(2,5-Dioxoimidazolidin-4-ylidene)aminocarbonylcarbamic Acid as a Precursor of Parabanic Acid, the Singlet Oxygen-Specific Oxidation Product of Uric Acid

Article abstract of DOI:10.1021/acs.joc.9b00163

Previously, we identified that parabanic acid (PA) and its hydrolysate, oxaluric acid (OUA), are the singlet oxygen-specific oxidation products of uric acid (UA). In this study, we investigated the PA formation mechanism by using HPLC and a time-of-flight mass spectrometry technique and identified unknown intermediates as (2,5-dioxoimidazolidin-4-ylidene)aminocarbonylcarbamic acid (DIAA), dehydroallantoin, and 4-hydroxyallantoin (4-HAL). DIAA is the key to PA production, and its formation pathway was characterized using ¹⁸O₂ and H₂¹⁸O. Two oxygen atoms were confirmed to be incorporated into DIAA: the 5-oxo- oxygen from singlet oxygen and the carboxylic oxygen from water. Isolated DIAA and 4-HAL gave PA stoichiometrically. A plausible reaction scheme in which two pathways branch out from DIAA is presented, and the potential for PA as an endogenous probe for biological formation of singlet oxygen is discussed.

Full text of DOI:10.1021/acs.joc.9b00163

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Article
(2,5-Dioxoimidazolidin-4-ylidene)aminocarbonylcarbamic acid as a precursor
of parabanic acid, the singlet oxygen-specific oxidation product of uric acid
Sayaka Iida, Yorihiro Yamamoto, and Akio Fujisawa
J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.9b00163 • Publication Date (Web): 26 Feb 2019
Downloaded from http://pubs.acs.org on February 28, 2019
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(
2,5-Dioxoimidazolidin-4-ylidene)aminocarbonylcarbamic acid as a
precursor of parabanic acid, the singlet oxygen-specific oxidation
product of uric acid
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Sayaka Iida, Yorihiro Yamamoto, and Akio Fujisawa*
School of Bioscience and Biotechnology, Tokyo University of Technology, 1404-1 Katakura-cho, Hachioji, Tokyo 192-0982,
Japan
ABSTRACT: Previously we identified that parabanic acid (PA) and its hydrolyzate, oxaluric acid (OUA), are the singlet oxygen-
specific oxidation products of uric acid (UA). In this study we investigated the PA formation mechanism by using HPLC and a
time-of-flight mass spectrometry technique and identified unknown intermediates as (2,5-dioxoimidazolidin-4-
ylidene)aminocarbonylcarbamic acid (DIAA), dehydroallantoin, and 4-hydroxyallantoin (4-HAL). DIAA is the key to PA produc-
1
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tion and its formation pathway was characterized using
2 2
O and H O. Two oxygen atoms were confirmed to be incorporated into
DIAA: the 5-oxo- oxygen from singlet oxygen and the carboxylic oxygen from water. Isolated DIAA and 4-HAL gave PA stoichi-
ometrically. A plausible reaction scheme in which two pathways branch out from DIAA was presented and the potential for PA as
an endogenous probe for biological formation of singlet oxygen was discussed.
INTRODUCTION
4-hydroxyallantoin (4-HAL) by using time-of-flight mass
spectrometry (TOFMS). Two oxygen atoms were confirmed to
The ability to detect reactive oxygen species (ROS) or reac-
tive nitrogen species (RNS) in pathological events is highly
desired, since they are considered to play a key role in many
diseases. Monitoring end products of these reactive species
and substrates, such as lipids, proteins, and nucleic acids, is a
reasonable way for identifying ROS and RNS in vivo because
these species are too unstable to detect directly. We believe
that uric acid (UA) is an ideal endogenous probe for this pur-
pose because it exists ubiquitously in human bodily fluids at
18
be incorporated into the DIAA molecule by using
2
O and
1
8
1
2 2
H O; one from O and the other from water. Moreover, we
isolated DIAA and 4-HAL and found that they gave PA and
OUA as final products. We then presented a plausible reaction
scheme.
1
relatively high concentrations (e.g., ~300 µM in plasma ) and
reacts with ROS and RNS, yielding different products (see
additional details later in this section).
UA is an interesting molecule. It is a terminal metabolite of
purine in primates including humans and also an essential
water-soluble antioxidant. UA gives specific oxidation prod-
ucts after its reaction with various ROS and RNS. Allantoin
1,2
(AL) is produced by free radical-induced oxidation, triuret
3,4
–
5
(
TU) by ONOO , 6-aminouracil (6-AU) by NO, 5-N-
carboxyimino-6-N-chloroaminopyrimidine-2,4(3H)-dione
(CCPD) by hypochlorite, and parabanic acid (PA) by singlet
oxygen ( O ) (Figure 1). Obviously, the products are different
2
depending on the ROS and RNS; therefore UA can function as
6
7
1
an endogenous probe for each ROS and RNS.
7
In our previous study, we identified PA and its hydrolyzate,
1
oxaluric acid (OUA), as the O
2
-specific oxidation products of
UA, and confirmed no other ROS and RNS gave PA and OUA
as products. However, the PA formation pathway remains
unclear. We also found an intermediate (U3, m/z –199.01072)
but couldn’t fully characterize it. In this study, we succeeded
Figure 1. The oxidation products of UA induced by various ROS
and RNS. AL is produced by free radical-induced oxidation; 6-
–
–
AU is by ·NO; TU by ONOO ; CCPD by ClO ; and PA and its
hydrolyzate OUA by O .
2
in
identifying
U3
as
(2,5-dioxoimidazolidin-4-
1
ylidene)aminocarbonylcarbamic acid (DIAA) and two other
intermediates (U4 and U5) as dehydroallantoin (DHAL) and
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Figure 2. HPLC chromatograms of reaction mixture after 60 min of UA photooxidation induced by 10 µM Rose Bengal and UVA irradia-
tion (1.12 mW/cm ) and MS spectra of observed products U3, U4, and U5. (A) The chromatograms, monitored at absorption of 210 nm
2
(upper panel) and total ion intensity (lower panel) measured by optimized LC/TOFMS. The MS spectra of U3 (B), U4 (C), and U5 (D) and
their possible chemical structures according to the accurate m/z values of the predominant ions with TFA as an internal standard.
RESULTS and DISCUSSION
Photooxidation of UA. In the presence of Rose Bengal (10
µM), an aqueous solution of UA (200 µM) was photoirradiat-
2
ed by UVA (1.12 mW/cm ) for 2 hours. UA and its oxidation
products were measured by LC/TOFMS every 30 min during
photooxidation. Figure 2A shows chromatograms of the reac-
tion mixture after 60 min irradiation, monitored by the absorb-
ance at 210 nm, and total ion intensity. In addition to UA, AL
(
(
impurity), PA (U1), OUA (U2), and unidentified products
U3, U4, and U5) were observed; the retention times of the
unknowns were 5.4, 7.6, and 13.7 min, respectively. These
compounds were also formed by NEPO-initiated UA oxida-
tion (data not shown) indicating that U3, U4, and U5 were
1
2
O -induced UA oxidation products.
MS spectra of U3, U4, and U5 are shown in Figure 2B-D
and their accurate m/z values were determined to be –
199.01121, –155.01855, and –173.03023, respectively, using
TFA as an internal standard. The molecular formulas of U3,
U4, and U5 were determined as C
, indicating that they were a peroxide derivative of
UA, DHAL, and 4-HAL. Similar structures of DHAL and 4-
5 4 4 5 4 4 4 3
H N O , C H N O , and
4 6 4 4
C H N O
1
HAL were proposed in the
oxoguanine.
2
O -mediated oxidation of 8-
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Figure 3. MS spectra of PA (A), U3 (B), DHAL (C), and 4-HAL (D) formed by UA photooxidation conducted under 18
O in H O. Meas-
2 2
urements were carried out by LC/TOFMS and the m/z values were standardized against TFA.
Photooxidation of UA in 18O
and H
18O. In order to char-
2
2
acterize U3 more fully, we conducted the photooxidation in
18
2
O . Figure 3B shows that the m/z value for U3 shifted to –
18
2
01.01459, indicating one atom of O was incorporated. It is
noteworthy that the peak at m/z –199.01 was absent, suggest-
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ing that all air was replaced by
O
2
and little
2
O was left.
Similarly, PA, DHAL, and 4-HAL were monoisotopic (Fig-
1
8
ures 3A, 3C, and 3D: Insets show plausible locations for
insertion).
O
Next, we conducted the photooxidation in H
2
18O (Figure 4).
PA, U3, DHAL, and 4-HAL were formed during UA oxida-
tion similar to other oxidations. However, PA, U3, and 4-HAL
were monoisotopic (Figures 4A, 4B, and 4D: Insets show
1
8
plausible locations for O insertion), whereas DHAL was not
monoisotopic (Figure 4C). These results indicate that an O
1
atom incorporated to DHAL is derived from not water but O
2
.
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18O. Meas-
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Figure 4. MS spectra of PA (A), U3 (B), DHAL (C), and 4-HAL (D) formed by UA photooxidation conducted under air in H
urements were carried out by LC/TOFMS and the m/z values were standardized against TFA.
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Scheme 1. A plausible mechanism of PA production by O
2
-induced UA oxidation
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Taken together, these data confirm that U3 incorporated two
oxygen atoms from the outside: one from O and the other
2
1
from water. From these results, a plausible reaction scheme
8
based on a proposal by Duarte et al. was proposed (Scheme
1
1
). O
2
adds to UA, yielding UA-4,5-dioxetane (1). The 4,5-
dioxetane is converted to UA-5-hydroperoxide (2). The nucle-
ophilic addition of water on the C6 carbonyl carbon of UA-5-
OOH results in cleavage of the peroxide and the C5-C6 bond
to yield U3. U3 was determined to be DIAA. This scheme
1
suggests that an oxygen atom from O
2
is incorporated in the
5-oxo- group and another oxygen atom from water is located
on the carboxylic group of DIAA. DIAA is then converted
either to DHAL by decarboxylation or to (4-hydroxy-2,5-
dioxoimidazolidin-4-yl)aminocarbonylcarbamic
acid
(4-
hydroxyDIAA) (3) by hydration. DHAL is hydrolyzed to 4-
HAL, which decomposes to PA and urea. HDAA is rapidly
converted to PA and N-carboxyurea.
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Figure 5. Fragmentation patterns of the DIAA measured by optimized LC/TOFMS. (A) The speculated 18O atom location in DIAA mole-
1
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cule derived from [ O
2 2
] (left arrow) or H O (right arrow). The observed fragmentation of DIAA formed by UA photooxidation under
1
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normal air and in H O (B), under
2
2 2 2
O and H O (C), and under normal air and in H O (D). The negative ionization was carried out by ESI
with –2000 V of ionization potential. The m/z values were standardized against TFA as an internal standard and postulated formulas
showed their calculated m/z values to be within 5 milli-mass units from the observed values.
Analysis of DIAA fragmentation. To confirm the above
and DIAA production requires water as an oxygen donor, and
these observations also support the proposed scheme.
1
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scheme, fragmentation patterns of DIAA formed in
O
2
and in
1
8
H
2
O were compared. If the 5-oxo- and carboxylic oxygen
1
2
atoms of DIAA are derived from O and water, respectively
(
Figure 5A), their fragmentations can be distinguished. Colli-
sion-induced dissociation (CID) of DIAA (m/z –199.00952)
formed in normal air and water gave two typical fragment ions
that were caused by decarboxylation (m/z –155.02090) and
further desorption of isocyanic acid (m/z –112.01578) (Figure
5
B). Figure 5C shows the fragmentation of the monoisotopic
1
8
DIAA (m/z –201.01460) formed in
2
O . A monoisotopic
fragment of decarboxylation (m/z –157.02368) and a non-
isotopic isocyanate desorption ion (m/z –112.01543) were
1
8
observed, indicating O was incorporated in the dissociated
isocyanate fragment. On the other hand, both of the fragment
ions of the monoisotopic DIAA (m/z –201.01576) produced in
1
8
18
2
H O were non-isotopic (Figure 5D), suggesting that O lo-
cated on the carboxylic group was primarily desorbed. These
fragmentations were consistent with the proposed chemical
structure and formation scheme of DIAA. Furthermore, UA
photooxidation in DMSO led to UA decrease without PA and
DIAA formation (data not shown). It was suggested that PA
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Figure 6. PA formation from the isolated DIAA and 4-HAL. (A) The chromatograms of DIAA immediately after isolation (upper panel)
and after 60 minutes under neutral conditions (lower panel). (B) The time course of changes in the concentrations of PA plus OUA (■) and
DIAA () during storage at room temperature under neutral conditions. (C) The chromatograms of 4-HAL immediately after isolation (up-
per panel) and after 80 minutes under neutral conditions (lower panel). (D) The time course of changes in the concentrations of PA plus
OUA (■), urea (△) and 4-HAL (□) during storage at room temperature under neutral conditions.
betes, such as HbA1c, glucose, and insulin-related indices.9
Isolation and incubation of DIAA and 4-HAL. For further
confirmation of the proposed scheme, DIAA and 4-HAL were
isolated using HPLC since they were relatively stable. Incuba-
tion of these isolated compounds at neutral pH gave PA and
OUA stoichiometrically as shown in Figure 6B and 6D. On
the other hand, isolated DHAL was immediately converted
into 4-HAL (data not shown).
Furthermore, Hillered et al. showed that PA levels were ele-
1
0
vated in serious aneurysmal subarachnoid hemorrhage.
These data suggest that O
1
2
may be produced in vivo under
chronic diabetic conditions or acute severe brain damage. We
1
expect that PA will be a good indicator for monitoring O
2
production in vivo and its importance will increase in the clin-
ical fields.
The formation of urea was also observed during 4-HAL in-
cubation, indicating that 4-HAL was the precursor of urea and
PA. When DIAA was decomposed under weak acidic condi-
tions (pH 5-6), DHAL production was observed in addition to
PA formation (data not shown). It was shown that DIAA is the
precursor of not only PA but also DHAL.
CONCLUSION
In the current study, the mechanism of PA formation with
1O
-induced UA oxidation was fully characterized. We identi-
2
fied the chemical structure and formation pathway of DIAA,
which is the key intermediate for PA production. Moreover,
there are two pathways that branch out from DIAA leading to
1
Biological significance. Recently O
2
-induced oxidation in
vivo has attracted much interest. Umeno et al. reported that 10-
and 12-(Z,E)-hydroxyoctadecadienoic acids (HODE), which
2
PA production: H O addition and subsequently N-carboxyurea
1
are O
2
-induced oxidation products of linoleic acid (LA), were
elimination directly yield PA, and decarboxylation gives
DHAL. DHAL is converted into 4-HAL, which is another
precursor for PA formation. This characteristic mechanism is
detected in human plasma by LC/MS/MS analysis and the
ratio of 10- and 12-(Z,E)-HODE to LA correlated significantly
with several important clinical values for the diagnosis of dia-
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-initiated UA oxida- kyo, Japan, 5 µm, 250 mm × 2.0 mm) as a separation column
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plausible for PA formation induced by O
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tion.
and an aqueous mobile phase adjusted to pH 3.5 by formic
acid delivered at 0.2 mL/min.
EXPERIMENTAL SECTION
AUTHOR INFORMATION
Chemicals. UA, PA, Rose Bengal, H 1
dihydro-1,4-epidioxy-4-methyl-1-naphthyl)propionic
NEPO) were purchased from Wako Pure Chemical Industries,
8O and 3-(1,4-
2
Corresponding Author
* afujisawa@stf.teu.ac.jp
acid
(
Ltd. (Osaka, Japan), Tokyo Chemical Industry Co., Ltd. (To-
kyo, Japan) or Waken B Tech Co., Ltd. (Kyoto, Japan) and
Notes
used as received. OUA was prepared by hydrolysis of PA as
The authors declare no competing financial interest.
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described previously. A super dehydrated grade (H
2
O content
< 0.001%) of dimethyl sulfoxide (DMSO) was used. The puri-
REFERENCES
1
2
ty of the NEPO, which is a chemical O generator, was de-
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id provides an antioxidant defense in humans against oxidant- and
radical-caused aging and cancer: a hypothesis. Proc. Natl. Acad. Sci.
U. S. A. 1981, 78 (11), 6858–6862.
termined as 78% by the comparison of the UV absorption at
2
88 nm before and after the thermal decomposition of a meth-
1
8
anolic solution of NEPO. Pure N
2
and pure
2
O gases were
(
2) Hicks, M.; Wong, L. S.; Day, R. O. Identification of products
purchased from Tomoe Shokai Co., Ltd. (Tokyo, Japan).
from oxidation of uric acid induced by hydroxyl radicals. Free Radi-
cal Res. 1993, 18 (6), 337-351.
(3) Robinson, K. M.; Morré, J. T.; Beckman, J. S. Triuret: a novel
product of peroxynitrite-mediated oxidation of urate. Arch. Biochem.
Biophys. 2004, 423 (1), 213-217.
(4) Gersch, C.; Palii, S. P.; Imaram, W.; Kim, K. M.; Karumanchi,
S. A.; Angerhofer, A.; Johnson, R. J.; Henderson, G. N. Reactions of
peroxynitrite with uric acid: formation of reactive intermediates, al-
kylated products and triuret, and in vivo production of triuret under
conditions of oxidative stress. Nucleosides, Nucleotides Nucleic Acids
UA photooxidation. In order to identify the oxidation prod-
ucts, the photooxidation of UA was carried out under various
conditions. An aqueous solution containing 200 µM UA and
1
0 µM Rose Bengal was prepared and irradiated by UVA
2
(1.12 mW/cm ) for 2 hours. During photooxidation, the mix-
ture was analyzed every 30 min by a LC/TOFMS equipped
1
8
with a UV detector. When the photooxidation under
mosphere was conducted, deoxygenation with N bubbling
was performed prior to the oxidation. The aqueous reaction
mixture was poured into an airtight vial connected to a N gas
cylinder and a vacuum pump. N gas was delivered into the
well-stirred solution with vigorous bubbling for 3 hours. Next,
2
O at-
2
2
009, 28 (2), 118-149.
2
(5) Gersch, C.; Palii, S. P.; Kim, K. M.; Angerhofer, A.; Johnson,
2
R. J.; Henderson, G. N. Inactivation of nitric oxide by uric acid. Nu-
cleosides, Nucleotides Nucleic Acids 2008, 27 (8), 967-978.
(6) Iida, S.; Yamamoto, Y.; Susa, C.; Tsukui, K.; Fujisawa, A. 5-N-
Carboxyimino-6-N-chloroaminopyrimidine-2,4(3H)-dione as a hypo-
chlorite-specific oxidation product of uric acid. J. Clin. Biochem. Nutr.
2018, 63 (2), 85-89.
1
8
2
O gas was induced into the vial using a vacuum pump. In
1
8
the photooxidation in H
2
O, the experimental solution was
O as a solvent instead of water.
NEPO-initiated UA oxidation. UA was also oxidized by
1O
generated from thermal decomposition of NEPO as de-
18
prepared with H
2
(
7) Iida, S.; Ohkubo, Y.; Yamamoto, Y.; Fujisawa, A. Parabanic
2
acid is the singlet oxygen specific oxidation product of uric acid. J.
Clin. Biochem. Nutr. 2017, 61 (3), 169-175.
7
scribed previously . Briefly, a methanolic solution of NEPO
was added to UA aqueous solution. The reaction mixture con-
taining 200 µM UA and 8 mM NEPO was incubated at 35 ºC
and analyzed by LC/TOFMS.
(
8) Duarte, V.; Gasparutto, D.; Yamaguchi, L. F.; Ravanat, J. L.;
Martinez, G. R.; Medeiros, M. H. G.; Mascio, P. D.; Cadet, J. Ox-
aluric acid as the major product of singlet oxygen-mediated oxidation
of 8-oxo-7,8-dihydroguanine in DNA. J. Am. Chem. Soc. 2000, 122
HPLC analysis. A reverse phase HPLC equipped with UV
detector was used for measuring and isolating UA oxidation
metabolites. Water adjusted to pH 3.5 by formic acid was de-
livered at a rate of 1.0 mL/min as an isocratic mobile phase. A
Develosil C30-UG (Nomura Chemical Co., Ltd., Tokyo, Ja-
pan), 5 µm, 250 mm × 4.6 mm, was used as a separation col-
umn.
(
51), 12622-12628.
(9) Umeno, A.; Shichiri, M.; Ishida, N.; Hashimoto, Y.; Abe, K.;
Kataoka, M.; Yoshino, K.; Hagihara, Y.; Aki, N.; Funaki, M.; Asada,
Y.; Yoshida, Y. Singlet oxygen induced products of linoleates, 10-
and 12-(Z,E)-hydroxyoctadecadienoic acids (HODE), can be potential
biomarkers for early detection of type 2 diabetes. PLoS One 2013, 8
(
5), e63542.
(10) Hillered L; Persson L. Parabanic acid for monitoring of oxy-
gen radical activity in the injured human brain. Neuroreport 1995, 6
(13), 1816-1820.
LC/TOFMS analysis. In order to obtain accurate mass-to-
charge ratios (m/z) of UA oxidation products, an HPLC sys-
tem (Agilent 1100 series, Agilent, Santa Clara, CA, USA)
equipped with a TOFMS (JMS-T100LC, JEOL Ltd., Tokyo,
Japan) was used. Negative electrospray ionization (ESI) was
performed at an ionization potential of –2000 V. The opti-
mized applied voltages to ring lens, outer orifice, inner orifice,
and ion guide were –5 V, –10 V, –5 V, and –500 V, respec-
tively.
For measurements of fragmentation, those potentials were
adjusted to –10 V, –50 V, –10 V, and –500 V. Trifluoroacetic
acid (TFA) was used as an internal standard for m/z calibration.
HPLC separation was performed in the isocratic elution mode
using a Develosil C30-UG (Nomura Chemical Co., Ltd., To-
8
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