4594 J. Am. Chem. Soc., Vol. 122, No. 19, 2000
Chatgilialoglu et al.
are limited to polyunsaturated fatty acid (PUFA) peroxida-
tion,11,12 and to a recently reported homolytical cleavage of
lysophospholipids.13
be either incorporated into the bilayer, or dissolved in the
aqueous phase. Amphiphilic HOCH2CH2SH was used without
any concern about the partition of thiol between hydrophobic
and hydrophilic regions.25 However, two other biologically
related thiols of different lipophilicity such as glutathione (GSH)
and cysteine (CySH) were also used.25
The lipophilic initiators azobis(isobutyronitrile) (AIBN) and
azobis(dimethylvaleronitrile) (AMVN) and the hydrophilic
azobis(2-amidinopropane) hydrochloride (AAPH) were used at
71, 54, and 37 °C, respectively, taking into account their half-
life times (eq 1).
From a chemical perspective, the cis to trans conversion of
double bonds is a thermodynamic favored process,14 which can
also occur by the reversible addition of a free radical. During
our study on the isomerization of double bonds caused by radical
species,15 we found that thiyl radicals are among the most
efficient isomerizing agents.16 Since then, we have been attracted
by the idea of a similar reaction taking place in the case of
naturally occurring unsaturated phospholipids containing cis-
residues. This event should change the double bond geometry,
thus leading to the thermodynamically more stable trans-isomer.
We report herein that thiyl radicals are indeed able to induce
the isomerization of cis-monounsaturated phospholipids (Scheme
1).20,21 This process was observed using lipid solutions and
liposome vesicles under anoxic or aerobic conditions. We
showed that phospholipids containing trans-fatty acid residues
are produced in high yield.
Radiolysis of neutral water leads to the species eaq-, HO•,
and H• as shown in eq 2, where the values in parentheses
represent the yields expressed in terms of G-values (µmol J-1).26
Results and Discussion
Thiyl radicals were generated by irradiating N2O-saturated
solutions containing i-PrOH at natural pH. The presence of N2O
efficiently transforms eaq- into the HO• radical (eq 3, k3 ) 9.1
× 109 M-1 s-1). Hydrogen abstraction from i-PrOH by HO•
Generation of Radicals. Selectively generated thiyl radicals
were produced by the reaction of an alkyl radical with the
corresponding thiol. Alkyl radicals were generated by either
thermal decomposition of azo derivatives or γ-irradiations. The
choice of thiol was based on the experimental conditions. In a
homogeneous system where t-BuOH was used as a solvent,
benzenethiol (PhSH) and â-mercaptoethanol (HOCH2CH2SH)
were chosen. In a heterogeneous system (vesicles), thiols can
and H• produces (CH3)2 COH (eqs 4 and 5, k4 ) 1.9 × 109
•
M-1 s-1, k5 ) 7.4 × 107 M-1 s-1). The (CH3)2 COH in turn
•
reacts with the thiol to give the corresponding thiyl radical (eq
6, k6 = 2 × 108 M-1 s-1).26,27
(11) For some representative reviews, see: Porter, N. A. Acc. Chem.
Res. 1986, 19, 262-268. Niki, E. In Organic Peroxides; Ando, W., Ed.;
Wiley: New York; 1992; pp 764-787. Barclay, L. C. R. Can. J. Chem.
1993, 33, 1-16.
(12) For the autoxidation of methyl oleate, see: Porter, N. A.; Mills, K.
A.; Carter, R. L. J. Am. Chem. Soc. 1994, 116, 6690-6696.
(13) Muller, S. N.; Batra, R.; Senn, M.; Giese, B.; Kisel M.; Shadyro,
O. J. Am. Chem. Soc. 1997, 119, 2795-2803.
(14) For example, see: Sonnet, P. E. Tetrahedron 1980, 36 557-604.
Saltiel, J.; Sears, D. F., Jr.; Ko, D.-H.; Park, K.-M. In CRC Handbook of
Organic Photochemistry and Photobiology; Horspool, W. H., Song, P. S.,
Eds.; CRC Press: Boca Raton, 1995; Chapter 1.
(15) Chatgilialoglu, C.; Ballestri, M.; Ferreri, C.; Vecchi, D. J. Org.
Chem. 1995, 60, 3826-3831.
(16) The ability of thiyl radicals to isomerize double bonds was first
reported 40 years ago17 and has since attracted considerable attention
particularly in organic synthesis.18 On the other hand, thiols and the
corresponding thiyl radicals are of considerable importance in biological
processes, where they can act as repairing and as damaging agents,
respectively.19
(17) (a) Pallen, R. H.; Sivertz, C. Can. J. Chem. 1957, 35, 723-733.
(b) Walling, C.; Helmreich, W. J. Am. Chem. Soc. 1959, 81, 1144-1148.
(c) For a review of early work, see: Kice, J. L. In Free Radicals; Kochi,
J. K., Ed.; Wiley: New York, 1973; Vol. 2, pp 711-740.
(18) For recent reviews, see: (a) Chatgilialoglu, C.; Guerra, M. In
Supplement S: The Chemistry of Sulfur-containing Functional Groups;
Patai, S., Rappoport, Z., Eds.; Wiley: London, 1993; Chapter 8, pp 363-
394. (b) Chatgilialoglu, C.; Bertrand, M. P.; Ferreri, C. In S-Centered
Radicals; Alfassi, Z. B., Ed.; Wiley: Chichester, 1999; pp 312-354.
(19) Sulfur-centered ReactiVe Intermediates in Chemistry and Biology;
Chatgilialoglu, C., Asmus, K.-D., Eds.; Plenum Press: New York, 1990.
(20) For a preliminary communication, see: Ferreri, C.; Costantino, C.;
Landi, L.; Mulazzani, Q. G.; Chatgilialoglu, C. Chem. Commun. 1999, 407-
408. In this communication, Fig 2b refers to MLVs with 75 mM of
HOCH2CH2SH and not to LUVETs with 7 mM of HOCH2CH2SH, as
euroneously reported.
Radiolysis of t-BuOH leads to the species esol-, and
•CH2C(CH3)2OH as shown in eq 7. In N2O-saturated solutions
-
esol is transformed into the HO• radical (eq 8). Hydrogen
abstraction from t-BuOH by HO• produces •CH2C(CH3)2OH (eq
9, k9 ) 6.0 × 108 M-1 s-1) and therefore, we can consider the
G[•CH2C(CH3)2OH] to be ca. 0.65 µmol J-1 28
. It is known that
•
in water the CH2C(CH3)2OH abstracts hydrogen from the
HOCH2CH2SH with a rate constant of k10 ) 8.2 × 107 M-1
s-1 to give the corresponding thiyl radical (eq 10).26,27 The rates
of thiol trapping of alkyl radicals are solvent dependent.30 The
(22) Scho¨neich, C.; Bonifacic, M.; Dillinger, U.; Asmus, K.-D. In Sulfur-
Centered ReactiVe Intermediates in Chemistry and Biology; Chatgilialoglu,
C., Asmus, K.-D., Eds.; Plenum Press: New York, 1990; pp 367-376.
(23) Schwinn, J.; Sprinz, H.; Dro¨âler, K.; Leistner, S.; Brede, O. Int. J.
Radiat. Biol. 1998, 74, 359-365.
(24) Jiang, H.; Kruger, N.; Lahiri, D. R.; Wang, D.; Vate`le, J.-M.; Balazy,
M. J. Biol. Chem. 1999, 274, 16235-16241.
(25) Newton, G. L.; Aguilera, J. A.; Kim, T.; Ward, J. F,; Fahey, R. C.
Radiat. Res. 1996, 146, 206-215 and references therein.
(26) Buxton, G. V.; Greenstock, C. L.; Helman, W. P.; Ross, A. B. J.
Phys. Chem. Ref. Data 1988, 17, 513 and references therein.
(27) Ross, A. B., Mallard, W. G.; Helman, W. P.; Buxton, G. V.; Huie,
R. E.; Neta, P. NDRL-NIST Solution Kinetic DatabasesVer. 3; Notre Dame
Radiation Laboratory: Notre Dame, IN; NIST Standard Reference Data:
Gaithersburg, MD, 1998.
(21) It has been reported that oleic acid shows no measurable reaction
with alkyl thiyl radicals by pulse radiolysis techniques, and consequently,
an addition of RS• to the double bonds seems to be of minor if any
importance.22 Recently cis-trans isomerization of polyunsaturated fatty acid
residues in phospholipids by free radicals has also been reported. Schwinn
et al. proposed that cis-trans isomerization in linoleate moieties is the fate
of the pentadienyl radical which reacts with thiols in different conforma-
tions.23 On the other hand, Balazy and co-workers proposed a reversible
(28) On the basis of the total G ) 0.61 µmol J-1 in water (eq 2) and
G(•CH2OH)
)
0.67 µmol J-1 in methanol,29 the assumption of
G[•CH2C(CH3)2OH] ) 0.65 µmol J-1 seems reasonable.
(29) Spinks, J. W. T.; Woods, R. J. An Introduction to Radiation
Chemistry, 3rd ed.; Wiley: New York, 1990; p 421.
(30) Tronche, C.; Martinez, F. N.; Horner, J. H.; Newcomb, M.; Senn,
M.; Giese, B. Tetrahedron Lett. 1996, 33, 5845-5848.
•
addition of NO2 radicals to arachidonic moieties.24