Efficient phosphodiester cleaving nanozymes resulting from multivalency and local medium polarity control

Article abstract of DOI:10.1021/ja411969e

The self-organization of Zn(II) complexes on the surface of 1.6-nm diameter gold nanoparticles (nanozymes) allows the spontaneous formation of multiple bimetallic catalytic sites capable to promote the cleavage of a RNA model substrate. We show that by tu

Full text of DOI:10.1021/ja411969e

Communication
pubs.acs.org/JACS
Efficient Phosphodiester Cleaving Nanozymes Resulting from
Multivalency and Local Medium Polarity Control
Marta Diez-Castellnou, Fabrizio Mancin,* and Paolo Scrimin*
Department of Chemical Sciences, University of Padova, via Marzolo 1, 35131 Padova, Italy
S
* Supporting Information
remarkable cleavage efficiency of phosphate esters thanks to the
ABSTRACT: The self-organization of Zn(II) complexes
on the surface of 1.6-nm diameter gold nanoparticles
(nanozymes) allows the spontaneous formation of multi-
ple bimetallic catalytic sites capable to promote the
cleavage of a RNA model substrate. We show that by
tuning the structure of the nanoparticle-coating mono-
layer, it is possible to decrease the polarity of the reaction
site, and this in turn generates remarkable increments of
the cleavage efficiency.
cooperation between several identical active species (“nano-
zymes”).8 In this communication we report how by taking full
advantage of the modular structure of a nanozyme, which
allows the easy modification of the organic coating monolayer,
we were able to obtain a nanosystem capable of accelerating in
water the cleavage of a RNA model substrate (2-hydroxypropyl-
p-nitrophenyl phosphate, HPNP, Chart 1) more efficiently than
any other artificial hydrolytic agent so far reported in this
solvent.
Chart 1. (Top) HPNP Cleavage by Nanozyme Mechanism
and (Bottom) Metal Chelating Thiols Used in This Work,
Represented in Their Nanoparticle-Bound Form
he obtainment of synthetic agents capable of reproducing
Tthe reactivity of phosphate ester cleaving enzymes has
attracted considerable efforts over the years.1 Indeed, such
systems not only would be suitable for many important
applications in the biomedical field, but also represent a
stimulating intellectual challenge. In fact, nucleases and related
enzymes, often containing metal ions in their active sites,2 are
among the most efficient enzymes known, producing
accelerations up to 16 orders of magnitude of the background
reaction.3 Activity of different metal ions and simple
monometallic complexes in promoting the hydrolytic cleavage
of phosphate esters, mainly by Lewis acid catalysis, has been
known for decades, but the accelerations obtained are modest.4
The problem has been addressed over the years by rationally
designing metal complexes where more than one (usually two)
metal ions and/or appropriate organic groups are preorganized
to achieve optimal cooperation.1 However, even the most
effective synthetic agents produced in this way are far from
reaching enzymes’ activity. Somehow, it appears that there is an
intrinsic limit to the acceleration that can be achieved using
small molecule catalysts in water.5 On the other hand, the
investigations performed by Brown’s group reveal that when
the reaction is performed in low polarity solvents, which
reproduce the dielectric constant of enzymes active site,6
impressive accelerations may be reached.7 Under these
conditions, even poorly preorganized bimetallic complexes
produce rate accelerations of 12 orders of magnitude,
approaching those achieved by enzymes. These observations
suggest that key elements of nuclease catalysis may be not only
the precise preorganization of the functional groups participat-
ing in the reaction, but also the ability to place the negatively
charged transition state in a low polarity environment, where
the electrostatic stabilization exerted by the positively charged
metal ions is maximized.
Thiol 1 (Chart 1), bearing the 1,4,7-triazacyclononane metal
chelating moiety, was used previously to prepare efficient
nanoparticles for the cleavage of HPNP in the presence of
Zn(II).8d As in micellar catalysis, the cleavage reaction occurs at
the interface between the bulk aqueous solution and the
pseudophase formed by the nanoparticle coating monolayer.9 It
occurred to us that the structure of thiol 1 allows its easy
modification by the insertion of different diamine spacers
between the metal chelating and the surface anchoring
We previously showed that the assembly of Zn(II) complexes
on the surface of monolayer-passivated nanoparticles leads to
Received: August 15, 2013
Published: January 9, 2014
© 2014 American Chemical Society
1158
dx.doi.org/10.1021/ja411969e | J. Am. Chem. Soc. 2014, 136, 1158−1161

Journal of the American Chemical Society
Communication
portions, which should consent the tuning of the properties of
the nanoparticle-coating monolayer. With this in mind, we
prepared10 gold nanoparticles AuNp1−4 (1.6-nm diameter of
the metal core) coated by thiols 1−4 and investigated their
ability to promote HPNP cleavage in the presence of Zn(II)
ions.
Interestingly, modification of the spacer unit in thiols 1−4
produces remarkable effects on the nanoparticles reactivity as
highlighted by Figure 1, which reports the dependence of the
Table 1. Michaelis−Menten Parameters for HPNP Cleavage
in the Presence of AuNp1-4 and Zn(II) in Water at pH 7.5
a
and 40° C
b
b
c
d
e
np kcat (s−1
)
KM (mM) k2 (M−1 s−1
)
krel
kNBIC (s−1
)
1
2
3
4
0.036
0.212
0.019
0.196
0.58
0.40
0.38
0.30
62
437
50
1.8 × 105
1.1 × 106
9.5 × 104
9.8 × 105
3.0 × 10−5
1.1 × 10−4
6.8 × 10−5
3.0 × 10−4
638
a
b
Errors are within 5%. Normalized for the theoretical concentration
c
d
of bimetallic sites, i.e., [Zn(II)]/2. k2 = kcat/KM. krel = kcat/kuncat, kuncat
e
= 2 × 10−7 s−1, ref 8d. First order rate for the NBIC spontaneous
decarboxylation in the presence of AuNp1−4 and Zn(II) in water at
pH 7.5 and 25 °C, extrapolated at [NBIC] = 0. (Conditions: [NP, in
thiol units] = 2.0 × 10−5 M, [Zn(II)] = 2.0 × 10−5 M, [buffer] = 1.0 ×
10−2 M).
pseudophase formed by the nanoparticle coating monolayer,
where the reaction occurs. In line with Brown’s observations,7
the lower is the polarity of the monolayer pseudophase, the
higher is the nanozyme reactivity. This means that, in this
reaction, the stabilization of the transition state is higher than
that of the ground state as polarity decreases.11 A strong point
in support of such a hypothesis is the comparison of the
reactivity of AuNp2 and AuNp3 nanoparticles. Here the length
of the coating thiols is identical, with the only difference being
the presence of two ether oxygens instead of two methylenes in
the structure of thiol 3. As already stressed, such a difference,
which should bring about both a higher conformation flexibility
and a higher polarity of the nanoparticle coating monolayer, has
no effect on the nanoparticle ability to bind the substrate but
produces a 10-fold difference on the reactivity.
To get more clues on such a behavior we investigated the
temperature dependence of the reaction rate. Figure 2
summarizes the ΔH‡ and ΔS‡ values obtained from the Eyring
plots for the kcat values measured in the interval 30−52 °C.12
Even though the differences are small, as expected for rate
accelerations spanning 1 order of magnitude, Figure 2 appears
Figure 1. Rate of HPNP cleavage promoted by AuNp1−4 and Zn(II)
●
○
as a function of the concentration of HPNP ( , AuNp4; , AuNp2;
■
□
, AuNp1; , AuNp3). Conditions: [AuNp, in thiol units] = 2.0 ×
10−5 M, [Zn(II)] = 2.0 × 10−5 M, [buffer] = 1.0 × 10−2 M, pH = 7.5,
40 °C.
initial rate of HPNP cleavage (at a fixed AuNp-Zn(II)
concentration) as a function of the substrate concentration.
Inspection of the curves clearly reveals that the reactivity of the
nanoparticles coated with the longer alkyl-spaced thiols 2 and 4
is substantially higher than that of 1-coated nanoparticles.
Saturation profiles are followed by all nanoparticles diagnostic
of a mechanism involving the formation of a complex between
the nanosystem and the substrate before the cleavage reaction.
As previously reported, at least two Zn(II) ions are involved in
the substrate binding and cleavage,8a,d and a pseudoternary
complex is formed at the reaction site, which is likely placed at
the interface between the coating monolayer and the bulk
aqueous solvent. Accordingly, experiments performed at
different Zn(II)/nanoparticle ratios show sigmoidal reactivity
profiles, clearly diagnostic of the cooperation of two metal ions
in the reaction (see Supporting Information).
Fitting of the reactivity profiles reported in Figure 1 with the
Michaelis−Menten equation provides the kinetic parameters
reported in Table 1, which reveal several interesting aspects of
nanoparticles reactivity. First of all KM values are similar for all
nanoparticles. This indicates that elongation and modification
of the metal chelating thiols, while potentially introducing a
greater conformational flexibility, are not affecting substantially
the binding site preorganization. This is true even for the
nanoparticles coated with the highly flexible thiol 3 featuring an
oligo(oxyethylene) spacer.
The different activities of the four nanozymes hence arise
only from the different reactivity of the fully formed substrate-
catalyst complex. In other words, being the structure of the
reaction site similar for all nanoparticles, as witnessed by the
similar KM values, what differentiates the nanozymes is their
ability to stabilize the dianionic transition state of the reaction.
Such ability appears to arise from the properties of the
Figure 2. ΔH‡ (blue) and −TΔS‡ (red, T = 318 K) values obtained
from the Eyring plots of kcat values measured in the interval 25−50 °C
for AuNp1−4 coated nanoparticles and Zn(II). Conditions: [AuNp, in
thiol units] = 2.0 × 10−5 M, [Zn(II)] = 2.0 × 10−5 M, [buffer] = 1.0 ×
10−2 M, pH = 7.5.
1159
dx.doi.org/10.1021/ja411969e | J. Am. Chem. Soc. 2014, 136, 1158−1161

Journal of the American Chemical Society
Communication
to indicate that the larger reactivity of AuNp2 and AuNp4 has a
prevalent enthalpic origin, the activation entropy being quite
similar in all cases. These results suggest that in AuNp2 and
AuNp4 there is a stronger interaction between the transition
state and the metal complexes (and, consequently, an higher
stabilization), as one would expect in a low polarity
environment.
To independently assess the different monolayer polarity of
the nanoparticles investigated we used the well-known 6-
nitrobenzisoxazole-3-carboxylate (NBIC) probe.13 The rate of
the spontaneous decarboxylation of NBIC is very sensitive to
solvent polarity, and this reaction has been used to assess the
medium effect in several systems, including micelles, polymers
and macrocyclic hosts.13 We measured the rate of NBIC
decarboxylation in the presence of the different nanozymes and
the first order rate values, extrapolated at zero NBIC
concentration,14 are reported in Table 1. Remarkably, the
reaction rate in the presence of AuNp2 and AuNp4 is sensibly
larger than that measured in the presence of AuNp1 and
AuNp3. Moreover, the latter values are very similar to those
measured in buffer only, indicating that in these nanoparticles
the polarity at the monolayer-water interface is very similar to
that of bulk water.
Taken all together, the results here reported provide an
insightful picture of the potential of monolayer-coated
nanoparticles as enzyme mimetic systems. On one hand they
allow the self-assembly of the bimetallic catalytic site, while on
the other, the simple modification of the structure of the
coating thiol by the insertion of longer alkyl chains leads to the
obtainment of a microenvironment with a polarity lower than
that of bulk water. In such conditions the ability of the metal
ions to stabilize, likely by electrostatic interaction (coordina-
tion), the dianionic transition state formed during the HPNP
cleavage is increased, and consequently, the reaction is faster.
Indeed, to the best of our knowledge, kcat values for its cleavage
corresponding to t1/2 less than 20 s, like those we observe with
AuNp4, have never been reported previously for Zn(II)-based
catalysts working in water.
The reactivity data for the best performing bimetallic systems
so far reported and for the nanozymes here described are
summarized in Figure 3 (note that 25 °C rates for nanozymes
AuNp1 and AuNp4 are reported to allow the comparison with
the literature data). Richard and Morrow bimetallic complex 5
is based on the same triazacyclonane metal chelating unit we
used for our nanozymes.15 When its reactivity is compared with
that of AuNp1, it appears evident that the higher activity of the
nanoparticles arises substantially from higher substrate affinity.
As recently demonstrated by Prins, such a decrease in KM
values is due to the multivalency of the nanoparticles.8d,16 The
number of potential bimetallic binding sites is statistically
increased and this causes an apparent increase in binding
affinity.
Beside increased affinity, the intrinsic reactivity of the two
systems is similar indicating that in both the cases the optimal
(or the best possible) reactive site organization is reached and
that the reaction occurs in a similar environment. It should be
noted that such an optimally preorganized active site is reached
in the nanoparticle monolayer without the need of the bridging
oxygen present in 5. In methanol such structural element has a
detrimental effect on the catalyst reactivity. In fact, complex 7 is
37000 times more reactive than the equivalent one comprising
an alkoxy bridge.17 The bridge may decrease the net charge of
the catalyst (decreasing both Lewis acidity and transition state
Figure 3. Structure and reactivity parameters (at 25 °C) for different
HPNP cleaving bimetallic systems reported in literature or studied in
this work. But for catalyst 7, all the rest were studied in water.
stabilization) or reduce the flexibility of the complex preventing
its reorganization during the reaction.18 In water, the presence
of such a bridging alkoxy group is necessary to keep the two
metal ions in close proximity, as demonstrated by the fact that 7
becomes a rather poor catalyst in these conditions. It is,
however, still unclear whether this gain in organization of the
catalysts pays a reactivity cost also in this solvent.17 The fact
that the kcat value we here measured for AuNp1 is similar to
that of 5 indicates that the detrimental effect of the alkoxy
bridge may be smaller in water than in methanol.
Eventually, when the reactivity of AuNp4 is examined, the
advantage of nanozymes clearly emerges, as the decrease of
medium polarity is awarded by a 10-fold increase of kcat.
Remarkably, the reactivity parameters of these nanoparticles
are, although to not such a large extent, better also when
compared with Williams bimetallic complex 6, the current
benchmark for HPNP cleavage in water, where the reactivity
arises from highly efficient cooperation between metal ions and
H-bond donors.19 Admittedly, when the comparison is made
with Brown bimetallic complex 7 in methanol,7f as already
pointed out, the reactivity of AuNp4 is at least 3 orders of
magnitude lower. Interestingly, when Michaelis−Menten
parameters for 5 and 7 are compared, it appears quite evident
that moving the reaction from water to methanol affects more
reactivity (kcat) than binding (KM).20 The same occurs in the
nanoparticles series AuNp1−4, supporting the hypothesis that
the activity increase observed is related to a decrease of local
medium polarity at the reaction site.21 The huge difference
observed between kcat values of AuNp4 (in water) and catalyst
7 (in methanol) indicates that such a polarity decrease is quite
small, as it may be expected since the reaction is still occurring
at the monolayer/water interface, a still highly hydrated
region.22 One may speculate that an higher activity could be
obtained by moving the reaction site more deeply in the
nanoparticle coating monolayer.23
In conclusion, we have demonstrated that nanoparticles can
be used not only to easily assemble active units capable to
cooperate in a reaction taking advantage of their multivalency,
1160
dx.doi.org/10.1021/ja411969e | J. Am. Chem. Soc. 2014, 136, 1158−1161

Journal of the American Chemical Society
Communication
(8) (a) Manea, F.; Houillon, F. B.; Pasquato, L.; Scrimin, P. Angew.
Chem., Int. Ed. 2004, 43, 6165−6169. (b) Bonomi, R.; Selvestrel, F.;
Lombardo, V.; Sissi, C.; Polizzi, S.; Mancin, F.; Tonellato, U.; Scrimin,
P. J. Am. Chem. Soc. 2008, 130, 15744−15745. (c) Bonomi, R.;
Scrimin, P.; Mancin, F. Org. Biomol. Chem. 2010, 8, 2622−2626.
(d) Zaupa, G.; Mora, C.; Bonomi, R.; Prins, L. J.; Scrimin, P. Chem.
Eur. J. 2011, 17, 4879−4889.
but also to modify the local medium in order to tune the
reactivity. In this way, nanoparticles offer the possibility to
create artificial “binding sites”, something long sought in
micellar catalysis24 and rarely found because of the very fast
monomer exchange occurring in such aggregates. An analogous
medium effect has been also observed with synzymes, metal-
based HPNP-cleaving polymers, but with far lower efficiency
due probably to the lack of cooperativity between metal ions
shown by these systems.25 The fact that the rate accelerations
here observed in water, although very high, are lower than
those reported by Brown while working in nonaqueous
solvents7 indicates that there is still room for improvement of
nanozymes reactivity.
(9) Zaramella, D.; Scrimin, P.; Prins, L. J. J. Am. Chem. Soc. 2012,
134, 8396−8399.
(10) Manea, F.; Bindoli, C.; Polizzi, S.; Lay, L.; Scrimin, P. Langmuir
2008, 24, 4120−4124.
(11) For an interesting related approach, see: (a) Breslow, R.;
Groves, K.; Maye, M. U. Org. Lett. 1999, 1, 117−120. (b) Breslow, R.;
Groves, K.; Mayer, M. U. J. Am. Chem. Soc. 2002, 124, 3622−3635.
(12) Such a limited temperature interval is the result of a
compromise between a reasonable rate of the reaction and the
stability of the nanoparticle.
ASSOCIATED CONTENT
* Supporting Information
■
S
(13) Ferris, D. C.; Drago, R. S. J. Am. Chem. Soc. 1994, 116, 7509−
7514.
Synthesis, characterization of 1−4 and AuNp1−4, and
additional kinetic experiments. This material is available free
of charge via the Internet at http://pubs.acs.org.
(14) This is required to remove the contribution to the
decarboxylation rate by unbound NBIC. As shown in the Supporting
Information, as the concentration of NBIC increases its decarbox-
ylation rate converges to that observed in buffered water.
(15) (a) Iranzo, O.; Kovalevsky, A. Y.; Morrow, J. R.; Richard, J. P. J.
Am. Chem. Soc. 2003, 125, 1988−1993. See also: (b) Morrow, J. R.;
Amyes, T. L.; Richard, J. P. Acc. Chem. Res. 2008, 41, 539−548.
(16) Zaupa, G.; Scrimin, P.; Prins, L. J. J. Am. Chem. Soc. 2008, 130,
5699−5709.
(17) Mohamed, M. F.; Neverov, A. A.; Brown, R. S. Inorg. Chem.
2009, 48, 11425−11433.
(18) Maxwell, C. I.; Mosey, N. J.; Brown, R. S. J. Am. Chem. Soc.
2013, 135, 17209−17222.
(19) Feng, G. Q.; Natale, D.; Prabaharan, R.; Mareque-Rivas, J. C.;
Williams, N. H. Angew. Chem., Int. Ed. 2006, 45, 7056−7059.
(20) This consideration is unbiased even by keeping into
consideration the negative effect of the alkoxy bridge. Second order
rate constant for HPNP cleavage by the di-Zn(II) complex of 2-
propoxy analogue of 7 in CH3OH at 25 °C is 7.6 M−1 s−1; see ref 17.
(21) But for small effects due to mobility changes of the monolayer.
(22) We have unsuccessfully tried to carry out the reaction with
AuNp2 in methanol for comparison. Regrettably, upon addition of the
substrate, substantial precipitation occurs, rendering the experiment
unfeasible.
AUTHOR INFORMATION
Corresponding Author
paolo.scrimin@unipd.it; fabrizio.mancin@unipd.it
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
Funded by the EU-FP7 Marie Curie-ITN Project PhosChem-
Rec (M.D.C. and P.S., Grant 238679) and by the ERC Starting
Grants Project MOSAIC (F.M., Grant 259014). The authors
̀
thank Prof. Raimondo Germani (Universita di Perugia) for a
generous gift of NBIC. M.D.C. thanks the PhosChemRec
Project for a fellowship.
REFERENCES
■
(1) Selected reviews (a) Mancin, F.; Scrimin, P.; Tecilla, P. Chem.
Commun. 2012, 48, 5545−5559. (b) Aiba, Y.; Sumaoka, J.; Komiyama,
M. Chem. Soc. Rev. 2011, 40, 5657−5668. (c) Lonnberg, H. Org.
Biomol. Chem. 2011, 9, 1687−1703.
(2) (a) Dupureur, C. M. Metallomics 2010, 2, 609−620. (b) Cowan,
J. A. Chem. Rev. 1998, 98, 1067−1087.
(23) It has been suggested that the decrease of the polarity of the
monolayer caused by the substrate may also affect the reaction
pathway. See: Pengo, P.; Baltzer, L.; Pasquato, L.; Scrimin, P. Angew.
Chem., Int. Ed. 2007, 46, 400−404.
(3) (a) Jarvinen, P.; Oivanen, M.; Lonnberg, H. J. Org. Chem. 1991,
56, 5396−5401. (b) Schroeder, G. K.; Lad, C.; Wyman, P.; Williams,
N. H.; Wolfenden, R. Proc. Natl. Acad. Sci. U. S. A. 2006, 103, 4052−
4055.
(24) Mancin, F.; Scrimin, P.; Tecilla, P.; Tonellato, U. Coord. Chem.
Rev. 2009, 253, 2150−2165.
(25) Avenier, F.; Hollfelder, F. Chem.Eur. J. 2009, 15, 12371−
(4) Korhonen, H.; Mikkola, S.; Williams, N. H. Chem.Eur. J. 2012,
12380.
18, 659−670.
(5) Morrow, J. R. Comments Inorg. Chem. 2008, 29, 169−188.
(6) (a) Cleland, W. W.; Frey, P. A.; Gerlt, J. A. J. Biol. Chem. 1998,
273, 25529−25532. (b) Czerwinski, R. M.; Harris, T. K.; Massiah, M.
A.; Mildvan, A. S.; Whitman, C. P. Biochemistry 2001, 40, 1984−1995.
(c) Cachau, R. E.; Garcia-Moreno, E. B. J. Mol. Biol. 1996, 255, 340−
343.
(7) (a) Mohamed, M. F.; Sanchez-Lombardo, I.; Neverov, A. A.;
Brown, R. S. Org. Biomol. Chem. 2013, 10, 631−639. (b) Brown, R. S.;
Lu, Z. L.; Liu, C. T.; Tsang, W. Y.; Edwards, D. R.; Neverov, A. A. J.
Phys. Org. Chem. 2010, 23, 1−15. (c) Neverov, A. A.; Liu, C. T.; Bunn,
S. E.; Edwards, D.; White, C. J.; Melnychuk, S. A.; Brown, R. S. J. Am.
Chem. Soc. 2008, 130, 6639−6649. (d) Liu, C. T.; Neverov, A. A.;
Brown, R. S. J. Am. Chem. Soc. 2008, 130, 13870−13872. (e) Liu, C.
T.; Neverov, A. A.; Brown, R. S. J. Am. Chem. Soc. 2008, 130, 16711−
16720. (f) Bunn, S. E.; Liu, C. T.; Lu, Z.-L.; Neverov, A. A.; Brown, R.
S. J. Am. Chem. Soc. 2007, 129, 16238−16248. (g) Neverov, A. A.; Lu,
Z.-L.; Maxwell, C. I.; Mohamed, M. F.; White, C. J.; Tsang, J. S. W.;
Brown, R. S. J. Am. Chem. Soc. 2006, 128, 16398−16405.
1161
dx.doi.org/10.1021/ja411969e | J. Am. Chem. Soc. 2014, 136, 1158−1161