Ternary oxovanadium(IV) complexes of ONO-donor Schiff base and polypyridyl derivatives as protein tyrosine phosphatase inhibitors: synthesis, characterization, and biological activities.

Abstract

A series of oxovanadium complexes with mixed ligands, a tridentate ONO-donor Schiff base ligand [viz., salicylidene anthranilic acid (SAA)], and a bidentate NN ligand [viz., 2,2'-bipyridine (bpy), 1,10-phenanthroline (phen), dipyrido[3,2-d:2',3'-f]quinoxaline (dpq), dipyrido[3,2-a:2',3'-c]phenazine (dppz), or 7-methyldipyrido[3,2-a:2',3'-c]phenazine (dppm)], have been synthesized and characterized by elemental analysis, electrospray ionization mass spectrometry, UV-vis spectroscopy, Fourier transform IR spectroscopy, EPR spectroscopy, and X-ray crystallography. Crystal structures of both complexes, [V(IV)O(SAA)(bpy)].0.25bpy and [V(IV)O(SAA)(phen)].0.33H(2)O, reveal that oxovanadium(IV) is coordinated with one nitrogen and two oxygen atoms from the Schiff base and two nitrogen atoms from the bidentate planar ligands, in a distorted octahedral geometry (VO(3)N(3)). The oxidation state of V(IV) with d(1) configuration was confirmed by EPR spectroscopy. The speciation of VO-SAA-bpy in aqueous solution was investigated by potentiomtreic pH titrations, and the results revealed that the main species are two ternary complexes at a pH range of 7.0-7.4, and one is the isolated crystalline complex. The complexes have been found to be potent inhibitors against human protein tyrosine phosphatase 1B (PTP1B) (IC(50) approximately 30-61 nM), T-cell protein tyrosine phosphatase (TCPTP), and Src homology phosphatase 1 (SHP-1) in vitro. Interestingly, the [V(IV)O(SAA)(bpy)] complex selectively inhibits PTP1B over the other two phosphatases (approximate ninefold selectivity against SHP-1 and about twofold selectivity against TCPTP). Kinetics assays suggest that the complexes inhibit PTP1B in a competitive and reversible manner. These suggest that the complexes may be promising candidates as novel antidiabetic agents.

Full text

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ORIGINAL PAPER
Ternary oxovanadium(IV) complexes of ONO-donor Schiff
base and polypyridyl derivatives as protein tyrosine phosphatase
inhibitors: synthesis, characterization, and biological activities
Caixia Yuan Æ Æ Liping Lu Æ Æ Xiaoli Gao Æ Æ Yanbo Wu Æ Æ
Maolin Guo Æ Æ Ying Li Æ Æ Xueqi Fu Æ Æ Miaoli Zhu
Received: 3 December 2008/Accepted: 4 March 2009/Published online: 17 March 2009
? SBIC 2009
Abstract
ligands, a tridentate ONO-donor Schiff base ligand [viz.,
salicylidene anthranilic acid (SAA)], and a bidentate NN
ligand [viz., 2,20-bipyridine (bpy), 1,10-phenanthroline
(phen), dipyrido[3,2-d:20,30-f]quinoxaline (dpq), dipyrido
[3,2-a:20,30-c]phenazine (dppz), or 7-methyldipyrido[3,2-
a:20,30-c]phenazine (dppm)], have been synthesized and
characterized by elemental analysis, electrospray ionization
A series of oxovanadium complexes with mixed
mass spectrometry, UV–vis spectroscopy, Fourier trans-
form IR spectroscopy, EPR spectroscopy, and X-ray
crystallography. Crystal structures of both complexes,
[VIVO(SAA)(bpy)]?0.25bpy
0.33H2O, reveal that oxovanadium(IV) is coordinated with
one nitrogen and two oxygen atoms from the Schiff base
and two nitrogen atoms from the bidentate planar ligands, in
a distorted octahedral geometry (VO3N3). The oxidation
state of V(IV) with d1configuration was confirmed by EPR
spectroscopy. The speciation of VO–SAA–bpy in aqueous
solution was investigated by potentiomtreic pH titrations,
and the results revealed that the main species are two ter-
nary complexes at a pH range of 7.0–7.4, and one is the
isolated crystalline complex. The complexes have been
found to be potent inhibitors against human protein tyrosine
phosphatase 1B (PTP1B) (IC50approximately 30–61 nM),
T-cell protein tyrosine phosphatase (TCPTP), and Src
homology phosphatase 1 (SHP-1) in vitro. Interestingly, the
[VIVO(SAA)(bpy)] complex selectively inhibits PTP1B
over the other two phosphatases (approximate ninefold
selectivity against SHP-1 and about twofold selectivity
against TCPTP). Kinetics assays suggest that the complexes
inhibit PTP1B in a competitive and reversible manner.
These suggest that the complexes may be promising
candidates as novel antidiabetic agents.
and[VIVO(SAA)(phen)]?
Keywords
tyrosine phosphatase 1B ? Src homology phosphatase 1 ?
T-cell protein tyrosine phosphatase ? Phosphatase inhibitor
Oxovanadium(IV) complexes ? Protein
Abbreviations
BMOV
bpy
dppm
DFT
Bis(maltolato)oxovanadium
2,20-Bipyridine
7-Methyldipyrido[3,2-a:20,30-c]phenazine
Density functional theory
Electronic supplementary material
article (doi:10.1007/s00775-009-0496-6) contains supplementary
material, which is available to authorized users.
The online version of this
C. Yuan ? L. Lu (&) ? X. Gao ? Y. Wu ? M. Zhu (&)
Institute of Molecular Science,
The Key Laboratory of Chemical Biology and Molecular
Engineering of Education Ministry,
Shanxi University, 92 Wucheng Road,
030006 Taiyuan, China
e-mail: luliping@sxu.edu.cn
M. Zhu
e-mail: miaoli@sxu.edu.cn
L. Lu ? M. Guo (&)
Department of Chemistry and Biochemistry,
University of Massachusetts Dartmouth,
Dartmouth, MA 02747, USA
e-mail: mguo@umassd.edu
Y. Li ? X. Fu (&)
Edmond H. Fischer Signal Transduction Laboratory,
College of Life Sciences,
Jilin University,
130023 Changchun, China
e-mail: fxq@jlu.edu.cn
M. Zhu
State Key Laboratory of Coordination Chemistry,
Nanjing University,
210093 Nanjing, China
123
J Biol Inorg Chem (2009) 14:841–851
DOI 10.1007/s00775-009-0496-6

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DMSO
dppz
dpq
ESI-MS
IPTG
LB
2-ME
MOPS
pNPP
phen
PTP
PTP1B
SAA
SHP-1
TCPTP
Tris
Dimethyl sulfoxide
Dipyrido[3,2-a:20,30-c]phenazine
Dipyrido[3,2-d:20,30-f]quinoxaline
Electrospray ionization mass spectrometry
Isopropyl b-D-thiogalactopyranoside
Luria–Bertani
2-Mercaptoethanol
3-Morpholinopropanesulfonic acid
p-Nitrophenol phosphate
1,10-Phenanthroline
Protein tyrosine phosphatase
Protein tyrosine phosphatase 1B
Salicylidene anthranilic acid
Src homology phosphatase 1
T-cell protein tyrosine phosphatase
Tris(hydroxymethyl)aminomethane
Introduction
Protein tyrosine phosphatases (PTPs) are enzymes that
catalyze protein tyrosine dephosphorylation. In humans,
over 100 PTPs have been identified that function either as
negative or positive modulators in various signal trans-
duction pathways [1, 2]. Dysregulation of PTP activities
contributes to the pathogenesis of several human diseases,
including diabetes, obesity, cancer, and immune disorders
[3–5]. The importance of PTPs in diverse pathogenesis has
made them promising targets for drug discovery [6, 7].
Among various members in the PTP superfamily, human
PTP 1B (PTP1B) plays a major role in modulating both
insulin sensitivity and fuel metabolism and has emerged as
an important drug target for the treatment of diabetes and
obesity [1, 3, 8]. Recent studies have validated the role of
PTP1B as a major contributor to insulin resistance, there-
fore providing a potential pharmaceutical target for
treatment of type II diabetes and obesity [9, 10]. PTP1B
inhibitors have been pursued to develop novel antidiabetic
drugs [11–16]. A major problem with the current inhibitors
is their lack of selectivity towards various PTPs because
their active sites are highly conserved, hampering their
pharmaceutical development [1]. Thus, potent and specific
inhibitors of PTP1B are expected to have high therapeutic
value.
Vanadium compounds have long been recognized as
phosphatase inhibitors since vanadate has a structure
similar to that of phosphate [17]. Numerous studies have
demonstrated that vanadium compounds have insulin-
enhancing effects and can improve the symptoms of
diabetes in a variety of animal models [17–20]. Two
inorganic vanadium salts, vanadyl sulfate and sodium
metavanadate, have entered phase I clinical trials, but
further development is impeded by the poor bioavailability
and severe irritation of the digestive tract [21]. Organo-
vanadium complexes were subsequently developed, in
which the organic ligands may provide ways for tuning the
chemistry and bioactivities of vanadium, thereby mini-
mizing the adverse effects without sacrificing important
benefits [22–24]. A pharmacokinetic study performed
with carrier-added bis(maltolato)oxovanadium (BMOV)
and VOSO4has demonstrated improved tissue uptake of
vanadium from the maltolato-complexed vanadyl com-
pared with its inorganic congener [25]. A BMOV
derivative is currently in phase II clinical trials as an
antidiabetic drug [26]. The mechanism of action for
vanadium’s insulin-sensitizing effects is not well under-
stood but a few studies indicated that association with PTP
inhibition is implicated [27–29]. Therefore, how vanadium
compounds directly inhibit PTPs and then influence
metabolism needs more investigation.
We have been exploring novel vanadium complexes with
tridentateSchiffbaseligandsasPTP1Binhibitors[30].Here
we report the bioactivities in inhibition of three PTPs of five
novel mixed-ligand oxovanadium complexes containing a
tridentate Schiff base (salicylidene anthranilic acid, SAA)
[31]andbidentatepolypyridylligands (2,20-bipyridine,bpy;
1,10-phenanthroline, phen; dipyrido[3,2-d:20,30-f]quinoxa-
line, dpq; dipyrido[3,2-a:20,30-c]phenazine, dppz; and 7-
methyldipyrido[3,2-a:20,30-c]phenazine, dppm). The potent
inhibitory activity and good selectivity towards PTP1B
suggest that these complexes may be promising candidates
for novel antidiabetic drug development.
Materials and methods
Reactants
All reagents and solvents were purchased commercially
and used without further purification unless specially
noted. Double-distilled water was used to prepare buffer
solutions. The ligands (SAA, dpq, dppz, and dppm) were
synthesized as previously described [32–34].
Physical measurements
Elemental analyses were carried out with a VARI-EL
elemental analyzer. IR spectra (4,000–400 cm-1) were
recorded using a Shimadzu Fourier transform IR-8300
spectrometer in KBr disks. The electronic spectra were
recorded with a Hewlett-Packard HP-8453 Chemstation
spectrophotometer in dimethyl sulfoxide (DMSO) solu-
tions. The EPR spectrum was obtained in DMSO solution
at 110 K using a Bruker-ER 200-D-SRC spectrometer.
842J Biol Inorg Chem (2009) 14:841–851
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Electrospray ionization mass spectra were recorded with a
Quattro Micro API instrument (Waters, USA) in methanol
aqueous solution (1:9, v/v). The X-ray data were collected
using a Bruker SMART APEX 1K CCD diffractometer.
Bioactivity assays of the complexes were carried out with a
Bio-Rad model 550 microplate reader.
Potentiometric titration
pH-potentiometric titration was performed to study the
species formed in aqueous solution. The protonation con-
stant of the ligand (SAA) and the stability constants of the
VO–SAA–bpy complex were determined by pH-potentio-
metric titrations of 40-mL samples. Measurements of the
pH values were carried out at 298 ± 0.1 K and at a con-
stant ionic strength of 0.1 M NaCl with a PHS-3TC pH
meter with a combined glass electrode. The electrode was
calibrated by standard buffer solutions. The titrations were
performed with a carbonate-free NaOH solution of known
concentration (0.09495 M) by using a microsyringe under
a nitrogen atmosphere in a sealed jacketed vessel. The
protonation constants of the ligand and the equilibrium
constants of the complexes were calculated with the aid of
the SUPERQUAD program [35]. The values of pKwof
water and pKaof bpy used in the calculations were 13.78
[36] and 6.6 [37], respectively. The overall formation
constant of the ternary complex is denoted as the loga-
rithm of bpqrs= [VOpSAAqbpyrHs]/[VO]p[SAA]q[bpy]r[H]s.
The conventional notation has been used: negative indices
for H in the formulas indicate either the dissociation
of groups which do not deprotonate in the absence of
VO(IV) coordination or hydroxo ligands. The following
hydroxo species of VO2?were taken into account in
the calculations: [VO(OH)]?(log b100-1= -5.94) and
[(VO)2(OH)2]2?(log b200-2= -6.95), with the stability
constant calculated from the data of Henry et al. [38] and
corrected for the different ionic strengths by use of the
Davies equation [39], and [(VO)2(OH)5]-(log b200-5=
-22.0) [40].
Preparation of oxovanadium(IV) complexes
Five new oxovanadium complexes, each containing a tri-
dentate Schiff base ligand and a bidentate NN ligand, were
prepared by a two-step procedure as shown in Scheme 1.
[VIVO(SAA)(bpy)]?H2O
A stirring solution of salicylaldehyde (2 mmol) in 5 mL of
methanol was added dropwise to a mixture of anthranilic
acid (2 mmol) and KOH (2 mmol), which were dissolved
in 10 mL of methanol and stirred at 333 K. After 1 h, a hot
aqueous VOSO4solution (2 mmol) was added. Then the
reaction mixture was neutralized by adding aqueous KOH
solution. After 2 h of refluxing, a solution of bpy (2 mmol)
in 10 mL of methanol was added dropwise to this mixture.
The solution on further refluxing for another 3 h gave a
reddish-brown precipitate. The solid powder was isolated,
washed with distilled water, methanol, and diethyl ether,
respectively, and dried in vacuo. Yield: 0.48 g (51%).
Anal. data for C24H19N3O5V ([VIVO(SAA)(bpy)]?H2O,
1?H2O)— calcd: C 60.01, H 3.99, N 8.75%; Found: C
60.62, H 4.02, N 8.96%; formula weight (1): 462.35.
Electrospray ionization mass spectrometry (ESI-MS): m/z
463.4 [1 ? H]?, 485.3 [1 ? Na]?. Selected IR data [KBr,
mmax(cm-1)]: 3,447br, 1,632s, 1,604vs, 1,581w, 1,530m,
1,461w, 1,439m, 1,344m, 1,188w, 1,151w, 957s, 876m,
761m, 733w, 611w, 419w (br, broad; vs, very strong; s,
strong; m, medium; w, weak). When excess bpy (3 mmol)
was employed in the last step of the synthesis, reddish-
brown crystals of 1?0.25bpy were obtained by slow evap-
oration of the reaction solution at room temperature for
2 weeks. Suitable dimensional crystals were selected for
X-ray single-crystal diffraction.
[VIVO(SAA)(phen)]?H2O
This complex was synthesized following the same general
procedures as those described for 1?H2O, with phen in
N
N
= bpy, phen, dpq, dppz, dppm
OH
OHC
+
COOH
NH2
KOH
reflux 1h
HC
OH
N
COOH
VOSO4
N
N+
pH = 7, reflux 5h
+
HC
O
N
C
V
O
O
O
N
N
SAA
1-5
Scheme 1 The synthesis of the
complexes. SAA salicylidene
anthranilic acid,
bpy 2,20-bipyridine,
phen 1,10-phenanthroline,
dpq dipyrido
[3,2-d:20,30-f]quinoxaline,
dppz dipyrido[3,2-a:20,
30-c]phenazine,
dppm 7-methyldipyrido
[3,2-a:20,30-c]phenazine
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place of bpy. Yield: 0.54 g (53%). Anal. data for
C26H19N3O5V([VIVO(SAA)(phen)]?H2O,
calcd: C 61.91, H 3.80, N 8.33%; Found: C 62.39, H 3.99,
N 8.16%; formula weight (2): 486.37. ESI-MS: m/z 487.44
[2 ? H]?, 509.5 [2 ? Na]?. Selected IR data [KBr,
mmax(cm-1)]: 3,454br, 1,627s, 1,603vs, 1,578s, 1,531m,
1,457w, 1,422m, 1,359m, 1,182w, 1,144w, 1,032w, 966s,
842m, 777m, 724 m, 614w, 429w. Reddish-brown crystals
were obtained by slow evaporation of the reaction solution
at room temperature for 2 days.
2?H2O)—
[VIVO(SAA)(dpq)]?CH3OH
This complex was synthesized following the same general
procedures as those described for 1?H2O, with dpq in place
of bpy. Yield: 0.52 g (46%). Anal. data for C29H21N5O5V
([VIVO(SAA)(dpq)]?CH3OH, 3?CH3OH)— calcd: C 61.06,
H 3.71, N 12.28%; Found: C 60.81, H 3.675, N 12.30%;
formula weight (3): 538.41. ESI-MS: m/z 539.3 [3 ? H]?,
561.4 [3 ? Na]?. Selected IR data [KBr, mmax(cm-1)]:
3,432br, 1,647s, 1,603vs, 1,576s, 1,532m, 1,437w, 1,422m,
1,354m, 1,182w, 1,144w, 1,032w, 970s, 842m, 777m,
724m, 614w, 429w.
[VIVO(SAA)(dppz)]
This complex was synthesized following the same general
procedures as those described for 1?H2O, with dppz in
place of bpy. Yield: 0.47 g (40%). Anal. data for
C32H19N5O4V ([VIVO(SAA)(dppz)], 4)— calcd: C 65.31,
H 3.25, N 11.90%; Found: C 64.86, H 3.31, N 11.93%;
formula weight (4): 588.47. ESI-MS: m/z 589.4 [4 ? H]?,
611.9 [4 ? Na]?. Selected IR data [KBr, mmax(cm-1)]:
3,434br, 1,637s, 1,600vs, 1,576m, 1,532s, 1,494m, 1,461m,
1,436m, 1,338m, 1,188w, 1,148w, 972s, 875m, 765m,
736m, 616w, 415w.
[VIVO(SAA)(dppm)]?H2O
This complex was synthesized following the same general
procedures as those described for 1?H2O, with dppm in
place of bpy. Yield: 0.53 g (43%). Anal. data for
C33H23N5O5V ([VIVO(SAA)(dppm)]?H2O,
calcd: C 63.88, H 3.74, N 11.29%; Found: C 63.36, H
3.741, N 11.27%; formula weight (5): 602.49. ESI-MS: m/z
603.5 [5 ? H]?, 626.6 [5 ? Na]?. Selected IR data [KBr,
mmax(cm-1)]: 3,434br, 1,635s, 1,605vs, 1,578m, 1,532s,
1,495m, 1,461m, 1,438m, 1,358m, 1,183w, 1,148w, 962s,
876w, 756m, 738m, 613w, 419w.
5?H2O)—
X-ray crystallography
Single crystals of complexes 1?0.25bpy or 2?0.33H2O were
mounted on glass fibers for data collection. Cell parameters
and an orientation matrix for data collection were obtained
by least-squares refinement of diffraction data from 1,402
and 2,201 reflections with h of 2.1–25.0? for 1?0.25bpy and
2?0.33H2O using a Bruker SMART APEX 1K CCD auto-
matic diffractometer. Data were collected at 298 K using
Mo Ka radiation (k = 0.71073 A˚) and the x-scan tech-
nique, and corrected for Lorentz and polarization effects
(SADABS) [41]. The structures were solved by direct
methods (SHELXS-97) [42] and subsequent difference
Fourier maps and then refined on F2by a full-matrix least-
squares procedure using anisotropic displacement parame-
ters [43]. Solvent molecule water in 2?0.33H2O was found
to be disordered with 33% occupancies. After several cycles
of refinement, hydrogen atoms attached to carbon atoms
were located at their calculated positions (C–H, 0.93A˚) and
were refinedusinga riding model. Hydrogen atoms attached
to oxygen atoms (water) in 2?0.33H2O were located from
difference Fourier maps and refined their global Uisovalue.
Molecular graphics are from SHELXTL [44].
Density functional theory calculations and molecular
modeling
By using the Gaussian 03 program [45], we performed
density functional theory (DFT) structural optimizations on
the complexes at the B3LYP/LANL2DZ level [46–48].
The imaginary frequencies and DFT wavefunction insta-
bilities were checked at the same theoretical level. The
starting structures of 1 and 2 were derived from the crystal
structure and those of other complexes were built on the
basis of the crystal structure of complex 2. The molecular
modeling (seeing the electronic supplementary informa-
tion) was performed with an SGI workstation with the
insight II software package. The main calculation program
was DISCOVER 98 with default settings. An extensible
and systematic force field was used with its default
parameters.
Expression and purification of recombinant human PTP
enzymes
Expression and purification of human PTP1B
Plasmids pET/PTP1B encoding human PTP1B catalytic
domain 1–321 and a C-terminal His-tag sequence [30]
were transformed into Escherichia coli BL21 (DE3) cells
844J Biol Inorg Chem (2009) 14:841–851
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and subsequently plated on Luria–Bertani (LB) plates. A
single colony of an overnight culture was inoculated into
1 L fresh liquid LB medium containing 100 mg L-1
ampicillin at 310 K. Expression of PTP1B was induced by
isopropyl b-D-thiogalactopyranoside (IPTG) and the His-
tagged fusion protein was purified by nickel nitrilotriacetic
acid chromatography similarly as described previously for
the periplasmic heme-transport protein [49]. The fractions
were collected and analyzed by sodium dodecyl sulfate
polyacrylamide gel electrophoresis. The peak fractions
containing the pure fusion protein were pooled, and then
dialyzed against3-morpholinopropanesulfonic
(MOPS) buffer (20 mM MOPS, 0.5 M NaCl, pH 7.4) and
stored at 253 K. Protein concentration was determined by
the Bradford method using bovine serum albumin as a
standard.
acid
Expression and purification of human T-cell PTP
and Src homology phosphatase 1
A single clone of E. coli (DE3) cells transformed by the
plasmid pT7-DTCPTP [50] containing the human T-cell
PTP (TCPTP) catalytic domain was inoculated into LB
medium containing 100 lg mL-1ampicillin, 34 lg mL-1
chloramphenicol at 310 K. When the culture reached to an
optical density at 600 nm of approximately 0.4, 80 lM
IPTG was added to induce the expression of TCPTP at
301 K overnight. The cells were harvested by centrifuga-
tion (5,000 rpm, 277 K, 10 min). The pellet was washed
with phosphate-bufferedsaline
tris(hydroxymethyl)aminomethane (Tris) buffer [25 mM
Tris, 10 mM 2-mercaptoethanol (2-ME), pH 7.5, including
0.1% phenylmethylsulfonyl fluoride and 0.1% phospho-
enolpyruvate] and lysed by sonication. The cell debris was
removed by centrifugation (12,000 rpm, 277 K, 30 min).
The supernatant was directly loaded to an FFQ column and
eluted with buffer Q (25 mM Tris, 2 mM 2-ME, pH 7.5)
containing 50 mM NaCl. The active peak fraction was
loaded onto a SP-Sephadex column and eluted with buffer
S (20 mM MeS, 2 mM 2-ME, pH 6.0) containing 150 mM
NaCl. The active peak fraction was collected and analyzed
by sodium dodecyl sulfate polyacrylamide gel electropho-
resis, then concentrated and kept at 253 K. Protein
concentration was determined by the Bradford method
using bovine serum albumin as a standard.
Src homology phosphatase 1 (SHP-1) constructed as the
pT7-DSHP-1 expression vector containing human SHP-1
catalytic domain [51] was expressed and purified by
almost the same procedure as that for TCPTP. The only
difference was that the active peak fraction was eluted
from the SP-Sephadex column with buffer S containing
450 mM NaCl.
and resuspendedin
PTP inhibition assays
PTP activities were measured similarly as described by
Montalibet et al. [52] using p-nitrophenol phosphate
(pNPP) as the substrate. The assays were performed in
20 mM MOPS buffer, pH 7.2, containing 50 mM NaCl and
2 mM L-glutathione. The complexes were dissolved in
DMSO (10-2M), and diluted to various concentrations,
then further diluted 10 times into enzyme–MOPS buffer
solutions for activity studies. Inhibition assays were per-
formed in the same buffer on a 96-well plate in 100-lL
volumes. The PTPs were diluted to final concentrations of
60, 250, and 200 nM for PTP1B, TCPTP, and SHP-1
respectively. Then 10 lL of the complex with various
concentrations was mixed with 83 lL of enzyme solution
for 30 min. Then 2 lL of pNPP (0.1 M) substrate was
added. After incubation for 30 min at room temperature,
the assays were terminated by the addition of 5 lL of 2 M
NaOH. A405was measured on a microplate reader. IC50
values were obtained by fitting the concentration-depen-
dent inhibition curves using the program Origin. All data
points were obtained in triplicate. Solutions of the oxova-
nadium complexes were all freshly prepared before each
experiment.
The inhibiting kinetic analysis was performed according
to Eq. 1 [53]:
m ¼
VmaxS ½ ?
I ½ ?
Km 1 þ
Ki
??
þ S ½ ?
ð1Þ
where Vmax is the maximum initial velocity, Kmis the
corresponding constant for substrate, S is the substrate, I is
the inhibitor, and Kiis the inhibition constant at various
substrate concentrations, derived from the slope of the
Lineweaver–Burk plots.
Inhibition constants were determined by measuring
initial hydrolysis rates at different substrate and inhibitor
concentrations. The apparent Kivalues measured at the
various inhibitor concentrations were plotted against con-
centration of the inhibitor to calculate the Kivalues [54].
Results and discussion
Description of molecular structures of complexes 1
and 2
Complexes 1?0.25bpy and 2?0.33H2O crystallize in space
groups P1and R?3; respectively. Detailed information on the
crystal data and structure determination is summarized in
Table S1. Selected bond lengths and angles are shown in
Table S2. As shown in Fig. 1, the structures of the com-
plexes consist of a monomeric vanadium(IV) species with
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a VO2?moiety bonded to a dianionic tridentate Schiff base
ligand and an N,N-donor bpy (or phen) base. The com-
plexes have a VIVO3N3coordination geometry, with the
V=O distanceof1.595(3) A˚,
1.589(3) A˚(2), typical for a double bond (the ‘‘normal’’
V=O bonds in other oxovanadium complexes are com-
monly in the range from 1.57 to 1.62 A˚) [55–57]. The
Schiff base is bound through the phenolate oxygen, the
imine nitrogen, and the carboxylate oxygen of the benzo-
formic acid. The Schiff base exhibits a meridional binding
mode, leaving the N,N-donor base bound at the axial–
equatorial positions. Owing to the ‘‘trans effect’’ from the
oxo ligand, the V–N bond trans to the V=O group is sig-
nificantly longer[2.322(4) A˚,
2.365(4) A˚(2)] than the other V–N bonds (approximately
2.091–2.141 A˚), which is well comparable to that in
complexes with a similar coordination sphere [58]. The
V–O distances involving the Schiff bases are in the range
1.928(4)–1.967(3) A˚in 1 [1.957(3) and 1.978(3) A˚in 2],
typical for single bonds. The lattice water molecule in
1.601(3) A˚(1),and
2.321(4) A˚(1),and
complex 2 shows intermolecular hydrogen-bonding inter-
actions with the carboxylate oxygen of the Schiff base. The
cell packing of both complexes is shown in Figs. S1 and
S2.
DFT calculations were performed for complexes 1 and 2
and the other complexes using the crystal structure of
complex 2 as a starting model. The calculated structures
are shown in Fig. 2. Selected bond lengths and angles are
summarized in Table 1. A comparison of the calculated
geometry of complexes 1 and 2 with the structures
obtained from single X-ray diffraction indicates a good
agreementbetweencomputational
results. The DFT calculations suggest that all the com-
plexes adopt a geometry around the vanadyl similar to that
in complexes 1 and 2.
andexperimental
IR spectra
The vanadium complexes 1–5 exhibit a sharp band at
approximately 966 cm-1
due to the m(V=O) mode,
Fig. 1 ORTEP view of 1 (left)
and 2 (right) showing atom
labeling with 30% probability
thermal ellipsoids
Fig. 2 The optimized structures
of the complexes
[VO(SAA)(NN)] (SAA is
salicylidene anthranilic acid) at
the B3LYP/LANL2DZ level
(a NN is 2,20-bipyridine (bpy),
b NN is 1,10-phenanthroline,
c NN is dipyrido[3,2-d:20,30-f]
quinoxaline, d NN is
dipyrido[3,2-a:20,30-c]
phenazine, e NN is 7-
methyldipyrido[3,2-a:20,30-c]
phenazine)
846J Biol Inorg Chem (2009) 14:841–851
123

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indicating that the oxovanadium is in a hexacoordinated
environment [54]. The band near 620 cm-1is assigned to
m(V–N) and the one near 420 cm-1is assigned to m(V–O)
[59, 60]. The C=N stretching frequencies for the ligands
occurring at approximately 1,620 cm-1are shifted to lower
frequencies (1,600–1,605 cm-1) in the complexes, indi-
cating the coordination of the azomethine/ring nitrogen to
vanadium [61]. A distinct positive shift of m(C–O) phenolic
oxygen compared with the free Schiff base ligand is
observed owing to the formation of the V–O (phenolic
oxygen) bond (cf. in the complexes at 1,338–1,359 cm-1
and in the free ligand at 1,234 cm-1) [62]. Furthermore,
the peaks at 1,695 and 1,598 cm-1, due to masym(COO-)
and msym(COO-) of the Schiff base ligand, appear in the
complexes at 1,627–1,647 and 1,530–1,578 cm-1. The
shifts of these two bands suggest coordination of the car-
boxylic group oxygen to vanadium in all these complexes
[63]. In addition, a broad band appears at approximately
3,400 cm-1, which may be ascribed to hydrogen-bonded
m(O–H) and/or m(N–H), and may also include m(C–H).
EPR spectrum
EPR is a useful tool in investigating the nature of bonding
in oxovanadium(IV) complexes [64]. The X-band EPR
spectrum of complex 2 in 100% DMSO at 110 K is shown
in Fig. 3. Similar to the spectra of many other oxovana-
dium(IV) complexes [62, 65, 66], the spectrum exhibits an
axially symmetrical signal of tetravalent vanadium, split
into a number of hyperfine lines which originate from the
d1electron interaction with nuclear spin I = 7/2. The
spectrum displays well-resolved
lines (g||[g\) and the parameters (g||= 1.989 and
g\= 1.9541) were obtained from the simulated spectrum.
It is clear that the g values obtained are indicative for the
presence of vandal ions (VO2?) in a distorted octahedrally
coordinated environment [67]. The EPR data are consistent
51V (I = 7/2) hyperfine
with the solution structure being the same as that charac-
terized in the solid state by X-ray crystallography.
Electronic absorption spectra
The complexes were insoluble in water and only moder-
ately soluble in methanol, but highly soluble in DMSO.
The spectral data of all ligands and the oxovanadium(IV)
complexes are displayed in Table S3. The spectra of the
ligands show several sharp absorption maxima in the UV
region, assignable to p ? p* and n ? p* transitions.
Upon complexation, the p ? p* transitions shifted to
longer wavelengths, while the n ? p* transition merged
with an additional broad intense band at approximately
401 nm due to ligand-to-metal charge transfer from the
phenolate oxygen to an empty d orbital of vanadium
(Fig. 4). In addition, all neutral complexes display two
absorptions at 770 and 510 nm with low intensities,
assignable to a metal-centered
remaining bands in the UV region are assignable to the
d–d transition. The
Table 1 Selected geometrical parameters for crystal structure of complexes 1 and 2 and those from density functional theory calculations for the
vanadium complexes (A˚,o)
N1–VN2–VN3–VO1–VO2–VO3–VN2–V–O3
C19–N3–C20–C21
[VO(SAA)(bpy)]
[VO(SAA)(bpy)]a
2.1562.3832.0901.6051.9601.93475.63 143.69
2.1312.3212.0631.5951.9421.96777.00 144.20
[VO(SAA)(phen)]
[VO(SAA)(phen)]a
2.1482.4402.0871.6041.9581.931 75.46142.96
2.141 2.3652.091 1.5891.957 1.978 76.31144.10
[VO(SAA)(dpq)]2.1502.4332.0851.6031.957 1.931 75.42142.93
[VO(SAA)(dppz)]2.1502.427 2.0861.604 1.9581.93175.60 143.15
[VO(SAA)(dppm)] 2.1492.4282.0861.604 1.9581.931 75.57143.24
SAA salicylidene anthranilic acid, bpy 2,20-bipyridine, phen 1,10-phenanthroline, dpq dipyrido[3,2-d:20,30-f]quinoxaline, dppz dipyrido[3,2-
a:20,30-c]phenazine, dppm 7-methyldipyrido[3,2-a:20,30-c]phenazine
aX-ray crystal values
Fig. 3 X-band EPR spectrum of complex 2 in dimethyl sulfoxide
(DMSO) at 110 K
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intraligand transitions [55, 58, 68]. The stabilities of the
complexes in DMSO were also studied at 298 K by
UV–vis spectroscopy. No changes were observed for the
complexes in DMSO for over 72 h, indicating that the
complexes are quite stable in DMSO (Fig. 4).
ESI-MS analysis
The complexes were further characterized by ESI-MS in
methanol aqueous solution (1:9, v/v). Species correspond-
ing to the proton adducts and the sodium adducts were
observed for all five complexes (Fig. S3). The composition
of the complexes deduced from the elemental analysis was
confirmed by the ESI-MS study and the presence of the
corresponding parent peak in the positive ion ESI-MS
excluded immediate hydrolysis of complexes in methanol
aqueous solution [69].
Potentiometric titrations
To obtain information about the complexes formed in
aqueous solution, which is relevant to their bioactivities,
potentiometric titration was performed for VO2?cation
and two ligands (SAA and bpy). As a preliminary step for
studying the VO2?–SAA–bpy system, the protonation
constants of SAA were determined and the estimated pKa
values were 4.24(14) and 8.90(9). The protonation con-
stants of the bpy ligand and the stability constants of the
hydroxo complex of VO2?were obtained from the litera-
ture [37, 38, 40]. Then the titration curves for the binary
systems of VO2?–SAA (1:2) and VO2?–bpy (1:3) and the
ternary system VO2?–SAA–bpy (1:1:1) were analyzed and
the species were suggested according to the data shown in
Table 2. On the basis of these data, the species distribution
diagram as a function of pH is shown in Fig. 5. The
distributioncurvessuggest
oxovanadium species [VO(SAA)(bpy)] and [VO(SAA)
(bpy)H-1]-are the predominant species (their percentage is
above 90%) in the pH range 7–7.4. The species
[VO(SAA)(bpy)] agrees with the structural model from the
X-ray structure studies.
that the mixed-ligand
PTP1B inhibition assays
The inhibitory activities of all the [VIVO(SAA)(NN)]
complexes on PTP1B were measured using pNPP as the
substrate. The enzyme and complexes were preincubated
for 30 min prior to the initiation of the enzymatic reaction
by the addition of the substrate pNPP. IC50values were
Fig. 4 UV–vis spectra of
complexes 1 (a) and 4 (b)
recorded at 298 K in DMSO
over 72 h with 8-h intervals
Table 2 pKaof the ligand SAA and VO(IV) (log b) stability con-
stants for the ligands and complexes studied
SpeciespKa/log b
SpeciespKa/log b
H2SAA 4.24 (14)
8.90 (9)
[VO(bpy)2]2?
10.90 (9)
[VO(SAA)H]?
13.43 (8) [VO(bpy)2H-1]?
[VO(bpy)2H-2]
[VO(bpy)(SAA)]
[VO(bpy)(SAA) H-1]-
[VO(bpy)(SAA)H]?
6.14 (6)
[VO(SAA)2H2]
[VO(SAA)]
[VO(SAA)H-1]-
[VO(bpy)]2?
25.04 (12)-2.47 (9)
7.67 (8)16.05 (6)
-2.97 (15)8.91 (5)
5.75 (12)20.64 (8)
298 K, I = 0.10 M (NaCl)
Fig. 5 Species distribution as a function of pH for the VO2?–SAA–
bpy (1:1:1) system (0.9 mM VO2?)
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obtained by fitting the dose-dependent inhibition curves
based on the means of several replicate experiments. IC50
values of the ligands were determined to be over 1 mM. As
shown in Fig. 6, the five oxovanadium complexes demon-
strated strong inhibition against PTP1B, with IC50ranging
from 30 to 61 nM. Complex 1 shows the most potent
inhibition, with the lowest IC50value (30 nM). With the
increase in size of the polypyridyl ligands, the inhibitory
activity gradually decreased, with complex 5 showing the
lowest activity (IC50= 61 nM). It appears that the spatial
bulk of the polypyridyl moieties of the complexes weakly
diminishes the PTP1B inhibition activities in this series.
Inhibitions of SHP-1 and TCPTP
As an initial assessment of its specificity, complex 1 was
also tested for its inhibitory activities against another two
tyrosine phosphatases, SHP-1 and TCPTP, in addition to
PTP1B. A comparison of the dose-dependent inhibition of
the three PTPs and the IC50values is shown in Fig. 7.
Complex 1 is found to be a potent inhibitor for all three
PTPs, with IC50values in the nanomolar to submicromolar
range. Interestingly, the differences in IC50values suggest
that complex 1 appears to be more selective in inhibiting
PTP1B than the other two PTPs, with an IC50 value
approximately half of that for TCPTP and one ninth of that
for SHP-1. Therefore, complex 1 appears to be a PTP1B
selective inhibitor, compared with SHP-1 and TCPTP. This
interesting selectivity may offer advantages for antidiabetic
drug development [15].
Kinetic analysis of PTP1B inhibition
To elucidate the modes of PTP1B inhibition by the oxo-
vanadium complexes, kinetic studies of the inhibitions
against PTP1B by complexes 1, 2, and 4 were performed in
more detail. Six different concentrations of the substrate
pNPP (0.3, 0.5, 1.0, 2.0, 4.0, and 8.0 mM) and five different
concentrations of the inhibitors (0, 15, 30, 50, and 80 nM)
were used in the steady state kinetics assays. Figure 8
shows the Lineweaver–Burk double-reciprocal plot of the
kinetics data in the presence of various of concentrations
of complex 1. It appears that the lines converged at an
intersection on the y-axis above the x-axis, implying a
competitive inhibition mode versus pNPP. Similar phe-
nomena were observed with the other oxovanadium
complexes, suggesting that these complexes are likely to be
classic competitive inhibitors against PTP1B. A similar
inhibition mode has been reported for BMOV [27]. The Ki
values were determined to be 23 ± 1, 24 ± 1, and
35 ± 3 nM for complexes 1, 2 and 4, respectively.
Fig. 6 Concentration-dependent inhibitions of protein tyrosine phos-
phatase 1B by complexes 1–5. The inset shows IC50values
Fig. 7 Concentration-dependent inhibitions of three tyrosine phos-
phatases by complex 1. The inset shows IC50values
Fig. 8 Lineweaver–Burk plot of 1/m (min lM-1) versus the recipro-
cal of the p-nitrophenol phosphate concentration (mM-1) at five fixed
concentrations of complex 1
J Biol Inorg Chem (2009) 14:841–851849
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Biological relevance
Type 2 diabetes, characterized by insulin resistance, affects
over 200 million people and the epidemic is exploding
worldwide [70]. Recently, PTP1B was identified as a key
enzyme related to insulin resistance [1–3]. PTP1B inhibi-
tion has emerged as an important approach to enhance
insulin sensitivity. Various inorganic and organovanadium
compounds have been demonstrated to enhance insulin
sensitivity in animal models [21]. Organic ligands on
vanadium can influence its bioactivity, by affecting either
the potency of the phosphatase enzyme inhibition, the
selectivity for various phosphatases and kinases, or the
bioavailability of the vanadium ion. Careful design of
the organic ligands on vanadium could affect their bioac-
tivities significantly, thus making them more suitable for
clinical usages in fighting diabetes. Our PTP inhibition data
show that the mixed-ligand oxovanadium compounds are
highly potent, competitive inhibitors for PTP1B, with IC50
values at the nanomolar level. Moreover, complex 1 dem-
onstrated good selectivity for PTP1B compared with the
other two human PTPs. It is worth mentioning that
achieving selectivity between PTP1B and TCPTC is
extremely challenging [71], because they are approximately
80% homologous in the catalytic domains and share very
similar active sites [71]. Our preliminary molecular mod-
eling studies suggest that the oxovanadium complex
[VIVO(SAA)(phen)] can be accommodated into the active-
site cleft of PTP1B (see the electronic supplementary
material). The computer model suggests that the oxovana-
dium itself can be well fitted into the active-site cleft of
PTP1B. In the model, the vanadyl oxygen is close to the
sulfur atom of the active-site cysteine (Cys215) (Fig. S4), at
a distance of 3.38 A˚. Meanwhile, two hydrogen bonds may
also be formed between the two oxygens of the carboxyl of
the complex and the nitrogens of the arginine residue
(Arg221) in the enzyme (Ocarboxyl oxygen–NArg221, 3.13 A˚
and Ocarbozyl
observations suggest that the mixed-ligand oxovanadium
complexes may be promising candidates as novel antidia-
betic agents or lead compounds for further development.
oxygen–NArg221, 3.07 A˚) (Fig. S5). These
Conclusion
A series of novel ternary oxovanadium(IV) complexes
were synthesized and characterized. The structures of
complexes 1 and 2 were determined by X-ray crystal
analysis. Potentiometric studies suggest that, in the physi-
ological pH range, VO(IV) can form stable complexes with
SAA and bpy. Interestingly, the complexes display potent
inhibition of PTP1B, with IC50values in low nanomolar
range. Kinetic analysis indicates that these complexes are
reversible competitive inhibitors of PTP1B. Moreover,
complex 1 appears to be a PTP1B selective inhibitor,
compared with SHP-1 and TCPTP. These observations
make the oxovanadium(IV) complexes promising candi-
dates for novel antidiabetic drug development.
Acknowledgments
National Natural Science Foundation of China (grant no. 20471033),
the Natural Science Foundation of Shanxi Province (grant no.
20051013), the Overseas Returned Scholar Foundation of Shanxi
Province of China in 2006 and 2008, and University of Massachusetts
Dartmouth, MA, USA.
This work was supported financially by the
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