Dephosphorylation reactions of mono-, Di-, and triesters of 2,4-dinitrophenyl phosphate with deferoxamine and benzohydroxamic acid

Article abstract of DOI:10.1021/jo302374q

This work presents a detailed kinetic and mechanistic study of biologically interesting dephosphorylation reactions involving the exceptionally reactive nucleophilic group, hydroxamate. We compare results for hydroxamate groups anchored on the simple molecular backbone of benzohydroxamate (BHA) and on the more complex structure of the widely used drug, deferoxamine (DFO). BHA shows extraordinary reactivity toward the triester diethyl 2,4-dinitrophenyl phosphate (DEDNPP) and the diester ethyl 2,4-dinitrophenyl phosphate (EDNPP) but reacts very slowly with the monoester 2,4-dinitrophenyl phosphate (DNPP). Nucleophilic attack on phosphorus is confirmed by the detection of the phosphorylated intermediates formed. These undergo Lossen-type rearrangements, resulting in the decomposition of the nucleophile. DFO, which is used therapeutically for the treatment of acute iron intoxication, carries three hydroxamate groups and shows correspondingly high nucleophilic activity toward both triester DEDNPP and diester EDNPP. This result suggests a potential use for DFO in cases of acute poisoning with phosphorus pesticides.

Full text of DOI:10.1021/jo302374q

Article
pubs.acs.org/joc
Dephosphorylation Reactions of Mono‑, Di‑, and Triesters of 2,4-
Dinitrophenyl Phosphate with Deferoxamine and Benzohydroxamic
Acid
Michelle Medeiros,† Elisa S. Orth,†,⊥ Alex M. Manfredi,† Paulina Pavez,‡ Gustavo A. Micke,†
Anthony J. Kirby,§ and Faruk Nome*,†
†INCT-Catal
́ ́
ise, Departamento de Química, Universidade Federal de Santa Catarina, Florianopolis, SC, 88040-970, Brasil
‡Pontificia Universidad Catol
́
ica de Chile, Av. Vicuna Mackenna 4860, Santiago 6094411, Chile
̃
§University Chemical Laboratory, Cambridge CB2 1EW, U.K.
S
* Supporting Information
ABSTRACT: This work presents a detailed kinetic and
mechanistic study of biologically interesting dephosphorylation
reactions involving the exceptionally reactive nucleophilic
group, hydroxamate. We compare results for hydroxamate
groups anchored on the simple molecular backbone of
benzohydroxamate (BHA) and on the more complex structure
of the widely used drug, deferoxamine (DFO). BHA shows
extraordinary reactivity toward the triester diethyl 2,4-
dinitrophenyl phosphate (DEDNPP) and the diester ethyl
2,4-dinitrophenyl phosphate (EDNPP) but reacts very slowly
with the monoester 2,4-dinitrophenyl phosphate (DNPP). Nucleophilic attack on phosphorus is confirmed by the detection of
the phosphorylated intermediates formed. These undergo Lossen-type rearrangements, resulting in the decomposition of the
nucleophile. DFO, which is used therapeutically for the treatment of acute iron intoxication, carries three hydroxamate groups
and shows correspondingly high nucleophilic activity toward both triester DEDNPP and diester EDNPP. This result suggests a
potential use for DFO in cases of acute poisoning with phosphorus pesticides.
normal treatment for patients receiving blood transfusions.12
INTRODUCTION
■
Numerous side effects are known, including the inhibition of
DNA synthesis in human lymphocytes.13,14 DFO has been
examined as an antiproliferative drug in cancer chemotherapy,15
and studies have shown that DFO shows antitumor activity in the
treatment of neuroblastoma, leukemia, bladder and hepatocel-
lular carcinoma, and brain cancer.16 Furthermore, this natural
hydroxamate can also be used to suppress the oxidative
degradation of DNA, via complexation of iron in a nonreactive
form.17 Thus, DFO is an extremely powerful bacterial side-
rophore, containing three bidentate oxygen-containing ligands,
perfectly distributed and well-suited for the chelation of metal
cations such as high-spin, octahedral ferric ion. However, these
bidentate oxygen-containing ligands are hydroxamic acids
(Scheme 2), with anions known to be exceptionally powerful
nucleophiles, particularly toward the phosphorus centers of
phosphate esters.18
The importance of phosphate esters in biological processes is the
basis of the continuing interest in the study of phosphoryl
transfer mechanisms. The hydrolysis and other nucleophilic
substitution reactions of phosphoesters are studied in order to
understand the mechanisms of these reactions.1 α-Effect
nucleophiles such as hydroxylamine,2 hydrazine,3 and hydroxa-
mic acids4,5 all act as effective dephosphorylating agents. We
recently reported a detailed study of products and intermediates
involved in the dephosphorylation of bis(2,4-dinitrophenyl)
phosphate anion (BDNPP) by the benzohydroxamate anion
(BHA−), which showed competing nucleophilic attack on
phosphorus and on the aromatic carbon.6 Attack on carbon
gives an intermediate, which was detected, but slowly
decomposes to aniline and 2,4-dinitrophenol, whereas attack
on phosphorus gives an unstable intermediate that undergoes a
Lossen rearrangement to urea, amine, isocyanate, and carbamyl
hydroxamate.7,8 (Scheme 1)
We have reported the reaction of DFO with the phosphate
diester BDNPP, which involves nucleophilic attack by the
hydroxamate groups of DFO specifically on phosphorus, as
confirmed by the detection of two important phosphorylated
intermediates. Furthermore, its reaction with plasmid DNA
Thus, BHA behaves as a self-destructive molecular scissor,
which loses its nucleophilicity upon reaction. The reaction with
BHA (0.05 mol L−1) is over 105 times faster than the
spontaneous hydrolysis of BDNPP (1.9 × 10−7 s−1).
Deferoxamine (DFO, Scheme 2) is a naturally occurring
hydroxamate metal chelator,9 therapeutically indicated in iron10
and aluminum11 overload disorders. It has for many years been a
Received: October 25, 2012
Published: November 20, 2012
© 2012 American Chemical Society
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Scheme 1. Nucleophilic Attack of BHA− on Phosphorus
Scheme 2. DFO Structure and Acid Dissociation Constants of
its Four Ionizing Groups19
confirmed the ability of DFO to cleave DNA strands.20 It is
known that the reactivity of phosphate di- and triesters depends
strongly on the nature of the nucleophile and leaving group, with
significant participation of the “spectator” groups in the reaction
of triesters, where the two nonleaving groups are found to play
important roles, accounting for the substantial difference in
reactivity between triaryl and dialkyl aryl phosphate triesters.21 In
diesters, on the other hand, it seems that the nonleaving group
has minimal effect on reactivity, because bis-2,4-dinitrophenyl
phosphate is hydrolyzed at 39 °C less than 3 times faster than the
methyl 2,4-dinitrophenyl ester, little more than the statistical
factor of 2, despite the very large pKa difference.22 To better
understand the effects of substituent groups on the kinetics and
mechanisms of reaction of different phosphate esters with
different nucleophiles, we have now extended the kinetic studies
of the reactions of BHA and DFO to a complete series of 2,4-
dinitrophenyl phosphate esters: the triester diethyl 2,4-
dinitrophenyl phosphate (DEDNPP), the diester ethyl 2,4-
dinitrophenyl phosphate (EDNPP), and the monoester 2,4-
dinitrophenyl phosphate (DNPP) (Scheme 3).
■
Figure 1. pH−rate profiles for the reactions of (A) DEDNPP ( ) and
●
(B) EDNPP ( ) with 0.05 M BHA, μ = 1.0 M, 25 °C. The spontaneous
hydrolysis reactions for the esters are shown for comparison: DEDNPP
( ) and EDNPP ( ).
22,23
Scheme 3. Reactions Studied: DFO and BHA with DEDNPP,
EDNPP and DNPP
□
○
show that their reactions are accelerated many thousand-fold. In
contrast, under conditions similar to those shown below for the
di- and triester, the reaction of DNPP is accelerated only
modestly, with a rate increase of about 20%. (Full data sets for
these reactions are given in the Supporting Information (Tables
S1 and S9).
Data for the reactions with BHA were analyzed using eq 1,
which is consistent with Scheme 4, as described previously.6
RESULTS AND DISCUSSION
■
Kinetic Studies. Reactions were followed spectrophoto-
metrically, in water, by monitoring the appearance of 2,4-
dinitrophenoxide at 400 nm. We compared the reactivities of the
simple hydroxamic acid BHA (benzohydroxamic acid, 0.05 M)
and the more complex DFO (0.01 M) as nucleophiles toward the
three phosphate esters. pH−rate profiles for the reactions of
BHA and DFO with DEDNPP and EDNPP (Figures 1 and 2)
kobs = k0 + kOH[OH] + kN[BHA]χ
−
(1)
BHA
The rate constants k0, kOH, and kN correspond to the first-order
rate constant for the spontaneous hydrolysis and the second-
order rate constants for the reaction of the di- and triesters with
hydroxide ion and the hydroxamate nucleophile, respectively.
−
The term χBHA represents the molar fraction of benzohydrox-
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Table 1. Kinetic Parameters, Activation Parameters, and
Kinetic Solvent Isotope Effects for the Reactions of DEDNPP
and EDNPP with BHA, pH 10.5, μ = 1.0 M, and 25 °C
DEDNPP
EDNPP
a
k0, s−1
(8.00 0.50) × 10−6
(4.20 0.20) × 10−1
2.74 0.03
9.16
(3.60 0.60) × 10−8
(4.90 0.50) × 10−5
(2.70 0.04) × 10−3
a
kOH
kN, M−1 s−1
,
M−1 s−1
a
pKa (BHA)
b
ΔH⧧, kcal/mol
+8.9 0.2
+16.8 0.8
−20.5 2.6
22.78
b
ΔS⧧, cal
−33.1 0.7
18.75
b
ΔG⧧, kcal/mol
kH/kD
1.14
1.08
a
b
Consistent with literature values.22,23 Calculated at pH 10.5 with
eqs. S4−S6 and ΔG⧧ at 25 °C.
The experimental data for DFO were fitted to an extended
version (eq 2) of this equation, which takes account of the
multiple acid dissociations of DFO, with the subscripts 1 to 4
referring to the neutral, monoanionic, dianionic, and trianionic
species of DFO (Scheme 2).20 The kinetic parameters obtained
are given in Tables 1 and 2, for BHA and DFO, respectively.
kobs = k0 + kOH[OH] + (k1χ + k2χ + k3χ + k4χ4)
1
2
3
× [DFO]
(2)
Table 2. Kinetic Results for the Reactions of DEDNPP and
a
EDNPP with DFO at 25 °C
■
Figure 2. pH−rate profiles for the reactions of (A) DEDNPP ( )and
●
(B) EDNPP ( ) with 0.01 M DFO, μ = 1.0 M, 25 °C. The spontaneous
hydrolysis reactions for the esters are shown for comparison: DEDNPP
DEDNPP
EDNPP
k1, M−1 s−1
k2, M−1 s−1
k3, M−1 s−1
k4, M−1 s−1
(1.65 0.51) × 10−1
(3.48 0.55) × 10−1
1.40 0.03
1.38 0.03
8.96
−
−
22,23
□
○
( ) and EDNPP ( ).
(2.12 0.82) × 10−4
(1.37 0.09) × 10−3
Scheme 4. Reactions of BHA with DEDNPP and EDNPP
b
DFO, pKa2
DFO, pKa3
DFO, pKa4
b
b
9.55
10.79
a
b
Values of k0 and kOH are given in Table 1. Values taken from ref 19.
The results show BHA to be one of the most powerful known
nucleophiles toward phosphate esters such as DEDNPP and
EDNPP. Toward the model diester EDNPP only the more basic
hydroperoxide and hydroxylamine anions are known to be more
reactive.24 The nucleophilic center of rigid benzohydroxamate is
exposed, while those of DFO are surrounded by an extended
conformationally mobile structure that could offer some steric
hindrance to reaction, with the most basic center reacting fastest
(see Tables 1 and 2). It is interesting to note that because DFO is
already used as a drug, the high reactivity against triesters
suggests a further potential use for DFO, in cases of acute
poisoning with phosphorus pesticides such as paraoxon
derivatives (see below for some kinetic results reinforcing this
hypothesis). In terms of absolute reactivity the triester DEDNPP
is the most reactive, as expected. However, the diester EDNPP is
not the least reactive in the series, because (i) the reaction with
the monoester DNPP involves the electrostatically unfavorable
attack of the BHA monoanion on a dianion and (ii) α-effects are
minimal for reactions of monoester dianions.25 Table 1 includes
thermodynamic activation parameters and kinetic solvent
isotope effects for reaction with BHA. The large and negative
entropies of activation and low solvent deuterium isotope effects
kH/kD indicate that the reactions are at least primarily
amate ion.6 The reaction showed a first-order dependence in
relation to BHA, as shown by the linear plot of kobs versus [BHA]
(see Supporting Information). The pH−rate profiles for the
formation of DNP shown in Figure 1A and 1B were fitted by
using eq 1, considering initially the reaction in the absence of
BHA, which allowed us to estimate rate constants for the
spontaneous hydrolysis of the esters (k0), and the reactions
(kOH) with OH− to generate dinitrophenoxide ion. These
reactions are relatively unimportant at pH 9−10, where the
products for the reaction of BHA were examined, and their
contribution to the observed rate constant are negligible:
allowing a convenient fitting of the data with eq 1 and a
confident determination of the rate constant kN. The kinetic
parameters obtained for the reactions of BHA with DEDNPP
and EDNPP are given in Table 1.
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nucleophilic. In the reaction of DNPP with BHA and DFO there
is a small acceleration of about 20% in relation to the rate
constant determined for the hydrolysis reaction: kobs= 1.80 ×
10−5 s−1 at 25 °C, pH 9, and ionic strength 1.0 M26 (experimental
data given in Tables S1 and S9 in the Supporting Information).
Products of the Reactions. To test if the reactions are
nucleophilic and to elucidate mechanisms for all these reactions,
we carried out two series of spectroscopic (1H and 31P NMR,
ESI-MS) investigations. The results indicate that the reactions of
BHA with phosphate diester EDNPP and triester DEDNPP
follow parallel paths (Scheme 5), with hydroxamate attack on the
plicities, and coupling constants for the species detected are
shown in Table S12). Peaks were assigned by comparison with
spectra of the pure compounds, and available literature data.2,3
1
To confirm the structural assignment of product 3, H NMR
spectra were recorded for the reaction of BHA with 1-chloro-2,4-
dinitrobenzene (CDNB), which involves exclusive attack on the
aromatic ring. NMR spectra obtained as a function of time for
this reaction (Figure S13, Supporting Information) demonstrate
the formation of 3 over the first 5 min: 3 subsequently breaks
down to form phenyl isocyanate, aniline, and DNP. Thus, the
aromatic intermediate 3, aniline 7, and DNP could all be detected
1
by H NMR for the reactions of BHA with DEDNPP and
Scheme 5. Nucleophilic Attack of BHA− by Two Reaction
Paths: (A) at Phosphorus and (B) on the Aromatic Ring
EDNPP, according to Scheme 5. In the case of the reaction of
BHA with the monoester DNPP (Table S14, Supporting
Information), the aromatic intermediate 3 is not observed,
consistent with the rather small acceleration observed, which
probably proceeds exclusively by attack on phosphorus.
Figure 3A and 3B shows the courses of the reactions of BHA
with EDNPP and DEDNPP, respectively, according to Scheme
aromatic ring (path B), giving an aromatic intermediate (3) and a
less reactive phosphate ester (1 or 2). Hydroxamate attack on
phosphorus (path A) generates dinitrophenol (DNP) and a
phosphorylated intermediate (4 or 5). Intermediates 4 and 5
undergo rapid Lossen rearrangements to produce phenyl
isocyanate (6), which is hydrolyzed to aniline (7). Phenyl
isocyanate can then also react with BHA or with a molecule of the
aniline produced, giving the carbamyl derivative 8 and
diphenylurea (9), respectively. Thus, BHA reacts as a self-
destructive molecular scissor, which loses its nucleophilicity
upon reaction. The Lossen rearrangement has been observed
previously for the reactions of hydroxamic acid with sulfonyl and
phosphoryl halides,7,27 and more recently for BDNPP,6 as the
first evidence of this type of reaction in dephosphorylation
reactions.
Figure 3. Relative concentration vs time profiles obtained by 1H NMR
for the reaction of BHA (0.105 M at pH = 10) with 5 × 10−3 M of (A)
EDNPP and (B) DEDNPP, at 25 °C.
1
5, as followed continuously by H NMR, in terms of relative
concentration vs time profiles for the most important species,
namely the rapid disappearance of EDNPP and DEDNPP and
the appearance of DNP and the aromatic intermediate 3. DNP
forms from the start, and more is generated during the reaction,
presumably from the breakdown of 3. As expected, the
phosphorylated intermediates are too unstable to be detected
by this technique, but attack of BHA on phosphorus (path A,
Scheme 5) is demonstrated by the initial formation of DNP,
which must involve path A. The formation of aniline (7) is
consistent with the mechanism proposed. For the reactions with
DEDNPP, the substrate disappears after 10 min, with aniline
reaching a concentration maximum at about 20 min, followed by
a slow transformation to other Lossen degradation products, and
the corresponding decrease in concentration. By contrast, aniline
continues to appear throughout the much slower reaction with
the diester EDNPP. It was possible to derive percentages of
attack by BHA on phosphorus and the aromatic center (Table 3),
Following Reactions by NMR. We followed the dephos-
1
phorylation reactions involving BHA and DFO by H NMR.
Because of the complex nature of the intermediates and products
formed in the reactions of DFO,20 we could make NMR
assignments accurately only for BHA. The conclusions we reach
for BHA can be extended with reasonable confidence to reactions
with DFO. Sequential 1H NMR spectra for the reactions of BHA
with DEDNPP and EDNPP show the formation of important
species of Scheme 5, as expected from previous results on the
reaction of BHA with BDNPP.6 (For details, see Figures S10 and
S11 of the Supporting Information. Chemical shifts, multi-
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because the DNP detected is produced exclusively by attack at
phosphorus.
this spectrum, a series of key anions was detected and identified
including (i) the (deprotonated) intermediate 3 of m/z 302, (ii)
the BHA dimer of m/z 273, (iii) the (deprotonated) carbamyl
derivative 8 of m/z 255, produced by a Lossen rearrangement,
(iv) the phenoxide DNP of m/z 183, (v) the anion of the reagent
BHA of m/z 136, (vi) and (deprotonated) aniline of m/z 92. The
same species were detected for the reaction of BHA (0.05 M)
with EDNPP.
ESI-MS/MS was then used to characterize some of these
important species via collision-induced dissociation (CID). The
resulting tandem ESI-MS/MS(−) for carbamyl derivative 8 (m/z
255) shows (Figure 5) that it dissociates to BHA (m/z 136) and
the aniline anion (m/z 92). ESI-MS/MS spectra of other species
detected in the reactions of BHA with DEDNPP and EDNPP
appear in the Supporting Information (Figures S15 and S16).
This ESI-MS study is crucial to the investigation because species
such as the carbamyl derivative 8 were detected that could not be
observed by NMR.
We also recorded ESI-MS spectra for the reaction of BHA with
DNPP, where we observed very small amounts of (i) carbamyl
derivative 8 (deprotonated) of m/z 255, (ii) the phenoxide DNP
of m/z 183, (iii) the BHA anion of m/z 136, and (iv) aniline
(deprotonated) of m/z 92. The absence of the deprotonated
intermediate 3 (of m/z 302) indicates that the small contribution
(rate increase of ≈20%) to the observed hydrolysis due to the
reaction of BHA with DNPP probably proceeds by attack on
phosphorus. ESI-MS spectra for species detected in the reactions
of BHA with DNPP are shown in Figure S17.
Table 3. Percentages of Attack by BHA on Phosphorus and
the Aromatic Center in the Reactions with DEDNPP and
EDNPP
DEDNPP
EDNPP
C
P
%
%
45
55
34
66
N
N
Fitting the data of Figure 3A and 3B to standard coupled
differential equations for consecutive first-order reactions28 (see
the Supporting Information) gave a first approximation of the
kinetic parameters for the various reactions the values are
included in Table 4. Scheme 6 summarizes the full set of reaction
Table 4. Parameters Obtained for Fitting of Data of Figure 3A
and 3B to Equations S20−S24 for Reaction of BHA with
DEDNPP and EDNPP
DEDNPP
EDNPP
a
(k1 + k3) , M−1 s−1
2.74
2.70 × 10−3
k2, s−1
k4, s−1
k5, s−1
k6, s−1
(3.5 1.8) × 10−3
(5.0 2.3) × 10−4
(1.8 0.9) × 10−3
(2.0 0.8) × 10−4
(8.3 3.4) × 10−4
(4.0 1.8) × 10−5
(3.3 1.7) × 10−6
a
Taken from Table 1.
Reaction of DFO with Methyl Paraoxon. We performed
our full kinetic study with the model triester DEDNPP because
of its good leaving group (2,4-dinitrophenol, pKa = 4.07).
However, because DFO is already used as a drug, our results
suggest a further potential use for DFO, in cases of acute
poisoning with phosphorus pesticides. One such pesticide is
paraoxon, with 4-nitrophenoxide as leaving group (pKa = 7.14).
To check this possibility, we followed the reaction of DFO with
methyl paraoxon and once again observed a rate enhancement
(Table 5), substantial enough to confirm that our conclusions
about dephosphorylation of DEDNPP can be extended to
organophosphorus pesticides.
paths considered in the fitting equations. The scheme includes
the nucleophilic reactions at phosphorus and aromatic carbon,
and the decomposition of the intermediates so formed.
Mass Spectrometric Analysis. Hydroxamate attacks both
the aromatic ring and the phosphorus centers of EDNPP and
DEDNPP, as confirmed by ESI-MS in the negative mode, which
monitors the course of reaction by collecting snapshots of its
anionic composition. Reagents, intermediates, and products
present as anions are expected to be transferred directly from the
reaction solution to the gas phase and then detected by ESI-MS.
Figure 4 shows a ESI-MS(−) spectrum recorded after 2 min for
the reaction of BHA (0.05 M) with DEDNPP (8 × 10−3 M). In
Scheme 6. Reaction Paths Considered in Fitting the Data of the Figure 3A and 3B
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Figure 4. ESI-MS after 2 min for the reaction of BHA (0.05 M) with DEDNPP (8 × 10−3 M), pH 10.5, and 25 °C.
Figure 5. ESI-MS/MS(−) of the carbamyl derivative 8 of m/z 255.
Table 5. Kinetic Results for the Reaction of DFO with Methyl Paraoxon at 25° C, pH 11, and μ = 1.0 M
(DFO), are extremely reactive toward phosphate esters. In
general, BHA and DFO follow similar reactivity patterns, with
reactions proceeding much faster with the triester diethyl 2,4-
dinitrophenyl phosphate (DEDNPP) than with the diester ethyl
CONCLUSIONS
■
The results show that simple benzohydroxamate (BHA) and the
more complex derivative, the widely used drug, deferoxamine
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Present Address
2,4-dinitrophenyl phosphate (EDNPP). The reactions with the
monoester 2,4-dinitrophenyl phosphate (DNPP) are much
slower and proceed almost exclusively by attack at phosphorus.
For the reactions of DEDNPP and EDNPP, attack on
phosphorus was confirmed by the detection of the phosphory-
lated intermediates, which undergo Lossen-type rearrangements,
resulting in the decomposition of the nucleophile. DFO, which is
used therapeutically for the treatment of acute iron intoxication,
showed high nucleophilic activity toward the triester DEDNPP
and the diester EDNPP, a result which suggested a potential use
for DFO in cases of acute poisoning with phosphorus pesticides.
The rapid detoxification of the pesticide methyl paraoxon
supports this suggestion.
⊥Department of Chemistry, Universidade Federal do Parana
(UFPR), CP 19081, CEP 81531-990, Curitiba, PR, Brazil.
́
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
We are grateful to INCT-Catal
and CAPES for support of this work and to FONDECYT
1100640.
■
́
ise, PRONEX, FAPESC, CNPq,
REFERENCES
■
(1) Cleland, W. W.; Hengge, A. C. Chem. Rev. 2006, 106, 3252.
(2) Domingos, J. B.; Longhinotti, E.; Bunton, C. A.; Nome, F. J. Org.
Chem. 2003, 68, 7051.
EXPERIMENTAL SECTION
■
(3) Domingos, J. B.; Longhinotti, E.; Brandao, T. A. S.; Santos, L. S.;
Eberlin, M. N.; Bunton, C. A.; Nome, F. J. Org. Chem. 2004, 69, 7898.
(4) Bunton, C. A.; Gillitt, N. D.; Foroudian, H. J. Langmuir 1998, 14,
4415.
(5) Simanenko, Y. S.; Prokop’eva, T. M.; Popov, A. F.; Bunton, C. A.;
Karpiche, E. A.; Savelona, V. A.; Ghosh, K. K. Russ. J. Phys. Chem. 2004,
40, 1337.
(6) Orth, E. S.; da Silva, P. L. F.; Mello, R. S.; Bunton, C. A.; Milagre, H.
M. S.; Eberlin, M. N.; Fiedler, H. D.; Nome, F. J. Org. Chem. 2009, 74,
5011.
(7) Hurd, C. D.; Bauer, L. J. Am. Chem. Soc. 1954, 76, 2791.
(8) Samuel, D.; Silver, B. L. J. Am. Chem. Soc. 1963, 85, 1197.
(9) Kiss, T.; Farkas, E. J. Inclusion Phenom. Mol. Recognit. Chem. 1998,
32, 385.
(10) Maggio, A. Br. J. Hamaetol. 2007, 138, 407.
(11) Kontoghiorghes, G. J. Toxicol. Lett. 1995, 80, 1.
(12) Cappellini, M. D.; Pattoneri, P. Annu. Rev. Med. 2009, 60, 25.
(13) Abetz, L.; Baladi, J. F.; Jones, P.; Rofail, D. Health Qual. Life
Outcomes 2006, 4.
(14) Lederman, H. M.; Cohen, A.; Lee, J. W. W.; Freedman, M. H.;
Gelfand, E. W. Blood 1984, 64, 748.
(15) Becton, D. L.; Roberts, B. Cancer Res. 1989, 49, 4809.
(16) Dayani, P. N.; Bishop, M. C.; Black, K.; Zeltzer, P. M. J.
Neurooncol. 2004, 67, 367.
Materials. Distilled water was used throughout these experiments.
Deferoxamine (DFO), 2,4-dinitrophenol (DNP), and 1-chloro-2,4-
dinitrobenzene (CDNB) were of the highest purity and were used as
received. BHA,6 DEDNPP,29 EDNPP,29 and DNPP30 were prepared
according to procedures described in the literature.
Kinetic Measurements. Reaction rates were followed by UV−vis
spectrophotometry by monitoring the appearance of DNP at 400 nm in
the thermostatted cell holder of a diode-array spectrophotometer
maintained at 25.0 0.1 °C. Reaction was initiated by injecting 30 μL of
the substrate (1 × 10−3 M) from a stock solution into 3 mL of the
reaction mixture, with a large excess (>0.01 M) of the nucleophile
ensuring first-order-kinetics for the initial nucleophilic attacks upon the
substrates. Ionic strength was maintained at 1.0 M with KCl. The pH
was controlled using external Tris, Na2CO3, and phosphate buffers (0.01
M). Absorbance versus time data were stored directly on a micro-
computer and first-order rate constants kobs obtained from linear plots of
ln(A∞ − At) against time for at least 90% of reaction by using an iterative
least-squares program; correlation coefficients were >0.999 for all
kinetic runs.
1H NMR Experiments. 1H NMR spectra were recorded in D2O with
sodium 3-(trimethylsilyl)propionate (TMSP) as internal reference
except for reactions with 1-chloro-2,4-dinitrobenzene, where 10%
CD3CN was used. Some aromatic 1H signals were obscured by signals of
the excess BHA−. The pDs of the solutions were corrected considering
pD = pHread + 0.4 at 25 °C.
Mass Spectrometry. To identify intermediates and reaction
products of phosphate esters with BHA, direct infusion electrospray
ionization mass spectrometry analyses were performed using a hydrid
triple quadrupole linear ion-trap mass spectrometer.6 A microsyringe
pump delivered the reagent 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 mode. Main conditions: curtain gas
nitrogen flow = 20 mL min−1; ion spray voltage = −4500 eV;
declustering potential = −21 eV; entrance potential = −10 eV; collision
cell exit potential = −12 eV. The carbamyl derivative 8 detected by ESI-
MS was subjected to ESI-MS/MS by using collision-induced
dissociation (CID), with nitrogen and collision energies ranging from
5 to 45 eV. Other MS analyses were performed to detect nonanionic
products in the reaction medium.
(17) Daniels, J. S.; Gates, K. S. J. Am. Chem. Soc. 1996, 118, 3380.
(18) Miller, M. J. Chem. Rev. 1989, 89, 1563.
(19) Farkas, E.; Enyedy, E. A.; Csoka, H. Polyhedron 1999, 18, 2391.
(20) Orth, E. S.; Medeiros, M.; Bortolotto, T.; Terenzi, H.; Kirby, A. J.;
Nome, F. J. Org. Chem. 2011, 76, 10345.
(21) Mora, J. R.; Kirby, A. J.; Nome, F. J. Org. Chem. 2012, 77, 7061.
(22) Kirby, A. J.; Younas, M. J. Chem. Soc. B 1970, 1165.
(23) Orth, E. S.; Wanderlind, E. H.; Medeiros, M.; Oliveira, P. S. M.;
Vaz, B. G.; Eberlin, M. N.; Kirby, A. J.; Nome, F. J. Org. Chem. 2011, 76,
8003.
(24) Kirby, A. J.; Manfredi, A. M.; Souza, B. S.; Medeiros, M.; Priebe, J.
P.; Brandao, T. A. S.; Nome, F. ARKIVOC (Gainesville, FL, U. S.) 2009,
28.
(25) Kirby, A. J.; Varvoglis, A. G. J. Chem. Soc. B 1968, 135.
(26) Bunton, C. A.; Fendler, E. J.; Fendler, J. H. J. Org. Chem. 1967, 89,
1221.
(27) Yale, H. L. Chem. Rev 1943, 33, 209.
ASSOCIATED CONTENT
■
(28) McQuarrie, D. A.; Simon, J. D. Physical Chemistry: A Molecular
Approach; University Science Books: Mill Valley, CA, 1997.
(29) Moss, R. A.; Ihara, Y. J. Org. Chem. 1983, 48, 588.
(30) Rawji, G.; Milburn, R. M. J. Org. Chem. 1981, 46, 1205.
S
* Supporting Information
Tables and figures giving kinetic, ESI-MS(/MS), and 1H and 31P
NMR data. This material is available free of charge via the
Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*Tel.: +55-48-3721-6849; fax: +55-48-3721-6850; e-mail: Faruk.
Nome@ufsc.br.
10913
dx.doi.org/10.1021/jo302374q | J. Org. Chem. 2012, 77, 10907−10913