been prepared by incorporating Cu
II complexes into biomacromolecules covalently
or non-covalently.
Reetz and coworkers inserted Cu
II− phtalocyanine into serum albumins including
BSA and HSA [151, 152]. Palomo et al. generated an heterogeneous artificial
Diels–Alderase by assembling Cu
II -phenanthroline-lipase at the surface of Sepabeads™ [153, 154]. Cu
II -1,10‐phenanthroline was also coupled to a testosterone
anchor and inserted into the neo-carzinostatin variant NCS‐3.24, thanks to its
affinity for the testosterone moiety [155]. (η
6 -arene)-Ru
II complexes were covalently anchored into papain [156] whereas Cu
II -phenanthroline and terpyridine
complexes were covalently anchored into F119C and Y26C mutants of dimeric
double chain (A 3 ) [157] and dimeric single chain (A3_A3’) [158] synthetic
alpha-repeat proteins. Cu
II -pyrenyl and -terpyridine complexes were also anchored,
respectively, into Nb and FhuA [159]. Deuss et al. also covalently bound Cu
II -
phenanthroline (Phen) and -dipicolylamine complexes in two sterol carrier protein
type 2 like domain (SCP‐2L) mutants [160]. Finally, the Cu
II -1,10-phenanthroline
complex was also bound to the LmrR M89C mutant [161]. All of the prepared
ArMs catalyzed the Diels–Alder cycloaddition using benchmark substrates
2-azachalcone and cyclopentadiene, providing up to four isomer products. Yields
ranging between 11 and 98% could be obtained, whereas the endo isomer was
always the major isomer with endo/exo ratios ranging from 66/34 to 96/4. The
enantiomeric excesses varied greatly with values between 0 and 97%. The most
efficient of these enzymes were the Cu
II -Phen-SB-Lys-GTL*/196 catalyst [154] and
the Cu
II -Phen-LmrR-M89C [161] that provided 98 and 93% yield and 97% ee with
an endo/exo ratio of about 95:5, respectively. Directed evolution was used by Reetz
et al. to design a Cu(II)-specific binding site in the thermostable protein tHisF. The
introduction of a His-His-Asp coordinating triad for the Cu
II ion into this F
affording a copper enzyme catalyzing the Diels–Alder reaction of the benchmark
substrates with 73% yield, 93/7 endo/exo ratio and 46% ee [152], [162]. The
substitution of the native Fe(II) of the His-His-Asp coordinating triad of
1-aminocyclopropane carboxylic acid oxidase by Cu
II afforded the most efficient
artificial Diels–Alderase ever reported with a quantitative yield and >99 % ee
[163].
Finally, Ghattas et al. used the A 2A adenosine receptor embedded in the cytoplasmic membrane of living human cells as a platform to build artificial
metallo-Diels Alderases. The metalloenzymes were assembled by inserting in the
WT receptor conjugates of a strong antagonist covalently bound to Cu(II) catalysts.
The resulting cells enantio selectively catalyzed the abiotic Diels–Alder cycloaddition reaction with up to 42% yield, an 82/18 endo/exo ratio, and 14% ee (Fig. 27)
[164]. The prospects of this strategy lie in the in vivo preparation of organ-confined
receptor-based artificial metalloenzymes for the catalysis of reactions exogenous to
the human metabolism. This strategy could be used for the targeted synthesis of
either drugs or deficient metabolites and for the activation of prodrugs, leading to
therapeutic tools with unforeseen applications.
Current Applications of Artificial Metalloenzymes …
399
II complexes into biomacromolecules covalently
or non-covalently.
Reetz and coworkers inserted Cu
II− phtalocyanine into serum albumins including
BSA and HSA [151, 152]. Palomo et al. generated an heterogeneous artificial
Diels–Alderase by assembling Cu
II -phenanthroline-lipase at the surface of Sepabeads™ [153, 154]. Cu
II -1,10‐phenanthroline was also coupled to a testosterone
anchor and inserted into the neo-carzinostatin variant NCS‐3.24, thanks to its
affinity for the testosterone moiety [155]. (η
6 -arene)-Ru
II complexes were covalently anchored into papain [156] whereas Cu
II -phenanthroline and terpyridine
complexes were covalently anchored into F119C and Y26C mutants of dimeric
double chain (A 3 ) [157] and dimeric single chain (A3_A3’) [158] synthetic
alpha-repeat proteins. Cu
II -pyrenyl and -terpyridine complexes were also anchored,
respectively, into Nb and FhuA [159]. Deuss et al. also covalently bound Cu
II -
phenanthroline (Phen) and -dipicolylamine complexes in two sterol carrier protein
type 2 like domain (SCP‐2L) mutants [160]. Finally, the Cu
II -1,10-phenanthroline
complex was also bound to the LmrR M89C mutant [161]. All of the prepared
ArMs catalyzed the Diels–Alder cycloaddition using benchmark substrates
2-azachalcone and cyclopentadiene, providing up to four isomer products. Yields
ranging between 11 and 98% could be obtained, whereas the endo isomer was
always the major isomer with endo/exo ratios ranging from 66/34 to 96/4. The
enantiomeric excesses varied greatly with values between 0 and 97%. The most
efficient of these enzymes were the Cu
II -Phen-SB-Lys-GTL*/196 catalyst [154] and
the Cu
II -Phen-LmrR-M89C [161] that provided 98 and 93% yield and 97% ee with
an endo/exo ratio of about 95:5, respectively. Directed evolution was used by Reetz
et al. to design a Cu(II)-specific binding site in the thermostable protein tHisF. The
introduction of a His-His-Asp coordinating triad for the Cu
II ion into this F
affording a copper enzyme catalyzing the Diels–Alder reaction of the benchmark
substrates with 73% yield, 93/7 endo/exo ratio and 46% ee [152], [162]. The
substitution of the native Fe(II) of the His-His-Asp coordinating triad of
1-aminocyclopropane carboxylic acid oxidase by Cu
II afforded the most efficient
artificial Diels–Alderase ever reported with a quantitative yield and >99 % ee
[163].
Finally, Ghattas et al. used the A 2A adenosine receptor embedded in the cytoplasmic membrane of living human cells as a platform to build artificial
metallo-Diels Alderases. The metalloenzymes were assembled by inserting in the
WT receptor conjugates of a strong antagonist covalently bound to Cu(II) catalysts.
The resulting cells enantio selectively catalyzed the abiotic Diels–Alder cycloaddition reaction with up to 42% yield, an 82/18 endo/exo ratio, and 14% ee (Fig. 27)
[164]. The prospects of this strategy lie in the in vivo preparation of organ-confined
receptor-based artificial metalloenzymes for the catalysis of reactions exogenous to
the human metabolism. This strategy could be used for the targeted synthesis of
either drugs or deficient metabolites and for the activation of prodrugs, leading to
therapeutic tools with unforeseen applications.
Current Applications of Artificial Metalloenzymes …
399