Oligonucleotide functionalization by a novel alkyne-modified nonnucleosidic reagent obtained by versatile building block chemistry

Article abstract of DOI:10.1080/15257770.2013.787147

A convenient synthetic strategy has been designed to prepare an alkyne-modified synthon for automated DNA synthesis. It is based on the key O-DMTr-protected 4-(2-hydroxyethyl)morpholin-2,3-dione and building blocks obtained by its functionalization by var

Full text of DOI:10.1080/15257770.2013.787147

This article was downloaded by: [Florida State University]
On: 04 May 2013, At: 07:59
Publisher: Taylor & Francis
Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered
office: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK
Nucleosides, Nucleotides and Nucleic
Acids
Publication details, including instructions for authors and
subscription information:
http://www.tandfonline.com/loi/lncn20
Oligonucleotide Functionalization by a
Novel Alkyne-Modified Nonnucleosidic
Reagent Obtained by Versatile Building
Block Chemistry
Maxim S. Kupryushkin a , Dmitrii A. Konevetz a , Svetlana V. Vasilyeva
a , Anastasia S. Kuznetsova b , Dmitry A. Stetsenko a & Dmitrii V.
Pyshnyi a c
a Institute of Chemical Biology and Fundamental Medicine, Siberian
Branch of the Russian Academy of Sciences , Novosibirsk , Russia
b Astafiev Krasnoyarsk State Pedagogical University , Krasnoyarsk ,
Russia
c Novosibirsk State University , Novosibirsk , Russia
To cite this article: Maxim S. Kupryushkin , Dmitrii A. Konevetz , Svetlana V. Vasilyeva , Anastasia
S. Kuznetsova , Dmitry A. Stetsenko & Dmitrii V. Pyshnyi (2013): Oligonucleotide Functionalization
by a Novel Alkyne-Modified Nonnucleosidic Reagent Obtained by Versatile Building Block Chemistry,
Nucleosides, Nucleotides and Nucleic Acids, 32:6, 306-319
To link to this article: http://dx.doi.org/10.1080/15257770.2013.787147
PLEASE SCROLL DOWN FOR ARTICLE
Full terms and conditions of use: http://www.tandfonline.com/page/terms-and-conditions
This article may be used for research, teaching, and private study purposes. Any
substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing,
systematic supply, or distribution in any form to anyone is expressly forbidden.
The publisher does not give any warranty express or implied or make any representation
that the contents will be complete or accurate or up to date. The accuracy of any
instructions, formulae, and drug doses should be independently verified with primary
sources. The publisher shall not be liable for any loss, actions, claims, proceedings,
demand, or costs or damages whatsoever or howsoever caused arising directly or
indirectly in connection with or arising out of the use of this material.

Nucleosides, Nucleotides and Nucleic Acids, 32:306–319, 2013
Copyright © Institute of Chemical Biology and Fundamental Medicine,
Siberian Branch of the Russian Academy of Sciences
ISSN: 1525-7770 print / 1532-2335 online
DOI: 10.1080/15257770.2013.787147
OLIGONUCLEOTIDE FUNCTIONALIZATION BY A NOVEL
ALKYNE-MODIFIED NONNUCLEOSIDIC REAGENT OBTAINED BY
VERSATILE BUILDING BLOCK CHEMISTRY
Maxim S. Kupryushkin,1 Dmitrii A. Konevetz,1 † Svetlana V. Vasilyeva,1
Anastasia S. Kuznetsova,2 Dmitry A. Stetsenko,1 and Dmitrii V. Pyshnyi1,3
1Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian
Academy of Sciences, Novosibirsk, Russia
2Astafiev Krasnoyarsk State Pedagogical University, Krasnoyarsk, Russia
3Novosibirsk State University, Novosibirsk, Russia
A convenient synthetic strategy has been designed to prepare an alkyne-modified synthon for au-
2
tomated DNA synthesis. It is based on the key O-DMTr-protected 4-(2-hydroxyethyl)morpholin-2,3-
dione and building blocks obtained by its functionalization by various aliphatic amines. A respective
nonnucleosidic phosphoramidite monomer containing a terminal alkyne in the side-chain was syn-
thesized, and corresponding oligothymidylates incorporating the modification in various positions
were prepared. The presence of the alkyne group was confirmed by Cu(I)-catalyzed azide–alkyne
cycloaddition (CuAAC) between the functionalized oligonucleotide and an azide derivative of 7-
nitro-2,1,3-benzoxadiazole.
Keywords
Nonnucleosidic phosphoramidite; fluorescent oligonucleotide probe;
alkyne-azide click chemistry
INTRODUCTION
Modified oligonucleotides are widely used as molecular tools for cutting-
edge research in life sciences, in particular, in molecular diagnostics. Auto-
mated solid-phase phosphoramidite DNA synthesis is an established method
for oligonucleotide preparation. A majority of chemical modifications can
be routinely introduced into DNA sequences during the synthesis by using
nonnucleoside phosphoramidite monomers bearing the required functional
groups.[1–3] It should be noted that the preparation of those monomers may
be labor-consuming in some cases. Thus, the development of versatile and
Received 13 February 2013; accepted 14 March 2013.
†Deceased 25.10.2011.
Address correspondence to Dr Dmitrii Pyshnyi, Doctor of Science, Laboratory of Bionanotech-
nology, Institute of Chemical Biology and Fundamental Medicine of the Siberian Branch of the Rus-
sian Academy of Sciences (SB RAS), Lavrenteva Ave 8, Novosibirsk 630090, Russia. E-mail: pyshnyi@
niboch.nsc.ru
306

Oligonucleotide Functionalization
307
convenient methods for the preparation of nonnucleoside phosphoramidite
synthons equipped with desired functionalities is an important task.
We describe herein a novel approach to the design of nonnucleosidic
DNA functionalization reagents based on a merger of two methodologies:
one utilizing the ring-opening of lactones by aliphatic amines [4] and another
that uses activated oxalate esters[5] for oligonucleotide synthesis and deriva-
tization. It also takes into account our previous experience in the synthesis
and application of oligonucleotides with nonnucleotidic inserts.[6]
We have applied our new building block strategy for the preparation
of achiral nonnucleoside phosphoramidite synthons bearing various func-
tional groups.[7,8] In accordance with the proposed approach, two separately
functionalized building blocks have been synthesized: a core block bearing
functional groups for incorporation into DNA chain and a functional block
bearing functional groups required to obtain oligonucleotide derivatives
with specific properties. The core block can be used as a common reagent
for the preparation of a family of monomers with varying functionalities.
In each case, DNA modification would be accomplished by using an appro-
priately functionalized synthon. The structure of a functional block would
be defined by the desired oligonucleotide modification. An aliphatic amine
bearing the required functionality would be a suitable candidate for a func-
tional block. Coupling of the two building blocks is performed immediately
before the final phosphitylation step that yields a nonnucleosidic phospho-
ramidite monomer (Scheme 1).
SCHEME 1 A general scheme of building block strategy for the preparation of nonnucleoside
phosphoramidite monomers. R, R1—a desired functional group or H, CEP–2-cyanoethoxy-N ,N -
diisopropylaminophosphinyl.
In this work, we demonstrate the applicability of our approach to the
preparation of a phosphoramidite synthon suitable for the introduction of
a terminal alkyne group into either terminal or internal positions within an
oligonucleotide chain. The alkyne moiety is widely used in click chemistry[9]
allowing for mild postsynthetic conjugation with functionalized azides via
copper(I)-catalyzed azide–alkyne cycloaddition (CuAAC).

308
M. S. Kupryushkin et al.
RESULTS AND DISCUSSION
Preparation of Click Chemistry Components
Synthesis of the Core Block
Synthesis of the core block 2 was carried out in two high-yielding steps
(Scheme 2). A reaction between diethanolamine and diethyl oxalate results
in a key lactone 4-(2-hydroxyethyl)morpholin-2,3-dione (1).
SCHEME 2 Synthesis of the core block. (i) diethyl oxalate, PriOH; (ii) DMTrCl,pyridine.
Very small excess of 4,4ꢀ-dimethoxytrityl chloride (DMTrCl) (1.05 equiv.)
was enough to quantitatively convert 1 into a monodimethoxytritylated core
block (2). A convenient extraction without column chromatography was
sufficient. Trace amount of DMTrOH in 2 does not interfere with further re-
actions (data not shown). It should be noted that extraction with aq NaHCO3
results in quantitative hydrolysis of the lactone 2. To avoid this, phosphate
buffer (0.3 M KH2PO4, pH 6.5) was used as an aqueous phase.
Synthesis of an Alkyne Functional Block
Synthesis of a functional block 4-(2-propynyloxy)butylamine bearing the
terminal alkyne group was carried out in accordance with a previously pub-
lished protocol[10] starting from 1,4-butanediol and 1,2,3-tribromopropane
(Scheme 3).
SCHEME 3 Synthesis of the alkyne-containing functional block 4-(2-propynyloxy)butylamine: (i) 1,4-
butanediol, KOH, ButOH; (ii) SOCl2; (iii) CF3CONH2, Cs2CO3, DMF; (iv) aq NaOH.

Oligonucleotide Functionalization
309
Synthesis of an Azide-Functionalized Fluorophore
As a fluorophore for the postsynthetic labeling of oligonucleotides via
the CuAAC reaction, we have used an azido derivative of 7-nitro-2,1,3-
benzoxadiazole.[11] The compound was prepared by a series of transfor-
mations from commercially available 4-chloro-7-nitro-2,1,3-benzoxadiazole
(Scheme 4). The overall yield of the reaction was acceptable due to opti-
mization of procedures.
SCHEME 4 Synthesis of N-(2-azidoethyl)-N -methyl-7-nitro-2,1,3-benzoxadiazol-4-ylamine. (i) N -methy-
laminoethanol, EtOH, 20◦C, 40 minutes; (ii) PhSO2Cl, pyridine, 90◦C, 5 hours; (iii) NaN3, DMSO, 65◦C,
8 hours.
Preparation of an Alkyne-Functionalized Phosphoramidite Synthon
The reaction between the core block (2) and functional block (4) has
produced an alkyne-functionalized alcohol 8. Noteworthy, such a conversion
does not require any condensing agents. After the phosphitylation of the
alcohol (8), we have obtained the required phosphoramidite monomer 9
for the incorporation of a terminal alkyne into oligonucleotides (Scheme 5).
SCHEME 5 A scheme for the preparation of an alkyne-functionalized phosphoramidite. (i)
DMAP, pyridine; (ii) 2-cyanoethyl-N ,N ,N ꢀ,N ꢀ-tetraisopropylphosphordiamidite, 1H-tetrazole, N ,N -
diisopropylethylamine, MeCN.
The obtained monomer 9 is derived from diethanolamine, which
is a convenient scaffold for construction of nonnucleosidic inserts for
oligonucleotide functionalization. There are two published approaches
to prepare diethanolamine-based nonnucleosidic synthons. One requires

310
M. S. Kupryushkin et al.
prior functionalization of the amino group of diethanolamine by a de-
sired substituent followed by dimethoxytritylation and phosphitylation
of the obtained symmetrical diol.[12–14] Another approach utilizes initial
O-monodimethoxytritylation of diethanolamine followed by N -function
alization and phosphitylation.[15] In some cases, additional temporary pro-
tection for another hydroxyl group of diethanolamine is required to in-
crease the yield of the desired synthon. Approaches of both type require the
preparation of intermediate mono-O-protected diol precursors that are ac-
companied by inevitable formation of substantial amount of di-O-substituted
byproduct.
Our scheme of conversion of diethanolamine into compound 1 solves
this problem by simultaneous N -functionalization and O-activation. The core
precursor 2 already has a temporary ester protecting group for one of the two
hydroxyl group of diethanolamine, which is stable under neutral conditions
and yet able to react with primary and secondary aliphatic amines without any
additional activation.[7] Highly specific acylation of an appropriately func-
tionalized aliphatic amine with concomitant deprotection of the hydroxyl
group followed by final phosphitylation step furnishes the desired phospho-
ramidite synthon. The results obtained allow us to conclude that the func-
tionalized synthon obtained by our approach could be inserted into any po-
sition of an oligonucleotide chain by standard automated synthesis protocol.
Synthesis of Alkyne-Tethered Oligonucleotides and Their
Fluorescent Labeling via Click Chemistry
By the use of monomer 9 (Scheme 5), we have been able to obtain
model oligonucleotides functionalized with alkyne-containing nonnucleo-
tidic inserts at either internal or terminal position and an oligonucleotide
post-synthetically labeled with an azide-tethered fluorophore.
It was shown that compound 9 effectively reacts with the growing DNA
chain under standard coupling conditions in a DNA synthesizer. A series of
oligothymidylates bearing the alkyne group at either terminal or internal
position was obtained in good yields including an example of three con-
secutive incorporations of the modification. All the oligonucleotides after
deprotection followed by RP-HPLC purification were analyzed for homo-
geneity by denaturing gel electrophoresis visualized by staining with Stains-
AllꢁR . MALDI-TOF mass-spectrometry was applied to analyze the molecular
mass of the products obtained (Table 1).
Various conditions for cleaving the oligomers from solid support
and deprotecting them were utilized. There was no degradation of the
alkyne-containing oligothymidylates when either mild (tert-butylamine in aq
methanol) or standard (conc aq ammonia) protocols were applied. The only
exception was the case of the 5ꢀ-modified oligonucleotide which was freed
from the 5ꢀ-terminal DMTr group before alkaline treatment. A product with

Oligonucleotide Functionalization
311
TABLE 1 MALDI TOF analysis of the oligonucleotides prepared
Results of MALDI-mass analysis (found values), m/z
Sequence
Exact mass4
Positive mode
Ion form
Negative mode
Ion form
1
XT6
XT6
XT6
2412
2110
2110
3108
3022
2110
2373
2149.1
2149.0
1930.5
3111.9
3024.6
2111.0
2374.6
[M − DMTr + K]+
[M + K]+
2411.0
2109.3
1928.0
3107.1
3024.4
2108.7
2373.8
[M − H]−
[M − H]−
[M − H]−
[M − H]−
[M − H]−
[M − H]−
[M − H]−
2
3
[M + H]+
1
X3T6
[M + H]+
T3XT6
T3XT3
T3YT3
[M + H]+
[M + H]+
[M + H]+
1
Synthesized in the DMTr-on mode and analyzed by MALDI-TOF after purification without manual
detritylation.
2
Synthesized in the DMTr-on mode and analyzed by MALDI-TOF after purification and manual
detritylation.
3 Synthesized in the DMTr-off mode and analyzed by MALDI-TOF after purification.
4 The sum of atomic masses of the most common isotope of each element in the compound calculated
ꢁR
using ISISDraw software.
X, Y—nonnucleosidic insert introduced into the oligonucleotide using phosphoramidite 9 before (X)
and after (Y) click reaction. For structures see Scheme 7.
the expected mass was not detected during RP-HPLC purification. A similar
result has been observed earlier with oligonucleotides modified at the 5ꢀ-end
by a substituted aminoethanol residue.[14] We ascribe the observed degra-
dation of the oxalamide proximal to O-unprotected ethanolamine during
alkaline deprotection step to anchimeric effect (Scheme 6). In contrast with
previous results,[14] we have been able to identify the product of such a
base-catalyzed degradation. It is the oligonucleotide derivative bearing the
5ꢀ-terminal N -deacylated diethanolamine monophosphate, i.e. the product
of oxalamide bond cleavage assisted by the neighboring free hydroxyl group.
In contrast, di-O-phosphorylated N -oxalyl diethanolamine in the middle of
an oligonucleotide is not sensitive to alkaline treatment. MALDI-TOF data
provide the evidence of the reaction (see Table 1 and footnotes therein).
However, intact 5ꢀ-alkyne-tethered oligonucleotides can be obtained by re-
taining the 5ꢀ-terminal DMTr group during ammonia deprotection step and
removing it afterwards by aqueous acetic acid treatment (80% AcOH, 10 min-
utes, ambient temperature) (Table 1). The product is stable under neutral
condition and can be easily purified by RP-HPLC.
SCHEME 6 Degradation of the oxalyl diethanolamine unit at the 5ꢀ-end during ammonia deprotection.

312
M. S. Kupryushkin et al.
SCHEME 7 Scheme of click chemistry conjugation. R—oligonucleotide, R1 —oligonucleotide or
hydrogen.
Conjugation of Alkyne-Functionalized Oligonucleotide
with Azide-Tethered Fluorophore
An alkyne-tethered oligonucleotide containing the nonnucleotidic insert
in the middle of the sequence T3XT3 was chosen for the postsynthetic con-
jugation with a model azide by click chemistry. A deprotected and desalted
FIGURE 1 HPLC profiles of oligonucleotide derivatives: lines 1 and 3— labeled oligonucleotide T3YT3;
line 2—alkyne-modified precursor oligonucleotide T3XT3. Lines 1 and 2 recorded at 260 nm. Line 3
recorded by fluorescence detector λem 550 nm, λex 450 nm. Insert: Spectral analysis of the oligonucleotide
T3YT3. Fluorescence spectra (left axis), excitation (λ 540 nm, line 1), and emission (λ 480 nm, line
em
ex
2). Line 3: UV-Vis spectrum of the product obtained by click reaction (right axis).

Oligonucleotide Functionalization
313
sample of alkyne-modified oligomer reacts very efficiently with azidofluo-
rophore (7). The reaction was carried out in the presence of TBTA (tris-
[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine) ligand (for full protocol see
Experimental section). According to gel electrophoresis (data not shown)
and HPLC, the yield of click conjugation was ∼90%. MALDI-TOF, UV/VIS
and fluorescence spectra allow us to conclude that the fluorophore was suc-
cessfully incorporated into the model oligonucleotide (Scheme 7, Figure 1,
Table 1).
Conjugation of azide-substituted 2,1,3-benzoxadiazole with alkyne-tethe
red oligomer slightly increases the hydrophobicity of the oligonucleotide.
UV-VIS spectra of the product obtained display two characteristic maxima
at 350 and 490 nm, which are typical for substituted 2,1,3-benzoxadiazole.
Emission and excitation spectra of the labeled conjugate show the expected
fluorescent behavior of the incorporated fluorophore.[11]
CONCLUSIONS
The data obtained show that our building block approach to the prepa-
ration of nonnucleosidic reagents is both versatile and convenient. A novel
alkyne-modified phosphoramidite monomer suitable for terminal as well as
internal oligonucleotide modification by achiral diethanolamine units was
synthesized. The proposed synthetic scheme is efficient and allows for high-
yielding preparation of the target product. The synthesis of a functional
block which is an alkyne-functionalized aliphatic amine would be the most
time-consuming step of the synthesis. The core lactone block can be pre-
pared in bulk quantity and stored until needed. The yields are commonly
in the range of 60–90% (see Experimental section). Alkyne-functionalized
oligothymidylates and a conjugate with an azide-containing fluorescent dye
were obtained. These results together with our earlier reports[7,8] indicate
that a promising technique for DNA modification has been designed. It
could be easily applied for the synthesis of various functionalized nonnucle-
osidic phosphoramidites. This would be the aim of our further work, which
would be published in due course.
EXPERIMENTAL
General Information
All the reagents and solvents used were of the highest available purity
obtainable from their respective commercial supplier. 1,2,3-Tribromopro
pane, cesium carbonate, trifluoroacetamide, thionyl chloride, 1,4-butane
diol, benzenesulfonyl chloride, diethanolamine were purchased from Sigma-
Aldrich (USA). 4-Chloro-7-nitro-2,1,3-benzoxadiazole (NBD-Cl, 98%),

314
M. S. Kupryushkin et al.
1H-tetrazole, N ,N -diisopropylethylamine, diethyl oxalate were from
Acros Organics (USA). 4-N ,N -dimethylaminopyridine, triethylamine, N -
methylimidazole were bought from Fluka (Switzerland). 4,4ꢀ-Dimethoxytrityl
chloride was from ChemGenes (USA). Sodium azide was from Serva
(Germany). Nucleoside 3ꢀ-β-cyanoethyl-N ,N -diisopropyl phosphoramidites
were obtained from Glen Research (USA). 2-Cyanoethyl-N ,N ,N ꢀ,N ꢀ-
tetraisopropylphosphordiamidite was a generous gift of Dr V. A. Ryabinin
(LMC, ICBFM SB RAS). Solvents were dried by standard methods and stored
˚
over either 3 A molecular sieves or calcium hydride pellets.
1H, 13C, and 31P NMR spectra were recorded on Bruker AV-300, AV-
400, and DRX-500 NMR spectrometers. Chemical shifts (δ) are reported in
ppm relative to residual solvent signals. For 31P NMR, external standard
of 85% aq H3PO4 was used. Coupling constant (J ) values are reported
in Hertz (Hz), and signal multiplicities are given as s (singlet), d (dou-
blet), t (triplet), q (quartet), m (multiplet), app.t (apparent triplet), and br
(broad). Matrix-assisted laser desorption ionization—time of flight (MALDI-
TOF) mass spectra were run on Reflex III, Autoflex Speed (Bruker, Ger-
many) with 3-hydroxypicolinic acid as a matrix. DFS High Resolution GC-MS
and Finnigan MAT 8200 mass spectrometers were also used for molecular
mass measurements. Processing of mass analysis results was performed using
mMass software.[16] Elemental analyses were performed on EURO EA 3000.
The presence of the azide group was confirmed by recording IR spectra
on a Bruker Tensor 27 IR-spectrometer. Emission and excitation spectra
were recorded on Cary Eclipse (Agilent, USA) using 10−6 M solution of the
oligonucleotide T3YT3. Processing of analytical results was performed using
Spin-Works software. Spectra were recorded at the Center for Collective Use
of Analyses of Organic Compounds and Materials NIOC SB RAS.
Thin layer chromatography was carried out using DC-Alufolien Kieselgel
60 F254 TLC plates (Merck, Germany) in appropriate solvent mixtures and
was visualized by UV irradiation and stained by ninhydrin (0.1% in EtOH)
or 10% sulfuric acid. Column chromatography was carried out on a 100 cm3
column using Kieselgel 60 (Merck, Germany).
Oligonucleotide synthesis. Oligonucleotides were synthesized on an au-
tomated DNA synthesizer ASM-800 (Biosset, Russia) using standard nucleo-
side phosphoramidites (Glen Research, USA) at 0.1 M concentration in abso-
lute MeCN. Alkyne-modified nonnucleotidic monomer 9 was used at 0.15 M
concentration in absolute MeCN. During condensation of the modified
monomer, the volumes of activator (0.5 M 1H-tetrazole in absolute MeCN)
and monomer 9 were increased from 20 to 80 μL, and condensation time
was increased from 1 to 3 minutes as compared with the standard method.
The removal from support and deprotection of the oligonucleotides
were carried out either by a standard way (ca. 33% aq ammonia, 20◦C,
1 hour) or with tert-butylamine–methanol–water (1:1:2 v/v/v) for 5 hours at
37◦С.

Oligonucleotide Functionalization
315
Modified oligonucleotides were purified by reverse-phased HPLC on an
Agilent 1200 series chromatograph using a gradient of acetonitrile (0–40%)
in 0.02 M triethylammonium acetate, pH 7 for 30 minutes, flow rate of 2 cm3
min−1 using a Zorbax SB-C18 (5 μm) column (4.6 × 150 mm). Absorption
spectra were recorded at wavelengths from 220 to 600 nm.
4-(2-Hydroxyethyl)morpholin-2,3-dione (1). Diethanolamine (24.9 g,
0.24 mol) was dried by coevaporation in isopropanol (3 × 10 cm3), dis-
solved in isopropanol (20 cm3), and added dropwise for a period of 3 hours
to a well-stirred solution of diethyl oxalate (32 cm3, 0.2 mol) in isopropanol
(48 cm3) at ambient temperature. The resulting milky solution was stirred
for 12 hours. The precipitate was collected on a sintered glass filter and
washed with isopropanol (2 × 10 cm3). Final drying under reduced pressure
afforded 1 (86%) as a white powder. TLC (CH2Cl2—EtOH 1:1 v/v): Rf = 0.6.
1H NMR (300 МHz, DMSO-d6, δ, ppm): 4.49 (2H, t, J = 5.1, OCH2CH2N);
3.71 (2H, t, J = 5.1, OCH2CH2N); 3.53 (2H, t, J = 5.5, NCH2CH2OH); 3.43
(2H, t, J = 5.5, NCH2CH2OH). 13C-NMR (300 MHz, DMSO-d6, δ, ppm):
45.99, 49.45, 58.25, 65.84, 154.01, 157.77.
4-[2-(4,4ꢀ-Dimethoxytrityloxy)ethyl]morpholin-2,3-dione (2). Compo-
und 1 (1.55 g, 9.85 mmol) was dried by coevaporation with absolute pyridine
(3 × 10 cm3) and dissolved in abs. pyridine (30 cm3). 4,4ꢀ-Dimethoxytrityl
chloride (3 g, 8.85 mmol) was added to the solution, and the reaction mix-
ture was stirred at ambient temperature for 3 hours. Then the solution was
evaporated to an oil, dissolved in 200 cm3 of CH2Cl2, and the organic layer
was washed with freshly prepared saturated NaHCO3 solution (3 × 200 cm3),
dried over anhydrous Na2SO4, filtered, and evaporated to an oil. Com-
pound 2 was used in the next reaction without purification. TLC (CH2Cl2
1
– EtOH 96:4 v/v): Rf = 0.7 (purity ∼95%). H NMR (300 MHz, DMSO-
d6, δ, ppm): 6.85–8.66 (13H, m, ArH); 4.49 (2H, t, J = 4.9, OCH2CH2N);
3.73 (3H, s, OMe); 3.68 (2H, t, J = 4.9, OCH2CH2N); 3.59 (2H, t, J = 5.3,
NCH2CH2ODMTr); 3.16 (2H, t, J = 5.3, NCH2CH2ODMTr).
N-[4-(2-Propynyloxy)butyl]trifluoroacetamide (3). Potassium hydroxide
(100 g, 1.78 mol) was dissolved in 500 cm3 of tert-butanol containing 144 g
(1.6 mol) of 1,4-butanediol. The mixture was stirred vigorously under reflux
while 153 g (0.55 mol) of 1,2,3-tribromopropane were added dropwise. The
reaction mixture was stirred at 80◦C for 36 hours, then allowed to cool down
to ambient temperature. Precipitated KBr was filtered off and washed twice
with tert-butanol. The washes were added to the filtrate, evaporated under re-
duced pressure at 60◦C, and the resulting oil was cooled and filtered. Thionyl
chloride (300 cm3) was slowly added to the crude 4-hydroxybutyl propargyl
ether. The reaction mixture was stirred for 18 hours at ambient tempera-
ture then refluxed for 6 hours and filtered. The filtrate was evaporated and
distilled under reduced pressure. The crude 4-chlorobutyl propargyl ether
(32.3 g) was collected at 70–74◦C (13 torr). Trifluoroacetamide (11.3 g,

316
M. S. Kupryushkin et al.
0.1 mol), 14.66 g (0.1 mol) of crude 4-chlorobutyl propargyl ether, and
32.6 g (0.1 mol) of cesium carbonate were mixed in 100 cm3 of DMF and
stirred for 6 days at ambient temperature. The reaction mixture was cooled
to −12◦C, and salts were filtered off, DMF was evaporated, and the residue
was distilled under reduced pressure. The fraction boiling at 45◦C (0.5 torr)
was collected and redistilled. The yield of 3 was 7.44 g (13.2% from 1,2,3-
1
tribromopropane), bp 95–97◦C (0.5 torr). H NMR (400 MHz, CDCl3, δ,
ppm): 7.24 (1H, br. t, NH); 4.09 (2H, d, HC≡CCH2, J = 2.4); 3.53 (2H, t,
OCH2CH2CH2CH2, Jt = 5.7); 3.33 (2H, dt, OCH2CH2CH2CH2, Jt = 6.0, Jd =
6.2); 2.40 (1H, t, HC≡C, J = 2.3); 1.67–1.61 (4H, m, OCH2CH2CH2CH2).
13C NMR (100.6 MHz, CDCl3, δ, ppm): 157.15; 117.2; 79.2; 77.2; 69.3; 57.9;
39.49; 26.6; 25.67. Mass spectra were recorded on a DFS High Resolution
GC-MS spectrometer. Calculated for C9H12F3NO2 m/z 223.0815. Found m/z
+
184.0582 (C6H9F3NO2 ). There is a fragment with m/z 39 in the spectrum,
so we have assumed that it is a breakdown product of the molecular ion.
4-(2-Propynyloxy)butylamine (4). N -[4-(2-Propynyloxy)butyl]trifluoroa
cetamide 3 (160 mg, 0.72 mmol) was dissolved in 1 cm3 of 1 M NaOH
and incubated for 2 hours at ambient temperature. The reaction mixture
was evaporated to 0.2 cm3 under reduced pressure, and the amine 4 was ex-
tracted with diethyl ether. After removal of ether in vacuo, the product was
obtained with 58% yield. 1H NMR (300 MHz, СDCl3, δ, ppm): 4.06 (d, 2H,
HC≡CCH2, J = 2.34); 3.47 (t, 2H, OCH2CH2CH2CH2, J = 6.1); 2.65 (t, 2H,
OCH2CH2CH2CH2, J = 7.0); 2.36 (t, 1H, HC≡C, J = 2.4); 1.60–1.50 (m,
2H, OCH2CH2CH2CH2); 1.48–1.40 (m, 2H, OCH2CH2CH2CH2). 13C NMR
(75.47 MHz, CDCl3, δ, ppm): 79.67; 73.96; 69.7; 57.76; 41.6; 30.05; 26.59.
MALDI-TOF: calculated for C7H13NO [M+H]+ 127.10, observed 128.10.
2-(N-Methyl-N-7-nitro-2,1,3-benzoxadiazol-4-ylamino)ethanol (5). A mix-
ture of 0.99 g (5.0 mmol) 4-chloro-7-nitro-2,1,3-benzoxadiazole, 0.56 cm3
(7.0 mmol) 2-methylaminoethanol in 10 cm3 of EtOH was stirred at 20◦C
for 40 minutes. The orange precipitate was collected by filtration and dried
to yield 0.92 g (78%) of 5. 1H NMR (500 MHz, DMSO-d6, δ, ppm): 8.51 (d,
1H, H6, J = 9.0); 6.49 (d, 1H, H5, J = 9.0); 4.50 (s, 2H, CH2OH); 3.99
(t, 2H, CH2CH2N, J = 6.0); 3.50 (s, 3H, NMe). MS (Finnigan MAT 8200):
calculated for C9H10N4O4 m/z 238, observed m/z 238.
N-2-Chloroethyl-N-methyl-7-nitro-2,1,3-benzoxadiazol-4-ylamine (6). A
mixture of 0.5 g (2.1 mmol) of 5, 0.51 cm3 (4.0 mmol) benzenesulfonyl
chloride, and 10 cm3 pyridine was stirred at 90◦C for 5 hours. After cool-
ing, the mixture was poured into crushed ice–water. The orange precipitate
1
was collected by filtration and dried to yield 0.42 g (82%) of 6. H NMR
(500 MHz, DMSO-d6, δ, ppm): 8.51 (d, 1H, H6, J = 9.0); 6.49 (d, 1H, H5,
J = 9.0); 4.50 (s, 2H, CH2Cl); 3.99 (t, 2H, CH2CH2N, J1 = 6.00, J2 = 6.00);
3.50 (s, 3H, Me). MS (Finnigan MAT 8200): calculated for C9H9ClN4O3 m/z
256, observed m/z 256.

Oligonucleotide Functionalization
317
A
N-2-Azidoethyl-N-methyl-7-nitro-2,1,3-benzoxadiazol-4-ylamine (7).
mixture of 0.3 g (1.2 mmol) N -(2-chloroethyl)-N -methyl-7-nitro-2,1,3-
benzoxadiazol-4-ylamine 6, 0.13 g (2.0 mmol) sodium azide, and 5 cm3
DMSO was stirred at 65◦C for 8 hours. After cooling, the mixture was poured
into crushed ice – water. Orange precipitate was collected by filtration and
dried to yield 0.22 g (74%) of 7. IR: N3 2100 cm−1. Elemental analysis (%):
calculated for C9H9N7O3 C, 41.06; H, 3.42; N, 37.26; found C, 40.97; H,
3.30; N, 37.73. 1H NMR (500, DMSO-d6, δ, ppm): 8.51 (d, 1H, H6, J = 9.0);
6.48 (d, 1H, H5, J = 9.0); 4.34 (br.s, 2H, CH2N3); 3.77 (t, 2H, CH2NMe,
J1 = 6.00, J2 = 6.00); 3.48 (s, 3H, Me). MS (DFS High Resolution GC-MS):
calculated for C9H10N7O3 m/z 263, observed m/z 263.
N-4-(2-Propynyloxy)butyl-Nꢀ-2-(4,4ꢀ-dimethoxytrityloxy)ethyl-Nꢀ-2-
hydroxyethyl oxalamide (8). 4-(2-Propynyloxy)butylamine 4 (250 mg,
1.96 mmol) and DMAP (465 mg, 3.9 mmol) were dried by coevaporation
with absolute pyridine (3 × 5 cm3) and dissolved in abs. pyridine (20 cm3).
Compound 2 (1530 mg, 3.3 mmol) was added to the solution, and the
reaction was stirred at ambient temperature overnight. Then the solution
was evaporated to an oil, dissolved in 100 cm3 CH2Cl2, organic layer washed
with 0.3 M KH2PO4, pH 7 (3 × 100 cm3), dried over anhydrous Na2SO4,
filtered and evaporated to dryness. The crude product was purified by flash
chromatography on a silica gel column eluted with CH2Cl2 + 1% Et3N
yielding 8 as a yellow foam (58%). TLC (CH2Cl2 – EtOH 96:4 v/v): Rf =
0.4. 1H NMR (300 MHz, DMSO-d6, δ, ppm): 6.84–8.65 (13H, Ar); 4.08 (2H,
m, HC≡CCH2O); 3.73 (6H, s, OMe); 3.35–3.63 (8H, m, NCH2CH2OH
+ NCH2СH2ODMTr + HC≡CCH2OCH2); 3.05–3.17 (4H, m, CH2NH +
CH2ODMTr); 2.07 (1H, s, HC≡C); 1.4–1.55 (4H, m, CH2CH2CH2CH2).
13C NMR ((DMSO-d6, δ, ppm): 25.52; 26.33; 38.01; 45.56; 48.22; 50.67;
55.01; 57.29; 58.22; 59.59; 61.03; 62.12; 68.74; 76.90; 80.49; 85.58; 85.75;
113.22; 126.66; 127.64; 127.87; 129.62; 135.59; 144.89; 158.05; 162.93;
163.41; 164.43; 164.94.
N-4-(2-Propynyloxy)butyl-Nꢀ-2-(4,4ꢀ-dimethoxytrityloxy)ethyl-Nꢀ-2-(2-
cyanoethoxy-N,N-diisopropylaminophosphinyloxy)ethyl]oxalamide (9). 1H-
Tetrazole (35 mg, 1.2 mmol) and DIPEA (0.54 cm3, 3.1 mmol) were
dissolved in anhydrous MeCN (2.5 cm3), 2-cyanoethyl-N ,N ,N ꢀ,N ꢀ-
tetraisopropylphosphordiamidite (0.44 cm3, 1.3 mmol) was added dropwise,
and the mixture was stirred for 30 minutes at ambient temperature. Com-
pound 8 (409 mg, 0.52 mmol) was dried by coevaporation with anhydrous
MeCN (3 × 5 cm3), dissolved in anhydrous MeCN (2.5 cm3), and added to
the solution of 2-cyanoethyl-N ,N ,N ꢀ,N ꢀ-tetraisopropylphosphordiamidite.
The mixture was stirred at ambient temperature for 2 hours, then diluted
with CH2Cl2 (100 cm3), organic layer washed with 0.3 M KH2PO4, pH 7
(3 × 100 cm3), dried over anhydrous Na2SO4, filtered, and evaporated
to dryness. Crude product was purified by flash chromatography on silica
gel column eluted with toluene + 1% Et3N yielding 9 as a yellow foam

318
M. S. Kupryushkin et al.
1
(59%). TLC (toluene – EtOAc 75:25 v/v): Rf = 0.8. H NMR (300 MHz,
acetone-d6, δ, ppm): 6.85–7.91 (13H, Ar); 4.12 (2H, m, HC≡CCH2O);
3.79 (6H, s, OMe); 3.46–3.77 (8H, m, NCH2CH2OP + NCH2СH2ODMTr
+ HC≡CCH2OCH2 + CH2CH2CN + (CH3)2CH); 3.23–3.35 (4H, m,
CH2NH + CH2ODMTr); 2.71–2.93 (2H, m, CH2CN); 2.31 (1H, s, HC≡C);
1.54–1.69 (4H, m, CH2CH2CH2CH2); 1.16–1.24 (12H, m, (CH3)2CH). 13
C
NMR (300 MHz, DMSO-d6, δ, ppm): 19.37; 23.55; 23.62; 25.51; 26.27; 38.14;
42.25; 42.51; 46.93; 47.93; 54.18; 57.02; 57.87; 58.33; 60.01; 61.00; 61.85;
62.07; 62.39; 68.64; 74.24; 85.66; 112.58; 117.63; 126.17; 126.21; 127.28;
127.62; 129.51; 135.55; 144.77; 144.86; 158.23; 161.41; 161.70; 162.98;
163.15. 31P NMR (300 МHz, acetone-d6, δ, ppm): 148.67, 148.72.
Click labeling. Copper(I)-catalyzed alkyne-azide [3+2]cycloaddition was
carried out in accordance with published protocol.[17] To 25 μL of 6.7 mM
oligonucleotide in water, 15 μL of azide 7 (11.5 mM, 1 equiv) in DMSO was
added followed by 10 μL aq СuSO4 (840 mM, 50 equiv), 4.4 mg TBTA (50
equiv), 10 μL aq ascorbic acid (840 mM, 50 equiv) and 20 μL of 0.2 M tri-
ethylammonium acetate, pH 7. After incubation overnight oligonucleotide
material was precipitated by 2% LiClO4 in acetone, and the product was
isolated by RP-HPLC (Fig. 1).
ACKNOWLEDGMENTS
The authors thank Marat F. Kassakin (ICBFM SB RAS) for recording
MALDI-TOF spectra. This work was supported by the RFBR (10-04-01 492
and 13-04-01176), the MCB Programme of the Presidium of the Russian
Academy of Sciences, and partially by interdisciplinary grants from Siberian
Branch of the Russian Academy of Sciences and the Ministry of Educa-
tion and Science of the Russian Federation (Agreement No. 8535 from
07.09.2012).
REFERENCES
1. Deleavey, G.F.; Damha, M.J. Designing chemically modified oligonucleotides for targeted gene
silencing, Chem. Biol. 2012, 19, 937–954.
2. Kashida, H.; Liang, X.; Asanuma, H. Rational design of functional DNA with a non-ribose acyclic
scaffold, Curr. Org. Chem. 2009, 13, 1065–1084.
3. Loakes, D. Nucleotides and nucleic acids; oligo- and polynucleotides, in Organophosphorus Chemistry
(Vol. 41), ed. D.W. Allen, D. Loakes, J.C. Tebby, The RSC, Cambridge, 2012, pp. 169–250.
4. Dioubankova, N.N.; Malakhov, A.D.; Stetsenko, D.A.; Korshun, V.A.; Gait, M.J. (R)-2,4-
Dihydroxybutyramide seco-pseudonucleosides: New versatile homochiral synthons for synthesis of
modified oligonucleotides, Org. Lett. 2002, 4, 4607–4610.
5. Morocho, A.M.; Karamyshev, V.N.; Polushin, N.N. Methoxyoxalamido chemistry in the synthesis of
novel amino linker and spacer phosphoramidites: A robust means for stability, structural versatility,
and optimal tether length. Bioconjugate Chem. 2004, 15, 569–575.
6. Kupryushkin, M.S.; Pyshnyi, D.V. Oligonucleotide derivatives in nucleic acid hybridization analysis.
III. Synthesis and investigation of properties of oligonucleotides, bearing bifunctional non-nucleotide
insertion. Russ. J. Bioorg. Chem. 2012, 38, 625–638 (Russian version Bioorg. Khim. 2012, 38, 706–720).

Oligonucleotide Functionalization
319
7. Kupryushkin, M.S.; Pyshnyi, D.V. A convenient method for the synthesis of phosphoramidite non-
nucleotide inserts for preparation of functionalized oligonucleotides. Russ. J. Bioorg. Chem. 2012, 38,
662–666 (Russian version Bioorg. Khim. 2012, 38, 745–750).
8. Kupryushkin, M.S.; Pyshnyi, D.V. Approach to obtain phosphoramidite monomer of achiral non-
nucleosidic insert for oligonucleotide modification. Russian patent RU2460721, priority from
25.02.2011.
9. Kolb, H.C.; Finn, M.G.; Sharpless, K.B. Click chemistry: Diverse chemical function from a few good
reactions. Angew. Chem. Int. Ed. 2001, 40, 2004–2021.
10. Vasilyeva, S.V.; Budilkin, B.I.; Konevetz, D.A.; Silnikov, V.N. Synthesis of novel nucleoside 5ꢀ-
triphosphates and phosphoramidites containing alkyne or amino groups for the postsynthetic func-
tionalization of nucleic acids. Nucleosides Nucleotides Nucleic Acids. 2011, 30, 753–767.
11. Seiichi, U.; Tomofumi, S.; Takeshi, F.; Hiroshi, H.; Kazuhiro, I. Effects of the substituent groups at
the 4- and 7-positions on the fluorescence characteristics of benzofurazan compounds. J. Chem. Soc.
Perkin Trans. 2. 1998, 2165–2173.
12. Lin, K.; Matteucci, M. Hybridization properties of deoxyoligonucleotides containing anthraquinone
pseudonucleosides. Nucleic Acids Res. 1991, 19, 3111–3114.
13. Korshun, V.A.; Pestov, N.B.; Nozhevnikova, E.V.; Prokhorenko, I.A.; Gontarev, S.V.; Berlin, Y.A.
Reagents for multiple non-radioactive labelling of oligonucleotides. Synth. Commun. 1996, 26,
2531–2547.
14. Poulsen, C.S.; Pedersen, E.B.; Nielsen, C. DNA conjugated phenoxyaniline intercalators: Synthesis
of diethanolaminoacetamide-type linkers. Acta Chem. Scand. 1999, 53, 425–431.
15. Okamoto, A.; Ichiba, T.; Saito, I. Pyrene-labeled oligodeoxynucleotide probe for detecting base
insertion by excimer fluorescence emission. J. Am. Chem. Soc. 2004, 126, 8364–8365.
16. Strohalm, M.; Kavan, D.; Novak, P.; Volny, M.; Havlicek, V. mMass 3: A cross-platform software
environment for precise analysis of mass spectrometric data. Anal Chem. 2010, 82, 4648–4651.
17. Ustinov, A.V.; Dubnyakova, V.V.; Korshun, V.A. A convenient ‘click chemistry’ approach to perylene
diimide oligonucleotide conjugates. Tetrahedron. 2008, 64, 1467–1473.