Receiver-amplifier, self-immolative dendritic device

Article abstract of DOI:10.1002/chem.200601263

Self-immolative dendrimers disassemble through a domino-like chain fragmentation initiated by a single cleavage at the dendrimer core. We have designed and synthesized dendritic molecules that resemble dendritic architectures present in nature. The unique

Full text of DOI:10.1002/chem.200601263

DOI: 10.1002/chem.200601263
Receiver–Amplifier, Self-Immolative Dendritic Device
[a]
Roey J. Amir, Eyal Danieli, andDoron Shabat*
Abstract: Self-immolative dendrimers
disassemble through domino-like
to be transferred convergentlyto a
focal point. The signal is divergently
amplified through to the other side of
the dendrimer, reporter units are re-
leased, and fluorescence is emitted.
During signal propagation, the dendrit-
ic molecule disassembles in a self-im-
molative manner into small fragments.
These compounds are the longest den-
a
chain fragmentation initiated bya
single cleavage at the dendrimer core.
We have designed and synthesized den-
dritic molecules that resemble dendritic
architectures present in nature. The
unique design allows a cleavage signal
received byanyone of the multiple
triggers on one side of the dendrimer
dritic system ever reported to dis
ACHTREUNGas-
A
ACHTREUNG
tive reactions. The synthesized dendrit-
ic molecules have an architecture and
signal-conducting activityrelated to
that of neurons.
Keywords: dendrimers · enzymes ·
fluorescence
·
prodrugs
·
self-immolative
Introduction
Dendritic architectures[1] are often used in nature to achieve
divergent or convergent conducting effects. For example, the
structural properties of a tree allow it to transfer water and
nutrients from the trunk toward the branches and the
leaves. The structural design of nerve cells is another strik-
ing example of a dendritic architecture that acts as a signal
transduction system. Neurons are known to send out a
series of long specialized processes that will either receive
electrical signals (dendrites) or transmit these electrical sig-
nals (axons) to their target cells (Figure 1).
Figure 1. Schematic representation of the dendritic architecture of
a
neuron. The electrical signal is transferred in a convergent manner from
the dendrites towards the axon where it diverges to the synaptic termi-
nals.
The dendritic architecture of neurons inspired us to
design dendrimers with a signal transduction pathwaysimi-
lar to that of a nerve cell. Here we report the design and
synthesis of a self-immolative dendritic molecule that is ca-
pable of transferring a cleavage signal in a convergent
manner to the core and then amplifying it divergently to the
periphery. This synthetic system is analogous to the signal
to release all of their tail units through a domino-like chain-
fragmentation process that is initiated bya single cleavage
at the dendrimer core.[6–9] Incorporation of drug molecules
as the tail units and use of an enzyme substrate as the trig-
ger[10] generates a multiple-release prodrug unit that is initi-
ated bya single enzmy atic cleavage. Dendritic prodrugs
have a significant advantage in tumor-cell-growth inhibition
compared with classic monomeric prodrugs.[11,12] We have
also designed and synthesized fully biodegradable dendri-
[2]
transduction pathwayof a neuron.
Self-immolative dendrimers have recentlybeen intro-
duced as a potential platform for single-triggered, multiple-
release prodrugs.[3–5] These unique dendrimers are designed
[a] R. J. Amir, E. Danieli, Prof. D. Shabat
Department of Organic Chemistry, School of Chemistry
Facultyof Exact Sciences, Tel Aviv University
Tel Aviv 69978 (Israel)
mers that are disassembled through multienzymatic trigger-
[13]
ing followed byself-immolative chain fragmentation.
The
concept of a multitriggered, self-immolative dendron was re-
centlyapplied to the snythesis of a prodrug activated
Fax : (+972)3-640-9293
E-mail: chdoron@post.tau.ac.il
812
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FULL PAPER
through a molecular “OR” logic trigger (a dual-input trigger
activated byeither one of two different enzymes).
molecule, the signal transduction is programmed to initiate
through enzymatic cleavage of the phenylacetamide trigger
byusing penicillin G amidase (PGA). 6-Aminoquinoline
was used on one end as a reporter unit because its release
can be monitored byemploying fluorescence spectroscop.y
[14]
Results andDiscussion
Upon release of 6-aminoACHTREUqNG uinAHCTREUNGoACHRTElUNG ine from the dendrimer, the
Molecular design of the dendritic system: To construct a
dendritic architecture with signal-conducting activitysimilar
to that of a neuron, we used a multitriggered self-immola-
tive dendron[13] as a receiver and linked it through a short
spacer to a single-triggered self-immolative dendron[3] that
acts as an amplifier. In this design (shown schematicallyin
Figure 2), a signal is received through activation of either of
the triggers. The signal is transferred to the focal point,
where it is divergentlyamplified through one of the den-
drons, and both reporter units are released. During signal
propagation, the dendritic molecule is disassembled into
small fragments.
conjugation of its amine functional group with the quinoline
p system is increased and a new band at l=460 nm appears
in the fluorescence emission spectrum.[15] Polyethylene
glycol 400 (PEG-400) oligomers were attached to the other
end (right-hand side in Figure 2) of the dendritic molecules
to make them sufficientlysoluble in aqueous solution to
allow enzymatic activation.
The signal-transfer mechanism of the first-generation den-
dritic molecule (1) is illustrated in Scheme 1. Enzymatic
cleavage of either one of the phenylacetamide groups by
PGA gives intermediate 3, which contains an exposed
amine group. The amine group is then cyclized to initiate a
series of self-immolative fragmentations that releases phenol
4 along with several other short fragments. Phenol 4 is disas-
sembled through a double quinone methide type rearrange-
ment to release carbon dioxide, compound 5, and most im-
portantlyfree the two fluorescent molecules of 6-aminoquin-
oline. The second-generation dendritic molecule 2 disassem-
bles by employing a similar mechanism (Scheme 2). Enzy-
matic cleavage of one of the four phenylacetamide groups
byPGA releases amine intermediate 6, which initiates the
signal transfer through self-immolative fragmentations. The
output is expressed in the form of a fluorescence signal as a
result of the release of four 6-aminoquinoline molecules.
Based on the design illustrated in Figure 2, we synthesized
two dendritic molecules: compound 1 (first generation) and
compound 2 (second generation) shown in Figure 3. In each
Synthesis of the dendritic molecules 1 and 2: First-genera-
tion dendritic molecule 1 was synthesized according to
Figure 2. Graphical illustration of a receiver–amplifier dendritic mole-
cule.
Figure 3. Chemical structures of first-generation (1) and second-generation (2) self-immolative, receiver–amplifier dendritic molecules with an enzymatic
trigger (blue), cleaved byPGA and 6-aminoquinoline (red) reporter groups.
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D. Shabat et al.
Scheme 1. Signal transduction mechanism for dendritic molecule 1 byusing a self-immolative reaction sequence.
Scheme 2. Signal transduction pathwayfor dendritic molecule 2 byemploying a self-immolative reaction sequence.
Scheme 3. 4-Hydroxybenzoic acid was coupled with propar-
gylamine to form amide 7, which was treated with paraform-
aldehyde to generate dibenzyl alcohol 8. The latter was sub-
sequentlytreated with two equivalents of tert-butyldimeth-
ylsilyl chloride (TBSCl) to afford phenol 9, which was acy-
lated with p-nitrophenyl chloroformate to give carbonate 10.
Reaction of 10 with mono-Boc-protected N,N’-dimethyl-
ACHTREUNGethylenediamine (Boc=t-butoxycarbonyl) generated com-
lents of p-nitrophenyl chloroformate afforded dicarbonate
13, which was then treated with two equivalents of 6-amino-
quinoline to give compound 14. Deprotection with trifluoro-
acetic acid (TFA) afforded an amine salt, which was treated
in situ with compound 15[13] to yield dendritic molecule 16.
Compound 16 was treated with commerciallyavailable
PEG-400 azide to perform a copper(I)-catalyzed Huisgen
cycloaddition[16] reaction to generate first-generation den-
dritic molecule 1.
AHCTREUNG
A
G
ACHTREUNG
AHCTREUNG
ACHTREUNG
pound 11, which was deprotected in the presence of Amber-
lyst-15 to give diol 12. Acylation of diol 12 with two equiva-
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Self-Immolative Dendrimers
FULL PAPER
Scheme 3. Synthesis of first-generation dendritic molecule 1.
The synthesis of second-generation dendritic molecule 2 is
shown in Schemes 4 and 5. Acylation of phenol 17[12] with p-
nitrophenyl chloroformate afforded carbonate 18. Reaction
of 18 with mono-Boc-protected N,N’-dimethylethylenedia-
mine generated compound 19, which was deprotected in the
presence of Amberlyst-15 to give diol 20. Acylation of diol
20 with two equivalents of p-nitrophenyl chloroformate af-
forded dicarbonate 21.
Two equivalents of dendron 14 were deprotected with
TFA to afford an amine salt, which was treated in situ with
compound 21 to yield dendritic molecule 22. The latter was
treated with TFA to afford an amine-salt that was treated in
situ with compound 23 (see Experimental Section) to yield
dendritic molecule 24. Compound 24 was treated with com-
merciallyavailable PEG-400 azide to perform a copper(I)-
catalyzed Huisgen cycloaddition reaction to generate
second-generation dendritic molecule 2.
Enzymatic activation of dendritic molecules 1 and 2: To pre-
pare aqueous solutions of 1 and 2, the compounds were ini-
tiallydissolved in dimethlysulfoxide (DMSO)/Cremophor
EL (4:1) and then added to water. The final composition of
the solution was 10% organic and 90% aqueous. Dendritic
molecules 1 and 2 were then incubated with PGA in phos-
Scheme 4. Synthesis of intermediate molecule 21.
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D. Shabat et al.
Scheme 5. Synthesis of second-generation dendritic molecule 2.
phate-buffered saline (PBS,
pH 7.4) solution at 378C. Con-
trol solutions were incubated in
buffer without the enzyme. The
sequential fragmentation illus-
trated in Schemes 1 and 2 was
monitored through the release
of 6-aminoquinoline. Free 6-
aminoquinoline is generated
upon addition of PGA to a so-
lution of 1 or 2, as shown in
Figure 4. The fluorescence spec-
tra of 1 and 2 exhibit one emis-
sion band at l=390 nm that
disappeared during dendrimer
fragmentation. The generation
of a new band at l=460 nm in-
dicates the formation of free 6-
aminoquinoline. To evaluate
the kinetic behavior of the se-
quential fragmentation, the in-
tensities of the bands at 390
and 460 nm were plotted as a
function of time (Figure 4b and
Figure 4. Emission fluorescence spectra (lex =250 nm) of 1 (a, b) and 2 (c, d) upon addition of PGA
d). The release of 6-aminoquin- (0.1 mgmLÀ1). The concentrations of dendritic molecules 1 and 2 were 25 and 12 mm, respectively.
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Self-Immolative Dendrimers
FULL PAPER
A
G
Conclusion
plete in approximately4 h, whereas the fragmentation of
second-generation dendritic molecule 2 required over 50 h.
No release of 6-aminoquinoline was observed when com-
pounds 1 and 2 were incubated in the buffer without PGA
In conclusion, we designed and synthesized new dendritic
molecules that act as receiver–amplifier devices. A cleavage
signal received byone side of the dendritic molecule is
transferred in a convergent manner to the core and then
amplified divergentlytoward the other side. The signal is
propagated through self-immolative sequential fragmenta-
tions to release reporter molecules that are observed by
means of fluorescence spectroscopy. There are similarities
between this system and the dendritic architecture and func-
tion of neurons and other dendritic transduction pathways
in nature. Dendritic molecule 2 is the longest dendritic
system ever reported to date to disassemble through sequen-
tial self-immolative reactions. Learning to control and opti-
mize the kinetics of self-immolative reactions is currentlya
major focus of our laboratory.
(data not shown). The fragmentation mechanism was previ-
[13]
ouslydescribed for both the receiver
and the amplifier
dendrons.[3]
PGA is a specific proteolytic enzyme that selectively
cleaves phenylacetamide groups.[17] Dendritic molecule 25
with t-butylcarbamate as a trigger instead of the phenyl
ACHTREaUNG cet-
ACHTREUNG
pound 25 was found to be stable when incubated with PGA
and no degradation was observed. However, when 25 was
first deprotected bytreatment with TFA and then incubated
in PBS at pH 7.4 the release of 6-aminoquinoline was ob-
served (Scheme 6). This result clearlyproves that activation
of dendrimers 1 and 2 byPGA is specific and that the re-
lease process is indeed trigger-dependent.
Dendrimer fragmentation occurs through enzymatic
cleavage, followed by cyclization, quinone methide type re-
arrangement, and decarboxylation. Previous studies have
shown that the slow step in self-immolative reactions is the
Experimental Section
General: All reactions requiring anhydrous conditions were performed
under an argon or nitrogen atmosphere. Chemicals and solvents were
either A.R. grade or purified byemploying standard techniques. Thin-
layer chromatography (TLC) was performed on silica gel plates Merck
60 F254; compounds were visualized byirradiation with UV light and/or
cyclization.[3] The signal transfer is significantlyslower in
2
than in 1. This observation is not unexpected because four
cyclization steps are needed to complete the disassembly of
the second-generation molecule, whereas onlytwo are
needed in the first-generation dendrimer. In addition, the
trigger groups in 2 are less accessible to the activating
enzyme owing to increased steric hindrance in comparison
with 1. This phenomenon was observed in dendrimer-bound
peptides that had increased stabilitytowards enzymatic deg-
radation when compared with monomeric peptides.[18] We
are currentlyattempting to snythesize dendritic molecules
that will disassemble without a cyclization step. The signal
transfer is expected to be significantlyfaster in the absence
of this slow step.
bytreatment with
a solution of phosphomolbydic acid (25 g), Ce-
AHCTRENU(G SO4)2·H2O (10 g), concd H2SO4 (60 mL), and H2O (940 mL), and devel-
oped byheating. Flash chromatographywas performed byusing silica gel
Merck 60 (particle size 0.040–0.063 mm) and the eluent given in paren-
theses. 1H NMR spectroscopywas performed byusing a Bruker AMX
200 or 400 instrument. The chemical shifts are expressed in d relative to
tetramethylsilane (TMS) (d=0 ppm) and the coupling constants J in Hz.
The spectra were recorded byusing CDCl 3 or CD3OD as a solvent at RT.
All reagents, including salts and solvents, were purchased from Sigma-Al-
drich. PEG-400 azide was purchased from Polypure (Norway). tris(1-
BenACHTERzUNG yl-1H-[1,2,3]triazol-4-ylmethyl)amine (TBTA) was kindly received
from the Sharpless laboratory(The Scripps Research Institute, La Jolla,
USA).
Scheme 6. Activation studies for dendritic molecules 25 and 26.
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D. Shabat et al.
Synthesis of first-generation self-immolative dendritic molecule 1
80.2, 78.8, 72.0, 59.9, 46.4, 46.1, 36.4, 35.9, 35.1, 29.8, 28.3, 25.7, 18.2,
À5.5 ppm; MS (FAB): m/z: 700.4 [M+Na]+.
Compound 7: Commercially available 4-hydroxybenzoic acid (2.0 g,
14.5 mmol) was dissolved in DMF (10 mL) before N-(3-dimethylamino-
propyl)-N’-ethylcarbodiimide (EDC) (3.3 g, 17.4 mmol), 1-hydroxybenzo-
triazole (HOBT) (1.0 g, 7.3 mmol), and propargylamine (1.0 mL,
14.5 mmol) were added. The mixture was stirred overnight and moni-
tored byTLC (EtOAc/He x 2:3). Upon completion of the reaction, the
solvent was removed under reduced pressure. The crude product was pu-
rified byusing column chromatographyon silica gel (EtOAc/Hex 2:3) to
give 7 as a yellowish oil (1.8 g, 70%). 1H NMR (200 MHz, CDCl3): d=
7.70 (d, J=6.8 Hz, 2H), 6.81 (d, J=6.8 Hz, 2H), 4.11 (d, J=2.5 Hz, 2H),
2.71 ppm (t, J=2.5 Hz, 1H); 13C NMR (100 MHz, CDCl3): d=167.9,
160.6, 128.8, 124.4, 114.5, 79.5, 70.3, 28.3 ppm; MS (FAB): m/z: 176.0
[M+H]+.
Compound 12: Compound 11 (1.5 g, 2.2 mmol) was dissolved in MeOH
(10 mL) and Amberlyst-15 was added. The reaction was stirred at RT for
2 h and monitored byTLC (EtOAc). Upon completion of the reaction,
the solution was filtered to remove Amberlyst-15 and the solvent was re-
moved under reduced pressure. The crude product was purified byusing
column chromatographyon silica gel (EtOAc/MeOH 19:1) to give 12 as
a white solid (500 mg, 56%). 1H NMR (200 MHz, CD3OD): d=7.78 (s,
2H), 4.57 (brs, 4H), 4.25–4.23 (m, 2H), 3.60–3.40 (m, 4H), 3.20 (s, 2H),
3.10 (s, 1H), 2.96 (s, 3H), 2.40 (brs, 1H), 1.59–1.54 ppm (m, 9H);
13C NMR (100 MHz, CD3OD): d=169.8, 158.9, 157.5, 152.1, 134.1, 130.3,
130.0, 83.2, 83.0, 74.4, 62.5, 50.3, 49.0, 38.7, 37.9, 37.6, 31.3 ppm; HRMS
(MALDI-TOF): m/z calcd for C22H31N3O7: 472.2015; found: 472.2059
[M+Na]+.
Compound 8: Compound 7 (1.8 g, 10.2 mmol) was added to a cold (08C)
12% aqueous NaOH (12 mL) solution. The mixture was kept at 08C and
formaldehyde (37% in water; 10 mL) was added. The reaction was stir-
red at 558C for 3 d and monitored byTLC (EtOAc/MeOH 95:5). Upon
completion of the reaction, the solution was diluted with EtOAc and
washed with saturated ammonium chloride solution. The aqueous layer
was washed twice with EtOAc. The combined organic layer was dried
over MgSO4 and the solvent was removed under reduced pressure. The
crude product was purified byusing column chromatographyon silica gel
(EtOAc/MeOH 19:1) to give 8 as a white solid (1.9 g, 80%). 1H NMR
(200 MHz, CD3OD): d=7.80 (s, 2H), 4.91 (s, 4H), 4.26 (d, J=2.5 Hz,
2H), 2.70 ppm (t, J=2.5 Hz, 1H); 13C NMR (100 MHz, CD3OD): d=
168.1, 156.7, 126.8, 126.0, 124.4, 79.4, 70.2, 60.3, 28.3 ppm; MS (FAB):
m/z: 236.0 [M+H]+.
Compound 13: Compound 12 (300 mg, 0.67 mmol) was dissolved in dry
THF (6 mL) and was cooled to 08C before diisopropyl ethyleneamine
(DIPEA) (945 mL, 5.4 mmol) followed by p-nitrophenyl chloroformate
(800 mg, 4.0 mmol) and pyridine (27 mL, 0.33 mmol) were added. The re-
action was allowed to warm to RT and was monitored byTLC (EtOAc/
Hex 3:1). Upon completion of the reaction, the solution was diluted with
EtOAc and washed with satACHTERuUNG rated aqueous NH4Cl and NaHCO3 solu-
tions. The organic layer was dried over MgSO4 and the solvent was re-
moved under reduced pressure. The crude product was purified byusing
column chromatographyon silica gel (EtOAc/Hex 7:3) to give 13 as a
white solid (430 mg, 82%). 1H NMR (200 MHz, CDCl3): d=8.23 (d, J=
9.0 Hz, 4H), 7.94 (s, 2H), 7.34 (d, J=9.0 Hz, 4H), 5.28 (s, 4H), 4.23 (m,
2H), 3.62–3.43 (m, 4H), 3.18–3.00 (m, 3H), 2.92–2.83 (m, 3H), 2.27 (brs,
1H), 1.45–1.42 ppm (m, 9H); 13C NMR (100 MHz, CDCl3): d=166.2,
156.6, 154.1, 153.1, 146.3, 132.9, 130.4, 130.0, 126.1, 122.6, 122.5, 80.8,
78.9, 73.0, 66.2, 48.5, 47.8, 46.8, 36.1, 35.6, 30.7, 29.2 ppm; HRMS
(MALDI-TOF): m/z calcd for C36H37N5O15: 802.2178; found: 802.2112
[M+Na]+.
Compound 9: Compound 8 (713 mg, 3.0 mmol) was dissolved in DMF
(10 mL) and cooled to 08C before imidazole (408 mg, 6.0 mmol) and
TBS-Cl (910 mg, 6.0 mmol) were added. The reaction was stirred at RT
for 2 h and monitored byTLC (EtOAc/Hex 2:8). Upon completion of
the reaction, the solution was diluted with diethyl ether and washed with
saturated ammonium chloride solution. The organic layer was dried over
MgSO4 and the solvent was removed under reduced pressure. The crude
product was purified byusing column chromatographyon silica gel
(EtOAc/Hex 15:85) to give 9 as a colorless oil (1.12 g, 80%). 1H NMR
(400 MHz, CDCl3): d=7.57 (s, 2H), 4.87 (s, 4H), 4.23 (dd, J=5.0, 2.5 Hz,
2H), 2.17 (t, J=2.5 Hz, 1H), 0.95 (s, 18H), 0.13 ppm (s, 12H); 13C NMR
(100 MHz, CDCl3): d=166.7, 156.4, 126.1, 124.5, 79.6, 71.7, 62.7, 29.6,
25.8, 25.6, 18.2, À5.5 ppm; MS (FAB): m/z: 464.2 [M+H]+.
Compound 10: Compound 9 (1.12 g, 2.4 mmol) was dissolved in dryTHF
(20 mL) before NEt3 (1.0 mL, 7.2 mmol) was added and the mixture was
cooled to 08C. p-Nitrophenyl chloroformate (581 mg, 2.9 mmol) dissolved
in dryTHF (10 mL) was then added dropwise before the reaction was
stirred for 1 h at RT and was monitored byTLC (EtOAc/Hex 2:8). Upon
completion of the reaction, the solution was filtered, the solvent was
evaporated, and the crude product was purified byusing column chroma-
tographyon silica gel (EtOAc/Hex 15:85) to give 10 as a colorless oil
(1.35 g, 90%). 1H NMR (200 MHz, CDCl3): d=8.43 (d, J=8.1 Hz, 2H),
8.02 (s, 2H), 7.63 (d, J=8.1 Hz, 2H), 7.01 (m, 1H), 4.91 (s, 4H), 4.38 (dd,
J=2.5, 2.6 Hz, 2H), 2.41 (t, J=2.5 Hz, 1H), 1.08 (s, 18H), 0.29 ppm (s,
12H); 13C NMR (100 MHz, CDCl3): d=166.4, 155.2, 149.4, 147.7, 145.5,
133.9, 132.2, 126.3, 125.3, 121.5, 79.2, 71.8, 60.3, 31.5, 25.8, 18.2,
À5.5 ppm; HRMS (MALDI-TOF): m/z calcd for C31H44N2O8Si2:
651.2528; found: 651.2562 [M+Na]+.
Compound 14: Compound 13 (430 mg, 0.55 mmol) was dissolved in
DMF (2 mL) before 6-aminoquinoline (320 mg, 2.2 mmol), a catalytic
amount of HOBT (5 mg), and DIPEA (240 mL, 1.4 mmol) were added.
The reaction was heated to 508C, stirred overnight, and monitored by
TLC (MeOH/EtOAc 1:9). Upon completion of the reaction, the solvent
was removed under reduced pressure. The crude product was purified by
using column chromatographyon silica gel (MeOH/EtOAc 2:8) to give
1
14 as a white solid (270 mg, 62%). H NMR (200 MHz, CDCl3): d=8.69–
8.67 (m, 2H), 7.98–7.88 (m, 8H), 7.54–750 (m, 2H), 7.25–7.22 (m, 2H),
5.08 (brs, 4H), 4.13 (s, 2H), 3.52–3.36 (m, 4H), 3.05–2.76 (m, 6H), 2.17
(brs, 1H), 1.38–1.30 ppm (m, 9H); 13C NMR (100 MHz, CDCl3): d=
166.8, 154.4, 154.1, 149.8, 149.7, 145.9, 137.0, 136.3, 132.3, 131.1, 130.9,
129.9, 129.6, 123.4, 122.3, 114.8, 81.2, 80.1, 72.7, 63.3, 47.6, 46.4, 36.6, 35.2,
30.6, 29.2 ppm; HRMS (MALDI-TOF): m/z calcd for C42H43N7O9:
812.3061; found: 812.3014 [M+Na]+.
Compound 16: Compound 14 (64 mg, 0.08 mmol) was dissolved in TFA
(1 mL) and stirred for few minutes before the excess acid was removed
under reduced pressure and the crude amine salt was dissolved in DMF
(0.5 mL). Compound 15[13] (53 mg, 0.08 mmol) and NEt3 (0.1 mL) were
then added, and the reaction was monitored byTLC (MeOH/CH 2Cl2
1:9). Upon completion of the reaction, the solvent was removed under
reduced pressure. The crude product was purified byusing column chro-
matographyon silica gel (MeOH/EtOAc 1:9) to give 16 as a white solid
(45 mg, 46%). 1H NMR (200 MHz, CDCl3): d=8.85–8.55 (m, 2H), 8.10–
7.80 (m, 10H), 7.67–7.45 (m, 2H), 7.29–6.71 (m, 14H), 5.09–5.01 (m,
6H), 4.13–4.05 (m, 2H), 3.67–3.30 (m, 16H), 3.04–2.90 (m, 6H), 2.23 ppm
(s, 1H); 13C NMR (100 MHz, CDCl3): d=172.1, 166.5, 155.3, 153.7, 153.5,
150.9, 148.9, 145.0, 136.6, 135.8, 135.0, 133.6, 103.7, 130.5, 130.2, 129.9,
129.4, 129.3, 129.0, 128.9, 128.8, 128.5, 127.3, 122.9, 121.8, 121.7, 80.0,
71.9, 71.8, 62.2, 53.6, 48.5, 43.6, 38.8, 32.1, 31.7, 30.0, 29.8 ppm; HRMS
(MALDI-TOF): m/z calcd for C66H64N10O13: 1227.4547; found: 1227.4656
[M+Na]+.
Compound 11: Compound 10 (1.5 g, 2.3 mmol) was dissolved in DMF
(7 mL). Previouslydescribed mono-Boc-protected N,N’-dimethylethyl-
ACHTREUNGeneACHTREUNG
diamine[3] (541 mg, 2.9 mmol) was added. The reaction was stirred at
RT for 1 h and monitored byTLC (EtOAc/Hex 1:1). Upon completion
of the reaction, the solvent was removed under reduced pressure and the
crude product was purified byusing column chromatographyon silica gel
(EtOAc/Hex 2:8) to give 11 as a colorless oil (1.45 g, 90%). 1H NMR
(400 MHz, CDCl3): d=7.79 (s, 2H), 6.32 (m, 1H), 4.68–4.67 (m, 4H),
4.27–4.25 (m, 2H), 3.61–3.43 (m, 4H), 3.24 (s, 2H), 3.12 (s, 1H), 2.96 (s,
3H), 2.32 (brs, 1H), 1.51–1.46 (m, 9H), 0.92 (s, 18H), 0.08 ppm (s, 12H);
13C NMR (100 MHz, CDCl3): d=167.2, 153.1, 153.0, 134.6, 130.8, 125.1,
Compound 1: Compound 16 (16 mg, 0.013 mmol) was dissolved in DMF
(0.5 mL) before PEG-400 azide (6.3 mg, 0.016 mmol), copper sulfate
(2 mg, 0.013 mmol), and TBTA (7.5 mg, 0.0133 mmol) were added. Final-
818
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Chem. Eur. J. 2007, 13, 812 – 821

Self-Immolative Dendrimers
FULL PAPER
ly, a few copper turnings were added before the reaction was stirred over-
night at RT. The reaction was monitored byHPLC, and upon completion
of the reaction the solution was filtered and the solvent was removed
under reduced pressure. The crude product was purified byusing column
chromatographyon silica gel (MeOH/CH 2Cl2 1:9) to give 1 as a white
solid (17.7 mg, 83%). HPLC: C18 reverse-phase column; l=250 nm;
flow: 1 mLminÀ1; gradient program: t=0 (30% MeCN/70% H2O), t=
20–25 min (100% MeCN); tR =8.26 min (16); tR =7.38 min (1). HRMS
(MALDI-TOF): m/z calcd for C82H97N13O21: 1622.6814; found: 1622.6797
[M+Na]+.
2.94–2.85 (m, 3H), 1.43–1.41 (m, 9H), 1.38 ppm (t, J=7.0 Hz, 3H);
13C NMR (100 MHz, CDCl3): d=165.8, 156.2, 154.1, 153.0, 152.5, 152.4,
146.3, 132.0, 129.6, 126.1, 122.8, 122.6, 80.7, 66.4, 62.3, 48.3, 46.9, 35.6,
32.3, 29.1, 14.9 ppm; HRMS (MALDI-TOF): m/z calcd for C35H38N4O16
:
793.2175; found: 793.2148 [M+Na]+.
Compound 22: The Boc group of compound 14 (200 mg, 0.25 mmol) was
removed with TFA (1 mL). The excess TFA was removed under reduced
pressure and the residue was dissolved in DMF (1 mL). Compound 21
(90 mg, 0.12 mmol) and NEt3 (1 mL) were then added and the solution
was stirred for 3 h. Upon completion of the reaction, DMF was removed
under reduced pressure and the crude product was purified byusing
Synthesis of second-generation self-immolative dendritic molecule 2
Compound 18: Compound 17[12] (780 mg, 1.7 mmol) was dissolved in
CH2Cl2 (20 mL) before NEt3 (870 mL, 6.0 mmol) and a catalytic amount
of dimethylaminopyridine (DMAP; 5 mg) were added. The reaction was
cooled to 08C and p-nitrophenyl chloroformate (520 mg, 2.6 mmol) was
added. The reaction was stirred at RT for 1 h and monitored byTLC
(EtOAc/Hex 1:9). Upon completion of the reaction, the solution was di-
luted with CH2Cl2, and washed with saturated aqueous NH4Cl and with
brine. The organic layer was dried over MgSO4 and the solvent was re-
moved under reduced pressure. The crude product was purified byusing
column chromatographyon silica gel (EtOAc/Hex 5:95) to give com-
pound 18 as a colorless oil (790 mg, 75%). 1H NMR (200 MHz, CDCl3):
d=8.29 (d, J=9.0 Hz, 2H), 8.11 (s, 2H), 7.45 (d, J=9.0 Hz, 2H), 4.75 (s,
4H), 4.35 (q, J=7.0 Hz, 2H), 1.36 (t, J=7.0 Hz, 3H), 0.90 (s, 18H),
0.07 ppm (s, 12H); 13C NMR (100 MHz, CDCl3): d=166.5, 156.1, 150.16,
149.26, 146.4, 134.5, 129.9, 129.6, 126.2, 122.1, 61.9, 61.2, 26.7, 19.1, 15.1,
À4.5 ppm; HRMS (MALDI-TOF): m/z calcd for C30H45NO9Si2:
642.2525; found: 642.2482 [M+Na]+.
column chromatographyon silica gel (CH Cl2/MeOH 9:1) to give 22 as a
2
white powder (130 mg, 58%). 1H NMR (200 MHz, CD3OD): d=8.59
(brs, 4H), 8.02–7.34 (m, 22H), 7.34–7.28 (m, 4H), 5.11–5.00 (m, 16H),
4.09 (brs, 4H), 3.57–3.41 (m, 12H), 3.10–2.57 (m, 18H), 1.82 (brs, 1H),
1.24–1.14 ppm (m, 9H); HRMS (MALDI-TOF): m/z calcd for
C97H98N16O24: 1893.6832; found: 1893.6937 [M+Na]+.
Compound 23a (Scheme 7): Compound 15[13] (587 mg, 1 mmol) was dis-
solved in DMF (3 mL) before diethylenetriamine (51.6 mg, 0.5 mmol)
was added and the reaction was stirred at RT for several hours. Upon
completion of the reaction, p-hydroxybenzyl p-nitrophenyl carbonate
(150 mg, 0.52 mmol) and NEt3 (65 mL, 0.5 mmol) were added. The reac-
tion was monitored byTLC (EtOAc). Once the reaction was complete,
the solvent was removed under reduced pressure and the crude product
was purified byusing column chromatographyon silica gel (EtOAc) to
1
give 23a as a white powder (332 mg, 52%). H NMR (400 MHz, CDCl3):
d=7.32–7.06 (m, 26H), 6.94 (d, J=8.2 Hz, 4H), 6.86 (d, J=8.3 Hz, 2H),
6.58 (brs, 2H), 5.9–5.5 (m, 2H), 5.01 (s, 2H), 4.98 (s, 2H), 4.49 (s, 2H),
3.45–3.24 ppm (m, 32H); 13C NMR (100 MHz, CDCl3): d=172.8, 172.6,
157.4, 156.0, 151.7, 151.0, 139.5, 135.8, 135.7, 130.7, 130.1, 129.5, 128.5,
127.9, 122.5, 122.3, 66.8, 65.0, 49.1, 49.0, 44.21, 40.6, 39.4 ppm; HRMS
(MALDI-TOF): m/z calcd for C70H77N9O15: 1306.5431; found: 1306.5529
[M+Na]+.
Compound 19: Compound 18 (750 mg, 1.2 mmol) was dissolved in DMF
(5 mL) and
mono-Boc-protected
N,N’-dimethylethylenediamine[3]
(280 mg, 1.45 mmol) was added. The reaction was stirred at RT and
monitored byTLC (EtOAc/Hex 1:3). Upon completion of the reaction,
the solution was diluted with EtOAc, and washed with saturated aqueous
NH4Cl solution and with brine. The organic layer was dried over MgSO4
and the solvent was removed under reduced pressure. The crude product
was purified byusing column chromatographyon silica gel (EtOAc/Hex
1:4) to give 19 as a viscous oil (630 mg, 78%). 1H NMR (200 MHz,
CDCl3): d=8.10 (s, 2H), 4.64 (s, 4H), 4.32 (q, J=7.0 Hz, 2H), 3.55–3.42
(m, 4H), 3.12–3.00 (m, 3H), 2.92–2.89 (m, 3H), 1.53–1.45 (m, 9H), 1.38
(t, J=7.0 Hz, 3H), 0.9 (s, 18H), 0.07 ppm (s, 12H); 13C NMR (100 MHz,
CDCl3): d=167.0, 153.7, 149.4, 135.1, 128.8, 126.8, 116.3, 80.6, 61.6, 60.2,
48.1, 47.1, 36.2, 35.9, 29.2, 26.6, 19.1, 14.9, À4.5 ppm; HRMS (MALDI-
TOF): m/z calcd for C33H60N2O8Si2: 691.3780; found: 691.3748 [M+Na]+.
Compound 23: Compound 23a (125 mg, 0.098 mmol) and DIPEA
(25 mg, 0.195 mmol) were dissolved in CH2Cl2 (3 mL). p-Nitrophenyl
chloroformate (39 mg, 0.195 mmol) and DMAP (3 mg) were then added
before the reaction was monitored byTLC (EtOAc). Upon completion
of the reaction, the solution was diluted with EtOAc and washed with
satACHTREuUGN rated aqueous NH4Cl solution and brine. The organic layer was dried
over MgSO4, the solvent removed under reduced pressure, and the crude
product was purified byusing column chromatographyon silica gel
(EtOAc) to give 23 as a yellowish powder (92 mg, 65%). 1H NMR
(400 MHz, CDCl3): d=8.24 (d, J=8.2 Hz, 2H), 7.41–7.18 (m, 28H), 7.10
(d, J=8.4 Hz, 2H), 6.99 (d, J=7.5 Hz, 4H), 6.60 (brs, 2H), 6.31 (brs,
2H), 5.23 (s, 2H), 5.04 (s, 2H), 5.02 (s, 2H), 3.49–3.29 ppm (m, 32H);
13C NMR (100 MHz, CDCl3): d=172.7, 172.4, 157.3, 156.2, 156.0, 153.2,
152.4, 151.8, 151.7, 146.2, 135.7, 135.6, 130.8, 130.7, 130.3, 130.1, 129.6,
128.0, 126.0, 122.8, 122.6, 71.1, 66.9, 49.4, 49.2, 44.3, 40.6, 39.5 ppm;
HRMS (MALDI-TOF): m/z calcd for C77H80N10O19: 1471.5493; found:
1471.5544 [M+Na]+.
Compound 20: Compound 19 (570 mg, 0.85 mmol) was dissolved in
methanol (15 mL) and Amberlyst-15 was added. The reaction was stirred
at RT for 5 h and monitored byTLC (EtOAc). Upon completion of the
reaction, the solution was filtered to remove Amberlyst-15 and the sol-
vent was removed under reduced pressure. The crude product was puri-
fied byusing column chromatographyon silica gel (EtOAc) to give 20 as
a white solid (270 mg, 71%). 1H NMR (200 MHz, CDCl3): d=8.03 (s,
2H), 4.55 (s, 4H), 4.35 (q, J=7.0 Hz, 2H), 3.59–3.44 (m, 4H), 3.13–3.00
(m, 3H), 2.90–2.85 (m, 3H), 1.44–1.39 (m, 9H), 1.34 ppm (t, J=7.0, 3H);
13C NMR (100 MHz, CDCl3): d=166.6, 156.8, 155.6, 155.4, 135.1, 131.5,
129.2, 81.2, 61.9, 61.0, 47.5, 47.1, 36.9, 35.8, 29.1, 15.1 ppm; HRMS
(MALDI-TOF): m/z calcd for C21H32N2O8: 463.2051; found: 463.2087
[M+Na]+.
Compound 24: Compound 22 (100 mg, 0.053 mmol) was dissolved in
TFA (1.5 mL) and stirred for a few minutes before excess TFA was re-
moved under reduced pressure and then the crude amine salt was redis-
solved in DMF (0.5 mL). Compound 23 (77.5 mg, 0.053 mmol) and NEt3
(0.1 mL) were added and the reaction was monitored byTLC (MeOH/
CH2Cl2 1:9). Upon completion of the reaction, the solvent was removed
under reduced pressure. The crude product was purified byusing column
chromatographyon silica gel (MeOH/CH 2Cl2 1:9) to give 24 as a white
powder (65 mg, 40%). 1H NMR (400 MHz, CDCl3): d=8.71 (s, 4H),
8.04–7.76 (m, 16H), 7.70–7.28 (m, 4H), 7.27–7.15 (m, 26H), 7.14–6.70 (m,
12H), 5.15–4.90 (m, 18H), 4.30–4.02 (m, 6H), 3.63–3.19 (m, 44H), 3.07–
2.70 (m, 18H), 2.25 (brs, 2H), 0.89–0.85 ppm (m, 3H); HRMS (MALDI-
Compound 21: Compound 20 (75 mg, 0.17 mmol) was dissolved in dry
THF (2 mL) and was cooled to 08C before DIPEA (270 mL, 1.44 mmol),
p-nitrophenyl chloroformate (220 mg, 1.1 mmol), and pyridine (7 mL,
0.09 mmol) were added. The reaction was allowed to warm up to RT and
was monitored byTLC (EtOAc/Hex 1:1). Upon completion of the reac-
tion, the solution was diluted with EtOAc and washed with saturated
aqueous NH4Cl and NaHCO3 solutions. The organic layer was dried over
MgSO4 and the solvent was removed under reduced pressure. The crude
product was purified byusing column chromatographyon silica gel
(EtOAc/Hex 2:3) to give 21 as a white solid (100 mg, 75%). 1H NMR
(400 MHz, CDCl3): d=8.24–8.20 (m, 6H), 7.36 (d, J=7.0 Hz,4H), 5.31 (s,
4H), 4.38 (q, J=7.0 Hz, 2H), 3.60–3.45 (m, 4H), 3.20–3.02 (m, 3H),
TOF): m/z calcd for
C163H165N25O38: 3103.1640; found: 3103.1723
[M+Na]+.
Compound 2: Compound 24 (15 mg, 4.9 mmol) was dissolved in DMF
(0.5 mL), and PEG-400 azide (4.6 mg, 11.7 mmol), copper sulfate (1.6 mg,
9.7 mmol), and TBTA (5.5 mg, 9.7 mmol) were added. Subsequentlya few
copper turnings were added and the reaction was stirred overnight at RT.
Chem. Eur. J. 2007, 13, 812 – 821
ꢀ 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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819

D. Shabat et al.
Scheme 7. Synthesis of intermediate molecule 23.
The reaction was monitored byHPLC and upon its completion, the mix-
ture was filtered and the solvent was removed under reduced pressure.
The crude product was purified byusing column chromatographyon
silica gel (MeOH/CH2Cl2 2:9) to give 2 as a white solid (16 mg, 85%).
HPLC: C18 reverse-phase column; l=250 nm; flow: 1 mLminÀ1; gradi-
ent program: t=0 (10% MeCN/90% H2O), t=23–27 min (100%
MeCN); tR =15.66 min (24); tR =15.01 min (2); HRMS (MALDI-TOF):
m/z calcd for C195H231N31O54: 3893.6175; found: 3893.6046 [M+Na]+.
Compound 16a: Compound 14 (100 mg, 0.13 mmol) was dissolved in
TFA (1.5 mL) and stirred for few minutes. The excess acid was then re-
moved under reduced pressure and the crude amine salt was dissolved in
DMF (0.5 mL). Subsequently, compound 15c (78 mg, 0.13 mmol) and
NEt3 (0.1 mL) were added and the reaction was monitored byTLC
(EtOAc). Upon completion of the reaction, the solution was diluted with
EtOAc and washed with saturated NH4Cl solution and with brine. The
organic layer was dried over MgSO4 and the solvent was removed under
reduced pressure. The crude product was purified byusing column chro-
matographyon silica gel (EtOAc) to give 16a as a white solid (131 mg,
88%). 1H NMR (200 MHz, CDCl3): d=8.78–8.77 (m, 2H), 8.10–7.96 (m,
8H), 7.63–7.60 (m, 2H), 7.34–7.32 (m, 2H), 7.05–6.95 (m, 4H), 5.12–4.97
(m, 6H), 4.28–4.23 (m, 2H), 3.55–3.38 (m, 12H), 3.17–2.97 (m, 6H),
Synthesis of first-generation dendritic molecule 25 (Scheme 8)
Compound 15b: Compound 15a[19] (1.14 g, 3.75 mmol) was dissolved in
DMF (10 mL) before the p-nitrophenyl carbonate of 4-hydroxybenzyl al-
cohol (1.64 g, 5.62 mmol) and NEt3 (780 mL, 5.62 mmol) were added. The
reaction was stirred at RT for 2 h and monitored byTLC (EtOAc/Hex
3:1). Upon completion of the reaction, the solution was diluted with
EtOAc and washed with water and with brine. The organic layer was
dried over MgSO4 and the solvent was removed under reduced pressure.
The crude product was purified byusing column chromatographyon
silica gel (EtOAc/Hex 3:1) to give compound 15b as a white solid
(1.20 g, 70%). 1H NMR (400 MHz, CDCl3): d=7.27 (d, J=8.4 Hz, 2H),
7.03 (d, J=8.4 Hz, 2H), 5.24 (brs, 2H), 4.57 (s, 2H), 3.48–3.25 (m, 8H),
1.39 (s, 9H), 1.37 ppm (s, 9H); 13C NMR (100 MHz, CDCl3) d=157.2,
156.9, 156.3, 151.1, 139.3, 128.5, 122.4, 80.2, 80.1, 65.1, 48.8, 40.0, 29.2,
29.1 ppm.
2.31 ppm (brs, 1H); HRMS (MALDI-TOF): m/z calcd for C60H68N10O15
1191.4758; found: 1191.4767 [M+Na]+.
:
Compound 25: Compound 16a (28 mg, 0.024 mmol) was dissolved in
DMF before PEG-400 azide (11.4 mg, 0.029 mmol), copper sulfate (4 mg,
0.024 mmol), and TBTA (13 mg, 0.024 mmol) were added. Subsequently,
a few copper turnings were added and the reaction was stirred overnight
at RT. The reaction was monitored byHPLC and upon its completion,
the mixture was filtered and the solvent was removed under reduced
pressure. The crude product was purified byusing column chromatogra-
phyon silica gel (MeOH/CH Cl2 1:9) to give 25 as a white solid (16.7 mg,
2
44%). HPLC: C18 reverse-phase column; l=250 nm; flow: 1 mLminÀ1
;
Compound 15a: Compound 15b (122 mg, 0.27 mmol) was dissolved in
THF (5 mL) before DIPEA (188 mL, 1.1 mmol), 4-nitrophenyl chlorofor-
mate (163 mg, 0.81 mmol), and a catalytic amount of pyridine (5.4 mL,
0.07 mmol) were added. The reaction was stirred at RT for 1 h and moni-
tored byTLC (EtOAc/Hex 1:1). Upon completion of the reaction, the
solution was diluted with EtOAc and washed with 1m aqueous HCl solu-
tion and with brine. The organic layer was dried over MgSO4 and the sol-
vent was removed under reduced pressure. The crude product was puri-
fied byusing column chromatographyon silica gel (EtOAc/Hex 1:1) to
give 15c as a white powder (97 mg, 60%). 1H NMR (400 MHz, CDCl3):
d=8.26 (d, J=9.1 Hz, 2H), 7.44 (d, J=8.4 Hz, 2H), 7.37 (d, J=9.1 Hz,
2H), 7.18 (d, J=8.4 Hz, 2H), 5.28 (s, 2H), 5.09 (brs, 2H), 3.57–3.37 (m,
8H), 1.44 (s, 9H), 1.41 ppm (s, 9H); 13C NMR (100 MHz, CDCl3): d=
157.1, 156.8, 156.3, 156.0, 153.2, 152.5, 146.2, 132.1, 130.7, 126.1, 122.9,
122.6, 80.2, 71.1, 49.0, 40.1, 29.2, 29.1 ppm.
gradient program: t=0 (30% MeCN/70% H2O), t=20–25 min (MeCN);
tR =16.70 min (16a); tR =15.60 min (25); HRMS (MALDI-TOF): m/z
calcd for C76H101N13O23: 1586.7026; found: 1586.7300 [M+Na]+.
Dendrimer activation protocol and fluorescence measurements for 6-ami-
noquinoline: PGA (56 mgmLÀ1) was purchased from Sigma. Stock solu-
tions of dendrimers 1, 1a, and 2 were prepared in DMSO with 20% Cre-
mophor EL to yield 250 mm stock solutions of 1 and 1a, and a 100 mm
stock solution of 2. The stock solutions (100 mL) were diluted with either
900 mL PBS at pH 7.4 (control) or with 882 mL PBS at pH 7.4 and
18 mL PGA (56 mgmLÀ1) to give final concentrations of 25 mm of den-
drimers 1 and 1a and 10.0 mm of dendrimer 2. The final concentration of
PGA was 1.0 mgmLÀ1 (14 mm). All solutions were kept at 378C and their
fluorescence spectra were measured byusing a SpectraMax M2 spectro-
photometer (Molecular Devices). Standard Costar 96-well plates were
820
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ꢀ 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2007, 13, 812 – 821

Self-Immolative Dendrimers
FULL PAPER
Scheme 8. Synthesis of first-generation dendritic molecule 25.
used with sample volumes of 150 mL. The spectra were measured byex-
citation at 250 nm and the emitting fluorescence between 360–660 nm
was recorded. The relative fluorescence units (RFU) values at 390 and
460 nm were used for the kinetic analysis of the release of 6-aminoquino-
line from the dendrimers.
[5] E. W. Meijer, M. H. P. van Genderen, Nature 2003, 426, 128.
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Chem. 2003, 115, 341; Angew. Chem. Int. Ed. 2003, 42, 327.
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1726.
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44, 716.
Acknowledgements
D.S. thanks the Israel Science Foundation, the Israel Ministryof Science
“Tashtiot” program and the Israel Cancer Association for financial sup-
port.
[13] R. J. Amir, D. Shabat, Chem. Commun. 2004, 1614.
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[1] a) D. A. Tomalia, J. M. J. FrØchet J. Polym. Sci. Part A: Polym.
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[2] The analogyof our dendritic molecules to neurons refers onlyto the
similarityof the signal-transduction direction (initiallyconvergent
and then divergent). We do not claim to have synthesized an artifi-
cial neuron. Furthermore, unlike a neuron our dendritic system is
disassembled during the signal transduction.
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115, 4632; Angew. Chem. Int. Ed. 2003, 42, 4494.
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Received: September 3, 2006
Published online: October 31, 2006
Chem. Eur. J. 2007, 13, 812 – 821
ꢀ 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
821