Design of artificial enzymes by supramolecular strategies

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ScienceDirect Design of artificial enzymes by supramolecular strategies Tingting Wang1, Xiaotong Fan1, Chunxi Hou and Junqiu Liu Enzymes are biomacromolecules with three-dimensional structures composed of peptide polymers via supramolecular interactions. Owing to the incredible catalytic efficiency and unique substrate selectivity, enzymes arouse considerable attention. To rival natural enzymes, various artificial enzymes have been developed over the last decades. Since supramolecular interactions play important roles in both substrate recognition and the process of enzymatic catalysis, designing artificial enzymes using supramolecular strategies is undoubtedly significant. Here we discuss the recent advances in constructing artificial enzymes using supramolecular platforms.

Address State Key Laboratory of Supramolecular Structure and Materials, College of Chemistry, Jilin University, Changchun 130012, China Corresponding author: Liu, Junqiu ([email protected]) These authors contributed equally.

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Current Opinion in Structural Biology 2018, 51:19–27 This review comes from a themed issue on Engineering & design Edited by Giovanna Ghirlanda and Ivan Korendovych

https://doi.org/10.1016/j.sbi.2018.02.003 0959-440X/ã 2018 Elsevier Ltd. All rights reserved.

Introduction Enzymes are biomacromolecules which act as biological catalysts. Enzymes participate in almost all metabolic processes in cells with substrate specificity and unbelievable catalytic efficiency. The wisdom of nature prompts researchers to explore the underlying rules of the nature and develop artificial analogues. Great attention has been paid to investigate the structure and the catalytic mechanism of natural enzymes. In order to explore the catalytic process and observe the intermediates occurring during the enzymatic reactions, various methods such as nuclear magnetic resonance (NMR) [1–3], single-molecule kinetics [4], steady-state kinetics [5] and molecular dynamic simulation [6] are developed. However, to date we still cannot give an accurate answer how enzymes have substrate selectivity and high catalytic efficiency. www.sciencedirect.com

Despite a plenty of studies on natural enzymes, artificial enzymes provide an alternative choice for researchers to understand the behaviors of enzymes and considerable progress has been made in the field of mimicking natural enzymes [7–11]. As far as we know, supramolecular interactions, including hydrophobic interaction, electrostatic attraction, hydrogen bonding, van der Waals interaction and metal–ligand coordination, play important roles in both substrate recognition and the process of enzyme catalysis. So constructing artificial enzymes based on supramolecular strategies attracts much attention and excellent work has been done. In this critical review, we mainly focus on the examples published in the past three years. Different approaches for designing artificial enzymes from supramolecular strategies are presented in Figure 1.

Macrocyclic molecules for the design of artificial enzymes It is believed that a suitable microenvironment is indispensable when an enzymatic reaction is carried out. An eligible pocket could not only segregate substrates and active sites of enzymes from the surroundings, but also provide the microenvironment for accomplishing the substrates recognition and enzymatic reaction. Cavitycontaining molecules, such as cyclodextrins, cucurbiturils, calixarenes, container molecules, with the advantage that is similar to the pocket of enzyme, attract researchers’ attention and have been extensively studied [12–16]. These macrocyclic molecules can protect the enzymatic reaction from the surroundings similar to enzymes. The related researches have confirmed that modifications on cavity or portals of these macrocyclic molecules will influence the catalytical ability and substrate selectivity. Many artificial enzymes have been developed based on this unique characteristic of macrocyclic molecules with examples provided by Bender [17], Bleslow [18], our group [19–22], Bols [23–24] and so on. Recently, Matt and co-workers published a review summarizing the coordination chemistry based on cyclodextrins and phosphorus (III), and discussed the catalytic properties of these cavity-shaped ligands [25]. Bols’ group also reported an artificial metalloenzyme using cyclodextrin diacids in an unprecedented simple method [26]. They synthesized cyclodextrin diacids and studied the binding to various metal ions, such as iron, zinc and copper. When the catalyst was used that was generated from Fe(ClO4)2 and cyclodextrin diacids, 100–1000 times of oxidation rate of substituted benzyl alcohol was observed, but no Current Opinion in Structural Biology 2018, 51:19–27

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Figure 1

Macrocyclic molecule as scaffold

Container molecule as scaffold

Artificial enzymes

GSSG

C 2H 6

GSH

C2H5OH

Assembly as scaffold Protein as acaffold OMe

DNA as scaffold Current Opinion in Structural Biology

Different approaches for designing artificial enzymes using supramolecular strategies. Macrocyclic molecules: copyright 2017 Wiley-VCH. Container molecules: copyright 2015 American Chemical Society. Protein: copyright 2015 American Chemical Society. DNA: copyright 2016 American Chemical Society. Assembly: copyright 2015 American Chemical Society.

acceleration was observed for catalysts from Zn(ClO4)2 and cyclodextrin diacids or Cu(ClO4)2 and cyclodextrin diacids. When they substitute succinic acids for cyclodextrin diacids to form iron complex, the catalytic rate rarely increases, which suggests the cyclodextrin cavity is essential for catalysis. As for cucurbiturils, an oligomer of glycolurils. Werner and co-workers recently provided a delicate review about the synthesis and high-affinity binding of cucurbiturils, describing the catalytic application of cucurbiturils in detail [27]. It is well known that cucurbituril (CB6) can form stable 1:1 complexes via host–guest interaction with diprotonated diaminopentane or diaminobutane. However, the host–guest interaction decreases significantly when the diprotonated state is destroyed. Considering for this, our group successfully constructed a pH-responsive artificial glutathione peroxidase (GPx) which was a wellknown selenoenzyme via using a cucurbit [6]uril-pseudorotaxane-based molecular switch and an organoselenium compound [28]. When pH was below 6 because of two nitrogen atoms of compound 1 diprotonated, the GPx Current Opinion in Structural Biology 2018, 51:19–27

mimic formed a 1:1 host–guest pseudorotaxane complex. Therefore, compound 1 did not show apparently GPx activity due to encapsulation of the active site of compound 1 into CB [6]. At pH above 7, the two nitrogen atoms of compound 1 were deprotonated and the binding ability of CB [6] with compound 1 was significantly reduced, so it caused a gradually increased GPx activity. As shown in Figure 2, the activity of selenoenzyme can be turned on/off between pH = 7 and pH = 6. Calixarenes are also used to construct artificial enzymes. For example, Schatz’s recent study indicated that sulfocalixarenes could boost the catalytic activity of Grubbs-type catalysts in the olefin metathesis reaction [29].

Container molecules for the design of artificial enzymes Despite the classic host molecules, researchers have aimed at constructing container molecules which mimick the enzymatic pockets by providing the microenvironment for enzymatic reactions [30]. In the last two years, Fujita [31], Nitschke [32], Nolte [33,34], and Raymond [35–38] have made great progress in constructing www.sciencedirect.com

Design artificial enzymes Wang et al. 21

Figure 2

‘OFF’ state (a)

(b) N H

Se

N H

2 GSH + H2O2

= GSSG + 2 H2O Selenoenzyme active site

Compound 1

OH-

=

H+

CB[6]

‘ON’ state 2 GSH + H2O2

+ GSSG + 2 H2O = CB[6] Current Opinion in Structural Biology

When pH  6, compound 1 containing imino groups is diprotonated, and form stable 1:1 complexes with CB6, the artificial enzyme is at ‘OFF’ state. Although when pH  7, the diprotonated of Compound 1 is destroyed and the host–guest complexes decompose, the artificial enzyme is at ‘ON’ state [28]. Copyright 2016 Royal Social of Chemical.

container molecules to simulate natural enzymes. Recently, Bergman, Raymond and co-workers found that the assembled tetrahedral cage could catalyze the bimolecular aza-Prins cyclization with the feature of transannular 1, 5-hydride transfer [39]. The tetrahedral M4L6 as supramolecular cage was fabricated through metal coordination in which naphthalene spacers served as the ligands. The hydrophobic cavity promotes the formation of iminium ions, and the encapsulation of transient iminium ions in the cavity is rate-limiting step. The mechanism of the reaction is analyzed using kinetic analysis and labeling studies. However, the reaction pathway goes to another direction to generate alcohol without the participation of the supramolecular cage as an artificial enzyme. In order to assess the influence of the K12Ga4L6 tetrahedral cavity architecture on the intramolecular reactions and product selectivity during the catalytic process, Raymond et al. modified the aromatic spacers and chelating moieties of the host molecules [40]. The variation of chelating moieties has obvious effects on Prins cyclization reaction rate, while the variation does not affect the substrate selectivity. In contrast, the selectivity and reaction efficiency both make changes with the variation of aromatic spacers. In specific in the tetrahedral catalysts, larger spacer of pyrene analogue-based catalyst could catalyze conversion of an achiral substrate to more amount of trans product (98:2) than naphehalene-based catalyst, providing evidence that the cavity size of the supramolecular enzyme plays a key role in the product stereoselectivity. www.sciencedirect.com

Protein complexes for the design of artificial enzymes Many protein enzymes with the binding pockets for substrates recognition have been well studied. The binding pockets of proteins can incorporate various functional ligands by using noncovalent anchoring strategies, resulting in forming stable supramolecular complexes. Based on protein–ligand complexes, supramolecular artificial enzymes have been extensively constructed [41–43]. Recently, great progress involved in protein–ligand based supramolecular artificial enzymes has been made by many researchers, such as Watanabe [44,45], Pan [46], Ward [47–51], and Roelfes [52]. The specific recognition between proteins and their ligands are increasingly utilized to anchor the metal complexes to the protein scaffolds. One successful example is the biotin–streptavidin technology, as the metal-containing biotinylated pre-catalyst could be firmly attached to streptavidin to form a catalytic hybrid [53]. To make Suzuki reaction happen, Ward and colleagues reported an enantioselective artificial Suzukiase by introducing a biotinylated palladium complex into streptavidin [51]. They selected 2-methoxy1-naphthaleneboronic acid with 1-iodonaphthalene 6 as the model reaction. The enantioselectivity and activity of this newly developed artificial metalloenzyme could be optimized when moderate mutagenesis is made. This catalyst works for various aryl iodides and the yield is up to 90% ee. Except for the biotin–avidin system, many other anchoring strategies are used to construct artificial enzymes for ester hydrolysis [54], hydrogenation [55], sulfoxidation [56] and more reactions. Current Opinion in Structural Biology 2018, 51:19–27

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Besides biotin–(strept) avidin technology, other supramolecular anchoring strategies also contribute to exploiting artificial enzymes. Roelfes and co-workers created a novel artificial metalloenzyme through the supramolecular binding of Cu (II) complex with catalytic activity to the dimer interface of Lactococcal multidrug resistance Regulator (LmrR), which carries a huge hydrophobic cavity in the dimer interface [52]. Comparing with other anchoring strategies, this one is conveniently prepared through self-assembly. It is should be noted that the authors do not make accurate design in the process of enzyme preparation and catalysis in this study, just let substrates and active sites find suitable orientation themselves. The catalytic capacity of this novel artificial metalloenzyme is testified in Friedel–Crafts alkylation reactions with excellent ee’s.

1 resulting in the formation of product 3a, ligands dmbipy, phen, and L1 embedding into DNA scaffolds exhibit different enantioselectivity. And among them ligand dmbipy combined with DNA results in high conversion with a promising ee of 59%. Surprisingly, using ligand dmbipy alone in this reaction, almost no conversion is acquired, which indicates a very strong acceleration effect in this reaction based on DNA complexes. Recently, chemical reactions catalyzed by DNA-based metalloenzymes were extended to oxidative process by Li and colleagues. They firstly reported an example of enantioselective sulfoxidation reaction using DNA-based metalloenzyme composed of DNA G-quadruplex and Cu (II) complexes [65]. And up to 77% ee was achieved.

Assemblies for artificial enzymes Nucleic acid complexes for artificial enzymes The wonderful nucleic acid structures of DNA duplex, aptamers, G-quadruplexes can serve as quality platforms for engineering artificial enzymes via supramolecular strategies [42,57,58]. This new concept of DNA-based asymmetric catalysis is based on hybrid catalysts formed by transition-metal complexes embedding in DNA scaffolds using non-covalent anchoring strategies. DNAbased catalysts have been successfully used in various catalytic reactions with enantioselectivity and highly effective catalytic capacity because of their well-defined and stable supramolecular architectures [59,60]. Owing to these features, DNA has gotten much more attention for designing hybrid catalysts. The second-coordination-sphere interactions are recognized to play key roles in constructing highly active catalysts based on DNA scaffolds [61–63]. Inspired by this, recently, Roelfes et al. developed a novel DNAbased hybrid catalyst which was made up of DNA and an iron (III) complex of cationic porhyrins [58]. The newly exploited artificial enzyme could effectively catalyze carbene-transfer reactions under a mild condition, demonstrating that styrene derivatives and ethyl diazoacetate (EDA) are the carbine precursors. As a feature of this catalytic reaction, a dimer EDA was observed, accompanied with a significant acceleration of the reaction induced by DNA. The novel DNA/cationic-iron-porphyrin-based hybrid catalyst enabled highly DNA accelerated catalysis of carbene-transfer reactions and the reason was that ortho-substituted N-methypyridinium pophyrin (instead of meta and para isomers) could bind to the DNA groove to allow access of active site to substrate and the reaction energy barrier was subsequently decreased. Shortly before, Garcı´a-Fernandez et al. reported a novel study of DNA-based catalysis of Friedel–Crafts conjugate addition/enantioselective protonation reaction in aqueous solution [64]. As shown in Figure 3, for the Friedel– Crafts conjugate addition of indole 2a with thiazol Current Opinion in Structural Biology 2018, 51:19–27

Enzymes with three-dimensional architectures are formed by peptide polymers via supramolecular interactions, which can be used to construct a variety of nanometer scale assemblies, for example, vesicles, micelles, nanowires, and nanotubes [66–70]. The diverse assemblies can serve as excellent platforms for designing artificial enzymes through introducing functional groups into the assemblies. Over the past decades, many artificial enzymes base on nanometer scale assemblies have been developed on the basis of designing the building blocks rationally [9,19–21]. Recently, the aggregates for artificial enzymes have been extended to protein assemblies [22,71,72]. Hou and coworkers have designed and constructed supramolecular enzymes by CB [8]-based host guest interactions [73]. By engineering a FGG tag to the N-terminal of glutation transferase (GST), C2-symmertric GST assembled into nanowires in the presence of host molecule of cucurbit [8] uril due to the binding of FGG to CB[8] at 2:1. The supramolecular protein assemblies was converted to supramolecular enzymes by incorporation of catalystic group selenocysterin. The supramolecular enzymes displayed 50% higher in activity after 18 days compared with monomers due to the better stability provided by the supramolecular effect. As an example, Sun et al. utilized generation 5 PAMAM dendrimer (PD5) with positive charge to induce the assembly of the negative charged cricoid proteins (stable protein one, SP1) through electrostatic interaction, leading to the generation of protein nanowires [72]. As shown in Figure 4, by introducing the active sites (selenocysteine for glutathione peroxidase, manganese porphyrin for superoxide dismutase) of glutathione peroxidase (GPx) and superoxide dismutase (SOD) into SP1 and PD5, respectively, the authors successfully constructed a supramolecular artificial enzyme with both SOD and GPx activities. This example indicates that one of the developing trend of designing artificial enzymes is to build multienzyme-cooperative system. www.sciencedirect.com

Design artificial enzymes Wang et al. 23

Figure 3

O N

H N

+

OMe

S 1

2a

Cu2+ H2O OMe O N S

H

NH

3a

N

N

N

dmbipy

N

N

N H

MeO N

N phen

OMe

L1 Current Opinion in Structural Biology

Friedel–Crafts conjugate addition catalyzed by artificial metalloenzyme and ligands. Substrates indole (1) and thiazole (2a) can be converted to 3a under the condition of the Cu (II) complex containing ligands dmbipy, phen or L1 combined with DNA [64]. Copyright 2016 American Chemical Society.

We know that substrate selectivity, which needs a suitable microenvironment, is a notable characteristic of enzymes. However, when researchers construct artificial enzymes, they usually focus on solving the problem of active sites, and neglect the microenvironment for recognition. Recently, Shi and co-workers made great progress in constructing assemblies for artificial enzymes [74–76]. A micelle-hydrogel system with both substrate selectivity and catalytic capacity was exploited by them [76]. Micelles are assembled from supramolecular amphiphiles with hemin as the active sites of horseradish peroxidase (HRP), and alginate hydrogels, which enhance the activities of enzymes, provide the microenvironment for substrate recognition and selectivity. The hemin-micelle was prepared by cooperative self-assembly of the block copolymer poly (ethylene glycol)-block-poly(1-vinylimidazole) (PEG-b-PVIm) and hemin, and the block copolymer was designed with high mass ratio of hydrophilic/hydrophobic block. Hence, the micelle provided a soluble carrier for hemin and a similar microenvironment for it as that in horseradish peroxidase. The production of specific and www.sciencedirect.com

hydrophobic microenvironments of artificial enzymes with amphipathic supramolecules was achieved by a self-assembly method. In addition, peptide assemblies for mimicking natural enzymes have garnered tremendous attention [77–80]. Recently, Ulijn et al. constructed a pH-switchable artificial hydrolase by means of introducing catalytic histidine residues into the pH-responsive peptides [81]. The peptides can self-assemble into fibrils with esterase activity in an alkaline condition. While in an acid condition, the fibrils disassembled into random coils which were catalytically inactive.

Conclusions and future directions Natural enzymes mainly composed of peptide polymers via supramolecular interactions can catalyze a wide variety of chemical reactions with substrate specificity and unbelievable catalytic efficiency. Inspired by the wisdom of nature, biologists and chemists have done much work to explore the mechanism of enzymatic reaction and develop various Current Opinion in Structural Biology 2018, 51:19–27

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Figure 4

GPx Center

a)

GPx Centers SP1

SeSP1

on SP1

90˚ Partial View

Top View

Side View

b) SOD Centers on PD5

PD5

MnPD5

Partial View

c)

Self-assembly

+ Dual-Enzyme SeSP1

Cooperative Nanowire

MnPD5

Current Opinion in Structural Biology

SeSP1 and MnPD5 assemble into protein nanowires through electrostatic interactions. The protein nanowires exhibit both excellent superoxide dismutase and glutathione peroxidase activities [72]. Copyright 2015 American Chemical Society.

artificial analogues. However, it is still a great challenge to unravel the mystery of natural enzymes completely. Because supramolecular interactions play important roles in both substrate recognition and the process of enzyme catalysis, a large quantity of artificial enzymes based on supramolecular platforms have been developed. Many complexes ranging from assembled nanometer scale objects to synthetic macromolecules are used to design artificial enzymes through supramolecular interactions. Owing to the exciting properties, enzymes are anticipated to be promising green catalysts in future. However, the high cost and low stability limit the application of natural enzymes. Artificial enzymes with catalytic capacity and substrate selectivity attract chemists’ much attention. Although most artificial enzymes designed using supramolecular strategies still cannot compete with natural enzymes on substrate selectivity and catalytic efficiency, the designed artificial

Current Opinion in Structural Biology 2018, 51:19–27

enzymes mentioned in this critical review overcome some shortcomings of natural enzymes, for example, high cost and low stability. Further studies will focus on developing multifunctional enzymes and exploiting the multiple application of enzymes in different areas, for instance, life science, environment, and materials.

Acknowledgements This work was supported by the National Natural Science Foundation of China (21420102007, 21574056, 91527302), and the Chang Jiang Scholars Program of China.

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Current Opinion in Structural Biology 2018, 51:19–27