Comparative studies of the cellular uptake, subcellular localization, and cytotoxic and phototoxic antitumor properties of ruthenium(II)-porphyrin conjugates with different linkers

Article abstract of DOI:10.1021/bc300201h

Six water-soluble free-base porphyrin-Ru(II) conjugates, 1-3, and Zn(II) porphyrin-Ru(II) conjugates, 4-6, with different linkers between the hydrophobic porphyrin moiety and the hydrophilic Ru(II)-polypyridyl complex, have been synthesized. The linear and two-photon-induced photophysical properties of these conjugates were measured and evaluated for their potential application as dual in vitro imaging and photodynamic therapeutic (PDT) agents. Conjugates 1-3, with their high luminescence and singlet oxygen quantum yields, were selected for further study of their cellular uptake, subcellular localization, and cytotoxic and photocytotoxic (under linear and two-photon excitation) properties using HeLa cells. Conjugate 2, with its hydrophobic phenylethynyl linker, was shown to be highly promising for further development as a bifunctional probe for two-photon (NIR) induced PDT and in vitro imaging. Cellular uptake and subcellular localization properties were shown to be crucial to its PDT efficacy.

Full text of DOI:10.1021/bc300201h

Article
pubs.acs.org/bc
Comparative Studies of the Cellular Uptake, Subcellular Localization,
and Cytotoxic and Phototoxic Antitumor Properties of
Ruthenium(II)−Porphyrin Conjugates with Different Linkers
Jing-Xiang Zhang,†,# Jun-Wei Zhou,§,# Chi-Fai Chan,†,# Terrence Chi-Kong Lau,*,§ Daniel W. J. Kwong,†
Hoi-Lam Tam,‡ Nai-Ki Mak,∥ Ka-Leung Wong,*,† and Wai-Kwok Wong*,†
‡
∥
†Department of Chemistry, Department of Physics, and Department of Biology, Hong Kong Baptist University, Kowloon Tong,
Hong Kong SAR
§Department of Biology and Chemistry, City University of Hong Kong, Tat Chee Avenue, Kowloon Tong, Hong Kong SAR
S
* Supporting Information
ABSTRACT: Six water-soluble free-base porphyrin-Ru(II)
conjugates, 1−3, and Zn(II) porphyrin-Ru(II) conjugates,
4−6, with different linkers between the hydrophobic porphyrin
moiety and the hydrophilic Ru(II)-polypyridyl complex, have
been synthesized. The linear and two-photon-induced photo-
physical properties of these conjugates were measured and
evaluated for their potential application as dual in vitro
imaging and photodynamic therapeutic (PDT) agents.
Conjugates 1−3, with their high luminescence and singlet oxygen quantum yields, were selected for further study of their
cellular uptake, subcellular localization, and cytotoxic and photocytotoxic (under linear and two-photon excitation) properties
using HeLa cells. Conjugate 2, with its hydrophobic phenylethynyl linker, was shown to be highly promising for further
development as a bifunctional probe for two-photon (NIR) induced PDT and in vitro imaging. Cellular uptake and subcellular
localization properties were shown to be crucial to its PDT efficacy.
two-photon absorption (TPA) cross section, σ2, from <20 GM
INTRODUCTION
■
to 178 GM, permitting their potential use as TPA-PDT agents
Photodynamic therapy (PDT) has recently emerged as a
against solid tumors. Furthermore, these conjugates became
promising alternative therapy against cancer, with several PDT
luminescent upon visible/near-infrared excitation in vitro, thus
agents approved and used clinically in the past two decades.1−3
enabling their development as bifunctional tumor imaging and
In PDT, three key elements are required: a photosensitizer
PDT agents.25,26 Nonetheless, to be an efficacious photo-
(PS), which when excited by light of an appropriate
chemotherapeutic agent, its cellular uptake and subcellular
wavelength, converts molecular oxygen in tissues to the
localization properties are important parameters to consider as
cytotoxic singlet oxygen (1O2).4−13 A porphyrin-based
well.27
compound, shown to accumulate preferentially in solid tumors,
In this work, we report the synthesis of six amphiphilic
was the first PDT agent approved by the U.S. Food and Drug
Ru(II) appended porphyrins (1, 2, 3) and Zn(II) porphyrins
Administration as a noninvasive antitumor treatment modal-
(4, 5, 6) using linkers with different physicochemical (e.g.,
hydrophobic, electronic) properties and compare their
luminescent (via linear and two-photon excitations), cellular
ity.14−16 Ruthenium and organoruthenium complexes have also
been shown to be effective chemotherapeutic agents against a
variety of cancer cells.17−21 Recently, several hydrophilic
uptake, subcellular localization, and (dark) cytotoxic and
Ru(II)-porphyrin conjugates were synthesized and reported
photocytotoxic properties in order to determine the optimal
to have potent cytotoxic, as well as phototoxic activities toward
linker properties in these Ru(II)-porphyrin conjugates for TPA-
tumors.22,23 The IC50 values of these conjugates obtained under
photoirradiation (λirr = 590−700 nm) were found to be lower
by 1 order of magnitude than those obtained in the dark.
PDT application.
EXPERIMENTAL SECTION (MATERIALS AND
■
However, the strongest absorptions of these Ru(II) conjugates
METHODS)
were at ca. 420 nm, with less than 3% of their maximum
Synthesis of Compounds 1 to 6. All analytical-grade
solvents were dried by standard procedures, distilled, and
absorption intensities found in the tissue-transparent region
(>650 nm). To overcome this problem, we have designed some
Ru(II)-porphyrin conjugates with substantial absorption under
two-photon excitation at λex ≥ 800 nm.22−24 In these
conjugates, the linkage of the Ru(II)-polypyridyl complex, a
charge-transfer moiety, to the tetraphenylporphyrin raised its
Received: April 10, 2012
Revised: May 23, 2012
Published: July 9, 2012
© 2012 American Chemical Society
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Figure 1. Schematic structures of ruthenium-porphyrin conjugates 1−6.
a
Scheme 1. Synthetic Route for Conjugates 1 and 4
a
Reactions and conditions: (a) propionic acid, 140 °C, 15%; (b) BrCH2CONH-phen, Cs2CO3, DMF, 80 °C, 42%; (c) cis-Ru(bpy)2Cl2, THF/
ethanol, 81%; (d) Zn(OAc)2·2H2O, MeOH, 98%.
deaerated before use. NMR spectra were recorded on a Bruker
Ultrashield 400 plus NMR spectrometer. The 1H NMR
chemical shifts were referenced to tetramethylsilane, TMS (d
= 0.00). High-resolution mass spectra were obtained on a
Bruker Autoflex MALDI−TOF mass spectrometer. IR spectra
were recorded on Nicolet Magna 550 Series II. Preparations of
cis-Ru(bpy)2Cl2,28 5-bromo-1,10-phenanthroline,29 and di-
(ethylene glycol) di-p-toluenesulfonate30 were performed
according to literature procedures. The synthetic routes of
compounds 1−6 are listed in Schemes 1, 2, and 3.
5,10,15-Tris(3′,4′,5′-trimethoxylphenyl)-20-(4′-hydoxyl-
phenyl)-21H,23H-porphyrin (p-OH Por). A solution of pyrrole
(4.556 g, 0.068 mol) in 100 mL of propionic acid was added
dropwise into a solution of 3,4,5-trimethoxylbenzaldehyde
(10.00 g, 0.051 mol) and 4-hydroxylbenzaldehyde (2.075 g,
0.017 mol) in 300 mL of propionic acid at 120 °C. When it is
completed, the temperature was raised up to 140 °C. After
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b
Scheme 2. Synthetic Route for Conjugates 2 and 5.
b
Reactions and conditions: (a) propionic acid, 140 °C, 18%; (b) Zn(OAc)2·2H2O, CHCl3, MeOH, 97%; (c) TBAF, THF; (d) Pd(PPh3)4, CuI,
THF, diisopropylamine, 5-bromo-1,10-phen, 51% (two steps); (e) HCl, CH2Cl2, RT, 90%; (f) cis-Ru(bpy)2Cl2, THF/ethanol, 85%; (g) cis-
Ru(bpy)2Cl2, THF/ethanol, 81%.
refluxing for 3 h, the propionic acid was distillated out under
reduced pressure until about 80 mL solution was left. Then,
100 mL of ethanol was added into the flask and the solution
was cooled overnight in the refrigerator. The precipitate was
filtered out and washed with ethanol for several times. The
product (2.297 g, 0.003 mol) was obtained in the second band
as a purple solid after column chromatography (CHCl3 as
eluent), yield 15%. UV−visible (CHCl3), λabs/nm (log ε) 422
(5.56), 518 (4.35), 553 (3.86), 592 (3.80), 648 (3.75). IR
(KBr), ν/cm−1: 2925 (s), 2847 (w), 1576 (s), 1495 (s), 1460
(s), 1401 (s), 1356 (s), 1245 (s), 1168 (m), 1119 (s), 996 (m),
(200 mg, 0.222 mmol) in anhydrous DMF (10 mL). The
solution was stirred for 24 h at 80 °C, and then the solvent was
removed; the residue was extracted with CHCl3 and washed
with water. The organic solvent was collected and removed
under reduced pressure. The crude product was purified by
column chromatography (CHCl3/MeOH (v/v) = 30:1); yield:
106 mg, 42%. UV−visible (CHCl3), λabs/nm (log ε) 422
(5.51), 518 (4.30), 553 (3.87), 592 (3.70), 651 (3.68). IR
(KBr), ν/cm−1: 2917 (s), 2843 (w), 1699 (m), 1576 (s), 1499
(s), 1458 (s), 1401 (s), 1356 (m), 1233 (s), 1180 (m), 1119
(s), 1004 (m), 967 (w), 927 (m), 792 (s), 730 (m), 628 (w),
1
1
972 (w), 918 (s), 792 (m), 730 (m), 587 (m). H NMR
583 (w). H NMR (CDCl3) δ −2.76 (s, 2H), 3.98 (s, 18H),
(CDCl3) δ −2.79 (s, 2H), 3.96 (s, 18H), 4.18 (s, 9H), 5.30 (s,
1H), 7.22 (d, 2H, J = 8.6 Hz), 7.46 (d, 6H, J = 1.6 Hz), 8.07 (d,
2H, J = 8.4 Hz), 8.88 (d, 2H, J = 4.7 Hz), 8.94 (d, 6H, J = 4.7
Hz). HRMS (MALDI-TOF) ([M]+, m/z): Calcd for
C53H48N4O10, 900.9; Found for [M+H]+, 901.3.
Amide-Linked Porphyrin-Phen (L1). 2-Bromo-N-(1,10-phe-
nanthrolin-5-yl) acetamide (421 mg, 1.332 mmol) and Cs2CO3
(64 mg, 0.333 mmol) were added into a solution of p-OH Por
4.19 (s, 9H), 5.16 (s, 2H), 7.48 (d, 6H, J = 2.0 Hz), 7.52 (d,
2H, J = 8.2 Hz), 7.69−7.72 (m, 1H), 7.80−7.84 (m, 1H), 8.27
(d, 2H, J = 8.4 Hz), 8.33−8.35 (dd, 1H, J = 1.6 Hz, 8.1 Hz),
8.44−8.46 (dd, 1H, J = 1.5 Hz, 8.4 Hz), 8.57 (s, 1H), 8.88 (d,
2H, J = 4.7 Hz), 8.98 (s, 6H), 9.14 (s, 1H), 9.20−9.22 (dd, 1H,
J = 1.7 Hz, 4.4 Hz), 9.32 (dd, 1H, J = 1.7 Hz, 4.1 Hz). HRMS
(MALDI-TOF) ([M]+): Calcd for C67H57N7O11, 1136.2;
Found for [M]+, 1136.4.
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c
Scheme 3. Synthetic Route for Conjugates 3 and 6
c
Reactions and conditions: (a) Di(ethylene glycol) di-p-toluenesulfonate, K2CO3, DMF, 55%; (b) 4,7-diol-1,10-phen, NaH, DMF, 30%; (c) cis-
Ru(bpy)2Cl2, acetic acid, reflux, 56%; (d) Zn(OAc)2·2H2O, MeOH, 88%.
Amide-Linked [(Porphyrin-Phen)Ru(bpy)2][Cl]2 (1). Por-
phyrin ligand L1 (50 mg, 0.044 mmol) and cis-Ru(bpy)2Cl2
(85 mg, 0.176 mmol) were added into a solution of 15 mL
THF and 15 mL ethanol, and then the solution was bubbled
with N2 for a few minutes, and the reaction temperature was set
to 85 °C. After refluxing 15 h, the solvent was removed under
vacuum and the residue was chromatographed on Al2O3 several
times, the eluent in turn is CHCl3, (CHCl3:MeOH (v/v) =
12:1), yield 58 mg, 81%. UV−visible (CHCl3), λabs/nm (log ε)
288 (4.91), 424 (5.58), 517 (4.29), 554 (3.98), 592 (3.79), 646
(3.65). IR (KBr), ν/cm−1: 2925 (m), 2819 (w), 1580 (s), 1540
(m), 1499 (s), 1466 (s), 1405 (s), 1356 (m), 1233 (s), 1176
(m), 1119 (s), 1004 (m), 976 (m), 932 (m), 845 (w), 800 (m),
763 (m), 726 (m), 566 (m). 1H NMR (d6-DMSO) δ −2.91 (s,
2H), 3,89 (s, 18H), 3.99 (s, 9H), 5.39 (s, 2H), 7.36−7.40 (m,
2H), 7.50 (m, 8H), 7.58−7.62 (m, 4H), 7.84−7.89 (m, 3H),
7.94−7.98 (m, 1H), 8.06−8.14 (m, 3H), 8.16−8.23 (m, 5H),
8.78 (s, 1H), 8.82−8.91 (m, 7H), 8.94 (s, 6H), 9.18 (m, 1H),
11.31 (s, 1H). HRMS (MALDI−TOF) ([M−2Cl]+, m/z):
mg, 0.022 mmol) in 20 mL of methanol at 65 °C for 3 h. The
crude product was washed with water and then dried in
vacuum. The pure product was obtained by flash chromatog-
raphy on Al2O3, yield: 31 mg, 98%. UV−visible (CHCl3), λabs/
nm (log ε) 288 (4.94), 431 (5.67), 560 (4.28), 603 (4.32). IR
(KBr), ν/cm−1: 2917 (s), 2847 (w), 1593 (m), 1580 (s), 1540
(m), 1507 (s), 1458 (s), 1409 (s), 1343 (s), 1237 (s), 1176
(m), 1123 (s), 996 (m), 943 (m), 800 (m), 763 (m), 722 (m),
1
620 (m). HNMR (d6-DMSO) δ 3.90 (s, 18H), 4.00 (s, 9H),
5.33 (s, 2H), 7.35−7.38 (m, 2H), 7.43 (d, 6H, J = 6.2 Hz), 7.52
(d, 2H, J = 8.5 Hz), 7.58−7.63 (m, 4H), 7.83−7.88 (m, 3H),
7.94−7.99 (m, 1H), 8.06−8.15 (m, 5H), 8.19−8.24 (m, 3H),
8.75 (s, 1H), 8.79−8.87 (m, 7H), 8.89 (d, 6H, J = 4.4 Hz), 9.05
(d, 1H, J = 8.5 Hz), 11.06 (s, 1H). HRMS (MALDI−TOF)
([M−2Cl]+, m/z): Calcd for [C87H71N11O11RuZn]+, 1613.4;
Found for [M−2Cl]+, 1612.1.
5,10,15-Tris(3′,4′,5′-trimethoxylphenyl)-20-[4′-(2″-
trimethylsilylethyl)phenyl]-21H,23H-porphyrin (p-Acetylene
Por). A solution of pyrrole (3.646 g, 0.054 mol) in propionic
acid (100 mL) was added dropwise into a solution of 3,4,5-
trimethoxylbenzaldehyde (8.00 g, 0.041 mol) and 4-[2′-
(trimethylsilyl)ethynyl]benzaldehyde (2.748 g, 0.014 mol) in
propionic acid (300 mL) at 120 °C. When it was completed,
+
Calcd for [C87H73N11O11Ru] , 1549.6; Found for [M−2Cl]+,
1549.4.
Amide-Linked [(Zn-porphyrin-Phen)Ru(bpy)2][Cl]2 (4). 1
(30 mg, 0.0185 mmol) was treated with Zn(OAc)2·2H2O (5
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the temperature was raised up to 140 °C. After refluxing for 3 h,
the propionic acid was distilled out completely under reduced
pressure. The residue was dissolved in CHCl3 and filtered
through a short silica gel column and then the filtrate was
washed with water. The crude product was purified by column
chromatography using CHCl3 as eluent. Purple solid was
obtained. Yield: 2.400 g, 18%. UV−visible (CHCl3), λabs/nm
(log ε) 422 (5.65), 516 (4.31), 553 (3.90), 591 (3.83), 648
(3.81). IR (KBr), ν/cm−1: 2925 (s), 2823 (m), 1576 (s), 1499
(s), 1470 (s), 1405 (s), 1352 (s), 1225 (s), 1176 (m), 1123 (s),
1017 (m), 967 (w), 918 (m), 861 (m), 792 (m), 730 (m), 653
(w), 567 (w). 1H NMR (CDCl3) δ −2.80 (s, 2H), 0.38 (s, 9H),
9.36 (s, 18H), 4.18 (s, 9H), 7.45 (s, 6H), 7.86 (d, 2H, J = 8.2
Hz), 8.16 (d, 2H, J = 8.3 Hz), 8.91 (d, 2H, J = 4.7 Hz), 9.08 (m,
6H). HRMS (MALDI-TOF) ([M]+, m/z): Calcd for
C58H56N4O9Si, 981.2; Found for [M]+, 981.2.
5,10,15-Tris(3′,4′,5′-trimethoxylphenyl)-20-[4′-[2″-
(trimethylsilyl)ethynyl]-phenyl]-21H,23H porphyrinato zinc-
(II) (p-Acetylene ZnPor). Zn(OAc)2·2H2O (107 mg, 0.490
mmol) and porphyrin p-Acetylene Por (400 mg, 0.408 mmol)
were dissolved in a mixture of CHCl3 and MeOH, and the
reaction was maintained at 60 °C for 3 h. The solvent was
removed and the residue was purified on a silica gel using
CHCl3 as eluent to give 7 as purple solid. Yield: 413 mg, 97%.
1H NMR (CDCl3) 0.39 (s, 9H), δ 3.92 (s, 18H), 4.12 (s, 9H),
7.45 (s, 6H), 7.86 (d, 2H, J = 8.1 Hz), 8.07 (d, 2H, J = 8.0 Hz),
8.93 (d, 2H, J = 4.7 Hz), 9.07 (m, 6H).
Phenylacetylene-Linked Zinc-Porphyrin-Phen (ZnL2). p-
Acetylene ZnPor (380 mg, 0.364 mmol) was dissolved in
anhydrous CH2Cl2 in N2 atmosphere, TBAF solution (400 μL,
1 M in THF) was injected into the CH2Cl2 solution and the
resultant solution was stirred at room temperature for 30 min.
The reaction solution was filtered by flash chromatography and
the deprotected ethynyl-zinc-porphyrin was dried in vacuum. 5-
Bromo-1,10-phenanthrolin (62.8 mg, 0.242 mmol) and then 20
mL of anhydrous THF was added in the above flask. The
Sonogashira coupling reaction was administered in N2
atmosphere using Pd(PPh3)4 (28 mg, 0.024 mmol) and CuI
(2.3 mg, 0.012 mmol) as catalysts and diisopropylamine (6
mL) as base. The reaction mixture was heated at 45 °C for 8 h.
The solvent was removed and the residue was purified on silica
gel using CHCl3/MeOH (v/v = 20:1) as eluent, yield 142 mg,
51%. UV−visible (CHCl3), λabs/nm (log ε) 278 (4.20), 426
(5.58), 553 (4.30), 595 (3.95). IR (KBr), ν/cm−1: 2921 (s),
2851 (w), 1617 (s), 1576 (s), 1556 (m), 1535 (w), 1503 (s),
1458 (s), 1405 (s), 1348 (s), 1237 (s), 1127 (s), 992 (m), 935
(w), 796 (m), 718 (m), 669 (m), 567 (s). 1H NMR (CDCl3) δ
3.97 (s, 18H), 4.19 (s, 9H), 7.49 (d, 6H, J = 1.7 Hz), 7.68−7.71
(m, 1H), 7.81−7.88 (m, 1H), 8.08 (d, 2H, J = 8.1 Hz), 8.27 (s,
1H), 8.29−8.31 (m, 3H), 8.97 (d, 2H, J = 4.7 Hz), 9.04−9.08
(m, 7H), 9.18 (d, 1H, J = 3.6 Hz), 9.25 (d, 1H, J = 2.8 Hz).
HRMS (MALDI−TOF) ([M]+, m/z): Calcd for
C67H52N6O9Zn, 1148.3; Found for [M+H]+, 1149.3.
(s), 1580 (s), 1511 (m), 1454 (m), 1405 (w), 1356 (m), 1241
(s), 1119 (s), 1070 (w), 1004 (w), 927 (s), 804 (m), 726 (w),
665 (w), 575 (w). 1H NMR (CDCl3) δ −2.75 (s, 2H), 3.98 (d,
18H, J = 4.2 Hz), 4.18 (d, 9H, J = 4.2 Hz), 7.49 (m, 6H), 7.69
(m, 1H), 7.84 (m, 1H), 8.10 (m, 2H), 8.27 (d, 1H, J = 3.4 Hz),
8.30 (m, 3H), 8.91 (d, 2H, J = 4.4 Hz), 8.99−9.06 (m, 7H),
9.22−9.26 (m, 1H), 9.29−9.33 (m, 1H). HRMS (MALDI−
TOF) ([M]+, m/z): Calcd for C67H54N6O9, 1086.4; Found for
[M+H]+, 1087.4.
Phenylacetylene-Linked [(Zinc-Porphyrin-Phen)Ru(bpy)2]-
[Cl]2 (5). Zinc-porphyrin ligand ZnL2 (50 mg, 0.044 mmol) and
cis-Ru(bpy)2Cl2 (63 mg, 0.131 mmol) were added in a mixture
of THF (15 mL) and ethanol (15 mL). The solution was then
bubbled with N2 for a few minutes, and heated to 85 °C. After
refluxing 15 h, the solvent was removed under vacuum and the
residue was chromatographed on Al2O3 several times, and the
eluent in turn was CHCl3, (CHCl3:MeOH (v/v) = 12:1).
Yield: 60 mg, 85%. UV−visible (CHCl3), λabs/nm (log ε) 288
(4.92), 431 (5.55), 562 (4.46), 606 (4.31). IR (KBr), ν/cm−1:
2921 (s), 2843 (w), 1625 (s), 1572 (s), 1482 (m), 1462 (s),
1405 (s), 1348 (s), 1229 (s), 1115 (s), 1061 (w), 988 (s), 935
1
(m), 792 (w), 763 (m), 722 (m), 543 (m). H NMR (d6-
DMSO) δ 3.91 (d, 18H, J = 11.7 Hz), 4.06 (d, 9H, J = 10.6
Hz), 7.37−7.40 (m, 2H), 7.51 (d, 6H, J = 4.8 Hz), 7.57−7.61
(m, 2H), 7.74−7.79 (m, 4H), 7.83−7.87 (m, 1H), 7.91 (d, 2H,
J = 7.2 Hz), 7.95−7.99 (m, 3H), 8.08−8.12 (m, 2H), 8.18−8.24
(m, 3H), 8.31 (d, 1H, J = 5.1 Hz), 8.61 (s, 1H), 8.65−8.68 (m,
3H), 8.73−8.79 (m, 4H), 8.89 (d, 2H, J = 4.7 Hz), 8.94−8.97
(m, 4H), 9.15 (d, 1H, J = 8.4 Hz). HRMS (MALDI-TOF)
([M−2Cl]+, m/z): Calcd for C87H68N10O9RuZn, 1563.9;
Found for [M−2Cl]+, 1563.2; [M−2Cl−bpy]+, 1408.1.
Phenylacetylene-Linked [(Porphyrin-Phen)Ru(bpy)2][Cl]2
(2). Porphyrin ligand L2 (50 mg, 0.046 mmol) and cis-
Ru(bpy)2Cl2 (67 mg, 0.138 mmol) were dissolved in a mixture
of THF (15 mL) and ethanol (15 mL). The solution was then
bubbled with N2 for a few minutes before heating to 85 °C.
After refluxing 15 h, the solvent was removed under vacuum
and the residue was chromatographed on Al2O3 several times;
the eluent in turn was CHCl3 (CHCl3:MeOH (v/v) = 12:1).
Yield: 58 mg, 81%. UV−visible (CHCl3), λabs/nm (log ε) 288
(4.66), 425 (5.33), 517 (4.12), 554 (3.91), 593 (3.72), 646
(3.66). IR (KBr), ν/cm−1: 2925 (s), 2843 (w), 1643 (s), 1609
(s), 1576 (w), 1503 (m), 1450 (m), 1405 (s), 1380 (m), 1237
(w), 1119 (s), 993 (m), 800 (m), 767 (w), 661 (w), 561 (m).
1H NMR (d6-DMSO) δ −2.98 (s, 2H), 3.82 (s, 18H), 3.91 (s,
9H), 7.32−7.37 (m, 2H), 7.45 (d, 6H, J = 7.8 Hz), 7.54−7.57
(m, 2H), 7.61 (d, 2H, J = 5.4 Hz), 7.80 (s, 2H), 7.88−7.91 (m,
1H), 7.99−8.03 (m, 1H), 8.06−8.11 (m, 2H), 8.13−8.21 (m,
6H), 8.31 (d, 2H, J = 8.0 Hz), 8.81−8.93 (m, 14H), 9.21 (d,
1H, J = 8.6 Hz). HRMS (MALDI-TOF) ([M−2Cl]+, m/z):
Calcd for C87H70N10O9Ru, 1500.6; Found for [M−2Cl]+,
1500.4; [M−2Cl +DHB]+, 1653.3.
1-[4-[5,10,15-Tris(3,4,5-trimethoxylphenyl)-20-porphyrin-
yl]-phenoxy]-5-(p-tolylsulfonyloxy)ethoxy-ethane (p-PEG-
OTs Por). Di(ethylene glycol) di-p-toluenesulfonate (345 mg,
0.832 mmol) and porphyrin p-OH Por (300 mg, 0.332 mmol)
were dissolved in anhydrous DMF (10 mL), and then K2CO3
(68 mg, 0.498 mmol) was added into the solution. The reaction
solution was heated at 65 °C for 24 h. DMF was removed and
the residue was washed with water several times and extracted
with CH2Cl2. The organic phase was collected and purified on
silica gel to give p-PEG-OTs Por as purple solid. Yield: 208 mg,
55%. UV−visible (CHCl3), λabs/nm (log ε) 422 (5.68), 518
Phenylacetylene-Linked Porphyrin-Phen (L2). Demetalation
of ZnL2 was achieved by dissolving ZnL2 (60 mg, 0.052 mmol)
in a mixture of concentrated HCl (2 mL) and CH2Cl2 (10 mL).
After stirring for 1 h, the solvent was washed by water and the
organic phase was neutralized by NaHCO3. The solvent was
dried by sodium sulfate and removed under reduced vacuum.
Purification was done on silica gel to give purple−red solid of
L2. Yield: 51 mg, 90%. UV−visible (CHCl3), λabs/nm (log ε)
282 (4.18), 424 (5.57), 518 (4.27), 554 (3.83), 592 (3.83), 648
(3.76). IR (KBr), ν/cm−1: 2913 (s), 2855 (w), 1638 (s), 1613
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(4.41), 553 (3.87), 592 (3.87), 648 (3.80). IR (KBr), ν/cm−1:
2921 (s), 2847 (w), 1707 (s), 1646 (w), 1597 (s), 1580 (s),
1495 (s), 1462 (m), 1413 (s), 1352 (s), 1327 (s), 1233 (s),
1172 (s), 1131 (s), 1000 (m), 923 (m), 812 (m), 763 (m), 661
water and then dried under vacuum. The pure product was
obtained by column chromatography on Al2O3 (CHCl3:MeOH
(v/v) = 10:1 as eluent). Yield: 16 mg, 88%. UV−visible
(CHCl3), λabs/nm (log ε) 288 (4.72), 431 (5.64), 562 (4.28),
600 (4.01). IR (KBr), ν/cm−1: 2921 (s), 2847 (w), 1625 (s),
1576 (m), 1503 (m), 1454 (s), 1405 (s), 1343 (m), 1282 (w),
1229 (s), 1131 (s), 1119 (s), 1053 (m), 1004 (m), 967 (w),
1
(m), 555 (m). H NMR (CDCl3) δ −2.78 (s, 2H), 2.45 (s,
3H), 3.92 (m, 2H), 3.97 (s, 18H), 4.15 (m, 2H), 4.17 (s, 9H),
4.31 (m, 2H), 4.37 (m, 2H), 7.30 (d, 2H, J = 8.7 Hz), 7.47 (d,
6H, J = 1.5 Hz), 7.78 (d, 2H, J = 8.3 Hz), 7.86 (d, 2H, J = 8.3
Hz), 8.11 (d, 2H, J = 8.4 Hz), 8.87 (d, 2H, J = 4.7 Hz), 8.94 (d,
6H, J = 5.3 Hz). HRMS (MALDI−TOF) ([M]+, m/z): Calcd
for C64H62N4O14S, 1143.2; Found for [M]+, 1143.4.
1
759 (s), 628 (w), 555 (m). H NMR (d6-DMSO) δ 3.83 (d,
18H, J = 2.5 Hz), 3.93 (d, 9H, J = 2.2 Hz), 4.03−4.07 (m, 4H),
4.39−4.46 (m, 4H), 6.00 (d, 1H, J = 6.8 Hz), 6.64 (d, 1H, J =
6.8 Hz), 7.24 (d, 1H, J = 6.4 Hz), 7.28−7.35 (m, 3H), 7.36−
7.39 (m, 7H), 7.42−7.46 (m, 3H), 7.52 (d, 1H, J = 6.2 Hz),
7.72 (d, 1H, J = 5.8 Hz), 7.76−7.78 (m, 2H), 7.80−7.83 (m,
2H), 7.93−7.98 (m, 4H), 8.01−8.06 (m, 2H), 8.14 (d, 1H, J =
9.0 Hz), 8.68−8.76 (m, 6H), 8.83−8.86 (m, 6H). HRMS
(MALDI−TOF) ([M−2Cl]+ , m/z): Calcd for
[C89H76N10O13RuZn]+, 1660.1; Found for [M−2Cl−H]+,
1658.9.
Linear-Induced Photophysical Properties. UV−visible
absorption spectra in the spectral range 200−1100 nm were
recorded by an HP UV-8453 spectrophotometer. Single-
photon luminescence spectra were recorded using an
Edinburgh Instrument FLS920 combined fluorescence lifetime
and steady state spectrophotometer that was equipped with a
visible to near-infraed-sensitive photomultiplier by in-nitrogen
flow-cooled housing. The spectra were corrected for detector
response and stray background light phosphorescence. The
emission quantum yields of all the compounds were measured
by a demountable 142 mm (inner) diameter barium sulfide-
coated integrating sphere supplied with two access ports in
Edinburgh Instrument FLS920. pH titrations of porphyrin-Ru
homometallic complexes 1−3 were performed on 10 μM
(DMSO) samples dissolved in aqueous solution whose pH was
adjusted by small addition of 1 M NaOH or HCl.
Ethoxyethane-Linked Porphyrin-Phen (L3). 1,10-Phenan-
throline-4,7-diol (74 mg, 0.350 mmol) was dissolved in 5 mL
anhydrous DMF, and then NaH (17 mg, 0.7 mmol) was added
to the solution. The solution was stirred for 20 min, then a
DMF solution of porphyrin p-PEG-OTs Por (200 mg, 0.175
mmol) was added dropwise into the reaction mixture. The
reaction temperature was maintained at 50 °C for 8 h. DMF
was removed and CH2Cl2 was added. The unreacted 1,10-
phenanthroline-4,7-diol was filtered out and the organic phase
was washed by water for several times. The product was
purified on silica gel with the mixed solvent (CHCl3/MeOH
(v/v) = 25:1) as eluent. Yield: 62 mg, 30%. UV−visible
(CHCl3), λabs/nm (log ε) 330 (4.30), 422 (5.72), 516 (4.43),
554 (3.89), 591 (3.88), 648 (3.80). IR (KBr), ν/cm−1: 2917
(s), 2847 (w), 1625 (m), 1580 (s), 1499 (s), 1458 (s), 1405
(s), 1352 (m), 1303 (w), 1282 (w), 1233 (s), 1168 (m), 1119
(s), 1049 (m), 1004 (m), 967 (m), 914 (w), 792 (s), 628 (w),
1
555 (m). H NMR (CDCl3) δ −2.78 (s, 2H), 3.96 (s, 18H),
4.18 (s, 9H), 4.21 (m, 2H), 4.27 (m, 2H), 4.48 (m, 2H), 4.54
(m, 2H), 6.48 (d, 1H, J = 7.3 Hz), 7.03 (d, 1H, J = 5.2 Hz),
7.29 (d, 2H, J = 8.5 Hz), 7.46 (s, 6H), 7.73 (m, 1H), 8.08−8.14
(m, 3H), 8.37 (d, 1H, J = 9.0 Hz), 8.75 (d, 1H, J = 5.2 Hz),
8.84 (d, 2H, J = 4.6 Hz), 8.92−8.95 (m, 6H), 10.41 (s, 1H).
HRMS (MALDI−TOF) ([M]+, m/z): Calcd for C69H62N6O13,
1183.2; Found for [M]+, 1183.4.
Two-Photon Induced Emission and Two-Photon
Absorption Cross-Section Measurement. Two-photon
absorption spectra of 1−6 were measured at 850 nm by the
open-aperture Z-scan method31 using 100 fs laser pulses with a
peak power of 276 GW cm−2 from an optical parametric
amplifier operating at a 1 kHz repetition rate generated from a
Ti:Sapphire regenerative amplifier system. The laser beam was
split into two parts by a beam splitter. One was monitored by a
photodiode (D1) as the incident intensity reference, I0, and the
other beam was detected by the photodiode (D2) as the
transmitted intensity. After passing through a lens with f = 20
cm, the laser beam was focused and passed through a quartz
cell. The position of the sample cell, z, was moved along the
laser-beam direction (z axis) by a computer-controlled
translatable table so that the local power density within the
sample cell could be changed under the constant incident
intensity laser power level. Finally, the transmitted intensity
from the sample cell was detected by the photodiode D2
interfaced to a computer for signal acquisition and averaging.
Each transmitted intensity data represent an average of over
100 measurements. Assuming a Gaussian beam profile, the
nonlinear absorption coefficient β can be obtained by curve
fitting to the observed open-aperture traces, T(z), with eq 1
Ethoxyethane-Linked [{Porphyrin-(5-hydroxyl-Phen)Ru-
(bpy)2}][Cl]2 (3). Porphyrin ligand L3 (50 mg, 0.042 mmol)
and cis-Ru(bpy)2Cl2 (61 mg, 0.127 mmol) were added to acetic
acid (10 mL). Then, the reaction solution was refluxed under
N2 atmosphere for 3 h. The solvent was removed under
vacuum and the residue was chromatographed on Al2O3 several
times; the eluents in turn were (CHCl3:MeOH (v/v) = 20:1),
(CHCl3:MeOH (v/v) = 10:1). Yield: 39 mg, 56%. UV−visible
(CHCl3), λabs/nm (log ε) 288 (4.71), 424 (5.48), 517 (4.30),
554 (3.98), 592 (3.97), 646 (3.71). IR (KBr), ν/cm−1: 2921
(s), 2847 (m), 1629 (s), 1576 (m), 1508 (m), 1462 (s), 1404
(s), 1343 (m), 1282 (w), 1231 (s), 1131 (s), 1119 (s), 1053
(m), 1010 (m), 955 (w), 759 (s), 628 (w), 555 (m). 1H NMR
(d6-DMSO) δ −2.92 (s, 2H), 3.89 (d, 18H, J = 2.0 Hz), 3.99
(d, 9H, J = 2.6 Hz), 4.09−4.12 (m, 4H), 4.44−4.51 (m, 4H),
6.14 (d, 1H, J = 6.8 Hz), 6.74 (d, 1H, J = 6.8 Hz), 7.26 (d, 1H, J
= 6.3 Hz), 7.37−7.45 (m, 5H), 7.47−7.51 (m, 8H), 7.58 (d,
1H, J = 6.1 Hz), 7.77 (d, 1H, J = 5.7 Hz), 7.81−7.84 (m, 2H),
7.87−7.91 (m, 2H), 7.96−8.03 (m, 2H), 8.05−8.12 (m, 4H),
8.27−8.30 (m, 1H), 8.70−8.80 (m, 6H), 8.93−8.96 (m, 6H).
HRMS (MALDI-TOF) ([M−2Cl]+, m/z): Calcd for
C89H78N10O13Ru, 1596.7; Found for [M−2Cl]+, 1596.4.
Ethoxyethane-Linked [{Zn-porphyrin-(5-hydroxyl-Phen)}-
Ru(bpy)2][Cl]2 (6). Ethoxyethane-linked [{porphyrin-(5-hydrox-
yl-Phen)}Ru(bpy)2][Cl]2 (3) (18 mg, 0.011 mmol) was treated
with Zn(OAc)2·2H2O (3 mg, 0.013 mmol) in methanol (20
mL) at 65 °C for 3 h. The crude product was washed with
βI0(1 − e−α 3
)
0
T(z) = 1 −
2a0[1 + (z/z0)]2
(1)
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where a0 is the linear absorption coefficient, l is the sample
length (1 mm quartz cell), and z0 is the diffraction length of the
incident beam.
After obtaining the nonlinear absorption coefficient β, the
two-photon absorption cross section σ2 of the sample molecule
(in units of GM, where 1 GM = 10−50 cm4 s photon−1) can be
calculated using eq 2
cytometry. The cells were excited with 488 nm argon laser and
emission was collected with FL-3 (equipped with 650 nm long
pass filter) and 10000 events were analyzed.
Photocytotoxicity Assay. HeLa cells (2 × 104/well) were
incubated in wells of a 96-well plate overnight. The cells were
treated with 1, 2, and 3 for 6 h in the dark. The culture medium
was then replaced with fresh medium and the cells were
exposed to yellow light (1−4 J/cm2) produced from a 400 W
tungsten lamp fitted with a heat-isolation filter and a 500 nm
long-pass filter. The fluence rate was 4 mW/cm2. Cell viability
was determined by the MTT reduction assay at 24 h post-
PDT.34 The cell monolayers were rinsed twice with phosphate-
buffered saline (PBS) and then incubated with 250 μg/mL
MTT solution at 37 °C for 3 h. The formazan crystal formed
was dissolved in DMSO and the absorbance of dissolved
formazan crystal at 540 and 690 nm was measured using a 96-
well plate reader (ELx800 Absorbance Microplate Reader).
Two-Photon-Induced Confocal Microscopic Imaging
of 1−3. HeLa cells (1 × 105) were seeded onto coverslip in 35-
mm culture dishes overnight. The cells were initially incubated
with 1−3 for 6 h in the dark. The two-photon-induced
fluorescent signals of 1−3 were captured using the Leica SP5
(upright configuration) confocal microscope equipped with a
femtosecond-pulsed Ti:Sapphire laser (Libra II, Coherent)
inside the tissue culture chamber (5% CO2, 37 °C). The
excitation beam produced by the femtosecond laser, which was
tunable from 680 to 1050 nm, was focused on the adherent
cells through a 40× oil immersion objective. For the evaluation
of effectiveness of photodynamic therapy, the time-lapse images
(0−32 min, one laser shot per 8 min) were obtained with
femtosecond laser excitation at 850 nm (laser power ∼8 mW).
1000βhv
σ2 =
N d
(2)
A
where NA is the Avogadro constant, d is the concentration of
the sample compound in solution, h is the Planck constant, and
v is the frequency of the incident laser beam.
Singlet Oxygen Quantum Yield Measurement. Singlet
oxygen was detected directly by its phosphorescence emission
at 1270 nm using an InGaAs detector on a PTI QM4
luminescence spectrometer. The singlet oxygen quantum yields
(ΦΔ) of the test compounds were determined in CHCl3 by
comparing the singlet oxygen emission intensity of the sample
solution to that of a reference compound (H2TPP, ΦΔ = 0.55
in CHCl3)32 according to eq 333
2
GΔS
AREF
AS
⎛
⎞
⎟
nS
n
ΦΔS = ΦΔREF
×
×
⎜
⎝
REF ⎠ GΔREF
(3)
where ΦΔ is the singlet oxygen quantum yield, GΔ is the
integrated emission intensity, A is the absorbance at the
excitation wavelength, n is the refractive index of the solvent.
Superscripts REF and S correspond to the reference and the
1
sample, respectively. In all measurements, the O2 emission
spectra were obtained using an excitation with the absorbance
set at 0.05 in order to minimize reabsorption of the emitted
light.
RESULTS AND DISCUSSION
■
Synthesis and Characterization. (Scheme 1) The
substitution reaction of 2-bromo-N-(1,10-phenanthrolin-5-yl)-
acetamide and 5,10,15-tris(3,4,5-trimethoxylphenyl)-20-(4-hy-
doxylphenyl)-21H,23H-porphyrin (p-OH Por) in the presence
of Cs2CO3 in DMF afforded the L1 porphyrin ligand with a
yield of 42%. The identity of L1 was confirmed by 1H NMR, in
which the two internal NH pyrrole protons appeared as a sharp
singlet at ca. −2.7 ppm, the two CH2 protons appeared as a
singlet at ca. 5.1 ppm, the seven protons of 1,10-phenanthroline
occurred at ca. 8.2−8.5 ppm (4 protons) and 9.1−9.5 ppm (3
protons), respectively. High-resolution mass spectrum of L1
showed two peaks at m/z 1136.4, which corresponded to the to
the peak [M+H]+ (cf. calculated [M]+, m/z 1135.4) and m/z
901, which corresponded to the loss of the 2-bromo-N-(1,10-
phenanthrolin-5-yl)acetamide fragment. Ru(II) complexation
was carried out with cis-Ru(bpy)2Cl2 in mixed solvent THF/
CH3CH2OH (1:1), giving compound 1 with 81% yield.
Metalation of the porphyrin in 1 with zinc acetate afforded
compound 4 in good yield. Both 1 and 4 were characterized by
Cell Culture. Human cervical carcinoma (HeLa) cells were
maintained in an RMPI 1640 medium supplemented with 10%
fetal bovine serum (FBS) and 1% penicillin and streptomycin in
5% CO2. Culture medium in each dish was changed prior to
exposure to the test compounds. Stock solutions of the test
compounds (1 mM) were prepared in aqueous solution and
stored in the dark at room temperature. These compounds,
when used in the imaging and bioassay experiments involving
cultured cells, were diluted with the corresponding culture
media to appropriate concentrations.
Confocal Microscopic Imaging of Compounds 1−6.
HeLa cells (1 × 105) were seeded onto coverslip in 35 mm
culture dishes for overnight. The cells were initially incubated
with compounds 1−6 (1 μM) for 30 min in the dark. For
colocalization experiments, the cells were then washed and
stained with 100 nM mitochondria-specific probe Mito Tracker
Green FM dye M7514 or lysosome-specific probe Lyso Tracker
Green DND-26 L7526, for 30 min. The emitted fluorescent
signals of tested compounds and the organelle-specific probes
were examined using the Leica SP5 (upright configuration)
confocal microscope equipped with argon laser, HeCd laser and
a femtosecond-pulsed Ti:Sapphire laser (Libra II, Coherent)
inside the tissue culture chamber (5% CO2, 37 °C). A 40× oil
immersion objective and pinhole size of 110 μm was used for
image capturing.
1
1H NMR, HRMS, IR, and UV−vis spectroscopies. In their H
NMR spectra (Figure S1 and S3), the two internal NH pyrrole
protons of 1 appeared as a relatively broad singlet at −2.9 ppm
and the bipyridine protons in both 1 and 4 appeared as
multiplets in the downfield region. The high-resolution mass
spectra of 1 and 4 are given in Figures S2 and S4, respectively.
Figure S2 shows a single peak at m/z 1549.4, which
corresponds to the molecular ion peak of 1 (calculated [M]+,
m/z 1549.6), and Figure S4 shows a single peak at m/z 1612.1,
which corresponds to the loss of a proton in either the pyrrole
ring or the amide bond of 4 (calculated [M]+, m/z 1613.4).
Flow Cytometric Cellular Uptake. HeLa cells cells (105
per sample) were seeded to 35 m Petri dish for overnight and
then the cells were incubated with drugs at 2 mM for 1 and 6 h.
Cells were trypsinized and washed with PBS for twice. The
drug’s uptakes by the HeLa cells were analyzed with flow
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In Scheme 2, Sonogashira coupling reaction between p-
acetylene-ZnPor and 5-bromo-1,10-phenanthroline under N2
atmosphere afforded the zinc porphyrin, ZnL2, in moderate
yield. Demetalation of ZnL2 in concentrated HCl solution gave
1
the free base porphyrin, L2. H NMR spectra of L2 and ZnL2
show the Phen proton signals at δ ≈ 8.2−8.4 ppm (4 protons)
and 9.2−9.5 ppm (3 protons), respectively. High-resolution
mass spectra of L2 and ZnL2 show their respective [M+H]+
peaks at m/z 1087.4 (calculated [M]+, m/z 1086.4) and m/z
1149.3 (calculated [M]+, m/z 1148.3). Reaction of L2 and ZnL2
with cis-Ru(bpy)2Cl2 gave conjugates 2 and 5, respectively, in
1
good yields. These products were characterized by H NMR,
1
HRMS, IR, and UV−vis spectra. The H NMR spectra (in d6-
DMSO) of 2 displayed slightly broader peaks and overlapping
resonances for the bipyridine protons can be observed in the
region 8.0−8.5 ppm. The two internal pyrrole NH protons also
located in the upfield region (δ = −2.9 ppm). High-resolution
mass spectrum of 2 shows two peaks at m/z 1500.4 (M+) and
Figure 2. UV−vis absorption spectra of conjugates 1−3 (upper) and
4−6 (lower) in DMSO (1 μM). The inset shows the Q bands between
500 and 700 nm.
1
1653.3 ([M+DHB]+). H NMR spectrum of 5 has a relatively
well-resolved pattern, possibly because of its more symmetric
structure. High-resolution mass spectrum of 5 also shows two
peaks, one at m/z 1563.2 (M+) and the other at m/z 1408.1
([M-bipyridine]+).
In Scheme 3, the reaction between p-OH Por and diethylene
glycol di-p-toluenesulfonate in 1:1 ratio gave p-PEG-OTs Por as
the main product. Reaction of p-PEG-OTs Por with 4,7-diol-
1,10-phenanthroline in the ratio of 1:2 gave L3 with a relatively
low yield, owing to the poor solubility of 4,7-diol-1,10-
phenanthroline in the reaction media. The complexation of
L3 with cis-Ru(bpy)2Cl2 gave 3, which upon metalation with
Zn(OAc)2 afforded 6. These final products, 3 and 6, were also
1
characterized by H NMR, HRMS, IR, and UV−vis spectros-
copies. 1H NMR spectra of 3 and 6 are quite similar with well-
resolved peaks. High-resolution mass spectra of both 3 and 6
show single peaks at m/z 1596.4 and 1658.9, which match
favorably to the calculated m/z values of their respective
molecular ions (for 3, calculated [M]+, m/z 1596.7; and for 6,
calculated [M]+, m/z 1660.1).
Photophysical Properties. The objective of this work is to
develop a dual probe capable of in vitro imaging and
photodynamic therapeutic treatment. Herein, we report the
relevant photophysical properties, which include both the linear
and multiphoton-induced photophysical properties, singlet
oxygen quantum yields, and so forth, of the porphyrin-Ru(II)
conjugates synthesized.
Figure 3. Emission spectra (λex = 430 nm) of 1 μM of 1−3 (upper
panel) and 4−6 (lower panel) measured in DMSO at 298 K.
5,10,15,20-tetrakis(4-methoxyphenyl)porphyrin,36 are given in
Table 1. From this table, it can be seen that the emission
quantum yields of conjugates 1, 2, and 3 are strongly influenced
by the length and conjugation of the linker between the
porphyrin moiety and the Ru(bpy)2(phen)2+ complex. By
elongating the linkage using an ethoxyethane group in 3, we
observed a dramatic reduction in the emission quantum yields
from 5.30% (1) and 4.96% (2) to 1.93% (3). This result
indicates that the observed porphyrin emissions are due to an
UV−visible Absorption. The UV−vis absorption spectra of
the six conjugates (1 to 6) were recorded in DMSO. Figure 2
shows the electronic spectra of free-base porphyrin-Ru(II)
conjugates 1 to 3, with the intense π to π* transitions, the Soret
band, and the four weak Q-bands of the porphyrin found at
∼300 nm, ∼430 nm, and 500−700 nm, respectively.
Introduction of a Zn(II) ion to the porphyrin in conjugates 4
to 6, resulted in small (a few nm) red shift of the Soret band
and the number of Q bands were reduced from four to two. 35
Linear (One-Photon) Emission Spectra. The emission
spectra of the six Ru(II)-porphyrin conjugates, excited at 430
nm and measured in DMSO, are shown in Figure 3. The free-
base porphyrin-Ru(II) conjugates, 1, 2, and 3, show intense
emission bands at ∼655 and ∼718 nm (Figure 3, upper panel).
These two bands are assigned to the Q(0−0) and Q(0−1)
transitions of the free-base porphyrins based on their emission
wavelengths and lifetimes. The emission quantum yields of
these conjugates, measured relative to the reference standard of
energy transfer from the excited Ru(II)-polypyridyl moiety
abs
(λ ≈ 450 nm) to the porphyrin.
max
The Ru(II)−Zn(II) porphyrin conjugates, 4, 5, and 6, give
emission peaks at 610, 659, and 760 nm (Figure 3, lower panel)
with the first two peaks assigned to the fluorescent emissions of
the Zn(II) porphyrin. The 760 nm peak was assigned to the
phosphorescence of the Zn(II) porphyrin.37 The significant
reduction in emission intensity observed in 6 is due to the
2+
longer ethoxyethane linkage between Ru(phen)(bpy)2 and
Zn(II) porphyrin, making energy transfer between these two
chromophores less efficient.
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Table 1. Photophysical Properties of the Ru(II)-Porphyrin Conjugates 1 to 6
a
b
compound
λAbs/nm (log ε)
emission/nm (τ/ns)
Φem /%
ΦΔ /%
σ/GM
1
2
3
4
5
6
288(4.91), 424(5.58), 517(4.29), 554(3.98), 592(3.79), 646(3.65)
288(4.66), 425(5.33), 517(4.12), 554(3.91), 593 (3.72), 646(3.66)
288(4.71), 424(5.48), 517(4.30), 554(3.98), 592(3.97), 646(3.71)
288(4.94), 431(5.67). 560(4.28), 603(4.32)
607(weak) 655(10.70), 718(8.78)
607(weak) 654(11.6), 718(9.54)
607(weak) 653(4.23), 718(3.82)
610(10.50), 659(3.88) 760(weak)
610(3.74), 659(3.06) 760(weak)
5.30
4 90
43
58
38
52
73
177
168
144
172
228
106
1.93
0.58
288(4.92), 431(5.55), 562(4.46), 606(4.31)
0.30
288(4.72), 431(5.64), 562(4.28), 600(4.01)
610(3.04), 659(3.50) 770(weak)
0.16
45
a
b
c
1
The emission quantum yields of 1 to 6 (λem = 550−800 nm, λex = 430 nm). The O2 quantum yields of 1 to 6 (λex = 430 nm). Two-photon
absorption cross sections of 1 to 6 (GM = 10−50 cm4 s photon−1 molecule−1, λex = 800 nm), with an average measurement uncertainty of 23.5 GM.
Figure 4. Emission spectral response of 10 μM of free-base porphyrin-Ru(II) conjugates 1 (a), 2 (b), and 3 (c) as a function of pH (pH = 3−7)
measured in aqueous solution (295 K, <5% DMSO, λex = 428 nm for 1 and 2, 435 nm for 3). Inset: Variation of the intensity ratio of the emission at
659 and 718 nm of 1−3 with pH in aqueous solution. The data were fitted with a sigmoidal function (shown as red curves) to give the following
apparent pKa values: 3.75 (7) for 1, 3.26 (8) for 2, and 4.39 (0) for 3.
The emission spectra of the free-base porphyrin-Ru(II) (1, 2,
and 3) and the Zn(II) porphyrin-Ru(II) (4, 5, and 6) series of
conjugates are seen to be quite similar to those of the
Ru(bpy)2-(pyridylporphyrin)2 hybrid (1:2) complexes synthe-
sized and studied by Kon et al.37 These authors showed that the
excitation energy transfer mechanism in the Ru(II)-porphyrin
conjugates corresponded to an energy transfer from the excited
reverse energy transfer going from free-base porphyrin-Ru(II)
to Zn(II) porphyrin-Ru(II) conjugates, the emission quantum
yield variations among these two systems are obviously
different compared to other systems in the literature.38−40
pH-Dependence Emission Change. It is well-known that
the cellular uptake of an acidic organelle probe used to stain
lysosomes as well as other acidic subcellular compartments,
such as trans-Golgi vesicles, endosomes, and so forth,41−45 is
driven by the plasma membrane H+ gradient.46−48 Free-base
porphyrin (H2P), with two imine nitrogens which can be
protonated at low pH to give the mono-(H3P+) and dications
(H4P2+), can potentially serve as an in vitro probe for the
subcellular acidic organelles and compartments.
1
3
Ru(II) MLCT state via its MLCT state to the porphyrin B1
singlet state. However, in Zn(II) porphyrin, the energy level of
the porphyrin singlet state becomes destabilized, as shown by a
blue shift of ca. 45 nm (∼1126 cm−1), resulting in the
porphyrin singlet state of these conjugates being higher in
3
energy than the Ru(II) MLCT excited state. This led to a
reverse energy transfer in conjugates 4−6, where the energy
migrates from the Soret-excited porphyrin to the Ru(II)
chromophore. Even though this is not the first example of
In principle, two distinct protonation reactions can occur on
a free-base porphyrin, but the proton affinity of this macrocycle
is generally represented by an averaged protonation constant,
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Figure 5. The two-photon induced emission spectra of conjugates 1−3 (left) and 4−6 (right) in aqueous DMSO (10 μM, λex = 800 nm). Inset:
Plots of the observed emission intensities (1−3, λem = 650 nm; 4−6, λem = 610 nm) against the laser excitation (λex = 800 nm) power used.
pK, as often only one pK value can be determined in pH
titration experiments. The pK values measured for most water-
soluble porphyrins were in the range 2−449 and were shown to
depend on a complex interplay of the following factors:
electronic effects of the substituents, the degrees of planarity
and extended conjugation,50−52 and the peripheral charges,
which provide electrostatic shielding to the protonation site,
surrounding the porphyrin.53,54 In this work, we attempted to
evaluate the potential application of the free-base porphyrin-
Ru(II) conjugates, 1−3, as probes for subcellular acidic
compartments by studying their spectral changes under
different pH conditions and measure their pK values.
Absorption spectra of the deprotonated (free-base) form of
1−3 in aqueous solution (pH = 10.0) showed their Soret bands
at ∼428 nm (Figure S13) and the characteristic four-band
pattern in the Q-band region, which is typical for
tetraarylporphyrins. At pH 2, the fully protonated form of 1−
3 (i.e., porphyrin dication) exhibited a red-shifted Soret band
(λmax ≈ 450 nm) with increased intensity and a single Q-band
pattern (λmax ≈ 665 nm) due to the higher symmetry of the
porphyrin dication. The fluorescence spectra of 1−3 (Figure
S14) also show clear changes upon protonation. The emission
spectra of these free-base porphyrins show two well-defined
peaks, a strong emission at 660 nm and a weak emission at 726
nm at pH 10. However, when fully protonated at pH 2 (i.e.,
dicationic form), only one broad emission band at around 715
nm was seen.
Figure 4 shows the emission spectral changes of the
conjugates 1−3 in the pH range 3.0−7.0. The change in the
fluorescence intensity ratio at 660 and 726 nm (I660/I726) for
1−3 from pH 3.0 to 7.0 is shown as insets in Figure 4 as well.
The fluorescence intensity of these conjugates at 660 nm
increases with increasing pH, corresponding to the conversion
of the porphyrin from its dicationic form to the free-base form.
Fitting the I660/I726 vs pH curve (insets, Figure 4) with a
Sigmoidal function of the Hasselbach equation55 gave the
following protonation constants pK for these conjugates: ∼3.75
(1), ∼3.26 (2), and ∼4.39 (3). At first glance, the relatively
lower pK values in 1 and 2 can be explained in terms of the
more extensive π-conjugation in these conjugates with
conjugative linkers which decreases the porphyrin pK via
delocalization of the core electron density and reduces the
intrinsic basicity of the macrocycle.50 However, the perpendic-
ular orientation adopted by the phenyl ring in the linkers of 1
and 2 relative to the porphyrin ring does not seem to support a
significant extension of π-conjugation beyond the macrocycle
(vide infra). Thus, at this point we have no adequate
explanation for the significantly lower pK values observed in
1 and 2 as compared to 3.
1O2 Quantum Yield. To assess their potential application as
PDT agents, the 1O2 production yields of these Ru(II)-
porphyrin conjugates were measured based on the phosphor-
1
escence intensities of the O2 produced upon photoirradiation
1
(at 430 nm) of these compounds. Figure S15 shows the O2
phosphorescence spectra of the Ru(II)-porphyrin conjugates,
1−6, together with tetraphenylporphyrin, H2TPP, used as a
1
reference, in CHCl3. On the basis of the O2 quantum yield of
H2TPP (ΦΔ = 0.55 0.11),35 the relative ΦΔ values of these
conjugates are given in Table 1. Conjugates 2 and 5, with their
relative ΦΔ estimated to be 0.58
0.11 and 0.73
0.11,
respectively, exhibited the highest 1O2 yields in their respective
series of free-base and Zn(II) porphyrin-Ru(II) conjugates. A
comparison of the trend of 1O2 quantum yields with that of the
emission quantum yields for the two series of conjugates (1−3
and 4−6) shows no apparent correlation, suggesting that the
energy transfer pathways leading to emission and 1O2
production are perhaps distinct. A more detailed investigation
of these photophysical processes by transient absorption
spectroscopy will be undertaken.
Two-Photon Induced Emission and Absorption. Photo-
excitation in the near-infrared (NIR) region followed by
luminescence at a shorter wavelength in the visible region is
known as NIR-induced visible emission. This is a rather unusual
process because apparently low-energy photons are “converted”
to higher energy photons. This process is accomplished by the
simultaneous absorption of two or three NIR photons via
virtual intermediate states to produce a real excited state, which
then returns to the ground state by the emission of a single
photon in the visible region. The study of two- or three-photon
induced emission processes on novel organic materials has
received considerable attention recently because of potential
applications in bioimaging and photodynamic therapy, optical
data storage, and microfabrication.56−58
Figure 5 shows the emission spectra of a series of Ru(II)-
porphyrin conjugates, 1−6, in aqueous DMSO (0.4 mM)
excited by near-infrared femtosecond laser (λex = 800 nm). The
emission spectral profiles (i.e., band shape, position, and width)
obtained bear a close resemblance to the corresponding single-
photon emission spectra (shown in Figure 3) of these
conjugates. To confirm the involvement of two photons in
the observed induced emission, the emission peak intensities of
these conjugates were plotted against the incident laser
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excitation power at 800 nm (inset of Figure 5). A slope of 1 in
this plot would indicate that the emission resulted from one-
photon absorption, whereas a slope of 2 is indicative of a two-
photon absorption process. The slopes obtained for these
conjugates in these plots (1.81 for 1, 1.88 for 2, 1.85 for 3, 1.87
for 4, 1.88 for 5, 1.83 for 6) clearly demonstrate the two-
photon nature of their induced emissions.
Open-aperture Z-scan method (λex = 800 nm) was used to
measure their two-photon absorption properties of conjugates
1−6. The Z-scan traces and the derived two-photon absorption
cross sections, σ2 (average uncertainty 23.5 GM), of these
conjugates are shown in Figure 6 and Table 1, respectively.
and amide linkages. This interpretation is consistent with the
substantially higher σ2 value (555 GM) observed in a similar
2+
free-base porphyrin-Ru(phen)(bpy)2 conjugate connected
through a β-ethynyl only linkage (i.e., no intervening phenyl
ring).32
In Vitro Studies. On the basis of the observed photo-
1
physical properties (i.e., stronger emission and O2 quantum
efficiency) of the Ru(II)-porphyrin conjugates studied, the free-
base porphyrin-Ru(II) conjugates, 1, 2, and 3, appear to be
more suitable for further investigation as potential dual probes
for imaging and photodynamic therapy. The following in vitro
experiments, i.e., flow cytometry for cellular uptake, MTT assay
for cytotoxicity (dark and light), subcellular localization
imaging, and two-photon PDT experiments, were carried out
to evaluate the potential biological and clinical applications of
these compounds.
Cellular Uptake. In the past decades, many luminescent
transition metal complexes have been extensively studied for
potential in vitro and in vivo applications. However, most
research work have been devoted to the screening of these
complexes for biological applications, with few studies focusing
on the effects of a systematic variation of particular structural
elements on particular in vitro and/or in vivo properties of
these complexes.60−62 Puckett et al.63 measured and compared
the cellular uptake, a crucial property for any bioprobe and drug
molecule, of Ru(II) complexes with different polypyridyl
ligands by human cervical carcinoma HeLa cells and
investigated the underlying transport mechanism as well.42 In
this work, we examined the cellular uptake properties of these
porphyrin-Ru(II) conjugates with different linkers by HeLa
cells using flow cytometry. The results obtained are shown in
Figure 7.
Figure 6. Open-aperture Z-scan traces of 0.4 mM of free-base
porphyrin-Ru conjugates (1−3) and Zn(II) porphyrin-Ru conjugates
(4−6) excited at 800 nm in DMSO. The average power of the laser
beam was 0.271 mW.
From Figure 7, conjugates 1 and 2, with relatively
hydrophobic linkers between the porphyrin and the Ru(II)-
polypyridyl complex, are seen to be taken up by the HeLa cells
much more efficiently than conjugate 3, which contains a
relatively more hydrophilic ethoxyethane linker. For 3, the
luminescence signal observed, which was indicative of the
number of 3-loaded cells, after 6 h of incubation, was even less
than those observed after 1 h of incubation with 1 and 2. Thus,
even taking into account that 3 is ca. 2−3-fold less luminescent
(i.e., lower Φem) than 1 and 2, the data support the following
order of cellular uptake rates: 1 ∼ 2 ≫ 3.
The σ2 value measured for conjugate 1 (177 23.5 GM) was
found to be virtually identical to that of a structurally very
similar Ru(II)-porphyrin conjugate, Ru−P (178 26.8 GM),25
indicating that hydrophobic perturbation of the porphyrin
substituent (3,4,5-trimethoxyphenyl in 1 vs phenyl in Ru−P)
exerts no effect on the two-photon absorption cross section of
these conjugates. Since σ2 is strongly dependent on the π-
conjugation length of a molecule, the lack of substantial
enhancement in the σ2 of conjugates 1 and 2 relative to 3 must
be due to the perpendicular orientation of their meso-
substituted phenyl linkers with respect to the porphyrin ring,
which does not support extending the porphyrin π-conjugation
to the polypyridyl ligand of the Ru(II) complex via the ethynyl
Subcellular Localization. Localization of these porphyrin-
Ru(II) conjugates (2 μM) within the HeLa cells after
incubation for 6 h was examined by laser confocal microscopy
Figure 7. Flow cytometric analysis of the cellular uptakes of 2 μM of 1 (a), 2 (b), and 3 (c) by HeLa cells (105 cells per sample) after incubation for
1 h (green curve) and 6 h (pink curve) in dark. The luminescence signal (λex = 488 nm) was collected using FL-2 channels equipped with a long pass
filter (>650 nm). At least 10 000 events were counted. Live cells were differentiated by their low To-Pro-3 emission.
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Figure 8. Confocal fluorescent microscopic images of the free-base porphyrin-Ru(II) conjugates 1 (a), 2 (d), and 3 (g) (dosed concentration = 2
μM, λex = 860 nm, incubation time = 6 h), together with their corresponding bright-field images (1 − b; 2 − e; 3 − h), linear (λex = 430 nm) and
two-photon induced (λex = 860 nm) in vitro emission spectra in HeLa cells (resolution = 4 nm).
(Figure 8). Cells loaded with conjugates 1 (Figure 8a,b,c), 2
(Figure 8d,e,f), and 3 (Figure 8g,h,i) were excited by visible
argon-ion (λex = 432 nm, Figures S18 and S19) and near-
infrared femtosecond lasers (λex = 860 nm, Figure 8). The in
vitro emission profiles of the three conjugates are similar under
linear or two-photon excitation using the same dosed
concentration and time duration.
The subcellular localizations of conjugates 1 and 2 were
determined by costaining experiments using organelle-specific
trackers. After 3 h of incubation, conjugate 1 showed specific
localization in lysosomes (Figure S18), whereas conjugate 2
showed specific localization in mitochondria (Figure S19).
Furthermore, when the in vitro luminescence of these
conjugates was monitored hourly, 90% of the maximum
luminescence (measured after 3 h) was observed after 2 h of
incubation, showing a fairly rapid intracellular trafficking of
these conjugates to their targeted organelles. As for conjugate 3,
it appeared to reside in the cytoplasm with no obvious
organelle localization (Figure 8).
The dose−response curves obtained by incubation of these
conjugates in the dark at various concentrations ranging from 1
to 250 μM with HeLa cells are shown in Figure 9a. From these
curves, the IC50 of 1, 2, and 3 were estimated to be 118, 175,
and >250 μM, respectively. The lower cytotoxicity seen in 3 is
presumably related to its lower cellular uptake rate by the cells.
The photocytotoxicity of conjugates 1, 2, and 3 was
measured at 1 μM under varying light doses from 1.5 to 12.5
J/cm2. The light dose−response curves obtained are shown in
Figure 9b. Conjugate 2 showed the strongest photocytotoxicity
(LD50 = 2 J/cm2), followed by conjugate 1 (LD50 = 6.5 J/cm2),
with conjugate 3 showing the weakest photocytotoxicity (LD50
= 11.5 J/cm2) toward HeLa cells. This trend does not seem to
correlate with either the cellular uptake rates of these
1
conjugates (1 ∼ 2 ≫ 3) or their O2 quantum yields (Table
1, 1 ∼ 2 > 3), but appears to correlate better with their
subcellular localization properties. As 2 exhibits mitochondria-
localizing property, its photoexcitation in this organelle
1
produces O2-mediated oxidative stress which can readily elicit
Dark Cytotoxicity and Photocytotoxicity. MTT assay was
apoptotic cell death. Since 1 is shown to localize in lysosomes,
its photoexcitation in this organelle produces O2 that can
used to measure the cytotoxicity of conjugates 1, 2, and 3.37
1
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Figure 9. Dose−response curves of the dark cytotoxicity (left) and photocytotoxicity (right) of conjugates 1, 2, and 3. Dark cytotoxicity curves were
obtained using dosed concentration from 1 to 250 μM. Photocytotoxicity curves were obtained using 1 μM of conjugates and various light doses
from 0 to 12.5 J/cm2. MTT assay was carried out after incubation for 24 h. The results were expressed as the mean SD of three separate trials.
Table 2. Summary of the in Vitro Properties of the Free-Base Porphyrin-Ru(II) Conjugates 1, 2, and 3
cytotoxicity (IC50)
cellular uptake
a
b
c
c
compound
dark (μM)
light (J/cm2)
flow cytometry (1 h)
flow cytometry (6 h)
subcellular localization
pK
1
2
3
118
175
6.5
2
80-fold
80-fold
10-fold
500-fold
500-fold
20-fold
lysosome
3.75
3.26
4.39
mitochondria
cytoplasm
>250
11.5
a
Dark cytotoxicities (IC50) of 1, 2, and 3 toward HeLa cells were determined from the dose−response curve obtained after 24 h incubation with
b
various concentrations of the conjugates. Photocytotoxicities of 1, 2, and 3 toward HeLa cells was determined using 1 μM of the conjugates under
various light doses. MTT assay was performed on the HeLa cells after 24 h incubation. The luminescence signal measured (relative to the control)
c
was used as the parameter for comparing the cellular uptake rates of the conjugates.
damage the lysosomal membrane, causing the release of the
lysosomal enzymes which leads to necrosis. Conjugate 3, which
is more hydrophilic, distributes in the cytoplasm. When
photoexcited, 3 produces 1O2 in the cytosol where a substantial
1O2 population could become deactivated before inflicting
damage on any vital subcellular targets to cause cell death. The
results of the in vitro properties of these conjugates, 1−3,
toward HeLa cells, which include their cellular uptake,
subcellular localization, and dark cytotoxicity and photo-
cytotoxicity, are summarized in Table 2.
Two-Photon Induced Imaging and Cytotoxicities of 1, 2,
and 3. HeLa cells were incubated with 5 μM of conjugates 1, 2,
or 3 separately for 6 h. The cells were then excited at 850 nm,
at which the three conjugates 1, 2, and 3 showed a two-photon
absorption cross section of 177, 168, and 144 GM, respectively.
The confocal images were captured at one laser shot per minute
for a total of 32 min. Figure 10 shows the confocal images of
these cells after two-photon laser irradiation for time t = 0, 8,
16, and 32 min. To demonstrate the potential use of conjugates
1, 2, and 3 as two-photon excited imaging agents, these
conjugates stained HeLa cells were excited with a snap laser
flash at 860 nm and the images were captured under a confocal
microscope. A clear fluorescent image of the stained HeLa cells
was obtained (Figure 10a,e,i). The fluorescence intensities of
the 1- and 2-stained cells were greater than that of the 3-stained
cells. This observation is consistent with the flow cytometric
analysis (Figure 7) that the intracellular concentrations of 1 and
2 are greater than 3.
To further study the efficacy of these conjugates on two-
photon induced cytotoxicity, the cells were continuously
irradiated and the fluorescent images of the cells were captured
at 8 (Figure 10b,f,j), 16 (Figure 10c,g,k), and 32 min (Figure
10d,h,l) after irradiation. After 2PA-PDT, the patterns of
subcellular localization of conjugates 1 and 3 in the treated
HeLa cells were similar to those of the control cells (Figure
10a,i). A significant nuclear localization of 2 (Figure 10f,g,h)
was seen in cells at 8 to 32 min after irradiation. Cell shrinkage
was also apparent at 16 to 32 min in the PDT-treated cells,
indicating that conjugate 2-mediated PDT could effectively
damage the nuclear membrane and caused the cell death. The
nuclear localization of 2 is probably due to a redistribution of 2
from the cytoplasm (presumably released from the damaged
mitochondria during initial PDT) to the nucleus. In this short-
term cell imaging study, conjugate 2-mediated PDT is found to
be more effective than 1 and 3 in triggering the cell death
process. This result is also consistent with the result of
conventional one-photon PDT-induced cell death assay as
shown in Table 2.
CONCLUSION
■
Six water-soluble free-base porphyrin-Ru(II) conjugates, 1−3,
and Zn(II) porphyrin-Ru(II) conjugates, 4−6, have been
synthesized with satisfactory yields (>70%) and their potential
development as dual in vitro imaging and photodynamic
therapeutic agents investigated. Photoexcitation of conjugates
1−3 at 430 nm led to an energy transfer from the Ru(II)
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Figure 10. Confocal microscopic images of HeLa cells treated with 5 μM of 1 (a, b, c, d), 2 (e, f, g, h), and 3 (i, j, k, l) for 6 h. Top row: images
1
obtained for 1 (left), 2 (middle), and 3 (right) -treated cells after one snap laser flash (to avoid O2 generation) at 860 nm. Second row: images
obtained after 8 min of laser irradiation. Third row: images obtained after 16 min of laser irradiation. Bottom row: images obtained after 32 min of
laser irradiation.
1MLCT excited state to the porphyrin singlet state, resulting in
of the perpendicular orientation adopted by the phenyl-
containing linkers which does not support the extension of
the porphyrin π-conjugation to the polypyridyl ligand of the
Ru(II) complex.
emissions at ca. 659 and 718 nm. In contrast, photoexcitation of
the Zn(II) porphyrin-Ru(II) conjugates, 4−6, at 430 nm
resulted in an energy transfer from the Soret-excited porphyrin
to the Ru(II) due to an increase in the singlet energy level of
The in vitro properties of conjugates 1−3 toward HeLa cells
1
3
were further investigated as they gave higher emission and O2
Zn(II) porphyrin to above that of the Ru(II) MLCT state.
quantum yields. The IC50 measured for conjugates 1, 2, and 3
in the dark were 118, 175, and >250 μM, which is consistent
with their cellular uptake properties with 1 ∼ 2 ≫ 3, where 3,
which contained a more hydrophilic poly(ethylene glycol)
linkage, was the least efficient in cell penetration. The
photocytotoxicity of these conjugates, however, gave the
following light dose dependence at 1 μM concentration: 2 (2
J/cm2) > 1 (6.5 J/cm2) > 3 (11.5 J/cm2), which is explained by
their respective subcellular localizations at the mitochondria,
lysosomes and cytoplasm. These one-photon induced cytotox-
icity data is consistent with the observed two-photon PDT data,
which taken together strongly suggest the importance of
The emission quantum yields of these conjugates, 4−6, were ca.
10-fold lower than those of conjugates 1−3. An obvious
dependence of the energy transfer efficiency on the linker of
these conjugates was noted (1 ∼ 2 > 3 and 4 > 5 > 6), with the
efficiency reduced by 2−3-fold via the longer ethoxyethane
1
linkage. All conjugates gave O2 quantum yields (ranging from
0.73 to 0.38) adequate for potential application as PDT agents,
with no obvious difference between conjugates with different
linkers. The two-photon absorption cross sections, σ2, of these
conjugates ranged from 228 GM in 5 to 106 GM in 6, with no
expected enhancement from the “conjugative” phenylethynyl
and phenylamide linkers. This observation is explained in terms
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subcellular localization in affecting the in vitro PDT activity of a
drug. Among the three porphyrin-Ru(II) conjugates studied, 2,
with its high emission quantum yield, low dark cytotoxicity, and
high phototoxicity, is the most promising candidate for both in
vitro imaging and PDT activity.
ASSOCIATED CONTENT
* Supporting Information
■
S
Synthetic details, 1HNMR spectra, HRMS spectra, 1O2
phosphorescence spectra, comparative absorption and fluo-
rescence spectra of the porphyrin-Ru(II) conjugates in free-
base form (pH = 10.0) and dicationic form (pH = 2.0),
luminescence decay curves of the six porphyrin-Ru(II)
conjugates, costaining confocal microscopic images of 1 and
2. This material is available free of charge via the Internet at
http://pubs.acs.org.
(14) MacDonald, I. J., and Dougherty, J. (2001) Basic principles of
photodynamic therapy. J. Porphyrins Phthalocyanines 5, 105−129.
(15) Vicente, M. G. (2001) Porphyrin-based sensitizers in the
detection and treatment of cancer: recent progress. Curr. Med. Chem.:
Anti-cancer Agents 2, 175−194.
AUTHOR INFORMATION
Corresponding Author
■
(16) Schmitt, F., Govindaswamy, P., Suss-Fink, G., Ang, W. H.,
̈
*E-mail: wkwong@hkbu.edu.hk, klwong@hkbu.edu.hk,
chiklau@cityu.edu.hk. Fax: +852-3411-5862. Tel: +852-
34117011. Tel: +852-34112370.
Dyson, P. J., Juillerat-Jeanneret, L., and Therrien, B. (2008)
Ruthenium porphyrin compounds for photodynamic therapy of
cancer. J. Med. Chem. 51, 1811−1816.
Author Contributions
(17) Bergamo, A., Gaiddon, C., Schellens, J. H. M., Beijinen, J. H.,
and Sava, G. (2011) Approaching tumour therapy beyond platinum
drugs: status of the art and perspectives of ruthenium drug candidates.
J. Inorg. Biochem. 106, 90−99.
#The first to third authors contributed equally as co-1st author.
Notes
The authors declare no competing financial interest.
(18) Ang, W. H., Casini, A., Sava, G., and Dyson, P. J. (2011)
Organometallic ruthenium-based antitumor compounds with novel
modes of action. J. Organomet. Chem. 696, 989−998.
ACKNOWLEDGMENTS
■
The work described in this paper was partially supported by
grants from the Research Grants Council of the Hong Kong
SAR, P.R. China (HKBU 202509 and HKBU 202210) and a
starting grant from the Hong Kong Baptist University (KLW).
(19) Antonarakis, E. S., and Emadi, A. (2010) Ruthenium-based
chemotherapeutics: are they ready for prime time? Cancer Chemother.
Pharmacol. 66, 1−9.
(20) Suess-Fink, G. (2010) Arene ruthenium complexes as anticancer
agents. Dalton Trans. 39, 1673−1688.
(21) Bruijnincx, P. C. A., and Sadler, P. J. (2009) Controlling
platinum, ruthenium, and osmium reactivity for anticancer drug
design. Adv. Inorg. Chem. 61, 1−62.
(22) Gianferrara, T., Bergamo, A., Bratsos, I., Milani, B., Spagnul, C.,
Sava, G., and Alessio, E. (2010) Ruthenium−porphyrin conjugates
with cytotoxic and phototoxic antitumor activity. J. Med. Chem. 53,
4678−4690.
(23) Juzeniene, A., Peng, Q., and Moan, J. (2007) Milestones in the
development of photodynamic therapy and fluorescence diagnosis.
Photochem. Photobiol. Sci. 6, 1234−1245.
(24) Moan, J. J. (1990) On the diffusion length of singlet oxygen in
cells and tissues. Photochem. Photobiol. B: Biol. 6, 343−344.
(25) Poon, C. T., Chan, P. S., Man, C., Jiang, F.-L., Wong, R. N. S.,
Mak, N. K., Kwong, D. W. J., Tsao, S. W., and Wong, W. K. (2010) An
amphiphilic ruthenium(II)-polypyridyl appended porphyrin as poten-
tial bifunctional two-photon tumor-imaging and photodynamic
therapeutic agent. J. Inorg. Biochem. 104, 62−70.
ABBREVIATIONS:
■
PDT, photodynamic therapy; TPA, two-photon absorption;
NIR, near-infrared; phen, 1,10-phenanthroline; bpy, 2,2′-
bipyridine.
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