Synthesis of a naphthalene endoperoxide as a source of 18O-labeled singlet oxygen for mechanistic studies [4]

Full text of DOI:10.1021/ja0016452

10212
J. Am. Chem. Soc. 2000, 122, 10212-10213
Scheme 1. Release of 18[1O2] and the Parent Naphthalene
Derivative (DHPN) from the Thermodissociation of the
Hydrophilic Naphthalene-Labeled Endoperoxide (DHPN18O2)
Synthesis of a Naphthalene Endoperoxide as a
Source of 18O-labeled Singlet Oxygen for Mechanistic
Studies
Glaucia R. Martinez, Jean-Luc Ravanat,‡
Marisa H. G. Medeiros, Jean Cadet,‡ and Paolo Di Mascio*
Departamento de Bioqu´ımica, Instituto de Qu´ımica,
UniVersidade de Sa˜o Paulo, CP 26077, CEP 05513-970,
Sa˜o Paulo, SP, Brazil
ReceiVed May 13, 2000
ReVised Manuscript ReceiVed July 28, 2000
Singlet oxygen (1O2) exhibits a substantial reactivity toward
electron-rich organic molecules, leading to the formation of allylic
hydroperoxides, dioxetanes, or endoperoxides.1 Singlet oxygen
has been shown to be generated in biological systems.2 As possible
biological sources of 1O2, we may mention enzymatic processes
catalyzed by peroxidases or oxygenases,3 reactions of hydrogen
peroxide with hypochlorite4 or peroxynitrite,5 thermodecompo-
sition of dioxetanes,6 and photosensitization reactions.7
using appropriate methods. In this respect, HPLC coupled to mass
spectrometry is particularly relevant.
We report, in the present work, the chemical synthesis of the
[18O]-labeled endoperoxide of N,N′-di(2,3-dihydroxypropyl)-1,4-
naphthalenedipropanamide (DHPN18O2). In addition, the ability
for DHPN18O2 to release labeled singlet oxygen was checked
using the water-soluble disodium salt of anthracene-9,10-diyldi-
1
The reactions of 1O2 with unsaturated fatty acids, proteins, and
DNA have been extensively studied since this activated oxygen
species can induce various types of cell damage that are related
to aging, cancer, and other cytotoxic effects.8
ethyl disulfate (EAS) as a chemical trap of O2. The analysis of
the products of the reaction was achieved by mass spectrometry
measurement after HPLC purification.
The hydrophilic naphthalene carrier N,N′-di(2,3-dihydroxypro-
pyl)-1,4-naphthalenedipropanamide (DHPN) was prepared as the
following. In the first step, a double bromination of 1,4-
dimethylnaphthalene, in the presence of light, yields 1,4-dibro-
momethylnaphthalene, which was purified by recrystallization in
chloroform. Thereafter, 1,4-naphthalenedipropanoic acid (NDPA)
was synthesized by malonic synthesis, hydrolysis, and decar-
boxylation.11 NDPA was subsequently used for the synthesis of
diethyl-1,4-naphthalenedipropanoate (DENDP) by acid-catalyzed
esterification.12 This was achieved by refluxing during 2 h 21 g
(77 mmol) of NDPA and 1 mL of H2SO4 (95%) in ethanol. A
Dean-Stark trap was settled in the presence of toluene, and the
reflux was left for 4 h. The organic phase was washed with 5%
aqueous NaHCO3, dried, and evaporated to yield DENDP (22 g,
87%) as an oil. Finally, the amidation13,14 of the diester DENDP
with 3-amino-1,2-propanediol was made by stirring, under reflux,
a solution of DENDP (5.1 g, 15 mmol) and 3-amino-1,2-
propanediol (9 g, 99 mmol) in 80 mL of MeOH for 24 h. After
evaporation of the solvent, the residue was triturated with 100
mL of acetone. The colorless precipitate was filtered by suction,
rinsed with acetone, and recrystallized in MeOH, yielding 50%
DHPN. The final product, the nonionic carrier DHPN was
1
Aqueous sources of O2 are required in order to study the
1
reactivity of O2 toward biomolecules. In this respect, type II
photosensitization reactions have been widely used but this
approach may suffer for the presence of the type I competitive
reaction.7 Alternatively, several chemical compounds that are able
1
to convert, in the dark, an oxygen precursor into O2 almost
quantitatively1 were designed. However, conditions required by
biological media (i.e., an aqueous environment, a neutral pH, a
moderate temperature) are not compatible in most cases with the
application of the latter sources of 1O2. To overcome these
difficulties, alternative methods to generate pure 1O2 under mild
conditions that involve the thermolysis of hydrophilic naphthalene
endoperoxides have been employed.9,10 These compounds are
chemically inert and, upon heating, regenerate molecular oxygen,
partly in the singlet state, and the parent hydrocarbon.
The development of a water-soluble naphthalene endoperoxide
acting as a chemical source (Scheme 1) of [18O] isotopically
labeled singlet oxygen (18[1O2]) is of particular interest in order
1
to assess the reactivity of O2 toward chemical, biochemical, or
biological targets. Oxidation products thus formed will be labeled
with, at least, one oxygen atom. Therefore, the oxidation products
which contain the labeled oxygen can be detected and quantified
1
characterized by mass spectroscopy and H NMR analysis.
DHPN18O2 was prepared by photosensitization in the presence
of [18O]-labeled molecular oxygen (99%). Typically, in a 50-mL
flask, 210 mg of DHPN was suspended in 2.5 mL of deuterated
water that contained 0.5 mg of methylene blue. The solution,
maintained in the dark, was purged several times with argon and
gently heated to dissolve DHPN. Thereafter, the solution main-
tained under continuous stirring in an atmosphere of 18O2 (2 bar)
was cooled at 4 °C and irradiated during 6 h with a 500 W
tungsten lamp placed at a distance of 30 cm. Then, Chelex 100
cation-exchange resin was added to the blue mixture, which was
then stirred for 20 min at 4 °C until complete fixation of the
sensitizer onto the insoluble anionic polymer. Using a 5-mL
* Corresponding author. E-mail: pdmascio@iq.usp.br.
‡ Laboratoire “Le´sions des Acides Nucle´iques” Service de Chimie Inor-
ganique et Biologique, De´partement de Recherche Fondamentale sur la Matie`re
Condense´e, CEA Grenoble, 17 Avenue des Martyrs, F-38054 Grenoble Cedex
9, France.
(1) Frimer, A. A. Singlet O2; CRC Press: Boca Raton, FL, 1985; Vol. I,
II.
(2) Steinbeck, M. J.; Khan, A. U.; Karnovsky, M. J. J. Biol. Chem. 1993,
268, 15649.
(3) Cilento, G. In Chemical and biological generation of excited states;
Adam, W., Cilento, G., Eds.; Academic Press: New York, 1982.
(4) Held, A. M.; Halko, D. J.; Hurst, J. K. J. Am. Chem. Soc. 1978, 100,
5732.
(5) Di Mascio, P.; Bechara, E. J. H.; Medeiros, M. H. G.; Briviba, K.;
Sies H. FEBS Lett. 1994, 355, 287.
(6) Briviba, K.; Saha-Mo¨ller, C. R.; Adam, W.; Sies, H. Biochem. Mol.
Biol. Int. 1996, 38, 647.
(7) Foote, C. S. Photochem. Photobiol. 1991, 54, 659.
(8) Halliwell, B. E.; Gutteridge J. M. C. Free Radicals in Biology and
Medicine, 3rd ed.; Oxford University Press: New York, 1999.
(9) Aubry, J. M. In Membrane Lipid Oxidation; Vigo-Pelfrey, C., Ed.; CRC
Press: Boca Raton, FL, 1991; Vol. 2, p 65.
(10) Pierlot, C.; Hajjam, S.; Barthe´le´my, C.; Aubry, J. M. J. Photochem.
Photobiol., B 1996, 36, 31.
(11) Di Mascio, P.; Sies, H. J. Am. Chem. Soc. 1989, 111, 2909.
(12) Vogel, A. I. In Vogel’s Textbook of Pratical Organic Chemistry;
revised by Furniss, B. S.; Hannaford, A. J.; Smith, P. W. G.; Tatchell, A. R.;
John Wiley & Sons: New York, 1989; p 695.
(13) Dewilde, A.; Pellieux, C.; Pierlot, C.; Wattre´, P.; Aubry, J. M. Biol.
Chem. 1998, 379, 1377.
(14) Pierlot, C.; Aubry, J. M.; Briviba, K.; Sies, H.; Di Mascio, P. Methods
Enzymol. 2000, 319, 3.
10.1021/ja0016452 CCC: $19.00 © 2000 American Chemical Society
Published on Web 09/29/2000

Communications to the Editor
J. Am. Chem. Soc., Vol. 122, No. 41, 2000 10213
Figure 1. Mass spectrometry analysis of the DHPNO2 and DHPN18O2.
Figure 2. Mass spectrometry analysis of the EAS, EASO2, and EAS18O2.
Scheme 2. Chemical Trapping of 18[1O2] by the
Water-Soluble Anthracene-9,10-diyldiethyl Disulfate (EAS)
Yielding the Labeled Endoperoxide EAS18O2
mass spectrometry (Figure 2). The mass spectrum of EAS,
recorded in the negative mode, exhibits a major ion at m/z )
212. This corresponds to the doubly charged molecule [M -
2H]2- which has a molecular weight of 426. As expected, the
corresponding endoperoxide EASO2 (MW ) 458), produced by
methylene blue photosensitization, exhibits a [M - 2H]2- ion at
m/z ) 228. The EAS18O2 endoperoxide formed in the presence
of DHPN18O2 shows an intense [M - 2H]2- ion at m/z ) 230,
corresponding to a molecular weight of 462. This is strongly
indicative of the incorporation of two labeled oxygen atoms into
the molecule. The ion corresponding to the unlabeled endoper-
oxide at m/z ) 228 was also detected with a relative abundance
of about 15% compared with that of the labeled molecule. This
reaction demonstrated that DHPN18O2 is able to produce [18O]-
labeled singlet oxygen with an isotopic enrichment of 85%.
The endoperoxide DHPN18O2 is able to produce 18[1O2] as a
clean source and in a high yield (60%).13 In addition, the
naphthalene derivative is soluble in aqueous media and has been
shown to penetrate into cells.15 Therefore, DHPN18O2, in associa-
tion with a mass spectrometric detection method, will allow study
syringe, the solution was passed through a polymeric membrane
(porosity, 0.45 µm), and the filtrate was stored at -80 °C until
use. The residual blue resin was washed with 1 mL of deuterated
water to remove the remaining endoperoxide product. The
concentration (130 mM) and the yield of formation (close to 95%)
of the endoperoxide DHPN18O2 was determined using UV
spectroscopy.14
1
of the mechanistic features of the reaction of O2 with cellular
targets. All these characteristics contribute to make this new model
an excellent tool for mechanistic studies of the reaction of singlet
oxygen in biological media.
In addition, the isotopic purity of DHPN18O2 was determined
by electrospray mass spectrometry (Figure 1). The mass spectrum
of DHPNO2 exhibits, in the positive mode, a pseudomolecular
ion at m/z ) 451 corresponding to the expected molecular formula
C22H30N2O8. The main intense ion is observed for the sodium
adduct [M + Na]+ at m/z ) 473. The mass spectrum of
DHPN18O2 exhibits an intense ion at m/z ) 477 corresponding
to the sodium adduct [(M + 4) + Na]+ of the labeled endoper-
oxide. The sodium adduct of unlabeled DHPNO2 is also detectable
(m/z ) 473), indicating that the isotopic purity of DHPN18O2 is
close to 85% (Figure 1).
Acknowledgment. Supported by the “Fundac¸a˜o de Amparo a`
Pesquisa do Estado de Sa˜o Paulo”, FAPESP (Brazil), the “Conselho
Nacional para o Desenvolvimento Cient´ıfico e Tecnolo´gico”, CNPq
(Brazil), and the “Programa de Apoio aos Nu´cleos de Exceleˆncia”,
PRONEX/FINEP (Brazil), “Universidade de Sa˜o Paulo”/“Comite´ Franc¸ais
d'Evaluation de la Coope´ration Universitaire avec le Bre´sil” (USP-
COFECUB). We gratefully thank Colette Lebrun and Calogero Tornabene
for their contribution to mass spectrometry analyses. G.R.M. is a recipient
of FAPESP fellowship.
Supporting Information Available: 1H-RMN data of DHPN, HPLC
purification of EASO2,and the synthetic scheme for DHPN18O2 (PDF)
are presented. This material is available free of charge via the Internet at
http://pubs.acs.org. See any current masthead page for ordering informa-
tion and Web access instructions.
The generation of 18[1O2] by thermal decomposition of
DHPN18O2 was monitored using the water-soluble EAS, which
can react with 1O2, yielding the anthracene-9,10-diyldiethyl
disulfate endoperoxide (EASO2) as the specific oxidation product
(Scheme 2). For this purpose, EAS was incubated at 37 °C in the
presence of DHPN18O2. The resulting endoperoxide was purified
by reversed-phase HPLC and analyzed by electrospray ionization
JA0016452
(15) Klotz, L. O.; Pellieux, C.; Briviba, K.; Pierlot, C.; Aubry, J. M.; Sies,
H. Eur. J. Biochem. 1999, 260, 917.