reveals that the polar and proton-donating environment sur-
rounding the MnSalen inside the protein is critical for enhan-
cing reactivity and chemoselectivity. Examination of the
hydrophobicity of the residues near the substrate entrance
channel demonstrates that chemoselectivity is at least partially
controlled by modulating the polarity at the A71 position, and
occurs with minimal change in structure and reactivity. These
novel insights are valuable for the future rational design of
artificial biocatalysts.
We are grateful for the support of the National Science
Foundation (CHE-05-52008) and the National Institute of
Health (GM062211).
Fig. 3 Protein surfaces of the 1ꢀMb and 1ꢀMb(A71S) showing the
local hydrophobicity around the right side of the MnSalen cofactor.
White and blue colors represent hydrophobic and hydrophilic regions,
20
respectively. Rendered using VMD).
Notes and references
a
Table 2 Reactivity and selectivity of 1ꢀMb and 1ꢀMb(A71S)
1
V. P. Miller, G. D. DePillis, J. C. Ferrer, A. G. Mauk and P. R.
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ee of
sulfoxide (%)
Yield of Yield of
sulfoxide (%) sulfone (%)
2
Entry
Catalyst
3
047–3080.
b
0
1
2
a
1ꢀMb
60 ꢂ 1.2
49.4 ꢂ 1.8
50.0 ꢂ 1.2
Determined using the same method as in Table 1 in Hepes buffer
3 D. Qi, C. -M. Tann, D. Haring and M. D. Distefano, Chem. Rev.,
2001, 101, 3081–3111.
1ꢀMb(A71S) 58 ꢂ 0.9
2.1 ꢂ 0.8
4
5
6
M. T. Reetz, Tetrahedron, 2002, 58, 6595–6602.
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b
(
50 mM, pH = 7, 5% MeOH) at 4 1C for 10 min. Sulfone was
not detected.
9
7–103.
7
8
9
Y. Lu, Angew. Chem., Int. Ed., 2006, 45, 5588–5601.
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Computational modeling showed that mutation of A71 to S
results in increased hydrophilicity on the protein surface near
the substrate entrance (Fig. 3, white and blue colors represent
hydrophobic and hydrophilic residues, respectively). This new
biocatalyst MnSalen-Mb (T39C/L72C/A71S) called 1ꢀ
Mb(A71S) was then expressed and purified as described pre-
1
1
0 C. J. Reedy and B. R. Gibney, Chem. Rev., 2004, 104, 617–649.
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1
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4
2
1
viously and characterized by UV-Vis spectroscopy and ESI
mass spectrometry (see ESIw). As shown in Table 2, 1ꢀ
Mb(A71S) exhibits similar reactivity and enantioselectivity
1
15 M. T. Reetz and N. Jiao, Angew. Chem., Int. Ed., 2006, 45,
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2
1
1
1
1
2
2
(
entry 2, Table 2) to 1ꢀMb (entry 1, Table 2), with the
exception that the formation of sulfone was now observed
2.1 ꢂ 0.8%). A comparison of the GC traces of sulfoxidation
2
7 M. T. Reetz, J. J. P. Peyralans, A. Maichele, Y. Fu and M.
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USA, 2005, 102, 4683–4687.
(
catalyzed by 1ꢀMb(A71S) and 1ꢀMb (Fig. S1, ESIw) indicates
formation of sulfone only in the presence of 1ꢀMb(A71S).
Therefore we conclude that the increased polarity of the
protein surface in 1ꢀMb(A71S) allows the more polar sulfoxide
to enter the pocket for oxidation to the sulfone, while the more
hydrophobic surface presented by 1ꢀMb inhibited this side
reaction. These results demonstrate that tuning the hydropho-
bicity of the substrate entrance channel could affect the
chemoselectivity of an oxidation reaction.
9 M. Ohashi, T. Koshiyama, T. Ueno, M. Yanase, H. Fujii and Y.
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2
2
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1
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7
In conclusion, we have demonstrated the effectiveness of a
protein scaffold for enhancing the reactivity and chemoselec-
tivity of MnSalen in the sulfoxidation of thioanisole in an
artificial metalloenzyme. A comparison of MnSalen outside
the protein in aqueous media with that in organic solvents
3rd edn, 1992.
2
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This journal is ꢁc The Royal Society of Chemistry 2008
Chem. Commun., 2008, 1665–1667 | 1667