Biocatalytic amide condensation and gelation controlled by light

Article abstract of DOI:10.1039/c4cc01431f

We report on a supramolecular self-assembly system that displays coupled light switching, biocatalytic condensation/hydrolysis and gelation. The equilibrium state of this system can be regulated by light, favouring in situ formation, by protease catalysed

Full text of DOI:10.1039/c4cc01431f

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Biocatalytic amide condensation and gelation
controlled by light†
Cite this: Chem. Commun., 2014,
50, 5462
Jugal Kishore Sahoo, Siva Krishna Mohan Nalluri, Nadeem Javid, Hannah Webb
and Rein V. Ulijn*
Received 24th February 2014,
Accepted 25th March 2014
DOI: 10.1039/c4cc01431f
www.rsc.org/chemcomm
We report on a supramolecular self-assembly system that displays structure growth and amplification associated with catalysis
coupled light switching, biocatalytic condensation/hydrolysis and with both equilibrium8 and non-equilibrium9 approaches
gelation. The equilibrium state of this system can be regulated by demonstrated. Bing Xu’s group recently demonstrated a system
light, favouring in situ formation, by protease catalysed peptide that combined enzymatic and light responsiveness, where an
synthesis, of self-assembling trans-Azo-YF-NH2 in ambient light; enzymatic reaction was performed to generate a hydrogel that
however, irradiation with UV light gives rise to the cis-isomer, which showed light responsive assembly and disassembly.10
readily hydrolyzes to its amino acid derivatives (cis-Azo-Y + F-NH2)
Responsiveness in biological systems is commonly achieved by
molecular systems that are able to respond to applied stimuli by a
redistribution of components in coupled (networked) reactions.
with consequent gel dissolution.
Molecular self-assembly1 provides a methodology for the fabrica- A similar adaptive behaviour may be achieved in synthetic
tion of functional nanostructures for next generation healthcare systems, by coupling reactions. Conceptually, this idea fits with
and energy related technologies.2 Peptide based systems are the objectives of systems chemistry11 to make synthetic networked
attractive in this regard, as they combine chemical versatility molecular systems that are adaptive.
with ease of synthesis and biological relevance.3 Aromatic peptide
Herein, we demonstrate the coupling of light switching with
amphiphiles, comprising di- or tripeptides incorporating (non- biocatalytic amide condensation/hydrolysis. The equilibrium
biological) aromatic residues are being increasingly studied position of the amide reaction is controlled by the self-assembly
due to their simplicity and versatility.4 The interplay of kinetics propensity of the azo-peptide vs. its amino acid precursors,8
(self-assembly route)5 in addition to thermodynamics (optimised which in turn is regulated by the input of energy (light), thus
supramolecular interactions) contributes to the properties of indirectly affecting the equilibrium situation of the system
the assembled material. Methods to control the self-assembly (Scheme 1). The free energy change associated with molecular
process are therefore extensively researched.
self-assembly and gelation is sufficiently thermodynamically
Light has been used to control molecular assembly, typically by favourable to overcome the bias for peptide hydrolysis normally
cis-to-trans isomerisation and consequent differences in stacking observed in aqueous systems to facilitate condensation. This has
of the required light sensitive aromatic residues (typically deriva- been shown before for Fmoc-,8a naphthoxy8b peptides as well as
tives of azobenzene). Using light in this context is attractive due purely peptidic12 systems but has not yet been demonstrated for
to its non-invasiveness, wavelength selectivity and the possibility peptides linked to light responsive aromatic moieties. This rever-
of patterning using photo-masks. A number of light responsive sible biocatalytic condensation is combined with the well-known
peptide self-assembly systems have been reported including an cis-to-trans isomerisation for azobenzene, which has been used
azo-dipeptide.6
successfully in a range of light switchable systems.13 Xu’s system
Alternatively, biocatalytic control of molecular self-assembly,7,8 focuses on the enzymatic activation of the system which in itself is
i.e. enzymatic conversion of non-assembling precursors into self- irreversible. Our system is different in that the enzyme action and
assembling molecules, provides an attractive approach, combining self-assembly is coupled and reversibly influenced by light.
biological selectivity, a level of space-time control of nucleation and
Azobenzene functionalised tyrosine was prepared via a four
step synthetic procedure (Scheme S1, ESI†) to form Azo-Y (com-
pound 5 in Scheme S1, ESI†). First, we investigated, the enzymatic
formation (using thermolysin from Bacillus thermoproteolyticus
rokko) of different Azo-YX-NH2 derivatives (X = F-, L-, V-, represented
as YX) in order to select a system most suited to demonstrate
WestCHEM, Department of Pure and Applied Chemistry, Thomas Graham Building,
295 Cathedral Street, Glasgow G1 1XL, UK. E-mail: rein.ulijn@strath.ac.uk
† Electronic supplementary information (ESI) available. See DOI: 10.1039/
c4cc01431f
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increased substantially between 4 and 24 h. These results
indicate that the gelation-driven self-assembly is sufficiently
thermodynamically favourable to reverse peptide hydrolysis
substantially to condensation. The contributing interactions
are p–p stacking between trans-azobenzene moieties, as well as
the aromatic amino acids, combined with and hydrogen bonding
interactions between dipeptide units. The higher percentage
conversion obtained for YF is likely a result of its more favourable
self-assembly, due to additional aromatic stacking contributions
in F compared to V and L. Competitive sequence selection for self-
assembly under thermodynamic control for naphthalene-peptides
also showed YF as a preferred sequence.8b
Next, fluorescence spectroscopy was used to monitor any changes
in the fluorophore environment upon assembly. The fluorescence
emission peak of the starting mixture (Azo-Y + X-NH2) exhibits a
weak emission peak at 552 nm. The addition of thermolysin to this
mixture induces a red-shift (558 nm) accompanied by a substantial
increase in the relative emission intensity peak (Fig. 1B). This
aggregation-induced emission (AIE)14 behaviour indicates that self-
assembly of the dipeptide derivatives induces dramatic changes in
the electronic properties, most likely due to changes in p-stacking
interactions. In addition to spectroscopy, investigations into their
nanoscale architecture were carried out using transmission electron
microscopy (TEM). The starting materials, before enzyme addition,
showed micellar structure while after enzyme addition, the presence
of entangled nanofibres was observed for the three dipeptide
sequences investigated (Fig. 1C). The diameter of the nanofibres
was 20–30 nm and the length of the fibres was up to several
micrometers, in line with nanostructures observed for other aromatic
peptide amphiphiles.4 Fibres formed by YF appeared to be signifi-
cantly shorter compared to those found for YL and YV.
Scheme 1 (A) trans-Azobenzene substituted tyrosine derivatives (trans-
Azo-Y) upon enzymatic condensation with amide derivatives of amino
acids (F-, L-, V-NH2) generate the corresponding dipeptide hydrogelators
(Azo-YX-NH2, X = F, CH2–C6H5; L, CH2–CH–(CH3)2; V, CH–(CH3)2) in
presence of thermolysin at pH 8 in phosphate buffer under ambient
conditions. Exposure to UV-light induces Azo switching, resulting in
disassembly and hydrolysis of the gelator. (B) Proposed energy diagrams
for condensation and self-assembly of azo-peptide hydrogel for the trans-
and cis-isomer. Left: energy difference for self-assembly exceeds that of
hydrolysis, resulting in overall favourable condensation and self-assembly.
Right: self-assembly is not favoured resulting in hydrolysis, rather than
condensation of the azo-peptide.
the concept. The molecular self-assembly of building blocks upon
the addition of thermolysin, was macroscopically observed by
transformation from a translucent solution to a self-supporting
(yellow coloured) hydrogel. Of the systems tested, YF gelled rapidly
(within 2–5 min after the addition of enzyme) while YL and YV
gelled within periods of 15 and 30 min, respectively. The percentage
conversion to the dipeptide derivatives was determined using
reverse-phase high performance liquid chromatography (HPLC).
As shown in Fig. 1A, the dipeptide derivatives formed in good yields
of YF (84%), YL (59%) and YV (63%) at 24 h after enzyme addition.
While YL and YF appeared to have reached an equilibrium
conversion after 4 hours, the YV conversion was slower and still
The gel-like behaviour of the self-supporting hydrogels was
confirmed by oscillatory rheology (Fig. S2, ESI†). For the three
dipeptide derivatives, the elastic modulus (G0) is over 10 times
greater than the viscosity modulus (G00) confirming the exis-
tence of a strong hydrogel network for each system with similar
moduli observed for each system. The data suggest that YV has
more stiffness (higher G0) than YL and YF derivatives.
Among the three dipeptide derivatives tested, YF showed a
high conversion and rapid response time, and so was used to
investigate its light-responsive properties. When the hydrogel
(10 mM, 24 h after enzyme addition) was irradiated using a UV
lamp (365 nm), the hydrogel disintegrated and dissolved after
48–72 h of exposure.15 This gel–sol transition of YF is expected
due to the conformational change of azobenzene from planar
trans-(E) to non-planar (bent) cis-(Z) form, with the cis-isomer
prohibiting effective hydrophobic association and p–p stacking
between azobenzene chromophores.10 This unfavourable self-
assembly further results in a switch from favourable condensa-
tion in the self-assembling system (Scheme 1) to a situation
where the enzymatic peptide hydrolysis takes over (Fig. S5,
ESI†), resulting in degradation of the fibrous network. (HPLC
yields confirm the peptide hydrolysis, as discussed below.)
Upon heating and sonication for 60 s, the cis-isomer (sol),
reverts back to the trans-isomer after 48–72 h and gelation is
observed. This gel–sol–gel transition of the system upon UV
Fig. 1 (A) HPLC data, showing the percentage peptide conversion of
different dipeptide derivatives over time (l = 340 nm). (A) Fluorescence
spectroscopy of azobenzene based dipeptide hydrogels (Azo-YX-NH2, X =
F, L, V) before and 24 h after thermolysin addition (lexcitation = 343 nm).
(C) TEM images of the dipeptide derivatives.
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using a coupled system, involving biocatalytic condensation,
gelation and light switching. Influencing of biological pathways
by using light may provide important tools for the development of
adaptive nanotechnology, with possible therapeutic implications,
e.g. in the photo-modulation of cellular environments.17
The authors declare no competing financial interest.
We thank the BBSRC for funding through Award 120315.
The research leading to these results has received funding from
the European Research Council under the European Union’s
Seventh Framework Programme (FP7/2007-2013)/ERC grant
agreement no. 258775.
Fig. 2 Circular dichroism (CD) spectra of Azo-YF-NH2 (10 mM) before
(gel) and after (sol) UV irradiation. Corresponding HT (high tension) voltage
(Fig. S3, ESI†).
Notes and references
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Fig. S4 (ESI†), in the gel-phase YF arranges itself into a three
dimensional network of nanofibres which, upon UV irradia-
tion, converts to micellar aggregates. Under ambient light,
nanofibres reform over time (48–72 h).
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CD analysis reveals insights into changes in the chiral supra-
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phore.13,16 After UV induced gel dissolution, the system becomes
CD silent, which is in line with expectation for the formation of a
micellar aggregate system. 3 h after UV treatment, we observe
supramolecular chirality, which confirms that the isomerisation of
the cis- into trans-form favours the reformation of chiral nanofibres.
After 3 days, the CD signal reverts back to similar intensity, albeit,
with a change in the spectral shape, suggesting a slight difference
in the supramolecular arrangement compared to the initial gel
formed. HPLC analysis (Fig. S5, ESI†) was performed to assess the
influence of light on the condensation/hydrolysis equilibrium,
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To further confirm that UV irradiation not only switches the
self-assembly properties but also enables hydrolysis, the system
was started under UV illumination (Scheme 1B, right). To
this end, the starting solution (Azo-Y + F-NH2; 10 mM) was
isomerised under UV-light in solution state before addition of
the enzyme. The conversion of trans- to cis-isomer could be
monitored by UV-vis spectroscopy (Fig. S6, ESI†). The enzyme
was subsequently added and the biocatalytic self-assembly of
the dipeptide derivative monitored using HPLC, giving rise to a
7.6% conversion after 24 h (Fig. S7, ESI†) (instead of 84% for the
trans isomer), which indicates that the cis-isomer dis-favours
condensation and gelation.
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In summary, we demonstrated the ability to control and direct
enzymatic amide condensation using light. This is achieved by
5464 | Chem. Commun., 2014, 50, 5462--5464
This journal is ©The Royal Society of Chemistry 2014