Synthesis of monoconjugated and multiply conjugated oligonucleotides by "click thiol" thiol-Michael-type additions and by combination with CuAAC "click huisgen"

Article abstract of DOI:10.1002/ejoc.201201311

Monoconjugated and multiply conjugated oligonucleotides were efficiently synthesized by starting from mono-thiohexyl- or tetra-thiohexyl-oligonucleotides and treatment with acrylamide derivatives (carbohydrates, ferrocene, biotin, fluorescent dyes, deoxyc

Full text of DOI:10.1002/ejoc.201201311

FULL PAPER
DOI: 10.1002/ejoc.201201311
Synthesis of Monoconjugated and Multiply Conjugated Oligonucleotides by
“Click Thiol” Thiol-Michael-Type Additions and by Combination with CuAAC
“Click Huisgen”
Albert Meyer,[a] Jean-Jacques Vasseur,[a] and François Morvan*[a]
Keywords: Nucleic acids / Oligonucleotides / Carbohydrates / Click chemistry / Conjugation / Multiple conjugation
Monoconjugated and multiply conjugated oligonucleotides
were efficiently synthesized by starting from mono-
thiohexyl- or tetra-thiohexyl-oligonucleotides and treatment
with acrylamide derivatives (carbohydrates, ferrocene, bio-
tin, fluorescent dyes, deoxycholic acid). The thiol Michael-
type additions (TMTAs) occurred during the deprotection
and release of the oligonucleotides from the solid support, so
no additional time for conjugation was needed. Sequential
combination either of Copper-catalyzed Azide–Alkyne
Cycloaddition (CuAAC) on solid support and then TMTA or
of TMTA followed by CuAAC in solution allowed the synthe-
sis of several oligonucleotides decorated with two different
biomolecules.
tween thiol-modified oligonucleotides and maleimide deriv-
atives[8–10] or more recently between maleimide oligonucleo-
tides and thiol derivatives.[11] In contrast, these reactions
have not been widely applied to conjugation of oligonucleo-
tides with acrylamide derivatives.[12,13] However, acrylamide
derivatives are easily and rapidly obtained from virtually
any amine or carboxylic acid derivative under mild condi-
tions. A key advantage of acrylamide derivatives over male-
imide ones is their better stability in basic media.
Introduction
Hydrothiolation of alkenes occurs either by radical or
by nucleophilic mechanisms, corresponding either to thiol
radical additions to electron-rich enes (termed thiol-ene re-
actions) or to thiol Michael-type additions (TMTAs) to
electron-deficient enes, respectively. Both reactions are also
termed “click thiol” reactions. Thiol-ene reactions have
been applied for the synthesis of multiple structures, espe-
cially for polymer synthesis,[1–4] but also for the preparation
of glycoclusters.[5] The reactions usually proceed between
thiols and enes in the presence of radical initiators under
UV irradiation conditions. Although the reactions work
well, the conditions are not very convenient because specific
Results and Discussion
Here we report the application of TMTA to monocon-
equipment is required. In contrast, the use of activated enes jugation of a 5Ј-thiol oligonucleotide and different acryl-
bearing electron-withdrawing substituents (such as ester, amide derivatives (i.e., phenyl, mannose, ferrocene, dansyl,
amide or cyano) leads to TMTA under basic catalysis con- biotin or deoxycholic acid) and also for multiple conjuga-
ditions without the need for an activator or special equip- tion of a tetrathiol-based oligonucleotide and an acrylamide
ment.[3]
mannose derivative to afford a glycocluster. Finally, the or-
With regard to oligonucleotide applications, the litera- thogonality of TMTA with copper-catalyzed azide–alkyne
ture shows this strategy to be undeveloped, because reac- cycloaddition (CuAAC)[14,15] in application to oligonucleo-
tions under UV irradiation conditions can lead to photo- tides was studied. It was demonstrated that the two reac-
damage with the formation of a large variety of photoprod- tions could not be performed together, due to reaction be-
ucts, more specifically dimeric pyrimidine units.[6,7] TMTA tween thiol and copper, but could be performed sequen-
is therefore used, but is mostly reserved for reactions be- tially to allow the preparation of bis-conjugated oligonucleo-
tides (mannose-galactose, biotin-galactose, biotin-mannose
or dansyl-mannose).
The acrylamide derivatives 4a–f (Scheme 1) were synthe-
sized in a few steps with yields between 34 to 90%. Phen-
ethylamine was treated with acryloyl chloride in the pres-
ence of triethylamine to afford phenethylacrylamide (4a).
The 2,3,4,6-tetraacetyl-mannosyl-propyl-acrylamide 4b was
prepared from 3-azidopropyl 2,3,4,6-tetraacetylmannos-
ide,[16] which was firstly reduced to the amine by treatment
[a] Institut des Biomolécules Max Mousseron UMR 5247 CNRS-
UM1-UM2, Université Montpellier II,
Place E. Bataillon, CC1704, 34095 Montpellier cedex 5, France
Fax: +33-4-67042029
E-mail: morvan@univ-montp2.fr
Homepage: http://www.ibmm.univ-montp1.fr/
Oligonucleotides-Modifies,262.html
Supporting information for this article is available on the
WWW under http://dx.doi.org/10.1002/ejoc.201201311.
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with tris(2-carboxyethyl)phosphane (TCEP) and secondly, amine compound. After workup, the crude product was
after workup, further transformed by direct treatment of treated with acryloyl chloride to give the dansylacrylamide
the crude product with acryloyl chloride. The reaction be- derivative 4d. The biotin acrylamide derivative 4e was pre-
tween ferrocenaldehyde and N-acryloyl-ethylenediamine[17] pared as previously reported[17] by starting from biotin,
gave the Schiff base, which was reduced in situ with sodium which was converted into its N-hydroxysuccinimide deriva-
borohydride to afford the ferrocene acrylamide derivative tive. Treatment with N-acryloyl-ethylenediamine then gave
4c. Dansyl chloride was treated with 8 equiv. of ethylenedi- 4e. Finally, the same protocol was applied to deoxycholic
amine to form the corresponding monosulfonamide ethyl- acid, leading to deoxycholic acid acrylamide derivative 4f.
Scheme 1. Synthesis of acrylamide derivatives 4a–f.
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Conjugated Oligonucleotides by Click Reactions
The fully protected solid-supported 5Ј-S-acetylthiohexyl- meric form, due to rapid oxidation of the thiol function into
decanucleotide 2 (Scheme 2) was straightforwardly syn- the disulfide by air. Treatment with tris-(2-carboxylethyl)-
thesized from commercially available building blocks (nu- phosphane (TCEP) gave the expected 5Ј-thiol decamer with
cleosides and S-acetylthiohexyl phosphoramidites) by stan- good purity.
dard phosphoramidite chemistry[18]. After oligonucleotide
Our first attempted conjugation was performed with the
elongation, the cyanoethyl protecting groups of the phos- solid-supported thiohexyl phosphodiester decamer and N-
phates are removed by β-elimination during the ammonia phenethylacrylamide (15 equiv.) in methanol and aqueous
treatment, leading to the formation of acrylonitrile. Acrylo- ammonia (1:4, v/v) in the presence of TCEP at 55 °C for
nitrile reacts rapidly with thiols, so it is essential to remove 4 h and showed full conversion of the starting compound
it before the deprotection of the thiol function. For this into the expected conjugate. We envisioned that because
purpose, we applied a two-step deprotection procedure. both deprotection of the oligonucleotide and TMTA occur
Firstly, the solid-supported decamer was treated for 15 min in ammonia it should be possible to proceed by a deprotec-
with 10% piperidine/acetonitrile solution, allowing the β- tion/reaction cascade by adding all of the reagents together
elimination of cyanoethyl groups without the hydrolysis of (i.e., N-phenethylacrylamide, TCEP and ammonia) to the
the acetyl protecting group of the thiol. Secondly, after solid-supported thiohexyl phosphodiester decamer. By this
washing with acetonitrile to remove acrylonitrile, the solid- strategy, the expected conjugate was obtained easily with
supported phosphodiester decamer containing the S-acetyl- the same efficiency. It is noteworthy that the presence of
thiohexyl group was treated with ammonia to afford the 5Ј- TCEP was necessary, because oxidation to the disulfide was
thiohexyl decamer. After concentration, HPLC and mass more rapid here than TMTA.
spectrometry analyses showed it to exist mainly in its di-
When the reaction was performed with dansylacrylamide
derivative 4d we observed some instability in ammonia,
leading to some degradation of 4d. This result prompted
us to look for a milder basic treatment that would allow
deprotection and activation without degradation of the ac-
rylamide derivatives.
We therefore used a methanolic solution of potassium
carbonate, which is known to deprotect oligonucleotides
bearing easily labile protecting groups on their nucleo-
bases.[19] To test this approach, we synthesized the 5Ј-S-
acetyl-thiohexyl decamer 2 (Scheme 2) with benzoyl pro-
tecting groups for A and C and isopropylphenoxyacetyl
groups for G. Decamer 2 was treated with 10% piperidine
solution in acetonitrile to remove the cyanoethyl groups
and washed to afford 3.[20] Compound 3 was engaged in
TMTA with N-phenethylacrylamide (15 equiv.) either in the
presence of TCEP (8 equiv.) in concentrated aqueous am-
monia (1.0 mL) or in methanolic K2CO3 solution (50 mm,
0.5 mL) at 55 °C for 4 h. After concentration, HPLC analy-
ses showed the formation of the expected conjugate with a
slightly better purity for the reaction carried out with
K2CO3 (80% vs. 70%). For further conjugation we there-
fore used a methanolic K2CO3 solution.
The reaction was attempted with only 4 equiv. of acryl-
amide derivative and 8 equiv. of TCEP. After 3 h at 55 °C,
57% conversion had been achieved. After one night, only a
63% yield of conjugate had been produced and we observed
the presence of some dimerisation (ca. 10%) through di-
sulfide bond formation. Addition of further TCEP
(4 equiv.) and acrylamide derivative (4 equiv.) allowed full
conversion. Thus, to obtain rapid and full conversion of the
thiol oligonucleotide into its conjugate it is more efficient
to use 10 to 15 equiv. of acrylamide derivative.
To extend the scope of this reaction, we applied it to
conjugations of oligonucleotides with different acrylamide
derivatives bearing mannose (compound 4b),[21] ferrocene
(compound 4c), dansyl (compound 4d), biotin (compound
4e) or deoxycholic acid (compound 4f) moieties (Scheme 2).
To allow fast and complete reactions, TMTA was per-
Scheme 2. Synthesis of 5Ј-conjugate oligonucleotides 5a–f. SPOS:
solid-phase oligonucleotide synthesis; 1) phosphoramidite deriva-
tive + benzylthiotetrazole; 2) phenoxyacetyl anhydride, N-Me-
imidazole and 2,6-lutidine; 3) 0.1 m I2, THF/H2O/pyridine; 4) 2.5%
dichloroacetic acid, CH2Cl2. B* asterisk represents A or C with a
benzoyl protecting group and G with the isopropyl-phenoxyacetyl
protecting group, and B represents A, C, G or T.
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formed under the following conditions: the solid-supported
5Ј-thiol decamer 3 (ca. 0.25 μmol) was treated with 8 equiv.
of TCEP and 15 equiv. of 4b–f in 500 μL of 50 mm K2CO3
in methanol for 4 h at 55 °C. The completion of the reaction
was checked by MALDI-TOF MS. After filtration and
washing of the CPG beads with water (500 μL) the solution
was concentrated to half its original volume and subjected
to size exclusion chromatography (SEC) to remove excess
acrylamide derivative 4a–f, TCEP and K2CO3. HPLC
analyses of the crude products showed that the reactions
were clean, with purities from 75% to 95%, and the main
peaks were characterized by MS as the expected conjugates
(see Table S1 in the Supporting Information). Figure 1
shows MALDI-TOF and HPLC profiles of 5b (crude and
pure) as an example (see the Supporting Information for 5a
and 5c–f). It can be seen that the reaction is very clean,
effectively with the formation of only one product. The
non-conjugated oligonucleotide (m/z = 3189.2) was not ob-
served, showing that the TMTA was quantitative. The con-
jugates 5a–f were easily purified by reversed-phase HPLC
Figure 1. MALDI-TOF and HPLC profiles of conjugate 5b.
We then applied this efficient thiol click reaction proto-
and characterized by MALDI-TOF MS (Table S1 in the col, developed for monoconjugation, to multiple conjuga-
Supporting Information). tion to produce conjugate 12 (Scheme 3), a tetrathiol oligo-
Scheme 3. Synthesis of mannosyl-centered tetramannosyl-substituted oligonucleotide conjugate 12.
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Conjugated Oligonucleotides by Click Reactions
nucleotide containing a mannosyl-centred tetramannosyl- gate 17b (Scheme 4), containing both mannose and galac-
substituted motif. To this end, firstly 5Ј-pent-4-ynyl de- tose residues, was synthesized in two ways. By Route A,
camer 7 was synthesized on solid support from pent-4-ynyl CuAAC on the solid support[29] was applied first, and after
phosphoramidite 6.[22] Mannosylpropyl azide 8[23] was then washes TMTA was carried out. By Route B, TMTA was
conjugated by CuAAC in the presence of sodium ascorbate performed first and CuAAC was then carried out in solu-
and CuSO4 for 1 h at 60 °C, which allowed the introduction tion. To this end, a solid-supported oligonucleotide was
of a mannose core with four free hydroxy functions (com- successively elongated with monoalkyne phosphoramidite
pound 9).[23] Thiol phosphoramidite 1 was introduced onto 13[30] and thiol phosphoramidite 1 to afford 14, containing
each hydroxy group by use of double coupling (180 s). The an alkyne function and
a S-acetylthiohexyl group
resulting tetra-thiol-modified oligonucleotide 10 was (Scheme 4).
treated with piperidine to remove the cyanoethyl groups.
A portion of compound 14 was conjugated with tetra-
Finally, solid-supported phosphodiester oligonucleotide 11 acetyl-galacto-propyl azide 15 by CuAAC in the presence
(ca. 0.25 μmol) was engaged in a deprotection TMTA cas- of CuSO4 and sodium ascorbate (1 h 60 °C). The CPG be-
cade with 4b (20 equiv.) as described above to afford exclu- ads were then thoroughly washed with a saturated aqueous
sively the neoglyco-oligonucleotide conjugate 12 containing solution of EDTA to remove any trace of copper. Simple
the mannosyl-centred tetramannosyl motif. This result washing with water and organic solvents was not efficient,
demonstrated that multiple conjugation of oligonucleotides so traces of copper were still present and quenched the
by TMTA is also very efficient with use only of 5 equiv. of TMTA. After removal of cyanoethyl groups with piper-
4b per thiol function.
idine, yielding 16, the second conjugation with 4b was car-
The combination of TMTA and CuAAC reactions has ried out by TMTA as described above to afford 17b. Ex-
previously been reported in the polymer field,[24,25] but to cesses of reagents were mainly removed by SEC and the
the best of our knowledge this combination has never been final bis-conjugate was isolated by HPLC and characterized
applied for bis-conjugation of oligonucleotides. We had pre- by MS (see the Supporting Information).
viously shown that CuAAC could be combined with oxime
In parallel, another portion of 14 was treated with piper-
formation[26] or with amidative oxidation[27] to prepared idine and the first conjugation was performed by TMTA to
bis-conjugated oligonucleotides. Here, we studied condi- afford 18, within 1 h. Compound 18 was directly treated
tions for combining CuAAC and TMTA in the synthesis of with tetraacetyl-galactose azide 15[31] and CuSO4 for the
an oligonucleotide containing mannose and galactose resi- second conjugation by CuAAC. Because TCEP was pres-
dues. TMTA and CuAAC are not fully orthogonal, due to ent, there was no need to add sodium ascorbate to form CuI
the possibility of reaction between a thiol moiety and cop- in situ. Furthermore, because of the presence of K2CO3/
per,[28] so it is necessary to proceed sequentially. The conju- methanol, the acetyl groups of galactose were hydrolysed
Scheme 4. Synthesis of bis-conjugated oligonucleotide 17b by TMTA and CuAAC.
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A. Meyer, J.-J. Vasseur, F. Morvan
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Scheme 5. Synthesis of oligonucleotides bearing 5Ј-dansyl or 5Ј-biotin and 3-mannose groups by CuAAC and then TMTA.
during the reaction to afford 17b directly; it was treated as and methanol for 4 h at 55 °C to afford the bis-conjugated
described above.
oligonucleotides 21d and 21e, respectively, with dansyl or
Both strategies led to the formation of the bis-glyco-con- biotin groups at their 5Ј-ends and mannose at their 3Ј-ends.
jugated oligonucleotide 17b with the same efficacy. It
should be noted, however, that sequential CuAAC and
TMTA could not be performed in solution because the
presence of copper quenches the latter reaction.
Likewise, the 5Ј-biotin-galactose 17e oligonucleotide was
prepared from 16 by TMTA conjugation with biotin acryl-
amide 4e (see Scheme S1 in the Supporting Information).
Finally, oligonucleotides decorated at both ends with 5Ј-
dansyl and 3Ј-mannose (compound 21d, Scheme 5) or with
Conclusions
TMTA was efficiently applied to the synthesis of mono-
conjugated oligonucleotides and also, for the first time, for
the synthesis of a multiply conjugated oligonucleotide, un-
der very convenient and simple conditions. A great advan-
tage of TMTA with acrylamide derivatives for the synthesis
of oligonucleotide conjugates is that the reactions take place
5Ј-biotin and 3Ј-mannose (compound 21e) components
during the deprotection and release from the solid support,
were prepared by Route A with CuAAC on solid support
so no additional time is required for the conjugation. Such
a strategy could not be applied with maleimide derivatives
because they, unlike acrylamide derivatives, are not very
stable under basic conditions. Finally, sequential combina-
tion of CuAAC and TMTA allowed the synthesis of bis-
conjugated oligonucleotides. This result opens the way to
the synthesis of oligonucleotides decorated with different
labels.
first, to introduce a mannose residue from mannose propyl
azide 8 at the 3Ј-end, and then by divergent TMTA, with
either 4d or 4e, to introduce a dansyl or a biotin motif,
respectively, at the 5Ј-end (Scheme 5). An oligonucleotide
was thus elongated on monoalkyne solid support 18[32] and
S-acetyl-thiohexyl phosphoramidite 1 was coupled at the
5Ј-end to afford 19. CuAAC was carried out on solid sup-
port with mannose propyl azide 8, CuSO4 and sodium as-
corbate for 1 h at 60 °C to afford 20. After washing with
saturated aqueous EDTA solution, water and acetonitrile,
Experimental Section
cyanoethyl groups were removed by piperidine treatment.
One portion of the 3Ј-mannnose solid-supported oligonu-
cleotide was treated with dansylacrylamide 4d and another
N-Phenethylacrylamide (4a): Acryloyl chloride (80 μL, 1 mmol) was
added to a solution of phenethylamine (126 μL, 1 mmol) and anhy-
with biotin acrylamide 4e in the presence of TCEP, K2CO3 drous triethylamine (280 μL, 2 mmol) in dry toluene (5 mL). After
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Conjugated Oligonucleotides by Click Reactions
2 h stirring at room temp., the mixture was diluted with toluene
and poured into a saturated aqueous NaHCO3 solution (50 mL).
room temp. to a solution of ethylenediamine (134 μL, 2 mmol) and
anhydrous triethylamine (70 μL, 0.5 mmol) in anhydrous CH2Cl2
The compound was extracted with CH2Cl2 (3ϫ 15 mL). The or- (4 mL). The mixture was stirred for 1 h, diluted with CH2Cl2
ganic layer was then collected and dried with Na2SO4, and the
solvents were evaporated under vacuum. Purification by flash
chromatography on silica gel (AcOEt in cyclohexane 25% to 50%)
afforded 4a as a colourless oil (110.3 mg, 63%). TLC Rf = 0.6 (cy-
clohexane/AcOEt 1:4 v/v). H NMR (CDCl3, 300 MHz): δ = 2.79
(t, J = 6.9 Hz, 2 H, CH2), 3.54 (q, J = 6.9 Hz, 2 H, CH2NH), 5.48
(50 mL) and washed with cold water (2ϫ 25 mL). The organic
phase was dried with Na2SO4 and filtered, and the solvent was
evaporated under vacuum to afford a pale green powder. The crude
product (50 mg, 0.17 mmol) was dissolved in anhydrous triethyl-
amine (47 μL, 0.34 mmol) in anhydrous CH2Cl2 (2 mL), and acryl-
oyl chloride (14 μL, 0.17 mmol) was added. After 2 h stirring at
1
(br. s, 1 H, NH), 5.55 (dd, J = 1.4, 10.4 Hz, 1 H, CH=CH2,cis), room temp., the mixture was diluted with CH2Cl2 and poured into
5.95 (dd, J = 10.2, 17 Hz, 1 H, CH=CH2), 6.2 (dd, J = 1.5, 17 Hz,
1 H, CH=CH2,trans), 7.12–7.28 (m, 5 H, ar) ppm. 13C NMR
(CDCl3, 100 MHz): δ = 35.6, 40.7, 126.3, 126.5, 128.7, 128.8, 130.9,
138.8, 165.6 ppm. HRMS (ESI+): calcd. for C11H14NO [M + H]+
176.1075; found 176.1051.
a saturated aqueous NaHCO3 solution (50 mL). The organic layer
was then collected and dried with Na2SO4, and the solvents were
evaporated under vacuum. Purification by flash chromatography
on silica gel (MeOH in CH2Cl2 0 to 5%) yielded 4d as a pale green
1
powder (50 mg, 58%). TLC Rf = 0.5 (CH2Cl2/MeOH 9:1, v/v). H
NMR (CDCl3,300 MHz): δ = 2.94 (s, 6 H), 3.08–3.17 (m, 2 H,
-CH2NH-), 3.40–3.48 (m, 2 H, -CH2NHSO2-), 5.62 (d, J = 1.6,
8.6 Hz, 1 H, CH=CH2,cis), 5.83 (t, J = 5.8 Hz, 1 H, NH), 5.96 (dd,
J = 8.6, 16.8 Hz, 1 H, CH=CH2), 6.24 (dd, J = 1.6, 16.8 Hz, 1 H,
CH=CH2,trans), 6.30 (m, 1 H, NH), 7.24 (d, J = 7.6 Hz, 1 H, Ar),
7.6 (q, J = 8.4 Hz, 2 H, Ar), 8.3 (t, J = 8.6 Hz, 2 H, Ar), 8.6 (d, J
= 8.6 Hz, 1 H, Ar) ppm. 13C NMR (CDCl3, 100 MHz): δ = 35.4,
38.8, 41.4, 46.5, 111.3, 114.8, 119.2, 122.6, 124.5, 125.5, 125.5,
126.00, 126.4, 126.6, 130.6, 148.1, 162.7 ppm. HRMS (ESI+):
calcd. for C17H22N3O3S [M + H]+ 348.1379; found 348.1382.
2,3,4,6-O-Tetraacetyl-1-O-[3-(acryloylamino)propyl]-α-D-mannose
(4b): Tris(2-carboxyethyl)phosphane (TCEP, 330 mg, 1.15 mmol)
was added to a solution of 2,3,4,6-O-tetraacetyl-1-O-azidopropyl-
α-d-mannose[16] (100 mg, 0.23 mmol) in MeOH/H2O (8:2, v/v,
6 mL). The reaction was monitored by TLC. After 1.5 h stirring at
65 °C, the mixture was concentrated and filtered to remove the
white precipitate. The filtrate was evaporated to dryness to afford
a clear oil, which was diluted with anhydrous CH3CN (10 mL).
Anhydrous Et3N (64 μL, 2 mmol) and acryloyl chloride (19 μL,
0.23 mmol) were then added successively. The cloudy mixture was
stirred for 2 h at room temp. The reaction was then quenched with
saturated NaHCO3 solution (50 mL) and the mixture was extracted
with CH2Cl2 (2ϫ 50 mL). The organic layers were concentrated
and dried with Na2SO4, and the residue was purified by flash col-
umn silica gel (MeOH in CH2Cl2 0 to 5%) to afford 4b as a clear
oil (70 mg, 67%). TLC Rf = 0.45 (cyclohexane/AcOEt 1:1, v/v). 1H
NMR (CDCl3, 200 MHz): δ = 1.86–199 (m, 2 H, CH2CH2NH),
1.97, 2.05, 2.14 and 2.20 (4ϫs, each 3 H, 4ϫCOCH3), 3.37–4.38
(m, 7 H, OCH2 CH2CH2 N3 , OCHHCH2 CH2N3, 5-H and
OCHHCH2CH2N3, 6-Ha and 6-Hb), 4.85 (d, J = 1.4 Hz, 1 H, 1-
H), 5.28–5.35 (m, 3 H, 3-H, 2-H, 4-H), 5.69 (dd, J = 2.3, 9.5 Hz,
1 H, CH2=CH,cis), 6.14–6.31 (m, 3 H, CH2=CH, NH) ppm. 13C
NMR (CDCl3, 100 MHz): δ = 20.8, 20.9, 21.0, 29.2, 37.4, 62.77,
66.3, 66.8, 68.7, 69.2, 69.5, 97.7, 126.6, 130.9, 165.8, 169.8, 170.1,
170.2, 170.7 ppm. HRMS (ESI+): calcd. for C20H30NO11 [M +
H]+ 460.1817; found 460.1819.
N-[2-(Acrylamido)ethyl]biotinamide (4e) was prepared as described
in the literature.[17]
Deoxycholin Derivative 4f: Deoxycholic acid (392 mg, 1 mmol) and
N-hydroxysuccinimide (130 mg, 1.1 mmol) were dissolved in DMF
(10 mL), followed by addition of N,NЈ-dicyclohexylcarbodiimide at
room temp. After overnight stirring, the precipitate (DCU) was fil-
tered off and the filtrate was concentrated under vacuum. The
crude oil was washed with Et2O (3ϫ 50 mL) to afford a white
precipitate, which was diluted with anhydrous CH3CN (10 mL). A
solution of anhydrous Et3N (240 μL, 1.7 mmol) and N-acryloyl-
ethylenediamine[17] (90 mg, 0.8 mmol) was then added. The cloudy
mixture was stirred overnight at 50 °C and then concentrated under
vacuum. The residue was purified by flash column chromatography
(silica gel, MeOH in CH2Cl2 0 to 10%) to afford 4f as a clear oil
1
(166 mg, 34%). TLC Rf = 0.5 (CH2Cl2/MeOH 95:5 v/v). H NMR
(CD3OD, 600 MHz): δ = 0.65 (s, 3 H, CH3), 0.88 (s, 3 H, CH3),
0.95 (d, J = 9 Hz, 3 H CH3), 1.01–1.88 (m, 24 H, CH2, CH), 2.01–
2.24 (m, 2 H, C23, CH2), 3.25–3.27 (m, 4 H, CH2), 3.44–3.51 (m, 1
H, C3, CH), 3.90 (br, 1 H, C12, CH), 5.61 (dd, J = 5.4, 12 Hz, 1
H, CH2=CH,cis), 6.16–6.18 (m, 2 H, CH2=CH) ppm. 13C NMR
(CD3OD, 151 MHz): δ = 13.2, 17.7, 23.7, 24.8, 27.4, 28.4, 28.6,
29.9, 31.0, 33.2, 34.1, 34.8, 35.3, 36.4, 36.8, 37.2, 37.4, 39.9, 40.1,
43.6, 47.5, 48.0, 72.5, 73.9, 126.8, 132.0, 168.4, 177.2 ppm. HRMS
(ESI+): calcd. for C29H49N2O4 [M + H]+ 489.3692; found 489.3685.
N-[2-(Ferrocenylmethylamino)ethyl]acrylamide (4c): Ferrocenecarb-
aldehyde (214 mg, 1 mmol) was added to a solution of N-acryloyl-
ethylenediamine[17] (114 mg, 1 mmol) and triethylamine (140 μL,
1 mmol) in CH2Cl2/MeOH (5 mL). After the system had been
stirred for 1 h at room temp., NaBH4 (70 mg, 2 mmol) was added.
After 1 h, the mixture was diluted with CH2Cl2 and poured into
water (50 mL). After washing, the organic layer was collected and
dried with Na2SO4, and the solvents were evaporated under vac-
uum. The residue was purified by column chromatography (silica
gel, 2 to 10% MeOH in CH2Cl2) to afford 4c as a yellow powder
General Procedure for the Synthesis of Oligonucleotides: Oligo-
nucleotides were synthesized on a 1 μmol scale with a DNA synthe-
sizer and use of standard phosphoramidite chemistry. For the cou-
pling step, benzylmercaptotetrazole (BMT, 0.3 m in anhydrous
CH3CN) was used as the activator, commercially available nucleo-
side phosphoramidites (with benzoyl for A and C and isopropyl-
phenoxyacetyl for G, 0.09 m in anhydrous CH3CN) were intro-
duced with 20 s coupling times, and S-acetyl-thiohexyl phos-
phoramidite 1 (0.15 m in anhydrous CH3CN), pent-4-ynyl phos-
phoramidite 6[22] and 1-O-(4,4Ј-dimethoxytrityl)-2-propargyloxy-
methyl-2-methyl-3-O-[(2-cyanoethyl)-N,N-diisopropylphosphor-
1
(150 mg, 48%). TLC Rf = 0.55 (CH2Cl2/MeOH 8:2 v/v). H NMR
(CDCl3, 200 MHz): δ = 2.94 (br. s, 2 H, CH2NH), 3.49 (br. s, 2 H,
NHCH2), 3.88 (s, 2 H, FeCH2NH), 4.11 (s, 5 H, cyclopent), 4.16
(s, 2 H, cyclopent), 4.4.37 (s, 2 H, cyclopent), 5.58 (t, J = 5.4 Hz,
1 H, CH2=CH,cis), 6.19 (d, J = 5.8 Hz, 2 H, CH2=CH), 8.15 (br.
s, 1 H, NH) ppm. 13C NMR (CDCl3, 100 MHz): δ = 18.4, 30.9,
36, 46.8, 47.8, 69, 69.7, 70.7, 75.4, 126.8, 130.8, 166.7 ppm. HRMS
(ESI+): calcd. for C16H21N2OFe [M + H]+ 313.0987; found
313.1003.
N-(2-Acrylamidoethyl)-5-(dimethylamino)naphthalene-1-sulfon- amidite]propane-1,3-diol (13[30], 0.09 m in anhydrous CH3CN) were
amide (4d): Dansyl chloride (136 mg, 0.25 mmol) was added at introduced with 60 s coupling times. The capping steps were per-
Eur. J. Org. Chem. 2013, 465–473
© 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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471

A. Meyer, J.-J. Vasseur, F. Morvan
FULL PAPER
formed with acetic anhydride in commercially available solutions
(Cap A: phenoxyacetyl anhydride, pyridine, THF 10:10:80 and
Cap B: 10% N-methylimidazole in THF) for 60 s. Oxidation was
performed with a commercially available solution of iodide (I2,
0.1 m, THF/pyridine/water 90:5:5, 13 s). Detritylation was per-
formed with DCA in CH2Cl2 (2.5%, 35 s).
Synthesis of Bis-conjugated Oligonucleotide 17b by TMTA and then
CuAAC (Route B): Compound 14 (≈0.25 μmol) was first treated
with piperidine (see above). 1-O-Propylacrylamide-2,3,4,6-O-tet-
raacetyl-α-mannose (4b, 3.75 μmol, 37 μL of a 100 mm solution in
MeOH), TCEP (2 μmol, 20 μL of a 100 mm solution in H2O) and
K2CO3 in MeOH (50 mm, 500 μL) were then added. The vial con-
taining the mixture was heated for 1 h at 55 °C. After completion
of the reaction (HPLC monitoring), 1-O-propyl-azide 2,3,4,6-tetra-
acetyl-α-galactose (15,[31] 100 mm, 1.0 μmol, 10 μL of a 100 mm
solution in MeOH) and a freshly prepared solution of CuSO4
(0.25 μmol, 6 μL of 40 mm solution in water) were added. The vial
was heated for 4 h at 35 °C. The mixture was then filtered, diluted
with water (500 μL) and concentrated to remove MeOH. Com-
pound 17b was obtained after desalting on NAP 10.
Protocol for the Removal of Cyanoethyl Groups: After elongation,
the solid-supported S-acetyl-thiolhexyl-oligonucleotides were
treated with a solution of piperidine in dry CH3CN (10%, 1.5 mL,
back and forth between two syringes for 15 min), washed with dry
acetonitrile and dried with nitrogen flushing.
General Protocol for TMTA: An acrylamide derivative (4a–f,
3.8 μmol, 15 equiv., 38 μL of a 100 mm solution in MeOH), TCEP
(2 μmol, 8 equiv., 20 μL of a 100 mm solution in H2O) and K2CO3
in MeOH (50 mm, 500 μL) were added to a solid-supported S-
acetyl-thiohexyl-oligonucleotide 3 (≈0.25 μmol). The vial contain-
ing the mixture was heated for 4 h at 55 °C. The mixture was fil-
tered off and desalted on NAP10.
Bis-conjugated oligonucleotides 17e, 21a and 21e were synthesized
by Route A.
Supporting Information (see footnote on the first page of this arti-
1
cle): H and 13C NMR spectra of 4a–d and 4f, HPLC profiles of
conjugates 5a–f, 12, 17b, 17e, 21d and 21e with MS spectra.
Synthesis of the Mannosyl-Centred Tetramannosyl-Substituted
Oligonucleotide 12: 1-O-Azidopropyl-α-mannose[16] (4 μmol, 40 μL
of a 100 mm solution in MeOH) and freshly prepared solutions of
CuSO4 (1 μmol, 24 μL of a 40 mm in water) and of sodium ascorb-
ate (5 μmol, 50 μL of a 100 mm in water) were added to a vial
containing the solid-supported 5Ј-(pent-4-ynyl)-oligonucleotide 7
(1 μmol) in H2O/MeOH (1:1, v/v, 200 μL). The vial was heated at
60 °C (oil bath) under magnetic stirring for 1 h. The solid-sup-
ported 5Ј-mannosyl-oligonucleotide 9 was then filtered off, washed
(2 mL) with water, MeOH and CH3CN, dried and loaded onto a
DNA synthesis column. Thiohexyl phosphoramidite 1 was conju-
gated by application of a double coupling with a 180 s wait between
each coupling. Compound 10 was treated over 15 min with a solu-
tion of piperidine in CH3CN (10%, 1.5 mL) and washed with
CH3CN, and a quarter of the beads (ca. 0.25 μmol) were transfer-
Acknowledgments
The authors thank Julie Mayen for the synthesis of some oligonu-
cleotides. This work was financially supported by the Agence Na-
tionale de la Recherche (ANR) (grant numbers ANR-08-BLAN-
0114-01, ANR-09-PIRI-0023-02) and by Eurobiomed. F. M. is a
member of Inserm.
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a vial. 1-O-Propylacrylamide-2,3,4,6-O-tetraacetyl-α-
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472
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Eur. J. Org. Chem. 2013, 465–473

Conjugated Oligonucleotides by Click Reactions
the deprotection reaction with a pentamer. Despite the 10-fold
excess of 4a, the HPLC profile showed the formation of 70%
of the S-cyanoethyl pentamer and only 30% of the expected
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Received: October 4, 2012
Published Online: December 4, 2012
Eur. J. Org. Chem. 2013, 465–473
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