Synthesis of 5-alkynylated uracil-morpholino monomers using Sonogashira coupling

Article abstract of DOI:10.1016/j.tetlet.2012.05.141

Sonogashira coupling of 5-iodo uracil-morpholino monomer with different terminal alkynes has been achieved for first time in good to excellent yields (67-86%). We have optimized the iodination conditions for the preparation of 5-iodo uracil-morpholino mon

Full text of DOI:10.1016/j.tetlet.2012.05.141

Tetrahedron Letters 53 (2012) 4179–4183
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Tetrahedron Letters
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Synthesis of 5-alkynylated uracil–morpholino monomers using
Sonogashira coupling
⇑
Sibasish Paul, Bappaditya Nandi, Sankha Pattanayak, Surajit Sinha
Department of Organic Chemistry, Indian Association for the Cultivation of Science, Jadavpur, Kolkata 700 032, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Sonogashira coupling of 5-iodo uracil–morpholino monomer with different terminal alkynes has been
achieved for first time in good to excellent yields (67–86%). We have optimized the iodination conditions
for the preparation of 5-iodo uracil–morpholino monomer under basic condition.
Ó 2012 Elsevier Ltd. All rights reserved.
Received 28 March 2012
Revised 28 May 2012
Accepted 30 May 2012
Available online 7 June 2012
Keywords:
Uracil–morpholino
Iodination
Detritylation
Alkynyl-derivatives
Sonogashira coupling
Morpholino (MO) antisense oligonucleotides (Gene Tools LLC1)
have emerged as potent candidates for evaluating the function of
a specific gene as they can inhibit the translation of the corre-
sponding target mRNA by steric blocking and have high resistance
to enzymatic degradations (Fig. 1).2–4 MO oligomers have been rou-
tinely used for reverse-genetic studies during embryogenesis in
developmental biology. The incorporation of T- or U-morpholino
unit(s) into DNA or siRNA has also been reported to obtain better
efficacy of these oligomers as antisense compounds.5–7 Similarly,
chimeric oligonucleotides with MO modification show better resis-
tance toward the nuclease activity.8 Due to the application of MO
triphosphates as chain terminating reagents in DNA sequencing,9
synthesis of these triphosphates has been reported as nucleoside
analogues.5 Apart from their antisense activity, these oligomers
have also found their growing applications in nanotechnology,10
in surface hybridization11,12 and as neutral DNA analogues. In spite
of having a wide application of morpholinos, so far there is no
report on the synthesis of functionalized monomers except Gene
Tools patent1 that also describes only the synthesis of regular
MOs. For academic interest as well as in continuation of our
research toward morpholino synthesis, we describe herein the syn-
thesis of 5-substituted uracil–MO monomers through C-5-alkyny-
lation of iodo-MO monomer using Sonogashira coupling reaction.
The 5-substituted uracil derivatives could be synthesized from
5-iodo uracil–MO unit 2, which in turn can be synthesized from ura-
cil–MO 1. Compound 1 was synthesized following our established
protocol.13 As there is no literature precedence toward the synthesis
of iodo-substituted monomer 2, we employed the procedures avail-
able for the iodination of uridine at C-5 and the results have been
summarized in Table 1. Acid-mediated halogenation method14 has
been avoided since our MO monomers have an acid sensitive trityl
group. Trityl-protection was used as it serves as a suitable protect-
ing group during oligomer synthesis and also known to give selec-
tive N-tritylated product in the presence of primary OH group
while synthesizing the monomer 1.1,15 Iodination methods reported
for uridine16a–e have been applied on 1 (entries 1–5) and in most of
the cases either starting material was recovered (entry 1) or the
detritylated compound was isolated in almost quantitative yield
(entries 2 and 3). In the case of NIS-mediated reaction under
O
H
N
O
Base
O
O
5
N
P
O
N
O
N
HO
O
N
O
N
Base
Ph
Ph
Ph
Uracil Morpholino
monomer
morpholino oligomer
Base= adenine, thymidine,
guanidine, cytidine
⇑
Corresponding author. Tel.: +91 33 2473 4971; fax: +91 33 2473 2805.
E-mail address: ocss5@iacs.res.in (S. Sinha).
Fig. 1. Morpholino monomer and oligomer.
0040-4039/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tetlet.2012.05.141

4180
S. Paul et al. / Tetrahedron Letters 53 (2012) 4179–4183
Table 1
Attempted optimization of iodination reaction
O
N
O
N
O
N
I
I
HN
HN
HN
O
O
O
X
O
N
O
N
O
N
HO
HO
HO
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
addition product;
, 3a
1
2
iodinated product;
X= N3
Entry
Iodinating agent
Solvent
Temp (°C)
Time (h)
Compound isolated
a
1.
2.
3.
4.
5.
I2/K2CO3
THF:H2O
CH3CN
CH3CN
DMF
rt
10
8
4
—
5
1
I2/CAN
NAI/CAN
NIS
80
80
65b
0
Detritylated product
Detritylated product
2, 12%
ICI/NaN3
CH3CN
3a, 30%
a
20 mol % DMAP was added as catalyst.
Reaction was carried out at 200 W power for 10 min.
b
NH4OH to give the corresponding iodo-derivative. Following their
condition detritylation was noticed in our case at rt. Then we
thought to introduce a base along with ICl/MeOH in the same reac-
tion pot and the results have been summarized in Table 2. Accord-
ingly, we tried with the combination of ICl and Et3N (entry 1) but
the starting material was recovered probably because Et3N was
forming a complex with ICl due to its Lewis base character.
Replacement of Et3N by K2CO3 gave the iodinated product 2 along
with the addition product 3b (entry 2). Increasing the equivalent of
ICl gave a better yield of the iodinated product 2 whereas the yield
of the addition product 3b was same (entries 3 and 4). We opti-
mized that the presence of 1.5 equiv of K2CO3 and 3.0 equiv of ICl
in MeOH gave the best result where the starting material was con-
sumed completely (entry 5). Though the addition product 3b was
isolated in 15% yield, 3b was readily converted into the desired
product 219 in the presence of Et3N in CH2Cl2 at rt. The combined
yield was found to be 83%. During the iodination we realized that
for every equivalent of ICl exactly half its equivalent of K2CO3 was
needed to obtain the iodination product and to stop detritylation.
Surprisingly, the combination of ICl/MeOH in the presence of
K2CO3 did not react with 20,30-dideoxy uridine18 whereas, it gave
the best result in case of morpholino unit.
Table 2
Optimization of ICl mediated iodination of 1
Entry Iodinating agent Base (equiv) Temp (°C) Product isolatedc (%)
(equiv)a
2
3b X=OMe
1
1.
2.
3.
4.
5.
ICI (1.2)
ICI (1.2)
ICI (2.0)
ICI (2.5)
ICI (3.0)
Et3N (1.2)
K2CO3 (1.2)
K2CO3 (1.2)
K2CO3 (1.25) 0 to 40
K2CO3 (1.5) 0 to 40
0 to 40b
0 to rt
0 to rt
—
23
48 10
64 10
73 15
—
8
92
60
35
20
—
a
b
c
Reaction was carried out in 0.5 mmol scale using MeOH as solvent.
ICI was added at 0 °C then raised to 40 °C.
Isolated yield.
microwave condition, the iodinated product 2 was obtained in 12%
yield and the starting material 1 was also recovered (78%). When
ICl/NaN3 was used as iodinating agent, the addition product 3a
17
was isolated in 30% yield, however, the conversion was 71% based
on the recovered starting material (entry 5). Interestingly, such
addition product is known to form during the iodination of uridine,
followed by the elimination of HN3 to give 5-iodo-uridine. Unfortu-
nately, in case of morpholino unit, the addition product 3a was sta-
ble under the reaction condition even elimination did not take place
in the presence of Et3N. When the reaction was carried out at room
temperature, formation of detritylated product was predominant.
Another procedure was reported18 for iodination of 20,30-dide-
oxy uridine involving the treatment with ICl/MeOH followed by
We next optimized the Sonogashira reaction of 2 with propargyl
alcohol 4 (Table 3). Our initial attempt of the Sonogashira reac-
tion20 of 2 with 4 gave exclusively the cyclized product 5 in the
presence of Pd(PPh3)2Cl2 (entry 1). When Pd(0) was employed as
Table 3
Optimization of Sonogashira condition
OH
O
N
O
OH
HN
N
O
N
O
Sonogashira
O
N
O
N
coupling
HO
HO
2
+
DMF
Ph
Ph
OH
Ph
Ph
4
Ph
Ph
6
5
Entry
Palladium catalyst (mol %)
Copper source (mol %)
Product
Yield (%)
1.
2.
3.
Pd(PPh3)2Cl2 (5)
Pd(PPh3)4 (5)
Pd(PPh3)4 (10)
CuI
CuI
CuI
5
6
6
63
38
86

S. Paul et al. / Tetrahedron Letters 53 (2012) 4179–4183
4181
Table 4
Sonogashira coupling of 5-iodo uracil–morpholino 2 with different terminal alkynes
H
N
R'
H
N
O
O
O
I
O
Pd(PPh3)4 (10 mol%)
CuI (20 mol%)
O
HO
N
O
N
O
R
HO
R,
HO
N
Et3N (2.0 equiv.)
DMF, rt
CPh3
N
N
CPh3
CPh3
Entry
Alkyne
Product, time (h)
Yield
Entry
Alkyne
Product, time (h)
Yield
O
F
F
OH
(1.5)
HN
O
6
OH
1
86
11
79
N
R
O
HN
16
(1)
N
R
O
NHCOCF3
CF3
CF3
O
HN
O
CF3
CF3
2
86
12
74
7 (1)
NHCOCF3
N
R
O
HN
17
(1)
N
R
O
NO2
Si
O
O
HN
Si
3
4
82
76
13
14
78
68
HN
NO2
18
(1)
8 (1)
N
R
O
N
R
O
CN
O
3
O
HN
CN
HN
9 (5)
3
19
O
N
R
(1.5)
N
R
O
CO2H
N
O
O
3
N
CO2H
3
HN
5
6
7
67
68
78
15
16
17
68
82
80
HN
10
(4)
20
(1)
N
R
O
N
R
O
HO
O
S
O
OH
S
HN
HN
11
(2)
21
(1)
N
R
O
O
N
R
O
O
Fe
Fe
HN
HN
12
13
(1.5)
(1.5)
N
R
O
N
R
22
O
(2)
HO
O
O
OH
HN
HN
8
77
18
83
N
R
O
N
R
OH
O
23
(3)
OH
(continued on next page)

4182
S. Paul et al. / Tetrahedron Letters 53 (2012) 4179–4183
Table 4 (continued)
Entry
Alkyne
Product, time (h)
Yield
77
Entry
Alkyne
Product, time (h)
Yield
3
O
9
HN
3
14 (1)
N
R
O
OCH3
O
10
77
HN
OCH3
15
N
R
(1)
O
3. Eisen, J. S.; Smith, J. C. Development 2008, 135, 1735.
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5. Abramova, T. V.; Bakharev, P. A.; Vasilyeva, S. V.; Silnikov, V. N. Tetrahedron Lett.
2004, 45, 4361.
a catalyst, we isolated the desired product 6 though in poor yield
(38%). Finally, 10 mol % of the catalyst, 20 mol % of CuI, and 2 equiv
of Et3N gave 621 in excellent yield.
6. Perez-Rentero, S.; Alguacil, J.; Robles, J. Tetrahedron 2009, 65, 1171.
7. Zhang, N.; Tan, C. Y.; Cai, P. Q.; Jiang, Y. Y.; Zhang, P. Z.; Zhao, Y. F. Tetrahedron
Lett. 2008, 49, 3570.
8. Zhang, N.; Tan, C. Y.; Cai, P. Q.; Zhang, P. Z.; Zhao, Y. F.; Jiang, Y. Y. Bioorg. Med.
Chem. 2009, 17, 2441.
9. Marciacq, F.; Sauvaigo, S.; Issartelte, J.-P.; Mouret, J. F. C.; Molko, D. Tetrahedron
Lett. 1999, 40, 4673.
10. Zhang, G.-J.; Luo, Z. H. H.; Huang, M. J.; Tay, G. K. I.; Lim, E.-J. A. Biosens.
Bioelectron. 2010, 25, 2447.
11. Tercero, N.; Wang, K.; Gong, P.; Levicky, R. J. Am. Chem. Soc. 2009, 131, 4953.
12. Wang, X.; Smirnov, S. ACS Nano 2009, 3, 1004.
13. Sinha, S.; Pattanayak, S.; Paul, S.; Nandi, B. PCT Int. Appl. 2011, WO 2011018798
A2 20110217.
14. Pesnot, T.; Jorgensen, R.; Palcic, M. M.; Wagner, G. K. Nat. Chem. Biol. 2010, 6,
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15. Shestopalov, I. A.; Sinha, S.; Chen, J. K. Nat. Chem. Biol. 2007, 3, 650.
16. (a) Krafft, M. E.; Cran, J. W. Synlett 2005, 1263; (b) Terrazas, M.; Kool, E. T
Nucleic Acids Res. 2009, 37, 346; (c) Kumar, V.; Yap, J.; Muroyama, A.; Malhotra,
S. Synthesis 2009, 23, 3957; (d) Asakura, I-J.; Robins, M. J. J. Org. Chem. 1990, 55,
4928; (e) Paolini, L.; Petricci, E.; Corelli, F.; Botta, M. Synthesis 2003, 7, 1039.
17. Kumar, R.; Wiebe, L. I.; Knaus, E. E. Can. J. Chem. 2005, 1994, 72.
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The optimized condition was then applied to the coupling of 2
with a wide range of electronically and structurally different al-
kynes (Table 4). Functionalized alkynes have been incorporated
at the 5-position of 2 as the products could be used for further
derivatization. Particularly propargylamine substituted monomer
7 (entry 2) is known to undergo guanidylation to give cationic olig-
omers.13 Trimethylsilylacetylene-substituted monomer 19 could
be further coupled at terminal alkyne after TMS-deprotection (en-
try 3). Arylalkynes containing both electron donating and electron
withdrawing groups underwent coupling smoothly to give the cor-
responding 5-substituted U–morpholino monomers in very good
yields (entries 7–14). In order to have a wide range of substrates,
we coupled 2 with heterocyclic acetylenes and obtained the prod-
ucts (20 and 21) in excellent yields (entries 15 and 16). Likewise,
ethynylferrocene and sterically hindered acetylene reacted in an
efficient manner with 2 to give the products 22 and 23 in excellent
yields (entry 17 and 18).
In summary, we are the first to report the synthesis of various
types of alkynyl-substituted uracil–morpholino monomers. Such
functionalized monomers could be useful for the synthesis of
functionalized MO oligomers and also could be the potential
nucleotide analogues for biomedical applications. In this direc-
tion, we also standardized the iodination method to obtain the
5-iodo U–MO monomer. Work toward the synthesis of function-
alized oligomer is now underway and results will be reported
in due course.
19. Preparation and characterization of compound 2: To a stirred solution of 1 (1.2 g,
2.5 mmol) in dry MeOH (10.0 mL) was added K2CO3 (0.53 g, 3.8 mmol) under
argon atmosphere. The reaction mixture was cooled to 0 °C, followed by
dropwise addition of ICl (7.5 mL, 1.0 M solution in CH2Cl2). The reaction mixture
was warmed to 40 °C and heated for a period of 1 h in dark. The reaction was
monitored by TLC. Reaction mixture was evaporated to dryness, extracted with
CH2Cl2 (2 ꢀ 30 mL). The organic layers were washed with H2O (2 ꢀ 15 mL), and
the excess iodine was removed by washing with half saturated sodium
thiosulfate (20 mL). The organic layer was further washed with water (20 mL),
half saturated brine (20 mL) and dried over Na2SO4. The organic extract was
concentrated in vacuo to obtain a colorless solid. The crude mass obtained was
dissolved in CH2Cl2 (20 mL) followed by the addition of Et3N (2.0 mL) and left for
overnight stirring. Reaction mixture was concentrated in vacuo and purified by
column chromatography (100–200 mesh silica gel) using MeOH in CH2Cl2 as
eluent to get compound 2 (1.26 g, 83%) as colorless solid. Rf (19:1, CH2Cl2/
Acknowledgements
MeOH) = 0.51. IR (neat):
m ;
3385, 1687, 1681, 1448, 1265, 750 cmꢁ1 1H NMR
S.S. thanks CSIR, India, for financial support by
a Grant
(500 MHz, CDCl3): d = 1.38–1.42 (1H, t, J = 9.5 Hz), 1.44–1.49 (1H, t, J = 11.0 Hz),
2.00 (1H, br s), 3.09–3.11 (1H, dd, J = 12.0, 4.0 Hz), 3.38–3.41 (1H, d, J = 11.5 Hz),
3.58–3.64 (2H, m), 4.27–4.31 (1H, m), 6.11–6.13 (1H, dd, J = 9.5, 2.0 Hz), 7.18–
7.21 (3H, m), 7.29–7.32 (6H, m), 7.46 (5H, br s), 7.59 (1H, s), 8.71 (1H, s); 13C NMR
(125 MHz, CDCl3): d = 48.92, 52.46, 63.74, 68.27, 78.21, 81.29, 92.37, 126.80,
128.12, 129.19, 144.38, 149.31, 159.66; HRMS (ESI) (M+Na)+ Calcd for
02(0012)/11/EMR-II. S.P., B.N. and S.P. are thankful to CSIR for their
fellowship. We thank Professor. B.C. Ranu, for providing us alkyne
compounds.
C
28H26IN3O4Na+ = 618.0866. Found 618.0868.
Supplementary data
20. Hobbs, F. W., Jr. J. Org. Chem. 1989, 54, 3420.
21. Preparation and characterization of compound 6: Compound
2 (0.184 g,
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.tetlet.2012.05.
141.
0.31 mmol) was dissolved in dry DMF (4.0 mL) followed by the addition of
propargyl alcohol (0.052 g, 0.92 mmol) and Et3N (85 L, 0.62 mmol). The
l
reaction mixture was degassed with argon for 15 min. To the reaction mixture
were added Pd(PPh3)4 (0.036 g, 0.031 mmol) and CuI (0.012 g, 0.062 mmol)
and stirred under argon atmosphere for a period of 1.5 h. The reaction mixture
was diluted with EtOAc (10 mL), washed with water (3 ꢀ 10 mL), brine (5 mL).
The reaction mixture was dried over Na2SO4, concentrated in vacuo and
purified by column chromatography (100–200 mesh silica gel) using MeOH in
CH2Cl2 as eluent to get compound 17 (0.146 g, 86%) as brown solid. Rf (1:1,
References and notes
1. Gene Tools LLC [http://www.gene-tools.com].
2. Corey, D. R.; Abrams, J. M. Genome Biol. 2001, 2, 1015.

S. Paul et al. / Tetrahedron Letters 53 (2012) 4179–4183
4183
petroleum ether/EtOAc) = 0.38, IR (neat/CHCl3):
m
= 3078, 3054, 2217, 1702,
7.45–7.52 (6H, m), 9.53 (1H, s); 13C NMR (125 MHz, CDCl3): d = 48.69, 51.19,
52.36, 63.55, 76.05, 77.94, 81.23, 92.87, 99.32, 126.73, 128.10, 129.30, 143.12,
148.89, 162.02. HRMS (ESI) (M+Na)+ Calcd for C31H29N3O5Na+ = 546.2005.
Found 546.2003.
1698, 1510, 1247, 750 cmꢁ1 1H NMR (500 MHz, CDCl3): d = 1.34–1.38 (1H, t,
;
J = 10.5 Hz), 1.41–1.46 (1H, t, J = 11.0 Hz), 2.11 (1H, br s), 3.03–3.05 (1H, d,
J = 11.5 Hz), 3.35–3.37 (1H, d, J = 11.0 Hz), 3.47–3.62 (2H, m), 4.24–4.28 (3H,
m), 6.08–6.10 (1H, dd, J = 8.0, 1.0 Hz), 7.16–7.18 (3H, m), 7.26–7.29 (6H, m),