Preparation of regio- and stereoisomeric di- and tetrahydrogeranylgeraniols and identification of esterifying groups in natural (bacterio)chlorophylls

Article abstract of DOI:10.1016/j.bmc.2017.10.002

All regioisomeric di- and tetrahydrogeranylgeraniols possessing the C2[dbnd]C3 double bond were prepared as authentic samples. The synthetic C₂₀-isoprenoid alcohols were separated well by gas chromatography. Based on the chromatographic analysis, the enzymatic reduction pathway of a geranylgeranyl group was investigated to identify the last stage of (bacterio)chlorophyll biosynthesis in phototrophs. The geranylgeranyl group was triply reduced to the phytyl group through the first regio- and stereospecific hydrogenation of C10[dbnd]C11 to C10H–C11(S)H, the second of C6[dbnd]C7 to C6H–C7(S)H, and the third of C14[dbnd]C15 to C14H–C15H. The identification of the reduction sequence completes the biosynthetic pathways for naturally occurring chlorophyll-a and bacteriochlorophyll-a bearing a phytyl group as the esterifying moiety in the 17-propionate residues.

Full text of DOI:10.1016/j.bmc.2017.10.002

Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Preparation of regio- and stereoisomeric di- and
tetrahydrogeranylgeraniols and identification of esterifying groups in
natural (bacterio)chlorophylls
⇑
Hitoshi Tamiaki , Kota Nomura, Tadashi Mizoguchi
Graduate School of Life Sciences, Ritsumeikan University, Kusatsu, Shiga 525-8577, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
All regioisomeric di- and tetrahydrogeranylgeraniols possessing the C2@C3 double bond were prepared
as authentic samples. The synthetic C20-isoprenoid alcohols were separated well by gas chromatography.
Based on the chromatographic analysis, the enzymatic reduction pathway of a geranylgeranyl group was
investigated to identify the last stage of (bacterio)chlorophyll biosynthesis in phototrophs. The geranyl-
geranyl group was triply reduced to the phytyl group through the first regio- and stereospecific hydro-
genation of C10@C11 to C10HAC11(S)H, the second of C6@C7 to C6HAC7(S)H, and the third of
C14@C15 to C14HAC15H. The identification of the reduction sequence completes the biosynthetic path-
ways for naturally occurring chlorophyll-a and bacteriochlorophyll-a bearing a phytyl group as the ester-
ifying moiety in the 17-propionate residues.
Received 7 September 2017
Revised 30 September 2017
Accepted 5 October 2017
Available online xxxx
Keywords:
Biosynthesis
Hydrogenation
Photosynthesis
Phytyl group
Regioisomer
Ó 2017 Elsevier Ltd. All rights reserved.
1. Introduction
diphosphate is transformed into phytyl diphosphate by GG
reductases, ChlP and BchP for oxygenic and anoxygenic
Isoprenoids, called also terpenes and terpenoids, are natural
compounds and consisted of an isoprene unit, its oligomers, and
their derivatives through oxidation, reduction, and cyclization.1
Photosynthetic organisms utilize a variety of isoprenoids as bioac-
tive components. For example, geranylgeranyl (GG) diphosphate,
one of the C20-isoprenoids (diterpenoids), is transformed directly
into tocotrienols and geranylgeranylated (bacterio)chlorophylls
[(B)Chls], and it dimerizes and successively dehydrogenates to give
phototrophs, respectively. Phytyl diphosphate is then reacted with
chlorophyllides and bacteriochlorophyllides by Chl and BChl syn-
thases (ChlG and BchG), respectively, to give (B)Chls possessing a
phytyl group. 2) Chlorophyllides and bacteriochlorophyllides are
first esterified with GG diphosphate by ChlG and BchG, and the GG
group of the resultant Chls and BChls is hydrogenated by ChlP and
BchP to afford (B)Chls bearing a phytyl group.
Although GG reductases stereospecifically hydrogenate the
double bonds of (DH/TH)GG substrates to give chiral (7S)- and
(11S)-configurations, their regioselectivity has not been com-
pletely determined yet (see Fig. 1).3,4 A previous investigation of
Chl pigments in etiolated oat seedlings showed that a 6,7,10,11-
THGG group was biosynthesized during the GG reduction, but a
regioisomeric DHGG moiety was not confirmed: both 6,7- and
10,11-DHGG were possible as the intermediate.5 One of the purple
photosynthetic bacteria, a Rhodopseudomonas sp. Rits strain, accu-
mulated a large amount of BChls-a possessing GG, DHGG, and
THGG groups in the 17-propionate residue as well as a mature
phytyl group (Fig. 2, middle). Spectroscopic analyses indicated that
the isolated BChl-a intermediates were esterified with 6,7-DHGG
and 6,7,10,11-THGG groups.6 This is the first report for the deter-
mination of a regioisomeric DHGG group, where the GG reduction
in BChl-a biosynthesis was speculated to occur in the order of GG
carotenoids with
p-conjugated polyene chromophores often after
further modifications. GG diphosphate is sequentially hydrogenated
by a GG reductase to afford phytyl diphosphate via dihydrogeranyl-
geranyl (DHGG) and tetrahydrogeranylgeranyl (THGG) diphos-
phates (see Fig. 1, XOH = diphosphoric acid). The resulting phytyl
diphosphate is enzymatically reacted with homogentidate, 1,4-
dihydroxy-2-naphthoate, and (bacterio)chlorophyllides to give
tocopherols (vitamin E), phylloquinone (vitamin K1), and phytylated
(B)Chls, respectively. Phytylated (B)Chls are alternatively biosyn-
thesized by the triply hydrogenation of geranylgeranylated (B)Chls
[Fig. 1, XOH = (bacterio)chlorophyllide].2 In the last stage of (B)Chl
biosynthesis, the following two pathways are possible.3 1) GG
⇑
Corresponding author.
E-mail address: tamiaki@fc.ritsumei.ac.jp (H. Tamiaki).
https://doi.org/10.1016/j.bmc.2017.10.002
0968-0896/Ó 2017 Elsevier Ltd. All rights reserved.
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H. Tamiaki et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
Fig. 1. Sequential hydrogenation of geranylgeranyl (GG) to phytyl moiety via di- and tetrahydrogeranylgeranyl (DHGG and THGG) moieties in a stereoselective manner: ROX
= C20-isoprenoid alcohols (X = H), diphosphates (XOH = diphosphoric acid), and (bacterio)chlorophylls [XOH = (bacterio)chlorophyllide].
2. Results and discussion
2.1. Synthesis of dihydrogeranylgeraniols
14,15-Dihydrogeranylgeraniol (14,15-DHGG-OH, 6a) was pre-
pared as shown in Scheme 1. Commercially available farnesol
(1a) was esterified with acetic anhydride in the presence of 4-(N,
N-dimethylamino)pyridine (DMAP) to give farnesyl acetate (2a)
in a 76% yield [step (i) in Scheme 1]. The resulting ester 2a was
treated using tert-butyl peroxide with selenium oxide as a catalyst
in the presence of salicylic acid at room temperature for 2 days
[step (ii)].15 The oxidation proceeded at the allyl positions and
Fig. 2. Molecular structures of naturally occurring chlorophylls possessing a series
of geranylgeranyl and its hydrogenated moieties as the esterifying group (R) in the
the less sterically hindered terminal methyl group was primarily
17-propionate residue: E = COOCH3.
oxidized to give 3a as the desired product, whereas the other allylic
positions were partially oxidized and a small amount of aldehyde
? 6,7-DHGG ? 6,7,10,11-THGG ? phytyl (6,7,10,11,14,15-hexa-
hydrogeranylgeranyl). Recently, it was reported that commercially
available diatom (Chaetoceros calcitrans) cultures contained Chls-a
bearing 10,11-DHGG and 6,7,10,11-THGG groups (Fig. 2, left) from
their NMR spectroscopic analyses.7 This observation shows
another possible route for the GG reduction pathway in Chl-a
biosynthesis: C10@C11 ? C6@C7 ? C14@C15. It is noteworthy
that all the sequential reduction pathways employ the hydrogena-
tion of the C14@C15 double bonds at the last step.
was obtained by the further oxidation of 3a. After separation from
the starting 2a (ca. 30%) and the by-products with flash column
chromatography (FCC), analytically pure 3a (27%) was isolated as
the regio- and stereoselectively oxidized product. Primary alcohol
3a was esterified by diethyl chlorophosphate with DMAP to yield
the corresponding phosphate 4a (64%) [step (iii)]. The coupling
The greening processes in the etiolated leaves of angiosperm
are useful for determining regiospecific GG reduction path-
ways.5,8–10 The temporally produced Chl intermediates are present
in low amounts and their physical properties are little differenti-
ated. Therefore, the following technique is effective for their regio-
isomeric determination. Chls possessing DHGG and THGG groups
are first isolated from greening leaves, then cleaved to chlorophyl-
lides and DHGG- and THGG-OHs (see Fig. 1, X = H), which are ana-
lyzed by gas chromatography (GC). Here, we report regio- and
stereospecific synthesis of 6,7-, 10,11-, and 14,15-DHGG-OHs as
authentic samples and preparation of 6,7,10,11-, 6,7,14,15-, and
10,11,14,15-THGG-OHs from naturally occurring (B)Chls. The pre-
sent GC analysis of DHGG- and THGG-OHs confirmed the sequen-
tial reduction pathway of a GG to a phytyl group in phototrophs
including barley, radish sprouts, diatom, and purple bacteria.
Moreover, some photosynthetic bacteria biosynthesize (B)Chls
specifically esterified with a THGG group.11–14 We note their
regiospecific reduction pathways from GG to THGG via DHGG.
Scheme 1. Synthesis of 14,15- and (11S)-10,11-dihydrogeranylgeraniols (6a and
6b): (i) Ac2O, DMAP/CH2Cl2; (ii) tBuOOH, Se2O, o-HOC6H4COOH/CH2Cl2; (iii) (EtO)2-
POCl, DMAP/CH2Cl2; (iv) Mg, I2/THF; (v) THF.
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H. Tamiaki et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
3
reaction16 of 4a with isopentylmagnesium bromide prepared
freshly from 5a [step (iv)] accompanying the cleavage of the
acetate ester by the Grignard reagent afforded 14,15-DHGG-OH
the above extract was treated with hexane in which the less polar
Chl-a was highly dissolved. The hexane phase was purified with
RP- and successive normal phase (NP)-HPLC to give a pure sample
of 10,11,14,15-tetrahydrogeranylgeranylated Chl-a. The separated
Chl-a molecule was treated with potassium hydroxide in
methanol20 to give 10,11,14,15-THGG-OH after purification by
RP-HPLC.
6a (28%) as the desired
the -attacked by-product by reverse-phase (RP) HPLC [step (v)].
Similar to the synthesis of 14,15-DHGG-OH 6a, enantiomeri-
a-attacked product after separation from
c
cally pure (11S)-10,11-dihydrogeranylgeraniol (10,11-DHGG-OH,
6b) was successfully prepared from geraniol 1b and (R)-citronellyl
bromide 5b: 1b ? 2b (71%) ? 3b (46%) ? 4b (64%) + 5b ? 6b
(15%). To obtain (7S)-6,7-dihydrogeranylgeraniol (6,7-DHGG-OH,
6c), the coupling of phosphate 4c from prenol 1c with the Grignard
reagent from 5c was applied: 5c was obtained by the bromination
of the corresponding tosylate (see below). The Grignard reaction
yielded no desired product 6c, which was ascribable to the low
reactivity of the sterically demanded Grignard reagent. Therefore,
the other pathway to 6c was examined as follows.
Halorhodospira halochloris is one of the purple bacteria species
and primarily produces BChl-b esterified with a 6,7,14,15-THGG
group (Fig. 2, right, R = 6,7,14,15-THGG).11,21 After culturing this
species, the harvested cells were extracted with acetone and
methanol. The extract was treated with petroleum ether, diethyl
ether, and distilled water at 0 °C. The separated ethereal phase
was purified with RP-HPLC to give 6,7,14,15-tetrahydrogeranylge-
ranylated BChl-b as the major fraction. The isolated BChl-b mole-
cule was methanolyzed (vide supra) to give 6,7,14,15-THGG-OH.
In the above procedures, strictly dark operation was required for
avoiding the decomposition of chemically fragile BChl-b.
The Rhodopseudomonas sp. Rits strain is a purple bacterium and
biosynthesizes BChls-a with a variety of GG and its hydrogenated
groups in the cells (Fig. 2, middle).6,22–24 The hydrogenation posi-
tions in the THGG group have been already determined to be at
C6@C7 and C10@C11 double bonds.6 Similar to the extraction
and purification of the above BChl-b molecule, BChls-a were
extracted from the cultured cells, then 6,7,10,11-tetrahydroge-
ranylgeranylated BChl-a was separated from non-, di-, and hexahy-
drogeranylgeranylated (phytylated) BChls-a and methanolyzed to
afford 6,7,10,11-THGG-OH.
According to reported procedures,17 farnesol 1a was stereo- and
regioselectively hydrogenated with a chiral ruthenium catalyst to
give 2,3-dihydrofarnesol 7 [step (i) in Scheme 2]. The product
was a 4:1 mixture of (3R) and (3S)-enantiomers from the 1H
NMR analysis of its diastereomeric ester with (R)-(-)-a-
methoxyphenylacetic acid (see Fig. S1). The isolated yield (80%)
and stereoselectivity (60% ee) were comparable to the reported
values (65% and 81% ee).17 The resulting alcohol 7 was converted
into the corresponding iodide 9 via tosylate 8 by conventional pro-
cedures [steps (ii) and (iii)]: 7 ? 8 (84%) ? 9 (70%). 2,3-Dihydro-
farnesyl iodide
9 [(3R):(3S) = 4:1] was reacted with ethyl
acetoacetate in the presence of sodium methoxide, followed by
hydrolysis and pyrolysis [step (iv)].18 The acetoacetic ester synthe-
sis gave methyl ketone 10 (32%). The Horner-Wadswarth-Emmons
reaction18 of 10 with ethyl 2-(diethoxyphosphoryl)acetate in the
2.3. Identification of regioisomeric di- and tetrahydrogeranylgeraniols
presence of sodium methoxide afforded
a,b-unsaturated methyl
The discrimination of three regioisomeric DHGG-OHs possess-
ing the C2@C3 double bond was examined. The 1H NMR spectrum
of 14,15-DHGG-OH showed a characteristic doublet peak for the
ester 11 (64%) [step (v)], which was reduced by Vitride reagent18
to yield (7S)-6,7-DHGG-OH 6c (80%, 60% ee) [step (vi)].
terminal methyl protons [C15H(CH3)2] and was different from
those of 6,7- and 10,11-DHGG-OHs (see Fig. S2). The latter two
spectra resembled each other, so their distinction was difficult
from the 1H NMR spectral analysis. The 13C NMR spectra of the
three regioisomers were partly different in CD3OD (see Fig. S3).
Especially, their three tertiary alkenyl carbons (ACH@) gave regioi-
somerically dependent peaks at around 125 ppm. A relatively large
amount of the samples was necessary for such NMR spectral mea-
surements. The authentic samples of the three DHGG-OH regioiso-
mers were submitted to GC and clearly separated as shown in the
left profiles of Fig. 3. For the GC analysis, a less volume of their
solutions was enough, then the present GC performance was useful
for the identification of the regioisomers. Regioisomeric 14,15-,
11,10-, and 6,7-DHGG-OHs were eluted in the order. The hydro-
genation of a double bond near the terminus position of the GG
group shortened the retention times (tR) of the resulting DHGG-
OHs.
Under the same conditions, the three regioisomeric THGG-OHs
were separated by the present GC (Fig. 3, right). Similarly as in
DHGG-OHs, the presence of a double bond near the THGG terminus
lengthened their tR. This observation is consistent with their GC
analysis reported previously.25 Geranylgeraniol and phytol eluted
at 14.4 and 8.4 min, respectively (see Fig. S4 and Table S1). The suc-
cessive hydrogenation of GG-OH shortened the tR.
2.2. Synthesis of tetrahydrogeranylgeraniols
Three regio- and stereoisomeric tetrahydrogeranylgeraniols,
6,7,10,11-, 6,7,14,15-, and 10,11,14,15-THGG-OHs, were prepared
by the methanolysis of naturally occurring (B)Chls. The specific
(B)Chls esterified with one of the THGG-OHs were isolated from
cultured phototrophs and the ester bonds in their 17-propionate
residues were cleaved under basic conditions to give the desired
THGG-OHs.
Green sulfur bacteria have Chl-a species as a primary electron-
acceptor in their reaction centers. The specific Chl-a molecule was
esterified with a 10,11,14,15-THGG group (see the left drawing of
Fig. 2, R = 10,11,14,15-THGG).12 Chlorobaculum tepidum,19 one of
the green sulfur bacterial species, was cultured and the harvested
cells were extracted with acetone and methanol (1:1). This species
produces a large amount of BChls-a/c, where the desired Chl-a is a
minor pigment. To roughly separate the more polar BChl pigments,
2.4. Characterization of esterifying groups in the 17-propionate
residue of chlorophyll-a
Scheme 2. Synthesis of (7S)-6,7-dihydrogeranylgeraniol (6c): (i) L*Ru(II)Cl2(p-
MeC6H4iPr), KOH/iPrOH, reflux; (ii) TsCl, DMAP/CH2Cl2, rt; (iii) NaI/THF, reflux;
(iv) AcCH2COOEt, MeONa–MeOH/THF, rt, then aq. KOH/iPrOH, 80 °C; (v) (EtO)2-
POCH2COOEt, NaOMe/C6H14, rt; (vi) NaAl(OCH2CH2OMe)2H2–PhMe, rt. L* = (S)-2,20-
[(p-MePh)2P]2–1,10-binaphytyl.
Recently, we reported that commercially available cultures of
Chaetoceros calcitrans, one of the diatoms, contained a variety of
Chls-a esterified with GG, 10,11-DHGG, and 6,7,10,11-THGG moi-
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H. Tamiaki et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
Fig. 3. GC profiles of authentic di- (left) and tetrahydrogeranylgeraniol regioisomers (right): see the analytical conditions in Section 4.4.
eties as well as a phytyl group from their spectral analyses and the
former three species were also photosynthetically active in the
cells.7 Their molecular structures were reconfirmed as follows.
From the Chaetoceros calcitrans cultures, Chl-a species were
extracted and analyzed by LC-MS. The first, second, third, and
fourth fractions (#1–#4 in Fig. 4, top) were assigned to Chls-a
esterified with GG, DHGG, THGG, and phytyl groups, respectively.
The second and third RP-HPLC fractions were separated and both
the isolated Chls-a were methanolyzed under the basic conditions
(vide supra). The resulting C20-isoprenoid alcohols, DHGG-OH and
THGG-OH, were analyzed by GC (Fig. 5, top). Regioisomeric 10,11-
DHGG-OH and 6,7,10,11-THGG-OH were exclusively detected, so
Chl-a species possessing sole 10,11-DHGG and 6,7,10,11-THGG
groups were present in the Chaetoceros calcitrans cultures. This
suggested that the GG group was biosynthetically reduced in the
hydrogenation order of the C10@C11, C6@C7, and C14@C15 double
bonds in the oxygenic phototroph primarily producing Chl-a with a
phytyl group.
During the greening in Hordeum vulgare, barley, substantial
amounts of three Chl-a species esterified with GG, DHGG, and
THGG moieties were temporally found as the precursors of mature
Chl-a with a phytyl group.10 The Chl-a pigment extract gave four
species as shown in the bottom panel of Fig. 4. After illumination
for 25 min to the etiolated leaves, the HPLC-isolated fractions #2
and #3 were chemically cleaved at the 17-propionate residue to
give DHGG-OH and THGG-OH, respectively. Their GC analyses indi-
cated the production of sole 10,11-DHGG-OH and 6,7,10,11-THGG-
OH (Fig. 5, upper middle). Their exclusive detection showed that a
GG group was hydrogenated during the Chl-a biosynthesis in bar-
ley through C10@C11 ? C6@C7 ? C14@C15, similarly as in the
diatom.
Prolonged illumination for 6 h gave a small amount of regioiso-
meric 6,7-DHGG-OH as well as 10,11-DHGG-OH, but no regioiso-
mers were detected other than 6,7,10,11-THGG-OH (Fig. 5, lower
middle). The observation can be rationalized by the following
two enzymatic pathways. 1) The C6@C7 double bond of a GG moi-
ety might be initially reduced in the late stage of the greening pro-
cess in Hordeum vulgare. 2) The 6,7,10,11-THGG moiety would be
reversibly dehydrogenated at the C10HAC11H position to give
6,7-DHGG-OH. The latter route is likely due to the favorable enzy-
matic regioselectivity at the C10@C11 over C6@C7, an increasing
amount of the THGG moiety, and an accumulation of NADP+ in
the late stage. Throughout the greening in Raphanus sativus, white
radish sprouts, 10,11-DHGG-OH and 6,7,10,11-THGG-OH were
exclusively detected after the modification of Chl-a intermediates
with dehydrogenated GG moieties (Fig. 5, bottom). Therefore, a
GG moiety is enzymatically reduced to give phytylated Chl-a via
10,11-DHGG and 6,7,10,11-THGG species in Raphanus sativus.
2.5. Characterization of esterifying groups in the 17-propionate
residue of bacteriochlorophylls
In full growth cultures of many purple bacteria, BChls esterified
with GG, DHGG, and THGG groups were observed as well as stan-
dard phytylated BChls.6,22–24 They work photosynthetically in the
light-harvesting and reaction center apparatuses. Rhodobacter
sphaeroides, one of the purple photosynthetic bacteria, produced
Fig. 4. RP-HPLC profiles of Chl-a species isolated from commercially available
Chaetoceros calcitrans cultures (top) and the greening leaves of Hordeum vulgare
(bottom, 25-min illumination): Cosmosil 5C18-AR-II 3.0 ꢀ 150 mm, methanol:
acetonitrile = 37:3, 1.0 ml/min.
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H. Tamiaki et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
5
Fig. 5. GC profiles of di- (left) and tetrahydrogeranylgeraniol regioisomers (right) derived from Chl-a in commercially available Chaetoceros calcitrans cultures (top) as well as
the greening leaves of Hordeum vulgare (upper and lower middle after illumination for 25 min and 6 h, respectively) and Raphanus sativus (bottom, 6-h illumination): see the
analytical conditions in Section 4.4. Peaks at the asterisk are ascribable to undetermined impurities.
BChls-a with GG, DHGG, THGG, and phytyl groups in the first, sec-
ond, third, and fourth eluted fractions, respectively in RP-HPLC
(Fig. 6, top).22,24 The structures of the DHGG and THGG moieties
were determined by HPLC-isolation of the BChl-a fractions #20/
#30, methanolysis to their alcohols, and successive GC analyses.
The top GC profiles of Fig. 7 proved the production of sole 10,11-
DHGG-OH and 6,7,10,11-THGG-OH. Therefore, phytylated BChl-a
in the purple bacterium was biosynthesized through sequential
enzymatic reduction of the C10@C11, C6@C7, and C14@C15 of a
GG moiety.
For the Rhodopseudomonas sp. Rits strain, another purple bac-
terium, larger amounts of BChl-a intermediates were accumulated
in its cultured cells.6,22–24 Based on the GC analysis (Fig. 7, middle),
the culture gave also the same BChl-a intermediates with the
10,11-DHGG and 6,7,10,11-THGG groups as in the above Rhodobac-
ter sphaeroides. The observation is partially different from our pre-
vious report6 that a specific strain produced BChls-a bearing 6,7-
DHGG and 6,7,10,11-THGG groups (see Introduction section). Their
molecular structures had been characterized by their 1H NMR
spectra. The structural conflict in the DHGG moiety may be depen-
dent on the culturing conditions. BChl-a esterified with a 6,7-
DHGG group might be produced by similar dehydrogenation of
the 6,7,10,11-THGG group as in the greening in Hordeum vulgare
(vide supra). From various stages of the cultured strain, no more
6,7-DHGG-OH could be detected in the present GC analysis. The
reason for the inconsistency is being pursued and will be reported
anywhere if fixed.
Halorhodospira halochloris, one of the thermophilic purple bac-
teria, produced predominantly BChl-b esterified with a 6,7,14,15-
THGG group.11,21 Based on LC-MS analyses, the cultured cells con-
tained small amounts of BChls-b with GG and DHGG moieties as
the first and second fractions, respectively (see Fig. 6, bottom),
without phytylated BChl-b.21 GC analyses of their esterifying alco-
hols (Fig. 7, bottom) clearly indicated that the cells biosynthesized
BChls-b with 6,7-DHGG and 6,7,14,15-THGG moieties. As a result,
the GG moiety in Halorhodospira halochloris was first reduced at
the C6@C7, followed by hydrogenation at the C14@C15. The reduc-
Fig. 6. RP-HPLC profiles of BChl-a isolated from Rhodobacter sphaeroides (top) and
BChl-b from Halorhodospira halochloris (bottom): Cosmosil 5C18-AR-II 4.6 ꢀ 150
mm, methanol:acetonitrile = 37:3 (top) and methanol:water = 19:1 (bottom), 1.0
ml/min.
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H. Tamiaki et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
Fig. 7. GC profiles of di- (left) and tetrahydrogeranylgeraniol regioisomers (right) derived from BChl-a in Rhodobacter sphaeroides (top) and Rhodopseudomonas sp. (Rits strain,
middle) as well as BChl-b in Halorhodospira halochloris (bottom): see the analytical conditions in Section 4.4.
tion sequence was the same as those described above, although the
initial hydrogenation at the C10@C11 observed in the other pho-
totrophs was inhibited. The C10@C11 double bond of a GG sub-
strate would be fully protected from the enzymatic reduction in
Halorhodospira halochloris cells. It cannot be ruled out that the
enzymatic active site is different from the others.
possessing the 6,7,14,15-THGG group and the biosynthetic route
was proposed to be the order of GG ? 6,7-DHGG ? 6,7,14,15-
THGG. This sequence is consistent with that in the above standard
purple bacteria, except for the complete suppression of the
C10@C11 reduction. Based on the final reduction at the
C14@C15, Chl-a bearing the 10,11,14,15-THGG group found in
green sulfur bacteria might be produced through the successive
reduction of C10@C11 ? C14@C15 of a GG moiety, where the
C6@C7 reduction is fully inhibited.12
3. Conclusions
To elucidate the hydrogenation (reduction) pathway from a GG
group to THGG and phytyl groups in the last stage of (B)Chl biosyn-
thesis, three possible DHGG-OH regioisomers were synthesized
through CAC couplings. The authentic DHGG-OHs as well as three
THGG-OHs obtained from naturally occurring and structurally
determined (B)Chls were analyzed by GC, showing well-resolved
peaks for the six C20-isoprenoid alcohols. Trace amounts of (B)Chl
intermediates produced temporally in phototrophs were isolated
by HPLC and successively methanolyzed to give DHGG- and
THGG-OHs. The resulting alcohols were characterized by the above
GC analyses and the molecular structures of the biosynthetic (B)
Chl precursors were identified.
During the greening processes in etiolated leaves of barley and
radish sprouts, 10,11-DHGG-OH and 6,7,10,11-THGG-OH were
observed from the derivatization of Chls-a isolated in the initial
stage and comparison of their GC peaks. The regioselectivity indi-
cated the following hydrogenation sequence. A GG moiety as the
esterifying group in the 17-propionate residue of Chl-a was first
reduced at the C10@C11 double bond, followed by the hydrogena-
tion of the C6@C7, and transformed to a phytyl moiety by the final
reduction of the C14@C15. The first hydrogenation position of the
GG group in the oxygenic phototrophs was confirmed by the pre-
sent work to be its C10@C11 and the biosynthetic pathway for
Chl-a was fully determined here. In addition, the same pathway
was proposed in a diatom, which was consistent with the recent
report investigated by spectroscopic analyses.7
4. Experimental
4.1. General
1H NMR spectra were recorded on a JEOL AL-400 (400 MHz) or
ECA-600 (600 MHz) spectrometer; residual CHCl3 (dH = 7.26 ppm)
or CD2HOD (dH = 3.31 ppm) was used as an internal reference.
13C NMR spectra were recorded on an ECA-600 (151 MHz) spec-
trometer; residual 13CD3OD (dC = 49.00 ppm) was used as an inter-
nal reference. High resolution mass spectra (HRMS) were recorded
on a Bruker micrOTOF II spectrometer: atmospheric pressure
chemical ionization (APCI) and positive mode in an acetonitrile
solution. FCC was performed with silica gel (Merck, Kieselgel 60,
40–63 lm, 230–400 mesh). HPLC was performed by a Shimadzu
LC-10ADVP pump, SPD-M10AVP diode-array detector, and
SCL-10AVP system controller using a packed ODS column (Nacalai
Tesque Cosmosil 5C18-AR-II) for RP or a packed silica gel column
(Cosmosil 5SL-II) for NP. LC-MS was obtained on a Shimadzu
LCMS-2010EV chromatograph with APCI and positive mode. GC
was done with
chromatograph with an AOC-20i auto-injector and an FID-2010
plus flame ionization detector.
Chaetoceros calcitrans cultures were purchased from Marintech
Co Ltd, and the Rhodobacter sphaeroides ATH2.4.1, Rhodopseu-
domonas sp. Rits, and Chlorobaculum tepidum ATCC49652 strains
were in our collection. All the reaction reagents were obtained
from commercial suppliers and utilized as supplied. Commercially
available solvents (Nacalai Tesque) were used for the synthetic
procedures and distilled water was prepared by a Yamato AutoStill
WG250 system. All HPLC solvents except distilled water were
obtained as HPLC grade from Nacalai Tesque and the mixed sol-
vents were degassed before use.
a Shimadzu GC-2010 plus high-end gas
Based on the aforementioned GC analysis, both the Rhodobacter
sphaeroides and Rhodopseudomonas palustris strains produced
BChls-a esterified with 10,11-DHGG and 6,7,10,11-THGG groups
in their full-growth cultures. Purple photosynthetic bacteria would
biosynthesize phytylated BChl-a through the same sequential
reduction of GG moiety as in the above angiosperm. Halorhodospira
halochloris, another purple bacterium, specifically produced BChl-b
Please cite this article in press as: Tamiaki H., et al. Bioorg. Med. Chem. (2017), https://doi.org/10.1016/j.bmc.2017.10.002

H. Tamiaki et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
7
4.2. Synthesis of dihydrogeranylgeraniols
in 70% yield: colorless oil; 1H NMR (CDCl3, 600 MHz) d = 5.11–5.08
(2H, m, 6-, 10-H), 3.27–3.15 (2H, m, CH2I), 2.09–1.86 (6H, m, 4-, 7-,
8-CH2), 1.68 (3H, s, 11-CH3 trans to C10ACH2), 1.60 (6H, s, 7-CH3,
11-CH3 cis to C10ACH2), 1.38–1.15 (5H, m, 1-CH2CHCH2), 0.89
(3H, d, J = 7 Hz, 3-CH3); see also the spectral data of its racemic
mixture in Ref. 18.
4.2.1. 6,7-Dihydrogeranylgeraniol (6c)
To
a solution of farnesol (1a, 50.0 mg, 225 mmol) in
2-propanol (22.5 ml, dried over molecular sieves 3A for 1 day)
were chloro[(S)-(-)-2,20-bis(di-p-tolyl-phosphino)-1,10-binaphytyl]
(p-cymene) ruthenium(II) chloride (22 mg, 24
l
mol) and potas-
The above iodide 9 (218 mg, 0.652 mmol) was dissolved in THF
(5 ml), to which were added 28% sodium methoxide in methanol
sium hydroxide (85%, 3.0 mg, 45 mol) and the mixture was
l
refluxed under nitrogen for 2 days. The reaction mixture was
cooled down to room temperature, to which were added distilled
water and hexane with stirring. The separated organic phase was
washed twice with distilled water and brine and dried over sodium
sulfate. After evaporation of the solvent, the residue was purified
with FCC (hexane:ethyl acetate = 4:1) to give 2,3-dihydrofarnesol
(7, 40.4 mg, 180 mmol) in 80% isolated yield (lit.17 65%) as a 4:1
mixture of (3R)- and (3S)-enantiomers (60% ee, see below; lit.17
81% ee): yellow oil; 1H NMR (CDCl3, 600 MHz) d = 5.12–5.08 (2H,
m, 6-, 10-H), 3.73–3.64 (2H, m, CH2O), 2.08–1.95 (6H, m, 4-, 7-,
8-CH2), 1.68 (3H, s, 11-CH3 trans to C10ACH2), 1.60 (6H, s, 7-CH3,
11-CH3 cis to C10ACH2), 1.42–1.15 (5H, m, 1-CH2CHCH2), 0.91
(3H, d, J = 7 Hz, 3-CH3) [The 1-OH was invisible.]; see the spectral
data of its racemic mixture in Ref. 18.
(0.3 ml, 1.5 mmol) and ethyl acetoacetate (441 ll, 450 mg, 3.46
mmol). The mixture was stirred at room temperature under nitro-
gen for 20 h, diluted with distilled water, and extracted with
diethyl ether. The ethereal phase was washed twice with distilled
water and brine and dried over sodium sulfate. After evaporation of
the solvents, the residue was dissolved in 2-propanol (2 ml), to
which was added aq. 50% sodium hydroxide. The mixture was
heated at 80 °C under nitrogen for 1 h, cooled down to room tem-
perature, acidified with aq. 0.1 M hydrogen chloride, and extracted
with diethyl ether. The ethereal phase was washed twice with aq.
0.1 M hydrogen chloride, aq. 4% sodium hydrogen carbonate, dis-
tilled water, and brine and dried over sodium sulfate. After evapo-
ration of the solvents, the residue was purified with FCC (hexane:
ethyl acetate = 9:1) to give 6,10,14-trimethylpentadeca-9,13-
dien-2-one (10, 55 mg, 0.208 mmol) in 32% yield: colorless oil;
1H NMR (CDCl3, 600 MHz) d = 5.11–5.08 (2H, m, 9-, 13-H), 2.41–
2.39 (2H, m, 1-COCH2), 2.13 (3H, s, COCH3), 2.08–1.91 (6H, m, 7-,
10-, 11-CH2), 1.68 (3H, s, 14-CH3 trans to C13ACH2), 1.62–1.50
(2H, m, 3-CH2), 1.60 (3H, s, 14-CH3 cis to C13ACH2), 1.59 (3H, s,
10-CH3), 1.43–1.11 (5H, m, 4-CH2CHCH2), 0.87 (3H, d, J = 7 Hz, 6-
CH3); see also the spectral data of its racemic mixture in Ref. 18.
The above alcohol
methoxyphenylacetic acid (18 mg, 110
dichloromethane (0.5 ml), to which N,N0-diisopropylcarbodiimide
(13.8 mg, 109 mol) and DMAP (6 mg, 50 mol) were added. The
7
(22 mg, 98
lmol) and (R)-(-)-a-
l
mol) were dissolved in
l
l
mixture was stirred at room temperature under argon for 12 h.
After addition of diethyl ether to the reaction mixture, the ethereal
phase was washed with aq. 1% hydrogen chloride, aq. 4% sodium
hydrogen carbonate, distilled water, and brine and dried over
sodium sulfate. All the solvents were evaporated and the residue
was purified with FCC (hexane:ethyl acetate = 10:1) to give 2,3-
To a solution of the above ketone 10 (50 mg, 189
ane (1 ml) were added sodium methoxide (80 mg, 1480
ethyl 2-(diethoxyphosphoryl)acetate (80 mg, 357 mol). The mix-
lmol) in hex-
l
mol) and
l
dihydrofarnesyl (R)-
a
-methoxyphenylacetate (20 mg, 54 mmol)
ture was stirred at room temperature under nitrogen for 2 h and
the reaction was quenched with distilled water. After extraction
with hexane, the organic phase was washed with twice with aq.
60% methanol and brine and dried over sodium sulfate. After evap-
oration of the solvent, the residue was purified with FCC (hexane:
ethyl acetate = 19:1) to give methyl 3,7,11,15-tetramethylhexa-
in 55% yield with 60% de: colorless oil; 1H NMR (CDCl3, 600
MHz) d (3R/3S = 4/1) = 7.33–7.44 (5H, m, 1-OCOCC6H5), 5.11–5.04
(2H, m, 6-, 10-H), 4.75 (1H, s, 1-OCOCH), 4.21–4.10 (2H, m,
CH2O), 3.41 (2H, s, OCH3), 2.09–1.85 (6H, m, 4-, 7-, 8-CH2), 1.68
(3H, s, 11-CH3 trans to C10ACH2), 1.60 (3H, s, 11-CH3 cis to
C10ACH2) 1.58 (3H, s, 7-CH3), 1.43–1.08 (5H, m, 1-CH2CHCH2),
0.81/0.84 (3H, d, J = 7 Hz, 3-CH3).
deca-2,10,14-trienoate (11, 39.0 mg, 122 lmol) in 64% yield: color-
less oil; 1H NMR (CDCl3, 600 MHz) d = 5.67–5.65 (1H, m, 2-H),
5.12–5.08 (2H, m, 10-, 14-H), 3.68 (3H, s, COOCH3), 2.15 (3H, s,
3-CH3), 2.13–1.92 (8H, m, 3-, 8-, 11-, 12-CH2), 1.68 (3H, s, 15-
CH3 trans to C13ACH2), 1.60 (3H, s, 15-CH3 cis to C13ACH2), 1.59
(3H, s, 11-CH3), 1.43–1.08 (7H, m, 4-CH2CH2CHCH2), 0.87 (3H, d,
J = 7 Hz, 7-CH3); see also the spectral data of its racemic mixture
in Ref. 18.
To a solution of (3R)-2,3-dihydrofarnesol (7, 60% ee, 159 mg,
709
lmol) in dichloromethane (10 ml) were added p-tosyl chloride
(200 mg, 1050
lmol) and DMAP (88 mg, 720 mol). The mixture
l
was stirred at room temperature under nitrogen for 16 h and the
reaction was quenched with distilled water. After extraction with
diethyl ether, the organic phase was washed twice with aq. 1%
hydrogen chloride, aq. 4% sodium hydrogen carbonate, distilled
water, and brine and dried over sodium sulfate. After evaporation
of the solvent, the residue was purified with FCC (hexane:ethyl
acetate = 9:1) to give 2,3-dihydrofarnesyl p-tosylate (8, 225.1 mg,
To a hexane solution (1 ml) of the above ester 11 (30 mg, 94
l
mol) was added a toluene solution of sodium bis(2-methoxy-
ethoxy)aluminum hydride (Red-Al, ꢁ70%, 118 l, 120 mg, ꢁ420
mol) and stirred at room temperature under nitrogen for 2 h. A
l
l
595
l
mol) in 84% yield: colorless oil; 1H NMR (CDCl3, 400 MHz)
small amount of acetone and aq. 50% acetic acid (1 ml) were added
to the reaction mixture and further stirred at room temperature
under nitrogen for 1 h. After extraction with diethyl ether, the
organic phase was washed with aq. 4% sodium hydrogen carbonate
and brine and dried over sodium sulfate. The solvents were
evaporated and the residue was purified with FCC (hexane:ethyl
d = 7.93 (2H, d, J = 8 Hz, o-H of Ts), 7.34 (2H, d, J = 8 Hz, m-H of
Ts), 5.10–5.04 (2H, m, 6-, 10-H), 4.10–4.04 (2H, m, CH2O), 2.45
(3H, s, p-CH3 of Ts), 2.09–1.86 (6H, m, 4-, 7-, 8-CH2), 1.68 (3H, s,
11-CH3 trans to C10ACH2), 1.60 (3H, s, 11-CH3 cis to C10ACH2),
1.57 (3H, s, 7-CH3), 1.48–1.07 (5H, m, 1-CH2CHCH2), 0.82 (3H, d,
J = 7 Hz, 3-CH3); see the spectral data of its racemic mixture in
Ref. 26.
A THF solution (20 ml) of the above tosylate 8 (665 mg, 1.76
mmol) and sodium iodide (3.0 g, 20 mmol) was refluxed under
argon for 2 h and cooled down to room temperature, followed by
the addition of distilled water. After extraction with diethyl ether,
the organic phase was washed twice with aq. 10% sodium thiosul-
fate, distilled water and brine and dried over sodium sulfate. After
evaporation of the solvents, the residue was purified with FCC
(hexane) to give 2,3-dihydrofarnesyl iodide (9, 410 mg, 1.23 mmol)
acetate = 6:1) to give 6,7-DHGG-OH 6c (22 mg, 75 lmol) in 80%
yield: colorless oil; 1H NMR (CD3OD, 600 MHz) d = 5.35 (1H, tq,
J = 7, 2 Hz, 2-H), 5.10, 5.09 (each 1H, br-t, J = 7 Hz, 10-, 14-H),
4.08 (2H, d, J = 7 Hz, CH2O), 2.08 (2H, q, J = 7 Hz, 12-CH2), 2.05–
1.94 (6H, m, 3-, 8-, 11-CH2), 1.67 (3H, s, 15-CH3 trans to
C13ACH2), 1.66 (3H, s, 3-CH3), 1.60 (6H, s, 11-CH3, 15-CH3 cis to
C13ACH2), 1.49–1.28, 1.18–1.08 (5H + 2H, m, 4-CH2CH2CHCH2),
0.88 (3H, d, J = 7 Hz, 7-CH3) [The 1-OH was invisible.]; 13C NMR
(CD3OD, 151 MHz) d = 139.73, 135.62, 132.05, 126.11, 125.46,
124.79, 59.42, 40.93, 40.87, 38.19, 37.67, 33.32, 27.73, 26.38,
Please cite this article in press as: Tamiaki H., et al. Bioorg. Med. Chem. (2017), https://doi.org/10.1016/j.bmc.2017.10.002

8
H. Tamiaki et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
26.28, 25.89, 20.05, 17.74, 16.13, 16.03; HRMS (APCI) found: m/z
291.2682, calcd for C20H35O: [MꢂH]+, 291.2682; see also the spec-
tral data of its racemic mixture in Ref. 18.
3-CH3), 1.61, 1.60 (each 3H, s, 7-CH3, 15-CH3 cis to C14ACH2),
1.47–1.25, 1.17–1.04 (5H+2H, m, 8-CH2CH2CHCH2), 0.87 (3H, d,
J = 7 Hz, 11-CH3); 13C NMR (CD3OD, 151 MHz) d = 139.44, 136.42,
131.84, 126.04, 125.10, 124.93, 59.44, 40.97, 40.75, 38.29, 37.52,
33.40, 27.40, 26.56, 26.37, 25.90, 20.08, 17.73, 16.26, 15.94; HRMS
(APCI) found: m/z 291.2678, calcd for C20H35O: [MꢂH]+, 291.2682;
see also the spectral data of the (11R)-epimer in Ref. 27.
4.2.2. 10,11-Dihydrogeranylgeraniol (6b)
To a solution of geraniol (1b, 4.45 g, 28.8 mmol) in dichloro-
methane (15 ml) were added DMAP (175 mg, 1.43 mmol) and acetic
anhydride (6.0 ml, 63 mmol) and the mixture was stirred at room
temperature under nitrogen for 1 h. After addition of methanol
(10 ml), the reaction mixture was further stirred for 1 h, diluted with
diethyl ether, washed twice with aq. 4% sodium hydrogen carbon-
ate, distilled water, and brine, and dried over sodium sulfate. The
solvents were evaporated and the residue was purified with FCC
(hexane:ethyl acetate = 20:1) to give geranyl acetate (2b, 4.04 g,
20.6 mmol) in 71% yield: colorless oil; 1H NMR (CDCl3, 400 MHz)
d = 5.34 (1H, t, J = 7 Hz, 2-H), 5.08 (1H, t, J = 7 Hz, 6-H), 4.58 (2H, d,
J = 7 Hz, CH2O), 2.13–2.03 (4H, m, 3-CH2CH2), 2.06 (3H, s, 1-
OCOCH3), 1.70 (3H, s, 3-CH3), 1.68 (3H, s, 7-CH3 trans to C6ACH2),
1.60 (3H, s, 7-CH3 cis to C6ACH2); see also its spectral data in Ref. 15.
The above acetate 2b (1.02 g, 5.20 mmol) was dissolved in
dichloromethane (10 ml) and cooled down to 0 °C, to which were
added an aqueous 70% tert-butyl hydroperoxide solution (1.8 ml,
13 mmol), salicylic acid (88 mg, 0.64 mmol), and selenium oxide
(30 mg, 0.27 mmol). The mixture was stirred at room temperature
for 2 days and the reaction was quenched with distilled water. After
extraction with diethyl ether, the organic phase was washed twice
with distilled water and brine and dried over sodium sulfate. All the
solvents were evaporated and the residue was purified with FCC
(hexane:ethyl acetate = 4:1) to give 8-hydroxy-farnesyl acetate
(3b, 505 mg, 2.38 mmol) in 46% isolated yield: colorless oil; 1H
NMR (CDCl3, 600 MHz) d = 5.38–5.33 (2H, m, 2-, 6-H), 4.58 (2H, d,
J = 7 Hz, CH2OAc), 3.99 (3H, s, 7-CH2), 2.19–2.08 (4H, m, 3-CH2CH2),
2.05 (3H, s, 1-OCOCH3), 1.71 (3H, s, 7-CH3), 1.66 (3H, s, 3-CH3) [The
8-OH was invisible.]; see also its spectral data in Ref. 15.
4.2.3. 14,15-Dihydrogeranylgeraniol (6a)
Similar to the synthesis of geranyl acetate (2b, see Section 4.2.2),
the esterification of farnesol (1a, 545 mg, 2.45 mmol) with acetic
anhydride (0.75 ml, 7.9 mmol) and DMAP (60 mg, 0.49 mmol) in
dichloromethane (20 ml) gave farnesyl acetate (2a, 490 mg, 1.85
mmol) in 76% yield: colorless oil; 1H NMR (CDCl3, 400 MHz) d =
5.34 (1H, t, J = 7 Hz, 2-H), 5.11–5.06 (2H, m, 6-, 10-H), 4.58 (2H,
d, J = 7 Hz, CH2O), 2.14–1.95 (8H, m, 3-, 4-, 7-, 8-CH2), 2.05 (3H,
s, 1-OCOCH3), 1.70 (3H, s, 3-CH3), 1.68 (3H, s, 11-CH3 trans to
C10ACH2) 1.60 (6H, s, 7-CH3, 11-CH3 cis to C10ACH2); see also
its spectral data in Ref. 15.
Similar to the synthesis of 8-hydroxy-farnesyl acetate (3b, see
Section 4.2.2), the above acetate 2a (490 mg, 1.85 mmol) in dichlo-
romethane (10 ml) was oxidized by an aqueous 70% tert-butyl
hydroperoxide solution (1.0 ml, 7.3 mmol), salicylic acid (60 mg,
0.43 mmol), and selenium oxide (13 mg, 0.12 mmol) for 1 day to
give 12-hydroxy-farnesyl acetate (3a, 142 mg, 0.506 mmol) in
27% isolated yield: colorless oil; 1H NMR (CDCl3, 600 MHz) d =
5.39 (1H, t, J = 7 Hz, 10-H), 5.34 (1H, t, J = 7 Hz, 2-H), 5.10 (1H, t, J
= 7 Hz, 6-H), 4.59 (2H, d, J = 7 Hz, CH2OAc), 3.99 (2H, s, 11-CH2),
2.15–2.00 (8H, m, 3-, 4-, 7-, 8-CH2), 2.05 (3H, s, 1-OCOCH3), 1.71
(3H, s, 11-CH3), 1.67 (3H, s, 3-CH3), 1.60 (3H, s, 7-CH3) [The 12-
OH was invisible.]; see also its spectral data in Ref. 15.
Similar to the synthesis of 8-diethoxyphosphoryloxy-geranyl
acetate (4b, see Section 4.2.2), the esterification of the above alco-
hol 3a (142 mg, 506
1100 mol) and DMAP (60 mg, 491
diethoxyphosphoryloxy-farnesyl acetate (4a, 134 mg, 322
l
mol) with diethyl chlorophosphate (190 mg,
mol) for 3 h gave 12-
mol)
The above alcohol 3b (60 mg, 283
dichloromethane (2 ml), to which were added diethyl chlorophos-
phate (103.2 mg, 598 mol) and DMAP (60 mg, 491 mol). The ice-
lmol) was dissolved in
l
l
l
l
l
in 64% yield: colorless oil; 1H NMR (CDCl3, 600 MHz) d = 5.48
(1H, t, J = 7 Hz, 10-H), 5.35 (1H, t, J = 7 Hz, 2-H), 5.11 (1H, t, J = 7
Hz, 6-H), 4.59 (2H, d, J = 7 Hz, CH2OAc), 4.39 (2H, s, 11-CH2O),
4.11 (4H, q, J = 7 Hz, POCH2 ꢀ 2), 2.15–2.00 (8H, m, 3-, 4-, 7-, 8-
CH2), 2.05 (3H, s, 1-OCOCH3), 1.70 (3H, s, 11-CH3), 1.69 (3H, s, 3-
CH3), 1.60 (3H, s, 7-CH3), 1.33 (6H, t, J = 7 Hz, POCCH3 ꢀ 2).
Similar to the synthesis of 10,11-dihydrogeranylgeraniol (6b),
isopentylmagnesium bromide prepared by magnesium (72 mg,
3.0 mmol), a piece of iodine, and 1-bromo-3-methylbutane (5a,
cooled mixture was stirred under nitrogen for 6 h and the reaction
was quenched with distilled water. After extraction with diethyl
ether, the organic phase was washed twice with aq. 0.1 M hydro-
gen chloride, distilled water, and brine and dried over sodium sul-
fate. All the solvents were evaporated and the residue was purified
with FCC (hexane:ethyl acetate:methanol = 50:50:1) to give 8-
diethoxyphosphoryloxy-geranyl acetate (4b, 63 mg, 181 lmol) in
64% yield: colorless oil; 1H NMR (CDCl3, 600 MHz) d = 5.48 (1H,
m, 6-H), 5.34 (1H, m, 2-H), 4.58 (2H, d, J = 7 Hz, CH2OAc), 4.39
(2H, m, 7-CH2O), 4.15–4.07 (4H, m, POCH2 ꢀ 2), 2.20–2.07 (4H,
m, 3-CH2CH2), 2.06 (3H, s, 1-OCOCH3), 1.70 (3H, s, 3-CH3), 1.69
(3H, s, 7-CH3), 1.33 (6H, t, J = 7 Hz, POCCH3 ꢀ 2).
isopentyl bromide, 313
ml) for 2 h, was reacted with the above phosphate 4a (134 mg,
322 mol) to give 14,15-DHGG-OH 6a (26 mg, 89 mol) in 28%
ll, 375 mg, 2.48 mmol) in dry THF (10+5
l
l
yield: colorless oil; 1H NMR (CD3OD, 600 MHz) d = 5.36 (1H, tq, J
= 7, 2 Hz, 2-H), 5.14 (1H, tq, J = 7, 2 Hz, 6-H), 5.10 (1H, tq, J = 7, 2
Hz, 10-H), 4.08 (2H, d, J = 7 Hz, CH2O), 2.13, 2.09 (each 2H, q, J =
8 Hz, 4-, 8-CH2), 2.04 (2H, t, J = 8 Hz, 3-CH2), 2.00 (2H, t, J = 8 Hz,
7-CH2), 1.95 (2H, t, J = 7 Hz, 11-CH2), 1.67 (3H, s, 3-CH3), 1.61
(3H, s, 7-CH3), 1.58 (3H, s, 11-CH3), 1.53 (1H, nonet, J = 7 Hz, 15-
H), 1.39 (2H, quintet, J = 7 Hz, 12-CH2), 1.14 (2H, q, J = 7 Hz, 13-
CH2), 0.88 (6H, d, J = 7 Hz, 15-CH3 ꢀ 2) [The 1-OH was invisible.];
13C NMR (CD3OD, 151 MHz) d = 139.44, 136.15, 136.13, 125.29,
125.26, 124.92, 59.43, 40.96, 40.81, 40.75, 39.72, 29.07, 27.53,
27.45, 26.84, 23.04 (15-C2), 16.26, 16.05, 15.96; HRMS (APCI)
found: m/z 291.2678, calcd for C20H35O: [MꢂH]+, 291.2682.
Dry THF (15 ml) was added to magnesium (170 mg, 7.0 mmol)
and a piece of iodine under argon. (R)-Citronellyl bromide (5b,
1.5 g, 6.8 mmol) was slowly dropped into the stirred mixture at
room temperature and THF (30 ml) was further added with stirring
for 1 h. To the THF solution of the resulting Grignard reagent was
gradually added the above phosphate (636 mg, 1.83 mmol). After
stirring for 4 h at room temperature, the reaction was quenched
with aq. 1% ammonium chloride and the mixture was extracted
with diethyl ether. The ethereal phase was washed with distilled
water and dried over sodium sulfate. All the solvents were evapo-
rated and the residue was purified with FCC (hexane:ethyl acetate
= 6:1) and RP-HPLC (acetonitrile) to give (11S)-10,11-DHGG-OH 6b
(80 mg, 273
l
mol) in 15% yield: colorless oil; 1H NMR (CD3OD, 600
4.3. Preparation of tetrahydrogeranylgeraniols
MHz) d = 5.36 (1H, tq, J = 7, 2 Hz, 2-H), 5.13 (1H, br-t, J = 7 Hz, 6-H),
5.10 (1H, br-t, J = 7 Hz, 14-H), 4.08 (2H, d, J = 7 Hz, CH2O), 2.13 (2H,
q, J = 7 Hz, 4-CH2), 2.04 (2H, t, J = 7 Hz, 3-CH2), 2.01–1.92 (4H, m,
7-, 12-CH2), 1.68 (3H, s, 15-CH3 trans to C14ACH2), 1.67 (3H, s,
4.3.1. 6,7,10,11-Tetrahydrogeranylgeraniol
Wet cells from a fully grown liquid culture of the Rhodopseu-
domonas sp. Rits strain22 were treated with a 1:1 (v/v) mixture of
Please cite this article in press as: Tamiaki H., et al. Bioorg. Med. Chem. (2017), https://doi.org/10.1016/j.bmc.2017.10.002

H. Tamiaki et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
9
acetone and methanol (2 ml). The filtrate was stirred with a
1:1 (v/v) mixture of petroleum ether and diethyl ether (2 ml) and
ice-cooled. After addition of ice-chilled distilled water (4 ml), the
mixture was stirred vigorously and centrifuged. The separated
ethereal phase was dried by a stream of nitrogen gas and the resi-
due was purified with RP-HPLC (methanol:acetone:water = 16:3:1)
to separate BChl-aTHGG from BChls-a esterified by the other
isoprenoid alcohols.6 The sample containing the tetrahydroge-
ranylgeranylated chlorophyll pigments was dissolved in methanol
(2 ml) with stirring, to which was dropped a 20% methanol
solution of potassium hydroxide (2 ml) at room temperature. After
stirred for 20 min, the reaction mixture was extracted twice with
hexane. The combined hexane phases were washed with distilled
water and dried by a stream of nitrogen gas. The residue was puri-
fied with RP-HPLC (methanol) to give (7S,11S)-6,7,10,11-THGG-
OH: Cosmosil 5C18-AR-II 6.0 ꢀ 250 mm, 1.0 ml/min, 9.8 min. After
evaporation of the eluted methanol, the residual sample was
dissolved in hexane and subjected to GC analysis.
dried by a stream of nitrogen gas. The residue was purified with
RP-HPLC to give Chl-a species.
Acknowledgements
We would like to thank Dr. Megumi Isaji and Prof. Masahiro
Kasahara of Ritsumeikan University and Mr. Shigeaki Shibamoto
of Shimadzu Corporation for their experimental assistance, Prof.
Hiroshi Shinokubo of Nagoya University for his valuable informa-
tion of CAC coupling, and Dr. Yusuke Kinoshita of Ritsumeikan
University for his aid of manuscript preparation. This work was
partially supported by JSPS KAKENHI Grant Numbers JP24107002
and JP17H06436 in Scientific Research on Innovative Areas.
A. Supplementary data
Supplementary data (1H NMR spectrum of chiral esters of asym-
metrically synthesized 2,3-dihydrofarnesol, 1H and 13C NMR spec-
tra of DHGG-OHs, and GC profiles of C20-isoprenoid alcohols with
their tR) associated with this article can be found, in the online ver-
sion, at https://doi.org/10.1016/j.bmc.2017.10.002.
4.3.2. 6,7,14,15-Tetrahydrogeranylgeraniol
As mentioned in Section 4.3.1, wet cells of the Halorhodospira
halochloris DSM1059 strain (DSMZ)21 were similarly treated to give
a pigment mixture. The mixture was purified with RP-HPLC
(methanol:water = 19:1) to give BChl-bTHGG as a major compo-
nent.11,21 Caution: all the above procedures must be performed
in the dark to avoid the decomposition of chlorophyll pigments.
The separated BChl-b was methanolyzed similarly as in Sec-
tion 4.3.1 to give (7S)-6,7,14,15-THGG-OH as the authentic sample
for GC analysis.
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