Synthesis and nucleic acid-binding properties of water-soluble porphyrins appending platinum(II) complexes

Article abstract of DOI:10.1248/cpb.49.1573

We synthesized two water-soluble porphyrins appending platinum(II) complexes |α,β(4a) and α,α-(4b) 5,15-bis(2-trans-[PtCl(NH₃)₂| N-2-aminoethylaminocarbonylphenyl) 2,3,7,8,12,13,17,18-octamethylporphyrin] and studied their reactions

Full text of DOI:10.1248/cpb.49.1573

December 2001
Chem. Pharm. Bull. 49(12) 1573—1580 (2001)
1573
Synthesis and Nucleic Acid-Binding Properties of Water-Soluble
Porphyrins Appending Platinum(II) Complexes
Hiroki MUNAKATA,* Toshiki KANZAKI, Shigeo NAKAGAWA, Hiroyasu IMAI, and Yoshio UEMORI
Faculty of Pharmaceutical Sciences, Hokuriku University, Ho-3, Kanagawa-machi, Kanazawa 920–1181, Japan.
Received July 27, 2001; accepted September 17, 2001
We synthesized two water-soluble porphyrins appending platinum(II) complexes [a,b-(4a) and a,a-(4b)
5,15-bis(2-trans-[PtCl(NH3)2]N-2-aminoethylaminocarbonylphenyl) 2,3,7,8,12,13,17,18-octamethylporphyrin] and
studied their reactions with a variety of nucleic acids [disodium adenosine-5؅-monophosphate (AMP), disodium
guanosine-5؅-monophosphate (GMP), disodium thymidine-5؅-monophosphate (TMP), disodium cytidine-5؅-
monophosphate (CMP), synthetic polymer poly(dG)–poly(dC), poly(dA)–poly(dT)] by 1H-NMR, UV–vis and
FAB-MS spectroscopies. Based on the denaturation experiments of synthetic nucleic acid polymers, we conclude
that the presence of the porphyrins (5.6 mM) does not cause significant changes in the melting temperature of
poly(dA)–poly(dT) (28 mM) (DT؍1 °C) and shows reannealing. On the other hand, gradual melting of
poly(dG)–poly(dC) (28 mM) occurs at a low temperature (DT؍؊27 °C) in the presence of the porphyrins (5.6 mM),
and the solutions do not show reannealing phenomena. The results of UV–vis and 1H-NMR experiments revealed
that the porphyrins bind to guanine bases and that the porphyrins bind to GMP more strongly than to the other
nucleotides. The binding modes between the porphyrins and synthetic nucleic acids are affected more by the co-
ordination of the nucleobase [poly(dG)–poly(dC)] to the Pt(II) in the porphyrins than by Coulomb and hy-
drophobic interactions.
Key words porphyrin; platinum complex; nucleic acid
There has been considerable interest in understanding the cleic acids [disodium adenosine-5Ј-monophosphate (AMP),
binding of the antitumor drug cis-dichlorodiammine plat- disodium guanosine-5Ј-monophosphate (GMP), disodium
inum(II) (CDDP) and its analogs to their putative target DNA thymine
-5Ј-monophosphate (TMP), disodium cytidine-5Ј-
in cancer cells.1) Platinum complexes can bind rapidly to nu- monophosphate (CMP), synthetic polymer poly(dG)–
1
cleic acids (including DNA) in aqueous solution and there- poly(dC), poly(dA)–poly(dT)] using H-NMR, UV–vis or
fore are useful as a functional group to design artificial re- FAB-MS spectroscopies.
ceptors for DNA. The anti-cancer activity of CDDP arises
from its ability to damage DNA by forming a major adduct Results and Discussion
with intrastrand d(GpG) and d(ApG) crosslinks.2) These
Syntheses and Characterization of Water-Soluble Por-
crosslinks of CDDP to d(GpG) and d(ApG) bend and unwind phyrins Appending Platinum(II) Complexes or Quater-
the duplex, and then the altered structure attracts high-mobil- nary Ammonium Groups Syntheses of the water-soluble
ity-group (HMG) domain proteins.3) This binding of HMG- porphyrins with two platinum(II) complexes are outlined in
domain proteins to CDDP-modified DNA has been postu- Fig. 1. Condensation of (tetramethyldipyrryl)methane7) with
lated to mediate the antitumor properties of the drugs.4)
o-phthalaldehydic acid gave porphyrinogen 1 in a good yield.
Studies on the binding of cationic porphyrins to DNA have Oxidation of the porphyrinogen to porphyrin 2 was accom-
been of great significance to those working in cancer re- plished by using o-chloranil. Because of the limited solubil-
search and gene technology.5) Some cationic porphyrins bear- ity of 2, the chromatographic separation of the two atropiso-
ing quaternary ammonium salt units such as ammonium or mers could not be accomplished; it was achieved after deriv-
pyridinium groups can act as binders for DNA. Several re- ing to aminoethylcarbamides 3a and 3b. Compound 3b was
searchers have investigated how cationic porphyrins bind to hydorolyzed with hydrochloric acid (6 M), and a treatment of
DNA in groove or outside binding on the basis of Coulomb the hydrolyzed product with 1,5-diaminopentane gave
and/or hydrophobic interactions.5a,c,6) We designed and strapped porphyrin 8 (Fig. 2), where the 1,5-diaminopentane
synthesized two water-soluble porphyrins appending plat- group is found to be strapping over the porphyrin,8) judging
1
inum(II) complexes and studied their reactions with a variety from the H-NMR data. The formation of the strapped por-
of DNA and nucleic acids. In the porphyrins, cationic groups phyrin indicates that 3b is assigned to be the a,a-isomer.9)
of the appended platinum(II) complex solubilize the por- The expected polarity of the porphyrins, which is that 3b is
phyrins to water and provide coordination ability for binding more polar than 3a, also agrees with the order of the Rf val-
to DNA. Furthermore, because the cationic porphyrins can ues of TLC (see Experimental section). We used trans-
also bind to DNA, the platinum(II) complexes appended by [PtCl(NH3)2(N,N-dimethylformamide (DMF))]NO3 as the
the porphyrins are expected to function additively with the appending platinum(II) complexes to 3a and 3b. The counter
porphyrins and then bind strongly to DNA.
ions of the porphyrins appending platinum(II) complexes
This report describes the synthesis of two derivatives of (4a, b) were exchanged from nitrates to chlorides by passing
5,15-bis(o-substituted phenyl)porphyrin appending two them through an ion-exchange column in the ClϪ form.
chlorodiammineplatinum(II) complexes at the ortho position
For comparison, we prepared two cationic porphyrins
of the two phenyl rings of the porphyrin (see 4a and 4b in bearing two quaternary ammonium salts a,b-(7a) and a,a-
Fig. 1). We then studied their reactions with a variety of nu- (7b) 5,15-bis((o-N,N,N-trimetylammoniummethylamido)-
To whom correspondence should be addressed. e-mail: h-munakata@hokuriku-u.ac.jp
© 2001 Pharmaceutical Society of Japan

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Vol. 49, No. 12
Fig. 1. Synthetic Route of Porphyrins Appending Platinum(II) Complexes
(a) o-Phthalaldehyde, p-toluenesulfonic acid, methanol; (b) (i) o-chloranil, tetrahydrofuran, (ii) triethylamine, methanol; (c) (i) oxalyl chloride, dichloromethane, (ii) ethylenedi-
amine, dichloromethane, (iii) silicagel column chlomatography; (d), (e) (i) trans-[PtCl(DMF)(NH3)2], DMF, (ii) anion-excange regin.
dimerized or aggregated in H2O. We also examined the tem-
perature dependence of the absorption spectrum for 4a in
aqueous solution (20 mM NaCl) (Fig. 6). Temperature rising
caused spectral changes with a blue shift of the Soret band,
and these spectral changes were reversible. The W1/2 of a
Soret band in this condition (20—60 °C, 20 mM NaCl) has a
Fig. 2. Structure of Strapped Porphyrin
large W1/2 compared with the value of the porphyrins in DMF
(monomer form). These results suggest that the synthesized
water-soluble porphyrins were also dimerized or aggregated
in 20 mM NaCl at the temperature range from 20 to 60 °C.
Melting and Reannealing Behavior of Synthetic Nu-
cleic Acid Polymers We studied the interactions between
porphyrins and synthetic nucleic acid polymers by measuring
the melting temperatures and observing the reannealing be-
havior. The reannealing phenomenon is what occurs when a
warmed solution containing denaturated duplexes is slowly
cooled to room temperature and the UV–vis spectrum of the
solution becomes the same as it was before denaturation. Be-
fore measuring the melting temperatures of the synthetic nu-
cleic acid polymer, the solutions containing the porphyrins
must be equilibrated at 20 °C. Figure 7 represents the time-
courses of the UV–vis spectra for the solution containing 4a
and poly(dG)–poly(dC) in 20 mM NaCl at 20 °C. The Soret
maximum at 425 nm of 4a shows a basochromic shift to
440 nm just after the mixing of 4a with poly(dG)–poly(dC).
Subsequently, a blue hyperchromic shift of the Soret maxi-
mum occurs gradually, and the spectrum reaches a steady
state after 15 h. The first changes were observed in the cases
of 4b or 7a, whereas the second change was not observed in
7a. Since such a gradual change in UV–vis spectra has been
accompanied by outside, groove, and/or intercalative bind-
ing, the binding interactions of 4a and 4b with poly(dG)–
poly(dC) may be different from that of 7a.
phenyl)-2,3,7,8,12,13,17,18-octamethylporphyrin (Fig. 3) from
5a and 5b, respectively. The precursors (6a, b) prepared from
5a and 5b with N-dimethylglycine were methylated by
methyl iodide to give 7a and 7b, respectively.
Some water-soluble porphyrins have a tendency to form
intermolecular aggregates in aqueous solution.10) The bind-
ing of the porphyrins to nucleic acids is often complicated by
the aggregation. UV–vis spectroscopy is a prominent and
simple tool that allows one to gain information on the aggre-
gates. It has been reported that porphyrin aggregates gener-
ally have large W1/2 (band width at half-height peak) of the
Soret band and that the aggregates are more stable in water
than in several organic solvents, such as DMF, dimethyl sul-
foxide (DMSO), MeCN or MeOH.10) The spectral data for
the Soret band of the porphyrins 4a, 4b, 7a and 7b in aque-
ous solution are given in Tables 1 and 2. The UV–vis spectral
changes of 4a at various concentrations in H2O are shown in
Fig. 4. The spectra for these porphyrins in the concentration
range from 0.17 to 10 mM do not obey Beer’s law in H2O at
25 °C (Table 2). Figure 5 represents the UV–vis spectra of 4a
in H2O, MeOH and DMF. Each porphyrin has a Soret band
with a large W1/2 in H2O, while in DMF each has a Soret
band with small W1/2 as shown in Table 3. These results sug-
gest that the synthesized water-soluble porphyrins were

December 2001
1575
Fig. 3. Synthetic Route of Porphyrins Appending Platinum(II) Complexes
(a), (c) (i) N,N-dimethylaminoacetic acid, oxalyl chloride, dichloromethane, (ii) dichloromethane, (iii) silica-gel column chlomatography; (b), (d) (i) iodomethane, dimethylfor-
mamide, (ii) ainion excange regin.
Table 1. UV–Vis Spectral Data for Porphyrins
Prophyrins
l
max, nm (log e)a)
W1/2
4ab)
4bb)
7ac)
7bc)
408.5 (5.03)
405.5 (4.98)
404.0 (5.20)
402.0 (4.99)
40
48
39
63
a) e (MϪ1 cmϪ1). b) Concentration of porphyrins is 8 mM at 25 °C. c) Concentra-
tion of porphyrins is 5 mM at 25 °C.
Table 2. UV–Vis Spectral Data for Porphyrinsa)
Porphyrins
Conc./mM
l
max, nm (log e)b)
4a
0.17
0.35
0.50
1.0
5.0
10
0.17
0.35
0.50
1.0
5.2
10
0.5
1.0
2.1
7.0
0.5
1.0
2.1
7.0
410 (sh)
423.5 (4.90)
420 (sh)
420 (sh)
Fig. 4. Spectral Changes of 4a at Various Concentrations in H2O
410.5 (4.88)
410.0 (4.89)
409.0 (4.81)
409.0 (4.93)
409.0 (5.00)
410 (sh)
408.5 (4.93)
408.5 (4.87)
407.0 (4.81)
404.0 (4.88)
401.0 (4.91)
406.0 (5.01)
404.0 (5.10)
403.0 (5.17)
402.5 (5.23)
1, 2, 3, 4, 5 and 6 are the spectra at 0.17, 0.35, 0.50, 1.0, 2.7 and 5.0 mM, respectively.
4b
423.0 (5.09)
427.0 (5.14)
7a
7b
427.0 (4.88)
426.5 (5.07)
426.5 (5.11)
426.5 (5.23)
a) In aqueous solution at 25 °C. b) e (MϪ1 cmϪ1).
The melting temperatures for poly(dA)–poly(dT) in the
presence of 4a, 4b and 7a are listed in Table 4. The melting
curves for poly(dG)–poly(dC) in the presence of 4a, 4b and
7a are also shown in Fig. 8. The presence of the porphyrins
——
— —
), MeOH ( ), and
Fig. 5. UV–Vis Spectra of 4a (8 mM) in DMF (
H2O (------) at 25 °C
·

1576
Vol. 49, No. 12
Table 3. UV–Vis Spectral Data for Porphyrins in Various Solventsa)
4ab)
4bb)
7ac)
7bc)
Solvent
l
max (nm)
W1/2 (nm)
l
max (nm)
W1/2 (nm)
l
max (nm)
W1/2 (nm)
l
max (nm)
W1/2 (nm)
H2O
MeOH
DMF
40.8.5
408.5
411.0
38
32
28
406.0
408.0
411.0
52
32
28
403.0
403.0
409.5
33
35
31
400.0
403.5
409.0
51
36
32
a) At 25 °C. b) Porphyrin concentration is 8 mM. c) Porphyrin concentration is 6 mM.
Table 4. Melting Temperatures (°C)a) of Poly(dA)–Poly(dT)b) in the Pres-
ence of Porphyrins
N/Pc)
7a
4a
4b
f)
10d)
5e)
61
58
55
57
56
a) The estimated errors in melting temperatures were Ϯ1 °C. b) Concentration of
poly(dA)–poly(dT) is 28 mM in H2O (NaClϭ20 mM). The melting temperature in the ab-
sence of porphyrin is 56 °C. c) Ratio of poly(dA)–poly(dT) concentration (in base
pairs) to porphyrin concentration. d) Concentrations of porphyrins are 2.8 mM. e)
Concentrations of porphyrins are 5.6 mM. f) Melting temperature was not determined.
Fig. 8. Melting Profiles at 260 nm of Poly(dG)–Poly(dC) (28 mM in Base
Pair Concentration) in the Presence of Porphyrins (5.6 mM) in 20 mM NaCl at
260 nm
A is the observed absorbance, AMIN is the absorbance of duplex poly(dG)–poly(dC)
and AMAX is the absorbance of denaturated duplex poly(dG)–poly(dC). Porphyrin:
——
(
—
—
— —
— —
) 4b, ( ᭺ ) 7a.
) non, (
᭹
) 4a, (
ϫ
did not cause a significant change in the melting temperature
of poly(dA)–poly(dT), and reannealing was observed with
and without the porphyrins. Since groove or outside binding
should not affect the melting temperature or UV–vis spectral
behavior,6) the binding of 7a to both poly(dA)–poly(dT) and
poly(dG)–poly(dC) will be in these binding modes. Further,
the binding modes of 4a and 4b with poly(dA)–poly(dT) will
be similar to that of 7a, and these binding modes are affected
by Coulomb and hydrophobic interaction. Contrary to this,
gradual melting of poly(dG)–poly(dC) occurs at low temper-
atures (ca. 65 °C) in the presence of 4a and 4b, and these so-
lutions do not show reannealing. The destabilization of the
poly(dG)–poly(dC) by 4a and 4b, together with the spectral
changes of the solution at 20 °C (Fig. 7), suggests that the
binding mode for 4a, 4b to poly(dG)–poly(dC) should be dif-
ferent from that for 7a to poly(dG)–poly(dC). To explore the
binding mode of 4a and 4b to poly(dG)–poly(dC) polymers,
we measured the UV–vis spectra of the porphyrins with four
nucleotides (GMP, AMP, CMP, TMP).
Fig. 6. Spectral Changes of 4a (8 mM) at Various Temperatures in H2O
(20 mM NaCl)
1, 2, 3, 4 and 5 are the spectra at 20, 30, 40, 50 and 60 °C, respectively.
Reactions of Porphyrins with Nucleotides The time-
course of the reaction between 4a and GMP is shown in Fig.
9. As in the case of 4a with poly(dG)–poly(dC), the spectral
changes consist of two steps. By adding GMP to the solution
containing 4a, a change of the first step occurs immediately
(the spectral change between spectrum 0 and 1 in Fig. 9).
After the first step, a gradual change is observed as the sec-
ond step over a period of 10 h (the spectral changes between
spectrum 1 and 7 in Fig. 9). The first step is also observed in
all cases of the porphyrins with the four nucleotides. The
spectral change in the first step will be due to the dissociation
of a porphyrin dimmer to monomers11) and subsequently to
Fig. 7. Time-Dependent UV–Vis Spectra of the Reaction between 4a
(5.6 mM) and Poly(dG)–Poly(dC) (28 mM) at 20 °C in 20 mM NaCl
1 is the spectrum of 4a. 2, 3, 4, 5, and 6 are the spectra at 0, 2, 8, 16, and 20 h, re-
spectively, after mixing of 4a with Poly(dG)–poly(dC).

December 2001
1577
Fig. 11. Possible Structures of the Guanine Base Adducts 4a
for H-8 and H-1Ј protons for guanosine appeared at 8.63 and
5.93 ppm, respectively. Subsequently, the signals were con-
verted to 8.79 ppm for H-8 and to 5.70 and 5.97 ppm for H-
1Ј, respectively. These changes can be explained by succes-
sive binding of two guanosine molecules to 4a and not by
non-bonding association of guanosine with 4a, as observed
for many aromatic compounds with porphyrins, for the fol-
lowing reasons: (1) The association reaction of aromatic
compounds with porphyrins reaches to equilibrium within a
few minutes,13) but the observed changes are continued over
a period of 14 d, and (2) the proton signals for the aromatic
compounds shift to a high field by the ring current upon as-
sociation,14) but the H-8 proton signal for guanosine shifts to
a low field.
Fig. 9. Time-Dependent UV–Vis Spectra of the Reaction between 4a
(7.8 mM) and GMP (19 mM) at 40 °C in 10% MeOH
0 is the spectrum of 4a. 1, 2, 3, 4, 5, 6, and 7 are the spectra at 0, 2, 4, 6, 8, 10, and
12 h, respectively, after mixing of 4a with GMP.
FAB-MS spectra provide information on the products
formed in the solution. The spectrum for the DMF solution
containing 4a and guanosine shows peaks at m/zϭ1852 and
1656, which correspond to the formation of a bis-guanosine
adduct to 4a (ϭ2 guanosineϩ4aϪ2Cl) and a mono-guano-
sine adduct to 4a (ϭguanosineϩ4aϪCl), respectively. This
result also supports the successive binding of two guanosine
molecules to 4a. The binding site of guanosine to 4a is tenta-
tively assignable to N-7, in analogy with the reaction of
trans-[Pt(NH3)2Cl2] with guanosine,12) and a proposed struc-
ture is shown in Fig. 11. The spectrum for the solution con-
taining 4a and adenosine does not show any peak corre-
sponding to a mono- or bis-adenosine adduct to 4a. These re-
sults indicate that guanosine coordinates to Pt(II) but adeno-
sine does not coordinate and that formation of the coordina-
tion bond can be detected by the FAB-MS spectra. On the
other hand the FAB-MS spectrum for the solution containing
4b and guanosine shows a peak corresponding to mono-
guanosine adduct of 4b (ϭguanosineϩ4bϪCl, FAB-MS m/z:
Fig. 10. 1H-NMR Spectra of the Mixture of 4a (7.5 mM) and Guanosine
(15 mM) at 27 °C in 1 : 1 Mixtures between D2O and DMF-d7 (1 : 1)
The signal marked with an asterisk is due to the solvent.
the association of the porphyrin monomer with GMP.6e) The 1565 [Mϩ]) but does not show a peak corresponding to bis
second step is not observed in any other combination of the guanosine adduct. This is unexpected from the UV–vis spec-
porphyrins with the nucleotides except for 4a with poly(dG)– trum of 4b and GMP where the second step change observed
poly(dC). Since a gradual change similar to the second step for the case of 4a was not detected. However, the FAB-MS
has been reported in the case of CDDP with GMP,12) we pro- spectrum demonstrates the formation of the mono-guanosine
pose that the second step is due to coordination of GMP to Pt adduct. From these results, it is clear that 4a reacts with two
in 4a.
guanosine molecules successively and 4b reacts with one
To determine the binding mode of 4a with poly(dG)– guanosine molecule.
1
poly(dC) or GMP, we measured H-NMR spectra of 4a and
Binding Modes of Porphyrins with Synthetic Nucleic
guanosine in DMF-d7–D2O mixed solvent (Fig. 10). Since Acid Polymer In conclusion, we wish to note the following
the solubility of GMP in DMF–D2O is low and the mixing of significant findings of this investigation. The UV–vis spectral
the DMF-d7–D2O solution containing GMP with the por- changes of the second step observed for 4a with GMP can be
phyrin gave precipitation, we used guanosine instead of GMP. explained by the selective binding of the appended plat-
The solution containing guanosine (15 mM) was added to a inum(II) complexes to two guanine bases. For the case of 4b
porphyrin solution (7.5 mM) dissolved in DMF-d7 : D2Oϭ with GMP, the lack of the spectral changes of the second step
1
1 : 1, and the reaction was monitored by using H-NMR at may be due to the fact that 4b binds only one guanine base,
27 °C. The spectral changes occurred over a period of 14 d. A as exemplified by the binding of 4b with guanosine. Further-
day after from the mixing of 4a with guanosine, new signals more, it is reasonable to speculate that the two appended

1578
Vol. 49, No. 12
The crude porphyrinogen 1 (6.5 g, 9.7 mmol) was dissolved in tetrahydro-
furan (200 ml) and o-chloranil (13.0 g, 52.9 mmol) in tetrahydofurane (50
ml) was added to the solution. The solution was stirred at room temperature
for 8 h. The purple solid was recovered by filtration and the solid was redis-
solved in methanol (100 ml). To the methanol solution, triethylamine (60 ml)
platinum(II) complexes in the porphyrins react with guanine
bases in poly(dG)–poly(dC) as in the case of the CDDP.1,15)
Finally, the destabilization of poly(dG)–poly(dC) by 4a and
4b, suggests that the binding mode for 4a, 4b to poly(dG)–
poly(dC) should be different from that for 7a to poly(dG)– was added to precipitate the porphyrin. The product was collected by filtra-
tion and was washed with methanol (yield, 5.30 g, 52.2%).
Syntheses of a,b- and a,a-5,15-Bis((o-aminoethylaminocarbonyl)-
phenyl)-2,3,7,8,12,13,17,18-octamethylporphyrin (3a, b) Compound 2
poly(dC). It has been reported that coordination of CDDP to
adjacent guanosines destabilizes the double helix.16) The
gradual melting of poly(dG)–poly(dC) in the presence of 4a
(1.00 g, 1.51 mmol) was dissolved in dichloromethane (20 ml) and was
or 4b can also be explained by the selective binding of the
appended platinum(II) complexes with guanine bases in the
duplex. The binding modes between the porphyrins and syn-
thetic nucleic acids are affected more by coordination of the
nucleobase (poly(dG)–poly(dC)) to the Pt(II) in the por-
phyrins than by Coulombic interaction between cations to the
porphyrins and anions on nucleic acids and/or by hydropho-
bic interaction between the porphyrin ring and the nucle-
obase.
cooled to 5 °C. Oxalyl chloride (3.0 ml, 37 mmol) was carefully added to the
solution with cooling and the reaction mixture was stirred for 2 h. The sol-
vent and excess oxalyl chloride was removed in vacuo. The residue was then
redissolved in a small amount of dichloromethane, and the solvent was again
removed in vacuo. The residue in dichloromethane (30 ml) was added drop
wise to a solution of ethylenediamine (1.0 g, 16.7 mmol) in dichloromethane
(20 ml) under N2. After the addition was completed (ca. 2 h), the mixture
was stirred for 8 h at 0 °C, then the solution was poured into water. The
product was extracted with dichloromethane (3ϫ200 ml). The organic layer
was washed with water, 2% aqueous sodium bicarbonate, and water, in turn.
The organic layer was dried over anhydrous sodium sulfate. Evaporation of
the solvent gave a purple residue which was chromatographed on a silica-gel
column (dichloromethane, 3fϫ50 cm). Elution with 10% methanol in
dichloromethane gave the a,b-isomer 3a first. The a,a-isomer 3b was
eluted with 25% methanol in dichloromethane. From 1.0 g of a mixture of
3a and 3b was isolated 598 mg (53.1%) of 3a and 348 mg (30.9%) of 3b.
a,b-Isomer 3a: Rf valueϭ0.51 (CHCl3 : CH3OHϭ5 : 1). 1H-NMR
(CDCl3) d: Ϫ2.40 (2H, s, pyrrole-NH), 0.54 (4H, br, –CH2–NH2), 2.16 (4H,
m, –CO–NH–CH2–), 2.49 (12H, s, 2, 8, 12, 18-CH3), 3.54 (12H, s, 3, 7, 13,
17-CH3), 5.20 (2H, br, –CH2–NH2), 7.82 (2H, t, ph-H), 7.94 (2H, t, ph-H),
8.02 (2H, d, ph-H), 8.39 (2H, d, ph-H), 10.26 (2H, s, 10, 20-H). IR (KBr)
cmϪ1: 694 (pyrrole-NH), 2962, 2926, 1160, 1137 (pyrrole–CH3), 1650
(–CONH–), 3401 (–NH2). Anal. Calcd for C46H50N8O2·H2O·1/3CHCl3: N,
13.97; C, 69.33; H, 6.57. Found: N, 13.74; C, 69.38; H, 6.62. FAB-MS m/z:
746 [Calcd for C46H50N8O2 746 Mϩ].
Experimental
Measurements of UV–vis spectra and the DNA denaturation experiments
were carried out on a Hitachi U-3000 spectrophotometer with a temperature
controller Hakke F423 or on a Shimadzu UV-2500PC with a temperature
controlled cell holder Shimadzu TCC-240A. 1H-NMR spectra were mea-
sured on a JEOL JNM-GSX-400 (400 MHz) spectrometer with tetramethyl-
silane (TMS) as the internal standard and chemical shifts were expressed in
d value. Signal multiplicities are represented by s (singlet), d (doublet), t
(triplet), dd (double doublet), and m (multiplet). FAB-MS spectra were mea-
sured on a JEOL JMS-SX-102A mass spectrometer using magic bullet as
matrix. IR spectra were recorded on a Perkin-Elmer Spectrum 2000 spec-
trophotometer. Elemental analyses were performed on a Perkin-Elmer 240C
elemental analyzer.
All chemicals were purchased commercially and were used as received
without further purification unless otherwise noted. Silica gel (Wakogel C-
200 Wako Pure Chemicals Industries, Ltd.) was used for column chromatog-
raphy. Precoated silica gel plates (Merck Kieselgel 60F254) or reversed phase
silica gel plates (Merck Kieselgel RP-8 F254) with a layer thickness of
0.25 mm were used for TLC to determine Rf values. Distilled water was pur-
chased from CAMBEX Company. Synthetic nucleic acids polymers,
poly(dG)–poly(dC), and poly(dA)–poly(dT) were purchased from Sigma and
were used without further purification. Ethyenediamine was used after distil-
lation. (3,3,4Ј,4Ј-tetramethyl-2,2Ј-dipyrryl) methane,7e) 5,15-(2-amino)-
diphenyloctamethylporphyrin (5a, b)9) were prepared according to the litera-
ture. trans-[PtCl(NH3)2(DMF)]NO3 was prepared according to the publish
method.17)
UV–Vis Analysis To obtain the time-course of the UV–vis spectra of
the solution containing porphyrin and synthetic nucleic acid polymer in
20 mM NaCl at 20 °C, the sample solution was prepared by adding a solution
of porphyrin to a solution of synthetic nucleic acid polymer in a cuvette (the
pathlength is 1 cm) via a micro syringe. The sample solution to measure the
melting curves was prepared by a similar method for the measurements of
the time-course of the UV–vis spectra. The sample solution containing 5 mM
(in base pair) synthetic nucleic acid polymer in 20 mM NaCl was used after
standing the solution at 20 °C over 15 h.
Synthetic Nucleic Acid Polymer Denaturation Experiments The
melting curves of both free synthetic nucleic acid polymers (poly(dA)–
poly(dT) and poly(dG)–poly(dC)) and their reactants with porphyrins in
20 mM NaCl (pH 7) were obtained by measuring the hypochromicity of poly-
mers at 260 nm as a function of temperature. The temperature was scanned
from 20 to 99 °C at a speed of 1 °C per min. The melting temperature (Tm)
was taken as the mid-point of the hyperchromic transition.
a,a-Isomer 3b: Rf valueϭ0.22 (CHCl3 : CH3OHϭ5 : 1). 1H-NMR (CDCl3)
d: Ϫ2.46 (2H, s, pyrrole-NH), 0.38 (4H, br, –CH2–NH2), 2.24 (4H, br,
–CO–NH–CH2–), 2.49 (12H, s, 2, 8, 12, 18-CH3), 3.53 (12H, s, 3, 7, 13, 17-
CH3), 5.23 (2H, br, –CH2–NH2), 7.82 (2H, t, ph-H), 7.94 (2H, t, ph-H), 8.04
(2H, d, ph-H), 8.33 (2H, d, ph-H), 10.26 (2H, s, 10,20-H). IR (KBr) cmϪ1
:
694 (pyrrole-NH), 2955, 2926, 1161, 1137 (pyrrole–CH3), 1644 (–CONH–),
3401 (–NH2). Anal. Calcd for C46H50N8O2·CHCl3·4H2O: N, 11.94; C,
60.16; 6.34. Found: N, 12.58; C, 60.18; H, 6.12. FAB-MS m/z: 746 [Calcd
for C46H50N8O2 746 Mϩ].
Synthesis of a,b-5,15-Bis(2-trans-[PtCl(NH3)2]N-2-aminoethylaminocar-
bonylphenyl)2,3,7,8,12,13,17,18-octamethylporphyrin (4a) trans-[PtCl-
(NH3)2(DMF)]NO3 was prepeared in situ by reacting trans-[PtCl2(NH3)2]
(48.5 mg, 0.0162 mmol) with 0.97 eq of AgNO3 (26.6 mg, 0.0157 mmol) in
DMF (10 ml) solution for 24 h at room temperature. The a,b-isomer 3a
(50.0 mg, 0.0670 mmol) was dissolved in DMF (10 ml) and was cooled to
Ϫ18 °C. The trans-[PtCl(NH3)2(DMF)]NO3 solution was added to the por-
phyrin solution below Ϫ18 °C and the reaction mixture was stirred for 3 d at
room temperature. The solution was then evaporated to dryness under high
vacuum. The residue was dissolved in methanol (20 ml), and the solution
was kept overnight at 4 °C. The solid was filtered off, then washed with di-
ethyl ether (5 ml), chloroform (5 ml), and methanol (5 ml), in turn. The re-
sultant solid was dissolved in methanol and chromatographed on a cation-
exchange resin column (amberlist A-21 chloride form: 2fϫ25 cm) to ex-
change the counter ion to ClϪ. The column was eluted with methanol. The
eluate was evaporated to dryness and the residue was crystallized from
methanol/diethyl ether, and dried in vacuo to afford the a,b-isomer 4a
(47.6 mg, 52.9%).
Rf valueϭ0.64 (n-C4H9OH : (CH3)2CO : H2O : NH4OHϭ5 : 3 : 1 : 1) RP-8.
1H-NMR (DMF-d7) d: Ϫ2.25 (2H, s, pyrrole-NH), 2.53 (12H, s, 2, 8, 12,
18-CH3), 2.75 (4H, br, –CH2–NH2), 2.90 (4H, br, –CO–NH–CH2–), 3.56
(12H, s, 3, 7, 13, 17-CH3), 4.04 (12H, br, Pt-NH3), 5.84 (4H, br, –CH2–
NH2), 7.87—7.98 (6H, m, ph-H), 8.36 (2H, d, ph-H), 8.90 (2H, br, –CO–
NH–), 10.27 (2H, s, 10,20-H). IR (KBr) cmϪ1: 695 (pyrrole-NH), 2970,
2926, 1161, 1137 (pyrrole–CH3), 1642 (–CONH–), 3401 (–NH2), 327 (Pt-
Cl), 3250 (Pt-NH3). Anal. Calcd for C46H62Cl2N12O2Pt2·(Cl2)·CHCl3: N,
11.46; C, 38.50; H, 4.33. Found: N, 11.39;C, 38.91; H, 4.50. FAB-MS m/z:
Method for Studying the Interactions between Porphyrin Appending
Platinum(II) Complexes and Nucleotides During incubation of a mixture
of porphyrin and nucleotide in 10% methanol aqueous solution at 40Ϯ0.1
°C, UV–vis spectra were measured.
Synthesis of 5,15-Bis(o-carboxyphenyl)-2,3,7,8,12,13,17,18-octamethyl-
porphyrin (2) A solution of (3,3,4Ј,4Ј-tetramethyl-2,2Ј-dipyrryl)methane
(3.10 g, 15.3 mmol), o-phthalaldehydic acid (2.40 g, 16.0 mmol), and p-tolu-
ensulfonic acid monohydrate (1.00 g, 5.26 mmol) in methanol (200 ml) was
allowed to stand at 20 °C for 5 h. The brown solid was recovered by filtration
and was washed with methanol.
ϩ
H2O
max
1275 [Calcd for C46H62Cl2N12O2Pt2 1275 M ]. UV–visible spectrum l
nm (log e); 408.5 (5.06), 512.0 (3.95), 547.0 (3.70), 571.5 (3.79), 620.5
(3.34).

December 2001
1579
C50H60N8O2·(Cl2)·2H2O·CH3OH: N, 11.87; C, 64.89; H, 7.06. Found: N,
Synthesis of a,a-5,15-Bis(2-trans-[PtCl(NH3)2]N-2-aminoethylaminocar
-
11.72; C, 64.92; H, 7.06. FAB-MS m/z: 803 [Calcd for C50H60N8O2 804
bonylphenyl)-2,3,7,8,12,13,17,18-octamethylporphyrin (4b) This por-
phyrin was prepared by a similar procedure to that of the a,b-isomer (4a)
from 3b (50.0 mg, 0.670 mmol) and trans-[PtCl(NH3)2(DMF)]NO3 (pre-
pared in situ from 48.5 mg, 0.160 mmol of trans-[PtCl2(NH3)2] and AgNO3)
in 10 ml of DMF. Yield 21.9 mg (24.3%).
ϩ
H2O
(MϪH) ]. UV–visible spectrum l
nm (log e): 403.0 (5.18), 507.0 (3.95),
max
545.0 (3.69), 565.5, (3.79), 616.5 (3.29).
Synthesis of a,a-5,15-Bis((o-N,N,N-trimetylammoiummethylamido)-
phenyl)-2,3,7,8,12,13,17,18-octamethylporphyrin (7b) This porphyrin
was prepared from 6b (105 mg, 0.136 mmol), by a similar procedure to that
of the corresponding a,b-isomer (7a), yielding 77.4 mg (65.3%).
Rf valueϭ0.50 (n-C4H9OH : (CH3)2CO : H2O : NH4OHϭ5 : 3 : 1 : 1) RP-8.
1H-NMR (DMF-d7) d: Ϫ2.26 (2H, s, pyrrole-NH), 2.53 (12H, s, 2,8,12,18-
CH3), 2.79 (4H, br, –CH2–NH2), 2.95 (4H, br, –CO–NH–CH2–), 3.58 (12H,
s, 3,7,13,17-CH3), 3.88 (12H, br, Pt-NH3), 5.53 (4H, br, –CH2–NH2), 7.97
(2H, t, ph-H), 8.19 (2H, d, ph-H), 8.25 (2H, d, ph-H), 8.51 (2H, br, –CO–
NH–), 10.30 (2H, s, 10,20-H). IR (KBr) cmϪ1: 695 (pyrrole-NH), 2955,
2926, 1161, 1137 (pyrrole–CH3), 1642 (–CONH–), 3400 (–NH2), 327 (Pt-
Cl), 3249 (Pt-NH3). Anal. Calcd for C46H62Cl2N12O2Pt2·(Cl2)·2CHCl3: N,
10.60; C, 36.36; H,4.07. Found: N, 11.25; C, 35.89; H, 4.41. FAB-MS m/z:
1
Rf valueϭ0.37 RP-8 (H2O : CH3COOH : CH3OH : C5H5Nϭ2 : 2 :1 : 1). H-
NMR (CD3OD) d: 2.55 (12H, s, 2, 8, 12, 18-CH3), 2.66 (18H, s, N-(CH3)3),
3.30 (4H, m, –NH–CO–CH2–), 3.54 (12H, s, 3, 7, 13, 17-CH3), 7.70 (2H, t,
ph-H), 7.90—7.95 (4H, m, ph-H), 8.28 (2H, d, ph-H), 10.31 (2H, s, 10,20-
H). IR (KBr) cmϪ1: 693 (pyrrole-NH), 2962, 2926, 1160, 1135 (pyrrole–
CH3), 3238, 1695 (–CONH–), 3410 (NϩϪCH3). Anal. Calcd for
C50H60N8O2·5H2O: N, 11.60; C, 62.16; 7.30. Found: N, 11.59; C, 62.62; H,
6.95. FAB-MS m/z: 803 [Calcd for C50H60N8O2 804 (MϪH)ϩ]. UV–visible
ϩ
H2O
1275 [Calcd for C46H62Cl2N12O2Pt2 1275 M ]. UV–visible spectrum l max
nm (log e); 406.0 (4.91), 514.0 (3.95), 547.5 (3.49), 575.0 (3.63), 622.5
(3.00).
H2O
spectrum l max nm (log e): 400.0 (4.93), 511.5 (3.76), 547.0 (3.47), 573.5
(3.59), 621.0 (3.00)
Synthesis of a,a-5,15-Bis((o-pentaneaminocarbonyl)phenyl)-2,3,7,8,
12,13,17,18-octamethylporphyrin (8) A mixture of 3b (1.00 g, 1.51
mmol) and 6 M HCl (100 ml) was heated at 60 °C with stirring for 8 h. The
solution was cooled, diluted with water (100 ml), and neutralized with 5 M
aqueous ammonia (ca. 90 ml). The precipitate was recoverd by filtration and
the solid was washed with water, and dried in vacuo to afford the hydrolyzed
product. The product was dissolved in dichloromethane (20 ml) and was
cooled to 5 °C. Oxalyl chloride (3.0 ml, 37 mmol) was carefully added below
10 °C and the reaction mixture was stirred for 2 h. The solvent and excess
oxalyl chloride was removed in vacuo. The residue was then redissolved in a
small amount of dichloromethane, and the solvent was again removed in
vacuo. The resultant acid chloride of porphyrin in dichloromethane (30 ml)
was added to dichloromethane solution (20 ml) containing 1,5-diaminopen-
tane (0.20 g, 0.20 mmol) and the mixture was stirred for 12 h at 0 °C. Then
the solution was poured into water and was extracted with dichloromethane
(3ϫ200 ml). The organic layer was washed with water, 2% aqueous sodium
bicarbonate, and water, in turn. The organic layer was dried over anhydrous
sodium sulfate. Evaporation of the solvent gave a purple solid which was
chromatographed on a silica-gel column (3fϫ50 cm) eluted with chloro-
form. The main brown fraction was collected to yield 550 mg of 8 (50.0%).
Strapped Porphyrin 8: 1H-NMR (CDCl3) d: Ϫ4.7—Ϫ4.5 (4H, m,
–CH2–CH2–NHCO–), Ϫ2.28 (2H, s, pyrrole-NH), Ϫ1.40—Ϫ1.34 (4H, m,
–CH2–NHCO–), Ϫ1.53 (2H, m, –CH2–CH2–CH2–NHCO–), 2.52 (12H, s, 2,
8, 12, 18-CH3), 3.55 (12H, s, 3, 7, 13, 17-CH3), 7.86—7.97 (6H, m, ph-H),
8.63—8.65 (2H, m, ph-H), 10.21 (2H, s, 10,20-H). Anal. Calcd for
C47H48N6O2·CHCl3·H2O: N, 9.70; C, 66.85; 5.93. Found: N, 9.39; C, 66.85;
H, 5.80. FAB-MS m/z: 729 [Calcd for C47H48N6O2 729 Mϩ].
Synthesis of a,b-5,15-Bis((o-N,N-dimetylaminomethylamido)phenyl)-
2,3,7,8,12,13,17,18-octamethylporphyrin (6a) N,N-dimethylglycine hy-
drochloride (940 mg, 6.76 mmol) was dissolved in dichloromethane (5 ml)
and cooled to 5 °C. Oxalyl chloride (3.0 ml, 37 mmol) was carefully added to
the solution below 10 °C and the reaction mixture was stirred for 2 h. The
solvent and excess oxalyl chloride were removed in vacuo. The residue of
acid chloride was then redissolved in a small amount of dichloromethane,
and the solvent was again removed in vacuo. The residue was dissolved
in dichloromethane (30 ml) and the solution was added slowly to
dichloromethane (50 ml) containing aminoporphyrin 5a (1.00 g, 1.65 mmol),
and the reaction mixture was stirred at 0 °C. After the addition was complete
(ca. 2 h), triethylamine (4 ml) was added to the solution, and the mixture was
stirred for 8 h at 0 °C. Then the solution was poured into water and was ex-
tracted with dichloromethane (3ϫ200 ml). The organic layer was washed
with water, 2% aqueous sodium bicarbonate, and water, in turn. The organic
layer was dried over anhydrous sodium sulfate. Evaporation of the solvent
gave a purple residue which was chromatographed on a silica-gel column
(3fϫ50 cm) eluted with 10% diethyl ether in chloroform, yielding 6a,
570 mg (44.3%).
a,b-Isomer 6a: Rf valueϭ0.78 (CHCl3 : C6H6 : C2H5OHϭ20 : 20 : 1). 1H-
NMR (CDCl3) d: Ϫ2.51 (2H, s, pyrrole-NH), 1.33 (4H, s, –NH–CO–CH2–),
2.11 (12H, s, N-(CH3)2), 2.35 (12H, s, 2, 8, 12, 18-CH3), 3.34 (12H, s, 3, 7,
13, 17-CH3), 7.33 (2H, t, ph-H), 7.65 (2H, t, ph-H), 7.72 (2H, d, ph-H), 8.61
(2H, d, ph-H), 8.85 (2H, br, –NH–CO–), 10.01 (2H, s, 10,20-H). IR (KBr)
cmϪ1: 693 (pyrrole-NH), 2963, 2926, 1160, 1136 (pyrrole–CH3), 3273, 1688
(–CONH–). Anal. Calcd for C48H54N8O2·1/2(C2H5)2O: N, 13.80; C, 73.95;
H, 7.32. Found: N, 13.84; C, 73.53; H, 7.07. FAB-MS m/z: 775 [Calcd for
C48H54N8O2 775 Mϩ].
Synthesis of a,a-5,15-Bis((o-N,N-dimetylaminomethylamido)phenyl)-
2,3,7,8,12,13,17,18-octamethylporphyrin (6b) This porphyrin was pre-
pared from 5b (1.00 g, 1.65 mmol) by a similar procedure to that of the cor-
responding a,b-isomer (6a), yielding 370 mg (28.8%).
References
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1999, pp. 73—157.
2) Eckstein F., Lilley D. M. J. (ed.), “Nucleic acids And Molecular Biol-
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Rf valueϭ0.55 (CHCl3 : C6H6 : C2H5OHϭ20 : 20 : 1). 1H-NMR (CDCl3) d:
Ϫ2.25 (2H, s, pyrrole-NH), 1.61 (12H, s, N-(CH3)2), 2.99 (4H, s, –NH–
CO–CH2–), 2.62 (12H, s, 2, 8, 12, 18-CH3), 3.61 (12H, s, 3, 7, 13, 17-CH3),
7.63 (2H, t, ph-H), 7.93 (2H, t, ph-H), 8.06 (2H, d, ph-H), 8.83 (2H, d, ph-
H), 9.10 (2H, br, –NH–CO–), 10.28 (2H, s, 10,20-H). IR (KBr) cmϪ1: 693
(pyrrole-NH), 2962, 2924, 1160, 1136 (pyrrole–CH3), 3217, 1689
(–CONH–). Anal. Calcd for C48H54N8O2·1/2H2O: N, 14.29; C, 73.54; 7.07.
Found: N, 14.07; C, 73.53; H, 7.12.
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Synthesis of a,b-5,15-Bis((o-N,N,N-trimetylammoniummethylamido)-
phenyl)-2,3,7,8,12,13,17,18-octamethylporphyrin (7a) Iodomethan (0.20
ml, 3.2 mmol) was added to a DMF solution (20 ml) containing 6a (105 mg,
0.136 mmole) at room temperature and the solution was stirred for 3 h. Ether
(20 ml) was added to the reactant to produce a solid. After filteration, the re-
sultant solid was dissolved in methanol and chromatographed on a caion-ex-
change resin column (Amberlist A-21: 2fϫ25 cm) to exchange the counter
ion to ClϪ. The column was eluted with methanol. The eluate was evapo-
rated to dryness and was recrystallized from methanol/acetone, yielding
107 mg (90.0%).
Rf valueϭ0.52 RP-8 (H2O : CH3COOH : CH3OH : C5H5Nϭ2 : 2 : 1 : 1). 1H-
NMR (CD3OD) d: 2.24 (18H, s, N-(CH3)3), 2.47 (12H, s, 2, 8, 12, 18-CH3),
3.09 (4H, s, –NH–CO–CH2–), 3.44 (12H, s, 3, 7, 13, 17-CH3), 7.66 (2H, t,
ph-H), 7.82 (2H, t, ph-H), 7.98 (2H, d, ph-H), 8.08 (2H, d, ph-H), 10.24 (2H,
s, 10,20-H). IR (KBr) cmϪ1: 693 (pyrrole-NH), 2963, 2926, 1160, 1136
(pyrrole–CH3) 3276, 1686 (–CONH–), 3413 (NϩϪCH3). Anal. Calcd for
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1580
Vol. 49, No. 12
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