Bis- and tris-alkyne phosphoramidites for multiple 5′-labeling of oligonucleotides by click chemistry

Article abstract of DOI:10.1002/ejoc.201101763

Three new phosphoramidites exhibiting two or three alkyne functions were prepared and introduced at the 5′ end of oligonucleotides. The resulting bis- and tris-alkyne oligonucleotides were conjugated with acetylthiohexyl, ferrocenecarboamide hexyl, or car

Full text of DOI:10.1002/ejoc.201101763

FULL PAPER
DOI: 10.1002/ejoc.201101763
Bis- and Tris-Alkyne Phosphoramidites for Multiple 5Ј-Labeling of
Oligonucleotides by Click Chemistry
Caroline Ligeour,[a] Albert Meyer,[a] Jean-Jacques Vasseur,[a] and François Morvan*[a]
Keywords: Oligonucleotides / Nucleic acids / Conjugation / Carbohydrates / Alkynes / Click chemistry
Three new phosphoramidites exhibiting two or three alkyne
functions were prepared and introduced at the 5Ј end of oli-
gonucleotides. The resulting bis- and tris-alkyne oligonu-
carboamide hexyl, or carbohydrate-propyl azides by using
click chemistry (CuAAC) to afford polyconjugated oligonu-
cleotides. Conjugations were performed either in solution or
cleotides were conjugated with acetylthiohexyl, ferrocene- on solid support with high efficiency.
thiol anchor conjugates[8] to be synthesized. As an alterna-
Introduction
tive, we present herein the synthesis of three new phos-
phoramidites containing two or three alkyne functions (Fig-
ure 1). Indeed, the azide alkyne 1,3-dipolar cycloaddition
catalyzed by copper(I) (CuAAC)[12,13] is a very efficient and
popular method used to label biomolecules, especially oli-
gonucleotides.[14] Although many kinds of alkyne phos-
phoramidites have been reported in the literature, only a
few of them contain multiple alkyne functions.[15–19] In con-
trast, numerous alkyne phosphoramidites can be incorpo-
rated several times, but in these cases, the labels could not
be close to each other.[14,20] Finally, our strategy allows the
multiple 5Ј-end labeling either on solid support or in solu-
tion; hence, it is possible to introduce base-sensitive mole-
cules.
Oligonucleotide conjugates are widely used for various
applications in biology, biotechnology, and medicine. Sev-
eral applications require multiple labeling of oligonucleo-
tides. This is the case of multiple redox tags for enhanced
signals by electrochemistry,[1–3] of the introduction of sev-
eral carbohydrates for cluster effects,[4–7] or several sulfur
atoms for immobilization on a gold surface.[8,9] To address
this point, the use of a “trebler” phosphoramidite for the
introduction of three new alcohol functions for subsequent
couplings has been reported, and this method allows oligo-
nucleotide dendrimers,[10] glycoconjugates,[11] or multiple
Results and Discussion
Phosphoramidites 1 and 5 both contain two alkyne func-
tions and phosphoramidite 7 contains three alkyne func-
tions (Figure 1). Compound 1 was prepared in one step by
reaction of diisopropylphosphoramidous dichloride with
pent-4-yn-1-ol (2 equiv.) in the presence of triethylamine. It
was isolated in 83% yield after purification by flash
chromatography (Scheme 1).
Figure 1. Structure of bis-alkyne phosphoramidites 1 and 5 and
tris-alkyne phosphoramidite 7.
[a] Institut des Biomolécules Max Mousseron UMR 5247 CNRS-
UM1-UM2, Université Montpellier 2,
Scheme 1. Synthesis of bis-pent-4-ynyl phosphoramidite 1.
place E. Bataillon, CC1704, 34095 Montpellier cedex 5, France
Fax: +33-4-67042029
Bis-alkyne derivative 5 was synthesized by bis-alkylation
of 1-(4,4Ј-dimethoxytrityloxy)-2,2-bis(hydroxymethyl)prop-
ane (2)[21] with propargyl bromide in the presence of so-
E-mail: morvan@univ-montp2.fr
Supporting information for this article is available on the
WWW under http://dx.doi.org/10.1002/ejoc.201101763.
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C. Ligeour, A. Meyer, J.-J. Vasseur, F. Morvan
FULL PAPER
dium hydride in THF to afford 3, which was directly en- galactopyranoside propyl azide 11c[24] was performed with
gaged in the next step (Scheme 2). The DMTr group was purified 10. Copper(I) was generated in situ by using
removed by treatment with a solution of 3% trichloroacetic CuSO4 (5 equiv.) reduced by sodium ascorbate (25 equiv.)
acid in dichloromethane to give 4, which was purified by in a mixture of water/methanol (1:1). The reactions pro-
silica gel flash chromatography (80%, two steps). Finally, ceeded at room temperature and were finished within 2.5 h.
pure compound 4 was phosphitylated by using diisopropyl We observed that 5 equiv. of copper was required for a ra-
cyanoethyl chlorophosphoramidite in the presence of diiso- pid CuAAC reaction and avoided degradation of the the
propylethylamine in dry dichloromethane to afford ex- oligonucleotide without the use of a CuI chelator like tris-
pected phosphoramidite 5 bearing two alkyne functions in (benzyltriazolylmethyl)amine (TBTA).[25] Furthermore, we
82% yield (66% overall yield).
worked with a degassed aqueous solution and avoided the
presence of oxygen that would lead to degradation of the
oligonucleotide. The mixture was desalted by size-exclusion
chromatography (SEC), which also removed the excess
amount of the azide derivatives. The HPLC profiles of the
crude showed very clean reactions with the formation of
expected bis-conjugated oligonucleotides 12a–c with a high
purity (Figure 2). Bis-conjugates 12a and 12b were obtained
from crude 10 and purified by C18 HPLC. The yields were
between 80 and 87%, respectively. For the synthesis of 13c,
we started with purified 10, which was conjugated with 11c
leading to pure conjugate 12c after SEC. Finally, the acetyl
groups on the galactose moieties of 12c were removed by
ammonia treatment (1 h) affording pure bis-galactosyloli-
gonucleotide 13c (93%) without further purification (Fig-
ure 2). Each conjugate was characterized by MALDI-TOF
mass spectrometry (see the Supporting Information).
Bis-alkyne phosphoramidite 5 and tri-alkyne phos-
phoramidite 7 were introduced at the 5Ј end of oligonucleo-
tide 14 under conditions similar to those used for 1, leading
to bis- and tris-alkynyl solid-supported oligonucleotides 15
and 16, respectively (Scheme 5). Half of the amount of each
was treated with concentrated ammonia (55 °C, overnight)
for deprotection and release, affording, in solution, 5Ј-bis-
and 5Ј-tris-propargyl oligonucleotides 17 and 18, respec-
tively. Each alkynylated oligonucleotide (100 nmol) was
conjugated with S-acetylthiohexyl azide 11b (5.5 equiv./
alkyne) by CuAAC with the use of CuSO4 (5 equiv.) and
sodium ascorbate (25 equiv.) in methanol/water. The reac-
tions were complete in 1 and 2 h leading to bis- and tris-S-
acetylthiohexyl conjugates 19b and 20b, respectively. The
mixtures were desalted by SEC and purification was per-
formed by HPLC (73 and 78%). The conjugates were
characterized by MALDI-TOF MS.
Scheme 2. Synthesis of bis-alkyne phosphoramidite 5.
The preparation of tris-alkyne phosphoramidite 7[22] pro-
ceeded similarly with the use of tris-propargyl pentaerythrit-
ol derivative 6 prepared from pentaerythritol according to
the literature[23] and 2-cyanoethyl tetraisopropylphos-
phorodiamidite activated with diisopropylammonium tetra-
zolide in dry dichloromethane (94%, Scheme 3).
Oligonucleotide (12-mer) 8 was synthesized by a stan-
dard phosphoramidite protocol on a DNA synthesizer by
using a commercially available solid support and nucleoside
phosphoramidites. Then, bis-pent-4-ynyl phosphoramidite
1 was coupled at the 5Ј-end according to the same elong-
ation cycle, except a coupling time of 60 s was used to en-
sure good coupling. The expected solid-supported 5Ј-bis-
pent-4-ynyl 12-mer 9 was deprotected and released from the
solid support by treatment with ammonia (55 °C overnight)
to afford 10 (Scheme 4). The CuAAC reaction was carried
out on part of crude bis-alkyne oligonucleotide 10 with
azide derivatives 11a and 11b (11 equiv.) exhibiting a ferro-
cenamide hexyl and an S-acetylthiohexyl group, respec-
tively, whereas the CuAAC reaction with tetraacetyl-β-d-
Similarly, solid-supported bis-alkynyl oligonucleotide 15
was conjugated with ferrocenyl azide derivative 11a. How-
ever, we observed a strong degradation of the bis-ferrocenyl
oligonucleotide due to the ammonia treatment likely
Scheme 3. Synthesis of tris-alkyne phosphoramidite 7.
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Bis- and Tris-Alkyne Phosphoramidites by Click Chemistry
Scheme 4. Synthesis of oligonucleotides bis-conjugated with ferrocenecarboamide hexyl, S-acetylthiohexyl, or galactose propyl motifs by
using bis-pent-4-ynyl phosphoramidite 1. SPOS: solid-phase oligonucleotide synthesis. Conditions: (1) 3% trichloroacetic acid (TCA) in
CH2Cl2; (2) phosphoramidite derivative + benzylthiotetrazole (BTT); (3) Ac2O, NMe-imidazole, 2,6-lutidine; (4) 0.1 m I2 THF/H2O/pyr-
idine. Asterisk (*) represents protecting group on nucleobase (benzoyl for A and C or isobutyryl for G).
(70%). (see the Supporting Information). This result
showed that this kind of ferrocenyl derivative must be con-
jugated in solution to avoid degradation during basic treat-
ment.
Mannose moieties were introduced on an oligonucleotide
working either on solid support or in solution (Scheme 5).
To this end, solid-supported bis- and tris-alkyne oligonu-
cleotides 15 and 16 were treated with tetraacetyl β-d-
mannose propyl azide 21[26] by using CuSO4 (1 equiv.) and
sodium ascorbate (5 equiv.) for 45 min at 60 °C to afford 22
and 23, respectively, which were treated with ammonia for
deprotection and release from the solid support to give bis-
and tris-mannosylated oligonucleotide 26 and 27, respec-
tively. In parallel, a CuAAC reaction was performed in
solution with bis- and tris-alkynyl oligonucleotides 17 and
18 by using azide 21 for 1 h at room temperature leading to
acetylated conjugates 24 and 25, which were desalted by
SEC and treated with ammonia for 1 h to give bis- and tris-
mannosylated oligonucleotide 26 and 27, respectively. Both
CuAAC reactions performed on solid support and in solu-
tion yielded similar results with an HPLC purity of the
Figure 2. HPLC profiles of bis-alkyne oligonucleotide 10 and its
conjugates with 11a–c.
crude material between 77 and 84%. Each conjugate was
characterized by MALDI-TOF mass spectrometry.
Finally, to extend the scope of multiple conjugations, a
through a reaction on the ferrocenyl moiety. Thus, the last conjugate exhibiting four alkyne functions was synthe-
CuAAC reaction was carried out in solution by using bis- sized on a DNA synthesizer by derivatization and introduc-
alkynyl oligonucleotide 17 and ferrocenyl azide derivative tion of two molecules of bis-pentynyl phosphoramidite (1,
11a affording expected bis-ferrocenyl oligonucleotide 19a Scheme 6). Thus, solid-supported oligonucleotide 28 was
Eur. J. Org. Chem. 2012, 1851–1856
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C. Ligeour, A. Meyer, J.-J. Vasseur, F. Morvan
FULL PAPER
Scheme 5. Synthesis of bis- and tri-alkynyl oligonucleotides by using 5 and 7 phosphoramidites, respectively, and their conjugates by
CuAAC using ferrocenyl, acetylthiohexyl, or mannosyl azide derivatives.
Scheme 6. Synthesis of tetramannosylated oligonucleotide conjugate.
synthesized and special phosphoramidite 29,[27] prepared anol and water affording tetramannosyl oligonucleotide
from tris-(hydroxymethyl)ethane and bearing two hydroxy conjugate 33, which was desalted by SEC and purified by
functions protected with a dimethoxytrityl group, was in- HPLC.
troduced to afford 30. Then a thymidine phosphoramidite
was introduced on each hydroxy group, as linkers, and fi-
nally two phosphoramidite moieties 1 were introduced lead-
ing to tetraalkyne oligonucleotide 31 after ammonia treat-
Conclusions
ment. The CuAAC reaction was performed in solution with
Using three new phosphoramidite bearing two or three
β-d-mannose propyl azide 32[24] (4 equiv./alkyne) by using alkyne functions, we were able to synthesize several oligo-
CuSO4 (5 equiv.) and sodium ascorbate (25 equiv.) in meth- nucleotides conjugated with thiohexyl group, a ferrocenyl
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Bis- and Tris-Alkyne Phosphoramidites by Click Chemistry
13C NMR (75 MHz, CDCl3): δ = 17.3, 40.4, 58.7, 68.3, 74, 74.4,
79.7 ppm. HRMS (ESI+): calcd. for C11H17O3 [M + H]+ 197.1175;
found 197.1178.
moiety, or carbohydrate residues by using their azide deriv-
atives. These phosphoramidites contribute to the elabora-
tion of new building blocks for the multiple conjugation of
oligonucleotides by the very efficient CuAAC reaction.
2,2-Bis(propargyloxymethyl)propyl 2-Cyanoethyl N,N-Diisoprop-
ylphosphoramidite (5): To a solution of anhydrous 2,2-bis(propargyl-
oxymethyl)propan-1-ol (4; 2.7 mmol, 530 mg) and diisopropyl-
ethylamine (4.05 mmol, 705 μL) in anhydrous CH2Cl2 (30 mL) was
added 2-cyanoethyl chlorophosphoroamidite (3.25 mmol, 720 μL).
The resulting mixture was stirred for 45 min at room temperature.
Then, the reaction was quenched with H2O (1 mL), diluted with
CH2Cl2 (100 mL), and washed with NaHCO3 (3ϫ40 mL). The or-
ganic layer was dried (Na2SO4), filtered, and concentrated. The
crude product was purified by silica gel column chromatography (0
to 30% CH2Cl2 in cyclohexane containing 5% triethylamine) to
afford 5 (0.87 g, 82%) as clear oil. Rf = 0.5 (cyclohexane/AcOEt/
Et3N, 7:2:1). 1H NMR (200 MHz, CDCl3): δ = 1.02 (s, 3 H, CH3C),
1.23 (d, J = 6.8 Hz, 12 H, CH3 ipr), 2.45 (t, J = 2.4 Hz, 2 H, CCH),
2.69 (t, J = 6.4 Hz, 2 H, CH2CN), 3.44 (s, 4 H, CCH2Opropargyl),
3.51–3.91 [m, 6 H, CH(ipr), OCH2CH2, CCH2OP], 4.17 (d, J =
2.4 Hz, 4 H, CH2CCH) ppm. 13C NMR (100 MHz, CDCl3): δ =
17.3, 20.4, 24.5, 24.7, 40.8, 43.0, 43.2, 58.2, 58.4, 58.6, 66.0, 72.5,
74.1, 80.0, 117.6 ppm. 31P NMR (81 MHz, CDCl3): δ = 147.7 ppm.
HRMS (ESI+): calcd. for C20H34N2O4P [M + H]+ 397.2256; found
397.2246
Experimental Section
Bis(pent-4-ynyl) N,N-Diisopropylphosphoramidite (1): To a solution
of anhydrous pent-4-yn-1-ol (5.4 mmol, 500 μL) and anhydrous
Et3N (6 mmol, 826 μL) in dry CH2Cl2 (5 mL), stirred at 0 °C under
an atmosphere of argon, was added a solution of diisopropylphos-
phorodichloridite (2 mmol, 550 mg) in dry CH2Cl2 (5 mL). The re-
sulting mixture was stirred for 4 h at room temperature, diluted
with CH2Cl2 (100 mL), filtered to remove the triethylammonium
chloride salt, and washed with NaHCO3 (1ϫ150 mL). The organic
layer was dried (Na2SO4), filtered, and concentrated. The crude
product was purified by silica gel column chromatography (0 to
50% CH2Cl2 in cyclohexane containing 10% triethylamine) to af-
ford the title compound (0.50 g, 83%) as a clear oil. Rf = 0.60
(cyclohexane/AcOEt/Et3N, 5:4:1). 1H NMR (300 MHz, CD3CN):
δ = 1.18 (d, J = 6.8 Hz, 12 H, 4 CH3), 1.73–1.82 (m, 4 H, 2
CH2CH2CH2), 2.16–2.19 (t, J = 2.6 Hz, 2 H, 2 CCH), 2.29 (dt, Jd
= 2.6 Hz, Jt = 7.1 Hz, 4 H, 2 CH2CH2CCH), 3.54–3.77 (m, 6 H, 2
OCH2, 2 CHMe2) ppm. 13C NMR (300 MHz, CDCl3): δ = 14.2,
23.6, 29.8, 42.3, 61.2, 68.6, 83.5 ppm. 31P NMR (121 MHz,
CD3CN): δ = 146.25 ppm. HRMS (ESI+): calcd. for C16H29O2NP
[M + H]+ 298.1915; found 298.1936.
2-Cyanoethyl 2,2,2-Tris(propargyloxymethyl)ethyl N,N-Diisoprop-
ylphosphoramidite (7): To a solution of anhydrous 2,2,2-tris(prop-
argyloxymethyl)ethan-1-ol (6;[23] 2.8 mmol, 700 mg) and diisoprop-
ylammonium tetrazolide (1.4 mmol, 240 mg) in anhydrous CH2Cl2
(10 mL) was added 2-cyanoethyl tetraisopropylphosphorodiamidite
(3.4 mmol, 1.1 mL). The resulting mixture was stirred for 5 h at
room temperature, diluted with CH2Cl2 (50 mL), and washed with
brine (2ϫ150 mL). The organic layer was dried (Na2SO4), filtered,
and concentrated. The crude product was purified by silica gel col-
umn chromatography (15 to 50% CH2Cl2 in cyclohexane contain-
ing 5% triethylamine) to afford 7 (1.0 g, 94%) as clear oil. Rf = 0.45
(cyclohexane/CH2Cl2/Et3N, 6:3:1). 1H NMR (400 MHz, CDCl3): δ
= 1.18–1.20 (dd, J = 7 Hz, 12 H, 4 CH3), 2.41 (t, J = 2.3 Hz, 3 H,
3 CCH), 2.63–2.67 (m, 2 H, CH2CN), 3.53 (s, 6 H, 3 CCH2O),
3.55–3.67 (m, 4 H, POCH2C, 2 CHMe2), 3.81–3.89 (m, 2 H,
OCH2CH2), 4.14 (d, J = 2.3 Hz, 6 H, 3 OCH2CCH) ppm. 13C
NMR (100 MHz, CDCl3): δ = 20.4, 24.7, 43.2, 45.1, 58.2, 58.4,
58.7, 68.6, 74.2, 80.0, 117.73 ppm. 31P NMR (121 MHz, CDCl3): δ
= 147.6 ppm. HRMS (ESI+): calcd. for C23H36N2O5P [M + H]+
451.2362; found 451.2373.
1-(4,4Ј-Dimethoxytrityloxymethyl)-2,2-bis(propargyloxymethyl)prop-
ane (3): To a cold solution of 1-(4,4Ј-dimethoxytrityloxymethyl)-
2,2-bis(hydroxymethyl)propane[21] (13.25 mmol, 5.6 g) in anhy-
drous THF (200 mL) was added NaH (60% in oil, 200 mmol,
8.0 g), and after 10 min propargyl bromine (80% in toluene,
106 mmol, 11.8 mL) was added. The mixture was stirred at 40 °C
overnight. Then, the reaction was cooled to 0 °C, quenched with
H2O (20 mL), and poured into H2O (400 mL). The aqueous layer
was extracted with CH2Cl2 (3ϫ150 mL), and the organic layers
were pooled, washed with brine, and concentrated. The crude oil
was used directly for the next step or purified by silica gel column
chromatography (50 to 100% CH2Cl2 in cyclohexane) to afford the
title compound as a clear oil. Rf = 0.6 (cyclohexane/AcOEt, 1:1).
1H NMR (300 MHz, CDCl3): δ = 0.89 (s, 3 H, CCH3), 2.30 (t, J
= 2.3 Hz, 2 H, CCH), 2.89 (s, 2 H, CCH2ODMTr), 3.37 (d, J =
3.6 Hz, 4 H, CCH2Opropargyl), 3.8 (s, 6 H, OCH3), 4.0 (d, J =
2.4 Hz, 4 H, OCH2CC), 6.72–7.36 (m, 13 H, aromatic) ppm. 13C
NMR (75 MHz, CDCl3): δ = 17.8, 40.7, 55.1, 58.6, 64.7, 72.9, 74.0,
80.2, 85.4, 112.9, 126.5, 127.6, 128.3, 130.2, 136.4, 145.4,
158.3 ppm. HRMS (ESI+): calcd. for C32H34O5Na [M + H]+
521.2304; found 521.2308.
General Procedure for the Synthesis of Oligonucleotides: Oligonu-
cleotides were synthesized on a 1 μmol-scale on a DNA synthesizer
(ABI 394) starting from a commercially available solid support by
standard phosphoramidite chemistry. For the coupling step, benz-
ylmercaptotetrazole (BMT) was used as the activator (0.3 m in an-
hydrous CH3CN), commercially available nucleoside phosphor-
amidites (0.075 m in anhydrous CH3CN) was introduced with a 20 s
coupling time, alkyne phosphoramidite 1 (0.2 m in anhydrous
CH3CN) was introduced with a 60 s coupling time, and alkyne
phosphoramidites 5 and 7 (0.1 m in anhydrous CH3CN) was intro-
duced with a double coupling of 40 s each. The capping step was
performed with acetic anhydride using commercial solutions (Cap
A/Ac2O, pyridine, THF 10:10:80 and Cap B/10% N-methylimid-
azole in THF) for 15 s. Oxidation was performed with a commer-
cial solution of iodide (0.05 m I2 in THF/pyridine/water, 90:5:5) for
13 s. Detritylation was performed with 3% TCA in CH2Cl2 for 35 s.
2,2-Bis(propargyloxymethyl)propan-1-ol (4): To a cold solution of
[1-(4,4Ј-dimethoxytrityloxy)-2,2-bis(propargyloxymethyl)propane
(13.25 mmol, crude oil) in CH2Cl2 (100 mL), whilst stirring, was
added a solution of 3% TCA in CH2Cl2 (100 mL). The red mixture
was stirred for 15 min and quenched by the slow addition of an
aqueous saturated solution of NaHCO3. The aqueous layer was
extracted with CH2Cl2 (3ϫ100 mL), and the organics layers were
pooled, dried (Na2SO4), and concentrated. The crude oil was puri-
fied by silica gel column chromatography (0% to 5% MeOH in
CH2Cl2) to afford 4 (2.1 g, 80%, two steps) as a brown oil. Rf =
0.25 (cyclohexane/AcOEt, 7:3). Rf = 0.5 (MeOH/CH2Cl2, 5:95). 1H
NMR (300 MHz, CDCl3): δ = 0.84 (s, 3 H, CH3), 2.37 (t, J =
2.4 Hz, 2 H, CCH), 3.43 (d, J = 1.5 Hz, 4 H, CCH2Opropargyl), General Procedure for the Deprotection of Solid-Supported Oligonu-
3.5 (s, 2 H, CCH2OH), 4.08 (dd, J = 2.4 Hz, 4 H, OCH2CC) ppm. cleotides: CPG beads were treated with concentrated aqueous am-
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monia (1.5 mL) for 18 h at room temperature for 9 or for 5 h at
55 °C for 15 and 16. The supernatant was withdrawn and the sol-
vents were evaporated to dryness. The residue was dissolved in
water.
General Procedure for CuAAC Reaction
In Solution: To bis- or tris-alkyne oligonucleotide (1 equiv.) in H2O
(50 μL) and azide derivative (4 equiv./alkyne in MeOH) was added
a freshly prepared and degassed aqueous solution of CuSO4
(5 equiv.), sodium ascorbate (25 equiv.), and water/MeOH (1:1) up
to a volume of 200 μL. The tube containing the resulting prepara-
tion was sealed and magnetically stirred for 1 h at room tempera-
ture. Upon completion of the reaction, the solution was desalted
on NAP10 and the solvents were evaporated.
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On Solid Support: To the solid-supported bis- or tris-alkyne oligo-
nucleotide (1 equiv.) in H2O (50 μL) and azide derivative (4 equiv./
alkyne in CH3OH) was added a freshly prepared and degassed
aqueous solution of CuSO4 (1 equiv.), sodium ascorbate (5 equiv.),
and water/MeOH up to 200 μL. The tube containing the resulting
preparation was sealed and placed for 45 min at 60 °C in an oil
bath under magnetic stirring. After the reaction, the CPG beads
were filtered off, washed with H2O (3ϫ2 mL) and MeOH
(3ϫ2 mL), and dried.
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Acknowledgments
This work was financially supported by the Agence Nationale de
la Recherche (ANR-08-BLAN-0114-01, ANR-09-PIRI-0023-02),
Lyon Biopole, and Eurobiomed. C. L. thanks the Ministère de
l’éducation nationale de la recherche et de la technologique
(MENRT) for the award of a research studentship. F. M. is from
Inserm.
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Received: December 7, 2011
Published Online: Feburary 6, 2012
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