A versatile long-wavelength-absorbing scaffold for Eu-based responsive probes

Article abstract of DOI:10.1002/chem.201203957

Coumarin-sensitized, long-wavelength-absorbing luminescent Eu ^III-complexes have been synthesized and characterized. The lanthanide binding site consists of a cyclen-based chelating framework that is attached through a short linker to a 7-hydroxycoumarin, a 7-B(OH)₂-coumarin, a 7-O-(4-pinacolatoboronbenzyl)-coumarin or a 7-O-(4-methoxybenzyl)-coumarin. The syntheses are straightforward, use readily available building blocks, and proceed through a small number of high-yielding steps. The sensitivity of coumarin photophysics to the 7-substituent enables modulation of the antenna-absorption properties, and thus the lanthanide excitation spectrum. Reactions of the boronate-based functionalities (cages) with H₂O ₂ yielded the corresponding 7-hydroxycoumarin species. The same species was produced with peroxynitrite in a ×10⁶-10 ⁷-fold faster reaction. Both reactions resulted in the emergence of a strong ≈407 nm excitation band, with concomitant decrease of the 366 nm band of the caged probe. In aqueous solution the methoxybenzyl caged Eu-complex was quenched by ONOO^-. We have shown that preliminary screening of simple coumarin-based antennae through UV/Vis absorption spectroscopy is possible as the changes in absorption profile translate with good fidelity to changes in Eu^III-excitation profile in the fully elaborated complex. Taken together, our results show that the 7-hydroxycoumarin antenna is a viable scaffold for the construction of turn-on and ratiometric luminescent probes. A visible change: Eu complexes can be rendered analyte responsive by exploiting the sensitivity of a coumarin antenna to the nature of the 7-substituent (see figure). A boronate switch installed in the 7-position afforded complexes that responded rapidly to μM concentrations of peroxynitrite; the product of this reaction being the corresponding phenol. The analogous reaction with H ₂O₂ proceeds at a significantly lower rate. As the reaction shifts the absorption to longer wavelengths, the construction of ratiometric probes is possible. Copyright

Full text of DOI:10.1002/chem.201203957

FULL PAPER
DOI: 10.1002/chem.201203957
A Versatile Long-Wavelength-Absorbing Scaffold for Eu-Based
Responsive Probes
Csongor Szꢀjjꢁrtꢂ,[a] Elias Pershagen,[a, b] Nadia O. Ilchenko,[b] and K. Eszter Borbas*[a, b]
Dedicated to Professor Jꢀzsef Rꢁbai
Abstract: Coumarin-sensitized, long-
wavelength-absorbing luminescent
tenna-absorption properties, and thus
the lanthanide excitation spectrum. Re-
actions of the boronate-based function-
alities (cages) with H2O2 yielded the
corresponding 7-hydroxycoumarin spe-
cies. The same species was produced
with peroxynitrite in a ꢀ106–107-fold
faster reaction. Both reactions resulted
in the emergence of a strong ꢀ407 nm
excitation band, with concomitant de-
crease of the 366 nm band of the caged
probe. In aqueous solution the me-
thoxybenzyl caged Eu-complex was
G
ACHTUNGTRENNUNG
EuIII-complexes have been synthesized
and characterized. The lanthanide
binding site consists of a cyclen-based
chelating framework that is attached
through a short linker to a 7-hydroxy-
coumarin, a 7-B(OH)2-coumarin, a 7-
O-(4-pinacolatoboronbenzyl)-coumarin
or a 7-O-(4-methoxybenzyl)-coumarin.
The syntheses are straightforward, use
readily available building blocks, and
proceed through a small number of
high-yielding steps. The sensitivity of
coumarin photophysics to the 7-sub-
stituent enables modulation of the an-
quenched by ONOOÀ. We have shown
that preliminary screening of simple
coumarin-based antennae through UV/
Vis absorption spectroscopy is possible
as the changes in absorption profile
translate with good fidelity to changes
in EuIII-excitation profile in the fully
elaborated complex. Taken together,
our results show that the 7-hydroxycou-
marin antenna is a viable scaffold for
the construction of turn-on and ratio-
metric luminescent probes.
Keywords: coumarins · cyclen · eu-
ropium · fluorescent probes · lan-
thanides · reactive oxygen species
Introduction
substituents (“antennae”) can increase the brightness of the
complex by several orders of magnitude.[2]
Lanthanide-based luminescent probes have come a long way
since their first applications as spectroscopically active Ca
substitutes in proteins.[1] Complexes of increasing sophistica-
tion are applied to monitor biologically relevant targets in
vitro and in cellulo.[2] The popularity of lanthanide-based
probes can be ascribed to their exceptionally long excited-
state lifetimes, which enables the filtering-out of sample au-
tofluorescence and scattered excitation light.[2] Whereas lan-
thanide absorption coefficients are typically ꢀ1–10, multi-
dentate chelating frameworks that can shield the metal from
Responsive lanthanide probes can be obtained by analyte-
dependent regulation of antenna photophysics,[2b–d,f,3] the an-
tenna-lanthanide energy-transfer,[2b–d,f,3] or the Ln-emission
lifetime.[4] Turn-on probes are preferred to those for which
detection is accompanied by a change in signal intensity due
to their inherently larger sensitivity. However, such probes
require calibration before they can report on analyte con-
centrations. Additional factors, for example, antenna bleach-
ing, and fluctuations in excitation light intensity and probe
concentration can complicate the situation. These unpredict-
able variations can be eliminated by the use of ratiometric
probes.
À
solvent O H oscillators in combination with light-harvesting
Of all the known antennae a few stand out as particularly
attractive: Fluorescein and rhodamine are good sensitizers
for the near infrared (NIR) emitting Yb and Nd,[5] and strat-
egies exist to transpose the rich chemistry of fluorescein-
based responsive probes into the lanthanide probes
domain.[6] A major drawback is the awkward synthesis of
fluorescein derivatives.[7] Additionally, most laboratories are
not equipped to perform NIR emission spectroscopy, and
fluorescein does not sensitize the visible-emitting lantha-
nides (Eu, Tb). Parker and co-workers have developed Eu-
sensitizing azaxanthones and azathioxanthones.[8] Precursors
to these antennae are readily available on multigram scale
in a two-step process from substituted (thio)phenols and
[a] Dr. C. Szꢁjjꢂrtꢃ,+ E. Pershagen,+ Dr. K. E. Borbas
Department of Chemistry, BMC
Uppsala University
Box 576, 75123 Uppsala (Sweden)
Fax : (+)46-18-4714988
E-mail: eszter.borbas@kemi.uu.se
[b] E. Pershagen,+ N. O. Ilchenko, Dr. K. E. Borbas
Department of Organic Chemistry
Arrhenius Laboratory
Stockholm University
10691, Stockholm (Sweden)
[+] These authors contributed equally to this work.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.201203957.
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various 2-chloronicotinic acids.[8b] Complexes incorporating
such antennae have been used for ratiometric oxygen detec-
tion in vitro, as well as the monitoring of changes in intracel-
lular bicarbonate concentration and lysosomal pH.[8j,n,o]
However, the drawback of these probes is that the sensing
mechanism seems to limit them to the handful of reported
analytes.
410 nm.[13] Additionally, the redshift in absorption maximum
upon OH deprotonation would enable the construction of
EuIII-based pH indicators. Encouragingly, the triplet energy
levels of 7-O-alkyl, 7-OH and 7-phenolate coumarins were
all reported to be above ꢀ20000 cmÀ1.[14–16] Efficient sensiti-
zation requires an antenna triplet-lanthanide excited state
energy gap of ꢁ2000 cmÀ1. This puts the red end of the exci-
tation of EuIII complexes at around 430 nm, given that the
most likely EuIII acceptor level is at 17200 cmÀ1.[2f] Coumar-
ins,[17,18] and their N-analogous carbostyrils[19] have been em-
ployed for the sensitization of both EuIII and TbIII. Respon-
sive probes incorporating coumarin antennae have been re-
ported that rely on selective coumarin excited state quench-
ing by nucleotides, dsDNA, or a proximal ketone.[18] Our
group has reported a strategy relying on the in situ analyte-
induced formation of the sensitizing coumarin antenna.[20]
However, the potential of controlling the coumarin excited
state through the 7-substituent to regulate lanthanide emis-
sion has not been explored so far.
Esterified, amidated, or carbamoylated phenol or aniline
antennae can undergo chemoselective ester, amide, or car-
bamate cleavage with concomitant changes in the absorp-
tion/excitation spectrum, and/or lanthanide sensitization ef-
ficiency.[9] Reactivity-based turn-on or ratiometric probes
have been constructed to detect H2O2 and to measure the
activity of enzymes such as phosphatases or peptidases.[9]
Analyte-dependent installation of the OH group has been
C [10]
reported for the detection of OH . A serious limitation of
these probes is their excitation maxima, which rarely extend
beyond 300 nm. Such high-energy light can be damaging to
biomolecules, and is poorly suited for in vivo work.
This work was carried out to investigate coumarin-sensi-
tized Eu-complexes for reactivity-based analyte detection.
In addition to absorbing at >320 nm and emitting light at
>400 nm with high fluorescence quantum yield, coumarins
are also easily synthesized from readily available precur-
sors,[11] and possess appreciable two photon absorption.[12]
Coumarin absorption and emission spectra are sensitive to
the electronic properties of the 7-substituent, with electron-
donating groups affecting a bathochromic shift (Figure 1).[11]
We decided to demonstrate the feasibility of our approach
by the creation of responsive luminescent probes for reac-
tive oxygen species (ROS). ROS and reactive nitrogen spe-
cies (RNS) are attractive targets due to their roles in the
emergence of a variety of pathologies (e.g., neurodegenera-
tive diseases, asthma, stroke) on the one hand, and essential
physiological functions on the other.[21] Key to such discover-
ies was the development of tools to monitor ROS with high
sensitivity and selectivity.[22] In addition to the ones detect-
[9,20,23]
C[10]
ing HO
and H2O2
mentioned above, lanthanide-
[26]
based probes specific for NO,[24] ONOOÀ,[25] 1O2
known.
are
Results and Discussion
Preliminary screening of alkylated coumarins: 7-Hydroxy-
coumarin derivatives O-alkylated with H2O2-cleavable 4-p-
pinacolatoboron benzyl bromide, or one of three other
benzyl bromides with differing electron-rich moieties were
prepared. It was expected that by fine-tuning the substitu-
tion pattern in the benzyl ether we could discover caging
groups that can be selectively cleaved by one of the ROS in
preference of the others. Alternatively, differential responses
to ROS might be exploited to obtain “fingerprints” for the
different species. Such a strategy is reminiscent of the pat-
tern-based recognition of peptides,[27a,b] terpenes,[27c] flavo-
noids[27d], and plastic explosives[27e] based on UV/Vis absorp-
tion changes. Alkylated coumarins 3a–d were prepared
from 7-hydroxycoumarin 1 (Scheme 1). Alkylation with the
appropriate benzyl bromides or chlorides in the presence of
K2CO3 afforded 2a–d in good yields. Exposure to NaOH in
a mixture of H2O and MeOH yielded the carboxylic acids
after aqueous/organic work-up and precipitation from meth-
anolic solutions with Et2O.
Figure 1. a) Responsive luminescent probes based on analyte-modulated
changes in the photophysical properties of the coumarin antenna.
b) Energy diagram of 7-O-alkylated and 7-phenolate coumarins and the
most likely accepting levels of EuIII. The location of the coumarin triplet-
states in relation to the EuIII 5D2 level is not known.
If coupled to lanthanide emission, such changes offer the
possibility of ratiometric luminescent probe development.
Such a design was supported by literature data that placed
the absorption maxima of 7-O-alkylcoumarins at 330–
360 nm, and that of the corresponding phenolate at ꢀ370–
Methanolic solutions of 2a–d and 3a–d were treated with
À
CÀ
C
various ROS or RNS (H2O2, O2 , HO , NO2 , the Support-
ing Information, Figures S1–S3). Boronate-caged 2a and 3a
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Eu-Based Responsive Probes
FULL PAPER
tain protected carboxylic acids
that can later stabilize the lan-
thanide complex, as well as a
primary amine that could serve
as the point of attachment for
the antennae. Additionally, they
are readily prepared on a multi-
gram scale (see the Supporting
Information for the synthesis of
4Et).[30] The primary amines in
4Et or 4tBu and the carboxylic
acid in 5 were reacted with
HATU (1-[bis(dimethylamino)-
Scheme 1.
reacted with H2O2, as evidenced by a decrease in absorption
at 332 nm and a concomitant increase at 387 nm. The spec-
trum of dimethoxybenzyl-caged 3c did not change apprecia-
bly under these conditions, that of 3d decreased by 25% in
the presence of KO2. Exposure of 4-methoxybenzyl-caged
3b resulted in a decrease in absorption at 332 nm and the
emergence of a shoulder that slowly developed into a new
peak at 447 nm. Based on these results we decided to incor-
porate 7-(4-pinacolatoboronbenzyl)-coumarin and 7-(4-me-
thoxybenzyl)-coumarin antennae into the Eu complexes.
methylene]-1H-1,2,3-triazoloACTHNGUETRNNU[G 4,5-b]pyridinium 3-oxid hexa-
fluorophosphate) as the coupling reagent in the presence of
DIPEA. Other activation methods (acid chloride, EDCI/
HOBt) were inferior to HATU/DIPEA, resulting in either
no product formation, or irreproducible yields. Key inter-
mediates 6Et and 6tBu were isolated in 67 and 65% yields,
respectively. Exposure of 6tBu to a 1:1 mixture of TFA and
CH2Cl2 for 18 h cleaved the tert-butyl esters quantitatively.
Heating with EuCl3 in MeOH, followed by precipitation
with Et2O afforded EuLOH in 57% yield as a pale yellow
solid (a discussion of the coordination environment of the
EuIII is given below).
Coumarin-appended lanthanide complexes: One of our aims
was a readily accessible core structure that can serve as the
starting point for luminescent probe development. Thus we
developed a scalable, high yielding and short synthetic
route. Initial attempts involved the synthesis of 4-azidometh-
yl-7-hydroxycoumarin (Cou-Az)[28] with the plan of intro-
ducing it into alkynyl-lanthanide complex Eu-Alk[29a]
(Scheme 2) through a Cu-catalyzed azide–alkyne cycloaddi-
tion.[29] This route was particularly attractive due to its high
convergence. However, upon exposure to a variety of cyclo-
addition reaction conditions, only complete decomposition
of Cou-Az was observed. The lability of Cou-Az was con-
firmed by its rapid decomposition when we attempted to
reduce the azide to the amine [PPh3/H2O, H2/Pd(C)].
The 7-hydroxycoumarin-bearing 6Et was functionalized at
the 7-OH position. Alkylation with 4-pinacolatoboron
benzyl bromide or 4-methoxybenzyl chloride afforded
8Bpin or 8OMe cleanly in good yield (69–83%). Overal
ACHTUNGTRENNUNGkyl-
AHCTUNGTRENNUNG
1
by ESI-MS analysis or H NMR analysis. The reaction could
be monitored by TLC analysis despite the small differences
in the polarities of starting materials and products by follow-
ing the disappearance of the characteristic blue 7-OH cou-
marin-fluorescence. This diversification step on an advanced
intermediate enables the efficient synthesis of a broad varie-
ty of complexes. Cleavage of the ethyl esters was possible
with an excess of NaOH in a mixture of H2O/MeOH/THF.
Treatment of 9Bpin or 9OMe with EuCl3 in hot MeOH, fol-
lowed by precipitation with diethyl ether and freeze-drying
from water yielded EuLBpin or
Cyclen derivatives 4Et and 4tBu[30,31] were chosen as al-
ternative scaffolds (Scheme 3). Both of these structures con-
EuLOMe in 84 and 73% yield,
respectively.
The synthesis of analogous
complexes equipped with two
photon absorbing 6,8-dibromo-
coumarin antennae[32] was at-
tempted starting from 3,5-di-
bromo-2,4-dihydroxybenzalde-
hyde (the Supporting Informa-
tion, Scheme S1).[33] However,
this route was ultimately aban-
doned due to the instability of
some of the intermediates. A
discussion of the synthesis is
given in the Supporting infor-
Scheme 2.
mation.
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K. E. Borbas et al.
ture and an aqueous/organic
work-up. The crude product
was >95% pure as shown by
1H NMR analysis. In situ acid
chloride formation with SOCl2
and catalytic DMF,[34] followed
by the addition of excess tert-
butanol afforded 13 in 61%
yield as a pale yellow solid. Al-
ternative methods for the tert-
butyl ester formation were less
satisfactory. EDCI/DMAP me-
ACHUTNGRENdNUG iACTHNGUTRENNUG
ated esterification[35] with tert-
butanol gave the isolated 13 in
29–41% yield (48% based on
recovered starting material).
Reaction of 12 with tert-butyl
trichloroacetimidate and cata-
[36]
lytic BF3·OEt2 afforded 13 in
54% yield, however, the purifi-
cation of this product was tedi-
ous due to the presence of co-
eluting impurities. Alternative
carboxylic acid activation with
carbonyl-diimidazole in DMF
in the presence of DBU and
tert-butanol[37] afforded 76% of
a compound that was tentative-
ly assigned as the decarboxylat-
ed derivative of 12.
Miyaura borylation[38] (condi-
tions: (Bpin)2, PdCl2ACHTUNGTRENNUNG(dppf),
KOAc, dioxane, and 908C) pro-
vided 14 in good yield. Cleav-
age of the tert-butyl ester was
quantitative in 1 h with TFA/
CH2Cl2 (1:1). We found it nec-
essary to dilute the reaction
mixture with an equal volume
of toluene prior to removal of
the volatile components to
Scheme 3.
avoid hydrolysis of the boro-
nate ester. Carboxylic acid 15
The effect of masking the 7-OH functionality as a boronic
was then coupled to 4tBu in the presence of HATU and
DIPEA to afford 16 in 62% yield after column chromatog-
raphy. Quantitative cleavage of the tert-butyl esters to
obtain 17 necessitated overnight exposure of 16 to a 1:1 mix-
ture of TFA and CH2Cl2, which resulted in complete hydrol-
ysis of the boronate ester to the boronic acid. Treatment of
acid on the photophysical properties of the coumarin anten-
na and the corresponding Eu complex was also studied.
Coumarin-7-boronic acid-functionalized EuLB(OH)2 was pre-
pared as shown in Scheme 4. The base-labile ethyl ester in
7-bromocoumarin 11 was replaced with acid-cleavable tert-
butyl ester in a two-step procedure. This was deemed neces-
sary for synthetic tractability, as the boronate ester moiety
might be destroyed when exposed to high concentrations of
aqueous base. Whereas not detrimental per se, a mixture of
poorly defined boronic acid and partially protected boronate
ester species was expected to complicate further synthetic
steps. Hydrolysis with aqueous NaOH provided carboxylic
acid 12 in 80% yield after acidification of the reaction mix-
ligand 17 with EuACTHNUTRGNEUNG
(OTf)3 gave EuLB(OH)2 in 79% yield after
precipitation from MeOH with Et2O.
Photophysical characterization of lanthanide complexes:
The UV/Vis absorption spectra of coumarins, ligands and
Eu-complexes were recorded in MeOH (the Supporting In-
formation, Figures S4 and S5). All 7-O-benzylated species
had strong (eꢀ ꢀ104) absorption bands in the 335–348 nm
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Eu-Based Responsive Probes
FULL PAPER
mation, Figures S7 and S8).
This shows that Eu can be sen-
sitized by both the neutral and
anionic forms of 7-hydroxycou-
marin. It is worth noting that
the pKa of 7-hydroxycoumarins
are easily variable between
ꢀ3.7 and ꢀ7.8, which provides
a straightforward way to in-
crease the proportion of the
long-wavelength-absorbing phe-
nolate at physiological pH.[39c]
The lifetimes of the com-
plexes in H2O and D2O were
measured (Table 2). From this,
the number of Eu-bound water
molecules (q) were calculated
using the equation q=1.2ꢀ[1/
tHÀ1/tDÀ(0.25+0.075x)],
in
which x=exchangeable NH-os-
cillators.[42,43] All complexes
have similar hydration states
(q<1), which indicates that in
addition to the three carboxy-
lates, the amide carbonyl
carbon in the linker moiety can
also coordinate to the metal.
The hydration state does not
change upon cleavage of the
H2O2-responsive
cages.
boronate
The quantum yields of the
uncomplexed ligands and the
Eu-complexes were determined
(Table 2). The overall Eu quan-
tum yields were 0.39, 0.45, and
2.70% for EuLBpin, EuLOMe, and
Scheme 4.
region, attributed to a p–p* transition. The lmax of this band
depended on the 3-substituent (carboxylic acid, ethyl ester,
or amide). The absorption spectra of coumarin-phenols 7
and EuLOH and that of EuLB(OH)2 were pH-sensitive. Increas-
ing the pH resulted in the emergence of a redshifted peak
attributed to the phenolate[39] or boronate anion. For EuLOH
this new peak emerged at ꢀ405 nm, with an isosbestic point
at 375 nm. The pKa of EuLOH was determined to be ꢀ6.8 by
spectrophotometric titration (the Supporting Information,
Figure S6), a typical value for a 3-carboxy-7-hydroxycoumar-
in.[39c] The spectra of the O-benzylated ligands and their Eu-
complexes were similar to that of their respective antennae,
but blueshifted by 10–12 nm (Table 1).
The excitation spectra of all Eu complexes were recorded
in HEPES buffer (Figure 2). The spectra were similar to the
absorption spectra, consisting of a single peak around
360 nm for EuLBpin and EuLOMe. The excitation spectrum of
EuLOH consisted of a broad peak centered at 356 nm below
pH 6.5. Above pH 7 this peak disappeared, and a new peak
at 408 nm from the anion emerged. This redshifted peak ex-
tended up to ꢀ430 nm (Figure 3 and the Supporting Infor-
Figure 2. Excitation spectra of EuLBpin, EuLOMe, EuLB(OH)2 and EuLOH
(lem =700 nm, Eu complex (10 mm) in HEPES (10 mm), pH 7, RT).
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K. E. Borbas et al.
Table 1. Absorption and emission data for Eu complexes and their pre-
cursors.[a]
Table 2. Photophysical properties of Eu complexes.
Entry
tH O[ms]
tD O [ms]
q
F
2
2
Entry
lmax
e
lem [nm]
ACHTUGNERTN(NUGN lex/[nm])
(k
A
(k
A
[%][e]
[nm]
A
]
EuLOH
0.59Æ0.02
(1.69)
0.66Æ0.02
À0.2 (0.2)[c,d] 2.70[f]
1
5
351[b]
–
406[b]
(1.52)
74.9[g]
339, 380[c] 4.21, 3.80[c] 450 (385)[c]
0.41Æ0.02[c]
(68.3)[h]
6Et
368, 409[d] 3.86, 4.09
355
407 (370)[d]
EuLB(OH)2 1.15Æ0.03
(0.870)
3.49Æ0.30
(0.287)
0.3 (0.6)[c] n.d.[i]
EuLOH
4.04
410, 578, 587, 592, 615, 653, 700
(320)[a]
1.14Æ0.02[b]
3.12Æ0.27
407, 578, 587, 592, 615, 653, 700
(373)[e]
(0.321)
EuLBpin
EuLOMe
1.11Æ0.04
(0.901)
2.61Æ0.14
(0.383)
0.2 (0.5)[c] 0.39[f]
2.60[g]
2a
348
4.20
407 (370)
3a
347
4.20
407 (374)
1.02Æ0.03[b]
1.16Æ0.04
(0.862)
(3.95)[h]
8Bpin
EuLBpin
2b
338
290, 334
351
4.00
3.72, 3.88
3.98
407 (363)
2.50Æ0.17
0.2 (0.5)[c] 0.45[f]
0.90%[g]
450, 578, 587, 615, 700 (367)[f]
407 (373)
(0.400)
0.81Æ0.04[b]
(14.6)[h]
3b
350
4.20
413 (372)
[a] lex =338, lem =614 nm in the solvent indicated. [b] Under an Ar at-
mosphere. [c] Calculated without correction for the proximal NH-oscilla-
tors by using q=1.05ꢀ(1/tHÀ1/tD). [d] lex =363 nm. [e] In methanol,
lex =360 nm. [f] Eu-quantum yield. [g] Coumarin quantum yield in com-
plex. [h] Coumarin quantum yield in ligand. [i] n.d.=not determined.
8OMe
EuLOMe
14
338
4.01
3.76, 3.94
3.99, 3.68
404 (363)
284, 338
297, 335
411, 578, 591, 615, 700 (359)[e]
422 (350)
15
297, 335[d] 4.28, 4.02
425 (365)
17
302, 336
3.91, 3.75
3.85, 3.84,
3.25
409 (367)
EuLB(OH)2 312, 346,
443, 578, 587, 592, 595, 615, 688,
408
700 (346)[e]
[a] In MeOH. [b] From Ref. [40]. [c] From Ref. [41]. [d] In DMSO. [e] In
HEPES (10 mm) at pH 7. [f] In H2O.
to 74.9% (EuLOH). The increase in coumarin quantum yield
upon EuIII complexation compared with the free ligand may
be down to a change in coumarin OH pKa upon coordina-
tion of the amide carbonyl to the metal; the coumarin phe-
nolate is significantly more emissive than the neutral form.
Eu complexation decreased the coumarin fluorescence com-
pared with that of the free ligand for EuLBpin and EuLOMe, in
the latter case quite dramatically, from 14.6 to 0.90%. This
decrease could be due to the external heavy-atom effect of
Eu.[45] Photoinduced electron-transfer from the antenna to
EuIII forming the relatively accessible EuII may also contrib-
ute to a diminished antenna quantum yield.[45]
The instrumentation available for us did not enable the
determination of the triplet levels of the antennae. Howev-
er,
a lower estimate for the anionic triplet state of
19300 cmÀ1 (518 nm) can be obtained by monitoring the
sensitivity of Eu-emission to quenching by oxygen. The pres-
ence of atmospheric oxygen did not appreciably affect the
europium emission intensities and lifetimes of EuLOH, Eu-
LBpin and EuLOMe. This invariance indicates that if energy
transfer happens from the coumarin triplet state, it is fast
and irreversible. Thus the triplet state is >2000 cmÀ1 above
the 17300 cmÀ1 EuIII excited state (the Supporting Informa-
tion, Figure S9–S11, Table 2). The literature values of
(22000Æ300) cmÀ1 for the triplet state of 3-chloro-4-methyl-
7-methoxycoumarin (analogous to the antenna in EuLBpin
and EuLOMe),[15] and 21320 cmÀ1 for 7-hydroxycoumarin[14]
are consistent with this finding.
Figure 3. pH-dependence of the emission spectrum of EuLOH (lex
=
410 nm, EuLOH (10 mm) in HEPES (10 mm), RT, delay 50 ms, sample
window 1050 ms, pH values: 4.0, 5.0, 6.4, 7.0, 7.5, 8.0, 8.3, 9.3, 10).
EuLOH, respectively. These values are typical for coumarin-
sensitized Eu-complexes,[17a] and are in the range reported
for several well-performing responsive probes.[8c,9a,c,20,44] The
coumarin fluorescence increased dramatically when going
from O-alkylated- to free hydroxyl 7-substituents. This
change is analogous to that observed for 7-substituted cou-
marins carrying modestly and strongly electron-donating
groups. The same effect is seen upon phenol deprotonation.
Both these observations are explained by the contribution
of the electron-donating 7-substituent on the charge-transfer
The Eu-emission intensity of EuLB(OH)2 decreased in the
presence of oxygen compared with that of a deareated solu-
tion, indicating the sensitivity of the antenna excited state to
quenching by triplet oxygen (the Supporting Information,
Figure S12). A possible explanation is that the triplet state
of EuLB(OH)2 lies below that of EuLOH, and thus closer to the
Eu excited state, making the back energy-transfer from the
lanthanide to the antenna possible. The detailed photophysi-
character of the
ACHTUNGTRENNUNG
1(p–p*) level. From 2.60% (EuLBpin) and
0.90% (EuLOMe), coumarin fluorescence quantum yield rose
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Eu-Based Responsive Probes
FULL PAPER
cal investigation of all Eu complexes is in progress, and will
be reported separately.
Reactions of Eu-complexes with ROS: Aryl boronates are
well-established H2O2-selective switches. Boronate cages are
often incorporated into ROS-sensitive prodrugs,[46] pro
ACHTUNGTRENNUNG
ACHTUNGTRENNUNGlatACHTUNGTRENNUNG
Scheme 5.
formation of the corresponding phenols when exposed to
H2O2. The addition of H2O2 to a 1 mm solution of EuLBpin
yielded a gradual decrease in Eu emission when the excita-
tion was performed at 361 nm, and an increase upon excita-
tion at 397 nm (Figure 4), indicative of the formation of
disappearance of CH3CH2O2C-signals (t, dꢀ1.30 ppm and
q, ꢀ3.77 ppm) and emergence of signals associated with
free EtOH (t, dꢀ1.17 ppm and q, ꢀ3.62 ppm).[48] 1H NMR
spectroscopy confirmed that 2b did not react with H2O2
even under forcing conditions (1 mm H2O2, several hours).
We subjected EuLBpin and EuLB(OH)2 to a range of ROS
and RNS (H2O2, OClÀ, tBuOOH, ·OH, NO2 , NO3 ,
À
À
1
À
ONOOÀ, O2, NO, O2 , Figure 5 and the Supporting Infor-
mation, Figures S17–S19). Both probes displayed good selec-
tivity for H2O2 over most ROS. Peroxynitrite was an excep-
tion, producing an instantaneous, robust decrease in the
labs =360 nm absorption, with a concomitant increase at
labs =405 nm (Figure 5 and the Supporting Information, Fig-
Figure 4. Changes in the ratio of the 698 nm Eu emission intensity with
361 nm and 397 nm excitation wavelengths in a sample of EuLBpin in the
presence of 1 mm H2O2. I: intensity (a.u.) with lex =397 nm, I’: intensity
(a.u.) with lex =361 nm (EuLBpin (10 mm), pH 7 HEPES buffer (10 mm),
RT).
EuLOH. The complex EuLB(OH)2 behaved similarly to EuLBpin
.
The ratio of the 689 nm-peak intensities (excitation at 320
and 400 nm) increased from 0.54 to 1.50 in the presence of
100 mm H2O2 (the Supporting Information, Figures S13 and
S14). These data reflect the differences in the excitation
spectrum of EuLB(OH)2 on the one hand and EuLOH on the
other. The former is excitable at 320–340 nm, and not at
400 nm. EuLOH is readily excitable at ꢀ400 nm and only
modestly at 320–360 nm.
Figure 5. Reaction of Eu complexes with highly reactive ROS/RNS at
pH 7.5 in the presence of catalase (Eu complex (10 mm), ROS/RNS
(200 mm) in HEPES buffer (200 mL, 10 mm, pH 7.5, containing
0.1 mgmLÀ1 catalase), lex =356, lem =615 nm, 50 ms delay, 1050 ms sample
C
window). OH was generated in the Fenton system from FeSO4 and
H2O2. A stock solution of peroxynitrite was generated by mixing aqueous
solutions of H2O2 (0.65m) and KNO2 (0.6m), the resulting solution was
treated with MnO2 to eliminate excess H2O2. The concentration of per-
NMR/ESI studies: We followed the reactions of alkylated
coumarins 2a and 2b with ROS by ESI-MS spectrometry
and 1H NMR spectroscopy. In accordance with the litera-
ture, the UV/Vis spectroscopic studies and the fluorescence
measurements, compound 2a reacted with H2O2 to afford
the corresponding phenol 1. The initial product arising from
the reaction of 2a and KO2 in DMSO was tentatively identi-
fied as 18 (Scheme 5) by ESI-MS (m/z [M+K+H2O]+
409.3, [M+Na+H2O]+ 393.3; the Supporting Information,
Figures S15 and S16). In methanolic solution this species
slowly hydrolyzed to the corresponding free acid 19 (m/z
[M+Na]+ 363.1), and to 7-OH coumarin 1 (m/z [M+Na]+
AHCTUNGERTGoNNUN xynitrite was determined by measuring its absorption at 302 nm (e=
1670). Singlet oxygen was prepared by mixing equimolar amounts of
H2O2 and NaOCl in water. NO was generated from diethylenetriamine/
nitric oxide adduct. The superoxide solution was generated from a mix-
ture of xanthine (0.1m) and xanthine oxidase (0.1 unitmLÀ1).
ure S20). These data, in combination with ESI-MS analysis
of the crude reaction mixture (m/z found: 728.7695; calcd
for [EuLOH +H]+: 728.1434; m/z found: 750.7965; calcd for
[EuLOH +Na]+: 750.1254) suggest that the product of the re-
action of ONOOÀ is EuLOH, the same as that with H2O2. In
fact, Kalyanaraman and co-workers have recently found
1
257.0). H NMR spectroscopy in CD3OD clearly showed the
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K. E. Borbas et al.
that peroxynitrite reacted at least a million fold faster than
H2O2 with alkyl and aryl boronates giving the corresponding
alcohols.[49] Building on these findings, Yu et al. have devel-
oped the first ONOOÀ-selective fluorescent probe consisting
of pyreneboronic acid. This probe could be used to monitor
ONOOÀ levels in PMA-stimulated macrophages.[50]
Peroxynitrite induced concentration-dependent changes in
the Eu-emission of EuLBpin and EuLB(OH)2 at low micromolar
concentrations (Figure 6 and the Supporting Information,
zyl-caged 2a reacted 5.2-fold faster than aryl pinacolatobo-
AHCTUNGTRENNUNG
ron-based 2e (0.900mÀ1 sÀ1 (R2 =0.96) vs. 0.173mÀ1 sÀ1, (R2 =
0.96), respectively). These results are in line with those of
Cohen and co-workers, who reported similar differences be-
tween boronates attached through benzyl ether self-immola-
tive linkers and aryl boronates.[52] Pseudo-first-order rate
constants for the reactions of 2a and 2e were measured
using a stopped-flow instrument in fluorescence emission
mode (the Supporting Information, Figures S23 and S24).
This was required as nitrite absorbs at 354 nm, which inter-
fered with the coumarin absorption measurements. Further-
more, it was necessary to use peroxynitrite as the limiting
reagent, as at high peroxynitrite concentrations a decrease
in emission output was recorded after an initial fluorescence
emission intensity increase (the Supporting Information,
Figure S25). The decrease in coumarin fluorescence is pre-
sumably due to uncontrolled oxidation of the relatively elec-
tron-rich hydroxycoumarin core, and is similar to that ob-
served previously with other fluorescent redox indicators.[49b]
Under these conditions, pseudo-first-order rate constants of
3.00ꢀ103 mÀ1 sÀ1 (R2 =0.95) and 1.68ꢀ105 mÀ1 sÀ1 (R2 =0.97)
were obtained for 2a and 2e, respectively, corresponding to
a 1 million-fold faster reaction for 2e with ONOOÀ com-
pared with that with H2O2. The change in order of reactivity
with ONOOÀ compared to that with H2O2 presumably re-
À
flects a change in rate-determining step from C B bond oxi-
dation to quinone methide elimination for 2a.
Figure 6. The Eu-intensity change of EuLBpin in the presence of various
concentrations of ONOOÀ (30 min incubation time, EuLBpin (10 mm),
HEPES buffer (10 mm), pH 7.5, lex =356, lem =615 nm, 50 ms delay,
1050 ms sample window). A stock solution of peroxynitrite was generated
by mixing aqueous solutions of H2O2 (0.65m) and KNO2 (0.6m), the re-
sulting solution was treated with MnO2 to eliminate excess H2O2. The
concentration of the peroxynitrite stock solution was determined by
measuring its absorption at 302 nm (e=1670).
Peroxynitrite is a strong, but surprisingly selective oxidant
with involvement in inflammation, stroke and neurodegener-
ation.[53] Low concentrations of peroxynitrite have also been
shown to modulate cell signaling.[54] Based on the above re-
sults, EuLBpin and EuLB(OH)2 are selective probes for
ONOOÀ. However, it should be noted that the different in-
tracellular lifetimes (H2O2: ꢀ10 ms[55] vs. ONOOÀ: 10–
20 ms),[56] concentrations (H2O2: low micromolar to nano-
molar[57] vs. ONOOÀ: nanomolar),[53,54] and localization pro-
files of these two species mean that H2O2 could potentially
interfere with the detection of ONOOÀ.
Figure S20). The Eu-emission from methoxybenzyl ether-
functionalized EuLOMe was quenched by ONOOÀ (the Sup-
porting Information, Figure S20). This result is interesting,
as EuLOMe does not react with H2O2, thus giving a potential-
ly very selective response to a single ROS/RNS. Whereas a
slow reaction was observed for the corresponding 7-me-
A
E
Conclusion
KO2, the corresponding Eu complex was inert to this ROS
in aqueous buffers, presumably due to the rapid dismutation
Europium complexes equipped with coumarin antennae
have been prepared in short, concise, and scalable syntheses.
The complexes can be rendered analyte-responsive through
modulation of the electronic properties of the 7-position of
the coumarin antenna. We have demonstrated this principle
by the introduction of ROS/RNS-cleavable caging groups.
Preliminary screening of potential antennae was possible as
changes in the coumarin absorption/emission properties
translated well to changes in the corresponding Eu-complex
photophysics. This way both ratiometric and turn-on probes
could be accessed. 7-Boronate or 7-O-(4-pinacolatoboron-
benzyl) cages were rapidly cleaved by peroxynitrite to the
corresponding phenol, furnishing the first ratiometric single-
component luminescent probes for this RNS. We wish to
point out that the design is not limited to the analytes used
CÀ
of O2 . Quenching of Eu-emission was concentration de-
pendent, and persisted long after the lifetime of ONOOÀ
under the experimental conditions (t1/2 ꢀ1 s at pH 7.4).[51]
This suggests that EuLOMe undergoes
a reaction with
ONOOÀ, however, we have so far been unable to identify
the reaction product. There is one known lanthanide-based
ONOOÀ-responsive ratiometric system, which consists of a
mixture of methoxyphenyl-terpyridine TbIII/EuIII complexes.
Sensing was achieved by selective Tb-emission quenching
through a charge-transfer mechanism.[25]
Pseudo first-order rate constants were determined for the
reactions of 2a and 2e with excess H2O2 by following the
changes in the coumarin absorption at 405 nm (the Support-
ing Information, Figures S21 and S22). Pinacolboronatoben-
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Eu-Based Responsive Probes
FULL PAPER
thank Dr. James I. Bruce and Dr. Luke Pilarski for critical reading of the
manuscript and Dr. Marcin Kalek for helpful advice on the kinetics ex-
periments.
in this work. A large number of biologically and environ-
mentally relevant species install phenols selectively (for ex-
ample, enzymes, metal ions (PdII/0, HgII), small molecules
(H2S)),[20,58] making the current ligands potentially very ver-
satile.
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Experimental Section
General Procedures: 1H NMR (300 MHz, 400 MHz or 500 MHz) and
13C NMR (75 MHz, 100 MHz or 125 MHz) spectra were recorded on a
Varian 300, a Varian 400 or a Bruker 500 MHz instrument, respectively.
Chemical shifts were referenced to residual solvent peaks and are given
as follows: chemical shift (d, ppm), multiplicity (s, singlet; br, broad sin-
glet; d, doublet, t, triplet; q, quartet; m, multiplet), coupling constant
(Hz), integration. IR experiments were performed on a PerkinElmer
Spectrum-100 FT-IR spectrometer equipped with an ATR accessory. HR-
ESI-MS analyses were performed on a Bruker MicroTOF ESI mass spec-
trometer. For accurate mass determination of Eu complexes the 153Eu
isotope was used. Compounds 1,[13d] 4tBu,[30] Eu-Alk,[29] Cou-Az,[28] 4-bro-
mosalicylaldehyde,[59] S1[33] and S7,[30] were synthesized following litera-
ture methods. All other chemicals were from commercial sources and
used as received. Microwave heating was performed with a Biotage Ini-
tiator instrument. Titrations, selectivity measurements and kinetic experi-
ments were carried out at least twice (independent), either representative
or averaged data are shown.
Chromatography: Preparative chromatography was performed using
silica (230–400 mesh). Thin layer chromatography was performed on
silica-coated aluminum plates. Samples were visualized by UV-light (254
and 356 nm), or staining with KMnO4/K2CO3 or cerium ammonium mo-
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Cary 300 instrument; (sh) denotes shoulder to a peak. Steady state and
time-resolved emission spectra were collected at room temperature using
a Horiba Scientific FluoroMax 4 instrument equipped with a flash lamp
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GEMINI EM Dual Scanning Microplate Spectrofluorometer in fluores-
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the Supporting Information, S6–S8, S14, and S17–S20). Hydration states
(q) were determined according to the method developed by Horrocks,[42]
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ˇ
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,
and EuLB(OH)2 with H2O2 were determined following the procedure of
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Acknowledgements
This work was supported by the Swedish Research Council, Carl Tryggers
Stiftelse, and the Departments of Organic Chemistry, Stockholm Univer-
sity and Chemistry, BMC, Uppsala University. We thank Prof. Gunnar
von Heijne for access to a plate reader, Prof. Peter Brzezinski and Prof.
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Received: November 5, 2012
Published online: && &&, 2012
Chem. Eur. J. 2013, 00, 0 – 0
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www.chemeurj.org
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ÞÞ
These are not the final page numbers!

K. E. Borbas et al.
A visible change: Eu complexes can be
rendered analyte-responsive by
Fluorescent Probes
exploiting the sensitivity of a coumarin
antenna to the nature of the 7-substitu-
ent (see figure). A boronate switch
installed in the 7-position afforded
complexes that responded rapidly to
mm concentrations of peroxynitrite; the
product of this reaction being the cor-
responding phenol. The analogous
reaction with H2O2 proceeds at a sig-
nificantly lower rate. As the reaction
shifts the absorption to longer wave-
lengths, the construction of ratiometric
probes is possible.
C. Szꢂjjꢁrtꢀ, E. Pershagen,
N. O. Ilchenko, K. E. Borbas* &&& — &&&
AVersatile Long-Wavelength-
Absorbing Scaffold for Eu-Based
Responsive Probes
&
12
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www.chemeurj.org
ꢄ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 0000, 00, 0 – 0
ÝÝ
These are not the final page numbers!