Efficient covalent capture of 8-nitroguanosine via a multiple hydrogen-bonded complex

Article abstract of DOI:10.1021/ol500452r

A novel 1,3-diazaphenoxazine nucleoside derivative (nitroG-Grasp) bearing a thiol group with a urea linker forms multiple hydrogen-bonded complexes with 8-nitroguanosine and efficiently displaces the nitro group; thus, it is the first molecule that can covalently capture 8-nitroguanosine.

Full text of DOI:10.1021/ol500452r

Letter
pubs.acs.org/OrgLett
Efficient Covalent Capture of 8‑Nitroguanosine via a Multiple
Hydrogen-Bonded Complex
Yasufumi Fuchi and Shigeki Sasaki*
Graduate School of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan
S
* Supporting Information
ABSTRACT: A novel 1,3-diazaphenoxazine nucleoside de-
rivative (nitroG-Grasp) bearing a thiol group with a urea linker
forms multiple hydrogen-bonded complexes with 8-nitro-
guanosine and efficiently displaces the nitro group; thus, it is
the first molecule that can covalently capture 8-nitroguanosine.
eactive oxygen species (ROS) and reactive nitrogen
Rspecies (RNOS) are representative chemical sources of
oxidative stress for cells and cause nucleic acid damage, such as
8-oxoguanine and 8-nitroguanine derivatives.1−3 They are
highly mutagenic and are regarded as biomarkers of oxidative
stress.4 8-Nitroguanosine (8-nitroG) is generated frequently in
inflammatory or infected tissues.5 Despite its conventional
classification as a genotoxic material, 8-nitroguanosine 3′,5′-
cyclic monophosphate (8-nitro-cGMP) has recently attracted
Figure 1. Selective complex formation with 8-Oxo-dG (A) and G or 8-
significant interest due to its unique biological properties.6−9
nitroG (B), in which single hydrogen bonding is responsible for
selectivity.
For instance, 8-nitro-cGMP is produced in response to iNOS
activity and reacts with sulfhydryl groups of specific proteins
(protein S-guanylation).10 8-Nitro-cGMP also reacts with
highly nucleophilic H2S/HS− to form 8-SH-cGMP,11 but its
scheme, in which access of the thiol group is possible due to the
fixed conformation of the urea-alkyl linker in the multiple
reactivity is low for glutathione, an abundant thiol in cells.10
hydrogen-bonded complex through which the compound
These findings have led to a proposal for a new metabolic cycle
undergoes S-guanylation (Figure 2).
or signaling pathway mediated by 8-nitro-cGMP.10,12 Thus,
In this study, 8-nitro-2′,3′,5′-tri-O-acetylguanosine (triAc-8-
selective molecules for 8-nitroG derivatives are needed for
nitroG) was used as a target molecule and synthesized
further understanding their biological functions. However,
according to previous literature.8 The nitroG-Grasp derivatives
except for antibodies,13 there is no recognition compound for
(3a−c) were synthesized through a reaction between the amino
8-nitroguanosine derivatives. This paper reports for the first
group of the 3′,5′-O-diTBDMS-G-clamp unit as a common
time new 1,3-diazaphenoxazine nucleoside derivatives bearing a
intermediate and the S-trityl protected mercaptoalkylamido-
thiol arm that exhibits efficient covalent capture of 8-
nitroguanosine via a multiple hydrogen-bonded complex.
Our early studies demonstrated that oxoG-clamp (1) is a
selective fluorescent binder of 8-oxo-dG and that a carbamate
group of 1 is responsible for the selective recognition of 8-oxo-
dG (Figure 1A, Ks: 8-oxo-dG/dG = 10).14−16 1H NMR and the
structure-binding relationship of 1 have indicated that the
complex is formed through multiple hydrogen bonds as shown
in Figure 1.14,17 Compound 2 became selective for dG because
of the substitution of a urea for the carbamate in 1 (Figure 1B,
R = H, Ks: 8-oxo-dG/dG = 0.04).17 A preliminary investigation
revealed that 8-nitroG formed more stable complexes with 2
than with 1 (Ks in CHCl3, 1: 0.9 × 106 M−1 vs 2: 3.6 × 106 M−1
Figure 2. Design of a new reactive molecule (NitroG-Grasp) for the
covalent capture of 8-NitroG.
(Figure S1). Thus, we designed new recognition molecules for
8-nitroG such that the thiol group attached to a urea linker
could reach the 8-position to displace the nitro group (3a−c).
The compound was named nitroG-grasp based on its reaction
Received: February 12, 2014
Published: March 7, 2014
© 2014 American Chemical Society
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Letter
imidazole, followed by the deprotection of the S-trityl group by
TFA in the presence of triethylsilane (Scheme S1). As a control
compound, the carbamate derivative 4 was synthesized using
the 3′,5′-O-diTBDMS-G-clamp unit and S-trityl-protected
mercaptopropyloxycarbonylimidazole. The control compound
4 lacks the hydrogen-bonding site for the N7 atom of
nitroguanosine.
The reaction of the nitroG-Grasp derivatives with triAc-8-
nitroG was performed in MeCN and monitored by HPLC. An
equimolar mixture of C3-nitroG-Grasp (3b) and triAc-8-nitroG
formed an adduct quantitatively as a single product within 20
min at 30 °C in MeCN containing Et3N (Figure 3). This
Figure 4. Comparison of the time courses of the reactions with
nitroG-Grasp 3a−c or the control compounds (4 and Z-NH-
(CH2)3SH). Product yields were obtained at the indicated time
points by HPLC analysis, as described in the footnote to Figure 3.
adduct under the same conditions. The guanylation of 3b
occurred in aqueous media (CH3CN−H2O) buffered with TEA
and AcOH at pH 7.0, which was not largely inhibited by
glutathione (Figure S4). These results exemplify the con-
tribution of the complexation of 3b for efficient reactivity.
To clarify the origin of the differences in reactivity observed
for 3a−c, we next evaluated the reaction kinetics by monitoring
the reaction by UV−vis spectroscopy. The absorbance at 368
nm due to the 8-nitroguanine base19 gradually decreased with
time (Figure 5). Simultaneously, the absorbance at 260 nm due
Figure 3. Reaction of C3-nitroG-Grasp (3b) with triAc-8-nitroG
monitored by HPLC and the structure of the guanylated-product (5b).
The reaction was performed using 1 mM each of 3b and 8-nitroG in
the presence of 10 mM Et3N in CH3CN at 30 °C. HPLC conditions:
column: Xbridge C8 3.5 μm, 3.0 mm × 100 mm; solvents: (A) 0.1 M
TEAA buffer and (B) CH3CN, A/B = 20:80; flow rate: 0.5 mL/min;
monitored by UV at 254 nm.
Figure 5. UV−vis spectral changes due to the desorption of the nitro
group of triAc-8-nitroG. Example spectra were measured at the
indicated time intervals using 3b (50 μM) and triAc-8-nitroG (50 μM)
in CH3CN containing Et3N (500 μM).
product was isolated, and its structure was confirmed to be that
depicted in 5b by 1H NMR, ESI-MS, and HMBC. The
chemical shifts of the methylene protons adjacent to the sulfur
atom were shifted from 2.5 to 3.2 ppm (Supporting
Information). The HMBC spectrum revealed the correlation
between the 8-carbon atom of the 8-nitroguanine unit and the
methylene protons next to the sulfur atom, as depicted in 5b in
Figure 3. The released HNO2 was confirmed by the formation
of the azo dye compound using Bratton−Marshall reagents
(Figure S2).18
The time courses of the reaction between the nitroG-clamp
derivatives (3a−c, 4) and triAc-8-nitroG are compared in
Figure 4. C3-nitroG-Grasp (3b) exhibited the most efficient
reactivity; moderate reactivity was observed with C2-nitroG-
Grasp (3a), and relatively slow reactions were observed with
C4-nitroG-Grasp (3c). The reaction by 3b proceeded
efficiently at concentrations as low as 1 μM (Figure S3). In
contrast, the control compound 4, which lacks a hydrogen-
bonding site for the N7 of 8-nitroguanine due to its carbamate
linker, exhibited much slower reactivity (Figure 4). N-Cbz-
aminopropanethiol (ZNH(CH2)3SH), used as a control
without a binding site for 8-nitroguanosine, did not yield any
to the 8-thioguanine base20 increased with time. These changes
agree with the time course obtained by HPLC (Figure 4).
Thus, the absorbance at 400 nm was monitored in the presence
of excess nitroG-Grasp derivatives to obtain the kinetic
parameters of the pseudo-first-order reaction. The obtained
k
obs values were in the order 3b (0.0336 s−1) > 3a (0.0256 s−1)
> 3c (0.0064 s−1) > 4 (0.0008 s−1), agreeing with the results
determined using HPLC (Figure 4A).
The kobs values increased proportionally with the concen-
trations of the nitroG-Grasp derivatives without exhibiting
saturation phenomena (Figure S5), suggesting that the
formation of the hydrogen-bonded complex is not a rate-
determining step under these conditions. The reaction was
performed at different temperatures, and kinetic parameters
were obtained by Arrhenius plots (Table 1, Figure S6). The
reaction of 3b exhibited the lowest values of ΔG‡ and ΔH‡.
Although its ΔS‡ term was in the average range, the highly
favorable ΔH‡ term produced the lowest ΔG‡ value.
Interestingly, the ΔH‡ value of 3c is relatively low, but the
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Letter
a
of 4 has the highest reactivity in terms of its nucleophilicity, but
its displacement reactivity is diminished because of its limited
access to the reactive site. Thus, we concluded that the highest
displacement reactivity of 3b toward 8-nitroG is due to both
the limited flexibility of the urea linker in the multiple
hydrogen-bonded complex and the relatively facile formation
of the thiolate.
Table 1. Kinetic Parameters for the Guanylation Reaction
b
R−SH kobs (s−1
)
Ea (kJ/mol) ΔG‡ (kJ) ΔH‡ (kJ) ΔS‡ (J/K)
3a
3b
3c
4
0.0256
21.6
16.2
17.8
12.0
85.5
81.8
87.3
91.4
19.1
13.7
15.4
9.5
−224
−231
−244
−272
0.0336
0.0064
0.0008
a
The pseudo-first-order rate constants (kobs) were obtained by
In this study, we designed new thiol-containing recognition
molecules 3a−c for the covalent capture of 8-nitroguanosine
based on the G-clamp skeleton by introducing thioalkylurea
linker units of varying length. Compounds 3a−c efficiently
displaced the nitro group of 8-nitroG to form a sulfide bond
with the guanine residue (guanylation). Among them, 3b
exhibited the most efficient guanylation even at low
concentrations, supporting the selective complex formation
for efficient reactivity for 8-nitroG. In the control experiments,
ZNH(CH2)3SH did not show reactivity; 4 with the carbamate
linker displayed only low reactivity. The design principles, in
which binding selectivity can be controlled by one of multiple
hydrogen bonds and efficient reactivity can be achieved due to
the close proximity in the complex, have been validated through
the efficient reactivity of 3b, and may be applied to develop
recognition molecules for other 8-substituted guanine deriva-
tives such as 8-chloro- or 8-thioguanosine, etc. Because interest
in 8-nitroguanosine derivatives is increasing due to their
biological roles in signal transduction pathways, the nitroG-
Grasp molecule is expected to be a potential platform structure
to develop specific molecules for 8-nitroG. In our group,
systematic studies are now ongoing to develop nitroG-Grasp
derivatives conjugated with a variety of phosphates binding
moiety,22 aiming to attain selective affinity to 8-nitro-cGMP in
aqueous media, which will be reported in due course.
monitoring the absorbance at 400 nm using R−SH (200 μM) and
b
triAc-8-nitroG (10 μM). The rate constants at 30 °C.
unfavorable ΔS‡ term leads to a high ΔG‡ value. In addition,
although the ΔH‡ value of 4 is the lowest, it possessed the most
unfavorable ΔS‡ term, making its ΔG‡ value the highest.
As the intrinsic nucleophilicity of thiolate is several orders of
magnitude greater than that of the corresponding thiol, the
ΔH‡ values of 3a−c may reflect the ionization of their thiol
group. The pKa values of the thiol of 3a−c and 4 were
simulated using quantum mechanical calculations and plotted
against the corresponding ΔH‡ values. A clear linear relation-
ship was obtained, as depicted in Figure 6A, suggesting that the
ASSOCIATED CONTENT
■
S
* Supporting Information
Experimental details include the synthesis, HNO2 detection,
NMR spectra of the nitroG-Grasp derivatives, reaction
monitoring, and kinetic evaluation. This material is available
free of charge via the Internet at http://pubs.acs.org.
Figure 6. (A) Plot of ΔH‡ against the calculated pKa. (B) Plot of ΔS‡
against the number of atoms between the NH and SH groups. (C)
The numerals in parentheses represent the pKa values predicted by the
quantum mechanical calculation (pKa calculation protocol installed in
Jaguar) for the partial structure of the thiol units corresponding to 3a−
c and 4. The numerals marked on the structures represent the number
of atoms from the NH to the SH group.
AUTHOR INFORMATION
■
Corresponding Author
*E-mail: sasaki@phar.kyushu-u.ac.jp.
Notes
ΔH‡ values include the process of thiolate formation.21
Acceleration by TEA also supports that the thiolate is a
nucleophile (Figure S7). As 4 showed the lowest reactivity
despite the lowest ΔH‡ value, the ΔH‡ value is not the
determinant of the selectivity. In other words, the selectivity of
3b is not due to the acidity of the thiol. When the ΔS‡ values
were plotted against the number of atoms from the NH to the
SH group, a linear relationship was obtained for 3a−c (Figure
6B). Assuming that the two NH groups of the urea unit have
limited flexibility in the hydrogen-bonded complex in the
transition state, as shown in Figure 2, it is reasonable that the
ΔS‡ value reflects the freedom of the N-alkyl chain. The ΔS‡
value of 4 with the carbamate linker was more unfavorable
(closed circle) than that expected based on the number of
linker atoms (open circle), as depicted in Figure 6B. It is likely
that the strength of one hydrogen bond is insufficient,
increasing the flexibility of the entire linker, including the NH
group. In other words, the thiol group of the carbamate linker
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We are grateful for support provided by a Grant-in-Aid for
Scientific Research (S) (No. 21229002) and a Grant-in-Aid for
Challenging Exploratory Research (No. 24659008) from the
Japan Society for the Promotion of Science (JSPS). S.S. and
Y.F. also acknowledge the Astellas Foundation for Research on
Metabolic Disorders and a Research Fellowship for Young
Scientists from JSPS, respectively.
REFERENCES
■
(1) Jena, N. R. J. Biosci. 2012, 37, 503−517.
(2) Yermilov, V.; Rubio, J.; Ohshima, H. FEBS Lett. 1995, 376, 207−
210.
(3) Masuda, M.; Nishino, H.; Ohshima, H. Chem. Biol. Interact. 2002,
139, 187−197.
1762
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Letter
(4) A recent review: Loft, S.; Danielsen, P.; Løhr, M.; Jantzen, K.;
Hemmingsen, J. G.; Roursgaard, M.; Karotki, D. G.; Møller, P. Arch.
Biochem. Biophys. 2012, 518, 142−150.
(5) A recent review: Hiraku, Y. Environ. Health Prev. Med. 2009, 63−
72.
(6) Akaike, T.; Okamoto, S.; Sawa, T.; Yoshitake, J.; Tamura, F.;
Ichimori, K.; Miyazaki, K.; Sasamoto, K.; Maeda, H. Proc. Natl. Acad.
Sci. U.S.A. 2003, 100, 685−690.
(7) Sawa, T.; Ohshima, H. Nitric Oxide 2006, 14, 91−100.
(8) Saito, Y.; Taguchi, H.; Fujii, S.; Sawa, T.; Kida, E.; Kabuto, C.;
Akaike, T.; Arimoto, H. Chem. Commun. 2008, 5984−5986.
(9) Saito, Y.; Ito, C.; Fujii, S.; Sawa, T.; Akaike, T.; Arimoto, H.
ChemBioChem 2013, 14, 1068−1071.
(10) Sawa, T.; Zaki, M. H.; Okamoto, T.; Akuta, T.; Tokutomi, Y.;
Kim-Mitsuyama, S.; Ihara, H.; Kobayashi, A.; Yamamoto, M.; Fujii, S.;
Arimoto, H.; Akaike, T. Nat. Chem. Biol. 2007, 3, 727−735.
(11) Ahmed, K. A.; Sawa, T.; Akaike, T. Amino Acids 2011, 41, 123−
130.
(12) Sawa, T.; Ihara, H.; Ida, T.; Fujii, S.; Nishida, M.; Akaike, T.
Nitric Oxide 2013, 34, 10−18.
(13) Yoshitake, J.; Akaike, T.; Akuta, T.; Tamura, F.; Ogura, T.;
Esumi, H.; Maeda, H. J. Virol. 2004, 78, 8709−8719.
(14) Nakagawa, O.; Ono, S.; Li, Z.; Tsujimoto, A.; Sasaki, S. Angew.
Chem., Int. Ed. 2007, 46, 4500−4503.
(15) Nakagawa, O.; Ono, S.; Tsujimoto, A.; Li, Z.; Sasaki, S.
Nucleoside Nucleotide Nucleic Acids 2007, 26, 645−649.
(16) Nasr, T.; Li, Z.; Nakagawa, O.; Taniguchi, Y.; Ono, S.; Sasaki, S.
Bioorg. Med. Chem. Lett. 2009, 19, 727−730.
(17) Li, Z.; Nakagawa, O.; Koga, Y.; Taniguchi, Y.; Sasaki, S. Bioorg.
Med. Chem. 2010, 18, 3992−3998.
(18) Bratton, A. C.; Marshall, K. E. J. Biol. Chem. 1939, 128, 537−
550.
(19) Bhamra, I.; Compagnone-Post, P.; O’Neil, I. A.; Iwanejko, L. A.;
Bates, A. D.; Cosstick, R. Nucleic Acids Res. 2012, 40, 11126−11138.
(20) Holmes, R. E.; Robins, R. K. J. Am. Chem. Soc. 1964, 86, 1242−
1245.
(21) A recent example where nucleophilicity of thiol in protein is
discussed in relation to its pKa value: Sardi, F.; Manta, B.; Portillo-
Ledesma, S.; Knoops, B.; Comini, M. A.; Ferrer-Sueta, G. Anal.
Biochem. 2013, 435, 74−82.
(22) Hargrove, A. E.; Nieto, S.; Zhang, T.; Sessler, J. L.; Anslyn, E. V.
Chem. Rev. 2011, 111, 6603−6782.
1763
dx.doi.org/10.1021/ol500452r | Org. Lett. 2014, 16, 1760−1763