Dendritic chain reaction: Responsive release of hydrogen peroxide upon generation and enzymatic oxidation of methanol

Article abstract of DOI:10.1016/j.bmc.2010.02.038

Signal amplification dramatically increases the sensitivity of diagnostic methods. Recently, we introduced a new technique for signal amplification that uses a distinctive dendritic chain reaction (DCR) to generate exponential evolution of a diagnostic signal. In this report, we demonstrate how the modular design of our DCR probe can be used to improve the detection sensitivity. We synthesized a new probe based on a methyl carbonate linkage, which has superior stability in aqueous media. Triggered release of methanol, which was oxidized by alcohol oxidase present in the solution, produced hydrogen peroxide that used as a reagent in the DCR amplification technique. The new probe exhibited higher sensitivity in detection of hydrogen peroxide than our previously reported probe.

Full text of DOI:10.1016/j.bmc.2010.02.038

Bioorganic & Medicinal Chemistry 18 (2010) 3643–3647
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Dendritic chain reaction: Responsive release of hydrogen peroxide upon
generation and enzymatic oxidation of methanol
*
Michal Avital-Shmilovici, Doron Shabat
Department of Organic Chemistry, School of Chemistry, Raymond and Beverly Sackler Faculty of Exact Sciences, Tel-Aviv University, Tel Aviv 69978, Israel
a r t i c l e i n f o
a b s t r a c t
Article history:
Signal amplification dramatically increases the sensitivity of diagnostic methods. Recently, we introduced
a new technique for signal amplification that uses a distinctive dendritic chain reaction (DCR) to generate
exponential evolution of a diagnostic signal. In this report, we demonstrate how the modular design of
our DCR probe can be used to improve the detection sensitivity. We synthesized a new probe based on
a methyl carbonate linkage, which has superior stability in aqueous media. Triggered release of methanol,
which was oxidized by alcohol oxidase present in the solution, produced hydrogen peroxide that used as
a reagent in the DCR amplification technique. The new probe exhibited higher sensitivity in detection of
hydrogen peroxide than our previously reported probe.
Received 21 December 2009
Revised 17 February 2010
Accepted 21 February 2010
Available online 25 February 2010
Keywords:
Self-immolative dendrimer
Enzyme
Hydrogen peroxide
Amplification
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
the DCR assay. In this report, we demonstrate the advantages of
the modular design and characterize a probe with higher sensitiv-
Molecular probes, used for identification of various analytes,
commonly depend on responsive release of a chromogenic mole-
cule.1 The analyte of interest usually interacts with the probe
through a specific reaction that activates a latent chromophore.2,3
Recently, we introduced a new technique for signal amplification4,5
that can be used in diagnostic detection of analytes6–10, which is
based on a distinctive dendritic chain reaction (DCR).11 This tech-
nique depends on the disassembly properties of a self-immolative
dendrimer12,13 to generate an exponential evolution of a diagnostic
signal. The analyte of interest reacts with the trigger of the DCR
probe to release a reporter unit and reagents. Upon release, the re-
agents acquire the chemical reactivity of the analyte and thus can
activate two additional probe molecules (Fig. 1). This process re-
peats itself to ultimately release all of the reporter units from the
DCR probe.
The DCR technique was demonstrated for detection of hydrogen
peroxide as the analyte of choice. Hence, the probe was equipped
with a phenylboronic acid trigger14 that can be cleaved by hydro-
gen peroxide, 4-nitroaniline reporter and two choline molecules as
reagent units (compound 2, Fig. 2). In order to generate hydrogen
peroxide upon release of the choline molecules, the assay was per-
formed in the presence of choline oxidase. The modularity of the
probe molecule allows replacing structural elements like the cho-
line substrate with other compounds that can be oxidized to pro-
duce hydrogen peroxide and thereby to apply other oxidases in
ity in detection of hydrogen peroxide than our previously reported
probe.
2. Results and discussion
2.1. Design of the DCR probe
The detection limit of the analyte is dependent on the signal-to-
noise ratio.15 The sensitivity of the DCR assay is limited by the
spontaneous hydrolysis of the probe. This background signal is
likely generated through hydrolysis of the carbonate linkage of
the choline in dendron 2. In general, carbonates are more reactive
when they are constructed from alcohols with low pKa. The pKa of
choline is 13.9, whereas that of methanol is 15.5, more than one or-
der of magnitude higher. Thus, by replacing the choline with meth-
anol, the carbonate linkage in dendron 3 should be less likely to
spontaneously hydrolyze and the background signal of the DCR as-
say is expected to be lower.
The methyl carbonate in dendron 3 is much more hydrophobic
than the choline carbonate of dendron 2. Since the DCR assay is
performed under aqueous conditions, we replaced the 4-nitroani-
line reporter with 5-amino-2-nitrobenzoic acid. The addition of
ionized carboxylic acid group should compensate for the loss of
polarity in the reagent units.
The disassembly pathway of dendron 3 is presented in Figure 3.
Oxidation of the phenylboronic acid with hydrogen peroxide fol-
lowed by hydrolysis generates phenol 3a, which undergoes 1,6
elimination to release phenol 3b. The latter is known to undergo
* Corresponding author. Tel.: +972 (0) 3 640 8340; fax: +972 (0) 3 640 9293.
E-mail address: chdoron@post.tau.ac.il (D. Shabat).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.02.038

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M. Avital-Shmilovici, D. Shabat / Bioorg. Med. Chem. 18 (2010) 3643–3647
Reagent
Analyte
Analyte Activity
Trigger
Adaptor
Reagent
Reporter
1
Reagent
Reagent
Reporter
Signal
Figure 1. Graphical illustration of the DCR amplification cycle.
N
OCH3
O
O
O
O
O
O
O
HO
HO
HO
HO
B
B
HN
O
NO2
COOH
O
HN
O
NO2
O
O
O
O
O
O
2
3
OCH3
N
Figure 2. Chemical structure of DCR probes for detection of hydrogen peroxide.
OCH3
O
O
H2O2
Dendron 3
HO
HN
O
NO2
H2O/OH-
O
O
3a
COOH
O
O
O
OCH3
OCH3
H2N
NO2
O
O
COOH
HN
O
NO2
COOH
HO
O
CH3OH
CH3OH
3b
O
O
OCH3
Figure 3. Disassembly pathway of dendron 3 upon reaction with hydrogen peroxide.
three subsequent elimination reactions to release the reporter and
two methanol units.
2.2. Chemical synthesis of the DCR probe
As illustrated in Figure 4, the released methanol units are oxi-
dized in presence of alcohol oxidase (AOX) to produce two mole-
cules of hydrogen peroxide, which can then activate two
additional molecules of DCR probe 3. The amplification cycle re-
peats itself until all reporter molecules are released.
Dendron 3 was synthesized as shown in Figure 5. Amine 3c16
was treated with triphosgene to generate isocyanate 3d, which
was coupled in situ with alcohol 3e (prepared as previously re-
ported11) to afford carbamate 3f. Selective deprotection of the t-
butyldimethyl-silyl groups of compound 3f in the presence of p-

M. Avital-Shmilovici, D. Shabat / Bioorg. Med. Chem. 18 (2010) 3643–3647
3645
H2O2
OCH3
O
O
HO
B
HO
HN
O
NO2
O
O
COOH
3
H2O2
O
O
OCH3
Alcohol
Oxidase
COOH
CH3OH
CH3OH
H2N
NO2
Signal
Figure 4. The amplification cycle of DCR probe 3 for detection of hydrogen peroxide.
OTBS
OTBS
O
B
O
O
OH
O
O
3e
H2N
TMS
OCN
TMS
TMS
triphosgene
O
O
NO2
NO2
3d
3c
TMS
OH
OH
OTBS
O
O
O
B
B
p-TsOH
O
O
O
O
O
O
O
O
O
O
O
HN
NO2
HN
NO2
3f
OTBS
3g
OCH3
TMS
O
O
O
O
methyl chloroformate
TBAF
B
Dendron 3
O
O
O
O
O
O
O
HN
NO2
3h
OCH3
Figure 5. Chemical synthesis of dendron 3.
toluenesulfonic acid generated diol 3g, which was acylated with
methyl-chloroformate to afford dicarbonate 3h. Deprotection of
the trimethylsilylethanol ester and the pinacol ester groups with
tetrabutylammonium-fluoride afforded dendron 3.
5-amino-2-nitrobenzoic acid was monitored at a wavelength of
405 nm (Fig. 6). When 1.0 equiv of hydrogen peroxide was added,
the system reached complete disassembly within 2 h. Under these
conditions, there was no amplification since the analyte interacted
with the probe through a stoichiometric reaction. The signal gener-
ated from 1.0 equiv of hydrogen peroxide was used as a reference
when analyzing smaller amounts of analyte. As expected, the
system reached complete disassembly after a longer period of time
when less hydrogen peroxide was used. The sigmoidal plots
obtained when the reaction was analyzed using various equiva-
lents of H2O2 are characteristic of the exponential progress of the
2.3. Evaluation of the signal amplification obtained by the DCR
probe upon activation with hydrogen peroxide
In order to evaluate the amplification activity of DCR probe 3, it
was incubated in phosphate buffered saline, pH 7.0 (PBS) with
hydrogen peroxide in the presence of AOX and the release of

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M. Avital-Shmilovici, D. Shabat / Bioorg. Med. Chem. 18 (2010) 3643–3647
Figure 6. The release of 5-amino-2-nitrobenzoic acid from 500 lM dendron 3 in the presence of 0.1 mg/mL alcohol oxidase in PBS 7.0 upon addition of buffer only (B.G.) or
the indicated equivalents of H2O2. The reaction was monitored at 405 nm.
reaction (Fig. 6). In the absence of H2O2, DCR probe 3 exhibited
background signal that was significantly lower than the signal ob-
served in the presence of 0.01 equiv of H2O2.
peroxide (vs. the concentration of the probe) were evaluated with
DCR probe 3, the observed signal was 70-fold stronger than that
obtained by a classic probe. When the same experiment was per-
formed with DCR probe 2, the observed signal was only 53-fold
stronger than that obtained by a classic probe.11
In order to obtain an ideal DCR probe, one should design a mol-
ecule that is highly stable to spontaneous hydrolysis but yet can
undergo rapid disassembly upon relation with the analyte. The
higher stability of the current probe reduces to some extent its rate
of disassembly. In this case, the obtained increase in sensitivity
was accompanied with a longer response time. In principle numer-
ous analytes that have cleavage reactivity toward a specific trigger
could be incorporated into the dendritic platform. Thus, the DCR
amplification technique could be used to detect a variety of clini-
cally or environmentally important analytes.
The increased sensitivity of the DCR technique is clearly demon-
strated when signals obtained with and without the addition of
AOX are compared (Fig. 7). When 1.0 equiv of the analyte was ap-
plied and no AOX was added, dendron 3 exhibited the behavior of a
classic probe, a molecule constructed from a triggering protecting
group attached to a reporter. Signal levels at 1 equiv hydrogen per-
oxide were identical with and without enzyme. However, when
less hydrogen peroxide was evaluated, the signal measured in
the presence of AOX was significantly larger than that obtained
in the absence of enzyme. In fact, the ratio between the signal with
and without enzyme was increased as analyte concentration de-
creased. When 0.01 equiv of hydrogen peroxide was evaluated,
the signal of the DCR technique is 70-fold stronger than that ob-
tained by a classic probe.
3. Conclusions
The self-immolative dendritic platform used in the DCR tech-
nique has a modular design. Thus, some structural elements can
be replaced in order to optimize the probe sensitivity. By replacing
the choline unit in dendron 2 with methanol, a carbonate linkage
was generated (dendron 3) that was less likely to undergo sponta-
neous hydrolysis. This adjustment reduced the background signal
of the DCR assay and to some extent improved the signal-to-noise
ratio in comparison to DCR probe 2. When 0.01 equiv of hydrogen
In summary, we designed and synthesized a DCR probe for
amplification of a diagnostic signal that is generated by hydrogen
peroxide. The probe was triggered by hydrogen peroxide that
was produced through release and oxidation of methanol mole-
cules by alcohol oxidase. The switch from choline-based carbonate
to a methyl carbonate derivative generated a linkage with higher
stability, which reduced the noise of the reaction. The new DCR
probe exhibited slightly higher sensitivity in detection of hydrogen
peroxide than our previously reported probe. Other modifications
in the modular structure could yield even more sensitive DCR
probes for detection of hydrogen peroxide or other analytes.
4. Experimental
4.1. General
All reactions requiring anhydrous conditions were performed
under an Argon atmosphere. All reactions were carried out at room
temperature unless stated otherwise. Chemicals and solvents were
either A.R. grade or purified by standard techniques. Thin layer
chromatography (TLC): Silica Gel plates Merck 60 F254; compounds
were visualized by irradiation with UV light and/or by treatment
with a solution of phosphomolybdic acid (20 wt % in ethanol), fol-
lowed by heating. Flash chromatography (FC): Silica Gel Merck 60
(partical size 0.040–0.063 mm), eluent given in parentheses. 1H
NMR: Bruker AMX 200 or 400 instrument. 13C NMR: Bruker AMX
200 or 400 instrument. The chemical shifts are expressed in d rel-
Figure 7. Comparison of signals measured after complete disassembly (1.00 equiv
H2O2—2 h, 0.10 equiv H2O2—12 h, 0.01 equiv H2O2—24 h) in the presence of AOX by
the DCR amplification technique (blue) versus signals measured using dendron 3 as
a classic probe (i.e., without AOX) (red). The background signal (in the absence of
the analyte) was subtracted from the values shown.

M. Avital-Shmilovici, D. Shabat / Bioorg. Med. Chem. 18 (2010) 3643–3647
3647
ative to TMS (d = 0 ppm) and the coupling constants J in Hz. The
4.6. Compound 3
spectra were recorded in CDCl3 as a solvent at room temp unless
stated otherwise. All reagents, including alcohol oxidase, salts
and solvents, were purchased from Sigma–Aldrich.
To a solution of compound 3g (103 mg, 0.15 mmol) in dry THF
(2 mL) and pyridine (153 L, 1.90 mmol) was added methyl-chlo-
roformate (138 L, 1.78 mmol) dropwise with a syringe at 0 °C.
l
l
4.2. Compound 3c
The reaction mixture was stirred for 1 h, while warming to room
temperature and monitored by TLC (EtOAc/Hex 2:3). Upon comple-
tion, the reaction mixture was diluted with EtOAc, and washed
with saturated solution of NH4Cl. The organic layer was separated,
dried over MgSO4, and evaporated under reduced pressure. The
crude product was purified by column chromatography on silica
gel (EtOAc/Hex 2:3) to give compound 3h (92 mg, 77%) as a yellow
oil. The compound was (92 mg, 0.11 mmol) directly dissolved in
Compound 3c was synthesized as previously reported.16
4.3. Compound 3e
Compound 3e was synthesized as previously reported.11
4.4. Compound 3f
dry THF (2 mL) under Argon atmosphere and TBAF (340 lL, 1 M
in THF) was added. The reaction was stirred in room temperature
for 1 h and was monitored by C-18 RP-HPLC (gradient: 30–100%
ACN in water over 20 min). Upon completion of the reaction, the
solvent was removed under reduced pressure and the crude prod-
uct was purified by using preparative RP-HPLC (gradient: 30–100%
ACN in water over 20 min) to give compound 3 (40 mg, 50%) as a
white powder.
Toluene (15 mL) was heated to reflux (110 °C) under Argon
atmosphere and triphosgene (705 mg, 2.37 mmol) was added.
Then, a solution of compound 3c (560 mg, 1.98 mmol) in 5 mL tol-
uene was slowly added dropwise with a syringe. The reaction was
stirred for 30 min at reflux and monitored by 1H NMR (200 MHz,
CDCl3). After the isocyanate derivative (compound 3d) was ob-
served, the solvent was removed under reduced pressure. A solu-
tion of compound 3e (950 mg, 1.52 mmol) in 9 mL dry THF under
1H NMR (400 MHz, CD3CN): d = 8.48 (1H, br s); 7.99 (1H, d,
J = 8.8 Hz); 7.84–7.82 (3H, m); 7.75 (1H, dd, J = 8.8, 2.2 Hz); 7.54
(2H, s); 7.49 (2H, d, J = 8 Hz); 5.24 (4H, s); 5.21 (2H, s); 4.98 (2H,
s); 3.74 (6H, s). 13C NMR (50 MHz, CD3OD): d = 164.3, 155.6,
152.3, 144.1, 142.8, 142.3, 141.8, 133.3, 132.7, 130.4, 129.7,
128.7, 126.8, 125.1, 118.6, 117.2, 77.0, 65.8, 64.2, 53.9. MS (ESI):
m/z calcd for C28H27BN2O15: 642.1; found: 641.0 [MÀH]À.
Argon, followed by the addition of 25 lL DBTL, was added to the
isocyanate residue. The reaction mixture was allowed to warm to
55 °C and was stirred for 1 h. The reaction was monitored by TLC
(EtOAc/Hex 15:85). The solvent was removed under reduced pres-
sure and the crude product was purified by using column chroma-
tography on silica gel (EtOAc/Hex 1:9) to give compound 3f (1.03
gr, 60%) as a yellow oil.
5. Spectroscopic assay conditions
1H NMR (200 MHz, CDCl3): d = 8.01 (1H, d, J = 10.3 Hz); 7.80
(2H, d, J = 8 Hz); 7.65–7.59 (2H, m); 7.38–7.34 (4H, m); 7.20 (1H,
br s); 5.19 (2H, s); 4.86 (2H, s); 4.68 (4H, s); 4.44–4.35 (2H, m);
1.32 (12H, s); 1.14–1.03 (2H, m); 0.87 (18H, s); 0.04 (21H, s). 13C
NMR (100 MHz, CDCl3): d = 179.2, 152.7, 142.9, 141.4, 139.4,
135.1, 134.4, 131.7, 130.7, 129.7, 128.5, 127.1, 125.8, 119.0,
117.7, 115.3, 83.9, 67.1, 65.2, 60.4, 25.5, 24.7, 24.6, 22.6, À1.6,
À3.7. MS (FAB): m/z calcd for C47H73BN2O11Si3: 936.4; found: 959
[M+Na]+.
The signal developed by the DCR amplification technique was
monitored with a spectrophotometer using a 96-well plate reader.
A total volume of 100 lM incubation mixture was used for each
well. The absorbance was measured upon addition of various
amounts of H2O2 [1.00, 0.10 and 0.01 equiv] at 405 nm.
Acknowledgments
D.S. thanks the Israel Science Foundation (ISF) and the Bina-
tional Science Foundation (BSF) for financial support.
4.5. Compound 3g
Compound 3f (300 mg, 0.32 mmol) was dissolved in 3 mL
MeOH. Catalytic amount of p-TsOH was added to the suspension
and the reaction mixture was stirred at room temperature for
20 min, and monitored by TLC (EtOAc/Hex 1:1). After completion,
the solvent was removed under reduced pressure and the crude
product was purified by using column chromatography on silica
gel (EtOAc/Hex 1:1) to give compound 3g (143 mg, 63%) as a white
powder.
References and notes
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1H NMR (200 MHz, MeOD): d = 7.99 (1H, d, J = 10.3 Hz); 7.76
(2H, d, J = 8 Hz); 7.64–7.59 (2H, m); 7.38–7.34 (4H, m); 5.13 (2H,
s); 4.88 (2H, s); 4.61 (4H, s); 4.41–4.29 (2H, m); 1.29 (12H, s);
1.08–0.99 (2H, m); 0.03 (9H, s). 13C NMR (100 MHz, MeOD):
d = 176.3, 150.7, 144.6, 140.4, 135.8, 135.3, 132.8, 131.5, 128.9,
127.9, 126.6, 119.7, 118.4, 84.7, 65.9, 60.7, 30.4, 25.5, 17.7, -0.9.
MS (FAB): m/z calcd for C35H45BN2O11Si: 708.3; found: 731
[M+Na]+.