Exponential diagnostic signal amplification via dendritic chain reaction: The dendritic effect of a self-immolative amplifier component

Article abstract of DOI:10.1039/c1nj20486f

Signal amplification techniques are commonly used to boost diagnostic signals. We have recently developed a new approach for exponential signal amplification obtained through a distinctive dendritic chain reaction. Here we report evaluation of the effect of the self-immolative dendritic amplifier component on the signal amplification. Four dendrons with various numbers of end-units were evaluated for the ability to produce exponential signal amplification through self-immolative disassembly pathways. The dendron composed of a first-generation platform with three end-units exhibited the best characteristics with rapid disassembly rate and good stability under aqueous conditions. This study demonstrates the efficiency of molecules based on dendritic structures.

Full text of DOI:10.1039/c1nj20486f

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PAPER
Exponential diagnostic signal amplification via dendritic chain reaction:
the dendritic effect of a self-immolative amplifier componentwz
Naama Karton-Lifshin and Doron Shabat*
Received (in Victoria, Australia) 6th June 2011, Accepted 27th August 2011
DOI: 10.1039/c1nj20486f
Signal amplification techniques are commonly used to boost diagnostic signals. We have recently
developed a new approach for exponential signal amplification obtained through a distinctive
dendritic chain reaction. Here we report evaluation of the effect of the self-immolative dendritic
amplifier component on the signal amplification. Four dendrons with various numbers of end-units
were evaluated for the ability to produce exponential signal amplification through self-immolative
disassembly pathways. The dendron composed of a first-generation platform with three end-units
exhibited the best characteristics with rapid disassembly rate and good stability under aqueous
conditions. This study demonstrates the efficiency of molecules based on dendritic structures.
this strategy has been demonstrated for detection of two different
Introduction
analytes: hydrogen peroxide and ubiquitous sulfhydryl
compounds.17 In the 2CDCR approach the self-immolative
Development of new signal amplification techniques is needed in
order to improve the ability to detect trace amounts of a wide
dendron is used as an amplifier moiety and the other component
variety of analytes. Therefore, there is a considerable interest in
as the signal generator. Hence, the rate of amplification should in
the development of new molecularly based amplification
part depend on the number of end-groups on the self-immolative
methodologies.1–10 Recently, we reported on a new distinctive
dendron. Here we report an evaluation study of the dendritic
molecular system that generates exponential signal amplification
effect on the signal produced by the 2CDCR technique.
through a dendritic chain reaction (DCR).11,12 The system is
To evaluate the dendron-generation effect on the 2CDCR, we
based on the unique disassembly properties of self-immolative
chose a model system designed for detection of hydrogen
dendrimers, where a single stimulus event by a specific analyte at
peroxide (Fig. 1). Dendron 1 is composed of two glucose units
the dendron focal point is translated into release of multiple
peripheral end units.13–15 When the end units are released, they
and a phenylboronic-acid trigger, which is cleaved upon reaction
with hydrogen peroxide.5 Probe 2 is composed of the reporter
acquire the chemical reactivity of the analyte of interest and
5-amino-2-nitrobenzoic acid attached to the phenylboronic-acid
thereby can then activate additional dendrons to produce a chain
trigger. The two-component DCR amplification cycle of dendron 1
reaction that progresses through an exponential disassembly
and probe 2 is illustrated in Fig. 1. Cleavage of the trigger of
mode. The system disassembly is monitored by a reporter
dendron 1 by a hydrogen peroxide molecule will release two
molecule, which is also released from the self-immolative
glucose molecules. The two free glucose units will be oxidized
dendron to produce exponential signal growth. A simpler version
by glucose oxidase (GOX) present in solution to produce two
of the DCR system was later developed, in which a separated
molecules of hydrogen peroxide, which then activate additional
chromogenic probe and a self-immolative dendron were used
dendrons. The rate of disassembly should exponentially increase
together to produce exponential signal amplification through a
until all the reporter molecules have been released. The signal can
two-component dendritic chain reaction (2CDCR).16 The modular
be detected with a spectrophotometer by monitoring the yellow
design of the DCR probes allows introduction of detection
color of the released 5-amino-2-nitrobenzoic acid.
capabilities for various compounds through incorporation of a
Glucose is used as a precursor for hydrogen peroxide since it
trigger that is reactive with the analyte of interest. Thus far,
can be easily oxidized by the commercially available enzyme
GOX after its release from the dendritic platform. In addition,
the hydrophilicity of glucose increases the aqueous solubility
Department of Organic Chemistry, 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 9293; Tel: +972 (0)3 640 8340
w Electronic supplementary information (ESI) available: Full experi-
mental details, characterization data of all new compounds (PDF). See
DOI: 10.1039/c1nj20486f
of the relatively lipophilic dendrons.
Results and discussion
In order to employ the proposed dendritic chain reaction for
detection of hydrogen peroxide, two major chemical
z This article is part of the themed issue Dendrimers II, guest edited by
Jean-Pierre Majoral
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Fig. 1 A two-component DCR system to detect hydrogen peroxide; glucose reagent units and 5-amino-2-nitrobenzoic acid reporter are indicated.
Fig. 2 Disassembly mechanism of compound 1a and probe 2. PGA-responsive release of covalently linked glucose to generate hydrogen peroxide is shown.
reactivities must be in hand: a ‘‘disassembly’’ chemical path-
way that enables the release of a covalently linked glucose and
a specific protecting group that is cleaved by a reaction with
hydrogen peroxide. These two reactions are illustrated in
Fig. 2. The release of a covalently linked glucose was achieved
through the design of molecules like compound 1a (see ESIw
for synthesis). This compound is composed of a glucose
molecule linked through a self-immolative linker to phenyl-
acetamide—a substrate for penicillin-G-amidase (PGA). When
PGA, a model-cleavage reagent, was incubated with the
compound 1a, the generated aniline undergoes 1,6-elimination
to release intermediate 1b, which is further disassembled to
release the glucose molecule. The free glucose is then oxidized
by GOX, also present in the solution, to form hydrogen
peroxide, which then can activate probe 2 to release the
5-amino-2-nitrobenzoic acid reporter.
Probe 2 and model-compound 1a were incubated in PBS
(pH 7.4), in the presence of GOX with or without PGA. The
release of the 5-amino-2-nitrobenzoic acid was monitored by
UV-Vis spectroscopy. As expected, in the presence of PGA
and compound 1a, the reporter was released from probe 2; no
release was observed in the absence of the enzyme (Fig. 3). The
proposed disassembly pathway clearly occurred, and release of
a covalently linked glucose produced hydrogen peroxide,
which activated a phenylboronic acid protecting group-
based probe.
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Four different self-immolative dendrons were synthesized,
each equipped with
a phenylboronic-acid as a trigger
with various numbers of glucose end-units. Dendrons 3
(AB2, attached to two glucose units) and 4 (AB3, attached to
three glucose units) are based on a first-generation dendritic
platform, whereas dendrons 5 (AB4, attached to four glucose
units) and 6 (AB6, attached to six glucose units) are based on a
second-generation dendritic platform (Fig. 4).
Dendrons 3, 4, 5 and 6 were synthesized by linking the
glucose units through carbamate linkages generated in situ
from acylazide 9 (Fig. 5). The synthesis of molecule 9 was
performed by coupling of compound 718 with N-hydroxy-
succinimide (NHS) generated NHS-ester derivative 8. The latter
was reacted with sodium azide to afford acylazide 9.
Fig. 3 PGA-catalyzed release of 5-amino-2-nitrobenzoic acid
through sequential disassembly of compounds 1a and 2. Conditions:
wavelength, 405 nm; 1a and 2 [500 mM] in PBS (pH 7.4) and PGA
[0.01 mg mLꢀ1]; 25 1C.
Next, we synthesized AB2 dendron 3 that contains two
glucose units and phenylboronic acid as a trigger unit.
Fig. 4 Chemical structures of self-immolative dendrons equipped with a phenylboronic-acid trigger and various numbers of glucose end-units.
Fig. 5 Chemical synthesis of glucose derivative 9.
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Fig. 6 Chemical synthesis of self-immolative AB2 dendron 3.
Synthesis of compound 3 was performed according to the
synthetic strategy presented in Fig. 6. Curtius rearrangement of
four equivalents of acylazide 9 generated an isocyanate, which
was reacted in situ with diol 1012 to afford compound 11.
Deprotection of the acetate groups using a catalytic amount of
potassium carbonate in methanol afforded AB2 dendron 3.
The AB3 dendron was synthesized using a procedure similar
to that described for the AB2 system (Fig. 7). Dendron 1216
was treated with p-TsOH to afford triol 13. The latter was
reacted with six equivalents of compound 9 to give the
protected form of dendron 14. Deprotection of the acetate
groups afforded AB3 dendron 4.
dendron 22 and further reaction with eight equivalents of
compound 9 gave compound 23. Deprotection of the acetate
groups as previously described afforded dendron 6.
With four self-immolative dendrons in hand, we evaluated the
ability of the first dendron (AB2 platform) to produce a
two-component DCR reaction. Dendron 3 and probe 2 were
incubated with various amounts of hydrogen peroxide in the
presence of GOX and the release of the 5-amino-2-nitro-benzoic
acid reporter was monitored at a wavelength of 405 nm (Fig. 10).
When 1.0 equivalent of hydrogen peroxide (vs. dendron 3) was
used, the system reached complete disassembly within 50 min. As
expected, disassembly was slower when less hydrogen peroxide
was used (Fig. 10). The exponential progress of the system
disassembly is demonstrated by the sigmoidal plots obtained
for reaction with various equivalents of H2O2. The background
signal obtained due to some spontaneous hydrolysis was also
amplified; therefore, the sensitivity of this system for detection of
H2O2 is limited to the low mM range. As described for dendron 3,
we evaluated the ability of dendrons 4, 5 and 6 to produce a
two-component DCR reaction. As expected, all dendrons
disassembled to produce exponential signal growth upon incubation
with H2O2, although rates differed (see ESIw).
The synthesis of AB4 dendron 5 was performed according to
the strategy shown in Fig. 8. Iodination of diol 10 with sodium
iodide and chlorotrimethylsilane generated benzyliodide deri-
vative 15. Etherification of the latter with compound 1612 gave
dendron 17. The tert-butylsilyl protecting groups were then
removed using a catalytic amount of p-TsOH to afford
dendron 18, which was further treated with eight equivalents
of compound 9 to yield compound 19. Deprotection of the
acetate groups as previously described afforded dendron 5.
Synthesis of AB6 dendron 6 was achieved as presented in
Fig. 9. Etherification of phenol 20 by using benzyliodide
derivative 15 and potassium carbonate gave dendron 21.
Removal of the tert-butylsilyl protecting groups afforded
The 2CDCR disassembly behaviors of the self-immolative
dendrons were then compared at the same concentration of
H2O2. Due to the higher number of glucose units released in
Fig. 7 Chemical synthesis of self-immolative AB3 dendron 4.
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Fig. 8 Chemical synthesis of self-immolative AB4 dendron 5.
each disassembly cycle, AB6 dendron 6 had the fastest ampli-
fication rate, whereas the AB2 dendron 3 was the slowest
(Fig. 11). The amplification rate observed for AB4 dendron 5,
despite release of a higher number of glucose units, was slower
than that produced by AB3 dendron 4. A possible explanation
for this phenomenon could lie in the number of elimination
reactions that occurred during the release of the glucose units
from the dendrons. AB3 dendron 4 is based on a first-generation
dendritic platform, which undergoes two 1,4 eliminations
and one 1,6 elimination in order to release three glucose units.
AB4 dendron 5 is based on a second-generation dendritic
platform, which undergoes six 1,4 eliminations in order to
release four glucose units. AB6 dendron 6 is based on a second-
generation dendritic platform that undergoes the highest
number of elimination reactions (in comparison to the other
dendrons) in order to release its glucose units. However, since
in every disassembly cycle six glucose units are released, this
dendron had the highest amplification rate for the 2CDCR
systems tested.
The disassembly behaviors of each of the four dendrons were
also compared with 0.1 equivalents and 0.01 equivalents of
H2O2. The amplification effect increased as the hydrogen
peroxide concentration decreased. This observation further
demonstrates the capability of the 2CDCR amplification effect,
which is more significant at lower analyte concentrations.
In order to determine the most efficient system for the
2CDCR amplification, we also sought to examine the hydro-
lytic stability of our dendrons. Disassembly of the self-immolative
dendrons as a result of hydrolysis generated a background
reaction that decreased the signal-to-noise ratio of the probe
system. For example, although the AB6 dendron had the
highest rate of disassembly, its hydrolytic stability may be
relatively low due to the high number of chemical bonds that
can undergo spontaneous hydrolysis. To compare the signal-
to-noise ratios of the different 2CDCR systems, the back-
ground noise was subtracted from the measured signal and the
obtained net-signal was plotted as a function of time for the
same hydrogen peroxide concentration (Fig. 12).
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Fig. 9 Chemical synthesis of AB6 dendron 6.
Fig. 10 The release of 5-amino-2-nitrobenzoic acid from probe 2 [500 mM] in the presence of dendron 3 [1000 mM] and GOX [0.1 mg mLꢀ1] in
PBS (pH 8.3) upon addition of buffer only (background) or the indicated equivalents of H2O2. The reaction progress was monitored at 25 1C, a
wavelength of 405 nm for the indicated time period.
Dendrons that disassemble more rapidly are expected to
and this phenomenon is reflected in the Tmax values. An addi-
tional desirable characteristic for a probe system with a large
signal-to-noise ratio is derived from the value of the net-signal.
Dendrons with better hydrolytic stability are expected to exhibit
larger values of net-signal. The AB3 dendron 4 seems to be the
exhibit curve-maximum (Tmax) at shorter time scales. Indeed,
the curve produced by AB6 dendron 6 had the shortest Tmax
,
whereas the curve produced by AB2 dendron 3 was the longest.
AB3 dendron 4 had a faster disassembly rate than AB4 dendron 5
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Fig. 12 Comparison of signals measured in the presence of the
indicated dendrons vs. time. The background signal observed as a
function of time was subtracted from the measured values of the
corresponding 2CDCR systems.
Fig. 11 Comparison of signals measured due to the release of 5-amino-
2-nitrobenzoic acid from probe 2 [500 mM] in the presence of the
indicated dendrons [1000 mM] and GOX [0.1 mg mLꢀ1] in PBS
(pH 8.3) upon incubation with 0.1 and 0.01 equivalents of H2O2. The
reaction progress was monitored at 25 1C, a wavelength of 405 nm for the
indicated time period.
ideal component for the 2CDCR system based on these
characteristics. This dendron exhibited a high hydrolytic stability
with a relatively fast disassembly rate, which is expressed in its
Tmax value.
Measuring of dendritic effects in the 2CDCR system further
supported the amplification advantage observed for AB3
dendron 4. The concentration of each dendron was calibrated
to the obtained identical released glucose units in each
2CDCR system and the disassembly rate was measured for a
fixed hydrogen peroxide concentration (Fig. 13).
Under these conditions, in the absence of dendritic effects,
all dendrons are expected to exhibit similar disassembly
behavior. As observed in Fig. 11, AB4 dendron 5 showed a
negative dendritic effect, whereas AB3 dendron 4 had a more
positive dendritic effect than AB2 dendron 3. The negative
dendritic effect observed for the AB4 dendron can be explained
by the ratio between the numbers of elimination reactions that
occur in each disassembly cycle and the number of glucose
units that are released. The AB4 dendron undergoes three
more elimination reactions than does the AB3 dendron; however,
only one more glucose unit is released in each disassembly cycle.
The AB4 dendron undergoes two less elimination reactions than
the AB6 dendron, but also two less glucose units are released in
each disassembly cycle.
Fig. 13 Comparison of signals measured due to the release of
5-amino-2-nitrobenzoic acid from probe 2 [500 mM] in the presence
of GOX [0.1 mg mLꢀ1] in PBS (pH 8.3) at 25 1C, upon addition of the
0.1 equivalents of H2O2. The concentrations of the dendrons that gave
identical glucose release are indicated.
Conclusions
In summary, we have demonstrated a new 2CDCR probe
system for detection of hydrogen peroxide, which exponentially
amplifies a visual diagnostic signal. The system is composed of
a simple chromogenic probe and a self-immolative dendron
component that acts as a molecular amplifier. Four dendrons
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with various numbers of glucose end-units were evaluated for
the ability to produce exponential signal amplification through
their self-immolative disassembly pathway. The release of free
glucose molecules and their oxidation by GOX to produce
hydrogen peroxide generated a chain reaction, which amplified
a diagnostic signal. The self-immolative dendron based on an
AB3 platform exhibited the best characteristics of a probe
system with rapid disassembly rate and good stability under
aqueous conditions. The modularity and flexibility of a
two-component detection system should allow extension for
detection of other analytes.
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Acknowledgements
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D.S. thanks the Israel Science Foundation (ISF), the Binational
Science Foundation (BSF) and the German–Israeli Foundation
(GIF) for financial support.
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