Self-immolative comb-polymers: Multiple-release of side-reporters by a single stimulus event

Article abstract of DOI:10.1002/chem.200800917

A new self-immolative polymer based on a polyurethane backbone was developed. The polymer was prepared by simple polymerization of phenylcarbamate monomers, followed by capping of the polymer head with trigger. The polymer head was capped with a trigger designed for removal by penicillin-G amidase (PGA). The enzyme initiates the polymer disassembly to release its reporter units by a single cleavage of the phenylacetamide moiety, followed by 1,6-elimination and decarboxylation reactions. The polymer was incubated in phosphate buffer saline (PBS), pH 7.4, in the presence and in the absence of PGA and the release of 4-nitroaniline was monitored by RP-HPLC. The release of 4-nitroaniline was completed within 6 hours when PGA was added to the polymer solution. Use of drug molecules instead of the reporter units will generate a polymeric drug delivery system that can selectively release a high payload of drug upon simulus by specific enzyme.

Full text of DOI:10.1002/chem.200800917

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DOI: 10.1002/chem.200800917
Self-Immolative Comb-Polymers:
Multiple-Release of Side-Reporters by a Single Stimulus Event
Roy Weinstain, Amit Sagi, Naama Karton, and Doron Shabat*[a]
Chemists have developed several sophisticated strategies
in order to achieve amplification of molecular signals.[1–3]
Self-immolative molecular systems are molecules that disin-
tegrate into their building blocks through domino-like reac-
tions upon a single triggering stimulus. We and others have
previously reported on self-immolative dendritic molecules
that amplify a single triggering reaction at the focal point
into release of multiple peripheral reporter groups.[4–6] How-
ever, the number of groups that can be incorporated on a
dendrimer periphery is limited due to steric hindrance and
higher dendrimer generation requires additional synthetic
steps.[7] In order to overcome such disadvantages, we recent-
ly introduced a novel kind of linear “smart” polymer:[8,9]
These polymers respond to external stimuli by undergoing
head-to-tail disassembly through a self-immolative fragmen-
tation once the head-trigger is activated.[10] Disassembly of
this type of polymer to release monomers with strong fluo-
rescence signal was demonstrated in context of a molecular
sensor with large signal-to-noise ratio.[10] Here, we report
synthesis of the next generation of self-immolative polymers,
which represent a significant step forward. Appropriate in-
corporation of side reporters (R) on each monomer and a
trigger at the head-monomer generated a self-immolative
comb-polymer with releasable side reporters. (Figure 1) Re-
moval of the trigger initiated polymer disassembly along the
backbone into its building block, followed by spontaneous
release of the reporters from each monomer.
Self-immolative comb-polymers can be designed based on
building blocks such as 1 (Scheme 1). This molecule can un-
dergo double-elimination reactions to release its two report-
ers. The disassembly is initiated by cleavage of the trigger to
unmask amine 2. The latter spontaneously undergoes 1,6-
elimination to release a reporter (R1) and azaquinone me-
thide 3. This reactive intermediate is rapidly trapped by an
available nucleophile to form amine 4. Additional 1,6-elimi-
nation, this time at the vinylogous ortho-benzyl position, re-
leases a second reporter unit (R2) and compound 5, which
subsequently reacts with additional nucleophile to form
amine 6. The azaquinone–methide rearrangement can take
place either through an ortho-benzyl[11] or through a vinylo-
gous ortho-benzyl position. Our preference to use for a vi-
nylogous ortho-benzyl substituent is based on the synthetic
strategy presented in the Supporting Information.
We designed self-immolative comb-polymer 7 based on 1
(Scheme 2). The polymeric backbone is constructed of poly-
urethane. 4-Nitroaniline was used as a reporter molecule
Figure 1. Illustration of the disassembly of a self-immolative comb-polymer.
and 4-hydroxy-2-butanone, which is designed for removal
through a b-elimination reaction by piperidine, was applied
as a model trigger.[10] Removal of the trigger of the polymer
head should initiate the polymer disassembly along its back-
bone and the release of multiple reporter units.
[a] R. Weinstain, A. Sagi, N. Karton, Prof. D. Shabat
School of Chemistry
Raymond and Beverly Sackler Faculty of Exact Sciences
Tel-Aviv University, Tel Aviv 69978 (Israel)
Fax : (+972)3-640-9293
In order to study the disassembly mechanism presented in
Scheme 1, we synthesized monomeric system 8 (Scheme 3).
This molecule has two different reporters (1-naphthylamine
E-mail: chdoron@post.tau.ac.il
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.200800917.
Chem. Eur. J. 2008, 14, 6857 – 6861
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Scheme 1. Design and disassembly mechanism of a self-immolative unit of a comb-polymer.
Next, we sought to study the
disassembly of a self-immola-
tive comb polymer. Polymer 7
was synthesized by polymeri-
zation of monomer 9 according
to a procedure we recently de-
veloped (Scheme 4).[10] The po-
lymer head was capped with
the trigger 4-hydroxy-2-buta-
none to generate a polymeric
compound with an average of
11 building blocks (determined
by NMR). The polymer was in-
cubated in MeOH/DMSO so-
lution with or without piperi-
Scheme 2. Piperidine initiates disassembly of self-immolative polymer 7 to release multiple copies of the
4-nitroaniline reporter units.
and 4-nitroaniline) and a 4-hydroxy-2-butanone trigger.
Compound 8 was incubated with piperidine in methanol to
activate the trigger and disassembly to release 1-naphthyla-
mine and 4-nitroaniline was monitored by RP-HPLC.
The 1,6-elimination to release 1-naphthylamine occurred
rapidly (within less than 1 h), whereas the second elimina-
tion through the vinylogous ortho-benzyl position prolonged
over 20 h to release the second reporter 4-nitroaniline
(Figure 2). Importantly, the proposed double elimination of
2 to release two reporters occurred as expected. In a control
reaction with an identical compound and a trigger that is
not cleaved by piperidine (4-methylbenzylalcohol), almost
no release of the reporters was observed (data not shown).
These results support the disassembly pathway of 1 suggest-
ed above (Scheme 3).
dine/acetic acid and the release of 4-nitroaniline was moni-
tored by RP-HPLC (Figure 3).
The concentration of free 4-nitroaniline gradually in-
creased over 48 h when piperidine was present in the poly-
mer solution. In the absence of piperidine, almost no free 4-
nitroaniline was observed (about 10% after 50 h). As a con-
trol experiment we used similar polymer with a 4-methyl-
benzylalcohol capping group instead of 4-hydroxy-2-buta-
none (see polymer 7a in the Supporting Information). Since
this capping group does not undergo b-elimination in the
presence of piperidine, almost no release of 4-nitroaniline
was observed in this control reaction.
The disassembly of polymer 7 was evaluated in organic
solvents and was found to be relatively slow. Previous re-
sults showed a faster elimination rate for azaquinone–me-
thide generation under aque-
ous conditions.[12] In addition,
we sought to evaluate the po-
tential of our polymers as drug
delivery systems. Therefore, we
developed a water-soluble ver-
sion of
a
self-immolative
comb-polymer. Monomer 10
was designed from two aniline
subunits. The first contains a
Scheme 3. Piperidine initiates disassembly of self-immolative compound 8 to release two reporter units:
1-naphthylamine and 4-nitroaniline.
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Self-Immolative Comb-Polymers
COMMUNICATION
&
^
Figure 2. Release of 1-naphthylamine ( ) and 4-nitroaniline ( ) from
~
Figure 3. Disassembly ( ) of self-immolative comb-polymer 7 upon treat-
compound 9 upon exposure to piperidine/acetic acid.
ment with piperidine/AcOH to release 4-nitroaniline. Polymer 7 without
^
treatment of piperidine/AcOH ( ). Polymer 7a upon treatment with pi-
&
peridine/AcOH ( ).
building block and thus is expected to be soluble under
physiological conditions.
Self-immolative comb-polymer 12 was prepared according
to Scheme 5 from a monomer that carries 4-nitroaniline as a
reporter unit (a polymer with an average of 7 building
blocks was obtained). The polymer head was capped with a
trigger designed for removal by penicillin-G amidase
(PGA).[13] The enzyme initiates the polymer disassembly to
release its reporter units by a single cleavage of the phenyla-
cetamide moiety, followed by 1,6-elimination and decarbox-
ylation reactions (Scheme 6).
Scheme 4. Synthesis of self-immolative comb-polymer 7.
releasable side-reporter and the second has a tert-butylacry-
late substituent that can be deprotected with TFA after the
polymerization to afford polymer 11. Polymer 11 has an ion-
ized carboxyl functional group on every other aromatic
Scheme 5. Synthesis of a water-soluble self-immolative comb-polymer with a trigger.
Chem. Eur. J. 2008, 14, 6857 – 6861
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D. Shabat et al.
sues. The growth of the tumor
creates a constant need for the
continuous supply of new
blood vessels. This process,
termed angiogenesis, often re-
sults in the construction of ves-
sels with leaky walls, which
allow enhanced permeability
of macromolecules within the
tumor. In addition, poor lym-
phatic drainage at the tumor
site promotes accumulation of
large molecules. Self-immola-
tive comb-polymers loaded
with a chemotherapeutic drug
Scheme 6. PGA-catalyzed the disassembly of self-immolative comb-polymer 12 to release 4-nitroaniline.
The polymer was incubated in phosphate buffer saline
(PBS), pH 7.4, in the presence and in the absence of PGA
and the release of 4-nitroanilne was monitored by RP-
HPLC (Figure 4). The polymer disassembly indeed occurred
faster under aqueous conditions. The release of 4-nitroanilne
was completed within 6 h (vs 48 h in organic solvent) when
PGA was added to the polymer solution. About 20% re-
lease was observed in the background reaction as a result of
spontaneous hydrolysis of the relatively labile 4-nitroanilne
carbamate derivative. Additional example for activation and
disassembly of a self-immolative comb-polymer with BSA
(bovine serum albumin) is described in the Supporting In-
formation (polymer 25).
and a trigger that is activated by a specific enzyme overex-
pressed in tumor cells (such as a lysosomal protease) could
be used as an efficient drug delivery system. The polymer
should selectively accumulate at the tumor site due to the
EPR effect and will then be activated by a lysosomal pro-
tease upon endocytosis into cancerous cells. The slow re-
lease of the drug molecule is expected to occur inside the
cell and, therefore, nonspecific drug release should not
occur.
In conclusion, we have developed a novel self-immolative
polymer based on a polyurethane backbone. This comb-po-
lymer underwent complete disassembly to release multiple
reporter groups upon a single activation event at its head.
The polymer was prepared by simple polymerization of phe-
nylcarbamate monomers, followed by capping of the poly-
mer head with trigger. This synthetic technique allowed us
to rapidly obtain a polymeric molecule with large number of
reporter units. A water-soluble prototype of a self-immola-
tive comb-polymer was activated under physiological condi-
tions by the protease PGA. Use of drug molecules instead
of the reporter units will generate a polymeric drug delivery
system that can selectively release a high payload of drug
upon stimulus by specific enzyme. We are currently develop-
ing self-immolative polymeric systems with faster release
mechanism.
Acknowledgements
^
Figure 4. Disassembly ( ) of self-immolative comb-polymer 12 upon ad-
dition of PGA to release 4-nitroaniline. Control reaction: polymer 12 in
D.S. thanks the Israel Science Foundation (ISF) and the Binational Sci-
ence Foundation (BSF) for financial support. Special thanks go to Dr.
Gabriel Lemcoff, Mr. Charles Diesendruck and Mr. Yuval Vidavsky for
their help with the GPC analysis.
&
PBS, pH 7.4( ).
The protracted release of the side reporters can be appli-
cable in drug delivery systems that require a slow-release
procedure. Incorporation of a drug molecule instead of the
reporter in the polymeric molecule will afford a comb-poly-
mer that can release multiple drug units upon a single cleav-
age event. Polymeric molecules with molecular weight over
20 kD are known to accumulate selectively at tumor sites
due to the enhanced permeability and retention (EPR)
effect.[14] This effect occurs due to the difference between
the vasculature physiology of solid tumors and normal tis-
Keywords: dendrimers · drug delivery · polymers
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Received: May 14, 2008
Published online: June 25, 2008
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