Cationic Amphiphiles Bearing a Diacetylenic Function in the Headgroup: Aggregative Properties and Polymerization

Article abstract of DOI:10.1021/acs.langmuir.0c01735

In the wide panorama of diacetylenic lipids, the photoresponsive conjugated 1,3-diyne function is usually encased into the hydrocarbon chain of the amphiphile at a variable distance from the headgroup. Therefore, the polydiacetylene network obtained by polymerization upon UV irradiation of the corresponding liposomes, exploited as sensing function, is embedded in the hydrophobic region of liposomes. Structurally related cationic diacetylenic amphiphiles featuring the conjugated triple bonds proximate to charged nitrogen were synthesized and evaluated in their ability to polymerize under aggregative conditions. The occurrence of polymerization only in certain aggregating conditions was rationalized by nuclear magnetic resonance (NMR) and Langmuir trough experiments.

Full text of DOI:10.1021/acs.langmuir.0c01735

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Article
Cationic amphiphiles bearing a diacetylenic function in
the headgroup: aggregative properties and polymerization
Alessandro Mauceri, Luisa Giansanti, donatella capitani, anatoly sobolev, Luciano Galantini,
Mauro Bassetti, Maria Grazia nemi, Denise Gradella Villalva, Sara Battista, and Giovanna Mancini
Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.0c01735 • Publication Date (Web): 24 Sep 2020
Downloaded from pubs.acs.org on September 26, 2020
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Cationic amphiphiles bearing a diacetylenic function
in the headgroup: aggregative properties and
polymerization
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Alessandro Mauceri,§ Luisa Giansanti, ‡ Donatella Capitani,†
†
Anatoly Sobolev,† Luciano Galantini, Mauro Bassetti, Maria
§
#
#
§ †
Sara Battista, ‡
Grazia Nemi, Denise Gradella Villalva,
Giovanna Mancini†*
§Dipartimento di Chimica, Università di Roma “Sapienza”, P.le A.
‡
Moro 5, 00185 Roma (Italy); Dipartimento di Scienze Fisiche e
Chimiche, Università degli Studi dell’Aquila, Via Vetoio 10,
†
6
7010 Coppito (Italy); CNR - Istituto per i Sistemi Biologici,
#
Via Salaria km 29.300, 00016 Monterotondo Scalo, RM, Italy; CNR,
Istituto per i Sistemi Biologici c/o Dipartimento di Chimica,
Università di Roma “Sapienza”, P.le A. Moro 5, 00185 Roma
(Italy).
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KEYWORDS chain length; effect of surfactants concentration;
Langmuir monolayer; micelle polymerization; polydiacetilenic
amphiphile; twin surfactant
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ABSTRACT
In the wide panorama of diacetylenic lipids the photo-responsive
conjugated 1,3-diyne function is usually encased into the
hydrocarbon chain of the amphiphile at variable distance from the
headgroup. Therefore, the polydiacetylene network obtained by
polymerization upon UV irradiation of the corresponding liposomes,
exploited as sensing function, is embedded in the hydrophobic
region of liposomes. Structurally related cationic diacetylenic
amphiphiles featuring the conjugated triple bonds proximate to
charged nitrogen were synthesized and evaluated in their ability
to polymerize in aggregative conditions. The occurrence of
polymerization only in certain aggregating conditions was
rationalized by NMR and Langmuir trough experiments.
INTRODUCTION
Polydiacetylenes (PDAs) are ene-yne conjugated polymers formed
upon 1,4-photopolymerization by UV or -light irradiation of
diacetylenes, which only proceeds when diacetylenic molecules are
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well aligned and closely packed in ordered structures such as
crystal lattices or self-assembled systems.1,2 In almost fifty years
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from the first preparation reported by Wegner in 1969, PDAs have
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been extensively investigated due to their unique optical and
structural properties.4-6 In particular, the capability of PDAs to
undergo modifications in their emission and/or absorption spectra
upon exposure to certain stimuli such as specific molecular
recognition7-9 or changes in the external conditions such as
temperature,10,11 pH12 or solvent,13,14 has designated them as very
promising molecular devices for sensor applications.
Diacetylenic amphiphiles constitute an elected class of molecules
to obtain PDAs thanks to their ability to self-assemble into
molecular films, micelles or vesicles, in which the geometrical
requirements for the occurrence of polymerization are satisfied.
A wide amount of diacetylenic amphiphiles has been synthesized15-30
and most of them bear the photo-responsive diacetylenic function
encased into the hydrocarbon chain of the amphiphile at variable
distance from the head group, whereas few examples of amphiphiles
showing the diacetylenic function in proximity of the hydrophilic
headgroup have been reported.28-30
As mentioned above, polymerization can occur if the diacetylenic
units are closely packed, in particular they should be preorganized
at a distance d that matches the repeat distance of the polymer,
i.e. 4.9 Å, with C(1)-C(4) distance (s) of 3.4 Å and a tilt angle
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of 45º (Scheme 1). A position in the middle of hydrophobic chain
might favor polymerization; however, efficient packing of
hydrophobic chains is not sufficient, per se, to guarantee
polymerization since other parameters such as hydrogen bonding
between headgroups and the extent of headgroup hindrance are
crucial.30 The occurrence of a high extent of polymerization is
reflected by the appearance of a variety of colors, from deep blue
to orange. The most fascinating feature of PDAs is their above
mentioned chromism in response to external stimuli, in particular
their blue to red color transition associated with a change in
their backbone conformation from planar to non planar. The
colorimetric response strongly depends on the position of the ene-
yne backbone along the hydrophobic chain, and it was observed
stronger when the polymeric chromophore is close to the
headgroups.30 The minor response of the ene-yne backbone to
external stimuli in correspondence of a deeper positioning along
the hydrophobic chain was attributed to a cohesive energy of the
alkyl chain between the diacetylene group and the headgroup.30
Hence, it is evident that the placement of polymerizable residues
close to the headgroups is a delicate issue that requires molecular
structures addressing a compromise between topological conditions
guaranteeing, on the one hand, polymerization and, on the other,
a high chromatic response.
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Scheme 1. Favourable geometric parameters of monomers in crystal
lattice (d = 4.9 Å, s = 3.4 Å, γ ~ 45 °) for topochemical
polymerization.
Here we report on the synthesis and the physicochemical
characterization
of
three
structure-analogues
cationic
diacetylenic amphiphiles (CDs) 1, 2 and 3 (Chart 1), characterized
by an unprecedented molecular structure in which the diacetylenic
moiety is not included in the hydrocarbon chain but embedded
between two hydrophilic groups, i.e. a quaternary nitrogen and a
hydroxyl group. Besides the structural analogues CDs 1-3,
constituted by a hydroxyhexadiynyl moiety linked to the ammonium
head group and a hydrophobic chain of different length (i.e. C16,
C18 and C20), another novel amphiphile bearing the diacetylenic
function included in one of the two hydrophobic chains, CD 4, in
analogous proximity to the charged nitrogen, was synthesized, for
comparison with CDs 1-3. The aggregative properties of CDs 1-4 in
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aqueous solution were investigated and their capability to
polymerize was ascertained by UV-vis and 1H-NMR spectroscopies.
Furthermore, surface pressure-area isotherm analysis of Langmuir
monolayers formed by CDs 1-3 was performed and compared to that of
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two
cationic
surfactants,
i.e.
N-hexadecyl-N,N,N-
trimethylammonium bromide (CTAB) and cetyl-N-(2-hydroxyethyl)-
N,N-dimethylammonium bromide (CDHB), with the purpose to obtain
additional information on the organization of the aggregates
formed by the newly synthetized amphiphiles.
Chart 1. Molecular structures of the newly synthetized CDs 1-4.
EXPERIMENTAL SECTION
Instrumentation. 1H- and
13
C H-NMR spectra were recorded on a
1
1
Bruker Avance II 300 spectrometer (operating at 300 for H and 75
MHz for 13C) or on a Bruker Avance 600 spectrometer (operating at
600.13 MHz for 1H and 150.62 MHz for 13C). Chemical shifts are
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reported in δ values relative to tetramethylsilane, with reference
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to the solvent as internal standard for H-NMR (CDCl at 7.26 ppm;
3
CD OD at 3.34 ppm) and for 13C (CDCl3 at 77.0 ppm; CD OD at 49.9
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ppm), with coupling constants (J) given in Hz. HRMS-ESI spectra
were recorded on a LTQ Orbitrap XL instrument. UV-vis measurements
were carried out on Perkin Elmer Lambda 18 double beam
spectrophotometer, absorbance normalized for optical path length
and concentration was reported. Conductivity measurements were
carried out on a HI-9932 Hanna conductivity meter equipped with a
thermostat apparatus, in the temperature range 4–60 °C. All
measurements were carried out in a jacketed cell maintained at the
appropriate temperature (± 0.1 °C). Ultrasonic processor UP100H,
ultrasonic power 100 Watts, titanium sonotrode MS 0.5 (tip diameter
7
mm), frequency 30 kHz, automatic frequency tuning system,
amplitude adjustable 20-100%, pulse 0-100% was used for liposomes
preparation. DLS measurements were performed with a Malvern Nano-
ZetaSizer, equipped with a 5 mW HeNe laser (wavelength = 632.8 nm)
and a digital logarithmic correlator. The normalized intensity
autocorrelation functions were measured at an angle of 173° at
25.0 ± 0.1 °C. The autocorrelation functions were analyzed by using
the cumulant fit. The first cumulant was used to obtain the
apparent diffusion coefficients D of the particles, further
converted into apparent hydrodynamic diameters, d , by using the
h
Stokes–Einstein relationship d =k T/3D, where k T is the thermal
h
B
B
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energy and  the solvent viscosity. The inverse Laplace transform
(CONTIN) was also used to resolve correlation functions of
multimodal system. Gel permeation chromatography (GPC) analyses were performed on
a Hewlett Packard Series 1050 HPLC system equipped with a 1047A RI detector and a TSK gel
alpha-4000 GPC column (Tosoh, Japan), using DMF with LiBr 0.1% w/w as the mobile phase.
The system was coupled to Clarity software version 6.2 (DataApex, Prague, The Czech Republic)
for signal processing. Surface pressure (π) measurements were carried out
by means of a Wilhelmy plate (39.24 mm of perimeter) technique
using a Langmuir Minitrough, KSV Instruments Ltd., Helsinki in
Teflon with 325 mm of length and 75 mm width and total area of 24
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80 mm2 enclosed in
a
plexiglass box to reduce surface
contamination.
Materials. PBS (Aldrich, 0.01 M phosphate buffer 0.0027 M KCl,
0.137 M NaCl, pH 7.4), pyrene, LiBr, CTAB and all reagents employed
for the synthesis of CDs 1-4 were purchased from Sigma-Aldrich.
31
CDHB was prepared according to a reported procedure. The synthesis
of compound 6 was performed according to the method reported in
the literature.32 CH Cl was dried by distillation over P O under
2
2
2 5
nitrogen.
-Bromohexa-2,4-diyn-1-ol (5). 1.0 g (9 mmol) of hexa-2,4-diyne-
6
1,6-diol and 3.0 g (9 mmol) of CBr were suspended in 15 mL of dry
4
CH Cl under nitrogen. The mixture was cooled to 0 °C and a solution
2
2
of 2.5 g (9 mmol) of PPh in 15 mL of anhydrous CH Cl were added
3
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drop-wise. The reaction was quenched after two hours by addition
of MeOH and the solvent was removed under reduced pressure. The
oily residue was purified by chromatography on silica gel (eluent
hexane/AcOEt=5/1) to afford 0.48 g (2.8 mmol) of 5 (yield 31%).
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H-NMR (CDCl , 300 MHz): δ = 4.354 (s, 2H, CH OH); 3.951 (s, 2H,
3
2
13
1
CH Br); 2.572 (s, 1H, OH) ppm.
C H-NMR (CDCl , 75 MHz): δ =
2
3
80.10, 75.35, 71.83, 71.07, 52.85, 5.36 ppm.
N-hexadecyl-N-(6-hydroxyhexa-2,4-diynyl)-N,N-dimethylammonium
bromide (CD1). 0.85 mL (2.5 mmol) of N,N-dimethyl-N-hexadecylamine
and 0.44 g (2.5 mmol) of 5 were dissolved in 30 mL of acetone and
the mixture was heated to reflux for one day. The reaction was
allowed to cool to room temperature obtaining 0.86 g (1.9 mmol) of
crystallized product 1 (yield 76%) that resulted pure by elemental
1
analysis. H-NMR (CD OD, 300 MHz): δ = 4.501 (s, 2H, NCH C≡ C),
3
2
4
.298 (s, 2H, CH OH), 3.385-3.456 (m, 2H, CH CH N), 3.172 (s, 6H,
2 2 2
N(CH ) ), 1.690-1.844 (m, 1H, CH CH N), 1.252-1.423 (m, 26H,
3
2
2
2
3
13
1
CH (CH ) CH CH N); 0.901 (t, J = 6.2 Hz, 3H, CH CH ) ppm.
C H-
3
2
13
2
2
HH
3
2
NMR (CD OD, 75 MHz): δ = 82.40, 76.38, 67.76, 66.92, 65.81, 55.53,
3
51.24, 50.88, 33.04, 30.73, 30.72, 30.68, 30.49, 30.14, 27.27,
-
+
2
3.72, 23.61, 14.40 ppm. HRMS: calculated for C H NO [M-Br ] :
24 44
362.3423; found: 362.3979.
-Bromopropargyl alcool (6). 2.0 mL (39 mmol) of Br were added
3
2
to a solution of 8.0 g (0.143 mol) of KOH in 65 mL of water. After
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cooling to 0 °C, 2.0 mL (35 mmol) of propargyl alcohol were added
and the mixture was stirred at 0 °C for 3 h in the dark. Then the
reaction mixture was extracted three times with diethyl ether and
the combined organic layers were dried over Na SO , filtered and
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concentrated under reduced pressure to afford 3.9 g (29 mmol) of
1
6
(yield 83%). H-NMR (CD OD, 300 MHz): δ = 2.475 (s, 1H, OH) 4.282
3
13
1
(s, 2H, CH ) ppm.
C H-NMR (CDCl , 75 MHz): δ = 78.21, 51.84,
2
3
45.83 ppm.
6-(Dimethylamino)hexa-2,4-diyn-1-ol (7). 14 mg (0.14 mmol) of
CuCl were added to 6.5 mL of a 30 % n-BuNH2 aqueous solution
previously deoxygenated under Argon flush. After the blue mixture
turned yellow by adding a few amount (tip of a spatula) of
hydroxylamine hydrochloride, 0.91 mL (8.4 mmol) of N,N-
dimethylpropargyl amine were added to the mixture. The mixture was
cooled to 0 °C and 1.0 g (7 mmol) of 6 was added at once. More
crystals of hydroxylamine hydrochloride were added throughout the
course of reaction, as necessary, to prevent the solution from
turning blue. After the reaction completion, the mixture was
extracted three times with diethyl ether and the combined organic
layers were dried over Na SO , filtered and concentrated under
2
4
reduced pressure. The crude product was purified by chromatography
on silica gel (eluent from CH Cl to CH Cl /AcOEt = 4/1) to afford
2
2
2
2
1
0
.71 g (5.1 mmol) of 7 (yield 74%). H-NMR (CDCl , 300 MHz): δ =
3
4
.331 (s, 2H, CH OH), 3.412 (s, 2H, CH N), 2.444 (s, 1H, OH), 2.357
2
2
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(
s, 6H, NMe ) ppm.
C H-NMR (CDCl , 75 MHz): δ = 75.91, 74.93,
2
3
6
9.90, 69.88, 51.37, 48.22, 43.86 ppm.
CD 2 and 3. 0.50 g (3.6 mmol) of 7 and 1 equiv of alkyl bromide
either octadecyl for CD2 or icosyl for CD3) were dissolved in 35
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
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0
1
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1
2
3
4
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6
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8
9
0
1
2
3
4
5
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7
8
9
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2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(
mL of acetone and the mixture was heated to reflux for one week.
The reaction was cooled to room temperature and the residue
obtained by removal of the solvent under reduced pressure was
purified by crystallization in acetone/Et O providing 0.89 g (1.9
2
mmol) and 0.77 g (1.5 mmol) of amphiphiles 2 (yield 53%) and 3
(yield 43%), respectively.
N-(6-hydroxyhexa-2,4-diynyl)-N,N-dimethyl-N-octadecylammonium
1
bromide (CD 2). H-NMR (CD OD, 300 MHz): δ = 4.534 (s, 2H, NCH C≡
3
2
C), 4.321 (s, 2H, CH OH), 3.419-3.496 (m, 2H, CH CH N), 3.211 (s,
2
2
2
6H, NMe ), 1.755-1.863 (m, 2H, CH CH N), 1.257-1.422 (m, 30H,
2
2
2
3
13
1
CH (CH ) CH CH N), 0.933 (t, J = 6.2 Hz, 3H, CH CH ) ppm. C H-
3
2
15
2
2
HH
3
2
NMR (CD OD, 75 MHz): δ = 82.70, 76.61, 67.85, 66.94, 66.06, 54.63,
3
5
3
[
1.42, 50.90, 33.01, 30.75, 30.67, 30.65, 30.51, 30.44, 30.37,
0.01, 27.32, 23.77, 23.68, 14.36 ppm. HRMS: calculated for C H NO
26 48
-
+
M-Br ] : 390.3736; found: 390.4272.
N-icosyl-N-(6-hydroxyhexa-2,4-diynyl)-N,N-dimethylammonium
1
bromide (CD 3). H-NMR (CD OD, 300 MHz):  = 4.491 (s, 2H, NCH C≡
3
2
C), 4.302 (s, 2H, CH OH), 3.394-3.463 (m, 2H, CH CH N), 3.187 (s,
2
2
2
6H, NMe ), 1.758-1.863 (m, 2H, CH CH N), 1.255-1.424 (m, 34H,
2
2
2
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2
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3
13
1
CH (CH ) CH CH N), 0.931 (t, J = 6.2 Hz, 3H, CH CH ) ppm.
C H-
3
2
17
2
2
HH
3
2
NMR (CD OD, 75 MHz): δ = 82.52, 76.51, 67.82, 67.03, 65.99, 55.75,
3
51.44, 50.90, 33.01, 30.70, 30.65, 30.54, 30.40, 30.36, 30.00,
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
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9
0
1
2
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5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
- +
2
7.21, 23.66, 14.31 ppm. HRMS: calculated for C H NO [M-Br ] :
28 52
4
18.4049; found: 418.4695.
Pentadeca-2,4-diyn-1-ol (8). Compound 8 (0.60 g, yield 37%) was
prepared from compound 6 (1.00 g, 7.4 mmol) according to the
procedure described above for the preparation of compound 7. 1H
NMR (CDCl , 300 MHz, 298 K):  = 4.321 (s, 2H, CH OH), 2.282 (t, J
3
2
=
6.8 Hz, 2H, -CH CC), 1.604 (s, 1H, OH), 1.513 (qt, J = 6.8 Hz,
2
2H, CH CH CC), 1.196−1.458 (m, 14H, -(CH ) -), 0.881 (t, J = 7.5
2
2
2 7
13
1
Hz, 3H, CH ) ppm.
C H-NMR (CDCl , 75 MHz, 298 K):   82.02,
3
3
7
3.43, 71.05, 64.33, 51.68, 31.94, 29.52, 29.51, 29.38, 29.12,
28.84, 28.13, 22.72, 19.32, 14.11 ppm.
N-hexadecyl-N,N-dimethyl-N-(pentadeca-2,4-diynyl)-ammonium
bromide (CD4). Compound 8 (0.46 g, 2.1 mmol) was dissolved in dry
CH Cl (15 mL) under nitrogen. The mixture was cooled to 0 °C, and
2
2
a solution of PBr (0.20 mL, 2.13 mmol) in CH Cl (3 mL) was added
3
2
2
dropwise. After two hours, the solvent was removed under reduced
pressure and the oily residue was purified by chromatography on
silica gel (eluent: CHCl ) to afford the desired product (0.52 g,
3
1.8 mmol, yield 89%) that was dissolved in acetone (5 mL) with
N,N-dimethylhexadecylamine (0.68 mL, 2.0 mmol). The mixture was
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heated to reflux for one day. After cooling to room temperature,
the precipitate was isolated and crystallized from Et O to afford
2
amphiphile CD 4 (0.38 g, yield 60%) that resulted pure by elemental
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
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3
4
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5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
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2
3
4
5
6
7
8
9
0
1
2
3
4
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7
8
9
0
1
analysis. H NMR (CDCl , 300 MHz, 298 K):  = 4.901 (s, 2H, -CH N),
3
2
3
.594 (m, 2H, CH CH N), 3.463 (s, 6H, NMe ), 2.291 (t, J = 7.4 Hz,
2 2 2
2H, -CH CC), 1.734 (s, 2H, -CH CH N), 1.546 (qt, J = 7.4 Hz, 2H, -
2
2
2
CH CH CC),
1.192−1.413
(m,
40H,
CH (CH ) CH CH CC,
2
2
3
2
7
2
2
13
1
CH (CH ) CH CH N), 0.864 (t, J = 6.5 Hz, 6H, -CH CH ) ppm. C H-
3
2
13
2
2
2
3
NMR (CDCl , 75 MHz, 298 K):  = 84.61, 64.24, 63.53, 62.38, 55.46,
3
50.65, 31.91, 31.87, 29.72, 29.61, 28.96, 27.9, 26.2, 22.94, 22.73,
+
1
9.31, 14.10 ppm. HRMS: calculated for C H N [M-Br] : 472.4923;
33 62
found: 472.5401.
Methods
Determination of Krafft point (T ) and Krafft temperature of CDs
K
1-3. TK values of CDs 1-3 were determined by measuring the
temperature-scan conductivity on 20 mL sample of 20 mM surfactant
solutions in the temperature range 10-60 °C according to a
described procedure.33 During the measurements, the solution was
continuously stirred and the temperature was raised at a rate of
0.2 °C/min. The error in the conductivity measurements was within
±
0.1%. The T was defined as the temperature at which conductivity
k
begins to rise abruptly, whereas the Krafft temperature relative
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6
to the chosen concentration is the temperature above which the
specific conductivity remains constant.
Determination of critical micellar concentration (cmc) of CD 1-3. Known volumes of stock
solutions of CD 1-3 were added at 60 °C to 30 mL of deionized water to carry out conductivity
measurements after mixing and temperature equilibration. The cmc value for each amphiphile is
determined by the intercept point of the two linear trends obtained by plotting the specific
conductivity versus the concentration of amphiphile.
0
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7
8
9
0
Determination of the critical aggregation concentration (cac) of CD 4 by fluorescence
measurements. The cac of CD 4 was measured at 25 °C by a reported procedure that exploits the
variation in the intensity of the vibronic fine structure of pyrene monomer emission spectrum upon
3
4
association with aggregates. Aqueous solutions (3 mL) of CD 4 between 10 μM and 80 μM were
added to a defined amount of pyrene in order to obtain a final concentration of ~0.5 μM (prepared
from 7 μL of a 160 μM stock solution of pyrene in absolute ethanol degassed under a nitrogen
flux). The solutions were kept at 40 °C, under stirring, for 12 h. Emission spectra of the solutions
were acquired in the range 350–450 nm (λ = 335 nm). The cac was defined as the concentration
exc
where the plot of intensity ratio of the third (I , 380 nm) and first (I , 370 nm) vibronic peaks of
3
1
pyrene I /I versus CD 4 concentration begins to rise.
3
1
Preparation of CD 4 liposomes. Liposomes formed by CD 4 were prepared according to the
method of injection/sonication.35 Briefly, a proper amount of CD 4 was dissolved in
dimethylsulfoxide and injected into an opportune volume of water under heating at 80 °C and
vigorous stirring to give a 5mM liposomal suspension. Liposome suspension was exposed to
acoustic energy from a tip probe sonicator in order to reduce the multilamellarity of the vesicles
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and homogenize their dimensions. The exposure time to sonication (15 minutes, 70 Watts,
3
6
pulsed-power mode) determines the size of the vesicles. Lastly, before polymerization
liposomes were stored at 4 °C for at least 6 h to obtain crystallization of lipid membranes.
1
1
1
1
1
1
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2
2
2
2
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0
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2
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2
3
4
5
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1
2
3
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8
9
0
Determination of aggregates size by DLS. Measurements were
performed on micellar samples containing sodium bromide as added
electrolyte to minimize the effects of counterion cloud
fluctuations on the dynamic of ionic micelles in solution.37-38 The
concentration of added electrolyte was observed to affect the size
of the aggregates and is was adjusted depending on the surfactant
type and concentration to optimize the scattered light intensity.
Temperature scans revealed a decreasing trend of the aggregate
size of CD1-3 by heating, as observed for several ionic surfactants
self-assembling in wormlike micelles.39 The data at the highest
accessible temperature (60 °C), far from the Krafft point were
reported for these samples. DLS measurements were carried out on
aqueous micellar solutions of CD 1 [75 mM] at 60 °C in NaBr 100
mM, CD 2 [25 mM] at 60 °C in NaBr 50 mM, CD 3 [5 mM] at 60 °C in
NaBr 15 mM and on CD 4 liposomes. All aggregates were analyzed by
DLS measurements soon after preparation, and after irradiation.
Measurements were repeated after 48 h.
Irradiation of CDs in aggregative conditions. Micellar solutions
of amphiphiles 1-3 at 60 °C were irradiated at 254 nm for 2 h by
using
a
low-pressure mercury UV-lamp (Spectroline EF-140C).
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6
Polymerization was carried out on a) aqueous solutions of 0.06 mM
CD 1(2, 3), b) aqueous solutions of 76 mM CD 1(2, 3) and 150 mM
NaBr and c) deuterated aqueous solutions of 76 mM CD 1(2, 3) and
150 mM NaBr.
0
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0
GPC elution. Samples for GPC analysis were prepared dissolving
either the monomer or polymerized CD 1 in DMF with 0.1% LiBr w/w
(final concentration of ~1 mg/mL). The polymerized CD 1 sample was
obtained by i) irradiating at 254 nm 10 mL of 0.06 mM aqueous
solution for 2 h at 60 °C, ii) removing the water under high
vacuum.
Langmuir trough measurements. CD 1(3-4), CTAB and CDHB monolayers
were prepared using PBS as subphase. The minitrough was
thermostated in a 25 ºC bath. A volume of 10 µL of lipid solution
(
1.0 mg/mL dissolved in CHCl ) was spread over the aqueous subphase
3
using a Hamilton microsyringe. After the deposition, the solvent
was allowed to evaporate for 5 minutes at an initial pressure lower
then 1 mN/m before starting three cycles of compression/expansion
up to π = 3 mN/m to stabilize lipid monolayer by moving the nylon
barriers at a constant rate of 50 mm/min. The isotherms were
registered closing completely the barriers (target to stop at π =
100 N/m). Different isotherms on independent samples were carried
out several times to verify their reproducibility. The reported
isotherms are the average of at least three different and
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independent
runs.
The
experimental
error
for
the
final
concentration of each sample was estimated by means of the error
propagation, being in the order of ± 2.5 % for the reported values.
For calculation, we considered the error of the analytical balance
to be ± 0.5∙10-4 g, and the error of Hamilton syringe of ±1.25 % of
nominal volume.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
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2
3
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2
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5
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7
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0
1
2
3
4
5
6
7
8
9
0
RESULTS AND DISCUSSION
Synthesis and characterization of CDs 1-4. CDs 1-4 are characterized by a
novel molecular structure where the polymerizable diacetylenic moiety is located close to the
ammonium headgroup. To prepare CD 1 (Scheme 2), the commercially
available hexa-2,4-diyne-1,6-diol was mono-brominated according to
a reported procedure carried out in Apple’s conditions40 and the
resulting product was used for the alkylation of the N,N-
dimethylhexadecylamine. The latter reaction was carried out at
reflux in acetone, which was found to be a good solvent for the
subsequent crystallization of CD 1.
Scheme 2. Synthetic pathway to obtain amphiphile CD 1
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4
4
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5
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5
5
5
5
6
For the preparation of CDs 2-3 (Scheme 3), a different procedure
was
followed
because
N,N-dimethyloctadecyl-
and
N,N-
dimethylicosylamine are not commercially available. Therefore the
diacetylenic moiety was obtained through Cadiot-Chodkiewicz
0
1
2
3
4
5
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3
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9
0
coupling between 3-bromopropargyl alcohol 6, obtained by
a
reported
procedure,32
and
commercially
available
N,N-
dimethylpropargyl amine. Coupling adduct 7 was then alkylated with
octadecyl- or icosylbromide, in the same conditions used for
obtaining CD 1, to afford CD 2 and CD 3, respectively. However, in
these cases, the quaternization reaction required much longer time
probably because of the lower electrophilicity of the used
alkylbromides with respect to that of compound 5 involved in the
preparation of CD 1.
Bromo-alkyne 6 was also involved in the preparation of amphiphile
CD 4 through Cadiot-Chodkiewicz coupling with 1-dodecyne to afford
alcohol 8 that was successively brominated with PBr and used as
3
alkylating agent of N,N-dimethylhexadecylamine to obtain CD 4.
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0
1
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2
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0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Scheme 3. Synthetic pathways to obtain amphiphiles CDs 2-4
In order to define the temperature/concentration domain in which
CDs 1-3 form aggregates, Krafft point-temperature T and cmc (at T
K
>
TK) of amphiphiles were measured by conductivity experiments. As
expected, the value of T and that of cmc increases and decreases,
K
respectively, as the hydrophobic chain becomes longer (Table 1).
Table 1. Physicochemical features of CDs
CD
1
TK / °Ca
cmc/cac a [M]
(8.0 ± 0.4)·10-6 [60 °C]
(5.5 ± 0.3)·10-6 [60 °C]
D
8
h
(nm)b
[60 °C]
PDIb
0.24
0.48
0.22
0.21
27
35
42
<4
2
20 [60 °C]
33 [60 °C]
-6
3
(1.0 ± 0.3)·10 [60 °C]
(7.0 ± 0.5)·10-6 [25 °C]
310 [25 °C]
d
4
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6
a1-4 Krafft point (T ) and cmc/cac were determined in deionized
K
b
water; experimental conditions of DLS measurements: 75 mM CD 1 at
60 °C in 100 mM NaBr; 25 mM CD 2 at 60 °C in 50 mM NaBr; 5 mM CD
0
1
2
3
4
5
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0
1
2
3
4
5
6
7
8
9
0
3 at 60 °C in 15 mM NaBr; 5 mM CD 4 at 25 °C in H O; hydrodynamic
2
diameters D and polydispersity indexes (PDI) were obtained from
h
cumulant analysis, typical CONTIN distributions for some samples
are reported in the supplementary information. T were determined
K
with an accuracy of ± 0.1 °C; error in determination of Dh is
d
within ± 5% of the value. Reported data refer to CD4 liposomes.
DLS analyses of CD solutions above cmc and T reported in Table
K
1
indicated the presence of aggregates consistent with spherical
or worm-like shaped micelles in the case of CDs 1-3 (D values of
h
8, 20 and 33 nm, respectively) and with vesicular aggregates in
the case of CD 4 (D ~ 300 nm). In the case of CD 4, the value at which
h
lipids begin to self-assembly into bilayers, properly named critical aggregative concentration (cac),
was assessed by a method that exploits pyrene as fluorescent probe.34
Irradiation of CDs and assessment of polymerization by UV-vis
measurements. The photopolymerization of CDs in aggregating
conditions (micellar and vesicular aggregates formed by CDs 1-3
and CD 4, respectively) was investigated.
Extended diacetylene polymerization is known to yield colored
samples due to extended π-conjugated ene-yne backbone with
4
absorption at high frequencies. However, in the case of micellar
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aggregates, diacetylene polymerization was not expected to yield
a colored solution, because the low aggregation number of micelles
would limit the extent of the conjugated backbone.41 Therefore, in
the case of micellar aggregates, the occurrence of polymerization
is generally assessed by the appearance of new bands in the UV
spectrum. Concentration was expected to play a major role in the
occurrence of polymerization by controlling the state of phase of
aggregates and hence the relative topology of diacetylene residues
within the micelle. Micelle solutions of amphiphiles CDs 1-3 at
different concentrations - ranging from 0.06 to 60 mM - were
irradiated for two hours at 60 °C (i.e. above the TK of
amphiphiles). Surprisingly the evidence of PDA formation was
observed, for all amphiphiles, in the case of 0.06 and 0.6 mM
samples, whereas polymerization occurred at a lower or negligible
extent for more concentrated solutions (Figure 1).
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5
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5
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5
6
Figure 1. Absorption spectra of aqueous CD 1 solutions recorded
after two hours of irradiation carried out at 60 °C and different
concentrations: 0.06 (blue dotted trace), 0.6 (green dashed dotted
trace), 6.0 (red dashed trace) and 60 (purple dashed trace) mM;
black solid trace is referred to the non-irradiated sample 0.06
mM. An analogue behavior was observed in the case of CD 2 and CD
0
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9
0
3.
Actually, in agreement with literature reports on PDA micelles,41,
42
two broad bands appeared in the region between 220 and 350 nm
of the absorption spectra upon irradiation of 0.06 and 0.6 mM
samples, the highest values of absorbance being observed in the
case of 0.06 mM sample. Polymerization of 0.06 mM samples was
investigated by recording the UV spectra over time during
irradiation. A net change of the UV spectra was detectable yet
after 15 minutes of irradiation for all three amphiphiles. The
highest extent of polymerization occurred within one hour (Figure
2), then polymerization rate decreased and no significant changes
were detected in the next hour (Figure 2d). Absorption maxima were
observed at slightly different wavelengths in the case of the three
amphiphiles: ~280 nm for CD 1, and ~302 nm for both CD 2 and CD 3
(Figure 2a-2c). This result could indicate
a
more extended
conjugation in the case of CDs 2-3 with respect to CD 1. Further,
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in the case of CD 3, the maximum of absorbance of the PDA-
associated band was the most intense (i.e. 0.17); this result could
be related to the lowest value of cmc.
1
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9
0
1
2
3
4
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6
7
8
9
0
Figure 2. Absorption spectra of 0.06 mM aqueous solutions of a)
CD1, b) CD2 and c) CD3 recorded at fixed times of irradiation at
60 °C; d) evolution of intensity of absorption maximum as a
function of irradiation time.
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A further evidence of the occurrence of polymerization was
provided by GPC performed on monomeric and polymerized samples of
CD 1. In fact, the chromatogram relative to the non-irradiated
sample displayed a sharp peak (retention time ~16 min) of the
monomer whereas that relative to the irradiated sample showed a
slightly broad peak at lower retention time (i.e. ~14 min)
attributable to the polymer (Figure 3).
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0
1
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6
7
8
9
0
Figure 3. GPC chromatograms relative to monomeric (black solid
trace) and polymerized (blue dashed trace) samples of CD 1 (eluent
DMF with LiBr 0.1% w/w).
Polymerization upon UV irradiation of CD 4 vesicular aggregates
prepared according to the method of injection/sonication was
investigated in different aqueous media, i.e. water, PBS 150 mM
and PBS 15 mM. DLS analysis before and after irradiation indicated
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the presence of a stable monomodal distribution of vesicles
featuring a hydrodynamic diameter of ~300 nm in both water and PBS
buffers. In all polymerization experiments, both the lack of color
detectable with naked eye and the absence of typical bands in the
UV spectrum indicated that polymerization did not occur. The
failure of polymerization of CD 4 liposomes as well as that of CDs
1
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2
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9
0
1-3 aggregates at high concentration should be due to an
unfavorable arrangement of diacetylene moieties.
Investigation on CD 1 arrangement in aggregating conditions.
The extension of solid-state reactivity conditions to colloidal
systems suggests that polymerization of the diacetylenic function
into an ene-yne polymer requires a specific topology/alignment of
the diacetylenic moieties. Therefore, further investigations were
carried out to assess the topology of the diacetylenic functions
of amphiphile CDs 1-3 within their micellar aggregates, aimed at
understanding the results of polymerization experiments. In fact,
the hydrophobic diacetylenic function could be folded in the
hydrophobic region of the micelle or, due to the presence of the
terminal hydroxyl group, lay at the water/micelle interface,
perpendicular to the hydrophobic chains, thus being exposed to
water. ROESY NMR experiments aimed at elucidating this point were
carried out on 0.06 and 60 mM (i.e. in proximity of and largely
above the cmc, respectively) aqueous solutions of CD 1 chosen as
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testing molecule at 45 °C (i.e. above the T ). Further, a long
K
1
13
range
H- C heteronuclear correlation experiment (HMBC) was
carried out to univocally assign the signals of methylenic protons
adjacent to diacetylenic function. In fact, such protons give
0
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similar NMR signals in terms of multiplicity and chemical shift in
1
H NMR spectrum (i.e. singlets at 4.46 and 4.69 ppm in D O). The
2
HMBC spectrum allowed to attribute the signal at 4.69 ppm to the
propargylic protons next to the charged nitrogen by means of three-
bond coupling with the carbons adjacent to the quaternary nitrogen,
i.e. those of the two methyl groups and the first methylene of the
hexadecyl chain.
ROESY spectrum of 60 mM aqueous solution of CD 1 shows a
correlation peak between the signal due to the methylene adjacent
to the hydroxyl group and those due to methylene groups of the
hexadecyl chain, thus indicating that the hydroxyhexadiynyl
substituent is folded toward the hydrophobic region of the micelle
(Figure 4).
In the case of 0.06 mM sample, the absence of an analogous
correlation peak did not provide a definite indication, because it
could be ascribed to the occurrence of loose aggregates where the
distance between lipid components does not allow a NOE effect.
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0
a
N
N
b
b
a
OH
OH
Figure 4. Region of ROESY spectrum of the 60 mM sample of CD1 in
D2O and representation of the topology of amphiphiles within the
aggregates; folding of hydroxyhexadiynyl group in the hydrophobic
region of the micelle is consistent with the presence of the
correlation peak between the methylene group adjacent to the
hydroxyl group (4.46 ppm) and those of the hexadecyl chain.
Langmuir trough experiments. To support the hypothesis of the folding of the
hydroxyhexadiynyl chain suggested by the results of the ROESY experiment, we carried out
Langmuir trough experiments to obtain Langmuir compression isotherms of CD 1, CTAB and
CDHB. Non-polymerizable CTAB and CDHB were chosen because the only
difference between each of them and CD 1 concerns a substituent on
the ammonium nitrogen, namely hydroxyhexadiynyl (CD 1), methyl
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(
CTAB) and hydroxyethyl (CDHB), and the differences in their π/A
compression isotherms can be directly related to their different
substituents; therefore they can be goodcomparison systems to investigate the
topology of the hydroxyhexadiynyl residue in the lipid film. In fact, in the lipid monolayer at the
air water interface the hydroxyethyl residue of CDHB should be exposed to the water phase thus
giving a compression isotherm similar to CTAB. If the hydroxyhexadiynyl residue of CD 1 were
exposed to water the compression isotherm of its monolayer should be analogous to those of CTAB
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and CDHB. The obtained /mean molecular area (A ) isotherms are reported in Figure
h
5A.
The obtained monolayers were stable and in equilibrium
conditions, as proven by the fact that the isotherms obtained by
the compression/expansion cycles process were pretty similar to
those obtained by closing completely the barriers.
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Figure 5. π−A isotherms of monolayers of CD 1 (black line in A
and B) and A) CTAB (purple line), CDHB (blue line); B) CD 2 (red
line) and CD 3 (green line) monolayers.
1
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It can be clearly observed that all the isotherms of CD 1, CTAB
and CDHB show a similar behavior (Figure 5A), undergoing the
transition from the liquid-expanded (LE) phase to the liquid-
condensed (LC) phase up to the closure of the barriers. However,
in the isotherm of CD 1 π rises at higher A , in fact in
h
correspondence of the same A the π of the isotherms of CTAB and
h
CDHB monolayers is lower with respect to π of the isotherm of CD
1. Furthermore, in the case of CD 1 it seems that the monolayer
does not reach the collapse condition differently from the
monolayers of CTAB and CDHB. This finding indicates that the extent
of lipid packing in CD 1 monolayer is reduced with respect to CTAB
and CDHB ones, thus suggesting that the diacetylenic segment does
not protrude toward the subphase, as it happens for the methyl
group of CTAB and the hydroxylethyl group of CDHB, but is rather
folded toward the hydrophobic region (Figure 6), in agreement with
what suggested by NMR experiments.
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