Biocatalytic reduction of β,δ-diketo esters: A highly stereoselective approach to all four stereoisomers of a chlorinated β,δ-dihydroxy hexanoate

Article abstract of DOI:10.1002/1521-3765(20011105)7:21<4562::AID-CHEM4562>3.0.CO;2-4

A stereoselective chemoenzymatic synthesis of all four stereoisomers of tert-bulyl 6-chloro-3,5-dihydroxyhexanoate (6a) is presented. The key step of the sequence is a highly regio-and enantioselective single-site reduction of tert-butyl 6-chloro-3,5-dioxohexanoate (1a) by two enantiocomplementary biocatalysts. Alcohol dehydrogenase from Lactobacillus brevis (recLBADH) afforded a 72% yield of enantiopure tert-butyl (5)-6-chloro-5-hydroxy-3-oxohexanoate [(S)-2a]. The enantiomer (R)-2a was prepared with 90-94% ee by Baker's yeast reduction in a biphasic system (50% yield). Both biotransformations were performed on a gram scale. The β-keto group of the enantiomeric δ-hydroxy-β-keto esters 2a thus obtained was reduced by synand anti-selective borohydride reductions. Permutation of the reduction methods yielded all four stereoisomers of the crystalline target compound 6a (≥99.3% ee, dr≥205:1), which is a versatile 1,3-diol building block. recLBADH accepts a variety of β,δ-diketo esters as was determined in a photometric assay. tert-Butyl 3,5-dioxohexanoate (1b) and tert-butyl 3,5-dioxoheptanoate (1c) were reduced on a preparative scale as well to afford the corresponding δ-hydroxy-β-keto esters (R)-2b and (R)-2c with 99.4% ee and 98.1% ee, respectively. Wiley-VCH Verlag GmbH, 2001.

Full text of DOI:10.1002/1521-3765(20011105)7:21<4562::AID-CHEM4562>3.0.CO;2-4

FULL PAPER
Biocatalytic Reduction of b,d-Diketo Esters: A Highly Stereoselective
Approach to All Four Stereoisomers of a Chlorinated b,d-Dihydroxy
Hexanoate
Michael Wolberg,[a] Werner Hummel,[b] and Michael Müller*[a]
Abstract: A stereoselective chemoen-
zymatic synthesis of all four stereoiso-
mers of tert-butyl 6-chloro-3,5-dihydroxy-
hexanoate (6a) is presented. The key
step of the sequence is a highly regio-
and enantioselective single-site reduc-
tion of tert-butyl 6-chloro-3,5-dioxohex-
anoate (1a) by two enantiocomplemen-
tary biocatalysts. Alcohol dehydrogen-
90 ± 94% ee by Bakerꢀs yeast reduction
in a biphasic system (50% yield). Both
biotransformations were performed on a
gram scale. The b-keto group of the
enantiomeric d-hydroxy-b-keto esters
2a thus obtained was reduced by syn-
and anti-selective borohydride reduc-
tions. Permutation of the reduction
methods yielded all four stereoisomers
of the crystalline target compound 6a
(ꢀ99.3% ee, drꢀ 205:1), which is a
versatile 1,3-diol building block.
recLBADH accepts a variety of b,d-
diketo esters as was determined in a
photometric assay. tert-Butyl 3,5-dioxo-
hexanoate (1b) and tert-butyl 3,5-dioxo-
heptanoate (1c) were reduced on a
preparative scale as well to afford the
corresponding d-hydroxy-b-keto esters
(R)-2b and (R)-2c with 99.4% ee and
98.1% ee, respectively.
ase
from
Lactobacillus
brevis
(recLBADH) afforded a 72% yield of
enantiopure tert-butyl (S)-6-chloro-5-
hydroxy-3-oxohexanoate [(S)-2a]. The
enantiomer (R)-2a was prepared with
Keywords: alcohols
´
asymmetric
ketones
catalysis
´
enzymes
´
´
reduction
enantiomeric excess.[4] A notable exception to this is repre-
sented by the recent work of Carpentier et al., who succeeded
in controlling the reduction of methyl 3,5-dioxohexanoate
(1 f) at the initial step, namely the reduction of the b-keto
group. The achieved enantiomeric excess was, nevertheless,
limited to 78% at best.[4a] Highly enantioselective reduction of
ethyl 6-benzyloxy-3,5-dioxohexanoate by ADH (alcohol
dehydrogenase) of Acinetobacter calcoaceticus has been
reported (97 ± > 99% ee).[5] Regioselectivity was not encoun-
Introduction
Optically active b,d-dihydroxy esters C have found wide-
spread application in the stereoselective synthesis of natural
products, polyol fragments, and chiral drugs.[1] Both the syn-
and the anti-configured b,d-dihydroxy esters C can be inde-
pendently prepared from the corresponding chiral d-hydroxy-
b-keto esters B by means of highly diastereoselective reduc-
tion protocols.[2] Several syntheses of optically active hydroxy
keto esters B have been described in the literature.[3] The
regio- and enantioselective reduction of b,d-diketo esters A is
a straightforward and flexible approach. All four stereo-
isomers of a dihydroxy ester C can be derived from the same
precursor A by this strategy (Scheme 1).
OH OH
O
O
O
O
R
R
R
R
OR'
OR'
OR'
OR'
syn-C
OH
O
O
R
However, only a few publications dealing with this subject
can be found in the literature. Hydrogenation of diketo
esters Awith chirally modified ruthenium catalysts resulted in
mixtures of syn- and anti-dihydroxy esters C with varying
OR'
B
OH OH
anti-C
O
O
O
R
OR'
[a] Dr. M. Müller, Dipl.-Chem. M. Wolberg
Institut für Biotechnologie2
OH OH
syn-C
A
Forschungszentrum Jülich GmbH, 52425 Jülich (Germany)
Fax : (‡49)2461-613870
OH
O
O
R
E-mail: mi.mueller@fz-juelich.de
OR'
OH OH
anti-C
B
[b] Priv.-Doz. Dr. W. Hummel
Institut für Enzymtechnologie
Heinrich-Heine-Universität Düsseldorf
52426 Jülich (Germany)
Scheme 1. Regio- and enantioselective reduction of b,d-diketo esters A.
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Chem. Eur. J. 2001, 7, No. 21

4562±4571
tered, however, as was the case in the reduction of a variety of
b,d-diketo esters A with Bakerꢀs yeast.[6] The low to moderate
enantiomeric excess (6 ± 67% ee) of the prevailing b-hydroxy-
d-keto products suggests a mutual activity of at least two
Bakerꢀs yeast enzymes with opposite enantioselectivities.
Nevertheless, the application of biocatalysis in a regio- and
enantioselective reduction of b,d-diketo esters A seemed
most promising to us. Lack of selectivity in ADH catalyzed
ketone reductions is the exception rather than the rule,
especially when purified enzymes are applied.[7] In the present
paper, we describe a highly regio- and enantioselective
biocatalytic reduction of several b,d-diketo esters A.[8] Ac-
cording to the strategy outlined in Scheme 1, all four stereo-
isomers of a chlorinated dihydroxy hexanoate C were synthe-
sized in virtually enantiopure form.
Table 1. Relative reaction rates of the reduction of b,d-diketo esters by
recLBADH (photometric assay).
substrate
O
O
O
R
OR''
R''
R'
1
R
R'
c [mmolL
]
rel. rate [%]
1a
1b
1c
1d
1e
1 f
1g
1h
1i
1j
1k
1l
1m
1n
1o
1p
rac-1q
Cl
H
Me
Et
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Me
tBu
tBu
tBu
tBu
tBu
Me
Et
nPr
allyl
nhex
Bn
2[a]
10
10[b]
10[b]
0.5[b]
10
10
10
10
0.5[b]
2
10
10
10
10
5
10
10
77
86
18
4
ˆ
(E)-PhCH CH
H
H
H
H
H
H
H
0
64
74
99
93
54
106
69
92
0
0
0
70
100
iPr
F
tBu
Me
tBu
Me
tBu
MeO
MeO
BnO
H
Results and Discussion
Biocatalyst screening and enzymatic reduction of b,d-diketo
esters: In a photometric assay, that is, by monitoring
NAD(P)H consumption at 340 nm, diketo esters 1a and 1b
were added to ADH of Lactobacillus brevis, Thermoanaer-
obium brockii, horse liver, Bakerꢀs yeast, Rhodococcus
erythtropolis, and carbonyl reductase of Candida parapsilosis,
all of which are known to be capable of reducing a variety of
ketones.[7b, c] However, only NADP-dependent ADH of
Lactobacillus brevis (LBADH) accepted diketo esters 1a
and 1b as substrates. This stable enzyme is easily available in
the form of a crude cell extract (recLBADH) from a
recombinant E. coli strain.[9] As can be seen from Table 1,
recLBADH accepts a broad range of b,d-diketo esters A.
While the activity of recLBADH rapidly decreases on chain
elongation at C-6 (compounds 1b ± e), the enzyme is less
sensitive to variation of the ester group. All unsubstituted
alkyl 3,5-dioxohexanoates are accepted with good activity
(compounds 1b, f ± l), and only a slight preference for li-
pophilic ester alcohols can be noticed. The lower activity for
hexyl ester 1j is a consequence of the poor solubility of this
substrate rather than a consequence of lower acceptance by
the enzyme. Remarkably, the branched diketo ester rac-1q is
also a good substrate for recLBADH. The S enantiomer of
this compound is accepted preferentially, which allows a
dynamic kinetic resolution.[10] Oxy-substitution at C-6 is not
tolerated (compounds 1n ± p), in contrast to substitution by
chlorine and fluorine (1a and 1m).
acetophenone (standard)
[a] Higher concentration of substrate interferes at 340 nm. [b] Substrate
not completely dissolved.
O
O
O
OH
O
O
recLBADH
R
R
OtBu
OtBu
1a-c
2a-c
NADPH
NADP+
O
OH
recLBADH
Scheme 2. Reduction of b,d-diketo esters 1a ± c by recLBADH (R ˆ Cl,
H, Me).
Table 2. Products of the recLBADH catalyzed reduction of diketo
esters 1a ± c.
R
yield [%]
ee[a] [%]
> 99.5
99.4
98.1
[a]2D5
ref. [a]D [% ee]
(S)-2a
(R)-2b
(R)-2c
Cl
H
Me
72
77
61
24.9
40.1
36.0
23.0 (>97), ref. [12]
39.6 (99), ref. [13a]
35.6 (99), ref. [13b]
[a] Determined by HPLC (ChiracelOB) at the stage of the d-lactones
4a ± c.
(S)-3 (Scheme 3). Neither the b-hydroxy-d-keto regioisomer
nor b,d-dihydroxy esters could be detected by NMR and GC-
MS analysis of the crude enzymatic products.
To identify the keto group(s) reduced by recLBADH, we
performed the reaction with diketo esters 1a ± c on a 1 ±
10 millimole scale, by using substrate-coupled regeneration
of NADPH. The cosubstrate 2-propanol was applied in excess
to shift the equilibrium in the desired direction (Scheme 2).
Comparison of analytical data (1H NMR spectroscopy, [a])
of the products (R)-2b and (R)-2c with literature data clearly
proved the formation of R-configured d-hydroxy-b-keto
isomers of high optical purity (Table 2). In the case of the
chlorinated product (S)-2a, authentic material was prepared
by an independent route. Additionally, the enzymatic product
(S)-2a was transformed into the known[11] b-keto d-lactone
Precise determination of the enantiomeric excess was
accomplished after transformation of hydroxy keto esters
2a ± c to a,b-unsaturated d-lactones 4a ± c (Scheme 3), the
O
O
OH
O
O
b, c
a
O
O
R
OtBu
Cl
R
2a-c
O
4a-c
(S)-3
Scheme 3. Synthesis of d-lactones 3 and 4a ± c (R ˆ Cl, H, Me). a) cat.
TsOH, CH2Cl2, RT, 4 d, 67%; b) NaBH4, EtOH, 08C, 30 min; c) cat.
TsOH, toluene, D, 2 h, 60 ± 70%.
Chem. Eur. J. 2001, 7, No. 21
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4563

M. Müller et al.
FULL PAPER
enantiomers of which are easily separable by HPLC (Chir-
alcelOB). In the case of the chlorinated lactone (S)-4a, the
amount of R enantiomer was below the detection limit.
To the best of our knowledge, recLBADH is the first
catalyst that allows the preparation of d-hydroxy-b-keto
esters by highly regio- and enantioselective reduction of b,d-
diketo esters. Additionally, the presented enzymatic reaction
is of enormous preparative value. The substrates are readily
available, and the reaction only requires a simple batch
technique, which is easy to scale up. Reduction of the
chlorinated compound 1a is routinely performed on a 75 g
scale in our laboratory (8 L fed batch).[14]
Application of the resin considerably facilitated the work-
up, and formation of furanone 5 was found to be suppressed
close to the detection limit, which is an additional benefit of
the biphasic system. The reaction was carried out on a gram
scale without difficulty.
Diastereoselective reduction of tert-butyl 6-chloro-5-hydroxy-
3-oxo-hexanoate (2a): For the preparation of both enantio-
mers of dihydroxy ester syn-6a Prasadꢀs syn-selective boro-
hydride reduction was applied (Scheme 6).[2a, 20] This method
At the beginning of our investigations we were concerned
about the susceptibility of diketo ester 1a to furanone
formation, which is a known reaction of other g-chloro-b-
diketones as well.[15] Indeed, stirring compound 1a in mere
pH 7.0 buffer/ethanol solution resulted in complete conver-
sion to furanone 5 (Scheme 4).
OH OH
syn-(3R,5S)-6a
OH OH
O
Cl
Cl
a, b
c
OtBu
OtBu
OH
O
O
Cl
OtBu
O
(S)-2a
>99.5 % ee
anti-(3S,5S)-6a
We found this cyclization to be significantly suppressed in
the enzymatic reduction of diketo ester 1a by application of
pH 5.5 buffer. The remaining fraction of by-product 5 reflects
the high (>90%) yet incomplete enzymatic conversion. On
the other hand, unreacted diketo ester 1a would lower the
enantiomeric excess in the subsequent diastereoselective
borohydride reduction (see below). The formation of furan-
one 5 can be regarded as a convenient in situ elimination of
unreacted starting material since it does not interfere with the
borohydride reduction.
OH OH
syn-(3S,5R)-6a
OH OH
O
Cl
Cl
a, b
c
OtBu
OtBu
OH
O
O
Cl
OtBu
O
(R)-2a
90-94 % ee
anti-(3R,5R)-6a
Scheme 6. Diastereoselective reduction of hydroxy keto esters (S)-2a and
(R)-2a. a) 1. B(OMe)Et2, THF/MeOH (80:20 v/v), 708C, 20 min;
2. NaBH4, 708C, 3 h; b) 1. H2O2, THF/water (70:30 v/v, pH 9), 08C !
RT, 30 min; c) 1. Me4N[B(OAc)3H], MeCN/AcOH (50:50 v/v), 258C, 5 h.
O
O
O
O
O
a
Cl
OtBu
quantitatively gave dihydroxy esters syn-6a in a diastereo-
meric ratio (dr) syn-6a/anti-6a ˆ 28:1 to 45:1. The enantio-
mers of dihydroxy ester anti-6a were synthesized according to
Evansꢀ method,[2b] which resulted in a dr (anti-6a/syn-6a) ˆ
14:1 to 18:1 (Scheme 6). Advantageously, all dihydroxy
esters 6a solidified, and a single crystallization step raised
the diastereomeric ratios to ꢀ187:1. In the case of the two
dihydroxy esters derived from the Bakerꢀs yeast reduction
product most of the minor enantiomer was removed by this
procedure as well. Further recrystallization lowered the
amount of minor stereoisomers below the detection limit.
Assignment of the relative configuration was supported by the
known downfield shift of the carbinolic 13C-resonances of syn-
1,3-diols compared with those of the anti-diastereoisomers
(Table 3).[2a, 21]
The diastereomeric ratios were determined by GC on chiral
stationary phase (Cyclodex beta-1/P) at the stage of the
acetonides 7a (Scheme 7). All four stereoisomers can be
separated by this method, and thus we could also investi-
gate the enantiomeric excess of each individual dihydroxy
ester 6a. The ee thus determined for the crude products
confirmed the values for (S)-2a and (R)-2a, which were
measured at the stage of the lactones (S)-4a and (R)-4a by
HPLC (see above).
OtBu
O
1a
5
Scheme 4. Cyclization of diketo ester 1a. a) phosphate buffer (pH 7.0)/
ethanol (67:33 v/v), RT, 20 h, 79%.
In order to synthesize the R enantiomer of hydroxy keto
ester 2a, we reinvestigated the Bakerꢀs yeast reduction of b,d-
diketo esters described by the Japanese working group at
Mitsubishi Kasei.[6] In contrast to 1b and 1c, the chlorinated
diketo ester 1a was reduced with high regioselectivity at C-5
by Bakerꢀs yeast.[16] Only small amounts of the regioisomer
and double-site reduction products (<5% total) could be
detected by GC-MS. The enantiomeric excess (48%) of the
product (R)-2a was greatly enhanced by application of a
biphasic system (hexane/water or AmberliteXAD-7/wa-
ter).[17] Furthermore, we tested cell preparations from differ-
ent suppliers and found that a dried Bakerꢀs yeast gave the
best results with regard to enantioselectivity and conver-
sion.[18] A combination of the two parameters enhanced the
enantiomeric excess of the product (R)-2a from 48% to 90 ±
94% (Scheme 5).[19]
OH
O
O
O
O
O
a
Cl
Cl
OtBu
OtBu
(R)-2a
90-94 % ee
1a
Nucleophilic substitution of chlorine: A two-step conversion
of acetonide (3R,5S)-7a to hydroxy compound (3R,5S)-8 is
known from the patent literature.[12a] This compound is an
Scheme 5. Reduction of diketo ester 1a by Bakerꢀs yeast. a) dried Bakerꢀs
yeast, substrate 1a adsorbed on XAD-7, water, RT, 24 h, 50%.
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Chem. Eur. J. 2001, 7, No. 21

Reduction of b,d-Diketo Esters
4562±4571
Table 3. Diastereoselective reduction of hydroxy keto esters (S)-2a (>99.5% ee) and (R)-2a (90 ± 94% ee).
difference in vicinal coupling
compared with those of the
scale [mmol]
dr[a] syn/anti
ee[a] [%]
yield [%]
d
13C C-3/C-5
isomeric
(Figure 1).
tetrahydrofuranes
syn-(3R,5S)-6a
syn-(3S,5R)-6a
anti-(3S,5S)-6a
anti-(3R,5R)-6a
86 ± 217
3 ± 12
3
205:1 (28:1)
187:1 (45:1)[b]
1:211 (1:14)
1:316 (1:18)
> 99.5
62
52
70
68
68.4/71.6
68.4/71.6
65.5/68.9
65.5/68.9
98.0 (90.3)[b]
> 99.5
99.3 (93.8)
Epoxy esters like (3R,5S)-9
are also favorable intermedi-
ates in the synthesis of HMG-
CoA reductase inhibitors since
the epoxide can be regioselec-
tively opened by carbon nucle-
ophiles, for example, cup-
rates.[25]
1 ± 4
[a] Diastereomeric ratio and enantiomeric excess of crystallized product and crude product (in brackets);
determined by GC (Cyclodex beta-1/P) at the stage of the acetonide 7a; ªsynº and ªantiº refer to the major pair
of diastereomers homochiral at C-5. [b] ee > 99.5% and dr> 400:1 after recrystallization.
O
O
O
OH OH
O
a
Cl
Cl
OtBu
Conclusion
OtBu
6a
7a
Scheme 7. Synthesis of acetonide 7a. a) 2,2-Dimethoxy-propane, cat. CSA
(camphorsulfonic acid), 258C, 4 ± 7 h, 100% conv.
All four stereoisomers of the chlorinated dihydroxy ester 6a
can be synthesized in an enantiopure form on a preparative
scale by a flexible stereoselective two-step reduction se-
quence. For the crucial first step, two enantiocomplementary
biocatalysts are employed, namely recLBADH and Bakerꢀs
yeast, which allow the highly regio- and enantioselective
reduction of the d-keto group of diketo ester 1a. The keto
group of the products (S)-2a and (R)-2a can be reduced either
syn-selective or anti-selective by application of known boro-
hydride reduction protocols. The carbon chain of the resulting
dihydroxy ester 6a is terminated with two compatible func-
tional groups of distinguishable reactivity, which makes this
compound a valuable 1,3-diol building block.
advanced intermediate in the synthesis of HMG-CoA reduc-
tase inhibitors.[1j] Iodide (3R,5S)-10 has been utilized for this
purpose, too.[1i] We were able to substitute the chlorine of
acetonide (3R,5S)-7a by iodine in a single step under
advanced halogen exchange conditions (52% yield;
Scheme 8).[22] However, conversion was incomplete, and
The reduction of b,d-diketo esters with ADH of L. brevis is
a reaction of broad applicability. A great variety of diketo
Scheme 8. Nucleophilic substitution of the chlorine of acetonide (3R,5S)-
7a. a) see ref. [12a, b]; b) KI, [18]crown-6, toluene, D, 4 d, 52%.
the remaining starting material could not be removed from
the product (3R,5S)-10. Furthermore, complete decomposi-
tion was encountered in several experiments.
Alternatively, epoxide (3R,5S)-9 was regioselectively
opened with lithium iodide on silica.[23] The crude product
was immediately subjected to acetonide protection which
afforded the desired iodide (3R,5S)-10 with a 58% yield (44%
from (3R,5S)-6a; Scheme 9).
Scheme 9. Synthesis and regioselective opening of epoxide (3R,5S)-9.
a) DBU, CH2Cl2, D, 24 h, 66%; b) KOH, Et2O, 0 8C, 1 h, 46%; c) 1. LiI,
SiO2, RT, 1 h; 2. 2,2-dimethoxy-propane, cat. CSA, RT, 24 h, 58%.
Epoxide (3R,5S)-9 was easily obtained from dihydroxy
ester syn-(3R,5S)-6a by treatment with DBU (1,8-diazobicy-
clo[5.4.0]undec-7-ene, 66% yield) or KOH (46% yield). The
amount of tetrahydrofurane (2R,4S)-11 from competing
5-exo-tet ring closure was limited to 10 ± 13% in both
experiments (1H NMR analysis). The two resulting isomers
were distinguished by correlation of their NMR data with data
of closely related compounds.[24] The diagnostic proton
resonances of the epoxides show a distinct upfield shift and
Figure 1. 1H NMR spectroscopy of oxy-substituted epoxides and tetrahy-
drofuranes (relative configuration shown).[24]
Chem. Eur. J. 2001, 7, No. 21
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M. Müller et al.
FULL PAPER
2.07 (s, 3Hef; H-6), 2.25 (s, 3Hkf, H-6), 3.33 (s, 2Hef; H-2), 3.55 (s, 2Hkf;
H-2), 3.74 (s, 2Hkf; H-4), 4.08 (t, J ˆ 6.7 Hz, 2Hkf; OCH2) overlapping with
4.09 (t, J ˆ 6.7 Hz, 2Hef; OCH2), 5.61 (s, 1Hef; H-4), 15.11 (brs, 1Hef; OH);
13C NMR (75.5 MHz, CDCl3, 208C): enol form: d ˆ 10.4 (CH2CH3), 22.0
(CH2CH3), 24.5 (C-6), 45.3 (C-2), 67.3 (OCH2), 100.7 (C-4), 167.8 (C-1),
esters are accepted by this readily available enzyme, and the
required reaction technique is remarkably simple. As a result
of the mild conditions, the reaction can be carried out without
special care even in the case of sensitive compounds like 1a.
No serious limitations with regard to a further scale-up are
apparent. The hydroxy keto esters thus available are com-
pounds of high functionalization, and a variety of chemical
modifications are known from the literature. We are currently
investigating new applications of these compounds in the
synthesis of chiral drugs and natural products.
‡
187.4, 190.3 (C-3, C-5); MS (70 eV, EI): m/z (%): 186 (21) [M] , 144 (27),
‡
129 (19), 127 (29) [M OnPr] , 126 (43), 111 (15), 102 (13), 98 (32), 85
‡
(100) [M CH2COOnPr] , 84 (16), 69 (13); HRMS (EI) calcd for C9H14O4
‡
[M] : 186.0892; found: 186.0902.
n-Hexyl 3,5-dioxohexanoate (1j): Prepared from acetoacetylated Mel-
drumꢀs acid (1.14 g, 5.0 mmol) and 1-hexanol (1.53 g, 15 mmol) as described
in the example above. Kugelrohr distillation (10 3 mbar, 1208C) and
subsequent flash chromatography on acid-washed silica gel (ethyl acetate/
n-hexane 20:80 v/v) gave title compound 1j as a light-yellow oil. Yield:
0.59 g (52%).
Experimental Section
1H NMR (300 MHz, CDCl3, 208C): ratio enol form (ef):keto form (kf) ˆ
83:17; d ˆ 0.87 (brt, J ˆ 6.9 Hz, 3Hef‡kf; CH2CH3), 1.23 ± 1.39 (m, 6Hef‡kf
;
General: All solvents were HPLC grade or analytical grade. When
necessary, solvents were dried prior to use: THF was distilled from sodium
(benzophenone ketyl as indicator). Methanol and dichloromethane were
stored over 4 Š molecular sieves. Acetonitrile was distilled from calcium
hydride. Acetic acid was refluxed with acetic acid anhydride (3%) and then
subjected to fractional distillation. Air- and/or moisture-sensitive reactions
were carried out under an atmosphere of dry nitrogen in oven-dried
glassware. Silica gel60F254 precoated aluminium sheets (Merck) were used
for analytical thin layer chromatography. Visualization was accomplished
by UV light or by dipping the plate into a solution of p-anisaldehyde (1 mL)
in acetic acid/concd sulfuric acid (100 mL, 98:2 v/v) followed by heating.
Flash chromatography was performed on silica gel60 (40 ± 63 mm, Merck).
Acid-washed silica gel was used for b,d-diketo esters.[26] Melting points
were determined with a BüchiB-540 melting point apparatus. NMR spectra
were recorded with a BrukerAMX-300 spectrometer at 208C. 1H NMR:
300 MHz, residual undeuterated solvent as internal standard (CHCl3: d ˆ
7.27). 13C NMR: 75.5 MHz, deuterated solvent as internal standard (CDCl3:
d ˆ 77.2), broadband proton decoupling. Assignments were supported by
additional DEPT-135 experiments. GC-MS analysis was carried out with a
Hewlett-PackardHP6890 GC system (HP-5MS column) coupled to an
HP5973 mass selective detector.[27] GC on chiral stationary phase was
performed by a ChrompackCP9002 equipped with a flame ionization
detector. HPLC on chiral stationary phase was performed with a Hewlett-
PackardHP1100 system (UV-DAD). Optical rotations were determined
(CH2)3CH2CH3), 1.58 ± 1.68 (m, 2Hef‡kf; CH2CH3), 2.06 (s, 3Hef; H-6), 2.25
(s, 3Hkf; H-6), 3.32 (s, 2Hef; H-2), 3.54 (s, 2Hkf; H-2), 3.74 (s, 2Hkf; H-4),
4.11 (t, J ˆ 6.8 Hz, 2Hkf; OCH2) overlapping with 4.12 (t, J ˆ 6.7 Hz, 2Hef;
OCH2), 5.60 (s, 1Hef; H-4), 15.10 (brs, 1Hef; OH); 13C NMR (75.5 MHz,
CDCl3, 208C): enol form: d ˆ 14.1 (CH2CH3), 22.7 (CH2CH3), 24.5 (C-6),
25.6, 28.5, 31.5 ((CH2)3CH2CH3), 45.3 (C-2), 65.8 (OCH2), 100.7 (C-4), 167.8
‡
(C-1), 187.4, 190.3 (C-3, C-5); MS (70 eV, EI): m/z (%): 228 (23) [M] , 186
‡
(10), 145 (35), 129 (24), 127 (54) [M OnHex] , 126 (67), 111 (10), 103
‡
(18), 102 (15), 98 (27), 85 (100) [M CH2COOnHex] , 84 (13), 56 (10), 55
‡
(10); HRMS (EI) calcd for C12H20O4 [M] : 228.1362; found: 228.1370.
Diketo esters a) 1a, 1c, 1d, 1e,[31] 1q[10] and b) 1n, 1o, 1p,[4b] were prepared
by acylation of acetoacetate Na,Li-bisenolates[32] with a) Weinreb amides[33]
and b) methyl 2-alkoxyacetates. Alternatively, chlorinated diketo ester 1a
was synthesized by acylation of tert-butyl acetoacetate Li,Li-bisenolate
with methyl 2-chloroacetate. Fluoro compound 1m was prepared in a
similar manner with TMEDA (N,N,N',N'-tetramethyl 1,2-ethanediamine)
additionally present.[34]
General procedure for the acylation of acetoacetate Na,Li-bisenolates:
Acetoacetate was added dropwise (Tmax ˆ 108C) to an ice-cooled suspen-
sion of sodium hydride (60% suspension in oil) in anhydrous THF, and the
mixture was stirred for an additional ten minutes. The clear solution was
cooled to 108C, and n-butyl lithium (1.6 molL 1 in n-hexane) was added
dropwise (Tmax ˆ 08C). After stirring for an additional 10 min, the acylation
reaction was carried out as indicated. Hydrolysis was performed by pouring
the solution into a vigorously stirred ice-cooled mixture (50:50) of ethyl
acetate and hydrochloric acid (2 molL 1, HCl (4 ± 6 equiv) with regard to
the bisenolate). The aqueous phase was separated and extracted twice with
ethyl acetate. The unified organic phases were washed with aq. NaHCO3
(5%), water, and brine. After drying over Na2SO4, the solvent was
removed under reduced pressure, and the product was purified as
indicated.
with
a Perkin-Elmer241 polarimeter (1 dm cell). Mass spectroscopy
(KratosAEIMS50) and elemental analyses (Heraeus, VarioEL) were
performed in the analytical department of the Kekule Institut für
Organische Chemie und Biochemie (Universität Bonn). Mass peaks with
Â
‡
relative intensity <10% are omitted unless they correspond to [M] or
some other informative fragment. Spectrophotometric enzyme assays were
performed with a Pharmacia Ultrospec2000 spectrophotometer in dispos-
able cuvettes (1.5 mL, Brandt). Commercially available reagents were used
as delivered unless otherwise stated. Tetramethylammonium triacetoxy-
tert-Butyl 6-chloro-3,5-dioxohexanoate (1a)
‡
borohydride and NADP were purchased from Fluka, 2-chloro-N-meth-
Method A: A solution of Na,Li-bisenolate was prepared as described in the
general procedure from tert-butyl acetoacetate (0.79 g, 5.0 mmol), sodium
hydride (0.20 g of a 60% suspension in oil, 5.0 mmol), and n-butyl lithium
oxy-N-methylacetamide and AmberliteXAD-7 from Aldrich. Dried
emulsifier-free Bakerꢀs yeast (Bio-Zentrale GmbH, Stubenberg, Germany)
was purchased from a local supermarket.
1
(3.1 mL of a 1.6 molL solution in n-hexane, 5.0 mmol) in THF (15 mL).
Preparation of b,d-diketo esters: Diketo esters 1 f and 1g were prepared
from dehydroacetic acid as described in the literature and purified by
distillation.[28] Esters 1b, 1i, 1k, and 1l were prepared from acetoacetylated
Meldrumꢀs acid[29] as described in the literature and purified by Kugelrohr
distillation and subsequent flash chromatography on acid-washed silica
gel.[30] The derivatives 1h and 1j were obtained by the same method as
follows.
The solution was cooled to 758C, and a solution of 2-chloro-N-methoxy-
N-methylacetamide (0.69 g, 5.0 mmol) in anhydrous THF (4 mL) was
syringed into the flask (Tmax
ˆ
608C). The solution was stirred at this
temperature for 45 minutes, warmed to 308C over a period of 30 minutes,
stirred for an additional 15 minutes at this temperature, and then recooled
to 758C. Workup was carried out as described in the general procedure.
Flash chromatography on acid-washed silica gel (ethyl acetate/n-hexane
20:80 v/v) gave title compound 1a as an light-yellow oil. Yield: 0.68 g
(58%).
n-Propyl 3,5-dioxohexanoate (1h): Acetocetylated Meldrumꢀs acid (1.14 g,
5.0 mmol) and 1-propanol (0.90 g, 15 mmol) were refluxed in toluene
(10 mL) for three hours. After removal of volatiles under reduced pressure,
the residue was treated with dichloromethane and filtered. The filtrate was
concentrated under reduced pressure, and the residue was subjected to
Kugelrohr distillation (0.1 mbar, 708C). Flash chromatography of the
distillate on acid-washed silica gel (ethyl acetate/n-hexane 20:80 v/v) gave
title compound 1h as a light-yellow oil. Yield: 0.45 g (48%).
1H NMR (300 MHz, CDCl3, 208C): ratio enol form (ef):keto form (kf) ˆ
88:12; d ˆ 1.48 (s, 9Hef‡kf; C(CH3)3), 3.31 (s, 2Hef; H-2), 3.49 (s, 2Hkf; H-2),
3.92 (s, 2Hkf; H-4), 4.06 (s, 2Hef; H-6), 4.21 (s, 2Hkf; H-6), 5.97 (s, 1Hef;
H-4), 14.76 (brs, 1Hef; OH); 13C NMR (75.5 MHz, CDCl3, 208C): enol
form: d ˆ 28.1 (C(CH3)3), 44.4, 46.0 (C-2, C-6), 82.6 (C(CH3)3), 98.9 (C-4),
‡
166.5 (C-1), 187.0, 187.3 (C-3, C-5); MS (70 eV, EI): m/z (%): 234 (1) [M] ,
1H NMR (300 MHz, CDCl3, 208C): ratio enol form (ef): keto form (kf) ˆ
198 (6) [M HCl] , 179 (10), 161/163 (30/10) [M OtBu] , 129 (26), 119/
‡
‡
‡
‡
89:11; d ˆ 0.93 (t, J ˆ 7.4 Hz, 3Hef‡kf ; CH2CH3), 1.66 (m, 2Hef‡kf; CH2CH3),
121 (34/11) [M CH2COOtBu] , 103 (15), 85 (11), 57 (100) [C4H9] .
4566
ꢁ WILEY-VCH Verlag GmbH, D-69451 Weinheim, 2001 0947-6539/01/0721-4566 $ 17.50+.50/0 Chem. Eur. J. 2001, 7, No. 21

Reduction of b,d-Diketo Esters
4562±4571
Method B: tert-Butyl acetoacetate (7.9 g, 50 mmol) was added dropwise to a
solution of LDA [prepared from diisopropyl amine (10.6 g, 105 mmol) and
n-butyl lithium (64 mL of a 1.6 molL 1 solution in n-hexane, 102 mmol) in
300 mL THF] in THF at 158C, and the solution was stirred at this
temperature for ten minutes after addition was complete. The solution was
cooled to 758C and methyl 2-chloroacetate (5.4 g, 50 mmol) was added
general procedure. Kugelrohr distillation (0.04 mbar, 808C) gave title
compound 1d as a light-yellow oil. Yield: 0.64 g (70%).
1H NMR (300 MHz, CDCl3, 208C): ratio enol form (ef):keto form (kf) ˆ
86:14; d ˆ 0.91 (t, J ˆ 7.4 Hz, 3 Hkf; H-8) overlapping with 0.94 (t, J ˆ 7.4 Hz,
3Hef; H-8), 1.46 (s, 9Hef‡kf; C(CH3)3), 1.61 (m, 2Hef‡kf; H-7), 2.26 (t, J ˆ
7.5 Hz, 2Hef; H-6), 2.49 (t, J ˆ 7.4 Hz, 2Hkf; H-6), 3.24 (s, 2Hef; H-2), 3.46 (s,
2Hkf; H-2), 3.70 (s, 2Hkf; H-4), 5.58 (s, 1Hef; H-4), 15.18 (brs, 1Hef; OH);
13C NMR (75.5 MHz, CDCl3, 208C): enol form: d ˆ 13.8 (C-8), 19.3 (C-7),
28.1 (C(CH3)3), 39.8 (C-6), 46.7 (C-2), 82.0 (C(CH3)3), 99.9 (C-4), 167.0 (C-
dropwise (Tmax
ˆ
658C). The solution was stirred at this temperature for
25 minutes after addition was complete. Workup was carried out as
described in the general procedure, and unreacted starting materials were
removed in vacuo. A yellow oil was obtained (11.0 g, 94%) that consisted
mainly (approx. 95%) of the desired product 1a as determined by 1H NMR
spectroscopy.
‡
1), 188.2, 193.1 (C-3, C-5); MS (70 eV, EI): m/z (%): 228 (1) [M] , 173 (11),
‡
172 (32), 155 (43) [M OtBu] , 154 (19), 144 (41), 129 (64), 113 (78) [M
CH2COOtBu] , 111 (21), 85 (10), 84 (17), 71 (42), 69 (11), 57 (100) [C4H9] .
‡
‡
tert-Butyl 3,5-dioxoheptanoate (1c)
Methyl 6-methoxy-3,5-dioxo-hexanoate (1n): A solution of Na,Li-bisenol-
ate was prepared as described in the general procedure from methyl
a) N-Methoxy-N-methylpropionamide: Pyridine (1.69 mL, 21 mmol) at 08C
was added dropwise to a solution of O,N-dimethyl-hydroxylamine hydro-
chloride (1.02 g, 10.5 mmol) and propionyl chloride (0.93 g, 10 mmol) in
anhydrous dichloromethane (50 mL). The solution was stirred at room
temperature for 3.5 h, washed with hydrochloric acid (0.5 molL 1) twice,
and concentrated under reduced pressure to a volume of approximately
20 mL. Diethyl ether (30 mL) was added, and the solution was washed with
aq. NaHCO3 (5%) and brine. After drying over MgSO4, the solvent was
removed under reduced pressure, and the amide was left as a colorless oil
that was used for acylation without further purification. Yield: 1.04 g
(89%).
acetoacetate (0.70 g, 6.0 mmol), sodium hydride (0.24 g of
a
60%
1
suspension in oil, 6.0 mmol), and n-butyl lithium (3.8 mL of a 1.6 molL
solution in n-hexane, 6.1 mmol) in THF (30 mL). The solution was cooled
to 758C, and methyl 2-methoxyacetate (0.31 g, 3.0 mmol) was added in
one portion. The solution was warmed to 08C and stirred for two hours at
this temperature. Workup was carried out as described in the general
procedure. Kugelrohr distillation (10 1 mbar, 758C) and flash chromatog-
raphy on acid-washed silica gel (ethyl acetate/n-hexane 30:70 v/v) gave title
compound 1n as a yellow oil. Yield: 0.14 g (25%).
1H NMR (300 MHz, CDCl3, 208C): ratio enol form (ef):keto form (kf) ˆ
85:15; d ˆ 3.38 (s, 2Hef; H-2), 3.39 (s, 3Hkf; CH2OCH3), 3.41 (s, 3Hef;
CH2OCH3), 3.58 (s, 2Hkf; H-2), 3.73 (s, 3Hef‡kf, COOCH3), 3.76 (s, 2Hkf;
H-4), 4.00 (s, 2Hef; H-6), 4.02 (s, 2Hkf; H-6), 5.88 (s, 1Hef; H-4), 14.95 (brs,
1Hef; OH); 13C NMR (75.5 MHz, CDCl3, 208C): enol form: d ˆ 44.8 (C-2),
52.6 (COOCH3), 59.5 (CH2OCH3), 73.3 (C-6), 97.8 (C-4), 168.0 (C-1), 186.7,
1H NMR (300 MHz, CDCl3, 208C): d ˆ 1.09 (t, J ˆ 7.5 Hz, 3H; H-3), 2.40
(q, J ˆ 7.5 Hz, 2H; H-2), 3.13 (s, 3H; NCH3), 3.64 (s, 3H; OCH3); 13C NMR
(75.5 MHz, CDCl3, 208C): d ˆ 8.8 (C-3), 25.3 (C-2), 32.3 (NCH3), 61.2
‡
(OCH3), 174.9 (C-1); MS (70 eV, EI): m/z (%): 117 (17) [M] , 87 (10), 61
‡
(100) [NH(OMe)Me] , 57 (97).
‡
191.0 (C-3, C-5); MS (70 eV, EI): m/z (%): 188 (2) [M] , 158 (10), 143 (100)
b) Acylation reaction: A solution of Na,Li-bisenolate was prepared as
described in the general procedure from tert-butyl acetoacetate (0.79 g,
5.0 mmol), sodium hydride (0.20 g of a 60% suspension in oil, 5.0 mmol),
and n-butyl lithium (3.1 mL of a 1.6 molL 1 solution in n-hexane, 5.0 mmol)
in THF (20 mL). N-Methoxy-N-methylpropionamide (0.59 g, 5.0 mmol)
was syringed into the flask at 108C. The solution was stirred at this
temperature for 15 minutes, warmed to room temperature, and stirred for
an additional 30 minutes. Workup was carried out as described in the
general procedure. After removal of unreacted tert-butyl acetoacetate by
Kugelrohr distillation (0.2 mbar, 408C), the residue was subjected to flash
chromatography on acid-washed silica gel (ethyl acetate/n-hexane 15/85
v/v); this yielded title compound 1c (0.78 g, 73%) as a light-yellow oil.
‡
‡
[M CH2OCH3] , 115 (24) [M CH2COOMe] , 111 (15), 101 (63), 69
(31), 69 (13).
tert-Butyl 6-methoxy-3,5-dioxohexanoate (1o):
A solution of Na,Li-
bisenolate was prepared as described in the general procedure from tert-
butyl acetoacetate (1.58 g, 10.0 mmol), sodium hydride (0.40 g of a 60%
1
suspension in oil, 10.0 mmol), and n-butyl lithium (6.3 mL of a 1.6 molL
solution in n-hexane, 10.1 mmol) in THF (30 mL). The solution was cooled
to 758C, and methyl 2-methoxyacetate (0.52 g, 5.0 mmol) was added in
one portion. The solution was warmed to 08C over a period of 45 minutes
and stirred for an additional two hours at this temperature. Workup was
carried out as described in the general procedure. Kugelrohr distillation
(10 2 mbar, 858C) and flash chromatography on acid-washed silica gel
(ethyl acetate/n-hexane 20:80 v/v) gave title compound 1o as a yellow oil.
Yield: 0.91 g (79%).
1H NMR (300 MHz, CDCl3, 208C): ratio enol form (ef):keto form (kf) ˆ
82:18; d ˆ 1.07 (t, J ˆ 7.3 Hz, 3Hkf; H-7), 1.15 (t, J ˆ 7.5 Hz, 3Hef; H-7), 1.47
(s, 9Hef‡kf; C(CH3)3), 2.35 (q, J ˆ 7.5 Hz, 2Hef; H-6), 2.55 (q, J ˆ 7.3 Hz,
2Hkf; H-6), 3.25 (s, 2Hef; H-2), 3.48 (s, 2Hkf; H-2), 3.73 (s, 2Hkf; H-4), 5.60
(s, 1Hef; H-4), 15.15 (brs, 1Hef; OH); 13C NMR (75.5 MHz, CDCl3, 208C):
enol form: d ˆ 9.7 (C-7), 28.1 (C(CH3)3), 31.2 (C-6), 46.4 (C-2), 82.1
(C(CH3)3), 99.2 (C-4), 167.0 (C-1), 187.4, 194.7 (C-3, C-5); MS (70 eV, EI):
1H NMR (300 MHz, CDCl3, 208C): ratio enol form (ef):keto form (kf) ˆ
85:15; d ˆ 1.47 (s, 9Hef‡kf; C(CH3)3), 3.28 (s, 2Hef; H-2), 3.41 (s, 3Hkf;
OCH3), 3.43 (s, 3Hef; OCH3), 3.48 (s, 2Hkf; H-2), 3.76 (s, 2Hkf; H-4), 4.01 (s,
2Hef; H-6), 4.04 (s, 2Hkf; H-6), 5.89 (s, 1Hef; H-4), 15.03 (brs, 1Hef; OH);
13C NMR (75.5 MHz, CDCl3, 208C): enol form: d ˆ 28.2 (C(CH3)3), 46.3
(C-2), 59.6 (OCH3), 73.5 (C-6), 82.3 (C(CH3)3), 97.7 (C-4), 166.8 (C-1),
‡
‡
m/z (%): 214 (2) [M] , 159 (12), 158 (29), 141 (36) [M OtBu] , 140 (17),
‡
129 (40), 111 (13), 99 (52) [M CH2COOtBu] , 85 (14), 84 (10), 57 (100)
‡
‡
187.3, 191.2 (C-3, C-5); MS (70 eV, EI): m/z (%): 230 (1) [M] , 185 (18), 157
[C4H9] .
‡
‡
(21) [M OtBu] , 144 (16), 129 (100), 115 (45) [M CH2COOtBu] , 111
tert-Butyl 3,5-dioxooctanoate (1d)
‡
(27), 84 (10), 57 (65) [C4H9] .
a) N-Methoxy-N-methylbutyramide: This compound was prepared as
described in the preceding example from O,N-dimethyl-hydroxylamine
hydrochloride (1.02 g, 10.5 mmol), butyryl chloride (1.07 g, 10 mmol), and
pyridine (1.69 mL, 21 mmol) with 89% yield.
tert-Butyl 6-fluoro-3,5-dioxohexanoate (1m): tert-Butyl acetoacetate
(0.35 g, 2.2 mmol) was added dropwise to a solution of LDA [prepared
from diisopropyl amine (0.45 g, 4.4 mmol) and n-butyl lithium (2.7 mL of a
1
1.6 molL solution in n-hexane, 4.3 mmol) in 20 mL THF] in THF at
1H NMR (300 MHz, CDCl3, 208C): d ˆ 0.94 (t, J ˆ 7.4 Hz, 3H; H-4), 1.64
(sext, J ˆ 7.4 Hz, 2H; H-3), 2.38 (t, J ˆ 7.4 Hz, 2H; H-2), 3.16 (s, 3H;
NCH3), 3.66 (s, 3H; OCH3); 13C NMR (75.5 MHz, CDCl3, 208C): d ˆ 14.0
(C-4), 18.2 (C-3), 32.2 (NCH3), 33.9 (C-2), 61.3 (OCH3), 174.7 (C-1); MS
108C, and the solution was stirred for ten minutes. After cooling to
758C, ethyl 2-fluoroacetate (0.21 g, 2.0 mmol) and TMEDA (0.26 g,
2.2 mmol) were added. The solution was stirred for five minutes, warmed to
558C over a period of 30 minutes, stirred for an additional ten minutes at
this temperature, and then recooled to 758C. Workup was carried out as
described in the general procedure. After removal of excess tert-butyl
acetoacetate by Kugelrohr distillation (0.2 mbar, 408C), the residue was
subjected to flash chromatography on acid-washed silica gel (ethyl acetate/
n-hexane 10:90 v/v), and this yielded title compound 1m as a light-yellow
oil. Yield: 0.25 g (58%).
‡
‡
(70 eV, EI): m/z (%): 131 (20) [M] , 71 (98), 61 (100) [NH(OMe)Me] .
b) Acylation reaction: A solution of Na,Li-bisenolate was prepared as
described in the general procedure from tert-butyl acetoacetate (0.63 g,
4.0 mmol), sodium hydride (0.18 g of a 60% suspension in oil, 4.5 mmol),
and n-butyl lithium (2.6 mL of a 1.6 molL 1 solution in n-hexane, 4.2 mmol)
in THF (30 mL). N-Methoxy-N-methylbutyramide (0.54 g, 4.1 mmol) was
syringed into the flask at
temperature for 15 minutes, warmed to room temperature, and stirred
for an additional 60 minutes. Workup was carried out as described in the
108C. The solution was stirred at this
1H NMR (300 MHz, CDCl3, 208C): ratio enol form (ef):keto form (kf) ˆ
90:10; d ˆ 1.47 (s, 9Hef‡kf; C(CH3)3), 3.31 (s, 2Hef; H-2), 3.48 (s, 2Hkf; H-2),
3.86 (d, 4J(H,F) ˆ 3.8 Hz, 2Hkf; H-4), 4.86 (d, 2J(H,F) ˆ 46.8 Hz, 2Hef;
Chem. Eur. J. 2001, 7, No. 21
ꢁ WILEY-VCH Verlag GmbH, D-69451 Weinheim, 2001
0947-6539/01/0721-4567 $ 17.50+.50/0
4567

M. Müller et al.
FULL PAPER
H-6), 4.88 (d, 2J(H,F) ˆ 47.3 Hz, 2Hkf; H-6), 5.93 (d, 4J(H,F) ˆ 3.0 Hz, 1Hef;
H-4), 14.80 (brs, 1Hef; OH); 13C NMR (75.5 MHz, CDCl3, 208C): enol
form: d ˆ 28.1 (C(CH3)3), 45.9 (C-2), 81.3 (d, 1J(C,F) ˆ 183.1 Hz, C-6), 82.5
(C(CH3)3), 97.0 (d, 3J(C,F) ˆ 5.4 Hz, C-4), 166.5 (C-1), 186.7 (C-3), 189.8 (d,
J ˆ 17.7, 3.2 Hz, 1H; H-4), 2.91 (brs, 1H; OH), 3.38 (s, 2H; H-2), 4.27 (m,
1H; H-5), traces of enol form visible; 13C NMR (75.5 MHz, CDCl3, 208C):
d ˆ 22.6 (C-6), 28.1 (C(CH3)3), 51.2, 51.3 (C-2, C-4), 63.9 (C-5), 82.4
(C(CH3)3), 166.4 (C-1), 204.4 (C-3). The 1H NMR spectrum is in agreement
with published data.[13a]
‡
2J(C,F) ˆ 21.3 Hz, C-5); MS (70 eV, EI): m/z (%): 218 (1) [M] , 163 (17),
‡
‡
145 (37) [M OtBu] , 129 (15), 125 (12), 103 (48) [M CH2COOtBu] , 57
(100) [C4H9] .
tert-Butyl (R)-5-hydroxy-3-oxoheptanoate [(R)-2c]: In a round-bottom
flask diketo ester 1c (150 mg, 0.7 mmol) and 2-propanol (0.5 mL,
6.5 mmol) were added to phosphate buffer (35 mL, 250 mmolL 1, MgCl2
1 mmolL 1, pH 6.5), and the mixture was vigorously stirred for ten minutes.
‡
Preparation and assay of recLBADH: A suspension of wet cells (1.0 g) of
[9]
recombinant E. coli strain recADH-HB101 ‡
in phosphate buffer
‡
(4.0 mL, 100 mmolL 1, trace of poly(propylene glycol) anti-foam, pH 7.5)
was distributed over eight plastic vials (1.5 mL size), and glass beads (8 Â
1.2 g, 0.2 mm) were added to each vial. The vials were thoroughly vortexed
and cooled in ice. Disruption was carried out in a mixer mill at 08C (2 Â
5 min with intermediate cooling).[35] Cell debris and glass beads were
removed by centrifugation (12000 rpm, 10 min), the supernatants were
unified, and MgCl2 was added (final concentration 1.0 mmolL 1). Typical
yield: 3 mL, 1100 UmL 1. The enzyme solution thus obtained could be
stored for weeks at 48C without significant loss of activity. The enzyme
The stirring speed was lowered to 60 rpm, and NADP (15 mg, 18 mmol;
90% purity) and recLBADH (54 U) were added. The mixture was stirred
‡
at 208C, and recLBADH (50 U) and NADP (10 mg, 13 mmol) were added
after 24 and 48 hours each. After a total reaction time of 96 hours, the
solution was saturated with NaCl and extracted with ethyl acetate three
times. The unified organic phases were washed with aq. NaHCO3 (5%),
water and brine, dried over MgSO4, and the solvent was evaporated under
reduced pressure. Flash chromatography on silica gel (ethyl acetate/
isohexane 30:70 v/v) gave title compound (R)-2c as a colorless oil. Yield:
92 mg (61%).
activity was determined at 258C as follows. A cuvette was charged with a
1
solution (990 mL) of acetophenone (10 mmolL
)
and NADPH
[a]2D5
ˆ
36.0 (c ˆ 1.0 in CHCl3); ref. [13b]: [a]D
ˆ
35.6 (c ˆ 1 in CHCl3),
(0.25 mmolL 1) in phosphate buffer (100 mmolL 1, MgCl2 1.0 mmolL
,
1
99% ee; 1H NMR (300 MHz, CDCl3, 208C): d ˆ 0.94 (t, J ˆ 7.4 Hz, 3H;
H-7), 1.43 ± 1.56 (m, 2H; H-6) overlapping with 1.46 (s, 9H; C(CH3)3), 2.61
(dd, J ˆ 17.5, 8.9 Hz, 1H; H-4), 2.73 (dd, J ˆ 17.5, 3.0 Hz, 1H; H-4), 2.90
(brs, 1H; OH), 3.38 (s, 2H; H-2), 3.99 (m, 1H; H-5), traces of enol form
visible; 13C NMR (75.5 MHz, CDCl3, 208C): d ˆ 10.0 (C-7), 28.1 (C(CH3)3),
29.4 (C-6), 49.2, 51.4 (C-2, C-4), 69.0 (C-5), 82.4 (C(CH3)3), 166.4 (C-1),
204.6 (C-3). The 1H NMR spectrum is in agreement with published data.[13b]
pH 6.5), and the reaction was started by addition of enzyme solution [10 mL
1
of
a
200-fold dilution in phosphate buffer (100 mmolL
,
MgCl2
1 mmolL 1, pH 7.5)]. NADPH consumption was monitored at 340 nm
(eNADPH ˆ 6.22 cm2 mmol 1) over a period of one minute. One unit (U) of
activity is defined as the amount of recLBADH that catalyzes the oxidation
of 1 mmol NADPH per minute under these conditions. Relative rates for
the reduction of b,d-diketo esters (determined at the concentrations
indicated in Table 1) were calculated by setting the rate of acetophenone
reduction arbitrarily to 100%.
Bakerꢀs yeast reduction of diketo ester 1a
tert-Butyl (R)-6-chloro-5-hydroxy-3-oxohexanoate [(R)-2a]: Commercially
available AmberliteXAD-7 resin was washed prior to use with water,
acetone, and ethyl acetate, and dried under reduced pressure until the
weight remained constant. AmberliteXAD-7 (9.0 g) was added to a
solution of diketo ester 1a (2.00 g, 8.5 mmol) in ethyl acetate (70 mL), and
the solvent was thoroughly evaporated under reduced pressure. The
charged resin was added to a suspension of dried Bakerꢀs yeast (85 g) in
deionized water (400 mL), and the mixture was shaken at 208C (130 rpm,
1 L shake flask, horizontal shaker). After 15 hours, the resin was collected
on a sintered glass funnel (porosity ª0º), washed with a minimal amount of
water, and extracted with acetone (4 Â 50 mL) and ethyl acetate (50 mL).
The extract was concentrated under reduced pressure, and the residue
dissolved in ethyl acetate. The solution was washed with aq. NaHCO3 (5%)
and brine, dried over MgSO4, and the solvent evaporated under reduced
pressure. Flash chromatography on silica gel (ethyl acetate/isohexane 40:60
v/v) gave title compound (R)-2a as a light-yellow oil. Yield: 1.01 g (50%).
recLBADH reduction of b,d-diketo esters
tert-Butyl (S)-6-chloro-5-hydroxy-3-oxohexanoate [(S)-2a]: In a round-
bottom flask, a solution of diketo ester 1a (2.53 g, 10.8 mmol) in 2-propanol
(8.3 mL, 108 mmol) was added to phosphate ± citrate ± NaOH buffer
1
1
(530 mL, Na2HPO4 250 mmolL
, citric acid 125 mmolL , MgCl2
1 mmolL 1, pH 5.5), and the mixture was vigorously stirred for five
minutes. The stirring speed was lowered to 60 rpm, and NADP (92 mg,
‡
108 mmol, 90% purity) and recLBADH (1400 U) were added. After
stirring at 208C for 14 hours, the mixture was filtered, saturated with NaCl,
and extracted with ethyl acetate three times. The unified organic phases
were washed with saturated aq. NaHCO3 and brine, dried over MgSO4, and
the solvent was evaporated under reduced pressure. Flash chromatography
on silica gel (ethyl acetate/isohexane 40:60 v/v) gave title compound (S)-2a
as a light-yellow oil. Yield: 1.84 g (72%).
[a]2D5
ˆ
24.9 (c ˆ 1.4 in CHCl3); ref. [12]: [a]D25
ˆ
23.0 (c ˆ 1.5 in CHCl3),
[a]2D5 ˆ ‡22.8 (c ˆ 1.6 in CHCl3); [a]2D5
ˆ
24.9 (c ˆ 1.4 in CHCl3) for the S
1
>97% ee; H NMR (300 MHz, CDCl3, 208C): d ˆ 1.47 (s, 9H; C(CH3)3),
2.83 (dd, J ˆ 17.5, 7.3 Hz, 1H; H-4), 2.90 (dd, J ˆ 17.5, 5.0 Hz, 1H; H-4), 3.10
(brs, 1H; OH), 3.41 (s, 2H; H-2), 3.57 (dd, J ˆ 11.2, 5.0 Hz, 1H; H-6), 3.62
(dd, J ˆ 11.2, 5.1 Hz, 1H; H-6), 4.31 (m, 1H; H-5), traces of enol form
visible; 13C NMR (75.5 MHz, CDCl3, 208C): d ˆ 28.1 (C(CH3)3), 46.6, 48.4,
51.3 (C-2, C-4, C-6), 67.6 (C-5), 82.7 (C(CH3)3), 166.2 (C-1), 202.9 (C-3); MS
(70 eV, EI): m/z (%): 236 (1) [M] , 163/165 (27/9) [M OtBu], 144 (11),
131 (41), 121/123 (20/6) [M CH2COOtBu] , 102 (13), 85 (10), 59 (18), 57
(100) [C4H9] ; elemental analysis calcd (%) for C10H17ClO4 (236.7): C
50.74, H 7.24; found: C 50.81, H 7.29.
enantiomer, >99.5% ee (see above). NMR data were in agreement with
the data described for (S)-2a.
Preparation of d-lactones
(S)-6-Chloromethyl-5,6-dihydropyran-2,4-dione [(S)-3]: A catalytic amount
of TsOH monohydrate was added to a solution of hydroxy keto ester (S)-2a
(0.24 g, 1.0 mmol) in dichloromethane (15 mL), and the solution was stirred
at room temperature for four days. Dichloromethane was replaced by ethyl
acetate. The solution was washed with water and brine, dried over MgSO4,
and the solvent was evaporated under reduced pressure. Flash chromatog-
raphy on silica gel (ethyl acetate/diethyl ether/acetic acid 45:55:0.1) gave
title compound (S)-3 as a white solid. Yield: 0.11 g (67%).
‡
‡
‡
tert-Butyl (R)-5-hydroxy-3-oxohexanoate [(R)-2b]: In
a round-bottom
flask, a solution of diketo ester 1b (1.98 g, 9.9 mmol) in 2-propanol
(5.1 mL, 66 mmol) was added to 330 mL triethanolamine ± HCl buffer
(250 mmolL 1, MgCl2 1 mmolL 1, pH 7.0), and the mixture was vigorously
stirred for ten minutes. The stirring speed was lowered to 60 rpm, and
[a]2D5
ˆ
80.6 (c ˆ 1.1 in MeOH); ref. [11a]: [a]D25
ˆ
83.4 (c ˆ 1.07 in
MeOH), ꢀ98% ee; 1H NMR (300 MHz, CDCl3, 208C): d ˆ 2.72 (dd, J ˆ
18.2, 11.1 Hz, 1H; H-5), 2.89 (dd, J ˆ 18.2 Hz, 3.2 Hz, 1H; H-5), 3.51 (d, J ˆ
19.1 Hz, 1H; H-3), 3.61 (d, J ˆ 19.1 Hz, 1H; H-3), 3.82 (d, J ˆ 4.9 Hz, 2H;
CH2Cl), 4.90 (m, 1H; H-6). The NMR spectrum is in agreement with data
published for the racemate.[11b]
‡
NADP (28 mg, 33 mmol; 90% purity) and recLBADH (330 U) were
added. After stirring at 208C for 24 hours, the solution was saturated with
NaCl and extracted with ethyl acetate three times. The unified organic
phases were dried over MgSO4, and the solvent was evaporated under
reduced pressure. Flash chromatography on silica gel (ethyl acetate/
isohexane 40:60 v/v) gave title compound (R)-2b as a colorless oil. Yield:
1.54 g (77%).
General procedure for the preparation of a,b-unsaturated d-lactones and
determination of the enantiomeric excess
a) Ketone reduction: NaBH4 (0.6 equiv) was added portionwise over a
period of ten minutes to an ice-cooled solution of the hydroxy keto ester
(1 equiv) in ethanol (10 mL per mmol ester), and the mixture was stirred at
this temperature for an additional 30 minutes. Acetic acid (2.4 equiv) was
added dropwise, and stirring was continued for five minutes. Ethanol was
[a]2D5
ˆ
40.1 (c ˆ 1.9 in CHCl3); ref. [13a]: [a]D26
ˆ
39.6 (c ˆ 2 in CHCl3),
99% ee; 1H NMR (300 MHz, CDCl3, 208C): d ˆ 1.21 (d, J ˆ 6.4 Hz, 3H;
H-6), 1.48 (s, 9H; C(CH3)3), 2.64 (dd, J ˆ 17.7, 8.5 Hz, 1H; H-4), 2.74 (dd,
4568
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Chem. Eur. J. 2001, 7, No. 21

Reduction of b,d-Diketo Esters
4562±4571
replaced by ethyl acetate. The solution was washed with aq. NaHCO3 (5%)
and brine, dried over MgSO4, and the solvent was evaporated under
reduced pressure.
was determined at the stage of the acetonide (see below). Stereoisomers
with (5R) configuration could not be detected. Crystallization from
isohexane/ethyl acetate gave title compound syn-(3R,5S)-6a as light-yellow
crystals (m.p. 50.8 ± 53.08C, ref. [12a]: 50 ± 528C (hexane/ethyl acetate)).
Yield: 12.8 g (62%).
b) Lactonization and dehydration: A catalytic amount of TsOH monohy-
drate was added to a solution of the crude dihydroxy ester in toluene
(10 mL), and the mixture was heated to reflux for two hours. After cooling,
ethyl acetate was added. The solution was washed with aq. NaHCO3 (5%)
and brine, dried over MgSO4, and the solvent was evaporated under
reduced pressure. The product was analyzed by HPLC on chiral stationary
phase (Daicel ChiracelOB, 250 Â 4.6 mm with guard column 50 Â 4.6 mm;
[a]2D5
ˆ
27.0 (c ˆ 1.4 in CHCl3), >99.5% ee, diastereomeric ratio syn-
6a:anti-6a ˆ 205:1; 1H NMR (300 MHz, CDCl3, 208C): d ˆ 1.45 (s, 9H;
C(CH3)3), 1.59 ± 1.79 (m, 2H; H-4), 2.42 (d, J ˆ 6.2 Hz, 2H; H-2), 3.51 (dd,
J ˆ 11.1, 5.3 Hz, 1H; H-6), 3.55 (dd, J ˆ 11.1, 5.5 Hz, 1H; H-6), 3.71 (brs,
1H; OH), 3.81 (brs, 1H; OH), 4.08 (m, 1H; H-5), 4.24 (m, 1H; H-3);
13C NMR (75.5 MHz, CDCl3, 208C): d ˆ 28.3 (C(CH3)3), 39.5, 42.5, 49.2 (C-
2, C-4, C-6), 68.4, 71.6 (C-3, C-5), 81.9 (C(CH3)3), 172.2 (C-1); MS (70 eV,
1
258C; 1.0 mLmin isohexane/isopropyl alcohol 80:20 v/v for 4a,b, 85:15
for 4c; 215 nm). To verify separation performance, spiking experiments
were carried out with rac-4a ± c.[36] Retention times: (S)-4a 24.3 min; (R)-
4a 20.9 min; (R)-4b 23.8 min; (S)-4b 17.6 min; (R)-4c 29.1 min; (S)-4c
18.7 min.
‡
‡
EI): m/z (%): 182/184 (9/3) [M C4H8] , 165/167 (49/16) [M OtBu] , 147
(19), 145 (14), 133 (36), 129 (15), 127 (11), 123 (18), 115 (50), 105 (12), 97
‡
(14), 89 (20), 87 (17), 59 (13), 57 (100) [C4H9] , 56 (11); elemental analysis
calcd (%) for C10H19ClO4 (238.7): C 50.32, H 8.02; found: C 50.20, H 8.35.
(S)-6-Chloromethyl-5,6-dihydropyran-2-one [(S)-4a]: Prepared according
to the general procedure from hydroxy keto ester (S)-2a (270 mg,
1.1 mmol). Flash chromatography on silica gel (ethyl acetate/isohexane
50:50 v/v) gave title compound (S)-4a as a light-yellow oil. Yield: 100 mg
(60%).
tert-Butyl syn-(3S,5R)-6-chloro-3,5-dihydroxyhexanoate [syn-(3S,5R)-6a]:
Prepared from hydroxy keto ester (R)-2a (2.89 g, 12.2 mmol, 90% ee) as
described in the preceding example. The crude product was obtained in
quantitative yield and had a diastereomeric ratio syn-6a:anti-6a ˆ 45:1 as
determined at the stage of the acetonide (see below). Crystallization from
isohexane/ethyl acetate gave the product (1.51 g, 52%) in the form of
orange crystals (drˆ 187:1, ee ˆ 98.0%). An analytical sample was
recrystallized to give the title compound syn-(3S,5R)-6a as colorless
crystals (m.p. 51.7 ± 52.78C).
[a]2D5
ˆ
158.6 (c ˆ 0.5 in CHCl3), >99.5% ee; ref. [11a]: [a]D23
ˆ
144.8
(c ˆ 3.09 in CHCl3), ꢀ98% ee; 1H NMR (300 MHz, CDCl3, 208C): d ˆ
2.50 ± 2.65 (m, 2H; H-5), 3.71 (dd, J ˆ 11.7, 6.0 Hz, 1H of CH2Cl), 3.76 (dd,
J ˆ 11.7, 4.8 Hz, 1H of CH2Cl), 4.67 (m, 1H; H-6), 6.07 (dt, J ˆ 9.9, 1.9 Hz,
1H; H-3), 6.93 (m, 1H; H-4); 13C NMR (75.5 MHz, CDCl3, 208C): d ˆ 26.9
(C-5), 44.9 (CH2Cl), 76.5 (C-6), 121.3 (C-3), 144.7 (C-4), 163.2 (C-2). The
1H NMR spectrum is in agreement with published data.[37]
[a]2D5 ˆ ‡27.6 (c ˆ 1.3 in CHCl3), >99.5% ee, diastereomeric ratio syn-
6a:anti-6a > 400:1. NMR data were in agreement with the data described
for syn-(3R,5S)-6a.
(R)-6-Chloromethyl-5,6-dihydropyran-2-one [(R)-4a]: Prepared from hy-
droxy keto ester (R)-2a according to the preceding example. HPLC
analysis of the crude product indicated 94% ee.
tert-Butyl anti-(3S,5S)-6-chloro-3,5-dihydroxyhexanoate [anti-(3S,5S)-6a]:
Hydroxy keto ester (S)-2a (0.76 g, 3.2 mmol, >99.5% ee, dissolved in 5 mL
anhydrous acetonitrile) was added dropwise to a solution of tetramethyl-
ammonium triacetoxyborohydride (6.74 g, 25.6 mmol) in anhydrous
acetonitrile/acetic acid (30 mL, 50:50 v/v) at 258C. After stirring at this
temperature for five hours, aq. sodium potassium tartrate (35 mL,
0.5 molL 1) was added dropwise, and the mixture was warmed to room
temperature. Saturated aq. Na2CO3 (70 mL) was added, and the mixture
was extracted with ethyl acetate four times. The unified organic phases
were washed with saturated aq. Na2CO3 and brine, dried over MgSO4, and
the solvent was evaporated under reduced pressure. The crude product was
obtained in quantitative yield and had a diastereomeric ratio anti-6a:syn-
6a ˆ 14:1 as determined at the stage of the acetonide (see below).
Crystallization from isohexane/ethyl acetate gave title compound anti-
(3S,5S)-6a as colorless crystals (m.p. 73.3 ± 75.08C). Yield: 0.54 g (70%).
(R)-6-Methyl-5,6-dihydropyran-2-one [(R)-4b]: Prepared according to the
general procedure from hydroxy keto ester (R)-2b (205 mg, 1.0 mmol). The
crude product (79 mg, 70%) was not further purified; 99.4% ee.
1H NMR (300 MHz, CDCl3, 208C): d ˆ 1.37 (d, J ˆ 6.4 Hz, 3H; CH3),
2.17 ± 2.39 (m, 2H; H-5), 4.51 (m, 1H; H-6), 5.93 (ddd, J ˆ 9.8, 2.5, 1.2 Hz,
1H; H-3), 6.83 (ddd, J ˆ 9.8, 5.8, 2.7 Hz, 1H; H-4); 13C NMR (75.5 MHz,
CDCl3, 208C): d ˆ 20.7 (CH3), 31.0 (C-5), 74.4 (C-6), 121.1 (C-3), 145.3 (C-
4), 164.6 (C-2). The 1H NMR spectrum is in agreement with published data.[4b]
(R)-6-Ethyl-5,6-dihydropyran-2-one [(R)-4c]: Prepared according to the
general procedure from hydroxy keto ester (R)-2c (130 mg, 0.6 mmol). The
crude product (49 mg, 65%) was not further purified; 98.1% ee.
1H NMR (300 MHz, CDCl3, 208C): d ˆ 0.98 (t, J ˆ 7.5 Hz, 3H; CH3), 1.60 ±
1.85 (m, 2H; CH2CH3), 2.20 ± 2.39 (m, 2H; H-5), 4.32 (m, 1H; H-6), 5.96
(ddd, J ˆ 9.8, 2.4, 1.4 Hz, 1H; H-3), 6.86 (m, 1H; H-4); 13C NMR
(75.5 MHz, CDCl3, 208C): d ˆ 9.3 (CH3), 27.9, 28.9 (C-5, CH2CH3), 79.3
(C-6), 121.3 (C-3), 145.4 (C-4), 164.8 (C-2). The 1H NMR spectrum is in
agreement with data published for the racemate.[38]
[a]2D5 ˆ ‡7.2 (c ˆ 1.4 in CHCl3), >99.5% ee, diastereomeric ratio anti-
6a:syn-6a ˆ 211:1; 1H NMR (300 MHz, CDCl3, 208C): d ˆ 1.46 (s, 9H;
C(CH3)3), 1.66 ± 1.71 (m, 2H; H-4), 2.42 (d, J ˆ 6.3 Hz, 2H; H-2), 3.05 (brs,
1H; OH), 3.60 (brs, 1H; OH) overlapping with 3.53 (dd, J ˆ 11.0, 6.5 Hz,
1H; H-6), 3.62 (dd, J ˆ 11.0, 5.0 Hz, 1H; H-6), 4.12 (m, 1H; H-5), 4.30 (m,
1H; H-3); 13C NMR (75.5 MHz, CDCl3, 208C): d ˆ 28.3 (C(CH3)3), 39.3,
42.2, 49.6 (C-2, C-4, C-6), 65.5, 68.9 (C-3, C-5), 81.9 (C(CH3)3), 172.5 (C-1);
tert-Butyl (4-oxo-4,5-dihydrofuran-2-yl)-acetate (5): Phosphate buffer
(20 mL, 250 mmolL 1, pH 7.0) was added to a solution of diketo ester 1a
(234 mg, 1.0 mmol) in ethanol (10 mL), and the solution was stirred at room
temperature for 20 h. Ethanol was removed under reduced pressure.
Hydrochloric acid (5 mL, 2 molL 1) was added, and the mixture was
extracted twice with ethyl acetate. The unified organic phases were washed
with aq. NaHCO3 (5%) and brine, dried over Na2SO4, and the solvent was
removed under reduced pressure. Flash chromatography on silica gel (ethyl
acetate/isohexane 40:60 v/v) gave title compound 5 as a light-yellow oil that
solidified upon storage at 48C. Yield: 157 mg (79%).
‡
MS (70 eV, EI): m/z (%): 182/184 (9/3) [M C4H8] , 165/167 (48/16) [M
‡
OtBu] , 147 (16), 145 (11), 133 (30), 129 (15), 127 (11), 123 (19), 115 (45),
‡
105 (10), 97 (13), 89 (21), 87 (12), 59 (12), 57 (100) [C4H9] , 56 (11);
elemental analysis calcd (%) for C10H19ClO4 (238.7): C 50.32, H 8.02;
found: C 49.92, H 7.95.
tert-Butyl anti-(3R,5R)-6-chloro-3,5-dihydroxyhexanoate [anti-(3R,5R)-6a]:
Prepared from hydroxy keto ester (R)-2a (1.00 g, 4.2 mmol, 94% ee) as
described in the preceding example. The crude product had a diastereo-
meric ratio anti-6a:syn-6a ˆ 18:1 as determined at the stage of the
acetonide (see below). Crystallization from isohexane/ethyl acetate gave
title compound anti-(3R,5R)-6a as colorless crystals (m.p. 74.5 ± 76.08C).
Yield: 0.69 g (68%).
1H NMR (300 MHz, CDCl3, 208C): d ˆ 1.47 (s, 9H; C(CH3)3), 3.48 (s, 2H;
CH2COOtBu), 4.53 (s, 2H; H-5), 5.68 (s, 1H; H-3); 13C NMR (75.5 MHz,
CDCl3, 208C): d ˆ 28.1 (C(CH3)3), 38.3 (CH2COOtBu), 75.5 (C-5), 82.9
(C(CH3)3), 106.4 (C-3), 165.9 (COOtBu), 187.0 (C-2), 202.7 (C-4); MS
‡
‡
[a]2D5
ˆ
6.7 (c ˆ 1.3 in CHCl3), 99.3% ee, diastereomeric ratio anti-6a:syn-
(70 eV, EI): m/z (%): 198 (11) [M] , 125 (77) [M OtBu] , 97 (25), 98 (10),
‡
67 (20), 59 (13), 57 (100) [C4H9] .
6a ˆ 316:1. NMR data were in agreement with the data described for anti-
(3S,5S)-6a.
Diastereoselective reduction of hydroxy keto ester 2a
tert-Butyl syn-(3R,5S)-6-chloro-3,5-dihydroxyhexanoate [syn-(3R,5S)-6a]:
This compound was prepared from crude hydroxy keto ester (S)-2a (21.5 g,
86 mmol, approximately 95% purity, >99.5% ee) according to the
procedure described in ref. [12a]. The crude product was obtained in
quantitative yield and had a diastereomeric ratio syn-6a:anti-6a ˆ 28:1 as
Determination of the diastereomeric ratio syn-6a:anti-6a and the enantio-
meric excess of dihydroxy ester 6a at the stage of the acetonide 7a:
Dihydroxy ester 6a (10 mg) was dissolved in 2,2-dimethoxy-propane
(1 mL), and a catalytic amount of camphorsulfonic acid was added. After
shaking at 258C for 4 ± 7 h, GC-MS analysis indicated complete conversion,
Chem. Eur. J. 2001, 7, No. 21
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0947-6539/01/0721-4569 $ 17.50+.50/0
4569

M. Müller et al.
FULL PAPER
and the solution was investigated by gas chromatography on chiral
stationary phase. Column: CSFS-Cyclodex beta-1/P, 50 m  0.32 mm ID.
Injector: split, 2508C. Carrier gas: hydrogen, 0.95 bar (constant pressure).
Temperature program: 1358C isothermal. Retention times: anti-(3R,5R)-
7a 48.8 min; anti-(3S,5S)-7a 50.2 min; syn-(3R,5S)-7a 52.1 min; syn-
(3S,5R)-7a 53.8 min. To verify peak indentity and separation performance,
spiking experiments were carried out with acetonide rac-(3RS,5RS)-7a that
was prepared from hydroxy keto ester rac-2a[36] by sodium borohydride
reduction and subsequent acetonide formation.
light-yellow oil that consisted of 90 mol% title compound (3R,5S)-9 and
10 mol% tetrahydrofurane (2R,4S)-11 according to 1H NMR analysis.
Yield: 103 mg (46%).
tert-Butyl syn-(3R,5S)-6-iodo-3,5-(isopropylidenedioxy)-hexanoate [syn-
(3R,5S)-10]
Method A (halogen exchange): Acetonide syn-(3R,5S)-7a (2.01 g,
7.2 mmol), [18]crown-6 (2.85 g, 10.8 mmol), and finely powdered potassium
iodide (23.7 g, 143 mmol) were added to anhydrous p-xylene (50 mL), and
a stream of nitrogen was introduced for 15 minutes to remove dissolved
oxygen. The mixture was vigorously stirred and heated to reflux for three
days, whereupon another portion of potassium iodide (5.98 g, 36 mmol)
and [18]crown-6 (0.95 g, 3.6 mmol) was added. Heating was continued for
18 hours. Water (50 mL) was added, and the resulting mixture was filtered.
The phases were separated, and the aqueous phase was extracted with ethyl
acetate. The unified organic phases were washed with aq. NaHSO3 (20%),
saturated aq. NaHCO3 and brine, dried over Na2SO4, and volatiles were
removed under reduced pressure. Flash chromatography on silica gel (ethyl
acetate/isohexane 20:80 v/v) gave a yellow oil that consisted of title
compound (3R,5S)-10 (86 mol%) and unreacted acetonide syn-(3R,5S)-7a
tert-Butyl syn-(3R,5S)-6-chloro-3,5-(isopropylidenedioxy)-hexanoate [syn-
(3R,5S)-7a]: The compound 2,2-dimethoxy-propane (26 mL, 214 mmol)
and a catalytic amount camphorsulfonic acid at room temperature were
added to a solution of dihydroxy ester syn-(3R,5S)-6a (5.10 g, 21.4 mmol,
>99.5% ee, dr syn-6a:anti-6a ˆ 205:1) in acetone (17 mL). After stirring
the solution for 1.5 h, the volatiles were removed under reduced pressure,
and the residue was dissolved in ethyl acetate (60 mL). The solution was
washed with saturated aq. NaHCO3 and brine, dried over Na2SO4, and the
solvent was evaporated under reduced pressure, and pure product syn-
(3R,5S)-7a (5.83 g, 98%) was left as a colorless oil that solidified upon
storage at 48C.
1
(14 mol%) according to H NMR analysis. Yield: 1.61 g (52%).
[a]2D5 ˆ ‡3.1 (c ˆ 1.3 in CHCl3); 1H NMR (300 MHz, CDCl3, 208C): d ˆ
1.24 (m, 1H; H-4), 1.39 (s, 3H; CH3), 1.45 (s, 9H; C(CH3)3), 1.46 (s, 3H;
CH3), 1.76 (dt, J ˆ 12.6, 2.5 Hz, 1H; H-4), 2.33 (dd, J ˆ 15.2, 6.1 Hz, 1H;
H-2), 2.45 (dd, J ˆ 15.2, 7.1 Hz, 1H; H-2), 3.40 (dd, J ˆ 11.0, 5.8 Hz, 1H;
H-6), 3.51 (dd, J ˆ 11.0, 5.6 Hz, 1H; H-6), 4.06 (m, 1H; H-5), 4.28 (m, 1H;
H-3); 13C NMR (75.5 MHz, CDCl3, 208C): d ˆ 19.7 (CH3), 28.1 (C(CH3)3),
29.8 (CH3), 33.9, 42.5, 47.1 (C-4, C-2, C-6), 65.9, 69.2 (C-3, C-5), 80.7
(C(CH3)3), 99.2 (C(CH3)2), 170.0 (C-1); MS (70 eV, EI): m/z (%): 263/265
1H NMR (300 MHz, CDCl3, 208C): d ˆ 1.16 (dt, J ˆ 12.5, 11.5 Hz, 1H;
H-4), 1.40 (s, 3H; CH3), 1.45 (s, 12H; C(CH3)3) and CH3), 1.87 (dt, J ˆ 12.5,
2.4 Hz, 1H; H-4), 2.33 (dd, J ˆ 15.1, 6.1 Hz, 1H; H-2), 2.46 (dd, J ˆ 15.2,
7.0 Hz, 1H; H-2), 3.10 (dd, J ˆ 10.1, 6.1 Hz, 1H; H-6), 3.17 (dd, J ˆ 10.1,
5.7 Hz, 1H; H-6), 3.88 (m, 1H; H-5), 4.27 (m, 1H; H-3); 13C NMR
(75.5 MHz, CDCl3, 208C): d ˆ 9.4 (C-6), 19.8 (CH3), 28.2 (C(CH3)3), 29.9
(CH3), 36.3, 42.5 (C-4, C-2), 66.2, 69.1 (C-3, C-5), 80.8 (C(CH3)3), 99.6
‡
(C(CH3)2), 170.1 (C-1); MS (70 eV, EI): m/z (%): 355 (50) [M CH3] , 299
‡
‡
(37/12) [M CH3] , 207/209 (70/24), 165 (12), 147/149 (61/18), 129 (18), 115
(28), 239 (100), 197 (31), 129 (13), 111 (15), 59 (23), 57 (77) [C4H9] ; HRMS
‡
‡
(EI) calcd for [M CH3] : 355.0406; found: 355.0397.
(11), 111 (12), 105 (17), 59 (53), 57 (100) [C4H9] ; HRMS (EI) calcd for
‡
[M CH3] : 263.1050; found: 263.1037; elemental analysis calcd (%) for
Method B (opening of epoxide): Epoxide (3R,5S)-9 (1.69 g, 7.3 mmol, 87%
purity), anhydrous lithium iodide (3.33 g, 24.9 mmol), and silica gel (2.08 g)
were mixed in dichloromethane, and the solvent was evaporated at reduced
pressure. The charged silica gel was kept at room temperature for 1 h,
extracted with ethyl acetate, and the extract concentrated under reduced
pressure. The compound 2,2-dimethoxy-propane (20 mL) and a catalytic
amount of camphorsulfonic acid were added, and the solution was stirred at
ambient temperature for 24 hours. Volatiles were evaporated under
reduced pressure. The residue was dissolved in dichloromethane, and the
solution was washed with aq. Na2S2O3 (5%), saturated aq. NaHCO3, and
brine. After drying over Na2SO4, the solvent was evaporated under reduced
pressure. Flash chromatography on silica gel (ethyl acetate/isohexane 20:80
v/v) gave title compound (3R,5S)-10 as a yellow oil. Yield: 1.55 g (58%).
C13H23ClO4 (278.8): C 56.01, H 8.32; found: C 56.06, H 8.33.
tert-Butyl syn-(3R,5S)-5,6-epoxy-3-hydroxyhexanoate [syn-(3R,5S)-9]
Method A: DBU (3.83 g, 25.1 mmol) was added to a solution of dihydroxy
ester syn-(3R,5S)-6a (3.00 g, 12.6 mmol, >99.5% ee, dr syn-6a:anti-6a ˆ
205:1) in dichloromethane (130 mL), and the solution was heated to reflux
for 24 hours. The solution was washed with saturated aq. NH4Cl (2Â),
saturated aq. NaHCO3 and brine, dried over MgSO4, and the solvent was
evaporated under reduced pressure. The crude product was obtained as
yellow oil that consisted of 87 mol% title compound (3R,5S)-9 and
13 mol% tetrahydrofurane (2R,4S)-11 according to 1H NMR analysis.
Yield: 1.94 g (66%). An analytical sample was purified by flash chroma-
tography on silica gel (ethyl acetate/isohexane 50:50 v/v).
[a]2D5 ˆ ‡11.2 (c ˆ 2.3 in CHCl3). NMR data were in agreement with the
[a]2D5
ˆ
30.1 (c ˆ 1.7 in CHCl3); 1H NMR (300 MHz, CDCl3, 208C): d ˆ
data of the major product obtained by method A.
1.45 (s, 9H; C(CH3)3), 1.63 ± 1.78 (m, 2H; H-4), 2.45 (d, J ˆ 6.2 Hz, 2H;
H-2), 2.50 (dd, J ˆ 5.0, 2.7 Hz, 1H; H-6), 2.77 (brt, J ꢁ 4.5 ± 5.0 Hz, 1H;
H-6), 3.09 (m, 1H; H-5), 3.33 (d, J ˆ 3.3 Hz, 1H; OH), 4.19 (m, 1H; H-3);
13C NMR (75.5 MHz, CDCl3, 208C): d ˆ 28.3 (C(CH3)3), 39.0, 42.1, 46.8 (C-
2, C-4, C-6), 49.7 (C-5), 66.4 (C-3), 81.7 (C(CH3)3), 172.4 (C-1); MS (70 eV,
EI): m/z (%): 146 (5) [M C4H8] , 145 (14), 129 (21) [M OtBu] , 127
(18), 111 (21), 110 (13), 89 (20), 87 (49) [M CH2COOtBu] , 71 (11), 69
(15), 59 (15), 57 (100) [C4H9] . The more polar tetrahydrofurane (2R,4S)-
Acknowledgements
Financial support of this work by the Deutsche Forschungsgemeinschaft
(SFB380) is gratefully acknowledged. We are grateful to S. Bode, P.
Geilenkirchen, and Dipl.-Ing. R. Feldmann for skilful technical support.
We thank ProfessorDr. C. Wandrey for his generous support.
‡
‡
‡
‡
11 was obtained on further eluation as a colorless oil. 1H NMR (300 MHz,
CDCl3, 208C): d ˆ 1.45 (s, 9H; C(CH3)3), 1.74 (ddd, J ˆ 13.4, 9.5, 5.5 Hz,
1H; H-3), 2.00 (brs, 1H; OH) overlapping with 2.08 (dd, J ˆ 13.4, 5.9 Hz,
1H; H-3), 2.40 (dd, J ˆ 15.3, 6.3 Hz, 1H of CH2COOtBu), 2.55 (dd, J ˆ 15.3,
6.8 Hz, 1H of CH2COOtBu), 3.72 (brd, J ˆ 9.9 Hz, 1H; H-5), 4.00 (dd, J ˆ
9.9, 4.5 Hz, 1H; H-5), 4.46 ± 4.53 (m, 2H; H-2 and H-4); 13C NMR
(75.5 MHz, CDCl3, 208C): d ˆ 28.3 (C(CH3)3), 41.5, 41.7 (C-3, CH2COO-
tBu), 72.7, 74.6 (C-2, C-4), 75.6 (C-5), 81.0 (C(CH3)3), 170.6 (C-1); MS
Â
[1] For recent examples see: a) G. Solladie, L. Gressot, F. Colobert, Eur. J.
Â
Org. Chem. 2000, 357 ± 364; b) G. Solladie, N. Wilb, C. Bauder, Eur. J.
Org. Chem. 1999, 3021 ± 3026; c) D. A. Evans, P. H. Carter, E. M.
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Krause, U. Schacht, E. Baader, W. Bartmann, G. Beck, A. Bergmann,
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Schüssler, K. Wagner, J. Med. Chem. 1991, 34, 2962 ± 2983; j) G. Wess,
K. Kesseler, E. Baader, W. Bartmann, G. Beck, A. Bergmann, H.
‡
‡
(70 eV, EI): m/z (%): 146 (37) [M C4H8] , 145 (13), 129 (37) [M OtBu] ,
‡
128 (29), 127 (48), 102 (27), 98 (15), 97 (10), 87 (100) [M CH2COOtBu] ,
81 (10), 69 (14), 57 (46) [C4H9] ; HRMS (EI) calcd for [M C4H8] :
‡
‡
146.0579; found: 146.0575.
Method B: Finely powdered potassium hydroxide (364 mg, 6.5 mmol) was
added to an ice-cooled solution of dihydroxy ester syn-(3R,5S)-6a (240 mg,
1.0 mmol, >99.5% ee, diastereomeric ratio syn-6a:anti-6a ˆ 205:1) in
diethyl ether (10 mL). After stirring at this temperature for one hour, the
solution was filtered through a pad of anhydrous MgSO4, and the solvent
evaporated under reduced pressure. The crude product was obtained as
4570
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0947-6539/01/0721-4570 $ 17.50+.50/0
Chem. Eur. J. 2001, 7, No. 21

Reduction of b,d-Diketo Esters
4562±4571
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Â
Ï
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Â
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Â
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Â
Â
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compound in turn by sodium borohydride reduction of commercially
available ethyl 4-chloro-3-oxo-butyrate (0.5 equiv NaBH4, EtOH,
08C, 1 h, 84%). Compounds rac-2b and rac-2c were prepared from
tert-butyl acetoacetate bisenolate and acetaldehyde or propionalde-
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Received: January 29, 2001
Revised: May 3, 2001 [F3029]
Chem. Eur. J. 2001, 7, No. 21
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0947-6539/01/0721-4571 $ 17.50+.50/0
4571