Molecular recognition of the antiretroviral drug abacavir: Towards the development of a novel carbazole-based fluorosensor

Article abstract of DOI:10.1007/s10895-010-0798-7

Due to their optical and electro-conductive attributes, carbazole derivatives are interesting materials for a large range of biosensor applications. In this study, we present the synthesis routes and fluorescence evaluation of newly designed carbazole fluorosensors that, by modification with uracil, have a special affinity for antiretroviral drugs via either Watson-Crick or Hoogsteen base pairing. To an N-octylcarbazole-uracil compound, four different groups were attached, namely thiophene, furane, ethylenedioxythiophene, and another uracil; yielding four different derivatives. Photophysical properties of these newly obtained derivatives are described, as are their interactions with the reverse transcriptase inhibitors such as abacavir, zidovudine, lamivudine and didanosine. The influence of each analyte on biosensor fluorescence was assessed on the basis of the Stern-Volmer equation and represented by Stern-Volmer constants. Consequently we have demonstrated that these structures based on carbazole, with a uracil group, may be successfully incorporated into alternative carbazole derivatives to form biosensors for the molecular recognition of antiretroviral drugs. The Author(s) 2011. This article is published with open access at Springerlink.com.

Full text of DOI:10.1007/s10895-010-0798-7

J Fluoresc (2011) 21:1195–1204
DOI 10.1007/s10895-010-0798-7
ORIGINAL PAPER
Molecular Recognition of the Antiretroviral Drug Abacavir:
Towards the Development of a Novel
Carbazole-Based Fluorosensor
Krzysztof Ryszard Idzik & Piotr J. Cywinski &
Charles G. Cranfield & Gerhard J. Mohr &
Rainer Beckert
Received: 29 September 2010 /Accepted: 28 December 2010 /Published online: 11 January 2011
#
The Author(s) 2011. This article is published with open access at Springerlink.com
Abstract Due to their optical and electro-conductive
attributes, carbazole derivatives are interesting materials
for a large range of biosensor applications. In this study, we
present the synthesis routes and fluorescence evaluation of
newly designed carbazole fluorosensors that, by modifica-
tion with uracil, have a special affinity for antiretroviral
drugs via either Watson–Crick or Hoogsteen base pairing.
To an N-octylcarbazole-uracil compound, four different
groups were attached, namely thiophene, furane, ethyl-
enedioxythiophene, and another uracil; yielding four dif-
ferent derivatives. Photophysical properties of these newly
obtained derivatives are described, as are their interactions
with the reverse transcriptase inhibitors such as abacavir,
zidovudine, lamivudine and didanosine. The influence of
each analyte on biosensor fluorescence was assessed on the
basis of the Stern–Volmer equation and represented by
Stern–Volmer constants. Consequently we have demon-
strated that these structures based on carbazole, with a
uracil group, may be successfully incorporated into alter-
native carbazole derivatives to form biosensors for the
molecular recognition of antiretroviral drugs.
:
K. R. Idzik (*) R. Beckert
Institute of Organic and Macromolecular Chemistry,
Friedrich-Schiller-University Jena,
Humboldstraße 10,
07743 Jena, Germany
e-mail: krzysztof.idzik@pwr.wroc.pl
:
P. J. Cywinski G. J. Mohr
Institute of Physical Chemistry,
Friedrich-Schiller-University Jena,
Lessingstrasse 10,
.
.
.
Keywords HIV HAART Antiretroviral drugs
07743 Jena, Germany
.
.
Carbazole Base pairing Fluorescence spectroscopy
P. J. Cywinski (*)
Department of Physical Chemistry, Institute of Chemistry,
University of Potsdam,
Karl-Liebknecht-Strasse 24-25,
14476 Potsdam-Golm, Germany
e-mail: piotr.cywinski@uni-potsdam.de
Introduction
Highly Active Anti-Retroviral Therapy (HAART) is cur-
rently the process by which human immunodeficiency virus
(HIV) infection is controlled. The basic principle of
HAART is to use a mixture of at least three antiretroviral
drugs in combination. The basic regimen is to administer
two nucleoside reverse transcriptase inhibitors plus one
non-nucleoside reverse transcriptase inhibitor; or two
nucleoside reverse transcriptase inhibitors plus one protease
inhibitor. The aim of therapy is the long-term inhibition of
HIV replication. Commonly patients undergoing HARRT
C. G. Cranfield
Biomolecular Photonics Group, University Hospital Jena,
Nonnenplan 2-4,
07740 Jena, Germany
G. J. Mohr
Fraunhofer Institute for Reliability and Microintegration (IZM),
Department of Polytronic Systems, Workgroup Sensor Materials,
Josef-Engert-Straße,
93053 Regensburg, Germany

1196
J Fluoresc (2011) 21:1195–1204
therapy suffer from numerous side effects including
abdominal pain, diarrhea, liver failure, hepatitis, and
xeroderma. The ability to monitor antiretroviral drug
concentration on an individual basis will therefore allow
practitioners to create a patient specific regimen to reduce
individual suffering. This would be particularly relevant to
child patients where the clearance rate for drugs like
abacavir has been reported to be twice that of adults [1, 2].
Normally, the pharmacokinetics of nucleotide analog
reverse transcriptase inhibitors (NARTIs) such as abacavir
are determined by the use of liquid chromatography, mass
spectrometry, or the tandem use of both [3–5]. However
these methods are typically slow, expensive and require a
high degree of experimental expertise. A combination of
these methods with optical techniques such as absorbance
or fluorescence detection has also been reported [6–8], and
in a few cases electrochemical detection methods have also
been applied [9, 10]. The increased use of fluorescence
spectroscopy for detection of NARTIs would introduce
numerous advantages, as fluorescence based techniques
have higher sensitivity when compared to absorbance
techniques, and are more specific and less susceptible to
interferences due to low concentrations the fluorosensor
and/or analyte [11]. When compared to other analytical
techniques, such as gas chromatography or HPLC, fluores-
cence based techniques can also exhibit increased cost
efficiency. Consequently, molecular sensors utilizing fluo-
rescence are fast, reliable and can provide high sensitivity.
We report here the synthesis routes and fluorescence
properties of newly synthesized carbazole derivatives
functionalized with a uracil moiety. As guanine is present
in the structure of abacavir, this uracil moiety would have
an affinity to abacavir via mimicking either Watson–Crick
or Hoogsteen base pairing [12, 13].
Carbazole is a well-known hole-transporting and elec-
troluminescent organic molecule. Carbazole derivatives,
because of their chromic and electroconductive effects, are
interesting materials for a large range of electrical, optical
and electro-optical applications [14, 15]. The use of
conducting polymers in biosensor architecture has also
attracted a lot of attention over the years [16]. Carbazole
derivatives are also endowed with fluorescent absorptions
in the ultra-violet spectrum, and emission in the visible part
of the spectrum, making them useful for both single and
multi-photon fluorescence detection [17].
When a long aliphatic chain is attached to the N atom in
the five-membered central ring of carbazole, the resulting
molecule can be easily dissolved in most organic solvents,
with a decrease in the melting temperature [18, 19]. For this
reason we created N-octylcarbazole to act as the base
structure for the carbazole-uracil derivatives presented here.
Four different groups were attached to the opposite
aromatic ring of this N-octylcarbazole-uracil derivative;
namely, thiophene (U1), furane (U2), ethylenedioxythio-
phene (U3) and an extra uracil (U4). We assume the
interaction between our biosensor and the potential analyte
is by the formation of a triplex, as depicted in Scheme 1,
which shows the proposed interaction between the U1 and
U4 derivatives and abacavir. The introduction of a uracil
moiety to the carbazole core alters the photophysical
properties of carbazole by altering the charge distribution
within the molecule, which can then be observed by the shift
of fluorescence towards the red. The uracil moiety then, via
hydrogen binding, enables the molecule to play the role of an
Scheme 1 Proposed formation
of (a) the 1:1 complex of 6-(5-
a
S
uracilyl)-N-octylcarbazole (U1)
and (b) the 1:2 complex 3,6-bis
(5-uracilyl)-N-octylcarbazole
(U4) that are formed with aba-
cavir via Hoogsteen triple
bonding
HO
N
N
N
H
N
O
N
N
H
N
N
H
H
O
N
H
b
O
O
H
H
N
N
N
N
N
N
N
N
H
H
N
N
N
N
N
O
O
OH
N
H
H
N
N
HO
H
N
H
C8H17

J Fluoresc (2011) 21:1195–1204
1197
electron acceptor, with the NARTIs being the electron donors.
The efficiency of this electron transfer can then be seen by
fluorescence quenching of the fluorosensor [20].
[24]. Bromination of structure 2 at the 3,6-positions was
achieved by adding N-bromosuccinimide (NBS) in chloro-
form. A palladium-catalyzed Stille-type coupling reaction
was employed to obtain 3-aryl,6-bromo-N-octylcarbazole
(4) from (2-tributylstannyl)aryl derivatives. In short, the
brominated N-octylocarbazole (1 g, 2.29 mmol) (3) was
placed in a 250 mL round two-bottom flask, under argon, in
anhydrous toluene (100 mL). To this was added the 2-
(tributylstannyl)aryl (2.29 mmol) compounds and Pd
(PPh3)4 (0.230 g, 0.2 mmol). The resulting mixtures were
stirred for 96 h at 120 °C. After this time mixtures were
cooled to room temperature. Water was then added and the
resulting solutions were extracted with 50 mL portions of
CHCl3. The combined organic layers were washed with
50 mL of brine, dried over MgSO4 and evaporated to a dark
brown oil. The crude products were then purified over a
chromatographic silica gel column (hexane/AcOEt, 10:1).
Suzuki coupling was employed in the next step to obtain
the compounds at structure 5. The structure 4 compounds
(1.60 mmol) were dissolved in 100 mL of absolute THF
under argon. This solution was cooled to −78 °C before
1.14 mL of sec-Butyllithium (sec-BuLi, 1.4 M in hexane,
1.60 mmol) was added. The reaction mixture was stirred for
20 min and 2-Isopropoxy-4,4,5,5-tetramethyl-1,3,2-dioxa-
borolane (0.33 mL, 1.60 mmol) was added. After 24 h the
reaction mixture was allowed to warm to room temperature
and 40 mL of water was added. The solutions were
extracted with 50 mL portions of ethyl acetate. The
combined organic layers were washed with 20 mL of brine,
dried over MgSO4 and evaporated to a brown oil. The
products were purified by column chromatography (eluent:
hexane/AcOEt, 9:1).
The functionalization of N-octylcarbazole at the 3 or 6
positions by side groups such as thiophene, furane or
ethylenedioxythiophene improves the chemical stability of
carbazole and increases the mobility of the charge carrier in
regioregular systems [21–23] which demonstrates that
polymers based on polythiophene derivatives are good
materials to be employed as active electrodes in electro-
chemical/electronic devices. It was to compare the photo-
physical properties, and to choose the best derivative for
our purpose, that we employed thiophene, ethylenedioxy
thiophene, and furane, as well as an extra uracil group, to
conjugate to the N-octylcarbazole moiety.
Photophysical properties including absorbance and fluo-
rescence emission spectra, fluorescence lifetime, and
quantum yield were determined for all these N-octylcarba-
zole derivatives. The sensitivities of all the developed
biosensors were tested against the NARTIs abacavir,
lamivoudine, didanosine and zidovudine, in 0.1 M Hepes
Buffer (pH 7.4). The quenching of the N-octylcarbazole-
uracil derivatives as a result of binding to each analyte was
assessed by means of steady-state fluorescence spectrosco-
py. The efficiency of quenching was assessed according to
the Stern–Volmer equation and represented by the Stern–
Volmer quenching constant.
Experimental Section
Materials
In the last step structure 5 compounds (1.03 mmol) were
then mixed with 5-bromouracil (134 mg, 0.70 mmol), Pd
(PPh3)4 (95 mg, 0.082 mmol), and 2 M Na2CO3 aqueous
solution (15 mL, 30 mmol) in toluene (100 mL), and stirred
at reflux under an argon atmosphere for 120 h. Water
(150 ml) and CHCl3 (150 ml) were then added. The organic
phases were separated and the water phases were extracted.
Organic phases were collected together and washed with
water, then brine solution, dried over anhydrous MgSO4,
filtered, and the solvents left to evaporate off. The crude
products were then purified over a chromatographic silica
gel column (hexane/AcOEt, 3:1) to obtain the final
structures of: U1, 3-(2-thienyl)-6-(5-uracilyl)-N-octylcarba-
zole; U2, 3-(2-furyl)-6-(5-uracilyl)-N-octylcarbazole; and
U3, 3-[2-(3,4-ethylenedioxythienyl)]-N-octylcarbazole; (6).
In Scheme 3 we present a two-step synthesis of U4, 3,6-
bis(5-uracilyl)-N-octylcarbazole (8). From structure 3 in
Scheme 2, 3,6-dibromo-N-octylcarbazole reacts according
to the Suzuki-type reaction with an excess of borolane
derivatives to obtain structure 7. The reaction between 3,6-
bis(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-N-octyl-
Abacavir was purchased from Sequoia Research Products
(Pangbourne, UK) while Didanosine was obtained from
TCI Europe (Eschborn, Germany). All other chemicals
were purchased from Sigma-Aldrich (Germany) and used
without any further purification. All solvents, both for
chromatography and spectroscopy, as well as labware, were
from Roth (Karlsruhe, Germany).
Synthesis of Fluorosensors
All chemicals, reagents, and solvents were used as received
from commercial sources without further purification,
except tetrahydrofuran (THF) and toluene, which were
distilled over sodium/benzophenone. Scheme 2 illustrates
the synthetic routes to obtain novel N-octyl-3-(2-aryl),6-(5-
uracilyl)carbazoles (U1–U3). These compounds were syn-
thesized by a five-step procedure. Carbazole (1) was
alkylated by 1-bromooctane in the presence of benzyltrie-
thylammoniumchloride (BTEAC) as a phase-transfer cata-
lyst to yield N-octylcarbazole (2) as described previously

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J Fluoresc (2011) 21:1195–1204
Scheme 2 Reaction conditions
for the production of U1, U2
and U3: (i) C8H17Br (1 eq),
BTEAC (0.4 eq), Benzene/
NaOHaq; 90 °C, 12 h; (ii) NBS
(2.2 eq), CHCl3; 40 °C, 8 h; (iii)
2-(Tributylstannyl)aryl (1 eq),
Pd(PPh3)4 (0.08 eq), Toluene
120 °C, 96 h; (iv) 2-Isopropoxy-
4,4,5,5-tetramethyl-1,3,2-dioxa-
borolane (1.2 eq), sec-BuLi
(1.2 eq), THF, −78 °C, 24 h; (v)
5-Bromouracil (1.4 eq), Pd
Br
Br
Ar
Br
(i)
(ii)
(iii)
N
N
N
N
H
1
(PPh3)4 (0.08 eq), Na2CO3aq
,
2
3
4
Toluene, 90–100 °C, 120 h
O
H
N
O
O
NH
Ar
B
O
Ar
(iv)
(v)
N
N
6
5
Ar
:
4a,
5a,
6a,
S
4b,
5b,
6b,
O
S
4c,
5c,
6c.
O
O
carbazole (7) and 5-bromouracil finally yields U4; 3,6-bis
(5-uracilyl)-N-octylcarbazole (8).
organic layers were washed with 20 mL of brine, dried over
MgSO4 and evaporated to a brown oil. The product was
purified by column chromatography (eluent: hexane/
AcOEt, 9:1). The yield after purification by column
chromatography was 0.87 g (82%) of a light yellow solid.
3,6-bis(5-uracilyl)-N-octylcarbazole (8) was prepared by
mixing 7 (0.4 g, 0.75 mmol) with 5-bromouracil (0.403 g,
2.1 mmol), Pd(PPh3)4 (138 mg, 0.12 mmol), and 2 M
Na2CO3 aqueous solution (20 mL, 40 mmol) in toluene
(100 mL), and stirring at reflux under a argon atmosphere
for 120 h. Water (150 ml) and CHCl3 (150 ml) were then
added. The organic phase was separated and the water
phase was extracted with 3×50 mL of chloroform. Organic
For the preparation of 3,6-bis(4,4,5,5-tetramethyl-1,3,2-
dioxaborolan-2-yl)-N-octylcarbazole (7), N-octylocarbazole
(2) (0.874 g, 2.0 mmol) was dissolved in 100 mL of
absolute THF under argon gas. This solution was cooled to
−78 °C before 3.43 mL of sec-BuLi (1.4 M in hexane,
4.8 mmol) was added. The reaction mixture was stirred for
20 min and 2-isopropoxy-4,4,5,5-tetramethyl-1,3,2-dioxa-
borolane (0.98 mL, 4.8 mmol) was added. After 24 h the
reaction mixture was allowed to warm to room temperature
and 50 mL of water was added. The solution was extracted
with 3×50 mL portions of ethyl acetate. The combined

J Fluoresc (2011) 21:1195–1204
1199
O
Scheme 3 Reaction conditions
for the production of 3,6-bis(5-
uracilyl)-N-octylcarbazole, U4:
(vi) 2-Isopropoxy-4,4,5,5-tetra-
methyl-1,3,2-dioxaborolane
(2.4 eq), sec-BuLi (2.4 eq),
THF, −78 °C, 24 h; (vii) 5-
Bromouracil (2.8 eq), Pd(PPh3)4
(0.12 eq), Na2CO3aq, Toluene,
90–100 °C, 120 h
O
H
H
N
N
O
O
O
O
HN
NH
B
B
O
Br
Br
O
(vi)
(vii)
N
N
N
7
3
8
phases were collected and washed with water, then brine
solution, dried over anhydrous MgSO4, filtered, and the
solvents left to evaporate off. The crude product was
purified over a chromatographic silica gel column (hexane/
AcOEt, 3:1) to give U4, (8).
model and the resulting parameters. The fluorescence
decays were measured using an excitation wavelength of
275 nm, and fluorescence emission was collected at the
maximum of the steady-state fluorescence emission spectra,
being 410 nm (U2), 430 nm (U3), 475 nm (U1) and 480 nm
(U4).
Methods
Sample Preparation
1H NMR spectra were recorded in deuterated chloroform
(CDCl3) on a Brucker 250 spectrometer. Preparative
column chromatography was carried out on glass columns
of different sizes packed with silica gel 60 (Merck) (0.035–
0.070 mm). Mass spectra were recorded on a MAT SSQ
710 from Finnigan. Fluorescence spectra of U1–U4 were
measured in a FluoroLog-3 spectrofluorometer (Horiba
Jobin Yvon, Bensheim, Germany). Fluorescence was
excited at the last excitation band and recorded in the usual
rectangular configuration in a 1-cm cubic quartz cuvette
(Hellma, Mühlheim, Germany) placed in a cuvette holder,
whose temperature was maintained at 25 0.2 °C. Both,
excitation and emission slits were set to 5 nm bandpass.
Fluorescence quantum yield was measured on a C9920-02
Absolute PL Quantum Yield Measurement System (Hama-
matsu) with an integrate sphere unit and a Xenon lamp as
the monochromatic excitation light source. The decays of
fluorescence were collected using an Edinburgh Instru-
ments CD900 single photon counting spectrometer
equipped with a hydrogen-filled coaxial flash lamp as an
excitation source. The measurements were carried out with
the emission monitored at a 90° angle to the excitation. The
data were collected in 1,023 channels with 10,000 counts at
the peak, and the time calibration was 0.053 ns per channel.
The data were analyzed by a least squares reconvolution
procedure using the software package provided by Edin-
burgh Instruments. Goodness of fit was judged in terms of
χ2 value and residuals distribution, and the values for χ2 of
1.3 or lower indicated the appropriateness of the kinetic
Previously prepared solutions of each of the studied
fluorosensors (5×10−4 M) in DMF were used for studying
their interaction with the various analytes. An aliquot
(50 μL) of each solution was dissolved in 5 ml of HEPES
buffer 0.1 M at pH 7.4 (5×10−6 M, OD≤0.1) and then 3 ml
of this solution was transferred to a 1×1 cm quartz cuvette
(Hellma GmbH). Small aliquots (10 μl the smallest) of the
analytes (up to a total volume of 300 μL) were then added
to the cuvette. The pH was controlled using a pH meter
(Hanna Instruments), and the value remained constant
throughout all the measurements.
Results and Discussion
Photophysical Properties of Fluorosensors
Electronic absorbance peaks (λabs), fluorescence emission
spectra (λem), molar absorptivity (ε), quantum yields (ff)
and fluorescence lifetimes (τf) were separately determined
for all the fluorosensors in 0.1 M HEPES buffer. Electronic
absorbance, fluorescence excitation and emission spectra of
10 μM solution of the U1 derivative are shown in Fig. 1a.
A mono-exponential fit of U1’s fluorescence lifetime is
presented in Fig. 1b. The fluorescence lifetimes for all
derivatives, measured at their emission peaks, were in the
range of 7.2 to 8.5 ns in 0.1 M HEPES buffer (excitation
275 nm). Characteristically these compounds show absor-

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J Fluoresc (2011) 21:1195–1204
O
a
1.0
0.8
0.6
0.4
0.2
0.0
NH
N
Absorbance
Excitation
Emission
NH
N
N
O
O
N
HO
N
NH2
HO
N N NH
Abacavir
Zidovudine
NH2
N
O
N
N
N
O
N
S
N
H
250
340
430
520
610
700
O
HO
HO
O
Wavelength [nm]
Lamivudine
Didanosine
b
10000
1000
100
10
Scheme 4 Chemical structures of the nucleotide analog reverse
transcriptase inhibitors used to characterise the N-octylcarbazole
derivatives
Apparatus response
Fluorescence decay
Monoexponential fit
Base Paring Between Fluorosensors and NARTIs
The structures of NARTIs used within the study are shown
in Scheme 4. The fluorosensors U1–U4 were each
incubated with an increasing concentration (up to 50 μM)
of each quencher, and their emission spectra were recorded.
These N-octylcarbazole derivatives exhibited a marked
decrease in fluorescence upon addition of the quencher as
a consequence of base pairing (Figs. 2 and 3). The drop in
fluorescence is attributed to its quenching by photo-induced
electron transfer between the biosensor and nucleotide after
the hydrogen bonds are formed. This mechanism of
interaction between fluorescent dyes and quenchers has
been long established and is well described [26]. The
Stern–Volmer constant, KSV, was used to evaluate the
fluorescence quenching efficiency, according to the Stern–
Volmer equation:
1
0
20
40
60
80
100
Time [ns]
Fig. 1 a Electronic absorbance, fluorescence excitation and emission
spectra; b fluorescence lifetime decay of U1
bance in the range from 250 to 400 nm. In the case of U1,
the fluorescence emission spectrum is broad and positioned
in the blue-green range with maxima at 475 nm and
500 nm, which is characteristic for carbazole moieties [25].
All the photophysical properties of all tested biosensors are
gathered in Table 1.
I0
¼ 1 þ kqt0ðQÞ ¼ 1 þ KSV ðQÞ
ð1Þ
I
Table 1 Photophysical properties of studied fluorosensors. Maxima
of absorbance (λabs), extinction coefficient (ε), maxima of fluores-
cence emission (λem), quantum yield (ff), fluorescence lifetime (τf),
and goodness of fit of the fluorescence lifetime decays (χ2)
characteristic for each of the fluorosensors
Carbazole
λabs (nm)
ε (L mol−1 cm−1
)
λem (nm)
ff
τf (ns)
χ2
U1(tio)
310, 380
275, 295
278, 375
272, 330
14200
15920
14500
17540
480, 510
430
0.276
0.243
0.343
0.397
8.1 1.0
7.2 0.7
8.2 1.2
8.5 1.1
1.119
1.107
1.125
1.091
U2 (furan)
U3 (ditiof)
U4 (diura)
435, 485
485, 515

J Fluoresc (2011) 21:1195–1204
1201
a
where kq is the rate of biosensor quenching, I is
fluorescence intensity in the presence of the quencher (Q),
I0 and τ0 are the fluorescence intensity and lifetime in the
absence the quencher respectively.
1,0
no abacavir
0,8
0,6
0,4
0,2
0,0
The fluorescence spectra of U4 in the presence of an
increasing concentration of abcavir are shown in Fig. 2a.
A decrease in fluorescence intensity (more than 3 fold) is
observed upon the addition of up to 50 μM abacavir,
producing a Stern–Volmer constant (KSV) of 4.6×
104 M−1. The Stern–Volmer plots corresponding to the
fluorescence quenching of U4 in the presence of all the
NARTIs studied are presented in Fig. 2b. This was done to
evaluate the selectivity of the fluorosensor to abacavir
over didanosine, zidovudine and lamivudine since they are
commonly administrated in combination with abacavir.
The Stern–Volmer constants for all studied N-octylcarba-
zole derivatives, with all quenchers, are presented in
Table 2.
50 μM
450
500
550
600
650
700
Wavelength [nm]
b
2,5
Abacavir
The highest KSV values, and consequently the strongest
quenching, were found for U4, which possesses two uracil
moieties. Typically, U4 quenching was 3–5 fold stronger
than for the single-uracil N-octylcarbazoles, being in the
range of 6.2×103 M−1 for zidovudine to 45.3×103 M−1 for
abacavir. For the derivatives U1–U3 the Stern–Volmer
constants for all of the studied analytes were in the range
of 3.7–18.3×103 M−1 for U1, 0.6–9.5×103 M−1 for U2 and
0.9–13.2×103 M−1 for U3. We attribute this enhanced
selectivity of the fluorosensor to abacavir to the presence
of guanine in its structure. It has already been demonstrated
using NMR spectrosopy that guanine binds to uracil via
triple hydrogen bonding stronger than with thymine, adenine
or even cytosine [27, 28]. In the case of thymine and
cytidine, only weak hydrogen interactions are observed; and
in the case of adanine, uracil is only cabable of forming
wobble base pairing. We also tested the interaction of the
fluorosensor against metal ions such as Na+, K+, Ca2+ and
pyrophosphate PPi. We observed up to 5% (PPi) and 10%
(K+ and Na+) of abacavir induced quenching at 150 mM
concentration of each of the possible metal interferents. In
the case of Ca2+, the induced quenching was 18%, possibly
due to coordination of uracil by the calcium ion [29, 30].
Lamivudine
Didanosine
Zidavoudine
2,0
1,5
1,0
0,5
0,0
0
10
20
30
40
50
Drug concentration [μM]
Fig. 2 a Fluorescence emission spectra (λexc=350 nm) for the
quenching of U4 by abacavir.; b Stern–Volmer plots (λem=515 nm)
for all the studied nucleotide analog reverse transcriptase inhibitors.
The concentration of abacavir changes from 0 to 50 μM
2,5
U4
U1
2,0
U3
U2
1,5
1,0
0,5
0,0
Table 2 Stern–Volmer constants (KSV) × 103 [M−1] of the fluoro-
sensors when exposed to the various nucleotide analog reverse
transcriptase inhibitors
Analyte
U1 (tio)
U2 (furan)
U3 (ditio)
U4 (diura)
Abacavir
18,3 0,3
6,2 0.2
4,8 0,1
3,7 0,1
9,5 0,1
2,6 0,1
1,3 0,1
0,6 0,1
13,2 0,2
4,5 0,2
3,1 0,1
0,9 0,1
45,3 0,7
10,3 0,2
8,1 0,2
6,2 0.1
0
10
20
30
40
50
Lamivudine
Didanosine
Zidovudine
[abacavir] [μM]
Fig. 3 Stern–Volmer plots for fluorosensors U1–U4 interacting with
abacavir. The concentration of abacavir changes from 0 to 50 μM

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J Fluoresc (2011) 21:1195–1204
Table 3 Limits of detection (LOD) [μg/ml] of the fluorosensors when
exposed to the various nucleotide analog reverse transcriptase
inhibitors
When the fluorosensor had already been presented to another
analyte by hydrogen bonding, for example in the presence of
abacavir, no further effect was observed. On the other hand,
when abacavir was was added in the presence of PPi or the
other metal ions, there was a marked increase in fluorescence
quenching. This would indicate that abacavir exerts a
stronger influence on the fluorescence response of the
fluorosensors than do metal ions or phosphates that might
also be present in the aqueous media.
Analyte
U1 (tio)
U2 (furan)
U3 (ditio)
U4 (diura)
Abacavir
0.9
2.7
3.5
5.1
2.1
7.2
1.6
4.5
5.5
9.5
0.2
1.1
1.5
1.9
Lamivudine
Didanosine
Zidovudine
9.6
12.3
Quenching as a result of adding benzene, which has no
heteroatoms, was also tested. No appreciable change in
fluorescence intensity was observed (results not shown).
This gives clear evidence that the uracil moiety interacts
with the drugs by formation of hydrogen bonds, which is
what subsequently affects the fluorescence of the N-
octylcarbazole unit. To confirm the formation of the
complex between fluorosensors and abacavir we performed
titration experiments using steady-state fluorescence spec-
troscopy. The Job’s plots for the titration of U1 and U4 with
abacavir are shown in Fig. 4. One can easily distinguish the
formation of a 1:1 complex in the case of the U1:abacavir;
while for U4 possessing two uracil moieties, a 1:2
stoichiometry can be concluded.
Hydrogen binding by its nature is considered as a
mechanism which does not affect the lifetime of fluores-
cence [31]. We made an attempt, nevetheless, to confirm
this mechanism by measuring fluorescence lifetime during
the quenching of the fluorosensors. Analysis of the chemo-
sensor fluorescence decays upon addition of the various
drug concentrations revealed no change in the lifetime;
however the time of measurment was elongated, indicating
a loss of fluorescence intensity. This is clear evidence of the
static character of the quenching, and the formation of
stable complexes in the ground state [25].
In patients, after the administration of a 600 mg oral
dose of abacavir, its concentration, together with its
metabolites, ranges from 0.5 to 7 mg/ml in human fluids
[32, 33]. In order to verify whether our fluorosensors are
able to detect drugs in this range, we determined the limits
of detection (LOD) for each of the NARTIs used according
to the standard formula:
a
1,2
0,8
0,4
0,0
0,0
0,2
0,4
0,6
0,8
1,0
Xabacavir
b
1,2
0,9
0,6
0,3
LOD ¼ 3s=A
Where σ is a standard deviation of the fluorescence
signal produced by the fluorosensor in the absence of a
quencher; and A is the slope of the dependence between the
Table 4 Tolerance limits (TL) in [mM] for the detection 0.1 mM
abacavir in the presence of other nucleotide analog reverse transcrip-
tase inhibitors using the carbazole fluorosensors
Analyte
Tolerance limits [mM]
0,0
0,2
0,4
0,6
0,8
1,0
U1–U3
U4
2.1 0.2
Xabacavir
Lamivudine
Didanosine
Zidovudine
1.2 0.2
2.5 0.4
4.1 0.9
3.2 0.5
5.5 1.1
Fig. 4 Job plots for the complexation of U1 (a) and U4 (b) with
abacavir in 0.1 M HEPES buffer pH 7.4, (λexc=350 nm). The data
presented are an average of 300 measurements

J Fluoresc (2011) 21:1195–1204
1203
Open Access This article is distributed under the terms of the
Creative Commons Attribution Noncommercial License which per-
mits any noncommercial use, distribution, and reproduction in any
medium, provided the original author(s) and source are credited.
fluorosensor fluorescence and the concentration of the
quencher.
The LOD for abacavir was found to be 200 ng/ml
(4.2 μM) being, advantageously, below concentration of
abacavir usually found in real samples (500 ng/ml). All the
LOD can be found in Table 3.
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