Synthesis of methyl (132R/S)-alkyl-pyropheophorbide a and a non-epimerized chlorophyll a mimic

Article abstract of DOI:10.1016/j.bmc.2015.09.016

The (13²R/S)-methoxycarbonyl group of methyl pheophorbides a/a′ (chlorophyll a/a′ derivatives) was converted to methyl, ethyl, propyl, and isopropyl groups through the C13²-alkylation under basic conditions followed by pyrolysis in 2,4,6-collidine with lithium iodide. All the resulting products, methyl 13²-alkyl-pyropheophorbides a, predominantly gave the (13²R)-stereoisomers with about one tenth of the (13²S)-epimers. Their stereochemistry was determined by 1D/2D NMR and their optical properties were characterized by visible absorption and circular dichroism spectroscopy. Methyl (13²R)-propyl-pyropheophorbide a was converted to (13²R)-propyl-pyrochlorophyll a by ester exchanging and magnesium chelating reactions. The synthetic chlorophyll a analogue showed non-epimerization at the 13²-position in pyridine-d₅ at 40 °C, while naturally occurring chlorophyll a was easily epimerized under the same conditions to give its epimeric mixture.

Full text of DOI:10.1016/j.bmc.2015.09.016

Accepted Manuscript
Synthesis of methyl (132 R/S)-alkyl-pyropheophorbide a and a non-epimerized
chlorophyll a mimic
Shin Ogasawara, Hitoshi Tamiaki
PII:
S0968-0896(15)30041-9
DOI:
http://dx.doi.org/10.1016/j.bmc.2015.09.016
BMC 12563
Reference:
Bioorganic & Medicinal Chemistry
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Received Date:
Revised Date:
Accepted Date:
11 August 2015
7 September 2015
8 September 2015
Please cite this article as: Ogasawara, S., Tamiaki, H., Synthesis of methyl (132 R/S)-alkyl-pyropheophorbide a and
a non-epimerized chlorophyll a mimic, Bioorganic & Medicinal Chemistry (2015), doi: http://dx.doi.org/10.1016/
j.bmc.2015.09.016
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Synthesis of methyl (132R/S)-alkyl-pyropheophorbide a
and a non-epimerized chlorophyll a mimic
Shin Ogasawara, Hitoshi Tamiaki*
Graduate School of Life Sciences, Ritsumeikan University, Kusatsu, Shiga 525-8577,
Japan
Received
Accepted
2015
Keywords:
Chlorophyll
Epimerization
Pyrolysis
Stereochemistry
*Corresponding author. FAX: +81 77 561 2890.
E-mail address: tamiaki@fc.ritsumei.ac.jp (H. Tamiaki).
1

ABSTRACT
The (132R/S)-methoxycarbonyl group of methyl pheophorbides a/a’ (chlorophyll a/a’
derivatives) was converted to methyl, ethyl propyl, and isopropyl groups through the
C132-alkylation under basic conditions followed by pyrolysis in 2,4,6-collidine with
lithium iodide. All the resulting products, methyl 132-alkyl-pyropheophorbides a,
predominantly gave the (132R)-stereoisomers with about one tenth of the (132S)-epimers.
Their stereochemistry was determined by 1D/2D NMR and their optical properties were
characterized by visible absorption and circular dichroism spectroscopy. Methyl
(132R)-propyl-pyropheophorbide a was converted to (132R)-propyl-pyrochlorophyll a by
ester exchanging and magnesium chelating reactions. The synthetic chlorophyll a
analogue showed non-epimerization at the 132-position in pyridine-d5 at 40 °C, while
naturally occurring chlorophyll a was easily epimerized under the same conditions to
give its epimeric mixture.
1. Introduction
Chlorophylls [see Fig. 1 (a)] and their derivatives have been widely investigated
for elucidation of photosynthetic mechanisms1 as well as application to photochemical
devices2 and photodynamic therapy.3 A variety of the substitutions at the 2-, 3-, 7-, 8-,
131-, and 20-carbons have been performed by many researchers,4 while the
C132-substitution has been limited.5,6 Natural chlorophylls always have (some)
asymmetric carbon(s) in each molecule.7 In particular, C132-stereochemisrty affects the
structure and function of a primary electron-donor in reaction centers, such as a pair of
chlorophyll a and a’ in the photosystem I (PS I) of Thermosynechococcus elongatus
[Fig. 1 (b)]. Their stereochemistry also influences biological activities of chlorophyll
a/a’ including the photophysical properties8 and the metabolic pathways.9 For instance,
2

chlorophyll synthase, one of the enzymes for chlorophyll biosynthesis, recognizes only
(132R)-epimerically pure substrates to esterify their C172-carboxy groups.10 Thus, the
C132-stereochemistry is the important factor for biochemical researches on natural
chlorophylls. The chiral center in the -keto-ester moiety is easily epimerized under
basic conditions to give an epimeric mixture [see Fig. 1 (a)]; typically, chlorophyll a
(132R) : chlorophyll a’ (132S) = 4 : 1 in pyridine.11 The epimerization is induced via
deprotonation at the 132-position under basic conditions, and the resulting
C132-carbanion is also susceptible to oxidation (allomerization) leading to E ring
degradation.12 Hence, controlling the C132-stereochemistry and reactivity is an
interesting and important issue in (bio)organic, biological, and (bio)physical chemistry.
Our preliminary studies showed that the C132-alkylations of methyl pheophorbides a/a’
(R = H at left in Fig. 2), chlorophyll a/a’ derivatives, were achieved through a simple
SN2 reaction process in the presence of a base.13,14 We succeeded in the pyrolysis of
methyl 132-methyl-pheophorbides a/a’ (R = Me at left in Fig. 2) in 2,4,6-collidine
containing sodium hydroxide to afford its C132-demethoxycarbonylated product, methyl
(132R)-methyl-pyropheophorbide a (R = Me at right in Fig. 2) with a low yield; a trace
amount of the (132S)-epimer was detected.13
Figure 1. (a) Configuration formulae of natural occurring chlorophylls a/a’ and (b) the
crystal structure of PS I derived from Thermosynechococcus elongatus with a pair of
3

chlorophyll a and a’ (based on the PDB crystallographic data of 3PCQ).
Here, we performed pyrolysis of methyl 132-methyl-, ethyl-, propyl-, and
isopropyl-pheophorbide a/a’ (left in Fig. 2) in 2,4,6-collidine in the presence of lithium
iodide. All the pyrolyzed products were given in higher yields than previously. Based on
1D/2D
NMR
spectral
analyses,
the
resulting
products,
methyl
132-alkyl-pyropheophorbides a, were confirmed to be a 9:1 mixture of (132R)- and
(132S)-epimers (right in Fig. 2). The (132R)-propyl substituted product was
ester-exchanged and magnesium-chelated to synthesize (132R)-propyl-pyrochlorophyll a.
Since a propyl group is sterically similar to a methoxycarbonyl group, the synthetic
magnesium complex possessing the propyl group is a structural model of chlorophyll a.
Due to the exchange of electron-withdrawing COOMe to electron-donating Pr as the
C132-substituent (-keto-ester to -alkyl-ketone), the chlorophyll a mimic was
chemically more stable under basic conditions. No epimerization at the 132-position of
the semi-synthetic mimic was observed in heated pyridine where chlorophyll a was
quickly epimerized.
NH
N
N
NH
N
N
H
R
O
R
O
COOMe
(132R)-epimer
HN
132
HN
(132S)-epimer
R
O
O
O
R
O
O
O
O
O
H
R
(-alkyl-)ketone
O
R
O
-alkyl-)-keto-ester
COOMe
(132S)-epimer
(132R)-epimer
(R = H, Me, Et, Pr, iPr)
(R = H, Me, Et, Pr, iPr)
*Except R = H
Methyl (132R/S)-(alkyl-)pheophorbides a/a'
Methyl (132R/S)-(alkyl-)pyropheophorbides a
Figure 2. Configuration formulae with C132-stereochemistry of methyl
132-alkyl-pheophorbides (left) and their pyrolyzed products (right).
4

2. Results and discussion
2.1. Synthesis of methyl 132-alkyl-pyropheophorbides a
According to our previous reports,14 methyl (132R/S)-methyl-, ethyl-, propyl-,
and isopropyl-pheophorbides a/a’ [(R)/(S)-2a, (R)/(S)-2b, (R)/(S)-2c, and (R)/(S)-2d,
respectively, see Scheme 1] were synthesized from an epimeric mixture of methyl
pheophorbides a [(R)-1] and a’ [(S)-1] [step (i) of Scheme 1], which were prepared by
chemical modification of chlorophyll a.15,16
Usual pheophorbides are demethoxycarbonylated at the 132-position by
pyrolysis in refluxing 2,4,6-collidine [step (ii) of Scheme 1]; smooth transformation of
(R)/(S)-1 to methyl pyropheophorbide a (4).16 Under the same conditions, methyl
132-alkyl-pheophorbides a/a’ [(R)/(S)-2a–d] were inactive and the starting materials
were recovered. As mentioned in our preliminary report [step (iii) of Scheme 1],13
sodium hydroxide promoted the 132-demethoxycarbonylation of (R)/(S)-2a with partial
hydrolysis
of
the
C172-COOMe,
to
give
predominantly
methyl
(132R)-methyl-pyropheophorbide a [(R)-3a] with a low yield after methyl esterification.
In this study, epimeric mixtures of (R)/(S)-2a–d were dissolved in
2,4,6-collidine (10 mM), to which two equivalents of LiI with 1.28 H2O was added [step
(iv) of Scheme1]. The mixture was refluxed until it completely consumed the starting
pheophorbides (monitored by TLC and/or TOF-MS). After removing 2,4,6-collidine
under a reduced pressure at 50 °C, the residues were dissolved in dichloromethane, and
the solution was washed with a 4wt% sodium hydrogen carbonate solution and distilled
water. The target products were separated by flash column chromatography (FCC) with
a mixture of dichloromethane and diethyl ether as the eluent. The products were further
5

purified by reprecipitation from dichloromethane and hexane as well as
dichloromethane and methanol to give pure samples (ca. 30–80% yields). The values
1
were much larger than that aforementioned (≤6%). Because the H NMR spectra of all
the products showed one set of C132-H signals (major/minor = 9/1), they were
(132R/S)-epimeric mixtures (R)/(S)-3a–d.
(ii)
(iii)
NH
N
N
(R) / (S)-2a
(R) / (S)-2b
(R) / (S)-2c
(R) / (S)-2d
(R) / (S)-3a
(R) / (S)-3b
(R) / (S)-3c
(R) / (S)-3d
HN
132
(i)
(iv)
(R) / (S)-1
17
R2
R1
(ii)
172
O
O
4
O
Compound
R1
R2
H
Compound
R1
H
R2
H
(R)-1
COOMe
H
4
(S)-1
COOMe
(R)-2a
(S)-2a
(R)-2b
(S)-2b
(R)-2c
(S)-2c
(R)-2d
(S)-2d
COOMe
Me
Me
COOMe
Et
(R)-3a
(S)-3a
(R)-3b
(S)-3b
(R)-3c
(S)-3c
(R)-3d
(S)-3d
Me
H
H
Me
H
COOMe
Et
Et
H
COOMe
Pr
Et
H
COOMe
Pr
Pr
H
COOMe
iPr
Pr
H
COOMe
iPr
iPr
H
COOMe
iPr
Scheme 1. Synthetic routes of methyl (132R/S)-alkyl-pyropheophorbides a
[(R)/(S)-3a–d]: (i) NaOMe (5 equiv.), MeI/EtI/PrI/iPrI (excess), dry THF, reflux; (ii)
2,4,6-collidine, reflux; (iii) NaOH, 2,4,6-collidine, reflux and H2SO4, MeOH [for
6

[(R)/(S)-2a]; (iv) LiI·1.28H2O (2 equiv.), 2,4,6-collidine, reflux.
The epimers of the methylated and ethylated adducts (R)/(S)-3a and (R)/(S)-3b
were separated by reverse phase (RP) HPLC on a packed octadecylated silica gel
column with methanol/acetonitrile [1/10 (v/v)] and methanol/acetonitrile/water [50/45/5
(v/v)], respectively. The minor epimers were eluted more rapidly than the major epimers,
indicating that the formers have higher lipophilicities than the latters. Propylated and
isopropylated epimers (R)/(S)-3c and (R)/(S)-3d were completely separated by
successive preparative layer chromatography (PLC) on silica gel with a mixture of
dichloromethane and diethyl ether as the eluent. The major epimers gave lower Rf values
than the minors in PLC. Similar RP-HPLC analyses as in (R)/(S)-3b (vide supra)
showed that the retention times of major epimers were longer than those of minor
epimers in the propylated and isopropylated epimers (R)/(S)-3c and (R)/(S)-3d. This
order is consistent with that in (R)/(S)-3a and (R)/(S)-3b.
2.2. C132-stereochemistry of methyl 132-alkyl-pyropheophorbides a
HH-ROESY spectroscopy showed the specific NOE correlations between
C132-alkyl protons/C132-H and C17-CH2CH2/C17-H (Fig. S1–S7), and revealed the
stereostructures of the separated major and minor epimers. Since the NOE correlation
between C132-H and C17-H was observed in all the epimers due to their close atomic
distance in a molecule, it could not be used for their structural determination.
The HH-ROESY spectra of the major epimer of the C132-Me/H product
showed the NOE correlations between the C132-H and C17-CH2 as well as between the
C132-CH3 and C17-H, indicating that the major epimer was assigned to be (R)-3a.13 The
NOE correlation between the C132-CH3 and C17-CHCH in the minor epimer clearly
7

showed (S)-3a (Fig. S1). In the C132-Et/H product, the major epimer gave the NOE
correlations between the C132-H and C17-CH2CH as well as between the C132-CH2CH3
and C17-H and was assigned to be (R)-3b (Fig. S2), while the minor epimer afforded
the NOE correlation between the C132-CHCH3 and C17-CH and was confirmed to be
(S)-3b (Fig. S3). Since the NOE correlations of the major epimer in the C132-Pr/H
product were observed between the C132-H and C17-CHCH2 as well as between the
C132-CH2CHCH3 and C17-H (Fig. S4), the major epimer was assigned to be (R)-3c, and
the minor was (S)-3c due to the NOE correlation between the C132-CH and C17-CH2 as
well as between the C132-CCH and C17-CH (Fig. S5). The major epimer of the
C132-iPr/H showed the NOE correlations between the C132-H and C17-CH2CH2 as well
as between the C132-CHCH3 and C17-H (Fig. S6) and was assigned to be (R)-3d, while
the minor epimer showed the NOE correlation between the C132-CHCH3 and
C17-CHCH to be (S)-3d (Fig. S7).
1
From the above H NMR spectroscopic analyses, all the major and minor
epimers were (R)-3a–d and (S)-3a–d, respectively. The pyrolyzed products [step (iv) of
Scheme 1] predominantly gave the (132R)-epimer with less steric hindrance between the
C132- and C17-substituents. It is noteworthy that the C132-methyl, ethyl, and propyl
groups were primarily inverted around the 132-position during the pyrolysis
(demethoxycarbonylation) as shown in Fig. 3 (their epimeric ratios are summarized in
Table S1). The methylation, ethylation, and propylation of (R)/(S)-1 [step (i) of Scheme
1] mainly afforded the (132R)-epimeric adducts (R)-2a–c possessing the syn-oriented
C132-alkyl groups for the C17-propionate residue due to the sterically-demanding
C132-methoxycarbonyl group [Fig. 3(a)].14 In contrast, the C132-isopropylation yielded
(132S)-epimer (S)-2d bearing the anti-oriented C132-isopropyl group for the
C17-propionate residue, indicating that the isopropyl group is more sterically bulky than
the methoxycarbonyl group. After removing the C132-methoxycarbonyl group, the
C132-alkyl groups of all the major products (R)-2a–d direct anti-orientation for the
8

C17-propionate residue as shown in Fig. 3(b). Hence, the C132-substituted compounds
tend to take one of the epimeric configurations with less steric hindrance between the
C132-substituents and C17-propionate residue. Molecular modeling simulation
(MM+/PM3)17 indicated that the estimated total energies of the (R)-3a–d were lower by
3–5 kcal mol-1 than those of the corresponding (S)-3a–d as shown in Table 1, which
qualitatively corresponded with the predominant production of the (132R)-epimers in the
above pyrolysis but could not estimate the observed 9:1 ratios. Such steric factors
between the 132-alkyl group and the 17-propionate residue could affect the molecular
packing in their solid states; the melting points of the (132S)-epimers were ca. 20–50 °C
lower than those of the (132R)-epimers (see experimental section).
(a)
E
H
E
H
E
H
E
H
E
H
E
H
E
H
E
H
CH3
E
E
E
E
132
17
(R)-2a
(R)-2b
(R)-2c
(S)-2d
(b)
H
H
H
H
CH3
(R)-3a
(R)-3b
(R)-3c
(R)-3d
Figure 3. Schematic representations of the positional relationships between C132-alkyl
groups and C17-propionate residue of the major epimers; (a) before and (b) after
removing C132-methoxycarbonyl group: E = COOMe.
9

Table 1
The differences Ga (kcal mol-1) of estimated total energy between the (132R)- and
(132S)-epimers
Compound (132-alkyl group)
G
(R)/(S)-3a (methyl)
(R)/(S)-3b (ethyl)
–4.9
–3.1
–3.3
–4.8
(R)/(S)-3c (propyl)
(R)/(S)-3d (isopropyl)
a G = GR–GS; GR and GS are total energies of (R)-3a–d and (S)-3a–d, respectively.
2.3. Optical properties of methyl 132-alkyl-pyropheophorbides in dichloromethane
All of the products (R)/(S)-3a–d showed similar visible absorption spectra in
dichloromethane (Fig. 4). The absorption maxima at the Soret, Qx, and Qy bands of
(R)/(S)-3a–d showed almost the same wavelengths and were also similar to those of
(R)/(S)-1 and their derivative 4 lacking any C132-substituent. Close inspection shows
that Qy absorption maxima of (S)-3a–d were slightly shifted to longer wavelengths (1–2
nm) than those of (R)-3a–d (Table 2). In our previous report,18 derivatives of (S)-1
possessing C132-methoxy-, ethoxy-, isopropoxy-carbonyl groups also gave red-shifted
Qy maxima (0.4–1.1 nm), compared to those of the corresponding (132R)-epimers. All
the red-shifts could be due to the chlorin ring distortions induced by steric repulsing
between the C132-substituents and C17-propionate residue (vide infra).
10

Figure 4. Visible absorption spectra of methyl 132-(un)substituted (pyro)pheophorbides
a in dichloromethane; (a) 132R-epimers, (b) 132S-epimers. All the spectra were
normalized at Soret maxima.
Table 2
Visible absorption maxima (nm) of (R)/(S)-3a–d in CH2Cl2
R
S
Compound
Qx
Qy
Qx
Qy
Soret
Soret
(0,1) (0,0) (0,1) (0,0)
508 538 609 666
508 539 610 667
509 539 610 668
509 538 610 667
(0,1) (0,0) (0,1) (0,0)
509 539 610 668
509 540 611 669
510 540 611 669
510 540 611 669
414
414
414
414
413
414
414
414
3a
3b
3c
3d
Circular dichroism (CD) spectra of methyl 132-alkyl-pyropheophorbides a in
dichloromethane are dependent on the C132-stereochemistry as shown in Fig. 5, which
is consistent with the CD spectral change in (R)/(S)-1 reported earlier.19 (R)-3a/b
showed positive and negative CD bands at the Soret and Qy regions, respectively, and
gave no peak at the Qx region. In (S)-3a/b, more intense CD bands were observed and
11

positive CD peaks were visible at the Qx region. The epimeric control is ascribable to
the chlorin ring distortions induced by steric repulsing between the C132-methyl/ethyl
group and C17-propionate residue in (S)-3a/b. The standard deviations () of the
composite 22 atoms (= C1–16 + C19–20 + N21–24) from their mean plane were
calculated on the basis of the optimized molecular models by MM+/PM3, and the
-values of (S)-3a/b were larger than those of (R)-3a/b (Table S2). Due to the bulkier
(iso)propyl group at the 132-position, (R)-3c/d with larger -values than (R)-3a/b
showed a negative CD peak at the Qx region as well as positive and negative bands at the
Soret and Qy regions, respectively. Their epimeric (S)-3c/d showed opposite positive and
intense CD peaks at the Qx region with enhanced CD bands at the Soret and Qy regions.
The CD bands at the Soret region of the isopropylated (R)-3d was largely different from
those of the other (R)-3a–c, while (S)-3a–d gave similar CD spectra and no substitution
effect was observed, which is consistent with similar -values in (S)-3a–d (Table S2).
Figure 5. CD spectra of methyl 132-alkyl-pyropheophorbides a (R)/(S)-3a–d in
12

dichloromethane; (R)-3a–d (solid line) and (S)-3a–d (broken line).
2.4. Synthesis of (132R)-propyl-pyrochlorophyll a as a chlorophyll-a mimic and its
non-epimerization
Epimerically
pure
(R)-3c
was
reacted
with
phytol
and
bis[dibutylchlorotin(IV)]oxide in refluxing dry toluene under N2 flow in the dark6 to
give (132R)-propyl-pyropheophytin a (Pr-pPhe-a) possessing a phytyl ester group in a
yield of 89% [see step (i) of Scheme 2]. Free base Pr-pPhe-a was treated with an excess
amount of magnesium perchlorate in refluxing pyridine under N2 flow in the dark20 to
afford (132R)-propyl-pyrochlorophyll a (Pr-pChl-a) after purification with FCC and
HPLC [step (ii) of Scheme 2]. It was noteworthy that no epimerization was observed
during the ester-exchange of methyl to phytyl group [(R)-3c  Pr-pPhe-a) and
magnesium chelation of free base (Pr-pPhe-a  Pr-pChl-a) under the present
conditions.
Scheme 2. Synthesis of (132R)-propyl-pyrochlorophyll a: (i) phytol (10 equiv.),
(Bu2ClSn)2O, toluene, reflux; (ii) Mg(ClO4)2 (excess), pyridine, reflux.
The optical properties of Pr-pChl-a were compared with the original
13

chlorophyll a. As shown in Fig. 6(a), the visible absorption spectrum of Pr-pChl-a was
almost similar to that of chlorophyll a in diethylether. The relative intensities of the
Soret maxima (normalized at the Qy maxima) of Pr-pChl-a, interestingly, was larger
than that of chlorophyll a, and the wavelength of the Qy maximum of Pr-pChl-a (659
nm) was slightly blue-shifted than that of chlorophyll a (661 nm). The slight changes
were probably ascribable to the electron-donating C132-propyl group in Pr-pChl-a and
the electron-withdrawing C132-methoxycarbonyl group in chlorophyll a. The CD
spectra of Pr-pChl-a and chlorophyll a were almost similar, except for the peak
intensities at < 400 nm [Fig. 6(b)].
Figure 6. Visible absorption (a) and CD spectra (b) of chlorophyll mimic Pr-pChl-a
(solid line) and chlorophyll a (broken line) in diethylether; visible absorption spectra
were normalized at the Qy maxima.
The epimerization of chlorophyll a and its mimic Pr-pChl-a was examined
14

under basic conditions as previously reported.11 Their pyridine-d5 solutions were
incubated at 40 °C in the dark for 3 h, and the epimerization was monitored by 1H NMR
spectroscopy. Chlorophyll a, which was extracted from cyanobacterial (Spirulina) cells
and purified by traditional methods,21,22 was readily epimerized under the above
conditions to give one set of proton signals at the meso(5,10,20)-H, 132-H, and P1-H in
the 1H NMR spectra [Figure 7(a)] indicating an epimeric mixture of chlorophylls a and
a’ (87:13), which corresponds with ref 11. In contrast, only one signal assigned to the
meso(5,10,20)-H, 132-H, and P1-H of (132R)-Pr-pChl-a was found in the 1H NMR [Fig.
7(b)] indicating that (132R)-Pr-pChl-a was not converted to its (132S)-epimer under the
above basic conditions and was highly tolerant to such epimerization. These results
indicated that Pr-pChl-a was chemically more stable for the basic epimerization than
chlorophyll a, in spite of slight structural difference: C132-COO (chlorophyll a) 
C132-CH2CH2 (Pr-pChl-a). This is due to a lower acidity at the 132-position as the 
carbon of keto-type Pr-pChl-a (C132-CH2CH2CH3) than that of -ketoester-type
chlorophyll a (C132-COOCH3).
15

Figure 7. The specific proton signals in the 1H NMR (600 MHz) spectra of chlorophyll
a (a) and Pr-pChl-a in pyridine-d5 (b) incubated at 40 °C for 0 h (lower) and 3 h (upper).
The arrows indicate the corresponding protons of the (132S)-epimer (chlorophyll a’).
16

3. Conclusion
Methyl 132-alkyl-pyropheophorbides a (R)/(S)-3a–d were synthesized by
modifying naturally occurring chlorophyll a, and their stereochemistry were determined
by 1D/2D NMR spectroscopic technique. The pyrolysis of methyl
132-alkyl-pheophorbides a/a’ (R)/(S)-2a–d afforded a 9:1 mixture of (R)-3a–d and
(S)-3a–d. The ratios did not depend on the 132R/S ratios of the starting materials
(R)/(S)-2a–d but were controlled by the thermodynamic stability (steric factor around
the 132-substituent) of the products. All the (132R)-epimers possess higher lipophilicity
than the corresponding (132S)-epimers from their RP-HPLC analyses. The
hydrophobicity would be due to the direction of the lipophilic C132-alkyl groups; the
anti-oriented C132-alkyl groups to C17-propionate residue in the (132R)-epimers are
effective
for
hydrophobicity
in
a
molecule.
Epimerically
pure
(132R)-propyl-pyrochlorophyll a (Pr-pChl-a) was produced as the structural mimic of
chlorophyll a from methyl 132(R)-propyl-pyropheophorbide a [(R)-3c]. The visible
absorption and CD spectra of Pr-pChl-a in diethylether were almost similar to those of
chlorophyll a except for some relative intensities. Under the present basic conditions, no
epimerization was observed in Pr-pChl-a, while chlorophyll a was readily epimerized
to chlorophyll a’. This indicates that the electron-donating C132-propyl group
suppresses the production of the 132 carbanion under basic conditions. This study will
expand to control C132-stereochemistry and to produce chlorophyll derivatives tolerant
to undesired oxidations.
17

4. Experimental
4.1. General
2,4,6-Collidine, 1.28-hydrated lithium iodide, pyridine, and magnesium
perchlorate were purchased and used without further purification. FCC and PLC were
performed on silica gel (Merck, Kieselgel 60, 40–63 μm, 230–400 mesh for free base,
and WakogelⓇ C-300, 45–75 m for magnesium complex) and silica gel plate (Merck,
PLC Silica gel 60F254, 1 mm thickness, 20×20 cm), respectively. HPLC was performed
on Cosmosil (Nacalai tesque) and Inertsil columns (GL Sciences) with HPLC-grade
methanol, acetonitrile, ethyl acetate, and water as eluents.
HPLC was done with a Shimadzu LC-10AS pump and SPD-10AV
photodiode-array detector. 1D and 2D NMR spectra in CDCl3 were recorded on a JEOL
ECL-600 spectrometer (600 MHz for 1H and 150 MHz for 13C); CHCl3 (δH = 7.26 ppm)
13
and CDCl3 (δC = 77.00 ppm) were used as internal references. Visible absorption and
CD spectra were measured with a Hitachi U-3500 and Jasco J-720W spectrometers,
respectively. Time-of-flight mass spectra (TOF-MS) were recorded on a Shimadzu
AXIMA CFR-plus: laser deposition ionization and positive mode. High resolution mass
spectra (HRMS) were recorded on a Bruker micrOTOF II spectrometer: atmospheric
pressure chemical ionization (APCI) and positive mode in a MeOH solution for free
base pyropheophorbides, and electron spray ionization (ESI) and positive mode in an
acetonitrile solution for magnesium complex.
18

4.2. Synthetic procedure
4.2.1. Preparation of methyl pheophorbides a/a’ [(R)/(S)-1] and methyl
pyropheophorbide a (4)
According to the previous procedures,15,16 an epimeric mixture of pheophytins
a/a’ including other cellular contents were obtained from a commercially available dry
cyanobacterial cells of Spirulina containing chlorophyll a. The extracted and
pheophytinized mixture was treated with methanol and sulfuric acid at rt in the dark
overnight. The reaction mixture was purified by FCC with hexane, then
dichloromethane with 0 to 3 % of diethyl ether and precipitation from dichloromethane
and hexane to give an epimeric mixture of (R)/(S)-1 as a dark purple powder.15,16
Subsequently, (R)/(S)-1 was refluxed in 2,4,6-collidine and was purified by precipitating
from dichloromethane and methanol to give 4 as a dark purple powder.16
4.2.2. Synthesis of methyl 132-alkyl-pheophorbides a/a’ [(R)/(S)-2a–d]
An epimeric mixture of (R)/(S)-1 was reacted with an excess amount of methyl,
ethyl, propyl, and isopropyl iodide in the presence of sodium methoxide in refluxing dry
THF under N2 atmosphere in the dark to give an epimeric mixture of (R)/(S)-2a–d as
dark purple powders according to our previous reports.13,14
4.2.3. General procedure for synthesis of methyl 132-alkyl-pyropheophorbides a
[(R)/(S)-3a–d]
An epimeric mixture of methyl 132-alkyl-pheophorbides a/a’ [(R)/(S)-2a–d, 1.0
equiv.] was dissolved in 2,4,6-collidine (10 mM solution) and refluxed with 2.0 equiv.
of LiI·1.28H2O in the dark. The heating was stopped just after complete consumption of
the starting (R)/(S)-2a–d, which was monitored by TLC and/or TOF-MS. After
19

removing 2,4,6-collidine in vacuo, the residue was dissolved in dichloromethane and
washed with an aqueous 4% NH4Cl solution (×1) and water (×3). The organic layer was
dried by Na2SO4 and filtered off. The mixture was purified by FCC [CH2Cl2/Et2O =
100/5 (v/v)] and precipitation from dichloromethane and hexane, then from
dichloromethane and methanol to give dark purple powders as an epimeric mixture in
78% [(R)/(S)-3a], 61% [(R)/(S)-3b], 41% [(R)/(S)-3c], and 33% yields [(R)/(S)-3d].
The epimeric mixture of methyl/ethyl adducts (R)/(S)-3a/b was separated by
RP-HPLC [Cosmosil 5C18-AR-II, 20×250 mm, methanol/acetonitrile = 1/10 (v/v) for
(R)/(S)-3a and methanol/acetonitrile/water = 50/45/5 (v/v) for (R)/(S)-3b, 9.0 mL min-1].
In
the
analytical
RP-HPLC
[Cosmosil
5C18-AR-II,
4.6×150
mm,
methanol/acetonitrile/water = 50/45/5 (v/v) , 1.0 mL min-1], major epimer (R)-3a was
eluted at 14.0 min and minor epimer (S)-3a was eluted at 12.6 min, while major (R)-3b
and minor (S)-3b were eluted at 14.0 and 13.3 min, respectively. The epimeric mixture
of propyl/isopropyl adducts (R)/(S)-3c–d was separated by repeated PLC
[dichloromethane/diethyl ether = 100/1 (v/v) for (R)/(S)-3c, and 50/1 (v/v) for
(R)/(S)-3d: Rf values were 0.18/0.23 for (R)/(S)-3c and 0.25/0.31 for (R)/(S)-3d]. In the
same analytical RP-HPLC, major epimers (R)-3c/d moved more slowly (16.2/15.2 min)
than the corresponding minor (S)-3c/d (15.5/13.9 min).
4.2.4. Synthesis of 132-propyl-pyropheophytin a (Pr-pPhe-a)
Epimerically pure (R)-3c (60.0 mg, 102 mol) in dry toluene (10 mL) was
refluxed
with
phytol
(303
mg,
1.02
mmol,
10.0
equiv.)
and
bis[dibutylchlorotin(IV)]oxide (19.6 mg, 34.7 mol, 0.34 equiv.) under N2 atmosphere
in the dark for 3 h. After removing solvent in vacuo, the mixture was purified by FCC
with a mixture of dichloromethane and diethyl ether [100/2 (v/v)] as an eluent.
Precipitating from a mixture of dichloromethane and methanol gave Pr-pPhe-a as a
dark purple solid in a 89% yield.
20

4.2.5. Synthesis of 132-propyl-pyrochlorophyll a (Pr-pChl-a)
Free base Pr-pPhe-a (12.6 mg, 14.7 mol) was reacted with Mg(ClO4)2 (328
mg, 1.47 mmol, 100 equiv.) in refluxing pyridine (3 mL) under Ar bubbling in the dark
for 1 h. After removing the solvent, the residue was dissolved in diethyl ether and
washed with 1 M phosphate buffer. The organic layer was dried by Na2SO4 and filtered
off. The mixture was roughly purified by FCC [Wakogel C-300,
dichloromethane/diethyl ether = 100/3 (v/v)], and further purified by normal-phase
HPLC [Inertsil ODS-P, 10×250 mm, acetonitrile/methanol/ethyl acetate = 40/50/10
(v/v), 5.0 mL min-1] to give Pr-pChl-a as a green solid in a 28% yield.
4.3. Spectral data
4.3.1. Methyl (132R)-methyl-pyropheophorbide a [(R)-3a]
Mp 232–233 °C; the spectroscopic data were reported in 13.
4.3.2. Methyl (132S)-methyl-pyropheophorbide a [(S)-3a]
1
Mp 186–187 °C; H NMR (CDCl3) δ / ppm = 9.49 (1H, s, 10-H), 9.34 (1H, s,
5-H), 8.50 (1H, s, 20-H), 7.99 (1H, dd, J = 18, 11 Hz, 3-CH), 6.29 (1H, dd, J = 18, 1 Hz,
31-CH trans to 3-C–H), 6.16 (1H, dd, J = 11, 1 Hz, 31-CH cis to 3-C–H), 5.25 (1H, q, J
= 8 Hz, 132-H), 4.47–4.44 (1H, m, 18-H), 4.24–4.22 (1H, m, 17-H), 3.69 (2H, dq, J = 8,
3 Hz, 8-CH2), 3.66 (3H, s, 12-CH3), 3.64 (3H, s, 172-COOCH3), 3.39 (3H, s, 2-CH3),
3.23 (3H, s, 7-CH3), 2.71–2.66, 2.44–2.39 (each 1H, m, 171-CH2), 2.60–2.55, 2.17–2.11
(each 1H, m, 17-CH2), 2.26 (3H, d, J = 8 Hz, 132-CH3), 1.69 (3H, t, J = 8 Hz, 81-CH3),
1.56 (3H, d, J = 8 Hz, 18-CH3), 0.42, –1.44 (each 1H, s, NH×2); Vis (CH2Cl2) λmax / nm
= 668 (relative intensity, 0.41), 610 (0.08), 539 (0.09), 509 (0.10), 413 (1.00), 323
(0.19); HRMS (APCI) found: m/z = 563.3017, calcd for C35H39N4O3: MH+, 563.3017.
21

4.3.3. Methyl (132R)-ethyl-pyropheophorbide a [(R)-3b]
1
Mp 218–219 °C; H NMR (CDCl3) δ / ppm = 9.55 (1H, s, 10-H), 9.40 (1H, s,
5-H), 8.60 (1H, s, 20-H), 8.01 (1H, dd, J = 18, 11 Hz, 3-CH), 6.30 (1H, dd, J = 18, 1 Hz,
31-CH trans to 3-C–H), 6.17 (1H, dd, J = 11, 1 Hz, 31-CH cis to 3-C–H), 5.43 (1H, dd, J
= 6, 3 Hz, 132-H), 4.50–4.46 (1H, m, 18-H), 4.40–4.38 (1H, m, 17-H), 3.71 (3H, s,
12-CH3), 3.70 (2H, q, J = 8 Hz, 8-CH2), 3.61 (3H, s, 172-COOCH3), 3.42 (3H, s, 2-CH3),
3.24 (3H, s, 7-CH3), 2.95–2.88, 2.77–2.71 (each 1H, m, 132-CH2), 2.69–2.63, 2.55–2.49
(each 1H, m, 171-CH2), 2.27–2.20 (2H, m, 17-CH2), 1.88 (3H, d, J = 8 Hz, 18-CH3),
1.70 (3H, t, J = 8 Hz, 81-CH3), 0.80 (3H, t, J = 8 Hz, 132-CCH3), 0.20, –1.77 (each 1H, s,
13
NH×2); C NMR (CDCl3) δ / ppm = 199.78, 173.49, 170.66, 160.59, 155.27, 150.67,
149.14, 144.99, 141.19, 137.79, 136.18, 136.05, 135.68, 131.53, 130.40, 129.21, 127.98,
122.49, 109.44, 103.99, 97.14, 92.96, 58.61, 51.67, 50.98, 49.83, 30.88, 29.69, 23.51,
23.45, 19.48, 17.47, 12.13, 12.13, 11.26, 9.73; Vis (CH2Cl2) λmax / nm = 667 (relative
intensity, 0.43), 610 (0.07), 539 (0.09), 508 (0.10), 414 (1.00), 323 (0.19); HRMS
(APCI) found: m/z = 577.3195, calcd for C36H41N4O3: MH+, 577.3173.
4.3.4. Methyl (132S)-ethyl-pyropheophorbide a [(S)-3b]
1
Mp 196–197 °C; H NMR (CDCl3) δ / ppm = 9.50 (1H, s, 10-H), 9.35 (1H, s,
5-H), 8.49 (1H, s, 20-H), 8.00 (1H, dd, J = 18, 11 Hz, 3-CH), 6.30 (1H, dd, J = 18, 1 Hz,
31-CH trans to 3-C–H), 6.17 (1H, dd, J = 11, 1 Hz, 31-CH cis to 3-C–H), 5.36 (1H, dd, J
= 5, 4 Hz, 132-H), 4.47–4.44 (1H, m, 18-H), 4.22–4.20 (1H, m, 17-H), 3.70 (2H, q, J = 8
Hz, 8-CH2), 3.66 (3H, s, 12-CH3), 3.65 (3H, s, 172-COOCH3), 3.40 (3H, s, 2-CH3), 3.24
(3H, s, 7-CH3), 2.99–2.94, 2.76–2.72 (each 1H, m, 132-CH2), 2.71–2.65, 2.39–2.34
(each 1H, m, 171-CH2), 2.54–2.49, 2.19–2.12 (each 1H, m, 17-CH2), 1.69 (3H, t, J = 8
Hz, 81-CH3), 1.51 (3H, d, J = 8 Hz, 18-CH3), 0.81 (3H, t, J = 8 Hz, 132-CCH3), 0.41,
–1.44 (each 1H, s, NH×2); Vis (CH2Cl2) λmax / nm = 669 (relative intensity, 0.41), 611
22

(0.08), 540 (0.10), 509 (0.11), 414 (1.00), 323 (0.22); HRMS (APCI) found: m/z =
577.3159, calcd for C36H41N4O3: MH+, 577.3173.
4.3.5. Methyl (132R)-propyl-pyropheophorbide a [(R)-3c]
Mp 220.5–221.0 °C; 1H NMR (CDCl3) δ / ppm = 9.55 (1H, s, 10-H), 9.40 (1H,
s, 5-H), 8.60 (1H, s, 20-H), 8.01 (1H, dd, J = 18, 11 Hz, 3-CH), 6.29 (1H, dd, J = 18, 1
Hz, 31-CH trans to 3-C–H), 6.17 (1H, dd, J = 11, 1 Hz, 31-CH cis to 3-C–H), 5.43 (1H,
dd, J = 7, 3 Hz, 132-H), 4.50–4.46 (1H, m, 18-H), 4.40–4.38 (1H, m, 17-H), 3.71 (3H, s,
12-CH3), 3.70 (2H, q, J = 8 Hz, 8-CH2), 3.60 (3H, s, 172-COOCH3), 3.42 (3H, s, 2-CH3),
3.24 (3H, s, 7-CH3), 2.86–2.80 (1H, m, 132-CH), 2.69–2.63 (2H, m, 132-, 17-CH),
2.54–2.49 (1H, m, 171-CH), 2.28–2.18 (2H, m, 17-CHCH), 1.89 (3H, d, J = 8 Hz,
18-CH3), 1.70 (3H, t, J = 8 Hz, 81-CH3), 1.36–1.28, 1.20–1.11 (each 1H, m, 132-CCH2),
0.87 (3H, t, J = 7 Hz, 132-C2CH3), 0.18, –1.78 (each 1H, s, NH×2); 13C NMR (CDCl3) δ
/ ppm = 199.82, 173.46, 170.60, 160.52, 155.25, 150.66, 148.89, 144.98, 141.17, 137.81,
136.15, 136.05, 135.67, 131.51, 130.19, 129.22, 128.07, 122.47, 109.98, 103.96, 97.14,
92.96, 57.98, 51.65, 51.09, 49.81, 32.92, 30.91, 29.72, 23.42, 19.48, 19.06, 17.47, 14.62,
12.13, 12.12, 11.26; Vis (CH2Cl2) λmax / nm = 668 (relative intensity, 0.43), 610 (0.07),
539 (0.08), 509 (0.10), 414 (1.00), 322 (0.18); HRMS (APCI) found: m/z = 591.3326,
calcd for C37H43N4O3: MH+, 591.3330.
4.3.6. Methyl (132S)-propyl-pyropheophorbide a [(S)-3c]
1
Mp 202–203 °C; H NMR (CDCl3) δ / ppm = 9.48 (1H, s, 10-H), 9.34 (1H, s,
5-H), 8.50 (1H, s, 20-H), 7.99 (1H, dd, J = 18, 11 Hz, 3-CH), 6.29 (1H, dd, J = 18, 1 Hz,
31-CH trans to 3-C–H), 6.16 (1H, dd, J = 11, 1 Hz, 31-CH cis to 3-C–H), 5.35 (1H, dd, J
= 5, 4 Hz, 132-H), 4.48–4.44 (1H, m, 18-H), 4.22–4.20 (1H, m, 17-H), 3.68 (2H, q, J = 8
Hz, 8-CH2), 3.66 (3H, s, 12-CH3), 3.64 (3H, s, 172-COOCH3), 3.40 (3H, s, 2-CH3), 3.23
(3H, s, 7-CH3), 2.90–2.84 (1H, m, 132-CH), 2.71–2.60 (2H, m, 132-, 171-CH), 2.49–2.44,
23

2.19–2.13 (each 1H, m, 17-CH2), 2.40–2.35 (2H, m, 171-CH), 1.69 (3H, t, J = 8 Hz,
81-CH3), 1.52 (3H, d, J = 7 Hz, 18-CH3), 1.42–1.34, 1.04–0.97 (each 1H, m, 132-CCH2),
0.87 (3H, t, J = 7 Hz, 132-C2CH3), 0.40, –1.45 (each 1H, s, NH×2); Vis (CH2Cl2) λmax
/
nm = 669 (relative intensity, 0.41), 611 (0.08), 540 (0.10), 510 (0.10), 414 (1.00), 323
(0.20); HRMS (APCI) found: m/z = 591.3330, calcd for C37H43N4O3: MH+, 591.3330.
4.3.7. Methyl (132R)-isopropyl-pyropheophorbide a [(R)-3d]
1
Mp 231–232 °C; H NMR (CDCl3) δ / ppm = 9.57 (1H, s, 10-H), 9.43 (1H, s,
5-H), 8.63 (1H, s, 20-H), 8.03 (1H, dd, J = 18, 11 Hz, 3-CH), 6.30 (1H, dd, J = 18, 1 Hz,
31-CH trans to 3-C–H), 6.17 (1H, dd, J = 11, 1 Hz, 31-CH cis to 3-C–H), 5.29 (1H, d, J
= 3 Hz, 132-H), 4.49–4.43 (2H, m, 17-, 18-H), 3.72 (2H, q, J = 8 Hz, 8-CH2), 3.70 (3H,
s, 12-CH3), 3.61 (3H, s, 172-COOCH3), 3.43 (3H, s, 2-CH3), 3.26 (3H, s, 7-CH3),
3.18–3.12 (1H, m, 132-CH), 2.66–2.61 (1H, m, 17-CH), 2.50–2.45 (1H, m, 171-CH),
2.22–2.15 (2H, m, 17-CHCH), 1.94 (3H, d, J = 7 Hz, 18-CH3), 1.75, 0.76 [each 3H, d, J
= 7 Hz, 132-C(CH3)2], 1.70 (3H, t, J = 8 Hz, 81-CH3), 0.05, –1.87 (each 1H, s, NH×2);
13C NMR (CDCl3) δ / ppm = 199.41, 173.46, 170.33, 160.39, 155.11, 150.60, 149.08,
144.94, 140.97, 137.79, 136.06, 136.05, 135.61, 131.46, 130.75, 129.26, 127.85, 122.44,
110.92, 103.89, 97.17, 92.92, 63.11, 51.66, 50.95, 49.92, 32.79, 30.80, 29.67, 23.26,
22.15, 19.51, 17.85, 17.49, 12.15, 12.06, 11.27; Vis (CH2Cl2) λmax / nm = 667 (relative
intensity, 0.42), 610 (0.07), 538 (0.08), 509 (0.10), 414 (1.00), 323 (0.17); HRMS
(APCI) found: m/z = 591.3347, calcd for C37H43N4O3: MH+, 591.3330.
4.3.8. Methyl (132S)-isopropyl-pyropheophorbide a [(S)-3d]
1
Mp 188–189 °C; H NMR (CDCl3) δ / ppm = 9.49 (1H, s, 10-H), 9.36 (1H, s,
5-H), 8.50 (1H, s, 20-H), 8.00 (1H, dd, J = 18, 11 Hz, 3-CH), 6.30 (1H, dd, J = 18, 1 Hz,
31-CH trans to 3-C–H), 6.16 (1H, dd, J = 11, 1 Hz, 31-CH cis to 3-C–H), 5.23 (1H, d, J
= 4 Hz, 132-H), 4.47–4.44 (1H, m, 18-H), 4.21–4.19 (1H, m, 17-H), 3.70 (2H, q, J = 8
24

Hz, 8-CH2), 3.67 (3H, s, 172-COOCH3), 3.64 (3H, s, 12-CH3), 3.40 (3H, s, 2-CH3), 3.24
(3H, s, 7-CH3), 3.21–3.16 (1H, m, 132-CH), 2.70–2.65 (1H, m, 171-CH), 2.48–2.37 (2H,
m, 17-CHCH), 2.17–2.11 (1H, m, 17-CH), 1.85, 0.82 [each 3H, d, J = 7 Hz,
132-C(CH3)2], 1.69 (3H, t, J = 8 Hz, 8-CH3), 1.51 (3H, d, J = 7 Hz, 18-CH3), 0.29, –1.50
(each 1H, s, NH×2); Vis (CH2Cl2) λmax / nm = 669 (relative intensity, 0.41), 611 (0.07),
540 (0.09), 510 (0.10), 414 (1.00), 324 (0.18); HRMS (APCI) found: m/z = 591.3321,
calcd for C37H43N4O3: MH+, 591.3330.
4.3.9. 132-Propyl-pyropheophytin a (Pr-pPhe-a)
1
Mp 111–112 °C; H NMR (CDCl3) δ / ppm = 9.54 (1H, s, 10-H), 9.39 (1H, s,
5-H), 8.60 (1H, s, 20-H), 8.01 (1H, dd, J = 18, 11 Hz, 3-CH), 6.29 (1H, dd, J = 18, 1 Hz,
31-CH trans to 3-C–H), 6.16 (1H, dd, J = 11, 1 Hz, 31-CH cis to 3-C–H), 5.44 (1H, dd, J
= 7, 3 Hz, 132-H), 5.20 (1H, t, J = 7 Hz, P1-CH), 4.56 (1H, dd, J = 13, 7 Hz,
172-COOCH), 4.51–4.46 (2H, m, 172-COOCH, 18-H), 4.41–4.39 (1H, m, 17-H), 3.71
(3H, s, 12-CH3), 3.69 (2H, q, J = 8 Hz, 8-CH2), 3.42 (3H, s, 2-CH3), 3.23 (3H, s, 7-CH3),
2.86–2.81 (1H, m, 132-CH), 2.71–2.63 (2H, m, 132-, 171-CH), 2.54–2.48 (1H, m,
171-CH), 2.29–2.18 (2H, m, 17-CH2), 1.96–1.88 (2H, m, P3-CH2), 1.89 (3H, d, J = 7 Hz,
18-CH3), 1.70 (3H, t, J = 8 Hz, 81-CH3), 1.62 (3H, s, P3-CH3), 1.54–1.47 (1H, m,
P14-CH), 1.40–0.95 (20H, 132-CCH2, P4-, P5-, P7-, P8-, P9-, P11-, P12-, P13-CH2, P6-,
P10-CH), 0.87 (3H, t, J = 7 Hz, 132-C2CH3), 0.857, 0.856 [each 3H, d, J = 7 Hz,
P15-(CH3)2], 0.81, 0.79 (each 3H, d, J = 7 Hz, P7-, P11-CH3) 0.18, –1.79 (each 1H, s,
13
NH×2); C NMR (CDCl3) δ / ppm = 199.84, 173.06, 170.63, 160.64, 155.21, 150.64,
148.89, 144.94, 142.91, 141.15, 137.81, 136.13, 136.01, 135.63, 131.47, 130.22, 129.22,
128.04, 122.43, 117.71, 110.02, 103.93, 97.11, 92.97, 61.50, 57.99, 51.12, 49.81, 39.80.
39.33, 37.37, 37.31, 37.23, 36.61, 32.92, 32.74, 32.60, 31.14, 29.74, 27.95, 24.98, 24.76,
24.40, 23.42, 22.70, 22.61, 19.71, 19.64, 19.47, 19.08, 17.46, 16.31, 14.63, 12.12, 12.11,
11.24; Vis (CH2Cl2) λmax / nm = 667 (relative intensity, 0.43), 610 (0.07), 539 (0.09), 509
25

(0.10), 414 (1.00), 322 (0.19); HRMS (APCI) found: m/z = 855.6138, calcd for
C56H78N4O3: MH+, 855.6147.
4.3.10. 132-Propyl-pyrochlorophyll a (Pr-pChl-a)
Mp 82–83 °C; 1H NMR (1% pyridine-d5/CDCl3) δ / ppm = 9.58 (1H, s, 10-H),
9.33 (1H, s, 5-H), 8.33 (1H, s, 20-H), 8.03 (1H, dd, J = 18, 11 Hz, 3-CH), 6.21 (1H, dd,
J = 18, 1 Hz, 31-CH trans to 3-C–H), 6.00 (1H, dd, J = 11, 1 Hz, 31-CH cis to 3-C–H),
5.37 (1H, dd, J = 6, 3 Hz, 132-H), 5.16 (1H, t, J = 7 Hz, P1-CH), 4.46 (2H, dd, J = 14, 7
Hz, 172-COOCH2), 4.39–4.36 (1H, m, 18-H), 4.26–4.24 (1H, m, 17-H), 3.75 (2H, q, J =
8 Hz, 8-CH2), 3.67 (3H, s, 12-CH3), 3.34 (3H, s, 2-CH3), 3.26 (3H, s, 7-CH3), 2.78–2.72,
2.59–2.52 (each 1H, m, 132-CH2), 2.51–2.45, 2.23–2.17 (each 1H, m, 17-CH2),
2.34–2.29 (1H, m, 171-CH), 1.94–1.86 (3H, m, 171-CH, P3-CH2), 1.691 (3H, t, J = 8 Hz,
81-CH3), 1.690 (3H, d, J = 7 Hz, 18-CH3), 1.59 (3H, s, P3-CH3), 1.53–1.46 (1H, m,
P14-CH), 1.38–0.93 (20H, 132-CCH2, P4-, P5-, P7-, P8-, P9-, P11-, P12-, P13-CH2, P6-,
P10-CH), 0.842, 0.841 (each 3H, d, J = 7 Hz, P15-(CH3)2), 0.80, 0.78 (each 3H, d, J = 7
Hz, P7-, P11-CH3), 0.77 (3H, t, J = 7 Hz, 132-C2CH3); 13C NMR (1%
pyridine-d5/CDCl3) δ / ppm = 200.42, 173.12, 167.01, 161.46, 154.80, 153.96, 151.53,
147.64, 147.60, 145.74, 143.89, 142.75, 138.77, 134.96, 133.66, 133.66, 132.12, 130.51,
119.69, 117.73, 109.23, 107.19, 99.86, 92.21, 61.36, 58.56, 50.21, 48.88, 39.79, 39.30,
37.35, 37.28, 37.21, 36.60, 33.02, 32.72, 32.58, 30.54, 29.71, 27.92, 24.97, 24.73, 24.39,
23.62, 22.67, 22.58, 19.68, 19.61, 19.60, 19.13, 17.54, 16.27, 14.62, 12.82, 12.45,
11.13; Vis (C2H5OC2H5) λmax / nm = 659 (relative intensity, 0.67), 614 (0.10), 577 (0.05),
533 (0.03), 429 (1.00), 408 (0.54), 382 (0.33), 322 (0.18); HRMS (ESI) found: m/z =
877.5843, calcd for C56H76MgN4O3: MH+, 877.5841.
26

4.4. Epimerization of Pr-pChl-a and chlorophyll a
According to the previous procedures,21,22 chlorophyll a containing other
cellular contents was extracted from Spirulina by acetone. The mixture was roughly
purified by FCC [Wakogel C-300, petroleum ether/2-propanol = 100/6 (v/v)], and
further purified by normal phase HPLC [Cosmosil 5SL-II, 10×250 mm,
hexane/2-propanol/methanol = 100/0.8/0.4 (v/v), 5 mL min-1] and precipitation from a
7/1 mixture of methanol and 1,4-dioxane with aliquot water to give analytically pure
chlorophyll a as a green solid: Vis (C2H5OC2H5) λmax / nm = 661 (0.77), 614 (0.12), 576
(0.06), 532 (0.03), 429 (1.00), 409 (0.65), 381 (0.40), 325 (0.21).
The epimerization condition in this study was based on ref 11. Epimerically
pure Pr-pChl-a and chlorophyll a were dissolved in pyridine-d5 and their solutions were
1
added to NMR sample tubes. As soon as possible, the samples were analyzed by H
NMR, and the results were as the spectra at 0 h. The sample solutions in the tubes were
incubated at 40 °C in water baths in the dark. After 3 h, the tubes were taken out and
1
cooled down to rt. The samples were analyzed by H NMR again, and the results were
as the spectra at 3 h. C5H5N (the chemical shift at the highest magnetic field: δH = 7.19
ppm) was used as an internal reference.
Acknowledgements
Drs. Tadashi Mizoguchi, Yusuke Kinoshita, and Sunao Shoji of Ritsumeikan
University gave us useful advice and technical support. This work was partially
supported by a Grant-in-Aid for Scientific Research on Innovative Area “Artificial
photosynthesis (AnApple)” (No. 24107002) from the Japan Society for the Promotion
27

of Science (JSPS) and Research Promoting Program from Ritsumeikan University
Research Foundation (Japan).
Supplementary data
Supplementary data associated with this article (132-epimeric ratios and
1
estimated -distortions as well as HH-ROESY, H NMR and 13C NMR spectra of
products) can be found, in the online version, at http://dx.doi.org/10.10161j.bmc.2015.
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