Journal of Inorganic Biochemistry 105 (2011) 1323–1328

Contents lists available at ScienceDirect

Journal of Inorganic Biochemistry

j ourna l homepage: www.e lsev ie r.com/ locate / j inorgb io

Ternary oxovanadium(IV) complexes with amino acid-Schiff base andpolypyridyl derivatives: Synthesis, characterization, and protein tyrosinephosphatase 1B inhibition

Liping Lu a, Jinjun Yue a, Caixia Yuan a, Miaoli Zhu a,⁎, Hong Han a, Zhiwei Liu b, Maolin Guo b,⁎a Institute of Molecular Science, the Key Laboratory of Chemical Biology and Molecular Engineering of Education Ministry, Shanxi University, Taiyuan, 030006, Chinab Department of Chemistry and Biochemistry, University of Massachusetts Dartmouth, UMass Cranberry Health Research Center, Dartmouth, MA 02747, USA

⁎ Corresponding authors. Tel.: +86 351 7017974.E-mail addresses: miaoli@sxu.edu.cn (M. Zhu), mgu

0162-0134/$ – see front matter © 2011 Elsevier Inc. Aldoi:10.1016/j.jinorgbio.2011.07.008

a b s t r a c t

a r t i c l e i n f o

Article history:Received 22 February 2011Received in revised form 19 July 2011Accepted 20 July 2011Available online 28 July 2011

Keywords:Oxovanadium(IV) complexesProtein tyrosine phosphatase 1BPhosphatase inhibitorAmino acid-Schiff basePolypyridyl

To investigate the structure–activity relationship of vanadium complexes in inhibiting protein tyrosinephosphatase1B (PTP1B), eight mixed-ligand oxovanadium(IV) complexes, [VIVO(SalAla)(NN)] (H2SalAla forsalicylidene alanine, NN for N,N′-donor heterocyclic base, namely, 2,2′-bipyridine (bpy, 1), 1,10-phenanthroline(phen, 2), dipyrido[3,2-d:2′,3′-f]quinoxaline (dpq, 3), dipyrido[3,2-a:2′,3′-c]phenazine (dppz, 4)), [VIVO(SalLys)(dpq)] (5), [VIVO(SalLys)(dppz)] (6), [VIVO(SalAsp)(dppz)], (7) and [VIVO(SalTrp)(dppz)] (8)), of which 3–8 arenew, have been prepared and characterized by elemental analysis, infrared, UV–visible, electrospray ionizationmass spectrometry and conductivity. The molar conductance data confirmed the non-electrolytic nature of thecomplexes in DMSO solution. The coordination in [VIVO (SalAla)(phen)] (2) was confirmed by X-ray crystalstructure analysis. The oxidation state of V(IV) with d1 configuration in 2was confirmed by EPR. The speciationof VO–SalAla–phen in aqueous solution was investigated by potentiometric pH titrations. The results indicatethat the main species are two ternary complexes at the pH range 7.0–7.4. Biochemical assays demonstratethat themixed-ligandoxovanadium(IV) complexes are potent inhibitors of PTP1Bwith IC50 values in the range of62–597 nM, approximately 3–10 fold weaker in potency than those of similar mixed-ligand oxovanadium(IV)complexes of salicylidene anthranilic acid (SAA) derivative with polypyridyl ligands, except complex 8, whichexhibits comparable or better inhibition activity than those of the mixed-ligand oxovanadium(IV) complexes ofSAA derivative with polypyridyl ligands. The results demonstrate that the structures of vanadium complexesinfluence the PTP1B inhibition activity. Kinetics assays reveal that complex 2 inhibits PTP1B in a competitivemanner.

o@umassd.edu (M. Guo).

l rights reserved.

© 2011 Elsevier Inc. All rights reserved.

1. Introduction

Protein tyrosine phosphatases (PTPs) are a large family of diverseenzymes that regulate various cellular processes by dephosphorylat-ing phosphotyrosines on proteins [1]. Many human diseases such asdiabetes, obesity, cancer, and immune disorders are involved in thedysregulation of PTP activities [1–5]. As potential therapeutic targets,PTPs have attracted great attention in both academia and industrialin past decades [6,7].

PTP1B plays key roles in the negative regulation of insulin receptorand leptin receptor mediated signaling pathways. The knockout ofPTP1B in mice results in insulin hypersensitivity, even in a high fatdiet [8]. PTP1B antisense oligonucleotide lowers PTP1B proteinexpression, normalizes blood glucose, and improves insulin sensitiv-ity in diabetic mice [9]. PTP1B therefore emerged as a potentialpharmaceutical target for the treatment of type II diabetes and

obesity. It is highly desirable to design and exploit potent and specificPTP1B inhibitors as drug candidates [10,11].

Vanadium has demonstrated its usefulness in the treatment oftype I and type II diabetes in various animal models as well as limitedhuman trials [12]. Vanadium complexes with appropriate organicligands can improve absorption, tissue uptake, potency, and decreasetoxicity of the metal [13]. The mechanism of vanadium's insulin-sensitizing effects has been demonstrated due at least partly to theinhibition of PTPs which dephosphorylate the insulin receptor,resulting in prolongation of insulin signaling [14,15]. Recent studiesindicate that not only vanadium complexes directly inhibit some PTPsincluding PTP1B, but also the structures of ligands on vanadium mayinfluence the inhibitory effects on different PTPs, i.e., displaying somekind of selectivity [15–23]. Designing potent and selective vanadium-based PTP1B inhibitors with lower side effects may be achieved bymodifying the organic ligand moieties on vanadium.

To investigate the structure–activity relationship of vanadiumcomplexes in inhibiting PTP1B, eight oxovanadium(IV) complexes ofSal-aa Schiff base and polypyridyl derivatives, of which 6 are newcomplexes, have been designed, synthesized and characterized as a

1324 L. Lu et al. / Journal of Inorganic Biochemistry 105 (2011) 1323–1328

continuing research effort on ternary oxovanadium(IV) complexesof ONO-donor Schiff base and polypyridyl derivatives [19,21]. ThePTP1B inhibition assay results clearly indicate that the structures ofsal-aa Schiff bases influence the enzyme inhibition activity.

2. Experimental section

2.1. Materials

All reagents and solvents were obtained from commercial sourcesand used without further purification unless specially noted. Doubledistilled water was used to prepare buffer solutions.

2.2. Physical measurements

The C, H and N analyses were performed on a VARI-EL elementalanalyzer. IR spectra on KBr pellets were recorded on a Shimadzu FTIR-8300 spectrometer in the range of 4000–400 cm−1(KBr disks). Theelectronic spectra were recorded on a Hewlett–Packard HP-8453Chemstation spectrophotometer. Electrospray ionization mass spec-tra (ESI-MS) were recorded with a Quattro Micro API instrument(Waters, USA) in methanol solution. Molar conductivity of thecomplexes was measured with a DDS-307 conductivity meter inDMSO (1.0×10−3 M) at 298 K. EPR spectrum was obtained in DMSOsolution at 110 K on a Bruker-ER 200-D-SRC spectrometer. The X-raydata were collected on a Bruker SMART APEX 1K CCD diffractometer.Bioactivity assays (IC50 values) of the complexes were carried outon a Bio-RAD model 550 microplate reader as previously described[19–22].

2.3. Synthesis of the complexes

Polypyridyl ligands (dpq and dppz) were prepared according tothe reported methods from phen [24,25], and SalAla Schiff base ligand(used for pH-potentiometric titration) was prepared according toreference [26] from alanine and salicylaldehyde. They were charac-terized by NMR, IR and melting points.

For the synthesis of the complexes, Schiff bases condensated withsalicylaldehyde and L-amino acids were synthesized in solution anddirectly used in the next step for the synthesis of oxovanadiumcompounds (Chart 1). The oxovanadium complexes [VO(Sal-aa)(NN)]

Chart 1. Schematic route for the synthesis of the complexes 1–8. [VIVO(Sal-Ala)(bpy)] (1), [VIV

(dpq)] (5), [VIVO(Sal-Lys)(dppz)] (6), [VIVO(Sal-Asp)(dppz)] (7), [VIVO(Sal-Trp)(dppz)] (8).

(1–8), where Sal-aa for a Schiff base condensatedwith salicylaldehydeand an L-amino acid [viz. salicylidene alanine (SalAla), salicylidenelysine (SalLys), salicylidene asparagine (SalAsp), salicylidene tyrosine(SalTrp)], and NN for a N, N-donor heterocyclic base [viz. 2,2 -bipyridine(bpy), 1,10-phenanthroline (phen), pyrazino[2,3-f][1,10]phenanthroline (dpq), dipyrido[3,2-a:2 ,3 -c]phenazine (dppz)], wereprepared by methods analogous to our reported procedures[19,21](Chart 1).

Briefly, a methanolic solution of salicylaldehyde (1.0 mmol)was added dropwise to a mixture of L-Ala (1.0 mmol) and KOH(1.0 mmol) in 5 ml methanol and stirred at 333 K. The resultingsolution was refluxed for 1 h, followed by the addition of a hotaqueous VOSO4 solution (1.0 mmol). Then the reaction mixture wasneutralized by adding aqueous KOH solution. After 2 h of refluxing, asolution of 1.0 mmol 2,2 -bpy (1, 1,10-phen for 2, dpq for 3, and dppzfor 4) in 5 ml of methanol was added dropwise to this mixture. Afterfurther refluxing for another 3 h, the solution gave reddish-brownprecipitates. The solid powder was isolated by filtration, washed withdistilled water, methanol and diethyl ether, respectively, and driedin vacuo. Complexes 5 and 6 were synthesized following the samegeneral procedures as those described above, with Lys in place ofAla and with dpq (5, dppz for 6) in place of bpy. Complexes 7 and8 were synthesized following the same general procedures as thosedescribed for complex 6, with Asp (7, Trp for 8) in place of Lys. Theproducts, [VIVO(SalAla)(bpy)] (1), [VIVO(SalAla)(phen)] (2), [VIVO(SalAla)(dpq)] (3), [VIVO(SalAla)(dppz)] (4), [VIVO(SalLys)(dpq)] (5),[VIVO(SalLys)(dppz)] (6), [VIVO(SalAsp)(dppz)] (7) and [VIVO(SalTrp)(dppz)] (8), were characterized by elemental analysis andIR (Table 1). The other data of the complexes, such as ESI-MS, IR, UV–visible (UV–Vis) in DMSO and ΛM(DMSO), were given in ElectronicSupplementary Material (EMS). Reddish-brown crystal of 2 wasobtained by slow evaporation of the reaction solution at roomtemperature for two days.

2.4. X-ray crystallography

Single crystals of the complex 2⋅0.5H2O were mounted on glassfibers for data collection. Cell parameters and an orientationmatrix fordata collectionwere obtained by least-square refinement of diffractiondata from 3629 reflections with a 1.7–25.0° of θ for 2⋅0.5H2O using aBruker SMART APEX 1K CCD automatic diffractometer. Data were

O(Sal-Ala)(phen)] (2), [VIVO(Sal-Ala)(dpq)] (3), [VIVO(Sal-Ala)(dppz)] (4), [VIVO(Sal-Lys)

Table 1Synthesis and analysis of complexes.

Complex Yield(%, on V)

Element analysis(%), calcd/found IR (cm−1)

C H N νC=N,νV=Oa

1⋅0.67H2Ob 93 56.34/56.13 4.34/4.43 9.86/9.93 1612s, 959s2⋅H2Oc 85 57.90/57.42 4.20/4.24 9.21/9.18 1621vs, 956s3 44 58.78/58.24 3.49/3.49 14.28/14.29 1612s, 953s4·2CH3OH 40 59.61/60.05 4.50/5.08 11.59/11.32 1621vs, 964s5 44 59.24/59.42 4.42/4.42 15.35/15.42 1620vs, 963s6 38 62.31/62.02 4.39/4.31 14.06/13.23 1625vs, 961s7 54 59.05/58.64 3.35/3.59 11.87/12.23 1623vs, 985s8 60 65.96/66.24 3.69/3.77 12.82/13.05 1605vs, 982s

a 995 cm−1 in VOSO4, s for strong, vs for very strong.b Ref. [27].c Ref. [37].

1325L. Lu et al. / Journal of Inorganic Biochemistry 105 (2011) 1323–1328

collected at 298 K using MoKα radiation (λ=0.71073 Å) and theω-scan technique, and corrected for Lorentz and polarization effects(SADABS) [28]. The structures were solved by direct methods(SHELXS-97) [29] and subsequent difference Fourier maps and thenrefined on F2 by a full-matrix least-squares procedure using aniso-tropic displacement parameters [28]. Solvent molecule water in2⋅0.5H2O was found to be disordered with 25% occupancies. C(methyl) and Cα are in disorder with 62% and 38% occupancies,respectively. After several cycles of refinement, hydrogen atomsattached to C atoms were located at their calculated positions with C(sp2)-H 0.93 and C(sp3)-H 0.96 Å and were refined using a ridingmodel. H atoms attached to O atom in 2⋅0.5H2O were located fromdifference Fourier maps and refined their global Uiso value. Moleculargraphics were from SHELXTL [30].

2.5. Potentiometric titrations

pH-potentiometric titration was performed to study the speciesformed in aqueous solution. The protonation constants of ligandsand the stability constants of the complexes VOSO4 with ligands weredetermined by pH-potentiometric titrations of 50-mL samplescontaining the ligand being acidificated by HCl. High-purity nitrogengas was used to remove carbon dioxide and molecular oxygen fromsamples and to provide an inert gas atmosphere during all measure-ments. Measurements of the pH values were carried out at 298±0.2 Kand at a constant ionic strength of 0.1 M NaCl on a PHS-3TC pH meterwith a combined glass electrode. The electrode was calibrated bystandard buffer solutions. The titrations were performed with acarbonate-free NaOH solution of known concentration (0.09495 M)by using a microsyringe under a nitrogen atmosphere in a jacketedvessel. The protonation constants of ligands and the equilibriumconstants of the complexes were calculated with the aid of theSUPERQUAD program [31]. The values of pKw of water and pKa of phenused in the calculations are 13.78 [32] and 6.9 [33], respectively. Theoverall formation constant of the complex is denoted as the logarithmof βpqrs=[VOpSalAlaqphenrHs]/[VO]p[SalAla]q[phen]r[H]s. The con-ventional notation has been used: negative indices for H in theformulas indicate either the dissociation of groups which donot deprotonate in the absence of V(IV)O coordination or hydroxoligands. The following hydroxo species of VO2+ were taken intoaccount in the calculations: [VO(OH)]+ (log β100-1=−5.94) and[(VO)2(OH)2]2+ (log β200-2=−6.95) [34], [VO(OH)3]− (log β100-3=−18.0) and [(VO)2(OH)5]− (log β200-5=−22.0) [35]. All solutionswere fresh prepared before use. For SalAla (concentration at0.750 mM with twice acidification by HCl(SalAla⋅2HCl), noted pre-cipitates would be observed during the titrations when concentrationof SalAla was greater than 0.8 mM), more than 300 data points inthe pH range of 2.49–11.50 were used for subsequent analysis, ForVO–phen ([VOSO4]=0.750 mM and [phen⋅2HCl]=1.50 mM), VO–SalAla ([VOSO4]=0.750 mM and [SalAla⋅2HCl]=1.50 mM), and VO–

SalAla–phen ([VOSO4]=[SalAla⋅2HCl]=[phen⋅2HCl]=0.750 mM),more than 210, 280, 240 data points, respectively, in the pH rangeof 2.66 – 11.50 were used for subsequent analysis.

2.6. Protein tyrosine phosphatase inhibition assays

Human PTP1B was expressed and purified as described previously[19]. PTP activities were measured using p-nitrophenol phosphate(pNPP) as the substrate. The assays were performed in 20 mM MOPSbuffer, pH 7.2, containing 50 mMNaCl and 2 mM GSH. The complexes1–8 were dissolved in DMSO (10−2 M), and diluted to variousconcentration gradients, then further diluted 10 times into enzyme-MOPS buffer solutions for activity evaluations. Inhibition assayswere performed in the same buffer on a 96-well plate in 100 μlvolumes. The PTP1B was diluted to a final concentration of 60 nM.Then 10 μl of complex with various concentrations was mixed to 83 μlenzyme solution for 30 min. Then 2 μl of pNPP (0.1 M) substratewas added to initiate enzyme reactions. After incubation for 30 minat room temperature, the reactions were terminated by the additionof 5 μl of 2 M NaOH. The optical density at 405 nm was measuredon a microplate reader. IC50 values were obtained by fitting theconcentration-dependent inhibition curves using the Origin pro-gram. All data points were carried out in triplicates. Solutions ofthe oxovanadium complexes were all freshly prepared before eachexperiment.

The inhibiting kinetic analysis was performed according to Eq. (1),where Vmax is the maximum initial velocity, Km for the correspondingMichaelis–Menten constant, S for the substrate, I for the inhibitor, Ki

for the inhibition constant at varied substrate concentrations, derivedfrom the slope of the Lineweaver–Burk plots.

ν =Vmax S½ �

Km 1 + I½ �Ki

� �+ S½ �

: ð1Þ

Inhibition constants were determined by measuring initial hydro-lysis rates at different concentrations of substrate and inhibitor. Theapparent Kapp values measured at the various inhibitor concentrationswere plotted against concentration of the inhibitor to calculate theKi values.

3. Results and discussion

3.1. Synthesis and general aspects

The oxovanadium complexes are prepared from a typical syntheticprocedure, in which oxovanadium(IV) sulfate is reacted in situ withdianionic α-amino acid Schiff bases and polypyridyl in aqueousmethanol as shown in Chart 1, in which the intermediate Schiffbases were not isolated. All complexes were well characterized byelemental and ESI-MS analysis, IR and conductivity. Complex 2 as arepresentative of them was further characterized by X-ray singlediffraction, EPR and pH-potentiometric titration. Selected physico-chemical data for the complexes are given in ESM (Tables S1–S3). Allthe complexes are remarkably soluble in DMSO and DMF, solublein methanol, and insoluble in water as well. The molar conductivitiesin DMSO solution indicate that these complexes are neutral nonconducting compounds. The compositions of the complexes deducedfrom the elemental analysis were confirmed by ESI-MS study (ESMTable S1). In the infrared spectra (ESM Table S2), the bands at ca.1605–1625 cm−1 can be assigned to the C=N vibration involvingcoordination through the nitrogen of the azomethine group. The V=Ostretching frequencies of the complexes occur in the range of953–985 cm−1, which is consistent with previous reported rangeobserved for the majority of oxovanadium(IV) complexes [19–22].The electronic absorption spectra of the oxovanadium(IV) complexes

V1

O1O3

O4

O2

N3

C20

C22

N1C1

C2

C3C4

C7

C8

C9

C5C6

N2

C12

C11

C10

C19

C18

C17

C16

C15

C14

C13

Fig. 2. ORTEP view of complex 2 showing atom labeling with 30% probability thermalellipsoids, disordered water, minor parts C20 and C22 were omitted for clarity.

1326 L. Lu et al. / Journal of Inorganic Biochemistry 105 (2011) 1323–1328

in DMSO show a progress of ligand-to-metal charge transfer (LMCT)fromoccupanceπ (n) orbitals of the chelated ligand to emptydorbitalsof the vanadiumnear 370–390 nm (ESMTable S2).Molar conductivityof 1–8 was measured in 0.001 M DMSO solutions at 298 K andthe values were located in range of 1.37 to 4.33 μS cm2 mol−1 (ESMTable S3), which confirmed the vanadium complexes to be the non-electrolytes [36].

In order to ascertain the oxidation state of the vanadium, electronparamagnetic resonance was employed. As shown in Fig. 1, theX-band EPR spectrum of complex 2 in DMSO at 110 K exhibits anaxially symmetrical signal of tetravalent vanadium, split into anumber of hyperfine lines which originate from the d1 electroninteraction with a nuclear spin I=7/2. The spectrum displays well-resolved 51V (I=7/2) hyperfine lines.

3.2. Molecular structure of complex 2

The crystal structure of complex 1 was reported by Cavaco andco-workers [27], supporting the structures we proposed for thecomplexes in this study. The crystal structure of complex 2 had notbeen available but was reported [37] while this manuscript was underreview. After a careful comparison of our structure and the structurereported recently [37], we found that the overall coordination issimilar in the two structures but the reported crystallographic dataare in poor quality and the reported structure contains a fewunreasonable structural features and bond length/angles, as revealedby Checkcif report. Because our crystallographic data is of much betterquality and Checkcif report gives no problem for our structure, weprovided the details of our crystallographic and structure data inElectronic Supplementary Material (ESM S2, Tables S4 and S5). Forthe convenience of the readers, an ORTEP view of our structure of

Fig. 1. EPR spectra of complex 2 (A at 298 K, B at 110 K).

complex 2 is shown in Fig. 2. Briefly, the complex has a VIVO3N3

distorted octahedron geometry with the V=O distance of 1.598(3) Å,typical for a double bond. The Schiff base is bound through thephenolate oxygen (O2), the imine nitrogen (N3), and the carboxylateoxygen (O3) of the SalAla, which cooperated with N1 atom of phen toform a the equatorial plane and O1 and N2 atoms locate at the axialpositions. Due to the strong trans influence of the oxido ligand ofvanadium(IV), the V1–N2 bond trans to the V=O group is signif-icantly longer [2.359(3)Å] than the other both V–N bonds (Table S5),which is well constituent with similar coordination sphere [19,21].The V–O distances involving the Schiff bases are in range of 1.944(2)to 1.998(2) Å in 2, typical for single bonds.

3.3. Species and distribution in solution

The complexes were further characterized by ESI-MS in methanolsolution. Species corresponding to the proton adducts and the sodiumadducts were observed for all the 8 complexes (Table S1). The com-position of the complexes deduced from the elemental analysis wasconfirmed by the ESI-MS study and the presence of the corresponding

Table 2Stoichiometry, notation, pKa of the ligands and logarithm of cumulative stabilityconstant (log β) of oxovanadium complex species in the VO2+-(SalAla)-phen systema.

p(VO2+) q(SalAla2−) r(phen) s(H+) Species logβ or pKa

1 1 [(phen)H]+ 6.9b

1 1 [(SalAla)H]− 8.87(8)c

1 2 [(SalAla)H2] 17.66(7)c

1 3 [(SalAla)H3]+ 19.91(12)c

1 1 [(VO)(phen)]2+ 10.13(7)1 1 −1 [(VO)(phen)H−1]+ 10.53(5)1 1 −2 [(VO)(phen)H−2] −2.90(7)1 2 [(VO)(phen)2]2+ 15.66(9)1 1 1 [(VO)(SalAla)H]+ 21.49(10)1 1 [(VO)(SalAla)] 16.61(6)1 1 −1 [(VO)(SalAla)H−1]− 7.15(3)1 1 1 1 [(VO)(SalAla)(phen)H]+ 28.27(7)1 1 1 [(VO)(SalAla)(phen)] 21.65(14)1 1 1 −1 [(VO)(SalAla)(phen)H−1]− 12.84(14)1 1 1 −2 [(VO)(SalAla)(phen)H−2]2− 3.49(13)

a 298 K, I=0.10 M NaCl.b Taken from Ref [25].c As compared with two solvents to increase dissolution of SalAla, see 9.10, 15.98,

18.96, in DMSO(30%)–Water(70%), 8.81, 16.10, 20.02 in DMSO(50%)–Water(50%),298 K, I=0.10 M NaCl [40]; 5-Cl-SalA: 9.69, 17.45, 19.65 and 5-BrSalA: 10.25, 18.34,20.56 in a dioxane–water (1:1), 298 K, I=0.10 M KCl [41].

Fig. 3. Species distribution as a function of pH for VO2+-(SalAla)-phen (1:1:1, 0.75 mM)systems.

Table 3IC50 values of the oxovanadium(IV) complexes in inhibiting PTP1Ba.

Complexes IC50 (nM)±r.d. Complexes IC50 (nM)±r.d.

VO(Sal-Ala)(bipy)(1) 107±31 VO(SalLys)(dpq) (5) 552±20VO(SalAla)(phen)(2) 217±31 VO(SalLys)(dppz)(6) 598±46VO(SalAla)(dpq) (3) 409±38 VO(SalAsp)(dppz)(7) 411±22VO(SalAla)(dppz)(4) 287±24 VO(SalTrp)(dppz) (8) 62±7

a Concentration intervals are from 10−3 to 10−9 or 10−10 M. All data points werecarried out in triplicates. Fit curves of eight complexes were shown in Fig. 4 and Fig. S3(see Electronic Supplementary Material).

1327L. Lu et al. / Journal of Inorganic Biochemistry 105 (2011) 1323–1328

parent peak in the positive ion ESI-MS excluded immediate hydrolysisof complexes in methanol solution [38].

To further probe the stability of these complexes in the enzymeactivity assay buffer, the DMSO solutions (10−2 M) of the complexeswere diluted into the buffer (10−4 M) and kinetics was followed byUV–Vis for about 1 h (Fig. S2). The UV–Vis data showed no changes forcomplexes 3 and 7 while only very minor decreases in intensity forthe other complexes, suggesting that the complexes are quite stable inthe assay system.

To obtain species distribution of the complexes formed in aqueoussolution,which is relevant to their bioactivities, potentiometric titrationwas performed for the system of VO2+ cation and two ligands (SalAlaand phen). As a preliminary step for studying the VO2+–SalAla–phensystem, the protonation constants of SalAla were determined and theestimated pKa values were 8.87(8), 8.79(7) and 2.25(12) at [SalAla⋅2HCl]=0.750 mM. The protonation constants of the phen ligand andthe stability constants of the hydroxo complex of VO2+were cited fromthe literature [33,35,39]. Then the titration curves for the binary systemsof VO2+–SalAla (1:2), VO2+–phen (1:2) and the ternary system VO2+/SalAla/phen (1:1:1) were analyzed respectively. The species weresuggested according to the data shown in Table 2. On the base of thesedata, the species distribution diagram as a function of pH is shown inFig. 3. The distribution curves suggest that the mixed-ligand oxovana-dium species [VO(SalAla)(phen)] and [VO(SalAla)(phen)H]+ are thepredominant species in the pH range 7.0–7.4. The species [VO(SalAla)(phen)] agrees with the structural model from the X-ray structurestudies.

Fig. 4. Concentration-dependent inhibition of PTP1B (60 nM) by complex 1. IC50=107±31 nM.

3.4. Inhibition of PTP1B

All the complexes were tested for their abilities in inhibitingPTP1B, using pNPP as the substrate. Typical inhibition curves areshown in Fig. 4 and Fig. S3; the IC50 values for the complexes are listedin Table 3. The IC50 (62–597 nM) values indicate that all thecomplexes exhibit strong inhibition against PTP1B but 3–10 foldweaker than those of similar mixed-ligand oxovanadium(IV) com-plexes of SAA derivative with polypyridyl ligands [19,21]. Onlycomplex 8 displays high inhibition activity (IC50=62 nM) compara-ble to that of the mixed-ligand oxovanadium(IV) complexes of SAAderivative with polypyridyl ligands, indicating that the aryl of sal-aamay increase the bonding of the complexes to PTP1B. Also, as thosein the mixed-ligand oxovanadium(IV) complexes of SAA derivativewith polypyridyl ligands, with the increase in size of the polypyridylligands (complexes 1–4), the inhibitory activity decreased by 3-fold,suggesting that the spatial bulk of polypyridyl moieties of thecomplexes has moderate effects on PTP1B inhibition [19,21]. Inaddition, increasing in size of sal-aa ligands (complexes 3vs5, 4vs6and 7) also slightly decreases the inhibition activity by 1–2 fold.Interestingly, complex 8, with Trp as a co-ligand, exhibits significantlybetter potency than the other analogues (complexes 4, 6 and 7),suggesting that the Trp aryl groupmay play a significant role in PTP1Bbinding/inhibition. All these results demonstrate that the ligands onvanadium influence PTP1B inhibition activity.

The PTP1B inhibition mode of complex 2 was investigated further.As shown in Fig. 5, the Lineweaver–Burk plot of complex 2 exhibitedclassical kinetic behavior for a competitive inhibitor. The apparentinhibition constant (Ki) was calculated to be 283 nM (Fig. 5, inset).This result is consistent with our previous results on similar mixed-ligand oxovanadium(IV) complexes [19,21], suggesting that complex2 may bind to PTP1B at the catalytic site in the deep tyrosinephosphate binding pocket.

Fig. 5. Lineweaver–Burk plot of 1/ν (min μM−1) versus 1 (pNPP concentration)(mM−1) at 0 (□), 100 (○), 200 (△), 300 (▽) and 700 (◇) nM of complex 2 (PTP1B,80 nM). Inset for the plot of apparentMichaelis constant (Kapp) versus the concentrationof inhibitor to determine the inhibition constant (Ki=283 nM).

1328 L. Lu et al. / Journal of Inorganic Biochemistry 105 (2011) 1323–1328

4. Conclusion

In summary, we have synthesized and characterized eightoxovanadium(IV) complexes (of which 6 are new) of Sal-aa(aminoacid) Schiff base and polypyridyl derivatives. The inhibitory activitiesof the complexes against PTP1B indicated that the complexes maycompetitively inhibit PTP1B, but the potency is 3–10 fold weaker thanthose of the similar mixed-ligand oxovanadium(IV) complexes ofsalicylidene anthranilic acid derivative and polypyridyl ligands exceptcomplex 8 which is more potent and exhibits activity comparable tothose of themixed-ligand oxovanadium(IV) complexes of salicylideneanthranilic acid derivative and polypyridyl ligands. The resultsdemonstrate that the structures of vanadium complexes influencePTP1B inhibition activity. Aryl group may increase the binding of thecomplexes to PTP1B. We believe this result may be helpful for furtherdesign of potent and selective vanadium-based PTP inhibitors.

AbbreviationsBpy 2,2′-bipyridinedppz dipyrido[3,2-a:2′,3′-c]phenazinedpq dipyrido[3,2-d:2′,3′-f]quinoxalineEA Elemental analysisESI-MS Electrospray ionization mass spectraESM Electronic Supplementary MaterialGSH L-glutathione(reduced)Kapp apparent Michaelis–Menten constantKi inhibition constantKm Michaelis–Menten constant,MOPS 3-Morpholinopropanesulfonic acidNN N,N′-donor heterocyclic basePhen 1,10-phenanthrolinepNPP p-Nitrophenol phosphatePTP1B Protein tyrosine phosphatase 1BPTPs protein tyrosine phosphatasesSAA salicylidene anthranilic acidSalAla salicylidene alanineSalAsp salicylidene asparagineSalLys salicylidene lysineSalTrp salicylidene typtophen

X-ray crystallographic data in cif format of complex 2 (CCDC795204). These data can be obtained free of charge via http://www.ccdc.cam.ac.uk/conts/retrieving.html, or from the Cambridge Crystal-lographic Data Centre, 12 Union Road, Cambridge CB2 1EZ, UK; fax:(+44) 1223-336-033; or e-mail: deposit@ccdc.cam.ac.uk. ESI-MSdata and analysis, IR and UV data, molar conductance, X-ray crystalstructure of complex 2, UV–Vis spectra of complexes 1–8 in MOPSbuffer (pH=7.2), and PTP1B inhibition curves of complexes 2–8.Supplementary materials related to this article can be found onlineat doi:10.1016/j.jinorgbio.2011.07.008.

Acknowledgements

This work was supported financially by the National NaturalScience Foundation of China (20471033 and 21001070), the ScientificResearch Foundation for the Returned Overseas Chinese Scholars,State Education Ministry (20093602), the China Scholarship Council,the Natural Science Foundation of Shanxi Province (20051013,

2010011011-2, 2011011009-1 and 2011021006-2), the 100 TalentsProgram of Shanxi Province, the Overseas Returned Scholar Founda-tion of Shanxi Province of China in 2008, University of MassachusettsDartmouth, Cranberry Research Program, and UMass Science &Technology Initiatives Fund, MA, USA.

References

[1] J.N. Andersen, P.G. Jansen, S.M. Echwald, O.H. Mortensen, T. Fukada, R.D. Vecchio,N.K. Tonks, N.P.H. Møller, FASEB J. 18 (2004) 8–30.

[2] M. Stuible, K.M. Doody, M.L. Tremblay, Cancer Metastasis Rev. 27 (2008) 215–230.[3] K.M. Heinonen, F.P. Nestel, E.W. Newell, G. Charette, T.A. Seemayer, M.L. Tremblay,

W.S. Lapp, Blood 103 (2004) 3457–3464.[4] L.I. Pao, K. Badour, K.A. Siminovitch, B.G.Neel, Annu. Rev. Immunol. 25 (2007)473–523.[5] T. Vang, A.V. Miletic, Y. Arimura, L. Tautz, R.C. Rickert, T. Mustelin, Annu. Rev.

Immunol. 26 (2008) 29–55.[6] M.A.T. Blaskovich, Cur. Med. Chem. 16 (2009) 2095–2176.[7] P. Heneberg, Cur. Med. Chem. 16 (2009) 706–733.[8] M. Elchebly, P. Payette, E. Michaliszyn, W. Cromlish, S. Collins, A.L. Loy, D.

Normandin, A. Cheng, J. Himms-Hagen, C.C. Chan, C. Ramachandran, M.J. Gresser,M.L. Tremblay, B.P. Kennedy, Science 283 (1999) 1544–1548.

[9] B.A. Zinker, C.M. Rondinone, J.M. Trevillyan, R.J. Gum, J.E. Clampit, J.F. Waring, N.Xie, D. Wilcox, P. Jacobson, L. Frost, P.E. Kroeger, R.M. Reilly, S. Koterski, T.J.Opgenorth, R.G. Ulrich, S. Crosby, M. Butler, S.F. Murray, R.A. McKay, S. Bhanot, B.P.Monia, M.R. Jirousek, Proc. Natl. Acad Sci. USA 99 (2002) 11357–11362.

[10] S. Zhang, Z.-Y. Zhang, Drug Discov. Today 12 (2007) 373–381.[11] A.J. Nichols, R.D. Mashal, B. Balkan, Drug Develop. Res. 67 (2006) 559–566.[12] K.H. Thompson, C. Orvig, J. Inorg. Biochem. 100 (2006) 1925–1935.[13] J.H. McNeill, V.G. Yuen, S. Dai, C. Orvig, Mol. Cell. Biochem. 153 (1995) 175–180.[14] D.C. Crans, J.J. Smee, E. Gaidamauskas, L. Yang, Chem. Rev. 104 (2004) 849–902.[15] K.G. Peters, M.G. Davis, B.W. Howard, M. Pokross, V. Rastogi, C. Diven, K.D. Greis, E.

Eby-Wilkens, M. Maier, A. Evdokimov, S. Soper, F.J. Genbauffe, J. Inorg. Biochem.96 (2003) 321–330.

[16] B.I. Posner, R. Faure, J.W. Burgess, A.P. Bevan, D. Lachance, G. Zhang-Sun, I.G.Fantus, J.B. Ng, D.A. Hall, B.S. Lum, J. Biol. Chem. 269 (1994) 4596–4604.

[17] G. Huyer, S. Liu, J. Kelly, J. Moffat, P. Payette, B. Kennedy, G. Tsaprailis, M.J. Gresser,C. Ramachandran, J. Biol. Chem. 272 (1997) 843–851.

[18] F. Nxumalo, N.R. Glover, A.S. Tracey, J. Biol. Inorg. Chem. 3 (1998) 534–542.[19] C.X. Yuan, L.P. Lu, X.L. Gao, Y.B. Wu, M.L. Guo, Y. Li, X.Q. Fu, M.L. Zhu, J. Biol. Inorg.

Chem. 14 (2009) 841–851.[20] X.L. Gao, L.P. Lu,M.L. Zhu, C.X. Yuan, J.F.Ma, X.Q. Fu, Acta. ChimSin 67 (2009) 929–936.[21] C.X. Yuan, L.P. Lu, Y.B. Wu, Z.W. Liu, M.L. Guo, S. Xing, X.Q. Fu, M.L. Zhu, J. Inorg.

Biochem. 104 (2010) 978–986.[22] L.P. Lu, S.L. Wang, M.L. Zhu, Z.W. Liu, M.L. Guo, S. Xing, X.Q. Fu, BioMetals 23

(2010) 1139–1147.[23] L. Lu, M. Zhu, Anti-Cancer Agents Med. Chem. 11 (2011) 164–171.[24] J.E. Dickeson, L.A. Summers, Aust. J. Chem. 23 (1970) 1023–1027.[25] E. Amouyal, A. Homsi, J.C. Chambron, J.P. Sauvage, J. Chem.Soc., Dalton Trans.

(1990) 1841–1845.[26] D. Heinert, A.E. Martell, J. Am. Chem. Soc. 84 (1962) 3257–3263.[27] I. Cavaco, J.C. Pessoa, D. Costa, M.T. Duarte, R.D. Gillard, P. Matias, J. Chem. Soc.

Dalton Trans. (1994) 149–157.[28] G.M. Sheldrick, Correction Software, University of Gotingen, Germany, 1996.[29] G.M. Sheldrick, Program for the Solution of Crystal Structure, University of

Gotingen, Germany, 1997.[30] G.M. Sheldrick, SHELXTL/PC Version 5.1, Bruker AXS Inc., Madison, Wisconsin,

USA, 1999.[31] P. Gans, A. Sabatini, A. Vacca, J. Chem. Soc. Dalton Trans. (1985) 1195–1200.[32] G. Schwarzenbach, H. Flaschka, Complexometric Titrations, Methuen, London,

1969, p. 289.[33] A.E. Martell, S. Chaberek Jr., R.C. Courtney, S. Westerback, H. Hyytiainen, J. Am.

Chem. Soc. 79 (1957) 3036–3041.[34] B. Gyurcsik, T. Jakusch, T. Kiss, J. Chem. Soc. Dalton Trans. (2001) 1053–1057.[35] E.L. Chruscinska, D. Sanna, E. Garribba, G. Micera, Dalton Trans. (2008) 4903–4916.[36] W.J. Geary, Coord. Chem. Rev. 7 (1971) 81–122.[37] Y.-Z. Cao, H.-Y. Zhao, F.-Y. Bai, Y.-H. Xing, D.-M. Wei, S.-Y. Niu, Z. Shi, Inorg. Chim.

Acta 368 (2011) 223–230.[38] I.N. Stepanenko, A.A. Krokhin, R.O. John, A. Roller, V.B. Arion, M.A. Jakupec, B.K.

Keppler, Inorg. Chem. 47 (2008) 7338–7347.[39] R.P. Henry, P.C.H. Mitchell, J.E. Prue, J. Chem. Soc. Dalton Trans. (1973) 1156–1159.[40] A. Doğan, İ. Şakıyan, E. Kılıc, J. Solution Chem. 33 (2004) 1539–1547.[41] N. Sari, P. Gürkan, S. Çete, I. Şakiyan, Russian J. Coord. Chem. 32 (2006) 511–517.