Halogenated silicon(IV) phthalocyanines with axial poly(ethylene glycol) chains. Synthesis, spectroscopic properties, complexation with bovine serum albumin and in vitro photodynamic activities

Article abstract of DOI:10.1039/b307934a

A new series of unsubstituted and halogenated silicon(IV) phthalocyanines with two axial poly(ethylene glycol) (PEG) chains having an average molecular weight of 550 or 750 (PEG₅₅₀ or PEG₇₅₀) have been synthesised by treating the corresponding silicon phthalocyanine dichloride with PEG methyl ether in the presence of NaH. The compounds have been unambiguously characterised with ¹H NMR and MALDI-TOF mass spectrometry. With two bulky polymeric substituents, the compounds are essentially non-aggregated in common organic solvents. The longer PEG ₇₅₀ chain enhances the hydrophilicity of the phthalocyanine ring and is more effective to prevent aggregation and fluorescence quenching by Cu(OAc)₂. Substitution with heavier halogen atoms on the periphery of the ring leads to a reduction in fluorescence emission and an increase in singlet oxygen quantum yield, as a result of heavy atom effect. The compounds Si(PcX₈)(PEG₇₅₀)₂ [X = H (4b), Cl (4c), Br (4d)] are photocytotoxic towards HepG2 human hepatocarcinoma cells and J774 mouse mammary tumour cells. Although halogenation results in an increase in singlet oxygen quantum yield, the general photocytotoxicity follows the order 4b > 4d > 4c. This can be attributed to the opposite effect of aggregation, which follows the order 4a < 4b < 4c in the growth medium. The interactions of 4b-d with bovine serum albumin (BSA) have also been investigated by a fluorescence quenching method and a non-covalent conjugate of 4b and BSA has been prepared. Conjugation with BSA leads to a higher photocytotoxicity against J774 cells, which have a BSA-loving macrophage origin.

Full text of DOI:10.1039/b307934a

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P A P E R
Halogenated silicon(IV) phthalocyanines with axial poly(ethylene
glycol) chains. Synthesis, spectroscopic properties, complexation with
bovine serum albumin and in vitro photodynamic activitiesy
ab
a
a
c
d
Jian-Dong Huang, Shuangqing Wang, Pui-Chi Lo, Wing-Ping Fong, Wing-Hung Ko and
a
Dennis K. P. Ng*
a
Department of Chemistry, The Chinese University of Hong Kong, Shatin, N.T., Hong Kong,
China. E-mail: dkpn@cuhk.edu.hk
Institute of Research on Functional Materials, Department of Chemistry, Fuzhou University,
Fuzhou 350002, China
Department of Biochemistry, The Chinese University of Hong Kong, Shatin, N.T., Hong Kong,
China
Department of Physiology, The Chinese University of Hong Kong, Shatin, N.T., Hong Kong,
China
b
c
d
Received (in Montpellier, France) 11th July 2003, Accepted 13th November 2003
First published as an Advance Article on the web 27th January 2004
A new series of unsubstituted and halogenated silicon(IV) phthalocyanines with two axial poly(ethylene glycol)
(
treating the corresponding silicon phthalocyanine dichloride with PEG methyl ether in the presence of NaH.
PEG) chains having an average molecular weight of 550 or 750 (PEG550 or PEG750) have been synthesised by
1
The compounds have been unambiguously characterised with H NMR and MALDI-TOF mass spectrometry.
With two bulky polymeric substituents, the compounds are essentially non-aggregated in common organic
solvents. The longer PEG750 chain enhances the hydrophilicity of the phthalocyanine ring and is more effective
to prevent aggregation and fluorescence quenching by Cu(OAc) . Substitution with heavier halogen atoms on
2
the periphery of the ring leads to a reduction in fluorescence emission and an increase in singlet oxygen quantum
yield, as a result of heavy atom effect. The compounds Si(PcX
8
)(PEG750
)
2
[X ¼ H (4b), Cl (4c), Br (4d)] are
photocytotoxic towards HepG2 human hepatocarcinoma cells and J774 mouse mammary tumour cells.
Although halogenation results in an increase in singlet oxygen quantum yield, the general photocytotoxicity
follows the order 4b > 4d > 4c. This can be attributed to the opposite effect of aggregation, which follows the
order 4a < 4b < 4c in the growth medium. The interactions of 4b–d with bovine serum albumin (BSA) have also
been investigated by a fluorescence quenching method and a non-covalent conjugate of 4b and BSA has been
prepared. Conjugation with BSA leads to a higher photocytotoxicity against J774 cells, which have a
BSA-loving macrophage origin.
To increase drug circulating half-life and uptake by abnor-
mal tissues, poly(ethylene glycol) (PEG) has been widely used
Introduction
Photodynamic therapy (PDT) is a promising approach for the
treatment of cancer and certain non-cancerous conditions
that involve overgrowth of unwanted or abnormal cells.
4
as a pharmaceutical vehicle. While a substantial number of
PEG-derivatised (or pegylated) drugs have been reported, con-
jugation of this polymeric material to photosensitisers for
targeted PDT remains little studied. m-Tetrahydroxyphenyl-
1
The treatment involves systemic administration of
a
5,6
photosensitising agent, which has selective affinity for malig-
nant tissues and produces reactive oxygen species (ROS),
such as singlet oxygen, when excited by red light of appropri-
ate power. The therapeutic effect depends greatly on the beha-
chlorin (mTHPC), in particular, has been examined for the
effects of pegylation. It has been found that the biodistribu-
6
tion of the pegylated and non-pegylated mTHPC is remark-
ably different and the former generally exhibits enhanced
photosensitising properties compared to the latter. Although
phthalocyanines substituted with PEG chains on the periph-
eral positions have long been known, mainly for their liquid
1
viour of the photosensitisers. Photofrin , which is a complex
mixture of hematoporphyrin derivatives, is still widely used
clinically despite deficiencies such as complex composition,
poor absorption of tissue-penetrating red light, and pro-
longed retention. As a result, there has been a great demand
for new photosensitisers that show superior efficiency and
7
crystalline properties, reports on the photodynamic activities
8
of pegylated phthalocyanines are extremely rare. Bellemo
and co-workers reported a silicon(IV) 2,3-naphthalocyanine
with two axial PEG1900 chains, which showed little tumour
selectivity and no phototherapeutic activity towards a MS-2
2
fewer side effects. Owing to the desirable photophysical
and hotochemical properties, ease of chemical modification,
and low dark toxicity, phthalocyanines are promising candi-
dates for this application. Substantial progress has recently
been made in the development of phthalocyanine-based
8a
fibrosarcoma transplanted in Balb/c mice. In contrast, the
aluminum analogue AlPc(PEG2000) was found to be highly
photocytotoxic against the EMT-6 mouse mammary tumour
cells and the colon carcinoma Colo-26. It was found that
2
,3
photosensitisers.
8b
the additional hydrophilic axial ligand changed the in vitro
and in vivo kinetics, but did not reduce the photo-
dynamic activity of the parent photosensitiser. Non-covalent
y Dedicated to Prof. Malcolm L. H. Green on the occasion of his
retirement, with our warmest congratulations.
T h i s j o u r n a l i s Q T h e R o y a l S o c i e t y o f C h e m i s t r y a n d t h e
C e n t r e N a t i o n a l d e l a R e c h e r c h e S c i e n t i f i q u e 2 0 0 4
3
48
N e w . J . C h e m . , 2 0 0 4 , 2 8 , 3 4 8 – 3 5 4

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2 2 n
CH O) Me] fragment, each separa-
þ
þ
encapsulation of phthalocyanines in PEG-coated poly(lactic
acid) nanoparticles and PEG-liposomes has also been reported
and the efficiency of these delivery systems has been evalu-
ion M and the [M ꢀ O(CH
ted by 44 mass units corresponding to the repeating unit of PEG.
These signals were relatively weak for the chloro and bromo
analogues, for which an envelope due to the Na adduct of
9
þ
ated. We have recently employed PEG as a dispersing agent
to promote the formation of monomeric phthalocyanine in
2 2 n
the –O(CH CH O) Me fragment appeared as the base peak.
1
0
water. We describe herein the synthesis, spectroscopic prop-
erties, and in vitro photodynamic activities of a series of silico-
n(IV) phthalocyanines covalently linked with two short PEG
Electronic absorption and photophysical properties
1
1
The electronic absorption spectra of 4a–d in N,N-dimethyl-
formamide (DMF) were typical for non-aggregated phthalo-
cyanines, showing a B (or Soret) band at 354–360 nm, an
intense and sharp Q band at 671–682 nm, together with two
vibronic bands at 604–613 and 641–652 nm. All these absorp-
tions shifted slightly to the red upon halogenation as shown in
Table 1. Monomeric spectra were also observed for these com-
chains, namely PEG550 or PEG750 , at the axial positions.
Their complexation with bovine serum albumin (BSA) and
the effects of halogen substitution on the PDT efficiency are
also reported.
Results and discussion
3
pounds in MeOH, EtOH, tetrahydrofuran (THF) and CHCl .
Preparation and characterisation
To briefly examine the aggregation behaviour, the absorption
spectra of 4a and 4b in DMF were recorded in different con-
centrations. It was found that 4a did not follow the Lam-
bert–Beer law at concentrations higher than ca. 5 mM, while
Scheme 1 shows the synthetic pathway to the PEG-containing
phthalocyanines 4a–d. The unsubstituted and halogenated
derivatives were prepared in a similar manner. Starting from
the dichloro- (1b) or dibromophthalonitrile (1c), bubbling of
dry ammonia in the presence of NaOMe led to the respective
4
b gave a perfect straight line in the plot of absorbance vs. con-
12
centration up to ca. 10 mM. This observation indicates that
the longer PEG750 is more effective than PEG550 to hinder
the aggregation of phthalocyanine. The longer PEG750 also
enhances the hydrophilicity of the phthalocyanine ring. While
1,3-diiminoisoindoline 2b or 2c. Upon treatment with SiCl
4
in quinoline, these compounds were converted to the corre-
sponding silicon phthalocyanines 3b and 3c. Due to their poor
solubility in common organic solvents, the macrocycles could
only be purified by Soxhlet extraction.
4
a did not give a noticeable Q band absorption and fluores-
cence emission in water, the absorption spectrum of 4b in
water showed a rather intense Q band at 682 nm. Upon excita-
tion at 610 nm, this compound also showed a fluorescence
emission at 687 nm with a quantum yield of 0.25. It is worth
noting that due to the strong aggregation and hydrophobic
interactions, fluorescence of phthalocyanines is rarely observed
Treatment of 3a–c with PEG550 or PEG750 methyl ether in
the presence of NaH led to axial substitution, giving 4a–d in
moderate yields (Scheme 1). These polymeric materials are
highly soluble in various organic solvents and could be purified
readily by column chromatography. Reaction of diiodophtha-
lonitrile with ammonia also afforded a 1,3-diiminoisoindoline,
but attempts to prepare the octaiodo analogue of 4a–d follow-
13
in aqueous media. The chloro and bromo analogues 4c and
d have a lower solubility in water. The absorption spectra
4
1
ing the same route were not successful. The H NMR spectrum
in water showed broad Q bands due to the aggregated species
and no fluorescence signal was observed.
Compounds 4a and 4b were highly fluorescent in DMF and
the data are summarised in Table 1. Upon addition of
of the product showed several signals instead of a singlet in the
aromatic region. This indicated that some of the iodo groups
were cleaved under the strong basic conditions.
Apart from the signal(s) for the phthalocyanine ring pro-
tons, the H NMR spectra of 4a–d showed up to 8 well-
Cu(OAc)
of these compounds decreased gradually and the data could be
analysed by the Stern–Volmer equation: I
2
, which acts as a quencher, the fluorescence intensity
1
separated and upfield-shifted virtual triplets for the chain
methylene protons nearest to the ring centre. Due to the shield-
ing effect by the ring current, these signals were significantly
shifted upfield (up to d ꢀ 1.9). Compounds 4b–d were also
o
/I ¼ 1 þ KSV [Q],
where I and I are the fluorescence intensities in the absence
o
and presence of the quencher, respectively, [Q] is the
concentration of the quencher, and KSV is the Stern–Volmer
quenching constant. Under similar conditions ([fluorophore] ¼
1
1
characterised by MALDI-TOF mass spectrometry. The
spectrum of 4b showed two major envelopes for the molecular
1
.2–1.6 mM in DMF, [Q] varied from 0 to 3.7 mM), both
compounds gave a linear Stern–Volmer plot from which
the value of KSV was found to be 590 M for 4a and 310 M
ꢀ
1
ꢀ1
for 4b. This shows that the longer PEG750 imposes a more
hindered environment to prevent the approach of the quencher.
To evaluate the photosensitising efficiency of these PEG-
containing phthalocyanines, their singlet oxygen quantum
yields (F
,3-diphenylisobenzofuran (DPBF) as the scavenger. The con-
centration of the quencher was monitored spectroscopically at
11 nm with time, from which the values of F could be deter-
mined. As shown in Table 1, the value of F follows the
D
) were determined by a steady-state method using
1
4
D
1
4
D
Table 1 Electronic absorption and photophysical data for 4a–d in
DMF
a
b
F
c
Compound
lmax/nm
lem/nm
F
F
D
4
4
4
4
a
b
c
354, 604, 641, 671
355, 606, 644, 672
358, 611, 650, 679
360, 613, 652, 682
677
676
684
690
0.82
0.80
0.73
0.34
0.16
0.20
0.38
0.52
d
a
b
Excited at 610 nm. Relative to ZnPc (F
F
¼ 0.30 in 1-chloro-
c
naphthalene). Relative to ZnPc (F
D
¼ 0.55 in DMF).
Scheme 1 Preparation of pegylated phthalocyanines.
T h i s j o u r n a l i s Q T h e R o y a l S o c i e t y o f C h e m i s t r y a n d t h e
C e n t r e N a t i o n a l d e l a R e c h e r c h e S c i e n t i f i q u e 2 0 0 4
N e w . J . C h e m . , 2 0 0 4 , 2 8 , 3 4 8 – 3 5 4
349

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order 4a ꢁ 4b < 4c < 4d. The trend is in accord with the heavy
atom effect, which suggests that substitution with heavy atoms
enhances intersystem crossing, leading to a higher singlet oxy-
gen quantum yield. This concomitantly decreases the fluores-
cence quantum yield as found for these compounds (Table 1).
Interactions with BSA
BSA is a common protein carrier for anticancer drugs to
1
5
improve their passive targeting properties. With the goal of
enhancing the biocompatibility and selectivity of these phtha-
locyanine-based photosensitisers, attempts were made to pre-
pare the BSA conjugates of these dyes. To this end, we
firstly investigated the interactions of 4b–d with BSA by a
fluorescence quenching method. Fig. 1 shows the change in
fluorescence spectrum of BSA upon titration with 4d in an
aqueous solution containing 50 mM tris(hydroxymethyl)ami-
nomethane and 0.1 M NaCl, which has been adjusted to pH
Fig. 2 Elution profiles of 4b-BSA as monitored by (L) absorbance at
6
2
80 nm and (K) fluorescence emission at 340 nm upon excitation at
80 nm.
7
3
.4 with HCl(aq) (Tris-HCl buffer). The emission band at
43 nm decreases in intensity and shifts gradually to 327 nm.
As shown in the inset of Fig. 1, the quenching data follow
the Stern–Volmer equation, giving a KSV of 3.9 ꢂ 10 M .
Compound 4c behaved similarly except that the rate of
5
ꢀ1
phthalocyanine and BSA. Since a large excess of 4b was used
as also shown in Fig. 2), it was expected that the fraction
(
would be free from uncomplexed BSA. The protein content in
the resulting conjugatewas determined with the Bio-Radprotein
assay kit using BSA as standard. The concentration of 4b was
quenching was significantly faster. At a phthalocyanine to
BSA molar ratio of 5, for example, the I /I value increased
o
1
6
from 12 (for 4d) to 40 (for 4c). The Stern–Volmer plot, how-
ever, was curved upward, preventing the determination of
the KSV value. The non-halogenated analogue 4b could also
quench the fluorescence of BSA, but interestingly two instead
of one emission bands, at 339 and 372 nm, were observed dur-
ing the titration. It seems that this compound interacts with
BSA and changes its micro-environment in a different manner
compared with the halogenated counterparts. The Stern–Vol-
mer plot for this compound was also curved upward and the
apparent rate of quenching lay between those of 4c and 4d.
In view of the strong interactions between these phthalocya-
nines and BSA, attempts were made to prepare their non-cova-
lent conjugates. The 4b-BSA conjugate was prepared by
stirring a mixture of 4b and BSA (with a molar ratio of 20)
in a Tris-HCl buffer, followed by chromatography on a G-
measured from the Q band absorbance in a diluted DMF solu-
M
5
ꢀ1 ꢀ1
tion (e672 ¼ 2.1 ꢂ 10
cm ). The molar ratio of 4b to
BSA was found to be ca. 1:1 in this conjugate. Attempts to pre-
pare the BSA conjugates of 4c and 4d using a similar procedure
were not successful. The phthalocyanines form large aggregates
in the aqueous solution, of which the elution volumes (ca. 18–20
mL) are close to those of free BSA and the conjugates if formed,
making separation a difficult task.
The absorption spectrum of 4b-BSA in phosphate buffered
saline (PBS) is given in Fig. 3, which also displays the spectrum
of 4b in PBS for comparison. The latter shows a very sharp
and intense Q band at 681 nm, which strictly obeys the Lam-
bert–Beer law up to 10 mM. The spectrum remained virtually
unchanged upon standing for a few days. These observations
suggest that the compound exists mainly in monomeric form
and is stable in this solution. A rather sharp Q band was also
seen for the conjugate, indicating that the phthalocyanine is
also relatively non-aggregated in the conjugate.
1
4 3
00 Sephadex column using aqueous NH HCO (pH 8.3) as
eluent. Fig. 2 shows the elution profile monitored by absorp-
tion (for the phthalocyanine absorption at 680 nm) and fluor-
escence (for the BSA emission at 340 nm) spectroscopy. For
comparison, the elution profiles of pure 4b and BSA were also
obtained, which showed a maximum at an elution volume of
In vitro photodynamic activities
37 and 16 mL, respectively. Thus, it can be seen in Fig. 2 that
at an elution volume of 16–17 mL, the fraction contains both
The photodynamic activities of 4b–d and the BSA conjugate of
4b against HepG2 and J774 cell lines were examined. Fig. 4
Fig. 1 Change in fluorescence spectrum of BSA (6.4 mM, excited at
280 nm) in a Tris-HCl buffer upon titration with 4d. The inset shows
the corresponding Stern–Volmer plot.
Fig. 3 Absorption spectra of (—) 4b (3.3 mM) and (---) 4b-BSA
conjugate (3.7 mM) in PBS.
T h i s j o u r n a l i s Q T h e R o y a l S o c i e t y o f C h e m i s t r y a n d t h e
C e n t r e N a t i o n a l d e l a R e c h e r c h e S c i e n t i f i q u e 2 0 0 4
3
50
N e w . J . C h e m . , 2 0 0 4 , 2 8 , 3 4 8 – 3 5 4

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Fig. 6 Comparison of the photocytotoxicities of (white) 4b, (gray) 4c
and (black) 4d (all in Cremophor EL emulsion) towards HepG2. The
Fig. 4 (L) Dark- and (K) photocytotoxicities of 4b (in PBS) towards
HepG2. For the latter, the cells were illuminated with a red light
ꢀ2
cells were illuminated with a red light (l > 610 nm, 40 mW cm , 48
ꢀ2
ꢀ2
ꢀ2
J cm ). Values are expressed as mean ꢃ SD (n ꢄ 3).
(
l > 610 nm, 40 mW cm , 48 J cm ). Values are expressed as
mean ꢃ SD (n ꢄ 3).
substitution. Uptakes by HepG2 cells of 4b in PBS and 4b–d
in Cremophor EL emulsion, after incubation for 2 h, were
determined by absorption spectroscopy. Due to the weak
fluorescence emission of 4c and 4d, the commonly used fluor-
escence method could not be applied. It was found that 4b in
PBS has the highest uptake (0.52 ꢃ 0.04%), while the values
for all three phthalocyanines in Cremophor EL emulsion are
comparable (0.22 ꢃ 0.05% for 4b, 0.27 ꢃ 0.07% for 4c, 0.18 ꢃ
shows the survival curve for HepG2 using 4b in PBS as the
photosensitiser. While this system was essentially non-toxic
in the absence of light, it exhibited a very high photocytotoxi-
city with a LD50 (the extracellular dye concentration required
to kill 50% of the cells) of 0.75 mM. All the cells were basically
killed upon treatment with 4 mM of 4b. Figs. 5(a) and (b) show
the microscopic observations of the HepG2 cells after treat-
ment with 4b, both in the absence and presence of light. While
no significant morphological change was observed in the
absence of light, the cells became globular and shrunken after
the photo-treatment, indicating cell death. The chloro and
bromo analogues 4c and 4d had limited solubility in the
medium and required Cremophor EL to formulate. These
compounds were also non-toxic in the dark, but the
photocytotoxicity was significantly lower than that of 4b (also
in Cremophor EL emulsion) and followed the order
0
.08% for 4d). The higher uptake of 4b in PBS than in Cremo-
phor EL emulsion may account for the higher photocyto-
toxicity. Fig. 7 shows a fluorescence microscopic image of
HepG2 cells after 2 h incubation with 4b in PBS. It is clear that
the dye enters into the cell, causing substantial fluorescence in
the cytoplasm. Fig. 8 shows the absorption spectra of 4b–d in
the growth medium in the presence of 1 mM Cremophor EL. It
can be seen that while a sharp Q band appears for 4b, this band
is split and broaden upon halogenation. It appears that the
content of monomeric phthalocyanine decreases in the
order 4b > 4c > 4d. By considering the fact that heavy atom
substitution will enhance intersystem crossing, leading to a
higher singlet oxygen quantum yield (see Table 1 for the data
in DMF), it is expected that the bromo analogue 4d may also
be a better singlet oxygen generator in the medium. Since the
4
b > 4d > 4c. The data are compared in Fig. 6. Compound
b exhibits a slightly higher photocytotoxicity in PBS solution
4
than in Cremophor EL emulsion (Figs. 4 and 6).
The difference in photoactivity of these compounds can
be attributed to different cellular uptake and extent of
aggregation of the photosensitiser, and the effects of halogen
Fig. 5 Microscopic views of HepG2 after incubation with 4b:
(a) without illumination; (b) upon illumination. The corresponding
pictures for J774 are shown in (c) and (d), respectively.
Fig. 7 Fluorescence microscopic image of HepG2 after incubation
with 4b for 2 h.
T h i s j o u r n a l i s Q T h e R o y a l S o c i e t y o f C h e m i s t r y a n d t h e
C e n t r e N a t i o n a l d e l a R e c h e r c h e S c i e n t i f i q u e 2 0 0 4
N e w . J . C h e m . , 2 0 0 4 , 2 8 , 3 4 8 – 3 5 4
351

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HepG2 and J774 cancer cells. Halogen substitution on the peri-
pheral positions does not increase the PDT efficiency of the
phthalocyanine core even though halogenated phthalocyanines
have a higher singlet oxygen quantum yield. Other factors
such as cellular uptake and the aggregation state of the
photosensitiser also play an important role.
Experimental
General
Reactions were performed under an atmosphere of nitrogen
unless otherwise stated. Toluene and THF were distilled from
sodium and sodium benzophenone ketyl, respectively. DMF
was pre-dried over barium oxide and distilled under reduced
pressure. Quinoline was pre-dried over anhydrous sodium sul-
fate and fractionally distilled from zinc dust in vacuo. Chroma-
tographic purifications were performed on silica gel columns
(
All other solvents and reagents were of reagent grade and used
Macherey-Nagel, 70–230 mesh) with the indicated eluents.
Fig. 8 Absorption spectra of 4 mM of (—) 4b, (---) 4c and (ꢅ ꢅ ꢅ) 4d in
the growth medium in the presence of 1 mM Cremophor EL.
18
as received. 4,5-Dichlorophthalonitrile (1b), 4,5-dibromo-
1
phthalonitrile (1c), and silicon phthalocyanine dichloride
9
2
3a) were prepared according to literature procedures.
H NMR spectra were recorded on a Bruker DPX 300 spec-
0
(
cellular uptake of these three compounds is comparable, the
observed trend of photoactivity, 4b > 4d > 4c, may be a result
of the two opposing effects of aggregation and heavy atom
substitution.
1
trometer (300 MHz) in CDCl
stated. Chemical shifts are relative to internal SiMe
3
solutions unless otherwise
(d 0).
4
Electron impact (EI) mass spectra were measured on a Thermo
Finnigan MAT 95 XL mass spectrometer. Matrix-assisted
laser desorption/ionisation time-of-flight (MALDI-TOF)
spectra were obtained on a Bruker bench TOF mass spectro-
meter equipped with a standard UV-laser desorption source,
using 2,5-dihydroxybenzoic acid as matrix. Elemental analyses
were performed by Medac Ltd., Brunel Science Centre, UK.
UV-Vis and steady-state fluorescence spectra were taken on
a Cary 5G UV-Vis-NIR spectrophotometer and a Hitachi
F-4500 spectrofluorometer, respectively. The fluorescence
Compound 4b in PBS was also highly photocytotoxic
towards J774 cells (Fig. 9). The microscopic observation of
these cells is shown in Figs. 5(c) and (d), in which the effect
of light leading to cell death can be clearly seen. Because of
the monocyte-macrophage origin of these cells, which have a
high affinity for BSA, the photodynamic activity of 4b-BSA
conjugate against J774 was also investigated. It can be seen
in Fig. 9 that the conjugation leads to a substantial enhance-
ment in photocytotoxicity. At a dose of 0.9 mM of 4b, for
example, the cell viability decreases from ca. 50% to 6%. The
photoactivity of 4b–d in Cremophor EL emulsion towards
J774 was also compared. When 8 mM of photosensitiser was
used, the cell viability was found to be 5% for 4b, 81% for
1
7
quantum yields were determined by the equation:
2
sample
2
ref
21
F
sample ¼ (Fsample/Fref)(Aref/Asample)(n
/n )Fref
,
where
F, A and n are the measured fluorescence (area under the emis-
sion peak), the absorbance at the excitation position (610 nm)
and the refractive index of the solvent, respectively. Unsubsti-
tuted zinc(II) phthalocyanine (ZnPc) in 1-chloronaphthalene
4c, and 23% for 4d. The PDT efficiency thus again follows
the order 4b > 4d > 4c.
2
2
was used as the reference (FF ¼ 0.30). To minimise re-
absorption of radiation by the ground-state species, the emis-
sion spectra were obtained in very dilute solutions where the
absorbance at 610 nm was less than 0.03. Singlet oxygen quan-
Conclusion
We have prepared a series of four silicon(IV) phthalocyanines
with two axial PEG chains and a BSA conjugate of one of
these compounds, all of which are photocytotoxic against
tum yields (F ) were measured by the method of chemical
D
quenching of DPBF described by W o¨ hrle and co-workers,
14
except that the light intensity of our system was not deter-
mined. All measurements were performed in DMF using ZnPc
as a reference (FD ¼ 0.55).
Syntheses
Dichloro-1,3-diiminoisoindoline (2b). Sodium (0.06 g, 2.61
mmol) was dissolved in methanol (50 mL), to which 4,5-
dichlorophthalonitrile (1b; 1.05 g, 5.33 mmol) was added.
Ammonia was bubbled at a moderate rate into this mixture
at ambient temperature for 40 min. The mixture was then
brought to reflux for 3 h with continued stirring and addition
of ammonia. Upon cooling, the green solid formed was filtered
and washed with diethyl ether. The crude product was further
purified by recrystallisation from methanol to afford greenish
1
yellow crystals (0.42 g, 37%). H NMR (DMSO-d ) d 8.4–9.1
6
(
br s, 3 H, NH), 8.08 (s, 2 H, ArH). HRMS (EI) m/z calcd
for C H Cl ClN (M ) 212.9855, found 212.9857. Anal.
3
5
5
37
þ
8
3
calcd for C
4
8 5 2 3
H Cl N : C, 44.89; H, 2.35; N, 19.63; found: C,
4.46; H, 2.36; N, 18.94.
Fig. 9 (Filled) Dark- and (open) photocytotoxicities of (L, K) 4b and
Dibromo-1,3-diiminoisoindoline (2c). According to the above
procedure, 4,5-dibromophthalonitrile (1c; 1.14 g, 3.99 mmol)
(
/, N) 4b-BSA towards J774. Values are expressed as mean ꢃ SD (nꢄ 3).
T h i s j o u r n a l i s Q T h e R o y a l S o c i e t y o f C h e m i s t r y a n d t h e
C e n t r e N a t i o n a l d e l a R e c h e r c h e S c i e n t i f i q u e 2 0 0 4
3
52
N e w . J . C h e m . , 2 0 0 4 , 2 8 , 3 4 8 – 3 5 4

View Article Online
þ
was mixed with sodium (0.06 g, 2.61 mmol) in methanol (50
mL). After being stirred at ambient temperature for 40 min
under an ammonia atmosphere, the mixture was heated at
(calcd for M : 2286.7) and 1550.7 {calcd for
[M ꢀ O(CH CH O) Me] 1551.2} for n ¼ 16.
þ
2
2
n
ꢆ
60 C for 2 h with continued stirring and addition of ammonia.
Upon cooling, the orange solid formed was filtered and
washed with diethyl ether. The crude product was further pur-
8 750 2
Si(PcBr )(PEG ) (4d). By using the above procedure, 3c
(0.14 g, 0.11 mmol) was treated with PEG750 methyl ether
(average M ꢁ 750; 0.28 g, ca. 0.4 mmol) and NaH (25 mg,
n
1
ified by recrystallisation from methanol (0.32 g, 27%).
NMR (DMSO-d
ArH). HRMS (EI) m/z calcd for C
02.8825, found 302.8823.
H
1.04 mmol) in toluene (30 mL) to give the product as a green
1
6
) d 9.1–9.5 (br s, 3 H, NH), 8.25 (s, 2 H,
solid (0.16 g, 54%). Rf [CHCl –EtOH (9:1)] ¼ 0.45.
3
H
7
5
9
81
þ
8
H
Br BrN
3
(M )
NMR d 9.83 (s, 8 H, Pc–H ), 3.58–3.64 (m, ca. 80 H, CH ),
a
2
3
2 2
3.54–3.56 (m, 4 H, CH ), 3.48–3.50 (m, 4 H, CH ), 3.38 (s, 6
H, Me), 3.27–3.29 (m, 4 H, CH ), 3.05–3.07 (m, 4 H, CH ),
2
2
Si(PcCl
doline (2b; 0.16 g, 0.75 mmol) and SiCl (0.12 mL, 1.05 mmol)
in quinoline (20 mL) was heated at reflux for 2 h. The mixture
was then poured into benzene (80 mL) to give a blue paste,
which was filtered and washed thoroughly with benzene,
methanol and acetone. The resulting blue sold was then Soxh-
8
)Cl
2
(3b). A mixture of dichloro-1,3-diiminoisoin-
2 2
2.55–2.57 (m, 4 H, CH ), 1.79–1.81 (m, 4 H, CH ), 0.40 (vt,
4
J ¼ 6.0 Hz, 4 H, CH ), ꢀ1.94 (vt, J ¼ 6.0 Hz, 4 H, CH );
2
2
MS (MALDI-TOF) isotopic clusters peaking at m/z 2666.0
(calcd for [M þ Na] : 2665.2) and 1904.7 {calcd for
þ
þ
[M ꢀ O(CH CH O) Me] : 1906.9} for n ¼ 16.
2
2
n
let extracted with a mixture of these solvents plus CHCl
days to give a shiny blue solid (0.13 g, 80%).
3
for 2
4b-BSA conjugate. A solution of 4b in THF–EtOH (4:1; 1.4
mL, 2.1 mM) was added dropwise to a solution of BSA (40 mL,
3
.6 mM) in Tris-HCl buffer. The mixture with a molar ratio of
Si(PcBr
8
)Cl
doline (2c; 0.76 g, 2.51 mmol) and SiCl
2
(3c). A mixture of dibromo-1,3-diiminoisoin-
(0.50 mL, 4.36 mmol)
4b:BSA of 20 was stirred at ambient temperature overnight,
then chromatographed on a G-100 Sephadex column (Sigma,
dry bead diameter ¼ 40–120 mM; 1.8 ꢂ 21 cm) using 20 mM
aqueous NH HCO as eluent. The conjugate collected as the
4
in quinoline (35 mL) was heated at reflux for 1.5 h. By using
the above work-up procedure, a shiny blue solid was obtained
4
3
(
0.55 g, 72%).
4 3
first blue fraction was lyophilised to remove NH HCO and
water. The protein content was determined with the Bio-Rad
1
6
Si(PcH )(PEG550
8
)
2
(4a). A mixture of Si(PcH
8
)Cl
2
(3a; 0.10
protein assay kit using BSA as standard. The phthalocyanine
concentration was calculated from the Q band absorbance in a
g, 0.16 mmol), PEG550 methyl ether (average M ꢁ 550; 0.20 g,
n
5
ꢀ1
ꢀ1
ca. 0.4 mmol) and NaH (20 mg, 0.83 mmol) in toluene (10 mL)
was refluxed for 3 days. The solvent was then removed in vacuo
and the residue was subjected to column chromatography
diluted DMF solution (e672 for [4b] ¼ 2.1 ꢂ 10 M cm ).
In vitro studies
using CHCl –EtOH (9:1) as eluent. The crude product was
3
re-dissolved in ethyl acetate and the solution was extracted
with water to remove the unreacted PEG550 methyl ether.
The organic portion was dried over anhydrous MgSO
rotary evaporated to give a green solid (86 mg, 32%). R
For in vitro studies, a series of phthalocyanine-containing solu-
1
2
tions were first prepared. (a) A 2 mM solution of 4b in THF–
EtOH (4:1) was diluted with PBS to give a 80 mM solution.
After passing through a 0.45 mm filter, the solution was further
diluted with the following medium to give a series of solutions
with different concentrations of 4b. (b) A solution of 4b-BSA
conjugate in PBS was prepared. The concentration of 4b was
adjusted to 8 mM as monitored by the Q band absorbance in
4
and
f
1
[
CHCl
Pc–H
CH
.17 (vt, J ¼ 4.8 Hz, 4 H, CH
CH
), 2.43 (vt, J ¼ 4.8 Hz, 4 H, CH
H, CH
), 0.38 (vt, J ¼ 5.7 Hz, 4 H, CH
J ¼ 5.7 Hz, 4 H, CH ).
3
–EtOH (9:1)] ¼ 0.55. H NMR d 9.60–9.63 (m, 8 H,
a
), 8.30–8.35 (m, 8 H, Pc–H
b
), 3.51–3.63 (m, ca. 68 H,
), 3.36 (s, 6 H, Me),
), 2.95 (vt, J ¼ 4.8 Hz, 4 H,
), 1.65 (vt, J ¼ 4.8 Hz,
), ꢀ1.92 (vt,
2
), 3.44 (vt, J ¼ 4.8 Hz, 4 H, CH
2
3
2
5
ꢀ1 ꢀ1
DMF (e672 ¼ 2.1 ꢂ 10
M
cm ). This solution was then
2
2
diluted with the medium to appropriate concentrations. (c)
For studies using Cremophor EL emulsions, 2 mM solutions
of 4b–d were diluted to 80 mM with a ca. 0.01 M aqueous solu-
tion of Cremophor EL (Sigma, 0.5 g in 100 mL of water). The
solutions were clarified with a 0.45 mm filter, then diluted with
the medium to appropriate concentrations.
The HepG2 human hepatocarcinoma cells and J774 mouse
mammary tumour cells (both from ATCC) were maintained
in RPMI medium 1640 (Life Technologies) supplemented with
4
2
2
2
Si(PcH )(PEG ) (4b). By using the above procedure, 3a
8
750 2
(
(
5
0.64 g, 1.05 mmol) was treated with PEG750 methyl ether
average M ꢁ 750; 1.79 g, ca. 2.4 mmol) and NaH (0.14 g,
n
.83 mmol) in toluene (50 mL) to give the product as a green
1
H
solid (1.32 g, 62%). Rf [CHCl –EtOH (9:1)] ¼ 0.53.
3
a
NMR d 9.60–9.63 (m, 8 H, Pc–H ), 8.32–8.34 (m, 8 H, Pc–
4
H ), 3.54–3.72 (m, ca. 84 H, CH ), 3.44 (vt, J ¼ 4.8 Hz, 4
10% fetal calf serum (Invitrogen). About 2 ꢂ 10 (for HepG2)
b
H, CH
2
4
2
), 3.37 (s, 6 H, Me), 3.20 (vt, J ¼ 4.8 Hz, 4 H, CH
2
),
or 3 ꢂ 10 (for J774) cells per well in this medium were inocu-
ꢆ
2
.95 (vt, J ¼ 4.8 Hz, 4 H, CH ), 2.43 (vt, J ¼ 4.8 Hz, 4 H,
lated in 96-multiwell plates and incubated overnight at 37 C
2
CH
2
), 1.65 (vt, J ¼ 4.8 Hz, 4 H, CH
2
), 0.37 (vt, J ¼ 5.4 Hz,
2
under 5% CO . The cells were rinsed with PBS and incubated
4 H, CH ), ꢀ1.92 (vt, J ¼ 5.4 Hz, 4 H, CH ); MS (MALDI-
TOF) isotopic clusters peaking at m/z 2010.1 (calcd for M :
with 100 mL of the above solutions for 2 h under the same con-
ditions. The cells were then rinsed again with PBS and re-fed
with 100 mL of the growth medium before being illuminated
at ambient temperature. The light source consisted of a 300
W halogen lamp, a water tank for cooling and a colour glass
filter (Newport), cut-on 610 nm. The fluence rate (l > 610
2
2
þ
þ
2
1
012.0) and 1276.2 {calcd for [M ꢀ O(CH CH O) Me] :
2
2
n
275.6} for n ¼ 16.
Si(PcCl )(PEG750) (4c). By using the above procedure, 3b
8
2
ꢀ2
(
(
1
0.10 g, 0.11 mmol) was treated with PEG750 methyl ether
average M
ꢁ 750; 0.19 g, ca. 0.3 mmol) and NaH (25 mg,
.04 mmol) in toluene (30 mL) to give the product as a green
nm) was 40 mW cm . An illumination of 20 min led to a total
ꢀ2
n
fluence of 48 J cm .
Cell survival was determined by means of the colourimetric
1
H
23
MTT assay. After illumination, the cells were incubated at
solid (0.14 g, 55%). R
NMR d 9.64 (s, 8 H, Pc–H
.47–3.49 (m, 4 H, CH ), 3.40–3.42 (m, 4 H, CH
H, Me), 3.26–3.28 (m, 4 H, CH ), 3.05–3.07 (m, 4 H, CH
.56–2.58 (m, 4 H, CH ), 1.80–1.83 (m, 4 H, CH
J ¼ 6.0 Hz, 4 H, CH ), ꢀ1.94 (vt, J ¼ 6.0 Hz, 4 H, CH
f
[CHCl
), 3.54–3.64 (m, ca. 80 H, CH
), 3.37 (s, 6
3
–EtOH (9:1)] ¼ 0.60.
ꢆ
a
2
),
37 C under 5% CO
2
overnight. An MTT (Sigma) solution in
ꢀ1
3
2
2
PBS (50 mL, 3 mg mL ) was added to each well followed
by incubation for 2 h (for HepG2) or 5 h (for J774) under
the same environment. A solution of sodium dodecyl sulfate
(SDS; Sigma) in 0.04 M HCl(aq) (100 mL, 10% by weight) was
2
2
),
2
2
2
), 0.43 (vt,
2
2
);
ꢆ
MS (MALDI-TOF) isotopic clusters peaking at m/z 2295.5
then added to each well. The plate was incubated in a 60 C
T h i s j o u r n a l i s Q T h e R o y a l S o c i e t y o f C h e m i s t r y a n d t h e
C e n t r e N a t i o n a l d e l a R e c h e r c h e S c i e n t i f i q u e 2 0 0 4
N e w . J . C h e m . , 2 0 0 4 , 2 8 , 3 4 8 – 3 5 4
353

View Article Online
(a) H. Ali and J. E. van Lier, Chem. Rev., 1999, 99, 2379; (b) R.
Bonnett, J. Heterocycl. Chem., 2002, 39, 455.
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(b) C. M. Allen, W. M. Sharman and J. E. van Lier, J. Porphyrins
oven for 30 min, then 80 mL of iso-propanol was added to each
well. The plate was agitated on a Bio-Rad microplate reader at
ambient temperature for 20 s before the absorbance at 540 nm
of each well was taken. The average absorbance of the blank
wells, which did not contain the cells, was subtracted from
the readings of the other wells. The cell survival was then
2
3
Phthalocyanines, 2001, 5, 161; (c) A. C. Tedesco, J. C. G. Rotta
and C. N. Lunardi, Curr. Org. Chem., 2003, 7, 187.
4
5
6
(a) Poly(ethylene glycol) Chemistry and Biological Applications,
eds. J. M. Harris and S. Zalipsky, American Chemical Society,
Washington, DC, 1997; (b) G. Molineux, Cancer Treatment
Rev., 2002, 28, 13; (c) R. B. Greenwald, Y. H. Choe, J. McGuire
and C. D. Conover, Adv. Drug Delivery Rev., 2003, 55, 217.
(a) S. W. Young, K. W. Woodburn, M. Wright, T. D. Mody, Q.
Fan, J. L. Sessler, W. C. Dow and R. A. Miller, Photochem.
Photobiol., 1996, 63, 892; (b) F. Mitzel, S. FitzGerald, A. Beeby
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FitzGerald, A. Beeby and R. Faust, Chem. Eur. J., 2003, 9, 1233.
(a) P. Westermann, T. Glanzmann, S. Andrejevic, D. R.
Braichotte, M. Forrer, G. A. Wagnieres, P. Monnier, H. van
den Bergh, J.-P. Mach and S. Folli, Int. J. Cancer, 1998, 76,
determined by the equation: Cell Survival (%) ¼ [S(A
i
/
¯
A
control ꢂ 100)]/n, where A
i
is the absorbance of the ith data
¯
(
i ¼ 1, 2, . . ., n), Acontrol is the average absorbance of the con-
trol wells, in which the phthalocyanine was absent, and n (ꢄ3)
is the number of data points.
Cell uptake
6
HepG2 cells were plated at a density of 1 ꢂ 10 cells per well in
6
-multiwell plates in the growth medium (5 mL in each well).
Following an overnight incubation, the medium was removed
and the cells were rinsed with PBS, then incubated with 5 mL
of a 2 mM phthalocyanine dilution in the medium for 2 h. The
cells were then rinsed with PBS and re-fed with an SDS
solution in 0.04 M HCl(aq) (0.5 mL per well, 10% by weight).
8
42; (b) R. Hornung, M. K. Fehr, J. Monti-Frayne, B. J.
Tromberg, M. W. Berns and Y. Tadir, Br. J. Cancer, 1999, 81,
31; (c) H.-B. Ris, T. Krueger, A. Giger, C. K. Lim, J. C. M.
6
Stewart, U. Althaus and H. J. Altermatt, Br. J. Cancer, 1999, 79,
1061; (d ) R. Hornung, M. K. Fehr, H. Walt, P. Wyss, M. W.
Berns and Y. Tadir, Photochem. Photobiol., 2000, 72, 696;
ꢆ
The plate was incubated in a 60 C oven for 1 h, then 0.5 mL of
(e) T. Reuther, A. C. K u¨ bler, U. Zillmann, C. Flechtenmacher
iso-propanol and 2 mL of DMF were added to each well. The
solution was transferred to a 2 mL test tube, which was soni-
cated and centrifuged. The absorption spectrum of the solution
was recorded, from which the concentration of the phthalocya-
nine could be determined by the Q band absorbance
and H. Sinn, Lasers Surg. Med., 2001, 29, 314; ( f ) T. Krueger,
H. J. Altermatt, D. Mettler, B. Scholl, L. Magnusson and H.-B.
Ris, Lasers Surg. Med., 2003, 32, 61.
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Z. Ali-Adib, Macromolecules, 1996, 29, 913; (b) G. J. Clarkson,
B. M. Hassan, D. R. Maloney and N. B. McKeown, Macro-
molecules, 1996, 29, 1854; (c) T. Toupance, P. Bassoul, L. Mineau
and J. Simon, J. Phys. Chem., 1996, 100, 11 704.
(a) C. Bellemo, G. Jori, B. D. Rihter, M. E. Kenney and M. A. J.
Rodgers, Cancer Lett., 1992, 65, 145; (b) N. Brasseur, R. Ouellet,
C. La Madeleine and J. E. van Lier, Br. J. Cancer, 1999, 80, 1533.
(a) E. All e´ mann, N. Brasseur, O. Benrezzak, J. Rousseau, S. V.
Kudrevich, R. W. Boyle, J.-C. Leroux, R. Gurny and J. E. van
Lier, J. Pharm. Pharmacol., 1995, 47, 382; (b) J. C. Leroux, E.
All e´ mann, F. DeJaeghere, E. Doelker and R. Gurny, J. Controlled
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S. V. Kudrevich, K. Lewis and J. E. van Lier, Int. J. Cancer, 1996,
7
5
ꢀ1
ꢀ1
5
ꢀ1
M
(
cm
e
672 ¼ 2.1 ꢂ 10
M
cm
for 4b, e679 ¼ 2.9 ꢂ 10
ꢀ
1
5
ꢀ1
ꢀ1
cm for 4d). Each
for 4c, e6 ¼ 1.7 ꢂ 10 M
experiment was repeated two times.
82
8
9
Microscopic studies
Before the MTT assay, the cells in the 96-multiwell plates were
viewed with a Zeiss Axiovert 135 microscope (200ꢂ) and the
photographs were taken with a Nikon D-100 digital camera.
4
For the fluorescence imaging study, about 6 ꢂ 10 HepG2 cells
in the medium (0.6 mL) were loaded on a coverslip and incu-
. After removing the
66, 821; (d ) A. Gijsens, A. Derycke, L. Missiaen, D. de Vos, J.
Huwyler, A. Eberle and P. de Witte, Int. J. Cancer, 2002, 101, 78.
ꢆ
bated overnight at 37 C under 5% CO
2
medium, the cells were rinsed with PBS and incubated in the
medium containing 4b in PBS ([4b] ¼ 8 mM) for 2 h under
the same conditions. The cells were then rinsed again with
PBS and viewed with an Olympus 1 ꢂ 70S8F inverted micro-
scope. The excitation light source (at 380 nm) was provided
by a multi-wavelength illuminator (Polychrome IV, TILL
Photonics). The emitted fluorescence ( > 520 nm) was collected
using a digital cooled CCD camera (Quantix, Photometrics).
Images were digitised and analysed using MetaFluor V.6.0
10 (a) T. Ngai, G. Zhang, X.-Y. Li, D. K. P. Ng and C. Wu,
Langmuir, 2001, 17, 1381; (b) Z. Sheng, X. Ye, Z. Zheng, S. Yu,
D. K. P. Ng, T. Ngai and C. Wu, Macromolecules, 2002, 35, 3681.
1
1
A preliminary communication has appeared reporting the pre-
paration and photophysical properties of three of these com-
pounds (4b–d): P.-C. Lo, S. Wang, A. Zeug, M. Meyer, B.
R o¨ der and D. K. P. Ng, Tetrahedron Lett., 2003, 44, 1967.
12 To simplify the reporting of data and to facilitate comparison, the
molecular weights of 4a–d were estimated by assuming the mass of
the polymeric fragments PEG550 and PEG750 methyl ether to be
exactly 550 and 750, respectively.
(
Universal Imaging).
1
3
(a) J. Vacus and J. Simon, Adv. Mater., 1995, 7, 797; (b) M.
Kimura, K. Nakada, Y. Yamaguchi, K. Hanabusa, H. Shirai
and N. Kobayashi, Chem. Commun., 1997, 1215; (c) A. C. H.
Ng, X.-Y. Li and D. K. P. Ng, Macromolecules, 1999, 32,
Acknowledgements
5
292.
We thank Prof. Dominic Chan, Yi-Man Fung and Prof. Zong-
wei Cai for the mass spectrometric measurements. The Crou-
cher Foundation is acknowledged for a Chinese Visitorship
to Dr. J.-D. Huang. This work was supported by The Chinese
University of Hong Kong (Direct Grant 2002–03), the Natural
Science Foundation of China (Grant No. 20201005) and the
Foundation for University Key Teachers by the Ministry of
Education, China.
1
4
W. Spiller, H. Kliesch, D. W o¨ hrle, S. Hackbarth, B. R o¨ der and G.
Schnurpfeil, J. Porphyrins Phthalocyanines, 1998, 2, 145.
15 G. Stehle, A. Wunder, H. H. Schrenk, G. Hartung, D. L. Heene
and H. Sinn, Anti-Cancer Drugs, 1999, 10, 785.
1
1
6
7
M. M. Bradford, Anal. Biochem., 1976, 72, 248.
N. Brasseur, R. Langlois, C. La Madeleine, R. Ouellet and J. E.
van Lier, Photochem. Photobiol., 1999, 69, 345.
D. W o¨ hrle, M. Eskes, K. Shigehara and A. Yamada, Synthesis,
1993, 194.
1
8
9
1
C. C. Leznoff, Z. Li, H. Isago, A. M. D’Ascanio and D. S.
Terekhov, J. Porphyrins Phthalocyanines, 1999, 3, 406.
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T h i s j o u r n a l i s Q T h e R o y a l S o c i e t y o f C h e m i s t r y a n d t h e
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3
54
N e w . J . C h e m . , 2 0 0 4 , 2 8 , 3 4 8 – 3 5 4