Synthesis and characterization of mannosylated oligoribonucleotides

Article abstract of DOI:10.1016/j.carres.2009.08.033

Oligoribonucleotide (RNA)-carbohydrate conjugates bearing mono- and divalent mannosides were readily obtained using 3,4-diethoxy-3-cyclobutene-1,2-dione as the linking agent in the presence of trace amount of triethylamine. The glycoconjugates were purifi

Full text of DOI:10.1016/j.carres.2009.08.033

Carbohydrate Research 344 (2009) 2137–2143
Contents lists available at ScienceDirect
Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
Synthesis and characterization of mannosylated oligoribonucleotides
*
Yuyan Zhao, Kha Tram, Hongbin Yan
Department of Chemistry, Brock University, 500 Glenridge Ave., St. Catharines, Ontario, Canada L2S 3A1
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 5 June 2009
Received in revised form 13 August 2009
Accepted 25 August 2009
Available online 31 August 2009
Oligoribonucleotide (RNA)–carbohydrate conjugates bearing mono- and divalent mannosides were read-
ily obtained using 3,4-diethoxy-3-cyclobutene-1,2-dione as the linking agent in the presence of trace
amount of triethylamine. The glycoconjugates were purified by HPLC and characterized by electrospray
mass spectroscopy.
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
Carbohydrate
Drug delivery
Glycoconjugate
Lectin
Oligoribonucleotide
1. Introduction
We previously reported a method that allows for easy incorpo-
ration of carbohydrate moieties into the oligodeoxynucleotides
In recent years, nucleic acids have found a wide range of thera-
peutic applications such as gene therapy, antisense and antigene
oligonucleotides, aptamers, and RNA interference.1,2 One common
problem that these therapeutic nucleic acids face is their poor
pharmacokinetic properties, such as low bioavailability and poor
selectivity. A number of approaches have been under intense
investigation in order to improve the bioavailability of nucleic
acids as therapeutic agents, such as the use of lipids, liposomes,
peptides, viral vectors, and antibodies as delivery vehicles.3–12
It is known that lectins exist on cell surfaces and that they bind
specifically to certain carbohydrates.13 Sugars may therefore serve
as recognition determinants to promote delivery of biologically
active compounds. The concept of using lectin–glycan interactions
to mediate cell targeting and cellular uptake of molecules, an
approach known as glycotargeting,14 has been under intense inves-
tigation during the past two decades.15,16 A number of mammalian
lectins with different carbohydrate specificities have been identi-
fied.13 These lectins were found to direct their specific ligands to
intracellular compartments. This effect has been demonstrated
with various forms of nucleic acids of the deoxy series, for exam-
ple, antisense oligonucleotides, plasmids, and chromosomal
DNA.17,18 In many of these studies, interactions between glycans
and lectins improved the cellular uptake of nucleic acids. The same
approach with RNAs has not been demonstrated.
(the DNA series) under mild conditions.19 The chemistry involves
the use of 3,4-diethoxy-3-cyclobutene-1,2-dione 1 (diethyl squa-
rate)20 as the linking reagent. First, a glycan containing an amino
group reacts with diethyl squarate 1 to form an activated glycan,
which further reacts with an oligonucleotide bearing a primary
amine to form a glycoconjugate under slightly basic conditions.
When this conjugation chemistry is applied to oligoribonucleotides
(the RNA series), a few choices would have to be made to ensure
successful reactions and subsequent purification. For instance,
due to the instability of RNAs caused by the 20-hydroxyls, conjuga-
tion reactions will have to be carried out under conditions that are
mild enough to avoid degradation and migration of RNAs. We here-
in report a conjugation protocol that is suitable for the preparation
of glycan–oligoribonucleotide conjugates. Thus, conjugation reac-
tions were carried out while the oligoribonucleotides were still
protected at the 20-OH with the 1-(4-chlorophenyl)-4-ethoxypi-
peridin-4-yl (Cpep) group. Such an approach ensures the integrity
of RNA during the conjugation reactions. In this study, mannosides
were chosen as the glycan because they are recognized by the mac-
rophage mannose receptors on the surface of dendritic cells21 that
play important roles in immune responses.
2. Results and discussion
Fully protected 2-chloroethyl D-mannopyranoside 2 was pre-
pared from mannose via the trichloroacetimidate chemistry.22
Substitution by sodium azide gave fully protected 2-azidoethyl
* Corresponding author. Tel.: +1 9056885550x3545; fax: +1 9056829020.
E-mail address: tyan@brocku.ca (H. Yan).
D-mannopyranoside 3. Deacylation of 3 followed by the reduction
0008-6215/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2009.08.033

2138
Y. Zhao et al. / Carbohydrate Research 344 (2009) 2137–2143
OAc
OAc
OH
OAc
O
OAc
O
OH
O
a
b
AcO
AcO
AcO
AcO
HO
HO
3
O
4
2
O
O
Cl
N3
N3
c
d
OH
OAc
OH
OAc
O
O
O
O
HO
HO
AcO
AcO
TFA
NH3
5
7
O
O
EtO
OEt
1
NH2
e
OH
OH
O
O
HO
HO
O
6
N
O
H
OEt
Scheme 1. Reagents and conditions: (a) NaN3, DMF, 55 °C, 3 d; (b) NaOMe, MeOH; (c) Pd/C, H2, MeOH, TFA; (d) Pd/C, H2, H2O; (e) H2O, MeOH, NEt3, rt, 5 min.
O
HO
NH
2
H N
2
NH
NH
NHCbz
a
b
NHCbz
O
O
N
N
N
H N
2
NH
2
H N
2
9
8
10
HO
O
OH
OH
O
OAc
OAc
O
c
HO
HO
AcO
AcO
O
O
O
N
O
N
H
H
d, e
NH
NH
OH
NH
NH
NH
2
OH
O
O
O
OAc
NHCbz
OAc
O
N
HO
HO
N
O
AcO
AcO
O
O
N
O
N
H
H
O
O
12
11
OH
OH
O
f
HO
HO
N
O
O
O
PF
6
O
N
N
H
N(CH )
N
3 2
NH
O
P
N(CH )
3 2
OH
NH
OEt
O
O
OH
O
N
BOP
N(CH )
3 2
HO
HO
NH
O
N
H
O
13
Scheme 2. Reagents and conditions: (a) PhCH2OCOCl, CH2Cl2; (b) succinic anhydride, C5H5N; (c) 7, BOP, (i-Pr)2NEt, CH2Cl2; (d) NaOMe, MeOH; (e) Pd/C, H2, MeOH; (f) H2O,
MeOH, NEt3, rt, 7 min.
of the azido compound 4 gave 2-aminoethyl
D-mannopyranoside 5,
succinic anhydride to furnish bis-carboxylic acid 10. Compound 10
which was readily transformed into its squarate analogue 6 using
further reacted with the trifluoroacetate salt of 2-aminoethyl
2,3,4,6-tetra-O-acetyl-a-D-mannopyranoside 7 to give the fully
procedures (Scheme 1) reported previously.19
It is well known that interactions between lectins and their gly-
can ligands are relatively weak; therefore it is desirable to intro-
duce multiple (multivalent) mannosides in order to harness the
cluster glycoside effect.23 In this study, tris(2-aminoethyl)amine
8 was used as the core structure (Scheme 2) to build multivalent
mannosides. Upon protection of tris(2-aminoethyl)amine by the
benzyloxycarbonyl (Cbz) group, the product 9 was condensed with
protected divalent mannoside 11. Removal of O-acetates and Cbz
groups from 11 yielded bimannoside 12, which was subsequently
Salt 7 was prepared by hydrogenation of 2-azidoethyl 2,3,4,6-tetra-O-acetyl-a-D-
mannopyranoside 3 in the presence of trifluoroacetic acid to avoid trans-acetylation
of the reduced primary amine.

Y. Zhao et al. / Carbohydrate Research 344 (2009) 2137–2143
2139
DMTrO
O
The chemistry was similarly efficient when the bimannoside 13
was allowed to conjugate with U10-mer 17b. Reverse phase HPLC
profiles of the reaction mixture indicated that the conjugation
was complete in 9 h (Fig. 1). Conjugates 18 and 19 were purified
by reverse phase HPLC and identified by ESI mass spectrometry.
The Cpep-protected conjugates 18 and 19 were then deprotec-
ted (removal of Cpep) by incubation in a mixture of DMA–triethyl-
ammonium formate buffer (pH 2.52) (6:4 v/v) to give the
unprotected conjugates (20 and 21, Scheme 5). The final mono-
and divalent conjugates 20 and 21 were identified by ESI mass
spectrometry and analyzed by anion exchange chromatography
on a DNAPac PA100 column (Fig. 2). These conjugates can be
readily purified further by anion exchange chromatography if
necessary.
U
DMTrO
U
O
O
a
O
OCpep
NC
HO
OCpep
P
15
14
NEt2
N(i-Pr)2
O
EtO
CN
P
Cpep =
N
Cl
O
O
F3C
N
6
H
16
O
N
Ph
NH
DMTr =
U =
MeO
OMe
O
When the stability of mono- and dimannosyl U10-mers 20 and
21 was compared with that of unmodified U10-mer in the presence
Scheme 3. Reagents and conditions: (a) Et2NP(Cl)OCH2CH2CN, (i-Pr)2NEt, CH3CN.
O
O
HO
3'
5'
HO
O
O
O
O
O
H2N
O
O
O
O
B U
9
a
P
P
6
HO
HO
U U
9
O
NH
HN
17a; B = U
b; B = U
18
6
Scheme 4. Reagents and conditions: (a) 6, MeOH, H2O, NEt3. U: uridine protected at 20-position by Cpep; U: fully-unprotected uradine.
activated by reacting with diethyl squarate 1. The product 13 was
ready for conjugation with oligoribonucleotides.
of ribonuclease A and alkaline phosphatase, no significant differ-
ence was observed. Both the modified and unmodified U10-oligo-
mers were degraded by the enzymes at a similar rate under the
same conditions.
Oligoribonucleotides were prepared by solid phase phospho-
ramidite chemistry using the 1-(4-chlorophenyl)-4-ethoxypiperi-
din-4-yl (Cpep as shown in Scheme 3)à to protect the
20-hydroxyls.24 The Cpep chemistry was chosen because it allows
for stable oligoribonucleotide ‘precursor’, that is, oligoribonucleo-
tides protected only at 20-positions with Cpep, to be prepared for
conjugation with carbohydrates. The preparation of uridine phos-
phoramidite 15 is illustrated in Scheme 3. N,N-Diethyl phosphorami-
dites instead of N,N-diisopropyl phosphoramidites were used due to
their higher reactivity (unpublished results).
3. Experimental
3.1. General methods
Reverse phase HPLC was carried out on a 250 ꢀ 4.6 mm LiChro-
spher 100Ò RP-18 5
l column: the column was eluted with triethyl-
ammonium acetate buffer (TEAA)–acetonitrile mixtures [linear
gradient of TEAA buffer (0.1 M, pH 7.0)–acetonitrile (70: 30 v/v
to 20: 80 v/v) over 15 min and then by isocratic elution. Flow rate
1.0 mL/min]. Anion exchange chromatography was carried out on a
250 ꢀ 4 mm DNAPac PA100 column: the column was eluted with
the following program: eluent 1: TrisCl (0.1 M, pH 8.0, 10%), eluent
2: water, eluent 3: NaCl (1.0 M); flow rate: 1.5 mL/min. Concave
gradient of NaCl from 0.1 M to 0.55 M over 20 min.
Dichloromethane was purified by Pure-Solv Solvent Purification
Systems (Innovative Technology), and stored over activated 4 Å
molecular sieves. Triethylamine, Hünig’s base, and pyridine were
dried by heating, under reflux, in the presence of calcium hydride
and then distilled under nitrogen. (N-Trifluoroacetyl)aminohexyl-
2-cyanoethyl N,N-diisopropylphosphoramidite, uridine, and aden-
osine resins for solid phase synthesis were purchased from Glen
Research. The other chemicals were purchased from Aldrich and
used without further purification unless stated otherwise.
U10 homodecamers were used to establish the conjugation
chemistry. The decamers were modified at the 50-termini using
the standard phosphoramidite chemistry by a C6-amino modifier
16 that is commercially available (Glen Research).
We then carried out the conjugation reactions between
squarate-activated mono- and divalent mannosides and U10-mer
bearing a C6-amino modifier. Two routes were attempted. First,
conjugations were carried out by using amino-modified U10 homo
decamers 17a, in which all the Cpep-protecting groups were re-
moved, and the conjugations took place in a triethylammonium
acetate buffer (TEAA, pH 8.8, 0.1 M). Second, conjugations were
performed in a DMA–TEAA buffer, and amino-modified U10 homo
decamer 17b (all the uridines except the 30-terminal one were pro-
tected by Cpep at the 20-OH, as indicated by the italic U in Scheme
4) was used as a precursor. Neither approach was efficient to give
the conjugates in reasonable yields. In the first approach, less than
50% conversion was observed after the incubation proceeded for
five days, as indicated by anion exchange chromatography on a
DNA Pac PA100 column. The second route did not give any signif-
icant amount of the product after 10 days. However, in the latter
approach, upon addition of trace amount of triethylamine, the
reaction proceeded smoothly in aqueous methanol. Thus, when
Cpep-protected U10-mer 17b was treated with squarate-activated
monomannoside 6, the reaction was complete after 5 h at room
temperature as indicated by both ESI mass spectrometry and
reverse phase HPLC.
3.2. 2-Chloroethyl 2,3,4,6-tetra-O-acetyl-a-D-mannopyranoside
(2)
2,3,4,6-Tetra-O-acetyl
D-mannopyranosyl a-trichloroacetimi-
date25 (5.31 g, 10.8 mmol) was co-evaporated with dry toluene
(2 ꢀ 10 mL) and then dissolved in dry dichloromethane (20 mL).
Freshly distilled 2-chloroethanol (1.88 mL, 28.0 mmol) and 4 Å
activated molecular sieve powder (1.0 g) were added, and the mix-
ture was cooled to ꢁ78 °C (dry ice–acetone). To this solution was
added a pre-cooled solution (ꢁ78 °C) of boron trifluoride diethyl
etherate (2.3 mL, 18.3 mmol) in dry dichloromethane by cannula-
à
Cpep-protected RNA monomers are commercially available at Rasayan Inc., CA.

2140
Y. Zhao et al. / Carbohydrate Research 344 (2009) 2137–2143
1
WVL:260 nm
WVL:260 nm
WVL:260 nm
WVL:260 nm
WVL:260 nm
1
2
2
3
3
4
4
5
5
0.0
1.3
2.5
3.8
5.0
6.3
7.5
8.8
10.8
Retention Time [min]
Figure 1. Stack plot of reverse phase HPLC profiles of the conjugation reaction. (1) U10-mer 17b modified at the 50-terminus by a C6-amino modifier; (2) conjugation reaction
mixture after 3 h; (3) conjugation reaction mixture after 6 h; (4) conjugation reaction mixture after 9 h; (5) purified conjugate 19. Samples were run on a C18 RP column. The
peak with a tR of 2.2 min represents the excess of squarate-activated bimannoside 13.
1,000
800
280
mAU
WVL:260 nm
mAU
WVL:260 nm
200
150
100
50
600
3.66
2.70
400
200
min
min
-20
-5
0.00
1.00
2.00
3.00
4.00
5.00
6.10
0.00
1.00
2.00
3.00
4.00
5.00
6.00
Retention Time [min]
Retention Time [min]
Figure 2. Anion exchange chromatography (DNAPac PA100, Dionex) profiles of fully-deprotected (a) mono mannosyl U10-mer 20 and (b) dimannosyl U10-mer 21.
tion. After 1 h, the mixture was warmed up to room temperature,
and then extracted with saturated aqueous sodium hydrogen car-
bonate (100 mL). The layers were separated and the aqueous layer
was back-extracted with dichloromethane (2 ꢀ 20 mL). The com-
bined organic layers were dried (MgSO4) and concentrated under
reduced pressure. The residue was purified by column chromatog-
raphy on silica gel. The appropriate fractions, which were eluted
with hexane–ethyl acetate (3:2 v/v) were combined and evapo-
rated under reduced pressure to give the title compound as a white
dC[CDCl3]: 20.6 (CH3), 20.7 (CH3), 20.9 (CH3), 42.4 (OCH2CH2Cl),
62.4 (C-6), 66.0 (CH), 68.6 (OCH2CH2Cl), 68.9 (CH), 69.0 (C-5), 69.4
(CH), 97.8 (C-1), 169.8 (C@O), 169.9 (C@O), 170.0 (C@O), 170.6
(C@O).
Rf: 0.43 (hexane–ethyl acetate, 3:2 v/v).
3.3. 2-Azidoethyl 2,3,4,6-tetra-O-acetyl-a-D-mannopyranoside
(3)
glass (3.17 g, 71.5%).
2-Chloroethyl 2,3,4,6-tetra-O-acetyl-a-D-mannopyranoside 2
FAB-MS found [M+H]+ = 411.10556, C16H24ClO10
requires
(2.00 g, 4.98 mmol) was dissolved in DMF (30 mL) followed by
the addition of sodium azide (1.59 g, 24.5 mmol). The mixture
was heated at 50 °C for 3 d. The products were then cooled to room
temperature and filtered. The filtrate was concentrated under
reduced pressure and the residue was purified by column chroma-
tography on silica gel. The appropriate fractions, which were eluted
with hexane–ethyl acetate (3:2 v/v) were combined and evapo-
þ
411.10580.
dH[CDCl3]: 2.00 (3H, s, CH3), 2.06 (3H, s, CH3), 2.11 (3H, s, CH3),
2.17 (3H, s, CH3), 3.67 (2H, t, J = 5.6, OCH2CH2Cl), 3.83 (1H, dt,
J = 5.3 and 10.6, OCH2CH2Cl), 3.93 (1H, dt, J = 5.9 and 11.3,
OCH2CH2Cl), 4.12 (1H, m, H-5), 4.16 (1H, m, H-6), 4.30 (1H, m, H-
60), 4.88 (1H, d, J = 1.1, H-1), 5.26–5.38 (3H, m, H-2, H-3, and H-4).

Y. Zhao et al. / Carbohydrate Research 344 (2009) 2137–2143
2141
OH
dH[D2O]: 2.74 (2H, dd, J = 5.5 and 14.4, OCH2CH2NH2), 3.44
(1H, m, OCH2CH2NH2), 3.53–3.81 (6H, m, H-3, H-4, H-5, H-6,
H-60, and OCH2CH2NH2), 3.87 (1H, m, H-2), 4.77 (1H, d, J = 1.6,
H-1).
dC[D2O]: 39.8 (OCH2CH2NH2), 60.9 (C-6), 66.7 (CH), 68.5
(OCH2CH2NH2), 69.9 (C-2), 70.5 (CH), 72.7 (CH), 99.8 (C-1).
OH
O
HO
HO
O
O
O
O
O
O
O
O
N
H
P
U U
9
NH
NH
OH
OH
NH
HN
O
O
6
O
N
HO
HO
19
3.5. Monovalent mannosyl squarate (6)
O
N
H
2-Aminoethyl
dissolved in distilled water (200
methanol (1.8 mL), 3,4-diethoxy-3-cyclobutene-1,2-dione 1 (140
L, 0.95 mmol), and triethylamine (5 L). After 5 min, the solvents
a
-
D
-mannopyranoside 5 (35.5 mg, 0.16 mmol) was
O
lL), followed by the addition of
a
OH
OH
O
l
l
HO
HO
were quickly removed under reduced pressure. The residue was di-
luted with distilled water (1.0 mL) and purified by size exclusion
chromatography on BioGel P-2 gel (fine, 1.6 ꢀ 75 cm), eluted with
milliQ water. The appropriate fractions were combined and freeze-
dried to give the title compound as a light yellow amorphous solid
O
O
O
O
O
O
N
P
H
O
O
U
10
NH
OH
OH
O
NH
HN
O
6
N
HO
HO
20
O
(23.0 mg, 41.4%).
NH
FAB-MS found [M+H]+ = 348.12946, C14H22NO9
requires
þ
O
N
H
348.12765.
O
dH[D2O, recorded at 10 °C]: 1.15 (t, J = 7.0, –OCH2CH3), 1.17 (t,
J = 7.0, –OCH2CH3) (these two signals integrate 3H), 3.26–3.63
(10H, m, H-2, H-3, H-4, H-5, H-6, H-60, and CH2), 4.44 (q, J = 7,
–OCH2CH3), 4.47 (q, J = 7, –OCH2CH3) (these two signals integrate
2H), 4.58 (1H, d, J = 6.0, H-1) (the CH3 and CH2 each splits over
two regions presumably due to the pseudo-amide nature of the
compound).
O
O
HO
O
O
O
O
HO
O
P
U
10
HO
HO
O
NH
HN
6
21
Scheme 5. Reagents and conditions: (a) DMA–Et3N+HꢂHCOOꢁ buffer (pH 2.52),
40 °C, 6 h.
Rf: 0.69 (chloroform–methanol–water, 65:35:5 v/v).
3.6. 2-(Benzyloxycarbonyl)tris(2-aminoethyl)amine (9)
rated under reduced pressure to give the title compound as a white
Tris(2-aminoethyl)amine 8 (2.92 g, 20.0 mmol) was dissolved in
dry dichloromethane (50 mL) and cooled (ice-water bath). A solu-
tion of benzyl chloroformate (1.03 mL, 7.22 mmol) in dry dichloro-
methane (100 mL) was added over a period of 1 h. The mixture was
allowed to warm up to room temperature slowly. After an addi-
tional 1 h, the solvent was removed under reduced pressure. The
residue was purified by column chromatography on silica gel.
The appropriate fractions, which were eluted with chloroform–
methanol–ammonium hydroxide (10:4:1 v/v) were combined
and evaporated under reduced pressure to give the title compound
as a white amorphous solid (1.25 g, 61.8% based on benzyl
glass (1.94 g, 95.3%).
FAB-MS found [M+H]+ = 418.14481, C16H24N3O10 requires
þ
418.14617.
dH[CDCl3]: 2.00 (3H, s, CH3), 2.06 (3H, s, CH3), 2.11 (3H, s, CH3),
2.17 (3H, s, CH3), 3.48 (2H, m, CH2), 3.68 (1H, m, OCH2CH2N3), 3.88
(1H, m, OCH2CH2N3), 4.05 (1H, m, H-5), 4.13 (1H, dd, J = 2.3 and
12.3, H-6), 4.30 (1H, dd, J = 5.3 and 12.3, H-60), 4.88 (1H, s, H-1),
5.26–5.39 (3H, m, H-2, H-3, and H-4).
dC[CDCl3]: 20.6 (CH3), 20.7 (CH3), 20.8 (CH3), 20.9 (CH3), 50.3
(OCH2CH2N3), 62.4 (CH2, C-6), 66.0 (CH), 67.0 (OCH2CH2N3), 68.8
(2 ꢀ CH), 69.4 (CH), 97.7 (CH, C-1), 169.7 (C@O), 169.8 (C@O),
170.0 (C@O), 170.6 (C@O).
chloroformate).
FAB-MS found [M+H]+ = 281.19946, C14H25N4O2
requires
þ
Rf: 0.43 (hexane–ethyl acetate, 4:6 v/v).
281.19775.
dH[CDCl3]: 2.51 (4H, t, J = 6, –NCH2CH2NH2), 2.56 (2H, t, J = 5.9,
CH2), 2.72 (4H, t, J = 6, –NCH2CH2NH2), 3.26 (2H, br, CH2), 4.78
(4H, br, NH2), 5.10 (2H, s, –CH2Ph), 6.05 (1H, br, NH), 7.31–7.37
(5H, m, aromatic).
3.4. 2-Aminoethyl
a
-
D
-mannopyranoside (5)
-mannopyranoside 3 (0.61
2-Azidoethyl 2,3,4,6-tetra-O-acetyl-
a-D
g, 1.46 mmol) was dissolved in methanol (8.0 mL) followed by the
additionofasolutionofsodiummethoxideinmethanol(25 wt %solu-
dC[CDCl3]: 39.4 (CH2), 39.6 (CH2), 53.7 (CH2), 57.0 (CH2), 66.5
(CH2), 128.0 (CH), 128.1 (CH), 128.5 (CH), 136.8 (C), 156.7 (C@O).
Rf: 0.42 (chloroform–methanol–ammonium hydroxide, 10: 4:1
v/v).
tion in methanol, 79 lL, 0.36 mmol). After 30 min, pre-washed
Amberlite IR 120 resin (H+ form) (1.45 g) was added and stirred for
2 min. The resin was removed by filtration and the filtrate was con-
centrated under reducedpressuretogive 2-azidoethyl
a-D-mannopy-
3.7. 2,20-Bissuccinoyl-200-(benzyloxycarbonyl)tris(2-
aminoethyl)amine (10)
ranoside 4 as a colorless gum (0.283 g).
A portion of the above product (0.142 g, 0.57 mmol) was dis-
solved in water (10 mL) followed by the addition of Pd/C
(20.0 mg, 5% Pd on charcoal). The mixture was stirred under an
atmosphere of hydrogen for 6 h at room temperature. The products
were filtered through a bed of Celite. The filtrate was concentrated
under reduced pressure to give the title compound as a colorless
gum (0.109 g, 66.9% over two steps).
2-(Benzyloxycarbonyl)tris(2-aminoethyl)amine 9 (1.88 g, 6.71
mmol) was co-evaporated with dry toluene (2 ꢀ 10 mL) and then
dissolved in dry pyridine (30 mL). Succinic anhydride (1.47 g,
14.7 mmol) was added at room temperature. After 40 min, the
products were concentrated under reduced pressure. The residue
was dissolved in methanol (5 mL) and added dropwise to diethyl
ether (300 mL). The product was collected by filtration as a white
amorphous solid (2.71 g, 84.1%).
FAB-MS found [M+H]+ = 224.11771, C8H18NO6 requires 224.
þ
11341.

2142
Y. Zhao et al. / Carbohydrate Research 344 (2009) 2137–2143
ESI-MS found [MꢁH]ꢁ = 479.2, C22H31N4O8 requires 479.2.
dC[D2O]: 31.2 (CH2), 31.3 (CH2), 37.2 (CH2), 39.2 (C-6), 52.62
(CH2), 61.2 (CH2), 66.1 (CH2), 67.0 (C-4), 70.3 (C-2), 70.8 (C-3),
73.1 (C-5), 99.9 (C-1), 175.0 (C@O).
ꢁ
dH[CDCl3]: 2.37 (4H, t, J = 6.0, CH2), 2.41 (4H, t, J = 6.2, CH2), 3.26
(6H, m), 3.39 (5H, m), 5.01 (2H, –CH2Ph), 7.25–7.40 (5H, aromatic).
Rf: 0.48 (chloroform–methanol–water, 65:35:5 v/v).
Rf: 0.20 (methanol–ammonium hydroxide, 4:1 v/v).
3.8. Trifluoroacetate salt of 2-aminoethyl 2,3,4,6-tetra-O-acetyl-
3.11. Squarate-activated bivalent-mannoside (13)
a
-
D
-mannopyranoside (7)
Bivalent mannoside 12 (17.9 mg, 23.7
distilled water (100 L), followed by the addition of methanol
(900 L), 3,4-diethoxy-3-cyclobutene-1,2-dione (21 L, 142
mol), and triethylamine (3 L). After 7 min, the solvents were
lmol) was dissolved in
2-Azidoethyl 2,3,4,6-tetra-O-acetyl-
a-D
-mannopyranoside
3
l
(600.00 mg, 1.44 mmol) was dissolved in methanol (25 mL) fol-
lowed by the addition of Pd/C (42.0 mg, 5% Pd on charcoal) and tri-
l
1
l
l
l
fluoroacetic acid (200
l
L, 2.65 mmol). The reaction mixture was
quickly removed under reduced pressure. The residue was diluted
with distilled water (1.0 mL) and purified by size exclusion chro-
matography on Bio-Gel P2 resin (fine, 1.6 ꢀ 75 cm), eluted with
milliQ water. The appropriate fractions were combined and
freeze-dried to give the title compound as a light yellow amorphous
solid (11.6 mg, 55.6%).
stirred under an atmosphere of hydrogen for 5 h at room temper-
ature. The products were filtered through a bed of Celite. The fil-
trate was concentrated under reduced pressure and the product
was used for the next reaction without further purification.
FAB-MS found [M+H]+ = 881.38400, C36H61O19N6 requires
þ
3.9. Fully protected bivalent-mannoside (11)
881.39915.
2,20-Bissuccinoyl-200-(benzyloxycarbonyl)tris(2-aminoethyl)amine
10 (0.230 g, 0.48 mmol) was dissolved in dry dichloromethane
(10 mL). Then (benzotriazol-1-yloxy)tris(dimethylamino)phos-
phonium hexafluorophosphate (0.424 g, 0.96 mmol) followed by
Hünig’s base (0.75 mL, 4.31 mmol) was added at room temperature.
After 20 min, a solution of the trifluoroacetate salt of 2-aminoethyl
dH[D2O, recorded at 5 °C]: 1.07 (t, J = 7.2, –OCH2CH3), 1.08 (7,
J = 7.2, –OCH2CH3) (these two signals integrate 3H), 2.16–2.18
(8H, m, br, CH2), 2.36 (4H, s, br, CH2), 2.45 (2H, s. br, CH2), 2.94
(4H, s, br, CH2), 2.99–3.02 (2H, m, H-6), 3.09–3.12 (2H, m, H-60),
3.19–3.30 (6H, m, H-4, H-5, and CH2), 3.35–3.45 (6H, m, H-3 and
CH2), 3.51 (2H, d, J = 12.0, CH2), 3.57 (2H, s, br, H-2), 4.35 (q,
J = 7.1, –OCH2CH3), 4.39 (q, J = 7.1, –OCH2CH3) (these two signals
integrate 2H), 4.49 (2H, s, H-1).
2,3,4,6-tetra-O-acetyl-a-D-mannoside 7 (1.44 mmol) in dry dichlo-
romethane (5 mL) was added. The reaction was allowed to proceed
at room temperature for 12 h. The products were then concentrated
under reduced pressure. The residue was purified by column chro-
matography on silica gel. The appropriate fractions, which were
eluted with dichloromethane–methanol (95:5 v/v) were combined
and evaporated under reduced pressure to give the title compound
as a white glass (309.0 mg, 52.5%).
Rf: 0.45 (methanol–ammonium hydroxide, 4:1 v/v).
3.12. General procedures for the synthesis of 20-O-[1-(4-chloro-
phenyl)-4-ethoxypiperidin-4-yl]-50-O-(4,40-dimethoxytrityl)-
ribonucleoside-30-(2-O-cyanoethyl) N,N-diethylphosphorami-
dites
FAB-MS found [M+H]+ = 1227.60795, C54H79O26N6 requires
1227.50441.
dH[CDCl3] include the following signals: 2.01 (CH3), 2.07 (CH3),
2.12 (CH3), 2.17 (CH3), 4.00 (2H, br, H-5Man), 4.12 (2H, dd, J = 2.0
and 12.0, H-6Man), 4.29 (2H, dd, J = 5.0 and 12.0, H-60Man), 4.82
(2H, s, H-1Man), 5.10 (2H, s, –CH2Ph), 5.28–5.31 (6H, br, m, H-2,
H-3, H-4Man), 7.36 (5H, aromatic).
20-O-[1-(4-Chlorophenyl)-4-ethoxypiperidin-4-yl]-50-O-(4,40-
dimethoxytrityl) nucleoside24 (1.28 mmol) was co-evaporated
with dry toluene (2 ꢀ 5 mL). The residue was redissolved in dry
acetonitrile (15.0 mL) followed by the addition of N,N-diisopropyl-
ethylamine (6.32 mmol). After 5 min, N,N-diethyl-2-O-cyanoethyl
phosphorochloridite (2.54 mmol) was added. After a further period
of 30 min, the products were evaporated under reduced pressure.
The residue was purified by column chromatography on silica gel
to give the phosphoramidite as a colorless glass (80–85%).
Rf: 0.51 (dichloromethane–methanol, 90:10 v/v).
3.10. Fully deprotected bivalent-mannoside (12)
Fully protected bivalent-mannoside 11 (0.194 g, 0.158 mmol) was
3.13. Solid phase synthesis of oligoribonucleotides
dissolved in methanol (3.0 mL) followed by the addition of a solution
of sodium methoxide in methanol (25 wt %, 9
lL, 0.042 mmol). After
Solid phase synthesis of oligoribonucleotides was performed on
1 h, pre-washed Amberlite IR 120 resin (H+ form, 300 mg) was added
and stirred for 5 min. The resin was removed by filtration and the
filtrate was concentrated under reduced pressure. The residue was
redissolved in methanol (15.0 mL) followed by the addition of Pd/C
(30.0 mg, 5% Pd on charcoal). The reaction mixture was stirred in
an atmosphere of hydrogen at room temperature for 3 h. The prod-
ucts were filtered through a bed of Celite. The filtrate was concen-
trated under reduced pressure and the residue was purified by size
exclusion chromatography on Bio-Gel P2 gel (fine, 1.6 ꢀ 75 cm),
eluted with aqueous ammonium bicarbonate buffer (30 mM). The
appropriate fractions were combined and freeze-dried to give
the title compound as a colorless glass (0.115 g, 96.2%).
ABI 3400 DNA synthesizer. Standard 1.0 lmol RNA cycle condi-
tions were used. After synthesis was complete, the resins were
incubated in concentrated aqueous ammonia (28%) at 55 °C for
12 h. The supernatant was subsequently lyophilized to give par-
tially protected (20-Cpep-protected) oligoribonucleotides.
3.14. Man-U9U (18)
Partially protected (all the uridines except the 30-terminal one
were protected by Cpep at the 20-OH, as indicated by the italic U
in Scheme 4) U10-mer bearing a C6-amino modifier at the 50-end
17b (0.75
(200 L), followed by the addition of an aqueous solution (70
of squarate-activated monomannoside 6 (3.75 mol, 5 mol equiv)
and triethylamine (3 L). The reaction mixture was incubated at
37 °C. After 2 h, additional aqueous solution of squarate-activated
monomannoside 6 (37 L, 2 mol equiv) was added to the reaction
lmol, 1 mol equiv) was first dissolved in methanol
FAB-MS found [M+H]+ = 757.40622, C30H58O16N6 requires
l
lL)
þ
757.38311.
l
dH[D2O]: 2.66 (8H, s, br, CH2), 2.80 (4H, s, br, CH2), 2.85 (2H, s,
br, CH2), 3.05 (2H, s, br, CH2), 3.41 (4H, s, br, CH2), 3.50–3.53 (2H,
m, H-6), 3.57–3.60 (2H, m, H-60), 3.71–3.75 (4H, m, CH2 and H-
5), 3.78 (2H, t, J = 9.6, H-4), 3.86–3.93 (6H, m, CH2 and H-3), 4.01
(2H, d, J = 12, CH2), 4.07 (2H, s, H-2), 4.99 (2H, s, H-1).
l
l
mixture. The reaction was allowed to proceed for another 3 h.
The reaction mixture was lyophilized and the residue was purified

Y. Zhao et al. / Carbohydrate Research 344 (2009) 2137–2143
2143
by semi-preparative C18 RP-HPLC. The appropriate fractions were
collected and lyophilized to give the purified Man-U9U 18 as a
of stock solution, 5
of stock solution, 5
l
L) and alkaline phosphatase (100 ꢀ dilution
lL) were added. The stock solutions of Rnase
white solid.
A were prepared by dissolving Rnase A (0.092 unit) in 0.1 M Tris
chloride buffer (pH 8.0, 1.0 mL) and bacterial alkaline phosphatase
was obtained by dissolving ca. 2 units of the enzyme in 1.0 mL of
the same buffer. The mixture was incubated at 37 °C. Aliquots
Rt (C18): 7.63 min.
ESI-MS found
5618.6.
ꢁ = 5619.4, C225H283Cl9N31O98P10 requires
ꢁ
M
(20
40, and 60 min. To the aliquots was added an aqueous solution
of aluminon (5 M, 20 L) and the mixtures were frozen in liquid
lL) were withdrawn at the following time, 0, 2, 5, 10, 15, 20,
3.15. Bi-Man-U9U (19)
l
l
Partially protected U10-mer bearing a C6-amino modifier at the
nitrogen immediately. All aliquots were analyzed with anion
exchange HPLC (DNAPac PA100).
50-end 17b (0.5
l
mol) was dissolved in methanol (300
lowed by the addition of an aqueous solution (73 L) of squa-
rate-activated bivalent-mannoside 13 (2 mol) and triethylamine
(5 L). The reaction mixture was incubated at 37 °C. Additional
portions of aqueous solution of squarate-activated bivalent-man-
noside 13 (37 L, 2 mol equiv) were added at 3 and 6 h, respec-
lL), fol-
l
l
4. Conclusion
l
In summary, an efficient chemistry that is suitable for the prep-
aration of carbohydrate–RNA conjugates by using the squarate
linker was demonstrated. This chemistry is currently used to pre-
pare carbohydrate-siRNA conjugates for bioavailability studies.
l
tively. After 9 h, the products were lyophilized to give a white
solid. The residue was purified by semi-preparative C18 RP-HPLC.
The appropriate fractions were collected and lyophilized to give
Bi-Man-U9U 19 as a white solid.
Acknowledgments
Rt (C18): 7.34 min.
ꢁ
ESI-MS found
6152.2
M
ꢁ = 6152.6, C247H322Cl9N36O108P10
,
requires
This work is supported by Research Corporation, Natural Sci-
ences and Engineering Research Council of Canada, and Canada
Foundation for Innovation. The authors thank Anne Noronha for
performing the melting studies, and Tim Jones and Razvan Simione-
scu for assistance with mass and NMR spectroscopy, respectively.
3.16. General procedures for the removal of Cpep from 18 and
19
Substrate (18 or 19) was dissolved in DMA (300
the addition of triethylammonium formate buffer (TEAF, 200
l
L) followed by
L,
References
l
1. Patil, S. D.; Rhodes, D. G.; Burgess, D. J. AAPS J. 2005, 7, E61–E77.
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added, followed by vortex and centrifugation. The organic layer
was discarded and the aqueous layer was further extracted with
chloroform (2 ꢀ 300
a light yellow gel. The residue was redissolved in water (30
lowed by the addition of n-butanol (500 L). After vortexing, the
lL). The aqueous layer was lyophilized to give
l
L), fol-
l
mixtures were frozen in liquid nitrogen and then centrifuged for
10 min. The butanol layer was discarded. The pellet was dissolved
in water (500
lL) and lyophilized to give fully deprotected conju-
gate (20 or 21).
Man-U10 (21): Rt (DNAPac PA100): 3.66 min. ESI-MS found
ꢁ = 3479.1, C108H139N22O89P10ꢁ, requires 3479.1.
M
M
Bi-Man-U10 (20): Rt (DNAPac PA100): 2.70 min. ESI-MS found
ꢁ = 4012.6, C130H178N27O99P10 requires 4012.7.
ꢁ
U10: ESI-MS found Mꢁ = 2998.4, C90H110N20O78P9 requires:
ꢁ
2998.7. Rt (DNAPac PA100): 2.79 min.
18. Zatsepin, T. S.; Oretskaya, T. S. Chem. Biodivers. 2004, 1, 1401–1417.
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A10: ESI-MS found Mꢁ = 3229.2, C100H120N50O58P9 requires:
3229.1. Rt (DNAPac PA100): 2.95 min.
ꢁ
22. Schmidt,
R.
R.;
Jung,
K.-H.
Oligosaccharide
Synthesis
with
3.17. Enzymatic stability of U10, Man-U10, and Bi-Man-U10
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Ed.; Marcel Dekker Inc.: New York, 1997; pp 283–312.
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Chem. Soc., Perkin. 1 2000, 165–176.
0.183 ODU of substrate (unmodified U10-mer, monomanosyl-
U10-mer 21, and divalent mannosyl-U10-mer 20) was dissolved
25. Mori, M.; Ito, Y.; Ogawa, T. Carbohydr. Res. 1990, 195, 199–224.
in 200
l
L of Tris chloride buffer (pH 8.0). Rnase A (100 ꢀ dilution