Photochemistry and Photobiology, 2005, 81 667
Isomerization of BPhe g
was added to 10 mL of an acetone solution of BPhe g
nm, Abs[k 5 748 nm] 5 2.7/cm) (10). The mixture was stirred at room
temperature for 30 min in the dark, poured in H O, extracted with CHCl
washed with H O three times, dried over Na SO and filtered. After
evaporation, the residue was purified by the above described HPLC scheme
to give pure ‘‘pheophytin a ’’ (Phe a ).
Transesterification of ‘‘Phe a ’’ to ‘‘Phe a
sulfuric acid solution (6.4 M, 1.0 mL) was added to a 2.0 mL methanol
solution of ‘‘Phe a ’’ (Abs[k 5 666 nm] 5 3.5/cm) (8,15,16). The mixture
was stirred at room temperature for 90 min in the dark, poured into ice
water, extracted with CH Cl , washed with H O twice, dried over Na SO
and filtered. After evaporation, the residue was purified by HPLC to give
pure ‘‘Phe a .’’
Preparation of Mosher’s ester of 8 -OH-Phe a
and optically active a-methoxy-a-(trifluoromethyl)phenylacetyl chloride
MTPA-Cl, 10 lL, Tokyo Kasei Kogyo Co. Ltd., Tokyo, Japan) was added
F
to ‘‘Phe a
F
’’. Aqueous HCl (4.6 M, 500 lL)
F
(absorbance at 748
2
3
,
2
2
4
F
F
F
M
’’. An ice-chilled methanolic
F
2
2
2
2
4
M
1
F
. Commercially available
(
1
F
in the dark to 100 lL of a dry pyridine solution of 8 -OH-Phe a (ca 0.38
mg) (17). After stirring at room temperature for 1 h, N,N-dimethyl-1,3-
propandiamine (15 lL; Nacalai Tesque) was added. The mixture was stirred
3 2 2
for 15 min, poured into aqueous 4% NaHCO , extracted with CH Cl ,
washed with H O twice, dried over Na SO and filtered. After evaporation,
2
2
4
the residue was purified by HPLC to give the corresponding desired ester.
7
8
Figure 1. Expected molecular structures of BChl g
F 3
and 8 -OH–Chl a (C 5C –*CH(OH)CH ) from Hbt. modesticaldum (a).
F
(*C H–C 5CHCH
3
)
1
7
8
Their partial structure is shown at ring-B (b) and at ring-D and -E (c). The
numbers on carbon atoms are given by IUPAC-IUB nomenclature.
Optically active carbon atoms are indicated by asterisks.
RESULTS AND DISCUSSION
1
Molecular structures of BChl g
F
and 8 -OH-Chl a
F
from
Hbt. modesticaldum
1
and 8 -OH-
Here, we first report the stereochemistry of BChl g
Chl a
F
1
and 8 -OH-Chl
Figure 1 shows the molecular structure of BChl g
a in which F denotes the farnesyl group at the 17 -position.
F
Optically active carbon atoms are indicated by asterisks. BChl g is
F
F
extracted from Hbt. modesticaldum without ambiguity. On
the basis of the present stereochemical findings, we discuss the
4
1
structural relationship between BChl gF and 8 -OH-Chl a ,
F
F
characterized by the ethylidene group at the 8-position (4), which
especially for the configurational correlation between the ethyl-
1
and the 1-hydroxyethyl group of 8 -OH-
P
was also seen in BChl b from Blc. viridis (8,9). The configurations
idene group of BChl g
F
of the ethylidene group shown in the upper part of Fig. 1b and
2
at the chiral 13 -, 17- and 18-positions shown in Fig. 1c had
Chl a , and we also propose possible biosynthetic routes for
introduction of the 8-ethylidene and 8-(1-hydroxyethyl) groups (5).
F
1
been undetermined. 8 -OH-Chl a
F
is also characterized by the
-hydroxyethyl group at the 8-position (7). The absolute config-
1
urations of the 1-hydroxyethyl group shown in the lower part of
2
Fig. 1b and at the 13 -, 17- and 18-positions shown in Fig. 1c had
MATERIALS AND METHODS
General methods. HPLC was performed with a Shimadzu LC-10AD liquid
chromatograph equipped with a Shimadzu SPD-M10A diode-array detector
2þ
also remained unsolved. Replacement of the central Mg in these
B)Chls by two protons gives the corresponding (B)Phe deriva-
(
spectra were recorded with a JEOL GCmate II spectrometer (JEOL Ltd.,
Shimadzu Ltd., Kyoto, Japan). Fast atom bombardment mass (FAB-MS)
(
1
1
F F
tives: BPhe g and 8 -OH-Phe a .
Akishima, Japan); m-nitrobenzyl alcohol was used as a matrix. H-NMR
3
spectra in CDCl (99.8% CEA, Gif-Sur-Yvette, France) were recorded at
room temperature with a JEOL JNM-A400 Fourier transform NMR
spectrometer; tetramethylsilane (TMS) was used as an internal standard.
HPLC of extracted pigments from Hbt. modesticaldum
1
1
1
1
Spectra from H– H correlation spectroscopy (COSY) and H– H rotating-
frame Overhauser effect spectroscopy (ROESY; s 5250 ms) were recorded
Figure 2 shows a set of representative HPLC analyses of pigments
extracted from the cells of Hbt. modesticaldum (panel a) and after
acid treatment (panel b). In Fig. 2a, five peaks were resolved at the
region before 23 min of retention time. On the basis of their visible
spectra, each peak was assigned as peak 1 5 a carotenoid, peaks 2
and 3 5 BPhe g species and peaks 4 and 5 5 BChl g species. An
additional small peak was observed at 116 min (peak 6) shown in
m
to aid in assignment of the proton signals. Visible absorption and circular di-
chroism (CD) spectra were measured with a Hitachi U-3500 spectrophotom-
eter (Hitachi Ltd., Tokyo, Japan) and a JASCO J-720W spectropolarimeter
(
JASCO Ltd., Hachioji, Japan), respectively. All solvents were used without
further purification except dry pyridine for preparation of Mosher’s esters.
Hbt. modesticaldum was grown in a pyruvate–yeast extract medium (11)
at 458C instead of the optimal temperature of 50–528C for 24 h to avoid
accumulation of appreciable amounts of lysed cell (12). The wet cells were
extracted with a mixture of methanol and acetone (1:10 vol/vol) under
nitrogen, then the solution was evaporated in a rotary evaporator to dryness.
The residue was dissolved in acetone, treated with aqueous diluted HCl
the insert of Fig. 2a. This highly polar peak has a visible spectrum
1
similar to Chl a and is assigned as 8 -OH-Chl a
F
(7).
The stereoisomers at the 13 -position exhibit a general order of
2
normal phase HPLC elution: Chl a (Phe a) from higher plants and
BChl a (BPhe a) from purple bacteria possessing the 13 -(R)-
2
configuration tend to elute more slowly than the corresponding 13 -
(
30 mM) (13) and poured into aqueous 4% NaHCO
3
. After evaporation,
) were
);
2
1
1
resulting BPhe g
F
and 8 -hydroxy-pheophytin a
F
(8 -OH-Phe a
F
isolated by the following HPLC conditions: normal phase column (SiO
2
cosmosil 5SL-II 6.0 mm / 3 250 mm (Nacalai Tesque Inc., Kyoto, Japan);
eluent, hexane:2-propanol:methanol5100:2.0:0.2 (vol/vol) for analysis and
hexane:acetone 5 4:1 (vol/vol) for preparation; flow rate, 1.5 mL/min for
(S)-epimer, Chl a9 and BChl a9, respectively (13,18). Furthermore,
2
nonprimed types, 13 -(R)-isomers are always the major component,
2
and primed types, 13 -(S)-isomers are minor in higher plants, algae
and cyanobacteria (19,20) as well as in green sulfur bacteria (18). As
analysis and 3.0 mL/min for preparation. Methyl pheophorbide a (Phe a
as an authentic sample was prepared from the demetallation and
transesterification of Chl a extracted from cyanobacterium, Spirulina
geitleri as reported previously (14).
M
)
in the case of Hbt. modesticaldum, one can predict that a set of
2
stereoisomers at the 13 -position should appear in the general
P
Mizoguchi, Tadashi
