Synthesis of new benzo[a]phenoxazinium probes possessing carboxylic ester, hydroxyl and amino functional groups: Photophysical studies in dry ethanol and conjugation with CdTe quantum dots

Article abstract of DOI:10.1016/j.dyepig.2014.04.006

A new series of benzo[a]phenoxazinium chlorides possessing hydroxyl, ethyl ester and amino functional groups as terminal substituents at the 9- and 5-positions of the tetracyclic aromatic system in different combinations, was synthesized. A photophysical

Full text of DOI:10.1016/j.dyepig.2014.04.006

Dyes and Pigments xxx (2014) 1e11
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Dyes and Pigments
journal homepage: www.elsevier.com/locate/dyepig
Synthesis of new benzo[a]phenoxazinium probes possessing
carboxylic ester, hydroxyl and amino functional groups: Photophysical
studies in dry ethanol and conjugation with CdTe quantum dots
,
B. Rama Raju a b, Arlindo M. Fontes Garcia b, A. Luísa S. Costa a, Paulo J.G. Coutinho b,
,
M. Sameiro T. Gonçalves a
*
a Centre of Chemistry, University of Minho, Campus of Gualtar, 4710-057 Braga, Portugal
b Centre of Physics, University of Minho, Campus of Gualtar, 4710-057 Braga, Portugal
a r t i c l e i n f o
a b s t r a c t
Article history:
A new series of benzo[a]phenoxazinium chlorides possessing hydroxyl, ethyl ester and amino functional
groups as terminal substituents at the 9- and 5-positions of the tetracyclic aromatic system in different
combinations, was synthesized. A photophysical study was carried out in anhydrous ethanol and water,
as a function of solution pH. Acid dissociation constants were estimated and found to depend on the
nature of the terminal groups. Experimental evidence for an additional molecular form is presented and
is compatible with J-aggregates of the deprotonated form of some of the earlier studied benzo[a]phe-
noxazinium chlorides. The benzo[a]phenoxazinium chloride possessing the amino terminal group was
reacted with the carboxylic acid of CdTe quantum dots (QDs) to obtain a conjugate of the dye and QD.
Initial photophysical characterization indicates both photoinduced electron and energy transfer between
the QD and the attached benzo[a]phenoxazinium chloride.
Received 17 February 2014
Received in revised form
2 April 2014
Accepted 4 April 2014
Available online xxx
Keywords:
Nile Blue
Benzo[a]phenoxazinium dyes
CdTe quantum dots
Cationic dyes
Ó 2014 Elsevier Ltd. All rights reserved.
Near-infrared fluorescent probes
Fluorescent dyes
1. Introduction
continuous absorption bands with narrow and symmetric emission
for multicolour capability, and many biofunctionalisation strate-
Synthesis of fluorescent molecules is an enthusiastic topic of
interest due to their labelling ability in various analytical applica-
tions and biosciences [1e6]. In this context, the phenoxazines and
benzo[a]phenoxazinium compounds are of prime importance due
to their better photostability, high molar absorption coefficient and
non-toxicity compared to other dyes [7e9]. The oxazine core in
benzo[a]phenoxazinium compounds has its own unique synthetic
conciseness and spectral properties. Owing to the synthetic and
analytical importance of these heteroaromatic compounds, various
researchers have reported different types of compounds with a
variety of combinations [10e14].
Semiconductor nanocrystals, also called quantum dots (QDs),
are an emerging new class of powerful and versatile biomedical
imaging probes. Their fluorescence is unique in comparison with
the former traditional organic fluorophores. QDs exhibit high
quantum yields, high photostability, large absorption coefficients,
gies. Most of the preparation techniques use a tri-n-octylphosphine
(TOPO) based system in which the reagents are injected into a high
coordinating solvent at high temperature (200e400 ꢀC) under ni-
trogen or argon moisture free atmosphere [15]. Direct aqueous
synthesis of semiconductor nanocrystals by employing different
short-chain thiols as stabilizing agents provides a useful alternative
[16]. The ability of CdTe QDs to interact with neighbouring mole-
cules enables Förster Resonance Energy Transfer (FRET) and/or
photoinduced electron transfer processes that are the basis of
several sensing applications [17e19]. Recently a NADH-sensitive
quantum dots were reported [20] in which the fundamental pro-
cess was an electron transfer between NADH and a Nile Blue (NB)
molecule attached to the QD. This process interrupted an energy
transfer channel from the quantum dot to Nile Blue resulting in
increased emission from the QD.
Our main research interest is focused on the design, synthesis
and applications of new Nile Blue derivatives with different sub-
stituents at 5- and 9-positions [21e30]. The present work is aimed
towards the synthesis of a new set of (bi)functionalised benzo[a]
phenoxazinium chlorides possessing hydroxyl, carboxylic ester
* Corresponding author.
E-mail address: msameiro@quimica.uminho.pt (M.S.T. Gonçalves).
http://dx.doi.org/10.1016/j.dyepig.2014.04.006
0143-7208/Ó 2014 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Raju BR, et al., Synthesis of new benzo[a]phenoxazinium probes possessing carboxylic ester, hydroxyl and
amino functional groups: Photophysical studies in dry ethanol and conjugation with CdTe quantum dots, Dyes and Pigments (2014), http://
dx.doi.org/10.1016/j.dyepig.2014.04.006

2
B.R. Raju et al. / Dyes and Pigments xxx (2014) 1e11
and amino groups as terminal of substituents at 5- and 9-positions
of the heteroaromatic system, and the evaluation of its photo-
physical behaviour as a function of pH medium. The presence of an
amino terminal group allowed the coupling of the benzo[a]phe-
noxazinium chloride with CdTe quantum dots. This conjugated
system was developed with the aim of devising an efficient pH
sensor which can be used for measuring the NADH levels.
Furthermore, due to their relative ease of structural modifications,
benzo[a]phenoxazinium chlorides are useful for bioconjugation.
N(CH2CH2CH2OH)2), 3.66 (4H, t, J ¼ 6.8 Hz, N(CH2CH2CH2OH)2),
3.77 (4H, t, J ¼ 7.6 Hz, N(CH2CH2CH2OH)2), 7.0 (1H, d, J ¼ 8.4 Hz, 6-
H), 7.13e7.19 (2H, m, 2-H and 4-H), 7.43e7.49 (1H, m, 5-H) ppm. 13
C
NMR dC (CD3OD, 100.6 MHz), 28.44 (N(CH2CH2CH2OH)2), 58.05
(N(CH2CH2CH2OH)2), 59.70 (N(CH2CH2CH2OH)2), 110.20 (C-2),
113.91 (C-6), 118.48 (C-4), 132.56 (C-5), 139.78 (C-3), 160.00 (C-1)
ppm. HRMS: m/z (EI): Found [M]þ: 225.1352; C12H19NO3 requires
[M]þ: 225.1365.
2.2.3. Ethyl 4-((3-hydroxyphenyl)amino)butanoate 1b
2. Experimental section
To a solution of 3-aminophenol (0.436 g, 4.0 mmol) in ethanol
(2 mL), ethyl 4-bromobutanoate (0.854 g, 4.4 mmol) was added,
and the resulting mixture was heated at 78 ꢀC during 6 h. The
progress of reaction was monitored by TLC (dichloromethane/
methanol, 9.5:0.5). After completion of the reaction, the solvent
was evaporated and the mixture was purified by column chro-
matography on silica using dichloromethane as the eluent.
Compound 1b was obtained as brown oil (0.633 g, 71%). TLC
2.1. Material and instruments
All melting points were measured on a Stuart SMP3 melting
point apparatus. TLC analysis was carried out on 0.25 mm thick
precoated silica plates (Merck Fertigplatten Kieselgel 60F254) and
spots were visualized under UV light. Chromatography on silica gel
was carried out on Merck Kieselgel (230e240 mesh). IR spectra
were determined on a BOMEM MB 104 spectrophotometer. UVeVis
absorption spectra (200e800 nm) were obtained using Shimadzu
UV/3101PC spectrophotometer and fluorescence spectra with Spex
Fluorolog spectrofluorometer. NMR spectra were obtained on a
Varian Unity Plus Spectrometer at an operating frequency of
300 MHz for 1H and 75.4 MHz for 13C or a Bruker Avance III 400 at
an operating frequency of 400 MHz for 1H and 100.6 MHz for 13C
using the solvent peak as internal reference at 25 ꢀC. All chemical
shifts are given in ppm using dH Me4Si ¼ 0 ppm as reference and J
values are given in Hz. Assignments were made by comparison of
chemical shifts, peak multiplicities and J values and were supported
by spin decoupling-double resonance and bidimensional hetero-
nuclear correlation techniques. Low and high resolution mass
spectrometry analysis were performed at the “C.A.C.T.I. e Unidad
de Espectrometria de Masas”, at University of Vigo, Spain.
Commercially available reagents were used as received.
(dichloromethane/methanol, 9.5:0.5): Rf
¼
0.66. IR (Neat):
nmax ¼ 3398, 2981, 2938, 2873, 1715, 1655, 1616, 1600, 1515, 1499,
1475, 1446, 1415, 1374, 1343, 1301, 1236, 1184, 1160, 1106, 1028,
994, 949, 881, 830 cmꢁ1. 1H NMR dH (CDCl3, 400 MHz), 1.26 (3H, t,
J
¼
6.8 Hz, OCH2CH3), 1.92 (2H, quin,
J
¼
6.8 Hz,
NHCH2CH2CH2CO2Et),
NHCH2CH2CH2CO2Et),
2.41
3.12
(2H,
(1H,
t,
t,
J
J
¼
7.2
7.2
Hz,
Hz,
¼
NHCH2CH2CH2CO2Et), 4.16 (2H, q, J ¼ 7.2 Hz, OCH2CH3), 6.12 (1H,
t, J ¼ 2.4 Hz, 2-H), 6.16e6.23 (2H, m, 4-H and 6-H), 7.00 (1H, t,
J
¼
8.0 Hz, 5-H). 13C NMR dC (CD3OD, 100.6 MHz), 14.11
(OCH2CH3),
24.47
(NHCH2CH2CH2CO2Et),
31.85
(NHCH2CH2CH2CO2Et), 43.30 (NHCH2CH2CH2CO2Et), 60.72
(OCH2CH3), 99.84 (C-2), 104.69 (C-6), 105.78 (C-4), 130.14 (C-5),
149.46 (C-1), 156.97 (C-3), 173.98 (CO2Et) ppm. HRMS: m/z (EI):
Found [M]þ: 224.12808; C12H18NO3 requires [M]þ: 224.12812.
2.2.4. Diethyl 4,40-((3-hydroxyphenyl)azanediyl)dibutanoate 2b
In the above reaction, this compound was also isolated as brown
oil (0.297 g, 22%). TLC (dichloromethane/methanol, 9.5:0.5):
Rf ¼ 0.78. IR (Neat): nmax ¼ 3417, 2980, 2939, 1731, 1714, 1621, 1575,
1504, 1463, 1372, 1300, 1165, 1097, 1030, 970, 919, 823, 756, 690,
2.2. Synthetic methods for the preparation of precursors 1 and 2
2.2.1. 3-((3-Hydroxypropyl)amino)phenol 1a
To a solution of 3-aminophenol (0.50 g, 4.58 mmol) in ethanol
(2 mL), 3-bromopropan-1-ol (0.696 g, 5.04 mmol) was added, and
the resulting mixture was heated at 65 ꢀC during 8 h. The progress
of reaction was monitored by TLC (dichloromethane/methanol,
9:1). After completion of the reaction, the solvent was evaporated
and the mixture was purified by column chromatography on silica
using dichloromethane/methanol, mixtures of increasing polarity,
as the eluent. Compound 1a was obtained as brown oil (0.513 g,
67%). TLC (dichloromethane/methanol, 9:1): Rf ¼ 0.56. IR (Neat):
nmax ¼ 3320, 2950, 2923, 1616, 1489, 1461, 1316, 1288, 1232, 1150,
1056, 1000, 946, 859, 780, 748, 686, 665 cmꢁ1. 1H NMR dH (CD3OD,
400 MHz), 1.83 (2H, quin, J ¼ 6.8 Hz, NHCH2CH2CH2OH), 3.16 (2H, t,
665 cmꢁ1
.
1H NMR dH (CDCl3, 400 MHz), 1.25 (6H, t, J ¼ 7.2 Hz,
(2 ꢂ OCH2CH3), 1.83e1.92 (4H, m, N(CH2CH2CH2CO2Et)2), 2.32 (4H,
t, J ¼ 7.2 Hz, N(CH2CH2CH2CO2Et)2), 3.26 (4H, t, J ¼ 7.6 Hz,
N(CH2CH2CH2CO2Et)2), 4.14 (4H, q, J ¼ 7.2 Hz, 2 ꢂ OCH2CH3), 6.18
(1H, dd, J ¼ 7.2 and 1.2 Hz, 4-H), 6.22e6.26 (2H, m, 6-H and 2-H),
7.02 (1H, t, J ¼ 8.0 Hz, 5-H). 13C NMR dC (CDCl3, 100.6 MHz), 14.00
(2
ꢂ
OCH2CH3),
22.22
(N(CH2CH2CH2CO2Et)2),
31.35
(N(CH2CH2CH2CO2Et)2), 50.03 (N(CH2CH2CH2CO2Et)2), 60.49
(2 ꢂ OCH2CH3), 99.20 (C-6), 103.20 (C-4), 104.38 (C-2), 129.92 (C-5),
149.10 (C-1), 157.24 (C-3), 173.58 (CO2Et). HRMS: m/z (EI): Found
[M]þ: 337.1905; C18H27NO5 requires [M]þ: 337.1889.
J
¼
6.8 Hz, NHCH2CH2CH2OH), 3.69 (2H, t,
J
¼
6.8 Hz,
2.3. Synthetic methods for the preparation of nitrosophenols 3 and 4
NHCH2CH2CH2OH), 6.09e6.14 (2H, m, 2-H and 4-H), 6.16e6.20 (1H,
m, 6-H), 6.92 (1H, t, J ¼ 8.0 Hz, 5-H) ppm. 13C NMR dC (CD3OD,
100.6 MHz), 33.11 (NHCH2CH2CH2OH), 42.03 (NHCH2CH2CH2OH),
61.03 (NHCH2CH2CH2OH), 100.96 (C-2), 105.25 (C-6), 106.36 (C-4),
130.73 (C-5), 151.78 (C-3), 159.24 (C-1) ppm. HRMS: m/z (EI): Found
[M]þ: 167.0951; C9H13NO2 requires [M]þ: 167.0946.
2.3.1. 5-((3-Hydroxypropyl)amino)-2-nitrosophenol hydrochloride
3a
To an ice-cold solution of 3-((3-hydroxypropyl)amino)phenol 1a
(0.150 g, 0.898 mmol) in ethanol (2 mL), 2M hydrochloric acid
(0.36 mL) was added and stirred during 5 min. The solution of so-
dium nitrite (0.068 g, 0.988 mmol) in water (0.1 mL) was then
added drop-wise within an interval of 15e20 min. The resulting
mixture was stirred for 3 h and monitored by TLC (dichloro-
methane/methanol, 9:1 and 5:5). After evaporation of the reaction
mixture, compound 3a was obtained as an orange oily solid
(0.177 g) and was used in the following step without any
purification.
2.2.2. 3,30-((3-Hydroxyphenyl)azanediyl)bis(propan-1-ol) 2a
In the above reaction, this compound was also isolated as brown
oil (0.124 g, 16%). TLC (dichloromethane/methanol, 9:1): Rf ¼ 0.36.
IR (Neat): nmax ¼ 3370, 2956, 2890, 2691, 2587, 1619, 1600, 1489,
1431,1375,1318,1282,1240,1143,1059, 999, 987, 942, 921, 873, 784,
695, 665 cmꢁ1 1H NMR dH (CD3OD, 400 MHz), 1.77 (4H, br s,
.
Please cite this article in press as: Raju BR, et al., Synthesis of new benzo[a]phenoxazinium probes possessing carboxylic ester, hydroxyl and
amino functional groups: Photophysical studies in dry ethanol and conjugation with CdTe quantum dots, Dyes and Pigments (2014), http://
dx.doi.org/10.1016/j.dyepig.2014.04.006

B.R. Raju et al. / Dyes and Pigments xxx (2014) 1e11
3
2.3.2. Ethyl 4-((3-hydroxy-4-nitrosophenyl)amino)butanoate
hydrochloride 3b
with dichloromethane and dichloromethane/methanol 8.0:2.0 to
give compound 6b as a blue solid (0.235 g, 53%). mp > 300 ꢀC.
Rf ¼ 0.10 (dichloromethane/methanol, 9:1). IR (KBr 1%) nmax ¼ 3233,
1638, 1589, 1547, 1524, 1496, 1454, 1434, 1326, 1155, 1123, 1058,
1009, 949, 876, 831, 778, 757, 743, 705 cmꢁ1. 1H NMR dH (CD3OD,
400 MHz), 1.94e2.00 (2H, m, NHCH2CH2CH2OH), 2.23e2.31 (2H, m,
Starting from ethyl 4-((3-hydroxyphenyl)amino)butanoate 1b
(0.140 g, 0.627 mmol) in ethanol (1 mL), and 2M hydrochloric acid
(0.24 mL), using sodium nitrite (0.047 g, 0.69 mmol) in water
(0.1 mL), and following the same procedure as described before for
the preparation of 3a (TLC, dichloromethane/methanol, 9.7:0.3 and
5:5), compound 3b was obtained as an orange oily solid (0.180 g)
and was used in the following step without any purification.
NHCH2CH2CH2NH2.HBr),
NHCH2CH2CH2NH2.HBr),
3.20
3.52
(2H,
(2H,
t,
t,
J
J
¼
7.2
7.2
Hz,
Hz,
¼
NHCH2CH2CH2NH2.HBr), 3.75 (2H, t, J ¼ 6.0 Hz, NHCH2CH2CH2OH),
3.88 (2H, t, J ¼ 7.2 Hz, NHCH2CH2CH2OH), 6.81 (1H, s, 8-H), 7.03 (1H,
s, 6-H), 7.15 (1H, d, J ¼ 9.6 Hz, 10-H), 7.78 (1H, d, J ¼ 9.2 Hz, 11-H),
7.81 (1H, t, J ¼ 7.2 Hz, 3-H), 7.91 (1H, t, J ¼ 8.0 Hz, 2-H), 8.47 (1H, d,
J ¼ 8.0 Hz, 4-H), 8.61 (1H, d, J ¼ 8.4 Hz, 1-H) ppm. 13C NMR dC
(CD3OD, 100.6 MHz), 27.60 (NHCH2CH2CH2NH2.HBr), 32.52
2.3.3. Diethyl 4,40-((3-hydroxy-4-nitrosophenyl)azanediyl)
dibutanoate hydrochloride 4
Starting from diethyl 4,40-((3-hydroxyphenyl)azanediyl)dibuta-
noate 2b (0.100 g, 0.296 mmol) in ethanol (1 mL) and 2 M hydro-
chloric acid (0.12 mL), using sodium nitrite (0.023 g, 0.326 mmol) in
water (0.1 mL), and following the same procedure as described
before for the preparation of 3a, compound 4 was obtained as an
orange oily solid (0.109 g) and was used in the following step
without any purification.
(NHCH2CH2CH2OH),
(NHCH2CH2CH2NH2.HBr),
38.43
(NHCH2CH2CH2NH2.HBr),
(NHCH2CH2CH2OH),
41.68
60.09
42.48
(NHCH2CH2CH2OH), 94.31 (C-8), 95.41 (C-6), 120.53 (AreC), 124.16
(C-4), 124.66 (C-10), 125.45 (C-1), 125.97 (AreC), 129.53 (AreC),
130.82 (C-3), 132.77 (AreC), 132.88 (C-2), 134.08 (C-11), 147.21 (Are
C), 149.10 (AreC), 158.50 (C-5), 159.0 (C-9) ppm. HRMS: m/z (EI):
Found [Mþ1]þ: 377.19680; C22H25N4O2 requires [Mþ1]þ:
377.19720.
2.4. General procedure for the synthesis of benzo[a]phenoxazines 6
and 7
To a cold solution (ice bath) of nitrosophenols 3 or 4, in ethanol
(2 mL), precursors 5 (0.5 eq.) and concentrated hydrochloric acid
(0.27 mL) were added. The mixture was refluxed during the time
mentioned below, and monitored by TLC. After evaporation of the
solvent and column chromatography purification on silica gel with
dichloromethane and dichloromethane/methanol, mixtures of
different polarity, as the eluent, the required dye 6 or 7 was ob-
tained as a blue solid.
2.4.3. 4-Ethoxy-4-oxo-N-(5-(propylamino)-9H-benzo[a]
phenoxazin-9-ylidene)butan-1-aminium chloride 6c
The product of the reaction of 3b (0.114 g, 0.448 mmol) in
ethanol (3 mL) and concentrated hydrochloric acid (1.2 ꢂ 10ꢁ3 mL)
with N-propylnaphthalen-1-amine 5a (0.041 g, 0.22 mmol) (reflux
time 8.5 h), was chromatographed with dichloromethane and
dichloromethane/methanol 9.6:0.4, to give compound 6c as a blue
solid (0.121 g, 59%). mp 115.6e116.7 ꢀC. Rf ¼ 0.40 (dichloromethane/
methanol, 9:1). IR (KBr 1%): nmax ¼ 3450, 2926, 2855, 1728, 1641,
1591, 1548, 1495, 1456, 1434, 1384, 1282, 1160, 1122, 1094, 821,
2.4.1. 3-Hydroxy-N-(5-(propylamino)-9H-benzo[a]phenoxazin-9-
ylidene)propan-1-aminium chloride 6a
777 cmꢁ1
.
1H NMR dH (CD3OD, 400 MHz), 1.13 (3H, t, J ¼ 7.2 Hz,
The product of the reaction of 3a (0.138 g, 0.677 mmol) in
ethanol (2 mL) and concentrated hydrochloric acid (1.9 ꢂ 10ꢁ3 mL)
with N-propylnaphthalen-1-amine 5a (0.0627 g, 0.338 mmol)
(reflux time 8.5 h), was chromatographed with dichloromethane
and dichloromethane/methanol 9.3:0.7 to give compound 6a as a
blue solid (0.059 g, 21%). mp 178.7e180.2 ꢀC. Rf ¼ 0.27 (dichloro-
methane/methanol, 9:1). FTIR (KBr 1%): nmax ¼ 3389, 2961, 2928,
1637,1590,1549,1519,1460, 1435,1384, 1314,1289, 1258, 1181, 1127,
NHCH2CH2CH3), 1.30 (3H, t, J ¼ 7.2 Hz, NHCH2CH2CH2CO2CH2CH3),
1.86e1.91 (2H, m, NHCH2CH2CH3), 1.98e2.05 (2H, m,
NHCH2CH2CH2CO2CH2CH3), 2.55, (2H, t,
J
¼
6.4 Hz,
NHCH2CH2CH2CO2CH2CH3), 3.38 (2H, t, J ¼ 6.8 Hz, NHCH2CH2CH3),
3.63 (2H, t, J ¼ 6.8 Hz, NHCH2CH2CH2CO2CH2CH3), 4.20 (2H, q,
J ¼ 7.2 Hz, NCH2CH2CH2CO2CH2CH3), 6.63 (1H, s, 8-H), 6.76 (1H, s, 6-
H), 7.0 (1H, d, J ¼ 8.4 Hz, 10-H), 7.59 (1H, d, J ¼ 8.8 Hz, 11-H), 7.72
(1H, t, J ¼ 7.6 Hz, 3-H), 7.80 (1H, t, J ¼ 7.6 Hz, 2-H), 8.22 (1H, d,
J ¼ 7.6 Hz, 4-H), 8.63 (1H, d, J ¼ 8.4 Hz, 1-H) ppm. 13C NMR dC
1062, 1005, 874, 832, 772, 738, 703 cmꢁ1 1H NMR dH (CD3OD,
.
400 MHz), 1.14 (3H, t, J ¼ 7.2 Hz, NHCH2CH2CH3), 1.88e1.99 (4H, m,
NHCH2CH2CH3 and NHCH2CH2CH2OH), 3.52 (2H, t, J ¼ 6.8 Hz,
NHCH2CH2CH3), 3.70 (2H, t, J ¼ 6.8 Hz, NHCH2CH2CH2OH), 3.77 (2H,
t, J ¼ 6.0 Hz, NHCH2CH2CH2OH), 6.80 (1H, s, 8-H), 6.92 (1H, s, 6-H),
7.01 (1H, d, J ¼ 5.6 Hz, 10-H), 7.74 (1H, br s, 11-H), 7.78 (1H, t,
J ¼ 7.6 Hz, 3-H), 7.87 (1H, t, J ¼ 7.2 Hz, 2-H), 8.31 (1H, d, J ¼ 8.0 Hz, 4-
H), 8.68 (1H, d, J ¼ 8.0 Hz, 1-H) ppm. 13C NMR dC (CD3OD,
100.6 MHz), 11.73 (NHCH2CH2CH3), 23.07 (NHCH2CH2CH3), 31.92
(CD3OD,
(NHCH2CH2CH2CO2CH2CH3),
100.6
MHz),
11.78
23.04
(NHCH2CH2CH3),
(NHCH2CH2CH3),
14.58
24.94
(NHCH2CH2CH2CO2CH2CH3), 32.07 (NHCH2CH2CH2CO2CH2CH3),
43.80 (NHCH2CH2CH3), 47.42 (NHCH2CH2CH2CO2CH2CH3), 61.75
(NHCH2CH2CH2CO2CH2CH3), 94.27 (C-8), 95.60 (C-6), 123.82 (C-4),
124.54 (C-10), 125.37 (C-1), 130.88 (C-3), 131.72 (2 ꢂ AreC), 132.20
(2 ꢂ AreC), 132.85 (C-2), 133.76 (C-11), 134.42 (2 ꢂ AreC), 157.83
(C-5), 159.11 (C-9), 175.21 (CO2Et) ppm. HRMS: m/z (EI): Found
[Mþ1]þ: 418.21327; C25H28N3O3 requires [Mþ1]þ: 418.21324.
(NHCH2CH2CH2OH),
42.64
(NHCH2CH2CH3),
47.73
(NHCH2CH2CH2OH), 60.78 (NHCH2CH2CH2OH), 95.24 (C-6), 95.97
(C-8), 122.01 (AreC), 124.20 (C-4), 124.96 (C-10), 125.70 (AreC),
125.84 (C-1), 129.61 (AreC), 131.18 (C-11), 131.35 (C-3), 133.43 (Are
C), 134.54 (C-2), 148.14 (AreC), 151.88 (C-5), 153.24 (AreC), 160.31
(C-9) ppm. HRMS: m/z (EI): Found [Mþ1]þ: 362.18706; C22H24N3O2
requires [Mþ1]þ: 337.1889.
2.4.4. N-(5-(3-aminopropylamino)-9H-benzo[a]phenoxazin-9-
ylidene-4-ethoxy-4-oxobutan-1-aminium chloride 6d
The product of the reaction of 3b (0.114 g, 0.448 mmol) in
ethanol (3 mL) and concentrated hydrochloric acid (1.2 ꢂ 10ꢁ3 mL)
with N1-(naphthalen-2-yl)propane-1,3-diamine hydrobromide 5b
(0.063 g, 0.224 mmol) (reflux time 6 h 40 min), was chromato-
graphed with dichloromethane and dichloromethane/methanol
9:1, to give compound 6d as a blue solid (0.168 g, 68%). mp ¼ 124.5e
125.6 ꢀC. Rf ¼ 0.14 (dichloromethane/methanol, 9:1). IR (KBr 1%):
nmax ¼ 3423, 3237, 1719, 1640, 1590, 1548, 1523, 1497, 1456, 1409,
1328, 1283, 1259, 1156, 1124, 1094, 1007, 843, 776 cmꢁ1. 1H NMR dH
2.4.2. N-(5-((3-aminopropylamino)-9H-benzo[a]phenoxazin-9-
ylidene)-3-hydroxypropane-1-aminium chloride hydrobromide 6b
The product of the reaction of 3a (0.177 g, 0.898 mmol) in
ethanol (2 mL) and concentrated hydrochloric acid (2.4 ꢂ 10ꢁ3 mL)
with N1-(naphthalen-2-yl)propane-1,3-diamine hydrobromide 5b
(0.126 g, 0.449 mmol) (reflux time 13 h), was chromatographed
(CD3OD,
400
MHz), 1.29
(3H,
t,
J
¼
7.2
Hz,
Please cite this article in press as: Raju BR, et al., Synthesis of new benzo[a]phenoxazinium probes possessing carboxylic ester, hydroxyl and
amino functional groups: Photophysical studies in dry ethanol and conjugation with CdTe quantum dots, Dyes and Pigments (2014), http://
dx.doi.org/10.1016/j.dyepig.2014.04.006

4
B.R. Raju et al. / Dyes and Pigments xxx (2014) 1e11
(NHCH2CH2CH2CO2CH2CH3),
1.37e1.44
(2H,
m,
745, 706 cmꢁ1. 1H NMR dH (CD3OD, 400 MHz), 1.30 (6H, t, J ¼ 7.2 Hz,
NHCH2CH2CH2CO2CH2CH3), 2.13 (2H, br s, NHCH2CH2CH2NH2.HBr),
2.37 (2H, br s, NHCH2CH2CH2CO2CH2CH3), 2.65 (2H, t, J ¼ 6.4 Hz,
2
2
2
ꢂ
ꢂ
ꢂ
NHCH2CH2CH2CO2CH2CH3),
NHCH2CH2CH2CO2CH2CH3),
NHCH2CH2CH2CO2CH2CH3),
1.96e2.03
2.49e2.56
(4H,
(4H,
m,
m,
s,
(NHCH2CH2CH2NH2.HBr),
NHCH2CH2CH2NH2.HBr),
3.42e3.48
(2H, t,
(2H,
¼
m,
Hz,
7.2 Hz,
3.26
(2H,
(2H,
br
3.62
J
8.0
NHCH2CH2CH2CO2CH2CH3),
3.52
br
s,
NHCH2CH2CH2CO2CH2CH3), 3.73 (2H, q,
J
¼
NHCH2CH2CH2CO2CH2CH3), 4.20 (4H, q,
J
¼
7.2 Hz,
NHCH2CH2CH2CO2CH2CH3), 6.55 (1H, s, 8-H), 6.80 (1H, s, 6-H), 7.01
(1H, d, J ¼ 8.0 Hz, 10-H), 7.45 (1H, d, J ¼ 7.6 Hz, 11-H), 7.70 (1H, t,
J ¼ 7.6 Hz, 3-H), 7.79 (1H, br s, 2-H), 8.28 (1H, d, J ¼ 7.6 Hz, 4-H), 8.42
(1H, d, J ¼ 6.8 Hz, 1-H) ppm. 13C NMR dC (CD3OD, 100.6 MHz), 18.32
(NHCH2CH2CH2CO2CH2CH3), 24.83 (NHCH2CH2CH2CO2CH2CH3),
27.55 (NHCH2CH2CH2NH2.HBr), 29.99 (NHCH2CH2CH2CO2CH2CH3),
31.79 (NHCH2CH2CH2NH2.HBr), 43.90 (NHCH2CH2CH2NH2.HBr),
2 ꢂ NHCH2CH2CH2CO2CH2CH3), 6.31 (1H, s, 8-H), 6.47 (1H, s, 6-H),
6.77-6.87 (1H, m, 10-H), 7.25 (1H, d, J ¼ 6.8 Hz, 11-H), 7.51 (1H, br s,
3-H), 7.59 (1H, t, J ¼ 6.8 Hz, 2-H), 7.91 (1H, br s, 4-H), 8.22 (1H, d,
J ¼ 7.6 Hz, 1-H) ppm. 13C NMR dC (CD3OD, 100.6 MHz), 14.47
(2
ꢂ
NHCH2CH2CH2CO2CH2CH3),
24.66
(NHCH2CH2CH2CO2CH2CH3), 24.89 (NHCH2CH2CH2CO2CH2CH3),
31.95 (NHCH2CH2CH2CO2CH2CH3), 32.51
(NHCH2CH2CH2CO2CH2CH3), 43.86 (NHCH2CH2CH2CO2CH2CH3),
44.91 (NHCH2CH2CH2CO2CH2CH3), 61.75
58.26
(NHCH2CH2CH2CO2CH2CH3),
61.22
(NHCH2CH2CH2CO2CH2CH3), 94.25 (C-8), 97.21 (C-6), 123.95
(2 ꢂ AreC), 124.05 (C-4), 124.94 (C-1), 130.59 (C-3), 131.55 (AreC),
132.33 (C-10), 132.72 (C-2), 133.31 (AreC), 133.66 (C-11), 151.98
(2 ꢂ AreC), 157.84 (C-5), 158.31 (C-9), 175.17 (CO2Et) ppm. HRMS:
m/z (EI): Found [Mþ1]þ: 433.22420; C23H25N4O3 requires [Mþ1]þ:
433.22417.
(NHCH2CH2CH2CO2CH2CH3), 61.89 (NHCH2CH2CH2CO2CH2CH3),
93.96 (C-8), 95.13 (C-6), 123.65 (2 ꢂ AreC), 123.97 (C-4), 124.97 (C-
1), 130.63 (C-3), 131.51 (2 ꢂ AreC), 131.54 (C-2), 132.62 (C-10),
133.41 (C-11), 149.32 (AreC), 151.84 (AreC), 155.52 (C-5), 158.40 (C-
9), 174.82 (CO2Et) ppm. HRMS: m/z (EI): Found [Mþ1]þ: 490.23435;
C
28H32N3O5 requires [Mþ1]þ: 490.23430.
2.4.5. 4-Ethoxy-N-(5-((3-hydroxypropyl)amino)-9H-benzo[a]
phenoxazin-9-ylidene)-4-ethoxy-4-oxobutan-1-aminium chloride
6e
2.4.7. 4-Ethoxy-N-(4-ethoxy-4-oxobutyl)-4-oxo-N-(5-
(propylamino)-9H-benzo[a]phenoxazin-9-ylidene)butan-1-
aminium chloride 7
The product of the reaction of 3b (0.114 g, 0.448 mmol) in
ethanol (3 mL) and concentrated hydrochloric acid (1.2 ꢂ 10ꢁ3 mL)
The product of the reaction of 4 (0.109 g, 0.297 mmol) in ethanol
(2 mL) and concentrated hydrochloric acid (8.0 ꢂ 10ꢁ3 mL) with N-
propylnaphthalen-1-amine 5a (0.028 g, 0.148 mmol) (reflux time
9.5 h), was chromatographed with dichloromethane and
dichloromethane/methanol 9.5:0.5, to give compound 7 blue solid,
(0.087 g, 50%). Mp 192.3e195.1 ꢀC. Rf ¼ 0.67 (dichloromethane/
methanol, 9:1). IR (KBr 1%): nmax ¼ 2923, 2853, 1731, 1721, 1637,
1589, 1545, 1499, 1435, 1375, 1334, 1322, 1290, 1230, 1182, 1162,
1146, 1127, 1100, 1054, 1001, 918, 807, 753, 728, 665 cmꢁ1. 1H NMR
dH (CD3OD, 400 MHz), 1.13 (3H, t, J ¼ 7.2 Hz, NHCH2CH2CH3), 1.33
(6H, t, J ¼ 7.2 Hz, N(CH2CH2CH2CO2CH2CH3)2), 1.86e1.92 (2H, m,
NHCH2CH2CH3), 1.99e2.03 (4H, m, N(CH2CH2CH2CO2Et)2), 2.53 (4H,
with 3-(naphthalen-1-ylamino)propan-1-ol 5c (0.045
g,
0.224 mmol) (reflux time
7 h), was chromatographed with
dichloromethane and dichloromethane/methanol 9:1, to give
compound 6e as a blue solid, (0.100 g, 47%). mp ¼ 127.9e130.8 ꢀC.
Rf
¼
0.35 (dichloromethane/methanol, 9:1). IR (KBr 1%):
nmax ¼ 3419, 2928, 1718, 1640, 1591, 1548, 1495, 1458, 1434, 1384,
1325, 1282, 1177, 1158, 1126, 1090, 826, 776 cmꢁ1 1H NMR dH
.
(CD3OD,
400
MHz), 1.30
(3H,
t,
J
¼
7.2
Hz,
m,
NHCH2CH2CH2CO2CH2CH3),
1.98e2.02
(2H,
NHCH2CH2CH2CO2CH2CH3), 2.04e2.07 (2H, m, NHCH2CH2CH2OH),
2.52 (2H, t, J ¼ 7.2 Hz, NHCH2CH2CH2CO2CH2CH3), 3.37 (2H, br s,
NHCH2CH2CH2OH), 3.72 (2H, t, J ¼ 5.6 Hz, NHCH2CH2CH2OH), 3.81
(2H, t, J ¼ 5.6 Hz, NHCH2CH2CH2CO2CH2CH3), 4.21 (2H, q, J ¼ 7.2 Hz,
NHCH2CH2CH2CO2CH2CH3), 6.51 (1H, s, 8-H), 6.66 (1H, s, 6-H), 6.88
(1H, d, J ¼ 8.0 Hz, 10-H), 7.48 (1H, d, J ¼ 9.2 Hz, 11-H), 7.64 (1H, t,
J ¼ 7.6 Hz, 3-H), 7.73 (1H, t, J ¼ 7.2 Hz, 2-H), 8.07 (1H, d, J ¼ 8.0 Hz, 4-
H), 8.50 (d, J ¼ 8.0 Hz, 1H, 1-H) ppm. 13C NMR dC (CD3OD,
t,
J
¼
6.8 Hz, N(CH2CH2CH2CO2Et)2), 3.59e3.66 (6H, m,
NHCH2CH2CH3 and N(CH2CH2CH2CO2Et)2), 4.23 (4H, q, J ¼ 7.2 Hz,
N(CH2CH2CH2CO2CH2CH3)2), 6.76 (1H, s, 6-H), 6.89 (1H, s, 8-H), 7.27
(1H, d, J ¼ 8.0 Hz, 10-H), 7.62 (1H, d, J ¼ 8.0 Hz, 11-H), 7.69 (1H, t,
J ¼ 7.2 Hz, 3-H), 7.79 (1H, t, J ¼ 7.2 Hz, 2-H), 8.22 (1H, d, J ¼ 8.0 Hz, 4-
H), 8.59 (1H, d, J ¼ 8.0 Hz, 1-H). 13C NMR dC (CD3OD, 100.6 MHz),
11.81 (NHCH2CH2CH3), 14.64 (N(CH2CH2CH2OCH2CH3)2), 23.13
100.6
MHz),
14.60
(NHCH2CH2CH2CO2CH2CH3),
24.93
(NHCH2CH2CH2CO2CH2CH3), 30.80 (NHCH2CH2CH2OH), 32.10
(NHCH2CH2CH2CO2CH2CH3), 43.39 (NHCH2CH2CH2OH), 43.83
(NHCH2CH2CH2OH), 60.45 (NHCH2CH2CH2CO2CH2CH3), 61.77
(NHCH2CH2CH2CO2CH2CH3), 94.10 (C-8), 95.74 (C-6), 123.63
(2 ꢂ AreC), 124.31 (C-4), 125.28 (C-1), 130.57 (C-3), 130.68 (2 ꢂ Are
C), 130.79 (C-10), 132.77 (C-2), 134.11 (C-11), 149.68 (AreC), 149.75
(AreC), 157.75 (C-5), 158.76 (C-9), 174.79 (CO2Et) ppm. HRMS: m/z
(EI): Found [Mþ1]þ: 434.20743; C25H28N3O4 requires [Mþ1]þ:
434.20733.
(NHCH2CH2CH3),
(N(CH2CH2CH2CO2Et)2),
23.38
47.53
(N(CH2CH2CH2CO2Et)2),
(N(CH2CH2CH2CO2Et)2),
31.51
52.0
(NHCH2CH2CH3), 61.82 (N(CH2CH2CH2CO2CH2CH3)2), 94.59 (C-6),
97.42 (C-8), 116.55 (C-10), 123.95 (2 ꢂ AreC), 124.53 (C-4), 125.47
(C-1), 131.06 (AreC), 132.10 (AreC), 133.04 (C-2), 133.80 (C-11),
135.15 (AreC),149.11 (AreC),152.86 (AreC),155.80 (C-5),159.37 (C-
9), 174.73 (CO2Et) ppm. HRMS: m/z (EI): Found [Mþ1]þ: 532.28056;
C
31H38N3O5 requires [Mþ1]þ: 532.28060.
2.4.6. 4-Ethoxy-N-(5-((4-ethoxy-4-oxobutyl)amino)-9H-benzo[a]
phenoxazin-9-ylidene)-4-oxobutan-1-aminium chloride 6f
The product of the reaction of 3b (0.114 g, 0.448 mmol) in
ethanol (3 mL) and concentrated hydrochloric acid (1.2 ꢂ 10ꢁ3 mL)
with ethyl 4-(naphthalen-1-ylamino)butanoate 5d (0.058 g,
2.5. Procedure for the synthesis of CdTe quantum dots
A 6 ꢂ 10ꢁ4 M solution of cadmium nitrate was deoxygenated by
bubbling nitrogen for 30 min. Then, a 0.02 M aqueous solution of
mercapto-propionic acid (MPA) was added in order to achieve a
[MPA]/[Cd] molar ratio of 1.1. An appropriate amount of a poly-
telluride solution, with a concentration of 0.2 M in telluride, was
added and the resulting solution was heated at 85 ꢀC during 1 h.
The polytelluride solution was obtained from tellurium powder
upon addition of hydrazine and a 25% solution of sodium meth-
oxide in methanol. The resulting CdTe QDs aqueous solution had an
0.224 mmol) (reflux time
7 h), was chromatographed with
dichloromethane and dichloromethane/methanol 9.7:0.3, to give
compound 6f as a blue solid (0.104 g, 44%). mp 112.1e114.5 ꢀC.
Rf
¼
0.58 (dichloromethane/methanol, 9:1). IR (KBr 1%):
nmax ¼ 3433, 3225, 2984, 1727, 1641, 1590, 1546, 1495, 1455, 1435,
1383, 1326, 1282, 1183, 1158, 1127, 1090, 1032, 1007, 949, 826, 775,
Please cite this article in press as: Raju BR, et al., Synthesis of new benzo[a]phenoxazinium probes possessing carboxylic ester, hydroxyl and
amino functional groups: Photophysical studies in dry ethanol and conjugation with CdTe quantum dots, Dyes and Pigments (2014), http://
dx.doi.org/10.1016/j.dyepig.2014.04.006

B.R. Raju et al. / Dyes and Pigments xxx (2014) 1e11
5
excitonic absorption peak at 575 nm and an emission at 620 nm
with a fluorescence quantum yield of 0.065.
(naphthalen-1-ylamino)propan-1-ol 5c and ethyl 4-(naphthalen-1-
ylamino)butanoate 5d possessing the methyl, amine, hydroxyl and
ester groups as terminals of the aliphatic chain was achieved by
alkylation of naphthlen-1-amine with 1-bromopropane, 3-
bromopropan-1-amine hydrobromide, 3-bromopropan-1-ol and
ethyl 4-bromobutanoate respectively, in ethanol under reflux
conditions as previously reported [25,27,28]. After purification by
silica gel column chromatography, intermediates 5aed were iso-
lated as oils and solid (5a,b) in moderate yields, and spectroscopic
data confirmed the expected structures, being in accordance with
data published [25,27,28].
2.6. Typical procedure for the coupling of CdTe quantum dots with
benzo[a]phenoxazinium chloride 6d
A solution of CdTe QD’s in water (1 mL, w6 ꢂ 10ꢁ4 M) was
pippeted to an eppendorf tube, and N-(3-dimethylaminopropyl)-
ꢀ
N-ethylcarbodiimide hydrochloride (EDC) (0.1917 mg) was added.
The above solution was heated at 40 ꢀC for 15 min, and then a
0.1 mM ethanolic solution of benzo[a]phenoxazinium chloride 6d
(200
m
L) was added and the reaction mixture was heated at the
The reaction of 5-((3-hydroxypropyl)amino)-2-nitrosophenol
hydrochloride 3a with N-propylnaphthalen-1-amine 5a or N1-
(naphthalen-1-yl)propane-1,3-diamine hydrobromide 5b in an
acidic medium (concentrated hydrochloric acid), using ethanol as
solvent, and after silica gel column chromatography purification
gave the corresponding benzo[a]phenoxazinium chlorides 6a,b,
possessing the hydroxyl groups as the terminal substituents at the
9-positions and methyl or amine in the 5-position, respectively.
Similarly, reaction between ethyl 4-((3-hydroxy-4-nitrosophenyl)
amino) butanoate hydrochloride 3b and N-propylnaphthalen-1-
amine 5a, N1-(naphthalen-1-yl)propane-1,3-diamine hydro-
bromide 5b, 3-(naphthalen-1-ylamino)propan-1-ol 5c or ethyl 4-
(naphthalen-1-ylamino)butanoate 5d resulted in compounds 6c-f,
with an ester functionality at 9-position and methyl, amine, hy-
droxyl and ester groups as the terminal substituents at the 5-
position of the benzo[a]phenoxazine core.
same temperature with continuous stirring for 1 h. After the pre-
scribed time, the reaction mixture was cooled to room temperature
and centrifuged (10,000 rpm for 15 min). The QDs conjugated with
compound 6d precipitated from the solution. The supernatant
liquid was discarded and the quantum dots were washed thor-
oughly with distilled water for 3 times and the residual particles
remained in the bottom were dried and used for further physical
studies.
2.7. Typical procedure for drying of ethanol
The reagent grade absolute ethanol was further dried in accor-
dance with a reported procedure [31]. A 3 A molecular sieves (50 g)
ꢁ
was weighed in a round bottomed flask containing a reagent grade
absolute ethanol (250 mL). The flask was closed tight and ethanol
was left to stand over the sieves for 5 days. The dry ethanol was
filtered by a Whatman resist 13/0.45 PTFE filter unit and used for
the photophysical measurements.
In addition, 9-position bifunctionalised benzo[a]phenoxazinium
chloride 7 was also obtained by using 3 diethyl 4,40-((3-hydroxy-4-
nitrosophenyl)azanediyl)dibutanoate hydrochloride 4 and N-pro-
pylnaphthalen-1-amine 5a. These new set of compounds were
obtained as blue solids in moderate yields and were fully charac-
terized by high resolution mass spectrometry, IR and NMR (1H and
13C) spectroscopy (Scheme 1).
2.8. Typical procedure for preparation of buffer solutions of variable
pH
Buffer solutions of pH ¼ 2, 7, 11 were prepared following the
procedure reported in Ref. [32], by mixing appropriate amounts of a
mixed acidic solution (0.2 M boric acid and 0.05 M citric acid) and a
0.1 M sodium phosphate solution.
The 1H NMR spectra showed the methyl group for the carboxylic
ester functionality (6c-f, 7) as a triplet (1.29e1.33 ppm) and the
aliphatic methyl (6a, 6c, 7) (d 1.13 or 1.14 ppm), the methylene
protons directly linked to the nitrogen atom at 5- and 9-positions
(NHCH2 or NCH2) displayed as triplets, broad singlets and multi-
3. Results and discussion
plets centred around (
methylene groups, NHCH2CH2 or NCH2CH2 as multiplets and broad
singlet ( 1.37e2.31 ppm), and NHCH2CH2CH2 or NCH2CH2CH2,
which appeared as triplets, broad singlet and multiplet ( 2.37e
d 3.26e3.88 ppm), as well as the adjacent
3.1. Synthetic methods
d
d
Benzo[a]phenoxazinium chlorides 6aef and 7 were obtained by
the condensation reaction of nitrosophenol precursors 3a,b and 4
with naphthyl derivatives 5aed. The selection of these precursors is
important, since they determine the substituents present at the 5-
and 9- positions of the tetracyclic aromatic system of the target
compounds.
3.75 ppm). In addition to the signals corresponding to the aliphatic
N-substituents in the heterocycle, the 13C NMR spectra displayed
the expected aromatic carbons, that we highlighted C-8 (
97.42 ppm), C-6 ( 95.13e97.21 ppm), C-11 ( 131.18e134.11 ppm)
and C]O (
d 93.96e
d
d
d
174.73e175.21 ppm). Bands of the ester C]O bond
(1718e1731 cmꢁ1), hydroxyl and amine (3233e3419 cmꢁ1) groups,
as well as a strong band of the C]N bond (1545e1549 cmꢁ1) due to
the fused oxazine ring were observed in their IR spectra.
The reaction of 3-aminophenol with 3-bromopropan-1-ol fol-
lowed by silica gel column chromatography purification afforded
the mono- and di-N-alkylated derivatives, namely 3-((3-
hydroxypropyl)amino)phenol 1a and 3,30-((3-hydroxyphenyl)aza-
nediyl)bis(propan-1-ol) 2a, respectively. Similarly, 3-aminophenol
was reacted with ethyl 4-bromobutanoate to afford ethyl 4-((3-
hydroxyphenyl)amino)butanoate 1b and diethyl 4,40-(3-
hydroxyphenyl)azanediyl)dibutanoate 2b in moderate yields. By
the nitrosation of above compounds with sodium nitrite in hy-
drochloric acidic solution [33], the required 5-((3-hydroxypropyl)
amino)-2-nitrosophenol hydrochloride 3a, ethyl 4-((3-hydroxy-4-
nitrosophenyl)amino)butanoate hydrochloride 3b, and diethyl
3.2. Photophysical studies
From our previous research work [8] it was found that, in
ethanolic medium, the benzo[a]phenoxazinium chlorides show
acidebase equilibria between the benzo[a]phenoxazinium cation
(AHþ) and the corresponding deprotonated neutral form (A) which
manifests itself as spectral variations with dye concentration. The
basic form is characterized by an absorption band around 500 nm
and a broad emission band near 600 nm with low fluorescence
quantum yield (w0.01) while the acidic form showed absorption
near 620 nm and emission around 650 nm with high fluorescence
quantum yield (>0.1). In aqueous medium, non-emissive H-ag-
gregates of the acidic form are observed as a blue shoulder around
4,40-((3-hydroxy-4-nitrosophenyl)azanediyl)dibutanoate
hydro-
chloride 4 was obtained.
The intermediates 5, namely N-propylnaphthalen-1-amine 5a,
N1-(naphthalen-1-yl)propane-1,3-diamine hydrobromide 5b, 3-
Please cite this article in press as: Raju BR, et al., Synthesis of new benzo[a]phenoxazinium probes possessing carboxylic ester, hydroxyl and
amino functional groups: Photophysical studies in dry ethanol and conjugation with CdTe quantum dots, Dyes and Pigments (2014), http://
dx.doi.org/10.1016/j.dyepig.2014.04.006

6
B.R. Raju et al. / Dyes and Pigments xxx (2014) 1e11
Scheme 1. Synthesis of benzo[a]phenoxazinium chlorides 6aef and 7.
600 nm. Recently [22], the presence of a tautomeric equilibrium
(proton displacement resulting in localization of the positive
charge in one of the 5- or 9-amino positions) was shown to occur
when these compounds were tested in water-in-oil (w/o) micro-
colour was observed in all the solutions. The solutions turned
reddish instead of the blue colour typically observed in “reagent
grade absolute” ethanol.
Electronic absorption and emission spectra of 4 ꢂ 10ꢁ6 M so-
lutions in dried ethanol and in buffered aqueous solutions of the
fluorophores 6aef and 7 was then measured. The ethanolic media
was either acidified with trifluoroacetic acid (TFA) or basified by
methanolic solution of tetraethylammonium hydroxide (TEAH) at
1.5 M. Table 1 shows summarized data of this study alongside with
Figs. 1e3. The fluorescence quantum yields were determined in
acidic media at 575 nm excitation in order to maximize the
contribution of the AHþ form in both emission and absorption
spectra. Oxazine 1 was used as a standard (VF ¼ 0.11 in ethanol)
[35] at 575 nm excitation. Table 1 shows that compound 7 has the
greatest absorption and emission maxima. This is mainly a result of
the double alkylation in the 9-amino position as previously
observed [8,28]. Compounds 6b and 6f showed the highest
emulsions. The tautomer loses a part of the
p conjugation and
appeared near 540 nm in the emission spectrum. For benzo[a]
phenoxazinium chlorides with non-functionalized terminals [34] it
was possible to obtain the deprotonation equilibrium constant in
absolute ethanol.
Benzo[a]phenoxazinium chlorides (6aef and 7) reported in this
work exhibit absorption spectrum in ethanol that varies with dye
concentration showing a different proportion of acidic and basic
form, but in an inconsistent and irreproducible way (data not
shown). Also ethanol from different sources resulted in distinct
spectra. Considering, the amount of water dissolved in absolute
ethanol depends on ethanol batch and on atmospheric conditions,
we decided to dry the ethanol prior to its use. Overall, a different
Please cite this article in press as: Raju BR, et al., Synthesis of new benzo[a]phenoxazinium probes possessing carboxylic ester, hydroxyl and
amino functional groups: Photophysical studies in dry ethanol and conjugation with CdTe quantum dots, Dyes and Pigments (2014), http://
dx.doi.org/10.1016/j.dyepig.2014.04.006

B.R. Raju et al. / Dyes and Pigments xxx (2014) 1e11
7
Table 1
Yields and photophysical data of compounds 6aef and 7 in ethanol acidified with trifluoroacetic acid (TFA) and buffered aqueous solution at pH ¼ 2 (C ¼ 4 ꢂ 10ꢁ6 M).
Cpd
Dry ethanol acidified with TFA
Buffer solution pH ¼ 2
Yield (%)
labs (nm)
3
(Mꢁ1cmꢁ1
)
lem (nm)
Dl (nm)
FF
labs (nm)
3
(Mꢁ1cmꢁ1
)
lem (nm)
Dl (nm)
FF
6a
6b
6c
6d
6e
6f
7
21
53
59
68
47
44
50
615
629
623
626
623
625
634
77,375
10,850
97,925
66,100
87,900
77,025
125,950
647
646
648
646
647
647
671
32
17
25
20
24
22
37
0.39
0.42
0.37
0.29
0.38
0.41
0.31
606
619
615
620
617
619
631
39,250
5650
650
652
653
652
653
654
677
44
33
38
32
36
35
46
0.21
0.16
0.15
0.17
0.15
0.14
0.55
45,500
48,775
46,175
54,025
66,650
fluorescence efficiency in ethanolic media, while in water the dia-
lkylated compound 7 is the one with highest quantum yield.
Notably, a greater Stokes shift in aqueous media was also observed
which clearly indicates the stronger solventefluorophore
interactions.
compounds 6c, 6d and 6f through a red shift (540 nm instead of
the usual w500 nm) and sharper absorption of the expected
neutral form. The only distinctive structural characteristic of
these compounds is the existence of ethyl ester terminals in 9-
amino position, with compound 6f having an additional one in
the 5-amino position. The exceptions are compound 6e that
either have a very small effect or the presence of the hydroxyl
group in the other end (5-amino position) disrupts this specific
interaction and compound 7 where there are two ethyl ester
terminals in 9-amino position transforming this amine into a
tertiary one.
Under the applied acidic conditions (Fig. 1(C)) it is seen that,
with an exception of compound 7, it was not possible to completely
displace the acidebase equilibria. Further, an additional emission is
observed near 540 nm especially for compound 6f. According to a
previous study this band probably corresponds to a tautomeric
form with the excitation spectrum showing absorption at w510 nm
and w480 nm.
Fig. 1(A) shows emission spectra at 575 nm excitation, where
only the acidic AHþ form is excited, whereas Fig. 1(B)e(D) plot
emission spectra at 470 nm excitation where other possible
molecular forms of the studied benzo[a]phenoaxine compounds
can also contribute to the fluorescence spectrum. In dry ethanol
medium at 470 nm excitation, the acidic form fluorescence is still
observed, but depending on the structure of the compound,
other bands of different intensities also appear. Near 600 nm for
compounds 6a, 6b, 6e and 7 and near 580 nm for compounds 6c,
6d and 6f. Fig. 1(C) and (D) shows emission and excitation
spectra either in acidic (Fig. 1(C)) or basic (Fig. 1(D)) conditions.
These bands are only seen in basic conditions (Fig. 1(D)) and,
from excitation spectra further distinction is observed for
Fig. 1. Emission and excitation spectra of compounds 6aef and 7 in dried ethanol media. A e In dried ethanol with excitation at 575 nm; B e In dried ethanol with excitation at
470 nm; C e In acidified dried ethanol at 470 nm excitation; D e In basified dried ethanol at 470 nm excitation; the excitation spectra were collected at 605 nm emission
wavelength.
Please cite this article in press as: Raju BR, et al., Synthesis of new benzo[a]phenoxazinium probes possessing carboxylic ester, hydroxyl and
amino functional groups: Photophysical studies in dry ethanol and conjugation with CdTe quantum dots, Dyes and Pigments (2014), http://
dx.doi.org/10.1016/j.dyepig.2014.04.006

8
B.R. Raju et al. / Dyes and Pigments xxx (2014) 1e11
Fig. 2. Absorption spectra of compounds 6f (A) and 7 (B) in dried ethanol media at concentrations from 2
mM to 20 mM (solid lines e experimental; grey lines e fitted spectrum); the
dotted line is the spectrum of 4 M concentration in acidified ethanol; the dashed line is the spectrum correspondent to the basic form that, in the case of compound 6f (A) was
m
corrected with a fraction of the spectrum represented by the dash-dotted line. Panel C shows the obtained fraction of basic form of compounds 6aef and 7 in dried ethanol media.
(*) Compounds in which an additional band around 540 nm was required in the fitting procedure.
Fig. 2 shows molar absorptivity of compounds 6f (panel A)
and 7 (panel B) as a function of dye concentration. In the case of
compound 7 the spectra could be fitted to a sum of a fraction of
the spectrum obtained in basic conditions (dashed line) with the
complementary fraction of the spectrum obtained in acid (dotted
line) conditions. The same was possible for compounds 6a, 6b
and 6e (data not shown). However, for compound 6f (Fig. 2(A))
an additional sharp absorption near 540 nm band had to be taken
into account. The spectrum in basic conditions was a sum of the
spectrum plotted as a dashed line with 12% of the band at
540 nm (dash-dotted line). The criteria used to obtain this value
was that the resulting basic form spectrum (dashed line) should
be similar to that observed for compound 7, namely with a
smooth descent. It was then possible to fit the experimental
spectra a sum of three components: the acid form; the corrected
basic form and the additional benzo[a]phenoxazine molecular
form that corresponds to the 540 nm sharp band. Interestingly,
the absorption bands corresponding to the tautomeric form
(w520 nm and 480 nm) were not apparent which mean that
their concentration is very low. The same procedure was
successful for compounds 6c and 6d (data not shown) with a
contribution of, respectively, 12% and 9% of the additional band to
the spectrum obtained in basic conditions. Considering the
sharpness of the additional absorption band at 540 nm and
corresponding emission at 580 nm it is possible to put forward
the possibility of J-aggregate formation of the neutral form of
compounds 6c, 6d and 6f promoted by the presence of ethyl ester
groups, especially in the 9-amino position.
Supposing that the neutral basic molecular population is in
equilibrium, as a whole, with the acid cationic subpopulation, it is
possible to estimate a dissociation equilibrium constant from the
variation basic form fraction with compound concentration.
Following a procedure similar to that used in a previous study [34]
the fitted curves in Fig. 2(C) are defined by the equilibrium disso-
ciation constants, Ka, shown in Table 2 and by an ethanol self-
dissociation constant of 1.75 ꢂ 10ꢁ11 M2, obtained globally for all
data in Fig. 2(C). This value is higher than expected as for methanol
the corresponding value is 1.2 ꢂ 10ꢁ17 M2 [36]. This could be the
consequence of specific interactions of the studied benzo[a]phe-
noxazine dyes with ethanol solvating molecules.
Please cite this article in press as: Raju BR, et al., Synthesis of new benzo[a]phenoxazinium probes possessing carboxylic ester, hydroxyl and
amino functional groups: Photophysical studies in dry ethanol and conjugation with CdTe quantum dots, Dyes and Pigments (2014), http://
dx.doi.org/10.1016/j.dyepig.2014.04.006

B.R. Raju et al. / Dyes and Pigments xxx (2014) 1e11
9
Fig. 3. Emission spectra at 470 nm excitation of compounds 6f (A) and 7 (B) in aqueous buffered solutions at pH ¼ 2 (solid line) pH ¼ 7 (dotted line) and pH ¼ 11 (dashed line). Panel
C shows the absorption spectra of compound 6f (black lines) and compound 7 (grey lines) at pH ¼ 2 (solid line), pH ¼ 7 (dotted line) and pH ¼ 11 (dashed line).
The obtained values of Ka show that the presence of an OH
terminal in 9-amino position (compounds 6a and 6b) favours the
dissociation process. This is consistent with a main deprotonation
site at 5-amino position as the OH group would stabilize the
localization of positive charge at the 9-amino position. In previous
work [37] additional evidence for preferential deprotonation at the
5-amino position was already given. The presence of NH2 terminal
at 5-amino position disfavours deprotonation as can be concluded
by comparing compounds 6b and 6a. On the contrary, by
comparing compound 6c with 6e and 6f, the presence of an OH or
an ethyl-ester group at the 5-amino position seems to increase Ka.
Compound 7 can only be deprotonated at 5-amino position and the
disubstitution with ethyl ester terminals seems to increase the
dissociation constant (Ka(7)>Ka(6c)).
Fig. 3 shows the emission at 470 nm excitation and absorption
spectra of compounds 6f and 7 in aqueous media at controlled pH
values using suitable buffers. Huge changes with pH are observed.
At pH ¼ 2, the tautomeric form emission appears for compound 6f
but in much less amount for compound 7. This is consistent with
the result in acidified ethanol media (Fig. 1(C)). In basic conditions
(pH ¼ 11) low emission is observed. While for compound 6f, the
band at w580 nm is still observed but 10 times less intense than in
basified ethanol (Fig. 1(D)), for compound 7 there is practically no
emission in the 500e620 nm spectral region. The absorption
spectra plotted in Fig. 3(C) show that there is much less absorption
in basic than in acidic conditions. Further the fact that the baseline
is inclined indicates the presence of light dispersion that originates
from extended aggregation of neutral benzo[a]phenoxazine mole-
cules that are expected to have low solubility in water. The addi-
tional band at 540 nm is now much more pronounced for
compounds 6f than what was observed in basified ethanol
(Fig. 2(A)). This constitutes further evidence that this band can
correspond to J-aggregates of neutral benzo[a]phenoxazine
molecules.
As the studied compounds show high environment sensibility
and have functional groups that can be used to couple with other
molecular entities and following recent published research on the
behaviour of Nile Blue when conjugated with quantum dots [20],
initial attempts on coupling bifunctional benzo[a]phenoxazine
derivatives with CdTe quantum dots was performed.
The CdTe quantum dots were prepared in aqueous media using
mercapto-propionic acid (MPA) as a capping agent. It was expected
Table 2
Equilibrium dissociation constants of compounds 6aef and 7 in dried ethanol.
Compound
6a
6b
6c(a)
6d(a)
6e
6f(a)
7
Ka (10ꢁ5Mꢁ1
)
14.2
11.5
1.28
0.32
2.19
2.08
4.95
a
Obtained considering an additional absorption band near 540 nm.
Please cite this article in press as: Raju BR, et al., Synthesis of new benzo[a]phenoxazinium probes possessing carboxylic ester, hydroxyl and
amino functional groups: Photophysical studies in dry ethanol and conjugation with CdTe quantum dots, Dyes and Pigments (2014), http://
dx.doi.org/10.1016/j.dyepig.2014.04.006

10
B.R. Raju et al. / Dyes and Pigments xxx (2014) 1e11
Fig. 4. A e Emission spectra at 470 nm excitation (solid lines) and at 575 nm (dotted lines) of the 1st (black lines) and 2nd (grey lines) supernatant resulting from washing CdTe-6d
quantum dot conjugates. B e Emission spectra at 470 nm excitation (solid lines) and at 575 nm (dotted lines) of CdTe-6d quantum dot conjugates in dried ethanol (black lines),
acidified ethanol (grey lines) and basified ethanol (dark grey lines). The dashed black line corresponds to CdTe quantum dots emission. C e Absorption spectrum of CdTe-6d
quantum dot conjugates in dried ethanol (black line); acidified ethanol (grey line); basified ethanol (dark grey line) and in water at pH ¼ 4 (dotted line).
that the carboxylic acid functional groups at the surface of the
quantum dot can be activated and coupled with aliphatic terminal
amines using standard peptide coupling procedures. In this
context, compound 6d was chosen and additionally to the amine
terminal at the 5-amino position, the chemical function at the 9-
amino position is relatively inert in the coupling reaction condi-
tions. Fig. 4(A) shows the emission of the aqueous media obtained
upon successive particle centrifugation, supernatant removal and
water addition. It can be concluded that any benzo[a]phenoxazine
emission arising from the resulting particles results from attached
molecules rather than from physically adsorbed ones. Fig. 4(B)
shows that the CdTe emission is extensively quenched by the
proximity of 6d molecules probably through a photoinduced elec-
tron transfer process. In acid conditions the remaining emission of
CdTe quantum dots is completely lost, whereas it is still observable
in neutral and basic conditions. This is an indication that energy
transfer from the excited quantum dots to the acid form of the
benzo[a]phenoxazine occurs. The results also showed the response
of CdTe-6d conjugate was highly dependent on pH of the medium.
The absorption spectra in Fig. 4(C) showed less light scattering
in acidified ethanol medium and in water at pH ¼ 4. This indicates
that when compound 6d molecules are protonated there is a lower
tendency to quantum dot aggregation although the reverse effect is
expected from the protonation of the remaining carboxylic acid
groups at the CdTe surface. Also very noticeable is the absorption
band of compound 6d when the CdTe-6d conjugates are in acidified
ethanol, although in water at pH ¼ 4 it almost disappear.
4. Conclusion
A new set of (bi)functionalized benzo[a]phenoxazinium chlo-
rides possessing carboxylic ester, hydroxyl and amino functional
groups was synthesized. The photophysical behaviour of these
compounds was evaluated in dry ethanol and it was found to be
distinct from solvent grade absolute ethanol. A benzo[a]phenox-
azinum chloride possessing an amine functional group at the ter-
minal of the 5-amino position was coupled with CdTe quantum
dots and its photophysical studies revealed the evidence of
photoinduced electron and energy transfer to the attached dye.
This type of conjugated systems can function as pH sensor and/or
for measuring the NADH levels.
Acknowledgements
Thanks are due to the Fundação para a Ciência e Tecnologia (FCT,
Portugal) for financial support to the NMR portuguese network
(PTNMR, Bruker Avance III 400-Univ. Minho), FCT and FEDER (Eu-
ropean Fund for Regional Development)-COMPETE-QREN-EU for
Please cite this article in press as: Raju BR, et al., Synthesis of new benzo[a]phenoxazinium probes possessing carboxylic ester, hydroxyl and
amino functional groups: Photophysical studies in dry ethanol and conjugation with CdTe quantum dots, Dyes and Pigments (2014), http://
dx.doi.org/10.1016/j.dyepig.2014.04.006

B.R. Raju et al. / Dyes and Pigments xxx (2014) 1e11
11
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financial support to Research Centres CQ/UM [PEst-C/QUI/UI0686/
2013 (FCOMP-01-0124-FEDER-037302)] and CFUM [PEst-C/FIS/
UI0607/2013 (F-COMP-01-0124-FEDER-022711)]. Post-doctoral
grant to B. R. Raju (SFRH/BPD/62881/2009) is also acknowledged
to FCT, POPH-QREN, FSE.
[19] Li J, Bao D, Hong X, Li D, Li J, Bai Y, et al. Luminescent CdTe quantum dots and
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Please cite this article in press as: Raju BR, et al., Synthesis of new benzo[a]phenoxazinium probes possessing carboxylic ester, hydroxyl and
amino functional groups: Photophysical studies in dry ethanol and conjugation with CdTe quantum dots, Dyes and Pigments (2014), http://
dx.doi.org/10.1016/j.dyepig.2014.04.006