Amino-functionalized oligonucleotides with peptide internucleotide linkages

Article abstract of DOI:10.2174/157017812800221799

Oligonucleotide analogs with L-and D-lysine residues incorporated in internucleotide linkages were synthesized and their affinity toward complementary DNA was studied. Stability of the duplexes formed by the modified oligonucleotides and their wild-type c

Full text of DOI:10.2174/157017812800221799

106
Letters in Organic Chemistry, 2012, 9, 106-113
Amino-Functionalized Oligonucleotides with Peptide Internucleotide
Linkages
Anna M. Varizhuk, Svetlana V. Kochetkova, Alexander U. Fedotov, Natalia A. Kolganova, Edward
N. Timofeev and Vladimir L. Florentiev*
Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, ul. Vavilova 32, Moscow, 119991 Russia
Received December 14, 2010: Revised March 29, 2011: Accepted November 29, 2011
Abstract: Oligonucleotide analogs with L- and D-lysine residues incorporated in internucleotide linkages were
synthesized and their affinity toward complementary DNA was studied. Stability of the duplexes formed by the modified
oligonucleotides and their wild-type complements appeared to be close to that of the isosequential unmodified duplex,
oligonucleotides carrying D-lysine residues forming generally more stable duplexes than L-lysine derivatives.
Keywords: Analogs, hybridization, oligonucleotides, peptide internucleotide linkages, synthesis, thermal stability.
O
N
O
N
INTRODUCTION
Modified oligonucleotides (ONs) have become an
important instrument in molecular biology experiments. ON
analogs are extensively used for manipulation of gene
expression via antisense or RNA interference mechanisms
[1-8]. However, the current ON analogs display limitations
and there is a strong demand for new modified ONs. Much
effort has been recently devoted to the problem of
introduction of aminoalkyl side chains in ONs. This
modification finds application in functionalization of nucleic
acid aptamers and catalysts as well as nucleic acid labeling
[9-11]. Furthermore, aminoalkyl-ONs display cationic side
chains at appropriate pH and therefore can form with native
DNA duplexes or triplexes with increased stability under
certain conditions [12-15]. Most popular sites for appending
amino- or aminoalkyl functionalities are 5-position of
pirimidines [16, 17] and 2'-position of ribose [18, 19]. In this
paper, we describe ON analogs in which aminoalkyl
functionalities are introduced via peptide internucleotide
linkages. Peptide fragments are known to protect ON
analogs from nuclease digestion and facilitate their cellular
uptake, thus helping to overcome the two main impediments
in the use of ON-based therapeutics [20, 21]. In our previous
work we have reported a general method for incorporation of
hydrophobic amino acid residues in ON strands [22, 23].
Here we describe adaptation of this method for preparation
of L- and D-lysine derivatives (Scheme 1).
NH
NH
O
O
O
O
O
O
O
N
O
N
NH
NH
R
H
NH
NH
H
R
O
O
HN
O
HN
O
O
O
O
O
O
O
A
B
R = (CH2)4NH2
Scheme 1. Fragments of oligonucleotides with L-lysine (A) and D-
lysine (B) residues in internucleotide linkages.
Compounds 3a, 3b, key intermediates in the synthesis of
target dinucleosides 9a, 9 b, were prepared via condensation
of 3'-amino-3'-deoxy-5'-O-dimethoxytritylthymidine 1 with
L- and D-lysine derivatives 2a, 2b in the presence of DCC
and NHS (4 °C, 24 h). The usage of DCC and NHS enables
avoidance of epimerization at ꢁ-carbon atoms of amino acid
residues, observed when using other condensing agents
(about 30% epimerization, determined by 1H-NMR, occurred
when BOP was used). Compounds 3a, 3b were obtained in a
yield of 83%. Optical purity of 3a, 3b was no less than 98%
(determined by 1H-NMR spectroscopy). Trifluoroacetyl
protection was removed by treatment with K2CO3 in a
methanol/water mixture under reflux (4 h) to give
compounds 4a, 4b, bearing free ꢁ-amino groups, in a yield
of 71-80%. The latter were condensed with 3'-O-(t-
butyldimethylsilyl)thymidine 5'-carboxylc acid 5 (prepared
following the published procedure [22]) in the presence of
BOP and TEA in CH2Cl2 (20 °C, 1 h) to give dinucleoside
analogs 6a, 6b in a yield of 84-89%. Epimerization via the
5(4H)-oxazolone mechanism not being possible at this stage,
RESULTS AND DISCUSSION
Dinucleoside phosphoramidite blocks with trifluoro-
acetyl-protected ꢀ-amino functionalities (Scheme 2) were
prepared and used in automated ON synthesis. Treatment
with NH3 at the end of the ON synthesis yielded ON analogs
with free ꢀ-amino groups.
*Address correspondence to this author at the Engelhardt Institute of
Molecular Biology, Russian Academy of Sciences, ul. Vavilova 32,
Moscow, 119991 Russia; Tel: +7 (499) 135-2182; Fax: +7(499)135-1405;
E-mail: flor@imb.ac.ru
1875-6255/12 $58.00+.00
© 2012 Bentham Science Publishers

Amino-Functionalized Oligonucleotides with Peptide Internucleotide
Letters in Organic Chemistry, 2012, Vol. 9, No. 2
107
O
N
O
O
N
NH
O
NH
NH
HO
O
O
O
NH
DMTrO
DMTrO
N
O
O
O
O
5
O
OH
DMTrO
a
b
OTBDMS
N
O
+
O
c
O
NH
O
NH
(H2C)4
NHCOCF3
NHCbz
NH2
(H2C)4
NHCOCF3
(H2C)4
NH2
2a,b
1
NHCbz
NHCbz
3a,b
4a,b
O
N
O
N
O
N
O
NH
NH
NH
NH
DMTrO
DMTrO
DMTrO
DMTrO
O
O
O
O
N
O
O
O
O
O
d
e
f
O
N
O
N
O
N
O
N
O
NH
NH
O
NH
NH
O
NH
NH
NH
NH
NH
NH
NH
NH
(H2C)4
O
(H2C)4
NH
O
(H2C)4
NH
O
(H2C)4
NH
O
O
O
O
O
O
O
O
O
NHCbz
COCF3
COCF3
COCF3
O
OTBDMS
OTBDMS
OH
8a,b
O
NC
P
6a,b
7a,b
9a,b
N
a: L-lysine, b: D-lysine derivatives
Scheme 2. Synthesis of the dinucleoside blocks containing L- and D-lysine residues in internucleoside linkages.
Reagents and conditions: (a) DCC, NHS, CH2Cl2, 4°C; (b) K2CO3, methanol/water, reflux; (c) BOP, TEA, CH2Cl2; (d) i - NH4COOH, Pd/C,
methanol, reflux; ii - CF3COOEt, TEA, methanol; (e) TBAF, THF; (f) NCCH2CH2OP(NPri2)2, 1H-tetrazole, pyridine, CH2Cl2.
BOP was chosen as a condensing agent since it affords
higher yields of 6a, 6b. Benzyloxicarbonyl protection was
substituted for trifluoroacetyl protection without isolation of
intermediate dinucleosides with free ꢀ-amino groups.
Dinucleosides 6a, 6b were deprotected by hydrogenation
with ammonium formate in methanol in the presence of 10%
Pd/C (reflux, 3h). Subsequent treatment with ethyl
trifluoroacetate in the presence of TEA in methanol gave
triflouroacetylated dinucleosides 7a, 7b in a yield of 80-
82%. The silyl protection was removed with 0.5 M TBAF in
THF (20°C, 4 h). Resulting dinucleosides 8a, 8b with free 3'-
hydroxyl groups were treated with 2-cyanoethyl-N,N,N',N'-
tetraisopropylphosphoramidite in the presence of terazole
and pyridine in dichloromethane (20°C, 2 h) to give amidites
9a, 9b in a yield of 64-65%. Dinucleosides 6a,b – 9a,b were
characterized by MALDI-TOF mass spectrometry and the
structures of 6a,b - 8a,b were confirmed by COSY NMR
the modified ONs, isosequential and complementary wild-
type ONs were synthesized. Purification of the ONs was
carried out by reverse-phase HPLC. All the ONs were
characterized by MALDI-TOF mass spectrometry (Table 1).
Thermal dissociation of the modified duplexes and their
wild-type counterpart was measured. The resulting curves
enabled evaluation of melting temperatures of the duplexes.
The results are summarized in Table 2.
As evident from Table 2, ONs carrying D-lysine residues
generally form more stable duplexes than L-lysine
derivatives (ꢁꢀm = +0.4°C and -0.3°C per modification, on
an average, respectively1). The effect of the modification on
duplex stability depends on its position. Terminal
modifications tend to increase duplex stability, while
modifications in the middle of the strand have small
destabilizing effect, more significant in the case of the L-
lysine derivative.
spectroscopy.
COSY-spectra
enabled
unambiguous
assignment of signals to protons of lysine, thymidine 5'-
carboxylc acid and 3'-amino-3'-deoxythymidine moieties
(Fig. 1).
CONCLUSION
In summary, we have described the facile synthesis of the
novel ON analogs carrying L-and D-lysine residues in
internucleotide linkages. The effect of the modifications on
Dinucleoside amidite blocks 9a, 9b were used in the
synthesis of modified ONs on an automated synthesizer
following standard phosphoramidite protocols. Coupling
time was increased to 15 minutes for modified
phosphoramidites. No decrease in coupling efficiency was
observed (98-99% step-wise coupling yields). Along with
1 Average ꢁꢀm per modification was calculated as a sum of all corresponding ꢁꢀm
values reported in Table 2 divided by the total number of modifications.

108 Letters in Organic Chemistry, 2012, Vol. 9, No. 2
Varizhuk et al.
Fig. (1). COSY NMR spectrum of compounds 8a, 8b. Abbreviations: cT - thymidine 5'-carboxylc acid moiety, Ta - 3'-amino-3'-
deoxythymidine moiety.

Amino-Functionalized Oligonucleotides with Peptide Internucleotide
Letters in Organic Chemistry, 2012, Vol. 9, No. 2
109
Table 1. MALDI-TOF Mass Spectra of the Modified ONs
m/z, found (calculated for [M – H]ꢀ)ꢀ
Sequence
(5'ꢁ3')*ꢀ
X = L-Lysꢀ
X = D-Lysꢀ
TTAACTTCTTCACAXCꢀ
XAACTTCTTCACATTCꢀ
TTAACTTCXCACATTCꢀ
XAACTTCTTCACAXCꢀ
XAACTTCXCACAXCꢀ
-ꢀ
5131.6 (5130.51)ꢀ
5135.1 (5130.51)ꢀ
5132.3 (5130.51)ꢀ
5191.0 (5191.70)ꢀ
5254.3 (5253.89)ꢀ
5131.2 (5130.51)ꢀ
5129.9 (5130.51)ꢀ
5192.0 (5191.70)ꢀ
5251.5 (5253.89)ꢀ
Notes: *X – modified dinucleoside fragments containing L- or D- lysine residues in internucleotide linkages.
Table 2. Melting Temperatures of the Modified and Wild-Type Duplexes (Duplex Concentration 2.5·10-6 M)
Sequence
ꢀm, °ꢂ 0.5, (ꢃꢀm, °ꢂ**)ꢀ
(5'ꢁ3')*ꢀ
X = L-Lysꢀ
X = D-Lysꢀ
TTAACTTCTTCACAꢂꢂCꢀ
TTAACTTCTTCACAXCꢀ
XAACTTCTTCACAꢂꢂCꢀ
TTAACTTCXCACAꢂꢂCꢀ
XAACTTCTTCACAXCꢀ
XAACTTCXCACAXCꢀ
50.3ꢀ
-ꢀ
51.0 (+0.7)ꢀ
50.6 (+0.3)ꢀ
50.2 (-0.1)ꢀ
51.7 (+1.4)ꢀ
51.3 (+1.0)ꢀ
50.9 (+0.6)ꢀ
48.9 (-1.4)ꢀ
50.4 (+0.1)ꢀ
49.2 (-1.1)ꢀ
Notes: *X – modified dinucleoside fragments containing L- or D- lysine residues in internucleotide linkages.
**Tm difference between modified and natural duplexes.
duplex stability has been assessed. Melting temperatures of
the modified duplexes appeared to be close to that of the
isosequential native duplex. Thus, the novel analogs meet the
stability criterion set for modified ONs and could become a
promising tool for the development of gene-targeted
therapeutics. Considering that ꢀ-amino groups of lysine
residues in the novel analogs are accessible for conjugation
reactions, such as fluorescent labeling, we believe, these
analogs could also find application in diagnostics and in the
study of biological functions of nucleic acids.
1H NMR data were processed and presented using
MestReNova version 6.2.0 (Mestrelab Research SL).
MALDI-TOF mass spectra were acquired on a Kratos MS-
30 spectrometer using linear flight path.
ONs were synthesized on an Aplied Biosystem DNA
synthesizer 3400 using standard phosphoramidite protocols
and purified using preparative scale reverse-phase HPLC on
a 250 mm x 4.0 mm2 Hypersil C18 column with detection at
260 nm. Chromatography of dimethoxytrytil-protected ONs
was performed using 10-50% gradient of CH3CN in 0.05 M
TEAA. Melting curves of the duplexes were recorded on a
Shimadzu UV 160-A spectrophotometer, equipped with a
thermostatic system, in 20 mM sodium phosphate buffer,
100 mM NaCl, 01 mM EDTA, pH 7.0, concentration of each
duplex being 2.5·10-6M. Samples were denatured at 95°C for
5 min and slowly cooled to 20°C prior to measurements. A260
(duplex absorbance) was measured as a function of
temperature. It was registered every 0.5°C from 20 to 70°C.
Thermodynamic parameters of duplex formation were
obtained by performing nonlinear regression analysis using
DataFit version 9.0.059 (Oakdale Engineering, US). The
calculation method taking into account temperature
dependence of UV absorbance of duplexes and single strands
was applied.
EXPERIMENTAL
All reagents were commercially available and used
without further purification. 3'-Azydo-3'-deoxythymidine
was provided by Association AZT (Russia). Dichloro-
methane was dried by distillation from phosphorus
pentaoxide, pyridine was distilled from calcium hydride, and
THF was distilled from lithium alumohydride prior to use.
Silica gel flesh column chromatography was performed on
silica gel Kieselgel 60 (0.040-0.063 mm, Merck). TLC was
performed on silica gel Kieselgel 60 F254 precoated plates
(Merck) with detection by UV using the following solvent
systems (compositions expressed as v/v): ethanol
-
methylene chloride 1:65 (A), 1:32 (B), 1: 19 (C), 1:12 (D),
1
N-ꢄ-Benzyloxycarbonyl-N-ꢅ-trifluoroacetyl-L-lysine
(2a). To a stirred suspension of N-ꢀ-benzyloxycarbonyl-L-
lysine [24] (2.8 g, 10 mmol) in 30 mL of abs. MeOH, ethyl
trifluoroacetate (2.84 g, 2.4 mL, 20 mmol) and TEA (2.02 g,
2.8 mL, 20 mmol) were added. The mixture was stirred for
24 h. Solvent was evaporated, the residue was dissolved in
50 mL of ethyl acetate, washed with water, 10% NaHSO4,
1:9 + 0.1% TEA (E). H NMR spectra were recorded on a
Bruker AMXIII-400 NMR spectrometer on solutions in
deuterated dimethyl sulfoxide (DMSO-d6). Chemical shifts
are given in parts per million (ppm). The coupling constants
(J) are given in Hz. The signals were assigned using COSY
experiments. Abbreviations used: cT (thymidine 5'-carboxylc
acid moiety), Ta (3'-deoxy-3'-aminothymidine moiety). The

110 Letters in Organic Chemistry, 2012, Vol. 9, No. 2
Varizhuk et al.
water, dried over Na2SO4 and evaporated. Compound 2a was
3.023 (2 H, q, J 5.9, ꢃ-CH2 D-Lys), 2.429 – 2.316 (1 H, m,
1
2
obtained as a colourless viscous oil. Yield 3.5 g (93%). H
H2'a Ta), 2.175 (1 H, dt, J1', 2'b 6.2, J3', 2'b 6.2, J2'a, 2'b 13.1,
NMR: 12.898 (1 H, s, COOH), 9.621 (1 H, d, J 7.7, ꢀ-NH),
7.397 – 7.271 (5 H, m, Ar-H Ph), 7.206 (1 H, t, J 5.5, ꢁ-NH),
5.004 (2 H, s, CH2 Bn), 4.209 (1 H, ddd, J 9.8, 7.7, 4.7, ꢀ-
CH), 2.986 (2 H, q, J 6.4, ꢁ-CH2), 1.877 – 1.667 (2 H, m, ꢂ-
CH2), 1.463 – 1.363 (2 H, m, ꢃ-CH2), 1.341 – 1.249 (2 H, m,
ꢄ-CH2).
H2'b Ta), 1.767 – 1.635 (2 H, m, ꢄ-CH2 D-Lys), 1.429 (3 H,
4
d, J 1.1, 5-CH3 Ta), 1.429 – 1.358 (2 H, m, ꢅ-CH2 D-Lys),
1.324 – 1.196 (2 H, m, ꢆ-CH2 D-Lys).
(5S)-Benzyl 6-{(5'-O-(4,4'-dimethoxytrityl)-3'-deoxy-
thymidine-3'-yl)amino}-6-oxo-5-aminohexylcarbamate
(4ꢂ). Nucleoside 3a (730 mg, 0.809 mmol) was dissolved in
methanol (15 mL), water (1.5 mL) and K2CO3 (550 mg, 4
mmol) were added. The mixture was refluxed for 4 h, then
cooled, diluted with water (50 mL) and extracted with
chloroform (3ꢀ50 mL). Combined organic layers were
washed with water (30 mL), dried over Na2SO4, filtered and
evaporated. The residue was purified by CC in 7 - 10% of
ethanol in CH2Cl2 + 0.1% TEA to give 4a as a hard foam
N-ꢀ-Benzyloxycarbonyl-N-ꢁ-trifluoroacetyl-D-lysine
(2b) was prepared following the previous procedure from 2.8
g (10 mmol) of N-ꢃ-benzyloxycarbonyl-D-lysine [24]. Yield
3.4 g (90.3%). 1H NMR: 12.889 (1 H, s, COOH), 9.625 (1 H,
d, J 7.7, ꢀ-NH), 7.401 – 7.268 (5 H, m, Ar-H Bn), 7.216 (1
H, t, J 5.7, ꢁ-NH), 5.008 (2 H, s, CH2 Bn), 4.213 (1 H, ddd, J
9.9, 7.7, 4.6, ꢀ-CH), 2.991 (2 H, q, J 6.6, ꢁ-CH2), 1.885 –
1.662 (2 H, m, ꢂ-CH2), 1.466 – 1.365 (2 H, m, ꢃ-CH2), 1.344
– 1.251 (2 H, m, ꢄ-CH2).
1
(468 mg, 71.7% yield). Rf = 0.37 (Solvent system E). H
NMR: 8.359 (1 H, d, J 7.4, 3'-NH Ta), 7.559 (1 H, d, 4J 1.0,
H6), 7.447 – 6.805 (18 H, m, Ar-H), 7.170 (1 H, t, J 5.5, ꢃ-
NH L-Lys), 6.226 (1 H, t, J 6.4, H1' Ta), 4.988 (2 H, s, CH2
Bn), 4.586 – 4.458 (1 H, m, H3' Ta), 3.968 – 3.881 (1 H, m,
H4' Ta), 3.726 (6 H, s, CH3O DMTr), 3.234 (2 H, br. s, 5'-
CH2 Ta), 3.213 – 3.153 (1 H, m, ꢁ-CH L-Lys), 2.940 (2 H, q,
J 5.9, ꢃ-CH2 L-Lys), 2.401 – 2.290 (1 H, m, H2'a Ta), 2.220
(5S)-Benzyl 6-{(5'-O-(4,4'-dimethoxytrityl)-3'-deoxy-
thymidine-3'-yl)amino}-6-oxo-5-(2,2,2-trifluoracetamido)
hexylcarbamate (3ꢂ). To a stirred solution of 2a (206 mg,
0.55 mmol) in dry CH2Cl2 (5 mL), nucleoside 1 (272 mg, 0.5
mmol) and NHS (63 mg, 0.55 mmol) were added. The
mixture was cooled to 0 °C in an ice-water bath with stirring,
and DCC (113 mg, 0.55 mmol) was added. The mixture was
kept overnight at 4 °C, then filtered. The filtrate was diluted
with water (30 mL) and extracted with ethyl acetate (3 x 30
mL). Combined organic layers were washed with saturated
NaHCO3 (30 mL) and water (30 mL). The organic layer was
dried over Na2SO4, filtered and evaporated. The residue was
purified by silica gel flesh column chromatography in 3% of
ethanol in CH2Cl2 + 0.1% TEA to give 3a as a hard foam
2
(1 H, dt, J1', 2'b 6.6, J3', 2'b 6.6, J2'a, 2'b 13.2, H2'b Ta), 1.574 –
4
1.453 (2 H, m, ꢄ-CH2 L-Lys), 1.461 (3 H, d, J 1.0, 5-CH3
Ta), 1.428 – 1.301 (2 H, m, ꢅ-CH2 L-Lys), 1.295 – 1.138 (2
H, m, ꢆ-CH2 L-Lys).
(5R)-Benzyl 6-{(5'-O-(4,4'-dimethoxytrityl)-3'-deoxy-
thymidine-3'-yl)amino}-6-oxo-5-aminohexylcarbamate
(4b) was prepared following the previous procedure from
1.143 g (1.56 mmol) of nucleoside 3b. Yield 1.07 g (83.0%).
Rf 0.31 (Solvent system E). 1H NMR: 8.219 (1 H, d, J 7.2, 3'-
1
(390 mg, 83.3% yield). Rf = 0.48 (Solvent system B). H
4
NMR: 11.303 (1 H, s, H3), 9.518 (1 H, d, J 7.8, ꢂ-NH L-
NH Ta), 7.613 (1 H, d, J 1.2, H6), 7.506 – 6.853 (18 H, m,
4
Lys), 8.508 (1 H, d, J 7.8, 3'-NH Ta), 7.552 (1 H, d, J 1.0,
Ar-H), 7.227 (1 H, t, J 5.6, ꢃ-NH D-Lys), 6.284 (1 H, t, J
6.4, H1' Ta), 5.057 (2 H, s, CH2 Bn), 4.636 – 4.502 (1 H, m,
H3' Ta), 4.026 – 3.964 (1 H, m, H4' Ta), 3.785 (6 H, s, CH3O
H6), 7.427 – 6.838 (18 H, m, Ar-H), 7.199 (1 H, t, J 5.2, ꢃ-
NH L-Lys), 6.229 (1 H, t, J 6.4, H1' Ta), 4.995 (2 H, s, CH2
Bn), 4.594 – 4.487 (1 H, m, H3' Ta), 4.251 – 4.160 (1 H, m,
ꢂ-CH L-Lys), 3.927 – 3.846 (1 H, m, H4' Ta), 3.728 (6 H, s,
CH3O DMTr), 3.223 (2 H, br. s, 5'-CH2 Ta), 2.947 (2 H, q, J
5.9, ꢃ-CH2 L-Lys), 2.423 – 2.322 (1 H, m, H2'a Ta), 2.183 (1
2
DMTr), 3.322 (1 H, dd, J4', 5'a 4.7, J5'a, 5'b 10.5, H5'a Ta),
3.258 (1 H, dd, J4', 5'b 2.4, 2J5'a, 5'b 10.5, H5'b Ta), 3.127 (1 H,
t, JCH, CH2 6.12, ꢁ-CH D-Lys), 3.023 (2 H, q, J 5.9, ꢃ-CH2 D-
Lys), 2.441 – 2.341 (1 H, m, H2'a Ta), 2.272 (1 H, dt, J1', 2'b
6.7, J3', 2'b 6.7, 2J2'a, 2'b 13.3, H2'b Ta), 1.615 – 1.520 (2 H, m,
2
H, dt, J1', 2'b 6.6, J3', 2'b 6.6, J2'a, 2'b 13.2, H2'b Ta), 1.677 –
4
4
1.525 (2 H, m, ꢄ-CH2 L-Lys), 1.449 (3 H, d, J 1.0, 5-CH3
ꢄ-CH2 D-Lys), 1.522 (3 H, d, J 1.0, 5-CH3 Ta), 1.477 –
Ta), 1.421 – 1.324 (2 H, m, ꢅ-CH2 L-Lys), 1.312 – 1.085 (2
H, m, ꢆ-CH2 L-Lys).
1.377 (2 H, m, ꢅ-CH2 D-Lys), 1.380 – 1.242 (2 H, m, ꢆ-CH2
D-Lys).
(5R)-Benzyl 6-{(5'-O-(4,4'-dimethoxytrityl)-3'-deoxy-
thymidine-3'-yl)amino}-6-oxo-5-(2,2,2-trifluoracetamido)
hexylcarbamate (3b) was prepared following the previous
procedure from 1.087 g (2 mmol) of 3'-amino-3'-deoxy-5'-O-
dimethoxytritylthymidine (1). Yield 1.503 g (83.2%). Rf 0.45
(Solvent system B). 1H NMR: 11.302 (1 H, s, H3), 9.484 (1
H, d, J 7.8, ꢂ-NH D-Lys), 8.516 (1 H, d, J 7.4, 3'-NH Ta),
N-[(2S)-6-Benzyloxycarboxamido-1-{(5'-O-(4,4'-dime-
thoxytrityl)-3'-deoxythymidine-3'-yl)amino}-1-oxohexan-
2-yl]-3'-O-(tert-butyldimethylsilyl)thymidine-5'-carboxa-
mide (6ꢂ). Nucleoside 4a (1 g, 1.2 mmol) was suspended in
dry CH2Cl2 (12 mL), 5'-carboxynucleoside 5 (489 mg, 1.3
mmol), TEA (392 ꢇL, 2.8 mmol) and BOP (619 mg, 1.4
mmol) were added. The mixture was stirred for 1 h at 20 °ꢈ,
then diluted with water (50 mL) and extracted with ethyl
acetate (3ꢀ40 mL). Combined organic layers were washed
with water (30 mL) and saturated NaHCO3 (30 mL), dried
over Na2SO4, filtered and evaporated. The residue was
purified by silica gel flesh column chromatography in 4% of
ethanol in CH2Cl2 + 0.1% TEA to give 6a as a hard foam
4
7.539 (1 H, d, J 1.1, H6), 7.423 – 6.827 (18 H, m, Ar-H),
7.229 (1 H, t, J 5.4, ꢃ-NH D-Lys), 6.225 (1 H, t, J 6.5, H1'
Ta), 4.997 (2 H, s, CH2 Bn), 4.555 – 4.463 (1 H, m, H3' Ta),
4.254 – 4.184 (1 H, m, ꢂ-CH D-Lys), 3.944 – 3.882 (1 H, m,
H4' Ta), 3.725 (6 H, s, CH3O DMTr), 3.307 – 3.252 (1 H, m,
2
H5'a Ta2), 3.204 (1 H, dd, J4', 5'b 2.4, J5'a, 5'b 10.1, H5'b Ta),
1
(1.218 g, 87.6% yield). Rf = 0.35 (Solvent system C). H
NMR: 11.295 (2 H, s, H3 Ta, H3 cT), 8.840 (1 H, d, J 7.8,
3'-NH Ta), 8.225 (1 H, d, J 8.1, ꢁ-NH L-Lys), 7.948 (1 H, d,
2H5'a Ta signal is hidden under HOD signal. Identification by COSY-spectrum.

Amino-Functionalized Oligonucleotides with Peptide Internucleotide
Letters in Organic Chemistry, 2012, Vol. 9, No. 2
111
4
4J 0.9, H6 cT), 7.549 (1 H, d, J 1.0, H6 Ta), 7.439 – 6.812
8.425 (1 H, d, J 7.8, 3'-NH Ta), 8.224 (1 H, d, J 8.1, ꢃ-NH
L-Lys), 7.955 (1 H, s, H6 cT), 7.556 (1 H, s, H6 Ta), 7.462 –
6.800 (13 H, m, Ar-H), 6.302 – 6.167 (2 H, m, H1' cT, H1'
Ta), 4.562 – 4.482 (1 H, m, H3' Ta), 4.473 (1 H, dt, J3', 2'a 5.2,
(18 H, m, Ar-H), 7.160 (1 H, t, J 5.4, ꢂ-NH L-Lys), 6.296 –
6.175 (2 H, m, H1' cT, H1' Ta), 4.976 (2 H, s, CH2 Bn),
4.595 – 4.483 (1 H, m, H3' Ta), 4.473 (1 H, dt, J3', 2'a 5.1, J3',
2'b, J3', 4' 2.1, H3' cT), 4.302 (1 H, d, J3', 4' 2.0, H4' cT), 4.263 –
4.175 (1 H, m, ꢃ-CH L-Lys), 3.917 – 3.842 (1 H, m, H4' Ta),
3.715 (6 H, s, CH3O DMTr), 3.209 (2 H, br. s, 5'-CH2 Ta),
2.931 (2 H, q, J 5.9, ꢂ-CH2 L-Lys), 2.451 – 2.357 (2 H, m,
H2'a Ta, H2'a cT), 2.178 (1 H, dt, J1', 2'b 6.7, J3', 2'b 6.7, 2J2'a, 2'b
J
3', 2'b, J3', 4' 2.2, H3' cT), 4.299 (1 H, d, J3', 4' 1.6, H4' cT),
4.289 – 4.184 (1 H, m, ꢃ-CH L-Lys), 3.919 – 3.859 (1 H, m,
H4' Ta), 3.729 (6 H, s, CH3O DMTr), 3.215 (2 H, br. s, 5'-
CH2 Ta), 2.931 (2 H, q, J 5.9, ꢂ-CH2 L-Lys), 2.402 – 2.256
(2 H, m, H2'a Ta, H2'a cT), 2.183 (1 H, dt, J1', 2'b 6.4, J3', 2'b
2
2
13.8, H2'b Ta), 2.062 (1 H, ddd, J1', 2'b 5.9, J3', 2'b 2.1, J2'a, 2'b
6.4, J2'a, 2'b 13.1, H2'b Ta), 2.076 (1 H, ddd, J1', 2'b 5.8, J3', 2'b
4
13.5, H2'b cT), 1.754 (3 H, d, J 0.9, 5-CH3 4cT), 1.590 –
2.3, 2J2'a, 2'b 13.4, H2'b cT), 1.760 (3 H, s, 5-CH3 cT), 1.609 –
1.498 (2 H, m, ꢄ-CH2 L-Lys), 1.492 – 1.407 (2 H, m, ꢅ-CH2
L-Lys), 1.455 (3 H, s, 5-CH3 Ta), 1.278 – 1.126 (2 H, m, ꢉ-
CH2 L-Lys), 0.860 (9 H, s, t-BuSi), 0.095 (3 H, s, CH3Si),
0.084 (3 H, s, CH3Si). MS: m/z 1120.0. Calc. 1119.25 [M –
H]ꢆ (ꢊ55ꢋ67F3N7O13Si).
1.465 (2 H, m, ꢄ-CH2 L-Lys), 1.431 (3 H, d, J 1.0, 5-CH3
Ta), 1.407 – 1.311 (2 H, m, ꢅ-CH2 L-Lys), 1.278 – 1.114 (2
H, m, ꢉ-CH2 L-Lys), 0.873 (9 H, s, t-BuSi), 0.092 (3 H, s,
CH3Si), 0.081 (3 H, s, CH3Si). MS: m/z 1158.2. Calc.
1157.37 [M – H]ꢆ (ꢊ61ꢋ74N6O14Si).
N-[(2R)-6-Benzyloxycarboxamido-1-{(5'-O-(4,4'-
dimethoxytrityl)-3'-deoxythymidine-3'-yl)amino}-1-
oxohexan-2-yl]-3'-O-(tert-butyldimethylsilyl)thymidine-
5'-carboxamide (6b) was prepared following the previous
procedure from 284 mg (0.35 mmol) of nucleoside 4b. Yield
N-[(2R)-1-{(5'-O-(4,4'-Dimethoxytrityl)-3'-deoxythy-
midine-3'-yl)amino}-1-oxo-6-trifluoracetamidohexan-2-
yl]-3'-O-(tert-butyldimethylsilyl)thymidine-5'-carboxa-
mide (7b) was prepared following the previous procedure
from 498 mg (0.43 mmol) of dinucleoside 6b. Yield 385 mg
(80%). Rf 0.51 (Solvent system A). 1H NMR: 11.300 (2 H, s,
H3 Ta, H3 cT), 9.350 (1 H, t, J 5.6, ꢂ-NH D-Lys), 8.527 (1
H, d, J 7.3, 3'-NH Ta), 8.356 (1 H, d, J 8.1, ꢃ-NH D-Lys),
1
322 mg (84.4%). Rf 0.30 (Solvent system C). H NMR:
11.354 (2 H, s, H3 Ta, H3 cT), 8.570 (1 H, d, J 7.4, 3'-NH
Ta), 8.412 (1 H, d, J 8.1, ꢃ-NH D-Lys), 8.093 (1 H, d, 4J 1.1,
4
4
4
H6 cT), 7.590 (1 H, d, J 1.1, H6 Ta), 7.473 – 6.880 (18 H,
8.027 (1 H, d, J 1.1, H6 cT), 7.541 (1 H, d, J 1.1, H6 Ta),
7.420 – 6.777 (13 H, m, Ar-H), 6.338 (1 H, dd, J1', 2'a 9.1, J1',
2'b 6.0, H1' cT), 6.245 (1 H, t, J 6.6, H1' Ta), 4.568 – 4.471 (1
H, m, H3' Ta), 4.487 – 4.439 (1 H, m, H3' cT), 4.371 (1 H, d,
J3', 4' 1.1, H4' cT), 4.338 – 4.227 (1 H, m, ꢃ-CH D-Lys),
3.992 – 3.900 (1 H, m, H4' Ta), 3.772 (6 H, s, CH3O DMTr),
m, Ar-H), 7.244 (1 H, t, J 5.9, ꢂ-NH D-Lys), 6.392 (1 H, dd,
J1', 2'a 9.1, J1', 2'b 5.5, H1' cT), 6.297 (1 H, t, J 6.6, H1' Ta),
5.047 (2 H, s, CH2 Bn), 4.618 – 4.543 (1 H, m, H3' Ta),
4.547 – 4.489 (1 H, m, H3' cT), 4.427 (1 H, d, J3', 4' 1.0, H4'
cT), 4.361 – 4.281 (1 H, m, ꢃ-CH D-Lys), 4.013 – 3.954 (1
H, m, H4' Ta), 3.779 (6 H, s, CH3O DMTr), 3.412 – 3.366 (1
H, m, H5'a Ta3), 3.263 (1 H, dd, J4', 5'b 2.2, 2J5'a, 5'b 10.4, H5'b
Ta), 3.032 (2 H, q, J 6.1, ꢂ-CH2 D-Lys), 2.492 – 2.389 (1 H,
m, H2'a Ta), 2.298 – 2.195 (2 H, m, H2'b Ta, H2'a cT), (1 H,
2
3.321 (1 H, dd, J4', 5'a 4.4, J5'a, 5'b 10.9, H5'a Ta), 3.205 (1 H,
2
dd, J4', 5'b 2.4, J5'a, 5'b 10.9, H5'b Ta), 3.159 (2 H, q, J 6.0, ꢂ-
CH2 D-Lys), 2.437 – 2.336 (1 H, m, H2'a Ta), 2.244 – 2.125
(2 H, m, H2'b Ta, H2'a cT), 2.043 (1 H, ddd, J1', 2'b 5.6, J3', 2'b
1.8, 2J2'a, 2'b 13.5, H2'b cT), 1.654 (3 H, d, 4J 1.1, 5-CH3 cT),
2
ddd, J1', 2'b 5.5, J3', 2'b 1.8, J2'a, 2'b 13.5, H2'b cT), 1.710 (3 H,
4
4
d, J 1.1, 5-CH3 cT), 1.702 – 1.588 (2 H, m, ꢄ-CH2 D-Lys),
1.655 – 1.553 (2 H, m, ꢄ-CH2 D-Lys), 1.481 (3 H, d, J 1.1,
4
1.466 (3 H, d, J 1.1, 5-CH3 Ta), 1.500 – 1.456 (2 H, m, ꢅ-
5-CH3 Ta), 1.437 – 1.406 (2 H, m, ꢅ-CH2 D-Lys), 1.319 –
1.194 (2 H, m, ꢉ-CH2 D-Lys), 0.883 (9 H, s, t-BuSi), 0.110
(3 H, s, CH3Si), 0.095 (3 H, s, CH3Si). MS: m/z 1120.2.
Calc. 1119.25 [M – H]ꢆ (ꢊ55ꢋ67F3N7O13Si).
CH2 D-Lys), 1.380 – 1.240 (2 H, m, ꢉ-CH2 D-Lys), 0.936 (9
H, s, t-BuSi), 0.158 (3 H, s, CH3Si), 0.145 (3 H, s, CH3Si).
MS: m/z 1157.8. Calc. 1157.37 [M – H]ꢆ (ꢊ61ꢋ74N6O14Si).
N-[(2S)-1-{(5'-O-(4,4'-Dimethoxytrityl)-3'-deoxythy-
midine-3'-yl)amino}-1-oxo-6-trifluoracetamidohexan-2-
yl]-3'-O-(tert-butyldimethylsilyl)thymidine-5'-carboxa-
mide (7ꢀ). To a stirred solution of dinucleoside 6a (659 mg,
0.56 mmol) in dry methanol (10 mL), ammonium formate
(150 mg, 2.3 mmol) and 10 % Pd/C (66 mg) were added.
The mixture was refluxed for 3 h, then cooled to r.t. and
filtered through Celite. Ethyl trifluoroacetate (203 mL, 1.70
mmol) and TEA (236 mL, 1.70 mmol) were added to a
filtrate and the mixture was stirred for 2 h at 20 °ꢊ.
Methanol was evaporated, the residue was dissolved in ethyl
acetate (50 mL), washed with water (30 mL) and saturated
NaHCO3 (30 mL). The organic layer was dried over Na2SO4,
filtered and evaporated. The residue was purified by silica
gel flesh column chromatography in 3% of ethanol in
CH2Cl2 + 0.1% TEA to give 7a as a hard foam (520 mg,
N-[(2S)-1-{(5'-O-(4,4'-Dimethoxytrityl)-3'-deoxythy-
midine-3'-yl)amino}-1-oxo-6-trifluoracetamidohexan-2-
yl]thymidine-5'-carboxamide (8ꢀ). To a stirred solution of
dinucleoside 7a (570 mg, 0.5 mmol) in dry THF (1 mL), 1M
solution of tetrabutylammonium fluoride in dry THF (1 mL)
was added. The mixture was stirred for 4 h at 20 °ꢊ, diluted
with saturated NaHCO3 (50 mL) and extracted with CHCl3
(3ꢀ40 mL). Combined organic layers were washed with
water, dried over Na2SO4, filtered and concentrated. The
residue was purified by silica gel flesh column
chromatography in 7-9% of ethanol in CH2Cl2 + 0.1% TEA
to give 8a as a hard foam (492 mg, 95.8% yield). Rf = 0.57
1
(Solvent system D). H NMR: 11.329 (1 H, s, H3 cT),
11.315 (1 H, s, H3 Ta), 9.393 (1 H, t, J 5.5, ꢁ-NH L-Lys),
8.4775 (1 H, d, J 7.9, 3'-NH Ta), 8.359 (1 H, d, J 8.0, ꢂ-NH
L-Lys), 8.150 (1 H, s, H6 cT), 7.588 (1 H, s, H6 Ta), 7.466 –
6.879 (13 H, m, Ar-H), 6.363 (1 H, dd, J1', 2'a 8.5, J1', 2'b 6.0,
H1' cT), 6.266 (1 H, t, J 6.3, H1' Ta), 5.610 (1 H, d, J 4.2, 3'-
OH cT), 4.621 – 4.522 (1 H, m, H3' Ta), 4.400 (1 H, s, H4'
cT), 4.371 – 4.312 (1 H, m, H3' cT), 4.303 – 4.218 (1 H, m,
ꢂ-CH L-Lys), 3.962 – 3.887 (1 H, m, H4' Ta), 3.762 (6 H, s,
1
82% yield). Rf = 0.53 (Solvent system A). H NMR: 11.302
(2 H, s, H3 Ta, H3 cT), 9.336 (1 H, t, J 5.6, ꢂ-NH L-Lys),
3 H5'a Ta signal is hidden under HOD signal. Identification by COSY-spectrum.

112 Letters in Organic Chemistry, 2012, Vol. 9, No. 2
Varizhuk et al.
CH3O DMTr), 3.258 (2 H, br. s, 5'-CH2 Ta), 3.159 (2 H, q, J
5.9, ꢁ-CH2 L-Lys), 2.447 – 2.354 (1 H, m, H2'a Ta), 2.234 (1
ABBREVIATIONS
BOP
=
(benzotriazol-1-
yloxy)tris(dimethylamino)phosphonium
hexafluorophosphate
2
H, dt, J1', 2'b 6.3, J3', 2'b 6.3, J2'a, 2'b 12.7, H2'b Ta), 2.179 –
2.098 (2 H, m, H2'a cT, H2'b cT), 1.792 (3 H, s, 5-CH3 cT),
1.669 – 1.517 (2 H, m, ꢃ-CH2 L-Lys), 1.548 – 1.454 (2 H, m,
ꢄ-CH2 L-Lys), 1.481 (3 H, s, 5-CH3 Ta), 1.414 – 1.216 (2 H,
m, ꢉ-CH2 L-Lys). MS: m/z 1005.6. Calc. 1004.99 [M – H]ꢅ
(ꢊ49ꢋ53F3N7O13).
DCC
EDTA
NHS
ON
=
=
=
=
=
=
N,N’-dicyclohexylcarbodiimide
ethylenediaminetetraacetic acid
N-hydroxysuccinimide
oligonucleotide
N-[(2R)-1-{(5'-O-(4,4'-Dimethoxytrityl)-3'-deoxythy-
midine-3'-yl)amino}-1-oxo-6-trifluoracetamidohexan-2-
yl]thymidine-5'-carboxamide (8b) was prepared following
the previous procedure from 420 mg (0.37 mmol) of
dinucleoside 7b. Yield 370 mg (98.1%). Rf 0.56 (Solvent
system D). 1H NMR: 11.301 (2 H, s, H3 cT), 11.273 (2 H, s,
H3 Ta), 9.371 (1 H, t, J 5.5, ꢁ-NH D-Lys), 8.509 (1 H, d, J
7.4, 3'-NH Ta), 8.195 (1 H, d, J 8.0, ꢂ-NH D-Lys), 8.119 (1
TEA
triethylamine
TEAA
triethylammonium acetate
TBAM = tetrabutylammonium fluoride
THF tetrahydrofuran
=
4
4
H, d, J 1.1, H6 cT), 7.537 (1 H, d, J 1.0, H6 Ta), 7.447 –
6.799 (13 H, m, Ar-H), 6.374 (1 H, t, J 7.3, H1' cT), 6.239 (1
H, t, J 6.6, H1' Ta), 5.556 (1 H, d, J 4.1, 3'-OH cT), 4.579 –
4.458 (1 H, m, H3' Ta), 4.370 (1 H, s, H4' cT), 4.335 – 4.289
(1 H, m, H3' cT), 4.271 – 4.185 (1 H, m, ꢂ-CH D-Lys),
3.969 – 3.886 (1 H, m, H4' Ta), 3.718 (6 H, s, CH3O DMTr),
3.351 – 3.274 (1 H, m, H5'a Ta), 3.199 (1 H, dd, J4', 5'b 2.4,
2J5'a, 5'b 10.8, H5'b Ta), 3.172 (2 H, q, J 5.9, ꢁ-CH2 D-Lys),
2.451 – 2.292 (1 H, m, H2'a Ta), 2.246 – 2.114 (1 H, m, H2'b
Ta), 2.106 – 1.993 (2 H, m, H2'a cT, H2'b cT), 1.668 (3 H, d,
4J 1.1, 5-CH3 cT), 1.703 – 1.553 (2 H, m, ꢃ-CH2 D-Lys),
1.544 – 1.412 (2 H, m, ꢄ-CH2 D-Lys), 1.429 (3 H, d, 4J 1.0,
5-CH3 Ta), 1.326 – 1.122 (2 H, m, ꢉ-CH2 D-Lys). MS: m/z
1005.8. Calc. 1004.99 [M – H]ꢅ (ꢊ49ꢋ53F3N7O13).
DISCLOSURES
Part of information included in this chapter has been
previously published in Nucleosides, Nucleotides and
Nucleic Acids Volume 30, Issue 1, 2011.
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ACKNOWLEDGEMENT
The work was supported by a grant from the Russian
Foundation for Basic Research No 11-04-0031-a.

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