Production of bacteriopurpurin-18 phytyl ester from bacteriopheophytin a via allomerization by contact with titanium oxides in the presence of molecular oxygen

Article abstract of DOI:10.1016/j.bmcl.2014.12.002

Incubation of bacteriopheophytin (BPhe) a, which was a demetalated pigment of bacteriochlorophyll a in photosynthetic bacteria, in CH₂Cl₂ in the presence of TiO₂ particles with bubbling O₂ in the dark produced a pigment absorbing 814 nm. Detailed characterization of the novel pigment isolated from the CH₂Cl₂ suspension revealed that bacteriopurpurin-18 phytyl ester possessing an anhydride-type six-membered exocyclic E-ring was majorly formed by the treatment with TiO₂ particles under oxygenic conditions. Oxidation of the bacteriochlorin ring in BPhe a, namely formations of derivatives of 3-acetyl pheophytin a and 3-acetyl protopheophytin a, can barely be detected through the conversion processes.

Full text of DOI:10.1016/j.bmcl.2014.12.002

Accepted Manuscript
Production of bacteriopurpurin-18 phytyl ester from bacteriopheophytin a via
allomerization by contact with titanium oxides in the presence of molecular
oxygen
Yoshitaka Saga, Atsushi Ishitani, Naoya Takahashi, Kenji Kawamura
PII:
S0960-894X(14)01299-2
DOI:
http://dx.doi.org/10.1016/j.bmcl.2014.12.002
BMCL 22247
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
11 November 2014
29 November 2014
2 December 2014
Please cite this article as: Saga, Y., Ishitani, A., Takahashi, N., Kawamura, K., Production of bacteriopurpurin-18
phytyl ester from bacteriopheophytin a via allomerization by contact with titanium oxides in the presence of
molecular oxygen, Bioorganic & Medicinal Chemistry Letters (2014), doi: http://dx.doi.org/10.1016/j.bmcl.
2014.12.002
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Production of bacteriopurpurin-18 phytyl ester from
bacteriopheophytin a via allomerization by contact with titanium
oxides in the presence of molecular oxygen
Yoshitaka Saga*, Atsushi Ishitani, Naoya Takahashi, Kenji Kawamura
Department of Chemistry, Faculty of Science and Engineering, Kinki University, Higashi-Osaka,
Osaka 577-8502, Japan
Keywords: allomerization, bacteriochlorophyll, bacteriopheophytin, near-infrared, titanium oxide
*Corresponding author. Tel: +81 6 6730 5880; fax: +81 6 6723 2721.
E-mail address: saga@chem.kindai.ac.jp (Y. Saga).
1

ABSTRACT
Incubation of bacteriopheophytin (BPhe) a, which was a demetalated pigment of bacteriochlorophyll
a in photosynthetic bacteria, in CH2Cl2 in the presence of TiO2 particles with bubbling O2 in the dark
produced a pigment absorbing 814 nm. Detailed characterization of the novel pigment isolated from
the CH2Cl2 suspension revealed that bacteriopurpurin-18 phytyl ester possessing an anhydride-type
six-membered exocyclic E-ring was majorly formed by the treatment with TiO2 particles under
oxygenic conditions. Oxidation of the bacteriochlorin ring in BPhe a, namely formations of
derivatives of 3-acetyl pheophytin a and 3-acetyl protopheophytin a, can barely be detected through
the conversion processes.
2

In photosynthesis, cyclic tetrapyrrole pigments such as chlorophylls (Chls) and
bacteriochlorophylls (BChls) play essential roles in the processes of light-harvesting and charge
separation. Chlorin-type cyclic tetrapyrroles (17,18-dihydroporphyrins) are photofunctional
moieties of photosynthetic pigments such as Chls a and b in oxygenic photosynthetic organisms. In
contrast, bacteriochlorin-type pigments (7,8,17,18-tetrahydroporphyrins) function as light-harvesting
and charge separation in bacterial photosynthesis.1,2 Fig. 1A shows the molecular structure of BChl
a, which is a major pigment in purple photosynthetic bacteria. Owing to reduction of the bonds
between the 7- and 8-positions as well as between 17- and 18-positions in the cyclic tetrapyrrole ring,
BChl a exhibited an intense Qy absorption band in the near-infrared (NIR) region, whose peak
position is red-shifted by approximately 100 nm compared with chlorin-type pigments.1–3 Such
unique spectral features of bacteriochlorin-type photosynthetic pigments allow us to utilize them for
developments of NIR-responsive pigments, which have attracted considerable attentions in the
research area of life sciences and materials chemistry.4–7 In particular, BChl a and its derivatives
have been fascinated as photosensitizers in photodynamic therapy (PDT) because of their efficient
absorption abilities of NIR light that can penetrate tissues.5–13 Henderson et al. first attempted to use
BChl a as PDT sensitizers to tumors.8 However, BChl a was degraded in vivo and such degradation
affected PDT activities. To improve stabilities and PDT activities of BChl a derivatives, substitution
of central metals and peripheral groups in the bacteriochlorin macrocycle has been extensively
examined.9–13
Photosynthetically active chlorophyllous pigments have a five-membered exocyclic E-ring
with a 132-methoxycarbonyl group. The change of the E-ring to six-membered rings such as
anhydride and imide-type exocyclic rings, namely conversions to purpurin- and purpurinimide-type
pigments, is one of the promising strategies to regulate optical and physicochemical properties of
chlorophyllous pigments.14–20 Pandey and coworkers have developed various bacteriopurpurin- and
3

bacteriopurpurinimide-type functional pigments from natural BChl a and demonstrated their utilities
as stable PDT photosensitizers that can work effectively by excitation of NIR light.21–27
Bacteriopurpurin-18 (the molecular structure is shown in Fig. 1B) is known to be a key
compound in the synthesis of bacteriopurpurinimides. Bacteriopurpurin-18 is generally synthesized
under strong alkaline conditions with O2 from bacteriopheophytin (BPhe) a and its derivatives. In
this process, however, hydrolysis of an ester group at the 17-propionate residue of chlorophyllous
pigments is inevitable and thus re-esterification is required in many cases. Additionally, undesirable
side-reactions sometimes occur in the step of re-esterification in the synthesis of purpurin-type
pigments.28 In such situations, we previously found that pheophorbide a methyl ester consisting of
the chlorin -macrocycle was converted to purpurin-18 methyl ester by contact with TiO2 particles
under oxygenic conditions in the dark.20 This reaction encourages us to attempt one-step conversion
from BPhe a to bacteriopurpurin-18 phytyl ester under mild conditions. In this study, we examine
behaviors of BPhe a by contact with TiO2 particles with O2-bubbling in the dark and characterize a
major product isolated from the reaction mixture.
BPhe a was prepared by demetalation of natural BChl a, which was extracted from a purple
photosynthetic bacterium Rhodobacter sphaeroides, under acidic conditions.29 Anatase-type TiO2
particles (diameter, 5 m; purity, 99.9%; Wako Chemical Industries, Ltd.) were added to a
dichloromethane solution of BPhe a, followed by stirring with bubbling O2 in the dark at 25 C.
Visible absorption spectra of CH2Cl2 solutions after removal of TiO2 particles by filtration were
measured at a regular interval. Spectral measurements were performed within ca. 4 h, since CH2Cl2
significantly decreased by O2-bubbling for a long period.
Fig. 2 compares visible absorption spectra of BPhe a incubated for 4 h with TiO2 particles
in CH2Cl2 by bubbling O2 in the dark (thick solid curve) with those in control experiments (thin solid
and broken curves) and before incubation (dotted curve). BPhe a before incubation exhibited a Qy
4

absorption band at 753 nm in CH2Cl2 (dotted curve). When a CH2Cl2 suspension containing BPhe a
was bubbled by O2 in the presence of TiO2 particles, a new Qy absorption band appeared at 814 nm
(thick solid curve), indicating that a new pigment absorbing 814-nm NIR light (hereafter denoted 1)
was produced from BPhe a under the oxygenic conditions. On the contrary, no absorption band was
detected above 800 nm by bubbling N2 in the presence of TiO2 particles (thin solid curve) and by
bubbling O2 without TiO2 particles (broken curve). These indicate that TiO2 particles were
necessary for this conversion under the oxygenic conditions. It is worth noting that absorbance
increase between 600 and 700 nm, which is derived from formation of the chlorin and porphyrin
-macrocycles by oxidation of the bacteriochlorin ring, barely increased under the present conditions.
To characterize the product 1 absorbing 814 nm in detail, 1 was purified by preparative
HPLC using a reverse-phase column (5C18-AR-II, Nacalai Tesque) with methanol/chloroform (8/2)
after filtration of the reaction suspension and evaporation under reduced pressure. This pigment had
the molecular ion peak at m/z 860.5429 in the high-resolution mass spectrum, which was identical to
the molecular weight of bacteriopurpurin-18 phytyl ester (860.5452). Fig. 3 depicts 1H NMR
spectra of 1 and intact BPhe a. BPhe a showed two singlet peaks at 6.09 and 3.85 ppm, which were
ascribed to 132-H and 134-CH3, respectively (Fig. 3A). These signals disappeared in the 1H NMR
spectrum of 1 (Fig. 3B), indicating the lack of both the proton and the methoxycarbonyl group at the
132-position in this compound. The 17-H signal of 1 was shifted to the lower field by approximately
1.1 ppm compared with that of BPhe a. Such a large shift is characteristic of (bacterio)purpurin-18
type pigments.20,26 Given the molecular structure of bacteriopurpurin-18 phytyl ester (Fig. 1B), all
the signals in the 1H NMR spectrum of 1 can be assigned as described in Supplementary Data. Fig. 4
shows a FTIR spectrum of the compound 1. This pigment exhibited a vibrational band at 1720 cm-1,
which can be attributed to the anhydride group in the exocyclic E-ring of (bacterio)purpurin-type
chlorophyllous pigments, in the lower-wavenumber side of the stretching vibrational band of the
5

172-C=O in the ester moieties at 1746 cm-1. The stretching vibrational band of the 13-keto group in
intact BPhe a around 1700 cm-1 disappeared in the FTIR spectrum of 1. The bands at 1670-cm-1 and
around 1600–1620 cm-1 were ascribable to the C=O stretching vibrational band in the 3-acetyl group
and skeletal vibrational bands of the bacteriochlorin -macrocycle, respectively. These
characterizations proved that the pigment 1, which was mainly produced by contact with TiO2
particles in the presence of O2, was bacteriopurpurin-18 phytyl ester. The yield of 1 was estimated to
be 7% in the present reaction, which was lower than the conversion efficiency from pheophorbide a
methyl ester to purpurin-18 methyl ester (32%).20 One possible reason for the difference in the
conversion efficiency might be adsorption behaviors on TiO2 particles; more hydrophobic
bacteriopheophytin a phytyl ester tended to be less adsorbed on TiO2 surfaces. The adsorption
abilities of chlorophyllous pigments are responsible for the yields in this type of reaction, since the
contact of substrates on TiO2 surfaces is necessary for the conversion into (bacterio)purpurin-type
pigments.
In the conventional synthesis of purpurin-18 and bacteriopurpurin-18, the enolate anions
were generated from chlorophyllous pigments by subtraction of the 132-proton in the E-ring in the
action of hydroxide ions, followed by oxidation with O2 to form the six-membered anhydride
ring.30–32 The terminal and/or bridging hydroxides33–35 would be responsible for the formation of the
enolate anions in the early stage of the present conversion, followed by oxidation in the same manner
as the conventional conversions. Interactions of the 13-keto group in the E-ring of chlorophyllous
pigments with TiO2 surfaces36 might contribute to smooth formation and/or stabilization of the
enolate anions.
The present conversion from BPhe a to bacteriopurpurin-18 phytyl ester proceeded under
mild conditions without cleavage of the phytyl ester at the 17-propionate residue. This is in sharp
contrast to the conventional synthesis of bacteriopurpurin-18 derivatives under strong alkaline
6

conditions, accompanying inevitable hydrolysis of ester moieties. In addition, a work-up procedure
in this conversion, namely filtration and evaporation after the reactions, is significantly facile
compared with that in the conventional synthesis. This reaction, therefore, would be advantageous
from the viewpoints of green chemistry and will be useful for developments of NIR-light responsive
photofunctional pigments from BChl a in photosynthetic bacteria.
Acknowledgements
We thank Prof. Tomohiro Miyatake, Ryukoku University, and Mr. Keiya Hirota, Kinki
University, for HRMS measurements and experimental assistance, respectively. This work was
partially supported by Grants-in Aid for scientific Research on Innovative Areas ‘Artificial
Photosynthesis (AnApple)’ (No. 25107525) from the Japan Society for the Promotion of Sciences
(JSPS) and from the Tokuyama Science Foundation.
Supplementary data
Supplementary data associated with this article can be found, in the online version, at.
REFERENCES
1. Scheer, H. In Chlorophylls and Bacteriochlorophylls: Biochemistry, Biophysics, Functions and
7

Applications; Grimm, B., Porra, R. J., Rüdiger, W., Scheer, H., Eds.; Springer: Dordrecht, 2006;
pp 1–26.
2. Tamiaki, H.; Kunieda, M. In Handbook of Porphyrin Science; Kadish, K. M., Smith, K. M.,
Guilard, R., Eds.; World Scientific: Singapore, 2011; Vol.11, pp 223–290.
3. Linnanto, J.; Korppi-Tommola, J. Phys. Chem. Chem. Phys. 2006, 8, 663.
4. Wang, X.-F; Tamiaki, H. Energy Environ. Sci. 2010, 3, 94.
5. Sternberg, E. D.; Dolphin, D. Tetrahedron 1998, 54, 4151.
6. Nyman, E. S.; Hynninen, P. H. J. Photochem. Photobiol. B: Biol. 2004, 73, 1.
7. Chen, Y.; Li, G.; Pandey, R. K. Curr. Org. Chem. 2004, 8, 1105.
8. Henderson, B. W.; Sumlin, A. B.; Owczarczak, B. L.; Dougherty, T. J. J. Photochem. Photobiol.
B: Biol. 1991, 10, 303.
9. Kessek, D.; Smith, K. M.; Pandey, R. K.; Shiau, F.-Y.; Henderson, B. Photochem. Photobiol.
1993, 58, 200.
10. Fiedor, J.; Fiedor, L.; Kammhuber, N.; Scherz, A.; Scheer, H. Photochem. Photobiol. 2002, 76,
145.
11. Vakrat-Haglili, Y.; Weiner, L.; Brumfeld, V.; Brandis, A.; Salomon, Y.; Mcllroy, B.; Wilson, B.
C.; Pawlak, A.; Rozanowska, M.; Tadeusz, S.; Scherz, A. J. Am. Chem. Soc. 2005, 127, 6487.
12. Limantara, L.; Koehler, P.; Wilhelm, B.; Porra, R. J.; Scheer, H. Photochem. Photobiol. 2006,
82, 770.
13. Dandler, J.; Wilhelm, B.; Scheer, H. Photochem. Photobiol. 2010, 86, 182.
14. Hoober, J. K.; Sery, T. W.; Yamamoto, N. Photochem. Photobiol. 1998, 48, 579.
15. Lee, S. J. H.; Jagerovic, N.; Smith, K. M. J. Chem. Soc. Perkin Trans. 1 1993, 2369.
16. Kozyrev, A. N.; Zheng, G.; Lazarou, E.; Dougherty, T. J.; Smith, K. M.; Pandey, R. K.
Tetrahedron Lett. 1997, 38, 3335.
8

17. Zheng, G.; Potter, W. R.; Sumlin, A.; Dougherty, T. J.; Pandey, R. K. Bioorg. Med. Chem. 2000,
10, 123.
18. Pandey, S. K.; Sajjad, M.; Chen, Y.; Pandey, A.; Missrt, J. P.; Batt, C.; Yao, R.; Nabi, H. A.;
Oseroff, A. R.; Pandey, R. K. Bioconjugate Chem. 2009, 20, 274.
19. Wang, X.-F.; Wang, L.; Tamai, N.; Kitao, O.; Tamiaki, H.; Sasaki S. J. Phys. Chem. C 2011, 115,
24394.
20. Saga, Y.; Otono, T. Chem. Lett. 2012, 41, 360.
21. Kozyrev, A. N.; Zheng, G.; Zhu, C.; Dougherty, T. J.; Smith, K. M.; Pandey, R. K. Tetrahedron
Lett. 1996, 37, 6431.
22. Chen, Y.; Graham, A.; Potter, W.; Morgan, J.; Vaughan, L.; Bellnier, D. A.; Henderson, B. W.;
Oseroff, A.; Dougherty, T. J.; Pandey, R. K. J. Med. Chem. 2002, 45, 255.
23. Kozyrev, A. N.; Chen, Y.; Goswani, L. N.; Tabaczynski, W. A.; Pandey. R. K. J. Org. Chem.
2006, 71, 1949.
24. Gryshuk, A. L.; Chen, Y.; Potter, W.; Ohulchansky, T.; Oseroff, A.; Pandey, R. K. J. Med. Chem.
2006, 49, 1874.
25. Gryshuk, A. L.; Chen, Y.; Goswami, L. N.; Pandey, S.; Missert, J. R.; Ohulchanskyy, T.; Potter,
W.; Prasad, P. N.; Oseroff, A.; Pandey, R. K. J. Med. Chem. 2007, 50, 1754.
26. Goswani, L. N.; Chen, Y.; Missert, J.; Li, G.; Pallenberg, A.; Pandey, R. K. Heterocycles 2007,
71, 1929.
27. Chen, Y.; Potter, W. R.; Missert, J. R.; Morgan, J; Pandey, R. K. Bioconjugate Chem. 2007, 18,
1460.
28. Kozyrev, A. N.; Alderfer, J. L.; Dougherty, T. J.; Pandey, R. K. Chem. Commun. 1998, 1083.
29. Saga, Y.; Miura, R.; Sadaoka, K.; Hirai, Y. J. Phys. Chem. B 2011, 115, 11757.
30. Hynninen, P. H.; Leppäkases, T. S.; Mesilaakso, M. Tetrahedron Lett. 2006, 47. 1663.
9

31. Hynninen, P. H. In Chlorophylls; Scheer, H., Ed.; CRC Press: Boca Raton, 1991; pp 145–209.
32. Hynninen, P. H.; Hyvärinen, K. J. Org. Chem. 2002, 67, 4055.
33. Hadjiivanov, K. I.; Klissurski, D. G. Chem. Soc. Rev. 1996, 25, 61.
34. Connor, P. A.; Dobson, K. D.; McQuillan, A. J. Langmuir 1999, 15, 2402.
35. Sano, K.; Shiba, K. J. Am. Chem. Soc. 2003, 125, 14234.
36. Nakajima, K.; Noma, R.; Kitano, M.; Hara, M. J. Phys. Chem. C 2013, 117, 16028.
FIGURE CAPTION
Fig. 1 (A) Molecular structures of BChl a (M = Mg) and BPhe a (M=2H). (B) Molecular
structures of bacteriopurpurin-18 (R = H) and bacteriopurpurin-18 phytyl ester (R = phytyl).
10

Fig. 2. Visible absorption spectra of CH2Cl2 solutions containing BPhe a after incubation for 4 h by
bubbling O2 with TiO2 particles (thick solid curve), by bubbling N2 with TiO2 particles (thin solid
curve), by bubbling O2 without TiO2 particles (broken curve), and before incubation (dotted curve).
Spectra were normalized at the Soret peaks.
Fig. 3. 1H NMR spectra of BPhe a (A) and the product 1 isolated from a CH2Cl2 solution after
incubation with TiO2 particles and O2 (B) in CDCl3 between 3 and 7 ppm.
Fig. 4. FTIR spectrum of the product 1 isolated from a CH2Cl2 solution after incubation with TiO2
particles and O2 in CH2Cl2 between 1570 and 1850 cm-1.
11

31
(B)
O
O
(A)
7
8
N
N
NH
N
N
M
N
N
HN
18
17
132
O
O
O
O
O
134 OCH3
O
O
O
O
R
phytyl
P1
phytyl =
P3
P5
P7
P9
P11
P13
P15
P2
P4
P6
P8
P10
P12
P14
Saga et al.
Fig. 1

753
814
300
400
500
600
700
800
Wavelength / nm
Saga et al.
Fig. 2

2-,31-,12-
CH3
134-CH3
8-,17-H
7-,18-H
P1-H
132-H
P2-H
(A)
8-H
4
17-H
(B)
7
6
5
3
δ / ppm
Saga et al.
Fig. 3

1670
1720
1746
1850 1800 1750 1700 1650 1600
Wavenumber / cm-1
Saga et al.
Fig. 4

O
O
NH
N
N
NH
N
N
contact with TiO2
under oxygenic conditions
HN
HN
O
O
O
O
O
OCH3
O
O
O
O
phytyl
phytyl
Saga et al.
Graphical abstract