Notes and references
z Procedure for asymmetric epoxidation of 1a with StyAB2: 1a
(12 mg) was added to the suspension of recombinant E. coli
BL21 wet cells (2.0 g, wet weight) harboring StyAB2 in 20 ml of
potassium phosphate buffer (100 mM, pH 6.5). The mixture was
incubated at 30 1C with shaking at 240 rpm. Aliquots were taken at
different time points, centrifuged, and extracted by ether. The
organic layers were dried over anhydrous Na2SO4 and concentrated
under reduced pressure. All the samples were analyzed with reverse-
phase and chiral HPLC to determine the yield and enantiomeric
purity of products. After
2 h, (1R,2R)-phenyl glycidol (2a)
was obtained (yield 50%, ee >99%, dr 98 : 2) and enantiopure
(R)-1a was obtained (ee >99%). The products were purified
by silica gel column chromatography, and identified by NMR
analysis.
Fig. 2 Two different orientations of (S)-1a docked into the putative
active site of SMO (PDB code 3IHM). The substrate is shown in the
stick mode. The figure was generated using the program Autodock and
visualized using the program Pymol.
1 A. Mooney, P. Ward and K. O’Connor, Appl. Microbiol. Biotechnol.,
2006, 72, 1–10.
2 K. Otto, K. Hofstetter, M. Rothlisberger, B. Witholt and
A. Schmid, J. Bacteriol., 2004, 186, 5292–5302.
3 S. Bernasconi, F. Orsini, G. Sello, A. Colmegna, E. Galli and
G. Bestetti, Tetrahedron Lett., 2000, 41, 9157–9161.
4 S. Panke, M. G. Wubbolts, A. Schmid and B. Witholt, Biotechnol.
Bioeng., 2000, 69, 91–100.
5 S. Bernasconi, F. Orsini, G. Sello and P. Di Gennaro,
Tetrahedron: Asymmetry, 2004, 15, 1603–1606.
6 S. M. Park, J. W. Bae, J. H. Han, E. Y. Lee, S. G. Lee and S. Park,
J. Microbiol. Biotechnol., 2006, 16, 1041–1046.
7 D. Kuhn, M. A. Kholiq, E. Heinzle, B. Buhler and A. Schmid,
Green Chem., 2010, 12, 815–827.
Table 2 Epoxidation of substrates 1a and 1p using the mutantsa
Mutation Substrate dr
ee (%) de (%) Yield (%)
E
Y73F
T200V
T200V
1a
1a
1p
98 : 2
92 : 8
75 : 25
>99
99
75
97
87
59
50
36
37
>200
23
5
a
The reactions were carried out for 48 h. Other conditions and
analytical methods were the same as those listed in Table 1.
8 S. Panke, M. Held, M. G. Wubbolts, B. Witholt and A. Schmid,
Biotechnol. Bioeng., 2002, 80, 33–41.
9 D. Tischler, D. Eulberg, S. Lakner, S. R. Kaschabek, W. J. H.
van Berkel and M. Schlomann, J. Bacteriol., 2009, 191,
4996–5009.
10 E. W. van Hellemond, D. B. Janssen and M. W. Fraaije, Appl.
Environ. Microbiol., 2007, 73, 5832–5839.
11 H. Lin, J. Qiao, Y. Liu and Z.-L. Wu, J. Mol. Catal. B: Enzym.,
2010, 67, 236–241.
12 K. B. Sharpless, C. H. Behrens, T. Katsuki, A. W. M.
Lee, V. S. Martin, M. Takatani, S. M. Viti, F. J.
Walker and S. S. Woodard, Pure Appl. Chem., 1983, 55,
589–604.
13 K. B. Sharpless, Angew. Chem., Int. Ed., 2002, 41,
2024–2032.
14 M. M. Hussain and P. J. Walsh, Acc. Chem. Res., 2008, 41,
883–893.
15 W. Zhang, A. Basak, Y. Kosugi, Y. Hoshino and H. Yamamoto,
Angew. Chem., Int. Ed., 2005, 44, 4389–4391.
The result clearly demonstrated the significance of Thr200
in the chiral recognition of the epoxidation of secondary allylic
alcohols. However, its contribution to the diastereoselectivity
is most probably small as judged by the incomplete loss of
selectivity for mutant T200V. The excellent diastereo- and
enantio-selectivity might be largely attributed to the overall
architecture of the substrate binding cavity of the enzyme,
which is able to lead to high enantio-selectivity towards the
natural substrate styrene, which possesses no hydroxyl group.
Elucidation of the molecular basis for the selectivity of StyAB2
awaits further work on structural and mutational analysis.
In summary, we have developed a highly diastereo- and
enantio-selective enzymatic synthesis of glycidol derivatives with
contiguous stereogenic centers. The simple and efficient catalyst
system provides the first green alternative to classic chemical
synthesis. The novel substrate spectrum of SMO demonstrates
its great potential in chiral synthesis, and the identification of
selectivity-related amino acid residues provides a way for further
manipulation and functional extension of this enzyme.
16 Z. Li, W. Zhang and H. Yamamoto, Angew. Chem., Int. Ed., 2008,
47, 7520–7522.
17 M. Frohn, X. M. Zhou, J. R. Zhang, Y. Tang and Y. Shi, J. Am.
Chem. Soc., 1999, 121, 7718–7719.
18 M. E. Jung and Y. H. Jung, Tetrahedron Lett., 1989, 30,
6637–6640.
We thank G. T. Gassner for sharing the details of the NSMOA
crystal structure in advance of official release. This work was
supported by National Natural Science Foundation of China
(20802073), 100 Talents Program of the Chinese Academy of
Sciences and Sichuan Province Science Foundation for Young
Scholars 08ZQ026-023.
19 A. A. Qaed, H. Lin, D.-F. Tang and Z.-L. Wu, Biotechnol. Lett.,
2010, DOI: 10.1007/s10529-010-0472-9.
20 U. E. Ukaegbu, A. Kantz, M. Beaton, G. T. Gassner and
A. C. Rosenzweig, Biochemistry, 2010, 49, 1678–1688.
21 G. M. Morris, D. S. Goodsell, R. S. Halliday, R. Huey,
W. E. Hart, R. K. Belew and A. J. Olson, J. Comput. Chem.,
1998, 19, 1639–1662.
c
2612 Chem. Commun., 2011, 47, 2610–2612
This journal is The Royal Society of Chemistry 2011