Cholesterol-diaryl ketone stereoisomeric dyads as models for "clean" type i and type II photooxygenation mechanisms

Article abstract of DOI:10.1039/b718068c

Cholesterol (Ch) is a major target for oxidative degradation in cell membranes, a process which can occur by two mechanisms: Type I (via free radicals) and Type II (mediated by ¹O₂). In the present work, several dyads have been synthesized from β- and α-Ch and ketoprofen (KP) or tiaprofenic acid (TPA). Upon irradiation under anaerobic conditions, KP-α-Ch dyads were efficiently photolyzed, via intramolecular hydrogen abstraction from C-7. By contrast, KP-β-Ch, TPA-α-Ch, and TPA-β-Ch remained unchanged after prolonged irradiation. The transient absorption spectra of KP-α-Ch were assigned to the short-lived biradicals resulting from intramolecular hydrogen abstraction. Interestingly, the spectra and lifetimes obtained for the TPA-derived dyads were very similar to those of the TPA triplet excited state. For the KP-α-Ch dyads, generation of singlet oxygen was expectedly negligible. Conversely, for TPA-α-Ch a Φ_Δ value as high as 0.5 was determined. Thus, KP-based dyads are appropriate models for clean type I Ch oxidation, whereas the TPA derivatives are suitable systems for investigation of the purely type II process. The Royal Society of Chemistry.

Full text of DOI:10.1039/b718068c

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www.rsc.org/obc | Organic & Biomolecular Chemistry
Cholesterol–diaryl ketone stereoisomeric dyads as models for “clean” type I
and type II photooxygenation mechanisms†
Inmaculada Andreu,a Isabel M. Morera,a Francisco Bosca´,a Laura Sanchez,b Pelayo Campsb and
Miguel A. Miranda*a
Received 22nd November 2007, Accepted 4th January 2008
First published as an Advance Article on the web 31st January 2008
DOI: 10.1039/b718068c
Cholesterol (Ch) is a major target for oxidative degradation in cell membranes, a process which can
occur by two mechanisms: Type I (via free radicals) and Type II (mediated by 1O2). In the present work,
several dyads have been synthesized from b- and a-Ch and ketoprofen (KP) or tiaprofenic acid (TPA).
Upon irradiation under anaerobic conditions, KP–a-Ch dyads were efficiently photolyzed, via
intramolecular hydrogen abstraction from C-7. By contrast, KP–b-Ch, TPA–a-Ch, and TPA–b-Ch
remained unchanged after prolonged irradiation. The transient absorption spectra of KP–a-Ch were
assigned to the short-lived biradicals resulting from intramolecular hydrogen abstraction. Interestingly,
the spectra and lifetimes obtained for the TPA-derived dyads were very similar to those of the TPA
triplet excited state. For the KP–a-Ch dyads, generation of singlet oxygen was expectedly negligible.
Conversely, for TPA–a-Ch a UD value as high as 0.5 was determined. Thus, KP-based dyads are
appropriate models for clean type I Ch oxidation, whereas the TPA derivatives are suitable systems for
investigation of the purely type II process.
2-benzoylthiophene chromophore. In contrast to KP, TPA has a
Introduction
p,p* lowest triplet excited-state,6,12 and thereby the contribution
It is well established that cholesterol (cholest-5-en-3b-ol, Ch), one
of type II oxidation mechanism is enhanced.
of the most important structural components of cell membranes,
In a preliminary communication we have reported the efficient
is a major target for oxidative degradation, a process which can
and selective photogeneration of 7-allyl Ch radicals by intramolec-
result in potentially pathological consequences.1 Two mechanisms
ular hydrogen abstraction in (S)- and (R)-KP–a-Ch model dyads.13
Such dyads are reminiscent of Breslow’s biomimetic systems
constructed by esterification of cholestanol, androstanol and
related compounds with achiral benzophenone derived carboxylic
acids,14 which were designed to study the remote photooxidation
of steroids. This type of system was selected as a mimic of the
cytochrome P-450 enzyme to achieve a selectivity dominated by
the geometry of the catalyst–substrate complex. In this process,
benzophenone removes the C-14 hydrogen, giving rise to an olefin
and a mixture of cyclic products in the C or D rings. However,
none of the employed systems contained a double bond in ring B
(as Ch).14 On the other hand, isolation and structural elucidation
of the photoproducts was not attempted, and laser flash photolysis
studies to investigate transient species such as triplets and biradi-
cals were not performed. In the present work, we have completed
these studies and extended them to the analogous excited state
interactions between Ch and TPA. For this purpose, several
ketoprofen–cholesterol (KP–Ch) and tiaprofenic acid–cholesterol
(TPA–Ch) dyads have been synthesized through conjugation of b-
and a-Ch with the corresponding NSAID (Scheme 1). Inclusion of
KP–b-Ch and TPA–b-Ch in the series was done for comparison,
as these dyads should not be reactive in the intramolecular process
due to the extended relationship between the two active moieties,
which prevents their close approach. Interestingly, singlet oxygen
measurements by time-resolved near infrared emission have shown
that the KP-based dyads are appropriate models for clean type I
Ch oxidation, whereas the TPA-derivatives are suitable systems
for the investigation of purely type II process.
have been considered for Ch oxidation: Type I (via free radicals)
and Type II (mediated by O2). The former involves hydrogen
abstraction from Ch and could be promoted, in principle, by UVA-
irradiation in combination with photosensitizing agents.1,2
1
Ketoprofen (KP) has been recognized to be the strongest
photosensitizer among non-steroidal anti-inflammatory drugs
(NSAID).3–5 This property is attributable to the benzophenone
chromophore, whose lowest-lying triplet state is n,p* in nature;6
accordingly, KP is a typical Type I photosensitizer7 and is
known to induce photoperoxidation of polyunsaturated fatty
acids. In the course of model studies, we have found a significant
stereodifferentiation in the intramolecular hydrogen abstraction
from enantiomerically pure 1,4-cyclohexadienes (as simple sources
of double allylic hydrogens) by (S)-KP, where chiral discrimination
was demonstrated by measurement of the very short triplet
lifetimes of the benzophenone chromophore (18–31 ns).8,9
Tiaprofenic acid (TPA) is also a benzophenone-derived NSAID
with proven photosensitizing potential,4,10,11 which contains the
aDepartamento de Qu´ımica-Instituto de Tecnolog´ıa Qu´ımica UPV-
CSIC. Universidad Polite´cnica de Valencia, Camino de Vera s/n, Apdo
22012, 46071, Valencia, Spain. E-mail: mmiranda@qim.upv.es; Fax: +34
963877809; Tel: +34 963877807
bLaboratori de Qu´ımica Farmace`utica (Unitat Associada al CSIC), Univer-
sidad de Barcelona, Avda. Diagonal 643, 08028, Barcelona, Spain
† Electronic supplementary information (ESI) available: Further experi-
mental details and spectra. See DOI: 10.1039/b718068c
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Scheme 2 Synthesis of (R)-KP: a) PCl5, CCl4; b) AcOH, HCl 2 N.
Scheme 3 Synthesis of a-Ch: a) PPh3, ClCH2CO2H, diisopropyl azodi-
carboxylate (DIAD), THF; b) MeOH, K2CO3.
Dyads of interest (1–6) were synthesized by esterification of a- or
b-Ch with the appropriate arylpropionic acid following standard
procedures.8 Allnew compounds were fully characterizedby NMR
and MS data.
The esterification reaction between racemic TPA and a-Ch
afforded the expected diastereomeric mixture; however, when
crystallization from hexane–ethyl acetate was attempted only
one of the stereoisomers precipitated (4). On the other hand,
elimination of the solvent from the filtered solution gave rise to a
viscous oil; its spectroscopic properties were identical to those of
5 obtained by direct esterification of (S)-TPA and a-Ch.
Scheme 1 Structures of dyads 1–6.
Steady-state photolysis under anaerobic conditions
Results and discussion
Irradiations were performed with monochromatic light at kmax
=
Synthesis of the dyads
266 nm in CH2Cl2 (ca. 10−5 M solutions), under inert atmosphere,
and monitored by UV-spectrophotometry, following the decrease
in the absorption at 266 nm. The irradiation wavelength was
selected because it corresponded to the intersection of the UV-
spectra of KP- and TPA-derived dyads at the same concentration
(see, for example, Fig. 1A); in other words, the molar absorption
coefficient of all dyads was the same at 266 nm. The results of the
irradiations are shown in Fig. 1B.
The employed (S)-KP was commercially available, and its (R)
enantiomer was obtained in two ways: i) for preparation of small
amounts of compound suitable for photophysical and photochem-
ical studies in the early stages, it was separated by chiral HPLC
from the racemic mixture and ii) in order to obtain larger amounts
of the compound for further studies, an asymmetric synthesis was
carried out starting from racemic ketoprofen, using (R)-3-hydroxy-
4,4-dimethyl-1-phenyl-2-pyrrolidinone as a chiral auxiliary;15 this
led to the corresponding ester 7 with high diastereoselectivity.
Subsequent acidic hydrolysis under controlled conditions afforded
(R)-KP with high enantioselectivity (Scheme 2). The (R) and (S)
enantiomers of TPA were separated by chiral HPLC from the
racemic mixture.
In agreement with their different abilities to adopt a folded
conformation allowing a close approach between the active
moieties, dyads 1 and 2 were efficiently photolyzed while 3
remained almost unchanged after prolonged irradiation.13 On the
other hand, as shown in Fig. 1B, the KP-based dyads 1 and 2
were clearly more photoreactive than their TPA analogs 4 and
5. Moreover, in the case of the photoreactive dyads 1 and 2 it
can be observed that (R)-KP–a-Ch (2) was significantly more
photoreactive than its (S)-stereoisomer 1. Hence, as a general
observation, the photoreactivity of the dyads was markedly
For the preparation of (S)-KP–a-Ch and (R)-KP–a-Ch as
well as (R)-TPA–a-Ch and (S)-TPA–a-Ch inversion of the b-
cholesterol configuration was accomplished by a Mitsunobu
reaction (Scheme 3).16
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Fig. 1 A) UV-spectra of dyads 1 and 5 in CH2Cl2 (10−5 M solutions).
B) Degradation of dyads (1–6) as a function of the irradiation time at
266 nm.
dependent on the electronic nature of the excited states (n,p* vs.
p,p*) and on the configuration of the asymmetric carbons.
Photoproduct formation was investigated by steady-state ir-
radiation of more concentrated (ca. 10−2 M) dichloromethane
solutions under nitrogen, through Pyrex, with a 400 W medium
pressure mercury lamp. Dyads 4 and 5 were markedly unreactive
and did not give rise to any isolable photoproduct. This was
in agreement with the lack of reactivity observed in the kinetic
photodegradation experiments shown in Fig. 1B. Conversely,
dyads 1 and 2 progressively disappeared upon irradiation, af-
fording well-defined photoproducts. Their photomixtures were
submitted to silica gel column chromatography, using hexane–
ethyl acetate–dichloromethane (70 : 20 : 10 v/v/v) as eluent.
Spectral analysis of the separated fractions revealed the formation
of two diastereomeric compounds 8 and 9 from (S)-KP–a-Ch (1),
while in the case of (R)-KP–a-Ch (2) only one photoproduct (10)
was obtained (Scheme 4).13
Compounds 8–10 are the result of intramolecular hydrogen
abstraction from the C-7 position of Ch and subsequent C–C
coupling of the generated biradicals. It is interesting to note that
this photocyclization occurs at the activated allylic position of the
B ring, but not at the 4 allylic position of the A ring, obviously
due to steric hindrance. The nonactivated C and D rings (where
reaction was reported to occur in saturated benzophenone–steroid
systems) remained unaffected.14
Scheme 4 Formation of products 8–10 upon photolysis of dyads 1 and 2.
The structures of the photoproducts were unambiguously
assigned on the basis of their NMR spectroscopic data (1H,
13C, DEPT, COSY 45, HMQC, NOEDIFF). Mass spectrometry
(including high resolution measurements) supported the assign-
ments. Due to the rigidity of the polycyclic skeletons, NOE
experiments were essential to assign the stereochemistry of the
new asymmetric carbons, generated upon C–C coupling of the two
radical termini. The most significant interactions were observed
between the ortho-protons of the phenyl group and the allylic
protons at C-7.13
In view of the existing literature precedents on related steroid–
benzophenone systems,14 it appeared interesting to compare the
circular dichroism (CD) spectra of the KP-derived dyads with
those of the pure (S)- and (R)-KP enantiomers. In principle, the
relative photoreactivities could be correlated with CD changes
associated with ground state conformational differences.14 Unfor-
tunately, although clear CD spectra were obtained in all cases,
no significant differences were found between the dyads (see
supporting information).
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Table 1 Photophysical parameters of dyads 1, 2, 4 and 5
In view of the difficulty to achieve direct observation of the
short-lived triplet excited states, another set of experiments were
performed to obtain accurate values for the triplet lifetimes of
(S)-KP–a-Ch and (R)-KP–a-Ch by the well-established energy
transfer method, using naphthalene (NP) as acceptor.8,19 Thus,
laser flash photolysis of dyads 1 and 2 was performed at the
excitation wavelength of 355 nm in the presence of increasing
amounts of NP, and the triplet–triplet absorption of NP was
observed and monitored at 415 nm.
(S)-KP–a-Ch
(1)
(R)-KP–a-Ch
(2)
(R)- or (S)-TPA–a-Ch
(4, 5)
Parameter
sT/ls
0.010 0.002
1.0 × 108
0.012 0.002
1.2 × 108
5.500 0.100
1.0 × 104
<1.0 × 104
<1.0 × 104
<0.01
kiq/s−1
kH/s−1
0.8 × 108
1.0 × 108
kp/s−1
0.2 × 108
0.2 × 108
Uketyl radical
sbiradical/ls
0.80 0.02
0.28 0.01
0.80 0.02
0.22 0.01
—
When the reciprocal transient absorbance at 415 nm was plotted
against the reciprocal of concentration of NP two straight lines
were obtained [see Fig. 3 and eqn (1)].
Transient spectroscopic studies
1/A415 = a + a/(kq × sT × [NP])
(1)
The transient absorption spectra obtained upon 355 nm excitation
of (S)-KP–a-Ch (1) and (R)-KP–a-Ch (2), 20 ns after the laser
pulse, were very similar; they did not correspond to their triplet
excited states but to the corresponding biradicals.13 This is shown
in the insets in Fig. 2 for the case of (S)-KP–a-Ch (top) and
(R)-KP–a-Ch (bottom). The assignment was based on the typical
bands with maxima at ca. 340 nm and 545 nm (relative intensities
ca. 5 : 1).4,17,18 Interestingly, the lifetimes of the diastereomeric
biradicals were significantly different, namely 280 ns for (S)-KP–a-
Ch vs. 220 ns for (R)-KP–a-Ch (see Table 1). They were unaffected
by the temperature and were quenched by oxygen (see Fig. 2) with
kq ca. 3.6 × 109 M−1 s−1.
A415 is the absorbance of the triplet of NP at 415 nm, before
significant decay takes place, kq is the bimolecular rate constant
for triplet quenching by NP, sT is the triplet lifetime of 1 or 2
in the absence of NP, and a is a constant. The Stern–Volmer
parameters (kq × sT) were obtained from the intercept-to-slope
ratios [Fig. 3 and eqn (1)]. They were found to be 79 M−1 for
1 and 94 M−1 for 2. On the other hand, the intermolecular kq
determined in dichloromethane for (S)-KP was 8 × 109 M−1 s−1,
which was assumed to be the same for the dyads. With these data,
the values calculated for the triplet lifetimes of (S)-KP–a-Ch and
(R)-KP–a-Ch were 10 ns and 12 ns respectively.
Fig. 3 Double reciprocal plot for quenching of dyads (S)-KP–a-Ch and
(R)-KP–a-Ch triplet excited state by NP in CH2Cl2.
The intramolecular quenching rate constants (kiq) were deter-
mined by using eqn (2), where si are the lifetimes of the ketone
triplets in compounds (S)-KP–a-Ch, (R)-KP–a-Ch and (S)-KP–
b-Ch, while s0 is the (S)-KP triplet lifetime. The obtained values
were used to calculate the rate constants for hydrogen abstraction
(kH) and for physical quenching by the p system (kp), (see Table 1)
using eqn (3) and (4).
kiq = 1/si − 1/s0
kH = kiqUketyl radical
kiq = kH + kp
(2)
(3)
(4)
Fig. 2 Decays of the biradicals generated from dichloromethane solutions
of dyads 1 (top) and 2 (bottom), upon laser flash photolysis under different
conditions, monitored at 545 nm. The insets show the transient absorption
spectra of the biradicals.
By contrast, the triplet excited state of the extended diastereoiso-
meric form 3 (Fig. 4A) was much longer-lived, so it was possible
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Fig. 4 A) Transient absorption spectrum for dyad 3, 0.5 ls after the laser
pulse (kexc = 355). B) Decays of the triplets generated from (S)-KP and
dyad 3 monitored at 520 nm.
Fig. 5 A) Transient spectrum for dyad 6, 1 ls after the laser pulse (kexc
355 nm). B) Decays of the triplets generated from dyads 4 and 5, monitored
=
at 600 nm.
to record its absorption spectrum, which showed the typical T–T
benzophenone bands with maxima at ca. 330 nm and 525 nm.4,17,18
Direct determination of its lifetime (1.69 ls) showed that it was
essentially coincident with that obtained for (S)-KP under the
same conditions (1.70 ls, see Fig. 4B).
In this context, the transient absorption spectra obtained by
laser flash photolysis experiments from all the tiaprofenic acid-
derived dyads, (S)-TPA–a-Ch, (R)-TPA–a-Ch and TPA–b-Ch,
were very similar to that described for the tiaprofenic acid triplet
excited state (typical bands with maxima at ca. 360 nm and 600 nm
and relative intensities of ca. 2 : 1).4,12 For example, Fig. 5A shows
the T–T absorption spectrum of TPA–b-Ch. In addition, from the
decay traces of the triplets generated upon laser flash photolysis
of 4 and 5 (Fig. 5B), it was observed that the triplet excited state
lifetimes of the two dyads (ca. 5.5 ls) were also very close to each
other.
Fig. 6 Time-resolved emission at 1270 nm upon 308 nm excitation of
dyads 1–6, using perinaphthenone as standard for comparison.
Singlet oxygen quantum yields
Time-resolved near infrared emission studies were carried out on
the dyads, in order to assess their ability to photosensitize the
production of excited singlet molecular oxygen (1O2 or 1Dg). For-
mation of this species was detected by time-resolved measurements
of the luminescence at 1270 nm, in dichloromethane, using an
appropriate diode as detector. Fig. 6 shows that, while dyads 1
and 2 produced negligible luminescence, the b isomer 3 gave rise
to singlet oxygen with a quantum yield (UD) of 0.2, in the range
of the values described for ketoprofen (KP) in organic solvents.20
These results can be understood on the basis of the very fast
intramolecular hydrogen abstraction process occurring in dyads
1 and 2, which is not possible in 3. On the other hand, the UD
for dyads 4–6 was ca. 0.5 in all cases, close to that described for
tiaprofenic acid (TPA).20
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As regards the singlet oxygen lifetime in dichloromethane, it was
measured using perinaphthenone as standard and was 76 ls, in
reasonable agreement with the literature.21,22 Likewise, the lifetime
obtained in the presence of dyads 4–6 was ca. 64 ls, reasonably
close to the expected value.
Conclusions
The present work has demonstrated the different behavior between
the a-dyads derived from KP (an np* diaryl ketone) and those de-
rived from TPA (a heterocyclic analog with pp* character). Thus,
KP–a-Ch dyads are appropriate systems to generate biradicals by
intramolecular hydrogen abstraction from the 7-allyl Ch position;
in addition, in these dyads the generation of singlet oxygen is
expectedly negligible. By contrast, the corresponding TPA–a-Ch
dyads are unreactive via intramolecular hydrogen abstraction, but
1O2 is produced with UD = 0.5. Therefore, KP-based dyads are
appropriate models for clean type I Ch oxidation, whereas the
TPA-derivatives are suitable systems for investigation of the purely
type II process.
Type I vs. Type II photooxidation mechanisms
The nature of the involved excited triplet states showed a dramatic
influence on the photobehavior. Thus, when cholesterol was
attached to KP (an np* diaryl ketone) the photoreactivity under
anaerobic conditions was remarkable. This was clearly evident
from the steady-state studies, where a-dyads 1 and 2 were efficiently
photodegraded. Accordingly, in the laser flash photolysis experi-
ments a very short triplet lifetime was estimated for the a-dyads
1 and 2 due to efficient photogeneration of 7-allyl Ch biradicals
by intramolecular H abstraction. Thus, product studies as well as
biradical detection support the high np* photoreactivity. By con-
trast, a quite different situation was observed for the photostable
dyads derived from TPA, a diaryl ketone with pp* character.
Likewise, the electronic configuration of the excited triplet
state has a strong influence on the photooxidation mechanism.
This can be conveniently discussed on the basis of Scheme 5,
which summarizes all the relevant steps involved in the overall
process. Thus, the Type I mechanism would involve intramolecular
hydrogen abstraction (ii), followed by oxygen quenching of the
resulting biradicals (iv). As stated above, the rate constants
determined for step ii are ca. 108 s−1 for the KP–a-Ch dyads (np*)
and lower than 104 s−1 for the TPA–a-Ch analogs (pp*). Step (iv)
was also very fast in the KP derivatives; its rate constant was nearly
diffusion-controlled (kq(iv) = 3.6 × 109 M−1 s−1).
Experimental
General
(S)-Ketoprofen [(S)-2-(3-benzoylphenyl)propionic acid, (S)-KP]
and b-cholesterol [cholest-5-en-3b-ol, Ch] were commercially
available. The preparation of (R)-ketoprofen and a-cholesterol is
detailed in the Supporting Information. The two enantiomers of
tiaprofenic acid [(R)-and (S)-2-(5-benzoylthiophen-2-yl)propionic
acid, TPA) were obtained by chiral HPLC separation of the
racemic mixture, using hexane–methyl tert-butyl ether–acetic acid
(70 : 30 : 0.1 v/v/v) as the mobile phase; flow-rate 2 mL min−1.
Chromatographic HPLC separation was performed coupled with
a chiral detector. Samples were injected onto a semipreparative
column (Kromasil CHI-TBB). Irradiations with 266 nm light
were carried out with the Xe lamp of a Photon Technology
spectrofluorometer, equipped with a monochromator. The 1H
NMR and 13C NMR spectra were recorded in CDCl3 as solvent at
300 or 500 and 75 MHz, respectively; chemical shifts are reported
in ppm downfield from an internal solvent peak. Exact mass
was obtained by fast atom bombardment (FAB) recorded in a
VG Autospec high-resolution mass spectrometer (HRMS). All
reactions were monitored by analytical TLC with silica gel 60
F254 (Merck) revealed with cerium ammonium sulfate–ammonium
molybdate reagent. The residues were purified through silica gel
60 (0.063–0.2 mm).
Laser flash photolysis measurements
A pulsed Nd:YAG laser (SL404G-10 Spectrum Laser Systems)
was used for the excitation at 355 nm. The single pulses were ∼10 ns
duration and the energy was from 10 to 1 mJ per pulse. The laser
flash photolysis apparatus consisted of the pulsed laser, the Xe
lamp, a monochromator and a photomultiplier made up of a tube,
housing and power supply. The output signal from the oscilloscope
was transferred to a personal computer. All experiments were
carried out at room temperature. The samples were dissolved
in dichloromethane to have an absorbance ca. 0.05 at 355 nm.
Solutions were deaerated by bubbling nitrogen (when specified).
As naphthalene (NP) does not absorb at 355 nm, under these
conditions more than 99% of the light was always absorbed by
the dyads. The rate constants of triplet excited state quenching by
oxygen, NP and cholesterol Ch were determined by the Stern–
Volmer equation (1/s = 1/s0 + k[Quencher]). Concentrations
between 0.5 and 20 mM were used for NP, from 1 to 50 mM for Ch
Scheme 5 Cholesterol oxidation mechanisms intramolecularly photosen-
sitized by diaryl ketones.
Alternatively, the Type II mechanism would occur following
steps (v) and (vi), becoming the only significant pathway in the
case of TPA–a-Ch dyads. The rate constant for triplet quenching
by oxygen was kq(v) = 0.7 × 109 M−1 s−1 for 3 and 0.6 × 109 M−1 s−1
1
for dyads 4–6. As a result of such quenching, O2 was actually
produced with UD = 0.5; its reaction with the dyads is shown in
step vi.
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and 1.27 mM and 0.27 mM for oxygen (concentrations of pure O2
gas and air at room temperature, respectively). An energy transfer
reaction from (R or S)-KP–a-Ch to NP was used to calculate
the triplet state lifetimes of these dyads. These experiments were
carried out using dichloromethane solutions of the (R or S)-KP–
a-Ch dyads (0.5 mM and 2 mM–20 mm).
19.0, 20.7, 22.6, 22.8, 23.8, 24.2, 26.0, 28.0, 28.2, 31.7, 31.8, 33.6,
35.7, 36.0, 36.1, 36.9, 39.5, 39.6, 42.0, 42.2, 50.0, 56.0, 56.5, 71.9,
122.5, 126.0, 128.3, 129.1, 132.1, 134.6, 137.7, 138.0, 142.0, 153.1,
171.7, 187.7. HRMS (FAB) C41H56O3S m/z calcd: 628.39502 [M+];
found 628.39624.
Compound 5. 1H NMR (300 MHz, CDCl3) d = 0.64 (s, 3H),
0.86 (d, J = 6.5 Hz, 6H), 0.88 (d, J = 6.5 Hz, 3H), 0.96 (s, 3H),
1.60 (d, J = 7.2 Hz, 3H), 0.90–2.02 (complex signal, 26 H), 2.15
(broad d, J = 15.0 Hz, 1H), 2.40 (broad d, J = 15.0 Hz, 1H), 3.99
(q, J = 7.2 Hz, 1H), 4.97 (m, 1H), 5.08 (broad d, J = 4.8 Hz,
1H), 7.00 (d, J = 3.6 Hz, 1H), 7.47–7.57 (m, 4H), 7.84 (d, J =
6.9 Hz, 2H); 13C NMR (75 MHz, CDCl3) d = 11.8, 18.6, 18.7,
18.8, 20.7, 22.5, 22.8, 23.7, 24.1, 26.1, 28.0, 28.1, 31.6, 31.8, 33.6,
35.7, 36.0, 36.1, 36.8, 39.5, 39.6, 41.8, 42.2, 50.0, 56.0, 56.5, 71.8,
122.5, 125.9, 128.3, 129.0, 132.1, 134.5, 137.7, 138.0, 142.0, 153.1,
171.6, 187.6. HRMS (FAB) C41H56O3S m/z calcd: 628.39502 [M+];
found 628.39381.
Singlet oxygen measurements
The luminescence (1270 nm) from singlet oxygen was detected by
means of an Oriel 71614 germanium photodiode (5 mm2) coupled
to the laser photolysis cell in right-angle geometry. An excimer
laser (LEXTRA50 Lambda Physik) was used for the excitation at
308 nm (laser excitation at 5 low-pulse energies for each molecule).
A 5 mm thick (5 cm diameter) 1050 nm cut-off silicon filter and a
1270 nm interference filter were placed between the diode and the
cell. The photodiode output current was amplified and fed into
a TDS-640A Tektronix oscilloscope via a Co-linear 150 MHz,
20 dB amplifier. The output signal from the oscilloscope was
transferred to a personal computer for study. Thus, the singlet
oxygen quantum yield (UD) of the dyads was determined in
dichloromethane solutions using the same absorbance value (0.30)
at 308 nm for each compound. A singlet oxygen quantum yield
(UD) of 0.95 for perinaphthenone in dichloromethane was used as
standard.23
3b-Cholesteryl 2-(5-benzoylthienyl)propanoate (TPA–b-Ch, 6)
To a solution of racemic 2-(5-benzoylthien-2-yl)propanoyl chlo-
ride (ca. 100 mg, 0.36 mmol) in CH2Cl2 (15 mL), b-cholesterol
(150 mg, 0.39 mmol) in CH2Cl2 (3 mL) was added dropwise, and
the mixture was heated under reflux for 7 h. The reaction mixture
was cooled to room temperature and then it was washed with
water (3 × 10 mol) and brine (10 mol). The organic phase was dried
over Na2SO4, evaporated and purified by column chromatography
(eluent: hexane–dichloromethane–ethyl acetate 90 : 5 : 5 v/v/v) to
give the corresponding ester TPA–b-Ch (189 mg, 0.30 mmol, 83%).
1H NMR (300 MHz, CDCl3) d = 0.67 (s, 3H), 0.84 (d, J = 6.6 Hz,
6H), 0.90 (d, J = 6.6 Hz, 3H), 1.01 (s, 3H), 1.60 (d, J = 7.2 Hz,
3H), 0.90–2.05 (complex signal, 26 H), 2.32 (m, 2H), 3.99 (q, J =
7.2 Hz, 1H), 4.66 (m, 1H), 5.37 (m, 1H), 7.02 (d, J = 3.9 Hz, 1H),
7.45–7.57 (m, 4H), 7.83 (d, J = 6.9 Hz, 2H); 13C NMR (75 MHz,
CDCl3) d = 11.7, 18.7, 19.2, 19.3, 21.0, 22.5, 22.8, 23.8, 24.3, 28.0,
28.2, 31.8, 31.9, 35.8, 36.2, 36.6, 36.9, 37.8, 38.0, 39.5, 39.7, 41.9,
42.3, 50.0, 56.1, 56.7, 75.1, 122.8, 125.8, 128.3, 129.1, 132.1, 134.8,
138.0, 139.3, 142.2, 152.9, 171.8, 188.0. HRMS (FAB) C41H56O3S
m/z calcd: 628.39502 [M+]; found 628.39293.
Synthesis of the dyads 4 and 5
To
a cold solution of racemic 2-(5-benzoylthiophen-2-yl)-
propanoic acid (100 mg, 0.38 mmol) in CH2Cl2 (1.5 mL),
dicyclohexylcarbodiimide (DCC, 136 mg, 0.66 mmol) was added
portionwise, and the mixture was stirred at 0 ◦C for 30 min.
Then, a solution of a-cholesterol (131 mg, 0.34 mmol) and 4-
dimethylaminopyridine (DMAP, 4 mg, 0.033 mmol) in CH2Cl2
(1.2 mL) was added, and stirring was continued for 8 h at the same
temperature. The reaction mixture was filtered through a pad of
R
Celiteꢀ, brine (5 mL) added to the filtrate, and the mixture was
extracted with CH2Cl2 (3 × 5 mL). The combined organic layers
were washed with water, the organic phase was dried over Na2SO4
and evaporated and further purified by column chromatography
(eluent: hexane–dichloromethane–ethyl acetate 90 : 5 : 5 v/v/v) to
yield a diastereomeric mixture of the corresponding esters. After
crystallization of the above mixture from hexane–ethyl acetate
(1 : 1 v/v), one of the stereoisomers precipitated as a white solid
(88 mg, 0,14 mmol, 41%, 4); the other isomer was obtained by
elimination of the solvent from the filtered solutions as a colorless
oil (79 mg, 0,12 mmol, 37%, 5). Alternative synthesis of the
corresponding ester from enantiomerically pure (S)-TPA obtained
by chiral HPLC separation of the racemic mixture was used for
stereochemical assignment.
Steady-state photolysis of the dyads 1, 2, 4 and 5
Deaerated dichloromethane (20 mL) solutions of (S)- or (R)-KP–
a-Ch dyads and (S)- or (R)-TPA–a-Ch dyads (100 mg, 0.16 mmol)
were irradiated through Pyrex with a 400 W medium pressure
mercury lamp. Reactions were monitored by TLC and NMR, and
only KP derived dyads (1 and 2) were found to be reactive. After
4 hours, the reaction mixtures were concentrated under reduced
pressure and submitted to silica gel column chromatography, using
hexane–ethyl acetate–dichloromethane (eluent: 70 : 20 : 10 v/v/v).
This afforded the pure photoproducts 8–10.13
Data for (R)-TPA–a-Ch (4) and (S)-TPA–a-Ch (5)
Compound 4. 1H NMR (300 MHz, CDCl3) d = 0.64 (s, 3H),
0.86 (d, J = 6.5 Hz, 6H), 0.88 (d, J = 6.5 Hz, 3H), 0.97 (s, 3H),
1.61 (d, J = 7.2 Hz, 3H), 0.90–1.99 (complex signal, 26 H), 2.20
(broad d, J = 15.0 Hz, 1H), 2.45 (broad d, J = 15.0 Hz, 1H), 3.99
(q, J = 7.2 Hz, 1H), 4.97 (m, 1H), 5.19 (broad d, J = 5.1 Hz,
1H), 7.00 (d, J = 3.9 Hz, 1H), 7.48–7.58 (m, 4H), 7.84 (d, J =
7.2 Hz, 2H); 13C NMR (75 MHz, CDCl3) d = 11.8, 18.7, 18.8,
Acknowledgements
Financial support from the Spanish Government (Grant
CTQ2004–03811 and CTQ2007–67010), Generalitat Valenciana
(GV06/099), Vicerrectorado de Investigacio´n Desarrollo e Inno-
vacio´n de la UPV (PAID-06–06) and Juan de la Cierva contract
to I. A., are gratefully acknowledged.
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