Dyes and Pigments 102 (2014) 35e45

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Dyes and Pigments

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A pH sensitive and selective ratiometric PAMAM wavelength-shifting

bichromophoric system based on PET, FRET and ICT
Nikolai I. Georgiev a, Abdullah M. Asiri b, c, Abdullah H. Qusti b, Khalid A. Alamry b,
Vladimir B. Bojinov a, b, *
a
Department of Organic Synthesis, University of Chemical Technology and Metallurgy, 8 Kliment Ohridsky Str., 1756 Sofia, Bulgaria
b
Chemistry Department, Faculty of Sciences, King Abdulaziz University, P.O. Box 80203, Jeddah 21589, Saudi Arabia
c
Center of Excellence for Advanced Materials Research (CEAMR), King Abdulaziz University, P.O. Box 80203, Jeddah 21589, Saudi Arabia

a r t i c l e i n f o a b s t r a c t

Article history: A fluorescent poly(amidoamine)-based wavelength-shifting bichromophore was synthesized using

Received 30 August 2013 divergent strategy and characterized photophysically. Novel compound was configured as a light-har-
Received in revised form vesting antenna where the systems surface is labelled with yellow-green emitting 4-(N-piperazinyl)-1,8-
30 September 2013
naphthalimide “donor” units capable of absorbing light and efficiently transferring the energy to a focal
Accepted 3 October 2013
Available online 25 October 2013
Rhodamine 6G “acceptor”. The energy transfer was calculated to 91%. Moreover, the 1,8-naphthalimide
periphery of the system was designed on the “fluorophoreespacerereceptor” format able to act as a
molecular fluorescence photoinduced electron transfer based probe. Due to the both effects, photoin-
Keywords:
1,8-Naphthalimide
duced electron transfer in the periphery and pH dependent spirolactam to ring-opening amide equi-
Rhodamine 6G librium of rhodamine core, novel bichromophoric system acts as a selective ratiometric pH fluorescence
PAMAM dendrimer probe. Thus, the distinguishing features of light-harvesting (fluorescence resonance energy transfer)
Selective fluorescence pH probes systems were successfully combined with the properties of classical photoinduced electron transfer and
Light-harvesting FRET-based system ring-opening sensing systems.
Photoinduced electron transfer (PET) Ó 2013 Elsevier Ltd. All rights reserved.

1. Introduction two different dye molecules in which excitation is transferred from

In the past few decades, a great interest has been focused on a
supramolecular fluorescent systems in which fluorescence
switches between “on” and “off” states as these can be modulated,
or tuned, by employing external sources such as ions, molecules,
light, etc. [1,2].
The widespread use of fluorescent sensors was prompted by the
increasing need of fast and reliable sensing of chemical species in
many areas of human activity [3e9]. Different design strategies are
being employed in the development of molecular fluorescent sen-
sors, for instance PET (photoinduced electron transfer) based sensors
[10,11], CT (charge transfer) sensor [12], ET (energy transfer) sensors
[13], ring-opening sensors [14,15]. PET using the “fluorophoree
spacerereceptor” format is the most commonly exploited approach
for the design of molecular fluorescent sensors and switches [1].
Fluorescence resonance energy transfer (FRET) is a distance-
dependent interaction between the electronic excited states of
* Corresponding author. Department of Organic Synthesis, University of Chemical
Technology and Metallurgy, 8 Kliment Ohridsky Str., 1756 Sofia, Bulgaria. Tel.: þ359
2 8163206.
E-mail address: vlbojin@uctm.edu (V.B. Bojinov).
a donor molecule to an acceptor molecule without emission of a
photon [16,17]. Fluorescent properties of the FRET-based multi-
fluorophoric systems are very promising because of the long
communication wavelengths exhibited by these molecules. Long-
wavelength excitation reduces problems of autofluorescence and
scattering during fluorescent sensing within many biological and
industrial matrices [18]. Also it would be possible to fabricate a
ratiometric probe based on the FRET mechanism, in which the ratio
of the fluorescent intensities at two different wavelengths provides
a built-in correction for environmental effects, and stability under
illumination [19]. This method allows precise and quantitative
analysis and imaging even in complicated systems [20].
Molecular systems capable of light-harvesting and efficiently
transferring absorbed radiation unidirectionally over a nanometer
distance are of great interest [21]. The most attractive artificial
light-harvesting systems are the dendritic assemblies because of
their unique structures, reminiscent of the architecture of natural
light-harvesting complexes [21e27]. The globular shape of den-
dritic architectures provides a large surface area that can be deco-
rated with chromophores, resulting a large absorption cross section
and efficient capture of photons. Furthermore, the use of the flex-
ible aliphatic PAMAM bone as a scaffold for light-harvesting

0143-7208/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.dyepig.2013.10.007
36 N.I. Georgiev et al. / Dyes and Pigments 102 (2014) 35e45
antennae could give new systems with high efficiency of energy
transfer [28].
Particularly useful for PAMAM antennae are fluorophores such
as Rhodamine 6G and 1,8-naphthalimide [29e35]. Because of their
excellent fluorescence properties and good photostability, 1,8-
naphthalimide dyes were used extensively in a number of areas,
including chemosensing materials [36e58]. On the basis of the
spirolactam (non-fluorescent) to ring-open amide (fluorescent)
equilibrium of rhodamine, series of rhodamine-based dyes with
excellent “offeon” switching of fluorescence upon encountering
the correct target have been synthesized [20,59e69].
In this paper, we report on the design, synthesis and photo-
physical properties of a novel ratiometric fluorescence “offeon”
light-harvesting antenna based on a core and peripherally func-
tionalized PAMAM dendron (Scheme 1). The decorated with
yellow-green emitting 1,8-naphthalimide units dendron periphery
is capable of absorbing light and efficiently transferring the energy
to a focal Rhodamine 6G. Also, the peripheral 1,8-naphthalimides
were designed on a “fluorophoreespacerereceptor” format thus
providing PET based sensing properties of the novel light-har-
vesting antenna.
2. Experimental
2.1. Materials
Commercially available Rhodamine 6G 1, ethylenediamine, N-
methylpiperazine and methyl acrylate (Aldrich, Merck) were used
without purification. The starting 4-nitro-1,8-naphthalic anhydride
5 was prepared according to the reported procedure [70]. All sol-
vents (Fluka, Merck) were pure for analysis or of spectroscopy
grade. NaOH and HCl were supplied by Merck (Germany).
Ammonia buffer solution (ammonia/ammonium chloride, pH 10,
Aldrich), metal stock solutions (1  104 M) of Zn(NO3)2, Cu(NO3)2,
Ni(NO3)2, Co(NO3)2, Pb(NO3)2, Fe(NO3)3, Cd(NO3)2, AgNO3 and
Hg(NO3)2 in DMF (all Aldrich salts at p.a. grade) and working dye
solutions (1  105 M) were prepared daily.
2.2. Methods
FT-IR spectra were recorded on a Varian Scimitar 1000 spec-
trometer. The 1H NMR spectra (chemical shifts are given as d in
ppm) were recorded on a Bruker DRX-250 spectrometer, operating
at 250.13 MHz. Absorption spectra were recorded on a Hewlett
Packard 8452A spectrophotometer. Fluorescent spectra were
N N
N O O N
N N
O O
NH HN
O O
N
NH
N O
O 3); 7.44 (m, 2H, 9-Ph H-4 and 9-Ph H-5); 7.05 (m, 1H, 9-Ph H-6);
NH
7 N(CH2)3); 1.89 (s, 6H, 2  ArCH3); 1.32 (t, 6H, J ¼ 7.1 Hz, 2  CH2CH3).
Scheme 1. PAMAM light-harvesting dendron 7, core and peripherally functionalized
with Rhodamine 6G and 4-(N-methylpiperazinyl)-1,8-naphthalimides.
recorded on a Scinco FS-2 fluorescence spectrophotometer. The
excitation source was a 150 W Xenon lamp. Excitation and emission
slits width were 5 nm. Fluorescence measurement was carried out
in right angle sample geometry. A 1  1 cm quartz cuvette was used
for the spectroscopic analysis. Relative fluorescence quantum yields
(FF) were measured using Rhodamine 6G (FF ¼ 0.95 in ethanol
[71]) or Coumarin 6 (FF ¼ 0.78 in ethanol [72]) as standards. All
experiments were performed at room temperature. The spectral
data were collected using FluoroMaster Plus 1.3 and further pro-
cessed by OrginPro 6.1 software. A pH meter Metrohm 704 coupled
with combined pH electrode was used for pH measurements. The
commercial standard buffers for pH 2, 7 and 10 (Aldrich) were used
for calibration. TLC was performed on silica gel, Fluka F60 254,
20  20, 0.2 mm. The melting points were determined by means of
a Kofler melting point microscope.
The absorption and fluorescence properties were studied as a
function of pH by multiple additions of NaOH and HCl aqueous
solutions to 400 mL 1  105 M solution of examined compounds in
water/DMF (4:1, v/v). The addition was limited to 1 mL so that
dilution remains insignificant. The solution pH, absorption and
fluorescence spectra were recorded at each addition. The effect of
the metal cations was examined by addition of 0.2 mL of ammonia/
ammonium chloride buffer (pH 10) solution to 2 mL water/DMF
(4:1, v/v) solution of the corresponding fluorophore (1  105 M)
and 20 mL of the metal stock solution (1  102 M).
2.3. Synthesis of Rhodamine 6G core (2)
To a solution of Rhodamine 6G 1 (2.3 g, 4.6 mM) in 90 mL of
absolute ethyl alcohol, 1.8 mL of ethylenediamine (28 mM) was
added dropwise at room temperature. The resulting solution was
stirred at reflux for 5 h. After cooling to room temperature the solid
precipitated was filtered off, washed with water and dried to give
1.9 g 1.9 g (88%) of 2 as pale pink crystals (m.p. >250  C, Rf ¼ 0.45 in
a solvent system chloroform/ethylacetate/ethanol ¼ 1:1:1). FT-IR
(KBr) cm1: 3220 (nNH and nNH2); 2942, 2848 (nCH); 1678 (nC]
O); 1634, 1528 and 1484 (nAr]CH). 1H NMR (CDCl3-d, 250.13 MHz)
ppm: 7.93 (m, 1H, 9-Ph H-3); 7.47 (m, 2H, 9-Ph H-4 and 9-Ph H-5);
7.06 (m, 1H, 9-Ph H-6); 6.34 (s, 2H, Rhodamine H-4 and H-5); 6.22
(s, 2H, Rhodamine H-1 and H-8); 3.51 (br.s, 2H, 2  ArNH); 3.21 (m,
6H, 2  CH2CH3 and CH2NCO); 2.35 (t, 2H, J ¼ 6.8 Hz, CH2NH2); 1.90
(s, 6H, 2  ArCH3); 1.32 (m, 8H, 2  CH2CH3 and NH2). Elemental
analysis: Calculated for C28H32N4O2 (MW 456.58) C 73.66, H 7.06, N
12.27%; Found C 74.19, H 7.08, N 12.36%.
2.4. Synthesis of ester-functionalized Rhodamine 6G (3)
To a suspension of 2 (1.82 g, 4 mM) in 40 mL of cooled to 0  C
methanol, a solution of methyl acrylate (3.6 mL, 40 mM) in 4 mL of
methanol was added dropwise over a period of 30 min. The reac-
tion mixture was allowed to warm slowly to room temperature and
then stirred for 3 days. The final product was obtained after evap-
oration of methyl acrylate and methanol under vacuum as white
crystals (yield e 2.46 g (98%), m.p. 134e136  C, Rf ¼ 0.66 in a solvent
system toluene/ethanol ¼ 2:1). FT-IR (KBr) cm1: 3324 (nNH); 2942,
2896 (nCH); 1728 (nCOOMe); 1670 (nC]O); 1622, 1518 and 1450
(nArCH). 1H NMR (CDCl3-d, 250.13 MHz) ppm: 7.89 (m, 1H, 9-Ph H-
6.35 (s, 2H, Rhodamine H-4 and H-5); 6.20 (s, 2H, Rhodamine H-1
and H-8); 3.59 (s, 6H, 2  OCH3); 3.48 (br.s, 2H, 2  ArNH); 3.21 (q,
4H, J ¼ 7.1 Hz, 2  ArNHCH2); 2.58 (t, 4H, J ¼ 7.2 Hz,
2  CH2COOCH3); 2.22 (t, 2H, J ¼ 7.2 Hz, CH2NCOAr); 2.19 (m, 6H,
Elemental analysis: Calculated for C36H44N4O6 (MW 628.76) C
68.77, H 7.05, N 8.91%; Found C 68.96, H 6.99, N 8.96%.
 N.I. Georgiev et al. / Dyes and Pigments 102 (2014) 35e45
2.5. Synthesis of amino-functional Rhodamine 6G dendron (4)
To a solution of ethylenediamine (12.5 mL, 18 mM) in 10 mL of
cooled to 0  C methanol, a suspension of ester-functionalized
rhodamine 3 (1.88 g, 3 mM) in 15 mL of methanol was added
over a period of 30 min. The resulting mixture was stirred for 7 days
at room temperature. Then the solvent and the ethylenediamine
excess were distilled under vacuum. Final traces of excess ethyl-
enediamine were removed azeotropically using a 9:1 toluene/
methanol (v/v) solution. The final compound was obtained as white
crystals (yield - 1.98 g (96%), m.p. 88e90  C, Rf ¼ 0.27 in a solvent
system n-propanol/ammonium hydroxide ¼ 1:1). FT-IR (KBr) cm1:
3314 and 3204 (nNH and nNH2); 2922, 2878 (nCH); 1644 (nC]O);
1636 and 1490 (nAr]CH). 1H NMR (CDCl3-d, 250.13 MHz) ppm: 7.88
(m, 1H, 9-Ph H-3); 7.47 (m, 2H, 9-Ph H-4 and 9-Ph H-5); 7.34 (t, 2H,
J ¼ 5.7, 2  NHCO); 7.06 (m, 1H, 9-Ph H-6); 6.36 (s, 2H, Rhodamine
H-4 and H-5); 6.16 (s, 2H, Rhodamine H-1 and H-8); 3.64 (m, 4H,
2  CONHCH2CH2NH2); 3.55 (br.s, 2H, 2  ArNH); 3.21 (m, 8H,
2  NH2 and 2  ArNHCH2); 3.11 (m, 2H, CH2NCOAr); 2.73 (m, 4H,
2  CH2NH2); 2.55 (t, 4H, J ¼ 6.1 Hz, 2  CH2CH2CONH); 2.15 (m, 6H,
N(CH2)3); 1.90 (s, 6H, 2  ArCH3); 0.93 (t, 6H, J ¼ 7.2 Hz,
2  CH2CH3). Elemental analysis: Calculated for C38H52N8O4 (MW
684.87) C 66.64, H 7.65, N 16.36%; Found C 66.96, H 7.49, N 16.58%.
2.6. Synthesis of light-harvesting dendron (7)
To a solution of 0.97 g (4 mM) of 4-nitro-1,8-naphthalic anhy-
dride 5 in 40 mL of boiling methanol, a solution of 1.37 (2 mM) of
Rhodamine 6G dendron 4 in 25 mL of methanol was added drop-
wise under stirring over a period of 2 h. The resulting solution was
refluxed for 6 h. After cooling the brown precipitate was filtered off,
treated with 50 mL of 5% aqueous sodium hydroxide to give after
filtration and drying an intermediate 8. Then to the solution of the
obtained intermediate 8 (0.68 g, 0.6 mM) in 12 mL of DMF, N-
methylpiperazine (0.48 g, d ¼ 0.91, 4.8 mM) was added portion wise
at room temperature. After 48 h the resulting solution was poured
into water. The crude product that precipitate was filtered off,
washed with fresh water and dried. Silica gel column chromatog-
raphy (methanol/chloroform ¼ 1:2, v/v) afforded 0.60 g (81%) yel-
low solid of light-harvesting dendron 7 (81%, m.p. 188e190  C,
Rf ¼ 0.54 in a solvent system n-propanol/ammonium
hydroxide ¼ 1:1). FT-IR (KBr): 3384 (nNH); 2966, 2936 (nCH); 1692
(nasNeC]O); 1654 (nsNeC]O). 1H NMR (CDCl3-d, 250.13 MHz)
ppm: 8.30 (d, 2H, J ¼ 7.5 Hz, 2  naphthalimide H-5); 8.22 (d, 2H,
J ¼ 8.6 Hz, 2  naphthalimide H-2); 8.09 (d, 2H, J ¼ 8.4 Hz,
2  naphthalimide H-7); 7.84 (m, 1H, 9-Ph H-6); 7.56 (m, 2H,
2  NHCO); 7.45 (m, 4H, 2  naphthalimide H-6, 9-Ph H-4 and 9-Ph
H-5); 7.01 (m, 3H, 2  naphthalimide H-3 and 9-Ph H-3); 6.34 (s,
2H, Rhodamine H-4 and H-5); 6.14 (s, 2H, Rhodamine H-1 and H-8);
4.17 (t, 4H, J ¼ 6.4 Hz, 2  (CO)2NCH2); 3.56 (m, 4H, 2  CONHCH2);
3.20 (m, 16H, 2  Rhodamine ArNHCH2, 2  Rhodamine ArNHCH2,
CH2NCOAr and 4  piperazine ArNCH2); 2.70 (m, 8H, 4  piperazine
MeNCH2); 2.48 (m, 10H, 2  CH2CH2CONH and 2  piperazine CH3);
2.16 (m, 6H, N(CH2)3); 1.84 (s, 6H, 2  Rhodamine ArCH3); 1.28 (t,
6H, J ¼ 7.0 Hz, 2  Rhodamine CH2CH3). Calculated for C72H80N12O8
(MW 1241.48) C 69.66, H 6.50, N 13.54%; Found C 70.01, H 6.67, N
13.78%.
3. Results and discussion
3.1. Design and synthesis of light-harvesting antenna 7
The desired PAMAM dendron 7 was configured as a FRET-based
light-harvesting antenna, core and peripherally decorated with a
rhodamine acceptor and 1,8-naphthalimide donors, respectively.
37
The choice of 1,8-naphthalimide for the fluorescence modification
of PAMAM periphery was prompted by its chemical stability and
high fluorescence efficiency [73]. On the basis of the spirolactam
(non-fluorescent) to ring-open amide (fluorescent) equilibrium of
rhodamine, rhodamine-based dyes are excellent “offeon” fluores-
cence sensing molecules [59e68]. A requirement for efficient en-
ergy transfer is that there should be a spectral overlap between the
emission of the peripheral donor dyes and the absorbance of the
focal acceptor chromophore. It is well-known that rhodamines are
orange-red emitting fluorophores with maximal absorption in the
visible region at about lA ¼ 520e530 nm, where the emission of 4-
alkylamino-1,8-naphthalimides is appeared [30,31]. Consistent
with this requirement, 4-alkylamino-1,8-naphthalimides and
rhodamine dyes are suitable fluorescence donor-acceptor pair for
construction of wavelength-shifting bichromophoric systems
[19,61,66].
In order to impart “offeon” properties to the novel light-har-
vesting dendron, yellow-green emitting 4-(N-methylpiperazinyl)-
1,8-naphthalimides were used as peripheral donors. The 4-(N-
methylpiperazinyl)-1,8-naphthalimides are well known “offeon”
fluorescent probes based on “fluorophoreespacerereceptor”
format, which fluorescence is quenched and can be switched “on”
by coupling with the analyte (protons or different metal ions)
[73,74]. In this particular case, it could be predicted that a PET
process (an electron transfer from the receptor to the excited state
of the peripheral fluorophore) would quench fluorescence of the
peripheral 1,8-naphthalimide unit. Because of the quenched fluo-
rescence of the periphery the energy transfer in light-harvesting
dendron 7 is disabling and this would represent the “off-state” of
the system. Protonation of the N-methylpiperazine receptor would
increase its oxidation potential, and as such, thermodynamically
disallow the electron transfer to the peripheral 1,8-naphthalimides.
Consequently the energy transfer in light-harvesting dendron 7
would be “switched on” (Scheme 2).
Due to the “offeon” sensing properties of yellow-green emitting
periphery and pH sensitive character of Rhodamine 6G acceptor
dye, we expect the fluorescence signal of the novel system to be
function of pH. At pH >6 the Rhodamine 6G derivatives are in
colourless spirolactam closed form and the energy transfer from
the peripheral 1,8-naphthalimes to the rhodamine core in the light-
harvesting antenna is not feasible (Scheme 2). It could be expected
that under alkaline conditions due to the spirolactam closed form of
Rhodamine 6G and PET effect in the periphery the emission of
light-harvesting dendron 7 will be quenched. In acid media
(pH ¼ 2e6) the Rhodamine 6G spirolactam ring is opened and the
PET process in periphery is disallowed. Thus the energy of the
peripheral 1,8-naphthalimides in antenna 7 will be transferred to
the focal Rhodamine and the systems will emit orange-red fluo-
rescence signal.
The novel light-harvesting antenna was prepared in three basic
steps (Scheme 3): synthesis of amino-functional Rhodamine 6G
core, PAMAM dendronization of the amino-functional core to an
amino-terminated Rhodamine 6G PAMAM dendron and peripheral
decoration of the latter with yellow-green emitting 4-(N-methyl-
piperazinyl)-1,8-naphthalimide units to the desired antenna.
In brief, the synthesis of Rhodamine 6G core 2 was performed by
reaction of Rhodamine 6G 1 with ethylenediamine under reflux in
absolute ethyl alcohol. Then the rhodamine core 2 was subse-
quently converted into the PAMAM dendron 4 via divergent strat-
egy involves initial Michael addition of 2 with methyl acrylate
followed by exhaustive amidation of the resulting ester 3 with a
large excess of ethylenediamine. The target light-harvesting an-
tenna 7 was synthesized in two steps as shown in Scheme 3. First,
the intermediate dendron 6 with 4-nitro-1,8-naphthalimide pe-
riphery was obtained by reaction of 4-nitro-1,8-naphthalic
38 N.I. Georgiev et al. / Dyes and Pigments 102 (2014) 35e45

FRET N FRET NH
X - X e-

X
N e N

O O
HN O NH N HN O NH N
O O O O
NH NH
N H (pH 6) N
N FLUORESCENCE N FLUORESCENCE
O O
NH OH NH
O O O O
N N
O O
FLUORESCENCE FLUORESCENCE
X X
FRET N e- FRET N e-

X
7 7
N NH

FRET NH

e-

X
N

O H (pH 2-6)
HN O NH N
O O OH
NH
HN
N FLUORESCENCE
O
NH
O O
N
O
FLUORESCENCE

FRET N e-
X

7
NH

Scheme 2. Photophysical behaviour of antenna 7 as a function of pH after excitation within the spectral region of maximal absorption of the peripheral fluorophores.

anhydride 5 and PAMAM dendron 4 under reflux in methanol so-
lution. Finally, the yellow-green emitting periphery of the system
was obtained after substitution of the nitro groups in the inter-
mediate 6 with N-methylpiperazinyl moieties in DMF at room
temperature.
The synthesized compounds were characterized (melting point,
TLC retention value Rf) and identified by conventional techniques e
elemental analysis data, UVeVis, fluorescence, FT-IR and 1H NMR
spectroscopy. For instance, in the 1H NMR (CDCl3-d, 250.13 MHz)
spectrum of antenna 7 a resonance at 4.17 ppm (Fig. 1, A) was
observed. This is characteristic for the methylene protons coupled
to N-position of 1,8-naphthalimide moiety, which proves the
presence of 1,8-naphthalimide units in the dendron periphery. Also
the 1H NMR spectrum of antenna 7 contains two resonances at
6.34 ppm and 6.14 ppm that are typical for the core Rhodamine 6G
protons at position C-1, C-4, C-5 and C-8 (Fig. 1, C). The resonances
at 4.17 ppm, 6.14 ppm and 6.34 ppm are in ratio 2:1:1 suggesting
that in the novel dendron rhodamine core is surrounded by two
1,8-naphthalimide units. Furthermore, a resonance at 7.01 ppm
(Fig. 1, B) appears which is characteristic for the proton in position
C-3 of the yellow-green emitting 1,8-naphthalimide, substituted in
position C-4 with an electron-donating piperazine group.
This resonance is rather different from the corresponding value for
a non-substituted 4-nitro-1,8-naphthalimide moieties (8.35e
8.70 ppm) [17,75], which is a solid evidence that the peripheral 4-
nitro-1,8-naphthalimide units in the intermediate dendron 6 were
completely converted in yellow-green emitting donor periphery.
Moreover the 1H NMR spectra contain all requisite peaks for
rhodamine and 1,8-naphthalimide moieties as well as peaks in the
range of 2.3e3.5 ppm, attributed to the protons in the peripheral N-
methylpiperazine.
3.2. Photophysical characterization of antenna 7
Photophysical properties of the synthesized compounds 2e4
and 6e7 were determined in water/DMF (4:1, v/v) solution. Under
these conditions the rhodamine moiety adopts a closed, non-
fluorescent spirolactam form. At ca. pH 2 the spirolactam ring of
rhodamine is opened, which results in a new absorption (rhoda-
mine) band between 450 and 575 nm with maximum at 530e
532 nm. The listed in Table 1 absorption data for compounds 2e4
and 6 are common for Rhodamine 6G derivatives [76,77]. The
presented data show that the different alkylamino substituents in
the 9-phenyl amide of Rhodamine 6G (compounds 2e6) have a
small effect on the energy and the shape of the dyes’ absorption
bands.
In alkaline solution antenna 7 do not emit light due to the PET
quenching process in peripheral 1,8-naphthalimides and non-
fluorescent spirolactam form of the focal rhodamine (Scheme 2).
This was the reason to investigate the photophysical properties of
 N.I. Georgiev et al. / Dyes and Pigments 102 (2014) 35e45 39

HN O NH Cl HN O NH HN O NH
O
H2N O
OMe
NH2 OMe
O N N
EtOH, Reflux NH2 MeOH, 20 C o N
O O O
OMe
O
1 NO2 2 3

O
NO2
O O
HN O NH N HN O NH NH2
O O O O
NH NH NH2
N 5 N H2N
N N
O MeOH, Reflux O MeOH, 20 oC
NH NH
O O O
4
N NH2
6 O
N

N
NO2

O
HN O NH N
O O
HN N NH
N
N
DMF, 20oC O
NH
O O
N
7 O

Scheme 3. Synthesis of PAMAM light-harvesting antenna 7.

antenna 7 at pH 6.5 where the PET is not feasible and also at pH 2
where the rhodamine is in ring-opened form. At pH 6.5 light-
harvesting dendron 7 showed only one longest-wavelength ab-
sorption band in a range of about 340e520 nm, which is attributed
to an internal charge transfer process in the 1,8-naphthalimide
chromophores. After acidification to ca. pH 2 where the rhoda-
mine spirolactam form is opened, as expected the absorption
spectrum of light-harvesting system 7 showed two bands (Table 1)
corresponding to the absorption location of the peripheral 1,8-
naphthalimide donor chromophores (lA ¼ 394 nm) and the focal
rhodamine acceptor unit (lA ¼ 534 nm).
At pH 2 and excitation at 510 nm, compounds 2e4, 6 and 7 show
typical for Rhodamine 6G fluorescence spectra with maxima at
about 560 nm [51,52], suggesting that the substituents at 9-phenyl
amide do not affect the energy of the dyes’ fluorescence maximum.
The fluorescence spectra of light-harvesting antenna 7 in water/
DMF (4:1, v/v) solution at pH 6.5, recorded after excitation within
the spectral region of maximal absorption of the donor fluo-
rophores (lex ¼ 400 nm), showed emission band at 522 nm, cor-
responding to the emission band of the donor 1,8-naphthalimide
fragments in the donor-acceptor systems. In contrast, when the
fluorescence spectra was recorded at ca. pH 2, the observed emis-
sion was shifted to 561 nm (Table 1), which can be attributed to the
energy transfer from the donor 1,8-naphthalimide to the ring-
opened (fluorescence) form of the rhodamine acceptor.
It is well known, that the spectral overlap is the main factor that
predetermines the efficiency of FRET. Peripheral 1,8-naphthalimide
fluorophores in antenna 7 showed a broad emission band in the
visible region (450e650 nm) that practically covers completely a
rhodamine absorption band (Fig. 2).
The efficiency of the energy transfer ET in antenna 7 was
calculated to be 91% by applying Eq. (1) [16,78], where FDA and FD
are the normalized to the optical density fluorescence intensities of
the donor in the presence of acceptor (1,8-naphthalimide part in
the examined antenna at pH 2) and of the free donor without
acceptor (1,8-naphthalimide part at pH 6.5 e FRET process to the
acceptor is not faceable), respectively. The calculated energy
transfer clearly shows that the novel light-harvesting antenna is
able to act as an efficient wavelength-shifting chromophore.
ET ¼ 1  ðFDA =FD Þ (1)
The Stokes’ shift (nA  nF) is an important parameter for the
fluorescent compounds that indicates the differences in the prop-
erties and structure of the fluorophores between the ground state
S0 and the first excited state S1. The calculated Stokes’ shift (nA  nF)
values [57] for compounds 2e4 and 6 as well as for the individual
fluorophores in antenna 7 are typical for the rhodamines and 1,8-
naphthalimides [76,77]. The value of 6220 cm1 for the periph-
eral 1,8-naphthalimides and 876e947 cm1 for the focal
40 N.I. Georgiev et al. / Dyes and Pigments 102 (2014) 35e45

Fig. 1. 1H NMR spectrum (CDCl3-d, 250.13 MHz) of antenna 7.

Table 1 As can be seen from the data in Table 1, the quantum yield of
Photophysical characteristics of compounds 2e4 and 6 (lex ¼ 510 nm) and 7 fluorescence of the rhodamine core is decreasing with the increase
(lex ¼ 400 nm) in water/DMF (4:1, v/v).
of the molecular weight of the compounds under study. A similar
Compound lA (nm) 3 (l mol1 cm1) lF (nm) nA  nF (cm1) FF effect was reported before for the core functional PAMAM dendrons
2 530a 56 308a 558a 947a 0.92a using 1,8-naphthalimide and perylene-3,4,9,10-tetracarboxylic
3 532a 46 738a 558a 876a 0.72a diimide units [29,79]. This is probably due to the more flexible
4 532a 42 988a 560a 940a 0.55a PAMAM scaffold in the larger molecules which induces energy
6 532a 42 764a 560a 940a 0.52a loses thus reducing the quantum yield of fluorescence.
7 394a,b 17 564a,b 522a 6 220a 0.28a
534b 48 735b 561b 900b 0.43b
a
3.3. Influence of pH on the photophysical properties of antenna 7
Photophysical data recorded at pH 6.5.
b
Photophysical data recorded at pH 2.0 to avoid a rhodamine spirolactam closed
(non-fluorescent) form. The light-harvesting system under study was designed as a
molecular fluorescence sensor for determination of pH changes
rhodamine, do not indicate remarkable changes in the fluorophore over a wider pH scale. This was the reason to investigate their
excited state due to their incorporation in the dendritic systems. photophysical behaviour in water/DMF (4:1, v/v) solution at
The quantum yields of fluorescence were calculated using different pH values. In order to receive a more complete compar-
Rhodamine 6G (FF ¼ 0.95 in ethanol [71]) or Coumarin 6 (FF ¼ 0.78 ative picture for the influence of the dendron bone to the focal
in ethanol [72]) as standards according to Eq. (2), where Aref, Sref, nref rhodamine, compounds 2e4 were involved in the present study.
and Asample, Ssample, nsample represent the absorbance at the exited Fig. 3 presents the changes of absorption spectra of 3 at different pH
wavelength, the integrated emission band area and the solvent values as a typical example for the influence of pH on the absorp-
refractive index of the standard and the sample, respectively. tion spectra of the examined compounds 2e4. As can be seen, the
! ! ! decrease of pH results in increase of the absorbance at about
Ssample Aref n2sample 532 nm due to the ring opening reaction of rhodamine core.
FF ¼ Fref (2) Taking the part of the graphs located between pH 2 and 6, the
Sref Asample n2ref
pKa values of 2e4 have been calculated by Eq. (3) [18]. The calcu-
lated pKa value for amino-functional rhodamine 2 was 4.0 and 3.9
for the branching compounds 3 and 4. Probably the decrease of pKa
values is a result of the core protective role of the PAMAM scaffold.

log½ðAmax  AÞ=ðA  Amin Þ ¼ pH  pKa (3)

Fig. 2. Normalized absorption spectrum of dendron 4 at pH 2 and emission spectrum
of antenna 7 at pH 6.5 (lex ¼ 400 nm) in water/DMF (4:1, v/v).
The absorption spectrum of light-harvesting antenna 7 does not
show significant pH-dependent changes in a pH window 8e10 as
the 1,8-naphthalimide fluorophores do not affect their ICT excited
states. In contrast, upon acidification from pH 8 to ca. pH 2 the
absorption maximum of peripheral 1,8-naphthalimides is blue
shifted of about 20 nm (Fig. 4). One of the reasons for this effect is
that the protonation of the amine receptors exerts some weak
charge repulsion on the 4-amino moiety of the fluorophores. On the
other hand in very acidic conditions the pushepull character of the
ICT state is partially reduced due to the protonation of the aromatic
4-amino moiety.
When the pH decreases from 6 to ca. 2 a novel band corre-
sponding to the absorption of the focal rhodamine due to the ring
opening reaction is appeared. From the absorption changes at
534 nm the titration curve of antenna 7 was obtained (Inset in
Fig. 4). Analysis of these changes according to Eq. (3) gives two pKa
 N.I. Georgiev et al. / Dyes and Pigments 102 (2014) 35e45
Fig. 3. Absorption changes of 3 as a function of pH at concentration 1  105 mol L1 in
water/DMF (4:1, v/v). Inset: Titration plots of compounds 2e4 in a pH range of ca. 10e2.
values (3.8 and 2.5). The pKa of 3.8 is a typical for the rhodamine
core, while pKa of 2.5 suggests reversible aggregation processes at
alkaline pH values.
The fluorescence spectra of compounds 2e4 and 7 were also
recorded in water/DMF (4:1, v/v) solution at different pH values. In
alkaline solution rhodamines 2e4 are in spirolactam closed form
and do not emit light. However, upon acidification an emission
signal in range between 500 and 700 nm was gradually increased
(Fig. 5).
Analysis of the fluorescence changes at 560 nm according to Eq.
(4) [6] gives the pKa values 4.0 for amino-functional rhodamine 2
and 3.9 for the compounds 3 and 4. These pKa values are the same
as these calculated according to the absorption changes and should
be attributed to the spirolactam opening reaction.
log½ðIFmax  IF Þ=ðIF  IFmin Þ ¼ pH  pKa (4)
As can be seen (Figs. 3 and 5), the increase of rhodamine
absorbance and fluorescence intensity occurs at pH values less than
5, which is only due to the usual for the rhodamine derivatives ICT
ring opening reaction. Obviously, the presence of a PET process
would reflect in emission changes at pH values more than 5, but
actually such changes are absent.
Fig. 4. Absorption changes of antenna 7 as a function of pH at concentration
1  105 mol L1 in water/DMF (4:1, v/v). Inset: Titration plot of antenna 7 in a pH
range of ca. 10e2.
41
Family of fluorescence spectra of antenna 7 (lex ¼ 400 nm) at pH
window 6.5e10 in water/DMF (4:1, v/v) are presented in Fig. 6. As
could be expected in alkaline media the novel compound showed
very weak fluorescence intensity in range of 450e650 nm with
maximum at 522 nm (1,8-naphthalimide emission). However, upon
acidification an emission signal was gradually increased. The in-
crease in fluorescence intensity on transition from alkaline to
slightly acidic pH indicates that the peripheral fluorescence of the
examined antenna is being quenched by PET process from the
tertiary amine receptor to the 1,8-naphthalimide fluorophore. Upon
recognition of the analyte (protons) the oxidation potential of the
receptor is increased thermodynamically thus disallowing PET
process and as such, the fluorescence of the periphery is recovered.
Furthermore, as demonstrated experimentally by Liu and de
Silva, only the receptor that is directly attached to the 1,8-
naphthalimide 4-amino moiety (the “lower” moiety) is capable of
quenching the fluorophore excited state [80]. The different
behaviour of the receptors in C-4 and N-position of the peripheral
1,8-naphthalimides can be easily rationalized according to Scheme
4. The 4-aminonaphthalimide fluorophore is a “pushepull” p
electron system with the C-4 amine group as a donor and the 1,8-
naphthalimide as an acceptor. This leads to strong internal charge
transfer (ICT) in the lowest excited singlet state and considerable
dipole character (positive pole at the 4-amino terminus). A large
dipole moment in an excited state gives rise to a strong photo-
generated electric field. Such a molecular electric field can,
depending on its sign and magnitude, inhibit or accelerate a tran-
siting electron in the 1,8-naphthalimide compounds. Thus the
fluorescence quenching PET process is observed only if the electron
leaving the unprotonated amine donor can enter the space of the 4-
aminonaphthalimide fluorophore across C-4 position with its
attractive electric field (Scheme 4). The corresponding PET path
from the unprotonated amino receptor in N-position is just as
feasible thermodynamically but requires the electron to enter the
fluorophore across the imide moiety with its repulsive electric field
and is not observed.
The enhancement of the fluorescence emission FE was used as a
qualitative parameter for the evaluation of the sensing potential of
the synthesized antenna 7. The FE ¼ I/Io was determined as ratio
between the maximum of fluorescence intensity I at pH ca. 6.5 and
the minimum fluorescence intensity Io at about pH 10. Upon acid-
ification the fluorescence intensity of the yellow-green emitting
periphery had enhanced by over an order of magnitude (FE ¼ 11).
These changes are of such magnitude that they can be considered as
Fig. 5. Fluorescence changes (lex ¼ 510 nm) of rhodamines 2e4 as a function of pH (ca.
7e2) at concentration 1  105 mol L1 in water/DMF (4:1, v/v).
42 N.I. Georgiev et al. / Dyes and Pigments 102 (2014) 35e45

Fig. 6. Fluorescence changes (lex ¼ 400 nm) of antenna 7 as a function of pH (ca. 10e
6.5) at concentration 1  105 mol L1 in water/DMF (4:1, v/v). Inset: Titration plot of
antenna 7 in a pH range of ca. 6.5e10.

representing two different “states”, where the fluorescence emis-

sion is “switched off” in alkaline solution and “switched on” in
neutral to slightly acidic media.
As can be seen from the Inset of Fig. 6, peripheral 1,8-
naphthalimide fluorophores switch between “off” and “on” states
in pH interval of ca. 8.5e6.5. The calculated using Eq. (4) pKa value
of 7.15 for antenna 7 is in agreement with the data for 4-
piperazinyl-1,8-naphthalimide derivatives [7,34,48,50].
Further acidification from pH 6.5 to ca. pH 2 opens the rhoda-
mine spirolactam form, which allows the energy transfer from
periphery to the acceptor moiety (lex ¼ 400 nm). This results in
fluorescence quenching of the peripheral fluorescence and
remarkable fluorescence intensity enhancement (FE ¼ 32) in the
rhodamine emission region at 560 nm (Fig. 7).
Analysis of the fluorescence changes of antenna 7 at 520 nm and Fig. 7. Fluorescence changes of antenna 7 as a function of pH (6e2) at concentration
560 nm after excitation at 400 nm (Inset of Fig. 7A) according to Eq. 1  105 mol L1 in water/DMF (4:1, v/v) excited at lex ¼ 400 nm (A) and lex ¼ 510 nm
(B). Insets: Titration plot of antenna 7 in a pH range of ca. 6e2.
(4) gives pKa values of 3.8 and 2.5. The same results were obtained
after direct core excitation at 510 nm (Inset of Fig. 7B) suggesting
that only the spirolactam reaction of the rhodamine core is probe for pH determination. We expect that the method will serve
responsible for the pH sensing properties of the novel FRET system. as a practical tool for analysis of environmental samples and bio-
Thus, the changes in the fluorescence intensity of antenna 7 in the logical studies.
presence of protons are due to the PET process in the periphery of
the system and of the rhodamine core ICT ring opening reaction.
These results suggest that the novel wavelength-shifting 3.4. Influence of metal cations on the fluorescence intensity of the
bichromophoric system is able to act as a ratiometric fluorescent antenna 7

With regard to the potential application of antenna 7 as a

Repulsion Attractive fluorescent probe for recognition of different cations, its signalling
Field Field
properties in the presence of transition metal ions were studied in
e X
e DMF and in water/DMF (4:1, v/v) solution. DMF has been chosen
δ− δ+ since it is able as a polar solvent to stabilize the dye charge sepa-
N O rated state thus favouring the fluorescence switching by PET pro-
O cess. Also DMF guarantees a good solubility of the dye ligands, used
HN N N
O N metal salts and the respective complexes. Experiments have been
performed in the presence of different metal cations: Cd2þ, Co2þ,
O Cu2þ, Fe3þ, Ni2þ, Pb2þ, Zn2þ, Hg2þ and Agþ.
In DMF solution of antenna 7, considerable photophysical
X

changes were observed only upon addition of Cu2þ and Pb2þ, while
hν1 hν2 (fluorescence) the other metal ions produced a negligible effect (Fig. 8). The
presence of Cu2þ and Pb2þ induces a hypsochromic shift of the 1,8-
fluorophore sp. receptor
naphthalimide absorption maximum of DlA ¼ 10 nm for Cu2þ and
Scheme 4. Feasible photoinduced electron transfer in the 1,8-naphthalimide periph- 12 nm for Pb2þ (Fig. 8A). This means that the both nitrogen atoms of
ery of antenna 7. the N-methylpiperazine substituent at C-4 of the peripheral 1,8-
 N.I. Georgiev et al. / Dyes and Pigments 102 (2014) 35e45
Fig. 8. Absorption (A) and fluorescence (B) changes of antenna 7 (1  105 mol L1) in
the presence of metal cations (1  104 mol L1) in DMF.
naphthalimides are subjected to coordination with metal cations
[66]. The addition of Cu2þ and Pb2þ also results in enhancement
only of the peripheral fluorescence intensity (1,8-naphthalimides)
of antenna 7. The enhancement of the fluorescence emission was
FE ¼ 16.0 for Cu2þ and FE ¼ 16.7 for Pb2þ (Fig. 8B).
2 1
Fig. 9. Effect of metal ions (1  10 mol L ) on the fluorescence intensity of antenna
7 (1  105 mol L1) in water/DMF (4:1, v/v) in the presence ammonia/ammonium
chloride buffer (pH 10).
43
The examined metal ions do not induced any changes in the
photophysical properties of the antenna core even in a higher
cation concentration (1  102 M), suggesting that only protons are
able to open the spirolactam cycle. These results showed that
during the experiments the metal salts do not generate protons
from partial hydrolysis of the salts and as such do not provoke
unusual fluorescence enhancement of the rhodamine core.
It is well known that in aqueous solutions, especially in the
presence of an anionic buffer, coordination of the metal ions with
the receptor fragments competes with a number of other anions
and nucleophiles. That is why it was of interest to investigate the
emission behaviour of antenna 7 in the presence of metal ions in
aqueous medium in regard to its potential for practical application.
To avoid the effect of proton generation due to the partial hydro-
lysis of metal salts the experiments were conducted in buffered
solution. The influence of various metal ions on the fluorescence
behaviour of antenna 7 in water/DMF (4:1, v/v) solution in the
presence of ammonia/ammonium chloride buffer (pH 10) is pre-
sented in Fig. 9. As can be seen, the addition of metal ion solution
(even up to 1  102 mol L1) to the antenna solution
(1  105 mol L1) does not caused any noticeable changes, sug-
gesting that in water/DMF (4:1, v/v) the metal cations are not able
to coupling with the receptors of the peripheral PET based
naphthalimides.
The results obtained show excellent selectivity of antenna 7
toward protons over representative metal ions in aqueous medium.
The high selectivity and ratiometric pH sensitivity of the novel
antenna may be beneficially for monitoring pH variations in com-
plex samples.
4. Conclusions
A PAMAM light-harvesting dendron, core and peripherally
functionalized with Rhodamine 6G and 1,8-naphthalimide fluo-
rophores, respectively, was synthesized based on a divergent
approach. Novel compound was designed as a wavelength-shifting
chromophore capable of absorbing light by its periphery and effi-
ciently transferring the energy to a single acceptor dye in the focal
point of the system. The calculated energy transfer of 91% indicates
that the selected fluorophores are suitable donor-acceptor pair for
constructing light-harvesting molecules. Furthermore, the yellow-
green emitting periphery of the system was designed on the “flu-
orophoreespacerereceptor” format, making the antenna capable
to act as a molecular fluorescence PET-based pH sensor. After
excitation within the spectral region of maximal absorption of
peripheral 1,8-naphthalimides the synthesized antenna enhanced
its peripheral fluorescence intensity by over an order of magnitude
(FE ¼ 11) in a pH range of ca. 6.5e10, while upon acidification to pH
2 the fluorescence intensity of the core was enhanced more than 30
times with the decrease of the peripheral emission. Novel antenna
also showed excellent selectivity toward protons over representa-
tive metal ions in aqueous medium. Thus the distinguishing fea-
tures of the synthesized light-harvesting dendron were
successfully combined with the properties of classical PET and ring-
opening sensor systems. These results suggest that the novel an-
tenna is able to act as a highly selective and pH sensitive ratiometric
fluorescence probe, indicating that it could be used for analysis of
environmental samples and biological studies.
Acknowledgements
This work was supported by the National Science Foundation of
Bulgaria (project DDVU-02/97). Authors also acknowledge the
Science Foundation at the University of Chemical Technology and
Metallurgy (Sofia, Bulgaria).
44 N.I. Georgiev et al. / Dyes and Pigments 102 (2014) 35e45

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