OFF-ON fluorescent detection of thymidine nucleotides by a zinc(II)-cyclen complex bearing two diagonal pyrenes

Article abstract of DOI:10.1002/chem.200902734

Of a Zn^II-cyclen complex bearing two diagonal pyrenes (see picture) has been developed that has a high affinit for thymidine nucleotides, and a much greater enhancement in excimer emission for thymidine triphosphate,-diphosphate and-mono-phosphate than for the other DNA bases and a v of nucleotides.

Full text of DOI:10.1002/chem.200902734

COMMUNICATION
DOI: 10.1002/chem.200902734
OFF–ON Fluorescent Detection of Thymidine Nucleotides by a Zinc(II)–
Cyclen Complex Bearing Two Diagonal Pyrenes
[
a]
Zhanghua Zeng and Leone Spiccia*
[
8b]
DNA/RNA nucleotides are molecules that play important
ing in thymidine nucleotide detection. It consisted of: 1) a
[1]
II
roles in cellular metabolism. For example, thymidine nu-
cleotides, including thymidine-monophosphate (TMP), -di-
phosphate (TDP) and -triphosphate (TTP), which are syn-
thesised in vivo from thymidine in reactions catalysed by
dinuclear Zn –N,N’-di(2-picolyl)amine linked to an efficient
III
II
Ir energy acceptor; and 2) a Zn –cyclen-N,N’-dicarbazolyl-
[8b]
3,5-benzene complex (energy donor). Although the emis-
sion enhancement for TTP binding versus the other nucleo-
tide triphosphates was modest (ca. 20%), the selectivity was
four-fold when the energy transfer efficiency was taken into
[2]
thymidine kinase, phosphokinase and diphosphokinase,
are essential building blocks in DNA replication and cell di-
[3]
[8b]
vision. Imbalances in the cellular level of thymidine-tri-
phosphate have been implicated in the development of dis-
eases characterised by defects in the repair or replication of
mitochondrial DNA (mtDNA). These include Alpers syn-
drome, progressive external ophthalmoplegia, mtDNA de-
pletion syndrome and recessive mitochondrial neurogas-
account. This sensor exploited the high affinity of thymi-
dine derivatives for Zn –cyclen complexes (cyclen=
1,4,7,10-tetraazacyclododecane), demonstrated by Kimura
et al., which arises from strong binding of the deprotonat-
ed imide of the thymine base to Zn coupled with comple-
mentary hydrogen bonding between two diagonal NH
groups of cyclen and the two carbonyl oxygen atoms on the
II
[7]
II
[4]
trointestinal encephalomyopathy. As a further example,
the thymidine derivative, 3-azido-3-deoxythymidine (AZT)
is an important HIV-1 RT inhibitor that enters cells mainly
by passive diffusion and is converted into the thymidine nu-
[7]
thymine base.
We report the development of a new monomolecular, flu-
orescent chemosensor for thymidine nucleotides that con-
tains two pyrene moieties located diagonally on a cyclen
macrocycle (CydiPy in Scheme 1). Our objective was to se-
lectively detect thymidine phosphates by exploiting changes
in the monomer and excimer emission properties of the two
pyrene moieties. Indeed, binding of these guests to the
zinc(II) complex of CydiPy, denoted Zn-CydiPy (Scheme 2),
brings the pyrene molecules close together and strongly en-
hances the static pyrene excimer emission at 470 nm (OFF–
ON sensing, vide infra).
[5]
cleotides by cellular enzymes. Due to their many vital
functions, artificial receptors that are selective for thymidine
nucleotides would, therefore, aid efforts to elucidate the
mechanism of DNA replication, the mode of action of HIV
therapeutics and related biological activities.
[9]
Reports on receptors for thymidine nucleotides are very
rare when compared with receptors for adenosine and gua-
[6]
nosine nucleotides. NMR spectroscopy and potentiometric
[7]
titration have been applied but generally have low sensi-
[8]
tivity. Detection by fluorescence emission and, in particu-
lar, via the enhancement in the fluorescence emission from
a chromophore molecule on binding of the nucleotide to the
receptor (OFF–ON sensor) is attractive as the signal is gen-
The synthetic route to the free ligand CydiPy is summar-
ised in Scheme 1. Details of the synthesis and characterisa-
tion of CydiPy, intermediates leading to CydiPy and the
zinc(II) compound, [Zn ACHTUNGRETNN(UNG CydiPy)] ACHTNUGTRENNU(GN ClO ) , are provided as
4 2
[8b]
erated by the binding event. Hong and co-workers report-
ed a two-component receptor that applied this type of sens-
Supporting Information.
II
The interaction between the Zn complex and the DNA
bases or their nucleotides was investigated by measuring
changes in the fluorescence emission of the pyrene units in
the presence of an excess of each analyte. Fluorescence data
obtained for the DNA bases and their nucleotides (thymine
[
a] Dr. Z. Zeng, Prof. L. Spiccia
School of Chemistry, Monash University
Victoria, 3800 (Australia)
(
T); adenine (A); cytosine (C); guanine (G); thymidine
Fax : (+61)39905-4597
mono-, di- and triphosphate (TMP, TDP, TTP); adenosine
mono-, di- and triphosphate (AMP, ADP, ATP); cytidine
mono-, di- and triphosphate (CMP, CDP, CTP); and guano-
E-mail: leone.spiccia@sci.monash.edu.au
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.200902734.
Chem. Eur. J. 2009, 15, 12941 – 12944
ꢀ 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
12941

Figure 1. Fluorescent emission spectra of Zn-CydiPy (5.0 mm, excitation
at 350 nm) measured in 1:9 mixture of CH CN/HEPES buffer
10.0 mm, pH 7.4, T=20.08C) for solutions containing 100.0 mm of TMP,
TDP and TTP and 150.0 mm of the other guests (see text).
a
3
(
[
8b]
TTP.
Given that TMP, TDP and TTP bind strongly to
[7]
zinc(II)–cyclen derivatives, we propose that the enhance-
ment in excimer emission involves coordination of these
species to Zn-CydiPy. Binding of TTP to the Zn-CydiPy
complex also causes minor
Scheme 1. Synthesis of CydiPy.
changes in the UV/Vis spec-
trum, in the form of a decrease
in the pyrene p!p* transition
at 342 nm (see Figure S1 in the
[10]
Supporting Information).
The pyrene excimer emission
at 470 nm increases systemati-
cally with the concentration of
TTP (Figure 2). Fitting of the
data to Equation (1), which as-
sumes 1:1 binding ([Eq. (2)]),
has allowed the determination
of the binding constants (Kb)
for each thymidine derivative at
pH 7.4, yielding K values of
b
ꢀ1
Scheme 2. Proposed binding of thymidine phosphates to Zn-CydiPy.
5
(
0
1
1.46ꢁ0.15)ꢁ10 m ,
(1.52ꢁ
.15)ꢁ10 m and (3.17ꢁ0.26)ꢁ
0 m in CH CN/HEPES (1:9)
5
ꢀ1
5
ꢀ1
3
5
ꢀ1
sine mono-, di- and tri-phosphate (GMP, GDP, GTP)) and
inorganic salts (triphosphate, pyrophosphate (PPi), phos-
buffer solution and (1.41ꢁ0.14)ꢁ10 m , (1.47ꢁ0.15)ꢁ
5
ꢀ1
5
ꢀ1
10 m and (3.11ꢁ0.25)ꢁ10 m in CH CN/Tris-HCl (1:9)
3
ꢀ
ꢀ
phate (Pi), F and Cl ) are summarised in Figure 1. The ad-
dition of 20 equivalents of the thymidine nucleotides (TMP,
TDP and TTP) strongly enhances the pyrene excimer emis-
sion at 470 nm and decreases the monomer emission at
buffer solution for TMP, TDP and TTP, respectively (fluo-
rescence spectra, data fitting and confirmation of the 1:1 sto-
ichiometry of adducts are shown in Figure S2 and S3 in the
Supporting Information). The agreement in the Kb values
determined for the two buffers confirms that binding is un-
375 nm. The enhancement in excimer emission was greater
for TTP (10-fold), than for TDP (6-fold) and TMP (5-fold).
Only minor fluorescence enhancements, if any, were found
when 30 equivalents of the other analytes were added to
Zn-CydiPy. In comparison, Hong and co-workers also found
significant increases in fluorescence emission for the other
nucleotide phosphates (ATP, CTP and GTP) in addition to
affected by chloride (7.4 mm) present in the CH CN/Tris-
HCl buffer mixture.
3
ðFꢀF
Þ=F
¼F
K
½TTPꢂ=F
f1 þK
½TTPꢂg
ð1Þ
ð2Þ
0
0
b
b
0
b
Kb
Zn-CydiPy þTTP !ƒ½Zn-CydiPyðTTPÞꢂ
ƒ
12942
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ꢀ 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2009, 15, 12941 – 12944

Fluorescent Detection of Thymidine Nucleotides
COMMUNICATION
ꢀ
excess of triphosphate, Cl , TDP, TMP, T, PPi and GTP
added to the Zn-CydiPy/TTP host–guest complex. Displace-
ment of TTP from the host–guest complex would be expect-
ed to significantly decrease the excimer emission intensity
but these decreases were small (compare entries 1–5 with
entry 6 for TTP only in Figure 3). The greatest decrease
(
10%) was observed for TDP. The results are consistent
with the binding constants (vide supra), supporting the bind-
ing order: TTP> TDP ꢃTMP. Importantly, the Zn-CydiPy
receptor shows some selectivity for TTP over the other
binding agents.
The potential hydrolysis of thymidine nucleotides needs
II
to be considered as Zn –cyclen complexes can cleave the
[11]
phosphodiester bonds in DNA/RNA sequences. It is also
possible for the ester bonds in the receptor to hydrolyze. A
test of fluorescence stability for a solution containing a 1:1
Figure 2. Change in fluorescence emission spectra of Zn-CydiPy (5.0 mm,
excitation at 350 nm) upon addition of TTP (0, 0.1, 0.2, 0.4, 0.6, 0.7, 1.0,
ratio of Zn-CydiPy and TTP in CH CN/HEPES (1:9) buffer
3
(
10.0 mm, pH 7.4, T=20.08C) showed negligible change
1
.5, 2.5 and 4.0 equiv) in CH
3
CN/HEPES (1:9) buffer solution (10.0 mm,
over a 48 h period (see Figure S4 in the Supporting Informa-
tion).
pH 7.4, T=20.08C).
To account for the enhanced excimer emission in the pres-
ence of the thymidine nucleotides, we propose, as in previ-
ous studies, that these guests bind to Zn via a deprotonat-
F and F are the fluorescence intensities in the absence
0
[7]
II
and presence of the guest, F is the maximum fluorescence
b
intensity.
ed thymine (imide), forming a ZnꢀN bond; this binding
Given that TTP binds more strongly to the Zn-CydiPy re-
ceptor than the other DNA bases and corresponding nucleo-
tides, competition experiments were undertaken to explore
the selectivity of the receptor for TTP. Figure 3 plots the
change in excimer emission intensity relative to that of Zn-
CydiPy; for comparison, the fluorescence enhancement
caused by each guest molecule is included (entries up to
TTP). The top seven entries in Figure 3 show the excimer
emission intensity measured for solutions with a 10-fold
being strengthened by H-bonding (Scheme 2). Interactions
at multiple levels act cooperatively to lock the host–guest
complex into a conformation that brings the pyrene unit
into close proximity leading to strong excimer emission
[13,14]
(static) and quenching monomer emission.
The other
guests studied either do not bind to Zn-CydiPy or, if they
do, they do not promote pyrene–pyrene interactions. For
these guests, the weak excimer emission (dynamic) could be
indicating that, on average, a small fraction of the pyrene
units on the same or different
receptor molecules maybe in-
teracting.
For TMP, TDP and TTP, we
postulate that, at the pH used
in this study (7.4), hydrogen
bonding between the anionic
phosphates and the protonated
pendant amines linking the pyr-
enes to the macrocycle in Zn-
CydiPy (Scheme 2) cause the
enhancement in excimer emis-
sion by bringing these units in
close proximity. Indeed, exci-
mer emission from the 1:1 Zn-
CydiPy:TTP adduct, measured
in the pH range 6.5 to 11.3 (see
Figure S5 in the Supporting In-
formation), substantially de-
creases in the pH range 8–9
Figure 3. Fluorescence change ((FꢀF
0 0
)/F ) at 470 nm for Zn-CydiPy (5.0 mm, excitation at 350 nm) on addition
of 5.0 mm of Cl , F , Pi, PPi, triphosphate, A, G, C, T, AMP, GMP, CMP, TMP, ADP, GDP, CDP, TDP, ATP,
GTP, CTP, TTP; and a mixture of 50.0 mm PPi, GTP, T, TMP, TDP, chloride, triphosphate and 5.0 mm TTP in
(
pK 8.6) confirming the impor-
a
ꢀ
ꢀ
tance of protonation of the
pendant amines in bringing
about this emission.
CH
3 0
CN/HEPES (1:9) buffer solution (10.0 mm, pH 7.4, T=20.08C). F and F are the fluorescence intensities
in the absence and presence of the guest, respectively. The value for Zn-CydiPy is normalised to zero.
Chem. Eur. J. 2009, 15, 12941 – 12944
ꢀ 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
12943

L. Spiccia and Z. Zeng
3
1
P NMR spectroscopy allows mutual interactions be-
tween small receptor molecules and phosphates in DNA/
Acknowledgements
[12]
This work was supported by the Australian Research Council through
the Australian Centre of Excellence for Electromaterials Science. We
thank Prof. S. J. Langford for providing access to the fluorescence spec-
trophotometer.
RNA nucleotides to be probed. NMR titrations of 1.0 mm
TTP, TDP and TMP solutions (1:9 CD CN/HEPES buffer
pD 7.6)) confirmed a 1:1 adduct stoichiometry in each case
Figure 4), in keeping with the fluorescence experiments.
3
(
(
Keywords: fluorescence · hydrogen bonds · macrocycles ·
sensors · thymidine nucleotides · zinc
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Received: October 5, 2009
Published online: October 27, 2009
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Chem. Eur. J. 2009, 15, 12941 – 12944