Solvent effects in hydrogen abstraction from cholesterol by benzophenone triplet excited state

Article abstract of DOI:10.1021/ol2016059

Hydrogen abstraction from the C-7 position of cholesterol (Ch) by triplet excited benzophenone (BZP) exhibits remarkable solvent-dependence in product studies. Kinetic measurements on the intramolecular version of the process in dyads containing covalently linked Ch and BZP units reveal important solvent effects and significant stereodifferentiation.

Full text of DOI:10.1021/ol2016059

ORGANIC
LETTERS
2011
Vol. 13, No. 15
4096–4099
Solvent Effects in Hydrogen Abstraction
from Cholesterol by Benzophenone Triplet
Excited State
Inmaculada Andreu, Fabrizio Palumbo, Fedele Tilocca, Isabel M. Morera,
ꢀ
Francisco Bosca,* and Miguel A. Miranda*
´
Instituto de Tecnologıa Quımica UPV-CSIC, Universidad Politecnica de Valencia,
Consejo Superior de Investigaciones Cientıficas, Avda. de Los Naranjos s/n,
´
ꢀ
´
46022 Valencia, Spain
fbosca@itq.upv.es; mmiranda@qim.upv.es
Received June 15, 2011
ABSTRACT
Hydrogen abstraction from the C-7 position of cholesterol (Ch) by triplet excited benzophenone (BZP) exhibits remarkable solvent-dependence in
product studies. Kinetic measurements on the intramolecular version of the process in dyads containing covalently linked Ch and BZP units
reveal important solvent effects and significant stereodifferentiation.
Oxidative activation of cholesterol (Ch) at the C-7
position is a process of considerable chemical and biologi-
cal interest. Thus, the major pathway for the synthesis of
bile acids from Ch is initiated by C-7 hydroxylation, which
is achieved by the 7R-hydroxylase (CYP7A1), a member of
the P450 family of metabolic enzymes.1 It has also been
claimed that C-7 oxidation is involved in the interception
of blood and plasma oxidants by Ch, which thereby may
act as an effective in vivo antioxidant.2 In addition, Ch is a
major target for oxidative damage.3,4 This process can
occur by a Type I mechanism (via free radicals) through
abstraction of an allylic hydrogen from Ch by photosensi-
tizing agents upon UVA light activation.5
Despite the importance of Type I Ch photooxidation,
the key hydrogen abstraction step has not been previously
subjected to mechanistic studies. In this context, we wish
now to report on the photoreaction between the benzo-
phenone (BZP) triplet excited state and Ch, a paradigmatic
process of general interest.
Cholesterol is an essential structural component of cell
membranes, where it is required to achieve proper perme-
ability and fluidity. Inthe body, Chcan alsobe found in the
liver or intestines, as well as in the bloodstream, where it is
transported within lipoproteins. In addition, Ch is present
(1) Post, S. M.; Twisk, J.; Van Der Fits, L.; De Wit, E. C. M.;
Hoekman, F. M.; Mager, W. H.; Princen, H. M. G. Biochem. J. 1999,
341, 339–346.
(2) Smith, L. L. Free Radical Biol. Med. 1991, 11, 47–61.
(3) Lercker, G.; Rodriguez-Estrada, T. In Cholesterol and Phytoster-
ol Oxidation Products; Guardiola, F., Dutta, P. C., Codony, R., Savage, G. P.,
Eds.; AOCS Press: Champaign, IL, 2002; Chapter 1, pp 1ꢀ25.
(4) (a) Girotti, A. W. Antioxid. Redox Signal 2004, 6, 301–310. (b)
Girotti, A. W. J. Photochem. Photobiol., B 1992, 13, 105–118. (c) Foote,
C. S. Free Radicals in Biology; Academic Press: New York, 1976; Vol. 2, pp
85ꢀ134. (d) Girotti, A. W; Korytowski, W. Methods Enzymol. 2000,
319, 85–100.
ꢀ
(5) (a) Andreu, I.; Bosca, F.; Sanchez, L.; Morera, I. M.; Camps, P.;
Miranda, M. A. Org. Lett. 2006, 8, 4597–4600. (b) Andreu, I.; Morera,
ꢀ
I. M.; Bosca, F.; Sanchez, L.; Camps, P.; Miranda, M. A. Org. Biomol.
Chem. 2008, 6, 860–867.
r
10.1021/ol2016059
Published on Web 07/11/2011
2011 American Chemical Society

in all foods containing animal fat. Hence, it makes sense to
investigate the generation of Ch-derived radicals in lipo-
philic media of different characteristics. In this connection,
a recent discussion has arisen on whether hydrogen ab-
straction from carbon is subjected to significant solvent
effects. Specifically, controversial results have been re-
ported on the solvent-dependence of H-abstraction reac-
tivity from CꢀH donors such as 1,4-dienes by cumyloxyl
radical.6
With this background, the present work deals with the
solvent effects on abstraction of the Ch C-7 allylic hydro-
gens by the triplet excited state of BZP. This issue has been
addressed by performing both steady-state and time-re-
solved studies in different organic solvents.
To investigate photoproducts formation, steady-state
photolysis of BZP/Ch mixtures (1.5:1 molar ratio) was
performed under anaerobic conditions, in dichloro-
methane and acetonitrile, using a multilamp photoreactor
equipped with UVB-lamps (λmax = 300 nm, Gaussian
distribution). In dichloromethane, no Ch-derived product
was obtained, whereas in acetonitrile two diastereomeric
photoproducts 1 and 2 were isolated.
(see Figure 1, insert). The quenching rate constant was
s
ꢀ1. However, under these
found to be 2.2 ꢁ 108 Mꢀ1
conditions extensive H-abstraction from the solvent was
observed. This is clear from the comparison of the long-
lived component in the kinetic traces at 545 nm (ascribed
to the ketyl radical formed by hydrogen abstraction from
the solvent) obtained in the presence and in the absence of
Ch (Figure 1). In acetonitrile quenching was faster, but
the kinetic parameters could not be accurately deter-
mined due to solubility limitations; nonetheless, the rate
constant, estimated from the initial slope of the nonlinear
plot (not shown), was ca. 1 order of magnitude higher.
Similar solubility problems were found in other organic
solvents such as methanol or dioxane, which in addition
are better hydrogen donors than acetonitrile. Thus, the
major difficulties in obtaining reliable data for the inter-
molecular photoreaction between Ch and BZP are related
to the low solubility of Ch and the possibility of hydrogen
abstration from the solvent by triplet BZP. To circum-
vent these problems, a possible strategy could be to
examine the intramolecular version of the process, using
covalently linked Ch and BZP units. This would prevent
the need for high quencher concentrations and should
result in a dramatic decrase of the triplet lifetimes, mini-
mizing hydrogen abstraction from the solvent. Moreover,
the covalent link between the Ch and BZP units
would completely block radical cage escape, favoring
CꢀC coupling.
Scheme 1. Formation of Photoproducts 1 and 2 upon Photolysis
of Ch and BZP in Acetonitrile
As shown in Scheme 1, formation of 1 and 2 has to be
explained by intermolecular hydrogen abstraction from
the C-7 position of Ch, followed by CꢀC coupling of the
generated radical pair. The structures of the photopro-
ducts were unambiguously assigned on the basis of their
NMR spectroscopic data (1H, 13C). The stereochemistry of
the newchiralcenterwas determinedby NOE experiments;
the most significant interactions were observed between
the phenyl groups and the protons of the convex β-face in
photoproduct 2. The details are given in the Supporting
Information.
Figure 1. Trace obtained at 545 nm upon laser excitation of BZP
in dichloromethane solutions in the absence (red) and in the
presence of Ch (black, 0.02 M). Inset: SternꢀVolmer plot
showing the effect of Ch addition on the rates of BZP triplet
decay at 610 nm.
Following this idea, diasteromeric dyads, 3a and 3b
(Scheme 2), were submitted to laser flash photolysis
(λexc = 355 nm) under a variety of conditions, in order
to determine the kinetic parameters and the quantum
yields of the relevant processes.
We have previously demonstrated that the transient
absorption spectra of 3a and 3b in dichloromethane do
not correspond to the triplet excited states but instead to
the corresponding biradicals, generated by intramolecular
hydrogen abstraction. Subsequent cyclization gives rise to
CꢀC coupling photoproducts in a diastereoselective
The reaction kinetics was studied by laser flash photo-
lysis experiments at 355 nm. Thus, when the reciprocal
triplet lifetime was plotted against Ch concentration a
linear relationship was found in dichloromethane as solvent
(6) (a) Koner, A. L.; Pischel, U.; Nau, W. M. Org. Lett. 2007, 98,
2899–2902. (b) Bietti, M.; Salamone, M. Org. Lett. 2010, 12, 3654–3657.
(c) El-Sheshtawy, H. S.; Pischel, U.; Nau, W. M. Org. Lett. 2011, 13,
2694–2697. (d) Salamone, M.; Giammarioli, I.; Bietti, M. J. Org. Chem.
2011, 76, 4645–4651.
Org. Lett., Vol. 13, No. 15, 2011
4097

manner.5 Here, the transient absorption spectra of 3a,b
were recorded in different solvents (dichloromethane,
acetonitrile, dioxane, methanol, and octanol) 50 ns after
the 355 nm laser pulse. In all cases, the transients displayed
similar spectral features.
the transient absorbance at 545 nm immediately after the
laser pulse. This was achieved using BZP as standard and
assuming the same molar absorption coefficient for the
biradicals and for the longer-lived BZP ketyl radical
formed by intermolecular hydrogen abstraction from the
solvent. It is interesting to note that, even in good hydro-
gen-donating solvents (dioxane, methanol), the intra-
molecular process (φBR = 0.65ꢀ0.73) was by far more
Scheme 2. Structures of 3aꢀc and Products Resulting from
Irradiation of Dyads 3a and 3b
efficient than its intermolecular counterpart (φKR
=
0.05ꢀ0.14). As expected, all radicals arising from hydro-
gen abstraction reactions were quenched by oxygen with
.
rate constants in the range of (1.5ꢀ5.0) ꢁ 109 Mꢀ1 sꢀ1
As an example, Figure 2 shows the spectrum of dyad 3a
in methanol (top), which corresponds essentially to the
biradical, with a lifetime in the submicrosecond time scale.
For comparison, the results obtained for 3c, which also
contains the Ch and BZP units but cannot adopt the folded
conformation due to its 3β configuration, are shown in
Figure 2 (bottom). In the latter, the typical BZP tripletꢀ
triplet absorption was initially observed; the ketyl radical
formed by intermolecular H-abstraction from methanol
was found to develop at a later stage and did not decay
within several microseconds.
As a consequence of the spectral overlap of triplets and
biradicals and because of the short triplet lifetimes, it was
not possible to achieve direct observation of the TꢀT
absorption of dyads 3a and 3b in different media for reliable
kinetic measurements. Hence, further experiments were
performed to obtain accurate values for the triplet lifetimes
(τT) by the well-established energy transfer method, using
naphthalene as the acceptor (see Supporting Information).7,8
From these data the intramolecular quenching rate con-
stants (kiq) were determined; the values (ranging between
2.3 ꢁ 107 and 1.0 ꢁ 108 Mꢀ1 sꢀ1) are given in Table 1.
Direct kinetic analysis of the decay traces at 545 nm led
to determination of biradical lifetimes (τBR). The quantum
yields of biradical formation (φBR) were estimated by the
comparative method,9 from the short-lived component of
Figure 2. Transient absorption spectrum of dyad 3a (top) and 3c
(bottom) under N2 in methanol at different times after the laser
pulse (λexc = 355 nm).
After establishing the values of kiq and φBR, the rate
constants for intramolecular hydrogen abstraction (kH)
and physical quenching (kπ) were determined in all sol-
vents taking into account that kH = kiq ꢁ φBR and kiq =
kH þ kπ. The obtained results are summarized in Table 1.
Although deriving clear-cut correlations between the kH
values and the solvent properties was not straightforward,
important solvent effects and significant stereodifferentia-
tion were indeed noticed. The observed trends serve as
examples for the presently debated existence of solvent
effects on CH hydrogen abstractions. Thus, a much faster
process (ca. 4ꢁ) occurred in dichloromethane than in
dioxane or methanol, whereas an intermediate situation
was found for acetonitrile. In most solvents, a significant
stereodifferentiation was also observed, with higher kH
values for 3b than for 3a.
(7) Martı
11066–11070.
(8) (a) Miranda, M. A.; Martı
´
nez, L. J.; Scaiano, J. C. J. Am. Chem. Soc. 1997, 119,
ꢀ
nez, L. A.; Samadi, A.; Bosca, F.;
´
ꢀ
Morera, I. M. Chem. Commun. 2002, 280–281. (b) Bosca, F.; Andreu, I.;
Morera, I. M.; Samadi, A.; Miranda, M. A. Chem. Commun. 2003, 1592–
1593.
(9) Carmichael, I.; Gordon, L. H. J. Phys. Chem. Ref. Data 1986, 15,
1–250.
4098
Org. Lett., Vol. 13, No. 15, 2011

Table 1. Photophysical and Photochemical Parameters of Triplet Excited States of Dyads 3a and 3b in Different Solventsa
e
e
f
τT (μs)
kiq/106 (sꢀ1 c
)
kH/106 (sꢀ1 d
)
kπ/106 (sꢀ1 d
)
φBR
τBRd (μs)
φKR
φPR
b
3a
3a
3a
3a
3b
3b
3b
3b
CH2Cl2
0.010
0.025
0.030
0.030
0.010
0.020
0.020
0.020
100
33
80
26
19
20
80
37
33
31
20
7
0.80
0.80
0.70
0.69
0.80
0.75
0.73
0.65
0.28
0.60
0.66
0.80
0.22
0.56
0.62
1.10
0.00
0.00
0.14
0.05
0.00
0.00
0.13
0.05
0.30
0.70
0.76
0.76
0.47
0.70
0.76
0.64
CH3CN
Dioxane
CH3OH
23
4
28
8
b
CH2Cl2
100
50
20
13
6
CH3CN
Dioxane
CH3OH
39
45
14
a Relative errors lower than 5% of the stated values. b Data reported in part in ref 5. c The intramolecular quenching rate constants were obtained by
using the equation kiq = 1/τι ꢀ 1/τ0, where τi are the triplet lifetimes of compounds 3a and 3b and τ0 is the BZP triplet lifetime in the different solvents.
d The rate constants for hydrogen abstraction and physical quenching by the π system were obtained using the following equations: kH = kiq ꢁ φBR and
kiq = kH þ kπ. e The biradical quantum yields were determined by the comparative method, using BZP as standard and assuming the same molar
absorption for the biradicals and the BZP ketyl radical.9 f Photodegradation quantum yield was determined using N-methyldiphenylamine as
actinometer.10
To investigate the influence of solvents on the overall
photoreduction process, solutions of dyads 3a and 3b in
dichloromethane, acetonitrile, dioxane, and methanol
were photolyzed under identical conditions, and the
disappearance of the carbonyl chromophore with irradia-
tion time was monitored by the progressive decrease
of the 254 nm absorption band. Some representative
results are shown in Figure 3. The photoreduction quan-
tum yields were determined using N-methyldiphenylamine
as actinometer10 (see Table 1). Again, important solvent
effects and significant stereodifferentiation were observed.
Noteworthy, the less efficient photoreaction was found for
dichloromethane, in spite of the fact that the highest kiq, φBR
Figure 3. Photoreduction of dyads 3a and 3b in different deaer-
ated media as a function of the irradiation time. The percentage
of unreacted compound was monitored by UV-absorption
spectroscopy at 254 nm. Green: 3a in CH2Cl2. Blue: 3b in
CH2Cl2. Orange: 3a in MeOH. Magenta: 3b in MeOH.
,
and kH values were measured in this solvent. Actually,
biradical formation does not necessarily lead to the CꢀC
coupling products. This is because biradicals can also follow
alternative decay pathways, for instance back oxygen-to-
carbon hydrogen transfer to regenerate the starting materi-
als, a process that would be retarded by stabilization of the
1-hydroxy-biradicals via hydrogen bonding to the solvents.
In summary, hydrogen abstraction from the C-7 posi-
tion of Ch by triplet excited BZP is a process of consider-
able chemical and biological interest, which exhibits a
remarkable solvent-dependence. In the intermolecular ver-
sion, a striking difference is observed in product studies
between dichloromethane and acetonitrile, although ki-
netic effects are difficult to evaluate due to solubility
limitations. By contrast, covalent linking of the Ch and
BZP units in dyads 3aand 3b has made it possible to obtain
a complete set of kinetic measurements under different
conditions. This has revealed important solvent effects and
significant stereodifferentiation in most of the involved
processes and particularly in the rate constant of intra-
molecular hydrogen abstraction.
Acknowledgment. Financial support from the MI-
CINN (Grants CTQ2009-13699 and CTQ2010-19909),
from the Generalitat Valenciana (GV/2009/104) and
from Carlos III Institute of Health (Grant RIRAAF,
RETICS program) is gratefully acknowledged. Dedi-
ꢀ
cated to Prof. Dr. Carmen Najera on the occasion of her
60th birthday.
Supporting Information Available. Spectroscopic data
and experimental details. This material is available free of
charge via the Internet at http://pubs.acs.org.
€
(10) Forster, E. W.; Grellman, K. H.; Linschitz, H. J. Am. Chem. Soc.
1973, 95, 3108–3115.
Org. Lett., Vol. 13, No. 15, 2011
4099