Cellular uptake and photocytotoxicity of glycoconjugated porphyrins in HeLa cells

Article abstract of DOI:10.1562/2004-03-07-RA-103.1

Thirty-two glycoconjugated porphyrins were synthesized by a modification of Lindsey method in the presence of Zn(OAc)₂·₂H ₂O as a template. The Zn²⁺ ion template strategy improved the yield about three-fold in the case of meta-substituted tetraphenylporphyrins. In addition, free-base porphyrins were obtained almost quantitatively by demetalation with 4 M HCl. Sixteen deacetylated glycoconjugated porphyrins were tested as candidate photodynamic therapy (PDT) drugs using HeLa cells. Most of the deacetylated glycoconjugated porphyrins showed higher cellular uptake than tetraphenylporphyrin tetrasulfonic acid (TPPS), and 5,10,15, 20-tetrakis[4-(β-D-arabinopyranosyloxy)phenyl] porphyrin (p-5d) in particular showed 18.5-fold higher uptake than TPPS. The photocytotoxicity of 5,10,15,20-tetrakis[4-(β-D-glucopyranosyloxy)phenyl] porphyrin (p-5a), p-5d and TPPS was examined with HeLa cells, using a light dose of 16 J/cm². These photosensitizers had no cytotoxicity in the dark, but their photocytotoxicity increased in the order of TPPS < p-5a < p-5d. These results suggest p-5d is a good candidate for a PDT drug.

Full text of DOI:10.1562/2004-03-07-RA-103.1

Cellular Uptake and Photocytotoxicity of Glycoconjugated Porphyrins in HeLa
Cells
Author(s): Shiho Hirohara, Makoto Obata, Atsuhiro Saito, Shin-ichi Ogata, Chikara Ohtsuki, Suguru
Higashida, Shun-ichiro Ogura, Ichiro Okura, Yuko Sugai, Yuji Mikata, Masao Tanihara, and Shigenobu
Yano
Source: Photochemistry and Photobiology, 80(2):301-308.
Published By: American Society for Photobiology
DOI: http://dx.doi.org/10.1562/2004-03-07-RA-103.1
URL: http://www.bioone.org/doi/full/10.1562/2004-03-07-RA-103.1
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Photochemistry and Photobiology, 2004, 80: 301–308
Cellular Uptake and Photocytotoxicity of Glycoconjugated Porphyrins in
HeLa Cells{
Shiho Hirohara1, Makoto Obata*1, Atsuhiro Saito2, Shin-ichi Ogata2, Chikara Ohtsuki2, Suguru Higashida3,
Shun-ichiro Ogura4, Ichiro Okura4, Yuko Sugai1, Yuji Mikata1, Masao Tanihara2 and Shigenobu Yano*1
1Division of Material Science, Graduate School of Human Culture, Nara Women’s University, Nara, Japan
2Graduate School of Materials Science, Nara Institute of Science and Technology, Nara, Japan
3Department of Industrial Chemistry, Osaka Prefectural College of Technology, Osaka, Japan
4Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Kanagawa, Japan
Received 7 March 2004; accepted 9 June 2004
side effects, such as nausea and hair loss, PDT can be accurately
targeted by controlling the field of irradiation. Even though the
ABSTRACT
Thirty-two glycoconjugated porphyrins were synthesized by a
modification of Lindsey method in the presence of
Zn(OAc)2ꢀ2H2O as a template. The Zn21 ion template strategy
improved the yield about three-fold in the case of meta-
substituted tetraphenylporphyrins. In addition, free-base
porphyrins were obtained almost quantitatively by demetala-
tion with 4 M HCl. Sixteen deacetylated glycoconjugated por-
phyrins were tested as candidate photodynamic therapy (PDT)
drugs using HeLa cells. Most of the deacetylated glycocon-
jugated porphyrins showed higher cellular uptake than
tetraphenylporphyrin tetrasulfonic acid (TPPS), and 5,10,15,
20-tetrakis[4-(b-D-arabinopyranosyloxy)phenyl]porphyrin
(p-5d) in particular showed 18.5-fold higher uptake than TPPS.
The photocytotoxicity of 5,10,15,20-tetrakis[4-(b-D-glucopyra-
nosyloxy)phenyl]porphyrin (p-5a), p-5d and TPPS was exam-
ined with HeLa cells, using a light dose of 16 J/cm2. These
photosensitizers had no cytotoxicity in the dark, but their
photocytotoxicity increased in the order of TPPS < p-5a < p-5d.
These results suggest p-5d is a good candidate for a PDT drug.
mechanism of PDT involved is not fully understood, much effort has
beendevoted tothe developmentofsuperiorphotosensitizers forPDT.
Porphyrins, including chlorins and bacteriochlorins (5–10), as well as
phthalocyanines (11–13) and d-aminolaevulinic acid (14–16) are
considered promising photosensitizers. PhotofrinÒ is a potent first-
generation photosensitizers used for photodynamic treatment of
esophageal cancer and early- and late-stage lung cancer (4,17,18).
However, PhotofrinÒ must be used at a high dose and requires a long
irradiation time (19). To avoid serious side effects, such as
photosensitivity, it is desirable for photosensitizers to have high
tumor selectivity (i.e. low accumulation in normal tissues) and potent
photosensitization ability. The usage of a cellular recognition element
is a promising strategy to improve photosensitizers. In this context,
many glycoconjugated porphyrins have been synthesized not only to
improve water solubility but also because of the biological activity of
the sugar unit (20–24). Glycoconjugated porphyrins have been
synthesizedfromglycosylated benzaldehyde withpyrrole by meansof
the modified Lindsey method. However, the yields of m-substituted
glycoconjugatedporphyrinsare relatively low, presumablybecause of
steric hindrance of the saccharide moiety (3,21). Recently, we have
applied a zinc ion–templated condensation to the synthesis of
glycoconjugated porphyrins. The resulting zinc porphyrins readily
afford the corresponding free-base porphyrins in moderate yield on
treatment with hydrochloric acid, even in the case of meta-substituted
glycoconjugated porphyrins. The versatility of this method for the
synthesis of para- and meta-substituted glycoconjugated porphyrins
was examined with glucose, galactose, xylose and arabinose. This
method allows us to explore systematically the role of the
glycopyanosyl group and the effect of the substitution position on
the cellular uptake and the photodynamic effect. Here, we report
a systematic investigation of the synthesis of 32 glycoconjugated
porphyrins, a survey of the cellular uptake of 16 deprotected
glycoconjugated porphyrins in HeLa cells and a study of the
photocytotoxicity in HeLa cells of glycoconjugated porphyrins
selected on the basis of the cellular uptake data.
INTRODUCTION
Recently, much attention has been focused on photodynamic therapy
(PDT), in which tumor-localizing photosensitizers are administered
and stimulated by light to initiate a chemical reaction leading to the
death of tumor cells, as an effective and selective treatment for cancer
(1–4). Unlike chemotherapy, which often causes undesirable systemic
{Posted on the website on 23 June 2004.
*To whom correspondence should be addressed: Division of Material
Science, Graduate School of Human Culture, Nara Women’s University,
Nara 630-8506, Japan. Fax: 81-742-20-3392; e-mail: mobata@cc.nara-
wu.ac.jp (MO), yano@cc.nara-wu.ac.jp (SY)
Abbreviations: BF3ꢀOEt2, boron trifluoride diethyl ether complex; DMEM,
Dulbecco modified Eagle medium; DMSO, dimethyl sulfoxide; ESI,
elecrospary ionization; FAB, fast atom bombardment; FCS, fetal calf
serum; HPLC, high-performance liquid chromatography; HRMS, high-
resolution mass spectrometry; NaOMe, sodium methoxide; NMR,
nuclear magnetic resonance; 1O2, singlet oxygen; PBS(ꢁ), phosphate-
buffered saline (ꢁ); PDT, photodynamic therapy; PME, protoporphyrin
IX dimethyl ester; PpIX, protoporphyrin IX; THF, tetrahydrofuran;
TPPS, tetraphenylporphyrin tetrasulfonic acid.
MATERIALS AND METHODS
Measurements
1H and 13C nuclear magnetic resonance (NMR) spectra were recorded on
Bruker AVANCE 800 (800 MHz), Bruker DRX600 (600 MHz), JEOL
Ó 2004 American Society for Photobiology 0031-8655/04 $5.00þ0.00
301

302 Shiho Hirohara et al.
JNM-AL400 (400 MHz) and VARIAN GEMINI 2000 (300 MHz)
instruments. Following abbreviations were used for NMR assignment: P,
porphyrin ring; R, saccharide residue; Ar, phenyl group of benzaldehyde or
bridging phenylene group between P and R. Electronic absorption spectra
in dimethyl sulfoxide (DMSO) were recorded on a JASCO V-570
spectrophotometer. Mass spectra were recorded on MS 700T JEOL, JEOL
JMS-700 and JMS-100LC JEOL (ESI mode) instruments. Fluorescence
intensity was determined by the plate reader (SPECTRA Fluor Plus, Tecan,
Maennedorf, Switzerland).
(ESI-HRMS) (m/z)
2083.5103; found, 2083.5117.
5
[M
þ
Na]þ calcd for C100H100N4O40ZnNa,
Zinc(II)5,10,15,20-tetrakis[3-(2,3,4,6-tetra-O-acetyl-b-D-glucopyranosy-
loxy)phenyl]porphyrin (m-2a). Recrystallization from CHCl3/EtOH gave
a red solid m-2a (yield: 93%). Purity (HPLC): 96.1%. UV–vis (DMSO):
kmax (nm) (e 3 10ꢁ3/Mꢁ1 cmꢁ1) 5 428 (58.6), 560 (1.97), 598 (7.88). 1H
NMR (CDCl3, 300.07 MHz): d (ppm) 5 8.97 (8H, br, b-P), 7.97–7.85 (8H,
m, 2,6-Ar), 7.72–7.62 (4H, m, 5-Ar), 7.47–7.21 (4H, m, 4-Ar), 5.40–5.25
(12H, m, 1,2,3-R), 5.20–5.25 (4H, m, 4-R), 4.25–4.12 (4H, m, 6-R), 4.12–
4.00 (4H, m, 6-R), 3.89–3.77 (4H, m, 5-R), 2.16–1.99 þ 1.53–1.34 (48H,
m, Me). 13C NMR (CDCl3, 75.46 MHz): d (ppm)5170.56, 170.37, 169.52
(CO 33), 155.32 (3-Ar), 150.11 (a-P), 144.37 (1-Ar), 132.17 (b-P), 129.90
(5-Ar), 127.67 (6-Ar), 122.93 (4-Ar), 120.39 (meso-P), 116.31 (2-Ar),
99.20 (1-R), 72.65 (3-R), 72.00 (5-R), 71.21 (2-R), 68.20 (4-R), 61.87 (6-
R), 20.57, 20.44, 20.38, 19.83 (Me 3 4). ESI-HRMS (m/z) 5 [M þ Na]þ
calcd for C100H100N4O40ZnNa, 2083.5103; found, 2083.5090.
Zinc(II)5,10,15,20-tetrakis[4-(2,3,4,6-tetra-O-acetyl-b-D-galactopyrano-
syloxy)phenyl]porphyrin (p-2b). Recrystallization from CHCl3/EtOH gave
a red solid p-2b (yield: 53%). Purity (HPLC): .99%. UV–vis (DMSO):
kmax (nm) (e 3 10ꢁ3/Mꢁ1 cmꢁ1) 5 430 (60.2), 563 (3.37), 602 (2.64). 1H
NMR (CDCl3, 300.07 MHz): d (ppm)58.81 (8H, s, b-P), 8.13 (8H, d, 3J5
8.6 Hz, 2,6-Ar), 8.74 (8H, d, 3J 5 8.1 Hz, 3,5-Ar), 5.86 (4H, m, 2-R), 5.33
(8H, m, 1,4-R), 4.62–4.56 (4H, m, 3-R), 4.28–4.08 (12H, m, 5,6-R), 2.27,
2.23, 2.20, 2.04 ((12H, s, Me) 3 4). 13C NMR (CDCl3, 75.46 MHz): d
(ppm) 5 170.51, 170.32, 169.69 (CO 3 3), 156.57 (4-Ar), 150.39 (a-P),
137.81 (1-Ar), 135.44 (2,6-Ar), 132.02 (b-P), 120.31 (meso-P), 114.92
(3,5-Ar), 99.57 (1-R), 71.10 (5-R), 70.87 (2-R), 68.72 (3-R), 66.86 (4-R),
61.34 (6-R), 20.70, 20.65, 20.55, 20.52 (Me 34). ESI-HRMS (m/z) 5[M þ
Na]þ calcd for C100H100N4O40ZnNa, 2083.5103; found, 2083.5083.
Zinc(II)5,10,15,20-tetrakis[3-(2,3,4,6-tetra-O-acetyl-b-D-galactopyrano-
syloxy)phenyl]porphyrin (m-2b). Recrystallization from CHCl3/EtOH gave
a red solid m-2b (yield: 72%). Purity (HPLC): 96.9%. UV–vis (DMSO):
kmax (nm) (e 3 10ꢁ3/Mꢁ1 cmꢁ1) 5 429 (32.1), 560 (1.50), 599 (7.84). 1H
NMR (CDCl3, 600.13 MHz): d (ppm) 5 8.95 (8H, br, b-P), 7.91–7.88 (8H,
m, 2,6-Ar), 7.68–7.64 (4H, m, 5-Ar), 7.44 (4H, m, 4-Ar), 5.59–5.57 (4H, m,
2-R), 5.42–5.40 (4H, m, 4-R), 5.34–5.32 (4H, m, 1-R), 5.14–5.12 (4H, m,
3-R), 4.14–4.01 (12H, m, 5,6-R), 2.15–2.00þ1.37–1.28 (48H, m, Me). 13C
NMR (DMSO-d6, 75.46 MHz): d (ppm) 5 170.38, 170.28, 169.62 (CO 3
3), 155.40 (3-Ar), 150.17 (a-P), 144.25 (1-Ar), 132.25 (b-P), 129.81 (5-Ar),
127.72 (6-Ar), 122.70 (4-Ar), 120.55 (meso-P), 116.33 (2-Ar), 99.80 (1-R),
71.10 (5-R), 70.79 (3-R), 68.68 (2-R), 66.89 (4-R), 61.40 (6-R), 20.72,
20.50, 19.71 (Me 3 3). ESI-HRMS (m/z) 5 [M þ Na]þ calcd for
C100H100N4O40ZnNa, 2083.5103; found, 2083.5110.
Materials
General chemicals. All chemicals used were of analytical grade. The
chemicals 4- and 3-(2,3,4,6-tetra-O-acetyl-b-D-glucopyranosyloxy)benzal-
dehyde (p- and m-1, respectively) were prepared according to the literature
(22,25). Tetraphenylporphyrin tetrasulfonic acid (TPPS) was purchased
from Sigma Aldrich Japan Corporation (Tokyo, Japan).
General procedure for synthesis of zinc(II) glycoconjugated porphyrins 2.
4-(2,3,4,6-Tetra-O-acetyl-b-D-glucopyranosyloxy)benzaldehyde (p-1a) (1.9
g, 3.9 mmol), pyrrole (0.27 mL, 3.9 mmol) and dry CHCl3 (430 mL)
were stirred with Ar flushing for 30 min. Boron trifluoride diethyl ether com-
plex (BF3ꢀOEt2) (0.2 mL, 0.10 mmol) and Zn(OAc)2ꢀ2H2O (3.8 g, 17 mmol)
were added, and the mixture was stirred for 24 h at room temper-
ature. To the solution was added chloranil (0.73 g, 2.9 mmol), and the whole
was refluxed for 1 h. The solution was dried over Na2SO4 and evaporated
under reduced pressure. The crude product was purified by column
chromatography (silica gel, CHCl3/acetone [15/1–8/1]), followed by re-
crystallization from CHCl3/EtOH to give p-2a (1.1 g, yield 87%) as a red
solid.
Generalprocedure for synthesis offree-baseglycoconjugatedporphyrins4.
4-(2,3,4,6-Tetra-O-acetyl-b-D-glucopyranosyloxy)benzaldehyde (p-1a) (1.9
g, 3.8 mmol), pyrrole (0.27 mL, 3.8 mmol) and dry CHCl3 (430 mL) were
stirred with Ar flushing for 30 min. BF3ꢀOEt2 (0.06 mL, 0.48 mmol) and
Zn(OAc)2ꢀ2H2O (2.5 g, 11 mmol) were added, and the mixture was stirred
for 24 h at room temperature. To the solution was added chloranil (0.44 g,
1.7 mmol), and the whole was refluxed for 1 h. The solution was washed
with 4 M HCl (100 mL 3 3), distilled water (100 mL 3 2) and saturated
NaHCO3 (aq) (100 mL 3 2). The solution was dried over Na2SO4, and the
solvent was taken off under reduced pressure. The crude product was
purified by column chromatography (silica gel, CHCl3/acetone [15/1–8/1]),
followed by recrystallization from CHCl3/EtOH to give p-4a (1.2 g, 64%) as
a red solid.
General procedure for deprotection of 2 and 4. p-2a (0.25 g, 0.12
mmol) was dissolved in tetrahydrofuran (THF) (30 mL). To the solution
was added sodium methoxide (NaOMe) (pH 9). This mixture was stirred for
24 h at room temperature, then 25% AcOH (aq) was added to neutralize it.
The solvent was evaporated, and the crude product was purified by gel
filtration on a Sephadex LH 20 column eluted with MeOH. Finally, the
product was lyophilized to yield p-3a (0.15 g, 94%).
Zinc(II)5,10,15,20-tetrakis[4-(2,3,4-tri-O-acetyl-b-D-xylopyranosyloxy)-
phenyl]porphyrin (p-2c). Recrystallization from CHCl3/EtOH gave a red
solid p-2c (yield: 84%). Purity (HPLC): .99%. UV–vis (DMSO): kmax
(nm) (e 3 10ꢁ3/Mꢁ1 cmꢁ1) 5 429 (40.6), 560 (2.86), 599 (2.15). H NMR
1
3
(CDCl3, 300.07 MHz): d (ppm) 5 8.96 (8H, s, b-P), 8.14 (8H, d, J 5 8.7
Hz, 2,6-Ar), 7.39 (8H, d, 3J 5 8.4 Hz, 3,5-Ar), 5.54 (4H, t, 3J 5 6.0 Hz, 2-
R), 5.39–5.37 (8H, m, 1,3-R), 5.15 (4H, m, 4-R), 4.43 (4H, dd, 3J54.8 Hz,
2J 5 12.3 Hz, 5-R), 3.73 (4H, dd, 3J 5 7.8 Hz, 2J 5 12.3 Hz, 5-R), 2.22,
2.17, 2.13 ((9H, s, Me) 3 3). 13C NMR (CDCl3, 75.46 MHz): d (ppm) 5
170.22, 170.09, 169.72 (CO 3 3), 156.40 (4-Ar), 150.47 (a-P), 137.64
(1-Ar), 135.53 (2,6-Ar), 132.07 (b-P), 120.47 (meso-P), 114.91 (3,5-Ar),
98.63 (1-R), 70.76 (3-R), 70.25 (2-R), 68.54 (4-R), 62.04 (5-R), 20.76,
20.72 (Me32). Fast atom bombardment (FAB)–HRMS (m/z)5[M]þ calcd
for C88H84N4O32Zn, 1772.436; found, 1772.4304.
The purity of the acetylated compounds 2 and 4 was estimated by liquid
chromatography (CLASS-VP ver. 5.02 system, Shimadzu, Kyoto, Japan)
equipped with silica gel-ODS (Mightysil RP-18 4.6-20, Kanto, Tokyo,
Japan) column and a diode array detector (SPD-M10A VP) using MeOH/
H2O 5 8/2 as an eluent. The purity of the deacetylated compounds 3 and 5
was estimated by liquid chromatography (L-6000, Hitachi, Tokyo, Japan)
equipped with silica gel-ODS (Hitachi, HITACHI GEL 4 mm/ 3 150 mm)
column and a diode array detector (Hitachi L-4000) using CH3CN/H2O51/
1 as an eluent.
Zinc(II)5,10,15,20-tetrakis[3-(2,3,4-tri-O-acetyl-b-D-xylopyranosyloxy)-
phenyl]porphyrin (m-2c). Recrystallization from CHCl3/EtOH gave a red
solid m-2c (yield: 84%). Purity (HPLC): .99%. UV–vis (DMSO): kmax
(nm) (e 3 10ꢁ3/Mꢁ1 cmꢁ1) 5 429 (40.2), 560 (2.86), 599 (2.08). H NMR
1
Zinc(II)5,10,15,20-tetrakis[4-(2,3,4,6-tetra-O-acetyl-b-D-glucopyranosy-
loxy)phenyl]porphyrin (p-2a). Recrystallization from CHCl3/EtOH gave
a red solid p-2a (yield 87%). Purity (high-performance liquid chromatogra-
phy [HPLC]): .99%. UV–vis (DMSO): kmax (nm) (e 3 10ꢁ3/Mꢁ1 cmꢁ1) 5
429 (26.8), 561 (1.49), 601 (1.02). 1H NMR (CDCl3, 600.13 MHz): d
(CDCl3, 300.07 MHz): d (ppm) 5 8.97 (8H, br, b-P), 7.93–7.87 (8H, m,
2,6-Ar), 7.68 (4H, m, 5-Ar), 7.43 (4H, m, 4-Ar), 5.38 (4H, m, 2-R), 5.25
(8H, m, 1,3-R), 5.05 (4H, m, 4-R), 4.26 (4H, m, 5-R), 3.57 (4H, m, 5-R),
2.12, 2.06, 2.04 ((9H, s, Me)33). 13C NMR (CDCl3, 75.46 MHz): d (ppm)
5 170.07, 169.96, 169.59 (CO 3 3), 155.03 (3-Ar), 150.14 (a-P), 144.37
(1-Ar), 132.14 (b-P), 129.76 (5-Ar), 127.67 (6-Ar), 123.16 (4-Ar), 120.42
(meso-P), 116.09 (2-Ar), 98.63 (1-R), 70.58 (3-R), 70.09 (2-R), 68.36
(4-R), 61.81 (5-R), 20.59 (Me). FAB-HRMS (m/z) 5 [M]þ calcd for
C88H84N4O32Zn, 1772.436; found, 1772.4303.
3
(ppm) 5 8.89 (8H, s, b-P), 8.12 (8H, d, J 5 8.7 Hz, 2,6-Ar), 7.38 (8H, d,
3J 58.4 Hz, 3,5-Ar), 5.48–5.42 (12H, m, 1,2,3-R), 5.30 (4H, t, 3J 59.6 Hz,
4-R), 4.40 (4H, dd, 3J512.3 Hz, 2J55.4 Hz, 6-R), 4.31 (4H, dd, 3J512.3
2
Hz, J 5 2.4 Hz, 6-R), 4.06 (4H, m, 5-R), 2.21, 2.11, 2.10, 2.09 ((12H, s,
Me) 3 4). 13C NMR (CDCl3, 75.46 MHz): d (ppm) 5 170.80, 170.49,
169.65 (CO 3 3), 156.63 (4-Ar), 150.45 (a-P), 137.88 (1-Ar), 135.47 (2,6-
Ar), 132.09 (b-P), 120.39 (meso-P), 114.99 (3,5-Ar), 99.18 (1-R), 72.81 (3-
R), 72.23 (5-R), 71.29 (2-R), 68.35 (4-R), 62.04 (6-R), 20.70, 20.65, 20.55,
20.52 (Me 3 4). Elecrospary ionization–high-resolution mass spectrometry
Zinc(II)5,10,15,20-tetrakis[4-(2,3,4-tri-O-acetyl-b-D-arabinopyranosy-
loxy)phenyl]porphyrin (p-2d). Recrystallization from CHCl3/EtOH gave
a red solid p-2d (yield: 63%). Purity (HPLC): 96%. UV–vis (DMSO): kmax
(nm) (e 3 10ꢁ3/Mꢁ1 cmꢁ1) 5 430 (46.8), 563 (2.26), 603 (2.01). H NMR
1

Photochemistry and Photobiology, 2004, 80 303
3
(CDCl3, 300.07 MHz): d (ppm) 5 8.85 (8H, s, b-P), 8.14 (8H, d, J 5 8.4
(3-Ar), 143.65 (a-P, 1-Ar), 132.20 (b-P), 129.83 (5-Ar), 128.25 (6-Ar),
123.22 (4-Ar), 119.48 (meso-P), 116.28 (2-Ar), 98.66 (1-R), 70.59 (3-R),
70.28 (2-R), 68.38 (4-R), 61.86 (5-R), 20.61 (Me). FAB-HRMS (m/z)5[M
þ H]þ calcd for C88H87N4O32, 1711.52979; found, 1711.52702.
Hz, 2,6-Ar), 7.41 (8H, d, 3J58.7 Hz, 3,5-Ar), 5.58 (4H, dd, 3J56.3 Hz, 3J
5 8.7 Hz, 3-R), 5.44 (8H, m, 1,4-R), 5.15 (4H, dd, 3J 5 9.0 Hz, 2-R), 4.32
(4H, dd, 3J 5 3.6 Hz, 2J 5 11.4 Hz, 5-R), 3.96 (4H, d, 3J 5 11.4 Hz, 5-R),
2.28, 2.24, 2.17 ((9H, s, Me)33). 13C NMR (CDCl3, 75.46 MHz): d (ppm)
5 170.53, 170.40, 169.77 (CO 3 3), 156.47 (4-Ar), 150.47 (a-P), 137.62
(1-Ar), 135.50 (2,6-Ar), 132.06 (b-P), 120.46 (meso-P), 114.89 (3,5-Ar),
98.87 (1-R), 69.88 (3-R), 69.09 (2-R), 67.25 (4-R), 62.96 (5-R), 20.83,
20.65 (CH3 3 2). FAB-HRMS (m/z) 5 [M]þ calcd for C88H84N4O32Zn,
1772.436; found, 1772.4337.
Zinc(II)5,10,15,20-tetrakis[3-(2,3,4-tri-O-acetyl-b-D-arabinopyranosy-
loxy)phenyl]porphyrin (m-2d). Recrystallization from CHCl3/EtOH gave
a red solid m-2d (yield: 60%). Purity (HPLC): .99%. UV–vis (DMSO):
kmax (nm) (e 3 10ꢁ3/Mꢁ1 cmꢁ1) 5 430 (34.0), 560 (2.89), 599 (2.10). 1H
NMR (CDCl3, 300.07 MHz): d (ppm) 5 8.97 (8H, br, b-P), 7.90 (8H, m,
2,6-Ar), 7.67 (4H, m, 5-Ar), 7.43 (4H, m, 4-Ar), 5.54 (4H, m, 3-R), 5.34
(8H, m, 1,4-R), 5.16 (4H, m, 2-R), 4.12 (4H, m, 5-R), 3.73 (4H, m, 5-R),
3.44–3.40 (4H, m, 5-R), 2.11, 2.09, 2.03 ((9H, s, Me) 3 3). 13C NMR
(CDCl3, 75.46 MHz): d (ppm) 5 170.40, 170.25, 169.60 (CO 3 3), 155.19
(3-Ar), 150.17 (a-P), 144.30 (1-Ar), 132.15 (b-P), 129.69 (5-Ar), 127.65
(6-Ar), 123.09 (4-Ar), 120.49 (meso-P), 116.07 (2-Ar), 98.95 (1-R),
69.80 (3-R), 68.98 (2-R), 67.05 (4-R), 62.81 (5-R), 20.72 (Me).
FAB-HRMS (m/z) 5 [M]þ calcd for C88H84N4O32Zn, 1772.436; found,
1772.4303.
5,10,15,20-Tetrakis[4-(2,3,4-tri-O-acetyl-b-D-arabinopyranosiloxy)phe-
nyl]porphyrin (p-4d). p-4d was synthesized by the procedure same as that
of reference. The pure product was crystallized from CHCl3/EtOH to give
a red solid (yield: 36%). Purity (HPLC): .99%. UV–vis (DMSO): kmax
(nm) (e 3 10ꢁ3/Mꢁ1 cmꢁ1) 5 422 (36.9), 518 (2.21), 554 (1.53), 592 (0.88),
1
644 (0.82). H NMR (CDCl3, 300.07 MHz): d (ppm) 5 8.85 (8H, s, b-P),
3
8.14 (8H, d, J 5 8.4 Hz, 2,6-Ar), 7.41 (8H, d, 3J 5 8.7 Hz, 3,5-Ar), 5.58
(4H, dd, 3J56.3 Hz, 3J58.7 Hz, 3-R), 5.44 (8H, m, 1,4-R), 5.15 (4H, d, 3J
59.0 Hz, 2-R), 4.32 (4H, dd, 3J53.6 Hz, 2J511.4 Hz, 5-R), 3.96 (4H, dd,
2J 5 11.4 Hz, 5-R), 2.28, 2.24, 2.17 ((9H, s, Me) 3 3), ꢁ2.82 (2H, br, NH).
13C NMR (CDCl3, 75.46 MHz): d (ppm) 5 170.53, 170.41, 169.77 (CO 3
3), 156.61 (a-P and 1-Ar), 136.96 (4-Ar), 135.68 (2,6-Ar), 131.12 (b-P),
119.47 (meso-P), 115.02 (3,5-Ar), 98.84 (1-R), 69.88 (3-R), 69.09 (2-R),
67.23 (4-R), 62.96 (5-R), 20.88, 20.85, 20.68 (Me 3 3). FAB-HRMS (m/z)
5 [M þ H]þ calcd for C88H87N4O32, 1711.52979; found, 1711.53042.
5,10,15,20-Tetrakis[3-(2,3,4-tri-O-acetyl-b-D-arabinopyranosiloxy)phe-
nyl]porphyrin (m-4d). Recrystallization from CHCl3/EtOH gave a reddish
purple solid m-4d (yield: 37%). Purity (HPLC): .99%. UV–vis (DMSO):
kmax (nm) (e 3 10ꢁ3/Mꢁ1 cmꢁ1) 5 430 (34.0), 514 (2.02), 550 (1.25), 589
(1.07), 644 (0.84). 1H NMR (CDCl3, 300.07 MHz): d (ppm) 5 8.88 (8H, s,
b-P), 7.95 (8H, m, 2,6-Ar), 7.68 (4H, m, 5-Ar), 7.48 (4H, m, 4-Ar), 5.70
(4H, t, 3J 5 8.7 Hz, 3-R), 5.37 (8H, m, 1,4-R), 5.16 (4H, m, 2-R), 4.16 (4H,
m, 5-R), 3.79 (4H, m, 5-R), 2.14, 2.09, 2.06 ((9H, s, Me) 3 3), ꢁ2.78 (2H,
br, NH). 13C NMR (CDCl3, 75.46 MHz): d (ppm)5170.27, 170.12, 169.52
(CO 3 3), 155.16 (3-Ar), 143.51 (a-P and 1-Ar), 131.21 (b-P), 129.63 (5-
Ar), 127.75 (6-Ar), 123.11 (4-Ar), 119.42 (meso-P), 116.27 (2-Ar), 98.86
(1-R), 69.69 (3-R), 68.85 (2-R), 67.05 (4-R), 62.81 (5-R), 20.62, 20.44,
5,10,15,20-Tetrakis[4-(2,3,4,6-tetra-O-acetyl-b-D-glucopyranosyloxy)-
phenyl]porphyrin (p-4a). Recrystallization from CHCl3/Et2O gave a red
solid p-4a (yield: 64%). 1H and 13C NMR spectra agreed with the literature
(3,21,23).
5,10,15,20-Tetrakis[3-(2,3,4,6-tetra-O-acetyl-b-D-glucopyranosyloxy)-
phenyl]porphyrin (m-4a). Recrystallization from CHCl3/EtOH gave a red
solid m-4a (yield: 68%). Purity (HPLC): .99%. H and 13C NMR spectra
agreed with the literature (3,21,23).
1
20.30 (Me 3 3). FAB-HRMS (m/z) 5 [M þ H]þ calcd for C88H87N4O32
,
5,10,15,20-Tetrakis[4-(2,3,4,6-tetra-O-acetyl-b-D-galactopyranosyloxy)-
phenyl]porphyrin (p-4b). Recrystallization from CHCl3/EtOH gave a pur-
ple solid p-4b (yield: 62%). Purity (HPLC): .99%. 1H and 13C NMR
spectra agreed with the literature (3,21,23).
1711.52979; found, 1711.52918.
Zinc(II)5,10,15,20-tetrakis[4-(b-D-glucopyranosyloxy)phenyl]porphyrin
(p-3a). Yield: 94%. Purity (HPLC): .99%. UV–vis (DMSO): kmax (nm) (e
3 10ꢁ3/Mꢁ1 cmꢁ1) 5 431 (52.4), 560 (0.18), 601 (1.39). 1H NMR (CD3OD,
3
5,10,15,20-Tetrakis[3-(2,3,4,6-tetra-O-acetyl-b-D-galactopyranosyloxy)-
phenyl]porphyrin (m-4b). Recrystallization from CHCl3/EtOH gave a pur-
ple solid m-4b (yield: 57%). Purity (HPLC): 98%. UV–vis (DMSO): kmax
(nm) (e 3 10ꢁ3/Mꢁ1 cmꢁ1) 5 419 (38.0), 513 (1.67), 548 (0.63), 589 (0.54),
646 (0.62). 1H NMR (CDCl3, 300.07 MHz): d (ppm) 5 8.89 (8H, br, b-P),
7.95–7.84 (8H, m, 2,6-Ar), 7.73–7.62 (4H, m, 5-Ar), 7.50–7.42 (4H, m,
4-Ar), 5.68–5.54 (4H, m, 2-R), 5.54–5.38 (4H, m, 4-R), 5.38–5.20 (4H, m,
1-R), 5.20–4.93 (4H, m, 3-R), 4.16–4.04 (12H, m, 5,6-R), 2.17–2.01 þ
1.29–1.22 (48H, m, Me), ꢁ2.88 (2H, br, NH). 13C NMR (CDCl3, 75.46
MHz): d (ppm) 5 170.37, 170.27, 169.60 (CO 3 3), 155.45 (3-Ar), 150.13
(a-P), 143.54 (1-Ar), 131.42 (b-P), 129.84 (5-Ar), 127.85 (6-Ar), 122.59
(4-Ar), 119.52 (meso-P), 116.59 (2-Ar), 99.76 (1-R), 71.11 (5-R), 70.76
(3-R), 66.64 (2-R), 66.87 (4-R), 61.40 (6-R), 20.68, 20.49, 20.43, 19.62
300.07 MHz): d (ppm) 5 8.83 (8H, s, b-P), 8.10 (8H, d, J 5 8.4 Hz, 2,6-
Ar), 7.49 (8H, d, 3J 5 8.4 Hz, 3,5-Ar), 5.26 (4H, d, 3J 5 4.5 Hz, 1-R), 4.01
3
2
3
2
(4H, dd, J 5 1.8 Hz, J 5 12.0 Hz, 6-R), 3.80 (4H, dd, J 5 5.1 Hz, J 5
11.7 Hz, 6-R), 3.69–3.48 (16H, m, 2,3,4,5-R). 1H NMR (DMSO-d6, 300.07
MHz): d (ppm) 5 8.81 (8H, s, b-P), 8.08 (8H, d, 3J 5 8.1 Hz, 2,6-Ar), 7.44
(8H, d, 3J 5 7.8 Hz, 3,5-Ar), 5.51 (4H, d, 3J 5 5.1 Hz, 2-ROH), 5.21 (4H,
m, 2-R), 5.21 (4H, br, 1-R), 5.11 (8H, br, 3,4-ROH), 4.88–4.64 (4H, m, 6-
ROH), 3.80 (4H, m, 6-R), 3.62–3.42 (20H, m, 2,3,4,5,6-R). 13C NMR
(DMSO-d6, 75.46 MHz): d (ppm) 5 157.54 (4-Ar), 149.95 (a-P), 136.62
(1-Ar), 135.43 (2,6-Ar), 131.95 (b-P), 120.24 (meso-P), 114.52 (3,5-Ar),
100.97 (1-R), 77.54 (3-R), 77.02 (5-R), 73.77 (2-R), 70.10 (4-R), 61.09 (6-
R). ESI-HRMS (m/z) 5 [MþH]þ calcd for C68H67N4O24Zn, 1387.3437;
found, 1387.3391.
(Me 3 4). ESI-HRMS (m/z) 5 [M ꢁ H]ꢁ calcd for C100H101N4O40
,
Zinc(II)5,10,15,20-tetrakis[3-(b-D-glucopyranosyloxy)phenyl]porphyrin
(m-3a). Yield: 89%. Purity (HPLC): .99%. UV–vis (DMSO): kmax (nm)
(e 3 10ꢁ3/Mꢁ1 cmꢁ1) 5 429 (50.6), 560 (0.19), 600 (0.87). 1H NMR
(CD3OD, 600.13 MHz): d (ppm)5 8.90–8.84 (8H, m, b-P), 7.95–7.85 (8H,
m, 2,6-Ar), 7.68–7.64 (4H, m, 5-Ar), 7.53–7.49 (4H, m, 4-Ar), 5.22–5.20
(4H, m, 1-R), 3.82–3.81 (4H, m, 6-R), 3.68–3.66 (4H, m, 6-R), 3.56–3.43
(12H, m, 2,3,5-R), 3.39 (4H, m, 4-R). 1H NMR (DMSO-d6, 300.07 MHz):
d (ppm)58.84 (8H, m, b-P), 7.81–7.79 (8H, m, 2,6-Ar), 7.73–7.70 (4H, m,
5-Ar), 7.51–7.48 (4H, m, 4-Ar), 5.42 (4H, d, 3J 5 4.2 Hz, 2-ROH), 5.19
(4H, m, 3- or 4-ROH), 5.11 (4H, m, 1-R), 5.01 (4H, br, 3- or 4-ROH), 4.60
(4H, m, 6-ROH), 3.82 (4H, d, 2J512.0 Hz, 6-R), 3.67 (4H, dd, 3J55.4 Hz,
2J 5 13.2 Hz, 6-R), 3.62–3.31 (16H, m, 2,3,4,5-R). 13C NMR (DMSO-d6,
75.46 MHz): d (ppm) 5 155.12 (3-Ar), 149.60 (a-P), 144.41 (1-Ar), 132.06
(b-P), 128.87 (5-Ar), 127.84 (6-Ar), 122.79 (4-Ar), 120.22 (meso-P),
115.48 (2-Ar), 100.85 (1-R), 77.31 (3-R), 76.85 (5-R), 73.72 (2-R), 69.95
1997.5992; found, 1997.5996.
5,10,15,20-Tetrakis[4-(2,3,4-tri-O-acetyl-b-D-xylopyranosyloxy)phenyl]-
porphyrin (p-4c). Recrystallization from CHCl3/EtOH gave a red purple
solid p-4c (yield: 64%). Purity (HPLC): .99%. UV–vis (DMSO): kmax
(nm) (e 3 10ꢁ3/Mꢁ1 cmꢁ1) 5 422 (47.0), 517 (2.58), 554 (1.72), 592 (1.08),
649 (0.97). 1H NMR (CDCl3, 800.03 MHz): d (ppm) 5 8.85 (8H, br, b-P),
8.13 (8H, d, 3J 5 8.7 Hz, 2,6-Ar), 7.39 (8H, d, 3J 5 8.4 Hz, 3,5-Ar), 5.53–
5.55 (4H, m, 1-R), 5.40–5.38 (8H, m, 2,3-R), 5.17–5.15 (4H, m, 4-R), 4.44
(4H, dd, J 5 4.5 Hz, J 5 11.9 Hz, 5-R), 3.74 (4H, dd, J 5 7.6 Hz, J 5
11.9 Hz, 5-R), 2.23, 2.18, 2.14 ((9H, s, Me) 3 3), ꢁ2.815 (2H, br, NH). 13C
NMR (CDCl3, 75.46 MHz): d (ppm) 5 170.22, 170.11, 169.73 (CO 3 3),
156.55 (4-Ar), 136.99 (a-P and 1-Ar), 135.71 (2,6-Ar), 130.10 (b-P),
119.45 (meso-P), 115.02 (3,5-Ar), 98.58 (1-R), 70.752 (3-R), 70.24 (2-R),
68.52 (4-R), 62.04 (5-R), 20.71 (Me). FAB-HRMS (m/z) 5 [M þ H]þ calcd
for C88H87N4O32, 1711.52979; found, 1711.53108.
3
2
3
2
(4-R), 60.91 (6-R). ESI-HRMS (m/z)
68H68N4O24Zn, 1389.2845; found, 1389.2853.
Zinc(II)5,10,15,20-tetrakis[4-(b-D-galactopyranosyloxy)phenyl]porphyr-
5
[M
þ
H]þ calcd for
5,10,15,20-Tetrakis[3-(2,3,4-tri-O-acetyl-b-D-xylopyranosiloxy)phenyl]-
porphyrin (m-4c). Recrystallization from CHCl3/EtOH gave a red solid m-
4c (yield: 86%). Purity (HPLC): .99%. UV–vis (DMSO): kmax (nm) (e 3
10ꢁ3/Mꢁ1 cmꢁ1) 5 420 (41.2), 514 (2.72), 549 (1.46), 589 (1.28), 644
C
in (p-3b). Yield: 90%. Purity (HPLC): .99%. UV–vis (DMSO): kmax (nm)
(e 3 10ꢁ3/Mꢁ1 cmꢁ1) 5 431 (52.4), 563 (3.33), 602 (2.64). 1H NMR
(CD3OD, 300.07 MHz): d (ppm) 5 8.84 (8H, s, b-P), 8.10 (8H, d, 3J 5 8.4
Hz, 2,6-Ar), 7.49 (8H, d, 3J 5 8.4 Hz, 3,5-Ar), 5.20 (4H, d, 3J 5 7.8 Hz, 1-
R), 4.02–3.80 (20H, m, 2,4,5,6-R), 3.74–3.70 (4H, m, 3-R). 1H NMR
(DMSO-d6, 300.07 MHz): d (ppm) 5 8.81 (8H, s, b-P), 8.08 (8H, d, 3J 5
8.4 Hz, 2,6-Ar), 7.45 (8H, d, 3J58.1 Hz, 3,5-Ar), 5.37 (4H, d, 3J55.1 Hz,
1
(1.09). H NMR (CDCl3, 300.07 MHz): d (ppm) 5 8.88 (8H, s, b-P), 7.94
(4H, m, 2-Ar), 7.87 (4H, m, 6-Ar), 7.68–7.43 (8H, m, 4,5-Ar), 5.38 (4H, m,
2-R), 5.25 (8H, m, 1,3-R), 5.05 (4H, m, 4-R), 4.26 (4H, m, 5-R), 3.57 (4H,
m, 5-R), 2.10, 2.06, 2.04 ((9H, s, Me) 3 3), ꢁ2.87 (2H, br, NH). 13C NMR
(CDCl3, 75.46 MHz): d (ppm) 5 170.10, 169.98, 169.60 (CO 3 3), 155.16

304 Shiho Hirohara et al.
2-ROH), 5.11 (4H, d, 3J 5 7.5 Hz, 1-R), 4.99 (4H, d, 3J 5 5.4 Hz, 3-ROH),
4.77 (4H, m, 6-ROH), 4.63 (4H, d, 3J 5 4.5 Hz, 4-ROH), 3.83–3.77 (12H,
m, 4,2,6-R), 3.77–3.63 (8H, m, 6,5-R), 3.63–3.50 (4H, m, 3-R). 13C NMR
(DMSO-d6, 75.46 MHz): d (ppm) 5 157.64 (4-Ar), 149.95 (a-P), 136.54
(1-Ar), 135.43 (2,6-Ar), 131.93 (b-P), 120.25 (meso-P), 114.57 (3,5-Ar),
101.66 (1-R), 76.00 (5-R), 73.71 (3-R), 70.79 (2-R), 68.56 (4-R), 60.81 (6-
R). ESI-HRMS (m/z) 5 [M ꢁ H]ꢁ calcd for C68H67N4O24Zn, 1387.3437;
found, 1387.3418.
149.63 (a-P), 144.39 (1-Ar), 132.05 (b-P), 128.70 (5-Ar), 127.84 (6-Ar),
122.84 (4-Ar), 120.30 (meso-P), 115.88 (2-Ar), 101.39 (1-R), 72.78 (3-R),
70.78 (2-R), 67.93 (4-R), 66.07 (5-R). FAB-HRMS (m/z) 5 [M þ Na]þ
calcd for C64H60N4O20ZnNa, 1291.29846; found, 1291.29759.
5,10,15,20-Tetrakis[4-(b-D-glucopyranosyloxy)phenyl]porphyrin (p-
5a). Yield: 91%. Purity (HPLC): 96%. 1H and 13C NMR spectra agreed
with the literature (3,21,23).
5,10,15,20-Tetrakis[3-(b-D-glucopyranosyloxy)phenyl]porphyrin (m-
5a). Yield: 96%. Purity (HPLC): 98%. 1H and 13C NMR spectra agreed
with the literature (3,21,23).
5,10,15,20-Tetrakis[4-(b-D-galactopyranosyloxy)phenyl]porphyrin (p-
5b). Yield: 87%. Purity (HPLC): .99%. 1H and 13C NMR spectra agreed
with the literature (3,21,23).
5,10,15,20-Tetrakis[3-(b-D-galactopyranosyloxy)phenyl]porphyrin (m-
5b). Yield: 76%. Purity (HPLC): .99%. UV–vis (DMSO): kmax (nm) (e
3 10ꢁ3/Mꢁ1 cmꢁ1) 5 420 (45.4), 515 (3.11), 550 (2.02), 591 (1.87), 645
(1.73) nm. 1H NMR (CD3OD, 300.07 MHz): d (ppm) 5 8.92 (8H, br, b-P),
7.97 (4H, br, 2-Ar), 7.85 (4H, m, 6-Ar), 7.67 (4H, m, 5-Ar), 7.57 (4H, m, 4-
Ar), 5.17 (4H, d, 3J 5 7.5 Hz, 1-R), 3.93–3.87 (8H, m, 2,4-R), 3.71 (8H, m,
6-R), 3.65–3.58 (8H, m, 3,5-R). 1H NMR (DMSO-d6, 300.07 MHz): d
(ppm) 5 8.89 (8H, br, b-P), 7.89 (4H, br, 2-Ar), 7.82 (4H, br, 6-Ar), 7.73
(4H, m, 5-Ar), 7.51 (4H, m, 4-Ar), 5.29 (4H, d, 3J 5 4.8 Hz, 2-ROH), 5.14
(4H, d, 3J57.5 Hz, 1-R), 4.88 (4H, d, 3J55.4 Hz, 3-ROH), 4.55–4.53 (8H,
m, 4,6-ROH), 3.68 (8H, br, 2,4-R), 3.64–3.44 (16H, m, 3,5,6-R), ꢁ2.97
(2H, br, NH). 13C NMR (DMSO-d6, 75.46 MHz): d (ppm)5156.35 (3-Ar),
142.74 (a-P and 1-Ar), 131.56 (b-P), 128.71 (5-Ar), 128.24 (6-Ar), 122.65
(4-Ar), 119.94 (meso-P), 116.26 (2-Ar), 101.23 (1-R), 75.700 (5-R), 73.53
(3-R), 70.68 (2-R), 68.34 (4-R), 60.51 (6-R). ESI-HRMS (m/z) 5 [M ꢁ H]ꢁ
calcd for C68H69N4O24, 1325.43027; found, 1325.4304.
5,10,15,20-Tetrakis[4-(b-D-xylopyranosyloxy)phenyl]porphyrin (p-
5c). Yield: 94%. Purity (HPLC): 98%. UV–vis (DMSO): kmax (nm) (e 3
10ꢁ3/Mꢁ1 cmꢁ1) 5 423 (29.6), 518 (2.01), 555 (1.80), 592 (0.87), 649
(0.91). 1H NMR (CD3OD, 300.07 MHz): d (ppm)58.85 (8H, br, b-P), 8.10
(8H, d, 3J 5 8.4 Hz, 2,6-Ar), 7.48 (8H, d, 3J 5 8.4 Hz, 3,5-Ar), 5.24 (4H, d,
3J 5 7.2 Hz, 1-R), 4.11–4.05 (4H, m, 5-R), 3.81–3.51 (16H, m, 2,3,4,5-R).
1H NMR (DMSO-d6, 300.07 MHz): d (ppm) 5 8.86 (8H, s, b-P), 8.14 (8H,
m, 2,6-Ar), 7.52–7.43 (8H, m, 3,5-Ar), 5.24 (4H, d, 3J 5 7.2 Hz, 1-R),
5.77–4.80 (12H, m, 2,3,4-ROH), 3.90 (4H, d, 3J 5 6.3 Hz, 5-R), 3.77–3.34
(16H, m, 2,3,4,5-R), ꢁ2.92 (2H, br, NH). 13C NMR (DMSO-d6, 75.46
MHz): d (ppm) 5 157.61 (4-Ar), 135.64 (a-P and 1-Ar), 135.09 (2,6-Ar),
131.18 (b-P), 119.94 (meso-P), 114.91 (3,5-Ar), 101.24 (1-R), 76.86 (3-R),
73.55 (2-R), 69.74 (4-R), 66.15 (5-R). FAB-HRMS (m/z) 5 [M þ H]þ calcd
for C64H63N4O20, 1207.40302; found, 1207.40263.
Zinc(II)5,10,15,20-tetrakis[3-(b-D-galactopyranosyloxy)phenyl]porphyr-
in (m-3b). Yield: 90%. Purity (HPLC): 98%. UV–vis (DMSO): kmax (nm)
(e 3 10ꢁ3/Mꢁ1 cmꢁ1) 5 429 (44.9), 559 (0.92), 597 (0.71). 1H NMR
(CD3OD, 300.07 MHz): d (ppm) 5 8.87 (8H, br, b-P), 7.96 (4H, br, 2-Ar),
7.60 (4H, m, 6-Ar), 7.81 (4H, m, 5-Ar), 7.56 (4H, m, 4-Ar), 5.17 (4H, d, 3J
5 7.5 Hz, 1-R), 3.93–3.87 (8H, m, 2,4-R), 3.87–3.71 (8H, m, 6-R), 3.71–
3.59 (8H, m, 3,5-R). 1H NMR (DMSO-d6, 300.07 MHz): d (ppm) 5 8.84
(8H, br, b-P), 7.83–7.72 (8H, m, 2,6-Ar), 7.72–7.61 (4H, m, 5-Ar), 7.49–
7.47 (4H, m, 4-Ar), 5.28 (4H, d, 3J54.8 Hz, 2-ROH), 5.13 (4H, d, 3J56.6
3
Hz, 1-R), 5.01–4.72 (4H, d, J 5 2.7 Hz, 3-ROH), 4.58–4.52 (8H, m, 4,6-
ROH), 3.73–3.32 (24H, m, 2,3,4,5,6-R). 13C NMR (DMSO-d6, 75.46
MHz): d (ppm)5156.15 (3-Ar), 149.62 (a-P), 144.39 (1-Ar), 131.95 (b-P),
128.79 (5-Ar), 127.81 (6-Ar), 122.68 (4-Ar), 120.27 (meso-P), 115.69 (2-
Ar), 101.40 (1-R), 75.70 (5-R), 73.58 (3-R), 70.79 (2-R), 68.27 (4-R), 60.44
(6-R). ESI-HRMS (m/z) 5 [M ꢁ H]ꢁ calcd for C68H67N4O24Zn, 1387.3437;
found, 1387.3433.
Zinc(II)5,10,15,20-tetrakis[4-(b-D-xylopyranosyloxy)phenyl]porphyrin
(p-3c). Yield: 98%. Purity (HPLC): .99%. UV–vis (DMSO): kmax (nm) (e
3 10ꢁ3/Mꢁ1 cmꢁ1) 5 431 (38.9), 560 (1.51), 602 (0.87). 1H NMR (CD3OD,
3
300.07 MHz): d (ppm) 5 8.81 (8H, s, b-P), 8.05 (8H, d, J 5 8.4 Hz, 2,6-
Ar), 7.37 (8H, d, 3J58.7 Hz, 3,5-Ar), 5.18 (4H, d, 3J57.5 Hz, 1-R), 4.06–
4.00 (8H, m, 5-R), 3.76–3.43 (12H, m, 2,3,4-R). 1H NMR (DMSO-d6,
300.07 MHz): d (ppm) 5 8.82 (8H, s, b-P), 8.10 (8H, br, 2,6-Ar), 7.45 (8H,
br, 3,5-Ar), 5.54 (4H, br, 2- or 4-ROH), 5.23 (8H, br, 1-R, 3-ROH), 5.17
(4H, br, 2- or 4-ROH), 3.91 (8H, br, 5-R), 3.91–3.58 (12H, m, 2,3,4-R). 13C
NMR (DMSO-d6, 75.46 MHz): d (ppm) 5 157.20 (4-Ar), 149.94 (a-P),
136.67 (1-Ar), 135.44 (2,6-Ar), 131.90 (b-P), 120.20 (meso-P), 114.56
(3,5-Ar), 101.26 (1-R), 76.88 (3-R), 73.56 (2-R), 69.78 (4-R), 66.12 (5-R).
FAB-HRMS (m/z) 5 [M]þ calcd for C64H60N4O20Zn, 1268.30869; found,
1268.30900.
Zinc(II)5,10,15,20-tetrakis[3-(b-D-xylopyranosyloxy)phenyl]porphyrin
(m-3c). Yield: 92%. Purity (HPLC): 95%. UV–vis (DMSO): kmax (nm) (e
3 10ꢁ3/Mꢁ1 cmꢁ1) 5 429 (37.2), 559 (2.53), 602 (1.30). 1H NMR (CD3OD,
300.07 MHz): d (ppm)58.88 (8H, s, b-P), 7.94–7.87 (8H, m, 2,6-Ar), 7.70–
7.65 (4H, m, 5-Ar), 7.51–7.49 (4H, m, 4-Ar), 5.15 (4H, d, 3J57.5 Hz, 1-R),
3.94–3.89 (4H, m, 5-R), 3.63–3.36 (16H, m, 2,3,4,5-R). 1H NMR (DMSO-
d6, 300.07 MHz): d (ppm) 5 8.81 (8H, s, b-P), 7.79 (8H, br, 2,6-Ar), 7.70–
7.67 (4H, m, 5-Ar), 7.48–7.45 (4H, m, 4-Ar), 5.47 (4H, br, 2-ROH), 5.14
(8H, br, 1-R, 3-ROH), 5.05 (4H, m, 4-ROH), 3.82–3.66 (4H, m, 5-R), 3.51–
3.17 (16H, m, 2,3,4,5-R). 13C NMR (DMSO-d6, 75.46 MHz): d (ppm) 5
155.88 (3-Ar), 149.57 (a-P), 144.36 (1-Ar), 132.01 (b-P), 128.81 (5-Ar),
127.82 (6-Ar), 122.82 (4-Ar), 120.22 (meso-P), 115.79 (2-Ar), 101.42 (1-R),
76.65 (3-R), 73.56 (2-R), 69.65 (4-R), 65.94 (5-R). FAB-HRMS (m/z) 5
[M]þ calcd for C64H60N4O20Zn, 1268.30869; found, 1268.30957.
Zinc(II)5,10,15,20-tetrakis[4-(b-D-arabinopyranosyloxy)phenyl]por-
phyrin (p-3d). Yield: 98%. Purity (HPLC): 99%. UV–vis (DMSO): kmax
5,10,15,20-Tetrakis[3-(b-D-xylopyranosyloxy)phenyl]porphyrin (m-
5c). Yield: 91%. Purity (HPLC): 95%. UV–vis (DMSO): kmax (nm) (e 3
10ꢁ3/Mꢁ1 cmꢁ1) 5 423 (20.2), 514 (2.81), 552 (1.37), 589 (1.09), 645
1
(1.00). H NMR (DMSO-d6, 300.07 MHz): d (ppm) 5 8.88 (8H, br, b-P),
7.85 (8H, m, 2,6-Ar), 7.77–7.71 (4H, m, 5-Ar), 7.56–7.46 (4H, m, 4-Ar),
5.17 (4H, d, 3J56.9 Hz, 1-R), 3.75–3.73 (4H, m, 5-R), 3.73–3.25 (16H, m,
2,3,4,5-R), ꢁ2.96 (2H, br, NH). 13C NMR (DMSO-d6, 75.46 MHz): d
(ppm) 5 156.12 (3-Ar), 124.80 (a-P, 1-Ar), 131.72 (b-P), 128.76 (5-Ar),
128.27 (6-Ar), 122.82 (4-Ar), 119.91 (meso-P), 116.35 (2-Ar), 101.45 (1-
R), 76.64 (3-R), 73.46 (2-R), 69.65 (4-R), 65.99 (5-R). FAB-HRMS (m/z)5
[M ꢁ H]ꢁ calcd for C64H63N4O20, 1207.40302; found, 1207.40359.
5,10,15,20-Tetrakis[4-(b-D-arabinopyranosyloxy)phenyl]porphyrin (p-
5d). Yield: 96%. Purity (HPLC): 99%. UV–vis (DMSO): kmax (nm) (e 3
10ꢁ3/Mꢁ1 cmꢁ1) 5 423 (20.9), 518 (2.12), 556 (1.86), 590 (1.36), 649
(1.80). 1H NMR (CD3OD, 300.07 MHz): d (ppm) 5 8.89 (8H, s, b-P), 8.11
(8H, d, 3J 5 8.7 Hz, 2,6-ArH), 7.51 (8H, d, 3J 5 8.1 Hz, 3,5-Ar), 5.21 (4H,
d, 3J 5 6.0 Hz, 1-R), 4.11–3.77 (20H, m, 2,4,3,5-R). 1H NMR (DMSO-d6,
1
(nm) (e 3 10ꢁ3/Mꢁ1 cmꢁ1) 5 431 (22.6), 563 (2.01), 603 (2.06). H NMR
(DMSO-d6, 300.07 MHz): d (ppm) 5 8.81 (8H, s, b-P), 8.09 (8H, d, 3J 5
8.4 Hz, 2,6-Ar), 7.42 (8H, d, 3J 58.4 Hz, 3,5-Ar), 5.39 (4H, d, 3J 54.8 Hz,
2-ROH), 5.19 (4H, d, 3J 5 6.9 Hz, 1-R), 4.95 (4H, d, 3J 5 5.4 Hz, 3-ROH),
3
4.76 (4H, d, J 5 3.9 Hz, 4-ROH), 3.92–3.89 (4H, m, 5-R), 3.81 (12H, br,
2,4,3-R), 3.60 (4H, br, 5-R). 13C NMR (DMSO-d6, 75.46 MHz): d (ppm)5
157.28 (4-Ar), 149.94 (a-P), 136.56 (1-Ar), 135.48 (2,6-Ar), 131.88 (b-P),
120.20 (meso-P), 114.61 (3,5-Ar), 101.32 (1-R), 73.01 (3-R), 70.83 (2-R),
68.06 (4-R), 66.27 (5-R). FAB-HRMS (m/z) 5 [M þ Na]þ calcd for
3
300.07 MHz): d (ppm) 5 8.87 (8H, s, b-P), 8.14 (8H, d, J 5 8.1 Hz, 2,6-
Ar), 7.45 (8H, d, 3J 5 8.4 Hz, 3,5-Ar), 5.41 (4H, br, 2-ROH), 5.21 (4H, d,
3J 5 7.2 Hz, 1-R), 4.98 (4H, m, 3-ROH), 4.77 (4H, m, 4-ROH), 3.92–3.50
(20H, m, 2,3,4,5-R), ꢁ2.92 (2H, br, NH). 13C NMR (DMSO-d6, 75.46
MHz): d (ppm) 5 157.72 (4-Ar), 135.67 (a-P and 1-Ar), 134.96 (2,6-Ar),
131.80 (b-P), 119.99 (meso-P), 114.98 (3,5-Ar), 101.26 (1-R), 72.93 (3-R),
70.75 (2-R), 68.08 (4-R), 66.33 (5-R). FAB-HRMS (m/z) 5 [M þ H]þ calcd
for C64H63N4O20, 1207.40302; found, 1207.40339.
C64H60N4O20ZnNa, 1291.29846; found, 1291.29604.
Zinc(II)5,10,15,20-tetrakis[3-(b-D-arabinopyranosyloxy)phenyl]por-
phyrin (m-3d). Yield: 96%. Purity (HPLC): 99%. UV–vis (DMSO): kmax
1
(nm) (e 3 10ꢁ3/Mꢁ1 cmꢁ1) 5 429 (23.6), 558 (2.37), 598 (2.00). H NMR
(CD3OD, 300.07 MHz): d (ppm) 5 8.86 (8H, s, b-P), 7.92 (4H, br, 2-Ar),
7.87–7.84 (4H, m, 6-Ar), 7.69–7.64 (4H, m, 5-Ar), 7.52–7.49 (4H, m, 4-
Ar), 5.13 (4H, d, 3J 5 7.2 Hz, 1-R), 3.96–3.34 (20H, m, 2,3,4,5-R). 1H
NMR (DMSO-d6, 300.07 MHz): d (ppm) 5 8.83 (8H, s, b-P), 7.81 (8H, br,
2,6-Ar), 7.73–7.68 (4H, m, 5-Ar), 7.49–7.46 (4H, m, 4-Ar), 5.53–4.54
5,10,15,20-Tetrakis[3-(b-D-arabinopyranosyloxy)phenyl]porphyrin (m-
5d). Yield: 98%. Purity (HPLC): .99%. UV–vis (DMSO): kmax (nm) (e
3 10ꢁ3/Mꢁ1 cmꢁ1) 5 429 (23.6), 515 (2.12), 555 (1.74), 590 (1.56), 647
(1.40). 1H NMR (CD3OD, 300.07 MHz): d (ppm)58.91 (8H, br, b-P), 7.94
(4H, br, 2-Ar), 7.85 (4H, m, 6-Ar), 7.70 (4H, m, 5-Ar), 7.56 (4H, m, 4-Ar),
5.13 (4H, d, 3J 5 7.2 Hz, 1-R), 3.94–3.84 (12H, m, 2,4,5-R), 3.69–3.63
3
(12H, br, 2,3,4-ROH), 5.13 (4H, d, J 5 6.6 Hz, 1-R), 3.76–3.17 (20H, m,
2,3,4,5-R). 13C NMR (DMSO-d6, 75.46 MHz): d (ppm) 5 155.96 (3-Ar),

Photochemistry and Photobiology, 2004, 80 305
In vitro studies
(8H, m, 3,5-R). 1H NMR (DMSO-d6, 300.07 MHz): d (ppm) 58.89 (8H, s,
b-P), 7.84 (4H, br, 2-Ar), 7.74 (4H, br, 6-Ar), 7.60 (4H, m, 5-Ar), 7.49 (4H,
m, 4-Ar), 5.30 (4H, d, 3J54.5 Hz, 2-ROH), 5.14 (4H, d, 3J 57.2 Hz, 1-R),
4.87 (4H, d, 3J 5 5.4 Hz, 3-ROH), 4.67 (4H, d, 3J 5 4.2 Hz, 4-ROH), 3.71
(8H, m, 2,5-R), 3.66–3.50 (12H, m, 3,4,5-R), ꢁ2.98 (2H, br, NH). 13C
NMR (DMSO-d6, 75.46 MHz): d (ppm) 5 156.17 (3-Ar), 142.77 (a-P and
1-Ar), 131.46 (b-P), 128.65 (5-Ar), 128.21 (6-Ar), 122.81 (4-Ar), 119.98
(meso-P), 116.39 (2-Ar), 101.26 (1-R), 72.75 (3-R), 70.73 (2-R), 67.87 (4-
Photosensitizers. Stock solutions of photosensitizers (TPPS and 16
deprotected glycoconjugated porphyrins) were prepared by weighing the
dried compounds and dissolving them in DMSO (Wako Pure Chemical
Industries, Ltd., Osaka, Japan). Stock solutions of photosensitizers were
kept in the freezer (ꢁ308C).
Cellular uptake of glycoconjugated porphyrins. HeLa cells were kindly
provided by Professor Jun-ya Kato (Graduate School of Biological Science,
Nara Institute of Science and Technology, Japan). They were grown in
Dulbecco modified Eagle medium (DMEM) with phenol red and 10% fetal
R), 66.04 (5-R). FAB-HRMS (m/z) 5 [M þ H]þ calcd for C64H63N4O20
,
1207.40302; found, 1207.40118.

306 Shiho Hirohara et al.
Table 1. Synthesis of glycoconjugated porphyrins
Yield (%)
BF3ꢀOEt2
BF3ꢀOEt2/Zn2þ
p-3a
m-3a
p-3b
m-3b
p-3c
m-3c
p-3d
m-3d
53.2*
26.0*
20
—à
—à
—à
—à
—à
64
68
62
57
64
86
36
37
*Kohara et al. (21).
Mailippe et al. (22).
àNo report is available.
calf serum (FCS) (Hyclone Laboratories, Inc., Logan, UT) (26). Cellular
uptake of glycoconjugated porphyrins was determined as follows: HeLa
cells (53104 cells/well) were plated onto a 24-well plate in 500 lL per well
of DMEM with phenol red and 10% FCS, and the plates were incubated for
24 h (378C, 5% CO2). The cells were further incubated for 24 h with 500 lL
Figure 2. Light dose–dependent photocytotoxicity of p-5a in HeLa cells.
[p-5a] 5 5 3 10ꢁ6 M. The light intensity applied was 24 mW/cm2 using
a halogen lamp (100 W, k . 500 nm). The percentage of cell survival was
determined by Cell Titer-GloÔ Luminescent assay after 24 h incubation.
The values represent the means 6 SD of six replicate experiments.
of 1 3 10ꢁ5 M (2% DMSO) photosensitizers in DMEM (c 5 5 3 10ꢁ6
M
[1% DMSO]). The medium was removed, and the cells were then washed
with Dulbecco phosphate-buffered saline (PBS)(ꢁ) (13.7 mM NaCl, 2.7
mM KCl, 8.1 mM Na2HPO4ꢀ12H2O and 1.5 mM KH2PO4, pH 7.4) (500 lL
32) (Wako Pure Chemical Industries, Ltd.). The cells were lysed in DMSO
(100 lL), and the lysate was pipetted to extract the photosensitizers. The
fluorescence intensity from photosensitizers was measured on a plate reader
using excitation and emission wavelengths of 430 and 635 nm, respectively
(27–31). The amount of photosensitizer uptake was calculated on the basis
of calibration data in each case.
Then, this 96-well plate was subjected to luminescence measurement using
a
plate reader. The percentage of cell survival was calculated by
normalization with respect to the value for no drug treatment.
Statistical analysis. All statistical evaluations were performed using
Student’s t-test. All values for cellular uptake and cytotoxicity are expressed
as mean 6 standard deviation.
General procedure for in vitro photocytotoxicity test. HeLa cells (5 3
103 cells/well) were plated in 100 lL of DMEM containing 10% FCS and
incubated overnight (378C, 5% CO2) in a 96-well plate. The cells were then
incubated for 24 h with 100 lL of 1 3 10ꢁ5 M (2% DMSO) photosensitizer
in DMEM containing 10% FCS (c 5 5 3 10ꢁ6 M [1% DMSO]). The cells
were washed in PBS(ꢁ) (100 lL32), and microliters of DMEM containing
10% FCS was added. The cells were then exposed to light from a 100 W
halogen lamp (Ushio, Tokyo, Japan KBEX-102A, equipped with a water
filter and a Y-50 cutoff filter [k . 500 nm]) for an appropriate time (0 min
for evaluation of cytotoxicity in the dark) (27–31). For all experiments, the
light intensity was varied from 0 to 24.5 mW/cm2 by using a UV–vis power
meter (ORION/TH, Ophir, Saituma, Japan). The light dose was simply
calculated as the product of light intensity (mW/cm2) and irradiation time,
and irradiation time was adjusted to obtain the desired light dose of 0, 16,
20, 30 or 40 J/cm2. Cell viability was measured after 24 h by means of
CellTiter-GloÔ Luminescent Assay (Promega Corporation, Madison, WI).
RESULTS AND DISCUSSIONS
Synthesis of glycoconjugated porphyrins
Glycoconjugated porphyrins were synthesized as shown in Scheme
1. p- or m-Hydroxybenzaldehyde was allowed to react with
peracetylated 1-bromoglycopyranosides according to the Halazy or
Knorr method in ca 50% yield (22,25). The condensations of 1 with
pyrrole were conducted using BF3ꢀOEt2 in the presence of
Zn(OAc)2ꢀ2H2O. After addition of BF3ꢀOEt2 to initiate the
condensation, the reaction mixture slowly became pale red,
Figure 3. Cytotoxicity in the dark (a) and photocytotoxicity (b) of p-5a, p-
5d and TPPS in HeLa cells. [Photosensitizer] 5 5 3 10ꢁ6 M. The light
intensity applied was 24 mW/cm2 using a 100 W halogen lamp (k . 500
nm). The percentage of cell survival was determined by Cell Titer-GloÔ
Luminescent assay after 48 h incubation. The values represent the means 6
SD of six replicate experiments. *Significant difference, P , 0.05.
Figure 1. The cellular uptake of glycoconjugated porphyrins 3a–d and 5a–
d by HeLa cells after incubation for 24 h. The values represent the mean 6
SD of three replicate experiments. àThe values for m-5c and p-5b could not
be quantified because of precipitation. **The value of p-5d was statistically
significantly (P , 0.01) different from that of p-5a.

Photochemistry and Photobiology, 2004, 80 307
indicating chelation with Zn2þ ion. This suppressed the formation of
polymeric by-products (e.g. oligopyrroles) at the porphyrinogen
formation stage. Theoxidation of the porphyrinogen with p-chloranil
gave the glycoconjugated porphyrins 2 in 53–93% yield. The zinc
ion in 2 was easily removed by washing with 4 M HCl to give the
free-base porphyrins 4 in 36–86% yields (Table 1). The yield of zinc
porphyrin depends on the amount of zinc(II) acetate used. To
synthesize the zinc porphyrin 2, hence, the large excess amount of
zinc(II) acetate was used to suppress the formation of free-base
porphyrin. In the synthesis of free-base porphyrin 4, however, the
excess amount of zinc(II) acetate reduces the overall yield, probably
because of the additional separation step. The detailed condition on
the synthesis of the glycoconjugated porphyrins slightly depends on
the glycoconjugated benzaldehyde. The amount of BF3ꢀOEt2 and p-
chloranil was determined by repeating trial. The Zn2þ ion template
strategy improved the yield about three-fold in the case of meta-
substituted tetraphenylporphyrins, and free-base porphyrins were
easily obtained by demetalation with 4 M HCl (32). As a result, this
procedure affords higher yields than the modified Lindsey method, in
spite of the additional step. Deprotection of 2 and 4 was carried out
using NaOMe in THF or in a mixture of MeOH and CH2Cl2 (20–24).
Eventually, 16 glycoconjugated porphyrins 3 and 5 were obtained in
moderate yield. The identity of these compounds was confirmed by
1H and 13C NMR, mass, IR and UV–vis spectroscopy. The purity of
all compounds was .95% as evaluated by HPLC using an
octadecylsilyl-supported silica gel column.
First, the photocytotoxicity of p-5a was examined using
a halogen lamp (100 W, k . 500 nm) and a thermostated stage
to find an appropriate light dose. Figure 2 shows the light dose
dependency of the photocytotoxicity of p-5a. The cell survival
decreased from 110.5% to 17.9% as the light dose increased, and
the lethal dose to 50% of the population was around 16 J/cm2.
In vitro study of relationship between cellular uptake
and photocytotoxicity
The cytotoxicity of p-5a, p-5d and TPPS to HeLa cells in the dark
was examined. Figure 3a shows the percentage of cell survival after
48 h incubation without light irradiation. These photosensitizers (p-
5a, p-5d and TPPS) showed no cytotoxicity in the dark.
The photocytotoxicity of p-5a, p-5d and TPPS was examined
with HeLa cells. The above results show that the light dose of 16 J/
cm2 was appropriate to examine differences of photocytotoxicity
among p-5a, p-5d and TPPS. Figure 3b shows the percentage of cell
survival after 24 h incubation. Photocytotoxicity of p-5a (35.6%)
and p-5d (27.6%) is significantly higher than that of TPPS (69.9%).
Interestingly, the order of photocytotoxicity (p-5d . p-5a . TPPS)
seems to be in accord with the order of cellular uptake. The low
photocytotoxicity of TPPS could thus be attributable to the lower
cellular uptake as compared with p-5a and p-5d. In contrast, the
difference in photocytotoxicity between p-5d and p-5a was small,
even though the cellular uptake of p-5d was almost twice as that of
p-5a. It was reported that the singlet oxygen (1O2)–producing ability
was independent of the kind of sugar units (23,31). Photosensitizers
accumulated in mitochondria or lysosomes (or both) induce
Cellular uptake of glycoconjugated porphyrins
1
apoptosis by O2 production (34), so p-5a may produce a higher
The cellular uptake of porphyrins by HeLa cells was examined
after incubation with photosensitizers (c 5 5 3 10ꢁ6 M) for 24 h.
Figure 1 shows the relative value of the cellular uptake against
TPPS as a standard. Among the 16 deprotected glycoconjugated
porphyrins, precipitation of m-5c and p-5b was detected by optical
microscopy. Because the precipitates were hardly removed from
the well, cellular uptake of m-5c and p-5b could not be determined.
The other glycoconjugated porphyrins 3 and 5 show higher uptake
than TPPS. The order of the cellular uptake amount was
determined as p-5d . p-5a . m-5a . m-5d . p-3d . m-5b,
p-5c . m-3d . m-3c . p-3c . m-3a . p-3b . p-3a . m-3b .
m-5b . TPPS. Interestingly, p-5d showed an uptake 18-fold
greater than that of TPPS. The xylopyranosyl (c) and arabinopyr-
anosyl (d) groups are much more effective in improving cellular
uptake than glucopyranosyl (a) and galactopyranosyl (b). This may
reflect the lack of one hydroxymethyl group. In addition, para-
substituted free-base porphyrins showed higher uptake than meta-
substituted ones, and free-base compounds 5 showed higher uptake
than the corresponding zinc compounds 3. These differences might
be related to the shape of the species in water, namely, the globular
structure of meta-substituted porphyrins and the dimeric form
of the zinc porphyrins (3,33). These findings indicate that glyco-
pyranosyl groups modulate the cellular uptake of porphyrin.
concentration of 1O2 in mitochondria or lysosomes (or both) than p-
5d. This could occur if p-5a is more readily concentrated around
these organelles as compared with p-5d. Differences in accumula-
tion might be related to the kinds of sugar units.
Recently, Yee et al. (35) reported the cellular uptake, subcellular
localization and photocytotoxicity of protoporphyrin IX (PpIX) and
protoporphyrin IX dimethyl ester (PME). Although PME always
showed a higher cellular uptake than PpIX, there was no significant
difference of photocytotoxicity between PpIX and PME under the
maximum cellular uptake condition (incubation time was 9 h). Under
those conditions, both PME and PpIX accumulated in the mitochon-
1
dria. Hence, mitochondrial damage due to O2 may be a critical
determinant of the photodynamic effect. High efficiency in cellular
uptake can reduce the necessary drug dose, which would be an
important requirement for a PDT drug. However, not only cellular
uptake but also selective accumulation of photosensitizer is
important.
In conclusion, the introduction of glycopyransyl groups on the
periphery of tetraphenylporphyrins improves both cellular uptake
and photocytotoxicity in HeLa cells. However, the cellular uptake
is not connected simply with the photocytotoxicity, indicating that
the subcellular localization may dominate the photodynamic effect.
Acknowledgements—This research was supported by a Grant-in-Aid for
Scientific Research on Priority Areas (417) from the Ministry of Educa-
tion, Culture, Sports, Science and Technology (MEXT) of Japanese Gov-
ernment, grants from OSAKA GAS and San-Ei Gen Foundation for
Food Chemical Research.
Light dose dependence of the effect of
glycosylated porphyrin
The above results indicate a unique effect of the glycopyranosyl
moiety on the interaction with HeLa cells. A very interesting
question then arises, namely, whether the differences in cellular
uptake of photosensitizers affect their photocytotoxicity. To
examine this issue, the photocytotoxicity of p-5a, p-5d and TPPS
was assayed in vitro.
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