Solid-supported 2-O-glycoconjugation of oligonucleotides by azidation and click reactions

Article abstract of DOI:10.1021/bc200097g

2′-O-[(2-Bromoethoxy)methyl]cytidine and 2′-O-[(2-azidoethoxy) methyl]cytidine have been prepared and introduced as appropriately protected 3′-phosphoramidite (1) and 3′-(H-phosphonate) (2) building blocks, respectively, into 2-O-methyl oligoribonucleotid

Full text of DOI:10.1021/bc200097g

TECHNICAL NOTE
pubs.acs.org/bc
Solid-Supported 20-O-Glycoconjugation of Oligonucleotides by
Azidation and Click Reactions
Anu Kiviniemi,*,† Pasi Virta,† Mikhail S. Drenichev,‡ Sergey N. Mikhailov,*,‡ and Harri L€onnberg†
†Department of Chemistry, University of Turku, FIN-20014 Turku, Finland
‡Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Vavilov Street 32, Moscow 119991, Russia
S Supporting Information
b
ABSTRACT: 20-O-[(2-Bromoethoxy)methyl]cytidine and 20-
O-[(2-azidoethoxy)methyl]cytidine have been prepared and
introduced as appropriately protected 30-phosphoramidite (1)
and 30-(H-phosphonate) (2) building blocks, respectively, into
20-O-methyl oligoribonucleotides. The support-bound oligonu-
cleotides were subjected to two consecutive conjugations with
alkynyl-functionalized monosaccharides. The first saccharide
was introduced by a Cu(I) promoted click reaction with 2 and
the second by azidation of the 2-bromoethoxy group of 1
followed by the click reaction. The influence of the 20-glycoconjugations on hybridization with DNA and 20-O-methyl RNA
targets was studied. Two saccharide units within a 15-mer oligonucleotide had a barely noticeable effect on the duplex stability, while
introduction of a third one moderately decreased the melting temperature.
’ INTRODUCTION
were prepared and incorporated into 20-O-methyl oligoribonu-
cleotides. The azido group of the nucleoside units 2 was first
subjected to a click reaction,20,21 with a propynyl functionalized
monosaccharide on-support. The bromo substituent of unit 1 was
then displaced with azide ion on-support, as shown previously by
Pourceau et al.,14 and another click reaction was carried out. The
hybridization efficiency of the conjugates with cDNA- and 20-O-
Me-RNA-oligonucleotides was assessed by UV-melting studies
and compared to the hybridization of the corresponding unmo-
dified sequence.
Among various oligonucleotide conjugates, the 20-conjugates
have received special interest for the reason that upon formation
of an A- or B-type duplex, the conjugate group is accommodated
in the minor or major grooves, respectively, and hence, the
duplex is not markedly destabilized.1,2 Introduction of reactive
functionalities in the 20-position of nucleosides, incorporation of
the resulting monomers into oligonucleotides, and their subse-
quent reactions with appropriately functionalized conjugate
groups has been rather recently reviewed.3 Somewhat surpris-
ingly, such a stepwise protocol has only once been utilized for
solid phase-synthesis of 20-glycoconjugates, potentially useful for
the delivery and targeting of therapeutic oligonucleotides,4ꢀ8
although several procedures for the derivatization of other
intrachain positions, including the C40-position,9,10 internucleo-
sidic phosphoramidate linkage,11,12 or intrachain non-nucleosi-
dic units,13ꢀ18 have been described. Very recently,19 saccharide
ligands have been conjugated by Cu(I) promoted click reaction20,21
to 20-O-(2-propynyl) groups on-support. Otherwise 20-glycoconju-
gates have been obtained by converting prefabricated 20-O-glycosyl
nucleosides with all four natural heterocyclic bases22 into phosphor-
amidite building blocks and introducing them into oligonucleotides
on a solid-support.23ꢀ26 The cis diol of the 20-O-glycosyl group,
when oxidized to dialdehyde with sodium periodate, has been
shown to allow affinity labeling of different enzymes.27ꢀ29
’ EXPERIMENTAL PROCEDURES
General Remarks. Synthetic reactions were monitored by
TLC (Merck DC Kieselgel 60F254), using UV light, iodine
vapors, or heating with 10% H2SO4 (aq) for detection. Column
chromatography was performed on Silica gel 60 (Fluka, 0.040ꢀ
0.063 mm). The NMR spectra were recorded at 400 or 500 MHz.
Chemical shifts are given in parts per million (ppm) and measured
relative to the solvent signals (1H and 13C). Appropriate 2D NMR
methods (COSY and HSQC) were used for peak assignment.
Solvents weredried with 4 Å molecular sieves (DCM, pyridineand
MeCN) or by distillation and storage over CaH2 (Et3N). The
mass spectra were recorded using ESI ionization methods. RP
HPLC analysis of the oligonucleotide conjugates was performed
on a Thermo ODS Hypersil C18 (250 ꢁ 4.6 mm, 5 μm) analytical
column, applying a gradient elution from 0 to 50% MeCN in
We now report on a solid-supported conjugation method that
enablesthe attachmentoftwo different carbohydrateligands to the
20-position at desired sites within the chain. For this purpose, two
building blocks, viz. 20-O-[(2-bromoethoxy)methyl]-50-O-(4,40-
dimethoxytrityl)cytidine 30-(2-cyanoethyl-N,N-diisopropylpho-
phoramidite) (1) and 20-O-[(2-azidoethoxy)methyl]-50-O-(4,40-
dimethoxytrityl)cytidine 30-(H-phosphonate) (2) monomers,
Received: February 25, 2011
Revised:
April 11, 2011
Published: May 03, 2011
r
2011 American Chemical Society
1249
dx.doi.org/10.1021/bc200097g Bioconjugate Chem. 2011, 22, 1249–1255
|

Bioconjugate Chemistry
TECHNICAL NOTE
Scheme 1a
a Reagents: (i) Bu4NF, THF; (ii) DMTrCl, pyridine; (iii) 2-cyanoethyl N,N-diisopropylphosphoramidochloridite, Et3N, DCM; (iv) diphenyl H-
phosphonate, pyridine; (v) Et3N, H2O.
0.1 mol Lꢀ1 TEAA (aq, Et3NHOAc) buffer over 60 min, at a flow
rate of 1 mL minꢀ1, and detection at 260 nm.
163.1 (C-4), 155.4 (C-2), 146.2 (C-6), 133.3, 129.0, 128.0 (Bz),
97.3 (C-5), 95.5 (OCH2O), 91.2 (C-10), 84.9 (C-40), 79.9 (C-
20), 68.2 (C-30), 67.4 (OCH2CH2N3), 60.4 (C-50), 50.9
(OCH2CH2N3).
20-O-(2-Bromoethoxymethyl)-N4-benzoylcytidine
(4a).
Nucleoside 3a (1.30 g, 1.79 mmol), prepared as described earlier,30
was dissolved in Bu4NF trihydrate in THF (0.25 mol Lꢀ1; 4.8 mL),
kept for 10 min at 20 °C, evaporated to dryness, coevaporated
with chloroform (2 ꢁ 10 mL), and applied onto a column with
silica gel (25 g). The column was washed with DCM (100 mL),
DCM-EtOH (97:3, v/v), and then eluted with DCM-EtOH
(95:5) to obtain 4a (490 mg, 57%) as a white amorphous foam
(Scheme 1). 1H NMR [400 MHz, CDCl3/CD3OD (9:1)] δppm
8.47 (d, 1H, J = 7.5 Hz, H-6), 7.98ꢀ7.40 (m, 6H, Bz, H-5), 5.88
20-O-(2-Bromoethoxymethyl)-50-O-(4,40-dimethoxytrityl)-
N4-benzoylcytidine (5a). Nucleoside 4a (357 mg, 0.74 mmol)
was dried by coevaporation with pyridine (2 ꢁ 20 mL). The
residue was dissolved in dry pyridine (10 mL), 4,40-dimethoxy-
trityl chloride (351 mg, 1.04 mmol) was added, and the resulted
solution was kept in the dark for 16 h at 20 °C. MeOH (1 mL)
was added and after 30min, the mixture was concentrated in vacuo
to near dryness. The residue was dissolved in DCM (50 mL) and
washed with a 10% aqueous solution of sodium bicarbonate
(20 mL) and water (2 ꢁ 20 mL). The organic layer was dried
over anhydrous Na2SO4, the solvent was removed in vacuo,
and the residue was coevaporated with toluene (2 ꢁ 10 mL)
and purified by column chromatography on silica gel (20 g).
The column was washed with a mixture of DCM, hexane, and
Et3N (75:23:2, v/v/v, 200 mL), followed by DCM, and the
product was eluted with a mixture of DCM and Et3N (98:2, v/v)
(d, 1H, J = 2.2 Hz, H-10), 5.02 (d, 1H, J = 6.5 Hz, OCHHO), 4.92
0
0
0
(d, 1H, J = 6.5 Hz, OCHHO), 4.33 (dd, 1H, J2 ,3 = 5.0 Hz, H-2 ),
0
0
0
0
0
0
0
4.26 (dd, 1H, J3 ,4 = 7.5 Hz, H-3 ), 4.07 (dt, 1H, J4 ,5 a = J4 ,5 b
=
2.2 Hz, H-40), 3.99 (dd, 1H, J5 a,5 b = 12.8 Hz, H-50a), 3.89 (dd,
2H, J = 6.2 Hz, 5.9 Hz, OCH2CH2Br), 3.81 (dd, 1H, H-50b),
3.44 (dd, 2H, OCH2CH2Br). 13C NMR (CDCl3/CD3OD
(9:1)) δppm 167.1 (CdO), 163.1 (C-4), 146.0 (C-6), 133.3,
129.0, 128.0 (Ph), 97.3 (C-5), 95.3 (OCH2O), 90.9 (C-10), 84.7
(C-40), 79.9 (C-20), 68.8 (C-30), 68.0 (OCH2CH2Br), 60.1 (C-
50), 30.5 (OCH2CH2Br).
0
0
1
to give 5a (412 mg, 71%) as a foam. H NMR (500 MHz,
CD3CN) δppm 9.63 (br s, 1H, NH), 8.53 (d, 1H, J = 7.5 Hz, H-6),
8.00ꢀ7.94 (m, 2H, Bz), 7.60 (m, 1H, Bz), 7.52ꢀ7.46 (m, 4H, Bz,
DMTr), 7.41ꢀ7.32 (m, 6H, DMTr), 7.29ꢀ7.22 (m, 2H, DMTr,
H-5), 6.94ꢀ6.88 (m, 4H, DMTr), 5.92 (s, 1H, H-10), 5.17 (d,
1H, J = 6.5 Hz, OCHHO), 4.95 (d, 1H, J = 6.5 Hz, OCHHO),
4.58 (m, 1H, H-30), 4.38 (d, 1H, J = 5.0 Hz, H-20), 4.15 (m, 1H,
H-40), 4.00ꢀ3.89 (m, 2H, OCH2CH2Br), 3.78 (s, 6H, DMTr-
OMe), 3.56ꢀ3.46 (m, 4H, OCH2CH2Br, H-50). 13C NMR
(125 MHz, CD3CN) δppm 167.2 (Bz-CdO), 163.0 (C-4),
158.8, 158.8 (DMTr), 154.9 (C-2), 144.9 (C-6), 144.5, 136.0,
135.6 (DMTr), 133.5, 132.9 (Bz), 130.2, 130.0 (DMTr), 128.7,
128.3, 128.1, 128.1 (DMTr, Bz), 127.1, 113.3 (DMTr), 96.5 (C-5),
20-O-(2-Azidoethoxymethyl)-N4-benzoylcytidine
(4b).
Analogous to the preparation of 4a, the desilylation of 3b
(1.12 mg, 1.63 mmol) yielded 4b (683 mg, 94%) as a white
1
amorphous foam. H NMR (400 MHz, CDCl3) δppm 8.48 (d,
1H, J = 7.5 Hz, H-6), 8.00ꢀ7.40 (m, 6H, Bz, H-5), 5.88 (d, 1H,
J = 2.5 Hz, H-10), 5.03 (d, 1H, J = 6.5 Hz, OCHHO), 4.93 (d, 1H,
OCHHO), 4.48 (dd, 1H, J= 5.3Hz, H-20), 4.41 (dd, 1H, J= 6.5 Hz,
H-30), 4.16 (ddd, 1H, J4 ,5 a = 1.9 Hz, J4 ,5 b = 1.5 Hz, H-40), 4.08
0
0
0
0
(dd, 1H, J5 a,5 b = 12.5 Hz, H-50a), 3.92 (dd, 1H, H-50b),
3.82ꢀ3.71 (m, 2H, OCH2CH2N3), 3.46ꢀ3.37 (m, 2H,
OCH2CH2N3). 13C NMR (MHz, CDCl3) δppm 167.2 (CdO),
0
0
1250
dx.doi.org/10.1021/bc200097g |Bioconjugate Chem. 2011, 22, 1249–1255

Bioconjugate Chemistry
TECHNICAL NOTE
94.3 (OCH2O), 89.8 (C-10), 86.8(DMTr-Cq), 82.6 (C-40), 78.9(C-
20), 68.3 (OCH2CH2Br), 68.0 (C-30), 61.2 (C-50), 55.0 (DMTr-
OMe), 31.5 (OCH2CH2Br). HRMS(ESI): [MH]þ C40H41Br-
N3O9 requires 786.2021, found 786.2019.
Chart 1
20-O-(2-Azidoethoxymethyl)-50-O-(4,40-dimethoxytrityl)-
β-D-N4-benzoylcytidine (5b). Analogous to the preparation of
5a, the tritylation of nucleoside 4b (778 mg, 1.74 mmol) yielded
5b (940 mg, 72%) as a foam. 1H NMR (500 MHz, CD3CN) δppm
9.29 (br s, 1H, NH), 8.48 (d, 1H, J = 7.5 Hz, H-6), 8.00ꢀ7.95 (m,
2H, Bz), 7.66 (m, 1H, Bz), 7.57ꢀ7.52 (m, 2H, Bz), 7.51ꢀ7.48
(m, 2H, DMTr), 7.41ꢀ7.35 (m, 6H, DMTr), 7.30 (m, 1H,
DMTr), 7.18 (br d, 1H, J = 6.5 Hz, H-5), 6.96ꢀ6.91 (m, 4H,
DMTr), 5.91 (d, 1H, J = 1.0 Hz, H-10), 5.16 (d, 1H, J = 7.0 Hz,
OCHHO), 4.94 (d, 1H, J = 6.5 Hz, OCHHO), 4.52 (m, 1H,
H-30), 4.32 (d, 1H, J = 5.0 Hz, H-20), 4.11 (ddd, 1H, J = 9.0 Hz,
2.5 Hz, 2.5 Hz, H-40), 3.88ꢀ3.76 (m, 8H, DMTr-OMe,
OCH2CH2N3), 3.53ꢀ3.37 (m, 5H, H-50, OCH2CH2N3,
30ꢀOH). 13C NMR (125 MHz, CD3CN) δppm 167.2 (Bz-
CdO), 162.9 (C-4), 158.8, 158.8 (DMTr), 154.8 (C-2), 144.9
(C-6), 144.5, 136.0, 135.6 (DMTr), 133.5, 132.9 (Bz), 130.2,
130.0 (DMTr), 128.7 (Bz), 128.2, 128.1, 128.1 (DMTr, Bz),
127.1, 113.3 (DMTr), 96.3 (C-5), 94.4 (OCH2O), 89.8 (C-10),
86.7 (DMTr-Cq), 82.6 (C-40), 79.0 (C-20), 68.0 (C-30), 66.9
(OCH2CH2N3), 61.2 (C-50), 55.0 (DMTr-OMe), 50.7 (OCH2-
CH2N3). HRMS(ESI): [MH]þ C40H41N6O9 requires 749.2930;
found, 749.2935.
(0.6 mL) were added, and the reaction mixture was stirred for an
additional 15 min. After evaporation, the residue was partitioned
between DCM (20 mL) and 5% aq NaHCO3 (20 mL). The
organic layer was dried with Na2SO4, evaporated, and purified by
silica gel chromatography (5ꢀ10% MeOH in DCM þ 0.1%
1
Et3N), yielding product 2 (0.39 g, 86%) as a white foam. H
NMR (500 MHz, CDCl3/CD3OD (1:1)) δppm 8.53 (m, 1H,
H-6), 7.99ꢀ7.94 (m, 2H, Bz), 7.61 (m, 1H, Bz), 7.55ꢀ7.49 (m,
2H, Bz), 7.47ꢀ7.40 (m, 2H, DMTr), 7.37ꢀ7.14 (m, 8.5H,
DMTr, H-5, PH (0.5H)), 6.92ꢀ6.85 (m, 4H, DMTr), 6.19
(br, 0.5H, PH), 6.09 (s, 1H, H-10), 5.05 (d, 1H, J = 7.0 Hz,
OCHHO), 4.97 (d, 1H, J = 7.0 Hz, OCHHO), 4.92 (m, 1H,
H-30), 4.52 (m, 1H, H-20), 4.35 (d, 1H, J = 7.0 Hz, H-40),
3.87ꢀ3.76 (m, 8H, DMTr-OMe, OCH2CH2N3), 3.64 (d, 1H,
J = 11.0 Hz, H-50a), 3.52 (d, 1H, J = 11.0 Hz, H-50b), 3.43 (m, 2H,
OCH2CH2N3), 3.13 (br q, 6H, J = 6.5 Hz, CH2 of Et3N), 1.31
(t, 9H, J = 7.3 Hz, CH3 of Et3N). 13C NMR (125 MHz, CDCl3/
CD3OD, 1:1) δppm 167.3 (Bz-CdO), 163.3 (C-4), 158.8 (DMTr),
156.2 (C-2), 144.8 (C-6), 143.9, 135.4, 135.1 (DMTr), 133.1, 133.0
(Bz), 130.2, 130.1 (DMTr), 128.7 (Bz), 128.3, 127.9, 127.9
(DMTr, Bz), 127.1, 113.2 (DMTr), 97.6 (C-5), 94.9 (OCH2O),
89.4 (C-10), 87.2 (DMTr-Cq), 82.0 (C-40), 78.5 (C-20), 70.1 (C-
30), 67.1 (OCH2CH2N3), 60.9 (C-50), 54.9 (DMTr-OMe), 50.7
(OCH2CH2N3), 46.3 (CH2 of Et3N), 8.3 (CH3 of Et3N). 31P
NMR (200 MHz, CDCl3/CD3OD, 1:1) δppm 2.53. HRMS(ESI):
[M]ꢀ C40H40N6O11P requires 811.2498; found, 811.2488.
Synthesis of 20-O-Me-oligoribonucleotides. Oligonucleo-
tides incorporating 1 and 2 were synthesized in a 1.0 μmol scale
using commercial 500 Å CPG-succinyl-20-O-Me-adenosine sup-
port. For the synthesizer, a protocol including both the phosphor-
amidite/tetrazole and H-phosphonate/pivaloyl chloride couplings
was generated. For the coupling of phosphoramidite block 1 (0.13
mol Lꢀ1 solution in dry MeCN) and the commercial 20-O-Me-
nucleoside phosphoramidites, standard RNA phosphoramidite
protocol was applied, with the exception of an extra detritylation
step for monomer 1. For the H-phosphonate monomer 2 [35
mmol Lꢀ1 solution in dry MeCN/pyrine (1:1, v/v)], a previously
generated protocol was utilized.9 Accordingly, the coupling time
was 570 s on using pivaloyl chloride at a concentration of 100
mmol Lꢀ1 in dry MeCN/pyrine (1:1, v/v). An additional
detritylation step was used also for monomer 2. After the
oligonucleotide assembly, the support-bound material was re-
20-O-(2-Bromoethoxymethyl)-50-O-(4,40-dimethoxytrityl)-
β-N4-benzoylcytidine 30-(2-cyanoethyl-N,N-diisopropylpho-
sphoramidite) (1). Compound 5a (0.29 g, 0.37 mmol) was
predried over P2O5 in a vacuum desiccator and dissolved in
DCM (5 mL). Et3N (260 μL, 1.85 mmol) and 2-cyanoethyl N,N-
diisopropylphosphoramidochloridite (99 μL, 0.44 mmol) were
added under nitrogen. After 2 h, the mixture was filtered through
a short dried silica gel column (60ꢀ70% EtOAc in hexane and
0.1% Et3N). The mixture of phosphoramidite 1 RP- and SP-
diastereomers (0.26 g, 71%) was obtained as a colorless oil. 1H
NMR (500 MHz, CD3CN) δppm 9.22 (br, 1H, NH), 8.50 [d,
0.5H, J = 7.5 Hz, H-6 (diast. I)], 8.45 [d, 0.5H, J = 7.0 Hz, H-6
(diast. II)], 8.00ꢀ7.94 (m, 2H, Bz), 7.66 (m, 1H, Bz), 7.58ꢀ7.48
(m, 4H, Bz, DMTr), 7.43ꢀ7.34 (m, 6H, DMTr), 7.31 (m, 1H,
DMTr), 7.11 [br, 0.5H, H-5 (diast. I)], 7.04 [br, 0.5H, H-5 (diast.
II)], 6.96ꢀ6.90 (m, 4H, DMTr), 5.99 (br s, 1H, H-10), 5.09 [d,
0.5H, J = 6.5 Hz, OCHHO (diast. I)], 5.06 [d, 0.5H, J = 6.5 Hz,
OCHHO (diast. II)], 5.01 [d, 0.5H, J = 6.5 Hz, OCHHO (diast.
II)], 4.96 [d, 0.5H, J = 6.5 Hz, OCHHO (diast. I)], 4.60 (m, 1H,
H-30), 4.47 [m, 0.5H, H-20 (diast. I)], 4.41 [m, 0.5H, H-20 (diast.
II)], 4.28 [m, 0.5H, H-40 (diast. I)], 4.23 [m, 0.5H, H-40 (diast.
II)], 4.05ꢀ3.85 [m, 2.5H, OCH2CH2Br, OCHHCH2CN (0.5H
diast. II)], 3.84ꢀ3.46 [m, 13.5H, DMTr-OMe, OCH2CH2CN
(2 ꢁ 0.5H diast. I), OCHHCH2CN (0.5H diast. II), NCH(CH3)2,
OCH2CH2Br, H-50], 2.71ꢀ2.67 [m, 2 ꢁ 0.5H, OCH2CH2CN
(diast. II)], 2.58ꢀ2.53 [m, 2 ꢁ 0.5H, OCH2CH2CN (diast. I)],
1.21ꢀ1.12 [m, 12 ꢁ 0.5H (diast. I), 6 ꢁ 0.5H (diast. II),
NCH(CH3)2], 1.06 [d, 6 ꢁ 0.5H, J = 7.0 Hz, NCH(CH3)2
(diast. II)]. 31P NMR (200 MHz, CD3CN) δppm 148.6 (diast. I),
150.5 (diast. II). HRMS(ESI): [MH]þ C49H58BrN5O10P re-
quires 986.3099; found, 986.3078.
moved from the synthesizer, oxidized with iodine (0.1 mol Lꢀ1
)
in wet pyridine (98:2, v/v) at rt for 1 h, washed with pyridine/
MeCN/MeOH, and dried in vacuum. The oligonucleotides were
released from the support and deprotected with concentrated
ammonia (33% aqueous NH3) at 55 °C overnight.
On-Support Click Conjugation. Alkynyl-functionalized li-
gands 69 and 731 (Chart 1) were conjugated to the azido groups
of the support-bound oligonucleotides as follows. The ligands in
MeOH (50 mmol Lꢀ1, 15 equiv/azido group) were added onto
the support. After that, CuSO4 and tris[(1-benzyl-1,2,3-triazol-4-
yl)methyl]amine (TBTA-ligand) were added as a 25 mmol Lꢀ1
20-O-(2-Azidoethoxymethyl)-50-O-(4,40-dimethoxytrityl)-
N4-benzoylcytidine 30-(H-phosphonate) Triethylammonium
Salt (2). Compound 5b (0.37 g, 0.5 mmol) was dissolved in
dry pyridine (2 mL), and diphenyl H-phosphonate (0.7 mL,
3.6 mmol) was added. After 45 min, water (0.6 mL) and Et3N
1251
dx.doi.org/10.1021/bc200097g |Bioconjugate Chem. 2011, 22, 1249–1255

Bioconjugate Chemistry
TECHNICAL NOTE
Figure 1. Solid-supported introduction of two different sugar ligands into 20-positions within a 20-O-Me-oligoribonucleotide (10).
solution in a 12:3:1 (v/v/v) mixture of water, DMSO, and
2-butanol (7.5 equiv/azido group). Finally, sodium ascorbate
(15 equiv/azido group) as a 0.1 mol Lꢀ1 aqueous solution was
added. The mixture was shaken at room temperature for 5 h. The
support was collected by filtration and washed first with water,
DMF, and MeOH. To remove the copper salt and TBTA ligand as
quantitatively as possible, the support was then treated for 10 min
with aqueous EDTA solution (0.1 mol Lꢀ1), washed with water,
and then for another 10 min with an aqueous solution of neomycin
trisulfate (0.1 mol Lꢀ1). Finally, the support was washed with
water, DMF, DCM, MeOH, and MeCN.
On-Support Azidation. The support-bound oligonucleotides
(0.5 μmol) were treated with a solution of tetramethylguanidinium
azide (TMGA, 15.8 mg, 200 equiv) and NaI (15.0 mg, 200 equiv) in
dry DMF (500 μL) for 1.5 h at 65 °C.13 The support was collected
by filtration and washed with DMF, DCM, water, and MeOH.
Characterization of the Conjugates. The identity of the
sugar conjugates 10 and 11 was verified by ESI-MS. Observed
masses are calculated from [(M-4H)/4]. ON10 requires an
isotopic mass of 5527.08; found, 5527.15. ON11 requires an
isotopic mass of 5830.19; found, 5830.18.
Melting Temperature Studies. The melting curves (absorbance
versus temperature) were measured at 260 nm on a Perkin-Elmer
Lambda 35 UVꢀvis spectrometer equipped with a multiple cell
holder and a Peltier temperature controller. The temperature was
changed at a rate of 0.5 °C/min (from 15 to 90 °C). The
measurements were performed in 10 mmol Lꢀ1 potassium phos-
phate buffer (pH 7) containing 0.1 mol Lꢀ1 or 1.0 mol Lꢀ1 NaCl.
The oligonucleotide conjugates and their cDNA or 20-O-methyl
RNA targets were used at a concentration of 2 μmol Lꢀ1. Tm values
were determined as the maximum of the first derivate of the
melting curve.
’ RESULTS AND DISCUSSION
Synthesis of Monomeric Building Blocks. An O-glycosyla-
tion approach30,32 was applied to the preparation of the 20-O-(2-
bromoethoxy)methyl (3a) and 20-O-(2-azidoethoxy)methyl (3b)
substituted cytidine monomers. Accordingly, 30,50-O-(1,1,3,3-
tetraisopropyldisiloxane-1,3-diyl)-N4-benzoylcytidine33,34 was
treated with (2-bromoethoxy)methyl acetate in the presence of
tin tetrachloride at ꢀ12 °C.30 Nucleoside 3a was isolated in 88%
yield, and conversion to the corresponding 20-O-(2-azidoethoxy)
methyl derivative (3b) by displacement of the bromo substituent
with NaN3 in DMF was virtually quantitative. The 30,50-cyclic
silyl protecting group was removed from 3a and 3b with Bu4NF
in THF, and the 50-OH of the deprotected nucleosides (4a,b)
was protected with a 4,40-dimethoxytrityl group to obtain 5a,b.
Finally, 5a and 5b were converted to 30-(2-cyanoethyl-N,N-
diisopropylphosphoramidite) (1) and 30-(H-phosphonate) (2),
respectively. Owing to the presence of the azido group, 5b
undergoes a Staudinger reaction upon phosphitylation, which
prevents its conversion to a phosphoramidite reagent.9,35
The alkynyl-derivatized monosaccharide ligands, viz. propar-
gyl 2,3,4,6-tetra-O-acetyl-R-D-mannopyranoside (6) and propar-
gyl 2,3,4,6-tetra-O-acetyl-β-D-galactopyranose (7), used in the
click conjugation were prepared from peracetylated R-D-manno-
pyranose and β-D-galactopyranose by boron trifluoride etherate
promoted glycosidation with propargyl alcohol. The compounds
1
exhibited H and 13C NMR chemical shifts identical to those
reported earlier.9,31
Oligonucleotide Synthesis and Click Conjugation. The
appropriately protected phosphoramidite (1) and H-phospho-
nate (2) building blocks were incorporated into 15-mer 20-O-
methyl oligoribonucleotides: 50-(CAU C2UG GUU C1UA
1252
dx.doi.org/10.1021/bc200097g |Bioconjugate Chem. 2011, 22, 1249–1255

Bioconjugate Chemistry
TECHNICAL NOTE
Figure 2. RP HPLC traces of the crude and purified oligonucleotide glycoconjugates 10 and 11, respectively. For the chromatographic conditions, see
the Experimental Procedures Section.
Table 1. Melting Temperatures for the Duplexes of Oligonucleotide Conjugates 10 and 11 with Their Complementary DNA and
20-O-Methyl-RNA Sequencesa
compl. DNA Tm/°C and (Δ Tm/°C)
0.1 mol Lꢀ1 NaCl 1.0 mol Lꢀ1 NaCl
compl. 20-O-methyl RNA Tm/°C and (Δ Tm/°C)
0.1 mol Lꢀ1 NaCl
50-CAU CUG GUU CUA CGA
50-CAU XUG GUU YUA CGA (10)
50-CAU YUG GUU XUA YGA (11)
50.1
61.7
72.0
50.6 (þ0.5)
47.0 (ꢀ3.1)
60.6 (ꢀ1.1)
60.2 (ꢀ1.5)
71.5 (ꢀ0.5)
68.2 (ꢀ3.8)
a X = monomer C2 conjugated with 6; Y = monomer C1 conjugated with 7.
CGA)-30 (8) and 50-(CAU C1UG GUU C2UA C1GA)-30 (9)
(C1 and C2 refer to nucleosidic units derived from 1 and 2,
respectively). The oligomers were assembled by phosphorami-
dite chemistry, with the exception of the insertion of 2, which was
carried out by pivaloyl chloride promoted H-phosphonate cou-
pling. As also shown previously, the presence of an azido group in
the growing solid-supported oligonucleotide chain does not
disturb the phosphoramidite coupling of subsequent monomers.9
The coupling yield of building block 1 was ∼99%, but the H-
phosphonate block 2 was coupled in only 85% yield.
After completion of the chain assembly, oligonucleotides 8
and 9 were conjugated with the two alkynyl-functionalized
monosaccharide ligands (6 and 7) (Figure 1). The azido group
of monomeric unit 2 was first subjected to Cu(I) promoted click
reaction with propargyl R-D-mannopyanoside tetraacetate (6),
using a mixture of CuSO4 and sodium arcorbate in the presense
of tris[(1-benzyl-1,2,3-triazol-4-yl)methyl]amine (TBTA-
ligand) as the source of Cu(I) ion (5 h, r.t.). The bromo
substituent of monomeric unit 1 was then displaced with an
azide ion, as reported previously by Pourceau et al.13,14 Both
tetramethylguanidinium azide36 (TMGA/NaI/DMF, 65 °C)
and less soluble NaN3 (NaN3/NaI/DMF, 65 °C) were used as
a source of azide ions. The second click reaction was then
performed using propargyl β-D-galactopyranoside tetraacetate
(7) as the alkyne component. The oligonucleotide conjugates
obtained (10 and 11) were deprotected and released from the
support by standard ammonolysis (33% aq NH3, 55 °C, over-
night). The crude products were purified by RP-HPLC, and their
identity was verified by ESI-MS. Figure 2 shows the HPLC traces
of the crude conjugates and the homogeneity of the products
after purification.
Melting Temperature Studies. It has been shown previously
that oligonucleotides bearing a β-D-ribofuranosyl group at an
intrachain 20-O form stable complexes with RNA (ΔTm = 0 °C)
and maintain A-type helical geometry with the additional 20-O-
ribose moiety located in the minor groove.2,24 Consistent with
these findings, the sugar ligands attached in the present study to
20-O via a [2-(4-hydroxymethyl-1H-1,2,3-triazol-1-yl)ethoxy]-
methyl linker only slightly destabilize the duplex. Table 1 records
the melting temperatures (Tm) for the duplexes of oligonucleo-
tide conjugates 10 and 11 with the full cDNA and 20-O-methyl
RNA sequences at pH 7.
The saccharide ligands accommodated quite well in the duplex
structure. Oligonucleotide 10 bearing two saccharide units in an
1253
dx.doi.org/10.1021/bc200097g |Bioconjugate Chem. 2011, 22, 1249–1255

Bioconjugate Chemistry
TECHNICAL NOTE
intrachain position exhibited even a slight stabilization of the
(3) Zatsepin, T. S., Romanova, E. A., and Oretskaya, T. S. (2004)
Nucleosides and oligonucleotides containing 20-reactive groups: synth-
esis and applications. Russ. Chem. Rev. 73, 701–733.
(4) Ugarte-Uribe, B., Pꢀerez-Rentero, S., Lucas, R., Avi~nꢀo, A., Reina,
J. J., Alkorta, I., Eritja, R., and Morales, J. C. (2010) Synthesis, cell-surface
binding, and cellular uptake of fluorescently labeled glucose-DNA
conjugates with different carbohydrate presentation. Bioconjugate Chem.
21, 1280–1287.
(5) Zhu, L., and Mahato, R. I. (2010) Targeted delivery of siRNA to
hepatocytes and hepatic stellate cells by bioconjugation. Bioconjugate
Chem. 21, 2119–2127.
(6) L€onnberg, H. (2009) Solid-phase synthesis of oligonucleotide
conjugates useful for delivery and targeting of potential nucleic acid
therapeutics. Bioconjugate Chem. 20, 1065–1094.
duplex with the DNA target at a low ionic strength (ΔTm
=
þ0.5 °C) and a slight destabilization at a high ionic strength
(ΔTm = ꢀ1.1 °C). Addition of a third saccharide ligand to obtain
conjugate 11 resulted in a moderate destabilization even at a
low ionic strength (ΔTm = ꢀ3.1). At high ionic strength, the
destabilization was less prominent (ΔTm = ꢀ1.5 °C). With the
20-O-Me RNA target, two saccharide ligands within conjugate 10
were similarly well tolerated (ΔTm = ꢀ0.5 °C), but with 11,
containing three saccharides, Tm again dropped significantly
(ΔTm = ꢀ3.8 °C). A tentative explanation for the marked
destabilizing influence of the third saccharide, especially at low
ionic strength, is that two of the sugars are rather close to each
other within conjugate 11, viz. in positions 10 and 13.
(7) Zatsepin, T. S., and Oretskaya, T. S. (2004) Synthesis and applica-
tions of oligonucleotide-carbohydrate conjugates. Chem. Biodiversity
1, 1401–1417.
’ CONCLUSIONS
(8) Yan, H., and Tram, K. (2007) Glycotargeting to improve cellular
delivery efficiency of nucleic acids. Glycoconjugate J. 24, 107–123.
(9) Kiviniemi, A., Virta, P., and L€onnberg, H. (2008) Utilization of
intrachain 40-C-azidomethylthymidine for preparation of oligodeoxyr-
ibonucleotide conjugates by click chemistry in solution and on a solid
support. Bioconjugate Chem. 19, 1726–1734.
(10) Kiviniemi, A., Virta, P., and L€onnberg, H. (2010) Solid-sup-
ported synthesis and click conjugation of 40-C-alkyne functionalized
oligodeoxyribonucleotides. Bioconjugate Chem. 21, 1890–1901.
(11) Chevolot, Y., Bouillon, C., Vidal, S., Morvan, F., Meyer, A.,
Cloarec, J.-P., Jochum, A., Praly, J.-P., Vasseur, J.-J., and Souteyrand, E.
(2007) DNA-based carbohydrate biochips: a platform for surface glycol-
engineering. Angew. Chem., Int. Ed. 46, 2398–2402.
(12) Bouillon, C., Meyer, A., Vidal, S., Jochum, A., Chevolot, Y.,
Cloarec, J.-P., Praly, J.-P., Vasseur, J.-J., and Morvan, F. (2006) Micro-
wave assisted click chemistry for the synthesis of multiple labeled-
carbohydrate oligonucleotides on solid support. J. Org. Chem. 71,
4700–4702.
(13) Pourceau, G., Meyer, A., Chevolot, Y., Souteyrand, E., Vasseur,
J.-J., and Morvan, F. (2010) Oligonucleotide carbohydrate-centered
galactosyl cluster conjugates synthesized by click and phosphoramidite
chemistries. Bioconjugate Chem. 21, 1520–1529.
A convenient and reliable method for solid-phase introduction
of two different types of saccharide ligands in desired positions of
an oligonucleotide has been developed. The underlying principle
is regiospecific introduction of azido- and bromo-functionalized
building blocks into the oligonucleotide upon chain assembly
and their subsequent derivatization on the support. A propynyl-
functionalized saccharide is first attached to the azido groups
by Cu(I) promoted 1,3-dipolar cycloaddition, and the same
reaction is utilized to conjugate the second propynyl saccharide
after conversion of the bromo substituents to azido groups by
displacement with the azide ion. For the chain assembly, con-
ventional phosphoramidite chemistry may be applied, except for
the introduction of the azido functionalized building blocks that
have to be coupled as H-phosphonates. The saccharide ligands
are quite well accommodated to the duplexes formed with both
DNA and 20-O-Me RNA targets
’ ASSOCIATED CONTENT
(14) Pourceau, G., Meyer, A., Vasseur, J.-J., and Morvan, F. (2009)
Synthesis of mannose and galactose oligonucleotide conjugates by bi-
click chemistry. J. Org. Chem. 74, 1218–1222.
(15) Ketom€aki, K., and Virta, P. (2008) Synthesis of aminoglycoside
conjugates of 20-O-methyl oligoribonucleotides. Bioconjugate Chem.
19, 766–777.
S
Supporting Information.
ESI-MS data and melting
b
temperature curves for oligonucleotides 10 and 11. This material
is available free of charge via the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION
(16) Morvan, F., Meyer, A., Jochum, A., Sabin, C., Chevolot, Y.,
Imberty, A., Praly, J.-P., Vasseur, J.-J., Souteyrand, E., and Vidal, S.
(2007) Fucosylated pentaerythrityl phosphodiester oligomers (PePOs):
automated synthesis of DNA-based glycoclusters and binding to Pseu-
domonas aeruginosa lectin (PA-IIL). Bioconjugate Chem. 18, 1637–1643.
(17) Karskela, M., Virta, P., Malinen, M., Urtti, A., and L€onnberg, H.
(2008) Synthesis and cellular uptake of fluorescently labeled multivalent
hyaluronan disaccharide conjugates of oligonucleotide phosphorothio-
ates. Bioconjugate Chem. 19, 2549–2558.
Corresponding Author
*(A.K.) Fax: þ35823336700. E-mail: anu.kiviniemi@utu.fi.
(S.N.M.) Fax: þ074991359751. E-mail: smikh@eimb.ru.
’ ACKNOWLEDGMENT
Financial support from the Russian Foundation for Basic
Research, Russian Academy of Sciences (Program “Molecular
and Cell Biology”), and the Academy of Finland is gratefully
acknowledged.
(18) Katajisto, J., Virta, P., and L€onnberg, H. (2004) Solid-phase
synthesis of multiantennary oligonucleotide glycoconjugates utilizing
on-support oximation. Bioconjugate Chem. 15, 890–896.
(19) Yamada, T., Peng, C. G., Matsuda, S., Addepalli, H., Jayaprakash,
K. N., Alam, M. R., Mills, K., Maier, M. A., Charisse, K., Sekine, M.,
Manoharan, M., and Rajeev, K. G. (2011) Versatile site-specific conjuga-
tion of small molecules to siRNA using click chemistry. J. Org. Chem.
76, 1198–1211.
(20) Rostovtsev, V. V., Green, L. G., Fokin, V. V., and Sharpless, K. B.
(2002) A stepwise huisgen cycloaddition process: Copper(I)-catalyzed
regioselective “ligation” of azides and terminal alkynes. Angew. Chem.,
Int. Ed. 41, 2596–2599.
’ REFERENCES
(1) Manoharan, M. (1999) 20-Carbohydrate modifications in anti-
sense oligonucleotide therapy: importance of conformation, configura-
tion and conjugation. Biochim. Biophys. Acta 1489, 117–130.
(2) Luyten, I., Esnouf, R. M., Mikhailov, S. N., Efimtseva, E. V.,
Michiels, P., Heus, H. A., Hilbers, C. W., and Herdewijn, P. (2000)
Solution structure of a RNA decamer duplex, containing 9-[(2-O-(β-D-
ribofuranosyl)-(β-D-ribofuranosyl]adenine, a special residue in lower
eukaryotic initiator tRNAs. Helv. Chim. Acta 83, 1278–1289.
(21) Tornoe, C. W., Christensen, C., and Meldal, M. (2002)
Peptidotriazoles on solid phase: [1,2,3]-triazoles by regiospecific
1254
dx.doi.org/10.1021/bc200097g |Bioconjugate Chem. 2011, 22, 1249–1255

Bioconjugate Chemistry
TECHNICAL NOTE
copper(I)-catalyzed 1,3-dipolar cycloadditions of terminal alkynes to
azides. J. Org. Chem. 67, 3057–3064.
(22) Mikhailov, S. N., Efimtseva, E. V., Gurskaya, G. V., Zavodnik,
V. E., De Bruyn, A., Rozenski, J., and Herdewijn, P. (1997) An efficient
synthesis and physico-chemical properties of 20-O-β-D-ribofuranosyl-
nucleosides, minor tRNA components. J. Carbohydr. Chem. 16, 75–92.
(23) Efimtseva, E. V., Kulikova, I. V., and Mikhailov, S. N. (2007)
Disaccharide nucleosides and their incorporation into oligonucleotides.
Curr. Org. Chem. 11, 337–354.
(24) Efimtseva, E. V., Bobkov, G. V., Mikhailov, S. N., Van Aerschot,
A., Schepers, G., Busson, R., Rozenski, J., and Herdewijn, P. (2001)
Oligonucleotides containing disaccharide nucleosides. Helv. Chim. Acta
84, 2387–2397.
(25) Mikhailov, S. N., Efimtseva, E. V., Rodionov, A. A., Shelkunova,
A. A., Rozenski, J., Emmerechts, G., Schepers, G., Van Aerschot, A., and
Herdewijn, P. (2005) Synthesis of RNA containing O-β-D-
ribofuranosyl-(100-20)-adenosine-500-phosphate and 1-methyladenosine,
minor components of tRNA. Chem. Biodiversity 2, 1153–1163.
(26) Andreeva, O. I., Golubeva, A. S., Kochetkov, S. N., Van Aerschot
A., Herdewijn, P., Efimtseva, E. V., Ermolinsky, B. S., and Mikhailov, S. N.
(2002) Anadditional20-ribofuranose residue ata specific position ofDNA
primer prevents its elongation by HIV-1 reverse trancriptase. Bioorg. Med.
Chem. Lett. 12, 681–684.
(27) Tunitskaya, V. L., Rusakova, E. E., Memelova, L. V., Kochetkov,
S. N., Van Aerschot, A., Herdewijn, P., Efimtseva, E. V., Ermolinsky, B. S.,
and Mikhailov, S. N. (1999) The mapping of T7 RNA polymerase active
site with novel reagents - oligonucleotides with reactive dialdehyde
groups. FEBS Lett. 442, 20–24.
(28) Efimtseva, E. V., and Mikhailov, S. N. (2002) Disaccharide
nucleosides and oligonucleotides on their bases. New tools for the study
of enzymes of nucleic acid metabolism. Rev. Biochem. (Moscow) 67,
1136–1144.
(29) Gritsenko, O. M., Koudan, E. V., Mikhailov, S. N., Ermolinsky,
B. S., Van Aerschot, A., Herdewijn, P., and Gromova, E. S. (2002)
Affinity modification of EcoRII DNA methyltransferase by the dialde-
hyde-substituted DNA duplexes: mapping the enzyme region that
interacts with DNA. Nucleosides, Nucleotides Nucleic Acids 21, 753–764.
(30) Bobkov, G. V., Mikhailov, S. N., Van Aerschot, A., and
Herdewijn, P. (2008) Phosphoramidite building blocks for efficient
incorporation of 20-O-aminoethoxy(and propoxy)methyl nucleosides
into oligonucleotides. Tetrahedron 64, 6238–6251.
(31) Mereyala, H. B., and Gurrala, S. R. (1998) A highly diaster-
eoselective, practical synthesis of allyl, propargyl 2,3,4,6-tetra-O-acetyl-
β-D-gluco, β-D-galactopyranosides and allyl, propargyl heptaacetyl-β-
D-lactosides. Carbohydr. Res. 307, 351–354.
(32) Efimtseva, E. V., Kulikova, I. V., and Mikhailov, S. N. (2009)
Disaccharide nucleosides as an important group of natural compounds.
Mol. Biol. (Russia) 43, 301–312.
(33) Markiewicz, W. T. (1979) Tetraisopropyldisiloxane-1,3-diyl
group for simultaneous protection of 30- and 50-hydroxy functions of
nucleosides. J. Chem. Res. (S), 24–25.
(34) Bobkov, G. V., Brilliantov, K. V., Mikhailov, S. N., Rozenski, J.,
Van Aerschot, A., and Herdewijn, P. (2006) Synthesis of oligoribonu-
cleotides containing pyrimidine 20-O-[(hydroxyalkoxy)methyl]-
ribonucleosides. Collect. Czech. Chem. Commun. 71, 804–819.
(35) Coppola, C., Simeone, L., De Napoli, L., and Montesarchio, D.
(2011) On the compatibility of azides in phosphoramidite-based
couplings: synthesis of a novel, convertible azido-functionalized Cy-
PLOS analogue. Eur. J. Org. Chem. 6, 1155–1165.
(36) Papa, A. J. (1966) Synthesis and azidolysis of 2-chlorotetra-
methylguanidine. Synthetic utility of hexa- and tetramethylguanidinium
azide. J. Org. Chem. 31, 1426–1430.
1255
dx.doi.org/10.1021/bc200097g |Bioconjugate Chem. 2011, 22, 1249–1255