Functionalising the azobenzene motif delivers a light-responsive membrane-interactive compound with the potential for photodynamic therapy applications

Article abstract of DOI:10.1039/c5ob00465a

When adorned with n-octyl chains azobenzene is able to disrupt a variety of calcein-loaded phospholipid liposomes. The levels of lysis observed are dependent both on the lipid headgroup and the conformation of the azobenzene compound. In all cases studied

Full text of DOI:10.1039/c5ob00465a

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Functionalising the azobenzene motif delivers a
light-responsive membrane-interactive compound
with the potential for photodynamic therapy
applications†
Cite this: DOI: 10.1039/c5ob00465a
Theodore J. Hester,a Sarah R. Dennison,b Matthew J. Bakerc and Timothy J. Snape*b
When adorned with n-octyl chains azobenzene is able to disrupt a variety of calcein-loaded phospholipid
liposomes. The levels of lysis observed are dependent both on the lipid headgroup and the conformation
of the azobenzene compound. In all cases studied, it has been shown that the cis-conformer is more
membrane-interactive than the trans-conformer, suggesting that this class of molecule could be
optimised for photo-dynamic therapy applications against infectious pathogens.
Received 9th March 2015,
Accepted 25th June 2015
DOI: 10.1039/c5ob00465a
www.rsc.org/obc
way of designing new photodynamic therapeutic agents for
infectious diseases.
Introduction
The use of light for the photoisomerisation of azobenzene
(1, Scheme 1) and its derivatives has been exploited in numer-
ous biomedical applications.1 For example, light as a trigger
has been used to induce changes in a molecular assembly for
the controlled release of chemical compounds;2 to drive func-
tional changes in peptides, proteins, nucleic acids, lipids, and
carbohydrates;3 and for the in vivo light-activation of ion chan-
nels for the remote control of neuronal firing which has poten-
tial applications in controlling activity downstream from sites
of neural damage or degeneration.4 Moreover, proteasome
inhibitors, widely used as cytotoxic agents in cancer treatment,
have been prepared through the introduction of a photoswitch-
able azobenzene motif into the molecular structures studied,
giving rise to chemotherapeutic agents that can be switched
on and off with light.5
The precedent for azobenzene derivatives to be exploited as
light-triggered molecular switches in biological systems
suggested that such a practice might be combined with our
current interests studying the nature of non-covalent inter-
actions between conformationally distinct benzanilides (e.g. 2)
and diarylureas with phospholipid monolayers,6–9 if suitable
changes to the azobenzene motif could be incorporated, as a
Results and discussion
Scheme 1 outlines the approach taken to merge the desired
structural features of 1 and 2 and the subsequent synthesis of
the target compound (4, 73%) using a TEMPO/CuBr-based oxi-
dative coupling of 4-n-octylaniline (3).10 With suitable quan-
tities of the lipophilic azobenzene derivative 4 in hand,
attempts to determine its spectroscopic and conformational
properties were undertaken, prior to studying the interaction
of any isolable conformers with phospholipid membranes.
To determine the response of 4 to UV light, a 0.125 mM
stock solution was prepared in a suitable solvent or water :
solvent mix and spectra recorded every 10 s using 365 nm
irradiation (ESI†). In the absence of UV irradiation, an intense
π–π* transition band at around 330 nm and a weaker n–π*
transition band around 440 nm is observed for trans-4.3
aSchool of Forensic and Investigative Sciences, University of Central Lancashire,
Preston, PR1 2HE, UK
bSchool of Pharmacy and Biomedical Sciences, University of Central Lancashire,
Preston, PR1 2HE, UK. E-mail: tjsnape@uclan.ac.uk; Tel: +44 (0)1772 895805
cWestCHEM, Department of Pure and Applied Chemistry, University of Strathclyde,
Glasgow, G1 1XL, UK. E-mail: matthew.baker@strath.ac.uk;
Tel: +44 (0)141 548 4700
†Electronic supplementary information (ESI) available: Synthesis, characteris-
ation, UV and membrane lysis experiments. See DOI: 10.1039/c5ob00465a
Scheme 1 Azobenzene (1), a lipid-interactive benzanilide (2) and the
conformationally similar azobenzene derivatives studied here (4 and 5),
and their synthesis.
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Following irradiation of 4 with 365 nm light for 180 s a spec- pendent single-lipid liposomes were used which were com-
trum indicative of cis-5 was recorded which possessed a n–π* posed of dimyristoyl phosphatidylethanolamine (DMPE),
transition with a higher intensity compared to the trans- dimyristoyl phosphatidylglycerol (DMPG), dimyristoyl phos-
isomer.3 Despite this difference, a photostationary equilibrium phatidylserine (DMPS) and dimyristoyl phosphatidylcholine
is set up meaning that a maximum of ∼85% of the cis-isomer (DMPC), phospholipids which are the main constituents in,
forms upon irradiation at 330 nm, an observation that was and can therefore act as simple membrane mimics of, Escheri-
confirmed by 1H NMR (see below).
chia coli (DMPE) and Staphylococcus aureus (DMPG)14 or cancer
To ensure that the observed conformational change was cells (DMPS and DMPC).15 The total lipid concentration used
fully reversible, a relaxation experiment was carried out, as in the assay was 100 μM, with a P/L ratio ranging from ∼1 : 4
above, using a 532 nm laser as the light source and the now (24 μM) to ∼1 : 33 (3 μM).
“excited” sample of 5 (0.125 mM solution). In this case, after
The results (Fig. 1) demonstrate that the compounds do
irradiation with the 532 nm laser using 30 s intervals for interact with phospholipid membranes, and that there is a sig-
10 min, an identical time-course spectral series was recorded nificant difference between the two conformers, with cis-5
to that of the original trans-sample (4), indicating that the being more membrane-interactive than trans-4 across all lipids
n-octyl-functionalised azobenzene (4) was able to switch con- tested, results which mimic the trend of the benzanilide
formations, as anticipated.11
analogue too.6,8
Corroboration of the light-induced conformational switch
The maximal lysis observed occurred with cis-5 and lipo-
was achieved when the experiment was repeated in CDCl3 and somes consisting of DMPS, where a plateau was reached at
the irradiated sample analysed directly by NMR and compared 12 μM (∼64% lysis) in 1 h, a value that was not superseded
to the non-irradiated sample. Similar changes in the UV even at double that concentration. These results compare well
spectra to those seen with the other solvents above were to those of cis- and trans-benzanilide (2)6 against DMPE and
obtained in CDCl3. The changes in the 1H NMR of 4 were com- DMPG liposomes where, even at the higher concentration of
pared prior to irradiation, after 21 h of 365 nm irradiation, 400 μM, the maximum levels of lysis were 25.0 0.3% and
and after exposure of the irradiated sample to darkness and 42.9
0.1% for the trans- and cis-conformers respectively
heat or 532 nm light (to promote relaxation back to trans-4). against liposomes composed of DMPE lipid, and 21.1 0.6%
The spectra (ESI†) show an expected upfield shift occurs upon and 41.9 0.2% for the trans- and cis-conformers respectively
irradiation of trans-4,12 giving a spectrum which is consistent against liposomes composed of DMPG lipid. These can be
with the cis-conformation (5). Similarly, a switch back to the compared to the data shown in Fig. 1 (at 12 μM) of 10.41
trans-conformer (4) occurs with 532 nm light or at 60 °C in the 0.30% and 33.44
0.22% for trans-4 and cis-5 respectively
dark. In all spectra a small amount of the other conformer is against liposomes composed of DMPE lipid, and 28.58
visible, consistent with the UV experiments above. Combined,
these results support that a conformational switch occurs
between trans-4 and cis-5, whereby it switches from trans to cis
at 365 nm and switches back again at 532 nm (or 60 °C in the
dark).
Having confirmed that the azobenzene derivative 4 cleanly
undergoes the expected conformational switch, and that its
relaxation back to the trans-conformer is sufficiently slow
(>21 h) to enable assays to take place on the individual confor-
mers, attention turned to establish what, if any, interactions
the two conformers have with phospholipid membranes.
Moreover, if the two conformers were able to interact with
phospholipid membranes, it could be determined whether the
results mirror those of the benzanilide conformers of 2
studied previously,6,8 whereby the cis-conformer was shown to
be more membrane interactive than the trans-conformer, pre-
sumably a consequence of both lipid chains inserting into,
and disrupting the membrane, rather than predominantly only
one of the trans-compound.6,8 Furthermore, the light-switch-
ing advantage of the azobenzene series over the N-methylation
state “pseudo-switch” of the amide series means that the com-
pounds could be optimised for photodynamic therapy appli-
cations, especially for topical treatments.
Fig. 1 The ability of trans-4 (top) and cis-5 (bottom) to induce mem-
brane leakage using the lipids DMPE (solid black line), DMPG (solid dark
A concentration-dependent calcein-release assay was used
to determine the extent to which the two conformers are able
grey line), DMPS (solid light grey line) and DMPC (dotted black line). The
to lyse liposomes loaded with the fluorescent dye.13 Four inde- data shown are the average of three independent experiments.
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0.21% and 54.72
0.31% for trans-4 and cis-5 respectively group (i.e. ethanolamine, glycerol, serine and choline in
against liposomes composed of DMPG lipid, suggesting that, DMPE, DMPG, DMPS and DMPC, respectively), as does
in both cases, the cis-conformer is the most active form at the exact make-up of the charge, i.e. whether is it net neutral
lysing liposomes. Presumably such a trend occurs because in (zwitterionic) as seen in DMPE and DMPC, negative as seen in
the cis-conformation the lipid chains are able to insert into the DMPG, or net negative (zwitterionic) as seen in DMPS, and
lipid bilayer and disrupt the membrane packing. Since conver- this makes a difference in the levels to which the membrane is
sion of the isomers at or near the membrane is the core lysed.18,19
concept here, the behaviour of the isomers in the presence of
Such findings demonstrate that the properties of the lipid
lipids was ascertained (ESI†). A simple UV-Vis experiment was polar headgroup are important in such systems and the overall
performed in which a spectral series of the azobenzene deriva- amount of lysis is governed by a fine balance of interaction
tives were followed over time in the presence of the liposomes contributions.
made up from both DMPC and DMPE, and it was shown that
Having observed that the cis-conformer is more membrane-
identical series were obtained, in each case, to those of the interactive than trans-4 over a 1 h time period, we were inter-
series in the absence of the lipids, suggesting that the azo- ested in understanding what would happen over an extended
benzene derivatives are able to switch their conformations in time period of 24 h. This could be important since this is the
the presence of lipids, as required. Since compounds 2, 4 and 5 sort of time-scale in which a cis → trans relaxation may begin
contain the same lipid side-chains, the differences observed to take place, and thus if it did whilst the compound was
between the compounds and different liposomes studied is buried in the membrane, it would further increase the levels
therefore seemingly dependent on the specific interactions of membrane lysis observed, and its potential as a membrane-
between the phosphate headgroups and the motif linking the disruptive compound.
two adorned benzene rings. Although it is known that hydro-
Again, a concentration-dependent time course was studied
phobic molecules can disrupt phospholipid membranes, there against all four lipids (ESI†), but representative data can be
is a significant difference between trans-4 and cis-5 suggesting seen in Fig. 3 at 12 μM for simplicity.
that shape plays an important role here too.
As seen in Fig. 3, against all lipids the cis-conformer (5) is
As can be seen in Fig. 2, the size of the two conformers more membrane lytic than trans-4, sometimes as much as two-
varies such that the distance between the para-positions of the fold or more, and in most cases, maximal lysis occurs, and a
benzene rings (the location of the alkyl chains) is smallest in plateau is reached, around the 3 h time-point. Overall maximal
the cis-isomer (∼0.5 nm cf. ∼1.0 nm). This geometry means lysis occurs at ∼80% for cis-5 against DMPS, but interestingly,
that in the cis-conformation, the n-octyl chains are better posi-
tioned to interact with, and disrupt, the membrane packing
(Fig. 2, left – shown for clarity with one molecule, although in
reality clustering of the compounds in the membrane would
most likely occur). Combined with the larger dipole moment
of the cis-isomer enabling larger ion-dipole interactions to take
place between 5 and the negatively charged membrane, makes
the cis-isomer more membrane disruptive than the trans.
Whether studying the azobenzene derivative here, benz-
anilides6,8 ureas,7 or other constructs,16,17 the subtle differences
in the non-covalent interactions experienced between the par-
ticular molecular motif and the lipid headgroup should affect
the stability of the compound in the membrane, and hence
the level of lysis observed.18–24 For example, the membrane’s
charge varies depending the nature of the phosphate head-
Fig. 3 The kinetics of calcein release over time after addition of 12 µM
Fig. 2 (a) A schematic showing the proposed mechanism for mem-
brane disruption of cis-5 (left, shown, for clarity with one molecule), and
(b) the reversible isomerisation of azobenzene showing its geometry and
dipole changes (right).
(P/L = ∼1 : 8) trans-4 (top) and cis-5 (bottom) to 100 µM DMPE (solid
black line), DMPG (dark grey line), DMPC (dotted black line) and DMPS
(light grey line). After 7 hours the sample ceased calcein release and this
did not change over time.
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there appears to be very little, if any, selectivity for liposomes
composed of DMPC between the two conformers. If the differ-
ence between the levels of lysis caused by the cis and trans con-
formers for each time point are calculated, then it can be seen
that, for all cases except the 1 h time-point, both the neutral
(zwitterionic) lipids DMPE and DMPC have the smallest differ-
ence in lysis for the two conformers. This is in contrast to the
two negatively charged lipids DMPG and DMPS, which have
the greatest difference, and more specifically, the negative
(zwitterionic) lipid DMPS is the most lysed liposome of all
those studied. This difference provides a handle with which to
potentially prepare compounds which can be photochemically
switched to selectively interact with different phospholipid
membranes, and thus be optimised to be selective chemothera-
peutic agents.
Notes and references
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Conclusions
In conclusion, we have shown that an azobenzene motif
adorned with suitable n-octyl chains is able to disrupt a variety
of independent calcein-loaded liposomes composed of four
different phospholipids, and that the levels of lysis observed
are dependent both on the lipid headgroup and the confor-
mation of the molecules being studied. In all cases, it has
been shown that the cis-conformer is more membrane-interac-
tive than the corresponding trans-conformer, results which
mimic those of other structurally-related conformational
switches,6,8 suggesting that this class of molecule could be
optimised for photo-dynamic therapy applications.
Future work will look to focus on studying mixed lipid
systems and further adorning the benzene rings with water-
solubilising groups, groups which could also shift the wave-
length of light required to implement the conformational
switch to the visible spectrum,1 as well as introducing charge
to the molecules to increase their interaction with net negative
membranes, as has seen success recently with pyridinium
analogues.9
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Acknowledgements
The authors would like to thank the EPSRC UK National Mass
Spectrometry Facility (NMSF), Swansea.
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