A method for thermal generation of aryloxyl radicals at ambient temperatures: application to low-density lipoprotein (LDL) oxidation.

Full text of DOI:10.1002/1521-3773(20020301)41:5<804::AID-ANIE804>3.0.CO;2-6

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incubation, the fraction with tR ˆ 10.0 min yielded, after lyophilization,
17a,b ¥ HCOOH (unlabeled, 36.5 mg, 0.089 mmol, 4.5%), LC ESI-MS
(gradient:% MeOH (t [min]) 5(0)-95(30 35)-5(40 45), cone voltage
40 V). 17a: tR ˆ 8.3 min; m/z (%): 401 (3) [MK] , 385 (4) [MNa] , 363
A Method for Thermal Generation of Aryloxyl
Radicals at Ambient Temperatures:
Application to Low-Density Lipoprotein
(LDL) Oxidation**
‡
‡
‡
‡
(100) [MH] , 219 (8), 187(17); 17b: tR ˆ 8.5 min; m/z (%): 401 (4) [MK] ,
‡
‡
385 (6) [MNa] , 363 (100) [MH] , 219 (12), 187(14); accurate mass (mean
of 11 measurements Æ standard deviation): 17a,b: calculated for
Thomas Paul* and Keith U. Ingold*
‡
C18H27N4O4: 363.2032, found: m/z 363.2034 Æ 0.0007[ MH] . Preparation
(scaled down to one seventh) was repeated with [1-13C]d-glucose, and the
products were isolated analogously. For the d-arabinose incubation,
fractions with tR ˆ 10.5 and 11.3 min yielded, after lyophilization, 11 ¥
HCOOH (12.3 mg, 0.033 mmol, 1.6%) and 12 ¥ HCOOH (4.8 mg,
0.013 mmol, 0.6%), respectively, LC ESI-MS (for gradient and cone
Antioxidant phenols (ArOH) react with peroxyl radicals
.
(ROO ) and form relatively unreactive aryloxyl radicals
.
(ArO ) [Eq. (1)] which, in homogeneous solutions, then trap
a second peroxyl [Eq. (2)].[1, 2] a-Tocopherol (TocH, vit-
‡
voltage, see above). 11: tR ˆ 7.5 min; m/z (%): 371 (1) [MK] , 355 (2)
amin E) is the most active lipid-soluble antioxidant in
‡
‡
[MNa] , 333 (45) [MH] , 175 (100), 159 (30); 12: tR ˆ 8.3 min; m/z (%): 371
‡
‡
‡
(1) [MK] , 355 (1) [MNa] , 333 (100) [MH] , 315 (5), 189 (5), 187(4);
accurate mass (mean of 10 measurements Æ standard deviation): 11:,
calculated for C17H25N4O3: 333.1927, found: m/z 333.1931 Æ 0.0007
.
.
ArOH ‡ ROO À! ArO ‡ ROOH
(1)
(2)
.
.
‡
ArO ‡ ROO À! nonradical products
[MH] ; 12: calculated for C17H25N4O3: 333.1927, found: m/z 333.1935 Æ
‡
0.0010 [MH] .
mammals[2] but, surprisingly, it acts as a prooxidant in human
low-density lipoproteins (LDL).[3] Oxidatively modified LDL
may initiate atherosclerosis.[4] Various agents (e.g., enzymes,
transition metals) have been suggested to be responsible for
this modification of LDL in vivo.[5] The free radical initiated
oxidation of LDL in which TocH transfers radical character
from water-soluble peroxyls into the LDL has been studied
Received: September 5, 2001 [Z17857]
[1] F. Ledl, E. Schleicher, Angew. Chem. 1990, 102, 597 626; Angew.
Chem. Int. Ed. Engl. 1990, 29, 565 594.
[2] K. M. Biemel, O. Reihl, J. Conrad, M. O. Lederer, J. Biol. Chem. 2001,
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[3] a) D. B. Shin, F. Hayase, H. Kato, Agric. Biol. Chem. 1988, 52, 1451
1458; b) M. A. Glomb, C. Pfahler, Carbohydr. Res. 2000, 329, 515
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Egan, P. Ulrich, Nature 1996, 382, 275 278; d) R. H. Nagaraj, I. N.
Shipanova, F. M. Faust, J. Biol. Chem. 1996, 271, 19338 19345;
e) M. O. Lederer, R. G. Klaiber, Bioorg. Med. Chem. 1999, 7, 2499
2507; f) H. Odani, T. Shinzato, J. Usami, Y. Matsumoto, E. Brink-
mann-Frye, J. W. Baynes, K. Maeda, FEBS Lett. 1998, 427, 381 385.
[4] V. M. Monnier, R. R. Kohn, A. Cerami, Proc. Natl. Acad. Sci. USA
1984, 81, 583 587.
[5] a) A. M. Schmidt, O. Hori, J. Brett, S. D. Yan, J. L. Wautier, D. Stern,
Arterioscler. Thromb. 1994, 14, 1521 1528; b) T. Kislinger, C. F. Fu, B.
Huber, W. Qu, A. Taguchi, S. D. Yan, M. Hofmann, S. F. Yan, M.
Pischetsrieder, D. Stern, A. M. Schmidt, J. Biol. Chem. 1999, 274,
31740 31749; c) A. M. Schmidt, S. D. Yan, S. F. Yan, D. M. Stern,
Biochim. Biophys. Acta 2000, 1498, 99 111.
.
extensively. The resulting tocopheroxyl radical (Toc ) then
carries a lipid peroxidation chain within the LDL in a process
christened tocopherol-mediated peroxidation (TMP).[6] The
aryloxyl radical, tyrosyl, which is formed (in 25% yield) by
reaction of myeloperoxidase with tyrosine during the immune
response, can also initiate LDL peroxidation.[7] These two
examples of aryloxyl radical-induced biological damage high-
light the need for quantitative, in vitro studies of their
reactions using thermolabile compounds which would provide
.
™clean∫ and well-defined ArO fluxes. To design an aryloxyl
radical thermal source (ARTS) which would generate any
.
.
ArO and only that ArO radical is therefore a worthwhile and
exciting challenge.[8]
[6] J. W. Baynes, S. R. Thorpe, Diabetes 1999, 48, 1 9.
[7] C. A. L. S. Colaco, The Glycation Hypothesis of Atherosclerosis,
Springer, Heidelberg, 1997.
Hyponitrites, which are not subject to metal ion- or radical-
induced decomposition,[9, 10] decompose at ambient temper-
atures to give N2 and alkoxyl radicals. It appeared probable
that aryloxyalkoxyl radicals would undergo very fast b-
scission[11] to yield aryloxyl radicals [Eq. (3)]. A synthetic
route to aryloxyalkyl hyponitrites suitable for many different
[8] a) S. D. Yan, X. Chen, J. Fu, M. Chen, H. Zhu, A. Roher, T. Slattery, L.
Zhao, M. Nagashima, J. Morser, A. Migheli, P. Nawroth, D. Stern,
A. M. Schmidt, Nature 1996, 382, 685 691; b) A. Takeda, T. Yasuda,
T. Miyata, Y. Goto, M. Wakai, M. Watanabe, Y. Yasuda, K. Horie, T.
Inagaki, M. Doyu, K. Maeda, G. Sobue, Acta Neuropathol. 1998, 95,
555 558.
[9] See Supporting Information for: a) 1H and 13C NMR data for 7a d,
(Table 1); b) reasoning for the number of observable diastereoisomers
for 7 and assignment of their relative configuration (Tables 1 and 2
and Figure 1); c) 1H and 13C NMR data for 17a,b (Table 3); d) LC
chromatograms (Figure 2); e) 1H and 13C NMR data for 11 and 12
(Table 3).
D
b-scission
.
.
ˆ
ˆ
[ArOCR2ON ]2 À! ArOCR2O
ArO ‡ R2C O
(3)
À!
ÀN2
[*] Dr. T. Paul,[‡] Dr. K. U. Ingold
National Research Council of Canada
100 Sussex Drive
Ottawa, ON, K1A 0R6 (Canada)
Fax : (‡1)613-941-8447
[10] M. O. Lederer, H. P. B¸hler, Bioorg. Med. Chem. 1999, 7, 1081 1088.
[11] B. Huber, F. Ledl, Carbohydr. Res. 1990, 204, 215 220.
[12] M. S. Feather, T. G. Flynn, K. A. Munro, T. J. Kubiseski, D. J. Walton,
Biochim. Biophys. Acta 1995, 1244, 10 16.
E-mail: Thomas.Paul@avecia.com, Keith.Ingold@nrc.ca
‡
[ ] Current Address:
Avecia Ltd., P.O. Box 42, Hexagon House
Blackley, Manchester, M9 8ZS (UK)
Fax : (‡44)161-721-5240
[**] This work was partly supported by the National Foundation for
Cancer Research. We wish to thank M. C. Depew and J. K. S. Wan
(Queen×s University, Kingston, Canada) for their help in recording
ESR spectra and D. Leek for the NMR measurements.
Supporting information for this article is available on the WWW under
http://www.angewandte.com or from the author.
804
¹ WILEY-VCH Verlag GmbH, 69451 Weinheim, Germany, 2002
1433-7851/02/4105-0804 $ 17.50+.50/0
Angew. Chem. Int. Ed. 2002, 41, No. 5

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.
phenols was designed (Scheme 1)[9, 12] and used to prepare 3a
(Arˆ Ph; ARTS-Ph) as a potential phenoxyl radical source
at least some free ArO from both ARTS, this was confirmed
.
with 3b by the direct detection of Toc by ESR and UV/Vis
spectroscopy (Figure 1). The maximum steady-state concen-
tration of Toc ([Toc ]mss) was solvent dependent, with 1.1 mm
3b at 208C [Toc ]mss ꢀ 5.5 mm in chlorobenzene but was only
.
and 3b (Arˆ tocopheryl; ARTS-Toc) as a potential Toc
.
.
source.[13] Decomposition rates of 3a and 3b were measured
.
1
by H NMR spectroscopy and found to be almost identical,
Scheme 1. Synthesis of ARTSs. a) NaH in DMF, 08C, 30 min, ClCH2SCH3,
208C, 6 h; b) m-ClC6H4CO3H in CH2Cl2, 08C, 10 min; c) CH3COCl in
CH2Cl2, 0 !208C, 3 h; d) Ag2N2O2, 0 !208C, 1 5 h. For steps a c see
ref. [12] and for step d see ref. [9]. Physical data for new compounds are
given in the Supporting Information.
for example, at 378C: 9.1 Â 10À4 sÀ1 for ARTS-Ph (in CD3CN)
and 10 Â 10À4 sÀ1 for ARTS-Toc (in CDCl3). ARTS-Ph
decomposed at the same rate in CDCl3 and CD3CN:D2O
(1:1) as in dry CD3CN, that is, its decomposition is solvent
independent. The Arrhenius parameters for ARTS-Ph de-
composition (based on measurements at three temperatures
between 23 and 378C) were: EA ˆ 106 kJmolÀ1 and
log(A/sÀ1) ˆ 14.8.
The expected decomposition pathways for an ARTS are
outlined in Scheme 2. The initial geminate pair of alkoxyls,
4sc, will partition between diffusion from the solvent cage
(efficiency e) and an in-cage disproportionation to 5 and 6,
with the latter probably decomposing to ArOH and form-
aldehyde. Under most circumstances the vast majority of free
alkoxyls, 4, would be expected to undergo b-scission to yield
Figure 1. Thermal decomposition of ARTS-Toc. Top: Concentration of
.
Toc as determined by UV/Vis spectroscopy (l ˆ 424 nm)[14] during
.
the desired aryloxyls, ArO , with very little H-atom abstrac-
decomposition of 1 mm ARTS-Toc in chlorobenzene. Inset: UV/Vis
tion to form 6. Consistent with this scheme, thermolysis of 3a
gave PhOH, 5a, formaldehyde, biphenols, phenoxyphenols,
and polyphenols and thermolysis of 3b gave TocH, 5b,
formaldehyde, tocopheryl quinone, and various tocopherol
dimers.[14] These products are consistent with the formation of
.
&
^
spectra recorded at the maximum [Toc ]. Key: 208C ( ), 258C ( ), 378C
~
*
), and 508C ( ). Bottom: ESR spectrum recorded during decomposition
(
of ARTS-Toc (10 mm) at room temperature in benzene. Hyperfine splitting
constants determined by simulation: a(CH3) ˆ 0.644 mT, a(CH3) ˆ
0.492 mT, a(CH3) ˆ 0.104 mT, a(CH2) ˆ 0.162 mT, a(CH2) ˆ 0.016 mT
(linewidth: 0.015 mT).
.
half as large in cyclohexane and no Toc could be detected in
1,4-cyclohexadiene. The rate constant for b-scission (kb) in
nonpolar solvents can now be estimated since the rate of b-
scission in cyclohexane must be roughly equal to the rate of
H-atom abstraction from the cyclohexane, ks  [c-C6H12] ˆ
1.2  106 mÀ1 sÀ1[15] thus ks  [c-C6H12] ˆ 1.2  106 mÀ1 sÀ1[15]
Â
9.3m ˆ 1.1  107 sÀ1. More precisely, we estimate kb ꢀ 1.8 Â
107 sÀ1 in nonpolar solvents (see Supporting Information)
and this reaction will be even faster in polar solvents.[11, 15]
.
Indeed, [Toc ]mss in cyclohexene containing 1m methanol
.
([Toc ]mss ˆ 4 mm) was twice as large as in neat cyclohexene. It
is likely that all 4 would undergo b-scission in chlorobenzene.
The minimum cage escape efficiency (e) in chlorobenzene at
208C can therefore be calculated to be 5% based on the 3b
.
decomposition rate constant (1.8 Â 10À4 sÀ1), [Toc ]mss, and the
.
.
smallest reported rate constant for the Toc /Toc reaction
(2k ˆ 1000mÀ1sÀ1).[14] The presence of a TocH dimer as a
minor impurity causes a dramatic increase in the apparent
.
.
Scheme 2. Expected thermal decomposition pathways of ARTS.
rate constant of the Toc /Toc reaction[14] and since this dimer
Angew. Chem. Int. Ed. 2002, 41, No. 5
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may be formed during 3b decomposition the true value of e is
likely to be >5%.
Aryloxyl-radical induced oxidative stress has been largely
ignored because of the lack of suitable precursors. The present
synthesis of two ARTS overcomes this lack and provides a new
The phenoxyl radical could not be detected during the
thermal decomposition of 3a at 238C, presumably because the
.
tool for studying the effects of known fluxes of ArO radicals on
.
.
PhO /PhO reaction (2k ˆ 1 12  108 mÀ1 sÀ1)[16] is so much
biologically relevant targets. Currently, we are designing a
synthesis for water-soluble ARTSs which will enable the tyrosyl
radical to be generated in a controlled manner.
.
.
faster than the Toc /Toc reaction.[14] However, e for 3a, could
be estimated by assuming that the in-cage reaction of 4sc
would yield equal amounts of 5a and PhOH whereas GC
analysis showed an excess of PhOH and biphenols over 5a.
From the ™excess∫ phenol, e was estimated to be around 20%.
The aryloxyl radical-initiated peroxidation of LDL was
chosen to illustrate a biologically relevant in vitro application
of ARTS. To a freshly prepared LDL dispersion 3a was added
and incubated at 378C until decomposion was virtually
complete. The TocH consumption curve and the cholesteryl
ester hydroperoxide (CEOOH) formation curve are charac-
teristic of TMP in that peroxidation is faster while TocH is
present than after the TocH is consumed[6] (Figure 2).
Experimental Section
.
The Toc EPR spectrum was recorded at room temperature under N2 on a
Varian E104 spectrometer (9.5 GHz) with microwave powerˆ 2 mW,
modulation amplitude ˆ 0.04 mT, modulation frequence ˆ 100 kHz, scan
time ˆ 8 min. The hyperfine splitting constants were obtained using the
ESR simulation program WINSIM.[17] NMR spectroscopic data were
recorded on a Bruker 400-DRX spectrometer. Freshly isolated LDL,[18] was
dispersed in aerated PBS, mixed with a known amount of a 3a stock
solution in DMSO and then incubated for 1 h at 378C with the usual
analyses for CEOOH and TocH.[19]
Received: August 27, 2001 [Z17803]
[1] L. R. Mahoney, Angew. Chem. 1969, 81, 555 563; Angew. Chem. Int.
Ed. Engl. 1969, 8, 547 555.
[2] G. W. Burton, K. U. Ingold, Acc. Chem. Rev. 1986, 19, 194 201.
[3] V. W. Bowry, K. U. Ingold, R. Stocker, Biochem. J. 1992, 288, 341
344.
[4] D. Steinberg, S. Parthasarathy, T. E. Carew, J. C. Khoo, J. L. Witzum,
N. Engl. J. Med. 1989, 87, 915 924.
[5] J. W. Heinecke, Curr. Opin. Lipidol. 1997, 8, 268 274, and references
therein.
[6] V. W. Bowry, R. Stocker, J. Am. Chem. Soc. 1993, 115, 6029 6044.
[7] a) J. W. Heinecke, Atherosclerosis 1998, 141, 1 15; b) M. I. Savenko-
va, D. M. Mueller, J. W. Heinecke, J. Biol. Chem. 1994, 269, 20394
20400; c) J. W. Heinecke, W. Li, H. L. Daehnke III, J. A. Goldstein, J.
Biol. Chem. 1993, 268, 4069 4077.
.
[8] A few methods for thermal generation of ArO are known but either
they are limited to sterically hindered phenols (see for example: C. D.
Cook, M. Fraser, J. Org. Chem. 1964, 29, 3716 3719) or a second,
more reactive radical is formed simultaneously and stoichiometrically
(see e.g. P. M. Lahti, D. A. Modarelli, F. C. Rossitto, A. L. Inceli, A. S.
Ichimura, S. Ivatury, J. Org. Chem. 1996, 61, 1730 1738).
[9] C. A. Ogle, S. W. Martin, M. P. Dziobak, M. W. Urban, G. D.
Mendenhall, J. Org. Chem. 1983, 48, 3728 3733.
[10] K. U. Ingold, T. Paul, M. J. Young, L. Doiron, J. Am. Chem. Soc. 1997,
119, 12364 12365; T. Paul, Arch. Biochem. Biophys. 2000, 382, 253
261.
Figure 2. Concentration of cholesteryl ester hydroperoxides (CEOOH)
&
^
(
) and a-tocopherol (TocH) ( ) in 1.8 mm LDL dispersed in aerated
phosphate buffered saline (PBS; pH 7.4, 50 mm) after incubation for 1 h at
378C in the presence of the indicated initial ARTS-Ph concentrations.
ARTS-Ph was added as a solution in DMSO, the amount of which did not
exceed 1% of the 300 mL LDL dispersion.
[11] The thermochemically analogous b-scission of 2-phenylethoxyl has a
rate constant in benzene of 2.3 Â 107 sÀ1. See: G. D. Mendenhall, L. C.
Stewart, J. C. Scaiano, J. Am. Chem. Soc. 1982, 104, 5109 5114.
[12] È. Antonsen, T. Benneche, K. Undheim, Acta Chem. Scan. Ser. B
1988, 42, 515 523.
[13] See Supporting Information for physical data of new compounds.
[14] V. W. Bowry, K. U. Ingold, J. Org. Chem. 1995, 60, 5456 5467, and
references therein.
Furthermore, at low initial 3a concentrations where signifi-
cant amounts of TocH remain after the 1 h incubation, the
CEOOH was formed in a chain reaction, for example
.
d[CEOOH]/d[PhO ] ꢀ 16 at an initial [3a] ˆ 35 mm (see Sup-
porting Information). That is, TMP is initiated in LDL by
.
PhO attack on TocH [Eq. (4)]. TMP explains the earlier
[15] D. V. Avila, C. E. Brown, K. U. Ingold, J. Lusztyk, J. Am. Chem. Soc.
1993, 115, 466 470.
.
.
(PhO )aq ‡ (TocH)LDL À! (PhOH)aq ‡ (Toc )LDL
(4)
[16] J. A. Howard, J. C. Scaiano in Landolt-Bˆrnstein, New Series Vol. 13d
(Ed.: H. Fischer), Springer, Berlin, 1984, pp. 142 192.
[17] The WINSIM program was developed at the National Institute of
Environmental Health Sciences, National Institutes of Health,
Research Triangle Park, NC, USA and can be downloaded at:
epr.niehs.nih.gov.
[18] B. H. Chung, J. P. Segrest, M. J. Ray, J. D. Brunzell, J. E. Hokanson,
R. M. Krauss, K. Baudrie, J. T. Cone, Method Enzymol. 1986, 128,
181 209.
observation that tyrosyl radicals generated by myeloperox-
idase initiate LDL peroxidation in a process not inhibited by
TocH.[7b]
At low 3a concentration, approximately 0.2 molecules of
.
TocH are consumed per PhO generated (see Supporting
.
Information). This value would be 0.5 if all the PhO were
.
.
destroyed by TocH and it implies that PhO /PhO coupling
reactions are probably important under the present conditions.
[19] W. Sattler, D. Mohr, R. Stocker, Method Enzymol. 1994, 233, 469
489.
806
¹ WILEY-VCH Verlag GmbH, 69451 Weinheim, Germany, 2002
1433-7851/02/4105-0806 $ 17.50+.50/0
Angew. Chem. Int. Ed. 2002, 41, No. 5