Photochemical Control over Oscillations in Chemical Reaction Networks

Article abstract of DOI:10.1021/jacs.7b08109

Systems chemistry aims to emulate the functional behavior observed in living systems by constructing chemical reaction networks (CRNs) with well-defined dynamic properties. Future expansion of the complexity of these systems would require external control to tune behavior and temporal organization of such CRNs. In this work, we design and implement a photolabile probe, which upon irradiation strengthens the negative feedback loop of a CRN that produces oscillations of trypsin under out-of-equilibrium conditions. By changing the timing and duration of irradiation, we can tailor the temporal response of the network.

Full text of DOI:10.1021/jacs.7b08109

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Photochemical Control over Oscillations in Chemical Reaction Networks
Aleksandr A. Pogodaev, Albert S.Y. Wong, and Wilhelm T. S. Huck
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.7b08109 • Publication Date (Web): 17 Oct 2017
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Photochemical Control over Oscillations in Chemical Reac-
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Aleksandr A. Pogodaev, Albert S. Y. Wong, Wilhelm T. S. Huck*
Radboud University, Nijmegen, Institute for Molecules and Materials, Heyendaalseweg 135, 6525 AJ Nijmegen, the
Netherlands.
Supporting Information Placeholder
incorporation of simple designs into more complex out-of-
ABSTRACT: Systems chemistry aims to emulate the func-
equilibrium networks will make it ever more difficult to en-
sure that each reaction in the network has a suitable reaction
rate. Thus, we will require a method to regulate individual
rates in the network, without disrupting others.
tional behavior observed in living systems by constructing
chemical reaction networks (CRNs) with well-defined dy-
namic properties. Future expansion of the complexity of the-
se systems would require external control to tune behavior
and temporal organization of such CRNs. In this work, we
design and implement a photolabile probe, which upon irra-
diation strengthens the negative feedback loop of a CRN that
produces oscillations of trypsin under out-of-equilibrium
conditions. By changing the timing and duration of irradia-
tion, we can tailor the temporal response of the network.
Light is an attractive external control element, as it can
selectively and rapidly change reactions rates (in contrast to
changes in concentration, solvent quality, or temperature).
Research on the well-known BZ (Belousov-Zhabotinsky) sys-
tem has shown that light can be used to alter frequency and
phase of the oscillations18 and induce formation of specific
patterns.19
A discipline of ‘systems chemistry’ is developing, which
aims to capture the complexity observed in natural systems
within a synthetic chemical framework.1,2,3 In recent years,
significant progress has been made, including the design of
oscillating reaction networks,4,5 the formation of transient
gels6 and vesicles7, self-replicating systems,8,9 DNA-based
controllers and oscillators,10,11 self-organizing ensembles of
nanoparticles,12 and catalytic reactions with chemo-
mechanical feedback.13 A major challenge for systems chem-
istry is to translate the design principles of living systems,
based on feedback loops and reaction networks, into func-
tional synthetic equivalents.
We are inspired by the regularly recurring motifs of cou-
pled feedback loops generate much of the functional behav-
ior in biological networks.14,15 We recently reported an oscil-
lating out-of-equilibrium CRN, consisting of one positive
feedback loop (the autocatalytic production of enzyme tryp-
sin (Tr) from trypsinogen (Tg)) and one negative feedback
loop (Figure 1a).5 Figure 1a gives a schematic overview which
process dominates during different stages of the oscillation.
The negative feedback loop starts with the activation of a
pro-inhibitor (Pro-I), by Tr, into an intermediate inhibitor
that produces active inhibitor of Tr by the action of the en-
zyme aminopeptidase (Ap). The two-step negative feedback
loop produces a delay in Tr inhibition, which is a key re-
quirement for producing oscillations in flow conditions.14 We
can tailor the system-level properties of these networks (i.e
robustness and resilience of the functional output) by chang-
ing the precise structure of the small molecules.16,17 However,
Figure 1. Introduction of a control node. (a) Schematic
representation of the out-of-equilibrium system and the key
processes during oscillations, (b) Details of the original net-
work (light blue box) and the photochemical control node
(orange box).
Here, we demonstrate photochemical regulation of an
oscillating enzymatic reaction network. In our design, the
concentration of active trypsin (Tr) is expected to fully con-
trol the output of the network. In order to change the level of
active [Tr] instantaneously, external regulation is obtained
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Figure 2. Modelling of a short single irradiation pulse (a) Irradiation for 3 min. at different phases of oscillations: 0, 0.24π,
1π, 1.66π, solid lines depict simulations with irradiation and dashed gray lines without irradiation, orange stars indicate a
timepoint of irradiation. (b) Zoom with irradiations at 1.50π and 1.66π for different times. The conditions used for simulations:
[Tg]0=167 µM, [Pro-I] 0=1.5 mM, [Ap]0=0.28 U/ml, [Photo-I]0=100 µM, [Tr]0=0.2 µM, kf=0.37 h-1
via a photolabile inhibitor (Figure 1b). Upon irradiation with
light at 380 nm, the active inhibitor concentration is in-
creased, strengthening the final step in the negative feed-
back. We will demonstrate how light can be used to induce
delay, perturb or entrain oscillations, and synchronize os-
cillations in separate reactors.
system to different concentrations of the Photo-I, as well as
the duration and the precise timing of the irradiation. Figure
2a shows the simulated response of the network upon three
min. irradiation pulses at different phases (denoted here in
radians from 0 to 2π) of the oscillation. We note that only
upon irradiation during the rise in Tr concentration (around
1.66π; figure 2a – iv), we see a marked damping of the ampli-
tude followed by a slow recovery in the oscillations. In all
other cases the effect of a short pulse is quite similar and
results mostly in a small delay. A closer look at irradiation
around 1.66π (Figure 2b) shows that the system is rather sen-
sitive to the precise timing of irradiation, as the sustained
oscillations recover differently from perturbations at 1.50π
and 1.66π.
The photolabile inhibitor (Photo-I) consists of an inhibitor
moiety
coupled
to
a
well-known
methyl-6-
nitroveratryloxycarbonyl
(MeNVOC)
photoprotection
group,20,21 further functionalized with poly ethylene glycol
tails (PEG) to increase water solubility. The synthesis of Pho-
to-I was completed in 5 steps full experimental details and
characterization can be found in the Supporting Information
S2. Before introducing the Photo-I into the network, we stud-
ied the cleavage reaction as function of UV irradiation. We
first verified that Tr remains stable upon irradiation (S3.5).
Next, we determined the apparent rate constant of the cleav-
age reaction, and found a cleavage rate constant of 33 h-1. In
comparison to the typical time scales of our oscillations (pe-
riods ~5h), active inhibitor formation from the Photo-I can
First, we carried out a preliminary test of the photochemical
switching of the inhibitor in the full network. The full net-
work was studied experimentally in a flow reactor fed by four
different syringes with Tg, Tr, Ap, combined ProInh and Pho-
to-I. A computer-controlled circuit with three LEDs placed
around the flow reactor was used to control length and tim-
ing of irradiations. The outflow of the reactor was coupled to
a microfluidic chip that served as an online detection system
where active [Tr] was quantified in flow by a fluorogenic
be considered an instantaneous process. Furthermore, the
ꢀ#*/*ͯꢁ
inhibition by the uncleaved photolabile inhibitor (͟
Ɣ
$)#
1.4 mM-1h-1) is roughly 40 times slower in comparison with
Ɣ 52.7 mM).-1h-1). The
ꢁ
assay
based
on
benzoyl-L-arginine-7-amido-4-
inhibition by active inhibitor (͟
$)#
methylcoumarin (see S5 for details). As shown in Figure 3a,
the network shows sustained oscillations without UV expo-
sure, and complete inhibition of Tr during continuous irradi-
ation of the flow reactor.
apparent inhibition rate constant of the uncleaved photo-
inhibitor is similar to other background inhibition reactions
in the network (S3.4), and can therefore be treated as a back-
ground reaction. See Supporting Information S3 for full de-
tails on kinetic and spectral properties of Photo-I, including
the photo-cleavage rate, background inhibition, background
hydrolysis of the sulfonyl fluoride group, and absorbance
spectra.
Next, we confirmed the effect observed in simulations by
performing two experiments with exactly the same starting
conditions and irradiated for three minutes at different phas-
es of oscillation. Figure 3b shows an overlay of two irradiated
experiments in comparison with non-irradiated oscillations.
Although there are some fluctuations in the amplitude, we
observed that irradiating the reactor during the initial rise in
trypsin concentration, yielded a stronger perturbation, and a
similar slow recovery of the amplitude as observed in the
simulations shown in Figure 2b. Qualitatively, the phase de-
pendence can be explained by considering the amount of
Simulations are required to establish the starting concen-
trations of Tg, Tr, and small molecules, as well as suitable
flow rates and the importance of timing of irradiation. There-
fore, we added the kinetic parameters of the photochemical
production of inhibitor to the set of differential equations in
MATLAB (see S4) that we previously used to simulate the
dynamics of the network5. We simulated the response of the
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active Tr present in the system (see also Figure 1a). When no
active Tr is present, the formation of additional inhibitor will
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not have much impact. Similarly, when the oscillation is past
its peak, the negative feedback loop has already started to
outcompete the autocatalytic production of Tr, and hence
the impact of additional inhibitor will be small. Therefore,
the perturbation of the system will be largest during the (ini-
tial) rise in [Tr] (i.e. during the start of the autocatalytic posi-
tive feedback loop). A detailed, experimental study into the
underlying mechanisms of the sensitivity of the system to the
precise timing of irradiation is beyond the scope of this work.
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Figure 4. Demonstration of entrainment of oscilla-
tions. Experiment showing entrainment of damped oscilla-
tions by a series of 10 min irradiations.
Above, we have demonstrated how we can use light to
modulate the output of the network. As explained in the
introduction, future work will show the integration of multi-
ple networks. To demonstrate how light-induced inhibition
of Tr can be used in a control systems engineering approach
in coupled reaction networks, we ran two oscillating net-
works (containing Photo-I) in separate reactors and then
combined the outflow of the reactors in a Y-junction prior to
analyzing the functional output of the combined set (Figure
5a). In Figure 5b, we started the two oscillating reactions
with a delay of 3 hours, leading to a significant phase differ-
ence between the two reactors. The first part of Figure 5
shows the oscillations of both reactors combined, and one
can clearly distinguish peaks from both oscillating reactors
(reactor started first depicted as the solid blue line and se-
cond reactor as the dashed red line). By irradiating both re-
actors for 45 min, we flood both networks with active inhibi-
tor and thus bring the system back to a state where the con-
centration of active Tr is minimal. Subsequently, both reac-
tors ‘restart’ with the positive feedback loop initiating the
cycle, synchronizing the two reactors and producing a single
oscillation in the combined output.
Figure 3.Dependency on time of irradiation. Three sep-
arate experiments performed with identical starting condi-
tions, namely 1.4mM Pro-I, 0.05 mM Photo-I, 0.34 U/ml Ap,
kf=0.34 h-1. Irradiation pulse was 3 min. The orange stars de-
picts time of irradiation.
In a next experiment, we studied the effect of irradiation
on a network that operated outside the regime of sustained
oscillations. External pulsing with light might ‘rescue’ the
functionality of the system. For example, it is possible to
entrain a system that is in a regime of damped oscillations,
by a series of irradiation pulses, to produce a forced oscillat-
ing system (Figure 4). Simulations indicate (see S4.2) that
irradiation during the rise in Inh oscillations, which are shift-
ed by 0.5π phase from Tr oscillations, is most suitable if en-
trainment into sustained oscillations are desired. For the
damped oscillations shown in Figure 4, there is insufficient
active inhibitor to bring [Tr] back to baseline levels, leading
to a steady state [Tr] of ~1.5 µM. Upon each irradiation, the
additional active inhibitor lowers the active [Tr] to baseline
levels, and subsequently the autocatalytic, positive feedback
loop restarts. Remarkably, even after the irradiation pulses
have terminated, the system is able to oscillate for some time
more, before eventually reaching a damped state.
Figure 5. Phasing of two coupled oscillators. (a) schematic
representation of the experimental setup, (b) phasing of two
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oscillators by simultaneous irradiation of both reactor for 45
min..
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In summary, we have shown how a light-induced local
perturbation of the network (i.e. we only affect concentration
of a single component) can be used to gain external influence
over its out-of-equilibrium function. Experimental realiza-
tion of the photochemical control over the phase and ampli-
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synchronize multiple oscillators; a useful feature for more
integrated systems, where the timing of different modules
needs to be controlled. Our concept is general and we envi-
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small molecules4, synthetic gene networks22, or DNA PEN
toolbox23. We have also observed subtle changes in the re-
sponse of the network to short exposures to light at different
phases of the oscillation. These results indicate that we
might be able to study the dynamics of the network (robust-
ness, resilience) using photocleavage of Photo-I as a probe.
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ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website.
23. Gines, G.; Zadorin, A. S.; Galas, J. C.; Fujii, T.; Estevez-
Torres, A.; Rondelez, Y. Nat. Nanotech. 2017, 12, 351.
Synthesis data, kinetics, simulation info, flow experiment
details (PDF)
AUTHOR INFORMATION
Corresponding Author
*w.huck@science.ru.nl
Notes
The authors declare no competing financial interests.
ACKNOWLEDGMENT
We wish to thank W.M. Berkers for efforts in synthesis, S.
G. J. Postma and P.A. Korevaar for their comments and help
in the preparation of the manuscript. Our work is supported
by funding from the Dutch Ministry of Education, Culture
and Science (Gravity program 024.001.035).
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