Oligonucleotide analogs with peptide internucleotide linkages

Article abstract of DOI:10.1080/15257770.2010.542790

Oligonucleotide analogs containing one or a few glycine, L-, and D-alanine or L-and D-phenylalanine residues instead of phosphodiesterinternucleotide linkages were synthesized. The stability of the duplexes formed by modified oligonucleotides and their wi

Full text of DOI:10.1080/15257770.2010.542790

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Oligonucleotide Analogs with Peptide
Internucleotide Linkages
Anna Varizhuk a , Svetlana Kochetkova a , Natalia Kolganova a
Edward Timofeev a & Vladimir Florentiev a
a Engelhardt Institute of Molecular Biology , Moscow, Russian
Federation
,
Published online: 22 Jan 2011.
To cite this article: Anna Varizhuk , Svetlana Kochetkova , Natalia Kolganova , Edward Timofeev
& Vladimir Florentiev (2011) Oligonucleotide Analogs with Peptide Internucleotide Linkages,
Nucleosides, Nucleotides and Nucleic Acids, 30:1, 31-48, DOI: 10.1080/15257770.2010.542790
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Nucleosides, Nucleotides and Nucleic Acids, 30:31–48, 2011
Copyright ꢀC Taylor and Francis Group, LLC
ISSN: 1525-7770 print / 1532-2335 online
DOI: 10.1080/15257770.2010.542790
OLIGONUCLEOTIDE ANALOGS WITH PEPTIDE INTERNUCLEOTIDE
LINKAGES
Anna Varizhuk, Svetlana Kochetkova, Natalia Kolganova, Edward Timofeev,
and Vladimir Florentiev
Engelhardt Institute of Molecular Biology, Moscow, Russian Federation
Oligonucleotide analogs containing one or a few glycine, L-, and D-alanine or L-and D-
2
phenylalanine residues instead of phosphodiesterinternucleotide linkages were synthesized. The sta-
bility of the duplexes formed by modified oligonucleotides and their wildtype complements was stud-
ied. Oligonucleotides with D-alanine residues in internucleotide linkages form duplexes more stable
than native ones (ꢀTm +0.2◦C per modification), whereas other modifications destabilize the du-
plexes.
Keywords Oligonucleotides; analogs; synthesis; peptide internucleotide linkages; hy-
bridization; thermal stability
INTRODUCTION
Significant progress has been made over the last several decades
in the area of oligonucleotide technology. A great number of modi-
fied oligonucleotides (ONs) meant for selective suppression of gene ex-
pression via antisense or RNA interference mechanism[1–8] have been
synthesized.[9–13] However, only some of them (phosporthioate analogs,
2ꢁ-О-methoxyethyloligoribonucleotides, morfolino oligonucleotides, and
LNA) showed sufficient activity and were brought to clinical trials,[14–17] and
there is still a need to develop new ON analogs that can perform advanced
functions.
We have reported a novel class of modified ONs, carrying amino acid
residues in internucleoside linkages.[18] They are chirally pure, and a modi-
fied fragment can be incorporated into any predetermined position of a nu-
cleic acid chain. The novel ON analogs represent significant interest since
peptide internucleotide linkages can increase power and versatility of nu-
cleic acid receptors, ligands, and catalysts. Moreover, the novel analogs are
Received 6 October 2010; accepted 22 November 2010.
This work was supported by a grant from the Russian Foundation for Basic Research.
Address correspondence to Vladimir Florentiev, Engelhardt Institute of Molecular Biology, Vaviliva
st., 32, Moscow, 119991, Russian Federation. E-mail: flor@imb.ac.ru
31

32
A. Varizhuk et al.
likely to demonstrate improved nuclease resistance and enhanced cellular
uptake, characteristic of DNA-peptide conjugates.[19,20] The latter are ex-
tensively used to circumvent poor pharmacokinetic properties of antisense
olinucleotides and si-RNA.[21, 22] In this article, we describe a full detail of
the general method for facile and efficient synthesis of ONs with glycine,
L- and D-alanine and L- and D-phenylalanine residues in internucleotide
linkages (Scheme 1).
O
O
O
NH
NH
NH
O
O
O
N
O
N
O
N
O
O
O
O
O
N
O
O
N
HN
HN
HN
R
H
R
NH
NH
NH
H
O
O
O
HN
O
O
HN
O
O
HN
O
O
O
N
O
O
O
O
O
R = Me or Bn
SCHEME 1 Fragments of ONs with glycine, L- and D-alanine and D-phenylalanine residues in internu-
cleotide linkages.
RESULTS AND DISCUSSION
For the preparation of the modified ONs, a method for the synthesis of 3ꢁ-
phosphoramidite dinucleosideblocks (7a–7e) was developed (Scheme 2). 3ꢁ-
O-t-(Butyldimethylsilyl)thymidine 5ꢁ-carboxylc acid 1 was prepared following
the modified published procedure.[23] 3ꢁ-O-(t-Butyldimethylsilyl)thymidine
was oxidised by bis(acetoxy)iodobenzene in the presence of a catalytic
amount of 2,2,6,6-tetramethylpiperidine 1-oxyl (TEMPO) in acetonitrile-
water (1:1) mixture (20◦C, 3 hours) to give 1 in a yield of 93%. Acid 1
was coupled with amino acids esters 2a–2e using (benzotriazol-1-yloxy (tris-
(dimethyl amino)-phosphonium hexafluorophasphate) (BOP) as a con-
densing agent in the presence of triethylamine (TEA) in CH2Cl2 (20◦C, 1
hour). Esters 3a–3e were saponified with 0.5 M KOH in an ethanol-water
(3:1) mixture (20◦C, 2 hour). Acid 4a was obtained in a yield of 48%,
while the yields of L- and D-alanine as well as L- and D-phenylalane deriva-
tives 4b–4e were over 80%. This is consistent with the published data on
saponification of C-terminal glycine esters in peptides.[24] Acids 4a–4e were
coupled with 3ꢁ-amino-3ꢁ-deoxy-5ꢁ-O-dimethoxytritylthymidine to yield fully

Oligonucleotideanalogs with Peptide Internucleotidelinkages
33
O
N
O
N
O
N
O
N
O
OH
NH
DMTrO
R
R
O
NH
NH
NH
O
O
O
O
R
O
+
HO
O
O
HN
O
O
HN
O
O
O
O
O
b
NH2
c
a
NH2
2
OTBDMS
OTBDMS
OTBDMS
O
4
1
3
O
N
O
N
NH
NH
DMTrO
NH
N
O
O
DMTrO
DMTrO
O
O
O
O
O
HN
e
d
R
O
N
O
N
NH
HN
HN
O
R
R
NH
HN
O
O
NH
N
O
O
O
HN
O
O
HN
O
O
O
O
N
O
P
OTBDMS
OTBDMS
O
CN
6
5
7
a: R = H; b: R = (S)-CH3; c: R = (R)-CH3; d: R = (S)-Bn; e: R = (R)-Bn
SCHEME 2 Synthesis of the dinucleoside blocks containing glycine (5a), L- and D-alanine (5b, 5c) and
L- and D-phenylalanine (5d, 5e) residues. Reagents and conditions: (a) BOP, TEA, CH2Cl2; (b) KOH,
EtOH/H2O; (c) BOP, TEA, CH2Cl2 for 4a and DCC, NHS, CH2Cl2, 4◦C for 4b-4e; (d) TBAF, THF; (e)
NCCH2CH2OP(NPri2)2, tetrazole, pyridine, CH2Cl2.
protected dinucleoside analogs 5a–5e. The condensation conditions for 4a
(a glycine derivative) were the same as for 2a. In the case of alanine and
phenylalanine derivatives 4b–4e, N ,N -dicyclohexylcarbodiimide (DCC) and
N-hydroxysuccinimide (NHS) were used since this enables the avoidance of
epimerization at the α-carbon atom of an amino acid residue (about 15%
for alanine and about 30% for phenylalanine) observed when using BOP.
The optical purity of 5b–5e was confirmed by 1HNMR spectroscopy. The silyl
protection was removed with 0.5 M tetrabutylammonium fluoride (TBAF)
in tetrahydrofuran (THF) (20◦C, 4 hours). Resulting dinucleosides 6a–6e,
bearing free 3ꢁ-hydroxyl groups, were treated with 2-cyanoethyl-N ,N ,N ꢁ,N ꢁ-
tetraisopropylphosphoramidite in the presence of 1H-tetrazole and pyri-
dine in dichloromethane to give amidites 7a–7e. Dinucleosides 5a–e–7a–e
were characterized by MALDI-TOF mass spectrometry and the structures of
5a–5e and 6a–6e were confirmed by COSY NMR spectroscopy. COSY-spectra
enabled assignment of proton signals to thymidine 5ꢁ-carboxylc acid and
3ꢁ-amino-3ꢁ-deoxythymidine moieties (Figure 1).
Dinucleosideamidite blocks 7a–e were used for the synthesis of mod-
ified ONs on an automated synthesizer using standard phosphoramidite
protocols. Coupling time was increased to 15 minutes for modified phos-
phoramidites. No decrease in coupling efficiency was observed. Step-wise
coupling yields were about 98–99% for both modified and unmodified

34
A. Varizhuk et al.
FIGURE 1 COSY NMR spectrum of compound 6c. cT = 5ꢁcarboxylc acid moiety, Ta = 3ꢁ-amino-3ꢁ-
deoxythymidine moiety.
amidites. The overall yield of the oligonucleotide synthesis was about 80%.
Along with the modified ONs, isosequential and complementary wildtype
ONs were synthesized. The purification of the ONs was carried out by reverse-
phase HPLC. All the ONs were characterized by MALDI mass spectrometry
(Table 1).
Thermal dissociation of the modified duplexes and their wildtype coun-
terparts was measured (Figure 2).
The resulting curves enabled evaluation of the melting temperatures of
the duplexes. The results are summarized in Table 2.
As evident from Table 2, modifications D (L-phenylalanine) and E
(D-phenylalanine) significantly decrease duplex stability for both the ran-
dom sequence (ꢀТm = −1.8◦C and −1.0◦C per modification on an aver-
age, respectively (for each modification average ꢀТm per modification was

35

36
A. Varizhuk et al.
FIGURE 2 Melting curves of the modified and wildtype duplexes. Sequence (5ꢁ→3ꢁ) of a modified
strand: GXTTXTTXTTXG, where X-modified dinucleoside fragments containing glycine (curve 2), L-
alanine (curve 3), D-alanine (curve 4), L-phenylalanine (curve 5), or D-phenylalanine (curve 6) residues
respectively in internucleotide linkages. Curve 1: unmodified duplex.
calculated as a sum of all corresponding ꢀТm values reported in Table 2 di-
vided by the total number of modifications)) and the regular sequence(ꢀТm
= −3.4◦C and −2.6◦C per modification on an average, respectively). Mod-
ifications A (glycine) and B (L-alanine) cause insignificant destabilization.
(ꢀТm = −1.1◦C and −0.9◦C per modification on an average, respectively,
for the random sequence and ꢀТm = −1.9◦C and −2.3◦C per modification
on an average, respectively, for the regular sequence). In contrast to that,
modification C (D-alanine) has small stabilizing effect (ꢀТm = +0.3◦C per
TABLE 2 Melting temperatures of the modified and wildtype duplexes (duplex concentration 5∗10−6
M)
Тm
,
◦С 0.5, (ꢀТm
X = C
,
◦С∗∗
)
Sequence (5ꢁ→3ꢁ)∗
X = A
X = B
51.6
X = D
X = E
TTAACTTCACAТТC∗∗∗
TTAACTTCACAXC
XAACTTCACAXC
50.3 (−1.3) 50.8 (−0.8) 51.6 (0.0)
49.7 (−1.9)
50.0 (−1.6)
50.2 (−1.4)
49.7 (−1.9) 49.9 (−1.7) 52.5 (+0.9) 58.1 (−3.5)
44.3
GTTTTTTTTTTTTTTG∗∗∗
GXTTTTTTTTTTTTG
GTTTTTTXTTTTTTG
GTTTTTTTTTTTTXG
GXTTTTTTTTTTXG
GXTTTTXTTTTXG
GXTTXTTXTTXG
42.1 (−2.2) 42.0 (−2.3) 44.1 (−0.2) 39.7 (−4.6)
42.9 (−1.4) 42.8 (−1.4) 44.6 (+0.3) 41.5 (−2.8)
42.2 (−2.1) 42.1 (−2.2) 44.4 (+0.1) 40.9 (−3.4)
40.3 (−4.0) 39.1 (−5.2) 44.4 (+0.1) 35.9 (−8.4)
38.4 (−5.9) 36.8 (−7.5) 44.8 (+0.5) 34.4 (−9.9)
37.4 (−6.9) 35.3 (−9.0) 45.3 (+1.0) 32.6 (−11.7)
41.4 (−2.9)
42.2 (−2.1)
-
38.9 (−5.4)
36.7 (−7.6)
34.0 (−10.3)
Notes: ∗X = A, B, C, D or E—modified dinucleoside residues containing glycine, L-alanine, D-alanine,
L-phenylalanine, or D- phenylalanine respectively in an internucleotide linkage.
∗∗Tm difference between modified and natural duplexes.
∗∗∗Unmodified oligonucleotide. Corresponding duplex was used as a reference when calculating ꢀТm
.

Oligonucleotideanalogs with Peptide Internucleotidelinkages
37
modification on an average for the random sequence and ꢀТm = + 0.2◦C
per modification on an average for the regular sequence). Apparently, D-
amino acid derivatives tend to form generally more stable duplexes than
L-analogs.
CONCLUSION
In summary, we have described a novel promising class of oligonu-
cleotide analogs and a general strategy for their synthesis. Analogs with
glycine, L-, and D-alanine and L-and D-phenylalanine residues in internu-
cleotide linkages have been synthesized and their ability to form stable du-
plexes with natural complements has been studied. ONs with D-alanine
residues satisfy the stability criterion set for modified ONs. D-amino acid
derivatives seem generally more promising with respect to their hybridization
properties. This may be of great importance for in vivo applications of the
novel ON analogs. Nonnatural D-amino acids can be used to avoid enzymatic
degradation of peptide internucleotide linkages by carboxypeptidases.The
herein presented general approach to DNA modification potentially offers a
wide range of functional group incorporation. The synthetic strategy worked
out for hydrophobic amino acids can be optimized to design ON analogs
with additional functionalities, e.g. lysine-containing analogs. This study is
currently underway. Amino acid side chain functionalities can be used for
attaching various reporter groups.
EXPERIMENTAL
All reagents were commercially available (Fluka, Switzerland) and used
without further purification. 3ꢁ-Deoxy-3ꢁ-azidothymidine was provided by As-
sociation AZT (Russia). All solvents were purchased from Khimmid (Russia).
Dichloromethane was dried by distillation from phosphorus pentaoxide,
pyridine was distilled from calcium hydride, and THF was distilled from
lithium alumohydride prior to use. Flesh column chromatography (CC) was
performed on silica gel Kieselgel 60 (0.040–0.063 mm, Merck, Germany).
TLC was performed on silica gel Kieselgel 60 F254 precoated plates (Merck)
with detection by UV using the following solvent systems (compositions ex-
pressed as v/v): ethanol-methylene chloride 1:65 (A),1:32 (B), 1: 19 (C), 1:9
(D), 1:9 + 0.1% TEA.1HNMR spectra were recorded on a Bruker AMXIII-400
NMR spectrometer (Germany) on solutions in deuterated dimethyl sulfox-
ide (DMSO-d6). Chemical shifts are given in parts per million (ppm). The
coupling constants (J ) are given in Hz. Abbreviations used: cT (thymidine 5ꢁ-
carboxylc acid moiety), Ta(3ꢁ-amino-3ꢁ-deoxythymidine moiety). The signals
were assigned using COSY experiments. The 1HNMR data were processed us-
ingMestReNova version 6.1.1 (Mestrelab Research SL, Spain). MALDI TOF

38
A. Varizhuk et al.
mass spectra were acquired on a compact IV mass spectrometer (Kratos, UK)
using linear flight path.
ONs were synthesized on an ABI 3400 DNA synthesizer (Applied Biosys-
tems, USA) using standard phosphoramidite protocols and purified using
preparative scale reverse-phase HPLC on a 250 mm × 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 (triethylammonium acetate). Detritylated oligonucleotides were fur-
ther purified in 0–25% gradient of CH3CN in TEAA buffer. Melting curves
of the duplexes were recorded on a UV 160-A spectrometer (Shimadzu,
Japan), equipped with a thermostatic system, in 20 mM sodium phosphate
buffer, 100 mM NaCl, 01 mM ethylenediaminetetraacetic acid (EDTA). pH
7.0, concentration of each duplex being 5·10−6M. Samples were denatured
at 95◦C for 5 minutes 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 du-
plex formation were obtained by performing nonlinear regression analysis
using Data Fit version 9.0.059 (Oakdale Engineering, USA). The calculation
method taking into account temperature dependence of UV absorbance of
duplexes and single strands was applied.
3ꢀ-O-(tert-Butyldimethylsilyl)thymidine-5ꢀ-carboxylic acid (1)
3ꢁ-O-(tert-Butyldimethylsilyl)uridine (4.12 g, 11.6 mmol, prepared fol-
lowing the published procedure[25]) was dissolved in acetonitrile (25 mL).
Bis(acetoxy)iodo benzene (BAIB) (9.02 g, 28 mmol), TEMPO (0.5 g, 3.2
mmol) and water (25 ml) were added. The mixture was stirred for 2 hours
at 20◦С and gradually poured into 0.5 N KOH (190 mL). After stirring for
10 minutes at 20◦С the mixture was extracted with CH2Cl2 (3 × 50 mL).
Combined organic layers were washed with water (50 mL). Combined water
layers were acidified carefully under cooling to pH 2 with 2 N HCl. The
lade-down sediment was filtered, washed with water at filter and dried to
1
give compound 1 as white crystals. Yield 4.22 g, 98.2%. HNMR: 13.265 (1
H, s, COOH), 11.307 (1 H, s, H3), 8.004 (1 H, d, 4J 1.1, H6), 6.305 (1 H, dd,
J 1 , 2 a 8.5, J 1 ,2 b 5.9, H1ꢁ), 4.680–4.614 (1 H, m, H3ꢁ), 4.293 (1 H, d, J 3 , 4 1.1,
H4ꢁ), 2.162–2.034 (2 H, m, H2ꢁa, H2ꢁb), 1.786 (3 H, d, 4J 1.1, 5-CH3), 0.892
(9 H, s, t-BuSi), 0.125 (3 H, s, CH3Si), 0.121 (3 H, s, CH3Si).
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
General Procedures
General Procedure for Preparation of Compounds 3a–3e (Amide Formation
with BOP as a Condensing Reagent)
To a stirred solution of amino acid hydrochloride (1 mmol) in dry
CH2Cl2 (10 mL), TEA (0.47 mL, 3.4 mmol), acid 1 (0.41 g, 1.1 mmol)
and BOP (0.53 g, 1.2 mol) were added. The mixture was stirred for 1 hour at
20◦С, diluted with water (50 mL) and extracted with CH2Cl2 (3 × 30 mL).

Oligonucleotideanalogs with Peptide Internucleotidelinkages
39
Combined organic layers were washed with saturated NaHCO3(aqueous)
(2 × 30 mL) and water (30 mL). The organic layer was separated, dried over
Na2SO4, filtered, and concentrated. The residue was purified by silica gel
column chromatography (CC) using the solvent systems specified below to
give 3a–3e as colorless foam.
Methyl (3ꢀ-O-(tert-butyldimethylsilyl)thymidine-5ꢀ-carboxamido)acetate (3a)
CC solvent system: 3% of ethanol in CH2Cl2, yield 67.9%, Rf = 0.56
(Solvent system C).1HNMR: 11.315 (1 H, s, H3 Ta), 8.603 (1 H, t, J 5.9, NH
Gly), 7.861 (1 H, d, 4J 1.2, H6 Ta), 6.328 (1 H, dd, J 1 , 2 a 9.1, J 1 ,2 b 5.5, H1ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
Ta), 4.584–4.527 (1 H, m, H3ꢁ Ta), 4.278 (1 H, d, J 3 , 4 1.4, H4ꢁ Ta), 3.911
(2 H, dd, J 5.9, 1.2, CH2Gly), 3.654 (3 H, s, CH3O Gly), 2.229 (1 H, ddd,
ꢁ
ꢁ
J 1 , 2 a 9.1, J 3 , 2 a 5.1, 2J 2 a, 2 b 13.9, H2ꢁa Ta), 2.078 (1 H, ddd, J 1 , 2 b 5.5, J 3 , 2 b
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
2
4
1.6, J 2 a, 2 b 13.9, H2ꢁb Ta), 1.765 (3 H, d, J 1.0, 5-CH3 Ta), 0.890 (9 H, s,
ꢁ
ꢁ
t-BuSi), 0.119 (3 H, s, CH3Si), 0.107 (3 H, s, J 3.1, CH3Si).
Methyl (2S)-2-(3ꢀ-O-(tert-butyldimethylsilyl)thymidine-
5ꢀ-carboxamido)propanoate (3b)
CC solvent system: 1,5% of ethanol in CH2Cl2, yield 85.6%,Rf = 0.40
(Solvent systemА). 1HNMR: 11.301 (1 H, s, H3 Ta), 8.612 (1 H, d, J 7.3, NH
L-Ala), 7.928 (1 H, d, 4J 0.9, H6 Ta), 6.299 (1 H, dd, J 1 , 2 a 8.9, J 1 ,2 b 5.6, H1ꢁ
Ta), 4.525–4.473 (1 H, m, H3ꢁ Ta), 4.381 (1 H, quintet, J 7.32, CH L-Ala),
ꢁ
ꢁ
ꢁ
ꢁ
4.268 (1 H, d, J 3 , 4 1.0, H4ꢁ Ta), 3.636 (3 H, s, CH3O L-Ala), 2.307 (1 H,
ꢁ
ꢁ
ddd, J 1 , 2 a 8.9, J 3 , 2 a 5.1, 2J 2 a, 2 b 13.7, H2ꢁa Ta), 2.070 (1 H, ddd, J 1 , 2 b 5.6,
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
J 3 , 2 b 1.5, 2J 2 a, 2 b 13.7, H2ꢁb Ta), 1.761 (3 H, d, 4J 0.9, 5-CH3 Ta), 1.329 (3
H, d, J 7.3, CH3 L-Ala), 0.897 (9 H, s, t-BuSi), 0.128 (3 H, s, CH3Si), 0.117
(3 H, s, CH3Si).
ꢁ
ꢁ
ꢁ
ꢁ
Methyl (2R)-2-(3ꢀ-O-(tert-butyldimethylsilyl)thymidine-
5ꢀ-carboxamido)propanoate (3c)
CC solvent system: 1,5% of ethanol in CH2Cl2, yield 86.3%,Rf = 0.40
(Solvent system А). 1HNMR: 11.314 (1 H, s, H3 Ta), 8.612 (1 H, d, J 7.2, NH
D-Ala), 7.872 (1 H, d, 4J 0.9, H6 Ta), 6.302 (1 H, dd, J 1 , 2 a 8.8, J 1 , 2 b 5.6, H1ꢁ
Ta), 4.567–4.503 (1 H, m, H3ꢁ Ta), 4.341 (1 H, quintet, J 7.2, CH D-Ala),
ꢁ
ꢁ
ꢁ
ꢁ
4.263 (1 H, d, J 3 , 4 1.5, H4ꢁ Ta), 3.655 (3 H, s, CH3O D-Ala), 2.339 (1 H,
ꢁ
ꢁ
ddd, J 1 , 2 a 8.8, J 3 , 2 a 5.1, 2J 2 a, 2 b 13.6, H2ꢁa Ta), 2.088 (1 H, ddd, J 1 , 2 b 5.6,
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
J 3 , 2 b 2.0, 2J 2 a, 2 b 13.6, H2ꢁb Ta), 1.768 (3 H, d, 4J 0.9, 5-CH3 Ta), 1.329 (3
H, d, J 7.3, CH3 D-Ala), 0.897 (9 H, s, t-BuSi), 0.128 (3 H, s, CH3Si), 0.117
(3 H, s, CH3Si).
ꢁ
ꢁ
ꢁ
ꢁ
Methyl (2S)-2-(-3ꢀ-O-(tert-butyldimethylsilyl)thymidine-5ꢀ-carboxamido)-3-
phenylpropanoate (3d)
CC solvent system: 3% of ethanol in CH2Cl2, yield 85.2%, Rf = 0.51
(Solvent system A). 1HNMR (DMSO): 11.326 (1 H, s, H3 Ta), 8.593 (1 H, d,

40
A. Varizhuk et al.
J 7.7, NH L-Phe), 7.804 (1 H, d, 4J 1.1, H6 Ta), 7.314–7.149 (5 H, m, ArH),
6.218 (1 H, dd, J 1 , 2 a 9.1, J 1 , 2 b 5.4, H1ꢁ Ta), 4.564(1 H, ddd, J NH,α-H7.7,
ꢁ
ꢁ
ꢁ
ꢁ
J
α-H, β Ha6.0, J α-H, β-Hb8.8, α-H L-Phe), 4.363–4.282 (1 H, m, H3ꢁ Ta), 4.230 (1
H, d, J 3 , 4 1.3, H4ꢁ Ta), 3.655 (3 H, s, CH3O L-Phe), 3.106 (1 H, dd,J α-H, Ha
6.0, 2J Ha, Hb 13.9,β-Ha L-Phe), 3.010 (1 H, dd, J α-H, Hb 8.8, 2J Ha, Hb 13.9, β-Hb
ꢁ
ꢁ
L-Phe), 2.266–2.162 (1 H, m, H2ꢁa Ta), 2.035 (1 H, ddd, J 1 , 2 b 5.4, J 3 , 2 b
ꢁ
ꢁ
ꢁ
ꢁ
2
4
1.5, J 2 a, 2 b13.3, H2ꢁb Ta), 1.745 (3 H, d, J 1.1, 5-CH3 Ta), 0.876 (9 H, s,
ꢁ
ꢁ
t-BuSi), 0.085 (3 H, s, CH3Si), 0.075 (3 H, s, CH3Si).
Methyl (2R)-2-(3ꢀ-O-(tert-butyldimethylsilyl)thymidine-5ꢀ-carboxamido)-3-
phenylpropanoate (3e)
CC solvent system: 3% of ethanol in CH2Cl2, yield 84.7%, Rf = 0.57
(Solvent system A). 1HNMR (DMSO): 11.311 (1 H, s, H3 Ta), 8.672 (1 H, d,
J 8.1, NH D-Phe), 7.897 (1 H, d, 4J 1.1, H6 Ta), 7.310–7.174 (5 H, m, ArH),
6.268 (1 H, dd, J 1 , 2 a 9.3,J 1 , 2 b 5.4, H1ꢁ Ta), 4.641 (1 H, ddd, J NH,α-H 8.1,
J
ꢁ
ꢁ
ꢁ
ꢁ
α-H, β Ha 5.0, J α-H, β-Hb9.7, α-H D-Phe), 4.244 (1 H, br. s, H4ꢁ Ta), 4.227–4.193
(1 H, m, H3ꢁ Ta), 3.660 (3 H, s, CH3O D-Phe), 3.121 (1 H, dd,J α-H, Ha
5.0,2J Ha, Hb13.9, β-Ha D-Phe), 2.947 (1 H, dd, J α-H, Hb 9.7, 2J Ha, Hb13.9, β-Hb
D-Phe), 2.145–2.054 (1 H, m, H2ꢁa Ta), 1.989 (1 H, ddd, J 1 , 2 b 5.4, J 3 , 2 b 1.1,
ꢁ
ꢁ
ꢁ
ꢁ
2
4
J 2 a, 2 b12.7, H2ꢁb Ta), 1.753 (3 H, d, J 1.1, 5-CH3), 0.861 (9 H, s, t-BuSi),
ꢁ
ꢁ
0.058 (3 H, s, CH3Si), 0.041 (3 H, s, CH3Si).
Hydrolysis of Esters 3a–3e (General Procedure for Preparation
of Acids 4a–4e)
To a stirred solution of the methyl ester (2 mmol) in ethanol (6 mL),
2 N KOH (2 mL) was added. The mixture was stirred for 2 hours at 20◦С,
diluted with water (12 mL) and pH was adjusted to neutral with 2 N HCl.
The solution was evaporated to half its original volume and acidified to pH 2
with 2 N HCl. The lade-down sediment was filtered, washed with cold water
at filter and dried to give 3a–3c as white powder or 3d, 3e as colorless oil.
(3ꢀ-O-(tert-Butyldimethylsilyl)thymidine-5ꢀ-carboxamido)acetic acid (4a)
Yield 48%.1HNMR: 12.688 (1 H, s, COOH Gly), 11.313 (1 H, s, H3 Ta),
8.447 (1 H, t, J 5.7, NH Gly), 7.893 (1 H, d, 4J 0.7, H6 Ta), 6.338 (1 H, dd,
J 1 , 2 a 9.1, J 1 , 2 b 5.5, H1ꢁ Ta), 4.602–4.542 (1 H, m, H3ꢁ Ta), 4.274 (1 H, d,
ꢁ
ꢁ
ꢁ
ꢁ
J 3 , 4 0.8, H4ꢁ Ta), 3.852 (1 H, dd, J Ha, NH6.1, J Ha, Hb 17.5, Ha Gly), 3.767
2
ꢁ
ꢁ
2
(1 H, dd, J Hb, NH5.7, J Ha, Hb 17.5, HbGly), 2.259–2.165 (1 H, m, H2ꢁa Ta),
2.069 (1 H, ddd, J 1 , 2 b 4.4, J 3 , 2 b 1.7, 2J 2 a, 2 b 13.1, H2ꢁb Ta), 1.768 (3 H, d,
4J 0.7, 5-CH3 Ta), 0.891 (9 H, s, t-BuSi), 0.120 (3 H, s, CH3Si), 0.111 (3 H, s,
CH3Si).
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ

Oligonucleotideanalogs with Peptide Internucleotidelinkages
41
(2S)-2-(3ꢀ-O-(tert-Butyldimethylsilyl)thymidine-5ꢀ-carboxamido)propanoic
acid (4b)
Yield 81.5%. 1HNMR: 12.659 (1 H, s, COOH L-Ala), 11.299 (1 H, s, H3
Ta), 8.434 (1 H, d, J 7.4, NH L-Ala), 7.958 (1 H, d, 4J 1.0, H6 Ta), 6.295 (1
H, dd, J 1 , 2 a 8.9, J 1 , 2 b 5.6, H1ꢁ Ta), 4.615–4.470 (1 H, m, H3ꢁ Ta), 4.277 (1
ꢁ
ꢁ
ꢁ
ꢁ
H, qintet, J 6.8, CH L-Ala), 4.272 (1 H, d, J 3 , 4 1.1, H4ꢁ Ta), 2.306 (1 H, ddd,
ꢁ
ꢁ
J 1 , 2 a 9.0, J 3 , 2 a 5.1, 2J 2 a, 2 b 13.3, H2ꢁa Ta), 2.069 (1 H, ddd, J 1 , 2 b 5.2, J 3 , 2 b
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
1.2, 2J 2 a, 2 b 13.3, H2ꢁb Ta), 1.766 (3 H, d, 4J 1.0, 5-CH3 Ta), 1.318 (3 H, d, J
7.3, CH3 L-Ala), 0.893 (9 H, s, t-BuSi), 0.123 (3 H, s, CH3Si), 0.112 (3 H, s,
CH3Si).
ꢁ
ꢁ
(2R)-2-(3ꢀ-O-(tert-Butyldimethylsilyl)thymidine-5ꢀ-carboxamido)propanoic
acid (4c)
Yield 87.6%. 1HNMR: 12.737 (1 H, s, COOH D-Ala), 11.308 (1 H, s, H3
Ta), 8.472 (1 H, d, J 7.2, NH D-Ala), 7.911 (1 H, br. s, H6 Ta), 6.316 (1 H,
dd, J 1 , 2 a 8.4, J 1 , 2 b 5.7, H1ꢁ Ta), 4.615–4.470 (1 H, m, H3ꢁ Ta), 4.369–4.141
ꢁ
ꢁ
ꢁ
ꢁ
(2 H, m, H4ꢁ; CH D-Ala), 2.298–2.152 (1 H, m, H2ꢁa Ta), 2.088 (1 H, ddd,
2
J 1 , 2 b 5.2, J 3 , 2 b 1.2, J 2 a, 2 b 12.8, H2ꢁb Ta), 1.768 (3 H, br. s, 5-CH3 Ta),
1.329 (3 H, d, J 7.3, CH3 D-Ala), 0.897 (9 H, s, t-BuSi), 0.128 (3 H, s, CH3Si),
0.117 (3 H, s, CH3Si).
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
(2S)-2-(3ꢀ-O-(tert-Butyldimethylsilyl)thymidine-5ꢀ-carboxamido)propanoic
acid (4d)
A modified procedure was used. After stirring for 2 hours at 20◦С, the
mixture was diluted with water (100 mL) and pH was adjusted to 3 with
2 N HCl. The mixture was extracted with CHCl3 (3 × 30 mL). Combined
organic layers were washed with water (30 mL). The organic layer was dried
over Na2SO4, filtered and evaporated. Yield 94.7%.1HNMR: 12.831 (1 H, s,
COOH L-Phe), 11.334 (1 H, s, H3 Ta), 8.372 (1 H, d, J 8.0, NH L-Phe),
7.739 (1 H, d, 4J 1.2, H6 Ta), 7.310–7.157 (5 H, m, ArHL-Phe), 6.194 (1 H,
dd, J 1 , 2 a9.1, J 1 , 2 b5.5, H1ꢁ Ta), 4.541 (1 H, ddd, J NH,α-H8.0, J α-H, β Ha 5.1,
ꢁ
ꢁ
ꢁ
ꢁ
α-H, β-Hb9.1, α-H L-Phe),4.340–4.275 (1H, m, H3ꢁ Ta), 4.210 (1 H, d, J 3 , 4
ꢁ
ꢁ
J
2
1.4, H4ꢁ Ta), 3.147 (1 H, dd, J α-H, Ha 5.1, J Ha, Hb 13.9, β-Ha L-Phe), 3.010
2
ꢁ
ꢁ
(1 H, dd, J α-H, Hb9.1, J Ha, Hb 13.9, β-Hb L-Phe), 2.193 (1 H, ddd, J 1 , 2 a9.1,
J 3 , 2 a5.4, 2J 2 a, 2 b 13.8, H2ꢁa Ta), 2.098 (1 H, ddd, J 1 , 2 b 5.5, J 3 , 2 b 1.5, 2J 2 a, 2 b
13.8, H2ꢁb Ta), 1.728 (3 H, d, 4J 1.2, 5-CH3 Ta), 0.889 (9 H, s, t-BuSi), 0.079
(3 H, s, CH3Si), 0.071 (3 H, s, CH3Si).
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
(2R)-2-(3ꢀ-O-(tert-Butyldimethylsilyl)thymidine-5ꢀ-carboxamido)propanoic
acid (4e)
The compund was prepared by the same procedure as 4d. Yield
87.6%.1HNMR: 12.903 (1 H, s, COOH D-Phe), 11.299 (1 H, s, H3 Ta),
8.492 (1 H, d, J 8.5, NH D-Phe), 7.928 (1 H, d, 4J 1.0, H6 Ta), 7.336–7.116

42
A. Varizhuk et al.
(5 H, m, ArH D-Phe), 6.278 (1 H, dd, J 1 , 2 a 9.4, J 1 , 2 b 5.3, H1ꢁ Ta), 4.571 (1
H, ddd, J NH,α-H8.5, J α-H, β Ha 5.1, J α-H, β-Hb9.7, α-H L-Phe), 4.251 (1H, br. s,
ꢁ
ꢁ
ꢁ
ꢁ
H4ꢁ Ta), 4.228–4.184 (1 H, m, H3ꢁ Ta), 3.142 (1 H, dd, J α-H, Ha5.1, J Ha, Hb
2
2
13.9, β-Ha D-Phe), 2.917 (1 H, dd, J α-H, Hb 9.7, J Ha, Hb13.9, β-HbD-Phe),
2.138–2.031 (1 H, m, H2ꢁa Ta), 2.019- 1.922 (1 H, m, H2ꢁb Ta), 1.754 (3 H,
4
d, J 1.0, 5-CH3 Ta), 0.863 (9 H, s, t-BuSi), 0.058 (3 H, s, CH3Si), 0.039 (3
H, s, CH3Si).
N-[2-{(5ꢀ-O-(4,4ꢀ-Dimethoxytrityl)-3ꢀ-deoxythymidine-3ꢀ-yl)amino}-2-oxoethyl]-
3ꢀ-O-(tert-butyldimethylsilyl)thymidine-5ꢀ-carboxamide (5a)
The compound was prepared following the general procedure described
for 3a-3e(amide formation with BOP as a condensing reagent) from 3ꢁ-
amino-3ꢁ-deoxy-5ꢁ-O-dimethoxytritylthymidine (1 mmol) and acid 4a (1.1
mmol) using 1.2 mmol of BOP and 2.4 mmol of TEA. CC solvent system: 5%
of ethanol in CH2Cl2 + 0.1% TEA, yield 86.3%, Rf = 0.38 (Solvent system
C).1HNMR: 11.309 (2 H, s, H3 Ta, H3 cT), 8.376 (1 H, d, J 7.4, 3ꢁ-NH Ta),
4
8.293 (1 H, t, J 5.9, NH Gly), 7.937 (1 H, d, J 1.0, H6 cT), 7.544 (1 H, d,
4J 1.0, H6 Ta), 7.423–6.843 (13 H, m, ArHDMTr), 6.343 (1 H, dd, J 1 , 2 a 9.1,
ꢁ
ꢁ
J 1 , 2 b 5.5, H1ꢁ cT), 6.235 (1 H, t, J 1 , 2 a, b 6.6, H1ꢁ Ta), 4.612–4.567 (1 H, m,
ꢁ
ꢁ
ꢁ
ꢁ
H3ꢁ cT), 4.547–4.462 (1 H, m, H3ꢁ, Ta), 4.292 (1 H, d, J 3 , 4 1.1, H4 cT),
ꢁ
ꢁ
ꢁ
2
3.955–3.896 (1 H, m, H4ꢁ Ta), 3.834 (1 H, dd, J Ha, NH5.9, J Ha, Hb 16.5, Ha
2
Gly), 3.729 (6 H, s, CH3O DMTr), 3.692 (1 H, dd, J Hb, NH5.9, J Ha, Hb 16.5,
2
HbGly), 3.317 (1 H, dd, J 4 , 5 a 4.7, J 5 a, 5 b 10.4, H5ꢁa Ta), 3.210 (1 H, dd,
ꢁ
ꢁ
ꢁ
ꢁ
J 4 , 5 b 2.4, 2J 5 a, 5 b 10.4, H5ꢁb Ta), 2.430–2.331 (1 H, m, H2ꢁa Ta), 2.272–2.141
ꢁ
ꢁ
ꢁ
ꢁ
2
(2 H, m, H2ꢁb Ta, H2ꢁa cT), 2.048 (1 H, ddd, J 1 , 2 b 5.5, J 3 , 2 b 1.6, J 2 a, 2 b
12.0, H2ꢁb cT), 1.722 (3 H, d, 4J 1.0, 5-CH3cT), 1.470 (3 H, d, 4J 1.0, 5-CH3
Ta), 0.883 (9 H, s, t-BuSi), 0.108 (3 H, s, CH3Si), 0.098 (3 H, s, CH3Si). MS:
m/z 952.9. Calculated (calc.) 952.12 [M–H]− (С49Н59N6O12Si).
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
General Procedure for Preparation of Dinucleosides 5b–5e
(Condensation with DCC and NHS)
3ꢁ-Amino-3ꢁ-deoxy-5ꢁ-O-dimethoxytritylthymidine (0.41 g, 0.72 mmol)
was dissolved in dry CH2Cl2, acid (0.79 mmol) and NHS (0.09 g, 0.72 mmol)
were added. The mixture was cooled to 0◦C in an ice-water bath with stirring,
and DCC (0.16 g, 0.79 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 × 30 mL). Combined organic layers were washed with
saturated NaHCO3(aqueous) (30 mL) and water (30 mL). The organic layer
was dried over Na2SO4, filtered and evaporated. The residue was purified by
CC using the solvent systems specified below to give 5d–5e as hard foam.

Oligonucleotideanalogs with Peptide Internucleotidelinkages
43
N-[(2S)-1-{(5ꢀ-O-(4,4ꢀ-Dimethoxytrityl)-3ꢀ-deoxythymidine-3ꢀ-yl)amino}-1-
oxopropane-2-yl]-3ꢀ-O-(tert-butyldimethylsilyl)thymidine-5ꢀ-carboxamide
(5b)
CC solvent system: 3% of ethanol in CH2Cl2 + 0.1% TEA, yield 83.3%,
Rf = 0.48 (Solvent system B). (H5ꢁa Ta signal is hidden under the signal of
1
residual HOD. Identification by COSY-spectrum.) HNMR: 11.298 (2 H, s,
H3 Ta, H3 cT), 8.331 (1 H, d, J 7.8, 3ꢁ-NH Ta), 8.256 (1 H, d, J 7.6, NH L-
Ala), 7.952 (1 H, d, 4J 1.0, H6 cT), 7.557 (1 H, d, 4J 1.0, H6 Ta), 7.427–6.837
(13 H, m, ArHDMTr), 6.320 (1 H, dd, J 1 , 2 a 8.7, J 1 , 2 b 5.7, H1ꢁ cT), 6.225
ꢁ
ꢁ
ꢁ
ꢁ
(1 H, t, J 1 , 2 a, b 6.4, ꢁH1ꢁ Ta), 4.612–4.567 (2 H, m, H3ꢁ cT, H3ꢁ Ta), 4.294
ꢁ
ꢁ
ꢁ
ꢁ
(1 H, d, J 3 , 4 1.5, H4 cT), 4.293 (1 H, quintet, J 7.4, CH L-Ala), 3.921–3.862
(1 H, m, H4ꢁ Ta), 3.732 (6 H, s, CH3O DMTr), 3.250–3.190 (2 H, m, H5ꢁa
Ta, H5ꢁb Ta), 2.430–2.331 (2 H, m, H2ꢁa cT, H2ꢁa Ta), 2.223–2.131 (1 H, m,
H2ꢁb Ta), 2.063 (1 H, ddd, J 1 , 2 b 5.7, J 3 , 2 b 2.1, 2J 2 a, 2 b 13.4, H2ꢁb cT), 1.756
(3 H, d, 4J 1.0, 5-CH3cT), 1.464 (3 H, d, 4J 1.0, 5-CH3 Ta), 1.184 (1 H, d, J
7.3, CH3 D-Ala), 0.876 (9 H, s, t-BuSi), 0.103 (3 H, s, CH3Si), 0.093 (3 H, s,
CH3Si). MS: m/z 966.8. Calc. 966.15 [M–H]− (С50Н61N6O12Si).
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
N-[(2R)-1-{(5ꢀ-O-(4,4ꢀ-Dimethoxytrityl)-3ꢀ-deoxythymidine-3ꢀ-yl)amino}-1-
oxopropane-2-yl]-3ꢀ-O-(tert-butyldimethylsilyl)thymidine-5ꢀ-carboxamide (5c)
CC solvent system: 2% of ethanol in CH2Cl2 + 0.1% TEA, yield 87.5%,
Rf = 0.6 (Solvent system B).1HNMR: 11.296 (2 H, s, H3 Ta, H3 cT), 8.476 (1
H, d, J 7.3, 3ꢁ-NH Ta), 8.293 (1 H, d, J 7.5, NH L-Ala), 7.983 (1 H, d, 4J 0.9,
4
H6 cT), 7.537 (1 H, d, J 0.9, H6 Ta), 7.418–6.804 (13 H, m, ArHDMTr),
6.320 (1 H, dd, J 1 , 2 a 8.8, J 1 , 2 b 5.6, H1ꢁ cT), 6.248 (1 H, t, J 1 , 2 a, b 6.7, H1ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
Ta), 4.537–4.448 (2 H, m, H3ꢁ cT, H3ꢁ Ta), 4.311 (1 H, d, J 3 , 4 1.4, H4 cT),
ꢁ
ꢁ
ꢁ
4.273 (1 H, quintet, J 7.1, CH L-Ala), 3.961–3.922 (1 H, m, H4ꢁ Ta), 3.717 (3
ꢁ
ꢁ
H, s, CH3O DMTr), 3.712 (3 H, s, CH3O DMTr), 3.204 (1 H, dd, J 4 , 5 b 2.2,
2
J 5 a, 5 b 10.3, H5ꢁb Ta), 2.420–2.351 (1 H, m, H2ꢁa Ta), 2.224–2.151 (2 H, m,
ꢁ
ꢁ
H2ꢁa cT, H2ꢁb Ta), 2.038 (1 H, ddd, J 1 , 2 b 5.7, J 3 , 2 b 1.8, 2J 2 a, 2 b 13.3, H2ꢁb
cT), 1.656 (3 H, d, 4J 0.9, 5-CH3cT), 1.430 (3 H, d, 4J 0.9, 5-CH3 Ta), 1.237 (1
H, d, J 7.3, CH3 D-Ala), 0.876 (9 H, s, t-BuSi), 0.099 (3 H, s, CH3Si), 0.086 (3
H, s, CH3Si). MS: m/z 990.4. Calc. 990.14 [M + Na]+ (С50Н62N6O12SiNa).
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
N-[(2S)-1-{(5ꢀ-O-(4,4ꢀ-Dimethoxytrityl)-3ꢀ-deoxythymidine-3ꢀ-yl)amino}-1-oxo-3-
phenylpropane-2-yl]-3ꢀ-O-(tert-butyldimethylsilyl)thymidine-5ꢀ-carboxamide
(5d)
CC solvent system: 2% of ethanol in CH2Cl2+ 0.1% TEA, yield 89%, Rf =
0.42 (Solvent system B). 1HNMR:11.332 (1 H, s, H3 cT), 11.299 (1 H, s, H3
Ta), 8.451 (1 H, d, J 7.6, 3ꢁ-NH Ta), 8.319 (1 H, d, J 8.3, NH L-Phe), 7.740
4
(1 H, d, J 1.0, H6 cT), 7.537 (1 H, br. s, H6 Ta), 7.455–6.816 (18 H, m,
ArHDMTr, ArHL-Phe), 6.271–6.125 (2 H, m, H1ꢁ cT, H1ꢁ Ta), 4.620–4.509
(1H, m, α-H L-Phe), 4.498–4.383 (1 H, m, H3ꢁ Ta), 4.348–4.282 (1 H, m, H3ꢁ

44
A. Varizhuk et al.
ꢁ
ꢁ
ꢁ
ꢁ
cT), 4.231 (1 H, d, J 3 , 4 1.7, H4 cT), 3.719 (7 H, br. s, CH3O DMTr, H4 Ta),
3.234–3.154 (2 H, m, H5ꢁa Ta, H5ꢁb Ta), 2.962–2.756 (2 H, m, β-Ha, β-Hb
L-Phe), 2.414–2.278 (1H, m, H2ꢁa Ta), 2.268–2.096 (2H, m, H2ꢁa cT, H2ꢁb
Ta),2.033 (1 H, ddd, J 1 , 2 b5.4, J 3 , 2 b1.7, 2J 2 a, 2 b12.9, H2ꢁb cT), 1.719 (3 H, d,
4J 1.0, 5-CH3cT), 1.463 (3 H, d, 4J 0.7, 5-CH3 Ta), 0.861 (9 H, s, t-BuSi), 0.069
(3 H, s, CH3Si), 0.059 (3 H, s, CH3Si). MS: m/z1068.1. Calculated 1066.23
[M + Na]+ (С56Н66N6O12SiNa+).
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
N-[(2R)-1-{(5ꢀ-O-(4,4ꢀ-Dimethoxytrityl)-3ꢀ-deoxythymidine-3ꢀ-yl)amino}-1-oxo-
3-phenylpropane-2-yl]-3ꢀ-O-(tert-butyldimethylsilyl)thymidine-5ꢀ-carboxamide
(5e)
CC solvent system:% of ethanol in CH2Cl2 + 0.1% TEA, yield 92%, Rf =
0.51 (Solvent system B). 1HNMR:11.291 (2 H, br. s, H3 cT, Ta), 8.549 (1 H, d,
J 7.3, 3ꢁ-NH Ta), 8.462 (1 H, d, J 8.5, NH D-Phe), 8.009 (1 H, d, 4J 1.0, H6 cT),
7.563 (1 H, d, 4J 0.6, H6 Ta), 7.425–6.812 (18 H, m, ArHDMTr, ArH D-Phe),
6.279 (1H, dd, J 1 , 2 a9.2, J 1 , 2 b5.4, H1ꢁ cT), 6.209 (1 H, t, J 1 , 2 a, b 6.6, H1ꢁ Ta),
4.610 (1H, dt, J α-H,NH, J α-H, β-Hb 8.5, J α-H, Ha 5.2, α-H D-Phe), 4.541–4.440 (1
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
H, d, H3ꢁ Ta), 4.303 (1 H, br. s, H4ꢁ cT), 4.149 (1 H, d,J 3 , 2 a3.9, H3ꢁ cT),
ꢁ
ꢁ
3.948–3.885 (1H, m, H4ꢁ Ta), 3.716 (6 H, s, CH3O DMTr), 3.404–3.330
2
(1H, m, H5ꢁa Ta), 3.192(1 H, dd, J 4 , 5 b 2.4, J 5 a, 5 b 10.5, H5ꢁb Ta), 3.013
ꢁ
ꢁ
ꢁ
ꢁ
2
(1H, dd, J α-H, Ha5.2, J Ha, Hb 13.8, β-Ha D-Phe), 2.830 (1 H, dd, J α-H, Hb8.5,
2
J Ha, Hb 13.8, β-Hb D-Phe), 2.341 (1H, dd, J 1 , 2 a6.6, 2J 2 a, 2 b 12.5, H2ꢁa Ta),
ꢁ
ꢁ
ꢁ
ꢁ
2
2.102 (1H, dd, J 1 , 2 b 6.6, J 2 a, 2 b 12.3, H2ꢁb Ta), 2.018 (1H, dd, J 1 , 2 a9.2,
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
J 3 , 2 a3.9,2J 2 a, 2 b 12.5, H2ꢁa cT),1.954 (1H, dd, J 1 , 2 b 5.4, 2J 2 a, 2 b 12.5, H2ꢁb
cT),1.643 (3 H, d, 4J 1.0, 5-CH3cT), 1.441 (3 H, d, 4J 0.6, 5-CH3 Ta), 0.861
(9 H, s, t-BuSi), 0.061 (3 H, s, CH3Si), 0.031 (3 H, s, CH3Si). MS: m/z1067.1.
Calculated 1066.23 [M + Na]+ (С56Н66N6O12SiNa+).
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
General Procedure for Cleavage of 3ꢀ-O-tert-butyldimethylsilyl
Protection (Preparation of Dinucleosides 6a–6e)
To a stirred solution of a dinucleoside (0.73 mmol) in dry THF (1.5 mL),
1 M solution of tetrabutylammonium fluoride in dry THF (1.5 mL) was
added. The mixture was stirred for 4 hours at 20◦С, then TEA (0.2 mL) was
added, and the mixture was partitioned between water (30 mL) and CHCl3
(40 mL). The water layer was extracted with CHCl3 (2 × 40 mL). Combined
organic layers were washed with water, dried over Na2SO4, filtered, and
concentrated. The residue was purified by silica gel column chromatography
(CC) using the solvent systems specified below.
N-[2-{(5ꢀ-O-(4,4ꢀ-Dimethoxytrityl)-3ꢀ-deoxythymidine-3ꢀ-yl)amino}-2-oxoethyl]-
thymidine-5ꢀ-carboxamide (6a)
CC solvent system: 15% of ethanol in CH2Cl2 + 0.1% TEA, yield 86.1%,
Rf = 0.4 (Solvent system D). 1HNMR: 11.231 (1 H, s, H3 cT), 11.208 (1 H, s,

Oligonucleotideanalogs with Peptide Internucleotidelinkages
45
H3 Ta), 8.301 (1 H, d, J 7.4, 3ꢁ-NH Ta), 8.215 (1 H, t, J 5.9, NH Gly), 7.956
(1 H, d, 4J 0.9, H6 cT), 7.464 (1 H, d, 4J 0.9, H6 Ta), 7.364–6.747 (13 H, m,
ArHDMTr), 6.305 (1, dd, J 1 , 2 a 8.6, J 1 , 2 b 6.1, H1ꢁ cT), 6.155 (1 H, t, J 1 , 2 a, b
6.6, H1ꢁ Ta), 5.579 (1 H, d, J 4.2, 3ꢁ-OH cT), 4.479–4.377ꢁ(1 H, m, H3ꢁ Ta),
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
4.368–4.307 (1 H, m, H3ꢁ cT), 4.240 (1 H, d, J 3 , 4 0.8, H4 cT), 3.884–3.816
(1 H, m, H4ꢁ Ta), 3.725 (1 H, dd, J Ha, NH 6.1, 2J Ha, Hb 16.5, Ha Gly), 3.653 (6
H, s, CH3O DMTr), 3.635 (1 H, dd, J Hb, NH 3.1, 2J Ha, Hb 16.5, Hb Gly), 3.232
ꢁ
ꢁ
(1 H, dd, J 4 , 5 a 4.6, 2J 5 a, 5 b 10.3, H5ꢁa Ta), 3.133 (1 H, dd, J 4 , 5 b 2.2, 2J 5 a, 5 b
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
10.3, H5ꢁb Ta), 2.352–2.257 (1 H, m, H2ꢁa Ta), 2.152–2.064 (1 H, m, H2ꢁb
4
Ta), 2.053–1.957 (2 H, m, H2ꢁa cT, H2ꢁb cT), 1.647 (3 H, d, J 0.9, 5-CH3
cT), 1.393 (3 H, d, 4J 0.9, 5-CH3 Ta). MS: m/z 838.8. Calc. 837.86 [M–H]−
(С43Н45N6O12).
N-[(2S)-1-{(5ꢀ-O-(4,4ꢀ-Dimethoxytrityl)-3ꢀ-deoxythymidine-3ꢀ-yl)amino}-1-
oxopropane-2-yl]-thymidine-5ꢀ-carboxamide (6b)
CC solvent system: 10% of ethanol in CH2Cl2 + 0.1% TEA, yield 92%,
Rf = 0.47 (Solvent system C).1HNMR: 11.294 (1 H, s, H3 cT), 11.276 (1 H,
s, H3 Ta), 8.340 (1 H, d, J 7.9, 3ꢁ-NH Ta), 8.304 (1 H, d, J 7.5, NH L-Ala),
8.078 (1 H, d, 4J 1.1, H6 cT), 7.546 (1 H, d, 4J 0.9, H6 Ta), 7.427–6.833 (13
H, m, ArHDMTr), 6.338 (1 H, dd, J 1 , 2 a 8.9, J 1 , 2 b 5.7, H1ꢁ cT), 6.217 (1 H,
ꢁ
ꢁ
ꢁ
ꢁ
t, J 1 , 2 a, b 6.4, H1ꢁ Ta), 5.550 (1 H, d, J 4.2, 3ꢁ-OH cT), 4.548–4.452 (1 H,
m, H3ꢁ Ta), 4.358–4.221 (3 H, m, H3ꢁ cT, H4ꢁ cT, CH L-Ala), 3.904–3.844
(1 H, m, H4ꢁ Ta), 3.734 (6 H, s, CH3O DMTr), 3.242–3.184 (2, Hm, H5ꢁa
Ta, H5ꢁb Ta), 2.410–2.309 (1 H, m, H2ꢁa Ta), 2.222–2.113 (2 H, m, H2ꢁa cT,
ꢁ
ꢁ
H2ꢁb Ta), 2.077 (1 H, ddd, J 1 , 2 b 5.7, J 3 , 2 b 1.8, 2J 2 a, 2 b 12.5, H2ꢁb cT), 1.748
(3 H, d, 4J 1.1, 5-CH3 cT), 1.462 (3 H, d, 4J 0.9, 5-CH3 Ta), 1.175 (1 H, d, J
7.1, CH3 L-Ala). MS: m/z 852.7. Calc. 851.88 [M–H]− (С44Н47N6O12).
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
N-[(2R)-1-{(5ꢀ-O-(4,4ꢀ-Dimethoxytrityl)-3ꢀ-deoxythymidine-3ꢀ-yl)amino}-1-
oxopropane-2-yl]thymidine-5ꢀ-carboxamide (6c)
CC solvent system: 10% of ethanol in CH2Cl2 + 0.1% TEA, yield 94.5%,
Rf = 0.44 (Solvent system C).1HNMR: 11.266 (2 H, s, H3 cT, H3 Ta), 8.454
(1 H, d, J 7.4, 3ꢁ-NH Ta), 8.211 (1 H, d, J 7.4, NH D-Ala), 8.092 (1 H, d, 4J
0.9, H6 cT), 7.534 (1 H, d, 4J 0.9, H6 Ta), 7.422–6.822 (13 H, m, ArHDMTr),
6.373 (1 H, t, J 1 , 2 a, b 7.4, H1ꢁ cT), 6.246 (1 H, t, J 1 , 2 a, b 6.7, H1ꢁ Ta), 5.557
(1 H, d, J 3.4, 3ꢁ-OH cT), 4.540–4.446 (1 H, m, H3ꢁ Ta), 4.363–4.221 (2 H,
m, H3ꢁ cT, H4ꢁ cT), 4.271 (1 H, quintet, J 7.19, CH D-Ala), 3.966–3.910 (1
ꢁ
ꢁ
ꢁ
ꢁ
H, m, H4ꢁ Ta), 3.720 (3 H, s, CH3O DMTr), 3.716 (3 H, s, CH3O DMTr),
2
3.320 (1 H,dd, J 4 , 5 a 4.2, J 5 a, 5 b 10.4, H5ꢁa Ta), 3.206 (1 H, dd, J 4 , 5 b 2.4,
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
2
J 5 a, 5 b 10.4, H5ꢁb Ta), 2.431–2.342 (1 H, m, H2ꢁa Ta), 2.229–2.148 (2 H,
m, H2ꢁb Ta), 2.095–2.040 (2 H, m, H2ꢁa cT, H2ꢁb cT), 1.661 (3 H, d, 4J 0.9,
5-CH3 cT), 1.433 (3 H, d, 4J 0.9, 5-CH3 Ta), 1.236 (1 H, d, J 7.1, CH3 D-Ala).
MS: m/z 876.2. Calc. 875.87 [M + Na]+ (С44Н48N6O12SiNa).
ꢁ
ꢁ

46
A. Varizhuk et al.
N-[(2S)-1-{(5ꢀ-O-(4,4ꢀ-Dimethoxytrityl)-3ꢀ-deoxythymidine-3ꢀ-yl)amino}-1-oxo-3-
phenylpropane-2-yl]-thymidine-5ꢀ-carboxamide (6d)
CC solvent system: 7% of ethanol in CH2Cl2 + 0.1% TEA, yield 89.1%,
1
Rf = 0.59 (Solvent system D). HNMR: CC solvent system:% of ethanol in
1
CH2Cl2 + 0.1% TEA, yield 92%, Rf = 0.51 (Solvent system B). HNMR:
11.343 (1 H, s, H3 cT), 11.320 (1 H, s, H3 Ta), 8.510 (1 H, d, J 7.8, 3ꢁ-NH
Ta), 8.443 (1 H, d, J 8.2, NH L-Phe), 7.948 (1 H, d, 4J 0.9, H6 cT), 7.583 (1
H, br. s, H6 Ta), 7.480–6.835 (18 H, m, ArHDMTr, ArHL-Phe), 6.302 (1H,
t, J 1 , 2 a,b7.8, H1ꢁ cT), 6.241 (1 H, t, J 1 , 2 a, b 6.5, H1ꢁ Ta), 5.456 (1H, d, J 4.3,
3ꢁ-OH cT), 4.609–4.452 (2H, m, α-H D-Phe, H3ꢁ Ta), 4.322 (1 H, br. s, H4ꢁ
cT), 4.259 (1H, br. s, H3ꢁ cT), 3.826–3.701 (7H, m, H4ꢁ Ta, CH3O DMTr),
3.240 (2H, br. s, H5ꢁCH2 Ta), 2.931–2.775 (2H, m, β-Ha, β-Hb D-Phe), 2.393
ꢁ
ꢁ
ꢁ
ꢁ
(1H, ddd,J 1 , 2 a, 6.5,J 3 , 2 a, 7.0,2J 2 a, 2 b12.9, H2ꢁa Ta), 2.204 (1H, dt, J 1 , 2 b
,
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
2
J 3 , 2 b 6.5, J 2 a, 2 b12.9, H2ꢁb Ta), 2.139–2.056(2H, m, H2ꢁa, bcT), 1.753 (3
H, br. s, 5-CH3cT), 1.510 (3 H, br.s, 5-CH3 Ta). MS: m/z 952.3. Calculated
952.8 [M + Na]+ (С50Н52N6O12Na+).
ꢁ
ꢁ
ꢁ
ꢁ
N-[(2R)-1-{(5ꢀ-O-(4,4ꢀ-Dimethoxytrityl)-3ꢀ-deoxythymidine-3ꢀ-yl)amino}-1-oxo-
3-phenylpropane-2-yl]thymidine-5ꢀ-carboxamide (6e)
CC solvent system: 7% of ethanol in CH2Cl2 + 0.1% TEA, yield 96.3%,
Rf = 0.57 (Solvent system D). 1HNMR:11.314 (1 H, s, H3 cT), 11.259 (1 H,
s, H3 Ta), 8.499 (1 H, d, J 7.4, 3ꢁ-NH Ta), 8.281 (1 H, d, J 8.4, NH D-Phe),
4
4
8.039 (1 H, d, J 1.0, H6 cT), 7.503 (1 H, d, J 0.7, H6 Ta), 7.428–6.764
(18 H, m, ArHDMTr, ArH D-Phe), 6.312 (1H, dd, J 1 , 2 a 5.4, J 1 , 2 b 9.4, H1ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
cT), 6.201 (1 H, t, J 1 , 2 a, b 6.6, H1ꢁ Ta), 5.456 (1H, d, J 4.1, 3ꢁ-OH cT), 4.573
(1H, dt, J α-H,NH, J α-H, β-Hb8.5, J α-H, Ha 5.7, α-H D-Phe), 4.502–4.405 (1 H, m,
ꢁ
ꢁ
H3ꢁ Ta), 4.310 (1 H, br. s, H4ꢁ cT), 4.029 (1H, t, J 3 , 3 -OH, J 3 , 2 b 4.1, H3ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
cT), 3.956–3.869 (1H, m, H4ꢁ Ta), 3.712 (6 H, s, CH3O DMTr), 3.371–3.295
(1H, m, H5ꢁa Ta), 3.201 (1 H, dd, J 4 ,5 b2.3, J 5 a, 5 b 10.5, H5ꢁb Ta), 3.003
ꢁ
ꢁ
ꢁ
ꢁ
(1H, dd, J α-H, Ha 5.7, J Ha, Hb 13.7, β-Ha D-Phe), 2.830 (1 H, dd, J α-H, Hb8.5,
2
J Ha, Hb 13.7, β-HbD-Phe), 2.321 (1H, dtJ 1 , 2 a, J 3 , 2 a 6.6, J 2 a, 2 b13.3, H2ꢁa
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
Ta), 2.080 (1H,ddd,J 1 , 2 b6.6, J 3 , 2 b 6.3, J 2 a, 2 b13.3,H2ꢁb Ta), 1.980 (1H,
2
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
dd, J 1 , 2 a 5.4, J 2 a, 2 b 12.9, H2ꢁa cT), 1.880 (1H, ddd, J 1 , 2 b9.4, J 3 , 2 b4.1,
2
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
J 2 a, 2 b12.9, H2ꢁbcT), 1.663 (3 H, d, 4J 1.0, 5-CH3cT), 1.461 (3 H, d, 4J 0.7,
2
ꢁ
ꢁ
5-CH3 Ta). MS: m/z 952.3. Calculated 951.97 [M + Na]+ (С50Н52N6O12Na+).
General Procedure for Preparation of Amidites 7a–7e
To a stirred solution of a dinucleoside (0.8 mmol) in dry CH2Cl2 (3 mL),
pyridine (0.19 mL, 2.4 mmol) and tetrazole (0.11 g, 1.6 mmol) were added,
and the reaction mixture was stirred for 30 minutes at 20◦С. Then 2-
cyanoethyl-N,N,Nꢁ,Nꢁ-tetraisopropylphosphoramidite in CH2Cl2 (2 mL) was
added, and the mixture was stirred for 2 hours at 20◦С. TEA (0.1 mL) was

Oligonucleotideanalogs with Peptide Internucleotidelinkages
47
added, the mixture was partitioned between water (20 mL) and CH2Cl2
(30 mL). The water layer was extracted with CH2Cl2 (2 mL). Combined
organic layers were washed with water (30 mL), saturated NaCl (aqueous)
(30 mL). The organic layer was dried over Na2SO4, filtered and concen-
trated. The residue was purified by CC using the solvent systems specified
below.
N-[2-{(5ꢀ-O-(4,4ꢀ-Dimethoxytrityl)-3ꢀ-deoxythymidine-3ꢀ-yl)amino}-2-oxoethyl]-
3ꢀ-deoxy-3ꢀ-[(2-cyanoethoxy)(diisopropylamino)phosphinooxy]thymidine-5ꢀ-
carboxamide (7a)
CC solvent system: 5% of ethanol in CH2Cl2 + 0.1% TEA, yield 76%, Rf =
0.49 (Solvent system D).31P NMR: 150.79, 150.24.MS: m/z 1039.0. Calculated
1038.08 [M–H]− (С52Н62N8O13P).
N-[(2S)-1-{(5ꢀ-O-(4,4ꢀ-Dimethoxytrityl)-3ꢀ-deoxythymidine-3ꢀ-yl)amino}-1-
oxopropane-2-yl]- 3ꢀ-deoxy-3ꢀ-[(2-cyanoethoxy)(diisopropylamino)
phosphinooxy]thymidine-5ꢀ-carboxamide (7b)
CC solvent system: 4% of ethanol in CH2Cl2 + 0.1% TEA, yield 73%, Rf =
0.54 (Solvent system C). 31P NMR: 150.53, 150.20.MS: m/z 1053.2. Calculated
1052.10 [M–H]− (С53Н64N8O13P).
N-[(2R)-1-{(5ꢀ-O-(4,4ꢀ-Dimethoxytrityl)-3ꢀ-deoxythymidine-3ꢀ-yl)amino}-1-
oxopropane-2-yl]-3ꢀ-deoxy-3ꢀ-[(2-cyanoethoxy)(diisopropylamino)
phosphinooxy]thymidine-5ꢀ-carboxamide (7c)
CC solvent system: 4% of ethanol in CH2Cl2 + 0.1% TEA, yield 76%,
Rf = 0.56 (Solvent system C). 31P NMR: 150.51, 150.26. MS: m/z 1052.7.
Calculated 1052.10 [M–H]− (С53Н64N8O13P).
N-[(2S)-1-{(5ꢀ-O-(4,4ꢀ-Dimethoxytrityl)-3ꢀ-deoxythymidine-3ꢀ-yl)amino}-1-oxo-3-
phenylpropane-2-yl]-3ꢀ-deoxy-3ꢀ-[(2-cyanoethoxy)(diisopropylamino)
phosphinooxy]thymidine-5ꢀ-carboxamide (7d)
CC solvent system: 2% of ethanol in CH2Cl2 + 0.1% TEA, yield 76%, Rf =
0.79 (Solvent system B).31P NMR: 150.47, 150.25. MS: m/z 1152.7. Calculated
1152.19 [M–Na]+ (С59Н69N8O13PNa+).
N-[(2R)-1-{(5ꢀ-O-(4,4ꢀ-Dimetoxytrityl)-3ꢀ-deoxythymidine-3ꢀ-yl)amino}-1-oxo-3-
phenylpropane-2-yl]-3ꢀ-deoxy -3ꢀ-[(3ꢀ-deoxy-3ꢀ-[(2-cyanoethoxy)
(diisopropylamino)phosphinooxy]thymidine-5ꢀ-carboxamide (7e)
CC solvent system: 2% of ethanol in CH2Cl2 + 0.1% TEA, yield 77.2%,
Rf = 0.78 (Solvent system B).31P NMR: 151.21, 150.56. Mass spectrum: m/z
1128.7. Calculated 1128.20 [M+ H]− (С59Н69N8O13P).

48
A. Varizhuk et al.
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