The synthesis and study of fluorescent PAMAM-based dendritic molecules

Article abstract of DOI:10.1016/j.tet.2013.07.054

New water soluble and fluorescent PAMAM-based dendritic molecules based on the naphthalimide derivative 7H-benz[de]benzimidazo[2,1-a]isoquinoline-7-one as the fluorescent unit, have been prepared and their photophysical properties studied in aqueous solution over a wide pH range. The dendrons are all strongly fluorescent through an intramolecular charge transfer (ICT) excited state, but this can be modulated by photoinduced electron transfer (PeT) processes, which increases with higher PAMAM dendron generation.

Full text of DOI:10.1016/j.tet.2013.07.054

Tetrahedron 69 (2013) 8439e8445
Contents lists available at SciVerse ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
The synthesis and study of fluorescent PAMAM-based dendritic
molecules
,
,
Alaa M.M. El-Betany a b, Lenka Vachova a c, C. Grazia Bezzu a, Simon J.A. Pope a,
,
Neil B. McKeown a
*
a School of Chemistry, Cardiff University, PO Box 912, Cardiff CF10 3TB, UK
b Chemistry Department, Faculty of Science, Damietta University, New Damietta City 34517, Egypt
c Department of Pharmaceutical Chemistry and Drug Control, Faculty of Pharmacy in Hradec Kralove, Charles University in Prague, Heyrovskeho 1203,
Hradec Kralove 50005, Czech Republic
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 26 April 2013
Received in revised form 27 June 2013
Accepted 15 July 2013
Available online 20 July 2013
New water soluble and fluorescent PAMAM-based dendritic molecules based on the naphthalimide
derivative 7H-benz[de]benzimidazo[2,1-a]isoquinoline-7-one as the fluorescent unit, have been pre-
pared and their photophysical properties studied in aqueous solution over a wide pH range. The den-
drons are all strongly fluorescent through an intramolecular charge transfer (ICT) excited state, but this
can be modulated by photoinduced electron transfer (PeT) processes, which increases with higher PA-
MAM dendron generation.
Keywords:
PAMAM dendron
Ó 2013 Elsevier Ltd. All rights reserved.
Water soluble fluorescent dendritic
Photoinduced electron transfer
Fluorescence lifetime
Fluorescent pH sensor
1. Introduction
the focal point of a dendritic molecule (dendron). The use of a single
dendron substituent also allows the aromatic fluorophore to interact
There is continuing interest in water-soluble fluorescent den-
drimers for biological applications including visualization and di-
agnostic imaging.1e6 Fluorescent labels are required to have high
sensitivity, appropriate excitation and emission maxima, and specific
functionality. Therefore, it is important to develop new fluorescent
labels, which have high detection limits and appropriate fluorescent
behaviour.7 Self-quenching is a problem in aqueous media due to
aggregation of the hydrophobic fluorophore, hence, it has been
found beneficial to use bulky water-soluble dendritic structures to
hinder self-association.6 Fluorescent units can be incorporated
within a dendrimer as peripheral groups, as branch points or as the
core and it is clear that the resulting location of the fluorophore in-
fluences its properties. Importantly, the resulting position of the
fluorescent unit has a major role in directing the method and com-
plexity of the synthesis of the dendrimer.6 The loading of fluo-
rophores onto the periphery of dendrimers is still not trivial, with
problems related to stoichiometric control of synthesis,8,9 and
intramolecular fluorescent quenching. These disadvantages may be
avoided by loading the fluorophore as the core of a dendrimer or at
freely with biological systems (e.g., bind to DNA).
As well as being the first high-generation dendrimers and one of
the most studied,10 poly(amidoamine) (PAMAM) dendrimers are
also considered one of the best potential drug delivery carriers due
to their high biocompatibility. PAMAM dendrimers are very hy-
drophilic, can mimic globular proteins,10 are readily transported
across the epithelial barrier of the gut,11e13 inhibit proteineprotein
binding,14 and can be synthetically modified to possess fluorescent
units at their core or peripheries. While in general PAMAM den-
drimers are of biomedical interest, it is the anionic carboxylate-
terminated PAMAM dendrimers, which are considered to be of
greater potential as drug delivery vehicles because they have
the important advantage of being non-cytotoxic in contrast to
the cationic dendrimers,11,15 whilst also being highly aqueous sol-
uble and effective in transcellular transport and oral delivery
applications.15
The objective of this work was the synthesis and study of the
photophysical properties of novel PAMAM dendritic molecules that
contain
a single highly fluorescent unit at the focal point.
Carboxylate-terminated PAMAM dendrons were selected to enhance
solubility in water and, ultimately, biocompatibility.16,17 Because of
their strong fluorescence and good photostability, derivatives of 1,8-
naphthalimide are used as fluorescent markers in biology,18 light
* Corresponding author. Tel.: þ44 (0)2920875851; fax: þ44 (0)2920874030;
e-mail address: mckeownnb@cardiff.ac.uk (N.B. McKeown).
0040-4020/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tet.2013.07.054

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A.M.M. El-Betany et al. / Tetrahedron 69 (2013) 8439e8445
emitting diodes,19e21 photoinduced electron transfer sensors,22e26
fluorescent switches27e29 and photo-chemotherapeutic inhib-
itors.30e34 A number of 1,8-naphthalimide derivatives were screened
for fluorescence and 7H-benz[de]benzimidazo[2,1-a]isoquinoline-7-
one (BBIQ) selected for use as the fluorophore due to its strong
fluorescence.7,16,35e40
phenylenediamine and 4-bromo-1,8-naphthalenedicarboxylic an-
hydride using a literature procedure,7,36 allows facile derivatisation
of the BBIQ core by the replacement of the bromide via aromatic
nucleophilic substitution. Hence, the introduction of the primary
aliphatic amine functionality required for growth of the desired
PAMAM dendrons was achieved by the reaction between 1 and
ethylenediamine to give 3-(2-aminoethylamino)-7H-benz[de]ben-
zimidazo[2,1-a]isoquinoline-7-one 2 (Scheme 2). Two different
procedures were used (Methods A & B) both of which gave modest
but adequate yields.
2. Results and discussion
2.1. Synthesis
PAMAM synthesis, following a well-established method,16,37
was instigated from 2 with exclusive Michael addition of methyl
acrylate occurring at the aliphatic primary amine to yield 3
The precursor 3-bromo-7H-benz[de]benzimidazo[2,1-a]iso-
quinoline-7-one 1, which was readily prepared from o-
Scheme 1. Synthesis of 3e10. Reagents and conditions: (i) Methyl acrylate, MeOH, reflux 6 h, stirring 16 h, rt; (ii) MeOH/DCM, EDA, stirring 2 days/N2; (iii) Methyl acrylate, MeOH,
reflux 24 h; (iv) MeOH/DCM, EDA, stirring 5 days/N2; (v) Methyl acrylate, MeOH, reflux 2 days; (vi) Ethanolic NaOH, EtOH, (8) reflux 16 h; (9) and (10) stirring at rt for 24 h.

A.M.M. El-Betany et al. / Tetrahedron 69 (2013) 8439e8445
8441
Scheme 2. Synthesis of 1 and 2. Reagents and conditions: (i) AcOH, reflux 2 h; (A)
MeCN, EDA, reflux 3 days; (B) EDA, heating 130 ꢀC for 1 h.
(Scheme 1). Sequential amidations with ethylenediamine and Mi-
chael additions with methyl acrylate yielded dendritic molecules
4e7. Alkaline hydrolysis of oligoesters 3, 5 and 7, by stirring
with ethanolic NaOH at room temperature gave carboxylate ter-
minated dendritic molecules 8e10, respectively, in excellent yields
(93e97%).
The dendritic molecules terminated with methyl esters were
easily purified in good yield using column chromatography (eluent
DCM/MeOH) (3¼77%, 5¼70%, 7¼67%). The amine terminated den-
dritic molecules (4, 6) proved more difficult to purify but were
obtained sufficiently pure for further reaction by azeotropic re-
moval of excess ethylenediamine (bp¼118 ꢀC) using a toluene/
methanol mixture (9:1) followed by flash column chromatography
using a mixture of DCM and MeOH/NH4OH as eluent. The carbox-
ylate terminated dendritic molecules 8e10 were purified by simple
recrystallization from ethanol. The structures of 2e10 were verified
by 1H NMR, 13C NMR and FTIR spectroscopy and electron ionization
(EI) or matrix assisted laser desorption ionization (MALDI-TOF)
mass spectrometry with all results agreeing with the proposed
structures.37
Fig. 1. UV/vis adsorption (top) and fluorescence spectra (bottom) of compounds 2e10
in methanol solution. See Table 1 for details.
The integrated emission intensities of compounds 2e10 are
clearly influenced by the dendritic substituent at the C-3 position of
the fluorophore. The emission lifetimes are all short (<10 ns) and
characteristic of fluorescence in all cases. For the higher generation
dendrimers the lifetime decay profiles do not fit satisfactorily to
a single exponential decay and in these cases bi-exponential fits
proved more appropriate (the relative weightings of each compo-
nent are given in Table 1). The observation of dual component
decay characteristics suggests that there are two distinct excited
state environments for these higher generation species. Together
with the observed trend in integrated intensity of fluorescence
(2>>3w4>5>6>10), this effect is likely to be due to excited state
quenching originating from photoinduced electron transfer (PeT)
involving the multiple amine donors on the PAMAM dendrons,38
with the tertiary amines being particularly efficient at PeT.39e41
2.2. Photophysical characterization
For compounds 2e10, the lmax from the absorption spectra to-
gether with the emission wavelength and lifetimes of the lumi-
nescence bands are presented in Table 1. The lowest energy
absorption band (Fig. 1) associated with the chromophore is likely
to have significant intramolecular charge transfer (ICT) character
and are all in the range 500e511 nm.
Table 1
UV/vis and fluorescence data for selected compounds
Compound
Generation number
lmax absorptiona/nm
3
/Mꢁ1 cmꢁ1
lmax emissionb/nm
s
/nsc
2
3
4
5
6
8
9
10
0
0.5
1
1.5
2
0.5
1.5
2.5
445
458
457
445
450
446
455
457
3.5ꢂ104
5.8ꢂ104
4.1ꢂ104
5.7ꢂ104
2.3ꢂ104
4.3ꢂ104
4.3ꢂ104
3.2ꢂ104
504
508
507
511
509
500
506
507
6.2
2.0
1.3
0.7, 2.7 (77%)d
1.1, 3.8 (58%)d
2.0, 5.3 (83%)d
1.4, 5.2 (73%)d
1.3, 4.8 (72%)d
a
From MeOH solution.
b
(2e10) excitation at 455 nm (MeOH).
(2e10) excitation at 459 nm (MeOH).
Relative weighting of longer lifetime component.
c
d

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A.M.M. El-Betany et al. / Tetrahedron 69 (2013) 8439e8445
However, the retention of long lifetime components for 5e10 also
suggests that a proportion of their excited states are relatively un-
perturbed and this may be due to conformational rigidity on the
timescale of the lifetime experiments. Fluorescence intensity
was also found to decrease in the carboxylate salts 8>9>10 due
to the dendritic substituents (Fig. 1).42 It was found that the
emission intensity increased almost linearly with concentration for
aqueous solutions of 8, 9 and 10 indicating that there was no
self-quenching due to aggregation over the concentration range
3. Conclusions
A series of dendritic molecules that combine the highly fluo-
rescent 7H-benz[de]benzimidazo[2,1-a]isoquinoline-7-one unit
with small PAMAM side-groups were prepared. From a photo-
physical perspective, each of the compounds is visibly fluorescent
in solution (including aqueous media) through an ICT excited state.
The emission intensities and lifetimes are sensitive to the nature
and generation of the dendritic architecture and this is ascribed to
the well-understood PeT intramolecular quenching mechanism
involving the amine groups. The pH dependence of the fluores-
cence behaviour is consistent with this mechanism. Lifetime mea-
surements indicate that the PeT mechanism is likely to be
conformationally dependent. Overall, the photophysical behaviour
of these systems is roughly similar to that of the analogous 1,8-
naphthalimide systems although the fluorescence for the later oc-
curs at a significantly longer wavelength (w550 nm).17
0.83e5.75ꢂ10ꢁ4 mM Lꢁ1
.
The UV/vis absorption spectra of compounds 2, 4 and 8 revealed
similar pH dependence with a bathochromic shift of the main band
below pH 5, presumably due to the protonation of the chromo-
phore.36 The pH dependence of the fluorescence spectra of the
molecules 2, 4 and 8 was also investigated over the pH range 1e13.
For 2 the emission intensity is quenched at very low pH, but char-
acterized by two maxima at 490 and 560 nm; increasing the pH 7e8
sees a single maximum intensity at 510 nm. The emission lifetimes
at these pH values are relatively insensitive and all lie in the range
5.1e6.2 ns (Table 2). In contrast, 4 revealed two emissionpeaks at pH
1e4, an emission maximum intensity at pH 4, and a single emission
peak at pH 6e12. The emission lifetimes at pH 1e4 were signifi-
cantly extended (5.2e5.6 ns) and consistent with reduced
quenching and suppression of PeT; at pH >6 the lifetime decays are
dual component with very short (<0.5 ns) and short (<3 ns) con-
tributions (Table 2), suggesting that excited state quenching is en-
hanced at such pH regimes and dependent upon conformational
freedom. The behaviour of 8, shown in Fig. 2, shows a maximum
intensity of fluorescence at around pH¼6e8 at 500 nm; at very low
pH (<2) the emission is red-shifted to 560 and at very high pH
emission is weak because the PeT is operative due to the lack of
protonation of the amine groups within the dendritic substituent.
4. Experimental
4.1. Photoluminescence measurements
Fluorescence spectra: Photophysical data were obtained on
a JobinYvoneHoriba Fluorolog spectrometer fitted with a JY TBX
picoseconds photodetection module as MeOH or aqueous solutions
where appropriate. Emission spectra were uncorrected and exci-
tation spectra were instrument corrected. The pulsed source
was a Nano-LED configured for 372 nm output operating at
1 MHz. Luminescence lifetime profiles were obtained using the
JobinYvoneHoriba FluoroHub single photon counting module and
the data fits yielded the lifetime values using the provided DAS6
deconvolution software.
4.2. Materials and methods
Table 2
Emission lifetimes (relative weightings in parentheses) of compounds 2, 4 and 8 as
a function of solution pH
Precursors were purchased from SigmaeAldrich or Acros Or-
ganics/Fisher Scientific and were used without further purification.
Anhydrous dichloromethane was obtained by distillation over
calcium hydride under a nitrogen atmosphere. All reactions in-
volving air/moisture sensitive reagents were performed in oven-
dried glassware, under a nitrogen atmosphere. TLC analysis refers
to analytical thin layer chromatography, using aluminium-backed
plates coated with Merck Kieselgel 60 GF254. Flash chromatography
Compound 2
Compound 4
Compound 8
pH
s
ns
pH
sns
pH
s ns
1
3
5
7
8
10
12
5.29
5.39
5.68
6.24
6.20
5.15
3.50
1
2
5.17
5.39
1
2
4.68
4.74
4
5.60
3
5.11
6
8
10
12
1.03, 3.27 (69%)
0.45, 2.36 (80%)
0.20, 2.21 (82%)
0.38, 1.96 (82%)
4
6.32
5
6.24
6
6.35
was performed on silica gel 60A (35e70 m) chromatography grade
8
6.30
(Fisher Scientific). Melting points were recorded using a Gallen-
kamp Melting Point Apparatus. Infrared spectra were recorded in
the range 4000e600 cmꢁ1 using a PerkineElmer 1600 series FTIR
instrument either as a thin film or as a Nujol mull between sodium
chloride plates. UV absorption spectra were recorded on a JASCO
(V-570) UV/vis/NIR spectrophotometer. 1H and 13C NMR spectra
were recorded in MeOD or CDCl3 solution (as stated) using an
Avance Bruker DPX 400 instrument (400 MHz) or an Avance Bruker
9
10
11
6.24
6.31
0.27, 5.09 (60%)
DPX 500 (500 MHz). All chemical shifts (
d) are reported in parts per
million with referenced to TMS (d¼0.0). Low-resolution mass
spectrometric data were determined using a Fisons VG Platform II
quadrupole instrument using electrospray (ES) ionization unless
otherwise stated. High-resolution mass spectrometric data were
obtained in electrospray (ES) mode on a Waters Q-TOF micromass
spectrometer. Matrix Assisted Laser Desorption-time of flight Mass
Spectrometry (MALDI-TOF) is available using a Waters Maldi Micro
MX research grade instrument.
4.3. Synthetic details and characterization
4.3.1. 3-Bromo-7H-benz[de]benzimidazo[2,1-a]isoquinoline-7-one
Fig. 2. Emission spectra for compound 8 obtained at pH 0.63e13.26.
(1). The mixture of 4-bromo-1,8-naphthalic anhydride (0.277 g,

A.M.M. El-Betany et al. / Tetrahedron 69 (2013) 8439e8445
8443
0.001 mol) and o-phenylenediamine (0.2 g, 0.002 mol) was dis-
2.62 (t, J¼6.3 Hz, 4H), 2.56e2.49 (m, 2H), 2.30 (t, J¼6.3 Hz, 4H); 13
C
solved in acetic acid (50 mL). The resulting solution was allowed to
reflux for 2 h, the mixture was cooled to room temperature, and
then poured into distilled water to precipitate the product as
a bright yellow powder. Compound 1 (0.18 g, 54%) was obtained
after twice recrystallized from toluene. Rf (20% MeOH/DCM) 0.86;
mp: 253e254 ꢀC (lit. mp: 253e254);36 UV (lmax, nm): 390 (MeOH,
NMR (100 MHz, CDCl3):
d
172.7 (COO), 160.5 (C),150.6 (C),149.8 (C),
143.7 (C), 134.9 (CH), 131.9 (C), 128.3 (C), 126.8 (CH), 124.9 (CH),
124.8 (CH), 124.5 (C), 120.5 (CH), 119.9 (CH), 119.3 (C), 115.9 (CH),
109.4 (CH), 104.3 (CH), 51.5 (Me), 51.4 (CH2), 48.8 (CH2), 40.1 (CH2),
32.4 (CH2); LRMS (ESþ): m/z¼501.21 (MHþ, 100%), 502.22 (33%),
503.22 (6%); HRMS calcd for (C28H28N4O5): 500.2060, found
500.2139 (Mþ, 100%).
3
/L molꢁ1 cmꢁ1 1.2ꢂ104); IR (KBr, cmꢁ1): 1696 (C]O), 746 (CeBr);
1H NMR (500 MHz, CDCl3):
d
8.86 (dd, J¼17.4, 7.3 Hz, 1H), 8.65 (dd,
J¼11.3, 8.2 Hz, 1H), 8.61e8.48 (m, 2H), 8.10 (dd, J¼10.5, 7.9 Hz, 1H),
4.3.5. First generation PAMAM dendron (4). A solution of 3 (0.5 g,
0.001 mol) in (MeOH/DCM; 5:15 mL) was added dropwise over
30 min to a cooled (0 ꢀC) solution of ethylenediamine (13.38 mL,
0.2 mol) (100 equiv/COOMe) in MeOH (30 mL) and a small amount
of DCM was added until the solution cleared. The reaction was
stirred for 2 days under a nitrogen at room temperature. The bulk
of the solvent and excess ethylenediamine were removed via ro-
tary evaporation. Final traces of excess ethylenediamine were re-
moved azeotropically using a mixture of toluene and MeOH (9:1)
(this was repeated several times until all traces of ethylenediamine
had been removed). The product was dried thoroughly under high
vacuum (1 mmHg) then was purified by flash column chroma-
tography (DCM/MeOH/NH4OH; 9:1:0.2) to obtain (0.32 g, 57%) of
compound 4 as a pink oil. Rf (8:2:0.4 MeOH/DCM/NH4OH) 0.32; UV
7.96e7.85 (m, 2H), 7.54e7.46 (m, 2H); 13C NMR (125 MHz, CDCl3):
d
193.3 (C]O), 144.0 (C), 143.4(C), 141.3 (CH), 138.4 (C), 133.7 (CH),
133.1 (CH), 131.4 (C), 131.2 (C), 130.5 (C), 130.2 (CH), 128.9 (CH),
128.6 (C), 127.5 (CeBr), 127.4 (CH), 123.2 (CH), 115.3 (CH); HRMS
(EIþ): m/z¼347.9909 (M79Brþ, 100%), 349.9903 (M81Brþ, 98%).
4.3.2. 3-(2-Aminoethylamino)-7H-benz[de]benzimidazo[2,1-a]iso-
quinoline-7-one (2). Method A: Compound 1 (0.6099 g, 1.747 mmol)
and ethylenediamine (1.2 mL,18.02 mmol) were stirred in refluxing
MeCN (10 mL) for 3 days. The orange precipitate was collected by
hot filtration and stirred in ether for 16 h, then filtered and dried.
The orange solid was recrystallized from toluene to give (0.084 g,
14%) a pale orange powder 2 after flash column chromatography
(DCM/MeOH; 10:0 to 8:2).
(
lmax, nm): 445 (MeOH,
3
/L molꢁ1 cmꢁ1 4.1ꢂ104); IR (Neat, cmꢁ1):
Method B: Compound 1 (0.6022 g, 0.002 mol) was suspended in
ethylenediamine (6.68 mL, 0.1 mol) and the resulting suspension
was heated to 130 ꢀC for 1 h, the mixture was cooled to 95 ꢀC and
poured into distilled water at 90 ꢀC. After stirring and cooling to
room temperature, the deep red suspension was filtered and
washed with water. The solid was dried and recrystallized from
toluene twice to give (0.12 g, 19%) as deep red crystals 2 after flash
column chromatography (DCM/MeOH; 10:0 to 8:2). Rf (20% MeOH/
3284 (eNH2), 3075 (AreH), 2942 (eCH2e), 1675 (NeC]O), 1572
(NHeC]O); 1H NMR (500 MHz, MeOD):
d
8.08 (d, J¼7.8 Hz, 1H),
7.99 (d, J¼7.3 Hz, 1H), 7.82 (d, J¼8.3 Hz, 1H), 7.72 (d, J¼8.5 Hz, 1H),
7.53 (d, J¼7.7 Hz, 1H), 7.30 (dd, J¼11.0, 4.1 Hz, 1H), 7.24 (dd, J¼11.0,
4.1 Hz, 1H), 7.13 (t, J¼7.8 Hz, 1H), 6.07 (d, J¼8.8 Hz, 1H), 3.34e3.30
(m, 2H), 3.28 (t, J¼6.1 Hz, 4H), 3.08 (s, 2H), 2.81e2.73 (m, 6H), 2.70
(t, J¼6.2 Hz, 2H), 2.46 (t, J¼6.7 Hz, 4H); 13C NMR (125 MHz, MeOD):
d
173.6 (CONH), 159.5 (C), 150.9 (C), 149.0 (C), 142.5 (C), 134.4 (CH),
DCM) 0.26; mp: 212e213 ꢀC; UV
3
(eCH2e), 1677 (C]O); 1H NMR (500 MHz, MeOD):
(
lmax
,
nm): 433 (MeOH,
131.1 (C), 127.6 (C), 126.3 (CH), 125.1 (CH), 124.6 (CH), 123.8 (C),
123.7 (CH), 119.8 (CH), 118.2 (C), 115.4 (CH), 107.6 (CH), 103.6 (CH),
49.3 (CH2), 48.5 (CH2), 43.3 (CH2), 41.6 (CH2), 40.6 (CH2), 33.3
(CH2); HRMS calcd for (C30H36N8O3): 556.2983, found 556.2977
(Mþ, 100%).
/L molꢁ1 cmꢁ1 3.5ꢂ104); IR (KBr, cmꢁ1): 3530 (eNH2), 2900
d 8.82 (d,
J¼7.3 Hz, 1H), 8.61 (dd, J¼12.4, 7.9 Hz, 2H), 8.05 (d, J¼8.4 Hz, 1H),
7.90 (d, J¼6.3 Hz, 1H), 7.66 (t, J¼7.9 Hz, 1H), 7.50e7.45 (m, 2H), 6.71
(d, J¼8.5 Hz, 1H), 6.32 (s, 1H), 3.38 (dd, J¼10.7, 5.1 Hz, 2H), 3.17 (t,
J¼5.7 Hz, 2H), 1.29 (s, 2H); 13C NMR (125 MHz, MeOD):
d
142.5 (C),
4.3.6. PAMAM dendrondG1.5 [5]. Methyl acrylate (13.5 mL,
0.15 mol) was added to a solution of 4 (0.556 g, 0.001 mol) in MeOH
(30 mL) and the mixture was heated to reflux for 24 h, then stirred
for 16 h at room temperature. MeOH and excess of methyl acrylate
were eliminated by heating under reduced pressure to yield the 5
as a deep orange oil (0.63 g, 70%) after purification by flash column
chromatography (DCM/MeOH; 10:0e10:0.3). Rf (3% MeOH/DCM)
141.4 (C), 141.1 (C), 135.2 (C), 133.7 (CH), 130.6 (C), 128.4 (C), 127.6
(CH), 124.5 (CH), 124.0 (CH), 123.9 (C), 123.1 (CH), 122.0 (CH), 121.8
(C), 116.1 (CH), 111.9 (CH), 45.7 (CH2), 39.5 (CH2); HRMS (ESþ): m/
z¼329.1401 (MHþ, 100%).
4.3.3. General procedure for the synthesis of polyesters derivatives (3,
5, 7). Methyl acrylate (75 equiv/NH2 function) was added to a so-
lution of conjugate with amine terminal groups and the mixture
was heated to reflux. MeOH and the excess of methyl acrylate were
eliminated to yield the polyester after chromatography using silica
gel as substrate and a mixture of CHCl3 and MeOH (10:0.1) as
eluent.
0.34; UV (lmax, nm): 445 (MeOH,
/L molꢁ1 cmꢁ1 5.7ꢂ104); IR (KBr,
1729 (eCOOCH3), 1674 (NeC]O), 1589 (NHeC]O); 1H NMR
(400 MHz, MeOD):
d
8.16 (d, J¼7.5 Hz, 1H), 8.13 (d, J¼7.5 Hz, 1H),
7.93 (d, J¼0.54 Hz, 1H), 7.86 (d, J¼8.5 Hz, 1H), 7.49 (d, J¼8.5 Hz, 1H),
7.26e7.16 (m, 3H), 6.30 (d, J¼8.8 Hz, 1H), 3.40 (s, 12H), 3.03 (t,
J¼6.1 Hz, 2H), 2.98 (t, J¼6.4 Hz, 4H), 2.67 (t, J¼6.6 Hz, 4H), 2.57 (t,
J¼5.7 Hz, 2H), 2.43 (t, J¼6.7 Hz, 8H), 2.27e2.19 (m, 8H), 2.14 (t,
4.3.4. PAMAM dendrondG0.5 (3). Methyl acrylate (6.75 mL,
0.075 mol) was added to a solution of 2 (0.328 g, 0.001 mol) in
MeOH (40 mL) and the mixture was heated to reflux for 6 h, then
stirred for 16 h at room temperature, filtered, washing with
MeOH and dried. The orange solid was recrystallized from MeOH to
give (0.084 g, 77%) a bright orange solid 3 after flash column
chromatography (DCM/MeOH; 10:0 to 10:0.2). Rf (3% MeOH/DCM)
J¼6.6 Hz, 8H); 13C NMR (100 MHz, MeOD):
d 173.2 (CONH), 173.1
(COO), 159.9 (C), 151.3 (C), 149.4 (C), 142.7 (C), 134.8 (CH), 131.3 (C),
128.0 (C), 126.6 (CH), 125.6 (CH), 124.8 (CH), 124.0 (C), 120.2 (CH),
118.6 (CH), 118.3 (C), 115.5 (CH), 107.9 (CH), 104.0 (CH), 52.2 (CH2),
51.0 (CH2), 50.7 (Me), 49.3 (CH2), 48.9 (CH2), 40.9 (CH2), 37.0 (CH2),
33.3 (CH2), 32.0 (CH2); HRMS (EIþ) calcd for (C46H60N8O11):
900.4454, found 900.4453 (Mþ, 100%).
0.70; mp: 170e171 ꢀC; UV (lmax, nm): 445 (MeOH, /L molꢁ1 cmꢁ1
3
5.8ꢂ104); IR (KBr, cmꢁ1): 3413 (eNH), 3058 (AreH), 2950 (eCH2e),
2848 (eOCH3), 1734 (eCOOCH3), 1681 (NeC]O), 1581 (NHeC]O);
4.3.7. Second generation PAMAM dendron (6). A solution of 5 (0.9 g,
0.001 mol) in MeOH (30 mL) was added dropwise over 30 min to
a cooled (0 ꢀC) solution of ethylenediamine (26.76 mL, 0.4 mol)
(100 equiv/COOMe) in MeOH (70 mL). The reaction was stirred for 5
days under nitrogen at room temperature. The bulk of the solvent
1H NMR (400 MHz, CDCl3):
d
8.55 (d, J¼7.3 Hz, 1H), 8.47 (d,
J¼7.2 Hz, 1H), 8.24 (d, J¼8.5 Hz, 1H), 7.94 (d, J¼8.4 Hz, 1H), 7.74 (d,
J¼8.4 Hz, 1H), 7.38 (t, J¼7.9 Hz, 1H), 7.34e7.30 (m, 2H), 6.29 (d,
J¼8.6 Hz, 1H), 6.08 (s, 1H), 3.40 (s, 6H), 3.08e3.01 (m, J¼5.3 Hz, 2H),

8444
A.M.M. El-Betany et al. / Tetrahedron 69 (2013) 8439e8445
and excess ethylenediamine were removed via rotary evaporator.
Final traces of excess ethylenediame were removed azeotropically
using a mixture of toluene and MeOH (9:1) (this was repeated
several times until all traces of ethylenediamine had been re-
moved). The product was dried thoroughly under high vacuum
(1 mmHg) then was purified by flash column chromatography
(DCM/MeOH/NH4OH; 9:1:0.2) to obtain (0.57 g, 56%) of polyamine
6 as a red oil. Rf (8:2:0.4 MeOH/DCM/NH4OH) 0.08; UV (lmax, nm):
(CH2); LRMS (ESþ): m/z¼473.18 (MHþ, 100%); HRMS calcd for
(C26H24N4O5): 472.1872, found 473.1840 (MHþ, 100%).
4.3.10. PAMAM dendrondG1.5 salt (9). To NaOH (1.0 g, 25 mmol),
dissolved in absolute EtOH (100 mL), was added a solution of 5
(2.23 g, 2.4 mmol) in EtOH (10 mL). The mixture was stirred for 24 h
at room temperature, and the precipitate was collected by filtration
and recrystallized from wet EtOH to yield 9 as a deep orange solid
(2.17 g, 97%). Rf (40% MeOH/H2O) 0.46; mp: 283 ꢀC; UV (lmax, nm):
433 (MeOH,
3
/L molꢁ1 cmꢁ1 2.3ꢂ104); IR (Neat, cmꢁ1): 3262
(eNH2), 3075 (AreH), 2929 (eCH2e), 1675 (NeC]O), 1557
445 (MeOH,
3
/L molꢁ1 cmꢁ1 4.3ꢂ104); IR (KBr, cmꢁ1): 3362
(NHeC]O); 1H NMR (500 MHz, MeOD):
d
8.22 (d, J¼6.7 Hz, 1H),
(eNHe), 3079 (AreH), 2968 (eCH2e), 1655 (eCOONa), 1681
8.12 (d, J¼6.6 Hz, 1H), 7.92 (d, J¼7.6 Hz, 1H), 7.84 (d, J¼7.0 Hz, 1H),
7.60 (d, J¼7.0 Hz, 1H), 7.37e7.32 (m, 1H), 7.32e7.28 (m, 1H),
7.23e7.17 (m, 1H), 6.13 (d, J¼7.1 Hz, 1H), 3.27e3.19 (m, J¼5.9 Hz,
12H), 3.13e3.03 (m, 2H), 2.81 (t, J¼29.7 Hz, 4H), 2.71 (t, J¼6.2 Hz,
(NeC]O), 1571 (NHeC]O); 1H NMR (400 MHz, MeOD):
d 8.48 (d,
J¼7.3 Hz, 1H), 8.41 (d, J¼7.2 Hz, 1H), 8.29 (d, J¼3.5 Hz, 1H), 8.26 (d,
J¼8.7 Hz, 1H), 7.71 (d, J¼6.8 Hz, 1H), 7.59e7.48 (m, 1H), 7.48e7.33
(m, 2H), 6.56 (d, J¼8.8 Hz, 1H), 3.49e3.36 (m, 2H), 3.36e3.29 (m,
4H), 3.03e2.91 (m, 4H), 2.89e2.78 (m, 8H), 2.62 (t, J¼5.9 Hz, 4H),
2.51 (t, J¼6.1 Hz, 4H), 2.43e2.23 (m, 10H); 13C NMR (100 MHz,
18H), 2.53e2.46 (m, 4H), 2.44e2.38 (m, 4H), 2.32e2.24 (m, 8H); 13
C
NMR (125 MHz, MeOD):
d 173.5 (CONH), 173.0 (CONH), 164.4 (C),
159.6 (C), 151.1 (C), 149.2 (C), 142.8 (CH), 134.6 (C), 131.3 (C), 127.8
(CH), 126.5 (CH), 126.4 (CH), 124.7 (C), 124.0 (CH), 120.0 (CH), 118.4
(C), 115.5 (CH), 107.8 (CH), 103.8 (CH), 52.1 (CH2), 49.7 (CH2), 49.4
(CH2), 48.6 (CH2), 43.6 (CH2), 41.7 (CH2), 40.8 (CH2), 37.3 (CH2), 33.4
(CH2); MALDI-TOF [LDþ] calcd for (C50H76N16O7): 1013.6170, found
1013.674 (Mþ, 100%).
MeOD): d 181.4 (COO), 174.7 (CONH), 162.7 (C), 153.2 (C), 150.9 (C),
144.1 (C), 142.3 (CH), 137.6 (C), 132.7 (C), 129.7 (CH), 127.3 (CH),
126.3 (CH), 125.7 (C), 123.4 (CH), 120.1 (CH), 119.7 (C), 117.0 (CH),
108.9 (CH),105.5 (CH), 58.3 (CH2), 53.3 (CH2), 52.4 (CH2), 50.8 (CH2),
41.6 (CH2), 38.5 (CH2), 36.4 (CH2), 34.6 (CH2); MALDI-TOF [LDþ]
calcd for (C42H52N8O11): 845.3756, found 845.2874 (Mþ, 100%).
4.3.8. PAMAM dendrondG2.5 (7). Methyl acrylate (27 mL,
0.30 mol) was added to a solution of 6 (1.01 g, 0.001 mol) in MeOH
(30 mL) and the mixture was heated to reflux for 2 days. MeOH and
excess of methyl acrylate were eliminated by heating under re-
duced pressure to yield the 7 as a deep orange oil (1.3 g, 76%) after
flash column chromatography (DCM/MeOH; 10:0 to 10:0.3). Rf (3%
4.3.11. PAMAM dendrondG2.5 salt (10). To NaOH (3.0 g, 75 mmol),
dissolved in absolute EtOH (150 mL), was added a solution of 7
(4.23 g, 2.4 mmol) in EtOH (10 mL). The mixture was stirred for 24 h
at room temperature and the precipitate was collected by filtration
and was recrystallized from EtOH to yield 10 as a deep orange solid
(3.9 g, 93%). Rf (40% MeOH/H2O) 0.30; mp: 269 ꢀC; UV (lmax, nm):
MeOH/DCM) 0.04; UV (lmax, nm): 445 (MeOH,
3
/L molꢁ1 cmꢁ1
445 (MeOH,
3
/L molꢁ1 cmꢁ1 3.2ꢂ104); IR (KBr, cmꢁ1): 3412 (eNHe),
1.6ꢂ104); IR (KBr, cmꢁ1): 3422 (eNH), 3043 (AreH), 2924 (eCH2e),
3091 (AreH), 2956 (eCH2e), 1655 (eCOONa), 1681 (NeC]O), 1571
2850 (eOCH3), 1736 (eCOOCH3), 1681 (NeC]O), 1581 (NHeC]O);
(NHeC]O); 1H NMR (500 MHz, MeOD):
d
8.52 (d, J¼7.0 Hz, 1H),
1H NMR (400 MHz, MeOD):
d
8.46 (d, J¼7.3 Hz, 1H), 8.41 (d,
8.44 (d, J¼7.5 Hz, 1H), 8.36e8.31 (m, 2H), 7.72 (d, J¼7.4 Hz, 1H),
7.59e7.53 (m, 1H), 7.45e7.40 (m, 2H), 6.60 (d, J¼8.7 Hz, 1H),
3.30e3.25 (m, 14H), 2.83e2.77 (m, 28H), 2.58e2.53 (m, 14H),
J¼7.2 Hz, 1H), 8.23 (dd, J¼13.7, 8.5 Hz, 2H), 7.73 (d, J¼7.3 Hz, 1H),
7.53e7.37 (m, 3H), 6.51 (d, J¼8.8 Hz, 1H), 3.62 (s, 24H), 3.27e3.22
(m, 4H), 3.22e3.18 (m, 8H), 2.93e2.87 (m, 4H), 2.86e2.81 (m, 2H),
2.74 (t, J¼6.8 Hz, 8H), 2.69 (t, J¼6.6 Hz, 16H), 2.56e2.51 (m, 4H),
2.51e2.43 (m, 14H), 2.39 (t, J¼6.6 Hz, 16H), 2.31 (t, J¼6.8 Hz, 8H);
2.34e2.30 (m, 28H); 13C NMR (125 MHz, MeOD):
d 181.4 (COO),
174.7 (CONH), 174.6 (CONH), 161.9 (C), 153.4 (C), 151.1 (C), 144.3 (C),
142.5 (CH), 136.9 (C), 132.9 (C), 128.7 (CH), 127.6 (CH), 126.4 (CH),
125.6 (C), 122.2 (CH), 120.4 (CH), 119.9 (C), 117.0 (CH), 109.4 (CH),
105.8 (CH), 58.3 (CH2), 53.3 (CH2), 52.0 (CH2), 50.9 (CH2), 42.2 (CH2),
38.5 (CH2), 38.4 (CH2), 36.5 (CH2), 34.8 (CH2), 34.5 (CH2); MALDI-
TOF [LDþ] calcd for (C74H108N16O23): 1588.7773, found 1588.27
(Mþ, 100%).
13C NMR (100 MHz, MeOD):
d 173.2 (CONH), 173.1 (CONH), 173.0
(COO), 160.0 (C), 151.4 (C), 149.5 (C), 142.8 (C), 135.0 (CH), 131.4 (C),
128.3 (C), 126.8 (CH), 125.8 (CH), 124.9 (CH), 124.4 (C), 124.2(CH),
120.4 (CH), 118.9 (C), 115.6 (CH), 108.1 (CH), 104.2 (CH), 52.3 (CH2),
52.0 (CH2), 51.1 (Me), 50.8 (CH2), 49.5 (CH2), 49.0 (CH2), 40.6 (CH2),
37.2 (CH2), 37.0 (CH2), 33.4 (CH2), 33.2 (CH2), 32.1 (CH2), 29.5 (CH2);
MALDI-TOF [LDþ] calcd for (C82H124N16O23): 1701.9554, found
1701.9690 (Mþ, 100%).
Acknowledgements
The authors would like to acknowledge the EPSRC Mass Spec-
trometry Facility (Swansea University) and the Egyptian Cultural
Affairs & Missions Sector (ECAMS) and Cardiff University for fi-
nancial support.
4.3.9. PAMAM dendrondG0.5 salt (8). To NaOH (0.5 g, 12.5 mmol),
dissolved in hot EtOH (40 mL), was added a solution of the 3
(1.2 g, 2.4 mmol) in EtOH (10 mL). The mixture was heated to
reflux for 16 h. After cooling, the precipitate was collected by
filtration and recrystallized from wet EtOH to yield 8 as a pale
yellow solid (0.92 g, 95%). Rf (40% MeOH/H2O) 0.66; mp>300 ꢀC
Supplementary data
decomposed at 315 ꢀC (TGA); UV
(lmax, nm): 445 (MeOH,
These data include the UV/vis adsorption spectra at different pH
of compound 9 and 10; corrected excitation spectra (lem¼500 nm)
for the compounds 1e10 obtained in MeOH and the MALDI-MS
spectrum for compound 7. Supplementary data related to this ar-
ticle can be found at http://dx.doi.org/10.1016/j.tet.2013.07.054.
3
/L molꢁ1 cmꢁ1 4.3ꢂ104); IR (KBr, cmꢁ1): 3380 (eNH), 3067
(AreH), 2956 (eCH2e), 1666 (eCOONa), 1681 (NeC]O), 1571
(NHeC]O); 1H NMR (400 MHz, MeOD):
d
8.47 (dd, J¼7.3, 0.7 Hz,
1H), 8.38 (d, J¼7.3 Hz, 1H), 8.33 (d, J¼8.7 Hz, 1H), 8.28 (d,
J¼8.6 Hz, 1H), 7.69 (d, J¼7.7 Hz, 1H), 7.56 (dd, J¼8.2, 7.6 Hz, 1H),
7.45e7.35 (m, 2H), 6.60 (d, J¼8.8 Hz, 1H), 3.44 (t, J¼6.4 Hz, 2H),
2.98e2.92 (m, 4H), 2.89 (t, J¼6.5 Hz, 2H), 2.46e2.40 (m, 4H); 13C
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