Vanadyl complexes with dansyl-labelled di-picolinic acid ligands: Synthesis, phosphatase inhibition activity and cellular uptake studies

Article abstract of DOI:10.1039/c5dt04753f

Vanadium complexes have been previously utilised as potent inhibitors of cysteine based phosphatases (CBPs). Herein, we present the synthesis and characterisation of two new fluorescently labelled vanadyl complexes (14 and 15) with bridged di-picolinic acid ligands. These compounds differ significantly from previous vanadyl complexes with phosphatase inhibition properties in that the metal-chelating part is a single tetradentate unit, which should afford greater stability and scope for synthetic elaboration than the earlier complexes. These new complexes inhibit a selection of cysteine based phosphatases (CBPs) in the nM range with some selectivity. Fluorescence spectroscopic studies (including fluorescence anisotropy) were carried out to demonstrate that the complexes are not simply acting as vanadyl delivery vehicles but they interact with the proteins. Finally, we present preliminary fluorescence microscopy studies to demonstrate that the complexes are cell permeable and localise throughout the cytoplasm of NIH3T3 cells.

Full text of DOI:10.1039/c5dt04753f

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Vanadyl complexes with dansyl-labelled di-picolinic acid ligands: sy
D
n
OI
t
:
h
10
e
.10
s
3
i
9
s
/C5DT04753F
,
phosphatase inhibition activity and cellular uptake studies
1
,2
1,2
1
1
1
Juliet Collins, Agostino Cilibrizzi, Marina Fedorova, Gillian Whyte, Lok Hang Mak ,
1
1,2,3
1,2
1,2
Inna Guterman , Robin Leatherbarrow,
Rudiger Woscholski, Ramon Vilar *
1
Department of Chemistry, Imperial College London, London SW7 2AZ, UK
Institute of Chemical Biology, Imperial College London, London SW7 2AZ, UK
2
3
Current address: Liverpool John Moores University, Egerton Court, Liverpool L1 2UA, UK
*
Corresponding author: r.vilar@imperial.ac.uk

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Abstract
DOI: 10.1039/C5DT04753F
Vanadium complexes have been previously utilised as potent inhibitors of cysteine based
phosphatases (CBPs). Herein, we present the synthesis and characterisation of two new
fluorescently labelled vanadyl complexes (14 and 15) with bridged di-picolinic acid ligand.
These compounds differ significantly from previous vanadyl complexes with phosphatase
inhibition properties in that the metal-chelating part is a single tetradentate unit, which should
afford greater stability and scope for synthetic elaboration then the earlier complexes. These
new complexes inhibit a selection of cysteine based phosphatases (CBPs) in the nM range
with some selectivity. Fluorescence spectroscopic studies (including fluorescence anisotropy)
were carried out to demonstrate that the complexes are not simply acting as vanadyl delivery
vehicles but they interact with the proteins. Finally, we present preliminary fluorescence
microscopy studies to demonstrate that the complexes are cell permeable and localise
throughout the cytoplasm of NIH3T3 cells.

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Introduction
DOI: 10.1039/C5DT04753F
Cysteine based phosphatases (CBPs) are a large family of enzymes that are defined by a
cysteine containing motif (CX R) in their active site and include the protein tyrosine
5
1
phosphatases (PTPs) and inositol lipid phosphatases . While the former counteract the action
2
,3
of tyrosine protein kinases the latter balance inositol lipid kinases. Both play an important
role in the regulation of phosphorylation in cells, which in turn impacts cellular signalling
3
,4
and function. Mutation or dysfunction of many members of this family of enzymes have
been implicated in a range of diseases including immune and neurodegenerative disorders,
5
,6
cancer, diabetes and obesity, as well as having a role in inflammatory responses. Given the
importance of CBPs in disease progression, they are regarded as interesting targets for drug
5
,7-14
development, with a variety of inhibitors being reported.
One subset of these inhibitors
are metal ions and their complexes including vanadium, zinc, copper and others, which have
11,15-19
shown potent inhibition of PTPs in vitro and in vivo.
In particular, a variety of
complexes with both vanadium(IV) and vanadium(V) centres have been developed and
7
,10,20-27
characterised for their CBP inhibition properties
including several examples with
2
8-33
picolinic acid and its derivatives as ligands.
The potency of these inhibitors against PTPs
and lipid phosphatases can be tuned as a function of the ligand, the vanadium coordination
IV
number and the oxidation state. For example, the vanadyl complexes V O(picolinic acid)
2
IV
and V O(3-hydroxypicolinic acid) (1 and 2, Fig.1) have been shown to inhibit specific
2
2
9,31
phosphatases at nanomolar concentrations.
Using as a starting point vanadyl-picolinate complexes such as 1 and 2, we were
interested in developing novel inhibitors for PTPs and lipid phosphatases with improved
features. We hypothesised that more robust vanadyl complexes could be obtained by using
ligands where the two picolinic acids were covalently linked by an appropriate spacer to yield
a tetradentate ligand (Fig. 2). Furthermore, the linker would offer the potential for further

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chemical functionalisation, such as allowing addition of a fluorophore to track cellular uptake
DOI: 10.1039/C5DT04753F
and localisation of the resulting vanadyl complexes. Thus, herein we report on the synthesis
and characterisation of two new fluorescently labelled di-picolinic acid ligands (8 and 13,
Fig. 2) and the corresponding vanadyl complexes (14 and 15, Fig. 3). We demonstrate that
addition of the complexes to PTPs and lipid phosphatase leads to inhibition (in the nM range)
of the enzymes’ activity. We also show by fluorescence microscopy that the complexes are
cell permeable.
Fig. 1 Known vanadyl complexes with PTP and lipid phosphatase inhibitory properties.
Results and Discussion
Synthesis of ligands
The two new tetradentate ligands (8 and 13) were synthesised as shown in Fig. 2. 5-
3
4
hydroxypicolinic acid methyl ester (3) was reacted with the corresponding Boc-protected
di-alcohol (4 or 9) via a Mitsunobu reaction using diethyl azodicarboxylate and
triphenylphosphine. In both cases, after initial purification by column chromatography, the
products (5 and 10) were obtained as a mixture with triphenylphosphine oxide. Optimisation
of chromatographic conditions reduced the amount of triphenylphosphine oxide but it could

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not be removed completely. The crude materials were used in the de-protection step which,
DOI: 10.1039/C5DT04753F
after purification, yielded the corresponding amines 6 and 11 as pure compounds (as
1
confirmed by H NMR spectroscopy and mass spectrometry). The dansyl fluorophore was
introduced via sulfonylation under anhydrous conditions in the presence of triethylamine to
obtain 7 and 12, which were taken forward to the final step for removal of the methyl
protecting groups to give the corresponding ligands 8 and 13. The formation of the desired
1
products was confirmed by H NMR spectroscopy (Fig. S1 and S2, Supporting Information)
through the disappearance of the methyl peaks (at ca. 3.9 ppm); mass spectrometry provided
further evidence for the formation of both 8 and 13. Fluorescence emission spectroscopy
confirmed the presence of the dansyl fluorophore in both the diethanolamine (8, Fig. S3
Supporting Information) and dipropanolamine (13) linked ligands with emission maxima at
5
38 nm and 528 nm respectively.
Fig. 2 Synthesis of fluorescently labelled ligands 8 and 13.

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DOI: 10.1039/C5DT04753F
Synthesis of vanadyl complexes
In order to prepare the vanadyl complexes, the corresponding ligand was dissolved in water
in the presence of NaHCO ) and an aqueous solution of vanadyl sulphate slowly added. A
(
3
clear change from a yellow solution to a dark green suspension was observed upon addition
of VOSO to the ligand. The new vanadyl complexes 14 and 15 were obtained as dark green
4
solids and were characterised by spectroscopic and analytical techniques. MALDI-TOF mass
+
spectrometry showed peaks corresponding to [M+Na] in each case. The IR spectra of the
-1
two complexes shows peaks for the ligand at 1645-1572 cm (which can be assigned to C=O,
-1
-1
C=C and C=N) as well as a strong peak at 965 cm and 961 cm (assigned to V=O) for 14
and 15 respectively (Table 1). The formulation and purity of the complexes was confirmed by
elemental analyses. Both vanadyl complexes are fluorescent (Table 1) although their
emission intensities are lower than those of the corresponding free ligands.
Fig. 3 Synthesis of vanadyl complexes 14 and 15 from the corresponding ligands (8 and 13) and
vanadyl sulphate.

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DOI: 10.1039/C5DT04753F
Table 1. Spectroscopic data for vanadyl complexes. The IR data was recorded using a solid sample
of the corresponding compound while UV/Vis and emission spectroscopic data was collected using
solutions in methanol (14 and 15 at 30 µM, 1 at 15 µM and 2 at 45 µM). Fluorescence data was
recorded in Tris buffer (30 µM).
UV-vis λmax /nm
IR
V=O)/cm
Fluorescence
λmax(em)/nm
Compound
-1
-1
-1
(
(
ε/ M cm )
1
1
1
2
4
5
965
961
962
954
263
568
566
-
4
(
4.86×10 )
262
4
(
7.37×10 )
266
3
(
7.78 ×10 )
305
-
3
(
9.8×10 )
Phosphatase inhibition assays
In order to characterise the inhibitory potency of 14 and 15, phosphatase inhibition assays
against a selection of PTPs (PTP1B, LMW-PTP, VHR and SHP-2 – see Table 2 for
abbreviations) as well as the inositol lipid phosphatase PTEN were carried out employing 3-
2
8
O-methylfluorescein phosphate (OMFP) as a substrate. As shown in Table 2 compounds 14
and 15 inhibit all PTPs tested in the mid to high nanomolar range, which is consistent with
2
9,31
the inhibitory potency previously reported for compound 1 (Fig 1).
The IC value for 14
50
against LMW-PTP and PTEN is significantly higher than for the other enzymes while
complex 15 has a significantly lower IC value for PTP1B than for any other of the tested
5
0
phosphatases. These observations suggest that the complexes show some selectivity towards
certain PTPs compared to others. There also seems to be an effect on the inhibitory properties
of the complex on increasing the length of the linker from diethanolamine (14) to
dipropanolamine (15) for VHR, SHP2, and LMW-PTP. The IC values for VHR and SHP2
5
0

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DOI: 10.1039/C5DT04753F
are around two to three fold higher for compound 15 than 14 whereas the IC is reduced
5
0
against LMW-PTP on increasing linker length. This suggests that varying the linker length
could be used as a method to tune selectivity of the complex as the effect differs across the
CBPs. As a control, we also carried out the inhibition studies with the free ligands (8 and 13)
using VHR and LMW-PTP as representative examples of the phosphatases. No inhibition
was observed up to a 1 μM concentration of the ligand and only minor effects were observed
at 10 μM (Fig. S6 and S7, Supporting Information); this indicates that the free ligand does not
contribute to the IC values reported in Table 2. We also carried out the inhibition studies
5
0
using VOSO as a control. As can be seen in Table 2, the inhibitor profile for VOSO did not
4
4
match with the one observed for compounds 14 and 15. For example, VOSO is more potent
4
against SHP2, but significantly less potent for PTP1B, which is not what one would expect if
the complexes are a delivery mechanism for vanadyl itself. Thus, the data suggests that 14
and 15 are not acting simply as a vanadyl delivery vehicle. This notion was explored in more
depth using spectroscopy means (see next section).
Table 2 IC values (nM) of vanadyl complexes 14 and 15 (the data shown is ±standard deviation of
50
triplicate repeats)
Compound
PTEN
211±33
222±49
-
PTP1B
94±43
84±23
151±16
VHR
72±10
SHP2
77±27
162±20
71±80
LMW-PTP
177±7
1
1
4
5
130±14
120±60
120±12
117±40
VOSO4
PTEN = Phosphatase and tensin homolog. PTP1B = Protein-tyrosine phosphatase 1. SHP-2 = Src homology
region 2 domain-containing phosphatase-2. LMW-PTP = Low molecular weight protein tyrosine phosphatase.
VHR = dual specificity protein phosphatase 3. For assay conditions see experimental section.

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DOI: 10.1039/C5DT04753F
Effects of enzyme on the fluorescence of vanadyl complexes
Having shown that the potency of 14, 15 and VOSO for the selected PTPs is close to each
4
other, it was of interest to corroborate the conclusion drawn from the data presented in table
2
. In order to investigate the integrity of the complexes in the enzyme assays, we made use of
the dansyl moiety within the ligand in a number of fluorescence spectroscopic studies. Upon
coordination of vanadyl to ligands 8 and 13, the emission of the dansyl decreases, which can
be employed to monitor the integrity of the complexes while interacting with the enzymes.
Therefore, we first studied whether the fluorescence of the ligand in complex 14 could be
recovered by removing the vanadyl from the corresponding complex with EDTA, a strong
2
3
vanadyl chelator. As can be seen in Figure 4, addition of an excess of EDTA (1 mM) to
complex 14 (30 µM), restored the fluorescence of the ligand in the absence of enzyme.
Having established that EDTA can indeed remove vanadyl from the complexes and in doing
so change the fluorescence of the ligand, we investigated the effect of LMW-PTP (as a
representative example of the PTPs) on the emission of complex 14. As can been seen in
Figure 4, incubation of 14 with LMW-PTP did not restore the emission of the ligand which
suggests that the vanadyl is still coordinated to ligand 8 in the presence of the enzyme. A
control experiment was carried out in which the compound was incubated with both EDTA
and the enzyme and the emission intensity compared to that of the corresponding ligand
under the same conditions. Interestingly, in this case full restoration of the emission was not
observed after 5 minutes incubation (see Fig. S4, Supplementary Information) in spite of the
presence of EDTA. Complete restoration was, however, observed after 2 hours incubation
(see Fig. 4). These experiments were also carried out with ligand 13 and complex 15 and the
same pattern of results was observed (see Fig S5, Supplementary Information, i.e. 2 hours
incubation).

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Taken together these observations imply that the enzyme LMW-PTP binds to the
DOI: 10.1039/C5DT04753F
intact complex (as defined by the vanadyl being near the fluorophore-ligand) and in doing so
protects’ the tightly bound complexes from EDTA. However, we cannot rule out the
‘
possibility that the vanadyl, while still bound to the ligand, may form new interactions with
the enzymes, which would imply that the vanadyl would exchange partially coordination sites
between ligand and enzyme. We should also note that the interaction between the vanadium
complexes and the Tris buffer used in these studies is unlikely to affect the observations.
3
5
Previous studies have shown that such interactions are weak (i.e. high mM). Furthermore,
as we have demonstrated in this study, EDTA is required to remove the vanadium centre
from the complex (in Tris buffer). Consequently, under the conditions of our studies, Tris is
an appropriate buffer.
Fig.4 Fluorescence intensity at 560 nm of a 30 µM solution of ligand 8 and complex 14 in the
presence or absence of LMW-PTP (Enz) and 1 mM EDTA in Tris buffer containing 1 mM DTT. Reading
was taken after 2 hours incubation at room temperature. Intensity is recorded ±standard deviation
of triplicate repeats. A statistical analysis has been performed showing that the difference between
st
rd
the fluorescence intensity of 8 and 14 incubated with enzyme in the absence of EDTA (1 and 3 bar
pairs in this figure) are statistically significant (*p < 0.05).
To investigate this further, we carried out fluorescence anisotropy studies of complex
1
4 in the presence and absence of LMW-PTP (as model enzyme). This type of studies is
widely used to investigate the interaction between an emissive molecule and large bio-

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molecules (such as proteins). This technique relies on the change in rotational correlation
DOI: 10.1039/C5DT04753F
time of the fluorophore (in this case our dansyl-labelled vanadyl complex) upon interaction
with a large molecule (in this case LMW-PTP). As can be seen in Fig. 5(a), addition of the
enzyme to the complex led to an increase in the fluorescence anisotropy (reaching a
maximum at ca. 2 equivalents of the enzyme) consistent with an interaction of the complex
with the enzyme.
We then investigated the effect that EDTA has on the fluorescence anisotropy of
complex 14 pre-bound to LMW-PTP. As Fig. 5(b) shows, the fluorescence anisotropy was
reduced within an hour to the level of the enzyme-free sample suggesting that when the
vanadyl is sequestered by the EDTA the fluorescent ligand no longer associates with the
enzyme. Control studies with the free ligand were also carried out. The fluorescence
anisotropy of 8 under the same experimental conditions (see caption in Fig. 5) was 0.20 and
in the presence of enzyme (36 μM) was 0.21. This clearly demonstrates that the free ligand
does not interact with the enzyme and therefore the changes observed with complex 14 are
due to binding of the complex with the protein.
Fig.5 (a) Fluorescence anisotropy of 14 (10 µM) in the presence of increasing concentrations of
LMW-PTP (0-36 µM). (b) Fluorescence anisotropy of 14 (10 µM) in the absence or presence of LMW-
PTP and over 100 minutes of incubation with the enzyme and 1 mM EDTA. Readings were taken in
1
3
00mM Tris buffer solution with 1mM DTT in a 200µL fluorescence cuvette. Excitation wavelength is
40 nm, emission recorded at 560 nm. The error is shown as the standard deviation of 6 readings
from two independent experiments. Significant differences (5% cut-off for significance) to the

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DOI: 10.1039/C5DT04753F
“
buffer + compound” data are indicated by stars (* p < 0.05; ** p < 0.01; *** p < 0.001). The blank
control used 50% glycerol in elution buffer to mimic the enzyme solution as the viscosity of the
mixture would have an effect on the polarisation.
The spectrofluorimetric data shown in Figures 4 and 5 clearly indicate that the
complex does not break completely apart in the presence of the enzyme under physiological
conditions. There have been extensive studies in the literature aimed at understanding the
mode of action of vanadium complexes in the context of their CBP inhibition properties.
There is considerable evidence suggesting that such complexes act as prodrugs releasing the
3
6
active vanadium species in biological media. Interestingly our data suggests that, under the
conditions used, the vanadium complexes do not break apart completely.
Cellular uptake studies
In order for the complexes 14 and 15 to be utilised as PTPs inhibitors in a cellular
environment their ability to be taken up into cells would have to be confirmed. Compound 14
was chosen as a representative complex and thus, NIH3T3 cells were incubated with three
different concentrations of 14 (1, 5 and 10 μM) for 24 hours at 37 ºC. The cells were
subsequently analysed by fluorescence microscopy. As can be seen in Fig. 6, the complex
(green) is widely taken up by cells. Under these conditions, the complex is localised
throughout the cell, but seems to be enriched in some structures within the nucleus and the
perinuclear region. This is in agreement with the known subcellular localisation of PTEN and
VHR, which are known to be present in the perinuclear region as well as the nuclear
3
7-39
compartment.
In contrast, other enzymes that might be targeted by 14 are known to
4
0
localise to the ER/nuclear envelope network (PTP1B), the cytosol and cytoskeletal

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4
1
39
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DOI: 10.1039/C5DT04753F
compartment (LMW-PTP), or the cytosolic localisation (SHP-2). While we cannot rule
out the possibility that the observed localisation of 14 is influenced by other cysteine based
phosphatases as well, it seems that in the cellular environment 14 is distributed throughout
the cell in a pattern that matches the phosphatases it is targeting.
Fig. 6 (a) Fluorescence microscopy images showing uptake of 14 (green) by NIH3T3. Cells were
incubated in media containing 0 μM (control), 1 μM, 5 μM or 10 μM of complex 14 and lastly fixed
with PFA before imaging. To provide a better perception of the nuclear and perinuclear localisation
of compound 14 three representative images are presented for each treatment in comparison with
merge channel images. (b) Quantification of fluorescence signal was performed with FIJI. Data for
the quantification are reported as ± SEM and were obtained from a minimum of 3 independent
experiments counting a total number of cells as follow: 10 (for control), 7 (for 1 μM), 10 (for 5 μM),

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DOI: 10.1039/C5DT04753F
1
8 (for 10 μM). Significant differences (5% cut-off for significance) to the control data are indicated
by stars ( *** p < 0.001).
Conclusions
Two new di-picolinic acid-based compounds containing a dansyl fluorophore have been
prepared and successfully used as ligands to prepare the two novel vanadyl complexes 14 and
1
5. Phosphatase inhibition assays showed that the complexes inhibited a selection of
phosphatases (PTP1B, SHP-2, LMW-PTP, VHR and PTEN) over a concentration range of
2–222 nM and some selectivity and effect of linker length was observed. The variability in
7
inhibition properties, together with fluorescence data for the complexes in the presence and
absence of EDTA and enzyme indicate that the complexes are not simply acting as vanadyl
delivery vehicles. On the other hand, based on their large size and saturated coordination
sphere, it is unlikely that the intact complexes are binding to the enzymes and causing the
inhibition of their activity. Therefore, we propose that derivatives of complexes 14 and 15 in
which the ligand (8 and 13 respectively) is partially de-coordinated from the vanadyl centre
are interacting with the enzymes. A likely possibility could be that one of the picolinate
‘arms’ of the ligands de-coordinates leaving the vanadyl coordinatevely unsaturated and
hence able to interact strongly with the enzymes and yet retain the fluorescent ligand (see
Figure 7) as supported by the fluorescence studies. It has also been shown that the complexes
are cell permeable and localize in the cytoplasm. The complexes represent a new class of
tetradentate vanadyl inhibitors that offer the potential for synthetic functionalization. The data
indicates that these new optically labelled vanadyl complexes have the potential to be
developed further into potent and potentially selective inhibitors and targeted probes.

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DOI: 10.1039/C5DT04753F
Fig. 7 Schematic representation of a potential binding mode between the vanadyl complexes and
CBPs. This proposed interaction – based on the de-coordination of one ‘arm’ of the ligand – is
consistent with the experimental observations herein presented.
Acknowledgements
This work was supported by Proxomics EPSRC Cross-Disciplinary Research Landscape
Award (EP/I017887/1). L.H.M. is grateful for financial support from the Lowe Syndrome
Trust UK. Dr David Gaboriau is acknowledged for his help with cell microscopy experiments
performed at “The Facility for Imaging by Light Microscopy” (FILM, Imperial College
London), supported by funding from the Wellcome Trust (grant P49828) and BBSRC (grant
P48528)”.
Experimental Details
General spectroscopy and materials. IR spectra were recorded on a Perkin Elmer FT–
-1
IR1720 Research Series spectrometer in the range 4000-600 cm . Electrospray (ESI+) mass
spectrometry was run at on a Waters Aquity UPLC I-CLASS coupled with Waters LCT
Premier (operating in ES+ or ES- mode). MALDI analyses were performed using MicroMass
MALDI microMX TOF operating in reflectron mode using a 337nm nitrogen laser. NMR

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6
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DOI: 10.1039/C5DT04753F
spectra were recorded on a Bruker spectrometer with d -DMSO (δ = 2.50 ppm, δ = 39.5
H
C
ppm), CD OD (δ = 3.31 ppm, δ = 49.2 ppm) and CDCl (δ = 7.26 ppm, δ = 77.2 ppm) as
3
H
C
3
H
C
internal references. UV-vis spectra were recorded on a Perkin-Elmer Lambda 25 UV-vis
spectrometer. Fluorescence data were collected using a Varian fluorescence spectrometer,
while fluorescence polarisation studies were performed with a Perkin-Elmer LS50B
Luminescence Spectrometer. Compounds 3, 4, and 9 were prepared according to reported
3
4,42,43
procedures.
Synthesis of 5. To a solution of 5-hydroxyoxypicolinic acid methyl ester 3 (312 mg, 2.03
mmol), triphenylphosphine (1.07 g, 4.07 mmol) and N-Boc diethanolamine 4 (209 mg, 1.02
mmol) in anhydrous THF (15 mL), stirred on ice under N , was added diethyl
2
azodicarboxylate (DEAD, 0.64 mL, 4.07 mmol) in anhydrous THF (1 mL). The resulting
reaction mixture was stirred at room temperature for 16 hours. The solvent was removed by
rotary evaporation to give the crude product 5 as an orange oil. The product was purified by
silica gel column chromatography (70% EtOAc/Hex – 3% MeOH/EtOAc) to yield a mixture
(ca. 1:0.1) of 5 and triphenylphosphine oxide. This compound was used without further
purification for the next step. The following spectroscopic data corresponds to the signals
1
associated to 5: H NMR (400 MHz, CDCl ): δ(ppm) 8.37 (d, J = 2.9 Hz, 2H), 8.10 (d, J =
3
8
.7 Hz, 2H), 7.26 (m, 2H), 4.24 (m, 4H), 3.98 (s, 1H,), 3.77 (t, J = 5.4 Hz, 4H), 1.46 (s, 9H).
1
3
C NMR (100 MHz, CDCl ): δ(ppm) 165.2 (C), 157.4(C), 155.3 (C), 138.5 (CH), 138.3
3
(
CH), 127.0 (C), 120.4 (CH), 67.4 (CH ), 52.9 (CH ), 48.2 (CH ), 28.6 (CH ). ESI-MS (+)
2
3
2
3
+
4
76 ([M+H] ) (100%).
Synthesis of 6. To a solution of 5 (477 mg, 0.87 mmol) in dichloromethane (1 mL) was
added trifluoroacetic acid (1 mL) and the reaction stirred at room temperature for 1 hour. The
solvent was removed under reduced pressure and the product purified by silica gel column

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DOI: 10.1039/C5DT04753F
chromatography (EtOAc then MeOH:dichloromethane: NEt 10:85:5) to give 6 (326 mg,
3
1
0
.87 mmol, 100%) as a pale pink solid. H NMR (400 MHz, CDCl ): δ(ppm) 8.33 (d, J = 2.8
3
Hz, 2H), 8.09 (d, J = 8.7 Hz, 2H), 7.52 (dd, J = 8.8, 2.9 Hz, 2H), 4.35 (t, J = 5.0 Hz, 4H), 3.91
1
3
(
s, 6H), 3.36 (t, J = 5.0 Hz). C NMR (100 MHz, CD OD): δ(ppm) 166.2 (C), 159.0 (C),
3
1
41.2 (CH), 139.5 (CH), 127.9 (C), 122.2 (CH), 67.5 (CH ), 53.0 (CH ), 47.9 (CH ). IR in
2 3 2
-1
cm υ(C-H)/υ(N-H) 3059-2955, υ(C=N)/υ(C=C) 1735, 1668,1623, 1578, υ(C-O) 1303. LC-
+
MS(+) 377 ([M+H] ) (100%).
Synthesis of 7. To a solution of 6 (100 mg, 0.27 mmol) and dansyl chloride (87 mg, 0.32
mmol) in anhydrous acetonitrile (8.5 mL) was added anhydrous triethylamine (0.08 mL, 0.54
mmol) and the reaction stirred at room temperature for 16 h. The solvent was removed by
rotary evaporation to give a yellow residue. The crude mixture was purified by silica gel
column chromatography (2% MeOH in dichloromethane) to give the desired product 7 (83
1
mg, 0.14 mmol, 50%) as a yellow/green oil. H NMR (400 MHz, CD OD): δ(ppm) 8.50 (dt, J
3
=
8.6, 1.0 Hz, 1H), 8.24 (dd, J = 7.3, 1.2 Hz, 1H), 8.21-8.18 (m, 1H), 8.01-7.90 (m, 4H), 7.52
(
(
(
(
(
(
dd, J = 8.5, 7.3 Hz, 1H) 7.41 (dd, J = 8.7, 7.6 Hz, 1H), 7.19 (dd, J = 8.8, 2.9 Hz, 2H), 7.13
1
3
dd, J = 7.7, 0.8 Hz, 1H), 4.21 (t, J = 5.2 Hz, 4H), 3.91 (m, 10H), 2.81 (s, 6H). C NMR
100 MHz, CD OD): δ(ppm) 166.2 (C), 158.8 (C), 153.2 (C), 140.8 (CH), 139.3 (CH), 136.1
3
C), 131.9 (C), 131.3, 131.2 (CH), 131.1 (CH), 129.2 (CH), 127.7 (C), 124.3 (CH), 121.8
CH), 120.5 (C), 116.4 (CH), 68.2 (CH ), 53.0 (CH ), 45.7 (CH ). ESI-MS (+) 609
2
3
2
+
-1
[M+H] ). IR in cm υ(C=N)/υ(C=C) 1715, 1573.
Synthesis of 8. To a solution of 7 (196 mg, 0.32 mmol) in THF:MeOH (0.8:0.3 mL) on ice
was added 6M NaOH (0.15 mL, 0.90 mmol) and the reaction stirred for 3 hours at room
temperature. The solvent was removed by rotary evaporation to give a bright yellow solid
which was diluted with water and extracted with ethyl acetate. The organic layer was set

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aside and the aqueous layer acidified with 2M HCl and re-extracted with EtOAc (4 x 10 mL).
DOI: 10.1039/C5DT04753F
The organic layers were combined, dried over Mg SO and the solvent removed by rotary
2
4
1
evaporation to give the desired product 8 (98.5 mg, 0.17 mmol, 53%) as a yellow solid. H
NMR (400 MHz, CD OD): δ(ppm) 8.51 (d, J = 8.6 Hz, 1H), 8.25 (d, J = 7.4, 1.2 Hz, 1H),
3
8
7
3
1
1
.20 (d, J = 8.7 Hz, 1H), 8.06-7.92 (m, 4H), 7.53 (dd, J = 8.5, 7.3 Hz, 1H), 7.42 (dd, J = 8.7,
.6 Hz, 1H), 7.21 (dd, J = 8.7, 2.9 Hz, 2H), 7.13 (d, J = 7.5 Hz, 1H), 4.21 (t, J = 5.2 Hz, 4H),
1
3
6
.93 (t, J = 5.2 Hz, 4H), 2.83 (s, 6H). C NMR (100 MHz, DMSO-d ): δ(ppm) 165.7 (C),
56.5 (C), 151.4 (C), 140.6 (CH), 137.7 (CH), 134.8 (C), 130.1 (C), 129.4, 129.2 (CH),
28.9 (CH), 128.1 (CH), 126.1 (C), 123.6 (CH), 120.6 (CH), 118.8 (C), 115.2 (CH), 66.4
+
-1
(
CH ), 46.8 (CH ), 45.0 (CH ). ESI-MS (+) 581 ([M+H] ). IR in cm υ(C=N)/υ(C=C) 1690,
2 3 2
1
573, υ(S=O) 1140. Fluorescence in DMF: λmax(ex) 358 nm, λmax(em) 538 nm.
Synthesis of 14. To a suspension of 8 (167 mg, 0.29 mmol) in water (6 mL) was added 10%
NaHCO (0.51 mL, 0.58 mmol) and the mixture stirred until all solid was dissolved. VOSO
3
4
(63 mg, 0.29 mmol) in water (4 mL) was added slowly over 2 minutes after which a
precipitate began to form. The reaction was left to stir for 2 hours. After 1 week without
stirring a green solid precipitated from the solution. This was collected by filtration and IR
indicated a vanadyl complex had formed. Further characterisation by mass spectrometry
indicated this was the desired complex 14 (13 mg, 0.02 mmol, 7%). ESI-MS (+) 668
+
-1
(
[M+Na] ). IR in cm υ(C=N)/υ(C=C) 1636, 1591, 1575, υ(S=O) 1136, υ(V=O) 965. Calc.
for C H O N SV·3H O: C 48.07, H 4.61, N 8.01 Found: C 48.07, H 4.39, N 7.72. UV-vis
2
8
26
9
4
2
-1
-1
4
4
(
MeOH/DMF) λ/nm (ε/M ·cm ) 263 (4.86×10 ), 337 (1.03×10 ). Fluorescence in Tris
buffer: λmax(ex) 340 nm, λmax(em) 568 nm.
Synthesis of 10. To a solution of 5-hydroxypicolinic acid methyl ester 3 (362 mg, 2.36
mmol), triphenylphosphine (1.24 g, 4.72 mmol) and N-Boc dipropanolamine 9 (276 mg, 1.18

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DOI: 10.1039/C5DT04753F
mmol) in anhydrous THF (18 mL), stirred on ice under N , was added diethyl
2
azodicarboxylate (DEAD, 0.74 mL, 4.72 mmol) in anhydrous THF (1.2 mL) and the reaction
was stirred at room temperature for 16 h. Solvent was removed by rotary evaporation to give
the crude product 10 as an orange oil. The product was purified by silica gel column
chromatography (80% EtOAc/Hex – 3% MeOH/EtOAc) to yield a mixture (ca. 1:0.2) of 10
and triphenylphosphine oxide. This compound was used without further purification for the
1
next step. The following spectroscopic data corresponds to the signals associated to 10: H
NMR (400 MHz, CDCl ): δ(ppm) 8.37 (d, J = 2.9 Hz, 2H), 8.10 (d, J = 8.7 Hz, 2H), 7.24 (dd,
3
J = 8.8, 2.9 Hz, 2H), 4.13-4.08 (m, 4H), 3.97 (s, 6H), 3.41 (t, J = 6.9 Hz, 4H), 2.16-2.03 (m,
4
H), 1.39 (s, 9H).
Synthesis of 11.To a solution of 10 (421 mg, 0.95 mmol) in dichloromethane (1 mL) was
added trifluoroacetic acid (1 mL) and the reaction stirred at room temperature for 1 hour. The
solvent was removed and the product 11 isolated pure by silica gel column chromatography
(
EtOAc then MeOH:dichloromethane:NEt 10:85:5) to give the desired product 11 (279 mg,
3
1
0
2
3
1
.67 mmol, 71%) as a pale pink solid. H NMR (400 MHz, d6-DMSO): δ(ppm) 8.37 (d, J =
.8 Hz, 2H), 8.03 (d, J = 8.7 Hz, 2H), 7.50 (dd, J = 8.8, 2.9 Hz, 2H), 4.21 (t, J = 6.2 Hz, 4H),
1
3
.84 (s, 6H), 2.91 (t, J = 7.1 Hz, 4H), 2.00 (m, 4H). C NMR (100 MHz, d6-DMSO): δ(ppm)
64.8 (C), 157.2 (C), 139.5 (CH), 139.4 (CH), 126.4 (C), 120.6 (CH), 66.1 (CH ), 52.1
2
-1
(
CH ), 44.9 (CH ), 27.1 (CH ). IR in cm υ(N-H) 2804, υ(C=N)/υ(C=C) 1732, 1669, 1574,
3 2 2
+
+
υ(C-O) 1316. ESI-MS (+) 404 ([M+H] ), 426 ([M+Na] ).
Synthesis of 12. To a solution of 11 (270 mg, 0.67 mmol) and dansyl chloride (217 mg, 0.80
mmol) in anhydrous acetonitrile (21 mL) was added anhydrous triethylamine (0.28 mL, 2.0
mmol) and the reaction stirred at room temperature for 48 h. The solvent was removed by
rotary evaporation to give a yellow residue. The crude mixture was purified by silica gel

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column chromatography (2- 3% MeOH in dichloromethane) to give the desired product 12
DOI: 10.1039/C5DT04753F
1
(
317 mg, 0.50 mmol, 75%) as a yellow solid.. H NMR (400 MHz, CD OD): δ(ppm) 8.26 (dt,
3
J = 8.6, 1.0 Hz, 1H), 8.21 (dd, J = 7.4, 1.2 Hz, 1H), 8.13 (d, J = 8.7, 0.9 Hz, 1H), 8.04 (d, J =
.9 Hz, 2H), 7.99 (d, J = 8.8 Hz, 2H), 7.54-7.46 (m, 2H), 7.12 (dd, J = 8.7, 2.9 Hz, 2H), 7.09
d, J = 7.7, 0.8 Hz, 1H), 3.96-3.93 (m, 10H), 3.59 (t, 4H, J = 6.7 Hz), 2.77 (s, 6H) 2.08 (m,
2
(
1
3
4
1
1
H). C NMR (100 MHz, CD OD): δ(ppm) 166.3 (C), 159.1 (C), 153.1 (C), 140.5 (CH),
3
39.3 (CH), 135.3 (C), 131.9 (C), 131.7, 131.2 (CH), 130.9 (CH), 129.2 (CH), 127.7 (C),
24.1 (CH), 121.6 (CH), 120.0 (C), 116.0 (CH), 66.7 (CH ), 53.0 (CH ), 45.7 (CH ), 43.6
2
3
2
+
+
(
CH ), 28.0 (CH ). ESI-MS (+) 637 ([M+H] ), 659 ([M+Na] ).
2 2
Synthesis of 13. To a solution of 12 (317 mg, 0.50 mmol) in THF:MeOH (1.3:0.5 mL) on ice
was added 6M NaOH (0.23 mL, 1.90 mmol) and the reaction stirred for 3 h at room
temperature. The solvent was removed by rotary evaporation to give a pale yellow solid
which was diluted with water and extracted with ethyl acetate. The organic layer was set
aside and the aqueous layer acidified with 6M HCl and re-extracted with EtOAc (3×6 mL)
and dichloromethane (1 × 6 mL). The organic layers were combined, dried over Mg SO and
2
4
the solvent removed by rotary evaporation to give the desired product 13 (157 mg, 0.26
1
mmol, 52%) as a yellow solid. H NMR (400 MHz, CD OD): δ(ppm) 8.27 (d, J = 8.5 Hz,
3
1
8
H), 8.22 (d, J = 7.4 Hz, 1H), 8.14 (d, J = 8.4 Hz, 1H), 8.06 (d, J = 2.8 Hz, 2H), 8.02 (d, J =
.8 Hz, 2H), 7.54-7.48 (m, 2H), 7.17 (dd, J = 8.7, 2.9 Hz, 2H), 7.12 (d, J = 7.6 Hz, 1H), 3.96
1
3
(
t, J = 5.8 Hz, 4H), 3.60 (t, 4H, J = 6.8), 2.79 (s, 6H), 2.12 – 2.05 (m, 4H). C NMR (100
6
MHz, d -DMSO): δ(ppm) 165.7 (C), 156.8 (C), 151.4 (C), 140.3 (CH), 137.7 (CH), 134.2
(C), 130.1 (C), 129.4, 129.3 (CH), 129.1 (CH), 128.1 (CH), 126.1 (C), 123.5 (CH), 120.4
(
CH), 118.5 (C), 115.0 (CH), 65.4 (CH ), 45.0 (CH ), 43.1 (CH ), 27.0 (CH ). ESI-MS (+)
2
2
2
2
+
6
09 ([M+H] ). Fluorescence in DMF: λmax(ex) 340 nm, λmax(em) 528 nm.

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Synthesis of 15. To a suspension of 13 (150 mg, 0.25 mmol) in water (6 mL) was added 10%
DOI: 10.1039/C5DT04753F
NaHCO (0.44 mL, 0.49 mmol) and the mixture stirred until all solid was dissolved. A
3
solution of VOSO (53.5 mg, 0.25 mmol) in water (3.5 mL) was added slowly over 2 minutes
4
after which a precipitate began to form. The reaction was left to stir for 2 hours. After 2 days
without stirring a green solid had precipitated from the reaction solution which was collected
by filtration and IR indicated a vanadyl complex had formed. Further characterisation by
mass spectrometry indicated this was the desired complex 15 (42 mg, 0.06 mmol, 25%). ESI-
+
-1
MS (+) 696 ([M+Na] ). IR in cm υ(C=N)/υ(C=C) 1645,1591, 1572, υ(S=O) 1134, υ(V=O)
9
7
61. Calc. for C H O N SV·3.5H O: C 48.92, H 5.06, N 7.61 Found: C 48.62, H 4.71, N
30 30 9 4 2
-1
-1
4
4
.39. UV-vis (MeOH/DMF) λ/nm (ε/M ·cm ) 262 (7.37×10 ), 340 (1.30×10 ). Fluorescence
in Tris buffer: λmax(ex) 340 nm, λmax(em) 566 nm.
CBP Expression and Purification. PTP1B, VHR, SHP2 and LMW-PTP were expressed as
GST-fusion proteins. The protocol followed was: the corresponding RNA was extracted
using PureLink® RNA Mini Kit from Invitrogen (according to manufacturer's protocol) from
HEK293 cells. First DNA synthesis was performed using ProtoScript® First Strand cDNA
Synthesis Kit from New England Biolabs according to manufacturer's protocol. In a second
round of PCR, gene specific primers were used to obtain the DNA for PTEN, PTP1B, VHR,
SHP2 and LMW-PTP, adding the restriction site BamHI at 5' and XhoI at the 3' end,
respectively. The obtained DNA fragments were then cloned into pGEX-6P-1 vector using
the aforementioned restriction sites. All four constructs were validated via DNA sequencing
to ensure in-frame cloning with the GST tag encoded by the pGEX-6P-1 vector.
Protein expression was induced in the E. coli strain DH5α for 24h using 1 mM IPTG at 23°
C. After growth the cells were harvested and stored at -20°C. The harvested cells were

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resuspended in lysis buffer containing 50 mM Tris (pH 7.4), 1% Triton X-100, 10 mM
DOI: 10.1039/C5DT04753F
benzamidine hydrochloride, 100 µg/mL soybean trypsin inhibitor, 1 mM 4-(2-
Aminoethyl)benzenesulfonyl fluoride hydrochloride and 2 mM DTT. Lysozyme was added
to the cell suspension at a concentration of 2 mg/mL and stirred for 1 h at 4°C. Lysis was
completed by sonication, followed by centrifugation at 18000 g for 30 min at 4°C. The
supernatant was loaded onto a glutathione sepharose column, pre-equilibrated with 50 mM
Tris (pH 7.4), 140 mM NaCl and 2.7 mM KCl. After loading, the column was washed twice
with 50 mM Tris (pH 7.4), 140 mM NaCl, 2.7 mM KCl and 2 mM DTT. Another two washes
were performed using the same buffer with 500 mM NaCl. The GST-tagged phosphatases
were eluted using 20 mM glutathione in 50 mM Tris (pH 7.4), 250 mM NaCl and 2 mM
DTT. 50 % glycerol was added and the proteins were stored at -80°C. Protein concentration
was determined using Bradford assay.
Phosphatase inhibition assays in the absence of EDTA. 3-O-methylfluorescein phosphate
(OMFP, 10 mM in DMSO) was diluted with a 1% DMSO solution to the required
concentration. The corresponding vanadyl complexes were dissolved in DMF or DMSO to 10
mM and further diluted in water containing 1% DMSO to the required concentrations. Assays
were run in 100 mM Tris (pH7.4) containing 1 mM DTT at room temperature. The inhibitor
solutions were incubated with the enzyme in the buffer for 10 minutes at room temperature
before reaction was initiated by addition of OMFP. Hydrolysis of OMFP to OMF was
monitored by measuring changes in fluorescence over 30 minutes at 60 s intervals (excitation
4
85 nm, emission 525 nm) using a Varian fluorescence spectrometer. Enzyme free blanks
were run to eliminate background effects of OMFP hydrolysis in solution.
Statistical analysis of enzyme inhibition data. Statistical comparison of the means (using
GraphPad Prism 5 or StatPlus, AnalystSoft Inc.) was performed by a two-tailed t-test. A 5%

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cut-off for significance was employed and the degree of significance indicated by stars (i.e. *
DOI: 10.1039/C5DT04753F
p < 0.05; ** p < 0.01; *** p < 0.001) within the corresponding figures.
Changes in fluorescence of complexes in presence of enzyme and/or EDTA. Vanadyl
compounds were dissolved in DMF to 10 mM and further diluted in buffer containing 1%
DMSO to 300 µM. Enzyme was added to buffer solution of 100 mM Tris (pH7.4) containing
1
mM DTT, with or without 1 mM EDTA. Enzyme free controls consisted of buffer solution
of 100 mM Tris (pH=7.4) containing 1 mM DTT, with or without 1 mM EDTA. In a 96-well
plate, the inhibitor solutions were added to wells containing one of the above mentioned
buffers to a final concentration of 30 µM. Fluorescence emission spectra were recorded in the
range of 380-650 nm after excitation at 340 nm with excitation slit widths of 5-10 nm and
emission slit widths of 10-20 nm using a Varian fluorescence spectrometer. Readings were
taken 5 minutes and 2 hours after addition of inhibitor solutions.
Fluorescence Anisotropy Measurements: Vanadium complexes were dissolved in DMSO
to 10 mM and further diluted in a 1% DMSO solution to the required concentration. A five-
fold excess of enzyme was diluted in an aqueous solution of 1 mM DTT and 100 mM Tris
(
pH 7.4) and incubated with the inhibitor for 10 mins. A 50:50 glycerol-elution buffer
100mM Tris, 250 mM NaCl, 20 mM reduced glutathione, 2 mM DTT, pH 7.4) mixture was
(
used for the enzyme free control. Samples were then analysed for polarised fluorescence
emission in a fluorescence cuvette (excitation 340 nm, emission 560 nM). 1mM EDTA was
then added to the enzyme containing solution and further readings were taken every 15 mins
for 60 mins and then a final reading at 100 mins.

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Fluorescence Anisotropy Measurements – enzyme titration: Vanadium complexes were
DOI: 10.1039/C5DT04753F
dissolved in DMSO to 10 mM and further diluted in a 1% DMSO solution to the required
concentration before mixing with an aqueous solution of 1 mM DTT and 100 mM Tris (pH
7
.4). A fluorescence anisotropy reading was taken at excitation 340 nM, emission 560 nM.
LMW-PTP was then titrated into the mixture increasing from 0.36 equivalent to 3.6
equivalents with a fluorescence anisotropy reading taken after 1 min incubation with each
enzyme addition.
Cell uptake studies. NIH3T3 cells were grown in Dulbecco’s modified Eagle’s medium
(
DMEM) supplemented with 10% newborn calf serum in an atmosphere of 10 % CO at
2
3
7ºC. Cells were seeded in a plastic 24-well plate. After 16 hours the compound was added
to the cells in fresh media at 1, 5 and 10 µM (final DMSO concentration 1 % v/v for all
wells), and incubated for 24 hours at 37ºC and 10% CO . The media was then removed and
2
the cells washed three times with sterile Dulbecco’s phosphate buffered saline (PBS, EDTA-
free). The cells were fixed by adding 4% paraformaldehyde (PFA) for 15 minutes at room
temperature. The coverslips were then washed a further three times with PBS and. processed
to fluorescence microscopy experiments using a Zeiss Axiovert 200 inverted microscope with
filter for FITC (excitation wavelength 465 – 495 nm, emission wavelength 515 – 555
nm). Images were acquired using a Hammamatsu Flash 4 (2048x2048 pixel) camera for each
channel separately and processed using Volocity Software.
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Dalton Transactions
Page 28 of 28
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DOI: 10.1039/C5DT04753F
Table of Contents Entry
Two new vanadyl complexes with a fluorescent label have been prepared and their ability to
inhibit phosphatases studied. The fluorescent label has allowed us to confirm that the
complexes are cell permeable.