Sulfhydryl-based dendritic chain reaction

Article abstract of DOI:10.1039/c0cc02195d

A new dendritic chain reaction probe system was demonstrated to produce exponential signal amplification for the detection of sulfhydryl compounds.

Full text of DOI:10.1039/c0cc02195d

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Sulfhydryl-based dendritic chain reactionw
Eran Sella,a Roy Weinstain,a Rotem Erez,a Noah Z. Burns,b Phil S. Baranb and
Doron Shabat*a
Received 29th June 2010, Accepted 22nd July 2010
DOI: 10.1039/c0cc02195d
A new dendritic chain reaction probe system was demonstrated
to produce exponential signal amplification for the detection of
sulfhydryl compounds.
New molecular approaches for signal amplification are
frequently applied to improve the sensitivity of chemical
sensors.1–5 A powerful method of signal amplification is
exponential generation of a target molecule.6–10 Recently, we
reported a new molecular probe for the detection of hydrogen
peroxide that is based on a distinctive dendritic chain reaction
Fig. 1 Disassembly mechanism of compound 1 and probe 2.
(DCR).11,12 The self-immolative dendritic probe13–15 was
equipped with a triggering group (based on phenylboronic
acid) that upon interaction with the analyte, hydrogen
peroxide, released a chromogenic molecule and two or more
reagent molecules (choline). The free choline was enzymati-
cally oxidized to produce additional hydrogen peroxide that
could then activate additional probe molecules. As a result,
this chain reaction produces an exponential increase in
enables the release of a covalently linked thiol and a specific
protecting group that is cleaved by reaction with a thiol. These
two reactions are illustrated in Fig. 1. The release of a
covalently linked thiol was achieved through the design of
molecules like compound 1. The phenylacetamide moiety of 1
is removed by penicillin-G-amidase (PGA) followed by 1,6
elimination and decarboxylation to release amine 1a. The
latter undergoes spontaneous elimination and disassembles
to methanimine and free mercaptoacetic acid. Protecting
groups cleaved by thiols have been described in the literature
before.21–26 We have used compound 2 with a benzoquinone
trigger24 that upon reaction with a thiol generates intermediate 2a.
This intermediate undergoes rapid elimination to release quinone
2b and a reporter molecule like 5-amino-2-nitrobenzoic acid.
In order to test if we can release a general thiol and use it to
activate a chromogenic probe, compounds 1 and 2 (syntheses
are described in the ESIw) were incubated together in PBS
(pH 7.4) with or without PGA and the release of the reporter
5-amino-2-nitrobenzoic acid was monitored by UV-Vis
spectroscopy. Reaction of PGA with compound 1 resulted in
the release of free mercaptoacetic acid that then activated probe
2 to release 5-amino-2-nitrobenzoic acid (Fig. 2, blue plot). No
release was observed in the absence of PGA (Fig. 2, red plot).
the concentration of the chromogenic molecule and
a
strong diagnostic signal is developed from a relatively small
starting amount of analyte.16 The choline/hydrogen-peroxide/
phenyboronic acid reactions work beautifully together, but to
be broadly useful, the DCR technique must be demonstrated
for other analytes. In principle, the DCR technique could be
applied for the detection of other analytes since its probe has a
modular structural design. Generally, any analyte of interest
that has cleavage reactivity toward a specific trigger could be
incorporated collectively with the specific triggering group in
the dendritic platform. In order to validate this claim, an
additional example of a DCR probe that is based on different
chemistry than that of hydrogen peroxide as an analyte must
be demonstrated. We now report a new dendritic chain reac-
tion for the detection of molecules with ubiquitous sulfhydryl
functional group.
Thiols are important biomarkers that participate in several
physiological functions and their plasma level can indicate
the diagnosis of disease states.17–19 Therefore, the design of
diagnostic probes for identification of thiols is of obvious
importance.20 In order to employ a dendritic chain reaction
for the detection of thiols, two major chemical reactivities
should be in hand: a ‘‘disassembly’’ chemical pathway that
a School of Chemistry, Raymond and Beverly Sackler Faculty of
Exact Sciences, Tel-Aviv University, Tel Aviv 69978, Israel.
E-mail: chdoron@post.tau.ac.il; Fax: +972 (0)3 640 5761;
Tel: +972 (0)3 640 8340
b Department of Chemistry, The Scripps Research Institute,
10550 North Torrey Pines Road, La Jolla, CA 92037, USA
w Electronic supplementary information (ESI) available: Experimental
details, synthetic schemes, additional supporting data including NMR
spectra of all new compounds. See DOI: 10.1039/c0cc02195d
Fig. 2 PGA-catalyzed the release of 5-amino-2-nitrobenzoic acid through
sequential disassembly of compounds 1 and 2. Conditions: wavelength,
405 nm; 1 and 2 [500 mM] in PBS 7.4 and PGA [0.01 mg mLÀ1].
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With these results in hand, we sought to incorporate
the described chemical reactivities into a thiol dendritic
chain reaction system. The two-component dendritic chain
reaction27 (2CDCR) is a simple methodology that provides
exponential amplification and thus was chosen for evaluation
of the thiol analyte. The first component is based on an AB2
self-immolative dendron28 equipped with two reagent units
and a trigger designed to react with a specific analyte. The
second component is a probe composed of the same trigger
attached to a reporter. The AB2 self-immolative dendron acts
as an amplifier moiety, and the other component acts as a
probe that releases a chromogenic molecule to produce a
diagnostic signal. The 2CDCR mode of action for the thiol-
based reactions is illustrated in Fig. 3. Dendron 3 was com-
posed of two mercaptoacetic acid units and a benzoquinone
moiety as a trigger, which is cleaved upon reaction with any
sulfhydryl. Probe 2 was composed of the 5-amino-2-nitrobenzoic
acid reporter attached to the benzoquinone trigger. Cleavage
of the trigger of dendron 3 by a thiol molecule will initially
generate intermediate 3a. The latter will undergo double
eliminations to release two mercaptoacetic acid molecules
that will then activate some AB2 dendrons and some probe
molecules. Since the concentration of dendron 3 is at least
twice that of probe 2, the rate of the system disassembly should
exponentially increase until all of the reporter molecules have
been released. The signal can be detected with a spectro-
photometer by monitoring the yellow color of the released
5-amino-2-nitrobenzoic acid.
Fig. 4 The release of 5-amino-2-nitrobenzoic acid from probe 2
[500 mM] in the presence of dendron 3 [1000 mM] in Tris buffer
(pH 7.2) upon addition of the indicated equivalents of ethyl-2-
mercaptoacetate (relative to probe 2). The reaction progress was
monitored at 405 nm for the indicated time period.
ethyl-2-mercaptoacetate. The background signal obtained due
to some spontaneous hydrolysis is also amplified; therefore,
the sensitivity of this detection system for sulfhydryl is limited
to the low micromolar range.
The increased sensitivity obtained by the 2CDCR technique
is demonstrated in Fig. 5. When three different sulfhydryls
were incubated with probe 2 (in the absence of dendron 3)
no amplification was expected and the observed signal was
stoichiometrically correlated to the amount of the analyte.
However, the net signal (after subtraction of the background
signal) measured in the presence of dendron 3 was significantly
larger than that obtained from probe 2 alone. In fact, the ratio
between the signal with and without dendron 3 was increased
as the analyte concentration decreased. When 0.05 equivalent
of sulfhydryl vs. probe 2 was evaluated, the signal from
the 2CDCR system was roughly 6-fold stronger than that
obtained by the probe in the absence of dendron 3.
In order to test the sulfhydryl two-component DCR system,
dendron 3 and probe 2 were incubated with various amounts
of ethyl-2-mercaptoacetate and the release of 5-amino-2-nitro-
benzoic acid reporter was monitored at a wavelength of
405 nm (Fig. 4). When 1.0 equivalent of ethyl-2-mercaptoacetate
(vs. probe 2) was used, the system reached complete disassembly
within 15 min. As expected, disassembly was slower when less
thiol was used, however, the signal level has indicated full
release of the 5-amino-2-nitro-benzoic acid reporter (Fig. 4).
The exponential progress of the system disassembly is indi-
cated by the sigmoidal shape of the plots of time vs. percent
conversion obtained for reactions with various equivalents of
In addition, the benzoquinone trigger was found to have
high selectivity towards addition reaction with thiols in the
presence of other nucleophiles.24
In the first example of our DCR amplification technique, we
designed a probe that had a detection activity for hydrogen
Fig. 5 Comparison of signals measured by the 2CDCR amplification
technique (with three different sulfhydryls) vs. signals measured using
probe 2 without dendron 3 (purple). The background signal present at
22 min was subtracted from the values shown.
Fig. 3 A two-component DCR system to detect sulfhydryl com-
pounds; mercaptoacetic acid reagent units and 5-amino-2-nitrobenzoic
acid reporter are indicated by red and blue color, respectively.
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peroxide.12 The generation of the analyte by the release and
enzymatic oxidation develops a chain reaction that results in
exponential amplification of a diagnostic signal. In this report we
showed that the distinctive pathway of the DCR amplification is
not limited to one analyte and can be extended to other reacti-
vities. The modular design of DCR probes allows us to introduce
detection capabilities for other compounds once the analyte of
interest that has cleavage reactivity toward a specific trigger would
be incorporated collectively with the specific triggering group. In
the sulfhydryl-based DCR system, the dendritic probe directly
releases the analyte of interest, a thiol, which can then initiate
additional diagnostic cycles. This is the first example of a DCR
amplification system that does not require any additional reagents
or enzymes in order to produce the chain reaction.
Notes and references
1 R. J. Amir, E. Danieli and D. Shabat, Chem.–Eur. J., 2007, 13,
812–821.
2 J. M. Nam, C. S. Thaxton and C. A. Mirkin, Science, 2003, 301,
1884–1886.
3 S. W. Thomas III, G. D. Joly and T. M. Swager, Chem. Rev., 2007,
107, 1339–1386.
4 Q. Wu and E. V. Anslyn, J. Am. Chem. Soc., 2004, 126,
14682–14683.
5 L. Zhu and E. V. Anslyn, Angew. Chem., Int. Ed., 2006, 45,
1190–1196.
6 C. M. Niemeyer, M. Adler and R. Wacker, Trends Biotechnol.,
2005, 23, 208–216.
7 V. Patzke and G. von Kiedrowski, ARKIVOC, 2007, 5,
293–310.
8 T. Sano, C. L. Smith and C. R. Cantor, Science, 1992, 258,
120–122.
9 K. Soai, T. Shibata and I. Sato, Acc. Chem. Res., 2000, 33,
382–390.
10 H. J. Yoon and C. A. Mirkin, J. Am. Chem. Soc., 2008, 130,
11590–11591.
11 M. Avital-Shmilovici and D. Shabat, Bioorg. Med. Chem., 2010,
18, 3643–3647.
12 E. Sella and D. Shabat, J. Am. Chem. Soc., 2009, 131, 9934–9936.
13 R. J. Amir, N. Pessah, M. Shamis and D. Shabat, Angew. Chem.,
Int. Ed., 2003, 42, 4494–4499.
14 A. Sagi, R. Weinstain, N. Karton and D. Shabat, J. Am. Chem.
Soc., 2008, 130, 5434–5435.
15 E. Sella and D. Shabat, Chem. Commun., 2008, 5701–5703.
16 M. A. Swiderska and J. L. Reymond, Nat. Chem., 2009, 1,
527–528.
17 K. S. McCully, Nat. Med., 1996, 2, 386–389.
18 A. Meister and M. E. Anderson, Annu. Rev. Biochem., 1983, 52,
711–760.
19 F. Michelet, R. Gueguen, P. Leroy, M. Wellman, A. Nicolas and
G. Siest, Clin. Chem. (Washington, DC), 1995, 41, 1509–1517.
20 X. Chen, Y. Zhou, X. Peng and J. Yoon, Chem. Soc. Rev., 2010,
39, 2120–2135.
The addition reaction of the thiol-analyte to the benzo-
quinone moiety of probe 2 or dendron 3 occurs very fast
(time-scale of seconds). The rate-determining step of the DCR
sequence is the release of the thiol units from dendron 3. Thus,
almost any thiol compound is expected to present similar
kinetic behavior upon reaction with the 2CDCR probe system.
It should be noted that although there are more sensitive
probes for the detection of thiols, this study represents a
general route for achieving exponential signal amplification,
which is based merely on reactions of small molecules. The
DCR technique is particularly useful in circumstances when a
reporter molecule with relatively weak spectroscopic signal is
used. In such example the exponential amplification leads to
strong observable diagnostic signal despite a low extinction
coefficient (in the case of UV-Vis reporter) or a low quantum
yield (in the case of a fluorescence reporter).
In summary, a new two-component DCR probe system for
the detection of sulfhydryl compounds was developed. The
probe is activated by a thiol analyte through a stoichiometric
reaction to generate a chain reaction that exponentially amplifies
a diagnostic signal. Importantly, this demonstrates the advan-
tage of our modular design of the two-component DCR system
and shows that versatile reactivities can be incorporated in order
to achieve exponential signal amplification for the detection of
various analytes. Additional DCR probes for the detection of
other analytes based on different chemistries are currently being
developed in our lab.
21 J. Bouffard, Y. Kim, T. M. Swager, R. Weissleder and
S. A. Hilderbrand, Org. Lett., 2008, 10, 37–40.
22 T. Fukuyama, M. Cheung, C.-K. Jow, Y. Hidai and T. Kan,
Tetrahedron Lett., 1997, 38, 5831–5834.
23 T. Fukuyama, C.-K. Jow and M. Cheung, Tetrahedron Lett., 1995,
36, 6373–6374.
24 S. T. Huang, K. N. Ting and K. L. Wang, Anal. Chim. Acta, 2008,
620, 120–126.
25 H. Maeda, H. Matsuno, M. Ushida, K. Katayama, K. Saeki and
N. Itoh, Angew. Chem., Int. Ed., 2005, 44, 2922–2925.
26 P. D. Senter, W. E. Pearce and R. S. Greenfield, J. Org. Chem.,
1990, 55, 2975–2978.
27 E. Sella, A. Lubelski, J. Klafter and D. Shabat, J. Am. Chem. Soc.,
2010, 132, 3945–3952.
28 R. Erez and D. Shabat, Org. Biomol. Chem., 2008, 6,
2669–2672.
D.S. thanks the Israel Science Foundation (ISF) and the
Binational Science Foundation (BSF) for financial support.
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Chem. Commun., 2010, 46, 6575–6577 | 6577