Synthesis of novel nucleoside 5′-triphosphates and phosphoramidites containing alkyne or amino groups for the postsynthetic functionalization of nucleic acids

Article abstract of DOI:10.1080/15257770.2011.595379

A series of novel nucleoside 5′-triphosphates and phosphoramidites containing alkyne or amino groups for the postsynthetic functionalization of nucleic acids were designed and synthesized. For this purpose, the new 3-aminopropoxypropynyl linker group was used. It contains two alternative functional capabilities: an amino group for the reaction of amino-alkynyl- modified oligonucleotides with corresponding activated esters and an alkyne group for the copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) reaction. It was shown that a variety of methods of the attachment of the new linker can be used to synthesize a diversity of modified pyrimidine nucleosides. Copyright Taylor and Francis Group, LLC.

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Synthesis of Novel Nucleoside 5′-
Triphosphates and Phosphoramidites
Containing Alkyne or Amino Groups for
the Postsynthetic Functionalization of
Nucleic Acids
Svetlana V. Vasilyeva a b , Boris I. Budilkin a , Dmitrii A. Konevetz a &
Vladimir N. Silnikov a b
a Institute of Chemical Biology and Fundamental Medicine,
Novosibirsk, Russia
b “NanoTech-S” LLC, Novosibirsk, Russia
To cite this article: Svetlana V. Vasilyeva , Boris I. Budilkin , Dmitrii A. Konevetz & Vladimir N.
Silnikov (2011) Synthesis of Novel Nucleoside 5′-Triphosphates and Phosphoramidites Containing
Alkyne or Amino Groups for the Postsynthetic Functionalization of Nucleic Acids, Nucleosides,
Nucleotides and Nucleic Acids, 30:10, 753-767, DOI: 10.1080/15257770.2011.595379
To link to this article: http://dx.doi.org/10.1080/15257770.2011.595379
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Nucleosides, Nucleotides and Nucleic Acids, 30:753–767, 2011
Copyright ꢀC Taylor and Francis Group, LLC
ISSN: 1525-7770 print / 1532-2335 online
DOI: 10.1080/15257770.2011.595379
SYNTHESIS OF NOVEL NUCLEOSIDE 5ꢀ-TRIPHOSPHATES
AND PHOSPHORAMIDITES CONTAINING ALKYNE OR AMINO
GROUPS FOR THE POSTSYNTHETIC FUNCTIONALIZATION
OF NUCLEIC ACIDS
Svetlana V. Vasilyeva,1,2 Boris I. Budilkin,1 Dmitrii A. Konevetz,1
and Vladimir N. Silnikov1,2
1Institute of Chemical Biology and Fundamental Medicine, Novosibirsk, Russia
2“NanoTech-S” LLC, Novosibirsk, Russia
A series of novel nucleoside 5ꢁ-triphosphates and phosphoramidites containing alkyne or amino
2
groups for the postsynthetic functionalization of nucleic acids were designed and synthesized. For
this purpose, the new 3-aminopropoxypropynyl linker group was used. It contains two alternative
functional capabilities: an amino group for the reaction of amino–alkynyl-modified oligonucleotides
with corresponding activated esters and an alkyne group for the copper(I)-catalyzed azide–alkyne
cycloaddition (CuAAC) reaction. It was shown that a variety of methods of the attachment of the
new linker can be used to synthesize a diversity of modified pyrimidine nucleosides.
Keywords Modified nucleosides; triphosphate analogues; nucleoside synthesis; modi-
fied oligonucleotides
INTRODUCTION
Modified nucleoside triphosphates and phosphoroamidite synthons are
of great importance for both enzymatic and chemical synthesis of modi-
fied DNA containing insertions of chemically active nucleotides. They can
be then postsynthetically modified to design new biologically active or la-
beled oligonucleotide derivatives for the fundamental research or medical
applications and probes for diagnostics and imaging.[1–6]
C5-amino-modified nucleosides are usually used for the incorporation
of different functional groups into DNA (or RNA). The procedure includes
Received 20 January 2011; accepted 6 June 2011.
The work was supported by the Interdisciplinary Integration Project of the Presidium of Siberian
Branch of the Russian Academy of Sciences no. 88.
Supplementary material is available online for this article. Go to the publisher’s online edition of
Nucleosides, Nucleotides and Nucleic Acids to view the free supplemental file.
Address correspondence to Svetlana V. Vasilyeva, Institute of Chemical Biology and Fundamental
Medicine, Lavrent’ev Ave 8, Novosibirsk, 630090, Russia. E-mail: svetlana2001@gmail.com
753

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S. V. Vasilyeva et al.
the attachment of the protected allyl–amino linker to the C5 position of
uridine or cytidine by conventional palladium-coupling chemistry by using
halogenated nucleosides.[7–9] However, it was shown[10] that the removal of
the protecting group leads to the formation of the bicyclic cytosine analogue.
With uridine derivatives, this reaction can become particularly prominent.
The propargylamine linker allows one to avoid the cyclization. Triphos-
phates containing this linker were shown to be substrates for a number of
polymerases.[4, 11, 12] However, some steric hindrances may arise due to the
small length of the propargylamine linker if the subsequent postsynthetic
introduction of a large hydrophobic group is necessary.
At the same time, the copper(I)-catalyzed azide–alkyne cycloaddition
(CuAAC) reaction introduced into the arsenal of bioorganic chemistry is
widely used for the functionalization of biopolymers.[13–15] One publication
out of many reports on the synthesis of alkyne modified nucleosides for
“click” reactions showed exclusive cyclization of an N -disubstituted propar-
gylamine analogue in the C5 position of uridine in the presence of Cu(I).[16]
We realized a need to create and utilize a universal linker to modify
DNA (or RNA) containing amino or alkyne functional groups. In addition,
this linker should be more extended and flexible than propargylamine in
order to avoid the steric hindrance during the postsynthetic modification
that follows.
RESULTS AND DISCUSSION
In our previous work, the new 3-aminopropoxypropynyl linker for DNA
(or RNA) modification was proposed.[17] This linker (1, Scheme 1) is flexible
and more hydrophilic due to the presence of oxygen; it has a more extended
structure than propargylamine and probably causes less steric hindrance
in the enzymatic synthesis and subsequent modifications of the amino or
alkyne groups. It can be introduced both in the C5 position of pyrimidine
nucleosides by the Sonogashira coupling reaction[18] and in the N 4 position
using the reactivity of the amino group.
The synthesis of N -3-phthalimidopropyl propargyl ether was de-
scribed in our previous publication.[17] The phthalimide protection of
the amino group was removed, and the product was isolated as hy-
drochloride of 1 or trifluoroacetamide 2 depending on the subsequent
use of the linker. Here we present a more convenient one-step way for
the synthesis of N -(3-propynyloxypropyl)trifluoroacetamide 2 and then
3-propynyloxypropylamine 1, without the phthalimide protection.
Sonogashira coupling was the Pd-catalyzed reaction of correspond-
ing 5-iodo nucleosides with N -(3-propynyloxypropyl)trifluoroacetamide (2,
Scheme 2). For the preparation of 5-iodo nucleosides, a number of described
procedures was examined.[19–23] The best yields of 5-iodo-2ꢁ-deoxycytidine 3

Nucleoside 5ꢁ-Triphosphates and Phosphoramidites
755
SCHEME 1 Synthesis of 3-aminopropoxypropynyl linker group: (i) KOH/t-BuOH; (ii) SOCl2; (iii)
CF3C(O)NH2 + Cs2CO3/DMF; (iv) NaOH/H2O.
SCHEME 2 Synthesis of C5-substituted pyrimidine derivatives by Sonogashira coupling reaction:
(i) (Ph3P)4Pd, CuI, DMF, Et3N, N -(3-propynyloxypropyl)trifluoroacetamid 2, argon, yield 58%–65%;
(ii) POCl3/[MeO]3PO/N(C4H9)3, 0◦C, 10 minutes, (Bu4N)2H2P2O7 in acetonitrile, N(C4H9)3,
0◦C, 10 minutes, TEAB 0.1 M (pH = 7), 2 hours, yield 54%; (iii) N(C4H9)3 in acetonitrile,
then 2,2ꢁ-dithiodipyridine/Ph3P/N -Me-Im/DMF/DMSO, then [(C4H9)3N]2H2P2O7 in acetonitrile,
then NH3/H2O, yield 68%; (iv) DMTrCl, pyridine, DMAP; (v) ((iPr)2N)2PO(CH2)2CN/tetrazole-
NH(iPr)2/CH2Cl2, yield 70%.
and 5-iodo-2ꢁ-deoxyuridine 5 were achieved in the presence of iodic acid,
as described in the literature[19, 20] (up to 80% compared with 40%[21]).
However, we were not able to reproduce the iodination reactions using pro-
cedures described in the literature.[22, 23] Deoxycytidine 5ꢁ-monophosphate,
as was shown,[19] can also be iodinated in the presence of iodic acid. Because

756
S. V. Vasilyeva et al.
of their greater solubility than the deoxyribonucleosides in polar solvents,
the best method for the isolation of product 4 is an anion exchange chro-
matography. As a result, 4 was obtained in a yield of up to 95%.
3-Trifluoroacetamidopropoxypropynyl was also successfully attached to
the C5 position of cytidine, cytidine-5ꢁ-monophosphate, and uridine het-
erocyclic bases in 58%–65% yields (compounds 6, 7, and 8, respectively,
Scheme 2). Reduced yields of the desired products are probably caused by
the formation of the furano[2,3-d]pyrimidine derivatives, which are com-
mon byproducts in the Pd-catalyzed reaction of corresponding 5-iodo uri-
dine with alkines.[24] However, the formation of byproducts was not the
subject of our study.
Another way of introducing the new linker into pyrimidine nucleoside
is the use of the amino group reactivity. In this case, we obtained modified
pyrimidine nucleosides bearing the alkyne group, which can be used for the
functionalization of nucleic acids by the CuAAC reaction.
It was shown that the introduction of an alkyl substituent into the 4-amino
group of 2ꢁ-deoxycytidine did not prevent the hydrogen bond formation dur-
ing complementary interactions.[25] For the synthesis of the N 4-substituted 2ꢁ-
deoxycytidine and 5-methyl-2ꢁ-deoxycytidine analogues, bisulfite-mediated
transamination and the method of the replacement of O4-benzoyl protection
with alkyl amine were examined.[26, 27] Unfortunately, these reactions did not
give acceptable yields of the desired product. Perhaps, this is due to a some-
what reduced reactivity of the amino group in 3-propynyloxypropylamine
as hydrochloride compared with aliphatic diamines, which were used in the
original method. Finally, the approach that utilizes triazolyl derivatives was
chosen.[28, 29] The desired compounds were synthesized from 5ꢁ,3ꢁ-O-diacetyl-
2ꢁ-deoxyuridine 14a and 5ꢁ,3ꢁ-O-diacetyl-thymidine 14b by the reaction
of its 4-triazolyl-derivatives with 3-propynyloxypropylamine hydrochloride
(Scheme 3). We obtained N 4-(3-propynyloxypropyl)-2ꢁ-deoxycytidine 16a
and N 4-(3-propynyloxypropyl)-5-methyl-2ꢁ-deoxycytidine 16b in 70%–80%
yields.
For the synthesis of pyrimidine derivatives bearing the linker with
the alkyne functional group at the C5 position, we chose the convenient
procedure recommended by the related literature.[30] The original method
involves the reaction between protected 5-bromomethyl-2ꢁ-deoxyuridine
and alcohol. The starting material, 3ꢁ,5ꢁ-O-diacetyl-5-bromomethyl-2ꢁ-
deoxyuridine (19, Scheme 4), was synthesized according to the referenced
method.[31] The treatment of 19 with propargyl alcohol in dry dimethyl-
formamide (DMF) afforded 3ꢁ,5ꢁ-O-diacetyl-5-propargyloxymethyl-2ꢁ-
deoxyuridine 20a. This reaction occurred in 18 hours. After removing the
protective groups, 5-propargyloxymethyl-2ꢁ-deoxyuridine 21a was obtained
in
a
60% yield. The analogous treatment of 19 with 3-
propynyloxypropylamine hydrochloride in the presence of triethy-
lamine afforded 3ꢁ,5ꢁ-O-diacetyl-5-(3-propynyloxypropyl)aminomethyl-2ꢁ-
deoxyuridine 20b. The reaction mixture was analyzed by high performance

Nucleoside 5ꢁ-Triphosphates and Phosphoramidites
757
SCHEME 3 Synthesis of N 4-substituted pyrimidine derivatives bearing the alkyne functional group:
(i) Ac2O, pyridine, yield (a) 82%, (b)76%; (ii) POCl3, Et3N, 1,2,4-triazole, acetonitrile; (iii) 3-
propynyloxypropylamine hydrochloride, Et3N, acetonitrile; (iv) NH3/H2O, overall yield 72%; (v)
DMTrCl, pyridine, DMAP; (vi) ((iPr)2N)2PO(CH2)2CN/tetrazole-NH(iPr)2/CH2Cl2, yield 69%–75%.
liquid chromatography (HPLC). The reaction time was 4–6 hours at room
temperature. After deprotection of 20b by the treatment with 24% aqueous
ammonia, 5-(3-propynyloxypropyl)aminomethyl-2ꢁ-deoxyuridine 21b was
obtained in a 43% yield.
Thus, four N 4- and C5-modified pyrimidine nucleosides bearing the
alkyne functional group (16a, 16b, 21a, and 21b, Schemes 3 and 4)
were obtained along with C5-alkynyl-modified uridine, cytidine, and 5ꢁ-
monophospho-cytidine derivatives bearing the protected amino group (6,
7, and 8, Scheme 2). Each of these modified nucleosides may be con-
verted into general-purpose triphosphates or phosphoramidite synthones
for oligonucleotide synthesis. Since the attachment of the amino linker to
the C5 position through the triple bond guarantees the preservation of the
substrate specificity of corresponding triphosphates to polymerases, we con-
verted compounds 6, 7, and 8 to triphosphates. Nucleosides 16a, 16b, 21a,
and 8 were converted into corresponding phosphoramidites synyhones.
5-(3-Fluoroacetamidopropoxyprop-1-ynyl)-5ꢁ-O-(4,4ꢁ-dimethoxytrityl)-3ꢁ-
(2-cyanoethyl-N,N -diisopropylamino)-2ꢁ-deoxyuridine (10, Scheme 2),
N 4-(3-propynyloxypropyl)-5ꢁ-O-(4,4ꢁ-dimethoxytrityl)-3ꢁ-(2-cyanoethyl-N,N -
diisopropylamino)-2ꢁ-deoxycytidine, N 4-(3-propynyloxypropyl)-5-methyl-5ꢁ-

758
S. V. Vasilyeva et al.
SCHEME 4 Synthesis of C5-substituted pyrimidine derivatives bearing the alkyne functional group:
(i) (a) Propargyl alcohol, (b) 3-propynyloxypropylamine hydrochloride, Et3N, DMF; (ii) NH3/H2O,
overall yield 72%–43%; (iii) DMTrCl, pyridine, DMAP; (iv) ((iPr)2N)2PO(CH2)2CN/tetrazole-
NH(iPr)2/CH2Cl2, yield 69%–75%.
O-(4,4ꢁ-dimethoxytrityl)-3ꢁ-(2-cyanoethyl-N,N -diisopropylamino)-2ꢁ-deoxyc-
ytidine (18a,b, Scheme 3), and 5-propargyloxymethyl-5ꢁ-O-(4,4ꢁ-dimeth-
oxytrityl)-3ꢁ-(2-cyanoethyl-N,N -diisopropylamino)-2ꢁ-deoxyuridine
(23a,
Scheme 4) were prepared following procedures described in the litera-
ture[32, 33] in yields of 70%, 69%, 76%, and 79%, respectively, from 8 and
preprotected 5ꢁ-O-(4,4ꢁ-dimethoxytrityl)-2ꢁ-deoxynucleosides 17a,b and 22a.
1
All phosphoramidite derivatives were characterized by H, 31P NMR, and
mass spectroscopy. The data are summarized in Table 1 (supplementary
material is available online).
Various methods were used for the synthesis of 5ꢁ-triphosphates of
nucleoside derivatives 6, 7, and 8, (Scheme 2). For the conversion of 5-(3-
fluoroacetamidopropoxyprop-1-ynyl)-2ꢁ-deoxycytidin-5ꢁ-monophosphate 7
into its 5ꢁ-triphosphate derivative 9a, we used the previously reported
synthetic procedure.[34] Activation of the terminal phosphate group was
achieved under Mukaiyama conditions in the presence of a nucleophilic
catalyst (68% yield). We also used Ludwig’s method[35] for the synthe-
sis of 5ꢁ-triphosphates 9b and 11 from unprotected 5-(3-trifluoroacet-
amidopropoxyprop-1-ynyl)-2ꢁ-deoxycytidine and 5-(3-trifluoroacetamid-
opropoxyprop-1-ynyl)-2ꢁ-deoxyuridine, respectively, in trimethylphosphate

Nucleoside 5ꢁ-Triphosphates and Phosphoramidites
759
FIGURE 1 dCTP analogue 5-(biotinyl-6-amidocaproyl-3-amidopropoxyprop-1-ynyl)-2ꢁ-deoxycytidine-5ꢁ-
triphosphate.
(50% and 54% yields). It was shown that iodination of the C5 position
in 2ꢁ-deoxycytidine 5ꢁ-phosphate occurred at a higher extent than that in
2ꢁ-deoxycytidine, so compound 4 was obtained in a much higher yield (up
to 95%) as compared with compounds 3 and 5. Furthermore, the formation
of 5ꢁ-triphosphate 9a,b from compound 7 by activation of the terminal phos-
phate group is carried out in a higher and reproducible yield as compared
to the formation of the same triphosphate from a nucleoside derivative 6
using Ludwig’s method. Therefore, the synthesis of modified triphosphates
starting from a nucleotide seems to be the most attractive. Some reporter,
photoreactive, and functional groups can be introduced after deprotection
of the amino group in the synthesized amino–alkynyl-modified nucleotides.
As an example of functionalization with corresponding activated esters,
the deoxycytidine triphosphate (dCTP) analogue (12) containing the
biotin residue covalently bound to the C5 position of the pyrimidine ring
through the 6-amidocaproyl-3-amidopropoxyprop-1-ynyl arm (Figure 1)
was synthesized.[36, 37] All 5ꢁ-triphosphate derivatives were characterized by
1H, 31P NMR, and mass spectroscopy. The data are summarized in Table 1
(supplementary material is available online).
CONCLUSION
The universal hydrophilic linker to modify DNA (or RNA) was proposed.
As was shown, a variety of methods of the attachment of the new linker can
be used to synthesize a number of pyrimidine nucleosides containing a
functional alkyne or amino group. A series of modified 5ꢁ-triphosphates and
phosphoramidites containing the new alkynyl–amino linker for the function-
alization of nucleic acids was synthesized. The synthesis of amino–alkynyl-
modified nucleotides was optimized due to both the new linker and the pro-
posed way of its 5ꢁ-triphosphate synthesis through 5ꢁ-phosphate derivatives.
EXPERIMENTAL
The following reagents were used: 1,2,3-tribromopropane, cesium
carbonate, trifluoroacetamide, thionylchloride, bis(tri-n-butylammonium)

760
S. V. Vasilyeva et al.
pyrophosphate, 2,2ꢁ-dithiodipyridine, 1,3-propanediol (Sigma-Aldrich,
USA); 4-(N,N-dimethylamino)pyridine, triphenylphosphine (Fluka Chemie
AG, Buchs, Switzerland); 4,4ꢁ-dimethoxytrityl chloride and 1,2,4-triazole
(Chem-IMPEX, USA). Nucleosides, their protected derivatives, and 5ꢁ-
monophosphorylated 2ꢁ-deoxycytidine were purchased from ChemGenes
Corporation (USA). N-Hydroxysuccinimidyl biotinyl-6-amidocaproate was
purchased from NanoTex-C (Novosibirsk, Russia). All other reagents were
from Sigma-Aldrich (Milwaukee, WI, USA). Organic solvents were dried and
purified by standard procedures. 1H and 31P NMR spectra were recorded on
Bruker AV-400 and AV-300 spectrometers. The chemical shifts (δ) are re-
ported in ppm relative to the residual solvent signals. In case of 31P, an
external standard of 85% H3PO4 was used. Coupling constant (J ) values
are estimated in hertz (Hz) and spin multiples are given as s (singlet), d
(doublet), t (triplet), q (quartet), m (multiplet), app.t (apparent triplet),
and br (broad singlet). Matrix-assisted laser desorption/ionization-time of
flight (MALDI-TOF) mass spectra were run on Reflex III (Bruker Dalton-
ics, Germany) in a positive detector mode with dihydroxybenzoic acid as a
matrix. A 6410 Agilent LCMS triple quadrupole mass spectrometer [liquid
chromatography–mass spectrometry (LC-MS) with an electrospray ioniza-
tion (ESI) interface was also used for molecular weight measurements. El-
emental analyses were performed on the Elemental Analyzer Model 1106
(Carlo Erba, Italy). UV-absorption spectra were recorded on a Specord
M40 spectrophotometer (Carl Zeiss, Jena, Germany). The product yields
(Schemes 1–3, Figure 1) were evaluated as described in the figure legends.
Aliquots of the reaction mixtures were taken off at the appropriate time, di-
luted 10-fold with water or ethanol, centrifuged, and analyzed by analytical
anion exchange HPLC or analytical reverse phase HPLC. The analytical an-
ion exchange chromatography was performed on a Milichrom-4 chromato-
graph (Econova, Russia) using a 2.5 × 60 mm column packed with Polysil SA,
15 µm (Vector, Russia). A linear gradient (flow rate 50 µL/minute) from 0
to 0.8 M of K2HPO4/KH2PO4 (pH 7.0) was used. The peaks were integrated
from chromatographic profiles generated by the chromatography system
software. Preparative anion exchange chromatography was performed with
Polysil SA, 15 µm (Vector, Russia), or DEAE Sephadex A-25, 40–120 µ,
(Pharmacia Fine Chemicals, Sweden). Analytical reverse phase HPLC was
performed using a Milichrom A-02 chromatograph (Econova, Russia) and a
2 × 75 mm column packed with ProntoSIL 120-5C18 AQ (Bischoff, Leon-
berg, Germany). Preparative reverse phase chromatography was performed
on Polygoprep C18, 50–100 mkm (Macherey-Nagel, Germany). Thin layer
chromatography was performed using Alufolien Kieselgel 60 F254 plates
(Merck, Darmstadt, Germany) in appropriate solvent mixtures and was visu-
alized by UV irradiation, ninhydrin (for amine groups), or cystein/aqueous
sulphuric acid (for nucleosides). All evaporations were performed under
reduced pressure.

Nucleoside 5ꢁ-Triphosphates and Phosphoramidites
761
Synthesis of N-(3-Propynyloxypropyl)Trifluoroacetamide (2)
Potassium hydroxide [100 g (1.78 mol)] was dissolved in 500 mL of tert-
butanol containing 122 g (1.6 mol) of 1,3-propanediol. The mixture was
intensively stirred under reflux followed by the dropwise addition of 153 g
(0.55 mol) of 1,2,3-tribromopropane. The reaction mixture was stirred at
80◦C for 36 hours and then allowed to cool to room temperature. Precipi-
tated KBr was filtered off and washed twice with tert-butanol. The wash liquor
was added to the filtrate and evaporated under reduced pressure at 60◦C,
and the resulting oil was cooled and filtered. Thionyl chloride (300 mL) was
slowly added to crude 3-hydroxypropyl propargyl ether. The reaction mix-
ture was then stirred for 18 hours at room temperature and then refluxed for
6 hours. Next, the mixture was filtered, and the filtrate was evaporated under
reduced pressure and distilled in vacuo. The fraction boiling up to 66◦C (13
torr) was collected and redistilled. The crude 3-chloropropyl propargyl ether
(29.2 g) was collected at 68◦C–78◦C under 13 torr. Trifluoroacetamide (11.3
g, 0.1 mol), 13.25 g (0.1 mol) of crude 3-chloropropyl propargyl ether, and
32.6 g (0.1 mol) of caesium carbonate was suspended in 100 mL DMF and
stirred for 6 days at room temperature. The reaction mixture was cooled to
−12◦C, and the salts were filtered off. DMF was evaporated, and the residue
was distilled in vacuo. The fraction boiling at 45◦C (0.5 torr) was collected
and redistilled. The yield of target product 2 was 6.97 g (13.2% calc. for
1,2,3-tribromopropane), b.p. 90◦C (0.5 torr).
Synthesis of 3-Propynyloxypropylamine (1)
N -(3-propynyloxypropyl)trifluoroacetamide 2 [150 mg (0.72 mmol),
Scheme 1] was dissolved in 1 mL of 1 M NaOH and incubated for 2
hours at room temperature. The reaction mixture was evaporated to 0.2
mL in vacuo, and the amine was extracted with ether. After the ether
was evaporated, the yield of target amine 1 was 58%. An analytical sam-
ple was obtained as a picrate, m.p. (for picrate) 128–130 (decomposition).
3-Propynyloxypropylamine hydrochloride was obtained for subsequent syn-
theses.
Synthesis of C5-Substitued Pyrimidine Derivatives by
Sonogashira Coupling Reaction:
5-(3-Fluoroacetamidopropoxyprop-1-ynyl)-2ꢀ-Deoxycytidine (6),
5-(3-Fluoroacetamidopropoxyprop-1-ynyl)-2ꢀ-Deoxycytidin-5ꢀ-
Phosphate (7), 5-(3-Fluoroacetamidopropoxyprop-1-ynyl)-
2ꢀ-Deoxyuridine (8)
5-Iodo-derivative (3 or 4 or 5, Scheme 2; 1.0 mmol) was dissolved in
degassed, anhydrous DMF (8 mL). Copper (I) iodide (0.030 g, 0.1 mmol)
was added, and the reaction mixture was stirred under an inert atmosphere

762
S. V. Vasilyeva et al.
in the absence of light until copper (I) iodide was dissolved. Freshly dis-
tilled triethylamine (0.3 mL, 2.0 mmol) was added to the reaction mixture
followed by the addition of N -(3-propynyloxypropyl)trifluoroacetamide 2
(1.13 g, 10 mmol) and tetrakis(triphenylphosphine)-palladium(0) (0.116 g,
0.1 mmol). The reaction mixture was stirred for 18 hours in the absence of
light at room temperature under an inert atmosphere. Methanol (8.0 mL),
dichloromethane (8.0 mL), and the excess of Dowex AG1×4 (bicarbonate
form) were then added to the reaction mixture and the resulting suspension
was stirred for 30 minutes, filtered, and concentrated by evaporation. The
resulting brown oil was then suspended in an ethanol–water mixture, and
the desired products 6 and 8 were isolated by reverse-phase chromatography
in yields of 64%–65%. Crude product 7 was purified by anion exchange chro-
matography on DEAE Sephadex A-25 using a linear gradient of NH4HCO3
(0 → 1 M) in 20% ethanol in a yield of 58%.
Synthesis of 5ꢀ-Triphosphate Derivatives by Ludwig’s Method[35]
5-(3-Trifluoroacetamidopropoxyprop-1-ynyl)-2ꢀ-Deoxycytidin-5ꢀ-
Triphosphate (9b) and 5-(3-Trifluoroacetamidopropoxyprop-1-
ynyl)-2ꢀ-Deoxyuridin-5ꢀ-Triphosphate (11)
:
The synthesis of 9b and 11 was carried out from unprotected nucleosides
(6 and 8, respectively; 0.23 mmol). Purification of the 5ꢁ-triphosphate deriva-
tives was performed by anion exchange chromatography using a column
packed with Polisil SA. A linear gradient of NaCl concentration (0→1 M)
in 0.1% AcOH was used. Fractions containing the product were combined,
diluted 10-fold with water, and applied to a column packed with DEAE
Sephadex A-25. Elution was performed in the linear gradient of NH4HCO3
(concentration, 0 → 1 M) in 20% ethanol. Appropriate fractions were pooled
and evaporated. The residue was co-evaporated several times with aqueous
ethanol to remove buffer traces. 5ꢁ-Triphosphates 9b and 11 were precipi-
tated as trilithium salts by the addition of a 10-fold volume of 4% LiClO4 in
acetone to aqueous solutions of the products. Yields of 9b and 11 were 50%
and 54%, respectively.
Synthesis of 5ꢀ-Triphosphate from 5ꢀ-Monophosphate Derivative:
5-(3-Aminopropoxyprop-1-ynyl)-2ꢀ-Deoxycytidin-5ꢀ-Triphosphate
(9a)
The synthesis was carried out by the procedure described in the litera-
ture[34] from 5-(3-aminopropoxyprop-1-ynyl)-2ꢁ-deoxycytidin-5ꢁ-phosphate 7
(0.140 g, 0.16 mmol). 5ꢁ-Triphosphate was precipitated by the addition of
6% LiClO4 in acetone (100 mL). The precipitate was washed with acetone
and ether, purified by reverse phase chromatography on Polygoprep C18,

Nucleoside 5ꢀ-Triphosphates and Phosphoramidites
763
with a gradient of acetonitrile (0 → 10%) in water in the presence of 0.1 M
TEA–AcOH (pH 7.0). Appropriate fractions were pooled and evaporated.
The resulting protected 5ꢁ-triphosphate was subjected to the standard depro-
tection protocols (concentrated aqueous ammonia for the trifluoroacetic
protective groups). After the deprotection, target 5ꢁ-triphosphate 9a was
purified by anion exchange chromatography using a column packed with
DEAE Sephadex A-25. Elution was performed with a linear gradient of 300
mL each of 20% EtOH and 1 M NH4HCO3 in 20% EtOH. Appropriate frac-
tions were pooled and evaporated. The residue was co-evaporated several
times with aqueous ethanol to remove traces of buffer. 5ꢁ-Triphosphate 9a
was precipitated as trilithium salts by the addition of a 10-fold volume of 4%
LiClO4 in acetone to the aqueous solutions of the products in a yield of 0.1
mmol (68%).
Synthesis of 5-(Biotinyl-6-Amidocaproyl-3-Amidopropoxyprop-
1-ynyl)-2ꢀ-Deoxycytidin-5ꢀ-Triphosphate (12)
N -Hydroxysuccinimidyl biotinyl-6-amidocaproate (0.25 mmol) was dis-
solved in 1.5 mL of dimethyl sulfoxide (DMSO) and added to the solu-
tion of 5-(3-aminopropoxyprop-1-ynyl)-2ꢁ-deoxycytidine-5ꢁ-triphosphate 9a
or 9b (0.025 mmol; previously deprotection was carried out by 24% aqueous
ammonia) in 1.5 mL of 1 M aqueous NaHCO3/Na2CO3 (pH 9.0). After
1.5 hours, the product was precipitated by the addition of a 10-fold volume
of 2% LiClO4 in acetone, washed with acetone, and dried. The target com-
pound was purified by reverse phase chromatography on Polygoprep C18
in a gradient of acetonitrile (0 → 30%) in water in the presence of 0.1 M
TEA-HOAc (pH 7.0). The yield of 12 was 70%.
Synthesis of N4-Substitued Pyrimidine Derivatives:
N4-(3-Propynyloxypropyl)-2ꢀ-Deoxycytidine (16a),
N4-(3-Propynyloxypropyl)-5-Methyl-2ꢀ-Deoxycytidine (16b)
The synthesis was carried out as described in the literature[28]
from 5ꢁ,3ꢁ-O-diacetyl-2ꢁ-deoxyuridine 14a and 5ꢁ,3ꢁ-O-diacetyl-thymidine 14b
(0.194 mmol each), respectively, and 3-propynyloxypropylamine hydrochlo-
ride (3.88 mmol; Scheme 3). After the subsequent treatment with concen-
trated aqueous ammonia (30 mL), the reaction mixture was evaporated
and the residue was purified by reverse phase chromatography on Polygo-
prep C18 in a gradient of ethanol (0 → 20%) in water. The target fractions
were pooled and evaporated. The yields of 16a and 16b were 70 and 80%,
respectively.

764
S. V. Vasilyeva et al.
Synthesis of C5-Substitued Pyrimidine Derivatives Containing a
Functional Alkyne Group: 5-Propargyloxymethyl-2ꢀ-Deoxyuridine
(21a), 5-(3-Propynyloxypropyl)aminomethyl-2ꢀ-Deoxyuridine (21b)
5ꢁ,3ꢁ-O-diacetyl-5-bromomethyl-2ꢁ-deoxyuridine (19, Scheme 4; 0.400 g,
0.98 mmol), synthesized as described in the literature[31], was dissolved
in dried DMF (4 mL). Propargyl alcohol (0.550 g, 9.8 mmol) or 3-
propynyloxypropylamine hydrochloride (9.8 mmol) and triethylamine (1.37
mL, 9.8 mmol) were added, and the reaction mixture was stirred for 16 hours
at room temperature and concentrated by evaporation. The residue was dis-
solved in chloroform (20 mL) and washed with water (2 × 20 mL) and 5
M sodium chloride (20 mL). The organic phase was dried under anhydrous
sodium sulfate and evaporated. Concentrated aqueous ammonium (15 mL)
was then added to the residue, the reaction mixture was stirred for 16 hours
at room temperature and concentrated by evaporation. The resulting oil
was then suspended in an ethanol-water mixture and the desired products
(21a and 21b, Scheme 4) were purified by reverse phase chromatography
in a gradient of ethanol in water 0 → 50%. Appropriate fractions were
pooled and evaporated. The yields of 21a and 21b were 60% and 43%,
respectively.
General Procedure for the Introduction of the 4,4ꢀ-Dimethoxytrityl
Protecting Group
The corresponding nucleoside (1 mmol) was dissolved in dry pyri-
dine (5 mL), and 4,4ꢁ-dimethoxytrityl chloride (1.15 mmol) was added
at stirring. After the reaction was completed (2–3 hours), the reac-
tion mixture was evaporated and the residue was dissolved in methy-
lene chloride and applied onto a silica gel column. Elution with a gra-
dient of methanol (0% → 12%) in methylene chloride supplemented
with 1% pyridine led to the target product fractions. They were evapo-
rated, and the product was precipitated from the solution in methylene
chloride by a tenfold volume of hexane. 5ꢁ-O-(4,4ꢁ-dimethoxytrityl)-N4-
(3-propynyloxypropyl)-2ꢁ-deoxycytidine (17a), 5ꢁ-O-(4,4ꢁ-dimethoxytrityl)-
N4-(3-propynyloxypropyl)-5-methyl-2ꢁ-deoxycytidine (17b), and 5ꢁ-O-(4,4ꢁ-
dimethoxytrityl)-5-propargyloxymethyl-2ꢁ-deoxyuridine (22a) were obtained
in yields of 45%, 53%, and 43%, respectively.
General Procedure for Obtaining Phosphoroamidite Reagents for
Oligonucleotide Synthesis
2-Cyanoethyl-N,N,Nꢁ,Nꢁ-tetraisopropylphosphoroamidite
(4.5
mL,
14 mmol, and, after 2 hours, 2.2 mL, 6.9 mmol) was added portionwise
under stirring to the solution of the protected nucleoside (10 mmol) and di-
isopropylammonium tetrazolide (900 mg, 4.47 mmol) in dichloromethane

Nucleoside 5ꢁ-Triphosphates and Phosphoramidites
765
(50 mL). After 3–7 hours (thin layer chromatography monitoring), the
reaction mixture was evaporated to dryness, covered with hexane, and
left overnight. The hexane was then decanted, and the residue was chro-
matographed on a silica gel column. Elution with a gradient of methanol
(0 → 10%) in dichloromethane and the evaporation of the target fractions
gave the product, which was precipitated from dichloromethane by hexane
and dried in a vacuum.
5-(3-Fluoroacetamidopropoxyprop-1-ynyl)-5ꢁ-O-(4,4ꢁ-dimethoxytrityl)-3ꢁ-
(2-cyanoethyl-N,N -diisopropylamino)-2ꢁ-deoxyuridine (10), N4-(3-propynyl-
oxypropyl)-5ꢁ-O-(4,4ꢁ-dimethoxytrityl)-3ꢁ-(2-cyanoethyl-N,N -diisopropylami-
no)-2ꢁ-deoxycytidine (18a), N4-(3-propynyloxypropyl)-5-methyl-5ꢁ-O-(4,4ꢁ
-dimethoxytrityl)-3ꢁ-(2-cyanoethyl-N,N -diisopropylamino)-2ꢁ-deoxycytidine
(18b), and 5-propargyloxymethyl-5ꢁ-O-(4,4ꢁ-dimethoxytrityl)-3ꢁ-(2-cyanoe-
thyl-N,N -diisopropylamino)-2ꢁ-deoxyuridine (23a) were obtained with yields
of 70%, 79%, 69%, and 76%, respectively.
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