Photo-enhanced hydrolysis of bis(4-nitrophenyl) phosphate using Cu(II) bipyridine-capped plasmonic nanoparticles

Abstract

We show that the hydrolysis of bis(4-nitrophenyl) phosphate by a Cu(II) bipyridine complex was enhanced by 1000-fold when covalently attached to 10 nm gold nanoparticles (AuNP) and irradiated with a 120 mW green laser at 532 nm when compared to an unsupported vinyl-substituted Cu bipyridine analog. The catalytic reaction was monitored by UV-vis spectroscopy in 20 mM MOPS buffer at pH 8 and at room temperature by observing the growth of the product, 4-nitrophenolate, at 405 nm. Initial rate data was analyzed using a Michaelis–Menten formalism. Control experiments suggested that the photo-enhanced hydrolysis reaction required that the Cu metal center be attached to the AuNPs via a thiolated bpy ligand. At higher laser power, the initial rate data deviated from the Michaelis–Menten formalism. Possible mechanisms are discussed.

---

Introduction

The extraordinary ability to couple light with matter using the surface plasmon resonances (SPR) in metallic nanostructures has received intense interest recently with widespread application in the fields of biosensing,^1–3 photocatalysis,^4,5 medicine⁶ and spectroscopic study of chemical processes.⁷ The use of plasmonic metal nanoparticle-semiconductor assemblies for improvement of energy conversion efficiency and enhancement of sunlight-harvesting is also a well-known phenomenon.⁸ Another emerging area of research is the exploitation of plasmon heating for chemical reactions and organic synthesis.^9,10 The use of “hot electrons” generated from plasmon decay (as distinct from photoelectron emission) can drive chemical processes such as the classical example of the plasmon driven oxidation of p-aminothiophenol to give dimercaptoazobenzene (DMAB).¹¹

We recently described the surface plasmon resonance promotion of supported homogeneous catalytic thiophosphate ester degradation using 10 nm gold nanoparticles which were surface modified with a copper(II) bipyridine complex (1).¹² Irradiation of the plasmon band of the gold nanoparticle with a low-power (120 mW) green laser at 532 nm resulted in an increase in the rate of hydrolysis of the insecticide thiophosphate ester methyl parathion (MeP) to nitrophenolate (Scheme 1). In this particular case, the increase in rate of reaction could not be attributed to a thermal process at the nanoparticle surface i.e. simple localized plasmon heating at the gold surface was not responsible for the increase in rate of degradation of MeP. In the 2014 report we suggested one possible mechanism involving splitting of a hydroxy-bridged catalytically inactive Cu(II)–OH–Cu(II) dimer resulting in the formation of an active [(bipy)Cu(OH₂) (OH)]⁺ species anchored onto the monolayer surface. This process could involve a light mediated Cu–O bond scission although conclusive evidence for such a phenomenon has yet to be established and at this time this remains as only one of many possible mechanisms under investigation.

Scheme 1

The catalytic hydrolysis of phosphate esters using metal complexes continues to be an active area of research with recent focus primarily directed towards organophosphorus chemical warfare agent (OP-CWA) degradation,¹³ and DNA/RNA hydrolysis.¹⁴ The use of the phosphate diester bis(4-nitrophenyl) phosphate (BNPP) as a model substrate for catalysis is well known. Not only is kinetic data for a large number of catalysts readily available but BNPP does not suffer from potential oxidation or isomerization side-reactions as found for example in the degradation of the thiophosphate ester MeP. BNPP cleanly hydrolyzes to give 4-nitrophenolate which absorbs intensely in the visible region of the spectrum. Examples of highly active copper(II)-based homogeneous catalysts for organophosphate ester hydrolysis using bis(4-nitrophenyl) phosphate (BNPP) as the model substrate are known. For example, the neutral Schiff-base complex [CuL(OAc)] (L = (E)-1-((quinolin-8-ylimino)methyl)naphthalen-2-ol) hydrolyzes BNPP at modest rates, in addition to promoting plasmid DNA cleavage.¹⁵ Burstyn has reported a number of active Cu(II) catalysts including the azomacrocycle complex [Cu(i-Pr₃[9]aneN₃) (OTf) (OH₂)]OTf.¹⁶ Substituted bipyridine and phenanthroline ligands form complexes which are some of the most active copper-containing artificial nucleases.¹⁷ Numerous examples of other metal ions which are active for phosphate ester hydrolysis including Zr(IV),¹⁸ Ce(IV)¹⁸ La(III)¹⁸ and Co(II)¹⁹ have also been reported recently. Strategies for increasing the rates of catalytic activity include the incorporation of substrate binding sites to catalytic centers, attachment of ligands for increasing Lewis acidity and use of surfactants and micelles for improving substrate solubility. In view of the quest for even higher rates for catalytic OP-CWA hydrolysis and DNA cleavage we investigated the SPR mediated acceleration of BNPP hydrolysis using a low-power green laser and a copper bipyridine complex supported on 10 nm Au nanoparticles. The work described herein provides a valuable comparison with our previously reported catalytic hydrolysis of the neutral sulphur containing thiophosphate ester MeP and gives further insight into the mechanism of the SPR promoted homogeneous catalysis.

Results

The capped 10 nm AuNPs were prepared and characterized as described in our previous published work.¹² In addition to the thiolated copper bipyridine complex 1 (Scheme 1), the presence of a thiolated oligoethylene glycol ligand 1-(mercaptohex-6-yl)tri(ethylene glycol) 2 helps to stabilize the AuNPs and prevents aggregation in buffered solutions. In Fig. 1A, the UV-vis spectra of 10 nm capped AuNPs are shown with either 1, a 1 : 1 ratio of 1 and 2, and 2 only. Each spectrum clearly shows the surface plasmon absorption band at ca. 535 nm. For the samples containing copper complex 1, the peaks in the UV region of the spectra at 305 nm correspond to absorption due to π–π transitions of the copper complexed bipyridine. The coverage of complex 1 on the AuNPs was determined to be 2400 Cu/AuNP using UV-vis spectra data and the extinction coefficient of Cu complex assuming 10 nm. This coverage was confirmed by determining Au and Cu ratios of isolated nanoparticle samples using ICP-OES. In this work, we tested AuNPs capped with 1 for the hydrolysis of BNNP. The reactions were monitored spectrophotometrically at 405 nm to measure the product (4-nitrophenolate) formation as a function of time at 25 °C in buffered solutions (20 mM MOPS, pH = 8.0). During the course of the reaction, the absorbance of the 4-nitrophenolate is clearly seen at 405 nm and increases with time. One example of the spectral changes is shown in Fig. 1B for the reaction in the dark using AuNPs capped with 1 with [BNPP] = 2 × 10⁻³ M. In Fig. 2A, product formation normalized to [Cu] vs. time are shown for a single BNPP concentration comparing various control experiments. When Cu is chelated to a bipyridine ligand and attached to the AuNPs in the form of complex 1, there is a significant increase in product formation which is further enhanced by laser excitation at 532 nm.

Fig. 1 A. UV-vis absorbance spectra of 10 nm AuNPs capped with 1, 2 or both in water. B. UV-vis spectral changes of the reaction of AuNPs capped with 1 + BNPP in the dark. [AuNP] = 6.7 × 10¹⁴ particles per L, [Cu] = 2.5 × 10⁻⁶ M, buffer = 0.02 M MOPS at pH 8.0, and [BNPP] = 2 × 10⁻³ M. Temperature = 25 °C.

Fig. 2 (A) [PNP] vs. time normalized to [Cu] for different conditions listed in the legend. Other parameters include: buffer = 0.02 M MOPS at pH 8.0, [Cu(NO₃)₂] = 1 × 10⁻⁵ M, [AuNPs capped with 1] = 6.7 × 10¹⁴ particles per L with[Cu] = 2.5 × 10⁻⁶ M, and laser power = 120 mW. (B) Initial rate of product formation vs. [BNPP] under two conditions. With (i) and without (ii) laser excitation for AuNPs capped with 1. For all fits, R² = 0.99. Error bars are the average of 2 data sets.

A number of control experiments were then performed. Under laser irradiation no acceleration of BNPP hydrolysis is observed over a 6 hour period under the following conditions: (a) AuNPs capped with OEG-thiol 2; (b) a copper(II) bipyridine complex [(5-methyl-2,2′-bipyridine)Cu]Cl₂ 3 or Cu(NO₃)₂ in solution in the absence of AuNP and (c) copper bipyridine complex 3 in the presence of AuNPs but not covalently attached to the nanoparticle surface. Clearly, the prerequisite for the phenomenon is the presence of a copper bipyridine complex located in close proximity to the gold nanoparticle surface via an organic linker.

To further describe this effect, we analysed the kinetics for the hydrolysis of BNPP using Michaelis–Menten formalism as shown in eqn (1) where k_cat is turnover number i.e. the maximum number of substrate molecules converted to product per catalyst molecule per second and K_M is the Michaelis constant.

Initial rate = [Cu][BNPP]k_cat/(K_M + [S]) (1)

In Fig. 2B, the initial rates are plotted vs. substrate concentration between 0.05 mM and 2 mM for the laser irradiation and dark reactions with the two different AuNPs having different coverages of 1. The data shows typical Michaelis–Menten kinetic curves in which the rate increases linearly at low concentrations of substrate and then begins to saturate at higher concentrations. The apparent Michaelis–Menten parameters, k_cat and K_m, are listed in Table 1 from the fit of eqn (1) and compared with values for other copper(II) catalysts reported from our laboratory including the independently prepared [(5-methyl-2,2′-bipyridine)Cu]Cl₂ complex 3 (see Experimental section). Although the primary focus of this work is rate acceleration we also investigated the nature of the active nanomaterial. TEM images before and after interaction with the BNPP substrate indicate that the nanoparticle morphology is essentially unchanged (see ESI†). Close inspection of the plasmon band for the gold nanoparticles at ca. 535 nm as the reaction proceeded also confirmed the morphology of the particles is being maintained during catalysis. We were not however, able to develop a suitable re-cycle protocol for the catalyst although the means for achieving this without loss of activity is currently under investigation.

Table 1 Apparent Michaelis–Menten parameters for the hydrolysis of BNPP

Conditions^a	kcat, s^−1	KM, M^−1
a 25 °C, pH 8.0, 20 mM MOPS buffer. Non-linear regression analysis of the data was performed using Solver in Excel and the error bars were calculated from the macro Solver Statistics.^23b mby = 5-methyl-2,2′-bipyridine.c vby = 5-vinyl-2,2′-bipyridine.
Cu(II)mbpy^b	3.1 ± 0.7 × 10^−6	5 ± 1 × 10^−3
Cu(II)vbpy^20^c	1.0 × 10^−6	2.3 × 10^−3
10 nm AuNP capped with 1	8.3 ± 0.4 × 10^−6	2.1 ± 0.4 × 10^−4
Cu(II)vbpy polymer^20	2.3 × 10^−5	1.0 × 10^−4
10 nm AuNP capped with 1 + 120 mW 532 nm laser	1.3 ± 0.06 × 10^−4	2.6 ± 0.4 × 10^−4

---

We also investigated the laser power dependence on the initial rate of hydrolysis. As shown in Fig. 3, at two higher laser powers (400 and 800 mW) there is a linear dependence of the initial rate of hydrolysis vs. [BNPP] which is a deviation from the classical Michaelis Menten kinetics displayed with the lower power laser or in dark conditions which suggests a different mechanism may be in play at higher laser powers.

Fig. 3 Initial rate of product formation vs. [BNPP] at two different power levels of the laser. Reaction conditions are the same as in Fig. 2B.

Discussion

Several comparisons are worth noting. First, by simply attaching a Cu(II) bipyridine (Cubpy) catalyst 1 to 10 nm AuNP, we have increased k_cat/K_M ∼ 90–64 fold compared to literature results for a vinyl-substituted Cu(II)bpy complex in solution under similar conditions²⁰ and the analogous methyl substituted complex respectively. The enhancement is reflected in both a decrease in K_M i.e. an increase in the binding of the BNPP substrate to 1 at the surface of the AuNP and a slightly larger k_cat. Related detailed studies of a triazacyclonane Zn(II) catalytic unit attached to AuNPs catalysing a transphosphorylation reaction have reported the source of enhanced catalysis on AuNPs as an intrinsic property of the self-assembled monolayer in which the enhancement originated from cooperative effects between two neighbouring catalytic units.²¹ Similar behaviour is likely to be in effect here considering the proximity of metal complexes on the surface of the spherical gold nanoparticle, however, the necessity of two metal centers for catalysis is not a prerequisite for the catalytic hydrolysis using copper bipyridine systems.

More significantly for this study, k_cat/K_M is increased by an additional ∼12-fold via irradiation of the plasmon absorption band of AuNP using a 120 mW green laser. In this case, the enhancement is primarily shown in k_cat (i.e. a significantly higher turnover number during laser irradiation) since K_M is similar within experimental error between the laser and dark reactions (2.3 × 10⁻³ and 2.6 × 10⁻³ M⁻¹). The deviation from Michaelis–Menten kinetics at higher laser power suggests a different mechanism in which saturation kinetics are not observed with increasing substrate concentration. This would dictate that a strong substrate–catalyst interaction is not required. One possible mechanism would include photoinduced electron transfer of “hot” electrons from the AuNPs to the BNPP through the attached Cubpy by a 2nd order process. However, the electrochemical reduction of BNPP has been reported in organic solvents generating 4,4-dinitrobiphenyl, but not PNP and so this mechanism is unlikely.²²

From our previous work, for the hydrolysis of the thiophosphate triester MeP, the modulation of the hydrolysis rate by the irradiation of the plasmon absorption band of AuNP using a low power green laser was sensitive to the surface concentration of the Cu(II)bpy catalyst, and we showed the effect is attenuated by dilution of the Cu(II) catalyst at the AuNP surface.

We suggested a possible mechanism for catalytic enhancement involving the dissociation of catalytically inactive hydroxy-bridged Cu(II) dimer formed at the higher surface concentrations.¹² In this work, laser enhancement for the hydrolysis of BNPP is greater than that reported for MeP. One possible reason for the enhancement may be found in the greater affinity of BNPP vs. MeP for the surface bound Cu(II)bpy complex since BNPP is negatively charged at the pH of the experiment. In fact, K_M is smaller (higher binding) for BNPP (K_M = 2.1 ± 0.4 × 10⁻⁴) compared to MeP (K_M = 4.3 ± 2 × 10⁻⁴)¹² when comparing the apparent Michaelis–Menten parameters for the hydrolysis of both substrates using Cu(II)bpy capped 10 nm AuNPs under similar reaction conditions.

Conclusions

In summary, catalytic reactions, such as those involving hydrolytic mechanisms, remain an important area of research. The ability to activate catalysis by laser light is an important opportunity to add to the fundamental understanding of how the unique properties of gold nanoparticles can be further exploited to open up new fields in nanotechnology.²⁴ Studies on surface plasmon band assisted supported homogeneous catalysis for a number of catalytic reactions with a view to exploring the scope of the technique are currently being investigated and will be reported in due course.

Acknowledgements

This work received support from the Defense Threat Reduction Agency-Joint Science and Technology Office for Chemical and Biological Defense (MIPR #HDTRA1516012).

Notes and references

1. Y. Hirakawa, T. Yamasaki, E. Watanabe, F. Okazaki, Y. Murakami-Yamaguchi, M. Oda, S. Iwasa, H. Narita and S. Miyake, J. Agric. Food Chem., 2015, 63, 6325–6330.
2. J. N. Anker, W. P. Hall, O. Lyandres, N. C. Shah, Z. Jing and R. P. Van Duyne, Nat. Mater., 2008, 7, 442–453.
3. S. Kim and H. J. Lee, Anal. Chem., 2015, 87, 7235–7240.
4. X. Lang, X. Chen and J. Zhao, Chem. Soc. Rev., 2014, 43, 473–486.
5. W. Hou and S. B. Cronin, Adv. Funct. Mater., 2013, 23, 1612–1619.
6. Z. A. Nima, A. Biswas, I. S. Bayer, F. D. Hardcastle, D. Perry, A. Ghosh, E. Dervishi and A. S. Biris, Drug Metab. Rev., 2014, 46, 155–175.
7. S. S. E. Collins, M. Cittadini, C. Pecharromán, A. Martucci and P. Mulvaney, ACS Nano, 2015, 9, 7846–7856.
8. M. Lee, J. U. Kim, K. J. Lee, S. Ahn, Y.-B. Shin, J. Shin and C. B. Park, ACS Nano, 2015, 9, 6206–6213.
9. J. R. Adleman, D. A. Boyd, D. G. Goodwin and D. Psaltis, Nano Lett., 2009, 9, 4417–4423.
10. C. Fasciani, C. J. B. Alejo, M. Grenier, J. C. Netto-Ferreira and J. C. Scaiano, Org. Lett., 2011, 13, 204–207.
11. Z. Zhang, L. Chen, M. Sun, P. Ruan, H. Zheng and H. Xu, Nanoscale, 2013, 5, 3249–3252.
12. D. A. Knight, R. Nita, M. Moore, D. Zabetakis, M. Khandelwal, B. D. Martin, J. Fontana, E. Goldberg, A. R. Funk, E. L. Chang and S. A. Trammell, J. Nanopart. Res., 2014, 16, 1–12.
13. R. Zboril, M. Andrle, F. Oplustil, L. Machala, J. Tucek, J. Filip, Z. Marusak and V. K. Sharma, J. Hazard. Mater., 2012, 211–212, 126–130.
14. R. F. Brissos, A. Caubet and P. Gamez, Eur. J. Inorg. Chem., 2015, 2633–2645.
15. N. Kozlyuk, T. Lopez, P. Roth and J. H. Acquaye, Inorg. Chim. Acta, 2015, 428, 176–184.
16. K. M. Deck, T. A. Tseng and J. N. Burstyn, Inorg. Chem., 2002, 41, 669–677.
17. C. Wende, C. Lüdtke and N. Kulak, Eur. J. Inorg. Chem., 2014, 2014, 2597–2612.
18. T. K. N. Luong, G. Absillis, P. Shestakova and T. N. Parac-Vogt, Eur. J. Inorg. Chem., 2014, 2014, 5276–5284.
19. M. Zhao, S.-S. Xue, X.-Q. Jiang, L. Zheng, L.-N. Ji and Z.-W. Mao, J. Mol. Catal. A: Chem., 2015, 396, 346–352.
20. C. M. Hartshorn, A. Singh and E. L. Chang, J. Mater. Chem., 2002, 12, 602–605.
21. G. Zaupa, C. Mora, R. Bonomi, L. J. Prins and P. Scrimin, Chem.–Eur. J., 2011, 17, 4879–4889.
22. K. S. V. Santhanam and A. J. Bard, J. Electroanal. Chem. Interfacial Electrochem., 1970, 25, A6–A9.
23. E. J. Billo, Excel for chemists a comprehensive guide, John Wiley & Sons, Hoboken, N.J., 3rd edn, 2011.
24. J. C. Scaiano and K. Stamplecoskie, J. Phys. Chem. Lett., 2013, 4, 1177–1187.

---

Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c6ra07119h

---