ISSN 1070-3632, Russian Journal of General Chemistry, 2018, Vol. 88, No. 3, pp. 452–461. © Pleiades Publishing, Ltd.

, 2018.
Original Russian Text © Yu.V. Martynenko-Makaev, V.V. Udodova, O.L. Sharko, V.V. Shmanai, 2018, published in Zhurnal Obshchei Khimii, 2018, Vol. 88,
No. 3, pp. 425–433.

Synthesis of Pentaerythritol-Based Branching Reagents

for Modification of Proteins and Nucleic Acids
by [2+3] Dipolar Cycloaddition Reaction
Yu. V. Martynenko-Makaev*, V. V. Udodova, O. L. Sharko, and V. V. Shmanai
Institute of Physical Organic Chemistry of the National Academy of Sciences of Belarus,
ul. Surganova 13, Minsk, 220072 Belarus
*e-mail: yrmart@gmail.com

Received October 12, 2017

Abstract—Alkylation of pentaerythritol symmetrically substituted with propylene glycol with propargyl

bromide afforded compounds containing two or three alkyne moieties. Amidophosphite reagents and solid
supports were prepared for the introduction of two and three acetylene fragments into oligonucleotides at the 3'-
and 5'-positions and inside the chain under conditions of automated solid-phase oligonucleotide synthesis.
Based on the trialkynyl derivative, an N-hydroxysuccinimide ester was obtained which can be used to modify
biomolecules attacking the amino group. Conjugates obtained can be used for multiple modifications by [3+2]
dipolar cycloaddition reaction.
Keywords: pentaerythritol, linkers, cycloaddition, click-reaction, biomolecules modification

DOI: 10.1134/S1070363218030118

The intensive development of methods of
molecular biology and bioorganic chemistry in recent
decades has made available synthetic proteins and
nucleic acids, which are representatives of a new
generation of highly effective drugs. However, as a
rule, biomolecules require various chemical modifica-
tions for the use in medicine, which required applica-
tion of fine organic synthesis procedures. Modified
biomolecules are used in medicine as therapeutic
agents [1–8], in the diseases diagnostics [9, 10], in
molecular biology [11, 12], as well as for creating new
materials [13–16].
To perform modifications the reaction of azide-
alkyne [3+2] cycloaddition (click-chemistry) has
become widespread [22]. It proceeds in mild condi-
tions, is highly selective and bioorthogonal, i. e., it can
proceed within living systems without affecting natural
biochemical processes.
Several modifications of a biomolecule [17, 18] are
sometimes urgent. However, it can lead to a significant
decrease in its biological activity. In this regard,
branching reagents can be used, which require only one
biomolecule site for introducing several modifications.
Amino group is one of the most versatile sites for
bioconjugation. The reaction between the amino group
and N-hydroxysuccinimide ester is used to modify
proteins, liposomes, to build dendrimeric structures, as
well as to introduce synthetic polymers into biomole-
cules [19]. To obtain modified oligonucleotides used
as DNA probes and gene therapy agents [20], amido-
phosphite reagents are used under conditions of
automated solid phase synthesis [21].
This work comprises the synthesis of branching
reagents containing two or three terminal alkyne groups
that allow modification to be introduced by the azide-
alkyne [3+2] cycloaddition (click-chemistry) reaction,
as well as an amidophosphite group or a bond with a
solid support for the modified oligonucleotides
synthesis. The preparation of N-hydroxysuccinimide
ester is also used for conjugation with proteins or other
biomolecules and materials at the amino group.
Alkyne derivatives based on pentaerythritol were
prepared according to the previously described
procedures from tetraalcohol 1 (Scheme 1) [28, 29]. It
was found that the best yield of the disubstitution
product 3 can be achieved using propargyl bromide in

452
 SYNTHESIS OF PENTAERYTHRITOL-BASED BRANCHING REAGENTS 453

the NaH–THF system, and the most suitable system The yields of compounds 2–5 under various reaction conditions
for the preparation of the trisubstitution product is
Reaction Yield, %
propargyl bromide in dimethyl sulfoxide using NaOH
as base (see the table). conditions 2 3 4 5
In addition to the desired products of di- and tri- NaH–THF 0–2% 25–30% 27–28% 10–12%
substitution 3 and 4, potentially useful compounds 2 DMSO–NaOH 1–5% 18–20% 37–39% 7–8%
and 5 were obtained as by-products.
Such reagents are used to create dendritic-like branched action of dimethoxytrityl chloride (Dmt-Cl) in pyridine
oligonucleotides [30, 31] and DNA-conjugates [32]. in 49% yield (Scheme 2). Phosphitylation of 6 with 2-
The presence of two hydroxyl groups in the mole- cyanoethoxy-N,N'-diisopropylchlorophosphoramidite
cule of 3 makes it is possible to prepare reagents for (CEP-Cl) in the presence of diisopropylethylamine
introducing two terminal acetylene groups in different (DIPEA) yielded the desired reagent 7 (32%).
positions of the oligonucleotide chain (3',5'-positions Controlled pore glass (CPG), N,N-diisopropyl-
and within the chain) on its basis. carbodiimide (DIC), and N,N-dimethylaminopyridine
Compound 6 with a dimethoxytrityl protective (DMAP) were used to prepare a solid phase reagent
group (Dmt) was prepared from compound 3 by the allowing modification to be introduced at the 3' posi-

Scheme 1.

HO OH O n
Br
O O O
O O O
HO OH OH
1
4 _n

n = 1 (2), 2 (3), 3 (4), 4 (5).

Scheme 2.

O OH
O O
O O
O OH
3
O

O O
a O O
O
O O
O OR

6: R = H, 49%
b 7: R = P(N-iPr)O(CH2)2CN, 32%
c
8: R = CPG-CO.
a, Dmt-Cl, pyridine, 15 h; b, CEP-Cl, DIPEA, CH2Cl2; c, CPG-COOH, DIC, DMAP, pyridine, DMF, 48 h.

RUSSIAN JOURNAL OF GENERAL CHEMISTRY Vol. 88 No. 3 2018

454 MARTYNENKO-MAKAEV et al.

Scheme 3.

O O
O O
O O
O OH
4

O O
CEP_Cl, DIPEA, CH2Cl2
O O
75%
O O
O O
P O

N CN

Scheme 4.

O O O O
O O a O O
O O 65% O O
O OH O O
O
4 10 O

O O DmtO H
N
b O O
O O c OH
O O
OH 75%
O
O O
O O
O O OH
11 O O
N
O
O

O O
12

a, tert-butyl acrylate, 40% aq. NaOH, DMSO; b, CF3CO2H, CH2Cl2; c, EDCI, DIPEA, HOBt.

tion of the oligonucleotide. The loading of the
functional groups in material 8 was determined from
the optical density of the Dmt cation formed by the
addition of a catalytic amount of trifluoroacetic acid to
the sample [33], and it was 35.5 μmol per 1 g.
Compound 4 containing three alkynyl groups was
used for obtaining a number of reagents for
introduction of modifications into oligonucleotides at
the terminal positions and inside the chain. Compound
4 was modified with a branching reagent based on

RUSSIAN JOURNAL OF GENERAL CHEMISTRY Vol. 88 No. 3 2018

 SYNTHESIS OF PENTAERYTHRITOL-BASED BRANCHING REAGENTS 455

Scheme 5.

O O
O O ODMTr
O O
O O
N
OH
12 O

O
O O
a O O O
64% O O
O O
N
O
O P
O N

13 NC

O O
b O O ODMTr
O O
O O CPG
N
O
14 O
_
CPG controlled pore glass.

a, tert-butyl acrylate, 40% aq. NaOH, DMSO; b, CF3CO2H, CH2Cl2; c, EDCI, DIPEA, HOBt.

(2S,4R)-4-hydroxyprolinol, a derivative of natural amino
acid 4-L-hydroxyproline. The presence of a prolinol
fragment allows Dmt-protection to be left at the 5'-end
of the synthesized oligonucleotides, which facilitates
the isolation and purification of such oligonucleotides
by reversed-phase chromatography [21, 34, 35].
To modify oligonucleotides at the 5'-position, amido-
phosphite 9 (Scheme 3) was obtained in 75% yield by
phosphitylation of alcohol 4 with CEP-Cl.
For the synthesis of the other amidophosphite
reagents from the trialkyl derivative 4, compound 12
containing a fragment of the Dmt-protected 5-(hyd-
roxymethyl)pyrrolidin-3-ol was obtained (Scheme 4).
Hydrolysis of tert-butyl ester 10 with trifluoroacetic
acid in methylene chloride proceeded in good yield
and without the formation of the reaction by-products,
which allowed the acid 11 to be introduced into further
transformations without purification.
Phosphitylation of compound 12 with CEP-Cl
furnished reagent 13 in the total yield of 31% over
4 stages (Scheme 5). Solid support 14 was prepared
using controlled pore glass with carboxyl groups on
the surface (CPG-COOH). N,N-Diisopropylcarbo-
diimide in the presence of DMAP was used as a
condensing agent. The loading of the functional groups
in compound 14 determined by measuring the optical
density of the cleavable Dmt cation was 49 μmol per
1 g.
To modify the protein or other biomolecules
through amino groups acid 11 was converted into N-
hydroxysuccinimide ester 15 in 68% yield (Scheme 6),
which is a reactive acylating agent [19]. The reaction

RUSSIAN JOURNAL OF GENERAL CHEMISTRY Vol. 88 No. 3 2018

456 MARTYNENKO-MAKAEV et al.

Scheme 6.

O O
O O
O O
O O
OH
O
11

O O
DSC, DMAP, CH2Cl2 O O
68% O O
O O
O O
N
O
15 O

was carried out in methylene chloride using di- Alkylation of 3,3'-[(2,2-bis[(3-hydroxypropoxy)-

succinimidyl carbonate (DSC) and DMAP.
The structure of the obtained compounds was
confirmed by 1H, 13C and 31P NMR spectra.
The resulting reagents can be used to introduce two
and three terminal alkynyl groups in different positions
of the oligonucleotide chain for subsequent post-
synthetic modification using azide-alkyne [3+2] cyclo-
addition of azide derivatives of the modifiers. Oligo-
nucleotides modified with amidophosphite reagents
with Dmt fragments at the 5'-position can be purified
by reversed-phase chromatography. The N-hydroxy-
succinimide ester of a trialkyne derivative can be more
accurately used for amino group modification of
proteins or other biomolecules and materials.
In summary, a method for the synthesis of com-
pounds that allows introducing two or three terminal
alkyne groups into biomolecules at one site for
subsequent modification by the azide-alkyne [3+2]
cycloaddition reaction was developed.
EXPERIMENTAL
NMR spectra were taken on a Avance-500 Bruker
spectrometer. Chemical shifts were measured relative
to residual proton and carbon signals of the deuterated
solvents (CDCl3, DMSO-d6, CD3CN). Optical density
was determined using a Solar CM 2203 spectro-
fluorimeter. Merck silica gel (0.040–0.063 mm) was
used for the column chromatography. Merck plates
were used for thin layer chromatography.
methyl]propane-1,3-diyl}bis(oxy)]bis(propan-1-ol)-
with 3-bromoprop-1-yne. a. To a 0.25 M. solution of
alcohol 1 (15 g, 40.8 mmol) in anhydrous tetra-
hydrofuran was added 4 equiv of sodium hydride (60%
in mineral oil, 6.5 g, 163.2 mmol), then 9 mL of an
80% solution of propargyl bromide (81.6 mmol) in
toluene. After stirring for 30 min at 40°C the solvents
were distilled off, an excess of sodium hydride was
neutralized with 10 mL of isopropanol. The mixture
was diluted with 80 mL of water and extracted with
diethyl ether (3 × 50 mL). The organic phases were
combined, dried with anhydrous magnesium sulfate
and filtered. The solvent was evaporated in a vacuum,
the residue was chromatographed on silica gel eluting
with a toluene–acetone mixture (9 : 1 → 6 : 4). Product
3 was obtained as a colorless oil in 30% yield (5.39 g),
and also compounds 4 (4.1 g, 23%) and 5 (2.16 g,
12%) were isolated.
b. To a solution of compound 1 (10 g, 27 mmol) in
28 mL of DMSO was added 18.5 mL of a 40%
aqueous solution of sodium hydroxide. After 30 min,
30 mL of an 80% solution of propargyl bromide
(270 mmol) in toluene was added with stirring in one
portion. The mixture was left standing at room
temperature for 3 h. Next, 275 mL of diethyl ether was
added to the reaction mixture. The organic layer was
separated, washed with water (2 × 95 mL), dried with
sodium sulfate, and evaporated at a reduced pressure.
The residue was chromatographed on silica gel in a
petroleum ether–ethyl acetate system (7 : 3 → pure

RUSSIAN JOURNAL OF GENERAL CHEMISTRY Vol. 88 No. 3 2018

 SYNTHESIS OF PENTAERYTHRITOL-BASED BRANCHING REAGENTS
ethyl acetate). Compound 4 was obtained as colorless
oil in a yield of 39% (5.06 g).
In addition, compounds 2 (0.64 g, 5%), 3 (2.59,
20%) and 5 (1.04, 8%) were isolated.
3,3'-[(2-[(3-Hydroxypropoxy)methyl]-2-{[3-(prop-
2-yn-1-yloxy)propoxy]methyl}propane-1,3-diyl)bis-
(hydroxy)]bis(propan-1-ol) (2). Rf 0.17 (CH2Cl2–
MeOH, 19 : 1). 1H NMR spectrum (CD3CN), δ, ppm:
1.69 quintet (6H, CH2, 3JHH = 6.1 Hz), 1.76 m (2H,
CH2), 2.69 t (1H, C≡CH, 4JHH = 2.4 Hz), 3.31 s (2H,
CCH2O), 3.33 s (6H, CCH2O), 3.41 t (2H, CH2, 3JHH =
6.2 Hz), 3.47 t (6H, CH2, 3JHH = 6.0 Hz), 3.53 t (2H,
CH2, 3JHH = 6.5 Hz), 3.57 t (6H, CH2, 3JHH = 6.1 Hz),
4.10 d (2H, CH2C≡CH, 4JHH = 2.4 Hz). 13С NMR spec-
trum (CD3CN), δC, ppm: 29.56, 32.38, 45.09, 57.58,
59.49, 66.79, 67.87, 68.86, 69.59, 69.89, 74.49, 80.24.
3,3'-[(2,2-Bis{[3-(prop-2-yn-1-yloxy)propoxy]-
methyl}propane-1,3-diyl)bis(oxy)]bis(propan-1-ol) (3).
Rf 0.2 (acetone–toluene, 1 : 3). 1H NMR spectrum
(CDCl3), δ, ppm: 1.74–1.79 m (4H, CH2), 1.79–1.85 m
(4H, CH2), 2.42 t (2H, С≡CH, 4JHH = 2.3 Hz), 3.34 s
(4H, 2CCH2O), 3.38 s (4H, 2CCH2O), 3.44 t (4H,
2CH2, 3JHH = 6.2 Hz), 3.53–3.58 m (8H, CH2), 3.72 t
(4H, CH2, 3JHH = 5.4 Hz), 4.11 d (4H, CH2C≡CH, 4JHH =
2.3 Hz). 13C NMR spectrum (CDCl3), δC, ppm: 29.81,
31.83, 45.07, 58.19, 61.85, 67.25, 68.27, 70.20, 70.75,
71.14, 74.34, 80.03.
3-(3-[3-(Prop-2-yn-1-yloxy)propoxy]-2,2-bis-{[3-
(prop-2-yn-1-yloxy)propoxy]methyl}proproxy)-
propan-1-ol (4). Rf 0.47 (CH2Cl2–MeOH, 19 : 1). 1H
NMR spectrum (CD3CN), δ, ppm: 1.70 quintet (2H,
CH2, 3JHH = 6.1 Hz), 1.76 quintet (6H, CH2, 3JHH =
6.3 Hz), 2.68 t (3H, C≡CH, 4JHH = 2.4 Hz), 3.31 s (6H,
CCH2O), 3.33 s (2H, CCH2O), 3.42 t (6H, CH2, 3JHH =
6.2 Hz), 3.47 t (2H, CH2, 3JHH = 6.1 Hz), 3.51–3.59 m
(8H, CH2), 4.10 d (6H, CH2C≡CH, 4JHH = 2.4 Hz). 13С
NMR spectrum (CD3CN), δC, ppm: 30.59, 33.44,
46.26, 58.56, 60.61, 67.81, 68.82, 69.97, 70.36, 70.66,
75.42, 81.23.
10,10-Bis{[3-(prop-2-yn-1-yloxy)propoxy]methyl}-
4,8,12,16-tetraoxanonadeca-1,18-diene (5). Rf 0.85
(acetone–toluene, 1 : 3). 1H NMR spectrum (CD3CN),
δ, ppm: 1.76 quintet (8H, CH2, 3JHH = 6.3 Hz), 2.68 t
(4H, C≡CH, 4JHH = 2.4 Hz), 3.31 s (8H, CCH2O), 3.42
t (8H, CH2, 3JHH = 6.2 Hz), 3.54 t (8H, CH2, 3JHH =
6.5 Hz), 4.10 d (8H, CH2C≡CH, 4JHH = 2.4 Hz). 13C
NMR spectrum (CD3CN), δС, ppm: 30.58, 46.31,
58.54, 67.78, 68.74, 70.14, 75.40, 81.20.
RUSSIAN JOURNAL OF GENERAL CHEMISTRY Vol. 88 No. 3 2018
457
2,2-Bis[(3-propargyloxy)propoxymethyl]-1-
[(propan-3-ol)-1-oxy]-3-(4,4'-dimethoxytrityl)pro-
poxypropane (6). Compound 3 (2.26 g, 5.1 mmol)
was co-evaporated with pyridine (2 × 20 mL) at a
reduced pressure to remove traces of water, then
dissolved in 20 mL of pyridine and cooled in an ice
bath to 0°C. Then, 4,4'-Dmt-Cl (1.58 g, 5.1 mmol) was
added with stirring. The mixture was stirred for 15 h at
room temperature, then pyridine was distilled off in a
vacuum. The residue was diluted with 150 mL of
dichloromethane, washed with 30 mL of water, 30 mL
of a saturated solution of sodium hydrogen carbonate
and 30 mL of a saturated solution of sodium chloride.
The organic phase was dried with anhydrous sodium
sulfate, and the solvent was evaporated in a vacuum.
The residue was chromatographed eluting with toluene–
triethylamine–acetone mixture (99 : 1 : 0 → 59 : 1 : 40).
Yield 1.26 g (49%), pale yellow oil, Rf 0.13 (toluene–
acetone–triethylamine, 94 : 5 : 1). 1.77 quintet (2H,
CH2, 3JHH = 5.6 Hz), 1.81 quintet (4H, CH2, 3JHH =
6.4 Hz), 1.85 quintet (2H, CH2, 3JHH = 6.4 Hz), 2.41 t
(2H, C≡CH, 4JHH = 2.4 Hz), 2.80–2.85 m (1H,
CH2OH), 3.11 t (2H, CH2, 3JHH = 6.4 Hz), 3.31 s (4H,
CCH2O), 3.34 s (2H, CCH2O), 3.35 s (2H, CCH2O),
3.41 t (4H, CH2, 3JHH = 6.4 Hz), 3.49–3.54 m (4H,
CH2), 3.56 t (4H, CH2, 3JHH = 6.4 Hz), 3.69–3.75 m
(2H, CH2), 3.78 s (6H, OCH3), 4.11 d (4H, CH2C≡CH,
4
JHH = 2.4 Hz), 6.80–6.84 m (4H, HAr), 7.17–7.22 m
(2H, HAr), 7.25–7.29 m (1H, HAr), 7.29–7.33 m (4H,
HAr), 7.40–7.45 m (2H, HAr). 13C NMR spectrum
(CDCl3), δС, ppm: 29.84, 30.34, 31.90, 45.23, 55.28,
58.18, 60.71, 62.49, 67.31, 68.17, 68.82, 70.01, 70.12,
71.06, 71.30, 74.32, 80.06, 85.84, 113.06, 126.68,
127.81, 128.28, 130.10, 136.71, 145.40, 158.39.
3-(3-{3-[Bis(4-methoxyphenyl)(phenyl)methoxy]-
propoxy}-2,2-bis{[3-(prop-2-ynyloxy)propoxy]-
methyl}propoxy)propyl-2-cyanoethyldiisopropylphos-
phoramidite (7). Compound 6 (1.26 g, 1.75 mmol)
was co-evaporated with dichloromethane (2 × 20 mL)
at a reduced pressure to remove traces of water, then
dissolved in 20 mL of dichloromethane. DIPEA
(360 μL, 2.1 mmol) and 2-cyanoethoxy-N,N-diisopropyl-
aminochlorophosphine (480 mg, 2.03 mmol) was
added to the solution with stirring. After 1.5 h, the
mixture was diluted to 100 mL with dichloromethane,
washed with a mixture of brine and saturated solution
of sodium hydrogen carbonate (2 × 50 mL), dried with
anhydrous sodium sulfate. The solvent was evaporated
in a vacuum. The residue was chromatographed
eluting with toluene–acetone–triethylamine mixture
458 MARTYNENKO-MAKAEV et al.
(98 : 1 : 1) system. Yield 520 mg (32%), colorless oil,
Rf 0.41 (toluene–acetone-–triethylamine, 94 : 5 : 1). 1H
NMR spectrum (CD3CN), δ, ppm: 1.14 d [6H,
CH(CH3)2, 3JHH = 5.1 Hz], 1.15 d [6H, CH(CH3)2, 3JHH =
5.1 Hz], 1.74 quintet (2H, CH2, 3JHH = 6.4 Hz), 1.70
quintet (4H, CH2, 3JHH = 6.4 Hz), 1.78 quintet (2H,
CH2, 3JHH = 6.1 Hz), 2.62 t (2H, CH2CN, 3JHH =
5.9 Hz), 2.66 t (2H, С≡CH, 4JHH = 2.3 Hz), 3.08 t (2H,
CH2, 3JHH = 6.4 Hz), 3.22 s (4H, CCH2O), 3.23 s (2H,
CCH2O), 3.26 s (2H, CCH2O), 3.27 m (2H, CH2), 3.34
t (4H, CH2, 3JHH = 6.3 Hz), 3.38 t (2H, CH2, 3JHH =
6.1 Hz), 3.46 t (2H, CH2, 3JHH = 6.1 Hz), 3.50 t (4H,
CH2, 3JHH = 6.3 Hz), 3.54–3.70 m [4H, CH(CH3)2,
OCH2CH2CN], 3.75 s (6H, OCH3), 4.08 d (4H,
CH2С≡CH, 4JHH = 2.3 Hz), 6.83–6.88 m (4H, HAr),
7.19–7.24 m (1H, HAr), 7.20–7.27 m (6H, HAr), 7.41–
7.44 m (2H, HAr). 13C NMR spectrum (CD3CN), δC,
ppm: 30.56, 30.98, 32.26, 46.26, 55.87, 58.53, 59.22,
59.37, 61.32, 61.55, 67.75, 68.48, 68.70, 69.00, 70.08,
70.20, 75.42, 81.18, 113.94, 127.64, 128.74, 128.96,
129.30, 130.89, 137.52, 146.60. 31P NMR spectrum
(CD3CN): δР 164.47 ppm.
Modification of controlled pore glass with
dialkyne compound 6. To a solution of 6 in a mixture
of anhydrous pyridine (2 mL) and dimethylformamide
(2 mL) were added DMAP (17 mg, 0.14 mmol),
255 mg CPG with carboxy groups on the surface, and
diisopropylcarbodiimide (240 μL, 1.51 mmol). The
mixture was kept for 48 h at room temperature, then a
solution of pentafluorophenol (150 mg, 0.81 mmol) in
1 mL of pyridine was added. After 24 h the solid phase
support was filtered off, washed with 10 mL of
pyridine, 10 mL of acetonitrile, and dried in a vacuum.
230 mg of modified glass 8 were obtained with a
specific content of modifying reagent of 35.5 μmol/g.
General procedure for determining the specific
content of the modifying reagent in a solid-phase
support. The modified solid-phase support was
suspended in a solution of trifluoroacetic acid in
methylene chloride (25 mL, 3% by volume) and
stirred. Optical density of the solution was measured at
505 nm using the formula:
L = 1000AV/εm,
where A is the optical density, V is the solution
volume (mL), ε is the molar extinction coefficient
(78 000 L mol–1 cm–1), m is the sample mass (g).
6,6-Bis{[3-(prop-2-ynyloxy)propoxy]methyl}-
4,8,12,16-tetraoxanonadec-18-ynyl-2-cyanoethyldiiso-
RUSSIAN JOURNAL OF GENERAL CHEMISTRY Vol. 88 No. 3 2018
propylphosphoramidite (9). Compound 4 (1.52 g,
3.16 mmol) was co-evaporated with dichloromethane
(2 × 40 mL) at a reduced pressure to remove traces of
water, then dissolved in 40 mL of anhydrous
methylene chloride, and diisopropylethylamine
(0.686 mL, 3.95 mmol) was added under an argon
atmosphere. 2-Cyanoethoxy-N,N-diisopropylamino-
chloro-phosphine (0.86 g, 3.64 mmol) was added in
one portion with stirring at room temperature. After
1 h, the reaction mixture was diluted with 20 mL of
methylene chloride, washed with a saturated solution
of sodium hydrogen carbonate (5 × 40 mL), then with
a saturated sodium chloride solution (40 mL). The
organic layer was dried for 1 h with stirring over
sodium sulfate. The desiccant was filtered off and the
solvents were removed at a reduced pressure. The
mixture was chromatographed on 75 mL of silica gel
eluting with toluene–triethylamine mixture (19 : 1).
Yield 1.6 g (75%), Rf 0.77 (toluene–acetone–
triethylamine, 18 : 1 : 1). 1H NMR spectrum (CD3CN),
δ, ppm: 1.17 d [12H, CH(CH3)2, 3JHH = 6.8 Hz], 1.78
m (8H, CH2), 2.64 t (2H, CH2CN, 3JHH = 6.0 Hz), 2.68
t (3H, С≡CH, 4JHH = 2.4 Hz), 3.32 m (8H, CCH2O),
3.37–3.48 m (8H, CH2), 3.54 т (6H, CH2, 3JHH =
6.5 Hz), 3.56–3.85 m [6H, CH2, OCH2CH2CN,
CH(CH3)2], 4.10 d (6H, CH2С≡CH, 4JHH = 2.4 Hz). 13C
NMR spectrum (CD3CN), δC, ppm: 30.57, 32.22,
32.28, 43.69, 43.79, 46.30, 58.54, 58.54, 61.34, 61.48,
67.77, 68.54, 68.75, 70.22, 75.42, 81.18, 119.58. 31P
NMR spectrum (CD3CN): δP 146.96 ppm.
tert-Butyl 10,10-bis[(3-(propyn-2-yl-1-oxy)pro-
poxy)methyl]-4,8,12,16-tetraoxanonadecane-18-oate
(10). To a solution of compound 4 (2 g, 4.15 mmol) in
3.3 mL of DMSO was added 0.207 mL of 5 M
aqueous solution of sodium hydroxide. After 15 min,
t-butyl acrylate (1.21 mL, 8.3 mmol) was added in one
portion with stirring. The reaction mixture was stirred
overnight at room temperature. The reaction mixture
was diluted three times with water and washed once
with ethyl acetate (5 mL). The organic phase was
washed with water (2 mL) and saturated sodium
chloride solution (2 mL). The organic layer was dried
with sodium sulfate, then the solvent was removed at a
reduced pressure. The residue was chromatographed
on silica gel eluting with methylene chloride–methanol
mixture (100 : 0 → 97 : 3). Yield 1.64 g (65%), Rf 0.67
(CH2Cl2–MeOH, 24 : 1). 1H NMR spectrum (CD3CN),
δ, ppm: 1.43 s [9H, C(CH3)3], 1.69–1.80 m (8H, CH2),
2.68 t (3H, С≡CH, 4JHH = 2.2 Hz), 3.30 s (2H,
CCH2O), 3.31 s (6H, CCH2O), 3.38–3.47 m (12H,
 SYNTHESIS OF PENTAERYTHRITOL-BASED BRANCHING REAGENTS
CH2), 3.54 t (6H, CH2, 3JHH = 6.4 Hz), 3.59 t (2H,
CH2, 3JHH = 6.2 Hz), 4.10 d (6H, CH2С≡CH, 4JHH =
2.2 Hz). 13C NMR spectrum (CD3CN), δC, ppm: 28.35,
30.63, 30.77, 37.17, 46.34, 58.57, 67.25, 67.82, 68.53,
68.80, 68.92, 70.18, 70.24, 75.42, 80.86, 81.23,
171.86.
10,10-Bis{[3-(propyn-2-yl-1-hydroxy)propoxy]-
methyl}-4,8,12,16-tetraoxanonadecyne-18-oic acid
(11). To a solution of compound 10 (1.46 g, 2.4 mmol)
in 14 mL of methylene chloride was added 5.6 mL of
trifluoroacetic acid. The reaction mixture was stirred
overnight at room temperature. The solvent was
removed, and the reaction mixture was co-evaporated
with toluene (2 × 20 mL) and dried in a vacuum. Yield
1.301 g (98%), Rf 0.59 (CH2Cl2–MeOH, 19 : 1). The
product 11 was used in subsequent conversions
without further purification.
1-[(2S,4R)-2-{[Bis(4-methoxyphenyl)(phenyl)
methoxy]methyl}-4-hydroxypyrrolidin-1-yl]-10,10-
bis{[3-(propyn-2-yl-1-oxy)propoxy]methyl}-
4,8,12,16-tetraoxanonadec-18-yne-1-one (12).
DIPEA (1.07 ml, 6.1 mmol), 1-hydroxybenzotriazole
(933 mg, 6.1 mmol), N-(3-dimethylaminopropyl)-N'-
ethylcarbodiimide hydrochloride (778 mg, 4.06 mmol)
and (3R,5S)-5-{[bis(4-methoxyphenyl)(phenyl)
methoxy]methyl}pyrrolidine-3-ol (1.024 g, 2.44 mmol)
were added to a solution of compound 11 (1.126 g,
2.03 mmol) in 25 mL of anhydrous dichloromethane.
The reaction mixture was stirred overnight at room
temperature. The solvents were removed at a reduced
pressure. The resulting mixture was diluted with
120 mL of ethyl acetate and washed with water (2 ×
40 mL), then with 5% citric acid solution (2 × 40 mL),
with saturated sodium hydrogen carbonate solution
(2 × 40 mL), and saturated sodium chloride solution
(40 mL). The organic layer was dried with sodium
sulfate, the solvents were removed at a reduced
pressure. The mixture was chromatographed on silica
gel eluying with dichloromethane–triethylamine–
methanol mixture (100 : 1 : 0 → 90 : 1 : 9). Yield 1.46
g (75%), Rf 0.59 (CH2Cl2–MeOH, 19 : 1). 1H NMR
spectrum (CD3CN), δ, ppm: 1.68–1.79 m (8H, CH2),
2.40–2.57 m (2H, CH2), 2.68 t (3H, С≡CH, 4JHH =
2.4 Hz), 3.03–3.18 m (2H, CH2), 3.22–3.27 m (1H,
CH), 3.29 s (2H, CCH2O), 3.30 s (6H, CCH2O), 3.33–
3.44 m (13H, CH2, CH) 3.46 t (2H, CH2, 3JHH = 6.5
Hz), 3.53 t (6H, 3CH2, 3JHH = 6.5 Hz), 3.63 t (2H, CH2,
3
JHH = 6.5 Hz), 3.76 s (6H, OCH3), 4.10 d (6H,
CH2С≡CH, 4JHH = 2.4 Hz), 6.81–6.89 m (4H, HAr),
7.18–7.42 m (9H, HAr). 13C NMR spectrum (CD3CN),
RUSSIAN JOURNAL OF GENERAL CHEMISTRY Vol. 88 No. 3 2018
459
δC, ppm: 30.63, 30.78, 34.72, 36.17, 37.42, 38.96,
46.35, 55.92, 55.95, 58.57, 64.44, 67.54, 67.83, 67.83,
68.66, 68.81, 68.99, 69.43, 70.23, 70.50, 70.50, 75.46,
81.24, 114.00, 114.13, 127.73, 127.87, 128.78, 128.89,
128.95, 130.96, 159.59.
(3R,5S)-1-(10,10-Bis{[3-(propyn-2-yl-1-hydroxy)-
propoxy]methyl}-4,8,12,16-tetraoxanonadecan-18-
oyl)-5-{[bis(4-methoxyphenyl)(phenyl)methoxysilyl-
methyl}pyrrolidin-3-yl-(2-cyanoethyl)diisopropylphos-
phoramidite (13). Compound 12 (0.836 g,
0.875 mmol) was co-evaporated with dichloromethane
(2 × 10 mL) at a reduced pressure to remove traces of
water, then dissolved in 11 mL of anhydrous
methylene chloride. Next, 190 μL of diisopropyl-
ethylamine was added, and then 2-cyanoethoxy-N,N-
diisopropylaminochlorophosphine (238 mg, 1.01 mmol)
was added in one portion with stirring. After 30 min,
100 mg (0.42 mmol) of 2-cyanoethoxy-N,N-diiso-
propylaminochlorophosphine was added. After the
reaction completed the mixture was diluted with
20 mL of methylene chloride, washed with a saturated
solution of sodium hydrogen carbonate (10 × 2 mL),
saturated sodium chloride solution (10 mL), and dried
for 1 h with stirring over sodium sulfate. After
filtration and evaporation the product was chromato-
graphed on silica gel eluting with toluene–acetone–
triethylamine mixture (14 : 5 : 1). Yield 0.829 mg
(82%), Rf 0.66 (toluene–acetone–triethylamine,
80 : 15 : 5). 1H NMR spectrum (CD3CN), δ, ppm: 1.12–
1.19 m [12H, CH(CH3)2], 1.63–1.72 m (2H, CH2), 1.75
quintet (6H, CH2, 3JHH = 6.3 Hz), 2.47–2.52 m (2H,
CH2), 2.60–2.65 m (2H, CH2CN), 2.68 t (3H, С≡CH,
4
JHH = 2.4 Hz), 3.01–3.10 m (2H, CH2), 3.24–3.28 m
(1H, CH), 3.29 s (2H, CCH2O), 3.30 s (6H, CCH2O),
3.33–3.39 m (7H, CH2, CH), 3.41 t (6H, CH2, 3JHH =
6.2 Hz), 3.44 t (2H, CH2, 3JHH = 6.5 Hz), 3.53 t (6H,
CH2, 3JHH = 6.5 Hz), 3.56–3.65 m [6H, CH2,
OCH2CH2CN, CH(CH3)2], 3.77 s (6H, OCH3), 4.09 d
(6H, CH2С≡CH, 4JHH = 2.4 Hz), 6.82–6.88 m (4H,
HAr), 7.33–7.13 m (9H, HAr). 13C NMR spectrum
(CD3CN), δC, ppm: 30.59, 30.76, 30.90, 34.71, 36.12,
36.62, 36.73, 43.92, 44.02, 46.30, 55.32, 55.89, 55.92,
58.55, 64.44, 67.43, 67.65, 67.78, 68.57, 68.64, 68.76,
68.95, 70.16, 75.47, 81.21, 113.98, 114.11, 119.58,
127.72, 127.86, 128.79, 128.90, 128.96, 130.93,
159.61. 31P NMR spectrum (CD3CN): δР 147.40 ppm.
Modification of controlled pore glass with tri-
alkyne compound 12. To a solution of 12 in a mixture
of anhydrous pyridine (1.2 mL) and dimethylform-
amide (1.2 mL) were added DMAP (0.047 g, 383 mmol),
460 MARTYNENKO-MAKAEV et al.
393 mg of CPG with carboxy groups on the surface
and diisopropylcarbodiimide (920 μL, 5.895 mmol).
The suspension was kept for 3 days at room
temperature in a dark, after which pentafluorophenol
(325 mg, 1.77 mmol) was added. After 24 h, the solid
was filtered off and washed successively with 10 mL
of pyridine, 10 mL of acetonitrile, 10 mL of tetrahyd-
rofuran, 10 mL of acetone, and 10 mL of methylene
chloride. The solvents were removed at a reduced
pressure. 395 mg of the modified solid-phase support
14 were obtained with a specific content of the
modifying reagent of 49 μmol/g.
2,5-Dioxopyrrolidin-1-yl-10,10-bis{[3-(propyn-2-
yl-1-hydroxy)propoxy]methyl}-4,8,12,16-tetraoxano-
nadecyne-18-oate (15). DMAP (127 mg, 1.04 mmol)
and bis(2,5-dioxopyrrolidin-1-yl) carbonate (279 mg,
1.04 mmol) were added to a solution of compound 11
(175 mg, 0.315 mmol) in anhydrous dichloromethane
(10 mL). The mixture was stirred at room temperature
overnight, then diluted with 100 mL of methylene
chloride and washed rapidly with water (3 × 30 mL),
30 mL of saturated sodium chloride solution and dried
with sodium sulfate. The solvent was evaporated, and
the residue was chromatographed on silica gel eluting
with methylene chloride–methanol mixture (100 : 0 →
95 : 5). Yield 109 mg (53%), Rf 0.57 (CH2Cl2–MeOH,
93 : 7). 1H NMR spectrum (DMSO-d6), δ, ppm: 1.66–
1.75 m (8H, CH2), 2.84–2.87 m (3H, С≡CH), 2.90 t
(2H, CH2, 3JHH = 5.9 Hz), 3.26 s (8H, CCH2O), 3.35–
3.41 m (12H, CH2), 3.42–3.52 m (8H, CH2), 3.66 t
(2H, CH2, 3JHH = 5.9 Hz), 4.09 d (6H, CH2С≡CH, 4JHH =
2.4 Hz). 13C NMR spectrum (DMSO-d6), δC, ppm:
25.45, 29.27, 29.33, 31.62, 45.03, 57.39, 64.91, 66.33,
67.38, 67.56, 67.82, 68.65, 68.94, 76.88, 80.47, 167.38,
170.12.
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