Trans-bisglycoconjugation is an efficient and robust architecture for PDT photosensitizers based on 5,10,15,20-tetrakis(pentafluorophenyl)porphyrin derivatives

Article abstract of DOI:10.1246/bcsj.20130131

To examine the versatility of the trans-bisglycoconjugation architecture for 5,10,15,20-tetrakis(pentafluorophenyl) porphyrin (TFPP) derivative-based photodynamic therapy (PDT), 25 porphyrin derivatives bearing D-glucopyranose moieties were prepared, and their in vitro photocytotoxicities were examined. trans-Bisglycoconjugated TFPP derivatives exerted the best photocytotoxicity among TFPP derivatives in the same category, regardless of the central metal ion and the type of glycoconjugation. trans-Bisglycoconjugated free-base TFPP bearing β-D-glucopyranosylthio groups (trans- H₂2 _SGlc) was found to be the most potent photosensitizer, not only in HeLa cells (EC₅₀ < 5 nM), but also in highly metastatic cancer cell lines such as B16-BL6 melanoma cells (EC₅₀ < 10 nM). UVvis and dynamic light-scattering measurements suggested that trans- bisglycoconjugated TFPP derivatives formed relatively large J-aggregates in an aqueous solution. trans-Bisglycoconjugated TFPP derivatives exhibited greater uptake by HeLa cells than those with different glycoconjugation patterns, with the exception of Zn(II) complexes. trans-Bisglycoconjugated TFPP derivatives efficiently generated hydrogen peroxide and hydroxyl radical by a type I photoreaction, while no significant differences were found in the efficiency of singlet oxygen generation by a type II photoreaction. These unique characteristics of transbisglycoconjugated TFPP derivatives support the conclusion that trans-bisglycoconjugation is a highly efficient and robust architecture for TFPP-based PDT development.

Full text of DOI:10.1246/bcsj.20130131

© 2013 The Chemical Society of Japan
Bull. Chem. Soc. Jpn. Vol. 86, No. 11, 1295-1308 (2013) 1295
trans-Bisglycoconjugation is an Efficient and Robust
Architecture for PDT Photosensitizers Based on
5,10,15,20-Tetrakis(pentafluorophenyl)porphyrin Derivatives
Shiho Hirohara,*1 Kohei Sharyo,2 Yuji Kawasaki,2 Masayasu Totani,2 Ayumi Tomotsuka,2
Ryota Funasako,2 Nobutaka Yasui,2 Yasuchika Hasegawa,3 Junpei Yuasa,2 Takuya Nakashima,2
Tsuyoshi Kawai,2 Chio Oka,4 Masashi Kawaichi,4 Makoto Obata,*5 and Masao Tanihara2
1Department of Chemical and Biological Engineering, Ube National College of Technology,
2-14-1 Tokiwadai, Ube, Yamaguchi 755-8555
2Graduate School of Materials Science, Nara Institute of Science and Technology,
8916-5 Takayama, Ikoma, Nara 630-0192
3Division of Materials Chemistry, Faculty and Graduate School of Engineering, Hokkaido University,
N13 W8, Kita-ku, Sapporo, Hokkaido 060-8628
4Graduate School of Biological Sciences, Nara Institute of Science and Technology,
8916-5 Takayama, Ikoma, Nara 630-0192
5Interdisciplinary Graduate School of Medicine and Engineering, University of Yamanashi,
Takeda, Kofu, Yamanashi 400-8510
Received May 11, 2013; E-mail: hirohara@ube-k.ac.jp, mobata@yamanashi.ac.jp
To examine the versatility of the trans-bisglycoconjugation architecture for 5,10,15,20-tetrakis(pentafluorophen-
yl)porphyrin (TFPP) derivative-based photodynamic therapy (PDT), 25 porphyrin derivatives bearing D-glucopyranose
moieties were prepared, and their in vitro photocytotoxicities were examined. trans-Bisglycoconjugated TFPP derivatives
exerted the best photocytotoxicity among TFPP derivatives in the same category, regardless of the central metal ion and
the type of glycoconjugation. trans-Bisglycoconjugated free-base TFPP bearing β-D-glucopyranosylthio groups (trans-
H22SGlc) was found to be the most potent photosensitizer, not only in HeLa cells (EC50 < 5 nM), but also in highly
metastatic cancer cell lines such as B16-BL6 melanoma cells (EC50 < 10 nM). UV-vis and dynamic light-scattering
measurements suggested that trans-bisglycoconjugated TFPP derivatives formed relatively large J-aggregates in an
aqueous solution. trans-Bisglycoconjugated TFPP derivatives exhibited greater uptake by HeLa cells than those with
different glycoconjugation patterns, with the exception of Zn(II) complexes. trans-Bisglycoconjugated TFPP derivatives
efficiently generated hydrogen peroxide and hydroxyl radical by a type I photoreaction, while no significant differences
were found in the efficiency of singlet oxygen generation by a type II photoreaction. These unique characteristics of trans-
bisglycoconjugated TFPP derivatives support the conclusion that trans-bisglycoconjugation is a highly efficient and
robust architecture for TFPP-based PDT development.
In photodynamic therapy (PDT), a lethal photochemical
reaction is initiated in malignant cells by the irradiation of
photosensitizers with light.1-7 The efficacy of PDT critically
depends on the photosensitizers,1-5 which must meet the
following requirements: (1) no cytotoxicity in the dark, (2)
efficient generation of reactive oxygen species (ROS) upon
photoirradiation, (3) rapid clearance, (4) reasonable water
solubility, and (5) tumor-targeting characteristics. Porphyrin
derivatives including reduced porphyrin derivatives are the de
facto standard photosensitizing functionality because of their
excellent photosensitizing ability and their inherent accumu-
lation in the tumor region.8 Because porphyrin derivatives are
highly hydrophobic molecules, hydrophilic substituents are
frequently introduced to develop practical porphyrin-based PDT
photosensitizers. The water-soluble anionic porphyrin deriva-
tives, sulfonated tetraphenylporphyrin derivatives (TPPSx series
in Chart 1) were examined as PDT photosensitizers. Kessel
et al. reported that the photocytotoxicity of TPPSx exhibited
significant dependence on the degree and pattern of sulfona-
tion, and cis-TPPS2 was found to be the most potent TPPSx
photosensitizer.9 Later, Berg et al. also concluded that TPPS1
and cis-TPPS2 exerted greater photocytotoxicity than other
TPPSx’s due to their high cellular uptake in combination with
their high efficiency of photoinactivation.10 In another ap-
proach, Jensen et al. synthesized water-soluble cationic porphy-
rin derivatives, 5,10,15,20-tetraphenylporphyrin (TPP) bearing
trimethylammonio groups at the 4-position of the phenyl groups
(TPPNx series in Chart 1), to examine the effect of the overall
charge and the charge distribution on the photocytotoxicity
in HEp2 cells.11 They found that TPPN1 is the most promising
Published on the web August 24, 2013; doi:10.1246/bcsj.20130131

1296 Bull. Chem. Soc. Jpn. Vol. 86, No. 11 (2013)
trans-Bisglycoconjugated Photosensitizers
Chart 1.
PDT photosensitizer. Interestingly, the photocytotoxicity of
bis-substituted TPPs depends on the substitution pattern, i.e.,
cis-TPPN2 exerted a much higher photocytotoxicity than trans-
TPPN2, a similar finding to the TPPSx series. These two
examples clearly demonstrate that the number of hydrophilic
functionalities and their pattern of substitution in TPP deriva-
tives are important factors for the development of efficient TPP-
based PDT photosensitizers.
the influence of the central metal ion), O-glycoconjugated
H2TFPPs (to study the influence of the ligating moiety) using a
post-glycoconjugation approach based on the click technique
and examining the in vitro photocytotoxicity of each of these
compounds in several cancer cells.
Experimental
Materials.
All chemicals were of analytical grade.
Carbohydrates are essential molecules that sustain life as
energy sources and exert many biological functions. Glyco-
conjugated photosensitizers have been investigated to improve
their water-solubility as well as to enhance tumor-specificity. In
a pioneering study, Momenteau et al. synthesized TPP deriva-
tives bearing glucose, galactose, maltose, and glucosamine by a
glycoconjugation-first approach based on glycoconjugation of
benzaldehyde prior to the synthesis of the porphyrin ring.12
They examined the photocytotoxicity of β-D-glucopyranosyl-
oxy groups at the 4-position of the phenyl groups (TPPOGlcx
series in Chart 1). Their results also indicated that a cis-
bisglycoconjugated porphyrin, cis-TPPOGlc2, exerted a higher
photocytotoxicity than trans-bisglycoconjugated porphyrins,
similarly to the TPPSx and TPPNx series. In 1999, Boyle et al.
reported the synthesis of glycoconjugated porphyrin by means
of anomeric S-arylation of 5,10,15,20-tetrakis(pentafluoro-
phenyl)porphyrin (H2TFPP) by thiol-substituted molecules.13
Recently, we synthesized the S-glycoconjugated H2TFPP
derivatives (H21SGlc, cis-H22SGlc, trans-H22SGlc, H23SGlc, and
H24SGlc as shown in Scheme 1) and examined the effects of
the number and pattern of carbohydrate moieties on cellular
uptake and photocytotoxicity.14 Unlike TPP derivatives such
as TPPSx, TPPNx, and TPPOGlcx, the trans-bisglycoconjugated
derivative, trans-H22SGlc, exerted approximately 20-fold higher
photocytotoxicity than the other S-glycoconjugated H2TFPPs.
We examined the versatility of the trans-bisglycoconju-
gated architecture in the TFPP-based PDT photosensitizers by
preparing S-glycoconjugated TFPP metal complexes (to study
Benzaldehyde, benzophenone, and sodium iodide were pur-
chased from Wako Pure Chemical Industries, Ltd. (Osaka,
Japan). Pyrrole, pentafluorobenzaldehyde, bromobenzaldehyde,
p-methoxybenzaldehyde, m-hydroxybenzaldehyde, TPPS, and
hematoporphyrin (HP) were purchased from Sigma-Aldrich
Japan (Chiba, Japan). 4¤,6-Diamidino-2-phenylindole di-
hydrochloride hydrate (DAPI) was purchased from Molecular
Probes (Eugene, OR). Hydroxyphenyl Fluorescein (HPF) was
purchased from Sekisui Medical Co., Ltd. (Tokyo, Japan).
H2TFPP,15 5,10,15,20-tetrakis(pentafluorophenyl)porphinato-
zinc(II) (ZnTFPP),15 5,10,15,20-tetrakis(pentafluorophenyl)-
porphinatopalladium(II) (PdTFPP),16 5,10,15,20-tetrakis(penta-
fluorophenyl)porphinatoplatinum(II) (PtTFPP),16 2,3,4,6-
tetra-O-acetyl-β-D-glucopyranosyl thioacetate (AcGlcSAc),17
2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl azide (AcGlcN3),18
and 2,3,4,6-tetra-O-acetyl-D-glucopyranose (AcGlcOH)19 were
prepared according to previous reports in the literature. 5-[4-(β-
D-Glucopyranosylthio)-2,3,5,6-tetrafluorophenyl]-10,15,20-
tris(pentafluorophenyl)porphyrin (H21SGlc),14 5,10-bis[4-(β-
D-glucopyranosylthio)-2,3,5,6-tetrafluorophenyl]-15,20-bis-
(pentafluorophenyl)porphyrin (cis-H22SGlc),14 5,15-bis[4-(β-
D-glucopyranosylthio)-2,3,5,6-tetrafluorophenyl]-10,20-bis-
(pentafluorophenyl)porphyrin (trans-H22SGlc),14 5,10,15-tris[4-
(β-D-glucopyranosylthio)-2,3,5,6-tetrafluorophenyl]-20-penta-
fluorophenylporphyrin (H23SGlc),14 5,10,15,20-tetrakis[4-
(β-D-glucopyranosylthio)-2,3,5,6-tetrafluorophenyl]porphyrin
(H24SGlc),14,20 5,10,15,20-tetrakis[4-(β-D-glucopyranosylthio)-
2,3,5,6-tetrafluorophenyl]porphinatozinc(II) (Zn4SGlc),20 and

S. Hirohara et al.
Bull. Chem. Soc. Jpn. Vol. 86, No. 11 (2013) 1297
Scheme 1.
5,10,15,20-tetrakis[4-(β-D-glucopyranosylthio)-2,3,5,6-tetra-
fluorescence intensity of each well were determined using
plate readers (Multiscan JX, Thermo Fisher Scientific Co.,
Yokohama, Japan and SPECTRA Fluor Plus, TECAN Group
Ltd., Seestrasse, Switzerland, respectively). Bright field and
fluorescence images of cells were taken using a confocal laser-
scanning microscope (CLSM, Model LSM 510, Carl Zeiss,
Jena, Germany).
20
fluorophenyl]porphinatoplatinum(II) (Pt4SGlc
)
as shown in
Scheme 1 were prepared according to our previous papers.
Preparative gel permeation chromatography (GPC) was per-
formed on a high-performance liquid chromatography (HPLC)
system consisting of a PU-2086 Plus Intelligent Pump (Jasco
Co., Ltd., Tokyo, Japan) and an RI-2031Plus Intelligent RI
Detector (Jasco Co., Ltd.) equipped with a Megapak GEL 201C
column (GPC, 25 mmº © 300 mm, Jasco Co., Ltd.) using
CHCl3 as an eluent. The purities of the photosensitizers were
determined on an HPLC system (1515 Isocratic HPLC system,
Waters Co., MA) equipped with a UV-vis detector (2487 Dual
- Absorbance Detector, Waters Co.) and a silica gel column
(COSMOSIL 5SL-II packed column, 4.6 mmº © 150 mm,
Nacalai tesque, Inc., Kyoto, Japan) at 30 °C or an octadecyl-
silyl-bonded silica gel column (Mightysil RP-18, 4.6 mmº ©
150 mm, KANTO CHEMICAL Co., Inc., Tokyo, Japan) at
40 °C. Elemental analysis was performed on a Perkin-Elmer
PE2400 Series II CHNS/O Analyzer (Perkin-Elmer Co.,
Anomeric S-Arylation Route. Syntheses of the Glyco-
conjugated Porphyrins; Zn1SAcGlc, cis-Zn2SAcGlc, trans-
Zn2SAcGlc, and Zn3SAcGlc
:
ZnTFPP (434 mg, 402 ¯mol),
AcGlcSAc (350 mg, 862 ¯mol), and diethylamine (44.1 ¯L,
42.7 ¯mol) were dissolved in N,N-dimethylformamide (DMF)
(30 mL). The reaction mixture was stirred at room temperature
for 24 h. The reaction mixture was diluted with CH2Cl2 (100
mL) and washed with distilled water (100 mL © 5). The extract
was dried over Na2SO4, and the solvent was removed under
reduced pressure. The crude product was roughly purified by
column chromatography (silica gel, CH2Cl2/ethyl acetate =
10/0-7/3, v/v). cis- and trans-Zn2SAcGlc were separated by
preparative HPLC (Porasil silica·, Waters Co., MA, CH2Cl2/
ethyl acetate = 8/2, v/v). Finally, all compounds were purified
by preparative GPC, yielding Zn1SAcGlc (139 mg, 24.9%), cis-
Zn2SAcGlc (162 mg, 23.4%), trans-Zn2SAcGlc (104 mg, 14.9%),
and Zn3SAcGlc (223 mg, 26.9%) as pink solids.
1
Ltd., Shelton, CT). H, 13C, and 19F NMR, COSY, and HMQC
spectra were recorded on JNM-EC600 (600 MHz, JEOL Ltd.,
Tokyo, Japan) and AVANCE 400 (400 MHz, Bruker Biospin
K.K., Yokohama, Japan) instruments. UV-vis spectra were
recorded on a V-570 spectrophotometer or a V-560 spectro-
photometer (Jasco Co., Ltd.). The particle sizes of the photo-
sensitizers in phosphate buffered saline (PBS) solution con-
taining 1 vol % dimethyl sulfoxide (DMSO, Wako Pure Chem-
ical Industries, Osaka, Japan) were measured on a Nano-ZS
Zetasizer (Malvern Instruments, Worcestershire, U.K.). Photo-
sensitizer stock solutions were prepared by weighing the dried
photosensitizers and dissolving them in DMSO and were
stored in a freezer (¹30 °C) until use. The absorbance and the
Syntheses of the Glycoconjugated Porphyrins; Zn1SGlc
,
cis-Zn2SGlc, trans-Zn2SGlc, and Zn3SGlc The typical synthe-
:
sis procedure was as follows. Zn1SAcGlc (106 mg, 87.3 ¯mol)
was dissolved in CH2Cl2 (20 mL) and CH3OH (20 mL).
Sodium methoxide was added to adjust the pH to 9. The mix-
ture was refluxed for 15 min at 50 °C, and then neutralized with
acetic acid. After the solvent was removed, the crude product
was washed with distilled water (10 mL © 10). The crude

1298 Bull. Chem. Soc. Jpn. Vol. 86, No. 11 (2013)
trans-Bisglycoconjugated Photosensitizers
product was purified by column chromatography (Cosmosilμ,
Nacalai tesque, Inc., Kyoto, Japan, CH3CN/H2O = 8/2, v/v)
and washed with distilled water to yield Zn1SGlc (79.6 mg,
75.1%) as a pink-red solid.
Click Route. Syntheses of the Clickable Porphyrins;
Pd1p, cis-Pd2p, trans-Pt2p, Pd3p, and Pd4p: The typical
procedure for Pd1p, Pd3p, and Pd4p synthesis was as follows.
PdTFPP (300 mg, 279 ¯mol), propargyl alcohol (16.3 ¯L, 280
¯mol), and K2CO3 (309 mg, 2.24 mmol) were dissolved in
DMSO (60 mL) and shaken for 14 h at 50 °C. The solution
was diluted with CH2Cl2 (100 mL) and washed with distilled
water (100 mL © 2), 1 M HCl(aq) (100 mL) and distilled
water (100 mL © 2). The solution was dried over Na2SO4,
and the solvent was removed under reduced pressure. The
crude product was purified by column chromatography (silica
gel, CH2Cl2/hexane = 2/8-5/5, v/v) to yield Pd1p (93.3 mg,
30.0%) as a red solid. The typical synthesis procedure for
cis-Pd2p and trans-Pd2p was as follows. PdTFPP (151 mg,
140 ¯mol), propargyl alcohol (16.2 ¯L, 278 ¯mol), and potas-
sium tert-butoxide (62.6 mg, 557 ¯mol) were dissolved in DMF
(15 mL) and were stirred for 15 min at ¹15 °C. The reaction
mixture was neutralized with 1 M HCl(aq) and extracted with
CH2Cl2 (100 mL). The extract was washed with 1 M HCl(aq)
(100 mL © 3) and distilled water (100 mL © 2). The solution
was dried over Na2SO4, and the solvent was removed under
reduced pressure. The crude product was purified by column
chromatography (silica gel, CH2Cl2/hexane = 7/3-5/5, v/v)
followed by preparative HPLC (Porasil silica·, CH2Cl2/
hexane = 4/6, v/v) to yield cis-Pd2p (19.9 mg, 12.3%) and
trans-Pd2p (9.9 mg, 6.2%) as red solids.
Syntheses of the Glycoconjugated Porphyrins; Pt1SAcGlc
,
cis-Pt2SAcGlc, trans-Pt2SAcGlc, and Pt3SAcGlc The typical
:
synthesis procedure was as follows. PtTFPP (49.8 mg, 42.7
¯mol), AcGlcSAc (17.1 mg, 42.1 ¯mol), and diethylamine (44.1
¯L, 42.7 ¯mol) were dissolved in DMF (30 mL). The solution
was stirred at room temperature for 24 h. The reaction mixture
was diluted with CH2Cl2 (100 mL) and washed with distilled
water (100 mL © 5). The extract was dried over Na2SO4, and
the solvent was removed under reduced pressure. The crude
product was purified by column chromatography (silica gel,
CH2Cl2/ethyl acetate = 10/0-9/1, v/v) followed by prepara-
tive GPC to yield Pt1SAcGlc (28.4 mg, 44.0%) as a red solid.
Syntheses of the Glycoconjugated Porphyrins; Pt1SGlc
,
cis-Pt2SGlc, trans-Pt2SGlc, and Pt3SGlc The typical synthesis
:
procedure was as follows. Pt1SAcGlc (114 mg, 75.4 ¯mol) was
dissolved in CH2Cl2 (20 mL) and CH3OH (20 mL). Sodium
methoxide was added to adjust the pH to 9. The mixture was
refluxed for 15 min at 50 °C and then neutralized with acetic
acid. After the solvent was removed, the crude product was
washed with distilled water (10 mL © 10). The crude product
was purified by column chromatography (Cosmosilμ, CH3CN/
H2O = 8/2, v/v). After the solvent was removed, the residue
was washed with distilled water to yield Pt1SGlc (59.8 mg,
42.4%) as a red solid.
Syntheses of the Glycoconjugated Porphyrins; Pd1pAcGlc
,
cis-Pd2pAcGlc, trans-Pd2pAcGlc, Pd3pAcGlc, and Pd4pAcGlc
:
Anomeric O-Arylation Route. Syntheses of the Glyco-
conjugated Porphyrins; H21OAcGlc, cis-H22OAcGlc, trans-
The typical procedure for Pd1pAcGlc, Pd3pAcGlc, and Pd4pAcGlc
synthesis was as follows. Pd1p (195 mg, 175 ¯mol), AcGlcN3
(131 mg, 349 ¯mol), and CuBr (24.9 mg, 175 ¯mol) were
dissolved in DMF (50 mL) and stirred for 16 h at 70 °C under
argon. The reaction mixture was diluted with CH2Cl2 (100 mL),
washed with distilled water (100 mL © 2), 1 M HCl(aq) (100
mL) and then distilled water again (100 mL © 2). The solution
was dried over Na2SO4, and the solvent was removed under
reduced pressure. The crude product was purified by column
chromatography (silica gel, CH2Cl2/ethyl acetate = 10/0-9/1,
v/v) followed by preparative GPC to give Pd1pAcGlc (156 mg,
61.9%) as a red solid. The typical procedure for the synthesis
of cis-Pd2pAcGlc and trans-Pd2pAcGlc was as follows. cis-Pd2p
(99.7 mg, 88.6 ¯mol), AcGlcN3 (129 mg, 346 ¯mol), CuBr
(49.7 mg, 346 ¯mol), and N,N,N¤,N¤¤,N¤¤-pentamethyldiethyl-
enetriamine (PMDETA, 72.4 ¯L, 346 ¯mol) were dissolved in
DMF (30 mL) and then stirred at room temperature for 1 h
under argon. The solution was diluted with CH2Cl2 (100 mL)
and washed with 1 M HCl(aq) (30 mL © 3) and distilled water
(30 mL © 2). The solution was dried over Na2SO4, and the
solvent was removed under reduced pressure. The crude
product was purified by column chromatography (silica gel,
CH2Cl2/ethyl acetate = 10/0-5/5, v/v) followed by prepara-
tive GPC to yield cis-Pd2pAcGlc (121 mg, 73.4%) as a red solid.
H22OAcGlc, H23OAcGlc, and H24OAcGlc
:
TFPP (208 mg, 214
¯mol), AcGlcOH (226 mg, 650 ¯mol), and K2CO3 (125 mg,
906 ¯mol) were dissolved in dimethyl sulfoxide (DMSO) (60
mL) and shaken for 8 h at 50 °C. The solution was diluted with
ethyl acetate (100 mL) and washed with cold 1 M HCl(aq) (100
mL © 1) and brine (100 mL © 3). The solution was dried over
Na2SO4, and the solvent was removed under reduced pressure.
The crude product was initially purified by column chromatog-
raphy (silica gel, gradient from CH2Cl2 to CH2Cl2/ethyl ace-
tate = 9/1-6/4, v/v). For cis-H22OAcGlc and trans-H22OAcGlc
,
further purification was performed by preparative HPLC (silica
gel, CH2Cl2/ethyl acetate = 8/2, v/v) to separate each other.
Finally, all compounds were purified by preparative GPC
to yield H21OAcGlc (14.4 mg, 5.2%), cis-H22OAcGlc (39.3 mg,
11.3%), trans-H22OAcGlc (26.4 mg, 7.6%), H23OAcGlc (67.3 mg,
16.1%), and H24OAcGlc (18.6 mg, 3.8%) as purple solids.
Syntheses of the Glycoconjugated Porphyrins; H21OGlc
,
cis-H22OGlc, trans-H22OGlc, H23OGlc, and H24OGlc The
:
typical synthesis procedure was as follows. H21OAcGlc (116 mg,
88.6 ¯mol) was dissolved in CH2Cl2 (10 mL) and CH3OH
(10 mL). Sodium methoxide was added to adjust the pH to 9.
This mixture was refluxed for 5 min at 50 °C and neutralized
with acetic acid. After solvent removal, the crude product was
washed with distilled water (10 mL © 10). The crude product
was purified by column chromatography (silica gel, ethyl
acetate/acetone = 6/4, v/v) followed by column chromatog-
raphy (Sephadex· LH-20, CH3OH). After the solvent was
removed, the residue was washed with hexane and distilled
water to yield H21OGlc (30.3 mg, 30.1%) as a purple solid.
Syntheses of the Glycoconjugated Porphyrins; Pd1pGlc
,
cis-Pd2pGlc, trans-Pd2pGlc, Pd3pGlc, and Pd4pGlc: The typical
synthesis procedure was as follows. Pd1pAcGlc (149 mg, 99.9
¯mol) was dissolved in CH2Cl2 (10 mL) and CH3OH (10 mL).
Sodium methoxide was added to adjust the pH to 9. This
mixture was refluxed for 5 min at 50 °C and then neutralized
with acetic acid. After removal of the solvent, the crude product

S. Hirohara et al.
Bull. Chem. Soc. Jpn. Vol. 86, No. 11 (2013) 1299
was washed with distilled water (10 mL © 10). The crude
product was purified by column chromatography (Cosmosilμ,
CH3CN/H2O = 85/15, v/v). After the solvent was removed,
the residue was washed with distilled water to yield Pd1pGlc
(86.0 mg, 65.2%) as a red powder.
octadecylsilyl-bound silica gel (R-18F254s, Merck Japan Ltd.,
Tokyo, Japan) and a mixture of CH3OH and H2O (85/15, v/v)
as the eluent, and calculated as follows,
k0 ¼ 1=Rf
ð2Þ
Quantum Yield of Singlet Oxygen (Φ¦).
The photo-
Benzophenone (log P = 3.18),23 p-bromobenzaldehyde
(2.57),24 m-hydroxybenzaldehyde (1.70),24 and p-methoxy-
benzaldehyde (1.25)24 were used as standards to calibrate the
RP-TLC system.
sensitizer under study was dissolved in D2O containing 0.1
vol % DMSO. The absorbance of the solution was adjusted to
be 0.4 at the appropriate excitation wavelength (see Supporting
Information, the light intensity was substantially constant in
the wavelength range from 381 to 449 nm) and purged with
oxygen gas for 1 min. Luminescence spectra of singlet oxygen
generated by the photoirradiation of the solution were recorded
on a spectrometer (Jobin Yvon SPEX fluorolog3, HORIBA,
Ltd., Kyoto, Japan) equipped with a photomultiplier (NIR-
PMT R5509-72, Hamamatsu Photonics K.K., Shizuoka, Japan)
cooled at 193 K.
Relative Quantum Yield of Hydroxyl Radicals (º•OH).
The photosensitizers under study, HP (c = 1.00 ¯M) and HPF
(c = 2.00 ¯M) were dissolved in PBS solution containing
1 vol % DMSO and placed into a sample tube. Oxygen gas was
introduced to the solution for 1 min prior to photoirradiation.
The solution was then exposed to light from a 100 W halogen
lamp (KBEX-102A, USHIO Inc., Tokyo, Japan) through a Y-
50 cutoff filter (- > 500 nm, Toshiba Co., Tokyo, Japan) at
37 °C. The initial rate of increase in HPF-fluorescence intensity
at 535 nm was monitored using a plate reader (SPECTRA
Fluor Plus, TECAN Group Ltd., Seestrasse, Switzerland) at the
excitation wavelength of 485 nm. The apparent rate constant
was estimated from the first-order plot of the HPF-fluorescence
intensity versus the photoirradiation time. The relative quan-
tum yield (º•OH) was evaluated by dividing the initial rate of
increase in HPF-fluorescence intensity by the oscillator strength
in the wavelength region of photoirradiation, i.e., ( f>500 nm),
and was normalized to the HP value.
Cell Culture. HeLa human cervical cells (ATCC CCL-2)
were obtained from Dainippon-Sumitomo Pharmaco. Ltd.
(Osaka, Japan). The murine melanoma cell lines B16 (ATCC
CRL-6322), B16F1 (ATCC CRL-6323), B16F10 (ATCC CRL-
6475), and B16-BL6 (ATCC CRL-6322) were generously pro-
vided by Dr. Akihiko Itoh (Department of Pathology, Faculty
of Medicine, Kinki University), and murine 4T1 breast cancer
cells (ATCC CRL-2539) were generously provided by Dr.
Hisataka Sabe (Department of Physiological Science, Graduate
School of Medicine, Hokkaido University). HeLa cells were
grown in Dulbecco’s modified Eagle’s medium (DMEM)
containing 10 vol % fetal calf serum (FCS, Hyclone Laborato-
ries, Inc., Logan, UT). Other cells (B16, B16F1, B16F10,
B16-BL6, and 4T1 cells) were grown in DMEM (Sigma-
Aldrich Japan, Chiba, Japan) containing 10 vol % FCS, 2 mM
L-glutamine (Life Technologies Japan Ltd., Tokyo, Japan) and
penicillin/streptomycin (Life Technologies Japan Ltd.).
Cellular Uptake Measurement in HeLa Cells. HeLa cells
(1 © 106 cells/well) were plated in 1.5 mL of culture medium
in a 6-well plate (Nalge Nunc International, Naperville, IL,
USA) and incubated for 24 h (37 °C, 5% CO2). Then, 1.5 mL of
1.00 ¯M photosensitizer in culture medium containing 2 vol %
DMSO was added to each well, and the cells were incubated
for 24 h. The final photosensitizer concentration in the culture
medium was 0.5 ¯M (the final DMSO content was 1 vol %
in all cases). The cells were washed twice with Dulbecco’s
phosphate-buffered saline (PBS). The cells were lysed in 150
¯L of DMSO. The absorbance of the extracts at the Soret
band was measured by a UV-vis spectrophotometer. The con-
centration of the photosensitizer was calculated based on the
calibration plot obtained for each photosensitizer in DMSO.
The cellular uptake is reported as the mean of three replicates.
Photocytotoxicity in HeLa Cells. HeLa cells (5 © 103
cells/well) were plated in 100 ¯L of culture medium in 96-well
plates (Nalge Nunc International) and incubated for 24 h
(37 °C, 5% CO2). Each well received 100 ¯L of photosensitizer
in culture medium containing 2 vol % DMSO, and the plate was
then incubated for 24 h in the presence of the photosensitizer.
The photosensitizer concentration was varied from 1 to 1000
nM (the final DMSO content was 1 vol % in all cases). The
cells were washed twice with PBS, and 100 ¯L of fresh culture
medium was added. The cells were exposed to light from a
100 W halogen lamp equipped with a water jacket and a Y-50
cutoff filter (- > 500 nm). The light intensity was measured
using a UV-vis power meter (ORION/TH, Ophir Optronic
Ltd., Jerusalem, Israel). The irradiation time was adjusted to
obtain the desired light dose of 16 J cm¹2. The mitochondrial
NADH dehydrogenase activity of the cells in each well was
measured 24 h after photoirradiation using the WST-8μ reagent
(10 ¯L) from Cell Counting Kit-8 (Dojindo Molecular Tech-
Relative Quantum Yield of Hydrogen Peroxide (ºH O ).
2
The photosensitizers under study, HP (c = 1.00 ¯M) and2NaI
(c = 200 mM) were dissolved in PBS containing 1 vol %
DMSO and placed in a sample tube. Oxygen gas was intro-
duced to the solution for 1 min in prior to photoirradiation.
The solution was subsequently exposed to light from a 100 W
halogen lamp through a Y-50 cutoff filter (- > 500 nm) at
37 °C.21 The concentration of H2O2 was monitored by measur-
¹
ing the absorbance at 365 nm (absorption band of the I3
species, ¾max = 2.5 © 104 M¹1 cm¹1).22 The apparent rate con-
stant was estimated from the first-order plot of the H2O2 concen-
tration versus photoirradiation time. The relative quantum yield
¹
(ºH O ) was evaluated by the initial rate of I3 formation divided
2
2
by the f>500 nm value and was normalized to the HP value.
Hydrophobicity Parameters Determined by RP-TLC.
The hydrophobicity parameter (logarithm of the partition co-
efficient between n-octanol and water; log P) is well-correlated
with the capacity factor k¤ in reverse-phase (i.e., partition)
chromatography as follows:
log P ¼ a log k0 þ b
ð1Þ
where a and b are constants for a given chromatographic
system. The capacity factor k¤ was determined by the Rf value
in reverse-phase thin layer chromatography (RP-TLC) using an

1300 Bull. Chem. Soc. Jpn. Vol. 86, No. 11 (2013)
trans-Bisglycoconjugated Photosensitizers
nologies, Inc., Tokyo, Japan) according to the manufacturer’s
instructions. The absorbance at 450 nm was measured using
a plate reader. The percentage cell survival was calculated by
normalization with respect to the absorbance for untreated cells
(vehicle).
Photocytotoxicities of trans-H22SGlc and H24SGlc in B16,
B16F1, B16F10, B16-BL6, and 4T1 Cells. Cells (5 © 103
cells) in 100 ¯L of culture medium were plated in a 96-well
plate and incubated for 24 h (37 °C, 5% CO2). Each well
received 100 ¯L of photosensitizer in culture medium contain-
ing 2 vol % DMSO. Then, the cells were incubated for 24 h in
the presence of the photosensitizer. The photosensitizer con-
centration was varied from 0.1 to 1000 nM (the final DMSO
content was 1 vol % in all cases). The cells were washed twice
with PBS, and then 100 ¯L of the fresh culture medium was
added. Then, each well was photoirradiated under the same
conditions as described for the HeLa cells.
Subcellular Localization of trans-H22SGlc and H24SGlc in
HeLa Cells. HeLa cells (1 © 104 cells) were plated in 500 ¯L
of culture medium in 24-well plates (Nalge Nunc International)
and incubated for 24 h (37 °C, 5% CO2). Each well received
500 ¯L of 0.6 ¯M trans-H22SGlc or 2.0 ¯M H24SGlc in the culture
medium containing 2 vol % DMSO, and then cells were incu-
bated for 24 h in the presence of the photosensitizers. The final
fluorophenyl)porphyrin (TFPP) derivative-based PDT, zinc(II),
and platinum(II) complexes of S-glycoconjugated TFPPs were
synthesized by nucleophilic S-glycosylation of 5,10,15,20-
tetrakis(pentafluorophenyl)porphinatozinc(II) (ZnTFPP) and
-platinum(II) (PtTFPP) by 2,3,4,6-tetra-O-acetyl-β-D-gluco-
pyranosyl thioacetate (AcGlcSAc) using a similar procedure
as reported in our previous papers.14,20 All five S-glycoconju-
gated TFPP metal complexes, namely, M1SAcGlc, cis-M2SAcGlc
,
trans-M2SAcGlc, M3SAcGlc, and M4SAcGlc (Scheme 1), in which
M represents the metal ion, the Zn(II) or Pt(II) ions in this case,
were successfully separated by chromatographic techniques.
The deacetylated porphyrins M1SGlc, cis-M2SGlc, trans-M2SGlc
,
M3SGlc, and M4SGlc were obtained with reasonable purities after
the acetyl groups were removed by alkali hydrolysis and
purified by reverse-phase chromatography.
To examine the influence of the ligating moiety between the
porphyrin and D-glucopyranosyl groups on the superiority of
trans-bisglycoconjugation in TFPP-based PDT, the O-glyco-
conjugation of 5,10,15,20-tetrakis(pentafluorophenyl)porphy-
rin (H2TFPP) with 2,3,4,6-tetra-O-acetyl-D-glucose (AcGlcOH)
were also carried out in N,N-dimethylformamide (DMF) at
50 °C using potassium carbonate as a base (Scheme 2). In
1H NMR spectra of all O-glycoconjugated H2TFPPs in CDCl3,
the spin coupling constants of anomeric protons (1-GlcH)
were found to be approximately 3.5 Hz, indicating that the
D-glucopyranosyloxy group had the α-anomer configuration.
In addition, no β-anomer derivatives were observed, even in
the synthesis of H24OAcGlc, indicating the very high α-anomer
selectivity of the anomeric O-arylation. The protecting acetyl
groups were removed by saponification using sodium meth-
oxide to give the porphyrins H21OGlc, cis-H22OGlc, trans-
H22OGlc, H23OGlc, and H24OGlc with reasonable yields.
S- and O-Glycoconjugation approaches require careful opti-
mization of the chromatographic separation to yield trans-
bisglycoconjugated TFPP. Hence, it is difficult to extend this
synthetic route to the preparation of various trans-bisglyco-
conjugated TFPPs. Porphyrin precursors bearing easily con-
nectable functionalities at trans-positions would be highly
useful for efforts to develop more sophisticated trans-bisglyco-
conjugated TFPPs. To develop a universal approach to the
synthesis of trans-bisglycoconjugated TFPPs, we adopted a
copper(I)-catalyzed Huisgen cycloaddition, the so-called click
reaction,25 between porphyrin bearing propargyl groups and
glycosyl azide. The palladium(II) complex of TFPP (PdTFPP)
was selected as a photosensitizing molecule to avoid the
insertion of the copper(II) ion during the click reaction. Five
derivatives of propargyloxy-substituted PdTFPP were suc-
cessfully prepared by means of nucleophilic substitution by
propargyl alcohol using potassium carbonate at 50 °C or potas-
sium tert-butoxide at ¹15 °C (Scheme 3). The crude products
were successfully purified by column chromatography to afford
five derivatives of the clickable porphyrin precursors, namely,
Pd1p, cis-Pd2p, trans-Pd2p, Pd3p, and Pd4p. The glycocon-
jugations of these clickable porphyrin precursors were per-
formed with 2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl azide
(AcGlcN3) in DMF using CuBr and N,N,N¤,N¤¤,N¤¤-pentameth-
yldiethylenetriamine (PMDETA) as a catalyst at room temper-
ature. After the removal of the acetyl groups and chromato-
graphic purification, five glycoconjugtated PdTFPPs, namely,
concentrations of the photosensitizers were 0.3 (trans-H22SGlc
)
and 1 ¯M (H24SGlc). The cells were washed twice with 500 ¯L
of PBS, and then 500 ¯L of 250 nM MitoTracker Green· in
Hank’s balanced salt solution (HBSS) was added. The cells
were incubated for 30 min (37 °C, 5% CO2). After coincubation
with MitoTracker Green·, the cells were washed twice with
500 ¯L of PBS and fixed by adding PBS containing 4 wt %
paraformaldehyde. One hour after fixation, fluorescence images
of the cells were taken with the CLSM using an excitation
wavelength of 480 nm and emission wavelengths from 505 to
530 nm (mitochondria) and 650 nm (photosensitizer).
ROS Detection in HeLa Cells Photosensitized in the
Presence of trans-H22SGlc and H24SGlc
.
HeLa cells (1 © 104
cells) were plated in 500 ¯L of culture medium in 24-well plates
and incubated for 24 h (37 °C, 5% CO2). Each well received
500 ¯L of 0.2 ¯M photosensitizer (trans-H22SGlc or H24SGlc) in
the culture medium with 2 vol % DMSO, and then incubation
was continued for 24 h in the presence of the photosensitizers.
The final photosensitizer concentration was 0.10 ¯M (1 vol %
DMSO). The cells were washed twice with 500 ¯L of PBS, and
500 ¯L of 10 ¯M HPF in HBSS containing 0.2 vol % DMF was
added and incubated for 30 min (37 °C, 5% CO2). Then, each
well was photoirradiated under the same conditions as described
for the photocytotoxicity test. Ten minutes after photoirra-
diation, fluorescence images of HPF-loaded cells were taken
with the CLSM using an excitation wavelength of 488 nm and
emission wavelength from 505 to 530 nm.
Statistical Analysis.
All statistical evaluations were
performed using ANOVA. All values for cellular uptake and
cytotoxicity are expressed as the means « standard deviation.
Results and Discussion
Syntheses of Glycoconjugated Porphyrin Derivatives.
To examine the influence of the central metal ion on the superi-
ority of trans-bisglycoconjugation in 5,10,15,20-tetrakis(penta-

S. Hirohara et al.
Bull. Chem. Soc. Jpn. Vol. 86, No. 11 (2013) 1301
Scheme 2.
Scheme 3.
Pd1pGlc, cis-Pd2pGlc, trans-Pd2pGlc, Pd3pGlc, and Pd4pGlc, were
obtained with reasonable yields and acceptable purities. These
glycoconjugated porphyrins also provide a good platform to
examine the synergistic influence of the central metal ion and
the ligating moiety on the superiority of the trans-bisglycocon-
jugation in TFPP-based PDT.
The structures of all porphyrin derivatives including the
substitution numbers and orientations (cis- or trans-), were
confirmed by means of 1H, 19F, and 13C NMR spectroscopy. As
reported in our previous paper,14 cis- and trans-isomers can be
easily assigned by the aromatic region in the 1H NMR spectrum
recorded in C6D6. Due to the anisotropic solvent effect, the cis-

1302 Bull. Chem. Soc. Jpn. Vol. 86, No. 11 (2013)
trans-Bisglycoconjugated Photosensitizers
Figure 1. Photocytotoxicity to HeLa cells of S-glycoconjugated free-base porphyrins (H21SGlc, cis-H22SGlc, trans-H22SGlc, H23SGlc
,
and H24SGlc) (a), S-glycoconjugated porphyrin zinc(II) complexes (Zn1SGlc, cis-Zn2SGlc, trans-Zn2SGlc, Zn3SGlc, and Zn4SGlc) (b), S-
glycoconjugated porphyrin platinum(II) complexes (Pt1SGlc, cis-Pt2SGlc, trans-Pt2SGlc, Pt3SGlc, and Pt4SGlc) (c), O-glycoconjugated
free-base porphyrins (H21OGlc, cis-H22OGlc, trans-H22OGlc, H23OGlc, and H24OGlc) (d), click-glycoconjugated porphyrin palladium(II)
complexes (Pd1pGlc, cis-Pd2pGlc, trans-Pd2pGlc, Pd3pGlc, and Pd4pGlc) (e), and photocytotoxicity of trans-H22SGlc to B16, B16F1,
B16F10, B16BL6, and 4T1 cells (f).
isomer exhibits two sets of doublet peaks and two sets of
singlet peaks, while the trans-isomer only shows two sets
of doublet peaks. After alkali hydrolysis and purification
by reverse-phase column chromatography, 25 glycoconjugated
porphyrin derivatives were prepared as shown in Schemes 1-3.
The purities of all glycoconjugated porphyrin derivatives were
confirmed as >96% by means of reverse-phase high-perform-
ance liquid chromatography (HPLC) using an octadecylsilyl-
bounded silica gel column.
Photocytotoxicity. The photocytotoxicities of all glyco-
conjugated porphyrin derivatives shown in Schemes 1-3 were
examined in HeLa cells under the same conditions. None of
the glycoconjugated porphyrin derivatives exerted cytotoxicity
in the dark under these conditions. Figure 1 shows the cell
survival rate (%) of HeLa cells at 24 h after photoirradiation as
a function of the concentration of the glycoconjugated porphy-
rin derivatives. The trans-bisglycoconjugated porphyrin deriva-
tives (filled circles in Figure 1) exerted the highest photo-
cytotoxicities of each category of glycoconjugated porphyrin
derivatives. The superiority of trans-bisglycoconjugated por-
phyrin derivatives was not influenced by the central metal ions
(Zn(II), Pt(II), and Pd(II)) or by the type of glycoconjugation.
Hence, trans-bisglycoconjugation is a highly efficient and
robust architecture for the development of efficient TFPP-
based PDT photosensitizers. The drug concentrations inducing
50% cell death (EC50 values) of the trans-bisglycoconjugated
porphyrin derivatives decreased in the following order, trans-
Zn2SGlc (50 nM) > trans-Pt2SGlc (10 nM) > trans-Pd2pGlc (<10
14
nM) > trans-H22OGlc and trans-H22SGlc (<5 nM). To exam-
ine the versatility of trans-H22SGlc, the most efficient photo-
sensitizer, with other malignant cells, the photocytotoxicity of
trans-H22SGlc was examined in weakly metastatic B16, B16F1
melanoma cells, metastatic B16F10 melanoma cells, highly
metastatic B16-BL6 melanoma cells and metastatic 4T1 breast
cancer cells. Figure 1f shows the dose-dependent photocyto-
toxicity of trans-H22SGlc in these malignant cell lines. The EC50
values fell in the narrow range from 5 to 10 nM in these cell
lines. Hence, trans-H22SGlc exerted excellent photocytotoxicity
not only in metastatic cell lines but also in highly metastatic
cancer cell lines.
The trans-bisglycoconjugated TFPP trans-H22SGlc was
found to be a highly efficient PDT photosensitizer. To char-

S. Hirohara et al.
Bull. Chem. Soc. Jpn. Vol. 86, No. 11 (2013) 1303
Figure 2. Fluorescence (a and b) and bright field images (c) of MitoTracker Green·-stained HeLa cells after a 24 h incubation
in the presence of trans-H22SGlc. The localizations of MitoTracker Green· (a) and trans-H22SGlc (b) were captured by detecting
fluorescence at 505-530 and 650 nm, respectively, at an excitation wavelength of 488 nm. The fluorescence intensity profile (d) was
recorded along the white line on the fluorescence image (a).
acterize the mechanism of the photodynamic effects of trans-
H22SGlc, the intracellular localization of trans-H22SGlc was
examined in HeLa cells using the inherent luminescence from
trans-H22SGlc detected by confocal laser-scanning micros-
copy. Figure 2 shows bright field and fluorescence images of
MitoTracker Green·-stained HeLa cells at 24 h of incubation
in the presence of trans-H22SGlc. The intracellular localizations
of MitoTracker Green· and trans-H22SGlc were visualized by
detecting the luminescence in the range of 505-530 nm (green)
and at 650 nm (red), respectively. Figure 2b indicates that
trans-H22SGlc localized at the periphery of the nuclear region.
In addition, the fluorescence profile of trans-H22SGlc over-
lapped well with that of MitoTracker Green·, indicating that
the primary intracellular target of trans-H22SGlc was the mito-
chondria. Thompson et al. reported a confocal microscopic
it enhances cellular uptake. It is well-known that the hydro-
phobicity parameter, namely, the log P value, is closely related
to the cellular uptake. The log P values of the glycoconjugated
porphyrin derivatives were evaluated by reverse-phase thin
layer chromatography. For all glycoconjugated porphyrin
derivatives, the log P values decreased monotonically in the
order of mono-, trans-bis-, cis-bis-, tris-, and tetrakis-glyco-
conjugated porphyrin derivatives (Table 1). Figure 4 shows
the plot of the relative cellular uptake as a function of log P.
Glycoconjugated porphyrin derivatives with log P values of
3.5-4.5 tend to exhibit relatively high levels of cellular uptake.
However, no simple relationship was found between log P
values and cellular uptake, indicating that the enhancement of
the cellular uptake of trans-glycoconjugated compounds is not
simply due to the adjustment of hydrophobicity.
image of MitoTracker Green·-stained MDA-MB-231 cells
Physicochemical Aspects of trans-Bisglycoconjugated
Porphyrin Derivatives. Table 2 reports the UV-vis spectral
data of the glycoconjugated porphyrin derivatives in phosphate
buffered saline (PBS) solution containing 1 vol % dimethyl
sulfoxide (DMSO). All glycoconjugated porphyrin derivatives
showed one intense peak at approximately 410 nm (Soret
band) and two or four weak peaks from 510 to 640 nm (Q
bands). Interestingly, all trans-bisglycoconjugated porphyrin
derivatives (trans-H22SGlc, trans-Zn2SGlc, trans-Pt2SGlc, trans-
H22OGlc, and trans-Pd2pGlc) showed a red-shifted Soret band
in PBS solution, suggesting the formation of J-aggregates.28
26
coincubated with H24SGlc
.
The image indicated very poor
overlap of the MitoTracker Green· and H24SGlc localizations,
and they concluded that H24SGlc mainly localized in endoplas-
mic reticulum (ER). Thus, the intracellular localization of
trans-H22SGlc is very different from the localization pattern of
the fully glycoconjugated photosensitizer H24SGlc. The mito-
chondria is well-known as a key component in the initiation
of the signaling cascade leading to apoptotic cell death.27 The
characteristic localization of trans-H22SGlc could be one of the
reasons for its highly efficient photodynamic action.
The last two sections of the paper focus on the biological and
physicochemical aspects of trans-bisglycoconjugated porphy-
rin derivatives in an attempt to understand why they afford
higher in vitro photocytotoxicity than compounds with other
glycoconjugation patterns.
Biological Aspects of trans-Bisglycoconjugated Porphy-
rin Derivatives. The photocytotoxicity testing clearly showed
that trans-bisglycoconjugation is a highly efficient and robust
architecture for TFPP-based PDT photosensitizers. One possi-
ble mechanism by which trans-bisglycoconjugation could
improve photocytotoxicity is by enhancing cellular uptake.
Figure 3 shows the profiles of cellular uptake and cell survival
(%) as functions of the glycoconjugation pattern for each
glycoconjugated porphyrin derivative. Except for the zinc(II)
complexes, trans-bisglycoconjugated porphyrin derivatives led
to the highest cellular uptake of each category. Thus, one of the
reasons for the superiority of trans-bisglycoconjugation is that
The size distributions of H21SGlc, cis-H22SGlc, trans-H22SGlc,
H23SGlc, and H24SGlc aggregates in PBS solution containing
1 vol % DMSO were determined by means of dynamic light-
scattering (DLS) measurements (Figure 5). As expected, the
maximum aggregate size decreased with an increase of the
number of the sugar units. Interestingly, trans-H22SGlc resulted
in the formation of larger aggregates than cis-H22SGlc. The
size of the trans-H22SGlc aggregates was determined to be 319
nm, approximately twice as large as that of the cis-H22SGlc
aggregates. The UV-vis spectroscopy and DLS measurement
indicated that trans-glycoconjugated porphyrin derivatives
form relatively large J-aggregates. The formation of J-aggre-
gates of trans-disubstituted porphyrin derivatives was also
reported by Villari et al. using trans-bisPEGylated tetraphen-
ylporphyrin.29 The aggregate size and shape could affect cellu-
lar uptake. Recently, Hao et al. studied the HeLa cell uptake
of mesoporous silica nanoparticles (MSNs, with diameters of

1304 Bull. Chem. Soc. Jpn. Vol. 86, No. 11 (2013)
trans-Bisglycoconjugated Photosensitizers
Figure 3. The relative cellular uptake at 0.5 ¯M (arb., left axis, open circle) and the cell survival (%, right axis, filled circle) of HeLa
cells treated with S-glycoconjugated free-base porphyrins (H21SGlc, cis-H22SGlc, trans-H22SGlc, H23SGlc, and H24SGlc; 80 nM for
the photocytotoxicity test) (a), S-glycoconjugated porphyrin zinc(II) complexes (Zn1SGlc, cis-Zn2SGlc, trans-Zn2SGlc, Zn3SGlc, and
Zn4SGlc; 500 nM for the photocytotoxicity test) (b), S-glycoconjugated porphyrin platinum(II) complexes (Pt1SGlc, cis-Pt2SGlc, trans-
Pt2SGlc, Pt3SGlc, and Pt4SGlc; 100 nM for the photocytotoxicity test) (c), O-glycoconjugated free-base porphyrins (H21OGlc, cis-
H22OGlc, trans-H22OGlc, H23OGlc, and H24OGlc; 100 nM for the photocytotoxicity test) (d), click-glycoconjugated porphyrin
palladium(II) complexes (Pd1pGlc, cis-Pd2pGlc, trans-Pd2pGlc, Pd3pGlc, and Pd4pGlc; 100 nM for the photocytotoxicity test) (e). The
values are the means « standard deviation of three and six experiments for cellular uptake and photocytotoxicity, respectively.
may differ from other types of glycoconjugated porphyrin
derivatives. From the photophysical point of view, aggregate
formation results in a decrease in the lifetime of the excitation
state, diminishing the quantum yield of reactive oxygen species
such as singlet oxygen (1O2). Recently, Verma et al. reported
ultrafast excitation dynamics of J- and H-aggregates of
porphyrin-catechol in an aqueous solution.31 They found that
the excitation dynamics of aggregates depends on the config-
uration of the aggregates. J-Aggregates have longer emission
lifetimes (180 ps) than H-aggregates (70 ps) due to the large
number of vibronic states in H-aggregates, resulting in inter-
molecular phonon modes. Thus, trans-bisglycoconjugated por-
phyrin has an advantage in the formation of J-aggregates rather
than H-aggregates in terms of the lifetime of the excited state.
The efficiency of generation of reactive oxygen species
(ROS) is a crucial factor in the success of PDT photosensitizers.
1O2 is formed by a type II photoreaction and is thought to
be the major cytotoxic species because of its relatively long
lifetime. Nevertheless there are several reports that hydrogen
peroxide (H2O2) and hydroxyl radical (•OH) can be produced
by photosensitization using photosensitizers such as hemato-
porphyrin,32 meso-tetrakis[4-(carboxymethoxy)phenyl]porphy-
rin,33 and cationic metalloporphyrin.34 Mroz et al. reported that
their cationic imidazole metalloporphyrin produced •OH upon
photoirradiation in HeLa cells.34 They proposed the following
Table 1. Hydrophobicity Parameters (log P) of Glycoconju-
gated Porphyrin Derivativesa)
log P
log P
log P
H21SGlc 5.1b)
Zn1SGlc 5.7
cis-Zn2SGlc 4.8
Pt1SGlc 5.7
cis-Pt2SGlc 4.8
cis-H22SGlc 4.0b)
trans-H22SGlc 3.5b) trans-Zn2SGlc 4.3 trans-Pt2SGlc 4.3
H23SGlc 3.0b)
H24SGlc 0.8b)
Zn3SGlc 3.4
Zn4SGlc 2.2
Pt3SGlc 3.4
Pt4SGlc 2.2
H21OGlc 5.1
Pd1pGlc 6.7
cis-H22OGlc 4.0
cis-Pd2pGlc 5.5
trans-H22OGlc 3.4 trans-Pd2pGlc 4.7
H23OGlc 2.4
H24OGlc ¹1.1
Pd3pGlc 3.6
Pd4pGlc 1.8
a) Determined by RP-TLC. b) Data cited from Ref. 14.
approximately 100 nm) with various aspect ratios (1, 2, and
4).30 They concluded that the mechanism of cellular uptake
of MSNs depended on the aspect ratio, with MSNs with
small aspect ratios predominantly taken by a clathrin-mediated
process, while MSNs with large aspect ratios were taken up by
a caveolae-mediated mechanism. Because only trans-bisglyco-
conjugated porphyrin derivatives condense with specific J-type
alignments that could affect the overall aggregate shape, the
mechanism of cellular uptake and the fate of theses aggregates

S. Hirohara et al.
Bull. Chem. Soc. Jpn. Vol. 86, No. 11 (2013) 1305
Thus, •OH radical species were generated in the cellular
environment by photoirradiation in the presence of trans-
H22SGlc and H24SGlc, and the º•OH value determined in PBS
containing 1 vol % DMSO reflected the efficiency of •OH gen-
eration in the cellular environment. The efficient generation of
•OH species partly explains the superiority of trans-bisglyco-
conjugated porphyrin derivatives over other glycoconjugation
patterns. However, the free-base porphyrins (trans-H22SGlc
and trans-H22OGlc) tend to exert greater photocytotoxicities
than metalloporphyrins (trans-Zn2SGlc, trans-Pt2SGlc, and trans-
Pd2pGlc), even though the metalloporphyrins are much better
•OH generators than the free-base porphyrins. Thus, •OH
species is not likely to be the dominant species in the photo-
dynamic action of glycoconjugated porphyrins.
In summary, this study demonstrates that trans-bisglycocon-
jugation is an efficient and versatile architecture for TFPP-
based PDT photosensitizers. The superiority of this architecture
is insensitive to the central metal ion (Zn, Pd, and Pt) and to
the glycoconjugation methodology (S-arylation, O-arylation,
and click route). The superiority of trans-bisglycoconjugated
porphyrin derivatives could be due to the following reasons
(Table 4): (1) enhanced cellular uptake, (2) characteristic sub-
cellular localization in the mitochondria, (3) formation of
relatively large J-type aggregates in aqueous media resulting in
a longer lifetime of the excited state compared with H-type
aggregates, and (4) moderately efficient 1O2 production and
excellent •OH production.
Figure 4. Plots of the relative cellular uptake by HeLa cells
as a function of the log P values of S-glycoconjugated
free-base porphyrins (red), S-glycoconjugated porphyrin
zinc(II) complexes (blue), S-glycoconjugated porphyrin
platinum(II) complexes (green), O-glycoconjugated free-
base porphyrins (purple), and click-glycoconjugated por-
phyrin palladium(II) complexes (black). The values repre-
sent the means « standard deviation of three experiments.
a
Data cited from Ref. 14.
Conclusion
type I mechanism; First photoexcited photosensitizer (PS*)
accepts an electron from reducing agent to form radical anion
(PS•¹). Then radical anion PS•¹ reduces molecular oxygen (O2)
We systematically synthesized 25 glycoconjugated porphy-
rin derivatives with all five patterns of glycoconjugation. The
in vitro photocytotoxicity test clearly showed the superiority
of trans-bisglycoconjugation of porphyrin regardless of the
central metal ion and the type of glycoconjugation. Considering
their biological interaction, most trans-bisglycoconjugated
porphyrin derivatives were taken up by cells at higher levels
than the other glycoconjugated porphyrin derivatives in the
same category. This enhancement of cellular uptake could be
one of the reasons for the superiority of trans-glycoconjuga-
tion. Considering their physicochemical characteristics, UV-
vis spectroscopy and DLS measurements suggest that trans-
bisglycoconjugated porphyrin derivatives tend to aggregate in
J-type form in aqueous solution. In aqueous media, all glyco-
conjugated porphyrin derivatives tested in this article generate
•¹
to form superoxide anion (O2 ).35 Superoxide anion can
dismute to H2O2 and further converted to •OH by Fenton
chemistry. In this study, therefore, the production of 1O2 as well
as H2O2 and •OH have been investigated. The quantum yield
1
1
of O2 production (Φ¦) was determined by means of the O2
luminescence measurement at 1270 nm in O2-saturated D2O
containing 0.1 vol % DMSO using TPPS (Φ¦ = 0.72)36 as a
standard. On the other hand, the relative quantum yields of
H2O2 and •OH were evaluated by the initial rate constant of the
¹
formation of I3 and the initial rate of increase in HPF-fluo-
rescence intensity,37 respectively, both of which were divided
by the oscillator strength in the region over 500 nm ( f>500 nm
)
1
and normalized to the value for hematoporphyrin (HP). The
relative quantum yields of each ROS are listed in Table 3. All
glycoconjugated porphyrin derivatives have lower Φ¦ values
(<0.53) than the water-soluble porphyrin, TPPS (Φ¦ = 0.72).
Interestingly, trans-bisglycoconjugated porphyrin derivatives
showed lower Φ¦ values than other glycoconjugated com-
pounds, while trans-bisglycoconjugated porphyrin derivatives
efficiently generated •OH radical species. To clarify whether the
•OH species was generated in the cellular environment, HPF
treatment of HeLa cells was carried out 30 min before photo-
irradiation in the presence of trans-H22SGlc (º•OH = 7.7) and
H24SGlc (º•OH = 2.0). Figure 6 shows bright field and fluores-
cence images of HPF-stained HeLa cells. The fluorescence
intensity of HPF-stained HeLa cells treated with trans-H22SGlc
H2O2 and •OH as well as O2. trans-Bisglycoconjugated por-
1
phyrin derivatives have slightly smaller quantum yields of O2
1
and much greater relative quantum yields of •OH. While O2
may be the dominant species responsible for photodynamic
activity, •OH contributes to the photocytotoxicityt as well. This
could also contribute to the superiority of trans-glycoconjuga-
tion. The clickable precursor, namely 5,10,15,20-tetrakis(per-
fluorophenyl)porphyrinatopalladium(II) bearing two propargyl
groups at the trans position (trans-Pd2p), can be used to
develop sophisticated glycoconjugated porphyrin derivatives
with the superior characteristics resulting from trans-bisglyco-
conjugation.
The authors thank Professor Atsushi Ikeda and Dr. Motofusa
Akiyama (Graduate School of Materials Science, Nara Institute
was obviously higher than that of cells treated with H24SGlc
.

1306 Bull. Chem. Soc. Jpn. Vol. 86, No. 11 (2013)
trans-Bisglycoconjugated Photosensitizers
Table 2. UV-vis Spectral Data of Glycoconjugated Porphyrin Derivatives in PBS Containing 1 vol % DMSO
at 25 °Ca)
¹1
-
max/nm (¾ © 10¹3/M¹1 cm
)
b)
f>500 nm
Soret band
Q bands
H21SGlc
cis-H22SGlc
trans-H22SGlc
H23SGlc
421 (13.5)c)
422 (14.2)c)
441 (20.5)c)
433 (13.1)c)
410 (29.5)c)
509 (2.3)c)
509 (2.2)c)
519 (3.2)c)
516 (2.2)c)
510 (2.4)c)
553 (0.24)c)
554 (0.23)c)
554 (0.66)c)
556 (0.35)c)
556 (0.32)c)
584 (0.8)c)
583 (0.8)c)
587 (1.1)c)
584 (0.8)c)
579 (0.8)c)
639 (0.13)c)
639 (0.10)c)
639 (0.18)c)
639 (0.12)c)
639 (0.09)c)
0.12c)
0.11c)
0.18c)
0.12c)
0.13c)
H24SGlc
Zn1SGlc
cis-Zn2SGlc
trans-Zn2SGlc
Zn3SGlc
427 (12.0)
430 (13.6)
429 (13.8)
431 (13.4)
424 (22.8)
555 (2.0)
555 (2.2)
554 (2.2)
555 (2.1)
558 (2.1)
588 (0.5)
588 (0.4)
588 (0.4)
588 (0.4)
591 (0.5)
0.13
0.13
0.13
0.13
0.14
Zn4SGlc
Pt1SGlc
cis-Pt2SGlc
trans-Pt2SGlc
Pt3SGlc
392 (10.5)
387 (11.4)
414 (10.8), 362 (5.0) 517 (1.6)
397 (8.0), 374 (4.1)
393 (9.9), 375 (4.2)
511 (1.3)
509 (1.6)
542 (1.7)
543 (1.7)
543 (1.3)
542 (1.1)
541 (1.2)
0.08
0.14
0.14
0.09
0.09
551 (1.8)
513 (1.1)
509 (1.1)
Pt4SGlc
H21OGlc
cis-H22OGlc
trans-H22OGlc
H23OGlc
427 (4.78)
419 (10.5)
437 (7.9)
409 (11.0)
409 (29.4)
510 (1.1)
510 (1.9)
516 (1.6)
510 (1.8)
508 (1.7)
586 (0.4)
584 (0.7)
588 (0.6)
584 (0.6)
577 (0.6)
0.08
0.12
0.13
0.12
0.09
H24OGlc
Pd1pGlc
cis-Pd2pGlc
trans-Pd2pGlc
Pd3pGlc
412 (5.35)
412 (5.30)
416 (6.01)
412 (5.82)
411 (7.54)
522 (0.9)
522 (0.9)
523 (0.9)
522 (0.8)
520 (1.3)
557 (0.76)
556 (0.66)
557 (0.71)
556 (0.57)
555 (0.80)
0.05
0.05
0.04
0.04
0.06
Pd4pGlc
a) [Photosensitizer] = 1.00 ¯M. b) Oscillator strength in the range above 500 nm estimated as 4.32 © 10¹9 ∫¾(¯) d¯.
c) Spectral data were cited from Ref. 14.
Table 3. Quantum Yield of Singlet Oxygen (Φ¦) and Relative Quantum Yield of Hydrogen Peroxide (º
Hydroxyl Radical (º•OH) of Glycoconjugated Porphyrin Derivativesa)
) and
H2O2
b)
c)
c)
b)
c)
c)
Φ¦
ºH O
º•OH
Φ¦
ºH O
2
º•OH
2
2
2
H21SGlc
cis-H22SGlc
trans-H22SGlc
H23SGlc
n.d.
1.7
1.5
0.8
1.6
1.2
2.6
2.3
7.7
1.6
2.0
H21OGlc
cis-H22OGlc
trans-H22OGlc
H23OGlc
0.40
0.53
0.22
0.32
1.5
1.5
1.0
1.4
1.5
4.4
5.0
11.3
1.9
0.22
0.16
0.22
0.25
e)
H24SGlc
H24OGlc
®
0.5
e)
Zn1SGlc
cis-Zn2SGlc
trans-Zn2SGlc
Zn3SGlc
n.d.
4.7
10.3
7.7
18.3
6.0
12.9
16.3
17.3
16.2
12.5
Pd1pGlc
cis-Pd2pGlc
trans-Pd2pGlc
Pd3pGlc
®
®
6.5
12.1
7.8
14.7
5.6
12.1
18.6
20.4
20.4
8.7
e)
0.09
0.05
0.15
0.33
e)
®
®
e)
e)
Zn4SGlc
Pd4pGlc
®
Pt1SGlc
cis-Pt2SGlc
trans-Pt2SGlc
Pt3SGlc
0.09
0.13
n.d.d)
0.17
0.21
2.2
3.8
4.1
1.9
2.0
3.0
2.6
28.7
6.1
Pt4SGlc
1.2
a) The ºH O and º•OH values were evaluated by the initial rate of formation of I3¹ and the initial rate of increase in
HPF-fluor2es2cence intensity, respectively, both divided by the f>500 nm value and normalized to the values for HP.
1
b) In O2-saturated D2O containing 0.1 vol % DMSO. c) In O2-saturated PBS containing 1 vol % DMSO. d) O2
1
luminescence was not detected. e) O2 luminescence could not be measured due to the second-order diffraction of
the intense luminescence of the porphyrin derivative.

S. Hirohara et al.
Bull. Chem. Soc. Jpn. Vol. 86, No. 11 (2013) 1307
Figure 6. Bright field (upper) and fluorescence (lower)
images of HPF-stained HeLa cells after photoirradia-
tion in the presence of vehicle (a), trans-H22SGlc (b), and
H24SGlc (c). [Photosensitizer] = 100 nM. [HPF] = 10 ¯M.
The excitation and emission wavelengths were 488 and
530 nm, respectively.
Table 4. Summary of Characteristics of trans-Bissubstituted
Porphyrin
trans-2
Others
Low
Photocytotoxicity
Very high
Aggregation in aqueous media
Cellular uptake
Subcellular localization
1O2 Production efficiency
•OH Production efficiency
J-Type
High
Mitochondria
Relatively low
High
®
Low
®
High
Low
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