Niobium(V) peroxo α-amino acid complexes: Synthesis, stability and kinetics of inhibition of acid phosphatase activity

Article abstract of DOI:10.1016/j.poly.2016.09.057

The present work was undertaken with an objective to investigate some biologically relevant attributes of peroxoniobium (pNb) derivatives, using a set of heteroleptic pNb compounds containing biogenic species as co-ligands. New peroxoniobium complexes cor

Full text of DOI:10.1016/j.poly.2016.09.057

Accepted Manuscript
Niobium(V) peroxo α-amino acid complexes: synthesis, stability and kinetics
of inhibition of acid phosphatase activity
Sandhya Rani Gogoi, Gangutri Saikia, Kabirun Ahmed, Rituparna Duarah,
Nashreen S. Islam
PII:
S0277-5387(16)30487-9
DOI:
http://dx.doi.org/10.1016/j.poly.2016.09.057
POLY 12245
Reference:
Polyhedron
To appear in:
Received Date:
Revised Date:
Accepted Date:
10 May 2016
14 September 2016
28 September 2016
Please cite this article as: S. Rani Gogoi, G. Saikia, K. Ahmed, R. Duarah, N.S. Islam, Niobium(V) peroxo α-amino
acid complexes: synthesis, stability and kinetics of inhibition of acid phosphatase activity, Polyhedron (2016), doi:
http://dx.doi.org/10.1016/j.poly.2016.09.057
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Niobium(V) peroxo α-amino acid complexes: synthesis, stability and kinetics of
inhibition of acid phosphatase activity
Sandhya Rani Gogoi, Gangutri Saikia, Kabirun Ahmed, Rituparna Duarah and
Nashreen S. Islam*
*To whom correspondence should be addressed:
Fax: +91-3712-267006
E-mail: nsi@tezu.ernet.in
nashreen.islam@rediffmail.com
Address: Prof. Nashreen S. Islam
Dept. of Chemical Sciences
Tezpur University
Napaam, Tezpur-784028
Assam, India
Phone : +91-9435380222 (Off.)
+91-3712-267007
1

Niobium(V) peroxo α-amino acid complexes: synthesis, stability and kinetics of
inhibition of acid phosphatase activity
Sandhya Rani Gogoi, Gangutri Saikia, Kabirun Ahmed, Rituparna Duarah and
Nashreen S. Islam*
Abstract
The present work was undertaken with an objective to investigate some
biologically relevant attributes of peroxoniobium (pNb) derivatives, using a set of
heteroleptic pNb compounds containing biogenic species as co-ligands. New
peroxoniobium complexes corresponding to the general formula, Na2[Nb(O2)3L]
(L=alanine or valine) were synthesized and comprehensively characterized by
1
elemental analysis, IR, Raman, 13C NMR, H NMR, AAS and UV-Vis spectral
studies, as well as thermogravimetric analysis. The structures of the compounds have
been studied by density functional theory (DFT) method and their stability in solution
at physiological pH has been ascertained. These compounds, along with our
previously reported arginine and nicotinate complexes, Na2[Nb(O2)3(arg)]·2H2O and
Na2[Nb(O2)3(nic)(H2O)]·H2O, respectively were screened for their activities with two
different types of enzymes viz., catalase and acid phosphatase (ACP). Employing
wheat thylakoid ACP as the model enzyme, it has been demonstrated for the first time
that peroxoniobium derivatives serve as active inhibitors of phosphatase (IC50 values
2-9 µM). Enzyme kinetics data revealed that compounds exert mixed type of
inhibition on ACP activity (Kii>Ki). Each of the tested pNb species exhibited
significant resistance to degradation under the effect of catalase vis-a-vis the native
substrate of the enzyme, H2O2.
Keywords: Peroxoniobium complex, ACP inhibition, Catalase resistance, Heteroleptic
peroxoniobate
2

1. Introduction
The contemporary interest on peroxo compounds of niobium mainly originates
from their utility as ideal water soluble precursors to obtain Nb-based oxide
materials for high technology applications [1-3], in addition to their use as efficient
and versatile oxidation catalysts [1,4-6]. It is intriguing to note that although Nb
belongs to the same periodic group as vanadium, very few studies dealing with
exploration of biochemical potential of peroxo compounds of niobium [7,8] are
available in contrast to peroxovanadates (pV). Compounds of vanadium including
the peroxovanadates have gained a special status in medicinal inorganic chemistry
mainly as antineoplastic and insulin-mimetic agents [9-22], despite the fact that
most of the synthetic pV compounds are unstable under physiological condition
and are toxic [10,11,13,17]. It is notable that Nb has been known to be practically
non-toxic to animals in comparison to vanadium [23]. Compounds of Nb have also
been documented to exhibit anti-cancer activity [7,24] and niobium substituted
heteropolytungstates were found to be potent anti-HIV-1 agents while being
minimally cytotoxic [25]. According to a recent report, vanadate and niobate are
strong inhibitors of the sarcoplasmic reticulum (SR) Ca2+-ATPase that can affect
calcium homeostasis, cell signalling, and many other cellular processes [26].
The process of enzyme inhibition has already been identified as an important
pathway for action of inorganic drugs in recent years [27]. For instance, insulin-
like activity of vanadium compounds has been linked to the inhibitory effect of
V-compounds on phosphatases [28]. In fact, phosphohydrolases have become a
key target for studying metabolism [29,30], for modifying cell signalling
3

[30,31,32] and for treatment of diseases owing to the pivotal role of phosphatases
in signal transduction pathways [29,30,33]. The efficacy of vanadium based
phosphatase inhibitors have been comprehensively reviewed by McLauchlan and
co-workers recently [30], in which it has been observed that, notwithstanding the
existence of numerous studies dealing with relative inhibitory potency such as
EC50 of V as well as Mo and W derivatives, the reports in which mode of
inhibition and Ki values have been defined are still very limited. Surprisingly, no
reports appear to exist so far, on in vivo or in vitro effect of discreet pNb
complexes on phosphohydrolases. Pertinent here is to mention that we have
previously investigated several bio-relevant aspects of peroxometallates [34-36]
including detailed inhibition kinetics of phosphatases involving monomeric [37-
39], dimeric [37,40] as well as macromolecular complexes of V, Mo and W [41-
43]. In the present study, in continuation of our work on peroxometallates, we
deemed it imperative to examine some of the heretofore unexplored biologically
important features of pNb derivatives including their activity with phosphatase
using wheat thylakoid membrane ACP as model enzyme. Apart from playing vital
roles in diverse biological phenomena, these enzymes serve as excellent models to
probe the metal induced inhibitory effect in membrane proteins [44-46].
We have also considered it important to study the effect of catalase, the
enzyme responsible for the decomposition of H2O2 to H2O and O2 in the
intercellular peroxisomes, on hetero as well as homoleptic pNb complexes.
Hydrogen peroxide, being a reactive oxygen species (ROS) has long been known
as a major contributor to DNA damage, protein oxidation and lipid peroxidation,
which can ultimately lead to cell death [47-49]. In recent years however, the view
that H2O2 is a toxic waste product has changed following the reports documenting
4

its importance as a key signal transducing agent which regulate a variety of cellular
processes [20,21,35,50-52]. Rapid decomposition of cellular H2O2 by catalase and
glutathione peroxidase has been found to be a hindrance for studying the cellular
effects of H2O2 [20,21,35,50]. This has led to a search for peroxide derivatives that
can substitute for H2O2, which would be less susceptible to degradation by
catalase, yet efficient in their action. A fascinating finding of our previous studies
has been the resistance exhibited by some of the metal bound peroxides towards
degradation under catalase action [37-42]. Diperoxovanadate has already been
recognized as a useful tool in studies of cellular effects mediated by H2O2
[35,50,53].
In this paper, we present the synthesis and characterization of two new
members of heteroleptic pNb complexes with amino acids, alanine and valine as
ancillary ligands. In nature, amino acids usually promote solubility of metals in
aqueous environment and impart stability by complexation and increase their
bioavailability. One of the factors believed to be responsible for the paucity of
information related to biological studies of Nb compounds, is the restricted
aqueous chemistry of the metal which is confined to very few available water
soluble compounds [1,2,7,8,54-61]. We have recently gained an access to a pair of
new triperoxoniobium complexes, Na2[Nb(O2)3(arg)]·2H2O (arg = arginate) (NbA)
and Na2[Nb(O2)3(nic)(H2O)]·H2O (nic = nicotinate) (NbN) as auxiliary ligands
which proved to be excellent catalysts for selective oxidation of thioethers with
H2O2, in neat water [62]. Here, we report our findings on hydrolytic stability,
steady state kinetic studies of the effect of the pNb complexes, NbA, NbN, NbAla
and NbVal as well as the parent complex, Na3[Nb(O2)4]·13H2O (NaNb) on acid
phosphatase in vitro. Our results confirm that apart from being highly potent
5

inhibitors of the model enzyme, the compounds exhibit significant resistance to
degradation under catalase action.
2. Experimental
2.1 Materials and methods
Niobium pentoxide, p-nitrophenyl phosphate (p-NPP), catalase, wheat
thylakoid membrane acid phosphatase (ACP) used in this study were obtained
from Sigma–Aldrich Chemical Co., Milwaukee, USA. Sodium thiosulphate,
potassium hydrogen phosphate, potassium dihydrogen phosphate, citric acid,
glacial acetic acid were purchased from Merck, India. L-alanine and L-valine were
obtained from SRL, Mumbai, India and hydrogen peroxide (30%), sodium citrate
(dihydrate) from Rankem, India.
The niobium content of the synthesized compounds was determined by atomic
absorption spectroscopy (AAS) using Thermo iCE 3000 series atomic absorption
spectrophotometer model analyst 200, as well as by gravimetric estimation [63].
Elemental analyses for C, H and N was performed on an elemental analyzer
(Perkin-Elmer 2400 series II). Peroxide content for the compounds were
determined, as has been described in earlier paper [64], by adding a weighed
amount of the compound to a cold solution of 1.5% boric acid (w/v) in 0.7 M
sulfuric acid (100 mL) and titration with a standard cerium (IV) solution. The
sodium content was measured by AAS and also with an ionometer (Orion
Versaster).
The IR spectra were recorded by making pressed pellets of samples with KBr
using Perkin-Elmer spectrum 100 FTIR spectrophotometer. Raman spectra of the
compounds were recorded using a Renishaw InVia Raman microscope equipped
6

with an argon ion laser with an excitation wavelength of 514 nm and a laser
maximum output power of 20 mW. Thermogravimetric analysis (TGA) was
carried out on a SHIMADZU TGA-50 system at a heating rate of 10 °C min-1
under N2 atmosphere using an aluminium pan. 1H NMR and 13C NMR spectra with
D2O as solvent were recorded using a JEOL JNM-ECS400 spectrometer with the
following parameters for 13C NMR spectra: a carbon frequency of 100.5 MHz,
4096 X-resolution points, 1000-20000 number of scans, 1 s of acquisition time,
and 30° pulse length. Cary model Bio 100 spectrophotometer equipped with a
Peltier-controlled constant temperature cell was used for spectroscopic
determinations of the initial rate of ACP catalyzed hydrolysis of p-NPP at 30 °C.
2.2 Na3[Nb(O2)4]·13H2O (NaNb) synthesis [6]
The precursor complex, Na3[Nb(O2)4]·13H2O, was prepared by fusing 1 g of
Nb2O5 with 1.85 g of NaOH in a nickel crucible at 700 °C, as has been reported
previously. The solid product was cooled and subsequently dissolved in 100 mL of
1 M aqueous H2O2. Unreacted Nb2O5 was filtered off and the filtrate was allowed
to stand at 5 °C for 24 h to obtain the crystals of Na3[Nb(O2)4]·13H2O.
2.3 Synthesis of Na2[Nb(O2)3(Ala)], NbAla (Ala
Na2[Nb(O2)3(Val)], NbVal (Val = L-valine)
=
L-alanine) and
In
a
typical reaction, 0.6550
g
(1.25 mmol) of solid sodium
tetraperoxoniobate, Na3[Nb(O2)4]·13H2O (NaNb) was dissolved in minimum
volume (4 mL, 25 mmol) of 30% H2O2 in a 250 mL beaker. Maintaining the
temperature of the reaction solution below 4 °C in an ice bath, the respective amino
acid (1.25 mmol) was added gradually to it with constant stirring. The molar ratio
of Nb: ligand was maintained at 1:1. The mixture was stirred for ca. 15 min till all
7

solid dissolved and a clear solution was obtained, which was subsequently allowed
to stand for 3 h. The pH of the resultant solution was recorded to be ca. 6. On
addition of pre-cooled acetone to the reaction solution with constant stirring, a
white pasty product separated out which on repeated treatment with acetone under
scratching turned into microcrystalline solid. The compound was separated by
centrifugation and dried in vacuo over concentrated sulfuric acid.
The complexes, Na2[Nb(O2)3(arg)]·2H2O (arg = arginate) (NbA) and
Na2[Nb(O2)3(nic)(H2O)]·H2O (nic = nicotinate) (NbN) were prepared according to
procedures reported earlier [62].
2.4 Computational details
The Gaussian09 programme [65] at the B3LYP/LANL2DZ level of theory has
been used to perform density functional theory (DFT) [66] calculations. The
ground-state geometry of the two niobium complexes were obtained in the gas
phase, and the minima of the optimized structures were verified by the absence of
imaginary frequencies.
2.5 Stability of the complexes in aqueous solution
In order to assess the stability of the compounds in solution, a stock solution
(100 mL) was prepared for each of the compounds maintaining the initial compound
concentration as 0.2 mM, by adding weighed amount of the respective compound
NaNb (0.105 mg/mL), NbA (0.089 mg/mL), NbN (0.079 mg/mL), NbAla (0.065
mg/mL) or NbVal (0.070 mg/mL). The peroxide content was then determined in
aliquots drawn from the stock solution at different time intervals by the procedure
described above under Section 2.1, for a period of 12 h. Stability of the compounds in
solution at pH 4.6, pH 7.0 and 8.0, respectively, maintained by using phosphate buffer
(50 mM, pH 4.6, pH 7.0 or 8.0), at pH 1.2 and 2.1 (50mM KCl/HCl buffer), and at pH
1
3.1 (50 mM citrate buffer) was similarly measured. Moreover, electronic, H NMR
8

and 13C NMR spectra of the compounds were monitored at 30 min time intervals for
any possible change over a period of 12 h, as a measure of stability of the compounds
in solution.
2.6 Effect of catalase on the pNb complexes
The extent of catalase induced decomposition of the compounds was evaluated
by determining the change in peroxide content of the compounds in a solution
consisting of the pNb compound in phosphate buffer and the enzyme, at specified
time intervals as has been reported earlier [37-42]. The test solution containing the
phosphate buffer (50 mM, pH 7.0) and catalase (40 µg/mL) was incubated at 30 oC.
The volume of the test solution was kept at 100 mL. The weighed amount of the
respective pNb compound as mentioned in Table 6, was then added to the test
solution and aliquots of 5 mL were drawn out at an interval of 5 min from the
starting of reaction and its peroxide content was determined after quenching the
reaction by adding it to cold sulfuric acid (0.7 M, 100 mL).
2.7 Acid phosphatase activity
The acid phosphatase activity was assayed spectrophotometrically employing
p-nitrophenyl phosphate (p-NPP) as a substrate [43,45,67,68]. In the standard
assay, the reaction mixture contained acetate buffer (0.1 M, pH = 4.6), the enzyme
(18.38 µg protein mL-1) and different concentrations of pNb species (concentration
varies between 1-12 µM, as shown in Fig. 7 and Fig. 8) or the free ligand. The
reaction was started by addition of the substrate p-NPP to the test solution which
was pre-incubated for 5 min. After 30 min of incubation of the reaction solution at
o
30 C, the reaction was terminated by addition of 0.9 mL of 0.5 M NaOH solution
and the absorbance at 405 nm was recorded in order to determine the p-NP
produced (molar extinction coefficient of the p-nitrophenolate = 18000 M-1 cm-1)
9

[69]. The enzyme activity in the absence of the inhibitors was determined which
was used as the control. The half-maximal inhibitory concentration of the inhibitor
species giving 50% inhibition (IC50) was determined graphically. All experiments
were performed in triplicate. The data in the figures are presented as the means
SE from three separate experiments.
2.8 Determination of the kinetic constants
The kinetic constants, maximum velocity (Vmax) and Michaelis constant (Km)
were determined from Lineweaver–Burk plots following a rearrangement of the
Michaelis–Menten equation [68,70,71] using Cary 100 Bio Enzyme Kinetics
software.






K m
Vmax [S ] Vmax
1
1
=
+
V
In the present case, the following equation was used to characterize the mode
of inhibition, which was found to be of mixed type:








V max × [S ]
V =




1 + [I ]
K i
1 + [ I ]
K ii














K m
+ [S ]




where V is the velocity, [S] is the p-NPP concentration and [I] denotes the inhibitor
concentration, Ki and Kii are inhibitory constants for the competitive part and non
competitive part, respectively. The enzyme inhibitor constants were determined
from secondary plots of the initial rate data by a linear regression analysis. In order
to obtain Ki, the slopes from the Lineweaver plots were re-plotted against the
inhibitor concentration values from the x-intercepts of these re-plots. The Kii was
10

obtained from the plot of intercepts that has been found from the Lineweaver plots
against the inhibitor concentration values from the x-intercepts of these re-plots.
3. Results and Discussions
3.1 Synthesis and characterization
The white microcrystalline pNb compounds, Na2[Nb(O2)3(Ala)] (NbAla) and
Na2[Nb(O2)3(Val)] (NbVal) were obtained by stabilizing peroxoniobium species in
a ligand sphere of L-alanine or L-valine in presence of excess H2O2. Water soluble
precursor complex, Na3[Nb(O2)4]·13H20, used as the source of niobium, has been
prepared by the literature method [6]. The maintenance of pH of ca. 6 and limiting
the amount of water to that contributed by 30% H2O2 appeared to be the key
parameters responsible for the successful synthesis of the compounds in an
aqueous medium. The near neutral pH apparently favoured the formation of the
triperoxoNb species and co-ordination of the amino acids in their anionic form,
facilitating the synthesis of the desired complexes. It is pertinent to mention that
ligands with carboxylate functional group have been identified as excellent
co-ligands for stabilizing peroxoNb(V) species [1,8,54-61].
From the elemental analysis data (Table 1), the ratio of Nb: O22- content in the
title complexes was found to be 1:3 whereas, the C, H, N analysis results indicated
the Nb: amino acids ratio as 1:1 in each of them. The values are consistent with the
<Table 1>
formation of triperoxoniobium species with one amino acid co-ligand in the
co-ordination sphere and are in accord with their formulation as Na2[Nb(O2)3L]
(L= alanine or valine). Occurrence of Nb in its +5 oxidation state in the
11

synthesized compounds has been confirmed from the magnetic susceptibility
measurements, which showed the compounds to be diamagnetic.
3.2 IR, Raman and electronic spectral studies
The IR and Raman spectra of the compounds NbAla and NbVal displayed
sufficiently well resolved spectral patterns main features of which are summarized
in Table 2. The spectra of both NbAla and NbVal (Fig. 1) exhibited three bands in
the 850-800 cm-1 region, which are typical of ν(O-O) modes of η2-peroxo groups
belonging to a triperoxoniobium species [1,72]. The presence of three side-on
<Table 2>
bound peroxo groups in the complexes has been corroborated further by the
complementary Raman spectra. The νas(Nb-O2) and νs(Nb-O2) absorptions were
observed in the expected 500-600 cm-1 region, respectively in the IR as well as
Raman spectra [1,73].
<Fig. 1>
Presence of co-ordinated amino acids in the complexes was also evident from
their IR and Raman spectra which displayed characteristic shifts in the positions of
the vibrations attributable to amino acids, relative to the corresponding free ligand
values. The assignments were made on the basis of relevant literature data [74-79].
The free alanine spectrum exhibited νas(COO-) and νs(COO-) vibrations at 1586 and
1409 cm-1, respectively with a frequency difference ∆ν = 177 cm-1 [∆ν = νas(COO-)
– νs(COO-)]. In case of free valine, these bands were located at 1570 and 1395
cm-1. A definite shift of νas(COO-) to a higher frequency and that of νs(COO-) to a
lower frequency (Table 2) and the resulting frequency difference of 266 and 234
12

cm-1 found in NbAla and NbVal, respectively are typical of unidentately
co-ordinated non-protonated carboxylate groups (Table 2) [74,75].
The participation of amino groups of the ligands in co-ordination was
indicated by the shift of ν(NH) frequencies to a lower wave number in comparison
to the free amino acid bands. The well-resolved medium intensity band observed in
the spectra of NbAla at 3081 cm-1 and the two bands occurring at 3058 and 3162
cm-1 for NbVal have been ascribed to the NH stretchings of co-ordinated amino
acid [74]. The δ(NH2) vibrations appeared at ca. 1510 cm-1 in the spectra of both
the complexes. A band at 490 cm-1 has been observed in the free alanine zwitterion
+
for NH3 torsion [78]. On complexation, the peak disappeared and additional peaks
have been obtained at 477 and 411 cm-1 in the IR spectrum of NbAla which may
be assigned to νas(Nb-N) and νs(Nb-N) stretchings, respectively [74]. Similarly, the
presence of 430 and 405 cm-1 peaks owing to νas(Nb-N) and νs(Nb-N) stretchings in
the spectrum of NbVal constitutes an additional valuable indication for the
involvement of NH2 group in coordination [74]. The intense peak observed
between 2980-2880 cm-1 in the Raman spectra occurring as a medium intensity
peak in the IR spectra of both the complexes have been assigned to C-H stretching
vibration.
The electronic spectra of complexes NbAla and NbVal displayed a broad
absorption between 300 and 350 nm which is characteristic of peroxo to metal
charge transfer transition of peroxo derivatives of niobium [7,59,61,62].
3.3 1H and 13C NMR studies
1
The H NMR spectra for the pNb complexes NbVal and NbAla in D2O are
presented in Fig. 2 and Fig. S1 (Supplementary Information), respectively and the
13

corresponding chemical shifts for the complexes and free amino acids are listed in
1
Table 3. A close resemblance was observed in the H NMR spectra of the
complexes and the spectra of the respective amino acid displaying the well-
resolved resonances with expected integration and peak multiplicities. The spectra
<Fig. 2>
however, showed distinct up field shift of the proton signals attached to carbon
atoms C-2 and C-3 relative to the free ligand, consistent with the occurrence of
metal ligand co-ordination [62,80,81]. Similar upfield shift of the proton signals
have been observed earlier in case of arginine containing complex NbA [62] as
well, which appears to be common feature among the reported metal compounds
containing co-ordinated amino acids [80-82]. The position of H-4 and H-5 protons
in NbVal remains practically unaffected which may not be unexpected as these
atoms are well-separated from the actual ligand donor sites.
<Table 3>
13
The C NMR spectra of ligands L-alanine and L-valine exhibited resonances
due to the carboxylate carbon atoms at 175.81 and 174.24 ppm, respectively, apart
from the expected peaks corresponding to the side chain carbon atoms [80]. As
seen in Fig. S2 (Supplementary Information) and Table 4, all the signals appear
with downfield shift to varying degrees, vis-a-vis the free amino acids, obviously
<Table 4>
resulting from the deshielding of carbon atoms as a consequence of complexation
[41,62,80]. The most significant co-ordination induced shift ∆δ (δcomplex − δfree
carboxylate) ≈ 7.02 ppm for NbAla (8.55 ppm for NbVal) occurred as expected for
14

the metal linked carboxylate carbon, suggesting a strong metal-ligand interaction
[41]. Appearance of carboxylate carbon resonance as a single peak in the spectra of
the compounds reflected a single carbon environment for co-ordinated carboxylate
[41]. Thus, in conformity with the 1H NMR evidence, 13C NMR spectra too, testify
to the presence of only one complex species in solution confirming that the
compounds did not hydrolyze and remained intact in solution. Thus apart from
valuable structural information, the NMR analysis data provided persuasive
evidence in support of the stability of the compounds in solution.
3.4 TGA-DTG analysis
The TGA-DTG plots [Fig. 3 (a) and (b)] obtained for the title compounds on
o
heating up to a temperature of 700 C, revealed a close analogy among their
decomposition patterns. Each of the compounds, as has been observed in case of
previously reported complexes, NbAla and NbVal undergoes continuous
degradation and do not explode on heating [55,59,62,83-85]. A significant
common feature shared by the two pNb compounds, as evident from the respective
thermograms, is the absence of water molecule in the compounds.
<Fig. 3>
Although existence of homo- or heteroleptic pNb complexes without free or
bound water molecule is not unprecedented [1,54,83,86,87], majority of
heteroligand pNb complexes including the ones reported previously by us (NbA
and NbN) [62], and sodium tetraperoxoniobate, NaNb [6] contain either outer
sphere or co-ordinated, or both types of water molecules [1,54,55,59,83]. The first
decomposition stage occurs in NbAla and NbVal in the temperature ranges of 135-
210 °C and 140-205 °C, respectively attributable to loss of co-ordinated peroxo
15

groups from the complexes. Subsequently, ligand degradation takes place in the
temperature range of 243-480 °C in case of NbAla (241-389 °C for NbVal) and
further continues up to a final decomposition temperature of 700 °C. The residue
remaining from the pNb compounds after their complete degradation was
characterized to be oxoniobate species. The IR spectra of the residue showed the
characteristic ν(Nb=O) absorptions and complete disappearance of the signature
peaks pertaining to peroxo as well as amino acids of the original compounds. The
TGA-DTG analysis data for the compounds thus furnished further endorsement in
support of the composition and formula assigned to the compounds.
Taking into account the above collective evidences the structure of the
complex ions may be represented by eight co-ordinated polyhedra of the type
shown in Fig. 4, comprising of three terminal peroxo groups and an amino acid
occurring as a bidentate ligand, bonded to Nb(V) via carboxylate and amino
groups.
<Fig. 4>
3.5 Theoretical investigation
The viability of the proposed structures for the complexes, NbAla and NbVal
was further supported by the results of density functional theory (DFT) [66]
calculations at the B3LYP/LANL2DZ level of theory. The initial structures of the
two complexes were modelled on the basis of the experimentally derived structural
information. Presented in Fig. 5 are the optimized geometries of the niobium
<Fig. 5>
16

complexes which show coordination spheres of the two complexes comprising of
central metal atom (Nb) surrounded by three η2-peroxo groups and deprotonated
amino acid ligand bonded via O(carboxylate) and N(amino) atoms. While one of
the peroxo groups occupies a trans position to the co-ligand, the other two are in
cis configuration. The coordination polyhedron around the niobium atom in each of
the complexes is a dodecahedron which is in accord with the structures of majority
of the reported pNb complexes [1,59], including the ones studied herein viz., NbA
and NbN [62]. Moreover, the geometrical parameters such as the bond angles and
bond lengths obtained from DFT calculations (Table 5) are within the range
typical of the heteroleptic peroxoniobium(V) complexes. The bond distances
associated with the peroxo groups viz., Nb-O and O-O bonds vary between 2.023
to 2.086 Å and 1.552 to 1.557 Å, respectively. The geometrical parameters derived
from the theoretical calculations are in close agreement with the reported
crystallographic data obtained for other triperoxoniobium(V) complexes consisting
of N,O-donor ligands in the co-ordination sphere [1,6,54-61].
<Table 5>
We have also calculated vibrational frequencies for the optimized geometries
of the pNb compounds NbAla and NbVal. The theoretically obtained IR and
Raman data presented in Table 2, correlated well with the respective
experimentally determined frequencies. The small deviations observed between the
calculated and experimental spectral data appear to be within the acceptable limit
and are not unusual as the calculated spectral data were obtained from gas phase
optimized geometries of the complexes. The reported average error for frequencies
calculated with the B3LYP functional was of the order 40-50 cm-1 for inorganic
molecules [88]. Thus the results of our theoretical studies substantiate the
17

experimental observations and impart additional validity to the proposed
geometries for the complexes NbAla and NbVal.
3.6 Stability of the complexes toward decomposition in solution
We have examined the stability of the pNb compounds in solution under
varying pH conditions (Fig. 6). Compound stability was verified with respect to
loss of co-ordinated peroxo groups by determining the peroxide content and
monitoring the electronic spectra for any possible change at specified time
intervals. In addition to pH of 9-10 which is the natural pH of the synthesized pNb
<Fig. 6>
compounds in solution, the stability was tested at pH values ranging from 1.2 to
8.0. We found that neither peroxide content of the compounds under investigation
nor the position or intensity of their electronic spectral absorptions undergo any
1
significant change during a period of 12 h. Furthermore, 13C and H NMR spectra
of the pNb compounds when monitored over a period of 12 h remained practically
unaltered (Fig. 2). These results are mutually consistent and collectively corroborate
that the compounds retain their solid state structure in solution.
3.7 Interaction of pNb compounds with catalase
The effect of catalase on the hetero-ligand pNb complexes NbA, NbN, NbAla
and NbVal, as well as the parent complex, NaNb are shown in Fig. 6. The data
presented in Table 6 demonstrate that in presence catalase at pH 7, each of the
heteroligand pNb compounds undergoes gradual degradation with loss of peroxide.
<Table 6>
18

On the other hand, addition of catalase to a 0.1 mM solution of its native substrate,
H2O2 leads to rapid degradation of H2O2 with release of half equivalent of oxygen
(molecular basis) with a rate of 430 µM/min as expected from the
disproportionation reaction, and the process is completed within ca. 2 min [53].
This difference in rates of degradation between H2O2 and the pNb species indicates
that the compounds are at least 30-50 fold weaker as substrate to the enzyme, with
respect to H2O2. From the above results it may be surmised that co-ordination of
peroxo ligand to Nb enhances its resistance towards degradation under catalase
action. It is also notable that the initial rates of degradation of the tested pNb
compounds vary from compound to compound (Table 6) suggesting that the
ability of the complexes to withstand catalase action is influenced by the auxiliary
ligand environment. On comparing these values with those obtained from some of
the previously reported monomeric hetero-ligand peroxo complexes of d0 metal
ions viz., V and Mo, it emerged that peroxo groups attached to Nb show
comparable degree of resistance with respect to pV compounds [37-42] whereas,
these are several fold more resistant to catalase action than the molybdenum
containing analogues [41].
On the basis of the peroxo groups present in these complexes, total peroxide
loss was expected to be 0.6 mM for the triperoxo compounds and 0.8 mM for
tetraperoxo compound from a solution of 0.2 mM compound concentration. We
have previously observed that the catalase action on compounds of V, Mo and W
led to nearly complete loss of their peroxide content within ca. 30 min of
incubation as anticipated [37-42]. In contrast, as seen in Fig. 6 in case of the pNb
complexes, after the initial loss of two molecules of peroxo groups of the
triperoxoNb complexes, within ca. 30 min of incubation, a unique feature observed
19

for each of the pNb complexes is that, the compounds continued to retain one
molecule of peroxide even beyond a period of ca. 1 h. The formation of a
monoperoxo species of Nb and its high resistance to catalase action is thus evident
from our data.
It is pertinent to mention that, we have preferred to employ phosphate buffer
in the present study, as the use of near neutral phosphate buffers has proven to be
most satisfactory in several previous investigations dealing with peroxovanadates
[41,53,89,90] as well as peroxo compounds of Mo(VI) and W(VI) [41,43], studied
in our laboratory. Although vanadate has been reported to react with most of the
compounds used in buffer including phosphate [89,91], pV compounds are known
to be fairly inert and do not react with non-reducing buffers such as phosphate
[53,89,90]. It is important to note that we have also conducted similar experiments
using Hepes as buffer in lieu of phosphate buffer (Fig. S3 and Fig. S4,
Supplementary Information). The rates of catalase induced degradation of the pNb
compounds derived from these experiments showed no significant variation (17.0,
11.0, 9.4 and 11.9 µM/min for NaNb, NbA, NbN and NbVal, respectively) from
the results obtained using phosphate buffer (Table 6), suggesting that these buffer
systems have no observable influence on the stability or the degradation profile of
the compounds in solution, under the employed experimental condition.
3.8 Inhibitory effect of pNb compounds on acid phosphatase
We have investigated the inhibitory mechanism of the pNb complexes on ACP
activity in vitro. The phosphatase inhibitory effect of the triperoxoniobium
compounds viz., NbA, NbN, NbAla and NbVal, as well as the parent
tetraperoxoniobate, NaNb upon activity of the wheat thylakoid membrane ACP
20

was evaluated employing standard enzyme assay system and p-NPP as substrate. A
variety of enzyme assays have been made use of previously, to measure
phosphatase inhibition by metal compounds such as vanadates, molybdates and
tungstates [30,41,43,45,68,71]. The mode of enzyme inhibition such as
competitive, non-competitive or mixed type can usually be ascertained [92] by
varying the inhibitor concentration and measuring the enzyme activity at various
substrate concentrations. The findings of our investigation on dose response
inhibition of the model enzyme activity by the pNb complexes are illustrated in
Fig. 7. The inhibitor potential of the test compounds was quantified in terms of the
half-maximal inhibitory concentration (IC50). The IC50 values recorded, being
within the range of 2-9 µM, implied that the compounds are highly potent
inhibitors of the enzyme (Fig. 7, Table 7). While the IC50 value of tetraperoxo
species was observed to be comparable to that displayed by triperoxoNb
complexes with arginine (NbA) and nicotinic acid (NbN) as co-ligands, the
compounds NbVal and NbAla was found to be relatively less potent. Since
individually the free ligands had no observable effect on the enzyme activity, under
the employed assay conditions, the above observations indicate that the inhibitory
<Fig. 7>
effect of the pNb compounds indeed originate from the interaction of the intact
metal complexes with the enzyme and the ligand environment influences the
inhibitor potency of the tested intact complexes.
<Table 7>
To establish the mechanism of inhibition for the acid phosphatase catalyzed
hydrolysis of p-NPP by the inhibitor species, the steady state kinetics of this
21

process were investigated and kinetic parameters Km and Vmax were determined
[68,70,71]. The L-B plots of the reciprocal initial velocity versus the reciprocal
substrate concentration in the absence and presence of the inhibitor complexes at
various concentrations are shown in Fig. 8 (for NaNb, NbN and NbAla) and
Fig. S5 [(for NbA and NbVal) (Supplementary Information)]. Kinetic
measurements yielded straight lines with point of intersection in the second
quadrant in each case. As can be readily seen in Fig. 8 as well as in Fig. S5
(Supplementary Information), both Vmax and Km values are affected by the
presence of each of the inhibitors showing an increase in Km and decrease in
velocity Vmax values with increasing inhibitor concentration. This behaviour is
characteristic of mixed type of inhibition combining competitive and
non-competitive modes. We have also determined the inhibitor constants Ki and Kii
<Fig. 8>
in order to assess the affinity of the enzyme for the inhibitors. The inhibitor
constant Ki is a measure of the affinity of the inhibitor for the free enzyme
whereas, Kii reflects its affinity for the enzyme-substrate complex. The ratio of Kii
and Ki values were used to assess the relative competitiveness of each inhibitor. A
larger Kii/Ki value indicates a mixed type of inhibition that is relatively more
competitive in its action, whereas a lower Kii/Ki reflects the mixed type of
inhibition that is more uncompetitive. On the other hand, when Kii = Ki, the mode
of inhibition becomes noncompetitive. In the present study, as shown in the inset to
Fig. 8 and Fig. S5 (Supplementary Information), the inhibitor constant Ki for the
competitive part of inhibition, was obtained from the secondary plot of the slope of
the primary plot (1/V versus 1/[S]) against the inhibitor concentration with the
intercept on the inhibitor axis being -Ki. The value of Kii, inhibitor constant for
22

non-competitive inhibition, was obtained from the replot of slope versus inhibitor
concentration, with the inhibitor axis intercept being equivalent to -Kii. The kinetic
results presented in Table 7 show that for each of the inhibitors Kii > Ki,
confirming that pNb species exert mixed type of inhibition on ACP activity.
A competitive inhibitor is known to share a close structural resemblance to the
natural substrate of the enzyme whereas, a non-competitive inhibitor binds
reversibly at a site far removed from the active site causing a conformational
change in the overall three-dimensional shape of the enzyme [43]. Acid
phosphatases, isolated from various plant and animal sources, contain dinuclear
iron active sites and highly conserved amino acid sequences with a histidine
residue at the active site [44,45,93-96]. Although, oxy anions of V, Mo and W
having penta or hexa co-ordinated structures have mostly been known to be
competitive inhibitors of phosphatases due to their structural analogy with
phosphate, however with respect to inhibition of acid phosphatases, different
oxometallates have been shown to display different mechanistic preferences
ranging from competitive, non-competitive to uncompetitive patterns of inhibition
[43,97-100]. The work of Averill and co-workers demonstrated that oxyanions
such as molybdate and tungstate being relatively larger in size, bind in a
non-competitive fashion by bridging the two iron atoms in the binuclear active site
of mammalian ACP, while the smaller anions may also bind at a single iron atom
in a competitive manner [93]. Oxidative interaction between the highly oxidative
compounds with the Fe2+ centre resulting in its oxidation to ferric form and
consequent inactivation of the enzyme in a non-competitive manner was suggested
as an additional possibility of inhibitory effect of these complexes [93]. In view of
these observations as well as our findings on efficient oxidising ability displayed
23

by the test compounds [62], it is reasonable to expect that in the present study too,
a similar mechanism involving oxidation of Fe2+ centre by the pNb species is
perhaps operative, contributing to their mixed type of inhibition of ACP function,
combining competitive and non-competitive pathways. However, in absence of
direct evidence and due to the complicated chemistry and intricacy of the
interactions between pNb species and complex biomolecules involved in the
present study, it is difficult to comment on the exact mechanism of inhibition at
this stage. Nevertheless, there are ample evidences in the literature highlighting the
importance of redox properties of pV compounds in inhibition of protein
phosphatases [28,101] which lend credence to our hypothesis.
4. Conclusions
The present work afforded a set of water soluble pNb complexes which
contain species familiar to biological environment as co-ligand. The spectral and
chemical data obtained have provided satisfactory evidences for the composition of
the ligand sphere and the likely mode of ligation to the Nb(V) centre, which has
been substantiated by the results of DFT calculations performed on the complexes.
Our experiments confirm that the pNb complexes tested, induce strong inhibitory
effect on acid phosphatase. Detailed inhibition kinetics studies clearly
demonstrated that each of the compounds is a potent mixed-type of inhibitor of the
enzyme, combining competitive and non-competitive modes of inhibition. The
remarkable stability of the compounds in aqueous solution of a wide range of pH
values, their ability to partially retain the coordinated peroxo groups even beyond 1
h of incubation with the ROS mopping enzyme catalase, are other distinctive
attributes of the compounds.
24

Acknowledgements
Financial support from the Department of Biotechnology, New Delhi, India, is
gratefully acknowledged. We are thankful to Dr. K. K. Bania, Department of
Chemical Sciences, Tezpur University, for help in theoretical investigation and
Dr. Gayatri Ramakishna, Cancer Biology Laboratory, Centre for DNA
Fingerprinting and Diagnostics (CDFD), Hyderabad for valuable discussion. We
are grateful to the Council of Scientific and Industrial Research (CSIR), New
Delhi, India for providing Senior Research Fellowship to S.R.G.
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