Glycosyl-nucleolipids as new bioinspired amphiphiles

Article abstract of DOI:10.3390/molecules181012241

Four new Glycosyl-NucleoLipid (GNL) analogs featuring either a single fluorocarbon or double hydrocarbon chains were synthesized in good yields from azido thymidine as starting material. Physicochemical studies (surface tension measurements, differential scanning calorimetry) indicate that hydroxybutanamide-based GNLs feature endothermic phase transition temperatures like the previously reported double chain glycerol-based GNLs. The second generation of GNFs featuring a free nucleobase reported here presents a better surface activity (lower_γlim) compared to the first generation of GNFs.

Full text of DOI:10.3390/molecules181012241

Molecules 2013, 18, 12241-12263; doi:10.3390/molecules181012241
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Glycosyl-Nucleolipids as New Bioinspired Amphiphiles
Laurent Latxague 1,2,†, Amit Patwa 1,2,†, Eric Amigues 1,2 and Philippe Barthélémy 1,2,
*
1
INSERM U869, Bordeaux, F-33076, France
2
Université de Bordeaux, Bordeaux, F-33076, France
†
These authors contributed equally to this work.
* Author to whom correspondence should be addressed; E-Mail: philippe.barthelemy@inserm.fr.
Received: 28 August 2013; in revised form: 25 September 2013 / Accepted: 25 September 2013 /
Published: 30 September 2013
Abstract: Four new Glycosyl-NucleoLipid (GNL) analogs featuring either a single
fluorocarbon or double hydrocarbon chains were synthesized in good yields from azido
thymidine as starting material. Physicochemical studies (surface tension measurements,
differential scanning calorimetry) indicate that hydroxybutanamide-based GNLs feature
endothermic phase transition temperatures like the previously reported double chain
glycerol-based GNLs. The second generation of GNFs featuring a free nucleobase reported
here presents a better surface activity (lower lim) compared to the first generation of GNFs.
Keywords: nucleolipid; nucleoside; glycosyl; fluorocarbon; amphiphiles; surface tension
1. Introduction
The chemical combination of biological molecules such as nucleic acids [1–3], aminoacids [4],
peptides [5–8] or sugar [9–11] with lipids, remains an amazing approach to create new hybrid
amphiphilic structures. Owning to their biocompatibility properties and biological functions, hybrid
amphiphiles hold considerable potential for biomedical and biotechnological applications. For
example, the association of nucleic acid moieties with lipids was realized to enhance the cellular
uptake and/or bio-distribution characteristics of nucleoside drugs [12] for cancer therapies. It has been
demonstrated that the chemical combination leading to nucleolipids [13–16] is of particular interest
due to the supramolecular properties of these molecules and their potential applications in the
biomedical field [17–21]. Interestingly, additional moieties were recently attached to the nucleolipid
platforms, including aminoacids [22–24] and sugars [25,26]. Among these amphiphiles, glycosyl-

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nucleolipids (GNLs) [27] hold a special place as nucleoside derivatives. The GNL structure
corresponds to the covalent association of a lipid, a nucleoside and a sugar. Each moiety is separated
from the other by a triazole linker resulting from the click-chemistry used for their synthesis (Figure 1a).
Figure 1. (a) Retrosynthetic pathway leading to the GNLs in to steps, (b) Fluorinated GNL 1
also designed by the acronym GNF (Glycosyl-nucleoside fluorinated amphiphile).
(c) Double chain GNLs based on a glycerol backbone.
Most of the GNL amphiphiles exhibit the ability to form gels with either water or organic solvents
giving rise to vesicles, nanofibers networks or nanoparticules as evidenced by TEM studies [28,29].
GNLs represent a new class of low molecular weight gelators (LMWGs) [30], which are of particular
interest for biomedical applications due to their intrinsic properties, in particular the non-covalent
nature of the molecular interactions at work (- stacking between the nucleobases and/or the triazole
moieties, intermolecular hydrogen bond, etc). The Hydrophilic-Lipophilic balance (HLB) is also an
important parameter to take into account for the design of new soluble LMWG. The lipophilicity
parameter can be modulated via the insertion of fluorocarbon chains [31] in the amphiphilic structures.
Interestingly, this modification provides fluorinated GNL featuring an absence of cytotoxicity (MTT
and neutral red assays) [32]. For example, the fluorinated GNL 1 (aka GNF, Figure 1b) in its hydrogel
form has been used as a scaffold for stem cell culture and differentiation [33], and many
complementary studies are still underway to take advantage of this particular property. In contrast, the
hydrocarbon single chain GNLs were cytotoxic above 100 µM in human hepatocarcinoma cell line for
example [29], which was attributed to a detergent effect of the single chain on the cell membrane, leading
to fracture initiation and then cellular death. Consequently, in parallel to the fluoroalkyl (F-alkyl) chain, we
also synthesized GNLs featuring a double H-alkyl C18 chain linked to a glycerol backbone (Figure 1c),
which was expected to avoid any toxicity towards cells. As expected, GNL 2a was not cytotoxic
(Figure 1c). Moreover, it promotes liposome internalization in adipose stem cells [25] demonstrating
again the inherent relevance of GNLs in biomedical applications.

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Further investigations in this domain led us to synthesize new GNLs featuring either a single F-alkyl
chain or a double H-alkyl chain as hydrophobic moieties. In this contribution we report the synthesis of
these new compounds, which have been characterized by NMR, MS, tensiometry and DSC experiments.
2. Results and Discussion
2.1. Synthesis of New GNLs
Previous unpublished results from our group obtained with single H-alkyl GNLs, showed that a
more stable gel was formed with an amido vs. an ether linkage. This prompted us to synthesize new
GNLs with modified glycerol backbone structures in favour of a hydroxybutanamide synthon (Figure 2).
Figure 2. New GNL structure; dotted box highlighting the hydroxybutanamide synthon.
The synthetic pathway leading to different GNLs is described in Scheme 1. The acetonide-ester 3 is
the key starting material for the synthesis of all three GNLs and was therefore required in significant
quantities. Although commercially available, acetonide-ester 3 is relatively expensive, so it was
synthesized, as was the intermediate dimethyl (S)-malate 3b, from (S)-malic acid 3a according to a
literature procedure [34]. Aminolysis of unactivated ester 3 with propargylamine turned out to be less
effective. The use of sodium methoxide in toluene for amidation of unactivated esters [35] failed.
However, aminolysis of acetonide-ester 3 was successfully performed with 1,2,4-triazole anion as
catalyst [36], but in a moderate yield, to provide N-propargylamide derivative of acetonide 4. The
acetal deprotection of 4 was carried out with Dowex® 50 resin in methanol to give the key intermediate
5 in good yield. Different GNLs were synthesized in four steps starting from compound 5. Briefly, the
fatty acid chain grafting was achieved by means of a DCC/DMAP coupling reaction to afford the
lipidic derivatives 6a–c. The first ‘click’ reaction with 5'-azido-5'-deoxythymidine [37] in the presence
of CuSO4 in a THF/H2O mixture provided the expected lipid-triazole-thymidine 7a–c. This
lipid-nucleoside intermediates 7a–c were treated with propargyl bromide in the presence of K2CO3 to
afford N-propargylated thymidine derivatives 8a–c, which were reacted with 1-azido-1-deoxy-β-D-
glucopyranoside in the presence of CuSO4 in a THF/H2O mixture following a second ‘click’ reaction
to provide the expected GNLs 9a–c in an average 85% yield.
It must be emphasized that other GNL structures can be envisioned by connecting the G, N and L
moieties differently. Accordingly, the free 3'-ribose position of the nucleoside was used to attach the
sugar moiety as depicted below (Figure 3). This new connectivity provides a second generation of
GNL featuring a free nucleobase.

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Scheme 1. General scheme for synthesis of double chains GNLs 9a–c.
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Figure 3. Schematic representation of 2nd generation GNL structure.
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This new structure could offer several advantages:
-
The GNL synthesis is no more “thymidine-dependent” because any nucleobase can now be used
to increase the number of available GNLs, therefore extending the scope of the synthesis.
Unmodified nucleobases retain their native H-bonding ability, particularly with a complementary
base, which may lead to potentially new supramolecular assemblies.
-
As a preparatory step to a more detailed consideration of this issue, the first so-called
“2nd generation” GNL we wanted to investigate is the analog of GNF (2nd GNF). The synthetic route
for the preparation of “2nd generation” GNL is shown in Scheme 2. It relies on the preparation of
3'-propargyl thymidine. First, we selectively protected the 5'-hydroxyl group of thymidine with
TBDMSCl to obtain 5'-OTBDMS-thymidine 11. Then the 3'-hydroxy group was propargylated with
NaH (2.5 equiv.) and propargyl bromide (2.5 equiv.) in THF at room temperature [38,39]. The
selective 3'-O-alkylated product 12 was obtained in 90% yield after purification. The TBDMS
protection was then removed with TBAF to afford 3'-O-propargylated thymidine 13. Then, 1-azido-
2,3,4,6-tetra-O-acetyl-β-D-glucopyranoside [40] was reacted with the propargyl derivative 13 in the
presence of CuSO4 following a first ‘click’ reaction to provide click product 14. In contrast with the
1st generation GNL synthesis, the use of a protected glucose here allowed the exclusive formation of
the desired mesylate intermediate which was directly displaced with sodium azide to yield the azido
derivative 15 in 92% yield. Following a second ‘click’ reaction, the N-propargyl-1H,1H,2H,2H-
perfluoroundecanoyl amide [29] was reacted with compound 15 in the presence of CuSO4 in a
THF/H2O mixture to afford the expected acetyl protected GNF 16. Finally, deacetylation of glucose
moiety was carried out with sodium methoxide in methanol at room temperature to give the final
compound 17 in 93% yield.
2.2. Physicochemical Studies
One of the goals of the present study was to determine the impact of structural modifications, on the
self-assembly properties of the new GNLs. The phase transition temperature (Tm) and phase transition
enthalpies of the new double chains GNLs were determined by differential scanning calorimetry
(DSC). The Tm values of −16.1, 51.7 and 62.1 °C were measured for 9a, 9b and 9c, respectively (Table 1).

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Scheme 2. Synthesis of 2nd generation GNF 17 (2nd GNF).

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Except for compound 9a, which possess unsaturated oleyl chains, these values are similar to the
glycerol based GNL 2a, suggesting that the hydroxybutanamide moiety does not affect the melting
temperature of the GNLs. The phase transition enthalpies for the new GNLs 9a, 9b and 9c and the
glycerol based compound 2a have been also analyzed by DSC. For all compounds, the evolution
towards the fluid state of the alkyl chains is endothermic reflecting that amphiphiles possess different
organizations below and/or above the Tm. Note that no phase transition was observed for both first and
second generation GNFs due to their rigid fluorocarbon chain.
Table 1. Melting temperatures and phase transition enthalpies of GNLs (n.o.: not observed).
GNLs
9a
Chains
Tm in °C
−16.3 ± 2.8 °C
51.7 ± 1.8 °C
51,9 ± 1.8 °C
56.1 ± 2.4 °C
n.a.
ΔH (KJ/mol)
300 ± 15
26.5 ± 5
Double Oleyl
9b
Double saturated C12
Double saturated C8
Double saturated C18
Single Fluorocarbon
9c
0.46 ± 0.02
1.03 ± 0.06
n.a.
2a
17
The critical aggregation concentrations (CAC’s) of GNFs were determined by air-solution surface
tension (γ) measurements as a function of amphiphile concentration (C). Examples of (γ) versus (C)
curves, measured at 25 °C, are shown in Figure 4. The GNF first and second generation 17 gave breaks
in c versus Log (C) characteristic of CAC’s of roughly 11 µM for both GNFs indicating that the CAC
depends mainly on the hydrophobic segment. Unexpectedly, the structural modification of polar head
induces a decrease of the γlim value (30 mN/M, 20 mN/M, for 1st and 2nd generations GNFs,
respectively) likely due to a tight packing of the molecule at the air water interface in the case of the
2nd generation GNF 17. In contrast with the 1st GNF, which forms gels in water at very low
concentration (0.1% w/w), 2nd GNF 17 induces the formation of very viscous colloidal suspensions in
similar conditions.
Figure 4. Air–water interfacial tension γ versus concentration for 1st and 2nd generations
GNFs (blue square and orange diamond) at 25 °C.

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3. Experimental
3.1. General
All compounds were purchased from Sigma-Aldrich (Saint Quentin Fallavier, France), and Alfa
Aesar (Schiltigheim, France) unless otherwise mentioned. Solvents for reactions were purchased from
Sigma-Aldrich in the highest quality and from VWR (Fontenay sous Bois, France) for other uses. All
the reactions were run under nitrogen atmosphere unless otherwise stated. Analytical thin layer
chromatography (TLC) was performed on pre-coated silica gel F254 plates with fluorescent indicator
from Merck (Fontenay sous Bois, France). The detection of compounds was accomplished using a UV
light (254 nm) and visualized on TLC plates by subsequent spraying with 10% conc. H2SO4 solution in
ethanol, followed by heating. Column chromatography was performed with flash silica gel (0.04–0.063 mm)
1
13
from Merck. All the compounds were characterized using H and C Nuclear Magnetic Resonance
(NMR) spectroscopy. These NMR spectra were recorded (in CDCl3/DMSO-d6/CD3OD obtained from
Eurisotop, (Gif sur Yvette, France) on a BRUKER Avance DPX-300 spectrometer (1H at 300.13 MHz
and 13C at 75.46 MHz). The chemical shifts (δ) are given in parts per million (ppm) relatively to
tetramethylsilane or residual solvent peaks (CHCl3: 1H: 7.26, 13C: 77.0). The coupling constants J are given
in Hertz (Hz); the peak multiplicity is reported as follows: s = singlet, bs = broad singlet, d = doublet,
t = triplet, m = multiplet. High resolution electronspray ionization mass spectra (HR ESI-MS) were
performed by the CESAMO (Bordeaux, France) on a QSsat Elite Applied Biosystems mass
spectrometer. The instrument is equipped with an ESI source and spectra were recorded in negative
mode. The electrospray needle was maintained at 4500 V and operated at room temperature. Samples were
introduced by injection through a 10 μL sample loop into a 200 μL/min flow of methanol from the
LC pump.
3.2. Tensiometer
The critical aggregation concentrations (CACs) of GNFs were determined using a DCAT11
tensiometer from Dataphysics (Voisins le Bretonneux, France). To determine the CAC value of the
amphiphiles, freshly prepared solutions of GNFs were added dropwise into distilled water. All
additions and measurements were automated and performed at room temperature.
3.3. Differential Scanning Calorimetry (DSC)
DSC scans were made with a Mettler-Toledo (Viroflay, France) DSC calorimeter. The GNLs
(10 mg) were dissolved in 100 μL of water and hermetically sealed in an aluminum pan. The
temperature was increased at 0.5 °C/min to 70 °C where it was held for 2 min. The temperature was
then reduced to 10 °C and held at this temperature for 2 min. This heating-cooling cycle was repeated
one more times before the sample was held isothermal at 10 °C for 20 min. In the case of oleyl
derivative the heating-cooling cycle was done between −30 °C and to 70 °C. The data collected on the
second cycle were analyzed. Integrating under the heat flow curve of the main peak afforded the
enthalpy of the transition. Mettler Toledo STARe computer program was used to analyze the data.

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3.4. Syntheses
Dimethyl (S)-malate (3b). Compound 3b was synthesized according to the literature procedure [34].
1
The data agreed with the literature values. H-NMR (300 MHz, CDCl3): δ in ppm 2.73–2.89 (m, 2H,
-CH2-C=O), 3.32 (bs, 1H, -OH), 3.69 (s, 3H, -CH3-O-C(=O)-CH2-), 3.78 (s, 3H, -CH3-O-C(=O)-CH-),
4.47–4.51 (m, 1H, -CH-OH).
Methyl (4S)-(+)-2,2-dimethyl-1,3-dioxolane-4-acetate (3). Compound 3 was synthesized according to
the literature procedure [34]. The data agreed with the literature values. 1H-NMR (300 MHz, CDCl3):
δ in ppm 1.33 (s, 3H, -C-CH3-), 1.39 (s, 3H, -C-CH3-), 2.46–2.54 (m, 1H, -CH'H≡-C=O), 2.66–2.73
(m, 1H, -CH'H≡-C=O), 3.60–3.65 (m, 1H, -O- CH'H≡-), 3.68 (s, 3H, -O-CH3), 4.11–4.16 (m, 1H, -O-
CH'H≡-), 4.45 (quintet, 1H, -O-CH-, J = 6.45 Hz).
(S)-2-(2,2-Dimethyl-1,3-dioxolan-4-yl)-N-(prop-2-ynyl)acetamide (4). A reaction mixture consisting of
propargyl amine (2.95 g, 3.5 mL, 53.63 mmol, 1.0 equiv), methyl (4S)-(+)-2,2-dimethyl-1,3-dioxolane-
4-acetate (3, 11.2 g, 64.36 mmol, 1.2 equiv), 1,2,4-triazole (740 mg, 10.73 mmol, 0.20 equiv.) and DBU
(1.63 g, 1.6 mL, 10.73 mmol, 0.20 equiv.) was stirred at room temperature for 48 h and then applied
directly to a silica gel chromatographic column (diethyl ether-TEA 98:2). Product 4 was then isolated
1
as a colourless oil. Yield: 4.77 g (45.2%). Rf: 0.5 (diethyl ether). H-NMR (300 MHz, CDCl3): δ in
ppm 1.35 (s, 3H, CH3), 1.43 (s, 3H, CH3), 2.22 (t, 1H, -C≡CH, J = 2.5 Hz), 2.45–2.54 (m, 2H,
-CH2-C=O), 3.59-3.64 (m, 1H, -O-CH′H″), 3.91–4.07 (m, 2H, -NH-CH2-C≡), 4.09–4.14 (m, 1H,
13
-O-CH′H″), 4.36–4.45 (m, 1H, -CH-), 6.43 (bs, 1H, -NH-C=O-). C-NMR (75 MHz, CDCl3): δ in
ppm 25.5 Η(-CH3), 26.8 (-CH3), 29.0 (-CH2-NH), 40.1 (-CH2-C=O), 68.9 (-O-CH2), 71.5 (≡CH), 72.2
(-CH), 79.4 (-C≡), 109.5 (O-C-O), 169.6 (-NH-C=O). High-resolution ESI-MS m/z calcd. for
C10H15NO3Na 220.0944 [M+Na]+, found 220.0952.
(S)-3,4-Dihydroxy-N-(prop-2-ynyl)butanamide (5). A solution of compound 4 (4.2 g, 21.32 mmol) in
methanol (100 mL) was treated with Dowex® 50X2 (10 g) at room temperature until completion of the
reaction (4 to 5 h) as evidenced by TLC (methanol-ethyl acetate: 1:9). After removal of the solvent
under reduced pressure, the crude deprotected product applied directly to a silica gel chromatographic
column (methanol-ethyl acetate 1:9). Product 5 was isolated as a white solid. Yield: 3.1 g (92.8%).
1
Rf: 0.33 (methanol-ethyl acetate 1:9). H-NMR (300 MHz, CD3OD): δ in ppm 2.26–2.45 (m, 2H,
-CH2-C=O), 2.59 (t, 1H, -C≡CH, J = 2.5 Hz), 3.48 (d, 2H, J = 5.5, -CH2OH), 3.95–3.99 (m, 2H,
13
NH-CH2-C≡), 4.00-4.09 (m, 1H, -CH-). C-NMR (75 MHz, CD3OD): δ in ppm 29.4 (-CH2-NH),
40.9 (-CH2-C=O), 66.8 (-CH2-OH), 70.4 (-CH-OH), 72.2 (≡CH), 80.5 (-C≡), 173.6 (-NH-C=O).
High-resolution ESI-MS m/z calcd. for C7H11NO3Na 180.0631 [M+Na]+, found 180.0625.
General procedure for synthesis of coupling products 6a, 6b and 6c. To a solution of compound 5
(1 equiv.) in anhydrous dichloromethane was added DCC (3 equiv.), DMAP (3 equiv.) and the
appropriate fatty acid 5a or 5b or 5c (3 equiv.), and the mixture was stirred overnight at room
temperature. The reaction mixture was filtered and the filtrate was concentrated under reduced
pressure. The crude product was purified by column chromatography on silica gel eluting with
different ratios of ethylacetate/hexane to obtain pure product 5a, 5b and 5c.

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(Z)-((S)-4-Oxo-4-(prop-2-ynylamino)butane-1,2-diyl) dioleate (6a). Anhydrous dichloromethane (15 mL),
compound 5 (60 mg, 0.382 mmol, 1.0 equiv.), DCC (236 mg, 1.146 mmol, 3.0 equiv.), DMAP (140
mg, 1.146 mmol, 3.0 equiv.), oleic acid (5a, 324 mg, 364 μL, 1.146 mmol, 3.0 equiv.). Product 6a was
isolated after purification on silica gel (ethyl acetate-hexane 1:3) as a white solid. Yield: 230 mg
(87.7%). Rf: 0.32 (ethyl acetate-hexane 1:3). 1H-NMR (300 MHz, CDCl3): δ in ppm 0.87 (t, 6H, 2 CH3,
J = 6.6 Hz), 1.26–1.29 (m, 40H, 20CH2 of the dioleyl chain), 1.51–1.62 (m, 4H, 2CH2-CH2-C=O),
1.88–2.09 (m, 8H, 2CH2-CH=CH-CH2), 2.23 (t, 1H, -C≡CH, J = 2.5 Hz), 2.27–2.33 (m, 4H,
2CH2-C=O of the dioleyl chain), 2.53 (d, 2H, CH2-C=O, J = 6.4 Hz), 4.02–4.05 (m, 2H, NH-H2-C≡),
4.12–4.18 (m, 1H, -O-CH′H″), 4.31-4.36 (m, 1H, -O-CH′H″), 5.28–5.35 (m, 4H, 2CH=CH of the
dioleyl chain), 5.37–5.42 (m, 1H, -CH-), 5.93 (bs, 1H, -NH-C=O-). 13C-NMR (75 MHz, CDCl3): δ in ppm
14.1 (2CH3), 22.7 (2CH2-CH3), 24.8 (2CH2-CH2-C=O), 27.1 (CH2-CH=CH-CH2), 27.2 (CH2-CH=CH-
CH2), 29.0–29.7 (CH2 of the dioleyl chain and -CH2-NH), 31.9 (2CH2-CH2-CH3), 34.0 (CH2-C=O of
chain), 34.2 (CH2-C=O of chain), 37.9 (-CH2-C=O), 64.2 (-CH2-O-), 68.4 (≡CH), 71.8 (-CH-O-), 79.1
(-C≡), 129.7 (-CH=CH-), 130.0 (-CH=CH-), 168.2 (-NH-C=O), 172.9 (-O-C=O), 173.3 (-O-C=O).
High-resolution ESI-MS m/z calcd. for C43H75NO5Na 708.5537 [M+Na]+, found 708.5532.
(S)-4-Oxo-4-(prop-2-ynylamino)butane-1,2-diyl didodecanoate (6b). Anhydrous dichloromethane (20 mL),
compound 5 (150 mg, 0.95 mmol, 1.0 equiv.), DCC (590 mg, 2.86 mmol, 3.0 equiv.), DMAP (349 mg,
2.86 mmol, 3.0 equiv.), lauric acid (5b, 573 mg, 2.86 mmol, 3.0 equiv.). Product 6b was isolated after
purification on silica gel (ethyl acetate-hexane 3:7) as a white solid. Yield: 465 mg (93.3%). Rf: 0.38
(ethyl acetate-hexane 3:7). 1H-NMR (300 MHz, CDCl3): δ in ppm 0.87 (t, 6H, 2 CH3, J = 6.6 Hz), 1.25
(s, 32H, 16CH2 of the dilauryl chain), 1.51–1.69 (m, 4H, 2CH2-CH2-C=O), 2.23 (t, 1H, -C≡CH,
J = 2.54 Hz), 2.27–2.33 (m, 4H, 2CH2-C=O of the dilauryl chain), 2.53 (d, 2H, CH2-C=O, J = 6.4 Hz),
4.03–4.05 (m, 2H, NH-CH2-C≡), 4.12–4.18 (m, 1H, -O-CH′H″), 4.31–4.36 (m, 1H, -O-CH′H″), 5.35–5.44
13
(m, 1H,-CH-), 5.92 (bs, 1H, -NH-C=O-). C-NMR (75 MHz, CDCl3): δ in ppm 14.1 (2CH3), 22.6
(2CH2-CH3), 24.8 (2CH2-CH2-C=O), 29.0–29.6 (CH2 of the dilauryl chain and -CH2-NH), 31.9
(2CH2-CH2-CH3), 34.0 (CH2-C=O of chain), 34.2 (CH2-C=O of chain), 37.8 (-CH2-C=O), 64.1 (-CH2-O-),
68.4 (≡CH), 71.7 (-CH-O-), 79.1 (-C≡), 168.3 (-NH-C=O), 172.9 (-O-C=O), 173.4 (-O-C=O).
High-resolution ESI-MS m/z calcd. for C31H55NO5Na 544.3972 [M+Na]+, found 544.3978.
(S)-4-Oxo-4-(prop-2-ynylamino)butane-1,2-diyl dioctanoate (6c). Anhydrous dichloromethane
(20 mL), compound 5 (130 mg, 0.83 mmol, 1.0 equiv.), DCC (512 mg, 2.49 mmol, 3.0 equiv.), DMAP
(303 mg, 2.49 mmol, 3.0 equiv.), octanoic acid (5c, 359 mg, 395 μL, 2.49 mmol, 3.0 equiv.). Product
6c was isolated after purification on silica gel (ethyl acetate-hexane 3:7) as a colourless oil (which may
turn into a white solid upon standing). Yield: 310 mg (91.4%). Rf: 0.6 (ethyl acetate-hexane 2:3).
1H-NMR (300 MHz, CDCl3): δ in ppm 0.85 (t, 6H, 2 CH3, J = 6.6 Hz), 1.25 (s, 16H, 8CH2 of the
dioctanoyl chain), 1.56–1.60 (m, 4H, 2CH2-CH2-C=O), 2.22 (t, 1H, -C≡CH, J = 2.54 Hz), 2.25–2.32
(m, 4H, 2CH2-C=O of the dioctanoyl chain), 2.53 (d, 2H, CH2-C=O, J = 6.4 Hz), 4.00–4.03 (m, 2H,
NH-CH2-C≡), 4.11–4.17 (m, 1H, -O-CH′H″), 4.29–4.35 (m, 1H, -O-CH′H″), 5.34–5.42 (m, 1H, -CH-),
13
6.12 (bs, 1H, -NH-C=O-). C-NMR (75 MHz, CDCl3): δ in ppm 14.0 (2CH3), 22.5 (2CH2-CH3), 24.8
(2CH2-CH2-C=O), 28.9–29.2 (CH2 of the dioctanoyl chain and -CH2-NH), 31.6 (2CH2-CH2-CH3), 34.0
(CH2-C=O of chain), 34.2 (CH2-C=O of chain), 37.7 (-CH2-C=O), 64.1 (-CH2-O-), 68.3 (≡CH), 71.7

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(-CH-O-), 79.2 (-C≡), 168.3 (-NH-C=O), 172.9 (-O-C=O), 173.4 (-O-C=O). High-resolution ESI-MS
m/z calcd. for C23H39NO5Na 432.2720 [M+Na]+, found 432.2722.
General procedure for the synthesis of the first click products 7a, 7b and 7c. The above coupling
products 6a or 6b or 6c, sodium ascorbate and copper sulfate were added to 5'-azido-5'-
deoxythymidine [37] in a water/THF (1:1) mixture. The reaction mixture was maintained at 65 °C
under stirring for 10 h. After cooling to room temperature, the solvent was evaporated. The residual
solid was dissolved in DCM and successively washed with water and brine. The organic layer was
dried over Na2SO4 and evaporated under reduced pressure. The crude product was purified by column
chromatography on silica gel eluted with different ratios of methanol/ethyl acetate to obtain purified
compounds 7a, 7b or 7c.
(Z)-((S)-4-(1-(((2R,3S,5R)-3-Hydroxy-5-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)yl) tetrahydrofuran
-2-yl)methyl)-1H-1,2,3-triazol-4-ylamino)-4-oxobutane-1,2-diyl) dioleate (7a). Coupling product 6a
(210 mg, 0.306 mmol, 1.0 equiv.), 5'-azido-5'-deoxythymidine (81.7 mg, 0.306 mmol, 1.0 equiv.),
sodium ascorbate (31 mg, 0.153 mmol, 0.5 equiv.), copper sulfate (13 mg, 0.0765 mmol, 0.25 equiv.),
water/THF (1:1) (20 mL). Product 7a was isolated after purification on silica gel (methanol-ethyl
acetate:2:98) as a white solid. Yield: 248 mg (85.2%). Rf: 0.28 (methanol-ethyl acetate 2:98).
1H-NMR (300 MHz, CDCl3): δ in ppm 0.87 (t, 6H, 2 CH3 of the dioleyl chain, J = 6.7 Hz), 1.26–1.28
(m, 40H, 20CH2 of the dioleyl chain), 1.41–1.66 (m, 4H, 2CH2-CH2-C=O), 1.88 (s, 3H, CH3(base)),
1.96–2.09 (m, 8H, 2CH2-CH=CH-CH2), 2.23–2.42 (m, 6H, 2CH2-C=O of the dioleyl chain,
2H2′(sugar)), 2.47–2.60 (m, 2H, CH2-C=O), 3.38 (bs, 1H, -OH), 4.06–4.12 (m, 1H, NH-CH′H″-C=),
4.15–4.25 (m, 1H, -O-CH′H″), 4.28–4.36 (m, 1H, -O-CH′H″), 4.45–4.77 (m, 5H, NH-CH′H″-C=, H3′,
H4′, 2H5′), 5.27–5.35 (m, 4H, 2CH=CH of the dioleyl chain), 5.37–5.46 (m, 1H, -CH-), 6.00 (t, 1H,
J = 6.4 Hz, H1′), 6.98 (s, 1H, triazole), 7.17 (bs, 1H, -NH-C=O-), 7.77 (s, 1H, H-6(base)), 9.79 (s, 1H,
13
-NH(base)). C-NMR (75 MHz, CDCl3): δ in ppm 12.4 (CH3(base)), 14.1 (2CH3 of the dioleyl chain),
22.6 (2CH2-CH3), 24.9 (2CH2-CH2-C=O), 27.2 (2CH2-CH=CH-CH2), 29.0–29.7 (CH2 of the dioleyl
chain), 31.9 (2CH2-CH2-CH3), 34.0 (CH2-C=O of chain), 34.3 (CH2-C=O of chain), 34.7 (-CH2-NH),
37.6 (C2′), 38.6 (-CH2-C=O), 51.3 (C5′), 64.4 (-CH2-O-), 68.5 (C3′), 71.2 (-CH-O-), 83.8 (C4′), 87.1
(C1′), 111.3 (C5-thymine base), 124.5 (-CH triazole), 129.6 (-CH=CH-), 130.0 (-CH=CH-), 137.1
(C6-thymine base), 144.4 (=C- triazole), 150.3 (C=O(2) thymine base), 164.0 (C=O(4) thymine base),
169.3 (-NH-C=O), 173.2 (-O-C=O of the oleyl chain), 173.6 (-O-C=O of the oleyl chain).
High-resolution ESI-MS m/z calcd. for C53H88N6O9Na 975.6505 [M+Na]+, found 975.6548.
(S)-4-(1-(((2R,3S,5R)-3-Hydroxy-5-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)tetrahydrofuran-2-
yl)methyl)-1H-1,2,3-triazol-4-ylamino)-4-oxobutane-1,2-diyl didodecanoate (7b). Coupling product 6b
(350 mg, 0.67 mmol, 1.0 equiv.), 5'-azido-5'-deoxythymidine (180 mg, 0.67 mmol, 1.0 equiv.), sodium
ascorbate (66 mg, 0.34 mmol, 0.5 equiv.), copper sulfate (27 mg, 0.17 mmol, 0.25 equiv.), water/THF
(1:1) (15 mL). Product 7b was isolated after purification on silica gel (methanol-ethyl acetate 0:100
to 5:95) as a white solid. Yield: 490 mg (92.4%). Rf: 0.25 (methanol-ethyl acetate 2.5:97.5). 1H-NMR
(300 MHz, CDCl3:CD3OD(9.5:0.5)): δ in ppm 0.75 (t, 6H, 2 CH3 of the dilauryl chain, J = 6.6 Hz),
1.13 (s, 32H, 16CH2 of the dilauryl chain), 1.35–1.54 (m, 4H, 2CH2-CH2-C=O), 1.78 (s, 3H, CH3(base)),

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2.06–2.23 (m, 6H, 2CH2-C=O of the dilauryl chain, 2H2′(sugar)), 2.31–2.45 (m, 2H, CH2-C=O),
3.97–4.04 (m, 2H,-O-CH2), 4.15–4.21 (m, 2H, 2H5′(base)), 4.26 (s, 2H, NH-CH2-C=), 4.5–4.62 (m,
2H, H3′(sugar), H4′(sugar)), 5.22–5.34 (m, 1H, -CH-), 5.98 (t, 1H, J = 6.7 Hz, H1′(sugar)), 6.90 (s, 1H,
triazole), 7.65 (s, 1H, H-6(base)). 13C-NMR (75 MHz, CDCl3:CD3OD(9.5:0.5)): δ in ppm 12.1
(CH3(base)), 13.9 (2CH3 of the dilauryl chain), 22.5 (2CH2-CH3), 24.7 (2CH2-CH2-C=O), 28.9–29.4
(CH2 of the dilauryl chain), 31.7 (2CH2-CH2-CH3), 33.9 (CH2-C=O of chain), 34.1 (CH2-C=O of
chain), 34.2 (-CH2-NH), 37.2 (C2′), 38.5 (-CH2-C=O), 51.1 (C5′), 64.2 (-CH2-O-), 68.3 (C3′), 70.6
(-CH-O-), 83.5 (C4′), 86.0 (C1′), 111.1 (C5-thymine base), 124.5 (-CH triazole), 136.4 (C6-thymine
base), 144.3 (=C- triazole), 150.3 (C=O(2) thymine base), 164.2 (C=O(4) thymine base), 169.5
(-NH-C=O), 173.2 (-O-C=O of the lauryl chain), 173.6 (-O-C=O of oleyl chain). High-resolution
ESI-MS m/z calcd. for C41H68N6O9Na 811.4939 [M+Na]+, found 811.4972.
(S)-4-(1-(((2R,3S,5R)-3-Hydroxy-5-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)tetrahydrofuran-2-
yl)methyl)-1H-1,2,3-triazol-4-ylamino)-4-oxobutane-1,2-diyl dioctanoate (7c). Coupling product 6c
(450 mg, 1.1 mmol, 1.0 equiv.), 5'-azido-5'-deoxythymidine (294 mg, 1.1 mmol, 1.0 equiv.), sodium
ascorbate (44 mg, 0.22 mmol, 0.2 equiv.), copper sulfate (18 mg, 0.11 mmol, 0.1 equiv.), water/THF
(1:1) (20 mL). Product 7c was isolated after purification on silica gel (methanol-ethyl acetate 1:9) as a
1
white solid. Yield: 670 mg (90.1%). Rf: 0.33 (methanol-ethyl acetate 5:95). H-NMR (300 MHz,
CD3OD): δ in ppm 0.90 (t, 6H, 2 CH3 of the dioctanoyl chain, J = 6.7 Hz), 1.29 (s, 16H, 8CH2 of the
dioctanoyl chain), 1.48–1.67 (m, 4H, 2CH2-CH2-C=O), 1.90 (s, 3H, CH3(base)), 2.21–2.33 (m, 6H,
2CH2-C=O of the dioctanoyl chain, 2H2′ (sugar)), 2.43–2.60 (m, 2H, CH2-C=O), 4.03–4.18 (m, 2H,
-O-CH2), 4.32–4.46 (m, 4H, 2H5′(base), NH-CH2-C=), 4.62–4.79 (m, 2H, H3′(sugar), H4′(sugar)),
5.37–5.49 (m, 1H, -CH-), 6.19 (t, 1H, J = 6.4 Hz, H1′), 7.32 (s, 1H, triazole), 7.88 (s, 1H, H-6(base)).
13C-NMR (75 MHz, CD3OD): δ in ppm 12.5 (CH3(base)), 14.5 (2CH3 of the dioctanoyl chain), 23.7
(2CH2-CH3), 26.0 (2CH2-CH2-C=O), 30.1-30.2 (CH2 of the dioctanoyl chain), 32.9 (2CH2-CH2-CH3),
34.9 (CH2-C=O of chain), 35.1 (CH2-C=O of chain), 35.6 (-CH2-NH), 38.2 (C2′), 39.6 (-CH2-C=O),
52.8 (C5′), 65.5 (-CH2-O-), 69.9 (C3′), 72.5 (-CH-O-), 85.6 (C4′), 87.0 (C1′), 111.9 (C5-thymine base),
125.4 (-CH triazole), 138.2 (C6-thymine base), 146.1 (=C- triazole), 152.1 (C=O(2) thymine base),
166.3 (C=O(4) thymine base), 171.7 (-NH-C=O), 174.4 (-O-C=O of the octanoyl chain), 174.9
(-O-C=O of octanoyl chain). High-resolution ESI-MS m/z calcd. for C33H52N6O9Na 699.3687
[M+Na]+, found 699.3683.
General procedure for the synthesis of the N-propargyl derivatives of click products 8a, 8b and 8c.
Anhydrous DMF was added under nitrogen to the click products 7a, 7b or 7c, followed by K2CO3.
Propargyl bromide was then added at room temperature, and the reaction mixture was stirred overnight.
DMF was removed under reduced pressure, the residual solid was dissolved in ethyl acetate
(or dichloromethane for 8a) and was washed twice with water and once with brine. The organic phase
was dried over Na2SO4 and evaporated under reduced pressure. The crude product was purified by
column chromatography on silica gel eluted with different ratios of methanol-ethyl acetate to afford
the purified click products 8a, 8b or 8c.

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(Z)-((S)-4-(1-(((2R,3S,5R)-3-Hydroxy-5-(5-methyl-2,4-dioxo-3-(prop-2-ynyl)-3,4-dihydropyrimidin-1(2H)-yl)
tetrahydrofuran-2-yl)methyl)-1H-1,2,3-triazol-4-ylamino)-4-oxobutane-1,2-diyl) dioleate (8a). Click
product 7a (190 mg, 0.2 mmol, 1 equiv.), propargyl bromide (59.5 mg, 43.1 μL (80 wt.% solution in
toluene), 0.4 mmol, 2 equiv.), K2CO3 (54 mg, 0.4 mmol, 2 equiv.), dry DMF (10 mL). Product 8a was
isolated after purification on silica gel (methanol-ethyl acetate 0:100 to 2:98) as a colourless oil. Yield:
185 mg (83.6%). Rf: 0.41 (ethyl acetate). 1H-NMR (300 MHz, CDCl3): δ in ppm 0.87 (t, 6H, 2 CH3 of
the dioleyl chain, J = 6.7 Hz), 1.26–1.28 (m, 40H, 20CH2 of the dioleyl chain), 1.44–1.69 (m, 4H,
2CH2-CH2-C=O), 1.95 (s, 3H, CH3(base)), 1.97–2.04 (m, 8H, 2CH2-CH=CH-CH2), 2.23–2.39 (m, 7H,
2CH2-C=O of the dioleyl chain, 2H2′(sugar), -C≡CH), 2.47–2.58 (m, 2H, CH2-C=O), 2.98 (bs, 1H,
-OH), 4.05–4.12 (m, 1H, -O-CH′H″-C=), 4.21–4.31 (m, 2H, -O-CH′H″, NH-CH′H″-C=), 4.41–4.47 (m,
3H, NH-CH′H″-C=, CH′H″-C≡, H3′), 4.63–4.76 (m, 4H, H4′, 2H5′, -CH′H″-C≡), 5.27–5.35 (m, 4H,
2CH=CH of the dioleyl chain), 5.37–5.44 (m, 1H, -CH-), 6.14 (t, 1H, J = 6.7 Hz, H1′), 6.82 (bs, 1H,
-NH-C=O-), 6.96 (s, 1H, triazole), 7.74 (s, 1H, H-6(base)). 13C-NMR (75 MHz, CDCl3): δ in ppm 13.1
(CH3(base)), 14.1 (2CH3 of the dioleyl chain), 22.6 (2CH2-CH3), 24.8 (2CH2-CH2-C=O), 27.2
(2CH2-CH=CH-CH2), 29.0–29.7 (CH2 of the dioleyl chain), 30.4 (-CH2-C≡), 31.9 (2CH2-CH2-CH3),
34.0 (CH2-C=O of chain), 34.2 (CH2-C=O of chain), 34.7 (-CH2-NH), 37.8 (C2′), 38.6 (-CH2-C=O),
51.4 (C5′), 64.3 (-CH2-O-), 68.4 (C3′), 70.9 (≡CH), 71.5 (-CH-O-), 78.1 (-C≡), 83.9 (C4′), 87.5 (C1′),
110.7 (C5-thymine base), 124.4 (-CH triazole), 129.6 (-CH=CH-), 130.0 (-CH=CH-), 135.0
(C6-thymine base), 144.3 (=C- triazole), 150.0 (C=O(2) thymine base), 162.2 (C=O(4) thymine base),
169.1 (-NH-C=O), 173.2 (-O-C=O of the oleyl chain), 173.6 (-O-C=O of oleyl chain). High-resolution
ESI-MS m/z calcd. for C56H90N6O9Na 1013.6661 [M+Na]+, found 1013.6711.
(S)-4-(1-(((2R,3S,5R)-3-Hydroxy-5-(5-methyl-2,4-dioxo-3-(prop-2-ynyl)-3,4-dihydropyrimidin-1(2H)-yl)
tetrahydrofuran-2-yl)methyl)-1H-1,2,3-triazol-4-ylamino)-4-oxobutane-1,2-diyl didodecanoate (8b).
Click product 7b (440 mg, 0.56 mmol, 1 equiv.), propargyl bromide (116 mg, 120 μL (80 wt.%
solution in toluene), 1.12 mmol, 2 equiv.), K2CO3 (154 mg, 1.12 mmol, 2 equiv.), dry DMF (15 mL).
Product 8b was isolated after purification on silica gel (methanol-ethyl acetate 0:100 to 2:98) as a
1
colourless oil. Yield: 385 mg (83.4%). Rf: 0.30 (methanol-ethyl acetate 2:98). H-NMR (300 MHz,
CDCl3): δ in ppm 0.86 (t, 6H, 2CH3 of the dilauryl chain, J = 6.6 Hz), 1.23 (s, 32H, 16CH2 of the
dilauryl chain), 1.46–1.65 (m, 4H, 2CH2-CH2-C=O), 1.93 (s, 3H, CH3(base)), 2.11–2.42 (m, 7H,
2CH2-C=O of the dilauryl chain, 2H2′(sugar), -C≡CH), 2.47–2.56 (m, 2H, CH2-C=O), 4.05–4.11 (m,
1H, -O-CH′H″-C=), 4.19–4.30 (m, 2H, -O-CH′H″, NH-CH′H″-C=), 4.37–4.49 (m, 3H, NH-CH′H″-C=,
CH′H″-C≡, H3′), 4.58–4.80 (m, 4H, H4′, 2H5′, -CH′H″-C≡), 5.35–5.40 (m, 1H, -CH-), 6.15 (t, 1H,
13
J = 6.7 Hz, H1′), 6.96 (bs, 2H, -NH-C=O, triazole), 7.73 (s, 1H, H-6(base)). C-NMR (75 MHz,
CDCl3): δ in ppm 13.1 (CH3(base)), 14.1 (2CH3 of the dilauryl chain), 22.6 (2CH2-CH3), 24.8
(2CH2-CH2-C=O), 29.0–29.6 (CH2 of the dilauryl chain), 30.4 (-CH2-C≡), 31.8 (2CH2-CH2-CH3),
34.0 (CH2-C=O of chain), 34.2 (CH2-C=O of chain), 34.7 (-CH2-NH), 37.7 (C2′), 38.5 (-CH2-C=O),
51.2 (C5′), 64.3 (-CH2-O-), 68.4 (C3′), 70.9 (≡CH), 71.3 (-CH-O-), 78.1 (-C≡), 83.9 (C4′), 87.2 (C1′),
110.7 (C5-thymine base), 124.3 (-CH triazole), 135.0 (C6-thymine base), 144.4 (=C- triazole), 150.0
(C=O(2) thymine base), 162.2 (C=O(4) thymine base), 169.1 (-NH-C=O), 173.2 (-O-C=O of the lauryl
chain), 173.6 (-O-C=O of oleyl chain). High-resolution ESI-MS m/z calcd. for C44H70N6O9Na 849.5096
[M+Na]+, found 849.5111.

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(S)-4-(1-(((2R,3S,5R)-3-Hydroxy-5-(5-methyl-2,4-dioxo-3-(prop-2-ynyl)-3,4-dihydropyrimidin-1(2H)-
yl)tetrahydrofuran-2-yl)methyl)-1H-1,2,3-triazol-4-ylamino)-4-oxobutane-1,2-diyl dioctanoate (8c).
Click product 7c (565 mg, 0.84 mmol, 1 equiv), propargyl bromide (248 mg, 180 μL (80 wt.% solution
in toluene), 1.67 mmol, 2 equiv.), K2CO3 (231 mg, 1.67 mmol, 2 equiv.), dry DMF (20 mL). Product
8c was isolated after purification on silica gel (methanol-ethyl acetate 5:95) as a colourless oil. Yield:
1
540 mg (90.5%). Rf: 0.45 (methanol-ethyl acetate 5:95). H-NMR (300 MHz, DMSO-d6): δ in ppm
0.84 (t, 6H, 2CH3 of dioctanoyl chain, J = 6.2 Hz), 1.23 (s, 16H, 16CH2 of the dioctanoyl chain),
1.37–1.57 (m, 4H, 2CH2-CH2-C=O), 1.87 (s, 3H, CH3(base)), 2.15–2.27 (m, 6H, 2CH2-C=O of the
dioctanoyl chain, 2H2′(sugar)), 2.44 (d, 2H, J = 6.6 Hz, CH2-C=O), 3.10 (t, 1H, J = 2.26 Hz, -C≡CH),
3.99–4.11 (m, 2H, -O-CH2), 4.22–4.30 (m, 4H, 2H5′(base), NH-CH2-C=), 4.52 (s, 2H, -CH2-C≡),
4.56–4.73 (m, 2H, H3′(sugar), H4′(sugar)), 5.26–5.34 (m, 1H, -CH-), 5.56 (d, 1H, J = 4.14 Hz,
-NH-C=O, ), 6.21 (t, 1H, J = 6.9 Hz, H1′), 7.49 (s, 1H, triazole), 8.49 (t, 1H, J = 5.47 Hz, H-6(base)).
13C-NMR (75 MHz, DMSO-d6): δ in ppm 12.7 (CH3(base)), 14.0 (2CH3 of the dioctanoyl chain), 22.1
(2CH2-CH3), 24.4–24.5 (2CH2-CH2-C=O), 28.4–28.5 (CH2 of the dioctanoyl chain), 30.1 (-CH2-C≡),
31.2 (2CH2-CH2-CH3), 33.4 (CH2-C=O of chain), 33.6 (CH2-C=O of chain), 34.2 (-CH2-NH), 36.4
(C2′), 38.0 (-CH2-C=O), 51.2 (C5′), 64.1 (-CH2-O-), 68.3 (C3′), 70.8 (≡CH) 73.1 (-CH-O-), 79.1 (-C≡),
84.3 (C4′), 85.3 (C1′), 109.1 (C5-thymine base), 123.6 (-CH triazole), 135.3 (C6-thymine base), 144.7
(=C- triazole), 149.8 (C=O(2) thymine base), 161.8 (C=O(4) thymine base), 168.3 (-NH-C=O), 172.1
(-O-C=O of the octanoyl chain), 172.6 (-O-C=O of the octanoyl chain). High-resolution ESI-MS m/z
calcd. for C36H54N6O9Na 737.3844 [M+Na]+, found 737.3823.
General procedure for the synthesis of GNLs 9a, 9b and 9c. The above N-propargyl derivative 8a, 8b or
8c, sodium ascorbate and copper sulfate were added to 1-azido-β-D-glucopyranoside in water/THF
(1:1) mixture. The reaction mixture was maintained at 65 °C under stirring for 10 h, then cooled to
room temperature and the solvent was evaporated. The residual solid was dissolved in DCM and
successively washed by water and brine. The organic layer was dried over Na2SO4 and evaporated
under reduced pressure. The crude product was purified by column chromatography on silica gel
eluted with different ratios of methanol/dichloromethane to obtain pure click product 9a, 9b or 9c.
(Z)-4-(1-(((2R,3S,5R)-3-Hydroxy-5-(5-methyl-2,4-dioxo-3-((1-((2R,3R,4S,5S,6R)-3,4,5-trihydroxy-6-
(hydroxymethyl)tetrahydro-2H-pyran-2-yl)-1H-1,2,3-triazol-4-yl)methyl)-3,4-dihydropyrimidin-1(2H)-
yl)tetrahydrofuran-2-yl)methyl)-1H-1,2,3-triazol-4-ylamino)-4-oxobutane-1,2-diyl
dioleate
(9a).
N-propargyl derivative 8a (160 mg, 0.16 mmol, 1.0 equiv), 1-azido-β-D-glucopyranoside (33 mg,
0.16 mmol, 1.0 equiv.), sodium ascorbate (16 mg, 0.2 mmol, 0.5 equiv.), copper sulfate (6.5 mg,
0.1 mmol, 0.25 equiv.), water-THF (1:1) (15 mL). Product 9a was isolated after purification on silica
gel (methanol-dichloromethane 15:85) as a white solid. Yield: 170 mg (88.1%). Rf: 0.4 (methanol-
1
dichloromethane 15:85). H-NMR (300 MHz, DMSO-d6): δ in ppm 0.84 (t, 6H, 2CH3 of the dioleyl
chain, J = 6.0 Hz), 1.23 (s, 40H, 20CH2 of the dioleyl chain), 1.38–1.57 (m, 4H, 2CH2-CH2-C=O),
1.88 (s, 3H, CH3(base)), 1.90–2.07 (m, 8H, 2CH2-CH=CH-CH2), 2.15–2.30 (m, 6H, 2CH2-C=O of the
dioleyl chain, 2H2′(sugar)), 2.44 (d, 2H, J = 6.2 Hz, CH2-C=O), 3.17–3.23 (m, 1H, -O-CH-
(glucose-C5)), 3.32–3.45 (m, 4H, -CH2-OH(glucose-C6), -CH(glucose-C4), -OH(glucose (C6)), 3.65–3.77
(m, 4H, -CH(glucose-C3), H3′, -OH(C3′-sugar), -OH(glucose-C3)), 4.00-4.14 (m, 3H, -O-CH2-,

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-OH(glucose-C4)), 4.26–4.28 (m, 5H, -OH(glucose-C2), 2H5′, -CH2-NH-C=O), 4.56–4.72 (m, 2H, H4′,
-CH(glucose-C2)), 5.05 (s, 2H, N-CH2-triazole), 5.25–5.35 (m, 5H, -CH-, 2CH=CH of the dioleyl
chain 5.48 (d, 1H, -N-CH-O (glucose-C1), J = 9.2 Hz), 6.22 (t, 1H, H1′, J = 6.7 Hz), 7.49 (s, 1H,
triazole-between nucleoside and glucose), 7.92 (s, 1H, triazole-between alkyl chain and nucleoside),
13
8.11 (s, 1H, H-6(base), 8.49 (bs, 1H, -NH-C=O-). C-NMR (75 MHz, DMSO-d6): δ in ppm 12.8
(CH3(base)), 14.0 (2CH3 of the dioleyl chain), 22.2 (2CH2-CH3), 24.4 (CH2-CH2-C=O), 24.5
(CH2-CH2-C=O), 26.6 (CH2-CH=CH-CH2), 26.7 (CH2-CH=CH-CH2), 28.5–29.2 (CH2 of the dioleyl
chain), 31.4 (2CH2-CH2-CH3), 33.4 (CH2-C=O of chain), 33.6 (CH2-C=O of chain), 34.2 (-CH2-NH),
36.2 (N-CH2-triazole), 36.4 (C2′), 38.1 (-CH2-C=O), 51.2 (C5′), 60.7 (-CH2-OH (glucose-C6)), 64.2
(-CH2-O-), 68.3 (C3′), 69.6 (-CH(glucose-C2)), 70.8 (-CH(glucose-C4)), 71.9 (-CH-O-), 77.0
(-CH(glucose-C3)), 80.0 (-CH(glucose-C5)), 84.2 (-CH(glucose-C1)), 85.2 (C4′), 87.4 (C1′), 109.2
(C5-thymine base), 122.5 (-CH triazole between thymidine and alkyl chain), 123.6 (-CH triazole
between glucose and thymidine), 129.6 (2-CH=CH-), 135.1 (C6-thymine base), 142.6 (=C- triazole
triazole between glucose and thymidine), 144.7 (=C- triazole between thymidine and alkyl chain),
150.3 (C=O(2) thymine base), 162.3 (C=O(4) thymine base), 168.3 (-NH-C=O), 172.0 (-O-C=O of the
oleyl chain), 172.5 (-O-C=O of oleyl chain). High-resolution FD-MS m/z calcd. for C62H102N9O14
1196.7546 [M]+, found 1196.7449.
4-(1-(((2R,3S,5R)-3-Hydroxy-5-(5-methyl-2,4-dioxo-3-((1-((2R,3R,4S,5S,6R)-3,4,5-trihydroxy-6-
(hydroxymethyl)tetrahydro-2H-pyran-2-yl)-1H-1,2,3-triazol-4-yl)methyl)-3,4-dihydropyrimidin-1(2H)-
yl)tetrahydrofuran-2-yl)methyl)-1H-1,2,3-triazol-4-ylamino)-4-oxobutane-1,2-diyl didodecanoate (9b).
N-propargyl derivative 8b (335 mg, 0.4 mmol, 1.0 equiv), 1-azido-β-D-glucopyranoside (83 mg,
0.4 mmol, 1.0 equiv.), sodium ascorbate (40 mg, 0.2 mmol, 0.5 equiv.), copper sulfate (16.2 mg, 0.1 mmol,
0.25 equiv.), water/THF (1:1) (20 mL). Product 9b was isolated after purification on silica gel
(methanol-dichloromethane 15:85) as a white solid. Yield: 374 mg (89.6%). Rf: 0.52 (methanol-
1
dichloromethane 15:85). H-NMR (300 MHz, DMSO-d6): δ in ppm 0.85 (t, 6H, 2CH3 of the dilauryl
chain, J = 6.2 Hz), 1.23 (s, 32H, 16CH2 of the dilauryl chain), 1.46–1.49 (m, 4H, 2CH2-CH2-C=O),
1.88 (s, 3H, CH3(base)), 2.07–2.27 (m, 6H, 2CH2-C=O of the dilauryl chain, 2H2′(sugar)), 2.45
(d, 2H, J = 6.6 Hz, CH2-C=O), 3.18–3.24 (m, 1H, -O-CH- (glucose-C5)), 3.33–3.46 (m, 4H, -CH2-
OH(glucose-C6), -CH(glucose-C4), -OH(glucose (C6)), 3.65–3.77 (m, 3H, -CH(glucose-C3), H3′,
-OH(C3′-sugar)), 4.00–4.11 (m, 3H, -O-CH2-, -OH (glucose-C3)), 4.26–4.28 (m, 4H, -OH(glucose-C4),
H5′, -CH2-NH-C=O), 4.57–4.73 (m, 3H, H4′, H5″, -CH(glucose-C2)), 5.05 (s, 2H, N-CH2-triazole),
5.29–5.36 (m, 2H, -CH-, -OH (glucose-C2)), 5.48 (d, 1H, -N-CH-O (glucose-C1), J = 9.2 Hz), 6.23
(t, 1H, H1′, J = 6.8 Hz), 7.49 (s, 1H, triazole-between nucleoside and glucose), 7.92 (s, 1H, triazole-
between alkyl chain and nucleoside), 8.10 (s, 1H, H-6(base), 8.48 (t,1H, -NH-C=O-, J = 5.4 Hz).
13C-NMR (75 MHz, DMSO-d6): δ in ppm 12.7 (CH3(base)), 13.9 (2CH3 of the dilauryl chain), 22.1
(2CH2-CH3), 24.4 (CH2-CH2-C=O), 24.5 (CH2-CH2-C=O), 28.4-29.1 (CH2 of the dilauryl chain), 31.3
(2CH2-CH2-CH3), 33.4 (CH2-C=O of chain), 33.6 (CH2-C=O of chain), 34.1 (-CH2-NH), 36.1
(N-CH2-triazole), 36.4 (C2′), 38.1 (-CH2-C=O), 51.1 (C5′), 60.7 (-CH2-OH (glucose-C6)), 64.1 (-CH2-O-),
68.3 (C3′), 69.5 (-CH(glucose-C2)), 70.7 (-CH(glucose-C4)), 71.9 (-CH-O-), 77.0 (-CH(glucose-C3)),
80.0 (-CH(glucose-C5)), 84.2 (-CH(glucose-C1)), 85.1 (C4′), 87.4 (C1′), 109.1 (C5-thymine base),
122.5 (-CH triazole between thymidine and alkyl chain), 123.5 (-CH triazole between glucose and

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thymidine), 135.1 (C6-thymine base), 142.6 (=C- triazole triazole between glucose and thymidine),
144.6 (=C- triazole between thymidine and alkyl chain), 150.3 (C=O(2) thymine base), 162.3 (C=O(4)
thymine base), 168.3 (-NH-C=O), 172.0 (-O-C=O of the lauryl chain), 172.5 (-O-C=O of the lauryl
chain). High-resolution ESI-MS m/z calcd. for C50H81N9O14Na 1054.5795 [M+Na]+, found 1054.5854.
4-(1-(((2R,3S,5R)-3-Hydroxy-5-(5-methyl-2,4-dioxo-3-((1-((2R,3R,4S,5S,6R)-3,4,5-trihydroxy-6-
(hydroxymethyl)tetrahydro-2H-pyran-2-yl)-1H-1,2,3-triazol-4-yl)methyl)-3,4-dihydropyrimidin-1(2H)-
yl)tetrahydrofuran-2-yl)methyl)-1H-1,2,3-triazol-4-ylamino)-4-oxobutane-1,2-diyl dioctanoate (9c).
N-propargyl derivative 8c (490 mg, 0.7 mmol, 1.0 equiv.), 1-azido-β-D-glucopyranoside (141 mg,
0.7 mmol, 1.0 equiv.), sodium ascorbate (28 mg, 0.14 mmol, 0.2 equiv.), copper sulfate (11 mg, 0.07 mmol,
0.1 equiv.), water/THF (1:1) (20 mL). Product 9c was isolated after purification on silica gel
(methanol-dichloromethane 15:85) as a white solid. Yield: 530 mg (84.1%). Rf: 0.46 (methanol-
1
dichloromethane 15:85). H-NMR (300 MHz, DMSO-d6): δ in ppm 0.85 (t, 6H, 2CH3 of the
dioctanoyl chain, J = 6.2 Hz), 1.23 (s, 32H, 16CH2 of the dioctanoyl chain), 1.36–1.60 (m, 4H, 2CH2-
CH2-C=O), 1.88 (s, 3H, CH3(base)), 2.16–2.28 (m, 6H, 2CH2-C=O of the dioctanoyl chain,
2H2′(sugar)), 2.45 (d, 2H, J = 6.8 Hz, CH2-C=O), 3.17–3.25 (m, 1H, -O-CH- (glucose-C5)), 3.32–3.47
(m, 3H, -CH2-OH(glucose-C6), -CH(glucose-C4), 3.64–3.78 (m, 2H, -CH(glucose-C3, H3′), 4.01–4.12
(m, 2H, -O-CH2-), 4.23–4.28 (m, 4H, -OH(glucose-C4), H5′, -CH2-NH-C=O,), 4.57–4.73 (m, 3H, H4′,
H5″,-CH(glucose-C2)), 5.05 (s, 2H, N-CH2-triazole), 5.14 (d, 1H, -OH(glucose (C6), J = 5.3 Hz), 5.24
(d, 1H, -OH (glucose-C3), J = 4.7 Hz), 5.29–5.31 (m, 1H, -CH-), 5.34 (d, 1H, -OH (glucose-C2),
J = 6.0 Hz) 5.48 (d, 1H, -N-CH-O (glucose-C1), J = 9.2 Hz), 5.53 (d, 1H, -OH(C3′-sugar), J = 4.2 Hz)
6.23 (t, 1H, H1′, J = 6.8 Hz), 7.49 (s, 1H, triazole-between nucleoside and glucose), 7.91 (s, 1H,
triazole-between alkyl chain and nucleoside), 8.10 (s, 1H, H-6(base), 8.48 (t, 1H, -NH-C=O-, J = 5.4 Hz).
13C-NMR (75 MHz, DMSO-d6): δ in ppm 12.7 (CH3(base)), 13.9 (2CH3 of the dioctanoyl chain), 22.1
(2CH2-CH3), 24.4 (CH2-CH2-C=O), 24.5 (CH2-CH2-C=O), 28.3–28.4 (CH2 of the dioctanoyl chain),
31.1 (2CH2-CH2-CH3), 33.4 (CH2-C=O of chain), 33.6 (CH2-C=O of chain), 34.1 (-CH2-NH), 36.1
(N-CH2-triazole), 36.4 (C2′), 38.1 (-CH2-C=O), 51.1 (C5′), 60.7 (-CH2-OH (glucose-C6)), 64.1 (-CH2-O-),
68.3 (C3′), 69.5 (-CH(glucose-C2)), 70.7 (-CH(glucose-C4)), 71.9 (-CH-O-), 77.0 (-CH(glucose-C3)),
80.0 (-CH(glucose-C5)), 84.2 (-CH(glucose-C1)), 85.2 (C4′), 87.4 (C1′), 109.1 (C5-thymine base),
122.5 (-CH triazole between glucose and thymidine), 123.6 (-CH triazole between thymidine and alkyl
chain), 135.1 (C6-thymine base), 142.6 (=C- triazole triazole between glucose and thymidine), 144.7
(=C- triazole between thymidine and alkyl chain), 150.3 (C=O(2) thymine base), 162.3 (C=O(4)
thymine base), 168.3 (-NH-C=O), 172.1 (-O-C=O of the dioctanoyl chain), 172.6 (-O-C=O of
the dioctanoyl chain). High-resolution ESI-MS m/z calcd. for C42H65N9O14Na 942.4543 [M+Na]+,
found 942.4562.
1-((2R,4S,5R)-5-((tert-Butyldimethylsilyloxy)methyl)-4-hydroxytetrahydrofuran-2-yl)-5-methylpyrimidine-
2,4(1H,3H)-dione (11). To a solution of thymidine 10 (5 g, 20.6 mmol, 1.0 equiv) in pyridine (125 mL)
was added DMAP (0.126 g, 1.03 mmol, 0.05 equiv.) and TBDMS-Cl (3.48 g, 23.1 mmol, 1.1 equiv.)
sequentially. The reaction mixture was stirred overnight at room temperature. After removal of the
solvent under reduced pressure, the crude reaction mixture was dissolved in DCM and successively
washed with water, aqueous NaHCO3 solution (5%) and brine. The organic layer was dried over

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Na2SO4 and evaporated under reduced pressure. The crude product was purified by column
chromatography on silica gel (ethyl acetate-hexane-TEA (79:20:1)) to give pure 11 as a white solid.
Yield: 5.4 g (73.5%). Rf: 0.45 (ethyl acetate-hexane-TEA 79:20:1). The data agreed with the literature
values [41,42]. 1H-NMR (300 MHz, CDCl3): δ in ppm 0.1 (s, 6H, -Si(CH3)2), 0.91 (s, 9H, -Si-C(CH3)2),
1.90 (s, 3H, CH3(base)), 2.04-2.12 (m, 1H, H2'(sugar)), 2.35-2.42 (m, 1H, H2 (sugar)), 3.12 (bs, 1H,
-OH(sugar)), 3.80–3.92 (m, 2H, 2H5'(sugar)), 4.02–4.11 (m, 1H, H3'(sugar)), 4.44 (bs, 1H, H4'(sugar)),
6.37–6.42 (m, 1H, 1H'(sugar)), 7.53 (s, 1H, H-6(base)), 9.39 (s, 1H, -NH(base)).
1-((2R,4S,5R)-5-((tert-Butyldimethylsilyloxy)methyl)-4-(prop-2-ynyloxy)tetrahydrofuran-2-yl)-5-
methylpyrimidine- 2,4(1H,3H)-dione (12). Anhydrous THF (100 mL) was added under nitrogen to
TBDMS protected thymidine 11 (5 g, 14.03 mmol, 1 equiv.). The mixture was cooled at 0 °C and NaH
(1.4 g (60% in oil), 35.06 mmol, 2.5 equiv.) was added by small portion. Propargyl bromide (4.69 g,
3.4 mL (80 wt.% solution in toluene), 31.55 mmol, 2.25 equiv) was then added at room temperature.
The reaction mixture was maintained at 50 °C under stirring for 24 h. The reaction was stopped by
addition of methanol (20 mL). The solvent was removed under reduced pressure. The residual mixture
was dissolved in dichloromethane and successively washed by water and brine. The organic layer then
dried over Na2SO4 and evaporated under reduced pressure. The crude product was purified by column
chromatography on silica gel (ethyl acetate-hexane-TEA 39:60:1 to 49:50:1). Product 12 was isolated
as a colourless oil. Yield: 4.95 g (89.5%). Rf: 0.44 (ethyl acetate-hexane-TEA (49:50:1)). The data
agreed with the literature values [39]. 1H-NMR (300 MHz, CDCl3): δ in ppm 0.09 (s, 6H, -Si(CH3)2),
0.89 (s, 9H, -Si-C(CH3)2), 1.88 (s, 3H, CH3(base)), 1.92–2.01 (m, 1H, H2'(sugar)), 2.40–2.47 (m, 2H,
H2 (sugar), -C≡CH), 3.75–3.90 (m, 2H, 2H5'), 4.07–4.24 (m, 3H, H3'(sugar), O-CH2-C≡), 4.31–4.33
(m, 1H, H4'(sugar)), 6.25–6.29 (m, 1H, 1H'(sugar)), 7.48 (s, 1H, H-6(base)), 9.76 (s, 1H, -NH(base)).
13C-NMR (75 MHz, CDCl3): δ in ppm −5.56 (-Si-CH3), −5.5 (-Si-CH3), 12.4 (CH3(base)), 18.2
(-C-(CH3)3), 25.8 (-C-(CH3)3), 37.5 (C2′), 56.2 (-O-CH2-C≡), 63.4 (C5'), 75.0 (≡CH), 78.4 (C3'), 79.0
(-C≡), 84.7 (C4'), 84.8 (C1'), 110.6 (C5-thymine base), 135.2 (C6-thymine base), 150.5 (C=O(2)
thymine base), 164.1 (C=O(4) thymine base). High-resolution ESI-MS m/z calcd. for C19H30N2O5NaSi
417.1816 [M+Na]+, found. 417.1834.
1-((2R,4S,5R)-5-(Hydroxymethyl)-4-(prop-2-ynyloxy)tetrahydrofuran-2-yl)-5-methylpyrimidine-
2,4(1H,3H)-dione (13). To a mixture of compound 12 (4.85 g, 12.3 mmol, 1.0 equiv.) in THF (40 mL)
was added 1M solution of TBAF (14.7 mL, 14.75 mmol, 1.2 equiv.) in THF and the resulting mixture
was stirred at room temperature for 1h. After removal of the solvent under reduced pressure, the crude
deprotected product was applied directly to a silica gel chromatographic column (methanol-ethyl acetate
0:100 to 5:95). Product 13 was isolated as a white solid. Yield: 3.2 g (93.0%). Rf: 0.55 (ethyl acetate).
1H-NMR (300 MHz, DMSO-d6): δ in ppm 1.77 (s, 3H, CH3(base)), 2.04–2.33 (m, 2H, H2'(sugar)),
3.47 (s, -C≡CH), 3.58 (bs 2H, 2H5'), 3.93 (bs, 1H, H3'(sugar)), 4.21 (bs, 3H, - OH, O-CH2-C≡), 5.15
(s, 1H, H4'(sugar)), 6.10 (bs, 1H, 1H'(sugar)), 7.70 (s, 1H, H-6(base)), 11.34 (s, 1H, -NH(base)).
13C-NMR (75 MHz, DMSO-d6): δ in ppm 12.4 (CH3(base)), 36.1 (C2′), 55.9 (-O-CH2-C≡), 61.6 (C5'),
77.4 (≡CH), 78.8 (C3'), 80.3 (-C≡), 83.9 (C4'), 84.5 (C1'), 109.7 (C5-thymine base), 136.1
(C6-thymine base), 150.6 (C=O(2) thymine base), 163.9 (C=O(4) thymine base). High-resolution
ESI-MS m/z calcd. for C13H16N2O5Na 303.0951 [M+Na]+, found. 303.0945.

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(2R,3R,4S,5R,6R)-2-(Acetoxymethyl)-6-(4-(((2R,3S,5R)-2-(hydroxymethyl)-5-(5-methyl-2,4-dioxo-3,4-
dihydropyrimidin-1(2H)-yl)tetrahydrofuran-3-yloxy)methyl)-1H-1,2,3-triazol-1-yl)tetrahydro-2H-
pyran-3,4,5-triyl triacetate (14). 1-Azido-2,3,4,6-tetra-O-acetyl-β-D-glucopyranoside (2.5 g, 6.69 mmol,
1 equiv.), sodium ascorbate (265.5 mg, 1.34 mmol, 0.2 equiv.) and copper sulfate (106.8 mg, 0.66
mmol, 0.1 equiv.) were added to compound 13 (1.87 g, 6.69 mmol, 1 equiv.) in water/THF (1:1)
mixture (100 mL). The reaction mixture was maintained at 65 °C under stirring for 10 h. The mixture
was cooled down to room temperature. After removal of the solvent under reduced pressure, the crude
product was applied directly to a silica gel chromatographic column (methanol-ethyl acetate 0:100 to 5:95).
Product 14 was isolated as a white solid (foam). Yield: 4.24 g (97.1%). Rf: 0.25 (ethyl acetate).
1H-NMR (300 MHz, CDCl3): δ in ppm 1.88 (s, 3H, CH3(base)), 1.89–2.08 (m, 12H, 4CH3-
C=O(glucose)), 2.24–2.33 (m, 1H, H2'(sugar)), 2.37–2.45 (m, 1H, H2 (sugar), 3.31 (bs, 1H, -OH),
3.73–4.17 (m, 5H, 2H5'(sugar), H3'(sugar), O-CH2-triazole), 4.25–4.34 (m, 2H, H4'(sugar),
-O-CH(glucose-C5)), 4.68 (q, 2H, -CH2-OAc(glucose), J = 12.7 Hz), 5.25 (t, 1H, -CH(glucose-C4),
J = 9.5 Hz), 5.34-5.48 (m, 2H, 2-CH(glucose-C2, C3)), 5.88 (d, 1H, -N-CH-O-(glucose), J = 8.85 Hz),
6.15 (t, 1H, 1H'(sugar), J = 6.8 Hz), 7.46 (s, 1H, triazole), 7.86 (s, 1H, H-6(base)), 9.10 (s, 1H,
-NH(base)). 13C-NMR (75 MHz, CDCl3): δ in ppm 12.4 (CH3(base)), 20.1–20.6 (4 -CH3 (acetyl)), 36.1
(C2′), 61.4 (-O-CH2-triazole), 62.0 (C5'), 62.2 (-CH2-OAc), 67.5 (-CH (glucose-C3)), 70.5 (-CH
(glucose-C2)), 72.3 (-CH (glucose-C4)), 74.9 (-CH (glucose-C5)), 78.0 (-CH (glucose-C1)), 84.9 (C3'),
85.5 (C4'), 86.0 (C1'), 110.8 (C5-thymine base), 121.4 (-CH triazole), 136.7 (C6-thymine base), 145.2
(=C- triazole), 150.5 (C=O(2) thymine base), 164.1 (C=O(4) thymine base), 169.3–169.9
(3 C=O(acetyl at C2, C3, C4)), 170.5 (-CH2-C=O(acetyl at C6)). High-resolution ESI-MS m/z calcd.
for C27H35N5O14Na 676.2072 [M+Na]+, found. 676.2089.
(2R,3R,4S,5R,6R)-2-(Acetoxymethyl)-6-(4-(((2R,3S,5R)-2-(azidomethyl)-5-(5-methyl-2,4-dioxo-3,4-
dihydropyrimidin-1(2H)-yl)tetrahydrofuran-3-yloxy)methyl)-1H-1,2,3-triazol-1-yl)tetrahydro-2H-
pyran-3,4,5-triyl triacetate (15). Anhydrous pyridine (80 mL) was added to click product 14 (4 g,
6.12 mmol, 1 equiv.). The mixture was cooled to 0 °C and methanesulfonyl chloride (0.92 g, 0.62 mL,
7.95 mmol, 1.3 equiv.) was added dropwise. The mixture was stirred at room temperature for 4 h. The
solvent was evaporated under reduced pressure and the residual compound was used directly in the
following step without further purification. To the above crude product, DMF (80 mL) and sodium
azide (1.99 g, 30.6 mmol, 5 equiv) were added. The reaction mixture was maintained at 80 °C under
stirring for 4 h. DMF was removed under reduced pressure. The residual solid was dissolved in ethyl
acetate and washed successively twice with aqueous 5% NaHCO3 solution and once with brine. The
organic phase was dried on Na2SO4 and evaporated under reduced pressure. The crude product was
purified by column chromatography on silica gel (ethyl acetate). Product 15 was isolated as a white
1
solid (foam). Yield: 3.85 g (92.7%). Rf: 0.45 (ethyl acetate). H NMR (300 MHz, CDCl3): δ in ppm
1.88 (s, 3H, CH3(base)), 1.92–2.08 (m, 12H, 4CH3-C=O(glucose)), 2.11–2.15 (m, 1H, H2'(sugar)),
2.38–2.46 (m, 1H, H2 (sugar), 3.54–3.73 (m, 2H, 2H5'(sugar), 3.99–4.19 (m, 4H, H3'(sugar), O-CH2-
triazol, -O-CH(glucose-C5)), 4.28–4.34 (m, 1H, H4'(sugar), 4.66 (q, 2H, -CH2-OAc(glucose),
J = 12.2 Hz), 5.24 (t, 1H, -CH(glucose-C4), J = 9.5 Hz), 5.35–5.48 (m, 2H, 2-CH(glucose-C2, C3)),
5.88 (d, 1H, -N-CH-O-(glucose), J = 8.86 Hz), 6.23–6.28 (m, 1H, 1H'(sugar)), 7.32 (s, 1H, triazole),
7.83 (s, 1H, H-6(base)), 8.95 (s, 1H, -NH(base)). 13C-NMR (75 MHz, CDCl3): δ in ppm 12.5

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(CH3(base)), 20.0-20.6 (4 -CH3 (acetyl)), 36.9 (C2′), 52.3 (C5'), 61.4 (-O-CH2-triazole), 62.5 (-CH2-OAc),
67.5 (-CH (glucose-C3)), 70.4 (-CH (glucose-C2)), 72.2 (-CH (glucose-C4)), 74.9 (-CH (glucose-C5)),
78.5 (-CH (glucose-C1)), 82.3 (C3'), 84.8 (C4'), 85.6 (C1'), 111.3 (C5-thymine base), 121.2 (-CH triazole),
135.3 (C6-thymine base), 145.0 (=C- triazole), 150.3 (C=O(2) thymine base), 163.8 (C=O(4) thymine
base), 168.9–169.8 (3 C=O(acetyl at C2, C3, C4)), 170.4 (-CH2-C=O(acetyl at C6)). High-resolution
ESI-MS m/z calcd. for C27H34N8O13Na 701.2137 [M+Na]+, found. 701.2139.
(2R,3R,4S,5R,6R)-2-(Acetoxymethyl)-6-(4-(((2R,3S,5R)-2-((4-((4,4,5,5,6,6,7,7,8,8,9,9,10,10,11,11,11-
heptadecafluoroundecanamido)methyl)-1H-1,2,3-triazol-1-yl)methyl)-5-(5-methyl-2,4-dioxo-3,4-
dihydropyrimidin-1(2H)-yl)tetrahydrofuran-3-yloxy)methyl)-1H-1,2,3-triazol-1-yl)tetrahydro-2H-
pyran-3,4,5-triyl triacetate (16). N-propargyl-1H,1H,2H,2H-perfluoroundecanoyl amide[29] (480 mg,
0.91 mmol, 1 equiv.), sodium ascorbate (36 mg, 0.18 mmol, 0.2 equiv.) and copper sulfate (14.5 mg,
0.091 mmol, 0.1 equiv.) were added to azido derivative 15 (616 mg, 0.91 mmol, 1 equiv.) in
water/THF (1:1) mixture (50 mL). The reaction mixture was maintained at 65 °C under stirring for 10 h.
The mixture was cooled down to room temperature. After removal of the solvent under reduced
pressure, the crude product applied directly to a silica gel chromatographic column (methanol-ethyl
acetate 0:100 to 5:95). Product 16 was isolated as a white sticky foam. Yield: 1.03 g (94.4%). Rf: 0.2
(ethyl acetate). 1H-NMR (300 MHz, CDCl3): δ in ppm 1.69 (s, 3H, CH3(base)), 1.80, 2.00 and 2.11 (s,
12H, 4CH3-C=O(glucose)), 2.13–2.73 (m, 6H, 2H2'(sugar), CF2-CH2-CH2-C=O), 4.07–4.45 (m, 6H,
H5≡(sugar), -CH2-OAc(glucose-C6), -O-CH(glucose-C5), triazole-CH'H≡-NH-C=O, H3'(sugar)),
4.61–4.70 (m, 3H, O-CH2-triazole, H5'(sugar)), 4.80–4.89 (m, 2H, H4'(sugar), triazole-CH'H -NH-
C=O), 5.40–5.51 (m, 2H, 2-CH(glucose-C3, C4)), 5.69–5.78 (m, 1H, -CH(glucose-C2)), 5.82–5.90
(m, 1H-N-CH-O-(glucose-C1)), 6.22–6.27 (m, 1H, 1H'(sugar)), 6.54 (s, 1H, triazole-between
nucleoside and glucose), 7.35 (bs, 1H, -NH-C=O-), 7.64 (s, 1H, triazole-between F-chain and
13
nucleoside) 8.35 (s, 1H, H-6(base)), 9.27 (s, 1H, -NH(base)). C-NMR (75 MHz, CDCl3): δ in ppm
11.9 (CH3(base)), 20.0–20.6 (4 -CH3 (acetyl)), 26.5 (-CH2-CH2-chain) 34.2 (triazole-CH2-NH) 35.2
(C2′), 49.8 (C5'), 61.5 (-O-CH2-triazole), 63.2 (-CH2-OAc), 67.8 (-CH (glucose-C3)), 70.8 (-CH
(glucose-C2)), 72.2 (-CH (glucose-C4)), 74.2 (-CH (glucose-C5)), 75.1 (-CH (glucose-C1)), 81.0 (C3'),
84.6 (C4'), 85.9 (C1'), 111.2 (C5-thymine base), 121.9 (-CH triazole-between F-chain and nucleoside),
124.3 (-CH triazole-between nucleoside and glucose) 136.3 (C6-thymine base), 144.4 (=C- triazole-
between nucleoside and glucose), 144.5 (=C- triazole-between F-chain and nucleoside), 150.3 (C=O(2)
thymine base), 163.7 (C=O(4) thymine base), 169.1–170.2 (3 C=O(acetyl at C2, C3, C4)), 170.3
(-CH2-C=O(acetyl at C6)), 170.6 (-NH-C=O). High-resolution FD-MS m/z calcd. for C41H42F17N9O14
1207.2580, found. 1207.2582.
4,4,5,5,6,6,7,7,8,8,9,9,10,10,11,11,11-Heptadecafluoro-N-((1-(((2R,3S,5R)-5-(5-methyl-2,4-dioxo-3,4-
dihydropyrimidin-1(2H)-yl)-3-((1-((2R,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-
2H-pyran-2-yl)-1H-1,2,3-triazol-4-yl)methoxy)tetrahydrofuran-2-yl)methyl)-1H-1,2,3-triazol-4-yl)methyl)
undecanamide (17). To a solution of double click product 16 (950 mg, 0.79 mmol, 1.0 equiv.) in
methanol (40 mL) was added sodium methoxide powder (850 mg, 15.74 mmol, 20 equiv.) and the
resulting mixture was stirred at room temperature for 2 h. After removal of the solvent under reduced
pressure, the crude deprotected product was applied directly to a silica gel chromatographic column

Molecules 2013, 18
12260
(methanol-dichloromethane 1:4). Product 17 was isolated as a white solid. Yield: 762 m g (93.2%). Rf:
1
0.35 (methanol-dichloromethane 1:4). H-NMR (300 MHz, DMSO-d6): δ in ppm 1.80 (s, 3H,
CH3(base)), 2.20–2.33 (m, 2H, 2H2'(sugar)), 2.43–2.56 (m, 4H, CF2-CH2-CH2-C=O), 3.15–3.26
(m, 1H, -O-CH- (glucose-C5)), 3.40–3.45 (m, 3H, -CH2-OH(glucose-C6), -CH(glucose-C4), 3.63–3.80
(m, 2H, -CH(glucose-C3), H3′), 4.20–4.40 (m, 4H, -OH (glucose-C4), H5′, -CH2-NH-C=O,), 4.58
(s, 2H, -O-CH2-triazole), 4.63–4.76 (m, 3H, H4′, H5″, -CH(glucose-C2)), 5.19 (d, 1H, -OH(glucose
(C6), J = 5.5 Hz), 5.33 (d, 1H, -OH (glucose-C3), J = 4.7 Hz), 5.44 (d, 1H, -OH (glucose-C2), J = 6.0 Hz),
5.53 (d, 1H, -N-CH-O (glucose-C1), J = 9.4 Hz), 6.12 (t, 1H, 1H'(sugar), J = 7.3 Hz), 7.45 (s, 1H,
triazole-between nucleoside and glucose), 7.98 (s, 1H, triazole-between F-chain and nucleoside) 8.33
13
(s, 1H, H-6(base)), 8.57 (t, 1H, -NH-C=O-, J = 5.46 Hz), 11.37 (s, 1H, -NH(base)). C-NMR (75
MHz, DMSO-d6): δ in ppm 12.0 (CH3(base)), 25.7–25.8 (-CH2-CH2-chain) 34.4 (triazole-CH2-NH)
35.1 (C2′), 51.4 (C5'), 60.8 (-O-CH2-triazole), 62.1 (-CH2-OH (glucose-C6)), 69.6 (-CH(glucose-C2)),
72.1 (-CH(glucose-C4)), 77.0 (-CH(glucose-C3)), 79.1 (C3′), 80.0 (-CH(glucose-C5)), 81.6
(-CH(glucose-C1)), 84.5 (C4′), 87.5 (C1′), 109.9 (C5-thymine base), 123.3 (-CH triazole between
thymidine and alkyl chain), 123.6 (-CH triazole between glucose and thymidine), 136.1 (C6-thymine
base), 143.5 (=C- triazole triazole between glucose and thymidine), 144.7 (=C- triazole between
thymidine and alkyl chain), 150.4 (C=O(2) thymine base), 163.7 (C=O(4) thymine base), 169.3
(-NH-C=O). MS m/z calcd. for C33H34F17N9O10Na 1062.6, found. 1062.3.
4. Conclusions
The objectives of the present investigation were to expand the current repertoire of glycosyl-
nucleolipids (GNLs) and study the effect of the structural modifications on their physicochemical
properties. In this contribution, two synthetic routes to a series of i) double chain hydroxybutanamide
based GNLs and ii) second generation GNF are described. Thermal experiments show that
hydroxybutanamide amphiphiles exhibit endothermic phase transitions at temperatures of 51.9 ± 1.8 °C,
51.7 ± 1.8 °C, 56.1 ± 2.4 °C and −16.3 ± 2.4 °C for C8, C12, C18 and oleyl double chains analogues,
respectively. In the case of GNFs, surface tension measurements show critical aggregation
concentrations (CACs) of 11 µM for both 1st GNF and 2nd GNF, indicating that the polar head
structure has a poor impact on the CAC values. The 2nd GNF features a better surface activity (γlim)
compared to the 1st GNF. The synthetic strategies developed in this report will be used in the future
for the design of new glycosyl-nucleolipids that self-assemble into soft materials for biomedical and
tissue engineering applications.
Supplementary Materials
Supplementary materials can be accessed at: http://www.mdpi.com/1420-3049/18/10/12241/s1.
Acknowledgments
This work has been supported by the French National Agency (ANR) in the frame of its programme
blanc (project GelCells, appel à projets Blanc SIMI 7-2010). All the authors greatly acknowledge
Gaelle Girard for achieving physicochemical experiments.

Molecules 2013, 18
12261
Conflicts of Interest
The authors declare no conflict of interest
References
1. Schade, M.; Knoll, A.; Vogel, A.; Seitz, O.; Liebscher, J.; Huster, D.; Herrmann, A.; Arbuzova, A.
Remote control of lipophilic nucleic acids domain partitioning by DNA hybridization and
enzymatic cleavage. J. Am. Chem. Soc. 2012, 134, 20490–20497.
2. McLaughlin, C.K.; Hamblin, G.D.; Sleiman, H.F. Supramolecular DNA assembly. Chem. Soc. Rev.
2011, 40, 5647–5656.
3. Patwa, A.; Gissot, A.; Bestel, I.; Barthélémy, P. Hybrid lipid oligonucleotide conjugates:
Synthesis, self-assemblies and biomedical applications. Chem. Soc. Rev. 2011, 40, 5844–5854.
4. Tang, C.; Ulijn, R.V.; Saiani, A. Effect of glycine substitution on Fmoc-diphenylalanine self-assembly
and gelation properties. Langmuir 2011, 27, 14438–14449.
5. He, C.; Han, Y.; Fan, Y.; Deng, M.; Wang, Y. Self-Assembly of Aβ-based peptide amphiphiles
with double hydrophobic chains. Langmuir 2012, 28, 3391–3396.
6. Ziserman, L.; Lee, H.-Y.; Raghavan, S.R.; Mor, A.; Danino, D. Unraveling the mechanism of
nanotube formation by chiral self-assembly of amphiphiles. J. Am. Chem. Soc. 2011, 133, 2511–2517.
7. Ku, T.-H.; Chien, M.-P.; Thompson, M.P.; Sinkovits, R.S.; Olson, N.H.; Baker, T.S.; Gianneschi, N.C.
Controlling and switching the morphology of micellar nanoparticles with enzymes. J. Am. Chem. Soc.
2011, 133, 8392–8395.
8. Dong, H.; Dube, N.; Shu, J.Y.; Seo, J.W.; Mahakian, L.M.; Ferrara, K.W.; Xu, T. Long-circulating
15 nm micelles based on amphiphilic 3-helix peptide-peg conjugates. ACS Nano 2012, 6, 5320–5329.
9. Chae, P.S.; Gotfryd, K.; Pacyna, J.; Miercke, L.J.W.; Rasmussen, S.G.F.; Robbins, R.A.; Rana,
R.R.; Loland, C.J.; Kobilka, B.; Stroud, R.; et al. Tandem facial amphiphiles for membrane
protein stabilization. J. Am. Chem. Soc. 2010, 132, 16750–16752.
10. Szilluweit, R.; Hoheisel, T.N.; Fritzsche, M.; Ketterer, B.; Fontcuberta I Morral, A.; Demurtas, D.;
Laporte, V.; Verel, R.; Bolisetty, S.; Mezzenga, R.; et al. Low-temperature preparation of tailored
carbon nanostructures in water. Nano Lett. 2012, 12, 2575–2578.
11. Méndez-Ardoy, A.; Guilloteau, N.; Di Giorgio, C.; Vierling, P.; Santoyo-González, F.;
Ortiz Mellet, C.; García Fernández, J.M. β-cyclodextrin-based polycationic amphiphilic “click”
clusters: Effect of structural modifications in their DNA complexing and delivery properties.
J. Org. Chem. 2011, 76, 5882–5894.
12. Rosemeyer, H. Nucleolipids: Natural occurrence, synthesis, molecular recognition, and
supramolecular assemblies as potential precursors of life and bioorganic materials. Chem. Biodivers.
2005, 2, 977–1062.
13. Barthélemy, P. Nucleoside-based lipids at work: From supramolecular assemblies to biological
applications. C. R. Chim. 2009, 12, 171–179.
14. Allain, V.; Bourgaux, C.; Couvreur, P. Self-assembled nucleolipids: From supramolecular
structure to soft nucleic acid and drug delivery devices. Nucl. Acid. Res. 2012, 40, 1891–1903.

Molecules 2013, 18
12262
15. Gissot, A.; Camplo, M.; Grinstaff, M.W.; Barthélémy, P. Nucleoside, nucleotide and
oligonucleotide based amphiphiles: A successful marriage of nucleic acids with lipids.
Org. Biomol. Chem. 2008, 6, 1324–1333.
16. Sun, Y.; Yan, Y.; Wang, M.; Chen, C.; Xu, H.; Lu, J.R. Controlled Release of Hydrophilic Guest
Molecules from Photoresponsive Nucleolipid Vesicles. ACS Appl. Mater. Interfaces 2013, 5,
6232–6236.
17. Simeone, L.; Mangiapia, G.; Irace, C.; Di Pascale, A.; Colonna, A.; Ortona, O.; De Napoli, L.;
Montesarchio, D.; Paduano, L. Nucleolipid nanovectors as molecular carriers for potential
applications in drug delivery. Mol. Biosyst. 2011, 7, 3075–3086.
18. Chabaud, P.; Camplo, M.; Payet, D.; Serin, G.; Moreau, L.; Barthélémy, P.; Grinstaff, M.W.
Cationic nucleoside lipids for gene delivery. Bioconjugate Chem.2006, 17, 466–472.
19. Khiati, S.; Luvino, D.; Oumzil, K.; Chauffert, B.; Camplo, M.; Barthélémy, P. Nucleoside-lipid-
based nanoparticles for cisplatin delivery. ACS Nano 2011, 5, 8649–8655.
20. Ceballos, C.; Prata, C.A.H.; Giorgio, S.; Garzino, F.; Payet, D.; Barthélémy, P.; Grinstaff, M.W.;
Camplo, M. Cationic nucleoside lipids based on a 3-nitropyrrole universal Base for siRNA
delivery. Bioconjugat. Chem. 2009, 20, 193–196.
21. Ceballos, C.; Khiati, S.; Prata, C.A.H.; Zhang, X.-X.; Giorgio, S.; Marsal, P.; Grinstaff, M.W.;
Barthélémy, P.; Camplo, M. Cationic nucleoside lipids derived from universal bases: A rational
approach for siRNA transfection. Bioconjugat. Chem. 2010, 21, 1062–1069.
22. Simeone, L.; Irace, C.; Di Pascale, A.; Ciccarelli, D.; D’Errico, G.; Montesarchio, D. Synthesis,
self-aggregation and bioactivity properties of a cationic aminoacyl surfactant, based on a new
class of highly functionalized nucleolipids. Eur. J. Med. Chem. 2012, 57, 429–440.
23. Yang, H.W.; Yi, J.W.; Bang, E.-K.; Jeon, E.M.; Kim, B.H. Cationic nucleolipids as efficient
siRNA carriers. Org. Biomol. Chem. 2011, 9, 291–296.
24. Tonelli, G.; Oumzil, K.; Nallet, F.; Gaillard, C.; Navailles, L.; Barthélémy, P. Amino
acid-nucleotide-lipids: Effect of amino acid on the self-assembly properties. Langmuir 2013, 29,
5547–5555.
25. Latxague, L.; Ziane, S.; Chassande, O.; Patwa, A.; Dalila, M.-J.; Barthélémy, P. Glycosylated
nucleoside lipid promotes the liposome internalization in stem cells. Chem. Commun. 2011, 47,
12598–12600.
26. Arigon, J.; Prata, C.A.H.; Grinstaff, M.W.; Barthélémy, P. Nucleic acid complexing glycosyl
nucleoside-based amphiphile. Bioconjugat. Chem. 2005, 16, 864–872.
27. Godeau, G.; Barthélémy, P. Glycosyl-nucleoside lipids as low-molecular-weight gelators.
Langmuir 2009, 25, 8447–8450.
28. Godeau, G.; Bernard, J.; Staedel, C.; Barthélémy, P. Glycosyl-nucleoside-lipid based
supramolecular assembly as a nanostructured material with nucleic acid delivery capabilities.
Chem. Commun. 2009, 5127–5129.
29. Godeau, G.; Brun, C.; Arnion, H.; Staedel, C.; Barthélémy, P. Glycosyl-nucleoside fluorinated
amphiphiles as components of nanostructured hydrogels. Tetrahedron Lett. 2010, 51, 1012–1015.
30. Hirst, A.R.; Coates, I.A.; Boucheteau, T.R.; Miravet, J.F.; Escuder, B.; Castelletto, V.; Hamley, I.W.;
Smith, D.K. Low-molecular-weight gelators: Elucidating the principles of gelation based on
gelator solubility and a cooperative self-assembly model. J. Am. Chem. Soc. 2008, 130, 9113–9121.

Molecules 2013, 18
12263
31. Krafft, M.P.; Goldmann, M. Monolayers made from fluorinated amphiphiles. Curr. Opin. Colloid
Interface Sci. 2003, 8, 243–250.
32. Latxague, L.; Dalila, M.-J.; Patwa, A.; Ziane, S.; Chassande, O.; Godeau, G.; Barthélémy, P.
Glycoside nucleoside lipids (GNLs): An intrusion into the glycolipids’ world? C. R. Chim. 2012,
15, 29–36.
33. Ziane, S.; Schlaubitz, S.; Miraux, S.; Patwa, A.; Lalande, C.; Bilem, I.; Lepreux, S.; Rousseau, B.;
Le Meins, J.-F.; Latxague, L.; et al. A thermosensitive low molecular weight hydrogel as scaffold
for tissue engineering. Eur. Cell. Mater. 2012, 23, 147–160.
34. Tararov, V.I.; König, G.; Börner, A. Synthesis and highly stereoselective hydrogenation of the
statin precursor ethyl (5S)-5,6-isopropylidenedioxy-3-oxohexanoate. Adv. Synth. Catal. 2006,
348, 2633–2644.
35. Ohshima, T.; Hayashi, Y.; Agura, K.; Fujii, Y.; Yoshiyama, A.; Mashima, K. Sodium methoxide:
A simple but highly efficient catalyst for the direct amidation of esters. Chem. Commun. 2012, 48,
5434–5436.
36. Yang, X.; Birman, V.B. Acyl transfer catalysis with 1,2,4-Triazole anion. Org. Lett. 2009, 11,
1499–1502.
37. Hiebl, J.; Zbiral, E.; Balzarini, J.; De Clercq, E. Synthesis and antiretrovirus properties of
5′-isocyano-5′-deoxythymidine, 5′-isocyano-2′,5′-dideoxyuridine, 3′-azido-5′-isocyano-3′,5′-
dideoxythymidine, and 3′-azido-5′-isocyano-2′,3′,5′-trideoxyuridine. J. Med. Chem. 1991, 34,
1426–1430.
38. Wu, J.-C.; Xi, Z.; Gioeli, C.; Chattopadhyaya, J. Intramolecular cyclization-trapping of carbon
radicals by olefins as means to functionalize 2'- and 3′-carbons in β-D-nucleosides. Tetrahedron
1991, 47, 2237–2254.
39. Teste, K.; Colombeau, L.; Hadj-Bouazza, A.; Lucas, R.; Zerrouki, R.; Krausz, P.; Champavier, Y.
Solvent-controlled regioselective protection of 5′-O-protected thymidine. Carbohyd. Res. 2008,
343, 1490–1495.
40. Sirion, U.; Kim, H.J.; Lee, J.H.; Seo, J.W.; Lee, B.S.; Lee, S.J.; Oh, S.J.; Chi, D.Y. An efficient
F-18 labeling method for PET study: Huisgen 1,3-dipolar cycloaddition of bioactive substances
and F-18-labeled compounds. Tetrahedron Lett. 2007, 48, 3953–3957.
41. Colombeau, L.; Zerrouki, R.; Krausz, P.; Champavier, Y. Dinucleoside analog synthesis via
microwave activated cross coupling metathesis. Lett. Org. Chem. 2005, 2, 613–615.
42. Roy, V.; Zerrouki, R.; Krausz, P. New dinucleoside analogues via cross-coupling metathesis.
Nucleos. Nucleot. Nucleic Acids 2005, 24, 289–301.
Sample Availability: Samples of compounds 2a, 9a, 9b, 9c and 17 are available from the authors.
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