Highly chemoselective palladium-catalyzed Sonogashira coupling of 5-iodouridine-5′-triphosphates with propargylamine: A new efficient method for the synthesis of 5-aminopropargyl-uridine-5′-triphosphates

Article abstract of DOI:10.1016/j.tetlet.2012.04.023

An efficient palladium-catalyzed Sonogashira coupling of 5-iodouridine-5′-triphosphates with propargylamine is described. The catalytic reaction is highly chemoselective that affords exclusively 5-aminopropargyl-uridine-5′-triphosphates in good yields with high purities (>99.8%).

Full text of DOI:10.1016/j.tetlet.2012.04.023

Tetrahedron Letters 53 (2012) 3070–3072
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Tetrahedron Letters
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Highly chemoselective palladium-catalyzed Sonogashira coupling of
5-iodouridine-50-triphosphates with propargylamine: a new efficient
method for the synthesis of 5-aminopropargyl-uridine-50-triphosphates
⇑
Anilkumar R. Kore , Annamalai Senthilvelan, Muthian Shanmugasundaram
Life Technologies Corporation, Bioorganic Chemistry Division, 2130 Woodward Street, Austin, TX 78744-1832, USA
a r t i c l e i n f o
a b s t r a c t
An efficient palladium-catalyzed Sonogashira coupling of 5-iodouridine-50-triphosphates with propargyl-
amine is described. The catalytic reaction is highly chemoselective that affords exclusively 5-aminoprop-
argyl-uridine-50-triphosphates in good yields with high purities (>99.8%).
Ó 2012 Elsevier Ltd. All rights reserved.
Article history:
Received 8 March 2012
Revised 3 April 2012
Accepted 4 April 2012
Available online 13 April 2012
Keywords:
Palladium
Sonogashira-coupling
Chemoselective
Non-radioactive probe
Aminopropargyl nucleotides
The chemical modification at C-5 position of the pyrimidine
moiety in uridine-50-triphosphates has been the subject of im-
mense interest in view of their wide applications in chemical biol-
ogy, structural biology, DNA sensing, and nanobiotechnology.1 In
particular, aminopropargyl nucleotides serve as a versatile molec-
ular biology tool for in vitro enzymatic introduction of functional
groups into a nucleic acid target of interest.2 The incorporation of
5-(3-aminopropargyl)-20-deoxyuridine-50-triphosphate into DNA
produces amine-modified DNA by conventional enzymatic incor-
poration methods such as reverse transcription, nick translation,
random primed labeling, or PCR and the resulting amine-modified
DNA can labeled with any amine-reactive dye or hapten.2 The
aminopropargyl nucleotide coupled with fluorescent dye has been
useful for generating labeled nucleic acids for various molecular
biology applications such as chromosome and mRNA fluorescence
in situ hybridization (FISH) experiment, gene expression, and
mutation detection on arrays and microarrays, and in situ PCR
and RT-PCR.3–5 The C-5 position of UTP and dUTP is not involved
in Watson–Crick base-pairing thereby minimizing any distortion
in the shape of the base pairs.6 The presence of unique alkynylami-
no linker provides a spacer between the nucleotide and the dye to
reduce the fluorophore’s interaction with enzymes or target bind-
ing sites. It is noteworthy that the use of such non-radioactive
labeling provides several advantages over traditional radioactive
labeling in terms of safety reasons, better stability and higher
labeling efficiency, and consistency, at reduced cost. The successful
molecular biology applications depend on the quality and purity of
aminopropargyl modified nucleotides.
The palladium-catalyzed Sonogashira coupling provides a pow-
erful strategy for the construction of a carbon–carbon triple bond
in organic synthesis.7 Although palladium-catalyzed Sonogashira
coupling involving halogenated nucleosides has been well docu-
mented in the literature,8,9 the use of halogenated nucleotides for
the coupling reaction has been limited.10 The cross-coupling reac-
tion involving iodo-nucleotides often results in poor yields due to
partial hydrolysis of the starting and final nucleotides to the corre-
sponding diphosphates. The current reported method to make
5-aminopropargyl nucleotide involves the Sonogashira coupling
of 5-iodouridine with protected propargylamine, followed by trip-
hosphorylation of protected aminopropargyl uridine using Ludwig
‘One pot, three step strategy’ and subsequent deprotection of the
propargylamine moiety.8 However, the reaction involves overall
three steps and the triphosphate step results in poor to moderate
yields. Given the potential molecular biology applications of these
aminopropargyl nucleotides, the development of a novel and effi-
cient gram-scale method would be highly valuable to meet the
application of nucleic acid chemistry demand. Our continuous
interest in nucleic acid chemistry11,12 prompted us to explore the
possibility of palladium-catalyzed Sonogashira coupling reaction
of 5-iodouridine-50-triphosphates with propargylamine for the
synthesis of 5-aminopropargyl-uridine-50-triphosphates. Herein,
⇑
Corresponding author. Tel.: +1 512 721 3589; fax: +1 512 651 0201.
E-mail address: anil.kore@lifetech.com (A.R. Kore).
0040-4039/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tetlet.2012.04.023

A. R. Kore et al. / Tetrahedron Letters 53 (2012) 3070–3072
3071
Table 1
we report the first example of a palladium-catalyzed Sonogashira
coupling of 5-iodouridine triphosphates with propargylamine,
leading to the formation of 5-aminopropargyl-uridine triphos-
phates in good yields with high purities.
Effects of solvent and palladium complex on the Sonogashira coupling of 5-iodo-dUTP
2a with propargyl aminea
Entry
Catalyst
Solvent
Yieldb (%)
The two step synthetic strategy to make aminopropargyl nucle-
otides such as 5-(3-aminopropargyl)-20-dexoyuridine-50-triphos-
phate 3a and 5-(3-aminopropargyl)-uridine-50-triphosphate 3b is
depicted in Scheme 1. The required starting material, 5-iodo-dUTP
2a for the palladium-catalyzed reaction was prepared by the iodin-
ation of 20-deoxyuridine-50-triphosphate, dUTP 1a with N-iodosuc-
cinimide in the presence of sodium azide using water as the
solvent. The iodination reaction is completely regioselective to
afford 5-iodo-dUTP 2a in 86% yield.11 The palladium-catalyzed
Sonogashira coupling of 5-iodo-dUTP 2a with propargylamine
using Pd(PPh3)4 and CuI as the catalysts and DMF as the solvent
afforded 5-(3-aminopropargyl)-20-dexoyuridine-50-triphosphate
3a in 85% isolated yield with >99.8% purity as evidenced by HPLC.13
The control experiments revealed that in the absence of either
palladium or copper catalyst, no reaction occurred.
1
2
3
4
5
6
7
8
9
Pd(OAc)2
K2PdCl4
PdCl2
Pd/C
Pd(OAc)2/PPh3
DMF
DMF
DMF
DMF
DMF
DMF
DMF
DMSO
CH3CN
THF
11
5
8
0
c
18
12
95
20
12
0
c
K2PdCl4/PPh3
Pd(PPh3)4
Pd(PPh3)4
Pd(PPh3)4
Pd(PPh3)4
Pd(PPh3)4
10
11
Toluene
0
a
Reactions of 5-iodo-dUTP (0.15 mmol) with propargyl amine (0.3 mmol) were
carried out at rt for 4 h in 5 mL of solvent by using palladium catalyst (0.06 mmol)
and CuI (0.128 mmol).
b
Yields were measured from crude products by using HPLC analysis.
0.15 mmol of PPh3 was used.
c
To further understand the nature of the catalytic reaction, the
catalytic activities of various palladium complexes for the reaction
of 5-iodo-dUTP 2a with propargylamine were tested and the re-
sults are delineated in Table 1. Phosphine-free palladium com-
plexes such as Pd(OAc)2, K2PdCl4, and PdCl2 exhibited low
catalytic activity, affording 3a in 11%, 5%, and 8% yields, respec-
tively, (entries 1–3). No reaction occurred using Pd/C as a catalyst
(entry 4). The addition of triphenylphosphine ligand to Pd(OAc)2 or
K2PdCl4 did not improve the catalytic activity, affording 3a in 18%
and 12% yields, respectively, (entries 5 and 6). The use of Pd(PPh3)4
showed the highest catalytic activity, giving 3a in 95% yield (entry
7). A brief examination of the effect of solvent using Pd(PPh3)4 as
the catalyst on the yield of 3a revealed that DMF was the solvent
of choice (entry 7). Other solvents such as DMSO and CH3CN were
less suitable, furnishing 3a in 20% and 12% yields, respectively, (en-
tries 8 and 9). No reaction occurred using THF or toluene as a sol-
vent (entries 10 and 11). From the above optimization studies, it is
clear that both the presence of phosphine ligand in the palladium
complex and the use of DMF as the solvent are important for the
successful Sonogashira coupling to proceed smoothly.
In a similar manner, the present Sonogashira coupling reaction
is successfully extended to the synthesis of 5-(3-aminopropargyl)-
uridine-50-triphosphate 3b. The iodination reaction of uridine tri-
phosphate 1b with N-iodosuccinimide in the presence of sodium
azide in water at 60 °C for 15 h furnished 5-iodo-UTP 2b in 79%
yield. Treatment of 2b with propargylamine using Pd(PPh3)4 and
CuI as the catalyst and DMF as the solvent afforded 5-(3-amino-
propargyl)-uridine-50-triphosphate 3b in 81% isolated yield with
>99.8% purity based on HPLC.
There are several interesting features that deserve comment
from the present Sonogashira coupling reaction. First, the nature
of salt associated with 5-iodouridine triphosphate plays a crucial
role for the successful reaction. It is to be mentioned that the
TEA salt of iodo triphosphate works very well for the Sonogashira
coupling reaction, whereas the sodium salt of uridine triphosphate
did not provide the desired product under our standard conditions.
These results strongly suggest that the pre-requisite for the 5-iod-
ouridine triphosphate is the presence of a hydrophobic counter ion.
Second, the propargylamine used in the reaction is highly chemo-
selective. The reaction affords exclusively single Sonogashira cou-
pling product and the other possible copper-catalyzed Ullmann–
Goldberg coupling product14 was not detected under our standard
conditions. Third, it is interesting to compare the present palla-
dium-catalyzed Sonogashira coupling reaction involving 5-iodo
nucleotides with the literature known palladium-catalyzed Sono-
gashira coupling involving 5-iodo nucleosides.8 Under our stan-
dard conditions, the present coupling reaction proceeds smoothly
with propargylamine and does not require protection of propargyl-
amine. This is in striking contrast to other palladium-catalyzed
Sonogashira coupling that requires protection of propargylamine
with trifluoroacetate group before coupling step.8 Fourth, the pres-
ent catalytic reaction tolerates a wide variety of functional groups
such as hydroxyl, phosphate, amide, and amine groups. Finally, the
purification procedure is simple, straightforward, and provides a fi-
nal pure product with extremely high purities, >99.8%.
In summary, we have developed a new palladium-catalyzed
Sonogashira coupling reaction of 5-iodouridine-50-triphosphates
O
O
O
N
I
HN
HN
I
O
P
O
P
O
P
O
P
O
O
P
O
O
P
O
O
O
N
O
O
N
O
, NaN3
O
O
O
O
O
O
O
O
O
O
O
water, 15 h
H
H
R
OH
R
OH
NH(Et)3 NH(Et)
3 NH(Et)3
1a,b
2a,b
O
NH2
HN
Propargylamine, Pd(PPh3)4,
CuI, Et3N, DMF, rt, 4 h
O
P
O
O
P
O
O
P
O
O
O
N
O
O
O
O
3a,b
a: R = H
H
R
OH
b: R = OH
Scheme 1. Synthesis of aminopropargyl-dUTP 3a and aminopropargyl-UTP 3b.

3072
A. R. Kore et al. / Tetrahedron Letters 53 (2012) 3070–3072
Woodroofe, C. C.; Lacenere, J.; Quake, S. R.; Stoltz, B. M. Chem. Commun. 2005,
4551–4553; (c) Vailyeva, S. V.; Budilkin, B. I.; Konevetz, D. A.; Silnikov, V. N.
Nucleosides, Nucleotides Nucleic Acids 2011, 30, 753–767.
with propargylamine that allows an efficient gram-scale synthesis
of 5-aminopropargyl-uridine-50-triphosphates in good yields with
high purities. The catalytic reaction is highly chemoselective and
tolerates a wide variety of functional groups present in the sub-
strates. The presence of hydrophobic counter ion in 5-iodouridine
triphosphate is crucial for the successful Sonogashira coupling
reaction. Further work is in progress to extend the scope of this
reaction and to study the application of these 5-aminopropargyl
nucleotides.
10. Kielkowski, P.; Pohl, R.; Hocek, M. J. Org. Chem. 2011, 76, 3457–3462.
11. Kore, A. R.; Shanmugasundaram, M. Tetrahedron Lett. 2012, 53, 2530–2532.
12. (a) Kore, A. R.; Shanmugasundaram, M. Bioorg. Med. Chem. Lett. 2008, 18, 880–
884; (b) Kore, A. R.; Shamugasundaram, M.; Vlassov, A. V. Bioorg. Med. Chem.
Lett. 2008, 18, 4828–4832; (c) Kore, A. R.; Shanmugasundaram, M.; Charles, I.;
Vlassov, A. V.; Barta, T. J. J. Am. Chem. Soc. 2009, 31, 6364–6365; (d) Kore, A. R.;
Charles, I.; Shanmugasundaram, M. Curr. Org. Chem. 2010, 14, 1083–1098.
13. Preparation of aminopropargyl-dUTP 3a: To a stirred solution of 5-iodo-20-
deoxyuridine-50-triphosphate 2a (5.0 g, 5.58 mmol) in dry DMF (80 mL),
palladium tetrakis triphenylphosphine (2.58 g, 2.23 mmol), copper iodide
(0.90 g, 4.73 mmol), propargylamine (0.61 g, 11.16 mmol), and triethylamine
(1.69 g, 16.74 mmol) were added and the reaction mixture was stirred at room
temperature for 4 h. The reaction mixture was poured into 500 mL of water
and stirred for 1 h. The reaction mixture was filtered. The collected aqueous
solution was adjusted to pH 6.5 and loaded on a DEAE Sepharose column. The
desired product was eluted using a linear gradient of 0–1 M TEAB and the
fractions containing the product were pooled, evaporated, and co-evaporated
with water (3 ꢀ 1000 mL). The final TEA salt residue was dissolved in water
(100 mL) and then poured into a solution of sodium perchlorate (10.0 g) in
acetone (700 mL). The resulting mixture was centrifuged and the supernatant
liquid was discarded. The solid was washed with acetone (200 mL) and the
resulting solid was dried in vacuum to give a sodium salt of aminopropargyl-
dUTP 3a (2.78 g, 85%). 1H NMR (D2O, 400 MHz) d 8.46 (s, 1H), 6.29 (t, J = 6.0 Hz,
1H), 4.65 (m, 1H), 4.32–4.19 (m, 3H), 4.02 (s, 2H), 2.44 (m, 2H); 31P NMR (D2O,
162 MHz) d ꢁ6.53 (d, J = 19.8 Hz, 1P), ꢁ10.19 (d, J = 20.1 Hz, 1P), ꢁ20.83 (t,
J = 20.3 Hz, 1P); MS (m/z): 520 [MꢁH]+.
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