Light-controlled out-of-equilibrium assembly of cyclodextrins in an enzyme-mediated dynamic system

Article abstract of DOI:10.1039/c9cc08452e

We show that the selective enzymatic synthesis of specific cyclodextrins can be modulated using light. We use enzyme-mediated dynamic combinatorial chemistry to generate a mixture of interconverting linear and cyclic α-1,4-glucans, and employ an azobenzene photoswitch as a template. Using UV or blue light to switch between photostationary states with different azobenzene cis/trans isomeric ratios, we can promote the out-of-equilibrium assembly of either α-cyclodextrin or β-cyclodextrin.

Full text of DOI:10.1039/c9cc08452e

Volume 55 Number 100 28 December 2019 Pages 15009–15150
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Sophie R. Beeren et al.
Light-controlled out-of-equilibrium assembly of
cyclodextrins in an enzyme-mediated dynamic system

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Light-controlled out-of-equilibrium assembly of
cyclodextrins in an enzyme-mediated dynamic
system†
Cite this: Chem. Commun., 2019,
55, 15037
Received 30th October 2019,
Accepted 22nd November 2019
Dennis Larsen,
Philip M. Bjerre and Sophie R. Beeren
*
DOI: 10.1039/c9cc08452e
rsc.li/chemcomm
We show that the selective enzymatic synthesis of specific cyclo- Specifically, we control the preferential enzyme-catalysed synth-
dextrins can be modulated using light. We use enzyme-mediated esis of a- or b-cyclodextrin via the photoisomerisation of an
dynamic combinatorial chemistry to generate a mixture of inter- azobenzene template (Fig. 1).
converting linear and cyclic a-1,4-glucans, and employ an azobenzene
Dynamic combinatorial chemistry is a now well-established
photoswitch as a template. Using UV or blue light to switch between methodology for the templated synthesis of complex molecular
photostationary states with different azobenzene cis/trans isomeric architectures, and the assembly of dynamic polymers and
ratios, we can promote the out-of-equilibrium assembly of either responsive systems from small building blocks under thermo-
a-cyclodextrin or b-cyclodextrin.
dynamic control.6 A few examples of dynamic combinatorial
libraries based on reversible organic reactions (e.g. disulfide
Light-responsive systems in living organisms exploit photo- and hydrazone exchange) that incorporate light-responsive
switchable co-factors to trigger the onset of enzyme-catalysed building blocks7 and templates8 have been reported.
reaction cascades to modulate growth and control essential
We recently described enzyme-mediated dynamic combinatorial
functions. For example, phototropic plants optimize their chemistry, wherein enzyme-catalysed reactions are used to reversibly
growth in response to sunlight via the judicious synthesis of link molecular building blocks and generate mixtures of
specific poly- and oligosaccharides,1 while the cis–trans photo- interconverting bio-oligomers.9 We employed cyclodextrin
isomerisation of retinal is responsible for visual transduction.2 glucanotransferase (CGTase)10 to generate a dynamic mixture
Inspired by Nature, chemists have developed artificial light-
responsive systems wherein a photo-activated conformation
change in a small molecule or molecular moiety leads to a
specific output from a chemical network, or altered physical
properties of a self-assembled material.3
A number of artificial systems have recently been reported in
which enzyme activity is controlled using light, either by direct
bioconjugation of enzymes with photo-responsive moieties,
or by photoisomerisation-based activation of a co-factor or
deactivation of an inhibitor.4 More complex systems use light-
controlled self-assembly of supramolecular structures for
compartmentalisation or release of co-factors or substrates, thus
stimulating enzyme activity.5 Here we apply a unique strategy to
control the outcome of an enzymatic process using light, which
relies upon the use of a photo-responsive template to select
different products in an enzyme-mediated dynamic system.
Fig. 1 Light-controlled, selective enzyme-mediated synthesis of cyclo-
dextrins. (a) CGTase acts on an a-1,4-glucan source to generate a dynamic
combinatorial library of cyclodextrins. Different isomers of the azobenzene
template promote the selective synthesis of different cyclodextrins. (b)
Azobenzenes switch from the trans- to the cis-isomer upon irradiation with
UV light and switch back thermally or with blue light.
Department of Chemistry, Technical University of Denmark, Kemitorvet 207,
DK-2800 Kongens Lyngby, Denmark. E-mail: sopbee@kemi.dtu.dk
† Electronic supplementary information (ESI) available: Experimental details, full
list of photoswitches screened, chromatographic data, UV-vis spectra, NMR
spectra. See DOI: 10.1039/c9cc08452e
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of linear a-1,4-glucans (maltooligosaccharides) and cyclic
a-1,4-glucans (cyclodextrins (CDs)). We outlined how CGTase
catalyses the fast, reversible transglycosylation and slow, irreversi-
ble hydrolysis of a(1 - 4)-glycosidic linkages, and thus generates a
complex dynamic chemical system, wherein cyclodextrins assemble
transiently out-of-equilibrium in a kinetically trapped subsystem.
This subsystem operates under pseudo-thermodynamic control
and by adding different templates, we were able to alter the
distribution of the different cyclodextrins formed to obtain
either a-CD, b-CD or g-CD, with high selectivities ranging from
89% to 99%.9
Azobenzene moieties have been extensively utilised to
achieve photo-responsive behaviour in functional molecular
and biomolecular systems.11 Irradiation with UV light (typically
340–380 nm) causes a photo-induced isomerisation from the
most stable trans isomer to the more sterically encumbered
and less stable cis isomer (Fig. 1b).12 Return to the trans
isomer occurs via a thermal back-reaction, or can be promoted
by irradiation with visible light (typically 430–550 nm). Sparsely
substituted azobenzenes are known to bind within the
hydrophobic cavities of cyclodextrins (in aqueous solution)
and the selective recognition of the cis or trans isomers
has been exploited to control self-assembly of functional
materials.13 For this study, we screened a number of different
substituted azobenzenes (Table S1, ESI†) and identified a set of
water soluble azobenzene photoswitches (1–5) exhibiting
markedly different cis/trans isomeric ratios in ambient light
and when irradiated with UV light at 365 nm (Table 1 and
Fig. S2, S3, ESI†).
Fig. 2 Screening of azobenzenes 1–5 as light-responsive templates for
preferential synthesis of different cyclodextrins in a CGTase-mediated
dynamic combinatorial library. (a) Representative chromatograms (HPLC-
ELS) depicting the relative concentrations of a-, b-, and g-CD formed
when CGTase acts on a-CD in the absence of template, and with 1 (2 mM)
in the dark or under UV-irradiation (365 nm). (b) Bar graph depicting the
cyclodextrin distributions produced in similar reactions templated with
azobenzenes 1–5. Reaction time: 2 hours. Conditions: 2 mg mLꢀ1 a-CD
used as starting a-glucan in 50 mM sodium phosphate buffer at pH 7.5.
To investigate the possibility of modulating the selective
synthesis of different cyclodextrins using light, we examined
the action of CGTase on a-CD (2 mg mLꢀ1 in 50 mM sodium
phosphate buffer at pH 7.5) in the presence of azobenzenes 1–5
(2 mM), in the dark, or with UV-irradiation (365 nm, for 1 hour
Pleasingly, in all cases, we observed different distributions
prior to addition of enzyme and continuously throughout the of cyclodextrin products depending on whether or not the
reaction). The composition of each library was analysed after reaction was irradiated with UV-light. HPLC chromatograms
2 hours, using high performance liquid chromatography with showing the differing distributions of cyclodextrins that are
evaporative light-scattering detection (HPLC-ELS), and com- formed in the absence of any template and in the presence of
pared with an untemplated reaction (Fig. 2 and Fig. S1, ESI†). photoswitch 1, in the dark and with UV irradiation, are shown
in Fig. 2a. In each of these reactions, a-CD and b-CD were the
major products (very little g-CD is produced at low concentra-
Table 1 Physicochemical properties of azobenzenes 1–5a
tions of a-1,4-glucan (2 mg mLꢀ1)). A clear increase in the
production of a-CD and concurrent decrease in the production
of b-CD was apparent upon addition of template 1 in the dark.
Conversely, an increase in the production of b-CD was observed
upon application of UV-light together with the template. In
the absence of template, UV-irradiation did not influence the
Photoswitch
R
lmax (nm)
cis/trans ratiob
distribution of products.
R0
trans
cis
No irr.
UV
For each of the azobenzenes 1–5, addition of the template
without irradiation generated higher concentrations of a-CD,
indicating that in each case the trans isomer binds preferentially
to a-CD (Fig. 2b). For photoswitches 2–5, irradiation led to a
reduction in the amplification of a-CD suggesting that either the
cis-isomers have little interaction with either of the cyclodextrins,
or there is a favourable interaction between the cis-isomers and
b-CD, but this is outweighed by the interaction between a-CD and
1
2
3
4
5
COOH
COOH
SO3Na
SO3Na
H
COOH
OAc
326
331
325
354
320
426
429
426
420
423
15 : 85
10 : 90
12 : 88
1 : 99
73 : 27
51 : 49
63 : 27
74 : 26
70 : 30
NHAc
18 : 82
a
b
At room temperature in 50 mM sodium phosphate buffer at pH 7.5.
By 1H NMR spectroscopy when kept in ambient light (no irr.) or after
irradiation with light (365 nm) for 1 hour (UV).
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any remaining trans-isomer (note that the photostationary state presence of UV irradiation. When the enzymatic reaction is
obtained under UV irradiation contains significant amounts of carried out in the dark, trans-1 predominates, and since it binds
the trans isomers for all of the azobenzene templates, Table 1). a-CD approximately twice as strongly as b-CD, the production of
Only photoswitch 1 caused the amplification of b-CD when a-CD is amplified. When exposed to UV light, cis-1 predominates,
irradiated, indicating that cis-1 binds preferentially to b-CD, while and higher production of b-CD results, because cis-1 binds
trans-1 binds more strongly to a-CD.
15 times more strongly to b-CD than to a-CD.
Determination of association constants for the binding of
To illustrate how the dynamic enzymatic synthesis of cyclo-
a guest that exists as a mixture of slowly interconverting dextrins can be coupled to the dynamic switching between
isomers is not straightforward, as the isomeric ratio is altered photostationary states of photoswitch 1, the CGTase-catalysed
by selective interaction with the host. As photoisomerization of interconversion of CDs was monitored over time under varying
azobenzenes does not give complete trans to cis conversion, the light conditions. Firstly, a reaction was started in the dark and
cis isomer is always contaminated with a minority of the trans kept in darkness for the first 80 minutes before being sub-
isomer even after photoirradiation. This means that simple mitted to UV-irradiation (365 nm) (Fig. 3a). The system adapted
titrations, and fitting to a 1-to-1 binding model, cannot be used quickly to the UV light, switching readily from 80 : 20 a-CD/b-CD
to accurately determine association constants for binding of ratio in the dark to the expected 40 : 60 ratio when under UV
cis-isomers, due to competitive binding by the trans isomer irradiation. A second reaction was begun under UV irradiation,
minority. We have previously described how an NMR titration and after 60 minutes, the irradiation was switched to blue light
can be used to simultaneously determine association constants (460 nm) (Fig. 3b). Irradiation with blue light returns the
for the binding of a host to a number of different guests present mainly cis-1 to trans-1, and thus the a-CD/b-CD ratio returned
in a mixture.14 This methodology does not require the concen- to approximately 80 : 20. Consecutive cycles of irradiation with
trations of the host and guests to be known, nor should one be UV-light (365 nm, 20 min) and blue light (460 nm, 15 min) were
kept constant. It requires only that the association constant (Ka) is performed, demonstrating the possibility to oscillate up to six
known for one of the guests. Here we show that the same times between CD mixtures of predominantly a-CD or b-CD
methodology can be used to determine the association constants (Fig. 3c).
for cis-1 and trans-1 binding to a-CD and b-CD. Solutions of
Due to the irreversible background hydrolysis of glycosidic
photoswitch 1 (1 mM in 50 mM sodium phosphate buffer at linkages, and the gradual accumulation of glucose (which
pH 7.5) were titrated with a-CD and b-CD, with each titration cannot act as a glycosyl donor in CGTase-mediated glycosyl
performed under two different sets of conditions: (1) in the dark transfer), a steady decrease in the total cyclodextrin concen-
and after heating at 80 1C for 2 days to obtain exclusively trans-1; tration is observed (Fig. 3 grey line and Fig. S6–S9, ESI†).
(2) in ambient light, such that a mixture of cis-1 and trans-1 were This explains the gradual rise in the a-CD concentration and
present. For the titrations performed in the dark, association concomitant descent in the b-CD concentration, as at lower
constants, Ka(trans) (Table 2) were obtained by fitting to a regular building block concentrations, shorter oligomers are favoured
1 : 1 binding isotherm (Fig. S4, ESI†). For the titrations in ambient in dynamic combinatorial libraries.15 Background hydrolysis
light, both cis-1 and trans-1 were present, and these interacted appears faster during UV-irradiation but this is presumably due
unequally with the cyclodextrins, leading to a change in the ratio to the overall weaker binding by both cyclodextrins to cis-1
of isomers present upon increasing the concentration of cyclo- compared to trans-1, and as we have previously shown, hydro-
dextrin. By plotting the changes in chemical shift observed for lysis of cyclodextrins is hindered by complexation.9
each isomer upon addition of cyclodextrin against one another In our enzyme-mediated dynamic system, cyclodextrins
(Ddcis vs. Ddtrans), and fitting to the equation below, we could are not formed under equilibrium conditions, but instead self-
obtain Ka(cis) for each cyclodextrin (Table 2 and Fig. S5, ESI†).
assemble transiently out-of-equilibrium and are slowly converted
to glucose, the thermodynamic product.9 Surprisingly, templates
can nevertheless be exploited to select for specific cyclodextrin
products, presumably because a-, b-, and g-CD are separated
only by low energy barriers and form a kinetically-trapped
subsystem operating under pseudo-thermodynamic control.
This azobenzene-templated system is doubly dynamic, as the
equilibria for both the cis/trans isomerization and the CGTase-
catalysed a-CD/b-CD interconversion influence one another.
The unconventional energy landscape for this synthetic dynamic
system is depicted in Fig. 4. Here we show that we can use
UV light to push the system further out-of-equilibrium and
transiently occupy an alternative, higher energy dissipative, but
also kinetically trapped state.16 Using different wavelengths of
light to switch between predominantly trans-1 or cis-1, we can
control which kinetically trapped regime is occupied, and
promote the selective formation of a-CD or b-CD, respectively.
DdmaxðcisÞDdtransK
DdmaxðtransÞKaðtransÞ ꢀ DdtransK aðcisÞþ DdtransK
Ddcis
¼
aðtransÞ
aðcisÞ
The relative affinities of cis-1 and trans-1 for a-CD and b-CD
can thus explain the different product distributions obtained
when CGTase catalyses the formation of CDs in the absence or
Table 2 Association constants, Ka (Mꢀ1), for a- and b-CD with trans and
cis isomers of 1a
Guest
Host
trans-1
cis-1
140 ꢁ 70
a-CD
b-CD
5000 ꢁ 400
2500 ꢁ 300
2200 ꢁ 300
a
In sodium phosphate buffer (pH 7.5, 50 mM) in D2O at 25 1C.
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Fig. 3 Light-controlled changes in CD distribution in CGTase-mediated dynamic cyclodextrin systems generated starting from a-CD (2 mg mLꢀ1) and
templated with photoswitch 1 (2 mM). (a) Dynamic system kept in the dark before exposure to UV light (365 nm). (b) Dynamic system started under UV
light irradiation (365 nm), then irradiated with blue light (460 nm). (c) Consecutive cycles of irradiation with UV light (365 nm, 20 minutes) and blue light
(460 nm, 15 minutes).
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Conflicts of interest
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There are no conflicts to declare.
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15040 | Chem. Commun., 2019, 55, 15037--15040
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