Chem. Res. Toxicol. 1993,6, 69-74
69
Reactions of Vitamin E and Its Model Compound 2,2,5,7,8 - Pentamethylc hroman-6- 01 with Ozone Daniel C. Liebler,'i+ Shigenobu Matsumoto,t Yoichi Iitaka,e and Mitsuyoshi Matsuo*J Department of Pharmacology and Toxicology, College of Pharmacy, University of Arizona, Tucson, Arizona 85721, Tokyo Metropolitan Institute of Gerontology, 35-2 Sakaecho, Ztabashi-ku, Tokyo 173, Japan, and Department of Biological Science, Nishi Tokyo University, Uenohara, Yamanashi 409-01, Japan Received September 2, 1992
Reaction of vitamin E [ (R,R,R)-a-tocopherol1with ozone in acetonitrile yielded a-tocopheryl quinone and its precursor 8a-hydroxytocopherone, which accounted for approximately 30 % of the products a t 6 0 % a-tocopherol oxidation. In addition, two novel products were identified as epimers of lO-acetyl-7-(4',8',12'- trimethyltridecy1)-3,4,7trimethyl-2-oxo-1,6-dioxaspiro[4.51deca-3,g-diene. These spiro products were formed in equal amounts in a combined yield of approximately 33 % after complete a-tocpherol oxidation. Ozonation of the vitamin E model compound 2,2,5,7,8-pentamethylchroman-6-01 yielded an analogous spiro product, 10-acetyl3,4,7,7-tetramethyl-2-oxo-1,6-dioxaspiro[4.5]deca-3,9-diene, whose structure was confirmed by X-ray crystallography. The spiro products may be formed by ozone addition to the chroman ring and subsequent rearrangement to ring-opened hydroxyacid products, which yield spiro products by ring closure due to dehydration. Novel spiro products formed by ozonation of vitamin E may be unique markers of ozone interaction with lipid structures that contain vitamin
E. Introduction
Vitamin E [principally (R&&)-a-tocopherol; la] is a principal inhibitor of lipid autoxidation in biological membranes and is an important protectant against oxidative damage to tissues (1,2). Compound la is thought
la R = CI6H, l b R I CH,
to function primarily by scavenging peroxyl radicals (31, but it also reacts with other oxidants. Indeed, the antioxidant versatility of la is reflected by the diversity of products formed by its many reactions with different oxidants. For example, peroxyl radicals oxidize la primarily to epoxides and 8a-substituted tocopherones (47),whereas benzoyloxy and alkoxy1 radicals oxidize la to 5-methyl-substituted alkoxy derivatives and dimers and trimers via a quinonemethide intermediate (8-11). Singlet oxygen oxidizes la to an 8a-hydroperoxide(12)and unique spiro products (13),whereas base-catalyzed oxygenations yield still different epoxides and other unique rearrangement products (14,151. Alkyl radicals convert la to 0and C-alkylated derivatives (16-19). Considerable evidence indicates that la protects tissues against oxidative damage caused by ozone, which is a major component of smog and a powerful oxidant (20-22). Ozone reacts rapidly with la in aqueous micelles (k = 1X lo6M-l + University of Arizona. f
Tokyo Metropolitan Institute of Gerontology.
I Nishi Tokyo University.
s-1) and somewhat more slowly in CCld (k = 5.5 X lo3M-' s-l) (23). In the same study, the tocopheroxylradical was detected by electron spin resonance spectroscopy as an intermediate in the oxidation, and the authors suggested that ozone reacts with la at least in part by electron transfer. a-Tocopheryl quinone (2) was identified as a product of la ozonation in CCb. However, the chemistry of reactions between la and ozone remains poorly understood, largely because ozonation products of la have not yet been characterized. Our continuing interest in the reactions of la with oxidants led us to examine the reaction of la with ozone. Because ozone displays unique characteristicsas an oxidant for organic compounds and because la yields different, distinctive products upon reaction with other oxidants, we postulated that ozonation of la also would produce unique products. Here we report that ozonation oxidizes la and its model compound 2,2,5,7,8-pentamethylchroman-6-01 (lb) to products observed previously with other oxidants and to unique ring cleavage products that have not previously been identified. Experimental Procedures General. IR and CD spectra were measured on JASCO IR-2 and J-500A spectrometers (Japan Spectroscopic Co., Tokyo, Japan), respectively, UV spectra on a Cary 219 spectrometer (Varian Associates, Palo Alto, CA), and mass spectra on a Finnegan MAT-90 instrument (Finnegan MAT, Bremen, Germany). Mass spectra of compound 4b were recorded on a JEOL JMS-DX 300 mass spectrometer (Japan Electron Optical Laboratories,Oome,Japan). 'H- and 13C-NMRspectrawere recorded on a Varian VXR-400s spectrometer operating at 399.95 MHz for 1H and at 100.58 MHz for l3C. Analytical HPLC was performed with a Hewlett-Packard 1090 Series II/M chromatograph equipped with a HP 1040M diode array detector; preparative HPLC was done with a Waters Mode1510chromatograph equipped with a Soma S-310A UV detector.
0893-228~/93/2706-0069$04.00/0 0 1993 American Chemical Society
Lieblet et al.
70 Chem. Res. Toxicol., Vol. 6, No. 1, 1993 (R,R,R)-a-Tocopherol (la) was prepared from (R,R,R)-atocopherol acetate (Sigma, St. Louis, MO) by reduction with lithium aluminum hydride. Compound l b was synethisized by the method of Nilsson et al. (24). Spectro-grade acetonitrile (Wako Fine Chemicals, Tokyo, Japan) was used as received. Ozonation of la. (Caution: Ozone is a very strong oxidant and is highly toxic! All procedures involving ozone should be
done in a well-ventilated hood equipped with a safety shield. Excess, unreacted ozone should be destroyed bypassage through a solution of potassium iodide.) Ozone was prepared by passing a stream of air through asilent arc discharge type ozone generator a t a flow rate of 20 mL mi+. The aidozone stream was bubbled through a 4 mM solution of la in acetonitrile (70 mL) a t ambient temperature. Although the ozone content of the air stream was not measured directly, the generator voltage and air flow were adjusted to achieve complete oxidation of la in approximately 3 h. Samples of the reaction mixture were removed periodically for reverse-phase HPLC analysis on a Hewlett-Packard Hypersil ODS 5-pm, 4.6- X 100-mm column eluted with methanol/water (955 v/v) at 1mL min-1. When consumption of la was complete, the ozone flow was stopped and the solvent was evaporated from the reaction mixture in vacuo. Products 4a were purified by reverse-phase preparative HPLC on a Waters Nova-Pak radial compression column (10 X 100mm) eluted with methanol/water (955 v/v) a t 5 mL min-l. Fractions containing 4al and 4az were collected, products were extracted from the mobile phase with hexane, and the extracts were evaporated in vacuo. Compound 4al: UV (CH3CN) A,, 219 nm ( 6 14 500);CD (CH3CN) X 214 (01, 226 (A6 -7.8) nm; IR (CCl4) vmlu 1780,1693 cm-l; MS (EI, 70 eV) m/z 460 (M+) (1,2), 415 (30.31, 398 (3.3), 235 (52.3), 193 (100.0); m/z 460.3536 (calcd for C29H4804,460.3553);'H- and 13C-NMR: (seeTable1). Compound4az: UV (CH3CN)Xm,219nm(e 14 700); CD (CH3CN) X 214 (O),226 (Ae +7.7) nm; IR (Cc14) vmax 1780, 1693 cm-1; MS (EI, 70 eV) m/z 460 (M+) (2.8), 415 (13.6), 398 (3.4), 235 (39.71, 193 (100.0); m/z460.3542 (calcd for C2gH4804, 460.3553); 1H- and 13C-NMR: (see Table I). Ozonation of lb. An ozone/air stream was bubbled through a 4 mM solution of l b in acetonitrile (100 mL) at ambient temperature. Disappearance of l b was monitored by HPLC. Immediately after l b was consumed, the ozone flow was stopped and the solvent was evaporated in vacuo. Products were purified by reverse-phase preparative HPLC on a Senshu Pak ODS-5251 5-pm, 20- x 250-mm column (Senshu Scientific Co., Ltd., Tokyo, Japan). Products were eluted with acetonitrile/water (35:65 v/v) at a flow rate of 7 mL min-l. A fraction eluted a t 41 min was collected and evaporated to dryness. The residue then was recrystallized from a mixture of hexane and ethyl ether. Compound 4b: UV (CH3CN) A,, 219 nm (e 15 700), (hexane) 214 nm (e 15 200); IR (CCL) v,, 1780,1693 cm-1; MS m/z 250.1185 (calcd for C14H1804, 250.1200); 'H- and W-NMR: (see Table I). X-ray Crystallography. A single crystal of 4b was subjected to X-ray diffraction analysis with a Nonius CAD4 diffractometer. The intensities of 2501 reflections (out of a possible 2961 reflections) were measured in the 28 range of 6- 150' above 1 . 5 ~ (0withgraphite monochromated Cu K a radiation. The structure was solved by the direct method and refined by the method of block-diagonal least squares to an R value of 0.058. The 14 carbon and 4 oxygen atoms were refined with the assumption of anisotropic thermal vibrations. The 18 hydrogen atoms were refined with the assumption of isotropic thermal vibrations. The hydrogen atoms were found on the difference electron-density map and located a t the calculated positions. The perspective view was drawn by the PLUTO program (25). The crystal data are as follows: C14Hl8O4;MW 250.3; crystal habit colorless flakes; crystal size 0.73 X 0.33 X 0.10 mm; space group P2dc; 2 = 4; ~ ; constraints crystal system monoclinic;Dedcd= 1.227g ~ m -lattice a = 12.4441 (7) A, b = 10.1065 (5)A, c = 11.435 (2) A; /3 = 109.549 (8)O; V = 1355.2 A3; p for Cu K a = 6.97 cm-l. Additional crystallographic data are available as supplementary material.
30
n
1
I1
la
0 0
400
2
4
6
8 1 0 1 2 1 4
!I
4
0
Q
200
0
2
4
6
8 1 0 1 2 1 4
time, min
Figure 1. Reverse-phase HPLC analysis of products formed by ozonation of la for 2.5 h in acetonitrile. Chromatograms were recorded by UV monitoring a t 270 nm (top) or 220 nm (bottom).
Results Ozonation of la. Compound la was oxidized by continuous passage of an ozone/air mixture through a solution of la in acetonitrile at ambient temperature. Samples of the solution were analyzed periodically by reverse-phase HPLC (Figure 1). UV monitoring at 270 nm indicated the presence of several products, one of which displayed elution characteristics identical to those of authentic 2. Nevertheless, diode array UV scanning indicated that the peak contained at least two components (Figure 2, top). Scans of the leading and trailing edges of the peak displayed UV maxima at 240 nm, whereas scans at the peak apex exhibited a maximum at 266 nm. The UV spectra of the leading and trailing edges are characteristic not of 2, but of 8a-hydroxytocopherone(3),which is a product of la oxidation in other systems and readily rearranges to 2 (7,26,27).The spectrum at the peak apex
I 2
3
appeared to represent superimposition of the spectra of 2 (which displays an absorbance maximum at 266 nm) and 3. The dominant appearance of 3 at the leading and tratling edges of the peak (but not at the peak apex) may be due to the elution of two diastereomers of 3. Indeed, the HPLC system used resolves diastereomers of other tocopherone products of la oxidation (7).To further test the hypothesis that 3 is the precursor to 2 in this system, la was ozonized in acetonitrile/5% HClOl(95:5 v/v). 8aHydroxytocopherone (3) readily rearranges to 2 under acidic conditions (26,27).HPLC analysis of the products again revealed a peak with retention characteristics identical to those of 2. Diode array scans of the leading and trailing edges and of the peak apex produced identical
Chem. Res. Toxicol., Vol. 6, No. 1, 1993 71
Ozonation of Vitamin E
-4 250
300
350
v
480
-8
sO l
I
220 250 280 wave length (nm)
888
0
C
4
P Q
Figure 3. CD spectra of products 4al and 4az.
680
500 408
308 208
I e0 250
300
358
400
wavelength, nm Figure 2. Diode array UV scans of HPLC peak eluting as compound 2 from ozonation of la in acetonitrile (top) or in acetonitrile/5% HCIOh(955 v/v) (bottom). Specta labeled a, b, and c, are scans of the leading edge, peak apex, and trailing edge of the peak, respectively.
spectra with a UV maximum at 266 nm (Figure 2,bottom), which is characteristic of 2. This result suggests that compound 3 is a precursor to 2 in this system, but that 3 is detected only in neutral reaction mixtures, where it rearranges to 2 comparatively slowly. Since the product peak from neutral reaction mixtures corresponding to 2 also contained variable amounts of 3, the yield of 2 was not quantified. However, the yield of 2 in acidic reactions was approximately 30% when la was 50% oxidized, but fell to 14%by the time la had been consumed completely. This indicated that 2 formed during the reaction was itself further oxidized (see below). Table I. Assignments of Resonances in
In reverse-phase HPLC analyses of reaction mixtures two other major products were detected by UV monitoring at 220 nm (Figure 1). These products were purified by reverse-phase preparative HPLC and identified as isomers of l0-acetyl-7-(4',8',12'-trimethyltridecyl)-3,4,7-trimethyl2-oxo-1,6-dioxaspiro[4.5ldeca-3,9-diene (4a1/4a2).These two products exhibited nearly identical UV, IR, 'H- and 13C-NMR,and mass spectra, and mirror image CD spectra (Figure 31, and apparently are epimers that differ in configuration at the spiro carbon. 'H- and 13C-NMR resonances for 4al and 4a2 are listed in Table I. Although product 4a was detected from the earliest stages of la oxidation, we examined the possibility that
it was formed by further oxidation of 2. Ozonation as described for la rapidly consumed 2, but product 4a was NMR
Spectra of Products 4al,4az, and 4b
chemical shift, DDm functional group
atom
nucleus
c-0 c=c
cz,c11
C=CH
c3,C4r ClO c9
COZ
cs
1 3 c (5) 13c (5) 13C (d) lH (m, 1 H) 1 3 c (5)
c7
13c (5)
CHz
CS
1 3 c lH (m, 2
CH3
C3a, c 4 a
co
(t)
Cle, C 7 b
H) 13C (9) 'H(s,3H) 13C (4) 'H ( s , 3 H) I3C
c 1 2
(9) 3 H)
lH ( 8 ,
isoprenoid chain CH CH2
C#, Ce., c12'
c1, c2, C3', C7', c g , C6.1 ClW c11, C4" C8,S C12'm c13'
cs,
CH3
13C (d) 1 3 c (t) 1 3 c (t) 13c (t) 1 3 c (t) 1%
(t)
I3C (9) 13C (9)
~
4b 173.1, 194.6 125.6,134.1,156.2 143.3 -7.23-7.26 102.0 72.0 36.9 -2.31-2.47 8.7, 10.6 1.68, 1.83 25.8, 30.2 1.34, 1.38 26.6 2.26
4al 172.9,194.5 125.7,134.0,156.1 143.0 -7.21-7.24 101.7 74.6 35.7 -2.38-2.58 8.9,10.6 1.68,1.83 27.1 1.28 26.6 2.26 28.0,32.8,32.9 37.6 22.1 37.1, 37.2,37.3, 37.4 24.5, 24.8 39.6 19.5, 19.7 22.6, 22.7
4az 173.2,194.6 125.6,134.2, 156.3 143.5 -7.24-7.27 102.0 74.0 34.8 -2.25-2.48 8.7,10.7 1.68,1.83 24.3 1.34 26.6 2.26 28.0, 32.7, 32.8 43.1 20.8 37.2 (X2), 37.3,37.4 24.4, 24.8 39.4 19.7 (X2) 22.6, 22.7
72 Chem. Res. Toricol., Vol. 6, No. 1, 1993
Liebler et al.
Scheme I la
03
I
I
Figure 4. X-ray molecular structure of 4b. not formed (data not shown). The rapid disappearance of 2 is consistent with its poor stability in oxidations of la, where the apparent yield of 2 declined as oxidation of la progressed (see above). In contrast to 2, the yield of 4a did not decline with increasing la oxidation. The yield of 4a after complete consumption of la was approximately 33%. Although compound 3, the precursor to 2, might also undergo further oxidation, this possibility was not studied as compound 3 tends to rearrange to 2 under the reaction conditions employed (see above). Ozonation of lb. Ozonation of lb under conditions identical to those described for ozonation of la yielded the analogous product 4b, which was purified by reversephase HPLC. As with 4a derived from la oxidation, the product was isolated as the spirolactone. UV-vis, IR, mass spectrometry, and NMR spectra of 4b were analogous to those of 4a, and X-ray analysis confirmed the assigned structure (Figure 4). Product 4b probably is a 1:lracemic mixture of C-5enantiomers, as a single crystal of 4b belongs to the space group P21/c (see crystal data) and a unit cell contains two pairs of enantiomers, with each pair positioned about a mutual center of symmetry. Discussion
Ozonation of vitamin E (la) yielded a-tocopheryl quinone (21, which is formed from la by other oxidants, and two epimeric spiro products 4a, which are reported here for the first time. These spiro products appear to be formed uniquely by ozone. This conclusion is drawn in part because other oxidants are not known to form these products from la and in part because product formation apparently involves cleavage of the chromanol ring, which is characteristic of the reaction of ozone with other phenols (see below). X-ray analysis of the corresponding product 4b from ozonation of the vitamin E model compound l b confirmed the unusual product structure. Other products of la ozonation remain to be identified, as the combined yields of spiro products 4a and quinone 2 did not exceed 60%. The formation of structurally distinct products 4a and 2 suggests that ozone reacts with la by at least two distinct mechanisms. The identification of 2 as a reaction product is consistent with the finding that 2 was an ozonation product of la in CCld (23). Whether the reaction is initiated by electron transfer from la to ozone, as suggested by Giamalva et al. (23),or by another mechanism is not clear. Two mechanisms may lead to 2 via the intermediate 3 (Scheme I). First, a hydrogen transfer followed by an electron transfer may oxidze la first to the tocopheroxyl radical and then to the tocopherone cation, which hydrates to 3 (pathway
A, Scheme I). Ambient moisture in the reaction solvent may be sufficient for the hydration reaction as freshly opened spectro-grade acetonitrile may contain up to0.01% (approximately 4 mM) HzO. Alternatively, ozone may add to the 8a-position of la and then eliminate oxygen to yield 3 directly (pathway B, Scheme I). This latter mechanism has been proposed previously to account for the hydroxylation of phenols (28). Another possible path to 3 is the abstraction of benzylic hydrogen by ozone (29), which could yield either the tocopheroxyl radical or a benzylic hydrotrioxide by recombination. Radicals formed by hydrotrioxide decomposition (29)could oxidize other molecules of la to 3. Although all of these mechanisms could explain the formation of 2 in our studies, the reaction depicted by pathway B is of interest because it may release singlet oxygen. Kanofsky and Sima recently have shown that ozonation of la in detergent/buffer mixtures produces singlet oxygen in about 15% yield (30). The reaction proposed in pathway B, Scheme I, may be somewhat analogous to the reaction of ozone with hindered olefins, in which ozone-dependent epoxidation is accompanied by release of molecular oxygen (31). Singlet oxygen generated in this manner may also oxidize la. Indeed, other unique spiro compounds recently have been reported as products of dye-sensitized photooxidation of la (23). However, these spiro products are different from those identified in our study. Similarly, other spiro compounds formed by the base-catalyzed oxidation of la also differ from those described here (14). The cleavage of la/b probably is initiated by addition of ozone across the 5,6-double bond of the chroman ring to form a trioxolane intermediate (Scheme 11). This initial step is analogous to that proposed for other phenols (32). Cleavage of the trioxolane then yields an acetyl substituent and a carboxyl oxide intermediate, which may exhibit zwitterionic or diradical character (31). Radical addition by the diradical form of the carboxyl oxide, followed by hydrogen atom abstraction from the heterocyclic ring, may form a hydroxyacid product, which dehydrates to 4a/b (Scheme 11, reaction A). Alternatively, abstraction of a proton from the heterocyclic ring yields a carbanion/ carboxyl oxide cation pair, which then collapses to the hydroxyacid precursor to 4a/b (Scheme 11,reaction B). If hydrolysis of the carboxyl oxide (32) occurs, it may not affect the yield of 4a, which was approximately 33% both in acetonitrile and in acetonitrile containing 5 % water. Further work will be necessary to establish the mechanism
Chem. Res. Toxicol., Vol. 6, No. 1, 1993 73
Ozonation of Vitamin E
Scheme I1
*I O
H
O
IB
H
k
V
'1
0
I
J
by which 4a and 3 are formed. Since 213 and 4a account for about 60% of la oxidized in these studies,other reaction products remain to be identified. Among reactions that could lead to other products are the "anomalousoozonation of phenols (32) and secondary oxidation of products 2,3, and 4a. Although the absolute configurationsof the compounds have not been established unambiguously,analysis of their NMR spectra indicates that the 13C shift of C ~(i.e., J the fiit carbon of the isoprenoid side chain) of 4al(37.6 ppm) is 5.7 ppm upfield of that for C1t in 4a2 (43.1 ppm). On the other hand, the 13Cshift for C7a (methyl carbon) in 4al (27.1 ppm) is 2.8 ppm downfield of that for C7a in 4a2 (24.3 ppm). These chemical shift differences may result from differences in the absolute configuration at CS (Le., the spiro carbon). Stereo models indicate that, in the 5S,7R epimer, the C1t methylene group is closelyjuxtaposed with the lactone ester oxygen. A steric compression shift (3335)therefore may increase shielding of C1t relative to that for C1t in the 5R,7R epimer. In the 5R,7R epimer, the 7a-methyl group, rather than the isoprenoid chain, is close to lactone ring and C7a is shielded by a steric compression effect. Consequently, we propose a tentative assignment of 481, in which Cy is shielded, as the 5S,7R epimer and 482, in which C7a is shielded, as the 5R,7R epimer. Interestingly, the 13Cresonances of the C7a and C7b methyl groups in 4b differ by 4.4 ppm, which suggests that the steric compression effect between C7 substituents and the lactone ring in 4a also occurs in 4b. A surprising property of spiro products 4a/b is their relatively low UV,, values (219 nm in acetonitrile). This W, is consistentwith the presence of an cu,P-unsaturated 5-membered cyclic lactone (36). However, the presence
of another conjugated carbonyl (Ce-Cio-Cii-0n.J containing one a-and one 8-alkyl substituent would be expected to produce another absorbance at 237 nm. X-ray analysis of product 4b indicates that the angle between planes defined by C.&e-Clo-Cr, and Clo-C11-011a-Clzis 1.7'. Thus, there appears to be conjugation between the Ce-Clo bond and the C11-Olla bond. We presume that the unusually low A, values for 4a and 4b are caused by some electronic effect that alters the behavior of the enone chromophore, rather than by steric hinderance that interrupts conjugation. In support of this explanation, we note that the ,A, value reported for l-acetylcyclohexeneis 232 nm (37), whereas that for l-acetyl-2-(methoxycarbonyl)cyclohexene is 220 nm (38). The formation of an ozone-specificproduct is interesting because ozonation may result in the concomitant generation of other reactive species such as radicals (29,39)and singlet oxygen (30), which also oxidize la/b. Although spiro products 4a/b apparently are formed directly by ozone, other products yet to be identified may result from oxidation by ozone-derived singlet oxygen. Ozonation of some substrates, such as polyunsaturated fatty acids, is accompanied by concomitant free radical-mediated autoxidation reactions (22,39). Such reactions produce 2 as well as other products. With the exceptionof a-tocopheryl quinone (2), which may be formed directly from ozone (see above), we have not identified products associated with free radical oxidations of la/b. Nevertheless, some free radical oxidation may accompany ozonation of lalb in our system. Giamalva et al. (23) have suggested that la does not react directlywith ozone in biological systems, but is consumed instead by free radical reactions secondary to ozonation. This assertion was supported by their observation that ozone reacted with la and polyunsaturated fatty acids at similar rates and by the assumption that the presence of equally reactive alternate substrates in great excess would preclude direct ozonation of la. An experimental test of this hypothesis would require that ozone-specific products could be detected and quantified. The results presented here indicate that it should be possible to distinguish la depletion by direct ozonation from la depletion by free radical reactions. Acknowledgment. We thank Prof. Naomichi Baba of the Okayama University for high-resolution mass spectral measurement for compound 4b and Mr. Peter F. Baker of the University of Arizona for mass spectral measurements of compounds 4a. We also thank Dr. Yoshiharu Nawata of the Chugai Pharmaceutical Co. for X-ray diffraction measurement and Mr. Shun-ichiroNakano for isolation of product 4a. This work was supported in part by USPHS Grant CA47943. Supplementary Material Available: Tables I-IV, listing bond lengths, bond angles, atomic coordinates, and thermal parameters, and Table V, listing observed and calculated F for compound 4b (10 pages). Ordering information is given on any current maethead page.
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