Biologically relevant chemical properties of peroxymonophosphate ({double bond, long}O3POOH)

Article abstract of DOI:10.1016/j.bmcl.2008.10.133

It has been suggested that peroxymonophosphate could serve as an endogenous hydrogen peroxide-derived regulator of cellular protein tyrosine phosphatase activity under physiological or pathophysiological conditions. To facilitate further consideration of

Full text of DOI:10.1016/j.bmcl.2008.10.133

Bioorganic & Medicinal Chemistry Letters 19 (2009) 218–221
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Biologically relevant chemical properties of peroxymonophosphate (@O3POOH)
*
Jason N. LaButti, Kent S. Gates
Departments of Chemistry and Biochemistry, University of Missouri, 125 Chemistry Building, Columbia, MO 65211, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
It has been suggested that peroxymonophosphate could serve as an endogenous hydrogen peroxide-
derived regulator of cellular protein tyrosine phosphatase activity under physiological or pathophysiolog-
ical conditions. To facilitate further consideration of the potential role of peroxymonophosphate in bio-
logical systems we present studies related to the preparation, characterization, stability, and fluorometric
detection of this agent.
Received 8 September 2008
Revised 22 October 2008
Accepted 27 October 2008
Available online 5 November 2008
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords:
Protein tyrosine phosphatase
Signal transduction
Hydrogen peroxide has emerged as a signaling agent that regu-
lates cellular responses to a variety of extracellular stimuli includ-
ing insulin, epidermal growth factor, platelet-derived growth
factor, endothelin-1, and B-cell receptor stimulation.1 H2O2 is gen-
erated by NADPH oxidase enzymes (Nox), which are activated by
the binding of these growth factors and cytokines to cell surface
receptors.2 Protein tyrosine phosphatases (PTPs) are important cel-
lular targets of hydrogen peroxide.1,3–6 PTPs work in tandem with
protein tyrosine kinases to regulate the phosphorylation status of
proteins involved in critical signal transduction pathways.7–9
Accordingly, peroxide-mediated inactivation of target PTPs, involv-
ing oxidation of the catalytic cysteine residue in these en-
zymes,3,4,10,11 has the potential to profoundly influence the
duration and intensity of cellular responses to various stimuli.
Cellular responses to hydrogen peroxide are generally rapid,
occurring within 5–10 min.12,13 Interestingly, kinetic measure-
ments on isolated PTPs suggest that, at the low concentrations gen-
erated during cell signaling processes, H2O2 is a rather slow PTP
inactivator.14 For example, the inactivation of the archtypal mem-
ber of the PTP class of enzymes, PTP1B,9 by a steady state concen-
phate, this would involve the reaction of hydrogen peroxide with
one of the many phosphoryl donors, such as adenosine triphos-
phate, that are present inside cells (Scheme 1). Phosphoryl transfer
to oxygen nucleophiles is a common reaction in biological sys-
tems.18 Phosphoryl transfer to H2O2 may be possible even in the
presence of vast excesses of water due to the exceptional nucleo-
philicity of peroxide.19 The hydrogen peroxide anion, HOOÀ, is
much more nucleophilic than HOÀ (kHOO–/kHO– ꢀ 100).19 Reactions
with peroxide are further facilitated by the fact that a greater frac-
tion exists in the reactive, anionic form at physiological pH (H2O2,
pKa = 11.6; H2O, pKa = 15.7).19
Importantly, we recently showed that peroxymonophosphate
inactivates PTP1B about 10,000 times faster than H2O2.17 Accord-
ingly, nanomolar concentrations of peroxymonophosphate (20–
40 nM) are capable of inactivating PTPs within the biologically
relevant time frame of 5–10 min. To facilitate further consideration
of the potential role of peroxymonophosphate in biological sys-
tems we present studies related to the preparation, characteriza-
tion, stability, and detection of this agent.
Peroxymonophosphate was prepared by a modification of the
methods described by Griffith and Battaglia.20,21 Briefly, an aque-
ous solution of potassium phosphate (4.12 M), potassium hydrox-
ide (6.6 mM), and potassium fluoride (3.9 M) was electrolyzed at
10ꢀC, 10 V, 400 mA, for 6 h on three consecutive days to generate
crude potassium peroxydiphosphate as a precipitate.21 Lithium
peroxydiphosphate was then prepared by a metathesis reaction
tration of 1 lM H2O2 is predicted to occur with a half-life of about
20 h. It is possible that co-localization of Nox and PTP enzymes in-
side cells produces a high local concentration of H2O2 near the
phosphatase target, thus leading to rapid enzyme inactivation.15,16
Alternatively, we have considered that the kinetic discrepancy
might be explained by a scenario in which intracellular hydrogen
peroxide is converted either spontaneously or enzymatically into
inorganic peroxides such as peroxymonophosphate, peroxymono-
sulfate, or peroxymonocarbonate that are more potent oxidizing
agents than the parent peroxide.17 In the case of peroxymonophos-
O
O
HO O P O–
O–
HO O–
–O P OR
O–
+
+
HOR
* Corresponding author. Tel.: +1 573 882 6763; fax: +1 573 882 2754.
Scheme 1. Phosphoryl transfer to hydrogen peroxide has the potential to produce
E-mail address: GatesK@missouri.edu (K.S. Gates).
peroxymonophosphate in cells.
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.10.133

J. N. LaButti, K. S. Gates / Bioorg. Med. Chem. Lett. 19 (2009) 218–221
219
and recrystallized from methanol–water at 45ꢀC to give pure mate-
rial.21 Incubation of a solution of lithium peroxydiphosphate
(200 mM) in perchloric acid (1 M) at 50ꢀC for 1 h yields a mixture
of lithium phosphate and lithium peroxymonophosphate.20 It is
worth noting that we found commercially available peroxydiphos-
phate to be unsuitable in this preparation due to the very impure
nature of the material.
The product was characterized by 31P NMR and mass spectrome-
try. The 31P NMR shift of the product is pH-dependent giving a reso-
nance at 4.27 ppm in 0.5 M HClO4 versus 85% phosphoric acid as an
external standard (Fig. 1). With careful selection of instrument set-
tings,22 it is possible to use 31P NMR to quantitatively measure the
concentration of peroxymonophosphate in solutions by comparison
to an internal diphenyl phosphate standard of known concentration.
Alternatively, iodometric titration can be used to measure the con-
centration of peroxymonophosphate in solutions.20 Using electro-
spray mass spectrometry operating in the positive ion mode, the
monoisotopic molecular ion was detected at 114.7 (M+H)+ (calcu-
lated 115.0). MS/MS analysis of the ion at 114.68 reveals consecutive
neutral losses of 17 mass units, as expected for a phosphate deriva-
tive. On a practical note, we prefer the preparation of peroxymono-
phosphate described above over a more recent method involving
reaction of 70% H2O2 with phosphorus pentoxide.23 We found that
the reaction using 70% H2O2 was difficult to control, sometimes lead-
ing to vigorous exotherms as noted by the original authors.23 More
importantly, 31P NMR analysis revealed that the 70% H2O2 process,
in our hands, produced mixtures that may include peroxydiphos-
phate and diperoxyphosphate derivatives alongside the desired
peroxymonophosphate.
2À
Figure 2. The stability of peroxymonophosphate (HPO5
)
in the presence of
2À
biologically relevant additives. A solution of HPO5 (10 mM final concentration)
was combined with additive and the pH was adjusted with concentrated NaOH.
2À
After standing for 1 h at room temperature, the
%
remaining HPO5
was
determined by quantitative 31P NMR (peak area) using a freshly made solution of
2À
HPO5 in 100 mM HClO4 as a control. Values shown are the averages of three
independent experiments and the error bars depict the standard error in the
measurements.
We examined the stability of peroxymonophosphate in the
presence of several commonly used buffers and biologically rele-
vant substrates. We find that peroxymonophosphate is quite stable
in HClO4 (100 mM) over the course of 1 h at 24ꢀC (Fig. 2). Similarly,
peroxymonophosphate is stable in sodium phosphate (100 mM, pH
7) and bis–tris buffer (100 mM, pH 7) under these conditions. In
contrast, peroxymonophosphate is completely destroyed upon
incubation with Hepes buffer (100 mM, pH 7) for 1 h at 24ꢀC. Sim-
ilarly, addition of the biological thiol, glutathione (10 mM), to a
sodium phosphate buffered solution leads to complete decomposi-
tion of the peroxymonophosphate. The sulfide-containing amino
acid methionine also destroys peroxymonophosphate. Tryptophan
and glycine lead to only small amounts of peroxymonophosphate
decrease in the concentration of peroxymonophosphate. Neither
1% dimethyl sulfoxide (140 mM) nor the hydrogen peroxide-
destroying enzyme catalase have significant effects on the stability
of peroxymonophosphate under these conditions.
Finally, we examined the potential of 3-oxo-3H-phenoxazin-7-
yl pinacolatoboron (PC-1, 1) to serve as a fluorescent sensor of per-
oxymonophosphate in biochemical and biological systems. Com-
pound
1 was designed by Chang and coworkers as an
intracellular sensor for H2O2.24 Their clever design capitalizes on
the fact that reaction of peroxide with the boronate ester group
in 1 leads to release of the highly fluorescent resorufin dye
(Scheme 2). Peroxymonophosphate is a more reactive oxidizing
agent than hydrogen peroxide thus, we anticipated that peroxy-
monophosphate might convert 1 to its fluorescent form. Indeed,
we find that peroxymonophosphate rapidly ‘‘lights up” solutions
of 1 in bis–tris buffer (50 mM, pH 7) at 22ꢀC. The rate constant
decomposition. Addition of FeSO4 (10 mM) results in
a 60%
for the reaction of peroxymonophosphate with
1
is
1447 52 MÀ1 sÀ1 (Fig. 3). For comparison, we determined that
H2O2 converts 1 to the fluorescent product with a rate constant
of 1.21 0.17 MÀ1 sÀ1
.
In summary, we have conducted the first survey of the reactiv-
ity of peroxymonophosphate under biologically relevant condi-
tions. Peroxymonophosphate is substantially more reactive than
hydrogen peroxide as an oxidant.17,25 Nonetheless, we find that
the selectivity of peroxymonophosphate towards reaction with
various biochemicals, in many regards, mirrors that of H2O2. Per-
oxymonophosphate is stable in perchloric acid, sodium phosphate
buffer, and bis–tris buffer. These results are consistent with those
of Battaglia and Edwards who reported the half-life of peroxy-
monophosphate to be 12.5 h in 4 M HClO4 (rate of decomposition,
k = 1.54 Â 10À5 sÀ1).20
Peroxymonophosphate is unstable in the presence of Fe(II). Pre-
sumably this breakdown involves a Fenton-type reaction analo-
gous to the well known metal-mediated decomposition of
H2O2.26 In addition, we showed that Hepes buffer, the biological
thiol glutathione, and the sulfide-containing amino acid methio-
Figure 1. 31P NMR of peroxymonophosphate in HClO4 (0.5 M). The NMR was
calibrated against an external standard of 85% phosphoric acid. Peroxymonophos-
phate appears at 4.27 ppm, phosphate at 0.59 ppm, and diphenyl phosphate added
as an internal standard at À8.35 ppm. The small peak at 3.13 ppm is assigned to a
trace of peroxydiphosphate. This impurity disappears as the sample ages.

220
J. N. LaButti, K. S. Gates / Bioorg. Med. Chem. Lett. 19 (2009) 218–221
ROO–
RO
O
O
B
O
B—
O
O
O
O
O
N
O
N
PC-1
(1)
O
O
B
H2O
O
O
O
O
O
N
OH
O
O
+
+
ROH
B
N
OH
Resorufin
Ex=550; λEm=585
λ
Scheme 2. Oxidative deboronation of PC-1 (1).
have reported reactions of peroxymonophosphate with sulfoxides
and indoles;31,32 however, these earlier studies were conducted
under conditions where the protonated species H3PO5 was pre-
dominant. In contrast, with pKa values of 1.0, 5.5, and 12.8, peroxy-
monophosphate exists primarily as the dianion under the
conditions of our experiments (pH 7).20,32 A number of studies
show that the reactivity of peroxymonophosphate is pH-depen-
dent, increasing at lower pH values as the oxygens become increas-
ingly protonated.25,30,31 A most striking difference between H2O2
and peroxymonophosphate, is revealed by our observation that
the H2O2-destroying enzyme catalase does not decompose peroxy-
monophosphate. The inability of catalase to decompose peroxy-
monophosphate is in alignment with the observation that
another bulky hydroperoxide, t-butyl hydroperoxide, is a poor sub-
strate for the enzyme.33,34
Peroxymonophosphate readily ‘‘lights up” the fluorescent per-
oxide sensor, PC-1. The reaction of PC-1 with peroxymonophos-
phate is approximately 1200 times faster than that with H2O2.
This finding highlights the possibility that boronate ester probes
might preferentially detect a secondary, H2O2-derived oxidant
such as peroxymonophosphate, if this species were generated in-
side cells. Taken together, elements of the work described here
provide a foundation for the development of assays designed to de-
tect spontaneous or enzyme-catalyzed conversion of H2O2 to per-
oxymonophosphate in biochemical or biological systems. After
incubation of H2O2 with a phosphoryl donor substrate (perhaps
in the presence of a putative enzymatic catalyst for the reaction),
catalase can be used to destroy excess H2O2. Catalase treatment
leaves peroxymonophosphate intact (see data in Fig. 2) and addi-
tion of the peroxide sensor PC-1 provides a means for highly sen-
Figure 3. Kinetics of the reaction of peroxymonophosphate with PC-1 (1). Various
amounts of peroxymonophosphate were added to a 100 nM solution of 1 in 50 mM
bis–tris, pH 7.0 at 22 °C. (A) The resulting pseudo-first-order rate of fluorescence
increase was immediately measured using an SLM-AMINCO model 8100 spectro-
fluorimeter (Ex: 550 nm, Em: 585 nm, 4 nm slit widths). (B) Replotting the pseudo-
first-order rates derived from this data gives the apparent second order rate of
reaction. Values shown in both graphs are averages of three independent
experiments in which the error bars depict the standard error in the measurements.
sitive detection of peroxymonophosphate produced under
a
given set of reaction conditions. This general approach might be
amenable to either a high-throughput microplate reader or in-gel
assays that search for peroxymonophosphate-producing enzymes
in a proteome. Such tools will help explore the possibility that per-
oxymonophosphate participates in the regulation or dysregulation
of cell signaling processes under physiological or pathophysiologi-
cal conditions.
nine completely decompose peroxymonophosphate. Again this is
analogous to the reactivity of H2O2 with Hepes,27 glutathione,28
and methionine.29 Our result with methionine in pH 7 buffer is
Acknowledgments
consistent with previous work showing
a facile reaction of
peroxymonophosphate with aryl sulfides in acetonitrile–water
mixtures.30 We observe that, DMSO glycine, and the indole-con-
taining amino acid tryptophan do not cause substantial decompo-
sition of peroxymonophosphate in neutral aqueous buffer. Others
We thank the National Institutes of Health for financial support
of this work (CA 83925 and CA 119131). We thank Wei Weicoff for
assistance with the 31P NMR experiments. We also gratefully

J. N. LaButti, K. S. Gates / Bioorg. Med. Chem. Lett. 19 (2009) 218–221
221
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