Synthesis of 6-formylpterin nucleoside analogs and their ROS generation activities in the presence of NADH in the dark

Article abstract of DOI:10.1039/b710466a

We demonstrated previously that 3-position-modified 6-formylpterin (6FP) derivatives produce reactive oxygen species (ROS) such as hydrogen peroxide (H₂O₂) from oxygen in the presence of NADH in the dark. It has been shown that 6FP d

Full text of DOI:10.1039/b710466a

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www.rsc.org/obc | Organic & Biomolecular Chemistry
Synthesis of 6-formylpterin nucleoside analogs and their ROS generation
activities in the presence of NADH in the dark
Mitsuru Nonogawa,a,b Seung Pil Pack,a,b Toshiyuki Arai,c Nobuyuki Endo,d Piyanart Sommani,a,b
Tsutomu Kodaki,a,b Yashige Kotakee and Keisuke Makino*a,b
Received 10th July 2007, Accepted 8th August 2007
First published as an Advance Article on the web 3rd September 2007
DOI: 10.1039/b710466a
We demonstrated previously that 3-position-modified 6-formylpterin (6FP) derivatives produce reactive
oxygen species (ROS) such as hydrogen peroxide (H2O2) from oxygen in the presence of NADH in the
dark. It has been shown that 6FP derivatives markedly generate ROS, which gives rise to their
particular physiological activities, such as induction of apoptosis in cellular and living systems,
suggesting that such compounds provide a hint for the design of a ROS controlling agent in vivo.
However, it is not well understood why such unique activities appear on chemical modification. In the
present study, in order to see the effect on ROS generation activity in the dark by the modification of
the 1-position in 6FP, we have developed a new synthetic procedure for nucleoside analogs of 6FP and
prepared 1-(b-D-ribofuranosyl)-2-(N,N-diethylaminomethyleneamino)-6-formylpteridin-4-one
(RDEF) and 1-(b-D-ribofuranosyl)-2-(piperidine-1-ylmethyleneamino)-6-formylpteridin-4-one (RPIF)
in which the 1-position of 6FP is glycosylated. At pH 7.4, NADH was spontaneously oxidized to
NAD+ in the presence of RDEF in the dark. Using electron paramagnetic resonance analysis coupled
with the spin trapping technique, we show that O2 was converted to H2O2 via superoxide anion radical
(•O2−) during this reaction. The modification of the 1-position of 6FP did not cancel ROS generation
activities, which were demonstrated in 3-position-modified 6FPs. Since the 6FP derivatives developed in
the present study have a ribose moiety, these compounds can be subjected to further derivatization,
such as incorporation into oligonucleotides, oligosaccharides, proteins, or any other compounds that
recognize and interact with specific biomolecules, and therefore would be useful in pharmaceutical
investigations that need generation of appropriate and controllable amounts of ROS in vivo.
6FP has been shown to be produced from folic acid in some
Introduction
pathological conditions, such as carcinoma.2 Furthermore, in
6-Formylpterin (6FP, Scheme 1) is a pteridine derivative produced
the past two decades, 6FP has been investigated in the field
from folic acid by photooxidation in the presence of O2.1 In vivo,
of photochemistry.3 Under UV irradiation, for instance, 6FP
activates O2, generates singlet oxygen, and also forms H2O2 from
O2.4 However, detailed reaction mechanisms for both examples
remain to be defined.
In previous studies, we have shown that reactive oxygen species
(ROS), such as H2O2, are generated by 6FP and that it induced
apoptosis in HL-60 cells, inhibited Fas-mediated apoptosis in
Jurkat cells, and suppressed activation of NF-jB, cytokine pro-
duction, and cell proliferation in PanC-1 and human blood T
cells.5 In addition, 6FP showed neuroprotective effects against
transient ischemia-reperfusion injury (IRI) in gerbils6 and similar
effects for retinal IRI in rats;7 however, the mechanism of such
physiological activities has not been elucidated to date. IRI occurs
in conditions, including cardiac infarction, brain infarction, and
organ transplantation, that result in apoptotic cell death and,
therefore, protective agents against IRI have been one of the
most important research targets in medicinal and pharmaceutical
studies. Although 6FP shows notable physiological activities, it has
not been used clinically, partly because of its poor water solubility
at neutral pH.8
Scheme 1 Structures of 6FP and 6FP derivatives.
aInstitute of Advanced Energy, Kyoto University, Gokasho, Uji 611-0011,
Japan. E-mail: kmak@iae.kyoto-u.ac.jp; Fax: +81-0774-38-3524; Tel: +81-
0774-38-3517
bCREST, JST (Japan Science and Technology Agency), Kyoto University,
Gokasho, Uji 611-0011, Japan
cDepartment of Anesthesia, Kyoto University Hospital, Sakyo-ku, Kyoto
606-8507, Japan
To overcome this drawback, 6FP derivatives with improved
water solubility at neutral pH have been developed and assessed
for their chemical activities in vitro.8,9 It is demonstrated that 6FP
dWakasa Wan Energy Research Center, Tsuruga 914-0129, Japan
eOklahoma Medical Research Foundation, Oklahoma City, OK 73104, USA
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derivatives, such as 2-(N,N-dimethylaminomethyleneamino)-6-
formyl-3-pivaloylpteridin-4-one (DFP), 2-(N,N-dimethylamino-
methyleneamino)-6-formyl-3-methylpteridin-4-one (DFM), and
2-amino-6-formyl-3-methylpteridin-4-one (FM) (Scheme 1), with
a modified 3-position can generate H2O2 through NADH to
NAD+ oxidation in the dark in phosphate-buffered saline (PBS) at
pH 7.4 without the aid of any biological systems.10 It is important
to explore ROS generation by 6FP derivatives in the absence of
light because most biological events occur in the dark. In addition,
DFP, one of the 3-position modified 6FP derivatives in which
hydrogen atoms on the 2- and 3-positions of 6FP are replaced by
N,N-dimethylaminomethylene and pivaloyl groups, respectively,
showed suppressed proliferation of PanC-1 cells.11 Although the
mechanism is not clearly understood yet, we speculate that ROS
generated by DFP in the presence of NADH is involved in the
mechanism.
It is now widely known that ROS are not only involved in cell
death but are also involved in the modulation of a variety of cell
functions,12 thus, it is important to understand the relationship
between ROS activity and biological events. 6FP derivatives that
can generate ROS in the dark through NADH oxidation at neutral
pH in the absence of any biological systems, can be potent
physiological compounds that generate appropriate amounts of
ROS in living systems. In addition, it is possible that by using these
compounds the above-mentioned physiological activities of 6FP
can be introduced in most biological systems. In the present study,
in order to determine the factors responsible for such activities,
we have developed a synthetic procedure for nucleoside analogs of
6FP in which the 1-position is modified. It should be noted that in
photochemistry, pteridine nucleoside analogs, other than the 6FP
derivatives, have been developed and used for analytical purposes
due to their unique fluorescence properties.13
Scheme 2 Chemical synthesis of RDEF and RPIF: (a) synthesis of
DF from 6FP using tert-butoxybis(dimethylamino)methane in DMF at
60 ◦C for 20 min, 95%; (b) glycosylation of DF using 1,8-diazabicyclo-
[5,4,0]undec-7-ene, trimethylsilyl trifluoromethanesulfonate, and 1-O-
acetyl-2,3,5-tri-O-benzoyl-b-D-ribofuranose in CH3CN at 60 ◦C for 14 h,
68%; (c) deprotection of BzRDF in MeOH containing 25% diethylamine
at rt for 24 h, 49%; (d) deprotection of BzRDF in MeOH containing 25%
piperidine at rt for 24 h, 25%.
Recently, a number of homing devices that direct physiological
compounds to specific cells or biomolecules, i.e., specific drug
delivery systems,14 have been developed. Nucleoside analogs of
6FP derivatives developed in the present work contain a ribose on
the pteridine ring and, therefore, can be incorporated into homing
devices such as oligonucleotides, oligosaccharides, proteins, or any
other compounds. These compounds could be directly introduced
into specific cellular systems to recognize and interact with specific
biomolecules and generate appropriate and controllable amounts
of ROS in some specific position in vivo because NADH, a central
intermediate in oxidative catabolism that acts as a convenient
source of readily transferable electrons in cells,15 is abundant.
reaction of BzRDF was carried out. When BzRDF was
stirred in methanol containing 25% diethylamine solution at
room temperature for 24 h, 1-(b-D-ribofuranosyl)-2-(N,N-
diethylaminomethyleneamino)-6-formylpteridin-4-one (RDEF)
was obtained in a yield of 49% as a nucleoside analog of a 6FP
derivative (Scheme 2). RDEF shows over 100 mM solubility
in PBS at pH 7.4 because of the hydrophilic ribose moiety. In
this reaction, the dimethylaminomethyleneamino group on the
2-position was substituted by N,N-diethylaminomethyleneamino.
To determine if this occurred generally, BzRDF was stirred
in methanol containing 25% piperidine solution at room
temperature for 24 h, and it was found that 1-(b-D-ribofuranosyl)-
2-(piperidine-1-ylmethyleneamino)-6-formylpteridin-4-one
(RPIF) was produced in a yield of 25% (Scheme 2).
Results and discussion
We have demonstrated previously that DFP suppresses prolif-
eration of PanC-1 cells without photo irradiation.11 We speculate
that the observation of DFP-induced suppression of proliferation
in PanC-1 cells may be due to ROS generation through NADH
oxidation by DFP. ROS generation in the presence of NADH in
the dark is one of the most prominent physiological activities
attributed to the 6FP derivatives because it is related to the
physiological activities of 6FP.5 Therefore, the ROS generation
activity through NADH oxidation by RDEF in the dark was
investigated.
When 6FP was reacted with tert-butoxybis(dimethylamino)-
methane in N,N-dimethylformamide (DMF) at 60 ◦C for 20 min,
2-(N,N-dimethylaminomethyleneamino)-6-formylpteridin-4-one
(DF)9 was obtained in a yield of 95%, as shown in Scheme 2. Then
the glycosylation reaction of DF was conducted. When DF was
reacted with 1-O-acetyl-2,3,5-tri-O-benzoyl-b-D-ribofuranose in
the presence of 1,8-diazabicyclo[5,4,0]undec-7-ene and trimethyl-
silyl trifluoromethanesulfonate in acetonitrile at 60 ◦C for
14 h, glycosylation reaction16 occurred at the 1-position of DF
and 1-(2,3,5-tri-O-benzoyl-b-D-ribofuranosyl)-2-(N,N-dimethyl-
aminomethyleneamino)-6-formylpteridin-4-one (BzRDF) was
obtained in a yield of 68% (Scheme 2). Next, the deprotection
PBS solutions (pH 7.4) containing 2 mM RDEF and 2 mM
NADH were stirred in the dark in an open system at room
temperature and the time-dependent concentration change of each
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component was analyzed by reversed-phase high performance
liquid chromatography (RP-HPLC). During the reaction, a de-
crease in NADH concentration and generation of NAD+ was
observed, as shown in Fig. 1. In this oxidation reaction, RDEF
concentration also gradually decreased, probably because of the
decomposition caused by ROS generated in this system. In the
absence of NADH, RDEF remained constant over 100 h (data not
shown). In addition, in the absence of RDEF, NADH remained
constant over 100 h (data not shown). From these results, we
conclude that RDEF, a 6FP derivative in which the 1-position is
glycosylated, oxidized NADH to NAD+ in the dark.
Fig. 2 EPR spectra obtained for PBS (pH 7.4) containing (a) 2 mM
RDEF, (b) 2 mM NADH, (c) 2 mM RDEF and 2 mM NADH, and (d)
2 mM RDEF and 2 mM NADH. The reactions were performed for 48 h
in the dark in an open system. After the reaction, 500 lM DETAPAC,
250 lM FeSO4, and 100 mM DMPO were added to the reaction mixtures,
which were diluted to 1/10 immediately prior to the EPR measurement.
Catalase (10 000 units ml−1) was also added to (d).
been shown to demonstrate excellent ability to trap •O2− and •OH
as compared with DMPO.18 PBS solutions (pH 7.4) containing
4 mM RDEF, 4 mM NADH, and 20 mM CYPMPO were
incubated at room temperature for 1 h in the dark in an open
system and measured using EPR spectroscopy. The results are
summarized in Fig. 3. The solutions containing either RDEF
or NADH and CYPMPO did not show EPR signals (data not
shown). The solutions containing RDEF, NADH, and CYPMPO
Fig. 1 Oxidation reaction of NADH by RDEF in the dark. The
time course of the change in RDEF (᭹), NADH (ꢀ), and NAD+
(ꢁ) concentrations in the dark in an open system was measured by
RP-HPLC. A PBS solution containing 2 mM RDEF and 2 mM NADH
was stirred in the dark at pH 7.4. In the absence of RDEF, NADH is
constant over the 100 h of this experiment.
showed a prominent EPR spectrum pattern of a mixture of
•
•O2 and OH adducts of CYPMPO18 (Fig. 3(a)). These signals
−
were completely quenched by superoxide dismutase (SOD), which
converts •O2− to H2O2 in the presence of a proton source (Fig. 3(b)).
•
−
Fig. 3(c) is a computer simulated spectrum of a mixture of O2
•
•
•
−
To identify the ROS generated in this reaction, electron
paramagnetic resonance (EPR) spectroscopy coupled with the
spin trapping technique, using 5,5-dimethyl-1-pyrroline-N-oxide
(DMPO) as a spin trap, was employed. PBS solutions (pH 7.4)
containing 2 mM RDEF and 2 mM NADH were incubated
for 48 h and diethylenetriaminepentaacetic acid (DETAPAC)
(500 lM), FeSO4 (250 lM), and DMPO (100 mM) were added
to the reaction mixture prior to EPR analysis. It should be noted
that in the presence of ferrous ion (Fe2+), hydroxyl radical (•OH)
is generated from H2O2 via the iron-catalyzed Fenton reaction.17
The results are summarized in Fig. 2. The solutions containing
either RDEF or NADH did not show EPR signals over 48 h,
as represented in Fig. 2(a) and 2(b). The solution containing
RDEF and NADH showed a quartet with the hyperfine splitting
constant (HFSC) of AN = 1.49 mT and AH = 1.49 mT (Fig. 2(c)).
This signal was quenched by adding catalase (10 000 units ml−1),
which eliminates H2O2, to the crude reaction solution (Fig. 2(d)).
Based on these observations, the quartet was identified as a DMPO
adduct of •OH (DMPO–OH), indicating that H2O2 was generated
in the dark in the solutions containing RDEF and NADH.
To identify the exact ROS species directly generated in the
reaction of RDEF and NADH, further EPR spectroscopy coupled
with the spin trapping technique, using a newly developed spin
trap, 5-(2,2-dimethyl-1,3-propoxy cyclophosphoryl)-5-methyl-1-
pyrroline-N-oxide (CYPMPO),18 was employed. CYPMPO has
and OH adducts of CYPMPO. Both O2 and OH adducts
are composed of two diastereomers respectively. The computer-
simulated spectrum showed good coincidence with the previously
•
•
−
reported spectrum of the mixture of O2 and OH adducts of
CYPMPO,18 and also with the spectrum in Fig. 3(a) in this study.
Fig. 3 EPR spectra obtained for PBS (pH 7.4) containing (a) 4 mM
RDEF, 4 mM NADH and 20 mM CYPMPO, (b) 4 mM RDEF, 4 mM
NADH, 20 mM CYPMPO and 2000 units ml−1 SOD. The reactions were
performed for 1 h at room temperature in the dark in an open system and
measured using EPR spectroscopy. (c) Computer-simulated spectrum for
a mixture of •O2 and •OH adducts of CYPMPO.
−
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Table 1 HFSCs of •O2 and •OH adducts of CYPMPO used for
−
reaction in the absence of light.4 Based on the present results and
previously reported data, it is possible that NADH was oxidized
by RDEF to NAD+ by hydride transfer,20 and then the reduced
form of RDEF thus generated would reduce O2 to a ROS such
as H2O2, through O2 formation. It should be emphasized that
all these reactions occurred in the dark without any biological
systems.
computer-simulation
•O2 adduct
•OH adduct
−
•
−
HFSC (AN, AH, AP)/mT
1.26, 1.11, 5.25
1.27, 1.04, 5.10
1.37, 1.37, 4.98
1.35, 1.23, 4.88
HFSCs used in this simulation are listed in Table 1. These results
clearly indicate that O2 was converted into •O2− in the presence of
RDEF and NADH.
Conclusions
•
−
We synthesized novel nucleoside analogs of 6FP. RDEF, which
To further confirm O2 generation in the reaction, a chemi-
•
−
luminescence method with the •O2 specific probe 2-methyl-
−
has a ribose on the 1-position of the pteridin ring, showed O2
and H2O2 generation activity through the oxidation of NADH to
NAD+ in the dark at pH 7.4 without any biological systems. This
indicates that, in addition to the modification at the 3-position, a
modification at the 1-position also introduces the activity of ROS
generation in the presence of NADH in the dark. The compounds
developed in the present study have a ribose moiety, and would
therefore be of help in designing physiologically active molecules
such as oligonucleotides, oligosaccharides, proteins, or any other
compounds that recognize and interact with specific biomolecules
in specific cells in living systems and generate appropriate and
controllable amounts of ROS. Further applications of these
nucleoside analogs of 6FP derivatives are now under investigation.
6-phenyl-3,7-dihydroimidazo[1,2-a]pyrazine-3-one (CLA)19 was
also employed. After mixing 2 mM RDEF and 100 lM CLA with
or without 700 units ml−1 SOD in PBS solution, the luminescence
was monitored with a luminescence reader. It should be noted
•
that if •O2− is generated in the reaction of RDEF and NADH, O2
−
reacts with CLA and chemiluminescence is observed. When 2 mM
NADH was added to the mixture containing RDEF and CLA,
enhancement of the luminescence was observed (Fig. 4(a)). When
NADH was added to the mixture containing RDEF, CLA, and
SOD, the luminescence was suppressed (Fig. 4(b)). These results
•
−
also indicate that O2 was generated in the presence of RDEF
and NADH.
Experimental
Materials and methods
6FP (Scheme 1) was obtained from Sankyo Kasei Kogyo (Hi-
ratsuka, Japan); tert-butoxybis(dimethylamino)methane, Sigma
Chemical Co. (St. Louis, MO, USA); N,N-dimethylformamide
(DMF), 1-O-acetyl-2,3,5-tri-O-benzoyl-b-D-ribofuranose, and
acetonitrile, Wako (Osaka, Japan); 1,8-diazabicyclo[5,4,0]undec-
7-ene (DBU), trimethylsilyl trifluoromethanesulfonate, and cata-
lase, Sigma-Aldrich Co. (St. Louis, USA); NADH, Oriental
Yeast (Tokyo, Japan); 5,5-dimethyl-1-pyrroline-N-oxide (DMPO),
Labotec Co. (Tokyo, Japan); FeSO4·7H2O (FeSO4), Kantokagaku
(Tokyo, Japan), diethylenetriaminepentaacetic acid (DETAPAC),
Nacalai Tesque (Kyoto, Japan), 2-methyl-6-phenyl-3,7-dihydro-
imidazo[1,2-a]pyrazine-3-one (CLA), Tokyo Kasei (Tokyo,
Japan), and 5-(2,2-dimethyl-1,3-propoxy cyclophosphoryl)-5-
methyl-1-pyrroline-N-oxide (CYPMPO), Radical Research Inc.
(Tokyo, Japan). Column chromatography was performed using
silica gel 60 from Nacalai Tesque (Kyoto, Japan). RDEF and
RPIF, were isolated using a reversed-phase high performance
liquid chromatography (RP-HPLC) system consisting of a CCPM
pumping system, and a PX-8010 system controller (TOSOH;
Tokyo, Japan): Column, ULTRON VX-ODS, Shinwa Chemical
Industries (Kyoto, Japan); column and particle size, 20.0 × 250 mm
Fig. 4 Detection of •O2 in PBS solutions containing RDEF and
−
NADH by the chemiluminescence method. PBS solutions containing (a)
2 mM RDEF and 100 lM CLA, (b) 2 mM RDEF, 100 lM CLA, and
700 units ml−1 SOD were incubated and the luminescence intensities (᭹
for (a), and ꢁ for (b)) were monitored every 30 s for 30 min. 2 mM NADH
was added to the solutions 90 s after the measurement was started.
In summary, we have shown that in the solutions containing
RDEF and NADH in the dark, RDEF oxidized NADH to NAD+
•
and simultaneously O2 was converted to •O2−. The generated O2
−
1
was finally converted to H2O2. The oxidation reaction of NADH
by RDEF was shown to be essential for •O2− and H2O2 generation.
In previous studies, it has been reported that biopterin, neopterin,
and 6-(hydroxymethyl)pterin activate O2 to H2O2 under UV
irradiation,4 andreactionmechanisms thatinvolveanintermediate
such as 6-formyl-5,8-dihydropterin, a reduced form of 6FP, have
been proposed. As evidence, such an intermediate was produced
from 6-(hydroxymethyl)pterin by UV irradiation, which activated
O2 to H2O2 and simultaneously converted 6FP by a thermal
and 5 lm, respectively; eluting with H2O–CH3CN (80 : 20). H
and 13C NMR spectra were recorded on a JEOL ECA-600 NMR
spectrometer (JEOL Ltd.; Tokyo, Japan) operating at 600 MHz
and 150 MHz, respectively. All chemical shifts are reported in ppm
downfield from tetramethylsilane. Peak multiplicities are denoted
by singlet (s), broad singlet (br s), doublet (d), broad doublet (br d),
triplet (t), and multiplet (m), with coupling constants (J) given in
Hz. Numberings of BzRDF, RDEF, and RPIF were as described
in Scheme 3. Mass spectra were recorded on a JEOL DX-300 at
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7.37–7.33 (6H, b), 7.46 (1H, d, J 2.4, r1), 6.43 (1H, br s, r2), 6.26
(1H, br s, r3), 4.86 (1H, br d, r5), 4.73 (1H, m, r4), 4.64 (1H, dd,
J 12.0, 4.8, r5), 3.21 (3H, s, 2ꢀ), 3.12 (3H, br s, 2ꢀ). dC (150 MHz;
CDCl3) 191.2 (S1), 166.6 (4), 166.2 (a), 165.4 (b), 165.3 (b), 160.9
(1ꢀ), 159.7 (2), 149.9 (9), 144.2 (7), 133.6 (6), 133.4 (b), 130.9 (10),
129.7–128.4 (b), 89.6 (r1), 78.2 (r4), 73.4 (r2), 70.6 (r3), 63.0 (r5),
42.2 (2ꢀ), 36.3 (2ꢀ). MS(FAB) m/z (M + H+), 691.2151.
1-(b-D-Ribofuranosyl)-2-(N,N-diethylaminomethyleneamino)-6-
formylpteridin-4-one (RDEF). BzRDF (69.1 mg, 0.1 mmol) was
allowed to stir in methanol (containing 25% diethylamine, 8 ml)
under nitrogen at room temperature for 24 h. The mixture was
evaporated in vacuo. Then, H2O (10 ml) was added and neutralized
by 0.6 N HCl. The product was isolated by RP-HPLC. The
fraction was evaporated in vacuo and the title compound was
obtained as a yellow solid (19.9 mg, 49%). dH (600 MHz; CDCl3)
10.14 (1H, s, S1), 9.05 (1H, s, 7), 8.90 (1H, s, 1ꢀ), 7.11 (1H, d, J
5.5, r1), 5.04 (1H, t, J 1.5, r2), 4.71 (1H, t, J 6.2, r3), 4.09 (1H, m,
r4), 3.87 (2H, ddd, J 76.6, 12.0, 2.8, r5), 3.71 (2H, ddd, J 6.9, 2ꢀ),
3.55 (2H, ddd, J 6.9, 2ꢀ), 1.37 (3H, t, J 6.9, 3ꢀ), 1.31 (3H, t, J 7.6,
3ꢀ). dC (150 MHz; CDCl3) 190.9 (s1), 166.4 (4), 161.0 (2), 159.8
(1ꢀ), 150.1 (9), 144.1 (7), 143.8 (6), 130.5 (10), 91.6 (r1), 84.7 (r4),
71.4 (r2), 67.0 (r3), 62.4 (r5), 48.1 (2ꢀ), 42.0 (2ꢀ), 14.4 (3ꢀ), 12.4 (3ꢀ).
MS(FAB) m/z (M + H+) 407.1691.
Scheme 3 The numbering of the 6FP nucleoside analogs synthesized in
this study.
30 eV for electron impact (EI) or fast atom bombardment (FAB)
conditions. The reaction in the dark was performed in a vessel
covered completely with foil. The EPR spectra were recorded on
a Model JES-TE300 spectrometer (JEOL Ltd.; Tokyo, Japan).
1-(b-D-Ribofuranosyl)-2-(piperidine-1-ylmethyleneamino)-6-for-
mylpteridin-4-one (RPIF). BzRDF (138.0 mg, 0.2 mmol) was
allowed to stir in methanol (containing 25% piperidine, 10 ml)
under nitrogen at room temperature for 24 h. The mixture was
evaporated in vacuo. Then, H2O (10 ml) was added and neutralized
by 0.6 N HCl. The product was isolated by RP-HPLC. The fraction
was evaporated in vacuo and the title compound was obtained as
a yellow solid (20.9 mg, 25%). dH (600 MHz; CDCl3) 10.11 (1H, s,
S1), 9.05 (1H, s, 7), 8.78 (1H, s, 1ꢀ), 7.06 (1H, d, J 4.8, r1), 5.12
(1H, br, r2), 4.69 (1H, t, J 5.7, r3), 4.09 (1H, m, r4), 3.87 (2H,
ddd, J 74.6, 12.1, 2.5, r5), 3.83 (2H, br d, 2ꢀ), 3.65 (2H, br, 6ꢀ),
1.80 (2H, br, 4ꢀ), 1.78 (2H, br, 5ꢀ), 1.75 (2H, br, 3ꢀ). dC (150 MHz;
CDCl3) 190.9 (s1), 166.3 (4), 161.0 (2), 158.9 (1ꢀ), 150.1 (9), 144.3
(7), 143.7 (6), 130.1 (10), 91.5 (r1), 84.7 (r4), 71.1 (r2), 69.9 (r3),
62.3 (r5), 53.0 (6ꢀ), 45.9 (2ꢀ), 26.5 (4ꢀ), 25.3 (3ꢀ), 23.9 (5ꢀ). MS(FAB)
m/z (M + H+) 419.1664.
Synthesis
2-(N,N -Dimethylaminomethyleneamino)-6-formylpteridin-4-
one (DF). DF was synthesized from 6FP according to the
procedure reported previously.9 6FP (570.0 mg, 3.0 mmol) and
tert-butoxybis(dimethylamino)methane (930 ll, 4.5 mmol) were
mixed in DMF (6 ml) with stirring under nitrogen for 20 min at
◦
60 C. During this time, the suspended solid 6FP dissolved. The
mixture was evaporated in vacuo and the resultant orange-tan oil
was purified by column chromatography, eluting with chloroform–
methanol (90 : 10), to give the title compound as a yellow solid
(700.5 mg, 95%). dH (600 MHz; CDCl3–DMSO-d6 = 4 : 1) 11.89
(1H, br s, NH), 10.17 (1H, s, CHO), 9.24 (1H, s, 7), 9.02 (1H, s,
NCHN), 3.28 (3H, s, CH3), and 3.24 (3H, s, CH3). dC (150 MHz;
CDCl3–DMSO-d6 = 4 : 1) 191.1, 162.0, 160.3, 160.2, 159.1, 148.2,
142.3, 130.9, 41.8, and 35.7. MS(EI) m/z M+, 246.
Oxidation reaction of NADH by RDEF in the dark
1-(2,3,5-Tri-O-benzoyl-b-D-ribofuranosyl)-2-(N,N -dimethyl-
aminomethyleneamino)-6-formylpteridin-4-one
(BzRDF). Tri-
PBS solutions (4 ml, pH 7.4) containing 2 mM RDEF and 2 mM
NADH were prepared. These solutions were kept in the dark
and stirred in an open system at room temperature. The time-
dependent concentration change of each component in the sample
solutions was analyzed by RP-HPLC consisting of a DP-8020
pumping system, a CO-8020 column oven, and a PX-8020 system
controller (TOSOH; Tokyo, Japan): Column, ULTRON VX-ODS,
Shinwa Chemical Industries; (Kyoto, Japan); column and particle
size, 4.6 × 150 mm and 5 lm, respectively; eluent, 10 mM TEAA
buffer (pH 7.0); gradient, CH3CN concentration in the liner
gradient mode (0 min: 0%; 30 min: 50%); flow rate, 1.0 ml min−1;
temperature, 37 ◦C. NAD+ in the RP-HPLC peak was identified
methylsilyl trifluoromethanesulfonate (1450 ll, 8.9 mmol) was
added slowly with ice cooling to a solution of 1-O-acetyl-
2,3,5-tri-O-benzoyl-b-D-ribofuranose (1006.5 mg, 2.24 mmol),
DF (550.2 mg, 2.24 mmol), and DBU (900 ll, 6.0 mmol) in
acetonitrile (10 ml). After stirring under nitrogen at 60 ◦C for
14 h, the mixture was evaporated in vacuo and the resultant
brown oil was purified by column chromatography, eluting with
chloroform–methanol (98 : 2), to give the title compound as a
yellow solid (939.3 mg, 68%). By heteronuclear multiple bond
correlation (HMBC) NMR analysis, it was observed that H at
C(r1) of the ribose ring has correlations with C(2) and C(9) of the
pterin ring. dH (600 MHz; CDCl3) 10.25 (1H, s, S1), 9.08 (1H, s,
7), 9.01 (1H, s, 1ꢀ), 7.97 (4H, b), 7.88 (2H, b), 7.56–7.53 (3H, b),
1
by UV absorption spectra, H- and 13C-NMR analysis, and RP-
HPLC was used for its quantitative analysis.
3318 | Org. Biomol. Chem., 2007, 5, 3314–3319
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EPR spectra measurement in PBS solutions containing RDEF and
NADH in the dark
Japan, and by CREST of the Japan Science and Technology
Corporation.
PBS solutions (pH 7.4) containing 2 mM RDEF and 2 mM
NADH were stirred for 48 h at room temperature in the dark
and measured using electron paramagnetic resonance (EPR)
spectroscopy combined with spin trapping with DMPO. The
reaction mixtures were diluted to 1/10, and 100 mM DMPO,
500 lM DETAPAC, and 250 lM FeSO4 were added before the
EPR measurement. The EPR settings were as follows: microwave
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Detection of •O2 in PBS solutions containing RDEF and NADH
−
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We acknowledge the substantial support of Dr Masatoshi
Nishi for his significant contribution in the identification of
nucleoside analogs of 6FP derivatives. This work was par-
tially supported by the Grants-in-Aid for Scientific Research
(No. 18350083) from the Ministry of Education, Culture, Sports,
Science and Technology (MEXT), Japan, and by the Center
of Excellence (COE) program for the “Establishment of COE
on Sustainable-Energy System.” This work was also supported
by a Grant-in-Aid for regional science and technology promo-
tion “Kyoto Nanotechnology Cluster” project from the MEXT,
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The Royal Society of Chemistry 2007
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