1092 J ournal of Natural Products, 2000, Vol. 63, No. 8
Itoh et al.
F igu r e 3. Labeling patterns of the phytyl side chain of chlorophyll a (1) formed in cultured cells of the hornwort A. punctatus.
heterophylla. The IPP-derived portion was less intensely
labeled, implying that endogenously biosynthesized IPP
had been incorporated into the phytyl side chain of 1. The
degree of efficiency of utilization of exogenous MVA in
hornworts is less than that in the liverworts. Preferential
labeling can be explained by the fact that cytoplasmic FPP
derived from exogenous MVA permeates the chloroplastidic
membrane and condenses with the endogenously formed
IPP. However, it has been reported that, in the biosynthesis
of geranylgeraniol in the chloroplasts of higher plants
(spinach), membrane fractions (thylakoid and the envelope
membrane) were unable by themselves to synthesize
geranylgeraniol.16 When stromal and membrane fractions
were combined, however, the capacity to synthesize ge-
ranylgeraniol was restored. Moreover, immunocytochemi-
cal staining showed that FPP synthase is localized in the
chloroplasts of rice, wheat, and tobacco.17 Thus, the bio-
synthesis of FPP from IPP and dimethylallyl diphosphate
and the condensation of FPP with IPP to geranylgeranyl
diphosphate (GGPP) may occur at different sites within the
chloroplasts of hornworts and liverworts. It is possible,
therefore, that cytoplasmic IPP may have been taken into
chloroplasts and condensed to FPP in a particular sub-
plastidic fraction in chloroplasts, followed by condensation
with chloroplastidically synthesized IPP in a different
subplastidic fraction.
(5 mm from the tip) of the gametophytes were sterilized
with Tween 80 and NaOCl and grown in MSK (modified
Murashige and Skoog)-3 medium19 supplied with 2%
glucose and 0.2% activated charcoal at 25 °C under
continuous light of 3000 lux. The calli were subcultured at
40-day intervals over two years prior to the feeding
experiment.
Ten or five callus cultures of A. punctatus were grown
in MSK-3 medium (70 mL each) with 2% glucose, 0.2%
activated charcoal, and 10 mM [2-13C]MVA or [2,2-
2H2]MVA, respectively. The calli were grown at 25 °C under
continuous light of 2500 lux. The calli were harvested 28
days after inoculation, and chlorophyll a (1) was isolated
as described in the following procedure (reported previ-
ously13).
Extr a ction a n d Isola tion of Ch lor op h yll a (1).
Freshly harvested calli were extracted with 5 vol of MeOH
(v/w) for 24 h (× 3). The MeOH solution was concentrated
in vacuo and dissolved in Et2O. The Et2O solution was
concentrated in vacuo, and the residue was redissolved in
Me2CO. The Me2CO solution was layered on a DEAE-
Sepharose CL-6B column and successively eluted with
Me2CO and Me2CO-MeOH (10:3, v/v). The Me2CO-MeOH
eluate containing 1 was concentrated and chromatographed
on a Sepharose CL-6B column and eluted first with
hexane-propan-2-ol (20:1, v/v) and then with hexane-
propan-2-ol (10:1, v/v). Chlorophyll a (1) was eluted in the
hexane-propan-2-ol (20:1) fraction, and it was concen-
trated to dryness to yield pure 1.
Hyd r olysis of Ch lor op h yll a (1) a n d Acetyla tion of
P h ytol (4). The biosynthetically labeled 1 was hydrolyzed
according to the following procedure. Aqueous Cs2CO3 (0.2
mL of 0.61 mM) was added to the 1, which was then
dissolved in 10 mL of MeOH and stirred at room temper-
ature for 2 h in an N2 atmosphere. Another 0.4 mL of 0.61
mM aqueous CS2CO3 was added, and after further stirring
overnight, the reaction solution was extracted with 10 mL
of pentane (× 3). The pentane extracts were combined,
dried over dry Na2SO4, filtered, and concentrated in vacuo.
The residue was chromatographed on a Si gel column and
eluted with hexane-EtOAc (4:1, v/v) to give pure 4. Phytol
(4) incorporating [2-13C]MVA was acetylated in the usual
manner. Acetic anhydride and pyridine were removed by
evaporation in vacuo at 60 °C to give phytyl acetate (5).
Phytol (4) incorporating [2,2-2H2]MVA was acetylated with
[2H6]acetic anhydride.
Exp er im en ta l Section
1
Gen er a l Exp er im en ta l P r oced u r es. The H, 2H{1H},
and 13C{1H} NMR spectra of biosynthetically labeled phytol
(4) were recorded on a J EOL EX-270 at 270 MHz (CHCl3
1
in CDCl3 as an internal standard, δ H 7.26 or CH3OD in
CD3OD as the internal standard, δ 1H 3.35), 41.3 MHz
(CDCl3 as the internal standard, δ 2H 7.26), and 67.8 MHz
(13CDCl3, δ 13C 77.0), respectively. Three 13C NMR mea-
surements were taken. Assignments of all of the 13C atoms
in 4 were carried out according to previous data.14,15 Optical
rotations were determined on a J ASCO DIP-370 polarim-
eter. HPLC separations were performed with a J ASCO 880
PU equipped with J ASCO UVIDEC-100V by monitoring
UV at 260 nm. [2-13C]- and [2,2-2H2]-MVA were prepared
by the published method.18 An authentic sample of (+)-
rosmarinic acid was purchased from Extrasynthe´se, France.
Ca llu s Cu ltu r e a n d F eed in g Exp er im en t. Gameto-
phytes of A. punctatus plants were obtained from Sandan-
kyo in Hiroshima Prefecture, J apan. The apical portions