the analog G4-DFtet. This problem was solved by optimizing the sequence of a
loop far from the active site [16]. The finally designed ArM catalyzed the O 2 -
dependent oxidation of a larger catechol derivative, 3,5-di-tert-butyl-catechol
(3,5-DTBC), to the corresponding quinone (3,5-DTBQ) (Fig. 1), with a catalytic
efficiency (k cat /K M = 6315 M
−1 min
−1 ) about 4.6 times higher than that observed
for a smaller substrate, 4-aminophenol (4-AP) (k cat /K M = 1380 M
−1 min
−1 ).
Finally, the group of T. R. Ward developed ArMs that were active for the
oxidation of secondary alcohols (phenethyl alcohol, benzyl alcohol, and cyclohexanol) in water using
t BuOOH as oxidizing agent. They used the above mentioned “Trojan Horse” strategy based on the non-covalent incorporation of
biotinylated d 6 piano stool ruthenium complexes into (strept)avidin. The best of
them catalyzed the oxidation of dry phenethyl alcohol with a yield greater than 90%
[19] in 90 h at room temperature.
2.1.2 Amine Oxidation
DeGrado et al. also used their metalloproteins, that were originally designed for the
oxidation of hydroquinones, in order to catalyze the selective N-hydroxylation of
arylamines [17]. For this purpose, on the one hand, they reshaped the access to the
substrate cavity by incorporating three mutations at different levels of the protein
and, on the other hand, they introduced an additional iron-binding histidine into the
active site in order to mimic the active site of the natural di-oxoxine p-aminobenzoate N-oxygenase which is the only structurally characterized N-oxygenase
known to contain a diiron catalytic center [20]. The resulting biohybrid proved to
efficiently convert p-anisidine to the corresponding hydroxylamine (Fig. 1). In
2015, spectroscopic studies demonstrated that the 4-aminophenol substrate directly
binds to the bi-ferrous site in the active site of the proteins. The actual active species
of the Due Ferri scaffolds were thus identified and mechanisms explaining their
different reactivities were suggested [18].
2.1.3 Sulfide Oxidation
The oxidation of sulfides by various oxidants catalyzed by ArMs, elaborated by all
of the strategies described in the introduction of this chapter, has been by far the
most widely investigated. One of the first reports was published by Sheldon et al.
who constructed an ArM for the catalysis of sulfide oxidation using phytase as a
protein scaffold (Fig. 2) [21]. Vanadium chloroperoxidases are non-heminic metalloenzymes that are more resistant to oxidative degradation than their heminic
analogs [22]. Unfortunately, these enzymes can only accommodate small substrates
in their relatively small active site, which impedes their potential use in asymmetric
synthesis. Since this active site shows very high similarities with that of metal-free
phytases, Sheldon et al. thought about building an ArM by inserting vanadate into
phytase. The new artificial metalloprotein showed a catalytic activity similar to that
of natural vanadium chloroperoxidase and catalyzed the quantitative sulfoxidation
of thioanisole by H 2 O 2 , with up to 66% ee [23]. Further experiments were performed to improve the system, both by varying the nature of the host protein (acid–
phosphatase, phospholipase, sulfatase, apo-ferritin, BSA) and of the metal moiety
368
J.-P. Mahy et al.
loop far from the active site [16]. The finally designed ArM catalyzed the O 2 -
dependent oxidation of a larger catechol derivative, 3,5-di-tert-butyl-catechol
(3,5-DTBC), to the corresponding quinone (3,5-DTBQ) (Fig. 1), with a catalytic
efficiency (k cat /K M = 6315 M
−1 min
−1 ) about 4.6 times higher than that observed
for a smaller substrate, 4-aminophenol (4-AP) (k cat /K M = 1380 M
−1 min
−1 ).
Finally, the group of T. R. Ward developed ArMs that were active for the
oxidation of secondary alcohols (phenethyl alcohol, benzyl alcohol, and cyclohexanol) in water using
t BuOOH as oxidizing agent. They used the above mentioned “Trojan Horse” strategy based on the non-covalent incorporation of
biotinylated d 6 piano stool ruthenium complexes into (strept)avidin. The best of
them catalyzed the oxidation of dry phenethyl alcohol with a yield greater than 90%
[19] in 90 h at room temperature.
2.1.2 Amine Oxidation
DeGrado et al. also used their metalloproteins, that were originally designed for the
oxidation of hydroquinones, in order to catalyze the selective N-hydroxylation of
arylamines [17]. For this purpose, on the one hand, they reshaped the access to the
substrate cavity by incorporating three mutations at different levels of the protein
and, on the other hand, they introduced an additional iron-binding histidine into the
active site in order to mimic the active site of the natural di-oxoxine p-aminobenzoate N-oxygenase which is the only structurally characterized N-oxygenase
known to contain a diiron catalytic center [20]. The resulting biohybrid proved to
efficiently convert p-anisidine to the corresponding hydroxylamine (Fig. 1). In
2015, spectroscopic studies demonstrated that the 4-aminophenol substrate directly
binds to the bi-ferrous site in the active site of the proteins. The actual active species
of the Due Ferri scaffolds were thus identified and mechanisms explaining their
different reactivities were suggested [18].
2.1.3 Sulfide Oxidation
The oxidation of sulfides by various oxidants catalyzed by ArMs, elaborated by all
of the strategies described in the introduction of this chapter, has been by far the
most widely investigated. One of the first reports was published by Sheldon et al.
who constructed an ArM for the catalysis of sulfide oxidation using phytase as a
protein scaffold (Fig. 2) [21]. Vanadium chloroperoxidases are non-heminic metalloenzymes that are more resistant to oxidative degradation than their heminic
analogs [22]. Unfortunately, these enzymes can only accommodate small substrates
in their relatively small active site, which impedes their potential use in asymmetric
synthesis. Since this active site shows very high similarities with that of metal-free
phytases, Sheldon et al. thought about building an ArM by inserting vanadate into
phytase. The new artificial metalloprotein showed a catalytic activity similar to that
of natural vanadium chloroperoxidase and catalyzed the quantitative sulfoxidation
of thioanisole by H 2 O 2 , with up to 66% ee [23]. Further experiments were performed to improve the system, both by varying the nature of the host protein (acid–
phosphatase, phospholipase, sulfatase, apo-ferritin, BSA) and of the metal moiety
368
J.-P. Mahy et al.