Transnitrosation of thiols from aliphatic N-nitrosamines: S-nitrosation and indirect generation of nitric oxide

Article abstract of DOI:10.1021/ja0658259

S-Nitrosothiols and heme nitrosyl species are nitric oxide (NO)-derived metabolites that provide an endogenous reservoir of NO and also play roles in protein S-nitrosation, that is, transnitrosation of thiols (or thiolates) in proteins, thereby regulating protein functions and signal transduction pathways. Intriguingly, endogenous N-nitrosamines are present in similar abundance to S-nitrosothiols, and though they are thought to play similar physiological roles to S-nitrosothiols, their transnitrosation reactivities and their contribution to biological events are little understood. Herein we report aliphatic N-nitroso derivatives of 7-azabicyclo[2.2.1]heptanes, which do not act as NO donors themselves, but can transnitrosate thiols. On the basis of the calculated activation energies of transnitrosation and the aorta smooth-muscle relaxation activities of these N-nitrosamines, we present a possible scenario of S-transnitrosation from aliphatic N-nitrosamines, leading to indirect generation of NO. Copyright

Full text of DOI:10.1021/ja0658259

Published on Web 01/04/2007
Transnitrosation of Thiols from Aliphatic N-Nitrosamines: S-Nitrosation and
Indirect Generation of Nitric Oxide
Takahiro Yanagimoto,† Takeshi Toyota,‡ Norio Matsuki,‡ Yumi Makino,† Seiichi Uchiyama,† and
Tomohiko Ohwada*,†
Laboratory of Organic and Medicinal Chemistry and Laboratory of Chemical Pharmacology, Graduate School of
Pharmaceutical Sciences, The UniVersity of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan
Received August 10, 2006; E-mail: ohwada@mol.f.u-tokyo.ac.jp
S-Nitrosothiols and heme nitrosyl species1 are nitric oxide (NO)-
derived metabolites that provide an endogenous reservoir of NO2
and also play roles in protein S-nitrosation, that is, transnitrosation
of thiols (or thiolates) in proteins,3-6 thereby regulating protein
functions and signal transduction pathways.7,8 Intriguingly, endog-
enous N-nitrosamines are present in similar abundance to S-
nitrosothiols,9 and though they are thought to play similar physi-
ological roles to S-nitrosothiols,6 their transnitrosation reactivities
and their contribution to biological events are little understood.
Aromatic N-nitrosamines, N-nitrosotryptophan derivatives, can
generate S-nitrosothiols10 and can also act as direct NO donors,11
but little is known about the S-transnitrosation reactivities of
aliphatic N-nitrosamines, such as proline derivatives. Herein we
describe the S-transnitrosation reaction of aliphatic N-nitroso
derivatives of 7-azabicyclo[2.2.1]heptanes (2-10), which resemble
conformationally constrained proline derivatives,12 and its chemical
features, that is, reactivity and chemoselectivity.
transnitrosation.14 All the bicyclic N-nitrosamines 2-10 were
positive in the Griess assay (Figures S1 and S2) whereas N-
nitrosamine 1 did not generate any dye at all.12 It was confirmed
that no NO was generated from the N-nitroso derivatives of
7-azabicyclo[2.2.1]heptanes at physiological pH (7.4, in PBS buffer)
by means of an ESR spin trap experiment (Figure S5).15 Thus, these
aliphatic N-nitrosamines are not direct NO donors under neutral
conditions.12
To examine the N-nitroso compound-dependent formation of
S-nitrosothiols from thiols, we studied the UV-visible spectroscopic
change upon transnitrosation in an aprotic solvent. We choose
triphenylmethylthiol as a model thiol (S-nucleophile), because
S-nitrosotriphenylmethylthiol (11) is sufficiently stable to be well
characterized.16 To study the chemoselectivity of transnitrosation,
pyrrolidine, a mimic of proline, was used as a model secondary
amine (N-nucleophile).
The bicyclic N-nitrosamine derivative 7 undergoes transnitro-
sation reaction with triphenylmethylthiol to afford the S-nitrosothiol
11 (eq a).17 This is consistent with the relevant spectral changes
(Figure 1b and the MNTS case (see Supporting Information, Figure
These N-nitroso derivatives of 7-azabicyclo[2.2.1]heptanes do not
act as NO donors themselves, but can transnitrosate thiols. On the
basis of the calculated activation energies of transnitrosation and
the aorta smooth-muscle relaxation activities of these N-nitro-
samines, we present a possible scenario of S-transnitrosation from
aliphatic N-nitrosamines, leading to indirect generation of NO.
The N-NO bond of the N-nitroso derivatives of 7-azabicyclo-
[2.2.1]heptanes tends to be weak,12 and this is reflected in reduced
rotational barriers of the N-NO bonds in solution and nitrogen-
pyramidal structures of the N-nitroso group in the solid state. To
increase the reactivity and hydrophilicity of these compounds, we
synthesized the benzo derivatives 2-10 (see Supporting Informa-
tion).
S3b)) and was also confirmed by ESI mass spectrometry (Figure
S4e). In contrast, the monocyclic aliphatic N-nitrosopyrrolidine 12,
a mimic of N-nitrosoproline derivatives, did not nitrosate triph-
enylmethylthiol in chloroform even during 50 h at 37 °C (eq b and
Figure S3d). These activities are consistent with the Griess assay
results. In contrast, no N-nitrosopyrrolidine was formed from
pyrrolidine by the action of 7 (Figure 1d (arrow) and eq c). Thus,
the transnitrosation reaction of 7 exhibited chemoselectivity, that
is, S-transnitrosation was preferred over N-transnitrosation. Similar
chemoselectivity was observed for the bicyclic N-nitrosamine 8
(data not shown). In aqueous solution (methanol/water (20:80) or
DMSO/PBS buffer (20:80)), the transnitrosation reaction of 8 also
occurred with glutathione (GSH), a ubiquitous endogenous thiol,
to form S-nitrosoglutathione (GSNO) (eq d), as indicated by the
absorption spectral change (Figure S3f) and the detection of the
formed GSNO by ESI mass spectroscopy (Figure S4f).
The reactivity of the N-NO bond of N-nitrosamines, or in other
words, the propensity to release NO+ (or NO) in acidic solution,
can be estimated with the Griess method.13 Well-studied nitrosating
reagents, such as N-nitrososulfonamides (e.g., N-methyl-N-nitroso-
p-toluenesulfonamide (MNTS)) were used as positive controls of
† Laboratory of Organic and Medicinal Chemistry.
‡ Laboratory of Chemical Pharmacology.
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10.1021/ja0658259 CCC: $37.00 © 2007 American Chemical Society

C O M M U N I C A T I O N S
Figure 2. Relaxation of rat aorta smooth-muscle strips with N-Nitrosamines
(n ) 3∼4); dose-dependent increase of relaxation. Asterisk indicates the
maximum relaxation induced by 3% DMSO.
NO via S-transnitrosation. Thus, the present compounds may
provide a lead for developing a new class of slow-release NO
donors, which generate NO indirectly through S-transnitrosation.
Figure 1. Absorption spectral changes in the presence of NO-donating
agent. (a) S-Nitrosotriphenylmethylthiol 11 (authentic, 1 mM, CHCl3, 37
°C); (b) 7 + triphenylmethylthiol in CHCl3, 37 °C; (c) MNTS + pyrrolidine
in CHCl3, 37 °C; (d) 7 + pyrrolidine in CHCl3, 37 °C.
Acknowledgment. This work was supported by the 21st COE
project from the Ministry of Education, Science, Sports, Culture
and Technology. T.O. is grateful for a Grant-in-Aid for Scientific
Research (No. 17109001) from Japan Society for the Promotion
of Science. We also thank Prof. Hiroshi Nishihara and Mr. Kousuke
Namiki, Department of Chemistry, Graduate School of Science of
our university, for assistance for ESR measurements.
To shed light on the feasibility of transnitrosation reaction of
the N-nitroso derivatives (2, 12, 13 (a model of MNTS), and 14)
DFT calculations were carried out, assuming an SN2-like mecha-
nism,4,14 because no transnitrosation reaction occurred in the absence
of a potent nucleophile, such as thiols. The calculated reaction
energies at the DFT levels are summarized in Figure S8 and Table
S1. The calculated transition states (TSs) for transnitrosation have
a four-membered cyclic arrangement of the atoms, involving proton
transfer from sulfur to nitrogen (e.g., TS-14-CH3SH).
Supporting Information Available: Detailed discussions and
experimental and calculation details. This material is available free of
charge via the Internet at http://pubs.acs.org.
References
(1) Stamler, J. S.; Jaraki, O.; Osborne, J.; Simon, D. I.; Keaney, J.; Vita, J.;
Singel, D.; Valeri, C. R.; Loscalzo, J. Proc. Natl. Acad. Sci. U.S.A. 1992,
89, 7674-7677.
(2) Alencar, J. L.; Lobysheva, I.; Chalupsky, K.; Geffard, M.; Nepveu, F.;
Stoclet, J.-C.; Muller, B. J. Pharmacol. Exp. Ther. 2003, 307, 152-159.
(3) Hogg, N. Anal. Biochem. 1999, 272, 257-262.
(4) Williams, D. L. H. Acc. Chem. Res. 1999, 32, 869-876.
(5) (a) Perissinotti, L. L.; Turjanski, A. G.; Estrin, D. A.; Doctorovich, F. J.
Am. Chem. Soc. 2005, 127, 486-487. (b) Zhao, Y.-L.; McCarren, P. R.;
Houk, K. N.; Choi, B. Y.; Toone, E. J. Am. Chem. Soc. 2005, 127, 10917-
10924.
A model S-nucleophile (methylthiol) and N-nucleophile (methyl-
amine) are used. The present computational results, i.e., magunitude
of activation energies and overall reaction energies (endotherm/
exotherm) are consistent with the experimentally observed high
transnitrosation reactivities and S-nucleophile selectivity of the
present bicyclic N-nitrosamines.17
(6) Bryan, N. S.; Rassaf, T.; Maloney, R. E.; Rodriguez, C. M.; Saijo, F.;
Rodriguez, J. R.; Feelisch, M. Proc. Natl. Acad. Sci. U.S.A. 2004, 101,
4308-4313.
(7) Jaffrey, S. R.; Erdjument-Bromage, H.; Ferris, C. D.; Tempst, P.; Snyder,
S. H. Nat. Cell Biol. 2001, 3, 193-197.
(8) Hess, D. T.; Matsumoto, A.; Kim, S.-O.; Marshall, H. E.; Stamler, J. S.
Nat. ReV. Mol. Cell Biol. 2005, 6, 150-166.
To investigate the ability of the bicyclic nitrosamines to induce
biological events, experiments were carried out with isolated rat
aortic strips. The tested compounds exhibited concentration-
dependent relaxing of aorta strips preconstricted with norepinephrine
(Figure 2). The N-nitroso derivatives of 7-azabicyclo[2.2.1]heptanes
(5-8) were effective, and 7 was as potent as sodium nitroprusside
(SNP), a positive control vasodilator. The monocyclic N-nitrosamine
1 essentially did not relax the aorta, while the vehicle, DMSO, itself
showed some relaxing activity. Both hemoglobin, a typical NO
scavenger, and methylene blue and LY83583, guanylate cyclases
inhibitors, inhibited the relaxation induced by compounds 7 (10
µM) and 8 (100 µM). These results indicate that the bicyclic
N-nitrosamine-dependent relaxation is mediated by activation of
guanylate cyclase via the induction of NO (see Figures S6 and S7).
In summary, we showed here that aliphatic N-nitroso derivatives
of 7-azabicyclo[2.2.1]heptanes, which are not direct NO donors
under physiological conditions (pH and temperature), exhibit
S-transnitrosation reactivity. These N-nitrosamines showed a smooth
muscle-relaxing effect, presumably through indirect generation of
(9) Rassaf, T.; Bryan, N. S.; Kelm, M.; Feelisch, M. Free Radical Biol. Med.
2002, 33, 1590-1596.
(10) Sonnenschein, K.; de Groot, H.; Kirsch, M. J. Biol. Chem. 2004, 279,
45433-45440.
(11) Turjanski, A. G.; Leonik, F.; Estrin, D. A.; Rosenstein, R. E.; Doctorovich,
F. J. Am. Chem. Soc. 2000, 122, 10468-10469.
(12) Ohwada, T.; Miura, M.; Tanaka, H.; Sakamoto, S.; Yamaguchi, K.; Ikeda,
H.; Inagaki, S. J. Am. Chem. Soc. 2001, 123, 10164-10172.
(13) Green, L. C.; Wagner, D. A.; Glogowski, J.; Skipper, P. L.; Wishnok, J.
S.; Tannenbaum, S. R. Anal. Biochem. 1982, 126, 131-138.
(14) Zhu, X.-Q.; Hao, W.-F.; Tang, H.; Wang, C.-H.; Cheng, J.-P. J. Am. Chem.
Soc. 2005, 127, 2696-2708.
(15) Akaike, T.; Yoshida, M.; Miyamoto, Y.; Sato, K.; Kohno, M.; Sasamoto,
K.; Miyazaki, K.; Ueda, S.; Maeda, H. Biochemistry 1993, 32, 827-832.
(16) Arulsamy, N.; Bohle, D. S.; Butt, J. A.; Irvine, G. J.; Jordan, P. A.; Sagan,
E. J. Am. Chem. Soc. 1999, 121, 7115-7123.
(17) The second-order rate constant for N-nitrosamine 6 and triphenyl-
methylthiol in chloroform was kobs ≈ (1.1 ( 0.25) × 10-1 M-1 s-1 at 20
°C, under a single component approximation of a second-order reaction.
In the case of 2, an Arrhenius plot of the rate constants of the reaction
with triphenylmethylthiol provided the activation energy, Ea ) 22.0 (
1.8 kcal/mol, which is in good agreement with the DFT calculated values
[21.4 kcal/mol (BB1K/6-31+G(d,p)); 27.5 kcal/mol (B3LYP/6-31+G-
(d)), in the case of a model thiol, methylthiol] (see Supporting Infomation).
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