Kinetics and mechanism of oxidation of hydroxyurea derivatives with hexacyanoferrate(III) in aqueous solution

Article abstract of DOI:10.5562/cca1799

Kinetics and mechanisms of the oxidation of methoxyurea and N-methylhydroxyurea were studied in neutral and basic aqueous solutions. The obtained pH dependences of the oxidation rates indicate that for both hydroxyureas the reactive species are the deprotonated ones. The second order rate constants, the activation enthalpies and the activation entropies for the reactions of methoxyurea (O-methylhydroxyurea) and N-methylhydroxyurea anions with Fe(CN)₆^3- at 25 °C, I = 2 mol dm^-3 (NaClO₄) were determined as (5.06 ± 0.01) × 10 ² mol^-1 dm³ s^-1, (1.92 ± 0.02) × 10⁴ mol^-1 dm³ s^-1, 27 ± 1 kJ mol^-1, 16 ± 1 kJ mol^-1,.101 ± 2 J mol^-1 K^-1, and.107 ± 4 J mol^-1 K^-1, respectively. The pKa value of methoxyurea at 25 oC and 2 mol dm-3 ionic strength was determined kinetically as 12.7 ± 0.1 and the thermodynamic parameters for the deprotonation reaction were determined as Δ_aH = 43 ± 1 kJ mol^-1, and.aS =.96 ± 4 J mol^-1 K^-1. When the kinetic results are compared with the data reported for hydroxyurea, an inverse dependence of the rate constants on the pKa of the hydroxyurea derivatives at 25 °C is observed. Such unexpected behaviour has been explained by the ab initio calculations and NBO analysis of HOMOs for all three hydroxyureates.

Full text of DOI:10.5562/cca1799

CROATICA CHEMICA ACTA
CCACAA, ISSN 0011-1643, e-ISSN 1334-417X
Croat. Chem. Acta 84 (2) (2011) 133–147.
CCA-3460
Original Scientific Article
Kinetics and Mechanism of Oxidation of Hydroxyurea Derivatives with
Hexacyanoferrate(III) in Aqueous Solution†
Ana Budimir, Tin Weitner, Ivan Kos, Davor Šakić,‡ Mario Gabričević,
*
Erim Bešić, and Mladen Biruš
University of Zagreb, Faculty of Pharmacy and Biochemistry, A. Kovačića 1, 10000 Zagreb, Croatia
RECEIVED NOVEMBER 17, 2010; REVISED JANUARY 4, 2011; ACCEPTED JANUARY 20, 2011
Abstract. Kinetics and mechanisms of the oxidation of methoxyurea and N-methylhydroxyurea were stud-
ied in neutral and basic aqueous solutions. The obtained pH dependences of the oxidation rates indicate
that for both hydroxyureas the reactive species are the deprotonated ones. The second order rate cons-
tants, the activation enthalpies and the activation entropies for the reactions of methoxyurea (O-methylhy-
droxyurea) and N-methylhydroxyurea anions with Fe(CN)6 at 25 C, I = 2 mol dm−3 (NaClO4) were
determined as (5.06 ± 0.01)  102 mol−1 dm3 s−1, (1.92 ± 0.02)  104 mol−1 dm3 s−1, 27 ± 1 kJ mol−1,
16 ± 1 kJ mol−1, 101 ± 2 J mol−1 K−1, and 107 ± 4 J mol−1 K−1, respectively. The pKa value of methoxy-
3−
o
o
urea at 25 C and 2 mol dm−3 ionic strength was determined kinetically as 12.7 ± 0.1 and the thermody-
namic parameters for the deprotonation reaction were determined as ΔaH = 43 ± 1 kJ mol−1, and
ΔaS = 96 ± 4 J mol−1 K−1. When the kinetic results are compared with the data reported for hydroxyurea,
o
an inverse dependence of the rate constants on the pKa of the hydroxyurea derivatives at 25 C is ob-
served. Such unexpected behaviour has been explained by the ab initio calculations and NBO analysis of
HOMOs for all three hydroxyureates. (doi: 10.5562/cca1799)
Keywords: methoxyurea, N-methylhydroxyurea, hexacyanoferrate, oxidation, kinetics, NBO, quantum
chemical study
INTRODUCTION
pathophysiological properties of HU.7 It was shown that
the formation of iron nitrosyl hemoglobin from the
Hydroxyurea (HU) (Chart 1) is a highly specific low
molecular weight inhibitor of ribonucleotide reductase,
and, therefore, of DNA synthesis,1 with a broad spec-
trum of anti-tumor effects,2 which also effectively im-
proves clinical outcomes in patients with sickle cell
disease.3,4
reaction of hydroxyureas and hemoglobin requires an
unsubstituted −NHOH group and that the nitrogen atom
of the non-N-hydroxy group must contain at least a
single hydrogen atom.8 It was also reported that the
selected hydroxyureas are oxidized by lipoxygenase to
form their corresponding nitroxides.9
Hydroxyureas belong to the family of hydroxamic
acids that may act as nitric-oxide donors under oxidative
conditions in vitro.5,6 In reactions with hemoglobin,
myoglobin, or hemin, NO may contribute to the overall
On the other hand, investigation of inhibitory ef-
fects of nitro-vasodilators and HU on DNA synthesis in
cultured human aortic smooth muscle cells indicates
that NO does not mediate the inhibitory action of HU in
this system.10 In line with that observation, we have
recently published papers dealing with the oxidation of
3−
HU by dioxovanadium(V) and Fe(CN)6 ions, which
have revealed that the oxidation of HU with those two
oxidizing agents does not afford the formation of
NO.11,12 These studies are extended in this work by the
investigation of the reaction mechanism of reduction of
the Fe(CN)63− ion13 with two derivatives of hydroxyurea
shown in the Chart 1, viz. N-methylhydroxyurea
Chart 1. Structures of hydroxyureas: hydroxyurea (HU): R1 =
R2 = H, N-methylhydroxyurea (NMHU): R1 = CH3, R2 = H,
and N-methoxyurea (MetU): R1 = H, R2 = CH3
†
This article belongs to the Special Issue Chemistry of Living Systems devoted to the intersection of chemistry with life.
Taken in part from Davor Šakić’s PhD Thesis.
‡
* Author to whom correspondence should be addressed. (E-mail: birus@pharma.hr)

134
A. Budimir et al., Kinetics and Mechanism of Oxidation of Hydroxyurea Derivatives
(NMHU) and methoxyurea (MetU).
urements buffered solutions of K3[Fe(CN)6] and NMHU
The hydroxamic acids contain the smallest
O=CNH unit that can bind to the DNA helix,14,15 and the
observed biological activities of hydroxamic acids
might be related to the similarity between the O=CNH
unit and analogous structural segments found in pro-
teins.16 Hence, knowledge of the favoured ionization
sites is important for understanding the role played by
hydroxamic acids in biological processes, but also in
metal ion complexation, since these processes could be
interrelated. A large number of papers dealing with
theoretical and experimental aspects of hydroxamic
acids’ acidity have been published,17 among which we
have recently published a thermodynamic study of the
acid-base properties of HU and NMHU.18 It was con-
cluded that the electronic properties of the functional
groups, R1 and R2, play a significant role in the ioniza-
tion of hydroxamic acids,19 but an unresolved dispute
whether hydroxamic acids in water deprotonate at the
oxygen or the nitrogen atom of hydroxamic moiety is
still open for debate.
Since the redox active species of hydroxyureas are
found to be their anions, and the pKa values of hydroxy-
ureas are necessary for interpretation of the redox kinet-
ics, herein we also report determination of the ionization
constant of MetU. In order to confirm the identity of the
favoured ionization site in MetU (NH2 or NHOCH3),
we also report the quantum chemical calculations of its
electronic structures and energies. Thus, besides im-
proving our understanding of the reaction mechanism of
oxidation of the hydroxyurea derivatives, this work also
advances understanding of the acid-base properties of
hydroxamic acids in general, presenting our contribu-
tion to answer the above question by studying MetU
molecule that has no –OH site available for deprotona-
tion.
were prepared by addition of appropriate volumes of
stock solutions of metal or ligand and buffer, except in
measurements with HEPES because of a slow reaction
of HEPES and K3[Fe(CN)6] observed by a yellowish
colour of K3[Fe(CN)6] disappearing within a few hours
(depending on the concentration of K3[Fe(CN)6]). These
solutions were prepared by the addition of double
amounts of buffer to NMHU solutions. In all solutions
pH was adjusted by the addition of a standard NaOH
solution. The ionic strength was maintained constant
with NaClO4. Care was taken in the preparation and
manipulation of hexacyanoferrate(III) solutions in order
to prevent decomposition upon light exposure and long
standing. Fresh solutions of hexacyanoferrate(III) were
prepared before each experiment and their concentra-
tions were calculated using the molar absorptivity of
1050 mol−1 dm−3 cm−1 at 420 nm.
Synthesis of Hydroxyureas
NMHU was prepared by drop wise addition of 5.125 g
KCNO (0.063 mol) dissolved in 50 ml water to solution
of 5.25 g (0.063 mol) N-methyl hydroxylamine hydro-
chloride dissolved in 50 ml of methanol. The solution
was mixed over night at room temperature and the sol-
vent was removed under reduced pressure afterwards.
The remaining oil was dissolved in diethyl-ether and
filtered to remove KCl. Diethyl-ether was then removed
under reduced pressure and the remaining oily sub-
stance began to crystallize in a vacuum desiccator over
P2O5. Crystallization and filtration from ethylacetate
1
gave the pure product (yield 4.465 g, 79 %, H-NMR
(DMSO-d6) δ/ppm: 9.27, 6.27 and 2.95; 13C-NMR
(DMSO-d6) δ/ppm: 162.45 and 38.26). Methoxyurea
was prepared according to published procedure and was
o
checked for purity by its melting point (81−84 C, lit.
value: 84 oC)20 and spectroscopic data: IR (KBr) ν/cm−1:
EXPERIMENTAL
Materials
3402, 3220, 2170, 1670, 1598, 1474, 1440, 1416, 1194,
1
1128, 1092, 810, 712, 602, 564; H NMR (DMSO-d6)
δ/ppm: 9.01 (NH), 6.40 (NH2), 3.52 (CH3); 13C NMR
(DMSO-d6) δ/ppm: 161.82 (C=O), 64.40 (CH3).
All chemicals used were commercially available, of
reagent grade and were used without further purifica-
tion. All water used was deionized and then twice dis-
tilled in an all-glass apparatus, first from an alkaline
solution of KMnO4. All solutions were prepared with
twice distilled and deionized water boiled for 1 hour and
cooled down under argon atmosphere (purified by a
SIGMA OXICLEAR cartridge) in order to exclude CO2 and
O2. NaClO4 was synthesized by addition of HClO4 to a
water solution of NaHCO3 until neutralized, then fil-
tered and recrystallized. A 7.2 mol dm−3 stock solution
of NaClO4 was standardized by passage through Amber-
lite IR120 strong acid cation exchange column in the H+
form, and titrated against standard NaOH. In all meas-
Instrumental Measurements
The UV-Vis absorption spectra were recorded on a
VARIAN CARY 50 spectrophotometer. Kinetic measure-
ments were performed by using an OLIS USA RSM
1000 stopped-flow spectrophotometer coupled to an on-
line data acquisition system and equipped with thermo-
statted cell compartment. The kinetic traces were evalu-
ated using the OLIS GLOBALWORKS program. Tem-
perature control was maintained with the use of a HA-
AKE DC10-K10 refrigerated circulator bath with tem-
perature accuracy of 0.1 C over the range 10−100 C.
Absorbance changes during the kinetics were followed
Croat. Chem. Acta 84 (2011) 133.

A. Budimir et al., Kinetics and Mechanism of Oxidation of Hydroxyurea Derivatives
135
at 420 nm, and each experimental rate constant reported
herein is an average of at least 5 runs carried out. pH
was measured using Mettler Toledo MP220 pH-meter
with InLab 413 combination electrode. Differences in
pH values before and after reaction were negligible. The
electron paramagnetic resonance (EPR) spectra were
recorded at room temperature with a Bruker Elexsys
E500 X-band spectrometer for both aqueous and heavy-
water solutions. The spectra were recorded immediately
after mixing Fe(CN)63− with NMHU.
the harmonic frequencies at the same levels of theory.
Solvent effects in water have been calculated by
means of the polarized continuum model with po-
larisable conductor parameters (CPCM)27 in which the
solvent is considered as a continuum dielectric, charac-
terized by a constant permittivity. The CPCM method
was directly applied to the neutral and anionic forms of
MetU, as implemented in the Gaussian 09 package,
using the same level of theory. The cavity radii were
those recommended for the UAHF model.28 The relative
solvent dielectric constant at 298.15 K has been set at
78.5 (Gaussian 09 default for water). Thus obtained
minima were then optimized at B2PLYP/6-311+
G(d,p),29 MP2/6-311+G(d,p)30 and G3B331 methods
with hydration effects calculated by the CPCM method
on the same levels of theory, except for G3B3, where
the hydration energy was computed on B3LYP/6-
311++G(d,p) level of theory. B3LYP was selected as
the most frequently used density functional theory
which has good concurrence with experimental results
and is a fairly cheap and fast method for large sets of
starting structures. MP2 method was selected as the
most used Møller-Plesset method, and B2PLYP was
used as a representative of the double hybrid method
which combines exact HF exchange with an MP2-like
correlation to a DFT calculation. G3B3 method involves
complex energy computations including several pre-
defined calculations on the specified molecular system
and is the most expensive in computer clock-time.
Computational Details
The Gaussian 09 program package21 has been em-
ployed in all the calculations. Initial geometries and
their different rotamers were built using Avogadro
program package.22 Initial geometries of the water
clusters of MetU species were generated by stochastic
search procedure, namely a modified Saunder's kick
procedure,23 including two important modifications: (i)
use of dynamic molecular fragments (viz. water mole-
cules) that are randomly rotated, and then kicked
around a stationary molecular fragment (viz. the MetU
species), versus the original all-atom displacement
approach (Addicoat and Metha24 also made the same
modification), and (ii) constraint of the kick relative to
the position of randomly selected atom of stationary
molecular fragment, thus avoiding the necessity to
define a box in which the kick is allowed. In other
words, defining the "kick-size" and "box-size" (e.g.
maximum displacement of atom from starting position
in specified space as defined by Saunders) is now
replaced with a value of maximum displacement of
dynamic molecular fragment from a randomly selected
atom of the stationary structure. Water molecules were
placed randomly in a variety of locations to sample the
various arrays of hydrogen-bonding networks available
between MetU/water and water/water molecules, re-
ducing the possible errors caused by the limitations of
our “chemical intuition”.
Batches of initial geometries were prepared, each
consisting of at least 100 different starting geometries.
At least three batches of every investigated cluster were
submitted for first optimization using the Becke three
parameter Hybrid functionals with Lee-Yang-Parr cor-
relation method25 with Pople’s 6-31+G(d) basis set26
(B3LYP/6-31+G(d)) without any conformational or
symmetry constraints. The five lowest-energy distinct
minima from each batch were compared and if no new
original minima were found it was assumed that a
global minimum had been found. Otherwise, if a new
distinct minimum with lowest energy was found, a new
batch was created and submitted in the same way. The
claim of the found global minimum was further tested
by a hand-modification of geometries while the nature
of the stationary point was verified by computations of
Natural Bond Analysis32 was performed to deter-
mine charge distribution and molecular orbital composi-
tion and shape, using NBO version 3, as implemented in
Gaussian 09 program package. For visualization of
orbitals Avogadro program package was used.
RESULTS
General Observations
After mixing the reactants in an aqueous solution within
a few milliseconds mixing time, a spectral maximum at
3−
420 nm, where only Fe(CN)6 absorbs the light, fades
out on stopped-flow time scale. The first recorded UV-
Vis spectrum of the reaction mixture corresponded to
Fe(CN)63−, whereas the spectrum measured after the
4−
reaction was completed corresponded to Fe(CN)6
(Figure S1). Since on that time-scale [Fe(CN)6]3− does
not undergo the observable aquation reaction,33 the
observed colour fading can be attributed to the reduction
3−
of Fe(CN)6 with hydroxyureas. A single exponential
decay Eq. (1) of the measured absorbance vs. time (Fig-
ure 1) was successfully fitted to the experimental data
(A is the total absorbance change while At and Ainf. are
the absorbance at time t and infinite time, respectively)
implying first-order dependence with respect to the
3−
absorbing species (counting here Fe(CN)6 and its
Croat. Chem. Acta 84 (2011) 133.

136
A. Budimir et al., Kinetics and Mechanism of Oxidation of Hydroxyurea Derivatives
Plots of absorbance against the mixing ratio first
showed a linear decrease and then constant values
describing a horizontal line. From the intersection of
straight lines shown in Figure 2, the stoichiometric
molarity ratio for NMHU (like for HU)12 is found to be
1:2 = NMHU:Fe(CN)63−, whereas for MetU the found
ratio is 1:1. These results indicate that, contrary to HU
and NMHU, which act as two-electron donors, MetU
acts as a one-electron donor in the reduction of
3−
Fe(CN)6
.
Free Radical Formation
EPR spectra of a free radical formed in the reaction of
Fe(CN)63− with NMHU in water and D2O were found to
be identical to the analogous spectra recorded when
NMHU was oxidized with dioxovanadium(V).34 An
attempt to record a free radical generated from MetU in
Figure 1. A typical kinetic trace of reaction between
Fe(CN)6 and NMHU followed at λ = 420 nm. Conditions:
c(K3Fe(CN)6) = 0.5 mmol dm−3, c(NMHU) = 2 · 10−2 mol
dm−3, pH = 8, c(HEPES) = 0.2 mol dm−3, I = 2 mol dm−3,  =
(25  0.2) C. Inset: the linear dependence of ln(A − Ainf.) vs.
time confirming the first-order condition.
3−
3−
reaction with Fe(CN)6 at room temperature by the
EPR spectroscopy technique failed, most probably due
to a too low concentration of the formed free radical.
Therefore, involvement of a free radical in that reaction
was assessed by initiating the polymerization of acryla-
mide: 0.09 g MetU, 0.033 g K3Fe(CN)6 and 0.39 g of
acrylamide were placed in a dry test tube and dearated
with Ar for 15 minutes. The test tube was sealed and 5
ml of degassed water was added with a syringe and
stirred for 10 min. This reaction mixture was added to a
double volume of cold methanol with the result of pro-
gressive formation of a white solid across the whole
solution. When the experiment was repeated in the ab-
sence of MetU or K3Fe(CN)6 under the same conditions
no white solid was formed.
outer-sphere ion-pairs).
A  Aexp k t  Ainf .
(1)


t
obs
In experiments dealing with NMHU buffer solu-
tions were prepared from MES and HEPES to cover the
whole investigated pH range. No significant buffer con-
centration dependence of kobs was observed (Figure S2).
Stoichiometry of the Redox Reactions
Mixtures having increasing hydroxyureas:Fe(CN)6
3−
molar ratios were allowed to react to completion, and
the reaction stoichiometries were determined spectro-
photometrically by measuring the 420 nm absorbance
of reaction solutions due to unreacted Fe(III) species.
Kinetics of oxidation
As mentioned above, the kinetics of the redox reaction
Figure 2. Absorbance of the solutions equilibrated at (25  0.1) °C of (left) NMHU and K3Fe(CN)6 as a function of
3−
NMHU/Fe(CN)6 molar ratio. Conditions: c(K3Fe(CN)6) = 1 mmol dm−3, 0.1 mol dm−3 NaOH, I = 2 mol dm−3 (NaClO4), l = 1
3−
cm, and (right) MetU and K3Fe(CN)6 at 420 nm as a function of MetU/Fe(CN)6 molar ratio. Conditions: c(K3Fe(CN)6) = 0.5
mmol dm−3, 0.01 mol dm−3 NaOH, I = 2 mol dm−3 (NaClO4), l = 1 cm.
Croat. Chem. Acta 84 (2011) 133.

A. Budimir et al., Kinetics and Mechanism of Oxidation of Hydroxyurea Derivatives
137
Figure 3. Plots of kobs vs. initial [K3Fe(CN)6] for redox reaction between Fe(CN)63− and hydroxyurea derivatives. (left) NMHU in
a molar excess over Fe(CN)63− at pH = 10. Conditions: c(NMHU) = 0.03 mol dm−3, c(buffer) = 0.2 mol dm−3, I = 2 mol dm−3,  =
(25  0.2) °C and (right) Fe(CN)63− in a molar excess over MetU at pH = 12. Conditions: c(MetU) = 0.03 mmol dm−3, I = 2 mol
dm−3 (NaClO4),  = (25  0.2) C.
3−
between Fe(CN)6 and hydroxyureas were studied
using the stopped-flow technique, monitoring a decrease
Fe(CN)63− was in molar excess over hydroxyurea deriva-
tives and in the second series the ratio was reversed. In
the latter case the obtained pseudo-first order rate con-
3−
in absorbance of Fe(CN)6 at 420 nm with time (under
3−
the pseudo-first order conditions and constant ionic
strength) to which the expression (1) was fitted.35 If an
inner-sphere species had been created, a bathocromic
shift of the absorption band, associated with the ligand-
metal charge transfer, could have been expected. An
absence of such a shift in the final UV-Vis spectrum,
which coincides with spectrum of Fe(CN)64−, proves
that this is the reduction product.
stants are independent of the Fe(CN)6 concentration,
while in the former case they exhibit a linear dependence
3−
on the initial Fe(CN)6 concentration (Figure 3). These
facts clearly demonstrate the first order dependence of
the redox reactions with respect to the concentration of
this reactant.
Effect of the Initial Concentration of Hydroxyureas
With the hydroxyurea derivatives in a large molar excess
over Fe(CN)63−, linear dependencies of the calculated
pseudo-first order rate constants, kobs, on the concentra-
tion of both hydroxyureas were observed (Figure 4). The
slopes at two different proton concentrations differ
significantly. The larger slopes at the higher base con-
Effect of the Initial Fe(CN)63− Concentration
In order to determine the rate order with respect to hexa-
cyanoferrate(III) concentration, two series of kinetic
measurements were carried out in which only the initial
concentration of K3Fe(CN)6 was varied. In the first series
Figure 4. Dependence of kobs of oxidation of hydroxyurea derivative on its concentration. (left) Conditions: c(K3Fe(CN)6) = 0.5
mmol dm−3, c(buffer) = 0.2 mol dm−3, I = 2 mol dm−3 (NaClO4),  = (25  0.1) °C. (right) Conditions: c(K3Fe(CN)6) = 0.25 mmol
dm−3, I = 2 mol dm−3 (NaClO4),  = (25  0.1) C.
Croat. Chem. Acta 84 (2011) 133.

138
A. Budimir et al., Kinetics and Mechanism of Oxidation of Hydroxyurea Derivatives
Figure 5. Dependence of the observed pseudo-first order rate constants (kobs) on the acidity of aqueous solutions. (left) Conditions:
c(NMHU) = 5 mmol dm−3, c(K3Fe(CN)6) = 0.2 mmol dm−3, c(buffer) = 50 mmol dm−3, I = 2 mol dm−3 (NaClO4),  = (25  0.1) °C.
(right) Conditions: c(MetU) = 7.5 mmol dm−3, c(K3Fe(CN)6) = 0.25 mmol dm−3, I = 2 mol dm−3 (NaClO4),  = (25  0.1) C.
centration are indicative of a more reactive anionic
species in comparison with the molecular forms. Hence,

 

 

kobs  a OH / 1 b OH
(3)





the dependence of the oxidation rates on the acidity of
the aqueous solution was studied with the reductants in
a large molar excess over hexacyanoferrate.
As shown in Figure 5 (left), a sigmoid dependence
was obtained for NMHU, which can be expressed by
equation (2),
which is equivalent to Eq. (4), i.e. the reduced form of
kinetic expression (2).

 

kobs  k K MetU
/
H
 Ka
(4)
a 



2
tot


 

 
kobs  k H k2 Ka NMHU / H  Ka
(2)






1
The parameters a and b in Eq. (3) equal the ratios
k2Ka[MetU]tot/Kw and Ka/Kw in Eq. (4), respectively.37


tot


Assuming Kw = 1.07  10−14 at 25 C and I = 2 mol
o
where kobs is the observed pseudo-first order rate con-
stant, k1 is the second order rate constant for the reaction
dm−3,38 Ka and k2 were calculated as 2.14(6) × 10−13
mol−1 dm3 and 5.06(1)  102 mol−1 dm3 s−1, respectively.
The calculated Ka value of MetU is approximately three
orders of magnitude smaller than the Ka values of other
two hydroxyureas studied. An attempt to determine the
Ka value by the combined pH-spectrophotometric titra-
tion failed, most probably due to the pKa value outside
the range of the glass electrode reliability.
3−
of Fe(CN)6 with protonated species of NMHU, k2 is
the second order rate constant for the reaction of
3−
Fe(CN)6 with deprotonated species of NMHU, and Ka
is the deprotonation equilibrium constant of NMHU
(paths assignation according to Scheme 1). Assuming a
first order with respect to the NMHU concentration, the
calculated values are: k1 = 1(1)  102 mol−1 dm3 s−1, k2 =
1.92(2)  104 mol−1 dm3 s−1, and pKa = 9.82(2).36 The
calculated pKa value is very similar to the value deter-
mined potentiometrically under the same experimental
conditions (pKa = 9.79,18 I = 2 mol dm−3 NaClO4,  = 25
°C). Because of a large standard deviation obtained for
k1, the reaction of Fe(CN)63− with the protonated species
should be considered as rather dubious. Nevertheless,
even if the calculated value were considered as an upper
limit, k2 would be at least three orders of magnitude
larger than k1. The observed difference can be explained
by the build-up of negative charge upon the deprotona-
tion of NMHU.
Effect of the Initial Fe(CN)64− Concentration
The effect of the concentration of hexacyanoferrate(II)
ions, as one of the reaction products initially added to
the reaction solutions, on the rate of the redox reaction
was also studied. The concentrations of hexacyanofer-
rate(II) ions were varied up to 1 mmol dm−3 at fixed
concentrations of the hydroxyurea derivatives,
Fe(CN)63− and NaOH, and at constant ionic strength I =
2 mol dm−3 (NaClO4). The rate remained constant upon
changing the concentration of hexacyanoferrate(II) ions,
thus ruling out “reversibility” of the rate determining
electron-transfer step. The observed dependence of rate
constants on the initial concentration of K4Fe(CN)6 is
presented in Figure S3.
A nonlinear dependence of kobs on hydroxide con-
centration obtained for MetU (Figure 5, right side) can
be expressed by equation (3),
Croat. Chem. Acta 84 (2011) 133.

A. Budimir et al., Kinetics and Mechanism of Oxidation of Hydroxyurea Derivatives
139
as the activation parameters for the redox reaction as
follows: ΔaH = +43(1) kJ mol−1, ΔaS = 96(4) J K−1
mol−1, ΔH‡ = 27(1) kJ mol−1, and ΔS‡ = 101(4) J K−1
mol−1.
It is obvious that since NMHU has pKa = 9.7, the
anionic form of NMHU is the dominant reactant in 0.01
M NaOH. Accordingly, as shown by levelling off in
Figure 5 (left), a slight change of pH caused by the dis-
solved NMHU and variation in temperature does not
affect the reaction rate. Therefore, the observed rate con-
stant corresponds to k2, and the Eyring plot shown in
Figure S4 should relate directly to the kinetics of the
redox reaction alone. The obtained activation parameters
are: ΔH‡ = 16(1) kJ mol−1 and ΔS‡ = 107(4) J K−1 mol−1.
Effect of the Ionic Strength
The observed rate constant for both hydroxyureas was
found to be strongly increased upon the increase of
ionic strength of the reaction medium (Figure 8), clearly
pointing to a reaction between similarly charged ions.
Since the experiments were carried out at a high ionic
strength, the extended Bjerrum–Brønsted equation was
used. However, while for MetU a nonlinear dependence
was obtained, for NMHU a linear dependence of log kobs
Figure 6. Dependence of the observed pseudo-first order rate
constants (kobs) on the acidity of aqueous solutions at (from
bottom) 10.8, 15.0, 20.2, 25.0, 29.7, 35.0 and 40.2 C. Condi-
tions: c(MetU)= 7.5 mmol dm−3, c(K3Fe(CN)6) = 0.25 mmol
dm−3, I = 2 mol dm−3 (NaClO4).
Effect of Temperature
Since the obtained pKa of MetU is 12.67, slight varia-
tions in pH and pKa with temperature should affect the
value of the observed rate constant measured as a func-
tion of temperature. Hence, the temperature effect on
the kinetics of the latter reaction had to be measured
simultaneously as a function of temperature and the
OH− concentration (Figure 6).
Ka and k2 were calculated from the data shown in
Figure 6, taking into account that within the studied
temperature range the ionic product of water increases
with temperature.39 The slopes and intercepts of plots
shown in Figure 7 afforded calculation of the reaction
enthalpy and entropy for deprotonation of MetU as well
vs. I 0.5(1+ I 0.5 −1 was obtained, as in the case of HU.12
)
The effect of ionic strength on Ka, and in turn on the rate
constant according to Eq. (4), becomes negligible only
for [H+] < Ka, since in that case the rate expression re-
duces to k2. In the case of NMHU, which has a pKa of
9.7, a 0.01 mol dm−3 solution of NaOH buffers the reac-
tion solution exactly in that range. Although somewhat
unexpected, the obtained linearity for HU and NMHU
could be justified by the annulations of certain contra-
effects on the reaction rate, and considering the charge
of the hydroxamate ion (–1) and the calculated slopes
(+1.5) for the reaction of NMHU, the hexacyanoferrate
charge can be speculated as 1.5. This can be explained,
Figure 7. Dependence of the ln Ka and ln (k2/T) on the inverse temperature. Conditions: c(K3Fe(CN)6) = 0.5 mmol dm−3, c(NaOH)
= 0.01 M, I = 2 mol dm−3. (left) c(MetU) = 7.5 mmol dm−3, (right) c(MetU) = 10 mmol dm−3.
Croat. Chem. Acta 84 (2011) 133.

140
A. Budimir et al., Kinetics and Mechanism of Oxidation of Hydroxyurea Derivatives
Figure 8. Dependence of the observed pseudo-first order rate constants (kobs) on the ionic strength. (left) Conditions:
c(K3Fe(CN)6) = 0.5 mmol dm−3, c(NMHU) = 5 mmol dm−3, pH = 12.0,  = (25  0.1) C; (right) Conditions: c(K3Fe(CN)6) = 0.25
mmol dm−3, c(MetU) = 7.5 mmol dm−3, pH = 12.0,  = (25  0.1) C. The theoretical curve of the right side figure is defined as:
log kobs = 1.05 + 2.62x  2.00x2, where x = I 0.5 (1 + I 0.5)−1.
as in the case of HU,13 by the formation of
The pKa values were calculated using equation (5),
pKa   G / RT ln10 – logcH O
Na+ ..Fe(CN)63− ionic associates.
x
The nonlinearity observed for MetU in the plot of
log kobs vs. I0.5(1+I0.5)−1 could be caused by a simultane-
ous effect of ionic strength on the ionization of MetU.
Such an effect cannot be eliminated by working within
the pH range where the anionic form dominates because
of a very high pKa value of MetU. According to Eq. (4),
the observed rate constant is essentially a product of
multiplication of the r.d.s. oxidation rate constant by a
fraction of the anionic form of methoxyurea (= Ka([H+] +
Ka)–1). Since in 0.01 mol dm−3 aqueous NaOH the ani-
onic form of MetU does not entirely predominate, the
ionic strength could affect its fraction by changing the
value of deprotonation constant. By combining the ex-
tended Debye-Hückel and Bjerrum-Brønsted equations
for k2 and Ka, the observed rate constant dependence on
ionic strength is rather complex and can be expressed as:
(5)


a
2
whereas the reaction Gibbs free energy for acid ioniza-
tion in water, ΔaG(aq.), was defined by equations (6) and
(7).
aG(aq.)  aG(g)  solv.GA (aq.)  solv.G
+ (aq.)
H3O
(6)
 solv.GHA(aq.)  solv.GH2O(aq.)
aG(g)  GA (g)  G  (g) GHA(g) GH O(g)
(7)
H3O
2
The respective standard Gibbs free energies of
H2O(g) and H3O+(g), and the energies of their solvation
were calculated at the B3LYP/6-31+G(d) theory level.
The calculation afforded the value of 1134.2 kJ mol−1
for the hydrated H+ ion, which favourably compares to
the reported value of 1129.6 kJ mol−1.40
The gas phase Gibbs free energies calculated at
the B3LYP/6-31+G(d) level reveal that the molecular
and anionic forms of MetU are mixtures of trans and cis
conformations. The calculated ΔG for the deprotonation
at amine (NHC) site was ca. 28 kJ mol−1 larger than for
the deprotonation at hydroxylamine (CNO) site. This
corresponds to ca. 5 units larger pKa for the NHC site in
the gas phase, indicating that only the deprotonation at
the CNO site should be considered.
log kobs = log Kak2 = –2Q(1 + ZAZB)I0.5 (1 + I0.5)−1
+
constant. At 25 oC, Q has a value of 0.51, so log kobs  –
(1 + ZAZB)I0.5 (1 + I0.5)−1 + constant. Additionally, since
at the high ionic strengths the Bjerrum-Brønsted equa-
tion and the Debye-Hückel limiting law fail, the only
meaningful parameter easy to obtain from Figure 8
(right) is the limiting slope. Its value is 2.62, which ac-
cording to the above equation is in agreement with the
hexacyanoferrate species bearing three negative charges,
and a strong ion-pair formation of this ion with Na+ ion
even at the lowest ionic strength used in this study.
Computation of the Thermodynamic Parameters for
Deprotonation of MetU
Furthermore, calculation of the hydration energy
of amidic monosubstituted anions (R−NH−) proved to
be a very difficult task in general. For the anions of
acetamide (−920.6 kJ/mol), urea (−2568.1 kJ mol−1),
hydroxyurea (−1598.8 kJ mol−1), methylamine (−2579.3
B3LYP method was used throughout this study for the first
optimizations and energy calculations, and then B2PLYP
and G3B3 were used on the optimized geometries.
Croat. Chem. Acta 84 (2011) 133.

A. Budimir et al., Kinetics and Mechanism of Oxidation of Hydroxyurea Derivatives
141
Table 1. Calculated standard Gibbs free energies, Gtot, for various forms of MetU at 298.15 K calculated in the gas phase and
water solution at various levels of theory
rG*
kJ mol1
G
tot / a.u.
% of cis or weighted average
trans form tot / a.u.
pKa
species
pK
(CPCM, 298 K)
G
(= pK − log 55.6)
B3LYP/6-31+G(d)
cis-H2NC(=O)NHOCH3
trans-H2NC(=O)NHOCH3
−339.8464782
−339.8492683
−339.3861657
−339.3856213
−76.9120724
−76.4800820
5
−339.84913
95
64
36
81.8
14.34
12.59
12.74
11.58
−
cis-H2NC(=O)NOCH3
−339.38597
−
trans-H2NC(=O)NOCH3
H3O+
H2O
G3B3
cis-H2NC(=O)NHOCH3
trans-H2NC(=O)NHOCH3
−339.58325409
−339.58508721
−339.10261665
−339.10127746
−76.87160210
−76.42059553
13
87
81
19
−339.58486
−339.10236
82.69
14.48
−
cis-H2NC(=O)NOCH3
−
trans-H2NC(=O)NOCH3
H3O+
H2O
B2PLYP/6-311+G(d,p)
cis-H2NC(=O)NHOCH3
trans-H2NC(=O)NHOCH3
−339.63404423
−339.63611679
−339.17104899
−339.16945147
−76.88569932
−76.44956552
10
−339.63591
−339.17080
90
84
16
76.07
13.33
−
cis-H2NC(=O)NOCH3
−
trans-H2NC(=O)NOCH3
H3O+
H2O
(a) The reaction free energy (given in kJ mol−1) corresponding to: HA + H2O ⇄A− + H3O+
kJ mol−1) and ammonia (−1144.1 kJ mol−1) calculations
afforded similar, unexpectedly large hydration energies.
These values are much larger than the ones calculated
for amidic disubstituted anions (R1R2N−) species, where
nitrogen is bonded to two fragments, as in anions of
N-methylacetamide (−284.6 kJ mol−1), N-methyl-
hydroxyurea (−288.6 kJ mol−1), N-methylmetoxyurea
(−311.5 kJ mol−1), and N1,N3-dimethyl-urea (299.2 kJ
mol−1) (Table S1). The calculated free energy of hydra-
tion for the MetU anion deprotonated at the NHC site
was calculated to be at least 140 kJ mol−1 larger than for
the anion deprotonated at the CNO site. Therefore, only
the CNO deprotonation site was considered further in
this paper.
Hydration of different conformations of neutral
and anionic forms of MetU was done treating the water
both implicitly as included in CPCM methods and as a
discrete solvent molecule combined with CPCM meth-
ods. On B3LYP/6-31+G(d) level of theory (Table 1),
when the Boltzmann distribution was taken into ac-
count, pKa was calculated as 12.54, which is in excellent
agreement with the experimentally obtained value. Us-
ing a model with one discrete water molecule added, the
calculated value of pKa was 14.84 (Table S2). The low-
est minima computed at B3LYP/6-31+G(d) level were
then used as a starting structures for G3B3 and B2PLYP
calculations. G3B3 afforded a value pKa = 12.73 for
implicitly included effect of water, but much smaller
pKa = 10.00 for explicitly added one water molecule.
B2PLYP afforded pKa = 11.55 for implicitly included
effect of water and pKa = 15.16 for explicitly added one
water molecule.
Comparing all three methods we can safely as-
sume that the implicit model, without the addition of a
discrete water molecule, is a satisfactory model for the
calculations on MetU with Gaussian09. B3LYP and
G3B3 gave similar results, while B2PLYP underesti-
mated pKa when no water molecule was explicitly added
and overestimated when one discrete water molecule
was added. The difference between the pKa values esti-
mated from the implicit model and the model with one
water molecule explicitly added can be attributed to the
over/underestimation of entropy effects by Gaussian
program.41 Therefore, the computations reveal that the
Croat. Chem. Acta 84 (2011) 133.

142
A. Budimir et al., Kinetics and Mechanism of Oxidation of Hydroxyurea Derivatives
Table 2. Equilibrium and kinetic parameters for deprotonation and oxidation of hydroxyureas with Fe(CN)63−, at 25 C and 2 M
o
ionic strength (NaClO4)
2H‡
2S‡
a H
a S
k 25 C
k 25 C
1 

2 

Parameter ⇒
pKa (25 oC)
kJ mol1 J K1 mol1
kJ mol1 J K1 mol1
mol1dm3s1
mol1dm3s1
Compound ⇓
96 ± 4
(5.06 ± 0.01)  102
101 ± 4
MetU
12.7 ± 0.1
10.69(a)
43 ± 1
27 ± 1

37.8(a)
7.6(b)
81(a)
3.6  103
76
HU
<0.15
27
10.15(b)
169(b)
9.82 ± 0.02
(1 ± 1)  102
(1.92 ± 0.02)  104
107 ± 4
NMHU
16 ± 1
9.79(b)
4.9(b)
171(b)
(a) Data from Ref. 12, parameters determined kinetically.
(b) Data from Ref. 18, parameters determined potentiometrically.
deprotonation site of MetU in aqueous solution is the
hydroxylamino nitrogen rather than the amide nitrogen
atom. Geometries of the most stable minima are given
in supplementary information (Table S4).
Based on the above arguments the following reac-
tion mechanism is proposed (Scheme 1).
DISCUSSION
In this study we investigated the mechanism of oxida-
tion of the hydroxyurea derivatives with hexacyanofer-
rate(III) in aqueous solutions. As already shown for HU,
for both derivatives of hydroxyurea that were studied,
the dependence of the rate of oxidation on the acidity of
aqueous solutions shows that the anions are much more
reactive than the molecular forms. The stoichiometric
ratio [Fe(CN)63−]/[NMHU] in the redox reaction be-
3−
tween Fe(CN)6 and NMHU was found to be 2, the
same as found for the reactions of HU with dioxovana-
3−
dium(V) and Fe(CN)6 ions, while the stoichiometric
ratio [Fe(CN)63−]/[MetU] in the redox reaction between
3−
Fe(CN)6 and MetU was found to be 1. These results
+
3−
indicate that neither VO2 nor Fe(CN)6 ions can oxi-
dize HU, NMHU, or MetU to NO because the conver-
sion of the hydroxyureas to NO is a three-electron oxi-
dation process. The obtained difference in stoichiomet-
ric coefficients of the hydroxyurea derivatives indicates
a rather different reaction mechanism operative in oxi-
dation of MetU compared to the oxidation of NMHU
and HU.
Scheme 1.
The first step represents a fast deprotonation pre-
equilibrium, followed by the rate determining step (ii).
Step (iii) allows for the obtained stoichiometric coeffi-
cients in the oxidation of MetU, probably representing
the termination step. On the other hand, step (iv) is
necessary for NMHU and HU, since these two com-
pounds act as two-electron donors in the studied redox
reactions. Table 2 lists all the calculated deprotonation,
thermodynamic, kinetic and activation parameters.
The data in Table 2 reveal that (i) the second order
rate constants of the redox reactions are inversely re-
lated to the basicities of the hydroxyureates, (ii) the rate
constant of redox reaction decreases or increases upon
the substitution of methyl group for H atom at the hy-
droxamate oxygen (MetU) or nitrogen (NMHU) atom of
The results reveal that free radicals are formed as
the reactive intermediates during the oxidation of all
three hydroxyureas. The obtained spectral parameters
of EPR measurements reveal that the same free radical
is formed in the oxidation of NMHU with both
3−
Fe(CN)6 and dioxovanadium(V) ions, as already
reported for the oxidation of HU with these two oxi-
dants. Our failure to detect the free radical derived
from MetU by EPR spectroscopy indicates that this
free radical is present at much lower concentration
than the free radicals generated either from HU or
NMHU. This could be a consequence of a much lower
stability of the former one, or, more probably, its much
slower formation.
Croat. Chem. Acta 84 (2011) 133.

A. Budimir et al., Kinetics and Mechanism of Oxidation of Hydroxyurea Derivatives
143
HU, respectively, and (iii) the substitution at the hy-
droxamate oxygen is only a slightly more effective than
the substitution at the nitrogen atom.
Since the charges of all three hydroxyureates are
equal (1), the ion-pair association constants between
the Fe(CN)63− and the anions mediated by Na+ ion(s) are
expected also to be nearly equal. Hence, one may rea-
sonably assume that the primary reason for the observed
decrease of rate constant upon the substitution of methyl
group for H atom is the bulkiness of the methyl sub-
stituent in MetU, but such an explanation does not com-
prehend the observed accelerating effect of the substitu-
tion in NMHU. The EPR spectroscopic characteriza-
tions of the HU and NMHU free radicals, showing that
the spin density is localized at the hydroxamate oxygen
atom, may help reconcile these apparently contradictory
results. It could be speculated that in order for the suc-
cessful reactive contact to occur, Fe(CN)6 should
approach the hydroxamate oxygen atom. If the same is
true for the free radical of MetU, which cannot be struc-
turally characterized because it was detected only by the
acrylamide polymerization test but not by EPR spec-
troscopy, then the electron donor site in MetU could
also be the oxygen atom and not the nitrogen atom
where the deprotonation occurs.
Figure 9. Plot of ΔaH vs. ΔaS for investigated hydroxamic
acids at I = 2 mol dm−3: squares; data from Refs. 17, 56, 57
and 58, circles; data from Ref. 18 and triangle; data point for
MetU, this work.
3−
follow the same reaction pattern proposed for other
monohydroxamic acids in the series. Making the depro-
tonation of MetU possible only at the N-site (by the
substitution of the methyl group for the hydrogen atom),
the observed deviation from the straight line addition-
ally corroborates the suggestion that the distinction
between the deprotonation sites of low molecular
weight hydroxamic acids in water is possible on the
basis of the linear relationship between the reaction
enthalpy and entropy.
Unlike NMHU, where the substitution of the
methyl group for the hydrogen atom at the hydroxamato
nitrogen atom causes the deprotonation to occur exclu-
sively at the oxygen atom, and HU, where the deproto-
nation occurs at both sites, in MetU the deprotonation is
forced to occur only at the less electronegative atom, i.e.
at one of the nitrogen atoms. The results of ab initio
computations point to the hydroxamate nitrogen atom as
the single deprotonation site in MetU.
The reduction of electron density at the redox re-
active site in methoxyureate compared to N-
methylhydroxyureate, caused by dislocation of the elec-
tron-transfer from the deprotonation sites, should retard
the rate determining electron transfer to hexacyanofer-
rate(III) ion. A slight increase of the electron density on
the hydroxamate oxygen atom of MetU, caused by the
substitution of the methyl group for the hydrogen atom,
obviously cannot compensate for that effect.
However, before any further discussion of this hy-
pothesis, the deprotonation site of MetU has to be ascer-
tained as much as possible. A large number of papers
dealing with theoretical and experimental aspects of
hydroxamic acids’ acidities has been pub-
lished14,15,19,42−54 in order to determine whether these
compounds are nitrogen or oxygen acids. The electronic
properties of the functional groups attached to the hy-
droxamate moiety were found to play a dominant role in
the ionization of hydroxamic acids. A plot of the reac-
tion enthalpies vs. entropies for a series of small mono-
hydroxamic acids shown in Figure 9 clearly illustrates
that the thermodynamic parameters determined for ioni-
zation of hydroxyureas, and particularly for MetU, ex-
hibit a significant deviation from the theoretical straight
line defined by Crumbliss and coworkers. Since the
hydroxamic acids used by Crumbliss and collaborators
were apparently O-acids, the deviation from theoretical
line could be explained by invoking the deprotonation at
the N-site. The observed deviation for HU was ex-
plained by the participation of the N-site in the deproto-
nation.55 An effort was made to explain a smaller but
evident deviation observed for NMHU by unexpectedly
negative reaction entropy for deprotonation at the O-
site, but the hypothesis was not confirmed by the per-
formed calculations.
Therefore, the inverse correlation of rate constants
of the electron transfer from the hydroxyureates to
3−
Fe(CN)6 with basicities of hydroxyureates, could be
Nevertheless, it is clear that the MetU data-point
deviates from the Crumbliss straight line by far more
than the data-points of other two hydroxyureas, indicat-
ing clearly that the deprotonation of MetU does not
speculated to occur mainly because the deprotonation
and the electron donating sites do not overlap each oth-
ers. In other words, whereas the electronic properties of
Croat. Chem. Acta 84 (2011) 133.

144
A. Budimir et al., Kinetics and Mechanism of Oxidation of Hydroxyurea Derivatives
Figure 10. HOMO orbitals of cis- and trans-H2NC(=O)NOCH3− obtained by NBO analysis.
the hydroxamate nitrogen atom play a crucial role in the
ionization of the methoxyurea molecule, a crucial role
in the kinetics of electron transfer is played by the elec-
tronic properties of the oxygen atom in methoxyureate.
In order to test this hypothesis, ab initio theoreti-
ticipation of the amine-nitrogen AO in the anion’s
HOMO was predicted by NBO analysis, but an unex-
pectedly weak participation of the hydroxyl-oxygen AO
was also found.
NBO analysis performed on N-methylhydroxyure-
ate (NMHU can deprotonate only the OH site) showed
that HOMO is located only on the hydroxyl oxygen
atom of each anion, cis- or trans- H2NC(=O)NCH3O−
(Figure 11). The charges on the hydroxamate oxygen
atom (0.75) and the carbonyl oxygen atom (0.74) are
almost the same, but the charge on the hydroxamate
nitrogen atom is much smaller (0.20).
A similar analysis carried out on HU reveals that
when deprotonation occurs at the hydroxamate nitrogen,
H2NC(=O)NOH− is produced and the HOMO is located
on the carbonyl oxygen atom of anion (Figure 12 top).
The charge distribution is similar to that in MetU, i.e.
similar charges on hydroxamate oxygen (0.64) and
nitrogen (0.54) atoms, and a larger negative charge on
the carbonyl oxygen (0.82). When the deprotonation of
HU occurs at the –OH site, H2NC(=O)NHO− is pro-
duced and the anion's HOMO is located on the hydroxyl
cal calculations were carefully analyzed for the three
hydroxyureates studied. As expected, the performed
calculations strongly indicated that the deprotonation
site in MetU is indeed the hydroxamate nitrogen atom
alone, because pKa of the amine group in methoxyurea
was estimated to be almost five units higher. The calcu-
lated distribution of the negative charge in methoxyure-
ate, based on the performed NBO analysis (Table S3),
indicated almost equal charges (0.48) on the hydrox-
amate nitrogen and oxygen atoms, and a much larger
negative charge on the carbonyl oxygen (0.79). The
calculated HOMO of methoxyureate, from which the
electron transfer to hexacyanoferrate(III) is expected to
occur, is composed mostly of the carbonyl-oxygen p
atomic orbital (> 80 %), while the participation of the
hydroxamate nitrogen p AO (ca. 5 %) in HOMO is
negligible (Figure 10). As expected, a negligible par-
Figure 11. HOMO orbitals of cis- and trans-H2NC(=O)NCH3O− obtained by NBO analysis.
Croat. Chem. Acta 84 (2011) 133.

A. Budimir et al., Kinetics and Mechanism of Oxidation of Hydroxyurea Derivatives
145
Figure 12. HOMO orbitals of (top) cis- and trans-H2NC(=O)NHO− and (bottom) cis- and trans-H2NC(=O)NOH− obtained by
NBO analysis.
Acknowledgements. The authors gratefully acknowledge fi-
nancial support from the Croatian Ministry of Science.
oxygen atom (Figure 12 bottom). The difference in
charge between the hydroxamate oxygen (−0.76) and
nitrogen (−0.32) atoms was computed, but an even larg-
er negative charge was found on the carbonyl oxygen
atom (−0.82).
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=
=
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]
)
tot
[
]
[
tot
Ka = b× Kw
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thermodynamic parameters defining the theoretical line in Figure
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would align much better to the theoretical line. However, the
Croat. Chem. Acta 84 (2011) 133.

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