Preparation of Dual-activity Enzyme-metal-nanoparticles Conjugate Catalysts for Cascade Processes

The fabrication of novel systems where enzymatic and metallic actives sites directly interact represents an elegant strategy for sustainable processes. One of the new strategies involves the in situ formation of metal nanoparticles on a protein network as scaffold. This biological entity is the key element because it conserves the biological catalytic efficiency and induces the final metallic nanoparticles generation on the structure. This method allows to obtain bifunctional enzyme-metallic catalysts with excellent versatility and efficiency in a different reaction and experimental conditions. In this concept article, the relevant aspects in terms of fabrication of enzyme-metal nano-particles hybrids and recently reported examples of successful application in cascade processes will be described.


Introduction
The development of novel combined catalytic systems is the best choice to obtain efficient sustainable processes, for a modern chemical industry.However, these two types of catalysts are often mutually inactivated and the reaction conditions for one cannot be applied to the other, for example some enzymes are not active in organic solvent or palladium salts are not soluble in water.Therefore, alternative strategies, where both systems conserved full activity, are necessary.[15][16][17][18][19] However, this required a timeconsuming in the preparation of the organometallic compound or required different modification of the protein for site-selective insertion of the metal.Another issue in this aspect is that mainly this artificial enzyme is water soluble as natural enzyme, presenting similar problems of compatibility in some experimental conditions or being required additional steps.[22][23] The formation of the nanoparticles is important and enzyme also can conserve the enzymatic activity after the nanobiohybrid formation. [21] Some of these results are the most relevant in the literature in the particular catalytic process, demonstrating the advantages to use nanotechnology vs bulk materials, besides the simple synthetic approach of them.The conservation of both activities, producing bifunctional (enzyme and metal) catalysts have been recently successfully used in complex chemical routes by cascade processes in very sustainable conditions, [28][29][30][31][32][33][34][35][36] and some of them will be discussed in this article (Figure 1).
Figure 1 must be just in this point at the end of this paragraph in the page number 1 as similar than in accepted paper version

Synthetic Considerations in the Preparation of Dual enzyme-metal Nanoparticles Biohybrids
Nanobiohybrids containing enzyme and metal nanoparticles sites have been made possible by a very fancy, simple and sustainable strategy (Figure 2).The key element of the approach is the use of enzymes as biological component to induce the in situ metal nanoparticles formation. [13]This criterion is the main aspect, which make possible to synthesize metal nanoparticles in aqueous media, room temperature and from simple metal salts.
Enzyme composition has also an influenced in the mechanism of nanohybrid formation, basically by a first enzyme-metal coordination, then the in situ nanoparticle formation and posterior growth of them to obtain the final nanostructure [21] The conservation of the protein three-dimensional structure in the enzyme is mandatory to the final formation of the hybrids and to conserve the enzymatic activity.Metal nanoparticles sizes and particle morphology are also marked importantly by the enzyme.A very clear example are the iron nanobiohybrids (Figure 3). [37]Iron is quite instable with a great tendency to oxidation.Indeed, in iron nanoparticles, the most typical synthesis involved the formation of iron oxides or derivative forms. [38]In this enzyme-induced synthetic approach, enzyme showed important differences.For example, the use of lipase from C. antarctica B, after optimized experimental conditions, allows to synthesize new nanobiohybrids containing superparamagnetic FeCO 3 nanorods, whereas using other en-zymes, the formation of other species (magnetite or goethite) and morphologies (nanoparticles or nanowires) were obtained. [37]nother parameter to consider in the aqueous synthesis is the effect of pH and buffer in the solution.The use or not of buffer to control the pH can be a critical aspect, for example, to control the formation of a particular metal species (Figure 4).This was observed in the case of Cu nanobiohybrids, where it was possible to obtain species from Cu (0) to Cu(II) depending on the solution conditions. [23]Increasing the pH from neutral to alkaline conditions showed an effect on a different coordination approach, in which, after a reducing step, mainly Cu(0)NPs embedded on the protein matrix could be obtained.When the phosphate buffer was in the medium at neutral pH, the only species formed was a Cu(II) (copper phosphate) species without reducing step or Cu 2 O in the case of using a reducing agent (Figure 4).
Recently, although the synthetic approach is well described at room temperature, the temperature phenomenon was evaluated in the case of Pd nanobiohybrids. [39]Synthesis at lower T (4 °C) demonstrated that protein structure modification has a major effect on the final PdNPs formation, where lower but smaller nanoparticles were found using C. antarctica B lipase as protein scaffold.The link between protein structure and temperature, as well as the influence of the latter, has been demonstrated to be    critical in the reducing capacity of the enzyme in the final Pd nanoparticle production, metal species, and even nanoparticle size.Thermal phenomena were also important in the final hybrid creation with more complicated enzymes.
Finally, one of the most critical aspects in achieving optimum stability and enzymatic activity has been the innovative creation of nanobiohybrids on solid-phase (Figure 5). [31,34]The site-specific immobilization of enzymes provides for the most stable and active form of these biocatalysts.Thus, the prior experimental conditions for metal nanoparticle formation can be applied utilizing this solid-phase enzyme.Solid support properties, such as unspecific adsorption of metal ions on their surface, must be studied in order to ensure that metal nanoparticles are generated exclusively on and by the enzyme.
Lipase immobilized fixing the open conformation on graphene derivative displayed the maximum activity, which is retained after the nanobiohybrid creation. [31,34]This property was recently expanded to other enzymes, including co-immobilization of two distinct enzymes on the same solid material. [31,34]Enzymatic activity was mostly preserved in all circumstances, such as when creating Cu nanoparticles or even Pd nanoparticles.
][42] The coating of the enzyme prior to hybrid formation is a well-established technique for enzyme stabilization because it reduces the possible distortion of the structure caused, for example, by an increase in T or the presence of a co-solvent.The effect of size on nanoparticle formation and metal species has been studied using tailor-made dextran derivatives such as dextran functionalized with aspartic acid or aldehydes (Figure 6).The activity of enzymes such as glucose oxidase and lipase, for example, was increased in synthetic Enzyme-Ag hybrids. [28]

Dual-active Enzyme-metal Nanoparticles Hybrids
One of the benefits of these nanobiohybrid systems is their ability to demonstrate two distinct catalytic activities in a single compartment.This is advantageous in terms of cooperation and efficiency.Enzyme active sites exhibit enantioselectivity or specificity, whereas metallic active sites exhibit significant catalytic versatility.Thus, tandem catalysis by dynamic kinetic resolution of amines is one of the more intriguing examples. [20,31,43]This is a particularly important reaction since it is possible to obtain > 99 % of a distinct enantiomer product, whereas kinetic resolution only allows for 50 %.Until now, combination Pd/lipase systems have proven to be more efficient than single catalysts, where a rapid racemization of the undesired enantiomer for the enzyme allow a faster enzymatic enantioselective amidation process.This nanobiohybrid showed excellent results in the production of (R)-benzylamide, a very important intermediate in the synthesis of a range of therapeutic drugs.A highly effective tandem process between the high selectivity of the enzyme and the high racemization capacity of PdNPs.Another advantage of the nanobiohybrid structure is its remarkable robustness, with enzyme and Pd working at 70 °C in toluene.
The dual efficiency of these systems we have been recently demonstrated in the arylative allenol cyclization. [44]A nanobiohybrid comprising glucose oxidase (GOx) and Pd nanoparticles was used as proof of concept.In this scenario, the combination of this hybrid with soluble chloroperoxidase allowed free enzyme to halocyclize allenic alcohol via the consumption of H 2 O 2 in situ produced by GOx.Then, consecutively, PdNPs on the hybrid catalyzed the Suzuki reaction, yielding a high enantiopurity product (Figure 7). These dual-active catalysts successfully synthesized various enantiomerically enriched 2,5-dihydrofurans from allenic acetates in aqueous solution and room temperature (Figure 8). [28]Using the DexAsp-conjugated Au-hybrids, high versatility was demonstrated (reaction from pure solvent to aqueous/solvent mixture) and dimerization event  Polymer-coated enzymes in nanobiohybrids synthesis.Adapted and reproduced under terms of the CC-BY license. [24]Copyright 2023, The Authors, published by Palomo JM and coworkers".leading to symmetrically fused dihydrofuran products that were observed with excellent enantio and diastereoselectivity (Figure 8).
The solid-phase enzyme-metal nanoparticles dual catalysts were successfully applied in both cases.This technique enables the production of completely active enzyme and metal active sites that are intramolecularly positioned at a position where a very efficient reaction can take place.
In one example, multilayer graphene-anchored Candida antarctica lipase (G@CALB)-PdNPs, CuNPs or PdCuNPs were synthesized and applied in different cascade processes. [31]Bifunctional capacity was clearly demonstrated in hydrolysis-reduction domino reaction from p-nitrophenylpropianate to p-aminophenol, combin-ing the hydrolytic capacity of the enzyme and reducing aspect of the metal, but most interesting, it was the application in the transformation of peracetylated glucal to novel disaccharides by CALB-CuNPs biohybrids in aqueous/solvent media and r.t.(Figure 9a).This reaction goes through a high regioselectivity of CALB in the monohydrolytic deprotection in C-3 of peracetylated glucal, followed by the oxidation by CuNPs of the double bond to afford an epoxide which was then directly attacked by the free OH of monodeprotected glucal to produce the new disaccharide product [31] (Figure 9a).
Furthermore, in this novel systems, the fabrication of a bimetallic CuPd alloy nanoparticles graphene-hybrid (CALBPd-CuNPs), exhibited a very high efficient in the DKR reaction in comparison with the similar Pd-hybrid, demonstrating a synergistic effect of copper on Pd. [31] In a second example of the applicability of this dual-activity catalysts in carbohydrate chemistry, a Graphene-Candida rugosa lipase (CRL)-CuNPs was prepared. [34]A one-pot parallel process combining a lipase-mediated regioselective hydrolytic monodeprotection with a metal-catalyzed oxidation in aqueous media was performed to synthesize 2,3,4-triacetyl-D-gluconic acid from αperacetylated-glucose.As in the first example, the enzyme was extremely selective for regiodeprotection at C-6 position of peracetylated glucose.In this case, CuNPs cause the oxidation of the molecule at anomeric position, in a parallel way to finally produce the di-hydrolyzed product [34] (Figure 9b).
These two examples show the potential utility of these bifunctional catalysts in carbohydrate chemistry, which can be extended to other more complicated glycoderivatives or glycan compounds with biological applications.

Summary and Outlook
Both high-efficiency systems for multiple steps in one compartment (robust enzymes, application of the concept to bimetallic systems (core-shell, alloying options), as we recently demonstrated that the PdCu bimetallic system enhanced metal activity compared to palladium alone.This concept will allow for greater catalytic adaptability as well as the progression to more accessible Figure 7. Design and application of novel metalloenzymes multiple active sites.Reaction performed by the enzymatic catalytic site (green).Reaction performed by the metallic catalytic site (red).Adapted and reproduced under terms of the CC-BY license. [21]Copyright 2019, The Authors, published by Palomo JM".multiactive systems, such as cascade reactions in which both metals exhibit different activities.These nanobiohybrid systems introduce a new concept in sustainable chemistry, a critical process in present and future industry, in which the biological entity enables chemical processes to be achieved under softer conditions, in addition to cascade reactions.This is a significant distinction, since where the biological entity can be a simple lattice with embedded and homogeneously dispersed metal nanoparticles, single atom enzyme-metal pairings can be formed.
Future lines of research in this approach, which are booming, will focus on designing methodologies that make it possible to obtain customized systems, the enzyme-metal NPs combination, preserving the enzymatic activity, keeping its environment of the active site intact and allowing the incorporation of controlled metal species as well as with a controlled size.One of the main objective in the near future will be to extend this technology to increase the number of different catalytic active sites in the same compartment.The use of polymer conjugations, computational technologies and directed evolution also will contribute in the novel design of the enzyme-metal catalysts.Furthermore, these systems could be applied in other fields besides organic synthesis, such as sensing and detection systems.

Figure 6 .
Figure 6.Polymer-coated enzymes in nanobiohybrids synthesis.Adapted and reproduced under terms of the CC-BY license.[24]Copyright 2023, The Authors, published by Palomo JM and coworkers".