Water-soluble self-assembled butadiyne-bridged bisporphyrin: A potential two-photon-absorbing photosensitizer for photodynamic therapy

Article abstract of DOI:10.1002/chem.200601205

We have synthesized a novel, two-photon-absorbing photosensitizer for two-photon-absorption photodynamic therapy (2PA-PDT). The molecule is a butadiyne-bridged porphyrin dimer terminated with two water-soluble porphyrin monomers connected through Zn-imida

Full text of DOI:10.1002/chem.200601205

FULL PAPER
DOI: 10.1002/chem.200601205
Water-Soluble Self-Assembled Butadiyne-Bridged Bisporphyrin: A Potential
Two-Photon-Absorbing Photosensitizer for Photodynamic Therapy
Joanne T. Dy, Kazuya Ogawa, Akiharu Satake, Atsushi Ishizumi, and
Yoshiaki Kobuke*[a]
Abstract: We have synthesized a novel,
two-photon-absorbing photosensitizer
for two-photon-absorption photody-
namic therapy (2PA-PDT). The mole-
cule is a butadiyne-bridged porphyrin
dimer terminated with two water-solu-
ble porphyrin monomers connected
through Zn-imidazolyl self-assembly
and covalently linked through olefin
metathesis. It has an effective two-
photon-absorption (2PA) cross-section
value, s(2), of 33000Æ4600 GM with 5-
ns pulses at 890 nm measured by using
the open-aperture Z-scan technique.
The compound was found to generate
singlet oxygen, cytotoxic for tumor
cells in photodynamic therapy (PDT),
under 2PA conditions by conducting
photobleaching experiments with an-
thracene-9,10-dipropionic acid sodium
salt (ADPA).
Keywords: photodynamic therapy ·
porphyrins · self-assembly · singlet
oxygen · two-photon absorption
Introduction
GM, which corresponds to 10À50 cm4 secmoleculeÀ1 photonÀ1.
In 2PA, the photon absorbed contains only half of the nomi-
nal excitation energy and, therefore, penetrates absorbing or
scattering media more effectively. The absorption at longer
wavelengths, especially in the near-infrared (NIR) region, is
vital for biological applications because it increases the pen-
etration of tissues by light.[7] In addition, the quadratic de-
pendence on the laser intensity permits spatial selectivity by
using a focused laser beam.[8]
Recent interdisciplinary attention has focused on nonlinear
optical materials, especially those with efficient two-photon
absorption due to their numerous potential applications
such as two-photon photodynamic therapy,[1] optical power
limiting,[2] ultrahigh-density optical-data storage,[3] two-
photon-excited fluorescence spectroscopy,[4] and three-di-
mensional (3D) microfabrication.[5]
Two-photon absorption (2PA) is a third-order nonlinear
phenomenon in which excitation occurs by the simultaneous
absorption of two photons at comparatively long wave-
lengths instead of by a single-photon excitation at a shorter
wavelength.[6] Typically, a chromophore reaches its excited
state (Sn) through excitation at its one-photon wavelength l.
However, Sn excitation is also possible at wavelength 2l by
using strong laser pulses, whereby two photons are simulta-
neously absorbed by the molecule. The 2PA efficiency is
quantified by the two-photon-absorption cross section s(2) in
A wide variety of 2PA materials have been developed by
using both organic and inorganic materials. The most effi-
cient of these materials are composed of donor and acceptor
molecules linked by a conjugated p bridge.[9] Some of the p
bridges that have been studied so far are trans-stilbene,[9a]
fluorene,[9b] dithienothiophene,[9c] and butadiyne[9d,e] linkers.
Increasing the length of the molecules also results in a sub-
stantial increase in s(2). Porphyrins that have highly conju-
gated p systems are also attractive candidates for 2PA mate-
rials. Another important feature of porphyrins is the easy
functionalization of their meso and b positions. Hydrophobic
or hydrophilic moieties can be affixed to alter the solubility
of the porphyrin ring without altering its vital properties.
However, monomeric porphyrins exhibit small cross-section
values of less than tens of GM.[10]
[a] J. T. Dy, Prof. K. Ogawa, Prof. A. Satake, Prof. A. Ishizumi,
Prof. Y. Kobuke
Graduate School of Materials Science
Nara Institute of Science and Technology
8916-5 Takayama, Ikoma, Nara 630-0101 (Japan)
Fax : (+81)743-72-6119
We have previously reported a butadiyne-linked bispor-
phyrin dimer with a strong s(2) value of 7600 GM with fem-
tosecond pulses at 870 nm.[11] Two porphyrins were connect-
ed by a butadiyne bridge that assumes a cumulenic structure
upon photoexcitation, allowing a highly conjugated planar
E-mail: kobuke@ms.naist.jp
Supporting information for this article is available on the WWW
under http://www.chemeurj.org/ or from the author and includes cal-
culations and detailed experimental diagrams and data.
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structure. We also discovered that the slipped cofacial ar-
rangement by complementary coordination of imidazolyl to
zinc[12] can effectively increase s(2) almost four fold. Ander-
son and co-workers have also investigated several acetylene-
bridged zinc-inserted porphyrin dimers with promising two-
photon-absorption cross sections in the near-infrared re-
gions, reaching values of 1”104 GM in the region of 820–
890 nm.[13]
10- and 20-meso positions. Herein, we report a different ap-
proach to building a water-soluble, two-photon-absorbing
porphyrin-based photosensitizer (1) as a potential candidate
for 2PA-PDT. A butadiyne-bridged bisporphyrin, known for
possessing large 2PA cross sections, was chosen as the heart
of this 2PA-PDT system. Unlike the previous compounds, a
dendritic residue was selected for hydrophilicity. Dissolving
a large organic structure such as porphyrin requires a rela-
tively large number of hydrophilic substituents. Thus, a por-
phyrin bearing six carboxylate units, as provided by the
Newkome-type dendrimer,[19a] was attached at both ends of
the butadiyne bisporphyrin through zinc-imidazolyl coordi-
nation to form a tetramer. To permanently link the coordi-
nated structure, the allyl ether side chains were introduced
to fix the structure through olefin metathesis.[19b] This overall
procedure results in an average of three carboxylate units
per porphyrin in the hope that this large porphyrin array is
not only soluble in water, but also that the bulkiness of the
terminal dendritic arms can increase hydrophilicity and
eventually help the delivery of the photosensitizer to the
tumor cells. In contrast to the previously reported com-
pounds, the hydrophilic groups in compound 1 are larger in
number and are located only at both ends of the tetramer.
These factors may potentially affect drug delivery to the
cells.
The field of photodynamic therapy (PDT) can benefit
enormously from 2PA. PDT is a light-activated treatment
used to destroy tumor cells, particularly in the skin, throat,
and lungs.[14] In PDT, a drug called a photosensitizer (PS) is
injected into the body and localizes to the tumor. On its
own, a photosensitizer is not active; however, upon irradia-
tion with laser light, it generates triplet states (3PS*) through
intersystem crossing. This, in turn, reacts with ground-state
molecular oxygen (3O2), which is in its triplet state, produc-
ing cytotoxic singlet-oxygen species responsible for the de-
struction of the tumor cells. One of the major problems of
currently available PSs is their excitation wavelength of
around only 630 nm, which reduces the cellular transmission
of laser light.[15] In 2PA, excitation can be performed at
longer wavelengths, which is beneficial for PDT because the
absorption within the optical window of biological tissues in
the range of 700–1500 nm is increased.[16] Moreover, the
quadratic dependence on the laser intensity requires a fo-
cused laser beam for excitation, which in turn allows high
spatial selectivity, keeping the healthy tissues safe and
intact. This ideal combination is known as two-photon-ab-
sorption photodynamic therapy (2PA-PDT).
Results and Discussion
Design and synthesis: The molecule is composed of two
Ogilby and co-workers have recently reported singlet-
oxygen generation from several
monomeric
parts that have different roles: the 2PA component, a zinc-
porphyrin-,[17a]
phenyl-vinylene-,[17b,c] and phen-
ylene-ethynylene-based[17d] pho-
tosensitizers by two-photon ex-
citation. Two-photon excitation
at 780 nm has also been applied
to cells treated with aminolevu-
linic acid and protoporphyrin
for photosensitization.[18a] Oh
and co-workers have demon-
strated the generation of singlet
oxygen by spatially controlled
two-photon excitation in fibro-
blasts by using 5-chloromethyl-
2’,7’-dichlorodihydrofluorescein
diacetate acetyl ester.[18b]
We also reported potential
2PA-PDT agents with large ef-
fective 2PA cross sections, good
yields for one-photon-excited
singlet-oxygen yields, and PDT
by using one-photon irradia-
tion.[14f] These compounds were
acetylene-bridged bisporphyrins
bearing carboxylates at their Scheme 1. Synthetic route to the 2PA (3Zn) and water-soluble (5Zn) components.
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Self-Assembled Butadiyne-Bridged Bisporphyrin
FULL PAPER
inserted butadiyne-bridged imi-
dazolylporphyrin dimer 3Zn,
and the water-soluble compo-
nent, a zinc-inserted isophthal-
amidoimidazolylporphyrin bear-
ing 12 carboxylic acid groups,
5Zn, The synthetic routes for
these components are shown in
Scheme 1.
The butadiyne-bridged por-
phyrin dimer was synthesized
À
by palladium-catalyzed C C
bond formation of 2 by reaction
with [Pd
A
ACHTREUNG
larsine to afford 3 in 64% yield.
Freebase 3 was treated with
zinc acetate to give the 2PA
component, a zinc-inserted bu-
tadiyne-bridged
porphyrin,
3Zn, in 90% yield. To increase
hydrophilicity, the water-soluble
component 4ZnH was append-
ed with a Newkome-type den-
drimer through BOP (benzo-
triazol-1-yloxytris(dimethylami-
no)phosphoniumhexafluoro-
phosphate)
condensation,
giving a total of six ester groups
for compound 5Zn in 90%
yield.
In noncoordinating solvents
such as CHCl3 and toluene,
these porphyrins 3Zn and 5Zn
exist as polymer (3Zn)n and as
dimer (5Zn)2, respectively, due
to the complementary coordi-
nation of imidazolyl to the cen-
Scheme 2. Synthetic route to the water-soluble 2PA porphyrin tetramer 1. (L=methanol or pyridine)
tral zinc metal of the porphyrin,
as shown in Scheme 2.[20] How-
ever, in coordinating solvents such as pyridine and metha-
nol, porphyrins 3Zn and 5Zn are in their monomeric form.
To form the desired tetramer 61, this coordination should in-
itially be dissociated by dissolving a 2:1 molar mixture of
5Zn/3Zn in pyridine. Reorganization took place as pyridine
was gradually removed by evaporation, wherein different
length arrays of (3Zn)n terminated with monomer 5Zn at
both ends were formed (6n). This oligomeric mixture, 6n,
exists in a statistical ratio, as observed from gel-permeation
chromatography (GPC) (Figure 1, left).
gradual reorganization, thus, it is imperative to permanently
fix the structure through metathesis of the allyl ether side
chains by using the Grubbs catalyst, to finally give the meta-
Tetrameric compound 61 was separated by using prepara-
tive GPC. In this study, only the tetramer fraction was se-
lected because of its better solubility in water after hydroly-
sis. Other fractions are interesting in view of the extended
length of the p systems and will be characterized in the
future. The collected fraction is relatively stable if dissolved
in noncoordinating solvents. However, it may still undergo
Figure 1. Analytical GPC charts of (left) (5Zn)2 and 6n (n=1,2,3, etc.), a
reorganized 2:1 mixture of 5Zn:3Zn; (right) 7, retention time=
12.85 min (JAIGEL 3H-A; eluent: CHCl3; flow rate: 1.2 mLminÀ1).
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Y. Kobuke et al.
thesized target compound 7. The length of the side chain
necessary for metathesis has been reported.[19b] The purity of
tetramer 7 is evident from the strong isolated peaks of its
analytical GPC (Figure 1, right) and MALDI-TOF mass
profiles (Figure 2). In Figure 1, tetramer 7 shows a single
Figure 3. Absorption spectra of (5Zn)2 (d), 5Zn+Im (b) and
3Zn+2Im (c) in CHCl3.
Figure 2. The MALDI-TOF mass spectrum of 7 shows the target peak at
m/z=4452.69.
peak with a retention time of 12.85 min excluded from other
peaks. The effective mass value of compound 7, m/z=
4452.69, is also in good agreement with the calculated mass
for C244H290N28O36Zn4 of 4452.20. This is further evidenced
in the 1H NMR peaks that were assigned according to
{1H,1H} COSY two-dimensional (2D) NMR spectra (see
Supplementary Information).
Because 7 possesses an average of three carboxylate
groups per porphyrin unit, it may be sufficient to dissolve
this large compound in water. The treatment of compound 7
with formic acid cleaves all twelve tBu groups to form the
carboxylic acid tetramer 7H and can be followed by treat-
ment with an equimolar amount of NaOH to finally yield
the water-soluble tetramer 1.
Photophysical characterization: 5Zn, 5Zn+Im, and 3Zn+
2Im (Im=methylimidazole) exhibit the typical absorption
spectra of a slipped-cofacial dimer,[12] a coordination-cleaved
monomer,[12] and a butadiyne porphyrin,[21] respectively, as
shown in Figure 3. However, the absorption spectrum of
metathesized tetramer 7 (Figure 4) is not a simple superposi-
tion of the two individual components. Its unique absorption
spectrum results from the excitonic coupling of the Bx, By,
and Bz dipole moments of the terminal monomer and cen-
tral dimer porphyrins. Peak P0 at 434 nm can be attributed
to the freely rotating face-to-face interactions of all the By
and Bz moments. It spans from the orthogonal conformation
of the butadiyne-linked bisporphyrin (B1yB2y and B’1zB’2z) to
the parallel conformation (B1yB2yB’2yB’1y), resulting in the
blue-shifted band. As mentioned previously, no interruption
of the conjugation path occurs between the two porphyrin
units of the butadiyne dimer through the butadiyne axis, and
its two interacting Bx dipole moments can be considered as
Figure 4. Absorption spectrum of 7 in CHCl3 (top) and excitonic coupling
of porphyrins in metathesized tetramer 7 (bottom).
a single “long” dipole moment (B2x). The most red-shifted
peak, P2, at 495 nm can be attributed to the head-to-tail in-
teractions of all the Bx dipole moments (i.e., B1xB2xB’1x). The
orientations of the porphyrins are unimportant because all
of the Bx moments are parallel to each other and all the
center-to-center distances of the adjacent Bx moments are
equal. Thus, the remaining peak P1 at 463 nm can be as-
signed to the B1xB2x interaction.
The emission spectra of compounds 5Zn+Im, 3Zn+
2Im, and 7 are shown in Figure 5. The emission intensities
are normalized according to the absorbance of the com-
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Self-Assembled Butadiyne-Bridged Bisporphyrin
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Two-photon absorption: The effective 2PA cross section was
measured by using an open-aperture Z-scan technique with
an Nd:YAG laser source. A 0.4-mm solution of compound 1
in water in a 1-mm cell was irradiated with 5-ns pulses at a
repetition rate of 10 Hz.
A typical Z-scan trace of compound 1 in water at 890 nm
with its theoretically fitted curve is shown in Figure 7, in
Figure 5. Fluorescence spectra of 5Zn+Im (b), 3Zn+2Im (d), and
metathesized tetramer 7 (c) in CHCl3.
pounds at their respective excitation wavelengths. At
566 nm excitation, porphyrin monomer 5Zn+Im gave two
emission peaks at 632 and 680 nm. On the other hand, stron-
ger and extremely red-shifted peaks at 734 and 817 nm were
observed for the butadiyne-bridged porphyrin dimer 3Zn+
2Im upon 577 nm irradiation. Interestingly, in the emission
spectrum of 7, the fluorescence component corresponding to
the terminal part observed in dimer 5Zn+Im was complete-
ly quenched and emitted from only the 2PA part, accompa-
nied by a slight increase in the peak intensity relative to that
of monomer 3Zn+2Im. Taking into account the complete
overlap of the emission bands of compound 5Zn+Im with
the absorption bands of compound 3Zn+2Im at around
600–700 nm, these effects are all indicative of fluorescence-
resonance-energy transfer from the terminal monomer to
the central porphyrin butadiyne-bridged dimer.[22] This is
further supported by the excitation spectrum for 7 (see Sup-
porting Information), which has a shape very similar to its
absorption spectrum.
*
Figure 7. Typical Z-scan trace ( ) and theoretically fitted curve (line) of
0.4 mm of 1, pH 7.
which the normalized transmittance is plotted as a function
of the sample position, Z. The curve fits were performed ac-
cording to the theoretical expression for the transmit-
tance.[9d,11] The effective 2PA spectrum of compound 1 in
water is shown in Figure 8.
Finally, the absorption and fluorescence spectral shape of
1 are different from those of 7 in the relative oscillator
strengths and emission intensities (Figure 6). This may be
caused by aggregation that frequently occurs with porphy-
rins in water.[23] This is now under further investigation.
Figure 8. Two-photon absorption spectrum of 0.4 mm 1 in water, pH 7.
The real 2PA maximum peak for compound 1 appeared
at 890 nm (11235 cmÀ1), with a value of 33000Æ4600 GM.
It should be noted that it is difficult to compare this 2PA
cross-section value with those obtained by different pulse
widths. For instance, acetylene-bridged porphyrins have
been reported to possess s(2) values in the range of
10000 GM by using femtosecond pulses.[11,13b] The nanosec-
ond values are approximately 30 times larger than the fem-
tosecond values for our previously reported compounds.[11b]
The large discrepancy between nanosecond and femtosec-
Figure 6. Absorption (c) and emission (b) spectra of 1 in water.
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Y. Kobuke et al.
ond values is attributed to excited-state absorption (ESA)
because of the longer pulse width in nanosecond lasers rela-
tive to those in femtosecond lasers. Furthermore, the ESA
component is extremely difficult to separate from the pure
2PA signal.
present in solution, s(2) is not aggregation-dependent for
compound 1.
Singlet-oxygen generation by two-photon excitation: In
PDT, cytotoxicity of the tumor cells is caused by singlet
oxygen that is usually generated by energy transfer from the
triplet state of the photosensitizer to ground-state oxygen.
The amount of singlet oxygen generated can be determined
quantitatively by using scavengers such as anthracene-9,10-
dipropionic acid sodium salt (ADPA), which reacts with
oxygen to form an endoperoxide. Therefore, a D2O solution
of ADPA and compound 1 was irradiated with 100-fs pulses
at 890 nm with a peak power of 6.1 GWcmÀ2. Although
ADPA absorbs in the same region as the sensitizers, it ex-
hibits characteristic vibrational peaks at 399, 378, 359, and
342 nm. Continuous photobleaching of anthracene absorp-
tion was observed for 3 h by using 890 nm excitation
(Figure 10). In contrast, almost no change was observed in
However, the effective values obtained herein are clearly
large compared to values of related compounds employing
the same nanosecond pulses. The effective s(2) value of com-
pound 1 is almost three orders of magnitude larger than
H2TPP, 150-fold that of zinc-imidazolyl-coordinated porphy-
rin dimer, and twice that of an alkyl meso-substituted bis-
porphyrinatozinc(II) monomer, which have values of 29 GM
(at 780 nm), 188 GM (at 780 nm), and 14000 GM (at
890 nm), respectively.[24] As reported previously, this in-
crease in 2PA can be attributed not only to the increased
number of porphyrins, but may also be due to the conjugat-
ed butadiyne p bridge that provides a pathway for strong
electronic conjugation between the porphyrins. Additionally,
the complementary coordination of porphyrins 3Zn and
5Zn also contributes to the increase in 2PA.[11] Moreover,
the donor–acceptor configuration of compound 1 induces
polarization within the molecule to enhance 2PA.[9]
Aggregation was probably present in the absorption spec-
trum because the Q bands were blue-shifted and the lower-
energy Soret bands, which are mainly from the butadiyne
bisporphyrin, showed an increase in oscillator strength upon
the addition of pyridine (see Supporting Information). To
determine the effect of aggregation on 2PA, the effective
s(2) value of compound 1 in water in the presence of excess
pyridine at 890 nm was measured under the same experi-
mental conditions. Compound 1 with pyridine gave a value
of 36000Æ5000 GM, which is almost equivalent to the value
measured in water only. In addition, 2PA of compound 1 in
water at different concentrations was measured. Figure 9
Figure 10. Photobleaching experiments of 1 after two-photon excitation
at 890 nm in D2O at RT. Inset: Magnification of continuous quenching of
ADPA absorbance during the course of a 3-h exposure.
the Q bands of compound 1, indicating that the sensitizer
itself is not affected during either two-photon excitation or
singlet-oxygen generation.
Almost no decrease in the anthracene absorbance was ob-
served in the solutions without the sensitizer. The same ex-
periment was undertaken by using tetrasulfonatophenylpor-
phyrin (TPPS), which has very weak 2PA within these wave-
length regions. The extent of quenching of the ADPA ab-
sorption peak at 379 nm by TPPS upon using compound 1
and TPPS is plotted in Figure 11a. It is clearly demonstrated
that compound 1 is an effective photosensitizing agent
under two-photon-excitation conditions. On the other hand,
TPPS totally lacks this activity. To confirm that the decay of
ADPA is due to the entrapment of singlet oxygen and not
to other mechanisms, similar experiments were also per-
formed in H2O (Figure 11b). It is known that singlet oxygen
in D2O has a longer lifetime (60 ms) than in H2O.[26,27] The
difference in ADPA quenching between D2O and H2O is
compatible with the expectation (Figures 11a and b, respec-
tively).
Figure 9. Linear dependence of the two-photon absorbance q0 on the
concentration of compound 1 in water, pH 7.
shows a plot of the two-photon absorbance q0 with respect
to concentration and clearly displays a linear relationship.
From Equations (3) and (5) of the s(2) calculations (see Sup-
porting Information), the two-photon absorbance q0 should
be linearly proportional to the number density N, and conse-
quently to the concentration. Thus, although aggregation is
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Self-Assembled Butadiyne-Bridged Bisporphyrin
FULL PAPER
to determine which type of structure and level of hydrophi-
licity will allow greater drug delivery into the tumor cells.
Experimental Section
General methods: 1H and 13C NMR spectra were obtained in CDCl3,
unless otherwise noted, with Me4Si as the internal standard and were re-
corded by using either a JEOL JNM EX270 or JEOL ECP 600 spectrom-
eter. IR spectra were obtained by using a Nicolet Avatar 320ES FTIR
spectrometer. UV/Vis spectra were obtained by using either a Shimadzu
UV-1650PC or UV-3100PC UV/Vis spectrophotometer. Fluorescence
measurements were performed by using a Hitachi F-4500 fluorescence
spectrophotometer. MALDI-TOF mass spectra were obtained by using a
Perseptive Biosystems Voyager DE-STR and
a Shimadzu/KRATOS
Axima-CFR Kompact MALDI with dithranol and a-cyano-4-hydroxy
cinnamic acid (Aldrich) as the matrix. Analytical GPC measurements
were performed by using an HP Series 1100 with
a JAIGEL 3H-A
column. Reactions were monitored on silica gel 60 F254 TLC plates
(Merck). The silica gel used for column chromatography was purchased
from Kanto Chemical Co.: Silica Gel 60N (spherical, neutral) 60–210 and
40–50 mm (Flash). The alumina used for column chromatography was
purchased from Merck: aluminum oxide 90 active basic.
All solvents and reagents were purchased and used without purification,
unless indicated otherwise. Dry tetrahydrofuran (THF) was obtained by
refluxing with sodium followed by distillation at 668C, atmospheric pres-
sure, and stored over dry 4A molecular sieve (Nacalai). Dry dimethylfor-
mamide (DMF) was obtained by stirring with calcium hydride overnight
and distilling in vacuo at 358C. Ethanol-free CHCl3 was obtained by
passing it through a short alumina column and was used only after stor-
age for a short time over 4A molecular sieve (Nacalai).
Synthesis: 1-methyl-1H-imidazole-2-carbaldehyde,[28a] meso-(3-allyloxy-
propyl)dipyrromethane,[28b] 5,15-bis(3-allyloxypropyl)-10-ethynyl-20-(1’-
methyl-2’-imidazolyl)porphyrin 2,[28c] 5-hydroxymethylisophthalic acid di-
ethyl ester,[28d] and tris(tert-butoxycarbonylpropyl)amine[19a] were synthe-
sized according to published procedures. Pyridinium chlorochromate
(PCC) was synthesized by reacting chromium trioxide (10 g, 0.1 mol)
with a vigorously stirred HCl solution (6n: 18.4 mL of 36% HCl in
11 mL H2O, 0.11 mol) in a 100-mL beaker. After 5 min, the mixture was
cooled to 08C and pyridine (8.1 mL, 0.1 mol) was added over 15 min
through a dropping funnel. Upon addition of pyridine, the brown-violet
solution coagulated into bright yellow-orange crystals. Pyridine was re-
moved by vacuum filtration and dabbing with filter paper several times.
It is important to remove all of the pyridine, otherwise, the obtained
product will discolor and the reactivity will be reduced (15 g, 70%).
Figure 11. Photobleaching of the ADPA peak at 379 nm after two-
photon-excited singlet-oxygen photosensitization at 890 nm: No photo-
~
&
^
sensitizer ( ), TPPS ( ), and 1 ( ) in a) D2O and; b) H2O.
Conclusion
A water-soluble two-photon-absorbing porphyrin tetramer 1
was successfully synthesized for the purpose of 2PA-PDT. A
maximum effective 2PA cross-section value of 33000Æ
4600 GM was observed at 890 nm (full-width at half-maxi-
mum (FWHM)=5 ns). This large effective s(2) value relative
to reference porphyrins measured under similar conditions
can be attributed mainly to the butadiyne linker, which
allows p conjugation between connected porphyrins. The in-
creased two-photon absorption at the NIR wavelength is
very beneficial for PDT applications, because the human
body is relatively transparent to wavelengths within this
region. Singlet oxygen was also successfully generated upon
laser irradiation of this low-energy wavelength, as evidenced
by ADPA photobleaching after two-photon excitation. Ef-
fective singlet-oxygen production upon two-photon irradia-
tion qualifies compound 1 as an attractive photosensitizer
candidate for two-photon photodynamic therapy, which will
allow deeper-site tumor treatments due to its strong 2PA ab-
sorption at a longer wavelength of 890 nm. Cell studies for
PDT are being actively pursued. Finally, it would be interest-
ing to conduct a comparative study between compound 1
and the previously reported acetylene-bridged porphyrins[14f]
1,3-Bis(5-
A
AHCTREUNG
solved in 5:1 v/v dry THF/TEA (11.4 mL). The solution was degassed
with nitrogen by means of freeze–thaw cycles. Tris(dibenzylideneacetone)
(chloroform)-di-palladium(0) ([Pd2(dba)3], 1.36 mg, 8.1 mmol) and triphe-
C
nylarsine (AsPh3, 3.22 mg, 64.8 mmol) were added to the mixture under
N2 atmosphere. After stirring overnight at 358C, water was added and
the mixture was extracted with CHCl3. The organic layer was washed
with brine, dried over anhydrous Na2SO4, and evaporated under reduced
pressure. The resulting crude product was subjected to silica-gel chroma-
tography for purification to elute compound 3 by using CHCl3/MeOH
(10:1 to 5:1 v/v); and CHCl3/acetone (20% v/v) to CHCl3/MeOH (10:1
v/v). Finally, the product was reprecipitated from CHCl3 solution by the
addition of n-hexane. The final product was vivid green in color (63.5 mg,
64%). Rf =0.5 (CHCl3/MeOH 10:1); 1H NMR (270 MHz, CDCl3): d=
10.01 (d, J=4.9 Hz, 2H; Por b), 9.63 (d, J=4.9 Hz, 2H; Por b), 9.47 (d,
J=4.9 Hz, 2H; Por b), 8.76 (d, J=4.9 Hz, 2H; Por b), 7.71 (d, J=1.1 Hz,
1H; imidazole H4), 7.49 (d, J=1.1 Hz, 1H; imidazole H5), 6.13 (ddt, J=
À
17.3, 10.5, 5.7 Hz, 2H; CH2=CH ), 5.46 (ddt, J=17.3, 3.2, 1.4 Hz, 2H;
À
À
CH2=CH ), 5.30 (ddt, J=10.5, 3.0, 1.4 Hz, 2H; CH2=CH ), 5.06 (brs,
À
4H; Por-CH2), 4.12 (dt, J=5.7, 1.4 Hz, 4H; CH2=CHCH2 ), 3.68 (t, J=
À
À
À
5.7 Hz, 4H;
A
OCH2 ), 3.42 (s, 3H; NCH3), 2.93–2.71 (m, 4H; Por-
Chem. Eur. J. 2007, 13, 3491 – 3500
ꢀ 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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3497

Y. Kobuke et al.
À
CH2CH2 ), À2.22 ppm (s, 2H; inner NH); UV/Vis (CHCl3): lmax =444,
lution (4 mL) of zinc acetate dihydrate in MeOH (500 mg/20 mL) was
added. After stirring for 1 h, the mixture was washed with sat NaHCO3
solution, brine, and water, and was dried over anhydrous Na2SO4. The re-
sulting crude product was purified by chromatography on silica gel with
CHCl3/acetone (1:0 to 5:1 v/v) to elute compound 4, giving a purple solid
(46.4 mg, 78%). Rf (CHCl3/acetone 5:1)=0.85; 1H NMR (270 MHz,
CDCl3): d=9.67 (d, J=4.6 Hz, 2H; Por b), 9.57–9.53 (m, 1H; Ph), 9.23–
9.20 (m, 1H; Ph), 8.99 (d, J=4.6 Hz, 2H; Por b), 8.95–8.92 (m, 1H; Ph),
8.90 (d, J=4.6 Hz, 2H; Por b), 6.17 (ddt, J=17.3, 10.5, 5.7 Hz, 2H; CH2=
475, 607, 714 nm; fluorescence (lex =444 nm, CHCl3): lem =721, 800 nm;
MALDI-TOF MS (dithranol): m/z calcd for C76H74N12O4: 1219.48
[M+H+]; found: 1219.8.
1,3-Bis(5-(15-(1-methyl-2-imidazolyl)-10,20-bis(3-allyloxypropyl)-zinc-
A
porphyrinyl))butadiyne (3Zn): Compound 6 (15 mg, 12.3 mmol) was dis-
solved in CHCl3 (10 mL), and a saturated solution (4 mL) of zinc acetate
dihydrate in MeOH (27 mg, 123 mmol) was added. After stirring for 2 h,
the mixture was washed with brine and water and dried over anhydrous
Na2SO4. During work-up, precipitates formed. These were washed thor-
oughly with water and were redissolved in pyridine. The solution was
then concentrated by rotary evaporation to give a metallic green-brown
filmlike solid (15 mg, 91%). The UV/Vis absorption and fluorescence
spectra of polymeric (3Zn)n were not measured because it was insoluble
in CHCl3. However, the UV/Vis absorption and fluorescence spectra of
compound 3Zn+2Im in CHCl3 were measured. The solid (3Zn)n was
dissolved in an excess amount of N-methylimidazole. Then a small
amount of this solution was dissolved in CHCl3 for measurements. UV/
Vis (CHCl3): lmax =455, 488, 577, 665, 724 nm; fluorescence (lex =444 nm,
CHCl3): lem =734, 817 nm.
À
À
CH ), 5.56 (d, J=1.4 Hz, 2H; CH2=CH ), 5.49 (d, J=1.1 Hz, 1H; imida-
zole H5), 5.44 (d, J=4.9 Hz, 2H; Por b), 5.34 (ddt, J=10.5, 3.0, 1.4 Hz,
À
2H; CH2=CH ), 5.25 (t, J=5.7 Hz, 4H; Por-CH2), 4.69 (q, J=7.3, 4H;
À
À
OCH2CH3), 4.48 (q, J=7.3, 4H; OCH2CH3), 4.24 (dt, J=5.7, 1.4 Hz,
À
À
À
4H; CH2=CHCH2 ), 4.01–3.86 (m, 4H; OCH2 ), 3.22–2.90 (m, 4H;
À
Por-CH2CH2 ), 2.14 (d, J=1.1 Hz, 1H; imidazole H4), 1.69 (s, 3H;
À
À
NCH3), 1.63 (td, J=7.3, 1.4 Hz, 6H; OCH2CH3), 1.38 ppm (td, J=7.3,
À
1.4 Hz, 6H; OCH2CH3); UV/Vis (CHCl3): lmax =416, 439, 568, 621 nm;
fluorescence (lex =439 nm, CHCl3): lem =624, 680 nm; MALDI-TOF MS
(dithranol): m/z calcd for C48H48N6O6Zn: 870.32 [M+H+]; found: 870.40.
5,15-Bis(3-allyloxypropyl)-10-[N,N’-bis-{1,1-(tert-butoxycarbonylethyl)-
tert-butoxycarbonylpropyl}isophthalamido]-20-(1’-methyl-2’-imidazolyl)-
porphyrinato zinc(II) (5Zn): Ester compound 4ZnEt (81 mg, 93.1 mmol)
was dissolved in 13:7 THF/MeOH (20 mL) and NaOH aqueous solution
(5 mL, 8n). After stirring for 3 h at RT, the solution was acidified with
4n HCl until a pH of 4 was reached, and the solution was extracted with
CHCl3. The acidic aqueous layer was neutralized with sat aq NaHCO3
and further extracted with 2:1 CHCl3/THF mixture. All organic layers
were dried over anhydrous Na2SO4 and concentrated by rotary evapora-
tion to give the carboxylic acid compound 4ZnH (68 mg, 90%). In a
50 mL flask, compound 4ZnH (78 mg, 95.7 mmol), benzotriazol-1-yloxy-
tris(dimethylamino)phosphoniumhexafluorophosphate (BOP, 84.6 mg,
191.4 mmol), and diisopropylethylamine (DIPEA, 37.1 mg, 287.1 mmol)
were dissolved in dry DMF (8 mL) at RT. After 1 h, tris(tert-butoxycar-
bonylpropyl) amine (100 mg, 239.25 mmol) and DIPEA (24.7 mg,
191.4 mmol) were added and the mixture was stirred for 2 h. After 2 h,
the mixture was concentrated, dissolved in CHCl3, washed with brine,
and dried over anhydrous Na2SO4. The organic layer was concentrated
and the crude product was purified by silica-gel column chromatography
eluting with CHCl3/acetone (20:1 v/v). After column chromatography,
the porphyrin was reprecipitated from a diethyl ether solution by the ad-
dition of n-hexane to give dendritic porphyrin 5Zn (138.2 mg, 90%). Rf
(Hex/EtOAc 1:1)=0.75; 1H NMR (600 MHz, CDCl3): d=9.64 (d, J=
4.2 Hz, 2H; Por b), 9.33 (s, 1H; Ph), 8.98 (d, J=4.2 Hz, 2H; Por b), 8.92
(d, J=4.2 Hz, 2H; Por b), 8.73 (s, 1H; Ph), 8.55 (s, 1H; Ph), 7.25 and
5-Formylisophthalic acid diethyl ester: In a 100-mL flask, which was
flushed with N2, 5-hydroxymethylisophthalic acid diethyl ester (3.6 g,
14.27 mmol) was dissolved in ethanol-free CHCl3 (100 mL) and 4A mo-
lecular sieve (eight crystals) was added. Pyridinium chlorochromate
(8.4 g, 39 mmol) was added in one portion and the reaction mixture was
stirred at RT for 1 h. The mixture was added to vigorously stirred diethyl
ether to coagulate the chromium reagent. The solvent was decanted off.
The residue was washed two more times with ether. All combined ether
layers were passed through Celite until the resulting solution was clear
and transparent. TLC of the filtrate showed the presence of the starting
material and the crude product was subjected to silica-gel column chro-
matography to elute the desired compound with n-hexane/ethyl acetate
(2:1 v/v) to give a pure white solid (2.45 g, 69%). Rf (n-hexane/ethyl ace-
tate 2:1)=0.95; 1H NMR: (270 MHz, CDCl3) d=10.14 (s, 1H; CHO),
8.92 (t, J=1.6 Hz, 1H; Ph-4,6), 8.71 (d, J=1.6 Hz, 2H; Ph-2), 4.46 (q, J=
7.3 Hz, 4H; CH2), 1.45 ppm (t, J=7.3 Hz, 6H; CH3).
5,15-Bis(3-allyloxypropyl)-10-(3’,5’-diethoxycarbonylphenyl)-20-(1’-
methyl-2’-imidazolyl) porphyrin (4): 1-Methyl-1H-imidazole-2-carbalde-
hyde (130 mg, 1.19 mmol), meso-(3-allyloxypropyl)dipyrromethane
(579 mg, 2.37 mmol), and 5-formylisophthalic acid diethyl ester (297 mg,
1.19 mmol) were dissolved in CHCl3 (500 mL). The mixture was degassed
by bubbling with N2 for 15 min. Then TFA (540 mg, 4.74 mmol) diluted
in CHCl3 was added. After stirring for 3.5 h, chloranil (1165 mg,
4.74 mmol) was added. The mixture was then stirred in the dark over-
night at RT. The mixture was washed with saturated NaHCO3 solution.
The aqueous layer was extracted with CHCl3 and the combined organic
layers were washed with brine and water. The resulting crude product
was washed with ether and hexane to remove tar and then subjected to
silica-gel chromatography to elute compound 4 by using CHCl3/acetone
(1:0 to 5:1 v/v). The resulting product was a red-violet solid (101 mg,
11%). However, at this stage, the product still contained some tar that
was very difficult to separate, and it was thought appropriate to do purifi-
cation after zinc insertion. Rf (CHCl3/MeOH 10:1)=0.54; 1H NMR
(270 MHz, CDCl3): d=9.54 (d, J=4.9 Hz, 2H; Por b), 9.50 (d, J=4.9 Hz,
2H; Por b), 9.16 (t, J=1.6 Hz, 1H; Ph), 9.60 (t, J=1.6 Hz, 1H; Ph), 8.96
(t, J=1.6 Hz, 1H; Ph), 8.79 (d, J=4.9 Hz, 2H; Por b), 8.73 (d, J=4.9 Hz,
2H; Por b), 7.69 (d, J=1.1 Hz, 1H; imidazole H4), 7.48 (d, J=1.1 Hz,
À
7.18 (s, 2H; CONH), 6.19 (ddt, J=17.4, 10.8, 5.4 Hz, 2H; CH2=CH ),
5.56 (d, J=1.8 Hz, 1H; imidazole H5), 5.53 (d, J=17.4, 3.0, 1.2 Hz, 2H;
À
CH2=CH ), 5.44 (d, J=4.2 Hz, 2H; Por b), 5.34 (ddt, J=10.8, 3.0,
À
1.2 Hz, 2H; CH2=CH ), 5.28–5.18 (m, 4H; Por-CH2), 4.24 (dd, J=5.7,
À
À
1.2 Hz, 4H; CH2=CHCH2), 3.99–3.88 (m, 4H; OCH2 ), 3.16–2.96 (m,
4H; Por-CH2CH2 ), 2.424 (dt, J=103.8, 7.8 Hz, 12H; NH2C=
2.189 (dt, J=93.6, 7.8 Hz, 13H; NH2C=
À
À
À
ACHTREUNG
À
À
ACHTREUNG
À
(s, 3H; NCH3), 1.53 (s, 27H; tBu), 1.22 ppm (s, 27H; tBu); UV/Vis
(CHCl3): lmax =416, 439, 566, 620 nm; fluorescence (lex =439 nm, CHCl3):
lem =623, 680 nm; MALDI-TOF MS (dithranol): m/z calcd for
C88H118N8O16Zn: 1609 [M+H+]; found: 1610.52.
Preparation of tetramer (61): Monomer 5Zn (30 mg, 22.3 mmol) and
dimer 3Zn (72 mg, 44.6 mmol) were dissolved in pyridine (400 mL) for
dissociation. After 1 h, pyridine was completely removed and the por-
phyrins were dissolved in CHCl3 for reorganization, during which, com-
pound (5Zn)2 and oligomeric mixtures 6n (n=1, 2, 3, etc.) were formed.
See Supporting Information for the analytical GPC spectrum. The target
tetramer 61 (n=1) was roughly separated by preparative GPC (JAI-
GEL 3H, eluent: CHCl3; flow rate: 3.5 mLminÀ1).
À
1H; imidazole H5), 6.08 (ddt, J=17.3, 10.5, 5.7 Hz, 2H; CH2=CH ), 5.41
À
(ddt, J=17.3, 3.2, 1.4 Hz, 2H; CH2=CH ), 5.26 (ddt, J=10.5, 3.0, 1.4 Hz,
À
2H; CH2=CH ), 5.10 (t, J=7.6 Hz, 4H; Por-CH2), 4.51 (q, J=7.3,
1.4 Hz, 4H; OCH2CH3), 4.08 (dt, J=5.7, 1.4 Hz, 4H; CH2=CHCH2 ),
3.66 (t, J=5.7 Hz, 4H; OCH2 ), 3.41 (s, 3H; NCH3), 2.85–2.72 (m,
À
À
À
À
À
À
À
4H; Por-CH2CH2 ), 1.43 (td, J=7.3, 1.4 Hz, 6H;
OCH2CH3),
À2.70 ppm (s, 2H; inner NH); MALDI-TOF MS (dithranol): m/z calcd
Olefin metathesis reaction for covalent linkage of 61 (7): Tetramer 61
(8.9 mg, 1.95 mmol) containing a small amount of dimer (5Zn)2 and hexa-
mer 62 was dissolved in CHCl3 (2 mL). The Grubbs catalyst (0.72 mg,
0.81 mmol) was added for the metathesis reaction. After 4 h, water was
added to terminate the reaction and the target material was extracted
for C48H50N6O6: 806.95 [M+H+]; found: 807.70.
5,15-Bis(3-allyloxypropyl)-10-(3’,5’-diethoxycarbonylphenyl)-20-(1’-
methyl-2’-imidazolyl) porphyrinatozinc(II) (4ZnEt): Compound
4
(55 mg, 74.35 mmol) was dissolved in CHCl3 (10 mL) and a saturated so-
3498
www.chemeurj.org
ꢀ 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2007, 13, 3491 – 3500

Self-Assembled Butadiyne-Bridged Bisporphyrin
FULL PAPER
with CHCl3. The combined organic layers were washed with water and
brine and dried over anhydrous Na2SO4. MALDI-TOF MS (dithranol):
m/z calcd for C172H228N16O32Zn2: 3162.52 (dimer) [M+H+]; found:
3164.75; m/z calcd for C244H290N28O36Zn4: 4452.20 (target tetramer 7)
[M+H+]; found: 4452.64; m/z calcd for C316H352N40O40Zn6: 5739.21
(hexamer) [M+H+]; found: 5742.76. Preparative GPC (JAIGEL 3H,
eluent: CHCl3; flow rate: 3.5 mLminÀ1) was performed for further purifi-
cation/isolation of 7 (7 mg, 80%). Analytical GPC peaks monitored at
300, 350, 420, and 480 nm (JAIGEL 3H-A, eluent: CHCl3; flow rate:
1.2 mLminÀ1): 12.85 min and (Tosoh TSKgel G2500HHR; eluent: pyri-
dine; flow rate: 1.2 mLminÀ1): 8.1 min. See Supporting Information for
analytical GPC spectra. 1H NMR, split signals due to isomers with re-
spect to two olefin moieties were observed in a ratio of about 1:3. Aster-
isks (*) and primes (’) indicate signals of major and minor isomers, re-
spectively. No mark indicates that peaks of the isomers (trans–trans, cis–
trans, cis–cis) were overlapped. 1H NMR (600 MHz, CDCl3): d=10.35–
10.28 (trans and cis, three d, J=4.2 Hz, 4H; Por b), 9.83–9.81 (trans and
cis, three d, J=4.2 Hz, 4H; Por b), 9.59–9.56 (trans and cis, three d, J=
4.2 Hz, 4H; Por b), 9.37* (trans–trans, s, 54%”2H; Ph-4), 9.35’ (cis–
trans, s, 40%”2H; Ph-4), 9.33’ (cis–cis, s, 6%”2H; Ph-4), 9.20–8.98
(trans and cis, six d*, 2”4H; Por b), 8.96–8.90 (trans and cis, three d*,
4H; Por b), 8.74 (brt, J=1.8 Hz, 2H; Ph-2,6), 8.57’ (cis–cis, s, 6%”2H;
Ph-2,6), 8.53’ (cis–trans, s, 40%”2H; Ph-2,6), 8.49* (trans–trans, s, 54%”
2H; Ph-2,6), 7.19’ (cis–cis, s, 6%”2H; CONH), 7.15’ (cis–trans, s, 40%”
2H; CONH), 7.11* (trans–trans, s, 54%”2H; CONH), 6.51* (trans, s,
switched Nd:YAG laser (Continuum Surelight I-10), frequency tripled
(l=355 nm) from the fundamental wavelength of 1064 nm to give 5-ns
pulses (FWHM) with a repetition rate of 10 Hz. The laser intensities
were attenuated with a filter to give a peak power of 17 GWcmÀ2. The
laser beam was focused by using a plano-convex lens with a focal length
of 100 mm. The samples were placed in a 1-mm quartz cuvette and scan-
ned at a range of 60 mm around the focal point. The data point at each Z
position was measured with 128 shots.
Singlet-oxygen generation upon two-photon irradiation by ADPA photo-
bleaching:[25,29] Singlet-oxygen generation was monitored by ADPA, a
well-known singlet-oxygen scavenger. Stock solutions of 5.0”10À5
m
ADPA in D2O, 10À4 m TPPS with ADPA in D2O, and 5.0”10À5 m com-
pound 1 with ADPA in D2O were prepared. The three samples were in-
dividually placed in a 1-cm quartz cuvette and illuminated with focused
100-fs pulses (beam waist was around 1 mm) of a mode-locked Ti:sap-
phire laser (Coherent Mira 900) with a repetition rate of 76 MHz. Two-
photon excitation was performed at 890 nm with
a peak power of
6.1 GWcmÀ2
.
Acknowledgements
This work was supported by Grant-in-Aid for Scientific Research (A)
(No. 15205020) and for Exploratory Research (No. 16655072) from the
Ministry of Education, Culture, Sports, Science, and Technology (Monbu
Kagakusho), by the Terumo Lifescience Foundation, and by the Iketani
Science Technology Foundation. K.O. acknowledges partial financial sup-
port from the Kinki-Chiho Invention Center.
À
À
78%”8H; CH=), 6.16’ (cis, s, 22%”8H; CH=), 5.69–5.44 (m, 19H;
À
imidazole-H5 (4H), Por b (2”4H), Por-CH2’ (44%”16H)), 5.33–5.11*
À
À
(br, 56%”16H; Por-CH2 ), 4.75’ (cis, broad, 24%”16H; OCH2CH=),
4.55–4.42* (trans, m, 76%”16H; OCH2CH=), 4.4–4.16 (m, 16H; Por-
À
À
À
(CH2)2CH2 ), 3.46–2.94 (brm, 16H; Por-CH2CH2 ), 2.59–2.04 (m, 52H;
À
À
amide ester CH2CH2 (48H), imidazole-H4 (4H)), 1.83–1.73 (trans and
A
cis, three d*, J=25.2 Hz, 12H; NCH3), 1.54 (brs, 54H; tBu), 1.24–
1.20 ppm (trans and cis, three s*, 54H; tBu). Two CONH protons that
are unaccounted for overlap with the CDCl3 peak. A * indicates that
peaks overlap, which makes the integration ratio difficult to determine.
The approximate ratio is 1:6:8. All of these doublet peaks have a cou-
pling constant of J=4.2 Hz; UV/Vis (CHCl3): lmax =423, 434, 463, 495,
566, 624, 667, 728 nm; fluorescence (lex =434 nm, CHCl3): lem =738,
821 nm; MALDI-TOF MS (dithranol): m/z calcd for C244H290N28O36Zn4:
4452.20 [M+H+]; found: 4452.69.
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Hydrolysis of 7 (7H) and formation of the sodium salt (1): Compound 7
(4.13 mg, 0.93 mmol) was dissolved in formic acid (1 mL). After stirring
for 12 h, formic acid was removed by vacuum evaporation. Toluene was
added to remove any remaining formic acid azeotropically. The residue
was not soluble in any solvent, even water. Therefore, NaOH (12 equiv
for the 12 carboxylic acid groups, 0.445 mg, 11.14 mmol) in water (0.1m)
was added to form the sodium salt, which became completely soluble in
water. The MALDI-TOF mass spectrum at this stage showed a partial
zinc demetalation. Zinc acetate dihydrate (6.12 mg, 27.81 mmol) in water
(2 mL) was added. Precipitates formed quickly due to the formation of
the zinc carboxylate salt of 7H. The precipitate was filtered by using a
Millipore filter. To obtain the sodium salt, an excess of NaOH (1 mg in
5 mL water) was added. The porphyrin dissolved in water was filtered by
using a Millipore filter. HCl (0.1n) was added very slowly and cautiously
to the porphyrin solution until the pH became 5–6 to precipitate com-
pound 7H in an acid form. The precipitates were collected and washed
well with water. Redissolving in alkaline conditions and precipitation in
slightly acidic conditions was undertaken twice to remove any water-solu-
ble impurities. Finally, NaOH (12 equiv for the 12 carboxylic acid groups,
0.445 mg, 11.14 mmol) in water (0.1m) was added and the water was
evaporated to afford 1 (3 mg, 85%). UV/Vis (water): lmax =429, 490, 571,
619, 670, 735 nm; fluorescence (lex =429 nm, water): lem =748 nm;
MALDI-TOF MS (a-cyano-4-hydroxy cinnamic acid): m/z calcd exact
mass for C196H194N28O36Zn4: 3779.36 [M+H+]; found: 3780.9.
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Received: August 19, 2006
Published online: January 16, 2007
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