Demetalation kinetics of the zinc chlorophyll derivative possessing two formyl groups:

Article abstract of DOI:10.1142/S1088424613500788

Demetalation kinetics of zinc chlorophyll derivative 1 possessing two formyl groups directly linked to the A- and B-rings of the chlorin macrocycle, which was synthesized from chlorophyll b, was examined under acidic conditions and compared with those of

Full text of DOI:10.1142/S1088424613500788

Journal of Porphyrins and Phthalocyanines
J. Porphyrins Phthalocyanines 2013; 17: 1120–1128
DOI: 10.1142/S1088424613500788
Published at http://www.worldscinet.com/jpp/
Demetalation kinetics of the zinc chlorophyll derivative
possessing two formyl groups: effects of formyl groups
conjugated to the chlorin macrocycle on physicochemical
properties of photosynthetic pigments
Kana Sadaokaa, Toru Obab, Hitoshi Tamiakic, Shigenori Kashimuraa
and Yoshitaka Saga*a◊
a Department of Chemistry, Faculty of Science and Engineering, Kinki University, Higashi-Osaka,
Osaka 577-8502, Japan
b Department of Material and Environmental Chemistry, Graduate School of Engineering, Utsunomiya University,
Utsunomiya, Tochigi 321-8585, Japan
c Graduate School of Life Sciences, Ritsumeikan University, Kusatsu, Shiga 525-8577, Japan
Received 8 May 2013
Accepted 2 June 2013
ABSTRACT: Demetalation kinetics of zinc chlorophyll derivative 1 possessing two formyl groups
directly linked to the A- and B-rings of the chlorin macrocycle, which was synthesized from chlorophyll
b, was examined under acidic conditions and compared with those of Zn chlorins 2 and 3 possessing a
single formyl group in the A- and B-ring, respectively, as well as Zn chlorin 4 lacking any formyl group
to unravel the substitution effects on demetalation properties of chlorophyllous pigments. Demetalation
kinetics of diformylated Zn chlorin 1 was slower than those of monoformylated Zn chlorins 2 and
3, indicating that the effect of the electron-withdrawing formyl group on demetalation kinetics was
amplified by introduction of two formyl groups to the chlorin macrocycle. High correlations were
observed between demetalation rate constants of Zn chlorins 1–4 and the sum of Hammett s parameters
of the 3- and 7-substituents on the chlorin macrocycle, indicating that the combination of electron-
withdrawing abilities of the substituents linked directly to the cyclic tetrapyrrole was responsible for
demetalation properties of zinc chlorophyll derivatives.
KEYWORDS: chlorophyll, formyl group, hammett constant, pheophytinization.
sunlight and charge separation using the light energy. Chl
INTRODUCTION
a has aliphatic substituents (2-methyl, 3-vinyl, 7-methyl,
Chlorophyll (Chl) molecules are crucial pigments in
and 8-ethyl groups) in the A- and B-rings of the chlorin
photosynthesis. Naturally occurring Chls have structural
macrocycle.
diversity in terms of the tetrapyrrole macrocycles and
Chl molecules possessing a formyl group directly
their peripheral substituents [1, 2]. Figure 1a depicts
linked to the chlorin macrocycle are of great importance
molecular structures of Chls, whose macrocycles are
in efficient light-harvesting activities in photosynthetic
chlorin-type (17,18-dihydroporphyrin), in oxygenic
organisms [1–5]. Three kinds of Chls, namely Chls
photosynthetic organisms. The most abundant Chl
pigment is Chl a, which functions in both capture of
b, d, and f, are present in oxygenic photosynthetic
organisms. In Chl b, the formyl group is occupied at
the 7-position in the B-ring of the chlorin macrocycle.
Such 7-formylated chlorin-type pigments are also found
◊SPP full member in good standing
as bacteriochlorophyll(BChl)s e and f in green sulfur
photosynthetic bacteria [6–8]. Chls d and f have the
*Correspondence to: Yoshitaka Saga, email: saga@chem.
kindai.ac.jp
Copyright © 2013 World Scientific Publishing Company

DEMETALATION KINETICS OF THE ZINC CHLOROPHYLL DERIVATIVE POSSESSING TWO FORMYL GROUPS 1121
R3
3
R7
7
R3
R7
7
analyze its physicochemical properties on removal of the
central metal from the chlorin macrocycle to afford the
corresponding free-base 1′.
3
R2
8
A
B
2
A
B
N
N
N
N
N
N
Demetalation of natural Chls called pheophytinization
is one of the key reactions in photosynthetic metabolisms.
Photosynthetically active (B)Chl molecules lacking
the central metal, namely pheophytin (Phe) a and
bacteriopheophytin (BPhe) a/b, function as the primary
electron acceptor in PS II-type photosynthetic reaction
centers [11–17], but biosynthesis of the (B)Phe molecules
has not completely been unraveled. Removal of the central
metal from chlorophyllous pigments is also of biological
importance in the early stage of Chl degradation, but this
mechanism is still enigma [11, 18–21]. Additionally,
pheophytinization of chlorophyllous pigments has also
attracted much attention for researchers engaged in
photosynthetic research, since this reaction is one of the
major denaturation phenomena in laboratories. Therefore,
physicochemical analyses of demetalation of natural
Chls and synthetic metallochlorins have extensively been
performed [22–39].
Formylgroupsdirectlylinkedtothechlorinmacrocycle
remarkably affect kinetic stability to demetalation of
chlorophyllous pigments [22–24, 27, 28, 30–32, 34, 35,
39]. Mackinney and Joslyn first reported the difference
of demetalation kinetics between Chl a possessing
the 7-methyl group and Chl b possessing the 7-formyl
group [22, 23], and this difference was confirmed by
other reports [24, 28, 32]. The effect of the 7-formyl
group on slow demetalation kinetics was also found in
the 132-stereoisomer of Chl b (Chl b′), BChl e in green
sulfur photosynthetic bacteria, and synthetic Zn methyl
pyropheophorbide b [27, 28, 30, 31, 34]. The formyl
group at the 3-position in the chlorin macrocycle is
also reported to contribute to resistance against removal
of the central metal [32, 35, 39]. In contrast to these
reports on demetalation properties of monoformylated
metallochlorins, there is no information on demetalation
properties of diformylated chlorophyllous pigments.
Comparison of demetalation properties of diformylated
Zn chlorin 1 with those of monoformylated Zn chlorins
2 and 3 as well as Zn chlorin 4 possessing no formyl
group allows us to understand effects of formyl groups
conjugated to the cyclic tetrapyrrole on removal of
central metal from the chlorin ring more deeply.
Mg
Zn
N
N
18
D
C
D
C
17
E
E
O
O
COOCH3
O
phytyl
O
O
OCH3
Pigment
Compound R3
R7
R2
R7
R3
Chl a CH3
1
2
3
4
CH=CH2 CH3
CHO
CHO
CHO
CH3
Chl b
CH3 CH=CH2 CHO
Chl d CH3
CHO
CH3
CH=CH2 CHO
CHO CH=CH2
CH3
Chl f
CH3
CH=CH2
(a)
(b)
Fig. 1. Molecular structures of natural chlorin-type chlorophylls
in oxygenic photosynthetic organisms (a) and synthetic Zn
chlorins 1–4 used in this study (b). The corresponding free-
bases of the latter were represented as 1′–4′
formyl group at the 3- and 2-positions, respectively, in
the A-ring [3–5]. The formyl groups conjugated to the
chlorin macrocycle are responsible for spectral and
physicochemical properties. In natural photosynthetic
systems, these formylated Chl molecules can harvest
light-energy, which is hardly absorbed by Chl a, because
of the shifts of the absorption bands compared with Chl a.
Therefore, physicochemical studies on formylated Chls
have attracted considerable attentions in the research area
of photobiology.
Recently, aChlmoleculepossessingtwoformylgroups
called 7-formyl-Chl d was successfully biosynthesized
in a mutant of Acaryochloris marina, in which a Chl a
oxygenase was expressed [9, 10]. This novel pigment
exhibits unique spectral features, in which the Soret
absorption band (470 nm in acetone) is shifted to a
longer wavelength than those in Chls b and d, whereas
the Qy-band (672 nm in acetone) is positioned between
those of Chls b and d. The analysis of photosystem(PS)-II
complexes isolated from the mutant demonstrated that
the diformylated Chl molecules occupied the binding
sites of Chl d and functioned as light-harvesting pigments
[10]. The physicochemical properties and metabolism of
such a diformylated Chl have emerged as contribution
for elucidation of photosynthetic mechanisms, as well
as developments of artificial photoactive nanodevices.
However, the content of the diformylated Chl in the
mutant of Acaryochloris marina is quite low (ca. 4%
of total biosynthesized Chls) at the stationary phase [9],
and its model pigments will be useful in this research
area. Here, we synthesize Zn chlorin 1 possessing two
formyl groups at the 3- and 7-positions (Fig. 1b) and
RESULTS AND DISCUSSION
Synthesis and spectral properties of diformylated
chlorins
The free-base chlorin 1′ possessing two formyl groups
at the 3- and 7-positions was synthesized by oxidation
of the 3-vinyl group in methyl pyropheophorbide b (3′),
followed by zinc insertion into its chlorin macrocycle to
afford diformylated Zn chlorin 1. The two compounds 1
Copyright © 2013 World Scientific Publishing Company
J. Porphyrins Phthalocyanines 2013; 17: 1121–1128

1122 K. SADAOKA ET AL.
and 1′ were identified by 1H NMR and
HRMS (see Experimental section). Visible
absorption spectra of Zn chlorin 1 and free-
base chlorin 1′ were compared with other
Zn chlorins 2–4 and the corresponding
free-base chlorins 2′–4′, respectively, in
Fig. 2. Zn chlorin 1 exhibited the Soret and
Qy absorption bands at 463 and 659 nm,
respectively, in acetone (Fig. 2a). The Soret
band of diformylated 1 was red-shifted
compared with monoformylated 2 and 3 by
20 and 12 nm, respectively. The Qy-band of 1
was shifted to a longer wavelength than that
of 7-formylated 3 by 22 nm, but was shifted to
Table 1. Summary of lmax differences between diformylated and
monoformylated pigments at the Soret and Qy positions
Dlmax (Soret), nm Dlmax (Qy), nm Reference
l
l
l
max(1) – lmax(2)
20
12
23
-21
22
this work
this work
max(1) – lmax(3)
max(7-formyl-Chl d)
–lmax(Chl d)
-21
[9] and [40]
l
max(7-formyl-Chl d)
– lmax(Chl b)
13
21
[1] and [9]
Spectral changes through demetalation
a shorter wavelength by 21 nm than that of 3-formylated
2. The relative ratio of the Qy absorbance over the Soret
absorbance of diformylated 1 was 0.26, which was
analogous to that of 7-formylated 3 (the relative ratio was
0.30). Such spectral features of 1 corresponded to those
of the novel 7-formyl-Chl d biosynthesized in the mutant
of Acaryochloris marina [9] as summarized in Table 1.
These indicate that diformylated Zn chlorin 1 should
be a good model pigment of 7-formyl-Chl d. Free-base
chlorin 1′ showed essentially the same tendencies as Zn
chlorin 1 as shown in Fig. 2b.
Demetalation of synthetic Zn 3,7-diformyl-chlorin 1
was kinetically analyzed in acidic aqueous acetone.
Figure 3 depicts a spectral change of Zn 3,7-diformyl-
chlorin 1 during the demetalation process in aqueous
acetone (acetone/water = 3/1, vol/vol) at the proton
concentrationof1.8×10-1 M.The472-nmSoretabsorption
bandofZn3,7-diformyl-chlorin1graduallydecreasedand
a new absorption band of demetalation product, namely
1′, appeared at 451 nm. The Qy absorbance of 1 at 666 nm
also decreased with an increase of the 676-nm Qy
absorbance of 1′. The small bands in the wavelength
range between 500 and 650 nm of Zn chlorin 1 also
gradually changed. This spectral change exhibited the
isosbestic points at 461, 508, 563, and 674 nm.
Spectral changes of the other Zn chlorins were
essentially the same as that of 1 (data not shown). The
Soret bands of demetalation products of 2–4 were shifted
to shorter wavelengths than those of the Zn chlorins (2:
450 → 430 nm, 3: 459 → 437 nm, 4: 430 → 414 nm).
These allow us to analyze their demetalation reactions
quantitatively by monitoring absorbance changes at the
Soret peak positions of Zn chlorins 1–4.
Fig. 2. Visible absorption spectra of Zn chlorins 1–4 (a) and free-
base chlorins 1′–4′ (b) in acetone. 1 and 1′: black solid curve. 2
and 2′: broken curve. 3 and 3′: gray solid curve. 4 and 4′: dotted
curve. The spectra were normalized at the Soret peak positions
Fig. 3. Spectral changes of Zn 3,7-diformy-chlorin 1 in acetone/
water (3/1, vol/vol) at the proton concentration of 1.8 × 10-1 M.
Spectra were measured from 0 to 23 h at a 30-min interval. The
arrows show the directions of absorbance changes
Copyright © 2013 World Scientific Publishing Company
J. Porphyrins Phthalocyanines 2013; 17: 1122–1128

DEMETALATION KINETICS OF THE ZINC CHLOROPHYLL DERIVATIVE POSSESSING TWO FORMYL GROUPS 1123
Demetalation kinetics
of Soret absorbance of Zn chlorins 1–3 to the following
kinetic equation:
Figure4depictstimecoursesofSoretpeakabsorbances
of Zn formylated chlorins 1–3 through demetalation at the
proton concentration of 1.8 × 10-1, 1.2 × 10-1, and 6.0 ×
10-2 M. All the kinetic plots exhibited linear relationships
between the logarithm of Soret absorbance and reaction
time. Therefore, the demetalation reactions of Zn chlorins
1–3 can be regarded as pseudo-first-order reactions.
These are consistent with the reaction conditions, in
which the proton concentration was much higher than
the concentration of Zn chlorins. Demetalation rate
constants, ks, were determined by fitting the time courses
ln [(A–A∞)/(A0–A∞)] = -kt,
(1)
where A0, A, and A∞ are Soret absorbance of 1–3 at the
onset of the measurement, at time t, and at the complete
demetalation, respectively. Demetalation rate constants
of Zn formylated chlorins 1–3, which were the averages
of more than three independent measurements, are
plotted in Fig. 5 against the proton concentrations. It is
worthy noting that the standard deviations were within 5,
4, and 10% of the averaged values of demetalation rate
constants for 1, 2, and 3, respectively. Demetalation rate
constants of 4 at the proton concentration of 6.0 × 10-2
M were also estimated from the kinetic plots in the same
manner as 1–3, and the averaged value is plotted in Fig. 5,
but kinetic analysis cannot be performed at higher proton
concentrations because of significantly fast demetalation
kinetics of 4. The logarithm of the demetalation rate
constants, log k, of Zn chlorins 1–3 increased linearly
with log [H+]. The slopes of log k of Zn formylated
chlorins 1–3 against log [H+] in Fig. 5 were estimated
to be 2.9, 3.0, and 2.9, respectively. These values were
slightly larger than those of Zn chlorin 2 (the slope was
2.6) in a previous report [39]. The difference of the slope
in log [H+]-log k plots between the present study and the
previous report [39] might be derived from the examined
0.0
(a)
-0.5
-1.0
-1.5
1
3
2
-2.0
0.0
0
0
0
20
40
60
80
(b)
-0.5
-1.0
-1.5
1
3
2
-2.0
0.0
50
100
150
200
250
(c)
1
-0.5
-1.0
-1.5
-2.0
3
2
500
1000
Time, min
1500
2000
Fig. 4. Kinetic plots for demetalation of Zn 3,7-diformyl-
chlorin 1 (open circle), Zn 3-formyl-chlorin 2 (open triangle),
and Zn 7-formyl-chlorin 3 (open square) in acetone/water (3/1,
vol/vol) at the proton concentration of 1.8 × 10-1 (a), 1.2 × 10-1
(b), and 6.0 × 10-2 M (c). Absorbance changes were monitored
at 472, 450, and 459 nm for 1–3, respectively. A0, A, and A∞ are
Soret absorbance of Zn chlorins at the onset of measurements,
at time t, and at the complete demetalation, respectively
Fig. 5. Demetalation rate constants of Zn 3,7-diformyl-chlorin
1 (open circle), Zn 3-formyl-chlorin 2 (open triangle), Zn
7-formyl-chlorin 3 (open square), and Zn chlorin 4 (open
diamond) in acetone/water (3/1, vol/vol) dependent on the
examined proton concentrations
Copyright © 2013 World Scientific Publishing Company
J. Porphyrins Phthalocyanines 2013; 17: 1123–1128

1124 K. SADAOKA ET AL.
proton concentrations, which were ca. 10-1 M and ca. 10-2
M, respectively.
Effects of formyl groups in the A- and B-rings
Comparison of demetalation kinetics among Zn
chlorins 1–4 revealed that formylated Zn chlorins 1–3
exhibited slower demetalation kinetics than 4 possessing
no formyl group. These results can be ascribed to the
electron-withdrawing ability of a formyl group, resulting
in decrease of electron densities on core nitrogen atoms
of the chlorin macrocycle [32, 35, 39]. Among the three
Zn formylated chlorins 1–3, the demetalation kinetics of
Zn 3,7-diformyl-chlorin 1 was the slowest at the proton
concentrations between 1.8 × 10-1 and 6.0 × 10-2 M, as
seen in Figs 4 and 5. The relative ratios of demetalation
rate constants of 1 to those of 2 and 3 were 0.013–0.014
and 0.062–0.065, respectively. These indicate that two
formyl groups directly linked to the chlorin macrocycle
result in significantly slower demetalation kinetics
than one formyl group. Therefore, the effects of an
electron-withdrawing formyl group on demetalation of
chlorophyllous pigments can be amplified by increase of
the number of formyl groups on the chlorin macrocycle.
Such a combination effect of two formyl substituents
linked to the different rings of the chlorin macrocycle on
demetalation kinetics of chlorophyllous pigments was
first demonstrated, although the effects of sole formyl
group have been reported [22–24, 27, 28, 30–32, 34, 35,
39]. The relative ratio of demetalation rate constants of
3-formylated 2 over 7-formylated 3 were estimated to be
4.6–5.0 under the acidic conditions. We reported that this
difference was ascribable to the aliphatic groups in the A-
and B-rings, namely the 7-methyl group in 2 and 3-vinyl
group in 3 [35]. Actually, dehydrogenation of the ethyl
group to the vinyl group makes demetalation kinetics of
metallochlorins slow down [35, 39].
To make the effects of formyl groups conjugated to
the chlorin macrocycle on demetalation properties more
clear, relationships between demetalation rate constants
of Zn chlorins 1–4 and the sum of Hammett s parameters
[41] of the 3- and 7-substituents were investigated.
Figures 6 and 7 show dependence of log k on the sum
of Hammett sm and sp parameters, respectively. High
correlations can be observed between log k of these
Zn chlorins and Hammett sm constants: correlation
coefficients |r| = 0.999, 0.998, and 0.990 at the proton
concentrations of 1.8 × 10-1, 1.2 × 10-1, and 6.0 × 10-2
M, respectively (Fig. 6). Hammett sp constants were also
correlated with log k: |r| = 0.989, 0.987, and 0.989 at the
proton concentration of 1.8 × 10-1, 1.2 × 10-1, and 6.0 ×
10-2 M, respectively (Fig. 7). Such high correlations
reveal that demetalation properties of chlorophyllous
pigments can be quantitatively explained by invoking
combination of inductive effects of substituents in the
A- and B-rings of the chlorin macrocycles. We reported
the high correlations of the demetalation rate constants
Fig. 6. Dependence of demetalation rate constants of Zn
3,7-diformyl-chlorin 1 (open circle), Zn 3-formyl-chlorin 2
(open triangle), Zn 7-formyl-chlorin 3 (open square), and Zn
chlorin 4 (open diamond) at the proton concentrations of 1.8 ×
10-1 (a), 1.2 × 10-1 (b), and 6.0 × 10-2 M (c) on the sum of
Hammett sm constants of the 3- and 7-substituents
and Hammett s parameters of the 3-substituents in Zn
chlorins [39]. This study suggests that the quantitative
effects of electron-withdrawing and -donating strength
of the substituents linked to the chlorin macrocycle
upon demetalation properties can apply to not only sole
group but also multiple substituents of chlorophyllous
pigments.
The slopes in Figs. 6 and 7 indicate susceptibility
of reactions to electronic effects, namely Hammett r
constants. Demetalation reactions of the Zn chlorins
exhibited that r values were -4.4, -4.4, and -5.4 at the
proton concentrations of 1.8 × 10-1, 1.2 × 10-1, and 6.0 ×
10-2 M, respectively, against sm parameters (Fig. 6).
Hammett r constants were estimated to be -3.0, -3.0,
and -3.7 at the proton concentrations of 1.8 × 10-1,
Copyright © 2013 World Scientific Publishing Company
J. Porphyrins Phthalocyanines 2013; 17: 1124–1128

DEMETALATION KINETICS OF THE ZINC CHLOROPHYLL DERIVATIVE POSSESSING TWO FORMYL GROUPS 1125
Fig. 8. HPLC elution patterns of Zn 3,7-diformyl-chlorin
1 before demetalation reaction (a), its reaction products
after demetalation reaction in acetone/water (3/1, vol/vol)
at the proton concentration of 1.8 × 10-1 M for 23 h (b), and
synthesized free-base 3,7-diformyl chlorin 1′ (c). Pigments
were eluted on a reverse-phase column 3C18-EB (4.6 mmf ×
150 mm) with methanol at a flow rate of 0.3 mL.min-1 at 40 °C.
Chromatograms (a), (b), and (c) were recorded at 469, 450, and
450 nm, respectively
HPLC analysis
The products after demetalation reactions under the
conditions in Fig. 3 were analyzed by reverse-phase
HPLC. Figure 8 depicts HPLC elution patterns of Zn
3,7-diformyl-chlorin 1 before incubation in acidic aqueous
acetone and its reaction products after the incubation. A
fraction at 9 min before incubation was assigned to Zn
3,7-diformyl-chlorin 1 (Fig. 8a). After incubation for
23 h, a main product, which had the Soret and Qy-bands
Fig. 7. Dependence of demetalation rate constants of Zn
at 450 and 677 nm, respectively, in the HPLC eluent, was
detected at 13 min (Fig. 8b). This fraction was ascribed
to free-base 3,7-diformyl-chlorin 1′, since synthesized 1′,
which was thoroughly characterized by 1H NMR, HRMS,
and visible absorption spectroscopy, was eluted at the same
retention time (Fig. 8c). A slight fraction, which would be a
by-product during the demetalation reaction, was detected
at 9 min in this chromatogram (Fig. 8b). HPLC analyses
of the products after demetalation reactions of the other
two monoformylated chlorins 2 and 3 have sole fraction
under the acidic conditions (data not shown). The HPLC
analysis revealed that few side-reactions occurred within
the reaction time in which demetalation rate constants
were estimated under the present reaction conditions.
3,7-diformyl-chlorin 1 (open circle), Zn 3-formyl-chlorin 2
(open triangle), Zn 7-formyl-chlorin 3 (open square), and Zn
chlorin 4 (open diamond) at the proton concentrations of 1.8 ×
10-1 (a), 1.2 × 10-1 (b), and 6.0 × 10-2 M (c) on the sum of
Hammett sp constants of the 3- and 7-substituents
1.2 × 10-1, and 6.0 × 10-2 M, respectively, against sp
parameters (Fig. 7). The negative r values indicate the
electrophilic attack of protons to core nitrogen atoms at
the rate-determining steps in these reactions. The r values
estimated in this study were somewhat different from
those estimated in previous reports [34, 39], in which
Hammett r values of the 3-substituted and 7-substituted
Zn chlorins were distributed between -6.1 and -6.7
against sm, and between -4.1 and -4.9 against sp. One
possible reason for the difference would be ascribable to
the difference in proton concentrations of demetalation
reactions. Further studies will be required to understand
effects of reaction conditions and molecular structures
on Hammett r constants in demetalation reactions of
chlorophyllous pigments.
EXPERIMENTAL
Apparatus
Visible absorption spectra were measured with a
Shimadzu UV-2450 spectrophotometer, where the
Copyright © 2013 World Scientific Publishing Company
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1126 K. SADAOKA ET AL.
reaction temperature was regulated with a Shimadzu
thermo-electric temperature-controlled cell holder TCC-
240A. High-performance liquid chromatography (HPLC)
was performed with a Shimadzu LC-20AT pump, an
SPD-M20A or SPD-20AV detector, and a CTO-20A
column oven. Mass spectra (MS) were measured with a
JEOL JMS700 spectrometer; m-nitorobenzyl alcohol was
used as a matrix. High-resolution mass spectra (HRMS)
were measured with a JEOL GC-mate II spectrometer;
m-nitorobenzyl alcohol and glycerol were used as
(1H, m, 18-H), 4.33–4.28 (1H, m, 17-H), 3.96–3.86 (2H,
m, 8-CH2), 3.58, 3.51, 3.32 (each 3H, s, 2-, 12-, 174-
CH3), 2.65–2.58, 2.56–2.44, 2.30–2.22 (1H + 2H + 1H,
m, 17-CH2CH2), 1.91 (3H, d, J = 6 Hz, 18-CH3), 1.67
(3H, t, J = 7 Hz, 81-CH3). UV-vis (acetone): lmax, nm
659 (relative intensity, 0.26), 606 (0.05), 558 (0.04), 463
(1.00), 437 (0.27). HRMS (FAB): m/z found 626.1555,
calcd. for C33H30N4O5Zn [M]+, 626.1508.
Synthesis of Zn chlorins 2–4. Zn methyl
pyropheophorbide d (Zn 3-formyl-chlorin 2) and Zn
methyl pyropheophorbide a (Zn chlorin 4) were prepared
from Chl a according to previous reports [34, 39, 43,
44]. Zn methyl pyropheophorbide b (Zn 7-formyl-
chlorin 3) was prepared from Chl b [34]. Zn 3-formyl-
chlorin 2. UV-vis (acetone): lmax, nm 680 (relative
intensity, 0.95), 630 (0.12), 586 (0.08), 541 (0.04), 443
(1.00), 390 (0.48). MS (FAB): m/z found 612.4, calcd.
for C33H32N4O4Zn [M]+, 612.2. Zn 7-formyl-chlorin
3. UV-vis (acetone): lmax, nm 637 (relative intensity,
0.30), 587 (0.07), 451 (1.00). MS (FAB): m/z found
624.4, calcd. for C34H32N4O4Zn [M]+, 624.2. Zn chlorin
4. UV-vis (acetone): lmax, nm 655 (relative intensity,
0.69), 607 (0.11), 568 (0.06), 524 (0.04), 425 (1.00), 405
(0.60), 379 (0.37). MS (FAB): m/z found 610.3, calcd. for
C34H34N4O3Zn [M]+, 610.2.
1
matrixes. H NMR spectra were measured with a JEOL
JNM-AL400 NMR spectrometer; chemical shifts were
expressed (in ppm) relative to chloroform (7.26 ppm) as
an internal reference.
Synthesis
Synthesis of diformylated Zn chlorin 1. To a THF
solution (20 mL) of methyl pyropheophorbide b (3, 16.7
mg), which was prepared from Chl b [34, 39], two pieces
of microcapsulated OsO4 [42], which was purchased
from Wako Chemical Industries, Ltd., was added at 0 °C.
A solution of NaIO4 (0.25 g) and CH3COOH (0.32 mL)
in H2O (2.0 mL) was dropped into the ice-chilled THF
solution, and stirred overnight at room temperature.
After extraction of reaction products with CH2Cl2 several
times, the combined organic phases were washed with
NaHCO3-saturated water and distilled water, dried
over anhydrous Na2SO4, and evaporated under reduced
pressure. The residue was purified by flash silica
gel column chromatography and recrystallized from
CH2Cl2 and hexane to give methyl 3-devinyl-3-formyl-
pyropheophorbide b. 3,7-diformyl-chlorin 1′. 8.7 mg,
52% yield. 1H NMR (CDCl3): d, ppm 11.44, 10.95 (each
1H, s, 3-, 7-CHO), 10.38, 9.36, 8.76 (each 1H, s, 5-,
10-, 20-H), 5.33, 5.17 (each 1H, d, J = 20 Hz, 132-H2),
4.57 (1H, dq, J = 2, 7 Hz, 18-H), 4.39 (1H, dt, J = 9, 3
Hz, 17-H), 3.86–3.74 (2H, m, 8-CH2), 3.69, 3.67, 3.63
(each 3H, s, 2-, 12-, 174-CH3), 2.84–2.75, 2.70–2.62,
2.45–2.27 (1H + 1H + 2H, m, 17-CH2CH2), 1.94 (3H,
d, J = 7 Hz, 18-CH3), 1.66 (3H, t, J = 8 Hz, 81-CH3),
-0.60, -2.49 (each 1H, s, NH). UV-vis (acetone): lmax, nm
675 (relative intensity, 0.20), 617 (0.04), 535 (0.08), 449
(1.00), 431 (0.48). HRMS (FAB): m/z found 564.2363,
calcd. for C33H32N4O5 [M]+, 564.2373. Free-base chlorin
1′ (17.7 mg) was dissolved in CH2Cl2 (9 mL) and
Zn(CH3COO)·2H2O-saturated methanol (4 mL) was
added to the CH2Cl2 solution, followed by stirring for 8 h.
The solution was neutralized by aq. 4% NaHCO3, washed
with distilled water several times, dried over anhydrous
Na2SO4, and evaporated under reduced pressure. The
residue was recrystallized from CH2Cl2 and hexane to
afford Zn methyl 3-devinyl-3-formyl-pyropheophorbide
Measurements of demetalation kinetics
A 3.0 mL acetone solution of Zn chlorins 1–4 (Soret
absorbance = 1.0 at the 1-cm pathlength) was mixed with
a 1.0 mL distilled water containing hydrochloric acid.
The proton concentration was calculated by assuming
complete dissociation of hydrochloric acid in a mixture
of acetone and water (3/1, vol/vol), since previous reports
ensured such dissociation at the proton concentration
up to ca. 10-1 M [27, 29]. Demetalation kinetics was
examined by monitoring the absorbances at the Soret
peak positions of Zn chlorins at the proton concentration
of 1.8 × 10-1, 1.2 × 10-1, and 6.0 × 10-2 M for 1–3, as
well as 6.0 × 10-2 M for 4 under the control of reaction
temperature at 25 °C.
Pigment analyses after demetalation reaction
After demetalation reactions, the solution was
neutralized by NaHCO3-saturated water. The reaction
products were extracted with CH2Cl2, washed with
NaCl-saturated water, and dried over anhydrous Na2SO4.
The solution was filtrated and dried with nitrogen gas.
The residue was analyzed by reverse-phase HPLC
column 3C18-EB (4.6 mmf × 150 mm, Nacalai Tesque,
Inc., Kyoto, Japan) with methanol at a flow rate of 0.3
mL.min-1 at 40 °C.
1
b. Zn 3,7-diformyl-chlorin 1. 14.8 mg, 75% yield. H
CONCLUSION
NMR (CDCl3): d, ppm 11.05, 10.70 (each 1H, s, 3-,
7-CHO), 10.04, 9.49, 8.53 (each 1H, s, 5-, 10-, 20-H),
5.01, 4.88 (each 1H, d, J = 20 Hz, 132-H2), 4.51–4.45
This study reveals effects of the formyl groups
conjugated to the A- and B-rings of the chlorin macrocycle
Copyright © 2013 World Scientific Publishing Company
J. Porphyrins Phthalocyanines 2013; 17: 1126–1128

DEMETALATION KINETICS OF THE ZINC CHLOROPHYLL DERIVATIVE POSSESSING TWO FORMYL GROUPS 1127
on kinetics stabilities against removal of the central
metal from the chlorin ring by using diformylated and
monoformylated Zn chlorins. Zn chlorins possessing
two formyl groups in both the A- and B-rings exhibited
significantly slow demetalation kinetics by combination
effects of both the formyl groups. The effects of the two
formyl groups in the chlorin macrocycle on demetalation
kinetics can be quantitatively rationalized by combination
of their electron-withdrawing ability, judged from high
correlations between the demetalation rate constants
and the sum of Hammett s parameters of the 3- and
7-substitutients on the chlorin macrocycle. The present
results will help to understand demetalation reactions of
natural Chl pigments and synthetic cyclic tetrapyrrole
molecules.
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We thank Prof. Tomohiro Miyatake and Mr. Yohei
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and Ms. Keiko Kinoshita, Kinki University, for
experimental assistance. This work was partially
supported by a Grant-in-Aid for Scientific Research
(C) (No. 23550201) (to YS) from the Japan Society for
the Promotion of Science. KS was supported by a JSPS
Fellowship for Young Scientists.
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