Chapter 1
Preparation of Artificial Metalloenzymes

Jared C. Lewis and Ken Ellis-Guardiola

Department of Chemistry, University of Chicago, 5735 S. Ellis Ave., Chicago, 60637, IL, USA

1.1 Introduction

Artificial metalloenzymes (ArMs) have the potential to merge key benefits of transition metal catalysts, particularly their ability to catalyze a wide range of challenging transformations, with those of enzymes, including their evolvability and capacity for molecular (i.e., substrate) recognition [1]. These topics and more are discussed in detail elsewhere in this volume, but their pursuit requires robust methods for ArM formation. Such methods are in and of themselves quite challenging to develop. Site-specific metal incorporation is required to ensure that single-site catalysts can be obtained. Compatibility with a wide range of metals and scaffolds is desirable to maximize the range of chemistries that can be explored. Compatibility with aqueous, ideally aerobic, reaction conditions and a wide range of functional groups, including those found in cellular milieu, are also important. An additional synthetic challenge is faced for ArMs generated from preformed catalysts, since these inherently reactive molecules must first be linked to scaffold anchoring moieties to generate ArM cofactors.

The hybrid nature of ArMs also complicates their characterization since distinct methods have conventionally been used for analysis of transition metal complexes and proteins. Various spectroscopies, including UV/Vis and electron paramagnetic resonance (EPR), can provide some insight into the metal primary coordination sphere [2], while dichroism spectrum (CD) and fluorescence spectroscopies can provide information on scaffold folding [3-5]. In some cases, NMR spectroscopy can also be used, but its utility is often limited by the high molecular weight of many scaffold proteins [6]. Inductively coupled plasma-mass spectrometry (ICP-MS) can be used to determine scaffold:metal stoichiometry, but not metal location within the scaffold [6]. High resolution MALDI and ESI MS can also be used to determine extent of cofactor incorporation and scaffold modification in general [4]. Of course, X-ray crystallography remains the best option for unambiguously charactering metal location and coordination environment within ArMs, but this technique is often complicated by conformational flexibility and variable occupancy of introduced metal centers [7].

Despite these challenges, a large number of methods have been developed that possess some or all of the properties noted above. The aim of this chapter is to provide an overview of key methodology developments. These will be broken into sections in which scaffold metalation is governed predominately by metal binding by scaffold residues (Section 1.2), non-covalent cofactor binding either to the catalyst itself or to a catalyst substituent (Section 1.3) and, finally, covalent scaffold modification using functionalized cofactors (Section 1.4). ArM formation often involves elements of multiple methods (e.g., ligation of a metal in a covalently linked cofactor or metalation of ligands that are introduced via a non-covalent scaffold binding), but this classification helps to address many unique features, advantages, and disadvantages of different methods of ArM formation.

1.2 ArM Formation via Metal Binding

A wide range of homogeneous metal catalysts can be prepared by combining appropriate quantities of a metal catalyst precursor (M) with one or more small molecule ligands (L) [8]. Several of the 20 canonical amino acids possess residues capable of binding to a wide range of transition metals via N, O, or S coordination. Protein scaffolds can organize these residues into well-defined three-dimensional chiral arrays metal binding sites. The reactivity conferred to metal centers by these binding sites has led to the evolution of metalloenzymes that catalyze a range of challenging organic transformations in nature [9], including nondirected C-H bond functionalization [10]. Inspired by the synthetic power of these natural metalloenzymes, researchers have explored the use of protein scaffolds as ligands for nonnative metal ions to generate ArMs that catalyze a variety of organic transformations (Figure 1.1) [11].

Figure 1.1 Approaches to generate ArMs via metal binding.

1.2.1 Repurposing Natural Metalloenzymes

Given their inherent metal-binding capabilities, natural metalloenzymes have obvious potential as scaffolds for ArM formation. In addition to metal binding, many metalloenzymes have active sites that evolved to bind small molecule substrates, providing additional space for unnatural substrates to bind. Of course, conditions must first be developed to extract native metal ions from a metalloenzyme of interest and to incorporate the desired metal ion or fragment without denaturing the scaffold. Once this is accomplished, however, it is often possible to incorporate a range of metal ions into the scaffold, and established methods for characterization of the native metalloenzyme can often be applied to the resulting ArM.

1.2.1.1 Carboxypeptidase A

Emil Kaiser's research group at the University of Chicago was one of the first to leverage the metal binding site of a natural metalloenzyme to form ArMs with novel reactivity. Carboxypeptidase A (CPA), a Zn(II)-containing metalloenzyme containing a His/His/Glu binding site, was dialyzed against 1,10-phenanthroline to generate the apoenzyme, which was subsequently metalated with a variety of metal(II) salts. The Cu(II)-CPA construct was found to catalyze the oxidation of ascorbic acid and to exhibit Michaelis-Menten kinetics, mimicking the activity of other Cu(II)-containing redox enzymes [12]. While this work established the potential for a metal binding site to be employed for nonnative metal binding and catalysis, unspecified spectroscopic characterization was reported to indicate significant perturbation of the coordination environment around the metal. This alteration was later confirmed by crystallographic studies using Hg(II)-CPA, which highlighted the importance of characterizing the primary coordination sphere of metal fragments incorporated into protein scaffolds [13].

1.2.1.2 Carbonic Anhydrase

Carbonic anhydrases (CAs), also Zn(II)-containing metalloenzymes but containing His3 binding sites, have subsequently been utilized for ArM formation by a number of researchers. As in the case of CPA, zinc(II) can be removed from CAs by dialysis against a chelating agent (1,10 phenanthroline or 2,6-pyridinedicarboxylate) to afford the apoproteins [14]. Incubation of the apoprotein with metal(II) salts results in metal-substituted CAs. These nonnative constructs were initially explored for their interesting spectroscopic and structural properties, including significantly distorted coordination geometries [15, 16]. Kazlauskas and Soumillion later demonstrated that substitution of bovine carbonic anhydrase (bCA) isoforms I and II and human carbonic anhydrase isoform II (hCAII) with manganese(II) afforded redox-active variants of the enzyme that exhibited peroxidase-like activity [14, 17]. Incubating apo-CA with substoichiometric quantities of Mn(OAc)2 or excess MnCl2 followed by dialysis against buffer provided ArMs free of free metal salts. Mn(II) loading was confirmed by loss of native CA activity and quantitated by ICP-AES. Alkene epoxidations catalyzed by these ArMs proceeded with generally low to moderate yields and enantioselectivities.

One of the challenges to preparing ArMs via metal substitution of apo-metalloenzymes is the possibility for nonspecific binding of metals to non-active site residues. For example, metalation of apo-hCA(II) with [Rh(cod)2]BF4 led to extensive nonspecific binding, with 6-8 rhodium ions bound to the protein monomer as determined by ICP-MS [18]. Unlike Mn(II) salts, which show low epoxidation activity relative to the corresponding CA ArMs, [Rh(cod)2]BF4 can efficiently catalyze the target reaction, enabling a nonselective reaction pathway that can compete to the detriment of the overall stereoselectivity of the transformation. To address this issue, Kazlauskas used mutagenesis to remove from hCAII several surface histidine residues that were hypothesized to be sites of nonspecific Rh binding. Mutating these histidine residues to arginine, phenylalanine, or alanine provided 9*His-hCAII-[Rh], which bound significantly fewer Rh ions (an average of 1.8 Rh/hCAII) and provided improved selectivity for hydrogenation of cis-stilbene relative to competing isomerization of this substrate to trans-stilbene. Kazlauskas later demonstrated that metalation of 9*His-hCAII with [Rh(CO)2(acac)] reduces the Rh/hCAII ratio to 1.2. The resulting ArM catalyzed styrene hydroformylation with improved selectivity for the linear aldehyde over free [Rh(CO)2(acac)] or wild-type hCAII-[Rh], indicating that surface-bound rhodium preferentially yields the branched aldehyde and negatively impacts the selectivity of the hybrid [19].

A more recent study provided additional insights into the preparation and characterization of Rh-substituted CAs [6]. Evaluating apo-hCAII metalation by a panel of Rh complexes revealed that the extent of nonspecific surface binding by the metal is determined not only by the presence of coordinating residues outside of the active site but also by the identity of the ancillary ligands on the Rh complex. Extent of metalation was confirmed by competitive metalation with Co(II), which, when bound to hCAII, is known to catalyze the hydrolysis of 4-nitrophenyl acetate, enabling...