Metalloporphyrin catalyzed oxidation of N-hydroxyguanidines: A biomimetic model for the H2O2-dependent activity of nitric oxide synthase

Article abstract of DOI:10.1016/S0960-894X(00)00345-0

A chemical model for the H₂O₂ promoted oxidation by nitric oxide synthase (NOS) has been developed. Biomimetic oxidations were carried out using H₂O₂ and tetrakis(perfluorophenyl)porphyrinato-iron(III) chloride (FeTPPF₂₀) as a catalyst. Similarly to NOS our model system produces N(δ)-cyanoornithine, citrulline and NO from NOHA and did not oxidize arginine itself. Based on these results we propose a peroxide shunt to be involved in the catalytic cycle of NOS. To the best of our knowledge this is the first chemical system that semiquantitatively mimics NOS activity. (C) 2000 Elsevier Science Ltd. All rights reserved.

Full text of DOI:10.1016/S0960-894X(00)00345-0

Bioorganic & Medicinal Chemistry Letters 10 (2000) 1775±1777
Metalloporphyrin Catalyzed Oxidation of N-Hydroxyguanidines:
A Biomimetic Model for the H2O2-Dependent Activity of
Nitric Oxide Synthase
Gyorgy M. Keseruꢀꢀ,a,b,* Gyorgy T. Baloghb and Tamas Karancsic
aChemical and Biotechnological R&D, Gedeon Richter Ltd, PO Box 27, H-1457 Budapest, Hungary
b
Â
Â
Department of Chemical Information Technology, Technical University of Budapest, Szt. Gellert ter 4, H-1111, Budapest, Hungary
cComGenex Inc., Bem rkp. 33-34, H-1024, Budapest, Hungary
Received 30 March 2000; accepted 15 June 2000
AbstractÐA chemical model for the H2O2 promoted oxidation by nitric oxide synthase (NOS) has been developed. Biomimetic
oxidations were carried out using H2O2 and tetrakis(per¯uorophenyl)porphyrinato-iron(III) chloride (FeTPPF20) as a catalyst.
Similarly to NOS our model system produces Nd-cyanoornithine, citrulline and NO from NOHA and did not oxidize arginine itself.
Based on these results we propose a peroxide shunt to be involved in the catalytic cycle of NOS. To the best of our knowledge this is
the ®rst chemical system that semiquantitatively mimics NOS activity. # 2000 Elsevier Science Ltd. All rights reserved.
Nitric oxide synthase (NOS) catalyzes the ®ve-electron
oxidation of arginine to citrulline (1) and nitric oxide (NO)1
via the formation of N-hydroxyarginine (NOHA, 2)
(Scheme 1).2 NO has numerous biological actions from
functioning as a key intercellular signal and defensive
cytotoxin in the nervous, muscular, cardiovascular and
immune system3 to its vasodilatory and antithrombotic
activity.4 Although the mechanism of the oxidation of
arginine to NOHA remains unclear, NOS oxidation of
NOHA is postulated to follow a pathway usual for heme
oxygenases.5 Since simple chemical models can be useful
to investigate mechanistic issues, Fukuto et al.6 reported
the use of organic and inorganic peracids to oxidize N-(N-
hydroxyamidino)piperidine (3)7 as a model for NOHA.
Although this study shed some light on the mechanism of
NOS-catalyzed oxidations, Clague et al. demonstrated8
the inability of NOS to catalyze enzymatic reactions with
peracids. The risk of over-simpli®cation of a biological
system by a chemical model can be signi®cantly reduced
using a biomimetic approach. Our good experience on
metalloporphyrin-catalyzed biomimetic oxidations9,10
prompted us to test this system for NOS-mediated reac-
tions using model compounds and the NOHA itself.
®rst investigated.11 Choosing an iron-porphyrin suitable
for modeling CP450 catalyzed oxidations12 tetrakis (pen-
ta¯uoro-phenyl)phorphirinato iron(III) chloride mediated
oxidation of an aromatic amidoxime (4)13 was carried out
(Scheme 2). Oxidation of 4 by H2O2 gave nicotinamide
(5) as the major product and also the corresponding
nitrile (6) as a minor component (Table 1). Next, we
oxidized N-(N-hydroxyamidino)piperidine (3), the
model compound of Fukuto et al.6 which yields almost
equal amounts of the corresponding amide (7) and 1-
piperidinecarbonitrile (8). Finally, H2O2 promoted oxi-
dation of NOHA (2) gave mainly Nd-cyanoornithine
(9)8 and also citrulline (1). This result is consistent with
the observation of Clague et al.8 who detected 55% of 9
and 45% of 1 in vitro for NOS. Similar to the biological
system8 H2O2 transformed NO to nitrite and nitrate
(NO2 /NO3 ) which was detected by spectrometry in a
stoechiometry of 0.8 relative to 1. Oxidation of arginine
in similar conditions and even in the presence of water-
soluble metalloporphyrins could not be carried out,
which is also in accordance with in vitro data obtained
by H2O2 dependent studies.14 Fair agreement obtained
for the oxidation of natural substrates (i.e., arginine and
NOHA) by the model system and NOS demonstrates
the biomimetic nature of our approach.
Since our biomimetic model was previously tested on
cytochrome P450 (CP450) catalyzed oxidations, a suitable
NO-donor model compound oxidized by this enzyme was
Discussions of the NOS mechanism is often based on its
proposed analogy to that of the P450-catalyzed reac-
tions.3 Although the recent X-ray structure of the oxy-
genase domain of iNOS15 revealed the overall fold of
*Corresponding author. Fax: +36-1-2623008; e-mail: gy.keseru@
richter.hu
0960-894X/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(00)00345-0

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G. M. Keseruꢀꢀ et al. / Bioorg. Med. Chem. Lett. 10 (2000) 1775±1777
A chemical model for the biomimetic oxidation of N-
hydroxyguanidines has been developed. This study
demonstrates the ability of the FeTPPF20/H2O2 system
to mimic the H2O2 dependent action of NOS on N-
hydroxyarginine. Comparing our results to those
obtained in vitro we can propose a P450-type peroxide
shunt in the catalytic cycle of NOS. Evaluation of this
biomimetic approach in chemistry-based pre-screen of
potential NO donor compounds is in progress.
Scheme 1.
Acknowledgements
The authors are grateful to Jianling Wang (Novartis,
US) for his help and also to the OTKA Foundation
(F030040) for ®nancial support. C.M.K. is a Bolyai
Research Fellow of the Hungarian Academy of
Sciences.
References and Notes
1. Marletta, M. A. J. Biol. Chem. 1993, 268, 12231.
2. Stuehr, D. J.; Kwon, N. S.; Nathan, C. F.; Grith, O. W.;
Feldman, P. L.; Wiseman, J. J. Biol. Chem. 1991, 266, 6259.
3. Grith, O. W.; Stuehr, D. J. Annu. Rev. Physiol. 1995, 57, 707.
4. Saavedra, J. M.; Southan, G. J.; Davies, K. M.; Lundell,
A.; Hanson, S. R.; Adrie, D.; Hurford, W. E.; Zapol, W. M.;
Keefer, L. K. J. Med. Chem. 1996, 39, 4361.
5. Korth, H. G.; Sustmann, R.; Thater, C.; Butler, A. R.;
Ingold, K. U. J. Biol. Chem. 1994, 269, 17776.
6. Fukuto, J. M.; Stuehr, D. J.; Feldman, P. L.; Bova, M. P.;
Wong, P. J. Med. Chem. 1993, 36, 2666.
7. Bailey, D. M.; deGarzia, C. G.; Lape, H. E.; Frering, R.;
Fort, D.; Skulan, T. J. Med. Chem. 1973, 16, 151.
8. Clague, M. J.; Wishnok, J. S.; Marletta, M. A. Biochem-
istry 1997, 36, 14465.
Scheme 2.
9. Keseruꢀꢀ, G. M.; Balogh, G. T.; Czudor, I.; Feher, A.; Ber-
tok, B. J. Agric. Food Chem. 1999, 47, 762.
Table 1. Distribution of products obtained by FeTPPF20/H2O2
e€ected biomimetic oxidations of 1, 4 and 7
10. Keseruꢀꢀ, G. M.; Balogh, G. T.; Bokotey, S.; Arvai, G.;
Bertok, B. Tetrahedron 1999, 55, 4457.
N-Hydroxy
Product distribution
Amide (%)
11. General procedure of biomimetic oxidations. N-Hydroxy
compounds (1.1 mmol) and 0.01 molar equiv of tetrakis(per-
¯uorophenyl)porphyrinato-iron(III) chloride (FeTPPF20) were
dissolved in 30 mL of methanol:dichloroethane (1:1) and 0.4
molar equiv of H2O2 was added. The reaction mixture was
stirred for 30 min at room temperature and the solvent was
evaporated. Formation of NO2 and NO3 oxidation products
of NO were monitored spectrophotometrically. Aliquots (0.2
mL) of the reaction mixtures were mixed with 0.2 mL of 1%
sulfanilamide in 4 N HCl and with 0.2 mL of 0.1% N-(1-
naphthyl)ethylenediamine in 0.4 N HCl and their absorption
at 543 nm was measured. All other products were identi®ed by
the analysis of reaction mixtures by HPLC-MS. A HPLC sys-
tem equipped with Symmetry C8 (Waters) 3.9Â150 column,
was used for these analyses using the eluent system of eluent A
(acetonitrile:water:formic acid 5:95:0.1) and eluent B (acetoni-
trile:water:formic acid 95:5:0.1) in a gradient of 100% A to
100% B in 20 min. Oxidation products were photometrically
detected at 254 nm by setting the ¯ow rate to 1 mL/min. Mass
spectrometry studies were performed by a VG QUATTRO
triplequad spectrometer (Micromass, Manchester, UK) with
electrospray ionization. Product ratios were calculated on the
basis of HPLC area% using authentic samples as internal
standards.
Compound
Nitrile
4
3
5 (61)
7 (48)
1 (37)
1 (45)
6 (39)
8 (52)
9 (63)
9 (55)
2
2 (in vitro)
this protein to be di€erent from CP450, Mansuy et al.
demonstrated16,17 that CP450 catalyzes the NO formation
from NOHA and other N-hydroxy compounds. P450
mediated processes involve NADPH to oxidize the sub-
strate. NADPH is necessary to form the high valent iron
oxo heme intermediate responsible for the oxidation of
substrates, but H2O2 can substitute NADPH to form
the iron oxo form directly from the resting state of the
heme (peroxide shunt). Formation of high valent iron
oxo complexes from metalloporphyrins and H2O2 is a
potential synthetic equivalent of this process. Since pro-
duct distributions are in good agreement with in vitro data,
our results suggest the presence of such a peroxide shunt in
the catalytic cycle of NOS as well. Considering that NOS
produces H2O2 when it oxidizes arginine to NOHA18 H2O2
mediated oxidation of NOHA cannot be ruled out as an
alternative to the NADPH assisted process.
12. Dolphin, D.; Taylor, T. G.; Xie, L. Y. Acc. Chem. Res.
1997, 30, 251.
13. Katagi, T.; Kataoka, H.; Takahashi, K.; Fujioka, T.;

G. M. Keseruꢀꢀ et al. / Bioorg. Med. Chem. Lett. 10 (2000) 1775±1777
1777
Kunitomo, M.; Yamaguchi, Y.; Fujiwara, M.; Inoi, T. Chem.
Pharm. Bull. 1992, 40, 2419.
14. Pufahl, R. A.; Wishnok, J. S.; Marletta, M. A. Biochem-
istry 1995, 34, 1930.
15. Crane, B. R.; Arvai, A. S.; Ghosh, D. K.; Wu, C.; Getzo€,
E. D.; Stuehr, D. J.; Tainer, J. A. Science 1998, 279, 2121.
16. Andronik-Lion, V.; Boucher, J. L.; Delaforge, M.; Henry,
Y.; Mansuy, D. Biochem. Biophys. Res. Commun. 1992, 185, 452.
17. Renaud, J. P.; Boucher, J. L.; Vadon, S.; Delaforge, M.;
Mansuy, D. Biochem. Biophys. Res. Commun. 1993, 192, 53.
18. Pfeifer, S.; Mayer, B.; Hemmens, B. Angew. Chem. Int.
Engl. Ed. 1999, 38, 1714.