Click-chemistry as a mix-and-match kit for amphiphile synthesis

Article abstract of DOI:10.1021/co300080g

A small library of amphiphilic compounds was synthesized in an array using the Huisgen 1,3-dipolar cycloaddition of terminal alkynes with azides (CuAAC or click reaction). The self-assembling properties of these compounds were evaluated by polarizing microscopy and synchrotron small-angle X-ray scattering analysis.

Full text of DOI:10.1021/co300080g

Research Article
pubs.acs.org/acscombsci
Click-Chemistry as a Mix-and-Match Kit for Amphiphile Synthesis
Oliver E. Hutt, Xavier Mulet, and G. Paul Savage*
CSIRO Materials Science and Engineering, Private Bag 10, Clayton South MDC, Vic 3169, Australia
S
* Supporting Information
ABSTRACT: A small library of amphiphilic compounds was synthesized in
an array using the Huisgen 1,3-dipolar cycloaddition of terminal alkynes with
azides (CuAAC or click reaction). The self-assembling properties of these
compounds were evaluated by polarizing microscopy and synchrotron small-
angle X-ray scattering analysis.
KEYWORDS: amphiphiles, cycloaddition, click chemistry, self-assembly, surfactants
tionally simple processes of copper-catalyzed, Huisgen 1,3-
dipolar cycloaddition of terminal alkynes with azides (CuAAC
or click reaction).12 This method lends itself to rapid
amphiphile library generation for high-throughput character-
ization.
INTRODUCTION
■
Amphiphilic compounds are industrially significant chemicals
that are typically composed of a polar, hydrophilic headgroup
and a nonpolar, hydrophobic tail. Commercially important
amphiphiles include surfactants, soaps, lipids, and amphiphilic
block copolymers, which are used extensively in food, paint,
adhesive, detergent, and cosmetic applications.1 When placed in
a solvent, most often of aqueous nature, amphiphilic molecules
will not only migrate to surfaces but also self-assemble into a
range of structures. These lyotropic liquid crystalline assemblies
include structures such as micelles, lamellar bilayers, and
hexagonal and cubic mesophases with a range of interfacial
curvatures.2 The nature of the aggregates formed by these
amphiphilic moieties varies considerably due to their molecular
architecture. The molecule’s inherent shape and polarity
properties will have a significant effect on the nanostructure
and long-range order of the self-assembled aggregates
formed.3−5 Variables, such as the balance of lipophilic and
hydrophilic strength of the nonpolar and polar moieties, the
critical packing parameter, and the composition of the
components, all influence the nature of the self-assembled
structure for a given amphiphilic system.6,7 Local environmental
properties will also play a significant role in the phase behavior
of these compounds.
We chose the hydrophobic tail as the alkyne partner for the
CuAAC cycloaddition reaction; primarily because terminal
alkynes are commercially available in all appropriate chain
lengths for amphiphile synthesis. C10, C12, C14, C16, C18,
and C20 straight-chain terminal alkynes were acquired from
commercial suppliers. The cationic, hydrophilic headgroup,
azide partners for the CuAAC click reaction were prepared by
reacting commercially available 4-azidophenacyl bromide 1
with tertiary amines in acetone. Four amines were selected:
triethylamine, N,N,N′,N′-tetramethylethylenediamine
(TMEDA), N-methylmorpholine (NMM), and N,N′-dimethyl-
piperazine (DMP). The product quaternary ammonium salts
2−5 precipitated immediately from acetone and could be
collected in essentially pure form by filtration (Scheme 1).
Scheme 1. Synthesis of Hydrophilic Head Group (Azide
Partners)
One of the more exciting applications for self-assembled
macromolecular structures is in the controlled delivery and
release of drugs.8,9 High-throughput techniques for screening
the self-assembly properties of amphiphilic molecules can
greatly accelerate the discovery of 3D nanostructured nano-
particles for drug delivery.10 One limitation on the rapid
development of this technology is the challenge in synthesis
and purification of diverse amphiphiles with which to tune the
molecular architecture of the self-assembled nanoparticles. This
is especially problematic for research groups without access to
specialist synthetic organic chemistry capabilities and equip-
ment. Herein, we describe a modular system of easily prepared,
polar head groups and commercially available, nonpolar tail
groups that are suitably functionalized to click11 together in any
combination. The final amphiphile assembly uses the opera-
Received: July 30, 2012
Revised: September 12, 2012
Published: September 22, 2012
© 2012 American Chemical Society
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The azide head groups 2−5 were then reacted with the
alkyne tail groups in the presence of copper metal with t-
butanol as solvent (for example, Scheme 2). The reactions were
temperature scans. Self-assembly was observed for all lipids in
the presence of water, with lamellar and micellar phases
typically observed. This is expected as the charged head groups
and straight saturated aliphatic chains will often lead to flat
interfaces. In general, the longer chain lipids formed lamellar
structures, while the shorter C10 and C12 lipids typically
formed micellar aggregates at room temperature under excess
water conditions.
Scheme 2. Click Reaction between Azide Head Group 2 and
C14 Alkyne Tail (1-Tetradecyne)
Several trends in self-assembly behavior were observed as a
function of increasing aliphatic chain length. Typically, the
shorter chained amphiphiles rapidly formed micelles at the
water interface at room temperature. At room temperature, a
clear phase sequence of water, micelles, lamellar phase, inverse
micelles, and a second lamellar phase (possibly a re-entrant
lamellar phase) were observed for samples 2-C10, 2-C12, 2-
C14, 3-C10, and 5-C10. Representative microscopy data for
these samples are provided in Figure 2 (all of the microscopy
carried out simultaneously in a six by four array of sample tubes
set in a machined aluminum block (Figure 1), heated at 35 °C
Figure 2. CPLM images of water penetration scans at 35 °C for (A) 2-
C10 and (B) 2-C20. Birefringent lamellar and isotropic domains are
highlighted.
data is available in the Supporting Information). This sequence
is as expected with decreasing water content. As the
hydrophobicity of the molecules increased, lamellar phases
were observed to exist in excess water and these retained their
integrity to relatively high temperatures (50−60 °C). As the
temperature was increased, a transition from a lamellar to a
micellar phase was observed. The systematic increase in chain
length led to a distinct increase in aggregate stability; the
lamellar−fluid isotropic phase transition temperature increased
with increasing chain length. Structural differences in the
headgroup of the amphiphile also affect the stability of the
lamellar aggregate with temperature. Series 3, 4, and 5 retained
their lamellar structure at lower chain lengths and higher
temperature than series 2. This may be correlated to the level of
hydrogen bonding and “headgroup to headgroup” or “head-
group to solvent” interactions as well as the relative
hydrophilicities of the head groups. Only the triethylamine
headgroup has one hydrogen bond donor/acceptor while the
others have at least two. Using the octanol−water partition
coefficient, it is possible to assess the hydrophilic nature of each
headgroup and relate this to the persistence of the lamellar
phase at higher temperatures. The most hydrophilic headgroup
is that of triethylamine (series 2) with an octanol−water
partition coefficient log ko/w 1.44. The other head groups,
tetramethylethylenediamine, N-methylmorpholine, and 1,4-
dimethylpiperazine, have octanol−water partition coefficients
of 0.30, −0.33, and −0.40, respectively. Therefore a lower log
Figure 1. Amphiphile library synthesis array. Azides 2−5 reacted with
terminal alkynes (C10 = 1-decyne, C12 = 1-dodecyne, C14 = 1-
tetradecyne, C16 = 1-hexadecyne, C18 = 1-octadecyne, C20 = 1-
icosyne).
for 24 h. The reactions proceeded to full conversion, as
monitored by LC−MS in purities ranging from 79 to 100%
(average 89%). The copper catalyzed azide−alkyne cyclo-
addition reaction is known to proceed regioselectively to give
1,4-cycloadducts.12 Excess copper was simply filtered off, then
the solvent was evaporated and the solid amphiphile products
were analyzed as described below. The triazole cycloadducts are
henceforth referred to by a combination of their headgroup
number (2−5) and precursor alkyne chain length (C10, C12,
C14, C16, C18, and C20).
Ultimately, as these amphiphiles are aimed at the creation of
self-assembled phases, the utility of the material library can be
assessed by considering each material’s aggregation properties
in aqueous solutions. Assessing self-assembly and the structures
formed can be performed via a range of techniques. Cross-
polarized light microscopy (CPLM) can differentiate between
isotropic and anisotropic liquid crystalline phases; characteristic
optical textures allow visual identification of certain phases.13
Definitive phase assignment is based on small-angle X-ray
scattering (SAXS) data which yields structural information as
well as unit cell size.2
The self-assembly in water of each amphiphile was initially
assessed using polarizing microscopy through water-penetration
ko/w is associated with increased stability of the liquid crystalline
(specifically lamellar) phases and their retention to higher
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temperatures. In addition, lamellar phases were observed at
longer chain amphiphiles. This trend was observed for all four
headgroup series in agreement with the CPLM data presented
above (see the Supporting Information for SAXS of all
molecules in 80% water conditions).
shorter chain lengths for molecules with a lower log ko/w
.
Synchrotron small-angle X-ray scattering (SAXS) was also
performed on these materials under excess water conditions to
confirm the microscopy findings and evaluate the effect of
molecular structure on the adopted mesophase and associated
lattice parameter. SAXS data confirmed the presence of lamellar
phases under excess water conditions (80 wt % water) observed
via CPLM for the longer chained lipids. A broad, diffuse peak,
characteristic of a fluid isotropic phase (potentially micellar or
liposomal material), was commonly present in these highly
hydrated samples at 25 °C.
In addition, for a particular headgroup, increasing chain
length also drives an increase in lattice parameter of the lamellar
phase (for constant hydration level and temperature). An
increase in lattice parameter was observed with increasing chain
length for the C16, C18, and C20 derivatives (see Figure 4).
Construction of self-assembling material libraries has been
previously attempted; however, generating sufficient numbers
of related materials with controlled structural differences
remains limited. For example, in the comprehensive review
by Fong et al., relatively few materials that form inverse phases
were identified, and these often had limited structural
information available.14 At the same time, the CuAAC click
reaction has been extensively used for generating libraries of
compounds, especially in the pharmaceutical15 and materials16
fields. It is therefore somewhat surprising that this versatile and
operationally simple method has not to date been exploited
more widely in assembling surfactant libraries. We are currently
exploring CuAAC click methodology to prepare amphiphile
libraries with diverse polar groups such as sugars, peptides, and
anionic and cationic head groups, along with a greater variety of
hydrophobic tail groups.
A clear increase in long-range ordering was observed with
increasing chain length for all head groups tested under excess
water conditions (80 wt % water). Figure 3 shows a typical
EXPERIMENTAL PROCEDURES
■
General synthetic procedures, and analytical instrumentation
were as previously described.17
Representative Procedure for Headgroup Synthesis.
2-(4-Azidophenyl)-N-(2-(dimethylamino)ethyl)-N,N-dimethyl-
2-oxoethan-aminium bromide 3. A flame-dried 2−5 mL
microwave vial was charged with 1-(4-azidophenyl)-2-bromoe-
thanone (100 mg, 0.42 mmol) and acetone (1 mL). The vial
was capped and the reaction cooled in an ice bath. N,N,N,N-
Tetramethylethane-1,2-diamine (0.42 mmol, 61 μL) was then
added, dropwise, whereupon a white precipitate formed almost
immediately. Acetone (0.5 mL) was added, and the reaction
stirred at room temperature for 2 h. The white precipitate was
Figure 3. Diffraction patterns of (A) 2-C10, (B) 2-C20, (C) 4-C10,
and (D) 4-C20 at 30 °C. Images A and B show broad micellar peaks at
low angle, with limited long-range ordering, while image C shows a
lamellar phase with a lattice parameter of 122 Å and image D a lattice
parameter of 133 Å.
progression from broad diffuse peaks indicative of micelle
formation observed for short-chain amphiphiles to distinct
Bragg reflections indicative of lamellar phase formation by
Figure 4. Diffraction patterns of compounds 5-C10 to 5-C20 at 30 °C. 5-C10, 5-C12, and 5-C14 show broad micellar diffraction peaks with very
limited long-range order while the C16−C20 derivatives adopt lamellar phases at 80 wt % water with lattice parameters of 120, 124, and 144 Å for
C16, C18, and C20, respectively. Traces have been artificially offset in the y-axis for clarity.
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then gently broken up with a glass pipet, collected, and washed
ASSOCIATED CONTENT
* Supporting Information
Experimental details, NMR spectra of compounds 2−5, HPLC
purity analysis, cross-polarized light microscopy data, and
synchrotron small-angle X-ray scattering data. This material is
available free of charge via the Internet at http://pubs.acs.org.
■
with acetone to give the bromide salt 3 (136 mg, 91%) as an
S
1
off-white solid. H NMR (D2O, 400 MHz) δ 7.85 (1H, d, J =
8.7 Hz); 7.13 (1H, d, J = 8.7 Hz); 3.67 (2H, t, J = 6.6 Hz); 3.29
(3H, s); 2.68 (2H, t, J = 6.6 Hz); 1.95 (3H, s). 13C NMR (D2O,
200 MHz) δ 188.8, 146.5, 130.5, 129.6, 119.3, 60.4, 53.3, 51.3,
43.5. HRMS (ESI) Found 276.1812; C14H22N5O+ requires
276.1824.
AUTHOR INFORMATION
Corresponding Author
*E-mail: paul.savage@csiro.au.
Author Contributions
GPS conceived the project, OEH and XM designed and
conducted experiments, GPS and XM co-wrote the manuscript.
Notes
■
Procedure for Amphiphile Library Synthesis. Each of
24 glass vials (18 mm × 50 mm) in a 4 × 6 array in an
aluminum reaction block was equipped with magnetic stirrer
bars. Each vial was charged with an amphiphilic headgroup (15
mg), tail group (1 equiv), t-BuOH (0.9 mL), and H2O (0.45
mL). The reaction block was heated to 35 °C, and the reactions
were stirred for 10 min to ensure complete dissolution of the
starting material. Copper powder (∼200 mg) was added to
each vial. The vials were then capped and the reactions stirred
at 35 °C for 24 h. After this time, EtOH (2 mL) was added to
each vial and the reactors stirred for an additional 10 min. The
reactions were then transferred to a Whatman Unifilter (10 mL,
24 well polypropylene GF/C filter) and the mother liquor
collected in 18 mm × 50 mm glass vials. The solvent was
removed in a Genevac EZ-2 Plus to give the desired
amphiphiles. The residues were subsequently placed in a
vacuum oven at 50 °C for 2 h, then analyzed for purity by LC−
MS (see the Supporting Information).
Synchrotron Small Angle X-ray Scattering. The internal
liquid crystalline structure of the hydrated phases was
determined using small-angle X-ray scattering (SAXS). Data
were collected using the SAXS/WAXS beamline at the
Australian Synchrotron using a beam with wavelength λ =
1.033 Å (12.0 keV) with a typical flux of 1013 photons/s. 2D
diffraction patterns were recorded on a Decris-Pilatus 1 M
detector of 10 modules. The detector was offset to access a
greater q-range. A silver behenate standard (λ = 58.38 Å) was
used to calibrate the reciprocal space vector. The samples were
loaded in special glass 1.5 mm capillaries (Hampton Research)
and positioned in a custom designed semihigh throughput
capillary holder capable of holding 40 capillaries with
temperature controlled to 0.1 °C between 20 and 75 °C.
Temperature control was via a recirculating water bath.
Exposure time for each sample was 1 s. Data were analyzed
using aXcess software created by Andrew Heron at Imperial
College, London.4 Samples, typically 3−4 mg of amphiphile
were hydrated with 80 wt % water 24 h prior to data collection.
Because of the small sample size, data could not be obtained at
higher temperatures due to sample drying.
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This research was, in part, undertaken on the SAXS/WAXS
beamline at the Australian Synchrotron, Victoria, Australia. We
thank Dr. Jessica A. Smith for technical assistance.
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