Synthesis of mannose and galactose oligonucleotide conjugates by bi-click chemistry

Article abstract of DOI:10.1021/jo802536q

Glyco oligonucleotide conjugates, each exhibiting two mannose and two galactose residues, were efficiently synthesized by two successive 1,3-dipolar cycloadditions (click chemistry). Two phosphoramidite derivatives were used: one bearing a bromoalkyl grou

Full text of DOI:10.1021/jo802536q

Synthesis of Mannose and Galactose Oligonucleotide Conjugates by
Bi-click chemistry
Gwladys Pourceau, 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 NoVember 14, 2008
Glyco oligonucleotide conjugates, each exhibiting two mannose and two galactose residues, were efficiently
synthesized by two successive 1,3-dipolar cycloadditions (click chemistry). Two phosphoramidite
derivatives were used: one bearing a bromoalkyl group as a precursor to azide functionalization and
another bearing a propargyl group. After a first cycloaddition with a mannosyl-azide derivative, the bromine
atoms were substituted with NaN3 and a second click reaction was performed with a 1′-O-propargyl
galactose, affording the heteroglyco oligonucleotide conjugate.
Introduction
functions. However, because some bacteria exhibit several
lectins recognizing different carbohydrate residues12-14 and also
a single lectin could recognize several carbohydrate residues,15
it is necessary to be able to synthesize such glycomimics bearing
different residues. Several strategies for coupling carbohydrates
to oligonucleotides are reported in the literature,16-21 and to
our knowledge only one describes the attachment of different
sugar moieties on the same scaffold.18 Recently, a synthesis
was reported for a heterobifunctional dendrimer exhibiting
mannose and fucose residues.22 Moreover, during this work,
Carbohydrates are involved in many biological events and
play crucial roles in various cellular recognition processes.1,2
Understanding their interactions with proteins is a major issue
for the development of new therapies. Glycoarrays have recently
emerged as a high-throughput tool for the study of carbohydrate-
lectin binding.3 We recently designed a new glycoarray using
DNA-directed immobilization4,5 and to this end have developed
very efficient strategies based on Cu(I) azide alkyne 1,3-dipolar
cycloaddition,6,7 or click chemistry, to synthesize oligonucleotide
carbohydrate conjugates5,8,9 and galactosylated10 or fucosylated
phosphodiester glycoclusters.11 In these syntheses, the same
carbohydrate (galactose, mannose, or fucose) was introduced
one to ten times onto a scaffold exhibiting one to ten alkyne
(11) 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.
(12) Mulvey, G.; Kitov, P. I.; Marcato, P.; Bundle, D. R.; Armstrong, G. D.
Biochimie 2001, 83, 841–847.
(13) Bernardi, A.; Arosio, D.; Potenza, D.; Sanchez-Medina, I.; Mari, S.;
Canada, F. J.; Jimenez-Barbero, J. Chem. Eur. J. 2004, 10, 4395–4406.
(14) Imberty, A.; Wimmerova, M.; Mitchell, E. P.; Gilboa-Garber, N.
Microbes Infect. 2004, 6, 221–228.
(15) Sabin, C.; Mitchell, E. P.; Pokorna, M.; Gautier, C.; Utille, J. P.;
Wimmerova, M.; Imberty, A. FEBS Lett. 2006, 580, 982–987.
(16) Matsuura, K.; Hibino, M.; Yamada, Y.; Kobayashi, K. J. Am. Chem.
Soc. 2001, 123, 357–358.
(1) Varki, A. Glycobiology 1993, 3, 97–130.
(2) Dwek, R. A. Chem. ReV. 1996, 96, 683–720.
(3) Horlacher, T.; Seeberger, P. H. Chem. Soc. ReV. 2008, 37, 1414–1422.
(4) Wacker, R.; Niemeyer, C. M. ChemBioChem 2004, 5, 453–459.
(5) 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.
(6) Rostovtsev, V. V.; Green, L. G.; Fokin, V. V.; Sharpless, K. B. Angew.
Chem., Int. Ed. 2002, 41, 2596–2599.
(7) Tornoe, C. W.; Christensen, C.; Meldal, M. J. Org. Chem. 2002, 67,
3057–3064.
(17) Dubber, M.; Frechet, J. M. J. Bioconjugate Chem. 2003, 14, 239–246.
(18) Katajisto, J.; Heinonen, P.; Lonnberg, H. J. Org. Chem. 2004, 69, 7609–
7615.
(8) 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.
(9) Meyer, A.; Bouillon, C.; Vidal, S.; Vasseur, J. J.; Morvan, F. Tetrahedron
Lett. 2006, 47, 8867–8871.
(10) Pourceau, G.; Meyer, A.; Vasseur, J. J.; Morvan, F. J. Org. Chem. 2008,
73, 6014–6017.
(19) Katajisto, J.; Virta, P.; Lonnberg, H. Bioconjugate Chem. 2004, 15, 890–
896.
(20) D’Onofrio, J.; de Champdore, M.; De Napoli, L.; Montesarchio, D.; Di
Fabio, G. Bioconjugate Chem. 2005, 16, 1299–1309.
(21) Singh, Y.; Renaudet, O.; Defrancq, E.; Dumy, P. Org. Lett. 2005, 7,
1359–1362.
(22) Deguise, I.; Lagnoux, D.; Roy, R. New J. Chem. 2007, 31, 1321–1331.
1218 J. Org. Chem. 2009, 74, 1218–1222
10.1021/jo802536q CCC: $40.75 2009 American Chemical Society
Published on Web 01/05/2009

Synthesis of Mannose and Galactose Oligonucleotide Conjugates
SCHEME 1. Synthesis of the Bromohexyl Phosphoramidite 4
Carell et al. developed a method23 to functionalize DNA with
one to three different labels. This strategy uses one to three
successive click reactions and is based on the orthogonal
protections of alkyne functions.
To compare the efficiencies of click conjugation on solid
support and in solution and to see if the presence of an
oligonucleotide hampered the substitution of bromide by azide,
the same trimannosylated dodecathymidine was synthesized by
two different ways.
We present herein a new strategy for adding two different
carbohydrate residues to a scaffold using successive azido- and
alkyne-carbohydrate derivatives. This strategy is based on the
use of a new derivative bearing a bromoalkyl group as the site
for azide substitution, in combination with already-reported
alkyne derivatives.10,24 It is not possible for alkyne and azide
functions to occur simultaneously on the same oligonucleotide
since they would participate in an intramolecular reaction leading
to cyclization.24-27 Instead, alkyne and azide functions borne
by the same oligonucleotide must be introduced or generated
successively in order to react specifically with azide and alkyne
derivates (respectively) by intermolecular cycloadditions. Thus,
we first synthesized an oligonucleotide using an automated DNA
synthesizer and introduced alkyne or bromohexyl functions at
its 3′- or 5′-end using the corresponding phosphoramidite
derivatives. Second, a cycloaddition was applied with an azido-
carbohydrate derivative, introducing the first type of sugar on
solid support. Third, the bromohexyl groups were converted into
azidohexyl groups, which were finally conjugated with alkyne
carbohydrate derivatives to introduce the second type of
carbohydrate on solid support or in solution.
For the cycloaddition on solid support, phosphoramidite 4
was coupled three times to a universal 1,3-dipropanol solid
support,28 and then one standard thymidine phosphoramidite was
added, affording 6 (Scheme 2). Treatment of 6 with a solution
of sodium azide and sodium iodide in DMF at 65 °C for 1 h 30
min yielded the corresponding azido derivative 7,29 which was
then conjugated with 1-O-propargyl-2,3,4,6-O-tetraacetyl man-
nose (8, 9 mol equiv) in a water/methanol solution of CuSO4
(0.4 mol equiv) and sodium ascorbate (2 mol equiv) for 30 min
at 60 °C8 to give 9.29 Finally, 11 thymidines were introduced,
and after treatment with ammonia, the 3′-trimannosyl-dode-
cathymidine 10 was obtained (recovery yield 8% from the solid
support after purification).
For the click reaction in solution, all of the elongation steps
were performed successively, affording 11, which was converted
into the azido derivative using the same treatment as above and
deprotected by an additional ammonia treatment to give 12
(recovery yield 53% from solid support, without purification).
Upon characterization of 12 by HPLC and MALDI-TOF MS,
we observed a full conversion of bromoalkyl groups into
azidoalkyl groups. Therefore, the presence of the 12-mer did
not hamper the substitution of the bromine atom by an azido
group. Intermediate 12 was conjugated with 8 (6 mol equiv) in
a water/methanol solution of CuSO4 (5 mol equiv) and sodium
ascorbate (12.5 mol equiv) for 30 min at 60 °C to afford 10
after an additional ammonia treatment (Figure 1) (100%
conversion rate determined by HPLC; recovery yield 16% from
solid support after HPLC purification). Indeed, we observed (as
have others30) that more equivalents of copper are needed to
perform the click reaction in solution with unprotected oligo-
nucleotides than on solid support with protected ones. We
hypothesize that some of the copper interacts with phosphodi-
ester linkages and hence is less available for the catalysis.
However, it is interesting to note that we did not observe this
phenomenon before, when a click reaction performed in solution
on an oligonucleotide bearing both alkyne and azide functions
afforded cyclic products.24
Results and Discussion
The first step in our procedure was to design a phosphora-
midite bearing a bromoalkyl group and to test the efficiency of
incorporating it into an oligonucleotide by phosphoramidite
chemistry on a DNA synthesizer. The second step was to set
up the conditions to convert the bromoalkyl group into an
azidoalkyl group and finally perform a click reaction with an
alkyne carbohydrate derivative.
The bromoalkyl phosphoramidite derivative was synthesized
with a three-step protocol (Scheme 1) from 1,1,1-tris(hydroxym-
ethyl)ethane 1, which was monoprotected with a DMTr group (2,
56%),24 monoalkylated with 1,6-dibromohexane and sodium hy-
dride in the presence of sodium iodide in THF (3, 69%), and finally
converted into the phosphoramidite derivative 4 (73%) using
cyanoethyl tetraisopropylphosphorodiamidite activated with diiso-
propylammonium tetrazolide in dichloromethane.
By both strategies, we obtained quite similar HPLC profiles
for the final product (Figure 1), and MALDI-TOF MS gave the
(23) Gramlich, P. M. E.; Warncke, S.; Gierlich, J.; Carell, T. Angew. Chem.,
Int. Ed. 2008, 47, 3442–3444.
(24) Lietard, J.; Meyer, A.; Vasseur, J. J.; Morvan, F. J. Org. Chem. 2008,
73, 191–200.
(25) Kumar, R.; El-Sagheer, A.; Tumpane, J.; Lincoln, P.; Wilhelmsson,
L. M.; Brown, T. J. Am. Chem. Soc. 2007, 129, 6859–6864.
(26) Nakane, M.; Ichikawa, S.; Matsuda, A. J. Org. Chem. 2008, 73, 1842–
1851.
(27) Grajkowski, A.; Cieslak, J.; Kauffman, J. S.; Duff, R. J.; Norris, S.;
Freedberg, D. I.; Beaucage, S. L. Bioconjugate Chem. 2008, 18, 1696–1706.
(28) Surprisingly, we were not able to perform an efficient substitution of
the bromine atom by the azide when compound 3 was directly loaded through
a standard succinyl linkage to the LCAA-CPG using 500 or 1000 A CPG. It
appeared that it is compulsory to have a longer spacer to obtain a complete
substitution.
(29) A few CPG beads were withdrawn from the DNA column and
deprotected by ammonia for HPLC and MALDI-TOF MS analyses showing a
complete reaction.
(30) Alvira, M.; Eritja, R. Chem. BiodiVersity 2007, 4, 2798–2809.
J. Org. Chem. Vol. 74, No. 3, 2009 1219

Pourceau et al.
SCHEME 2. Synthesis of 3-Trimannosyl-dodecathymidine 10 According to Click Reactions Performed on Solid Support or in
Solutiona
a SPS ) solid-phase synthesis: (1) 2.5% dichloroacetic acid CH2Cl2; (2) phosphoramidite derivative + benzylthiotetrazole; (3) Ac2O, N-Me imidazole,
2,6-lutidine; (4) 0.1 M I2 THF/H2O/pyridine.
of sodium ascorbate were used as previously, but total conjuga-
tion when these amounts were increased to 1 and 5 mol equiv,
respectively. The oligonucleotide was elongated by introducing
11 nucleotides (5′-GGA AAC GTC AC) to yield 18. Then,
bromine atoms were displaced by azide groups by treatment
with sodium azide and sodium iodide in DMF, and 19 was
obtained in solution after a subsequent treatment with ammonia
(recovery yield 7% from solid support after purification). A
second cycloaddition was carried out in solution under MW
for 45 min at 60 °C, with tetraacetyl mannose propargyl 8
FIGURE 1. HPLC profiles of purified trimannosyl-T12 10 obtained by
(6 mol equiv), CuSO4 (5 mol equiv), and sodium ascorbate (12.5
click reactions on solid support (left) and in solution (right).
mol equiv). A final ammonia treatment removed the acetyl
expected mass. However, the yield of the glyco oligonucleotide
conjugate was 2-fold greater for the solution-phase synthesis.
The main reason of this difference is that the solid-phase
synthesis requires more manipulations, with two transfers of
the supported material (7 was transferred from the column to
the microwave vial, and then 9 was retransferred to the DNA
column). In conclusion, converting 4 into an azide derivative
and using it to conjugate oligonucleotides by click chemistry is
very efficient and could be applied to the synthesis of more
complicated conjugates.
A second glyco oligonucleotide conjugate bearing bis-
alternate mannose and galactose residues at the 3′-end of the
sequence was synthesized using 4 and the phosphoramidite
derivative 14, which is an analog of 4 bearing a propargyl group
instead of a bromohexyl group. Starting from the solid support
13, which exhibits an alkyne function, 4, 14, and thymidine
were successively introduced by phosphoramidite chemistry to
afford 15 (Scheme 3). Then, a first click reaction was carried
out on solid support under microwaves assistance (MW) for 45
min at 60 °C to introduce two copies of the galactose azide
derivative 16,10 giving the digalactosylated species 17. Surpris-
ingly, with 10 mol equiv of 16, we observed incomplete
conjugation when only 0.4 mol equiv of CuSO4 and 2 mol equiv
groups and afforded the heteroglycomimic 3′-bis(mannose-
galactose) oligonucleotide conjugate 20 (100% conversion rate
determined by HPLC; recovery yield 5% from solid support
without purification).
Finally, we synthesized a last heteroglyco oligonucleotide
conjugate in which the mannose and galactose residues were
introduced at the 5′-end. For this purpose, the oligonucleotide
was elongated four times by incorporating the phosphoramidites
14 and 4, each twice and in alternation, to afford 21, which
bore two propargyl and two bromohexyl groups (Scheme 4).
Compound 21 was conjugated with 16 (10 mol equiv), CuSO4
(1.0 mol equiv), and sodium ascorbate (5 mol equiv), under
MW assistance for 45 min at 60 °C, to afford 22 with two
galactose residues, which was converted to the diazide derivative
23. The sample was then split in two: one part was transferred
into a MW vial and conjugated on solid support with tetraacetyl
propargyl mannose 8 (6 mol equiv), CuSO4 (0.4 mol equiv),
and sodium ascorbate (2 mol equiv), under MW assistance for
45 min at 60 °C to give 24. After removing the solution and
washing the beads, 24 was treated with ammonia to afford the
final heteroglyco oligonucleotide conjugate 25 (recovery yield
18% from solid support after purification).
1220 J. Org. Chem. Vol. 74, No. 3, 2009

Synthesis of Mannose and Galactose Oligonucleotide Conjugates
SCHEME 3. Synthesis of the 3′-End Heteroglycomimic Oligonucleotide 20 Exhibiting Two Mannose and Two Galactose
Residues
SCHEME 4. Synthesis of the 5′-End Heteroglycomimic Oligonucleotide 25 Exhibiting Two Mannose and Two Galactose
Residues
The other part was deprotected with ammonia (recovery yield
28% from solid support after purification); conjugated in solution
with 8 (6 mol equiv), CuSO4 (5 mol equiv), and sodium
ascorbate (12.5 mol equiv), under MW assistance for 45 min
at 60 °C; and treated again with ammonia (to remove the acetyl
groups of the mannosyl residues) to afford the expected
heteroglyco oligonucleotide conjugate 25 (100% conversion rate
determined by HPLC; recovery yield 26% from solid support
without purification). Both methods provided 25 with the same
efficiency and in similar amounts. Indeed, for the solid-phase-
only synthesis, only one additional transfer was necessary and
was made carefully to avoid loss of material.
We believed that this strategy could be applied for the
synthesis of other heteroconjugates of oligonucleotides.
Experimental Section
1-O-(4,4′-Dimethoxytrityl)-2-(6-bromohexyloxymethyl)-2-meth-
yl-1,3-propanediol 3. 2-[(4,4′-Dimethoxytrityl)oxymethyl]-2-me-
thylpropane-1,3-diol 224 (844 mg, 2 mmol), sodium iodide (30 mg,
0.2 mmol), and dibromohexane (1.54 mL, 10 mmol) were added
to a solution of sodium hydride (60% in oil, 240 mg, 6 mmol) in
anhydrous THF (30 mL) at 0 °C. After 19 h under magnetic stirring
at room temperature, the reaction was quenched by adding 50 mL
of CH2Cl2 and 2 mL of water. The organic layer was washed with
brine (2 × 100 mL), dried over Na2SO4, and evaporated. The crude
product was then purified by silica gel column chromatography
(0 to 30% acetone in cyclohexane containing 1% triethylamine) to
afford 3 (813 mg, 69%) as a colorless oil. Rf ) 0.58 (cyclohexane/
Conclusion
Two phosphoramidites, one bearing a propargyl group and
the other bearing a bromohexyl group, were successfully used
for the synthesis of heterocarbohydrate oligonucleotide conju-
gates by MW-assisted click chemistry. The oligocarbohydrate
could be introduced either at the 3′- or 5′-end of an oligonucle-
otide, and the bromine atom was easily substituted with an azide
group.
1
acetone 1:1 v/v). H NMR (CD3CN, 300 MHz): δ 0.89 (s, 3H),
1.34-1.99 (m, 15H), 2.67 (t, 1H, J) 5.7 Hz), 3.00-3.51 (m, 12H),
3.78 (s, 6H), 6.86-7.46 (m, 13H). 13C NMR (CD3CN, 300 MHz):
δ 16.8, 24.7, 26.6, 27.3, 28.9, 32.0, 32.2, 33.8, 33.93, 54.5, 65.0,
66.1, 70.7, 73.8, 85.0,112.5, 126.3, 127.4, 127.7, 129.7, 136.0,
136.1, 145.2, 158.2. HRMS ESI+ m/z calculated for C32H41O5Na1Br1
(M + Na)+ 607.2035, found 607.2083.
J. Org. Chem. Vol. 74, No. 3, 2009 1221

Pourceau et al.
solvent time
TABLE 1. Conditions Used for Each Click Reaction
click reaction
compound
scale
(µmol)
CuSO4
(mol equiv)
sodium ascorbate
(mol equiv)
clicka
8 (mol equiv)
16 (mol equiv)
volumeb (µL)
(min)
liquid
solid
solid
liquid
solid
solid
liquid
12f10
7f 9
15f17
19f20
21f22
23f24
26f25
1
1
1
1
1
0.5
0.5
5
0.4
1
5
1
12.5
2
5
12.5
5
2
3
3
0
3
0
3
3
0
0
5
0
5
0
0
400
200
300
400
300
200
400
30
30
45
45
45
45
45
0.4
5
12.5
a The click reaction was performed on solid support (solid) or in solution (liquid). b Water/methanol 1:1, v/v, under microwave assistance (60 °C, 100
W) and magnetic stirring. The solutions of CuSO4 and sodium ascorbate were prepared in water, and the carbohydrate derivatives 8 and 16 were
dissolved in methanol to 100 mM concentration.
1-O-(4,4′-Dimethoxytrityl)-2-(6-bromohexyloxymethyl)-2-meth-
yl-3-O-[(2-cyanoethyl)-N,N-diisopropyl-phosphoramidite]-1,3-
propanediol 4. 2-Cyanoethyl tetraisopropyl phophorodiamidite (457
µL, 1.4 mmol) was added to a solution of compound 3 (715 mg,
1.2 mmol) and diisopropylammonium tetrazolide (103 mg, 0.6
mmol) in anhydrous dichloromethane (15 mL). The resulting
mixture was stirred at room temperature for 3 h and then diluted
with CH2Cl2 (15 mL). The organic layer was washed with brine
(2 × 50 mL), dried (Na2SO4), filtered, and evaporated. The residue
was purified by silica gel chromatography (0 to 10% ethyl acetate
in cyclohexane containing 10% of triethylamine), affording the
phosphoramidite 4 (684 mg, 73%) as a colorless oil. Rf ) 0.54
(cyclohexane/AcOEt/Et3N 7:2:1 v/v/v). 1H NMR (CD3CN, 300
MHz): δ 0.96-0.99 (2s, 3H), 1.11-1.18 (m, 12H), 1.3-1.99 (m,
11H), 2.60 (m, 2H), 2.90-3.78 (m, 12H), 6.86-7.48 (m, 13H).
13C NMR (CD3CN, 300 MHz): δ 16.9, 19.7, 19.8, 23.5, 23.6, 23.65,
23.7, 24.8, 27.3, 28.9, 32.2, 33.9, 40.7, 40.8, 42.4, 42.5, 54.5, 57.7,
58.0, 64.6, 66.0, 66.2, 70.6, 72.6, 72.7, 85.0, 112.6, 118.13, 126.3,
127.4, 127.7, 129.7, 136.0, 145.3, 158.2. 31P NMR (CD3CN, 300
General Procedure for Azidation. The solid-supported bro-
mohexyl derivative (1 µmol) was treated with a solution of NaN3
(13 mg, 200 µmol) and NaI (30 mg, 200 µmol) in DMF (2 mL)
for 1 h 30 min at 65 °C. The CPG beads were washed with DMF
(2 × 1 mL) and CH2Cl2 (3 × 1 mL) and dried under vacuum.
General Procedure for Cu(I)-Catalyzed 1,3-Dipolar Cycload-
dition. A carbohydrate derivative (8 or 16) dissolved in methanol
and a freshly prepared solution of CuSO4 and sodium ascorbate in
water were added to solid-supported scaffolds or oligonucleotide
derivatives in solution. The vial containing the resulting mixture
was sealed and placed in a microwave synthesizer, with a 30 s
premixing time, for 30-45 min at 60 °C and 100 W. The
temperature was monitored with an internal infrared probe. For the
click performed on solid support, the solution was removed and
the CPG beads were washed with H2O (2 mL) and MeOH (2 mL)
and then dried. For the click performed in solution, the solution
was desalted on NAP10 and evaporated. The residue was treated
with concentrated aqueous ammonia at room temperature for 1 h
to remove acetyl groups.
MHz):
δ
147.04, 147.15. HRMS ESI+ m/z calcd for
General Procedure for Deprotection of Solid-Supported
Oligonucleotides. CPG beads were treated with concentrated
aqueous ammonia (3 mL) for 1 h 30 min at room temperature to
release the oligonucleotide from the solid support. The supernatant
was collected, warmed to 55 °C for 6 h, and then evaporated. The
residue was dissolved in water for subsequent analyses.
C41H59N2O6P1Br1 (M + H)+ 785.3294, found 785.3322.
Compounds 831 and 1610 were synthesized according to the
literature.
Synthesis of Oligonucleotides. The oligonucleotides were
synthesized at a 1-µmol scale on a DNA synthesizer (ABI 394),
using standard phosphoramidite chemistry, on commercially avail-
able thymidine solid support or on the supports 5 and 1324 using
alkyne phosphoramidite 14,10 bromohexyl phosphoramidite 4, and
commercially available nucleoside phosphoramidites (0.075 M in
anhydrous CH3CN). Benzylmercaptotetrazole was used as the
activator (0.3 M in anhydrous CH3CN). The capping step was
performed with acetic anhydride, using a commercial solution (Cap
A: Ac2O/pyridine/THF 10:10:80 v/v/v and Cap B: 10% N-
methylimidazole in THF) for 15 s. Oxidation was performed with
a commercial solution of iodide (0.1 M I2, THF, pyridine/water
90:5:5, v/v/v) for 13 s. Detritylation was performed with 2.5% DCA
in CH2Cl2 for 35 s.
Acknowledgment. We thank the Universite´ Montpellier 2
and CNRS “Programme interdisciplinaire Interface Physique
Chimie Biologie” for financial support. G.P. thanks the CNRS
and “Re´gion Languedoc Roussillon” for the award of a research
studentship. F.M. is from INSERM.
Supporting Information Available: HPLC profiles and
MALDI-TOF MS spectra of synthesized oligonucleotide con-
1
jugates 10, 20, and 25. H NMR, 13C NMR, and 31P NMR of
compounds 3 and 4. This material is available free of charge
via the Internet at http://pubs.acs.org.
(31) Hasegawa, T.; Numata, M.; Okumura, S.; Kimura, T.; Sakurai, K.;
Shinkai, S. Org. Biomol. Chem. 2007, 5, 2404–2412.
JO802536Q
1222 J. Org. Chem. Vol. 74, No. 3, 2009