A novel fluorescent chemosensor for Cu(II) in aqueous solution based on a β-aminobisphosphonate receptor

Article abstract of DOI:10.1016/j.tetlet.2007.09.029

A novel β-aminobisphosphonate receptor has been combined with naphthalene in the fluorophore-spacer-receptor format of a typical photoinduced electron transfer (PET) based sensor. The sensor was synthesised in two steps by first reacting aminomethylnaphth

Full text of DOI:10.1016/j.tetlet.2007.09.029

Tetrahedron Letters 48 (2007) 7756–7760
A novel fluorescent chemosensor for Cu(II) in aqueous
solution based on a b-aminobisphosphonate receptor
*
Sukanta Kamila, John F. Callan, Ray C. Mulrooney and Moira Middleton
School of Pharmacy, The Robert Gordon University, Aberdeen, Scotland AB10 1FR, UK
Received 14 August 2007; revised 27 August 2007; accepted 4 September 2007
Available online 8 September 2007
Abstract—A novel b-aminobisphosphonate receptor has been combined with naphthalene in the fluorophore–spacer–receptor
format of a typical photoinduced electron transfer (PET) based sensor. The sensor was synthesised in two steps by first reacting
aminomethylnaphthalene with diethyl vinylphosphonate to produce the parent tetraester, followed by deprotection with bromotri-
methylsilane to afford the desired sensor. The fluorescence emission of the sensor was observed to remain ‘On’ over a wide pH
range (2–10). Cu(II) was found to bind strongly to the sensor resulting in an ‘On–Off’ fluorescent response with sensitivity in the
lM range.
ꢀ
2007 Elsevier Ltd. All rights reserved.
Derivatives of bisphosphonates have found many appli-
cations in the diagnosis and treatment of disease states.
The aminobisphosphonates (NBP’s), for example, are
an important class of drugs used to treat bone metasta-
ses in patients suffering from breast and prostate can-
within the cell and can also contribute to algae growth
in seawater. The accurate measurement of transition
6
metals such as Cu(II) using PET type sensors can prove
difficult due to the acidic nature of their hydration shell.
This is a particular problem in organic solvents when
1
7
cer. The strong metal ion chelating ability of these
pH buffering is not possible. Certain examples of PET
compounds is well known, particularly for divalent
and trivalent ions. Gummiena-Kontecka et al. have
shown using electron paramagnetic spectroscopy
sensors where a fluorescent enhancement was attributed
2
8
to metal binding have later been shown to be due to
7
proton transfer from the coordinated water. Therefore,
(
EPR) that b-aminobisphosphonate derivatives bind
sensors for monitoring Cu(II) that prove robust to
changes in pH are advantageous.
3
strongly to Cu(II). Progressing from this we have devel-
oped a luminescent sensor incorporating b-amino-
bisphosphonate as a receptor using the well known
photoinduced electron transfer (PET) design principle.
Naphthalene was chosen as fluorophore due to its estab-
This study investigated the fluorescence response of 2
and its parent 1 to variations in pH. We also examined
the selectivity of 2 against common metal ions and pro-
pose 2 as a fluorescent chemosensor for Cu(II), operable
over a wide pH range.
4
5
lished photophysical properties. The receptor was con-
nected to the fluorophore via a methylene unit in the
conventional fluorophore–spacer–receptor format of
PET systems to produce {2-[naphthalen-1-ylmethyl-(2-
phosphono-ethyl)amino]ethyl}-phosphonic acid (2).
The synthesis of 2 was performed in two steps (Scheme
1). The first step involved a Michael addition reaction
between aminomethylnaphthalene and diethyl vinyl-
phosphonate to give (2-{[2-(diethoxy-phosphoryl)ethyl]-
naphthalen-1-ylmethylamino}ethyl)phosphonic acid di-
The desire to measure Cu(II) concentrations accurately
at the sub lM level is inspired by biological and environ-
mental importance of this ion. Cu(II) is involved in crit-
ical catalytic, biosynthetic and transport processes
9
ethyl ester (1) in 77% yield. The subsequent hydrolysis
of 1 with bromotrimethylsilane resulted in the formation
1
0
of 2 in 93% yield.
However, the synthesis of 1 was not as straightforward
as it appears. An attempt to synthesise it by alkylating
aminomethyl naphthalene with diethyl 2-bromoethyl-
phosphonate was unsuccessful. The basic conditions
Keywords: Fluorescence; b-Aminobisphosphonate; Copper; Naphtha-
lene; PET.
*
Corresponding author. Tel.: +44(0) 1224262546; fax: +44(0)
224262555; e-mail: j.callan@rgu.ac.uk
1
0040-4039/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2007.09.029

S. Kamila et al. / Tetrahedron Letters 48 (2007) 7756–7760
7757
O
P
O
EtO
EtO
HO
P
HO
NH2
N
N
P
O
P O
EtO
EtO
HO
HO
(i)
(ii)
7
7 %
93 %
(
1)
(2)
Scheme 1. Synthesis of {2-[naphthalen-1-ylmethyl-(2-phosphono-ethyl)amino]ethyl}-phosphonic acid (2). Reagents and conditions: (i) diethyl
vinylphosphonate/anhydrous MeOH, reflux 5 days; (ii) bromotrimethylsilane/anhydrous DCM, ambient, 60 min.
required for the reaction resulted in the conversion of
diethyl 2-bromoethylphosphonate to diethylvinyl phos-
phonate due to the acidity of the methylene protons
on the carbon a to the phosphorus atom (see Supple-
mentary data). Since this reduced the amount of diethyl
40000
3
3
2
5000
0000
5000
2
-bromoethylphosphonate available, the dialkylated
20000
15000
product was not observed with only small amounts of
the monoalkylated form, which was found to decom-
pose due to the acidic nature of the work-up (see Supple-
mentary data). Therefore a ‘neutral’ approach avoiding
the presence of acids or bases was required, and the
method outlined in Scheme 1 met this objective.
10000
5000
0
0
5
10
15
pH
The fluorescent spectrum of tetraester 1 recorded in
aqueous solution displayed an emission band centred
at 333 nm, typical of the naphthalenic fluorophore,
Figure 2. A plot of spectral area against pH for 1 (m) and 2 (r).
1
1
when excited at 283 nm (Fig. 1). There was no evi-
dence of intramolecular exciplex formation between
the naphthalene excited state and the proximal amine,
characterised by a broad longer wavelength emission
that has been reported in other similarly constructed
Àlog(F
À F)/(F À FMIN) against pH (where FMAX
MIN
MAX
is the maximum fluorescence intensity, F
the mini-
mum fluorescence intensity and F the measured fluores-
cence intensity) the pK of the tertiary amine was
a
1
5
1
2
calculated to be 4.99.
systems. The emission was observed to be strongly
dependent on pH, ‘switching on’ as the pH decreased,
with no noticeable change in wavelength. This is typical
of PET sensors where a basic amine forms part of the
As expected, the conversion of 1 to 2 resulted in no
change in the absorption or emission maxima. A plot
of fluorescence intensity against pH for 2 is again shown
in Figure 2, but resulted in a markedly different profile
1
3
receptor. A plot of fluorescent intensity against pH
for 1 is shown in Figure 2. A large fluorescence enhance-
ment (FE) over approximately 2 log units occurred with
decreasing pH. The enhancement is due to an increase in
the oxidation potential of the amine upon protonation,
removing the thermodynamic driving force for PET,
to 1, with evidence of two steps, one with a pK of
a
10.68 and the other at 4.98. In addition, fluorescence re-
mained ‘On’ much longer and did not fully switch ‘Off’
until ꢀpH 12. The reason for this is best explained as
follows: at high pH (>12) all the phosphate hydroxyls
are deprotonated, as illustrated in Scheme 2c, and
PET can happen in a similar manner as in 1, from the
tertiary amine to the fluorophore. As pH decreases,
the phosphate oxyanions become protonated enabling
the P–OH group to hydrogen bond with the amine lone
pair through the formation of a 6-membered ring
1
4
and switching fluorescence ‘On’. Using a plot of
9
8
7
6
5
4
3
2
1
00
00
00
00
00
00
00
00
00
0
(
Scheme 2b). This results in substantial proton transfer
responsible for partially switching fluorescence ‘On’. A
similar effect has been reported by Czarnik et al. for
an anthracene based PET sensor with a polyamine
16
receptor used to sense for pyrophosphate. At very
low pH the amine becomes protonated and fluorescence
is completely switched ‘On’ (Scheme 2a).
3
00
325
350
375
400
425
450
Wavelength (nm)
The wide pH window over which fluorescence remains
‘On’ for 2 does not lend itself to be an effective ‘Off–
On’ sensor, as an extremely high pH (>12) is required
Figure 1. Emission spectra of 1 upon varying pH from 2 (high
intensity) to 12 (low intensity).

7758
S. Kamila et al. / Tetrahedron Letters 48 (2007) 7756–7760
O
P
O
P
O
P
a
HO
HO
b
-O
c
-O
O
-
O
H
NH+
HO
N
N
P
O
P O
HO
-O
P O
HO
O
-
O
-
pH 2
High fluorescence
pH 9
Medium fluorescence
pH 12
Low fluorescence
Scheme 2. Anticipated forms of 2 at different pH values and associated fluorescence intensity.
for operation. However, this does mean that if 2 is used
as an ‘On–Off’ sensor then it can operate over a very
1
wide range, as fluorescence is strong from pH 2 to pH
0. It is well known that transition metals can quench
the excited state energy of organic fluorophores by
energy and/ or electron transfer. We determined the
selectivity of 2 against a selection of common mono
0.8
1
0
0
.6
.4
1
7
and divalent metal ions at
a
concentration of
0.2
0
À5
2
· 10 M, in 0.01 M HEPES buffer (pH 7.4), the
results of which are shown graphically in Figure 3.
3
4
5
6
7
8
9
10
-
log [Anion]
The alkali and alkali earth ions have little effect nor do
the closed shell Zn(II) and Hg(II). Ni(II), Co(II) and
Mn(II) quench fluorescence by about 60% of the origi-
nal intensity while Cu(II) and Fe(II) quench by over
Figure 4. Plot of relative intensity against Àlog[anion] for 2 with (m)
Cu(II) and (r) Fe(II) in 0.01 M HEPES buffered at pH 7.4. Metal ions
added as chloride salts, [2] = 8.58 · 10 M.
À7
9
5%. The sensitivity of 2 for Ni(II), Co(II), Mn(II),
Cu(II) and Fe(II) was determined by investigating
changes in the fluorescence intensity as a function of me-
tal ion concentration. This is illustrated in Figure 4
where relative intensity is plotted against Àlog[anion]
for Cu(II) and Fe(II). In each case a sigmoidal curve is
observed with quenching occurring over ca. 2 log units
Fe(II) can be cancelled by the addition of fluoride ion
3
À
19
to produce the colourless FeF6
, and as the sensitivity
of 2 for Mn(II), Co(II) and Ni(II) is quite low
(
ꢀ20 mM), this sensor could be viewed as a probe for
Cu(II) over a wide pH window in the lM range. The
sensitivity of 2 for Cu(II) is in the physiological range
meaning that it could potentially be used as an intra-
cellular probe. As expected, the parent 1 does not
respond to either Cu(II) or Fe(II) addition (see Supple-
mentary data).
1
8
suggesting a 1:1 binding between sensor and ion.
From the equation log(FMAX À F)/(F À FMIN) =
15
log[anion] À logb the binding constant (logb) for each
was calculated and is presented in Table 1.
These data suggest that Fe(II) binds more strongly to 2
than Cu(II), which in turn binds more strongly than
Mn(II), Co(II) and Ni(II). As the quenching effect of
A more detailed look at the binding of Cu(II) to 2 is pos-
sible with H NMR. Figure 5 shows the spectra of 2 with
1
different concentrations of Cu(II). A significant down-
field shift (0.7 ppm) was observed for the methylene pro-
tons adjacent to the phosphorus atom (b), with the other
protons remaining essentially unaffected. This downfield
shift suggests the involvement of this methylene group in
the binding of the Cu(II) ion. A possible explanation is
that a Cu(II) induced deprotonation of the methylene
group occurs resulting in the formation of a carbanion
that assists in chelation of the Cu(II), in a manner sim-
1
8
6
4
2
000
000
000
000
000
0
20
ilar to that of sulfonamide based sensors. More work
is ongoing to fully understand this interaction.
In summary, we have incorporated
a b-amino-
bisphosphonate receptor into a luminescent sensor
according to the PET format for the first time. The fluo-
rescence of the highly water soluble sensor remains ‘On’
over a wide pH range. Selectivity was demonstrated for
Cu(II) over other common metal ions with sensitivity
established at the lM level.
Metal Ion
Figure 3. Selectivity data for 2 against commonly available metal ions
as their chloride salts in 0.01 M HEPES buffered at pH 7.4. [Metal
À5
À7
ion] = 2 · 10 M, [2] = 8.58 · 10 M.

S. Kamila et al. / Tetrahedron Letters 48 (2007) 7756–7760
Table 1. Photophysical properties of 1 and 2
7759
À1
3
À1
a
Property
k
MAX
k
EM
e
MAX (mol dm cm
)
pK
a
(amino)
pK
a
(phosphonate)
U
FLU
logb
1
283
283
283
283
283
283
283
333
333
5788
5524
—
—
—
4.99
4.98
—
—
—
—
10.68
—
—
—
0.13
0.15
0.01
0.02
0.06
0.07
0.08
—
—
2
2
2
2
2
2
(unbound)
+ Cu(II)
+ Fe(II)
+ Mn(II)
+ Ni(II)
+ Co(II)
b
—
—
6.03
6.58
3.75
3.60
4.08
b
333
333
333
—
—
—
a
Quantum yield calculated with reference to tryptophan.
Accurate measurement not possible due to low intensity.
b
O
P
O
P
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1
Figure 5. H NMR spectra of 2 recorded in D
2
O at 400 MHz after
addition of increasing concentrations of Cu(II). The numbers on each
plot represent the molar equivalence of Cu(II) added. [2] =
.2 · 10 M.
À2
4
Acknowledgements
The authors would like to thank RGU and the Lever-
hulme Trust UK for financial support and Professor
A. P. de Silva for a helpful discussion. We also acknowl-
edge support from the EPSRC national mass spectrom-
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CODEN: PIXXD2 WO 2006080993 A1 20060803 CAN
Supplementary data
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/j.tetlet.
9
2
007.09.029.
1
45:201329 AN 2006:768269.
1
1
0. Analytical data for 1. H NMR (CDCl
3
, chemical shift d
in ppm relative to TMS): 1.30 (t, J = 6.4 Hz, –OCH CH ),
2
3
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1.90–2.01 {m, –CH
2
CH
2
P(O)(OEt
2
)
2
, 4H}, 2.80–2.90 (m,
3
–N, –OCH CH ,
–
N–CH –CH –, 4H), 3.91–4.10 (m, –CH
2
2
2
2
1
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1H, aromatic), 7.81 (dd, J = 2.4, 7.2 Hz, 1H, aromatic),
8.20 (dd, J = 2.4, 8.0 Hz, 1H, aromatic). C NMR
1
3
(

7760
S. Kamila et al. / Tetrahedron Letters 48 (2007) 7756–7760
3
, chemical shift d in ppm relative to TMS): 16.32 12. Galindo, F.; Becerril, M.; Burgette, I.; Luis, S. V.; Viagra,
(
(
CDCl
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3
3
), 25.08 (CH
2
), 25.47 (CH
2
), 42.6 (CH
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0.4 (CH ), 61.8 (CH ), 123.3 (CH), 125.3 (CH), 125.7
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2
(
CH), 126.3 (CH), 126.5 (CH), 128.2 (CH), 128.7 (CH),
3
1
1
31.6 (C), 133.8 (C), 134.0 (C), 175.2 (P@O). P NMR
(
with proton decoupling): d 30.28. ESMS: Calculated
+
for C H NO P [M ] = 486.2169; measured mass
23
37
6 2
+
1
[
(
2
M ] = 486.2170. Analytical data for 2.
CD OD, chemical shift d in ppm relative to TMS):
.10–2.11 {m, –CH CH P(O)(OEt ) , 4H}, 3.37–3.47
H
NMR
3
2
2
2 2
(
m, –N–CH
2
–CH
2 2
–, 4H), 4.89 (s, –CH N, 2H), 7.47–
7
.53 (m, 3H, aromatic), 7.6 (dd, J = 1.2, 7.2 Hz, 1H,
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1
3
(
(
(
(
CD
CH
3
2
OD, chemical shift d in ppm relative to TMS): 23.6
), 24.96 (CH ), 56.6 (CH ), 125.01 (CH), 127.69
2
2
CH), 128.99 (CH), 130.10 (CH), 131.49 (CH), 133.43
CH), 133.65 (C), 134.22 (C), 136.68 (C), 175.3 (P@O).
1
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