3,3′-dihydroxyisorenieratene, a natural carotenoid with superior antioxidant and photoprotective properties

Article abstract of DOI:10.1002/anie.200803668

(Figure Presented) The color of red smear cheeses arises from naturally occurring carotenoids like 3,3′-dihydroxyisorenieratene (1). This compound and its oxidation product 2, which have unusual phenolic and quinoid end groups, respectively, were synthesi

Full text of DOI:10.1002/anie.200803668

Communications
DOI: 10.1002/anie.200803668
Carotenoids
3,3’-Dihydroxyisorenieratene, a Natural Carotenoid with Superior
Antioxidant and Photoprotective Properties**
Hans-Dieter Martin,* Sebastian Kock, Roger Scherrers, Kaya Lutter, Tanja Wagener,
Claas Hundsdꢀrfer, Susanne Frixel, Klaus Schaper, Hansgeorg Ernst, Wolfgang Schrader,
Helmut Gꢀrner, and Wilhelm Stahl*
Carotenoids comprise a group of yellow to purple dyes widely
distributed in the plant and animal kingdoms. To date, more
than 750 carotenoids have been isolated. Not only do they
serve as colorants, they are also efficient antioxidants and
photoprotective agents that scavenge reactive oxygen species
such as singlet oxygen and free radicals. Thus, they protect
biologically important molecules from oxidative degradation.
In vitro and in vivo studies demonstrate that natural
carotenoids prevent UV-induced DNA damage and inflam-
mation.
3,3’-Dihydroxyisorenieratene (DHIR, 1), a carotenoid
with an unusual structure bearing methylated phenolic end
groups, was first isolated from the bacterium Streptomyces
mediolani. It is also present in the membrane of Brevibacte-
rium linens,[1] which is used in dairy industry for the
production of various red smear cheeses such as Munster,
Limburger, and Romadur cheeses.
We studied various carotenoids known to be excellent
antioxidants with four different model systems. DHIR (1)
proved to be superior to other carotenoids including astax-
anthin, cryptoxanthin, and the macula lutea pigments zea-
xanthin and lutein (for structural formulas see the Supporting
Information).[2] The data suggest that 1 acts as a bifunctional
radical scavenger owing to its polyenic and phenolic sub-
structures. Its activity in singlet oxygen quenching is com-
parable to that of lutein and other polyenic carotenoids; 1 acts
as a fast-quenching polyene and not like a slower-reacting
phenol. Studies in more complex systems such as liposomes
and human fibroblasts reveal that 1 prevents photo- and
photooxidative damage. The compound inhibits UV-induced
lipid oxidation, suppresses heme oxygenase-1 (HO-1)
expresssion, and prevents the formation of thymidine
dimers (eight assays are described in the Supporting Infor-
mation).
[*] Prof. H.-D. Martin, Dr. S. Kock, Dr. R. Scherrers, C. Hundsdꢀrfer,
Dr. S. Frixel
Compound 1 was synthesized previously,[1a] but this
approach did not provide sufficient amounts for extended
analytical, antioxidant, and biochemical studies. Scheme 1
describes a new total synthesis of 1 and the corresponding
quinone 2,[3] which had not been fully characterized.[3] In the
present synthesis protecting groups are not needed for the
final Wittig reaction (7 + 8!1), and the product is obtained in
62% yield (for details and characterization see the Exper-
imental Section and the Supporting Information).
Institute of Organic Chemistry and Macromolecular Chemistry,
Natural Compounds, and Photoprotection
Heinrich-Heine-University Dꢁsseldorf
Universitꢂtsstrasse 1, 40225 Dꢁsseldorf (Germany)
Fax: (+49)211-811-4324
E-mail: martin@uni-duesseldorf.de
wilhelm.stahl@uni-duesseldorf.de
K. Lutter, T. Wagener, Prof. W. Stahl
Institute of Biochemistry and Molecular Biology I
Heinrich-Heine-University Dꢁsseldorf
Antioxidant capacities can be compared easily by using
inhibition times determined by the cumene hydroperoxide
inhibition assay introduced by Terao[4] and later modi-
fied.[2b,d,5] For lutein (Figure 1) an inhibition time of 20 min
(2 ꢀ 10ꢀ4 m) was determined, whereas 1 is an outstanding
antioxidant with an inhibition time of 107 min at the same
concentration (Figure 1). Compound 1 can be considered an
efficient hybrid or bifunctional antioxidant with character-
istics between those of polyphenols and carotenoids. The
special character of 1 becomes even more distinct when the
inhibition times of other important carotenoids are plotted
against their concentrations (see Figure 1 and also Figure 2 in
the Supporting Information). The intermediary formation of
quinone 2 is evident in this assay from the change in color
from red to blue to yellow.
Universitꢂtsstrasse 1, 40225 Dꢁsseldorf (Germany)
Dr. K. Schaper
Institute of Organic Chemistry and Macromolecular Chemistry,
Organic Photochemistry, Heinrich-Heine-University Dꢁsseldorf
Dr. H. Ernst
GVF/A, BASF SE
67056 Ludwigshafen (Germany)
Dr. W. Schrader
Max-Planck-Institut fꢁr Kohlenforschung
45470 Mꢁlheim an der Ruhr (Germany)
Dr. H. Gꢀrner
Biophysicalische Chemie
Max-Planck-Institut fꢁr Bioanorganische Chemie
45413 Mꢁlheim an der Ruhr (Germany)
[**] The present work has been performed as a project of the SFB 663
(B1) at the Heinrich-Heine-University Dꢁsseldorf and is printed at
its instigation with financial support provided by the Deutsche
Forschungsgemeinschaft.
Compound 1 proved to be the best antioxidant in this
series. Blocking of the phenolic hydroxy groups by methyl-
ation (dimethoxyisorenieratene in Figure 1) leads to distinct
loss of antioxidant capacity. Thus, it is likely that oxidation of
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.200803668.
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Figure 2. Dependence of the relative rate of oxygen consumption on
carotenoid concentration and partial pressure of oxygen at 200 hPa
(150 torr) and 1013 hPa (760 torr) for 1.[2b,d,6] No prooxidation is seen
for 1 even at 1013 hPa (760 torr) partial pressure of oxygen.
Comparing the data of the established antioxidants
cryptoxanthin, zeaxanthin, lutein, and particularly astaxan-
thin, with that of 1 several features are apparent: Compound 1
gives rise to a much steeper decline in the rate of oxygen
consumption and a deeper minimum around zero, and the
rate does not recover (even at 760 Torr (1013 hPa), see
Figure 2) as with the three other carotenoids even at 150 Torr
(200 hPa). There is no evidence that 1 has prooxidant
behavior.[6]
Scheme 1. a) BBr3, 08C, CH2Cl2; b) (triphenylphosphaniumyl)propan-2-
on-1-ide, 1108C, toluene; c) vinylmagnesium bromide, ꢀ58C, THF;
d) triphenylphosphane hydrobromide, 08C, CH2Cl2; e) 1,2-epoxybutane,
reflux, ethanol; f) Ag2CO3, acetone. (The full synthesis is described in
the Supporting Information.)
A third assay, which was developed by Re et al.,[7]
measures the activity against non-oxygen-free radicals. The
ABTS assay (see the Supporting Information for details)
records the dependence on concentration and reaction time
+
of the antioxidant on the reduction of ABTSC radical
cations.[8] The results of this assay are given as TEAC
(trolox equivalent antioxidant capacity for specific times)
and/or RAA(AUC) (relative antioxidant activity as area
under curve for the whole time range).[7] Results for 1 are:
TEAC1 min = 1.46, TEAC5 min = 2.48, RAA(AUC)5 min = 1.86
(cf. RAA(AUC) of the phenol derivatives a-tocopherol
(0.90)[7] and luteolin (1.49),[7] and of the polyenes b-carotene
(2.50),[7] lycopene (3.04),[7] and lutein (1.35); mean ꢁ 1.9
Figure 3). Phenols yield lower values which may increase
somewhat over time, and carotenoids show higher values
from the start. Compound 1 behaves as a phenol at the start
and then as a polyene after minutes as a result of its
bifunctionality. For comparison, the mean of approximately
1.9 of the TEAC/RAA(AUC) of the five above-mentioned
compounds is similar to the RAA(AUC) of DHIR 1. Lutein is
less active, possibly because it has one conjugated double
bond less (Figure 3).
Figure 1. Plot of inhibition time of cumune hydroperoxide formation
against concentration. The antioxidant capacity decreases from the top
&
^
(1, ) to the bottom (lutein, ) of the sequence shown. The concept
of induction period, inhibition times, and chain-breaking antioxidants
is further explained in the Supporting Information.[5]
the phenolic groups to give the conjugated quinoid system 2 is
an additional option in 1 not feasable in typical nonphenolic
carotenoids.
We conducted a second assay, in which the dependence of
the rate of oxygen consumption on the carotenoid concen-
tration and partial pressure of oxygen is determined.[2b,d] This
assay has its origin in studies of Burton and Ingold,[6] and
allows a deeper insight into the oxygen consumption, the
influence of concentration, and the possible appearance of
prooxidative effects (Figure 2).
The fourth assay probes the activity against damaging
species in excited states, that is, O2(1Dg) singlet oxygen.[9,2a,c,d]
Most carotenoids quench singlet oxygen by a mechanism that
1
involves electronic energy transfer from a (1D·S0) encounter
complex that deactivates irreversibly by internal conversion
1
to an (3S·T1) encounter complex.[9c,d] It was of interest to
check whether 1 uses predominantly phenolic or polyenic
quenching behavior. The rate constant (kq) of quenching of
singlet molecular oxygen was determined as follows: O2(1Dg)
is formed upon generation of the triplet state of acridine as
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Figure 3. Plotting the percentage inhibition of absorbance of ABTSC+ at
Figure 4. MDA (malondialdehyde) levels in liposomes loaded with 1 or
734 nm[7,8] versus concentration yields a slope, which when plotted as
a function of time of reaction allows the calculation of the area under
the curve (AUC). The ratio between the area under the curve (AUC) for
the reaction of the specific antioxidant and that for trolox gives the
relative antioxidant activity RAA(AUC),[7] trolox RAA(AUC)[5min]: 1.00 (by
definition), lutein: RAA(AUC)[5min]: 1.35, 1[5min]: 1.86.
lutein after irradiation with UV-A light (27.5 Jcmꢀ2).
protective properties of 1 were further investigated in cell
culture with human dermal fibroblasts (ATCC-CRL-2076);
again lutein was applied for comparison. None of the
carotenoids was cell toxic (with and without UV irradiation)
as proven with the sulforhodamine B assay.[11] Cellular UV-A
response was determined by analyzing the expression of the
enzyme heme oxygenase 1 (HO-1) at the protein level by
Western blot analyses. Following UV-A-induced photooxida-
tion, HO-1 expression is increased. Compared to the irradi-
ated control (20 Jcmꢀ2), HO-1 levels were significantly
lowered by about 30% when the cells were preincubated
with 1.5 mm 1 for 24 h prior to irradiation. In contrast, no
photoprotection was found when the cells were pretreated
with similar amounts of lutein.
The formation of cyclobutane pyrimidine dimers is a
major mechanism of DNA damage in tissues exposed to UV-
B light. When human dermal fibroblasts are exposed to UV-B
light (300 mJcmꢀ2) thymidine dimers are formed and can be
visualized with specific antibodies (Figure 5A). If the cells are
preincubated for 24 h with 1 less dimer formation is observed
(Figure 5B) than in solvent controls. Quantitative evaluation
showed that the formation of thymidine dimers in the
presence of 1 is 25% lower than in the control; lutein had
no effect (Figure 6). Since the generation of cyclobutane
the sensitizer (lexc = 308 nm) and quenching of the triplet by
oxygen. The lifetime of singlet oxygen (ts = 1/kS) is solvent
dependent, and in dichloromethane tS is 0.03 ms. On addition
of a quencher, the rate constant (kS) increases with quencher
concentration. The molar absorption coefficient of 1 in
dichloromethane is e466 = 1.3 ꢀ 105 mꢀ1 cmꢀ1. Analysis of the
data with the Stern–Volmer relation (Figure 4 in the Support-
ing Information) yields kq(DHIR) = 9.7 ꢀ 109 mꢀ1 cmꢀ1. Com-
pared with other carotenoids (usual range kq ꢁ 1 ꢀ 109–
1010 mꢀ1 cmꢀ1
;
kq(lutein) = (12 ꢂ 4) ꢀ 109 mꢀ1 cmꢀ1)[9,2a,c,d] and
phenols (kq ꢁ 1 ꢀ 106 mꢀ1 cmꢀ1)[10] this is evidence that 1 uses
its polyene chain for efficient quenching and not the slower-
quenching phenolic subunits.[10] The appearance of quinone
2
[3] in the first assay (see above) can be followed visually, but
whether 2 plays a role in other assays remains unclear.
To determine the effects of 1 against photooxidation a
liposomal model was applied (for details see the Supporting
Information). Compound 1 and, for comparison, lutein were
incorporated into multilamellar liposomes composed of egg
yolk phospholipids. Photooxidation was initiated with UV
light (UV-A or UV-B light), and the decomposition product
malondialdehyde (MDA) was determined as a measure of
oxidative lipid damage. The result of a typical experiment is
shown in Figure 4. Upon irradiation of control liposomes with
UV-A light at 27.5 Jcmꢀ2, about 2.1 mmol MDA per mg
phospholipids forms (black line). When liposomes are loaded
with increasing amounts of 1 (0.01–50 nmolmgꢀ1 phospholi-
pids) MDA formation decreases significantly to about
0.5 mmol MDA per mg phospholipids, indicating antioxidant
activity. With lutein, only a moderate inhibition of lipid
oxidation was found. Protective effects of 1 were also
observed at doses of 10 Jcmꢀ2 UV-A light and less pro-
nounced at 0.5 and 1.5 Jcmꢀ2 UV-B light. The bifunctional
structure of 1, providing UV absorption and radical-scaveng-
ing properties, is also in this system likely responsible for the
superior activity of the compound. HPLC analyses revealed
that 1 was more stable than lutein under UV irradiation. UV-
Figure 5. Thymidine dimer (immunostaining, red) formation in human
skin fibroblasts; control (A), preincubated with 1 (B). DNA (DAPI
staining, blue), cytoskeleton-b-tubulin (immunostaining, green).
DAPI=4’,6-diamidino-2-phenylindole.
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pyrimidine dimers is a photochemical reaction of DNA bases
and not related to photooxidation it is likely that UV-B-
absorbing properties of 1 are responsible for the effect.
Keywords: carotenoids · photooxidation · photoprotection ·
quinones · singlet oxygen
.
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Experimental Section
Full procedures, general methods, characterization data and struc-
tural formulas of all compounds are given in the Supporting
Information.
Important data of 1 and 2: 1: Yield: 62%; m.p. 228–2308C; UV/
Vis (THF): lmax (e) = 280 (25970), 466 nm (130000); HRMS (Fin-
nigan MAT95/70 eV, EI): calcd for C40H48O2: 560.365057; found:
560.365431. 2: Yield: 61%; m.p. > 2508C; Vis (n-hexane): lmax (e) =
548 nm (151000); HRMS (ESIpos): calcd for C40H46O2Na:
581.339270; found: 581.338995.
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Vistica, J. T. Warren, H. Bokesch, S. Kenney, M. R. Boyd, J. Natl.
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Received: July 28, 2008
Published online: November 25, 2008
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