Asymmetric bioreduction of α,β-unsaturated nitriles and ketones

Article abstract of DOI:10.1016/j.tetasy.2008.05.023

Two applications for the asymmetric reduction of activated alkenes employing isolated enoate reductases are reported. A series of α,β-unsaturated nitriles were shown to be converted to the optically active nitrile products in high yields and excellent enantioselectivities (up to 99% ee). In addition, the reduction of 2,3-disubstituted cyclopentenones was shown to provide almost exclusively trans-2,3-disubstituted cyclopentanones in high yield and enantiopurity (94% ee).

Full text of DOI:10.1016/j.tetasy.2008.05.023

Tetrahedron: Asymmetry 19 (2008) 1403–1406
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Tetrahedron: Asymmetry
journal homepage: www.elsevier.com/locate/tetasy
Asymmetric bioreduction of a,b-unsaturated nitriles and ketones
*
Birgit Kosjek , Fred J. Fleitz, Peter G. Dormer, Jeffrey T. Kuethe, Paul N. Devine
Department of Process Research, Merck and Co., Inc., PO Box 2000, Rahway, NJ 07065-0900, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Two applications for the asymmetric reduction of activated alkenes employing isolated enoate reductases
are reported. A series of a,b-unsaturated nitriles were shown to be converted to the optically active nitrile
products in high yields and excellent enantioselectivities (up to 99% ee). In addition, the reduction of 2,3-
disubstituted cyclopentenones was shown to provide almost exclusively trans-2,3-disubstituted cyclo-
pentanones in high yield and enantiopurity (94% ee).
Received 29 February 2008
Accepted 19 May 2008
Available online 28 June 2008
Ó 2008 Elsevier Ltd. All rights reserved.
1. Introduction
aldehydes. Some examples of metal catalyzed hydrogenation exist,
but the enantioselective carbon–carbon bond reduction of conju-
The development of asymmetric bioreductive routes to enantio-
merically pure compounds has emerged in recent years to be-
come a key component in the synthesis of increasingly complex
pharmaceutical drugs, which often contain multiple stereocen-
ters.1 Bioreductions are of great industrial significance, because
the enzyme catalysts are highly chemo-, regio-, and stereoselec-
tive, preventing the formation of side products and avoiding pro-
tection/deprotection steps. The processes have low energy
requirements because of their ambient temperature and pressure
conditions. Enzymes are renewable resources and also biodegrad-
able, thus presenting a valuable tool for more environmentally
friendly industrial transformations.2
gated nitriles remains a challenge due to their inherent low reac-
tivity.10 For example, the reduction of
c
-cyano- ,b-unsaturated
a
ketones by a yeast species is known, but only product mixtures
of the saturated keto nitrile and the corresponding saturated alco-
hol were obtained due to the presence of ketone reductases in the
whole cells.11 Smallridge has described the reduction of 2-phenyl-
2-propenenitrile to (R)-phenylpropanenitrile in high enantiopurity
by employing baker’s yeast in petroleum ether.12 Herein, we report
the asymmetric bioreduction of a series of a,b-unsaturated nitriles
1–4 and 9 employing a library of commercially available enoate
reductases.13
We also investigated the enzymatic carbon–carbon double
The selective reduction of carbon–carbon double bonds pro-
bond reduction of a,b-disubstituted cyclopentenones. While
vides access to molecules with an
a
- and possibly an additional
reductions of cyclic enones are known, the substituents are con-
fined to protons, methyl, and ethyl groups.6,7,14 We describe a bio-
catalytic 1,4-reduction of 2,3-disubstituted cyclopenten-3-ones 11
and 12 as the asymmetric step in the synthesis of 1,2,3-trisubsti-
tuted cyclopentanes, which provide useful pharmaceutical
intermediates.15a
b-stereocenter, depending on the substitution pattern.3 Many
advances in the catalytic asymmetric hydrogenation of activated
alkenes have been reported using either chiral rhodium or
ruthenium phosphines or enoate reductase enzymes.3–7 The reduc-
tion mechanism for enoate reductases proceeds according to an
anti-addition of one hydride from the flavin cofactor, and one
hydrogen from the solvent. This stereo-complementary approach
to homogeneous chemical catalysts, which generally transfers
hydrogen to the olefin in syn fashion, provides access to chiral
intermediates with a trans-configuration.3,5–8
2. Results and discussion
2.1. Biocatalytic reduction of a,b-unsaturated nitriles 1–4 and 9
We were interested in evaluating bioreduction in systems
where the substitution patterns of the carbon–carbon double bond
had not previously been investigated. In particular, enantiopure ni-
triles are important synthetic precursors for the preparation of
pharmacologically important building blocks.9 Furthermore,
nitriles can easily be converted to carboxylic acids, amines, or
A series of para-substituted phenyl butenenitriles 1–4 were pre-
pared according to a literature protocol and were fully character-
ized by NMR spectroscopy.16 Phenyl butenenitriles 1–4 were
obtained in the Z-configuration as determined by NOE experi-
ments.17 Screening of compounds 1–4 was performed with
commercially available enoate reductases in the presence of
excess NAD(P)H (Fig. 1).18 The enzymes showed the enantioselec-
tive reduction of all phenyl butenenitriles 1–4, giving the corres-
ponding saturated products 5–8 in good yield and excellent
* Corresponding author.
E-mail address: birgit_kosjek@merck.com (B. Kosjek).
0957-4166/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetasy.2008.05.023

1404
B. Kosjek et al. / Tetrahedron: Asymmetry 19 (2008) 1403–1406
Table 2
R
R
Screening results for enzymatic reduction of substrates 11 and 12a
ERED
Enzymeb
13
% Conversionc
14
% Conversionc
CN
CN
3
Entry
% ee transd
% ee transe
NAD(P)H
NAD(P)
1
2
3
4
5
6
7
8
9
10
11
12
13
14
ERED101
ERED102
ERED103
ERED104
ERED105
ERED106
ERED107
ERED108
ERED109
ERED110
ERED111
ERED112
ERED113
ERED114
98
98
98
85 (1R,2R)
92 (1R,2R)
93 (1R,2R)
93 (1R,2R)
93 (1R,2R)
92 (1R,2R)
93 (1R,2R)
93 (1R,2R)
93 (1R,2R)
93 (1R,2R)
91 (1R,2R)
92 (1R,2R)
93 (1R,2R)
93 (1R,2R)
100
100
100
100
100
100
100
100
100
100
100
100
52
98 (1R,2R)
98 (1R,2R)
98 (1R,2R)
98 (1R,2R)
98 (1R,2R)
98 (1R,2R)
98 (1R,2R)
98 (1R,2R)
98 (1R,2R)
98 (1R,2R)
98 (1R,2R)
98 (1R,2R)
98 (1R,2R)
98 (1R,2R)
5 R=H
1 R=H
6 R=CH3
2 R=CH
3
7 R=OCH3
8 R=Cl
95
3 R=OCH
4 R=Cl
100
100
63
CN
CN
97
ERED
100
100
100
100
56
Cl
Cl
NAD(P)H
NAD(P)
NH
NH
9
10
100
100
a
Figure 1. Asymmetric reduction of
a,b-unsaturated nitriles 1–4, 9 using enoate
0.5 mg 11 or 12 per reaction in 0.5 ml 0.1 M KH2PO4 pH 7.0, 5 vol % DMSO, 5 mg
lyophilized enzyme preparation (ERED101-116, Biocatalytics, Inc.), 5 mg NAD(P)H.
30 °C, 3 h.
reductases (ERED). The absolute configuration of compound 10 is not specified.
b
ERED = enoate reductase.
c
mol % conversion determined by HPLC analysis. ERED115, 116 gave <15%
conversion.
enantioselectivities up to 99% ee (Table 1).19 All enzymes consis-
tently displayed the same facial selectivity giving the (R)-(+)-enan-
tiomer. This was established for compound 5 as determined by
comparison of the specific rotation of 5 from ERED112 to the
known (S)-(ꢀ)-phenyl butyronitrile.20 Given the structural similar-
ity of each of the substrates, we speculate that the (R)-(+)-enanti-
omers of 6–8 are also formed based on the enzyme mechanism
likely directing the reduction of analogous compounds with the
same facial selectivity when compared to 5.
Encouraged by the promising results from the para-substituted
phenyl butenenitriles 1–4, we examined the bioreduction of a
more complex pharmaceutical building block 9. Substrate 9 was
well accepted by five enzymes (Table 1, entries 3, 4, and 6–8)
and proceeded to give ꢁ85% conversion with enantioselectivities
up to 98% ee observed for compound 10 (Table 1).21 The absolute
configuration of 10 was not determined with absolute certainty.
The reaction likely follows the same facial selectivity as shown
for compound 5, but the sterically more demanding substitution
pattern could influence the enzyme’s stereopreference as examples
for a switch in stereopreference for an enoate reductase isozymes
that have been reported.22
d
Stereochemical assignments made based on comparison to Ref. 15.
Stereochemical assignment made based on results for analogous 13.
e
2.2. Biocatalytic reduction of 2,3-disubstituted cyclopenten-3-
ones 11 and 1215a,23
Having established that the biocatalytic reduction of a,b-unsat-
urated nitriles provided a means of preparing chiral nitriles in
excellent yield and enantioselectivity, we next examined the same
library of isolated enzymes against 2,3-disubstituted cyclopente-
nones 11 and 12 in the presence of excess NAD(P)H.18 These sub-
strates are particularly attractive for enoate reductases since the
alkene is conjugated to two electron-withdrawing carboxyl groups.
Most of the enzymes gave complete 1,4-reduction of the doubly
activated cyclopentenones 11 and 12 as shown in Table 2. In each
case, no detectable amount of 1,2-reduction of the ketone was ob-
served because of the excellent chemo- and regio-specificity of the
enoate reductases. The use of isolated enzymes avoids any interfer-
ing competitive ketone reduction by ketone reductases present in
some whole cell systems.3,14 Therefore, this bioreduction provides
The present results demonstrate the applicability of enoate
a valuable tool for the 1,4-reduction of
compounds. The corresponding cyclopentanones 13 and 14 were
obtained in a trans-configuration as the major product (13: ꢁ94%
a,b-unsaturated carbonyl
reductases for the reduction of more complex
nitriles.
a,b-unsaturated
Table 1
Screening results for enzymatic reduction of substrates 1–4 and 9a
Entry
Enzymeb
1
5
% ee
2
6
% ee
3
7
% ee
4
8
% ee
9
10
% ee
% Conversionc
% Conversionc
% Conversionc
% Conversionc
% Conversionc
1
2
3
4
5
6
7
8
9
10
11
12
13
ERED101
ERED102
ERED103
ERED104
ERED105
ERED106
ERED107
ERED108
ERED109
ERED110
ERED111
ERED112
ERED114
73
3
2
2
65
1
98 (R)
76
10
13
5
15
5
99
92
90
100
42
37
9
22
9
97
98
91
100
25
27
5
33
13
35
2
88
55
67
96
72
96
88
92
83
85
54
86
83
85
55
37
98
94
98
98
98
98
98
98
98 (R)
94 (R)
99
85
92
93
82
70
87
63
3
10
4
32
5
100
100
24
100
80
99 (R)
99 (R)
98 (R)
98 (R)
97 (R)
63
32
25
99
36
94
88
92
97
93
90
55
86
97
79
90
98
98
98
98
74
70
89
89
87
a
0.5 mg substrate 1–4 and 9 per reaction in 0.5 ml 0.1 M KH2PO4 pH 7.0, 5 vol % DMSO, 5 mg lyophilized enzyme preparation (ERED101-116, Biocatalytics, Inc.), 5 mg
NAD(P)H. 30 °C, 3 h.
b
ERED = enoate reductase.
mol % conversion determined by HPLC analysis.
c

B. Kosjek et al. / Tetrahedron: Asymmetry 19 (2008) 1403–1406
1405
trans, 14: ꢁ92% trans) which was determined by NMR spectroscopy
Acknowledgments
(NOE).24,25 This is consistent with the proposed mechanism by
Massey and Karplus, whereby anti-addition of H2 is observed and
the thermodynamically most favored configuration is isolated.3c,d,7
Good enantioselectivity was observed for 13 with 93% ee for the
(1R,2R)-enantiomer. Product 13 was converted to the known 2-
(4-fluorophenyl)-3-hydroxycyclopentane-1-carboxylic acid in or-
der to unequivably establish the absolute configuration of 13.15
The larger ester group of substrate 12 increased the enantioselec-
tivity of the enzymes yielding 14 in 98% ee (1R,2R). The stereo-
chemical assignment for 14 was based on analogous results
obtained for the bioreduction of 13 (Fig. 2).
The authors thank Drs. Thorsten Rosner, John Limanto and Rick
Sidler of Merck and Co., Inc. for helpful discussions and Mr. Adam
Beard of Merck and Co., Inc. for valuable assistance in the develop-
ment of chiral assays.
References
1. Pollard, D. J.; Woodley, J. M. Trends Biotechnol. 2007, 25, 66–73.
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3. (a) Stuermer, R.; Hauer, B.; Hall, M.; Faber, K. Curr. Opin. Chem. Biol. 2007, 11,
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29; (c) Abramowitz, A. S.; Massey, V. J. Biol. Chem. 1976, 251, 5327–5336; (d)
Xu, D.; Kohli, R. M.; Massey, V. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 3556–3561.
4. (a) Brown, J. M. In Comprehensive Asymmetric Catalysis; Jacobsen, E. N., Pfaltz, A.,
Yamamoto, H., Eds.; Springer: New York, 1999; Vol. 1, (b) Knowles, W. S.
Angew. Chem., Int. Ed. 2002, 41, 1998–2007; (c) Noyori, R. Angew. Chem., Int. Ed.
2002, 41, 2008–2022.
F
F
O
O
ERED
5. (a) Mueller, A.; Hauer, R.; Stuermer, B.; Rosche, B. Angew. Chem., Int. Ed. 2007,
46, 3316–3318; (b) Stueckler, C.; Hall, M.; Ehammer, H.; Pointner, E.; Kroutil,
W.; Macheroux, P.; Faber, K. Org. Lett 2007, 9, 5409–5411; (c) Grogan, G.;
Holland, H. J. . J. Mol. Catal. B: Enzym. 2000, 9, 1–32; (d) Shimoda, K.; Hirata, T. J.
Mol. Catal. B: Enzym. 2000, 8, 255–264; (e) Chaparro-Riggers, J. F.; Rogers, T. A.;
Vazquez-Figueroa, E.; Polizzi, K. M.; Bommarius, A. S. Adv. Synth. Catal. 2007,
349, 1521–1531; (f) Servi, S. Synthesis 1990, 1–25.; (g) Shimoda, K.; Kubota, N.;
Hamada, H.; Yamane, S.; Hirate, T. Bull. Chem. Soc. Jpn. 2004, 77, 2269–2272.
6. Hall, M.; Stueckler, C.; Kroutil, W.; Macheroux, P.; Faber, K. Angew. Chem., Int.
Ed. 2007, 46, 3934–3937.
CO2R
11 R=CH
CO2R
13 R=CH
NAD(P)H
NAD(P)
3
3
14 R=CH(CH )
12 R=CH(CH )
3 2
3 2
Figure 2. Asymmetric reduction of
reductases.
a,b-unsaturated ketones 11 and 12 using enoate
7. Swiderska, M. A.; Stewart, J. D. J. Mol. Catal. B: Enzym. 2006, 42, 52–54.
8. de Wildeman, S. M. A.; Sonke, T.; Shoemaker, H. E.; May, O. Acc. Chem. Res.
2007, 40, 1260–1266.
9. (a) Motokura, K.; Fujita, N.; Mori, K.; Mizugaki, T.; Jitsukawa, K.; Ebitani, K.;
Kaneda, K. Chem. Eur. J. 2006, 12, 8228–8239; (b) Im, D. S.; Cheong, C. S.; Lee, S.;
Youn, B. H.; Kim, S. C. Tetrahedron 2000, 56, 1309–1314.
10. Lee, D.; Kim, D.; Yun, J. Angew. Chem., Int. Ed. 2006, 45, 2785–2787.
11. Koul, S.; Crout, D. H. G.; Errington, W.; Tax, J. J. Chem. Soc. Perkin Trans. 1 1995,
2969–2988.
12. (a) Smallridge, A. J.; Ten, A.; Trewhella, M. A. Tetrahedron Lett. 1998, 39, 5121–
5124; (b) Dumanski, P. G.; Florey, P.; Knettig, M.; Smallridge, A. J.; Trewhella,
M. A. J. Mol. Catal. B: Enzym. 2001, 11, 905–908.
13. ERED101 to ERED116 were purchased from Biocatalytics, Inc., Pasadena, CA.
14. Kergomard, A.; Renard, M. F.; Veschambre, H. Tetrahedron Lett. 1978, 52, 5197–
5200.
15. (a) Kuethe, J. T.; Wong, A.; Wu, J.; Davies, I. W.; Dormer, P. G.; Welch, C. J.;
Hillier, M. C.; Hughes, D. L.; Reider, P. J. J. Org. Chem. 2002, 67, 5993–6000; For
the conversion of racemic 13 to 2-(4-fluorophenyl)-3-hydroxycyclopentane-1-
carboxylic acid, see: (b) Desai, R. C.; Cicala, P.; Meurer, L. C.; Finke, P. E.
Tetrahedron Lett. 2002, 43, 4569–4570.
We then focused on scalability and increasing the cost-effi-
ciency of two example reactions compared to the screening con-
ditions. ERED112 was chosen to demonstrate process
development on the reduction of 1 and ERED114 was selected
as catalyst for the reduction of 11. The first goal was accom-
plished by lowering the substrate to catalyst ratio by weight
from 1:10 in the screen to 1:1 for both processes. Reduction of
doubly activated 11 was complete within a 3 h time period sim-
ilar to the screening reactions, to provide 13 in similar yield and
ee. The conversion of 1 was complete within 18 h which is still a
feasible reaction time for scale-up.26 An in situ NAD(P)H cofactor
recycling system employing a sodium phosphite/phosphite dehy-
drogenase system was added to reduce the cost of the expensive
cofactor.27 This cofactor regeneration system offers the advan-
tage of a consistent pH throughout the reaction in contrast to
common glucose/glucose dehydrogenase systems where pH con-
trol is required.28 The improved bioreductions of compounds 1
and 11 were successfully demonstrated on a 200 mg scale, pro-
viding scope for efficient and scalable processes for the asym-
metric reduction of conjugated olefins with commercially
available enoate reductases.
16. Pepin, Y.; Nazemi, H.; Payette, D. Can. J. Chem. 1978, 56, 41–45. 2-Phenyl-but-2-
enenitrile 1. Light yellow oil: 1H NMR (CDCl3, 400 MHz) d 7.53 (m, 2H), 7.42–
7.34 (om, 3H), 6.91 (q, J = 7.2, 1H), 2.23 (d, J = 7.2, 3H); 13C NMR (CDCl3,
100 MHz) d 142.04, 133.48, 129.09, 128.98, 125.70, 117.27, 116.65, 17.96. 2-(4-
Methyl)-phenyl-but-2-enenitrile 2. Yellow oil: 1H NMR (CDCl3, 400 MHz) d 7.41
(m, 2H), 7.20 (m, 2H), 6.84 (q, J = 7.2, 1H), 2.37 (s, 3H), 2.20 (d, J = 7.2, 3H); 13
C
NMR (CDCl3, 100 MHz)
d 140.90, 138.99, 130.67, 129.73, 125.53, 117.06,
116.76, 21.25, 17.84. 2-(4-Methoxy)-phenyl-but-2-enenitrile 3. Yellow oil: 1H
NMR (CDCl3, 400 MHz) d 7.40 (m, 2H), 6.86 (m, 2H), 6.71 (q, J = 7.2, 1H), 3.78 (s,
3H), 2.14 (d, J = 7.2, 3H); 13C NMR (CDCl3, 100 MHz) d 160.22, 139.75, 126.93,
126.08, 116.84, 116.59, 114.44, 55.50, 17.79. 2-(4-Chloro)-phenyl-but-2-
enenitrile 4. Light yellow solid: 1H NMR (CDCl3, 400 MHz) d 7.44 (m, 2H),
7.35 (m, 2H), 6.88 (q, J = 7.2, 1H), 2.21 (d, J = 7.2, 3H). 13C NMR (CDCl3,
100 MHz) d 142.58, 134.86, 131.88, 129.59, 126.87, 116.19, 116.12, 17.94.
17. Observed NOE
3. Conclusion
Two applications of the asymmetric carbon–carbon double
bond reduction using commercially available enoate reductase
enzymes are reported. The reduction of a series of
a,b-unsatu-
rated nitriles was demonstrated in good yield and enantioselec-
tivity. This approach for the synthesis of optically active nitrile
compounds was also successfully applied to a pharmaceutical
intermediate. The enzymatic reduction of 2,3-disubstituted
cyclopentenones yielded valuable trans-cyclopentanone cores in
high diastereo- and enantioselectivities which demonstrates the
scope for these enzymes in the construction of more complex
R
%NOE:
R:
a: 0.5% b: 1.1%
H
CN
a: 1.0.%
CH3
CH3O
Cl
a
a: 1.2% b: 2.4%
a: 1.7% b: 0.9%
b
substrates. The described bioreductions of both
a,b-unsaturated
nitriles and ketones provide access to highly functionalized com-
pounds which may be elaborated to more structurally intriguing
pharmacophores. The complete scope and depth of the these
bioreductions is still under active investigation and the results
will be published in due course.
18. General screening conditions for the enoate reductases library with excess
NAD(P)H. NAD(P)(H) was purchased from Biocatalytics, Inc., Pasadena, CA.
5 mg lyophilized enzyme preparation was dissolved in buffer (0.4 ml 0.1 M
KH2PO4, pH 7) and combined with a solution of NADPH (5 mg, 6 lmol) in

1406
B. Kosjek et al. / Tetrahedron: Asymmetry 19 (2008) 1403–1406
mol) in 24. Observed NOE
KH2PO4 buffer (0.037 ml, 0.5 M, pH 7) and a solution of NADH (5 mg, 7
l
KH2PO4 buffer (0.0.37 ml, 0.5 M, pH 7). A solution of appropriate substrates
1–4, 9, 11, 12 (0.5 mg) in DMSO (0.025 ml) was added. The reaction was
incubated at 30 °C for 3 h.
F
O
19. Racemic 2-phenyl-butyronitrile was obtained from Aldrich. Racemic standards
for 6–8 were prepared according to Profitt, J. A.; Watt, D. S.; Corey, E. J. J. Org.
Chem., 1975, 40, 127. 2-(4-Methyl)-phenyl-butyronitrile 6. Colorless oil: 1H NMR
(CDCl3, 400 MHz) d 7.20 (m, 4H), 3.70 (t, J = 7.2, 1H), 2.35 (s, 3H), 1.93 (m, 2H),
1.07 (t, J = 7.2, 3H); 13C NMR (CDCl3, 100 MHz) d 138.00, 132.95, 129.86,
127.37, 121.11, 38.75, 29.40, 21.23, 11.66. 2-(4-Methoxy)-phenyl-butyronitrile
7. Yellow oil: 1H NMR (CDCl3, 400 MHz) d 7.24 (m, 2H), 6.91 (m, 2H), 3.82 (s,
3H), 3.69 (t, J = 7.2, 1H), 1.92 (m, 2H), 1.07 (t, J = 7.2, 3H); 13C NMR (CDCl3,
100 MHz) d 159.48, 128.58, 127.55, 121.18, 114.57, 55.52, 38.29, 29.40, 11.60.
2-(4-Chloro)-phenyl-butyronitrile 8. Light yellow oil: 1H NMR (CDCl3, 400 MHz)
d 7.37 (m, 2H), 7.28 (m, 2H), 3.73 (t, J = 7.2, 1H), 1.98–1.90 (m, 2H), 1.08 (t,
1.7%
H
CO2Me
25. trans-13: 1H NMR (CDCl3, 400 MHz) d 7.11 (m, 2H), 7.02 (m, 2H), 3.70 (dd,
J = 11.5, 1.2, 1H), 3.69 (s, 3H), 3.21 (td, J = 11.5, 6.4, 1H), 2.64 (m, 1H), 2.45 (m,
1H), 2.38 (ddd, J = 19.4, 10.3, 8.7, 1H), 2.10 (m, 1H). 13C NMR (CDCl3, 100 MHz)
J = 7.2, 3H). 13C NMR (CDCl3, 100 MHz)
d 134.43, 130.03, 129.41, 128.85,
120.46, 38.52, 29.30, 11.54. Conversions of 1–4 and the enantiomeric excesses
of 5–8 were measured by HPLC chromatography: Agilent HPLC system, tandem
d
214.70, 174.09, 162.32 (d, JCF = 245.7), 132.44 (d, JCF = 3.2), 130.61 (d,
Zorbax SB C18 (4.6 ꢂ 50 mm, 3.5 lm)-Chiralpak OJ-RH (4.6 ꢂ 150 mm, 5 lm),
JCF = 8.0), 115.86 (d, JCF = 21.7), 58.11, 52.42, 49.44, 37.87, 25.03. trans-14: 1H
NMR (CDCl3, 400 MHz) d 7.11 (m, 2H), 7.01 (m, 2H), 5.00 (septet, J = 6.4, 1H),
3.68 (d, J = 12.0, 1H), 3.14 (m, 1H), 2.62 (m, 1H), 2.44 (m, 1H), 2.36 (m, 1H), 2.09
(m, 1H), 1.21 (d, J = 6.4, 3H), 1.13 (d, J = 6.4, 3H). 13C NMR (CDCl3, 100 MHz) d
214.87, 173.08, 162.28 (d, JCF = 245.7 Hz), 132.50 (d, JCF = 3.6 Hz), 130.25 (d,
JCF = 8.0 Hz), 115.75 (d, JCF = 21.7 Hz), 68.79, 58.27, 49.85, 37.82, 24.85, 21.87.
Racemic standards 13 and 14 were prepared via reduction of 11 and 12 with H2
(1 mol equiv) and wet Pt/C in methanol. Conversion of 11 and 12 was
determined on an Agilent HPLC system using a Zorbax Extend C18 column
isocratic 40/60 MeCN/water (0.1% H3PO4), 1 ml/min, rt, 210 nm, 35 min.
17.1 min (1), 14.1 min (5, R), 16.1 min (5, S); 30.6 min (2), 22.4 min, 26.6 min
(6); 20.5 min (3), 13.9 min, 15.3 min (7); 30.4 min (4), 23.6 min, 25.1 min (8).
20. (a) Compound 5: ½a D20
¼ þ26:1 (c 7.08, methanol).; (b) Shibata, S.; Matsushita,
ꢃ
H.; Kaneko, H.; Noguchi, M.; Saburi, M.; Yoshikawa, S. Agric. Biol. Chem 1982,
46, 1271–1275; (c) Cram, D. J.; Haberfield, P. J. Am. Chem. Soc. 1961, 83, 2354–
2366.
21. Limanto, J.; Shultz, S. C.; Dorner, B.; Desmond, R. A.; Devine, P. N.; Krska, S. W. J.
Org. Chem. 2008, 73, 1639–1642. Compounds 9 and 10 were synthesized from
the corresponding N-Boc derivatives. Removal of the BOC group was performed
under acidic conditions (BF3ꢄEt2O in toluene) and standard aqueous work-up
followed by crystallization from tert-butyl methyl ether. Compound 9: Yellow
solid: 1H NMR (CDCl3, 400 MHz) d 7.57 (s, 1H), 7.39 (s, 1H), 7.36 (s, 1H), 3.21
(dt, J = 12.8, 3.6, 2H), 2.95 (td, J = 12.8, 2.8, 2H), 2.43 (s, 3H), 2.02 (ddd, J = 13.6,
12.0, 4.0, 2H), 1.41 (m, 2H). 13C NMR (CDCl3, 100MHz) d 154.39, 149.69, 136.64,
136.01, 134.14, 123.54, 123.02, 114.79, 114.47, 53.72, 44.34, 33.44, 20.39.
Compound: 10: Yellow solid: 1H NMR (CDCl3, 400 MHz) d 7.28 (s, 1H), 7.22 (s,
1H), 4.07 (t, J = 8.0, 1H), 3.09 (m, 2H), 2.80 (td, J = 12.8, 2.8, 2H), 2.65 (dd,
J = 12.8, 8.0, 1H), 2.38 (s, 3H), 2.28 (ddd, J = 12.8, 8.0, 1.2, 1H), 1.94 (td, J = 12.8,
4.0, 1H), 1.71–1.63 (om, 3H), 1.44 (dq, J = 13.2, 2.8, 1H). 13C NMR (CDCl3,
100MHz) d142.92, 136.04, 135.30, 134.92, 126.86, 124.09, 121.11, 46.82, 43.78,
43.51, 40.68, 38.14, 36.94, 31.82, 20.21. Conversion of 9 was determined on an
(4.6 ꢂ 50 mm, 3.5
lm) at a gradient from 20/80 MeCN/water (0.1% H3PO4) to
40/60 MeCN/water (0.1% H3PO4) over 4 min to 90/10 over 1 min, held for
2 min, 1 ml/min, rt, 215 nm. 5.5 min (11), 5.3 min (13 trans), 5.1 min (13 cis);
5.3 min (12), 5.2 min (14 trans), 5.0 min (14 cis). Enantiomeric excess of 13 and
14 was determined with a Berger SFC system employing a Chiralpak ADH
(250 ꢂ 4.6 mm), 4% methanol/CO2 for 4 min, ramp to 40% methanol/CO2 over
18 min, hold for 3 min, 1.5 ml/min, 200 bar, 215 nm, 35 °C. 5.3 min, 6.2 min
1R,2R (13); 4.4 min, 4.8 min (14). Alternatively, conversion of 11 and
enantiomeric excess of 13 can be measured using an Agilent HPLC system
equipped with tandem Zorbax Extend C18 (4.6 ꢂ 50 mm, 3.5
lm)-Chiralpak
AD-RH (4.6 ꢂ 150 mm, 5
l
m) at 40/60 MeCN/water (0.1% H3PO4) held for
5 min, ramp to 90/10 MeCN/water (0.1% H3PO4) over 8 min, held for 2 min,
1 ml/min, rt, 215 nm. 9.3 min (11), 9.1 min, 9.6 min (13).
26. A solution of 250 mg ERED114 (ERED112) in l8 ml 0.3 M Na2(PHO3) pH 7 was
Agilent HPLC system using a Zorbax SB C18 column (4.6 ꢂ 75 mm, 3.5
lm) at a
added to
a solution of 125 mg NADP and 125 mg PDH101 (phosphite
gradient from 2/98 MeCN/water (0.1% H3PO4) held for 1 min to 100/0 over
8 min at 1.5 ml/min, rt, 215 nm. 5.6 min (9), 5.3 min (10). Enantiomeric excess
of 10 was determined with a Berger SFC system employing a Chiralpak ADH
column (250 ꢂ 4.6 mm), isocratic 10% methanol (25 mM iso-butylamine)/CO2
@ 1.5 ml/min, 200 bar, 215 nm, 35 °C, 20 min. 13.6 min, 14.9 min (10).
22. Hall, M.; Stueckler, C.; Ehammer, H.; Pointner, E.; Oberdorfer, G.; Gruber, K.;
Hauer, B.; Stuermer, R.; Kroutil, W.; Macheroux, P.; Faber, K. Adv. Synth. Catal.
2008, 350, 411–418.
dehydrogenase, Biocatalytics, Inc.) in 220 ml 0.3 M Na2(PHO3) pH 7. To the
solution was added 250 mg of 11 (1) in 12 ml DMSO. The reaction was aged at
30 °C for 3 h (18 h). After extraction with 200 ml MTBE, the organic phase was
dried over MgSO4 and concentrated under reduced pressure to give 198 mg of
13 (176 mg 5).
27. (a) Relyea, H. A.; van der Donk, W. A. Bioorg. Chem. 2005, 33, 171–189; (b)
Johannes, T. W.; Woodyer, R. D.; Zhao, H. Appl. Environ. Microbiol. 2005, 71,
5728–5734; (c) Woodyer, R.; van der Donk, W. A.; Zhao, H. Comb. Chem. High
Throughput Screening 2006, 9, 237–245; (d) Johannes, T. W.; Woodyer, R. D.;
Zhao, H. Biotechnol. Bioeng. 2006, 96, 18–26.
28. (a) Pollard, D.; Truppo, M.; Pollard, J.; Chen, C.; Moore, J. Tetrahedron:
Asymmetry 2006, 17, 554–559; (b) Truppo, M. D.; Pollard, D.; Devine, P.
Organic Lett. 2007, 9, 335–338.
23. Compound 12 was prepared according to 11 described in Ref. 14. 12: Yellow
oil: 1H NMR (CDCl3, 400 MHz) d 7.30 (m, 2H), 7.07 (m, 2H), 5.10 (septet, J = 6.4,
1H), 2.92 (m, 2H), 2.64 (m, 2H), 1.17 (d, J = 6.4, 6H). 13C NMR (CDCl3, 100 MHz)
d
207.57, 165.47, 163.15 (d, JCF = 249.0 Hz), 157.92, 144.91, 131.19 (d,
JCF = 8.0 Hz), 126.47 (d, JCF = 3.6 Hz), 115.11 (d, JCF = 21.7 Hz), 69.64, 34.62,
21.17, 21.66.