C O M M U N I C A T I O N S
Table 2. DKR of ꢀ-Haloalcohols 4a
ization did not proceed and static kinetic resolution resulted, giving 5h
with 50% conversion and 72% ee of the epoxide (Table 2, entry 13).
To show the potential of this DKR as a preparative procedure, a number
of epoxides were isolated in good yields (entries 3, 6, 9, 11, and 12). The
lower isolated yields compared to the GC or HPLC yields, as well as the
variations in enantioselectivity, are partly due to the fact that a less
active but more enantioselective batch of enzyme was used.
In summary, we developed the first chemo-enzymatic DKR of
ꢀ-haloalcohols giving the corresponding epoxides in a single step,
with good yields and excellent enantioselectivities using a variety
of aromatic substrates. This required the development of iridacycle
3, one of the most effective racemization catalysts to date for
ꢀ-haloalcohols and compatible with water, and the use of HheC
mutant Cys153Ser Trp249Phe.
entry
substrate
R
X
product
convn (%)
ee (%)
1
2
4b
4c
4d
4d
4e
4f
4g
4h
4i
C6H5
C6H5
Cl
Br
Cl
Cl
Br
Cl
Br
Cl
Cl
Br
Cl
Cl
Cl
5a
5a
5b
5b
5b
5c
5c
5d
5e
5e
5f
90
57
98
56
3
4-NO2-C6H4
4-NO2-C6H4
4-NO2-C6H4
3-NO2-C6H4
3-NO2-C6H4
2-NO2-C6H4
4-CN-C6H4
4-CN-C6H4
3-MeO-C6H4
4-CF3-C6H4
C6H11
80 (67)b
76
95 (99)c
97
4d
5e
6
86
90
75 (65)b
79
97 (94)c
52
7
8
9
10
11f
12g
13
28
91
67 (59)b
89
95 (96)c
86
4j
4k
4l
Acknowledgment. We thank T. D. Tiemersma-Wegman for
technical support and H. J. Heeres, G. N. Kraai, C. Kronenburg,
and T. Sonke for valuable discussions. Financial support from The
Netherlands Organisation for Scientific Research (NWO-CW/STW),
the Dutch Ministry of Economic Affairs, Royal DSM N.V., and
N.V. Organon, administered through the IBOS program, and the
UCT program, is gratefully acknowledged.
64 (57)b
58 (51)b
50
85 (90)c
98 (99)c
72
5g
5h
4m
a Reactions were performed on 0.2 mmol scale, using 6 U (initial
activity) of enzyme. A general procedure can be found in the Supporting
Information. b Isolated yields in parentheses. c ee of the isolated product
in parentheses. d Run on a 1.0 mmol scale. e Reaction performed without
BSA. f Run using 15 U of HheC (instead of 6 U). g Run using 20 vol%
of DMSO (instead of 5 vol%); 30 U of HheC was used (instead of 6 U).
Supporting Information Available: All experimental procedures
and characterization data of new compounds. This material is available
mutations Cys153Ser, which increases the enzyme’s stability toward
oxidation,12 and Trp249Phe, which increases its enantioselectivity
especially for aromatic substrates.13
The optimal reaction conditions were determined and DKR was
performed as shown in Table 2.
References
(1) For recent reviews on DKR, see:(a) Ahn, Y.; Ko, S.-B.; Kim, M.-J.; Park,
J. Coord. Chem. ReV. 2008, 252, 647–658. (b) Pellissier, H. Tetrahedron
2008, 64, 1563–1601. (c) Mart´ın-Matute, B.; Ba¨ckvall, J.-E. Curr. Opin.
Chem. Biol. 2007, 11, 226–232. (d) Kim, M.-J.; Ahn, Y.; Park, J. Bull.
Korean Chem. Soc. 2005, 26, 515–522. (e) Pa`mies, O.; Ba¨ckvall, J.-E.
Trends Biotechnol. 2004, 22, 130–135. (f) Turner, N. J. Curr. Opin. Chem.
Biol. 2004, 8, 114–119. (g) Pa`mies, O.; Ba¨ckvall, J.-E. Chem. ReV. 2003,
103, 3247–3261.
(2) (a) Larsson, A. L. E.; Persson, B. A.; Ba¨ckvall, J.-E. Angew. Chem., Int.
Ed. Engl. 1997, 36, 1211–1212. (b) Dinh, P. M.; Howarth, J. A.; Hudnott,
A. R.; Williams, J. M. J.; Harris, W. Tetrahedron Lett. 1996, 37, 7623–
7626.
Typically, DKR experiments were performed at 0.2 mmol scale.
The reaction is biphasic, using 10 mL of 50 mM aqueous HEPES
buffer (pH 7.5 for bromides, pH 8.0 for chlorides) and 3 mL of
toluene. The racemization catalyst was activated separately using
1 equiv of KOtBu in freshly distilled toluene under an inert
atmosphere. The solution of activated catalyst was then added to
the reaction mixture over 6 h using a syringe pump.14 To solubilize
the substrates in the aqueous reaction medium, DMSO (5 vol%)
was used as a cosolvent. Finally, the addition of bovine serum
albumin (BSA, 3.5 mg/mL)15 was required to stabilize the enzyme.
The DKR of 4b gave the expected epoxide 5a with 90% conversion
and 98% ee (Table 2, entry 1).16 The equivalent bromoalcohol 4c gave
5a with only 57% conversion and 56% ee (Table 2, entry 2). The rate
of uncatalyzed ring closure of chloroalcohols is lower than that of
bromoalcohols, which explains the higher enantioselectivities. Com-
pound 4d gave an excellent result that could be reproduced on 1.0
mmol scale (Table 2, entries 3 and 4).
As expected, the corresponding bromoalcohol 4e was converted with
lower but still very high enantioselectivity (Table 2, entry 5). A similar
halide dependence was observed using 3-nitrophenyl-substituted 4f and
4g and 4-cyanophenyl-substituted 4i and 4j, as the chlorides could be
converted to the epoxides with excellent ee, whereas the bromides
led to lower selectivities (Table 2, entries 6 vs 7 and 9 vs 10). The
conversion of 2-nitro substituted 4h was low and in addition its product
5d was obtained with slightly lower ee (91%) compared to its meta-
and para-substituted equivalents 5c and 5b (97% ee, Table 2, entry 8
vs entries 4 and 6). We attribute this to the bulkiness of the ortho-
substituent. Substrate 4k was converted with slighly lower conversion
and enantioselectivity (Table 2, entry 11). Trifluoromethyl-substituted
substrate 4l was converted to 5g with an excellent ee of 98%, but
HheC seems to be less active toward this substrate and additional
enzyme was necessary to obtain a moderate yield (Table 2, entry 12).
When cyclohexyl-substituted chloroalcohol 4m was reacted, racem-
(3) Pa`mies, O.; Ba¨ckvall, J.-E. J. Org. Chem. 2002, 67, 9006–9010.
(4) Haak, R. M.; Tarabiono, C.; Janssen, D. B.; Minnaard, A. J.; De Vries,
J. G.; Feringa, B. L. Org. Biomol. Chem. 2007, 5, 318–323.
(5) For reviews on haloalcohol dehalogenases, see:(a) Janssen, D. B. AdV. Appl.
Microbiol. 2007, 61, 233–252. (b) De Jong, R. M.; Dijkstra, B. W. Curr.
Opin. Struct. Biol. 2003, 13, 722–730. (c) De Vries, E. J.; Janssen, D. B.
Curr. Opin. Biotechnol. 2003, 14, 414–420. (d) Fetzner, S.; Lingens, F.
Microbiol. ReV. 1994, 58, 641–685.
(6) Hasnaoui-Dijoux, G.; Majer´ıc Elenkov, M.; Lutje Spelberg, J. H.; Hauer,
B.; Janssen, D. B. ChemBioChem 2008, 9, 1048–1051.
(7) This evaluation is part of a forthcoming paper on racemization reactions
catalyzed by a range of iridacycles. See the Supporting Information for
racemization results using a number of ruthenium-based catalysts.
(8) Sortais, J.-B.; Pannetier, N.; Holuigue, A.; Barloy, L.; Sirlin, C.; Pfeffer,
M.; Kyritsakas, N. Organometallics 2007, 26, 1856–1867.
(9) Liu, J.; Wu, X.; Iggo, J. A.; Xiao, J. Coord. Chem. ReV. 2008, 252, 782–
809.
(10) Mart´ın-Matute, B.; Edin, M.; Boga´r, K.; Kaynak, F. B.; Ba¨ckvall, J.-E.
J. Am. Chem. Soc. 2005, 127, 8817–8825.
(11) Haloketone formation was not observed in subsequent DKR experiments
using a variety of haloalcohols.
(12) Tang, L.; Van Hylckama Vlieg, J. E. T.; Lutje Spelberg, J. H.; Fraaije,
M. W.; Janssen, D. B. Enzyme Microb. Technol. 2002, 30, 251–258.
(13) (a) Majer´ıc Elenkov, M.; Tang, L.; Hauer, B.; Janssen, D. B. Org. Lett.
2006, 8, 4227–4229. (b) Tang, L.; Torres Pazmin˜o, D. E.; Fraaije, M. W.;
De Jong, R. M.; Dijkstra, B. W.; Janssen, D. B. Biochemistry 2005, 44,
6609–6618. (c) Tang, L.; Van Merode, A. E. J.; Lutje Spelberg, J. H.;
Fraaije, M. W.; Janssen, D. B. Biochemistry 2003, 42, 14057–14065.
(14) Addition of the catalyst solution in one portion at the beginning of the
reaction led to poorer yields and enantioselectivities.
(15) Carter, D. C.; Ho, J. X. AdV. Protein Chem. 1994, 45, 153–203.
(16) In most cases conversion stopped at around 75-90%, which can be
attributed to the fact that the ring-closure reaction is reversible. See
also: Lutje Spelberg, J. H.; Van Hylckama Vlieg, J. E. T.; Bosma, T.;
Kellogg, R. M.; Janssen, D. B Tetrahedron: Asymmetry 1999, 10,
2863–2870.
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