Syntheses of nucleic acid mimics designed for metal-induced strand formation on DNA

Article abstract of DOI:10.1016/S0040-4020(02)00203-X

This paper describes design and syntheses of a new class of chelator-type nucleic acid mimics. Four nucleic acid mimics 7, 11, 15, 17 possess a nucleobase and two ethylenediamine moieties for intermolecular metal coordination. We expected that chelator-ty

Full text of DOI:10.1016/S0040-4020(02)00203-X

TETRAHEDRON
Pergamon
Tetrahedron 58 62002) 2965±2972
Syntheses of nucleic acid mimics designed for metal-induced
strand formation on DNA
Akihiko Hatano,a,² Kentaro Tanaka,b Motoo Shiroc and Mitsuhiko Shionoyab,p
aThe Graduate University for Advanced Studies, Myodaiji, Okazaki, Aichi 444-8585, Japan
bDepartment of Chemistry, Graduate School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113 8654, Japan
cRigaku Corporation, 3-9-12 Matsubaracho, Akishima, Tokyo 196-8666, Japan
Received 29 August 2001; accepted 25 February 2002
AbstractÐThis paper describes design and syntheses of a new class of chelator-type nucleic acid mimics. Four nucleic acid mimics 7, 11,
15, 17 possess a nucleobase and two ethylenediamine moieties for intermolecular metal coordination. We expected that chelator-type nucleic
acid mimics formed a strand in the presence of metal ion and then interacted with a single-stranded DNA. Ef®cient synthetic routes leading to
the four nucleic acid analogues have been achieved via the key intermediate compound 5 which was derived from 3,5-dimethylphenol in ®ve
steps. Compound 7 was treated with zinc nitrate and Zn complex 8 was isolated as crystals. 1H NMR study of Zn complex 8 in D2O showed
the single-strand formation. Job plots showed that mixture of 6dT)70 with Zn complex 8 at 58C reached a minimum absorbance at a mole ratio
of 2:1, and the hypochromic effect showed the p±p stacking interaction between the nucleobases. Thermal denaturation study at 2:1 mole
ratio of complexes between 6dT)70 and Zn complex 8 showed melting point at 278C. q 2002 Elsevier Science Ltd. All rights reserved.
1. Introduction
bonding and p-stacked base pairs. The backbone of each
strand is constructed by phosphodiester linkage. Recon-
struction of each structural component of DNA is expected
to generate a novel type of engineered DNAs with unique
structure and high functions.2
Recently, functionalization of DNA at the molecular level
has gained more and more attention from the viewpoint of
development of highly controlled materials.1 Originally,
DNA is a bio-informational molecule which codes protein
sequences by the order of four nucleotides. The double
helical structure of DNA contains complementary hydrogen
We envisioned that single-stranded oligonucleotides could
function as molecules which potentially record information
Chart 1.
Keywords: DNA; chelator-type nucleic acid mimics; intermolecular metal coordination.
Corresponding author. Tel./fax: 181-3-5841-8061; e-mail: shionoya@chem.s.u-tokyo.ac.jp
Present address: Department of Materials Science, Shizuoka Institute of Science and Technology, 2200-2, Toyosawa, Fukuroi, Shizuoka 437-8555, Japan.
p
²
0040±4020/02/$ - see front matter q 2002 Elsevier Science Ltd. All rights reserved.
PII: S0040-4020602)00203-X

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A. Hatano et al. / Tetrahedron 58 42002) 2965±2972
In this paper, we describe the convenient syntheses of four
metal chelator-type nucleic acid mimics 7, 11, 15, 17 6Chart
1), and the interaction of Zn complex 8 with a single-
stranded DNA 6dT)70 6Fig. 1).
2. Results and discussion
The syntheses of the nucleic acid mimics comprised of a
nucleobase and two chelating sites are given in Schemes
1±5. First, the common intermediate compound 5 was
synthesized from 3,5-dimethylphenol 6Scheme 1). The
phenolic hydroxyl group was protected by pivaloyl chloride
in CH2Cl2 to obtain pivaloyl ester 1. Bromination of the
resulting pivaloyl ester 1 was then carried out by heating
with N-bromosuccinimide 6NBS) in CCl4 at re¯ux to afford
dibromide 2.5 The compound 3 was synthesized by the
coupling reaction between compound 2 and N-Boc-ethyl-
enediamine.6 The two amino groups of compound 3 were
protected by Boc groups using di-t-butyl dicarbonate to
afford 4. Removal of the pivaloyl group of 4 was performed
in 1 M NaOH aqueous solution and THF 61:1) to obtain
compound 5 in 98% yield, which is the common inter-
mediate for four nucleobase derivatives.
Figure 1. Schematic representation of self-assembly of adenines on a
template single-stranded DNA via metal coordination.
to control the spatial arrangement of metal ions or functional
groups. We previously reported the use of a single-stranded
DNA template to control the assembly of the comple-
mentary adenine mimic 7,3,4 which has two metal binding
sites and an adenine base moiety. This adenine mimic can be
build upinto a single-stranded polymer by intermolecular
zinc coordination. The single-stranded Zn polymer 8 forms
an aggregate with complementary single-stranded 6dT)70.3
Scheme 1. Reagents and conditions: 6a) pivaloyl chloride, DMAP, 6iPr)2NEt in CH2Cl2, room temperature, 1 h 698%); 6b) NBS, benzoylperoxide in CCl4,
re¯ux, 22 h 658%); 6c) NH2CH2CH2NHBoc, 6iPr)2NEt in CH2Cl2, 508C, 2 h 645%); 6d) di-t-butyl dicarbonate, 6iPr)2NEt in CH2Cl2, 358C, 3 h 678%); 6e) 1 M
NaOH in H2O, THF, 608C, 6 h 698%).
Scheme 2. Reagents and conditions: 6f) 9-63-chloropropyl)adenine, tBuOK in DMF, room temperature, 10 days 659%); 6g) 1.0 M HCl in AcOH, room
temperature, 1 h 682%); 6h) IRA-400 6100%); 6i) Zn6NO3)2´6H2O in H2O, room temperature, 10 days 675%).

A. Hatano et al. / Tetrahedron 58 42002) 2965±2972
2967
Scheme 3. Reagents and conditions: 6j) 1,3-bis6p-toluenesulfonyloxy)propane, tBuOK in DMF, room temperature, 1.5 h 676%); 6k) 18-crown-6, tBuOK in
MeOH, 508C, 23 h 661%); 6l) 1.0 M HCl in AcOH, 08C, 15 min 682%).
The coupling reaction between 5 and 9-63-chloropropyl)-
adenine was carried out in the presence of potassium
t-butoxide 6tBuOK) in DMF 6Scheme 2)7 to afford 6 in
71% yield. The tetrahydrochloride salts of compound 7
were obtained by the treatment with 1 M HCl in acetic
acid. Ion exchange column chromatography 6IRA-400)
was carried out to obtain acid-free form 7.
purine with 1-bromo-3-chloropropane afforded 9H-2-
amino-6-chloro-9-63-chloropropyl)purine and 9H-2-amino-
6-chloro-7-63-chloropropyl)purine 6N9 alkylated purine±
N7 alkylated purineˆ26:1). Alkylation at the N9-position
was evidenced by X-ray crystallography.9 The coupling
between 5 and 9H-2-amino-6-chloro-9-63-chloropropyl)-
purine was carried out in the presence of tBuOK in DMF
6Scheme 4). The chloro groupof 12 was replaced by the
methoxy group6 13) using sodium methoxide in MeOH, and
subsequent treatment with 4 M NaOH in MeOH afforded
the guanine form 614). The Boc groups were readily
removed in 1 M HCl in acetic acid, and then recrystallized
from H2O/EtOH to afford 15.
The synthetic route for the thymine derivative 11 was differ-
ent from that for the adenine form 7, as shown in Scheme 3.
Alkylation of 5 with 1,3-bis6p-toluenesulfonyloxy)propane
afforded 9. The coupling reaction of 9 with thymine was
performed in MeOH in the presence of tBuOK and
18-crown-6 6Scheme 3). Deprotection of 10 leading to 11
was then carried out in a manner similar to that for
compound 7.
The synthesis of the cytosine monomer 17 was performed in
a manner similar to that of the adenine form. The coupling
between 5 and 3-chloropropylcytosine was carried out in the
presence of tBuOK to afford 16 in 30% yield. Deprotection
with 1 M HCl in acetic acid afforded compound 17 in 78%
yield.
Since selective alkylation of guanine base at the N9 position
was found to be dif®cult, 2-amino-6-chloropurine was used
as the starting material.8 Alkylation of 2-amino-6-chloro-
Scheme 4. Reagents and conditions: 6m) 9H-2-amino-6-chloro-9-63-chloropropyl)purine, tBuOK in DMF, 608C, 3 days 671%); 6n) MeONa in MeOH, room
temperature, 10 min 687%); 6o) 4 M NaOH aq. in MeOH, 708C, 1 week 655%); 6p) 1.0 M HCl in AcOH, room temperature, 30 min 637%).
Scheme 5. Reagents and conditions: 6q) 3-chloropropylcytosine, tBuOK in DMF, 408C, 10.5 h 630%); 6r) 1.0 M HCl in AcOH, 08C, 15 min 678%).

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A. Hatano et al. / Tetrahedron 58 42002) 2965±2972
Figure 4. UV melting curves for 2:1 molar ratio of Zn complex 8 and
6dT)70. [6dT)70]ˆ20 mM/base and [Zn complex 8]ˆ10 mM/base, Mops
buffer 61.0 mM, pH 7.0).
1
Figure 2. H NMR spectra of: 6a) compound 7 and 6b) Zn complex 8 in
D2O. All signals were assigned by NOESY experiment.
Reaction of 7 with Zn6NO3)2 in water afforded crystals of
[7-ZnII6OH2)2]6NO3)26H2O)10, 8, by slow evaporation of
water. The polymeric structure has been previously deter-
mined by single-crystal X-ray diffraction.3
6dT)70 and Zn complex 8 in a 2:1 ratio. It is well-known
that the formation of triple-stranded complexes 6dA)±6dT)2
lead to a hypochromic shift at 280 nm.10 Hypochromic
effects were observed at 280 nm with respect to Zn complex
8 and 6dT)70. This observation should be consistent with the
proposal that complex of Zn complex 8 and 6dT)70 were
three-stranded.
The solution behavior of Zn complex 8 was examined by 1H
NMR spectroscopy 6Fig. 2). The proton resonances of ethyl-
enediamine sites, b and c shifted to down®eld and signi®-
cantly broadened compared with those of the ligand itself.
This observation shows the decrease in electron density
around ethylenediamine moieties upon complexation. The
broadening of all signals implies the formation of the
oligomeric or polymeric structures by intermolecular
metal coordination. This result is in good agreement with
the polymeric structure clari®ed by X-ray analysis.3
The melting curve of a mixture of 6dT)70 and Zn complex 8
in a 2:1 ratio shows a biphasic transition at 278C 6Fig. 4).11 It
is possible that these results show the formation of the three-
stranded complex by hydrogen bonded interactions between
Zn complex 8 and 6dT)70.12,13
3. Conclusion
In this study, to determine the ratio of Zn complex 8 to
6dT)70 in their association form, we carried out Job plotting
6Fig. 3). UV absorption change at 260 nm was monitored at
58C for mixture of Zn complex 8 with 6dT)70 6total
baseˆ30 mM/base in different ratio). As a result, the hypo-
chromicity was maximum at a mole fraction of 0.67 6dT)70
to 0.33 Zn complex 8. This result clearly shows that the
presence of triple-stranded complexes are formed by
In this study, we have prepared the four chelator-type
nucleic acid mimics, and examined the interaction between
Zn complex 8 and a natural single-stranded DNA 6dT)70 by
the spectroscopic method. UV melting experiment clearly
showed the reversible association and dissociation
processes between Zn complex 8 and 6dT)70. In addition,
Job plotting for the extent of hypochromic effects further
indicated the possibility of the triple-strand formation
consisted of two natural strands and an oligomeric or poly-
meric Zn complex in aqueous solution with low ionic
strength.
4. Experimental
4.1. General
All solvents and reagents were of reagent-grade quality, and
used without further puri®cation. TLC analysis was carried
out on silica gel 60 F254 1.05554 6Merck). Column chroma-
tography was performed using Wakogel C-300 6silica gel,
Wako) or Silica gel 60 6Merck).
1H and 13C NMR spectra were recorded on either a JEOL
Lambda 500 or a Bruker DRX-500 spectrometer 6500 MHz
for 1H; 125.65 MHz for 13C). The spectra were referenced to
Figure 3. Job plotting for Zn complex 8 and 6dT)70. 1.0 mM Mops, pH 7.0,
260 nm at 58C, [Total base]ˆ30 mM/base.

A. Hatano et al. / Tetrahedron 58 42002) 2965±2972
2969
TMS in chloroform-d or DMSO-d6, and to TSP in D2O.
Chemical shifts 6d) are reported in ppm; multiplicity is
indicated by: s 6singlet), d 6doublet), t 6triplet), q 6quartet),
m 6multiplet), and br 6broad). Coupling constants, J, are
reported in Hz. FABMS, EIMS, ESIMS were recorded on
a Shimadzu KRATOS CONCEPT IS, a Shimadzu QP 1000
EX, and a PE SCIEX API-300 spectrometer, respectively.
Melting points were obtained on a Yanaco MP-500D.
UV spectra were measured on a Hitachi U-3500 spectro-
meter in a 1 cm quartz cell.
and diisopropylethylamine 61.56 g, 12.1 mmol) in CH2Cl2
640 mL) was added dropwise a solution of di-t-butyl dicar-
bonate 62.65 g, 12.1 mmol) over 15 min. The reaction
mixture was stirred for 3 h at room temperature and then
washed with water three times, dried over anhydrous
MgSO4, and evaporated. The crude product was chromato-
graphed on silica gel with n-hexane±AcOEt 62:1) to obtain
4 as a pale yellow foam 63.11 g, 78%). 1H NMR 6CDCl3): d
1.35 69H, s), 1.43 636H, s), 3.26 68H, br), 4.41 64H, br), 4.83
61H, br), 5.02 61H, br), 6.82 62H, br), 6.94 61H, br); 13C
NMR 6CDCl3): d 177.0, 156.1, 155.9, 151.6, 140.3, 123.4,
119.9, 119.2, 80.4, 79.1, 50.6, 50.0, 46.0, 39.1, 39.1, 28.4,
27.1; FABMS m/e 724 6M1H)1; HRMS calcd for
C37H63N4O10 723.4544, found 723.4540.
4.1.1. 3,5-Dimethylphenyl pivaloate -1). To a solution of
3,5-dimethyphenol 625.0 g, 0.21 mol) and 4-dimethyl-
aminopyridine 65.0 g, 0.041 mol), triethylamine 622.3 g,
0.22 mol) in CH2Cl2 640 mL) was added a cooled 6ice-
water bath) solution of pivaloyl chloride 626.5 g, 0.22
mol) in CH2Cl2 660 mL) dropwise over 1 h with a nitrogen
inlet. The reaction mixture was stirred for 1 h at room
temperature, and then poured into 600 mL CH2Cl2, washed
with water three times, and dried over anhydrous MgSO4.
After ®ltration, the solvent was evaporated. Puri®cation of
the residue by column chromatography on silica gel with
n-hexane±ethyl ether 61:1) gave 1 as a colorless syrup
4.1.5. Compound 5. To a solution of 4 61.94 g, 2.69 mmol)
dissolved in THF 620 mL) was added 1.0 M NaOH aqueous
solution 620 mL). The reaction mixture was heated at 608C
for 6 h. Then the solution was neutralized with Dowex
50W-X8 6Pyridinium form). The resin was removed by
®ltration and the ®ltrate was concentrated on a rotary
evaporator. The crude material puri®ed by silica gel
chromatography using n-hexane±AcOEt 62:1) afforded 5
1
1
642.5 g, 98%). H NMR 6CDCl3): d 1.34 69H, s), 2.30
as a pale yellow foam 61.72 g, 100%). H NMR 6CDCl3):
66H, s), 6.67 62H, s), 6.84 61H, s); 13C NMR 6CDCl3): d
177.3, 151.0, 139.2, 127.3, 119.1, 39.0, 27.2, 21.2; EIMS
m/e 206 6M1); HRMS calcd for C13H18O2 206.1307, found
206.1315.
d 1.43 636H, s), 3.32 68H, br), 4.44 64H, br), 5.01 61H, br),
5.16 61H, br), 6.55 61H, s), 6.60 62H, s); 13C NMR 6CDCl3):
d 157.4, 156.6, 156.3, 140.1, 139.8, 113.5, 113.2, 80.4, 79.3,
50.8, 50.1, 45.8, 39.2, 28.4, 27.2; FABMS m/e 637
6M2H)2; HRMS calcd for C32H53N4O9 637.3813, found
637.3800.
4.1.2. 3,5-Bis-bromomethyl)phenyl pivaloate -2). NBS
678.3 g, 0.44 mol) and benzoyl peroxide 60.10 g) were
added in 10 equal portions separately during 6 h to a re¯ux-
ing solution of 3,5-dimethylphenyl pivaloate 642.2 g, 0.20
mol) in CCl4 6412 mL). The reaction mixture was further
heated at re¯ux for 16 h, the resulting precipitate was
removed by ®ltration. The residual oil obtained after
evaporation was chromatographed on silica gel with
n-hexane±toluene 63.5:1) and then recrystallized from
n-hexane to afford 2 as pale yellow needles 642.2 g, 58%).
4.1.6. Compound 6. To a solution of 5 61.63 g, 2.55 mmol)
and tBuOK 60.35 g, 3.07 mmol) dissolved in DMF 650 mL)
was added 9-63-chloropropyl)adenine 60.48 g, 2.55 mmol)
in one portion. The reaction mixture was heated at 508C
for 10 days and then the solvent was concentrated on a
rotary evaporator. The residue was dissolved into 80 mL
CH2Cl2 and then washed with water twice, and dried over
anhydrous MgSO4, the solvent was removed on a rotary
evaporator. The crude material was puri®ed by silica-gel
column chromatography with CHCl3±MeOH 625:1) to
1
Mp44.0±45.0 8C; H NMR 6CDCl3): d 1.36 69H, s), 4.45
64H, s), 7.06 62H, s), 7.27 61H, s); 13C NMR 6CDCl3): d
176.8, 151.4, 139.7, 126.7, 122.2, 39.2, 32.0, 27.1; EIMS
m/e 364 6M1); HRMS calcd for C13H16Br2O2 363.9498,
found 363.9511.
1
afford 6 as a pale yellow foam 61.08 g, 59%). H NMR
6CDCl3): d 1.43 636H, s), 2.36 62H, q, Jˆ6.2 Hz), 3.22
68H, br), 3.93 62H, t, Jˆ5.6 Hz), 4.38 64H, s), 4.42 62H, t,
Jˆ6.7 Hz), 4.94 61H, br), 5.61 61H, br), 6.61 62H, s), 6.69
61H, s), 7.73 61H, s), 8.33 61H, s); FABMS m/e 815
6M1H)1; HRMS calcd for C40H64N9O9 814.4827, found
814.4867.
4.1.3. Compound 3. To a solution of N-Boc-ethylene-
diamine6 617.6 g, 110 mmol) and diisopropylethylamine
61.42 g, 11.0 mmol) dissolved in CH2Cl2 65 mL) was
added dropwise a solution of 2 62.0 g, 5.49 mmol) in
CH2Cl2 610 mL) over 1.5 h at room temperature. After
0.5 h, the reaction mixture was poured into CH2Cl2
6150 mL), washed with water twice, dried over anhydrous
MgSO4, and evaporated. The crude product was chromato-
graphed on silica gel with CHCl3±MeOH 620:1) to obtain 3
4.1.7. Compound 7´4HCl. Compound 6 61.01 g, 1.24
mmol) was placed in a ¯ask, to which 1.0 M HCl in acetic
acid 640 mL) was added. The solution was stirred in an ice-
water bath for 1 h. The solvent was removed in vacuo and
the resulting colorless solid was recrystallized from H2O/
iPrOH to afford 7´4HCl as colorless needles 60.569 g, 82%).
1
as a pale yellow foam 61.79 g, 45%). H NMR 6CDCl3): d
1
1.35 69H, s), 1.48 618H, s), 1.57 62H, br), 2.75 64H, br), 3.23
64H, br), 3.71 64H, s), 4.96 62H, br), 6.92 62H, s), 7.13 61H,
s); 13C NMR 6CDCl3): d 177.2, 156.2, 151.4, 141.9, 129.0,
128.2, 125.3, 125.0, 119.8, 79.2, 53.1, 48.7, 40.2, 39.1, 28.4,
27.2; FABMS m/e 523 6M1H)1; HRMS calcd for
C27H47N4O6 523.3496, found 523.3482.
Mp249.0±250.5 8C; H NMR 6D2O): d 2.26 62H, q, Jˆ
6.1 Hz), 3.27 64H, t, Jˆ5.7 Hz), 3.31 64H, t, Jˆ5.6 Hz),
4.01 62H, t, Jˆ5.6 Hz), 4.15 64H, s), 4.40 62H, t, Jˆ
6.6 Hz), 6.86 62H, s), 7.01 61H, s), 8.17 61H, s), 8.20 62H,
s); ESIMS m/e 414 6M)1, m/2e 207 6M)1.
4.1.8. Compound 7. The HCl salts of 7 6150 mg,
0.268 mmol) were passed through an ion exchange column
4.1.4. Compound 4. To a solution of 3 62.88 g, 5.51 mmol)

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A. Hatano et al. / Tetrahedron 58 42002) 2965±2972
6IRA-400, Organo Co. Japan) to obtain acid-free 7, which
was recrystallized from MeCN to afford 7 as colorless
prisms 6110 mg, 99%). Mp 109.5±110.58C; 1H NMR
6D2O): d 2.30 62H, q, Jˆ5.4 Hz), 2.56 64H, t, Jˆ6.6 Hz),
2.73 62H, t, Jˆ6.6 Hz), 3.58 64H, s), 4.09 62H, t, Jˆ4.8 Hz),
4.36 62H, t, Jˆ5.2 Hz), 6.29 62H, s), 6.75 61H, s), 7.86 61H,
s), 8.08 61H, s); 13C NMR 6D2O): d 158.9, 156.5, 153.1,
150.3, 143.9, 141.2, 122.3, 119.7, 114.4, 67.4, 53.1, 47.4,
43.4, 40.0, 29.4; ESIMS m/e 414 6M)1, m/2e 207 6M)1;
Anal. calcd for C20H31N9O: C, 58.09; H, 7.56; N, 30.48.
Found: C, 57.72; H, 7.34; N, 30.07.
60.756 g, 0.939 mmol) was placed in a ¯ask, to which
1.0 M HCl in acetic acid 620 mL) was added. The solution
was stirred in an ice-water bath for 15 min. The solvent was
evaporated in vacuo. The residue was then dissolved in
1.0 M NaClO4 aqueous solution 67.5 mL), and added to
iPrOH 65 mL). Recrystallization from H2O/iPrOH afforded
1
11 as colorless needles 60.424 g, 82%). H NMR 6D2O): d
2.11 62H, q, Jˆ5.9 Hz), 3.35 68H, m), 3.88 62H, t, Jˆ
6.3 Hz), 4.11 62H, t, Jˆ5.5 Hz), 4.22 64H, s), 7.01 62H, s),
7.08 61H, s), 7.39 61H, s); 13C NMR 6D2O): d 167.0, 158.9,
152.3, 143.5, 132.8, 123.5, 117.1, 110.6, 66.2, 51.1, 46.6,
43.9, 35.4, 26.9, 11.1; ESIMS m/e 405 6M)1, m/2e 202
6M)21; Anal. calcd for C20H37Cl3N6O16: C, 33.18; H, 5.15;
N, 11.61. Found: C, 33.63; H, 5.63; N, 11.55.
4.1.9. Zn complex 8. To a solution of 7 622.8 mg,
55.1 mmol) dissolved in distilled water 6100 mL) was
added 0.5 M Zn6NO3)2 aqueous solution 6110 mL). The
reaction mixture was ®ltered and the ®ltrate was slowly
evaporated to obtain the Zn complex 8 as colorless prisms
626.4 mg, 75%). 1H NMR 6D2O): d 1.91 62H, br), 2.77 64H,
br), 2.96 62H, br), 3.67 64H, s), 3.99 62H, br), 4.34 62H, br),
6.37 62H, s), 6.79 61H, s), 7.88 61H, s), 8.06 61H, s); Anal.
calcd for C20H39N11O9Zn: C, 37.60; H, 5.52; N, 24.11.
Found: C, 37.48; H, 5.65; N, 23.87 6for the sample com-
pletely dried).
4.1.13. Compound 12. To a solution of 5 6150 mg,
0.24 mmol) and tBuOK 628 mg, 0.25 mmol) dissolved in
DMF 62 mL) was added 9H-2-amino-6-chloro-9-63-chloro-
propyl)purine 661 mg, 0.25 mmol) in one portion. The
reaction mixture was heated at 608C for 3 days and then
the solvent was concentrated on a rotary evaporator. The
residue was dissolved in 80 mL CH2Cl2 and then washed
with water twice, dried over anhydrous MgSO4, and the
solvent was removed on a rotary evaporator. The crude
material was puri®ed by silica-gel column chromatography
with CHCl3±MeOH 625:1) to afford 12 as a pale yellow
4.1.10. Compound 9. To a solution of 5 62.03 g, 3.17 mmol)
and tBuOK 60.426 g, 3.80 mmol) dissolved in DMF 66 mL)
was added 1,3-di6p-toluenesulfonyloxy)propane 63.66 g,
5.73 mmol) in one portion and the reaction mixture was
stirred for 25 min at room temperature. After 1.5 h the
solvent was removed in vacuo. The residue was dissolved
in 150 mL CH2Cl2 and then washed with water twice, and
dried over anhydrous MgSO4, the solvent was removed on a
rotary evaporator. The crude material was puri®ed by silica-
gel column chromatography with n-hexane±AcOEt 63:1) to
1
foam 61.48 g, 71%). H NMR 6CDCl3): d 1.43 636H, s),
2.31 62H, br), 3.26 68H, br), 3.93 62H, t, Jˆ5.6 Hz), 4.32
62H, t, Jˆ6.5 Hz), 4.40 64H, br), 4.85 61H, s), 5.07 61H, s),
5.31 62H, s), 6.60 61H, s), 6.67 62H, s), 7.75 61H, s).
4.1.14. Compound 13. To a solution of 12 60.99 g,
1.16 mmol) dissolved in MeOH 65 mL) was added dropwise
a solution of 28% sodium methoxide in MeOH 62.4 mL,
11.8 mmol) over 10 min under argon at room temperature.
The reaction mixture was stirred at room temperature for
0.5 h, and then added to 120 mL CH2Cl2, washed with brine
three times, dried over anhydrous MgSO4 and concentrated.
Product 13 was obtained as a colorless syrup60.584 g, 87%)
1
afford 9 as a pale yellow foam 62.06 g, 76%). H NMR
6CDCl3): d 1.43 636H, s), 2.10 62H, br), 2.41 63H, s), 3.24
68H, br), 3.94 62H, br), 4.23 62H, t, Jˆ6.1 Hz), 4.37 64H, br),
4.74 61H, br), 5.00 61H, br), 6.58 62H, s), 6.65 61H, br), 7.30
61H, d, Jˆ7.3 Hz), 7.77 62H, d, Jˆ7.7 Hz); FABMS m/e
851 6M)1.
1
and used without further puri®cation for next reaction. H
NMR 6CDCl3): d 1.43 636H, s), 2.30 62H, s), 3.25 68H, br),
3.91 62H, t, Jˆ5.6 Hz), 4.08 63H, s), 4.29 62H, t, Jˆ6.5 Hz),
4.39 64H, br), 4.79 61H, s), 4.88 62H, s), 5.03 61H, s), 6.61
61H, s), 6.67 62H, s), 7.56 61H, s).
4.1.11. Compound 10. To a solution of thymine 60.355 g,
2.66 mmol), 18-crown-6 60.767 g, 2.90 mmol), and tBuOK
60.325 g, 0.290 mmol) in MeOH 65 mL) was added drop-
wise a solution of 9 62.06 g, 2.42 mmol) in MeOH 610 mL)
over 5 min under argon and at room temperature. The
reaction mixture was stirred for 23 h at 508C. After evapora-
tion of the solvent, the residue was poured into 150 mL
CH2Cl2, and the solution was washed with water twice,
dried over anhydrous MgSO4, and concentrated. The
crude material was puri®ed by silica-gel column chroma-
tography with n-hexane±AcOEt 61:3) to afford 10 as a pale
yellow foam 61.19 g, 61%). 1H NMR 6CDCl3): d 1.43 636H,
s), 1.87 63H, s), 2.15 62H, br), 3.25 68H, br), 3.91 62H, t,
Jˆ6.7 Hz), 3.99 62H, t, Jˆ5.5 Hz), 4.39 64H, br), 4.82 61H,
br), 5.04 61H, br), 6.63 61H, s), 6.68 62H, s), 7.02 61H, s),
8.67 61H, br); 13C NMR 6CDCl3): d 164.0, 159.0, 156.2,
155.9, 150.8, 140.9, 140.5, 140.2, 112.7, 112.2, 110.6,
80.3, 79.4, 63.7, 50.8, 50.2, 46.1, 39.1, 29.7, 28.4, 12.3,
FABMS m/e 806 6M1H)1; HRMS calcd for C40H65N6O11
805.4711, found 805.4748.
4.1.15. Compound 14. To a solution of 13 60.854 g,
1.01 mmol) dissolved in MeOH 68 mL) was added dropwise
4.0 M NaOH aqueous solution 68 mL) over 1 min under
argon at room temperature. The reaction mixture was stirred
at 708C for 1 week, and then poured into 120 mL CH2Cl2,
washed with water three times, and dried over anhydrous
MgSO4. After evaporation, the crude material was puri®ed
by silica-gel column chromatography with CHCl3±MeOH
1
625:1) to afford 14 as pale yellow foam 60.461 g, 55%). H
NMR 6CDCl3): d 1.42 636H, s), 2.27 62H, br), 3.23 68H, br),
3.94 62H, t, Jˆ5.3 Hz), 4.23 62H, t, Jˆ6.2 Hz), 4.39 64H,
br), 4.87 61H, br), 5.11 61H, br), 5.82 62H, br), 6.61 63H, m),
7.58 61H, s), 11.8 61H, br); 13C NMR 6CDCl3): d 159.2,
159.0, 156.2, 153.6, 152.0, 140.3, 138.0, 117.3, 80.3, 79.2,
64.4, 50.9, 50.3, 46.2, 40.6, 39.3, 29.6, 28.5.
4.1.16. Compound 15´5HCl. Compound 14 60.461 g,
0.56 mmol) was placed in a ¯ask, to which 1.0 M HCl in
4.1.12. Compound 11´3HClO4´H2O. Compound 10

A. Hatano et al. / Tetrahedron 58 42002) 2965±2972
2971
acetic acid 610 mL) was added. After stirring for 30 min at
room temperature, the reaction mixture was poured into
diethyl ether 610 mL) and the resulting colorless precipitate
was collected. The crude product was recrystallized from
H2O/EtOH to afford 15 as colorless needles 60.126 g, 37%).
1H NMR 6D2O): d 2.19 62H, q, Jˆ5.2 Hz), 3.35 68H, br),
4.08 62H, t, Jˆ5.9 Hz), 4.14 64H, s), 4.18 62H, t, Jˆ5.8 Hz),
6.67 62H, s), 6.97 61H, s), 7.71 61H, s); 13C NMR 6D2O): d
158.7, 158.6, 153.5, 151.9, 140.6, 132.8, 123.3, 116.6,
115.8, 66.6, 51.1, 44.0, 42.1, 35.6, 28.0; Anal. calcd for
C20H36Cl5N9O2: C, 39.26; H, 5.93; N, 20.60. Found: C,
39.04; H, 5.91; N, 20.40.
by Rigaku RAXIS-IV imaging plate diffractometer with
graphite monochromated Mo Ka radiation. For crystal
data of 9H-2-amino-6-chloro-9-63-chloropropyl)purine, see
Ref. 9.
Acknowledgements
This work is supported by Grant-in-Aids for Scienti®c
Research 6Nos. 13031018 and 13554024) form the Ministry
of Education, Science, Sports, and Culture, Japan.
4.1.17. Compound 16. To a solution of 5 6150 mg,
0.24 mmol) and tBuOK 628 mg, 0.25 mmol) dissolved in
DMF 62 mL) was added 3-chloropropylcytosine 661 mg,
0.25 mmol) in one portion. The reaction mixture was heated
at 608C for 3 days and then the solvent was concentrated on
a rotary evaporator. The residue was dissolved in 80 mL
CH2Cl2 and then washed with water twice, and dried over
anhydrous MgSO4, the solvent was removed on a rotary
evaporator. The crude material was puri®ed by silica-gel
column chromatography with CHCl3±MeOH 625:1) to
References
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1
afford 16 as a pale yellow foam 655.1 mg, 30%). H NMR
6CDCl3): d 1.42 636H, br), 2.20 62H, br), 3.22 68H, br), 3.94
64H, br), 4.38 64H, br), 4.88 61H, s), 5.13 61H, s), 5.69 61H,
d, Jˆ6.6 Hz), 6.65 63H, br), 7.20 61H, d, Jˆ6.9 Hz); 13C
NMR 6CDCl3): d 166.2, 159.1, 156.6, 156.0, 146.2, 140.3,
118.9, 112.4, 93.9, 80.3, 79.2, 64.5, 51.0, 47.7, 46.2, 39.2,
28.6.
2. 6a) Arya, D. P.; Bruice, T. C. J. Am. Chem. Soc. 1998, 120,
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Dempcy, R. O.; Bruice, T. C. Proc. Natl Acad. Sci. USA 1995,
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4.1.18. Compound 17´5HCl. To a solution of 16 655.1 mg,
0.068 mmol) was added 1.0 M HCl in acetic acid 61 mL).
The reaction mixture was stirred in an ice-water bath for
15 min and then poured into diethyl ether 65 mL). The
resulting colorless precipitate was collected and the ®ltrate
was evaporated in vacuo to afford 17 as a white precipitate
3. Hatano, A.; Tanaka, K.; Shiro, M.; Shionoya, M. Chem. Lett.
2000, 822±823.
4. Hatano, A.; Morishita, H.; Tanaka, K.; Shionoya, M. Nucl
Acids Symp. Ser. 1998, 39, 171±172.
1
623.1 g, 78%). H NMR 6D2O): d 2.11 62H, q, Jˆ6.2 Hz),
3.31 64H, t, Jˆ6.9 Hz), 3.38 64H, t, Jˆ6.9 Hz), 3.95 62H, t,
Jˆ6.7 Hz), 4.07 62H, t, Jˆ5.6 Hz), 4.21 64H, s), 6.03 61H, d,
Jˆ6.7 Hz), 7.05 62H, s), 7.09 61H, s), 7.75 61H, d, Jˆ
6.7 Hz).
5. 6a) Tashiro, M.; Yamato, T. J. Org. Chem. 1985, 50, 2939±
2942. 6b) Vinod, T.; Hart, H. J. Org. Chem. 1990, 55, 881±
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62, 411±417.
4.2. UV measurement and Job plotting -mixing curves)
7. When 9-63-bromopropyl)adenine was used instead as an
alkylating agent, an intramolecular cyclization product was
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Tetrahedron 1990, 46, 6903±6914.
In the temperature-dependence of UV absorbance at
260 nm, a solution containing an equimolar concentration
615 mM/base) of 6dT)70 and Zn complex 8 in 1.0 mM Mops
buffer at pH 7.0 was used for the measurement. The cuvette-
holding chamber was ¯ushed with a constant stream of N2
gas to avoid water condensation on the cuvette exterior. To
obtain the Job plotting for UV absorbance at 260 nm, solu-
tions of 6dT)70 and Zn complex 8 6total base concentrations
were 30 mmol/L) were mixed in a variety of concentrations
in 1.0 mM Mops buffer at pH 7.0. Each mixture was heated
at 808C for 20 min and then slowly cooled to 08C over 3
days.
9. Crystal data for 9H-2-amino-6-chloro-9-63-chloropropyl)-
purine; C8H9Cl2N5, FWˆ246.10, prismatic, space group Aba
Ê
6#41), aˆ23.98962), bˆ23.607, cˆ7.281964) A, Zˆ16, Dcalcd
ˆ
1.585 g/cm3, 66Mo Ka)ˆ6.01 cm21
0.123, Rwˆ0.133, R1ˆ0.067.
,
F6000)ˆ2016.00, Rˆ
10. Riley, M.; Maling, B.; Chamberlin, M. J. Mol. Biol. 1966, 20,
359±389.
11. The melting curve of a 1:1 mixture of Zn complex 8 with
6dT)70 was similar to that of natural DNA duplex.
4.3. Single-crystal X-ray analysis
12. In this ratio, the formed complex should be neutral, so that we
could not exclude the possibility of the formation of 2:1 poly-
ion complex of 6dT)70 with Zn complex 8. In the thermal
denaturation analysis of a mixture of natural DNA 6dA)70
9H-2-Amino-6-chloro-9-63-chloropropyl)purine. A single
crystal 60.50£0.10£0.05 mm3) grown from iPrOH was
used for the unit-cell determinations and the data collections

2972
A. Hatano et al. / Tetrahedron 58 42002) 2965±2972
and 6dT)70 in a 1:1 ratio in 100 mM NaCl, plots of absorbance
complex 8 with 6dT)70 in a 1:1 ratio, a biphasic transition
was observed at 408C when [NaCl]ˆ0, whereas no transition
when [NaCl]ˆ100 mM.
at 260 nm vs temperature exhibited a transition point at 598C.
However, the hypochromic effect was not observed in this
case without adding NaCl. On the other hand, since Zn
complex 8 possesses positive charges, it possibly interacts
with negatively charged DNA strands more strongly in solu-
tion with lower ionic strength. Indeed, in the case of Zn
13. In the thermal denaturation analysis of mixtures containing
Zn complex 8 and 6dA)70, 6dG)70, or 6dC)70 in a 1:1 ratio,
precipitation occurred in 1.0 mM Mops 6pH 7.0).