university of groningen artificial metalloenzymes bos, jeffrey · artircial metalloenzymes have...

University of Groningen

Artificial MetalloenzymesBos Jeffrey

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1Artificial metalloenzymes have emerged over the last decades as an attractive approach towards combining homogenous catalysis and biocatalysis A wide variety of catalytic transformations have been established by artificial metalloenzymes thus establishing proof of concept The field is now slowly transforming to take on new challenges This chapter describes design strategies of these artificial metalloenzymes and focusses on advances in this field over the last two years

Part of this chapter is an adaptation from the original paper J Bos and G Roelfes Curr Opin Chem Biol 2014 19 135-143

Artificial Metalloenzymes

phd_thesis_bookindb 1 7-5-2014 141015

2

Chapter 1

1

11 IntroductionThe concept of artificial metalloenzymes was introduced in 1978 by Wilson and

Whitesides[1] In their seminal paper a non-catalytically active protein was converted into an enzyme capable of performing an abiotic reaction This was achieved by the introduction of a transition metal complex into the protein environment of avidin In general artificial metalloenzymes aim to combine the attractive features of transition metal catalysis and bio-catalysis ie the broad scope of reactions catalyzed by transition metals and the high selectivities (both regio- as enantioselective) bioorthogonality and mild conditions achieved by bio-catalysis Essential for activity and high enantioselectivities obtained by artificial metalloenzymes is the second coordination sphere which is provided by the bioscaffold The second coordination sphere is defined as the combination of interactions that the bioscaffold provides to the substrate the transition state and metal complex eg hydrophobic electrostatic and hydrogen bonding interactions (figure 1) This is in contrast to conventional homogenous catalysis in which selectivity and rate acceleration are the result of an interplay between the transition metal and the ligand defined as the first coordination sphere

The use of artificial metalloenzymes was long considered a curiosity but after its revival in the early 2000rsquos the field has become very active and vibrant The proof of concept has been well established in the last decade and the field is currently slowly transforming to take on new challenges such as novel catalytic reactions cascade reactions and ultimately chemistry in vivo This chapter focuses on key examples

Interactions- hydrophopic H-bonding

electronic etc

Anchor metal complex- covalent

- non-covalent- dative

M Metal- transition metal

1st coordination sphere-synthetic ligand

2nd coordination sphere- bio-scaffold

M substrate

figure 1 Schematic representation of the concept of artificial metalloenzymes


3


1

and recent developments in the field of artificial metalloenzymes with the emphasis first on the design of such catalysts followed by recent new design and selected applications in catalysis A more comprehensive overview of this field can be found here[23]

12 DesignSeveral key parameters have to be considered for the construction of an

artificial metalloenzyme (figure 1) First depending on the reaction that has to be catalyzed a transition metal capable of performing this desired transformation has to be selected Second a decision has to be made about the anchoring strategy of the transition metal (complex) to the bioscaffold Finally a bioscaffold which provides the second coordination sphere has to be selected This section will describe the latter two key parameters and will provide key examples to illustrate them

121 Anchoring StrategiesThe transition metal (complex) can be anchored to the bioscaffold via several

anchoring strategies Three major classes are distinguished in this chapter covalent and non-covalent anchoring and the introduction of biosynthetically incorporated unnatural amino acids (UAA) capable of binding metals (figure 2)

1211 Covalent AnchoringCovalent attachment of the metal complex is generally achieved by creation

of a chemical bond between the bio-molecular scaffold and the ligand for the metal For this the bioscaffold needs to undergo a post-biosynthetic modification This strategy requires a unique reactive residue in the bioscaffold eg a nucleophile and an equivalent partner in the ligand eg an electrophile that can react with each other in a bio-orthogonal fashion As a consequence both reaction partners need to be orthogonal to the bioscaffold to achieve selective modification Several strategies that have been employed will be described below (scheme 1)

The most common bioconjugation strategy[4] used for the construction of artificial metalloenzymes entails alkylation of a cysteine residue For this a cysteine

M MM M

Synthetic

Bio-scaold

Synthetic Bio-scaold

M

covalent

M

non-covalent

M

dative

M

biosynthetically incorporated UAA

figure 2 Representation of anchoring strategies applied in the construction of artificial metalloenzymes


4

Chapter 1

1

needs to be introduced into the bioscaffold by mutation at the appropriate location or a native cysteine can be used As for its partner a variety of reactive electrophilic groups on the ligand can be applied For example α-halogenated carbonyl compounds can react with the cysteine to accomplish the bioconjugation[5-7] In an example Distefano introduced an iodoacetamide functionalized phenanthroline into the adipocyte lipid binding protein (ALBP) via this strategy[5] The resulting conjugate was used in the copper(II) catalyzed hydrolysis of amide bonds of several unactivated amino acid esters with eersquos up to 86 Maleimide substituted ligands is another class of electrophiles that is often used for cysteine alkylation[8-10] For example Reetz and coworkers used this method to couple a maleimide functionalized phenanthroline to the thermostable enzyme tHisF[8] The resulting artificial metalloenzyme was constructed as a platform for directed evolution but no catalysis was performed Lu and coworkers used a different approach to anchor a Mn(salen) complex to apomyoglobin[1112] Double anchoring of the complex was achieved by the reaction of cysteines introduced into the scaffold by mutation with methane thiosulfonate groups on the complex forming a disulfide bridge between the complex and the protein The resulting artificial metalloenzyme was used in the enantioselective and chemoselective sulfoxidation of thioanisole with eersquos up to 51 with no overoxidation of the product[11] By changing the position of dual attachment of the metal complex in the scaffold the ee increased up to 66[12] This result showed the importance of a correctly placed metal complex in a bioscaffold to achieve high selectivities For a long time these artificial metalloenzymes achieved the highest eersquos in catalysis of

Protein SR

O

O

RX

X = Br I

N

O

O

R

Protein SN

O

O

R

H3C S SO

O

R

Protein SS

R

N R

O

NProtein S R

O

N

O

O

NH

ONH2

O

H R

Protein SN

O

O HN

O

NR

1

2

OPO

REtO

O2N

Protein OPO

REtO

Protein Protein

OHSH

scheme 1 Overview of reaction types applied in the covalent anchoring of metal complexes in a bioscaffold in which R denotes the ligand for a metal


5


1

all artificial metalloenzymes created by covalent anchoring Kamer and coworkers developed a general two-step method to introduce phosphine ligands into a variety of bioscaffolds In the first step a cysteine of the protein was transformed into a hydrazine and then reacted with the phosphine ligand containing an aldehyde as reactive handle[13] This two-step method enabled the introduction of phosphine ligands that otherwise would react non-specifically with a cysteine residue No catalysis was reported with these conjugates

Nucleophilic residues other than cysteines can be used for anchoring the metal complex as well The native serine residue in serine hydrolases was used to introduce an organometallic NCN-pincer into the lipase cutinase[14] Known inhibitors of serine hydrolases ie phosphonates bearing a good leaving group were used to achieve the anchoring Only the conjugation of the NCN-pincer was demonstrated and no catalysis was reported

A disadvantage of the selective cysteine alkylation strategy is that no other cysteines can be present in the scaffold Removal of the other cysteines by mutagenesis can circumvent the selectivity problem However the structure of the scaffold can be affected by this removal Lewis and coworkers demonstrated the use of the unnatural amino acid p-azidophenylalanine to achieve unique selectivity of anchoring in the scaffold[15] The unnatural amino acid was introduced using the expanding genetic code concept[1617] and through strain-promoted azidendashalkyne cycloaddition several ligands were introduced in the thermostable protein tHisF The resulting artificial metalloenzymes catalyzed rhodium cyclopropanation reactions and Si-H insertion reactions However no enantioselectivity was observed and the activity of the artificial metalloenzyme was lower than that of the metal complex alone

1212 Non-covalent AnchoringNon-covalent or supramolecular anchoring of the metal complex to the

bioscaffold is based on a variety of supramolecular interactions eg hydrophobic interactions hydrogen bonds and electrostatic interactions This strategy puts some restraints on the bioscaffold as it should provide an environment in which binding can occur and still leave enough space for the reactants of the catalytic reaction In addition strong binding of the metal complex with the scaffold is required to avoid catalysis outside the scaffold resulting in lower selectivities On the other hand no post-biosynthetic modification of the scaffold is required and the artificial metalloenzyme is created by self-assembly

The most famous example of non-covalent anchoring is based on the tight binding of biotin to (strep)avidin Whitesides successfully used a biotinylated Rh-bisphosphine complex bound in avidin as the first artificial metalloenzyme for enantioselective hydrogenation albeit with moderate eersquos up to 41[1] Ward continued this approach of introducing biotinylated metal complexes into the protein scaffold but changed to streptavidin instead of avidin as key innovation[18] Chemogenetic


6

Chapter 1

1

approaches improved the hybrid catalyst ie using a longer spacer between the Rh complex and biotin and by introducing a S112G mutation in streptavidin As a result 96 ee was obtained in the catalytic enantioselective hydrogenation reaction Saturation mutagenesis of position S112 led to various artificial metalloenzymes which gave rise to both enantiomers of the product (lt95 ee) of the hydrogenation reaction[19] Since then more streptavidin-based artificial metalloenzymes have been constructed using biotinylated diamine-d6 transition metal piano stool complexes of Ru Rh and Ir These were applied successfully in several reaction types (see section 14 catalysis)[2021] Another strategy for the supramolecular anchoring of a metal complex for the construction of an artificial metalloenzymes involves the replacement of a ldquonaturalrdquo cofactor with a synthetical metal complex thus using the existing natural active site For example myoglobin a heme protein has been used extensively Watanabe demonstrated that Mn-salen Cr-salen and Ru-phebox complexes could be inserted into apomyoglobin and then applied in catalysis for example for catalytic sulfoxidations albeit with low enantioselectivities[22-25]

The examples discussed so far used a defined binding pocket in the scaffold to bind the metal complex Roelfes and coworkers have demonstrated that no pre-existing binding pocket is needed to construct an artificial metalloenzyme[2627] Their approach is based on anchoring a metal complex in the structure of DNA In the first generation DNA-based catalysts a catalytically active Cu(II) complex was linked to an acridine moiety that intercalates into DNA The DNA-based catalysts were applied in the enantioselective Diels-Alder reaction with moderate eersquos up to 53[26] In a second generation the Cu(II) metal complex eg based on bipyridine type ligands did not contain a separate DNA binding moiety These DNA-based catalysts were successfully applied in a variety of Lewis acid catalyzed enantioselective reactions (eersquos up to 99) including Diels-Alder Michael addition and Friedel-Crafts alkylation reactions

1213 Dative AnchoringDirect coordination of a metal ion to native residues of the bioscaffold that

can act as ligands ie N O and S functional groups is regarded as dative anchoring Natural metal binding enzymes use this strategy to bind their catalytically active metal ion[28] Replacement of the native metal ion by a nonnative metal ion is a strategy that has been employed for the construction of artificial metalloenzymes For example Kazlauskas and coworkers have converted human carbonic anhydrase (hCA) into a peroxidase by substituting the three histidine ligated Zn(II) ion by a Mn(II) ion[29] Moderate enantioselectivities were obtained in the epoxidations of a variety of styrene substrates ie up to 67 ee The use of naturally occurring metal binding sites can be a limiting factor in the type of metal ions to be used since different metals need different coordination environments thus limiting the catalytic scope However the engineering of metal binding sites in nonnative metal binding proteins


7


1

can expand the scope of coordination environments Reetz and coworkers have engineered a Cu(II) binding site into the thermostable protein tHisF by introducing a histidine-histidine-aspartic acid triad at an appropriate position and the resulting artificial metalloenzyme was used in the Cu(II) catalyzed Diels-Alder reaction although moderate enantioselectivities were obtained ie up to 46 ee[30]

1214 Non-natural Amino AcidsThe geometrically precise placement of residues to provide the correct

coordination environment for the metal ions is quite challenging This difficulty can be circumvented by the introduction of genetically encoded metal ligands using the expanding genetic code approach[17] Schultz and coworkers introduced the metal binding ligand bipyridylalanine (BpyAla) into the Z-domain protein using this strategy[31] An aminoacyl-tRNA synthetase pair from Methanococcus jannaschii was evolved to incorporate BpyAla by biosynthetic means This approach was used to construct an artificial metalloenzyme by the introduction of BpyAla in the Catabolite Activator Protein from E coli (CAP)[32] The resulting Cu(II) artificial metalloenzyme was capable of selectively cleaving DNA In another study the metal binding moiety 2-amino-3-(8-hydroxyquinolin-3-yl)propanoic acid (HQ-Ala) was introduced into a Z-domain protein[33] When metalated with Zn2+ ions the HQ-Ala residue in the protein scaffold acted as a fluorescent probe and as a heavy metal binding site for phasing in X-ray structures However artificial metalloenzymes constructed by this design have not been used in asymmetric catalysis to date

122 BioscaffoldsThe 2nd coordination sphere plays a key factor in artificial metalloenzymes and

is provided by the bioscaffold Therefore a few important factors needs be taken into consideration when choosing a bioscaffold The bioscaffold should show stability over a desired temperature and pH range Additionally the scaffold has to show tolerance towards organic solvent ie reactants used in catalysis often need a particular amount of organic solvent to be dissolved Another choice that has to be made is whether a scaffold has an existing active site or one has to be created

Artificial metalloenzymes constructed from a scaffold possessing an existing active site has advantages First the stability eg temperature and pH of the scaffold can be estimated from the apoform Second the 2nd coordination sphere is already in place However the binding site should be large enough to accommodate the metal complex and leave space for the reactants of the catalyzed reaction Most binding sites of proteins bind the binding partner andor reactant perfectly but do not provide the extra space that is needed Only a few bioscaffolds used successfully in enantioselective catalysis have such a binding site for the construction of artificial metalloenzymes for example avidinstreptavidin[134] bovine serum albumin (BSA)[35] and (apo)myoglobin[36] The creation of a new active site in a bioscaffold offers


8

Chapter 1

1

more freedom as the 2nd coordination sphere can be designed to fit the needs of the catalyzed reaction Moreover the number of potential scaffolds for the construction of artificial metalloenzymes can be greatly expanded However designing a whole protein scaffold from scratch in silico is still very challenging and requires a lot computer calculation power[37] This strategy has not been applied to the construction of an artificial metalloenzyme yet However redesigning existing sites using modeling approaches based on X-ray structural data has been applied for to construction of artificial metalloenzymes[3839] For example using computational design Lu and coworkers constructed an artificial nitric oxide reductase[38] A non-heme iron binding site was created by the introduction of two additional histidines and a glutamate into the distal pocket of myoglobin containing an iron heme complex The accuracy of the predicted model was confirmed by overlaying the X-ray structure of the predicted and experimental proteins

The creation of a new active site in a non-existing pocket can also be accomplished without de novo design of the active site Roelfes has reported the construction of an artificial metalloenzyme using DNA as bioscaffold The active site was created in the chiral groove of DNA by supramolecular anchoring of a Cu(II) complex The chiral information of the microenvironment created by the DNA was successfully transferred in several Lewis acid catalyzed reactions as described in the previous section[2640]

Peptides are small compared to proteins but are easier to design de novo and potentially still have enough functional diversity to generate a defined 2nd coordination sphere[41] For example Ball and coworkers have developed peptide based dirhodium complexes by employing as ligands the carboxylate groups of aspartic acid residues in a dimeric nonapeptide in a DxxxD motif[42] This metallopeptide was applied successfully in asymmetric Si-H insertion reactions with excellent eersquos ie up to 92 ee[43] (see more in section 132 Peptides as Bioscaffolds)

123 OptimizationArtificial metalloenzymes have the benefit that both the metal complex and the

bioscaffold can be optimized independently This optimization approach is referred to as the chemogenetic approach[44]

The optimization of the metal complex is often achieved by rational design In contrast to metal complexes used in homogenous catalysis no chiral version of the metal complex is needed which often simplifies the synthesis

On the other hand optimization of the scaffold can be achieved by several approaches[45] The empirical approach is based on visual inspection often using an X-ray structure of the non-functionalized protein or based on homology models to introduce mutations The theoretical approach uses computer algorithms to determine the effects of mutations on the active site The semi-theoretical approach is a combination of the approaches mentioned above However these three approaches


9


1

already require some knowledge of the structure of the scaffold Directed evolution of the bioscaffold is a non-biased approach for optimization

Optimization of artificial metalloenzymes is often accomplished using a semi- theoretical approach The resulting optimized model is checked by X-ray structures to refine the model As an example Ward and coworkers used this approach to optimize the streptavidinbiotin artificial metalloenzyme in an asymmetric transfer hydrogenation reaction (ATHse) Via computer modeling a histidine residue was introduced into the active site that coordinated with either Ir or Rh[46] A dual anchoring of the Ir or Rh metal with the ligand and the histidine residue improved catalytic efficiency (see more details in section 141 Asymmetric Transfer Hydrogenation)

Optimization by directed evolution for example by error prone PCR depends on random mutagenesis of the bioscaffold and screening of the resulting library for catalytic activity High-throughput screening is then needed to find a positive hit An artificial metalloenzyme constructed via supramolecular or dative anchoring speeds up the optimization as it does not require time consuming functionalization of the scaffold However random mutation of the bioscaffold needs a large library to include all possible mutations randomly distributed The library expands greatly as a function of the size of the bioscaffold and such a large library is difficult to screen This is because screening for enantiomeric excess of the catalyzed reaction requires high throughput GC or HPLC analysis since no color assay exists for these improvements For that reason a method was developed in which a more focused library was constructed The Combinatorial Active-site Saturation Test (CAST) focusses on the active site residues and was applied successfully to improve a streptavidinRh-biotin artificial metalloenzyme from 23 ee to 65 ee in three rounds of mutagenesis[47]

13 New Designs of Artificial MetalloenzymesThe key parameter in artificial metalloenzyme design is the second coordination

sphere provided by the biomolecular scaffold ie proteins peptides DNA etc which provides the supramolecular interactions that are expected to contribute to achieving enzyme-like rate accelerations and selectivities Hence the choice of biomolecular scaffold and the position and mode of anchoring of the transition metal complex are of the utmost importance as described in the previous sections Many of the examples of artificial metalloenzymes described in the literature to date rely on a limited number of protein scaffolds such as streptavidin[48] BSA and apomyoglobin[49] These have in common that they have a pocket that is large enough to accommodate the metal complex and leave enough space for the substrate This section describes new scaffolds and design approaches that have been introduced in the last two years


10

Chapter 1

1

131 Proteins as BioscaffoldsA new method for the construction of an artificial metalloenzyme by focusing

on the binding mode of substrates was reported by Meacutenage and coworkers (figure 3a)[50] The periplasmic nickel-binding protein NiKa was used as a host for iron catalyzed sulfoxidation reactions Based on the crystal structures of a hybrid of the NiKa protein with a bound iron complex of an N2Py2 ligand substrate molecules containing a C6H5-S-CH2-X motif were screened by molecular docking By constraining the distance between the Fe of the complex and the S of the substrate in accord with the suggested Fe-OS transition state a family of potential substrates with a Ph-S-CH2-CONH-Ph motif was identified Several members of this family were converted with high turnover numbers in the sulfoxidation reaction and no overoxidation was observed The experimental data were in agreement with the predicted relationship between the substrate and protein scaffold However only up to 10 ee was obtained

Using rational design based on X-ray data a Sc3+ binding site was constructed on the rigid tubular protein [(gp5βf)3]2 by Ueno and coworkers[51] The binding site

H2N

R

OH

NH

R

O

OHOH

Tyr residuesOH

OHSHOH

OHS

Sc3+

Sc3+

Cys introduction

Molecular

docking calculations

Sulfides

FeII

SNH

O

X

S NH

OO

X

NaOCl

TON up to 199

FeIICJBgtdockexe

A

B

figure 3 New scaffolds and design approaches for artificial metalloenzymes A) Design of an artificial metalloenzyme based on the binding mode of substrates Based on X-ray data of the nickel-binding protein NiKa sulfides where screened by molecular docking High turnover numbers in the sulfoxidation reaction were observed[50] B) A scandium binding side was created on the tubular protein [(gp5βf)3]2

[51] Tetradentate coordination of a Sc3+ ion was achieved by dative interactions with hydroxy groups of tyrosine and by an introduced bipyridine The resulting artificial metalloenzyme was used in the epoxide ring opening reaction of cis-stilbene oxide with aniline


11


1

on [(gp5βf)3] was created by a combination of a conjugated synthetic ligand and dative interactions with the amino acids of the scaffold (figure 3b) Tetradentate coordination of a Sc3+ ion was achieved by positioning a bipyridine ligand at an appropriate distance from the OH groups of Tyr pairs as estimated from the crystal structure This artificial metalloenzyme proved capable of catalyzing the epoxide ring opening reaction of cis-stilbene oxide with aniline derivatives with almost a 3 fold rate enhancement compared to a metal complex alone A small enantiomeric excess was observed using this design ie up to 17 ee

132 Peptides as BioscaffoldsPeptides are small compared to proteins but potentially still have enough

functional diversity to generate a defined 2nd coordination sphere Much effort has been devoted to the design of metallopeptides as functional mimics for metalloenzymes[52] However here the focus will only be on metallopeptides used for enantioselective catalysis

Ball and coworkers have developed de novo designed peptide-based di-rhodium complexes by employing as ligands the carboxylate groups of aspartic acid residues in a dimeric nonapeptide in a DxxxD motif[42] These were used before in asymmetric Si-H insertion reactions with excellent eersquos[43] However obtaining the opposite enantiomer of the product using these metallopeptides without turning to using D-amino acids often needs rigorous editing of the active site and is difficult to predict In this specific case predictions are especially problematic since these bis-peptides complexes were formed as mixtures of parallel and anti-parallel isomers Therefore a screening method was developed in which it was assumed that sequences optimized for monomeric peptide complexes are also selective in the dimer-complex form as was suggested in previous studies[5354] Hence libraries of monomeric peptides where synthesized on beads and screened in a high-throughput fashion Hits were identified and the corresponding bis-peptides prepared and tested in catalysis resulting in the discovery of metallopeptides that gave up to 97 ee of the si product in the catalyzed cyclopropanation reaction From the same library another metallopeptide was identified that gave 90 ee of the re product

Iridium-catalysed transfer hydrogenation reactions were performed using simple tripeptide Gly-Gly-Phe based iridium catalyst in aqueous media by rational design[55] These showed high turnover frequencies for the transfer hydrogenation of a variety of aldehydes ketones and imines (up to 391 h-1) Additionally the biologically important regeneration of NADH was demonstrated using this catalyst It was suggested that the tripeptides act as Noyori-type catalysts in which iridium binds the N-terminal amine and the adjacent amide group

A tetrapeptide containing a double methylated histidine was used to form an N-heterocyclic carbene that acted as a ligand for rhodium[5657] The sulphur atom of a methionine in the same peptide acted as chelator for the rhodium complex The


12

Chapter 1

1

resulting catalytic peptide was capable of hydrosilylation of 4rsquo-fluoroacetophenone in organic solvents with high chemoselectivities towards the silylether compared to the silylenolether (up to 83) but no enantioselectivities were obtained

The natural cyclic decapeptide gramicidin S served as chiral host for peptide-based bisphosphine ligands for rhodium and palladium catalysis in organic media[58] Modeling studies of the peptide were used to determine the positions for the bisphosphine ligands and the resulting catalysts were able to catalyse rhodium based transfer hydrogenation reactions up to 52 ee and asymmetric palladium catalysed allylic alkylations up to 15 ee

133 DNA as BioscaffoldSimilar to proteins and peptides DNA can offer a defined chiral 2nd

coordination sphere for the construction of an artificial metalloenzyme Roelfes and Feringa have introduced the concept of DNA-based asymmetrical catalysis[26] and applied it successfully to several Cu(II)-catalyzed reactions[40]

Two approaches to controlling the enantiomeric outcome of the catalyzed reaction have been reported By changing the denticity of the ligand coordinated to the copper(II) ion ie bipyridine versus terpyridine ligands the opposite enantiomers of Diels-Alder and Friedel-Crafts alkylation reaction products were obtained[59] In a study by Smietana and Arseniyadis ds-DNA made from L-nucleic acids instead of the natural occurring D-nucleic acids was used as a scaffold The resulting DNA-based catalyst gave rise to the mirror image products in the Cu(II)-catalyzed Friedel-Crafts alkylation and Michael addition reactions compared to using natural DNA[60]

A new DNA-based catalyst was created by covalent anchoring of a Cu(II) complex to double stranded DNA through a tethered cisplatin moiety The resulting hybrid catalyst was used successfully in Diels-Alder reactions and Friedel-Crafts alkylation reactions and could be recycled up to 10 times without loss of activity and enantioselectivity[61]

In addition to doubled stranded DNA G-quadruplex DNA has also been investigated as scaffold for DNA-based catalysis Human telomeric G-quadruplex DNA in combination with Cu2+ ions was found to catalyze the enantioselective Friedel-Crafts alkylation and Diels-Alder reactions with good enantioselectivities ie up to 75 and up to 74 respectively[6263] The opposite enantiomer of the products could be obtained by switching from the antiparallel to the parallel conformation of the G-quadruplex The hybrid of a Cu(II) porphyrin and G-quadruplex DNA resulted in a catalyst capable of performing Diels-Alder reactions (up to 69 ee) and residues which had an effect on catalysis were identified[64] Finally Cu(II) phenanthroline based ligands in combination with G-quadruplex DNA could be used in the intramolecular Friedel-Crafts alkylation reaction with moderate eersquos (up to 26)[65]


13


1

14 CatalysisInitially artificial metalloenzymes were constructed out of curiosity and their

catalytic capabilities were often tested with benchmark reactions These included simple oxidation reactions Lewis acid catalyzed Diels-Alder reactions etc that proved very effective in proof-of-principle studies[49] For an in-depth overview a number of review articles are available[349] The field is now slowly transforming and more challenging catalytic reactions can be performed including reaction types that have no equivalent in homogeneous or enzyme catalysis The next section will describe these advances for artificial metalloenzymes used in enantioselective catalytic transformations of the last two years (scheme 2)

141 Asymmetric Transfer HydrogenationSalmain and coworkers reported on two new asymmetric transfer

hydrogenationases (ATHase) metalloenzymes Ru(II) and Rh(III) d6-piano stool complexes were bound to papain via covalently attached 22rsquo-dipyridylamine ligands[66] These artificial metalloenzymes were employed in the transfer hydrogenation of trifluoracetophenone (TFAP) using formate as hydrogen source resulting in high conversions but low eersquos Additionally these papain constructs where used as artificial formate dehydrogenase for NAD(P)H regeneration[9]

Similar Ru(II) and Rh(III) complexes where anchored in a non-covalent fashion to bovine β-lactoglobulin (β-LG) by using a 22rsquo-dipyridylamine ligand equipped with a long aliphatic chain that can be bound by β-LG[67] Moderate eersquos were obtained in the transfer hydrogenation of TFAP using formate Based on X-ray structural information the observed enantioselectivities were explained by interactions of the complex with a loop in β-LG which restricts the number of conformations[68]

The most successful examples to date of artificial ATHases have been reported using the streptavidinbiotin systems Based on their experience with the ATH of ketones[69] the Ward group has focused on the ATH of imines[70] Screening revealed [CpIr(Biot-p-L)Cl] streptavidin as the most promising catalyst Both enantiomers of the reduction of a prochiral imine (1-methyl-34-dihydroisoquinoline) could be obtained by a single point mutation of S112 in streptavidin (R)-Selectivities up to 96 ee were obtained with a small amino acid at position 112 in the active site (glycine or alanine) In contrast cationic residues (lysine or arginine) at this position resulted in (S)-selectivities up to 78 ee Based on X-ray data Lys121 was identified as playing a role in the protonation step and it was proposed that both the ketone and imine reduction proceeds through the same mechanism

Next the activity of the artificial ATHase was further improved[71] The introduction of lipophilic residues (R84A-S112A-K121A) in the active site led to an 8-fold increase in catalytic efficiency compared to wild-type streptavidin as host and a 2-fold increase compared to the Ir-complex alone However only moderate eersquos


14

Chapter 1

1

were obtainedIn an alternative approach based on computational studies a histidine was

R

O

R

lowastR

OH

R

O

OH OH

ADPN+

NH2

O

O

OH OH

ADPN

NH2

OH H

SO

N2

O O

O

H

SO

O

R1

R2

R1

R2O

st-DNA Cu complex

MOPS pH 65

Xln10A Mn complex

Phosphate Buffer pH 70

Formate buffer pH 75 40degCβ-LG Ru complex

Phosphate buffer pH 7 HNCO2Na

β-LG Ru complex

NR lowast

NHR

(Strept)avidin IrRh-biotin complex

MOPS buffer HNCO2Na

hCA II Ir-biotin complex

MOPS buffer pH 75 HNCO2Na

NTos

N TosHCl pH 20 40 t-butanol

MjHSP Ru Grubbs-Hoveyda type catalyst

Acetate pH 40Streptavidin Ru Grubbs-Hoveyda type catalyst

α-chymotrypsin Ru Grubbs-Hoveyda type catalyst

OHHO

HO O

OH

N

O

OHHO

HO O

OH

N

OH2O

O

NH

OPivlowast

NH

O

R

streptavidin [RhCpbiotinCl2]2MOPS 20 MeOH

O

RN

N

Olowast

R2N

N

OR1

st-DNA Cu complex

MOPS pH 65 40 R1OH

R

ref [66]

ref [9]

ref [70] [46] [71]

ref [73]

ref [7]

ref [75]

ref [76]

ref [78]

ref [79]

ref [80]

ref [81]

NO

lowast

lowast

O

N

+

+

SCP-2L Cu complex

MES Buffer pH 60ref [10]

scheme 2 Reactions catalyzed by artificial metalloenzymes


15


1

introduced into the streptavidin scaffold either at positions 112 and 121 to activate and localize the metal-complex by formation of an additional dative bond with the metal[46] The modelled structures were confirmed by X-ray crystallography Both the enantiomers of the hydrogenation reaction could be obtained with up to 55 ee and 79 ee of the S- and R-enantiomer respectively depending on the position of the histidine residue Moreover the new artificial metalloenzyme displayed a 6-fold increase in turnover frequency compared to wild type streptavidin

It was demonstrated that the ATHase consisting of a biotinylated [CPIr(Biot-p-L)Cl] combined with streptavidin was still active when encapsulated in biocompatible polymersomes[72] This system remained active and proved to be stable under physiologically relevant conditions for several months indicating its potential for future applications in cells

Dative anchoring of an IrCp moiety in an genetically optimized human carbonic anhydrase II (hCAII) resulted in an artificial metalloenzyme capable of transfer hydrogenation of salsolidine with good activity and enantioselectivities up to 68 ee[73]

142 Olefin MetathesisCross metathesis (CM) could become an important tool for protein

modification due to its bio-orthogonal nature However large excesses of Grubbs-Hoveyda type metathesis catalysts are typically needed to perform these reactions in an aqueous environment Several artificial metalloenzymes capable of cross-metathesis reaction were reported Hilvert and coworkers attached the Grubbs-Hoveyda catalyst covalently to the heat shock protein from M jannaschii[74] whereas Ward and coworkers used the non-covalent strategy of biotin-(strept)avidin[75] and Matsuo and coworkers introduced the catalyst covalently to α-chymotrypsin through the intrinsic inhibition mechanism of α-chymotrypsin[76] While proof of concept was established in all cases the catalytic activity was not improved compared the metal complex alone

143 C-H Activation Cyclopentadienyl rhodium complexes such as [CpRhCl2]2 are versatile

catalysts for electrophilic aromatic C-H activation reactions For example dihydroisoquinolones can be prepared by the benzannulation reaction in good yield but no enantionselective version of this reaction existed[77] The problem lies in the fact that there is a negligible barrier for rotation of the Cp ligand and the use of a chiral Cp ligand would generate different conformations of almost the same energy Ward and Rovis reported a biotinylated [CpRhX2]2 bound in the chiral environment of streptavidin[78] Based on inspection of an auto-Dock model of biotinylated [Cp Rh(OAc)2]2 a carboxylate residue was introduced at position 112 which seemed crucial for high activity by acting as a general base The artificial metalloenzyme


16

Chapter 1

1

catalyzed the coupling of benzamides with alkenes resulting in dihydroisoquinolones in up to 86 ee An up to 92-fold acceleration compared to isolated rhodium complexes was observed This is a catalytic enantioselective reaction for which no obvious alternative ldquoconventionalrdquo approach exists

144 MiscellaneousDNA-based catalysis ie the supramolecular anchoring of a Cu(II) complex in

DNA as scaffold was used for the enantioselective oxa-Michael addition of alcohols to enones[79] Using achiral copper(II) complexes in combination with salmon testes DNA enantioselectivities up to 81 and 86 ee were achieved for the addition of methanol and propanol respectively to enones in aqueous media

Using the same strategy the intramolecular cyclopropanation of α-diazo-β-keto sulfones was also reported[80] Up to 84 ee was achieved using a hybrid of salmon testes DNA and an achiral Cu(I)complex The O-H bond insertion in H2O was observed as a major side reaction This represents the first example of DNA-based asymmetric organometallic catalysis

The introduction of an anionic manganese porphyrin into xylanase 10A from Streptomyces lividans (Xln10A) resulted in a catalyst for the enantioselective epoxidation of styrene derivatives by KHSO5 as oxidant[81] Electron donating groups on the styrene like the methoxy group at the para-position resulted in lower chemoselectivities ie 32 towards the expoxide but with the highest ee reported to date (80 R-selectivity) Differences in enantioselectivities were rationalized by docking experiments suggesting interactions of the substrate with residues in the active site

The cylindrically shaped hydrophobic cavity of the sterol carrier protein type 2 like domain (SCP-2L) was used by Kamer and coworkers to attach various nitrogen donor ligands covalently[10] Using a phenanthroline conjugate a moderate ee of 25 was obtained in the catalyzed Diels-Alder reaction

145 Cascade ReactionsCombined chemo and biocatalytic cascade reactions are highly desirable

However combining chemical and bio-catalysts is often complicated by mutual inactivation Nature solves this problem by compartmentalizing and thus spatially separating incompatible process Inspired by nature Hollmann Turner and Ward compartmentalized an iridium d6-piano stool complex within streptavidin to generate an artificial transfer hydrogenase (ATHase)[82] The ATHase was successfully included in several cascade reactions For example a double stereoselective deracemization of amines was achieved resulting in up to 99 ee Compatibility with other oxidases was demonstrated in a cascade reaction resulting in the formation of L-pipecolic acid with high enantioselectivity In addition the ATHase could act as a redox mediator to regenerate NADPH In all cases mutual inactivation of the metal catalysts and


17


1

enzymes was observed when the free Ir-complex was used demonstrating the power of encapsulation

In the Baumlckvall laboratory a cascade reaction was performed by immobilizing two catalysts namely the lipase CALB and palladium nanoparticles in siliceous mesocellular foams[83] This artificial metalloenzyme was used in the dynamic kinetic resolution of primary amines affording the product in quantitative yields and 99 ee This hybrid system was shown to have an enhanced efficiency in the dynamic kinetic resolution of an amine compared to the simple combination of the two components

15 Thesis AimsThe aim of this thesis was to develop a new design concept for artificial

metalloenzymes by creation of a novel active site at the subunit interface of dimeric proteins This concept can greatly expand the number of scaffolds applicable to the construction of artificial metalloenzymes

In a first approach we attempted the creation of an artificial metalloenzyme using a dimeric hormone peptide as scaffold[84] Despite achieving good enantioselectivities using this metallo-peptide as a catalyst the introduction of the transition-metal catalyst into the scaffold caused a significant disruption of the structure and loss of the dimerization affinity ie the catalyst predominantly existed in the monomeric form Therefore the dimeric protein Lactococcal multidrug resistance Regulator (LmrR) from Lactococcus lactis with a high dimerization affinity was selected as a new scaffold

LmrR was used for the construction of artificial metalloenzymes using the three anchoring strategies outlined in this chapter ie covalent and supramolecular attachment of a Cu(II) complex and dative anchoring of Cu(II) ions by genetically encoded non-natural amino acids The resulting hybrid catalysts were used in several enantioselective Lewis acid catalyzed reactions ie the Diels-Alder reaction the Friedel-Crafts alkylation reaction and the conjugate addition of water Furthermore the effect of the 2nd coordination sphere on the catalyzed reactions were probed by mutagenesis studies and important residues for catalysis were identified

151 OverviewChapter 2 will discuss the construction of an artificial metalloenzyme

based on LmrR using the covalent anchoring approach and its application in the enantioselective Diels-Alder reaction Chapter 3 will discuss the application of this LmrR based hybrid catalyst in the conjugate addition of water A subsequent mutagenesis study identified residues in the scaffold important for catalysis In chapter 4 a more comprehensive mutagenesis study is described in which the catalysis results of the enantioselective Diels-Alder reaction and the conjugate addition of water reaction are compared Chapter 5 describes the construction of


18

Chapter 1

1

an artificial metalloenzyme based on LmrR using the supramolecular anchoring approach of a Cu(II) complex The resulting hybrid was used in the enantioselective Friedel-Crafts alkylation reaction Chapter 6 describes the preliminary results of the construction of an artificial metalloenzyme based on LmrR in which aza-tryptophan residues where introduced ie a non-natural amino acid incorporated via a biosynthetic route Dative anchoring of Cu(II) ions led to the formation of a hybrid catalyst which was applied in the enantioselective Diels Alder reaction In chapter 7 conclusions will be given as well as perspectives for the field of artificial metalloenzymes

16 Reference[1] Wilson ME Whitesides GM Conversion of a protein to a homogeneous asymmetric

hydrogenation catalyst by site-specific modification with a diphosphinerhodium moiety J Am Chem Soc 1978 100306-307

[2] Ueno T Tabe H Tanaka Y Artificial metalloenzymes constructed from hierarchically-assembled proteins Chem Asian J 2013 81646-1660

[3] Lewis J C Artificial metalloenzymes and metallopeptide catalysts for organic synthesis ACS atal 2013 32954-2975

[4] Stephanopoulos N Francis MB Choosing an effective protein bioconjugation strategy Nat Chem Biol 2011 7876-884

[5] Davies RR Distefano MD A semisynthetic metalloenzyme based on a protein cavity that catalyzes the enantioselective hydrolysis of ester and amide substrates J Am Chem Soc 1997 11911643-11652

[6] Panella L Broos J Jin JF Fraaije MW Janssen DB Jeronimus-Stratingh M Feringa BL Minnaard AJ de Vries JG Merging homogeneous catalysis with biocatalysis papain as hydrogenation catalyst Chem Commun 2005 5656-5658

[7] Mayer C Gillingham DG Ward TR Hilvert D An artificial metalloenzyme for olefin metathesis Chem Commun 2011 4712068-12070

[8] Reetz MT Rentzsch M Pletsch A Taglieber A Hollmann F Mondiere RJG Dickmann N Hoecker B Cerrone S Haeger MC Sterner R A robust protein host for anchoring chelating ligands and organocatalysts ChemBioChem 2008 9552-564

[9] Haquette P Talbi B Barilleau L Madern N Fosse C Salmain M Chemically engineered papain as artificial formate dehydrogenase for NAD(P)H regeneration Org Biomol Chem 2011 95720-5727

[10] Deuss PJ Popa G Slawin AMZ Laan W Kamer PCJ Artificial copper enzymes for asymmetric Diels-Alder reactions ChemCatChem 2013 51184-1191

[11] Carey J Ma S Pfister T Garner D Kim H Abramite J Wang Z Guo Z Lu Y A site-selective dual anchoring strategy for artificial metalloprotein design J Am Chem Soc 2004 12610812-10813

[12] Garner DK Liang L Barrios DA Zhang J Lu Y The important role of covalent anchor positions in tuning catalytic properties of a rationally designed MnSalen-containing metalloenzyme ACS Catal 2011 11083-1089

[13] Deuss PJ Popa G Botting CH Laan W Kamer PCJ Highly efficient and site-selective phosphane modification of proteins through hydrazone linkage development of artificial metalloenzymes


19


1

Angew Chem Int Ed 2010 495315-5317[14] Kruithof CA Dijkstra HP Lutz M Spek AL Egmond MR Gebbink RJMK van Koten G Non-

tethered organometallic phosphonate inhibitors for lipase inhibition positioning of the metal center in the active site of cutinase Eur J Inorg Chem 2008 4425-4432

[15] Yang H Srivastava P Zhang C Lewis JC A general method for artificial metalloenzyme formation through strain-promoted azidendashalkyne cycloaddition ChemBioChem 2014 15223-227

[16] Wang Q Parrish AR Wang L Expanding the genetic code for biological studies Chem Biol 2009 16323-336

[17] Ryu Y Schultz P Efficient incorporation of unnatural amino acids into proteins in Escherichia coli Nat Methods 2006 3263-265

[18] Collot J Gradinaru J Humbert N Skander M Zocchi A Ward T Artificial metalloenzymes for enantioselective catalysis based on biotin-avidin J Am Chem Soc 2003 1259030-9031

[19] Klein G Humbert N Gradinaru J Ivanova A Gilardoni F Rusbandi U Ward T Tailoring the active site of chemzymes by using a chemogenetic-optimization procedure Towards substrate-specific artificial hydrogenases based on the biotin-avidin technology Angew Chem Int Ed 2005 447764-7767

[20] Letondor C Humbert N Ward T Artificial metalloenzymes based on biotin-avidin technology for the enantioselective reduction of ketones by transfer hydrogenation Proc Natl Acad Sci USA 2005 1024683-4687

[21] Ward TR Artificial metalloenzymes based on the biotin-avidin technology Enantioselective catalysis and beyond Acc Chem Res 2011 4447-57

[22] Ohashi M Koshiyama T Ueno T Yanase M Fujii H Watanabe Y Preparation of artificial metalloenzymes by insertion of chromium(III) Schiff base complexes into apomyoglobin mutants Angew Chem Int Ed 2003 421005-1008

[23] Ueno T Ohashi M Kono M Kondo K Suzuki A Yamane T Watanabe Y Crystal structures of artificial metalloproteins Tight binding of FeIII(Schiff- base) by mutation of Ala71 to gly in apo-myoglobin Inorg Chem 2004 432852-2858

[24] Ueno T Koshiyama T Ohashi M Kondo K Kono M Suzuki A Yamane T Watanabe Y Coordinated design of cofactor and active site structures in development of new protein catalysts J Am Chem Soc 2005 1276556-6562

[25] Satake Y Abe S Okazaki S Ban N Hikage T Ueno T Nakajima H Suzuki A Yamane T Nishiyama H Watanabe Y Incorporation of a phebox rhodium complex into apo-myoglobin affords a stable organometallic protein showing unprecedented arrangement of the complex in the cavity Organometallics 2007 264904-4908

[26] Roelfes G Feringa BL DNA-based asymmetric catalysis Angew Chem Int Ed 2005 443230-3232[27] Boersma AJ Coquiegravere D Geerdink D Rosati F Feringa BL Roelfes G Catalytic enantioselective

syn hydration of enones in water using a DNA-based catalyst Nat Chem 2010 2991-995[28] Ragsdale SW Metals and their scaffolds to promote difficult enzymatic reactions Chem Rev

2006 1063317-3337[29] Okrasa K Kazlauskas R Manganese-substituted carbonic anhydrase as a new peroxidase

Chem Eur J 2006 121587-1596[30] Podtetenieff J Taglieber A Bill E Reijerse EJ Reetz MT An artificial metalloenzyme creation

of a designed copper binding site in a thermostable protein Angew Chem Int Ed 2010 495151-5155


20

Chapter 1

1

[31] Xie J Liu W Schultz PG A genetically encoded bidentate metal-binding amino acid Angew Chem Int Ed 2007 469239-9242

[32] Lee HS Schultz PG Biosynthesis of a site-specific DNA cleaving protein J Am Chem Soc 2008 13013194-13195

[33] Lee HS Spraggon G Schultz PG Wang F Genetic incorporation of a metal-ion chelating amino acid into proteins as a biophysical probe J Am Chem Soc 2009 1312481-2483


[35] Kokubo T Sugimoto T Uchida T Tanimoto S Okano M The bovine serum albumin-2-phenylpropane-12-diolatodioxo-osmium(VI) complex as an enantioselective catalyst for cis-hydroxylation of alkenes J Chem Soc Chem Commun 1983769-770


[37] Kries H Blomberg R Hilvert D De novo enzymes by computational design Curr Opin Chem Biol 2013 17221-228

[38] Yeung N Lin Y Gao Y Zhao X Russell BS Lei L Miner KD Robinson H Lu Y Rational design of a structural and functional nitric oxide reductase Nature 2009 4621079-U144

[39] Podtetenieff J Taglieber A Bill E Reijerse EJ Reetz MT An Artificial Metalloenzyme Creation of a designed copper binding site in a thermostable protein Angew Chem Int Ed 2010 495151-5155

[40] Garciacutea-Fernaacutendez A Roelfes G Enantioselective catalysis at the DNA scaffold Met Ions Life Sci 2012 10249-68

[41] Zastrow ML Peacock AFA Stuckey JA Pecoraro VL Hydrolytic catalysis and structural stabilization in a designed metalloprotein Nat Chem 2012 4118-123

[42] Ball ZT Designing enzyme-like catalysts A rhodium(II) metallopeptide case study Acc Chem Res 2013 46560-570

[43] Sambasivan R Ball ZT Metallopeptides for asymmetric dirhodium catalysis J Am Chem Soc 2010 1329289-9291

[44] Pordea A Ward TR Chemogenetic protein engineering an efficient tool for the optimization of artificial metalloenzymes Chem Commun 2008 4239-4249

[45] Marshall NM Garner DK Wilson TD Gao Y Robinson H Nilges MJ Lu Y Rationally tuning the reduction potential of a single cupredoxin beyond the natural range Nature 2009 462113-U127

[46] Zimbron JM Heinisch T Schmid M Hamels D Nogueira ES Schirmer T Ward TR A dual anchoring strategy for the localization and activation of artificial metalloenzymes based on the biotin-streptavidin technology J Am Chem Soc 2013 1355384-5388

[47] Reetz MT Peyralans JJ- Maichele A Fu Y Maywald M Directed evolution of hybrid enzymes Evolving enantioselectivity of an achiral Rh-complex anchored to a protein Chem Commun 2006 4318-4320

[48] Ward TR Artificial metalloenzymes based on the biotin-avidin technology enantioselective catalysis and beyond Acc Chem Res 2011 4447-57

[49] Rosati F Roelfes G Artificial metalloenzymes ChemCatChem 2010 2916-927[50] Esmieu C Cherrier MV Amara P Girgenti E Marchi-Delapierre C Oddon F Iannello M Jorge-

Robin A Cavazza C Meacutenage S An artificial oxygenase built from scratch Substrate binding Site


21


1

identified using a docking approach Angew Chem Int Ed 2013 523922-3925[51] Inaba H Kanamaru S Arisaka F Kitagawa S Ueno T Semi-synthesis of an artificial scandium(III)

enzyme with a beta-helical bio-nanotube Dalton Trans 2012 4111424-11427[52] Zastrow ML Peacock AFA Stuckey JA Pecoraro VL Hydrolytic catalysis and structural

stabilization in a designed metalloprotein Nat Chem 2012 4118-123[53] Sambasivan R Ball ZT Screening rhodium metallopeptide libraries ldquoOn Beadrdquo Asymmetric

cyclopropanation and a solution to the enantiomer problem Angew Chem Int Ed 2012 518568-8572

[54] Sambasivan R Ball ZT Studies of asymmetric styrene cyclopropanation with a rhodium(II) metallopeptide catalyst developed with a high-throughput screen Chirality 2013 25493-497

[55] Mayer C Hilvert D A genetically encodable ligand for transfer hydrogenation Eur J Org Chem 2013 3427-3431

[56] Monney A Albrecht M A chelating tetrapeptide rhodium complex comprised of a histidylidene residue biochemical tailoring of an NHC-Rh hydrosilylation catalyst Chem Commun 2012 4810960-10962

[57] Monney A Nastri F Albrecht M Peptide-tethered monodentate and chelating histidylidene metal complexes synthesis and application in catalytic hydrosilylation Dalton Trans 2013 425655-5660

[58] Guisado-Barrios G Munoz BK Kamer PCJ Lastdrager B van der Marel G Overhand M Vega-Vazquez M Martin-Pastor M Cyclic decapeptide gramicidin S derivatives containing phosphines novel ligands for asymmetric catalysis Dalton Trans 2013 421973-1978

[59] Boersma AJ de Bruin B Feringa BL Roelfes G Ligand denticity controls enantiomeric preference in DNA-based asymmetric catalysis Chem Commun 2012 482394-2396

[60] Wang J Benedetti E Bethge L Vonhoff S Klussmann S Vasseur J Cossy J Smietana M Arseniyadis S DNA vs mirror image DNA A universal approach to tune the absolute configuration in DNA-based asymmetric catalysis Angew Chem Int Ed 2013 5211546-11549

[61] Gjonaj L Roelfes G Novel catalyst design by using cisplatin to covalently anchor catalytically active copper complexes to DNA ChemCatChem 2013 51718-1721

[62] Wang C Li Y Jia G Liu Y Lu S Li C Enantioselective Friedel-Crafts reactions in water catalyzed by a human telomeric G-quadruplex DNA metalloenzyme Chem Commun 2012 486232-6234

[63] Wang C Jia G Zhou J Li Y Liu Y Lu S Li C Enantioselective Diels-Alder reactions with G-Quadruplex DNA-based catalysts Angew Chem Int Ed 2012 519352-9355

[64] Wilking M Hennecke U The influence of G-quadruplex structure on DNA-based asymmetric catalysis using the G-quadruplex-bound cationic porphyrin TMPyP4 center dot Cu Org Biomol Chem 2013 116940-6945

[65] Park S Ikehata K Watabe R Hidaka Y Rajendran A Sugiyama H Deciphering DNA-based asymmetric catalysis through intramolecular Friedel-Crafts alkylations Chem Commun 2012 4810398-10400

[66] Madern N Talbi B Salmain M Aqueous phase transfer hydrogenation of aryl ketones catalysed by achiral ruthenium(II) and rhodium(III) complexes and their papain conjugates Appl Organometal Chem 2013 276-12

[67] Chevalley A Salmain M Enantioselective transfer hydrogenation of ketone catalysed by artificial metalloenzymes derived from bovine beta-lactoglobulin Chem Commun 2012 4811984-11986

[68] Cherrier MV Engilberge S Amara P Chevalley A Salmain M Fontecilla-Camps JC Structural basis for enantioselectivity in the transfer hydrogenation of a ketone catalyzed by an artificial


22

1

metalloenzyme Eur J Inorg Chem 2013 20133596-3600[69] Creus M Pordea A Rossel T Sardo A Letondor C Ivanova A Le Trong I Stenkamp RE Ward TR

X-ray structure and designed evolution of an artificial transfer hydrogenase Angew Chem Int Ed 2008 471400-1404

[70] Duumlrrenberger M Heinisch T Wilson YM Rossel T Nogueira E Knoumlrr L Mutschler A Kersten K Zimbron MJ Pierron J et al Artificial transfer hydrogenases for the enantioselective reduction of cyclic imines Angew Chem Int Ed 2011 503026-3029

[71] Schwizer F Koehler V Duumlrrenberger M Knoumlrr L Ward TR Genetic optimization of the catalytic efficiency of artificial imine reductases based on biotin-streptavidin technology ACS Catal 2013 31752-1755

[72] Heinisch T Langowska K Tanner P Reymond J Meier W Palivan C Ward TR Fluorescence-based assay for the optimization of the activity of artificial transfer hydrogenase within a biocompatible compartment ChemCatChem 2013 5720-723

[73] Monnard FW Nogueira ES Heinisch T Schirmer T Ward TR Human carbonic anhydrase II as host protein for the creation of artificial metalloenzymes the asymmetric transfer hydrogenation of imines Chem Sci 2013 43269-3274


[75] Lo C Ringenberg MR Gnandt D Wilson Y Ward TR Artificial metalloenzymes for olefin metathesis based on the biotin-(strept)avidin technology Chem Commun 2011 4712065-12067

[76] Matsuo T Imai C Yoshida T Saito T Hayashi T Hirota S Creation of an artificial metalloprotein with a Hoveyda-Grubbs catalyst moiety through the intrinsic inhibition mechanism of alpha-chymotrypsin Chem Commun 2012 481662-1664

[77] Hyster TK Rovis T Rhodium-catalyzed oxidative cycloaddition of benzamides and alkynes via C-HN-H Activation J Am Chem Soc 2010 13210565-10569

[78] Hyster TK Knoumlrr L Ward TR Rovis T Biotinylated Rh(III) complexes in engineered Streptavidin for accelerated asymmetric C-H activation Science 2012 338500-503

[79] Megens RP Roelfes G DNA-based catalytic enantioselective intermolecular oxa-Michael addition reactions Chem Commun 2012 486366-6368

[80] Oelerich J Roelfes G DNA-based asymmetric organometallic catalysis in water Chem Sci 2013 42013-2017

[81] Allard M Dupont C Munoz Robles V Doucet N Lledos A Marechal J Urvoas A Mahy J Ricoux R Incorporation of manganese complexes into xylanase New artificial metalloenzymes for enantioselective epoxidation ChemBioChem 2012 13240-251

[82] Koumlhler V Wilson YM Duumlrrenberger M Ghislieri D Churakova E Quinto T Knoumlrr L Haeussinger D Hollmann F Turner NJ Ward TR Synthetic cascades are enabled by combining biocatalysts with artificial metalloenzymes Nat Chem 2013 593-99

[83] Engstroumlm K Johnston EV Verho O Gustafson KPJ Shakeri M Tai C Baumlckvall J Co-immobilization of an enzyme and a metal into the compartments of mesoporous silica for cooperative tandem catalysis An artificial metalloenzyme Angew Chem Int Ed 2013 5214006-14010

[84] Coquiegravere D Bos J Beld J Roelfes G Enantioselective artificial metalloenzymes based on a bovine pancreatic polypeptide scaffold Angew Chem Int Ed 2009 485159-5162


1Artificial metalloenzymes have emerged over the last decades as an attractive approach towards combining homogenous catalysis and biocatalysis A wide variety of catalytic transformations have been established by artificial metalloenzymes thus establishing proof of concept The field is now slowly transforming to take on new challenges This chapter describes design strategies of these artificial metalloenzymes and focusses on advances in this field over the last two years

Part of this chapter is an adaptation from the original paper J Bos and G Roelfes Curr Opin Chem Biol 2014 19 135-143



2

Chapter 1

1





electronic etc






M substrate



3


1









M MM M

Synthetic

Bio-scaold


M

covalent

M

non-covalent

M

dative

M




4

Chapter 1

1


Protein SR

O

O

RX

X = Br I

N

O

O

R

Protein SN

O

O

R

H3C S SO

O

R

Protein SS

R

N R

O

NProtein S R

O

N

O

O

NH

ONH2

O

H R

Protein SN

O

O HN

O

NR

1

2

OPO

REtO

O2N

Protein OPO

REtO

Protein Protein

OHSH



5


1








6

Chapter 1

1






7


1








8

Chapter 1

1









9


1







10

Chapter 1

1




H2N

R

OH

NH

R

O

OHOH

Tyr residuesOH

OHSHOH

OHS

Sc3+

Sc3+

Cys introduction

Molecular


Sulfides

FeII

SNH

O

X

S NH

OO

X

NaOCl

TON up to 199

FeIICJBgtdockexe

A

B




11


1








12

Chapter 1

1









13


1









14

Chapter 1

1


R

O

R

lowastR

OH

R

O

OH OH

ADPN+

NH2

O

O

OH OH

ADPN

NH2

OH H

SO

N2

O O

O

H

SO

O

R1

R2

R1

R2O

st-DNA Cu complex

MOPS pH 65

Xln10A Mn complex




β-LG Ru complex

NR lowast

NHR


MOPS buffer HNCO2Na



NTos





OHHO

HO O

OH

N

O

OHHO

HO O

OH

N

OH2O

O

NH

OPivlowast

NH

O

R


O

RN

N

Olowast

R2N

N

OR1

st-DNA Cu complex

MOPS pH 65 40 R1OH

R

ref [66]

ref [9]

ref [70] [46] [71]

ref [73]

ref [7]

ref [75]

ref [76]

ref [78]

ref [79]

ref [80]

ref [81]

NO

lowast

lowast

O

N

+

+

SCP-2L Cu complex




15


1









16

Chapter 1

1










17


1










18

Chapter 1

1

















19


1




















20

Chapter 1

1






















21


1





















22

1



















2

Chapter 1

1





electronic etc






M substrate



3


1









M MM M

Synthetic

Bio-scaold


M

covalent

M

non-covalent

M

dative

M




4

Chapter 1

1


Protein SR

O

O

RX

X = Br I

N

O

O

R

Protein SN

O

O

R

H3C S SO

O

R

Protein SS

R

N R

O

NProtein S R

O

N

O

O

NH

ONH2

O

H R

Protein SN

O

O HN

O

NR

1

2

OPO

REtO

O2N

Protein OPO

REtO

Protein Protein

OHSH



5


1








6

Chapter 1

1






7


1








8

Chapter 1

1









9


1







10

Chapter 1

1




H2N

R

OH

NH

R

O

OHOH

Tyr residuesOH

OHSHOH

OHS

Sc3+

Sc3+

Cys introduction

Molecular


Sulfides

FeII

SNH

O

X

S NH

OO

X

NaOCl

TON up to 199

FeIICJBgtdockexe

A

B




11


1








12

Chapter 1

1









13


1









14

Chapter 1

1


R

O

R

lowastR

OH

R

O

OH OH

ADPN+

NH2

O

O

OH OH

ADPN

NH2

OH H

SO

N2

O O

O

H

SO

O

R1

R2

R1

R2O

st-DNA Cu complex

MOPS pH 65

Xln10A Mn complex




β-LG Ru complex

NR lowast

NHR


MOPS buffer HNCO2Na



NTos





OHHO

HO O

OH

N

O

OHHO

HO O

OH

N

OH2O

O

NH

OPivlowast

NH

O

R


O

RN

N

Olowast

R2N

N

OR1

st-DNA Cu complex

MOPS pH 65 40 R1OH

R

ref [66]

ref [9]

ref [70] [46] [71]

ref [73]

ref [7]

ref [75]

ref [76]

ref [78]

ref [79]

ref [80]

ref [81]

NO

lowast

lowast

O

N

+

+

SCP-2L Cu complex




15


1









16

Chapter 1

1










17


1










18

Chapter 1

1

















19


1




















20

Chapter 1

1






















21


1





















22

1



















3


1









M MM M

Synthetic

Bio-scaold


M

covalent

M

non-covalent

M

dative

M




4

Chapter 1

1


Protein SR

O

O

RX

X = Br I

N

O

O

R

Protein SN

O

O

R

H3C S SO

O

R

Protein SS

R

N R

O

NProtein S R

O

N

O

O

NH

ONH2

O

H R

Protein SN

O

O HN

O

NR

1

2

OPO

REtO

O2N

Protein OPO

REtO

Protein Protein

OHSH



5


1








6

Chapter 1

1






7


1








8

Chapter 1

1









9


1







10

Chapter 1

1




H2N

R

OH

NH

R

O

OHOH

Tyr residuesOH

OHSHOH

OHS

Sc3+

Sc3+

Cys introduction

Molecular


Sulfides

FeII

SNH

O

X

S NH

OO

X

NaOCl

TON up to 199

FeIICJBgtdockexe

A

B




11


1








12

Chapter 1

1









13


1









14

Chapter 1

1


R

O

R

lowastR

OH

R

O

OH OH

ADPN+

NH2

O

O

OH OH

ADPN

NH2

OH H

SO

N2

O O

O

H

SO

O

R1

R2

R1

R2O

st-DNA Cu complex

MOPS pH 65

Xln10A Mn complex




β-LG Ru complex

NR lowast

NHR


MOPS buffer HNCO2Na



NTos





OHHO

HO O

OH

N

O

OHHO

HO O

OH

N

OH2O

O

NH

OPivlowast

NH

O

R


O

RN

N

Olowast

R2N

N

OR1

st-DNA Cu complex

MOPS pH 65 40 R1OH

R

ref [66]

ref [9]

ref [70] [46] [71]

ref [73]

ref [7]

ref [75]

ref [76]

ref [78]

ref [79]

ref [80]

ref [81]

NO

lowast

lowast

O

N

+

+

SCP-2L Cu complex




15


1









16

Chapter 1

1










17


1










18

Chapter 1

1

















19


1




















20

Chapter 1

1






















21


1





















22

1



















4

Chapter 1

1


Protein SR

O

O

RX

X = Br I

N

O

O

R

Protein SN

O

O

R

H3C S SO

O

R

Protein SS

R

N R

O

NProtein S R

O

N

O

O

NH

ONH2

O

H R

Protein SN

O

O HN

O

NR

1

2

OPO

REtO

O2N

Protein OPO

REtO

Protein Protein

OHSH



5


1








6

Chapter 1

1






7


1








8

Chapter 1

1









9


1







10

Chapter 1

1




H2N

R

OH

NH

R

O

OHOH

Tyr residuesOH

OHSHOH

OHS

Sc3+

Sc3+

Cys introduction

Molecular


Sulfides

FeII

SNH

O

X

S NH

OO

X

NaOCl

TON up to 199

FeIICJBgtdockexe

A

B




11


1








12

Chapter 1

1









13


1









14

Chapter 1

1


R

O

R

lowastR

OH

R

O

OH OH

ADPN+

NH2

O

O

OH OH

ADPN

NH2

OH H

SO

N2

O O

O

H

SO

O

R1

R2

R1

R2O

st-DNA Cu complex

MOPS pH 65

Xln10A Mn complex




β-LG Ru complex

NR lowast

NHR


MOPS buffer HNCO2Na



NTos





OHHO

HO O

OH

N

O

OHHO

HO O

OH

N

OH2O

O

NH

OPivlowast

NH

O

R


O

RN

N

Olowast

R2N

N

OR1

st-DNA Cu complex

MOPS pH 65 40 R1OH

R

ref [66]

ref [9]

ref [70] [46] [71]

ref [73]

ref [7]

ref [75]

ref [76]

ref [78]

ref [79]

ref [80]

ref [81]

NO

lowast

lowast

O

N

+

+

SCP-2L Cu complex




15


1









16

Chapter 1

1










17


1










18

Chapter 1

1

















19


1




















20

Chapter 1

1






















21


1





















22

1



















5


1








6

Chapter 1

1






7


1








8

Chapter 1

1









9


1







10

Chapter 1

1




H2N

R

OH

NH

R

O

OHOH

Tyr residuesOH

OHSHOH

OHS

Sc3+

Sc3+

Cys introduction

Molecular


Sulfides

FeII

SNH

O

X

S NH

OO

X

NaOCl

TON up to 199

FeIICJBgtdockexe

A

B




11


1








12

Chapter 1

1









13


1









14

Chapter 1

1


R

O

R

lowastR

OH

R

O

OH OH

ADPN+

NH2

O

O

OH OH

ADPN

NH2

OH H

SO

N2

O O

O

H

SO

O

R1

R2

R1

R2O

st-DNA Cu complex

MOPS pH 65

Xln10A Mn complex




β-LG Ru complex

NR lowast

NHR


MOPS buffer HNCO2Na



NTos





OHHO

HO O

OH

N

O

OHHO

HO O

OH

N

OH2O

O

NH

OPivlowast

NH

O

R


O

RN

N

Olowast

R2N

N

OR1

st-DNA Cu complex

MOPS pH 65 40 R1OH

R

ref [66]

ref [9]

ref [70] [46] [71]

ref [73]

ref [7]

ref [75]

ref [76]

ref [78]

ref [79]

ref [80]

ref [81]

NO

lowast

lowast

O

N

+

+

SCP-2L Cu complex




15


1









16

Chapter 1

1










17


1










18

Chapter 1

1

















19


1




















20

Chapter 1

1






















21


1





















22

1



















6

Chapter 1

1






7


1








8

Chapter 1

1









9


1







10

Chapter 1

1




H2N

R

OH

NH

R

O

OHOH

Tyr residuesOH

OHSHOH

OHS

Sc3+

Sc3+

Cys introduction

Molecular


Sulfides

FeII

SNH

O

X

S NH

OO

X

NaOCl

TON up to 199

FeIICJBgtdockexe

A

B




11


1








12

Chapter 1

1









13


1









14

Chapter 1

1


R

O

R

lowastR

OH

R

O

OH OH

ADPN+

NH2

O

O

OH OH

ADPN

NH2

OH H

SO

N2

O O

O

H

SO

O

R1

R2

R1

R2O

st-DNA Cu complex

MOPS pH 65

Xln10A Mn complex




β-LG Ru complex

NR lowast

NHR


MOPS buffer HNCO2Na



NTos





OHHO

HO O

OH

N

O

OHHO

HO O

OH

N

OH2O

O

NH

OPivlowast

NH

O

R


O

RN

N

Olowast

R2N

N

OR1

st-DNA Cu complex

MOPS pH 65 40 R1OH

R

ref [66]

ref [9]

ref [70] [46] [71]

ref [73]

ref [7]

ref [75]

ref [76]

ref [78]

ref [79]

ref [80]

ref [81]

NO

lowast

lowast

O

N

+

+

SCP-2L Cu complex




15


1









16

Chapter 1

1










17


1










18

Chapter 1

1

















19


1




















20

Chapter 1

1






















21


1





















22

1



















7


1








8

Chapter 1

1









9


1







10

Chapter 1

1




H2N

R

OH

NH

R

O

OHOH

Tyr residuesOH

OHSHOH

OHS

Sc3+

Sc3+

Cys introduction

Molecular


Sulfides

FeII

SNH

O

X

S NH

OO

X

NaOCl

TON up to 199

FeIICJBgtdockexe

A

B




11


1








12

Chapter 1

1









13


1









14

Chapter 1

1


R

O

R

lowastR

OH

R

O

OH OH

ADPN+

NH2

O

O

OH OH

ADPN

NH2

OH H

SO

N2

O O

O

H

SO

O

R1

R2

R1

R2O

st-DNA Cu complex

MOPS pH 65

Xln10A Mn complex




β-LG Ru complex

NR lowast

NHR


MOPS buffer HNCO2Na



NTos





OHHO

HO O

OH

N

O

OHHO

HO O

OH

N

OH2O

O

NH

OPivlowast

NH

O

R


O

RN

N

Olowast

R2N

N

OR1

st-DNA Cu complex

MOPS pH 65 40 R1OH

R

ref [66]

ref [9]

ref [70] [46] [71]

ref [73]

ref [7]

ref [75]

ref [76]

ref [78]

ref [79]

ref [80]

ref [81]

NO

lowast

lowast

O

N

+

+

SCP-2L Cu complex




15


1









16

Chapter 1

1










17


1










18

Chapter 1

1

















19


1




















20

Chapter 1

1






















21


1





















22

1



















8

Chapter 1

1









9


1







10

Chapter 1

1




H2N

R

OH

NH

R

O

OHOH

Tyr residuesOH

OHSHOH

OHS

Sc3+

Sc3+

Cys introduction

Molecular


Sulfides

FeII

SNH

O

X

S NH

OO

X

NaOCl

TON up to 199

FeIICJBgtdockexe

A

B




11


1








12

Chapter 1

1









13


1









14

Chapter 1

1


R

O

R

lowastR

OH

R

O

OH OH

ADPN+

NH2

O

O

OH OH

ADPN

NH2

OH H

SO

N2

O O

O

H

SO

O

R1

R2

R1

R2O

st-DNA Cu complex

MOPS pH 65

Xln10A Mn complex




β-LG Ru complex

NR lowast

NHR


MOPS buffer HNCO2Na



NTos





OHHO

HO O

OH

N

O

OHHO

HO O

OH

N

OH2O

O

NH

OPivlowast

NH

O

R


O

RN

N

Olowast

R2N

N

OR1

st-DNA Cu complex

MOPS pH 65 40 R1OH

R

ref [66]

ref [9]

ref [70] [46] [71]

ref [73]

ref [7]

ref [75]

ref [76]

ref [78]

ref [79]

ref [80]

ref [81]

NO

lowast

lowast

O

N

+

+

SCP-2L Cu complex




15


1









16

Chapter 1

1










17


1










18

Chapter 1

1

















19


1




















20

Chapter 1

1






















21


1





















22

1



















9


1







10

Chapter 1

1




H2N

R

OH

NH

R

O

OHOH

Tyr residuesOH

OHSHOH

OHS

Sc3+

Sc3+

Cys introduction

Molecular


Sulfides

FeII

SNH

O

X

S NH

OO

X

NaOCl

TON up to 199

FeIICJBgtdockexe

A

B




11


1








12

Chapter 1

1









13


1









14

Chapter 1

1


R

O

R

lowastR

OH

R

O

OH OH

ADPN+

NH2

O

O

OH OH

ADPN

NH2

OH H

SO

N2

O O

O

H

SO

O

R1

R2

R1

R2O

st-DNA Cu complex

MOPS pH 65

Xln10A Mn complex




β-LG Ru complex

NR lowast

NHR


MOPS buffer HNCO2Na



NTos





OHHO

HO O

OH

N

O

OHHO

HO O

OH

N

OH2O

O

NH

OPivlowast

NH

O

R


O

RN

N

Olowast

R2N

N

OR1

st-DNA Cu complex

MOPS pH 65 40 R1OH

R

ref [66]

ref [9]

ref [70] [46] [71]

ref [73]

ref [7]

ref [75]

ref [76]

ref [78]

ref [79]

ref [80]

ref [81]

NO

lowast

lowast

O

N

+

+

SCP-2L Cu complex




15


1









16

Chapter 1

1










17


1










18

Chapter 1

1

















19


1




















20

Chapter 1

1






















21


1





















22

1



















10

Chapter 1

1




H2N

R

OH

NH

R

O

OHOH

Tyr residuesOH

OHSHOH

OHS

Sc3+

Sc3+

Cys introduction

Molecular


Sulfides

FeII

SNH

O

X

S NH

OO

X

NaOCl

TON up to 199

FeIICJBgtdockexe

A

B




11


1








12

Chapter 1

1









13


1









14

Chapter 1

1


R

O

R

lowastR

OH

R

O

OH OH

ADPN+

NH2

O

O

OH OH

ADPN

NH2

OH H

SO

N2

O O

O

H

SO

O

R1

R2

R1

R2O

st-DNA Cu complex

MOPS pH 65

Xln10A Mn complex




β-LG Ru complex

NR lowast

NHR


MOPS buffer HNCO2Na



NTos





OHHO

HO O

OH

N

O

OHHO

HO O

OH

N

OH2O

O

NH

OPivlowast

NH

O

R


O

RN

N

Olowast

R2N

N

OR1

st-DNA Cu complex

MOPS pH 65 40 R1OH

R

ref [66]

ref [9]

ref [70] [46] [71]

ref [73]

ref [7]

ref [75]

ref [76]

ref [78]

ref [79]

ref [80]

ref [81]

NO

lowast

lowast

O

N

+

+

SCP-2L Cu complex




15


1









16

Chapter 1

1










17


1










18

Chapter 1

1

















19


1




















20

Chapter 1

1






















21


1





















22

1



















11


1








12

Chapter 1

1









13


1









14

Chapter 1

1


R

O

R

lowastR

OH

R

O

OH OH

ADPN+

NH2

O

O

OH OH

ADPN

NH2

OH H

SO

N2

O O

O

H

SO

O

R1

R2

R1

R2O

st-DNA Cu complex

MOPS pH 65

Xln10A Mn complex




β-LG Ru complex

NR lowast

NHR


MOPS buffer HNCO2Na



NTos





OHHO

HO O

OH

N

O

OHHO

HO O

OH

N

OH2O

O

NH

OPivlowast

NH

O

R


O

RN

N

Olowast

R2N

N

OR1

st-DNA Cu complex

MOPS pH 65 40 R1OH

R

ref [66]

ref [9]

ref [70] [46] [71]

ref [73]

ref [7]

ref [75]

ref [76]

ref [78]

ref [79]

ref [80]

ref [81]

NO

lowast

lowast

O

N

+

+

SCP-2L Cu complex




15


1









16

Chapter 1

1










17


1










18

Chapter 1

1

















19


1




















20

Chapter 1

1






















21


1





















22

1



















12

Chapter 1

1









13


1









14

Chapter 1

1


R

O

R

lowastR

OH

R

O

OH OH

ADPN+

NH2

O

O

OH OH

ADPN

NH2

OH H

SO

N2

O O

O

H

SO

O

R1

R2

R1

R2O

st-DNA Cu complex

MOPS pH 65

Xln10A Mn complex




β-LG Ru complex

NR lowast

NHR


MOPS buffer HNCO2Na



NTos





OHHO

HO O

OH

N

O

OHHO

HO O

OH

N

OH2O

O

NH

OPivlowast

NH

O

R


O

RN

N

Olowast

R2N

N

OR1

st-DNA Cu complex

MOPS pH 65 40 R1OH

R

ref [66]

ref [9]

ref [70] [46] [71]

ref [73]

ref [7]

ref [75]

ref [76]

ref [78]

ref [79]

ref [80]

ref [81]

NO

lowast

lowast

O

N

+

+

SCP-2L Cu complex




15


1









16

Chapter 1

1










17


1










18

Chapter 1

1

















19


1




















20

Chapter 1

1






















21


1





















22

1



















13


1









14

Chapter 1

1


R

O

R

lowastR

OH

R

O

OH OH

ADPN+

NH2

O

O

OH OH

ADPN

NH2

OH H

SO

N2

O O

O

H

SO

O

R1

R2

R1

R2O

st-DNA Cu complex

MOPS pH 65

Xln10A Mn complex




β-LG Ru complex

NR lowast

NHR


MOPS buffer HNCO2Na



NTos





OHHO

HO O

OH

N

O

OHHO

HO O

OH

N

OH2O

O

NH

OPivlowast

NH

O

R


O

RN

N

Olowast

R2N

N

OR1

st-DNA Cu complex

MOPS pH 65 40 R1OH

R

ref [66]

ref [9]

ref [70] [46] [71]

ref [73]

ref [7]

ref [75]

ref [76]

ref [78]

ref [79]

ref [80]

ref [81]

NO

lowast

lowast

O

N

+

+

SCP-2L Cu complex




15


1









16

Chapter 1

1










17


1










18

Chapter 1

1

















19


1




















20

Chapter 1

1






















21


1





















22

1



















14

Chapter 1

1


R

O

R

lowastR

OH

R

O

OH OH

ADPN+

NH2

O

O

OH OH

ADPN

NH2

OH H

SO

N2

O O

O

H

SO

O

R1

R2

R1

R2O

st-DNA Cu complex

MOPS pH 65

Xln10A Mn complex




β-LG Ru complex

NR lowast

NHR


MOPS buffer HNCO2Na



NTos





OHHO

HO O

OH

N

O

OHHO

HO O

OH

N

OH2O

O

NH

OPivlowast

NH

O

R


O

RN

N

Olowast

R2N

N

OR1

st-DNA Cu complex

MOPS pH 65 40 R1OH

R

ref [66]

ref [9]

ref [70] [46] [71]

ref [73]

ref [7]

ref [75]

ref [76]

ref [78]

ref [79]

ref [80]

ref [81]

NO

lowast

lowast

O

N

+

+

SCP-2L Cu complex




15


1









16

Chapter 1

1










17


1










18

Chapter 1

1

















19


1




















20

Chapter 1

1






















21


1





















22

1



















15


1









16

Chapter 1

1










17


1










18

Chapter 1

1

















19


1




















20

Chapter 1

1






















21


1





















22

1



















16

Chapter 1

1










17


1










18

Chapter 1

1

















19


1




















20

Chapter 1

1






















21


1





















22

1



















17


1










18

Chapter 1

1

















19


1




















20

Chapter 1

1






















21


1





















22

1



















18

Chapter 1

1

















19


1




















20

Chapter 1

1






















21


1





















22

1



















19


1




















20

Chapter 1

1






















21


1





















22

1



















20

Chapter 1

1






















21


1





















22

1



















21


1





















22

1



















22

1



















university of groningen artificial metalloenzymes bos, jeffrey · artircial metalloenzymes have...

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