Cytidine- and guanosine-based nucleotide-lipids

Article abstract of DOI:10.1039/c8ob01023d

Hybrid nucleotide-lipids composed of a lipid covalently attached to purine and pyrimidine nucleobases exhibit supramolecular properties. The novel cytidine and guanosine derivatives are promising bioinspired materials, which can act as supramolecular gela

Full text of DOI:10.1039/c8ob01023d

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DOI: 10.1039/C8OB01023D
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PAPER
Cytidine- and guanosine-based nucleotide-lipids
a
a
a,b
a
c
Bruno Alies, Mohamed A. Ouelhazi , Amit Patwa, Julien Verget, Laurence Navailles, Valérie
a
a
Desvergnes, and Philippe Barthélémy
Received 00th January 20xx,
Accepted 00th January 20xx
Hybrid nucleotide-lipids composed of a lipid covalently attached or adenosine head groups covalently attached to 1,2-
2
0,21,22
to purine and pyrimidine nucleobases exhibit supramolecular dipalmitoyl-sn-glycerol.
properties. The novel cytidine and guanosine derivatives are hydrogels, resulting from the self-assembly of thymidine-3’-
promising bioinspired materials, which can act as supramolecular (1,2-dipalmitoyl-sn-glycero-3-phosphate) (diC16-3 -dT) salts,
gelators depending on both the nucleobase and the presence of were found to be effective soft materials for in vivo injection
Interestingly, biocompatible
′
2
3
salts. These supramolecular properties are of broad interest for and implantation.
biomedical applications.
The modulation of the polar head structure, in particular
the nature of the base could influence the aggregation
properties of the NLs. Therefore, we elected to design NLs with
a double dipalmitoyl glycerol phosphate linked to the 3’-
secondary hydroxyl of the four natural nucleosides. Indeed,
the diverse hydrogen bond donor-acceptor sites in all four
nucleobases (A: Adenine, T: Thymine C: Cytosine, and G:
Introduction
Supramolecular materials derived from biological structures
1
,2
3
4
carbohydrates, or nucleosides are
such as peptides,
attractive for various applications,
5
,6
including green
9,10
medicinal
7
chemistry, materials science, drug delivery,
,8
Guanine) are prone to form self-base pairing interactions that
24,25
1
1,12,13
chemistry,
1
medicine.
tissue engineering, or regenerative
15,16,17
stabilize supramolecular self-assemblies.
It is therefore
4
Nucleolipids,
which are biocompatible
valuable to explore the self-assembly properties of A, C, T and
G based nucleotide-lipids as supramolecular building blocks.
Specifically, we report the synthesis and the physicochemical
structures composed of a lipid covalently attached to a
nucleotide, are desirable candidates for the implementation of
supramolecular biomaterials as they easily engage non-
studies of a series of nucleoside-3’-monophosphate NLs
featuring palmitic double chains and A, T, C, G nucleobases.
1
8,19
covalent interactions.
Nucleotides-based lipids, in
particular, are unique in that they have multiple possibilities
for non-covalent interactions depending on the nature of the
bases and can self-associate into stable different type of self-
assembly, such as ribbons, bilayers, fibers etc. In previous
studies, we reported the self-assembly properties of several
nucleotide lipids (NLs) featuring 3’-monophosphate thymidine
Several examples of nucleoside-based amphiphiles have been
26,27,28
reported,
however to the best of our knowledge, there
are no previous examples of Cytosine and Guanosine
nucleoside-3 -monophosphate amphiphiles.
′
Results and discussion
a.
Inserm, U1212, Bordeaux, F-33076 France, Université de Bordeaux, F-33076,
Bordeaux, France. UMR CNRS CNRS 5320
SLSE, Navrachana University, Vasna Bhayali Road, Vadodara 391410, Gujarat,
India.
b.
Fig. 1. A) Chemical structure of the nucleotide-lipid (NLs). B) Nucleotide lipid
derivatives were incubated either in the absence or presence of sodium chloride.
The novel C and G nucleotide lipid aqueous suspensions exhibit different
macroscopic behaviors, the cytosine NL stabilizes hydrogels in the presence or
NaCl, whereas the guanosine derivative forms a gel in the absence of NaCl.
c.
Université de Bordeaux, F-33076, Bordeaux, France. Centre de Recherche Paul
Pascal UPR8641
Corresponding Author. E-mail: philippe.barthelemy@inserm.fr
Electronic Supplementary Information (ESI) available: Synthesis and
characterization data for nucleotide lipids, See DOI: 10.1039/x0xx00000x
†
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In this study, we prepared four purine and pyrimidine circular dichroism (CD) of NLs and deoxynucleosides was also
nucleotide-lipids via phosphoramidite strategy. The investigated (Fig. 2, right scale). Compared to deoxyadenosine
a
supramolecular assemblies obtained with A, T, C, G nucleotide (solid line), the CD spectra of diC16-3’-dA exhibited more
lipids either in the absence or presence of sodium chloride are pronounced peaks at 225 and 280 nm and the appearance of a
reported (Fig. 1). Depending on both the conditions and the band at 240 nm. With diC16-3’-dT, the band at 270nm is
nature of the base the stabilisation of supramolecular similar to thymidine. Preparation in NaCl (red dotted line)
hydrogels was observed. Likewise, as a proof of concept, in intensified the band. DiC16-3’-dG exhibited the appearance of
vitro drug release experiments show that a controlled delivery two new peaks, a positive band at 240 nm and a negative band
of a drug can be obtained thanks to the structure of the at 260 nm in addition to the peak at 225 nm observed in both
nucleolipid based gels.
CD spectra. With purine NLs (diC16-3’-dA & diC16-3’-dG) large
Several strategies have been used to synthesize nucleotide differences are seen between NLs and deoxynucleosides (Fig. 2
lipids, including coupling reactions of monoalkyl phosphate dashed vs solid lines). In NaCl solutions, the diC16-3’-dC CD
2
9
with nucleoside and enzyme catalysis,
phosphoramidite synthetic pathway. The novel NLs, diC16-3
dG 1a and diC16-3 -dC 1b, were synthesized using a similar green vs dotted green lines). These new bands were indicative
approach to that used for the synthesis of diC16-3’-LNA-A
see ESI, Scheme S1). This approach uses standard procedures, the case of well-hydrated alkali metal salts electrostatic
or via band at 266nm exhibited a substantial shift and increase
′
-
compared to diC16-3’-dC prepared in water solution (dashed
′
3
0
of the formation of chiral structures. It is hypothesized that in
(
routinely adopted in oligonucleotide synthesis via repulsions between the negatively charged phosphate would
phosphoramidite chemistry in the solid phase and adapted to occur, leading to the formation of low curvature aggregates. In
solution. Briefly, commercially available isobutyryl protected turn, less hydrated salts would result in higher curvature
guanosine
phosphoramidite
2a
(isobutyryl-dG-CE assemblies due to the stronger interaction among nucleosides.
phosphoramidite, see SI, Scheme S1) and acetyl protected In this direction, we compared the UV and the CD spectra of
cytosine phosphoramidite 2b (Ac-dC-CE phosphoramidite, see NLs in pure water and in pseudo-physiological conditions
SI, Scheme S1) were deprotected with methylamine solution in (NaCl: 154 mM equivalent to 0.9% w/v). Except for diC16-3’-dG
THF to achieve dG-CE phosphoramidite 3a and dC-CE and diC16-3’-dA, which were not soluble in pseudo-
phosphoramidite 3b, respectively. These phosphoramidites (3a physiological conditions, diC16-3’-dT and diC16-3’-dC did not
and 3b) were coupled with 1,2-dipalmitoyl-sn-glycerol in the show any significant change in their UV spectra. Whereas, in
presence of a tetrazole solution in acetonitrile (weakly acidic CD spectroscopy diC16-3’-dT exhibited a slight increase in CD
condition) to provide intermediate coupling product. The intensity but for diC16-3’-dC, a large increase in CD intensity
resulting phosphite intermediates were oxidized, with iodine was clearly observed with a red-shift to higher wavelength
solution in THF/pyridine/water mixture, to obtain cyanoethyl
protected phosphate intermediate. After deprotection of the
dimethoxytrityl (5’-DMTr) group under acidic conditions
(
trichloroacetic acid in DCM), the removal of the cyanoethyl
chain was achieved under basic conditions (triethylamine) to
yield diC16-3 -dG 1a and diC16-3 -dC 1b as their
triethylammonium salts (for detailed synthetic procedure, see
′
′
3
1
SI, Scheme S1). The NLs featuring thymidine (diC16-3
3
′
-dT 1c
)
2
and adenosinne (diC16-3′-dA 1d),
according to the literature procedure.
were synthesized
Spectroscopy studies
In order to study the inter-nucleobase interactions in the
NLs self-assemblies UV-visible and Circular Dichroism
experiments were performed. Figure 2 show the UV spectra of
each NLs in aqueous (dashed line) or NaCl solution (dotted
line) with their correspondent deoxynucleosides (solid line). It
is well known that UV spectra of aromatic rings are sensitive to
π-π interaction, when two or more rings are mutually close
they give rise to hypochromic effect. A decrease of about 20 %
in the absorbance values was observed for diC16-3’-dA, diC16-
3
’-dT and diC16-3’-dG compared with deoxyadenosine,
Fig. 2. UV (scale on left axis) and CD (scale on right axis) spectra of NLs prepared
in water (dashed line), in 0.9% w/v NaCl solution (diamond) with their
deoxynucleosides (solid line). The nature of the nucleic bases is indicated on the
graphs. DiC16-3’-dG and diC16-3’-dC are not soluble in NaCl solution therefore
their spectra are not displayed. All spectra were recorded at 100 μM in room
temperature.
thymidine and deoxyguanosine, respectively. In the case of
diC16-3’-dC, no significant decrease in absorbance and slight
bathochromic shift (5 nm) were observed compared to
deoxycytidine. In order to get further structural information,
2
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Fig. 2, bottom right, dashed vs dotted line). A different type of
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(
structure resulting from intermolecular nucleobase
interactions may explain this abrupt change. These results
indicate that in addition to the hydrophobic effect through the
dipalmitoyl double chains, inter-stacking and H-bonding
between nucleobases plays a major role in stabilizing NLs
aggregates. In addition, the counter ion used in these systems
strongly impact on the supramolecular organizations of diC16-
dT and diC16-3’-dC.
Taken together, UV-vis and CD
experiments show that the diC16-3’-dX series form
supramolecular assemblies, which can be modulated in the
presence of NaCl.
Salt effects on self-assemblies
We further investigated the supramolecular changes of NLs
either in the presence or absence of NaCl. Dynamic Light
Scattering (DLS) measurements were collected both in water
and in NaCl conditions. The mean diameters and the
polydispersity index (PDI) of the assemblies are reported as
well as the size distributions graphs (Table 1 down and Figure
ESI 1). In a first series of experiments, NLs were dispersed in
pure water under sonication. Results obtained by DLS
measurements indicated that sonication was not suitable for a
good dispersion of pyrimidines NLs (diC16-3’-dC and diC16-3’-
dT) giving a highly polydisperse size distribution. In contrast,
Fig. 3. DSC experiments of diC16-3′-dA (top left blue), diC16-3′-dT (top right red),
diC16-3′-G (bottom left black) & diC16-3′-C (bottom right green) in the absence
(
solid lines) or presence of 0.9% (w/v) NaCl (dotted lines). Cooling (positive signal)
and heating (negative signal) cycle are displayed. Conditions are arbitrary
separated and only one cycle is shown for clarity reason.
for purine NLs (diC16-3’-dG and diC16-3’-dA),
distribution was observed with a mean diameter at around 3’-dT) show a single distribution whereas purine based NLs
20 nm for both purine NLs with a relatively low PDI (0.23). (diC16-3’-dG and diC16-3’-dA) were not well dispersed as
a single are observed. Indeed, pyrimidine NLs (diC16-3’-dC and diC16-
2
The dispersion of NLs was also investigated in the absence of observed by DLS. These differences in behaviour illustrate the
sonication. Surprisingly, in this condition, opposite behaviours importance of the nucleobase in the dispersion of NLs. In NaCl
conditions, size distributions of diC16-3’-dC and diC16-3’-dT
Tables 1. Up) Transition temperatures by DSC of NLs obtained by DSC. The results
reported are the average of four independent experiments. ± symbol indicated
the standard deviation. ΔT(NaCl –Water) emphasize the increase of transition
temperatures in the presence of NaCl (0.9% w/v). Down) Z-average, diameters
and polydispersity index obtained by DLS for each diC16-3’-dX.
(only these NLs were soluble in these conditions) showed the
presence of a large size distribution with the appearance of
larger structures ranging between 180 to 1000 nm, supporting
the hypothesis that fibers formations occurred in the presence
of well-hydrated alkali metal. The results of the DLS
experiments indicated that the size of the supramolecular
structures depends on the nature of the NL nucleobase. In the
case of diC16-3’-dG and diC16-3-dA, bearing purine
nucleobases, higher sizes of the supramolecular assemblies
were observed with a tendency to form one size distribution
compared with NLs possessing pyrimidine nucleobases.
Transition Temperature (°C)
Heating Cycle
ΔT(NaCl – Water)
diC16-3’-dX
diC16-3’-dA
diC16-3’-dT
diC16-3’-dG
diC16-3’-dC
Water
NaCl
14.2 ±0.2
40.0 ±3.6
19.8 ±0.1
35.4 ±0.6
38.3 ±0.4
41.0 ±1.5
40.9 ±0.8
41.5 ±0.8
24
1
21
6
Cooling Cycle
ΔT(NaCl – Water)
diC16-3’-dX
diC16-3’-dA
diC16-3’-dT
diC16-3’-dG
diC16-3’-dC
Water
NaCl
To characterize the thermal reversibility of the hydrogels,
Differential Scanning Calorimetry (DSC) experiments were
performed with a total of three heating and cooling cycles in
pure water and in NaCl conditions (Fig. 3). Heating and cooling
cycles results from four independent runs of thermal analysis
are reported in Table 1 (up).
13.8 ±0.1
34.6 ±6.0
18.6 ±0.1
27.5 ±2.4
35.3 ±1.5
38.5 ±0.3
38.4 ±0.3
40.1 ±1.1
22
4
20
13
PolyDispersity
Z-average (nm) / Particles diameters (nm)
DSC experiments showed main phases transitions (Tm) at
various temperatures depending of nucleobases and
conditions (Fig 3). In water, transition occurs (heating/cooling)
at 15/14, 36/32, 20/19 and 35/27°C for diC16-3’-dA, diC16-3’-
dT, diC16-3’-dG, and diC16-3’-dC, respectively. The Tm
observed for NLs are similar to that for the natural di
palmitoyl-glycerol phosphocholine (DPPC) as well as synthetic
Index
diC16-3’-dX
diC16-3’-dG
diC16-3’-dA
With sonication
186 / 238
0.232
0.234
199 / 220
Without sonication
99 / 105
diC16-3’-dC
diC16-3’-dT
0.089
0.193
81 / 85
3
3
di palmitoyl uridine phosphocholine (DPUPC).
A clear
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hysteresis between heating and cooling cycle was observed for
diC16-3’-dC (8°C) and diC16-3’-dT (4°C), whereas only 1°C
difference occurred with diC16-3’-dG and diC16-3’-dA (See Fig
3
and Table 1 up). DiC16-3’-dG and diC16-3’-dA exhibited other
small transitions. In NaCl, the main phase transition
temperatures increased for all NLs and reached 39/37, 40/38,
42/39 and 42/40°C for diC16-3’-dA, diC16-3’-dT, diC16-3’-dG,
and diC16-3’-dC, respectively without any additional peaks.
Interestingly, a lower hysteresis between heating and cooling
cycle was observed with diC16-3’-dC and diC16-3’-dT (1°C) in
NaCl conditions. Note that for diC16-3’-dC and diC16-3’-dT in
water, broad peaks in heating and cooling cycle were
detected; this tendency did not appear in NaCl conditions. In
Fig. 4. Oscillatory strain sweeps for cytidine and guanosine nucleotide-lipids
based gels at 5% and 10%, respectively. Elastic, (G’, red) and viscous (G”, blue)
moduli versus strain in percent. Left) LVER of diC16-3’-dC (5%, w/v) in water
water condition, purine NLs (diC16-3’dT
showed a transition around 30°C whereas pyrimidine NLs
diC16-3’-dA & diC16-3’dG) exhibited a transition below 20°C.
This main phase transition disparity between nucleobases diC16-3’-dC gels rheological properties. For diC16-3’-dG, due
purine-pyrimidine) disappeared in NaCl conditions where all to the lack of solubility in the presence of NaCl, the LVER was
& diC16-3’-dC)
(
solid lines) or with 0.9% NaCl (symbols). Right) LVER of diC16-3’-dG (10%, w/v) in
(
(
NLs reached approximately the same thermal reversibility at determined only in water at 10%. At this concentration, the G
around 40°C. This observation indicates that the counter ion derivative shows similar properties to the diC16-3’-dC at 5% in
+
Na ) possess a stronger impact on the melting temperature of water (G’= 800 Pa, moduli inversion at 5%).
(
purine derivatives (diC16-3’-dA and diC16-3’-dG) than For diC16-3’-dT and diC16-3’-dC in NaCl conditions, the gel-sol
pyrimidine analogues (diC16-3’-dC and diC16-3’-dT). Although transition occurred instantaneously. Note that in pure water
purines bases were able to give hydrogen bonding, in water an interstate transition between the gel to solution was
condition, we observed that the transition temperatures of observed. This gel-solution transition difference was consistent
diC16-3’-dA and diC16-3’-dG were dramatically lower than with DSC results where broad peaks were observed in water
pyrimidine NLs. This could be explained by a higher steric conditions contrary to sharps peaks obtained in NaCl
hindrance with purine NLs causing difficulties to self-organise. conditions (Fig. 3, right panels). Such behaviour could explain
+
In the presence of a well-hydrated cation (Na ), more stable the broad peaks obtained in heating and cooling and the large
+
assemblies could be reached suggesting that Na would hysteresis obtained for diC16-3’-dC and diC16-3’-dT in pure
stabilize the supramolecular structure.
water. These results also indicated that the phase transition
behaviour depends mainly on the nucleobases type, the self-
binding affinity between nucleobases and the environmental
conditions, notably, the counter ion used.
Rheology and gel behaviors
In order to further characterize the physicochemical
properties of NLs at a macroscopic level, we performed gel
formation experiments (Fig. 1B). Depending of the conditions
Drug release experiments
(water or NaCl), striking differences were observed. In
Due to their unique properties, supramolecular gels are
presence of NaCl, a strong diC16-3’-dC gel was formed (Fig. 1B currently under investigation as drug delivery matrix.
left), whereas diC16-3’-dG precipitated without forming any Controlling the kinetics of drug release from such soft
gels. Following these qualitative tests, the mechanical materials via the molecular structure of gelators is highly
properties of new cytidine and guanosine nucleotide-lipids desirable since it would allow for a modulation of drug release
based gels were analyzed using oscillatory rheology to depending on the external conditions. To illustrate the
characterize the Linear Visco-Elastic Regime (LVER) and the usefulness of the novel nucleolipids in this field, in-vitro drug
strength of the gels. Figure 4 shows the oscillatory strain diffusion studies were carried out using diazepam as a drug.
sweeps for cytidine and guanosine nucleotide-lipids based gels Diazepam is a hydrophobic compound belonging to the
at 5% and 10%, respectively. The LVER, in which the moduli are benzodiazepine family, which are used as an anticonvulsant in
independent of the applied strain, of the new compounds the treatment of epilepsy. The data of percentage drug release
were determined up to 100%. For all hydrogels investigated, are shown in Fig. 5. For the kinetic study following plots were
the elastic G' consistently exceeds the viscous G" moduli, made: cumulative % drug release versus time for both diC16-
indicating the dominant elastic character of the nucleotide- 3’-dC and diC16-3’-dG in the presence or absence of NaCl. As a
lipid based Low Molecular Weight Gelators (LMWGs). In the general comment, a slower release is observed for both
case of diC16-3’-dC gels, the LVER was determined either in nucleolipids in the absence of NaCl (T1/2 > 400 minutes for both
the absence or presence of NaCl (0.9% w/v). The breaking compounds). After addition of NaCl, a faster release of
points measured by moduli inversion were found at 1 and 5% diazepam was observed for diC16-3’-dG (T1/2 = 20 min)
and the G’ moduli of 600 Pa and 2400 Pa, respectively, compared to diC16-3’-dC (T1/2 = 400 min), indicating that the
indicating that the presence of NaCl strongly impacts on the nature of the base is playing a major role in the release
4
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compounds was accomplished using a UV light (254 nm) and
visualized on TLC plates by subrsequent spraying with 10 % conc.
H
2
SO
4
solution in ethanol, followed by heating. Column
chromatography was performed with flash silica gel (0.04-0.063
1
mm) from Merck. All the compounds were characterized using H,
1
3
31
C and P Nuclear Magnetic Resonance (NMR) spectroscopy.
These NMR spectra were recorded (either in CDCl obtained from
3
1
13
Eurisotop) on BRUKER Avance DPX-300 ( H at 300 MHz, C at 75
1
13
MHz) and DPX-500 spectrometers ( H at 500 MHz, C at 125 MHz
3
1
and P at 121.49 MHz). The chemical shifts (δ) are given in parts
1
per million (ppm) relatively residual solvent peaks (CHCl
1
3
: H: 7.26,
3
C: 77.16). The coupling constants J are given in Hertz (Hz); the
Fig. 5. In vitro drug release experiments showing the cumulative release of
diazepam from the gel (% drug release) versus time (in minutes). Kinetics of
release for diC16-3’-dC (left) and diC16-3’-dG (right) with the addition of water or
sodium chloride solution (NaCl 0.9% w/v).
peak multiplicity is reported as The coupling constants J are given in
Hertz (Hz); the peak multiplicity is reported as follows: s = singlet,
brs = 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
mechanism from the supramolecular gels. Interestingly, these mass spectrometer (applied Biosystems). The instrument is
results demonstrate that the kinetic of the drug release can be equipped with an ESI source and spectra were recorded in negative
modulated via both the nature of the base and the presence of mode. The electrospray needle was maintained at 4500 V and
salts.
operated at room temperature. Samples were introduced by
introduced by injection through a 10 μL sample loop into a
2
00 μL/min flow of methanol from the LC pump. For more details,
Conclusion
see ESI.
In summary, a facile route for the synthesis of nucleoside- Preparation mode of diC16-3’-dX
3’-monophosphate featuring the four natural nucleobases has
DiC16-3’-dA, diC16-3’-dT, diC16-3’-dC, diC16-3’-dG were
been reported. Similarly, to thymidine and adenosine 3’-
nucleotide lipids previously published, both Cytosine and
Guanosine derivatives reported in this contribution exhibit
self-assembly properties. Taking together, the physico-
chemical studies indicate that the self-assembly properties
depend strongly on both the conditions and the nature of
synthesized by our laboratory (see details in ESI). The simple
procedure was followed for all compounds. Generic name of diC16-
dX is given to specificity either DiC16-3’-dA, diC16-3’-dT, diC16-3’-
dC or diC16-3’-dG. Exact mass around 6 mg of diC16-dX was
weighted on 0.1 mg precision balance and dissolved and mixed in
dichloromethane into 10mL volumetric flask. Aliquots were then
prepared by transferring 500µL into glass vial. Aliquots were then
evaporated until dryness and stored at -20°C. Hydration of the lipid
film is accomplished simply by adding 3 mL mΩ water or 3mL of a
NaCl solution (0.9% w/v; 154mM) The obtained solutions were then
heated for 30 minutes and sonicate for 5 min at temperature of
nucleobases.
The presence of NaCl induced NLs-NLs
interactions, but the specific supramolecular structures
achieved give rise to different states at the macrocopic level
depending on the nucleobase. The control of the lipid self-
assemblies via the molecular code of the four natural
nucleotides represent a powerful strategy, which could be
used to construct biocompatible soft materials such as gels. As
illustrated with the drug release experiments the kinetic of
6
0°C. Finally, the resulting solutions were directly assessed by either
Dynamic Light Scattering, CD or UV-Vis Spectroscopy.
release can be controlled by both the nature of the nucleobase CD / UV-Vis Spectroscopy
and the presence of salts. Their nucleic acid controlled self-
UV-Vis and CD spectra of diC16-3’-dX was performed on JASCO J-
500 Circular Dichroism (optical path length=1cm) in quartz cuvette
coupled with a Peltier device. All measurements were carry out at
5°C.
assembly is likely to greatly extend the potential of lipid-based
materials for biomedical applications, including drug delivery.
1
2
Differential Scanning Calorimetry
Experimental Section
Differential Scanning Calorimetry (DSC) was realized using METTLER
TOLEDO. A total of three heating and cooling cycles (5-65°C) on 2%
w/w in pure water and in NaCl conditions at a constant rate of 0.5°C
General Procedures for synthesis
All the compounds were purchased from Sigma-Aldrich, Fluka and
Alfa Aesar unless otherwise mentioned. Solvents for reactions were
purchased from Sigma-Aldrich in the highest quality and from VWR
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. The detection of
-1
min and 100µL aluminium crucible. Heating and cooling results of
thermal analysis are reported in Table 1.
Dynamic Light Scattering
Dynamic Light Scattering (DLS) measurements were performed
using Zetasizer 3000 HAS MALVERN at 25°C with 400 µL of a
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solution containing 100 µM of diC16dX prepared as explained thank the BIC Center for technical assistance during TEM
above. Measurements of Table 1 down have been performed in observations and Marc Biran from the CRMSB.
water with 5 minutes sonication for purine nucleolipids (diC16-3’-
dG and diC16-3’-dA) and without sonication for pyrimidines
nucleolipids (diC16-3’-dC and diC16-3’-dT).
Notes and references
Gel Formation
Water or a NaCl solution (0.9% w/v ;154mM) were added to diC16-
’-dC or diC16-3’-dG to obtain a solution at 2% w/v of diC16-3’-dX.
1
2
S. Zhang, Nat. Biotechnol., 2003, 21, 1171-1178.
K. S. Moon, H. J. Kim, E. Lee and M. Lee, Angew. Chem. Int.
Ed., 2007, 46, 6807-6810.
S. C. Lange, J. Unsleber, P. Drucker, H. J. Galla, M. P. Waller
and B. J. Ravoo, Org. Biomol. Chem., 2015, 13, 561-569.
G. M. Peters and J. T. Davis, Chem. Soc. Rev., 2016, 45, 3188-
3206.
3
Then the solution was heated at 60°C during 30 minutes and
sonicate the 5 last minutes. The solutions were allowed to cool
down at room temperature and gel formation were observed
3
4
5
(diC16-3’-dC water / NaCl & diC16-3’-dG / water) or not (diC16-3’-
dG NaCl). See Figure 1B.
Delin Pan, Jing Sun, Hongwei Jin, Yating Li, Liyu Li, Yun Wu,
,
Lihe Zhang and Zhenjun Yang, Chem. Commun., 2015, 51
469-472.
Drug release experiments
6
7
8
9
Nuthanakanti A, Srivatsan SG. ACS Appl Mater Interfaces,
017, , 22864–22874.
A. Nuthanakanti, S.G. Srivatsan, Nanoscale, 2016, 8, 3607-
3619.
A. Patwa, J. Labille, JY. Bottero, A. Thiéry, and P. Barthélémy,
Chem. Commun., 2015, 51, 2547-2550.
6
.0 mg of either diC16-3’-dG and diC16-3’-dC were accurately
2
9
weighted in 3 mL UV cuvette. 200 µL of diazepam solution at 500
µM are added in the cuvette. Cuvette is sealed and heated at 60°C
for 30 min then to cool down at room temperature for 30 min.
Appearance of gel is visually checked as solution become
J. A. Kaplan, P. Barthélémy and M. W. Grinstaff, Chem.
Commun., 2016, 52, 5860-5863.
opalescent and viscous. Cuvette is set in
a Jasco V630
spectrophotometer and UV spectra (400 nm to 200 nm) is recorded 10 P. Barthélémy, C. A. H. Prata, S. F. Filocamo, C. E. Immoos, B.
every 2 min after careful addition (without disturbing the gel at the
bottom of the cuvette) of 1600 µL of either water or 154 mM NaCl
solution. In this setting, the beam of the spectrophotometer pass
thought the bulk (above the gel) allowing to follow the kinetic of
diazepam release. Absorbance at 313 nm (specific for diazepam,
W. Maynor, S. A. N. Hashmi, S. J. Lee and M. W. Grinstaff,
Chem. Commun., 2005, 1261-1263.
1 N. Rana, S. Huang, P. Patel, U. Samuni, D. Sabatino, Bioorg
Med Chem Lett. 2016, 26, 3567-3571.
2 C. Riccardi, D. Musumeci, C. Irace, L. Paduano, and D.
1
1
Montesarchio, Eur. J. Org. Chem. 2017, 7, 1100–1119.
epsilon = 1700 cm .M ) was subtracted from baseline at 350 nm 13 C. Knies, K. Hammerbacher, G.A. Bonaterra, R. Kinscherf and
H. Rosemeyer, ChemistryOpen 2016, 5, 129 – 141
-1
-1
and converted into percentage.
1
4 L. Latxague, M. A. Ramin, A. Appavoo, P. Berto, M. Maisani,
C. Ehret, O. Chassande and P. Barthélémy, Angew. Chem. Int.
Ed., 2015, 54, 4517-4521.
Rheology experiments
Rheological measurements were carried out on a Malvern Kinexus 15 H. Rosemeyer, Chem. Biodiv., 2005,
2, 977–1062.
Pro+ rheometer with steel coneplate geometry (diameter: 40mm, 16 A. Gissot, M. Camplo, M. W. Grinstaff and P. Barthélémy,
Org. Biomol. Chem., 2008,
7 J. Baillet, V. Desvergnes, A. Hamoud, L. Latxague, P.
Barthélémy, Adv. Mater. 2018, 30 DOI:
0.1002/adma.201705078.
homogeneous temperature and to prevent water evaporation. The 18 M.Y. Arteta , D. Berti, C. Montis, R.A. Campbell, C. Eriksson,
6, 1324-1333.
angle: 1°). The lower plate is equipped with a Peltier temperature
control system, and all samples were studied at 25 ± 0.01°C unless
1
indicated otherwise.
A solvent trap was used to ensure
1
hydrogels were heated at 65°C and the resulting liquid was placed
into the rheometer and subjected to sinusoidal oscillations. The
experimental conditions to determine the linear visco elastic regime
L.A. Clifton , M.W. Skoda , O. Soltwedel, A. Koutsioubas, P.
Baglioni, T. Nylander. Soft Matter. 2015, 11, 1973-1990.
9 C. Montis, Y. Gerelli, G. Fragneto, T. Nylander, P. Baglioni, D.
Berti, Colloids Surf B Biointerfaces, 2016, 137, 203-213.
0 K. Sugiyasu, M. Numata, N. Fujita, S. M. Park, Y. J. Yun, B. H.
Kim and S. Shinkai, Chem. Commun., 2004, 1996-1997.
1 B. Desbat, N. Arazam, S. Khiati, G. Tonelli, W. Neri, P.
Barthélémy and L. Navailles, Langmuir, 2012, 28, 6816–6825.
2 A. Patwa, G. Salgado, F. Dole, L. Navailles and P. Barthélémy
Org. Biomol. Chem., 2013, 11, 7108
1
2
2
2
(
LVER) were determined by performing an amplitude strain sweep
-1
from 0.01 to 100% at an angular frequency of 1Hz (6.283rad.s ).
Conflicts of interest and acknowledgments
23 M. A. Ramin, K. R. Sindhu, A. Appavoo, K. Oumzil, M. W.
Grinstaff, O. Chassande, P. Barthélémy, Adv. Mater., 2017,
There are no conflicts to declare. This work was realized under
2
9, 1605227.
the frame of the Laboratory of Excellence AMADEus with the 24 A. N. Patwa, R. G. Gonnade, V. A. Kumar, M. M. Bhadbhade,
K. N. Ganesh, J. Org. Chem. 2010, 75, 8705-8708.
reference ANR-10-LABX-0042-AMADEUS, operated by the
2
2
2
5 J. Kumar, C. S. Purohit, S. Verma, Chem. Commun. 2008, 22,
2
“
“
Agence Nationale de la Recherche” under the program
Initiative for Excellence IdEx Bordeaux” (ANR-10-IDEX-0003-
526–2528.
6 S. Lena, S. Masiero, S. Pieraccini, GP. Spada, Chemistry 2009,
15(32), 7792-806
02). The work was also supported by INSERM and CNRS.
7 L. Simeone, G. Mangiapia, C. Irace, A. Di Pascale, A. Colonna,
O. Ortona, L. De Napoli, D. Montesarchio, L. Paduano, Mol
Biosyst. 2011, 7(11), 3075-86.
Aurore Beaurepaire, Candice Delcourt, Alexandra Gaubert are
gratefully acknowledged for their help with circular dichroism,
UV-vis and rheology experiments, respectively. The authors
6
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2
8 L. Simeone, D. Milano, L. De Napoli, C. Irace, A. Di Pascale, M.
Boccalon, P. Tecilla, D. Montesarchio, Chemistry 2011,
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9 D. Berti, P. L. Luisi, P. Baglioni, Colloids Surf. A 2000, 167, 95-
03.
0 A. Patwa, G. Salgado, F. Dole, L. Navailles and P. Barthélémy
Org. Biomol. Chem., 2013, 11, 7108 .
1 S. Khiati, N. Pierre, S. Andriamanarivo, M. W. Grinstaff, N.
Arazam, F. Nallet, L. Navailles and P. Barthélémy,
Bioconjugate Chem., 2009, 20, 1765–1772.
2
3
3
1
3
3
2 B. Desbat, N. Arazam, S. Khiati, G. Tonelli, W. Neri, P.
Barthélémy and L. Navailles, Langmuir, 2012, 28, 6816–6825.
3 L. Moreau, P. Barthélémy, Mohamed El Maataoui, and Mark
W. Grinstaff J. Am. Chem. Soc., 2004, 126, 7533-7539
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Pyrimidine
Purine
NH
2
O
N
T
O
C
G
NH
A
N
N
NH
2
N
NH
N
N
N
O
O
NH
2
N
O
N
N
HO
O
P
O
O
Lipid (diacyl glycerol)
O
O
O
H
O
O
Aꢀnucleoꢁde-lipidsꢀfamilyꢀfeaturingꢀtheꢀfourꢀnaturalꢀnucleobasesꢀ
wasꢀexploredꢀthroughꢀtheirꢀself-assemblyꢀproperꢁes.ꢀ