Semi-synthesis and HPLC analysis of (bacterio)chlorophyllides possessing a propionic acid residue at the C17-position

Article abstract of DOI:10.1142/S1088424618500347

Various chlorophyll and bacteriochlorophyll derivatives possessing a magnesium or zinc atom at the central position and a free carboxylic acid group at the C17²-position, also known as (bacterio)chlorophyllides, were synthesized through a combination of organic synthesis techniques and enzymatic steps. The semi-synthetic (bacterio)chlorophyllides were purified and analyzed using reversed-phase high-performance liquid chromatography with UV-vis spectroscopy and mass spectrometry. These free propionic acid-containing chlorophyllous pigments can be useful research materials for the study of (bacterio)chlorophyll metabolisms.

Full text of DOI:10.1142/S1088424618500347

Journal of Porphyrins and Phthalocyanines
J. Porphyrins Phthalocyanines 2018; 22: 1–14
DOI: 10.1142/S1088424618500347
Published at http://www.worldscinet.com/jpp/
Semi-synthesis and HPLC analysis of
(bacterio)chlorophyllides possessing a propionic acid
residue at the C17-position
Misato Teramura and Hitoshi Tamiaki*◊
Graduate School of Life Sciences, Ritsumeikan University, Kusatsu, Shiga 525-8577, Japan
Received 3 February 2018
Accepted 20 February 2018
ABSTRACT: Various chlorophyll and bacteriochlorophyll derivatives possessing a magnesium or
zinc atom at the central position and a free carboxylic acid group at the C172-position, also known as
(bacterio)chlorophyllides, were synthesized through a combination of organic synthesis techniques and
enzymatic steps. The semi-synthetic (bacterio)chlorophyllides were purified and analyzed using reversed-
phase high-performance liquid chromatography with UV-vis spectroscopy and mass spectrometry. These
free propionic acid-containing chlorophyllous pigments can be useful research materials for the study of
(bacterio)chlorophyll metabolisms.
KEYWORDS: bacteriochlorophyll, chlorophyll, enzymatic reaction, high-performance liquid
chromatography, hydrolysis, propionate residue.
group at the free C17-propionate (C17-propionic acid)
residue [15]. The biosynthesis of bacteriochlorophyll a
INTRODUCTION
Chlorophylls (Chls) play essential roles in the initial
(BChl a), which is the major photosynthetically active
stages of natural photosynthesis to capture sunlight,
pigment in anoxygenic bacteria, requires three additional
transfer the absorbed light energy, and carry out charge
steps: formation of a bacteriochlorin ring through the
separation [1, 2].The biologically important Chl pigments
C7=C8 reduction [16], introduction of the C3-acetyl
are biosynthesized through sequential enzymatic
group through hydration of the C3-vinyl group [17], and
reactions. The following facts are known regarding
oxidation of the resulting secondary alcohol [18]. In the
last step of BChl a biosynthesis, the C172-carboxylic
acid group of bacteriochlorophyllide a (BChlide a) is
esterified with a phytyl group to give BChl a [15].
In the above biosyntheses, (bacterio)chlorophyllides
[(B)Chlides], magnesium complexes of chlorophyllous
pigments possessing a free C172-carboxylic acid group,
are enzymatically modified, and esterification occurs
at the last step. To the contrary, hydrolysis at the C17-
propionate residue proceeds in the early stages of Chl
degradation in plants [19]. The esterified phytyl group is
not p-conjugated with the (bacterio)chlorin systems and
haslittleeffectontheiropticalproperties, butenhancesthe
lipophilicity of the molecules. Most photosynthetically
active (B)Chls have a long hydrocarbon chain [20]
that is useful for anchoring into photosystems through
hydrophobic interactions with proteins, lipids, and other
esterified chains.
the last steps for Chl a (the major Chl of oxygenic
phototrophs) biosynthesis (Fig. 1) [3–5]. Magnesium
is inserted into the central position of protoporphyrin
IX which is a common biosynthetic precursor to Chls
and hemes [6]. The C132-carboxylic acid group of
magnesium protoporphyrin IX is methyl-esterified by
S-adenosylmethionine (SAM) [7, 8]. This methyl ester
plays a key role in forming the isocyclic ring (E-ring) that
is characteristic of Chls [9, 10]. The resulting 3,8-divinyl-
protochlorophyllide a (DV-PChlide a) is subsequently
subjected to 8-vinyl hydrogenation [11] and C17=C18
reduction to give chlorophyllide a (Chlide a) [12–14].
Finally, Chl a is synthesized by esterification with a phytyl
◊ꢀSPP full member in good standing
*Correspondence to: Hitoshi Tamiaki, email: tamiaki@
fc.ritsumei.ac.jp, fax: +81-77-561-3729.
Copyright © 2018 World Scientific Publishing Company

2
M. TERAMURA AND H. TAMIAKI
Fig. 1. Biosynthetic pathway from protoporphyrin IX to (B)Chl a
Although (B)Chlides are essential as research
materials for studying (B)Chl metabolism and the effect
of esterified chains, the availability of (B)Chlides has
been limited. Pigments extracted from photosynthetic
organisms are usually esterified at the C17 position,
and a very small amount of (B)Chlides are available
from natural materials. Mutant strains accumulating
(B)Chlides can also be used as a resource, but the available
quantities and varieties of (B)Chlides are still limited
[10, 16, 21]. Another route for obtaining (B)Chlides is
through semi-synthesis from natural (B)Chls: (1) organic
synthetic methods [22] and (2) enzymatic modifications
[23]. Using photosynthetic chlorophyllous pigments
that are naturally abundant, a variety of (B)Chlides are
obtainable in an adequate amount. In this study, we report
the preparation of several (B)Chlides by combining the
above two techniques. The semi-synthetic (B)Chlides
were identified and purified by high-performance liquid
chromatography(–mass spectrometry) (HPLC(–MS))
and their retention times were compared.
RESULTS AND DISCUSSION
Synthesis of Chlide a and its derivatives
Chl a was extracted from commercially available,
dry cultured cells of the cyanobacterium Spirulina
geitleri with a mixture of petroleum ether and methanol
and purified according to previous procedures [24]
(see Fig. 2). The purified Chl a was converted into
Chlide a with one of the esterases, chlorophyllase
derived from Chenopodium album [25] as follows. The
chlorophyllase was overexpressed in E. coli, and its
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J. Porphyrins Phthalocyanines 2018; 22: 2–14

SEMI-SYNTHESIS AND HPLC ANALYSIS OF (BACTERIO)CHLOROPHYLLIDES POSSESSING A PROPIONIC ACID
3
Fig. 2. Synthesis of Chlide a and its derivatives from a Chl-a producing cyanobacterium
cell lysate was used for the following hydrolysis. Chl a
in acetone was dispersed in a mixture of aqueous Tris-
HCl buffer (pH 7.8) with sodium ascorbate, and stirred
at room temperature with an aliquot of the above
chlorophyllase solution. After incubation for 1 h, the
reaction was quenched by the addition of acetone, and
the mixture was centrifuged. The resulting supernatant
was washed with hexane, diluted with diethyl ether,
washed with distilled water, and concentrated to
dryness to give Chlide a [26]. The obtained Chlide a
was used without further purification as the starting
material in the following reactions. This enzymatic
hydrolysis should be carried out before the removal
of the C132-methoxycarbonyl group and the formation
of the porphyrin p-skeleton by dehydrogenation of
the C17H–C18H to the corresponding double bond
because of the chlorophyllase substrate specificity.
Chlide a was demethoxycarbonylated by refluxing in
collidine to yield pyrochlorophyllide a (pyroChlide a)
[27]. To obtain protochlorophyllide a (PChlide a),
a dry acetone solution of Chlide a was treated with
2,3-dichloro-5,6-dicyanobenzoquinone (DDQ), and the
chlorin p-system was oxidized to the fully p-conjugated
porphyrin moiety in PChlide a [28]. Chlide a in acetone
was acidified with an aqueous 5% (v/v) H2SO4 solution,
resulting in the smooth demetalation of Chlide a to
pheophorbide a (Pheide a). Pheide a in dichloromethane
was treated with zinc acetate dihydrate in acetone [29]
to yield zinc pheophorbide a (Zn-Pheide a).
ToanalyzeandpurifytheaboveChlides,reversedphase
(RP) HPLC was performed using a mixture of methanol
and aqueous 50 mM ammonium acetate solution (pH 5.2)
as a mobile phase. Chlide a and pyroChlide a were eluted
at 4.50 and 7.46 min, respectively, under the same HPLC
conditions: column, Cosmosil 2.5C18-MS-II 3.0 ϕ ×
75 mm octadecyl-silica gel (ODS); eluent, MeOH/
-1
.
50 mM AcONH4 = 80/20; flow rate, 0.6 mL min .
The slow elution of the 132-demethoxycarbonylation
product was ascribed to a decrease of the molecular
polarity, as supported by the estimated hydrophobicity,
calculated values of logP (ClogP) [30, 31]: ClogP = 8.88
(Chlide a) < 9.20 (pyroChlide a). The dehydrogenation
to the porphyrin p-skeleton as Chlide a → PChlide a
slowed down the elution of the pigment: retention time
(tR) = 3.30 (Chlide a) and 3.93 min (PChlide a); eluent,
MeOH/50 mM AcONH4 = 85/15. The change was also
consistent with the ClogP values: 8.88 (Chlide a) < 8.93
(PChlide a). Zn-Pheide a was eluted at 6.90 min later
than Chlide a at 4.50 min, whereas the former ClogP
value (8.46) was smaller than that of Chlide a (8.88). This
inconsistency might come from a higher coordination
ability of the central magnesium to the polar HPLC
eluent than the zinc of Zn-Pheide a [22].
Synthesis of BChl a derivatives
BChl a was extracted from the cultured cells of the
purplebacteriumRhodobactersphaeroideswithamixture
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M. TERAMURA AND H. TAMIAKI
Fig. 3. Synthesis of BChlides, Chlides, and PChlides from a BChl-a producing purple bacterium
of acetone and methanol and purified by RP-HPLC [32]
(see Fig. 3). BChl a, with a bacteriochlorin p-system, was
oxidized to 3-acetyl-chlorophyll a (3Ac-Chl a), with a
chlorin p-moiety, via dehydrogenation of C7H–C8H to
C7=C8 using DDQ in a manner similar to the Chlide a to
PChlide a transformation [33] (vide supra). The C3-acetyl
groups of BChl a and 3Ac-Chl a were reduced using tert-
butylamine borane in tetrahydrofuran (THF) to yield
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SEMI-SYNTHESIS AND HPLC ANALYSIS OF (BACTERIO)CHLOROPHYLLIDES POSSESSING A PROPIONIC ACID
5
3-(1-hydroxyethyl)-bacteriochlorophyll a (3HE-BChl a)
and 3-(1-hydroxyethyl)-chlorophyll a (3HE-Chl a) as
a 1:1 epimeric mixture of 31R/S-epimers [34]. BChl a,
3HE-BChl a, 3Ac-Chl a, and 3HE-Chl a in acetone
were hydrolyzed by chlorophyllase in aqueous Tris-HCl
buffer to BChlide a, 3HE-BChlide a, 3Ac-Chlide a, and
3HE-Chlide a, respectively. The enzymatic hydrolysis
reactions of the former two BChls were performed at
50°C owing to their lower reactivities when compared
with Chls. The hydrolysis was so mild that neither
demetalation to (bacterio)pheophorbides nor oxidation
of the bacteriochlorin to a chlorin ring was observed
in the reaction mixtures. The substrate specificity of
the present chlorophyllase was low enough that the
substitution of the C3-vinyl group with the C3-acetyl or
C3-(1-hydroxyethyl) group was tolerated. 3Ac-PChlide a
and 3HE-PChlide a were synthesized from 3Ac-Chlide a
and 3HE-Chlide a by selective oxidation of the chlorin
moiety to a porphyrin moiety using DDQ [28]. It is
noted that the DDQ-oxidation of bacteriochlorins gave
chlorins, followed by the formation of porphyrins
through the stepwise conversion of the first 7,8- and the
second 17,18-dehydrogenations. Zn-3Ac-Pheide a and
Zn-3HE-Pheide a were prepared from 3Ac-Chlide a
and 3HE-Chlide a by demetalation followed by a
Zn-metalation reaction (vide supra).
pigments (vide supra): ClogP = 6.91 (Zn-3HE-Pheide a)
< 7.37 (3HE-Chlide a) and 6.89 (3Ac-Chlide a) < 7.59
(Zn-3Ac-Pheide a).
Synthesis of BChl c/d derivatives
Homologous mixtures of BChl c and BChl d were
extracted with a mixture of acetone and methanol from
the cultured cells of green sulfur bacteria, Chlorobaculum
tepidum and Chlorobaculum parvum, respectively (see
Fig. 4). All the BChl c/d homologs were separated using
RP-HPLC (see Experimental). Chlorobaculum tepidum
gave almost exclusively the 31R-epimers of 8-ethyl-12-
methyl-([E,M])BChl c and 8,12-diethyl-([E,E])BChl c,
an approximate 9:1 mixture of the 31R- and 31S-epimers
of 8-propyl-12-ethyl-([P,E])BChl c, and exclusively the
31S-epimer of 8-isopropyl-12-ethyl([I,E])BChl c [26].
Chlorobaculum parvum gave epimeric/homologous
pigment compositions similar to Chlorobaculum tepidum,
but also contained a small amount of 8-propyl-12-methyl-
([P,M])BChl d [35]. [E,M]BChl c was converted into zinc
8-ethyl-12-methyl-bacteriopheophorbide c (Zn-[E,M]-
BPheide c) using the following steps. [E,M]BChl c in
THF was treated with aqueous H2SO4 to yield [E,M]-
BPheide c via demetalation of magnesium and hydrolysis
of the farnesyl ester [36]. [E,M]BPheide c was zinc-
metalated to Zn-[E,M]BPheide c as mentioned above.
The same procedures were used for the preparation of
Zn-[E,M]BPheide d, Zn-[E,E]BPheides c/d, Zn-[P,E]-
BPheides c/d, and Zn-[I,E]BPheides c/d.
Tosynthesizezinc3-vinyl-8-ethyl-12-methyl-bacterio-
pheophorbide c (Zn-3V-[E,M]BPheide c), [E,M]BChl c
in acetone was first treated with a diluted aqueous sulfuric
acid solution to effect central magnesium demetalation.
The resulting 8-ethyl-12-methyl-bacteriopheophytin c
([E,M]BPheo c) was refluxed in benzene containing
p-toluenesulfonic acid (PTSA) monohydrate to yield
3V-[E,M]BPheo c. 3V-[E,M]BPheo c was hydrolyzed
in THF with aqueous H2SO4 to 3V-[E,M]BPheide c and
subsequently subjected to zinc-metalation (vide supra) to
yield Zn-3V-[E,M]BPheide c [29]. Zn-3V-[E,M]BPheide
d, Zn-3V-[E,E]BPheides c/d, Zn-3V-[P,E]BPheides c/d,
and Zn-3V-[I,E]BPheides c/d were synthesized following
similar procedures.
In the HPLC analysis, pigments possessing the
same p-skeleton, but with different substituents at
the C3-position, were compared with respect to their
tR-values. 3HE-BChlide a and BChlide a were eluted at
7.21/7.99 (31R/S-epimers) and 9.08 min, respectively,
using MeOH/aqueous 50 mM AcONH4 = 65/35 at
-1
.
0.5 mL min . 3HE-Chlide a and 3Ac-Chlide a were
eluted at 10.1/11.2 and 15.0 min, respectively, using
-1
.
aqueous 65% (v/v) MeOH at 0.5 mL min . 3Ac-Chlide a
was eluted at 2.11 min, faster than Chlide a (tR
=
-1
.
3.30 min), using aqueous 85% MeOH at 0.6 mL min .
The same elution order was observed among the following
porphyrin pigments: tR = 1.77/1.98 (31R/S-epimers of
3HE-PChlide a) < 2.74 (3Ac-PChlide a) < 8.12 min
-1
.
(PChlide a) using aqueous 80% MeOH at 0.6 mL min .
These data show that pigments possessing the same
p-skeleton eluted in the order of 3-(1-hydroxyethyl)
(3HE), 3-acetyl (3Ac), and 3-vinyl (3V). This order
was consistent with the estimated hydrophobicity:
ClogP = 7.32 (3HE-BChlide a) < 7.53 (BChlide a), 7.37
(3HE-Chlide a) < 7.59 (3Ac-Chlide a) < 8.88 (Chlide a),
and 7.43 (3HE-PChlide a) < 7.65 (3Ac-PChlide a) <
8.93 (PChlide a). Zn-chelated 3HE/Ac-Pheides were
eluted slower than the corresponding Mg-chelated
pigments: tR = 10.1/11.2 (31R/S-epimers of 3HE-Chlide
a) < 16.5/18.0 min (31R/S-epimers of Zn-3HE-Pheide a)
Zn-(3V-)[E,M]BPheide
d was also synthesized
from Chl a through an alternative pathway [28]. Chl
a was first converted into methyl pyropheophorbide
a through demetalation and trans-esterification with
methanol under acidic conditions and a pyrolysis for the
132-demethoxycarbonylation product [37]. Zn-3V-[E,M]-
BPheide d (= zinc pyropheophorbide a) was obtained
from a subsequent hydrolysis at the propionate residue
and zinc-metalation. To prepare Zn-[E,M]BPheide d,
methyl pyropheophorbide a was hydrated at the C3-vinyl
group and concomitantly hydrolyzed at the C172-ester
with HBr in AcOH and with water, then zinc-metalated
[29]. Because the hydration was not stereoselective, the
and 15.0 (3Ac-Chlide a) < 31.3 min (Zn-3Ac-Pheide a)
-1
.
using aqueous 65% MeOH at 0.5 mL min . These
elution orders were opposite to the prediction based on
estimated lipophilicity. This inconsistency may have
resulted from the same reason as in the 3-vinylated
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M. TERAMURA AND H. TAMIAKI
Fig. 4. Synthesis of Zn-(3V-)BChlide c/d homologs from BChl-c/d producing green bacteria and by modification of Chl a
resulting products were 1:1 31R/S-epimeric mixtures of
(Zn-)[E,M]BPheide d. It is noted that other homologs,
Zn-(3V-)[E,E]/[P,E]/[I,E]BPheides c/d, could not be
synthesized from Chl a owing to synthetic difficulties in
the methylation of the C82- and C121-positions [38].
HPLC analysis showed that the above homologs
were eluted more slowly as the methylation level of
the peripheral substituents increased. This observation
paralleledthetrendinhydrophobicity.The20-methylation
of Zn-(3V-)BPheide d homologs to Zn-(3V-)BPheide c
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SEMI-SYNTHESIS AND HPLC ANALYSIS OF (BACTERIO)CHLOROPHYLLIDES POSSESSING A PROPIONIC ACID
7
gave larger tR-values compared with their corresponding
homologs: e.g. Zn-3V-[E,M]BPheide d was eluted at
9.74 min, and Zn-3V-[E,M]BPheide c was eluted at
11.3 min (see Experimental). The stepwise methylation
at the C82- and C121-positions also produced the same tR
effect, resulting in the elution order of [E,M] → [E,E] →
[P,E] → [I,E]BPheides c/d: e.g. tR = 11.3 (Zn-3V-
[E,M]BPheide c) < 13.2 (Zn-3V-[E,E]BPheide c) <
17.1(Zn-3V-[P,E]BPheide c) < 20.6 min (Zn-3V-[I,E]-
BPheide c). Isomerization at the C3-(1-hydroxyethyl)
group partially occurred during acidic hydrolysis, but
the 31-epimeric ratios of the semi-synthetic Zn-BPheide
c/d homologs were comparable to that of each isolated
BChl c/d homolog. Note also that the 31R-epimers of
the BChlide c/d and Zn-BPheide c/d homologs were
eluted faster than the corresponding 31S-epimers, which
is consistent with the RP-HPLC elution orders of the
other naturally occurring BChls c/d/e/f and their related
secondary alcohols [39, 40].
of DV-Chlide a led to non-regioselective hydration of
the C3- and/or C8-vinyl group(s) and simultaneously
removed the central magnesium. Therefore, the
synthesis of 3-(1-hydroxyethyl)-8-vinyl-chlorophyllide a
(3HE-8V-Chlide a) was accomplished through a regio-
selective hydration at the C3-vinyl group using
recombinant hydratase BchF from Chlorobaculum
tepidum in an aqueous buffer [26, 28, 43]. In the
enzymatic hydration reaction, the 31R-epimer was
produced as a major product owing to the stereoselec-
tivity of BchF. In the HPLC analysis, DV-Chlide a was
eluted at 4.89 min, slower than Chlide a (4.50 min),
-1
.
using aqueous 80% MeOH at 0.6 mL min . The same
elution trend was observed for PChlide a (3.93 min)
and DV-PChlide a (4.22 min) using aqueous 85%
-1
.
MeOH at 0.6 mL min .
Chl b and Chl d were extracted from commercially
available spinach powder with a mixture of petroleum
ether and methanol [44] and from Acaryochloris marina
cells with acetone and methanol [45], respectively (see
Fig. 5, lower). The pigment extracts were subjected to
RP-HPLC to give pure Chl b and Chl d. Hydrolysis at
the C17-propionate residues of phytylated Chl b and Chl
d was performed using chlorophyllase to yield Chlide
b and Chlide d, respectively. The chlorophyllase could
readily catalyze the hydrolysis of pigments possessing
a C7- or C3-formyl group. In comparison, between their
Synthesis of (bacterio)chlorophyllides
BChlide a and 3HE-BChlide a could be synthesized
from BChl a as mentioned above. However, 3V-BChlide
a could not be prepared through the chemical modification
of BChl a because the magnesium ion would be removed
from the center of the tetrapyrrole ring during the acidic
dehydration reaction. Therefore, to obtain 3V-BChlide a,
we used a mutant of Rhodobacter sphaeroides that
accumulated 3V-BChl a. A mixture of acetone and
methanol was added to the cultured cells of this 3-vinyl
hydratase gene bchF lacking mutant, and 3V-BChl a was
extracted [33] (see Fig. 5, upper). The resulting residue
was subjected to RP-HPLC purification and hydrolyzed
with chlorophyllase to yield 3V-BChlide a. 3V-BChlide
a should be carefully handled because it is significantly
less stable than BChlide a. BChlide a is stabilized against
oxidation and demetalation by the electron-withdrawing
C3-acetyl group [41]. The RP-HPLC elution order was
comparable to the dehydrogenation degree of the core
tetrapyrrole skeletons of the 3-vinylated pigments: tR = 2.74
tR-values under RP-HPLC conditions using aqueous
-1
.
80% MeOH at 0.6 ml min , the 7-formylated pigment
(Chlide b) eluted at 1.96 min, which was slightly faster
than the 3-formylated pigment (Chlide d) eluting at
2.05 min. Both pigments eluted faster than Chlide a (tR =
4.50 min), and the order could be rationalized by
their ClogP values: 8.09 (Chlide b) < 8.88 (Chlide a)
and 7.50 (Chlide d) < 8.88 (Chlide a). However,
between Chlide b and Chlide d, the elution order was
inconsistent with the estimated hydrophobicity, and the
coordination ability of these compounds to the eluent
may have affected the elution order: the estimated axial
coordination constant of Chl b is greater than that of
Chl d [41].
(3V-BChlide a) < 3.30 (Chlide a) < 3.93 min (PChlide a)
-1
.
using aqueous 85% MeOH at 0.6 mL min . This matched
EXPERIMENTAL
the trend predicted using ClogP values: 8.82 (3V-BChlide
a) < 8.88 (Chlide a) < 8.93 (PChlide a).
General
DV-Chl a was obtained from the cultured cells of a
cyanobacterium mutant Synechocystis sp. PCC6803
that lacked the divinyl reductase gene slr1923 [42] (see
Fig. 5, middle). DV-Chlide a was synthesized with the
aforementioned action of chlorophyllase, showing
that chlorophyllase was active for the 8-vinylated
substrate. DV-Chlide a was oxidized by DDQ (vide
supra) to DV-PChlide a. DV-Chlide a was incubated
with recombinant demethoxycarbonylase BciC from
Chlorobaculum tepidum in an aqueous buffer to yield
DV-pyroChlide a [33]. The aqueous acidic treatment
All the reactions on chlorophyllous pigments were
carried out at room temperature in the dark under an air
atmosphere unless otherwise stated.
Pigment purification and analysis by HPLC were
performed using a Shimadzu Prominence liquid chromato-
graphy system consisting of a CBM-20A system controller,
anSPD-M20Aphotodiodearray(PDA)detector,LC-20AD
pumps, a DGU-20A3 degasser and a CTO-20AC column
oven (Japan). For all HPLC analysis, the column oven was
set to 35°C, and the PDA detector was set to 300–800 nm.
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8
M. TERAMURA AND H. TAMIAKI
Fig. 5. Synthesis of 3V-BChlide a from the 3V-BChl-a producing DbchF mutant of a purple bacterium (upper), 8-vinylated
(P)Chlides a from the DV-Chl-a producing Dslr1923 mutant of a cyanobacterium (middle), and Chlides b/d from Chl-b producing
spinach and Chl-d producing cyanobacterium (lower)
Liquid chromatography–mass spectrometry (LCMS) was
performed using a Shimadzu LCMS-2010EV system
consisting of a quadrupole mass spectrometer equipped
with electrospray ionization. All the solvents used for
HPLC were of HPLC grade and purchased from Nacalai
Tesque or Wako Pure Chemical Ind.
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SEMI-SYNTHESIS AND HPLC ANALYSIS OF (BACTERIO)CHLOROPHYLLIDES POSSESSING A PROPIONIC ACID
9
-1
.
Synthetic procedures
MeCN/acetone/H2O = 70/15/15; flow rate, 2.5 mL min .
The mixture of [E,E] and [P,M]BChls d was separated
by a further NP-HPLC process: column, Cosmosil
Pigment extraction. Chl a was extracted from a
commerciallyavailablespirulinapowder(cyanobacterium
Spirulina geitleri) with a mixture of petroleum ether and
methanol (1:1, v/v) and purified according to the method
described previously [24].
DV-Chl a was extracted from the Dslr1923 mutant of
cyanobacterium Synechocystis sp. PCC6803 by stirring
its cultured cells in a mixture of acetone and methanol
(4:1, v/v) [42]. After the solvents were concentrated, the
residue was purified by RP-HPLC: column, Cosmosil
5C18-SL (6.0 ϕ × 250 mm); eluent, hexane/THF = 84/16;
-1
.
flow rate, 5 mL min .
Hydrolysis using chlorophyllase. Recombinant
chlorophyllase derived from Chenopodium album was
generously gifted by Dr. Yusuke Tsukatani of Japan
Agency for Marine-Earth Science and Technology. The
cell lysate of E. coli overexpressing chlorophyllase
was used for the following hydrolysis [23]. An acetone
solution (200 mL) of each substrate (Chl a, 3Ac-Chl a,
3HE-Chl a, DV-Chl a, Chl b, or Chl d) was dispersed in
25 mM Tris-HCl buffer (pH 7.8, 8 mL) containing 0.5 M
aqueous sodium L-ascorbate (50 mL). To this was added
5C18-AR-II (4.6 ϕ × 150 mm); eluent, MeCN/H2O =
-1
.
95/5; flow rate, 5 mL min .
Chl b was extracted from commercially available
spinach powder with a mixture of petroleum ether
and methanol (1:1, v/v) [44]. After centrifugation, the
resulting supernatant was partitioned between diethyl
ether and distilled water. The ether phase was washed
several times with distilled water and concentrated, and
Chl b was purified by RP-HPLC: column, Cosmosil 5C18-
-1
.
the chlorophyllase solution (5.7 mg mL , 500 mL), and
the mixture was stirred under nitrogen for 1 h. Similar
reactions were performed at 50°C for BChl a, 3HE-BChl
a, and 3V-BChl a. To stop the reaction, acetone (30 mL)
was added, and the mixture was centrifuged. The resulting
supernatant was washed with hexane to remove unreacted
(B)Chls, diluted with diethyl ether, and washed with
distilled water to give the corresponding carboxylic acids,
Chlide a, 3Ac-Chlide a, 3HE-Chlide a, DV-Chlide a,
Chlide b, Chlide d, BChlide a, 3HE-BChlide a, and
3V-BChlide a. For HPLC purification of these products,
see HPLC conditions mentioned in Spectral data section.
Acidic hydrolysis. Concentrated H2SO4 was added
dropwise to BChl c/d and 3V-BPheo c/d homologs in
THF and H2O (4/1, v/v) in an ice bath. The reaction was
monitored using NP-thin-layer chromatography with
diethyl ether and dichloromethane (1:9, v/v) as the eluent.
After the substrates were consumed, CHCl3 was added to
the reaction mixture, and the organic layer was washed
with distilled water. The organic layer was concentrated
to yield the corresponding BPheide c/d and 3V-BPheide
c/d homologs.
AR-II (4.6 ϕ × 150 mm); eluent, MeOH/H2O = 97/3; flow
-1
.
rate, 2.5 mL min .
Chl d was extracted from cyanobacterium Acaryo-
chloris marina cells using acetone and methanol (4:1,
v/v) [45] in a manner similar to the procedure used for
Chl b, and Chl d was purified by RP-HPLC: column,
Cosmosil 2.5C18-MS-II (3.0 ϕ × 75 mm); eluent, MeCN/
H2O = 95/5; flow rate, 2.0 mL min .
BChl a was extracted from the cultured cells of purple
bacterium Rhodobacter sphaeroides [32] in a manner
similar to the procedure used for Chl d. BChl a was
purified by normal−phase (NP) HPLC: column, Cosmosil
5SL-II (6.0 ϕ × 250 mm); eluent, hexane/acetone = 75/25
(v/v); flow rate, 6.0 mL min .
3V-BChl a was extracted from the cultured DbchF
mutant cells of Rhodobacter sphaeroides [33] in a manner
similar to the procedure used for BChl a. 3V-BChl a was
purified by RP-HPLC: column, Cosmosil 2.5C18-MS-II
(3.0 ϕ × 75 mm); eluent, MeOH/H2O = 85/15 (0–15 min),
then MeOH (15 min–end); flow rate, 0.6 mL min . The
fraction containing 3V-BChl a was dialyzed with 50 mM
phosphate buffer (pH 7.8) for the next chlorophyllase
reaction.
BChl c was extracted from green sulfur bacterium
Chlorobaculum tepidum [28] in a manner similar to the
procedure used for Chl d. Each BChl c homolog was
isolated from the mixture by RP-HPLC, and pure [E,M],
[E,E], [P,E], and [I,E]BChls c were obtained: column,
-1
.
-1
.
DDQ oxidation. The bacteriochlorin of BChl a was
oxidized to the chlorin (3Ac-Chl a), and the chlorins of
Chlide a, 3Ac-Chlide a, 3HE-Chlide a, and DV-Chlide
a were oxidized to the corresponding porphyrins ((un)
substituted PChlide a) using DDQ [28, 42]. DDQ,
dissolved in dry acetone, was added to a dry acetone
solution of each substrate. The oxidation was monitored
with UV-vis spectroscopy until the absorption peak
changed as follows: Qy absorption maxima = 770 (BChl
a) to 677 nm (3Ac-Chl a), 663 (Chlide a) to 623 nm
(PChlide a), 677 (3Ac-Chlide a) to 632 nm (3Ac-PChlide
a), 654 (3HE-Chlide a) to 616 nm (3HE-PChlide a),
and 663 (DV-Chlide a) to 625 nm (DV-PChlide a).
The reaction mixture of BChl a was poured into water
and extracted with dichloromethane. The organic layer
was washed with aqueous 4% NaHCO3 and water, and
concentrated to give 3Ac-Chl a. The other reaction
mixtures were suspended in 2-propanol and subjected
to RP-HPLC: for PChlide a, Inertsil ODS-P (4.6 ϕ ×
150 mm, GL Science Inc.) column and MeOH/50 mM
-1
.
Cosmosil 5C18-AR-II (10 ϕ × 250 mm); eluent, MeOH;
-1
.
flow rate, 2.0 mL min .
BChl d was extracted from green sulfur bacterium
Chlorobaculum parvum [43] in a manner similar to the
above preparation of BChl c homologs. The following
RP-HPLC purification yielded [E,M], [P,E], and [I,E]
BChls d as well as a mixture of [E,E] and [P,M]BChls d:
column, Cosmosil 5C18-AR-II (6.0 ϕ × 250 mm); eluent,
Copyright © 2018 World Scientific Publishing Company
J. Porphyrins Phthalocyanines 2018; 22: 9–14

10
M. TERAMURA AND H. TAMIAKI
-1
.
AcONH4 = 85/15 at 2.0 mL min ; for 3Ac-PChlide a
and for 3HE-PChlide a, Cosmosil 2.5C18-MS-II (3.0 ϕ ×
mixture was incubated at 35°C for 30 min and acetone
was added to stop the enzymatic reaction [43]. The
solution was concentrated and applied to RP-HPLC for
purification of 3HE-8V-Chlide a: column, Cosmosil
75 mm) column and MeOH/50 mM AcONH4 = 80/20 at
0.6 mL min ; for DV-PChlide a, Cosmosil 5C18-AR-II
-1
.
(4.6 ϕ × 150 mm) column and MeOH/50 mM AcONH4 =
2.5C18-MS-II (3.0 ϕ × 75 mm); eluent, MeOH/aqueous
-1
-1
.
.
85/15 at 1.0 mL min .
50 mM AcONH4 = 70/30; flow rate, 0.6 mL min .
Reduction of 3Ac to 3HE group. BChl a and 3Ac-Chl
a were reduced using tert-butylamine borane (140 mg) in
THF (10 mL) under nitrogen [34]. During the reduction,
their Qy absorption maxima were blue-shifted: from 771
(BChl a) to 722 nm (3HE-BChl a) and from 680 (3Ac-Chl
a) to 653 nm (3HE-Chl a). The reaction was quenched by
the addition of acetone and stirred for a further 30 min.
Extraction with diethyl ether and washing with distilled
water gave 3HE-BChl a and 3HE-Chl a.
Dehydration of 3HE to 3V group. Homologously pure
BPheo c/d were refluxed in benzene containing PTSA for
15 min [46]. After cooling down to room temperature,
CHCl3 was added to the reaction mixture, and the organic
layer was washed with distilled water. The organic layer
was concentrated to yield the corresponding 3V-BPheo
c/d homologs.
132-Demethoxycarbonylation by BciC. BciC deme-
thoxycarbonylase derived from Chlorobaculum tepidum
was generously gifted by Dr. Jiro Harada of Kurume
University School of Medicine. The cell lysate of
E. coli overexpressing BciC was used for the following
enzymatic reaction. DV-Chlide a in DMSO was
dispersed in 25 mM NaH2PO4 (pH 7.0) buffer containing
100 mM NaCl. After the addition of BciC protein, the
reaction mixture was incubated at 45°C for 30 min and
acetone was added to stop the enzymatic reaction [33].
The solution was concentrated and applied to RP-HPLC
for purification of DV-pyroChlide a: column, Cosmosil
2.5C18-MS-II (3.0 ϕ × 75 mm); eluent, MeOH/aqueous
-1
.
50 mM AcONH4 = 85/15; flow rate, 0.6 mL min .
Spectral data
Demetalation. Removal of the magnesium ion
coordinated to Chlide a, 3Ac-Chlide a, 3HE-Chide a,
and BChls c/d was achieved under acidic conditions.
Diluted aqueous H2SO4 was added to an acetone solution
of each magnesium-chelated pigment and the reaction
mixtures were stirred. The progress of the demetalation
reaction was monitored with UV-vis spectroscopy until
blue-shifts of the Soret maxima and red-shifts of the
Qy absorption maxima were observed. The reaction
mixtures were extracted using CH2Cl2, washed with
distilled water, and concentrated to yield the desired
metal-free pigments.
Chlorophyllide a (Chlide a). tR = 4.50 min (Cosmosil
2.5C18-MS-II, 3.0 ϕ × 75 mm, MeOH/aq. 50 mM
-1
.
AcONH4 = 80/20, 0.6 mL min , Fig. S1); VIS (MeOH/
aq. 50 mM AcONH4 = 80/20) lmax = 666 (relative
intensity, 1.00), 619 (0.24), 431 nm (0.94); MS (ESI)
found: m/z 615.5, calcd for C35H35N4O5Mg: MH+, 615.2
[23, 33, 47].
Chlorophyllide b (Chlide b). tR = 1.96 min (Cosmosil
2.5C18-MS-II, 3.0 ϕ × 75 mm, MeOH/aq. 50 mM
-1
.
AcONH4 = 80/20, 0.6 mL min , Fig. S2); VIS (MeOH/
aq. 50 mMAcONH4 = 80/20) lmax = 652 (relative intensity,
0.36), 604 (0.12), 469 nm (1.00); MS (ESI) found: m/z
629.5, calcd for C35H33N4O6Mg: MH+, 629.2 [47, 48].
Chlorophyllide d (Chlide d). tR = 2.05 min (Cosmosil
Zn metalation. An acetone solution containing zinc
acetate dihydrate was added to Pheide a, 3HE-Pheide a,
3Ac-Pheide a, BPheide c/d, or 3V-BPheide c/d in CH2Cl2
and stirred. After red-shifts of the Soret absorption
maxima and blue-shifts of the Qy absorption maxima
were observed, CHCl3 was added to the reaction mixture
and the organic layer was washed with distilled water and
concentrated to yield Zn-Pheide a, Zn-3HE-Pheide a,
Zn-3Ac-Pheide a, Zn-BPheide c/d homologs, or Zn-3V-
BPheide c/d homologs. For HPLC purification of these
products, see HPLC conditions mentioned in Spectral
data section.
2.5C18-MS-II, 3.0 ϕ × 75 mm, MeOH/aq. 50 mM
-1
.
AcONH4 = 80/20, 0.6 mL min , Fig. S3); VIS (MeOH/
aq. 50 mMAcONH4 =80/20)lmax = 702 (relative intensity,
1.00), 650 (0.21), 456 (0.72), 402 nm (0.72) [49]; MS
(ESI) found: m/z 617.6, calcd for C34H33N4O6Mg: MH+,
617.2.
3-Acetyl-chlorophyllide a (3Ac-Chlide a). tR = 15.0
min (Cosmosil 2.5C18-MS-II, 3.0 ϕ × 75 mm, MeOH/
-1
.
aq. 50 mM AcONH4 = 65/35, 0.5 mL min , Fig. S4);
Pyrolysis for 132-demethoxycarbonylation. Chlide a
was refluxed in collidine to yield pyroChlide a [27]. For
its HPLC purification, see HPLC conditions mentioned
in Spectral data section.
VIS (MeOH/aq. 50 mM AcONH4 = 65/35) lmax =
688 (relative intensity, 1.00), 634 (0.28), 442 (0.93),
397 nm (0.94); MS (ESI) found: m/z 631.4, calcd for
C35H35N4O6Mg: MH+, 631.2 [18].
Hydration of 3V group using BchF. BchF hydratase
derived from Chlorobaculum tepidum was generously
gifted by Dr. Jiro Harada of Kurume University School
of Medicine. The cell lysate of E. coli overexpressing
BchF was used for the following hydration. DV-Chlide a
in dimethyl sulfoxide (DMSO) was dispersed in 25 mM
Tris-HCl (pH 7.8) buffer containing BchF. The reaction
3-(1-Hydroxyethyl)-chlorophyllide a (3HE-Chlide a).
tR = 10.1/11.2 min for 31R/S-epimers (Cosmosil 2.5C18-
MS-II, 3.0 ϕ × 75 mm, MeOH/aq. 50 mM AcONH4 =
-1
.
65/35, 0.5 mL min , Fig. S5); VIS (MeOH/aq. 50 mM
AcONH4 = 65/35) lmax = 659 (relative intensity, 1.00),
614 (0.25), 426 (0.93), 414 nm (0.96); MS (ESI) found:
m/z 633.4, calcd for C35H37N4O6Mg: MH+, 633.3 [18, 26].
Copyright © 2018 World Scientific Publishing Company
J. Porphyrins Phthalocyanines 2018; 22: 10–14

SEMI-SYNTHESIS AND HPLC ANALYSIS OF (BACTERIO)CHLOROPHYLLIDES POSSESSING A PROPIONIC ACID
11
3,8-Divinyl-chlorophyllide a (DV-Chlide a). tR = 4.89
3-Acetyl-protochlorophyllide a (3Ac-PChlide a). tR =
min (Cosmosil 2.5C18-MS-II, 3.0 ϕ × 75 mm, MeOH/
2.74 min (Cosmosil 2.5C18-MS-II, 3.0 ϕ × 75 mm,
-1
-1
.
.
aq. 50 mM AcONH4 = 80/20, 0.6 mL min , Fig. S6);
MeOH/aq. 50 mM AcONH4 = 80/20, 0.6 mL min ,
VIS (MeOH/aq. 50 mM AcONH4 = 80/20) lmax = 668
(relative intensity, 0.82), 624 (0.20), 442 nm (1.00); MS
(ESI) found: m/z 613.5, calcd for C35H33N4O5Mg: MH+,
613.2 [33, 43, 50].
Fig. S14) [53]; VIS (MeOH/aq. 50 mM AcONH4 =
80/20) lmax = 643 (relative intensity, 0.17), 588 (0.04),
543 (0.03), 436 nm (1.00); MS (ESI) found: m/z 629.4,
calcd for C35H33N4O6Mg: MH+, 629.2.
3-(1-Hydroxyethyl)-8-vinyl-chlorophyllidea(3HE-8V-
3-(1-Hydroxyethyl)-protochlorophyllide
a
(3HE-
Chlide a). tR = 4.36/4.75 min for 31R/S-epimers (Cos-
PChlide a). tR = 1.77/1.98 min for 31R/S-epimers
mosil 2.5C18-MS-II, 3.0 ϕ × 75 mm, MeOH/aq. 50 mM
(Cosmosil 2.5C18-MS-II, 3.0 ϕ × 75 mm, MeOH/aq.
-1
-1
.
.
AcONH4 = 70/30, 0.6 mL min , Fig. S7); VIS (MeOH/aq.
50 mM AcONH4 = 80/20, 0.6 mL min , Fig. S15);
50 mM AcONH4 = 70/30) lmax = 660 (relative intensity,
0.88), 617 (0.19), 438 nm (1.00); MS (ESI) found: m/z
631.6, calcd for C35H35N4O6Mg: MH+, 631.2 [43].
VIS (MeOH/aq. 50 mM AcONH4 = 80/20) lmax = 626
(relative intensity, 0.16), 574 (0.06), 533 (0.04),
433 nm (1.00); MS (ESI) found: m/z 631.3, calcd for
C35H35N4O6Mg: MH+, 631.2.
Pyrochlorophyllide a (pyroChlide a). tR = 7.46 min
(Cosmosil 2.5C18-MS-II, 3.0 ϕ × 75 mm, MeOH/
3,8-Divinyl-protochlorophyllide a (DV-PChlide a).
tR = 4.22 min (Cosmosil 2.5C18-MS-II, 3.0 ϕ × 75 mm,
-1
.
aq. 50 mM AcONH4 = 80/20, 0.6 mL min , Fig. S8);
-1
.
VIS (MeOH/aq. 50 mM AcONH4 = 80/20) lmax = 666
(relative intensity, 1.00), 621 (0.22), 433 nm (0.97); MS
(ESI) found: m/z 557.6, calcd for C33H33N4O3Mg: MH+,
557.2 [26, 33].
MeOH/aq. 50 mM AcONH4 = 85/15, 0.6 mL min ,
Fig. S16); VIS (MeOH/aq. 50 mM AcONH4 = 85/15)
lmax = 630 (relative intensity, 0.15), 578 (0.06), 542
(0.03), 439 nm (1.00); MS (ESI) found: m/z 611.5, calcd
for C35H31N4O5Mg: MH+, 611.2 [33].
3,8-Divinyl-pyrochlorophyllide a (DV-pyroChlide a).
tR = 7.41 min (Cosmosil 2.5C18-MS-II, 3.0 ϕ × 75 mm,
Zinc pheophorbide a (Zn-Pheide a). tR = 6.90 min
-1
.
MeOH/aq. 50 mM AcONH4 = 85/15, 0.6 mL min ,
(Cosmosil 2.5C18-MS-II, 3.0 ϕ × 75 mm, MeOH/aq.
-1
.
Fig. S9); VIS (MeOH/aq. 50 mM AcONH4 = 85/15)
50 mM AcONH4 = 80/20, 0.6 mL min , Fig. S17);
l
max = 666 (relative intensity, 0.88), 622 (0.20), 441 nm
(1.00); MS (ESI) found: m/z 555.4, calcd for
VIS (MeOH/aq. 50 mM AcONH4 = 80/20) lmax = 661
(relative intensity, 0.89), 614 (0.21), 572 (0.12), 528
(0.05), 429 nm (1.00); MS (ESI) found: m/z 655.3, calcd
for C35H35N4O5Zn: MH+, 655.2 [54].
C33H31N4O3Mg: MH+, 555.2 [33].
3-Vinyl-bacteriochlorophyllide a (3V-BChlide a).
tR = 2.74 min (Cosmosil 2.5C18-MS-II, 3.0 ϕ × 75 mm,
MeOH/aq. 50 mM AcONH4 = 85/15, 0.6 mL min ,
Fig. S10); VIS (MeOH/aq. 50 mM AcONH4 = 85/15)
lmax = 725 (relative intensity, 0.77), 670 (0.34), 587
(0.29), 356 nm (1.00); MS (ESI) found: m/z 617.6, calcd
for C35H37N4O5Mg: MH+, 617.3 [16, 33, 43].
Bacteriochlorophyllide a (BChlide a). tR = 1.12
min (Cosmosil 2.5C18-MS-II, 3.0 ϕ × 75 mm, MeOH/
aq. 50 mM AcONH4 = 85/15, 0.6 mL min , Fig. S11);
Zinc 3-acetyl-pheophorbide a (Zn-3Ac-Pheide a).
-1
.
tR = 31.3 min (Cosmosil 2.5C18-MS-II, 3.0 ϕ × 75 mm,
-1
.
MeOH/aq. 50 mM AcONH4 = 65/35, 0.5 mL min ,
Fig. S18); VIS (MeOH/aq. 50 mM AcONH4 = 65/35)
lmax = 679 (relative intensity, 0.96), 630 (0.26), 437
(1.00), 388 nm (0.84); MS (ESI) found: m/z 671.2, calcd
for C35H35N4O6Zn: MH+, 671.2 [18].
Zinc-3-(1-hydroxyethyl)-pheophorbide a (Zn-3HE-
-1
Pheide a). tR = 16.5/18.0 min for 31R/S-epimers
.
VIS (MeOH/aq. 50 mM AcONH4 = 85/15) lmax
=
(Cosmosil 2.5C18-MS-II, 3.0 ϕ × 75 mm, MeOH/aq.
-1
.
773 (relative intensity, 0.88), 698 (0.26), 607 (0.27),
366 nm (1.00); MS (ESI) found: m/z 633.1, calcd for
C35H37N4O6Mg: MH+, 633.3 [47, 51].
50 mM AcONH4 = 65/35, 0.5 mL min , Fig. S19);
VIS (MeOH/aq. 50 mM AcONH4 = 65/35) lmax = 655
(relative intensity, 1.00), 608 (0.26), 570 (0.17), 425
(0.98), 412 nm (0.95); MS (ESI) found: m/z 673.4, calcd
for C35H37N4O6Zn: MH+, 673.2 [18].
3-(1-Hydroxyethyl)-bacteriochlorophyllide a (3HE-
BChlide a). tR = 7.21/7.99 min for 31R/S-epimers
(Cosmosil 2.5C18-MS-II, 3.0 ϕ × 75 mm, MeOH/aq.
Zinc 8-ethyl-12-methyl-bacteriopheophorbide c (Zn-
-1
50 mM AcONH4 = 65/35, 0.5 mL min , Fig. S12); VIS
[E,M]BPheide c). tR = 4.09/4.66 min for 31R/S-epimers
.
(MeOH/aq. 50 mMAcONH4 = 65/35) lmax = 709 (relative
intensity, 0.62), 667 (0.33), 576 (0.27), 346 nm (1.00);
MS (ESI) found: m/z 635.4, calcd for C35H39N4O6Mg:
MH+, 635.3 [52].
(Cosmosil 5C18-AR-II, 4.6 ϕ × 150 mm, MeOH/aq.
-1
.
50 mM AcONH4 = 85/15, 1.0 mL min , Fig. S20);
VIS (MeOH/aq. 50 mM AcONH4 = 85/15) lmax = 665
(relative intensity, 0.78), 623 (0.18), 434 nm (1.00); MS
(ESI) found: m/z 629.4, calcd for C34H37N4O4Zn: MH+,
629.2 [28].
Protochlorophyllide a (PChlide a). tR = 3.93 min
(Cosmosil 2.5C18-MS-II, 3.0 ϕ × 75 mm, MeOH/aq.
-1
.
50 mM AcONH4 = 85/15, 0.6 mL min , Fig. S13); VIS
Zinc 8-ethyl-12-ethyl-bacteriopheophorbide c (Zn-
(MeOH/aq. 50 mMAcONH4 = 85/15) lmax = 630 (relative
intensity, 0.17), 577 (0.05), 532 (0.03), 434 nm (1.00);
MS (ESI) found: m/z 613.3, calcd for C35H33N4O5Mg:
MH+, 613.2 [18, 28, 33, 43, 52].
[E,E]BPheide c). tR = 4.84/5.61 min for 31R/S-epimers
(Cosmosil 5C18-AR-II, 4.6 ϕ × 150 mm, MeOH/aq.
-1
.
50 mM AcONH4 = 85/15, 1.0 mL min , Fig. S21);
VIS (MeOH/aq. 50 mM AcONH4 = 85/15) lmax = 665
Copyright © 2018 World Scientific Publishing Company
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12
M. TERAMURA AND H. TAMIAKI
(relative intensity, 0.84), 624 (0.20), 433 nm (1.00); MS
(ESI) found: m/z 643.3, calcd for C35H39N4O4Zn: MH+,
643.2 [28].
Zinc 3-vinyl-8-ethyl-12-ethyl-bacteriopheophorbide
c (Zn-3V-[E,E]BPheide c). tR = 13.2 min (Cosmosil
5C18-AR-II, 4.6 ϕ × 150 mm, MeOH/aq. 50 mM
-1
.
Zinc 8-propyl-12-ethyl-bacteriopheophorbide c (Zn-
AcONH4 = 85/15, 1.0 mL min , Fig. S29); VIS
[P,E]BPheide c). tR = 5.69/6.70 min for 31R/S-epimers
(MeOH/aq. 50 mM AcONH4 = 85/15) lmax = 668
(relative intensity, 0.78), 630 (0.18), 435 nm (1.00); MS
(ESI) found: m/z 625.2, calcd for C35H37N4O3Zn: MH+,
625.2 [28].
Zinc3-vinyl-8-propyl-12-ethyl-bacteriopheophorbide
c (Zn-3V-[P,E]BPheide c). tR = 17.1 min (Cosmosil
(Cosmosil 5C18-AR-II, 4.6 ϕ × 150 mm, MeOH/aq.
-1
.
50 mM AcONH4 = 85/15, 1.0 mL min , Fig. S22);
VIS (MeOH/aq. 50 mM AcONH4 = 85/15) lmax = 665
(relative intensity, 0.80), 626 (0.18), 434 nm (1.00); MS
(ESI) found: m/z 657.2, calcd for C36H41N4O4Zn: MH+,
657.2 [28].
5C18-AR-II, 4.6 ϕ × 150 mm, MeOH/aq. 50 mM
-1
.
Zinc8-isobutyl-12-ethyl-bacteriopheophorbidec(Zn-
AcONH4 = 85/15, 1.0 mL min , Fig. S30); VIS (MeOH/
[I,E]BPheide c). tR = 6.09/7.29 min for 31R/S-epimers
aq. 50 mMAcONH4 =85/15)lmax = 668 (relative intensity,
0.77), 629 (0.18), 436 nm (1.00); MS (ESI) found: m/z
639.3, calcd for C36H39N4O3Zn: MH+, 639.2 [28].
(Cosmosil 5C18-AR-II, 4.6 ϕ × 150 mm, MeOH/aq.
-1
.
50 mM AcONH4 = 85/15, 1.0 mL min , Fig. S23);
VIS (MeOH/aq. 50 mM AcONH4 = 85/15) lmax = 664
(relative intensity, 0.75), 628 (0.18), 434 nm (1.00); MS
(ESI) found: m/z 671.2, calcd for C37H43N4O4Zn: MH+,
671.3 [28].
Zinc 3-vinyl-8-isobutyl-12-ethyl-bacteriopheophor-
bide c (Zn-3V-[I,E]BPheide c). tR = 20.6 min
(Cosmosil 5C18-AR-II, 4.6 ϕ × 150 mm, MeOH/aq.
-1
.
50 mM AcONH4 = 85/15, 1.0 mL min , Fig. S31);
Zinc 8-ethyl-12-methyl-bacteriopheophorbide d (Zn-
VIS (MeOH/aq. 50 mM AcONH4 = 85/15) lmax = 668
(relative intensity, 0.75), 630 (0.18), 436 nm (1.00); MS
(ESI) found: m/z 653.3, calcd for C37H41N4O3Zn: MH+,
653.3 [28].
[E,M]BPheide d). tR = 8.51/9.74 min for 31R/S-epimers
(Cosmosil 5C18-AR-II, 4.6 ϕ × 150 mm, MeOH/aq.
-1
.
50 mM AcONH4 = 75/25, 1.0 mL min , Fig. S24);
VIS (MeOH/aq. 50 mM AcONH4 = 75/25) lmax = 652
(relative intensity, 0.78), 610 (0.22), 425 nm (1.00); MS
(ESI) found: m/z 615.0, calcd for C33H35N4O4Zn: MH+,
615.2 [26, 43].
Zinc 3-vinyl-8-ethyl-12-methyl-bacteriopheophor-
bide d (Zn-3V-[E,M]BPheide d). tR = 9.74 min (Cos-
mosil 5C18-AR-II, 4.6 ϕ × 150 mm, MeOH/aq. 50 mM
-1
.
AcONH4 = 85/15, 1.0 mL min , Fig. S32); VIS (MeOH/
Zinc 8-ethyl-12-ethyl-bacteriopheophorbide d (Zn-
aq. 50 mMAcONH4 = 85/15) lmax = 660 (relative intensity,
0.88), 614 (0.19), 430 nm (1.00); MS (ESI) found: m/z
597.2, calcd for C33H33N4O3Zn: MH+, 597.2 [26, 43].
Zinc3-vinyl-8-ethyl-12-ethyl-bacteriopheophorbided
(Zn-3V-[E,E]BPheide d). tR = 12.2 min (Cosmosil 5C18-
[E,E]BPheide d). tR = 3.82/4.09 min for 31R/S-epimers
(Cosmosil 5C18-AR-II, 4.6 ϕ × 150 mm, MeOH/aq.
-1
.
50 mM AcONH4 = 85/15, 1.0 mL min , Fig. S25);
VIS (MeOH/aq. 50 mM AcONH4 = 85/15) lmax = 652
(relative intensity, 0.97), 607 (0.19), 425 nm (1.00); MS
(ESI) found: m/z 629.4, calcd for C34H37N4O4Zn: MH+,
629.2 [43].
AR-II, 4.6 ϕ × 150 mm, MeOH/aq. 50 mM AcONH4 =
-1
.
85/15, 1.0 mL min , Fig. S33); VIS (MeOH/aq. 50 mM
AcONH4 = 85/15) lmax = 660 (relative intensity, 0.87),
613 (0.19), 429 nm (1.00); MS (ESI) found: m/z 611.4,
calcd for C34H35N4O3Zn: MH+, 611.2 [43].
Zinc 8-propyl-12-ethyl-bacteriopheophorbide d (Zn-
[P,E]BPheide d). tR = 4.36/4.76 min for 31R/S-epimers
(Cosmosil 5C18-AR-II, 4.6 ϕ × 150 mm, MeOH/aq.
Zinc3-vinyl-8-propyl-12-ethyl-bacteriopheophorbide
d (Zn-3V-[P,E]BPheide d). tR = 15.9 min (Cosmosil
-1
.
50 mM AcONH4 = 85/15, 1.0 mL min , Fig. S26);
VIS (MeOH/aq. 50 mM AcONH4 = 85/15) lmax = 652
(relative intensity, 0.96), 607 (0.19), 426 nm (1.00); MS
(ESI) found: m/z 643.3, calcd for C35H39N4O4Zn: MH+,
643.2 [43].
5C18-AR-II, 4.6 ϕ × 150 mm, MeOH/aq. 50 mM
-1
.
AcONH4 = 85/15, 1.0 mL min , Fig. S34); VIS (MeOH/
aq. 50 mMAcONH4 = 85/15) lmax = 660 (relative intensity,
0.90), 612 (0.19), 430 nm (1.00); MS (ESI) found: m/z
625.5, calcd for C35H37N4O3Zn: MH+, 625.2 [43].
Zinc 8-isobutyl-12-ethyl-bacteriopheophorbide d (Zn-
[I,E]BPheide d). tR = 5.10/5.59 min for 31R/S-epimers
(Cosmosil 5C18-AR-II, 4.6 ϕ × 150 mm, MeOH/aq.
Zinc 3-vinyl-8-isobutyl-12-ethyl-bacteriopheophor-
bide d (Zn-3V-[I,E]BPheide d). tR = 19.7 min (Cosmosil
5C18-AR-II, 4.6 ϕ × 150 mm, MeOH/aq. 50 mM
-1
.
50 mM AcONH4 = 85/15, 1.0 mL min , Fig. S27); VIS
-1
.
(MeOH/aq. 50 mM AcONH4 = 85/15) lmax = 653 (relative
intensity, 0.95), 606 (0.20), 426 nm (1.00); MS (ESI) found:
m/z 657.4, calcd for C36H41N4O4Zn: MH+, 657.2 [43].
Zinc3-vinyl-8-ethyl-12-methyl-bacteriopheophorbide c
(Zn-3V-[E,M]BPheide c). tR = 11.3 min (Cosmosil 5C18-
AcONH4 = 85/15, 1.0 mL min , Fig. S35); VIS (MeOH/
aq. 50 mMAcONH4 = 85/15) lmax = 660 (relative intensity,
0.89), 612 (0.18), 430 nm (1.00); MS (ESI) found: m/z
639.6, calcd for C36H39N4O3Zn: MH+, 639.2 [43].
AR-II, 4.6 ϕ × 150 mm, MeOH/aq. 50 mM AcONH4 =
-1
.
85/15, 1.0 mL min , Fig. S28); VIS (MeOH/aq. 50 mM
CONCLUSIONS
AcONH4 = 85/15) lmax = 668 (relative intensity, 0.77),
629 (0.19), 436 nm (1.00); MS (ESI) found: m/z 611.4,
calcd for C34H35N4O3Zn: MH+, 611.2 [28].
Various(B)Chlidesweresuccessfullysemi-synthesized
from natural (B)Chls extracted from photosynthetic
Copyright © 2018 World Scientific Publishing Company
J. Porphyrins Phthalocyanines 2018; 22: 12–14

SEMI-SYNTHESIS AND HPLC ANALYSIS OF (BACTERIO)CHLOROPHYLLIDES POSSESSING A PROPIONIC ACID
13
organisms, using a combination of organic synthetic
techniques and enzymatic modifications. RP-HPLC on
an ODS column with an eluent mixture of methanol
and aqueous 50 mM ammonium acetate was useful for
purification, identification, and analysis of the semi-
synthetic (B)Chlides. The tR-values of these (B)Chlides
were dependent on the molecular lipophilicity and the
coordination ability of the central metal to the HPLC
eluent: (1) tR(bacteriochlorin) < tR(chlorin) < tR(porphyrin)
for p-conjugation of the tetrapyrrole skeleton, (2) tR(3HE)
< tR(3Ac) < tR(3V) for the 3-substituents, (3) tR(Mg) <
tR(Zn) for the central metal, and (4) tR(20-H) < tR(20-Me)
and tR([E,M]) < tR([E,E]) < tR([P,E]) < tR([I,E]) for 82-,
121-, and 20-methylation.
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Acknowledgments
We thank Dr. Yusuke Tsukatani for his generous gift
of chlorophyllase as well as Dr. Jiro Harada for BchF
hydratase and BciC demethoxycarbonylase. We also
thank Prof. Tadashi Mizoguchi of Ritsumeikan University
for his assistance in HPLC analyses. This work was
partially supported by JSPS KAKENHI Grant Number
JP17H06436 in the Scientific Research on Innovative
Areas “Innovation for Light-Energy Conversion (I4LEC)”
(to HT) as well as the Sasakawa Scientific Research Grant
from The Japan Science Society and JSPS KAKENHI
Grant Number JP17J08860 in the JSPS Research Fellow
program (to MT).
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Figures S1–S35 are given in the supplementary
material. This material is available free of charge via the
Internet at http://www.worldscinet.com/jpp/jpp.shtml.
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