Enzymatic activation of hydrophobic self-immolative dendrimers: The effect of reporters with ionizable functional groups

Article abstract of DOI:10.1016/j.bmcl.2009.03.002

Self-immolative dendrimers are uniquely structured molecules that release multiple tail units through a chain fragmentation initiated by a single cleavage at the dendrimer's core. Although bioactivation of self-immolative dendritic molecules with only two

Full text of DOI:10.1016/j.bmcl.2009.03.002

Bioorganic & Medicinal Chemistry Letters 19 (2009) 3959–3962
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Enzymatic activation of hydrophobic self-immolative dendrimers: The effect
of reporters with ionizable functional groups
*
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:
Self-immolative dendrimers are uniquely structured molecules that release multiple tail units through a
chain fragmentation initiated by a single cleavage at the dendrimer’s core. Although bioactivation of self-
immolative dendritic molecules with only two reporter groups was demonstrated, enzymatic activation
failed for self-immolative dendrimers with more reporters. These large and hydrophobic dendrimers
aggregated under aqueous conditions and enzyme did not efficiently trigger chain fragmentation. Here
we demonstrate a simple solution to the problem of enzymatic activation of hydrophobic self-immolative
dendrimers. The reporter units on the dendritic platform were equipped with ionizable functional group.
Polar interactions with water significantly decreased hydrophobicity of the dendrimers and prevented
aggregate formation. Consequently, hydrophobic self-immolative dendrons were effectively activated.
Ó 2009 Elsevier Ltd. All rights reserved.
Received 15 February 2009
Revised 1 March 2009
Accepted 3 March 2009
Available online 6 March 2009
Keywords:
Self-immolative dendrimer
Enzyme
Sensor
Amplification
Self-immolative dendrimers are designed to release multiple
peripheral end-units upon a single activation event at the den-
drimer’s focal point. This concept was first explored six years
ago, almost simultaneously, by three different research groups
including ours.1–3 All three groups exploited the fact that the
dendrimer skeleton can be constructed in such a way that the
entire molecule will disintegrate into known fragments once
the disintegration process is initiated. Incorporation of drug mol-
ecules as the tail units and use of an enzyme substrate as the
trigger generates a multi-prodrug that is activated with a single
enzymatic cleavage. These dendritic prodrugs offer significant
advantages in comparison with classic monomeric prodrugs in
inhibition of tumor cell growth.4,5 We have also designed and
synthesized fully biodegradable dendrimers that disassemble
through multi-enzymatic triggering followed by self-immolative
chain fragmentation.6,7 A practical application for such multi-
triggered self-immolative dendrons was demonstrated by the
concept of prodrug activation through a molecular OR logic trig-
ger.8 Self-immolative dendrimers were also shown to act as effi-
cient molecular amplifiers in the design of diagnostic probes.9,10
Recently, we harnessed these dendrimers as a platform for con-
trolled self-assembly of peptide nanotubes.11
mentation is inefficient. We recently demonstrated that enzymatic
activation of second-generation self-immolative dendrimers can
be achieved by conjugation of polyethylene glycol to the dendritic
platform.13 The polyethylene glycol tails significantly decrease the
hydrophobic properties of the dendrimers and prevent aggregate
formation. Here we report second approach to achieve enzymatic
activation of these dendrimers. We hypothesized that an ionizable
functional group introduced into the reporter unit would signifi-
cantly increase aqueous solubility and prevent aggregate forma-
tion. In order to evaluate our approach, we designed self-
immolative dendritic molecules 1, 2, and 3 (Fig. 1). The dendrons
were constructed with an enzymatic trigger that is activated by
Penicillin-G-Amidase(PGA)14 and the reporter groups were 5-ami-
no-2-nitrobenzoic acid.
The dendrons were designed to undergo complete disassembly
to release their reporter units upon a single cleavage event by PGA.
The disassembly mechanism of dendron 3 is presented in Scheme
1. Following the enzymatic cleavage, aza-quinonemethide is rap-
idly eliminated and decarboxylation occurs, leading to release of
amine 3a. The latter is disassembled through double 1,6 and 1,4-
eliminations to generate two equivalents of amine 3b, which is fur-
ther fragmented to release four equivalents of 5-amino-2-nitro-
benzoic acid.
The building blocks of self-immolative dendrimers are usually
hydrophobic aromatic molecules and, therefore, these molecules
have poor water solubility and tend to aggregate under aqueous
conditions.12 When aggregation occurs, access of the enzyme to
the substrate at the dendron’s focal point is restricted and frag-
Due to aggregate formation, analog dendrons equipped with 4-
nitroaniline reporter units instead of 5-amino-2-nitrobenzoic acid
were activated by PGA only for zeroth-generation molecules. The
addition of an ionized carboxylic acid to the reporters is expected
to inhibit aggregate formation otherwise observed in aqueous con-
ditions and thereby to allow activation of first and second-genera-
tion dendrons by the enzyme.
* Corresponding author. Tel.: +972 (0) 3 640 8340; fax: +972 (0) 3 640 9293.
E-mail address: chdoron@post.tau.ac.il (D. Shabat).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.03.002

3960
M. Avital-Shmilovici, D. Shabat / Bioorg. Med. Chem. Lett. 19 (2009) 3959–3962
O
OH
HN
NO2
O
O
O
OH
O
O
OH
O
H
N
O
O
N
H
NO2
O
O
N
H
NO2
O
N
H
N
H
O
1
2
O
OH
HN
O
NO2
O
O
OH
NO2
HN
O
HN
O
O
O
O
O
O
N
H
O
O
O
O
N
H
HN
HN
NO2
OH
O
3
O
O
HN
NO2
OH
O
Figure 1. Chemical structures of zeroth-generation (1), first-generation (2) and second-generation (3) self-immolative dendrons. The substrate for PGA is shown in red and
the 5-amino-2-nitrobenzoic acid reporter groups are shown in blue.
O
OH
HN
O
NO2
O
O
OH
1,6- and 1,4-elimination
OH
Enzymatic cleavage
H2O
HN
O
HN
O
NO2
Dedron 3
O
O
OH
O
H2N
O
CO2
O
2CO2
O
O
H2N
H2N
OH
HN
OH
HN
NO2
OH
3a
O
O
O
HN
NO2
OH
O
O
O
O
O
OH
OH
OH
OH
HN
NO2
HN
O
O
NO2
H2N
NO2
H2N
H2N
NO2
1,6- and 1,4-elimination
OH
O
O
O
H2N
HN
O
NO2
OH
O
H2N
HN
O
NO2
OH
OH
O
OH
O
2CO2
NO2
3b
O
H2N
3b
H2N
NO2
O
OH
Scheme 1. Disassembly pathway of second-generation dendritic molecule 3 triggered by enzymatic activation of PGA.
The synthesis of compound 1 was performed in a manner
similar to the previously published procedure (see Supplemen-
tary data).15 Dendron 2 was synthesized as shown in Scheme
2. Amine 2a15 was treated with triphosgene to generate isocya-
nate 2b, which was coupled in situ with alcohol 2c to afford car-
bamate 2d. Deprotection of carbamate 2d in the presence of p-

M. Avital-Shmilovici, D. Shabat / Bioorg. Med. Chem. Lett. 19 (2009) 3959–3962
3961
OTBS
O
OH
2c
O
N
H
p-TsOH
MeOH
76%
TBSO
H2N
OTBS
triphosgene
quant.
O
O
N
H
TBSO
OCN
OTBS
2b
OTBS
91%
N
H
2d
2a
TMS
O
O
O
OCN
TMS
OH
O
HN
O
NO2
TMS
O
NO2
O
O
2f
TBAF
65%
O
O
N
H
O
O
Dendron 2
OH
H
N
N
H
60%
O
O
N
H
NO2
O
N
H
O
2e
2g
Scheme 2. Chemical synthesis of dendron 2.
toluenesulfonic acid generated diol 2e. Then compound 2f (pre-
pared from the corresponded amine) was coupled in situ with
alcohol diol 2e to afford compound 2g. The latter was deprotec-
ted with tetrabutylammonium-fluoride to give first-generation
dendron 2.
The synthesis of second-generation dendron 3 is presented in
Scheme 3. Two equivalents of isocyanate 2b were coupled with
diol 2e to give dendron 3c. Deprotection of compound 3c in the
presence of p-toluenesulfonic acid generated tetra-ol 3d. An excess
of isocyanate 2f was coupled with tetra-ol 3d to afford compound
3e. The latter was deprotected by tetrabutylammonium-fluoride to
yield second-generation dendron 3.
Since the free reporter 5-amino-2-nitrobenzoic has an absor-
bance at wavelength of 405 nm, the disassembly of self-immola-
tive dendrons 1–3 can be monitored by Vis-spectroscopy.
Dendrons 2 and 3 were incubated in phosphate buffered saline
(PBS, pH 7.4) in the presence of PGA and disassembly was moni-
tored. The results are presented in Figure 2. Dendron 2 rapidly dis-
assembled; all reporter units were released within 30 min.
Disassembly of dendron 3 occurred over a longer time but, as ex-
pected, released double the number of reporter units in compari-
son to dendron 2. No release of 5-amino-2-nitrobenzoic was
0.5
0.4
0.3
0.2
0.1
0
0
30
60
90
120
150
180
Time (min)
Figure 2. PGA-catalyzed the release of 5-amino-2-nitrobenzoic acid from dendrons
2 ( ) and 3 ( ). Conditions: 250
M 2 or 3, 0.01 mg mLÀ1 PGA in PBS (pH 7.4).
Absorbance was monitored at 405 nm.
l
observed when either dendron was incubated in absence of PGA
(data not shown).
OTBS
OH
O
HN
O
O
N
H
OTBS
O
OH
N
H
O
O
p-TsOH
MeOH
2e
O
O
N
H
O
TBSO
OCN
OTBS
O
OTBS
N
H
81%
85%
HN
2b
3c
OTBS
O
TMS
O
O
HN
O
NO2
TMS
OH
O
O
HN
O
HN
O
HN
O
O
NO2
OH
OH
O
OCN
TMS
O
O
O
O
O
TBAF
57%
NO2
Dendron 3
O
O
N
H
O
2f
O
O
N
H
O
O
O
OH
O
O
N
H
60%
N
HN
HN
H
HN
NO2
O
O
O
3d
3e
O
HN
NO2
O
TMS
O
TMS
Scheme 3. Chemical synthesis of dendron 3.

3962
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M. Avital-Shmilovici, D. Shabat / Bioorg. Med. Chem. Lett. 19 (2009) 3959–3962
of this approach allowed enzymatic activation of hydrophobic
self-immolative dendrimers with four reporter units per dendron.
This type of dendritic molecule should be useful as a molecular
probe for detection of specific activity by introduction of the
appropriate trigger in the dendrimer focal point. The reporter unit
release can be conveniently monitored by UV-spectroscopy.16–20
The sensitivity of the molecular probe is enhanced at higher den-
drimer generations.
In summary, we have demonstrated a new approach to achieve
enzymatic activation of second-generation self-immolative dendri-
mers based on use of reporter units with ionizable functional
groups. We designed and synthesized zeroth-, first-, and second-
generation dendritic molecules with 5-amino-2-nitrobenzoic acid
as a reporter and a trigger that allows activation by PGA. All three
dendritic molecules, including the second-generation self-immola-
tive dendron was otherwise inactive due to aggregation, were
effectively cleaved by PGA in aqueous solution. The data presented
in this study demonstrate that self-immolative dendrimers, with
reporters monitored by UV, can be used as molecular probes for
detection of various enzymatic activities.
90
80
70
60
50
40
30
20
10
0
0
30
60
90
120
150
180
Time (min)
Figure 3. HPLC analysis PGA-catalyzed the release of 5-amino-2-nitrobenzoic acid
from compound 2 ( ) and 3 ( ). Conditions: 250
M 2 or 3, 0.01 mg mLÀ1 PGA in
PBS (pH 7.4). Absorbance was monitored at wavelength of 348 nm. Separation was
performed on a C-18 RP-column (gradient: 30–100% ACN in water over 20 min).
l
Acknowledgments
D.S. thanks the Israel Science Foundation (ISF) and the Bina-
tional Science Foundation (BSF) for financial support.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2009.03.002.
Figure 4. Change in absorbance (at 405 nm) as a function of PGA concentration for
the catalyzed release of 5-amino-2-nitrobenzoic acid from dendron 1 ( ), dendron
2 ( ), and dendron 3 ( ). The data shown are for the time point after disassembly
was complete at each concentration of enzyme. Conditions: 250 lM dendron 1, 2,
or 3 in PBS (pH 7.4) with indicated concentration of PGA.
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