Original Citation:
Photoswitchable catalysis by a nanozyme mediated by a lightsensitive cofactor
American Chemical Society
Publisher:
Published version:
DOI:
Terms of use:
Open Access
(Article begins on next page)
This article is made available under terms and conditions applicable to Open Access Guidelines, as
described at http://www.unipd.it/download/file/fid/55401 (Italian only)
Availability:
This version is available at: 11577/3227841 since: 2017-05-16T08:57:47Z
10.1021/jacs.6b12932
Università degli Studi di Padova
Padua Research Archive - Institutional Repository
Photoswitchable catalysis by a nanozyme mediated by a light-
sensitive cofactor
Simona Neri, Sergio Garcia Martin, Cristian Pezzato,† Leonard J. Prins*
Department of Chemical Sciences, University of Padova, Padova, Italy
Supporting Information Available
ABSTRACT:
The activity of a gold nanoparticle-based catalyst can be reversi-
bly up- and down-regulated by light. Light is used to switch a
small molecule between cis- and trans-isomers, which inhibit the
catalytic activity of the nanoparticles to different extents. The sys-
tem is functional in aqueous buffer, which paves the way for inte-
grating the system in biological networks.
The visual phototransduction process is a beautiful example of
the functional power of complex chemical networks.
1
The process
is initiated by the photon-triggered cis-trans isomerization of the
co-factor retinal, which activates a cascade of catalytic events
eventually leading to an electrical output signal for processing by
the nervous system. In the quest towards responsive synthetic
chemical systems of increasing complexity the availability of cat-
alysts that can be regulated using an external trigger is an essential
prerequisite.
2,3
The use of light is particularly attractive because it
can be delivered very efficiently and with high temporal and spa-
tial precision.
4
Although most attention has been focused on mo-
lecular catalysts equipped with light-sensitive molecular switch-
es,
5-10
there is an increasing number of examples of catalytic sys-
tems of higher complexity that can be regulated with light.
11,12
In
particular, nanoparticles functionalized with azobenzene-moieties
have turned out to be very effective, which is mainly caused by
the possibility to install innovative regulatory mechanism (e.g.
light-induced aggregation-dissociation) that are hard to achieve
with molecular catalysts.
13-15
It has been shown recently that na-
noparticle aggregation can also be controlled using an external
light-sensitive molecule, which liberates a proton upon light-
induced isomerization and in that way affects the aggregation
state of the nanoparticles.
16,17
Here, we apply a different strategy
to control the activity of catalytic nanoparticles relying on the use
of a small light-sensitive co-factor which inhibits catalytic activity
because it competes with the substrate for binding to the catalytic
monolayer (Figure 1). This mechanism, together with the fact
that the system functions in water, makes for a close analogy with
the visual phototransduction process and opens the way to light-
regulated hybrid networks composed of nanoparticles and en-
zymes.
Au NP 1 are gold nanoparticles (d = 1.6 ± 0.4 nm) passivated
with a monolayer of C
9
-thiols terminating with a 1,4,7-
triazacyclononane (TACN)·Zn
2+
head group (Figure 1).
18
Previ-
ously, we have shown that Au NP 1 and analogues catalyse very
efficiently the transphosphorylation of 2-hydroxypropyl-4-
nitrophenylphosphate (HPNPP), which is a model substrate for
RNA-hydrolysis.
19,20
The system has been referred to as a
nanozyme, because of its many analogies to enzymes: coopera-
tivity between TACN·Zn
2+
-complexes, Michaelis-Menten reac-
tion kinetics, and down-regulation of catalysis by inhibitors that
compete with the substrate for binding to Au NP 1.
21-24
In particu-
lar, this latter aspect stimulated us to exploit the reversible interac-
tion between a competitor and Au NP 1 as a tool to regulate cata-
lytic activity in a similar way as what happens in the visual photo-
transduction process. This would require the use of a small light-
sensitive molecule that would change the affinity for Au NP 1
upon photoisomerization. Our attention was drawn to commer-
cially available 4-(phenylazo)benzoic acid (2), because it com-
bines a photoresponsive azobenzene and a carboxylic acid group,
which is negatively charged at pH 7.0. UV-vis spectroscopy con-
firmed that also in the presence of Au NP 1 ([TACN·Zn
2+
] = 20
µM, [HEPES] = 10 mM, pH = 7.0) 2 can be reversibly switched
between two photostationary states (trans:cis = 35:65 after λ =
365 nm for 50 minutes, trans:cis = 77:23 after λ = 465 nm for 10
minutes) (Supporting Information). Throughout the manuscript,
cis-2 and trans-2 refer to the photostationary states enriched in
cis-2 (65%) and trans-2 (77%), respectively. Next, the relative
affinities of both cis- and trans-2 for Au NP 1 were determined by
means of a competition experiment with the fluorescent probe
6,8-dihydroxy-1,3-pyrenedisulfonic acid (3) (Figure 2a). In these
experiments, cis-2 and trans-2 were titrated separately to a buff-
ered aqueous solution of Au NP 1 ([TACN·Zn
2+
] = 20 µM) and 3
(8 µM). At this concentration nearly all 3 was bound to Au NP 1
resulting in a nearly complete quenching of its fluorescence by the
gold core.
25
The addition of increasing amounts of 2 resulted in
the displacement of 3 from the monolayer surface which could be
Figure 1. Light-induced cis-trans isomerization of 2
changes its
affinity for Au NP 1
, which affects the transphosphorylation rate
of HPNPP.
detected by an increase in fluorescence intensity. The resulting
displacement curves clearly showed that trans-2 has a higher af-
finity (∼2.2 times) for Au NP 1 compared to cis-2, which is a fun-
damental prerequisite for the use of 2 as a photoresponsive cofac-
tor (Figure 2b).
26
The lower affinity of cis-2 is ascribed to the in-
crease in polarity of azobenzene upon trans-cis isomerization,
which reduces favorable hydrophobic interactions with the apolar
part of the monolayer.
13,27
Importantly, the complete reversibility
of the interaction was demonstrated by measuring the fluores-
cence intensity after repetitive trans-cis and cis-trans isomeriza-
tions (Figure 2c). It is noted that the quantitative interpretation of
these measurements required a correction for the photobleaching
of probe 3 upon irradiation at 465 nm (Supporting Information).
Additional direct evidence that the cis- and trans-isomers of 2
displace a surface-bound molecule to a different extent was ob-
tained from ultrafiltration experiments followed by LC-MS meas-
urements.
28
In these experiments equal amounts of either cis- or
trans-2 (100 µM) were added to a solution of Au NP 1 saturated
with probe 4 (4.8 µM, Figure 2d) after which ultrafiltration using
a 10 kDa MW cutoff PES-membrane was used to separate free
from surface-bound molecules. LC-MS measurements of the fil-
trate showed a 2 times higher concentration of 4 in the sample to
which trans-2 was added compared to the cis-2 sample, which is
in agreement with the data obtained from fluorescence measure-
ments (inset of Figure 2d).
At this stage we also verified through a series of analysis that
Au NP 1 was not subject to structural alterations as a results of
extensive irradiation.
1
H NMR and Cu
2+
-titrations confirmed that
5 irradiation cycles did not affect the structural and functional
properties of the organic monolayer, whereas TEM, DLS and UV-
vis measurements confirmed the integrity of the inorganic core
(Supporting Information).
We then proceeded with a study of the inhibitory effect of the
cis- and trans-isomers of 2 on the catalytic activity of Au NP 1 in
the transphosphorylation of HPNPP (Figure 1). The reaction can
be conveniently followed by UV-vis spectroscopy by measuring
the increase in absorbance at 390 nm (which corresponds to the
isosbestic point of cis- and trans-2) originating from the liberated
p-nitrophenolate anion. It is noted that the Au nanoparticle metal
component itself is not involved in the reaction. During the course
of numerous studies using Au NP 1 or analogues, the reduction of
p-nitrophenol to p-nitroaniline, which is known to be catalyzed by
Au metal nanoparticles,
29
has never been observed.
19,20
Inhibition
studies were performed by measuring the initial reaction rate at
different concentrations of either cis- or trans-2. Comparison of
the inhibition curves showed an enhanced inhibitory capacity of
trans-2 compared to cis-2, which is in line with the results of the
displacement experiments (Figure 3a).
Figure 2. a) Schematic representation of the competition experiments between fluorophore 3 and cis- and trans-2 for binding to Au NP 1
.
b) Increase in fluorescence intensity as a function of the concentration of 2. To permit comparison, the fluorescence intensity of trans-2
was
corrected for the intrinsic difference in fluorescence intensity of 3 in the presence of trans- and cis-2. c) Fluorescence intensity after repeti-
tive irradiations (365 nm for 50 minutes, 465 nm for 10 minutes) of a solution containing Au NP 1, 2 and 3.
The intensity is corrected for
bleaching of 3 (see Supporting Information). d) Peak area of probe 4
(see Supporting Information) in the chromatograms of the dialysate
after ultrafiltration of a solution containing Au NP 1, probe 4, and either trans- and cis-2. All experimental conditions are given in the Sup-
porting Information.
In particular between 5 and 20 µM of 2 a significant difference
was observed. From within this range, a 20 µM concentration of 2
was chosen, because the lower reaction rates at higher inhibitor
concentrations would increase the available time frame for detect-
ing changes in activity upon irradiation. In an initial experiment
we followed the course of the reaction after adding HPNPP to a
buffered solution containing Au NP 1 and cis-2. After 7 minutes,
the cuvette was irradiated for 10 minutes at 465 nm after which
the measurement was continued together with a sample that was
non irradiated. Whereas prior to irradiation both rates were obvi-
ously the same, after irradiation a clear difference was observed
with a lower activity for the irradiated sample. This indicated that
the cis-trans isomerization of 2 resulted in a down-regulation of
Au NP 1 (Figure 3b). Objectively, the observed difference in rate
is not large, but it is in line with what can be expected based on
the relative affinities of cis- and trans-2 for Au NP 1. It is worth
reminding that 2 itself has a relatively low affinity for Au NP 1
and, also importantly, that the photostationary states are not very
well resolved. The fact that the system is any way able to respond
to the isomerization of 2 illustrates the sensitivity of this approach
to regulate the catalytic activity of Au NP 1 with light. The same
experiment performed in the absence of the inhibitor 2 obviously
resulted in higher rates, but, importantly, irradiation of the sample
had in this case no effect on the reaction rate (Supporting Infor-
mation). Yet, although promising, this procedure proved not very
suitable for multiple up- and down-regulation cycles. This was
mainly caused by the accumulation of the cleavage product p-
nitrophenolate in the system, which strongly absorbs in the same
region as azobenzene 2. As a result, we observed a strong reduc-
tion in the switching efficiency of 2 as the reaction proceeded. For
this reason, we performed the experiments in a different way by
adding the substrate HPNPP each time after isomerization of 2.
Thus, a large stock solution of Au NP 1 (20 µM) and 2 (20 µM) in
HEPES (10 mM, pH = 7.0) was prepared and subjected to irradia-
tion cycles to induce isomerization. After each irradiation a sam-
ple was transferred to a cuvette, HPNPP (100 µM) was added and
the increase in absorbance at 390 nm was measured. This had also
a second advantage that the initial rates could be quantitatively
compared between different cycles. In total, 3 cycles were per-
formed and the relative initial rates clearly demonstrated that the
catalytic activity of the system can be reversibly up- and down-
regulated using light (Figure 3c). Isomerization to the trans-
isomer in all cases resulted in an (expected) decrease in activity,
which was restored upon isomerization to the cis-isomer. In the
absence of 2 reversible up- and down-regulation was never ob-
served (Supporting Information). The slight decrease in perfor-
mance over multiple cycles is a side effect originating from a deg-
radation of the HEPES-buffer upon irradiation (Supporting Infor-
mation).
30,31
With that respect, we want to stress that this is one of
the few examples of a light-modulated Au NP system that is func-
tional in aqueous buffer
17
and thus marks a significant step for-
ward towards the integration of such systems in biological net-
works.
In conclusion, we have shown that a small light-sensitive mole-
cule can act as a co-factor to reversibly regulate the catalytic ac-
tivity of a nanosystem. Similar to what happens in the visual pho-
totransduction process, light irradiation causes a structural change
in the cofactor which affects the affinity for the catalytic site. Evi-
dently, the efficiently of the presented system is still less com-
pared to natural systems and also compared to previously reported
nanoparticle-based catalysts that operate in organic solvents.
However, the fact that the light-sensitive molecule interacts with
the nanoparticle through noncovalent interactions in water repre-
sents a significant step forward. The use of small, easily accessi-
ble molecule significantly facilitates optimization compared to
related systems in the literature in which the light-sensitive unit is
covalently attached to the monolayer. In addition, we have shown
that light can be used to displace surface bound molecules, which
is of interest for the development of innovative delivery systems
and materials.
ASSOCIATED CONTENT
Supporting Information
Materials and instrumentation, experimental protocols, control
experiments as mentioned in the manuscript. The Supporting In-
formation is available free of charge on the ACS Publications
website.
AUTHOR INFORMATION
Corresponding Author
leonard.prins@unipd.it
Notes
The authors declare no competing financial interests.
Present Address
† Department of Chemistry, Northwestern University, 2145 Sher-
idan Road, Evanston, Illinois 60208-3113, USA
ACKNOWLEDGMENT
Financial support from Marie Curie ITN READ (289723) and
COST Action CM1304 is acknowledged.
Figure 3. a) Plot of the initial rate (normalized on the rate for [2] = 0 µM) of the transphosphorylation of HPNPP as a function of the co
n-
centrations of cis- and trans-2. b)
Increase in absorbance as a function of time for samples that were (blue) and were not (red) irradiated at
465 nm for 10 minutes after 7 minutes of reaction. c) Plot
of the initial rates (normalized on the initial rate of the first kinetics) for several
irradiation cycles. Protocols and experimental conditions are given in the Supporting Information.
REFERENCES
(1) Ebrey, T.; Koutalos, Y., Prog. Retin. Eye Res. 2001, 20, 49-94.
(2) Blanco, V.; Leigh, D.A.; Marco, V. Chem. Soc.Rev. 2015, 44,
5341-5370.
(3) a) Wiester, M. J.; Ulmann, P. A.; Mirkin, C. A., Angew. Chem. Int.
Ed. 2011, 50, 114-137. b) Raynal, M.; Ballester, P.; Vidal-Ferran, A.; van
Leeuwen, P., Chem. Soc. Rev. 2014, 43, 1734-1787.
(4) Stoll, R. S.; Hecht, S., Angew. Chem. Int. Ed. 2010, 49, 5054-5075.
(5) Ueno, A.; Takahashi, K.; Osa, T., J. Chem. Soc., Chem. Commun.
1980, 837-838.
(6) Wurthner, F.; Rebek, J., Angew. Chem. Int. Ed. 1995, 34, 446-448.
(7) Sugimoto, H.; Kimura, T.; Inoue, S., J. Am. Chem. Soc. 1999, 121,
2325-2326.
(8) Sud, D.; Norsten, T. B.; Branda, N. R., Angew. Chem. Int. Ed.
2005, 44, 2019-2021.
(9) Peters, M. V.; Stoll, R. S.; Kuhn, A.; Hecht, S., Angew. Chem. Int.
Ed. 2008, 47, 5968-5972.
(10) Stoll, R. S.; Peters, M. V.; Kuhn, A.; Heiles, S.; Goddard, R.; Buhl,
M.; Thiele, C. M.; Hecht, S., J. Am. Chem. Soc. 2009, 131, 357-367.
(11) Niazov, T.; Shlyahovsky, B.; Willner, I., J. Am. Chem. Soc. 2007,
129, 6374-6375.
(12) Klajn, R.; Stoddart, J. F.; Grzybowski, B. A., Chem. Soc. Rev.
2010, 39, 2203-2237.
(13) Klajn, R.; Bishop, K. J. M.; Grzybowski, B. A., Proc. Natl. Acad.
Sci. 2007, 104, 10305-10309.
(14) Wei, Y. H.; Han, S. B.; Kim, J.; Soh, S. L.; Grzybowski, B. A., J.
Am. Chem. Soc. 2010, 132, 11018-11020.
(15) Zhao, H.; Sen, S.; Udayabhaskararao, T.; Sawczyk, M.; Kucanda,
K.; Manna, D.; Kundu, P. K.; Lee, J. W.; Kral, P.; Klajn, R., Nat.
Nanotechnol. 2016, 11, 82-88.
(16) Kundu, P. K.; Samanta, D.; Leizrowice, R.; Margulis, B.; Zhao, H.;
Borner, M.; Udayabhaskararao, T.; Manna, D.; Klajn, R., Nat. Chem.
2015, 7, 646-652.
(17) Samanta, D.; Klajn, R., Adv. Opt. Mater. 2016, 4, 1373-1377.
(18) Pieters, G.; Cazzolaro, A.; Bonomi, R.; Prins, L. J., Chem.
Commun. 2012, 48, 1916-1918.
(19) Manea, F.; Houillon, F. B.; Pasquato, L.; Scrimin, P., Angew.
Chem. Int. Ed. 2004, 43, 6165-6169.
(20) Zaupa, G.; Mora, C.; Bonomi, R.; Prins, L. J.; Scrimin, P., Chem.
Eur. J. 2011, 17, 4879-4889.
(21) Prins, L. J., Acc. Chem. Res. 2015, 48, 1920-1928.
(22) Wei, H.; Wang, E. K., Chem. Soc. Rev. 2013, 42, 6060-6093.
(23) Wang, X.; Guo, W.; Hu, Y.; Wu, J.; Wei, H. Nanozymes: Next
Wave of Artificial Enzymes. 2016, Springer-Verlag GmbH Berlin Heidel-
berg.
(24) Lin, Y. H.; Ren, J. S.; Qu, X. G., Acc. Chem. Res. 2014, 47, 1097-
1105.
(25) Sapsford, K. E.; Berti, L.; Medintz, I. L., Angew. Chem. Int. Ed.
2006, 45, 4562-4588.
(26) The difference in affinity is reflected by the 2.2-fold higher concen-
tration of cis-2 (compared to trans-2) required to displace the same
amount of 3.
(27) Pieters, G.; Pezzato, C.; Prins, L. J., Langmuir 2013, 29, 7180-
7185.
(28) Maiti, S.; Pezzato, C.; Martin, S. G.; Prins, L. J., J. Am. Chem. Soc.
2014, 136, 11288-11291.
(29) Haratu, M., Cattech, 2002, 6, 102-115.
(30) Zigler, J. S.; Lepezuniga, J. L.; Vistica, B.; Gery, I., In Vitro Cell.
Dev. Biol. 1985, 21, 282-287.
(31) Regrettably, the catalytic activity of Au NP 1 is completely inhibit-
ed by phosphate buffers such as PBS.
![Figure 3. a) Plot of the initial rate (normalized on the rate for [2] = 0 µM) of the transphosphorylation of HPNPP as a function of the concentrations of cis- and trans-2. b) Increase in absorbance as a function of time for samples that were (blue) and were not (red) irradiate t 465 nm for 10 minutes after 7 minutes of reaction. c) Plot of the initial rates (normalized on the initial rate of the first kinetics) for several irradiation cycles. Protocols and experimental conditions are given in the Supporting Information.](/figures/figure-3-a-plot-of-the-initial-rate-normalized-on-the-rate-3htcpa7h.webp)