Watanabe and coll. Used this strategy to prepare new ArMs. In particular, they
first inserted synthetic chromium salophens into apo-Mb and mutants. The artificial
metalloenzyme obtained from the H64D/A71G double mutant catalyzed the
stereoselective sulfoxidation of thioanisole, with rather low turnover frequencies
(TOF
0.13 min
−1 ) and enantiomeric excesses (ee
30% in favor of the (S)product) [37]. The 3D structures of two apo-Mb A71G mutant-Mn- and
Cr-salophen complexes revealed that the accessibility to the active site was sterically hindered by the bulky phenylenediamine moiety of the salophen cofactors.
They then used metal-salen cofactors instead, which had a rather low effect on the
efficiency of the chromium cofactors (TOF = 0.21 min
−1 ) but induced a noticeable
increase in the efficiency of the manganese cofactor (TOF = 2.7 min
−1 ). In addition, insertion of the later cofactor in the H64D/A71G double mutant led to a
slightly increased enantiomeric excess (30% ee in favor of the (S)-product) whereas
its insertion in the A71G single mutant led to an enantiomeric excess very similar to
that obtained with the WT protein (27% ee in favor of the (R)-product) [38].
Several other teams further inserted various Fe-tetrapyrrolic compounds into
apo-Mb to produce new biohybrids that catalyzed the oxidation of thioanisole by
H 2 O 2 and led to up to 38% ee in favor of the (S)-sulfoxide. The enantiomeric excess
could be increased either by covalent anchoring of metal complexes into apo-Mb
and mutants [39–41] or by changing the metal such as, for example, replacing iron
by manganese [41]. Accordingly, Lu et al. showed that a dual-point attachment of
manganese-salen complex to a double mutant of apo-Mb (apo-Mb Y103C/L72C)
led to an improved selectivity (51% ee) in thioanisole sulfoxidation relative to the
analogous single-point mutant (Y103C, 12% ee) (Fig. 5) [40, 41].
Xylanase A from Streptomyces lividans (Xln10A), a thermostable b1,4-endoxylanase glycoside hydrolase that hydrolyzes b-1,4 bonds in the main
chain of xylan [43], was also used to build up new artificial hemoproteins using the
host-guest strategy. This choice was guided by an early report from Nakamura and
Tsushida et al. that showed that xylanase possessed a wide enough active site to
accommodate an Fe(II)-a4-tetra-o-pivalamidophenylporphyrin. The resulting
heat-resistant hemoproteins were found to bind and release O 2 in aqueous medium
[44]. Metal complexes of synthetic tetraaryl porphyrins bearing negatively charged
substituents, such as Iron(III)tetra(4-carboxyphenyl)porphyrin (Fe(TpCPP)) and
iron(III)-meso-tetra(4-sulfonatophenyl)porphyrin (Fe(TpSPP)), were then inserted
into Xln10A to lead to new artificial hemoproteins that showed peroxidase activity
[12]. The catalytic activity of these Fe(TpCPP)-Xln10A and Fe(TpSPP)-Xln10A
biohybrids for the oxidation of thioanisole by H 2 O 2 (Fig. 6) was then investigated
and compared to that of Fe(TpCPP) and Fe(TpSPP) alone. These two Fe-porphyrin
complexes led, respectively to 45 and 33% yields in sulfoxide and to respective
TOFs of 0.56 and 0.41 min
−1 , but no enantiomeric excess could be detected. Use of
the Fe(TpCPP)- and Fe(TpSPP)-Xln10A biohybrids as catalysts led to a decrease in
the yields (about 24%) and turnover frequencies (about 0.30 min
−1 ), but enantiomeric excesses of up to 36% and 24% in favor of the (S)-sulfoxide could
respectively be observed. It is noteworthy that better yield and turnover frequencies
(85% and 1.09 min
−1 ) as well as a better enantiomeric excess in favor of the (S)372
J.-P. Mahy et al.
first inserted synthetic chromium salophens into apo-Mb and mutants. The artificial
metalloenzyme obtained from the H64D/A71G double mutant catalyzed the
stereoselective sulfoxidation of thioanisole, with rather low turnover frequencies
(TOF
0.13 min
−1 ) and enantiomeric excesses (ee
30% in favor of the (S)product) [37]. The 3D structures of two apo-Mb A71G mutant-Mn- and
Cr-salophen complexes revealed that the accessibility to the active site was sterically hindered by the bulky phenylenediamine moiety of the salophen cofactors.
They then used metal-salen cofactors instead, which had a rather low effect on the
efficiency of the chromium cofactors (TOF = 0.21 min
−1 ) but induced a noticeable
increase in the efficiency of the manganese cofactor (TOF = 2.7 min
−1 ). In addition, insertion of the later cofactor in the H64D/A71G double mutant led to a
slightly increased enantiomeric excess (30% ee in favor of the (S)-product) whereas
its insertion in the A71G single mutant led to an enantiomeric excess very similar to
that obtained with the WT protein (27% ee in favor of the (R)-product) [38].
Several other teams further inserted various Fe-tetrapyrrolic compounds into
apo-Mb to produce new biohybrids that catalyzed the oxidation of thioanisole by
H 2 O 2 and led to up to 38% ee in favor of the (S)-sulfoxide. The enantiomeric excess
could be increased either by covalent anchoring of metal complexes into apo-Mb
and mutants [39–41] or by changing the metal such as, for example, replacing iron
by manganese [41]. Accordingly, Lu et al. showed that a dual-point attachment of
manganese-salen complex to a double mutant of apo-Mb (apo-Mb Y103C/L72C)
led to an improved selectivity (51% ee) in thioanisole sulfoxidation relative to the
analogous single-point mutant (Y103C, 12% ee) (Fig. 5) [40, 41].
Xylanase A from Streptomyces lividans (Xln10A), a thermostable b1,4-endoxylanase glycoside hydrolase that hydrolyzes b-1,4 bonds in the main
chain of xylan [43], was also used to build up new artificial hemoproteins using the
host-guest strategy. This choice was guided by an early report from Nakamura and
Tsushida et al. that showed that xylanase possessed a wide enough active site to
accommodate an Fe(II)-a4-tetra-o-pivalamidophenylporphyrin. The resulting
heat-resistant hemoproteins were found to bind and release O 2 in aqueous medium
[44]. Metal complexes of synthetic tetraaryl porphyrins bearing negatively charged
substituents, such as Iron(III)tetra(4-carboxyphenyl)porphyrin (Fe(TpCPP)) and
iron(III)-meso-tetra(4-sulfonatophenyl)porphyrin (Fe(TpSPP)), were then inserted
into Xln10A to lead to new artificial hemoproteins that showed peroxidase activity
[12]. The catalytic activity of these Fe(TpCPP)-Xln10A and Fe(TpSPP)-Xln10A
biohybrids for the oxidation of thioanisole by H 2 O 2 (Fig. 6) was then investigated
and compared to that of Fe(TpCPP) and Fe(TpSPP) alone. These two Fe-porphyrin
complexes led, respectively to 45 and 33% yields in sulfoxide and to respective
TOFs of 0.56 and 0.41 min
−1 , but no enantiomeric excess could be detected. Use of
the Fe(TpCPP)- and Fe(TpSPP)-Xln10A biohybrids as catalysts led to a decrease in
the yields (about 24%) and turnover frequencies (about 0.30 min
−1 ), but enantiomeric excesses of up to 36% and 24% in favor of the (S)-sulfoxide could
respectively be observed. It is noteworthy that better yield and turnover frequencies
(85% and 1.09 min
−1 ) as well as a better enantiomeric excess in favor of the (S)372
J.-P. Mahy et al.