Azide solid support for 3′-conjugation of oligonucleotides and their circularization by click chemistry

Article abstract of DOI:10.1021/jo9014563

(Chemical Equation Presented) A solid support bearing an azido linker was used to synthesize a 3′-azido-alkyl-oligonucleotide by phosphoramidite chemistry. The resulting oligonucleotide was either conjugated by 1,3-dipolar cycloaddition on solid support o

Full text of DOI:10.1021/jo9014563

pubs.acs.org/joc
Azide Solid Support for 30-Conjugation of Oligonucleotides and
Their Circularization by Click Chemistry
Gwladys Pourceau, Albert Meyer, Jean-Jacques Vasseur, and Franc-ois Morvan*
ꢀ
Institut des Biomolecules Max Mousseron (IBMM), UMR 5247 CNRS, Universite Montpellier 1,
Universiteꢀ Montpellier 2, Place Eugeꢁne Bataillon, CC1704, 34095 Montpellier Cedex 5, France
ꢀ
morvan@univ-montp2.fr
Received July 8, 2009
A solid support bearing an azido linker was used to synthesize a 30-azido-alkyl-oligonucleotide by
phosphoramidite chemistry. The resulting oligonucleotide was either conjugated by 1,3-dipolar
cycloaddition on solid support or in solution with mannose-propargyl derivative and in solution with
dansyl propargyl. Besides, after introduction of an alkyne function at the 50-end, the resulting
oligonucleotide bearing both 30-azide and 50-alkyne functions was circularized.
Introduction
biomolecules such as peptides5-9or carbohydrates7,10-16
are commonly described. Most of the conjugation methods
require the introduction of functional groups to synthetic
ODNs by a DNA synthesizer. Amine or thiol functions are
from the most popular groups for the conjugation to the ends
of oligonucleotides via commercially available phosphora-
midite reagents or solid supports. Recently an alternative
approach using the azide/alkyne system arose as an efficient
and powerful method for conjugation. Both functions react
Oligonucleotide (ODN) conjugates are widely used for
various applications in biology, biotechnology, and medi-
cine. Many methods of conjugation are reported in the
literature.1-4 ODN conjugates with fluorescent dyes and
*To whom correspondence should be addressed. Phone: þ33/467 144 961.
Fax: þ33/467 042 029.
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DOI: 10.1021/jo9014563
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Published on Web 08/07/2009
J. Org. Chem. 2009, 74, 6837–6842 6837
2009 American Chemical Society

Pourceau et al.
JOCArticle
together through a Huisgen 1,3-dipolar cycloaddition17
called “click chemistry”.18 This reaction was found to be
catalyzed by the Cu(I) ion,19,20 leading to fast reactions.
Hence, a wide range of applications for bioconjugation have
been reported (for reviews see refs 21-27). It is generally
considered that both alkyne and azide functions are mainly
orthogonal with other functionalities so that the click reac-
tion is chemoselective and can be performed in water and
organic solvents. Many publications reported the introduc-
tion of the alkyne function into ODN using phosphoramidite
derivatives of modified nucleosides on the nucleobases,28-31
on the sugar at the 20 position32,33 or on the phosphorus
atom,34 or using non-nucleosidic derivatives.34-45 Methods
to introduce propargyl function through a phosphoramidate
linkage were also described.15,16,46
FIGURE 1. Schematic structure of phosphoramidite derivative
1-3 and solid support 4 bearing a bromohexyl group (CE: 2-
cyanoethyl).
In contrast only a few publications showed the introduc-
tion of the azide function into an oligonucleotide still on the
solid support. The main reason is that it was shown that a
nucleoside with an azide function reacts with the phosphor-
amidite derivative according to the Staudinger reaction when
both compounds are in solution.47 Likewise, van der Marel
et al.33 showed more recently that a nucleoside exhibiting
both phosphoramidite and an azide function rapidly decom-
posed in solution. Thus to avoid this side reaction the azide
function was mainly introduced by a time-consuming post-
elongation protocol in solution.33,38,48 As an alternative
Kool et al. reported the 50-iodination and subsequent treat-
ment with sodium azide to gain 50-azido-oligonucleotide49
but this approach was restricted since deoxyadenosine led to
a side reaction during the iodination.50 Along this line, the
use of building blocks with a bromine-alkyl group was
developed and introduced automatically by a DNA synthe-
sizer into a sequence34,51or at the 50-end.36,52 The bromo
function was then converted to an azido upon treatment with
sodium azide leading to azide oligonucleotides. However,
very recently Lonnberg et al. showed that the azide function
can be introduced by a 40-azidomethyl-thymidine-30-H-
phosphonate derivative and then the oligonucleotide can
be elongated with either H-phosphonate or phosphoramidite
chemistry without observing the Staudinger reaction.53
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Results and Discussion
(31) Nakane, M.; Ichikawa, S.; Matsuda, A. J. Org. Chem. 2008, 73,
1842–1851.
This recent result prompted us to design a solid support
allowing the introduction of an azide function for subse-
quent 30-conjugation by Cu(I) alkyne/azide cycloaddition
(CuAAC) and also the cyclization of an oligonucleotide
bearing both 30-azide and 50-alkyne functions.
(32) Grotli, M.; Douglas, M.; Eritja, R.; Sproat, B. S. Tetrahedron 1998,
54, 5899–5914.
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H. S.; van der Marel, G. A.; Rutjes, F. P. J. T.; van Delft, F. L. J. Org. Chem.
2008, 73, 287–290.
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73, 191–200.
To this end, two strategies were explored. The first one was
based on the use of a solid support bearing a bromohexyl
group 4 (Figure 1) as an extension of our previous work
where phosphoramidite derivatives 1-3 were used and the
bromine atom was easily substituted after elongation by
sodium azide affording the azido-oligonucleotides.34,51,52
However, with the solid support 4, prepared from CPG
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Imberty, A.; Praly, J. P.; Vasseur, J. J.; Souteyrand, E.; Vidal, S. Bioconjugate
Chem. 2007, 18, 1637–1643.
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2008, 73, 6014–6017.
˚
with 500 A pore size, we were unable to substitute the
bromine atom using either sodium azide or the more reactive
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2008, 64, 1467–1473.
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2009, 74, 1218–1222.
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O. S.; Wagenknecht, H. A. Bioconjugate Chem. 2009, 20, 558–564.
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Freedberg, D. I.; Beaucage, S. L. Bioconjugate Chem. 2008, 19, 1696–1706.
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Tetrahedron Lett. 2006, 47, 8867–8871.
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2007, 48, 8795–8798.
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6838 J. Org. Chem. Vol. 74, No. 17, 2009

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SCHEME 1. Synthesis of Azide Solid Support 10
tetramethylguanidine azide reagent. As the steric hindrance
due to the presence of the oligonucleotide was suspected,
we also tried to perform the substitution before the oligonu-
cleotide elongation directly on the solid support 4 but here
again without success. A last attempt with less loaded
ꢀ
˚
CPG with 1000 A pore size was also unsuccessful. These
results are surprising since we have already substituted bro-
mide atoms on solid supported oligonucleotides many
times.34,51,52 However, in these cases the alkyl bromide
chain was always borne on a phosphotriester linkage34,52
or between two phosphotriester linkages.51 We suspect a
detrimental long distance effect of the ester function linking
the bromine compound to the solid support, but we were
unable to find any information in the literature to support
this hypothesis.
The second strategy uses a solid support 10 bearing an
azide alkyl chain that was successfully applied to synthesized
30-azide alkyl-oligonucleotide according to the phosphora-
midite chemistry.
The solid support 10 was easily prepared by a 4-step
synthesis starting from dimethoxytrityl-1,1,1-tris(hydroxy-
methyl)ethane 634 that was alkylated with 1,4-dibromobu-
tane in the presence of NaH affording 7 as a racemic mixture
and then treated with sodium azide and sodium iodide to give
the azido derivative 8. This compound was converted into its
O-hydroquinone-O,O0-diacetyl hemiester54 derivative 9 and
FIGURE 2. HPLC profiles of 30-mannosylated CAT CGG GCT
TGG 13 obtained by CuAAC on solid support with MW assistance
or in solution with and without MW assistance and after puri-
fication. HPLC profiles of the crude 30-azide 9-mer 14 and its
30-conjugate 16 with dansyl obtained by CuAAC in solution with-
out MW.
oligonucleotide 14 (440 nmol, 54 OD260 nm). After evapora-
tion, the crude (18 OD260 nm for each) was used for CuAAC
reaction with dansyl-propargyl 1555 (5 molar equiv) or 1-O-
propargyl-2,3,4,6-O-tetraacetyl-R-mannose 12 (5 molar
equiv). The reactions were performed at room temperature,
using CuSO4 (2 molar equiv) and sodium ascorbate (10 molar
equiv) in methanol-water (1:1, v/v), and were complete
within 2 h affording the 30-dansyl oligonucleotide 16 (140 nmol,
17 OD260 nm) and the 30-tetraacetyl-mannose oligonucleotide,
respectively. The acetyl protections of the 30-tetraacetyl-man-
nose oligonucleotide were removed under ammonia to give 13
(115 nmol, 14 OD260 nm).
For comparison 11 (66 nmol) was used for CuAAC
reaction with 12 under MW assistance for 30 min at 60 °C,
using the same conditions as above. A complete cycloaddi-
tion was observed with a similar HPLC profile. A final
ammonia treatment gave conjugate 13 with a similar purity
to 13 obtained without MW assistance (Figure 2).
˚
finally anchored on LCAA-CPG by using EDC (500 A
loading of 65 μmol/g) (Scheme 1).
A first heptathymidylate was synthesized on solid support
10 by using standard phosphoramidite chemistry. Undeni-
ably, after deprotection upon ammonia treatment, the ex-
pected 30-azide-linker-T7 was obtained (data not shown). We
then synthesized on solid support 10 an oligonucleotide
exhibiting the four nucleobases (Scheme 2). One-third of
the resulting solid-supported dodecamer (CAT CGG GCT
TGG) bearing a 30-azide-linker group 11 was directly used
for a CuAAC with 1-O-propargyl-2,3,4,6-tetra-O-acetyl-R-
D-mannopyranose 12 (5 equiv) on solid support under
microwave (MW) activation15 at 60 °C for 30 min (2 equiv
of CuSO4, 10 equiv of Na ascorbate, methanol-water 1:1)
then deprotected with aqueous ammonia to provide the
30-mannosyl 12-mer 13 (29 OD260 nm 240 nmol, Figure 2).
The remaining two-thirds were treated with ammonia
to release in solution the fully deprotected 30-azide-linker
(55) Bolletta, F.; Fabbri, D.; Lombardo, M.; Prodi, L.; Trombini, C.;
Zaccheroni, N. Organometallics 1996, 15, 2415–2417.
(54) Pon, R. T.; Yu, S. Y. Nucleic Acids Res. 1997, 25, 3629–3635.
J. Org. Chem. Vol. 74, No. 17, 2009 6839

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SCHEME 2. Synthesis of 30-Mannose and 30-Dansyl Oligonucleotide Conjugatesa
aSPOS: solid-phase oligonucleotide synthesis: (1) 2.5% dichloroacetic acid CH2Cl2; (2) phosphoramidite derivative þ benzylthiotetrazole; (3) Ac2O,
N-Me imidazole, 2,6-lutidine; (4) 0.1 M I2 THF/H2O/pyridine. The asterisk represents protecting groups: benzoyl for A and C and isobutyryl for G.
We observed that it is quite compulsory to use MW
assistance when CuAAC reactions are carried out on solid
support to rapidly obtain (less than 30 min) a complete
reaction.15 In contrast, when CuAAC reactions were carried
out in solution, the MW assistance could be omitted and the
reaction is usually done within 2 h at rt but obviously
CuAAC reactions under MW assistance are more rapid since
the temperature is set at 60 °C.
SCHEME 3. Synthesis of Cyclic 9-Mer ACTGACATC 19
DNA Cyclization
Several chemical methods are described in literature to
circularize DNA or RNA.31,34,38,45,56-62 Among them, re-
cent examples of DNA circularization by CuAAC reaction
have been reported but in each case the protocol is not straight-
forward and requires an extra postelongation step.31,34,38,45,62
Along this line, we previously reported a universal method to
synthesize cyclic oligonucleotides starting from a monoalk-
yne solid support. After the oligonucleotide elongation the
introduction of phosphoramidite bromohexyl 1 was also
done with a DNA synthesizer. Then a postelongation treat-
ment on solid supported oligonucleotide with a NaN3 NaI
solution leads to the substitution of the bromide affording
the 50-azidooligonucleotide.34 After the ammonia treat-
ment to deprotect and release the 30-alkyne 50-azide oligo-
nucleotide from the solid support, this compound was finally
circularized in solution by CuAAC reaction. Here, we
proceeded in the reverse way, starting from the azide solid
support 10, synthesizing the oligonucleotide (ACTGA-
CATC) and introducing the alkyne function by means of
pentyn-4-oyl phosphoramidite (Scheme 3). After ammonia
deprotection the linear 5-alkyne 30-azide nonamer was
afforded in solution as a single peak. The CuAAC reaction
was efficiently performed with CuSO4 and sodium ascorbate
at room temperature for 3.5 h to give the cyclic nonamer as a
double peak due to the chiral carbon of the trishydroxy-
methylethane moiety. A similar behavior was already ob-
served on other cyclic oligonucleotides where a phosphorus
atom was the chiral center.34 The reaction was monitored by
reverse phase HPLC (Figure 3) and not by MALDI-TOF
MS since the mass of both linear and cyclic oligonucleotide is
the same. As observed before, the cyclic ODN exhibited a
lower retention time than the linear ODN due to the high
lipophilicity of the azide function. Both linear and cyclic
compounds were characterized by MALDI-TOF MS.
(56) De Napoli, L.; Galeone, A.; Mayol, L.; Messere, A.; Montesarchio,
D.; Piccialli, G. Bioorg. Med. Chem. 1995, 3, 1325–1329.
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Int. Ed. 1997, 36, 1506–1508.
(58) Micura, R. Chem.;Eur. J. 1999, 5, 2077–2082.
(59) Smietana, M.; Kool, E. T. Angew. Chem., Int. Ed. 2002, 41, 3704–3707.
(60) Edupuganti, O. P.; Defrancq, E.; Dumy, P. J. Org. Chem. 2003, 68,
8708–8710.
(61) Sturm, M. B.; Roday, S.; Schramm, V. L. J. Am. Chem. Soc. 2007,
129, 5544–5550.
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A. N.; Brown, T. ChemBioChem 2008, 9, 50–52.
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Pourceau et al.
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FIGURE 3. HPLC profiles of linear and cyclic 9-mer ACTGACATC 19.
The present method is more straightforward than all of the
previous ones since all the steps to obtain the linear oligo-
nucleotide bearing both alkyne and azide functions are
performed on a DNA synthesizer without any postelonga-
tion step.
74%) as a clear oil. Rf 0.5 (CH2Cl2/acetone 7.5:2.5 v/v); 1H NMR
(CDCl3, 400 MHz) δ 0.92 (s, 3H), 1.62-1.65 (m, 4H), 2.72 (m,
1H), 3.08-3.15 (m, 2H), 3.27-3.31 (m, 2H), 3.44-3.58 (m, 6H),
3.82 (s, 6H), 6.84-7.46 (m, 13H); 13C NMR (CDCl3, 100 MHz) δ
17.9, 25.8, 26.9, 40.8, 51.3, 55.2, 66.5, 69.4, 71.0, 75.9, 85.9, 113.1,
126.8, 127.8, 128.2, 130.1, 136.1, 145.0, 158.4; HRMS ESI (positive
mode) m/z calcd for C30H37O5N3Na [M þ Na]þ 542.2631, found
542.2646.
Conclusion
(4-CPG-long chain alkyl-carbamoylmethoxy-phenoxy)acetic
Acid 2-(4-Azido-butoxymethyl)-3-(4,40-dimethoxytrityloxymethyl)-
2-methylpropyl Ester 10. Compound 8 (250 mg, 0.48 mmol) was
dissolved in dry pyridine with hydroquinone-O,O0-diacetic acid
(130 mg, 0.58 mmol), DMAP (12 mg, 0.1 mmol), 1-(3-dimethyl-
aminopropyl)-3-ethylcarbodiimide DEC (92 mg, 0.4 mmol)
then the mixture was stirred at rt for 3 h. Pyridine was evapo-
rated and the residue was redissolved in CH2Cl2 and washed
with water (three times). After evaporation the crude hemiester
was directly anchored on CPG by using DMAP (10 mg), DEC
(120 mg), and dry Et3N (80 μL) in dry pyridine and shaken
overnight. After filtration and drying the solid support was
capped by using Cap A and Cap B solution for 1 h. Loading was
determined by trityl assay: 65 μmol/g
In conclusion, an azide solid support was efficiently pre-
pared. This solid support could be used with phosphorami-
dite chemistry allowing 30-end conjugation of oligonu-
cleotide either on solid support or in solution through
CuAAC reaction. Its use in combination with an alkyne
phosphoramidite derivative introduced at the 50-end af-
forded an oligonucleotide with both alkyne and azide func-
tions allowing a straightforward cyclization.
Experimental Section
1-O-(4,40-Dimethoxytrityl)-2-(4-bromobutoxymethyl)-2-methyl-
1,3-propanediol, 7. 2-[(4,40-Dimethoxytrityl)oxymethyl]-2-methyl-
propane-1,3-diol34 (1.7 g, 4 mmol), sodium iodide (62 mg, 0.1
mmol), and dibromobutane (1.54 mL, 10 mmol) were added to a
solution of sodium hydride (60% in oil, 645 mg, 4 mmol) in
anhydrous THF (16 mL) at 0 °C. After 5 h under magnetic stirring
at rt, the reaction was quenched by adding 50 mL of CH2Cl2 and
2 mL of water. The organic layer (300 mL) was washed with brine
(2 ꢀ 150 mL), dried over Na2SO4, and evaporated. The crude
product was purified by silica gel column chromatography (0 to
30% acetone in cyclohexane containing 1% triethylamine) to
afford a racemic mixture of 7 (1.4 g, 64%) as a clear oil. Rf 0.5
(cyclohexane/acetone 1:1 v/v); H NMR (CDCl3, 300 MHz) δ 0.89
(s, 3H), 1.68-1.92 (m, 4H), 2.70 (t, 1H, J = 5.9 Hz), 3.06-3.14 (m,
2H), 3.40-3.80 (m, 8H), 3.80 (s, 6H), 6.84-7.45 (m, 13H); 13C
NMR (CDCl3, 75 MHz) δ 16.9, 27.2, 28.6, 32.8, 39.8, 54.2, 65.4,
68.4, 69.7, 74.8, 84.9, 112.1, 125.9, 126.8, 127.1, 129.3, 135.1, 138.5,
144.0, 157.5. Mass spectrometry characterization was unsuccess-
ful.
1-O-(4,40-Dimethoxytrityl)-2-(4-azidobutoxymethyl)-2-meth-
yl-1,3-propanediol, 8. 1-O-(4,40-Dimethoxytrityl)-2-(4-bromo-
butoxymethyl)-2-methyl-1,3-propanediol (7; 0.36 mmol, 200 mg)
was dried three times by coevaporation with anhydrous acetoni-
trile and then dissolved in anhydrous acetonitrile (3 mL) before
addition of tetramethylguanidinium azide (0.72 mmol, 114 mg).
The reaction was stirred at rt for 5 h, and then diluted with 25 mL
of CH2Cl2. The organic layer was washed with brine (2ꢀ70 mL),
dried over Na2SO4, and evaporated. The crude product was
purified by silica gel column chromatography (0 to 3% acetone
in CH2Cl2 containing 0.5% triethylamine) to afford 8 (140 mg,
O-4-Pentynyl-O0-(2-cyanoethyl)-N,N-diisopropylphosphora-
midite, 17. To a solution of anhydrous 4-pentyn-1-ol (1.8 mmol,
151 mg) and diisopropylammonium tetrazolide (0.9 mmol, 154 mg)
in anhydrous CH2Cl2 (5 mL) was added 2-cyanoethyl diisopro-
pylphosphorodiamidite (1.8 mmol, 570 μL). The resulting mix-
ture was stirred for 5 h at room temperature, diluted with
CH2Cl2 (30 mL), and washed with brine (2ꢀ100 mL). The organic
layer was dried (Na2SO4), filtered, and evaporated to dryness.
The crude product was purified by silica gel column chroma-
tography (0 to 50% CH2Cl2 in cyclohexane containing 5%
triethylamine) to afford 17 (350 mg, 68%) as a clear oil. Rf 0.65
1
(cyclohexane/CH2Cl2/Et3N 5:4:1, v/v/v); H NMR (CDCl3, 200
MHz) δ1.18-1.21 (d, 12H, J = 6.4 Hz), 1.77-1.97 (m, 3H), 2.27-
2.36 (m, 2H), 2.62-2.69 (m, 2H), 3.55-3.88 (m, 6H); 13C NMR
(CDCl3, 100 MHz) δ 15.3, 20.4, 29.2, 42.1, 58.2, 58.4, 61.9, 68.9,
83.7, 117.6; 31P NMR (CDCl3 81 MHz) δ 148.8 ppm; HRMS ESI
(positive mode) m/z calcd for C14H26O2N2P [M þ H]þ 285.1732,
found 285.1732.
General Procedure for Cu(I)-Catalyzed 1,3-Dipolar Cycload-
dition: a. In Solution. A solution of 5-(dimethylamino)-N-(2-
propynyl)-1-naphthalene-sulfonamide 1555 (70 mM, 10 μL) or
1-O-propargyl-2,3,4,6-tetra-O-acetyl-R-D-mannopyranose 1251
(100 mM, 7.5 μL) in MeOH and freshly prepared solutions of
CuSO4 (40 mM 7.5 μL) and sodium ascorbate (100 mM, 15 μL)
in water were added to oligonucleotide 14 (0.146 μmol, 123 μL of
H2O) in solution. The coupling was monitored by HPLC. After
2 h, the mixture was desalted on NAP 10, affording the
oligonucleotide conjugate (16: 17 DO at 260 nm, 140 nmol,
J. Org. Chem. Vol. 74, No. 17, 2009 6841

Pourceau et al.
JOCArticle
97%; 13: 14 DO at 260 nm, 115 nmol, 81% after ammonia
treatment at 55 °C for 5 h).
(10 μL of a 40 mM solution in H2O), freshly prepared sodium
ascorbate (20 μL of a 100 mM solution in H2O), and methanol
(90 μL). After 3.5 h the solution was desalted on NAP 10.
b. On Solid Support. A solution of 1-O-propargyl-2,3,4,6-O-
tetraacetyl-R-mannose 12 (100 mM, 7.5 μL) in MeOH and freshly
prepared solutions of CuSO4 (40 mM, 7.5 μL) and sodium
ascorbate (100 mM, 15 μL) in water were added to the solid-
supported oligonucleotide (∼1/3 μmol). The vial containing the
resulting mixture was sealed and placed in a microwave synthesi-
zer, with a 30 s premixing time, for 30 min at 60 °C. The
temperature was monitored with an internal infrared probe. Then,
the CPG beads were filtered and washed with water (2 mL) and
MeOH (2 mL) and dried. A final ammonia treatment (55 °C for
5 h) removed all the protecting groups and released the oligonu-
cleotide conjugate in solution (29 DO at 260 nm, 239 nmol, 80%).
DNA Circularization by Cu(I)-Catalyzed 1,3-Dipolar Cy-
cloaddition Reaction. To the linear alkyne-azide oligonucleotide
18 (200 nmol) dissolved in 120 μL of water were added CuSO4
Acknowledgment. This work was financially supported by
the CNRS interdisciplinary program “Interface Physique
ꢁ
Chimie Biologie: soutien a la prise de risque”, ANR-08-
BLAN-0114-01, and Lyon Biopole. G.P. thanks the CNRS
ꢀ
and the Region Languedoc-Roussillon for the award of a
research studentship. F.M. is from Inserm.
Supporting Information Available: Protocol to synthesize
modified oligonucleotides, MALDI-TOF MS data of 13, 14, 16,
1
18, and 19, and H NMR, 13C NMR, and 31P NMR of com-
pounds 7, 8, and 17. This material is available free of charge via
the Internet at http://pubs.acs.org.
6842 J. Org. Chem. Vol. 74, No. 17, 2009