Ground-state elevation approach to suppress side reactions in gold-sensing systems based on alkyne activation

Article abstract of DOI:10.1021/ol5001389

A novel approach to suppress the side reactions observed in the reaction-based gold-sensing systems based on the alkyne activation is disclosed. By elevating steric strain around the reaction site, the gold ion promoted ring-opening process in rhodamine-l

Full text of DOI:10.1021/ol5001389

Letter
pubs.acs.org/OrgLett
Ground-State Elevation Approach To Suppress Side Reactions in
Gold-Sensing Systems Based on Alkyne Activation
Hyewon Seo,§,† Mi Eun Jun,§,† Koteeswari Ranganathan,† Kyung-Ha Lee,‡ Kyong-Tai Kim,‡
Woocherl Lim,† Young Min Rhee,† and Kyo Han Ahn*,†
†Department of Chemistry and Center for Electro-Photo Behaviors in Advanced Molecular System and ‡Department of Life Science,
POSTECH, San 31, Hyoja-dong, Pohang 790-784, Republic of Korea
S
* Supporting Information
ABSTRACT: A novel approach to suppress the side reactions
observed in the reaction-based gold-sensing systems based on
the alkyne activation is disclosed. By elevating steric strain
around the reaction site, the gold ion promoted ring-opening
process in rhodamine-lactam probes is significantly accelerated,
which also leads to suppression of those possible side
reactions. As a result, the probes show very high sensitivity
in addition to excellent selectivity toward gold species.
Furthermore, bioimaging of gold species in live cells was
demonstrated with a FRET version.
old complexes have been intensively used in various
Gresearch fields for their unique chemical and biological
properties. As a result of relativistic effect, the gold atom has the
6s orbital contracted and the 5d orbital expanded, which makes
gold ions strong π-bond acceptors;1 hence, they are used as
mild and efficient catalysts for activating carbon−carbon
multiple bonds.2 Gold complexes also exert cytotoxicity to
DNA and enzymes and thus have been investigatged as
anticancer and antirheumatoid agents.3 The rich chemistry and
biological activities of gold species demand efficient detection
systems; however, it is only in recent years that fluorescent
sensing systems appeared.
By following our reaction-based sensing approach to silver
ions,4 we recently reported a novel gold-sensing scheme.5
Figure 1. Known fluorescent probes 1 and 2 for gold species and a
new probe 3 and its FRET versions 5.
Others also concurrently reported related reaction-based
probes for gold ions.6 The initial probes such as 1a and 1b
(Figure 1) utilize the strong affinity of gold ions to the carbon−
that was reported by us7g and a related one by others.7f Soon
carbon triple bond to promote a chemical conversion that
brings turn-on fluorescence change. Subsequently, various types
of fluorescent probes for gold ions have been developed by
changing the alkyne activation mode.7 Although these probes
show high selectivity toward gold ions with turn-on
fluorescence response, they pose a serious drawback in the
gold ion-promoted chemical conversion: the activation of
alkyne by gold ions can involve side reactions including the
alkyne hydration,8 as we raised this issue earlier.7g Such side
reactions can complicate the quantification process because the
degree of side reactions would be dependent on the sensing
conditions (media, temperature, concentration, etc). In
addition, the nonsignaling processes lower the probe’s
sensitivity toward gold ions.
after, we recognized a potential weakness from such an ester-
type probe 2: the ester linkage is vulnerable to the ubiquitous
esterase activity in living systems.9 Our attempts to develop
ester-type probes that resist to esterase activity have not been
successful so far. In search of a reaction-based fluorescent probe
for gold ions that causes little side reactions and also is stable to
esterase activity, we have reinvestigated the initial rhodamine-
lactam ring-opening approach.5 In this paper, we disclose the
novel approach that alleviates the side reactions and also
improves the slow response rate and low sensitivity of the
previous probes. Furthermore, from the new sensing system we
can readily construct its fluorescence resonance energy transfer
One way to alleviate the side reactions is to separate the
reaction site from the signaling part, as in the case of probe 2
Received: January 15, 2014
Published: February 13, 2014
© 2014 American Chemical Society
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(FRET) system, which allows analyte quantification with
minimized interference from the environmental parameters.10
We reasoned that enhancing steric strain around the
rhodamine-lactam would accelerate the ring-opening process,
which might lead to suppression of the side reactions observed
in the previous N-propargylrhodamine-lactam probe:5,6 alkyne
hydration and unusual hydroarylation. The alkyne hydration
can be a serious side reaction for the gold sensing process, as
evidenced by our study on a model compound.11 A designed
probe is rhodamine-lactam 3 in which the N-(2-ethynlphenyl)
group is expected to raise steric strain around the lactam
moiety, which would lift the probe’s ground state energy and
thus accelerate the gold ion-promoted ring-opening process
(Scheme 1). To support the ground-state elevation in probe 3,
vinylgold intermediat Au-1 and subsequent protodeauration to
form oxazine 4 that is strongly fluorescent. Au-1 is also likely to
emit strong fluorescence from the fully conjugated xanthene
moiety.
In addition, a possible hydration of the ethynyl group in
probe 3 promoted by gold ions was carefully followed by TLC
and 1H NMR. Upon addition of AuCl3 to a solution of probe 3
(2.5 mM in CH3CN containing 30% water), a highly polar spot
appeared immediately with no other spots including that of the
probe. The simple hydration product, if any, is expected to
appear near or below the probe on silica gel TLC plate, but no
such spot appeared (Figure S6, Supporting Information). The
polar spot is likely to be vinylgold(III) intermediate Au-1,12
which is converted to oxazine 4 through a protodeauration
process, as followed by NMR spectral change for the vinylic
protons (Figure S7, Supporting Information). Formation of
oxazine 4 was supported by its M+ base peak at 543.29 (Figure
S8, Supporting Information). An unusual rhodamine-lactam
ring-opening product, as observed in the case of probe 1a,7g
seemed to be absent in the present case, plausibly owing to a
highly strained transition state expected.
Scheme 1. Turn-on Sensing Scheme of AuCl3 by Probe 3
Probe 3 responds to gold ions faster than the previous probe
1 (Figure 2a):5 the former showed an ∼4.5 times larger pseudo-
first-order rate constant than the latter (kobs = 0.13 min−1 for 3,
kobs = 0.029 min−1 for 1a; measured in pH 7 PBS buffer
containing 50% acetonitrile at 25 °C; Figures S9 and S10,
Supporting Information). It is thus proven that the elevation of
ground-state energy leads to suppression of the side reactions
while enhancing the reaction rate.
Probe 3 shows excellent selectivity toward gold ions over
various metal species examined (Mg2+, Cr2+, Mn2+, Fe2+, Fe3+,
Co2+, Ni2+, Cu2+, Zn2+, Pd2+, Ag+, Cd2+, Ba2+, Pt2+, Hg2+, and
Pb2+; as chloride salts except AgNO3) (Figure 3a).
we carried out DFT calculation on the rhodamine ring-opening
process by constrained optimization on the Au−C bond and
also pH dependent rhodamine ring-opening for probes 1a and
3 (Figures S1 and S2, Supporting Information). The energy
profiles show that the ring-opening reaction is faster in the case
of probe 3 than the case of probe 1a, and the difference
between the highest energy barriers in two probes is 12.88 kcal/
mol at Au−C bond distance of 2.8 Å and 2.9 Å for probe 1a
and probe 3, respectively. This rate acceleration in turn would
suppress the competing alkyne hydration that is a nonsignaling
process. Introduction of phenyl ring between the spirolactam
nitrogen and the ethynyl group would also make the unusual
cyclization process observed in probe 1a (hydroarylation
involving the diethylaminobenzene ring)7g unconceivable
because the distance between the two reaction sites becomes
so long (4.9 vs 6.0 Å, Figure S3, Supporting Information).
Probe 3 can be synthesized by direct coupling of rhodamine
B acid chloride with 2-ethynylaniline in 79% yield (Scheme S1,
Supporting Information). Probe 3 alone is nonfluorescent but
emits strongly upon addition of gold ions, both Au(I) and
Au(III), in aqueous media (pH 6 HEPES containing 30%
CH3CN at 36 °C, Figure 2b, Figure S4, Supporting
Information). The turn-on fluorescence response can be
explained by gold ion-triggered consecutive spirolactam ring-
opening and intramolecular cyclization processes to form
Figure 3. (a) Fluorescence response of probe 3 (10 μM) toward
various metal species (Mg2+, Cr2+, Mn2+, Fe2+, Fe3+, Co2+, Ni2+, Cu2+,
Zn2+, Pd2+, Ag+, Cd2+, Ba2+, Pt2+, Hg2+, and Pb2+ from the left; 10 μM
each). (b) Plot of fluorescence enhancement of probe 3 (10 μM)
depending on [AuCl3]. Both data were obtained in 30% CH3CN−
HEPES buffer (pH 6, 10 mM) at 36 °C after 60 min of mixing (λex
540 nm).
=
Furthermore, probe 3 shows a linear response with very high
sensitivity, enabling detection of AuCl3 down to 0.5 ppb level,
the lowest detection limit so far obtained (Figure S11,
Supporting Information, Figure 3b). Probe 3 is thus highly
promising for the quantification of gold residues in synthetic
products.7g Probe 3 showed pH-dependent fluorescent
response (Figure S12, Supporting Information), showing
pronounced fluorescence as the solution becomes acidic (pH
4−6).
A novel structural feature of probe 3 is that we can readily
functionalize the N-phenyl ring without significantly affecting
sensing properties. Next, we have synthesized two FRET
systems. A FRET probe is highly desirable in biological studies
Figure 2. (a) Fluorescence response of probes 1a and 3 (10 μM each)
toward 1 equiv of AuCl3 depending on time at 25 °C. (b) Time course
of fluorescence change of probe 3 toward AuCl3 (each at 10 μM) at 36
°C. Both data are obtained in 30% CH3CN−PBS buffer (pH 6.0, 10
mM).
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as it allows more reliable quantification of an analyte compared
to the intensity-based probe whose intensity change is affected
by environmental parameters such as pH, polarity, temperature,
emission collection efficiency, effective cell thickness in the
optical beam, and changes in the excitation intensity.10 So far,
only a few ratiometric probes for gold ions have been reported;
they are ICT-based sytems7b,j,l except for one FRET probe.7c
A naphthalimide dye is chosen as the FRET donor, as its
emission spectrum overlaps well with the absorption spectrum
of rhodamine B.12 To connect the donor to probe 3, we
introduced a carboxyl group to the N-phenyl ring. The resulting
FRET probe 5 can be readily synthesized, for example, by
coupling rhodamine-derived acid 6 with naphthalimide 7b that
contains an ammonium side group (Figure 1, Supporting
Information).
imaging of AuCl3 in cells met with failure, owing to its low cell
permeability (Figure S20, Supporting Information). To over-
come this problem, we synthesized probe 5b that contained a
quaternary ammonium group, which showed good cell
permeability. Fluorescent imaging data for N2A cells incubated
with probe 5b were obtained in the absence and presence of
AuCl3. Probe 5b alone in cells emits green fluorescence in
cytosol when excited at a fixed laser wavelength of 405 nm
(Figure 5a). When cells preincubated with probe 5b were
Fluorescent behavior of FRET probes 5a and 5b was
evaluated separately in pH 7.4 PBS buffer containing 10%
acetonitrile at 25 °C. Interestingly, these FRET probes show
faster response than probe 3; the fluorescence change is almost
complete within 20 min (Figure S13, Supporting Information).
It should be noted that most of the known probes based on
alkyne activation show rather slow response toward gold ions.
Probe 5a emitted bright green fluorescence centered at 520 nm
when excited at 396 nm (λmax of the naphthalimide moiety).
Significantly, upon addition of 1 equiv of AuCl3 to the probe
(10 μM), 5a for instance, an absorption band centered at 560
nm rose immediately with a concurrent color change from light
yellow to pink (Figure S14, Supporting Information). When the
solution was excited at 396 nm, a new fluorescence band at 587
appeared with a sudden fall of the band at 520 nm, with
corresponding emission color change from green to amber
(Figure 4, Figure S15, Supporting Information). These spectral
Figure 5. Fluorescent images of N2A cells treated with probe 5b (50
μM) only (the upper row) and the probe for 30 min followed by
AuCl3 (250 μM) for 1 h at 37 °C (the lower row): images observed
through (a, e) green channel (500−575 nm) and (b, f) red channel
(576−700 nm); (c, g) bright field images; (d, h) merged images. Scale
bar = 10 μm.
treated with AuCl3, red fluorescence emitted under excitation at
405 nm (Figure 5f, Figure S21, Supporting Information)
through FRET from the naphthalimide to the ring-opened
rhodamine dye. An MTT assay shows that probe 5b has low
cytotoxicity toward cells (Figure S22, Supporting Information).
In conclusion, we have disclosed a novel approach to
suppress side reactions observed in the reaction-based gold-
sensing systems based on alkyne activation. By elevating steric
strain around rhodamine-lactam nitrogen, the gold ion-
promoted ring-opening process is significantly enhanced. As a
result, the gold-sensing process proceeds as an exclusive
chemical conversion. As a result, the new sensing system
shows much higher sensitivity as well as faster response
compared with the previous ones, in addition to show excellent
selectivity toward Au(I) and Au(III) species. Furthermore, the
sensing system allows straightforward attachment of a donor
dye, leading to a novel FRET probe that enables fluorescent
imaging of gold species in cells. The ground-state elevation
approach demonstrated here holds a promise for the develop-
ment of other reactive sensing systems.
Figure 4. Fluorescence change of probe 5a (10 μM) upon addition of
AuCl3 (0−100 μM), measured in 10% CH3CN−PBS buffer (5 mM,
pH 7.4) at 25 °C (λex = 396 nm). Inset: a plot of the fluorescence
intensity vs [AuCl3]. All data were taken after 30 min of mixing at rt.
changes indicate that the spirolactam ring-opening by gold ions
activates FRET from the naphthalimide to the rhodamine
moiety. The FRET sensing system shows good ratiometric
behavior from pH 4−9 (Figure S16, Supporting Information).
As expected from the sensing characteristics shown by probe 3,
its FRET system 5a again shows excellent selectivity toward
gold ions among various metal ions (Figure S17, Supporting
Information). Also, in addition to AuCl and AuCl3, a stabilized
gold(I) species (CH3CN)Au[P(t-Bu)2(2-biphenyl)]SbF6 can
also be sensed with a linear ratiometric change depending on
the concentration of gold ions (Figures S18 and S19,
Supporting Information).
ASSOCIATED CONTENT
* Supporting Information
■
S
Fluorescence data and synthesis of new compounds. This
material is available free of charge via the Internet at http://
pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
■
*E-mail: ahn@postech.ac.kr.
Author Contributions
§These authors contributed equally to this work.
Finally, the FRET probes were applied for fluorescent
imaging of gold species in cells. An attempt to use probe 5a for
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Letter
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by grants from the EPB center (R11-
2008-052-01001) through the National Research Foundation,
Korea.
REFERENCES
(1) Gorin, D. J.; Toste, F. D. Nature 2007, 446, 395.
(2) (a) Hashmi, A. S. K.; Lothschutz, C.; Dopp, R.; Ackermann, M.;
De Buck Becker, J.; Rudolph, M.; Scholz, C.; Rominger, F. Adv. Synth.
Catal. 2012, 354, 133. (b) Rudolph, M.; Hashmi, A. S. K. Chem. Soc.
Rev. 2012, 41, 2448.
■
̈
̈
(3) (a) Finkelstein, A.; Walz, D.; Batista, V.; Mizraji, M.; Roisman, F.;
Misher, A. Ann. Rheum. Dis. 1976, 35, 2517. (b) Tomioka, R.; King, T.
E., Jr. Am. J. Respir. Crit. Care Med. 1997, 155, 1011. (c) Shaw, C. F.
Chem. Rev. 1999, 99, 2589.
(4) Chatterjee, A.; Santra, M.; Won, N.; Kim, S.; Kim, J. K.; Kim, S.
B.; Ahn, K. H. J. Am. Chem. Soc. 2009, 131, 2040.
(5) Egorova, O. A.; Seo, H.; Chatterjee, A.; Ahn, K. H. Org. Lett.
2010, 12, 401.
(6) (a) Jou, M. J.; Chen, X.; Swamy, K. M.; Kim, H. N.; Kim, H. J.;
Lee, S. G.; Yoon, J. Chem. Commun. 2009, 7218. (b) Yang, Y.-K.; Lee,
S.; Tae, J. Org. Lett. 2009, 11, 5610.
(7) (a) Do, J. H.; Kim, H. N.; Yoon, J.; Kim, J. S.; Kim, H.-J. Org. Lett.
2010, 12, 932. (b) Dong, M.; Wang, Y.-W.; Peng, Y. Org. Lett. 2010,
12, 5310. (c) Cao, X.; Lin, W.; Ding, Y. Chem.Eur. J. 2011, 17, 9066.
(d) Yuan, L.; Lin, W.; Yang, Y.; Song, J. Chem. Commun. 2011, 47,
4703. (e) Park, J. E.; Choi, M. G.; Chang, S.-K. Inorg. Chem. 2012, 51,
2880. (f) Patil, N. T.; Shinde, V. S.; Thakare, M. S.; Hemant Kumar,
P.; Bangal, P. R.; Barui, A. K.; Patra, C. R. Chem. Commun. 2012, 48,
11229. (g) Seo, H.; Jun, M. E.; Egorova, O. A.; Lee, K.-H.; Kim, K.-T.;
Ahn, K. H. Org. Lett. 2012, 14, 5062. (h) Wang, J. B.; Wu, Q. Q.; Min,
Y. Z.; Liu, Y. Z.; Song, Q. H. Chem. Commun. 2012, 48, 744.
(i) Emrullahoglu, M.; Karakus, E.; Ucuncu, M. Analyst 2013, 138,
3638. (j) Wang, B.; Fu, T.; Yang, S.; Li, J.; Chen, Y. Anal. Methods
2013, 5, 3639. (k) Yang, Y.; Yin, C.; Huo, F.; Chao, J. RSC Adv. 2013,
3, 9637. (l) Young Choi, J.; Kim, G.-H.; Guo, Z.; Yeon Lee, H.;
Swamy, K. M. K.; Pai, J.; Shin, S.; Shin, I.; Yoon, J. Biosens. Bioelectron.
2013, 49, 438. (m) Wang, J.; Lin, W.; Yuan, L.; Song, J.; Gao, W.
Chem. Commun. 2011, 47, 12506.
(8) Sanz, S.; Jones, L. A.; Mohr, F.; Laguna, M. Organometallics 2007,
26, 952.
(9) (a) Tian, L.; Yang, Y.; Wysocki, L. M.; Arnold, A. C.; Hu, A.;
Ravichandran, B.; Sternson, S. M.; Looger, L. L.; Lavis, L. D. Proc.
Natl. Acad. Sci. U.S.A. 2012, 109, 4756. (b) We have found that ester-
type probe 2 readily undergoes hydrolysis by several esterases:
unpublished results.
(10) (a) Fan, J.; Hu, M.; Zhan, P.; Peng, X. Chem. Soc. Rev. 2013, 42,
29. (b) Yuan, L.; Lin, W.; Zheng, K.; Zhu, S. Acc. Chem. Res. 2013, 46,
1462.
(11) Egorova, O. A.; Seo, H.; Kim, Y.; Moon, D.; Rhee, Y. M.; Ahn,
K. H. Angew. Chem., Int. Ed. 2011, 50, 11446.
(12) (a) Bojinov, V. B.; Venkova, A. I.; Georgiev, N. I. Sens. Actuators,
B 2009, 143, 42. (b) Luxami, V.; Verma, M.; Rani, R.; Paul, K.; Kumar,
S. Org. Biomol. Chem. 2012, 10, 8076. (c) Chereddy, N. R.;
Thennarasu, S.; Mandal, A. B. Analyst 2013, 138, 1334.
1377
dx.doi.org/10.1021/ol5001389 | Org. Lett. 2014, 16, 1374−1377