Selective fluorescence sensing of deoxycytidine 5′-Monophosphate (dCMP) employing a bis(diphenylphosphate)diimine ligand

Article abstract of DOI:10.1007/s10895-010-0703-4

A new bis(diphenylphosphate)diimine ligand (BP1) was prepared and evaluated for its ability for selective detection of deoxycytidine 5′-monophosphate (dCMP). BP1 exhibited off-type fluorescence in the presence of dCMP. The fluorescence of BP1 was signific

Full text of DOI:10.1007/s10895-010-0703-4

J Fluoresc (2011) 21:187–194
DOI 10.1007/s10895-010-0703-4
ORIGINAL PAPER
Selective Fluorescence Sensing of Deoxycytidine
5′-Monophosphate (dCMP) Employing a Bis
(diphenylphosphate)diimine Ligand
Weerachai Nasomphan & Pramuan Tangboriboonrat &
Srung Smanmoo
Received: 25 January 2010 /Accepted: 19 July 2010 /Published online: 12 August 2010
#
Springer Science+Business Media, LLC 2010
Abstract A new bis(diphenylphosphate)diimine ligand
(BP1) was prepared and evaluated for its ability for
selective detection of deoxycytidine 5′-monophosphate
(dCMP). BP1 exhibited off-type fluorescence in the
presence of dCMP. The fluorescence of BP1 was signifi-
cantly quenched upon the addition of 2.5×10−4 M dCMP
and the detection limit was 1.25×10−5 M in MeCN-H2O
(1:1, v/v). The binding ratio between BP1 and dCMP was
determined to be 1:1 with the binding constant of 3.98
0.60×10−3 M−1.
sciences since they provide excellent degree of selectivity
and sensitivity for the detection of biological and chemical
analytes [8–15]. In general, the design of chemosensors
bases on the incorporation of two functional moieties which
are: i) ionophore (the receptor part) and ii) chromophore
(the signal generating unit). The binding of the analyte to
the ionophore initiates the communication between the
receptor and the signaling unit to induce the change of
chromophore’s physical properties [16–18]. Physical
changes of the chromophore can be of various types, e.g.
chemiluminescence (CL), redox potentials, absorption and
fluorescence (FL) [19–34]. Of these, fluorescence chemo-
sensor receives much research attention since it provides
the detection of analytes with low cost sampling and high
degree of selectivity and sensitivity [35–37].
.
Keywords Fluorescence (FL) sensor Deoxycytidine
.
5′-monophosphate (dCMP) sensor Diimine ligand (DLs)
Introduction
Bisphosphonate and its derivatives are well recognized for
their applications as diagnostic tools in the cancer treatment
[38–42]. Particularly, aminobisphosphonate (NBP) is known
as an anti-cancer drug. The mode of action for an anti-cancer
activity of NBP is specific for prostate cancers. Apart from
its medical roles, NBP is known for its ability as a metal
chelator [43–45]. Gummiena and his group demonstrated the
ability of β-aminobisphosphonate derivatives as chelating
agents for copper (II) ion employing electron paramagnetic
spectroscopy (EPR) [46].
Analytes are usually present in minute amounts in
biological systems. Therefore, sensitive and selective
detection of analytes with real-time are highly desired [1–
7]. Fluorescence chemosensors have found their applica-
tions in various fields, e.g. environmental and biomedical
:
W. Nasomphan S. Smanmoo (*)
It is known that the bisphosphate moiety exhibits the
fluorescence quenching via the photon-induced electron
transfer (PET) mechanism. Diimine ligands (DLs) are
known as metal chelators and also as fluorophores due to
their photophysical properties [47]. Based on these facts,
the incorporation of these components would emerge a new
fluorescence chemosensor. Consequently, we have devel-
oped new bisphosphate derivatives based on diimine
ligands and evaluated for their FL sensing towards
Bioresources Research Unit,
National Center for Genetic Engineering and Biotechnology
(BIOTEC) Thailand Science Park,
Phaholyothin Road,
Klong Luang, Pathumthani 12120, Thailand
e-mail: srung.sma@biotec.or.th
:
W. Nasomphan P. Tangboriboonrat
Department of Chemistry, Faculty of Science, Mahidol University,
Rama 6 Road,
Phyathai, Bangkok 10400, Thailand

188
J Fluoresc (2011) 21:187–194
nucleotides. The introduction of bisphosphate group into DL
is aimed to facilitate the optimal hydrogen bonding between
the receptor and nucleotide substrates. The detection deox-
ycytidine 5detection deoxycytidine-monophosphate (dCMP)
is normally achieved employing high-performance liquid
chromatography (HPLC) [48]. Up to date, the fluorescent
(FL) chemosensor for dCMP is not yet reported. To the best
of our knowledge, the chemosensor based on diimine
bisphosphate has not been reported as a selective receptor
for dCMP.
solid was collected by filtration and crystallized from
methanol to obtain the final product in good yields.
3,3′-[1,2-Phenylenebis(nitrilomethylidene)]bis-phenol (DL1)
1
DL1 was obtained as white crystals (2.16 g, 74 %). H
NMR (DMSO-d6) δH 7.65 (d, 1H), 7.60 (m, 2H), 7.41 (q,
1H), 7.19 (m, 1H), 6.89 (d, 2H), 6.64 (d, 2H), 5.41 (s, 2H).
MS (ESI) [M+H]+ 317.13.
4,4′-[1,2-Phenylenebis(nitrilomethylidene)]bis-phenol
(DL2)
Experiments
1
DL2 was obtained as white crystals (2.40 g, 82 %). H
Apparatus
NMR (DMSO-d6) δH 8.94 (s, 2H), 7.67 (d, 2H), 7.46 (m,
4H), 7.41 (m, 4H), 6.97 (m, 4H). 13C NMR δ 117.6, 119.1,
119.4, 119.8, 127.9, 132.5, 133.5, 142.6, 161.5, 163.8. MS
(ESI) [M+H]+ 317.35.
Fluorescence measurements were carried out using a FP-
6300 spectrofluorometer (JASCO) equipped with a xenon
lamp source and a 1.0-cm quartz cell, and the scan speed
was 600 nmmin−1. H spectrum was recorded on Bruker
Synthesis of N,N′-Phenylenebis[2-hydroxybenzylidene-2-
1
DPX 400 MHz spectrometer in CDCl3 using TMS as the
internal standard. Mass spectra were recorded on Bruker
Esquire and Finnigan MAT INCOS 50 mass spectrometers.
benzylidenealdimine] (MP)
In a flame dried round bottom flask, DL1 (0.5 g, 1.58 mmol)
was added followed by addition of dry THF (5 mL). The
reaction mixture was stirred until the disappearance of DL1
and the solution was brought to −80 °C followed by addition
of potassium tert-butoxide (0.19 g, 1.74 mmol). The reaction
mixture was left stirring at room temperature for 3 h before
the reaction solution was cooled down to −80 °C followed
by the addition of diphenyl chlorophosphate (0.33 mL,
1.58 mmol). The reaction mixture was left stirring at room
temperature overnight before the addition of saturated
aqueous NH4Cl solution followed by extraction with EtOAc
(3×10 mL). The organic phase was washed with brine and
dried over MgSO4. The solvent was removed under a
vacuum and the crude product was chromatographed on a
silica gel column. Elution of the column with a mixture of
EtOAc and hexane (2:8) afforded the desired product as clear
Reagents
All reagents for the synthesis of diimine ligand phos-
phates obtained commercially were used without further
purification. The stock solution of diimine ligand phos-
phates was prepared in acetonitrile with the concentration
of 10 mM. The corresponding nucleotide monophosphates
[deoxycytidine 5´-monophosphate (dCMP); deoxyadeno-
sine 5´-monophosphate (dAMP); deoxycyclicadenosine
5´-monophosphate (dcAMP); deoxythymidine 5´-mono-
phosphate (dTMP); deoxyguanosine 5´-monophosphate
(dGMP); uridine 5´-monophosphate (UMP); adenosine
5´-monophosphate (AMP); guanosine 5´-monophosphate
(GMP); cytidine 5´-monophosphate (CMP); thymidine
5´-monophosphate (TMP)] used in this study were
purchased from Sigma Aldrich (USA) and used without
further purification. Methanol was used as a HPLC
grade. All other chemicals used were supplied from
Sigma Aldrich (USA) as analytical grade and used
without further purification. MilliQ water was used
throughout this study. The concentration of stock
solution of metal ions was 1 mM.
1
oil (0.54 g, 62 %). H NMR (DMSO-d6) δH 8.40 (s, 1H),
7.70 (d, 2H, J=8.51), 7.39 (d, 1H, J=8.50), 7.30 (t, 2H, J=
15.77), 7.22 (m, 2H), 7.08 (t, 2H, J=7.81 and 15.53), 6.92
(d, 1H, 7.49), 6.61 (d, 1H, J=7.74), 6.48 (d, 1H, J=7.49),
6.38 (s, 1H), 5.45 (s, 2H).13C NMR δ 111.13 (ArCH),
117.47 (ArCH), 119.57 (ArCH), 119.74 (ArCH), 119.79
(ArCH), 119.88 (ArCH), 119.92 (ArCH), 120.54 (ArCH),
121.38 (ArCH), 121.70 (ArCH), 122.61 (ArCH), 123.22
(ArCH), 123.66 (ArCH), 125.54 (ArCH), 125.94 (ArCH),
126.04 (ArCH), 126.36 (ArCH), 128.77 (ArCH), 129.35
(ArCH), 130.04 (ArCH), 130.19 (ArCH), 130.28 (ArCH),
130.76 (ArCH), 130.89 (ArCH), 131.90 (ArC), 135.91
(ArC), 139.37 (ArC), 142.53 (ArC), 149.64 (ArC), 149.71
(ArC), 150.17 (C=NH), 151.69 (C=NH). HRMS (ESI+)
[M+Na]+ found 571.1392 [M+H]+, calcd for C32H25N2O5P
General Synthesis of Diimine Ligands
DL was synthesized from the condensation between a
corresponding benzaldehyde (1 mmol) and o-phenylenedi-
amine (1 mmol) in the presence of distilled water (10 mL).
After the reaction mixture was left stirring for 6 h, the crude

J Fluoresc (2011) 21:187–194
189
Fig. 1 Chemical structures of
BP1, BP2 and MP. Conditions
and Reagents; (a) (i) 2.2 equiv
tert-BuOK, THF, room temper-
ature, 2 hrs (ii) 2 equiv ClP(O)
(OPh)2, −80 °C, overnight. (b)
(i) 1.1 equiv tert-BuOK, THF,
room temperature, 2 hrs (ii) 1
equiv ClP(O)(OPh)2, −80 °C,
overnight
(b)
(a)
N
N
N
N
N
N
OH
O
OH
HO
O
O
O P
P
O O P
OPh
OPh
OPh
PhO
OPh
OPh
BP1
MP
DL1
(a)
N
N
N
N
O
O
HO
OH
P
O
O P
PhO
OPh
OPh
OPh
DL2
BP2
548.1501. IR (KBr, cm−1) 3417, 3071, 2615, 1941, 1788,
1606, 1416, 1389, 1025, 964, 849, 777.
135.81 (ArC), 142.61 (ArC), 149.77 (ArC), 152.13
(C=NH). HRMS (ESI+) [M+Na]+ found 803.1687 [M+H]+,
calcd for C44H34N2O8P2 780.1790. IR (KBr, cm−1) 3064,
2929, 2870, 2611, 1942, 1867, 1783, 1730, 1588, 1487,
1449, 1389, 1298, 1186, 1161, 1090, 1072.
Synthesis of N,N′-Phenylenebis[2-benzylidenealdimine]
phosphate (BP1)
BP1 was synthesized according to the procedure described
for the synthesis of MP except 2 molar equiv of diphenyl
chlorophosphate was used. H NMR (DMSO-d6) δH 8.40
Synthesis of N,N′-Phenylenebis[3-benzylidenealdimine]
phosphate (BP2)
1
(s, 1H), 7.80 (d, 2H, J=8.67), 7.72 (d, 1H, J=8.96), 7.42
(m, 13H), 7.26 (m, 19H), 7.06 (d, 2H, J=8.61), 5.59 (s,
2H).13C NMR δ 111.14 (ArCH), 115.26 (ArCH), 119.36
(ArCH), 119.91 (ArCH), 119.97 (ArCH), 120.30 (ArCH),
120.35 (ArCH), 120.41 (ArCH), 121.76 (ArCH), 122.42
(ArCH), 122.94 (ArCH), 125.97 (ArCH), 126.08 (ArCH),
128.10 (ArCH), 128.79 (ArCH), 130.24 (ArCH), 130.32
(ArCH), 130.49 (ArCH), 131.16 (ArC), 134.53 (ArC),
BP2 was similarly prepared according to the procedure for
BP1 except DL2 was used as a starting material. H NMR
1
(DMSO-d6) δH 7.82 (d, 2H, J=8.65), 7.44–7.39 (m, 15H),
7.31–7.20 (m, 14H), 7.15 (d, 2H, J=7.15), 5.60 (s, 2H).13C
NMR δ 111.14 (ArCH), 115.26 (ArCH), 119.37 (ArCH),
119.87 (ArCH), 120.36 (ArCH), 121.80 (ArCH), 122.41
(ArCH), 125.61 (ArCH), 126.02 (ArCH), 128.10 (ArCH),
130.14 (ArCH), 131.16 (ArCH), 135.83 (ArCH), 142.64
Fig. 2 The effect of various
nucleotides on the FL intensities
of MP, BP1 and BP2 in MeCN:
H2O (1:1; v/v). [receptor] =
2.5×10−6 M. [nucleotides] =
2.5×10−4 M. λex=280 nm

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J Fluoresc (2011) 21:187–194
Fig. 3 FL changes of BP1 (2.5×10−6 M) in the presence and absence of nucleotides (a) BP1 + dAMP (b) BP1 + dcAMP (c) BP1 + dTMP (d)
BP1 + dGMP (e) BP1 + AMP (f) BP1 + GMP (g) BP1 + CMP (h) BP1 + TMP (i) BP1 + UMP and (j) BP1 + dCMP. [nucleotides] = 5×10−4
M
(ArCH), 142.77 (ArCH), 149.08 (ArCH), 149.83 (ArCH),
150.88 (ArCH), 151.51 (ArCH), 152.14 (ArC), 153.67
(ArC), 153.95 (ArC), 157.07 (ArC), 159.07 (ArC), 161
(C=NH). HRMS (ESI+) [M+Na]+ found 803.1681 [M+H]+,
calcd for C44H34N2O8P2 780.1790. IR (KBr, cm−1) 3451,
3268, 3070, 3007, 2881, 2512, 2249, 2123, 1996, 1828,
1768, 1621, 1374, 1223, 1056, 882, 759.
gen bonding between the receptor and analytes. The
synthesis of MP was achieved by reacting DL1 with 1
equiv of diphenylchlorophosphate. BP1 was prepared in
moderate yield (62%).
Fluorescence Screening of MP, BP1 and BP2 with Various
Nucleotide Substrates
Obtaining BP1, BP2 and MP, FL quenching experiments of
these receptors in the presence of various nucleotides were
investigated. With reference to the screened nucleotides, the
FL property of BP1 exhibited a significant quenching in the
presence of deoxycytidine 5′-monophosphate (dCMP)
(Fig. 2). It was shown that after the addition of 2.5×
10−4 M dCMP, the FL intensity of BP1 was significantly
inhibited. The presence of other nucleotides did not
significantly quench the FL of BP1. BP2 and MP showed
no degree of FL quenching upon treating with any
nucleotides. It was shown that the presence of bisphosphate
group at the meta position on the aromatic ring of BP1 is
necessary for proper chelation between BP1 and dCMP.
In the case of MP, it lacks a phosphate group which
resulted in an improper interaction between MP and dCMP.
This was evident that the presence of bisphosphate group
was important since no FL quenching of MP was observed
in the presence of any nucleotides. BP2 possesses the
Results and Discussion
Syntheses of MP, BP1 and BP2
BP1 was synthesized in two steps from the reaction
between DL1 with 2 equiv of diphenylchlorophosphate in
the presence of potassium tert-butoxide as base. BP2 was
also synthesized in order to investigate the positional effect
of bisphosphate group. The synthesis of BP2 was similar to
the procedure described for the synthesis of BP1 except
DL2 was used as the starting material. After purification,
BP1 and BP2 were obtained in good isolated yields (BP1=
72% and BP2=77%) (Fig. 1). Monodiphenylchlorophos-
phate diimine ligand (MP) was also synthesized as the
control ligand for the investigation of the effect of
bisphosphate group due to the fact that the presence of
bisphosphate group is important for facilitating the hydro-
0 equiv
400 equiv
Fig. 4 FL emission spectra of BP1 (2.5×10−6 M) in the presence of
Fig. 5 FL quenching of BP1 by various concentrations of dAMP,
dCMP, dTMP, and CMP
various concentrations of dCMP

J Fluoresc (2011) 21:187–194
1 H NMR spectra
191
Fig.
6
(400 MHz) of (a) free BP1
(10 mM) in DMSO (top); (b)
BP1 + 1 equiv of dCMP in
DMSO:D2O (1:1; v/v); (c)
dCMP in D2O
bisphosphate group which meets the requirement de-
scribed above for the optimal interaction between the
receptor and dCMP. The position of the bisphosphate
group on the BP2 aromatic ring is too remote for the
good binding between the receptor and dCMP. It was
obvious that BP1 exhibited high degree of selectivity
towards dCMP. The presence of the bisphosphate group
at the meta position on the aromatic ring of BP1 is a
prerequisite for the good recognition between the receptor
and dCMP. Therefore, BP1 was used as the optimal
receptor for subsequent experiments.
BP1 was shown to exhibit strong FL emission in
acetonitrile. The excitation wavelength (Ex) of BP1 was
280 nm while the emission wavelength (Em) was 360 nm.
Figure 3 shows the FL changes of BP1 upon the addition
of various nucleotides. The presence of various nucleotides
slightly to moderately affected the FL intensity of BP1. It
was clearly observed that 2.5×10−4 M of dCMP dramati-
cally quenched the FL of BP1.
FL emission of BP1 was measured at different concen-
trations of dCMP while fixed the concentration of BP1 at
2.5×10−6 M (Fig. 4). Increasing concentration of dCMP
resulted in the decrease of the FL emission of BP1.
When the concentration of dCMP reached 2.5×10−4 M,
the FL intensity of BP1 was significantly quenched. To
investigate more precisely, the quenching effect of BP1
with dCMP and other representing nucleotides at different
concentrations was studied. The FL intensity of BP1 was
measured in the presence of various concentrations of four
nucleotides, dAMP, dCMP, dTMP and CMP. At all
concentrations of dAMP, there was a little FL quenching
of BP1 (Fig. 5).
Addition of dTMP and CMP resulted in slight
decrease of BP1’s FL intensity. The FL intensity of
BP1 was significantly inhibited in the presence of dCMP
even at low concentration. Further increasing concen-
trations of dCMP resulted in a gradual FL quenching of
BP1. When the concentration of dCMP reached 400
Fig. 8 Stern-Volmer plot of BP1 with increasing concentrations of
dCMP. [BP1] = 2.5×10−6 M. [dCMP]/10−6 M=2.5, 5, 12.5, 25, 50,
300 (T=298 K)
Fig. 7 Job’s plot for BP1·dCMP. Y axis is fluorescence changes of
BP1

192
J Fluoresc (2011) 21:187–194
constant indicates the constant equilibrium of BP1·dCMP
complex in the static quenching process. It was shown in
Fig. 8 that the Stern-Volmer plot of BP1·dCMP complex
exhibited a linear relationship. The (F0/F) was directly
proportional to the increased concentration of dCMP with
the correlation coefficient of 0.9953. From the plot, BP1
had a weak binding affinity to dCMP (3.98 0.60×
10−3 M−1) and upon the binding between BP1 and dCMP
it formed the non-fluorescent complex.
Proposed Mechanism for BP1 Binding with dCMP
The mechanistic detail for the binding between BP1 and
dCMP is believed to involve the hydrogen bonding
between the bisphosphate group of BP1 and the
cytosine NH2 of dCMP. The good alignment for the
binding between BP1 and dCMP optimizes the interaction
of these two species. Upon binding between BP1 and
dCMP, the PET mechanism plays a significant role which
resulted in the FL quenching of the system. The steric
hindrance is also thought to play a role on the binding
between BP1 and dCMP. The steric hindrance could be
used to explain why the FL intensity of BP1 was not
quenched upon the addition with CMP. In the case of
CMP, the presence of the 2′-hydroxyl group causes the
structure more bulky to fit into the receptor’s pocket.
Therefore, the presence of the 2′-hydroxyl group in CMP
hindered proper binding between BP1 and CMP. This
explained the fact that CMP is not a good substrate for
BP1 due to the steric effect. The absence of the 2′-
hydroxyl group in dCMP facilitates the good binding
between BP1 and dCMP (Fig. 9).
Fig. 9 Energy minimized structure of the complex between BP1 and
dCMP
molar equiv of BP1, the FL intensity of BP1 was
significantly quenched.
1H NMR Titration of BP1 with dCMP
Of the mechanistic detail concerning interaction between BP1
and dCMP, 1H NMR experiments were used for the
investigation (Fig. 6). It was clearly shown that the addition
of 1 equiv dCMP into BP1 solution caused a slight downfield
shift of aromatic protons in BP1 indicating the disturbance of
the electron density within this area. Apart from the downfield
shift of protons in the aromatic region, the solution contained
both BP1 and dCMP exhibited the change of the multiplicity
for the designated cytosine proton. This effect was evident
with the appearance of the new peak at δ 6.15 ppm (indicating
as an asterisk in Fig. 6b). This new peak was the result from
the perturbation for the electronic environment of the cytosine
proton in dCMP due to the interaction with BP1.
Conclusions
In conclusion, we have discovered the selective FL
quenching of BP1 by dCMP. The FL quenching of BP1
exhibited excellent degree of selectivity towards dCMP
among various nucleotide substrates. FL and 1H NMR
experiments indicated the interaction between BP1 and
dCMP. Job’s plot analysis indicated the binding ratio
between BP1 and dCMP at 1:1 with the Stern-Volmer
constant of 3.98 0.60×10−3 M−1. It was suggested that the
FL quenching of BP1 by dCMP took place via the PET
mechanism. The recognition of dCMP by BP1 may be due
to the extended hydrogen bonding with the proper
alignment. The steric effect of the substrate also plays a
determining role for good binding between BP1 and dCMP.
Since BP1 shows high degree of selectivity and sensitivity
to dCMP over other nucleotides it could be regarded as a
new FL chemosensor for dCMP.
Determination of the Binding Ratio and Constant for BP1
and dCMP
Attempting to obtain the binding ratio between BP1 and dCMP
by mass spectrometry was unsuccessful. Therefore, Job’s plot
analysis was used to determine the binding ratio [49]. Job’s
plot analysis indicated a breaking point at 0.6 mol fraction of
dCMP indicating the formation of a 1:1 complex of
BP1·dCMP (Fig. 7). The binding constant between BP1 and
dCMP was determined using the Stern-Volmer plot (Ksv). The
Ksv was obtained from the Stern-Volmer Eq. (1) [50]
F0=F ¼ 1 þ KSV½Qꢀ
ð1Þ
Where [Q] is the concentration of the quencher (dCMP),
Ksv is the Stern-Volmer constant. The Stern-Volmer

J Fluoresc (2011) 21:187–194
193
Acknowledgements The Thailand Research Fund (TRF)/Commis-
sion on Higher Education (CHE)’s research grant to S.S. and P.T. The
Development and Promotion of Science and Technology Talents
Project’s scholarship to W.N. are gratefully acknowledged. We are
also grateful to the partial support from National Center for Genetic
Engineering and Biotechnology (BIOTEC).
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