Hetero-Click Conjugation of Oligonucleotides with Glycosides Using Bifunctional Phosphoramidites

Article abstract of DOI:10.1002/ejoc.201500165

Two phosphoramidite derivatives, 1 and 2, each bearing two orthogonal functions: alkyne/thioacetyl or alkyne/tosyl, respectively, were synthesized and used to generate heteroglyco 5′-oligonucleotide conjugates. After coupling, the first conjugation was pe

Full text of DOI:10.1002/ejoc.201500165

FULL PAPER
DOI: 10.1002/ejoc.201500165
Hetero-Click Conjugation of Oligonucleotides with Glycosides Using
Bifunctional Phosphoramidites
Albert Meyer,[a] Mathieu Noël,[a] Jean-Jacques Vasseur,[a] and François Morvan*[a]
Keywords: Carbohydrates / Oligonucleotides / Glycoclusters / Glycoconjugates / Click chemistry
Two phosphoramidite derivatives, 1 and 2, each bearing two
orthogonal functions: alkyne/thioacetyl or alkyne/tosyl,
respectively, were synthesized and used to generate hetero-
glyco 5Ј-oligonucleotide conjugates. After coupling, the first
conjugation was performed using CuAAC on solid support
with an azide-bearing carbohydrate. For 1, the second conju-
gation was performed in solution by a thiol “click” reaction
using a bromoacetamide galactoside, whereas for 2, the tosyl
group was converted into an azide and a second CuAAC was
applied on solid support. Accordingly, a deoxycholic-cen-
tered heteroglycocluster exhibiting two
-fucose motifs, conjugated to an oligonucleotide, was suc-
cessfully synthesized.
D-galactose and two
L
Introduction
With these possibilities in mind, one option for hetero-
glycocluster synthesis has involved a sequential process
wherein building blocks bearing the “click” reactive func-
tion are installed followed by the click reaction itself and
this order of steps repeated until the desired heterogly-
cocluster is completed.[15,23,25] Another approach requires
the combination of two orthogonal click reactions with the
introduction of two distinct building blocks, each bearing
an orthogonal functionality.[20,22,26,28–31]
As an alternative to these strategies, we report herein the
use of two variants of a single phosphoramidite building
block exhibiting two orthogonal functions that selectively
react with two different, suitably functionalized carbo-
hydrates. The main advantage of this novel strategy is that
the synthesis of a single intermediate is sufficient to gener-
Multivalent recognition between lectins and glycans oc-
curs in several biological processes such as those dealing
with cell–cell communication, innate immune response sys-
tems and pathogen infections.[1–3] Several synthetic multi-
valent glycoconjugates have been reported[4–7] in efforts to
compete with natural carbohydrate ligands and to control
or modulate these interactions; potential therapeutic appli-
cations are likely to result.[8,9] The resulting neoglycans are
expected to display significantly enhanced binding to their
targets relative to the sum of interactions of the constitutive
monomers, thanks to the so-called “cluster glycoside ef-
fect”.[10,11] Thus far, most synthetic glycoclusters have been
synthesized as homoglycoclusters. However, natural oligo-
saccharides consist of a variety of carbohydrates and use
this heterogeneity to achieve affinity and selectivity for spe-
cific lectins. It has been shown that the presence of non-
recognized carbohydrates increases the binding affinity of
those that are recognized.[12–15]
Despite earlier work, the synthesis of heteroglycoclusters
remains a challenging task; Fernandez et al. have recently
reviewed this area.[16] Early work relayed the use of a build-
ing block containing an orthogonal protecting group com-
bined with protected carbohydrate phosphoramidites to
generate heteroglycoclusters.[17] More recently, the synthesis
of heteroglycoclusters has employed “click chemistry” reac-
tions like Cu(I)-catalyzed azide–alkyne cycloaddition
(CuAAc),[18–23] oxime formation,[24–26] and “click thiol”
reactions[27] (thiol-ene,[13,14,18] thiol Michael-type ad-
dition,[28] and thiol–chloroacetyl coupling[26,29]).
[a] Institut des Biomolécules Max Mousseron UMR 5247 CNRS-
UM-ENSCM, Université de Montpellier,
Place E. Bataillon, CC1704, 34095 Montpellier cedex 5, France
E-mail: morvan@univ-montp2.fr
Supporting information for this article is available on the
WWW under http://dx.doi.org/10.1002/ejoc.201500165.
Scheme 1. Synthesis of bi-functional phosphoramidites 1 and 2.
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A. Meyer, M. Noël, J.-J. Vasseur, F. Morvan
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ate the desired heteroglycoclusters. In addition, the bifunc-
tional building blocks described here can be introduced sev-
eral times within a scaffold containing multiple hydroxy
Results and Discussion
Phosphoramidites 1 and 2 were synthesized in two steps
groups by using very efficient phosphoramidite chemistry. starting from propargyl-triethylene glycol 3 which was con-
Accordingly, two new phosphoramidites 1 and 2 were syn- verted into its phosphorodiamidite 4 by reaction with
thesized and evaluated (Scheme 1). The first building block bis(diisopropylamino)chlorophosphine in the presence of
(1) contains one alkyne function and one S-acetyl function, Et3N (Scheme 1). Compound 4 was then activated by diiso-
for subsequent CuAAC reactions and thiol-bromoacetyl propylammonium tetrazolide salt and reacted with either
coupling (TBC) chemistry, respectively. The second building S-acetyl triethylene glycol 5 or tosyl-triethylene glycol 6
block (2) displays one alkyne moiety and one tosyl func- leading to expected phosphoramidites 1 and 2, which were
tion; the latter is used as a proazide function group. Hence, purified by silica gel chromatography and obtained in good
after a first CuAAC reaction, the conversion of the tosyl yields (67% and 77% respectively).
into azide is performed followed by a second CuAAC reac-
The coupling efficiency of each phosphoramidite was
tion. Notably, the synthetic flexibilities embodied by 1 and evaluated on solid-supported oligonucleotide 7 synthesized
2 enable easy access to complex multivalent heteroglycocon- by standard phosphoramidite chemistry (Scheme 2). After
jugates.
synthesis of the oligonucleotide sequence, phosphoramid-
Scheme 2. Synthesis of oligonucleotide heteroglycoclusters by using a combination of CuAAC and bromoacetamide thiolation reactions
(8–16, left panel), or by using two sequential CuAAC reactions (9–19, right panel). SPOS: solid phase oligonucleotide synthesis, TCEP:
tris(2-carboxyethyl)phosphine, DBU: 1,8-diazabicycloundec-7-ene. * Represents the protecting group on the nucleobases: benzoyl for A
and C and isobutyryl for G.
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Hetero-Click Conjugation of Oligonucleotides with Glycosides
Figure 1. HPLC profiles of crude 14 [disulfide form (left)], 14 after TCEP addition, thiol form (middle) and MALDI-TOF MS with
TCEP, [M – H]– calcd. 3638.62 (right).
Figure 2. HPLC profiles of 16 crude (left), 16 pure (middle) and MALDI-TOF MS, [M – H]– calcd: 3949.92 (right).
ites 1 or 2 were introduced at the 5Ј-end of 7, using a cou- cided with formation of a new peak at 14.03 min corre-
pling time of 80 s to ensure completion of the coupling re- sponding to the thiol form.
action followed by a regular oxidation step leading to solid-
Finally, the galactose residue was introduced by thio-
supported, 5Ј-modified oligonucleotides 8 and 9, respec- lation of bromoacetamide 15 in the presence of Et3N and
tively. The first conjugation was performed, on solid sup- TCEP. TCEP was required to reduce the disulfide bridge of
port, by CuAAc using 3-azidopropyl 2,3,4-tri-O-acetyl-α,l- dimerized 14. Thiol condensation with 15 afforded hetero-
fucoside 10 for 8 and 3-azidopropyl 2,3,4,6-tetra-O-acetyl- glyco-oligonucleotide conjugate 16 bearing both d-galact-
β-d-galactoside 11 for 9, both in the presence of CuSO4 and ose and l-fucose motifs, as confirmed by HPLC and MS.
sodium ascorbate for 1 h at 60 °C with microwave assist- Notably, the acetyl groups of the galactose moiety were hy-
ance,[32] leading to monoconjugates 12 and 13, respectively. drolyzed during oligonucleotide conjugation (Figure 2).
Fucosylated oligonucleotide 12, bearing a thioacetyl
In the orthogonal pathway involving building block 2
function, was first treated on-support with a 1 m 1,8-diaza- (Scheme 2, right panel), the solid-supported tetra-O-acetyl-
bicycloundec-7-ene (DBU) solution in dry CH3CN for galactosylated oligonucleotide 13, bearing a tosyl function,
2 min to eliminate the phosphate-protecting cyanoethyl was treated with tetramethylguanidinium azide (TMGN3)
groups. The acrylonitrile generated during this reaction was in CH3CN for 1 h at 60 °C to generate corresponding solid-
removed by further washing of the solid support with dry supported azide oligonucleotide 17 (Scheme 2). The second
CH3CN. Indeed, acrylonitrile reacts readily with the free CuAAC was then performed, still on the solid support,
thiol function. Consequently, it was necessary to completely using propargyl 2,3,4-tri-O-acetyl-α,l-fucoside 18, CuSO4
remove residual acrylonitrile prior to thiol liberation via and sodium ascorbate. Finally, treatment with NH4OH led
deacetylation. Subjection of thioacetylated material to
NH4OH liberated the terminal thiol and effected oligonu-
cleotide release from the solid support; product 14 was ana-
lyzed and characterized by HPLC and MALDI-TOF MS
(Figure 1).
The formation of a disulfide bridge between two thiol-
modified oligonucleotides 14 was demonstrated by treat-
ment of a small amount of the crude product with tris(2-
carboxyethyl)phosphine (TCEP) and subsequent HPLC
analysis. The main peak at 14.84 min (representing dimer-
Figure 3. HPLC profiles of 19 crude (left), pure (middle) and
ized 14) underwent significant reduction in size which coin- MALDI-TOF MS, [M – H]– calcd: 3865.77 (right).
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A. Meyer, M. Noël, J.-J. Vasseur, F. Morvan
FULL PAPER
to heteroglyco-oligonucleotide conjugate 19 which was ana- either on solid support or in solution, making the approach
lyzed and purified by HPLC and ultimately characterized more versatile.
by MALDI-TOF MS (Figure 3).
As noted before, phosphoramidites 1 or 2 could be intro-
We have demonstrated that both phosphoramidites 1 and duced several times using a polyhydoxylated platform. Since
2 enable the efficient synthesis of bivalent heteroglyco-oli- the binding affinity of a carbohydrate to a lectin is generally
gonucleotide conjugates bearing both d-galactose and l-fu- low, multiple glycosylations of a scaffold usually increase
cose residues. Both reagents have certain advantages and the affinity of the final glycocluster in accord with the clus-
disadvantages. An advantage of 1 over 2 is that it does not ter glycoside effect.[11] Accordingly, we decided to introduce
require an extra step to generate the second click function- phosphoramidite 1 twice into a single deoxycholyl scaffold.
ality (tosylǞazide), as the thiol function is generated Indeed, deoxycholic acid can be easily derivatized through
simultaneously with the deprotection of the oligonucleotide, the carboxylic acid moiety and the OH moieties can be used
making this approach synthetically shorter. The advantage to anchor any tag using phosphoramidite chemistry. Since
of 2 over 1 is that the second CuAAC reaction can occur both hydroxyl groups are on the same face of the deoxy-
Scheme 3. Synthesis of N-(propargyl)deoxycholanamide 20.
Scheme 4. Synthesis of deoxycholyl-centered tetravalent heteroglycocluster oligonucleotide conjugate exhibiting two d-galactoside and
two l-fucoside motifs. SPOS: solid-phase oligonucleotide synthesis, TCEP: tris(2-carboxyethyl)phosphine, DBU: 1,8-diazabicycloundec-
7-ene.
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Hetero-Click Conjugation of Oligonucleotides with Glycosides
cholyl platform, carbohydrate motifs will likely be directed sponding building blocks bearing suitable and complemen-
in the same direction. To this end, deoxycholic acid was tary functions (i.e. azide, alkyne or bromoacetamide).
activated as its N-hydroxysuccinimide ester and then re-
acted with propargylamine affording desired N-(propargyl)-
deoxycholanamide[33] 20 (Scheme 3).
Experimental Section
Alkyne 20 was then immobilized onto azide solid sup-
port 21 by CuAAC affording 22 (Scheme 4). Phosphor-
amidite 1 was introduced using a double coupling step with
an extended time of 360 s to form bis-phosphorylated de-
O-(3,6,9-Trioxadodecan-11-yl)-N,NЈ-tetraisopropylphosphorodi-
amidite (4): To a solution of 3,6,9-trioxadodecan-11-yn-1-ol[34]
3
(1.55 g, 8.25 mmol) and Et3N (2.3 mL, 16.5 mmol) in dry Et2O
(45 mL) was added bis(diisopropylamino)chlorophosphine (2.64 g,
oxycholic scaffold 23, bearing the two bis-orthogonally 9.9 mmol) and the mixture stirred for 3 h at room temperature.
Then, the solution was diluted with Et2O and Et3N (9:1, v/v,
40 mL), and the salts were removed by filtration and washed. The
solution was evaporated to half and cyclohexane (20 mL) was
added. Et2O was removed by evaporation keeping cyclohexane in
solution. The solution was applied to a silica gel column (50 g) and
the compound was purified using cyclohexane containing 6% Et3N
(2.52, 73%). TLC: Rf = 0.70 in cyclohexane/AcOEt/Et3N, 6:3:1,
functionalized tethers, as well as a 4,4-dimethoxytrityl
(DMTr) protected hydroxy anchor. Following removal of
the DMTr group, the full sequence oligonucleotide was syn-
thesized by the standard protocol, affording 24. Notably,
the four click-functionalized conjugation tethers, already
present on solid support 23, were not affected by the 15
synthetic cycles needed for oligonucleotide construction.
Next, azido-fucoside derivative 10 was introduced twice by
CuAAC onto solid-supported 24, as described above, and
1
v/v/v. H NMR (300 MHz, CDCl3): δ = 1.19 (dd, J = 4.0, 6.7 Hz,
12 H, isopropyl), 2.46 (t, J = 2.2 Hz, 1 H, HCC-), 3.49–3.78 (m,
16 H, -CH2-O-P, -CH-, -O-CH2-CH2-O-), 4.24 (d, J = 2.4 Hz, 2 H,
the modified oligonucleotide was deprotected (first with CC-CH2O) ppm. 13C NMR (75 MHz, CDCl3): δ = 22.9, 23.0, 23.6,
23.7, 24.6, 24.7, [C(CH3)2], 44.2, 44.3, 45.1, 45.2, 45.3 (NCMe2),
58.4 (CC-CH2O), 62.4, 62.5 (POCH2), 69.1, 70.5, 70.6, 70.7
(CH2CH2OCH2CH2), 74.5 (CCH), 79.6 (CCH) ppm. 31P NMR
(121 MHz, CDCl3): δ = 127.1 ppm. HRMS (ESI/Q-TOF) m/z
calcd. for C21H44N2O4P [M + H]+ 419.3039, found 419.3052.
DBU and then with ammonia) affording bis-fucosylated
oligonucleotide conjugate 25 in solution. Generation of 25
was confirmed by MALDI-TOF MS analysis (data not
shown). Subsequently, bromoacetamide-containing galacto-
side 15 was introduced twice by TBC chemistry (as de-
scribed above and in the presence of TCEP) affording oligo-
nucleotide heteroglycocluster 26 with four pendant sugars,
two fucose and two galactose motifs. Final conjugate 26
was purified by HPLC and characterized by MS (Figure 4).
O-(3,6-Dioxanon-8-ethylthioacetyl)-OЈ-(3,6,9-trioxadodecan-11-
ynyl)-N,N-diisopropyl Phosphoroamidite (1): Dry 3,6-dioxanon-8-
ethylthioacetyl-1-ol[35] 5 (750 mg, 3.6 mmol) and dry diisoprop-
ylammonium tetrazolide (308 mg, 1.8 mmol) were dissolved in dry
CH2Cl2 (12 mL) and O-(3,6,9-trioxadodecan-11-yl)-tetraisopropyl
phosphorodiamidite 4 (1.26 g, 3 mmol) was added. After 3 h of
stirring at room temperature, the solution was diluted with CH2Cl2
and washed with brine (2ϫ 100 mL). The organic layer was dried
with Na2SO4 and the solvents evaporated. The crude oil was puri-
fied by chromatography (gradient 0 to 50% AcOEt in cyclohexane
containing 6% Et3N) to afford a clear oil (1.06 g, 67%). TLC: Rf =
0.45 in cyclohexane/AcOEt/Et3N, 5:4:1; v/v/v. 1H NMR (300 MHz,
CDCl3): δ = 1.1 (d, J = 6.8 Hz, 12 H, isopropyl), 2.27 (s, 3 H,
CH3CO-S), 2.36 (t, J = 2.4 Hz, 1 H, -CCH), 3.02 (t, J = 6.4 Hz, 2
H, -CH2S-), 3.48–3.8 (m, 24 H, -CH-, CH2-OP, -CH2-CH2-, -O-
CH2-), 4.14 (d, J = 2.4 Hz, 2 H, HCC-CH2-) ppm. 13C NMR
(75 MHz, CDCl3): δ = 24.5, 24.6 [C(CH3)2], 28.8 (CH3CO), 30.5
(CH2S), 42.7, 42.9 (NCMe2), 58.4, (CC-CH2O), 62.4, 62.6,
(POCH2), 69.1, 69.7, 70.3, 70.4, 70.5, 70.6, 71.2, 71.2, 71.3, 71.5,
(CH2 CH2 OCH2 CH2 ); 74.6, (CCH), 79.6 (CCH), 195.5
Figure 4. HPLC profiles of 26 crude (left), pure (middle) and
MALDI-TOF MS [M – H]– calcd. 7169.73 (right).
Conclusions
In summary, we have synthesized two new phosphor- (C=O) ppm. 31P NMR (121 MHz, CDCl3): δ = 147.6 ppm. HRMS
(ESI/Q-TOF) m/z calcd. for C23H45NO8PS [M + H]+ 526.2604
amidite building blocks, each bearing two linkers with or-
thogonal functions allowing the synthesis of heteroglyco-
found 526.2610.
oligonucleotide 5Ј-conjugates. These phosphoramidites can
O-(3,6,9-Trioxadodecan-10-p-tosylate)-OЈ-(3,6,9-trioxadodecan-
be introduced once or multiple times into a synthetic oligo- 11-ynyl)-N,N-diisopropyl Phosphoroamidite (2): Dry 3,6,9-trioxado-
decan-10-p-tosylate-1-ol[35] 6 (1.1 g, 3.6 mmol) and dry diisoprop-
ylammonium tetrazolide (308 mg, 1.8 mmol) were dissolved in dry
CH2Cl2 (12 mL) and 3,6,9-trioxadodecan-11-yl tetraisopropyl
phosphorodiamidite 4 (1.26 g, 3 mmol) was added. After 3 h of
stirring at room temperature, the solution was diluted with CH2Cl2,
washed with brine (2ϫ 100 mL). The organic layer was dried with
Na2SO4 and the solvents evaporated. The crude oil was purified by
chromatography (gradient 0 to 50% AcOEt in cyclohexane con-
nucleotide. This was demonstrated with the synthesis of het-
eroglycocluster oligonucleotide conjugates exhibiting both
d-galactose and l-fucose moieties in a monovalent or multi-
valent fashion. We believe that these bis-functional phos-
phoramidites 1 and 2 are not limited to the synthesis of
heteroglycan-oligonucleotide conjugates. Phosphoramidites
1 and 2 display wide versatility for the synthesis of different
monovalent or multivalent heteroconjugates of oligonucleo-
taining 6% Et3N) to afford a clear oil (1.44 g, 77%). TLC: Rf =
0.50 in cyclohexane/AcOEt/Et3N, 5:4:1 v/v/v. 1H NMR (400 MHz,
tides carrying, for example, various motifs such as fluores-
cent dyes, peptides, carbohydrates or biotin, using the corre- CDCl3): δ = 1.13 (dd, J = 2.2, 6.8 Hz, 12 H, isopropyl), 2.41 (br.
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A. Meyer, M. Noël, J.-J. Vasseur, F. Morvan
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s, 4 H, HCC, CH3), 3.52–3.79 (m, 24 H, -CH-, CH2-OP, -CH2- Thiol-Bromoacetyl Coupling (TBC): To a solution of crude 14 in
CH2-, -O-CH2-), 4.11–4.17 (m, 4 H, Ts-O-CH2-, HCC-CH2-), 7.32 H2O (≈ 125 nmol, 200 μL) was added galactoside bromoacetamide
(d, J = 8.1 Hz, 2 H), 7.76 (d, J = 8.2 Hz, 2 H) ppm. 13C NMR
(100 MHz, CDCl3): δ = 21.6 (CH3Ar), 24.6, 24.7 [C(CH3)2], 42.8,
derivative 15[38] (10 μL of 100 mm in MeOH, 1.0 μmol, 8 equiv.),
TCEP (25 μL of 100 mm in H2O, 2.5 μmol, 20 equiv.), and Et3N
42.9 (NCMe2), 58.4 (CC-CH2O), 62.4, 62.6 (POCH2), 68.7, 69.1, (200 μL, 1.5 μmol, 12 equiv.). The vial with the biphasic solution
69.3, 70.4, 70.6, 70.7, 70.8, 71.2, 71.3, 71.4, (CH2CH2OCH2CH2, was sealed and heated for 1 h at 60 °C with stirring. Et3N was then
CH2S) 74.6 (CCH), 79.7 (CCH), 128.0, 129.8, 133.1, 144.8 evaporated and the mixture was pre-purified by size exclusion
(Ar) ppm. 31P NMR (162 MHz, CDCl3): δ = 147.5 ppm. HRMS chromatography (NAP 10) and purified by C18 HPLC affording 16
(ESI/Q-TOF) m/z calcd. for C28H51NO11PS [M + H2O + H]+ (49 nmol).
640.2921 found 640.2920.
Synthesis of Heteroglycocluster Oligonucleotide 19
Deoxycholic Solid Support 22: To 1-(6-azidohexyl)-2-[(4,4Ј-dimeth-
CuAAC Coupling: Similar protocol than above using
9
(≈ 0.50 μmol) and azidopropyl 2,3,4,6-tetra-O-acetyl galactoside[39]
11 (8 equiv.).
oxytrityl)oxymethyl]-2-methyl-3-(succinic-LCAA CPG)propane-
1,3-diol[36] 21 (70 mg, 3 μmol) were added N-(propargyl)deoxychol-
anamide 20[33] (54 μL of 100 mm solution in MeOH, 5.4 μmol), a
mixture of H2O/MeOH (1.2 mL, 1:1, v/v), freshly prepared aque-
ous solutions of CuSO4 (30 μL, 40 mm, 1.2 μmol) and sodium as-
corbate (60 μL, 100 mm, 6 μmol). The vial containing the resulting
mixture was sealed and gently stirred for 1 h at 60 °C. Then, the
CPG beads were filtered off and washed with H2O (5 mL), MeOH
(5 mL), CH3CN (5 mL) and dried.
Azidation: Beads were treated with a solution of 0.3 m tetrameth-
ylguanidinium azide (TMG N3) in CH3CN (300 μL) for 1 h at
60 °C. Beads were then washed 5 times with CH3CN (2 mL) and
dried affording 17.
CuAAC Coupling: To compound 17 (≈ 0.5 μmol) was added prop-
argyl 2,3,4-tri-O-acetyl α-l-fucoside[37] 18 (40 μL of 100 mm in
MeOH, 4 μmol, 8 equiv.), freshly prepared CuSO4 (12 μL of 40 mm
in H2O, 1 equiv.), sodium ascorbate (25 μL of 100 mm in H2O,
5 equiv.), degassed H2O 150 μL and MeOH (150 μL). The tube
containing the resulting mixture was sealed and placed in a micro-
wave synthesizer at 60 °C for 60 min. The beads were filtered,
washed with water (1 mL), 0.1 m EDTA (1 mL), H2O (1 mL),
MeOH (1 mL), CH3CN (1 mL) and dried.
General Procedure for Solid-Phase Oligonucleotide Synthesis
(SPOS): The DNA sequences were synthesized starting from com-
mercially available thymidine solid support yielding 7 or from the
solid-supported deoxycholic scaffold with two thioacetylEG3/pro-
pargylEG3 phosphotriester moieties 23 yielding 24 at a 1 μmol scale
on a DNA synthesizer (ABI 394) by standard phosphoramidite
chemistry. For the coupling step, benzylmercaptotetrazole (BMT)
was used as activator (0.3 m in anhydrous CH3CN), commercially
available nucleosides phosphoramidites (0.075 m in anhydrous
CH3CN, 15 equiv.) were introduced with a 20 s coupling time. The
capping step was performed with acetic anhydride using commer-
cial solution (Cap A: Ac2O/pyridine/THF, 10:10:80 v/v/v and Cap
B: 10% N-methylimidazole in THF) for 15 s. Oxidation was per-
formed for 15 s using 0.1 m I2, THF/pyridine/H2O, 90:5:5. Detrityl-
ation was performed with 2.5% trichloroacetic acid (TCA) in
CH2Cl2 for 35 s.
Beads were heated with concentrated NH4OH 55 °C for 2 h afford-
ing 19 after evaporation which was purified by C18 HPLC
(220 nmol).
Synthesis of Heteroglycocluster Oligonucleotide 26: To 0.25 μmol of
oligonucleotide on solid-supported deoxycholic 24 was added fuco-
syl azide derivative 10 (100 mm, in MeOH, 20 μL, 4 equiv./site),
freshly prepared aqueous solutions of CuSO4 (40 mm, 6 μL,
1 equiv.) and sodium ascorbate (100 mm, 12.5 μL), H2O (50 μL)
and MeOH (50 μL). The vial containing the resulting mixture was
sealed and stirred for 1 h at 60 °C. Then, the CPG beads were fil-
tered and washed with H2O (5 mL), MeOH (5 mL), CH3CN
(5 mL) and dried. Then, CPG beads were treated with a solution
of DBU 1 m (300 μL in 1.7 mL of CH3CN) for 2 min, washed 5
times with 2 mL of CH3CN. Finally, the beads were treated with
concentrated NH4OH (1 mL) for 5 h at 55 °C to give 25.
General Protocol for Phosphorylation with Phosphoramidites 1 and
2:
i) At 5Ј-end: Phosphoramidite 1 or 2 (0.15 m in dry CH3CN,
30 equiv.) was coupled onto solid-supported oligonucleotide 7,
using a DNA synthesizer, with BMT as activator for 80 s, and then
standard oxidative step was applied affording 8 or 9.
To bis-fucosylated deoxycholic oligonucleotide 25 (30 nmol in
100 μL H2O) was added galactoside bromoacetamide derivative 15
(100 mm in MeOH, 2.4 μL, 240 nmol), TCEP (100 mm, 6 μL,
600 nmol, 20 equiv.) and Et3N (50 μL). The resulting preparation
was sealed and stirred for 1 h at 60 °C. The mixture was desalted
on NAP 10 size-exclusion cartridge and the solvents evaporated.
The deacetylated glycocluster oligonucleotide was dissolved in H2O
and purified by reversed-phase HPLC to give 10 nmol 26 (33%).
ii) On Deoxycholic Scaffold 22: Phosphoramidite 1 (0.15 m in dry
CH3CN) was coupled twice (30 equiv./OH) using BMT as activator
for 360 s (2ϫ180 s), then standard oxidative step was applied and
the capping step was extended to 180 s affording 23.
Synthesis of Heteroglycocluster Oligonucleotide 16
CuAAC Coupling: To solid-supported alkyne oligonucleotide 8
(≈ 0.50 μmol) was added azido-functionalized galactoside[37] 10
(20 μL of 100 mm in MeOH, 2 μmol, 4 equiv.), freshly prepared
CuSO4 (6 μL of 40 mm in H2O, 0.5 equiv.), sodium ascorbate
(25 μL of 100 mm in H2O, 5 equiv.), degassed H2O (150 μL) and
MeOH (150 μL). The tube containing the resulting mixture was
sealed and placed in a microwave synthesizer at 60 °C for 60 min.
The beads were filtered, washed with H2O (1 mL), 0.1 m EDTA
(1 mL), H2O (1 mL), MeOH (1 mL), CH3CN (1 mL) and dried.
Supporting Information (see footnote on the first page of this arti-
cle): 1H, 13C and 31P NMR spectra of all new compounds. Synthe-
sis procedures for compounds 5, 6 and 20.
Acknowledgments
This work was financially supported by the Agence Nationale pour
la Recherche (grant number ANR-12-BSV5-0020). F. M. is a mem-
ber of Inserm.
Deprotection: Beads were treated with 1 m DBU in dry CH3CN
(2 mL) for 2 min and washed 5 times with dry CH3CN (2 mL).
Finally, concentrated ammonia was added and the sealed vial was
heated at 55 °C for 2 h affording 14 after evaporation.
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