Tunneling in tocopherol-mediated peroxidation of 7-dehydrocholesterol

Article abstract of DOI:10.1039/c4ob02377c

The peroxidation of 7-dehydrocholesterol (7-DHC), a biosynthetic precursor to vitamin D₃ and cholesterol, has been linked to the pathophysiology of Smith-Lemli-Optiz syndrome (SLOS), a devastating human disorder. In SLOS, 7-DHC plasma and tissue levels are elevated because of defects in the enzyme that convert it to cholesterol. α-Tocopherol can mediate the peroxidation of 7-DHC under certain circumstances and this prompted us to investigate the kinetic isotope effect (KIE) during this process. Thus, 9,14-d₂-7-DHC was synthesized using a photochemical cyclization of deuterium-reinforced previtamin D₃ (retro to its biosynthesis). Subsequently, we carried out co-oxidation of 9,14-h₂-25,26,26,26,27,27,27-d₇- and 9,14-d₂-7-DHC in the presence of α-tocopherol under conditions that favor TMP. By monitoring the products formed from each precursor using mass spectrometry, the KIE for the hydrogen (deuterium) atom removal at C9 was found to be 21 ± 1. This large KIE value indicates that tunneling plays a role in the hydrogen atom transfer step in the tocopherol-mediated peroxidation of 7-DHC.

Full text of DOI:10.1039/c4ob02377c

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Biomolecular Chemistry
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Tunneling in tocopherol-mediated peroxidation
of 7-dehydrocholesterol†
Cite this: Org. Biomol. Chem., 2015,
13, 1249
H. Muchalski, L. Xu and N. A. Porter*
The peroxidation of 7-dehydrocholesterol (7-DHC), a biosynthetic precursor to vitamin D3 and choles-
terol, has been linked to the pathophysiology of Smith–Lemli–Optiz syndrome (SLOS), a devastating
human disorder. In SLOS, 7-DHC plasma and tissue levels are elevated because of defects in the enzyme
that convert it to cholesterol. α-Tocopherol can mediate the peroxidation of 7-DHC under certain cir-
cumstances and this prompted us to investigate the kinetic isotope effect (KIE) during this process. Thus,
9,14-d2-7-DHC was synthesized using a photochemical cyclization of deuterium-reinforced previtamin
D3 (retro to its biosynthesis). Subsequently, we carried out co-oxidation of 9,14-h2-25,26,26,26,27,27,27-
d7- and 9,14-d2-7-DHC in the presence of α-tocopherol under conditions that favor TMP. By monitoring
the products formed from each precursor using mass spectrometry, the KIE for the hydrogen (deuterium)
atom removal at C9 was found to be 21 1. This large KIE value indicates that tunneling plays a role in the
hydrogen atom transfer step in the tocopherol-mediated peroxidation of 7-DHC.
Received 10th November 2014,
Accepted 14th November 2014
DOI: 10.1039/c4ob02377c
www.rsc.org/obc
Introduction
Polyunsaturated fatty acids and esters undergo free radical
chain oxidation readily and this process, lipid peroxidation,
and the toxic products derived from it have been the subject of
intense scrutiny in recent years.1 Arachidonic acid is particu-
larly prone to radical chain oxidation and the products of per-
oxidation of this fatty acid and its esters have been used
extensively as biomarkers for oxidative stress in vivo.2 There is
a growing consensus that lipid peroxidation and oxidative
stress play an important role in the pathophysiology of a
number of human disorders.3
The rate-limiting step in radical chain oxidation is H-atom
transfer to a propagating peroxyl radical, eqn (1) in Fig. 1, and
polyunsaturated fatty acids are indeed highly reactive, the
rate constant for propagation, kp, of arachidonic acid being
197 M−1 s−1. It is therefore of some interest that the sterol
7-dehydrocholesterol (7-DHC) has a kp of 2260 M−1 s−1, a value
that is higher than the propagation rate constant measured for
any other lipid, making it a likely target for chain-carrying
peroxyl radicals.4
Fig. 1 Free radical peroxidation, inhibition and tocopherol-mediated
chain propagation.
7-DHC serves as the biosynthetic precursor to cholesterol
(kp = 11 M−1 s−1) and it plays a central role in one of the most
common autosomal recessive human disorders, Smith–Lemli–
Opitz syndrome (SLOS).5 SLOS is characterized by excessive
plasma and tissue levels of 7-DHC and lower than normal
levels of cholesterol, a consequence of mutations in the
enzyme 7-dehydrocholesterol reductase (DHCR7) that converts
7-DHC to cholesterol.5,6 A link between the pathology of SLOS
and the peroxidation of 7-DHC has been made with the discov-
ery that oxysterols derived from 7-DHC are found in tissues
Department of Chemistry, Vanderbilt University, 7330 Stevenson Center,
Station B 351822, Nashville, TN 37235, USA. E-mail: n.porter@Vanderbilt.Edu
†Electronic supplementary information (ESI) available: Full experimental details
including synthetic and analytical procedures, characterization of all new com-
pounds, and additional supplementary data can be found in the ESI. See DOI:
10.1039/c4ob02377c
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and fluids of SLOS mouse models and in skin fibroblasts and
plasma of SLOS patients.7 The fact that many of the 7-DHC-
derived oxysterols have potent biological activity provides
further support for the notion that SLOS has a significant oxi-
dative stress component.8
The free radical oxidation of 7-DHC is a complex process,
with over a dozen products formed in the azo-initiated solu-
tion oxidation of the lipid.9 Mechanistic studies suggest that
the hydrogen atoms at C9 and C14 are the reactive atoms.9 Co-
oxidation of 7-DHC in the presence of Nature’s major chain-
breaking antioxidant, α-tocopherol (kinh = 3.5 × 106 M−1 s−1),10
affects not only the rate of peroxidation but also the distri-
bution of products formed during the process.11 In fact, the
product mixture is greatly simplified if peroxidation of 7-DHC
is initiated in the presence of the antioxidant.11
Under certain circumstances, α-tocopherol can mediate the
peroxidation of reactive lipids like 7-DHC with the tocopheryl
radical abstracting a hydrogen atom from the reactive lipid,
see eqn (4) in Fig. 1.12 This process, tocopherol mediated peroxi-
dation (TMP), likely becomes a major propagation pathway
when radical intermediates are isolated in cellular organelles
or lipid particles such as low-density lipoproteins.12,13 Because
Scheme 2 Synthesis of ring A fragment 2: (a) ref. 16d; (b) ref. 16e; (c).
H2O2, K2CO3, H2O–MeOH (3 : 1), −20 °C, 1 h, 48% (d.r. ≥ 19 : 1 +16% of
the minor isomer); (d) (PhSe)2, NaBH4, 2-propanol, AcOH, 0 °C, 7,
15 min, 88%; (e) TBSCl, imidazole, DMF, 0 °C to rt, 12 h, 96%; (f) tri-
methylsilylacetylene, n-BuLi, THF, −78 °C, 30 min, 9, −78 °C, 3 h, 99%;
(g) Martin’s sulfurane dehydrating agent, CH2Cl2, rt, 2 h, 82% (d.r. =
4.5 : 1); (h) TBAF, THF, 0 °C to rt, 6 h, 56% (d.r. ≥ 19 : 1).
of the biological relevance of TMP of 7-DHC and the impor- co-workers determined that the quantum yield for ring closure
tance of this sterol in a devastating human syndrome, we have increases with wavelength applied (0.08 at 325 nm).15
focused our attention on the mechanism of this transform- However, the photoequilibrium is heavily in favor of previta-
ation. We report here that H-atom tunneling facilitates the min D3 at all wavelengths and the amount of 7-DHC present is
propagation step in this process.
very small. For this reason we developed a gram scale synthesis
of previtamin D3 with deuterium at C-9 and C-14 (Scheme 1).
Synthesis of previtamin D3 (1) is generally approached by
coupling of a CD-ring fragment derived from Grundmann’s
ketone with an appropriate ring A fragment. Okamura and co-
workers used this strategy to prepare several deuterated ana-
logues of previtamin D to study the kinetic isotope effects in
thermal [1,7]-sigmatropic shift.16 The synthesis of alkynol 2
with a deuterated methyl group was part of that work but the
introduction of CD3 group and late stage resolution made the
overall synthesis lengthy and not suitable for gram scale
needs. We prepared alkynol 2 from (+)-carvone as shown in
Scheme 2.†
With both cross-coupling partners in hand, standard Sono-
gashira conditions afforded the conjugated alkyne 12 which
was immediately reduced to a deutero-previtamin D3 (1–d2)
using deactivated Pd/CaCO3-quinoline semireduction protocol
(Scheme 3). Once appreciable amounts of the deuterated previ-
tamin D3 were secured, the construction of ring B of 9,14-d2-7-
DHC was achieved via a 6π conrotatory photocyclization of
1-d2. For practical reasons we employed a Hanovia 450 W
medium pressure mercury lamp equipped with a Vycor® filter
to cut off high energy UV and minimize formation of un-
desired isomers.17 A solution of 1-d2 in ethanol–hexanes was
irradiated at 0 °C for 50 min. Preparative HPLC separation of
the product mixture afforded 7-DHC-d2 in 4% yield.†
Results and discussion
To study the key atom-transfer step of TMP of 7-DHC, we
designed and synthesized deuterium-reinforced 7-DHC at the
reactive C9 and C14, 7-DHC-d2. Efforts to synthesize 7-DHC-d2
starting from 7-DHC proved unsuccessful; a bromination-
radical reduction strategy led to undesired diene products with
rearranged double bonds. We successfully achieved the syn-
thesis, however, using a photochemical reaction that is essen-
tially a retro-synthesis of previtamin D3 as shown in Scheme 1.
Thus, the construction of ring B utilizes a 6π conrotatory
photochemical cyclization of deutero-previtamin D3 1-d2
(Scheme 3). It is the reverse of the reaction by which vitamin
D3 is produced biosynthetically and industrially.14 Dauben and
TMP of 7-DHC occurs by abstraction of hydrogen (eqn (4) in
Fig. 1) at C9 or C14 of the lipid. Reaction at C9 proceeds via
the intermediate radical endo-B, see Scheme 4, and gives
7-keto-8-DHC and the two ketones, 3β,5α,9α-trihydroxy-cholest-
Scheme 1 Retrosynthesis of 7-DHC-d2.
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DHCDO.13b It is of some interest that these oxysterols have
been found in brain tissue of SLOS mouse models.18 Products
derived from H-atom removal at C14 of 7-DHC are formed via
the intermediate radical exo-B.11 Conjugated and non-conju-
gated diene hydroperoxides are formed, one of which, 14-
OOH-7-DHC, is shown in Scheme 4. This hydroperoxide is still
a highly oxidizable substrate, with a reactive hydrogen atom
remaining at C9 of the molecule.
To study the kinetic isotope effect (KIE) of TMP of 7-DHC, a
mixture having a defined ratio of 7-DHC-d7 and 7-DHC-d2,19
was oxidized in benzene in the presence of 0.9 M α-tocopherol
at 37 °C using 2,2′-azobis(4-methoxy-2,4-dimethylvaleronitrile
(MeOAMVN) as the radical initiator. The concentration of the
7-DHC-d2 was kept in large excess due to its resistance to oxi-
dation so that comparable levels of oxidation products from
both 7-DHC substrates were formed. A time-course study
suggested that 8 h of oxidation gave significant more products
(>10 times) than those at t = 0 while the consumption of the
starting sterols was still low. These conditions were chosen for
subsequent studies.†
Scheme 3 Synthesis of 7-DHC-d2: (a) Pd(OAc)2, PPh3, Et2NH, CuI, DMF,
rt, 30 min, degas (freeze–pump–thaw, 3 cycles) add 2 and 3-d2, DMF,
1 h in the dark, aqueous work-up; (b) Pd/CaCO3, quinoline, H2, EtOAc–
hexanes (3 : 7), 52%, 2 steps; (c) Medium pressure Hg lamp with a Vycor
filter, EtOH–hexanes (2 : 8 v/v, degassed), 50 min, 4% (+unreacted 1-d2
+ isomers).
An HPLC-APCI-MS chromatogram of the product mixture
formed from oxidation of a 10 : 1 mixture of 7-DHC-d2 and
7-DHC-d7 is shown in Fig. 2B (see Fig. S18 in ESI† for full chro-
matogram). By monitoring the ratio of the isotopically differen-
tiated products 7-keto-8-DHC-d7 and 7-keto-8-DHC-d1 and
knowing the ratio of their respective precursors, the kinetic
isotope effect for the removal of H(D)-9 could be determined.
The parent ion for 7-keto-8-DHC undergoes fragmentation
Scheme 4 Mechanistic pathway for the formation of 7-keto-8-DHC,
THCEO and DHCDO.
7-en-6-one (THCEO) and 3β,5α-dihydroxy-cholesta-7,9(11)-dien-
6-one (DHCDO) as shown in the Scheme.11 Likely intermedi-
ates in the formation of these products are a non-conjugated
hydroperoxide 13 formed by reaction of oxygen at the center
carbon of endo-B and the cyclic peroxide-hydroperoxide 14
that results from coupling with oxygen at either terminus of
endo-B. Dehydration and reduction of these intermediates
Fig. 2 Co-oxidation of deuterium-substituted 7-DHC. (A) Conditions of
oxidation and structures of isotopically labeled 7-DHCs. (B) Chromato-
gram of oxidation products obtained by normal phase HPLC-MS-MS
leads to 7-keto-8-DHC and THCEO, which dehydrates to give using selected reaction monitoring (SRM) (see ESI† for details).
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with loss of water and for the d7 compound this is observed at
m/z 406→388 while for the d1 product the transition is found
Experimental
at m/z 400→382. We conclude from these studies that kH/kD for In four screw cap vials containing solution of 7-DHC-d2,
removal of the hydrogen at C9 of 7-DHC by a tocopherol 7-DHC-d7 (ca. 10 : 1 ratio, 0.03 M), and α-tocopherol (0.9 M) in
radical is 21 1 (Table S3 in ESI†). Similar HPLC-MS analyses benzene (150 μL) was added the MeOAMVN initiator (10 μL,
were carried out on the d1- and d7-products of THCEO and 0.03 M). To the first vial was added PPh3 (0.5 M in benzene,
DHCDO and the KIE values of 22 1 and 29 4 were deter- 20 μL) and BHT (0.5 M in benzene, 20 μL) and benzene
mined, respectively. We note in passing that the KIE values (300 μL) and the vial was transferred to −80 °C freezer for
obtained from 7-keto-8-DHC (21 1) and THCEO (22 1) are storage. Three remaining vials were capped and incubated at
more reliable than that determined from DHCDO (29
4) 37 °C for 8 h and then was added PPh3 (0.5 M in benzene,
since the latter compound is a minor constituent of the pro- 20 μL) and BHT (0.5 M in benzene, 20 μL) and benzene
ducts formed from C9 H(D) abstraction (see Tables S4, S5, and (300 μL). A small fraction of each reaction was directly injected
Fig. S18†).
into MS via syringe pump at a flow rate of 20 μL min−1 (with a
Another significant observation in this study was a large make up flow of 1.0 mL min−1 by the HPLC) to obtain the ratio
KIE observed even in the t = 0 sample. The mixture of d2- and of the 7-DHC-d2 and 7-DHC-d7 by comparing the intensity of
d7-7-DHC was collected directly into a flask containing α-toco- m/z at 369 and 390, respectively. From each vial, 200 μL was
pherol which was kept at −78 °C but oxidation products were taken for LCMS analysis. HPLC-APCI-MS-MS analysis was
still observed after thawing and quenching the solutions with carried out similarly to the previously reported method for
triphenylphosphine. By analyzing the ratio of d1/d7-7-keto-8- 7-DHC-derived oxysterols with modification of the masses being
DHC in the t = 0 sample, a KIE of 20 was determined, similar monitored.7a,9,18 For example, for 7-DHC-d7-derived oxysterols,
to the value observed at 37 °C. This observation suggests that masses with 7 additional mass units relative to the non-deuter-
temperature does not have significant effect on the KIE of the ated oxysterols were monitored; for 7-DHC-d2-derived oxyster-
H-atom transfer from 7-DHC to the tocopheryl radical.
ols (giving d1-oxysterols after losing D-9 or D-14), masses with
The product distribution of 14-OOH-7-DHC and the regio- one additional mass unit were monitored. In general, selective
isomers formed from removal of H(D) at C14 (formation of reaction monitoring (SRM) was employed to monitor the dehy-
radical exo-B) was affected by isotopic substitution. The hydro- dration process of the ion [M + H]+ or [M + H − H2O]+ in the
peroxide intermediate, 14-OOH-7-DHC, retains
a reactive mass spectrometry. HPLC conditions (on Waters Alliance
C9 hydrogen atom and is still a highly oxidizable compound. 2695): 3 μm 150 × 4.6 mm silica column (Phenomenex, Inc.);
Subsequent free radical oxidation would be subject to a 10% 2-propanol in hexanes; 1.0 mL min−1. MS conditions (on
second KIE, preferentially removing 14-OOH-7-DHC-d7 from Thermo Finnigan TSQ Quantum Ultra): discharge current,
the product mixture since it bears an H at C9 while 14-OOH-7- 10 μA; sheath gas pressure, 20 mTorr; ion sweep gas pressure,
DHC-d1 that has D at C9 would be resistant to further oxi- 2 mTorr; auxiliary gas pressure, 15 mTorr; tube lens, 92 V;
dation, thus accounting for the product-directing effect skimmer offset, 6 V; collision pressure, 1.50 mTorr; collision
observed. This complication in reaction mechanism (i.e., sub- energy, 13 V; vaporizer temperature: 300 °C.
sequent H-atom transfer at C9) prevented determination of the
KIE of H/D transfer from C14. One can only speculate that a
large KIE for H(D) atom removal from C14 would be found
Conclusions
since the torsion angles between the reactive C-H bonds and
To summarize, this work is a first study of the KIE in TMP of a
the diene plane are similar for C9 and C14 (92.3° and 99.4°,
reactive sterol. The KIE obtained for the H-9 transfer is signifi-
respectively).11
cantly larger than classical KIE values reported for hydrogen
Historically, KIE values reported for the autoxidation of
atom transfer in peroxidation (typically <7).20,23 This result
hydrocarbons are less than 7, such as those reported for diphe-
suggests that tunneling is important in the H-transfer to a
nylmethane and cumene by Ingold and Russell,20 respectively.
tocopheryl radical in the TMP reaction, a conclusion that is
The large KIEs observed in this study (>20) suggest that tunnel-
consistent with our earlier finding in polyunsaturated fatty
ing is involved in the H-atom transfer from 7-DHC to toco-
acid peroxidations.13b As TMP is thought to play an important
pheryl radical. While tunneling is not uncommon in H-atom
role in the peroxidation of low-density lipoprotein (LDL) and
transfer processes catalyzed by enzymes, such as lipoxygen-
sterol esters, including those from 7-DHC, are major constitu-
ase,21 the phenomenon has not been observed in solution-
ents of LDL, our study suggests that tunneling contributes to
based inhibited lipid peroxidation reactions until our recent
the peroxidation of 7-DHC and hence to the pathophysiology
report on TMP of polyunsaturated fatty.13b Tunneling has also
associated with Smith–Lemli–Opitz syndrome in humans.
been suggested to play a role in regeneration of tocopherol
form tocopheryl radical by ubiquinol.22 These earlier studies
and our current report suggest that tunneling may be a
common phenomenon for H-atom transfer to tocopheryl
Acknowledgements
radical, irrespective of the H-atom donor being fatty acids, This work was supported by grants from the National Science
a sterol, or another phenolic antioxidant.
Foundation NSF (CHE-1057500 (N.A.P.)) and the National
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Institute of Health (R01 HD064727 (N.A.P.) and K99 HD073270
(L.X.)). We thank Alexander J. Levonyak for assistance with the
synthesis of Grundmann’s ketone.
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