Reactivities and products of free radical oxidation of cholestadienols

Article abstract of DOI:10.1021/ja5011674

7-Dehydrocholesterol (7-DHC) is the most oxidizable lipid molecule reported to date, with a propagation rate constant for free radical peroxidation that is 200 times that of cholesterol. To better understand the high reactivity of 7-DHC and elucidate the reaction mechanism, we synthesized conjugated and skipped nonconjugated cholestadienols that would give one of the two putative pentadienyl-radical intermediates formed in 7-DHC peroxidation. The additional dienols include 6,8(9)-dienol, 5,8(14)-dienol, 6,8(14)-dienol, and the biologically important 8-dehydrocholesterol (8-DHC; 5,8(9)-dienol). We found that all of the dienols are significantly (at least 40 times) more reactive than cholesterol. Among them, dienols leading to the formation of the pentadienyl radical in ring B (termed endo-B) of the sterol are more reactive than those leading to the pentadienyl radical spanning rings B and C (termed exo-B). By comparing the oxysterol profile formed from 7-DHC and those formed from 8-DHC and 5,8(14)-dienol, products formed from abstraction of the hydrogen atoms at C-9 and C-14 (H-9 or H-14 mechanism) were clearly differentiated. When the oxidation was carried out in the presence of the good hydrogen atom donor α-tocopherol, the oxysterol profile of 7-DHC peroxidation differed distinctly from the profile observed in the absence of the antioxidant and resembles more closely the profile observed in biological systems. This study suggests that oxidative stress and the accumulation of oxysterols should be considered as two key factors in cholesterol biosynthesis or metabolism disorders, where dienyl sterol intermediates are accumulated.

Full text of DOI:10.1021/ja5011674

Article
pubs.acs.org/JACS
Reactivities and Products of Free Radical Oxidation of
Cholestadienols
Libin Xu* and Ned A. Porter
Department of Chemistry and Vanderbilt Institute of Chemical Biology, Vanderbilt University, Nashville, Tennessee 37235, United
States
S
* Supporting Information
ABSTRACT: 7-Dehydrocholesterol (7-DHC) is the most
oxidizable lipid molecule reported to date, with a propagation
rate constant for free radical peroxidation that is 200 times that
of cholesterol. To better understand the high reactivity of 7-
DHC and elucidate the reaction mechanism, we synthesized
conjugated and skipped nonconjugated cholestadienols that
would give one of the two putative pentadienyl-radical
intermediates formed in 7-DHC peroxidation. The additional
dienols include 6,8(9)-dienol, 5,8(14)-dienol, 6,8(14)-dienol,
and the biologically important 8-dehydrocholesterol (8-DHC;
5,8(9)-dienol). We found that all of the dienols are
significantly (at least 40 times) more reactive than cholesterol.
Among them, dienols leading to the formation of the pentadienyl radical in ring B (termed endo-B) of the sterol are more
reactive than those leading to the pentadienyl radical spanning rings B and C (termed exo-B). By comparing the oxysterol profile
formed from 7-DHC and those formed from 8-DHC and 5,8(14)-dienol, products formed from abstraction of the hydrogen
atoms at C-9 and C-14 (H-9 or H-14 mechanism) were clearly differentiated. When the oxidation was carried out in the presence
of the good hydrogen atom donor α-tocopherol, the oxysterol profile of 7-DHC peroxidation differed distinctly from the profile
observed in the absence of the antioxidant and resembles more closely the profile observed in biological systems. This study
suggests that oxidative stress and the accumulation of oxysterols should be considered as two key factors in cholesterol
biosynthesis or metabolism disorders, where dienyl sterol intermediates are accumulated.
formation of two different pentadienyl radical intermediates
(C5−C6−C7−C8−C9 and C5−C6−C7−C8−C14) (Scheme
1). For convenience, we designate the C5−C6−C7−C8−C9
radical as endo-B since it is endocyclic in ring B of the sterol
and the C5−C6−C7−C8−C14 radical as exo-B since one arm
of this radical is exocyclic to ring B. The formation of both
endo-B and exo-B is followed by precedented free radical
transformations such as oxygen addition, 5-exo cyclization, and
intramolecular radical substitution (SHi)9 that lead to the
oxysterol products isolated and identified.12
To further understand the high reactivity of 7-DHC and
better define the reaction mechanisms, we synthesized
conjugated and methylene-skipped nonconjugated cholesta-
dien-3β-ols (abbreviated as “cholestadienols”) that would
readily give either the endo-B or exo-B pentadienyl radicals
upon abstraction of the reactive hydrogen atom(s) (Scheme 2).
Here we report the following: (i) the structure−reactivity
studies on the free radical oxidation of different cholestadienols
that include 7-DHC (5,7-dienol), 8-DHC (5,8(9)-dienol),
6,8(9)-dienol, 5,8(14)-dienol, and 6,8(14)-dienol; (ii) analysis
of oxidation products of these dienols and elucidation of the
INTRODUCTION
■
Lipid peroxidation plays important roles in the pathophysiology
of various human diseases, such as atherosclerosis,1 asthma,2
Alzheimer’s disease,3 and nonalcoholic fatty liver disease.4
Increasing research effort has been devoted to the oxidation
products of lipids as they not only serve as important
biomarkers for disease5 but also exert a variety of biological
activities.6−8 The rate-determining step of free radical-mediated
lipid peroxidation is the hydrogen atom transfer from a lipid
substrate to a propagating peroxyl radical.9 Previously we
determined the propagation rate constants (kp) of free radical
peroxidation of different lipids including polyunsaturated fatty
acids, cholesterol, and 7-dehydrocholesterol (7-DHC) in
solution and in liposomes.10 The kp for 7-DHC was
exceptionally large at 2260 M−1 s−1 in solution, a value that
is 200 times that for cholesterol,10 and 20 times that for the
simple 1,3-cyclohexadiene when normalized to the number of
reactive hydrogen atoms.11
Peroxidation of 7-DHC leads to the formation of over a
dozen oxysterols,12 and although a detailed reaction mechanism
has been proposed, the origin of some of the oxysterols is still
ambiguous.12 Briefly, the reaction of 7-DHC involves hydrogen
atom transfer from C-9 or C-14 of the sterol to a propagating
peroxyl radical (H-9 or H-14 mechanism), leading to the
Received: February 3, 2014
Published: March 13, 2014
© 2014 American Chemical Society
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Scheme 1. Formation of Two Pentadienyl Radicals after Losing H-9 or H-14 in the Initial Step of Free Radical Oxidation of 7-
Dehydrocholesterol
a
Scheme 2. Cholestadienols Synthesized in This Study That Serve As Precursors to endo-B or exo-B Pentadienyl Radicals
a
Rate constants for hydrogen atom transfer from the dienols to a linoleate peroxyl radical were shown (see Table 1).
reaction mechanism; (iii) the effect of an antioxidant, α-
tocopherol, on the product distribution of the free radical
oxidation of the dienols.
solved from the plot of trans,cis-/trans,trans-HODEs vs [R−H].
The propagation rate constants of the cholestadienols are
summarized in Table 1 and Scheme 2, and the clocking plots
RESULTS
Table 1. Free Radical Oxidation Propagation Rate Constants
■
of Different Unsaturated Sterols Determined by the
Structure−Reactivity Studies of Free Radical Oxida-
tion of Cholestadienols. Hydrogen atoms on bis-allylic
carbon centers are normally more reactive than monoallylic
hydrogen atoms toward reaction with peroxyl radicals because
of the lower bond dissociation enthalpy of the reactive C−H
bond.13,14 Thus linoleate (31 M−1 s−1 per H-atom) is much
more prone to give up a hydrogen atom to a peroxyl radical
than oleate (0.22 M−1 s−1 per H-atom).11,15 To compare the
reactivities of monoallylic and bis-allylic hydrogen atoms in the
ring system of cholesterol, we prepared a series of
cholestadienols with double bonds spanning ring B and/or C
as shown in Scheme 2 and measured their propagation rate
constants using the linoleate peroxyl radical clock.16 8-DHC,
5,8(14)-dienol, and 6,8(14)-dienol were synthesized as
previously reported.17−19 The 6,8(9)-dienol was synthesized
from TBS-protected 7-DHC via selective hydroboration/
oxidation at the C5 double bond,20 mesylation/elimination of
the resulting alcohol at C6, and deprotection (Scheme S1).
In the “slow” linoleate peroxyl radical clock, oxidation of
methyl linoleate gave trans,cis-hydroxyoctadecadienoates
(HODEs) and trans,trans-HODEs as the primary products
and the ratio of trans,cis-/trans,trans-HODEs is proportional to
the hydrogen atom transfer rate constant (kH) for a given
hydrogen atom donor (R−H) times the concentration of that
donor ([R−H]).16 Thus, under pseudo-first-order reaction
conditions, by varying the concentrations of R−H while
keeping the concentration of linoleate constant, kp can be
a
Linoleate Peroxyl Radical Clock
sterols (R−H)
kp (M−1 s−1
)
b
7-dehydrocholesterol (5,7-dienol)
6,8(9)-dienol
2260
1370 40
994 33
911 43
412 22
8-dehydrocholesterol (5,8-dienol)
5,8(14)-dienol
6,8(14)-dienol
b
cholesterol
11
a
b
At 37 °C in benzene or chlorobenzene; errors are 2σ. From ref 10.
are shown in Figure S1. As seen in the table, the reactivities of
the cholestadienols decrease in the order of 7-DHC (5,7-
dienol), 6,8(9)-dienol, 8-DHC (5,8-dienol), 5,8(14)-dienol,
and 6,8(14)-dienol.
Profiling the Oxysterols Formed from Free Radical
Oxidation of the Cholestadienols. In order to unequiv-
ocally elucidate the H-9 and H-14-mechanisms of 7-DHC
peroxidation and to identify the reactive hydrogen atoms in the
cholestadienols under study, peroxidation products of these
dienols were analyzed by HPLC-MS. Free radical oxidation of
the sterol dienols (0.01−0.08 M) in benzene at 37 °C was set
up as described previously using (2,2′-azobis(4-methoxy-2,4-
dimethylvaleronitrile) (MeOAMVN) as the radical initia-
tor.10,12 The product mixture was treated with PPh3 to reduce
hydroperoxides, and this product mixture was analyzed by
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Figure 1. Normal phase HPLC-APCI-MS-MS analysis of oxysterols formed from free radical oxidation of 7-DHC (A), 8-DHC (B), and cholesta-
5,8(14)-dienol (C). (D) Major oxysterols identified. D7-3,5-dihydroxycholest-7-en-6-one (DHCEO) was added to each sample as a standard for
proper comparison of the retention times between different chromatograms. The parent ion of each chromatogram and the MS/MS transition were
marked in the corresponding MS panel.
normal phase HPLC-MS. Typical chromatograms of oxidation
product mixtures of individual dienols are shown in Figures
1A−C and S2, and the structures of major oxysterols identified
are shown in Figure 1D. Based on the proposed reaction
mechanisms, oxidation of 8-DHC should exclusively give the
products formed from the H-9 mechanism (endo-B) while
oxidation of the 5,8(14)-dienol should only give products from
the H-14 mechanism (exo-B) after initial hydrogen abstraction
at the bis-allylic positions. Comparison of the oxysterol profile
from the peroxidation of 7-DHC with the profiles from these
two unconjugated dienols shows conclusively that compounds
1, 2a, and 2b are formed from the H-9 mechanism, while
compound 4 is formed from the H-14 mechanism and
compound 3 can be formed from either mechanism with the
H-14 mechanism being the primary route. Under the chromato-
graphic conditions used here, compound 2b coelutes with a
minor product of 7-DHC peroxidation, 3β,5α-dihydroxy-
cholesta-7,9(11)-dien-6-one (DHCDO; see Figure 2), as
shown previously.12 However, these two compounds can be
differentiated by observing the SRM transition 433→415,
which DHCDO lacks (see also Figure S4). Although 7-DHC-
5α,6α-epoxide can only form from 7-DHC via peroxyl radical
addition, we note that there is an oxysterol derived from the H-
14 mechanism (4.68-min peak in Figure 1C) that might have a
small contribution to the peak intensity of the epoxide (4.81
min) in Figure 1A.
and α-tocopherol is the major hydrophobic chain-breaking
phenolic antioxidant present in vivo.21 Studies of lipid oxidation
in the presence of α-tocopherol have biological relevance, and
we therefore carried out the free radical oxidation of the series
of cholestadienols, including 7-DHC, in the presence of α-
tocopherol (1.0 M). Under these conditions (i.e., high
concentration of α-tocopherol but low rate of initiation), the
tocopheryl radical becomes the main chain-propagating radical
even though at a much slower rate (0.10 M−1 s−1),22 i.e.,
tocopherol-mediated peroxidation.23,24 Typical HPLC-MS
chromatograms of product mixtures and the oxysterol products
identified in these oxidations are shown in Figures 2 and S3.
Oxidation of 7-DHC gave three partially overlapping peaks
with the mass of the dehydration ion of [7-DHC + 1O + H+]
and these products are the only products formed from the
oxidation of 5,8(14)-dienol in the presence of α-tocopherol,
suggesting that these three products were derived from exo-B
and the H-14 mechanism. Epoxidation of 7-DHC was not
observed under these conditions, likely because of rapid
trapping of peroxyl radicals by α-tocopherol before addition
to a CC bond can occur.
Three of the 7-DHC products are identical to the products of
peroxidation of 8-DHC, suggesting that they are formed from
endo-B and the H-9 mechanism. Two products observed in the
SRM panel of 415 → 397 were identified to be 3β,5α,9α-
trihydroxycholest-7-en-6-one (THCEO) and DHCDO (Figure
2), by comparing retention times and SRM transition
characteristics with standards isolated from free radical
oxidation of 7-DHC (Figures S4 and S5).12 Note that
Profiling Oxysterols Formed from the Free Radical
Oxidation of Cholestadienols in the Presence of α-
Tocopherol. Human tissues and cells are rich in antioxidants,
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Figure 2. Normal phase HPLC-APCI-MS-MS analysis of oxysterols formed from free radical oxidation of 7-DHC (A), 8-DHC (B), and cholesta-
5,8(14)-dienol (C) in the presence of 1.0 M α-tocopherol. (D) Structures of identified major oxysterols. D7-3,5-dihydroxycholest-7-en-6-one
(DHCEO) was added to each sample as a standard for proper comparison of the retention times between different chromatograms. The parent ion
of each chromatogram and the MS/MS transition were marked in the corresponding MS panel.
DHCDO does not display the SRM transition 433→415.
These two compounds were minor products of the free radical
oxidation of 7-DHC in the absence of α-tocopherol while
THCEO was identified as one of the major 7-DHC-derived
oxysterols observed in brain tissue of Dhcr7-KO mouse, a
genetic animal model for Smith−Lemli−Opitz syndrome
(SLOS).25,26 The product eluting in the peak in the SRM
panel of 399 → 381 was identified to be 7-oxo-5,8-dien-3β-ol
(7-keto-8-DHC) by comparing its SRM transition characteri-
sics and retention time with those of the known compound that
was synthesized from dehydration of 7-hydroperoxy-5,8-dien-
3β-ol,25 which was prepared in turn by photooxidation of 7-
DHC.27 The mechanism of its formation is discussed in the
following section.
Scheme 3. Competition between Peroxyl Radical Addition to
7-DHC (kadd) and Hydrogen Atom Transfer (kH)
DISCUSSION
atom transfer from tocopherol to the peroxyl radical (kH(α-
tocopherol) = 3.5 × 106 M−1 s−1) (Figure 2).16,28,29
■
In the absence of α-tocopherol, product analysis by HPLC-MS
suggests that 7-DHC undergoes free radical oxidation via three
pathways designated H-9 or H-14 abstraction and peroxyl
radical addition. The formation of the 7-DHC-5α,6α-epoxide
suggests that the addition of a propagating peroxyl radical to 7-
DHC can compete with abstraction of a hydrogen atom by the
same radical (Figure 1; Scheme 3). Peroxyl radical addition at
C-5 of 7-DHC eventually leads via an SHi process to the
formation of the epoxide and an alkoxyl radical that can
propagate the radical chain reaction. In the presence of α-
tocopherol (1.0 M), however, the addition pathway is
completely suppressed due to the competition from hydrogen
For each pentadienyl radical, molecular oxygen can add to
three positions that have high spin density, C-5, C-7, or C-9 for
endo-B and C-5, C-7, or C-14 for exo-B (Figure 3). Thus, in the
H-9 mechanism, three peroxyl radical regioisomers could form
as shown in Scheme 4. In our previous studies of 7-DHC
peroxidation, products derived from the bis-allylic peroxyl
radical were not observed likely due to the rapid β-
fragmentation of this radical to give back its precursor
pentadienyl radical.12,16 The rate constant of such a
fragmentation in an acyclic system (linoleic acid) was found
to be 2.6 × 106 s−1.16 In the presence of α-tocopherol, however,
a product derived from the bis-allylic peroxyl radical, 7-keto-8-
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Figure 4. Proposed concerted mechanism for the addition of oxygen
to endo-B.
Figure 3. Potential sites in the pentadienyl radicals endo-B and exo-B
for addition of molecular oxygen.
DHC, was observed as one of the major products formed. On
the other hand, even in the presence of 1.0 M α-tocopherol,
products formed from direct trapping of the 5- or 9-peroxyl
radical were not observed but rather those formed from 5-exo
cyclization were detected (see below) (Figure 2; Scheme 4),
suggesting that the 5-exo cyclization of these peroxyl radicals is
fast enough to compete with hydrogen atom transfer from α-
tocopherol (pseudo-first-order rate constant = kH(α-tocopher-
ol)·[α-tocopherol] = 3.5 × 106 s−1). This large peroxyl radical
5-exo cyclization rate constant is surprising since the rate
constant for the same reaction in an acyclic system was
estimated to be ca. 800 s−1 and the corresponding carbon
radical cyclization is only 4.1 × 105 s−1 at 37 °C.30,31 An
alternative mechanism to be considered for this transformation
is the concerted addition of oxygen to endo-B, leading directly
to the five-membered peroxide-allyl radical (Figure 4), although
a concerted transformation of this type has, to our knowledge,
no precedent. We note that oxygen-radical complex species
have been suggested as intermediates in the oxidation of acyclic
dienols such as linoleic acid, which proceeds via extended chain
pentadienyl radical intermediates.32 An oxygen-radical complex
of the ring constrained endo-B pentadienyl radical would
appear to be well on its way to the product allyl radical.
We suggest that the ketone products observed in the H-9
mechanism were formed via abstraction of the remaining bis-
allylic or allylic hydrogen atom at the α-position of hydro-
peroxide, followed by β-fragmentation of the peroxide bond
(Scheme 4). An alternative pathway could be that the
hydroperoxide was reduced by α-tocopherol to give an alkoxyl
radical, which is followed by elimination of the α-hydrogen
atom. Ketones formation has been observed in free radical
oxidation reactions, either from termination reactions of two
peroxyl radicals via the “Russell mechanism” or from
decomposition of hydroperoxides (via alkoxyl radical),33−35
such as those observed in the oxidation of linoleate.35−37
However, complete conversion of a peroxide to a ketone has
not been previously reported.
The formation of THCEO requires the reduction of the
cyclic peroxide bond, which may be achieved by proton-
coupled electron transfer from the most reducing agent in the
reaction, α-tocopherol, as shown in Scheme 4. We suggest that
even though cyclic peroxides are normally considered stable to
PPh3 and α-tocopherol, the strain of the 5,9-cyclic peroxide and
electron-withdrawing effect of the carbonyl group at C6 may
lead to higher reactivity of this peroxide toward reduction. One
such example is that isoprostanes bicyclic endoperoxides can be
readily reduced by PPh3 to give diols.38 THCEO was found to
be one of the major 7-DHC-derived oxysterols in the brain of
Dhcr7-KO mouse.25 We reported previously that THCEO is a
metabolite of compounds 1 and 2b in Neuro2a and human
fibroblast cells,25 and our results here suggest that THCEO
could form directly from 7-DHC via the H-9 mechanism in the
presence of α-tocopherol.
In the H-14 mechanism, oxygen can be added to three
different positions of the exo-B pentadienyl radical (Scheme 5).
However, after reduction by PPh3, only three products
corresponding to the molecular weight of [7-DHC + 1O]
were observed, possibly because 5-exo cyclization is not favored
for any of these peroxyl radical intermediates (Figure 2). We
speculate that these three products are the hydroxydienols
shown in Scheme 5. The peak eluting at 7.8 min in Figure 2
Scheme 4. H-9 Mechanism of the Free Radical Oxidation of 7-DHC in the Presence of α-Tocopherol (TOH)
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Scheme 5. H-14 Mechanism of the Free Radical Oxidation of 7-DHC in the Presence of α-Tocopherol (TOH)
Figure 5. Proposed radical intermediates formed from cholesta-6,8(9)-dienol (A) or cholesta-6,8(14)-dienol (B) via peroxyl radical addition (kadd
)
or hydrogen atom transfer mechanism (kH). Note that the peroxyl radical addition pathways are expected to be completely suppressed in the
presence of 1.0 M α-tocopherol as observed in the reaction of 7-DHC.
was tentatively assigned to the known compound 4 based on its
HPLC retention time.
formation of the pentadienyl radical endo-B are more reactive
than those leading to the pentadienyl radical exo-B. It is known
that cisoid 1,3-dienes have higher enthalpies than transoid 1,3-
dienes,39 which would imply a smaller activation energy of
hydrogen abstraction from the allylic positions of the cisoid
dienes, thus supporting our results on the reactivities of the
conjugated dienes: 5,7-dienol (7-DHC) > 6,8(9)-dienol ≫
6,8(14)-dienol.
It is unusual to observe that the Ring-B conjugated dienes, 7-
DHC and 6,8(9)-dienol, display significantly higher reactivity
than the dienols bearing a bis-allylic methylene group, 8-DHC
and 5,8(14)-dienol, since the bis-allylic C−H bond normally is
more reactive than the monoallylic C−H bond in acyclic
structures (i.e., in polyunsaturated fatty acids).11 We reported
previously that, in 7-DHC, the torsion angles between the
active C−H bond (at C9 or C14) and the diene plane are close
to 90°, so that the two hydrogen atoms are well aligned for
removal from the α-face of the steroid ring; i.e., maximum
delocalization stabilization is achieved in the hydrogen atom
In free radical oxidation of 6,8(9)-dienol or 6,8(14)-dienol
with or without α-tocopherol, additional products that were not
derived from the H-9 or H-14 mechanism were observed
(Figures S2 and S3). We speculate that these products were
derived from peroxyl radical addition to the double bond (to
give an epoxide; only in the absence of α-tocopherol) or from
hydrogen atom abstraction at the allylic position C-11 (for
6,8(9)-dienol) or C-17 (for 6,8(14)-dienol) (Figure 5).
We found that cholestadienols displayed much higher
reactivity than the acyclic dienes toward free radical oxidation
even though some of them do not possess a bis-allylic
methylene group. Even the least reactive dienol tested, the
6,8(14)-dienol, has a large propagation rate constant of 412
M
−1 s−1, a value that is ca. 40 times that of cholesterol (11 M−1
s−1) and ca. 7 times that of the methylene-skipped acyclic diene,
linoleate (62 M−1 s−1). The overall reactivity trend for the
cholestadienols suggests that the dienols that lead to the
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transfer transition state (Figure S6A).10 Similar molecular
mechanics calculation suggests that, in the 6,8(9)-dienol, the
torsion angle for H5−C5−C6−C7 is 79.2°, also close to 90°
(Figure S6B). In 8-DHC, the torsion angels for the C−H bonds
at C7 are 111.5° (C5−C6−C7−H7β) and 108.6° (H7β−C7−
C8−C9) for Hβ and 133.5° (C5−C6−C7−H7α) and 129.8°
(H7α−C7−C8−C9) for Hα, respectively (Figure S6C).
Although the diene in 8-DHC appears to be in the same
plane, the reactive C−H bonds are not positioned well for
hydrogen atom transfer. In the 5,8(14)-dienol, the diene does
not appear to be in the same plane, which would require
extensive molecular reorientation in order to achieve maximum
delocalization (Figure S6D). Further theoretical investigation
would be worthwhile to elucidate the differences in the
reactivities of these cholestadienols.
Implication in Cholesterol Biosynthesis and Metabo-
lism Diseases. Elevated dienyl cholesterol precursors have
been observed in a number of cholesterol biosynthesis and
metabolism disorders, such as 7-DHC and 8-DHC in
SLOS,40,41 7-DHC in cerebrotendinous xanthomatosis
(CTX),42 8-DHC in X-linked dominant chondrodysplasia
punctata (CDPX2),43 8(9),14-dienol in HEM dysplasia,44
etc.45 The high reactivity of cholestadienols suggests that
oxidative stress should be considered as a factor in disorders
where dienyl sterol intermediates are accumulated. Oxidation of
the reactive sterols would lead to the formation of oxysterols
with diverse structures and functions,8,12,46−50 metabolites that
may ultimately play significant roles in the pathophysiology of
these diseases.
literature, in which the compound was synthesized by a different
method.18
General Procedure for Clocking Experiments of Sterols
Using Methyl Linoleate. The reaction was carried out as previously
reported.16 Briefly, for each series of reactions, the same amount of a
stock solution of methyl linoleate in benzene was added to each
reaction vial, followed by the addition of a stock solution of the specific
sterol, an appropriate amount of benzene, and a stock solution of
MeOAMVN in benzene in order to make the total reaction volume
100 μL. The final concentrations for methyl linoleate, the sterol under
study, and MeOAMVN are 0.302 M, 0.01−0.08 M, and 0.001−0.002
M, respectively. The reaction was carried out at 37 °C for 1 h and was
quenched by the addition of BHT (50 μL; 0.1 M in benzene) and
PPh3 (50 μL; 0.1 M in benzene). The resulting mixture was blown dry
under nitrogen and redissolved in hexanes (500 μL) for the analysis of
HODEs by HPLC-UV as previously described.16 For oxysterol
analysis, the reactions were carried out similarly as described above,
but the resulting reaction mixtures were diluted to 1 mL with benzene
before being analyzed by HPLC-APCI-MS/MS using the method that
was described previously.12
General Procedure for Free Radical Oxidation of Sterols in
the Presence of α-Tocopherol. The oxidation was carried out
similarly as the clocking experiments, but with an additional reaction
component (1.0 M α-tocopherol) and a longer reaction time (2 h).
The reactions were quenched similarly and diluted to 1 mL with
benzene for the analysis of oxysterols using the same HPLC-MS
method.
ASSOCIATED CONTENT
* Supporting Information
■
S
Experimental procedures for the synthesis of 6,8(9)-dienol;
plots of clocking experiments; mass spectra of 2b, DHCDO,
THCEO, and 7-keto-8-DHC; HPLC-MS chromatograms of
oxysterols formed from oxidation of 6,8(9)- and 6,8(14)-
dienols in the absence and presence of α-tocopherol; NMR and
UV spectra of each cholestadienol. This material is available
free of charge via the Internet at http://pubs.acs.org.
SUMMARY
■
In this study, we found that dienes in the rigid cyclic systems
(cholestadienols) display much higher reactivities toward free
radical oxidation than the known acyclic dienyl fatty acids. By
comparing the oxysterol profiles from the oxidation of several
custom-designed cholestadienols with those of 7-DHC, the free
radical oxidation mechanism of 7-DHC was refined, with or
without the presence of α-tocopherol. From the reactions
carried out in the presence of α-tocopherol, evidence was
presented that supports either an unusually fast 5-exo peroxyl
radical cyclization or a concerted addition of molecular oxygen
to a pentadienyl radical, both of which are unprecedented. Also
notable was the complete conversion of a hydroperoxide to a
ketone during the process of free radical oxidation. The
knowledge of the reactivity and oxidation product profiles of
these cholestadienols revealed in this study contributes to the
overall understanding of the pathobiology of related cholesterol
biosynthesis/metabolism disorders.
AUTHOR INFORMATION
Corresponding Author
libin.xu@vanderbilt.edu
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by grants from the National Institute
of Health (K99HD073270 (L.X.), R01HD064727 (N.A.P.),
and NIH P30 ES000267 (Vanderbilt Center for Molecular
Toxicology)) and National Science Foundation (CHE-1057500
(N.A.P.)). We thank Frances Ding for the purification of
cholesta-6,8(9)-dienol by HPLC.
EXPERIMENTAL SECTION
■
REFERENCES
■
General Methods and Materials. The initiator, MeOAMVN, was
purchased from Wako Chemicals, dried under vacuum, and then
stored at −40 °C. 7-Dehydrocholesterol (>98%), tert-butyldimethyl-
silyl chloride, borane·THF (1.0 M in THF), and methanesulfonyl
chloride were purchased from Sigma-Aldrich Co. and were used
without further purification. Methyl linoleate (Nu-Chek-Prep, Inc.)
was purified on silica gel prior to use (10% ethyl acetate in hexane)
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HPLC grade hexanes and 2-propanol were purchased from Thermo
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synthesized following previously reported procedures.17−19 The
synthesis of 6,8(9)-dienol was described in the Supporting
Information, and the NMR spectra matched those reported in the
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