Electronic Effects on the Depropargylation Process in the Reaction-based Fluorescent Detection of Palladium Species: Benzocoumarin-based Ratiometric Sensing Systems

Article abstract of DOI:10.1002/bkcs.12173

The palladium catalyzed cross-coupling reactions are indispensable in organic synthesis of pharmaceutical compounds, demanding convenient tools for the analysis of residual palladium contents. Propargyl aryl ether-type fluorescent sensing systems that det

Full text of DOI:10.1002/bkcs.12173

Article
DOI: 10.1002/bkcs.12173
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S. W. Cho et al.
KOREAN CHEMICAL SOCIETY
Electronic Effects on the Depropargylation Process in the
Reaction-based Fluorescent Detection of Palladium Species:
Benzocoumarin-based Ratiometric Sensing Systems
*
Seo Won Cho, Ye Jin Reo, Sourav Sarkar, and Kyo Han Ahn
Department of Chemistry, POSTECH, Pohang 37673, Republic of Korea. *E-mail: ahn@postech.ac.kr
Received October 26, 2020, Accepted November 29, 2020, Published online December 29, 2020
The palladium catalyzed cross-coupling reactions are indispensable in organic synthesis of pharmaceutical
compounds, demanding convenient tools for the analysis of residual palladium contents. Propargyl aryl
ether-type fluorescent sensing systems that detect Pd(0)/Pd(II)/Pd(IV) species through depropargylation
require no additives. We investigated a ratiometric sensing system based on a benzocoumarin dye; thus,
three propargyl aryl ethers bearing electronically-different self-immolative linkers were synthesized and
their relative response rate toward Pd(0) and Pd(II) were compared. We found that the response rate
became faster when the self-immolative linker became more electron-deficient. The best-performing sys-
tem sensed Pd(0)/Pd(II) species selectively against various other metal species, which is also capable of
fluorescent imaging of Pd(0)/Pd(II) species in cells. The work provides us a clue to accelerate the catalytic
depropargylation step, which, when combined with a novel fluorophore, would enable us to develop out-
performing palladium sensing systems.
Keywords: Fluorescence, Palladium, Depropargylation, Ratiometric, Electronic effect
Introduction
propargyl ether can detect common Pd species, such as
PdCl2, PdCl2(CH3CN)2, Pd(OAc)2, (NH4)2PdCl2, and
Na2PdCl4, with high selectivity from other various metal spe-
cies. The depropargylation process seems to proceed through
two routes depending on the Pd species: in the case of
Pd(0) species, it proceeds through the allenyl palladium inter-
mediate; in the case of Pd(II) or Pd(IV) species, it proceeds
through the hydration route (Scheme 1).9 In both cases, the
end product is the depropargylated aryl alcohol (ArOH).
Therefore, it is possible to develop various fluorescent detec-
tion systems for Pd species at different oxidation states,
starting from fluorescent ArOH. Indeed, since this work, var-
ious aryl propargyl ether-type fluorescent detection systems
have been developed by other groups.10
Recently, we have developed push-pull type
benzocoumarin compounds as novel fluorophores. As
π-extended coumarin analogues, the linear-shape
benzocoumarins have promising photophysical properties
for bioimaging applications. Additionally, they are photo-
and chemo-stable, and show rather environment-insensitive
emission behavior in spite of the dipolar nature.11–16 In our
continuous efforts to explore fluorescent probes,17 we have
reinvestigated the palladium sensing system based on a
benzocoumarin dye, which responds to Pd(0)/Pd(II) species
with ratiometric fluorescence changes. Compared to the
fluorescein-based sensing system that provides turn-on fluo-
rescence response,8 this ratiometric sensing system may
find usefulness in the quantitative analysis of Pd species in
synthetic samples. In this work, to develop an efficient rati-
ometric sensing system based on the depropargylation
Palladium (Pd) species are widely used as important cata-
lysts in organic synthesis and fuel cells. For example, the
modern carbon–carbon cross-coupling reactions are fre-
quently conducted with Pd catalysts in academia as well as
in industry. The detection of residual Pd species in pharma-
ceutical products is crucial for their quality control.1,2 Con-
ventionally, inductively coupled plasma mass spectrometer
(ICP-MS) has been widely used for analysis of the residual
Pd species.3,4 ICP-MS needs complex sample preparation
steps, well experienced personnel, and high cost, demand-
ing an alternative and efficient analysis method. The fluo-
rescence detection method thus has received much attention
in recent years since the pioneering works by Koide and
coworkers who developed a fluorescein allyl ether as the
first reaction-based fluorescent sensing system for Pd spe-
cies.5 The fluorescein allyl ether undergoes the well-known
Pd(0)-catalyzed Tsuji–Trost oxidative insertion that causes
deallylation and release the fluorescent fluorescein derivative
(Scheme 1). As this sensing scheme detects catalytically
active Pd(0) species, additives such as tris(2-furyl)phosphine
and sodium borohydride are required to sense Pd(II/IV) spe-
cies.6 The sensing system can be also used to detect Pd(II) or
Pt(IV) directly through
a catalytic aromatic Claisen
rearrangement but at 50 ꢀC in dimethyl sulfoxide (DMSO)–
pH 10 buffer (1:4).7 Our group subsequently devised a dif-
ferent sensing scheme, the depropargylation route
(Scheme 1), which required no additives to detect Pd species
regardless of their oxidation states.8 This fluorescein
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Figure 1. Benzocoumarin (BC1) derived, ratiometric sensing sys-
tems for Pd species bearing different self-immolative moieties.
Synthesis. The three compounds (BC_Pd1, BC_Pd2, and
BC_Pd3) were synthesized from BC1 by an amide bond
formation with the corresponding (propargyloxy)benzyl
alcohol, the self-immolative moiety including the reactive
propargyl ether. The synthesis was straightforward and the
final products were fully characterized by nuclear magnetic
resonance and MS analysis.
Photophysical Properties. All the palladium sensing sys-
tems displayed their absorption bands with the same maxi-
mum wavelength (λabs) at 350 nm, as they share the
common fluorophore. The absorption spectrum of BC_Pd3
has tailing toward the longer wavelength region. Their
emission spectra also mostly overlapped with each other,
with the maximum emission peaks in 532–540 nm range.
The parent dye BC1 exhibited an absorption band with λabs
at 450 nm, a significantly longer wavelength from that of
the probes; it exhibited the maximum emission wavelength
(λem) at 600 nm, with substantial Stokes shifts of 60–68 nm
from the corresponding probe (Figure 2). Thus, the sensing
systems were expected to provide ratiometric signal
changes in detecting Pd species.
Scheme 1. Fluorescent detection of Pd species based on the cata-
lytic deallylation and the depropargylation schemes, and supposed
intermediates in the depropargylation process.
protocol, we focused on how to accelerate the
depropargylation reaction by changing the electron density
in the self-immolative linker, p-hydroxybenzyl alcohol. The
results indicate that the propargylation step proceeds faster
as the electron density of the phenyl group in the self-
immolative linker decreased.
Results and Discussion
As noted above, the benzocoumarin (BC) dye system has
proven to be a versatile dye platform for the development
of fluorescent probes. To develop BC-based fluorescent
probes with ratiometric signaling capability, we investi-
gated here three propargyl ethers (BC_Pd1, BC_Pd2, and
BC_Pd3) based on methyl 8-(methylamino)-2-oxo-2H-
benzo[g]chromene-3-carboxylate (BC1) via the carbamate
bond (Figure 1). It is well established that, upon
depropargylation, the p-hydroxybenzyl group will undergo
a fragmentation reaction to release BC1. This chemical
conversion will accompany a significant change in the
intramolecular charge transfer in the fluorophore and thus
an emission spectral change. Thus, we may realize a rati-
ometric sensing system for Pd species. The
depropargylation step seemed to be dependent on the elec-
tron density in the aryl ether moiety, a leaving group. To
investigate the electronic effect, in this work we varied the
phenyl group in the self-immolative moiety with different
electron densities: unsubstituted, mono- and dichloro-
substituted phenyl groups.
Relative Response Rate. A key concern in this work was
to realize faster response in detecting Pd species, from such
carbamate-linked propargyl ether type probes. We moni-
tored the time-course of fluorescence changes for BC_Pd1,
BC_Pd2, and BC_Pd3, each at 5.0 μM in acetonitrile–
water (1/9 by volume), in the presence of an equivalent
amount of PdCl2 as a Pd(II) species. We followed the time-
dependent changes of the emission peak height ratios
between the reaction product BC1 and probe, I600/I530. In the
case of BC_Pd1, the sensing process was slow, as indicated
by the slow ratio change (Figure 3(a)). On the other hand,
BC_Pd2, in particular, BC_Pd3 exhibited faster response, as
indicated by the steeper increase in the ratio value. We also
compared the relative response rate of the probes toward
(Ph3P)4Pd as a Pd(0) species, under otherwise the same con-
ditions (Figure 3(b)). Again, BC_Pd3 exhibited faster
response than the other probes, and BC_Pd1 responded little
in this case. When we compare the relative rate between the
Pd(II) and Pd(0) species, BC_Pd3 responds faster toward the
latter species (I600/I530 = 2.0 vs. 8.1). For the quantitative
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Benzocoumarin-based Ratiometric Sensing Systems
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4.0
3.5
3.0
2.5
2.0
1.5
1.0
0.5
(a)
(a)
BC_Pd1
BC_Pd2
BC_Pd3
BC1
BC_Pd1
BC_Pd2
BC_Pd3
PdCl2
1.0
0.8
0.6
0.4
0.2
0.0
0
50
100
150
200
Time (min)
(b) 10.0
7.5
Pd(Ph3P)4
BC_Pd1
BC_Pd2
BC_Pd3
350
400
450
500
550
600
Wavelength (nm)
(b)
1.2
5.0
BC_Pd1
BC_Pd2
BC_Pd3
BC1
1.0
0.8
0.6
0.4
0.2
0.0
2.5
0.0
0
20
40
60
80
100
120
Time (min)
Figure 3. (a) Time-course of fluorescence intensity ratio (I600/I530
)
changes of BC_Pd1, BC_Pd2, and BC_Pd3, each at 5.0 μM,
upon addition of an equivalent amount of (a) PdCl2 and (b) Pd(-
PPh3)4 in CH3CN–water (1/9) at 25 ꢀC, obtained under excitation
at 400 nm, the isosbestic point.
450
500
550
600
650
700
750
Wavelength (nm)
2x105
(a)
Figure 2. Normalized (a) absorption and (b) emission spectra of
BC_Pd1, BC_Pd2, BC_Pd3 and BC1, measured in CH3CN–
water (1/9, by volume) at 25 ꢀC under excitation at the maximum
absorption wavelength of each dye. A, absorbance; RFU, relative
fluorescence unit.
BC_Pd3
1 min
Pd(0)
3 min
5 min
10 min
1x105
20 min
30 min
45 min
60 min
5x104
90 min
120 min
analysis of residual Pd species, we need to compare the ratio
value at a given time with respect to those in a standard
curve/data.18
0
500
550
600
650
700
750
The fluorescence titration data with the best-performing
BC_Pd3 toward the two Pd species revealed its rati-
ometric detection behavior and the relative response rate
(Figure 4). The results suggest that the catalytic
depropargylation step (Scheme 1) proceeds faster with
Pd(0) species than with Pd(II) species. When we moni-
tored the ratio change of BC_Pd3 (5.0 μM) in the pres-
ence of varying concentration of (Ph3P)4Pd or PdCl2 for a
fixed reaction time of 2 h, the ratio signal became satu-
rated approximately at two equivalents of the
Pd(0) source. From the initial-stage data (Figure 5), the
detection limits of BC_Pd3 were determined to be 14 nM
and 55 nM for the Pd(0) and Pd(II) species, respectively.
It seems that the ratio value is dependent on the Pd source,
which suggests that the metal ligands also affect the emis-
sion intensity of the probe and its product.
Wavelength (nm)
BC_Pd3
1 min
5 min
(b)
5x104
3x104
Pd(II)
10 min
20 min
30 min
45 min
60 min
90 min
120 min
180 min
2x104
0
500
550
600
650
700
Wavelength (nm)
Figure 4. Fluorescence titration of BC_Pd3 (5 μM) with
(a) (Ph3)4Pd and (b) PdCl2, each at 5.0 μM in CH3CN–water (1/9,
by volume) at 25 ꢀC. The excitation wavelength was 400 nm.
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(a)
14
12
10
8
Pd(Ph3P)4
6
4
2
0
2
4
6
8
10
Pd(0), μM
(b)
PdCl
2
4
2
0
2
4
6
8
10
Pd(II), μM
Figure 5. Fluorescence intensity ratio (I600/I525) of BC_Pd3
(5.0 μM) versus increasing concentrations of the (a) Pd(0) and
(b) Pd(II) species in CH3CN–water (1/9). The ratio values were
acquired after 2 h incubation at 37 ꢀC. The excitation wavelength
was 400 nm.
Figure 7. Fluorescence images of A549 cells incubated with
Pd_BC3 (5.0 μM) in phosphate buffer saline (PBS) (10 mM,
pH 7.4) for 2.0 h; - with Pd(Ph3)4 (30 μM) for 30 min and then
with Pd_BC3 (5.0 μM) in PBS (10 mM, pH 7.4) for 2.0 h; - with
PdCl2 (30 μM) for 30 min and then with Pd_BC3 (5.0 μM) in
PBS (10 mM, pH 7.4) for 2.0 h at 36 ꢀC. (a) The images were
obtained under one-photon excitation at 405 nm and collection of
emissions through green (450–555 nm) and red (570–750 nm)
windows, respectively. The ratiometric images were constructed
from the pixel-to-pixel intensity ratio values between the red and
green (IRed/IGreen) images. The scale bar is 75 μm. (b) Respective
intensity plots of the ratio images.
Finally, we compared the sensing selectivity of BC_Pd3
toward other metal species. The ratio values (I600/I530),
observed in the presence of Cr(II), Ca(II), Co(II), Cu(II),
Ni(II), Mg(II), Hg(II), Mn(II), Cd(II), Ag(I), Ba(II), Zn(II),
Pb(II), Pt(II), Fe(III), Cr(III), Fe(II), Pt(IV), and Rh(III) spe-
cies, each at 10 equivalents, reveal that the probe is highly
selective to Pd(0) and Pd(II) species (Figure 6). The other
metal species caused slight increase or decrease in the ratio
value from the background ratio value (I600/I530 = 0.52 in
the case of the probe only), an inherent feature of the rati-
ometric sensing system whose emission peak overlaps.
Cellular Imaging of Pd Species. We briefly evaluated the
cellular imaging capability of BC_Pd3. A549 cells were
incubated with Pd(0) or Pd(II) species, each at 30 μM for
30 min, and then with the probe (5.0 μM) for 2 h were fluo-
rescently imaged by confocal laser scanning microscopy.
The cellular images were obtained by collecting of green
(450–555 nm) and red (570–750 nm) fluorescence under
Figure 6. (a) Fluorescence intensity ratio (I600/I530) changes of
BC_Pd3 (5.0 μM) toward various metal species (1, probe only;
2, Cr(II); 3, Ca(II); 4, Co(II); 5, Cu(II); 6, Ni(II); 7, Mg(II); 8, Hg(II);
9, Mn(II); 10, Cd(II); 11, Ag(I); 12, Ba(II); 13, Zn(II); 14, Pb(II);
15, Pt(II); 16, Fe(III); 17, Au(III); 18, Cr(III); 19, Fe(II); 20, Pt(IV);
21, Rh(III); 22, Pd(0); 23, Pd(II); as chloride salts except Pt(II), for
which H2PtCl6 was used) in CH3CN–water (1/9), measured after
2 h at 25 ꢀC.
excitation at 405 nm. The corresponding ratio (IRed/IGreen
)
images were significantly higher than that obtained for the
cells incubated with probe only (Figure 7). The results
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show that BC_Pd3 can be used to detect Pd(0)/Pd
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Acknowledgments. This work was financially supported
by the Global Research Laboratory Program (NRF-
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Conflict of Interest. The authors declare no conflict of
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