Systematic analysis of the demetalation kinetics of zinc chlorophyll derivatives possessing different substituents at the 3-position: Effects of the electron-withdrawing and electron-donating strength of peripheral substituents

Article abstract of DOI:10.1021/ic3016782

Removal of the central metal from chlorophyll (Chl) molecules is biologically important in terms of production of the primary electron acceptors in photosystem-II photosynthetic reaction centers and the early stage in Chl degradation. The physicochemical properties on demetalation of chlorophyllous pigments are useful in the understanding of such reaction mechanisms in photosynthetic organisms. Here we analyzed the demetalation kinetics of a series of Zn-Chl derivatives with a systematic variation in the electron-withdrawing and -donating substituents at the 3-position of the chlorin macrocycle under acidic conditions to elucidate thoroughly the substitution effects on the demetalation properties of chlorophyllous pigments. Dehydrogenation of the aliphatic group (CH₂CH₃ → CH-CH₂ → C? - CH) at the 3-position slowed the removal of the central zinc from the chlorin macrocycle. The gradual decrease in the demetalation rate constants of the three zinc chlorins originates from differences in the electron-withdrawing strength of the ethyl, vinyl, and ethynyl groups directly linked to the chlorin π macrocycle. Reduction of the 3¹-carbonyl groups significantly increased the demetalation rate constants, and the relative ratios of the demetalation rate constants of the zinc chlorins possessing a carbonyl group to those possessing the corresponding hydroxy group were analogous in the cases of 3-formyl- and 3-acetyl-zinc chlorins. The demetalation rate constants of the seven Zn-Chl derivatives possessing various electron-withdrawing and -donating groups exhibited good correlation with the Hammett σ parameters of the 3-position substituents.

Full text of DOI:10.1021/ic3016782

Article
pubs.acs.org/IC
Systematic Analysis of the Demetalation Kinetics of Zinc Chlorophyll
Derivatives Possessing Different Substituents at the 3‑Position:
Effects of the Electron-Withdrawing and Electron-Donating Strength
of Peripheral Substituents
Yoshitaka Saga,* Yuta Kobashiri, and Kana Sadaoka
Department of Chemistry, Faculty of Science and Engineering, Kinki University, Higashi-Osaka, Osaka 577-8502, Japan
S
* Supporting Information
ABSTRACT: Removal of the central metal from chlorophyll (Chl)
molecules is biologically important in terms of production of the
primary electron acceptors in photosystem-II photosynthetic reaction
centers and the early stage in Chl degradation. The physicochemical
properties on demetalation of chlorophyllous pigments are useful in
the understanding of such reaction mechanisms in photosynthetic
organisms. Here we analyzed the demetalation kinetics of a series of
Zn-Chl derivatives with a systematic variation in the electron-
withdrawing and -donating substituents at the 3-position of the
chlorin macrocycle under acidic conditions to elucidate thoroughly the substitution effects on the demetalation properties of
chlorophyllous pigments. Dehydrogenation of the aliphatic group (CH2CH3 → CHCH2 → CCH) at the 3-position slowed
the removal of the central zinc from the chlorin macrocycle. The gradual decrease in the demetalation rate constants of the three
zinc chlorins originates from differences in the electron-withdrawing strength of the ethyl, vinyl, and ethynyl groups directly
linked to the chlorin π macrocycle. Reduction of the 31-carbonyl groups significantly increased the demetalation rate constants,
and the relative ratios of the demetalation rate constants of the zinc chlorins possessing a carbonyl group to those possessing the
corresponding hydroxy group were analogous in the cases of 3-formyl- and 3-acetyl-zinc chlorins. The demetalation rate
constants of the seven Zn-Chl derivatives possessing various electron-withdrawing and -donating groups exhibited good
correlation with the Hammett σ parameters of the 3-position substituents.
electron acceptors in PS-II-type photosynthetic reaction
centers.
INTRODUCTION
■
Chlorophyll (Chl) and bacteriochlorophyll (BChl) molecules
play important roles in photosynthesis, which is an efficient
sunlight conversion system in nature.1,2 These chlorophyllous
pigments have tetrapyrrole macrocycles as the photofunctional
moieties and commonly possess a central magnesium in the π
macrocycle. The exceptions in photosynthetically active
pigments are Zn-BChl a in a purple bacterium Acidiphilium
rubrum3−5 and demetalated pigments of (B)Chl molecules in
photosystem (PS)-II-type photosynthetic reaction centers.6−11
PS-II reaction centers in oxygenic photosynthetic organisms
utilize pheophytin (Phe) a, which lacks the central magnesium
from Chl a, as the primary electron acceptor.6−9 In PS-II,
cofactors are positioned with pseudo-2-fold symmetry.9,12−15
Therefore, two Phe a molecules are present in the protein
scaffolds of PS-II, but only one Phe a participates in
photoinduced electron transfer. Biosynthesis of Phe a in PS-
II is completely unknown in spite of its biological importance.
Phe a would be produced from Chl a by its demetalation
because Chl synthase uses Chlide a as a substrate to esterify a
long hydrocarbon chain at the 17-propionate and cannot use
pheophorbide a lacking the central magnesium.16 From this
viewpoint, Chl demetalation is crucial for the in vivo
production of Phe molecules, which function as the primary
Another important event concerning demetalation of
chlorophyllous pigments is found in the Chl degradation
pathway. Chl degradation is one of the most large-scale
bioprocesses on earth and a major origin of beautiful
phenomena in autumn.17−21 Removal of the central magnesium
from the chlorin macrocycle occurs in the early stage of the
degradation process, but its mechanism has not been
thoroughly unraveled yet.
In vitro demetalation of natural Chl molecules and their
model compounds has been examined21−37 because the
physicochemical properties of Chl demetalation are clues to
understanding such biologically important reactions in photo-
synthetic organisms. Some of the previous studies on in vitro
Chl demetalation demonstrated that peripheral substituents on
the chlorin macrocycle had large effects on the demetalation
properties of chlorophyllous pigments.22,23,28,30−32,34−37 For
example, a comparison of the demetalation kinetics between
Chl a and b indicated that the 7-formyl group in Chl b was
responsible for significantly slow demetalation kinetics.22,23,28,32
Received: July 31, 2012
Published: December 11, 2012
© 2012 American Chemical Society
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of 1′ was hydrogenated in the presence of palladium charcoal under a
hydrogen atmosphere to give methyl 3-devinyl-3-ethyl-pyropheophor-
bide a (2′).45 The 3-vinyl group in 1′ was oxidatively cleaved by
sodium periodate and osmium tetraoxide to give methyl 3-devinyl-3-
formyl-pyropheophorbide a (4′).46,47 Subsequent reduction of the 3-
formyl group in 4′ by tert-butylamine borane complexes afforded
methyl 3-devinyl-3-hydroxymethyl-pyropheophorbide a (5′).46,48 The
formyl group in 4′ was converted to the ethynyl group to give methyl
3-devinyl-3-ethynyl-pyropheophorbide a (3′) by treatment with a
commercially available Bestmann−Ohira reagent [(MeO)2P(O)C-
(COMe)N2] in the presence of Cs2CO3.49,50 The 3-vinyl group in 1′
was converted to the hydroxyethyl group using hydrogen bromide,51,52
followed by the formation of methyl ester by treatment with
(trimethylsilyl)diazomethane53 to give methyl 3-devinyl-3-hydroxyeth-
yl-pyropheophorbide a (7′). The hydroxyethyl group in 7′ was
oxidized to produce methyl 3-devinyl-3-acetyl-pyropheophorbide a
(6′) possessing the 3-acetyl group by use of 4-methylmorpholine-N-
oxide and tetrapropylammonium perruthenate.54 Zinc was inserted
into free-base chlorins 1′−7′ by Zn(CH3COO)·2H2O to afford zinc
chlorins 1−7, respectively. The synthesized zinc chlorins were purified
by reverse-phase HPLC before measurements of the demetalation
kinetics.
We reported that formyl groups in other naturally occurring
pigments such as Chl d and BChl e also made their
demetalation kinetics slow.30−32 The effects of the formyl
groups in these pigments on the demetalation properties are
ascribable to their electron-withdrawing abilities. However, no
quantitative information is available on the relationship
between the electron-withdrawing/-donating strength of the
peripheral substituents and demetalation kinetics of chlor-
ophyllous pigments. We report herein the demetalation
properties of synthesized zinc chlorins 1−7, in which only 3-
position substituents are different from each other. Figure 1
Characteristic spectral data of 1−7 are described as follows (see also
refs 45−54). Zinc methyl pyropheophorbide a (1). UV−vis (acetone):
λmax = 655 nm (relative intensity 0.69), 607 (0.11), 568 (0.06), 524
(0.04), 425 (1.00), 405 (0.60). MS (FAB). Calcd for C34H34N4O3Zn
(M+): m/z 610.19. Found: m/z 610.26. Zinc methyl 3-devinyl-3-
ethylpyropheophorbide a (2). UV−vis (acetone): λmax = 643 nm
(relative intensity 0.77), 598 (0.12), 562 (0.06), 518 (0.03), 421
(1.00), 401 (0.65). MS (FAB). Calcd for C34H36N4O3Zn (M+): m/z
612.21. Found: m/z 612.28. Zinc methyl 3-devinyl-3-ethynyl-pyro-
pheophorbide a (3). UV−vis (acetone): λmax = 661 nm (relative
intensity 0.76), 613 (0.10), 571 (0.05), 527 (0.03), 428 (1.00), 408
(0.52). MS (FAB). Calcd for C34H32N4O3Zn (M+): m/z 608.18.
Found: m/z 608.22. Zinc methyl 3-devinyl-3-formyl-pyropheophor-
bide a (4). UV−vis (acetone): λmax = 680 nm (relative intensity 0.95),
631 (0.12), 586 (0.08), 541 (0.04), 443 (1.00). MS (FAB). Calcd for
C33H32N4O4Zn (M+): m/z 612.17. Found: m/z 612.25. Zinc methyl 3-
devinyl-3-hydroxymethyl-pyropheophorbide a (5). UV−vis (acetone):
λmax = 646 nm (relative intensity 0.76), 601 (0.12), 565 (0.06), 519
(0.04), 423 (1.00), 403 (0.64). MS (FAB). Calcd for C33H34N4O4Zn
(M+): m/z 614.19. Found: m/z 614.24. Zinc methyl 3-devinyl-3-
acetyl-pyropheophorbide a (6). UV−vis (acetone): λmax = 668 nm
(relative intensity 0.73), 619 (0.13), 579 (0.08), 533 (0.04), 432
(1.00). MS (FAB). Calcd for C34H34N4O4Zn (M+): m/z 626.19.
Found: m/z 626.23. Zinc methyl 3-devinyl-3-hydroxyethyl-pyropheo-
phorbide a (7). UV−vis (acetone): λmax = 645 nm (relative intensity
0.78), 600 (0.12), 565 (0.06), 519 (0.03), 422 (1.00), 402 (0.65). MS
(FAB). Calcd for C34H36N4O4Zn (M+): m/z 628.20. Found: m/z
628.30.
Measurements of the Demetalation Kinetics. A 3.0 mL
acetone solution of purified zinc chlorins (Soret absorbance = 1.0) was
mixed with 975 μL of distilled water. Then, 25 μL of aqueous
hydrochloric acid for volumetric analysis (Wako Pure Chemical
Industries, Ltd., Japan) was added to the solutions, and absorbance at
the Soret peak positions of zinc chlorins was measured under the
control of reaction temperatures at 25 °C.
Pigment Analysis after Demetalation. After demetalation
reactions, the solutions were neutralized by 4% aqueous NaHCO3.
The reaction products were extracted with dichloromethane, washed
with NaCl-saturated water, and dried over anhydrous Na2SO4. The
solutions were filtered and dried with nitrogen gas. The residues were
analyzed by reverse-phase HPLC.
Figure 1. Molecular structures of zinc chlorins 1−7 used in the
present study: 1, R = CHCH2; 2, R = CH2CH3; 3, R = CCH; 4,
R = CHO; 5, R = CH2OH; 6, R = COCH3; 7, R = CH(OH)CH3.
shows the molecular structures of zinc chlorins 1−7 used in this
study. Zinc chlorin 1 has a vinyl group at the 3-position and is a
good model compound of Chl a. The vinyl group is
hydrogenated (3-CHCH2 → 3-CH2CH3) and dehydrogen-
ated (3-CHCH2 → 3-CCH) in zinc chlorins 2 and 3,
respectively. Zinc chlorin 4 possessing a formyl group at the 3-
position is a derivative of Chl d, which is a unique
photosynthetic pigment in Acaryochloris marina.38,39 An acetyl
group in zinc chlorin 6 is a peripheral substituent characteristic
of BChl a and b in anoxygenic photosynthetic bacteria.1,2
Reduction of the 31-carbonyl groups in 4 and 6 affords zinc
chlorins 5 and 7, respectively. Zinc chlorins 5 and 7 possessing
a 31-hydroxy group have widely been used as model
compounds of light-harvesting BChl c and d in green
photosynthetic bacteria.40−44 A comparison of the demetalation
properties among 1−7 enables us to understand the inductive
effects of the 3-position substituents on the demetalation
properties of metallochlorins quantitatively without other
substitution effects.
EXPERIMENTAL SECTION
■
Apparatus. Visible absorption spectra were measured with a
Shimadzu UV-2450 spectrophotometer, where the reaction temper-
ature was regulated with a Shimadzu thermoelectric temperature-
controlled cell holder TCC-240A. High-performance liquid chroma-
tography (HPLC) was performed with a Shimadzu LC-20AT pump
and an SPD-M20A or SPD-20AV detector. Mass spectrometry (MS)
spectra were measured with a JEOL JMS700 spectrometer; m-
nitrobenzyl alcohol was used as a matrix.
RESULTS AND DISCUSSION
■
Spectral Changes. Demetalation of synthetic zinc chlorins
1−7 possessing a different substituent at the 3-position was
kinetically analyzed in acidic aqueous acetone. Figure 2 depicts
a spectral change of zinc chlorin 1 possessing the 3-vinyl group
Materials. Zinc chlorins 1−7 were synthesized from Chl a, which
was extracted from cyanobacterium Spirulina geitleri, according to
previous reports.2,45−54 Methyl pyropheophorbide a (1′) was
synthesized from Chl a through three steps.45,46 The 3-vinyl group
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Figure 2. Spectral change of zinc chlorin 1 in acetone/water [3:1 (v/
v)] at the proton concentration of 6.3 × 10−3 M. Spectra from 0 to
540 min at a 60-min interval. The arrows show the direction of the
absorbance changes.
during the demetalation process at the proton concentration of
6.3 × 10−3 M. Zinc chlorin 1 exhibited the Soret and Qy bands
at 430 and 659 nm before demetalation in this solution
[acetone:water = 3:1 (v/v)]. The Soret absorption band of 1
gradually decreased with an appearance of a new absorption
band at 411 nm under this condition. This 411-nm absorption
band was ascribable to the Soret band of the demetalation form
of 1, namely, 1′. The Qy absorbance of 1 at 659 nm decreased
simultaneously under acidic conditions. The small absorption
bands at 577 and 612 nm of 1 also became small gradually, and
the absorption bands at 507 and 537 nm, which was ascribable
to 1′, appeared. The isosbestic points were present at 421, 453,
554, and 667 nm in this spectral change.
Figure 3. Demetalation kinetics of 1−3 (A), 4 and 5 (B), and 6 and 7
(C) in acetone/water [3:1 (v/v)] at the proton concentration of 3.8 ×
10−2 M. Absorbance changes were monitored at 430, 424, 433, 450,
427, 438, and 426 nm for 1−7, respectively. A0, A, and A∞ are Soret
absorbances of zinc chlorins at the onset of measurements, at time t,
and at complete demetalation, respectively.
Essentially the same spectral changes were observed in
demetalation reactions of 2−7 in acidic aqueous acetone (data
not shown). The Soret and Qy absorption bands of zinc
chlorins 2−7 decreased and new Soret bands of the
corresponding free-base forms (2′−7′, respectively) appeared
at the shorter-wavelength side of the Soret band of zinc chlorins
with several isosbestic points. The differences of the Soret peak
positions between zinc chlorins 1−7 and the corresponding
free-base chlorins 1′−7′ were 15−24 nm (900−1300 cm−1).
This allows us to analyze their demetalation reactions
kinetically by monitoring the absorbance changes at the Soret
peak positions of 1−7.
1−7, which were the averages of three independent measure-
ments, were plotted in Figure 4 against the logarithm of the
Demetalation Kinetics. Figure 3 depicts time courses of
Soret absorbance of zinc chlorins 1−7 through demetalation at
the proton concentration of 3.8 × 10−2 M. All of the kinetic
plots exhibited linear relationships between the logarithm of the
Soret absorbance and reaction time. The demetalation reactions
of zinc chlorins 1−7, therefore, can be regarded as pseudo-first-
order. Such pseudo-first-order kinetics was also observed in
demetalation reactions of 1−7 at the lower proton concen-
trations between 3.1 × 10−2 and 6.3 × 10−3 M. These are in line
with the reaction conditions in which the proton concentration
was much higher than the concentration of zinc chlorins.
Demetalation rate constants, k's, were determined by fitting the
time courses of Soret absorbances of zinc chlorins 1−7 to the
following kinetic equation:
Figure 4. Demetalation rate constants of 1 (open circles), 2 (open
squares), 3 (open triangles), 4 (closed circles), 5 (open reverse-
triangles), 6 (closed squares), and 7 (open diamonds) in acetone/
water [3:1 (v/v)] depending on the examined proton concentrations.
proton concentrations, log [H+]. The logarithm of the
demetalation rate constants, log k, of 1−7 increased linearly
with log [H+]. The slopes of log k of 1−7 against log [H+] in
Figure 4 were estimated to be 2.5, 2.6, 2.6, 2.6, 2.6, 2.8, and 2.5,
respectively. These values were slightly larger than those of
natural and seminatural Chl molecules (magnesium chlorins)
reported previously: the slopes of these Chl molecules in the
ln(A − A∞)/(A0 − A∞) = −kt
where A0, A, and A∞ are Soret absorbances of zinc chlorins at
the onset of the measurement, at time t, and at complete
demetalation, respectively. The demetalation rate constants of
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log k versus log [H+] plots were estimated to be ca. 2 at proton
concentrations of around 10−3 M.27−29,37 One possible reason
for the differences of the slopes between zinc chlorins in this
study and (semi)natural Chl molecules in the previous reports
would be the effects of the central metals. Kobayashi et al.
reported that the slopes of zinc complexes (1.94 and 2.17 for
Zn-Chl a and Zn-BChl a, respectively) were larger than those
of the corresponding magnesium complexes (1.70 and 1.74 for
Mg-Chl a and Mg-BChl a, respectively).29 Therefore, slightly
larger values of the slopes in the plots of the demetalation rate
constants against the proton concentrations for zinc chlorins
1−7 are in line with the tendency in the previous work,
although further studies will be required for understanding the
origin of such differences.
Effects of the Aliphatic Groups on the Demetalation
Kinetics. Zinc chlorins 1−3 possess vinyl, ethyl, and ethynyl
groups, respectively, at the 3-position. Therefore, a comparison
of the demetalation rate constants among 1−3 allows us to
investigate the effects of the aliphatic groups on the
demetalation properties of chlorophyllous pigments systemati-
cally. The order of the demetalation rate constants is 2 > 1 > 3
under acidic conditions, as shown in Figures 3A and 4. The
relative ratio of the demetalation rate constants of 3 to those of
1, k(3)/k(1), were distributed from 5.5 × 10−2 to 7.3 × 10−2 at
the proton concentration range between 3.8 × 10−2 and 6.3 ×
10−3 M. These values were slightly smaller than the relative
ratio of k(1)/k(2), which were estimated within 1.0 × 10−1 and
1.5 × 10−1, under the corresponding conditions.
1.3 × 10−2, and 6.3 × 10−3 M, respectively. The demetalation
rate constants of 1−3 were also correlated with the Hammett σ
parameters of these groups,56 as described below. It was
reported that electron-withdrawing formyl and acetyl sub-
stituents on the chlorin π macrocycle slowed removal of the
central metal because such substituents decreased the electron
densities of the core nitrogen atoms and suppressed an
electrophilic attack of protons to the nitrogen atoms.31,32,34,35
This kinetic analysis clearly indicates that the electron-
withdrawing abilities of the aliphatic groups connected to the
chlorin macrocycle were also responsible for the demetalation
properties of Chl derivatives.
Effects of Reduction of the Carbonyl Groups on the
Demetalation Kinetics. The present study revealed that
reduction of the formyl and acetyl groups at the 3-position of
the chlorin macrocycle accelerated their demetalation kinetics.
The relative ratios of the demetalation rate constants of 4
possessing the 3-formyl group to those of 5 possessing the 3-
hydroxymethyl group were distributed from 3.4 × 10−3 to 4.3 ×
10−3 in the proton concentration range between 3.8 × 10−2 and
6.3 × 10−3 M and were similar to the cases of 6 possessing the
3-acetyl group and 7 possessing the 3-hydroxyethyl group [the
relative ratios of k(6)/k(7) ranged between 5.2 × 10−3 and 8.3
× 10−3 under the same conditions]. These suggest that the 3-
acetyl group affected the demetalation properties of metal-
lochlorins in a manner similar to that of the 3-formyl group. We
previously compared the demetalation kinetics between 7-
formyl- and 7-hydroxymethyl-metallochlorins: the relative ratio
of the demetalation rate constant of the 7-formyl-zinc chlorin to
that of the 7-hydroxymethyl-zinc chlorin was 5.3 × 10−3 at the
proton concentration of 3.8 × 10−2 M.34 This value is
analogous to the relative ratio of 4 to that of 5 under the
same conditions [k(4)/k(5) = 4.3 × 10−3], suggesting that
reduction of the formyl group in the B-ring of the chlorin
macrocycle had almost the same effects on the demetalation
properties as that in the A-ring.
The effects of dehydrogenation of the 3-aliphatic groups on
the demetalation kinetics can be rationalized by invoking
differences of the electron-withdrawing abilities among the
three groups. Figure 5 depicts the relationship between the
Correlation between the Demetalation Rate Con-
stants and Hammett σ Parameters. Hammett σ constants
are some of the most important parameters for interpretation of
organic reactions and have successfully been applied to
elucidation of various reaction mechanisms.56 The demetalation
rate constants of zinc chlorins 1−7 were plotted against
Hammett σm and σp parameters of the 3-position substituents
in Figures 6 and 7, respectively. The k values of 1−7 exhibited
high correlation coefficients |r| of 0.951−0.956 and 0.953−
0.964 with the Hammett σm and σp constants, respectively,
under acidic conditions. The correlation coefficients with σm
were quite similar to those with σp. These indicate that the
kinetic stabilities for removal of the central metal from
chlorophyllous pigments could be quantitatively rationalized
by invoking the electron-withdrawing and -donating strength of
the substituents directly linked to the chlorin π macrocycle. The
good correlation in the present study is in agreement with the
previous results of the oxidation potentials57 and monoproto-
nation equilibrium constants58 of β-substituted porphyrins, in
which these physicochemical properties were correlated with
the Hammett σ parameters of the β substituents.
Figure 5. Dependence of the demetalation rate constants of zinc
chlorins 1−3 at the proton concentrations of 3.8 × 10−2 (open
circles), 3.1 × 10−2 (open squares), 1.3 × 10−2 (open triangles), and
6.3 × 10−3 M (open diamonds) on the group electronegativities45 of
the substituents.
demetalation rate constants of 1−3 and the group electro-
negativities of the 3-position substituents in 1−3 estimated by
Inamoto and Masuda.55 Good correlations between their
demetalation rate constants and the group electronegativities
were observed under these conditions: the correlation
coefficients |r| were estimated to be 0.995, 0.998, 0.994, and
0.995 at the proton concentrations of 3.8 × 10−2, 3.1 × 10−2,
Recently, the metalation and demetalation properties of
chlorophyllous pigments have been reported.59−62 Several
factors such as the macrocyclic structures, peripheral sub-
stituents, and axial coordination were assumed to be
responsible for these properties. Orzel et al. reported that the
peripheral carbonyl groups suppressed the reactivity of
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Figure 6. Dependence of the demetalation rate constants of zinc
chlorins 1−7 at the proton concentrations of 3.8 × 10−2 (A), 3.1 ×
10−2 (B), 1.3 × 10−2 (C), and 6.3 × 10−3 M (D) on the Hammett σm
constants46 of substitution groups: 1 (open circles), 2 (open squares),
3 (open triangles), 4 (closed circles), 5 (open reverse-triangles), 6
(closed squares), and 7 (open diamonds).
Figure 7. Dependence of the demetalation rate constants of zinc
chlorins 1−7 at the proton concentrations of 3.8 × 10−2 (A), 3.1 ×
10−2 (B), 1.3 × 10−2 (C), and 6.3 × 10−3 M (D) on the Hammett σp
constants46 of substitution groups: 1 (open circles), 2 (open squares),
3 (open triangles), 4 (closed circles), 5 (open reverse-triangles), 6
(closed squares), and 7 (open diamonds).
metalation.60 Such effects would be consistent with the present
results. It is noted that the possibility of protonation at the 131-
carbonyl group in the isocyclic E-ring was ruled out under the
present reaction conditions, judged from the temporal changes
of visible absorption spectra through demetalation and HPLC
analyses of the reaction products (Figure 8). This is in line with
the lack of the 132-methoxycarbonyl group in zinc chlorins 1−
7.
HPLC Analysis of the Reaction Products. Figure 8 shows
HPLC elution patterns of the reaction products of 1−7 in
acetone/water (3:1) at the proton concentration of 3.8 × 10−2
M. After the demetalation reaction of 1, the main fraction was
observed at 42 min (Figure 8A). This fraction exhibited Soret
and Qy bands at 407 and 665 nm, respectively, in the HPLC
eluent, and was ascribed to the free-base form of 1, namely, 1′.
No other fractions were detected, indicating no side reactions
under the present conditions. HPLC analysis of the reaction
products of 2−7 gave similar results. In the HPLC chromato-
gram of the reaction products of 2, the sole fraction, which had
Soret and Qy bands at 404 and 654 nm, respectively, was
detected at 44 min (Figure 8B). This fraction could be assigned
to 2′. The reaction products of 3 gave the main fraction of 3′ at
30 min (λmax = 407 and 673 nm) with a slight byproduct at 13
min (Figure 8C). The chromatograms of reaction products of 4
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rate constants were estimated under the present reaction
conditions.
CONCLUSION
■
The present analysis of the demetalation kinetics of systemati-
cally substituted zinc chlorins 1−7 clearly indicates that the
demetalation rate constants of metallochlorins are correlated
with the group electronegativities and Hammett σ constants of
their peripheral substituents. Therefore, electron-withdrawing
and -donating strength of the substituents on the chlorin π
macrocycle provides useful information to understand the
physicochemical properties of Chl demetalation quantitatively.
These results will contribute to elucidation of the reaction
mechanisms of in vivo and in vitro demetalation of the Chl
molecules.
ASSOCIATED CONTENT
■
S
* Supporting Information
1H NMR and FAB-MS data of 1′−7′. This material is available
free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*E-mail: saga@chem.kindai.ac.jp. Fax: +81-6-6723-2721.
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Dr. Yuki Hirai, Nagahama Institute of Bio-Science
and Technology, and Yasuhiro Iida, Kinki University, for their
experimental assistance. This work was partially supported by a
Grant-in-Aid for Scientific Research (C) (No. 23550201 to
Y.S.) from the Japan Society for the Promotion of Science.
REFERENCES
■
(1) Scheer, H. In Chlorophylls and Bacteriochlorophylls: Biochemistry,
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Figure 8. HPLC elution patterns of the reaction products of 1 (A), 2
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the proton concentration of 3.8 × 10−2 M for 2, 0.25, 20, 23, 2, 22, and
1.5 h, respectively. The reaction products of 1−3 and 5−7 were eluted
on a reverse-phase column 5C18-AR-II (6 mm i.d. × 250 mm) with
methanol at a flow rate of 1.0 mL/min. The reaction products of 4
were eluted on a reverse-phase column 3C18-EB (4.6 mm i.d. × 150
mm) with methanol at a flow rate of 0.3 mL/min. Chromatograms A−
G were recorded at 407, 404, 407, 420, 404, 411, and 404 nm,
respectively.
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