Phosphorylimidazole derivatives: Potentially biosignaling molecules

Article abstract of DOI:10.1021/jo2017394

The phosphorylation of imidazole by two activated phosphate diesters and a triester gives phosphorylimidazole derivatives that are stable enough in aqueous solution to be observed and identified by ESI-MS/MS and NMR. Half-lives ranging from hours to days (in the case of the monoethyl ester) show that it is possible to design molecules with variable half-lives with potential to be used for biological intervention experiments as possible inhibitors of biosignaling processes or as haptens for the generation of antibodies.

Full text of DOI:10.1021/jo2017394

ARTICLE
pubs.acs.org/joc
Phosphorylimidazole Derivatives: Potentially Biosignaling Molecules
†
†
†
†
‡
Elisa S. Orth, Eduardo H. Wanderlind, Michelle Medeiros, Pedro S. M. Oliveira, Boniek G. Vaz,
‡
§
,†
Marcos N. Eberlin, Anthony J. Kirby, and Faruk Nome*
†
INCT-Catalise, Universidade Federal de Santa Catarina, Florian ꢀo polis, SC 88040-900, Brazil
‡
ThoMSon Mass Spectrometry Laboratory, Institute of Chemistry, University of Campinas, UNICAMP, Campinas, SP, Brazil
§
University Chemical Laboratory, University of Cambridge, Cambridge CB2 1EW, United Kingdom
S Supporting Information
b
ABSTRACT: The phosphorylation of imidazole by two activated phosphate diesters
and a triester gives phosphorylimidazole derivatives that are stable enough in aqueous
solution to be observed and identified by ESI-MS/MS and NMR. Half-lives ranging
from hours to days (in the case of the monoethyl ester) show that it is possible to
design molecules with variable half-lives with potential to be used for biological
intervention experiments as possible inhibitors of biosignaling processes or as
haptens for the generation of antibodies.
1
3
’
INTRODUCTION
DNAzyme with novel RNA cleaving catalytic activity. How-
ever, models in which an imidazole group acts as a nucleophile at
phosphate phosphorus are scarce in the literature, though much
effort has focused on preparing and characterizing phosphohis-
The histidine imidazole is the most versatile of the catalytic
groups available to protein enzymes. With a pK near 7, it can act
a
at physiological pH as a general base or nucleophile, as a Lewis
base to coordinate, position, and “tune” metal cations, and in
its protonated form as a general acid. Histidine imidazole plays
prominent roles not only in the active sites of hydrolytic enzymes
such as the serine proteases and ribonucleases, but also in
regulatory phosphorylation pathways.
The active site of ribonuclease A (RNase A) contains two
histidines, which work together to cleave (a relatively reactive
phosphodiester) RNA: most likely by a classical general acidꢀ
base pathway involving a neutral imidazole as a general base, with
a second, imidazolium group acting as a general acid to assist the
departure of the leaving group. An alternative route, involving
protonation of the phosphate oxyanion, could take advantage of
14,15
tidines.
phosphorylimidazole,
A handful of studies has evaluated the reactivity of N-
16,17
which is stable at high pH but hydrolyzed
below pH 6 with a half-life of 9.6 h at 39 °C. Furthermore,
phosphorylimidazole has been considered to be very reactive toward
nucleophilic attack. Analogues of phosphohistidine, derived from
pyrrole and furan rings, with improved stability have been proposed
recently as possible inhibitors of signaling processes, but these have
16
1
2
16
3
6
not yet been tested in vivo.
We recently reported results for the hydrolysis of a phosphate
diester, BMIPP, bearing two imidazole groups, Scheme 1.
This simple biomimetic model supported a remarkably efficient
intramolecular catalysis, such that BMIPP is hydrolyzed up to 10
1
8,19
4
9
4
times faster than diphenyl phosphate. We proposed a mechanism
involving general-acid catalysis of nucleophilic catalysis for the
hydrolysis of the reactive zwitterionic species and were able to
detect the cyclic intermediate INT as a short-lived species using
ESI-MS.
We are interested in evaluating mechanisms for the reactions
of imidazole with a range of phosphate esters and in particular the
stability of possible phosphorylated imidazole intermediates. We
present here a full set of kinetic, NMR, and ESI-MS data for the
reactions of imidazole with three activated esters: two diesters,
bis(2,4-dinitrophenyl) phosphate (BDNPP) and ethyl 2,4-dini-
trophenyl phosphate (EtDNPP), and the related triester diethyl
the enhanced reactivity of phosphate triesters. Histidine imida-
zole also acts as a nucleophile, as in the formation of the
phosphohistidine intermediates important in biological signaling
processes. The enhanced levels of reactivity of these intermedi-
ates support rapid and reversible phosphoryl group transfers5,
which play critical roles in regulating many aspects of cell life.
Studies involving phosphorylated histidine have proved some-
thing of a challenge, particularly in the design of inhibitors and of
haptens for the generation of antibodies, which require stable
6
ꢀ8
phosphohistidine analogues.
Biomimetic chemistry can provide benchmarks for a proper
9
understanding of biological reactions as well as offering insights
into the intelligent design of artificial enzymes as potential targets
for therapeutic applications. Several bifunctional mimics of
RNase A have been reported in the literature, typically featuring a
pair of imidazole groups attached to systems of interest such as
2
,4-dinitrophenyl phosphate (Et DNPP). The reactions proceed
via phosphorylimidazole derivatives, and the characterization of
2
10
Received: August 19, 2011
Published: August 23, 2011
11
12
oligonucleotides, cyclodextrins, and, more recently, a modified
r 2011 American Chemical Society
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|J. Org. Chem. 2011, 76, 8003–8008

The Journal of Organic Chemistry
ARTICLE
Scheme 1
Scheme 2
Table 1. Kinetic Parameters for the Reactions of BDNPP,
a
Et DNPP, and EtDNPP with Imidazole at 25 °C
2
Et
2
DNPP
BDNPP
EtDNPP
b
ꢀ6 ꢀ1
k
k
k
0
, 10
s
8.0 ( 0.50
34.0 ( 2.0
17.7 ( 0.10
7.10 ( 0.20
0.19 ( 0.04
2.90 ( 0.11
1.42 ( 0.03
0.036 ( 0.006
0.049 ( 0.005
0.10 ( 0.004
b
ꢀ3 ꢀ1 ꢀ1
OH, 10
M
s
c
ꢀ3 ꢀ1 ꢀ1
N
, 10
M
s
d
a
pK (IMZ)
a
b
Obtained by fitting experimental data in Figure 1 with eq 1. Con-
sistent with values from the literature. In agreement with k values
2
2
1 c
from linear plots of kobsd vs [imidazole], which confirms first-order
d
dependence, given in Supporting Information. Value confirmed by
potentiometric titration at 25 °C, I = 1.0, Supporting Information.
Table 2. Activation Parameters and Kinetic Solvent Isotope
Effects for the Reactions of BDNPP, Et DNPP, and EDNPP
2
a
with Imidazole (1 M) at pH 8.5
2
Et DNPP
BDNPP
EtDNPP
q
ΔH , kcal/mol
+9.90 ( 0.30
ꢀ32.08 ( 1.5
1.25
+10.15 ( 0.05
ꢀ32.20 ( 0.32
1.24
+12.64 ( 0.15
ꢀ39.83 ( 1.01
1.14
Figure 1. pHꢀrate profiles for the reactions of imidazole (1 M) with
q
ΔS , cal
Et DNPP (b, right y axes), BDNPP (9), and EtDNPP (2), all at 25 °C,
2
I = 1.0 (KCl). The spontaneous hydrolysis reactions for the esters are
kH2O/kD2O
21
a
shown for comparison (O, 0, and 4, respectively). Rates in water for
Et DNPP are experimental, and those for EtDNPP were estimated from
Experimental data, linear plots, and equations used are given in the
Supporting Information.
2
20
data for MeDNPP. Solid lines represent the fits to eq 1.
Indeed, k is 5 times slower for EtDNPP than for BDNPP,
their stability provides important information for the future
design of possible inhibitors of biosignaling processes or haptens
for the generation of antibodies.
0
2
0
compared with a factor of less than 3 for MeDNPP. On the
20
basis of previous results and from the kinetic parameters shown
in Table 1 for the reactions of the three compounds involved in
this work, it seems likely that the reactions with imidazole involve
SN2(P) mechanisms. New evidence, including thermodynamic
activation parameters and kinetic solvent isotope effects, is given
in Table 2. Entropies of activation are large and negative and thus
consistent with either nucleophilic or general-base catalysis, but
’
RESULTS AND DISCUSSION
Kinetics. Imidazole is an effective catalyst for the hydrolysis of
all three esters studied, as shown by the pHꢀrate profiles in
Figure 1. Experimental data in Figure 1 were fitted with eq 1,
the low solvent deuterium isotope effects k /k , which
range from 1.14 to 1.24, indicate that the reactions are at least
H O D O
2
2
k
¼ k0 þ k
½OHꢁ
þ kN½imidazoleꢁχ
obsd
OH
ð1Þ
primarily nucleophilic. We report results from two series of
imidazole
1
31
spectroscopic ( H and P NMR, ESI-MS) investigations that
support this conclusion.
which is based on Scheme 2, where χimidazole is the molar fraction
of the reactive neutral species of imidazole (pK = 7.0): the
kinetic parameters obtained are given in Table 1. Note the
different levels of absolute reactivity involved: the triester
Following Reactions by NMR. If the reactions with imidazole
are nucleophilic, they must involve phosphorylimidazole inter-
mediates (IMP, Schemes 3 and 4). In our work with BMIPP
(Scheme 1) the cyclic phosphorylimidazole intermediate (INT)
was too short-lived to be detected by NMR, but the acyclic
intermediates formed by nucleophilic attack on all three esters of
Schemes 3 and 4 were shown to be stable enough to be observed
a
Et DNPP, as expected, is the most reactive and BDNPP the
2
more reactive of the diesters (Table 1). The rate enhancements
brought about by 1 M imidazole are 2000-, 3000-, and 7500-fold
for Et DNPP, EtDNPP, and BDNPP, respectively. The differ-
2
1
31
ence probably reflects a small steric effect of the ethyl group(s).
directly. Reactions were followed by H NMR and P NMR, and
8
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The Journal of Organic Chemistry
ARTICLE
Scheme 3. Imidazole-Catalyzed Route for the Reactions with
a
Et DNPP and EtDNPP
2
a
ꢀ
2
,4-DNPO is 2,4-dinitrophenolate.
3
1
Figure 3. Typical progressive P NMR spectra obtained for the
reactions studied. Data for the reaction of imidazole (0.6 M) with
BDNPP (0.017 M) at pH 8.5 in D O and 10% CD CN at 25 °C.
2 3
Scheme 4. Imidazole-Catalyzed Route for the Reactions with
BDNPP
a
a
ꢀ
2
,4-DNPO is 2,4-dinitrophenolate.
Figure 4. Relative concentration vs time profiles for the species de-
tected by NMR, according to Scheme 3, for the reactions of imidazole
1
31
2
with (A) Et DNPP ( H NMR) and (B) EtDNPP ( P NMR).
Figure 5. Relative concentration vs time profiles for the species de-
tected by NMR, according to Scheme 4, for the reactions of imidazole
with BDNPP, followed by (A) H NMR and (B) P NMR, at 25 °C.
1
31
1
Figure 2. Typical progressive H NMR spectra obtained for the
reactions studied. Data for the reaction of imidazole (0.4 M) with
BDNPP (5 ꢂ 10ꢀ M) at pH 8.5 in D
3
2
O at 25 °C.
aromatic carbon, commonly observed in nucleophilic depho-
22
sphorylation reactions, can be ruled out since no reaction was
1
successive spectra obtained agree with Schemes 3 and 4. Typical
H and P NMR spectra obtained for the reaction of imidazole
observed (by H NMR, Supporting Information) for imidazole
1
31
with 1-chloro-2,4-dinitrobenzene.
with BDNPP are shown in Figures 2 and 3, and other spectra for
Figures 4 and 5 show the reaction course for each ester
1
EtDNPP and Et DNPP are given in the Supporting Information
according to Schemes 3 and 4, as followed continuously by H
2
31
(
Figures S14ꢀS16). The chemical shifts for the species detected
and P NMR, in terms of relative concentration vs time profiles
are presented in Table 3 (considering Schemes 3 and 4), where
the peaks were assigned by comparison with spectra of the pure
compounds and data given in the literature, when available.
measured in the presence of imidazole. Data for the reaction with
1
EtDNPP followed by H NMR are not shown in Figure 3, since
the peaks for IMP2 coincide with those of the product 4
(Scheme 3; chemical shifts are given in Table 3). Fitting data
of Figures 4 and 5 to the equations for consecutive first-order
1
Thus, IMP1 and IMP3 could be detected by H NMR, while
31
IMP2ꢀ5 were detected by P NMR. Nucleophilic attack on the
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ARTICLE
1
31
Table 3. H and P NMR Chemical Shifts of the Species Detected in the Reactions of Imidazole with the Phosphate Esters in D O
2
a
at pH 8.5 and 25 °C
1
31
compound
δ ( H NMR) (ppm)
δ ( P NMR) (ppm)
imidazole
EtDNPP
BDNPP
7.86 (s, 1H, Ar), 7.16 (s, 2H, Ar)
1.29 (t, 3H, CH ), 4.10 (q, 4H, CH
7.80 (d, 1H, Ar), 8.58 (dd, 1H, Ar), 8.95 (d, 1H, Ar)
1.38 (t, 6H, CH ), 4.39 (q, 4H, CH ), 7.80 (d, 1H, Ar), 8.64 (dd, 1H, Ar), 9.03 (d, 1H, Ar)
6.75 (d, 1H, Ar), 8.09 (dd, 1H, Ar), 8.83 (d, 1H, Ar)
1.22 (t, 3H, CH ), 3.83 (q, 2H, CH
7.88 (d, 1H, Ar), 8.52 (dd, 1H, Ar), 8.82 (d, 1H, Ar)
3
2
), 7.71 (d, 1H, Ar), 8.52 (dd, 1H, Ar), 8.89 (d, 1H, Ar)
ꢀ4.90
ꢀ13.56
2
Et DNPP
3
2
ꢀ
2
4
4
4
,4-DNPO
(EtDNPP)
(BDNPP)
3
2
)
4.28
0.56
2
(Et DNPP)
1.26 (t, 6H, CH
1.33 (t, 6H, CH
1.17 (t, 3H, CH
3
3
3
), 3.93 (q, 4H, CH
2
2
)
)
IMP1
IMP2
IMP3
IMP4
IMP5
), 4.30 (q, 4H, CH
), 3.89ꢀ3.83 (m, 2H, CH
2
)
ꢀ7.21
7.90 (d, 1H, Ar), 8.48 (dd, 1H, Ar), 8.91 (d, 1H, Ar)
ꢀ14.53
ꢀ18.54
ꢀ3.70
3.20
P
i
a
23ꢀ26
Assignments for BDNPP, Et
not reported in the literature, their formation was confirmed by ESI-MS/(MS).
2
DNPP, EtDNPP, DNP, 4, IMP1, IMP5, and P
i
agree with values found in the literarature.
For IMP2ꢀ4, which are
Table 4. Rate Constants for the Formation (k ) and Disap-
1
pearance (k ) of Phosphorylimidazole Intermediates
2
IMP1ꢀ5 Obtained from Fitting the Data of Figures 4 and 5 to
27
the Equation for Consecutive First-Order Reactions
Figure 6. ESI(ꢀ)-MS after 8 min of reaction of BDNPP (8 ꢂ 10ꢀ M)
5
with imidazole (1 M) at pH 8.5 and 25 °C.
Schemes 3 and 4 could be detected and characterized for each
reaction. Typical ESI-MS and ESI-MS/MS data are shown in
Figures 6 and 7 for the reactions with BDNPP: similar pairs of
2
7
reactions gives the rate constants summarized in Table 4, along
with the stabilities (half-lives) of the phosphorylimidazole inter-
mediates. Note that these data refer to pH 8.5, where the leaving
group for IMP1,2, imidazole, is almost exclusively unprotonated, since
spectra for the reactions with EtDNPP and Et DNPP are
2
presented in the Supporting Information (Figures S17ꢀS20).
Thus, the formation of the intermediates IMP1ꢀ4 is confirmed
by ESI-MS, though not that of IMP5, which was confirmed by
31
23
its pK should be below 6.50. Interestingly, in the reactions with
P NMR assignments from the literature (vide infra).
a
BDNPP (Scheme 4), for IMP3, the best leaving group (in terms of
In Figure 6, a series of major anions are detected and identified
ꢀ
pK ) is2,4-DNPO , and results show that IMP3 reacts with imidazole
as the anionic form of the reactant BDNPP of m/z 429, the
phosphorylimidazole intermediate IMP3 of m/z 313, and the
phenolic product DNP of m/z 183, as well as inorganic phos-
a
to give IMP4, which is subsequently hydrolyzed to IMP5 and Pi.
Mass Spectrometric Analysis. Drawing on our experience in
22
ꢀ
ꢀ
reaction mechanism studies using MS techniques, we mon-
itored the course of the reactions, in aqueous solution at pH 6.5
and 60 °C, using ESI-MS as well as its tandem version ESI-MS/
MS in the positive and negative ion modes. Reactants, inter-
mediates, and products were transferred directly from the reac-
tion solution to the gas phase, detected by ESI-MS, and then
characterized by ESI-MS/MS via their unimolecular dissoci-
ation chemistry. Using this approach, all the major anions from
phate in the form of PO
3
(m/z 79) and H
2
PO
4
(m/z 97).
ESI-MS/MS was then used to characterize these important
species via collision-induced dissociation. The intermediate
IMP4 (m/z 197) was detected by ESI-MS after 2 days of reaction
for BDNPP with imidazole and is presented in the Supporting
Information (Figure S21).
The resulting tandem ESI-MS/MS spectra for IMP3
(Figure 7) support these assignments: the phosphorylimidazole
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The Journal of Organic Chemistry
ARTICLE
hydrolyzed to imidazole and ethyl phosphate. IMP3 is the most
reactive intermediate, liberating 2,4-dinitrophenoxide to form
the more stable IMP4. This is hydrolyzed to the even more stable
IMP5, which is finally hydrolyzed to imidazole and inorganic
1
6
phosphate at a rate consistent with those from previous studies.
Note that the results regarding stability and reactivity of four
compounds (IMP1ꢀ4) among all five phosphorylimidazoles
studied here have not yet been reported and comprise an
interesting class of stable analogues of biologically important
intermediates. The new evidence for the differences in reactivity
of the phosphorylated imidazoles IMP1ꢀ4 furthers our under-
standing of the dynamics of the relevant signaling molecules,
which depending on their structure could be relatively stable in
biological systems and need enzymes for their rapid degradation.
These results establish that the phosphorylimidazole esters
IMPs generated in this work are reasonably stable species in
aqueous solution at 25 °C and pH 8.5 (Table 4). The inter-
mediates detected, especially the monoalkyl ester IMP2, are
promising models for the design of phosphorylimidazole analo-
gues important as possible inhibitors of biosignaling processes or
as haptens for the generation of antibodies.
Figure 7. ESI(ꢀ)-MS/MS of the ion of m/z 313, intercepted from the
ꢀ5
reaction solution of BDNPP (8 ꢂ 10 M) with imidazole (1 M) at
pH 8.5 and 25 °C.
’
EXPERIMENTAL SECTION
Materials. Inorganic salts were of analytical grade and were used
without further purification. Liquid reagents were purified by distillation.
The phosphate esters BDNPP, Et DNPP, and EtDNPP were prepared
2
22,28
by standard methods from POCl
3
, as described previously.
value of imidazole was
Potentiometric Titrations. The pK
a
determined using a digital pH meter and a combined glass electrode,
equipped with an automatic buret. Titrations were performed in a
1
2
50 mL thermostated cell under N at 25.0 °C, ionic strength 0.1 M KCl,
and [imidazole] = 1.0 mM (initial). The solution was titrated with small
increments of 0.1008 M CO -free KOH. All precautions were taken to
2
29
eliminate carbonate and CO
was used to calculate the dissociation constants, using the value of ꢀ13.78
for pK
2
during the titration. The program BEST7
w
.
Figure 8. Summary of the phosphorylimidazole derivatives detected
and their stabilities.
Kinetics. Reactions of BDNPP, Et DNPP, and EtDNPP were fol-
2
lowed spectrophotometrically by monitoring the appearance of 2,4-
dinitrophenolate (DNP) at 400 nm. Reactions were started by adding
10 μL of 10 mM stock solutions of the substrate in acetonitrile to 3 mL of
aqueous solutions to give a final concentration of the substrate of
66.7 μM. The temperatures of reaction solutions in quartz cuvettes
were controlled with a thermostated water-jacketed cell holder, and
ionic strengths were maintained at 1.0 M with KCl. Absorbance versus
time data were stored directly on a microcomputer, and observed first-
intermediate IMP3 anion of m/z 313 is found to dissociate to
DNP of m/z 183, which can lose two NO radicals sequentially,
giving the fragment ions m/z 153 and m/z 123. DNP of m/z 183
also loses NO and then CO to form the fragment ions of m/z
2
1
37 and m/z 109, respectively. Similar ions and dissociation
chemistry were observed for the reactions of EtDNPP and Et2-
DNPP with imidazole, where both phosphorylimidazole inter-
mediates IMP1,2 were detected (given in the Supporting In-
formation).
orderrate constants, kobsd, were calculated from linear plots of ln(A
against time for at least 90% of the reaction using an iterative least-squares
program; correlation coefficients were >0.999 for all kinetic runs. The pH
∞
ꢀ A
t
)
was maintained with 0.01 M buffers of CH
2
ClCOOH (pH 2ꢀ3),
HCOOH (pH 3ꢀ4.5), CH COOH (pH 4ꢀ5.5), NaH
3
2
PO
4
(pH 5.5ꢀ
6
.0), and KHCO (pH 10ꢀ11). In the pH range of 6ꢀ8, solutions were
’
CONCLUSIONS
3
self-buffered with imidazole.
The order of reactivity of the phosphorylimidazole intermedi-
Mass Spectrometry. To identify intermediates and reaction
products, direct infusion electrospray ionization mass spectrometry
analyses were performed with a hybrid triple-quadrupole linear ion trap
ates IMP1ꢀ5 is the same as that of the esters they are derived
from, as is to be expected, Figure 8. They are hydrolyzed at
pH 8.5 in the order of IMP3 > IMP1 > IMP4 > IMP5 > IMP2,
breaking down (after 5 half-lives at 25 °C) in 9 h, 30 h, 9 days, 48
days, and 4 months, respectively.
The neutral phosphorylimidazole IMP1 is moderately reac-
tive, with a half-life of nearly 6 h, decomposing to imidazole and
diethyl phosphate as reaction products. IMP2 is impressively
stable: the least reactive intermediate in this study, it is slowly
2
2
mass spectrometer. For typical electrospray ionization (ESI-MS)
conditions, the phosphate ester was reacted with imidazole (1 M) in
aqueous medium at pH 8.5. A microsyringe pump delivered the reagent
ꢀ
1
solution into the ESI source at a flow rate of 10 μL min . ESI and the
QqQ (linear trap) mass spectrometer were operated in the negative ion
ꢀ
1
mode. Main conditions: curtain gas nitrogen flow of 20 mL min , ion
spray voltage of ꢀ4500 eV, declustering potential of ꢀ21 eV, entrance
8
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The Journal of Organic Chemistry
ARTICLE
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anionic species detected by ESI-MS were subjected to ESI-MS/MS by
using collision-induced dissociation (CID) with nitrogen and collision
energies ranging from 5 to 45 eV.
2
009, 74, 5011.
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1
(
NMR Spectrometry. H spectra were monitored on a spectro-
1
meter (400 and 200 MHz) at 25 °C in D
2
O. Some aromatic H signals
were obscured by signals of the excess imidazole present, but none-
theless, the results obtained confirm the formation of the phosphor-
(
L. S.; Eberlin, M. N.; Bunton, C. A.; Nome, F. J. Org. Chem. 2004,
69, 7898.
1
ylimidazole intermediates. H chemical shifts are referred to internal
(
25) Kirby, A. J.; Manfredi, A. M.; Souza, B. S.; Medeiros, M.; Priebe,
J. P.; Brandao, T. A. S.; Nome, F. Arkivoc 2009, 28.
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A. M.; Nome, F. Chem. Commun. 2008, 4428.
27) McQuarrie, D. A.; Simon, J. D. Physical Chemistry: A Molecular
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sodium 3-(trimethylsilyl)propionate (TMSP), and pD was the observed
31
pH + 0.4 in D O at 25 °C. P NMR experiments were carried out on a
2
(
spectrometer at 200 MHz with 85% phosphoric acid as the external
1
reference. All reactions were followed by H NMR, and those for
(
31
BDNPP and EtDNPP by P NMR also. To obtain the plots of relative
concentration vs time for the species detected (Figures 4 and 5), the area
of the most representative signal for each species was calculated.
(
(29) Martell, A. E.; Smith, R. M.; Motekaitis, R. J. NIST Critical
Stability Constants of Metal Complexes Database: NIST Standard Refer-
ence Database 46; NIST: Gaithersburg, MD, 1993.
’
ASSOCIATED CONTENT
S
Supporting Information. Tables and figures giving kinetic,
b
1
31
potentiometric, H and P NMR, and ESI-MS(/MS) data. This
material is available free of charge via the Internet at http://pubs.
acs.org.
’
AUTHOR INFORMATION
Corresponding Author
*E-mail: faruk@qmc.ufsc.br.
’
ACKNOWLEDGMENT
We thank INCT-Cat ꢀa lise, INOMAT, and the Brazilian foun-
dations CNPq, FAPESC, and FAPESP for financial assistance.
’
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