Synthesis of fluorescent water-soluble functionalised benzo[a]phenoxazinium salts

Article abstract of DOI:10.1016/j.tetlet.2006.09.133

In order to develop long-wavelength functionalised oxazine dyes with good water solubility and high photostability for biological applications, a series of novel side-chain functionalised benzo[a]phenoxazinium salts were synthesised and characterised. The

Full text of DOI:10.1016/j.tetlet.2006.09.133

Tetrahedron Letters 47 (2006) 8567–8570
Synthesis of fluorescent water-soluble
functionalised benzo[a]phenoxazinium salts
Vaˆnia H. J. Frade, M. Sameiro T. Gonc¸alves* and Joao C. V. P. Moura
˜
Centro de Quı´mica, Universidade do Minho, Gualtar, 4710-057 Braga, Portugal
Received 7 July 2006; revised 20 September 2006; accepted 22 September 2006
Available online 17 October 2006
Abstract—In order to develop long-wavelength functionalised oxazine dyes with good water solubility and high photostability for
biological applications, a series of novel side-chain functionalised benzo[a]phenoxazinium salts were synthesised and characterised.
These polycyclic cationic compounds showed strong fluorescence in ethanol and water (pH 7.4), with an emission wavelengths
higher than 637 nm, as well as high quantum yields and moderate Stokes’ shifts. The photostability of the fluorophores synthesised,
under irradiation at 419 nm, was good to excellent in ethanol and moderate in water at physiological pH.
ꢀ 2006 Elsevier Ltd. All rights reserved.
1. Introduction
cationic dyes resulted from the cyclisation of 5-(ethyl-
amino)-4-methyl-2-nitrosophenol hydrochloride with
N-substituted-naphthylamine or 1-naphthylamine. All
compounds exhibit visible absorption in the 620–
630 nm region (in ethanol or water, pH 7.4) and show
strong fluorescence from 637 to 646 nm (in ethanol)
and at about 653 nm (in water, pH 7.4). Preliminary
evaluation of the photostability of the cationic poly-
cyclic heterocycles synthesised, in ethanol and water at
physiological pH, was carried out.
In recent years, the interest in the development of mono-
functional, water-soluble long-wavelength fluorochromo-
phores for the detection of biological and organic
molecules has dramatically increased.1 The sensitivity,
simplicity and selectivity of fluorescence-based tech-
niques make them particularly attractive compared to
radiochemical methods.
Among the long-wavelength absorbing and emitting
fluorophores are rhodamine,2,3 squaraine,4,5 cyanine6,7
and oxazine8–10 dyes. Excitation in the high wavelength
region is particularly interesting for in vitro and in vivo
cellular and molecular biology studies, because of
reduced cell damage.11 In addition, the presence of a
functional group in the dye is fundamental for covalent
labelling of analytes. The chemical and photochemical
stability of chromophores is also an important prerequi-
site for their use as probes in biological analysis.
The synthesis of functionalised 5,9-diaminobenzo[a]-
phenoxazinium chlorides 1 started with the preparation
of the nitroso intermediate 2 and the N-substituted-
naphthylamines 3 (except in the case of compound 3d).
The required 5-(ethylamino)-4-methyl-2-nitrosophenol
hydrochloride 2 was synthesised by the usual procedure
involving the treatment of the 3-ethylamino-4-methyl-
phenol with sodium nitrite in an acid solution.14
Alkylation of 1-naphthylamine with ethyl-4-bromobut-
yrate, followed by dry chromatography purification
produced ethyl-4-(naphthalen-1-ylamino)butanoate 3a
(46%).15 Hydrolysis of the ester group of this compound
(1 M NaOH/1,4-dioxane), yielded the corresponding
4-(naphthalen-1-ylamino)butanoic acid 3b in a quantita-
tive scale.
Bearing this in mind, and in connection with our preli-
minary work concerning the preparation of carboxyl-
ated 5,9-diaminobenzo[a]phenoxazinium salts,12,13 we
now present the efficient synthesis of new side-chain
functionalised 5,9-benzo[a]phenoxazinium salts. These
Condensation of the nitrosophenol 2 with the inter-
mediate 3a, in boiling ethanol and hydrochloric acid,
yielded the corresponding carboxylated benzo[a]phen-
oxazinium chloride 1a.16 When compound 2 reacted
Keywords: Near-infrared dyes; Functionalised probes; Benzo[a]phen-
oxazine; Fluorescent label; Fluorochromophores.
*
Corresponding author. Fax: +351 253 678 986; e-mail:
msameiro@quimica.uminho.pt
0040-4039/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2006.09.133

8568
V. H. J. Frade et al. / Tetrahedron Letters 47 (2006) 8567–8570
H3C
N
H3C
NO
OH
a
+
H
R
H
N
H3CH2C
H
R
H
+
N
O
N
N
H3CH2C Cl -
3
1
2
a R = (CH2)3CO2CH2CH3
b R = (CH2)3CO2H
c R = (CH2)3OH
d R = H
a i) H+/ EtOH (reflux) for compounds 1a, 1c and 1d; ii) H+/ DMF (70 ºC) for compound 1b.
H3C
N
O
b
1c
H
H
+
N
N
H3CH2C
Cl -
(CH2)3Cl
1e
b SOCl2, r.t.
Scheme 1.
with 4-(naphthalen-1-ylamino)butanoic acid 3b, in
acidic medium using DMF as a solvent and heating at
70 ꢁC, the (carboxypropylamino)-benzo[a]phenoxazi-
nium derivative 1b was obtained (Scheme 1). After dry
chromatography purification, cationic dyes 1a,b were
isolated as solid materials in 75% (1a) and 40% (1b)
yields (Table 1).
NMR (1H and 13C) and visible spectroscopies. In com-
pounds 1a and 1b, the 13C NMR spectra showed signals
at d 173.4 (1a) and 164.3 (1b) ppm due to the presence of
the carbonyl carbon, and IR of compound 1a also pre-
sented a band at 1727 cmÀ1, related to the ester group.
The IR of compounds 1c and 1d showed the bands
expected, due to the hydroxyl (3506 and 3241 cmÀ1
and amino functions (3319 cmÀ1), respectively.
)
Bearing in mind the synthesis of a fluorochromophore
with a hydroxyl group instead of carboxyl, the function-
alised intermediate 3c was prepared and used in the
cyclisation together with compound 2. Thus, 1-naph-
thylamine was alkylated with 3-bromo-1-propanol and
after purification by dry chromatography, 3-(naphthal-
en-1-ylamino)propanol 3c was obtained in 70%. By
reacting the nitrosophenol 2 with this intermediate, fol-
lowing the same procedure as described above for the
preparation of dye 1a, the desired hydroxypropylamino
benzo[a]phenoxazinium salt 1c was isolated in an excel-
lent yield (97%). Using 1-naphthylamine and the nitroso
compound 2, the amino derivative 1d was also prepared
(80%) (Scheme 1, Table 1).
The visible absorption spectra of 3 · 10À6 M solutions
of compounds 1a–e in degassed absolute ethanol
showed absorption peaks at 620 nm (1b,d), 625 nm
(1c,e) and 630 nm (1a) with e values ranging from
21,978 (1e) to 53,968 (1d) (Table 1).
The fluorescent properties of these compounds mea-
sured in the same solvent, using oxazine 1 as standard,
are summarised in Table 2. All the compounds exhibit
high levels of fluorescence, with quantum yields between
0.31 (1d) and 0.55 (1e) and showed a moderate Stokes’
shift (the highest 57 for 1d).
Considering the water solubility of the cationic dyes
(1a–c and 1e) synthesised, as well as the importance of
having fluorescent long-wavelength probes for biologi-
cal applications, their visible and fluorescent properties
were studied in water at physiological pH. The wave-
lengths of maximum absorption (kmax), excitation (kex)
and emission (kem) and also fluorescence quantum yields
of compounds 1a–e in water (pH 7.4) are shown in
Tables 1 and 2. The maximum absorption of these
compounds in water was at about 620–625 nm, which
was similar to kmax in ethanol. However, in the fluores-
cence emission, a slight bathochromic shift occurred in
water when compared to ethanol. Thus, maximum
emission wavelengths were at about 653 nm (in water)
and 637 to 646 nm (in ethanol). All compounds also
exhibited high levels of fluorescence in water, with quan-
tum yields ranging from 0.26 (1a) to 0.44 (1e) and mod-
erate Stokes’ shifts (61–71 nm), yet superior to ethanol
(46–57 nm).
In the synthesis of the chloropropylaminobenzo[a]phenox-
azinium chloride 1e, compound 1c, used as precursor, was
treated with thionyl chloride, at room temperature.
All compounds were fully characterised by elemental
analysis or high resolution mass spectrometry, IR,
Table 1. Yields and visible data for compounds 1a–e
Compound
Yield [%]
Visa
Visb
kmax [nm] (e)
kmax [nm] (e)
1a
1b
1c
1d
1e
75
40
97
80
55
630 (45,296)
620 (23,970)
625 (45,477)
620 (53,968)
625 (21,978)
625 (33,500)
620 (12,805)
620 (36,181)
—
620 (11,311)
a Spectra were measured in absolute ethanol.
b Spectra were measured in water (pH 7.4).

V. H. J. Frade et al. / Tetrahedron Letters 47 (2006) 8567–8570
Table 2. Fluorescence data for compounds 1a–e
8569
Compd
Fluorescencea
Stokes’ shift [nm]
Fluorescenceb
Stokes’ shift [nm]
kexc [nm]
kem [nm]
/
kexc [nm]
kem [nm]
/
1a
1b
1c
1d
1e
600
590
590
580
580
646
644
646
637
645
0.35
0.34
0.41
0.31
0.55
46
54
56
57
53
590
590
580
—
653
653
651
—
0.26
0.37
0.31
—
63
63
71
—
61
590
651
0.44
a Spectra were measured in absolute ethanol.
b Spectra were measured in water (pH 7.4).
cationic dyes, with absorption in the 620–630 nm range,
were highly fluorescent and revealed a maximum
emission wavelength between 637 and 653 nm. Their
photostability in ethanol (good to excellent) and water
(moderate), in connection with the above results,
strongly suggests that these oxazine derivatives constitute
promising fluorescent probes in biological applications.
7.00E+07
6.00E+07
1b
1c (Ethanol)
5.00E+07
4.00E+07
1c
3.00E+07
2.00E+07
1a
1.00E+07
0.00E+00
550
600
650
700
750
800
850
Acknowledgements
Wavelength (nm)
We thank the Fundac¸ao para a Cieˆncia e Tecnologia
˜
(Portugal) for its financial support to the Centro de
Figure 1. Fluorescence spectra of compounds 1a–c measured in water
(pH 7.4) and/or in ethanol.
´
Quımica (Universidade do Minho).
Figure 1 shows the comparison between the emission
spectra of compounds 1c in ethanol and water (pH
7.4) as well as the emission spectra of compounds 1a
and 1b in water.
References and notes
1. Haugland, R. P. Handbook of Fluorescent Probes and
Research Products, 9th ed.; Molecular Probes: Eugene,
OR, 2002.
2. Matveeva, E. G.; Gryczynski, Z.; Lakowicz, J. R. J.
Immunol. Methods 2005, 302, 26–35.
3. Waggoner, A. Curr. Opin. Chem. Biol. 2006, 10, 62–66.
4. Arun, K. T.; Ramaiah, D. J. Phys. Chem. A 2005, 109,
5571–5578.
One of the major limitations in the complete use of long-
wavelength dyes for several applications is the short
number of fluorophores with high fluorescence efficiency
and good stability.
5. Jyothish, K.; Avirah, R. R.; Ramaiah, D. Org. Lett. 2006,
8, 111–114.
6. Pham, W.; Medarova, Z.; Moore, A. Bioconjugate Chem.
2005, 16, 735–740.
7. Shindy, H. A.; El-Maghraby, M. A.; Eissa, F. M. Dyes
Pigments 2006, 68, 11–18.
Since our final purpose is that of the investigation of the
photostability of these fluorophores, we decided to
evaluate their behaviour under irradiation in the visible
region. In this study, solutions of compounds 1a–e in
ethanol or water at physiological pH (1 · 10À5 M) were
irradiated at 419 nm in a Rayonet RPR-100 reactor.17
The photostabilities of compounds 1a–e, in ethanol,
after seven hours of irradiation were in the order of
1e > 1d > 1c > 1a > 1b and the remaining absorption
varied from ꢀ98% (1e) to ꢀ80% (1b). In water, there
was a decrease in the photostability values: compounds
1a (ꢀ48% of remaining absorption) and 1e (ꢀ23% of
remaining absorption) were the most and least stable,
respectively. These results suggested that there was a
relationship between the functional group of the side
chain and the photostability of these benzo[a]phenox-
azine dyes. Further studies will be carried out for a
better comprehension of this correlation.
´
8. Marme, N.; Habl, G.; Knemeyer, J.-P. Chem. Phys. Lett.
2005, 408, 221–225.
9. Westman, G.; Jennsiche, E.; Hamberger, A. Eur. Pat.
104210A1, 2003.
10. Frangioni, J. V.; Onishi, S. U.S. Pat. 082423A2, 2005.
11. Weissleder, R.; Ntziachristos, V. Nat. Med. 2003, 9, 123–
128.
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Tetrahedron Lett. 2005, 46, 4949–4952.
13. Frade, V. H. J.; Gonc¸alves, M. S. T.; Coutinho, P. J. G.;
Moura, J. C. V. P. J. Photochem. Photobio. A 2006,
doi:10.1016/j.jphotochem.2006.06.013.
14. Synthesis of compound 2: Sodium nitrite (1.81 g;
26 mmol) was added in portions, with stirring, to a cold
solution (0 ꢁC) of 3-ethylamino-4-methylphenol (3.78 g;
25 mmol) in concentrated HCl (15 mL). The reaction
mixture was left stirring for 1 h with external cooling. The
precipitate was filtered off, washed with cold 1 M HCl and
dried in vacuum. 5-(Ethylamino)-4-methyl-2-nitrosophe-
nol hydrochloride 2 was obtained as a brown solid (4.05 g,
90%) and was used in the following reaction without
purification. Crossley, M. L.; Turner, R. J.; Hofmann, C.
2. Conclusion
In this work, 5,9-diaminobenzo[a]phenoxazinium dyes
1a–e, containing different functional groups, were syn-
thesised in good to excellent yields. These water-soluble

8570
V. H. J. Frade et al. / Tetrahedron Letters 47 (2006) 8567–8570
M.; Dreisbach, P. F.; Parker, R. P. J. Am. Chem. Soc.
1952, 74, 578–584.
monitored by TLC (silica: chloroform and chloroform–
methanol, 6:1). The solvent was removed under reduced
pressure and the crude mixture was purified by dry
chromatography (silica: chloroform–methanol, 5.5:0.5).
N-(5-(3-hydroxypropylamino)-10-methyl-9H-benzo[a]-
phenoxazin-9-ylidene) ethanaminium chloride 1c was
obtained as a blue solid (204 mg, 97%). Mp above
300 ꢁC. Rf 0.69 (silica: chloroform–methanol, 6:1). FTIR
(KBr, 1%): mmax 3506, 3241, 1641, 1592, 1561, 1544, 1521,
15. Typical procedure for the synthesis of compounds 3a and 3c
(described for 3c): To a solution of 1-naphthylamine (2 g;
14.0 mmol) in ethanol (5 mL), 3-bromo-1-propanol
(1.33 mL; 14.7 mmol) was added and the resulting mixture
was refluxed for 8 h and monitored by TLC (silica:
chloroform–methanol, 5.8:0.2). The solvent was removed
under reduced pressure and the crude mixture was purified
by dry chromatography (silica: chloroform and chloro-
form–methanol, 5.8:0.2). 3-(Naphthalen-1-ylamino) pro-
panol 3c was obtained as a pinkish oil (2.07 g, 70%). Rf
0.30 (silica: chloroform–methanol, 5.7:0.3). FTIR (neat):
1
1451, 1431, 1315, 1185, 1163, 1137, 1087, 1010 cmÀ1. H
NMR (CD3OD, 300 MHz): d 1.41 (2H, t, J 6.3 Hz,
NHCH2CH3), 2.11 (2H, br s, NHCH2CH2CH2), 2.39 (3H,
s, CH3), 3.50–3.68 (2H, m, NHCH2CH2CH2), 3.82 (2H, br
s, NHCH2CH2CH2), 3.87 (2H, br s, NHCH2CH3), 6.92
(1H, s, 8-H), 7.06 (1H, s, 6-H), 7.76 (1H, s, 11-H), 7.80–
7.90 (1H, m, 2-H), 7.96 (1H, t, J 7.5 Hz, 3-H), 8.33 (1H, br
s, 1-H), 8.98 (1H, d, J 7.5 Hz, 4-H) ppm. 13C NMR
(CD3OD, 75 MHz): d 14.2 (NHCH2CH3), 17.7 (CH3),
32.2 (NHCH2CH2CH2), 39.7 (NHCH2CH2CH2), 43.3
(NHCH2CH3), 60.4 (NHCH2CH2CH2), 93.9 (6-C), 94.5
(8-C), 123.6 (Ar–C), 124.7 (1-C), 125.5 (4-C), 128.7 (10-C),
130.8 (2-C), 132.0 (Ar–C), 132.4 (Ar–C), 132.7 (3-C), 132.8
(11-C), 134.3 (Ar–C), 149.2 (Ar–C), 152.8 (Ar–C), 156.6
(9-C), 158.6 (5-C) ppm. The assignments were supported
by HMQC technique. Anal. Calcd for C22H24N3O2Æ
3.5HCl: C, 53.92; H, 5.66; N, 8.58. Found: C, 53.99; H,
5.80; N, 8.37.
mmax 3529–3109, 3046, 2953, 2914, 2846, 1624, 1581, 1524,
1468, 1406, 1368, 1337, 1274, 1249, 1205, 1174, 1124,
1061 cmÀ1. 1H NMR (CDCl3, 300 MHz): d 1.96–2.10 (2H,
m, NCH2CH2CH2), 3.10–3.50 (1H, m, NH), 3.44 (2H,
t, J 6.0 Hz, NCH2CH2CH2), 3.90 (2H, t, J 5.7 Hz,
NCH2CH2CH2), 6.67 (1H, d, J 7.0 Hz, 4-H), 7.28 (1H,
d, J 6.6 Hz, 2-H), 7.38 (1H, t, J 7.5 Hz, 3-H), 7.43–7.51
(2H, m, 6-H and 7-H), 7.78–7.86 (2H, m, 8-H and 5-H)
ppm. 13C NMR (CDCl3, 75.4 MHz): d 31.2 (NCH2CH2-
CH2), 42.5 (NCH2CH2CH2), 61.8 (NCH2CH2CH2), 104.7
(4-C), 117.6 (2-C), 120.0 (5-C), 123.5 (4a-C), 124.7 (7-C),
125.7 (6-C), 126.5 (3-C), 128.5 (8-C), 134.2 (8a-C), 143.3
(1-C) ppm. The assignments were supported by HMBC
and HMQC techniques. HRMS: m/z (FAB): calcd for
C13H15NO [M+] 201.1154; found 201.1157.
17. Photofading of compounds 1a–e: Compounds 1a–e were
dissolved in ethanol or water (pH 7.4) with concentrations
of 1 · 10À5 M. The samples were irradiated at 419 nm, in a
Rayonet RPR-100 chamber reactor with 10 lamps. The
photostabilities were expressed in terms of the remaining
absorption (%) calculated from the change of absorption
intensities at the absorption maximum before and after
irradiation.
16. Typical procedure for the synthesis of 1a, 1c and 1d
(described for 1c): To a cold solution (ice bath) of 5-
(ethylamino)-4-methyl-2-nitrosophenol hydrochloride
2
(185 mg; 5.97 · 10À4 mol) in ethanol (2 mL), 3-(naphthal-
en-1-ylamino) propanol 3a (117 mg; 5.82 · 10À4 mol) and
concentrated hydrochloride acid (5.0 · 10À2 mL) were
added. The mixture was refluxed for 3 h and 30 min and