Solid-phase synthesis of oligonucleotide glycoconjugates bearing three different glycosyl groups: Orthogonally protected bis(hydroxymethyl)-N,N′- bis(3-hydroxypropyl)malondiamide phosphoramidite as key building block

Article abstract of DOI:10.1021/jo048984o

Diethyl O,O′-(methoxymethylene)bis(hydroxymethyl)malonate (3) was observed to undergo a stepwise aminolysis when treated with 3-aminopropanol. This allowed convenient preparation of bis(hydroxymethyl)-N,N′-bis(3- hydroxypropyl)malondiamide bearing orthogonal levulinyl (Lev) and tert-butyldiphenylsilyl (TBDPS) protections at the two N-hydroxypropyl groups (8). One of the hydroxylmethyl functions was then protected with a 4,4′-dimethoxytrityl (DMTr) group, and the other one was phosphitylated to obtain a methyl N,N-diisopropylphosphoramidite (1). This building block was used for the synthesis of oligonucleotide glycoconjugates (25 and 26) carrying three different sugar units. After conventional phosphoramidite chain assembly of the sequence containing 1, the 5′-terminal DMTr group was removed and an appropriate glycosyl 6-O-phosphoramidite was coupled. The remaining protections of the branching unit were removed in the order of Lev and TBDPS, and the exposed hydroxyl functions were reacted one after another with the desired glycosyl 6-O-phosphoramidites. Global deprotection and cleavage of the conjugate from the support were achieved by conventional ammonolysis.

Full text of DOI:10.1021/jo048984o

Solid -P h a se Syn th esis of Oligon u cleotid e Glycocon ju ga tes Bea r in g
Th r ee Differ en t Glycosyl Gr ou p s: Or th ogon a lly P r otected
Bis(h yd r oxym eth yl)-N,N′-bis(3-h yd r oxyp r op yl)m a lon d ia m id e
P h osp h or a m id ite a s Key Bu ild in g Block
J ohanna Katajisto,* Petri Heinonen, and Harri Lo¨nnberg
Department of Chemistry, University of Turku, FIN-20014 Turku, Finland
jokrka@utu.fi
Received J une 17, 2004
Diethyl O,O′-(methoxymethylene)bis(hydroxymethyl)malonate (3) was observed to undergo a
stepwise aminolysis when treated with 3-aminopropanol. This allowed convenient preparation of
bis(hydroxymethyl)-N,N′-bis(3-hydroxypropyl)malondiamide bearing orthogonal levulinyl (Lev) and
tert-butyldiphenylsilyl (TBDPS) protections at the two N-hydroxypropyl groups (8). One of the
hydroxylmethyl functions was then protected with a 4,4′-dimethoxytrityl (DMTr) group, and the
other one was phosphitylated to obtain a methyl N,N-diisopropylphosphoramidite (1). This building
block was used for the synthesis of oligonucleotide glycoconjugates (25 and 26) carrying three
different sugar units. After conventional phosphoramidite chain assembly of the sequence containing
1, the 5′-terminal DMTr group was removed and an appropriate glycosyl 6-O-phosphoramidite was
coupled. The remaining protections of the branching unit were removed in the order of Lev and
TBDPS, and the exposed hydroxyl functions were reacted one after another with the desired glycosyl
6-O-phosphoramidites. Global deprotection and cleavage of the conjugate from the support were
achieved by conventional ammonolysis.
In tr od u ction
of antisense oligonucleotides14 to a certain cell type and
thus improve their cellular intake by endocytosis. For
these purposes, noncovalent carriers and covalent con-
A number of important biological systems are known
to interact through multiple simultaneous carbohydrate-
protein binding events.1 This kind of multipoint attach-
ment to cell-surface lectins through several sugar ligands
covalently tethered to a natural scaffold, such as a protein
or lipid, ensures high affinity binding and provides the
molecule with cell- or tissue-specificity.2-6 Several mul-
tiantennary glycoclusters,7 glycodendrimers,8 glycopep-
tides,9 cyclodextrin-based glycoclusters,10 and glycopoly-
mers11 have been synthesized for this purpose. Most of
them contain a single type of sugar unit attached to a
polyfunctional scaffold. Many biological recognition pro-
cesses, however, rely on binding of several different types
of sugar moieties. Suprisingly, only few syntheses of such
diverse glycoconjugates have been reported. In fact, the
examples available are limited to the use of orthogonally
protected sugar-12 and pentaerythritol-based13 templates.
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Dubber, M. Org. Lett. 2001, 3, 4019-4022. (e) Roy, R.; Kim; J . M.
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otides has also gained increasing interest during recent
years as it offers a convenient way to enhance binding
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10.1021/jo048984o CCC: $27.50 © 2004 American Chemical Society
Published on Web 09/24/2004
J . Org. Chem. 2004, 69, 7609-7615
7609

Katajisto et al.
jugates have been prepared. The methods described
include electrostatic complexation of an oligonucleotide
with glycosylated polylysine;15 chemical ligation to ga-
lactosylated polylysine,16 to a cholane scaffold,17 and to
a neoglycopeptide;18 on-support glycosylation with trichlo-
roacetimidates19 or unprotected carbohydrates;20 and the
use of base moiety glycosylated nucleoside phosphora-
midites21 and glycoside phosphoramidites22 as building
blocks for the chain assembly. Some recent approaches
allow creation of structural diversity in the terms of the
number of sugar ligands and the distance between them.
These methods utilize pentaerythritol-based building
blocks as a branching unit either at the 5’-terminus23 or
at an intrachain position.24 The sugar units are coupled
to these polyvalent scaffolds as phosphoramidites23 or as
aldehydes via oxime bond formation with a solid-sup-
ported aminooxy group,24 respectively. Phosphoramidite
building blocks bearing orthogonally protected functional
groups may be expected to allow attachment of different
types of sugar moieties in a controlled manner and,
hence, the synthesis of more complex glyco-oligonucle-
otide conjugates. We now report on a facile method of
preparation of this kind of diverse oligonucleotide glyco-
conjugates. A phosphoramidite derived from bis(hy-
droxymethyl)-N,N′-bis(3-hydroxypropyl)malondiamide that
contains three orthogonally protected hydroxyl groups (1)
and is compatible with normal chain assembly has been
used as a key building block (Figure 1). Stepwise removal
of the 4,4′-dimethoxytrityl (DMTr), levulinyl (Lev), and
tert-butyldiphenylsilyl (TBDPS) protections followed by
coupling of a glycosyl-derived phosphoramidite to the
exposed hydroxyl group then gives the desired oligo-
nucleotide glycoconjugate. The applicability of this ap-
F IGURE 1.
proach has been demonstrated by preparing both T6 and
a heterosequence bearing three different sugar units, viz.,
mannose, galactose, and glucose.
Resu lts a n d Discu ssion
Syn th esis of Bis(h yd r oxym eth yl)-N,N′-bis(3-h y-
d r oxyp r op yl)m a lon d ia m id e P h osp h or a m id it e
1
(Sch em e 1). The orthogonally protected building block
1 was synthesized starting from commercially available
diethyl 2,2-bis(hydroxymethyl)malonate (2). Direct ami-
nolysis of 2 or its DMTr-ether is precluded, owing to their
tendency to rapidly release formaldehyde by retro-aldol
condensation in the presence of primary amines.25 Ac-
cordingly, 2 was first converted to its O,O′-methoxy-
methylene derivative (3). The next step, selective ami-
nolysis of one of the ethyl ester functions in an excess of
3-aminopropanol in THF was the crucial step of the
synthesis. A 48-h treatment at room temperature af-
forded exclusively monoamide 4 in a 36% yield. The
remaining unreacted starting material (64%) could be
easily recycled, which favorably compensated for the
relatively low yield of this step. The reaction was found
to be highly dependent on the conditions used. When the
reaction was performed in the absence of THF, when
aprotic solvents such as dichloromethane (DCM) or
dichloroethane were used as cosolvents, or when the
reaction mixture was heated to reflux, a mixture of mono-
and dialcohol products were obtained. The free hydroxy
function was then protected with a TBDPS group to
afford 5, and the remaining ester group was reacted with
3-aminopropanol (48 h at 50 °C) to yield 6. Protection of
the hydroxy function with a levulinyl group to obtain 7
was followed by acid-catalyzed hydrolysis of the ortho
ester protection, yielding 8. Selective protection of one
of the hydroxymethyl groups of 8 as a DMTr ether and
standard phosphitylation of the remaining hydroxyl
function with methyl N,N-diisopropylphosphoramido-
chloridite accomplished the synthesis of 1.
(12) (a) Patel, A.; Lindhorst, T. K. Eur. J . Org. Chem. 2002, 79-86.
(b) Tanaka, H.; Amaya, T.; Takahashi, T. Tetrahedron Lett. 2003, 44,
3053-3057.
(13) (a) Schmidt, M.; Dobner, B.; Nuhn, P. Synlett 2000, 1157-1159.
(b) Katajisto, J .; Karskela, T.; Heinonen, P.; Lo¨nnberg, H. J . Org. Chem.
2002, 67, 7995-8001.
(14) Wang, L.; Prakash, R. K.; Stein, C. A.; Koehn, R. K.; Ruffner,
D. E. Antisense Nucleic Acid Drug Dev. 2003, 13, 169-189.
(15) (a) Midoux, P.; Mendes, C.; Legrand, A.; Raimond, J .; Mayer,
R.; Monsigny, M.; Roche, A. C. Nucleic Acids Res. 1993, 21, 871-878.
(b) Liang, W.; Shi, X.; Deshpande, D.; Malanga, C. J .; Rojanasakul, Y.
Biochim. Biophys. Acta 1996, 1279, 227-234. (c) Erbacher, P.; Roche,
A. C.; Monsigny, M.; Midoux, P. Biochim. Biophys. Acta 1997, 1324,
27-36.
(16) (a) Bonfils, E.; Depierreux, C.; Midoux, P.; Thuong, N. T.;
Monsigny, M.; Roche, A. C. Nucleic Acids Res. 1992, 20, 4621-4629.
(b) Biessen, E. A. L.; Vietsch, H., Rump, E. T.; Fluiter, K.; Kuiper, J .;
Bijsterbosch, M. K.; Van Berkel, T. J . C. Biochem. J . 1999, 340, 783-
792.
(17) Maier, M. A.; Yannopoulos, C. G.; Mohamed, N.; Roland, A.;
Fritz, H.; Mohan, V.; J ust, G.; Manohara, M. Bioconjugate Chem. 2003,
14, 18-29.
(18) Hangeland, J . J .; Flesher, J . E.; Deamond, S. F.; Lee, Y. C.;
Ts’O, P. O. P. Antisense Nucleic Acid Drug Dev. 1997, 7, 141-149.
(19) Adinolfi, M.; Barone, G.; De Napoli, L.; Guariniello, L.; Iadonisi,
A.; Piccialli, G. Tetrahedron Lett. 1999, 40, 2607-2610.
(20) Sando, S.; Matsui, K.; Niinomi, Y.; Sato, N.; Aoyama, Y. Bioorg.
Med. Chem. Lett. 2003, 13, 2633-2636.
(21) (a) Hunziker, J . Bioorg. Med. Chem. Lett. 1999, 9, 201-204.
(b) Matsuura, K.; Hibino, M.; Yamada, Y.; Kobayashi, K. J . Am. Chem.
Soc. 2001, 123, 357-358. (c) De Kort, M.; Ebrahimi, E.; Wijsman, E.
R.; Van der Marel, G. A.; Van Boom, J . H. Eur. J . Chem. 1999, 2337-
2344.
Syn th esis of Glycosyl P h osp h or a m id ites (19-21).
The fully protected glycosyl phosphoramidites (19-21)
were obtained easily from commercially available methyl
R-D-glycopyranosides (galactose (10), mannose (11), and
glucose (12)) via a three-step procedure (Table 1). Methyl
R-D-glycopyranosides 10-12 were first subjected to two
sequential reactions in one pot. The trityl (Tr) protecting
group was introduced at the primary 6-O-position,26 and
the secondary hydroxyl groups were subsequently esteri-
fied with p-toluoyl chloride (13-15).22b The trityl protec-
tion was then removed with iodine in methanol (16-
(22) (a) Akhtar, S.; Routledge, A.; Patel, R. Tetrahedron Lett. 1995,
36, 7333-7336. (b) Sheppard, T. L.; Wong, C.; J oyce, G. F. Angew.
Chem., Int. Ed. 2000, 39, 3660-3663.
(23) Dubber, M.; Fre´chet, J . M. J . Bioconjugate Chem. 2003, 14,
239-246.
(25) Guzaev, A.; Salo, H.; Azhayev, A.; Lo¨nnberg, H. Bioconjugate
Chem. 1996, 7, 240-248.
(26) Du, Y.; Zhang, M.; Kong, F. Org. Lett. 2000, 2, 3797-3800.
(24) Katajisto, J .; Virta, P.; Lo¨nnberg, Bioconjugate Chem. 2004, 15,
890-896.
7610 J . Org. Chem., Vol. 69, No. 22, 2004

SPS of Diverse Glyco-Oligonucleotide Conjugates
SCHEME 1 a
a
Conditions: (i) (MeO)3CH, TsOH; (ii) 3-aminopropanol, THF; (iii) TBDPSCl, imidazole, DMF; (iv) 3-aminopropanol; (v) levulinic acid,
DCC, DMAP, pyridine, dioxane; (vi) 80% AcOH; (vii) DMTrCl, pyridine; (viii) methyl N,N-diisopropylphosphoramidochloridite, NEt3,
dichloromethane.
TABLE 1. Syn th esis of Glycosyl P h osp h or a m id ites 19-21
starting material
products
step Aa
step Ba
step Ca
10: R1 ) R4 ) R6 ) OH; R2 ) R3 ) R5 ) H
11: R2 ) R4 ) R5 ) OH; R1 ) R3 ) R6 ) H
12: R1 ) R4 ) R5 ) OH; R2 ) R3 ) R6 ) H
13, 16, 19: R1 ) R4 ) R6 ) OTol; R2 ) R3 ) R5 ) H
14, 17, 20: R2 ) R4 ) R5 ) OTol; R1 ) R3 ) R6 ) H
15, 18, 21: R1 ) R4 ) R5 ) OTol; R2 ) R3 ) R6 ) H
13: 86%
14: 89%
15:95%
16: 70%
17: 95%
18: 74%
19: 82%
20: 74%
21: 81%
a
Yields were determined after purification by silica gel chromatography.
18),27 and the exposed hydroxyl function was phos-
phitylated with methyl N,N-diisopropylphosphonamidic
chloride to afford the desired fully protected glycosyl
phosphoramidites 19-21.
DMT-cation assay, an acceptable 96% coupling efficiency
was obtained for 1.29 Next, the free 5′-terminal hydroxy
group of the solid-supported conjugate 22 was phosphi-
tylated with galactosyl 6-O-phosphoramidite (19) using
a prolonged coupling time of 600 s to afford 23. The
levulinyl protection was then removed by 30 min treat-
ment with 0.5 mol L-1 hydrazinium acetate, and man-
nosyl 6-O-phosphoramidite (20) was introduced to give
24. In this case, the coupling was somewhat retarded
compared to the coupling to the 5′-terminal hydroxyl
function, in all likehood owing to the steric hindrance
caused by the bulky TBDPS group. Accordingly, a longer
coupling procedure consisting of three successive injec-
tions of the mixture of the 6-O-phosphoramidite (20) and
activator (3 × 600 s) was applied. At this state, a small
aliquot of the solid support was withdrawn from the
synthesis column, and the completion of the last coupling
step and the formation of the desired conjugate 24 was
Syn th esis of Oligon u cleotid e Glycocon ju ga tes
(25, 26). The compatibility of phosphoramidite 1 with the
conventional oligonucleotide synthesis by the phosphora-
midite strategy was first examined by assembling a T6
sequence containing an internally linked 1 (22 in Scheme
2). A prolonged coupling time (600 s) was applied to the
nonnucleosidic building block 1. Nucleoside-3′-yl methyl
N,N-diisopropylphosphoramidites were used instead of
the more common 2-cyanoethyl phosphoramidites, be-
cause it has been previously shown that the latter are
not compatible with fluoride reagents required for re-
moval of the TBDPS protection.28 Otherwise the recom-
mended protocols were employed. On the basis of the
(27) Wahlstro¨m, J . L.; Ronald, R. C. J . Org. Chem. 1998, 63, 6021-
6022.
(28) Scaringe, S. A.; Wincott, F. E.; Caruthers, M. H. J . Am. Chem.
Soc. 1998, 120, 11820-11821.
(29) Atkinson, T.; Smith, M. Oligonucleotide Synthesis: A Practical
Approach; IRL Press: Oxford, U.K., 1984; p 48.
J . Org. Chem, Vol. 69, No. 22, 2004 7611

Katajisto et al.
calcd yield
SCHEME 2 a
TABLE 2. P r op er ties of Oligon u cleotid e
Glycocon ju ga tes
oligonucleotide retention time
obsd
entry glycoconjugate
(min)a
mass
mass
(%)b
1
2
25
26
8.32
11.5
2872.0 2871.0
5032.7 5032.6
52
50
a
On a ThermoHypersil C-18 column (4.6 mm × 150 mm, 5 µm),
a linear gradient from 0 to 100% B in 30 min, flow rate 1 mL
min-1. Buffer A: 0.05 mol L-1 NH4OAc (aq). Buffer B:
A
b
containing 65% MeCN. Percentage of the conjugate in the crude
reaction mixture on the basis of HPLC signal areas.
in THF, yielding the deprotected conjugate in 75% yield
(based on the relative peak areas of the protected and
deprotected conjugate in the HPLC chromatogram; for
the RP HPLC chromatogram, see Supporting Informa-
tion). Attempts to replace THF with solvents such as
DCM, water, or MeCN were unsuccessful, as was also
elevation of the reaction temperature. Finally, glucosyl
6-O-phosphoramidite (21) was coupled applying again a
prolonged reaction time of 600 s. Cleavage of the methyl
protections from the phosphate groups31 and subsequent
standard ammonolytic treatment (33% aqueous NH3, 7
h at 55 °C) released the desired crude oligonucleotide
glycoconjugate 25 in solution. No side reactions were
detected upon the ammonolytic deprotection. The main
product (25) was easily separated by RP HPLC from the
hydrophobic diglycosylated side product bearing still the
TBDPS protection (Table 2; entry 1, for the RP HPLC
chromatogram of purified 25, see Supporting Informa-
tion). The identity of conjugate 25 was verified by ESI-
MS (Table 2; entry 1)
The applicability of the procedure to the construction
of diverse oligonucleotide glycoconjugates was verified by
preparing an 11-mer heterosequence (26) carrying three
different sugar units essentially by the protocol described
above. The branching unit (1) was coupled to the 5′-
terminus of the solid-supported 11-mer; the protecting
groups were removed in the order DMTr, Lev, TBDPS;
and the exposed hydroxyl groups were phosphitylated
with 19, 20, and 21, respectively. The hydroxymethyl
branch was, however, elongated with one T before the
introduction of 19. Interestingly, a better 87% yield was
now obtained for the TBDPS deprotection step. Figure 2
shows the analytical RP HPLC chromatogram of the
crude product mixture of 26. After RP HPLC purification
(for the RP HPLC chromatogram of purified 26, see
Supporting Information), the authenticity of the desired
diverse oligonucleotide glycoconjugate was confirmed by
ESI-MS (Table 2; entry 2).
a
Conditions: (i) 19 in MeCN; (ii) hydrazinium hydrate, pyri-
dine, AcOH (0.124/4/1); (iii) 20 in MeCN; (iv) (TEA, 3HF), THF;
(v) 21 in MeCN; (vi) 1 M disodium-2-carbamoyl-2-cyanoethylene-
1,1-dithiolate (S2Na2) in DMF; (vii) aq NH3.
Melting temperature (Tm) measurements showed that
although the presence of a bulky glycocluster at the 5′-
terminus of an 11-mer heterosequence retarded the
hybridization, a reasonably stable duplex with a fully
complementary 11-mer deoxyribo-oligonucleotide was
still formed. The melting temperature of this duplex was
41 °C, when the concentration of 26 and its complemen-
tary 11-mer was 2 µmol L-1. Omission of the glycosylated
non-nucleosidic moiety from 26 increased the melting
point to 48 °C.
verified by RP HPLC and ESI-MS analysis after depro-
tection and cleavage from the solid support (for the RP
HPLC chromatogram of the crude product, see Support-
ing Information). The next step, removal of the TBDPS
protection by using triethylamine trihydrofluoride,30
turned out to be somewhat problematic, because quan-
titative cleavage of the TBDPS group could not be
achieved despite several attemps to optimize the reaction
conditions. The best results were obtained by repeating
an 18-h treatment with triethylamine trihydrofluoride
(31) M Disodium-2-carbamoyl-2-cyanoethylene-1,1-dithiolate (S2Na2)
in DMF in 30 min. Dahl, B. J .; Bjergarde, K.; Henriksen, L.; Dahl, O.
Acta Chem. Scand. 1990, 44, 639-641.
(30) Westman, E.; Stro¨mberg, R. Nucleic Acids Res. 1994, 22, 2430-
2431.
7612 J . Org. Chem., Vol. 69, No. 22, 2004

SPS of Diverse Glyco-Oligonucleotide Conjugates
(m, 3H); 13C NMR (CDCl3, 100 MHz) δ 169.3, 169.0, 168.4,
168.2, 110.0, 108.6, 64.3, 62.8, 62.6, 62.3, 62.0, 59.5, 59.3, 53.2,
53.0, 52.1, 51.7, 36.7, 36.6, 32.0, 31.8, 13.9; HRMS (ESI) [M +
Na]+ calcd 314.1210, obsd 314.1227.
E t h yl
2-Met h oxy-5-[3-(ter t-b u t yld ip h en ylsilyloxy)-
p r op ylca r ba m oyl]-1,3-d ioxa n e-5-ca r boxyla te (5). Com-
pound 4 (2.60 g, 8.90 mmol) was coevaporated twice with dry
MeCN. The residue was dissolved in dry DMF (30 mL)
together with imidazole (1.80 g, 26.8 mmol), and tert-butyl-
diphenylsilyl chloride (2.74 mL, 10.7 mmol) was slowly added.
After 16 h of treatment, the consumption of 4 was complete
according to TLC (3% MeOH in DCM). Diethyl ether was
added, and the mixture was washed with water and aqueous
NaHCO3. The organic phase was dried over Na2SO4 and
evaporated to dryness, and the product was isolated by silica
gel chromatography (3% MeOH in DCM) to afford 5 as a
colorless oil in a 74% yield (5.50 g): 1H NMR (CDCl3, 400 MHz)
δ 7.65 (m, 4H), 7.42 (m, 6H), 5.30, 5.29, 5.23, 5.20 (four s, total
1H), 4.43 (m, 2H), 4.07-4.30 (m, 4H), 3.69 (m, 2H), 3.36-3.48
(m, 5H), 1.69-1.81 (m, 3H), 1.06 (s, 9H); 13C NMR (CDCl3,
100 MHz) δ 169.6, 168.8, 167.8, 166.6, 135.5, 132.6, 129.7,
127.7, 109.5, 108.8, 63.7, 63.2, 62.8, 62.3, 62.2, 62.1, 61.9, 61.2,
53.0, 52.9, 52.8, 52.3, 51.8, 36.9, 36.8, 31.9, 31.8, 26.8, 19.1,
13.9; HRMS (ESI) [M + Na]+ calcd 552.2388, obsd 552.2380.
N -[3-(t er t -Bu t yld ip h e n ylsilyloxy)p r op yl]-N ’-(3-h y-
d r oxyp r op yl)-2-m eth oxy-1,3-d ioxa n e-5,5-d ica r boxa m id e
(6). The mixture of compound 5 (3.50 g, 6.61 mmol) and
3-aminopropanol (4.5 mL, 52.9 mmol) was stirred for 48 h at
50 °C. DCM was added to the reaction solution, and the
mixture was washed with water and dried with Na2SO4. The
solvent and the excess of 3-aminopropanol were removed under
reduced pressure. Separation of the crude product by silica
gel chromatography (3% MeOH in CH2Cl2) gave 6 as a clear
oil in a 79% yield (2.9 g): 1H NMR (CDCl3, 200 MHz) δ 7.64
(m, 4H), 7.36 (m, 6H), 5.30, 5.20 (two s, total 1H), 4.48 (d, 2H,
J ) 11.9 Hz), 4.12 (m, 3H), 3.59-3.70 (m, 5H), 3.21-3.49 (m,
7H), 2.95 (m, 1H), 1.63-1.78 (m, 4H), 1.06 (s, 9H); 13C NMR
(CDCl3, 50 MHz) δ 170.4, 169.9, 169.4, 168.8, 135.4, 133.6,
129.7, 127.7, 109.7, 65.3, 61.2, 59.3, 50.5, 36.9, 36.5, 31.9, 26.8,
19.2; HRMS (ESI) [M + Na]+ calcd 581.2654, obsd 581.2659.
N-[3-(ter t-Bu t yld ip h en ylsilyloxy)p r op yl]-N′-[3-(levu -
lin yloxy)p r op yl]-2-m et h oxy-1,3-d ioxa n e-5,5-d ica r b ox-
a m id e (7). A solution of N,N-dicyclohexylcarbodiimide (DCC;
2.13 g, 10.3 mmol) was slowly added to a solution of levulinic
acid (2.40 g, 20.6 mmol) in dry dioxane (40 mL). After the
mixture stirred for 4 h at room temperature, dicyclohexylurea
was filtered off, and compound 6 (2.90 g, 5.19 mmol) in dry
pyridine (15 mL) and a catalytic amount of DMAP were added.
The reaction was allowed to proceed for 2 h at room temper-
ature. The reaction mixture was diluted with DCM, and the
mixture was washed with aqueous NaHCO3 and dried with
Na2SO4. The organic phase was evaporated to dryness. Puri-
fication of the crude product on a silica gel (2% MeOH in DCM)
yielded 7 as a clear oil in a 80% yield (1.20 g): 1H NMR (CDCl3,
500 MHz) δ 7.64 (m, 4H), 7.40 (m, 6H), 4.46 (d, 2H, J ) 11.2
Hz), 4.12 (m, 4H), 3.71 (m, 2H), 3.30-3.46 (m, 7H), 2.75 (t,
2H, J ) 5.7 Hz), 2.58 (t, 2H, J ) 5.7 Hz), 2.18 (s, 3H), 1.71-
1.94 (m, 4H), 1.10 (s, 9H); 13C NMR (CDCl3, 125 MHz) δ 206.6,
172.8, 170.9, 168.9, 135.5, 133.6, 129.6, 127.7, 109.6, 65.2, 62.0,
61.2, 58.4, 53.3, 37.9, 36.9, 36.7, 32.5, 29.8, 28.3, 27.8, 19.1;
HRMS (ESI) [M + Na]+ calcd 679.3021, obsd 679.3049.
F IGURE 2. RP HPLC profile of crude 26 at 260 nm.
Notation: (a) conjugate 26, tR ) 11.5 min; (b) diglycosylated
side product carrying the TBDPS protection, tR ) 16.9 min.
For detailed chromatographic conditions, see footnote a in
Table 2.
In summary, a protocol for the attachment of three
different glycosyl phosphoramidites to the 5′-terminus of
an oligonucleotide on a solid support has been developed.
Alternatively, two different sugar moieties may be teth-
ered to any site within a given oligonucleotide sequence
and the third one to the 5′-terminus. Although the
conjugates have to be subjected to conventional HPLC
purification, owing to the incomplete removal of the
TBDPS group, these methods allow quite convenient
synthesis of combinatorial libraries.
Exp er im en ta l Section
Gen er a l Meth od s a n d Ma ter ia ls. See Supporting Infor-
mation for detailed information. The NMR spectra were
recorded at 200, 400, or 500 MHz in deuteriochloroform. The
chemical shifts are given in ppm from internal TMS, and the
coupling contants are reported in hertz. The mass spectra were
recorded using EI or ESI ionization methods.
Dieth yl 2-Meth oxy-1,3-d ioxa n e-5,5-d ica r boxyla te (3).
Compound 3 was prepared according to the procedure reported
previously.24 The product was obtained as a clear oil in 94%
yield. The 1H and 13C NMR spectra were in accordance with
the published data. HRMS (ESI) [M + Na]+ calcd 285.0945,
obsd 285.0950.
Eth yl 2-Meth oxy-5-(3-h yd r oxyp r op ylca r ba m oyl)-1,3-
d ioxa n e-5-ca r boxyla te (4). Compound 3 (10.0 g, 38.1 mmol)
and 3-aminopropanol (19.3 mL, 229 mmol) were dissolved in
dry THF (30 mL), and the mixture was stirred at ambient
temperature for 48 h. The solvent was removed in vacuo, and
the residue was dissolved in DCM, washed with water, dried
with Na2SO4, and evaporated to dryness. The oily residue was
purified by silica gel chromatrography (3% to 5% MeOH in
DCM), yielding compound 4 as a clear oil in a 36% yield (4.0
g): 1H NMR (CDCl3, 400 MHz) δ 5.33, 5.31, 5.29, 5.23 (four s,
total 1H), 4.45 (m, 2H), 4.08-4.30 (m, 5H), 3.65 (m, 2H), 3.43-
3.51 (m, 2H), 3.42, 3.39 (two s, total 3H), 1.71 (m, 2H), 1.29
2,2-Bis(h yd r oxym eth yl)-N-[3-(ter t-bu tyld ip h en ylsilyl-
oxy)pr opyl]-N′-[3-levu lin yloxy)pr opyl]-m alon diam ide (8).
Compound 7 (1.20 g, 1.83 mmol) was dissolved in 80% aqueous
AcOH (50 mL) and left for 2 h at room temperature. The
reaction mixture was concentrated to an oil and coevaporated
three times with water. Purification by silica gel chromatog-
raphy (10% MeOH in DCM) afforded 8 as a clear oil in a 91%
yield (1.0 g): 1H NMR (CDCl3, 500 MHz) δ 7.64 (m, 4H), 7.40
(m, 6H), 4.10 (t, 2H, J ) 5.9 Hz), 3.99 (t, 2H, J ) 7.4 Hz), 3.83
(d, 4H, J ) 7.4 Hz), 3.68 (t, 2H, J ) 5.9 Hz), 3.42 (q, 2H, J )
7.3 and 13.2 Hz), 3.30 (q, 2H, J ) 6.8 and 13.0 Hz), 2.88 (t,
J . Org. Chem, Vol. 69, No. 22, 2004 7613

Katajisto et al.
2H, J ) 7.8 Hz), 2.58 (t, 2H, J ) 7.8 Hz), 2.18 (s, 3H), 1.73-
1.85 (m, 4H), 1.04 (s, 9H); 13C NMR (CDCl3, 125 MHz) δ 206.7,
172.8, 171.5, 135.4, 133.6, 129.7, 127.7, 64.1, 61.7, 61.1, 57.2,
38.7, 36.6, 36.3, 32.5, 29.8, 28.3, 27.9, 26.8; HRMS (ESI) [M +
H]+ calcd 615.3096, obsd 615.3104.
21.7, 21.6. Anal. Calcd for C50H46O9: C, 75,93; H, 5.86.
Found: C, 77.35; H, 5.91.
Met h yl 6-O-Tr it yl-2,3,4-t r i-O-(p -t olu oyl)-r-D-m a n n o-
p yr a n osid e (14) a n d Meth yl 6-O-Tr ityl-2,3,4-tr i-O-(p-
tolu oyl)-r-D-glu cop yr a n osid e (15). See Supporting Infor-
mation.
2-Hyd r oxym et h yl-2-(4,4′-d im et h oxyt r it yloxym et h yl)-
N-[3-(ter t-b u t yld ip h en ylsilyloxy)p r op yl]-N′-[3-(levu lin -
yloxy)p r op yl]m a lon d ia m id e (9). Compound 8 (1.00 g, 1.63
mmol) was dried by repeated coevaporations with dry pyridine
and dissolved in dry pyridine (20 mL). 4,4′-Dimethoxytrityl
chloride (550 mg, 1.63 mmol) was added, and the solution was
stirred overnight at room temperature. Pyridine was removed
in vacuo, and the residue was subjected to a DCM/aq NaHCO3
workup. The organic phase was dried with Na2SO4, the
solution was evaporated, and the residue was coevaporated
twice with toluene. The product was separated by silica gel
chromatography (0 to 5% MeOH in DCM) to give 9 as a white
solid foam in a 51% yield (760 mg): 1H NMR (CDCl3, 400 MHz)
δ 7.62-7.65 (m, 4H), 7.34-7.43 (m, 9H), 6.80 (m, 4H), 4.05 (t,
2H, J ) 5.5 Hz), 3.77 (s, 6H), 3.65 (t, 1H, J ) 5.3 Hz), 3.44 (d,
2H, J ) 2,2 Hz), 3.21-3.42 (m, 4H), 2.71 (t, 2H, J ) 7.5 Hz),
2.54 (t, 2H, J ) 7.5 Hz), 2.16 (s, 3H), 1.68-1.79 (m, 4H), 1.04
(t, 9H); 13C NMR (CDCl3, 100 MHz) δ 206.6, 172.7, 170.7,
170.6, 158.6, 144.3, 135.5, 133.6, 130.0, 129.7, 127.9, 127.8,
127.7, 126.9, 86.3, 65.3, 63.9, 62.0, 61.1, 58.4, 55.2, 37.9, 36.8,
36.6, 32.1, 29.8, 28.4, 27.8, 26.8, 20.0; HRMS (ESI) [M + Na]+
calcd 939.4222, obsd 939.4167.
Meth yl 3-[(4,4′-Dim eth oxytr ityl)oxy]-2-{[3-(ter t-bu tyl-
diph en ylsilyloxy)pr opyl]car bam oyl}-2-{[3-(levu lin yloxy)-
p r op yl]ca r b a m oyl}p r op yl N,N-d iisop r op ylp h osp h or a -
m id ite (1). Compound 9 (148 mg, 0.16 mmol) was predried
overnight in vacuo over P2O5. Methyl N,N-diisopropylphos-
phoramidochloridite (43 µL, 0.23 mmol) was added to a
solution of 9 and triethylamine (112 µL, 0.81 mmol) in dry
MeCN (2 mL) under nitrogen. The reaction mixture was left
to stand at room temperature. The reaction was completed in
45 min, according TLC analysis (40% ethyl acetate in hexane).
The reaction mixture was applied onto a short dried silica gel
column and the pure compound was isolated by eluting with
a mixture of ethyl acetate, hexane, and triethylamine (40:59:
1, v/v/v). Phosphoramidite 1 was obtained as a white solid foam
in 80% yield (140 mg): 1H NMR (CDCl3, 400 MHz) δ 7.65 (d,
4H, J ) 6.7 Hz), 7.25-7.44 (m, 15H), 6.80 (d, 4H, J ) 8.1 Hz),
4.05-4.16 (m, 4H), 3.76 (s, 6H), 3.46-3.69 (m, 7H), 3.21-3.38
(m, 6H), 2.72 (t, 2H, J ) 6.5 Hz), 2.56 (t, 2H, J ) 6.6 Hz), 2.17
(s, 3H), 1.67-1.82 (m, 4H), 1.05-1.16 (m, 21H); 13C NMR
(CDCl3, 100 MHz) δ 206.6, 172.7, 170.1, 169.7, 158.5, 144.4,
135.4, 133.7, 130.1, 129.7, 128.1, 127.9, 127.7, 126.9, 113.3,
86.4, 64.7, 64.5, 64.0, 62.2, 61.5, 59.1, 55.2, 50.6, 50.5, 43.0,
42.1, 37.9, 36.9, 32.3, 29.8, 28.5, 27.9, 26.9, 24.6, 20.0; 31P NMR
(CDCl3, 400 MHz) δ 149.5.
Met h yl 6-O-Tr it yl-2,3,4-t r i-O-(p -t olu oyl)-r-D-ga la ct o-
p yr a n osid e (13). Methyl R-D-galactopyranoside (10) (5.00 g,
25.7 mmol) was dried by repeated coevaporations with dry
pyridine and dissolved in dry pyridine (50 mL). Trityl chloride
(7.90 g, 28.3 mmol) and a catalytic amount of (N,N-dimethy-
lamino)pyridine were added. The mixture was stirred at 80
°C overnight, and then p-toluoyl chloride (15.3 mL, 116 mmol)
was added. The reaction mixture was shaken overnight at 50
°C, slowly poured into ice-cold water, extracted with ethyl
acetate, washed with saturated NaHCO3, and dried over Na2-
SO4. The solution was concentrated in vacuo and coevaporated
to dryness with toluene. The oily residue was purified by silica
gel chromatography (DCM) to give 13 as a white amorphous
solid in a 86% yield (17.4 g): 1H NMR (CDCl3, 400 MHz) δ
7.86 (t, 4H), 7.69 (d, 2H), 7.34 (d, 6H), 7.18-7.30 (m, 13H),
7.05 (d, 2H), 5.95 (m, 2H), 5.53 (dd, 1H, J ) 3.7 and 10.2 Hz),
5.25 (d, 1H, J ) 3.6 Hz), 4.09 (t, 1H, J ) 6.6 Hz), 3.50 (s, 3H),
3.42 (m, 1H), 3.28 (dd, 1H, J ) 7.1 and 9.4 Hz), 2.46, 2.36 and
2.31 (each s, each 3H); 13C NMR (CDCl3, 100 MHz) δ 166.2,
165.6, 165.4, 146.9, 143.8, 129.9, 129.1, 128.9, 128.6, 127.9,
127.8, 126.8, 126.6, 97.5, 87.0, 69.6, 69.2, 68.3, 68.0, 61.8, 55.5,
Met h yl 2,3,4-Tr i-O-(p-t olu oyl)-r-D-ga la ct op yr a n osid e
(16). Compound 13 (17.4 g, 22.0 mmol) was stirred in a
mixture of methanolic iodine (200 mL, 1% iodine in methanol)
and DCM (100 mL) overnight at 60 °C. The mixture was
washed twice with aqueous Na2SO3 (10%, m/v), dried over Na2-
SO4, and evaporated to dryness. The product was isolated on
a silica gel column, eluting with a gradient of ethyl acetate
(20% to 50%) in petroleum ether, which afforded 16 as a white
amorphous solid in a 70% yield (8.50 g): 1H NMR (CDCl3, 400
MHz) δ 8.01, 7.89, 7.71, 7.30, 7.18 and 7.04 (each d, each 2H),
5.97 (dd, 1H, J ) 3.4 and 10.7 Hz), 5.83 (d, 1H, J ) 3.5 Hz),
5.69 (dd, 1H, J ) 3.6 and 10.7 Hz), 5.27 (d, 1H, J ) 3.6 Hz),
4.32 (m, 1H), 3.76 (m, 1H), 3.63 (m, 1H), 3.30 (s, 3H), 2.46,
2.37 and 2.31 (each s, each 3H); 13C NMR (CDCl3, 100 MHz)
δ 166.9, 166.3, 165.5, 144.6, 144.2, 143.9, 130.1, 129.9, 129.7,
129.4, 129.2, 129.0, 97.6, 70.0, 69.5, 69.2, 68.2, 60.8, 55.7, 21.8,
21.7, 21.6; HRMS (EI) [M - H]- calcd 547.2010, obsd 547.2010.
(Meth yl 2,3,4-Tr i-O-(p-tolu oyl)-r-D-m a n n op yr a n osid e
(17) a n d Meth yl 2,3,4-Tr i-O-(p-tolu oyl)-r-D-glu cop yr a n o-
sid e (18). See Supporting Information.
(Meth yl 2,3,4-Tr i-O-(p-tolu oyl)-r-D-ga la ctop yr a n osid e-
6-yl) Met h yl N,N-Diisop r op ylp h osp h or a m id it e 19. The
phosphoramidite 19 was prepared as described for 1, using
16 (217 mg, 0.40 mmol) as a starting material. The product
was obtained as a clear oil in a 82% yield (230 mg): 1H NMR
(CDCl3, 400 MHz) δ 7.97, 7.87, 7.68, 7.27, 7.16 and 7.03 (each
d, each 2H), 5.97 (m, 2H), 5.60 (dd, 1H, J ) 3.4 and 10.1 Hz),
5.30 (m, 1H), 4.10 (dd, 1H, J ) 6.1 and 11.7 Hz), 3.72-3.85
(m, 2H), 3.53-3.66 (m, 2H), 3.52 (s, 3H), 3.42, 3.39, 3.36 and
3.32 (each s, each 3H), 2.44, 2.35, 2.29 (each s, each 3H), 1.16
(m, 12H); 13C NMR (CDCl3, 100 MHz) δ 166.2, 165.5, 144.3,
143.6, 129.9, 129.8, 129.2, 129.1, 128.9, 97.5, 69.6, 69.1, 68.8,
68.7, 68.3, 61.8, 61.6, 55.5, 42.8, 42.3, 24.7, 21.6; 31P NMR
(CDCl3, 400 MHz) δ 149.2; MS (ESI) [M + Na]+ calcd 1100.5,
obsd 1100.5.
(Meth yl 2,3,4-Tr i-O-(p-tolu oyl)-r-D-m a n n op yr a n osid e-
6-yl) Meth yl N,N-Diisop r op ylp h osp h or a m id ite (20) a n d
(Meth yl 2,3,4-tr i-O-(p-tolu oyl)-r-D-glu cop yr a n osid e-6-yl)
Meth yl N,N-Diisop r op ylp h osp h or a m id ite (21). See Sup-
porting Information.
Im m obiliza tion of Th ym id in e to a Ten ta gel Su p p or t.
To 5′-O-(4,4′-dimethoxytrityl)thymidine (500 mg, 0.92 mmol)32
in dry pyridine (1.6 mL) were added (N,N-dimethylamino)-
pyridine (112 mg, 0.92 mmol) and succinic anhydride (147 mg,
1.47 mmol). The reaction mixture was stirred under nitrogen
atmosphere at room temperature for 20 h, and then water (200
µL) was added. The solution was stirred for 1 h and evaporated
to dryness, and the residue was coevaporated with toluene (3
× 2 mL). The reaction product was dissolved in DCM (80 mL)
and washed with 10% aqueous citric acid and water. The
organic phase was dried over Na2SO4 and evaporated in vacuo.
The residue was dissolved in a mixture of DCM and pyridine
(5 mL, 95:5, v/v), and the solution was added to the mixture
of anhydrous diethyl ether and hexane (200 mL, 25:75, v/v).
The mixture was centrifuged, and the precipitate was washed
with the mixture of diethyl ether and hexane (25:75, v/v) and
dried to give 420 mg of the 5′-O-(4,4′-dimethoxytrityl)thymi-
dine hemisuccinic acid ester. The crude product (420 mg, 0.65
mmol) and p-nitrophenol (81 mg, 0.65 mmol) were dissolved
in a mixture of dry dioxane (160 µL) and dry pyridine (40 µL).
Dicyclohexylcarbodiimide (134 mg, 0.65 mmol) in dry dioxane
(300 µL) was added dropwise to this solution under nitrogen.
After 4 h at room temperature, the insoluble N,N-dicyclohexy-
(32) Koga, M.; Wilk, A.; Moore, M. F.; Scremin, C. L.; Zhou, L.;
Beaucage, S. L. J . Org. Chem. 1995, 60, 1520-1530.
7614 J . Org. Chem., Vol. 69, No. 22, 2004

SPS of Diverse Glyco-Oligonucleotide Conjugates
lurea formed was filtered off and washed with anhydrous
dioxane. Filtrates were combined and added to 500 mg of
Tentagel S-NH2 (260 µmol/g) suspended in dry DMF (280 µL)
and triethylamine (60 µL). The suspension was shaken over-
night at ambient temperature. The support was then filtered
and washed with DMF, dioxane, methanol, and diethyl ether.
On the basis of the DMTr-cation assay,29 a loading of 240
µmol/g was obtained. The unreacted amino groups were capped
with Ac2O in THF containing N-methylimidazole and lutidine.
Finally the support was thoroughly washed with THF, DCM,
MeOH, and diethyl ether and vacuum-dried.
Oligod eoxyr ib on u cleot id e Syn t h esis. The oligodeoxy-
ribonucleotides were assembled on an Applied Biosystems 392
DNA synthesizer on a 1.0-µmol scale using the Tentagel
support described above (240 µmol/g) and methyl N,N-diiso-
propylphosphoramidite building blocks. Phosphoramidite 1
was used as a 0.15 mol L-1 solution in dry MeCN, the coupling
time being 600 s. After coupling of 1, the detritylation was
carried out by using two consecutive “no. 14 (acid solution) to
column” steps (2 × 60 s) separated by a trityl flush step (5 s).
Otherwise standard protocols were employed.
Syn th esis of Oligon u cleotid e Glycocon ju ga tes 25 a n d
26. Protected oligonucleotide sequences 5′-d(TTTXTTT)-3′ and
5′-d(TXTGACGATCTCAT)-3′, where X stands for the non-
nucleosidic building block 1, were synthesized as described
above. Galactosyl 6-O-phosphoramidite (19, 0.15 mol L-1
solution in dry MeCN) was reacted with the free 5′-terminal
hydroxyl group of the solid-supported conjugate, the coupling
time being 600 s. The support-bound conjugates were next
treated with 0.5 mol L-1 hydrazine acetate solution (0.124/4/
1, H2NNH2‚H2O/pyridine/AcOH, v/v/v) for 30 min, washed with
pyridine, MeOH, DCM and diethyl ether, and dried under
reduced pressure. The synthesis columns were transferred
back to the DNA synthesizer and mannosyl 6-O-phosphor-
amidite (20, 0.2 mol L-1 solution in dry MeCN) was introduced.
Reaction time of 600 s was used, and the coupling was repeated
three times. The supports were dried in vacuo and transferred
to microcentrifuge tubes. The TBDPS protection was removed
by shaking the resins for 18 h with a mixture of triethylamine
trihydrofluoride (30 µL) and dry THF (150 µL) at room
temperature. The reaction was repeated, after which the
support was filtered, washed with THF, MeOH, DCM, and
diethyl ether, and dried under vacuum. The supports were
then transferred again back to the DNA synthesizer and
glucosyl 6-O-phosphoramidite (21, 0.15 mol L-1 solution in dry
MeCN) was coupled using a reaction time of 600 s. Next, the
phosphate methyl protections were removed by treating the
support with disodium 2-carbamoyl-2-cyanoethylene-1,1-dithi-
olate (S2Na2) (1 M solution in DMF) for 30 min. The supports
were washed with DMF, MeOH, DCM, and diethyl ether,
dried, and finally treated with concentrated ammonia (33%
aqueous NH3, 7 h at 55 °C) to release and deprotect the
resulting oligonucleotide glycoconjugates. Evaporation, dis-
solution in water, HPLC purification, desalting, and charac-
terization by ESI-MS verified the formation of the expected
conjugates 25 and 26 (Table 2).
Meltin g Tem p er a tu r e Stu d ies of 26. The melting curve
(absorbance versus temperature) was measured at 260 nm on
a UV-vis spectrometer equipped with a temperature control-
ler, the heating rate being 1 °C min-1 (from 15 to 90 °C). The
experiments were performed in 10 mmol L-1 potassium
phosphate buffer (pH 7) containing 100 mmol L-1 NaCl. The
concentration of the oligonucleotide conjugate 26 and its
complementary sequence was 2 µmol L-1
.
Su p p or tin g In for m a tion Ava ila ble: Experimental de-
tails and spectral data for the compounds 1, 4-9, and 13-21
and HPLC analytica data and ESI-MS spectra for oligonucle-
otide glycoconjugates 25 and 26. This material is available free
of charge via the Internet at http://pubs.acs.org.
J O048984O
J . Org. Chem, Vol. 69, No. 22, 2004 7615