C O M M U N I C A T I O N S
to account for some S-nitrosothiol chemistry.9 Because of its
instability, 1 would decompose to form the sulfiliminosulfonium
ion 2. This mechanism is supported by the absence of a mass shift
when HcyN O was used instead of HcyN O (Table 1). Although
not isolated from the reaction of thiols with S-nitrosothiols,9
sulfiliminosulfonium ions have been characterized upon the reaction
of the isoelectronic N-chlorosulfimides with sulfides.10 The O atom
S-nitrosothiol inside a protein cavity may lead to reactions other
than transnitrosation.17 Covalent inhibitors for both currently known
mammalian DDAH isoenzymes and their homologue arginine
deiminase will be widely appreciated in both clinics and research.
Thus, HcyNO and the mechanism proposed herein pave the way
for the rational design of such inhibitors.
18
16
3,11
Acknowledgment. We thank Dr. Sergiy Chesnov and Dr. Peter
Hunziker for recording the mass spectra. This work was supported
by the “Forschungskommission und Nachwuchsf o¨ rderungskom-
mission der Universit a¨ t Z u¨ rich” (to M.K.), the Swiss National
Science Foundation Grant 3100A0-100246/1 (to M.V.), and the
2
inserted from H O may be best explained by the attack of a water
on the formally positively charged S atom of 2. In the enzyme
pocket, activated water molecules are present.11 Thus, the reaction
likely proceeds through the intermediate 3 to yield the N-thio-
sulfoximide 4.12 Similarly, the reaction of 1-thioglycerol with
nitrosobenzene in the presence of H O resulted in the formation of
2
an N-phenylsulfoximide.13
“
Jubil a¨ umsspende der Universit a¨ t Z u¨ rich” (to M.V.).
Supporting Information Available: Detailed experimental proto-
At this point, the question was addressed which of the two S
atoms of 2 would react with H O. Because of the electron-
withdrawing effect of the formal π-bond in eq 1
cols and additional figures. This material is available free of charge
via the Internet at http://pubs.acs.org.
2
References
+
+
DDAH1-S-NdS -Hcy T DDAH1-S dN-S-Hcy (1)
(1) (a) Stamler, J. S.; Lamas, S.; Fang, F. C. Cell 2001, 106, 675-683. (b)
Hogg, N. Annu. ReV. Pharmacol. Toxicol. 2002, 42, 585-600.
both S atoms can, in principle, be a target for nucleophilic attack.14
It should be noted that N-thiosulfoximides are stable in water-
containing solvents only at low pH.15 This property is in agreement
(2) (a) Petros, A.; Leone, A.; Moncada, S.; Bennett, D.; Vallance, P.
CardioVasc. Res. 1994, 28, 34-39. (b) Ashina, M.; Lassen, L. H.;
Bendtsen, L.; Jensen, R.; Olesen, J. Lancet 1999, 353, 287-289. (c).
Hobbs, A. J.; Higgs, A.; Moncada, S. Annu. ReV. Pharmacol. Toxicol.
1999, 39, 191-220.
with the observed pH stability of 4 in MS analysis (Figure 2). The
(3) (a) Vallance, P.; Leiper, J. Nat. ReV. Drug DiscoVery 2002, 1, 939-950.
b) Vallance, P. Fundam. Clin. Pharmacol. 2003, 17, 1-10. (c) Lu, X.;
Galkin, A.; Herzberg, O.; Dunaway-Mariano, D. J. Am. Chem. Soc. 2004,
(
1
26, 5374-5375. (d) Magalh a˜ es, B. S.; Harris, R.; Plevin, M. J.; Driscoll,
P. C. J. Biomol. Nucl. Magn. Reson. 2004, 29, 463-464. (e) Plevin, M.
J.; Magalh a˜ es, B. S.; Harris, R.; Sankar, A.; Perkins, S. J.; Driscoll, P. C.
J. Mol. Biol. 2004, 341, 171-184.
(
(
(
4) (a) MacAllister, R. J.; Parry, H.; Kimoto, M.; Ogawa, T.; Russel, R. J.;
Hodson, H.; Whitley, G. St. J.; Vallance, P. Br. J. Pharmacol. 1996, 119,
1
533-1540. (b) Ueda, S.; Kato, S.; Matsuoka, H.; Kimoto, M.; Okuda,
S.; Morimatsu, M.; Imaizumi, T. Circ. Res. 2003, 92, 226-233.
5) (a) Leiper, J.; Murray-Rust, J.; McDonald, N.; Vallance, P. Proc. Natl.
Acad. Sci. U.S.A. 2002, 99, 13527-13532. (b) Knipp, M.; Braun, O.;
Gehrig, P. M.; Sack, R.; Va sˇ a´ k, M. J. Biol. Chem. 2003, 278, 3410-
3416.
6) To avoid the formation of peroxynitrite all experiments were performed
under strictly anaerobic conditions. The interference of redox transition
metals was excluded by rendering all solutions metal-free through
treatment with Chelex 100 (BioRad) and the addition of 5 mM EDTA.
7) The product was found stable for at least 5 days at 4 °C. Interestingly,
S-nitroso-L-cysteine reacted differently. This and the biological implica-
tions of our findings will be published elsewhere.
(
(
(
8) Murray-Rust, J.; Leiper, J.; McAllister, M.; Phelan, J.; Tilley, S.; Santa
Maria, J.; Vallance, P.; McDonalds, N. Nat. Struct. Biol. 2001, 8, 679-
683.
9) (a) Singh, S. P.; Wishnok, J. S.; Keshive, M.; Deen, W. M.; Tannenbaum,
S. R. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 14428-14433. (b) Wong,
P. S.-Y.; Hyun, J.; Fukuto, J. M.; Shirota, F. N.; DeMaster, E. G.;
Shoeman, D. W.; Nagasawa, H. T. Biochemistry 1998, 37, 5362-5371.
(
c) Munro, A. P.; Williams, D. L. H. J. Chem. Soc., Perkin Trans. 2 2000,
1
794-1797. (d) Wang, K.; Wen, Z.; Zhang, W.; Xian, M.; Cheng, J.-P.;
Wang, G. W. Bioorg. Med. Chem. Lett. 2001, 11, 433-436.
(10) Furukawa, N.; Yoshimura, T.; Oae, S. Tetrahedron Lett. 1973, 23, 2113-
116.
Figure 2. Deconvoluted ESI Q-TOF mass spectra of DDAH-1 incubated
with 500 µM HcyNO in 20 mM NH4OAc/NH3 (pH 7.4) for 30 min at 37
C. Samples were injected in (A) 0.1% HCOOH, 50% CH3CN (pH 2.9),
B) 0.1% HOAc, 50% CH3OH (pH 3.5), and (C) 20 mM NH4OAc/NH3,
0% CH3CN (pH 9.0).
2
°
(
5
(
11) Galkin, A.; Kulakova, L.; Sarikaya, E.; Lim, K.; Howard, A.; Herzberg,
O. J. Biol. Chem. 2004, 279, 14001-14008.
(
12) The proposed mechanism is further supported by the determination of
the reaction stoichiometry of 1:1 between DDAH-1 and HcyNO (for details
see Supporting Information).
fact that the loss of modification completely recovered the native
mass of DDAH-1 indicates that the nucleophilic attack preferentially
takes place at Hcy:S according to Scheme 1. A preference of the
equilibrium of eq 1 to one site was also observed in the case of
asymmetric sulfiliminosulfonium salts.16 However, in the case of
DDAH-1 the strong selectivity for the formation of 4 likely
originates from the active-site structure.
To conclude, our results indicate that HcyNO forms the covalent
dead-end complex 4 with DDAH-1. The formation and stabilization
of 4 is accomplished by the protein structure. The results support
the recent proposal that the reaction between a thiol and a
(
13) Klehr, H.; Eyer, P.; Sch a¨ fer, W. Biol. Chem. Hoppe-Seyler 1985, 336,
755-760.
(14) Furukawa, N.; Akutagawa, K.; Oae, S. Phosphorous Sulfur 1984, 20,
1-14.
(
15) (a) Oae, S.; Iida, K.; Takata, T. Phosphorous Sulfur 1981, 12, 103-113.
(b) Oae, S.; Akutagawa, K.; Furukawa, N. Phosphorous Sulfur 1984, 19,
2
23-234. (c) Yoshimura, T.; Furukawa, N.; Akasaka, T.; Oae, S.
Tetrahedron 1977, 33, 1061-1067.
(16) Nishikawa, Y.; Matsuura, Y.; Kakudo, M.; Akasaka, T.; Fukurawa, N.;
Oae, S. Chem. Lett. 1978, 447-450.
(
17) Houk, K. N.; Hietbrink, B. N.; Bartberger, M. D.; McCarren, P. R.; Choi,
B. Y.; Voyksner, R. D.; Stamler, J. S.; Toone, E. J. J. Am. Chem. Soc.
2003, 125, 6972-6976.
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J. AM. CHEM. SOC.
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