8-Nitroguanosines as chemical probes of the protein S-guanylation

Article abstract of DOI:10.1039/b810771h

Azido- and fluoro- derivatives of 8-nitroguanosine were developed, and will contribute to the exploration of protein S-guanylation by endogenous nitrated nucleosides. The Royal Society of Chemistry.

Full text of DOI:10.1039/b810771h

View Article Online / Journal Homepage / Table of Contents for this issue
COMMUNICATION
www.rsc.org/chemcomm | ChemComm
8-Nitroguanosines as chemical probes of the protein S-guanylationw
Yohei Saito,a Hirobumi Taguchi,a Shigemoto Fujii,b Tomohiro Sawa,b Eriko Kida,a
Chizuko Kabuto,c Takaaki Akaike*b and Hirokazu Arimoto*a
Received (in Cambridge, UK) 25th June 2008, Accepted 22nd September 2008
First published as an Advance Article on the web 14th October 2008
DOI: 10.1039/b810771h
Azido- and fluoro- derivatives of 8-nitroguanosine were
developed, and will contribute to the exploration of protein
S-guanylation by endogenous nitrated nucleosides.
Nitric oxide (NO) has diverse physiological functions, such as
vascular and neuronal signal transduction, host defense, and
cell death regulation. NO activates soluble guanylate cyclase
(sGC), which leads to the production of guanosine 30,50-cyclic
monophosphate (cGMP).1 Moreover, reactive nitrogen spe-
cies are generated from nitric oxide (NO), and work as potent
nitrating agents for proteins, nucleic acids, and unsaturated
Fig. 1 The schematic reaction of an endogenous cGMP derivative 1
membrane lipids.2–4
with proteinous thiols (S-guanylation), which plays important roles in
We recently identified 8-nitro-cGMP 1, the first endogenous
cGMP derivative, in various cell lines.5 This compound mediates
a novel post-translational modification by reacting with protei-
NO-mediated signaling pathways.
(oo1% isolated yield),7 and thus the substitution of
8-bromoguanosine by nitrite in DMSO has been used for
preparative purposes.8,9 A drawback of this protocol is that
HPLC must be used to isolate the products from the high
boiling-point solvent and from the unreacted starting material.
The isolated yield of 2 remained moderate (10–20%),8 which
was in part due to the instability of 2 under the reaction
conditions. And indeed, a nitration of the guanine moiety
substantially destabilized the glycosyl bonds of guanosines,
and the half lives at pH 7 and 37 1C were reported to be 5 h for
8-nitroguanosine, and o 3 min for the 20-deoxy counterpart.7
We envisaged that use of proper protecting groups would
minimize the undesired depurination of nitrated guanosines in
polar solvents during the reactions and purifications. Thus, the
2-amino group of 8-bromo-20,30,50-tri-O-acetyl-guanosine 3
was protected as a 4, 40-dimethoxytrityl ether (DMTr) in
99% yield (Scheme 1). Nitration of 4 was then investigated
under the conditions summarized in Table 1. Acetonitrile was
first selected as solvent (entry 1), and the desired nitroguano-
sine 5 was obtained in 49% yield. The cleavage of the labile
glycoside bond seemed to be slow under these conditions.
N,N-Dimethylformamide was also investigated as a solvent
nous thiol in cells (Fig. 1). A specific antibody that recognizes
the SH modifications was prepared, and has already been used
for detection of the ’’protein S-guanylation’’ in cells.5 Our
previous efforts to uncover the role of the S-guanylations, led
to the identification of Keap1 (Kelch-like ECH-associated pro-
tein 1),6 a redox sensor, as one of the target proteins of 1.5
Keap1 (ca. 70 kDa) includes 25 Cys residues, and senses
oxidative stresses by their thiol modifications. Keap1 has also
been shown to be cytoprotective via HO-1 (heme oxygenase 1)
upregulation. These findings suggest that 8-nitro-cGMP 1 med-
iates a unique NO signaling pathway independent of cGMP.
Development of the 8-nitroguanosine based-probes should be
a promising approach to answer further biological questions
regarding S-guanylation at the molecular level. We describe here
the synthesis, activity, and reactions of these chemical probes.
We initiated our study by exploring an efficient synthesis of
8-nitroguanosine 2, which should constitute a basis for che-
mical studies of protein S-guanylation. Direct nitration of
guanosine by peroxynitrate was reported to give a poor yield
a Graduate School of Life Sciences, Tohoku University, Aoba-ku,
981-8555 Sendai, Japan. E-mail: arimoto@biochem.tohoku.ac.jp;
Fax: +81-22-717-8806; Tel: +81-22-717-8803
b Graduate School of Science, Tohoku University, Aoba-ku, 980-8578
Sendai, Japan
c School of Medicine, Kumamoto University, 860-0811 Kumamoto,
Japan. E-mail: takakai@gpo.kumamoto-u.ac.jp;
Fax: +81-96-362-8362; Tel: +81-96-362-8362
w Electronic supplementary information (ESI) available: Preparative
procedures for compounds, stability experiments of compounds 7 and
8, crystal structure of 7, procedures for the reaction of 8 with cell
lysate, and for detection of heme oxygenase-1 induction by com-
pounds 1, 2,
7
and
8
in cells. CCDC 692937. For ESI and
Scheme 1 Reagents and conditions: (i) DMTr-Cl, pyridine, room
temp., 10 h (99%); (ii) see Table 1; (iii) p-TsOH, CHCl3, room temp.,
25 min (quant.); (iv) MeNH2, MeOH, room temp., 12 h (83%).
crystallographic data in CIF or other electronic format see DOI:
10.1039/b810771h
ꢀc
This journal is The Royal Society of Chemistry 2008
5984 | Chem. Commun., 2008, 5984–5986

View Article Online
Table 1 Nitration of 8-bromoguanosine derivative 4
Reaction Yield
period/h
Recovery
(%)
Entry
Conditionsa Solvent
(%)
1
2
3
4
5
a
A
B
B
B
B
CH3CN
DMF
DMF
DMF
DMF
72
6
7
10
25
49
60
44
34
27
—
23
10
3
3
Condition A: KNO2 (6.5 eq.), 18-crown-6 (6.5 eq.), reflux; condition
B: KNO2 (10 eq.), 18-crown-6 (10 eq.), 100 1C.
Fig. 3 Molecular structure of 7 in crystal. Atom labels: hydrogen =
with varied reaction periods (entries 2–5). In this case, the
optimum isolated yield (60%, 78% based on recovered start-
ing material) was obtained for the reaction period of 6 h
(entry 2). Extension of the reaction period reduced the yield of
5 and the recovery of 4 (entries 3–5). The DMTr ether was
cleaved by exposure of 5 to p-toluenesulfonic acid in chloro-
form (quant.). Triacetates of 6 were then removed cleanly by a
methanolic methylamine solution at room temperature to give
crude 2. Since the only impurity was a trace amount of
N-methylacetamide, the obtained 2 could be used for most
purposes. The crude 2 was purified by ODS chromatography
at 4 1C (83%, the overall yield was 49% from 3 in 4 steps). The
described synthesis avoided the use of HPLC for purification
and would be easily scaleable to obtain larger amounts of the
sample for biological studies.
black, carbon = gray, nitrogen = purple, fluorine = green.
possibility of novel base-pair recognition patterns of nitro-
guanosines, although the guanine nitrations in nucleic acids
have not been fully uncovered in cells.
Having synthesized 8-nitroguanosines, we then turned our
attention to the biological activities of these compounds.
Induction of heme oxygenase-1 (HO-1) expression has been
associated with adaptive cytoprotection against a wide array
of cellular stresses such as nutrient starvation or hypoxia. It
has been suggested that carbon monoxide (CO), one of the
products of the HO-1 reaction, may play a role in this
protective activity.12 Our previous study5 suggested that
S-guanylation of the sensor protein Keap1 by endogenous
8-nitro-cGMP 1 upregulates HO-1 through a signaling path-
way.13 An important question here is whether the cGMP
substructure of 1 is indispensable for the upregulation.
Indeed, 8-nitroguanosine 2 and the 20-fluoro analogue 7,
which lack cyclic phosphate moieties could enhance the in-
duction of HO-1 in HepG2 cells, and protected the cells from
glucose deprivation-induced cytotoxicity14 (Fig. 4). The
50-azido analogue 8 also exhibited upregulation of HO-1
(Fig. S7 of ESIw). A more detailed interpretation of these
results will have to wait until both the full biological potential
of nitroguanosines and an complete structure-activity relation-
ships have been clarified. However, these findings do reveal
that, in principle, 8-nitroguanosine derivatives, which lack a
cyclic phosphate are useful as chemical probes for study of the
protein S-guanylations.
Considering the instability of 2 in water (half life: 5 h), we next
explored novel analogues suitable for biochemical research.
Similar synthetic schemes (Fig. S1 and S2 of ESI) were
applied for 20-deoxy-20-fluoro-8-nitroguanosine 7 and 50-azido-
50-deoxy-8-nitroguanosine 8 (Fig. 2). The half-lives of these
analogues in 0.05 M sodium phosphate buffer were examined
(pH 7.4, 37 1C). The half-lives were 72 d for 7 and 4.5 d for 8
(Fig. S3 and S4w). The stability of these analogues is an
important advantage for their use as chemical probes in biolo-
gical studies. Moreover, this characteristic allowed X-ray
crystallographic analysis of the stabilized 20-F-derivative 7. A
single-crystal of 7 was obtained from its water–ethyl acetate
solution.
Because this is the first crystallographic experiment con-
ducted among 8-nitroguanosine derivatives, important fea-
tures of the obtained structure should be mentioned here
(Fig. 3). (i) The bond length of the glycosidic C–N bond
(1.523 A) was remarkably longer than those of guanosine
(1.454 A)10 and 8-Br-guanosine (1.473 A).11 These results
would reflect a significant destabilization of the glycosidic
bond by an electron-withdrawing nitro group at the adjacent
carbon. (ii) The oxygen atoms of the nitro group were involved
in a hydrogen-bonding with NH2 of another molecule in a
crystal lattice (Fig. S5 of ESI). This fact might suggest the
So far, only Keap1 has been identified to be a physiological
target protein of S-guanylation.5 However, there may be other
important sensors or enzymes that are regulated by the
Fig. 4 Heme oxygenase-1 induction by 8-nitroguanosine 2 and its
fluorinated derivative 7: HepG2 cells were treated with indicated
concentrations of 2 or 7 under glucose-starved condition for 8 h,
and then were analyzed by Western blotting. As a control, expression
of b-actin is shown. The ratio represents relative intensity of HO-1 and
b-actin.
Fig. 2 Fluoro- and azido-analogues of 8-nitroguanosine.
ꢀc
This journal is The Royal Society of Chemistry 2008
Chem. Commun., 2008, 5984–5986 | 5985

View Article Online
Fig.
5
Fluorescence scan (left) and silver stain (right) of the
SDS-PAGE gel of the labeled C6 cell lysate 10 (Scheme 2). The
fluorescent labeled bands are indicated by arrows.
Grants-in-Aid (Nos. 16310150, 20651056) for Scientific
Research from the Ministry of Education, Sports, Science
and Technology (MEXT), Japan.
Scheme 2 Reaction of 8 with a cell lysate, and fluorescent labeling by
Huisgen reaction: Reagents and conditions: (i) cell lysate (C6 cell),
0.6 M Na2HPO4aq., room temp., 5 h; (ii) CuBr, 11, tris[(1-benzyl-1H-
1,2,3-triazole-4-yl)methyl]amine, 0.5 M sodium phosphate buffer,
pH 8, room temp., overnight.
Notes and references
1 L. J. Ignarro, Introduction and overview, in Nitric Oxide in Biology
and Pathobiology, ed. L. J. Ignarro, Academic, San Diego, 2000,
pp. 3–19.
2 F. J. Schopfer, P. R. Baker and B. A. Freeman, Trends Biochem.
Sci., 2003, 28, 646–654.
3 R. Radi, Proc. Natl. Acad. Sci. U. S. A., 2004, 101, 4003–4008.
4 J. P. Eiserich, M. Hristova, C. E. Cross, A. D. Jones, B. A.
Freeman, B. Halliwell and A. van der Vliet, Nature, 1998, 391,
393–397.
5 T. Sawa, M. H. Zaki, T. Okamoto, T. Akuta, Y. Tokutomi, S. K.
Mitsuyama, H. Ihara, A. Kobayashi, M. Yamamoto, S. Fujii, H.
Arimoto and T. Akaike, Nat. Chem. Biol., 2007, 3, 727–735.
6 (a) A. T. Dinkova-Kostova, W. D. Holtzclaw, R. N. Cole, K. Itoh,
N. Wakabayashi, Y. Katoh, M. Yamamoto and P. Talalay, Proc.
Natl. Acad. Sci. U. S. A., 2002, 99, 11908–11913; (b) N.
Wakabayashi, A. T. Dinkova-Kostova, W. D. Holtzclaw, M.-I.
Kang, A. Kobayashi, M. Yamamoto, T. W. Kensler and P.
Talalay, Proc. Natl. Acad. Sci. U. S. A., 2004, 101, 2040–2045;
(c) T. Yamamoto, T. Suzuki, A. Kobayashi, J. Wakabayashi, J.
Maher, H. Motohashi and M. Yamamoto, Mol. Cell. Biol., 2008,
28, 2758–2770.
7 R. S. Sodum and E. S. Flala, Chem. Res. Toxicol., 2001, 14,
438–450.
8 T. Akaike, S. Okamoto, T. Sawa, J. Yoshitake, F. Tamura, K.
Ichimori, K. Miyazaki, K. Sasamoto and H. Maeda, Proc. Natl.
Acad. Sci. U. S. A., 2003, 100, 685–690.
9 J. M. Lee, J. C. Niles, J. S. Wishnok and S. R. Tannenbaum, Chem.
Res. Toxicol., 2002, 15, 7–14.
10 U. Thewalt, C. E. Bugg and R. E. Marsh, Acta Crystallogr., Sect.
B: Struct. Sci., 1970, 26, 1089–1101.
guanylation. In this context, fluorecent labelings by 8-nitro-
guanosine-based probes could become powerful tools for the
rapid and comprehensive identification of potential target
proteins. Thus, we investigated the potential of the azide
derivative 8 as a labeling probe. Treatment of 8 with glu-
tathione (GSH: data not shown) or bovine serum albumin
(BSA: data not shown) were first examined to confirm its
reactivity to thiols. Whole rat glioma C6 cell lysate was then
treated with compound 8 (Scheme 2). The progress of the
reaction could be monitored by the release of NO2ꢁ ion by the
Griess method.15
The S-guanylated proteins 9 in the lysate were then labeled
by a fluorescent tag via a Huisgen reaction.16 Fluorescent
scanning of a SDS-PAGE gel (Fig. 5) confirmed the presence
of several labeled proteins. We are currently investigating the
guanylated proteins in a series of cell lines under defined
culture conditions using the protocols introduced above.
Enzymatic digestion and sequence analysis of the labeled
bands is also in progress. The results should provide an insight
into the possible physiological targets of protein S-guanyla-
tion, and the results will be described elsewhere.
In conclusion, 8-nitroguanosine and its stable analogues
were synthesized. The cyclic phosphate moiety of 8-nitro-
cGMP was proven unnecessary for cytoprotective HO-1 up-
regulation, because these analogues also induced the HO-1 in
cells. Successful fluorescent labeling of the cell lysate was
also described. Thus, the derivatives could become useful
chemical probes for biological or proteomic investigations into
protein-S-guanylations.
11 S. S. Tavale and H. M. Sobell, J. Mol. Biol., 1970, 48, 109–123.
12 (a) D. Yoshimura, K. Sakumi, M. Ohno, Y. Sakai, M. Furuichi, S.
Iwai and Y. Nakabeppu, J. Biol. Chem., 2003, 278, 37965–37972;
(b) M.-H. Li, J.-H. Jang, H.-K. Na, Y.-N. Cha and Y.-J. Surh,
J. Biol. Chem., 2007, 282, 28577–28586.
13 For the heme oxygenase-1 induction by 1: see Fig. S6 of ESIw.
14 Manuscript in preparation. A part of the study was presented at
the 3rd International Conference on the Biology, Chemistry and
Therapeutic Applications of Nitric Oxide & the 4th Annual
Scientific Meeting of the Nitric Oxide Society of Japan, Nara,
Japan, 24–28 May 2004. T. Akuta, T. Akaike, J. Yoshitake, K.
Kaneko, Y. Terasaki and H. Maeda, Nitric Oxide, 2004, 11, 98–99.
15 V. M. Ivanov, J. Anal. Chem., 2004, 59, 1109–1112.
16 H. C. Kolb, M. G. Finn and K. B. Sharpless, Angew. Chem., Int.
Ed., 2001, 40, 2004–2021.
We are grateful to Mr. Tadahiro Honda and Dr Zhaopeng
Liu (Nagoya University) for conducting the preliminary
experiments, and to Prof. Daisuke Uemura (Keio Univ.) for
his valuable suggestions. This work was supported in part by
ꢀc
This journal is The Royal Society of Chemistry 2008
5986 | Chem. Commun., 2008, 5984–5986