DNA sequence-specific ligands: XI. The synthesis and binding to DNA of bis-netropsins with the C-ends of their netropsin fragments tethered by tetra- or pentamethylene linkers

Article abstract of DOI:10.1023/A:1021241515208

Bis-Netropsins with the C-ends of their netropsin fragments tethered via tetra- or pentamethylene linkers and with Gly or L-Lys-Gly residues on their N-ends were synthesized. The footprinting technique was used to study the specificity of bis-netropsin bi

Full text of DOI:10.1023/A:1021241515208

Russian Journal of Bioorganic Chemistry, Vol. 28, No. 6, 2002, pp. 455–469. Translated from Bioorganicheskaya Khimiya, Vol. 28, No. 6, 2002, pp. 502–517.
Original Russian Text Copyright © 2002 by Grokhovsky, Nikolaev, Gottikh, Zhuze.
DNA Sequence-Specific Ligands: XI.* The Synthesis
and Binding to DNA of bis-Netropsins with the C-Ends
of Their Netropsin Fragments Tethered
by Tetra- or Pentamethylene Linkers
S. L. Grokhovsky1, 2,** V. A. Nikolaev1, B. P. Gottikh1, and A. L. Zhuze1
1Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, ul. Vavilova 32, Moscow, 119991 Russia
2Moscow Center of Medical Research of Oslo University, ul. Vavilova 32, Moscow, 119991 Russia
Received October 12, 2001; in final form, February 4, 2002
Abstract—Bis-Netropsins with the C-ends of their netropsin fragments tethered via tetra- or pentamethylene
linkers and with Gly or L-Lys-Gly residues on their N-ends were synthesized. The footprinting technique was
used to study the specificity of bis-netropsin binding to the specially constructed DNA fragments containing
various clusters of A · T pairs. It was found that the linker length affects the binding of bis-netropsins, with the
tetramethylene linker providing better protection than the pentamethylene linker. It was shown that the newly
synthesized bis-netropsins bind tighter to the 5'-A4T4-3' sequence, whereas the bis-netropsin with a linker
between the netropsin N-ends binds better to 5'-T4A4-3' sequences.
Key words: bis-netropsins, chemical synthesis, distamycin A, DNA sequence-specific ligands, footprinting,
netropsin
INTRODUCTION
clusters of A · T pairs. Subsequently, the sequence-spe-
cific compounds on the basis of various combinations
of pyrrole- and imidazolecarboxamide building blocks
were successfully developed in other laboratories [7,
17–20].
Natural antibiotics netropsin and distamycin A form
tight noncovalent complexes with A · T-enriched
regions of double-helical DNA [2–10]. A structure of
3
a new class of compounds that should preferably recog-
nize certain clusters of DNA A · T pairs was suggested
on the basis of a molecular model of netropsin– and dis-
tamycin A–DNA complexes, according to which anti-
biotic molecules are disposed in the complexes along
the DNA minor groove [4]. A general strategy for con-
structing the compounds of this type is the introduction
of various linkers between one or several pyrrolecar-
boxamide groups, which allows a definite orientation
and fixing of these fragments in the DNA complex.
Bis-Netropsins constituted of two molecules of a
netropsin analogue whose N-ends were tethered by the
residues of aliphatic dicarboxylic acids with various
numbers of methylene groups were the first compounds
of this class, synthesized in 1982 [11]. The study of
physicochemical [6, 12] and biochemical [6, 13–16]
properties of their complexes with DNA showed that
the netropsin fragments are specifically bound to two
We report herein the synthesis and the specificity of
DNA binding of bis-netropsins in which the C-ends of
netropsin fragments are linked by the residues of α,ω-
diaminoalkanes of various lengths and contain Gly or
L-Lys-Gly residues on their N-ends.
RESULTS AND DISCUSSION
Chemical formulas of netropsin, distamycin A, and
bis-netropsins we synthesized [(I), (Ia), (II), and (IIa)]
are shown in Fig. 1. Bis-Netropsins contain two netrop-
sin fragments that are built of two N-propylpyrrolecar-
boxamide blocks linked by five, (I) and (II), or four (Ia)
and (IIa), methylene groups. Each of the di-N-propyl-
pyrrolecarboxamide blocks differs from the netropsin
molecule not only in the structure of its C-terminal res-
idue and the substitution of n-propyl for methyl groups
at the pyrrole nitrogen atoms, but also in the substitu-
tion of Gly [in (I) and (Ia)] or L-Lys-Gly [in (II) and
(IIa) residues for the guanidylacetic acid residue.
According to the X-ray analysis of oligonucleotide
complexes with netropsin [21] or distamycin A [22],
these structural changes should not affect the specificity
of these compounds toward the DNA A T pairs, since
this specificity results from the formation of hydrogen
*For communication X, see [1].
**The author may be contacted by phone, +7 (095) 135-9718; fax,
+7 (095) 135-1405; or e-mail, grokhovs@imb.ac.ru.
3
Abbreviations: Apc, 1-propyl-4-aminopyrrole-2-carbonyl resi-
due; CDI, N,N'-carbonyldiimidazole; HOBt, 1-hydroxybenzotri-
azole; Im, imidazole residue; Npc, 1-propyl-4-nitropyrrole-2-car-
bonyl residue. All the nucleotide sequences are given in 5'
direction.
3'
1068-1620/02/2806-0455$27.00 © 2002 MAIK “Nauka /Interperiodica”

456
GROKHOVSKY et al.
H
H
C N
H2N
H2N
H
C N
O
H
C N
O
O
N
H
N
H
C N
O
H
C N
O
N
N
R
H
C N
O
H
C N
O
N
N
NH2
NH2
NH2
NH2
Netropsin
Distamycin
H
N
H
N
H
N
H
N
R
A
N
A
N
C
C
H
H
B
N
B
N
O
O
C N (CH2)n N C
O
O
C
O
Bis-netropsin (I) R =
Bis-netropsin (Ia) R =
H3N
H3N
, n = 5
C
, n = 4
O
H3N
H
N
Bis-netropsin (II) R =
Bis-netropsin (IIa) R =
, n = 5
C
O
C
H3N
O
H3N
H
, n = 4
N
C
C
H3N
O
O
H
N
O C
H
H
N
CH3
N
H2N
H2N Pt
H
N C
O
H3C
C
C
O
N
N
H
N
O
H
N
H3N NH3
H
C N
O
N
C
O
CH3
N
N
H
CH3
Pt-bis-netropsin
Fig. 1. Chemical structures of netropsin, distamycin, and bis-netropsins
bonds between nitrogen atoms of the ligand amide group enables its further modifications when obtaining
more complex DNA-specific ligands.
groups and acceptor groups of base pairs in the DNA
minor groove. In addition, the yields of N-propyl deriv-
atives of 4-nitro- and 4-aminopyrrole-2-carboxylic
acids are higher in comparison with the yields of the
corresponding N-methyl derivatives [3], and the
Synthesis of bis-Netropsins
Bis-Netropsins (I) and (II) with pentamethylene
replacement of the terminal guanidine group by amino linker were synthesized according to Scheme 1; a sim-
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DNA SEQUENCE-SPECIFIC LIGANDS: XI.
457
O2N
O2N
H
N
H
1. CDI
2. H2N–(CH2)n–NH2
C
O
C
O
N
H
N
N
C
O
OH
C N (CH2)5 NH2
O
N
N
N
(III)
O2N
(V)
NO2
H
H
C
N
N
C
O
H
H
N
O
C N (CH2)n N C
N
N
O
O
(IV), n = 5
(IVa), n = 4
1. H2/Pt
2. Boc-Gly-Im
3. TFA
H
H
H3N
C N
O
N C
O
NH3
H
N
H
N
C
O
C
O
H
H
N
N
C N (CH2)n N C
N
N
O
O
(I), n = 5
(Ia), n = 4
NH3
NH3
NH3
1. Boc-Lys(Z)-Im
2. TFA
3. H2/Pd
H
H
C N
O
H
H
C N
O
N C
O
N C
O
H3N
H
N
H
N
C
O
C
O
H
H
N
N
C N (CH2)n N C
N
N
O
O
(II), n = 5
(IIa), n = 4
Scheme 1.
ilar scheme was used for bis-netropsins with the tetra- protective groups were removed to give (I) and (Ia) in
32 and 24% yields, respectively.
methylene linker. Hereinafter, the homologous com-
pounds with 1,4-tetramethylendiamine (putrescine)
linker in place of a 1,5-pentamethylendiamine (cadav-
erine) linker are marked with index a. Imidazolide of
(III), synthesis of which was previously described [23],
was coupled with cadaverine in DMF. The yields of
dinitroderivative (IV) and its lower homologue (IVa)
were 59 and 46%, respectively. When synthesizing
(IV), we also isolated a monoacylcadaverine derivative
(V) in 12% yield. After reduction of nitro groups in
(IV) and (IVa) by hydrogenation in the presence of
Adams catalyst, the resulting diamino derivatives were
The resulting compounds were poorly soluble in
water under neutral pH. The introduction of two Lys
residues at Gly amino groups using N,N'-carbonyldiim-
idazole substantially improved the solubility of bis-
netropsins (II) and (IIa).
Bis-Netropsins (I) and (Ia) were also synthesized
according to Scheme 2. The coupling of 1-propyl-4-
nitropyrrole-2-carbonyl chloride with cadaverine or
putrescine and the subsequent reduction of intermedi-
ate (VI) and (VIa) over the Adams catalyst led to the
corresponding diaminoderivatives, which were coupled
coupled in situ with imidazolide of Boc-glycine. The in situ with Z-Gly-Apc-OH (VII) using the carbodiim-
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458
GROKHOVSKY et al.
O2N
O2N
NO2
H
H
C N (CH2)n N C
H2N–(CH2)n–NH2
N
N
C Cl
O
N
O
O
(VI), n = 5
(VIa), n = 4
1. H2/Pt
2. Z-Gly-Apc-OH, HPBt, DCC
(VII)
H
O C N
O
H
N C O
O
H
H
C N
O
N C
O
H
N
H
N
C
C
O
H
H
N
N
O
C N (CH2)n N C
N
N
O
O
(VIII), n = 5
(VIIIa), n = 4
1. H2/Pd
H3N
NH3
H
C N
O
H
N C
H
N
H
N
O
C
O
C
O
H
H
N
N
C N (CH2)n N C
N
N
O
O
(I), n = 5
(Ia), n = 4
Scheme 2.
ide method. Bis-Netropsins (I) and (Ia) were prepared depend on the mutual disposal of adenine and thymine
by catalytic hydrogenation of intermediate (VIII) and residues in complementary DNA strands [26]. The
(VIIIa), respectively, over a palladium catalyst. Overall tightest binding was observed for the sites containing
yields of bis-netropsin (I) according to Schemes 1 and thymine residues only in one of the strands and for the
2 were 19 and 28%, respectively; and those of bis- sites containing the AT sequence. The presence of the
netropsin (Ia), 11 and 24%, respectively.
TA sequence in the site weakens the binding [26]. Con-
formational calculations [30], X-ray data [31], and the
results of the DNA cleavage with hydroxyl radicals
[32] suggest that the minor grooves of the binding sites
containing the TT sequences or the AT sequence are
narrowed and their size corresponds to the thickness of
netropsin molecule. When being inserted in this
groove, netropsin forms numerous van der Waals con-
tacts, which give rise to a tight complex [24]. These
considerations are also valid for the case of distamycin
binding to DNA; the only exception is that it recognizes
five rather than four A · T pairs.
The Specificity of bis-Netropsin Binding to DNA
In order to determine the DNA sequences to which
bis-netropsins are most affine, the sequences with the
highest affinity to the netropsin fragment that consists
of two pyrrolecarboxamide blocks should preliminarily
be found. According to X-ray analysis of netropsin and
distamine complexes with various double-helical oli-
godeoxynucleotides, the amide groups of the antibiot-
ics can form a bifurcated (three-centered) hydrogen
bond with the neighboring N3 atoms of adenine and/or
O2 atoms of thymine in adjacent A · T pairs of comple-
The binding sites containing pyrimidine–purine TA
and/or GC sequences have the widened minor groove
mentary DNA strands [21, 22]. The NMR data [24] and because of small overlapping of neighboring base pairs.
footprinting analysis using DNase I [14, 25–28] and For distamycin and some of its analogues, the side-by-
hydroxyl radicals [29] suggest that netropsin binds to side parallel insertion of two antibiotic molecules into
the site composed of four A · T pairs in the DNA minor the widened minor groove was found using NMR spec-
groove. The constants of netropsin binding to such sites troscopy [33]. The monomeric binding of netropsin and
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DNA SEQUENCE-SPECIFIC LIGANDS: XI.
459
distamycin to the sequences containing G · C base pairs
Using statistical analysis of a large number of bind-
is energetically unfavorable because of steric hin- ing sites, we previously found that Pt-bis-netropsin in
drances resulting from the guanine 2-amino group situ- which two netropsin fragments were linked through
ated in the minor groove [4, 21], whereas the dimeric their N termini via cis-diammineplatinum(II) linker
binding is possible in the case of sites with widened (Fig. 1) forms the tightest complex with the T4A4
minor grooves [34].
sequence [40]. In bis-netropsin molecules (II) and
(IIa), the netropsin fragments linked by their C-ends
are oriented in opposite directions. Therefore, one can
expect that these bis-netropsins would form tighter
complexes with the A4T4 sequence.
The ligand molecule can be incorporated into the
minor groove in two orientations in the process of
monomeric binding to nonpalindromic DNA
sequences, for example, to the TTTT sequence [24].
The introduction into the terminal regions of netropsin
or distamycin molecules of a group capable of cleaving
the DNA sugar–phosphate backbone allows the deter-
mination of both the binding specificity and the orien-
tation of the molecule in the minor groove [35–37]. In
the case of the TTTT sequence, netropsin analogues are
predominantly oriented along the groove in such a
manner that their amidine moiety (the C-end) is situ-
ated at the 5'-end of this sequence. Therefore, the bis-
netropsins, in which two netropsin fragments are in dif-
ferent orientations, should have the highest affinity to
different sites. In addition, as follows from the behavior
of the duplexes containing octanucleotides with
reversed polarities (A4T4 and T4A4) using the gel
mobility shift assay in PAG [38] and molecular model-
ing [30, 39], the polarity of a nucleotide sequence can
be an important parameter that determine the DNA
structure and curvature, which can affect the binding of
extended ligands along the DNA minor groove.
In addition to the contribution of each of the two
netropsin fragments, the nature of linker also dramati-
cally affects the bis-netropsin affinity to various DNA
sequences. Several structures of DNA–bis-netropsin
complexes are possible in the minor groove:
(1) Both netropsin fragments form specific com-
plexes with DNA. To realize this possibility, DNA
should contain two closely situated recognition sites,
and linker should not prevent them from mutual bind-
ing. An excessively long linker can lead to the possibil-
ity of binding of the second half of bis-netropsin to sev-
eral closely situated sites. To reduce the number of pos-
sible variants, we chose comparatively short linkers,
although this increased the probability of preventing
the specific binding of one block by the binding of
another one.
We constructed plasmids bearing several potential
binding sites in the polylinker part in order to study the
binding of various bis-netropsins by DNA [28]. Each
site contains two netropsin binding sites that include
various sequences formed by four- or five A · T base
pairs either closely disposed or separated by one, two,
or three G · C pairs. These plasmid regions included the
sequences containing four A · T pairs in various combi-
nations and surrounded with the G · C-enriched clus-
ters. The sequences of two constructed DNA fragments
Ä and B are shown in Fig. 2.
The Specificity of Binding of bis-Netropsins (II) and
(IIa) to the DNA Fragment Ä
The patterns of cleavage of the upper strand of
radioactively labeled DNA fragment A by hydroxyl
radicals and by the DNase I in the presence of bis-
netropsins (II) and (IIa) at various concentrations are
shown in Figs. 3 and 4, respectively. One can see in the
patterns that all of the four above discussed variants of
the structures of bis-netropsin–DNA complexes appear
to be realized in the presence of bis-netropsins (II) and
(IIa). We observed that, at ligand concentrations of 8–
10 µM, which corresponds to approximately one ligand
molecule per four DNA base pairs, the whole fragment
A and even the GC sequences in it were strongly pro-
tected from the cleavage. Under these saturation condi-
tions, the formation of various types of dimeric or mul-
timeric [41] poorly specific complexes is possible.
If the bis-netropsin concentration did not exceed 4–
5 µM, they protected from cleavage only the sites con-
taining no less than four successive A · T pairs. Bis-
Netropsin (IIa) containing the tetramethylene linker
was a more potent protector than bis-netropsin (II) with
pentamethylene linker (Fig. 4).
(2) If there is only one binding site or the linker hin-
ders binding of the second half of bis-netropsin, only
one of the netropsin fragments would tightly bound by
DNA, whereas another one would either be expelled
out of the groove or be inside it, but would not form
specific contacts.
(3) For bis-netropsins, the side-by-side insertion in
the minor groove of two netropsin fragments and for-
mation of pins are possible.
These compounds are most tightly bound to the
sequence A5T5 [site (4)]: at the concentrations of 1.2
and 0.6 µM, respectively, bis-netropsins (II) and (IIa)
protect from cleavage about 7 bp. Bis-Netropsin (IIa)
at the concentration of 2.5 µM protects from cleavage
only 4-bp region situated at 3' end in sequences T5A5
[site (6)] and T5ëA5 [site (2)]. As in the case of the
TTAT sequence [site (3)], this suggests the formation of
(4) The binding and simultaneous side-by-side a hairpin-like structure [42]. An indirect proof of this
incorporation of netropsin fragments from different fact is the intensification of the effect of hydroxyl radi-
bis-netropsin molecules in the same binding site is also cals of the neighboring sites, which implies the broad-
possible.
ening of the minor groove. This intensification was not
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GROKHOVSKY et al.
Ä
(1)
(2)
5'
3'
(3)
(4)
(5)
(6)
(7)
(8)
(9)
3'
5'
B
(1)
(2)
(3)
(4)
5'
3'
(5)
(6)
(7)
(8)
(9)
(10)
3'
5'
Fig. 2. Nucleotide sequences of DNA restriction fragments A and B. The sites designated in Figs. 3–7 are marked and numbered.
observed in the case of the AAAT sequence [site (8)]; the minor groove and enables each of the netropsin
blocks to form tight specific contacts.
in this case, only one of the netropsin fragments is prob-
ably involved in the binding. In the ATATGGATAT
sequence [site (1)], (IIa) protects about 10 bp, with a
weak cleavage being observed in the center of the
sequence. In this case, two bis-netropsin molecules
appear to be bound in the form of hairpins [43].
The Binding Specificity for Netropsins (II) and (IIa)
to the DNA Fragment B
Since the analysis of the sites protected in fragment
A revealed that bis-netropsins (II) and (IIa) are bound
by the sequences containing alternating A · T and G · C
base pairs, we analyzed similarly the fragment B con-
taining many such sequences. Figures 5 and 6 present
the patterns of cleavage with DNase I of the upper and
lower strands, respectively, of radioactively labeled
fragment B at various concentrations of bis-netropsins
(II) and (IIa). The cleavage patterns of the lower strand
of fragment B with hydroxyl radicals are shown in
Fig. 7.
As a whole, these data suggest that bis-netropsins
(II) and (IIa) are more affine to the A5T5 than to the
T5A5 sequence. Nevertheless, this difference is much
weaker pronounced than in the case of Pt-bis-netropsin
containing netropsin fragments in opposite orientations
and specifically affine to the sequence T5A5 [40]. This
may be due to inability of the linkers under study to
ensure the optimal transition for “reading” twoAT clus-
ters with two netropsin fragments in opposite orienta-
tions. On the other hand, Pt-bis-netropsin not only
exhibits the optimal orientation of netropsin fragments,
but also has a bulky cis-diammineplatinum group that
One can see in Figs. 5–7 that bis-netropsin (IIa)
with tetramethylene linker, unlike bis-netropsin (II)
with pentamethylene linker, is bound, (e.g. Fig. 5), by
well incorporated into the TA-determined widening of TGGTACCAC [site (4)], AACCTTTT [site (3)],
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DNA SEQUENCE-SPECIFIC LIGANDS: XI.
461
TGTTGGT [site (2)], and some other similar sequences
at the concentration of 2.5–5.0 µM. The binding by
these sequences is most probably due to the formation
of hairpin-like structures. Thus, in fragment B, bis-
netropsin (IIa) exhibits a more strong protection from
cleavage than bis-netropsin (II) whose linker is only
one methylene unit longer.
(IIa)
(II)
0
0
1 2 3 4 5 6 7 8 9 101112
EXPERIMENTAL
5' → 3'
Solutions of substances in organic solvents were
dried over Na2SO4. Solvents were removed on a rotary
evaporator at 20°ë in the vacuum of a water-jet or oil
pumps. Compounds were dried in a vacuum over P2O5
and NaOH. Melting points were determined on a Boet-
hius device (Germany) and were not corrected. Hydro-
genation was carried out over the Adams catalyst [44]
at atmospheric pressure and 25°ë until a vigorous
hydrogen absorption was ceased. Purity of the resulting
compounds was monitored by TLC on Silufol UV-254
precoated plates (Czech Republic) in the following sys-
tems: 4 : 1 : 1 n-BuOH–AcOH–water (A); 7 : 1 : 3
PriOH–conc. ammonia–water (B); 3 : 7 1 M AcONH4
(pH 7.6)–EtOH (C); and 95 : 5 CHCl3–absolute EtOH
(D). The spots of substances were detected on chro-
matograms according to the UV-light absorption at
254 nm or with ninhydrin. If not stated otherwise, the
column chromatography was carried out on a Sephadex
LH-20 (Pharmacia, Sweden) column (3 × 150 cm)
eluted with methanol at 4°ë and a rate of 15 ml/h; 15-
ml fractions were collected. The elution was monitored
using a UV flowing densitometer Uvicord II-8300
(Sweden). Final purification of (I), (Ia), (II), and (IIa)
and analysis of their homogeneity was carried out by
the reversed-phase HPLC on a silica gel C-18 (6 µm)
column (250 × 15 mm) (SKB IOKh AN SSSR) under
elution by a linear gradient of acetonitrile in 0.1% TFA
(0–80% for 45 min; flow rate 4.8 ml/min). HPLC was
performed on a Gilson chromatograph (France) com-
prising model 302 and 303 pumps, a model 803C
manometer, a 811 mixer, a Data Master-621 control
unit, and a model 117 UV detector. The light absorption
of eluates was determined at 240 and 300 nm.
(1)
(1)
(2)
(2)
(3)
(3)
(4)
(4)
(5)
(5)
(6)
(6)
(7)
(7)
1H NMR spectra were registered on a Varian XL-
100 spectrometer (United States) with a working fre-
quency of 100 MHz at 33°ë in DMSO-d6 with Me4Si
as an internal standard (δ 0.00). Proton chemical shifts
(δ) are given in ppm. The symbols of pyrrole cycles (A,
B) are depicted in Fig. 1.
(8)
(8)
(9)
(9)
Mass spectra were registered on an MS-50TC spec-
trometer (Kratos, UK) using the fast atom bombard-
ment with xenon at 6–8 keV and glycerol as a matrix.
DNA fragments were prepared by digestion of a
modified plasmid pGEM7(f+) (Promega) containing an
insert of synthetic oligonucleotides within the
polylinker with restriction endonucleases XbaI and NsiI
(Promega) (fragment Ä) [28] or EcoRI and HindIII
(Promega) (fragment B) [16]. The radioactive label was
Fig. 3. The patterns of cleavage with hydroxyl radicals of
the upper DNA fragment A: 1, initial (untreated) fragment;
2, A + G, chemical cleavage at purine residues; 3 and 8, the
cleavage of the free fragment; 4–7 and 9–12, the fragment
cleavage in the presence of 8, 4, 2, and 1 µM bis-netropsins
(IIa) and (II), respectively.
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GROKHOVSKY et al.
(IIa)
(II)
0
0
1 2 3 4 5 6 7 8 9 10 11 12 1314 15 16 17 18
5' → 3'
(1)
(1)
(2)
(2)
(3)
(3)
(4)
(4)
(5)
(5)
(6)
(6)
(7)
(8)
(7)
(9)
(8)
Fig. 4. The patterns of cleavage with DNase I of the upper DNA fragment A: 1 and 10, initial (untreated) fragment; 2 and 11, chem-
ical cleavage at purine residues; 3 and 12, cleavage of the free fragment; 4–9 and 13–18, the fragment cleavage in the presence of
10, 5, 2.5, 1.25, 0.62, and 0.31 µM bis-netropsins (IIa) and (II), respectively.
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DNA SEQUENCE-SPECIFIC LIGANDS: XI.
463
(IIa)
(II)
0
0
1 2 3 4 5 6 7 8 9 10 1112 1314 151617 18
5' → 3'
(1)
(1)
(2)
(3)
(2)
(4)
(3)
(5)
(4)
(6)
(5)
(6)
(7)
(7)
(8)
(9)
(8)
(9)
(10)
Fig. 5. The patterns of cleavage with DNase I of the upper DNA fragment B: 1 and 10, initial (untreated) fragment; 2 and 11, chem-
ical cleavage at purine residues; 3 and 12, cleavage of the free fragment; 4–9 and 13–18, the fragment cleavage in the presence of
10, 5, 2.5, 1.25, 0.62, and 0.31 µM bis-netropsins (IIa) and (II), respectively.
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GROKHOVSKY et al.
(IIa)
(II)
0
0
1 2 3 4 5 6 7 8 9 10 11 12 13 14 1516 17 18
5' → 3'
(1)
(9)
(8)
(2)
(7)
(6)
(3)
(5)
(4)
(5)
(6)
(4)
(7)
(3)
(8)
(2)
(9)
(1)
Fig. 6. The patterns of cleavage with DNase I of the lower DNA fragment B: 1 and 10, initial (untreated) fragment; 2 and 11, chem-
ical cleavage at purine residues; 3 and 12, cleavage of the free fragment; 4–9 and 13–18, the fragment cleavage in the presence of
10, 5, 2.5, 1.25, 0.62, and 0.31 µM bis-netropsins (IIa) and (II), respectively.
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DNA SEQUENCE-SPECIFIC LIGANDS: XI.
465
(IIa)
(II)
0
0
1 2 3 4 5 6 7 8 9 10 11 12 13 14 151617 18
5' → 3'
(9)
(1)
(8)
(7)
(2)
(6)
(5)
(3)
(4)
(5)
(4)
(6)
(3)
(7)
(8)
(2)
(9)
(1)
Fig. 7. The patterns of cleavage by hydroxyl radicals of the lower DNA fragment B: 1 and 10, initial (untreated) fragment; 2 and
11, chemical cleavage at purine residues; 3 and 12, cleavage of the free fragment; 4–9 and 13–18, the fragment cleavage in the pres-
ence of 10, 5, 2.5, 1.25, 0.62, and 0.31 µM bis-netropsins (IIa) and (II), respectively.
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GROKHOVSKY et al.
A
(2 H, s, CONHB), 8.23 (2 H, d, H5A), 8.03 (2 H, t,
introduced into 3'-end of the fragment using
[α-33P]dCTP (fragment Ä) or [α-33P]dATP (fragment
B) (Isotop, St. Petersburg), unlabeled dNTP, and Kle-
now fragment of Escherichia coli DNA polymerase I,
(Boehringer Mannheim, Germany) [45]. DNA frag-
ments were isolated by PAGE in 5% PAG.
CONHCH2), 7.60 (2 H, d, H3A), 7.25 (2 H, d, H5B),
6.87 (2 H, d, H3B), 4.40 (4 H, t, NACH2), 4.25 (4 H, t,
NBCH2), 3.19 (4 H, m, NHCH2CH2), 1.70 (8 H, m,
CH2CH3), 1.48 (6 H, m, CH2), and 0.81 (12 H, m, CH3);
MS, m/z: 763 [M + H]+; calc. for C37H50N10O8: 762.87.
Footprinting by means of hydroxyl radicals [46].
To prepare the complex, a solution of the fragment
To isolate the monoacyl derivative of cadaverine
(5 µl, about 104 Bq) and 6 µM (per 1 kbp) unlabeled (V), the combined acidic extracts were neutralized with
DNA plasmid pGEM(f+) in 10 mM Tris-HCl (pH 7.5) NaHCO3, and extracted with ethyl acetate. The organic
was mixed with a ligand solution (5 µl) (concentrations
are given in figure captions) in the same buffer, and the
mixture was cooled to 0°ë. A solution (5 µl) containing
1 mM EDTA, 0.5 mM Moore salt, 20 mM sodium
ascorbate, and 10 mM Tris-HCl (pH 7.5) and 0.2%
solution of H2O2 (5 µl) were simultaneously added, and
the mixture was kept for 2 min at 0°ë. The reaction was
quenched by a solution (85 µl) containing 0.15 M
NaCl, 50 mM Tris-HCl (pH 7.5), 10 mM EDTA, and
10 µg/ml tRNA. The mixture was extracted with phe-
nol, the DNA was precipitated with ethanol, washed
with 70% ethanol, dried, and dissolved in 95% forma-
mide (1 µl) containing 15 mM EDTA (pH 8.0), 0.05%
Bromphenol Blue, and 0.05% Xylene Cyanol FF. The
mixture was heated for 1 min at 90°ë, rapidly cooled,
and loaded on a 8% denaturing PAG (40 cm long) with
a gradient thickness of 0.15–0.45 mm [45, 47]. PAGE
was performed for 70 min at 90 W (2.5 kV). Prior to
superimposition, the gel was fixed in 10% acetic acid
and dried on a glass plate preliminarily treated with
γ-methacrylpropyloxysilane (LKB, Sweden).
Footprinting with DNase I. To prepare the com-
plex, a solution of a fragment (5 µl, about 104 Bq) and
a solution of 6 µM (per 1 kbp) unlabeled DNA of plas-
mid pGEM7(f+) (Promega) in 10 mM Tris-HCl
(pH 7.5) containing 0.1 M NaCl were mixed with a
ligand solution (concentrations are given in figure cap-
tions) in the same buffer and cooled to 0°ë. A solution
(10 µl) of DNase I (1 µg/ml) (Sigma) in 10 mM Tris-
HCl (pH 7.5) containing 0.1 M NaCl and 5 mM MnCl2
were added, and the mixture was kept for 2 min at 0°ë.
Further treatment was performed as described in the
above procedure.
phase was dried and precipitated with a solution of 1 M
hydrogen chloride in ethyl acetate. The resulting oil
was separated, dissolved in methanol, and purified on a
Sephadex LH-20 column to give (V) hydrochloride
(1.6 g, 12%); mp 214–217°ë; Rf 0.48 (A) and 0.52 (B);
1H NMR: 10.36 (2 H, s, ACONHB), 8.25 (1 H, d, H5A),
8.09 (1 H, m, CONHCH2), 7.68 (1 H, d, H3A), 7.28 (1
H, d, H5B), 6.93 (1 H, d, H3B), 4.42 (2 H, t, NACH2),
4.26 (2 H, t, NBCH2), 3.18 (2 H, m, NHCH2CH2), 2.77
(2 H, m, CH2NH3), 1.68 (4 H, m, CH2CH3), 1.42 (6 H,
A
B
m, CH2), 0.85 (3 H, t, CH3 ), and 0.82 (3 H, t, CH3 ).
MS, m/z: 433 [M + H]+; calc. for C21ç32N6O4: 432.52.
Npc Apc NH(CH2)4NH Apc Npc (IVa)
was synthesized by a procedure similar to that used for
(IV) from (III) (10 mmol) and putrescine (5 mmol)
(Merck); yield 46%; mp 182–185°ë; Rf 0.91 (A) and
0.87 (B); 1H NMR: 10.23 (2 H, s, ACONHB), 8.23 (2 H,
d, H5A), 8.06 (2 H, t, CONHCH2), 7.60 (2 H, d, H3A),
7.25 (2 H, d, H5B), 6.88 (2 H, d, H3B), 4.40 (4 H, t,
NACH2), 4.26 (4 H, t, NBCH2), 3.23 (4 H, m,
NHCH2CH2), 1.70 (8 H, m, CH2CH3), 1.54 (4 H, m,
A
NHCH2CH2), 0.85 (6 H, t, CH3 ), and 0.81 (3 H, t,
B
CH3 ); MS, m/z: 749.4 [M + H]+; calc. for
C36H48N10O8: 748.84.
Gly Apc Apc NH(CH2)5NH Apc Apc
Gly (I). Compound (IV) (10.0 g, 13.1 mmol) was
hydrogenated over Adams catalyst (2 g) in a mixture of
ethanol (100 ml) and 6 M HCl (10 ml). The catalyst was
filtered off, triethylamine (8.4 ml, 60 mmol) was added
to the filtrate, and the mixture was evaporated. Anhy-
drous DMF (20 ml) was added to a mixture of Boc-Gly-
OH (5.3 g, 30 mmol) and CDI (5.0 g). After 10 min, the
resulting imidazolide solution was added to the amino
component, kept for 20 h at 20°ë, and precipitated with
0.5 M citric acid (200 ml). The precipitate was filtered
and twice chromatographed on a Sephadex LH-20 col-
umn. The fraction containing (I) were combined and
evaporated to give (I) as oil; yield 6.4 g. It was dis-
solved in TFA (50 ml), kept for 3 h at 25°ë, evaporated,
and twice chromatographed on a Sephadex LH-20 col-
umn followed by reversed-phase HPLC to give (I) bis-
trifluoroacetate (4.38 g, 32%); Rf 0.24 (A) and 0.75 (B).
Npc Apc NH(CH2)5NH Apc Npc (IV).
Anhydrous DMF (40 ml) was added to NpC-Apc-OH
(III) (10.0 g, 28.7 mmol) [23] and CDI (28.7 mmol),
and the mixture was stirred for 30 min at 20°ë. A solu-
tion of cadaverine (14.4 mmol, 1.7 ml) in DMF (10 ml)
was added in portions (2 ml/h) to the resulting imid-
azolide. The reaction mixture was kept for 70 h at 20°C
and evaporated. The residue was dissolved in ethyl ace-
tate (300 ml) and the solution was washed with 1 M
HCl (3 × 100 ml), water, and saturated NaHCO3 solu-
tion (3 × 100 ml); dried and evaporated. The residue
was dissolved in methanol (100 ml) and kept overnight
at –10°ë. The precipitate was filtered and washed with
methanol and ether to give (IV); yield 6.5 g (59%); mp
1H NMR: 10.67 (2 H, s, ACONHB), 9.94 (2 H, s,
CH2CONH), 8.34 (6 H, m, H3NCH2), 7.98 (2 H, t,
148–150°ë, Rf 0.97 (A) and 0.19 (D); 1H NMR: 10.29 NHCH2CH2), 7.28 (2 H, d, H5A), 7.24 (2 H, d, H5B),
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DNA SEQUENCE-SPECIFIC LIGANDS: XI.
467
6.97 (2 H, d, H3A), 6.88 (2 H, d, H3B), 4.28 (8 H, t,
(2 H, d, H3), 4.31 (4 H, t, NCH2), 3.18 (4 H, m,
NCH2), 3.72 (4 H, m, H3NCH2ëO), 3.22 (4 H, m, CONHCH2), 1.87–1.28 (10 H, m, CH2), and 0.80 (6 H,
t, CH3); MS, m/z: 462.5 [M + H]+; calc. for C21H30N6O6:
NHCH2CH2), 1.70 (8 H, m, CH2CH3), 1.47 [6 H, m,
NHCH2(CH2)3], and 0.82 (12 H, t, CH3); MS, m/z: 817
[M + H]+; calc. for C41H60N12O6: 816.99.
462.50.
Npc NH(CH2)4NH Npc (VIa) was prepared
similarly; yield 76%; mp 217–220°ë, Rf 0.93 (C) and
Gly Apc Apc NH(CH2)4NH Apc Apc
Gly (Ia) was prepared from (IVa) (2.5 mmol) by a pro-
cedure similar to that in the synthesis of (I); yield 24%;
0.74 (D); 1H NMR: 8.34 (2 H, t, CONH), 8.09 (2 H, d,
H5), 7.36 (2 H, d, H3), 4.32 (4 H, t, NCH2), 3.20 (4 H,
m, CONHCH2), 1.8–1.4 (8 H, m, CH2), and 0.79 (6 H,
1
Rf 0.15 (A) and 0.74 (B); H NMR: 10.69 (2 H, s,
t, CH3); MS, m/z: 449 [M + H]+; calc. for C20H28N6O6:
ΑCONHB), 9.92 (2 H, s, CH2CONH), 8.24 (6 H, m,
H3NCH2), 8.00 (2 H, t, NHCH2CH2), 7.27 (2 H, d,
H5A), 7.23 (2 H, d, H5B), 6.98 (2 H, d, H3A), 6.90 (2 H,
448.47.
Z-Gly-Apc-OH (VII). Triethylamine (18.4 ml,
d, H3B), 4.27 (8 H, t, NCH2), 3.74 (4 H, m, 131 mmol) and the imidazolide solution obtained from
Z-Gly-OH (13.6 g, 65 mmol) (Reanal), CDI (11.1 g,
65 mmol), and DMF (30 ml) were added for 10 min to
a solution of 4-aminopyrrole-2-carbonyl chloride
hydrochloride (13.4 g, 65.3 mmol) [23] in DMF
(100 ml). The mixture was kept for 48 h at 20°ë, evap-
orated, dissolved in water (300 ml), and acidified to pH
2 with conc. HCl. The precipitated oil was dissolved in
ethyl acetate (1 l), washed with 1 M HCl (4 × 200 ml)
and water, and dried. The solution was evaporated to
the volume of 70 ml, cooled to 0°ë; the resulting pre-
cipitate was filtered and washed with ethyl acetate and
ether to give (VII); yield 18.8 g (80%); mp 153–154°ë,
NH3CH2CO), 3.21 (4 H, m, NHCH2CH2), 1.68 (8 H, m,
CH2CH3), 1.55 [4 H, m, NHCH2(CH2)2], and 0.80
(12 H, t, CH3); MS, m/z: 803 [M + H]+; calc. for
C40H58N12O6: 802.98.
Lys Gly Apc Apc NH(CH2)5NH Apc
Apc Gly Lys (II). A solution of DCC (0.42 g,
2 mmol) in DMF (2 ml) was added to a mixture of Boc-
Lys(Z)-OH (0.76 g, 2 mmol) and HOBt (Fluka, 0.33 g,
2 mmol) in DMF (5 ml), and the reaction mixture was
kept for 30 min at 20°ë. The precipitate was filtered,
and the filtrate was added to (I) ditrifluoroacetate (0.52
g, 0.5 mmol). The reaction mixture was kept for 20 h at
20°ë and loaded onto a Sephadex LH-20 column. After
evaporation, the di-Boc-di-Z-derivative of (II) was
obtained as oil (0.55 g), which was dissolved in a mix-
ture of TFA (3 ml) and thioanisole (0.5 ml) (Fluka),
kept for 1 h at 25°ë, evaporated, and chromatographed
twice on a Sephadex LH-20 column. After evaporation
of the fractions containing di-Z-derivative of (II), it was
obtained as oil (0.51 g). Z-protective groups were
removed by hydrogenation over palladium black in
methanol, and the target (II) was purified on Sephadex
LH-20 column followed by reversed-phase HPLC;
yield 0.43 g (80%); Rf 0.09 (A) and 0.46 (B); MS, m/z:
1
Rf 0.96 (A) and 0.37 (D); H NMR: 9.78 (1 H, s,
CH2CONH), 7.43 (1 H, t, CONHCH2), 7.31 (5 H, s,
C6H5), 7.28 (1 H, d, H5), 6.71 (1 H, d, H3), 5.03 (2 H,
s, C6H5CH2), 4.19 (2 H, t, NCH2), 3.74 (2 H, m,
NHCH2CO), 1.66 (2 H, m, CH2CH3), and 0.80 (3 H, t,
CH3); MS, m/z: 360 [M + H]+; calc. for C18H21N3O5:
359.38.
Z-Gly Apc Apc NH(CH2)5NH Apc
Apc Gly-Z (VIII). A solution of DCC (3.81 g,
18.5 mmol) in DMF (10 ml) was added to a solution of
(VII) (6.47 g, 18 mmol) and HOBt (2.57 g, 19 mmol)
in DMF (20 ml). The mixture was kept for 1.5 h at
20°ë, the precipitate was filtered, and the filtrate was
added to a solution of amino component, which was
prepared by hydrogenation of (VI) (4.1 g, 8.9 mmol)
over the Adams catalyst (1.0 g) in a mixture of ethanol
(100 ml) and 6 M HCl (10 ml) followed by filtration
and evaporation of the filtrate, in the mixture of trieth-
ylamine (8.4 ml, 60 mmol) and DMF (30 ml). The reac-
tion mixture was kept for 24 h at 20°ë, the precipitate
was filtered, and the filtrate was evaporated. The resi-
due was dissolved in chloroform (300 ml), washed with
1 M HCl, water, and 9% NaHCO3 (2 × 100 ml), and
dried. After evaporation of the solvent, the residue was
dissolved in ethanol (150 ml) and allowed to stay for
several days at 0°ë. The resulting precipitate was fil-
tered, washed with ethanol and ether to give 5.5 g
(57%) of (VIII); mp 175–178°ë, Rf 0.89 (A) and 0.53
1073.6 [M + H]+; calc. for C53H84N16O8: 1073.35.
Lys Gly Apc Apc NH(CH2)4NH Apc
Apc Gly Lys (IIa) was similarly prepared from
(Ia) in yield of 67%; MS, m/z: 1059.6 [M + H]+; calc.
for C52H82N16O8: 1059.32.
Npc NH(CH2)5NH Npc (VI). 1-Propyl-4-
nitropyrrole-2-carboxylic acid [3] (20.0 g, 101 mmol)
was refluxed with SOCl2 (50 ml) and DMF (0.05 ml)
for 40 min. The solution was evaporated, the residue
was several times coevaporated with benzene and dis-
solved in ethanol (150 ml). Cadaverine (5.9 ml,
50 mmol) was added to the solution and then triethyl-
amine (15 ml, 104 mmol) was added dropwise under
vigorous stirring. After 2 h, the reaction mixture was
cooled to 0°ë, and the precipitate was filtered and
recrystallized from ethanol to give 20.9 g (89%) of
1
A
(D); H NMR: 10.71 (2 H, s, CONHB), 9.76 (2 H, s,
CH2CONHA), 7.91 (2 H, t, OCONH), 7.31 (10 H, s,
C6H5), 7.15 (4 H, m, H5A, B), 6.85 (2 H, d, H3A), 6.80
1
(VI); mp 204–206°ë; Rf 0.94 (C) and 0.82 (D); H
NMR: 8.29 (2 H, t, CONH), 8.06 (2 H, d, H5), 7.34
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GROKHOVSKY et al.
(2 H, d, H3B), 5.04 (4 H, s, C6H5CH2), 4.20 (8 H, t,
NCH2), 3.76 (4 H, d, NHCH2CO), 1.79–1.33 (14 H, m,
CH2CH3 + NHCH2CH2CH2CH2), and 0.79 (12 H, t,
CH3); MS, m/z: 1085 [M + H]+; calc. for C57H72N12O10:
1085.28.
12. Khorlin, A.A., Krylov, A.S., Grokhovsky, S.L.,
Zhuze, A.L., Zasedatelev, A.S., Gursky, G.V., and Got-
tikh, B.P., FEBS Lett., 1980, vol. 118, pp. 311–314.
13. Rechinskii, V.O., Bibilashvili, R.Sh., Khorlin, A.A.,
Grokhovsky, S.L., Krylov, A.S., Zasedatelev, A.S.,
Zhuze, A.L., Gursky, G.V., and Gottikh, B.P., Dokl.
Akad. Nauk SSSR, 1981, vol. 259, pp. 244–247.
14. Skamrov, A.V., Rybalkin, I.N., Bibilashvili, R.Sh., Got-
tikh, B.P., Grokhovsky, S.L., Gursky, G.V., Zhuze, A.L.,
Zasedatelev, A.S., Nechipurenko, Yu.D., and Khor-
lin, A.A., Mol. Biol. (Moscow), 1985, vol. 19, pp. 177–
195.
15. Stanchev, B.S., Grokhovsky, S.L., Khorlin, A.A., Got-
tikh, B.P., Zhuze, A.L., Skamrov, A.V., and Bibilash-
vili, R.Sh., Mol. Biol. (Moscow), 1986, vol. 20,
pp. 1614–1624.
Z-Gly Apc Apc NH(CH2)4NH Apc
Apc Gly-Z (VIIIa) was similarly synthesized from
(VIa) (2 mmol); yield 1.8 g (83%), Rf 0.85 (A) and 0.47
(D).
Z-groups were removed from (VIII) and (VIIIa) by
hydrogenation over palladium black, and the resulting
(I) and (Ia) were identical to those prepared according
to Scheme 1.
16. Sukhanova, A., Grokhovsky, S., Zhuze, A., Roper, D.,
and Bronstein, I., Biochem. Mol. Biol. Int., 1998, vol. 44,
pp. 997–1010.
17. Schultz, P.G. and Dervan, P.B., J. Am. Shem. Soc, 1983,
vol. 105, pp. 7748–7750.
18. Lown, J.W., Krowicki, K., Balzarini, J., Newman, R.A.,
and De Clercq, E., J. Med. Chem., 1989, vol. 32,
pp. 2368–2375.
19. Wyatt, M.D., Garbiras, B.J., Lee, M., Forrow, S.M., and
Hartley, J.A., Bioorg. Med. Chem. Lett., 1994, vol. 4,
pp. 801–806.
ACKNOWLEDGMENTS
The work was partly supported by the Russian Foun-
dation for Basic Research, project nos. 01-03-32699, 01-
04-48657, and 00-04-48240.
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