amine (-NHPr), or free amine
(-NH2), at the other end exhibited very weak fluorescence at physiological pH 7.4, but showed a
significant increase in fluorescence below pH 6.

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Figure 4. pH-Dependent fluorescence spectra of asymmetric and reduced xanthene fluorophores
(Normalized fluorescence intensity plotted against pH at 1.0 M) in buffers with pH ranging from 2
to 13. ( ) rhodol 13 = 472/532 nm) and rhodol 14 = 510/553 nm); ( ) fluorescein
em = 456/518 nm), rhodamine 17 em = 504/556 nm);
µ
A
(λ
/
λ
(λ /λ
ex em
B
3
ex em
(λ
ex
/
λ
(λ /λem = 490/537 nm) and rhodamine 18 (λ /λ
ex ex
(C
) rhodol
7
(λ
/
λ
= 473/535 nm), rhodol
8
(λ
/
λ
= 490/554 nm) and rhodol 12
(λ
/
λ
= 467/519
ex em
ex em
ex em
nm)..
We then investigated the solvent effect on the fluorescence emission of the asymmetric and
reduced xanthene fluorophores (Figure 5). It is well known that the fluorescence of xanthene dyes
is complicated by the presence of a solvent-dependent equilibrium, between the colored open form
bearing a zwitterion, and the colorless closed lactone form in protic solvents [30–32]. Urano’s group
reported a series of reduced rhodol and rhodamine fluorophores, and concluded that the lifetime of
the open form of reduced xanthene dyes is very important in determining fluorescence emission [11].
In addition to their results, we envisioned that the nucleophilicity of the benzylic alkoxide will shift the
equilibrium towards non-fluorescent closed form, as the spirocyclization of the open form is induced
by the nucleophilic addition of benzyl alkoxide (Figure 1B). As observed in many SN2 and nucleophilic
addition reactions, polar protic solvents, such as water and methanol, can stabilize the nucleophile via
solvation by hydrogen bonding and decrease its reactivity, whereas polar aprotic solvents, such as
DMSO (dimethyl sulfoxide), which has a strong dipole moment, but cannot form H-bonds, enhance
the reactivity of the nucleophile. Thus, we investigated the solvent effect on the fluorescence emission
of reduced xanthene dyes, in order to evaluate the nucleophilicity of benzyl alkoxide in equilibrium
between the closed and open forms (Figure 5). We measured the fluorescence emission of our reduced
xanthene fluoro◦phores (1.0
µ
M) in various solvents, including water (
ε
= 78.5 at 25 ◦C), methanol
r
◦
◦
(ε
ε
= 32.6 at 25 C), ethanol (
r = 47.0 at 25 ◦C). Most of the reduced xanthene dyes showed the strongest fluorescence in water and
ε
= 24.6 at 25 C), isopropanol (IPA,
ε
= 18.3 at 25 C), and DMSO
r
r
r
(
the weakest fluorescence in DMSO. The trends in the fluorescence emission, observed in polar protic
solvents, were proportional to the dipole moment of the solvent, except for reduced rhodol fluorophore
14. The fluorescence emission in polar protic solvents decreased with the decreasing dipole moment
of the solvent. Whereas, a polar aprotic solvent DMSO, with a dielectric constant between those
of water and methanol, showed lesser fluorescence emission, compared to the polar protic solvent
for all the reduced xanthene fluorophores. The solvent effect on fluorescence emission implies that
the nucleophilicity of benzyl alkoxide in the reduced xanthene fluorophores affects the equilibrium
between the fluorescent open form and non-fluorescent closed form. However, the structure-based
fluorescence emission of xanthene dyes in equilibrium is very complicated; for example, xanthene
dyes exist in several neutral and ionic forms in solution and their equilibria is susceptible to a variety
of factors, including concentration, pH, temperature, and solvent, etc. [30,31,33–36].

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Figure 5. Solvent effect of asymmetric and reduced xanthene fluorophores (1.0
EtOH, MeOH, and water. ( ) fluorescein = 456/518 nm); ( ) rhodol
nm); ( ) rhodol = 490/554 nm); ( ) rhodol 12 = 467/519 nm); ( ) rhodol 13
= 472/532 nm); ( = 510/553 nm); (G) rhodamine 17 (λ /λ = 490/537
ex em
µ
M) in DMSO, IPA,
7 (λ /λ = 473/535
ex em
A
3
(λ
/
λ
B
ex em
C
8
(
λ
/
λ
D
(
λ /λ
ex em
E
ex em
(
λ
/
λ
F) rhodol 14
(
λ
/
λ
ex em
ex em
nm); (H) rhodamine 18 (λex/λ = 504/556 nm).
em
Finally, we applied our asymmetric and reduced xanthene fluorophores as activatable fluorescent
probes targeting nitroreductase. We chose four representative fluorophores, including fluorescein
3,
rhodols 12 and 14, and rhodamine 18, to develop NTR-responsive fluorescent probes. Reduced rhodol
12 showed weaker fluorescence as compared to the other fluorophores, employed as NTR-responsive
probes. Nevertheless, we chose 12 as a fluorophore for activatable fluorescent probe, as the
concentration-dependent fluorescence calibration curve could be established, so that the concentration of
the fluorophore, released from the NTR-responsive fluorescent probe, could be determined. As reported
in our previous work on NTR-responsive fluorescent probes [6], xanthene fluorophores are linked to
the p-nitrobenzyl group via an ether (27 and 28) or carbamate (29 and 30) moiety, and reduction of the
nitro group in the probe triggers the release of the fluorophore via 1,6-rearrangement elimination of
the p-aminobenzyl group, leading to a turn-on fluorescent response.
The stability of the linkage in the NTR-responsive fluorescent probes (27–30) was assessed by
estimating the fluorescence emission under varying temperature and pH conditions (Figure 6). All the
probes showed weak, but stable, fluorescence emissions in the temperature range 25 to 45 ◦C and
pH range 5–13, implying that they can be applied as activatable fluorescent probes for sensing a
specific protein, NTR, under physiological conditions. Exceptionally, the strong fluorescence emission
of reduced rhodamine-based probe 30, containing a carbamate linkage, present in acidic pH range
between 2 to 4, is the result of the fluorophore via hydrolysis under acidic conditions, which is highly
correlated with the pH-dependent fluorescence spectrum of fluorophore 18 (Figure 4H).

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Figure 6. Stability of nitroreductase (NTR)-responsive fluorescent probes (1.0
in pH from 2 to 13 at 25 ◦C; ( ) Effect of variation in temperature (25, 28, 31, 34, 37, 43, and 45 ◦C) in
PBS (10 mM, pH 7.4).
µM). (A) Effect of variation
B
We performed kinetic studies (1.0
µM of probes) of probes during the NTR reaction, using
10
µg/mL of this protein as a function of time, in order to investigate the activatable response of probes
to NTR (Figure 7). Probe 27 and 28, which are fluorescein- and rhodol-based activatable fluorescent
probes, containing an ether linkage, showed strong fluorescence responses over time in the presence
of NTR (Figure 7A). On the other hand, probes 29 and 30, bearing a carbamate linkage, which were
prepared via N-functionalization of the reduced rhodol and rhodamine fluorophores containing an
-NH2 group, showed relatively weak fluorescence emission over time during the NTR reaction.
Figure 7. Kinetic study of NTR-responsive fluorescent probes and fluorescence calibration curves
of corresponding fluorophores. (
time in the presence of 10 g/mL of NTR, NADH (500
Calculated concentrations of the corresponding fluorophores released from NTR-responsive fluorescent
probes during NTR reaction; ( ) Calibration curves at concentrations of 0.005, 0.01, 0.02, 0.05, 0.1, 0.2,
0.5, and 1.0 M of fluorophore (Inset: Linear regression graph); Concentration-dependent changes in
A
) Plot of fluorescence emission of probes (1.0
µM) with reaction
µ
µ
M), PBS (pH 7.4), and incubation at 37 ◦C; (
B)
C–F
µ
the fluorescence emission of fluorophore 3 (C), 14 (D), 12 (E), and 18 (F).
We determined the concentration of the released fluorophores in the NTR reaction using the
concentration-dependent calibration curves obtained for the fluorophores (Figure 7B–F). Distinctly

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different results were obtained from the calibration curves: Probes 27 and 29 showed the completion of
the NTR reaction with almost 1.0 M of the released fluorophore ( and 12), whereas the concentration
of the fluorophore released from probes 28 and 30, reached only 0.167 M (14) and 0.065 M (18)
µ
3
µ
µ
(Figure 7B). Although, probe 29 gave 100% yield for the NTR reaction, the corresponding fluorophore
12 could not be applied as an activatable imaging probe at physiological pH due to its photochemical
nature, i.e., weak fluorescence emission at physiological pH. Rhodamine-based fluorophore 18 showed
enough fluorescence at physiological pH, but the corresponding NTR-responsive fluorescent probe
30 exhibited a poor release of the fluorophore, during the NTR reaction, demonstrating that 18 is
not a good candidate for an activatable fluorescent probe. The reduced fluorescein
3 and rhodol
14 fluorophores, bearing an -OH group showed strong fluorescence at physiological pH, and the
corresponding NTR-responsive fluorescent probes (27 and 28), also emitted strong fluorescence during
the NTR reaction. However, probe 28, based on the reduced rhodol gave a relatively low yield (~17%)
in the NTR reaction, as compared to probe 27. Taken together, the NTR reaction revealed that 3 and
14 are promising candidates for activatable fluorescent probes, and among all the asymmetric and
reduced fluorophores studied, fluorescein 3 is the best choice for an activatable fluorescent probe.
3. Materials and Methods
3.1. Materials and Instrumentation
All reagents and solvents were purchased from Sigma-Aldrich Chemical Co. (St. Louis, USA),
Tokyo Chemical Industries (Tokyo, Japan), Daejung Chemicals (Siheung-si, Korea), and Alfa Aesar
(Ward Hill, MA, USA) and used without any further purification. Anhydrous solvents were purchased
from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA), and all reactions were performed under
nitrogen atmosphere. Silica gel (ZEOprep 60 40–63 µm, Zeochem, Louisville, KY, USA) was used for
flash column chromatography, and silica gel plates (Kiesegel 60F254, Merck, Darmstadt, Germany)
were used for thin-layer chromatography. 1H and 13C-NMR spectra were measured on a JEOL
JNM-ECZ400s/L1 (400 MHz) spectrometer (Jeol, Tokyo, Japan), with CDCl3 or DMSO-d6 as the NMR
solvent (Cambridge Isotope Laboratories, Tewksbury, MA, USA). Chemical shifts are expressed in
parts per million (ppm), and the coupling constant J is reported in hertz (Hz). Chemical shifts (in
ppm) in 1H-NMR are based on the chemical shift of tetramethylsilane (
δ = 0 ppm) in CDCl3 as an
internal standard. The chemical shifts in 13C-NMR are reported in ppm relative to the centerline of
the triplet at 77.0 ppm observed for CDCl3 or 39.5 ppm for DMSO-d6. All in vitro enzyme assays
were performed by recording the absorbance and emission using a Synergy
™ H1 microplate reader
from BioTek Instruments (Winooski, VT, USA). Nitroreductase from Escherichia coli and NADH were
purchased from Sigma-Aldrich Chemical Co. The lyophilized nitroreductase powder was dissolved in
deionized water, fractionated, and immediately stored at −80 ◦C.
3.2. General Synthetic Procedures
3.2.1. General Procedure A: Alkylation
K2CO3 (2.5 eq.) was added to a solution of fluorescein (1.0 eq.) in DMF, and the reaction mixture
stirred at rt for 1 h under N2 atmosphere. Methyl iodide or chloromethyl methyl ether (3.0 eq.) was
added dropwise to the reaction mixture, using a syringe pump with the rate of 5 mL/1hr, and the
reaction mixture stirred at rt for 3–12 h. After completion of the reaction, ice water was added to the
reaction mixture and stirred at 0 ◦C for 30 min. The resulting yellow solid was filtered (in the case of
the MOM protection reaction, extraction was performed using ethyl acetate) and washed with water to
completely remove K2CO3. The resulting solid was dried or purified by column chromatography to
afford the desired compound.

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3.2.2. General Procedure B: LiAlH4 Reduction and p-Chloranil Oxidation
To a solution of compound
0 C. The reaction mixture was stirred at 0 C for 4 h. After completion of the reaction, sodium
sulfate decahydrate (5.6 eq.) was added to the reaction mixture at 0 C and then stirred at rt for
1
or
2
(1.0 e◦q.) in anhydrous THF was added LiAlH4 (2.0 eq.) at
◦
◦
30 min. The reaction mixture was filtered through a short pad of Celite, which was washed with
CH2Cl2. The filtrate was concentrated in vacuo, and the crude product used in the next reaction
without further purification. The crude compound, obtained from the LiAlH4 reduction, was dissolved
in MeOH, followed by the addition of p-chloranil (3.0 eq.), and stirred at rt for 2 h. The reaction
mixture was filtered, and the filtrate concentrated in vacuo. The residue was purified by flash column
chromatography on silica gel to give the desired product.
3.2.3. General Procedure C: Triflation
To a solution of the phenol derivative (1.0 eq.) in anhydrous CH2Cl2 or CH3CN was added
pyridine, or K2CO3 (4.0 eq.), respectively, and the reaction mixture stirred at 0 ◦C for 20 min. Triflic
anhydride or N-phenyl-bis(trifluoromethanesulfonimide) (2.0 eq.) was slowly added to the reaction
mixture over 30 min, and then, the mixture was allowed to warm to rt and stirred for 3 h. The reaction
was quenched with water and extracted with CH2Cl2. The organic layer was washed with aqueous
1 N HCl solution or saturated NH4Cl aqueous solution, followed by water and brine. The organic
layer was dried over Na2SO4, filtered and concentrated in vacuo. The residue was purified by flash
column chromatography on silica gel to give the desired product.
3.2.4. General Procedure D: Pd-Catalyzed Cross Coupling Reaction
All glassware was dried in an oven before use. To a solution of the corresponding triflate (1.0 eq.)
and amine (10 or 20 eq.) or benzophenone imine (1.2 eq.) in anhydrous toluene were added Pd(OAc)2,
Pd(PPh3)4, or Pd2(dba)3.CHCl3 (0.1 eq.), BINAP or Xantphos (0.16 eq.), and Cs2CO3 (3.0 eq.), and the
◦
reaction mixture was heated at 105 C for 4–12 h under N2 atmosphere. After completion of the
reaction, the mixture was filtered through a short pad of Celite and washed with CH2Cl2. The filtrate
was concentrated in vacuo, and the residue was purified by flash column chromatography on silica gel
to give the desired product.
3.2.5. General Procedure E: MOM-Deprotection
To a solution of the corresponding MOM protected compound (100 mg) in anhydrous CH2Cl2
(1 mL) at 0 ◦C, a solution of trifluoroacetic acid in CH2Cl2 [1 mL, TFA:CH2Cl2 = 1:1 (v/v)] was slowly
added dropwise at 0 ◦C. After the addition of TFA was complete, the reaction mixture was allowed
to warm to rt and stirred at rt for 1 h. The reaction was quenched with 1 N NaOH solution and
extracted with CH2Cl2. The organic layer was dried over Na2SO4, filtered, and concentrated in vacuo.
The residue was purified by flash column chromatography on silica gel to give the desired product.
3.3. Determination of Fluorescence Quantum Yield
The quantum yield (
by comparing the integrated area under the curve of the sample, excited at 490 nm, with the
reference fluorophore. Fluorescein ( r = 0.85, 0.1 N NaOH) is used as the reference fluorophore for
Φs) of the newly synthesized reduced xanthene fluorophores was determined
Φ
determining the quantum yield of reduced xanthene fluorophore. Absorption spectra and fluorescence
spectra of fluorophores were recorded on Synergy H1TM microplate reader (BioTek Instruments,
Winooski, VT, USA) and FluoroMate FS-2 fluorescence spectrometer (Scinco, Seoul, Korea), respectively.
The absorbance values are kept below 0.1 in order to minimize re-absorption effects. Fluorescence
quantum yield (Φs) of reduced xanthene fluorophores was calculated by using following Equation (1):

Molecules 2019, 24, 3206
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!
2
ꢀ
ꢁ
ꢀ
ꢁ
Ar
As
Fs
Fr
ηs
ηr
Φs = Φr ×
×
×
(1)
Φ
s = Quantum yield of sample; Ar = Absorbance of reference at excitation wavelength; As = Absorbance
of sample at excitation wavelength; Fr = Integrated area under emission curve of reference;
Fs = Integrated area under emission curve of sample; = Refractive index of solvent of reference;
η
r
η = Refractive index of solvent of sample; and Φr = Quantum yield of fluorescein.
s
3.4. Concentration-Dependent Fluorescence Study of Fluorophores
A concentration-dependent study was performed by incubating different concentration, such as
0.5, 1, 2, and 5 µ
M of the fluorophore in PBS (pH 7.4) at 25 ◦C, and recording the fluorescence spectra
at each concentration in 96-well microplate using Synergy H1 reader.
3.5. Effect of pH on Fluorescence Intensity of Fluorophores
A pH-dependent fluorescence change of fluorophore was performed by incubating 1 µM of the
probe in a range of pH buffer solutions (pH 2 to 13) at 25 ◦C, and recording the fluorescence at each pH
in 96-well microplate, using SynergyTM H1 (BioTek Instruments).
3.6. Solvent Effect on the Fluorescence Emission of Fluorophores
The effect of solvent polarity on the fluorescent intensity was measured by incubating 1 µM of
the fluorophore in different solvents, including water, methanol, ethanol, isopropanol, and DMSO.
The fluorescence was measured at the respective excitation wavelength of the fluorophore, in a 96-well
microplate, using SynergyTM H1 (BioTek Instruments).
3.7. In vitro Nitroreductase Assay
All spectroscopic readings were recorded on SynergyTM H1 (BioTek Instruments) using a 96-well
microplate. The NTR reaction was performed in a total volume of 200
of PBS (10 mM, pH 7.4), 10 L of probe stock solution (20 M in DMSO), 20
(5 mM in 0.01 M aq. NaOH), and an appropriate volume of NTR solution (10
water). The final volume was adjusted to 200
L using PBS. The plate was incubated at 37 ◦C for an
µ
L with the addition of 100
L of NADH solution
g/100 L in distilled
µL
µ
µ
µ
µ
µ
µ
appropriate length of time with continuous shaking, and the emission spectra were recorded at the
respective wavelengths with respect to time to prepare kinetic graph.
3.8. pH and Thermal Stability of Fluorescent Probes
A temperature-dependent assay was performed by incubating 1
µM of the probe in PBS (pH 7.4) at
different temperatures for 20 min, and recording the fluorescence at each temperature. A pH-dependent
study was performed by incubating 1 µM of the probe in a range of pH buffer solutions (pH 2 to 13) at
25 ◦C and recording the fluorescence at each pH in 96-well microplate, using SynergyTM H1 (BioTek
Instruments).
All synthetic procedures, 1H-NMR, 13C-NMR and HRMS of all compounds can be seen in the
Supplementary Materials.
4. Conclusions
In conclusion, we developed a highly efficient and versatile synthetic route to a series of
asymmetric and reduced xanthene fluorophores, including fluorescein, rhodol, and rhodamine
derivatives, which are representative xanthene scaffold-based dyes, and employed them as activatable
fluorescent probes for sensing nitroreductase. A variety of asymmetric and reduced xanthene
fluorophores, bearing an -OH or -NH2 group, capable of O- or N-functionalization to prepare
activatable fluorescent probes, were synthesized from commercially available fluorescein by continuous
Pd-catalyzed cross-coupling reactions. Their photochemical properties, including quantum yields,

Molecules 2019, 24, 3206
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fluorescence emission under various conditions (variation in concentrations, pH, and solvents) were
characterized. Two fluorophores, including fluorescein ( ) and rhodol (14) bearing an -OH group for
3
O-functionalization, and two other fluorophores, including rhodol (12) and rhodamine (18), containing
an -NH2 group for N-functionalization were subjected to develop NTR-responsive fluorescent probes.
These probes exhibited turn-on fluorescence by releasing the fluorophore in the NTR reaction. This work
demonstrates that the asymmetric and reduced xanthene fluorophores synthesized using our strategy
are useful in developing activatable fluorescent probes for diverse biological applications under
physiological conditions.
Supplementary Materials: Figures S1–S27: Synthetic procedures, 1H-NMR, 13C-NMR and HRMS of
all compounds.
Author Contributions: Conceptualization, D.-J.C.; methodology, T.-H.L. and J.K.; validation, K.N.M and H.Y.;
formal analysis, S.-T.Y; writing original draft preparation, K.N.M. and T.-H.L.; and supervision, D.-J.C.
Funding: This research was supported by the National Research Foundation of Korea (NRF) funded by
the Ministry of Science, ICT, and Future Planning (NRF-2016R1C1B1009901, NRF-2018R1D1A1B07050581 &
NRF-2019R1F1A1063304).
Conflicts of Interest: The authors declare no competing financial interest.
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2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).