Catalytic signal amplification using a heck reaction. An example in the fluorescence sensing of Cu(II)

Article abstract of DOI:10.1021/ja0401038

Catalytic signal enhancement using an organometallic reaction is demonstrated. The reactivity of a Heck cross-coupling reaction that creates a fluorophore is modulated by the addition of a polyazacyclam inhibitor. The inhibitor will complex with Cu(II), which restores the activity of the Pd(II). The addition of Cu(II) therefore leads to the generation of fluorescence, thereby creating a very sensitive assay for Cu(II). The rate of the Heck reaction is followed by monitoring emission as a function of time. The rate is proportional to the Cu(II) concentration and correlates to the affinity of the inhibitor to various metals. This strategy represents a general technique that can be exploited with other catalytic organometallic reactions. Copyright

Full text of DOI:10.1021/ja0401038

Published on Web 10/26/2004
Catalytic Signal Amplification Using a Heck Reaction. An Example in the
Fluorescence Sensing of Cu(II)
Qiaoyin Wu and Eric V. Anslyn*
Department of Chemistry and Biochemistry, The UniVersity of Texas at Austin, Austin, Texas 78712
Received April 12, 2004; E-mail: anslyn@ccwf.cc.utexas.edu
Scheme 1. Cu (II) Sensing Protocola
Signal amplification for ultrasensitive molecular sensing has
attracted considerable attention in the past decades.1 Among the
current literature methods, the “molecular wire” approach is
attractive because highly sensitive analyte detection results from
energy migration in conjugated semiconducting polymeric as-
semblies.2 This technique has found applications in the detection
of toxic ions3 and explosive compounds.4 Another amplification
routine exploits conformational changes in helical polymers that
result from chiral molecular interactions, giving discrimination of
the sense of chirality.5
The most common signal amplification routines involve bio-
chemical catalytic processes. For example, catalytic RNA cleavage
has been used to give both direct detection and quantification of
RNAs in situ.6 Similarly, nucleic acid-templated metal-catalyzed
ester hydrolysis has been used to amplify signals.7 In addition,
exponential amplification of RNA cleavage was achieved via
substrate deactivation, followed by seeding the cleavage reaction
with an initiator.8 Of course, enzyme-linked immunosorbent assay
(ELISA) is a renowned tool, proven to link antibody detection to
enzymatic signal amplification.9
a Fluorophore 2 is generated from nonfluorescent aniline 1 through Heck
reaction. When 1 equiv of PAC is introduced into the system, PAC-Pd(II)
is formed, which causes the absence of fluorescence. However, upon
addition of Cu(II) prior to Pd(II), the preferred PAC-Cu(II) complex is
formed, and fluorescence is catalytically “turned on”.
In contrast to the aforementioned methods using polymers and
bio-inspired systems, we have started exploring the use of common
organometallic reactions to amplify signals in molecular sensing
routines to detect and quantify various analytes in aqueous
solution.10 Our general signal amplification protocol relies upon
deliberate deactivation, by an exogenous ligand, of an organome-
tallic reaction that catalytically creates a fluorophore or chro-
mophore. The ligand also binds an analyte, and a competition is
set up between the analyte and the catalyst. To whatever extent
the deactivating ligand is sequestered by the analyte, the catalytic
reaction will proceed.
To test this strategy, we choose Cu(II) as the analyte and a
polyaza cyclam (PAC) as the deactivating ligand. Scheme 1 shows
the strategy in the context of a Heck cross-coupling reaction. In
our design strategy, an equivalent amount of ligand should result
in complete sacrifice of the Pd(II) due to creation of Pd(II)-PAC.
However, upon pretreatment of PAC with Cu(II), which has a larger
affinity than Pd(II), the deactivating ligand is only able to
fractionally capture the Pd(II), thereby leaving an equal amount of
Pd(II) to be reduced to the Pd(0) catalyst in a Heck coupling cycle.
Each equivalent of Cu(II) should free up a set amount of catalyst,
and as a result, the fluorescent product will be formed at a rate
linearly proportional to analyte concentration. Further, in such an
approach, the fluorescence is catalytically “turned-on”.
on a fluorescence quenching and lifetime reduction approach.10d
Further, 0.2 pM copper ion measurement was achieved using an
electrochemical sensor, in which the tripeptide Gly-Gly-His was
covalently attached to a modified gold electrode.12
The Heck reaction of 1 was chosen for our proof-of-principle
study because of its ease of operation, as well as the completely
different fluorescent activities between the starting material and
product. Through a Pd(0)-catalyzed Heck reaction,13 the nonfluo-
rescent starting material 2-iodo-N-allylaniline 1 can be converted
into the fluorescent indole product 2 (Φ ) 0.343).14 Excitation of
2 was performed at 275 nm (absorption maximum), and emission
was recorded at 351 nm (emission maximum). The reaction was
monitored by measuring fluorescence intensity as a function of time.
As expected, introduction of 1.0 equiv of ligand (PAC) deactivates
the Pd(II) by the formation of a Pd(II)-PAC complex. This
deactivating ligand nearly completely silences the emission over
time. However, when 1.0 equiv of Cu(II) is incubated with PAC
prior to addition of Pd(II), the remaining Pd(II) indeed yields a
successful Heck reaction with a rate equal to that of no ligand.
These control experiments are shown in Supporting Information.
To an acetonitrile-water (10:1 v/v) solution of 1 (0.5 mM), tri-
o-tolylphosphine (0.05 mM), PAC (0.025 mM), and TEA (1 mM)
under argon were added varied amounts of Cu(II), followed by
addition of Pd(II) (0.025 mM). A series of reactions at 60 °C were
performed in quartz cuvettes. To reduce the slight extent of
photocyclization of 1,15,16 the emission spectra were taken every
10 minutes, rather than using continual excitation. Figure 1 shows
the relationship between fluorescence intensity and time, with a
series of different Cu(II) concentrations (from 500 to 30 nM). The
slopes of the lines represent initial rate kinetics. As expected, when
Although several Cu(II) fluorescent chemosensors have been
reported, very few have sensitivity in the nanomolar range. In
addition, they involve fluorescence quenching processes rather than
fluorescence enhancement.11 To date, the most sensitive Cu(II)
detection method involves an optical fiber with an immobilized
fluorophore-tagged Cu(II) binding protein. The system reached an
astounding 0.1 pM Cu(II) detection limit, even though it was based
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C O M M U N I C A T I O N S
Figure 3. Selectivity analysis toward Cu(II), Co(II), Cd(II), Ni(II). Reaction
conditions are same as in Figure 1 except that Cu(II), Co(II), Ni(II), and
Cd(II) concentrations are 0.015 mM.
Figure 1. Initial rate analysis. Reaction conditions: [1] ) 0.5 mM, [PAC]
) 0.025 mM, [Pd(II)] ) 0.025 mM in CH3CN/H2O (10:1 v/v), under argon,
temperature ) 60 °C.
time, nearly 1.5 h. However, the study described here demonstrates
a new principle for signal enhancement in molecular sensing.
Clearly the use of faster organometallic reactions will significantly
improve the practicality of the general method.
Acknowledgment. This work was supported by a grant from
the National Institutes of Health (EB00549-5).
Supporting Information Available: Spectroscopic and controlling
experimental data and general procedure (PDF). This material is
available free of charge via the Internet at http://pubs.acs.org.
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Figure 2. Slope (initial rates) vs [Cu(II)]. [Cu(II)] was decreased from
500 to 30 nM. Reaction conditions are the same as in Figure 1.
decreasing the Cu(II) concentration, the initial rates dropped
correspondingly. A plot of slope values as a function of Cu(II)
concentration is linear, as shown in Figure 2. This linear relationship
implies that 1 equiv of Cu(II) frees a consistent amount of Pd(II).
Many transition metals have high affinities to poly-aza macro-
cyclic ligands.17 Therefore, we sought to test the selectivity of our
sensing protocol to various metals. Cu(II) has the highest affinity
to cyclam 1. Hence, we used the same protocol as with Cu(II)
described above, but for the metal ions Ni(II), Co(II), and Cd(II),
all of which have lower affinities. Figure 3 shows that the catalytic
production of fluorophore 2 tracks the affinities of the cyclam for
Cu(II), Ni(II), Co(II), and Cd(II). The higher the binding affinity,
the greater the initial rate observed. As indicated in Figure 3, when
Cu(II) was present in the system the initial rate was almost 3-fold
that of Co(II) and 6-fold those of Ni(II) and Cd(II). These relative
selectivities support the mechanism given herein for the sensing
method. While a particular metal ion in a mixture would be difficult
to distinguish with these small selectivity ratios, other ligands may
improve the selectivity.
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Supporting Information.
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sensitive sensing technique. Our specific example using a Heck
reaction has high sensitivity and reasonable selectivity for Cu(II).
Admittedly, to achieve a response for 30 nM Cu(II) required a long
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