Synthesis and self-assembly of zinc methyl bacteriopheophorbide-f and its homolog

Article abstract of DOI:10.1016/S0040-4020(00)00590-1

Zinc methyl bacteriopheophorbide-f, zinc 3-(1-hydroxyethyl)-7-formyl-chlorin, and its 3¹-demethyl derivative were prepared by modification of naturally occurring chlorophyll-b. The former complex was a 3¹-epimeric mixture and separated to pure stereoisomers by HPLC, while such a separation was unnecessary for the latter possessing the 3-hydroxymethyl group. The absolute configuration at the 3¹ -position of the separated samples was determined by transformation of the 7-formyl to the 7-methyl group because the 3¹ -stereochemistry of zinc methyl bacteriopheophorbide-d (3-(1-hydroxyethyl)-7-methyl-chlorin) has been confirmed. All the synthetic zinc chlorins self-aggregated in 0.5% (v/v) THF-hexane as well as in 6% (v/v) THF-water to form oligomers which absorbed longer-wavelength lights by the J-aggregation than the monomers in THF. Diastereomeric control in the self-aggregation of the 3¹-epimers was observed. The 3¹-demethyl compound interacted more tightly in the self-aggregates than the 3¹-epimers. Comparing with the corresponding 7-methyl analogs, zinc 7-formyl-chlorins have red-shifted Soret and blue-shifted Q(y) bands in both the monomeric and aggregated states. Visible, circular dichroism and resonance Raman spectra showed that the supramolecular structures of self-aggregates of the 7-formyl and 7-methyl derivatives were similar and highly ordered, and were built-up by 13-C = O···H(3-CHR)O···Zn and π-π interaction of the chlorin chromophores. The coordinatable 7-formyl group did not disturb the special bondings of the composite molecules in the self-aggregates. (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0040-4020(00)00590-1

TETRAHEDRON
Pergamon
Tetrahedron 56 (2000) 6245±6257
Synthesis and Self-Assembly of Zinc Methyl
Bacteriopheophorbide-f and its Homolog
²
*
Hitoshi Tamiaki, Masanobu Kubo and Toru Oba
Department of Bioscience and Biotechnology, Faculty of Science and Engineering, Ritsumeikan University, Kusatsu, Shiga 525-8577, Japan
Received 11 May 2000; accepted 30 June 2000
AbstractÐZinc methyl bacteriopheophorbide-f, zinc 3-(1-hydroxyethyl)-7-formyl-chlorin, and its 31-demethyl derivative were prepared by
modi®cation of naturally occurring chlorophyll-b. The former complex was a 31-epimeric mixture and separated to pure stereoisomers
by HPLC, while such a separation was unnecessary for the latter possessing the 3-hydroxymethyl group. The absolute con®guration at the
31-position of the separated samples was determined by transformation of the 7-formyl to the 7-methyl group because the 31-stereochemistry
of zinc methyl bacteriopheophorbide-d (3-(1-hydroxyethyl)-7-methyl-chlorin) has been con®rmed. All the synthetic zinc chlorins self-
aggregated in 0.5% (v/v) THF±hexane as well as in 6% (v/v) THF±water to form oligomers which absorbed longer-wavelength lights
by the J-aggregation than the monomers in THF. Diastereomeric control in the self-aggregation of the 31-epimers was observed. The
31-demethyl compound interacted more tightly in the self-aggregates than the 31-epimers. Comparing with the corresponding 7-methyl
analogs, zinc 7-formyl-chlorins have red-shifted Soret and blue-shifted Qy bands in both the monomeric and aggregated states. Visible,
circular dichroism and resonance Raman spectra showed that the supramolecular structures of self-aggregates of the 7-formyl and 7-methyl
derivatives were similar and highly ordered, and were built-up by 13-CvO´´´H(3-CHR)O´´´Zn and p±p interaction of the chlorin
chromophores. The coordinatable 7-formyl group did not disturb the special bondings of the composite molecules in the self-aggregates.
q 2000 Elsevier Science Ltd. All rights reserved.
Introduction
R8, R12, R20, R-groups and the absolute con®guration at the
31-position as shown in the left structure of Fig. 1, affects the
chlorosomal structures and functions5,7 and the ratios
among the composite pigments are easily changed by the
culturing conditions.13±15 Self-assemblies of BChls-c and d
(7-methyl-chlorins, R7ˆMe) were investigated in the
natural and arti®cial systems;5±7 far fewer reports are avail-
able for in vivo and in vitro self-aggregates of BChl-e
(7-formyl-chlorin, R7ˆCHO).16±25 Moreover, self-aggrega-
tion of BChl-f which is the 20-demethyl analog of BChl-e
has never been investigated because of the unavailability of
BChl-f in any bacteria.26
Photosynthetic reaction centers consist of pigment±protein
complexes and the supramolecular structures are similar in
all the present species.1,2 In contrast, several types of light-
harvesting antenna systems exist in plants, algae and
bacteria;3,4 inner- and extramembraneous apparatus, and
pigment±protein and pigment±pigment interaction. Green
bacteria have quite a unique antenna system on the inner
surface of the cytoplasmic membrane and the extra-
membraneous antennae are called chlorosomes.3,5 The
core part is constructed by self-aggregation of chlorophyl-
lous pigments without any protein scaffolds,5±8 which is
completely different from other core parts of structurally
determined antenna systems.9±11 The self-aggregative
chlorophylls in chlorosomes are mixtures of several
homologs of bacteriochlorophyll(BChl)s-c, d and e (see
Fig. 1),12 whereas other antennae usually have a sole struc-
tural formula as the chlorophyllous pigment in an apparatus.
Such a diversity in the molecular structures, variation in R7,
We earlier reported that zinc analogs of naturally occurring
magnesium complexes are good model compounds for
BChls-c and d.6,27±36 Here we report the synthesis of zinc
methyl 31-R- and 31-S-bacteriopheophorbides-f (Zn-1R and
Zn-1S), derivatives of one BChl-f homolog possessing
R8ˆEt and R12ˆMe and the 31-demethyl homolog Zn-2
by modi®cation of chlorophyll-b which is easily available
from spinach, and the self-aggregation of the synthetic zinc
31-hydroxy-7-formyl-131-oxo-chlorins Zn-1/2 in a non-
polar organic solvent as well as in an aqueous organic
solution. We particularly investigated the supramolecular
structures of self-aggregates of the synthetic zinc 7-formyl-
chlorins compared with those of the corresponding 7-methyl
analogs by their visible, circular dichroism, and resonance
Raman spectra.37
Keywords: aggregation; chlorophyll; stereochemistry; supramolecular
chemistry.
* Corresponding author. Fax: 181-77-561-2659;
e-mail: tamiaki@se. ritsumei.ac.jp
Present address: Department of Applied Chemistry, Faculty of Engineer-
²
ing, Utsunomiya University, Utsunomiya, Tochigi 321-8585, Japan
0040±4020/00/$ - see front matter q 2000 Elsevier Science Ltd. All rights reserved.
PII: S0040-4020(00)00590-1

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H. Tamiaki et al. / Tetrahedron 56 (2000) 6245±6257
Figure 1. Chlorosomal bacteriochlorophylls (left) and their model zinc complexes (right).
Results and Discussion
of 7-formyl-chlorin 5d. Heating of 3-vinyl-chlorin 5b in
30% HBr±AcOH for 3 h and successive treatment by ice
water and diazomethane gave desired methyl 71-hydroxy-
bacteriopheophorbide-d (6) possessing 3-CH(OH)Me in
10±20% yield after puri®cation by silica gel chromato-
graphy. The yield was lower than that in similar hydration
of 3-vinyl-7-methyl-chlorin 5a (50%40±86%33). The other
products gave no proton signal characteristic of 7-CH2OH at
around dˆ5.7 ppm in the 1H NMR spectra, indicating
that the 71-position was also attacked by HBr or AcOH
under the reaction conditions. To suppress formation of
the by-products, the hydration conditions were changed to
be mild; the reaction temperature in HBr±AcOH was
lowered. Reaction of 3-vinyl-7-hydroxymethyl-chlorin 5b
at 208C for 12 h gave the corresponding 3-(1-hydroxy-
ethyl)-chlorin 6 more ef®ciently (42%).
Synthesis of 31-hydroxy-7-formyl-chlorins
Preparation of methyl bacteriopheophorbide-f (1, see Fig. 2)
has been reported by Risch et al.38 and Simpson and Smith.39
Both the synthetic routes were based on hydration of the
3-vinyl group (R3ˆCHvCH2) in methyl pyropheo-
phorbide-b (5d) by treatment of hydrogen bromide in acetic
acid and successive hydrolysis of the resulting bromide. In
the former, heating at 1808C for 8 min was required and the
latter report gave no detailed procedures. First, we examined
the synthetic procedures described in the former report38 but
could not ®nd desired 1 in the reaction mixture. For the
hydration, we next used procedures similar to those used
in hydration of the 3-vinyl group in methyl pyropheo-
phorbide-a (5a, heating at 558C for 3 h),40 but again were
unable to get 1. Both the reaction mixtures after hydration
were very complex, and no proton signals characteristic of
the 7-formyl group were observed at around dˆ11 ppm in
the 1H NMR spectra of either mixture. These results indicate
that the 7-CHO reacted with HBr or AcOH under the above
conditions. Therefore, we attempted hydration of the
3-vinyl group in methyl 71-hydroxy-pyropheophorbide-a
(5b) possessing 7-CH2OH which was a synthetic precursor
Methyl 71-hydroxy-pyropheophorbide-a (5b) was given by
transformation of the phytyl ester in 71-hydroxy-pyropheo-
phytin-a (see left structure in top row of Scheme 1) which
was prepared by pyrolysis of 71-hydroxy-pheophytin-a
easily isolated from spinach extracts treated with
t-BuNH2BH3.41 The transesteri®cation by H2SO4 in MeOH
initially gave methyl ester 5b and the prolonged reaction
also afforded 7-CH2OMe in 5c. Over-reaction could be
avoided by careful check of the reaction mixture. However,
methyl 71-hydroxy-bacteriopheophorbide-d (6) could also
be prepared by reaction of 71-hydroxy-pyropheophytin-a
with HBr±AcOH at 208C followed by H2O and CH2N2
(see Scheme 1), because the phytyl ester was hydrolyzed
under the acidic conditions. The successive reactions were
simpler than the separated reactions (phytyl!methyl ester
and 3-vinyl!3-(1-hydroxyethyl)) and the yield of the
former (43%) was higher than the overall yield of the latter
(35%). The 7-hydroxymethyl group in 6 was selectively
oxidized by pyridinium dichromate (PDC)26 to give the
corresponding 7-formyl-chlorin 1 (79%). The selective
oxidation was dependent upon the reaction time. Prolonged
reaction induced oxidation of the 1-hydroxyethyl at the
3-position to afford 3-acetyl-7-formyl-chlorin 5h. The
process of the oxidation must be checked by measurement
of visible spectra of the reaction mixture; the Soret
peak moved from 420 nm to 440 nm by oxidation on the
Figure 2. Methyl bacterio- and pyropheophorbides.

H. Tamiaki et al. / Tetrahedron 56 (2000) 6245±6257
6247
Scheme 1. Synthesis of zinc methyl bacteriopheophorbide-f (Zn-1) and its homolog (Zn-2): (i) PDC; (ii) H2SO4/MeOH; (iii) HBr/AcOH, CH2N2; (iv)
HOCH2CMe2CH2OH±p-TsOH; (v) OsO4±NaIO4, t-BuNH2BH3; (vi) HCl; (vii) Zn(OAc)2.
7-position and the Qy peak moved from 650 nm to 680 nm
by oxidation on the 3-position.
prolonged reaction time. The synthetic route initiated by
oxidation via 5d is more effective for preparation of 1
from 71-hydroxy-pyropheophytin-a in both procedures and
total yield (41%ˆ0.75£0.88£0.62£100) than the route
initiated by hydration via 6 (34% yieldˆ0.43£0.79£100).
Mild addition of HBr to the 3-vinyl group in 7-hydroxy-
methyl-chlorin was effective for the preparation of 1
described above. Then, we attempted a similar mild
hydration of the 3-vinyl-7-formyl-chlorin. Methyl pyro-
pheophorbide-b (5d) was treated with 30% HBr±AcOH at
208C for 15 h, followed by water and diazomethane to give
1 in 62% yield. The reaction at 208C was effective for addi-
tion of HBr to the 3-vinyl group in any chlorins possessing a
reactive functional group. Methyl ester 5d was easily
synthesized by transformation (H2SO4 in MeOH) of the
phytyl ester in pyropheophytin-b (88%) which was readily
prepared by oxidation (PDC in C6H6) of 71-hydroxy-pyro-
pheophytin-a (75%). Neither the oxidation nor the trans-
esteri®cation afforded any over-reacted products for a
Finally, hydration of pyropheophytin-b by treatment of 30%
HBr±AcOH at 208C for 15 h, followed by H2O and CH2N2
was examined and gave 1 in 55% yield. The yield of the
direct hydration in the phytyl ester is the same as the overall
yield via methyl ester 5d (55%ˆ0.88£0.62£100, vide
supra). The former synthetic procedures was simpler than
the latter. Therefore, preparation of 1 by initial oxidation of
the 71-hydroxy-pyropheophytin-a (from spinach) followed
by acidic hydration (and concomitant acidic hydrolysis of
phytyl ester) and successive methyl-esteri®cation of the
resulting pyropheophytin-b was the best of the synthetic

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H. Tamiaki et al. / Tetrahedron 56 (2000) 6245±6257
routes examined here. The addition of HBr to the 3-vinyl
group was non-diastereoselective, which led to formation of
of desired 3-hydroxymethyl-chlorin 2 (major) and the regio-
isomeric 7-hydroxymethyl-chlorin 5f (minor).
1
an epimeric mixture of 31R (1R)/31S (1S)ˆ1/1 (from H
NMR spectral analysis). It is noted that benzene as the
reaction solvent is important for oxidation of the 7-hydro-
xymethyl group by PDC; any by-products (not determined)
which were absorbed at around 705 nm were produced for
oxidation in dichloromethane and could not be separated
from the desired 7-formyl-chlorin. Moreover, all the reac-
tions must be done in the dark; the light promoted formation
of any by-products in HBr-addition.
We then adopted the following synthetic route for prepara-
tion of 2: transformation of the 3-vinyl to the 3-hydroxy-
ethyl group after protection of the 7-formyl group.
The formyl group at the 7-position of methyl pyropheo-
phorbide-b (5d) was protected with alcohols by action of
an acid to give the acetal. When methanol was used as the
alcohol, the resulting dimethyl acetal was so unstable39 that
the acetal bonds were completely cleaved during a usual
workup such as washing the dichloromethane solution
with water. In the case of protection by propylene glycol,
the cyclic acetal was slightly deprotected in the workup. The
acetal by neopentyl glycol is stable43 enough to be puri®ed
by the workup and silica gel chromatography and the
protected acetal 5dP was given in 99% yield (see Scheme
1). Oxidation of the vinyl group at the 3-position of the 5dP
by OsO4±NaIO4 in aq. AcOH and THF gave the formyl
group and concomitantly there was partial deprotection of
the acetal. To suppress the undesired deprotection, the
reaction must be quenched with water before the oxidation
has been completed. The reaction mixture contained three
chlorins: 3-formyl-7-acetal-protected-formyl 5gP:3,7-difor-
myl 5g:3-vinyl-7-acetal-protected-formyl 5dPˆ72:16:12
Synthetic chlorin 1 is a 31-epimeric mixture and 1R and 1S
must be separated to get a pure sample. To avoid the separa-
tion, the 31-demethyl derivative 2 was prepared. Analog 2
has a hydroxymethyl group at the 3-position and the 31-
carbon is not asymmetric. Since we have already reported
that a similar 3-hydroxymethyl-chlorin 4 (7-methyl analog
of 2) is a good model for several naturally occurring esters of
bacteriopheophorbides-c and d possessing 3-CH(OH)Me,29
2 must be useful as a model of 1 (vide infra). To prepare
analog 2, we ®rst tried to oxidize selectively 3,7-
bis(hydroxymethyl)-chlorin 5e which was readily afforded
by transformation42 of the 3-vinyl group of 5b to the
3-hydroxymethyl group via the 3-formyl group as in 5f.
Treatment of the diol 5e with PDC gave a mixture of desired
7-formyl-chlorin 2 (major) and the regioisomeric 3-formyl-
chlorin 5f (minor) as the single oxidation products. These
regioisomers were dif®cult to separate. Next, we examined
the selective reduction of 3,7-diformyl-chlorin 5g prepared
by oxidation of 3-formyl-7-hydroxymethyl-chlorin 5f (the
intermediate in the synthesis of the above diol 5e, vide
supra) but the reduction by t-BuNH2BH3 gave a mixture
1
from the H NMR spectral analysis (see Scheme 2). These
compounds could not be separated easily and the mixture
was used for the following reduction without further separa-
tion. The mixture was reduced by t-BuNH2BH3 to afford a
mixture of 3-hydroxymethyl-7-acetal-protected-formyl-,
3,7-bis(hydroxymethyl)- and 3-vinyl-7-acetal-protected-
formyl-chlorins (2P, 5e and 5dP) which were easily
separated by silica gel chromatography because of the
Scheme 2. Synthesis of mono-alcohol 2P by modi®cation of 3-vinyl-chlorin 5dP: (i) OsO4±NaIO4; (ii) t-BuNH2BH3.

H. Tamiaki et al. / Tetrahedron 56 (2000) 6245±6257
6249
Figure 3. HPLC chromatogram of a 1:1 31-epimeric mixture of zinc methyl bacteriopheophorbide-f (Zn-1R/S); Cosmosil 5C18-AR, 6 mm f£250 mm,
Nacalai Tesque, MeOH/H2Oˆ4/1, 1.0 mL/min.
1
difference in the number of hydroxyl groups in the
molecule. The acetal protection of the separated mono-
alcohol 2P was cleanly cleaved by stirring aq. 1% HCl in
acetone to give desired 3-hydroxymethyl-7-formyl-
chlorin 2.
characterized by their H NMR, visible, infra-red and mass
spectra.
Separation and structure determination of 31-epimeric
chlorins
The metal-free chlorins 1 and 2 were zinc-metallated30 to
give zinc complexes Zn-1 and Zn-2, respectively, after
puri®cation by HPLC. All the synthetic chlorins were
Risch and his colleagues have reported38 that a 31-epimeric
mixture of metal-free chlorin 1 could be separated by
reversed-phase HPLC and the absolute con®guration was
Figure 4. HPLC chromatograms of (A) a reaction mixture prepared by reduction of epimerically pure zinc methyl bacteriopheophorbide-f (Zn-1, 138-min
eluted band in Fig. 3) with NaCNBH3 in MeOH and (B) 1:1 31-epimeric mixtures of zinc methyl 71-hydroxy-, 71-methoxy- and 71-unsubstituted-bacterio-
pheophorbides-d (Zn-6, Zn-7 and Zn-3).

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H. Tamiaki et al. / Tetrahedron 56 (2000) 6245±6257
Figure 5. Visible (A) and CD spectra (B) of zinc methyl 31R-bacteriopheophorbide-f (Zn-1R, solid line) and visible spectra of zinc methyl 31R-bacterio-
pheophorbide-d (Zn-3R, broken line of (A)) in THF.
estimated by the 1H NMR spectra of the ester derivatives of
a N-protected chiral phenylalanine; comparison of the
chemical shifts at the meso-protons in the d-Phe-1 deriva-
tive was made with those in the corresponding ester of
structurally determined 31-epimeric methyl bacteriopheo-
phorbide-d. According to the reported procedures (an
ODS column, MeOH/H2Oˆ4/1),38 we tried to separate a
1:1 mixture of 1 but obtained ill-resolved bands by a
single run of HPLC; the separation ratio was 0.5 at most.
A 31-epimeric mixture of zinc complex Zn-1 was partially
separated after a single HPLC run (Cosmosil 5C18-AR,
6 mm f£250 mm, Nacalai Tesque, MeOH/H2Oˆ2/1,
1.5 mL/min); the retention times were 132 and 138 min
and the separation ratio was about 0.8 (see Fig. 3). After
the repeated runs, each 31-epimer was obtained in pure
form. To determine the absolute con®guration of the
asymmetric 31-position unambiguously, the 31-epimerically
separated zinc chlorins Zn-1 were transferred to stereo-
chemistry-determined zinc methyl bacteriopheophorbide-d
(Zn-3).33 According to the procedures reported by
Scheumann and his colleagues,44 the second HPLC-eluted
epimer of Zn-1 was reduced by NaCNBH3 in methanol to
give three products as shown in a HPLC chromatogram of
the reaction mixture (Fig. 4A). Comparison with the
authentic samples (Fig. 4B) showed the 19-min band was
7-hydroxymethyl-chlorin Zn-6, the 27-min band was
7-methoxymethyl-chlorin Zn-7, and the 43-min was zinc
methyl 31S-bacteriopheophorbide-d (Zn-3S). Therefore,
the 31-con®guration of the ®rst (second) eluted band in
Zn-1 was determined to be R(S)-form. During the reduction,
no epimerization was observed and the R-epimers of all the
zinc chlorins examined were eluted faster than the corre-
sponding S-epimers under the HPLC conditions.
as an axial ligand to give a penta-coordinated zinc complex.
All three complexes Zn-1R, Zn-1S and Zn-2 gave almost
the same visible spectra in THF (solid line of Fig. 5A).
Sharp bands were obtained in 628 nm (Qy peak) and 448
(Soret peak), indicating that these compounds were mono-
meric in comparison with reported data of BChl-e.45 The Qy
and Soret peaks of zinc 7-formyl-chlorins are shifted to
shorter and longer wavelengths, respectively, than those
(645 and 423 nm) of the corresponding zinc 7-methyl-
chlorins Zn-3R, Zn-3S and Zn-4 in THF (broken line of
Fig. 5A). These shifts are observed in naturally occurring
chlorophylls: Chl-b (7-CHO, 644 and 455 nm) and Chl-a
(7-Me, 662 and 430 nm) in diethyl ether; BChl-e (7-CHO,
647 and 458 nm) and BChl-c (7-Me, 660 and 428 nm) in
acetone.46 Interestingly, the blue (red) shift in the Qy (Soret)
peak by Zn-3R (7-Me)!Zn-1R (7-CHO) is 420
(1300) cm21 which is the same as the shifted value in
Chl-a!Chl-b, and the consistency is due to the structural
similarity that all four metal-chlorins are unsubstituted at
the 20-position. The ratios of the peak intensities in
Zn-1R, Zn-1S and Zn-2 were 0.34 for Abs(Qy)/Abs(Soret),
while the ratios in Zn-3R, Zn-3S and Zn-4 were 0.77.
About half this ratio and the blue (red) shift in the Qy
(Soret) peak are characteristic features in visible spectra
of 7-formyl-chlorins, compared with those of 7-methyl-
analogs.
The above observation in visible spectra was made with
electronic spectra estimated by modeling calculation.
Calculation of THF-coordinating Zn-1R by succes-
sive MM1 and ZINDO/S (HyperChem 5.1)34 gave the
same wavelengths in allowed excitation (S0!Sn) as those
of Zn-1S and Zn-2. The ZINDO/S calculation based on
the molecular structure of THF´Zn-1R optimized by
the MM1 calculation predicted a HOMO to LUMO transi-
tion at 625 nm, in good agreement with the experimental
value of 628 nm in THF. The estimated Soret peak position
in the calculation (342 nm) was different from the experi-
mentally observed peak at 448 nm. Theoretically calculated
Zinc chlorins in a polar organic solvent
Synthetic zinc chlorins were highly soluble in polar organic
solvents as tetrahydrofuran (THF), pyridine and so on.
These solvent molecules coordinated with the central zinc

H. Tamiaki et al. / Tetrahedron 56 (2000) 6245±6257
6251
Figure 6. Fluorescence (excited at 448 nm, solid line) and visible spectra (broken line) of zinc methyl 31R-bacteriopheophorbide-f (Zn-1R) in THF.
shifts in the peaks by change of 7-Me to 7-CHO were quite
consistent with the experimental shifts; calculated and
measured red(blue)-shifts in Qy(Soret) peak were
350(1100) and 420(1300) cm21, respectively. The decrease
in the ratio of observed peak intensities by 7-Me!7-CHO
(0.77!0.34, see Fig. 5A) was also qualitatively predict-
able from the MM1 and ZINDO/S calculation, Int(Qy)/
Int(Soret)ˆ0.18 (7-Me)!0.16 (7-CHO).
Zinc chlorins in a non-polar organic solvent
We earlier reported that zinc 31-hydroxy-131-oxo-chlorins
possessing a methyl group at the 7-position as in Zn-3 and
Zn-4 self-aggregated in non-polar organic solvents to give
red-shifted visible absorption peaks.29,33 In 0.5% (v/v)
THF±hexane, visible bands of Zn-1 and Zn-2 were shifted
to longer wavelengths and were broader than those in THF
(Fig. 7), indicating that the zinc chlorins self-aggregated
even in the presence of the 7-formyl group in the molecule.
Absorption maxima of Qy peaks in self-aggregates of Zn-1
and Zn-2 possessing 7-CHO (673/677 and 714 nm) are situ-
ated at shorter wavelengths than those of Zn-3 and Zn-4
possessing 7-Me (ca. 700 and 740 nm), respectively. The
blue-shift in Qy peaks of self-aggregates (7-Me!7-CHO)
is ascribable to the blue-shift in the monomeric peaks
described above. Self-aggregates of Zn-1R showed slightly
less red-shifted and a sharper Qy peak (lmaxˆ673 nm,
Dˆ1250 cm21, broken line of Fig. 7) than those of the
epimeric Zn-1S (677 nm, 1530 cm21, dotted line of Fig.
7). Such a diastereoselective control in self-aggregation
has been reported in other 31-epimers as in Zn-3R and
Zn-3S.28,32,33 Zinc 3-hydroxymethyl-chlorin Zn-2 in 0.5%
(v/v) THF±hexane showed a more red-shifted Qy band at
714 nm (thick solid line of Fig. 7) than the corresponding
3-1-hydroxyethyl analogs Zn-1. The demethylation at the
31-position induced less steric repulsion and stronger
interaction among the composite molecules in the self-
aggregates because the hydroxyl group is a connecting
Synthetic zinc chlorin Zn-1R in THF gave small negative
and positive CD peaks in the Qy and Soret regions, respec-
tively (see Fig. 5B). When Zn-1R in THF was excited in the
Soret region, intense ¯uorescence lights were emitted at the
region of .600 nm and the emission spectrum was a mirror
image of the absorption spectrum (Fig. 6). Both CD spectra
and ¯uorescence emission spectra (excited at 500 nm) of
Zn-1R, Zn-1S and Zn-2 in THF are almost the same.
An additional methyl group and the stereochemistry at the
31-position scarcely affected CD or ¯uorescence spectra as
well as visible spectra in THF.
Resonance Raman spectra in THF (excited at 458 nm)
showed that all the monomers gave two peaks at 1700±
1690 and 1665±1660 cm21 in the region of 1800 to
1600 cm21 (vide infra). The former is assigned to a
13-CvO stretching vibrational band and the latter to
7-CHO stretching, comparing with reported data.20,27
These values indicate that the two carbonyl groups are
free from any interaction.
Figure 7. Visible spectra of zinc methyl 31R-bacteriopheophorbide-f (Zn-1R, broken line), 31S-bacteriopheophorbide-f (Zn-1S, dotted line), and 31-demethyl-
bacteriopheophorbide-f (Zn-2, thick solid line) in 0.5% (v/v) THF±hexane and Zn-2 in THF (thin solid line).

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H. Tamiaki et al. / Tetrahedron 56 (2000) 6245±6257
Figure 8. Visible spectra of zinc methyl 31R-bacteriopheophorbide-f (Zn-1R, A), 31S-bacteriopheophorbide-f (Zn-1S, B), and 31-demethyl-bacteriopheo-
phorbide-f (Zn-2, C) and CD spectra of Zn-1R (D), Zn-1S (E), and Zn-2 (F) in 6% (v/v) THF±water.
group as in 13-CvO´´´HO´´´Zn.29 Strong interaction and
high oligomerization of Zn-2 moved the Qy peak to a longer
wavelength and the red-shift by the self-aggregation was
1890 cm21. The value was comparable to that in Zn-4
(1990 cm21),29 indicating that the self-aggregates of zinc
31-hydroxy-7-formyl-131-oxo-chlorins would be similar to
those of the 7-methyl analogs.
solvents are similar to the in vivo and in vitro self-
aggregates of BChl-e.16 This means that the self-aggregates
of synthetic zinc chlorins Zn-1/2 would be good models for
natural chlorosomal aggregates of BChl-e. The self-
aggregates of 7-formyl-chlorins have intense absorbance
at around 500 nm which is the region absorbed by caro-
tenoids in a usual photosynthetic apparatus. This would
affect the content and variation of carotenoids in BChl-e
containing chlorosomes (and also living cells) of
anoxygenic photosynthetic brown-colored bacteria, e.g.
Chlorobium phaeobacteroides47,48 and phaeovibrioides.
Self-aggregates of Zn-1/2 have Soret bands in the region of
.500 nm, which are situated at longer wavelengths than
those of Zn-3/4 (lmaxˆ440±450 nm).29,33 The red-shift in
the oligomeric Soret peak is due to the red-shift in the
monomeric peak. As the oligomeric Soret band, typically,
Zn-2 absorbed the lights at the wide region of 400±550 nm
and at least two peaks were observed at 422 and 509 nm, as
estimated from the second derivative of the visible spec-
trum. A relatively large component at around 450±470 nm
in Zn-1 would be due to residual monomer in the solution,
which also shows less aggregativity of Zn-1 than of Zn-2
described above. Such a broad Soret band with two peaks is
characteristic of self-aggregates of zinc 7-formyl-chlorins.
Zinc chlorins in an aqueous organic solution
Solubility of Zn-1/2 in non-polar organic solvents is not so
high, and the concentrated solution easily changed to be
heterogeneous after the preparation and gave some precipi-
tates as in recrystallization. In aqueous organic solutions,
zinc chlorins self-aggregate true in non-polar organic
solvents.42,49 In 6% (v/v) THF±water,50 Zn-1 and Zn-2
gave similar red-shifted Qy peaks at 671 and 707 nm,
respectively, (Fig. 8A±C) as in 0.5% (v/v) THF±hexane.
These Qy bands of self-aggregates in the aqueous solution
These absorption spectra of Zn-1/2 in non-polar organic

H. Tamiaki et al. / Tetrahedron 56 (2000) 6245±6257
6253
Figure 9. Fluorescence spectra (excited at 500 nm) of zinc methyl 31R-bacteriopheophorbide-f (Zn-1R, broken line), 31S-bacteriopheophorbide-f (Zn-1S,
dotted line), and 31-demethyl-bacteriopheophorbide-f (Zn-2, solid line) in 6% (v/v) THF±water.
were sharper than those in non-polar organic solvents. In the
red-shifted Qy band region, large CD bands were observed
(Fig. 8D±F); a large band at a longer wavelength and a
small band at a shorter wavelength. The zero-crossing
points in the CD spectra are almost the same as the wave-
lengths of the Qy absorption maxima. Zn-1R gave a reverse
S-shaped CD spectrum and Zn-1S gave an S-shaped CD
spectrum. The difference in the 31-epimers is attributed to
diastereoselective control in the self-aggregation.
also due to the diastereoselective controlling aggregation in
the 31-epimers.
The CD couplet of Zn-2 in the Qy region is very similar to
that of Zn-1R (Fig. 8D and F), indicating that both Zn-1R
and Zn-2 give similar supramolecular structures in the self-
aggregates. Comparing the shapes of visible bands in the
Soret region (Fig. 8A and B), Zn-1S produced more residual
monomer absorbed at around 460 nm than Zn-1R in the
aqueous solution. Therefore, Zn-1R self-aggregated to
form more stable supramolecules than Zn-1S. The ¯uores-
cence spectrum of Zn-2 gave predominantly a 721-nm
band without an emission peak at around 650 nm from
the monomer (solid line of Fig. 9), indicating that Zn-2
self-aggregated more strongly than Zn-1 and left no residual
monomer in the solution.
The ¯uorescence spectrum of Zn-1R in the aqueous
solution (excited at 500 nm) shows 647- and 695-nm
peaks emitted from monomer (or small aggregates) and
aggregates, respectively (broken line of Fig. 9) and self-
aggregates of Zn-1S gave a longer wavelength emission
peak at 709 nm (dotted line of Fig. 9). This difference is
Figure 10. Resonance Raman spectra (excited at 458 nm) of zinc methyl 31-demethyl-bacteriopheophorbide-f (Zn-2) in THF (A) and in 6% (v/v) THF±water
(B).

6254
H. Tamiaki et al. / Tetrahedron 56 (2000) 6245±6257
Resonance Raman spectra (excited at 458 nm) of Zn-2 were
recorded (Fig. 10) to elucidate the supramolecular structure
of the self-aggregates. In THF, monomeric Zn-2 gave two
bands at 1698 and 1665 cm21 (Fig. 10A) assignable to
13-CvO and 7-CHO stretching vibrations, respectively,
both of which were free from any interaction (vide supra).
In 6% (v/v) THF±water, no Raman peak of the 13-carbonyl
group at 1700±1690 cm21 was observed in the aqueous
solution (Fig. 10B), indicating that the 13-CvO interacted
with any functional group in the self-aggregates of Zn-2. A
broad Raman band at 1680±1640 cm21 was observed in
self-aggregated Zn-2 and was composed of two bands at
1663 and 1652 cm21 from the band-®tting analysis. We
have reported that self-aggregates of Zn-4 in an aqueous
solution gave a 1650-cm21 Raman band for the 13-CvO
which was specially hydrogen-bonded with 31-OH as in
13-CvO´´´HO´´´Zn.35 Based on the similarity in red-shifts
of Qy peaks by self-aggregation of Zn-2 and Zn-4 (vide
supra), the Raman band at 1652 cm21 is proposed for
specially hydrogen-bonded 13-keto-carbonyl stretching in
self-aggregated Zn-2. The other band at 1662 cm21 is
attributed to the stretching of the 7-formyl group, indicating
that the 7-CHO in the self-aggregates is free from any inter-
action. These assignments in the arti®cial self-aggregates
are consistent with the previous report on natural chloro-
somal aggregates of BChl-e.20 Therefore, a coordinatable
formyl group at the 7-position in Zn-2 would not interact
with any other functional groups of the other molecules in
the self-aggregates, and did not disturb the formation of
highly ordered supramolecules which were built up mainly
by 13-CvO´´´H(3-CHR)O´´´Zn and p±p interaction of the
chlorin chromophores.
washed with brine and dried over Na2SO4. The solvents
were evaporated and the residue was puri®ed by FCC
(2.5% Et2O±CH2Cl2) and recrystallization from CH2Cl2
and MeOH to give pyropheophytin-b (13.8 mg, 75%); see
Ref. 41 for the spectral data.
Synthesis of methyl pyropheophorbide-b (5d)
Pyropheophytin-b (44 mg) was dissolved in a small amount
of dichloromethane (ca. 2 mL), to which methanol (72 mL)
was added. To the ice-chilled solution, an ice-cooled
methanol solution (4 mL) of sulfuric acid (1 mL) was
added dropwise under N2. After 16-h stirring at room
temperature, the reaction mixture was poured into ice-
water and CH2Cl2, washed with aqueous 4% NaHCO3
solution and dried over Na2SO4. Dichloromethane was
evaporated to give 5d (20 mg, 88%); brown solid; mp
218±2198C (lit.38 245±2468C); VIS (CH2Cl2) lmaxˆ656
(relative intensity, 0.21), 601 (0.07), 530 (0.09), 440 nm
1
(1.00); H NMR (CDCl3) dˆ10.86 (1H, s, CHO), 9.97,
9.07, 8.47 (each 1H, s, 5-, 10-, 20-H), 7.88 (1H, dd, Jˆ12,
18 Hz, 3-CH), 6.30 (1H, dd, Jˆ1, 17 Hz, 31-CH-trans to
C31-H), 6.17 (1H, dd, Jˆ1, 12 Hz, 31-CH-cis to C31-H),
5.19, 5.03 (each 1H, d, Jˆ20 Hz, 131-CH2), 4.45 (1H, dq,
Jˆ2, 7 Hz, 18-H), 4.25 (1H, dt, Jˆ9, 2 Hz, 17-H), 3.64 (2H,
q, Jˆ6 Hz, 8-CH2), 3.66, 3.45, 3.35 (each 3H, s, 2-, 12-CH3,
COOCH3), 2.55±2.70, 2.15±2.40 (each 2H, m, 17-
CH2CH2), 1.87 (3H, d, Jˆ7 Hz, 18-CH3), 1.61 (3H, t,
Jˆ8 Hz, 81-CH3), -0.01, -1.98 (each 1H, s, NH). MS
(FAB) found: m/z 563. Calcd for C34H35N4O4: MH1, 563.
Synthesis of methyl bacteriopheophorbide-f (1)
Via hydration of 71-hydroxy-pyropheophytin-a. 71-
Hydroxy-pyropheophytin-a (22 mg) was dissolved in 30%
hydrogen bromide in acetic acid (30 mL) and then stirred at
208C under N2 for 15 h. CAUTION, reaction in total
darkness is very important to suppress formation of any
undesired products. The solution was poured into ice-
water, extracted with CHCl3, washed with brine, and dried
over Na2SO4. The solvent was evaporated and the residue
was treated with excess ethereal CH2N2 solution at 08C
under N2. After 2-h stirring at room temperature, the solu-
tion was poured into brine, washed with brine, and dried
over Na2SO4. The solvent was evaporated and the residue
was puri®ed by FCC (1.7% MeOH±CH2Cl2) to give
31-epimeric mixtures of methyl 71-hydroxy-bacteriopheo-
phorbide-d (6, 6.7 mg, 43% yield, (31R)/(31S)ˆ1/1); black
solid (from CH2Cl2±hexane); mp 1328C; VIS (CH2Cl2)
lmaxˆ656 (rel. 0.38), 600 (0.07), 539 (0.07), 508 (0.09),
415 nm (1.00); 1H NMR (CDCl3) dˆ9.86/84, 9.44/43,
8.50/49 (each 1H, s, 10-, 5-, 20-H), 6.42/40 (1H, q, Jˆ
7 Hz, 3-CH), 5.74 (2H, s, 7-CH2), 5.16, 5.01 (each 1H, d,
Jˆ20 Hz, 131-CH2), 4.43 (1H, dq, Jˆ2, 8 Hz, 18-H), 4.21
(1H, dt, Jˆ9, 2 Hz, 17-H), 3.75 (2H, q, Jˆ7 Hz, 8-CH2),
3.62/61, 3.59/58, 3.40/38 (each 3H, s, 2-, 12-CH3,
COOCH3), 2.50±2.65, 2.20±2.35 (each 2H, m, 17-
CH2CH2), 2.13 (3H, d, Jˆ7 Hz, 31-CH3), 1.78 (3H, t, Jˆ
7 Hz, 81-CH3), 1.70 (3H, d, Jˆ8 Hz, 18-CH3), 0.19 (1H, br,
NH), -1.89 (1H, s, NH). MS (FAB) found: m/z 583. Calcd
for C34H39N24O5: MH1, 583.
Experimental
General
All of the apparatus used was described in our previous
reports.51,52 Chemical shifts (d) of H NMR spectra are
1
expressed in parts per million relative to CHCl3
(7.26 ppm) or CHD2OD (3.30 ppm) as an internal reference.
Zinc metallation of chlorins was done by reported pro-
cedures.30 THF and benzene were distilled from CaH2
before use. Flash column chromatography (FCC) was
performed with silica gel (Merck, Kieselgel 60, 9385).
HPLC was performed with a packed ODS column (Gelpack
GL-OP100, 6 mm f£150 mm, Hitachi Chemical Co. or
Cosmosil 5C18-AR, 6 mm f£250 mm, Nacalai Tesque).
Solvents for visible, CD and ¯uorescence spectra were
purchased from Nacalai Tesque (Grade for UV-spectro-
scopy). All synthetic procedures must be done in the dark!
Synthesis of pyropheophytin-b
According to procedures reported by Oba and his
colleagues,41 71-hydroxy-pyropheophytin-a was prepared
from spinach. Pyridinium dichromate (20 mg) was added
to a dry benzene solution (20 mL) of 71-hydroxy-pyropheo-
phytin-a (18.4 mg) under N2. After 3-h stirring at room
temperature, diethyl ether (20 mL) was added to the
reaction mixture, stirred and ®ltered. The ®ltrate was
To a dry benzene (20 mL) solution of 6 (6.7 mg) was added

H. Tamiaki et al. / Tetrahedron 56 (2000) 6245±6257
6255
pyridinium dichromate (22 mg) and the mixture was stirred
at room temperature under N2 for 3 h. Diethyl ether (40 mL)
was added to the mixture, stirred and ®ltered. The ®ltrate
was washed with brine and dried over Na2SO4. After
evaporation, the residue was puri®ed by HPLC (retention
timeˆ8.4 min, Cosmosil, MeOH, 1.0 mL/min) to give
31-epimeric mixtures of 138 (5.3 mg, 79% yield, (31R)/
(31S)ˆ1/1); brown solid (from CH2Cl2±hexane); mp
1118C; VIS (CH2Cl2) lmaxˆ649 (rel. 0.22), 594 (0.07),
Tamiaki and his colleagues,30 the above ketal 5dP
(23 mg) was oxidized for 23 h by OsO4 and NaIO4 to give
a mixture (22.7 mg) of the corresponding 3-formyl-chlorin
5gP [typical dˆ11.64 (1H, s, CHO), 10.72, 9.58, 8.81 (each
1H, s, 5-, 10-, 20-H)]: the over-reacted 3,7-diformyl-chlorin
5g [typical dˆ11.67, 11.26 (each 1H, s, CHO), 10.93, 9.76,
8.83 (each 1H, s, 5-, 10-, 20-H)]: unreacted 3-vinyl-chlorin
5dPˆ72: 12: 16 determined from the 1H NMR analysis. The
crude mixture without FCC puri®cation was used for the
following reduction.
1
524 (0.08), 436 (1.00), 415 nm (0.45); H NMR (CDCl3)
dˆ11.06 (1H, s, CHO), 10.45/44, 9.42, 8.51 (each 1H, s,
5-, 10-, 20-H), 6.52 (1H, q, Jˆ7 Hz, 3-CH), 5.22, 5.07 (each
1H, d, Jˆ20 Hz, 131-CH2), 4.47 (1H, dq, Jˆ2, 7 Hz, 18-H),
4.26 (1H, dt, Jˆ9, 2 Hz, 17-H), 3.87/86 (2H, q, Jˆ8 Hz,
8-CH2), 3.64/63, 3.59, 3.44 (each 3H, s, 2-, 12-CH3,
COOCH3), 2.50±2.65, 2.20±2.40 (each 2H, m, 17-
CH2CH2), 2.15 (3H, d, Jˆ7 Hz, 31-CH3), 1.82 (3H, d,
Jˆ7 Hz, 18-CH3), 1.73 (3H, t, Jˆ8 Hz, 81-CH3), 0.33,
-1.72 (each 1H, s, NH). MS (FAB) found: m/z 580. Calcd
for C34H36N4O5: M1, 580.
Also according to procedures similar to those reported by
Tamiaki and his colleagues,30 the above mixture (22.7 mg)
was reduced for 18 h by t-BuNH2BH3 to give a mixture of
3-hydroxymethyl-chlorin 2P, 3,7-bis(hydroxymethyl)-
chlorin 5e and 3-vinyl-chlorin 5dP. The crude mixture
was easily separated by FCC to give 5dP (6% Et2O±
CH2Cl2), 5e (2% MeOH±CH2Cl2, 2 mg) [selected spectral
data, dˆ5.74 and 5.61 (each 2H, s, 3-, 7-CH2) and m/z 568
(M1)], and the desired 2P (1.2% MeOH±CH2Cl2, 13.4 mg;
59% yield for the above two steps); black solid (from
CH2Cl2±hexane); mp 1198C; VIS (CH2Cl2) lmaxˆ655.5
(rel. 0.32), 600 (0.07), 541 (0.07), 509 (0.09), 417 nm
Via hydration of methyl pyropheophorbide-b (5d). Simi-
lar hydration of 5d (5.9 mg) by the above procedures gave
(31R)/(31S)ˆ1/1 mixtures of 1 (3.8 mg, 62% yield) after
puri®cation by FCC (1.0±1.2% MeOH±CH2Cl2).
1
(1.00); H NMR (CDCl3) dˆ10.10, 9.46, 8.51 (each 1H,
s, 5-, 10-, 20-H), 6.56 (1H, s, 7-CH), 5.90 (2H, s, 3-CH2),
5.21, 5.05 (each 1H, d, Jˆ20 Hz, 131-CH2), 4.44 (1H, dq,
Jˆ2, 7 Hz, 18-H), 4.23 (1H, dt, Jˆ9, 2 Hz, 17-H), 4.17, 4.07
(each 2H, d, Jˆ11 Hz, 71-OCH2), 3.82 (2H, q, Jˆ7.5 Hz,
8-CH2), 3.74, 3.60, 3.40 (each 3H, s, 2-, 12-CH3, COOCH3),
2.50±2.70, 2.25±2.40 (each 2H, m, 17-CH2CH2), 1.92, 1.05
(each 3H, s, 74-CH3), 1.79 (3H, d, Jˆ7 Hz, 18-CH3), 1.71
(3H, t, Jˆ7.5 Hz, 81-CH3), 0.10, -1.87 (each 1H, s, NH). MS
(FAB) found: m/z 653. Calcd for C38H45N4O6: MH1, 653.
Via hydration of pyropheophytin-b. Similar hydration of
pyropheophytin-b (9.9 mg) using the above procedures gave
(31R)/(31S)ˆ1/1 mixtures of 1 (3.8 mg, 55% yield) after
puri®cation by FCC (1.0±1.2% MeOH±CH2Cl2).
Synthesis of methyl 3-devinyl-3-hydroxymethyl-
pyropheophorbide-b (2)
3-Hydroxymethyl-7-neopentyl-ketal-protected-formyl-chlorin
2P (13.4 mg) was dissolved in acetone (20 mL), to which
aqueous 1% HCl solution (30 mL) was added. After 15-min
stirring at room temperature under N2, the reaction mixture
was poured into ice-water and dichloromethane, washed
with aqueous 4% NaHCO3 solution and dried over
Na2SO4. The solvents were evaporated and the residue
was puri®ed with HPLC (retention time was 8.3 min,
Cosmosil, MeOH, 1.0 mL/min) to give the titled compound
2 (11.3 mg, 97%); brown solid (from CH2Cl2±hexane); mp
115±1178C; VIS (CH2Cl2) lmaxˆ649.5 (rel. 0.20), 595.5
Methyl pyropheophorbide-b (5d, 20 mg) was dissolved in
dichloromethane (20 mL), to which neopentyl glycol
(22 mg) and p-toluene sulfonic acid (40 mg) were added.
After 3-h stirring at room temperature, the reaction mixture
was poured into pHˆ8.6 buffer solution prepared by
aqueous 0.5 M NaHCO3 (50 mL) and 0.05 M K2CO3
(50 mL) solutions. The separated dichloromethane solution
was washed with aqueous 4% NaHCO3 solution, washed
with brine and dried over Na2SO4. The solvent was evapo-
rated and the residue was puri®ed by FCC (5±6% Et2O±
CH2Cl2) and recrystallization from CH2Cl2 and hexane to
give neopentyl-ketal protected compound 5dP (23 mg,
99%); black solid; mp 112±1138C; VIS (CH2Cl2)
lmaxˆ660 (rel. 0.30), 605 (0.07), 543 (0.07), 512 (0.11),
419 nm (1.00); 1H NMR (CDCl3) dˆ10.10, 9.57, 8.52
(each 1H, s, 5-, 10-, 20-H), 8.01 (1H, dd, Jˆ11, 18 Hz,
3-CH), 6.56 (1H, s, 7-CH), 6.29 (1H, dd, Jˆ1, 18 Hz,
31-CH-trans to C31-H), 6.14 (1H, dd, Jˆ1, 12 Hz, 31-CH-
cis to C31-H), 5.26, 5.10 (each 1H, d, Jˆ20 Hz, 131-CH2),
4.48 (1H, dq, Jˆ2, 7 Hz, 18-H), 4.27 (1H, dt, Jˆ9, 2 Hz,
17-H), 4.16, 4.07 (each 2H, d, Jˆ11 Hz, 71-OCH2), 3.86
(2H, q, Jˆ7.5 Hz, 8-CH2), 3.67, 3.60, 3.39 (each 3H, s, 2-,
12-CH3, COOCH3), 2.50±2.70, 2.25±2.40 (each 2H, m,
17-CH2CH2), 1.85, 0.94 (each 3H, s, 74-CH3), 1.82 (3H, d,
Jˆ7 Hz, 18-CH3), 1.75 (3H, t, Jˆ7.5 Hz, 81-CH3), 0.37,
-1.98 (each 1H, s, NH). MS (FAB) found: m/z 649. Calcd
for C39H45N4O5: MH1, 649.
1
(0.06), 526 (0.07), 435.5 (1.00), 415 nm (0.49); H NMR
(CDCl3) dˆ10.82 (1H, s, CHO), 10.14, 9.07, 8.52 (each 1H,
s, 5-, 10-, 20-H), 5.86 (2H, s, 3-CH2), 5.21, 5.05 (each 1H, d,
Jˆ20 Hz, 131-CH2), 4.47 (1H, dq, Jˆ2, 7 Hz, 18-H), 4.25
(1H, dt, Jˆ9, 2 Hz, 17-H), 3.52 (2H, q, Jˆ7.5 Hz, 8-CH2),
3.65, 3.48, 3.40 (each 3H, s, 2-, 12-CH3, COOCH3), 2.55±
2.70, 2.25±2.40 (each 2H, m, 17-CH2CH2), 1.84 (3H, d,
Jˆ7 Hz, 18-CH3), 1.57 (3H, t, Jˆ7.5 Hz, 81-CH3), 0.07,
-1.84 (each 1H, s, NH). MS (FAB) found: m/z 567. Calcd
for C33H35N4O5: MH1, 567.
Transformation of zinc 7-formylchlorin to zinc 7-
methylchlorin
To a methanol solution (9 mL) of zinc methyl bacteriopheo-
phorbide-f (1, ca. 20 mg) was added NaBH3CN (4 mg) at
room temperature with stirring under N2. One molar
methanol solution of HCl was added dropwise to the
According to procedures similar to those reported by

6256
H. Tamiaki et al. / Tetrahedron 56 (2000) 6245±6257
mixture, which changed the Qy-peak from 635 to 647 nm.
After the complete shift in the visible peak, ethyl acetate and
aqueous 4% NaHCO3 solution was added to the reaction
mixture and the upper organic phase was separated. The
aqueous phase was re-extracted with ethyl acetate until
the solution was decolorized. The combined organic phase
was dried over Na2SO4. The solvent was evaporated and the
residue was zinc-metallated30 to give zinc methyl bacterio-
pheophorbide-d (3) after puri®cation of HPLC (retention
timeˆ39 (31R) and 43 min (31S), Cosmosil, MeOH/
H2Oˆ4/1, 1.0 mL/min).
449 nm (1.00); 1H NMR (CDCl3 Ð 0.2% C5D5N) dˆ11.20
(1H, s, CHO), 10.01, 9.60, 8.22 (each 1H, s, 5-, 10-, 20-H),
5.79 (2H, s, 3-CH2), 5.10, 4.97 (each 1H, d, Jˆ20 Hz, 131-
CH2), 4.29 (1H, dq, Jˆ2, 7 Hz, 18-H), 4.11 (1H, dt, Jˆ9,
2 Hz, 17-H), 4.09 (2H, q, Jˆ7.5 Hz, 8-CH2), 3.63, 3.59, 3.28
(each 3H, s, 2-, 12-CH3, COOCH3), 2.10±2.60 (4H, m, 17-
CH2CH2), 1.79 (3H, t, Jˆ7.5 Hz, 81-CH3), 1.69 (3H, d,
Jˆ7 Hz, 18-CH3). MS (FAB) found: m/z 628. Calcd for
C33H32N4O654Zn: M1, 628.
Acknowledgements
Spectral data
This work was partially supported by Research Grant from
Human Frontier Science Program and Grants-in-Aid for
Scienti®c Research (No. 09480144 and 12020259) from
the Ministry of Education, Science, Sports and Culture,
Japan.
Zinc methyl bacteriopheophorbide-f (31R/31Sˆ1/1) (Zn-
1). 91%; retention time was 8.1 min (Gelpack, MeOH,
1.0 mL/min); light brown solid (precipitated from hexane);
mp .3008C; VIS (THF) lmaxˆ629 (rel. 0.34), 581 (0.07),
448 nm (1.00); 1H NMR (CDCl3 Ð 0.2% C5D5N) dˆ11.22
(1H, s, CHO), 10.18/13, 9.59, 8.18 (each 1H, s, 5-, 10-, 20-
H), 6.41/37 (1H, q, Jˆ6.5 Hz, 3-CH), 5.09, 4.96 (1H, d,
Jˆ20 Hz, 131-CH), 4.32 (1H, dq, Jˆ2, 7 Hz, 18-H), 4.11
(1H, dt, Jˆ8, 2 Hz, 17-H), 4.07 (2H, q, Jˆ7 Hz, 8-CH2),
3.62, 3.59, 3.32/30 (each 3H, s, 2-, 12-CH3, COOCH3),
2.20±2.50 (4H, m, 17-CH2CH2), 2.12/11 (3H, d,
Jˆ6.5 Hz, 31-CH3), 1.80 (3H, d, Jˆ7 Hz, 18-CH3), 1.70
(3H, t, Jˆ7 Hz, 81-CH3). MS (FAB) found: m/z 642.
Calcd for C34H34N4O654Zn: M1, 642.
References
1. Lancaster, C. R. D.; Michel, H. Photosynth. Res. 1996, 48, 65±
74.
2. Klukas, O.; Schubert, W.-D.; Jordan, P.; Krauû, N.; Fromme,
P.; Witt, H. T.; Saenger, W. J. Biol. Chem. 1999, 274, 7361±7367.
3. van Grondelle, R.; Dekker, J. P.; Gillbro, T.; Sundstrom, V.
Biochim. Biophys. Acta 1994, 1187, 1±65.
Zinc methyl 31R-bacteriopheophorbide-f (Zn-1R).
Retention time was 130±133 min (Cosmosil, MeOH/
H2Oˆ2/1, 1.5 mL/min); light brown solid (precipitated
from hexane); VIS (THF) lmaxˆ628 (rel. 0.34), 580
4. Hu, X.; Damjanovic, A.; Ritz, T.; Schulten, K. Proc. Natl. Acad.
Sci. USA 1998, 95, 5935±5941.
5. Olson, J. M. Photochem. Photobiol. 1998, 67, 61±75.
6. Tamiaki, H. Coord. Chem. Rev. 1996, 148, 183±197.
7. Blankenship, R. E.; Olson, J. M.; Miller, M. Anoxygenic Photo-
synthetic Bacteria; Blankenship, R. E., Madigan, M. T., Bauer,
C. E., Eds.; Kluwer: Dordrecht, 1995, pp 399±435.
8. Sakuragi, Y.; Frigaard, N.-F.; Shimada, K.; Matsuura, K.
Biochim. Biophys. Acta 1999, 1413, 172±180.
1
(0.07), 448 nm (1.00); H NMR (CDCl3 Ð 0.2% C5D5N)
dˆ11.22 (1H, s, CHO), 10.13, 9.59, 8.17 (each 1H, s, 5-,
10-, 20-H), 6.41 (1H, q, Jˆ7 Hz, 3-CH), 5.08, 4.94 (1H, d,
Jˆ20 Hz, 131-CH), 4.28 (1H, dq, Jˆ2, 7.5 Hz, 18-H), 4.09
(1H, dt, Jˆ8, 2 Hz, 17-H), 4.00 (2H, q, Jˆ7 Hz, 8-CH2),
3.61, 3.57, 3.31 (each 3H, s, 2-, 12-CH3, COOCH3), 2.20±
2.50 (4H, m, 17-CH2CH2), 2.09 (3H, d, Jˆ7 Hz, 31-CH3),
1.79 (3H, d, Jˆ7.5 Hz, 18-CH3), 1.69 (3H, t, Jˆ7 Hz,
81-CH3).
9. Fenna, R. E.; Matthews, B. W. Nature 1975, 258, 573±577.
10. McDermott, G.; Prince, S. M.; Freer, A. A.; Hawthornthwaite-
Lawless, A. M.; Papiz, M. Z.; Cogdell, R. J.; Isaacs, N. W. Nature
1995, 374, 517±521.
11. Hofmann, E.; Wrench, P. M.; Sharples, F. P.; Hiller, R. G.;
Welte, W.; Diederichs, K. Science 1996, 272, 1788±1791.
12. Smith, K. M. Photosynth. Res. 1994, 41, 23±26.
13. Bobe, F. W.; Pfennig, N.; Swanson, K. L.; Smith, K. M.
Biochemistry 1990, 29, 4340±4348.
Zinc methyl 31S-bacteriopheophorbide-f (Zn-1S).
Retention time was 137±140 min (Cosmosil, MeOH/
H2Oˆ2/1, 1.5 mL/min); light brown solid (precipitated
from hexane); VIS (THF) lmaxˆ628 (rel. 0.34), 580
1
(0.07), 448 nm (1.00); H NMR (CDCl3 Ð 0.2% C5D5N)
14. Borrego, C. M.; Gerola, P. D.; Miller, M.; Cox, R. P.
Photosynth. Res. 1999, 59, 159±166.
dˆ11.22 (1H, s, CHO), 10.18, 9.58, 8.16 (each 1H, s, 5-, 10-
, 20-H), 6.37 (1H, q, Jˆ7 Hz, 3-CH), 5.08, 4.94 (1H, d,
Jˆ20 Hz, 131-CH), 4.29 (1H, dq, Jˆ2, 7.5 Hz, 18-H),
4.08 (1H, dt, Jˆ8, 2 Hz, 17-H), 4.03 (2H, q, Jˆ7 Hz, 8-
CH2), 3.61, 3.58, 3.28 (each 3H, s, 2-, 12-CH3, COOCH3),
2.20±2.50 (4H, m, 17-CH2CH2), 2.11 (3H, d, Jˆ7 Hz, 31-
CH3), 1.78 (3H, d, Jˆ7.5 Hz, 18-CH3), 1.67 (3H, t, Jˆ7 Hz,
81-CH3).
15. Baneras, L.; Borrego, C. M.; Garcia-Gil, L. J. Arch. Microbiol.
1999, 171, 350±354.
16. Smith, K. M.; Kehres, L. A.; Fajer, J. J. Am. Chem. Soc. 1983,
105, 1387±1389.
17. Otte, S. C. M.; van der Heiden, J. C.; Pfennig, N.; Amesz, J.
Photosynth. Res. 1991, 28, 77±88.
18. Causgrove, T. P.; Brune, D. C.; Blankenship, R. E.
J. Photochem. Photobiol. B: Biol. 1992, 15, 171±179.
19. Psencik, J.; Searle, G. F. W.; Hala, J.; Schaafsma, T. J.
Photosynth. Res. 1994, 40, 1±10.
Zinc methyl 3-devinyl-3-hydroxymethyl-pyropheophor-
bide-b(zinc methyl 31-demthyl-bacteriopheophorbide-f,
Zn-2). 90%; retention time was 6.9 min (Gelpack, MeOH,
1.2 mL/min) or 24 min (Cosmosil, MeOH/H2Oˆ2/1,
1.5 mL/min); light brown solid (precipitated from hexane);
mp .3008C; VIS (THF) lmaxˆ630 (rel. 0.32), 583 (0.06),
20. Feiler, U.; Albouy, D.; Lutz, M.; Robert, B. Photosynth. Res.
1994, 41, 175±180.
21. van Noort, P. I.; Francke, C.; Schoumans, N.; Otte, S. C. M.;
Aartsma, T. J.; Amesz J. Photosynth. Res. 1994, 41, 193±203.

H. Tamiaki et al. / Tetrahedron 56 (2000) 6245±6257
6257
22. Fetisova, Z. G.; Mauring, K.; Taisova, A. S. Photosynth. Res.
1994, 41, 205±210.
Effects, Garab, G., Ed.; Kluwer: Dordrecht, 1998; Vol. 1, pp
145±148.
È
38. Risch, N.; Koster, B.; Schormann, A.; Siemens, T.;
23. Kroh, P.; Psencik, J.; Polivka, T.; Engst, D.; Hala, J.
J. Luminescence 1997, 72, 593±594.
Brockmann, H. Liebigs Ann. Chem. 1988, 343±347.
39. Simpson, D. J.; Smith, K. M. J. Am. Chem. Soc. 1988, 110,
1753±1758.
24. Ma, Y.-Z.; AschenbruÈcker, J.; Miller, M.; Gillbro, T. Chem.
Phys. Lett. 1999, 300, 465±472.
40. Smith, K. M.; Bisset, G. M. F.; Bushell, M. J. Org. Chem.
1980, 45, 2218±2224.
È
25. van Walree, C. A.; Sakuragi, Y.; Steensgaard, D. B.; Bosinger,
C. S.; Frigaard, N.-U.; Cox, R. P.; Holzwarth, A. R.; Miller, M.
Photochem. Photobiol. 1999, 69, 322±328.
41. Oba, T.; Masada, Y.; Tamiaki, H. Bull. Chem. Soc. Jpn 1997,
70, 1905±1909.
26. Tamiaki, H.; Omoda, M.; Kubo, M. Bioorg. Med. Chem. Lett.
1999, 9, 1631±1632.
42. Oba, T.; Tamiaki, H. Photochem. Photobiol. 1998, 67, 295±
303.
27. Hildebrandt, P.; Tamiaki, H.; Holzwarth, A. R.; Schaffner, K.
J. Phys. Chem. 1994, 98, 2192±2197.
43. Osuka, A.; Marumo, S.; Wada, Y.; Yamazaki, I.; Yamazaki,
T.; Shirakawa, Y.; Nishimura, Y. Bull. Chem. Soc. Jpn 1995, 68,
2909±2915.
28. Tamiaki, H.; Takeuchi, S.; Tanikaga, R.; Balaban, S. T.;
Holzwarth, A. R.; Schaffner, K. Chem. Lett. 1994, 401±402.
29. Tamiaki, H.; Amakawa, M.; Shimono, Y.; Tanikaga, R.;
Holzwarth, A. R.; Schaffner, K. Photochem. Photobiol. 1996, 63,
92±99.
44. Scheumann, V.; Helfrich, M.; Schoch, S.; RuÈdiger, W. Z.
Naturforsch. 1996, 51c, 185±194.
45. Borrego, C. M.; Arellano, J. B.; Abella, C. A.; Gillbro, T.;
Garcia-Gil, J. Photosynth. Res. 1999, 60, 257±264.
46. Hoff, A. J.; Amesz, J. Chlorophylls; Scheer, H., Ed.; CRC:
Boca Raton, FL, 1991, pp 723±738.
30. Tamiaki, H.; Miyata, S.; Kureishi, Y.; Tanikaga, R. Tetrahedron
1996, 52, 12421±12432.
31. Tamiaki, H.; Miyatake, T.; Tanikaga, R.; Holzwarth, A. R.;
Schaffner, K. Angew. Chem. Int. Ed. Engl. 1996, 35, 772±774.
32. Balaban, T. S.; Tamiaki, H.; Holzwarth, A. R.; Schaffner, K.
J. Phys. Chem. B 1997, 101, 3424±3431.
47. Cox, R. P.; Miller, M.; AschenbruÈcjer, J.; Ma, Y.-Z.; Gillbro,
T. Photosynthesis: Mechanisms and Effects, Garab, G., Ed.;
Kluwer: Dordrecht, 1998; Vol. 1, pp 149±152.
48. Arellano, J. B.; Psencik, J.; Borrego, C. M.; Ma, Y.-Z.;
Guyoneaud, R.; Garcia-Gil, J.; Gillbro, T. Photochem. Photobiol.
2000, 71, 715±723.
33. Tamiaki, H.; Takeuchi, S.; Tsudzuki, S.; Miyatake, T.;
Tanikaga, R. Tetrahedron 1998, 6699±6718.
34. Kureishi, Y.; Tamiaki, H. J. Porphyrins Phthalocyanines
1998, 2, 159±169.
49. Oba, T.; Tamiaki, H. Photosynth. Res. 1999, 61, 23±31.
50. Uehara, K.; Hioki, Y.; Mimuro, M. Photochem. Photobiol.
1993, 58, 127±132.
35. Miyatake, T.; Tamiaki, H.; Holzwarth, A. R.; Schaffner, K.
Helv. Chim. Acta 1999, 82, 797±810.
51. Amakawa, M.; Tamiaki, H. Bioorg. Med. Chem. 1999, 7,
1141±1144.
36. Miyatake, T.; Tamiaki, H.; Holzwarth, A. R.; Schaffner, K.
Photochem. Photobiol. 1999, 69, 448±456.
37. Tamiaki, H.; Kubo, M. Photosynthesis: Mechanisms and
52. Tamiaki, H.; Kouraba, M. Tetrahedron 1997, 53, 10677±
10688.