Synthesis of Oligodeoxyribonucleotides Containing Oleylamine Moieties

Article abstract of DOI:10.1023/A:1011333421540

A method for directional introduction of oleylamine residues to any position of oligodeoxyribonucleotides during their automated synthesis was developed. The presence of oleylamine residues in 3′- or 5′-terminal nucleotides was shown to have no effect on

Full text of DOI:10.1023/A:1011333421540

Russian Journal of Bioorganic Chemistry, Vol. 27, No. 3, 2001, pp. 184–190. Translated from Bioorganicheskaya Khimiya, Vol. 27, No. 3, 2001, pp. 210–216.
Original Russian Text Copyright © 2001 by Andreev, Antsypovich, Volkov, Romanova, Hianik, Oretskaya.
Synthesis of Oligodeoxyribonucleotides Containing
Oleylamine Moieties
S. Yu. Andreev*, S. I. Antsypovich*, E. M. Volkov*, E. A. Romanova*,
1
T. Hianik**, and T. S. Oretskaya*
*Department of Chemistry and Belozersky Institute of Physico-Chemical Biology, Moscow State University,
Vorob’evy gory, Moscow, 119899 Russia
**Department of Biophysics and Chemical Physics, Comenius University,
Mlynska dolina F1, 84248 Bratislava, Slovak Republic
Received November 13, 2000; in final form, January 18, 2001
Abstract—A method for directional introduction of oleylamine residues to any position of oligodeoxyribonu-
cleotides during their automated synthesis was developed. The presence of oleylamine residues in 3'- or 5'-ter-
minal nucleotides was shown to have no effect on the thermodynamic stability of DNA duplexes formed by
such oligonucleotides and the complementary sequences. The rate of hydrolysis of oligonucleotides containing
oleylamine residues in the 3'-terminal units by the snake venom phosphodiesterase was shown to be markedly
lower than that of natural oligonucleotides.
Key words: modified oligodeoxyribonucleotides, solid phase synthesis, oleylamine
1
INTRODUCTION
The corresponding modification of individual units
during synthesis of an oligomer is a common method
for introducing hydrophobic substituents into oligonu-
cleotides. The methods for introduction of the unde-
canol [10] and vitamin E [11] residues to the oligonu-
cleotide 5'-end during the solid-phase synthesis, and
also for attachment of cholesterol to the 5'-terminal
phosphate group of protected oligonucleotides [12]
were reported.
In the last few years the field of chemistry of nucleic
acids dealing with their electrochemical properties has
intensively developed [1–5]. This is due to the possi-
bility of using electrodes with immobilized oligonucle-
otides for detection of nucleic acids of pathogenic
microorganisms [1, 6–8]. The development of effective
methods for immobilization of nucleic acids on elec-
trode surfaces is therefore a problem important for the
enhancement in technological effectiveness of analyti-
cal and diagnostic electrochemical procedures.
2
As a rule, the studies dealing with the modification
of 3'-terminal units of nucleotides during solid-phase
synthesis make use of CPG polymeric support modified
with bifunctional agents. This allows the reaction
between the 3'-modified oligonucleotide and the cho-
sen hydrophobic molecule to proceed after cleavage of
the oligonucleotide off the polymer. For example, 6-
aminohexan-1-ol was used for introducing a residue of
acridinylpropionic acid as a bifunctional agent to the 3'-
terminal unit [13]. Pyrrole derivatives N-substituted
with acridine and cholesterol were used for introduc-
tion of these residues [14].
A promising variant of immobilization is noncova-
lent binding of the residues of bulky hydrophobic mol-
ecules, such as lipids and fatty acids, introduced into
nucleotide chains to hydrophobic electrode surfaces
(e.g., graphite paste or lipid membranes) [1–5]. The
development of the methods for immobilization of
nucleic acid fragments on electrode surfaces requires
oligonucleotides with hydrophobic molecule residues
that must as a rule locate at the termini of the oligonu-
cleotide chain. Sometimes the process is accompanied
by the simultaneous modification of the electrode,
which allows the subsequent covalent immobilization
of oligonucleotides. Thus, octadecylamine and stearic
acid were included into graphite paste for this purpose
[9].
Postsynthetic methods for introduction of residues
of bulky hydrophobic molecules to oligonucleotides
have also been reported [15–17]. A cholesterol residue
containing a spacer with an aliphatic amino group was
attached to the nucleotide through the activation of the
terminal phosphate [15]. The attachment of the choles-
terol residue to the 3'-terminal phosphate group using
N-[(cholesteryloxy)carbonyl]ethylenediamine [16] or
to the 5'-terminal phosphate via the disulfide moiety
[17] was described.
1
To whom correspondence should be addressed; phone: +7 (095)
939-3148;
fax:
+7
(095)
939-3181;
e-mail:
oretskaya@genebee.msu.su.
2
Abbreviations: CPG, controlled pore glass; DMAP, 4,4-(N,N-
dimethyl)aminopyridine; TFA, trifluoroacetic acid.
1068-1620/01/2703-0184$25.00 © 2001 MAIK “Nauka /Interperiodica”

SYNTHESIS OF OLIGODEOXYRIBONUCLEOTIDES
185
Molecular masses of oligonucleotides (I)–(V) with oleylamine residues according to mass spectrometry data and melting tem-
peratures of their duplexes with complementary oligonucleotide (X)
Oligonuc-
leotides
Primary structure
(5' 3')
Mr (MALDI, m/z)
Duplex with (X)a
Tm, °Cb
calc/exp
(I)
CAC C T T G C T G AA A T T T T C C C*
CAC* C T T G C T G AA A T T T T C C C*
C*AC C T T G C T G AA A T T T T C C C*
CAC* C T T G C T G AA A T T T T C C C
C*AC C T T G C T G AA A T T T T C C C
AC* C T T G C T G AA A T T T T C C C*
6267.4/6266.0
6531.9/6529.6
6531.9/6526.7
6267.4/6265.8
6267.4/6268.4
A
B
C
D
E
41
33
38
34
41
(II)
(III)
(IV)
(V)
VI
VII
VIII
(IX)
AC
C T T G C T G AA A T T T T C C C*
AC* C T T G C T G AA A T T T T C C C*
CAC C T T G C T G AA A T T T T C C C
F (control)
41
4
Note: C* is N -oleyl-5-methyl-2'-deoxycytidine.
a
(3')GTG GAA CGA CTT TAA AAG GG(5') (X).
Thermodynamic stability of duplexes was determined in phosphate buffer at a duplex concentration of 3.3 µM within the temperature
range 5–50°C.
b
The methods suggested are not versatile and cannot
be used for simultaneous modification of 3'- and 5'-ter-
minal fragments nor for introduction of intrachain
modified units.
RESULTS AND DISCUSSION
To obtain oligonucleotides with oleylamine resi-
dues, we employed the method involving the reactive
thymidine derivative bearing a triazole residue in C4-
position. The 5'-é-protected thymidine (1) was modi-
fied by the standard procedure (Scheme 1) [18]. 1-(5'-
O-Dimethoxytrityl-2'-deoxy-β-D-ribofuranosyl)-4-(1-
triazolyl)-5-methyl-2-pyrimidone (2‡) was reacted
with oleylamine to give 5'-O-dimethoxytrityl-N4-oleyl-
5-methyl-2'-deoxycytidine (2b). In order to obtain
monomeric synthon (3b) for nucleotide synthesis, com-
pound (2b) was treated with N,N-diisopropylamido-β-
cyanoethylchlorophosphite (Scheme 2).
Here we propose a method for introduction of
hydrophobic residues to oligonucleotides that involves
introduction of the modified unit into any predeter-
mined position of the oligonucleotide chain during
automated phosphoramidite synthesis without modify-
ing the standard protocol. Thus, the oligonucleotide ter-
mini remain free. This widens the applicability of such
derivatives, because a possibility remains for introduc-
tion of radioactive or other labels into oligonucleotides
or incorporation of oligonucleotides modified at the
intrastrand units into longer fragments of nucleic acids.
Phosphoramidite derivative (3b) was introduced
We used the oleylamine residue as a hydrophobic sub- into automated oligonucleotide synthesis with an
stituent.
increase in the condensation time up to 4 min and in the
N
N
N
O
N
NH C18 H35
CH3
CH3
CH3
HN
O
N
N
O
O
N
O
N
DMTrO
DMTrO
DMTrO
pyridine
1) (CH3)3SiCl, gbhblby
triazole,
2) nhbfpjk, POCl3,
NH2–C18 H35
lbjrcfy
dioxane
O
O
(iPr)2NEt, CHCN
3
3) NH3/H2O/EtOH
OH
(1)
OH
OH
(2a)
(2b)
=
C18 H35
(CH2)8 CH CH (CH2)7 CH3
Scheme 1.
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 27 No. 3 2001

186
ANDREEV et al.
R
N
CH3
N
O
N (iPr)2
DMTrO
Cl
P
O
O(CH2)2CN
(iPr)2NEt, CHCl 2
2
R
N
O
CH3
N
P
NC(CH2)2O
N(iPr)2
O
(3a, b)
DMTrO
O
R
N
CH3
N
OH
O
O
(2a, b)
CPG
COOH
DMTrO
TPS-Cl, MeIm,
pyridine
N
a: R = N
CPG COO
(4a, b)
N
b: R = NH–C18 H35
Scheme 2.
concentration of (3b) to 0.12 M. The conversion at the
(3b) attachment step was no less than 95%.
In order to obtain oligonucleotides with a modified
unit at the 3'-end, nucleoside (2b) was immobilized on
the CPG-500 polymeric support according to Scheme 2
to yield (4b).
Using compound (3b) and/or polymeric support
(4b), oligodeoxyribonucleotides (I)–(VIII) with oleyl-
amine residues in the cytidine units were synthesized
(see table).
Note that the yields at the stage of treating activated
oligonucleotides with oleylamine (according to the
reverse-phase HPLC analysis data) were not quantita-
tive. In all cases HPLC showed the presence of addi-
tional peaks in the reaction mixtures, which probably
corresponded to oligonucleotides containing 5-methyl-
2'-deoxycytidine in the modification sites. Thus, the
first of the above approaches appeared to be more effec-
tive.
We also studied on the thermodynamic stability of
duplexes formed by modified oligonucleotides (I)–(V)
and unmodified oligonucleotide (IX) with complemen-
tary 20-unit oligonucleotide (X) (see table).
Molecular masses of modified nucleotides (I)–(V)
were determined by mass spectrometry (see table).
The target nucleotides are complementary to DNA
fragments of the pathogenic bacterium Salmonella. Oli-
gomers were isolated by reverse-phase HPLC using the
hydrophobic properties of the oleylamine residue.
HPLC showed the retention time of the target oligonu-
cleotides to vary depending on the nature and the
amount of the introduced functional groups (Fig. 1).
To estimate the effectiveness of the suggested
approach, we tried an alternative method for the synthe-
sis of oligonucleotides with oleylamine residues. Oli-
gonucleotides containing 1-(2'-deoxy-β-D-ribofurano-
syl)-4-(1-triazolyl)-5-methyl-2-pyrimidone were syn-
It appeared that the melting temperatures of
duplexes formed by nucleotides bearing hydrophobic
substituents in the 3'- or 5'-terminal units (duplexes A
and E, respectively) are not different from the melting
temperature of natural duplex F. In this connection, we
should expect oligonucleotides modified at the chain
ends to effectively hybridize with complementary DNA
sequences from solution upon immobilization on the
hydrophobic electrode surface.
On the other hand, the melting temperature of the
thesized using phosphoramidite derivative (3a) and duplex with modifications at both termini (duplex C)
polymeric support (4a). The oleylamine moieties were proved to be somewhat lower (the difference was 3°ë).
substituted for triazole residues after the completion of The presence of the modified unit inside the chain
oligonucleotide synthesis and before removing protec- (duplexes B and D) to some extent decrease the thermo-
tive groups and cleaving the oligomer off the polymeric dynamic stability. Thus, the melting temperature of
support.
duplex D is by 7°ë lower and of duplex B with modifi-
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 27 No. 3 2001

SYNTHESIS OF OLIGODEOXYRIBONUCLEOTIDES
187
∆A260
0.17
1
0.16
0.15
0.14
0.13
0.12
0.11
0.10
0.09
0.08
0.07
0.06
0.05
0.04
0.03
0.02
0.01
0
2
3
0
5
10
15
20
25
30
35
40
min
Fig. 1. HPLC analysis of (1) oligonucleotide (IX), (2) oligonucleotide (I), containing one oleylamine residue, and (3) of oligonu-
cleotide (III), containing two oleylamine residues (for conditions, see the Experimental section).
strate saturation (ëS = 1.2 × 10–5 M), the decrease in the
oligonucleotide concentration during enzyme hydroly-
sis is adequately described by an exponential time
dependence. The initial hydrolysis rates for nucleotides
(IX) and (III) calculated from the line slopes were equal
to (1.45 ± 0.18) × 10–7 and (2.6 ± 0.02) × 10–8 M s–1,
respectively. The ratio between the initial rates was
5.6 ± 0.6; that is, the presence of an oleylamine residue
in the 3'-terminal oligonucleotide unit causes a signifi-
cant delay of hydrolysis of the modified oligonucle-
otide as compared with that of the unmodified ana-
logue. This is typical of the majority of 3'-modified oli-
gonucleotides and is an obvious advantage for the DNA
hybridization analysis of natural objects.
cations both inside and at the termini is by 8°ë lower
than that of natural duplex F.
We studied the kinetics of enzyme hydrolysis of
unmodified oligonucleotide (IX) (Fig. 2a) and oligonu-
cleotide (III) with hydrophobic residues at both ends
(Fig. 2b). The resistance of oligonucleotides towards
snake venom phosphodiesterase was investigated under
conditions of hydrolysis to nucleosides with subse-
quent analysis of the hydrolyzate by reverse-phase
HPLC in a ion-pair mode. Uridine was used as an inter-
nal reference for minimization of the experimental
error upon the analysis of the reaction mixtures.
As shown in Fig. 2a, the snake venom phosphodi-
esterase hydrolysis of unmodified oligonucleotide (IX)
resulted in the formation of multiple intermediates of
various lengths, which underwent a further hydrolysis.
In the case of modified oligonucleotide (III) containing
oleylamine residues, no accumulation of the products
of partial hydrolysis was observed, as they were hydro-
lyzed more rapidly than the starting modified oligonu-
cleotide. This is clearly illustrated by Fig. 2b, where the
reaction mixture is represented by two major peaks (of
the starting oligonucleotide and the internal reference).
Thus, we have developed an effective scheme for
introduction of oleylamine-modified cytidine units into
predetermined position(s) of the oligonucleotide chain
during automated oligonucleotide synthesis. The high
yields of the condensation products upon introduction
of the modified units allow the suggested methods to be
used for the synthesis of oligonucleotides with two
hydrophobic substituents. In the near future we plan to
study the course of the immobilization of oleylamine-
containing oligonucleotides on the electrode surfaces
These data allowed us to compare the hydrolysis
rates for the modified (III) and unmodified (IX) nucle-
otides. In the concentration range distant from the sub- and to investigate their electrochemical behavior.
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 27 No. 3 2001

188
ANDREEV et al.
∆A260
(‡)
0.50
0.45
0.40
0.35
0.30
0.25
0.20
0.15
0.10
0.05
0
1
2
3
4
5
6
7
0
1
2
3
4
5
6
7
8
9
10 11 12 13 14 15 16 17 18
(b)
0.08
0.07
0.06
0.05
0.04
0.03
0.02
0.01
0
1
2
3
4
5
6
7
0
5
10
15
20
25
30
35
min
Fig. 2. HPLC analysis of the snake venom phosphodiesterase hydrolysis products of (a) unmodified oligonucleotide (IX) and
(b) modified nucleotide (III) after (1) 0, (2) 1, (3) 3, (4) 5, (5) 10, (6) 20, and (7) 30 min (for conditions, see the Experimental sec-
tion).
EXPERIMENTAL
triisopropylbenzenesulfonyl chloride (Aldrich, United
States); N-methylimidazole, (N,N-diisopropylamido)-
β-cyanoethylphosphite, DMAP, trimethylchlorosilane,
and triethylamine (Fluka, Switzerland); triazole and
tetrazole (Pharmacia, Sweden); diisopropylethylamine
(Merck, Germany), phosphorus (V) oxychloride and
5'-é-Dimethoxytrityl-2'-deoxyribonucleoside
3'-
(N,N-diisopropylamido)-β-cyanoethylphosphites (Glen
Research, United Kingdom); thymidine (Sigma, United
States); 5'-é-dimethoxytritylthymidine, dimethoxytri-
tyl chloride, oleylamine, succinic anhydride, and 2,4,6- TFA (Russia); abs. acetonitrile, pyridine, triethylamine,
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 27 No. 3 2001

SYNTHESIS OF OLIGODEOXYRIBONUCLEOTIDES
189
chloroform, methylene chloride, and ethanol were
used.
Succinic anhydride (100 mg, 0.86 mmol) and
DMAP (5 mg, 0.04 mmol) were added to a suspension
of aminopropyl-CPG (200 mg) with the amino group
load of 0.05 mmol/g in pyridine (500 µl). The reaction
was performed for 4 h at 20°ë with continual shaking.
The polymer was washed on a glass filter with pyridine
(3 × 5 ml), ethanol (3 × 5 ml), and diethyl ether (3 ×
5 ml) and dried in vacuum. Pyridine (500 µl) and tri-
methylchlorosilane (100 µl) were added to the support
modified with succinic anhydride, the polymer was
degassed and kept for 8 h at 20°ë. The support was
washed on a glass filter with pyridine (3 × 5 ml), etha-
nol (3 × 5 ml), and diethyl ether (3 × 5 ml) and dried in
vacuum. The resulting support was suspended in pyri-
dine (500 µl), 2,4,6-triisopropylbenzenesulfonyl chlo-
ride (30 mg, 0.1 mmol) and N-methylimidazole (23 µl,
0.3 mmol) were added followed by addition, after
15 min storage, of compound (2a) (25 mg, 0.04 mmol)
or compound (2b) (28 mg, 0.035 mmol). The reaction
was performed for 2 h at 20°ë; the support was treated
with methanol overnight and then washed with pyridine
(3 × 5 ml), ethanol (3 × 5 ml), and diethyl ether (3 ×
5 ml). The load of the polymeric support with a nucle-
oside was determined from the amount of dimethoxy-
trityl groups measured spectrophotometrically in a
solution containing the products of treating an aliquot
of the polymer with TFA. The resulting polymers (4a)
and (4b) were dried to give 200 mg of each polymer
with a nucleoside load of about 0.025 mmol/g.
TLC was performed on the Silica gel 60 F254
(Merck, Germany) precoated plates in (A) 95 : 5 chlo-
roform–ethanol, (B) 9 : 1 chloroform–ethanol, and (C)
98 : 2 methylene chloride-triethylamine systems. Col-
umn chromatography was carried out on Silica gel 60
(Merck, Germany) in (D) an ethanol gradient in chloro-
form [(D), 0
5% and (E) 0
15%].
Optical absorption and UV spectra were registered
on a Hitachi 150-20 spectrophotometer (Japan) in a
1-cm quartz cell. The temperature-dependences of UV
absorption at 260 nm were registered on the same
instrument supplied with a thermostated cell holder
upon a progressive temperature increase at a rate of
0.5°C/min.
Mass spectra were measured on a Finnigan MAT
VISION 2000 TOF (MALDI) mass spectrometer.
5'-O-Dimethoxytrityl-3'-O-trimethylsilylthymidine
was prepared as described in [19] and used without iso-
lation for the synthesis of (2‡).
1-(2'-Deoxy-5'-O-dimethoxytrityl-b-D-ribofura-
nosyl)-5-methyl-4-(1-triazolyl)-2-pyrimidone (2a).
Triazole (6.4 g, 93 mmol) was suspended in acetonitrile
(50 ml) and phosphorus (V) oxychloride (1.96 ml,
16.9 mmol) was added. Then triethylamine (15 ml,
110 mmol) was added dropwise at stirring and cooling
on ice, the reaction mixture was kept for 30 min at
20°ë, and a solution of 5'-O-dimethoxytrityl-3-O-trim-
Synthesis of oligonucleotides with modified units
ethylsilylthymidine (5 mmol) in acetonitrile (20 ml) was carried out on an Applied Biosystems 380B auto-
was added. The reaction was performed for 15 min at mated DNA synthesizer (United States) by the phos-
20°ë. After the reaction was over (TLC in system B), phoramidite method using 0.12 M solutions of modi-
the reaction mixture was washed with water. The fied phosphoramidites and the condensation time
organic layer was concentrated in vacuum to a foam- increased up to 4 min. To substitute the oleylamine res-
like residue and chromatographed on a Silica gel col- idue for the triazole when phosphoramidite derivative
umn in system E to yield 1.42 g (48%) of (2a); Rf 0.75 (3‡) or polymeric support (4‡) was used, the oligonu-
cleotides were treated, before deprotection and removal
from the polymeric support, with 1 M solution of oleyl-
amine in dioxane for 30 min. The polymer was washed
with dioxane (3 × 5 ml) and distilled water (3 × 5 ml).
After the synthesis was over, the resulting oligonu-
cleotides were deprotected as described in [20].
The study on the kinetics of enzyme hydrolysis of
oligonucleotides (III) and (IX) with snake venom
phosphodiesterase. Oligonucleotide (0.2 OD260) and
uridine (1.0 OD260) were dissolved in 16 µl of 0.2 M
Tris-HCl buffer solution (pH 8.5) containing 0.04 M
magnesium chloride, and 4 µl of a snake venom phos-
phodiesterase solution (1.0 × 104 U/ml) was added. The
reaction mixture was incubated at 37°ë. After certain
time intervals, 4-µl aliquots were taken, in which the
reaction was terminated by cooling in liquid nitrogen.
(A).
2'-Deoxy-5'-O-dimethoxytrityl-5-methyl-N4-oleyl-
cytidine (2b). Compound (2a) (0.3 g, 0.5 mmol) was
dissolved in dioxane (5 ml), and oleylamine (1.5 ml,
4.6 mmol) was added. The conversion at 20°C was mon-
itored by TLC (system B). After the reaction was over
(1 h), the reaction mixture was evaporated to an oily res-
idue and chromatographed on a Silica gel column in
system D to give 0.36 g (90%) of (2b); Rf 0.67 (B).
1-(2'-Deoxy-5'-O-dimethoxytrityl-b-D-ribofura-
nosyl)-5-methyl-4-(1-triazolyl)-2-pyrimidone
3'-
(N,N-diisopropylamido)-b-cyanoethylphosphite (3a)
and 2'-deoxy-5'-O-dimethoxytrityl-5-methyl-N4-ole-
ylcytidine 3'-(N,N-diisopropylamido)-b-cyanoeth-
ylphosphite (3b) were obtained from (2a) and (2b)
according to [20].
Immobilization of 1-(2'-deoxy-5'-O-dimethoxy-
trityl-b-D-ribofuranosyl)-5-methyl-4-(1-triazolyl)-
2-pyrimidone (2a) and 2'-deoxy-5'-O-dimethoxytri-
The analysis of the reaction mixtures of oligonucle-
otides was performed by reverse-phase HPLC in the
ion-pair mode on a Waters instrument (United States)
tyl-5-methyl-N4-oleylcytidine (2b) on the polymeric using a Diasorb S-16T (Russia) column and a 5–40%
support was performed by the method reported in [21]. logarithmic gradient of acetonitrile in buffer solution
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 27 No. 3 2001

190
ANDREEV et al.
8. Korri-Youssoufi, H., Garnier, F., Srivastava, P., and
Godillot, P., J. Am. Chem. Soc., 1997, vol. 119,
pp. 7388–7389.
(48 mM potassium phosphate and 2 mM tetrabutylam-
monium phosphate, pH 7.0) as a mobile phase.
Oligonucleotides with hydrophobic residues were
isolated by reverse-phase chromatography on a Gilson
instrument (United States) using a Beckman Ultra-
sphere Octyl (4.6 × 25 mm) column and a 0–80% linear
gradient of acetonitrile in 20 mM ammonium acetate as
a mobile phase (80 min).
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ACKNOWLEDGMENTS
12. Zarytova, V.F., Ivanova, E.M., and Chasovskikh, M.N.,
We are grateful to V.N. Tashlitskii for assistance in
performing HPLC analysis of modified nucleotides.
This work was supported by INCO-Copernicus
IC15-CT98-015 and the Russian Foundation for Basic
Research (project no. 00-04-48260).
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