New strategies for cyclization and bicyclization of oligonucleotides by click chemistry assisted by microwaves

Article abstract of DOI:10.1021/jo702177c

(Chemical Equation Presented) The synthesis of cyclic, branched, and bicyclic oligonucleotides was performed by copper-catalyzed azide-alkyne cycloaddition assisted by microwaves in solution and on solid support. For that purpose, new phosphoramidite buil

Full text of DOI:10.1021/jo702177c

New Strategies for Cyclization and Bicyclization of Oligonucleotides
by Click Chemistry Assisted by Microwaves
Jory Lietard, Albert Meyer, Jean-Jacques Vasseur, and Franc¸ois Morvan*
Institut des Biomole´cules Max Mousseron (IBMM), UMR 5247 CNRS, UniVersite´ Montpellier 1, UniVersite´
Montpellier 2, Place Euge`ne Bataillon, CC1704, 34095 Montpellier Cedex 5, France
morVan@uniV-montp2.fr
ReceiVed October 8, 2007
The synthesis of cyclic, branched, and bicyclic oligonucleotides was performed by copper-catalyzed azide-
alkyne cycloaddition assisted by microwaves in solution and on solid support. For that purpose, new
phosphoramidite building blocks and new solid supports were designed to introduce alkyne and bromo
functions into the same oligonucleotide by solid-phase synthesis on a DNA synthesizer. The bromine
atom was then substituted by sodium azide to yield azide oligonucleotides. Cyclizations were found to
be more efficient in solution than on solid support. This method allowed the efficient preparation of
cyclic (6- to 20-mers), branched (with one or two dangling sequences), and bicyclic (2 × 10-mers)
oligonucleotides.
SCHEME 1. Synthesis of the Pentenyl Phosphoramidite of
Thymidine
Introduction
DNA is a versatile material for the construction of nano-
structures (for recent review see refs 1 and 2). There is great
interest in constructs of oligonucleotides with unusual topolo-
gies. Among them, cyclic DNA and RNA were synthesized in
the late 1980s,3-7 and there appears to be a growing interest
for them in several recent works.8-14 Cyclic DNA and RNA
* Corresponding author. Tel: +33/467 144 961. Fax: +33/467 042 029.
(1) Feldkamp, U.; Niemeyer, C. M. Angew. Chem., Int. Ed. 2006, 45,
1856-1876.
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(3) Kool, E. T. J. Am. Chem. Soc. 1991, 113, 6265-6266.
(4) Capobianco, M. L.; Carcuro, A.; Tondelli, L.; Garbesi, A.; Bonora,
G. M. Nucleic Acids Res. 1990, 18, 2661-2669.
(5) Rao, M. V.; Reese, C. B. Nucleic Acids Res. 1989, 17, 8221-8239.
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Tetrahedron 1989, 45, 4523-4536.
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Marel, G. A.; Van Boom, J. H. Nucleic Acids Res. 1988, 16, 4607-4620.
(8) Roulon, T.; Le Cam, E.; Escude, C. ChemBioChem 2006, 7, 912-
915.
present unusual chemical and biological properties in compari-
son with linear DNA and have been evaluated for several
biological applications including antisense, triplex, and diag-
(9) Moggio, L.; De Napoli, L.; Di Blasio, E.; Di Fabio, G.; D’Onofrio,
J.; Montesarchio, D.; Messere, A. Org. Lett. 2006, 8, 2015-2018.
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Gonzalez, C. Chem. Eur. J. 2006, 12, 4035-4042.
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129, 5544 - 5550.
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L. M.; Brown, T. J. Am. Chem. Soc. 2007, 129, 6859-6864.
10.1021/jo702177c CCC: $40.75 © 2008 American Chemical Society
Published on Web 12/08/2007
J. Org. Chem. 2008, 73, 191-200
191

Lietard et al.
nostic applications.3,15-17 Several methods have been developed
for the synthesis of cyclic oligonucleotides using enzymatic or
chemical protocols leading to cyclic oligonucleotides with
phosphodiester,3,18-21 phosphorothiolate diester,13,22-24 disul-
fide,25 or oxime linkages.13,26
Herein, we present a method for the circularization of short-
to medium-size oligonucleotides (6- to 20-mers) that involves
a 1,3-dipolar cycloaddition between an alkyne and an azide
function, one borne on one end and the other on the other end
of an oligonucleotide, leading to a triazole linkage. This method
was then extended to the synthesis of branched and bicyclic
oligonucleotides. The copper-catalyzed azide-alkyne cycload-
dition (CuAAC) reaction27,28 applied to DNA appeared as a
useful reaction for anchored DNA on a solid support,29 for
preparation of DNA conjugates with carbohydrates30-33 and
recently for DNA ligation and DNA cyclization.14
diester linkage by means of carbon tetrachloride in the presence
of excess of propargylamine.32-34 This method requires the use
of H-phosphonate chemistry with pivaloyl chloride as a coupling
reagent to introduce H-phosphonate diester linkages and a
manual amidative oxidation. The oligonucleotide moiety is then
synthesized by phosphoramidite chemistry. This protocol is
efficient but requires the use of two chemistries on a DNA
synthesizer, consequently necessitating multiple manipulations.
In the present work, we developed a strategy using only the
phosphoramidite chemistry. Thus, an alkyne function was
introduced on the phosphorus atom of the 3′-end nucleoside by
means of the incorporation of a new thymidine phosphoramidite
2 bearing a pentenyl chain. To do so, 4-pentenyl tetraisopro-
pylphosphorodiamidite 1 was prepared from 4-penten-1-ol and
bis(diisopropylamino)chlorophosphine in dichloromethane in the
presence of triethylamine and was then reacted with com-
mercially available 5′-dimethoxytritylthymidine under activation
with diisopropylammonium tetrazolide affording 2 (Scheme 1).
DNA elongation was performed on an universal solid support
constituted of a disulfide linker35-37 that yields after its reduction
to an intramolecular elimination of episulfide with the formation
of a phosphodiester linkage. The solid-supported hexa- and
nonathymidines bearing a 3′-end alkyne function 4ab were
synthesized on this solid support with standard phosphoramidite
elongation cycle using the thymidine pentenyl phosphoramidite
2 and the commercially available cyanoethyl thymidine phos-
phoramidite (Scheme 2). A further, two-step 5′-azidation was
performed according to Kool et al.38 using first methyltriph-
enoxyphosphonium iodide39,40 and second sodium azide yielding
5′-azido solid-supported oligonucleotides 5ab (Scheme 2).
The corresponding solid-supported oligonucleotides were then
split in two parts. One part was left on the solid support and
the other part was treated with ammonia for 2 h at room
temperature to afford the 5′-azido-3′-alkyne homothymidylate
in solution. The HPLC analysis showed two major peaks that
were characterized by MALDI-TOF mass spectrometry as the
oligonucleotides with (6ab) and without (7ab) the disulfide
linker, due to some cleavage of the disulfide bond followed by
the elimination of episulfide yielding the phosphodiester linkage
(Figure 1a, Scheme 3).
Results and Discussion
We report an efficient method to prepare cyclic, bicyclic, and
branched oligonucleotides based on CuAAC reaction. Initially,
to set the conditions of cyclization the first reactions were
performed on hexa- and nonathymidylate to form the corre-
sponding cyclic oligonucleotides cT6 and cT9. In order to carry
out the reaction of cyclization on solid support as well as in
solution, both functionalities (i.e., akyne and azide) were
introduced into the oligonucleotide still on the solid support.
As a rule, it is more powerful to do so because excess reagents
can be used to push the reaction to completion, and excess is
removed by simple washings. The workups are easier and the
resulting compounds generally have better purity.
Previously, we designed a protocol to introduce an alkyne
function through an amidative oxidation of a H-phosphonate
(15) Dolinnaya, N. G.; Blumenfeld, M.; Merenkova, I. N.; Oretskaya,
T. S.; Krynetskaya, N. F.; Ivanovskaya, M. G.; Vasseur, M.; Shabarova, Z.
A. Nucleic Acids Res. 1993, 21, 5403-5407.
(16) Wang, S. H.; Kool, E. T. Nucleic Acids Res. 1994, 22, 2326-2333.
(17) Nilsson, M.; Malmgren, H.; Samiotaki, M.; Kwiatkowski, M.;
Chowdhary, B. P.; Landegren, U. Science 1994, 265, 2085-2088.
(18) Prakash, G.; Kool, E. T. J. Am. Chem. Soc. 1992, 114, 3523-3527.
(19) Alazzouzi, E.; Escaja, N.; Grandas, A.; Pedroso, E. Angew. Chem.,
Int. Ed. 1997, 36, 1506-1508.
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1594.
(21) Micura, R. Chem. Eur. J. 1999, 5, 2077-2082.
(22) Xu, Y. Z.; Kool, E. T. Tetrahedron Lett. 1997, 38, 5595-5598.
(23) Xu, Y. Z.; Kool, E. T. Nucleic Acids Res. 1998, 26, 3159-3164.
(24) Smietana, M.; Kool, E. T. Angew. Chem., Int. Ed. 2002, 41, 3704-
3707.
The 1,3-dipolar cycloaddition “click reaction” was performed
with the oligonucleotide in solution or still on the solid support.
Since this cycloaddition is catalyzed by Cu(I), we used CuSO4,
sodium ascorbate in water/methanol (1:1 v/v) to generate in situ
the Cu(I) species and the reaction was assisted by microwave
at 60 °C for 60 min.
The reactions were monitored by HPLC on a reversed-phase
column. HPLC profiles showed peaks with different retention
times for the starting linear oligonucleotides and the final cyclic
oligonucleotides. In each case, the cyclic oligonucleotide
displayed a peak at a lower retention time than the linear
oligonucleotide. Since there is no change of mass during the
(25) Gao, H.; Yang, M. H.; Patel, R.; Cook, A. F. Nucleic Acids Res.
1995, 23, 2025-2029.
(26) Edupuganti, O. P.; Defrancq, E.; Dumy, P. J. Org. Chem. 2003,
68, 8708-8710.
(27) Tornoe, C. W.; Christensen, C.; Meldal, M. J. Org. Chem. 2002,
67, 3057-3064.
(28) Rostovtsev, V. V.; Green, L. G.; Fokin, V. V.; Sharpless, K. B.
Angew. Chem., Int. Ed. 2002, 41, 2596-2599.
(29) Devaraj, N. K.; Miller, G. P.; Ebina, W.; Kakaradov, B.; Collman,
J. P.; Kool, E. T.; Chidsey, C. E. D. J. Am. Chem. Soc. 2005, 127, 8600-
8601.
(34) Meyer, A.; Bouillon, C.; Vidal, S.; Vasseur, J. J.; Morvan, F.
Tetrahedron Lett. 2006, 47, 8867-8871.
(35) Asseline, U.; Bonfils, E.; Kurfurst, R.; Chassignol, M.; Roig, V.;
Thuong, N. T. Tetrahedron 1992, 48, 1233-1254.
(36) Gupta, K. C.; Sharma, P.; Kumar, P.; Sathyanarayana, S. Nucleic
Acids Res. 1991, 19, 3019-3025.
(37) Kumar, P.; Bose, N. K.; Gupta, K. C. Tetrahedron Lett. 1991, 32,
967-970.
(38) Miller, G. P.; Kool, E. T. J. Org. Chem. 2004, 69, 2404-2410.
(39) Miller, G. P.; Kool, E. T. Org. Lett. 2002, 4, 3599-3601.
(40) Verheyden, J. P. H.; Moffatt, J. G. J. Org. Chem. 1970, 35, 2319-
2326.
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Soc. 2001, 123, 357-358.
(31) Burley, G. A.; Gierlich, J.; Mofid, M. R.; Nir, H.; Tal, S.; Eichen,
Y.; Carell, T. J. Am. Chem. Soc. 2006, 128, 1398-1399.
(32) Chevolot, Y.; Bouillon, C.; Vidal, S.; Morvan, F.; Meyer, A.;
Cloarec, J. P.; Jochum, A.; Praly, J. P.; Vasseur, J. J.; Souteyrand, E. Angew.
Chem., Int. Ed. 2007, 46, 2398-2402.
(33) Bouillon, C.; Meyer, A.; Vidal, S.; Jochum, A.; Chevolot, Y.;
Cloarec, J. P.; Praly, J. P.; Vasseur, J. J.; Morvan, F. J. Org. Chem. 2006,
71, 4700-4702.
192 J. Org. Chem., Vol. 73, No. 1, 2008

Cyclization and Bicyclization of Oligonucleotides by Click Chemistry
FIGURE 1. HPLC monitoring of click cyclization of 5′-azido-3′alkyne-T9 affording cT9: (A) crude linear T9 6b, 7b; (B) crude cT9 8b, 9b; (C)
crude cT9 9b after TCEP treatment; (D) pure cT9 9b.
SCHEME 2. Synthesis of Solid-Supported 5′-Azido-3′-alkyne Homothymidylatea
a Key: (a) 2.5% dichloroacetic acid CH2Cl2; (b) 2 or DMTr-thymidine phosphoramidite + benzylthiotetrazole; (c) Ac2O, N-Me imidazole, 2,6-lutidine;
(d) 0.1 M I2 THF/H2O/pyridine. SPS ) solid-phase synthesis.
cyclization, MALDI-TOF mass spectrometry is not the method
of choice to monitor it, but is useful to confirm that the peak at
lower retention time hypothesized to be the cyclic one has the
same mass as the starting oligonucleotide.
Cyclization in Solution. Before cyclization, the HPLC
chromatogram showed two peaks corresponding to the linear
oligonucleotide without (7b, 17.3 min) and with the disulfide
linker (6b, 20.9 min) (Figure 1a). The cyclization was performed
on the mixture, since a treatment with triscarboxylethylphos-
phine (TCEP) to reduce the disulfide would also reduce the azide
function to an amine. Click cyclization was carried out with
CuSO4, sodium ascorbate in water/methanol for 1 h at 60 °C
assisted by MW. After reaction, two main peaks were visualized
corresponding to the cyclic form without (9b, 15.3 min) and
with (8b, 17.3 min) the disulfide linker (Figure 1b). A treatment
with TCEP reduced the disulfide bound and afforded the
expected cyclic T9 (cT9) 9b (Figure 1c), which was finally
purified by HPLC (Figure 1d).
Furthermore, to confirm cyclization, a second reaction of 1,3-
dipolar cycloaddition was engaged with 5 molar equiv of
propargyl alcohol under the same conditions. In all cases, no
change of retention time in HPLC and no ion with +56 Da,
corresponding to the click reaction of propargyl alcohol, was
observed, suggesting that no azide function was still present.
In contrast, when a competitive 1,3-dipolar cycloaddition was
performed in solution with 5′-azido-3′-alkyne homothymidylate
in presence of 5 molar equiv of propargyl alcohol, two
compounds were obtained. The major one was the cyclic
oligonucleotide and the minor one the linear oligonucleotide
was reacted with propargyl alcohol (M + 56). Thus, although
the propargyl alcohol was in excess, the cycloaddition proceeded
preferentially through an intramolecular reaction.
Similar profiles were obtained for the cyclization of T6 (6a
and 7a) affording cT6 9a (see the Supporting Information).
Cyclization on Solid Support. The solid-supported 5′-azido-
3′-alkyne homothymidylate was treated with CuSO4 and sodium
J. Org. Chem, Vol. 73, No. 1, 2008 193

Lietard et al.
FIGURE 2. HPLC profile of click cyclization performed on solid support affording 9b (tR ) 15.3 min) and 12b (tR ) 16.2 min) and MALDI-TOF
MS spectra of the peak at 15.3 min (9b, left) and the peak at 16.2 min (12b, right).
SCHEME 3. Click Cyclization of T6 and T9 Performed in Solution
ascorbate in water/methanol (1:1 v/v) by microwave for 1 h at
60 °C. After filtration, the solid support was treated with
ammonia in the presence of TCEP to release the cyclic
oligonucleotide without the disulfide linker. HPLC profiles
showed a different pattern in comparison with the cyclization
done in solution, with a narrow peak corresponding to the
expected cT6 or cT9 cyclic oligo (9ab) and a broad peak at
higher retention time (Figure 2). The amount of the latter
increased with the length of the homothymidylate (41% for T6
and 71% for T9). Both peaks were isolated by HPLC and
characterized by MS. For the click reaction performed with T9,
the first peak at 15.3 min showed an ion at m/z ) 2842.86
corresponding to the cyclic cT9 and the second peak at 16.2
min showed an ion at m/z ) 5690.59 corresponding to a
dimerization of the oligonucleotide. Two possibilities were either
the production of a cyclic dimer with two triazole rings or a
linear dimer from a single cycloaddition and still one azide and
one alkyne functions (Scheme 4). To distinguish the two species,
a second “click” reaction was performed with propargyl alcohol,
and no extra ion was visualized by MS, confirming that only
cyclic dimer (12b) was present. Similar results were previously
reported for the cyclization of solid-supported peptides.41 It was
suggested that the click reaction on solid support could be
considered a reaction in a concentrated medium while the click
reaction in solution is performed in a diluted media. Hence,
intermolecular reaction is favored on solid support while only
the intramolecular reaction occurred in solution. This first result
showed that cyclization in solution was more efficient than on
solid support with only the formation of an intramolecular cyclic
oligonucleotide.
(41) Punna, S.; Kuzelka, J.; Wang, Q.; Finn, M. G. Angew. Chem., Int.
Ed. 2005, 44, 2215-2220.
194 J. Org. Chem., Vol. 73, No. 1, 2008

Cyclization and Bicyclization of Oligonucleotides by Click Chemistry
SCHEME 4. Click Reaction Performed on Solid-Supported Oligonucleotide 5ab Bearing One Azide and One Alkyne Functions
SCHEME 5. Synthesis of Monoalkyne Universal Solid Support 16
TABLE 1. Tm Values of cT20:An and Tn:An:Phosphate Buffer 10
mM, NaCl 100 mM pH 7, 1 µM of Each Oligonucleotide
SCHEME 6. Synthesis of Bromohexyl Phosphoramidite 17
A6
A7
A8
A9
A10
A20
cT20
Tn
25.2
0
25.9
<3
27.9
7.2
30.6
16.8
33.6
23.0
32.9
53.0
a
a n corresponds to the length of the homoadenylate.
an extra treatment of 2 h at 60 °C under MW. This result could
be explained by the fact that 3′-alkyne functions are poorly
accessible due to the steric hindrance of the long oligonucleotide.
Then, cyclic and linear T20 were cleaved from the solid support
by ammonia treatment and finally click cyclization was per-
formed in solution affording the expected cT20pro.
Since the disulfide bond was partially cleaved during the
ammonia treatment leading to complicated HPLC profiles, we
then performed the syntheses on another universal solid support
with a propanediol linker (pro).42 Three new oligonucleotides
were synthesized on it: a T6, a T20, and a heterooligonucleotide
(15-mer), according to the same procedure as described above.
Click cyclization was done in solution, and similar results were
obtained (see the Supporting Information). Only the intramo-
lecular cyclization occurred to afford cT6pro, cT20pro, and
cTTACACCCAATTCTTpro (c15-mer-pro). For the two shorter
oligonucleotides, HPLC profiles showed two peaks correspond-
ing to the two diastereoisomers due to the phosphotriester
linkage, while for cT20pro the two diastereoisomers were eluted
together. For the T20, click cyclization was also performed on
solid support. In that case, after 1 h at 60 °C under MW the
cyclization achieved only 50% and did not go further even after
Click without the MW. As previously observed in our hands,
click reaction on solid support was not efficient at all without
FIGURE 3. Schematic representation of cyclic branched oligonucle-
otides with one (B1) and two dangling sequences (B2).
(42) Seela, F.; Kaiser, K. Nucleic Acids Res. 1987, 15, 3113-3129.
J. Org. Chem, Vol. 73, No. 1, 2008 195

Lietard et al.
SCHEME 7. Universal Method for Cyclic Oligonucleotide Synthesis by MW-CuAAC Reactiona
a * corresponds to nucleobase protecting group: benzoyl for A and C and isobutyryl for G.
SCHEME 8. Synthesis of Bromohexyl Phosphoramidite of
Thymidine
phosphoramidites corresponding to each nucleoside (A, C, G,
and T) to introduce the alkyne function on the 3′-end. In
addition, the 5′-azidation is not efficient for all the nucleosides,
Kool et al. reported that the formation of 5′-iodide deoxyad-
enosine, the precursor of the 5′-azido deoxyadenosine, proceeded
with only a moderate yield (56%).39 In order to circumvent these
limitations, we designed a universal strategy using a new solid
support and a new phosphoramidite that could be used for any
sequence. Thus, we synthesized a new universal solid support
16 bearing an alkyne function (Scheme 5), and we prepared a
new 6-bromo-1-hexyl-phosphoramidite 17 for introduction of
an azide function on the 5′-end of any oligonucleotide (Scheme
6). After its coupling on the oligonucleotide, the bromine atom
was substituted by sodium azide in presence of sodium iodide
affording the 5′-azidohexyl oligonucleotide.
MW. In contrast, in solution, CuAAC click reaction carried out
at 65 °C without MW gave a 1,3-dipolar cycloaddition.
However, the results were less robust. Indeed, under the same
conditions (i.e., 65 °C with CuSO4 sodium ascorbate under
argon), the cyclization of T6 was incomplete after 24 h while
that of T9 and T20 was complete within 1 h 20 min to 2 h.
Universal Method for Preparation of Cyclic Oligonucle-
otides. The strategy described above if applied to the synthesis
of any sequence would require the synthesis of the four pentenyl
Alkyne universal solid support was elaborated with a three-
step protocol from tris(hydroxymethyl)ethane 13 which was
monoprotected with DMTr group (14, 56%), monoalkylated
with propargyl bromide and sodium hydride in THF (15, 74%),
and was finally loaded on a succinyl LCAA CPG using N-(3-
196 J. Org. Chem., Vol. 73, No. 1, 2008

Cyclization and Bicyclization of Oligonucleotides by Click Chemistry
SCHEME 9. Synthesis of Bicyclic Oligonucleotides 30 and 31
dimethylaminopropyl)-N′-ethylcarbodiimide (EDC) as coupling
reagent affording 16 (32.2 µmol/g) (Scheme 5).
Bromohexyl phosphoramidite 17 was obtained in one step
from 6-bromo-1-hexanol and cyanoethyl tetraisopropylphos-
phorodiamidite activated with diisopropylammonium tetrazolide
in dichloromethane (60%) (Scheme 6).
To evaluate the efficiency of both these building blocks, a
15-mer (ACT GAC ATC GTC GTA) was synthesized on the
alkyne universal solid support 16 applying a standard phos-
phoramidite elongation cycle, and likewise, the bromohexyl
phosphoramidite 17 was introduced at the 5′-end of the
oligonucleotide (Scheme 7). The 5′-azido-15mer was obtained
by treatment with a mixture of NaN3 and NaI in DMF for 1 h
at 65 °C. After ammonia treatment, the 5′-azidohexyl 3′-alkyne
oligonucleotide was obtained in solution and characterized by
HPLC and MALDI-TOF analyses (see the Supporting Informa-
tion).
min. HPLC profile after reaction showed the disappearance of
the starting linear oligonucleotide and a major peak of lower
retention time having the expected mass. Finally, cyclic oligo-
nucleotide 22 was obtained as pure material after HPLC
purification (see the Supporting Information).
Branched Cyclic Oligonucleotides. Then, we extended our
strategy to the synthesis of branched oligonucleotides corre-
sponding to cyclic oligonucleotides with a single dangling strand
B1 and with two single dangling strands B2 (Figure 3). The
B1 structure could be considered mimics of a lariat structure.43,44
The first branched oligonucleotide B1, corresponding to a
cyclic T20 with a dangling 3′-GCTCTCG sequence, was
synthesized on solid support using the pentyne phosphoramidite
2 introduced into the sequence after the synthesis of the
GCTCTCG sequence and followed by the elongation of 19
thymidines and finally the bromohexyl phosphoramidite 17 at
Cyclization of the oligonucleotide by CuAAC reaction
assisted by microwave at 60 °C was complete within 1 h 30
(43) Carriero, S.; Damha, M. J. Organic Lett. 2003, 5, 273-276.
(44) Carriero, S.; Damha, M. J. J. Org. Chem. 2003, 68, 8328-8338.
J. Org. Chem, Vol. 73, No. 1, 2008 197

Lietard et al.
FIGURE 4. HPLC profiles before and after cyclization affording the crude bicyclic oligonucleotides 30 (left) and 31 (right) and after purification.
the 5′-end. A subsequent treatment with NaN3 and NaI led to
its 5′-azidation. For the double-branched oligonucleotide B2,
we first synthesized a new 6-bromohexyl diisopropyl thymidine
phosphoramidite 24 in order to introduce an azide function into
the sequence. This compound was synthesized following the
same strategy used for the preparation of 2 starting from
6-bromo-1-hexanol by reaction with bis(diisopropylamino)
chlorophosphine to form the diamidite derivative 23, which
reacted with 5′-DMTr-thymidine under diisopropylammonium
tetrazolide catalysis (Scheme 8).
Thus, according to the phosphoramidite method on com-
mercial solid support, we synthesized the GTCTGCA sequence,
introduced phosphoramidite 2 with an alkyne function, followed
the synthesis with the AACGTCTG sequence, introduced the
phosphoramidite 24 with a bromo linker, and finished the
elongation with the ATCG sequence. A post-elongation treat-
ment with NaN3 and NaI led to an azidation on the hexyl linker
into the sequence.
GCTCTCG and ATCGT(azidohexyl)-AACGTCTGT(pentyne)-
GTCTGCA linear constructions were obtained in solution.
A CuAAC cyclization was performed in solution assisted by
MW for 1 h 30 min at 60 °C. Cyclization was monitored by
HPLC (see the Supporting information). Total disappearance
of starting linear oligonucleotide was always observed. Clear
monitoring was sometimes difficult since B1 and B2 (as well
as their precursors) are constituted of two and four diastereoi-
somers, respectively, due to the chiral phosphorus atom of the
phosphotriester linkages providing broad peaks. The HPLC
profiles of the crude showed some impurities that were assumed
to come from nucleophilic attack on the phosphotriester linkages
yielding oligonucleotides without the pentyne or the hexylazide
arm. This fact was emphasized when CuAAC cyclization to
obtain B1 was carried out without MW at 60 °C for several
hours. Indeed, the cyclization only achieved 49% after 3 h, and
during the time of consumption of starting compound (18 h)
several other compounds were formed, broadening the HPLC
peak of the cyclic compound. It is likely that the phosphotriester
functions were hydrolyzed affording a mixture of different linear
and cyclic oligonucleotides according to the cleavage of the
PO bond. Furthermore, it was reported that some degradations
Both oligonucleotides bearing azide and alkyne functions
were deprotected and cleaved from the solid support by an
ammonia treatment at room temperature for 18 h. This mild
treatment avoided strand cleavage due to nucleophilic attack
on the phosphotriester linkages (i.e., phosphorus bearing the
pentyne or the azidohexyl chain). Both N3(CH2)6T19T(pentyne)-
198 J. Org. Chem., Vol. 73, No. 1, 2008

Cyclization and Bicyclization of Oligonucleotides by Click Chemistry
occur due to the copper, even with degassed solvents.45
Nevertheless, both pure cyclic oligonucleotides B1 and B2 were
obtained after purification by HPLC and were characterized by
mass spectrometry. This is a clear advantage of the MW-assisted
click over the non-assisted reaction.
were always lower than those of cT20:An except for T20:A20
indicating a benefit of the cyclic partner able to form Hoogsteen
hydrogen bounds plus a lower entropic parameter.
Conclusion
Bicyclic Oligonucleotides. To our knowledge, there is only
one example of synthesis of bicyclic oligonucleotides reported
in the literature. They were prepared through the formation of
a disulfide bridge between two thymidine bases modified at the
C-5 position carrying a thiol at the end of an alkyl chain.46 In
our case, we developed another method based on a double
CuAAC cyclization from a construction bearing two alkyne and
two azide functions. For that purpose, a new solid support 25
bearing two alkyne functions was designed. It was prepared from
pentaerythritol according to the same procedure as above for
preparation of 16. Pentaerythritol was first monoprotected with
a dimethoxytrityl group (30%), alkylated twice by means of
propargyl bromide and sodium hydride in THF (89%),47 and
finally loaded on succinyl LCAA-CPG (40.0 µmol/g). Starting
from 25, a phosphoramidite derivative with two masked
hydroxyl functions 2648 was incorporated (Scheme 9). Then two
T10 were synthesized in parallel affording 27, and each 5′-end
was converted into azido. For comparison, the two azidation
protocols described above were applied leading to one construc-
tion with an azido function on the 5′-carbon of the oligonucle-
otide and the other one with an azido function on a hexyl linker
(Scheme 9). After release from the solid support by ammonia
treatment, HPLC and MS analysis of 28 and 29 showed that
azidation was almost complete (Figure 4). Then a click
cyclization assisted by MW was applied to both constructions
for 1 h 30 min at 60 °C affording the bicyclic oligonucleotides
30 and 31.
We applied the CuAAC reaction to the synthesis of cyclic
oligonucleotides, branched (lariat mimic) and bisbranched cyclic
oligonucleotides, and also bicyclic ones. For that purpose, we
designed different building blocks (i.e., phosphoramidites and
solid supports) allowing introduction of one or two alkyne and
azide functions into the same oligonucleotide. The CuAAC
reaction was found more efficient in solution than on solid
support. Furthermore, microwave assistance allowed the forma-
tion of the cyclic oligonucleotide within 1 to 1 h 30 min with
only little degradation. The evaluation of their biophysical
properties is in progress.
Experimental Section
General Remarks. All commercial chemicals were reagent grade
and were used without further purification. DNA synthesis reagents,
phosphoramidites, and benzylmercaptotetrazole (BMT) are com-
mercially available. Long-chain alkylamine controlled pore glass
(LCAA-CPG) 500 Å, 80-120 mesh, amino group 80-90 µmol/g
was from Sigma, and amino-SynBase CPG 1000, amino group 68
µmol/g was from Link Technologies.
4-Propyn-1-yl Tetraisopropylphosphorodiamidite (1). To a
solution of 4-propyn-1-ol (95 µL, 1.0 mmol) and Et3N (278 mL, 2
mmol) in dry diethyl ether (2.5 mL) was added bis(diisopropy-
lamino)chlorophosphine (267 mg, 1 mmol) and the mixture stirred
for 2 h at rt. The solution was diluted with diethyl ether/
triethylamine (9:1, v/v, 10 mL), and the salts were removed by
filtration and washed. The solution was evaporated to half, and
cyclohexane was added. Diethyl ether was removed by evaporation
keeping cyclohexane in solution. The solution was applied to a silica
gel column (25 g), and the compound was purified using cyclo-
hexane containing 6% Et3N: 280 mg, 89%, Rf 0.60 (cyclohexane/
HPLC profiles of the each crude showed complete disap-
pearance of the diazide starting oligonucleotide and a major
peak corresponding to the bicyclic oligonucleotide with some
impurities. These side products were attributed to the monocyclic
species after MALDI-TOF analysis with a 5′-OH function, a
product of incomplete azidation and the dimerization of two
monocyclic species. Each bicycle was finally purified by HPLC
and characterized by mass spectrometry.
1
CH2Cl2/Et3N, 6:3:1, v/v/v). H NMR (CDCl3, 200 MHz): δ 1.20
(24H, dd, J ) 10.4 and 13.0 Hz), 1.83 (2H, quint, J ) 6.6 Hz,),
1.95, (1H, t, J ) 2.6 Hz), 2.32 (2H, td, J ) 2.6 and 7.1 Hz,), 3.44-
3.77 (6H, m). 13C NMR (CD3CN,400 MHz): δ 14.9, 23.4, 24.1,
30.5, 44.2, 62.4, 68.7, 83.9, 117.1. 31P NMR (CD3CN,80 MHz): δ
125.0.
Hybridization Properties of Cyclic T20 (cT20). The cyclic
5′-O-Dimethoxytrityl-3′-O-[(4-propynyl)-N,N-diisopropylphos-
phoramidite]thymidine (2). Dry 5′-O-Dimethoxytritylthymidine
(544.5, 1 mmol) and dry diisopropylammonium tetrazolide (86 mg,
0.5 mmol) were dissolved in dry CH2Cl2 (8 mL), and 4-propyn-
1-ol tetraisopropylphosphorodiamidite 1 (377 mg, 1.2 mmol) was
added. After 3 h of stirring, the solution was diluted with ethyl
acetate (80 mL), and the solution was washed with brine (2 × 150
mL). The organic layer was dried over Na2SO4 and evaporated.
The compound was purified by flash chromatography on silica gel
using an increasing amount of CH2Cl2 (14 to 44%) in cyclohexane
containing 6% of Et3N: 650 mg, 85%, Rf 0.50 (cyclohexane/CH2-
Cl2/Et3N, 5:4:1, v/v/v). 1H NMR (CDCl3, 200 MHz): δ 1.07 (3H,
d, J ) 6.7 Hz), 1.18 (9H, d, J ) 6.8 Hz), 1.43 (3H, bs), 1.70-2.03
(3H, m), 2.20-2.36 (4H, m), 3.50-3.71 (6H, m), 3.81 (3H, s),
4.17-4.22 (1H, m) 4.65-4.68 (1H, m), 6.43-645 (1H, m), 6.83-
7.45 (13H, m), 7.68 (1H, d), 8.50 (1H, br s). 13C NMR (CD3CN,
400 MHz): δ 11.7, 15.1, 23.0, 24.5, 26.9, 30.0, 40.3, 43.1, 55.3,
61.9, 63.2, 68.7, 83.7, 84.8, 85.5, 85.9, 86.9, 111.2, 113.3, 127.2,
128.0, 128.2, 130.2, 135.3, 135.5, 135.7, 144.3, 150.4, 158.7, 163.9.
31P NMR (CD3CN, 80 MHz): δ 148.3 and 148.7.
T
20 was hybridized with hexa- to eicosa-adenylate (A6, A7, A8,
A9, A10, and A20), and the melting temperature (Tm) of each
complex was determined (Table 1). For comparison, the Tm of
Tn:An duplexes were also determined.
Tm values of cT20:An increased from A6 to A10 and then
reached a plateau. These results suggest that cT20 is able to
hybridize up to 10 nucleobases. It is known that such a cyclic
thymidylate forms a triplex helix with the cyclic oligo in a
dumbbell form with two loops of 3- to 5-nucleotide length.18
According to this structure, the cT20 could form a triplex with
five to seven TAT triplets. To explain the present result we
could hypothesize that extra nucleobases form base pairs with
the nucleobases of the loops. The Tm values of Tn:An duplexes
(45) Kanan, M. W.; Rozenman, M. M.; Sakurai, K.; Snyder, T. M.; Liu,
D. R. Nature 2004, 431, 545-549.
(46) Chaudhuri, N. C.; Kool, E. T. J. Am. Chem. Soc. 1995, 117, 10434-
10442.
(47) Morvan, F.; Meyer, A.; Jochum, A.; Sabin, C.; Chevolot, Y.;
Imberty, A.; Praly, J. P.; Vasseur, J. J.; Souteyrand, E.; Vidal, S.
Bioconjugate Chem. 2007, 18, 1637 -1643.
(48) Wijsman, E. R.; Filippov, D.; Valentijn, A. R. P. M.; van der Marel,
G. A.; van Boom, J. H. Recl. TraV. Chim. Pays-Bas 1996, 115, 397-401.
2-[(4,4′-Dimethoxytrityl)oxymethyl]-2-methylpropane-1,3-
diol (14). To 1,1,1-tris(hydroxymethyl)ethane (1.2 g, 10 mmol)
dissolved in dry pyridine (50 mL) was added dimethoxytrityl
chloride (2.4 g, 8 mmol) in two parts over 30 min. After 3 h of
J. Org. Chem, Vol. 73, No. 1, 2008 199

Lietard et al.
stirring at room temperature, the reaction was quenched with
methanol (2 mL). The mixture was treated with aqueous NaHCO3
(300 mL), and the title compound was extracted with CH2Cl2 (3 ×
150 mL). The organic layer was dried over Na2SO4 and evaporated
under reduced pressure. The oily residue was purified by silica gel
chromatography using an increasing amount of methanol (0-2%)
in CH2Cl2 containing 1% of Et3N affording 14 as a colorless oil:
1.9 g, 56% Rf ) 0.28 (CH2Cl2/MeOH, 19:1, v/v). 1H NMR (CDCl3,
400 MHz): δ 0.87 (3H, s), 2.42 (2H, br s), 3.16 (2H, s), 3.69 (2H,
d, J ) 25.2 Hz) 3.64 (2H, d, J ) 25.2 Hz), 3.82 (6H, s), 6.79-
7.49 (13H, m). 13C NMR (CDCl3, 100 MHz): δ 17.4, 41.1, 55.2,
67.2, 68.2, 86.3, 113.3, 126.9, 128.0, 130.0, 135.8, 144.7, 158.5.
HRFABMS (positive mode, nitrobenzylic alcohol) m/z: calcd for
C26H30O5 [M + H]+ 422.2093, found 422.2098.
42.9, 43.0, 43.1, 55.3, 63.4, 84.8, 86.9, 111.2, 113.3, 127.1, 128.0,
128.2, 130.1, 135.3, 135.4, 135.5, 135.8, 144.4, 150.2, 158.7, 163.7.
31P NMR (CDCl3, 80 MHz): δ 147.8 and 148.3.
1-O-(4,4′-Dimethoxytrityloxymethyl)-2,2-bis-propargyloxym-
ethyl-3-O-(succinic-LCAA CPG)-1,3-propanediol (26). LCAA-
CPG (1.00 g), 1-O-(4,4′-dimethoxytrityl)-2,2-bis-propargyloxymethyl-
1,3-propanediol 847 (0.114 g, 0.2 mmol), EDC (0.191 g, 1 mmol),
DMAP (0.012 g, 0.1 mmol), and Et3N (0.1 mL) were shaken in
anhydrous pyridine (5 mL) at room temperature for 48 h. Then
pentachlorophenol (135 mg, 0.5 mmol) was added, and the mixture
was shaken for 10 h. Piperidine (5 mL) was added, and after 5
min of shaking, the solid support was filtered off, washed with
CH2Cl2, and dried. A capping step with standard Cap A and Cap
B solutions was applied for 2 h, and the solid support was filtered
off, washed with CH2Cl2, and dried. Trityl assay indicated a loading
of 40 µmol/g.
Oligonucleotide Synthesis. Oligonucleotides (1 µmol scale) were
synthesized on an ABI 381A or 394 DNA synthesizer using a cycle
involving phosphoramidite chemistry. Detritylation was performed
with 2.5% DCA in CH2Cl2 for 60 s. Coupling step: BMT (0.3 M
in dry acetonitrile) was used as activator; propynyl and bromohexyl
phosphoramidites (0.09 M in CH3CN) were introduced with a 45
s coupling time; commercially available phosphoramidites (0.09M
in CH3CN) were introduced with a 30 s coupling time. The capping
step was performed with acetic anhydride using commercial solution
(Cap A: Ac2O, pyridine, THF 10/10/80 and Cap B: 10%
N-methylimidazole in THF) for 15 s. Oxidation was performed with
commercial solution of iodide (0.1 M I2, THF, pyridine/water 90/
5/5) for 10 s.
1-Propargyl-2-[(4,4′-dimethoxytrityl)oxymethyl]-2-methylpro-
pane-1,3-diol (15). Compound 14 (620 mg, 1.47 mmol) was
dissolved in anhydrous THF (6 mL), and sodium hydride (60% in
oil, 590 mg, 14.7 mmol) was added. After 10 min of stirring,
propargyl bromide (80% in toluene, 650 mL, 5.9 mmol) was added,
and the mixture was stirred at rt for 1 h. Then CH2Cl2 (100 mL)
and 2 mL of water were added. The organic layer was washed with
water (2 × 100 mL) and dried over Na2SO4. After evaporation,
the residue was purified by flash chromatography using an
increasing amount of ethyl acetate (10% to 50%) in cyclohexane
1
containing 1% of triethylamine: 500 mg 74%. H NMR (CDCl3,
200 MHz): δ 0.95 (3H, s), 2.58 (1H, t), 2.60 (1H, br s), 3.03 (1H,
d, J ) 20.7 Hz) 3.13 (1H, d, J ) 20.7 Hz) 3.58-3.6 (2H, m), 3.65
(2H, s), 3.82 (6H, s), 4.18 (2H, dd, J ) 0.9 and 1.4 Hz), 6.85-
7.54 (13H, m). 13C NMR (CDCl3, 100 MHz): δ 17.8, 40.8, 55.2,
58.7, 66.5, 68.9, 74.3, 74.5, 79.7, 86.0, 113.2, 126.7, 127.8, 128.1,
129.1, 130.1, 136.0, 145.0, 158.4. HRFABMS (positive mode,
nitrobenzylic alcohol) m/z: calcd for C29H32O5 [M + H]+ 460.2250,
found 460.2248.
General Procedure for Azidation. Azidation of 5′-hydroxyl
oligonucleotides was performed according to the procedure de-
scribed by Kool et al.38
1-Propargyl-2-[(4,4′-dimethoxytrityl)oxymethyl]-2-methyl-3-
(succinic-LCAA CPG)-propane-1,3-diol (16). Same protocol as
for 11. Loading 32.2 µmol/g for LCAA CPG 500 Å and 30.1
µmol/g for LCAA CPG 1000 Å.
Azidation from bromohexyl oligonucleotides was performed as
follows: A solution of NaN3 (13 mg) and NaI (30 mg) in dry DMF
(1.5 mL) was applied on the solid-supported bromohexyl oligo-
nucleotide for 1 h 15 min at 65 °C. Then the CPG beads bearing
the oligonucleotide were washed with DMF (2 × 1 mL) and CH2-
Cl2 (5 mL) and dried in a desiccator under reduced pressure for 30
min.
General Procedure for Deprotection. The beads were placed
into a sealed vial and treated with concentrated aqueous ammonia
(1 mL) for 24 h at room temperature for oligonucleotides containing
phosphotriester functions or 2 h at room temperature and then 5 h
at 55 °C for the others. The beads were filtered off, and the solution
was evaporated. The residue was dissolved in water for subsequent
analyses.
General Procedure for Cu(I)-Catalyzed 1,3-Dipolar Cycload-
dition. To azido-alkyne oligonucleotide (∼1 µmol) were added
CuSO4 (0.4 equiv, 0.4 µmol, 13.2 µL of a 20 mM solution in H2O),
freshly prepared (from degassed water) sodium ascorbate (2 equiv,
2 µmol, 13.2 µL of a 100 mM solution in H2O), methanol (100
µL), and water (23.6 µL). The tube containing the resulting
preparation was flushed with argon and sealed. The reaction was
placed in a microwave synthesizer Initiator from Biotage set at 100
W with a 30 s premixing time for 1 to 1 h 30 min at 60 °C.
Temperature was monitored with an internal infrared probe. The
solution was then desalted on NAP10.
6-Bromohexyl 2-cyanoethyl diisopropylphosphoramidite (17).
To a solution of anhydrous 6-bromo-1-hexanol (325 mg, 1.8 mmol)
and diisopropylammonium tetrazolide (154 mg, 0.9 mmol) in
anhydrous dichloromethane (5 mL) was added 2-cyanoethyl tet-
raisopropylphosphorodiamidite (570 µL, 1.8 mmol). The resulting
mixture was stirred for 5 h at room temperature, diluted with ethyl
acetate (30 mL), and washed with brine (2 × 100 mL). The organic
layer was dried (Na2SO4), filtered, and evaporated to dryness. The
residue was purified by flash column chromatography (silica gel;
cyclohexane with 3% Et3N) affording phosphoramidite 1 (400 mg
60% yield) as a colorless oil. TLC (cyclohexane/CH2Cl2/Et3N; 5/4/
1; v/v/v) Rf: 0.5. 1H NMR (CDCl3, 200 MHz): δ 1.17-1.21 (12H,
d), 1.44-1.88 (8H, m), 2.62-2.69 (2H, m), 3.39-3.87 (8H, m).
13C NMR (CDCl3, 100 MHz): δ 19.9, 24.0, 24.1, 24.2, 24.7, 27.3,
29.7, 30.4, 30.5, 32.2, 33.3, 42.4, 42.6, 57.7, 57.9, 62.9, 63.1, 117.1
31P NMR (CDCl3,80 MHz): δ 148.5 ppm.
6-Bromo-1-hexyl Tetraisopropylphosphorodiamidite (23). Us-
ing the same protocol as for 1 starting from 6-bromo-1-hexanol
1
271.6 mg 1.5 mmol gave 410 mg (90%). H NMR (CDCl3, 200
MHz): δ 1.16-1.20 (24H, dd), 1.42-1.47 (4H, m), 1.56-1.65
(2H, m), 1.85-1.92 (2H, m), 3.39-3.62 (8H, m). 13C NMR (CDCl3,
100 MHz): δ 23.6, 23.7, 24.6, 24.7, 27.9, 31.4, 31.5, 32.8, 33.7,
44.1, 44.4, 63.9, 64.1, 31P NMR (CDCl3,80 MHz): δ 125.3 ppm.
5′-O-Dimethoxytrityl-3′-O-[(6-bromohexyl)-N,N-diisopropy-
lphosphoramidite]thymidine (24). Using the same protocol as for
2 starting from 5′-dimethoxytritylthymidine (300 mg, 0.54 mmol)
and using 6-bromo-1-hexytetraisopropylphosphorodiamidite (23)
afforded 377 mg 82% of 24. 1H NMR (CDCl3, 200 MHz): δ 1.4-
1.20 (12H, m), 1.36-1.88 (9H, m), 2.50-2.65 (2H, m), 3.35-3.7
(8H, m), 3.81 (6H, s), 4.17-4.22 (1H, m), 4.61-4.69 (1H, m),
6.40-6.46 (1H, m), 6.83-7.67 (14H, m). 13C NMR (CDCl3, 100
MHz): 11.7, 24.5, 24.6, 25.1, 25.2, 27.8, 31.0, 32.7, 33.8, 339,
Acknowledgment. We thank the Universite´ Montpellier 2,
CNRS, and Inserm for financial supports.
Supporting Information Available: HPLC profiles and MALDI-
1
TOF MS spectra of oligonucleotides synthesized. H NMR, 13C
NMR, and 31P NMR spectra of all new compounds. This material
is available free of charge via the Internet at http://pubs.acs.org.
JO702177C
200 J. Org. Chem., Vol. 73, No. 1, 2008