Facile, divergent route to bis-Zn(II)dipicolylamine type chemosensors for pyrophosphate

Article abstract of DOI:10.1016/j.tet.2012.09.007

A new, two-step synthesis has been developed for a series of bis-DPA-type ligands whose dinuclear Zn(II) complexes function as fluorescent anion sensors. The Zn(II) complexes exhibit good selectivity for PP_i over other anions in aqueous medium

Full text of DOI:10.1016/j.tet.2012.09.007

Tetrahedron 68 (2012) 9435e9439
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Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Facile, divergent route to bis-Zn(II)dipicolylamine type chemosensors for
pyrophosphate
,
L. Gayathri Pathberiya a, Nicholas Barlow b, Tran Nguyen a, Bim Graham b, Kellie L. Tuck a
*
a School of Chemistry, Monash University, Clayton, VIC 3800, Australia
b Medicinal Chemistry, Monash Institute of Pharmaceutical Sciences, Parkville, VIC 3052, Australia
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 1 June 2012
Received in revised form 17 August 2012
Accepted 3 September 2012
Available online 10 September 2012
A new, two-step synthesis has been developed for a series of bis-DPA-type ligands whose dinuclear Zn(II)
complexes function as fluorescent anion sensors. The Zn(II) complexes exhibit good selectivity for PPi
over other anions in aqueous medium (pH 7.5) and may be used to monitor the extent of enzyme-
catalysed reactions, in which PPi is produced or consumed.
Ó 2012 Elsevier Ltd. All rights reserved.
Keywords:
Chemosensor
Fluorescence
Pyrophosphate
Di-(2-picolyl)amine
Hosteguest interactions
1. Introduction
been used to monitor the extent of DNA amplification in PCR,20 and,
in combination with a boronic acid, to distinguish PPi from nucle-
The development of chemosensors to detect anions has been
a growing area of research in recent years.1e8 Of particular interest
is the selective detection of pyrophosphate (PPi, P2O72ꢀ) over other
anions of biological relevance, principally adenosine triphosphate
(ATP), adenosine monophosphate (AMP) and phosphate (Pi,
PO42ꢀ).2,3 Pyrophosphate is extremely important in a number of
biological processdfor example, as a substrate or product of vari-
ous enzymes, and as a product of the cellular hydrolysis of ATP.9 Its
quantification can be used for real-time DNA sequencing, thereby
obviating of the need for electrophoresis.10
The di-(2-picolyl)amine (DPA) unit has been widely exploited as
a motif within metal complex-based anion sensor designs, partic-
ularly for pyrophosphate detection.2,8,11e19 Most notably, the Zn(II)
complex of DPA exhibits a high affinity for phosphate-bearing
moieties, which can lead to either an increase in fluorescence or
quenching of a tethered fluorophore, depending on the nature of
the fluorophore and the binding mode adopted by the interacting
anion. Selectivity for PPi is achievable using sensor designs that
feature two DPAeZn(II) units appropriately positioned to interact
favourably with the PPi anion. Hong et al., for example, recently
reported a 2-naphthalene-bearing bis-DPA ligand 1 (Fig. 1) the
dinuclear Zn(II) complex of which, 1-Zn2, is able to selectively de-
tect PPi in the presence of a range of other anions.15 This sensor has
oside triphosphates.21
Hong et al.’s synthesis of compound 1 requires four steps and
proceeds in 35% overall yield, beginning from a precursor that is not
commonly available (6-bromo-2,2-dimethyl-4H-1,3-benzodioxin-
8-methanol).15 Apart from the number of steps involved, the major
drawback of this synthetic route is that it does not allow for the
divergent production of analogues featuring other fluorescent
moieties in place of the 2-naphthyl group. In this paper we there-
fore present an alternate two-step synthesis of the 2-naphthalene
ligand 1, in which the fluorophore is introduced in the final step.
We also report the ready adaptation of this synthesis to the pro-
duction of two new, structurally-related ligands, 2 and 3, featuring
1-naphthyl and 9-anthracenyl moieties, respectively (Fig. 1). It was
hypothesised that the 1-naphthyl analogue might have different
fluorescent properties due to the different point of attachment to
the fluorophore, whilst anthracene is known to have a higher
quantum yield than naphthalene.22,23
This work was carried out as part of a program directed towards
the development of inhibitors of enzymes, which use or produce
PPi, for which we required a reliable means of assaying activity in
real-time. As such, we also report an exploration of the sensitivity
and selectivity of the Zn(II) complexes of 1e3 as chemosensors for
PPi under pseudo-biological conditions, and the use of one of the
chemosensors in monitoring the activity of inorganic pyrophos-
phatase as an example of a PPi-utilising enzyme.
* Corresponding author. E-mail address: Kellie.Tuck@monash.edu (K.L. Tuck).
0040-4020/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tet.2012.09.007

9436
L.G. Pathberiya et al. / Tetrahedron 68 (2012) 9435e9439
Fig. 1. Structures of Hong et al.’s ligand 115 and the 1-naphthyl and 9-anthracenyl analogues, 2 and 3, whose dinuclear Zn(II) complexes function as fluorescent pyrophosphate
sensors.
2. Results and discussion
The new synthetic route (Scheme 1) involved, firstly, a Mannich
reaction between p-bromophenol, DPA and formaldehyde to pro-
duce the bis-DPA intermediate 4 in good yield (87%). Ligands 1e3
were then furnished directly via a Suzuki reaction between 4 and
the boronic acid derivatives of the corresponding fluorophores
(46e69%). By this novel route, the desired chemosensor precursor
ligands were obtained in only two steps from readily available
starting materials. The dinuclear Zn(II) complexes,1-Zn2, 2-Zn2 and
3-Zn2 were then formed by addition of an aqueous solution of
Zn(NO3)2 (2 M equiv) to the respective ligands.
Fig. 2. Changes in fluorescence observed upon the addition of various anions (3 equiv)
to 1-Zn2 (25
m
M, Tris buffer (10 mM, pH 7.5)) (lex¼310 nm). Inset: fluorescence in-
tensity measured at 460 nm, (1) 1-Zn2 only, (2) þPPi, (3) þATP, (4) þAMP, (5) þPi.
the addition of a number of biologically and analytically important
anions (AMP, Clꢀ, Pi, CH3CO2ꢀ, and HCO3; 3 equiv), negligible
emission changes were observed (Fig. 4), whilst the addition of ATP,
GTP and ADP produced much less dramatic increase in fluorescent
intensity than PPi (addition of 3 equiv of ATP resulted in a ca. 2-fold
increase in fluorescence). For each of the latter anions, a bath-
ochromic shift of ca. 8 nm in lem was observed. These results are
15
also similar to those found for 1-Zn2 and indicate that 2-Zn2 is
Scheme 1. Synthesis of ligands 1e3.
In our hands, 1-Zn2 showed similar fluorescence changes, and
selectivity for PPi over a number of biologically important anions
(ATP, AMP and Pi), to those previously described by Hong et al.15
(Fig. 2). The 1-naphthyl analogue, 2-Zn2, showed a similar dose-
dependent response to the addition of PPi as 1-Zn2, with the fluo-
rescence output increasing by a factor of ca. 7-fold upon the addi-
tion of 1 equiv of PPi (Fig. 3). However, in this case the increase in
fluorescence was also accompanied by a bathochromic shift in the
wavelength of maximum emission intensity (lem) from 450 nm to
464 nm; no further changes in lem were observed after addition of
1.5 equiv of PPi. The apparent association constant (Ka), determined
from the fluorimetric titration curve, was about 7.2ꢁ105 Mꢀ1. Upon
Fig. 3. Change in fluorescence observed upon addition of PPi to 2-Zn2 (25 mM, Tris
buffer (10 mM, pH 7.5)) (lex¼310 nm). Inset: fluorescence intensity measured at
464 nm as a function of [PPi].

L.G. Pathberiya et al. / Tetrahedron 68 (2012) 9435e9439
9437
complete quenching of the native fluorescence of 3-Zn2, and
a bathochromic shift in lmax of 38 nm (Fig. 6). In contrast, addition
of 3 equiv of either AMP, Clꢀ, CH3CO2ꢀ, and HCO3ꢀ: resulted in only
a slight decrease in fluorescence, with no significant change to lem
(Fig. 6). The addition of 3 equiv ADP and ATP produced bath-
ochromic shifts in lmax of 8 nm and 20 nm, with significant
quenching occurring in both cases (Fig. 6). Pi addition (3 equiv)
yielded a less dramatic decrease in fluorescence and a bath-
ochromic shift of 16 nm.
Fig. 4. Changes in fluorescence intensity observed upon addition of various anions
(3 equiv) to 2-Zn2 (25
m
M, Tris buffer (10 mM, pH 7.5)) (lex¼310 nm). Inset: fluores-
cence intensity measured at 464 nm, (1) 2-Zn2 only, (2) þPPi, (3) þATP, (4) þGTP, (5)
þAMP, (6) þADP, (7) þClꢀ, (8) þPi, (9) þCH3CO2ꢀ, (10) þHCO3
:
ꢀ
a comparably selective chemosensor for PPi. The increase in fluo-
rescence observed upon the addition of PPi to either 1-Zn2 or 2-Zn2
can be attributed to a weakening of the bond between the Zn(II)
centres and the bridging phenolate oxygen upon PPi binding,
resulting in increased charge density on the phenolate oxygen and
an enhanced fluorescent output due to induced charge transfer.15
To further probe the selectivity of 2-Zn2 as a chemosensor for
PPi, we measured its fluorescence response to PPi in the presence of
ATP (the anion, which gave the greatest fluorescence response
other than PPi). Titration of PPi into a solution that contained 2-Zn2
and 3 equiv of ATP resulted in a dose-dependent increase in fluo-
rescence, with saturation occurring after the addition of 3 equiv of
PPi (Fig. 5). This is indicative of a substantially higher binding af-
finity for PPi and indicates that 2-Zn2 may be used to monitor PPi in
the presence of competing anions.
Fig. 6. Changes in fluorescence observed upon addition of various anions (3 equiv) to
3-Zn2 (10
m
M, Tris buffer (10 mM, pH 7.5)) (lex¼370 nm). Inset: fluorescence intensity
measured at 484 nm, (1) 3-Zn2 only, (2) þPPi, (3) þATP, (4) þAMP, (5) þADP, (6) þClꢀ
,
(7) þPi, (9) þCH3CO2ꢀ, (9) þHCO3
:
ꢀ
The addition of increasing amounts of PPi to 3-Zn2 resulted in
a dose-dependent decrease in the fluorescent output, with minimal
fluorescence observed after the addition of a single equivalent of PPi
(Fig. 7). The apparent association constant (Ka), determined from
the fluorimetric titration curve, was about 1.2ꢁ106 Mꢀ1
.
The quenching effect observed upon addition of PPi to 3-Zn2
contrasts starkly with the fluorescence increase observed for 1-Zn2
or 2-Zn2, however similar quenching effects have been observed
previously with sensors 524 and 617 (Fig. 8), which possess pyrenyl
and acridinyl fluorescent moieties, respectively. This may reflect an
We initially hypothesised that replacement of the 2-naphthyl
moiety with a 9-anthracenyl group might result in an increased
fluorescent output upon complexation to PPi. However, addition of
3 equiv of PPi to 3-Zn2 (10 mM) was instead found to result in near
Fig. 5. Change in fluorescence observed upon addition of PPi to 2-Zn2 complex (25
mM,
Fig. 7. Change in fluorescence observed upon addition of PPi to 3-Zn2 (10 mM, Tris
Tris buffer (10 mM, pH 7.5)) in the presence of ATP (3 equiv) (lex¼310 nm). Inset:
buffer (10 mM, pH 7.5)) (lex¼370 nm). Inset: fluorescence intensity measured at
fluorescence measured at 464 nm as a function of [PPi].
484 nm as a function of [PPi].

9438
L.G. Pathberiya et al. / Tetrahedron 68 (2012) 9435e9439
4. Experimental section
4.1. General
All reagents and inorganic pyrophosphatase from baker’s yeast
(Saccharomyces cerevisiae), were purchased from Sigma Aldrich and
used without furtheꢀr purification. ATP, GTP, ADP, AMP, PPi Pi, Clꢀ,
CH3CO2ꢀ, and HCO3 were used as the sodium salts. Organic solu-
tions were dried over MgSO4. The progress of reactions was moni-
tored by thin layer chromatography (TLC) on Merck 60 F240
precoated silica gel polyester plates. Flash chromatography was per-
formed with Davisil LC60A, 40e63 m
m silica media. Proton (1H) and
Fig. 8. Previously reported chemosensors whose fluorescence is quenched upon ad-
dition of PPi.
carbon (13C) NMR spectra were recorded on either a Bruker AV400 or
NMR spectra were recorded on a Bruker Ultrashield 400 Plus spec-
trometer operating 400 MHz, respectively, for proton nuclei, and
100 MHz for carbon nuclei. Chemical shifts (d) are expressed in parts
per million (ppm) and referenced to residual solvent signal as the
internal standard. Low resolution mass spectrometry was performed
on an Agilent 6120 single quadrapole LCMS system using electron
spray ionization. Highresolution electrosprayionisationmassspectra
were acquired using a Micromass Platform II single quadrupole mass
spectrometer equipped with an atmospheric pressure (ESI/APCI) ion
source. Analytical HPLC was performed on a Waters Alliance 2690
fitted with a Waters 5996 PDA detector and a Phenomenex Luna C8
ꢀ
column (5
m
m,100 A,150ꢁ4.60 mm). Analyses were conducted using
a gradient of 100% buffer A to 80% buffer B over 10 minwhere buffer A
was 0.1% trifluoroacetic acid in water and buffer B was 0.1% tri-
fluoroacetic acid, 80.0% acetonitrile and 19.9% water. Fluorescence
spectra were recorded on a Cary Eclipse fluorescence spectropho-
tometer and analysed with Cary WinUV software.
Fig. 9. Assay of pyrophosphatase activity using 2-Zn2. Conditions: 25
mM complex,
10 mM Tris buffer (pH 7.5), 10 mM MgCl2 and 4 equiv PPi (lex¼310 nm, lem¼460 nm).
4.1.1. 2,6-Bis((bis(2-pyridinylmethyl)amino)methyl)-4-bromo-phenol
(4). To a solution of p-bromophenol (509 mg, 2.94 mmol) in iso-
propanol (3 mL) and water (5 mL) was added di-(2-picolyl)amine
(2.0 mL, 11.1 mmol) and a 37% formaldehyde solution (1.6 mL,
19 mmol). The resulting solution was refluxed for 15 h. The iso-
propanol was removed from the reaction mixture in vacuo and
water (10 mL) was then added. The reaction mixture was extracted
with EtOAc (3ꢁ20 mL), the combined organic phases washed with
satd NaHCO3(aq) (20 mL), dried and the solvent removed in vacuo.
enhanced degree of photoinduced electron transfer (PET) from the
amines of the DPA units to the anthracene upon PPi binding, leading
to deactivation of the excited state. Charge transfer-derived fluo-
rescence, observed upon binding of PPi to 1-Zn2 or 2-Zn2, may in
this instance be prevented by the anthracene lying orthogonal to
the phenolate moiety, disrupting the
tween these groups.
p-conjugation pathway be-
To demonstrate the utility of the chemosensors for monitoring
enzymatic processes involving PPi, we tested one of them (2-Zn2) in
conjunction with inorganic pyrophosphatase, which catalyses the
cleavage of pyrophosphate into two phosphate groups. Chemo-
Flash column chromatography (95% CHCl3: 5% MeOH: 5% NH3(aq)
afforded the title compound as an oil (1.52 g, 87%). 1H NMR
(400 MHz, CDCl3)
8.50 (m, 4H), 7.66 (m, 4H), 7.50 (d, J¼7.8 Hz, 4H),
7.40 (s, 2H), 7.18 (m, 4H), 3.86 (s, 8H), 3.76 (s, 4H). 13C NMR
(100 MHz, acetone-d6) 160.0, 156.5, 149.7, 137.5, 127.9, 123.8,
)
d
sensor 2-Zn2 (25 mM), Tris buffer (10 mM, pH 7.5), MgCl2 (10 mM)
and PPi (4 equiv) were incubated at 30 ꢂC for 5 min, then pyro-
phosphatase added and the emission intensity at 460 nm moni-
tored as a function of time. As expected, the fluorescence intensity
decreased with time, accompanying consumption of the PPi sub-
strate (Fig. 9). The rate of reaction was also confirmed to increase
with increasing enzyme concentration.
d
123.0, 110.6, 60.2, 54.7. LCMS (ESI): m/z: 597.1 (100%), 595.2 (100%).
4.1.2. 2,6-Bis((bis(Pyridin-2-ylmethyl)amino)methyl)-4-(naph-
thalen-2-yl)phenol (1). A suspension of 2,6-bis((bis(pyridin-2-
ylmethyl)amino)methyl)-4-bromophenol (200 mg, 0.33 mmol),
naphthalene-2-boronic acid (70 mg, 0.40 mmol) and potassium
carbonate (70 mg) in water (1 mL) and dioxane (5 mL) was satu-
rated with nitrogen. To this mixture was added tetrakis(-
triphenylphosphine)palladium (10 mg, 0.0087 mmol) and the
suspension refluxed under nitrogen for 15 h. The solvent was re-
moved in vacuo and a third of the residue was purified by reverse
phase prep HPLC (0e30 % acetonitrile in water over 30 min, 0.1%
TFA throughout) to provide the title compound as a white solid
3. Conclusion
In conclusion, a facile route has been developed for the syn-
thesis of bis-DPA-type anion sensors, in which the fluorescent
moiety is introduced in the final step. This route has provided two
new ligands, 2 and 3, featuring 1-naphthyl and 9-anthracenyl
moieties, respectively. The corresponding dinuclear Zn(II) com-
plexes, like that of the previously reported ligand 1,15 exhibit good-
to-high selectivity for PPi over other anions in aqueous medium (pH
7.5). Fluorescence from the naphthyl moiety increases upon PPi
binding, whilst that from the anthracene group is diminished. We
have successfully employed 2-Zn2 for monitoring inorganic phos-
phatase activity and are now exploring the utility of the chemo-
sensors for assaying a range of other enzymes that feature PPi as
a substrate or product.
(30 mg, 46%). 1H NMR (400 MHz, DMSO)
d
8.60 (dd, J¼0.7, 5.0 Hz,
4H), 8.08 (bs, 1H), 7.87e8.01 (complex, 7H), 7.79 (dd, J¼1.8, 8.6 Hz,
1H), 7.73 (s, 2H), 7.50e7.58 (complex, 6H), 7.45 (m, 4H), 4.37 (s, 8H),
4.28 (s, 4H). HPLC: 95% pure. LCMS (ESI): m/z: 643.1(MþH, 100%).
4.1.3. 2,6-Bis((bis(pyridin-2-ylmethyl)amino)methyl)-4-(naph-
thalen-1-yl)phenol (2). A suspension of 2,6-bis((bis(pyridin-2-
ylmethyl)amino)methyl)-4-bromophenol (200 mg, 0.33 mmol),

L.G. Pathberiya et al. / Tetrahedron 68 (2012) 9435e9439
9439
naphthalene-1-boronic acid (74 mg, 0.40 mmol) and potassium
carbonate (70 mg) in water (1 mL) and dioxane (5 mL) was satu-
rated with nitrogen. To this mixture was added tetrakis(-
triphenylphosphine)palladium (10 mg, 0.0087 mmol) and the
suspension refluxed under nitrogen for 15 h. The solvent was re-
moved in vacuo and a third of the residue was purified by reverse
phase prep HPLC (0e30 % acetonitrile in water over 30 min, 0.1%
TFA throughout) to provide the title compound as a white solid
Zn(NO3)2$6H2O (2 equiv). After 30 min, this stock solution was then
diluted to the required concentration with Tris buffer (10 mM, pH
7.5) and the solution utilised for fluorescence experiments.
4.3. Pyrophosphatase assay
To a solution of the complex (25
(10 mM, pH 7.5), MgCl2 (10 mM) was added PPi (4 equiv). After
5 min at 30 ꢂC, pyrophosphatase (7
M stock in 10 mM Tris buffer)
mM, 2-Zn2) in Tris buffer
(35 mg, 50%). 1H NMR (400 MHz, MeOD)
d
8.44 (d, J¼4.9 Hz, 4H,
m
pyridinyl H6), 7.88 (d, J¼8.1 Hz, 1H, naphthalenyl H8), 7.81 (d,
J¼8.1 Hz, 1H, naphthalenyl H2), 7.75 (d, J¼8.6 Hz, 1H, naphthalenyl
H5), 7.69 (t, J¼7.6 Hz, 4H, pyridinyl H4), 7.55 (d, J¼7.6 Hz, 4H, pyr-
idinyl H4), 7.50e7.42 (m, 3H, naphthalenyl H3, H6 and H7), 7.30 (d,
J¼7.4 Hz, 2H, naphthalenyl H4), 7.27e7.21 (m, 6H, pyridinyl H5 and
phenolic H3), 3.92 (s, 8H, NCH2Py), 3.88 (s, 4H, 2-methylphenol CH2).
was added to the solutions such that the final enzyme concentra-
tion was 120, 240, or 480 nM. The fluorescence spectrum was pe-
riodically measured to monitor consumption of PPi by the enzyme.
Acknowledgements
13C NMR (100 MHz, MeOD)
d 159.9, 156.5, 149.5, 138.6, 135.5, 133.1,
The authors acknowledge the support of the School of Chem-
istry and Department of Medicinal Chemistry, Monash University,
as well as Monash University for providing L.G.P. with a MIPRS and
MGS.
132.5, 132.4, 129.3, 128.2, 127.9, 127.0, 126.8, 126.7, 126.4, 125.0,
124.7, 124.7, 123.8, 60.8, 56.1. HPLC: 95% pure. LRMS (ESI): m/z:
643.2 (MþH, 55%). HRMS (ESI): m/z: calcd for [MþH]þ C42H39N6O:
643.3180, found: 643.3203.
4.1.4. 4-(Anthracen-9-yl)-2,6-bis((bis(pyridin-2-ylmethyl)amino)
methyl)phenol (3). A suspension of 2,6-bis((bis(pyridin-2-ylmethyl)
amino)methyl)-4-bromophenol (50 mg, 0.084 mmol), anthracene-
9-boronic acid (25 mg, 0.072 mmol) and potassium carbonate (2.0 g)
in water (10 mL) and dioxane (20 mL) was saturated with nitrogen.
To this mixture was added tetrakis(triphenylphosphine)palladium
(5 mg, 0.0043 mmol) and the suspension refluxed under nitrogen
for 15 h. The solution was acidified by dropwise addition of 2 M HCl
and washed with dichloromethane. The aqueous layer was then
freeze-dried and the residue purified by reverse phase prep HPLC
(0e30% acetonitrile in water over 30 min, 0.1% TFA throughout) to
provide the title compound as a white solid (40 mg, 69%). 1H NMR
References and notes
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(400 MHz, MeOD)
d
8.66 (d, J¼7.8 Hz, 4H, pyridinyl H6), 8.55 (s, 1H,
anthracenyl H10), 8.08 (d, J¼8.5 Hz, 2H, anthracenyl H4), 7.99 (d,
J¼7.8 Hz, 4H, pyridinyl H4), 7.59 (d, J¼7.8 Hz, 4H, pyridinyl H3), 7.54 (t,
J¼7.8 Hz, 4H, pyridinyl H5), 7.47 (t, J¼8.5 Hz, 2H, anthracenyl H3), 7.43
(d, J¼8.5 Hz, 2H, anthracenyl H1), 7.35 (s, J¼8.5 Hz, 2H anthracenyl
H2), 7.29 (s, 2H, phenolic H3), 4.48 (s, 8H, NCH2Py), 4.36 (s, 4H, 2-
methylphenol CH2). 13C NMR (100 MHz, MeOD)
d 157.1, 154.5, 148.4,
141.2, 136.3, 132.9, 131.6,131.3, 129.7, 128.0, 127.3, 126.7, 126.2, 125.7,
125.6, 122.1, 59.0, 57.0. HPLC: 95% pure. MS (ESI): m/z: 693.1 (MþH,
100%). HRMS (ESI): m/z: calcd for [MþH]þ C46H41N6O: 693.3336,
found: 693.3348.
4.2. Formation of zinc(II) complexes
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To a solution of ligand 1, 2 or 3 (0.35 mg) in DMSO (40
added Tris buffer (1 mL, 10 mM, pH 7.5) and an aqueous solution of
mL) was