Single-triggered AB6 self-immolative dendritic amplifiers

Article abstract of DOI:10.1002/chem.200700142

Self-immolative dendrimers are a unique class of molecules that are able to disassemble upon undergoing a specific triggering reaction through domino-like fragmentations. We have designed and synthesized a novel AB₆ self-immolative dendritic adaptor that amplifies a single cleavage event into the release of six reporter units. The disassembly mechanism is based on a specifically triggered cleavage event followed by elimination of a cyclic urea derivative and six consecutive quinone methide eliminations. The system was disassembled under both organic and aqueous conditions by either chemical or enzymatic triggering. Various reporter molecules and triggering groups were introduced onto the dendritic adaptor to obtaining sensor molecules with enhanced properties for diagnostics and imaging.

Full text of DOI:10.1002/chem.200700142

FULL PAPER
DOI: 10.1002/chem.200700142
Single-Triggered AB6 Self-Immolative Dendritic Amplifiers
Marina Shamis and Doron Shabat*[a]
Abstract: Self-immolative dendrimers
are a unique class of molecules that are
able to disassemble upon undergoing a
specific triggering reaction through
domino-like fragmentations. We have
designed and synthesized a novel AB6
self-immolative dendritic adaptor that
amplifies a single cleavage event into
the release of six reporter units. The
disassembly mechanism is based on a
methide eliminations. The system was
disassembled under both organic and
aqueous conditions by either chemical
or enzymatic triggering. Various report-
er molecules and triggering groups
were introduced onto the dendritic
adaptor to obtaining sensor molecules
with enhanced properties for diagnos-
tics and imaging.
specifically triggered cleavage event
followed by elimination of a cyclic urea
derivative and six consecutive quinone
Keywords: dendrimers
cence molecular amplifiers
prodrugs · self-immolative
·
fluores-
·
·
Introduction
goes a double 1,4-quinone methide rearrangement to release
two reporter units, whereas dendron 3 undergoes a 1,6- and
a double 1,4-quinone methide rearrangement to release
three reporter units.
Amplification of molecular signals is an important task in
the fields of diagnostics,[1,2] imaging,[3] and drug delivery.[4] In
each of these fields there is a need for the release of active
signaling or drug molecules on specific demand. We, and
two other groups, have recently reported the design and syn-
thesis of dendrimers that fragment into their building blocks
in a self-immolative manner to release the tail-group units
after fragmentation has been initiated by a triggering reac-
tion.[5–7] The unique disassembly of these molecules was har-
nessed for the construction of self-immolative dendritic pro-
drugs[8] with single or multiple triggering modes of activa-
tion.[9–14] The basic building block of a first-generation den-
drimer is usually referred to as an ABn unit, in which A is
the head and B is the tail. Single-triggered self-immolative
dendritic adaptors AB1 (1), AB2 (2), and AB3 (3) were de-
veloped. The release of an active reporter molecule from
compound 1 is initiated by cleavage of the trigger, followed
by removal of a cyclic dimethylurea derivative, and a 1,6-
quinone methide rearrangement. Similarly, dendron 2 under-
[a] M. Shamis, Prof. D. Shabat
Department of Organic Chemistry
School of Chemistry
Faculty of Exact Sciences
Tel Aviv University
Tel Aviv 69978 (Israel)
Fax : (+972)(0)-3-640-9293
E-mail: chdoron@post.tau.ac.il
Elimination of reporter groups by the quinone methide
rearrangement was shown to be an efficient reaction to ex-
ploit when designing ABn self-immolative dendritic adap-
tors.[15] This elimination takes place when there is a good
Supporting information for this article is available on the WWW
under http://www.chemeurj.org/ or from the author.
Chem. Eur. J. 2007, 13, 4523 – 4528
ꢀ 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
4523

leaving group para or ortho to the phenolic oxygen. There-
fore, the maximum number of reporter units linked to one
benzene ring is limited to three because there are only two
ortho and one para positions available. Herein we report the
design, synthesis, and disassembly of a novel AB6 self-immo-
lative dendritic molecule in which the number of substitu-
ents that are conjugated to the phenolic oxygen has been
doubled. In this AB6 dendron, a single cleavage event re-
leases six reporter units.
Results and Discussion
To generate an AB6 self-immolative dendritic adaptor from
a benzene ring, we designed dendron 4. The number of sub-
stituents that were conjugated to the phenolic oxygen was
doubled through a short split extension. This molecular
design allows a single cleavage reaction to be amplified to
release six active reporter units.
The disassembly mechanism of dendron 4 is illustrated in
Scheme 1. Cleavage of the trigger initiates the cyclization of
a dimethylurea derivative to release phenol 5. The latter can
undergo 1,8-elimination followed by decarboxylation to re-
lease one reporter unit and generate quinone methide 6. In
the next step, a nucleophile (most likely a solvent molecule)
reacts with the highly electrophilic quinone methide to gen-
erate phenol 7. Similarly, we hypothesize that another 1,8-
elimination and four 1,6-eliminations occur to lead to the re-
lease of all six reporter units.
Next, we synthesized AB6 dendron 8 that contains six
molecules of aminomethylpyrene as the reporter units and
the t-butyloxycarbonyl (Boc) protecting group as the trigger
Scheme 1. Disassembly mechanism of AB6 self-immolative dendron 4.
4524
www.chemeurj.org
ꢀ 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2007, 13, 4523 – 4528

Self-Immolative Dendrimers
FULL PAPER
ate 13, which was further treat-
ed with six equivalents of ami-
nomethylpyrene to afford den-
dron 8.
To evaluate the disassembling
activity of dendron 8, the Boc
trigger was removed by using
trifluoroacetic acid (TFA) to
generate the corresponding am-
monium salt; this was incubated
in 1:1 MeOH/DMSO in a 2%
aqueous solution of tetrabutyl-
G
control reaction, dendron 8,
which contained an intact trig-
ger unit was incubated under
identical conditions. The release
of free aminomethylpyrene was
monitored by reverse-phase
HPLC (RP-HPLC). Figure 1
shows that the pyrene tail units
Scheme 2. Molecular structure of a self-immolative AB6 dendron with aminomethylpyrene reporter units and
a Boc protecting group as a trigger.
were
completely
released
within 6 h. Importantly, no re-
(Scheme 2). The dendron was designed to release its six tail
units upon removal of the protecting group.
The synthesis of 8 was performed by using the synthetic
strategy shown in Scheme 3. Trialdehyde 9 was prepared as
previously described.[16] Acylation of the phenol group of 9
by using the chlorocarbamoyl of N,N’-dimethylethylenedi-
lease was observed in the control solution. In the HPLC
chromatogram, the signal that corresponds to aminomethyl-
pyrene was directly observed from the disassembly of the
ammonium salt of dendron 8 without the formation of any
other intermediates. This observation suggests that the rate-
determining step of the disassembly process is the cycliza-
tion of the amine to release the dimethylurea derivative and
phenol 5 (Scheme 1). The latter has a short lifetime and the
six elimination reactions, which occur to release aminome-
thylpyrene that is observed are probably fast.
A
densed with three equivalents of diethyl malonate to give
hexaethyl ester 11, which was reduced with diisobutyl alumi-
numhydride to afford hexaol 12. Acylation of 12 with excess
p-nitrophenyl (PNP) chloroformate generated hexacarbon-
Scheme 3. Chemical synthesis of AB6 self-immolative dendron 8.
Chem. Eur. J. 2007, 13, 4523 – 4528
ꢀ 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
4525

M. Shamis and D. Shabat
from the dendritic platform, the band at l=470 nm disap-
peared from the spectrum and the band at l=408 nm,
which corresponds to free aminomethylpyrene, increased.
Similar to the results obtained from the HPLC assay, no de-
crease in the excimer band was observed for the control re-
action that contained dendron 8 with an intact Boc trigger
unit (data not shown). Similar photophysical behavior was
observed in the fluorescence spectrum of pyrene-functional-
ized poly(propylene imine) dendrimers.[17,18]
We also wanted to evaluate the disassembly of our den-
dritic system under physiological conditions. Thus, we syn-
thesized self-immolative AB6 dendron 14 that contained
water-soluble tryptophan tail units and a phenylacetamide
head as the trigger unit (Scheme 4) to evaluate disassembly
in aqueous conditions. The phenylacetamide is selectively
cleaved by the bacterial enzyme penicillin G amidase
(PGA).[19] The trigger was designed to disassemble through
an azaquinone methide rearrangement and cyclic dimethyl-
Figure 1. Release of aminomethylpyrene from dendron 8 upon removal
&
of the Boc-protecting group trigger ( ; 1:1 MeOH/DMSO mixture that
contains 2% aqueous solution of Bu4NOH). Control reaction under simi-
~
lar conditions with dendron 8 with the Boc trigger intact ( ).
Additional support for this disassembly mechanism was
obtained by monitoring the release of the pyrene tail units
by fluorescence spectroscopy. The confined nature of the
pyrene units in the dendritic molecule results in the forma-
tion of excimers. Excimer fluorescence generates a broad
band at a wavelength of 470 nm in the emission spectrum of
dendron 8 (Figure 2). Upon the release of the pyrene units
A
undergo six quinone methide elimination reactions to re-
lease the tryptophan tail units.
The synthesis of dendron 14 was performed by using the
synthetic strategy shown in Scheme 5. Hexacarbonate 13
was reacted with excess tryptophan to give dendron 15. The
Boc protecting group of 13 was removed by using TFA to
generate an ammonium salt, which was further treated with
linker 16 (prepared as reported in ref. [12]) to afford den-
dron 14.
Dendron 14 was incubated in phosphate buffer saline
(PBS, pH 7.4) in the presence of and in the absence of
PGA. The progress of the disassembly process was moni-
tored by RP-HPLC and the results are presented in
Figure 3. Tryptophan was gradually released from dendron
14 upon incubation with PGA. The release of tryptophan
was completed within 48 h in the presence of PGA, whereas
no release was observed for the control reaction in which
PGA was absent. Although disassembly of this dendron oc-
Figure 2. Emission fluorescence spectra (lex =340 nm) of dendron
[50 mm] upon removal of the Boc trigger upon incubation in a 1:1 MeOH/
DMSO mixture that contains 2% aqueous solution of Bu4NOH.
8
Scheme 4. Molecular structure of a self-immolative AB6 dendron with tryptophan tail units and phenylacetamide group as a trigger.
4526
www.chemeurj.org
ꢀ 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2007, 13, 4523 – 4528

Self-Immolative Dendrimers
FULL PAPER
Scheme 5. Chemical synthesis of self-immolative AB6 dendron 14.
Figure 4. Emission fluorescence spectra (lex =260 nm) of dendron 14
[12 mm] upon incubation in PBS, 378C with PGA [0.1 mgmLÀ1].
Figure 3. Release of tryptophan from dendron 14 upon incubation with
PGA. Conditions: dendron 14, [50 mm]; PGA, [0.1 mgmLÀ1], ( ). Control
*
~
reaction under similar conditions without PGA ( ).
AB6 dendritic system by including the two ortho and the
para substituents as dendritic arms. A single AB6 dendritic
subunit is able to give a sixfold amplification of a single trig-
gering event, whereas previous AB2 and AB3 self-immola-
tive dendritic subunits multiplied a triggering event only
two- or threefold, respectively. Thus, AB6-based self-immo-
lative dendrimers achieve higher degrees of signal amplifica-
tion at lower dendrimer generations. As higher generation
dendrimers are typically more difficult to synthesize, the
AB6-based dendrimer may be a more efficient dendritic am-
plification subunit.
The quinone methide intermediates generated during the
disassembly process are highly reactive electrophiles and
rapidly react with any available nucleophiles (methanol or
tetrabutylammonium hydroxide in organic solvents). We
could not isolate any significant amount of material that de-
rived from the core molecule, probably owing to the genera-
tion of a mixture of compounds by the addition of different
nucleophiles to the quinone methide.
curred more slowly under physiological conditions than dis-
assembly of dendron 8 in the MeOH/DMSO environment
(Figure 1), PGA cleaved the phenylacetamide from dendron
14 and the resulting amine intermediate was disassembled
to release a total of six molecules of tryptophan.
As tryptophan is also a fluorescent molecule, we moni-
tored the disassembly process through spectroscopic meas-
urements. It was found that within the AB6 dendron (14),
the tryptophan fluorescence was significantly quenched
owing to its confined nature, which is caused by the dendrit-
ic skeleton. Upon disassembly, we observed a gradual in-
crease in two bands at l=400 and 760 nm that correspond
to free tryptophan molecules (Figure 4).
The mechanism of double 1,8-elimination by azaquinone
methide rearrangement in a prodrug system was reported by
Firestone.[20] He reported the synthesis of an AB2 dendron
that was linked with two molecules of the anticancer drug
doxorubicin. The drug was released through enzymatic acti-
vation by using cathepsin B followed by double 1,8-elimina-
tion. We have extended that approach into a single-triggered
Chem. Eur. J. 2007, 13, 4523 – 4528
ꢀ 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
4527

M. Shamis and D. Shabat
[4] S. Muller, A. D. Schluter, Chem. Eur. J. 2005, 11, 5589–5610.
Conclusions
[5] R. J. Amir, N. Pessah, M. Shamis, D. Shabat, Angew. Chem. 2003,
115, 4632; Angew. Chem. Int. Ed. 2003, 42, 4494–4499.
[6] F. M. De Groot, C. Albrecht, R. Koekkoek, P. H. Beusker, H. W.
Scheeren, Angew. Chem. 2003, 115, 4628; Angew. Chem. Int. Ed.
2003, 42, 4490.
[7] M. L. Szalai, R. M. Kevwitch, D. V. McGrath, J. Am. Chem. Soc.
2003, 125, 15688–15689.
[8] D. Shabat, J. Polym. Sci., Part A: Polym. Chem. 2006, 44, 1569–
1578.
[9] R. J. Amir, E. Danieli, D. Shabat, Chem. Eur. J. 2007, 13, 812–821.
[10] R. J. Amir, M. Popkov, R. A. Lerner, C. F. Barbas, III, D. Shabat,
Angew. Chem. 2005, 117, 4452; Angew. Chem. Int. Ed. 2005, 44,
4378–4381.
[11] R. J. Amir, D. Shabat, Chem. Commun. 2004, 1614–1615.
[12] A. Gopin, S. Ebner, B. Attali, D. Shabat, Bioconjugate Chem. 2006,
17, 1432–1440.
[13] K. Haba, M. Popkov, M. Shamis, R. A. Lerner, C. F. Barbas, III, D.
Shabat, Angew. Chem. 2005, 117, 726; Angew. Chem. Int. Ed. 2005,
44, 716–720.
[14] M. Shamis, H. N. Lode, D. Shabat, J. Am. Chem. Soc. 2004, 126,
1726–1731.
In summary, we have developed a simple synthesis for a
newAB self-immolative dendritic adaptor. This molecule
6
acts as an amplifier for a cleavage reaction. Thus, a single
cleavage event in the dendron focal point is translated into
the release of six tail units. This is the first report of a mole-
cule that is capable of six consecutive elimination reactions
through a quinone methide rearrangement mechanism. The
disassembly process was performed in organic solvents by
using chemical activation and under aqueous conditions by
enzyme activation. Various reporter molecules and trigger-
ing groups could be introduced into the dendritic adaptor
and thereby create sensors with enhanced properties that
are suitable for use in diagnostics and imaging. Incorpora-
tion of drug molecules as the tail units and by using a specif-
ic enzymatic substrate as the trigger should generate a
single-triggered prodrug system that would be selectively ac-
tivated at the target site.
[15] E. E. Weinert, R. Dondi, S. Colloredo-Melz, K. N. Frankenfield,
C. H. Mitchell, M. Freccero, S. E. Rokita, J. Am. Chem. Soc. 2006,
128, 11940–11947.
[16] A. A. Anderson, T. Goetzen, S. A. Shackelford, S. Tsank, Synth.
Commun. 2000, 30, 3227–3232.
Acknowledgements
[17] L. A. Baker, R. M. Crooks, Macromolecules 2000, 33, 9034–9039.
[18] G. Pistolis, A. Malliaris, C. M. Paleos, D. Tsiourvas, Langmuir 1997,
13, 5870–5875.
[19] C. A. Claridge, A. Gourevitch, J. Lein, Nature 1960, 187, 237–238.
[20] R. Firestone, Patent Application: WO, 1998, 63pp, WO9813059.
D.S. thanks the Israel Science Foundation, the Israel Ministry of Science
“Tashtiot” program, and the Israel Cancer Association for financial sup-
port.
[1] Y. Okuhata, Adv. Drug Delivery Rev. 1999, 37, 121–137.
[2] S.-E. Stiriba, H. Frey, R. Haag, Angew. Chem. 2002, 114, 1385;
Angew. Chem. Int. Ed. 2002, 41, 1329–1334.
[3] W. J. M. Mulder, G. J. Strijkers, A. W. Griffioen, L. van Bloois, G.
Molema, G. Storm, G. A. Koning, K. Nicolay, Bioconjugate Chem.
2004, 15, 799–806.
Received: January 26, 2007
Published online: May 4, 2007
4528
www.chemeurj.org
ꢀ 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2007, 13, 4523 – 4528