A Molecular Chameleon with Fluorescein and Rhodamine Spectroscopic Behaviors

Article abstract of DOI:10.1021/acs.inorgchem.5b02147

A new class of fluorescein/rhodamine hybrids with two spirolactone rings was reported to exhibit dual-output fluorescent behaviors independently. Isolation and characterization for two diastereomers, trans-RhOH and cis-RhOH, have been made and their X-ray crystal structures determined. In a basic environment, the spirolactone ring on the hydroxyl side will be opened to give a fluorescein-like optical output with the lowest absorptions at 485 and 530 nm emission. On the other hand, a rhodamine-like optical output, i.e., 528 nm absorption and 575 nm emission, will be switched on by a H⁺ or a Hg²⁺ ion, attributed to the spirolactone ring opening on the amino side. In a methanol-buffer system with different pH values, the corresponding pK_a values for the hydroxyl and amino groups were determined as 5.7 and 2.3, respectively. Selective Hg²⁺-sensing properties have also been discussed, and log K_s values of about 3.60 and 3.73 were determined. Confocal microscopic images of Caenorhabditis elegans incubated with RhOH were found to show enhanced fluorescent intensity with a Hg²⁺ ion, demonstrating the potential application of RhOH for in vivo biological imaging.

Full text of DOI:10.1021/acs.inorgchem.5b02147

Article
pubs.acs.org/IC
A Molecular Chameleon with Fluorescein and Rhodamine
Spectroscopic Behaviors
Ling Li,†,‡ Chunyan Wang,†,‡ Jianjian Wu,§ Yu Chung Tse,*,§ Yue-Peng Cai,⊥
and Keith Man-Chung Wong*,†
§
†Department of Chemistry and Shenzhen Key Laboratory of Cell Microenvironment, Department of Biology, South University of
Science and Technology of China, No. 1088, Tangchang Boulevard, Nanshan District, Shenzhen 518055, P. R. China
⊥School of Chemistry and Environment, South China Normal University, Guangzhou 510006, P. R. China
S
* Supporting Information
ABSTRACT: A new class of fluorescein/rhodamine hybrids
with two spirolactone rings was reported to exhibit dual-output
fluorescent behaviors independently. Isolation and character-
ization for two diastereomers, trans-RhOH and cis-RhOH,
have been made and their X-ray crystal structures determined.
In a basic environment, the spirolactone ring on the hydroxyl
side will be opened to give a fluorescein-like optical output
with the lowest absorptions at 485 and 530 nm emission. On the other hand, a rhodamine-like optical output, i.e., 528 nm
absorption and 575 nm emission, will be switched on by a H+ or a Hg2+ ion, attributed to the spirolactone ring opening on the
amino side. In a methanol−buffer system with different pH values, the corresponding pKa values for the hydroxyl and amino
groups were determined as 5.7 and 2.3, respectively. Selective Hg2+-sensing properties have also been discussed, and log Ks values
of about 3.60 and 3.73 were determined. Confocal microscopic images of Caenorhabditis elegans incubated with RhOH were
found to show enhanced fluorescent intensity with a Hg2+ ion, demonstrating the potential application of RhOH for in vivo
biological imaging.
with two hydroxyl groups has recently been reported by us and
others.9 Such a class of compounds have been demonstrated to
show various spectroscopic behaviors arising from two
successive ring-opening processes, and their use as chemo-
dosimeters has been studied.9a Herein, we report a new class of
dual-output fluorescent probes based on a fluorescein/rhod-
amine hybrid, RhOH (Scheme 1), with two spirolactone rings.
The scaffold of this single molecule is from the fused structure
of fluorescein and rhodamine subunits, and the spectroscopic
outputs from the subunits would be independently switched on,
INTRODUCTION
■
Rhodamine and fluorescein are both the most commonly used
organic dyes, in particular of the development and application
of molecular probes for biological imaging.1−4 Rhodol, also
known as rhodaflour, is another kind of organic dye with the
structural hybrid of rhodamine and fluorescein.1−3 The
combination of the merits from rhodamine and fluorescein,
including high water solubility, photostability, strong fluo-
rescence, low excitation wavelength, and less sensitivity to pH
changes, renders rhodol as another suitable candidate for
fluorescent probing. On the other hand, rhodamine derivatives
were found to exist in ring-opened and ring-closed forms with
very distinguished colors and emission behaviors. The chemo-
sensing studies of rhodamine derivatives, with high selectivity
and sensitivity, have blossomed for over a decade for the
detection of various analytes, such as transition-metal cations of
Hg2+, Fe3+, Cu2+, Cr3+, and Au3+ as well as biologically related
compounds of nucleic acid, hydrogen peroxide, glucose,
peroxynitrite, nitric oxide, and hypochlorous acid.5,6 However,
the use of the same concept on the related studies of
fluorescein7 and rhodol3 is relatively less explored. The
combination of a single molecule tethered with individual
fluorescein and rhodamine by means of a linker or a spacer has
been shown to exhibit fluorescent Hg2+ sensing via a FRET
mechanism8a,b and a two-color fluorescent probe for intra-
cellular pH.8c
a
Scheme 1. Synthesis of RhOH
a
(i) 98% H2SO4, 100 °C, 3 h.
An interesting class of rhodamine derivatives with two
spirolactam moieties from resorcinol (1,3-dihydroxylbenzene)
Received: September 17, 2015
© XXXX American Chemical Society
A
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Article
depending on the position of ring opening. Detailed
spectroscopic responses in an acidic or a basic environment,
selective Hg2+-sensing behaviors, and related in vivo biological
imaging have also been studied.
Table 1. Crystal and Structure Determination Data of cis-
RhOH and trans-RhOH
cis-RhOH·2H2O
C38H31NO9
trans-RhOH·1.5C4H10O
empirical formula
fw
C44H41NO8.50
719.78
645.64
RESULTS AND DISCUSSION
■
temperature, K
wavelength, Å
cryst syst
space group
a, Å
298(2)
0.71073
orthorhombic
P2(1)2(1)2(1)
8.3836(12)
18.751(3)
23.344(3)
90
113(2)
Synthesis and Characterizations. According to our
previous work on the rhodamine derivatives with two
spirocyclic rings,9a the presence of two hydroxyl groups in
fluorescein was anticipated to construct a linear array with three
(X) or two (Y) spirocyclic rings (Scheme 1). Neither X nor Y
could be obtained under various reaction conditions and
stoichiometries. To our surprise, reaction of S1 and fluorescein
afforded an interesting compound, RhOH, with the structural
combination of fluorescein and rhodamine subunits. In order to
analyze and estimate the reaction site of fluorescein for the
electrophilic attack, natural bond orbital (NBO) analysis was
performed at the density functional theory (DFT)//B3LYP/6-
31G(d) level. Natural population analysis was used to
determine the electron density of an individual atom on
fluorescein. The electron density of the carbon atom at the 4′
(or 5′) position was found to be higher (Figure S1, upper)
relative to that at the 2′ (or 7′) position, indicative of a higher
susceptibility for electrophilic attack. On the other hand, the
results of the same analysis on rhodol suggested that the
electron density of the corresponding carbon atom is lower
(Figure S1, bottom), and therefore the corresponding linear
structure of a rhodamine derivative with two spirocyclic rings
was obtained.9a Such a theoretical calculation is supportive of
the experimental results in the synthesis of RhOH. In principle,
there are altogether four isomers, including two diastereomers,
trans-RhOH and cis-RhOH, and two enantiomers for each
diasteromer. Only one pair of diastereomers has been isolated
and characterized, while no attempt has been made to separate
the enantiomers. The identities of trans-RhOH and cis-RhOH
have been confirmed by 1H and 13C NMR (Figures S2−S5), as
well as low-resolution and high-resolution mass spectrometry
(MS).
1.54187
triclinic
P1
̅
8.2867(2)
9.9403(3)
23.3563(16)
89.016(6)
86.264(6)
73.891(5)
1844.44(15)
2
b, Å
c, Å
α, deg
β, deg
90
γ, deg
volume, Å3
90
3669.8(9)
4
Z
density (calcd),
g cm−3
1.169
1.296
cryst size, mm ×
mm × mm
0.21 × 0.17 × 0.12
0.30 × 0.10 × 0.10
index ranges
−10 ≤ h ≤ 9, −15 ≤ k ≤ −8 ≤ h ≤ 9, −11 ≤ k ≤
22, −27 ≤ l ≤ 28
11, −28 ≤ l ≤ 27
reflns collected/
unique
19012/6627 [R(int) =
0.0721]
22873/6518 [R(int) =
0.0992]
completeness (2θ = 100.0
25.242°), %
98.8
data/restraints/
param
GOF on F2
6627/2/431
6518/3/502
1.068
1.089
final R indices [I > R1 = 0.0504, wR2 =
R1 = 0.1426, wR2 =
0.3483
2σ(I)]
0.1273
largest diff peak
1.008 and −0.455
0.701 and −0.545
and hole, e Å−3
could be swtiched on independently in this single molecule of
RhOH. Spectroscopic studies of RhOH were performed with
various stimuli, i.e., in the presence of a H+, a OH−, or a Hg2+
ion. Figure 2 shows the electronic absorption and emission
spectra in two distinguished outputs. Identical spectroscopic
responses were observed for trans-RhOH and cis-RhOH,
indicating that the ring-opening behaviors of two stereoisomers
are almost the same. Under basic conditions, the solution color
of RhOH in methanol (MeOH) was changed from colorless to
yellow. Fluorescein-like optical properties were switched on,
leading to the emergence of vibronic-structured absorption
bands at 405, 431, 457, and 485 nm and an emission band at
530 nm. On the basis of the rich spectroscopic characteristics of
fluorescein derivatives, such absorption and emission are
attributed to the spirolactone ring opening as a result of
deprontonation on the hydroxyl group of RhOH in a basic
environment. Extended π conjugation over a xanthene moiety
with a ketone group was then formed. On the other hand, the
addition of a H+ or a Hg2+ ion would result in the absorption
bands at 433, 464, 495, and 528 nm, which is responsible for
the red color solution. At the same time, an emission at 575
nm, which resembles that of the rhodamine derivatives, was
observed. Similar to other rhodamine derivatives, which will
undergo a ring-opening process upon protonation or the
addition of certain transition-metal cations, this rhodamine-like
optical output is presumably due to the spirolactone ring
opening to give another extended π conjugation over a
xanthene moiety with an ammonium group.
Their molecular structures have also been determined by X-
ray crystallography, and all of the experimental details are given
in Table 1. The perspective drawing is shown in Figure 1, and
the selected bond distances (Å) and angles (deg) are tabulated
in Table 2. Both of them clearly reveal that they are in two ring-
closed conformations. The main backbone of RhOH consists of
five six-membered rings in an arrangement in which two
xanthene motifs share the same benzene ring with a turning
angle of 60°. Slightly distorted coplanarities are observed for
such five six-membered rings with interplanar angles of 4.64
and 9.58° between two xanthene moieties in trans-RhOH and
cis-RhOH, respectively. In trans-RhOH, the planes of two 3H-
isobenzofuran-1-ones are pointing in opposite directions and
are orthogonal to the xanthene plane with dihedral angles of
84.68−87.67°, while dihedral angles of 89.43−88.72° are
observed in cis-RhOH with the two 3H-isobenzofuran-1-one
planes aligned in the same direction. The C−C−O bond angles
and C−O bond distances about the spirolactone ring are in the
ranges of 101.0−102.2° and 1.494−1.513 Å, respectively, which
are comparable to those of rhodamine derivatives. Despite the
coplanarity along such five six-membered rings, no close π−π
stacking was observed probably because of the presence of
orthogonal 3H-isobenzofuran-1-one planes.
Dual-Output Responses. It is worth noting that dual
ouput of fluorescein-like or rhodamine-like optical properties
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Figure 1. Perspective drawings of cis-RhOH (left) and trans-RhOH (right) with an atomic numbering scheme. Hydrogen atoms and solvent
molecules are omitted for clarity. Thermal ellipsoids are drawn at the 25% probability level.
different wavelengths, which are characteristics of fluorescein-
like or rhodamine-like optical output depending on the nature
of the inputs (Figures S6−S9). The observation of fluorescein-
like absorption and emission bands in relatively low intensity in
MeOH in the absence of acid or base is ascribed to the
presence of a small amount of fluorescein ring-opened species
in such “neutral” conditions. For the formation of fluorescein-
like ring-opened species, about 10−15 equiv of OH− would be
required to reach saturation for the optical signals, whereas 60
equiv of H+ are required for rhodamine-like species. The
reversibility of such acid−base dual-output responses has been
tested by electronic absorption spectroscopy. Within five
alternating cycles upon the addition of p-toluenesulfonic acid
and triethylamine into cis-RhOH (concentration = 10−5 M) in
MeOH, a satisfactory reversible responsive output could be
obtained (Figure S10). By monitoring of the fluorescein-like
and rhodamine-like absorbances at 457 and 537 nm,
respectively, in a MeOH−buffer solution (1:1, v/v) with
different pH values (Figure 3), the corresponding pKa values of
Table 2. Selected Bond Lengths (Å) and Angles (deg) for cis-
RhOH and trans-RhOH with Estimated Standard Deviations
Given in Parentheses
cis-RhOH
trans-RhOH
Bond Lengths (Å)
C8−O2
C7−C8
C8−C9
C8−C16
O5−C21
C20−C21
C21−C22
C21−C29
1.511(6)
C8−O2
C7−C8
C8−C9
C8−C16
O5−C21
C20−C21
C21−C22
C21−C29
1.500(10)
1.515(11)
1.491(11)
1.517(11)
1.494(10)
1.516(11)
1.514(11)
1.503(11)
1.514(6)
1.499(6)
1.507(6)
1.513(5)
1.515(5)
1.475(6)
1.504(6)
Bond Angles (deg)
O2−C8−C7
O2−C8−C9
101.0(3)
109.2(4)
106.9(4)
110.5(3)
106.4(3)
101.2(3)
109.0(3)
109.7(3)
O2−C8−C7
O2−C8−C9
102.2(6)
107.9(7)
107.2(7)
111.7(7)
107.4(6)
102.0(6)
107.8(6)
110.0(7)
O2−C9−C16
C9−C8−C16
O5−C21−C20
O5−C21−C22
O5−C21−C29
C20−C21−C29
O2−C8−C16
C9−C8−C16
O5−C21−C20
O5−C21−C22
O5−C21−C29
C20−C21−C29
Figure 3. (Left) Electronic absorption spectra of trans-RhOH (50
μM) in different pH values and a MeOH−buffer solution (1:1, v/v).
(Right) Plots of the pH values against log[(Amax − A)/(A − Amin)] at
457 and 537 nm.
Figure 2. Schematic diagram (top) and electronic absorption and
emission spectra (bottom) for the dual outputs of cis-RhOH with
different inputs in MeOH. Concentration = 3 × 10−5 M.
RhOH for the hydroxyl and amino groups were determined as
5.7 and 2.3. The emission spectra of trans-RhOH (50 μM) in
different pH values in a MeOH−Buffer solution are depicted in
Figure S11. The fluorescein-like emission is relatively weak at
high pH value while the rhodamine-like emission is higher at
low pH value, suggesting that the emission quantum yield of
rhodamine-like species is higher than that of fluorescein-like
species. Such pKa values are comparable to those of rhodol
green (pKa = 5.6) and rhodamine derivatives (pKa = 3.1).10
Electronic absorption and emission titration studies of
RhOH in MeOH (in both the trans and cis forms) with
various concentrations of base or acid were investigated. In
general, the profiles of the spectroscopic changes showed a
gradual emergence of new absorption and emission bands at
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Carboxyseminaphthorhodafluor-1 (carboxy-SNARF-1) is a
related compound with a structural combination of fluorescein
and rhodamine containing only one spirocyclic ring,4b which
has been widely used as a intracelluar pH indicator by using
flow cytometry, microplate readers, confocal imaging, or
microspectrofluorometry. However, the relatively high pKa
(ca. 7.5) value limits application in an acidic or a cytosolic
environment. The occurrence of two pKa values for RhOH
arising from independent ring-opening processes leading to
distinguished fluorescein-like or rhodamine-like spectroscopic
properties provides a wider working pH range and is beneficial
for the development of an intracelluar pH indicator.
opening process with a Hg2+ ion to give rhodamine-like
spectroscopic behavior. The log Ks values for Hg2+ ion binding
were found to be 3.60 ( 0.12) and 3.73 ( 0.02) by electronic
absorption and emission spectroscopies, respectively. The
similar binding constant values are indicative of the same
origin for the new absorption and emission bands arising from
the formation of rhodamine-like ring-opened species. Detection
limits in the range of (2.1−5.7) × 10−5 M were also estimated
from the electronic absorption and emission titration results.
Compared to the related rhodamine derivatives with
spirolactam rings, the higher detection limits in RhOH are
attributed to the lack of an additional binding site to coordinate
a Hg2+ ion. The close resemblance of the experimental data to
the theoretical fit suggested the binding mode of 1:1
stoichiometry (Figure 5, insets). This 1:1 complexation mode
has been further confirmed by the method of continuous
variation showing a break point at a [RhOH]/([RhOH] +
[Hg2+]) mole fraction of 0.5 (Figure S13) and the observation
Selective Hg2+ Ion Sensing. In view of the spectral
changes from the effect of a Hg2+ ion, the potential use of
RhOH as a molecular probe was explored. Selective sensing
properties of RhOH were investigated for a series of metal
cations. A solution of trans-RhOH only with a Hg2+ ion could
exhibit a drastic color change from colorless to red (Figure 4)
of a peak of m/z 910 for the adduct of [RhOH + Hg2+
+
− +
ClO4 ] in the MS spectrum (Figure S14). The very similar IR
spectra of RhOH and RhOH + Hg2+ ion (Figure S15) indicate
the absence of the C···O bond involved in the binding process.
The proposed binding mode of RhOH with a Hg2+ ion is
depicted in Scheme 2.
Scheme 2. Proposed Binding Mode of RhOH with a Hg2+
Ion
Figure 4. Selectivity study of trans-RhOH (concentration = 5 × 10−5
M) in MeOH upon the addition of various metal ions (18 equiv).
Inset: Corresponding color change of trans-RhOH (concentration = 1
× 10−5 M) with various metal ions (100 equiv).
or the emergence of new absorption bands. Upon the addition
of a Hg2+ ion, RhOH was found to give electronic absorption
and emission spectral changes similar to those of H+ (Figure 5
It is anticipated that the third optical output could be
achievied for the formation of two ring-opened species, in
which there is an extension of the π conjugation across the
fused structure of five rings. An attempt has been made to open
two spirolactone rings of RhOH on both the hydroxyl and
amino sides, and a solution of a saturated fluoresein-like opened
form was first prepared by a OH− ion (or Et3N). However, the
subsequent addition of a Hg2+ ion would ring-close the
fluoresein-like opened form and eventually only the rhodamine-
like opened form could be obtained (Figure 6). In the reversed
sequence of addition, i.e., a Hg2+ ion first and then a OH− ion, a
Figure 5. Absorption (left) and emission (right) spectral changes of
cis-RhOH (concentration = 3 × 10−5 M) upon the addition of Hg2+
(concentration = 4 × 10−2 M) in MeOH. Insets: Plots of the
absorbance at 495 nm or emission intensity at 575 nm as a function of
the concentration of Hg2+ with a theoretical fit.
for cis-RhOH; Figure S12 for trans-RhOH). As in the acid
titration study, 530 nm emission was observed before the
addition of any Hg2+ ion because of the presence of a small
amount of fluorescein ring-opened species. According to the
spectroscopic response in an acidic environment, the
spirolactone ring-opening process on the amino side, leading
to the formation of extended π conjugation over a xanthene
moiety with an ammounium group, is responsible for such
spectral changes with a Hg2+ ion. Although there are two
spirolactone rings in RhOH, it is worth noting that only the
spirolactone ring near the amino group would undergo a ring-
Figure 6. Electronic absorption spectral changes of cis-RhOH
(concentration = 3 × 10−5 M) in MeOH upon the sequence of the
addition of OH− and then a Hg2+ ion.
D
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similar open−close−open process was observed to give the
fluoresein-like opened form at the end. The clear solution was
retained without observation of the HgO precipitate in the
aforementioned experiments. The ring-opening processes for
the formation of fluorescein-like and rhodamine-like species are
mutally exclusive, presumably because of the opposite charge
required for π conjugation of resonance structures. Although
the species with two opened rings could not be obtained, we
designed and synthesized a derivative of control, in order to
verify the spectroscopic behaviors of the fluoresein-like and
rhodamine-like opened forms as well as the two ring-opened
species. A mimic to the molecular structure of fluorescein-like
ring-opened species, control consists of an xanthene moiety
with a ketone group and only one spirolactone ring on the
amino side. The electronic absorption and emission spectra of
control and its corresponding opened form, control + Hg2+
ion, are depicted in Figure 7. The absorption and emission
Figure 8. Energy-level diagrams of molecular orbitals for four
compounds at B3LYP/6-31G(d).
Caenorhabditis elegans was incubated with cis-RhOH to
investigate whether RhOH can be taken up by nematodes,
and fluorescent signals are shown in the body. C. elegans is a
well-known biological model organism because it is transparent
and therefore ideal for fluorescent studies. Since RhOH was
found to be soluble and C. elegans was alive for 6 h of
incubation, 5% ethanol in double-distilled water was used as the
buffer system for RhOH studies. Control experiments were also
performed by incubating the worms in buffer only or
supplemented with a Hg2+ ion for 6 and 4 h, respectively. No
observable fluorescent signals (within 580−653 nm) were
detected by using spinning-disk confocal microscopy (Figure 9,
panels A and B; Figure 10), indicating that buffer and Hg2+
treatment were not able to increase the background signal in
the worm body. On the other hand, weak fluorescent patches
were observed in the body of worms after treatment with cis-
RhOH for 6 h (Figure 9, panel C, Figure 10). To further
investigate the cis-RhOH-labeled structures, 60× objective
lenses were used for high-resolution fluorescent imaging and
differential interference contrast (DIC) imaging. As shown in
Figure 9, panel G, the cis-RhOH-labeled structures were
superimposed with the intestine granules. Strikingly, the
intensity of these RhOH-labeled intestine granules was greatly
increased after exposure of C. elegans to 50 μM Hg2+ ion for 4 h
(Figure 9, panels D and H). According to quantitative analysis,
the integrated intensity of cis-RhOH in C. elegans treated with a
Hg2+ ion was found to increase about 9-fold (Figure 10). In
addition, the tissue in the buccal cavity was clearly stained by
cis-RhOH in Hg2+ treatment (Figure 9, panel I). This suggested
that RhOH was taken up by worms through the buccal cavity,
pharynx, then entered the gut, and eventually accumulated in
the intestine granules.
Figure 7. Electronic absorption and emission spectra of control and its
corresponding opened form, control + Hg2+ ion, in MeOH.
bands of control are at energies similar to those of the
fluoresein-like opened form of RhOH. The spirolactone ring
could be opened by the addition of a Hg2+ ion or an acid to give
the opened form of control. This opened control species
possesses an extended π conjugation across the fused structure
of two xanthene groups, similar to that of the two ring-opened
species of RhOH. The absorption and emission were found to
shift to the red, and the energies are lower than those of the
fluorescein-like and rhodamine-like opened forms of RhOH.
This could validate and confirm that only one spirolactone ring
was opened upon the addition of a Hg2+ ion, although one
could expect that both spirolactone rings could probably be
opened.
On the basis of the X-ray crystallographic data of cis-RhOH,
the full geometry optimizations of four compounds (two-
closed, −OH-opened, −NEt2-opened, and two-opened) were
calculated with the B3LYP hybrid functional of DFT (Figure
S16). From the energy-level diagrams of their molecular
orbitals (Figure 8), the band-gap values for π−π* transition
were found to decrease from the −OH opened form to the
−NEt2 opened form and would be further reduced in the two-
opened form. Time-dependent DFT (TD-DFT)//B3LYP/6-
31G(d) was also employed to obtain the nature and excitation
energies of the four compounds. The wavelength, oscillator
strength, and major contributions of the calculated transition
were tabulated in Table 3. The excitation energies from this
TD-DFT result give the same energy trend as the band-gap
values. These calculation data are in agreement with the
experimental findings in the spectroscopic properties of the
fluoresein-like opened form, rhodamine-like opened form, and
opened form of control.
In view of the fact that cis-RhOH is biofriendly and it can be
used to detect the accumulation of Hg2+ ions in the body of C.
elegans, this probe is anticipated to be used for biological studies
in a more advanced level. Fluorescence recovery after
photobleaching (FRAP) is a powerful optical technique to
characterize the kinetics of diffusion of cellular molecules within
living cells. Typically, the nonprotein cellular molecules are
made fluorescent by labeling with fluorescent dyes, while
proteins are fused with fluorescent proteins.11 In FRAP, the
fluorophore is irreversibly photobleached by a high-power laser
illumination in the region of interest (ROI). After that, the
Biological Imaging. In order to test the feasibility of
RhOH to act as a mercury sensor in living organisms, adult
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Table 3. Electronic Transition Data Obtained by TD-DFT for Four Compounds
compound
electron transition main transition configuration (CI coefficient) excitation energy (eV) calculated wavelength (nm) oscillator strength f
cis two-closed
S0−S15
S0−S3
S0−S3
S0−S4
HOMO−2 → LUMO+3 (0.47)
HOMO → LUMO or LUMO+5 (0.22)
HOMO → LUMO (0.49)
4.70
2.92
2.81
2.72
264.06
425.07
440.61
456.42
0.1704
cis −OH-opened
cis NR2-opened
cis two-opened
0.3017
0.4978
0.3862
HOMO−4 → LUMO (0.14)
HOMO−2 → LUMO (0.66)
HOMO−6 → LUMO (0.16)
HOMO−2 → LUMO (0.65)
HOMO−3 → LUMO (0.21)
Figure 11. Kinetics of cis-RhOH fluorescent signal after photo-
bleaching. Worms were treated with cis-RhOH and Hg2+, and images
were collected by a 63× objective lens. The ROIs (blue box) were
irradiated by a high-power laser to photobleach the cis-RhOH-labeled
intestine granule (left) and tissue in the buccal cavity (right).
Figure 9. Fluorescence confocal images (signal) and DIC images of C.
elegans treated with cis-RhOH and a Hg2+ ion. Worms were treated
with cis-RhOH (150 μM) and a Hg2+ ion (50 μM) as indicated.
Images were collected at 580−653 nm by using a 10× objective lens
(A−D) and a 63× objective lens (E−I).
intensity in the ROI was found to drop by about 30% after a
short pulse of a high-power laser and then recover to around
80% of the original level within 15 s (Figure 11, right, insets).
This suggested that some of the unbleached RhOH molecules
outside the ROI were able to recover the fluorescent intensity
by diffusion.
CONCLUSIONS
■
In conclusion, the present work represents the first report on
the synthesis and isolation of a novel dual-output fluorescent
sensor/probe, RhOH, based on a structural combination of
fluorescein and rhodamine with two spirolactone rings. Various
stimuli, such as a H+, a OH−, or a Hg2+ ion, could selectively
open a particular spirolactone ring, leading to the fluorescein-
like or rhodamine-like optical output. RhOH was found to
exhibit selective Hg2+-sensing behaviors. Confocal microscopic
images of C. elegans incubated with RhOH showed enhanced
fluorescent intensity with a Hg2+ ion, demonstrating the
potential application of RhOH in biological imaging. The pKa
values of 5.7 and 2.3, arising from the independent ring-
opening processes, provide wider working pH ranges, which
will be beneficial for the development of an intracelluar pH
indicator. The successful isolation of this class of complexes
should open up new avenues for the design and development of
molecular logic gates and fluorescent probes for biological
studies. Modification of the spirolactone rings into other
functionalities in order to alter the selectivity and sensitivity is
in progress.
Figure 10. Integrated intensity of C. elegans treated with cis-RhOH.
Worms were treated with cis-RhOH and Hg2+, and images were
collected by a 10× objective lens. The average number of pixels with
intensity over 3000 in a worm body was counted; n = number of
worms investigated.
intensity of fluorescence recovery over time within the ROI is
recorded, and then the dynamic behavior of the fluorescent
reporter is calculated by mathematical analysis. In order to
investigate the ability of RhOH for FRAP, we have applied a
high-energy laser to the RhOH-labeled intestine granules and
tissue in the buccal cavity. The diffusion behavior of RhOH was
also examined by measuring the fluorescent recovery over time
after photobleaching within a region of the structure (Figure
11). As shown in Figure 11, left, the fluorescence intensity of
RhOH-labeled intestine granules was completely lost after
photobleaching, and the whole granules did not show any
fluorescent recovery over time. This indicated that RhOH will
be photobleached irreversibly. Because the whole structure of
intestine granules was bleached, free molecules of RhOH were
not available to recover the intensity by diffusion. The
fluorescent signal of a portion of the RhOH-labeled tissue in
the buccal cavity, as shown in Figure 11, right, insets, was
erased followed by time-resolved image recording. The RhOH
EXPERIMENTAL SECTION
■
Materials and Reagents. Resorcinol (99%) and benzaldehyde
(98%) were purchased from Energy Chemical, and fluorescein (99%,
Shanghai Chemical Reagent Co., Ltd.), 3-(diethylamino)phenol (97%,
Sigma-Aldrich Chemical Co.), phthalic anhydride (Tianjin Fuchen
Chemial Reagents Factory), 2-methylresorcinol (98%, J&K Scientific
Ltd.), and other materials for the synthesis were used without further
F
DOI: 10.1021/acs.inorgchem.5b02147
Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry
Article
purification. All of the chemicals used for the synthesis were of
analytical grade. Methanol (MeOH) for analysis was of spectroscopy
grade. Copper(II) perchlorate, sodium(I) perchlorate, lead(II)
perchlorate trihydrate, cadmium(II) perchlorate hexahydrate, lithium-
(I) perchlorate, magnesium(II) perchlorate, cobalt(II) perchlorate,
and iron(II) perchlorate hydrate were purchased from Alfa Aesar with
RG grade, and zinc(II) perchlorate hexahydrate (RG, Aladdin
Chemical Co., Ltd.), barium(II) perchlorate (RG, Sigma-Aldrich
Chemical Co.), silver(I) perchlorate (AR, Energy Chemical), nickel-
(II) perchlorate hexahydrate, calcium(II) perchlorate tetrahydrate, and
mercury(II) perchlorate trihydrate were purchased from Strem
Chemicals, Inc., with over 99.0% purity.
153.47, 151.98, 151.86, 150.91, 150.32, 136.28, 135.86, 130.77, 130.62,
130.12, 129.26, 129.23, 127.90, 127.12, 125.51, 124.93, 124.83, 123.98,
113.97, 113.73, 113.69, 109.83, 109.61, 106.74, 105.22, 102.74, 97.76,
83.26, 78.48, 45.04, 12.73. HRMS (ESI). Calcd for C38H28NO7 ([M +
H]+): m/z 609.1788. Found: m/z 610.1871.
Characterization Data of cis-RhOH. 1H NMR (400 MHz,
CD3OD:CDCl3 = 1:1): δ 8.31−8.26 (m, 1H), 8.11−8.07 (m, 1H),
7.86−7.77 (m, 4H), 7.33−7.27 (m, 1H), 7.26−7.21 (m, 1H), 7.12 (d, J
= 8.9 Hz, 1H), 6.92 (d, J = 8.9 Hz, 1H), 6.69 (d, J = 9.6 Hz, 1H), 6.55
(dd, J = 4.0 and 2.0 Hz, 4H), 6.01−5.97 (m, 1H), 3.49 (q, J = 7.0 Hz,
4H), 1.30−1.26 (t, J = 7.0 Hz, 6H). 13C NMR (100 MHz,
CD3OD:CDCl3 = 1:1): δ 172.46, 170.72, 155.68, 153.80, 152.18,
151.85, 151.18, 150.30, 136.10, 135.82, 130.81, 130.57, 130.14, 129.27,
129.18, 127.92, 127.53, 125.58, 125.08, 124.80, 123.78, 114.19, 113.70,
109.82, 106.86, 105.25, 102.70, 97.76, 83.24, 78.62, 78.30, 77.97,
66.53, 45.05, 12.73. HRMS (ESI). Calcd for C38H28NO7 ([M + H]+):
m/z 609.1788. Found: m/z 610.1872.
Safety Precaution! Mercury(II) salt is hazard to health. Perchlorate
salts of metal ion are potentially explosive. Both of them should be handled
with care.
Instruments. The UV−vis absorption spectra were taken on Cary
60 UV−vis spectrophotometer. Steady-state emission spectra at room
temperature were recorded on an Edinburgh Instruments FLS980
fluorescence spectrometer. Quartz cuvettes (path length = 1 cm) were
used in all spectrophotometric and fluorometric measurements. NMR
spectra were recorded on a Bruker AVANCE 400 (1H NMR for 400
MHz and 13C NMR for 100 MHz) Fourier transform NMR
spectrometer with chemical shifts reported relative to tetramethylsi-
lane, (CH3)4Si. High-resolution MS spectra were performed on an
Orbitrap Fusion Tribrid mass spectrometer. The UV−vis absorption
spectra were obtained by utilizing a Cary 60 UV−vis spectropho-
tometer.
Synthesis of control. A mixture of benzaldehyde (1.06 g, 10 mmol)
and resorcinol (2.48 g, 20 mmol) in MeSO3H (50 mL) was heated at
70 °C for 24 h. After that, a dark solution was produced. The reaction
mixture was cooled to room temperature and poured into 400 mL of a
3 M NaOAc solution. The resulting dark-red solid was collected by
filtration to provide the crude product. The product was purified by
column chromatography on silica gel with dichloromethane/MeOH
(40:1, v/v) to afford the intermediate 6-hydroxy-9-phenyl-3H-xanthen-
3-one (0.89 g, 31%).
To a mixture of S1 (0.25 g, 0.8 mmol) and 6-hydroxy-9-phenyl-3H-
xanthen-3-one (0.23 g, 0.8 mmol) was added concentrated sulfuric
acid (6 mL) dropwise at 0 °C. The resulting suspension was heated at
100 °C for 3 h. After the mixture was cooled to room temperature and
poured into ice water (20 mL) with vigorous stirring, the pH of the
mixture was adjusted to ∼7. The mixture was extracted with
dichloromethane (20 mL) three times. The organic layers were
dried over anhydrous magnesium sulfate and evaporated to give the
crude product. Purification and separation of the product were
achieved by silica column chromatography, eluting with dichloro-
methane and MeOH (100:1) to give the pure form of control (0.11 g,
24%).
Ion-Binding Studies. Binding constants for 1:1 complexation
were determined by nonlinear least-squares fits to eq 1, in which the
derivations were described previously.12
Xlim − X
X = X0 +
0 {[M]T + [Hg2+] + 1/Ks
2[M]T
− [([M]T + [Hg2+] + 1/Ks)2 − 4[M]T[Hg2+]]1/2
}
(1)
where X0 and X are the absorbance (or luminescence intensity) of
RhOH at a selected wavelength in the absence and presence of the
Hg2+ ion, respectively, [M]T is the total concentration of RhOH,
[Hg2+] is the concentration of the Hg2+ ion, Xlim is the limiting value of
the absorbance (or luminescence intensity) in the presence of excess
HgII ion, and Ks is the stability constant.
Synthesis. Synthesis of cis-RhOH and trans-RhOH. To a solution
of (3-diethylamino)phenol (2.0 g, 12 mmol) in toluene (60 mL) was
added phthalic anhydride (1.8 g, 12 mmol). The suspension was
heated to reflux for 6 h. The resulting residue was filtered and washed
with MeOH to provide the intermediate S1 (2.1 g, 55%) for the next
step without further purification.
Characterization Data of control. 1H NMR (400 MHz, CDCl3): δ
7.87 (d, J = 7.6 Hz, 1H), 7.66 (td, J = 7.5 and 1.2 Hz, 1H), 7.61−7.37
(m, 1H), 7.48−7.37 (m, 2H), 7.30 (s, 1H), 7.27−7.20 (m, 2H), 7.17
(d, J = 7.6 Hz, 1H), 7.12 (d, J = 9.8 Hz, 1H), 6.93 (d, J = 7.7 Hz, 1H),
6.64−6.52 (m, 4H), 6.47 (d, J = 1.9 Hz, 1H), 6.41 (dd, J = 8.9 and 2.6
Hz, 1H), 3.40 (q, J = 7.1 Hz, 4H), 1.21 (t, J = 7.0 Hz, 6H). HRMS
(ESI). Calcd for C37H28NO5 ([M + H]+): m/z 565.1889. Found: m/z
566.1969.
To a mixture of S1 (0.313 g, 1.0 mmol) and fluorescein (0.332 g,
1.0 mmol) was added concentrated sulfuric acid (5 mL) dropwise at 0
°C. The resulting suspension was heated at 100 °C for 3 h. After the
mixture was cooled to room temperature and poured into ice water
(30 mL) with vigorous stirring, the pH of the mixture was adjusted to
∼7. The mixture was extracted with dichloromethane (30 mL) three
times. The organic layers were dried over anhydrous magnesium
sulfate and evaporated to give the crude product of a mixture of cis-
RhOH and trans-RhOH (0.37 g, 61%). Purification and separation of
the stereoisomer were achieved by silica column chromatography,
eluting with dichloromethane and MeOH (100:1) to give the pure
form of cis-RhOH (0.10 g, 27%) and trans-RhOH (0.11 g, 29.7%) as a
white solid in a ratio of about 1:1. Subsequent recrystallization of the
respective compounds by diffusion of diethyl ether vapor into a
solution of the product in dichloromethane afforded cis-RhOH and
trans-RhOH as single crystals, respectively.
X-ray Crystallography. Single-crystal X-ray diffraction analysis of
cis-RhOH was performed on a Bruker APEX-II CCD diffractometer
with graphite-monochromated Mo Kα radiation (λ = 0.71073 Å) at
room temperature. All absorption corrections were performed using a
multiscan. The structure was solved by direct methods and refined by
full-matrix least squares on F2 with the SHELXTL-97 program
package.13 The intensity data of trans-RhOH were collected on a
Rigaku Saturn944+ CCD diffractometer with graphite-monochro-
mated Cu Kα radiation (λ = 1.54178 Å) at 113 K. All absorption
corrections were performed using a multiscan. The structure was
solved by direct methods and refined by full-matrix least squares on F2
with the SHELXTL-2014 program package.13,14 CCDC 1407618 (cis-
RhOH) and 1407619 (trans-RhOH) contain the supplementary
crystallographic data for this paper. These data can be obtained free of
charge from the Cambridge Crystallographic Data Centre via www.
ccdc.cam.ac.uk/data_request/cif.
Characterization Data of trans-RhOH. 1H NMR (400 MHz,
CD3OD:CDCl3 = 1:1): δ 8.22−8.13 (m, 1H), 8.00 (dt, J = 7.6 and 1.0
Hz, 1H), 7.76 (td, J = 7.5 and 1.2 Hz, 1H), 7.72−7.65 (m, 3H), 7.25−
7.18 (m, 2H), 7.01 (d, J = 8.9 Hz, 1H), 6.84 (d, J = 8.9 Hz, 1H), 6.61
(d, J = 9.6 Hz, 1H), 6.54−6.42 (m, 4H), 6.10 (d, J = 2.0 Hz, 1H), 3.39
(q, J = 7.0 Hz, 4H), 1.19 (t, J = 7.0 Hz, 6H). 13C NMR (100 MHz,
CD3OD:CDCl3 = 1:1): δ 172.54, 170.81, 160.23, 155.58, 153.80,
Buffers in Different pH Values. Different buffer solutions were
prepared by using 50 mM potassium hydrogen phthalate (for the pH
1−5 buffer), 25 mM potassium dihydrogen phosphate (for the pH 6−
8 buffer), 10 mM sodium tetraborate (for the pH 9−10 buffer), and 50
mM sodium bicarbonate (for the pH 11−14 buffer). The pH was
adjusted by adding 0.1 M NaOH or 0.1 M HCl solutions.
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DOI: 10.1021/acs.inorgchem.5b02147
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Article
Notes
METHODOLOGY FOR BIOLOGICAL IMAGING
■
The authors declare no competing financial interest.
C. elegans Strain. C. elegans N2 strains were maintained on
nematode growth medium (NGM) plates using standard
procedures. This strain is provided by the Caenorhabditis
Genetics Center, which is funded by the National Center for
Research Resources, National Institutes of Health.
ACKNOWLEDGMENTS
■
K.M.-C.W. acknowledges receipt of the “Young Thousand
Talents Program” award and the start-up fund administrated by
South University of Science and Technology of China. This
project was also supported by the National Scientific
Foundation of China (NSFC; Grant 21471074). Y.C.T. and
J.W. thank the support National Scientific Foundation of China
(grant no. 31301114) and the Shenzhen Key Laboratory of Cell
Microenvironment (grant no. ZDSYS20140509142721429).
We thank X-ray service open platform of Peking University
Shenzhen Graduate School for structural determination of
trans-RhOH.
Worm Treatment, Imaging, and FRAP. N2 worms were
transferred from the NGM plate to a 150 μL solution (buffer,
5% ethanol in double-distilled water; Hg2+ solution, 50 μM
Hg2+ in buffer; cis-RhOH solution, 150 μM cis-RhOH in a
buffer) in a 1.5 mL centrifuge tube and incubated at 22 °C.
Worms were incubated for 4 h in a Hg2+ solution and 6 h for a
buffer and a cis-RhOH solution. After that, worms were
mounted on 3% agarose pads and sealed with Vaseline. For
confocal imaging, worms were imaged with Nikon Eclipse Ti
(Nikon, Japan) with 10× and 60× objective lenses, equipped
with a Yokogawa CSU-X1 spinning-disk unit (Nikon, Japan),
and illuminated with 50 mW 488 nm lasers (Andor, U.K.).
Images were captured on an iXon Ultra camera (Andor, Belfast,
U.K.) controlled by MetaMorph (Molecular Devices, Sunny-
vale, CA). For spectrum scanning, worms were imaged with
Leica TCS SP8 (Leica, Germany) with 63× objective lens every
5 nm from 500 to 705 nm. Image processing was performed
with ImageJ. For FRAP experiments, worms were treated and
imaged in the same conditions as the confocal imaging
experiments. The pulses of a high-energy laser were generated
by a Micropoint unit (Andor, U.K.) attached to the Nikon
Eclipse Ti (Nikon, Japan) system.
REFERENCES
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COMPUTATIONAL DETAILS
■
Calculations were performed with DFT and TD-DFT using the
Gaussian 09 program.15 Full geometry optimizations of systems in the
gas phase were calculated at hybrid DFT levels by the B3LYP
function16 with a 6-31G(d) basis set.17 The simulation of the
frequency based on the fully optimized geometries implied that
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ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.inorg-
chem.5b02147.
■
S
1H and 13C NMR spectra, titration spectra, and the
results of theoretical calculation (PDF)
X-ray crystallographic data of cis-RhOH in CIF format
(CIF)
X-ray crystallographic data of trans-RhOH in CIF format
(CIF)
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AUTHOR INFORMATION
Corresponding Authors
*E-mail: tseyc@sustc.edu.cn.
*E-mail: keithwongmc@sustc.edu.cn.
■
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‡L.L. and C.W. contributed equally to this work.
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DOI: 10.1021/acs.inorgchem.5b02147
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