Luminescent assays for ketones and aldehydes employing catalytic signal amplification

Article abstract of DOI:10.1039/b616765a

Herein we report the first use of transition metal catalytic signal enhancement for the analysis of small organic analytes. Two assays using Sonogashira and Suzuki cross-couplings have been used in the detection of ketones and aldehydes produce highly luminescent markers. The latter analysis utilizing the Suzuki coupling demonstrates the first use of peroxyoxalate initiated chemiluminescence in a sensing application. Chemiluminescent measurement revealed much higher sensitivity than fluorescence. The Royal Society of Chemistry and the Centre National de la Recherche Scientifique.

Full text of DOI:10.1039/b616765a

View Article Online / Journal Homepage / Table of Contents for this issue
PAPER
www.rsc.org/njc | New Journal of Chemistry
Luminescent assays for ketones and aldehydes employing catalytic signal
amplificationwz
Ronald J. T. Houk and Eric V. Anslyn*
Received (in Durham, UK) 16th November 2006, Accepted 15th January 2007
First published as an Advance Article on the web 21st February 2007
DOI: 10.1039/b616765a
Herein we report the first use of transition metal catalytic signal enhancement for the analysis of
small organic analytes. Two assays using Sonogashira and Suzuki cross-couplings have been used
in the detection of ketones and aldehydes produce highly luminescent markers. The latter analysis
utilizing the Suzuki coupling demonstrates the first use of peroxyoxalate initiated
chemiluminescence in a sensing application. Chemiluminescent measurement revealed much
higher sensitivity than fluorescence.
Cu(II) and various other transition metals was performed.
Introduction
When the Cu(II) was released from EDTA by a stronger
The trend in supramolecular chemistry, as with many other
binding metal, it was reduced in situ to a Cu(I) species. The
application driven fields, is to generate the greatest output with
Cu(I) then catalysed a Huisgen cyclization in which the azide
the minimum size. In molecular recognition, size translates to
and the alkyne precursors each contained one member of a
detection limit. For example, when attempting to sense a
FRET pair. Upon cyclization, the FRET pairs would be
disease marker, the earlier it can be discovered, the better.
brought together and the resulting emission shift was mon-
When attempting to decrease the limit of detection for an
itored. Sensitivity was reported for Pb(II) at millimolar con-
assay, two main questions must be addressed. The first is
centrations. Though not terribly sensitive, the assay
whether or not the recognition element has sufficient affinity
demonstrated the first use of a transition metal-catalyzed
for the target to attain favorable association at the desired
reaction being exploited to amplify signal output upon analyte
detection level. Supramolecular chemists have become very
recognition. Further studies in our group for the detection of
proficient at dealing with this problem, and most assays today
copper and cadmium using a Heck cross-coupling reaction
do not suffer from a lack of binding affinity.1,2 The second
were later reported.6,7
question that must be answered is whether one will be able to
Mirkin and coworkers have reported an assay for chloride
observe the desired recognition event. Reconciling low analyte
anions through the clever use of their signature ‘‘weak-link’’
approach to allosteric macrocyclic ring expansion/constriction.8
concentration with detectable signal output has received a
good deal of attention in recent years.3
In this assay, a heterometallic macrocycle containing two zinc(II)
One way that organic chemists have begun to deal with this
and two ruthenium(I) centers was created. In the presence of
problem is through the incorporation of a catalytic process to
chloride and CO the macrocycle adopts an open conformation,
increase signal output in an assay. To this end, biochemists
which fostered facile conversion of acetic anhydride and 4-
and microbiologists have been exploiting enzymatic processes
hydroxymethylpyridine to 4-acetylmethylpyridine and acetic acid
for a number of years. Perhaps the most famous use of this
via the catalytic zinc centers. In the presence of 9-(aminomethyl)
paradigm is the ELISA assay.4 Many standard disease screen-
anthracene, the creation of acetic acid protonated the appended
ings utilize this powerful method, which makes use of the
amine and disrupted the PET quenching mechanism. This led to
strong binding interactions between antibodies and their anti-
a turn on of the anthracene fluorescence.
gens. Though very powerful, these types of hybridization
Several recent reports have been published for the detection of
assays are only useful for bio-available analytes.
nucleic acids using transition metal based catalytic signal ampli-
fication as well.9,10 As yet, however, no instances have been
Recently, however, chemists have begun to exploit the vast
wealth of transition metal catalysts for sensing purposes. The
reported using transition metal catalysis to detect small organic
first instance of a metal catalyst used in a recognition assay for
analytes. In this paper, we discuss the first such assay to utilize
the detection of transition metal analytes was reported by our
these methods in the analysis of ketones and aldehydes. We also
group.5 Using EDTA as the host, a competition assay between
report the first instance of peroxyoxalate initiated chemilumines-
cence as a signaling motif in a supramolecular assay.11,12
Department of Chemistry and Biochemistry, The University of Texas
at Austin, 1 University Station A5300, Austin, TX 78712, USA.
E-mail: anslyn@ccwf.cc.utexas.edu; Fax: (512) 471-7791; Tel: (512)
Results and discussion
471-0068
w Electronic supplementary information (ESI) available: Excitation
Target choice and assay design
and emission spectra for all fluorophores and chemiluminescence
spectra for DPA. See DOI: 10.1039/b616765a
z Dedicated to Professor George Gokel on the occasion of his 60th
birthday.
All previously reported attempts to use transition metal
catalysis for signal enhancement describe analyses of inorganic
ꢀc
This journal is the Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2007
New J. Chem., 2007, 31, 729–735 | 729

View Article Online
or large biological substrates.13 The quandary which arises
when dealing with a small organic analyte is that the strength
of organic intermolecular noncovalent binding forces is gen-
erally much weaker than those associated with metal–organic
interactions. Hence, a competition assay between a specific
organic analyte and a metal based catalyst for an organic host/
inhibitor scaffold is difficult to design. To this end we realized
that a covalent interaction of our analyte with our host might
better suit our needs than a purely supramolecular approach.
The poisoning interaction between thiols and palladium
catalysts is well known.14 For process scale heterogeneous
hydrogenation catalysts, sulfur containing dopants are some-
times added to fresh catalysts to control the activity. The
porous palladium surface acts as a sponge for the soft sulfur
nucleophiles, which form very strong metal–sulfide bonds. The
usefulness of dithiols has long been known to organic che-
mists. Dithiols represent one of the first protecting groups a
student learns in introductory organic chemistry. When 1,3-
propanedithiol is condensed with aldehydes to form 1,3-
dithiolanes, so called ‘‘umpolung’’ reactions can be performed
by deprotonation of the otherwise intractable aldehyde pro-
ton.15 With both processes well established, we designed an
assay for ketones and aldehydes based on the thioacetal
condensation with a palladium-catalyzed Sonogashira cross-
coupling reaction as a signal amplification method (Scheme 1).
the initial amount of 2-butanone. To this mixture, triphenyl-
phosphine is added to displace the acetonitrile ligands for
more effective catalysis. To commence the Sonogashira cross-
coupling reaction, phenylacetylene and 9-bromo-10-(pheny-
lethynyl)anthracene are added and the mother liquor is
brought to a gentle reflux. Copper(I) iodide is added as a final
step along with 2,5-diphenyloxazole (PPO), an internal fluor-
escence standard.
The Sonogashira coupling described in the final step of
Scheme 1, produces the well known lumophore 9,10-bis(phe-
nylethynyl)anthracene (BPEA). As a fluorophore it has been
reported to have Fo = 1.0.16 At concentrations lower than
10ꢁ6 M, monomer lmaxem occurs near 470 nm in most
common organic solvents. At higher concentrations, BPEA
shows a strong tendency towards excimer formation with a
lmaxem near 500 nm. The excimer emits the characteristic lime
green color found in many commercially available glowsticks.
For this assay, we chose to focus mainly on the fluorescence
properties of BPEA rather than its chemiluminescent effects.
Due to PET quenching from the large amount of triethyl-
amine present in the reaction, we were unable to perform
direct fluorescence measurements on the system. Thus, the
assay was conducted on a semi-preparative scale with aliquots
taken at precise timed intervals over a two hour period. The
aliquots were acid washed to remove the offending amine base
and diluted in ethyl acetate to less than 10ꢁ6 M of fluorophore,
assuring a safe range of emission even at full conversion to
BPEA. Due to potential imprecision in aliquot acquisition and
solvent evaporation over the two hour reaction time at reflux,
we added the soluble internal standard 2,5-diphenyloxazole
(PPO). PPO has been shown to have extremely high quantum
efficiency and was an attractive choice as its lmaxex overlays
well with the troughs in the anthracene-dye excitation profiles
(see Supplementary Informationw). The use of this non-reac-
tive internal standard negates the need for rigorous attention
to volumetrics concerning aliquot acquisition (see derivation
Sonogashira assay
1,3-Propanedithiol is condensed with an analytical amount of
a representative ketone, 2-butanone, in acidic dichloro-
methane with the Lewis acid catalyst zinc(II) chloride to give
a mixture of the dithiolane and a remainder of free dithiol.
After quenching with triethylamine, one equivalent of bis
(acetonitrile)-palladium(II) chloride vs. original dithiol is
added. The residual dithiol chelates and poisons some of the
palladium precatalyst while the rest remains unspoiled. Hence,
the unpoisoned amount of palladium catalyst is the same as
Scheme 1 Description of the ketone assay based on a Sonogashira cross-coupling reaction. Pd(MeCN)2 and 1 are added in equamolar amounts,
and depending on the amount of 2-butanone present, a greater or lesser fraction of Pd(II) will remain unpoisoned towards the conversion of 4 to
BPEA.
ꢀc
This journal is the Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2007
730 | New J. Chem., 2007, 31, 729–735

View Article Online
below). The following discussion of the fluorescence based
kinetics relies heavily on the use of PPO in this assay.
eqn (10).
ꢄ
ꢅ
Z
Z
Z
x
FP
Fs
k0½catꢂ
½Sꢂk
x
d
¼
dt ¼ k00½catꢂ dt
ð9Þ
Derivation of kinetics
Determination of the sensitivity of this detection method
depends upon a good understanding of the kinetics of the
Sonogashira coupling. A proposed rate law is given by eqn (1)
where A is phenylacetylene, B is 4, P is BPEA, and ‘‘cat’’
represents the Pd-catalyst or alternatively the ketone/aldehyde
concentration.
½Aꢂ0½Bꢂ0
k00 ¼ k
ð10Þ
I0fPeP
½Sꢂ
I00 fSeS
The integrated rate law, eqn (11), shows that the rate can be
sufficiently described by a plot of the ratio of fluorescence
intensities of P to S vs. time.
dP
x
¼ k½catꢂ ½Aꢂ½Bꢂ
ð1Þ
FP
FS
dt
x
¼ k00½catꢂ t
ð11Þ
If we assume initial rate kinetics we can make the simplifica-
tion given in eqn (2). As mentioned above, a work-up step
prior to fluorescence measurement results in an unknown
amount of fluorophore loss. To solve this problem and
eliminate the need for any volumetric congruity between
aliquots, an internal standard was added to the reaction,
represented here as S. If we divide both sides of eqn (2) by
the concentration of the internal standard we arrive at a rate
law based on the change in the ratio of P to S, eqn (3).
Finally, a plot of the rates vs. catalyst/ketone concentration
will elucidate the order in catalyst and the apparent rate
constant, k00.
Rate studies
Rate studies were conducted by varying the amount of
2-butanone from 0% to 100% vs. 1,3-propanedithiol. This
represents a concentration range of roughly 1–10 mM of
2-butanone. Theoretically, a 0% assay should show no for-
mation of BPEA whereas a 100% assay should give very fast
conversion. A plot of the rate determinations for representa-
tive concentrations as a function of the ratio of product
fluorescence to PPO emission vs. time is shown in Fig. 1.
PPO is excited at 316 nm and read at 357 nm and BPEA is
excited at 451 nm and read at 472 nm. As expected with no
ketone present, no increase in product emission is observed
over the 2 hour reaction time. As the amount of analyte
increases so too does the rate of conversion to BPEA. At
85% ketone loading and above, the rates begin to plateau,
indicating a departure from initial rate kinetics at a time
dependent on the amount of catalyst present.
dP
x
¼ k0½catꢂ
ð2Þ
dt
where k0 = [A][B] and [A][B] = [A]0[B]0
ꢀ ꢁ
x
dP
dt
P
S
k0½catꢂ
½Sꢂ
d
¼
¼
ð3Þ
½Sꢂ
dt
However, since neither concentration is absolutely known,
the rate law needs to be expressed in terms of fluorescence
intensity. Eqn (4) represents the Beer–Lambert Law for
fluorescence where I0 is the intensity of the incident light,
f is the quantum yield of the fluorophore, e is the
molar absorptivity, c is the concentration, and b is the path
length.
The rate profile is shown in Fig. 2. The plot of rate vs.
ketone concentration, which directly translates to catalyst
concentration, shows a marked nonlinearity. This deviation
from linearity can be explained by the complex equilibrium
shown in Scheme 2, which can occur for the palladium–dithiol
interaction. It can be assumed that the strength of the initial
Pd–S bond, K1, will be very large due to the electron deficiency
of the Pd(II)Cl2 center. Upon binding the first sulfur, however,
the palladium center becomes much less electrophilic and the
F = I0 f(1–10ꢁecb
F = I0 f 2.3ecb
)
(4)
(5)
At low concentrations, only the first term of the expanded
Taylor series for eqn (4) remains important, and the Law can
be simplified to eqn (5). A ratio of the fluorescence of P to S is
given by eqn (6). Eqn (6) can then be reduced to eqn (7)
FP I0fP2:3ePcPb I0fPeP ½Pꢂ
¼
¼
ꢃ
ð6Þ
ð7Þ
FS I00 fS2:3eScSb I00 fSeS ½Sꢂ
FP
FS
½Pꢂ
½Sꢂ
¼ k
where k is a constant representing the ratio of I0fe.
ꢂ
ꢃ
ꢀ ꢁ
FP
FS
x
P
S
k0½catꢂ
½Sꢂ
d
d
¼
k ¼
ð8Þ
dt
dt
Substituting eqn (7) into eqn (3) gives eqn (8).
We now have a rate law expressed in terms of fluorescence
instead of concentration. Separating the derivative and com-
bining the constants sets up the integrated rate law given
in eqn (9) with the new apparent rate constant, k00, given by
Fig. 1 Rate studies over several different equivalencies of 2-buta-
none.
ꢀc
This journal is the Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2007
New J. Chem., 2007, 31, 729–735 | 731

View Article Online
ing of the Sonogashira by fluorescence again making the assay
less applicable. Collisional quenching also occurs from the
chloride anions released upon reduction of the palladium
catalyst. The catalyst itself is also less stable than some other
non-chloride palladium salts, introducing an unpredictable
source of error. Also, the ultimate goal of generating a
chemiluminescent sensing motif is impaired by the need for
an extended work up of the aliquots. All of these concerns lead
us to ponder a new potential catalytic system.
The Suzuki cross-coupling reaction solved many of the
problems associated with the previous assay. Suzuki’s are
often run in water, they require only a carbonate base, and
the catalyst, Pd(OAc)2 is comparatively cheap and very stable.
The product, 9,10-diphenylanthracene (DPA), of the Suzuki
coupling shown in Scheme 3, is similar to its counterpart,
BPEA, in many of its optical properties. A blue emitter with
F0 = 1.0, DPA is the lumophore found in most commercial
blue glowsticks. Unlike BPEA, however, DPA shows very
little tendency towards excimer formation and maintains
emission near 410 nm over a wide concentration range. Un-
fortunately, the precursor 10, and the intermediate 9-bromo-
10-phenylanthacene also both emit near 410 nm, however,
their apparent quantum efficiencies are much lower than that
of DPA which allows for the determination of emission
increase.
Fig. 2 A rate profile for the Sonogashira based assay shows a marked
nonlinearity with respect to catalyst concentration.
second bond formation occurs less readily, allowing a more
balanced equilibrium described by K2. It is proposed that K2
still favors the bidentate chelation such that at the high
concentrations of residual dithiol brought about by low
ketone equivalency, the equilibrium is driven towards 5. As
the concentration of dithiol drops relative to palladium, a
second equilibrium begins to build up in which the Pd : dithiol
binding becomes 2 : 1 as with 6. We propose that 6 still retains
a modest amount of catalytic activity.
The Suzuki based assay is described by Scheme 3. Using an
extremely simplified dithiane formation procedure reported by
Wakharkar, 1,2-ethanedithiol was condensed with cinnamal-
dehyde in water in the presence of hydrobromic acid.17 The
reaction is extremely fast and forms a white precipitate which
at low analyte concentrations, redissolves in the remaining
immiscible ethanedithiol over the course of the reaction time.
Upon completion, the reaction is quenched with 6N NaOH to
sequester the hydrobromic acid and deprotonate the dithiol to
allow homogenious mixing. Basifying the mixture also causes
any dissolved dithiane product, 8, to precipitate again.
Unfortunately, the low concentrations of the extremely
efficient fluorophore product required for safe fluorescence
determination again prevented this assay from direct monitor-
ing. At nanomolar catalyst concentrations, the Suzuki reaction
proceeds extremely slowly if at all. Thus, the reaction was
again run on a semi-prep scale. Analogous to the previous
assay, initial attempts were preformed using 9-bromo-10-
phenylanthracene as a starting material, however, results for
this reaction were erratic and inconclusive. 9,10-Dibromoan-
thracene, 10, gave much better results for both fluorescence
and chemiluminescence detection. The insolubility of the
anthracenes also prevented accurate aliquot measurement.
To overcome these difficulties two identical reactions were
set up in parallel, one as a control and one as the sample.
Both reaction vessels were charged with palladium acetate in
DMF and a small aliquot of the completed dithiane mixture
was added such that the palladium to original dithiol ratio was
unity. Then both flasks were given equal amounts of 10, PPO,
and sodium carbonate. The control flask was allowed to stir
for 20 minutes at 40 1C. The sample flask was charged with 3
eq. of phenylboronic acid and allowed to react for 2 hours at
40 1C. The Suzuki reaction conditions were modified from the
literature and run in a 7 : 5 : 14 mixture of DMF : THF :
Since this phenomenon occurred for both the Sonogashira
assay above and the Suzuki assay described below, we wanted
to test this theory. To that end, a test Suzuki reaction using
1 mmol of 9-bromoanthracene, 1.5 mmol phenylboronic acid,
2 mmol sodium carbonate, and 4.5 ꢃ 10ꢁ3 mmol each of
Pd(OAc)2 and 1-propanethiol, which can only monothiolate
one palladium, was run in a 3 : 3.5 acetone–water mix. The
reaction shows moderate conversion to 9-phenylanthracene
over a 2 hour period based on TLC and CI-MS analysis. Thus,
as the concentration of 6 increases due to decreasing dithiol
concentration, the fraction of active palladium increases non-
stoichiometrically.
Suzuki based assay
As a proof of concept, the Sonogashira assay worked well.
However, several pragmatic issues arise from the use of this
particular catalytic process. The first problem is the reaction’s
intolerance to water which makes the assay rather impractical
to apply. Another issue is the necessity of an amine base to
facilitate reduction of the Pd(II) salt to the active Pd(0) species.
The side effect of PET quenching prohibits the direct monitor-
Scheme 2 The complex equilibrium of Pd(II)–dithiol coordination.
ꢀc
This journal is the Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2007
732 | New J. Chem., 2007, 31, 729–735

View Article Online
Scheme 3 Description of the Suzuki based assay. Analogous to the Sonogashira assay, the rate of DPA production increases based on the
concentration of cinnamaldehyde.
H2O.18 THF was added for initial solubility of 10 before the
addition of water. Upon completion, the reactions were
directly diluted with ethyl acetate to partition the solvents
and dissolve the anthracenes. The organic layer was then
diluted for fluorescence and chemiluminescence measure-
ments.
dependence on the catalyst concentration appears to be pre-
sent in this assay as well. The reasons for this observation have
already been discussed. Fluorescence data was taken in THF
with an excitation of 393 nm and read at 407 nm and shows a
modest 13% intensity increase for the lowest concentration up
to nearly 200% increase for the highest concentration studied.
Perhaps even more interesting is the chemiluminescence data.
Chemiluminescence studies were performed in a fluorometer
with the excitation shutter closed. Using the Chemically
Initiated Electron Exchange Luminescence (CIEEL) mechan-
ism of initiation with bis(2-carbopentyloxy-3,5,6-trichlorophe-
nyl)oxalate and hydrogen peroxide, we were able to obtain the
data shown in Fig. 4. The increase in chemiluminescence is
nearly an order of magnitude higher than that detected by
fluorescence, with the lowest concentration tested giving an
outstanding 150% intensity increase. It is interesting to note
that even though 10, and the mono-coupled intermediate have
roughly the same excitation and emission profiles as DPA,
their chemiluminescence efficiency is essentially non-existant
as is evidenced by the large difference in intensity changes
Fluorescence vs. chemiluminescence
The solution resulting from the control experiment where no
phenylboronic acid was added was used as a time zero
measurement. A 120 minute measurement was taken with
the solution garnered from the full reaction flask. For fluor-
escence measurements, the samples were diluted to 5 mM of
total anthracene species. Chemiluminescence samples were
prepared at 200 mM of total anthracene containing species.
Data was observed over aldehyde loading from 0–90% vs. 1,2-
ethanedithiol. This represents a concentration range starting
at 10 mM. The results are plotted as a percent increase in
fluorescence (Fig. 3) and chemiluminescence (Fig. 4).
Though single point measurements are insufficient to fully
characterize the kinetics of the reaction, a similar nonlinear
Fig. 4 Chemiluminescence measurements show a much greater turn
Fig. 3 Rate profile for the Suzuki assay measured from fluorescence.
on for the Suzuki based assay.
ꢀc
This journal is the Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2007
New J. Chem., 2007, 31, 729–735 | 733

View Article Online
between fluorescence and chemiluminescence. This assay de-
monstrates the power of using chemiluminescent sensors for
high sensitivity outside the biological arena.
Palladium poisoning and sonogashira
Then 2.5 mL of an 8.60 ꢃ 10ꢁ3 M solution of Pd(MeCN)2Cl2
in dichloromethane is added to the quenched solution and
stirred for five minutes under argon. A 12.5 mL solution
containing 0.19 mmol 9-bromo-10-(phenylethynyl)anthracene
and 0.12 mmol 2,5-diphenyloxazole along with 5 mg neat CuI
are then added and the 30 mL solution is brought to gentle
reflux. Finally, 50 mL of phenylacetylene is added and 250 mL
aliquots are taken and worked up every 10 min for 2 h.
Conclusions
We have reported the first use of catalytic signal amplification
in the detection of a small organic analyte. Though these
particular assays may not be extremely sensitive, the concept
presented herein paves the road for the exploitation of transi-
tion metal catalysis for sensing purposes. Furthermore, these
assays demonstrate the use and power of peroxyoxalate–
CIEEL chemiluminescence in a sensing system, which to our
knowledge has not been reported to date.
Aliquot work-up
250 mL of the reaction liquor is partitioned between 4 mL
ethyl acetate and 2 mL 2N HCl. For fluorimetry, the organic
layer is then directly diluted by roughly 10ꢁ3 in ethyl acetate.
Preparation of the aliquot for chemiluminescence requires
further washing of the organic layer with 2M NaOH followed
by drying (Na2SO4).
Experimental
Materials and methods
Fluorescence
All reagents were obtained through Sigma-Aldrich or Acros
Fine Organics and used without further purification unless
Spectra were taken for 2,5-diphenyloxazole, 9-bromo-10-(phe-
nylethynyl)anthracene, and 9,10-bis(phenylethynyl) anthra-
cene in ethyl acetate exciting at 316 nm, 406 nm, and 451
nm, respectively for each aliquot. 2,5-Diphenylaoxazole was
read at 357 nm, 4 at 432, and BPEA at 471. Slit widths were set
at 2 nm for excitation and emission giving average fluorescence
intensities of 4105 counts per s. Data from each spectrum was
collected via integration from ꢁ5 to +5 nm about the
respective emission lmax using the Felix32 v1.0 software
package.
noted.
Bis(acetonitrile)palladium(II)
chloride,
9,10-di-
bromoanthracene, and 9-bromo-10-phenylanthracene were
prepared as described in the literature.19–21 Triethylamine
and dichloromethane were distilled over calcium hydride prior
to use. Fuorescence and chemiluminescence measurements
were performed on a Photon Technology International Quan-
taMaster Cuvet-Based spectrofluoromter.
9-Bromo-10-(phenylethynyl)anthracene (4). 4.0 g (12 mmol)
9,10-dibromoanthracene, 83.5 mg (0.12 mmol) bis(tripheny-
phosphine)palladium(II) chloride, and 23 mg (0.12 mmol)
copper(I) iodide were added to 250 mL of freshly distilled
triethylamine and brought to 40 1C. To this solution was
added dropwise over 6 hours 12.7 mmol phenylacetylene in 50
mL dry triethylamine. The reaction was allowed to stir over-
night at 40 1C after addition was complete. Upon completion
of the reaction by TLC, the mother liquor was filtered with
hexanes through celite and partitioned into 2N HCl. The
organic extract was washed again with 2N HCl until slightly
acidic by pH paper. The organic layer was then washed with
water, saturated NaCl, and dried (Na2SO4). 100 mg of chro-
matography grade silica gel was added to the dried organic
layer and the solvent was removed. The silica-adsorbed residue
was chromatographed on silica gel with 100% hexanes to give
2.2 g (6.1 mmol, 52% yield). Characterization is reported in
the literature and is concomitant with our analysis.22
Suzuki assay
The trial with 13.3% cinnamaldehyde is used as an example.
Dithiane condensation
To a 5 mL roundbottom flask are added 1 mL thoroughly
degassed water, 50 mL (0.6 mmol) ethanedithiol, 10 mL (0.08
mmol) cinnamaldehyde, and 25 mL 48% hydrobromic acid in
water. The reaction is stirred at 40 1C for 2 h during which
time a small amount of white precipitate forms and is redis-
solved in the remaining insoluble ethanedithiol. The reaction is
quenched with 3 mL of 6N sodium hydroxide which reforms
the white precipitate as the ethanedithiol is deprotonated and
dissolved in the aqueous layer. At this time the total concen-
tration of disulfur species (ethanedithiol and the condensed
dithiolane) is 0.147 M. This solution is labeled solution A.
Sonogashira assays
Suzuki coupling
The trial using 55% 2-butanone to catalyst is used as an
example procedure.
In a 10 mL volumetric flask was placed 101.3 mg (0.3 mmol)
9,10-dibromoanthracene and 25.3 mg (0.1 mmol) 2,5-diphe-
nyloxazole (PPO), which were then diluted to 10 mL with a
1 : 1 DMF : THF solution. The resulting solution was labeled
solution B. 83.5 mg (0.37 mmol) of palladium(II) acetate was
diluted to 50 mL in DMF in a separate volumetric flask and
labeled solution C. To each of two parallel reaction flasks were
added 3.5 mL degassed water, 0.5 mL of solution C, and 25 mL
of solution A. The solution turned from a light brown to burnt
orange instantaneously. To each of these solutions was then
added 2.5 mL of solution B which precipitated upon contact
Dithiolane formation
Under inert argon atmosphere, 2.5 mL of an 8.60 ꢃ 10ꢁ3
M
solution of 1,3-propanedithiol in dichloromethane is mixed with
2.5 mL of a 4.73 ꢃ 10ꢁ3 M solution of 2-butanone in dichlor-
omethane. To the solution is added 10 mL concentrated sulfuric
acid and 10 mg zinc chloride. The reaction is stirred under
argon at room temperature for 1 hour and then quenched by
adding 5 mL dichloromethane and 5 mL triethylamine.
ꢀc
This journal is the Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2007
734 | New J. Chem., 2007, 31, 729–735

View Article Online
with water. The reaction vessels were then brought to 40 1C.
To the first reaction flask labeled time 120, was added 31.6 mg
(0.3 mmol) sodium carbonate and 25.3 mg (0.2 mmol) phe-
nylboronic acid and the timer was started. To the second flask
labeled time 0, was added only 31.5 mg (0.3 mmol) sodium
carbonate. The time 0 flask was stirred for 20 min and the time
120 flask was stirred for 2 h. Final concentrations in the
reaction flask prior to reaction were as follows: 9,10-dibro-
moanthracene 0.012 M; PPO 0.004 M; palladium (II) acetate
5.7 ꢃ 10ꢁ4 M; phenylboronic acid 0.032 M; ethanedithiol
based on no dithiolane formation 5.7 ꢃ 10ꢁ4 M; sodium
carbonate 0.046 M. Upon completion of the reaction both
flasks were treated in the following manner. 7 mL of ethyl
acetate were added to quench the reaction giving a 10 mL
organic layer on the assumption that all DMF was parti-
tioned. From the organic layers, a 0.25 mL aliquot was diluted
to 10 mL with THF and labeled Lum0 and Lum120, respec-
tively.
Acknowledgements
We would like to acknowledge Ms. Himali Hewage of the
Anslyn research group for helpful discussions on chemilumi-
nescent methods and Joy Wu formerly of the Anslyn group
and Dr Lei Zhu of Florida State University for their initial
work on catalytic signal amplification and helpful insights on
developing these assays. Funding for this research was pro-
vided by The Welch Foundation and the National Institutes of
Health #GM65515-2.
References
1 Advances in Supramolecular Chemistry, ed. G. W. Gokel, Cerberus
Press Inc., Miami, FL, USA, vol. 9, 2003.
2 J. W. Steed and J. L. Atwood, Supramolecular Chemistry: A
Concise Introduction, Wiley, Indianapolis, IN, USA, 2000.
3 L. Prodi, New J. Chem., 2005, 29, 20–31.
Fluorescence
4 M. Los, Modern Bacteriophage Biology and Biotechnology, Re-
search Signpost, Trivandrum, India, 2006, pp. 131–152.
5 L. Zhu, V. M. Lynch and E. V. Anslyn, Tetrahedron, 2004, 60,
7267–7275.
6 Q. Wu and E. V. Anslyn, J. Am. Chem. Soc., 2004, 126,
14682–14683.
7 Q. Wu and E. V. Anslyn, J. Mater. Chem., 2005, 15, 2815
2819.
8 N. C. Gianneschi, S. T. Nguyen and C. A. Mirkin, J. Am. Chem.
Soc., 2005, 127, 1644–1645.
9 N. Graf, M. Goeritz and R. Kraemer, Angew. Chem., Int. Ed.,
2006, 45, 4013–4015.
10 A. Saghatelian, K. M. Guckian, D. A. Thayer and M. R. Ghadiri,
J. Am. Chem. Soc., 2003, 125, 344–345.
11 Z. Zhang, S. Zhang and X. Zhang, Anal. Chim. Acta, 2005, 541,
37–46.
12 C. V. Stevani, S. M. Silva and W. J. Baader, Eur. J. Org. Chem.,
2000, 4037–4046.
Fluorescence measurements were taken by diluting 50 mL of
Lum0 or Lum120, respectively to 2 mL with THF in a quartz
fluorescence cuvet. This dilution gives a maximum 4.9 ꢃ 10ꢁ6
M solution of fluorophore based on the initial amount of 9,10-
dibromoanthracene. Scans of each sample were taken with
excitations at 316 nm for PPO and 393 nm for the anthracene
fluorophores. The emission outputs at 357 and 407, respec-
tively were integrated as with the previous method and taken
as a ratio of product emission : PPO emission. The percent
fluorescence increase from time 0 to time 2 h was then
calculated and plotted vs. the initial concentration of cinna-
maldehyde.
Chemiluminescence
13 L. Zhu and E. V. Anslyn, Angew. Chem., Int. Ed., 2006, 45,
1190–1196.
Chemiluminescence measurements were conducted using
undiluted portions of Lum0 and Lum120 using the same
fluorometer. To a cuvet equipped with a magnetic stirring
rod was added 1.89 mL of the, respective sample, 50 mg (0.075
mmol) bis(2-carbopentyloxy-3,5,6-trichlorophenyl)oxalate, and
10 mL of 0.1 N sodium hydroxide. With the shutter to the
excitation lamp closed 100 mL of 30% hydrogen peroxide
solution was added followed quickly by initiation of a time-
based data collection protocol. When luminescence levels
plateaued, a spectrum scan was initiated, which took a 3-fold
average at each wavelength. The data was integrated as before
and the percent increase from time 0 to 2 h was calculated and
plotted vs. the initial concentration of cinnamaldehyde.
14 J. K. Dunleavy, Platinum Met. Rev., 2006, 50, 110.
15 D. Seeback and M. Kolb, Chem. Ind., 1974, 687–692.
16 H. Du, R.-C. A. Fuh, J. Li, L. A. Corkan and J. S. Lindsey,
Photochem. Photobiol., 1998, 68, 141–142.
17 P. D. Shinde, H. B. Borate and R. D. Wakharkar, ARKIVOC
(Gainesville, FL, USA), 2004, 110–117.
18 L. Liu, Y. Zhang and B. Xin, J. Org. Chem., 2006, 71, 3994
3997.
19 X. M. Zhang, F. G. Bordwell, J. E. Bares, J. P. Cheng and B. C.
Petrie, J. Org. Chem., 1993, 58, 3051–3059.
20 M. A. Andres, T. C. T. Chang, C. W. F. Cheng, L. V. Kapustay,
K. P. Kelly and M. J. Zweifel, Organometallics, 1984, 3,
1479–1484.
21 I. M. Heilbron and J. S. Heaton, Org. Synth., 1923, III, 41–43.
22 T. M. Swager, C. J. Gil and M. S. Wrighton, J. Phys. Chem., 1995,
99, 4886–4893.
ꢀc
This journal is the Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2007
New J. Chem., 2007, 31, 729–735 | 735