A reaction-based sensing scheme for gold species: Introduction of a (2-ethynyl)benzoate reactive moiety

Article abstract of DOI:10.1021/ol302291c

To alleviate side reactions identified in an N-propargyl-rhodamine lactam sensing system, we devised the novel reaction-based sensing scheme for gold species based on the alkynophilicity. A fluorescein (2-ethynyl)benzoate underwent Au(III)-promoted ester

Full text of DOI:10.1021/ol302291c

ORGANIC
LETTERS
2012
Vol. 14, No. 19
5062–5065
A Reaction-Based Sensing Scheme
for Gold Species: Introduction of a
(2-Ethynyl)benzoate Reactive Moiety
Hyewon Seo,† Mi Eun Jun,† Olga A. Egorova,‡ Kyung-Ha Lee,§ Kyong-Tai Kim,§ and
Kyo Han Ahn*,†
Department of Chemistry and Center for Electro-Photo Behaviors in Advanced
Molecular Systems, Pohang University of Science and Technology (POSTECH),
San 31, Hyoja-dong, Pohang, 790-784, Republic of Korea, Department of Analytic
Technology Research, Samsung Display, Tangjeong-meon, Asan-si, Chungnam,
336-841, Republic of Korea, and Department of Life Science, Pohang University
of Science and Technology (POSTECH), San 31, Hyoja-dong, Pohang, 790-784,
Republic of Korea
ahn@postech.ac.kr
Received August 17, 2012
ABSTRACT
To alleviate side reactions identified in an N-propargyl-rhodamine lactam sensing system, we devised the novel reaction-based sensing scheme
for gold species based on the alkynophilicity. A fluorescein (2-ethynyl)benzoate underwent Au(III)-promoted ester hydrolysis selectively over
other metal ions with high sensitivity, which accompanies a turn-on fluorescence change in pH 7.4 HEPES buffer. The work offers a versatile
reactive moiety for the development of gold probes with improved sensing properties.
Gold chemistry has been spotlighted in recent years
because of the unique optical properties of gold nano-
particles1 and catalytic2 and biological activities3 of
gold complexes. Widespread use of gold species in acade-
mia and industry thus needs efficient and convenient
detection methods to evaluate and monitor the residual
gold species in various circumstances. For analysis of gold
species, various spectroscopic (atomic absorption/emis-
sion spectroscopy with various atomizers), mass, and
electrochemical (anodic stripping voltammetry) methods
are available.4 In contrast, fluorogenic sensing systems for
gold ions have appeared very recently. Given that the
fluorescent method is sensitive and easy-to-use, it would
complement conventional analytical methods. Further-
more, the fluorescent probes are indispensible in the
bioimaging area.
† Department of Chemistry and Center for Electro-Photo Behaviors in
Advanced Molecular Systems, POSTECH.
‡ Samsung Display.
§ Department of Life Science, POSTECH.
Recently we developed a rhodamine-derived N-propar-
gyl-spirolactam P1 as a fluorescent probe for gold species.
P1 undergoes tandem spirolactam ring opening and
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r
10.1021/ol302291c
Published on Web 09/21/2012
2012 American Chemical Society

benzoxazole ring formation promoted by gold species, which
accompanies a turn-on fluorescence change (Scheme 1).5
This strategy was based on our previous reaction-based
approach to sense silver ions, that is, the silver ion-
promoted tandem spirolactam ring opening and oxazo-
line ring formation of an N-iodoethyl-rhodamine B lactam.6
which is nonfluorescent, has been identified by its single
crystal X-ray structure.10 This resolved structure also adds
a valuable piece of evidence for the participation of aryl
rings in the related gold-mediated cyclization reactions.11
If a reaction-based sensing protocol produces nonfluores-
cent side products, as in the case of P1, then the product
distribution would vary depending on the sensing condi-
Scheme 1. Reaction-Based Sensing of Gold Ions with P1
Scheme 2. Competing Reactions in the Gold Sensing Process
with P1 and Crystal Structure of I-1b (ORTEP Drawing with
20% Probability)
Concurrently, others have reported reaction-based
fluorescent probes for gold species based on the unique
alkynophilicity of gold ions.7,8 P1 provided fluorescent
formyloxazole 1, rather than the corresponding proto-
deauration products of the vinylgold intermediate (I-1a);
this unexpected result prompted us to characterize the
reaction intermediates in the gold-mediated cyclization of
N-propargylbenzamides as model compounds.9 We found
that the gold sensing process could also produce two types
of nonfluorescent compounds (Scheme 2), along with
formyloxazole 1 that constitutes 60ꢀ70% of the total
mass. Simple hydration of the acetylenic moiety promoted
by Lewis acidic gold species could compete with the
spirolactam ring-opening process, producing the corre-
sponding fluorescent compounds. Furthermore, an un-
usual ring-closing process is found to compete with the
sensing process: A tricyclic vinylgold(III) species I-1b,
tions, which would undermine the reliability of the quan-
tification data. Therefore, weset out toinvestigate arelated
but different approach that may alleviate the side reactions
observed in the rhodamine-based probe. We envisioned
that we could avoid fluorescence interference from the side
reactions by separating the reaction site from the fluoro-
phore. Herein, as a proof-of-concept we report such a
reaction-based sensing scheme for gold species that cir-
cumvents the aforementioned problems.
Our newly designed probes for gold species are (2-
ethynyl)benzoates of fluorescein, P2 (P2a, P2b) and P3,
where the reactive moiety, (2-ethynyl)benzoate, is sepa-
rated from the fluorophore. For example, activation of the
ethynyl group in P2 by Au3þ would generate the corre-
sponding oxonium intermediate I-2, which would subse-
quently undergo hydrolysis to regenerate the strongly
fluorescent fluorescein 2 (Scheme 3), along with isochro-
men-1-one 4. A related gold-promoted hydrolysis process
was ingeniously applied to a glycosylation reaction by Yao
and co-workers.12 According to the present sensing
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S. B.; Ahn, K. H. J. Am. Chem. Soc. 2009, 131, 2040–2041.
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H. N.; Yoon, J.; Kim, J. S.; Kim, H. J. Org. Lett. 2010, 12, 932–934. (d)
Dong, M.; Wang, Y. W.; Peng, Y. Org. Lett. 2010, 12, 5310–5313. (e)
Yuan, L.; Lin, W.; Yang, Y.; Song, J. Chem. Commun. 2011, 47, 4703–
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47, 12506–12508. (h) Wang, J. B.; Wu, Q. Q.; Min, Y. Z.; Liu, Y. Z.;
Song, Q. H. Chem. Commun. 2012, 48, 744–746. (i) Park, J. E.; Choi,
M. G.; Chang, S.-K. Inorg. Chem. 2012, 51, 2880–2884. For a different
class of gold probe, see: Park, J.; Choi, S.; Kim, T.-I.; Kim, Y. Analyst
2012, DOI: 10.1039/c2an35351b.
(8) For recent reviews on the reaction-based approach to fluorescent
probes, see: (a) Jun, M. E.; Roy, B.; Ahn, K. H. Chem. Commun. 2011,
47, 7583–7601. (b) Chen, X.; Pradhan, T.; Wang, F.; Kim, J. S.; Yoon, J.
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K. H. Angew. Chem., Int. Ed. 2011, 50, 11446–11450.
(10) Crystal data for I-1b (C31H33AuCl3N3O2): M = 782.92, mono-
˚
clinic, space group P21/n; a = 10.9486(11) A, b = 11.7414(12) A, c =
˚
3
˚
˚
24.580(3) A, β = 100.062(2)°, V = 3111.1(5) A ; Z = 4, T = 293(2) K, μ
(λ = 0.71073 A) = 5.019 mmꢀ1, Fcalcd = 1.671 g/cm3, 17 756 reflections
˚
measured, 6348 unique (Rint = 0.0686), R1 = 0.0412, wR2 = 0.1012 (I >
2σ(I)), R1 = 0.0597, wR2 =0.1104 (all data), GOF = 0.999, CCDC No.
747441.
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Org. Lett., Vol. 14, No. 19, 2012
5063

scheme, the hydration at the ethynyl group, ifany, does not
lead to the fluorescent compounds. Also, the unusual ring-
opening process observed in the case of the rhodamine
lactam is not conceivable.
(Figure S1). In addition, the fluorescence response of P2a
to AuCl3 was slower than that of P2b. In the case of P3,
it also showed a slower response to AuCl3 (Figure S2).
Furthermore, its sensing product 3 emitted weakly, giving
about one-fifth of the emission intensity of fluorescein
(Figure S3). Of particular note is that 3 barely emitted in
cells, giving poor fluorescent images (in the following).
Therefore, useof fluoresceinmethylether3 for cell imaging
purposes is not recommended in spite of some reported
probes based on it.15
Scheme 3. New Fluorescein-Derived Probes for Gold Species
(P2) Equipped with Outer Reactive Moiety of (2-ethynyl)-
benzoate, and Its Sensing Mechanism
A solution of P2b (5 μM) dissolved in pH 7.4 buffer
(5 mM HEPES containing 0.25% DMSO) emitted barely
when excited at 470 nm.16 Upon addition of AuCl3,
however, the solution of P2b emitted strong green fluo-
rescence with λmax at 508 nm. A time course of the fluores-
cence titration shows that the response is fast and complete
within 1 h at ambient temperature (Figure 1). These results
suggested that the AuCl3-promoted ester hydrolysis indeed
took place; the fluorescence was found to result from
fluorescein by comparison with an authentic sample
(Figure S5). Although it causes significant fluorescence
changes, we have not examined AuCl in detail, as Au(I)
readily undergoes a disproportionation into Au(III) and
Au(s) species in aqueous media [3Auþ(aq.) f Au3þ(aq.) þ
2Au0].17
Next, we evaluated the fluorescence response of P2b
toward various metal salts such as Mg(II), Ba(II), Cr(II),
Mn(II), Fe(III), Co(II), Ni(II), Pd(II), Cu(II), Ag(I), Zn-
(II), Cd(II), Hg(II), Pt(II), and Au(III) (as their chloride
salts except for AgNO3).
The mono- and bis-(2-ethynylbenzoate) of fluorescein
(P2a, P2b) and a methyl ether derivative P3 were readily
prepared by EDC-DMAP coupling of fluorescein or its
methyl ether derivative 3 with (2-ethynyl)benzoic acid,
respectively (see the Supporting Information). We have
found that (2-ethynyl)benzoic acid13 undergoes poly-
merization on standing, and thus its precursor, methyl
(2-ethynyl)benzoate, should be hydrolyzed prior to use.
Otherwise, 2-iodobenzoic acid can be first coupled with
fluorescein, and then the ethynyl group is introduced by
Sonogashira coupling.14 In both cases, the ester coupling
reactions yielded a mixture of mono- and bis-benzoates,
which were separable by silica gel column chromatogra-
phy. Similarly, P3 was synthesized from fluorescein methyl
ether 3. Fluorescein bis(benzoate) P2b thus could be
prepared in about 30% isolated yield under stoichiometric
reaction conditions.
Figure 1. Time-dependent fluorescence changes of P2b (5 μM) in
the presence of AuCl3 (2 equiv) in pH 7.4 buffer (5 mM HEPES
containing 0.25% DMSO) at ambient temperature, taken under
excitation at 470 nm. Inset: a plot of the fluorescence intensity vs
time.
P2b was found to be the probe of choice based on
preliminary evaluation studies for the 4 fluorescein benzo-
ates. P2b barely emitted, whereas P2a emitted fluores-
cence, albeit small, to give an apparent background signal
As shown in Figure 2, P2b responds only to the gold
species among the metal species examined. A competition
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Whorton, A. R.; Xian, M. Angew. Chem., Int. Ed. 2011, 50, 10327–
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(16) Excited at lower wavelength than the emission maximum of the
solution of P2b and AuCl3 (λmax = 490 nm, Figure S4) to avoid self-
absorption.
(13) (a) Thorand, S.; Krause, N. J. Org. Chem. 1998, 63, 8551–8553.
(b) Marchal, E.; Uriac, P.; Legouin, B.; Toupet, L.; Weghe, P. Tetra-
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Org. Lett., Vol. 14, No. 19, 2012

assay, however, showed that coexistence of some metal
species caused emission enhancement but Ag(I) and Pt(II)
species caused a small decrease in the fluorescence (Figure S6).
The reason for this interesting behavior is unclear at the
moment.
only near pH 7 but little at other pH values except at pH 10
where a substantial increase was observed (Figure S10). At
high pH conditions, nonspecific ester hydrolysis seems to
occur because P2b is an ester-type probe.
Finally, we briefly evaluated P2b for the fluorescent
imaging of AuCl3 in cells. Bright-field and fluorescent
microscopic images of HeLa cells incubated with both
P2b (10 μM) and AuCl3 (100 μM) showed green fluores-
cence. In contrast, little fluorescence was observed from
the cells treated only with the probe (Figure S11). How-
ever, wefound thathydrolysis ofthe ester probebyesterase
in the cells interferes with the gold-promoted sensing
process, which limits its application for the bioimaging
purpose. We also examined P3 in the fluorescent cell
imaging. As noted above, the corresponding reaction
product 3 gave poor fluorescence (Figure S12); hence,
fluorescein methyl ether 3 is not suitable for cell imaging.
As an alternative application of P2b, we used the probe to
quantify residual gold species in a AuCl3-catalyzed reac-
tion product that is purified by silica gel column chroma-
tography (Figure S13).
In summary, we have devised a new reaction-based
sensing scheme for gold species by utilizing their alkyno-
philicity. One of the (2-ethynyl)benzoates derived from
fluorescein underwent Au(III)-promoted hydrolysis, which
accompanies a turn-on fluorescence change in aque-
ous media. The sensing reaction is selectively promoted
by Au(III) species among various metal species exam-
ined. The new reactive moiety is promising for the
development of improved fluorescent probes for gold
species, as it alleviates the side reactions indentified in
the rhodamine-based probe. A further study to prevent
the ester-type probes’ sensitivity toward esterase is
underway and will be reported in due course.
Figure 2. Fluorescence response of P2b (5 μM) toward various
metal species (2 equiv) in pH 7.4 buffer (5 mM HEPES contain-
ing 0.25% DMSO) at ambient temperature, obtained after
20 min under excitation at 470 nm.
The fluorescence response of P2b was linear in the range
of 1ꢀ30 μM AuCl3 (Figure S7). On the basis of the signal-
to-noise ratio of 3, the detection limit of P2b for AuCl3 was
estimated to be below 0.4 μM (Figure S8).
Progress of the sensing reaction was followed by TLC,
and the product was identified by NMR analysis for a
reaction mixture containing P2b and AuCl3 at a higher
concentration (∼2 mM of substrates in the aqueous media).
Under the conditions, only the Au(III)-promoted ester
hydrolysis occurred predominantly, and other possible
side reactions such as simple hydration of the ethynyl
moiety were negligible (Figure S9). The results show that
the new approach alleviates the side reactions observed in
the rhodamine-based system.
Acknowledgment. This work was supported by grants
from the EPB Center (R11-2008-052-01001).
Interestingly, the sensing behavior of P2b toward AuCl3
was sensitive to pH. A solution of P2b and AuCl3 (2 equiv)
at different pH values (pH 1ꢀ10) showed strong emission
Supporting Information Available. Synthesis and char-
acterization of P2 and P3, pH-dependent fluorescence
behavior, and other fluorescence titration data. This
material is available free of charge via the Internet at
http://pubs.acs.org.
(17) (a) Gold. Progress in Chemistry, Biochemistry and Technology;
Schmidbaur, H., Ed.; Wiley: Chichester, U.K., 1999; pp 558ꢀ566. (b)
Kissner, R.; Welti, G.; Geier, G. J. Chem. Soc., Dalton Trans. 1997,
10, 1773–1777. Also, a solution of AuCl undergoes a faster reduction to
Au(0) compared to AuCl3.
The authors declare no competing financial interest.
Org. Lett., Vol. 14, No. 19, 2012
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