Self-immolative dendrimer biodegradability by multi-enzymatic triggering.

Article abstract of DOI:10.1039/b404946b

New self-immolative dendritic molecules have been designed and synthesized. The dendrons are built with a multi-enzymatic triggering mechanism, which initiates their biodegradation through a self-immolative chain fragmentation to release a reporter group

Full text of DOI:10.1039/b404946b

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Self-immolative dendrimer biodegradability by multi-enzymatic
triggering†
Roey J. Amir and Doron Shabat*
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
Received (in Cambridge, UK) 5th April 2004, Accepted 15th May 2004
First published as an Advance Article on the web 15th June 2004
New self-immolative dendritic molecules have been designed
and synthesized. The dendrons are built with a multi-enzymatic
triggering mechanism, which initiates their biodegradation
through a self-immolative chain fragmentation to release a
reporter group from the focal point. The dendritic backbone is
constructed from polycarbamate linkages, which are stable to
hydrolysis and enhance the dendrons’ solubility in water. The
degradation can readily take place under physiological condi-
tions on enzymatic triggering.
In order to evaluate our dendrimer biodegradation pathway we
synthesized G0, G1 and G2 dendrons (Scheme 2) with phenyl-
acetamide as a triggering substrate for penicillin-G-amidase16
(PGA) and 4-nitrophenol as a reporter group. 4-Hydroxybenzyl
alcohol was employed as a self-immolative linker to connect
between two amine groups through carbamate linkages.
Similarly to a G1-dendron, a G2-dendron (compound 3) can
disassemble to its building blocks through the described enzymatic
self-immolative fragmentation. The phenol which is released after
the first intra-cyclization undergoes 1,6-quinone-methide re-
arrangement to release carbamic acid from the benzylic carbon. The
quinone-methide species is rapidly trapped by a water molecule to
yield 4-hydroxybenzyl alcohol. The generated carbamic acid
undergoes spontaneous decarboxylation to form a free amine
group, which self-cyclizes to release the reporter group. Im-
portantly, only one enzymatic cleavage out of a possible four is
sufficient to initiate the domino breakdown that will release the
reporter group at the focal point of the dendrimer. The complete
degradation of the dendron to its building blocks is depicted in
Scheme 3.
The dendritic molecules were prepared straightforwardly as
shown in Scheme 4 and the ESI.† Thus, dendron 1 was obtained by
reaction of phenylacetyl chloride with mono-Boc-N-methyl-ethyle-
nediamine to afford compound 4, followed by Boc removal and
addition of dinitrophenyl carbonate. Dendron 2 was prepared by
reaction of diethylenetriamine with imidazol amide of phenylacetic
acid to afford compound 5, which was further reacted with
dinitrophenyl carbonate. Coupling of compound 5 with active
carbonate of 4-hydroxy-benzylalcohol afforded alcohol 6, which
was further activated with 4-nitrophenylchloroformate to give
compound 7. The latter (two equivalents) was reacted with
diethylenetriamine and a subsequent one pot reaction with
dinitrophenyl carbonate afforded dendron 3.
Dendrons 1–3 were incubated with PGA in PBS pH 7.4 at 37 °C.
Their biodegradation could be conveniently monitored by follow-
ing the formation of 4-nitrophenol with visible spectroscopy at a
wavelength of 405 nm. The kinetic release of 4-nitrophenol from
the dendrons is shown in Fig. 1. Upon addition of PGA to dendrons
1–3, free 4-nitrophenol was gradually formed, indicating that PGA
cleaves its phenylacetamide substrate and the degradation indeed
occurs as was predicted. As we expected the G1-dendron released
Degradable dendrimers have been attracting special interest in the
scientific community.1,2 They are particularly desirable in the field
of controlled drug delivery systems;3–6 after a dendritic platform
has ended its task as a drug carrier it needs to be cleared out from
circulation. Biodegradability of a dendrimer should speed up its
clearance and avoid undesired toxicity side effects.7,8 At this time,
there are only a few known examples of dendrimers that degrade by
controlled fragmentation.1,9 Recently, we and others have in-
troduced a new class of dendritic molecules which were termed
self-immolative dendrimers.10–13 These structurally unique den-
drimers can release all of their tail units, through a self-immolative
chain fragmentation, which is initiated by a single cleavage at the
dendrimer core.14 We now report a new enzymatic self-immolative
complete fragmentation of dendrimers, which is initiated by multi-
enzymatic triggering.
A recent report appeared in literature has described dendrimers
that can disassemble in organic solvents by benzyl-ether depoly-
merization, triggered by an allyl-ether deprotection.15 We have
extended this concept to fully biodegradable dendrimers, which
have reasonable solubility in water and are disassembled through
multi-enzymatic triggering followed by self-immolative chain
fragmentation. The dendrimer’s main building block is based on
diethylenetriamine, which has two primary and one secondary
amine functionalities. In a G1-dendron (Scheme 1) the secondary
amine is attached to a reporter group while the two primary amines
are linked to enzymatic substrates. The cleavage of either one of the
substrates by the enzyme, generates a free amine group which
initiates an intra-cyclization reaction to release the reporter
group.
Scheme 1 G1-dendron disassembly through a double triggering mecha-
nism. Cleavage of either triggers I or II will initiate the release of the
reporter group.
Scheme 2 Chemical structure of G0, G1 and G2 dendrons.
† Electronic supplementary information (ESI) available: experimental
procedure and UV–visible spectra of dendrons 1 and 2. See http://
www.rsc.org/suppdata/cc/b4/b404946b/
Scheme 3 PGA catalyzed fragmentation of a G3-dendron to its building
blocks.
1614
C h e m . C o m m u n . , 2 0 0 4 , 1 6 1 4 – 1 6 1 5
T h i s j o u r n a l i s © T h e R o y a l S o c i e t y o f C h e m i s t r y 2 0 0 4

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the 4-nitrophenol faster than the G0-dendron while the G2-dendron
released it relatively more slowly. The background control
reactions showed no release at all.
than the rate of the enzymatic substrate cleavage and therefore the
Kobs for dendron 3 is relatively smaller.
In conclusion, we have designed and synthesized new dendritic
The kinetic constants Kobs for the three reactions were calculated
by linear correlation with the measured plots (Table 1). The
phenomenon of dendron 2 releasing its reporter group faster than
dendron 1 occurs since the enzymatic substrate concentration in
dendron 2 is twice as high as in dendron 1. The following self-
cyclization step is relatively fast and therefore, the rate-limiting
step is cleavage of the enzymatic substrate. In dendron 3 additional
self-immolative reactions occur in order to complete the release of
the reporter group (another intra-cyclization and 1,6-quinone-
methide elimination). The overall rate of these reactions is slower
molecules with a multi-enzymatic triggering mechanism that
initiates their biodegradation through a self-immolative chain
fragmentation to release a reporter group from the focal point. For
the first time, the potential of diethylenetriamine was introduced as
a double trigger linker, which can be used as a building block for
constructing self-immolative dendrimers. The dendrons were found
to have fairly good (G0, G1) to moderate (G2) water solubility and
high stability to background hydrolysis under physiological
conditions. Their degradation readily occurs in aqueous medium
and can easily be monitored by generation of free reporter
molecule. Incorporation of different substrates on the dendron’s
periphery should allow the use of varying triggering enzymes.17
This concept may be particularly important in the field of prodrug
mono-therapy,18 if a drug molecule will be incorporated instead of
the reporter unit,19,20 especially in circumstances with more than
one tumor-associated or targeted enzyme with different catalytic
activity. Further studies of these dendritic molecules are under
progress.
Notes and references
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Fig. 1 UV absorbance at 405 nm as a function of time in the biodegradation
of the self-immolative dendrons. / Dendron 1 + PGA. 0 Dendron 2 +
PGA. : Dendron 3 + PGA. 8 Dendron 1 in PBS pH 7.4. . Dendron 2 in
PBS 7.4. Ω Dendron 3 in PBS pH 7.4 (substrate concentration is 200 mM
and 10 mM for PGA).
18 F. M. de Groot, E. W. Damen and H. W. Scheeren, Curr. Med. Chem.,
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Table 1 Kobs values for the reporter release reactions for dendrons 1–3
19 D. Shabat, C. Rader, B. List, R. A. Lerner and C. F. Barbas III, Proc.
Natl. Acad. Sci. USA, 1999, 96, 6925–6930.
Dendron 1
Dendron 2
Dendron 3
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C. F. Barbas III, Proc. Natl. Acad. Sci. USA, 2001, 98, 7528–7533.
K(obs)/min21
5.11
9.89
2.43
C h e m . C o m m u n . , 2 0 0 4 , 1 6 1 4 – 1 6 1 5
1615