Oligonucleotide derivatives in nucleic acid hybridization analysis. III. Synthesis and investigation of properties of oligonucleotides, bearing bifunctional non-nucleotide insertion

Article abstract of DOI:10.1134/S106816201206009X

Non-nucleotide phosporamidites were synthetized, having a branching backbone with different positions for functional groups. Phosphoramidite monomers obtained contain intercalator moiety, 6-chloro-2-methoxyacridine, and additional hydroxyl residue protected with dimethoxytrityl group or with the tertbutyldimethylsilyl group for post-synthetic modification. Oligothymidilates containing one or more modified units in different positions of the sequence were synthesized. The melting point and thermodynamic parameters of the formation of complementary duplexes formed by modified oligonucleotides were defined (change in enthalpy and entropy). The introduction of intercalating residue causes a significant stabilization of DNA duplexes. It is shown that the efficiency of the fluorescence of acridine residue in the oligonucleotide conjugate significantly changes upon hybridization with DNA. Pleiades Publishing, Ltd., 2012.

Full text of DOI:10.1134/S106816201206009X

ISSN 1068ꢀ1620, Russian Journal of Bioorganic Chemistry, 2012, Vol. 38, No. 6, pp. 625–638. © Pleiades Publishing, Ltd., 2012.
Original Russian Text © M.S. Kupryushkin, D.V. Pyshnyi, 2012, published in Bioorganicheskaya Khimiya, 2012, Vol. 38, No. 6, pp. 706–720.
Oligonucleotide Derivatives in Nucleic Acid Hybridization Analysis.
III. Synthesis and Investigation of Properties of Oligonucleotides,
Bearing Bifunctional NonꢀNucleotide Insertion
M. S. Kupryushkin and D. V. Pyshnyi1
Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences,
pr. Akademika Lavrent’eva 8, Novosibirsk, 630090 Russia
eꢀmail: pyshnyi@niboch.nsc.ru
Received December 19, 2011; in final form, January 20, 2012
Abstract—Nonꢀnucleotide phosporamidites were synthetized, having a branching backbone with different
positions for functional groups. Phosphoramidite monomers obtained contain intercalator moiety, 6ꢀchloroꢀ
2ꢀmethoxyacridine, and additional hydroxyl residue protected with dimethoxytrityl group or with the tert
ꢀ
butyldimethylsilyl group for postꢀsynthetic modification. Oligothymidilates containing one or more modiꢀ
fied units in different positions of the sequence were synthesized. The melting point and thermodynamic
parameters of the formation of complementary duplexes formed by modified oligonucleotides were defined
(change in enthalpy and entropy). The introduction of intercalating residue causes a significant stabilization
of DNA duplexes. It is shown that the efficiency of the fluorescence of acridine residue in the oligonucleotide
conjugate significantly changes upon hybridization with DNA.
Keywords: branched DNA, nonꢀnucleotide insertion, modified oligonucleotides, fluorescence probe, thermal staꢀ
bility
DOI: 10.1134/S106816201206009X
1
INTRODUCTION
ing various groups at the ends as well as within the
chain. Versatile use of phosphoramidite monomers,
providing an opportunity for the insertion of funcꢀ
tional residues in the inner part of the oligonucleotide
is obvious. These monomers contain two reactive cenꢀ
ters for incorporation of the residues in the synthesized
chain and additional groups (one or more) to realize
the possibility of the branching phosphodiester backꢀ
bone and/or introduction into the inner part of the oliꢀ
gonucleotide derivative of the desired residue with a
given functionality. The application of the branched
skeleton of a nonꢀnucleotide insertion based on the
substituted saturated hydrocarbons leads to the existꢀ
ence of chiral centers in the structure of such a link
[17–22]. However, for example, in [23] it was proved
that differences in the spatial orientation of an addiꢀ
tional group consisting of an oligonucleotide derivaꢀ
tive cause different hybridization properties of the corꢀ
responding enantiomer derivatives. The use of racemic
insertion is possible, but often functionally justified, as
synthons derived from optically pure isomers are
expensive.
The derivatives of oligonucleotides are widely used
as molecular tools in modern studies of living systems
and molecular diagnostics. At present, automatic synꢀ
thesis is a versatile method for obtaining fragments of
nucleic acids (NA). The vast majority of the modificaꢀ
tions are introduced into the synthesized oligonucleꢀ
otide chain in the process of building with the applicaꢀ
tion of appropriately functionalized phosphoramidite
synthons. Many of the functional groups are introꢀ
duced into the synthetic NA in the nonꢀnucleotide
inserts. The range of derivatives of this type is broad:
molecular probes in detection systems of point mutaꢀ
tions [1–3], dendritic structures and other supramoꢀ
lecular structures [4, 5], geneꢀdirected or antisense
agents [6–11], derived precursors for further functionꢀ
alization and/or modification of the oligonucleotide
structure requiring the “mild” reaction conditions
[12–16].
A wide range of commercially available synthons
was developed for obtaining the oligonucleotides bearꢀ
Abbreviations: CEPꢀ2, cyanoethoxy diisopropylaminophosphiꢀ
nyl; DMAP, 4ꢀN,Nꢀdimethylaminopyridine; DIPEA, diisoproꢀ
The disadvantages associated with the presence of a
chiral center in the insertion can be avoided by creatꢀ
ing the hydrocarbon structure bearing identical subꢀ
stituents or domains [3, 4, 24]. Obtaining the achiral
nonꢀnucleotide synthons, whose properties do not
depend on the nature of the structure of their substituꢀ
pylethylamine; DMTr, dimethoxytrityl; TBDMS, tretꢀbutyldimeꢀ
thylsilyl; TEA, triethylamine; Tz, 1Hꢀtetrazole. The prefix “d” in
the notation of oligodeoxyribonucleotides is omitted.
Corresponding author: phone: +7(383)363ꢀ51ꢀ51; eꢀmail:
pyshnyi@niboch.nsc.ru. Communication II see [1].
1
625

626
KUPRYUSHKIN, PYSHNYI
ents (functional groups), is possible using the benzene tional groups, one of which is able to stabilize the
ring and its analogues as a center of backbone branchꢀ duplex structure, while the other provides the ability
ing for insertion [25–27]. Structures containing nitroꢀ
gen atoms as centers of backbone branching are a funꢀ
damentally different type of three or more achiral
functionalized monomers [2, 28, 29].
for further modification of the oligonucleotide.
Aliphatic amines, which enable the formation of
branching centers due to the presence of nitrogen
atoms in them, were chosen as the basis of the
branched backbone of nonꢀnucleotide insertion
devoid of chiral centers [2, 28, 29]. A variety of comꢀ
mercially available amines and amino alcohols, the
relative simplicity and efficiency of their transformaꢀ
tion within protocols used in organic and bioorganic
chemistry, make this approach useful. For example,
The aim of this study is to analyze the possibility of
creating the achiral multifunctional nonꢀnucleotide
insertion (two functions for the introduction in the oliꢀ
gonucleotide chain and two additional functions) that
is suitable for automatic synthesis of NA derivatives
and has a relatively small size. The insertion prototype
must meet the following requirements: 1) the possibilꢀ
ity of introducing it into the oligonucleotide sequence
in the standard procedures for automated DNA synꢀ
thesis; 2) the absence of chiral centers to exclude the
possibility of ambiguous orientation of residues in the
modified DNA duplexes; 3) the presence of a group
for additional modifications of the oligonucleotide
chain; and 4) the presence of a group that compensates
for the “disturbing” effect of nonꢀnucleotide insertion
in the carbohydrate–phosphate backbone.
The availability of synthons of this type largely
determines the prospects for the development of a
number of approaches aimed at creating the geneꢀ
directed compounds, NAzymes, structural blocks for
NAꢀnanoarchitectonics. The establishment of such
structures may facilitate the formation of complex oliꢀ
gonucleotide constructs with spatially contiguous
fragments containing different functional groups.
diethanolamine,
Nꢀ(2ꢀhydroxyethyl) ethylenediꢀ
amine and other similar aliphatic amines and amino
alcohols can serve as convenient small blocks for the
introduction of branching points. Ethylene fragments
located between the heteroatoms (N and/or O), apparꢀ
ently, are the lowest possible linker fragments separatꢀ
ing the reaction and other functional centers in phosꢀ
phoramidite synthons. The secondary amino group of
these compounds may act as articulation points of
individual elements created for the branched backꢀ
bone. The application of residues of dicarboxylic
acids, a variety of precursors which is widely available,
is reasonable as the linkers that connect the functionꢀ
alized block of insertion. Preparation of amides from
secondary amines at the articulation points would
eliminate the availability of key sites (in the form of
tertiary amines) in the final structure of the synthon.
The presented strategy of forming the synthon of
insertion is universal, involving the carrying out of a
limited set of standard reactions: introduction of proꢀ
tective groups, phosphitylation of hydroxy compoꢀ
nents, functionalization of primary amine groups and
formation of an amide bond. At the same time, a variꢀ
ety of tetraꢀ (Scheme 1a) and triꢀfunctional monoꢀ
RESULTS AND DISCUSSION
Design of MultiꢀFunctional NonꢀNucleotide Insertion
In accordance with the objectives of the work, the
phosphoramidite synthon of achiral nonꢀnucleotide
insertion must contain four blocks: two groups to
ensure its inclusion in the growing chain of oligonuꢀ mers (Scheme 1b) can be obtained by combining a set
cleotide during automatic synthesis, and the two funcꢀ of blocks used.
(a)
(b)
(c)
O
O
O
O
R1
R1
R3
R3
8
9
1
4
2
OMe
linker
linker
7
N
N
N
N
H
3
R2
R2
R4
6
Cl
N
5
10
Scheme 1. Schematic representation of the structure of the tetraꢀ and triꢀfunctional monomers
and the residue structure of duplexꢀstabilizing agent used
.
We have previously described in detail the effect of varꢀ pletely described. Since the introduction of nonꢀnucleꢀ
ious nonꢀnucleotide insertions based on the oligoethyleꢀ otide fragments in the carbohydrate–phosphate backꢀ
neglycol and oligomethylenediols on the complexing bone of oligonucleotide markedly destabilizes the duplex
properties of the “bridge” oligonucleotides [30]. Influꢀ structure formed by them, we chose the residue of substiꢀ
ence of insertion based on diethylene glycol [31], which is tuted acridine, 6ꢀchloroꢀ2ꢀmetoxy acridine, intercalating
comparable in linear dimensions to diethanolamine, a into the double helix (Scheme 1c) as an important addiꢀ
basic element of insertion we created is sufficiently comꢀ tional function of the developed insertion. The employꢀ
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OLIGONUCLEOTIDE DERIVATIVES IN NUCLEIC ACID HYBRIDIZATION ANALYSIS
627
(а)
30
25
20
15
10
5
(b)
50
40
30
20
10
0
1
2
3
1
2
1
2
3
1
3
2
3
0
600 300
300
400
500
400
500
600
Wavelength, nm
(c)
Wavelength, nm
(d)
25
20
15
10
5
50
40
30
20
10
1
2
3
1
2
3
2
1
3
3
2
1
0
300
0
600 300
400
500
400
500
600
Wavelength, nm
Wavelength, nm
(e)
(f)
25
20
15
10
5
5
2.5
2.0
1.5
1.0
0.5
0
4
3
2
3
3
2
1
1
2
1
0
300
400
500
600
1
10
100
1000
Wavelength, nm
log(γ)
Fig. 1. Fluorescence spectra of (a–e) of acridineꢀcontaining oligothymidylates in the absence and presence of the complementary
–6
–6
strand and the corresponding curves of fluorometric titration (f). a, [T ZT ] = 2
×
10 M,
γ
: 0
(
γ
1
), 10 (
: 0 ), 10 (
γ is the excess of the matrix CA C with respect to the oligothymidylate derivative
2
), 40 (
), 230 (3), e, [YT YT] =
6
3); b, [ZT ] = 10 M,
3
3
6
–6
–7
γ
: 0
1.2
([CA C] / [oligothymidylate]); f, titration curves of the ZT
(
×
1
), 10 (
10 М,
2
), 20 (
3
(
), c, [YT ] = 10 М,
γ
: 0
(
1
), 5 (
2
) and 45 (
3
); d, [Y T ] = 8.8
×
10 M,
(1
2
6
2
6
–6
γ: 0
1), 11 (2), 130 (3), where
6
2
(1
), T ZT
(2), Y T
(3
), YT YT
(
4
) and YT ). Buffer solution: 1
(5
6
6
3
3
6
6
6
M NaCl, 0.01 M sodium phosphate (pH 7.3), 0.1 mM Na EDTA. (a–e). The excitation spectra (
λ
= 550 nm) (thin lines on
2
em
the left side of panels a–f); emission spectra (
λ = 400 nm) (bold lines on the right).
eх
ment of this residue as an effective duplexꢀstabilizing nucleotide. To do this, the created insertion must conꢀ
agent is described in detail in the literature [32–35].
An additional function of insertion is the ability a
tain a hydroxyl group blocked by the residue “orthoꢀ
gonal” to the 4,4'ꢀdimethoxy trityl group (DMTr)
postꢀsynthetic modification of the synthesized oligoꢀ commonly used in the automatic synthesis. For examꢀ
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 38
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628
KUPRYUSHKIN, PYSHNYI
ple, tertꢀbutyl dimethyl silyl group (TBDMS), which taric acid, which provides a spatial separation of bulk
is selectively removed in the presence of fluoride ions, residues in synthons, was chosen as the linker group.
meets this requirement [36].
Considering the aforesaid, we have chosen diethaꢀ
Synthesis of AcridineꢀContaining
nolamine and Nꢀ(2ꢀhydroxyethyl) ethylenediamine as
NonꢀNucleotide Insertions
the fragments of backbone insertion, ensuring the presꢀ
ence of required hydroxyl and amino functions in the
structure of the nonꢀnucleotide synthon. To join the
Synthesis of the first nonꢀnucleotide multifuncꢀ
tional phosphoramidite synthon was performed
functionalized blocks of backbone the residue of gluꢀ according to Scheme 2.
OH
OH
OTBDMS
HN
Cl
OMe
NH
NH
NH
NH
TBDMSꢀCl
Py
NH2
Py
Cl
N
OMe
OMe
Cl
N
Cl
N
(
I
)
(II)
OTBDMS
OTBDMS
O
O
O
N
OH
O
N
OH
N
O
O N
O
O
DCC
DMF
Py
O
O
O
O
HN
N
HN
N
OMe
OMe
Cl
Cl
(
III)
(IV)
OTBDMS
OH
OTBDMS
ODMTr
OH
OH
HN
N
O
N
N
N
DMTrꢀCl
Py
OH
Py
O
O
O
HN
N
OH
HN
N
OMe
OMe
VI
Cl
Cl
(
V
)
(
)
OTBDMS
ODMTr
N
O
N
i
2
(Pr N) POCH CH CN
2
2
2
Tz, DIPEA
CH CN
O
3
HN
OCEP
OMe
Cl
N
(
VII)
Scheme 2. Synthesis bifunctionalized synthon of nonꢀnucleotide insertion (VII).
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OLIGONUCLEOTIDE DERIVATIVES IN NUCLEIC ACID HYBRIDIZATION ANALYSIS
629
Initially, the addition of an agent capable of stabilizing
the duplex to the ꢀ(2ꢀhydroxyethyl) ethylenediꢀ selectively leading to the formation of an amide (
amine was carried out with the formation of amino
alcohol ( , yield 73%). It was found that a substitution
In our chosen conditions, this reaction proceeded
N
V),
even in the presence of free hydroxyl groups. The yield
after two stages of activation and condensation was
60%. After the standard procedures dimethoxytritylaꢀ
tion (yield (VI) 37%) and phosphilation the desired
product (VII) was obtained in 76% yield (Scheme 2).
I
reaction of the chlorine atom in position 9 of the acriꢀ
dine derivative by the primary amino group of the
amino alcohol is carried out in the selected conditions.
The hydroxyl group of the compound was blocked
with a silyl group by reaction with tertꢀbutyl dimethyl
chlorosilane (yield of compound (II) was 95%). This
position of the synthesized insertion can be further
used for further modification of the main chain of the
oligonucleotide construct. Branching of the backbone
was carried out by the treatment of glutaric anhydride
The transformation of the diol (V) in the (VI) derivaꢀ
tive turned out to be one of the least effective synthetic
steps in this scheme, due to the accumulation of the
corresponding diꢀOꢀsubstituted byꢀproduct during the
reaction. This partly explains the low total yield of the
desired product (VII) (9.4%).
It should be noted that the strategy of gradual
assembly of structures of the required synthon preꢀ
sented in Scheme 2 can be implemented in block
form. We tested the possibility of using a block syntheꢀ
sis for obtaining an alternative synthon of nonꢀnucleꢀ
derivative (
was obtained. Carboxylic acid (III) was converted to
an activated ester (IV) in the presence of ꢀhydroxꢀ
ysuccinimide and a condensing agent (DCC) and used
I), resulting in a derivative (III) (yield 80%)
N
it in the reaction with the second part of the amine otide insertion containing a smaller number of funcꢀ
backbone–diethanolamine.
tional groups (Scheme 3).
ODMTr
O
OH
ODMTr
ODMTr
O
O
O
N
OH
O
OH
O
N O
N
DMTrꢀCl
Py
O
DMF
O
HN
HN
N
Py
O
O
ODMTr
O
OH
ODMTr
VIII
ODMTr
(
)
(
IX)
(X)
OCEP
N
ODMTr
OH
ODMTr
i
2
N
(Pr N) POCH CH CN
2
N
N
(I)
Py
2
2
Tz, DIPEA
CH CN
3
O
O
O
O
HN
N
ODMTr
HN
N
ODMTr
OMe
OMe
Cl
Cl
(
XI
)
(XII)
Scheme 3. Obtaining the synthon of nonꢀnucleotide insertion of simplified structure (XII).
In this case, dimethoxytritylation of diethanolaꢀ developed schemes of receiving a nonꢀnucleotide
mine (compound VIII), acylation of the amino diol
substituted with glutaric anhydride (compound IX),
insertion on the basis of substituted aminoethanols
and tested various approaches to obtaining phosporaꢀ
midites of nonꢀnucleotide insertions contained bearꢀ
ing acridine residue.
activation of the carboxyl group by
imide in the presence of DCC (compound
lowed by acylation of acridine derivative (
N
ꢀhydroxysuccinꢀ
), folꢀ
) with a
X
I
It should be noted that the principal moments that
define the prospects of using such nonꢀnucleotide synꢀ
thons are the stability of the oligonucleotide derivaꢀ
tives bearing a developed nonꢀnucleotide insertion in
the synthesis and the deprotection of oligonucleotides,
and the preservation of the ability to form duplexes
compound (XI) were carried out sequentially. The
yield of compound (XI) in the four stages was 48%.
The phosphitylation reaction of the hydroxyl group in
the resulting alcohol led to an alternative synthon
(XII) with an 85% yield. Thus, the total yield in the
implementation of a simplified fiveꢀstep scheme for
obtaining the synthon was about 40%. So, we have with complementary sequences of NA.
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630
KUPRYUSHKIN, PYSHNYI
Table 1. Results of mass spectrometric analysis and electrophoretic mobility of selected products of synthesis of modified oliꢀ
gonucleotides
m/z
Desired product
Identified product
μ#
(calculation/exper.)
Z
T6
T3 T3
(T3)2Z2T3
Z
T6–[H+]
2485.5/2483.7
2485.5/2484.5
0.7
0.7
Z
T3
T3 + [H+]
Z
(T3)2Z2T3–[H+]
T3(T2)Z2T3–H+]
T3(T)Z2T3–[H+]
T3Z1T3–[H+]
3283.4/3277.7
2979.6/2975.4
2675.4/2670.5
2371.2/2366.2
0.75
0.74
0.72
0.71
YT6
Y
T6–[H+]
2372.2/2371.4
1843.2/1841.2
0.7
0.97
pT6 – [H+]
(Y
)
2Y1T6
pT6–[H+]
1843.2/1841.3
2284.1/2283.6
2371.2/2370.6
2980.3/2978.5
0.97
0.85
0.7
Y*T6–[H+]
Y
Y
T6–[H+]
2T6–[H+]
0.63
(T5)2Y1
T
T5
Y
T + [Li+]
2379.2/2379.1
763.1/764.3
0.76
0.52
Y
*T–[H+]
(T3)2Y1T3
pT3–[H+]
929.6/928.6
1731.5/1372.3
2371.2/2372.0
1.15
0.57
0.76
Y*T3–[H+]
T3Y
T3–[H+]
(Y
T6)2Y1
T
Y
Y
Y
T6
Y
T+[H+]
3286.4/3287.5
763.1/764.3
–
–
*T–[H+]
*T2–[H+]
((T2)2Y1T2)2Y1T2
1067.3/1068.3
–
Note:
Z and Y (Y*) are nonꢀnucleotide units introduced with the compounds (VII) and (XII), respectively.
3'
3'
5'
5'
Z
Y
Y*
O
O
P
O
O P O
RO
O
O
O
N
N
N
N
N
OH
Cl
O
O
Cl
Cl
OR
O
P
O
O
O
O
NH
O
O
NH
NH
N
3'
N
N
OMe
where R = –TBDMS (
OMe
), either the phosphodiester derivative residue (
OMe
). The arrow shows the direction
1
2
1
Z), –OH (
Z
,
Y
Z
,
Y
of synthesis of the oligonucleotide sequence on the polymer support. In this case, the conventional 3'ꢀ and 5'ꢀpositions of
nonꢀnucleotide insertion mean the place of articulation with the nucleotide or other nonꢀnucleotide units of chain (5'ꢀ and
3'ꢀ, respectively), # is the relative electrophoretic mobility in 20% denaturing polyacrylamide gel (8 M urea, pH 8.3) calcuꢀ
lated with respect to the electrophoretic mobility of bromophenol blue.
µ
Preparation of Model Oligonucleotide Derivatives
tively, to the presence of “Z” and “Y” residues within
the target oligonucleotide derivatives (Table 1).
The possibility of embedding the derived insertion
in oligonucleotides was tested, for example, in the
Bearing in mind that the presence of substituted
automatic synthesis of a series of oligodeoxyribonuꢀ acridine residue in the insertions may require the postꢀ
cleic thymidylate derivatives. Modified residue was synthetic deprotection of oligonucleotides, under
introduced as the 5'ꢀend of the chain, and in various mild conditions, different variants of the removal of
positions inside the oligomers. The introduction of the protective groups and oligonucleotide from the polyꢀ
insertion was performed with a modified protocol for meric carrier were tested. According to the analysis by
oligodeoxyribonucleotide synthesis: increased conꢀ RP HPLC, treatment oligomers for more than 3 h in
densation time (up to 5 min) and the concentration of concentrated aqueous ammonia at room temperature
nonꢀnucleotide monomers increased to 0.1 M. The resulted in a noticeable degradation of the product. As
use of synthons (VII) and (XII), should lead, respecꢀ alternative conditions were tested: 1) 0.4 M NaOH in
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OLIGONUCLEOTIDE DERIVATIVES IN NUCLEIC ACID HYBRIDIZATION ANALYSIS
631
methanol, 2) ethanolamine in water (1 : 1), 3) a satuꢀ It was shown that after selective removal of silyl proꢀ
rated solution of ammonia in methanol, 4) 0.05 M tection and attempts to introduce an additional trithyꢀ
К2СO3 in methanol, 5) 1,8ꢀdiazabicyclo [5.4.0]undecꢀ midilate fragment the yield of the desired branching
7ꢀene in methanol (1 : 10 by volume), and 6) tertꢀbutyꢀ product ((Т3)2Z2Т3, see note to Table 1) was extremely
lamine in methanol–water (1 : 1 : 2).
low. Along with an oligonucleotide (Т3)2Z2Т3 accordꢀ
ing to the mass spectrometric analysis the short oligoꢀ
nucleotides containing one or two additional trithymiꢀ
It was shown that the first three options, as well as
the standard way of deprotection by aqueous ammoꢀ
nia, leads to significant degradation of acridineꢀconꢀ
taining products until their complete destruction in
aqueous ethanolamine. The accumulation of the oliꢀ
gonucleotides lacking the intercalating fragment and
the corresponding acridine derivative was recorded in
all cases. Options 4 and 5 are allowed after the deproꢀ
tection within 5 h to obtain the expected product with
satisfactory yields, but further exposure of the oligonuꢀ
cleotide under these conditions led to the accumulaꢀ
tion of its degradation products. All tested variants and
the corresponding storage times were chosen in such a
way as to ensure complete cleavage of nucleotides
from the polymeric carrier. The lowest level of degraꢀ
dation of the desired product, even after incubation for
24 h, was observed when using the tertꢀbutylamine
solution in aqueous methanol, which was chosen as
the optimal deblocking solution.
dilate links in the side chain (Т3(Т2)Z2Т3 Т3(Т)Z2Т3
, )
were recorded among the products of synthesis of the
branched derivative. The yield of branched oligonuꢀ
cleotides was less than 10% of the total oligonucleotide
fractions, apparently due to the steric hindrances creꢀ
ated by neighboring hydroxyl group with additional
bulky substituents (acridine derivative).
We have synthesized a fullꢀsize structure only when
it was introduced at the 5'ꢀend of oligonucleotide
(
YТ6) of the planned derivatives of oligonucleotides
with insertion ꢀtype structure. We should also note
Y
that this insertion involves a parallel insertion of two
identical units at the stages following the introduction
of the nonꢀnucleotide unit in the synthesized nucleꢀ
otide chain. Attempts to obtain derivatives with a
branched target oligonucleotides were unproductive.
No increase in the number of nucleotide synthons
supplied or increase in the time of condensation allow
for enhancing the yield of branched products.
The homogeneity of the products obtained was
confirmed by electrophoretic analysis in denaturing
conditions (20% polyacrylamide, 8 M urea). The main
product of synthesis of each oligonucleotide derivative
and the most common byꢀproducts isolated by chroꢀ
matography were analyzed by TOF mass spectrometry
(MALDIꢀTOF). The list of characterized synthesis
products of modified oligothymidylates and a number
of typical byꢀproducts are presented in Table 1.
In all cases, the oligonucleotides in which the
building of the chain after the introduction insertion
took place in only one of a pair of the available
hydroxyl residues in the unlocked nonꢀnucleotide
unit: Y2Т6 instead of the planned sequence (Y)2Y1Т6
(see notes in Table 1), Т5YТ instead (Т5)2Y1Т, etc. corꢀ
responded to the isolated product with the maximum
molecular weight in the reaction mixtures. The most
likely causes reducing the efficiency of the resulting
synthon are steric constraints arising from contact of
phosphoramidites with the hydroxyl groups of the
unlocked nonꢀnucleotide insertion and/or rapid degꢀ
radation of the product of this reaction in the autoꢀ
matic synthesis.
The data RPꢀHPLC and mass spectrometry show
that the application of insertion developed allows one
to obtain only oligonucleotides with terminal modifiꢀ
cations with satisfactory yields (40–50%). In the case
of modification of the chain inside, the observed yield
of target products did not exceed 30%. It was found
that among the products of automatic synthesis of
modified oligonucleotides there is a significant
amount of 5'ꢀphosphorylated oligonucleotides, for
example, pT6 during the synthesis of YT6, and Т6 is
present only in trace amounts. This indicates the
effective addition of a nonꢀnucleotide unit to the
growing chain, but as a result of further reactions the
synthesized product is subjected to degradation at the
place of introduction of the nonꢀnucleotide insertion.
In the case where nonꢀnucleotide insertion is not a
terminal unit, a number of acridineꢀcontaining byꢀ
products were found other than those mentioned
5'ꢀphosphorylated oligonucleotides in reaction mixꢀ
tures.
In addition to the byꢀproducts shown above
(absence of condensation on one of the hydroxyl
groups), the products resulting from degradation of
the builtꢀin nonꢀnucleotide unit were found. For
example, the byꢀproducts with the degraded backbone
of insertion Y* were obtained in the synthesis of oligoꢀ
nucleotides with an internal localization of the nonꢀ
nucleotide unit for the separation of reaction mixtures
(Table 1). According to mass spectrometric analysis,
the structure of such a unit presumably corresponds to
carboxylic acid residue. It is important to note that
during the transition from the preparation of derivaꢀ
tives (Y)2Y1T6 and (T3)2Y1T3 to the synthesis of comꢀ
Considering the specifics of removal of tertꢀbutyl pounds (YT6)2Y1T and ((T2)2Y1T2)2Y1T2 with a large
dimethyl silyl protection (treatment of tetrabutylamꢀ number of units after the first injection of nonꢀnucleꢀ
monium fluoride), oligonucleotide Т3ZТ3 on the polyꢀ otide unit, the content of byꢀproducts having in their
styrene support was synthesized to test the possibility of composition degraded
Y*ꢀstructure in the postꢀsynꢀ
using an additional hydroxyl group of compound (VII). thetic mixtures increases. At the same time, these degꢀ
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 38
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2012

632
KUPRYUSHKIN, PYSHNYI
Table 2. Thermodynamic parameters of formation and melting points of DNA/DNA complexes of native oligonucleotides
and their acridineꢀcontaining derivatives
Δ
G
°
(37°C), kcal/mol
Compound
Δ
S
°
, cal/(mol K)
ΔH°, kcal/mol
*
Tmelt
°C
,
T6
–117.6
–114.0**
–113.8
–144.5
–149.8
–145.5
–161.0
–171.0
–38.8
–35.4**
–39.6
–50.1
–51.2
–49.9
–56.1
–57.9
–2.4
0.0**
–4.3
–5.2
–4.8
–4.8
–6.1
–4.9
6.8
–9.5**
20.5
29.1
26.7
26.6
T3^T3
T3ZT3
ZT6
YT6
Y2T6
YT6YT
T5YT
35.5
28.8
Note: ^ is nonꢀnucleotide insertion based on the phosphodiester diethylene glycol; * the melting point was calculated for the total conꢀ
–4
centration of oligonucleotide chains, taken in stoichiometric ratio equal to (1
× 10 M) in buffer 1 M NaCl, 10 mM sodium
phosphate (pH 7.3), 0.1 mM Na EDTA; ** thermodynamic parameters and magnitude of the melting point of the complex were
2
calculated by the method described in [31].
radation products were detected in the synthesis of a ified complex, and, according to the calculations,
derivative with a 5'ꢀterminal insertion YT6 (data not much greater than the stability of the duplex with nonꢀ
shown). The experiments conducted on the stability of nucleotide insertion on the basis of phosphodiester
the already isolated oligonucleotide derivatives diethylene glycol, which causes an almost equal
(
T3ZT3, ZT6, YT6YT) with repeated exposure of mixꢀ increase in the contour length of the oligonucleotide
ture of tertꢀbutylamine in aqueous methanol, showed backbone. Apparently, the acridine residue, effectively
no degradation.
interacting with the surrounding base pairs, provides
the observed effects.
The maximum stabilizing effect allows the presꢀ
ence of two intercalating residues in the duplex, and
only in case of modification of the terminal (3' and 5')
Summarizing the data obtained, it can be conꢀ
cluded that the partial degradation (or hidden modifiꢀ
cation) of oligomer chain in the case of application of
Y
ꢀtype synthon occurs during the automatic synthesis
of the oligomer.
sections. A pair of
duced at the 5'ꢀend (Y2Т6
Y
ꢀtype insertions successively introꢀ
/M
complex) has no addiꢀ
tional stabilizing effect in comparison with a single
insertion of the same type (Table 2).
Hybridization Properties of Model Oligonucleotides
It should be noted that the observed thermodyꢀ
The impact of the proposed acridineꢀcontaining
insertion on the efficiency of complexation of oligoꢀ
nucleotide derivatives obtained was characterized by
thermal denaturation of model duplexes formed with
the participation of the target oligonucleotide CA6C
(M). To ensure the uniqueness of the binding of derivꢀ
atives with the DNA target, the hexaadenylate fragꢀ
ment was flanked by cytidine residues.
namic effect of the introduction of
Zꢀ and Yꢀtype
insertions is associated with a pronounced increase in
the enthalpy of complex formation, which indicates
the implementation of stacking interactions of acriꢀ
dine residue with DNA bases [37]. The minimum
enthalpy contribution from the introduction of inserꢀ
tion was observed when it is introduced into the cenꢀ
tral part of the duplex (T3ZT3). In this case, the stabiꢀ
lizing effect is associated with the presence of the
modified residue in the structure of the positive charge
arising from protonation of the heterocyclic nitrogen
atom in the acridine residue [36].
The values of melting temperatures of complexes
and thermodynamic parameters of their formation are
presented in Table 2. Analysis of the data showed that
the introduction of proposed nonꢀnucleotide inserꢀ
tions, as in [32–35], in all cases leads to a significant
stabilization of duplexes formed. The presence of one
acridine residue increases the melting point of hexaꢀ
The resulting thermodynamic data fully confirm
the high efficiency of 6ꢀchloroꢀ2ꢀmethoxyacridine
residue as an agent for stabilizing the duplex structure,
and are in good agreement with the previously
described properties of various acridineꢀcontaining
oligonucleotides [32–35].
nucleotide complex by the value from 13.7 to 22.8°С.
Introduction of the insertion at the end of the chain or
the position of the penultimate unit of hexathymidyꢀ
late has a more pronounced stabilization of the short
complex compared to the case of localization in the
central part of duplex structure. The presence of acriꢀ
dine residue fully compensates of destabilizing effect
resulted from perturbation of the regular structure of
the carbohydrateꢀphosphate backbone paired with its
Fluorescent Properties of AcridineꢀContaining
Derivatives of Oligonucleotides
The change of fluorescence efficiency of the resiꢀ
extension. The stability of the Т3ZT3/M complex is due(s) of the dye upon complexation of oligonucleꢀ
higher than the stability of the corresponding unmodꢀ otide derivatives is well known. The formation of the
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 38
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2012

OLIGONUCLEOTIDE DERIVATIVES IN NUCLEIC ACID HYBRIDIZATION ANALYSIS
633
double helix with the labeled components can lead that detritylated hydroxyl groups of nonꢀnucleotide
both to an increase in the fluorescence of dyes, such as
the presence of ethidium residues [38] and its suppresꢀ
sion, as described, in particular, for the pyrene resiꢀ
dues [39]. The presence of such features in the molecꢀ
ular probes is often used to develop highly sensitive test
systems to identify a specific NAꢀtarget and local vioꢀ
lation of their structure [2].
insertion are poorly accessible for the addition of
phosphoramidites to them and, moreover, seem to
contribute to the degradation of nonꢀnucleotide inserꢀ
tion to the oligonucleotide chain of the subsequent
units during the addition (established for Yꢀtype inserꢀ
tion). In the latter case, the accumulation of oligomers
bearing terminal monophosphoester residue is
recorded, which may be the result of degradation of
the builtꢀin chain of the nonꢀnucleotide unit during
It has been previously described, that we use interꢀ
calating residue that changes its fluorescent properties
in hybridization with the NA [40]. In the course of this the subsequent cycle(s) of addition of additional units.
work, the properties of the obtained acridineꢀcontainꢀ
ing oligonucleotides were characterized. It was found
that the fluorescence of acridine residue in the formaꢀ
tion of a duplex structure can both increase and
decrease. Double quenching (by a factor of 2.3) occurs
upon complexation of oligonucleotide with an interꢀ
nal insertion (Т3ZТ3) (Fig. 1a). At the same time the
terminal insertion of intercalator hardly changes their
This conclusion is supported by the fact that the oligꢀ
othymidylates containing acridine residue, preꢀalloꢀ
cated and bearing the insertion, are stable both at the
end and in the inner part of the chain when kept in
conditions of postꢀsynthetic deprotection.
The establishment of the causes of accumulation of
products of the phosphodiester backbone cleavage
characteristics, the transition to the doubleꢀstranded requires additional analysis beyond the scope of this
state of the oligonucleotides of ZT6 (Fig. 1b) and YT6
(Fig. 1c) leads only to a slight decrease, by a factor of
1.3, in the efficiency of fluorescence. The presence of
two acridine residues has the opposite effect: the binding
of oligonucleotides Y2T6 (Fig. 1d), and YТ6YТ (Fig. 1e)
with the complementary sequence in both cases is
associated with the buildup of fluorescence by a factor
of 1.6 and 2.4 times, respectively. We note that more
pronounced changes are observed when the dye resiꢀ
dues are separated in the structure of an oligonucleꢀ
otide derivative.
work. However, it should be noted that there were no
similar features in the analysis of publications devoted
to the development and application of nonꢀnucleotide
insertions based on the diethanolamine. For example,
when creating the pyreneꢀcontaining insertions, the
backbone of which is the branched amide based on the
diethanolamine, Nꢀacylated with various diaminocarꢀ
boxylic acids, the authors avoid discussion of the
effectiveness of inclusion of phosphoramidites they
developed in the oligonucleotide chain and stability
analysis of the derivatives when removing them from
the substrate and deprotection [2].
Thus, the intercalating residue in the insertion can
play not only the role of a stabilizing agent duplex, but
also act as a sensor of transition of conjugates from the
free to the bound state (Figs. 1a–1e), which signifiꢀ
cantly increases the prospects of using multifunctional
units on receipt of new DNA structures.
The presented data suggest the existence of objecꢀ
tive problems in the creation and application of comꢀ
pact functionalized synthons involving branching of
the phosphodiester backbone of oligonucleotides.
Nevertheless, our findings indicate that the designed
nonꢀnucleotide insertions introduced in the carbohyꢀ
drate–phosphate backbone at least partially perform
the planned features. The acridine derivative residue,
as expected, increases the thermal stability of duplexes
with the complementary target, changing the effiꢀ
ciency of the intrinsic fluorescence relative to the
unbound state. The branched oligonucleotides were
not synthesized in sufficient quantities to conduct
studies of their physicoꢀchemical properties due to the
low efficiency of incorporation of the nucleotide unit
by means of the additional HOꢀfunction of the created
CONCLUSIONS
By optimization of the conditions of a sevenꢀstep
synthesis and isolation of intermediate products at
each stage, the phosphoramidite synthon (VII) for
compact nonꢀnucleotide insertion was obtained with a
satisfactory yield. Transition to the block method
instead of a continuous method of synthesis will
increase the efficiency of synthesis of the nonꢀnucleꢀ
otide synthon, and this is demonstrated by the examꢀ
ple of obtaining phosphoramidite (XII). Thus, we proꢀ
posed ways to obtain phosphoramidite synthons,
potentially allowing the introduction of the branching
unit in the synthesized oligonucleotide chain and, in
addition to this, carrying a residue that has a stabilizing
influence in the formation of the duplex.
Zꢀand Yꢀtype insertions. This situation requires furꢀ
ther study and should be taken into account when
designing similar nonꢀnucleotide synthons. We conꢀ
sider an orthogonal protecting group of additional
HOꢀcomponents and the linker fragment connecting
the disubstituted diethanol amine residues as the most
promising components for variations in the structure
of the multifunctional and compact synthons consiꢀ
It was found that the synthons (VII) and (XII
)
effectively incorporated into the growing chain end of
the oligomer, but lead to a drop in the reaction yield of
subsequent nucleotide residues. These data indicate dered.
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 38
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2012

634
KUPRYUSHKIN, PYSHNYI
EXPERIMENTAL
J
5.7, –NH–C
Acr–NH–CH2–C
NH–CH2–C 2–OH); 3.79 (2 H, t,
2–CH2–NH–); 3.94 (3 H, s, OC
1 9.l, 2 2.1, H7), 7.41 (1H, dd, 1 9.3,
7.62 (1H, s, H1), 7.82 (1 H, d, 9.3, H5); 7.86 (1H, s,
H4), 8.36 (1H, d, : 49.58
9.3, H8). 13C NMR
H
2–CH2–OH); 2.88 (2H, t,
2–NH–); 3.44 (2 H, t, 5.7, 5–
6.2, Acr–NH–
3); 7.32 (1H,
2 2.5, H3),
J 6.2,
H
J
The reagents used in this work were as follows:
H
J
phosphoramidites (GlenResearch, United States),
2ꢀcyanoethylꢀ ', ꢀtetraisopropylphosphoramidꢀ
C
dd,
H
H
N,N,N N'
J
J
J
J
ite (LMH, ICBFM SB RAS), 6,9ꢀdichloroꢀ2ꢀmethꢀ
oxyacridine, glutaric anhydride, diethanolamine,
J
J
δ
,
dicyclohexylcarbodiimide,
United States), ꢀ(2ꢀhydroxyethyl)ethylenediamine,
tertꢀbutyldimethylsilyl chloride, imidazole, 4ꢀ
dimethylaminopyridine, ꢀhydroxysuccinimide, triꢀ
ethylamine, ꢀmethylimidazole (Fluka, Switzerꢀ
Stainsꢀall
(Aldrich,
49.67, 51.62, 55.89, 60.67, 101.23, 115.28, 117.73,
122.94, 124.39, 126.9, 133.79, 148.13, 150.88, 155.37.
Mass spectrometry:
346.8 (С18H20N3O2Сl)
N
N,Nꢀ
m/z 346.4 [M
+ H]+. Calculated
.
N
N
land), 4,4'ꢀdimethoxytrityl chloride (ChemGenes,
United States), urea, tetrabutylammonium fluoride
(Merck, Germany), acrylamide (DiaM, Russia), tetꢀ
razole (Acros, United States), ethyl diisopropyl
amine, bromophenol blue, xilencianol FF (Sigma,
United States), reagents and solvents of chemically
pure and analytical pure grades. The dehydration of
solvents was performed by standard methods with furꢀ
ther aging over molecular sieves and calcium hydride.
The TLC plates were DCꢀAlufolien Kieselgel
60 F254 (Merck, Germany); the solvent system are ethꢀ
anol–triethylamine, 95 : 5 (A), dichloromethane–
ethanol–triethylamine, 69 : 30 : 1 (B), ethanol (C);
ethyl acetate–triethylamine, 95 : 5 (D); toluene–ethyl
acetate–triethylamine, 50 : 49 : 1 (E); dichloꢀ
romethane–ethanol, 97.5 : 2.5 (F); ethyl acetate–triꢀ
ethylamine, 99 : 1 (G).
For column adsorption chromatography the colꢀ
umn Kieselgel 60 (Merck, Germany) with volume of
100 mL filled to 70% of sorbent was used in all cases.
For evaporation and concentration of aqueous and
organic solutions Rotavapor R200, Rotavapor RE120
(Buchi, Switzerland) and Concentrator 5301 (Eppenꢀ
dorf, Germany) were used at a pressure of 10–15 mm
N
ꢀ[2ꢀ(tertꢀButyldimethylsilyloxy)ethyl]ꢀ
2ꢀmethoxyacridineꢀ9ꢀyl]ethylenediamine (II). A mixꢀ
ture of 1 g (2.9 mmol) of compound ( ) and 0.99 g
N 'ꢀ6ꢀchloroꢀ
I
(14.5 mmol) of dimethylaminopyridine was evapoꢀ
rated with abs. pyridine (10 mL), dissolved in 40 mL of
abs. pyridine. 2.18 g (14.5 mmol) TBDMSꢀCl was
added, and held for 1 h at 50°С, then the reaction mixꢀ
ture was evaporated to the oily condition, dissolved in
10 mL of ethanol, and 600 mL of 0.1 M NaOH was
added up to complete precipitation. The precipitate
was filtered, washed with cool bidistilled water, dried
to constant weight. The yield compound (II) was 1.33 g
(95%), Rf 0.9 (А). 1H NMR
δ
: –0.04 (6 H, s,
(CH3)2Si), 0.79 (9 H, s, (CH3)3CSi); 2.61 (2H, t, 5.8,
–NH 2–CH2–O–); 2.88 (2 H, t, 6.1, Acr–
NH–CH2–C 2–NH–); 3.57 (2 H, t, 5.8, –NH–
CH2 2–O–); 3.79 (2 H, t, 6.1, Acr–NH–CH2
CH2–NH–); 3.93 (3 H, s, OCH3 , 7.25–8.41 (6 H,
Ar). Mass spectrometry: 460.7 [
+ H]+. Calcuꢀ
lated 461.1 (С18H20N3O2Сl)
J
–CH
J
H
J
–
CH
J
–
)
m/z
M
.
4ꢀ{ ꢀ[2ꢀtertꢀButyldimethylsilyloxy)ethyl]ꢀNꢀ[2ꢀ(6ꢀ
N
chloroꢀ2ꢀmethoxyacridineꢀ9ꢀylamine)ethyl]carbamoyl}
butanoic acid (III). A mixture of 1.33 g (2.9 mmol) of
compound (II) and 0.32 g (2.9 mmol) DMAP were
Hg and a temperature of up to 40°С.
evaporated with abs. pyridine (
30 mL of abs. pyridine. 1.65 g (14.5 mmol) of glutaric
2 × 5 mL), dissolved in
Mass spectrometry (MALDIꢀTOF) analysis of the
investigated compounds was carried out on a ReflexꢀIII
or AutoflexꢀIII devices (Bruker Daltonics, Germany). anhydride was added, and held for 1 h at room temperꢀ
1H, 13C and 31P NMR spectra (
δ, ppm, J, Hz) were
ature. Further, the reaction mixture was evaporated to
the oily condition, dissolved in 10 mL of ethanol, and
600 mL of 1 M HCl was added to complete precipitaꢀ
tion. The precipitate was filtered and washed with cold
distilled water, and dried to constant weight. The yield
recorded on a Bruker AV 300/400 spectrometer at 300,
400 and 160 MHz, respectively, relative to an external
standard of tetramethylsilane (1H) and H3PO4 (31P) in
3–5% solution of DMSOꢀd6. The processing of the
analytical results was performed using SPINWORKS.
The spectra were recorded at the Center for the Colꢀ
lective Use of the Analysis of Organic Compounds and
Materials NIOC SB RAS.
1
compound (III) was 1.32 g (80%), Rf 0.8 (B). H
NMR,
CH3)3CSi); 1.73 (2 H, m, C(O)–CH2–C
COOH); 2.23 (2 H, m, C(O)–C 2–CH2–CH2–
COOH); 2.34 (2 H, m, C(O)–CH2–CH2–CH2
COOH); 3.39 (2 H, t, –NH–C 2–CH2–O–, 5.3),
3.67 (2 H, t, 5.3, –NH–CH2–C 2–O–); 3.7 (2 H, t,
5.9, Acr–NH–CH2–C 2–NH–); 3.9–4.0 (5 H, t,
Acr–NH–C 2–CH2–NH–, OCH3); 7.25–8.41
(6 H, Ar). 13C NMR,
: –5.25, 18.10, 20.44, 25.95,
δ: –0.04 (6 H, s, (CH3)2Si); 0.77 (9 H, s,
(
H2–CH2–
H
–
N
ꢀ(2ꢀhydroxyethyl)ꢀN'ꢀ(6ꢀchloroꢀ2ꢀmethoxyacridineꢀ
H
J
9ꢀyl)ethylenediamine (I). A mixture of 3 g (10.78 mmol)
of 6,9ꢀdichloroꢀ2ꢀmethoxyacridine and 11 mL (11.3 g,
J
H
J
H
107.8 mmol)
was evaporated with abs. pyridine (
solved in 120 mL of abs. pyridine and incubated 20 h
at 70 . The reaction mixture was kept for 12 h at
. The crystals were filtered and washed with cold
abs. pyridine, dried to constant weight. The yield of (
was 2.72 g (73%), Rf 0.5 (A). 1H NMR
: 2.61 (2 H, t, 574.84 [
N
ꢀ(2ꢀhydroxyethyl) ethylenediamine
H
2
×
15 mL), disꢀ
δ
31.91, 33.22, 46.01, 49.14, 50.27, 56.42, 59.21, 60.8,
101.53, 117.67, 120.94, 123.39, 127.45, 134.5, 139.09,
°С
5°С
155.46, 156.31, 174.45, 175.57. Mass spectrometry:
m/z
.
I)
δ
M
+ H]+. Calculated 575.2 (С29H40N3O5СlSi)
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 38
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2012

OLIGONUCLEOTIDE DERIVATIVES IN NUCLEIC ACID HYBRIDIZATION ANALYSIS
635
N
ꢀ[2ꢀtertꢀButyldimethylsilyloxy)ethyl]ꢀ
N
ꢀ[2ꢀ(6ꢀchloꢀ
⎯5.73, 17.67, 20.62, 25.55, 31.63, 31.93, 45.52, 46.54,
', 'ꢀbis [2ꢀ 47.80, 47.96, 49.66, 50.34, 54.93, 55.49, 58.71, 59.06,
roꢀ2ꢀmethoxyacridineꢀ9ꢀylamine)ethyl]ꢀ
N
N
hydroxyethyl]glutaramide (V). A mixture of 1.32 g 60.73, 61.56, 85.39, 100.01, 113.08, 122.24, 124.11,
(2.3 mmol) of compound (III) and 0.79 g (4.6 mmol) 126.53, 127.1, 127.56, 127.7, 129.52, 133.29, 135.47,
N
ꢀhydroxy succinimide was evaporated with abs. dimꢀ 144.69, 148.33, 149.86, 155.17, 157.97, 171.89,
ethylformamide (10 mL), dissolved in 40 mL of abs. 174.04.
dimethylformamide, 1.42 g (4.6 mmol) DCC was
N
ꢀ[2ꢀtertꢀButyldimethylsilyloxy)ethyl]ꢀ
chloroꢀ2ꢀmethoxyacridineꢀ9ꢀylamine)ethyl]ꢀ 'ꢀ[2ꢀ(4,4'ꢀ
dimethoxytrityloxy)ethyl]ꢀ 'ꢀ[2ꢀ(2ꢀcyanoꢀethyloxyꢀ
ꢀdiisopropylphosphoramiditeoxy)ethyl]glutaramide
(VII). A solution of compound (VI) 0.14 g (0.15
mmol) in abs. acetonitrile ( mL) was evaporated
Nꢀ[2ꢀ(6ꢀ
added, and reaction mixture was held for 6 h at room
temperature. Control: TLC, Rf (ref) 0.3, Rf (product)
0.6 (system C). Dicyclohexylurea was filtered off, 0.84 g
(6.9 mmol) of DMAP and 4.4 mL (4.8 g, 46 mmol) of
diethanolamine were added to the reaction mixture
and the mixture was kept for 6 h at room temperature.
The reaction mixture was filtered from the dicyclohexꢀ
ylurea remnants, and evaporated to the oily condition.
200 mL of CH2Cl2 was added and washed with 0.05 M
N
N
N,N
2
× 5
and the residue was dissolved in 2 mL of abs. acetoniꢀ
trile. A solution of 0.025 g (0.35 mmol) of tetrazole,
0.16 mL (0.12 g, 0.9 mmol) DIPEA, 0.13 mL (0.12 g,
0.38 mmol) of 2ꢀcyanoethylꢀN,N,N',N'ꢀtetraisoproꢀ
NaOH (3 × 200 mL). The organic phase was dried
pyl phosphoramidite in 0.72 mL of abs. acetonitrile
was kept for 30 min. To the resulting solution was
added dropwise a solution of compound (VI) for
2 min, and incubated for 1 h at room temperature. The
reaction mixture was evaporated to 3/4 volume; 100 mL
of СH2Cl2 was added and washed with 0.3 M KH2PO4
over anhydrous Na2SO4, concentrated by evaporation
to oil and dissolved in 10 mL of CH2Cl2. Further puriꢀ
fication was performed by column chromatography
using a step gradient: 200 mL of dichloromethane/triꢀ
ethylamine mixture (99 : 1), 150 mL dichloꢀ
romethane/ethanol/triethylamine mixture (94 : 5 : 1)
160 mL of dichloromethane/ethanol/triethylamine
mixture (84 : 15 : 1), 200 mL of dichloromethane/ethꢀ
anol/triethylamine mixture (69 : 30 : 1). The fractions
containing the product were evaporated, and the subꢀ
(3
× 150 mL). The organic phase was dried with anhyꢀ
drous Na2SO4, evaporated to dryness, dissolved in
5 mL of ethanol and placed in 300 mL of 0.3 M
KH2PO4. The precipitate was filtered, washed with
cold bidistilled water, and dried to constant weight.
stance dried to constant weight. The compound yield (
was 0.92 g (60%), f 0.45 (C). Mass spectrometry:
662.0 [
+ H]+. Calculated 662.3 (С29H40N3O5СlSi)
V)
m/z
The yield of compound (VII) was 0.12 g (76%),
(D). 1H NMR,
: –0.07 (6 H, s, (C 3)2Si); 0.76 (9 H, s,
(C 3)3CSi); 1.0–1.25 (12 H, m, ( 3)2CH–); 1.68
(2 H, m, 7.1, –C(O)–CH2–C 2–CH2–C(O)–);
2.33 (4 H, m, 7.1, C(O)–C 2–CH2–C
2.71 (2 H, t, –C
2–ODMTr); 3.33–3.68 (18 H, m, –C
2–CH2–CN, (CH3)2C
3.94 (3 H, s, –OCH3) 6.8–8.4 (19 H, aromatics). 31P
NMR, : 149.93, 150.11.
Rf 0.6
R
δ
H
M
.
H
CH
J
H
N
ꢀ[2ꢀtertꢀButyldimethylsilyloxy)ethyl]ꢀ ꢀ[2ꢀ(6ꢀ
chloroꢀ2ꢀmethoxyacridineꢀ9ꢀylamine)ethyl]ꢀ 'ꢀ[2ꢀ(4,4'ꢀ
dimethoxytrityloxy)ethyl]ꢀ 'ꢀ[2ꢀhydroxyethyl]gluꢀtaraꢀ
mide (VI). A solution of compound ( ) (0.92 g,
1.38 mmol) in abs. pyridine (10 mL) was evaporated,
the remnant was dissolved in 30 mL of abs. pyridine.
0.57 g (1.68 mmol). DMTrꢀCl was added, and incuꢀ
bated for 3 h at room temperature. The reaction mixꢀ
ture was evaporated, the oily residue was dissolved in
150 mL CH2Cl2 and washed with a saturated solution
N
J
H
H
2–C(O));
2CN); 3.04–3.16 (2 H, m, –CH2–
N
H
N
C
H
H
2–CH2–,
H–); 3.71 (6 H, s, –OCH3),
V
–CH
δ
N
,
N
ꢀBis[2ꢀ(4,4'ꢀdimethoxytrityloxy)ethyl]amine
(VIII). A solution of diethanolamine 0.52 g (5 mmol)
in abs. pyridine ( 15 mL) was evaporated, dissolved
in 20 mL of abs. pyridine. 1.4 g (11 mmol) DMTrꢀCl
was added, and incubated for 3 h at room temperature.
2
×
of NaCl, containing 5% NaHCO3 (3
organic phase was dried over anhydrous Na2SO4
× 200 mL). The
,
evaporated to oil with toluene and dissolved in 10 mL
of toluene. The reaction product was purified by colꢀ
umn chromatography using a linear gradient of ethaꢀ
nol concentration (0–7%) in toluene/triethylamine
mixture (99 : 1), the total volume of eluent of 600 mL).
The fractions containing the product were evaporated,
and dissolved in 6 mL of ethanol. 300 mL of 0.1 M
NaOH was added up to complete precipitation. The
precipitate was filtered, washed with bidistilled water,
R
f 0.5 (E). Further, we used the reaction mixture withꢀ
out isolation of the product.
4ꢀ{ ꢀBis[2ꢀ(4,4'ꢀdimethoxytrityloxy)ethyl]carꢀ
N,N
bamoyl}butanoic acid (IX). To the reaction mixture of
compound (VIII) was added 1.70 g (15 mmol) of gluꢀ
taric anhydride. The reaction was kept overnight at
room temperature. The reaction mixture was evapoꢀ
rated to the oily condition. 200 mL of CH2Cl2 was
added, and extracted with a saturated solution of
and dried to constant weight. The yield compound (VI
was 0.49 g (37%), : –0.07 (6 H,
f 0.3 (D). 1H NMR,
s, (CH3)2Si); 0.75 (9 H, s, (CH3)3CSi); 1.67 (2 H, m,
7.1, C(O)–CH2–C 2–CH2–C(O)); 2.20–2.40
(4 H, m, 7.1, C(O)–C 2–CH2–C 2–C(O)); 3.0–
3.1 (2 H, m, –CH2–C 2–ODMTr); 3.36–3.65 (14 H,
m, –C 2–C 2–); 3.71 (6 H, s, OCH3), 3.93 (3 H, s, dineꢀ9ꢀylamine)ethyl]ꢀ
OCH3); 6.8–8.4 (19 H, aromatics). 13C NMR,
oxy)ethyl]glutaramide (XI). Tenth of the reaction mixꢀ
)
NaCl, containing 5% NaHCO3 (3
× 200 mL). The
R
δ
organic phase was dried over Na2SO4, and evaporated
to oil. Rf 0.1 (E). The product was used in the subseꢀ
quent reaction without further purification.
J
H
H
J
H
H
N
ꢀ[2ꢀHydroxyethyl]ꢀ ꢀ[2ꢀ(6ꢀchloroꢀ2ꢀmethoxyacriꢀ
N
H
H
N',N'ꢀbis[2ꢀ(4,4'ꢀdimethoxytritylꢀ
δ:
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 38
No. 6
2012

636
KUPRYUSHKIN, PYSHNYI
ture of the compound (IX) (0.5 mmol) and 0.07 g
(0.58 mmol) ꢀhydroxysuccinimide was evaporated
Oligodeoxyribonucleotide derivatives were syntheꢀ
sized on an automated DNA synthesizer ASMꢀ800
(Biosset, Russia) using standard synthons (Glen
Research, United States) for phosphoramidite protoꢀ
col. The resulting synthons (VII and XII) were used as
0.1 M solutions in acetonitrile. At the stage of condenꢀ
sation of the synthesized insertions, the volumes of
solutions of activator (tetrazole 0.5 M) and the monoꢀ
N
with abs. dimethylformamide (10 mL) dissolved in
20 mL of abs. dimethylformamide. 0.21 g (1 mmol) of
DCC was added, and the reaction mixture was kept
overnight at room temperature. Control: TLC, Rf 0.6
(F). After filtration from dicyclohexylurea in the reacꢀ
tion mixture was added 0.13 g (10 mmol) DMAP and
mer supplied (from 20 to 80 μL), as well as the conꢀ
0.25 g (0.72 mmol) of compound (I) and incubated for
densation time (from 1 to 5 min) compared with the
standard method were increased. Removal of the polyꢀ
mer and deprotection of the protecting groups of
nitrogenous bases of the synthesized oligonucleotides
were carried out in a standard way, as well as through
various methods of “soft”, “very soft” and “fast” deproꢀ
tection listed on the site of Glen Research, United
States. For the same purposes, in the case of modified
oligonucleotide solution, the tertꢀbutylamine was used
6 hours at room temperature. The reaction mixture
was filtered from the dicyclohexylurea, and evaporated
to the oily condition. 200 mL of CH2Cl2 was added,
and extracted with 0.05 M NaOH (
organic phase was dried with anhydrous Na2SO4
3 × 200 mL). The
,
evaporated to oil and dissolved in 6 mL of toluene.
Further purification was performed by column chroꢀ
matography using a step gradient: 200 mL of toluꢀ
ene/triethylamine mixture (99 : 1), 400 mL of toluꢀ
ene/ethanol/triethylamine mixture (89 : 10 : 1). The
fractions containing the product were evaporated; the
substance was dried to constant weight. The yield of
compound (XI) was 0.32 g (48%), Rf 0.2 (A). 13C
in a methanol/water mixture (1 : 1 : 2) for 5 h at 37°С.
Removal of tertꢀbutyldimethylsilyl protection in
the synthesis of branched oligonucleotides was proꢀ
duced by keeping the polymer with a synthesized
sequence in a 1 M solution of tetrabutylammonium
fluoride in tetrahydrofuran for 1 h.
NMR, δ: 20.80, 31.84, 45.42, 46.81, 48.01, 48.45,
54.97, 55.53, 59.10, 61.57, 85.51, 100.51, 106.7,
113.15, 123.90, 125.21, 126.59, 127.18, 127.60,
128.20, 129.58, 133.40, 135.56, 144.77, 149.20,
149.98, 153.97, 155.15, 156.34, 158.02, 172.05,
174.02.
To analyze the homogeneity of the intermediate
products of synthesis of the desired nonꢀnucleotide
insertions, the analytical RP HPLC was carried out on
a Milichrome A02 chromatograph (EcoNova, Russia)
using a column (2 × 75 mm) with a sorbent Prontoꢀ
N
,
N
ꢀBis[2ꢀ(4,4'ꢀdimethoxytrityloxy)ethyl]ꢀ
(2ꢀcyanoethyloxyꢀN,Nꢀdiisopropyl diisopropylphosꢀ
phoramiditeoxy)ethyl]ꢀ 'ꢀ[2ꢀ(6ꢀchloroꢀ2ꢀmethoxyacriꢀ
N'ꢀ[2ꢀ
SILꢀ120ꢀ5ꢀC18 (EcoNova, Russia) and the concenꢀ
tration gradient of acetonitrile (0–90%) in 0.05 M aqueꢀ
ous solution LiClO4 (flow rate of 150 mcl/min, the therꢀ
mostat temperature of 50°С). Detection was performed
at four wavelengths: 300, 330, 350 and 360 nm.
N
dineꢀ9ꢀylamine)ethyl]glutaramide (XII). A solution of
compound (XI) 0.20 g (0.17 mmol) in abs. acetonitrile
(2 × 5 mL) was evaporated, the residue was dissolved
The modified oligonucleotides were isolated by
reverseꢀphase chromatography on a Agilent 1100
instrument in a concentration gradient of acetonitrile
(0–80%) in a solution of 0.05 M solution LiClO4 for
in 2 mL of abs. acetonitrile. A solution of 0.03 g
(0.4 mmol) of tetrazole, 0.18 mL (0.13 g, 1 mmol)
DIPEA, 0.16 mL (0.13 g, 0.43 mmol) of 2ꢀcyanoetꢀ
hylꢀN,N,N',N'ꢀtetraisopropyl phosphoramidite in
30 min, flow rate of 1.5 mL/min using a column (4.6
×
0.84 mL of abs. acetonitrile was kept for 30 min. To the
resulting solution was added dropwise over 2 min a
solution of compound (XI) and incubated for 1 h at
room temperature. The reaction mixture was evapoꢀ
rated to the 3/4 volume. 100 mL of СH2Cl2 was added,
and the extraction was carried out with 0.3 M KH2PO4
150 mm) with a sorbent Eclipse XDBꢀC8 (5 mcm)
(Agilent, United States), as well as on a Agilent 1200
series instrument in a gradient of acetonitrile (0–75%)
in 0.02 M solution of triethyl ammonium acetate for
30 minutes, the rate of 1.5 mL/min and a column (4.6
×
250 mm) with sorbent Zorbax SBꢀC18 (5 mcm). The
absorption spectra were recorded at wavelengths from
220 to 500 nm. Oligonucleotide material was precipiꢀ
tated by a 10ꢀfold volume of 2% solution of LiClO4 in
ether/acetone mixture (3 : 7). The supernatant was sepaꢀ
rated after centrifugation; the precipitate was washed
(3
× 150 mL). The organic phase was dried with anhyꢀ
drous Na2SO4, evaporated to oil with toluene and disꢀ
solved in 4 mL of toluene. Purification was performed
by flash chromatography, 200 mL of toluene/ethyl
acetate/triethylamine mixture (50 : 49 : 1). After evapꢀ
oration of acetonitrile, the product was dried to conꢀ
stant weight. The yield compound (XII) was 0.15 g
with acetone (2 × 1 mL) and dried at 2–4 mmHg.
(85%), Rf 0.6 (G). 13C NMR,
δ: 19.81, 20.69, 21.93,
The calculation of the concentration of oligonucleꢀ
otide derivatives was carried out, assuming molar
absorption coefficients of the modified oligonucleꢀ
otides, at 260 nm as the sum of the molar absorption
coefficients of the unmodified oligonucleotides and
24.25, 31.96, 42.31, 45.48, 46.63, 48.02, 48.53, 54.95,
55.51, 57.98, 61.67, 85.51, 100.01, 113.10, 113.70,
116.35, 118.81, 122.23, 124.10, 126.56, 127.22,
127.60, 129.57, 133.30, 135.54, 144.77, 145.83,
148.44, 149.80, 155.21, 158.02, 171.93, 174.07. 31P
the value of 23000 M
ing derivative. The ε260 value of the acridine derivative
–1 cm–1 for the acridineꢀcontainꢀ
NMR, δ: 146.95, 148.05.
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 38
No. 6
2012

OLIGONUCLEOTIDE DERIVATIVES IN NUCLEIC ACID HYBRIDIZATION ANALYSIS
637
was obtained by measuring the optical absorption of
the solution of ( ) in water with a known concentraꢀ
tion. The absorption spectra of aqueous solutions were
recorded on a UVꢀ2100 spectrophotometer (Shiꢀ
madzu, Japan).
ACKNOWLEDGMENTS
I
The authors thank A.A. Lomzov and V.V. Koval
(ICBFM SB RAS) for the analysis of the thermal staꢀ
bility of oligonucleotide duplexes and for the recordꢀ
ing of mass spectra, respectively. This work was supꢀ
ported by the RFBR (Project no. 10ꢀ04ꢀ01492ꢀa),
Program of the Russian Academy of Sciences Presidꢀ
ium “Molecular and cell biology” and partially by an
interdisciplinary grant from the Siberian Branch of the
Russian Academy of Sciences and the Ministry of
Education and Science of the Russian Federation.
Electrophoretic analysis was performed in 20%
polyacrylamide gel under denaturing conditions
(acrylamide:
N,N'ꢀmethylene bisacrylamide (30 : 1),
8 M urea, 89 mM Trisꢀborate, pH 8.3, 2 mM Na2ꢀ
EDTA) in TBE buffer (89 mM Trisꢀborate, pH 8.3,
2 mM Na2EDTA) at a voltage of 50 V/cm. The appliꢀ
cation of oligonucleotide samples on the gel was perꢀ
formed in a solution of 8 M urea containing 0.05%
xilencianol FF and 0.05% bromophenol blue.
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