Phosphoramidite reagents and solid-phase supports based on hydroxyprolinol for the synthesis of modified oligonucleotides

Article abstract of DOI:10.1134/S1068162017040148

The synthesis of phosphoramidite reagents and solid-phase supports based on hydroxyprolinol for the introduction of the residues of biotin, lipoic acid, amino groups, and terminal acetylene groups at different positions of the oligonucleotide chain has been described. The efficiency of the reagents and supports has been confirmed by the synthesis of the corresponding modified oligonucleotides.

Full text of DOI:10.1134/S1068162017040148

ISSN 1068-1620, Russian Journal of Bioorganic Chemistry, 2017, Vol. 43, No. 4, pp. 386–396. © Pleiades Publishing, Ltd., 2017.
Original Russian Text © M.Yu. Tatulchenkov, I.A. Prokhorenko, M.V. Kvach, M.E. Navakouski, I.A. Stepanova, N.V. Pilchenko, S.V. Gontarev, O.L. Sharko, V.A. Korshun,
V.V. Shmanai, 2017, published in Bioorganicheskaya Khimiya, 2017, Vol. 43, No. 4, pp. 377–387.
Phosphoramidite Reagents and Solid-Phase Supports Based
on Hydroxyprolinol for the Synthesis of Modified Oligonucleotides
M. Yu. Tatulchenkova, I. A. Prokhorenkob, c, M. V. Kvacha, M. E. Navakouskid, I. A. Stepanovab,
N. V. Pilchenkoa, d, S. V. Gontarevd, O. L. Sharkoa, V. A. Korshunb, c, and V. V. Shmanaia, 1
aInstitute of Physical Organic Chemistry, National Academy of Sciences of Belarus, Minsk, 220072 Belarus
bShemyakin–Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow, 117997 Russia
cGauze Institute of New Antibiotics, Moscow, 119021 Russia
dALC Primetech, Minsk, 220072 Belarus
Received November 8, 2016; in final form, November 16, 2016
Abstract⎯The synthesis of phosphoramidite reagents and solid-phase supports based on hydroxyprolinol for
the introduction of the residues of biotin, lipoic acid, amino groups, and terminal acetylene groups at differ-
ent positions of the oligonucleotide chain has been described. The efficiency of the reagents and supports has
been confirmed by the synthesis of the corresponding modified oligonucleotides.
Keywords: modified oligonucleotides, hydroxyprolinol, phosphoramidite reagents, oligonucleotide synthesis
DOI: 10.1134/S1068162017040148
ing, subsequent treatment, and purification of oligo-
nucleotides.
INTRODUCTION
Modified oligonucleotides containing various
reporter groups have found wide application in molec-
ular biology as a convenient tool for the specific detec-
tion of sequences, in DNA sequencing, and in other
modern molecular biology methods [1—3]. Biotin [4],
sulfur-containing (thiol and disulfide) functional groups
possessing the affinity for the surface of noble metals and
fluorescent nanocrystals (quantum dots) [5], terminal
alkyne for the bioconjugation through the [3 + 2] cyc-
loaddition of azides and alkynes [6], as well as the amino
group for the conjugation through carbodiimide con-
densation or the condensation with the use of acti-
vated esters [7], are often applied as modifications.
The introduction of a modification into the oligo-
nucleotide chain immediately during the automated
solid-phase oligonucleotide synthesis makes it possi-
ble to avoid additional condensation stages, attain
regioselectivity, and prevent side reactions. Here, we
describe the synthesis of new phosphoramidite
reagents and the preparation of the corresponding
solid-phase supports that enable one to perform this
functionalization in the automated regime. The appli-
cation of these reagents does not require changes in
the standard protocols of synthesis, as well as deblock-
RESULTS AND DISCUSSION
We chose (2S,4R)-4-hydroxyprolinol, a derivative
of the natural amino acid (2S,4R)-4-hydroxyproline,
as a structural basis for the preparation of modifying
reagents and supports (Fig. 1). Hydroxyprolinol has a
structural and spatial similarity to the deoxyribose res-
idue, which is a key structural component of DNA
chains. It contains a secondary amino group conve-
nient for the attachment of a target modification, as
well as the primary and secondary hydroxyl groups,
which can be functionalized similarly to the hydroxyl
groups of natural nucleosides with the conversion to
solid-phase supports and phosphoramidite reagents
for the single and multiple introduction of modifica-
tions at the 3'- and 5'-terminal and internucleotide
positions of an oligonucleotide during the automated
solid-phase synthesis. Hydroxyprolinol has been used
earlier in the synthesis of modifying reagents for the
introduction of fluorescent labels and functional
groups into oligonucleotides [8–11]. In the present
work, based on hydroxyprolinol, we improved the
method for obtaining a universal precursor reagent
and extended the range of functional groups intro-
duced with the use of this compound.
1
Corresponding author: phone/fax: +375 (17) 292-0-373; e-mail:
shmanai@ifoch.bas-net.by.
Abbreviations: CPG, controlled pore glass; DIPС, diisopropylcar-
bodiimide; DIPEA, diisopropylethylamine; DMAP, 4-dime-
thylaminopyridine; DMTr, dimethoxytrityl; PyBOP, benzotriazol-
1-yl-oxytripyrrolidinophosphonium hexafluorophosphate; minR,
minor rotamer; majR, major rotamer; dst, diastereomer.
(2S,4R)-4-Hydroxyprolinol (II) was obtained as a
hydrochloride from the readily available (2S,4R)-4-
hydroxyproline methyl ester (I) (Scheme 1). It is
known that esters are capable of being reduced with
386

L-HYDROXYPROLINOL-BASED REAGENTS AND SOLID-PHASE SUPPORTS
387
O
H
N
H
N
O
OH
HO
HO
HO
OH
Hydroxyproline
OH
Hydroxyprolinol
HO
Deoxyribose
Fig. 1. Hydroxyprolinol, a structural basis of modifying reagents, and its similarity to deoxyribose.
sodium borohydride in alcoholic medium, first to dioxane as the main solvent followed by the addition
aldehyde, which is quickly converted under reaction of methanol, which has not been described earlier.
conditions to alcohol [12]. Based on this method, we This made it possible to carry out the treatment of the
successfully performed the reduction of methyl ester reaction medium and isolation of the product without
hydrochloride (I) with sodium borohydride using using ion-exchange resins.
H
N
HO
H
N
MeO2C
H2N
i
·
·
HCl
HCl
OH
OH
(I)
(II)
O
O
H
N
CO2H
N
ii
F3C
O
O
(III)
O
(IV)
iii
CO2PFP
VI
CO2H
(V)
(
)
HO2C
PFPO2C
S
S
iii
HN
HN
(VII)
NH
NH
(VIII)
CO2H
O
O
NH2
iv
O
NH
CPG
(IX)
(X)
Scheme 1. Preparation of basic synthetic blocks for the assembly of target reagents. i: NaBH4, MeOH, dioxane, 70°C;
ii: trifluoroacetic anhydride, N-hydroxysuccinimide, CH2Cl2, 25°С; iii: pentafluorophenol, dicyclohexylcarbodiimide,
CH2Cl2, 0°C; iv: succinic anhydride, DMAP, pyridine, 25°С.
To provide the steric availability of functional tic anhydride in the presence of N-hydroxysuccin-
imide. Performing the reaction in methylene chlo-
ride substantially facilitated the treatment of the
reaction mixture and the isolation of the product in
comparison with the previously described protocol
[13] according to which the reaction was carried out
in DMF.
groups being introduced during the conjugation
with biomolecules and inorganic materials, it is
necessary to insert a spacer between the hydroxy-
prolinol skeleton and the functional group. For this
purpose, we chose the readily available 6-amino-
hexanoic acid (III), which was converted to acti-
vated ester (IV) with the simultaneous protection of
For the introduction of functional modifications,
the amino group by the treatment with trifluoroace- pentafluorophenyl esters of 5-hexynoic acid (VI) and
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388
TATULCHENKOV et al.
biotin (VIII) were synthesized. CPG porous glass cov-
The reaction of hydroxyprolinol (II) with activated
ered with amino groups (IX) was treated with succinic esters (IV) and (VI) led to amides (XI) and (XVI) from
anhydride to obtain the carboxylated support (X) which a series of precursors of modifying reagents
(Scheme 1).
were further synthesized (Scheme 2).
R2
R1O
i, (IV)
N
OH
(XI): R1 = H, R2 =
O
O
O
N
H
CF3
CF3
ii
O
N
H
(XII): R1 = DMTr, R2 =
iii
O
·
NH2
(XIII): R1 = DMTr, R2 =
HCl
HO
H
N
O
iv
O
O
(XIV): R1 = DMTr, R2 =
(XV): R1 = DMTr, R2 =
OH
N
H
(II)
v
S
S
O
N
H
S
HN
O
O
RO
NH
i, (VI)
N
OH
(XVI): R = H
ii
(XVII): R = DMTr
Scheme 2. Synthesis of precursor reagents based on hydroxyprolinol. i: Et3N/pyridine, 25°С; ii: DMTr-Cl/pyridine,
25°С; iii: K2CO3, MeOH–H2O (4 : 1), 25°С; iv: lipoic acid, PyBOP, CH2Cl2, 25°С; v: (VIII), pyridine, 25°С.
Owing to a high selectivity of 4,4′-dimethoxytrityl- polymerize on drying [14]. As a condensing agent, we
chloride (DMTr-Cl) in pyridine towards primary
alcohols, it is the primary hydroxyl that is predomi-
nantly tritylated in the presence of the equimolar
amount of the reagent in diols (XI) and (XVI), result-
ing in the formation of products (XII) and (XVII).
used PyBOP.
From compounds (XII), (XIV), (XV), and (XVII),
solid-phase supports and phosphoramidite reagents
(XVIII)–(XXV) (Scheme 3) were prepared, which
were then used in the automated DNA synthesis to
obtain 5′- and 3′-modified oligodeoxyribonucleotides
and sequences carrying an internal single and double
modifications. The time of the condensation of mod-
ifying reagents was increased from 20 s to 5 min. Typ-
ical HPLC profiles of 5′-modified oligonucleotides
After the protection of the primary hydroxyl, the
trifluoroacetyl N-protecting group was removed in an
alkaline medium. The resulting amine (XIII) was acy-
lated with lipoic acid or the activated ester of biotin to
form amides (XIV) and (XV). The lipoic acid residue
was introduced by direct condensation with the amino
group of compound (XIII), since its derivatives tend to carrying one modification are shown in Fig. 2.
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L-HYDROXYPROLINOL-BASED REAGENTS AND SOLID-PHASE SUPPORTS
389
O
O
51 µmol/g
CF3
N
H
(XVIII): R =
(XIX): R =
O
R
DMTrO
64 µmol/g
58 µmol/g
i
N
O
O
N
H
(XX): R =
S
S
O
O
O
O
N
S
(XXI): R =
H
54 µmol/g
NH
O
R
HN
DMTrO
NH
CF3
O
N
CPG
O
O
OH
N
(XXII): R =
H
(XII), (XIV),
(XV), (XVII)
O
[C CH]
[S−S]
(XXIII): R =
(XXIV): R =
(XXV): R =
R
DMTrO
O
N
O
ii or iii
N
H
S
S
O
O
O
O P
NC
N
[Biotin]
S
H
N
HN
O
NH
NH2
O
[NH2]
R =
Scheme 3. Synthesis of target phosphoramidite reagents and supports. i: (Х), DIPC, DMAP, pyridine–DMF, 25°С; ii:
N,N-diisopropylamino-2-cyanoethoxychlorophosphine, acetonitrile or CH2Cl2, 0°С; iii: bis(N,N-diisopropylamino-2-
cyanoethoxy)phosphine, CH2Cl2, 25°С. For solid-phase supports, the capacity is given to the right; for reagents, a condi-
tional designation of modifications is given, which is used in the table to indicate modifications being introduced into oli-
gonucleotides.
The efficiency of condensation of the phosphora-
midite reagents, calculated from the ratio of the area of
the peak of a modified reagent to the area of the peak
EXPERIMENTAL
The following reagents were used: РуВОР, suc-
cinic anhydride, (2S,4R)-4-hydroxyproline (Aldrich);
of an unmodified precursor, was 97% for the modify- 6-aminohexanoic
acid,
N-hydroxysuccinimide
(Fluka); lipoic acid (reagent purity grade), 4,4′-dime-
thoxytritylchloride (BASF), (N,N-diisopropyl-
ing phosphoramidite containing the amino group
(XXII) (amine) and more than 99% for reagents
(XXIII)–(XXV). However, due to the formation of
side products during the oligonucleotide synthesis, the
content of target oligonucleotides after the deblocking
decreased to 82% upon modification by lipoic acid; to
99% upon modification by biotin; to 90% by modifi-
cation with the aliphatic amino group; and to 96%
upon the modification with a terminal acetylene
group. The conjugates synthesized were purified by
amino)-(2-cyanoethoxy)chlorophosphine, bis(N,N-
diisopropylamino)-(2-cyanoethoxy)phosphine (ALC
Primetech, Belarus); aminopropyl-CPG (mean pore
size 500 Å, content of amino groups 78 μmol/g; Amer-
sham). Prior to experiments, solvents were purified by
distillation and, if necessary, absolutized by standard
methods.
The course of the reaction was monitored by TLC
on Kieselgel 60 F254 plates (Merck); for column chro-
HPLC and characterized by MALDI-TOF mass spec- matography, silica gel Kieselgel 60 (Merck) with a par-
trometry (Table 1).
ticle size of 40—63 μm was used. Solutions were evap-
orated on a rotary evaporator in the vacuum of a water-
jet pump at a bath temperature of 30—50°C. IR spec-
tra (ν, cm–1, KBr tablets) were recorded on a Protégé
460 spectrometer (Nicolet). NMR spectra (δ, ppm; J,
Hz) were recorded at 500 MHz (1H), 125.7 MHz
(13C), and 202.4 MHz (31P) on an AVANCE-500 spec-
Thus, we synthesized phosphoramidite reagents
and solid-phase supports that make it possible to
introduce single and multiple modifications at the 5′-
and 3′-terminal positions and inside the oligonucle-
otide chain.
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TATULCHENKOV et al.
T-10 alkyne
T-10 biotin
T-10 lipoic acid
T-10 NH2
T10
2
4
6
8
10
12
14
0
Retention time, min
Fig. 2. Typical HPLC profiles of unpurified 5′-modified oligonucleotides.
trometer (Bruker-Biospin). NMR spectra were HOCHaHb), 3.55 (dd, 1H, J1 12.4, J2 7.0, NHCH),
recorded in CDCl3, unless otherwise indicated. Spec-
tra were calibrated against residual signals of the pro-
tons of the solvent; chemical shifts are given relative to
SiMe4 (1H and 13C) and 85% aqueous H3PO4 (31P).
Melting temperatures were determined on a Boetius
heating table (not corrected). Oligonucleotides were
synthesized on a BiosSet ASM-800 device. MALDI-
TOF mass spectra were recorded on a Bruker Ultra-
flex spectrometer.
3.28 (dd, 1H, J1 12.7, J2 3.4, NHCHaHb), 3.16 (d, 1H,
J 12.6, NHCHaHb), 1.97 (dd, 1H, J1 14.1, J2 6.6,
CHCHaHbCH), 1.82–1.74 (m, 1H, CHCHaHbCH);
13C NMR (D2O): 70.07 (CHOH), 60.44 (CH2OH),
60.29 (NHCH), 53.19 (NHCH2), 35.21 (CHCH2CH);
IR: 3407.0, 3332.4, 3092.1, 2958.4, 1392.8, 1315.2,
1050.1 , 976.8.
N-Oxysuccinimide ester of 6-N-trifluoroacetylami-
nohexanoic acid (IV). N-hydroxysuccinimide (52.7 g,
0.46 mol) was added to a suspension of 6-aminohexa-
noic acid (50.0 g, 0.38 mol) in methylene chloride
(800 mL), and the mixture was cooled to 0°С. Pyri-
dine (70 mL) was added under stirring, and trifluoro-
acetic anhydride (132.6 mL, 0.70 mol) was added
dropwise for 2 h. The reaction mixture was heated to
room temperature and stirred for 30 min after which it
was washed with water (4 × 400 mL), a saturated
NaHCO3 solution (400 mL), and a saturated NaCl
solution (400 mL). The organic phase was dried over
anhydrous sodium sulfate and evaporated on a rotary
evaporator to dryness to yield 95.0 g (77%) of a white
crystalline substance; Rf 0.43 (acetone–CH2Cl2, 1 :
(2S,4R)-4-Hydroxyprolinol hydrochloride (II).
Sodium borohydride (57 g, 1.50 mol) was added to a
suspension of hydrochloride of (2S,4R)-4-hydroxy-
proline methyl ester (I) (55 g, 0.30 mol) in dioxane
(600 mL), the mixture was heated to boiling, and
methanol (200 mL) was added dropwise. The reaction
mixture was allowed to stand overnight at room tem-
perature under stirring. Then, concentrated HCl
(200 mL) was added, and the mixture was filtered. The
filtrate was evaporated on a rotary evaporator after
which 100 mL of isopropanol was added. The precipi-
tate insoluble in alcohol was filtered, and methanol
(150 mL) and concentrated HCl (20 mL) were added
to the filtrate. The mixture was refluxed for 30 min and
evaporated on a rotary evaporator. The residue was
recrystallized from isopropanol to give 44.8 g (96%) of
the product as a white powder; mp 122°C (i-PrOH);
1H NMR (D2O): 4.51–4.47 (m, 1H, CHOH), 3.89–
9); mp 85°C (CH2Cl2); 1H NMR: 6.68 (br s, 1H, NH),
3.38 (q, 2H, J 6.4, CH2NH), 2.84 (s, 4H,
COCH2CH2CO), 2.62 (t, 2H, J 7.0, COCH2), 1.80
(quin, 2H, J 7.5, CH2CH2NH), 1.63 (quin, 2H, J 7.3,
COCH2CH2),
1.49
(quin,
2H,
J
7.5,
3.82 (m, 1H, HOCHaHb), 3.76 (dd, 1H, J1 12.5, J2 3.6, COCH2CH2CH2); 13C NMR: 169.34 (2C,
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L-HYDROXYPROLINOL-BASED REAGENTS AND SOLID-PHASE SUPPORTS
391
Table 1. Properties of modified oligonucleotides synthesized using reagents (XVIII)–(XXV)*
Sequence (5′→3′)
MALDI-TOF mass spectra, mass found/calculated
[Biotin]-ТТТТТТТТТТ
[HC≡C]-ТТТТТТТТТТ
[S-S]-ТТТТТТТТТТ
3496.8/3496.7
3251.7/3251.6
3458.7/3458.7
3269.7/3270.6
3496.8/3496.7
3251.7/3251.6
3458.7/3458.7
3270.7/3270.6
3496.8/3496.7
3251.7/3251.6
3458.7/3458.7
3270.7/3270.6
4015.0/4014.9
3524.8/3524.7
3938.9/3938.8
3561.8/3562.7
[NH2]-ТТТТТТТТТТ
ТТТТТТТТТТ-[Biotin]
ТТТТТТТТТТ-[C≡CH]
ТТТТТТТТТТ-[S-S]
ТТТТТТТТТТ-[NH2]
ТТТТТ-[Biotin]-ТТТТТ
ТТТТТ-[C≡CH]-ТТТТТ
ТТТТТ-[S-S]-ТТТТТ
ТТТТТ-[NH2]-ТТТТТ
ТТТТТ-[Biotin][Biotin]-ТТТТТ
ТТТТТ-[C≡CH][C≡CH]-ТТТТТ
ТТТТТ-[S-S][S-S]-ТТТТТ
ТТТТТ-[NH2][NH2]-ТТТТТ
* In brackets, the type of a modification is indicated; in Scheme 3 it is shown near the formulas of the corresponding phosphoramidite
reagents that make it possible to introduce these modifications during the oligonucleotide synthesis.
COCH2CH2CO), 168.38 (1C, CO2), 157.30 (q, J 36.7, d6): 62.41 (CHNH), 60.76 (CHNH), 56.37 (SCH),
COCF3), 115.82 (q, J 287.6, CF3), 39.45 (CH2NH), 41.08 (SCH2), 33.38 (CH2CO), 29.16 (COCH2CH2),
30.77 (COCH2), 28.08 (CH2CH2NH), 25.56 (2C, 28.98 (CH2CHS), 25.47 (COCH2CH2CH2); IR:
COCH2CH2CO), 25.41 (COCH2CH2), 24.02 3254.0, 2940.2, 2872.2, 2850.2, 1794.9, 1708.4,
1520.5, 1469.5, 1112.6, 1094.6, 1078.4, 1021.5, 1005.9,
987.9.
(COCH2CH2CH2); IR: 3330.6, 1815.5, 1792.2, 1747.7,
1704.7, 1563.2, 1205.4, 1177.1, 1152.17, 1065.4, 1045.4,
873.9, 727.3, 655.0.
Preparation of a support containing carboxyl groups
(X) based on CPG. DMAP (1.08 g, 8.8 mmol) and suc-
cinic anhydride (3.0 g, 30.0 mmol) were added to a
suspension of the amino-containing support (2.5 g)
(aminopropyl-CPG, 500Å, the content of amino
groups 78 μmol/g) in pyridine (67 mL). The mixture
was allowed to stand at room temperature for 48 h, and
the support was filtered, washed successively with pyr-
idine, acetonitrile, methylene chloride, and ether
(50 mL each), and dried in vacuo.
Pentafluorophenyl ester of hex-5-ynoic acid (VI)
was synthesized by the method described earlier [15].
The product was obtained as a mobile yellow liquid.
Yield: 5.44 g (98%); Rf 0.74 (toluene); 1H NMR: 2.84
(t, J 7.4, 2H, OCCH2), 2.36 (dt, 2H, J1 6.9, J2 2.6,
CH2C≡CH), 2.03 (t, 1H, J 2.6, C≡CH), 1.99 (quin,
2H, J 7.2, CH2CH2CH2); 13C NMR: 169.01 (CO),
82.43 (C≡CH), 69.76 (C≡CH), 31.8 (COCH2), 23.31
(CH2C≡CH), 17.61 (CH2CH2CH2); IR: 1788.3,
1520.9, 1144.7, 1104.4, 1069.9, 1003.9, 996.7, 645.4.
(2S,4R)-4-Hydroxy-2-hydroxymethyl-1-(6-N-tri-
fluoroacetylaminohexanoyl) pyrrolidine (XI). Triethyl-
amine (55 mL) was added dropwise under stirring to a
suspension of hydrochloride (II) (30.7 g, 0.20 mol) in
pyridine (600 mL). Then, the N-oxysuccinimide ester
of 6-(N-(trifluoroacetyl)amino)hexanoic acid (IV)
(71.3 g, 0.22 mol) was added, and the mixture was left
to stand overnight under stirring. Pyridine was evapo-
rated on a rotary evaporator, and the residue was dis-
solved in ethyl acetate (900 mL), washed with an equal
volume of a saturated NaCl solution, and dried over
anhydrous sodium sulfate. Ethyl acetate was distilled
away. The product was purified by column chroma-
tography on silica gel using 5% ethanol in CH2Cl2 as
an eluent. Compound (XI) was obtained as a colorless
Pentafluorophenyl ester of biotin (VIII) was synthe-
sized by the method described earlier [16]. The prod-
uct was obtained as a white powder-like substance.
Yield: 9.96 g (73%); Rf 0.45 (MeOH–CH2Cl2, 1 : 5);
mp 187°C (petroleum ether–CH2Cl2, 1 : 1); 1H NMR
(acetone-d6): 5.91 (s, 1H, NH), 5.75 (s, 1H, NH),
4.53–4.40 (m, 1H, CHNH), 4.38–4.33 (m, 1H,
CHNH), 3.28–3.23 (m, 1H, SCH), 2.95 (dd, 1H, J1
12.5, J2 5.0, SCHaHb), 2.82 (t, 2H, J 7.5, COCH2),
2.71 (d, 1H, J 12.5, SCHaHb), 1.87–1.79 (m, 2H,
COCH2CH2), 1.73–1.63 (m, 2H, CH2CHS), 1.62–
1.52 (m, 2H, COCH2CH2CH2); 13C NMR (acetone-
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TATULCHENKOV et al.
oil. Yield: 46.6 g (72%); Rf 0.55 (EtOH–CH2Cl2, 1 :
(2S,4R)-1-(6-Aminohexanoyl)-4-hydroxy-2-(4,4′-
dimethoxytrityloxymethyl)pyrrolidine (XIII). Water
(50 mL) and K2CO3 (4.75 g, 34.42 mmol) were added
to a solution of compound (XII) (4.16 g, 6.62 mmol)
in methanol (200 mL) under stirring. The mixture was
allowed to stand overnight at room temperature after
which methanol was evaporated in a rotary evaporator,
and the residue was dissolved in water (50 mL) and
extracted with methylene chloride (4 × 50 mL). The
combined organic extracts were dried over anhydrous
sodium sulfate, filtered, and evaporated under low
pressure. The residue was chromatographed on silica
gel in a 1% Et3N/CH2Cl2 system using a gradient of
ethanol (the content of ethanol was increased by 1%
after each 100 mL of the eluent). The product was
obtained as a pale yellow substance in the form of
amorphous powder. Yield: 2.89 g (82%); Rf 0.10
4); 1H NMR (CD3CN): 7.47 (br s, 1H, HOCH2), 5.34
(1H, d, J 8.1, HOCH), 4.28 (1H, br s, NH), 4.20–4.13
1H, (m, CHOH), 3.57–3.49 (m, 2H, HOCH2), 3.45–
3.42 (m, 1H, NCH), 3.39 (t, J 11.4, 2H, CH2NH),
3.22–3.17 (m, 2H, NCH2), 2.26–2.11 (m, 3H,
COCH2, CHCHaHbCH), 1.61–1.50 (m, 3H,
CHCHaHbCH, CH2CH2NH), 1.47 (quin, 2H, J 7.2,
COCH2CH2),
1.26
(quin,
2H,
J
7.7,
COCH2CH2CH2); 13C NMR (CD3CN): 173.98 (CO),
68.44 (CHOH), 66.07 (CH2OH), 59.43 (NCH), 55.76
(NCH2), 38.92 (CH2NH), 36.32 (COCH2), 34.35
(CHCH2CH),
27.83
(CH2CH2NH),
25.62
(COCH2CH2), 23.52 (COCH2CH2CH2); IR: 3301.6,
2941.6, 1711.5, 1618.0, 1566.0, 1445.4, 1210.7, 1188.6,
1156.7.
(Et3N–EtOH–CH2Cl2, 1 : 20 : 79); 1H NMR
(DMSO-d6): 7.30–7.22 (m, 4H, ArH), 7.19–7.12 (m,
5H, ArH), 6.88–6.80 (m, 4H, ArH), 4.38–4.32 (m,
1H, CHOH), 4.14–4.04 (m, 1H, DMTr-OCHaHb),
3.69 (s, 6H, OCH3), 3.56–3.52 (m, 1H, DMTr-
OCHaHb), 3.24–3.19 (m, 1H, NCH), 3.16–3.08 (m,
1H, NCHaHb), 2.98–2.90 (m, 1H, NCHaHb), 2.51 (t,
1H, J 7.15, COCHaHb), 2.17 (t, 1H, J 7.2, COCHaHb),
2.04–1.94 (m, 1H, CHaHbNH2), 1.94–1.84 (m, 1H,
CHaHbNH2), 1.83–1.76 (m, 1H, CHCHaHbCH),
1.48–1.38 (m, 1H, CHCHaHbCH), 1.38–1.30 (m,
2H, CH2CH2NH2), 1.30–1.20 (m, 2H, COCH2CH2),
1.13–1.05 (m, 1H, COCH2CH2CHaHb), 1.02 (t, 1H, J
(2S,4R)-4-Hydroxy-2-(4,4'-dimethoxytrityloxymethyl)-
1-(6-N-trifluoroacetylaminohexanoyl)pyrrolidine (XII).
4,4′-Dimethoxytritylchloride (40.6 g, 0.12 mol) was
added to a solution of compound (XI) (32.6 g,
0.10 mol) in pyridine (300 mL) under stirring, and the
mixture was allowed to stand at room temperature
overnight. The solvent was evaporated in a rotary
evaporator after which the residue was dissolved in
ethyl acetate (300 mL) and washed with an equal vol-
ume of NaCl. The organic phase was separated and
dried over anhydrous sodium sulfate. Ethyl acetate was
distilled away. After the purification of the product by
column chromatography on silica gel using 1% tri-
ethylamine in CH2Cl2 as an eluent, compound (XII)
was obtained as a white amorphous powder. Yield:
39.1 g (62%); Rf 0.28 (Et3N–EtOH–CH2Cl2, 1 : 3 :
7.0, COCH2CH2CHaHb); 13C NMR (DMSO-d6):
171.47 (CO), 158.55 (2C), 145.67, 136.44, 136.32,
130.17 (4C), 128.36 (2C), 128.15 (2C), 127.17 (1C),
113.69 (5C), 85.69 (CH2ODMTr), 69.15 (CHOH),
63.92 (NCH2), 55.59 (3C, OCH3, NCH), 41.45
(CH2NH2), 36.87 (COCH2), 34.73 (CHCH2CH),
32.34 (CH2CH2NH), 26.64 (COCH2CH2), 24.86
(COCH2CH2CH2); IR: 2926.6, 2856.4, 1608.1,
1508.8, 1444.4, 1301.2, 1249.6, 1174.8, 1071.3, 1031.7,
827.4.
(2S,4R)-4-Hydroxy-2-(4,4'-dimethoxytrityloxy-
methyl)-1-(6-(N-(5-((R)-1,2-dithiolan-3-yl)pentanoyl-
аmino)hexanoyl)pyrrolidine (XIV). DIPEA (1.80 mL)
was added to a solution of lipoic acid (1.08 g,
5.26 mmol) and PyBOP (2.60 g, 5.00 mmol) in DMF
(50 mL) under stirring. After 10 min, a solution of
amine (XIII) (2.80 g, 5.26 mmol) in DMF (25 mL)
was added, and the mixture was left for 1 h at room
temperature. The reaction mixture was diluted with
water (300 mL) and extracted with an equal volume of
chloroform. The organic phase was washed with water
(2 × 300 mL), a saturated NaHCO3 solution
(300 mL), and a saturated NaCl solution (300 mL).
The mixture was dried over sodium sulfate and chro-
matographed on silica gel using acetonitrile as an elu-
96); 1H NMR: 7.37–7.33 (m, 2H, ArH) 7.28–7.22 (m,
6H, ArH), 7.21–7.16 (m, 1H, ArH), 6.83–6.77 (m,
4H, ArH), 4.64–4.58 (br s, 1H, NH), 4.42–4.34 (br s,
1H, HO), 4.14–4.08 (m, 1H, CHOH), 3.76 (s, 6H,
OCH3), 3.67 (dd, 1H, J1 10.9, J2 4.9, DMTrOCHaHb),
3.48 (dd, 1H, J1 10.7, J2 2.3, DMTr-OCHaHb), 3,41
(dd, 1H, J1 9.3, J2 4.5, CHaHbNH), 3.38–3.33 (m,
1H, NCH), 3,31 (q, 1H, J 6.4, CHaHbNH), 3.15–3.11
(m, 1H, NCHaHb), 3.08 (q, 1H, J 7.3, NCHaHb),
2.36–2.29 (m, 1H, COCHaHb), 2.27–2.16 (m, 2H,
COCHaHb, CHCHaHbCH), 1.67–1.61 (m, 1H,
CHCHaHbCH), 1.59 (quin, 2H, J 7.2, CH2CH2NH),
1.50 (quin, 1H, J 7.2, COCH2CHaHb), 1.42–1.33 (m,
3H, COCH2CHaHb, COCH2CH2CH2); 13C NMR:
172.49 (0.4C, CO, minR), 171.66 (0.6C, CO, majR),
158.33 (2C, OCH3), 144.99, 136.15, 135.63, 129.93
(4C), 127.98 (2C), 127.70 (2C), 126.69, 113.15
(Ar3CO), 112.98 (4C), 85.82 (CH2ODMTr), 70.36
(CHOH), 63.37 (NCH2), 55.76 (OCH3), 55.22
(OCH3), 55.15 (NCH), 39.15 (CH2NH), 36.60
(COCH2), 34.42 (CHCH2CH), 28.09 (CH2CH2NH),
25.98 (COCH2CH2), 23.35 (COCH2CH2CH2); IR:
3415.1, 2928.8, 1718.4, 1625.1, 1610.7, 1509.4, 1445.4, ent. The product was obtained as a yellow oily liquid,
1251.5, 1176.5, 1067.2, 1032.5. which is polymerized on long-term storage in a dry
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393
state. Yield: 3.6 g (95%); Rf 0.50 (Et3N–EtOH–CH2Cl2, (m, 1H, CHCHaHbCH), 2.25–2.10 (m, 4H, NCO-
1 : 10 : 89); 1H NMR (DMSO-d6): 7.12–7.04 (m, 4H,
ArH), 7.00–6.94 (m, 5H, ArH), 6.67–6.62 (m, 4H,
CH2, HNCOCH2), 2.10–1.94 (m, 1H, CHCHaHbCH),
1.77–1.53 (m, 6H, SCHCH2CH2CH2, CH2CH2NH),
ArH), 4.19–4.13 (m, 1H, CHOH), 3.95–3.86 (m, 1H, 1.53–1.47 (m, 2H, NCOCH2CH2), 1.47–1.31 (m, 4H,
NCOCH2CH2CH2, SCHCH2CH2); 13C NMR: 173.59
(NHCOCH2), 172.94 (0.3C, NCOCH2, minR),
172.36 (0.7C, NCOCH2, majR), 163.95 (NHCONH),
158.30 (2C), 145.06, 136.27, 136.13, 129.94 (4C),
128.03 (2C), 127.70 (2C), 126.66 (C), 113.12 (Ar3CO),
112.88 (4C), 85.64 (CH2O-DMTr), 69.98 (CHOH),
63.35 (NCH2), 63.03 (CHNH), 61.64 (CHNH), 60.07
(SCH), 55.22 (NCH), 55.17 (2C, OCH3), 45.79
(SCH2), 38.65 (CH2NH), 35.74 (NCOCH2), 34.72
(CHCH2CH), 33.05 (HNCOCH2), 28.88 (HNCO-
CH2CH2), 28.72 (CH2CHS), 27.96 (CH2CH2NH),
SCH), 3.50 (s, 6H, OCH3), 3.40–3.32 (m, 2H,
DMTrOCH2), 3.06–3.00 (m, 1Н, NCH), 2.96–2.90
(m, 2H, NCH2), 2.90–2.82 (m, 1H, SCHaHb), 2.81–
2.72 (m, 1Н, SCHaHb), 2.20–2.12 (m, 1Н,
SCH2CHaHb), 1.98 (t, 1H, J 7.30, CHaHbNHCO),
1.85–1.75 (m, 3H, CHaHbNHCO, NCOCH2), 1.64–
1.56 (m, 2H, HNCOCH2), 1.52–1.48 (m, 2H,
SCH2CHaHb, CHCHaHbCH), 1.46–1.48 (m, 1Н,
CHCHaHbCH), 1.34–1.20 (m, 4H, SCHCH2CH2CH2),
1.20–1.12 (m, 2H, CH2CH2NH), 1.12–0.86 (m, 6H,
SCHCH2CH2CH2,
NCOCH2CH2,
NCO-
CH2CH2CH2); 13C NMR (DMSO-d6): 171.66 (NCO), 26.28 (NCOCH2CH2), 25.68 (HNCOCH2CH2CH2),
170.82 (HNCO), 162.26 (2C), 145.03, 135.83, 135.67, 24.00 (NCOCH2CH2CH2); IR: 3385.4, 3284.9,
129.56 (4C), 127.74 (2C), 127.53 (2C), 126.54, 113.06
2924.6, 2854.7, 1702.7, 1630.9, 1608.6 , 1508.7,
1444.5, 1301.4, 1248.9, 1174.6, 1155.8, 1068.8, 1029.4,
(5C), 85.06 (DMTr-OCH2), 68.54 (CHOH), 63.28
(NCH2), 56.11 (SCH), 54.94 (4C(OCH3)2, NCH, 583.1.
SCH2), 45.80 (SCH2CH2), 38.21 (CH2NH), 36.25
(NCOCH2), 35.20 (CHCH2CH), 30.71 (HNCOCH2),
29.06 (SCHCH2CH2CH2), 28.30 (CH2CH2NH), 26.17
(HNCOCH2CH2), 25.91 (NCOCH2CH2), 25.05
(HNCOCH2CH2CH2), 24.14 (NCOCH2CH2CH2);
IR: 2931.4, 2863.0, 1643.5, 1621.4, 1609.2, 1508.6,
1444.0, 1249.9, 1176.4, 1072.9, 1032.5, 828.6.
(2S,4R)-4-Hydroxy-2-hydroxymethyl-1-(hex-5-
inoyl)pyrrolidine (XVI). Triethylamine (3.57 g, 35.3
mmol) was added dropwise under stirring to a suspen-
sion of (2S,4R)-4-hydroxyprolinol hydrochloride (II)
(2.7 g, 17.7 mmol) in pyridine (60 mL). Then, the pen-
tafluorophenyl ester (VI) (5.4 g, 19.4 mmol) was
added, and the mixture was left overnight at room
temperature under stirring. Pyridine was evaporated
on a rotary evaporator, and the residue was dissolved
in ethyl acetate (60 mL) and washed with an equal vol-
ume of a saturated NaCl solution. The mixture was
evaporated to dryness, and the residue was chromato-
graphed on silica gel in a system of 5% ethanol in CH2Cl2
to give 2.39 g (64%) of colorless crystals; Rf 0.61
(2S,4R)-4-Hydroxy-2-(4,4'-dimethoxytrityloxy-
methyl)-1-(6-(N-(5-((3aS,4S,6aR)-2-oxohexahydro-
1H-thieno[3,4-d]imidazol-4-ylpentanoylаmino)-hexa-
noyl)-pyrrolidine (XV). Triethylamine (1.7 g,
16.8 mmol) was added dropwise under stirring to a
solution of amine (XIII) (8.51 g, 16 mmol) in DMF
(62 mL). Then, a solution of pentafluorophenyl ester
of biotin (6.9 g, 16.8 mmol) in DMF (50 mL) was
added dropwise for 40 min. The reaction mixture was
left to stand overnight under stirring after which it was
diluted with methylene chloride (300 mL) and washed
with water (6 × 500 mL), a saturated NaHCO3 solu-
tion (500 mL), and a saturated NaCl solution (500 mL).
The mixture was dried over anhydrous sodium sulfate,
evaporated to dryness, and chromatographed in a sys-
tem of 1% triethylamine in CH2Cl2 using a gradient of
ethanol. The product was obtained as pale yellow
foam. Yield: 11.84 g (97%); Rf 0.53 (Et3N–EtOH–
(EtOH–CH2Cl2, 1 : 4); mp 79°C (CH2Cl2); 1H NMR:
5.45 (dd, 1H, J1 8.7, J2 2.0, HOCH2), 4.46–4.41 (br s,
1H, HOCH), 4.36–4.29 (m, 1H, HOCH), 3.67 (ddd,
1H, J1 11.7, J2 8.6, J3 2.2, HOCHaHb), 3.59–3.57 (m,
2H, NCH, NCHaHb), 3.69 (ddd, 1H, J1 11.7, J2 7.6, J3
1.8, HOCHaHb), 2.87 (d, 1H, J 3.6, NCHaHb), 2.48
(dt, 1H, J1 16.0, J2 7.3, COCHaHb), 2.41 (dt, 1H, J1
16.0, J2 7.4, COCHaHb), 2.29 (td, 2H, J1 6.8, J2 2.7,
CH2C≡CH), 2.09 (dddd, 1H, J1 13.6, J2 7.4, J3 2.4 , J4
1.4, CHCHaHbCH), 1.98 (t, 1H, J 2.7, C≡CH), 1.86
(quin, 2H, J 7.1, CH2CH2C≡CH), 1.71 (ddd, 1H, J1
13.7, J2 9.2, J3 4.6, CHCHaHbCH); 13C NMR: 173.98
(CO), 83.64 (C≡CH), 69.24 (C≡CH), 69.15 (CHOH),
66.75 (CH2OH), 59.98 (NCH), 56.19 (NCH2), 37.15
(CHCH2CH), 33.57 (COCH2), 23.41 (CH2C≡CH),
17.80 (CH2CH2CH2); IR: 3406.4, 3229.3, 2943.5,
2932.8, 2900.9, 1618.5, 1450.3, 1424.4, 1405.7, 1384.4,
1334.0, 1323.4, 1250.8 , 1214.3, 1195.6, 1080.8, 1061.0,
978.9, 859.0, 850.6, 711.9, 660.8, 538.8.
1
CH2Cl2, 1 : 20 : 79); H NMR: 7.38–7.30 (m, 2H,
ArH), 7.26–7.12 (m, 7H, ArH), 6.86–6.71 (m, 4H,
ArH), 6.63–6.54 (br s, 1H, NHCONH), 5.94–5.84
(br s, 1H, NHCONH), 4.54–4.47 (m, 1H, CHNH),
4.47–4.38 (br s, 1H, OH), 4.38–4.31 (m, 1H,
CHNH), 4.27–4.12 (m, 1H, CHOH), 3.75 (s, 3H,
OCH3), 3.73 (s, 3H, OCH3), 3.63–3.58 (br s, 1H,
NHCH2), 3.54–3.46 (m, 2H, DMTr-OCH2), 3.43–
3.37 (m, 1H, NCH), 3.25–3.12 (m, 2H, CH2NH),
3.12–3.04 (m, 3H, SCH, NCH2), 2.85–2.77 (m, 1H,
SCHaHb), 2.74–2.64 (m, 1H, SCHaHb), 2.40–2.31
(2S,4R)-4-Hydroxy-2-(4,4'-dimethoxytrityloxy-
methyl)-1-hex-5-ynoylpyrrolidine (XVII). Dimethoxy-
tritylchloride (4.06 g, 12 mmol) was added to a solu-
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394
TATULCHENKOV et al.
tion of compound (XVI) (2.3 g, 10.9 mmol) in pyri- (30 mL), methylene chloride (30 mL), and ether
dine (36 mL) under stirring, and the mixture was (30 mL), and dried in vacuo. From the absorbance of
allowed to stand overnight under stirring. Pyridine was the dimethoxytrityl cation in a solution of trifluoro-
evaporated after which the residue was dissolved in acetic acid (3%) in CH2Cl2, the load of solid-phase
ethyl acetate (60 mL) and washed with an equal vol-
ume of a saturated NaCl solution. The organic phase
was evaporated to dryness, and the residue was chro-
matographed on silica gel in a system of 1% triethyl-
amine in CH2Cl2. The product was obtained as white
foam. Yield: 3.7 g (66%); Rf 0.23 (Et3N–EtOH–CH2Cl2,
supports was determined, which was for different sam-
ples from 51 to 64 μmol/g.
(2S,4R)-4-(N,N-Diisopropylamino-2-cyanoethoxy-
phosphinyloxy)-2-(4,4'-dimethoxytrityloxymethyl)-1-
(6-N-trifluoroacetylamino)hexanoyl-pyrrolidine (XXII).
Diisopropylethylamine (1.23 g, 9.6 mmol) was added
to a solution of alcohol (XII) (5.48 g, 8.7 mmol) in
anhydrous methylene chloride (16 mL) under stirring,
argon was blown through the mixture, and the mixture
was cooled to 0°С. N,N-Diisopropylamino-2-cya-
noethoxychlorophosphine (2.27 g, 9.6 mmol) was
added to the cooled solution. The mixture was stirred
and heated to room temperature for 1 h. Methylene
1 : 3 : 96); 1H NMR: 7.38–7.32 (m, 2H, ArH), 7.30–
7.21 m, 6H, ArH), 7.21–7.15 (m, 1H, ArH), 6.84–6.76
(m, 2H, ArH), 4.67–4.60 (br s, 0.7H, HOCH, majR),
4.51–4.45 (br s, 0.3H, HOCH, minR), 4.41–4.34 (m,
0.7H, HOCH, majR), 4.20–4.13 (m, 0.3H, HOCH,
minR), 3.77 (s, 3H, OCH3), 3.76 (s, 3H, OCH3), 3.73
(dd, 1H, J1 11.0, J2 5.2, HOCHaHb), 3.49 (dd, 1H, J1
11.0, J2 3.4, HOCHaHb), 3.46–3.38 (m, 1H, NCH), chloride (80 mL) was added to the solution, and the
mixture was washed with a saturated NaHCO3 solu-
tion (100 mL) and a saturated NaCl solution
(100 mL). The organic phase was evaporated to dry-
ness, and the product was purified by column chroma-
tography in a system of 2% triethylamine in CH2Cl2.
The product was obtained as snow-white amorphous
foam. Yield: 6.5 g (90%); Rf 0.49 (Et3N–acetone–tol-
3.17–3.01 (m, 2H, NCH2), 2.43 (dt, 1H, J1 15.7, J2 7.4,
COCHaHb), 2.39 (dt, 1H, J1 15.7, J2 7.4, COCHaHb),
2.28 (td, 2H, J1 6.7, J2 2.5, CH2C≡CH), 1.96 (t, J 2.7,
1H, C≡CH), 1.85 (quin, J 7.2, 2H, CH2CH2C≡CH),
1.83–1.69 (m, 1H, CHCHaHbCH), 1.43–1.35 (m,
1H, CHCHaHbCH); 13C NMR: 171.86 (0.4C, CO,
minR), 171.07 (0.6C, CO, majR), 158.51 (0.6C,
minR), 158.34 (1.3C, majR), 145.03 (0.6C, majR),
144.55 (0.4C, minR), 136.19 (1C), 135.65 (1C),
129.94 (4C), 128.02 (1.4C, majR), 127.99 (0.6C,
minR), 127.87 (0.7C, minR), 127.72 (1.3C, majR),
126.89 (0.3C, minR), 126.68 (0.7C, majR), 113.15
(Ar3CO), 113.01 (4C), 85.82 (DMTrOCH2), 83.90
(C≡CH), 70.38 (C≡CH), 69.05 (CHOH), 63.44
(NCH), 55.73 (NCH2), 55.16 (2C, OCH3), 36.66
(CHCH2CH), 33.37 (COCH2), 23.48 (CH2C≡CH),
17.93 (CH2CH2CH2); IR: 3288.2, 2932.7, 1608.3,
1508.9, 1460.8, 1444.4, 1301.4, 1249.7, 1175.1, 1154.0,
1111.4, 1074.4, 1031.7, 828.3, 701.7, 582.9.
uene 1 : 10 : 39); 1H NMR (CD3CN): 7.67–7.58 (br s,
1H, NH), 7.41–7.37 (m, 2H, ArH), 7.32–7.28 (m, 2H,
ArH), 7.28–7.24 (m, 4H, ArH), 7.24–7.19 (m, 1H,
ArH), 6.88–6.82 (m, 4H, ArH), 4.75–4.64 (m, 0.7H,
CHOP, majR), 4.62–4.52 (m, 0.2H, CHOP, minR),
4.24–4.18 (m, 0.7H, NCHaHb, majR), 4.12–4.06 (m,
0.2H, NCHaHb, minR), 3.82–3.68 (m, 9H, (OCH3)2,
DMTOCH2, NCHaHb), 3.64–3.55 (m, 2H,
OCH2CH2CN), 3.32 (ddd, 1H, J1 17.8, J2 9.1, J3 4.6,
NCH), 3.28–3.20 (m, 2H, CH2NH), 3.04 (ddd, 1H,
J1 11.9, J2 9.1, J3 3.0, CHCHaHbCH), 2.65–2.59 (m,
2H, OCH2CH2CN), 2.29–2.23 (m, 1H, CHCHaHbCH),
2.23–2.13 (m, 4H, COCH2, N(CH(CH3)2)2), 1.60–
1.42 (m, 4H, COCH2CH2, CH2CH2NH), 1.39–1.30
(m, 2H, COCH2CH2CH2), 1.18 (d, 3H, J 1.3,
NCHCH3), 1.16 (d, 3H, J 1.3, NCHCH3), 1.16 (d, 3H,
Solid-Phase Supports (XVIII)–(XXI) for the Prepara-
tion of Oligonucleotides Modified at the 3'-Position.
A General Method
The support containing carboxyl groups (X) (0.50 g),
DMAP (30 mg), and diisopropylcarbodiimide (420 μL,
2.70 mmol) were added to a solution (0.4 mmol) of
one of precursor reagents (XII), (XIV), (XV), or
(XVII) in 6 mL of a pyridine–DMF mixture 1 : 1. The
J 2.0, NCHCH3), 1.14 (d, 3H, J 2.0, NCHCH3); 31P
NMR (CD3CN): 148.14 (sext, 0.38P, J 8.8, dst
1/majR), 147.85 (sext, 0.25P, J 9.0, dst 1/minR + dst
2/minR), 147.43 (sext, 0.37P, J 9.2, dst 2/majR).
(2S,4R)-4-(N,N-Diisopropylamino-2-cyanoethoxy-
mixture was left to stand for 72 h at room temperature phosphinyloxy)-2-(4,4'-dimethoxytrityloxymethyl)-1-
under periodic stirring after which a solution of penta- (hex-5-ynoyl)-pyrrolidine (XXIII). Diisopropylethyl-
fluorophenol (0.15 g) in pyridine (0.5 mL) was added, amine (0.73 g, 5.7 mmol) was added under stirring to
and the mixture was allowed to stand overnight. The a solution of alcohol (XVII) (2.42 g, 4.7 mmol) in
glass was filtered and suspended in a solution of piper- anhydrous methylene chloride (10 mL), argon was
idine in pyridine. After 5 min, the glass was filtered blown through the mixture, and the mixture was
again, washed with acetonitrile (11 mL), and sus- cooled to 0°С. N,N-Diisopropylamino-2-cyanoe-
pended in a solution containing N-methylimidazole thoxychlorophosphine (1.34 g, 5.7 mmol) was added
(0.45 mL), acetic anhydride (0.25 mL), and 2,6-luti- to the cooled solution, and the mixture was stirred and
dine (0.25 mL) in acetonitrile (4 mL). After 2 h, the heated to room temperature for 1 h. Methylene chlo-
support was filtered, washed with acetonitrile ride (40 mL) was added, and the solution was washed
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with a saturated NaHCO3 solution (50 mL) and a sat- NHCOCH2), 1.88–1.79 (m, 2H, CHCHaHbCH,
urated NaCl solution (50 mL). The organic phase was SCH2CHaHb), 1.61–1.47 (m, 6H, CH2CH2NH,
evaporated to dryness, and the product was purified by
column chromatography in a system of 2% triethyl-
amine in toluene. The product was obtained as a dense
oily liquid. Yield: 2.73 g (81%); Rf 0.48 (Et3N–ace-
SCHCH2CH2CH2), 1.47–1.39 (m, 2H, NCO-
CH2CH2), 1.39–1.27 (m, 4Н, SCHCH2CH2CH2,
NCOCH2CH2CH2), 1.16–1.08 (m, 12H, N(CH(CH3)2)2);
31P NMR (CD3CN): 164.96 (sext, 0.36P, J 9.0, dst
1/majR), 164.69 (sext, 0.26P, J 8.3, dst 1/minR + dst
2/minR), 164.28 (sext, 0.37P, J 9.2, dst 2/majR).
tone–toluene 1 : 10 : 39); 1H NMR (CD3CN): 7.41–
7.36 (m, 2H, ArH), 7.33–7.13 (m, 7H, ArH), 6.88–
6.82 (m, 4H, ArH), 4.76–4.65 (m, 0.7H, CHOP,
majR), 4.62–4.52 (m, 0.2H, CHOP, minR), 4.24–
4.18 (m, 0.7H, NCHaHb, majR), 4.13–4.07 (m, 0.2H,
NCHaHb, minR), 3.82–3.66 (m, 9H, (OCH3)2,
DMTrOCH2, NCHaHb), 3.64–3.53 (m, 2H,
OCH2CH2CN), 3.33 (ddd, 1H, J1 17.5, J2 9.2 Hz, J3
4.4, NCH), 3.02 (ddd, 1H, J1 12.1, J2 9.1 Hz, J3 3.0,
CHCHaHbCH), 2.65–2.59 (m, 2H, OCH2CH2CN),
2.40–2.31 (m, 2H, CHCHaHbCH, NCH(CH3)2),
2.25–2.20 (m, 2H, COCH2), 2.20–2.14 (m, 2H,
NCH(CH3)2, C≡CH), 2.13–2.01 (m, 2H,
CH2C≡CH), 1.75 (quin, 1H, J 7.2, COCH2CH2, dst
1), 1.74 (quin, 1H, J 7.2, COCH2CH2, dst 2), 1.19–
(2S,4R)-4-(N,N-Diisopropylamino-2-cyanoethoxy-
phosphinyloxy)-2-(4,4'-dimethoxytrityloxymethyl-1-
{6-N-5-((3aS,4S,6aR)-2-oxohexahydro-1H-thieno-
[3,4-d]imidazol-4-ylpentanoyl)-aminohexanoyl}pyrro-
lidine (XXV). Bis(N,N-diisopropylamino)-2-(cyanoe-
thoxy)phosphine (2.36 g, 7.83 mmol) and 5-(eth-
ylthio)-1Н-tetrazole (68 mg, 0.522 mmol) were added
to a solution of (XV) (3.96 g, 5.22 mmol) in methylene
chloride (7 mL). The reaction mixture was left to stand
overnight at room temperature after which it was
diluted with methylene chloride (40 mL) and washed
with equal volumes of a saturated NaHCO3 and NaCl
solutions. The mixture was dried over anhydrous
sodium sulfate and chromatographed on silica gel in a
system of 5% triethylamine in acetone. The product
was obtained as colorless amorphous foam. Yield: 3.92 g
1.12 (m, 12H, N(CH(CH3)2)2); 31P NMR (CD3CN):
147.46 (sext, 0.39P, J 8.9, dst 1/majR), 147.19 (sept,
0.25P, J 8.4, dst 1/minR + dst 2/minR), 146.88 (sext,
0.36P, J 9.1, dst 2/majR).
1
(78%); Rf 0.19 (Et3N–acetone, 1 : 19); H NMR
(CD3CN): 7.41–7.36 (m, 2H, ArH), 7.32–7.23 (m,
6H, ArH), 7.23–7.19 (m, 1H, ArH), 6.88–6.82 (m,
4H, ArH), 6.53 (t, 0.7H, J 5.7, NHCOCH2, majR),
6.49 (t, 0.2H, J 6.2, NHCOCH2, minR), 5.53–5.48
(br s, 1H, NHCONH), 5.20–5.15 (br s, 1H,
NHCONH), 4.74–4.64 (m, 0.7H, CHOP, majR),
4.62–4.51 (m, 0.2H, CHOP, minR), 4.39–4.35 (m,
1H, CHNH), 4.24–4.18 (m, 1H, CHNH), 3.82–3.66
(m, 9H, (OCH3)2, DMTrOCH2, NCH), 3.66–3.53
(m, 2H, OCH2CH2CN), 3.32 (ddd, 1H, J1 17.8, J2 9.2,
J3 4.6, NCHaHb), 3.17–3.06 (m, 3H, CH2NH, SCH),
3.03 (ddd, J1 12.1, J2 9.1, J3 3.0, 1H, NCHaHb), 2.85
(ddd, 1Н, J1 12.7, J2 5.0, J3 1.1, SCHaHb), 2.65–2.57
(m, 3H, OCH2CH2CN, SCHaHb), 2.29–2.22 (m, 2H,
CHCH2CH), 2.22–2.13 (m, 2H, N(CH(CH3)2)2),
2.13–2.03 (m, 4H, NCOCH2, NHCOCH2), 1.71–
1.50 (m, 6H, SCHCH2CH2CH2, CH2CH2NH), 1.50–
1.41 (m, 2H, NCOCH2CH2), 1.41–1.29 (m, 4H,
NCOCH2CH2CH2, SCHCH2CH2), 1.18–1.13 (m,
(2S,4R)-4-(N,N-Diisopropylamino-2-cyanoethoxy-
phosphinyloxy-2-)-2-(4,4'-dimethoxytrityloxymethyl)-
1-[6-N-5-((R)-1,2-dithiolan-3-ylpentanoylamino)-
hexanoyl]pyrrolidine (XXIV). Diisopropylethylamine
(166 μL 0.97 mmol) was added to a solution of (XIV)
(0.34 g, 0.88 mmol) in anhydrous acetonitrile (3 mL).
The mixture was purged with argon, cooled to 0°С,
and N,N-diisopropylamino-2-cyanoethoxy-chloro-
phosphine (0.23 g, 0.97 mmol) was quickly added
under stirring. The solution was purged with argon
once more and heated to room temperature after
which it was diluted with methylene chloride (20 mL)
and washed with a saturated NaHCO3 solution
(10 mL) and a saturated NaCl solution (2 × 10 mL).
The organic phase was dried over anhydrous sodium
sulfate, evaporated in a rotary evaporator, and chro-
matographed on silica gel using 1% triethylamine in
CH2Cl2 as an eluent. Fractions were evaporated, and
the residue was dissolved in benzene (3.5 mL) and lyo-
philized to give a pale-yellow oily substance. Yield:
325 mg (40%); Rf 0.80 (Et3N–CH2Cl2–MeCN, 2 :
12H, N(CH(CH3)2)2); 31P NMR (CD3CN): 147.46
(sext, 0.36P, J 9.1, dst 1/majR), 147.18 (sept, 0.26P, J
8.4, dst 1/minR + dst 2/minR), 146.75 (sext, 0.38P, J
1
49 : 49); H NMR (CD3CN): 7.39–7.33 (m, 2H,
ArH), 7.30–7.16 (m, 7H, ArH), 6.86–6.78 (m, 4H, 9.1, dst 2/majR).
ArH), 6.50–6.39 (m, 1H, NH), 4.24–4.16 (m, 1H,
Oligonucleotide synthesis was performed on a solid-
CHOP), 3.78–3.68 (m, 8H, (OCH3)2, DMTrOCH2),
3.60–3.42 (m, 4H, OCH2CH2CN, NCH, SCH),
3.39–3.32 (m, 1H, NCHaHb), 3.32–3.21 (m, 1H,
NCHaHb), 3.19–3.02 (m, 4Н, CH2NH, SCH2), 2.64–
2.58 (m, 2H, OCH2CH2CN), 2.44–2.34 (m, 1H,
SCH2CHaHb), 2.27–2.12 (m, 5H, N(CH(CH3)2)2,
NCOCH2, CHCHaHbCH), 2.09–2.04 (m, 2H,
phase support loaded with 200 nmol of an anchor
group in an automatic regime using standard phos-
phoramidites (dABz, dT, dCBz, dGIbu) according to the
protocols of the manufacturers of the device. The con-
densation of terminal phosphoramidites (XXII)–
(XXV) was carried out for 5 min using 0.1 М solutions
of phosphoramidites in absolute acetonitrile. The
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 43 No. 4 2017

396
TATULCHENKOV et al.
Awsiuk, K., Budkowski, A., and Kakabakos, S.E., Col-
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5. Dougan, J.A., Karlsson, C., Smith, W.E., and Gra-
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detachment of an oligonucleotide from the support
and the deblocking were performed with concentrated
(28%) ammonia (0.75 mL, overnight/60°С). After the
deblocking, the resulting solution was evaporated, and
the residue was dissolved in water (500 μL) and puri-
fied by reversed-phase HPLC. The starting eluent
(solution A) was 0.1 М AcONEt4 + 5% MeCN. The
supplementary solution (solution B) was MeCN.
Chromatography was carried out using a gradient of
2% solution B/min. Detection was with a flow-
through UV detector at λ 260 nm. Collected oligonu-
cleotide solutions were evaporated, and the residue
was dissolved in water (1 mL) and desalted on NAP-10
columns (Amersham Biosciences). The concentration
of an oligonucleotide was determined spectrophoto-
metrically by measuring the absorption at 260 nm. The
efficiency of the condensation of hydroxyprolinol
phosphoramidite reagents was estimated from the
HPLC data. During the registration of mass spectra of
oligonucleotides, a mixture of solutions of 2,6-dihy-
droxyacetophenone (40 mg in 1 mL of methanol) and
disubstituted ammonium citrate (80 mg in 1 mL of an
aqueous 50% acetonitrile solution) 1 : 1 (v/v) was used
as a matrix for ionization, which was prepared imme-
diately before each measurement.
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Translated by S. Sidorova
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY
Vol. 43
No. 4
2017