Novel class of DNA binding motifs based on bistetrahydrofuran and bisfuran skeleton with long alkyl chains

Article abstract of DOI:10.1081/NCN-100002331

Small molecules with DNA-binding affinity within the minor groove have become of great interest. In this paper, new DNA binding molecules; diaminobistetrahydrofuran (bisTHF) and diamino-bisfuran are reported. The bisTHF ligand with RR configuration at the amino groups and C8 alkyl chains (RR8) stabilized GC-rich duplex. In contrast, bisfuran compounds stabilized AT-rich duplex. The binding affinity of RR8 with 12 mer duplex DNA was determined by isothermal titration calorimetry to be 3.3 × 10⁸ M^-1.

Full text of DOI:10.1081/NCN-100002331

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NOVEL CLASS OF DNA BINDING MOTIFS BASED ON
BISTETRAHYDROFURAN AND BISFURAN SKELETON
WITH LONG ALKYL CHAINS
a
a
b
a
Shigeki Sasaki , Takayuki Shibata , Hidemine Torigoe , Yosinori Shibata & Minoru
a
Maeda
a
Graduate School of Pharmaceutical Sciences, Kyushu University , Fukuoka, 812-8582,
Japan
b
Tsukuba Life Science Center , Riken, Tsukuba City, Japan
Published online: 07 Feb 2007.
To cite this article: Shigeki Sasaki , Takayuki Shibata , Hidemine Torigoe , Yosinori Shibata & Minoru Maeda (2001) NOVEL
CLASS OF DNA BINDING MOTIFS BASED ON BISTETRAHYDROFURAN AND BISFURAN SKELETON WITH LONG ALKYL CHAINS,
Nucleosides, Nucleotides and Nucleic Acids, 20:4-7, 551-558, DOI: 10.1081/NCN-100002331
To link to this article: http://dx.doi.org/10.1081/NCN-100002331
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NUCLEOSIDES, NUCLEOTIDES & NUCLEIC ACIDS, 20(4–7), 551–558 (2001)
NOVEL CLASS OF DNA BINDING MOTIFS BASED
ON BISTETRAHYDROFURAN AND BISFURAN
SKELETON WITH LONG ALKYL CHAINS
Shigeki Sasaki,1 Takayuki Shibata, Hidemine Torigoe,
,∗
1
2
1
1
Yosinori Shibata, and Minoru Maeda
1Graduate School of Pharmaceutical Sciences, Kyushu University,
Fukuoka 812-8582, Japan
2
Tsukuba Life Science Center, Riken, Tsukuba City, Japan
ABSTRACT
Small molecules with DNA-binding affinity within the minor groove have be-
come of great interest. In this paper, new DNA binding molecules; diamino-
bistetrahydrofuran (bisTHF) and diamino-bisfuran are reported. The bisTHF
ligand with RR configuration at the amino groups and C8 alkyl chains (RR8)
stabilized GC-rich duplex. In contrast, bisfuran compounds stabilized AT-rich
duplex. The binding affinity of RR8 with 12 mer duplex DNA was determined
8
−1
.
by isothermal titration calorimetry to be 3.3 × 10 M
DNA-binding molecules with sequence specificity have become of great in-
terest, because of the potential application for the regulation of gene expression at
the specific site. Among DNA-binding molecules including triplex-forming oligo-
nucleotides or peptides (TFO or PNA) (1), low molecular weight ligands that bind
to DNA within the minor groove have shown rapid development recently (2).
Encouraging work has been reported by Dervan’s group, in which recognition of
full four-codes of DNA sequences within the minor groove has been demonstrated
with the use of synthetic pyrrole-imizazole polyamides (3). In this case, stacked- or
covalent dimer structures of binding motifs play a significant role for the formation
∗
Corresponding author.
551
Copyright ꢀC 2001 by Marcel Dekker, Inc.
www.dekker.com

ORDER
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5
52
SASAKI ET AL.
specific hydrogen bondings with each of the bases at the bottom of the minor
groove. On the other hand, other types of minor groove ligands such as Hoechst
33258 and CC-1065 bind to DNA as a monomer with sequence preference. In the
binding of Hoechst 33258 to DNA, hydrophobic interaction has been determined
as a major contributor of sequence selectivity (4). In the conceptual mechanism,
the first step of minor-groove binding may contain a hydrophobic transfer of the
ligand from aqueous solution to the DNA minor groove following arrangement of
specific interactions such as hydrogen bondings and van der Waals attraction (5).
The shape of the ligand isohelical with the curvature of the groove has been also
regarded as an important factor of the binding (6). We have been interested in the
development of new ligands based on an aliphatic non-planar structure, because
such binders would provide useful information for the design of new ligands. Here
we wish to report new high-affinity DNA binding molecules with an emphasis on
hydrophobic interactions.
We designed a series of new DNA binders based on a bistetrahydofuran
(bisTHF) skeleton bearing cationic groups and long alkyl chains. The idea was
originated from the facts that a helix-like conformation was suggested in studies
of naturally-occurring bisTHF compounds (7) or in oligoTHF compounds (8). A
molecular modeling of the bisTHF molecule also suggested that the dihedral angle
between the two tetrahydrofuran rings might be adjustable to fit into the minor
groove, and the molecular shape might be isohelical to the groove (Fig. 1).
We first synthesized diamino-bisTHF (1(RR8)) with (R,R)-configuration of
the amino groups (9). Interestingly, 1(RR8) raised the melting temperature of a
GC-rich duplex (G16-C16) by 13 degrees but showed no effect on an AT-rich du-
plex (A16-T16). Since 1(RR8) has neither aromatic systems nor planar structure
such as seen in most minor-groove binders, it seemed to be worthy for further in-
vestigation. Then, we systematically synthesized several bisTHF compounds (1–4)
having different stereochemistry, length and functionality of the alkyl chains. The
bisfuran compounds (5) were also synthesized for the comparison with bisTHF
ligands (Fig. 2).
H N
2
(CH ) CH
2 8 3
?
O
O
(
CH ) CH
3
2
8
H N
2
A
B
C
Figure 1.

ORDER
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NOVEL CLASS OF DNA BINDING MOTIFS
553
X
(A, B)
1(RR8) (R, R)
(RR7) (R, R)
(RR6) (R, R)
1(RR5) (R, R)
(RR4) (R, R)
n
8
7
6
5
4
0
8
8
8
8
8
X
R
H
H
H
H
H
H
H
H
A
NH
NH
NH
2
2
(
CH ) CH R
2 n 2
1
1
2
O
O
NH
2
2
1
NH
1
(RR0) (R, R)
NH
2
1(RS8)
(R, S)
(S, S)
(R, R)
(R, R)
(R, R)
NH2
1
(SS8)
NH
2
2
3
4
OH NH
2
(
CH ) CH R
B
2 n
2
NH OTHP
2
2
X
X
NH
OH
A
(
CH ) CH R
2 n 2
O
O
5
(meso) meso
8
NH
H
H
2
2
5
(racemi) racemi 8 NH
(
CH ) CH R
2
n
2
B
X
Figure 2.
The diamino-bisTHFs (1–4) were synthesized from respective diepoxy inter-
mediates (6) which were derived from diethyl isopropylidene D-tartrate (Scheme 1)
(10). Each epoxydes (6) was alkylated using Grignard reagents with catalytic
amount of CuBr to afford the secondary alcohols (7–8). Mitsunobu reaction of
these alcohols with HN gave diazido compounds involving the stereochemical in-
3
version at both hydroxyl groups. Finally, diamino-bisTHFs (1, 3, 4) were obtained
by reduction of the azido group with lithium aluminum hydride (LAH). The ligand
(2) was synthesized in a similar manner. For the synthesis of diamino-bisfurans,
5-bromo-2-furoic acid amyl ester was first coupled with Cu powder in DMF under
reflux to afford bisfuran building block 9. Diester 9 was transformed into a hy-
droxyalkyl intermediate 10. Hydroxyl groups of 10 were displaced by phthalimide
by Mitsunobu reaction to afford corresponding phthalimide derivatives as mixture
of stereoisomers. The isomers of dl- and meso form were separated by fractional
recrystallization, and the configuration of theless soluble derivative was determined
by X-ray crystallographic analysis to be meso. Finally, bothof meso and racemic
compounds were refluxed with hydrazine to furnish 5. The diamino-bidTHFs and
diamino-bisfurans were used as hydrochlorides in the following binding experi-
ments.
Interaction between the duplex DNA and the ligand was first evaluated by
measuring effect on the melting temperature (Tm) of the 16bp duplex (G16-C16
and A16-T16) (Table 1). Tm value of G16-C16 duplex was raised with 1(RR8)
by 13 degrees, whereas that of A16-T16 duplex was not affected, indicating that
1(RR8) stabilized G16-C16 selectively. Stereochemistry of the amino groups influ-
enced the stabilizing effect; order of the Tm values of G16-C16 duplex decreased
in the order of 1(RR8) > 1(RS8) > 1(SS8), suggesting that the stereochemistry

ORDER
REPRINTS
5
54
SASAKI ET AL.
X
HO
O
(
CH ) CH R
2 n 2
OPNB
OPNB
O
O
O
O
a
O
X
O
(CH ) CH R
2
n
2
HO
O
510
A, B)
6
7,8
(
n
X
R
7
7
7
7
7
7
7
7
a (R, R)
b (R, R)
c (R, R)
d (R, R)
e (R, R)
f (R, R)
g (R, S)
h (S, S)
8
OH
OH
OH
OH
OH
OH
OH
OH
H
H
H
H
H
H
H
H
7
6
5
4
0
8
8
8
b
1
c
3
4
2
b
d
8
(R, R)
OH OTHP
HO
O
OCH3
N
(CH ) CH3
2 8
Br
O
CH3
O
O
O
g
e
f
5
O
COOH
CH3
N
OCH3
(CH ) CH3
2 8
O
HO
9
10
a
a) RCH (CH ) MgBr, CuBr, THF, b) (1) HN , Ph P, DEAD, THF, (2)
2
2 n
3
3
LiAlH , THF, c) 1M HCl, THF.d) (1) TsOH, H O/EtOH/THF, (2)
4
2
phthalimide, Ph P, DEAD, THF, (3) hydrazine, EtOH, e) 1)
3
p-TsOH•EH O, PeOH, 2) Cu powder, DMF, 3) 1N NaOH , MeOH, 4)
2
aq
SOCl ,Pyridine, CH Cl , 5) N,O-Dimethylhydroxylamine, pyridine,
2
2
2
CH Cl , f)1) CH (CH ) MgBr, CH Cl 2) NaBH , CH Cl -EtOH, g) 1)
2
2
3
2 9
2
2,
4
2
2
phthalimide, Ph P, DEAD, THF, (2) hydrazine, EtOH.
3
Scheme 1.

ORDER
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NOVEL CLASS OF DNA BINDING MOTIFS
555
Table 1. Melting Temperature (T
m
) Changes by Diamino-
bisTHFs and Diamino-bisfurans
DNA
sequence
a)
◦
Compound
ꢀT
m
( C)
1
1
1
(RR8)b
(RS8)b
(SS8)b
G16-C16
A16-T16
G16-C16
A16-T16
G16-C16
A16-T16
13
0
7
0
0
0
5
5
(meso)c
G16-C16
A16-T16
G16-C16
A16-T16
0
4
0
9
(racemi)c
a) 1µM DNA in the buffer containing 100 mM KCL, 5 mM
Na HPO at pH 5.0 for G16-C16 or at pH 7.0 for A16-T16,
b) 20 µM 1 was used, c) 50 µM 5 was used.
2
4
G16-C16
Tm=62°C)
5' GGGGGAGGGGGCGGGA 3'
3' CCCCCTCCCCCGCCCT 5'
(
A16-T16
5
' AAAGAAAAAGAAAAAC 3'
(
Tm=40°C)
3' TTTCTTTTTCTTTTTG 5'
of the amino group has a key role in DNA stabilization. In contrast, diamino-
bisfuran 5(meso) and 5 (racemic) raised Tm of A16-T16 duplex selectively by 4 and
9
degrees, respectively. The stereochemistry of the amino groups of bisfuran also
affected DNA stabilization. It should be noted that bisTHF and bisfuran skeletons
apparently play a key role in sequence preference of the ligands.
We next evaluated binding affinity of the ligands by a competitive binding
assay using fluorescent ethidium bromide (ETBr). Emission intensity of ETBr is
increased by binding with duplex DNA, and is quenched in the presence of a
competitive binder. Concentration of the ligand to inhibit 50% of fluorescence
intensity of ETBr (C ) corresponds to a relative binding affinity of the ligand
5
0
(Table 2).
In this experiment, self-complementary 12bp duplexes CT12 and CA12 were
used, in which a GCGC or an AATT regions are included in the middle of CT12 or
CA12, respectively. High affinity of 1(RR8) to both duplexes was confirmed with
affinity as high as distamycin. Importance of the stereochemistry of 1 has been
again observed in this experiment, that is, the affinity decreased in the order of
1(RR8) > 1(RS8) > 1(SS8). The sequence preference of the bisTHF ligands was
not clear. The high affinity of 5(meso) as well as sequence preference to CA12 was
shown in this assay. Either the ligand 2 or the ligand 4 did not show significant
affinity. Protection of the hydroxyl group of 4 with tetrahydropyranyl group (3)
recovered binding affinity, indicating that hydrophobicity of the alkyl chain terminal

ORDER
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5
56
SASAKI ET AL.
Table 2. Comparison of the DNA Binding Affinitya)
50 (µM)
C
CT12
CA12
Compound
(K
ETBr = 2.4 × 106
)
(K
ETBr = 7.6 × 106
)
1
1
1
1
1
1
2
3
4
5
1
(RR8)
(RS8)
(SS8)
(RR7)
(RR6)
(RR5)
7
19
30
5.5
13
30
34
23
135
760
300
32
490
17
125
880
330
27
540
4
(meso)
(racemi)
>100
19
>100
>15
distamycin
a) 1.5 µM DNA and 1.5 µM ETBr were used in the buffer containing
9
.4 mM NaCl, 2.0 mM HEPES, 10 mM EDTA, pH 7.0.
CT12: 5' CGTAGCGCTACG 3' CA12: 5' CGCGAATTCGCG 3'
GCATCGCGATGC
GCGCTTAAGCGC
is also important for binding. These results have suggested that the hydrophobic
nature of the termini of alkyl chains is also important for binding affinity. It is
quite interesting that the linear correlation was between C values and the length
5
0
of alkyl chains of bisTHF ligand 1. These results clearly suggest that hydrophobic
interaction is the major contributor in the binding of bisTHF ligands.
A similar displacement assay was done with Hoechst33258, a strong minor-
8
groove binder with high affinity of about 3 × 10 to CA12 (4). In this assay, 5(meso)
displacedHoechst33258almostcompletelywithC valueof23µM, indicatingthat
5
0
the bisfuran ligand shares the binding site of CA12 with Hoechst33258, probably at
the AATT region, with relatively low affinity. Interestingly, addition of the bisTHF
ligand 1(RR8) to the complex between Hoechst33258 and CA12 changed the flu-
orescence spectra, indicating second binding between 1(RR8) and Hoechst33258•
CA12 complex. As 1(RR8) has been suggested to be GC-selective, 1(RR8) might
bind the terminal GCGC region of CA12. A similar displacement assay with CT12
and Hoechst33258 could not be done because of weak complexation between these
two. The binding affinity of 1(RR8) with CT12 was determined by isothermal
8
−1
titration calorimetric (ITC) experiments to be K = 3.36 × 10 M .
a
In conclusion, we have developed new DNA-binding molecules with unusual
binding motifs, diamino-bisTHF and diaminobisfuran. These molecules were ex-
pected to take a stable conformation with a crescent shape that seemed to be suitable
to fit to the minor groove. As a result, the binding affinity of 1(RR8) has been deter-
8
−1
mined to be as high as a binding constant of 10 M . It has been also clearly shown

ORDER
REPRINTS
NOVEL CLASS OF DNA BINDING MOTIFS
557
that both the stereochemistry of the amino groups and the alkyl chain length play
major roles for the DNA binding. The importance of the hydrophobic interaction
in DNA binding has been now clearly confirmed in this study. In the measurement
of melting temperature, diaminobisTHF 1(RR8) showed sequence preference to
GC-rich duplex, and diaminobisfuran 5 indicated preference to the AT-rich duplex.
Most minor-groove binders have preference to AT-tract, therefore, GC-preference
of the new ligand 1(RR8) will provide useful information for binding with GC-
rich sequence. A further investigation is now ongoing with footprinting analysis to
determine sequence selectivity in detail.
ACKNOWLEDGMENTS
This work was supported by Grant-in-Aid for Scientific Research from the
Ministry of Education, Science, Sports and Culture, Japan (C09672146 to SS). We
also grateful to Professor Tomofumi Miyamoto, Graduate School of Pharmaceutical
Sciences, Kyushu University, Japan, for X-ray analysis of the meso phthalimide
derivative of 10.
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9
Abbreviation in parentheses following compound number indicates the stereochem-
istry of the amino groups and the alkyl chain length. For example, 1(RR8) means that
the compound 1 has R, R configuration at the A and B position and a nonyl group
(
(CH2)8CH3) for alkyl chains. All of the bisTHF compounds used in this study have
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R,R,R,R) configuration as shown in Figure. 2.
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SASAKI ET AL.
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