Circular dichroism of neutral zinc porphyrin-oligonucleotide conjugates modified with flexiblelinker

Article abstract of DOI:10.1246/bcsj.82.1280

Neutral zinc porphyrin containing flexiblealkyllinker, 5-[4-(5- hydroxypentyloxy)phenyl]-10,15,20-tri-p-tolyl- porphyrinatozinc, was attached to the 5' ends of 20- and 30-bp oligodeoxynucleotides (ODN) by solid-phase synthesis. Zinc porphyrin-modified dou

Full text of DOI:10.1246/bcsj.82.1280

1280 Bull. Chem. Soc. Jpn. Vol. 82, No. 10, 1280–1286 (2009)
© 2009 The Chemical Society of Japan
Circular Dichroism of Neutral Zinc Porphyrin-Oligonucleotide
Conjugates Modified with Flexible Linker
Akira Onoda,2,³ Masahiro Igarashi,1 Satoshi Naganawa,1 Kiyomi Sasaki,1
Shinya Ariyasu,1 and Takeshi Yamamura*1
1Faculty of Science, Tokyo University of Science, 1-3 Kagurazaka, Shinjuku-ku, Tokyo 162-8601
2Department of Applied Chemistry, Graduate School of Engineering, Osaka University,
2-1 Yamadaoka, Suita, Osaka 565-0871
Received February 4, 2009; E-mail: tyamamur@rs.kagu.tus.ac.jp
Neutral zinc porphyrin containing flexible alkyl linker, 5-[4-(5-hydroxypentyloxy)phenyl]-10,15,20-tri-p-tolyl-
porphyrinatozinc, was attached to the 5¤ ends of 20- and 30-bp oligodeoxynucleotides (ODN) by solid-phase synthesis.
Zinc porphyrin-modified double-stranded DNA (dsDNAs), which included dsDNAs with porphyrin moieties on one end
and on both ends (ZnPor-ds20, ds20-ZnPor, ZnPor-ds20-ZnPor, ZnPor-ds30, ds30-ZnPor, and ZnPor-ds30-ZnPor),
were successfully prepared and were analyzed by variable-temperature UV-vis spectroscopy and CD measurements to
elucidate the interaction behavior of the porphyrin ring. Detailed investigation revealed that the zinc porphyrin-DNA
conjugates exhibited intra-duplex porphyrin-ODN interaction in a low-salt condition and inter-duplex porphyrin-
porphyrin interaction in a high-salt condition.
Porphyrins can serve as useful probe molecules in the
structural studies on biomolecules because of their exciton
chirality.1-6 Based on the measurements of circular dichroism
(CD), porphyrins and metalloporphyrins have been employed
in conformational and configurational investigation of bio-
molecules, such as peptides,7-9 oligosaccharides,10 glycol
lipids,11 and the marine neurotoxin brevetoxin B.12-14
electron-transfer property,30 and thermal stability of duplexes.31
Modification of porphyrin moiety via flexible linker will
provide not only intra-strand interaction but also inter-strand
interaction, which can be expanded to DNA-porphyrin
assemblies. The flexible linkages can lead to exciton-coupled
chirogenesis in porphyrin-DNA systems via three possible
interactions: intra-duplex short-range exciton interaction be-
tween a porphyrin and a nucleic acid, intra-duplex long-range
exciton interaction between the terminal porphyrins, and
intermolecular exciton interaction between two porphyrins or
between a porphyrin and a nucleic acid. Neutral porphyrins are
less potential to interact with anionic DNA and favorable for
assembling with other porphyrin molecules. The DNA con-
jugate having zinc porphyrin arrays in a close distance within a
duplex studied recently.32,33 To investigate the interactions with
the zinc porphyrin both in the intra-strand and in the inter-
strand, we synthesized series of zinc porphyrin-modified ODNs
at 5¤ end which comprise 20-bp and 30-bp dsDNAs having a
porphyrin moiety attached at both ends or at each 5¤ end of the
DNA via a flexible linker (Figure 1 and Scheme 1). Herein, we
report the studies on the CD signals of the zinc porphyrin-
ODN conjugates to elucidate the type of interaction that is
dependent on salt concentration.
As the demonstration of the photosensitized cleavage of a
nucleic acid with a interacting porphyrin moiety,15-17 a variety
of positively charged porphyrins and metalloporphyrins with
bulky aryl groups at the meso position have been shown
to bind to single-18,19 and double-stranded DNA20-23 (ssDNA
and dsDNA, respectively). At the same time, some studies
demonstrated the application of CD signals of porphyrin
chromophores in carrying out conformational analyses of DNA
molecules by noncovalent-24 and covalent-modification strat-
egies.25-28 For example, a study reported the use of a zinc
porphyrin as a chiroptical probe for the identification of B- and
Z-DNA.24 Covalent modification of DNAs with phenyltrispyri-
dylporphin and their analogs by using a rigid amide linker also
enabled the sensitive detection of conformational transition of
oligodeoxynucleotides (ODNs) between B- and Z-DNA; this
detection is due to the long-range exciton-coupled CD signal
that is generated by the interaction between the two terminal
porphyrins®with the distance between them reaching up to
40-50 ¡25,26®attached to the 5¤ ends of the DNA. On the ther
hand, modification of ODNs with flexible linker has been
carried out in studies conducted on DNA cleaving activity,29
Results and Discussion
Synthesis of Zinc Porphyrin ODNs.
Zinc porphyrin
ODNs were synthesized by the scheme shown in Figure 2. Zinc
tetraarylporphyrin with C5 linker (1) was attached to the 5¤ end
of ODN by carrying out solid-support synthesis. The zinc
porphyrin was O-phosphitylated by undergoing a coupling
reaction with 2-cyanoethyl diisopropylphosphoramidochlo-
ridite, which was used immediately after purification. In
³ Present address: Frontier Research Base for Global Young
Researchers, Graduate School of Engineering, Osaka University,
2-1 Yamadaoka, Suita, Osaka 565-0871
Published on the web October 8, 2009; doi:10.1246/bcsj.82.1280

A. Onoda et al.
Bull. Chem. Soc. Jpn. Vol. 82, No. 10 (2009) 1281
N
N
N
ZnII
Zn
N
CN
Figure 1. dsDNA molecule with porphyrin moieties at 5¤
end with flexible linker.
Cl
O
P
N
CN
ZnPor-ss20T
ss20B-ZnPor
ZnPor-ss30T
ss30B-ZnPor
ZnII
O
O
O
ZnII
O
O
OH
P
N
DIEA
DCM
CN
5'
ZnII
O
P
N
O
+
HO
5'
ZnII
O
O
O
O
P
(i) 1H-tetrazole
MeCN/DCM
O
(ii) I2, pyridine/H2O/THF
(iii) NH4OH
Figure 2. Synthesis of ODN containing Zn porphyrin at 5¤
end.
ZnPor-ds20
ds20-ZnPor
the 20-bp and 30-bp ODNs by porphyrin (ZnPor-ss20T,
ZnPor-ss20B, ZnPor-ss30T, and ss30B-ZnPor), the results
of which are shown in Figure 3. Each product showed the
characteristic Soret and Q bands of zinc porphyrin moieties at
430 nm and 560 and 600 nm, respectively; the intensities of
these bands were proportional to that of the characteristic band
of ODNs at 260 nm.
ZnPor-ds20-ZnPor
Variable-Temperature UV-Vis Spectroscopy. The results
of the variable-temperature experiments suggest that the
synthesis of singly and doubly modified DNA strands with
porphyrin ring via C5 linker allows duplex formation. Figure 4
shows the variable-temperature UV-vis spectra of ZnPor-ds20,
ds20-ZnPor, and ZnPor-ds20-ZnPor. The absorption maxima
of these modified duplexes found at the same wavenumber
shift in both the nucleic acid and porphyrin region. As the
temperature increased, the band at 260 nm of the modified
duplexes was observed to exhibit a typical hyperchromic shift,
which was also observed in the spectra of porphyrin-free
dsDNA. The Soret band at around 430 nm showed only a slight
shift or increase in intensity on heating. The absorption spectra
measured after carrying out the denaturation and renaturation of
modified dsDNAs exhibited spectral features identical to those
mentioned above indicating that the zinc-porphyrin modifica-
tion did not interfere with the formation of the duplexes; this
was observed in the case of all modified dsDNAs. The Tm value
of the porphyrin-conjugated duplexes was slightly lower than
that of the unmodified duplexes. It is known that an increase in
Tm indicates an increase in the stability of the double helix,
which is attributed to the intercalation of the porphyrin
moiety.20,34 Thus, we have verified that despite its hydrophobic
nature the pendant porphyrin moiety does not directly bind
to the dsDNA via processes such as intercalation or groove
binding.
ZnPor-ds30
ds30-ZnPor
ZnPor-ds30-ZnPor
Scheme 1. Attachment of ZnPor moiety to ss- and dsDNA.
general, the corresponding phosphoramidite of a metal-free
porphyrin is easily over oxidized to yield an unreactive
phosphate. However, the phosphoramidite of zinc porphyrin is
relatively stable after purification. The phosphoramide prepared
above was coupled to the ODN on a controlled pore glass
(CPG) resin used as a solid support. After cleavage and
deprotection, the crude modified ODNs were purified by
semi-preparative reverse-phase HPLC. The final product was
characterized by matrix-assisted laser-desorption ionization
time-of-flight (MALDI-TOF) MS. The UV-vis spectroscopy of
the products confirmed the successful covalent modification of

1282 Bull. Chem. Soc. Jpn. Vol. 82, No. 10 (2009)
Zinc Porphyrin-Oligonucleotide Conjugates
a)
4
system. The CD profile of different concentrations of ZnPor-
ds20-ZnPor is shown in Figure 5. The modified duplexes
exhibited negative (245 nm) and positive (275 nm) CD bands
characteristic of B-type DNA,35 and the spectral features of
the modified duplexes were identical to those observed in
the case of the unmodified duplex. In the CD profile of the
low-concentration sample, a strong positive band at 444 nm
and relatively weak bands at around 420 nm were observed.
Spectral changes observed in the case of higher sample
concentration were analyzed by measuring difference spectra
(Figure 5b); these spectra revealed that the changes at 415 and
425 nm are induced by the inter-duplex interaction. The
relatively small change in CD bands is presumably caused by
inter-duplex porphyrin-porphyrin interaction. Such spectral
change due to the inter-duplex interaction could not be
observed in low NaCl concentration. In higher salt concen-
tration, the interaction between hydrophobic aromatic mole-
cules is known to become stronger. Thus, our ZnPor-con-
jugated DNA is thought to give inter-strand porphyrin-
porphyrin interaction.
The intra-duplex interaction was also assessed by CD
measurements in a low-salt condition as shown in Figure 6.
Each modified duplex exhibited different spectral features; the
spectra of ZnPor-ds20, ds20-ZnPor, and ZnPor-ds20-ZnPor
showed positive CD bands near 425, 436, and 444 nm,
respectively. Further, it should be noted that we used non-
palindromic ODN sequence in the synthesis of dsDNA.
Therefore, the CD results suggest that the pendant porphyrin
with C5 linker attached to the 5¤ end of the DNA exhibits a
different porphyrin-nucleotide interaction at each terminus of
the duplex. CD spectra in the Soret-band region of the singly
and doubly modified porphyrin-DNA conjugates were com-
pared against each other to estimate the interaction behavior
of the porphyrin moiety in the two types of conjugates.
Figure 6a shows the CD profiles of ZnPor-ds20, ds20-ZnPor,
and ZnPor-ds20-ZnPor, while the bottom figure shows the
CD profile of ZnPor-ds20-ZnPor along with the combined
spectrum of ZnPor-ds20 and ds20-ZnPor in Figure 6c. CD
profile of ZnPor-ds20-ZnPor showed a strong positive band at
436 nm and a weak negative band at 425 nm, which are almost
identical to the bands observed in the combined spectrum of the
two singly modified dsDNAs. These results clearly indicate
that porphyrin-ODN interaction at each terminus of the duplex
is different from each other. It has been previously reported
that some porphyrin chromophores that are fixed rigidly on
both termini of the dsDNA exhibit long-range porphyrin-
porphyrin exciton interaction.25,26 However, in the case of our
modified dsDNAs, the zinc porphyrins attached to the DNA via
flexible linkers do not exhibit porphyrin-porphyrin exciton
interaction but exhibit interaction with nucleotides localized at
the termini.
3.5
3
2.5
2
1.5
1
0.5
0
200
300
400
500
600
700
Wavelength / nm
b)
4
3.5
3
2.5
2
1.5
1
0.5
0
200
300
400
500
600
700
Wavelength / nm
Figure 3. UV-vis spectra of (a) ZnPor-ss20T (blue) and
ss20B-ZnPor (red) and (b) ZnPor-ss30T (blue) and
ss30B-ZnPor (red) in aqueous solution.
The remarkable feature in our ZnPor-conjugated duplex is a
gradual increase of OD260 during the melting profiles. The
ds20 with similar DNA sequence gave a typical transition
around Tm (44.5) in 0 mM NaCl. In contrast, ZnPor-ds20
and ds20-ZnPor showed a smooth increase which is more
prominent in doubly modified ZnPor-ds20-ZnPor. This trend
is more obvious in the higher salt concentration, 100 mM NaCl
(Figure S4). The UV-vis analyses for the modified DNAs did
not indicate that the zinc porphyrins were not involved in the
strong interaction between the nucleobases. Weak interactions
with the zinc porphyrin moieties were investigated by the
following CD analyses.
CD Studies on Porphyrin-Conjugated dsDNAs.
By
carrying out CD experiments, we can elucidate the structural
aspects of DNA strands and their electronic interaction with
the porphyrin moiety. The appearance of CD bands in the
Soret-band region indicates the existence of electronic inter-
action of the porphyrin chromophore with other chromo-
phores, such as nucleobases or the other porphyrin ring. First,
we performed CD measurements on different concentrations
of the DNA conjugates and found that this electronic
interaction originates mainly from intra-duplex interaction in
a low-salt condition and that the effect of inter-duplex
interaction is also apparent in a high-salt condition on our
Variable-temperature CD experiments with porphyrin-con-
jugated 20-bp and 30-bp duplexes, ZnPor-ds20-ZnPor and
ZnPor-ds30-ZnPor, in the high-salt condition are shown in
Figure 7. The CD bands at 415 and 425 nm assigned to the
intermolecular ZnPor/ZnPor interactions are more prominent
in ZnPor-ds30-ZnPor. The results indicated that the longer
double-stranded DNA is more advantageous for the inter-
molecular ZnPor/ZnPor interaction at both termini.

A. Onoda et al.
Bull. Chem. Soc. Jpn. Vol. 82, No. 10 (2009) 1283
a)
b)
c)
d)
ZnPor-ds20
ds20-ZnPor
ZnPor-ds20-ZnPor
1
0.8
0.6
0.4
0.2
0
1
0.8
0.6
0.4
0.2
0
1
0.8
0.6
0.4
0.2
0
1
0.8
0.6
0.4
0.2
0
DNA
DNA
DNA
DNA
220
240
260
280
300
320
220 240 260 280 300 320
220 240 260 280 300 320
220
240
260
280
300
320
Wavelength / nm
Wavelength / nm
Wavelength / nm
Wavelength / nm
1
0.8
0.6
0.4
0.2
0
1
1
0.8
0.6
0.4
0.2
0
ZnPor
ZnPor
ZnPor
0.8
0.6
0.4
0.2
0
380 400 420 440 460 480
380
400
420
440
460
480
380
400
420
440
460
480
Wavelength / nm
Wavelength / nm
Wavelength / nm
Figure 4. Variable-temperature absorption spectra of (a) ZnPor-ds20, (b) ds20-ZnPor, (c) ZnPor-ds20-ZnPor, and (d) ds20. The
spectra were measured for 2 ¯M samples in 10 mM Tris-HCl solutions at 20, 30, 40, 50, 60, 70, and 80 °C.
and dissolved in CHCl3 for purification by using a silica gel
column (CHCl3:hexane = 7:3); this purification process was
Conclusion
repeated twice. The collected fraction was dried in vacuo to
obtained the final product. Yield, 3.5 g (4.1%). H NMR (CDCl3,
We have synthesized zinc porphyrin-DNA conjugates using
flexible alkyl linker and analyzed the interaction behavior of
the porphyrin moieties in these conjugates by carrying out UV-
vis spectroscopy and CD measurements. The results of the
detailed investigation suggested that the neutral zinc porphyrin
attached to the 5¤ end of dsDNA exhibited intra-duplex zinc
porphyrin-ODN interaction under low-salt condition and inter-
duplex zinc porphyrin/zinc porphyrin under high-salt condi-
tion. The intermolecular interaction of our ZnPor-conjugated
DNA led to form an aggregated species insoluble to an aqueous
solution. The strategy attaching a neutral zinc porphyrin moiety
with flexible linker provides responsive behavior in the pattern
of the interaction between the porphyrins and the DNA
duplexes.
1
500 MHz): ¤ 8.85-8.83 (8H, m, ¢-pyrrole), 8.21 (2H, d, J = 8.5
Hz, ArH), 8.1 (6H, d, J = 7.5 Hz, ArH), 7.55 (6H, d, J = 7.5 Hz,
ArH), 7.48 (2H, d, J = 8.5 Hz, ArH), 2.78 (2H, q, J = 7.6 Hz,
CH2), 2.70 (9H, s, CH3), 1.41 (3H, t, J = 8.0 Hz, CH3), ¹2.78 (2H,
s, NH). MALDI-TOF MS m/z calcd for C50H41N4O2 [M + H+]:
729.32, found 728.97.
5-[4-(5-Hydroxypentyloxy)phenyl]-10,15,20-tri-p-tolylporphin
(H2por-C5H10OH).
H2por-OCOEt (400 mg, 0.56 mmol) was
dissolved in 12 mL of DMF added to NaOH (720 mg, 17 mmol),
and the solution was stirred for 2 h at room temperature. This
¹1
mixture was added to 5-bromopentyl acetate (144 ¯L, 9.2 © 10
mmol), and the solution was stirred for 4 h. This mixed solution
was neutralized by adding 1 M HCl to obtain the product, H2Por-
C5H10OCOMe, which was extracted using CHCl3. The organic
layer was washed with aqueous NaHCO3 solution and then with
water, and the collected filtrate was evaporated to dryness. The
residue was dissolved in 22 mL of DMF and hydrolyzed by adding
5 M NaOH. The reaction mixture was neutralized, the product
was extracted with CHCl3, and the organic phase was washed
with water. The product was purified by column chromatography
(CHCl3:MeOH = 95:5) and recrystallized by CHCl3-hexane sol-
vent mixture to give H2por-C5H10OH. Yield, 419 mg (98%).
1H NMR (CDCl3, 500 MHz): ¤ 8.85-8.83 (8H, m, ¢-pyrrole),
8.10-8.07 (8H, m, ArH), 7.55 (6H, d, J = 7.5 Hz, ArH), 7.21 (2H,
d, J = 7.5 Hz, ArH), 4.26 (2H, t, J = 6.4 Hz, CH2), 3.78 (2H, td,
J = 6.4, 5.0 Hz, CH2), 2.70 (9H, s, CH3), 2.01 (2H, tt, J = 6.4,
7.5 Hz, CH2), 1.81-1.69 (4H, m, CH2), ¹2.78 (s, 2H, NH).
MALDI-TOF MS m/z calcd for C52H47N4O2 [M + H+]: 759.37,
found 758.07.
Experimental
Materials. All solvents were distilled over appropriate drying
agents and degassed prior to use. All starting reagents used were
of commercial grade. H2por-OCOEt (5-[4-(propionyloxy)phenyl]-
10,15,20-tri-p-tolylporphin) was synthesized by following
previously reported procedure with some modifications.36
a
5-[4-(Propionyloxy)phenyl]-10,15,20-tri-p-tolylporphin (H2por-
OCOEt). 4-Propionyloxybenzaldehyde (10.0 g, 56.2 mmol), p-
tolualdehyde (21.8 g, 181 mmol), and pyrrole (15.0 mL, 217 mmol)
were dissolved in 44 mL of propionic acid and 22.4 mL of acetic
anhydride. The mixture was refluxed for 2 h untill the solution
turned dark purple in color. The reaction mixture was cooled to
room temperature and concentrated to a volume of approximately
200 mL. The solution was made to stand overnight at ¹15 °C to
afford crystalline precipitates. These precipitates were collected

1284 Bull. Chem. Soc. Jpn. Vol. 82, No. 10 (2009)
Zinc Porphyrin-Oligonucleotide Conjugates
a)
b)
4
3
0.6
0.4
0.2
0
417 nm
0.3
DNA
0.2
0.1
450 nm
0
2
-0.1
432 nm
-0.2
1
-0.3
0
5
10
∆ µM
15
0
-1
-2
-3
-4
-0.2
-0.4
220
240
260
280
300
320
350
400
450
500
Wavelength / nm
Wavelength / nm
4
3
0.6
0.4
0.2
0
ZnPor
2
1
0
-1
-2
-3
-4
-0.2
-0.4
350
400
450
500
350
400
450
500
Wavelength / nm
Wavelength / nm
Figure 5. (a) Concentration-dependent CD spectra of ZnPor-ds20-ZnPor in 10 mM Tris-HCl, 1 mM EDTA, and 100 mM NaCl
solutions. The spectra were measured at sample concentrations of 10, 15, 20, 25, and 30 ¯M. Top; 220-320 nm, Bottom; 350-
500 nm. (b) Difference spectra at each sample concentration of 10, 15, 20, 25, and 30 ¯M. 10 mM Tris-HCl, 1 mM EDTA, and
100 mM NaCl solutions (top) and 10 mM Tris-HCl (bottom). The inset shows the intensity profile at 417, 432, and 450 nm.
5-[4-(5-Hydroxypentyloxy)phenyl]-10,15,20-tri-p-tolylpor-
phininatozinc (Znpor-C5H10OH) (1). H2por-OH (100 mg,
(hexane:ethyl acetate = 1:1). During this chromatography, the
solution was concentrated and charged into a silica gel column,
which was eluted with 5% TEA in the hexane-ethyl acetate solvent.
The purified band was evaporated to give a bluish purple powder of
ZnPor phosphoramidite. The purified product was used immedi-
ately in the solid-phase coupling reaction. 31P NMR (CDCl3):
0.13 mmol) and Zn(acac)2 (115 mg, 4.4 mmol) were dissolved
in 12 mL of CHCl3-MeOH solvent (CHCl3:MeOH = 9:1). The
reaction mixture was stirred for 1 h. Then, the reaction solution
was washed with water, and the organic layer was collected and
evaporated to dryness. The residue was purified by using a silica
gel column (CHCl3:MeOH = 95:5) and recrystallized to give blue
purple powder of 1. Yield, 90 mg (83%). The successful incorpo-
ration of Zn2+ ion in the porphyrin ring was verified by absorption
spectroscopy. 1H NMR (CDCl3, 500 MHz): ¤ 8.88-8.67 (8H, m, ¢-
pyrrole), 8.04-8.01 (8H, m, ArH), 7.47 (6H, d, J = 7.6 Hz, ArH),
7.21 (2H, d, J = 6.7 Hz, ArH), 4.20 (2H, t, J = 6.4 Hz, CH2), 3.63
(2H, td, J = 6.4, 5.0 Hz, CH2), 2.64 (9H, s, CH3), 1.94 (2H, tt,
J = 6.4, 7.5 Hz, CH2), 1.69-1.60 (4H, m, CH2). MALDI-TOF MS
m/z calcd for C52H44N4O2Zn [M + H+]: 821.28, found 821.10.
Znpor Phosphoramidite. 2-Cyanoethyl diisopropylphosphor-
amidochloridite (0.5 M in CH2Cl2) (24 ¯L, 0.0012 mmol) and
DIEA (N,N-diisopropylethylamine) (3.9 ¯L, 0.0024 mmol) were
dissolved in 0.5 mL of CH2Cl2, and after degassing, the solution
was mixed with Znpor-C5H10OH (1) (5.0 mg, 0.0061 mmol). The
reaction mixture was stirred for 1 h in Ar atmosphere protected
form light. The reaction was monitored by TLC (silica gel)
using 5% triethylamine (TEA) in hexane-ethyl acetate solvent
1
¤ 147.4 ppm. H NMR (CDCl3, 500 MHz): ¤ 8.88-8.85 (8H, m,
¢-pyrrole), 8.03 (2H, d, J = 8.7 Hz, ArH), 8.02 (6H, d, J = 7.6 Hz,
ArH), 7.47 (6H, d, J = 7.6 Hz, ArH), 7.27 (2H, d, J = 8.7 Hz, ArH),
4.19 (2H, t, J = 6.4 Hz, CH2), 3.87-3.60 (4H, m, CH2), 3.60-3.54
(2H, m, iPr-CH), 2.63 (9H, s, CH3), 2.60 (2H, t, J = 6.4 Hz, CH2),
1.95 (2H, tt, J = 6.4, 7.8 Hz, CH2), 1.78-1.72 (2H, m, CH2), 1.69-
1.64 (2H, m, CH2), 1.18-1.14 (12H, m, iPr-CH3).
Synthesis of Porphyrin-Modified ODNs. To the ODN, which
was supported on the CPG resin, ca. 4 mM CH2Cl2 solution
of porphyrin phosphoramidite (300 ¯L) was added with 0.1 M
acetonitrile solution of tetrazole (200 ¯L) in Ar atmosphere. The
reaction vessel was stirred for 2 h in the absence of light at room
temperature. This procedure was repeated twice. Then, the mixture
was washed with CH2Cl2 and acetonitrile five times, and 21 mL of
the oxidizing agent I2 (0.15 M, H2O:pyridine:THF = 10:10:1) was
added to the solution and the mixture was stirred for 2 min. The
solution was filtered and the solid support was washed with
acetonitrile five times. The porphyrin-modified resin was dissolved

A. Onoda et al.
Bull. Chem. Soc. Jpn. Vol. 82, No. 10 (2009) 1285
a)
4
ZnPor-ds20-ZnPor
a)
15
ZnPor-ds20
3
2
ds20-ZnPor
10
5
ZnPor-ds20-ZnPor
1
0
0
-1
-2
-3
-5
-10
200 250 300 350 400 450 500
250
300
350
400
450
500
Wavelength / nm
Wavelength / nm
b)
1
ZnPor-ds30-ZnPor
b)
15
10
5
ZnPor-ds20-ZnPor
0.5
0
ZnPor-ds20
ds20-ZnPor
0
-5
-0.5
-10
350
400
450
500
250
300
350
400
450
500
Wavelength / nm
Wavelength / nm
c)
1
0.5
0
Figure 7. Variable-temperature CD spectra of (a) ZnPor-
ds20-ZnPor and (b) ZnPor-ds30-ZnPor. The spectra were
measured for 20 ¯M samples in 10 mM Tris-HCl, 1 mM
EDTA, and 100 mM NaCl solutions at 20, 30, 40, 50, 60,
70, and 80 °C.
ZnPor-ds20-ZnPor
tetraethylammonium acetate (TEAA) (1:4-4:1).
MALDI-TOF MS. ZnIIPor-ss20T, found 7080, calcd 7069;
ss20B-ZnIIPor, found 6897, calcd 6891; ZnIIPor-ss30T, found
10272, calcd 10262 (+7Na); ss30B-ZnIIPor, found 10033, calcd
10041 (+2Na). Negative ion mode with CHCA matrix.
ZnPor-ds20 + ds20-ZnPor
Physical Measurements. The 1H NMR spectra were measured
using a JEOL JNM-LA500 or Bruker AVANCE 600 spectrometer.
The 31P NMR spectra were measured using the JEOL JNM-
-0.5
350
400
450
500
1
Wavelength / nm
LA500 spectrometer. H NMR spectra were measured using TMS
as an internal reference standard, and 31P spectra were measured
using 85% aqueous H3PO4 as an external reference standard.
The UV-vis spectra were recorded on a Shimadzu UV-2200A
spectrophotometer. The UV melting experiments were carried
out using a Beckman Coulter DU 800 spectrophotometer. The
fluorescence spectra were recorded on a Shimadzu RF5300-PC
fluorescence spectrometer. The CD spectra were measured on a
Jasco J-725 spectropolarimeter. The MALDI-TOF MS experi-
ments were performed using PerSpective Biosystems Voyager
LinerRD VDA-500, and the electrospray ionization time-of-
flight (ESI-TOF) MS experiments were performed using Bruker
micrOTOF spectrometer.
Figure 6. (a) and (b) CD spectra of ZnPor-ds20-ZnPor
(black), ZnPor-ds20 (blue), and ds20-ZnPor (red) in
10 mM Tris-HCl solution. (c) The spectrum of ZnPor-
ds20-ZnPor along with the combined spectrum of ZnPor-
ds20 and ds20-ZnPor (orange).
in 5 mL of 28% ammonia solution, and the mixture was stirred for
15 h at 55 °C. The reaction mixture was concentrated to dryness,
and the obtained residue was dissolved in H2O, while the cleaved
solid support was filtered off. The products were purified on C18
reverse-phase HPLC column with a gradient of acetonitrile:0.1 M

1286 Bull. Chem. Soc. Jpn. Vol. 82, No. 10 (2009)
Zinc Porphyrin-Oligonucleotide Conjugates
1315.
We greatly acknowledge Prof. Hidetaka Torigoe for permit-
ting us to carry out UV melting experiments. This study
was supported by a Grant-in-Aid for Scientific Research
(No. 1835034 to T.Y. and 16750148 to A.O.) from the Ministry
of Education, Culture, Sports, Science and Technology, Japan.
16 D. R. McMillin, K. M. McNett, Chem. Rev. 1998, 98, 1201.
17 S. Mettath, B. R. Munson, R. K. Pandey, Bioconjugate
Chem. 1999, 10, 94.
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Supporting Information
1H and 31P NMR spectra of ZnPor-C5H10OH phosphoramide.
MALDI-TOF MS, UV-vis, CD, and fluorescence spectra. This
material is available free of charge on the web at: http://
www.csj.jp/journals/bcsj/.
21 R. J. Fiel, B. R. Munson, Nucleic Acids Res. 1980, 8, 2835.
22 R. F. Pasternack, E. J. Gibbs, J. J. Villafranca, Biochemistry
1983, 22, 2406.
23 M. A. Sari, J. P. Battioni, D. Dupré, D. Mansuy, J. B. Le
Pecq, Biochemistry 1990, 29, 4205.
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