Preparation of enantiopure ketones and alcohols containing a quaternary stereocenter through parallel kinetic resolution of β-keto nitriles

Article abstract of DOI:10.1021/jo011092t

Racemic 1-methyl-2-oxocycloalkanecarbonitriles have been subjected to bioreduction by the fungus Mortierella isabellina NRRL 1757 through a parallel kineticresolution process. The u and l alcohols thus obtained (up to >99% ee) were easily separated and oxidized to the R and S ketones, respectively. The process can be then repeated so that both enantiomers of the ketone and two epimers of the alcohol can be obtained in their enantiopure forms.

Full text of DOI:10.1021/jo011092t

1716
J . Org. Chem. 2002, 67, 1716-1718
Sch em e 1
P r ep a r a tion of En a n tiop u r e Keton es a n d
Alcoh ols Con ta in in g a Qu a ter n a r y
Ster eocen ter th r ou gh P a r a llel Kin etic
Resolu tion of â-Keto Nitr iles
J uan R. Dehli and Vicente Gotor*
Departamento de Quı´mica Orga´nica e Inorga´nica,
Universidad de Oviedo, c/ J ulia´n Claverı´a, 8.
33006 Oviedo, Spain
vgs@sauron.quimica.uniovi.es
Received November 20, 2001
We decided then to merge both targets, and we chose
racemic 1-methyl-2-oxocycloalkanecarbonitriles 1 as sub-
strates for the bioreduction process. According to Prelog’s
rule,10 the formal introduction of the hydride would take
place from the re face of the carbonyl group, yielding the
corresponding alcohol of 2S configuration, which would
represent a parallel kinetic resolution (PKR): (R)-1 would
give u-2 and (S)-1, l-2 (see Scheme 1). Although this kind
of resolution is not completely new,11 the scarce biocata-
lytic examples described thus far do not allow for the
recuperation of the optically active substrate.12 In our
case, mild oxidation of the alcohol would let us recover
the enantioenriched ketone, which is what gives our
process additional value because compounds such as 1
are interesting building blocks for the preparation of
natural products13 and conformationally restricted amino
acids.14
Among the microorganisms tested at analytical scale15
for the bioreduction of (()-1a , the fungus Mortierella
isabellina NRRL 1757 gave the most satisfactory results.
Thus, after 2 h, a mixture of u-2a (>99% ee) and l-2a
(73% ee) was obtained in a 42:58 diastereomeric ratio.
To achieve efficient preparative-scale experiments at
gram scale, the substrate concentration was increased
to 10 g/L (75 mM) without any change as far as stereo-
chemistry was concerned. In a typical example, 1 g of
(()-1a was subjected to bioreduction using resting cells
grown in 100 mL of culture for 48 h16 and resuspended
in 100 mL of distilled water.17 After 7 h at 28 °C and 200
Abstr a ct: Racemic 1-methyl-2-oxocycloalkanecarbonitriles
have been subjected to bioreduction by the fungus Mortier-
ella isabellina NRRL 1757 through a parallel kinetic-
resolution process. The u and l alcohols thus obtained (up
to >99% ee) were easily separated and oxidized to the R and
S ketones, respectively. The process can be then repeated
so that both enantiomers of the ketone and two epimers of
the alcohol can be obtained in their enantiopure forms.
The asymmetric construction of molecules with qua-
ternary carbon stereocenters has represented a very
challenging and dynamic area in organic synthesis over
the past decade.1 Despite the numerous reports on
bioreduction of ketones, mainly by baker’s yeast,2 re-
ported in the literature, very few examples have made
use of bioreduction as a means to obtain optically active
compounds with fully substituted carbons, and in most
cases with only moderate success.3
On the other hand, the importance of optically active
â-hydroxy nitriles as suitable synthons for the prepara-
tion of γ-amino alcohols (like the antidepressant fluox-
etine)4 is steadily growing. Thus, recently, methodologies
have been developed to prepare these alcohols via clas-
sical kinetic resolution,5 dynamic kinetic resolution,6
reduction,7 alkylation-reduction,8 and addition9 pro-
cesses.
* Corresponding author.
(1) (a) Fuji, K. Chem. Rev. 1993, 93, 2037. (b) Corey, E. J .; Guzman-
Perez, A. Angew. Chem., Int. Ed. 1998, 37, 388.
(2) (a) Servi, S. Synthesis 1990, 1. (b) Csuk, R.; Gla¨nzer, B. I. In
Stereoselective Biocatalysis; Patel, R. N., Ed.; Marcel Dekker, Inc.: New
York, 2000; Chapter 19, p 527.
(3) (a) Brooks, D. W. J . Org. Chem. 1982, 47, 2820. (b) Brooks, D.
W.; Woods, K. W. J . Org. Chem. 1987, 52, 2036. (c) Brooks, D. W.;
Mazdiyasni, H.; Grothaus, P. G. J . Org. Chem. 1987, 52, 3223. (d)
Fuhshuku, K.; Funa, N.; Akeboshi, T.; Ohta, H.; Hosomi, H.; Ohba,
S.; Sugai, T. J . Org. Chem. 2000, 65, 129.
(10) Prelog, V. Pure Appl. Chem. 1964, 9, 119.
(11) (a) Vedejs, E.; Chen, X. J . Am. Chem. Soc. 1997, 119, 2584. (b)
Eames, J . Angew. Chem., Int. Ed. 2000, 39, 885. (c) Bertozzi, F.; Crotti,
P.; Macchia, F.; Pineschi, M.; Feringa, B. L. Angew. Chem., Int. Ed.
2001, 40, 930.
(12) See, for example: (a) Konigsberg, K.; Alphand, V.; Furstoss,
R.; Griengl, H. Tetrahedron Lett. 1991, 32, 499. (b) Petit, F.; Furstoss,
R. Tetrahedron: Asymmetry 1993, 4, 1341. (c) Mischitz, M.; Faber, K.
Tetrahedron Lett. 1994, 35, 81.
(4) Koenig, T. M.; Mitchell, D. Tetrahedron Lett. 1994, 35, 1339.
(5) (a) Itoh, T.; Takagi, Y.; Nishiyama, S. J . Org. Chem. 1991, 56,
1521. (b) Itoh, T.; Takagi, Y.; Murakami, Y. J . Org. Chem. 1996, 61,
2158.
(6) Pa`mies, O.; Ba¨ckvall, J .-E. Adv. Synth. Catal. 2001, 343, 726.
(7) (a) Itoh, T.; Fukuda, T.; Fujisawa, T. Bull. Chem. Soc. J pn. 1989,
62, 3851. (b) Mehmandoust, M.; Buisson, D.; Azerad, R. Tetrahedron
Lett. 1995, 36, 6461. (c) Florey, P.; Smallridge, A. J .; Ten, A.; Trewhella,
M. A. Org. Lett. 1999, 1, 1879. (d) Dehli, J . R.; Gotor, V. Tetrahedron:
Asymmetry 2000, 11, 3693.
(8) (a) Itoh, T.; Takagi, Y.; Fujisawa, T. Tetrahedron Lett. 1989, 30,
3811. (b) Gotor, V.; Dehli, J . R.; Rebolledo, F. J . Chem. Soc., Perkin
Trans. 1 2000, 307. (c) Dehli, J . R., Gotor, V. Tetrahedron: Asymmetry
2001, 12, 1485.
(9) Soai, K.; Hirose, Y.; Sakata, S. Tetrahedron: Asymmetry 1992,
3, 677.
(13) Enders, D.; Zamponi, A.; Raabe, G.; Runsink, J . Synthesis 1993,
725 and references therein.
(14) Westermann, B.; Walter, A.; Diedrichs, N. Angew. Chem., Int.
Ed. 1999, 38, 3384.
(15) Typically, the substrate (1 g/L), solvated in ethanol (1% v/v),
was added to a suspension of resting cells in 35 mL of distilled water.
This mixture was then shaken (200 rpm) at 28 °C, and the reaction
was monitored by chiral GC.
(16) (a) Quiro´s, M.; Rebolledo, F.; Liz, R.; Gotor, V. Tetrahedron:
Asymmetry 1997, 8, 3035. (b) Quiro´s, M.; Rebolledo, F.; Gotor, V.
Tetrahedron: Asymmetry 1999, 10, 473.
(17) Recently, special emphasis has been placed on the substitution
of plain water for buffers, which simplifies the downstream processing
of a hypothetical industrial application. See, for example: Genzel, Y.;
Archelas, A.; Broxterman, Q. B.; Schulze, B.; Furstoss, R. J . Org. Chem.
2001, 66, 538.
10.1021/jo011092t CCC: $22.00 © 2002 American Chemical Society
Published on Web 02/02/2002

Notes
J . Org. Chem., Vol. 67, No. 5, 2002 1717
has an ee value of only 73% (it consists of both (1S,2S)
from (S)-1a and (1R,2R) from (R)-1a ). Therefore, starting
from a ketone enriched in the S antipode (instead of the
racemic one), the l alcohol reaches a higher ee value.
The assignment of the relative configuration of the
alcohols 2 has been done on the basis of their 13C NMR
spectra, as had been previously done for similar struc-
tures.3c In the l isomers, the CN signal was substantially
deshielded (with regard to that of the u isomers) because
of a γ steric-compression effect of the vicinal hydroxyl
group.18 As expected, the opposite effect is observed for
the CH3 signal (see Experimental Section). The validity
of this assignment was confirmed by the transformation
of u-2a into its O-trimethylsilyl derivative and by com-
F igu r e 1. Preparative bioreduction of (()-1a by the fungus
M. isabellina: (b) conversion of (S)-1a , (() conversion of (R)-
1a , (2) ee of u-2a , (9) ee of l-2a .
1
paring its H- and 13C NMR spectra with those already
Sch em e 2
published.19 The absolute configuration of (R)-1a was
assigned by comparison of the sign of its optical rotation
with that of the value published in the literature.20 For
the cyclopentane analogues, the following correlation was
made: enantiopure u-2b was hydrolyzed to the carboxylic
acid and esterified with trimethylsilyl diazomethane.
Further oxidation with PCC led to the previously de-
scribed â-keto ester.21 This methodology was used to
unambiguously assign the configurations of all the
products reported herein.
In summary, the bioreduction of â-keto nitriles bearing
a quaternary stereocenter has been studied. A parallel
kinetic-resolution process takes place so that each enan-
tiomer yields a different diastereomer. The corresponding
optically active ketones have been efficiently recovered
by oxidation under mild conditions. The products that
were not obtained in their enantiopure forms can be
subjected to reduction again so that both enantiomers of
the ketone can be obtained (after a second oxidation) in
very good yield and excellent ee. To the best of our
knowledge, this simple and efficient method represents
the first biocatalytic approach to obtaining optically
active alcohols and ketones bearing quaternary centers
by using a parallel kinetic resolution. Further work
concerning an in-depth study of this process as well as
application to other sterically hindered ketones is cur-
rently in progress in our laboratory.
rpm, the reaction was complete. This reaction afforded,
after flash chromatography, u-2a (38% yield, >99% ee)
and l-2a (54% yield, 73% ee). As can be seen in Figure 1,
the difference in the rate of reduction of both enantiomers
is rather small but is no impediment to the high stereo-
selectivity of the process. This result confirms our initial
hypothesis of a PKR because after completion the prod-
ucts are obtained in high ee, contrary to what happens
to classical kinetic resolutions, which require conversion
values close to 50%. From these products, both antipodes
of ketone 1a (R and S respectively) were obtained by mild
oxidation with PCC without racemization (confirmed by
GC) and in quantitative yield.
Similar results were obtained when we used (()-1b as
the substrate: u-2b was obtained in 34% yield and >99%
ee, and l-2b, in 58% yield and 61% ee. u-2b and l-2b could
also be oxidized to the corresponding enantiomers of
ketone 1b.
Although in both cases the u isomer was obtained in
the enantiopure form, the ee of its epimer was unsatis-
tactory. We thought that it could then be easily increased
by iterating the process, making use of the easy recovery
of the enantioenriched ketone (S)-1 from l-2. Thus, in a
second bioreduction process, we obtained l-2a in 44%
yield and 96% ee (together with a small amount of
enantiopure u-2a , which increased its overall yield to
42%).
Exp er im en ta l Section
Gen er a l. Reagents were obtained from Aldrich Chemie. THF
was distilled over sodium and stored under nitrogen. Precoated
TLC plates of silica gel 60 F254 from Merck were used, while
for column chromatography, Merck silica gel 60/230-400 mesh
was used. NMR spectra were carried out in CDCl3. The ee and
dr values were determined by GC using a Rt-âDEXse (30 m ×
0.25 mm, Restek) capillary column and nitrogen as the carrier
gas (15 psia) (100 °C, 15 min; 1 °C min-1 until 170 °C). The
fungus M. isabellina NRRL 1757 was obtained from the NRRL
culture collection and was precultured and cultured as previously
described.16
Syn t h eses of (()-1-Met h yl-2-oxocycloa lk a n eca r b on i-
tr iles, (()-1. To a suspension of NaH (1.3 g, 55 mmol) in 100
mL THF was added 50 mmol of the corresponding alkanedini-
trile, and the mixture refluxed until the disappearance of the
To understand this increase in the ee, we undertook
an examination of the reduction of the enantiomerically
pure ketones (R)- and (S)-1a (see Scheme 2). In the first
case, the introduction of the hydride follows Prelog’s rule,
with moderate selectivity. Thus, the u alcohol was
obtained as the major isomer (u-l, 85:15). In the second
case, the reduction occurs with total diastereoselectivity,
and the l diastereomer was the only product detected by
GC.
(18) Grant, D. M.; Cheney, B. V. J . Am. Chem. Soc. 1967, 89, 5315.
(19) Imi, K.; Yanagihara, N.; Utimoto, K. J . Org. Chem. 1987, 52,
1013.
(20) [R]20 +263.8 (c 2.9, benzene, >99% ee S), lit. [R]25 +232.2 (c
D
D
1.04, benzene, 90% ee R), see ref 14.
(21) [R]20 +11.1 (c 1.0, CHCl3, >99% ee), lit. [R]25 -10.6 (c 1.15,
D
D
CHCl3, >96% ee R): Sato, T.; Maeno, H.; Noro, T.; Fujisawa, T. Chem.
Lett. 1988, 1739. It should be noted that because of the Cahn-Ingold-
Prelog priority rule (R)-nitrile led to (S)-ester.
By starting from (()-1a , enantiopure u-2a is obtained
(it consists only of the (1R,2S) enantiomer), whereas l-2a

1718 J . Org. Chem., Vol. 67, No. 5, 2002
Notes
1
substrate (TLC monitoring, eluent hexane/AcOEt 2:1). Then, the
reaction was allowed to reach room temperature, quenched with
30 mL of water, and extracted with diethyl ether. After drying
and elimination of the solvent, the enamine formed was hydro-
lyzed at room temperature with 50 mL of 3 N H2SO4 and
extracted again with diethyl ether and dried, and the solvent
was removed on the rotovapor. The residual oil was solvated in
a mixture of LiOH (55 mmol), water (25 mL), and methanol (25
mL). MeI (55 mmol) was added, and the solution was heated at
50 °C for 2 h. Extraction with diethyl ether, drying, and
elimination of the solvent on the rotovapor led to substantially
pure 1, which was further purified by bulb-to-bulb distillation.
GC analysis, tR (min): (S)-1a 15.9; (R)-1a 17.7; (S)-1b 15.5; (R)-
1b 17.1.
Syn th eses of th e Dia ster eom er ic Mixtu r es of (()-2. To
a solution of 1 mmol (()-1 in 5 mL of ethanol was added 25 mg
(0.7 mmol) of NaBH4, and the mixture was shaken at room
temperature for 6 h. After extraction with diethyl ether and
elimination of the solvent, a pale yellowish oil was obtained (95%
of theor yield) that was used without further purification as a
standard for chiral GC analyses. tR (min): (1S,2S)-2a 34.4;
(1R,2R)-2a 35.1; (1R,2S)-2a 41.6; (1S,2R)-2a 42.1; (1S,2S)-2b
28.7; (1R,2R)-2b 31.0; (1R,2S)-2b 38.2; (1S,2R)-2b 38.8.
P r ep a r a tive Biotr a n sfor m a tion s w ith M. isa bellin a
NRRL 1757. A 100 mL culture grown for 2 days at 28 °C and
200 rpm was filtered out and resuspended in the same volume
of distilled water, and the substrate (1 g) and ethanol (1 mL)
were added. The reaction was monitored by GC. When no more
substrate remained, cells were harvested by filtration and
washed with water. The combined aqueous phases were satu-
rated with NaCl and continuously extracted with CH2Cl2 for 12
h. After drying and evaporation of the solvent, the diastereomeric
mixture was efficiently separated by flash chromatography
(eluent hexane/Et2O 5:1).
ee); IR ν˜: 3059 (OH) and 2234 (CN) cm-1; H NMR δ: 1.36 (s,
3H, CH3), 1.6-1.85 (m, 4H), 2.0-2.25 (m, 2H), 2.57 (bs, 1H, OH),
4.25-4.35 (m, 1H, CH); 13C NMR δ: 18.0 (CH3), 20.2, 32.1, 35.7
(CH2), 41.9 (C), 77.8 (CH), 125.0 (CN); ESIMS m/z: 126 [(M +
H)+, 2], 148 [(M + Na)+, 100]. Anal. Calcd for C7H11NO: C, 67.17;
H, 8.86; N, 11.19. Found: C, 66.89; H, 8.99; N, 11.38.
(1S,2S)-2-H yd r oxy-1-m et h ylcyclop en t a n eca r b on it r ile,
l-2b: colorless oil; yield 58%; [R]20 +3.8 (c 1.0, CHCl3, 61% ee);
D
IR ν˜: 3060 (OH) and 2234 (CN) cm-1; H NMR δ: 1.35 (s, 3H,
1
CH3), 1.6-1.85 (m, 3H), 1.9-2.1 (m, 2H), 2.2-2.3 (m, 1H), 2.35
(bs, 1H, OH), 3.89 (m, 1H, CH); 13C NMR δ: 19.8 (CH2), 22.0
(CH3), 31.8, 35.4 (CH2), 44.6 (C), 79.8 (CH), 123.0 (CN); ESIMS
m/z: 126 [(M + H)+, <1], 148 [(M + Na)+, 100]. Anal. Calcd for
C7H11NO: C, 67.17; H, 8.86; N, 11.19. Found: C, 66.95; H, 9.14;
N, 11.12.
P r ep a r a tion of Op tica lly Active Keton es 1. To a solution
of the corresponding optically active hydroxy nitrile 2 (2 mmol)
in CH2Cl2 (5 mL), pyridinium chlorochromate (PCC) was added
(3 mmol), and the mixture was shaken at room temperature for
6 h. Then, it was filtered through Florisil and eluted with CH2-
Cl2. This crude residue (quantitative yield) was essentially pure
by TLC, GC, and NMR and could be used directly for a second
bioreduction. For analytical purposes, it was purified by flash
chromatography (eluent hexane/CH2Cl2 1:1).
1-Meth yl-2-oxocycloh exa n eca r bon itr ile, 1a : colorless oil;
yield 85% (from 1,7-heptanedinitrile) or quantitative (from 2a );
[R]20D +263.8 (c 2.9, benzene, >99% ee R); spectral data (IR, 1H-
and 13C NMR, and MS) match those already published.13 Anal.
Calcd for C8H11NO: C, 70.04; H, 8.08; N, 10.21. Found: C, 69.86;
H, 8.16; N, 10.13.
1-Meth yl-2-oxocyclop en ta n eca r bon itr ile, 1b: colorless oil;
yield 92% (from 1,6-hexanedinitrile) or quantitative (from 2b);
[R]20 +28.8 (c 1.3, CHCl3, >99% ee R); IR ν˜: 2235 (CN) and
D
(1R,2S)-2-H yd r oxy-1-m et h ylcycloh exa n eca r b on it r ile,
1756 (CO) cm-1; 1H NMR δ: 1.44 (s, 3H, CH3), 1.9-2.2 (m, 3H),
2.3-2.6 (m, 3H); 13C NMR δ: 19.0 (CH2), 20.3 (CH3), 35.9, 36.2
(CH2), 43.6 (C), 119.7 (CN), 209.5 (CO); ESIMS m/z: 136 (M +
H)+. Anal. Calcd for C7H9NO: C, 68.27; H, 7.37; N, 11.37.
Found: C, 68.01; H, 7.58; N, 11.12.
u -2a : colorless oil; yield 42%; [R]20 +31.5 (c 2.9, CHCl3, >99%
D
ee); IR ν˜: 3061 (OH) and 2234 (CN) cm-1; H NMR δ: 1.34 (s,
1
3H, CH3), 1.4-1.9 (m, 8H), 2.26 (bs, 1H, OH), 3.87 (m, 1H, CH);
13C NMR δ: 19.9 (CH3), 20.4, 21.3, 29.6, 32.2 (CH2), 38.3 (C),
71.0 (CH), 124.4 (CN); EIMS m/z: 139 (M+, <1), 110 (38), 68
(100). Anal. Calcd for C8H13NO: C, 69.03; H, 9.41; N, 10.06.
Found: C, 68.77; H, 9.68; N, 9.92.
Ack n ow led gm en t. Financial support of this work
by the Spanish Ministerio de Ciencia y Tecnolog´ıa
(Project BIO 98-0770) is gratefully acknowledged. J .R.D.
thanks the Spanish Ministerio de Educacio´n, Cultura
y Deporte for a predoctoral scholarship.
(1S,2S)-2-Hydr oxy-1-m eth ylcycloh exan ecar bon itr ile, l-2a:
colorless oil; yield 44%; [R]20 -22.4 (c 0.7, CHCl3, 96% ee); IR
D
ν˜: 3062 (OH) and 2235 (CN) cm-1
;
1H NMR δ: 1.2-1.36 (m,
2H), 1.42 (s, 3H, CH3), 1.5-1.7 (m, 3H), 1.75-1.87 (m, 1H), 1.9-
2.02 (m, 2H), 2.44 (d, 1H, J ) 5.1 Hz, OH), 3.24 (m, 1H, CH);
13C NMR δ: 22.4 (CH2), 23.6 (CH3), 24.2, 32.3, 36.1 (CH2), 42.1
(C), 75.2 (CH), 122.3 (CN); EIMS m/z: 139 (M+, <1), 110 (37),
68 (100). Anal. Calcd for C8H13NO: C, 69.03; H, 9.41; N, 10.06.
Found: C, 68.87; H, 9.62; N, 9.81.
Su p p or tin g In for m a tion Ava ila ble: 1H- and 13C NMR
spectra of 1a , 1b, l-2a , u-2a , l-2b, u-2b. This material is
available free of charge via the Internet at http://pubs.acs.org..
(1R,2S)-2-H yd r oxy-1-m et h ylcyclop en t a n eca r b on it r ile,
u -2b: colorless oil; yield 34%; [R]20 +43.8 (c 1.3, CHCl3, >99%
J O011092T
D