Synthesis of novel diselenide-linked porphyrin dimers under phase-transfer catalysis condition and their interactions with DNA

Article abstract of DOI:10.1002/cbdv.200800182

Novel diselenide-linked porphyrin dimers were synthesized under phase-transfer catalysis conditions. The targeted compounds were characterized by ¹H-NMR, high-resolution mass spectrometry, UV/VIS and fluorescence spectroscopies, redox-potential

Full text of DOI:10.1002/cbdv.200800182

CHEMISTRY & BIODIVERSITY – Vol. 6 (2009)
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Synthesis of Novel Diselenide-Linked Porphyrin Dimers under Phase-
Transfer Catalysis Condition and Their Interactions with DNA
by Zhi Xuea), Daniel Wei-Jing Kwongb), Ling-Wei Xuec), Qing Liua), An-Xin Hou*a), and
Wai-Kwok Wong*a)b)
a) Department of Chemistry, Wuhan University, Wuhan 430072, P. R. China
(phone: þ862768752323; fax: þ862768754067; e-mail: houanxin@yahoo.com)
b) Department of Chemistry, Hong Kong Baptist University, Kowloon Tong, Hong Kong SAR,
P. R. China (phone: þ85234117011; fax: þ85234117348; e-mail: wkwong@hkbu.edu.hk)
c) College of Chemistry and Chemical Engineering, Pingdingshan University, Pingdingshan 467002,
P. R. China
Novel diselenide-linked porphyrin dimers were synthesized under phase-transfer catalysis con-
1
ditions. The targeted compounds were characterized by H-NMR, high-resolution mass spectrometry,
UV/VIS and fluorescence spectroscopies, redox-potential measurements, and elemental analysis. The
interaction of the title compounds with DNA was studied using UV/VIS, fluorescence, and circular
dichroism (CD) spectroscopies. The relative rates of singlet-oxygen production from the diselenide-
linked porphyrin dimers upon photoirradiation were also measured.
Introduction. – Diselenide compounds such as dialkyl and diaryl diselenides have
aroused great interest over the past decades due to their diverse biological properties,
ranging from antioxidant [1], glutathione peroxidase (GSHPx)-like [2], anti-inflam-
matory, and antinociceptive activities [3] to chemopreventive properties towards colon
carcinogenesis [4] as well as antitumor properties [5] but with low long-term systemic
toxicity [6]. However, the biological effects of Se-containing compounds on cells
depend strongly on dosage as well as the specific chemical form used [7]. For instance,
at supranutritional dose, Se compounds exhibit chemopreventive action towards many
types of cancer [8]. But at higher concentrations, DNA-strand breaks occur, leading to
cytotoxic or possibly carcinogenic effects [9]. To avoid the undesirable effects due to an
overdosing of normal cells, selective uptake/retention of the Se compounds by the
targeted tumor cells is of crucial importance.
Porphyrins, which bind to DNA and exhibit antitumor and antimicrobial activities
upon photoirradiation [10][11], have been shown to localize selectively in tumor cells
and were used as photodynamic therapeutic (PDT) agents due to their ability to
produce singlet oxygen, which can cleave DNA and damage the tumor cells [12]. Even
though the mechanism of selective uptake/retention of porphyrins by tumor cells is not
completely known [13], attempts have been made to design highly selective tumor-
targeting drugs by linking a potent chemotherapeutic moiety, such as cisplatin and
taxol, to porphyrin to enhance their antitumor efficacies [14][15]. Furthermore,
selective targeting of specific cell types, such as hepatocytes, has been achieved in a
galactosylated porphyrin where the b-d-galactose is recognized and then internalized
ꢀ 2009 Verlag Helvetica Chimica Acta AG, Zꢁrich

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CHEMISTRY & BIODIVERSITY – Vol. 6 (2009)
by the asialoglycoprotein receptor (ASGR) present only on hepatocytes and several
hepatoma cell lines at high density [16].
We are interested in investigating the antitumor properties of diselenides
conjugated with porphyrins. In this work, we report a convenient method for the
preparation of such diselenide-linked porphyrin conjugates in which phase-transfer
catalyst (PTC) played an important role. The compounds synthesized were charac-
1
terized using high-resolution mass spectrometry, H-NMR, UV/VIS, and fluorescence
spectroscopies, physiologically relevant redox-potential measurements, and elemental
analyses. In addition, the interaction of the diselenide-linked porphyrin dimers with
herring-sperm DNA (HS DNA) was studied using UV/VIS absorption, fluorescence,
and circular dichroism (CD) spectroscopies. The relative rates of singlet-oxygen
production from these diselenide-linked porphyrin dimers upon photoirradiation were
also measured.
Results and Discussion. – Synthesis and Characterization of Diselenide-Linked
Porphyrin Dimers. The diselenide-linked porphyrin dimers were prepared for the first
time using PTC. The total synthetic route of the title compounds is given in the Scheme.
Briefly, Na2Se2 (1) was prepared by a modification of the procedure due to Klayman
and Griffin [17]. Selenium powder was first reduced in H2O by NaBH4 to 1.
Benzyl(triethyl)ammonium chloride (TEBA), used as the PTC, was added into the
aqueous solution of Na2Se2. The resulting dark-red solution was set aside for use in a
subsequent step. Monohydroxy-substituted porphyrin 2 was synthesized from PhCHO,
1H-pyrrole, and 4-hydroxybenzaldehyde. Using a modified literature procedure,
bromoalkyl-substituted porphyrins 3 were prepared by an addition of dibromoalkane
to 2 under moisture- and O2-free conditions [18]. Lastly, the aqueous solution of 1
containing the TEBA previously set aside was allowed to react with 3 under PTC
condition to afford the final products, diselenide-linked porphyrin dimers 4.
In this work, three key modifications were made to the literature procedures to
accomplish the synthesis of the title compounds 4. i) In the classical method for
preparing Na2Se2 (1), there was always considerable amount of Se powders remaining
unreacted [17]. As elemental Se is insoluble in H2O, addition of a PTC facilitates its
solubilization and allows the reaction to proceed smoothly at room temperature to
afford 1. ii) According to the literature synthetic procedure for 3 [18], dibromoalkane
was added to a suspension of 2 and anhydrous K2CO3 in dry DMF. The resulting
mixture was then stirred at 608 to afford 3. But, in this work, Schlenk technique was
applied to achieve a moisture- and O2-free condition in this synthesis, resulting in a
yield of up to 95%. iii) The synthesis of 4 was carried out under PTC condition.
Porphyrins 3 were not soluble in H2O, but 1 was in an aqueous solution. The use of PTC
such as TEBA was, therefore, necessary to bring about the reaction between 1 and 3,
resulting in the successful synthesis of the diselenide-linked porphyrin dimers.
The characterization of the title compounds 4a–4c was performed by high-
resolution mass spectrometry (HR-MS). The HR-MS data are presented in Fig. 1,
showing the molecular-ion peaks of these compounds, where their m/z values compared
favorably with the calculated ones (i.e., Dm <5 ppm). Further characterization of 4 by
1H- and 77Se-NMR, UV/VIS absorption, and fluorescence spectroscopies was
performed. The 77Se-NMR of one diselenide-linked porphyrin dimer, 4a, is given in

CHEMISTRY & BIODIVERSITY – Vol. 6 (2009)
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Scheme. Total-Synthetic Route for Diselenide-Linked Porphyrin Dimers
Fig. 2,a, where the major singlet observed at 20.888 ppm was due to 4a, and the minor
singlet observed at 32.669 ppm was due to its monomer, porphyrin-selenol (TPP-
(CH2)2-SeH). The presence of the monomeric porphyrin-selenol in the sample was
confirmed by MALDI-TOF-MS (observed m/z 739.150, [TPP-(CH2)2-SeHþH]þ ; calc.
739.197). Due to the symmetry of the compound, no Se,Se coupling, which gives direct

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CHEMISTRY & BIODIVERSITY – Vol. 6 (2009)
Fig. 1. HR-MS of 4a–4c (from top to bottom). For 4a: m/z 1475.3735 ([Mþ1]þ ; calc. 1475.3639, Dm ¼
ꢀ0.9489 ppm). For 4b: m/z 1503.4072 ([Mþ1]þ ; calc. 1503.3952, Dm ¼0.5986 ppm). For 4c: m/z
1531.4449 ([Mþ1]þ ; calc. 1531.4265, Dm ¼4.7014 ppm).
evidence of the presence of SeꢀSe bond, can be observed. But, the presence of two Se-
atoms in these compounds was evidenced by their elemental analysis and the Se-
isotope profile of the MALDI-TOF mass spectra of these compounds (see Fig. 2,b).

CHEMISTRY & BIODIVERSITY – Vol. 6 (2009)
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Fig. 2. a) 77Se-NMR Spectrum of the diselenide-linked porphyrin 4a in CDCl3 with Me2Se as the external
reference. The major singlet observed at d 20.888 was due to 4a, and the minor singlet at d 32.669 was due
to its monomer, porphyrin-selenol (TPP-(CH2)2-SeH). b) Selenium isotope profile of MALDI-TOF MS
of 4a. The solid vertical lines represent the expected (calculated) isotope mass profile of a compound
containing two Se-atoms, which matched closely with the peak profile observed for 4a.
Thus, redox-potential measurement to probe the redox reaction of the SeꢀSe bond of
the diselenide-linked porphyrin dimers 4 by cyclic voltammetry was also conducted.
Redox-Potential Characterization. To further characterize the compounds 4, the
redox properties of the three diselenide-linked porphyrin dimers were studied by cyclic
voltammetry. In this study, water-soluble multi-walled carbon nanotubes (MWCNT)-
modified gold (Au) electrode was chosen over other common electrodes (such as Pt
and glassy C electrodes), as it gave a better signal, presumably due to its larger surface
area and enhanced reactivity [19]. Fig. 3 shows the cyclic voltammograms of 4 and
5,10,15,20-tetraphenylporphyrin (H2TPP), which was included as a reference com-
pound for the porphyrin moiety in 4, on the modified Au electrode. For H2TPP, no

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CHEMISTRY & BIODIVERSITY – Vol. 6 (2009)
distinct redox peak was observed in the potential range from 0 to ꢀ1.0 V in 0.1m
phosphate buffer, pH 7.4 (Fig. 3,a). However, distinct reduction peaks were observed
for the three diselenide-linked porphyrin dimers, 4a–4c, adsorbed on the MWCNT-
modified Au electrode. The voltammograms of these diselenide-linked porphyrins
differ from each other only slightly in the number and potential values of their
reduction peaks (Fig. 3,b–d). More importantly, all of them exhibit an almost identical
oxidation peak at around ꢀ0.70 V, suggesting the same origin of the reduction peaks
for these compounds, i.e., the reduction of the SeꢀSe bond. The reduction peak
potential values observed (e.g., ꢀ0.79 V in 4b and ꢀ0.80 V in 4c) were comparable to
the range of ꢀ0.5 to ꢀ0.7 V observed for other diselenides [20]. The slight difference
in the reduction peaks for the three Se samples might arise from the different chemical
environments of the SeꢀSe bond in the three porphyrin dimers.
Fig. 3. Cyclic voltammograms of H2TPP (a) and the diselenide-linked porphyrin dimers 4a–4c (b–d,
resp.) adsorbed on MWCNT-modified gold electrode in 0.1m phosphate buffer solution (pH 7.4) measured
at a scan rate of 100 mV/s
UV/VIS- and Fluorescence-Spectroscopic Studies of Diselenide-Linked Porphyrin
Dimer–DNA Interactions. Most studies on porphyrin–DNA interactions were
performed using monoporphyrins; only few reports have appeared on porphyrin
dimers [21][22]. Here, we report the results of our spectrophotometric titration study
of the interactions between DNA and the diselenide-linked porphyrin dimers 4. Fig. 4
(left) shows the spectral changes of the Soret band of the porphyrins (4a–4c) at near
430 nm upon addition of HS DNA. Moderate red shift of the Soret band (Dl¼4–
9 nm), together with a significant hypochromic effect (55–74%), were observed for the
porphyrin dimer, e.g., a red shift up to 9 nm and a hypochromicity of 55% were
observed in 4b. The detailed spectral changes are collected in the Table.
In most porphyrin–DNA interactions studied, three distinct binding modes,
namely, intercalative binding, external groove binding, and outside binding with self-
stacking along the DNA helix have been documented [22]. Among these three binding

CHEMISTRY & BIODIVERSITY – Vol. 6 (2009)
1137
Fig. 4. Left: UV/VIS Absorption spectra of the diselenide-linked porphyrin dimers 4a–4c (10 mm) in the
presence of increasing concentrations of HS DNA, expressed as ratio of cDNA/cpor ¼0, 0.1, 0.2, 0.4, 0.8, 1.2,
1.6, 3.2, resp., at pH 7.4. Right: Fluorescence spectra of 4a–4c (10 mm) with increasing concentrations of
HS DNA, corresponding to the same cDNA/cpor ratios of those in the UV/VIS titration spectra.
modes, intercalative binding is characterized by a substantial red shift (>10 nm) and a
significant hypochromicity (up to 40%) of the Soret band. External groove binding is
characterized by a minor-to-insignificant spectral shift and an occasional hyper-
chromicity. As for those prophyrins that interact with DNA via the outside binding with
self-stacking mode, similar spectral characteristics as in the intercalative binding mode

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CHEMISTRY & BIODIVERSITY – Vol. 6 (2009)
Table. UV/VIS Absorption- and Fluorescence-Spectral Changes of the Diselenide-Linked Porphyrin
Dimers 4a–4c upon Titration with HS DNAa)
Porphyrin dimer
UV/VIS Spectral data of the Soret band
Fluorescence
Red shift [nm]
Hypochromicity [%]
Intensity decrease [%]
4a
4b
4c
4
9
6
65.7
55.0
73.5
26.9
58.7
52.5
a) The sample mixtures contained 10 mm of the porphyrin dimers, 4a–4c, and HS DNA in different molar
ratios, i.e., cDNA/cporphyrin ¼0 and 10. All experiments were performed at room temperature in 0.05m Tris·
HCl buffer, pH 7.4, containing 0.1m NaCl.
are seen [22]. According to this criterion, these diselenide-linked porphyrin dimers, 4a–
4c, appeared to bind to DNA via outside binding with self-stacking along the DNA
helix (vide infra).
For those porphyrin–DNA interactions showing significant red shift and hypo-
chromicity, an apparent binding constant (Kapp) can be calculated according to Eqn. 1:
ꢁ
ꢂ
½DNAꢁtotal
1
1
ꢀ
È
É
¼
½DNAꢁtotal
þ
(1)
ꢀ
ðjeb ꢀ efjÞ
ð eapp ꢀ efjÞ
Kappðjeb ꢀ efjÞ
where eapp, ef, and eb correspond to Aobs/[porphyrin], the extinction coefficient for the
free porphyrin, and the extinction coefficient for the porphyrin in the fully bound form,
respectively. From a plot of [DNA]total/(jeapp ꢀef j ) vs. [DNA]total, shown in Fig. 5, Kapp
is obtained by the ratio of the slope to the intercept [23]. The Kapp values of 4a–4c were
determined to be 2.57ꢂ105, 2.09ꢂ105, and 1.24ꢂ106 mꢀ1, respectively. These spectral
changes and Kapp values obtained for the diselenide-linked porphyrin dimers are
comparable to those observed in another porphyrin dimer, i.e., 4,4’-dicarboxy-2,2’-
bipyridine-5-(4-aminophenyl)-10,15,20-tripyridin-4-ylporphyrin (Dl¼9 nm, hypo-
chromicity H¼37%, Kapp ¼1.2 ꢂ106 mꢀ1) [24]. These Kapp values are also comparable
to those obtained in most monoporphyrin–DNA interactions, such as in cis- and trans-
Fig. 5. Plot of [DNA]total /(|eapp ꢀef|) vs. [DNAtotal] for 4a–4c in the presence of increasing concentrations
of HS DNA

CHEMISTRY & BIODIVERSITY – Vol. 6 (2009)
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bis(1-methylpyridinium-4-yl)diphenylporphyrin (Kapp ¼1.2 ꢂ106 and 1.4ꢂ106 mꢀ1
,
resp.), where intercalative binding modes with DNA were adopted [25].
Fluorescence spectroscopy was also applied to study the interactions of these
porphyrin dimers with DNA [26]. The fluorescence emission spectra of 4a–4c (lem,max
¼
659, 656, and 655 nm, resp.) obtained from titration with increasing concentrations of
HS DNA are shown in Fig. 4 (right). The fluorescence intensities of 4a–4c were
quenched to various extents with only minor spectral shifts upon DNA addition. These
data are listed also in the Table. Such observed fluorescence quenching is consistent
with the binding mode suggested by the Soret-band titration data, namely, outside
binding with self-stacking along the DNA helix. Actually, this binding mode with DNA
is expected for all porphyrins with bulky substituents [25]. In this case, the bulky
substituent present in 4a–4c is the second porphyrin moiety, which is quite capable of
self-stacking.
CD Spectra of Diselenide-Linked Porphyrin–HS DNA Interactions. To further
clarify the binding mode, the induced CD spectra of the porphyrins 4 were recorded in
the presence of HS DNA (Fig. 6). In the case of duplex DNA, a positive-induced CD
band in the Soret region indicates its groove binding, and a negative induced CD band
indicates intercalation [27]. Conservative (or bisignate) CD signal is a signature of self-
stacked porphyrins bound externally on the polymer surface [27]. As seen from Fig. 6,
porphyrins 4a–4c showed the typical conservative CD profiles. For instance, porphyrin
4a, upon binding to HS DNA, showed a strong positive peak centered at 418 nm and a
strong negative peak centered at 440 nm with the intensity of the two peaks being
almost identical. All these data were presented in the Table. No significant CD signal
was observed in DNA alone without the porphyrins or vice versa (data not shown).
Thus, the bisignate CD feature of 4 suggests an external binding mode with self-
stacking, presumably along the helical structure of DNA. This result corroborated with
the binding mode inferred from the absorption and fluorescence titration experiments.
Fig. 6. Induced CD spectra of 4a–4c (5 mm) in the presence of HS DNA (base-pair concentration 100 mm)
in 0.05m Tris·HCl buffer (pH 7.4), containing 0.1m NaCl
Singlet-Oxygen Production by Diselenide-Linked Porphyrin Dimers. The relative
rates of singlet-oxygen production by the diselenide-linked porphyrin dimers 4a–4c
upon photoirradiation were determined using the singlet-oxygen quencher, 1,3-

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CHEMISTRY & BIODIVERSITY – Vol. 6 (2009)
diphenylisobenzofuran (DPBF), in benzene. The consumption of DPBF (lmax 415 nm)
was monitored spectrophotometrically as function of photoirradiation time of 4a–4c
with H2TPP, whose singlet-oxygen yield was reported to be 0.62 in benzene, used as a
reference [28]. This result is shown in Fig. 7, from which we can see that the rates of
singlet-oxygen production from all three diselenide-linked porphyrin dimers were
comparable to that of the H2TPP reference, indicating that the diselenide linkage did
not alter the singlet-oxygen quantum yield of the porphyrin moiety significantly.
Fig. 7. Bleaching of 1,3-diphenylisobenzofuran (DPBF) by the singlet-oxygen produced from the
diselenide-linked porphyrin dimers, 4a–4c, and the tetraphenylporphyrin (H2TPP) reference as a function
of the photoirradiation time
Conclusions. – In this work, we reported a convenient synthesis of three diselenide-
linked porphyrin dimers. The success of this synthesis rests on the use of a PTC, which
facilitated i) the reduction of elemental Se to selenide in water and ii) the coupling of
porphyrin with Na2Se2. The binding mode of these porphyrin dimers with DNA was
consistent with outside binding with self-stacking along the DNA helix, based on UV/
VIS-absorption and fluorescence titration as well as induced CD studies. The singlet-
oxygen yields of these compounds were shown to be comparable to that of the H2TPP.
Further studies of the biological activities of these porphyrin dimers are currently
underway.
W.-K. W. thanks the Hong Kong Baptist University (FRG/05-06/II-03) and Hong Kong Research
Grants Council (HKBU 2023/04P) for financial support of this work.
Experimental Part
General. Selenium powder, NaBH4, 4-hydroxybenzaldehyde, 1H-pyrrole, propanoic acid, 1,2-
dibromoethane, 1,3-dibromopropane, 1,4-dibromobutane, DMF, toluene, and benzyl(triethyl)ammoni-
um chloride (TEBA), all of reagent grade, were obtained from Aldrich Chemical Company, and were
appropriately degassed before use. Solvents were dried by standard procedures, distilled, and deaerated
prior to use. UV/VIS Spectra: OLIS Cary 15 spectrophotometer, lmax in nm (log e). CD: JASCO J-810
1
spectrometer. H-NMR Spectra: JEOL JNM-EX 270 spectrometer (with TMS as internal standard in
CDCl3), d in ppm, J in Hz. 77Se-NMR Spectra: Bruker 400-MHz FT-NMR spectrometer AVANCE III
(77Se¼76.33 MHz) operating in Fourier-transform mode at 292.6 K. The sample was run in 5-mm NMR

CHEMISTRY & BIODIVERSITY – Vol. 6 (2009)
1141
tube. The 77Se chemical shifts were externally referenced to Me2Se (60% in CDCl3 at 292.2 K, d 0.0) by
the sample-tube replacement technique. Low-resolution mass spectra (LR-MS): Finnigan MAT SSQ-710
mass spectrometer, in positive-ion mode. HR-MS: Bruker Biflex III matrix-assisted laser desorption/
ionization time-of-flight (MALDI-TOF) mass spectrometer, in m/z. Fluorescence spectra: PTI QM-1
spectrofluorometer. Elemental analyses were run on a Thermo Finnigan Flash EA-1112 series analyzer.
Multi-walled carbon nanotubes (MWCNT; purity >95 wt%, Timesnano Co., Chengdu, China) were
used as received. Congo red (CR) was purchased from Shanghai Reagent Co., Shanghai, China. Cyclic
voltammetric (CV) measurements were conducted with an EG&G model 283 electrochemical
workstation (Princeton Applied Research, USA) controlled by a M270 software. The electrode system
consisted of a water-soluble MWCNT film-coated Au electrode, a Pt wire counter electrode, and a KCl-
saturated calomel reference electrode (SCE). All potential values in this work were reported against the
SCE value.
Synthetic Procedures. Synthesis of H2TPP. H2TPP was conveniently prepared according to the
procedure described in [18]. It served as a reference compound for the measurements of the redox
potentials and the singlet-oxygen production rates of the diselenide-linked porphyrin dimers.
Synthesis of Na2Se2 (1). Selenium powder (4.5 g, 56 mmol) and TEBA (0.1 g) were added to H2O
(25 ml) in a stoppered three-necked flask with magnetic stirring. NaBH4 (4.5 g, 119 mmol) dissolved in
H2O (25 ml) was added dropwise to the slurry of elemental Se. The flask was chilled in an ice bath to
prevent boiling. After all NaBH4 had been added, and the soln. had become colorless, additional Se
powder (4.5 g, 56 mmol) was added to the soln., which then turned reddish brown, characteristic of
Na2Se2. The flask was then stoppered and flushed with Ar.
Synthesis of 5-(4-Hydroxylphenyl)-10,15,20-triphenylporphyrin (2). Compound 2 was prepared by a
modification of the method described in [18]. A soln. of PhCHO (5.5 ml, 54 mmol) and 4-
hydroxybenzaldehyde (3.8 ml, 35 mmol) in propanoic acid (250 ml) was heated to reflux with mechanical
stirring. Then, freshly distilled 1H-pyrrole (5.0 ml, 72 mmol) in propanoic acid (50 ml) was added
dropwise to the soln. After reflux for 1/2 h, the mixture was cooled to r.t. MeOH (300 ml) was then added
to the mixture. The resulting soln. was kept in a refrigerator overnight. Filtration gave ca. 1.3 g of crude
product, which was redissolved in a minimum amount of CHCl3 and chromatographed on a silica-gel
column with CH2Cl2 as eluent. The second band gave 2 (1.10 g, 5%). FAB-MS (pos.): 631 ([MþH]þ ).
Synthesis of 5-[4-(2-Bromoethoxy)phenyl]-10,15,20-triphenylporphyrin (3a). Compound 3 was
prepared according to a modification of the procedure described in [18]. Anh. 2 (0.50 g, 0.8 mmol)
and K2CO3 (1.0 g) were placed in a Schlenk tube, and the mixture was heated to dryness in vacuum to
ensure an absolute moisture-free condition. Then, under N2, anh. DMF (20 ml) and 1,2-dibromoethane
(2 ml, 23.1 mmol) were injected into the Schlenk tube with a syringe. With magnetic stirring, the mixture
was heated at 608. The progress of the reaction was monitored by TLC. When 2 had been completely
converted, DMF in the tube was evaporated in vacuum. The residue was redissolved in CHCl3 and
chromatographed (silica-gel column; CHCl3/hexane 5 :1). The first band gave 3a (0.53 g, 90%). 1H-NMR
(CDCl3, 270 MHz): 8.80–8.87 (m, 8 H pyrrole); 7.26–8.22 (m, 19 arom. H); 4.39 (t, CH2O); 3.78 (t,
CH2Br); ꢀ2.77 (s, 2 NH). FAB-MS (pos.): 736 (Mþ ). Anal. calc. for C46H33BrN4O: C 74.90, H 4.51, N
7.59; found: C 74.82, H 4.60, N 7.52.
Synthesis of 5-[4-(3-Bromopropoxy)phenyl]-10,15,20-triphenylporphyrin (3b). Anh. 2 (0.50 g,
0.8 mmol) and K2CO3 (1.0 g) were placed in a Schlenk tube, and the mixture was heated to dryness in
vacuum. Then, under N2, anh. DMF (20 ml) and 1,3-dibromopropane (2 ml, 19.6 mmol) were injected.
1
As described for 3a, 3b (0.54 g, 90%) was obtained. H-NMR (CDCl3, 270 MHz): 8.78–8.87 (m, 8 H,
pyrrole); 7.26–8.22 (m, 19 arom. H); 4.25 (t, CH2O); 3.60 (t, CH2Br), 2.51 (m, CH2CH2CH2Br); ꢀ2.78 (s,
2 NH). FAB-MS (pos.): 750 (Mþ ). Anal. calc. for C47H35BrN4O: C 75.10, H 4.69, N 7.45; found: C 75.02,
H 4.76, N 7.49.
Synthesis of 5-[4-(4-Bromobutoxy)phenyl]-10,15,20-triphenylporphyrin (3c). Anh. 2 (0.50 g,
0.8 mmol) and K2CO3 (1.0 g) were placed in a Schlenk tube, and the mixture was heated to dryness in
vacuum. Then under N2, anh. DMF (20 ml) and 1,4-dibromobutane (2 ml, 16.7 mmol) were injected. As
described for 3a, 3c (0.56 g, 92%) was obtained. FAB-MS (pos.): 764 (Mþ ). 1H-NMR (CDCl3, 270 MHz)
8.87–8.88 (m, 8 H, pyrrole); 7.26–8.22 (m, 19 arom. H); 4.15 (t, CH2O); 3.55 (t, CH2Br); 2.24 (m,

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CHEMISTRY & BIODIVERSITY – Vol. 6 (2009)
OCH2CH2CH2CH2Br); 2.13 (m, OCH2CH2CH2CH2Br); ꢀ2.75 (s, 2 NH). Anal. calc. for C48H37BrN4O:
C 75.29, H 4.87, N 7.32; found: C 75.41, H 4.80, N 7.34.
Synthesis of Diselenide-Linked Porphyrin Dimer 4a from 3a. To a soln. of 3a (0.1 g, 0.14 mmol),
TEBA (0.01 g) in toluene (40 ml) and 1 were added in excess. The mixture was stirred at r.t., and the
reaction progress was monitored by TLC. After 4 h, the mixture was washed with H2O to remove the
diselenide anion. The org. phase was evaporated to dryness in vacuum, and the residue was dissolved in
CHCl3 and chromatographed (silica-gel column; CHCl3/hexane 1:1). The desired product 4a showed a
moderately higher Rf value than 3a. Yield: 49 mg (50%). UV/VIS (CH2Cl2): 647 (3.70), 591 (3.71), 551
1
(3.92), 516 (4.17), 485 (3.65), 420 (5.43). H-NMR (CDCl3, 270 MHz): 8.74–8.95 (m, 16 H, pyrrole);
7.25–8.22 (m, 38 arom. H); 4.34 (t, 2 CH2O), 3.42 (t, 2 CH2Se); ꢀ2.78 (s, 4 NH). HR-MS (MALDI-
TOF): 1475.3735 (calc. 1475.3639). Fluorescence: lex 422, lem 654. Anal. calc. for C92H66N8O2Se2: C 74.99,
H 4.51, N 7.60; found: C 74.80, H 4.42, N 7.48.
Synthesis of the Dimer 4b from 3b. As described for 4a, with 3b (100 mg, 0.13 mmol), excessive
amounts of TEBA (0.01 g) in toluene (40 ml), and 1. The desired product 4b showed a moderately higher
Rf value than 3b. Yield: 44 mg (45%). UV/VIS (CH2Cl2): 649 (3.87), 592 (3.91), 553 (4.04), 517 (4.21),
480 (3.97), 420 (5.35). 1H-NMR (CDCl3, 270 MHz): 8.78–8.88 (m, 16 H, pyrrole), 7.20–8.22 (m, 38
arom. H); 4.22 (t, 2 CH2O); 3.35 (t, 2 CH2Se); 2.40 (m, 2 OCH2CH2CH2Se); ꢀ2.80 (s, 4 NH). HR-MS
(MALDI-TOF): 1503.4072 (calc. 1503.3952). Fluorescence: lex 422, lem 653 nm. Anal. calc. for
C94H70N8O2Se2: C 75.19, H 4.70, N 7.46; found: C 75.24, H 4.59, N 7.38.
Synthesis of the Dimer 4c from 3c. As described for 4a, with 3c (100 mg, 0.13 mmol), TEBA (0.01 g)
in toluene (40 ml), and 1 was added in excess. The desired product 4c showed a moderately higher Rf
value than 3c. Yield: 41 mg (42%). UV/VIS (CH2Cl2): 651 (3.98), 594 (4.03), 553 (4.12), 519 (4.21), 420
1
(5.11). H-NMR (CDCl3, 270 MHz): 8.74–8.78 (m, 16 H, pyrrole); 7.12–8.11 (m, 38 arom. H); 4.09 (t,
2 CH2O); 3.27 (t, 2 CH2Se); 2.31 (m, 2 OCH2CH2CH2CH2Se); 2.23 (m, 2 OCH2CH2CH2CH2Se); ꢀ2.77
(s, 4 NH). HR-MS (MALDI-TOF): 1531.4449 (1531.4265). Fluorescence: lex 422 nm, lem 654. Anal. calc.
for C96H74N8O2Se2: C 75.38, H 4.88, N 7.33; found: C 75.42, H 4.77, N 7.29.
Redox-Potential Characterization. The preparation of water-soluble MWCNTs was carried out
according to a procedure described in [19]. Briefly, MWCNTs were mixed with Congo Red (CR) using a
certain weight ratio (e.g., MWCNTs/CR 5 :1) in an agate mortar and ground for 4 h with the addition of
little H2O to avoid agglomeration of the dry CR powder to produce a greenish black mixture of
MWCNTs and CR. The MWCNTs/CR mixture was dissolved in H2O, filtered through a polytetrafluoro-
ethylene (PTFE) filter disc (0.22-mm pore size), and then washed with H2O to remove the free CR until
the filtrate became colorless. The black product obtained was denoted as MWCNTs-CRwater, which was
immediately dissolved and stored in H2O after the washing to avoid the rebundling and the loss of the
exfoliated nanotubes when dried. Gold electrode (2.0 mm in diameter, from CHI, USA) was polished on
a slush of alumina (Al2O3, 0.05 mm), and then washed in H2O, HNO3/EtOH 1:1 under sonication, each
for 3 min. Then, 1 ml of 1.0 mg/ml water-soluble MWCNTs were cast onto the clean Au surface and air-
dried. To measure the redox potentials of the diselenide-linked porphyrin dimers 4, a few drops of a 1 mm
sample soln. (in DMF) was added onto the water-soluble MWCNTs-modified gold electrode surface and
air-dried. The resulting Se-loaded electrode was immersed directly in 0.1m phosphate buffer (pH 7.4),
and cyclic voltammetric measurement was then performed at a scan rate of 100 mV/s.
UV/VIS Absorption, Fluorescence and Induced CD Spectral Studies of the Diselenide-Linked
Porphyrin Dimer–DNA Interactions. Spectrophotometric titrations of porphyrins 4 with HS DNA were
performed in an aq. buffered soln. at 308. The porphyrin 4 stock solns. were initially prepared in DMF but
were diluted with H2O in preparing the sample solns. The concentration of the HS DNA stock soln. (in
base pairs) was determined spectrophotometrically based on the extinction coefficient of 1.31ꢂ104 mꢀ1
cmꢀ1 at 260 nm. Different amounts of HS DNA were mixed with the porphyrin 4, whose final
concentration was fixed at 10 mm, in 10 ml of buffer soln. (0.05m Tris·HCl, 0.1m NaCl, pH 7.4, with <5%
DMF) to prepare a series of porphyrin–HS DNA sample solns. of different molar ratios (r¼cDNA
/
cporphyrin) for spectrophotometric measurements in the range of 320–500 nm. Spectrofluorometric
titrations (lex 422, lem 630–700 nm) of porphyrins 4 and HS DNA were conducted in a similar manner.
Induced CD spectra of 4a–4c with HS DNA were recorded according to a standard procedure. The ratio
cDNA/cporphyrin was ranged from 0 to 20.0, and concentrations of porphyrins 4 were fixed at 5 mm.

CHEMISTRY & BIODIVERSITY – Vol. 6 (2009)
1143
Measurement of Singlet-Oxygen Production. A W lamp (power rating 50 W) equipped with an
optical filter (l>500 nm) was used as the photoirradiation source. The concentrations of DPBF and the
porphyrins used were 5ꢂ10ꢀ5 and 1ꢂ10ꢀ6 m, resp. The consumption of DPBF was monitored by its
absorbance at its lmax 415 nm as function of photoirradiation time of the porphyrin-DPBF mixture.
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Received May 8, 2008