Temperature-responsive self-assemblies of 'kinked' amphiphiles

Article abstract of DOI:10.1071/CH13278

The synthesis of novel norbornane-based amphiphiles and the thermal response of their corresponding colloids is presented. It was found that the hydrodynamic diameter (DH) expansion or contraction of 1-4 in response to increasing temperature was governed

Full text of DOI:10.1071/CH13278

RESEARCH FRONT
CSIRO PUBLISHING
Aust. J. Chem. 2013, 66, 899–909
Full Paper
http://dx.doi.org/10.1071/CH13278
Temperature-Responsive Self-Assemblies of ‘Kinked’
Amphiphiles
A
B C
,
D
Jennifer S. Squire, Gre´gory Durand,
Lynne Waddington,
E
A E F
, ,
Alessandra Sutti, and Luke C. Henderson
A
Strategic Research Centre for Chemistry and Biotechnology, Deakin University,
Pigdons Road, Waurn Ponds Campus, Geelong, Vic. 3216, Australia.
B
Unite´ Mixte de Recherche 5247, Centre National de la Recherche Scientifique and
Universite´s de Montpellier 1&2, Institut des Biomole´cules Max Mousseron, Faculte´ de
Pharmacie, 15 avenue Charles Flahault, F-34093 Montpellier Cedex 05, France.
C
Universite´ d’Avignon et des Pays de Vaucluse, Equipe Chimie Bioorganique et
Syste`mes Amphiphiles, 33 rue Louis Pasteur, F-84000 Avignon, France.
D
CSIRO Materials Science and Engineering, Bayview Avenue, Clayton, Vic. 3168,
Australia.
E
Institute for Frontier Materials, Deakin University, Pigdons Road, Waurn Ponds
Campus, Geelong, Vic. 3216, Australia.
F
Corresponding author. Email: luke.henderson@deakin.edu.au
The synthesis of novel norbornane-based amphiphiles and the thermal response of their corresponding colloids is
presented. It was found that the hydrodynamic diameter (DH) expansion or contraction of 1–4 in response to increasing
temperature was governed by the length of the hydrophobic region possessed by the amphiphile (a 12 or 16 carbon chain).
These data were used as a starting point to extend into an active tumour targeting system whereby two amphiphiles were
modified to incorporate the oestrogen receptor antagonist Tamoxifen at the polar head group. This was achieved by a
triazole moiety while both the C12 (18) or C16 (19) hydrophobic chains were incorporated as the hydrophobic region in an
attempt to retain the response to thermal stimuli observed in our preliminary findings. These functionalised novel
amphiphiles possessed critical aggregation concentration values of 510 and 19 mM, while aqueous self-assemblies of 56
and 106 nm for 18 and 19 were observed. Imaging by cryogenic transmission electron microscopy showed 18 to possess
liposomal morphology, while 19, bearing a C16 hydrophobic portion, formed non-defined amorphous aggregates. Finally,
the response to temperature of these assemblies was investigated with only the C12 variant 18 displaying a temperature
response in the 5–558C thermal window investigated.
Manuscript received: 31 May 2013.
Manuscript accepted: 16 July 2013.
Published online: 7 August 2013.
Introduction
disulfide bonds present in the amphiphile architecture. Alter-
natively, the method of release can be due to the destabilisation
of the lipid bilayer allowing for drug efflux, as proposed by
Torchilin[15b] or due to the formation of fine ‘grain boundaries’
which occur in the bilayer allowing for encapsulation release.[16]
The focus of this manuscript is thermally responsive self-
assemblies, a subject that has recently been reviewed by several
authors.[16–19] Self-assemblies that respond to temperatures just
above normal homeostatic biological temperature are of partic-
ular importance in cancer therapy as they represent a means to
selectively release therapeutic cargo specifically at the tumour
site, subsequently minimising toxicity to non-cancerous tissue.
In additional, localised mild hyperthermia has been found to
have synergistic effects when coupled with common chemo-
therapeutic agents such as Cisplatin and Doxorubicin.[20] The
synergistic effects arise due to the hyperthermic treatment
increasing blood flow to the tumour and increasing vascular
permeability, allowing deeper penetration into the tumour mass,
The self-assembly of amphiphilic compounds has been of great
interest to the scientific community and is at the core of many
cutting-edge technologies in areas of medicine, nutraceuticals,
gene-delivery, and in various aspects of materials science.[1–10]
Complementing this field is the development of stimuli-
responsive self-assemblies that are able to release encapsulated
cargo under specific conditions. Common stimuli that are used
to initiate cargo release include temperature, pH, ultrasound,
light, and redox-based systems.[11–15a] The release of encapsu-
lated cargo may be facilitated by either (i) the disassociation
between the amphiphiles within the self-assembly or (ii) the
breakdown of the individual amphiphiles of which the nanos-
tructure is composed. These stimuli are designed to take
advantage of potential changes that occur when the ‘loaded’
nanostructure passes from the extracellular to intracellular
environment, for example, the change in extracellular to intra-
cellular pH, or the increase in glutathione which cleaves
Journal compilation Ó CSIRO 2013
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900
J. S. Squire et al.
Receptor
Nucleus
Tumour
ACTIVE
TARGETING
Endothelial cell
PASSIVE TARGETING
Liposome
Blood
vessel
EPR EFFECT
Lymphatic
Vessel
Ineffective
lymphatic
drainage
Angiogenic
vessel
X
Fig. 1. Combined active and passive targeting for cancer therapy (EPR effect: enhanced permeation and retention effect).
and consequently increasing the chemotherapeutic effect. Simi-
larly, the increase in tumour permeability allows for the
increased accumulation of nanoparticles (such as liposomes)
within parts of the tumour not usually accessible due to a lack of
perfused vasculature. Therefore, temperature sensitive self-
assemblies have dual functionality as an adjuvant therapy with
hyperthermic treatment as they are exposed to more of the
tumour mass and can then be stimulated to release their
chemotherapeutic cargo.
Self-assemblies can be delivered to desired cells or cellular
mass (such as a tumour) by either passive or active targeting.
Passive targeting occurs predominantly through the enhanced
permeation and retention effect (EPR effect)[21–24] where
nanosized particles (typically , 400 nm) accumulate within a
tumour mass due to poorly constructed cellular vasculature
(Fig. 1), compounded by the poor lymphatic drainage typically
observed in tumours. Once the tumour is enriched with ‘loaded’
self-assemblies, the release of therapeutic cargo can be initiat-
ed by application of stimuli (e.g. heat) external to the body. The
use of passive targeting has had a great deal of success in
the treatment of tumours using a vast array of nanostructures
including micelles, liposomes, and nanoparticles of various
compositions.[21–24]
Active targeting is facilitated by the incorporation of ligands,
which possess a very high affinity for overexpressed cellular
receptors, onto the nanostructure surface. An excellent example
was demonstrated by Reddy and Banjeree[25] who used 17b-
oestradiol as a peripheral functionalisation on stealth liposomes
which target the GPR30 receptor on oestrogen dependent breast
cancer cells. In this scenario, the overexpressed receptors on the
cellular surface promoted uptake of the functionalised nanos-
tructures by receptor-mediated endocytosis and therefore pro-
vided a more focussed or ‘actively’ targeted therapy.
We recently reported preliminary investigations into a range
of amphiphiles that possessed a rigid norbornane structure
within the core of the molecule.[26] These amphiphiles displayed
a range of aggregate sizes and morphologies while their critical
aggregation concentration (CAC) values were relatively insen-
sitive to changes in the molecular structure. The unusual
behaviour of these amphiphiles was attributed to stacking
phenomena caused by the rigid core, similar to those proposed
by Matisons et al.[27] This manuscript presents the synthesis of
novel amphiphiles based on the norbornane scaffold which bear
Tamoxifen at the polar head group. These amphiphiles have
been physicochemically characterised, determining the CAC,
hydrodynamic diameter (DH), morphology (cryogenic transmis-
sion electron microscopy, cryo-TEM), and changes in DH in
response to increasing temperature.
Results and Discussion
Preliminary Temperature Response Investigation
Due to our experience with norbornane-derived amphiphiles
bearing an ammonium head group we began our investigation
by examining the changes in D of amphiphiles 1 4 formulated
]
H
into self-assemblies at a range of increasing temperatures
(Fig. 2). We chose to investigate the assembly behaviour from 5
to 558C as, from a therapeutic standpoint, temperatures above or
below these values are not practical.[16]
For this investigation, the amphiphiles were categorised
based on the value of m, which constitutes the hydrophobic
region of the amphiphiles. As can be seen in Fig. 3, amphiphiles
1 and 2 possessing an m value of 10 demonstrated a consistent
DH (30–60 nm) from 5 to 458C.
When heating from 45 to 558C a dramatic change in DH was
observed whereby each aggregate increased by a factor of ,3

Temperature-Responsive Self-Assemblies of Amphiphiles
901
(140–160 nm). To ensure this observation was not due to
temperature-induced accelerated equilibration of the aggre-
gates, we repeated this experiment employing the reverse
temperature gradient (cooling from 55 to 58C) in the same
108C increments.
Considering the hydrophobic region of these self-assemblies,
we attributed the observed changes in D , displayed by 1 4, to
be the result of a gel-to-liquid transition in the lipid phase. This
phenomenon has been observed in other self-assemblies and at
similar temperatures as reported by several groups.[28]
]
H
Interestingly, the exact same behaviour was observed for
both the forward and backward temperature directions indicat-
ing a very dynamic system with a reversible temperature
response. Repeating these experiments with amphiphiles 3 and
4 (possessing an m value of 14) the reverse trend was observed.
A sharp decrease in DH, by a factor of 2 and 6, occurred at
25–358C and 35–558C, respectively (Fig. 3).
The approximate 108C difference in the initiation of the
thermal transition demonstrated by these compounds can only
be attributed to the ethylene extension of the alkyl chain spacing
the ammonium head group from the norbornane core present in
4 relative to 3.
In an attempt to determine the nature of this thermal transi-
tion, a range of differential scanning calorimetry (DSC) studies
was undertaken on these amphiphiles over a wider temperature
range (5–1008C) at scan rates of 5 and 10 8C minꢀ1. DSC was
conducted on both the colloid and the solid amphiphiles as
powders, however, no thermal transitions were detected within
this temperature range at either scan rate. We attributed this to
the bulk solid material not being representative of the self-
assembly in an aqueous environment, while the aqueous colloid
may not contain enough of the amphiphile to give a measureable
thermal change by DSC. This latter point is highly likely since
the CAC values for compounds 1 4 are very low
]
(typically # 30 mM).[26]
O
O
Synthesis of Tamoxifen-Functionalised Amphiphiles
O
m
With these results in hand, it was considered that the temperature
response demonstrated by these amphiphiles would constitute a
suitable means to release encapsulated cargo. Our desire was to
transfer the thermal response observed in compounds 1–4 to an
active targeting colloid system based on the same scaffold. This
could be realised by grafting a small molecule that interacts with
overexpressed extracellular receptors onto the polar head group
on this class of amphiphile. A similar strategy has been suc-
cessfully used by incorporating Haloperidol or folic acid onto
the polar head group of liposomes. These ligands interact with
overexpressed sigma or oestrogen receptors, respectively, and
have demonstrated enhanced cellular uptake.[29,30]
N
n NH3Cl
O
1: n ꢁ 1, m ꢁ 10
2: n ꢁ 2, m ꢁ 10
3: n ꢁ 1, m ꢁ 14
4: n ꢁ 2, m ꢁ 14
Fig. 2. Norbornane amphiphiles 1–4 used in preliminary temperature
studies.
1
As 70 % of breast cancer cells overexpress oestrogen recep-
tors compared with healthy breast tissue, a focus on breast
cancer therapy was deemed advantageous.[25] The well estab-
lished oestrogen receptor antagonist, Tamoxifen, was chosen as
our targeting ligand to graft onto the polar head group of our
amphiphiles. Our choice of Tamoxifen was based on its rela-
tively simple chemical structure and when incorporated onto the
surface of a self-assembly it may possess a dual functionality (i)
as an active targeting moiety and (ii) simultaneously initiating a
chemotherapeutic response. We envisaged a series of com-
pounds conforming to the general structure presented in
Fig. 4. The effect on the CAC, DH, morphology, and thermal
response that the presence of Tamoxifen at the polar head group
induces on the colloid is of great interest given its lipophilic
nature.
2
3
4
200
180
160
140
120
100
80
60
40
20
Two variations of ‘linker’ (Fig. 4) to incorporate the Tamox-
ifen unit to the amphiphile structure were considered, the first
being directly by the methyl nitrogen unit on desmethyl-
Tamoxifen 16 (for preparation of 16, see below) and the second
by a copper azide–alkyne cycloaddition (CuAAC)-mediated
0
10
20
30
40
50
60
Temp [ꢀC]
Fig. 3. Hydrodynamic diameter of 1 4 with respect to increasing
]
temperature.
Active targeting
O
n
O
O
N
n ꢁ 10 or 14
To retain thermal response
N
O
Linker
O
O
Fig. 4. General structure of Tamoxifen conjugated amphiphiles.

902
J. S. Squire et al.
triazole.[31,32] Our synthesis began with the formation of nor-
bornane imide 6 with aminoethoxyethanol from anhydride 5
which, under microwave irradiation, gave 6 in very high yield
(88 %) and in . 95 % crude purity. Subsequent treatment with p-
toluenesulfonylchloride gave 7 in excellent (85 %) isolated
yield, followed by a dihydroxylation with OsO4/ N-methylmor-
pholine-N-oxide (NMO) in aqueous acetone (H2O/CH3COCH3,
1 : 4) to give the desired vic-diol 8 in an excellent (83 %) yield
(Scheme 1).
Treatment with para-toluenesulfonic acid (pTSA) and dode-
canal provided 9 in a 76 % yield and similarly, under the same
acidic conditions, the use of hexadecanal gave 10 in a similar
yield (73 %) (Scheme 1). The use of an acetal group as means of
installing the hydrophobic region is uncommon and has been
reported in an evaluated amphiphile system one other time to the
best of our knowledge.[33] Compounds 9 and 10 are extremely
versatile intermediates and with these in hand attention turned
towards the linking of Tamoxifen to this scaffold by a simple
amine group (Scheme 2). Treatment of 9 and 10 with desmethyl-
Tamoxifen 16 gave a complex mixture of products. Unfortu-
nately, isolation of 11 or 12 proved troublesome and only small
amounts of each could be recovered post chromatography. This
prevented further physicochemical evaluation due to the inade-
quate amount of material available, nevertheless, analytically
pure samples were obtained and fully characterised.
In light of these difficulties our attention turned to attachment
of the Tamoxifen unit by reliable CuAAC protocols. Substitu-
tion of the tosylate group for an azide moiety proceeded
smoothly for both 13 and 14 using a miscible organic/aqueous
system and sodium azide. These compounds were synthesised
on reasonable scales (1–2 g) with crude purities in excess of
1
85 % (by H NMR spectroscopy) and were used immediately
without further purification.
Using azides 13 and 14 in a CuAAC attachment strategy
required the propargylation of desmethyl-Tamoxifen 16 which
was accessed in excellent yield (95 %) using a demethylation
procedure described by Dreaden et al.[34] (Scheme 3). Treatment
of 16 with propargyl bromide in the presence of triethylamine
gave the desired alkyne 17 in a good isolated yield (73 %)
(Scheme 3).
Our initial attempts to tether the Tamoxifen group to our
amphiphiles focussed on using azide 14 (Scheme 4). A typical
reagent combination of CuSO4/sodium ascorbate in water
(Table 1, Entry 1) returned only unreacted starting material.
However, heating this reaction utilising microwave irradiation,
for 30 min and 1 h, showed an immediate improvement (Table 1,
Entries 2 and 3, 51 % and 40 %, respectively) with the shorter
reaction time being optimal. Changing the reaction solvent to
accommodate reactant solubility and examination of alternate
sources of copper (Table 1, Entries 4 and 5) demonstrated that
O
HO
(c) HO
n
O
O
(a)
(d)
O
O
O
N
O
N
OTs
OR
N
OTs
O
O
O
O
O
O
O
5
6 R ꢁ H
7 R ꢁ Ts
8
9 n ꢁ 10
(b)
10 n ꢁ 14
(a) Ethoxyethyleneamine, 20 min, MW, PhMe, 100ꢀC, 88 %; (b) TsCl, Et3N, CH2Cl2, 16 h, 85 %;
(c) OsO4, NMO, acetone/water 4:1, rt, 16 h, 83 %; (d) Dodecanal or hexadecanal, pTSA, MgSO4, CH2Cl2, 16 h, 76 %, 73 %, respectively
Scheme 1. Synthesis of norbornene–tosylates 9 and 10.
O
O
n
O
(a)
N
N
O
O
O
11 n ꢁ 10
12 n ꢁ 14
O
O
n
O
N
OTs
O
O
9 n ꢁ 10
10 n ꢁ 14
O
O
n
O
N
N3
O
(b)
O
13 n ꢁ 10
14 n ꢁ 14
(a) Desmethyl tamoxifen 16, K2CO3, THF, reflux, 16 h; n ꢁ 10, 21 %, n ꢁ 14, 16 %)
(b) NaN , MeCN/H O, 50ꢀC,16 h (ꢂ85 %, both cases)
3
2
Scheme 2. Synthesis of Tamoxifen conjugates 11 and 12 and norbornyl–azides 13 and 14.

Temperature-Responsive Self-Assemblies of Amphiphiles
903
Cu2O returned unreacted starting material while CuCl resulted
in a small amount of conversion (7 %) at room temperature
in the absence of heat.
Determination of CAC, DH, Thermal Response,
and Morphology for 18 and 19
Several means have been used to determine the CAC in aqueous
systems, such as surface tension, conductivity, NMR spectros-
copy, and fluorescence spectroscopy by encapsulation of lipo-
philic probes including pyrene.[36–38] We have found that the
fluorescence of encapsulated pyrene has been the most reliable
and reproducible methodology for this class of amphiphile.
The encapsulation of pyrene was carried out according to
literature procedures,[39] and the ratio of emission peaks at 386
and 393 nm (I386/I393) was monitored over a series of concen-
trations ranging from 1 to 0.0001 mg mLꢀ1. The CAC data for
all compounds by pyrene encapsulation are summarised in
Therefore, heating by microwave irradiation in combination
with CuCl in chloroform for 30 min at 708C and 1008C (Table 1,
Entries 6 and 7) gave the desired compound 19 in good yields of
61 % and 68 %, respectively. Repeating the latter reaction
conditions using azide 13 gave the desired compound 18 in
moderate yield (50 %) (Table 1, Entry 8). The two desired
Tamoxifen conjugated amphiphiles 18 and 19 were then con-
verted into their cationic form using gaseous HCl and formulat-
ed into aqueous colloids.[35]
(b)
R
N
N
O
O
15 R ꢁ Me
17
(a)
16 R ꢁ H.HCl
(a) α-Chloroethyl chloroformate, reflux, PhMe, 16 h, then MeOH reflux, 3 h, 95 %
(b) Propargyl bromide, NEt3, reflux, CHCl3,16 h, 73 %.
Scheme 3. Demethylation and propargylation of Tamoxifen.
O
O
n
O
N
N
O
N
17
O
N
N
O
O
NMe
Catalyst, solvent, time
O
n
O
O
N
N3
O
O
13 n ꢁ 10
14 n ꢁ 14
18 n ꢁ 10
19 n ꢁ 14
Scheme 4. Synthesis of Tamoxifen-conjugated amphiphiles 18 and 19.
Table 1. Optimisation of Cu azide alkyne coupling to furnish 18 and 19
]
Entry
Compound
CatalystA
AdditiveA
Solvent
Time [h]
Temp.B [8C]
Yield [%]
C
1
2
3
4
5
6
7
8
14
14
14
14
14
14
14
13
CuSO4
CuSO4
CuSO4
Cu2O
CuCl
Ascorbate
Water
Water
Water
CHCl3
CHCl3
CHCl3
CHCl3
CHCl3
16
0.5
1
25
100
100
70
—
Ascorbate
51
40
Ascorbate
C
—
—
—
—
—
—
0.5
16
0.5
0.5
0.5
25
7
61
68
50
CuCl
70
CuCl
100
100
CuCl
ALoading is 10 mol-%.
BHeated using microwave irradiation unless at room temperature.
CStarting materials were recovered.

904
J. S. Squire et al.
Table 2. In contrast to our previous work on norbornane-derived
scaffolds these compounds behave much more like traditional
surfactants, with the CAC value for amphiphile 18 being ,27
times higher than that of 19.
It has been noted in the literature that for each methylene unit
installed into the hydrophobic portion of an amphiphile a
corresponding ,5 fold decrease in CAC value is observed.[40]
Therefore, the two ethylene units distinguishing 18 from 19,
should result in an ,20 fold decrease in CAC for the latter which
is confirmed here with a critical micelle concentration (CMC)
value of the latter compound 27 times lower than that of the
former one.
The DHs of these compounds were determined by dynamic
light scattering (DLS) at room temperature and were found to be
56 and 106 nm for 18 and 19, respectively. Both compounds
showed a moderate size distribution (DLS output shown in
Supplementary Material).[35]
Attention then turned to evaluating any temperature response
these aggregates may possess. Conducting variable temperature
DLS showed that 18 possessed a similar temperature response to
1 and 2. In this case, 18 showed a steady increase in DH with
increasing temperature which became more pronounced when
heating the colloid from 35 to 558C, resulting in a rapid
transition from 79 to 118 nm (Fig. 5). This aggregate size
transition is presumably determined by the hydrophobic region
of the molecules as m ¼ 10 is the only common feature between
compounds 1, 2, and 18. Subjecting 19 to the same variable
temperature DLS showed no size transitions at all from 5 to
558C, even with the repetition of the experiment with an
increased temperature endpoint of 658C.
Visualisation of the aggregates was then undertaken to
determine the effect Tamoxifen at the cationic head group would
have on morphology. Cryo-TEM of 18 showed liposomal
aggregates (Fig. 6), some of which were fused to a spherical
self-assembly aggregate with no observable fine structure (see
Supplementary Material),[35] a feature consistent with those
observed by van Esch et al.[41] The lipid bilayers are ,4–5 nm
in accordance with our previous observations and those made by
Bordes et al. who incorporated a norbornane counterion into
various self-assemblies.[26,42,43]
Cryo-TEM images of 19 showed only the presence of diffuse
spherical amorphous aggregates with no defined lamellar,
micellar, or liposomal structure (images presented in the Sup-
plementary Material).[35] In all previous cases where a norbor-
nane motif has been incorporated into the amphiphile scaffold,
vesicles have been the preferred morphology.[26] We have
attributed the deviation from this trend demonstrated by 19 to
the modification of the polar head group. The hydrophobic
Tamoxifen group in concert with the hexadecyl aliphatic chain
may cause a large global increase in lipophilicity, thus causing
the compound to lose its amphiphilic nature. Being lipophilic at
both ends of the molecule would inhibit an ordered self-assem-
bly process causing a disordered amorphous assembly, similar
to those observed.
This latter result accounts for the lack of temperature
response displayed by 19 when monitored by DLS. If no defined
aggregate phase is formed then it would be expected that no
corresponding sharp transition temperature indicating a gel-to-
liquid phase shift would be observed.
Conclusions
Table 2. Critical aggregate concentration (CAC) and hydrodynamin
diameter (DH) data for compounds 18 and 19 by pyrene encapsulation
and dynaminc light scattering, respectively
We have demonstrated the thermal response properties of sev-
eral norbornane-based amphiphiles. A trend was observed when
the formulations of 1–4 were heated from 5 to 558C in 108C
increments. Compounds bearing a longer (14 methylene unit)
chain shrunk at 25 and 358C while compounds bearing a shorter
(10 methylene unit) tail expanded at temperature transitions
from 45 to 558C, both changes being attributed to gel-to-liquid
transitions in the lipophilic bilayer.
Entry
Compound
CACA
[mmol Lꢀ1
]
DBH [nm]
[mg Lꢀ1
]
1
2
18
19
432
18
510
19
56
106
This was used as the basis for the development of an active
targeting therapy colloid, whereby the oestrogen receptor antag-
onist Tamoxifen was grafted onto the polar head group of two
amphiphiles using a CuAAC approach. Functionalised amphi-
philes 18 and 19 were obtained in an efficient and high yielding
synthetic procedure. Physicochemical characterisation of these
amphiphiles showed a classical CAC relationship between
hydrocarbon length and changes in aggregation concentration
AResults are the average of triplicate experiments carried out at 188C in
Milli-Q water.
BDetermined at 10 times the CAC value at 258C with volume distribution.
120
110
100
90
80
70
60
0
10
20
30
40
50
60
Temperature [ꢀC]
Fig. 5. Changes in hydrodynamic diameter with respect to temperature for
aggregates of 18.
Fig. 6. Cryogenic transmission electron microscopy images of 18 showing
liposomal structures.

Temperature-Responsive Self-Assemblies of Amphiphiles
905
whereby the more hydrophobic 19 possessed a CAC value ,25
times lower than 18 (19 v. 510 mM). DLS showed the formation
of aggregates with a DH of 56 and 105 nm for 18 and 19.
Analysis of these aggregates by DLS with increasing tempera-
ture revealed an aggregate expansion from 79–118 nm correlat-
ing to an increase in temperature for 18, however, no response to
thermal stimulus for the more hydrophobic 19 was observed.
Finally, imaging by cryo-TEM suggested the formation of
liposomes for 18, while 19 showed only the formation of non-
defined amorphous assemblies. This was attributed to the
increase in overall lipophilicity of 19 once the Tamoxifen group
was grafted onto the polar head group. The formation of these
non-defined aggregates accounts for the lack of thermal
response observed for 19 at increasing temperature using
DLS. Current studies on the ability of 18 to interact with
oestrogen receptors, by surface plasmon resonance, and their
potential as transfection agents are currently being investigated
and will be disclosed in due course.
filter directly into the cuvette immediately before measurement
to avoid interference from dust particles. Measurements were
taken at 258C unless otherwise stated and repeated in triplicate.
Pyrene Encapsulation Measurements
The fluorescence properties of pyrene were determined using a
Varian Cary Eclipse fluorescence spectrophotometer. The sur-
factant solutions were prepared 24 h before the measurements
using milli-Q water (resistivity of 18.2 MO cm) at a range of
concentrations (1 to 0.0001 mg mLꢀ1). Excess crystalline pyr-
ene was then added to the surfactant aqueous solutions and
heated at 308C for 30 min in a sonic bath before allowing
equilibration at room temperature for 23 h. The solutions were
then filtered through a 0.45 mm filter before measurement. The
emission spectra of pyrene were acquired by exciting samples at
335 nm (Ex slit width 5 nm, Em slit width 5 nm). The spectra
were then used to determine the ratio of I386/I393.
Cryo-TEM
A laboratory-built humidity-controlled vitrification system was
used to prepare the samples for Cryo-TEM. Humidity was kept
close to 80 % for all experiments, and ambient temperature was
228C. A 200 mesh copper grid coated with perforated carbon
film (Lacey carbon film: ProSciTech, Qld, Australia) and
overlaid with a very thin continuous carbon film was glow
discharged in nitrogen to render it hydrophilic. The thin carbon
overlay was found to be necessary as the cationic particles were
consistently attracted to the perforacted film rather than being
suspended in the holes. Aliquots of 4 mL of the sample were
pipetted onto each grid before plunging. After 30 s adsorption
time the grid was blotted manually using Whatman 541 filter
paper for ,2 s. Blotting time was optimised for each sample.
The grid was then plunged into liquid ethane cooled by liquid
nitrogen. Frozen grids were stored in liquid nitrogen until
required. The samples were examined using a Gatan 626 cryo-
holder (Gatan, Pleasanton, CA, USA) and Tecnai 12 Trans-
mission Electron Microscope (FEI, Eindhoven, The
Netherlands) at an operating voltage of 120 kV. At all times low
dose procedꢀu2res were followed, using an electron dose of 8–10
Experimental
General
All 1H and 13C NMR spectra were recorded on a Jeol JNM-EX
270 MHz spectrometer as indicated. Samples were dissolved in
CDCl3 with the residual solvent peak used as an internal refer-
ence (CHCl3: dH 7.26 ppm). Proton spectra are reported as fol-
lows: chemical shift d (ppm), (integral, multiplicity (s ¼ singlet,
br s ¼ broad singlet, d ¼ doublet, dd ¼ doublet of doublets, t
¼ triplet, q ¼ quartet, m ¼ multiplet), coupling constant J (Hz),
assignment).
Thin-layer chromatography (TLC) was performed using
aluminium-backed Merck TLC Silica gel 60 F254 plates, and
samples were visualised using 254 nm ultraviolet (UV) light,
and potassium permanganate/potassium carbonate oxidising dip
(1 : 1 : 100 KMnO4/K2CO3/H2O, w/w/w).
Column chromatography was performed using silica gel 60
(70–230 mesh). All solvents used were of AR grade. Specialist
reagents were obtained from Sigma–Aldrich Chemical Co. and
used without further purification. Petroleum spirits (Pet) refers
to the fraction boiling between 40 and 608C.
˚
electrons A for all imaging. Images were recorded using an
Microwave reactions were conducted using a CM Discover
S-Class Explorer 48 Microwave Reactor, operating on a fre-
quency of 50/60 Hz and continuous irradiation power from 0 to
200 W or 20 W where specified. All reactions were performed in
10 mL septa vials with snap caps, with the following conditions:
pressure:17 bar, power max: off, and stirring: high.
Eagle 4k ꢁ 4k CCD camera (FEI, Eindhoven, The Netherlands)
using magnifications in the range 15 000–60 000ꢁ
Synthesis of Amphiphiles
For the synthesis of amphiphiles 1–4 and their corresponding
spectra please refer to previous publications from our group.[26]
Formulation of Self-Assemblies
Synthesis of 4,7-Methano-1H-isoindole-1,3(2H)-dione-
3a,4,7,7a-tetrahydro-2-[2-(2-hydroxy ethoxy)ethyl] 6
Freshly lyophilized amphiphiles were hydrated using MilliQ
water (resistivity of 18.2 MO cm) to the desired concentration.
The colloids were sonicated at room temperature for 30min and
left overnight at room temperature to stabilise. The colloids were
then extruded through a 0.45mm syringe filter before analysis.
To a 35 mL microwave vial containing 2-(2-aminoethoxy)eth-
anol (2.28 mL, 23.0 mmol), norbornene anhydride (2 g,
12.2 mmol) was added and dissolved with toluene (15 mL). The
solution was then subjected to microwave irradiation at 1008C
for 20 min. The resulting solution was diluted with CH2Cl2
(30 mL) and was transferred to a separating funnel where it was
washed with saturated aqueous NaCl (2 ꢁ 25 mL) followed by a
wash with HCl (2 M, 20 mL), and finally NaHCO3 (3 ꢁ 25 mL).
The combined organic phases were dried (MgSO4) and solvent
removed under vacuum to give a yellow viscous oil. 1H NMR
spectroscopy analysis showed it to be the desired ethoxy imide 6
(2.63 g, 88 %) in . 95 % purity which was then used without
further purification. dH (270 MHz, CDCl3) 6.09 (2H, t, J 2.7),
3.65–3.63 (2H, d, J 5.4), 3.59–3.47 (2H, m), 3.39–3.35 (2H, m),
Dynamic Light Scattering
The light scattering measurements were performed with a
Zetasizer Nano ZS (Malvern Instruments) at a scattering angle
of 1738. The concentrations of the surfactants were sufficiently
low enough (0.46–0.72 mM) to avoid multiple scattering from
the aggregates. Solutions were prepared at 10 ꢁ CMC from a
stock solution of 1 mg mLꢀ1 in Milli-Q water and sonicated for
30 min to ensure adequate dissolving before being left to
equilibrate overnight. All solutions were filtered with a 0.45 mm

906
J. S. Squire et al.
3.29–3.22 (2H, m), 2.59 (2H, br s), 1.83 (2H, br s), 1.74–1.69
(1H, dt, J 10.8, 16.2), 1.55–1.49 (1H, dt, J 8.1, 13.5);
dC (200 MHz, CDCl3) 178.10, 134.41, 72.22, 67.70, 61.58,
52.16, 45.81, 44.94, 37.86; m/z (HRMS ESI) Calc. for
[C13H18NO4]þ 252.12303, found 252.12400; nmax /cmꢀ1 3452
br, 2944 m, 2871 m, 1684 s.
stirred until dissolved. MgSO4 was then added, followed by the
addition of p-toluenesulfonic acid (577 mg, 3.0 mmol). The
resulting solution was warmed to 358C and stirred for 16 h
before being filtered and the solvent removed under vacuum to
give a light brown oil. Purification by silica gel column chro-
matography was performed with a 60 % petroleum spirit/40 %
EtOAc solution and the resulting caramel oil was shown by 1H
NMR spectroscopy analysis to be the desired dodecyl acetal 9
(348 mg, 76 %) in . 95 % purity. dH (270 MHz, CDCl3) 7.77–
7.74 (2H, d, J 8.1), 7.35–7.32 (2H, d, J 8.1), 4.58–4.54 (1H, t,
J 5.4), 4.08–4.05 (2H, m), 3.84 (2H, br s), 3.63–3.54 (2H, m),
3.10–3.08 (2H, m), 2.81–2.79 (2H, m), 2.43 (3H, s), 2.02–1.96
(4H, m), 1.69–1.57 (4H, m), 1.39–1.22 (18H, m), 0.87–0.83
(3H, t, J 5.4); dC (200 MHz, CDCl3) 174.41, 144.98, 132.98,
130.15, 129.82, 127.95, 104.27, 77.45, 69.12, 68.14, 67.17,
44.51, 42.90, 42.57, 37.68, 35.92, 32.76, 31.99, 29.64–29.41,
24.23, 22.76, 14.28; m/z (HRMS ESI) Calc. for [C32H48NO8S]þ
606.30951, found 606.31166; nmax /cmꢀ1 2942 m, 2854 m,
1700 s.
Synthesis of 2-(2-(1,3-Dioxo-1,3,3a,4,7,7a-hexahydro-
2H-4,7-methanoisoindol-2-yl)ethoxy)ethyl
4-Methylbenzenesulfonate 7
To a solution of ethoxy imide 6 (2.74 g, 10.8 mmol) in CH2Cl2
(30 mL), p-toluenesulfonyl chloride (3.09 g, 16.2mmol) was
added and stirred until dissolved. Triethyl amine (TEA) was
added (1.5mL, 10.8 mmol) and the resulting solution was stirred
under nitrogen for 16 h before being diluted with CH2Cl2 (30 mL)
and transferred to a separating funnel where it was washed with
saturated aqueous NaCl (3 ꢁ 25 mL). The combined organic
phases were dried (MgSO4) and solvent removed under vacuum
to give a beige viscous oil. Purification by silica gel column
chromatography was performed with a 50 % petroleum spirit/
50% EtOAc solution and the resulting oil was shown by 1H NMR
spectroscopy analysis to be the desired tosylate 7 (3.70 g, 85%)
in . 95 % purity. dH (270MHz, CDCl3) 7.76–7.73 (2H, d, J 8.1),
7.33–7.30 (2H, d, J 8.1), 6.01–6.59 (2H, t, J 8.1), 4.06–4.04
(2H, t, J 2.7), 3.57–3.53 (2H, m), 3.46–3.39 (2H, m), 3.32–3.31
(2H, m), 3.23–3.22 (4H, m), 2.41 (3H, s), 1.70–1.65 (1H, dt, J 8.1,
13.5), 1.51–1.47 (1H, d, J 10.8); dC (67.5 MHz, CDCl3) 177.71,
144.93, 134.48–134.36, 132.93, 130.06, 129.86, 69.22, 67.99–
67.31, 52.19, 45.85–45.01, 44.87, 37.30, 21.80; m/z (HRMS ESI)
Calc. for [C20H24NO6S]þ 406.13188, found 406.13400; nmax
/cmꢀ1 2946 m, 2873 m, 1697 s.
Synthesis of 2-(2-(-5,7-Dioxo-2-pentadecyloctahydro-6H-
4,8-methano[1,3]dioxolo[4,5-f]isoindol-6-yl)ethoxy)ethyl
4-Methylbenzenesulfonate 10
To a solution of hexadecyl aldehyde (295mg, 1.2 mmol) in
CH2Cl2 (30 mL), diol 8 (360mg, 0.82mmol) was added and
stirred until dissolved. MgSO4 was then added, followed by the
addition of p-toluenesulfonic acid (623mg, 3.3 mmol). The
resulting solution was warmed to 358C and stirred for 16h before
being filtered and the solvent removed under vacuum to give a
lightbrown oil. Purification bysilica gel column chromatography
was performed witha 60% petroleum spirit/40 % EtOAc solution
and the resulting caramel oil was shown by 1H NMR spectros-
copy analysis to be the desired hexadecyl acetal 10 (398mg,
73%) in . 95% purity. dH (400MHz, CDCl3) 7.79–7.76 (2H, d,
J 12.0), 7.36–7.34 (2H, d, J 8.0), 4.59–4.56 (1H, t, J 8.0),
4.10–4.07 (2H, m), 3.86 (2H, br s), 3.64–3.56 (2H, m), 3.11–3.10
(2H, m), 2.83–2.81 (2H, m), 2.45 (3H, s), 2.02–1.99 (4H, d,
J 12.0), 1.64–1.56 (4H, m), 1.40–1.24 (26H, m), 0.89–0.85 (3H, t,
8.0); dC (200MHz, CDCl3) 176.41, 144.98, 132.90,
130.07–129.90, 128.10, 127.94, 104.50, 77.42, 69.11, 68.15,
67.18, 44.50, 42.66, 37.69, 32.00, 29.74–29.44, 24.24, 22.78,
14.21; m/z (HRMS ESI) Calc. for [C36H56NO8S]þ 662.37211,
found 662.37125; nmax /cmꢀ1 2923m, 2853m, 1702s.
Synthesis of 2-(2-(-5,6-Dihydroxy-1,3-dioxooctahydro-1H-
4,7-methanoisoindol-2-yl)ethoxy) Ethyl-4-
methylbenzenesulfonate 8
Tosylate 7 (1.44 g, 3.0 mmol) was dissolved in a 4 : 1 solution of
acetone/water (30 mL) and NMO (628 mg, 5.0 mmol) was
added and stirred until dissolved. Osmium tetroxide (0.2 mL)
was added and the black solution stirred for 16 h at room tem-
perature. The reaction was quenched with sodium metabisulfite
(2 mL, 0.53 M) and the solution diluted with EtOAc (30 mL)
before being transferred to a separating funnel where it was
washed with saturated aqueous NaCl (3 ꢁ 25 mL). The organic
phase was dried (MgSO4) and solvent was removed under
vacuum to afford a yellow oil. Purification by silica gel column
chromatography was performed with 100 % EtOAc and the
resulting pale yellow oil was shown by 1H NMR spectroscopy to
be the desired diol 8 (1.09 g, 83 %). dH (400 MHz, CDCl3) 7.79–
7.77 (2H, d, J 8.0), 7.38–7.36 (2H, d, J 8.0), 4.12–4.07 (2H, m),
3.86 (2H, s), 3.67–3.58 (2H, m), 3.29 (2H, br s), 3.08–3.06 (3H,
m), 2.70 (3H, br s), 2.45 (3H, s), 2.16–2.13 (1H, d, J 12.0), 1.49–
1.46 (1H, d, J 12.0); dC (200 MHz, CDCl3) 177.06, 145.53,
130.20, 129.11, 128.30, 128.17, 128.15, 70.03, 69.92, 68.10,
67.29, 46.06, 45.81, 37.92, 36.18, 21.77; m/z (HRMS ESI) Calc.
for [C20H26NO8S]þ 440.13736, found 440.13965; nmax /cmꢀ1
3454 br, 2952 m, 1697 s.
Synthesis of 6-(2-(2-((2-(4-((Z)-1,2-diphenylbut-1-en-1-yl)
phenoxy)ethyl)(methyl)amino)ethoxy) Ethyl)-2-
undecylhexahydro-5H-4,8-methano[1,3]dioxolo[4,5-f]
isoindole-5,7(6H)-dione 11
The synthetic procedure was adapted from Dreaden et al.[1]
Briefly, Tosyl dodecyl acetal 9 (350 mg, 0.58 mmol) was added
to a solution of demethylated Tamoxifen 16 (152 mg,
0.39 mmol) in THF. Potassium carbonate was added (532 mg,
3.9 mmol) and the resulting solution was refluxed at 708C for
16 h. The solution was then diluted with CH2Cl2 and filtered
before being transferred to a separation funnel where it was
washed with saturated aqueous NaHCO3 (3 ꢁ 25 mL) and the
combined organic phases dried (MgSO4) and the solvent
removed under vacuum to give a pale yellow oil. Purification by
silica gel column chromatography was performed with a 5 %
MeOH in CH2Cl2 solution and the resulting yellow oil was
Synthesis of 2-(2-(-5,7-Dioxo-2-undecyloctahydro-6H-4,8-
methano[1,3]dioxolo[4,5-f]isoindol-6-yl)ethoxy)ethyl 4-
Methylbenzenesulfonate 9
1
To a solution of dodecyl aldehyde (0.34 mL, 1.5 mmol) in
CH2Cl2 (30 mL), diol 8 (333 mg, 0.76 mmol) was added and
shown by H NMR spectroscopy analysis to be the desired
hexadecyl Tamoxifen amphiphile 13 (69 mg, 21 %) in . 95 %

Temperature-Responsive Self-Assemblies of Amphiphiles
907
purity. dH (400 MHz, CDCl3) 7.36–7.08 (10H, m), 6.76–6.73
(2H, d, J 12.0), 6.54 -6.50 (2H, d, J 16.0), 4.59–4.56 (1H, t, J
4.0), 3.93–3.87 (2H, m), 3.63–3.49 (4H, m), 3.07–3.04 (2H, m),
2.82–2.80 (2H, m), 2.75–2.71 (2H, t, J 8.0), 2.63–2.56 (2H, m),
2.48–2.40 (4H, q, J 12.0), 2.30 (3H, s), 2.00–1.96 (2H, d, J 16.0),
1.69–1.56 (2H, m), 1.32–1.23 (18H, m), 0.93–0.83 (6H, m); dC
(200 MHz, CDCl3) 176.46, 143.79, 142.41, 138.33, 132.04,
130.87, 130.76, 129.77, 129.53, 128.20, 127.98, 127.85, 127.38,
126.64, 126.16, 125.76, 114.20, 104.17, 78.85, 67.44, 56.94,
56.34, 44.56, 43.12, 42.75, 35.85, 35.03, 32.75, 31.99, 29.69–
29.10, 24.25, 22.77, 14.20, 13.67; m/z (HRMS, ESI) Calc. for
[C50H67N2O6]þ 791.49936, found 791.49871; nmax /cmꢀ1
2924 m, 2854 m, 1702 s.
3.12–3.08 (2H, m), 2.84–2.83 (2H, m), 2.02–1.99 (2H, d, J 12.0),
1.64–1.59 (2H, m), 1.40–1.23 (18H, m), 0.87–0.85 (3H, t, J 4.0);
dC (200 MHz, CDCl3) 176.48, 104.14, 78.34, 69.45, 67.29,
50.82, 44.53, 42.77, 37.72, 35.88, 34.58, 32.75, 31.98, 29.70–
29.35, 24.26, 22.75, 14.19; m/z (HRMS, ESI) Calc. for
[C25H41N4O5]þ 477.30715, found 477.30950; nmax /cmꢀ1
2992 m, 2854 m, 2108 s, 1700 s.
Synthesis of 2-Pentadecylhexahydro-5H-6-(2-(2-
azidoethoxy)ethyl)-4,8-methano[1,3]dioxolo [4,5-f]
isoindole-5,7(6H)-dione 14
Hexadecyl acetal 10 (2.0 g, 3.0 mmol) was dissolved in a solu-
tion of 8 : 1 MeCN/H2O before the addition of sodium azide
(1.37 g, 21.1 mmol). The resulting solution was then refluxed for
16 h before being diluted with CH2Cl2 (30 mL) and transferred
to a separating funnel where it was washed with saturated
aqueous NaCl (3 ꢁ 25 mL) and the combined organic phases
dried (MgSO4) and the solvent removed under vacuum to give a
white solid. Several attempts at purification by silica gel column
chromatography were performed unsuccessfully and, as such,
Synthesis of 6-(2-(2-((2-(4-((Z)-1,2-diphenylbut-1-en-1-yl)
phenoxy)ethyl)(methyl)amino)ethoxy) Ethyl)-2-
pentadecylhexahydro-5H-4,8-methano[1,3]dioxolo[4,5-f]
isoindole-5,7(6H)-dione 12
The synthetic procedure was adapted from Dreaden et al.[1]
Briefly, hexadecyl acetal 10 (300 mg, 0.45 mmol) was added to a
solution of demethylated Tamoxifen 16 (119 mg, 0.30 mmol) in
THF. Potassium carbonate was added (417 mg, 3.02 mmol) and
the resulting solution was refluxed at 708C for 16 h. The solution
was then diluted with CH2Cl2 and filtered before being trans-
ferred to a separation funnel where it was washed with saturated
aqueous NaHCO3 (3 ꢁ 25 mL) and the combined organic phases
dried (MgSO4) and the solvent removed under vacuum to give a
pale yellow oil. Purification by silica gel column chromatog-
raphy was performed with a 5 % MeOH in CH2Cl2 solution and
1
the crude product that was shown by H NMR spectroscopy
analysis to be the desired hexadecyl azide 12 (1.45 g, 91 %) was
used in further steps. dH (400 MHz, CDCl3) 4.61–4.58 (1H, t,
J 4.0), 3.93 (4H, s), 3.66–3.64 (2H, t, J 4.0), 3.62–3.58 (2H, t,
J 8.0), 3.34–3.30 (2H, t, J 8.0), 3.10–3.08 (2H, m), 2.84–2.82
(2H, m), 1.99 (2H, s), 1.63–1.57 (2H, m), 1.40–1.19 (26H, m),
0.87–0.83 (3H, t, J 8.0); dC (200 MHz, CDCl3) 176.45, 104.26,
78.35, 69.46, 67.22, 50.83, 44.53, 42.86, 37.72, 32.76, 32.00,
29.74–29.44, 24.28, 22.78, 14.20; m/z (HRMS ESI) Calc. for
[C29H49N4O5]þ 533.36975, found 533.37242; nmax /cmꢀ1
2917 m, 2850 m, 2112 s, 1698 s.
1
the resulting yellow oil was shown by H NMR spectroscopy
analysis to be the desired hexadecyl Tamoxifen amphiphile 12
(41 mg, 16 %) in . 95 % purity. dH (400 MHz, CDCl3)
7.35–7.07 (10H, m), 6.76–6.74 (2H, d, J 8.0), 6.54–6.52 (2H, d,
J 8.0), 4.59–4.57 (1H, t, J 4.0), 3.92 (2H, br s), 3.65–3.56 (4H,
m), 3.08–3.06 (2H, m), 2.82–2.81 (4H, br s), 2.64 (2H, br s),
2.47–2.42 (4H, q, J 8.0, 16.0), 2.35 (3H, br s), 2.00–1.98 (2H, d,
J 8.0), 1.63–1.58 (2H, m), 1.37–1.23 (26H, m), 0.93–0.85
(6H, m); dC (200 MHz, CDCl3) 176.40, 143.89, 142.48, 141.43,
138.29, 131.95, 129.78, 129.54, 128.17, 127.95, 126.59, 126.10,
113.44, 104.09, 78.32, 67.02, 57.23, 56.64, 44.50, 43.43, 42.77,
37.82, 35.87, 35.01, 32.78, 32.01, 29.78–29.58, 24.27, 22.78,
14.21, 13.67; m/z (HRMS ESI) Calc. for [C54H75N2O6]þ
847.56196, found 847.56356; nmax /cmꢀ1 2924 m,
2853 m, 1703 s.
Synthesis of N-Desmethyl Tamoxifen 16
Using a method described by Dreaden et al.[1] a solution of
Tamoxifen 15 (500 mg, 1.4 mmol) and CH2Cl2 was cooled to
08C before the addition of chloroethyl chloroformate (0.23 mL,
2.0 mmol). The resulting solution was stirred for 15 min at 08C
before refluxing at 508C for 24 h. The solvent was then removed
under vacuum, and the resulting clear oil redissolved in MeOH
before being refluxed at 708C for a further 3 h. The solvent was
removed under vacuum to give a cream solid. Purification by
silica gel column chromatography was performed with a 10 %
MeOH in CHCl3 solution and the resulting white solid was
shown by 1H NMR spectroscopy analysis to be consistent with
literature values[24] for the desired demethylated Tamoxifen 16
(506 mg, 95 %).
Synthesis of 2-Undecylhexahydro-5H-6-(2-(2-azidoethoxy)
ethyl)-4,8-methano[1,3]dioxolo[4,5-f]
isoindole-5,7(6H)-dione 13
Synthesis of Ethanamine, 2-[4-[(1E)-1,2-Diphenyl-1-buten-
1-yl]phenoxy]-N,N-methyl-propargyl 17
Dodecyl acetal 9 (348 mg, 0.58 mmol) was dissolved in a
solution of 8 : 1 MeCN/H2O before the addition of sodium azide
(262 mg, 4.03 mmol). The resulting solution was then refluxed
for 16 h before being diluted with CH2Cl2 (30 mL) and trans-
ferred to a separating funnel where it was washed with saturated
aqueous NaCl (3 ꢁ 25 mL) and the combined organic phases
dried (MgSO4) and solvent removed under vacuum to give a
white solid. Several attempts at purification by silica gel column
chromatography were performed unsuccessfully and, as such,
the crude product that was shown by 1H NMR and mass spec-
troscopic analysis to contain the desired dodecyl azide 13
(243 mg, 89 %) was used in subsequent steps. dH (400 MHz,
CDCl3) 4.61–4.59 (1H, t, J 4.0), 3.93 (4H, s), 3.68–3.65 (2H, t,
J 12.0), 3.62–3.59 (2H, t, J 4.0), 3.34–3.32 (2H, t, J 4.0),
Propargyl bromide (0.18 mL, 2.03 mmol) was added to a solu-
tion of demethylated Tamoxifen 16 (400 mg, 1.01 mmol) in
PhMe and stirred. TEA was added before the solution was
stirred at 808C for 16 h before being diluted with CH2Cl2
(30 mL) and transferred to a separating funnel where it was
washed with saturated aqueous NaCl (3 ꢁ 25 mL) and the
combined organic phases dried (MgSO4) and the solvent
removed under vacuum to give a pale yellow solid. Purification
by silica gel column chromatography was performed with a
80 % petroleum spirit/20 % EtOAc solution and the resulting
white solid was shown by 1H NMR spectroscopy analysis to be
the desired propargylated Tamoxifen 17 (304 mg, 76 %) in
. 95 % purity. dH (400 MHz, CDCl3) 7.37–7.09 (10H, m),

908
J. S. Squire et al.
6.77–6.74 (2H, d, J 12.0), 6.57–6.53 (2H, d, J 16.0), 3.94–3.90
(2H, t, J 8.0), 3.41–3.40 (2H, d, J 4.0), 2.81–2.77 (2H, t, J 8.0),
2.49–2.40 (2H, q, J 12.0), 2.36 (3H, s), 2.21–2.19 (1H, t, J 4.0),
0.94–0.88 (3H, t, J 12.0); dC (200 MHz, CDCl3) 156.81, 143.91,
142.52, 141.44, 138.35, 135.72, 131.95, 129.81–127.97, 113.51,
78.56, 73.45, 65.80, 54.45, 46.06, 42.30, 29.81, 29.11, 13.71;
m/z (HRMS ESI) Calc. for [C28H30NO]þ 396.23219, found
396.23240; nmax /cmꢀ1 3291 m, 1704 m, 1572 s, 1173 s, 703 s.
J 8.0), 4.55–4.53 (1H, t, J 4.0), 4.43–4.41 (2H, t, J 4.0), 3.99 (2H,
br s), 3.85 (1H, s), 3.82–3.76 (4H, m), 3.62–3.69 (3H, t, J 4.0),
3.53–3.51 (3H, t, J 4.0), 3.09–3.08 (6H, m), 2.82 (2H, br s),
2.46–2.37 (2H, m), 2.00–1.97 (2H, d, J 12.0), 1.63–1.59 (3H, m),
1.38–1.24 (28H, m), 0.92–0.85 (6H, m); dC (200 MHz, CDCl3)
176.38, 156.68, 143.90, 138.27, 135.70, 131.93, 129.77, 129.53,
128.18, 127.96, 126.59, 126.11, 124.22, 113.43, 104.21, 78.31,
68.89, 67.52, 65.69, 55.33, 52.56, 50.18, 44.48, 42.73, 37.69,
35.83, 32.77, 32.00, 29.78–29.44, 29.09, 24.23, 22.77, 14.21,
13.67; m/z (HRMS ESI) Calc. for [C57H78N5O6]þ 928.59466,
found 928.59438; nmax /cmꢀ1 2923 m, 2853 m, 1701 s.
Synthesis of 6-(2-(2-(4-(((2-(4-((Z)-1,2-Diphenylbut-1-en-1-
yl)phenoxy)ethyl)(methyl)amino)methyl)-1H-1,2,3-triazol-
1-yl)ethoxy)ethyl)-2-undecylhexahydro-5H-4,8-methano
[1,3]dioxolo[4,5-f]isoindole-5,7(6H)-dione 18
Supplementary Material
1
All H and 13C NMR spectra for novel compounds, represen-
To a 35 mL microwave vial containing dodecyl azide 11
(258 mg, 0.54 mmol), CuCl (5.37 mg, 0.054 mmol) was added
and dissolved with CHCl3 (15 mL). Propargylated tamoxifen 17
(214 mg, 0.54 mmol) was added and the solution was then
subjected to microwave irradiation at 1008C for 30 min before
being diluted with CHCl3 (30 mL). The solution was then
transferred to a separating funnel where it was washed with 4 M
HCl (3 ꢁ 25 mL) followed by a wash with H2O (20 ml). The
combined organic phases were dried (MgSO4) and the solvent
removed under vacuum to give a green oil. Purification by silica
gel column chromatography was performed with 100 % EtOAc
followed by 100 % EtOH and the resulting yellow oil was shown
tative CMC determination, TEM images referred to in the text
and correlograms from DLS measurements during variable
temperature studies are available on the Journal’s website.
Acknowledgements
The authors would like to thank the CASS foundation and the Strategic
Research Centre for Chemistry and Biotechnology, Deakin University for
funding. Metabolomics Australia is thanked for the use of their HRMS
facilities and the authors also thank the Australian Government for the APA
scholarship for JS.
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