Oligo(2′-O-methylribonucleotides) and their derivatives: IV. Conjugates of oligo(2′-O-methylribonucleotides) with minor groove binders and intercalators: Synthesis, properties, and application

Article abstract of DOI:10.1134/S1068162013010081

Triplex forming 3′-protected pyrimidine oligo(2′-O- methylribonucleotides) containing minor groove binders (MGB) and triplex specific intercalator, benzoindoloquinoline (BIQ), at the 5′-terminus were synthesized. The resulting conjugates formed stable complexes with a target dsDNA due to the simultaneous binding to the minor and major grooves and BIQ intercalation. The dissociation constants and the thermal stability were evaluated for the conjugate complexes with the model dsDNA corresponding to the polypurine tract (PPT) of the nef and pol genes from the HIV proviral genome. The conjugation of oligo(2′-O-methylribonucleotides) with MGB and BIQ was shown to increase the stability of the triple complexes with dsDNA at pH 7.2 and 37 C. Moreover, the intercalator accelerates the process of the complex formation. The dosedependent arrest of the in vitro transcription was demonstrated when a 780-bp DNA fragment containing the polypurine tract was transcribed under the control of T7 promoter in the presence of different concentrations of conjugates of oligo(2′-O-methylribonucleotides) containing MGB and BIQ.

Full text of DOI:10.1134/S1068162013010081

ISSN 1068ꢀ1620, Russian Journal of Bioorganic Chemistry, 2013, Vol. 39, No. 2, pp. 138–152. © Pleiades Publishing, Ltd., 2013.
Original Russian Text © D.S. Novopashina, A.N. Sinyakov, V.A. Ryabinin, L. Perrouault, C. Giovannangeli, A.G. Venyaminova, A.S. Boutorine, 2013, published in Bioorganicheskaya
Khimiya, 2013, Vol. 39, No. 2, pp. 159–174.
Oligo(2'ꢀOꢀmethylribonucleotides) and Their Derivatives:
IV. Conjugates of Oligo(2'ꢀ
Oꢀmethylribonucleotides)
with Minor Groove Binders and Intercalators: Synthesis,
Properties, and Application
, 1
D. S. Novopashinaa , A. N. Sinyakova, V. A. Ryabinina, L. Perrouaultb,
C. Giovannangelib, A. G. Venyaminovaa, and A. S. Boutorineb
a Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of RAS,
pr. Lavrentieva 8, Novosibirsk, 630090 Russia
b Muséum National d’Histoire Naturelle (MNHN), CNRS UMR 7196, INSERM U565, 57,
rue Cuvier, BP 26, 75231, Paris, cedex 05 France.
Received February 29, 2012; in final form, May 1, 2012.
Abstract—Triplex forming 3'ꢀprotected pyrimidine oligo(2'ꢀOꢀmethylribonucleotides) containing minor
groove binders (MGB) and triplex specific intercalator, benzoindoloquinoline (BIQ), at the 5'ꢀterminus were
synthesized. The resulting conjugates formed stable complexes with a target dsDNA due to the simultaneous
binding to the minor and major grooves and BIQ intercalation. The dissociation constants and the thermal
stability were evaluated for the conjugate complexes with the model dsDNA corresponding to the polypurine
tract (PPT) of the nef and pol genes from the HIV proviral genome. The conjugation of oligo(2'ꢀOꢀmethylriꢀ
bonucleotides) with MGB and BIQ was shown to increase the stability of the triple complexes with dsDNA
at pH 7.2 and 37°C. Moreover, the intercalator accelerates the process of the complex formation. The doseꢀ
dependent arrest of the in vitro transcription was demonstrated when a 780ꢀbp DNA fragment containing the
polypurine tract was transcribed under the control of T7 promoter in the presence of different concentrations
of conjugates of oligo(2'ꢀOꢀmethylribonucleotides) containing MGB and BIQ.
Keywords: oligo(2'ꢀOꢀmethylribonucleotides), conjugates, minor groove binder, intercalator, DNA triple helix,
inhibition of in vitro transcription
DOI: 10.1134/S1068162013010081
1
INTRODUCTION
otides (TFO), which form a triple helix with genomic
DNA and can, thereby, block transcription or moduꢀ
late gene expression, are of particular interest [2, 3].
The use of TFO for targeting genomic DNA in cell
systems faced a number of obstacles, namely the rapid
degradation of oligodeoxyꢀribonucleotides in cells,
insufficient stability of triplex structures under physioꢀ
logical conditions, and poor penetration of oligonuꢀ
cleotides into living cells.
Compounds capable of selectively binding to speꢀ
cific nucleotide sequences of doubleꢀstranded DNA
(dsDNA) are used to study the structure and the funcꢀ
tion of genomic DNAs and the mechanisms of gene
expression, as well as to develop dsDNA detection sysꢀ
tems using sequenceꢀspecific probes. The ability of
these compounds to selectively inhibit gene expression
can be used to create a new generation of substances
for the diagnostics and the treatment of cancer, viral,
and genetic diseases. Triplexꢀforming oligonucleꢀ
One of the approaches to improve the stability of
oligonucleotides against nucleases is the introduction
of modifying groups into their structure [3–5]. As triꢀ
Abbreviations: BIQ, 6ꢀ[(3ꢀaminopropyl)amino]ꢀ11ꢀmethoxyꢀ
m
plexꢀforming oligonucleotides, we used oligo(2'ꢀ
Oꢀ
13
H
ꢀbenzo[6,7]indolo[3,2ꢀ
c
]quinoline; C , 2'ꢀ
O
ꢀmethylriboꢀ
cytidine; DMAP, 4ꢀN,N'ꢀdimethylaminopyridine;
nobutyric acid; HEPES, (4ꢀ(2ꢀhydroxyethyl)ꢀ1ꢀpiperazine
ethanesulfonic acid); Im, ꢀmethylimidazole; MES, 2ꢀ(
morpholino)ethanesulphonic acid; MGB, minor groove binder;
γ, γꢀamiꢀ methylribonucleotides) having a number of advanꢀ
tages. Their synthesis is simple and efficient; they are
resistant to nucleolytic degradation and form fairly
strong triple helices with dsDNA [6, 7]. The main
route of degradation of oligonucleotides in biological
media is known to be the 3'ꢀexonuclease cleavage [8].
N
Nꢀ
m
N , 2'ꢀ
O
ꢀmethylribonucleoside; Py,
Nꢀmethylpyrrole;
T, thyꢀ
inv
midine attached via 3'ꢀ3'ꢀphosphodiester internucleotide bond
(“inverted” thymidine); TEA, triethylamine; TFO, triplexꢀ
m
forming oligonucleotides; U , 2'ꢀ
Oꢀmethyluridine.
1
The 2'ꢀ
Oꢀmethylation prevents endonuclease degraꢀ
Corresponding author: phone: (383) 363ꢀ51ꢀ29; fax: (383) 363ꢀ
51ꢀ53; eꢀmail: danov@niboch.nsc.ru.
dation of oligoribonucleotides, but does not prevent
138

OLIGO(2'ꢀ
O
ꢀMETHYLRIBONUCLEOTIDES) AND THEIR DERIVATIVES
139
Table 1. Yields and nucleotide sequence of synthesized pyrimidine1
Code
Oligonucleotide (5'ꢀ3')
Yield, %1
Nucleoside content2
(III)
UmUmUmUmCmUmUmUmUmCmCmCmCmCmCmUminvT
pUmUmUmUmCmUmUmUmUmCmCmCmCmCmCmUminvT
12
8
Cm : Um : T = 6.7 : 9.2 : 1.1
Cm : Um : T = 6.8 : 9.0 : 1.3
Cm : Um : T = 6.8 : 9.0 : 1.3
Cm : Um : T = 6.7 : 8.9 : 1.1
, “inverted” thymidine.
(IV)
(V)
pL
1pUmUmUmUmCmUmUmUmUmCmCmCmCmCmCmUminvT
9
(VI)
pL
2pUmUmUmUmCmUmUmUmUmCmCmCmCmCmCmUminvT
10
m
m
C , 2'ꢀ
O
ꢀmethylribocytidine; U , 2'ꢀ
O
ꢀmethyluridine; L1
:
ꢀ(CH CH O) ꢀ; L2: ꢀ(CH CH O) ꢀ;
T
inv
2
2
3
2
2
6
1
The yield after isolation by HPLC relative to the first nucleoside unit attached to the polymer support.
According to data of RP HPLC of oligonucleotide hydrolysates obtained by the successive exhaustive hydrolysis with nuclease P1 and
alkaline phosphatase.
2
their cleavage with exonucleases. To overcome this
obstacle, oligo(2'ꢀ ꢀmethylribonucleotides) were
modified by the attachment to their 3'ꢀterminus of
“inverted” thymidine (invT, i.e., thymidine attached
through the 3'ꢀ3'ꢀphosphodiester bond) [9, 10].
We propose to use a combination of the advantages
of three components (TFO, MGB, and intercalator)
in the same conjugate. The goal of the presented work
is to design conjugates stable in biological media comꢀ
O
posed of pyrimidine oligo(2'ꢀOꢀmethylribonucleoꢀ
tides), minor groove binders, and intercalators and to
study their physicoꢀchemical properties and biological
effects in vitro. These conjugates should selectively
bind to polypurine sites in dsDNA forming a triple
helix due to the oligonucleotide component and to the
minor groove of the double helix in the adjacent
The introduction of triplexꢀspecific intercalators in
TFOs is used to enhance the stability of triplexes [11–
15]. For example, it was shown that the attachment of
intercalators of benzopyridoindole and benzopyriꢀ
doquinoxaline series to an oligonucleotide leads to the
significant increase in the stability of triplexes formed sequence due to MGB. The presence of the intercalaꢀ
tor in the conjugate should additionally stabilize the
resulting complex with dsDNA. We assume that these
constructions can efficiently inhibit gene expression.
with these conjugates at pH close to neutral values [14,
15]. Of the intercalators most selective to triplex strucꢀ
tures are benzoindoloquinolines (BIQ), particularly,
6ꢀ[(3ꢀaminopropyl)amino]ꢀ11ꢀmethoxyꢀ13Hꢀbenzo[6,
7]indolo[3,2ꢀ ]quinoline [16–18]. The other way to
enhance the triplex stability is the attachment of the
DNA minor groove binder (MGB) to TFO [18–20].
c
RESULTS AND DISCUSSION
When designing conjugates, we relied on the fact
that the simultaneous binding of an oligonucleotide
and ligand with the major and minor grooves of a tarꢀ
get DNA, respectively, should be ensured by the linker
MGB based on hairpin polyamides of
dazole and ꢀmethylpyrrole are capable of sequenceꢀ
specific binding to MGB regardless of pH and conꢀ
Nꢀmethylimiꢀ
N
centration of monovalent and divalent cations [21– with the length of at least 12 interatomic bonds [18,
19]. Oligoethylene glycols (starting with triethylene
glycol) have all features of an optimal linker between
an oligonucleotide and a ligand: enough length,
hydrophilicity, and flexibility. We developed a simple
method of the synthesis of 5'ꢀoligoethyleneglycolꢀ
phosphateꢀcontaining oligonucleotides using the
23]. Conjugates of TFO and MGB, having properties
of both components, are prospective selective tools for
targeting genomic DNA. These conjugates provide an
extension of the recognition site in a target DNA and,
thereby, the increase in the specificity and stability of
the complexes formed, which allows one to signifiꢀ
cantly reduce the effective conjugate concentration. Furꢀ
thermore, the covalent attachment to oligonucleotides
makes rather hydrophobic MGB more water soluble.
Hꢀphosphonate chemistry.
For this purpose, the corresponding
Hꢀphosphoꢀ
nates of dimethoxytritylꢀcontaining triꢀ and hexaethyꢀ
leneglycols ((IIa) and (IIb), respectively) and ꢀphosꢀ
Conjugates of oligo(2'ꢀOꢀmethylribonucleotides) conꢀ
H
taining one or two MGB residues at the 5'ꢀend were synꢀ
thesized and studied in our previous work [24].
phonate of sulfonyldiethanol (IIc) were synthesized by
the reaction of dimethoxytritylꢀcontaining derivatives
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140
NOVOPASHINA et al.
Table 2. Characteristics of the synthesized conjugates of oligo(2'ꢀOꢀmethylribonucleotides) with minor groove binders1
Molecular weight2
Code
Conjugate (5'ꢀ3')
Experiꢀ
Calculatꢀ
ed, m/z
mental,
m/z
*
(VIIa) (Py)3ꢀ
(VIIb) BIQꢀ(Py)3ꢀ
(VIIIa) (Py)4ꢀ ꢀ (Py)4ꢀ
(VIIIb) BIQꢀ(Py)4ꢀ ꢀ(Py)4ꢀ
(IXa) (Py)4ꢀ ꢀ(Py)4ꢀ ꢀNHꢀp
(IXb) BIQꢀ(Py)4ꢀ ꢀ(Py)4ꢀ ꢀNHꢀp
γ
ꢀ (Py)3ꢀ
ꢀ(Py)3ꢀ
ꢀNHꢀp
ꢀNHꢀp
2pUmUmUmUmCmUmUmUmUmCmCmCmCmCmCmUminvT
2pUmUmUmUmCmUmUmUmUmCmCmCmCmCmCmUminvT
γ
ꢀNHꢀp
L
2pUmUmUmUmCmUmUmUmUmCmCmCmCmCmCmUminvT
13.8 6769.81 6765.28
γ
γ
ꢀNHꢀp
L
2pUmUmUmUmCmUmUmUmUmCmCmCmCmCmCmUminvT
7038.08 7038.80
18.4
γ
γ
L
1pUmUmUmUmCmUmUmUmUmCmCmCmCmCmCmUminvT
6881.90 6888.25
14.0
γ
γ
L
1pUmUmUmUmCmUmUmUmUmCmCmCmCmCmCmUminvT
7150.17 7155.07
20.3
γ
γ
L
7014.06 7017.78
14.5
γ
γ
L
7282.33 7287.78
21.2
m
m
1BIQ, 6ꢀ[(3ꢀaminopropyl)amino]ꢀ11ꢀmethoxyꢀ13
L1, ꢀ(CH CH O) ꢀ; L2, ꢀ(CH CH O) ꢀ;
H
ꢀbenzo[6,7]indolo[3,2ꢀ
c
]quinoline; C , 2'ꢀ
O
ꢀmethylribocytidine; U , 2'ꢀ
O
ꢀmethyluridine;
T, “inverted” thymidine; p, phosphate group; the structure of the ligands is presented in Fig. 1.
2
2
3
2
2
6 inv
* According to mass spectrometry data.
(
Ia)–(Ic) [25, 26] with phosphorotriimidazolide, folꢀ were used resulting in pyrimidine oligo(2'ꢀ
lowed by the hydrolysis by analogy with the synthesis ribonucleotides) ( ) and (VI) with the phosphateꢀ
of nucleoside ꢀphosphonates [27] (Scheme 1). At containing linkers of the different length at the 5'ꢀend
the final stages of the solidꢀphase ꢀphosphonate synꢀ and “inverted” thymidine at the 3'ꢀend (Scheme 2,
thesis, oligoethylene glycol ꢀphosphonate (IIa) or Table 1). The resulting compounds were isolated by
IIb) and then sulfonyldiethanol
Oꢀmethylꢀ
V
H
H
H
(
Hꢀphosphonate (IIc
)
ionꢀexchange and reverseꢀphase HPLC.
P(Im)
3
O
OH
O
OH (CH O) TrCl
3
pyridine
2
(CH3O)2TrO
O
HO
O
(C H ) N
2 5 3
n
n
(Ia, b)
O
O
O
H
a
b
:
:
n
n
= 1
= 4
H O
2
(CH3O)2TrO
O
P
O−
+
n
(C2H5)3NH
(IIa, b)
(CH3O)2TrO
2
O
O
HO
OH
S
P(Im)
3
(C H ) N
OH
(CH O) TrCl
3
S
O
2
5 3
pyridine
O
(Ic)
O
O
O
H
(CH3O)2TrO
H O
2
S
P
O−
+
(C2H5)3NH
O
(IIc)
Scheme 1.
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OLIGO(2'ꢀ
O
ꢀMETHYLRIBONUCLEOTIDES) AND THEIR DERIVATIVES
OTr(OCH3)2
141
O
O n
O
O P O
H
(CH3O)2TrO
Base'
Base'
O
O
O
OCH3
O
O
OCH3
O
O P H
O
Base'
O P H
O
Base'
DCA (IIа) or (IIb)
CH Cl PivCl, pyridine
DCA
CH Cl
2
2
2
2
O
OCH3
O
OCH3
O P H
m O
O P H
m O
O
O
Thy
O
Thy
O
O
OTr(OCH3)2
O
S
O
O−
O P O−
O
H
O P O
O
O
O
n
On
O
O P O
H
O
O P O
O–
Base'
Base
O
O
O
OCH3
DCA
CH Cl
2
I
2
NH OH
4
conc.
O
OCH3
(IIc)
PivCl, pyridine
pyridine/H O
2
O P O−
2
O P H
O
Base'
Base
O
O
O
O
OCH3
O
OCH3
O P O−
O P H
m O
m
O
O
O
Thy
O
Thy
OH
(V) or (VI)
(Base) Base', (non)protected heterocyclic base Cyt or Ura
DCA, dichloroacetic acid, PivCl, pivaloyl chloride
m
= 15, n = 1 or 4
Scheme 2.
During ionꢀexchange chromatography, the retenꢀ as compared to oligonucleotide (V) containing the triꢀ
ethyleneglycol linker (data not shown).
tion times for oligomers (V) and (VI) containing the
5'ꢀoligoethyleneglycolꢀphosphate linkers were, as
expected, higher than for 5'ꢀphosphorylated oligomer
Based on 5'ꢀoligoethyleneglycolꢀphosphateꢀconꢀ
taining oligo(2'ꢀOꢀmethylribonucleotides) (V) and (VI)
with 3'ꢀ“inverted” thymidine, we synthesized a series
of conjugates bearing either only the MGB residue
(
IV) of the same sequence. During reverseꢀphase
HPLC, oligonucleotide (VI) containing the hexaetꢀ
hyleneglycol linker had the higher retention time value (the previously described oligoamides NH2ꢀγꢀ(Py)3ꢀγꢀ
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142
NOVOPASHINA et al.
O
O
HN
HN
NH
O
N
NH
O
O
N
O
N
N
N
N
O
NH
NH
H
N
HN
HN
O
O
N
N
O
O
NH
NH
O
O
N
N
N
H
HN
HN
N
N
N
N
H
H
N
N
(VIIa)
(VIIb)
O
O
O
O
HN P O
O−
O
O
HN P O
O−
NH
NH
O
O
O P O OLIGO
O P O OLIGO
6
6
O−
O−
(Py)3ꢀ
γ
ꢀ(Py)3ꢀ
γ
ꢀNHꢀpL2pꢀOLIGO
BIQꢀ(Py)3ꢀ
γ
ꢀ(Py)3ꢀγꢀNHꢀpL2pꢀOLIGO
O
O
HN
HN
NH
NH
O
N
O
N
N
N
O
O
N
O
NH
NH
HN
HN
O
O
O
N
N
N
O
NH
NH
H
N
HN
HN
O
O
N
N
NH
N
N
O
O
NH
O
O
N
N
N
H
HN
HN
N
H
N
N
N
H
N
N
O
O
O
O
HN P O
O−
O
O
HN P O
O−
(VIIIb) and (IXb)
NH
NH
(VIIIa) and (IXa)
O
O
O P O OLIGO
O P O OLIGO
n
= 3 or 6
n
n
O−
O−
(Py)4ꢀ
γꢀ(Py)4ꢀ
γ
ꢀNHꢀpLpꢀOLIGO
BIQꢀ(Py)4ꢀ
γ
ꢀ(Py)4ꢀ
γ
ꢀNHꢀpLpꢀOLIGO
m
m m m m m m m
Fig. 1. Conjugates of oligo(2'ꢀOꢀmethylribonucleotides) with MGB and MGBꢀBIQ. OLIGO, 5'ꢀU U U U C U U U ꢀ
m
m m m m m m m
U C C C C C C U
T.
inv
(Py)3ꢀBIQ or NH2ꢀ
γꢀ(Py)4ꢀ
γꢀ(Py)4ꢀBIQ containing
The synthesis of the conjugates was carried out
two fragments of 4ꢀaminoꢀ1ꢀmethylpyrroleꢀ2ꢀcarꢀ using the selective activation of the terminal phosꢀ
boxylic acid triꢀ and tetramer [30–32]), or the MGB phate of an unprotected oligonucleotide with the
residue and the intercalator; their structures have been triphenylphosphine/dipyridyldisulfide pair in the
confirmed. The structure of the resulting conjugates presence of dimethylaminopyridine followed by the
was confirmed by mass spectrometry and their characꢀ interaction of the activated oligonucleotide with the
teristics are presented in Fig. 1 and Table 2.
aminoꢀcontaining ligand [28, 29] (Scheme 3). Previꢀ
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OLIGO(2'ꢀ
O
ꢀMETHYLRIBONUCLEOTIDES) AND THEIR DERIVATIVES
143
ously described hexaꢀ or octa(
Nꢀmethylpyrrole)carꢀ NH2ꢀγꢀ(Py)4ꢀγꢀ(Py)4ꢀBIQ) were used as the aminoꢀ
boxamides [30–32] and the conjugates of BIQ with containing ligands.
these carboxamides (NH2ꢀγꢀ(Py)3ꢀγꢀ(Py)3ꢀBIQ and
1) Ph P/
3
/DMAP
O
O
O P O OLIGO
S
S
N
2) removal of activating agents
N
−O
P O
O−
O
n
O
(V), (VI)
n
= 2 or 5;
m
= 3 or 4
O
O
(Py)mꢀ
γ
ꢀ(Py)mꢀ
γ
ꢀ
NH P O
O P O OLIGO
O
O−
O–
n
(Py)m
ꢀ
γꢀ(Py)m
ꢀγ
ꢀNH2
or
(VIIa)–(IXa)
BIQꢀ(Py)m
ꢀγꢀ(Py)m
ꢀ
γꢀNH2
or
H2O/DMSO
O
O
BIQꢀ(Py)mꢀ
γ
ꢀ(Py)mꢀ
γ
ꢀ
NH P O
O P O OLIGO
O–
O
O−
n
(VIIb)–(IXb)
Scheme 3.
The conjugates were isolated by RP HPLC. Their by the conjugate with the dsDNA target is shown in
homogeneity was confirmed by gelꢀelectrophoresis in Fig. 2.
denaturing PAAG. The increase of the number of the
ꢀmethylpyrrole units in the ligand from six (VIIa) to
eight (VIIIa) led to the increase in the retention time
of the corresponding conjugates during chromatograꢀ
phy. As expected, the retention times for products
The formation of such triplexes was studied by both
the gel retardation method and the thermal denaturꢀ
ation method by measuring optical absorption of triꢀ
plexes at two wavelengths (260 and 330 nm) [35].
N
The process of the triplex formation between the
conjugates and radioactivelyꢀlabeled dsDNA over
time was studied by the gel retardation method at pH
6.0. In all cases, we observed the formation of the
complexes with lower electrophoretic mobility as
compared to the initial dsDNA target. Figure 3 demꢀ
onstrates, as an example, electrophoregrams of the
complexes of conjugates (VIIa) and (VIIb) and oligoꢀ
nucleotide (IV) with dsDNA target. Under the given
conditions, the complex is formed for about 3 h. The
introduction of BIQ in the conjugate led to the accelꢀ
eration of the process: the band of the duplex disapꢀ
peared within a few minutes after mixing the compoꢀ
nents. In the case of conjugate (VIIa) containing the
ligand, high molecular weight compounds of an uniꢀ
dentified nature were formed in addition to the triplex
(Fig. 3).
(
VIIb)–(IXb) containing the MGB residue and the
intercalator exceeded those for conjugates (VIIa)–
IXa) containing only MGB. The yields of the prodꢀ
(
ucts were 45–85%. The structure of the resulting
derivatives was confirmed by ESI QꢀTOF mass specꢀ
trometry. The electron spectra of these compounds
contained the peaks at 260 nm and 310–320 nm corꢀ
responding to the absorption of the oligonucleotide
part of the conjugate and the minor groove binder,
respectively [24].
The sequence found in the nef and pol genes of the
proviral human immunodeficiency virus containing
the polypurine tract (16 bp) with the adjacent regions
[33] was chosen as the target DNA. The ability of the
synthesized conjugates of pyrimidine oligo(2'ꢀOꢀ
methylribonucleotides) containing “inverted” thymiꢀ
dine to form triplexes was studied using the synthetic
duplex (29 bp) containing the same pyrimidine
The stability of triplexes consisted of the conjugates
(
VIIa), (IXa), (VIIb), and (IXb) and the dsDNA target
sequence [24, 34]. The peculiarity of this fragment lies was comparatively studied. Figure 4 demonstrates, as
in the presence of five А•Т pairs immediately adjacent an example, radioautographs of gels obtained while
to the polypurine region from the 5'ꢀend (Fig. 2). studying the stability of the triplexes involving conjuꢀ
These А•Т pairs are an ideal target for the binding of gate (VIIa) containing only the MGB residue and
the synthesized oligo(
Nꢀmethylpyrrole)carboxaꢀ conjugate (VIIb) containing the MBG and BIQ resiꢀ
mides. The schematic structure of the complex formed dues. The dissociation constants of the triplexes at pH
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NOVOPASHINA et al.
1
2
5'
3'
ꢀ(HIVꢀD1)
ꢀ(HIVꢀD2)
H3C
N
O
C
H
N
: BIQ ; Py:
;
:
;
C
O
N
H
L
= L1: ꢀ(OCH2CH2)3ꢀ or L2: ꢀ(OCH2CH2)6ꢀ.
Fig. 2. Structure of triple complex between DNA duplex and the conjugate containing minor groove binder (octa(
Nꢀmethylpyrꢀ
role)carboxamide) and intercalator BIQ. 1, polypurine region, 2, A/T binding site of oligo( ꢀmethylpyrrole)carboxamide.
N
(
IV)
(VIIa
)
(VIIb)
15 30 45 60 120 180 15 30 45 60 120 180 5 15 30 60 195 min
Fig. 3. Formation of triple complex between target DNA and oligonucleotide (IV) and conjugates (VIIa) and (VIIb). Native elecꢀ
trophoresis in 12% PAAG of ternary complexes between conjugates of 3'ꢀ“inverted” oligo(2'ꢀ
dsDNA depending on time of incubation. Concentrations of DNA duplex (HIVꢀ1)
M, respectively; the incubation time of the components of the triplex before loading onto gel is indicated above the lanes.
Buffer: 50 mM MES, pH 6.0, 50 mM NaCl and 5 mM MgCl ; 10
O
ꢀmethylribonucleotides) and
⋅
(HIVꢀ2) and the third strand are 60 nM and
2
μ
°C.
2
6.0 (10°С) and pH 7.2 (37°С) calculated from these dsDNA target, with the ligand and the intercalator sigꢀ
data are presented in Table 3. The formation of the triꢀ
plexes of the dsDNA target with oligonucleotides
nificantly stabilizing the triple complex under physioꢀ
logical conditions.
(
III)–(VI) containing neither MGB nor BIQ was
The stabilization of the triple complex with the
MGB and intercalator residues was also shown by the
thermal denaturation method. Differential curves of
the thermal denaturation of the triplexes involving
observed only at pH 6.0. The presence of oligoethyleꢀ
neglycol phosphate at the 5'ꢀend of oligonucleotides
(V) and (VI) somewhat stabilized their triplexes with
dsDNA. Conjugate (VIIb) containing the MGB and
BIQ residues, as well as conjugate (VIIa) containing
only the MGB residue formed complexes at both pH
6.0 and 7.2.
The attachment of one MGB residue to oligonuꢀ
cleotide (VIIa) stabilized the complex. The dissociaꢀ
tion constant of this complex at pH 7.2 was eight times
higher than at pH 6.0. Surprisingly, the introduction of
BIQ in conjugates (VIIb) and (IXb) insignificantly
increased the dissociation constant of the complex at
pH 6.0, while at pH 7.2 the dissociation constant of
the complex involving conjugate (VIIb) decreased by a
factor of almost seven. These results confirm the parꢀ
ticipation of both components of the conjugates (oliꢀ
conjugates of oligo(2'ꢀOꢀmethylribonucleotides) with
dsDNA were recorded at pH 6.0 (Fig. 5). An interestꢀ
ing feature of the melting curves is the absence, in
most cases, of the pronounced peak corresponding to
denaturation of the triple helix when the optical denꢀ
sity was recorded at 260 nm, in contrast to the melting
curve of the triplex formed with initial oligonucleotide
(
VI). However, the ternary complex was observed in all
cases in the gelꢀretardation experiments. This means
that, most likely, the melting of the triplex occurs
simultaneously with the melting of the target duplex,
and the triplex immediately dissociates into singleꢀ
gonucleotide and MGB) in the recognition of the stranded fragments.
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OLIGO(2'ꢀ
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ꢀMETHYLRIBONUCLEOTIDES) AND THEIR DERIVATIVES
145
(
VIIa
)
20 50 100 200 500 10 20 50 100 200 500 nМ
(VIIb)
k
(а)
(
VIIb
)
50 100 200 500 k nМ
(
10 50 100 200 500 10
VIIa
)
(b)
Fig. 4. Native gelꢀelectrophoresis of triplexes between dsDNA and conjugates of 3'ꢀ“inverted”oligo(2'ꢀ
VIIa) and (VIIb) in 12% PAAG. Concentration of DNA duplex is 60 nM; concentrations of the third strand are indicated above
the lanes; 50 mM MES (pH 6.0), 50 mM NaCl and 5 mM MgCl , 10°C (a); 50 mM HEPES (pH 7.2), 50 mM NaCl, 5 mM
Oꢀmethylribonucleotides)
(
2
MgCl , 37°C (b); k, control dsDNA.
2
It was previously shown [34] that the dissociation duplex (Fig. 5). On the basis of these data, we assume
that the conjugate containing MGB and BIQ stabiꢀ
lizes dsDNA and remains associated with it to the
complete dissociation of the complex. Similar results
were previously obtained in our study of thermal staꢀ
bility of the triplex formed from dsDNA and the conꢀ
jugates of oligo(2'ꢀOꢀmethylribonucleotides) containꢀ
ing two MGB residues [24].
of the complex of MGB with dsDNA leads to the
change in the absorption spectrum of the ligand,
namely, to the decrease in the absorption at 330 nm.
The negative peak in differential melting curves
recorded at 330 nm evidences the interaction of the
ligand with dsDNA. In the case of conjugate (VIIb
)
containing the ligand and the intercalator, the comꢀ
plete melting of the triple complex occurs at a temperꢀ
It should be noted that in control experiments,
ature which is 8°C higher than that of the initial DNA when unbound minor groove binders with or without
Table 3. Dissociation constants of triplexes of dsDNA with “3'ꢀinverted” oligo(2'ꢀ
jugates containing MGB and intercalator under different conditions1
O
ꢀmethylribonucleotides) and their conꢀ
Kd, nM
Code
Oligonucleotide/conjugate
pH 6.0,
10
pH 7.2,
37
°
C
°C
(IV)
(V)
pUmUmUmUmCmUmUmUmUmCmCmCmCmCmCmUminvT
308 52
291 35
187 30
–
–
pL
L
1pUmUmUmUmCmUmUmUmUmCmCmCmCmCmCmUminvT
2pUmUmUmUmCmUmUmUmUmCmCmCmCmCmCmUminvT
(VI)
p
n/d
(VIIa) (Py)3ꢀ
(VIIb) BIQꢀ(Py)3ꢀ
(IXa) (Py)4ꢀ ꢀ(Py)4ꢀ
(IXb) BIQꢀ(Py)4ꢀ ꢀ(Py)4ꢀ
γ
ꢀ(Py)3ꢀ
ꢀ(Py)3ꢀ
ꢀNHꢀp
ꢀNHꢀp
γ
ꢀNHꢀ
ꢀNHꢀp
2pUmUmUmUmCmUmUmUmUmCmCmCmCmCmCmUminvT
2pUmUmUmUmCmUmUmUmUmCmCmCmCmCmCmUminvT 218 24
p
L
2pUmUmUmUmCmUmUmUmUmCmCmCmCmCmCmUminvT
2pUmUmUmUmCmUmUmUmUmCmCmCmCmCmCmUminvT 150 60 90 20
85 40 600 140
γ
γ
L
γ
γ
L
157 70
–
γ
γ
L
–
m
m
1BIQ, 6ꢀ[(3ꢀaminopropyl)amino]ꢀ11ꢀmethoxyꢀ13Hꢀbenzo[6,7]indolo[3,2ꢀ
yluridine; L1, ꢀ(CH CH O) ꢀ; L2, ꢀ(CH CH O) ꢀ; , “inverted” thymidine;
c
]quinoline; C , 2'ꢀ
O
ꢀmethylribocytidine; U , 2'ꢀ
Oꢀmethꢀ
T
p
, phosphate group. Dissociation constants of triplexes
C and pH 6.0 (50 mM MES, 50 mM NaCl, 5 mM MgCl ) or at 37 C and pH 7.2
2
2
3
2
2
6
inv
were evaluated by the gel retardation method at 10
°
°
2
(50 mM HEPES, 50 mM NaCl, 5 mM MgCl ).
2
* Concentration of duplex was 60 nM; concentration of TFO was varied from 10 nM to 500 nM; n/d, no complex was detected up to
5 mM of TFO.
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NOVOPASHINA et al.
dA/dT
0.014
0.012
0.010
0.008
0.006
0.004
0.002
0
3260
2260
1260
2330
10
20
30
40
50
60
70
80
Temperature, °С
–0.002
–0.004
3330
Fig. 5. First derivatives of thermal denaturation curves of triple complexes of dsDNA and oligo(2'ꢀ
and its conjugates. , oligonucleotide (VI); , conjugate (VIIa);
late, pH 6.0, 100 mM NaCl, 5 mM MgCl , [HIVꢀD1] = [HIVꢀD2] = 1.3
O
ꢀmethylribonucleotide) (VI)
1
2
2
3, conjugate (VIIb). Melting conditions: 10 mM sodium cacodyꢀ
–6
–6
×
10 M, [conjugate] = 1.3
× 10 M. Rate of the
temperature change was 0.1°C/min. UV absorption was registered at 260 and 330 nm.
intercalator BIQ were added to dsDNA or triplexes of rine tract in the transcribed region. The restriction site
dsDNA with the initial oligonucleotides, the change was located at a distance of 660 bp from the start of
in the thermal denaturation curves was not observed.
transcription, and the polypurine tract was at a disꢀ
tance of 332 bp from this site [33]. Thus, it is the perꢀ
centage of the truncated 332ꢀmer RNA transcript in
the total pool of transcripts that was a measure of inhiꢀ
bition of transcription by the conjugate.
Thus, we demonstrated that the conjugates of pyriꢀ
midine oligo(2'ꢀOꢀmethylribonucleotides) bearing
3'ꢀ“inverted” thymidine and the MGB and BIQ resiꢀ
dues at the 5'ꢀend can form the ternary complex with
polypurine sequence of dsDNA of HIVꢀ1 even at neuꢀ
tral pH and close to physiological temperatures. It was
previously shown that TFO based on oligodeoxyriboꢀ
nucleotides and their modified analogues, as well as
their conjugates with MGB, can efficiently inhibit
RNA polymerase in the transcription complex [11, 36,
37]. Arrest of the enzyme at the site of the triplex forꢀ
mation leads to the suppression of RNA synthesis.
As expected, initial oligo(2'ꢀ
Oꢀmethylribonucleꢀ
otide) (III) and conjugates (VIIIa) and (IXa) containꢀ
ing one MGB residue very weakly inhibited transcripꢀ
tion at 5
μM concentration. Conjugates (VIIb) and
(
IXb) containing both MGB and BIQ were the most
efficient and stopped transcription at thepolypurine
sequence even at nanomolar concentrations, with the
inhibitory effect being dependent on the conjugate
concentration. The length of the linker between the
oligonucleotide and the ligand had almost no influꢀ
ence on the inhibition efficiency, although the inhibiꢀ
tory effect of conjugate (IXb) containing the hexaethꢀ
yleneglycol linker was somewhat higher than that of
conjugate (VIIIb) containing the triethyleneglycol
Analyzing the results obtained for conjugates
(
VIIa) and (VIIb), we came to the conclusion that in
living cells twoꢀcomponent conjugates of oligo(2'ꢀ
methylribonucleotides) and MGB (MGBꢀTFO)
VIIa)–(IXa) are unlikely to be effective inhibitors of
O
ꢀ
(
transcription because their complexes with dsDNA
are unstable at physiological pH. Threeꢀcomponent
constructions BIQꢀMGBꢀTFO (VIIb)–(IXb) can be
more suitable candidates for this purpose.
linker. The inhibition amounted to 75–78% at 1 μM
concentration of conjugates (VIIIb) and (IXb).
The efficient inhibition of transcription in vitro by
ꢀmethylribonucleotides) with
ꢀmethylribonucleotides) to inhibit transcription was minor groove binders and intercalators allows one to
The ability of the prepared conjugates of oligo(2'ꢀ conjugates of oligo(2'ꢀ
O
O
studied in the in vitro system. The fragment of plasmid consider them as prospective specific agents aimed at
pSGꢀF47 after the cleavage with restrictase BspEI was dsDNA. The proposed approach to construct triplexꢀ
transcribed in vitro under the control of promotor T7 forming conjugates of oligo(2'ꢀ
Oꢀmethylribonucleoꢀ
(Fig. 6). This fragment contained the model polypuꢀ tides) stable in biological media may find application
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OLIGO(2'ꢀ
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ꢀMETHYLRIBONUCLEOTIDES) AND THEIR DERIVATIVES
147
fullꢀlength transcript (660 nt)
truncated transcript (332 nt)
PPT
BspEI
T7
AAAAGAAAAGGGGGGA
C
CCCCCC
TTTT TTTT
T
Inhibition of transcription, %
80
(
(
(
(
(
III
)
70
60
50
40
30
20
10
0
VIIIа
)
)
VIIIb
IXb
)
)
IXа
5.00
5.00
0.10 0.25 0.50 1.00
0.10 0.25 0.50 1.00
5.00 µM
Fig. 6. Inhibition of transcription with conjugates of oligo(2'ꢀOꢀmethylribonucleotides) containing MGB and BIQ in vitro. The
top panel shows the experimental system. Inhibition of transcription was evaluated by the content of the truncated transcripts in
the total pool of RNA transcripts.
in designing therapeutic agents of a new generation for was kindly provided by C.H. Nguyen and P. Schmitt
the treatment of cancer, viral, and genetic diseases, as (Institut Curie, France). Minor groove binders and
well as to create highly specific probes for the detecꢀ
tion of dsDNA.
Bocꢀprotected derivatives BосꢀNHꢀ
γꢀ(Py)3ꢀγꢀ(Py)3ꢀ
COOH and BосꢀNHꢀ ꢀ(Py)4ꢀ ꢀ(Py)4ꢀCOOH were
γ
γ
prepared as described in [31, 32, 34]. Porous glass
CPGꢀ500 containing the anchor carboxyl groups
(Theoretical Practice, Russia) was used as the polymer
EXPERIMENTAL
support for the solidꢀphase
of oligonucleotides. The attachment of
dimethoxytritylꢀ2'ꢀ ꢀmethylribonucleosides
3'ꢀ ꢀdimethoxytritylthymidine to the modified polyꢀ
mer was carried out as described in [41]. The capacity
of the resulting nucleosideꢀbound polymers was 25–
Н
ꢀphosphonate synthesis
ꢀacylꢀ5'ꢀ
and
Materials
N
Оꢀ
We used reagents and solvents from SigmaꢀAldꢀ
richꢀFluka (United States), Merck (Germany) and
О
О
Reachim (Russia); radioactive
[γ
ꢀ32P]АТР and
[α
ꢀ32P]GТP were from Amersham Biosciences
(United States). Absolute solvents were prepared by
the standard methods [38].
45
μ
mol/g for the nucleoside unit.
Oligodeoxyribonucleotides
HIVꢀD1
(5'ꢀ
The syntheses of
tylꢀ2'ꢀ ꢀmethylribonucleosides and their 3'ꢀ
phonates [39], 3'ꢀ ꢀdimethoxytritylthymidine [40],
and 2ꢀ[ ꢀ(4,4'ꢀdimethoxytrityl]oxy]ethyl]sulfonylꢀ
ethanol [26] were carried out by the known methods.
6ꢀ[(3ꢀAminopropyl)amino]ꢀ11ꢀmethoxyꢀ13 ꢀbenꢀ
zo[6,7]indolo[3,2ꢀ ]quinoline (BIQ) prepared tocol for the solidꢀphase
according to the previously described method [14, 15] (Table 1).
N
ꢀprotected 5'ꢀ
О
ꢀdimethoxytriꢀ
CCACTTTTTAAAAGAAAAGGGGGG ACTGG)
and HIVꢀD2 (5'ꢀCCAGTCCCCCCTTTTCTTTꢀ
TAAAAAGTGG) were purchased from Eurogentec
О
Hꢀphosꢀ
О
[
O
(Belgium). Oligo(2'ꢀ
and (IV) were prepared according to the standard proꢀ
ꢀphosphonate synthesis
Оꢀmethylribonucleotides) (III)
H
c
Н
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 39
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148
NOVOPASHINA et al.
We used the following enzymes: nuclease Р1 Peniꢀ
Analytical and preparative RP HPLC of minor
cillium citrinum (EC 3.1.30.1) (100 U/mL) (Sigma, groove binders and oligonucleotide conjugates were
United States), alkaline phosphatase E. coli performed on a 1100 Agilent Technologies system
(EC 3.1.3.1) (34 U/mg) (Biolar, Litvania), phosphodꢀ (United States) on a column (7.8
× 300 mm) with
iesterase Сrotalus atros venom (EC 3.1.4.1) XꢀTerra Cꢀ18 (7 m, Waters, United States). Oligoꢀ
μ
(0.9 U/mL) (Sigma, United States), and T4 polynuꢀ carboxamides were isolated by chromatography in a
cleotide kinase (EC 2.7.1.78) (5000 U/mL) (Sibenꢀ gradient of concentrations of acetonitrile (0–90%) in
zyme, Russia).
the presence of trifluoroacetic acid; oligonucleotides
and their conjugates were subjected to chromatograꢀ
phy in a gradient of concentrations of acetonitrile (5–
40%) in 0.025 M NH4OAc.
Methods
Absorption spectra were recorded on a Kontron
923 Uvikon Instrument (BioTek, United States).
Radioactive gels were scanned on a PhosphorImager
(Molecular Dynamics, United States) and treated
with the ImageQuant program.
The analysis and identification of compounds were
performed by TLC on DCꢀAlufolien Kieselgel 60 F254
plates (Merck, Germany). Adsorption chromatograꢀ
phy was carried out using silica gel Kieselgel 60
(Merck, Germany).
Molar absorption coefficients of oligonucleotides
at 260 nm were calculated according to [42]. The valꢀ
ues of molar absorption coefficients of 2'ꢀ
Oꢀmethylriꢀ
bonucleotides were taken equal to those of corresponding
ribonucleotides. Molar absorption coefficients of
oligo(2'ꢀOꢀmethylribonucleotides) containing thymiꢀ
1H NMR spectra of the synthesized compounds
were recorded on an NMR spectrometer Bruker
AM400 in (СD3)2SO or CDCl3 using tetramethylsiꢀ
lane as a standard. 31P NMR spectra of the synthesized
compounds were recorded on an NMR spectrometer
Bruker AM400 in a pyridineꢀacetonitrile mixture (1 : 1)
using 85% H3PO4 as a standard.
dine attached through the 3'ꢀ3'internucleotide bonds
were taken equal to the sum of molar absorption coefꢀ
Molecular masses of the oligonucleotide conjuꢀ
gates were evaluated by mass spectrometry with elecꢀ
trospray ionization on a quadrupole mass spectromeꢀ
ter QꢀStar (Applied Biosystems, France) using the
QꢀTOF (ES QꢀTOF MS) technique in the aqueous
phase in the positive field (in the anion analysis mode).
ficients of the corresponding oligo(2'ꢀOꢀmethylriboꢀ
nucleotides) and thymidine. Molar absorption coeffiꢀ
cients of the conjugates with minor groove binders at
260 nm were taken equal to the sum of molar absorpꢀ
tion coefficients of the corresponding oligonucleꢀ
otides and the value of 36610 М–1 cm–1 for hexa(
Nꢀ
methylpyrrole)carboxamide or 51427 M–1 cm–1 for
octa( ꢀmethylpyrrole)carboxamide [43]. The contriꢀ
Oligo(2'ꢀOꢀmethylribonucleotides) were isolated
by preparative ionꢀexchange and reverseꢀphase HPLC
on a Waters chromatograph (United States) on colꢀ
N
bution of BIQ to the molar absorption coefficients of
conjugates at 260 nm was neglected.
umns (4.6
× 250 mm) with PolysilꢀSA (Theoretical
Practice, Russia) and LiChrosorb RPꢀ18 (Merck,
Germany), respectively. Elution during ion exchange
chromatography was performed in a gradient of conꢀ
centration of KH2PO4, pH 6.5 (0–0.3 M) in 30% aceꢀ
tonitrile for 100 min at a flow rate of 2 mL/min. Eluꢀ
tion during RP HPLC was performed in a gradient of
acetonitrile concentration (20–90%) in 0.05 M
LiClO4 for 30 min at a flow rate of 2 mL/min. Chroꢀ
matographically pure oligonucleotides were precipiꢀ
tated as lithium salts.
O
ꢀ(4,4'ꢀDimethoxytrityl)ꢀtriꢀ and hexaethyleneꢀ
glycols (Ia) and (Ib) were prepared starting from triethꢀ
yleneglycol (0.6 mL, 3.75 mmol) or hexaethyleneglyꢀ
col (0.95 mL, 3.75 mmol) and 4,4'ꢀdimethoxytrityl
chloride (1 g, 3 mmol) according to [25]. The monoꢀ
tritylated product with lower chromatographic mobilꢀ
ity was isolated by silica gel chromatography in a linear
gradient of concentrations of diethyl ether (0–5%) in
СН2Сl2 with 0.2% TEA. The chromatographically
pure product was evaporated to thick oil.
Oꢀ(4,4'ꢀDimethoxytrityl)ꢀtriethyleneglycol
Yield 65%.
f 0.29 (diethyl ether). 1H NMR (CDCl3):
.23–7.31 (13 H, m, aromatic protons), 6.77 (2 H, d,
aromatic protons in ortoꢀposition to CH3O group),
3.75 (6 Н, s, СН3О), 3.64 OCH2CH2O–), 1.97 (1H,
(Ia).
The oligonucleotides and their derivatives were
analyzed by RP HPLC on a Milichrom Aꢀ02 chromatoꢀ
R
7
graph (Econova, Russia) using a column (
with Nucleosil Cꢀ18 (5 m, MachereyꢀNagel, Gerꢀ
2 × 62 mm)
μ
many) and a gradient of acetonitrile concentrations
(0–80%) in 0.05 M LiClO4 at a flow rate of
100 μL/min. Analytical RP HPLC of oligonucleotide
hydrolysates was carried out in a gradient of concenꢀ
trations of acetonitrile (0–50%) in 0.01 M NaOAc at
45°C. The analytical ionꢀexchange HPLC was carried
t,
⎯ОН). Oꢀ(4,4'ꢀDimethoxytrityl)hexaethyleneglycol
1
(Ib). Yield 50%. Rf 0.39 (diethyl ether). H NMR
(CDCl3): 7.24–7.33 (13 H, m, aromatic protons),
6.79 (2 H, d, aromatic protons in ortoꢀposition to
CH3O group), 3.75 (6 H, s, СН3О), 3.62 (24 H, t,
OCH2CH2O–), 1.96 (1 H, t, ꢀOH).
out on a column (
gradient of concentrations of KH2PO4 (0–0.3 M) in
30% acetonitrile at a flow rate of 100 L/min.
2 × 62 mm) with Partisil 10 SAX in a
H
ꢀPhosphonates of
goethyleneglycols (IIa) and (IIb) were prepared by
Oꢀ(4,4'ꢀdimethoxytrityl)ꢀoliꢀ
μ
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 39
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OLIGO(2'ꢀ
O
ꢀMETHYLRIBONUCLEOTIDES) AND THEIR DERIVATIVES
149
analogy with the synthesis of nucleoside
nates [27]. Imidazole (1.35 g, 19.9 mmol) was disꢀ
H
ꢀphosphoꢀ yields and characteristics of oligonucleotides (IV)–
VI) are presented in Table 1. The presence of the 5'ꢀ
(
solved in 25 mL of abs. CH3CN under argon. PCl3 terminal phosphate was confirmed by the hydrolysis of
(0.54 mL, 6.2 mmol) and TEA (2.8 mL, 20 mmol) the oligonucleotides with alkaline phosphatase folꢀ
were quickly added under stirring to the resulting soluꢀ lowed by the analysis of the hydrolysate by analytical
tion cooled to 0°C. Stirring was continued for 15 min, ionꢀexchange HPLC.
followed by the dropwise addition of
dimethoxytrityl)ꢀtriethyleneglycol (Ia) (25 mL) or
(4,4'ꢀdimethoxytrityl)ꢀhexaethyleneglycol
Oꢀ(4,4'ꢀ
Analysis of nucleoside composition of oligo(2'ꢀ
methylribonucleotides) (IIIꢀVI). The nucleoside comꢀ
position of oligo(2'ꢀ ꢀmethylribonucleotides) conꢀ
Oꢀ
O
ꢀ
)
(
Ib
O
(1.37 mmol) in abs. CH3CN for 30 min. The reaction
was carried out under stirring for 90 min at room temꢀ
perature. The reaction was monitored by TLC in a
C2H5OH–CH2Cl2 mixture (4 : 6) containing 0.2% TEA.
After the completion of the reaction, H2O (15 mL) was
added and the mixture was evaporated to dryness, the
residue was dissolved in СН2Сl2 containing 0.2% TEA
and extracted two times with 5% aqueous solution of
NaCl and one time with water. The organic layer was
dried with anhydrous Na2SO4 and evaporated to oil.
The reaction mixture was subjected to silica gel chroꢀ
matography in a linear gradient of concentrations of
C2H5OH (0–30%) in СН2Сl2 containing 0.2% TEA.
The product with the lower mobility in TLC was colꢀ
lected and evaporated.
taining “inverted” thymidine was confirmed by the
exhaustive hydrolysis with nuclease P1 (0.03 M
NaOAc, pH 5.2, 1M ZnSO4, 16 h, 37°C) and alkaline
phosphatase (0.1M TrisꢀHCl, pH 7.8, 5 mM MgCl2,
4 h, 37°C) followed by the quantitative analysis of the
hydrolysate by analytical RP HPLC. Nucleosides were
identified by the retention time values on the column
and the spectral ratios in comparison with specially
prepared markers. The experimental ratios correꢀ
sponded to the calculating values.
Conjugates of minor groove binders with 6ꢀ[(3ꢀaminoꢀ
propyl)amino]ꢀ11ꢀmethoxyꢀ13
Hꢀbenzo[6,7]indolo[3,2ꢀ
c]quinoline (BIQ). 1ꢀHydroxybenzotriazole (5.1 mg,
0.033 mmol) and dicyclohexylcarbodiimide (7.5 mg,
0.03 mmol) were added to the solution of Bocꢀproꢀ
Products in the form of oil were dissolved in a small
amount of CH3CN, packed into tubes and dried at 1–
2 mmHg over P2O5. The yields were 50–60%.
tected derivative of hexaꢀ or octa(
role)carboxamide in the acid form (BосꢀNHꢀ
ꢀ(Py)3ꢀCOOH or BосꢀNHꢀ ꢀ(Py)4ꢀ ꢀ(Py)4ꢀCOOH)
N
γ
γ
γ
31P NMR (pyridineꢀCH3CN, 1 : 1):
O
ꢀ(4,4'ꢀdimethoxyꢀ
ꢀphosphonate (IIa): 4.77 (d,
ꢀ(4,4'ꢀdimethoxytrityl)ꢀhexaethyleneglyꢀ
ꢀphosphonate (IIb): 4.73 (d, PꢀH 933).
ꢀPhosphonate of 2ꢀ[2ꢀ(4,4'ꢀdimethoxytritylꢀ
oxy)ethylsulfonyl]ethanol (IIc) was prepared similar to
the synthesis described above starting from [2ꢀ[
(4,4'ꢀdimethoxytrityl)oxy]ethyl]sulfonyl]ethanol (Ic).
After silica gel chromatography, ꢀphosphonate(IIc
(0.03 mmol) in 0.5 mL of abs. DMF, and the mixture
was stirred for 12 h at room temperature. The reaction
was monitored by TLC in a СНСl3–СН3OH system
trityl)ꢀtriethyleneglycolꢀ
JPꢀH 933);
colꢀ
H
O
Н
J
(85 : 15). BIQ (11.1 mg, 0.03 mmol) and TEA (40 μL)
were added to the reaction mixture, and the stirring
was continued for another 12 h. The reaction mixture
was evaporated, the dry residue was dissolved in 3 mL
of a CF3COOH–СН2Сl2 mixture (1 : 2) and was kept
for 25 min at room temperature to remove the Boc
protecting group. The reaction mixture was evapoꢀ
rated to dryness, the residue was dissolved in a miniꢀ
mal volume of DMF and the resulting solution was
added in small portions to ethyl ether under stirring.
The residue was washed with ethyl ether and dried,
followed by its dissolution in water. The yields of the
H
O
ꢀ
H
)
in the form of a thick oil was dissolved in a small
amount of abs. CH3CN, packed into tubes and dried
at 1–2 mmHg over P2O5. The yield of (IIc) was 74%.
31P NMR (pyridine/CH3CN, 1 : 1): 2.63 (
JPꢀH 607).
Synthesis of oligo(2'ꢀ
containing oligoethyleneglycolꢀphosphate at the 5'ꢀend
Oꢀmethylribonucleotides)
products isolated by RP HPLC were 55% for NH2ꢀ
(Py)3ꢀ ꢀ(Py)3ꢀBIQ and 61% for NH2ꢀ ꢀ(Py)4ꢀ
(Py)4ꢀBIQ
1H NMR ((CD3)2SO):
NH2ꢀ ꢀ(Py)3ꢀ ꢀ(Py)3ꢀBIQ: 13.66 (1 H, s, –NH,
midine. Two last stages of the synthesis were the sucꢀ BIQ), 12.27 (1 H, s, –NH, BIQ), 9.90 (4 H, s,
γ
ꢀ
(IV)–(VI) was carried out according to the protocol of
γ
γ
γ
ꢀ
the solidꢀphase
polymer support bearing 1.4
3'ꢀ ꢀdimethoxytritylthymidine was used for the synꢀ
thesis of oligonucleotides containing “inverted” thyꢀ
H
ꢀphosphonate synthesis [39]. The
.
μ
mol of the attached
O
γ
γ
cessive attachment of dimethoxytritylꢀcontaining triꢀ
or hexaethyleneglycolꢀ ꢀphosphonate and [2ꢀ[
(4,4'ꢀdimethoxytrity)ꢀloxy]ethyl]sulphonyl]ethanolꢀ
phosphonate. After the standard treatments, oligonuꢀ BIQ,
cleotides were isolated by ionꢀexchange and RP m, BIQ), 7.88 (1 H, d, BIQ,
HPLC. Oligonucleotides were precipitated with aceꢀ 8.0), 7.71 (2 H, br.s, NH2–CH2CH2CH2–), 7.32
tone containing 2% LiClO4. The overall yield of oligoꢀ (1 H, d. 1.7, –CH–, Py), 7.21–7.15 (5 H, m, –CH–,
nucleotides relative to the first nucleoside unit Py), 7.05 (1 H, d, 1.7, –CH–, Py), 7.04 (1 H, d,
attached to the polymer support was 8–10%. The 1.7, –CH–, Py), 7.00 (1 H, d, J 1.7, –CH–, Py),
⎯CONH–), 9.89 (1 H, s, –CONH–), 9.88 (1 H, s,
H
O
ꢀ
⎯
BIQ,
ONH–), 8.69 (1 H, d, BIQ,
9.0), 8.30 (1 H, t, BIQ,
8.0), 8.18 (1 H, d, BIQ,
J
8.0), 8.57 (1 H, d,
Hꢀ
J
J
J 8.0), 8.22 (1 H, d,
8.0), 8.07–8.03 (2 H,
J
J
9.0), 7.81 (1 H, t, BIQ,
J
J
J
J
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 39
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2013

150
NOVOPASHINA et al.
6.91 (1 H, d,
J
1.7, –CH–, Py), 6.87 (1 H, d,
1.7, –CH–, Py), 4.04 containing minor groove binder and 6ꢀ[(3ꢀaminoproꢀ
(3 H, s, –OCH3), 3.91 (2 H, br. quartet, NH–CH2 pyl)amino]ꢀ11ꢀmethoxyꢀ13 ꢀbenzo[6,7indolo[3,2ꢀ
J
1.7,
Conjugates of oligo(2'ꢀOꢀmethylribonucleotides)
⎯
CH–, Py), 6.85 (1 H, d, J
–
H
c]
CH2CH2), 3.84 (3 H, s, –NCH3), 3.83 (3 H, s, –NCH3), quinoline (BIQ) (VIIb)–(IXb) were synthesized as
3.82 (6 H, s, –NCH3), 3.79 (3 H, s, –NCH3), 3.20 described above using as a ligand the conjugate of
(2 H, quartet,
(2 H, quartet,
br.t, CH2CH2–CH2–CO), 2.34 (2 H, t,
CH2CH2–CH2–CO), 2.27 (2 H, t, 6.0, CH2CH2– thesized oligonucleotide derivatives are presented in
CH2–CO), 2.02 (2 H, br. quintet, 6.0, CH2–CH2– Table 2.
CH2), 1.85–1.75 (4 H, m, CH2–CH2–CH2).
J
6.0, –NH–CH2–CH2CH2), 2.82 hexaꢀ or octa(
6.0, NH–CH2CH2–CH2), 2.55 (2 H, BIQ (NH2ꢀ ꢀ(Py)3ꢀ
6.0, (Py)4ꢀBIQ). The yields and characteristics of the synꢀ
N
J
γ
J
J
J
Thermal stability of triplexes. The thermal denaturꢀ
ation and renaturation of the complexes were perꢀ
formed according to [35] in 0.01 M cacodylate buffer,
pH 6.0, containing 0.1 M NaCl and 5 mM MgCl2. The
concentrations of the target duplex and the third strand in
NH2ꢀ ꢀ(Py)4ꢀ ꢀ(Py)4ꢀBIQ: 13.61 (1 H, s, –NH,
BIQ), 12.26 (1 H, s, –NH, BIQ), 9.92 (3 H, s,
CONH), 9.88 (3 H, s, –CONH), 9.83 (1 H, s,
CONH), 9.77 (1 H, s, –CONH), 8.70 (1 H, d, BIQ,
γ
γ
⎯
⎯
J
each sample were 1–1.3 μM and 1.3–1.7 μM, respecꢀ
8.0), 8.57 (1 H, d, BIQ,
J
9.0), 8.30 (1 H, t, BIQ,
8.0), 8.17 (1 H, d, BIQ,
8.0), 8.06–8.01 (2 H, m, BIQ), 7.89 (1 H, d, BIQ,
9.0), 7.80 (1 H, t, BIQ, 8.0), 7.70 (2 H, br.s, NH2
CH2CH2CH2–), 7.32 (1 H, d,
7.21–7.15 (7 H, m, –CH–, Py), 7.05–7.03 (3 H, m,
CH–, Py), 7.00 (1 H, d, 1.7, –CH–, Py), 6.94
(1 H, d, 1.7, –CH–, Py), 6.91 (1 H, d, 1.7, –CH–,
Py), 6.87 (1 H, d, 1.7, –CH–, Py), 6.85 (1 H, d, 1.7,
J
J
J
–
tively. The rate of the temperature change was
0.1°C/min, the absorption at 260, 330, and 580 nm
was recorded every 200 s on a Kontron Uvikon 940
spectrophotometer (BioTek, United States) adjusted
with nine thermostated 1ꢀcmꢀpathꢀlength cuvettes.
The melting curves were processed using the Kaleidaꢀ
Graph and Microsoft Excel programs.
8.0), 8.19 (1 H, d, BIQ,
J
J
J
1.7, –CH–, Py),
⎯
J
J
J
J
J
Dissociation constants of ternary complexes were
–CH–, Py), 4.04 (3 H, s, –OCH3), 3.91 (2 H, br. evaluated by the gel retardation method according to
quartet, NH–CH2–CH2CH2), 3.84 (15 H, s, –NCH3), [35] at pH 6.0 or 7.2 and at temperature of 10°C or
3.82 (6 H, s, –NCH3), 3.80 (3 H, s, –NCH3), 3.21 37°C varying the concentration of the triplexꢀforming
(2 H, quartet,
J
6.0, NH–CH2–CH2CH2), 2.79 (2 H, oligonucleotide (10, 50, 100, 200, and 500 nM). Each
quartet, 6.0, NH–CH2–CH2CH2), 2.55 (2 H, br.t, sample (10
J
μ
L) containing 0.6 pmol of the 5'ꢀ32Pꢀ
CH2CH2–CH2–CO), 2.35–2.15 (4 H, m, CH2CH2– labeled target in a 50 mM MES buffer, pH 6.0, conꢀ
CH2–CO), 2.02 (2 H, br. quintet, CH2–CH2–CH2), taining 50 mM NaCl and 5 mM MgCl2 or 50 mM
1.72 (2 H, br. quintet,CH2–CH2–CH2), 1.72 (2 H, br. HEPES, pH 7.2, containing 50 mM NaCl and 5 mM
quintet, CH2–CH2–CH2).
MgCl2 was denatured at 90°C for 3 min, then slowly
cooled to room temperature followed by the addition
of the third strand to the needed concentration. The
Conjugates of oligo(2'ꢀOꢀmethylribonucleotides)
containing minor groove binder (VIIa)–(IXa) were synꢀ
thesized according to protocols [28, 44]. Solutions of
5% solution (1
cyanol FF in 50% glycerol was added, and the reaction
mixture was incubated for 5–16 h at 10°C or 37°С.
μL) of bromophenol blue and xylane
triphenylphosphine (6.8 mg 25
DMSO and 2,2'ꢀdipyridyldisulfide (5.3 mg, 25
in 25 L of abs. DMSO were added to the solution of
oligonucleotide ( ) or (VI) in the form of cetyltrimeꢀ
μ
mol) in 25
μ
L of abs.
μmol)
Electrophoretic separation of the products was carried
out in 12% native PAAG at the corresponding temperꢀ
μ
V
ature. Dissociation constants of the triplexes (
Kd) were
thylammonium salts (10–15 OU260). The reaction
mixture was stirred for 15 min at 25°C. The activated
oligonucleotide was precipitated with 2% LiClO4 in
acetone, quickly washed with acetone, and dissolved
evaluated as described in [35].
Kinetic curves of triplex formation obtained by gel
retardation method. The kinetic experiments were carꢀ
ried out by the gel retardation method at pH 6.0 and
10°C. The target 5'ꢀ[32P]ꢀdsDNA (60 nM) was heated
in a buffer (50 mM MES, pH 6.0, containing 50 mM
NaCl, 5 mM MgCl2, 0.05% bromophenol, 0.05%
in water (5
hexa( ꢀmethylpyrrole)carboxamide)
in DMSO (100
μL). The corresponding ligand (octaꢀ or
N
(1
μmol)
μL) containing abs. TEA (5
μ
L) was
added to the activated oligonucleotide, and the reaction
mixture was kept for 2 h at room temperature, followed
by the precipitation with 2% LiClO4 in acetone and subꢀ
xylene cyanol FF, and 10% glycerol) at 90
slowly cooled to the room temperature followed by the
addition of the third strand to 2 M concentration.
°С for 3 min,
μ
sequent dissolution of the residue in water (100 μL). The
The reaction mixture was aliquoted after certain periꢀ
ods of time (0, 15, 30, 60, 120, and 180 min) and
applied to 12% native PAGE. The data were processed
using the ImageQuant and Sigma Plot 6.0 programs.
Inhibition of in vitro transcription with oligonucleꢀ
otide conjugates was performed as described in [33,
resulting conjugates were isolated by RP HPLC in a
gradient of concentrations of CH3CN (5–40%) in
0.025 M ammonium acetate (pH 6.0) followed by preꢀ
cipitation in the form of sodium salts. The yields and
characteristics of the synthesized oligonucleotide
derivatives are presented in Table 2.
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 39
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2013

OLIGO(2'ꢀ
O
ꢀMETHYLRIBONUCLEOTIDES) AND THEIR DERIVATIVES
151
11. Giovannangeli, C., Perrouault, L., Escude, C.,
Thuong, N.T., and Hélène, C., Biochemistry, 1996,
vol. 35, pp. 10539–10548.
36]. 780ꢀmer fragment of plasmid pSGꢀF47 (0.5
10 nmol) containing the polypurine tract was added to
25 L of the reaction mixture containing 40 mM Trisꢀ
HCl, pH 8.0, 6 mM MgCl2, 2 mM spermidine, 4 mM
dithiothreitol, 1 U/ L of RNase inhibitor, and the oliꢀ
gonucleotide or its conjugate to the concentration desꢀ
ignated in Fig. 6. The final concentration of nucleoside
μg,
μ
12. Arimondo, P.B. and Hélène, C., Curr. Med. Chem.
2001, vol. 1, pp. 219–235.
,
μ
13. Bentin, T. and Nielsen, P.E., J. Am. Chem. Soc., 2003,
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triphosphates in the reaction mixture was 500
AT P, C T P, a n d U T P, 1 0 0 M for GTP, and 0.3
ꢀ32P]GTP. T7 RNA polymerase (25 AU) was added
μ
M for
14. Silver, G.C., Nguyen, C.H., Boutorine, A.S., Bisagni, E.,
Garestier, T., and Hélène, C., Bioconj. Chem., 1997,
vol. 8, pp. 15–22.
μ
μM for
[α
to the resulting mixture, and the transcription 15. Silver, G.C., Sun, J.S., Nguyen, C.H., Boutorine, A.S.,
Bisagni, E., and Hélène, C., J. Am. Chem. Soc., 1997,
vol. 119, pp. 263–268.
occurred for 10 min at 37°C. The reaction was stopped
by precipitation with ethanol. The residue was disꢀ
solved in a loading buffer, and the transcription prodꢀ
ucts were analyzed by electrophoresis in denaturing
6% PAAG.
16. Nguyen, C.H., Marchand, C., Delage, S., Sun, J.S.,
Garestier, T., Hélène, C., and Bisagni, E., J. Am. Chem.
Soc., 1998, vol. 120, pp. 2501–2507.
17. Schmitt, P., Sun, J.S., Garestier, T., Hélène, C.,
Nguyen, C.H., Grierson, D.S., and Bisagni, E., Chem.
Commun., 2000, pp. 763–764.
ACKNOWLEDGMENTS
18. Sinyakov, A.N., Ryabinin, V.A., Grimm, G.N., and
Boutorine, A.S., Mol. Biol. (Moscow), 2001, vol. 35,
pp. 251–260.
The authors would like to thank J.P. Brouard,
A. Marie, and L. Dubost (Muséum National d’Hisꢀ
toire Naturelle, Paris, France) for recording mass
spectra and C.H. Nguyen and P. Schmitt (Institut
Curie, Paris, France) for kindly provided 6ꢀ[(3ꢀ
aminopropyl)amino]11ꢀmethoxyꢀ13Hꢀbenzo[6,7ꢀ
19. Szewczyk, J.W., Baird, E.E., and Dervan, P.B., Angew.
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nova, A., and Boutorine, A., Nucleosides Nucleotides
Nucleic Acids, 2003, vol. 22, pp. 1179–1182.
c
]quinoline. The work was supported by INTAS
(project no. 01ꢀ0638) , Russian Foundation for Basic
Research (project no. 12ꢀ04ꢀ91053ꢀNTsNI_a), and
the Ministry of Foreign Affairs of France (EGIDE
no. 04542ND).
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1999, vol. 3, pp. 688–693.
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