Synthesis and photophysical properties of novel succinimidyl benzazole derivatives, evaluated by Candida albicans ATCC 10231 fluorescent staining

Article abstract of DOI:10.1016/j.tetlet.2011.04.026

New fluorescent succinimidyl benzazole derivatives were synthesised and successfully used to stain Candida albicans ATCC 10231 cells. The dyes were characterised by means of infrared, ¹³C and ¹H NMR spectroscopies and elemental analy

Full text of DOI:10.1016/j.tetlet.2011.04.026

Tetrahedron Letters 52 (2011) 3048–3053
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Tetrahedron Letters
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Synthesis and photophysical properties of novel succinimidyl benzazole
derivatives, evaluated by Candida albicans ATCC 10231 fluorescent staining
Rosane Catarina dos Santos a, Nalva Vivian da Silva Faleiro a, Leandra Franciscato Campo a,
Maria Lúcia Scroferneker b, Valeriano Antonio Corbellini c, Fabiano Severo Rodembusch a, Valter Stefani a,
⇑
a Instituto de Química, Universidade Federal do Rio Grande do Sul, Av. Bento Gonçalves 9500, CP 15003, 91501-970 Porto Alegre-RS, Brazil
b Instituto de Ciências Básicas da Saúde, Universidade Federal do Rio Grande do Sul, Rua Sarmento Leite 500, 90050-170 Porto Alegre-RS, Brazil
c Departamento de Química e Física, Universidade de Santa Cruz do Sul (UNISC), Av. Independência 2293, 96815-900 Santa Cruz do Sul-RS, Brazil
a r t i c l e i n f o
a b s t r a c t
Article history:
New fluorescent succinimidyl benzazole derivatives were synthesised and successfully used to stain
Candida albicans ATCC 10231 cells. The dyes were characterised by means of infrared, 13C and 1H NMR
spectroscopies and elemental analysis. UV–Vis and steady-state fluorescence in solution were also
applied to characterise their photophysical behaviour. The novel dyes were fluorescent in the yellow–
green region by a phototautomerism in the excited state (ESIPT) with a large Stokes shift (9065–
10962 cmÀ1). Dual fluorescence could also be observed depending on the solvent polarity. The present
dyes were used as new probes by means of culture methodology or direct staining to study the micro-
morphology of Candida albicans.
Received 18 January 2011
Revised 1 April 2011
Accepted 6 April 2011
Available online 19 April 2011
Keywords:
Fluorescent probes
ESIPT
Succinimidyl dyes
Epifluorescence
Candida albicans
Ó 2011 Elsevier Ltd. All rights reserved.
Fluorescent probes with improved properties such as higher
light, air and temperature stability during storage, and which can
be used as a fast, economic and efficient method to stain fungal
cells always present great interest.1–8 Particular attention has been
given to benzazoles as fluorescent probes,9–12 since these com-
pounds show a large Stokes shift through an excited state intramo-
lecular proton transfer (ESIPT) mechanism.13,14 In the ESIPT
mechanism (Fig. 1), UV light absorption by the enol (E) produces
the excited enol (E⁄), which is quickly converted to an excited keto
tautomer (K⁄) by intramolecular proton transfer since the hydro-
gen becomes more acidic and the nitrogen becomes more basic
in the excited state. The excited keto tautomer (K⁄) decays, thereby
emitting fluorescence, to a keto tautomer in the ground state (K).
Since the enol conformer (E) is more stable than the keto tautomer
in the ground state, the initial enol form is regenerated without
any photochemical change.14
Fungemia is a frequent disease in humans, especially among
neonates, the elderly and HIV patients.15 Candida albicans is an
opportunistic fungal pathogen, and is the most common fungal
infection in hospitalised patients.16,17 C. albicans belongs to the nor-
mal human microflora and can drive systemic life-threatening dis-
ease in cancer, immunosuppressed, post-surgical and trauma
patients.18 Several fluorescent dyes have been used to indicate
Figure 1. ESIPT mechanism.
yeast physiological states, which are used in novel methods for
in vitro antifungal susceptibility tests.19–24 Morphological changes
in C. albicans can be followed by direct fluorescence staining with
suitable dyes to visualise the different features of cellular activi-
ties.25 However, pathogenic fungi, like C. albicans, require accurate
morphophysiological analysis for diagnostic and therapeutic pur-
poses. The study of C. albicans morphogenesis is very significant
since its ability to switch between its budding and filamentous
forms is crucial for its pathogenicity.26,27 In this way, the develop-
ment of new probes which are able to show morphological
changes, viability and metabolic activities is of extreme relevance.
⇑
Corresponding author. Tel.: +55 51 3308 7206; fax: +55 51 3308 7304.
E-mail address: vstefani@iq.ufrgs.br (V. Stefani).
0040-4039/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2011.04.026

Rosane Catarina dos Santos et al. / Tetrahedron Letters 52 (2011) 3048–3053
3049
Figure 2. Synthesis of the succinimidyl benzazole derivatives 5a–b and 8a–b.
Figure 3. Normalised UV–Vis absorption spectra of the succinimidyl benzazole derivatives 5a–b and 8a–b.
Succinimidyl derivatives28,29 have been used to label proteins in
biological processes,30 fluorescence microscopy31 and apoptosis
studies in disease.32–34 Although the benzazole dyes have been
used as fluorescent probes and in staining protocols, the major dif-
ficulty usually involves dye solubility with lower antifungal prop-
erties, which provide more safety as probes in fungal cell
cultures.10–12 In this way, we present in this Letter the synthesis
and photophysical characterisation of novel succinimidyl benzaz-
ole dyes, which could increase dye solubility in the aqueous phase
regarding non-substituted precursors, and the staining of C. albi-
cans cell structures using simple protocols.
The benzazole precursors were prepared using a previously de-
scribed methodology.14 The succinimidyl derivatives were pre-
pared according to the scheme in Figure 2. To a solution of the
corresponding benzazole dye in diethyl ether (20 ml) was added
an equimolar amount of the succinic anhydride (2) which was at
room temperature for 2 h. The obtained yellow precipitate was fil-
tered, dried and used without further purification. In a second
reaction step, this precipitate was heated in glacial acetic acid
(25 mL) at reflux for 8 h. The mixture was poured into ice and fil-
tered. The crude product was washed with water and cold diethyl
ether and dried at 60 °C. Purification was performed by column

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Rosane Catarina dos Santos et al. / Tetrahedron Letters 52 (2011) 3048–3053
Table 1
Relevant fluorescence emission data of the succinimidyl benzazole derivatives
Dye
Solvent
UV–Vis absorption
Fluorescence emission
kmax (nm)
emax (Â104Ál molÀ1 cmÀ1
)
Enol
Keto
kmax (nm)
D
kST (nm/cm-1
)
ufl
kmax (nm)
D
kST (nm/cmÀ1
)
ufl
5a
5b
8a
8b
Chloroform
Ethanol
1-Octanol
Chloroform
Ethanol
1-Octanol
Chloroform
Ethanol
1-Octanol
Chloroform
Ethanol
329
332
329
345
345
340
341
343
324
339
336
340
2.86
2.37
2.46
2.95
2.80
3.16
1.17
0.57
1.31
1.92
0.86
1.95
374
370
—
45/3657
38/3093
—
0.015
0.011
—
474
473
472
502
—
501
508
—
145/9298
141/8979
143/9209
157/9065
—
161/9452
167/9640
—
0.174
0.040
0.102
0.103
—
0.022
0.156
—
—
—
—
389
387
—
424
409
—
44/3279
47/3572
—
81/5570
85/6414
—
0.026
0.029
—
0.076
0.006
—
490
515
—
166/10456
176/10081
—
0.025
0.061
—
357
396
21/1751
56/4159
0.044
0.006
1-Octanol
542
202/10962
0.059
Figure 4. Normalised fluorescence emission spectra of the succinimidyl benzazole derivatives 5a–b and 8a–b.
chromatography using chloroform as the eluent or by recrystallisa-
tion using ethanol.
Figure 4 presents the fluorescence emission spectra of the succ-
inimidyl derivatives. As expected, a dual fluorescence emission
could be observed for all dyes in different solvents, since the ESIPT
mechanism is quite dependent on solvent polarity. Dye 5a presents
in chloroform and ethanol a main emission band located at 470 nm
and an additional one, blue shifted, located at 368 nm. One main
emission band located at 470 nm was observed in 1-octanol. Usu-
ally, the dual fluorescence emission presents a band at higher
wavelengths attributed to an excited keto tautomer (K), which
arises from the enol conformer (EI) in the excited state and a blue
shifted one due to the conformational forms which are stabilised in
solution and present normal relaxation (EII–IV) (Fig. 5).14,35
The UV–Vis absorption emission spectra were made in chloro-
form, ethanol and 1-octanol and were normalised (Fig. 3). 1-Octa-
nol was used in this protocol to simulate an apolar organic
environment, which could lead to selective staining of the hydro-
phobic portion of the cell wall. All the experiments were performed
at room temperature (25 °C) at a concentration of 10À6 M. The rel-
evant data from the UV–Vis and fluorescence emission are summa-
rised in Table 1 (see also Supplementary Tables 1 and 2).
As it can be seen in Figure 3, the dyes present an absorption
maximum located at 329–348 nm with molar absorptivity coeffi-
cient
e
values (104Ál molÀ1 cm-1) in agreement with
p
–
p⁄ transi-
Dye 5b presents one main band in chloroform and ethanol lo-
cated at 502 and 389 nm, ascribed to the ESIPT and the normal
band, respectively. On the other hand, a dual fluorescence emission
tions. The observed difference in these values can probably be
associated with the difference in planarity of the dyes.14

Rosane Catarina dos Santos et al. / Tetrahedron Letters 52 (2011) 3048–3053
3051
atom, as well as in the solvent polarity, play a fundamental role in
the photophysical behaviour of the succinimidyl benzazole
derivatives.
Vegetative cells of C. albicans ATCC 10231 were obtained by cul-
ture on YEPD agar medium (1% yeast extract, 1% peptone, 4% D-glu-
cose, 2% agar) with incubation at 37 °C for 48 h. The inoculum was
prepared for the experiment from 24 h cultures grown at the same
temperature. The C. albicans culture was activated by means of
three repetitive culture steps. A preliminary assay was performed
to observe cellular viability (24 h-old cultures) (Fig. 6F). Two differ-
ent protocols were used to analyse the cells by epifluorescence
microscopy.36
Both staining protocols presented similar behaviour. A small
difference could be observed in the fluorescence intensity distribu-
tion over the cellular structures. Figure 6 presents the images ob-
tained from Protocol A, with a better image resolution and
suitable fluorescence intensity. The results probably indicate a
higher specificity of the dyes for intracellular components from
the cellular wall or other membrane structures. The C. albicans
ovoid cells present fluorescence emission in the green or blue re-
gion after staining with the dyes presented in this work (Figs. 6
and 7). In a preliminary test to study cellular viability, intense fluo-
rescence could be observed in some cells (Fig. 6F), which possibly
indicates a selective characterisation of different cell metabolic
states.
Figure 5. Enol conformer stabilised in solution in the ground state.
could be observed in 1-octanol, with the main band at around
387 nm (normal band) and a red shifted one located at 501 nm
(ESIPT band). It is worth mentioning that the intensities of the
emission bands are changed in relation to those observed with
the dye 5a in chloroform and ethanol.
Derivatives 8a–b present in chloroform one main band located
at 508 nm (8a) and on at 515 nm (8b) ascribed to the ESIPT band.
Ethanol seems to stabilise the conformers which decay by normal
relaxation, since one main band, blue shifted in relation to the
ESIPT band, could be observed with the dyes 8a–b located at 424
and 350 nm, respectively. A dual fluorescence emission could be
observed using 1-octanol. The ESIPT bands are located at around
490–542 nm, while the blue shifted ones are located at 369–
409 nm. The observed results indicate that changes in the hetero-
Protocol B was more sensitive to dye concentration (Fig. 7A and
B). Higher dye content in relation to the one used in this protocol
led to irregular impregnation, with dye deposition on the fungal
structures and glass slide surfaces. A dye content of 65
lM pro-
duced better image resolution, where some topographic details
Figure 6. Candida albicans morphology examined by epifluorescence microscopy after staining with the dyes 3a (A), 7a (B), 5a (C), 5b (D), 8b (E) and 8b (F) (preliminary assay
to observe cellular viability) using Protocol A and a V-2A filter (Bar = 10 m).
l

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Rosane Catarina dos Santos et al. / Tetrahedron Letters 52 (2011) 3048–3053
Figure 7. Candida albicans morphology examined by epifluorescence microscopy after staining (Protocol B, dye concentration 65
filter). The unstained sample (C) and one stained with Calcofluor white (D) prepared with the same methodology used for the dyes 5a and 8b (V-2A filter) are also presented
for comparison (Bar = 10 m)
lM) with dyes 5a (A) and 8b (B) (V-2A
l
of the cell surface could be observed. In (Fig. 7C, an image from the
unstained slide is also shown which presents weak blue intrinsic
fluorescence. Calcofluor white was used for comparison (Protocol
B) under similar experimental conditions, however at a different
dye concentration, and presented worse resolution and excess
brightness. Since the experimental conditions used in the staining
protocols were inadequate for succinimidyl ring opening, the ben-
zazole derivatives were used in their ‘non-active’ forms (closed
ring), which forms non-covalent bonds between the dyes and the
C. albicans cell structures.
All the dye solutions used for staining C. albicans cells presented
photophysical stability when tested after six months of storage at
room temperature. The succinimidyl derivatives 5a–b and 8a–b
presented similar staining properties when compared to their pre-
cursors (Fig. 6A–B).The slides with the stained cells also kept their
fluorescence after four months of storage at room temperature.
This photophysical behaviour can be very useful for teaching meth-
odology in mycology studies. Additional studies are in progress to
use the succinimidyl benzazole derivatives in antifungal activity
assays to observe morphological changes associated with anti-
fungal effects, such as loss of cell viability and cell growth.
In conclusion, four new fluorescent succinimidyl benzazole
derivatives were synthesised and used to stain C. albicans ATCC
10231 cells. The new probes are fluorescent in the yellow-green re-
gion by an intramolecular proton transfer mechanism (ESIPT) with
a large Stokes shift (9065–10962 cmÀ1). The dyes were successfully
used as new dyes by means of a culture method or by direct stain-
ing to study the micromorphology of C. albicans.
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Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.tetlet.2011.04.026.

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concentrations: 5.0 and 2.5 mM and 65 lM. The C. albicans cells were directly
squashed with the dye solutions and dried at room temperature (25 °C). It is
worth mentioning that the selection of 1-octanol in this protocol was to
simulate an apolar organic environment, which could lead to selective staining
of the hydrophobic portion of the cell wall. The excess dye was removed by
washing with ethanol. In this step, an unstained slide was used for comparison.
A slide stained with Calcofluor white (1 mM) was also used as a standard to
detect chitin-rich areas of the cell wall.
36. Protocol
A (the culture protocol): An initial solution of the dyes in
dimethylsulphoxide (40 mM) was diluted in culture media (Sabouraud
dextrose agar, 1:100 v/v) at 60 °C. After its gelification, the C. albicans (24 h
culture) was inoculated and kept at 35 °C for 24 h. In the following stage, one
drop of distilled water was placed on the centre of a clean glass slide. The
stained treated cells were taken and spread over the drop of water and dried at