Effects of molecular structures on reduction properties of formyl groups in chlorophylls and pheophytins prepared from oxygenic photosynthetic organisms

Article abstract of DOI:10.1016/j.bmc.2011.05.039

Reduction of the 7-formyl groups in chlorophyll (Chl) b and its demetalated compound pheophytin (Phe) b was kinetically analyzed by using tert-butylamine-borane complex (t-BuNH₂·BH₃), and was compared with that of the 3-formyl groups in Chl d and Phe d. Reduction kinetics of the 7-formyl group in Chl b was similar to that in Phe b in dichloromethane containing 5 mM t-BuNH₂·BH₃. Little difference of the reduction kinetics of the 7-formyl groups between Chl b and Phe b was in sharp contrast to the reduction kinetics of the 3-formyl groups in Chl d and Phe d: the 3-formyl group in Phe d was reduced 5.3-fold faster than that in Chl d. The 7-formyl groups in Chl b and Phe b were reduced more slowly than the 3-formyl groups in Chl d and Phe d, respectively. The difference of the reactivity between the 3- and 7-formyl groups was in line with ¹³C NMR measurements of chlorophyllous pigments, in which the chemical shifts of carbon atoms in the 7-formyl groups of Chl b and Phe b were high-field shifted compared with those in the 3-formyl groups of Chl d and Phe d, respectively. These indicate that the 7-formyl groups in chlorophyllous pigments were less reactive for reduction to the corresponding hydroxymethyl groups than the 3-formyl groups due to the difference in electronic states of the formyl groups in the A- and B-rings of the chlorin macrocycle.

Full text of DOI:10.1016/j.bmc.2011.05.039

Bioorganic & Medicinal Chemistry 19 (2011) 3901–3905
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Effects of molecular structures on reduction properties of formyl groups in
chlorophylls and pheophytins prepared from oxygenic photosynthetic
organisms
⇑
Kana Sadaoka, Shigenori Kashimura, Yoshitaka Saga
Department of Chemistry, Faculty of Science and Engineering, Kinki University, Higashi-Osaka, Osaka 577-8502, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Reduction of the 7-formyl groups in chlorophyll (Chl) b and its demetalated compound pheophytin (Phe)
b was kinetically analyzed by using tert-butylamine–borane complex (t-BuNH2ꢀBH3), and was compared
with that of the 3-formyl groups in Chl d and Phe d. Reduction kinetics of the 7-formyl group in Chl b was
similar to that in Phe b in dichloromethane containing 5 mM t-BuNH2ꢀBH3. Little difference of the reduc-
tion kinetics of the 7-formyl groups between Chl b and Phe b was in sharp contrast to the reduction kinet-
ics of the 3-formyl groups in Chl d and Phe d: the 3-formyl group in Phe d was reduced 5.3-fold faster than
that in Chl d. The 7-formyl groups in Chl b and Phe b were reduced more slowly than the 3-formyl groups
in Chl d and Phe d, respectively. The difference of the reactivity between the 3- and 7-formyl groups was
in line with 13C NMR measurements of chlorophyllous pigments, in which the chemical shifts of carbon
atoms in the 7-formyl groups of Chl b and Phe b were high-field shifted compared with those in the 3-
formyl groups of Chl d and Phe d, respectively. These indicate that the 7-formyl groups in chlorophyllous
pigments were less reactive for reduction to the corresponding hydroxymethyl groups than the 3-formyl
groups due to the difference in electronic states of the formyl groups in the A- and B-rings of the chlorin
macrocycle.
Received 9 April 2011
Revised 17 May 2011
Accepted 19 May 2011
Available online 24 May 2011
Keywords:
Chlorophyll
Formyl group
Pheophytin
Photosynthesis
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
Qy absorption band of Chl d compared with Chl a. Because of such
spectral properties of Chl d, Acaryochloris can use far-red light for
Chlorophyll(Chl)s play crucial roles in photosynthesis. Chl mol-
ecules have cyclic tetrapyrrole moieties as the photofunctional
core, and peripheral substituents of the cyclic tetrapyrroles give
their structural diversity.1 Molecular structures of natural Chls a,
b, and d in oxygenic photosynthetic organisms are shown in Figure
1. The structural difference between Chls a and b is the substituent
at the 7-position of the chlorin macrocycle: methyl and formyl
groups are occupied at this position in Chls a and b, respectively.
The 7-formyl group of Chl b is responsible for the shift of its main
absorption bands, namely Soret and Qy absorption bands, to bath-
ochromic and hypsochromic, respectively, compared with those of
Chl a. Owing to such spectral properties, Chl b can capture photons
that are scarcely absorbed by Chl a in photosynthetic light-harvest-
ing complexes. Chl d is a major photosynthetic pigment in a cyano-
bacterium Acaryochloris,2,3 and play essential roles in both light-
harvesting complexes and reaction centers in this organism.4–11
Chl d has a formyl group at the 3-position of the chlorin macrocy-
cle, and the other substituents are the same as those of Chl a. This
structural difference results in the large red-shift of the monomeric
the photosynthetic activity.
Transformation of the formyl groups in Chls (or chlorophyl-
lide(Chlide)s) b and d is key reaction in biosynthesis and biodegrada-
tion of the chlorophyllous pigments. Formation and degradation of
R3
3
R7
7
8
A
B
N
N
N
N
Mg
D
C
E
131
132
O
COOCH3
133
O
O
Figure 1. Molecular structures of Chl a, Chl b, and Chl d. Chl a: R3 = CHCH2, R7 = CH3.
Chl b: R3 = CHCH2, R7 = CHO. Chl d: R3 = CHO, R7 = CH3. The bold lines show a major
18p-circuit.
⇑
Corresponding author. Tel.: +81 6 6730 5880; fax: +81 6 6723 2721.
E-mail address: saga@chem.kindai.ac.jp (Y. Saga).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.05.039

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K. Sadaoka et al. / Bioorg. Med. Chem. 19 (2011) 3901–3905
Chl b (or Chlide b) are based on interconversion between formyl and
methyl groups at the 7-position via a hydroxymethyl group, which is
called the Chl cycle.12–15 The 7-formyl group of Chl b-type molecules
are reduced to the corresponding hydroxymethyl group in the initial
step of the Chl b degradation by Chl b reductase, and this process has
been reported to be responsible for degradation of light-harvesting
complex II (LHC II) proteins in Arabidopsis.16 In the case of Chl d,
conversion of the 3-formyl group in bioprocesses has not been
unraveled yet. It is worth noting that biological formation of the
3-formyl group in Chl d has attracted considerable attention, and
the oxygen atom of the 3-formyl group was reported to be derived
from molecular oxygen.17 Therefore, reactivity of the formyl groups
conjugated to the chlorin macrocycle will be useful to understand
such biologically important processes in oxygenic photosynthetic
organisms.
Chemical reduction of the formyl group to the hydroxymethyl
group of natural Chls and synthetic chlorins has attracted much
attention from the viewpoints of in vivo transformation of formylat-
ed Chls described above and synthesis of photofunctional pig-
ments.18–32 Reduction of the 7-formyl groups in Chl b-type
pigments (Chl b, Phe b, and synthetic 7-formyl-chlorins) was first
performed by using sodium borohydride (NaBH4).19–21 A mild
reagent, sodium cyanoborohydride (NaBH3CN), was used in place
of NaBH4,22–24 since NaBH4 caused undesired reduction of the
carbonyl group at the 131-position. A much milder reagent, tert-
butylamine–borane complex (t-BuNH2ꢀBH3), has also been
employed for selective reduction of the 7-formyl group.25–27 In the
case of Chl d-type pigments (Chl d, Phe d, and synthetic 3-formyl-
chlorins), reagents such as NaBH4 and t-BuNH2ꢀBH3 have been used
for reduction of the 3-formyl groups.28–32 However, little informa-
tion is available, to the best of our knowledge, on physicochemical
properties of reduction of the formyl groups in natural Chls and their
model chlorins.30,32 Tamiaki et al. reported comparison of reactivity
between the 3- and 8-carbonyl groups in synthetic free-base chlo-
rins.30 We preliminarily analyzed reduction kinetics of the 3-formyl
groups in Chl d and Phe d.32 In this study, we report physicochemical
properties of reduction of the 7-formyl groups in Chl b and Phe b by
using t-BuNH2ꢀBH3, and compare their properties with the 3-formyl
groups in Chl d and Phe d. Additionally, we investigate the electronic
states of the formyl groups in the four chlorophyllous pigments pre-
pared from oxygenic photosynthetic organisms by means of 13C
NMR measurements. The present analysis enables us to elucidate
effects of central magnesium and the substituted positions of the
formyl groups on formyl reduction of naturally occurring chloro-
phyllous pigments.
Figure 2. Spectral changes of Chl b (A) and Phe b (B) in dichloromethane at the
concentration of t-BuNH2ꢀBH3 of 5 mM at 25 °C. Both spectra were measured from 0
to 1020 min at a 60-min interval. The arrows show the direction of the absorbance
changes.
Incubation of Chl d and Phe d possessing the 3-formyl group
with t-BuNH2ꢀBH3 showed similar spectral changes.32 Both Soret
and Qy absorption bands of the 3-formylated chlorophyllous pig-
ments decreased, and new bands appeared in the presence of sev-
eral isosbestic points. The new Qy bands in the spectral changes of
Chl d and Phe d were positioned in the shorter wavelength region
of the original 3-formylated pigments (Chl d: 692?657 nm, Phe d:
695?662 nm), which were different from the spectral changes of
the 7-formylated pigments described above. The new absorption
bands in the spectral changes of Chl d and Phe d were ascribed
to reduction of the 3-formyl groups, namely formation of 3-
deformyl-3-hydroxymethyl Chl d (3-OH Chl d) and 3-deformyl-3-
hydroxymethyl Phe d (3-OH Phe d) from Chl d and Phe d,
respectively.32
2. Results and discussion
Reaction products of Chl b and Phe b by incubation in dichloro-
methane containing 5 mM t-BuNH2ꢀBH3 were analyzed by reverse-
phase and normal-phase HPLC, respectively. Figure 3(A) depicts
typical HPLC elution patterns of Chl b before incubation and its
reaction products after incubation. A fraction at 16 min before
incubation was assigned to Chl b. After incubation for 240 min, a
main product was eluted at 13 min, accompanying the unreacted
Chl b at 16 min. This product at 13 min had Soret and Qy bands
at 437 and 661 nm in this HPLC eluent. FAB-MS analysis of the
product eluted at 13 min indicated that this main product gave a
molecular ion peak at m/z 908.5, which was the same as the calcu-
lated value ([M]+ = 908.5) for 7-deformyl-7-hydroxymethyl Chl b
(7-OH Chl b). This proved that the main product from Chl b by
incubation with t-BuNH2ꢀBH3 was 7-OH Chl b. Figure 3(B) shows
typical chromatograms of Phe b before incubation and its reaction
products. Phe b before incubation was eluted at 12 min. A main
product after incubation for 240 min, which was detected at
25 min, possessed the Soret and Qy bands at 414 and 664 nm in
Spectral changes of Chl b and Phe b possessing the 7-formyl
group by incubation in dichloromethane at the t-BuNH2ꢀBH3 con-
centration of 5 mM at 25 °C are depicted in Figure 2. Chl b had Sor-
et and Qy bands at 458 and 646 nm, respectively, as shown in
Figure 2(A). Incubation of Chl b in the solution containing t-
BuNH2ꢀBH3 decreased the Soret absorption band, accompanying
with a new absorption band at 434 nm. The 646-nm Qy absorption
band also decreased and a new Qy band appeared in the longer
wavelength region of the Qy band of Chl b. The isosbestic points
were present at 442, 602, 628, and 649 nm in this spectral change.
In the case of Phe b in Figure 2(B), the 438-nm Soret band of Phe
b decreased with appearance of a new Soret band at 418 nm in
dichloromethane containing t-BuNH2ꢀBH3. The 655-nm Qy absorp-
tion band also decreased with increase of a new 661-nm Qy band.
Small bands in the wavelength region between 500 and 600 nm
were also varied. This spectral change exhibited the isosbestic
points at 427, 494, 517, 604, 636, and 656 nm.

K. Sadaoka et al. / Bioorg. Med. Chem. 19 (2011) 3901–3905
3903
1.1
1.0
0.9
0.8
0.7
(A)
(B)
Chl b
(A)
7-OH
Chl b
x
0.6
0.5
1.1
1.0
Phe b
(B)
7-OH
Phe b
0.9
0.8
0.7
x
0
5
10
15
20
25
30
35
40
Retention time / min
0.6
0.5
Figure 3. HPLC elution patterns of Chl b (A) or Phe b (B) before incubation of t-
BuNH2ꢀBH3 (dotted line) and their reaction products after incubation (solid line) in
dichloromethane at the concentration of t-BuNH2ꢀBH3 of 5 mM at 25 °C for 240 min.
Chls and Phes were eluted on 5C18-AR-II (6 mm/ ꢃ 250 mm) with methanol and on
5SL-II (6 mm/ ꢃ 250 mm) with hexane/2-propanol (97:3), respectively, at a flow
rate of 1.0 mL minꢁ1. Chromatograms before and after incubation of Chl b were
recorded at 470 and 436 nm, respectively. Chromatograms before and after
incubation of Phe b were recorded at 434 and 412 nm, respectively. The signals
denoted by ꢃ were due to the injection of mixed solvents of HPLC eluents and
dichloromethane. The procedure of HPLC analysis of chlorophyllous pigments after
incubation was described in Section 3.
0
2
4
6
8
10
12
14
Time / 103 s
Figure 4. Kinetic plots for reduction of Chl b (A) and Phe b (B) in dichloromethane
at the concentration of t-BuNH2ꢀBH3 of 5 mM at 25 °C. Absorbance changes were
monitored at 459 and 438 nm for Chl b and Phe b, respectively. A0 and A are Soret
absorbances of chlorophyllous pigments at the onset of measurements and at time
t, respectively.
where A0, A, and A are Soret absorbances of Chl b and Phe b at the
1
the eluent. The unreacted Phe b was also detected at 12 min after
incubation for 240 min. A molecular ion peak at m/z 886.6 of this
main product at 25 min in FAB-MS measurements corresponded
to the calculated value ([M]+ = 886.6) for 7-deformyl-7-hydroxy-
methyl Phe b (7-OH Phe b). Therefore, 7-OH Phe b was predomi-
nantly formed from Phe b by incubation with t-BuNH2ꢀBH3. Slight
fractions were detected at 17 and 22 min in the chromatograms
of reaction products of Chl b and Phe b, respectively, as shown by
solid lines in Figure 3. Visible absorption spectra of these fractions
were almost identical to those of the main products. Their absorp-
tion spectra and elution patterns indicated that these slight prod-
ucts would be the 132-stereoisomers of 7-OH Chl b and 7-OH Phe
b, namely 7-OH Chl b0 and 7-OH Phe b0, respectively. A small frac-
tion at 10 min in the chromatogram of reaction products of Phe b
(solid line in Fig. 3(B)) was also assigned to Phe b0 by its absorption
spectrum and elution pattern. Such slight epimerization was also
observed in reduction of Chl d and Phe d under the same condi-
tion.32 No other reaction products such as a 131-hydroxylated com-
pound were observed, indicating that side reaction hardly occurred
under the present condition.
onset of measurements, at time t, and at the complete reduction,
respectively. The rate constants of reduction of the formyl groups
in Chl b and Phe b under the condition in Figure 4 were estimated
to be 3.5 ꢃ 10ꢁ5 and 3.4 ꢃ 10ꢁ5 sꢁ1, respectively. These values were
the averages of more than five independent measurements, where
the standard deviations were 14% and 6% of the averaged values
for Chl b and Phe b, respectively. The reduction rate constants of
the 7-formyl groups in Chl b and Phe b were significantly smaller
than those of the 3-formyl groups in Chl d and Phe d (1.2 ꢃ 10ꢁ4
and 6.3 ꢃ 10ꢁ4 sꢁ1, respectively) under the same reaction condition.
Therefore, the 7-formyl group in the B-ring of the chlorin macrocy-
cle was converted to the hydroxymethyl group more slowly than
the 3-formyl group in the A-ring. Lower reduction reactivity of for-
myl groups attached to the B-ring of the chlorin macrocycle than
that in the A-ring was also reported by using synthetic free-base
chlorin molecules.30 The difference of the reactivity between formyl
groups in the A-ring and those in the B-ring would be ascribed to
electronic properties of these formyl groups. The 3-formyl group
is connected to the chlorin 18p-conjugated system, whereas the
7-formyl group is attached to the rather isolated double bond be-
tween C7 and C8 atoms. As a result, the 7-formyl group would be
more strongly conjugated to the adjacent p-system than the 3-for-
myl group. Such difference should change the electron densities on
the carbon atoms of the 3-formyl and 7-formyl groups in the chlorin
macrocycle.
Reduction kinetics can be quantitatively analyzed by absor-
bance changes at the Soret peak positions for Chl b and Phe b, as
judged from their spectral changes in Figure 2. Time courses of Sor-
et absorbances of Chl b and Phe b incubated with 5 mM t-
BuNH2ꢀBH3 at 25 °C are shown in Figure 4. The reaction conditions
in which the concentration of t-BuNH2ꢀBH3 (5 mM) was much
13C NMR spectra of the four formylated Chls and Phes were
measured in a mixed solvent of methanol-d4/chloroform-d (1:19,
v/v) and chloroform-d, respectively, to investigate the electronic
states of the formyl groups of these chlorophyllous pigments. 13C
NMR signals were assigned by 1H–13C-HSQC, 1H–13C-HMBC, and
previous reports.33,34 Table 1 summarizes the chemical shifts ds
of carbon atoms in the formyl groups as well as carbonyl carbon
higher than the pigment concentration (14 lM) allows us to regard
these reactions as pseudo-first-order reactions.32 The reduction
rate constants, ks, can be estimated by fitting the logarithms of
the time courses of absorbance at Soret peak positions of Chl b
and Phe b for 600 s to the following kinetic equation,
ln½ðA ꢁ A Þ=ðA0 ꢁ A Þꢂ ¼ ꢁkt
1
1

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K. Sadaoka et al. / Bioorg. Med. Chem. 19 (2011) 3901–3905
Table 1
understanding the relationship between reactivity of the formyl
groups and redox potentials of chlorophyllous pigments.
Little difference of reduction properties of the 7-formyl group
between Chl b and Phe b might be in line with in vivo degradation
of Chl b in higher plants. In the initial step of Chl b degradation, the
7-formyl group in Chl b or Chlide b is reduced to the hydroxy-
methyl group, and subsequently converted to the methyl group,
followed by removal of central magnesium from the chlorin mac-
rocycle.12,13 It has been reported that Chl b-type molecules pos-
sessing the 7-formyl group exhibited higher resistance to
demetalation than chlorin molecules possessing the 7-methyl
and 7-hydroxymethyl groups.27,40,41 Such demetalation properties
of 7-formyl chlorins suggest potential difficulty of smooth demeta-
lation of Chl b or Chlide b. In contrast, the present results indicate
that possible biochemical reduction of the 7-formyl group would
similarly proceed in both Chl b and Phe b. Therefore, both the
reduction properties of the 7-formyl group and the demetalation
properties in the 7-formylated chlorophyllous pigments would
not contradict the biological process of Chl b degradation, where
the 7-formyl group is reduced before removal of central
magnesium.
Chemical shifts of carbonyl carbon atoms in Chl b, Chl d, Phe b, and Phe d
Pigments
d (C31)
d (C71)
d (C131)
d (C133)
Chl b
Chl d
Phe b
Phe d
—
189.02
—
188.29
—
187.57
—
189.97
189.71
189.46
189.44
171.99
172.29
169.26
169.31
188.27
Chls and Phes are dissolved in methanol-d4/chloroform-d (1:19) and in chloroform-
d, respectively.
atoms in the E-ring (C131 and C133 carbon atoms). The ds of carbon
atoms in the formyl groups in Chl b, Chl d, Phe b, and Phe d were
188.29, 189.02, 187.57, and 188.27 ppm, respectively. Both the
high-field shifts of the ds of the formyl carbon atoms in Chl
d?Chl
b
(189.02?188.29 ppm)
and
Phe
d?Phe
b
(188.27?187.57 ppm) indicated the C71 atoms of the 7-formylated
pigments were less electropositive than the C31 atoms of the 3-
formylated pigments. This is in line with the present results that
Chl b and Phe b are less reactive to a nucleophile than Chl d and
Phe d, respectively. Previous 13C NMR experiments of the synthetic
free-base chlorins30 also support such discussion.
In conclusion, we demonstrated that the 7-formyl groups in Chl
b and Phe b were less reactive to the reducing reagent than the 3-
formyl groups in Chl d and Phe d. Moreover, this study also indi-
cates that effect of central magnesium on reduction kinetics in 7-
formylated chlorophyllous pigments was small compared with
that in 3-formylated chlorin molecules. This work could be useful
for elucidation of biochemical conversion of formyl groups in pho-
tosynthetic pigments.
The reduction rate constant of the 7-formyl group in Chl b was
almost similar to that in Phe b under this condition. This is in sharp
contrast to reduction of the 3-formyl groups in Chl d and Phe d: the
reduction rate ꢁ1constant of the 3-formyl group in Phe
d
(k = 6.3 ꢃ 10ꢁ4
s ) was about 5.3-times larger than that in Chl d
(k = 1.2 ꢃ 10ꢁ4 sꢁ1) under the same condition. These indicate that
central magnesium has smaller effect on reduction kinetics of the
7-formyl group in Chl b-type pigments than that of the 3-formyl
group in Chl d-type pigments. This might originate from possible
stronger conjugation of the 7-formyl group to the C7–C8 double
3. Experimental
3.1. Apparatus
bond than the 18
might be more influenced by the 18
pared with the 7-formyl group. Such difference in connection of
the formyl groups with the adjacent -systems in chlorophyllous
p-aromatic circuit. In contrast, the 3-formyl group
p
-conjugated system com-
Visible absorption spectra were measured with a Shimadzu UV-
2450 spectrophotometer, where the reaction temperatures were
p
pigments is likely to change the effect of central magnesium on
reduction of the formyl groups linked to the chlorin macrocycle.
IR spectra of monomeric Chl b, Phe b, Chl d, and Phe d35–38 were
compared in order to examine the properties of the formyl groups
in natural chlorophyllous pigments. The stretching vibrational
bands of the 7-formyl carbonyl groups in Chl b and Phe b appeared
regulated with
a Shimadzu thermo-electric temperature-con-
trolled cell holder TCC-240A. High-performance liquid chromatog-
raphy (HPLC) was carried out with a Shimadzu LC-20AT pump and
an SPD-M20A or SPD-20A photodiode array detector. Mass spectra
were measured with a JEOL JMS700 spectrometer; m-nitrobenzyl
alcohol was used as a matrix. 1H and 13C NMR spectra were mea-
sured with a JEOL JNM-ECA500 or JNM-ECA700 NMR spectrome-
ter; chemical shifts of 1H and 13C were expressed (in ppm)
relative to chloroform (7.26 and 77.0 ppm, respectively) as internal
references.
at 1663 and 1667 cmꢁ1 35
peaks of the 3-formyl carbonyl groups in Chl d and Phe d were
.
In a while, the stretching vibrational
positioned at 1667 and 1677 cmꢁ1 37,38
Both the lower wavenum-
.
ber shifts in Chl d?Chl b (1667?1663 cmꢁ1) and Phe d?Phe b
(1677?1667 cmꢁ1) suggest that the 7-formyl groups in Chl b and
Phe b are more conjugated with the adjacent
p-system than the
3.2. Materials
3-formyl groups in Chl d and Phe d. Smaller difference of wave-
number shifts of the stretching vibrational peaks of the 7-formyl
Chls b and d were extracted from spinach leaves and harvested
cell pellets of Acaryochloris marina MBIC11017, respectively, with
acetone/methanol (1:1, v/v).32,41 The extracted solutions were di-
luted with diethyl ether and washed with NaCl–saturated water
(neutral pH) to remove water-soluble components, and then were
concentrated under reduced pressure. Chlorophyllous pigments
were extracted again from the concentrated solution with dichlo-
romethane to remove residual water, and were evaporated under
reduced pressure and dried with nitrogen gas. Chls b and d were
carbonyl groups between Chl b and Phe b (d
of the 3-formyl carbonyl groups between Chl d and Phe d
(d
= 10 cmꢁ1) implies that the 7-formyl groups would be less
influenced by central magnesium in the 18 -conjugated system
than the 3-formyl groups. These IR spectroscopic data of the four
pigments are in line with their reduction properties.
Reduction potentials of Chl b and Phe b in acetonitrile were re-
ported to be ꢁ1.02 and ꢁ0.64 V vs. SHE, respectively.39 These
reduction potentials are lower than those of Chl d (ꢁ0.91 V) and
Phe d (ꢁ0.63 V), respectively,39 suggesting that the 7-formylated
chlorophyllous pigments are less reactive to reduction than the
3-formylated pigments. Such assumption would not contradict
the present results on the reactivity of the 3-formyl and 7-formyl
groups. However, the order of reduction potentials of the four pig-
ments disagrees with the order of the reactivity of formyl reduc-
tion in this study. Further detailed studies will be necessary for
m
= 4 cmꢁ1) than that
m
p
finally purified on
a reverse-phase HPLC column 5C18-AR-II
(10 mm internal diameter (i.d.) ꢃ 250 mm, Nacalai Tesque).
Phes b and d were prepared from Chls b and d, respectively, by
treatment with aqueous HCl in dichloromethane and were purified
on a normal-phase HPLC column 5SL-II (6 mm i.d. ꢃ 250 mm, Nac-
alai Tesque). Purified Chls and Phes were stored in the dark at
ꢁ20 °C, and re-purified by HPLC just before measurements.

K. Sadaoka et al. / Bioorg. Med. Chem. 19 (2011) 3901–3905
3905
7. Tomo, T.; Kato, Y.; Suzuki, T.; Akimoto, S.; Okubo, T.; Noguchi, T.; Hasegawa, K.;
Tsuchiya, T.; Tanaka, K.; Fukuya, M.; Dohmae, N.; Watanabe, T.; Mimuro, M. J.
Biol. Chem. 2008, 283, 18198.
8. Allakhverdiev, S. I.; Tomo, T.; Shimada, Y.; Kindo, H.; Nagao, R.; Klimov, V. V.;
Mimuro, M. Proc. Natl. Acad. Sci. U.S.A. 2010, 107, 3924.
9. Schiller, H.; Senger, H.; Miyashita, H.; Miyachi, S.; Dau, H. FEBS Lett. 1997, 410,
433.
10. Chen, M.; Quinnell, R. G.; Larkum, A. W. D. FEBS Lett. 2002, 514, 149.
11. Kumazaki, S.; Abiko, K.; Ikegami, I.; Iwaki, M.; Itoh, S. FEBS Lett. 2002, 530, 153.
12. Rüdiger, W. Photosynth. Res. 2002, 74, 187.
3.3. Measurements of reduction kinetics
Dichloromethane solution of the purified pigment (28 lM),
whose concentration was the same as that of Chl d and Phe d in
the previous report,32 was mixed with the same amount of dichlo-
romethane solution of t-BuNH2ꢀBH3 (10 mM), and their visible
absorption spectra were measured at 25 °C.
13. Rüdiger, W. In Grimm, B., Porra, R. J., Rüdiger, W., Scheer, H., Eds.; Chlorophylls
and Bacteriochlorophylls: Biochemistry, Biophysics, Functions and
Applications; Springer: Dordrecht, 2006; pp 189–200.
3.4. HPLC analysis before and after reduction
14. Ito, H.; Tanaka, A. Plant Physiol. Biochem. 1996, 34, 35.
15. Tanaka, A.; Ito, H.; Tanaka, R.; Tanaka, N. K.; Yoshida, K.; Okada, K. Proc. Natl.
Acad. Sci. U.S.A. 1998, 95, 12719.
16. Horie, Y.; Ito, H.; Kusaba, M.; Tanaka, R.; Tanaka, A. J. Biol. Chem. 2009, 284,
17449.
17. Schliep, M.; Crossett, B.; Willows, R. D.; Chen, M. J. Biol. Chem. 2010, 285, 28450.
18. Rüdiger, W. In Porphyrin Handbook; Kadish, K. M., Smith, K. M., Guilard, R., Eds.;
Academic Press: San Diego, 2003; Vol. 13, pp 71–108.
19. Holt, A. S. Plant Physiol. 1959, 34, 310.
20. Porra, R. J.; Schäfer, W.; Cmiel, E.; Katheder, I.; Scheer, H. Eur. J. Biochem. 1994,
219, 671.
21. Ito, H.; Ohtsuka, T.; Tanaka, A. J. Biol. Chem. 1996, 271, 1475.
22. Boxer, S. G.; Bucks, R. R. J. Am. Chem. Soc. 1979, 101, 1883.
23. Bucks, R. R.; Boxer, S. G. J. Am. Chem. Soc. 1982, 104, 340.
24. Scheumann, V.; Helfrich, M.; Schoch, S.; Rüdiger, W. Z. Naturforsch. 1996, 51c,
185.
The purity of Chls and Phes before measurements of reduction
kinetics was checked on a reverse-phase HPLC column 5C18-AR-II
(6 mm i.d. ꢃ 250 mm, Nacalai Tesque) with methanol and a nor-
mal-phase HPLC column 5SL-II (6 mm i.d. ꢃ 250 mm, Nacalai Tes-
que) with hexane/2-propanol (97:3, v/v), respectively, at a flow
rate of 1.0 mL minꢁ1. After measurements of reduction kinetics,
the reaction mixtures were diluted with HPLC eluents, and the
solutions were filtrated with 0.45-lm pore size filter. The resulting
solutions were analyzed under the same conditions as the analysis
of pigments before measurements.
3.5. NMR measurements
25. Oba, T.; Masada, Y.; Tamiaki, H. Bull. Chem. Soc. Jpn. 1997, 70, 1905.
26. Oba, T.; Tamiaki, H. Supramol. Chem. 2001, 12, 369.
27. Hirai, Y.; Kashimura, S.; Saga, Y. Photochem. Photobiol. 2011, 87, 302.
28. Tomo, T.; Suzuki, T.; Hirano, E.; Tsuchiya, T.; Miyashita, H.; Dohmae, N.;
Mimuro, M. Chem. Phys. Lett. 2006, 423, 282.
1H and 13C NMR spectra of Chls and Phes were measured in a
mixed solvent of methanol-d4/chloroform-d (1:19, v/v) and chloro-
form-d, respectively. Chl b, Chl d, Phe b, and Phe d concentrations
were 15, 11, 15, and 14 mM, respectively, in NMR measurements.
29. Mizoguchi, T.; Shoji, A.; Kunieda, M.; Miyashita, H.; Tsuchiya, T.; Mimuro, M.;
Tamiaki, H. Photochem. Photobiol. Sci. 2006, 5, 291.
30. Tamiaki, H.; Hamada, K.; Kunieda, M. Tetrahedron 2008, 64, 5721.
31. Saga, Y.; Nakai, Y.; Tamiaki, H. Supramol. Chem. 2009, 21, 738.
32. Sadaoka, K.; Hirai, Y.; Kashimura, S.; Saga, Y. Chem. Lett. 2010, 39, 567.
33. Abraham, R. J.; Rowan, A. E. In Chlorophylls; Scheer, H., Ed.; CRC Press: Boca
Raton, 1991; pp 797–834.
Acknowledgments
We thank Dr. Toshie Minematsu and Dr. Yuki Hirai, Kinki Uni-
versity, for NMR measurements and kind advices, respectively.
34. Miyashita, H.; Adachi, K.; Kurano, N.; Ikemoto, H.; Chihara, M.; Miyachi, S. Plant
Cell Physiol. 1997, 38, 274.
35. Katz, J. J.; Dougherty, R. C.; Boucher, L. J. In The Chlorophylls; Vernon, L. P., Seely,
G. R., Eds.; Academic Press: New York, 1966; pp 185–251.
36. Lutz, M.; Mäntele, W. In Chlorophylls; Scheer, H., Ed.; CRC Press: Boca Raton,
1991; pp 855–902.
37. Morishita, H.; Tamiaki, H. Tetrahedron 2005, 61, 6097.
38. Morishita, H.; Mizoguchi, T.; Tamiaki, H. Photosynth. Res. 2010, 105, 257.
39. Kobayashi, M.; Ohashi, S.; Iwamoto, K.; Shiraiwa, Y.; Kato, Y.; Watanabe, T.
Biochim. Biophys. Acta 2007, 1767, 596.
40. Mazaki, H.; Watanabe, T.; Takahashi, T.; Struck, A.; Scheer, H. Bull. Chem. Soc.
Jpn. 1992, 65, 3212.
41. Hirai, Y.; Tamiaki, H.; Kashimura, S.; Saga, Y. Photochem. Photobiol. Sci. 2009, 8,
1701.
References and notes
1. Scheer, H. In Grimm, B., Porra, R. J., Rüdiger, W., Scheer, H., Eds.; Chlorophylls
and Bacteriochlorophylls: Biochemistry, Biophysics, Functions and
Applications; Springer: Dordrecht, 2006; pp 1–26.
2. Miyashita, H.; Ikemoto, H.; Kurano, N.; Adachi, K.; Chihara, M.; Miyachi, S.
Nature 1996, 383, 402.
3. Murakami, A.; Miyashita, H.; Iseki, M.; Adachi, K.; Mimuro, M. Science 2004,
303, 1633.
4. Sivakumar, V.; Wang, R.; Hastings, G. Biophys. J. 2003, 85, 3162.
5. Razeghifard, M. R.; Chen, M.; Hughes, J. L.; Freeman, J.; Krausz, E.; Wydrzynski,
T. Biochemistry 2005, 44, 11178.
6. Itoh, S.; Mino, H.; Itoh, K.; Shigenaga, T.; Uzumaki, T.; Iwaki, M. Biochemistry
2007, 46, 12473.