Functionalized graphene oxide as a nanocatalyst in dephosphorylation reactions: Pursuing artificial enzymes

Article abstract of DOI:10.1039/c4cc03262d

The present study reports for the first time the use of a thiol-functionalized graphene oxide nanocatalyst with impressive activity (>10⁵-fold) in dephosphorylation reactions. The innovative and recyclable nanocatalyst has potential in designing artificial enzymes with targeted multifunctionalities and in detoxification of organophosphorus agents.

Full text of DOI:10.1039/c4cc03262d

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Functionalized graphene oxide as a nanocatalyst
in dephosphorylation reactions: pursuing artificial
enzymes†
Cite this: Chem. Commun., 2014,
50, 9891
Received 1st May 2014,
Accepted 8th July 2014
Elisa S. Orth,* Jessica E. S. Fonsaca, Thomas Golin Almeida, Sergio H. Domingues,
´
Jose G. L. Ferreira and Aldo J. G. Zarbin
´
DOI: 10.1039/c4cc03262d
www.rsc.org/chemcomm
The present study reports for the first time the use of a thiol- covalent functionalization of the oxygenated groups on GO can
functionalized graphene oxide nanocatalyst with impressive activity enhance the properties of materials and extend further applica-
(4105-fold) in dephosphorylation reactions. The innovative and tions.6 Recently, we reported the thiolation of GO, by anchoring
recyclable nanocatalyst has potential in designing artificial enzymes cysteamine (CA) by amide bonds on the carboxylate sites, under
with targeted multifunctionalities and in detoxification of organo- mild conditions.7 The modified GO, namely GOSH, was thor-
phosphorus agents.
oughly characterized and showed impressive size controllable
features in nanocomposites with silver nanoparticles. Insofar,
Dephosphorylation reactions are vital in biological systems, the freely available thiol groups on GOSH can efficiently act as
such as in regulatory and signaling processes, which are nucleophiles in many reactions, although GOSH cannot be dis-
mediated by extraordinarily efficient enzymes. Particularly, persed in any solvent, especially in water. In this context, we report
reversible phosphorylation of protein dictates many cell life GOSH as a nanocatalyst in heterogeneous dephosphorylation
functionalities. Herein, the thiol group, mainly involved in reactions, using the activated triester diethyl 2,4-dinitrophenyl
oxidative thiol–disulfide pathways,1 also participates in the phosphate (DEDNPP) as a model (Fig. 1).8 The proposed nano-
rarely occurring thiol phosphorylation reactions, important in catalyst can be readily separated and recycled consecutively,
cysteine-based phosphatases, e.g., bacterial antibiotic resis- maintaining its catalytic activity.
tance.2 Moreover, many aspects regarding mechanistic elucida-
Solid GOSH was added to a buffered solution at different pH
tion and detection of intermediates in protein phosphorylation values, kept under controlled temperature (20 1C) and constant
reactions are still not clear.3 Therefore, mimicking enzymatic magnetic stirring, and the reaction was initiated by adding an
active sites is strategic for designing novel artificial enzymes aliquot of DEDNPP. Periodically, stirring was interrupted, the
and potential enzymatic inhibitors for therapeutic purposes.
solid was decanted and the remaining solution was monitored
Carbon-based nanomaterials, such as graphene, are multi- by UV-Vis spectroscopy, following the appearance of the product
functional and versatile, hence, the complex enzymatic archi- 2,4-dinitrophenolate (DNP) at 400 nm. Rate constants (kobs) were
tecture can be more readily mimicked, e.g., by anchoring obtained from the pseudo-first order kinetic profiles and corre-
different functionalities on the backbone of these materials. lated with the mass of the catalyst used and the local concen-
The two dimensional sheets with sp2 carbons of graphene tration of reactive thiol groups on the GO surface, obtained by
comprise many optimum chemical, mechanical and electrical thermogravimetric data (TGA, ESI†). It should be noted that the
properties,4 thus prompting promising applications in nano- highly reproducible pseudo first-order profiles are consistent
electronics, biomedicine, sensors and artificial enzymes.5 with heterogeneous catalytic reactions where kinetic control
A common graphene precursor is graphene oxide (GO), which overcomes diffusion contributions.9,10 Post-reaction characteri-
is obtained by oxidizing graphite, leading to a highly oxygenated zation was carried out using TGA (under air), infrared (FTIR) and
surface, followed by chemical reduction, yielding graphene (or
the so-called reduced-graphene oxide, rGO). Hence, targeted
´
Department of Chemistry, Universidade Federal do Parana (UFPR), CP 19081,
CEP 81531-990, Curitiba, PR, Brazil. E-mail: elisaorth@ufpr.br;
Fax: +55 41-33613186; Tel: +55 41-33613176
† Electronic supplementary information (ESI) available: Potentiometric titration
data, kinetic profiles, TGA curves and Raman spectra. See DOI: 10.1039/
c4cc03262d
Fig. 1 Reaction of DEDNPP with the nanocatalyst GOSH.
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Table 1 Kinetic parameters obtained for reactions of DEDNPP with GOSH
Rate constant, kGOSH
Comparativec
Mꢀ1 sꢀ1 a
gꢀ1 sꢀ1 b
kGOSH/kH O
kGOSH/kCA
2
pH 8
pH 9
pH 10
1.73 ꢂ 10ꢀ2
2.33 ꢂ 10ꢀ2
3.17 ꢂ 10ꢀ2
1.46 ꢂ 10ꢀ2
1.96 ꢂ 10ꢀ2
2.67 ꢂ 10ꢀ2
3 ꢂ 105
2 ꢂ 105
9 ꢂ 104
14
8
4
a
Second order rate constant: ratio of the pseudo-first order rate constant
and the concentration of thiol groups 8.42 ꢂ 10ꢀ4 mol Lꢀ1 mgꢀ1 of solid
b
GOSH, calculated by TGA analysis. Considering the total mass of the
catalyst used. Compared using the second order rate constant (Mꢀ1 sꢀ1
)
c
for the reaction of DEDNPP with GOSH, H2O (spontaneous reaction)8
and CA.
Fig. 2 Proposed mechanism for the reaction of GOSH with DEDNPP.
thiolate active species can attack the phosphorus atom leading
to an unstable intermediate (Int., Fig. 2) and the leaving
group DNP.
Raman spectroscopy. A full description of the characterization
techniques is given in the ESI.† Table 1 presents the most
representative kinetic data obtained, along with comparatives
to assign the catalytic efficiency of the reaction proposed.
Results show impressive second order constants (kGOSH, Mꢀ1 sꢀ1),
which increase with higher pH. Indeed, the alkaline hydrolysis of
phosphate esters11 contributes to the overall reaction at higher
pH, although results show that GOSH solely acts proficiently,
regardless of the parallel reaction with OHꢀ, as observed by the
high rate constants at pH 8 (kGOSH). Moreover, mild reaction
conditions (e.g. lower pH) are desirable when promoting greener
catalytic reactions, as performed by our proficient biological
enzymes. It should be noted that the reaction conversion is
100%, although no selectivity can be accounted, since only one
substrate was evaluated. In order to infer catalytic activity, rate
constants in the presence of GOSH were compared to the
Potentiometric titration was also carried out to determine
the pKa value for the thiol group on GOSH (see ESI†). Results
indicate an unexpected low pKa of 6.46 ꢁ 0.02 for the thiol
moiety of GOSH, in contrast to CA with a pKa of 8.35.17
Although, this lower pKa is common in complex systems
(proteins),18 with neighboring groups that can stabilize the
thiolate, such as the alcohol groups available on GO surfaces.
Hence, over the pH range studied, thiolate species are present
predominantly on GOSH, consistent with the proposed mecha-
nism. As in similar nucleophilic reactions,8 the putative phos-
phorylated surface of GOSH (Int., Fig. 2) easily hydrolyses,
regenerating the thiol sites for successive reactions. Another
possible mechanism is the nucleophilic attack by thiolate on
the aromatic carbon (C–O cleavage). Nevertheless, this path
should not contribute significantly to the overall reaction,
since the absorbance variation profile shows early formation
of DNP, consistent with attack on the phosphorus atom.19 Indeed,
on-going mechanistic studies involving the reaction of CA with
DEDNPP by mass spectrometry confirm the presence of the
phosphorylated intermediate formed via the thiolate attack that
should analogously occur herein.20 It should be noted that under
the reaction conditions used, only the thiol groups of CA are
analogous spontaneous hydrolysis reaction (kH O) of DEDNPP,
2
giving rate enhancements (kGOSH/kH O) over 105-fold, which are
2
among the highest increments reported for heterogeneous
dephosphorylation reactions.12,13 Comparing the proposed het-
erogeneous catalysis with an analogous homogenous reaction,
DEDNPP was reacted solely with CA, i.e., the reactive thiol group
present on GOSH. Surprisingly, rate constants in the presence
of the nanocatalyst GOSH are 14-fold higher than with CA
(kGOSH/kCA). Normally, a homogenous reaction is more efficient
3þ
reactive, since the nitrogen atom is protonated (pKaNH ¼ 10:86),
than its analogous heterogeneous reaction,12 thus corroborating hence, unavailable as a nucleophile.17 Due to this higher pKa of the
the prominent catalytic efficiency of the proposed nanocatalyst. potentially nucleophilic amine site, CA reactivity increases more
Also, one would expect CA (with the same concentration as that upon increasing pH, compared to GOSH. Additionally, the for-
of thiol groups on GOSH) to promote similar enhancements. mation of a disulphide bond, which would inhibit the reactivity of
These results indicate synergistic effects on the multifunctional GOSH, was discarded since previous characterization indicates
nanocatalyst. We propose that the additional effects contributing freely available thiol groups.7 For CA, the disulphide formation
to the overall catalysis can be due to attractive interactions of does not seem to contribute significantly during the reaction time
DEDNPP by the nanocatalyst, but further studies are needed to followed.20
corroborate this proposition. The substrate could adsorb on the
In order to infer recycling features, GOSH was recovered by
GO domains, common for carbon derived materials14,15 and this filtration and reused as a catalyst in new reactions with
process concentrates reactants, thus optimizing the catalysis, as DEDNPP (three cycles). Fig. 3 illustrates with photos how
in micellar catalysis.16 Comparing the rate constant per mass of recycling was carried out, along with rate constants for each
the catalyst (kGOSH, gꢀ1 ꢀ1) with similar studies is even more cycle evaluated.
s
impressive12 due to the low mass of the catalyst used (1–4 mg),
ideally desired in heterogeneous reactions.
Results confirm that the highly efficient nanocatalyst GOSH
maintains its catalytic properties over several reaction cycles,
Regarding the mechanism of the reaction, we propose that suggesting that its reactive groups remain intact. The slight
DEDNPP can adsorb on the GOSH surface and react with its decrease in reactivity upon recycling can be attributed to
functional groups, as shown in Fig. 2. The highly nucleophilic adsorbed species such as the phosphate inorganic product.
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and D0 (1607 cmꢀ1) bands.7 After recycling, these bands are
maintained and the intensity ratio between D and G bands
(ID/IG), associated with the defective nature of the material
(functionalized) is retained at B2.2, and no further major
change occurs on the GO surface. Also, the band at 2920 cmꢀ1
related to the CH2 asymmetric stretch of the thiol group was
detected after reaction.7 Therefore, post-reaction characteriza-
tion confirms the recycling features of GOSH, with no indication
of thiol leaching, which is expected since it is covalently attached
to the GO surface by highly stable amide bonds.
Fig. 3 Nanocatalyst recycling efficiency.
In conclusion, the present study reports for the first time the
use of GO-based materials with impressive catalytic activity in
dephosphorylation reactions. Additionally, the nanocatalyst
comprising reactive thiol groups, GOSH, can effectively be
recycled consecutively, without losing its catalytic activity
significantly. All characterization carried out confirmed that
the catalyst maintained its overall functionalities after reuse. A
mechanism involving nucleophilic attack by thiolate was
proposed, mimicking enzymatic reactions involving thiol-based
groups. Therefore, the nanocatalyst proposed has certainly inno-
vative features, particularly promising in designing artificial
enzymes, by exploring its multifunctionalities, e.g., coupling GOSH
with therapeutic agents, envisioning advances in genetic therapy.
In this sense, we believe that targeted functionalization of GO and
overall engineering of carbon nanomaterials broaden the field of
novel complex multifunctional catalysts which can combine
assembly characteristics and multiple catalyses (nucleophilic,
acid–base). It is noteworthy that these catalysts are also very
promising in the detoxification of chemical warfare and pesti-
cides from the phosphate ester family.21 There is great interest
in developing new and efficient methods to detoxify these
agents to eliminate stocks, treat population and contain
attacks. These concerns are evident by the 2013 Peace Nobel
Prize awarded to the Organisation for the Prohibition of
Chemical Weapons for its efforts in eliminating chemical
weapons.22 The nanocatalyst GOSH can be readily used for
detoxification purposes, since it effectively cleaved DEDNPP, a
substrate similar to known toxic agents (e.g. paraoxon). This
attribution is not unprecedented since it is common to evaluate
detoxification properties in model substrates. Finally, studies
that involve dephosphorylation processes by a clear-cut cataly-
sis are promising for both development of artificial enzymes
and detoxification of organophosphorus agents.
Although, even after the third cycle, GOSH is very effective:
2.6 ꢂ 105-fold increment, compared to the spontaneous reac-
tion, in contrast to 3 ꢂ 105-fold observed in the first cycle.
Overall, the nanocatalyst is easily recovered from reaction
medium and can be recycled consecutively, which is desired
in catalytic heterogeneous reactions.
After recovering the reused GOSH, the preservation of its
overall properties and functionalities was confirmed by various
characterization techniques: TGA, FTIR and Raman spectro-
scopy. FTIR spectra of GOSH taken before and after reaction
with DEDNPP are given in Fig. 4.
Results conclusively show typical bands for GOSH that are
preserved upon catalyst recycling: CQO stretching (1640 cmꢀ1),
N–H bending (3292 cmꢀ1), N–H stretching (1542 cmꢀ1), C–N
stretching (1427 cmꢀ1) and N–H wagging (686 cmꢀ1). Other
observed bands are attributed to GO domains.7 The weak band
due to S–H stretching (2569 cmꢀ1) is hardly distinguishable
after recycling GOSH, which is attributed to the predominance
of deprotonated thiolate (reaction pH 4 pKa). Even so, the
amide bond preservation confirms the thiol group on GOSH.
TGA analysis (ESI†) confirms the characteristic mass losses of
GOSH before and after recycling: (i) 120–230 1C, with B15%
mass loss due to oxygenated groups; (ii) 250–400 1C, with B25%
mass loss attributed to thiol groups attached to GO domains and
(iii) 400–550 1C, with B50% mass loss due to the GO carbon
backbone. Hence, the thiol moieties remain intact after reaction,
reiterating that the proposed recycling is legitimate.
Lastly, Raman spectra were obtained mainly to confirm the GO-
like nature. Indeed, results show (ESI†) intense bands, characteristic
of this kind of carbonaceous material: D (1350 cmꢀ1), G (1580 cmꢀ1
)
The authors acknowledge the financial support from CNPq,
˜
´
˜
CAPES, Fundaçao Araucaria, NENNAM (PRONEX Fundaçao
´
Araucaria/CNPq) and the National Institute of Science and
Technology of Carbon Nanomaterials (INCT-Nanocarbono).
Notes and references
1 C. E. Paulsen and K. S. Carroll, Chem. Rev., 2013, 113, 4633–4679.
2 A. Salmeen and D. Barford, Antioxid. Redox Signaling, 2005, 7, 560–577.
3 P. Cohen, Nat. Cell Biol., 2002, 4, E127–E130.
4 V. Singh, D. Joung, L. Zhai, S. Das, S. I. Khondaker and S. Seal, Prog.
Mater. Sci., 2011, 56, 1178–1271.
5 H. J. Jiang, Small, 2011, 7, 2413–2427.
6 T. Kuila, S. Bose, A. K. Mishra, P. Khanra, N. H. Kim and J. H. Lee,
Prog. Mater. Sci., 2012, 57, 1061–1105.
Fig. 4 FTIR spectra of GOSH before and after reaction with DEDNPP.
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7 E. S. Orth, J. E. S. Fonsaca, S. H. Domingues, H. Mehl, M. M. Oliveira 15 F. M. Koehler, A. Jacobsen, K. Ensslin, C. Stampfer and W. J. Stark,
and A. J. G. Zarbin, Carbon, 2013, 61, 543–550. Small, 2010, 6, 1125–1130.
8 E. S. Orth, E. H. Wanderlind, M. Medeiros, P. S. M. Oliveira, B. G. 16 T. Dwars, E. Paetzold and G. Oehme, Angew. Chem., Int. Ed., 2005,
Vaz, M. N. Eberlin, A. J. Kirby and F. Nome, J. Org. Chem., 2011, 76,
8003–8008.
9 S. Sawadjoon, A. Lundstedt and J. S. M. Samec, ACS Catal., 2013, 3,
635–642.
10 J. A. Widegren and R. G. Finke, J. Mol. Catal. A: Chem., 2003, 198, 317–341.
11 W. W. Cleland and A. C. Hengge, Chem. Rev., 2006, 106, 325–3278.
12 Y. C. Chang and D. H. Chen, React. Funct. Polym., 2009, 69, 601–605.
13 V. Chandrasekhar, A. Athimoolam, S. G. Srivatsan, P. S. Sundaram,
44, 7174–7199.
17 H. Fleischer, Y. Dienes, B. Mathiasch, V. Schmitt and D. Schollmeyer,
Inorg. Chem., 2005, 44, 8087–8096.
18 S. Vohnik, C. Hanson, R. Tuma, J. A. Fuchs, C. Woodward and
G. J. Thomas, Protein Sci., 1998, 7, 193–200.
19 E. S. Orth, P. L. F. da Silva, R. S. Mello, C. A. Bunton,
H. M. S. Milagre, M. N. Eberlin, H. D. Fiedler and F. Nome, J. Org.
Chem., 2009, 74, 5011–5016.
S. Verma, A. Steiner, S. Zacchini and R. Butcher, Inorg. Chem., 2002, 20 E. S. Orth, T. A. Golin, R. C. Campos, A. J. G. Zarbin, 2013,
41, 5162–5173. Unpublished work.
14 D. R. Dreyer, S. Park, C. W. Bielawski and R. S. Ruoff, Chem. Soc. 21 Y.-C. Yang, J. Baker and J. R. Ward, Chem. Rev., 1992, 92, 1729–1743.
Rev., 2010, 39, 228–240.
22 http://www.nobelprize.org/. (Accessed Feb 26, 2014).
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