Asymmetric reduction of ethynyl ketones and ethynylketoesters by secondary alcohol dehydrogenase from Thermoanaerobacter ethanolicus

Article abstract of DOI:10.1039/b001329n

Secondary alcohol dehydrogenase (SADH) from Thermoanaerobacter ethanolicus, an NADP-dependent, thermostable oxidoreductase, reduces ethynyl ketones and ethynylketoesters enantioselectively to the corresponding propargyl (propargyl = prop-2-ynyl) alcohols. Ethynyl ketones, in general, are reduced with moderate enantioselectivity (with the exception of 4-methylpent-l-yn-3-one, which gives the (S)-alcohol with >98% ee). Although ethynyl ketones bearing a small (up to n-propyl) alkyl substituent are reduced to (S)-alcohols, larger ethynyl ketones give (R)-alcohols. In contrast, ethynylketoesters are converted to (R)-ethynylhydroxyesters of excellent optical purity. Unexpectedly, isopropyl ethynylketoesters give higher chemical yields and higher enantioselectivities of ethynylhydroxyesters than methyl or ethyl ethynylketoesters. The optically pure ethynylhydroxyesters may serve as useful chiral building blocks for asymmetric synthesis.

Full text of DOI:10.1039/b001329n

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Asymmetric reduction of ethynyl ketones and ethynylketoesters by
secondary alcohol dehydrogenase from Thermoanaerobacter
ethanolicus†
1
Christian Heiss‡a and Robert S. Phillips*abc
a Department of Chemistry, b Department of Biochemistry and Molecular Biology, and
c Center for Metalloenzyme Studies, University of Georgia, Athens, Georgia 30602-2556,
USA. Phone: (706) 542-1996; Fax: (706) 542-9454;
E-mail: rsphillips@sunchem.chem.uga.edu
Received (in Cambridge, UK) 17th February, Accepted 23rd May 2000
Published on the Web 10th July 2000
Secondary alcohol dehydrogenase (SADH) from Thermoanaerobacter ethanolicus, an NADP-dependent,
thermostable oxidoreductase, reduces ethynyl ketones and ethynylketoesters enantioselectively to the
corresponding propargyl (propargyl = prop-2-ynyl) alcohols. Ethynyl ketones, in general, are reduced with
moderate enantioselectivity (with the exception of 4-methylpent-1-yn-3-one, which gives the (S)-alcohol
with >98% ee). Although ethynyl ketones bearing a small (up to n-propyl) alkyl substituent are reduced to
(S)-alcohols, larger ethynyl ketones give (R)-alcohols. In contrast, ethynylketoesters are converted to
(R)-ethynylhydroxyesters of excellent optical purity. Unexpectedly, isopropyl ethynylketoesters give higher
chemical yields and higher enantioselectivities of ethynylhydroxyesters than methyl or ethyl ethynylketoesters.
The optically pure ethynylhydroxyesters may serve as useful chiral building blocks for asymmetric synthesis.
Due to their high chemoselectivity, their environmentally
benign character, and their increasing availability as a result of
rapid progress in genetic engineering, enzymes play an increas-
ingly important role in organic synthesis. Dehydrogenases from
various sources have been widely applied to the asymmetric
reduction of ketones to give optically active secondary alcohols.
Among these enzymes, horse liver alcohol dehydrogenase
(HLADH)1 and yeast alcohol dehydrogenase (YADH)2
have been the most extensively studied. Unfortunately, both
HLADH and YADH exhibit some disadvantages that limit
their routine usage in asymmetric synthesis. HLADH gives
good results only with cyclic ketones, and YADH reduces
ketones with usually low, unreproducible, and hardly predic-
table enantioselectivity. Furthermore, both enzymes have low
thermal stability and tend to lose their activity in the presence
of organic solvents or upon immobilization. In contrast, the
NADP-dependent alcohol dehydrogenase from Thermoanaero-
bium brockii (TBADH),3 a highly thermophilic, ethanologenic
bacterium, is stable to these conditions and reduces ketones
with excellent enantioselectivity.
We have been studying a very similar NADP-dependent
enzyme, secondary alcohol dehydrogenase (SADH) from
Thermoanaerobacter ethanolicus, which was first described by
Bryant et al.4 We found that the enantioselectivity with which it
reduces ketones depends upon temperature,5 the nature of the
employed cofactor,6 and substrate structure.7 The reduction of
small ketones like butan-2-one, but-3-yn-2-one (1 in Scheme 1),
and pentan-2-one affords alcohols of low optical purity where-
as larger ketones and ketoesters (e.g. 3) give alcohols with ee’s
ranging from 92 to 99%.
Scheme 1
Scheme 2
the active site of TBADH that proposed a large and a small
alkyl binding pocket (Scheme 2). They suggested that the
substrate binds in an orientation determined by the best fit
of the two groups attached to the carbonyl into these pockets.
The nicotinamide cofactor then transfers its pro-R hydride to
the Re face of the ketone, producing secondary alcohols with
(S)-configuration (assuming that the larger substituent has
higher Cahn–Ingold–Prelog priority), i.e., the enzyme follows
Prelog’s rule.8
Based on the high degree of sequence identity (~99%) of
TBADH and SADH,9 Keinan’s active site model can also be
applied to SADH. On this basis, the high enantioselectivity
with which SADH reduces the various ketoesters together with
the low ee in the case of but-3-yn-2-one led us to believe that if
the triple bond in the ketone is “counterbalanced” with an ester
function on the opposite side of the carbonyl as in 5 (Scheme
3), it could be forced into the small pocket giving rise to
(R)-hydroxyalkynoates (6) in high optical purity. These repre-
This behavior of SADH is similar to results obtained from
reductions with TBADH. Keinan et al.3 put forth a model of
† Experimental details for the preparation of compounds 7a–k are
available as supplementary data. For direct electronic access see http://
www.rsc.org/suppdata/p1/b0/b001329n/
‡ Present address: McNeil Specialty Products Company, Athens,
Georgia.
DOI: 10.1039/b001329n
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This journal is © The Royal Society of Chemistry 2000
2821

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the same procedure11 was employed in the synthesis of the
methyl and ethyl esters, 5a,b,d, and f.
The isopropyl esters, 5c and e, had to be made in a differ-
ent manner (Scheme 5) because the acylation was accom-
Scheme 5
panied by formal ester hydrolysis (presumably by an A1
type mechanism). The resulting acids, 14a and b, however,
could easily be re-esterified with propan-2-ol yielding the
TMS-protected ethynylketoesters, 15a and b, and since they
are readily prepared from succinic or glutaric anhydride (13a
and b),12 respectively, this offered a convenient route to the
isopropyl ethynylketoesters.
Scheme 3
sent potentially useful chiral building blocks for asymmetric
syntheses, since they possess three functional groups that can
undergo further transformation independently of each other.
In order to obtain a more detailed model and greater predic-
tability of the scope and stereochemistry of SADH-reductions,
we also subjected simple alkyl-substituted ethynyl ketones (7) to
reduction by the enzyme. We were especially interested in the
minimum size of the alkyl substituent required to give pre-
dominantly (R)-alcohols (8), as well as how branching of the
alkyl group would affect the reaction.
Enzyme reactions
The enzyme reductions were carried out in aqueous buffer con-
taining 15% propan-2-ol, which serves as both co-solvent and
sacrificial substrate for coenzyme regeneration. We found that
ethynyl ketones require higher concentrations of SADH for
reaction than unconjugated ketones because they decompose
slowly (t1/2 ~ 1 day) under the reaction conditions, and, more
significantly, they cause irreversible inactivation of the enzyme.
We ran the reduction of isopropyl 4-oxohex-5-ynoate (5c) and
4-methylpent-1-yn-3-one (7c) with varying amounts of enzyme
and found that the minimum concentration of SADH to give
reasonable chemical conversions (>90%) is 54 U cmϪ3 for 5c
and less than 10 U mlϪ1 for 7c (compared to 2 U cmϪ3 for
unconjugated ketones7). The enzyme inactivation is possibly
caused by conjugate addition of a nucleophilic residue in the
active site of the enzyme to the triple bond of the substrate,
which is in conjugation with the carbonyl. Product inhibition
can be excluded as an explanation because the inactivation
takes place even if no measurable product is formed.
Results and discussion
Synthesis of ethynyl ketones and ketoesters
The ethynyl ketones (7a–k) were prepared by a modification of
the method by Walton and Waugh10 (Scheme 4). Bis(trimethyl-
Fortunately, the need for large amounts of enzyme poses no
major problem since Burdette et al. recently cloned SADH and
prepared a plasmid to overexpress it in E. coli.9 This new source
offers major advantages over cells of T. ethanolicus: E. coli does
not require high temperature anaerobic growth conditions and
can therefore be grown easily in large quantities; SADH is
produced in larger amounts in the recombinant cells than in
T. ethanolicus; and, due to the thermal stability of SADH, its
purification is greatly simplified because the E. coli proteins can
be selectively precipitated by heat treatment of the cell-free
extract, resulting in sufficiently pure enzyme for the reductions.
The reductions of the ethynyl ketones (7a–k) are summarized
in Table 1. Initially, increasing the size of the alkyl substituent
from methyl to ethyl to isopropyl (compounds 7a–c) leads to
higher amounts of the (S)-enantiomer, with the latter being the
only observed product when the alkyl group is isopropyl. Simi-
lar behavior was observed in TBADH-reductions of dialkyl
ketones by Keinan for TBADH,3 and was explained by assum-
ing that the small pocket has higher affinity for hydrophobic
groups. The observations that 7c is reduced with an ee >98%
and that 7i does not react indicate that the geometry of the
Scheme 4
silyl)acetylene (BTMSA) was acylated with the appropriate acyl
chloride (9) in the presence of AlCl3 to give the TMS-protected
ethynyl ketones (10), followed by phase-transfer catalyzed
removal of the TMS group with NaF and NBu4Cl. Essentially
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Table 1 SADH reduction of ethynyl ketones
yield, and the short reaction time in the case of 5c, cannot be
explained by simply invoking the size of the substituents.
Apparently, the ester group plays a significant role in substrate
binding to the active site. This assumption is supported by a
revised model of the active site of SADH published by Burdette
and Zeikus,13 which is based on evidence for acetyl-coenzyme
A being the physiological substrate for SADH. In this model,
the large alkyl binding pocket is replaced by an open-ended
channel that accommodates part of the pantotheine moiety of
acetyl-CoA. This part of acetyl-CoA possesses an amide func-
tion in the approximate position of the ester group in methyl
6-oxooct-7-ynoate. The notable influence of the isopropyl ester
on the enantioselectivity may be due to favorable hydrophobic
interaction with part of the binding channel of the enzyme.
We observed that 7c, which is reduced to (S)-8c, inactivates
the enzyme much more slowly than 5c, which yields (R)-6c. This
suggests that the putative nucleophilic residue responsible for
the inactivation by ethynyl ketones may be located in the small
alkyl binding pocket. However, we have found that mutation of
Cys295, a potential nucleophile in the small pocket, to alanine
does not prevent inactivation of SADH by ethynyl ketones.14
There is at least one hydroxyalkynoate that is obtained
in high yield and excellent optical purity for each length of
the tether between the carbonyl and ester groups (n = 2, 3, 4).
Therefore, by choosing the appropriate ester, potentially useful
chiral building blocks can be readily prepared with this enzym-
atic reduction methodology. The nature of the alkyl portion of
the ester group should have only minor influence on further
transformations. The achiral starting materials are obtained in
two (7a–k and 5a,b,d,f) or three (5c,e) steps from commercially
available compounds.
Yield
(%)
Abs.
conf.
Reaction
time/h
Compound
Ee (%)
7aa
7b
7c
7d
7e
7f
30
32
50
—
28
20
—
32
—
55
S
S
S
S
60
80
0.5
1
2
24
1
1
7
4
—
5
>98
85b
51
S
R
R
R
—
R
50
7g
7h
7i
66b
42
—
80
7k
a From ref. 7. b No product was isolated but the absolute configuration
and ee were determined by GC.
Table 2 SADH reduction of ethynylketoesters
Yield
(%)
Abs.
conf.
Reaction
time/h
Compound
Ee (%)
5a
5b
5c
5d
5e
5f
35
51
88
76
76
68
R
R
R
R
R
R
82
90
>98
97
>98
>98
2.5
1.5
1.0
1.5
1.5
2.5
α-carbon partly determines the binding orientation. It seems
likely that the α-substitution of the alkyl group leads to
unfavorable interaction with a putative steric restriction in the
large pocket close to the catalytic site. With its linear geometry,
the sp-carbon of the ethynyl group is less likely to be subject to
this kind of steric hindrance. Starting with 7e, substrates substi-
tuted with longer alkyl groups give increasingly higher amounts
of the (R)-enantiomer, as is to be expected from Keinan’s
model.
Whereas secondary carbons are tolerated well in substi-
tuents (compounds 7c, f, and k), tertiary carbons in the sub-
strates result in very slow reaction rates and marginal yields
(compounds 7d and g), the ketones decomposing before signifi-
cant conversion has taken place. The moderate yields in the
other cases can be ascribed to losses due to evaporation and to
some decomposition of the substrates under the reaction
conditions.
Table 2 lists the results of the SADH-reduction of the
ethynylketoesters (5a–f). The yields are higher the faster the
reactions take place, in accordance with both the slow decom-
position of the substrates and the competition of substrate
reduction with enzyme inactivation. As expected, the enantio-
selectivity of the reduction depends on the number of
methylenes between the carbonyl and the ester moiety as well as
on the size of the latter. Due to the high enzyme concentration
employed, the initial rates of reduction were too high to be
measured accurately. However, the time required for complete
conversion gives some indication of the relative reaction rates.
In contrast to earlier results involving the reduction of aliphatic
methyl ketones, where each additional methylene decreased the
reaction rate by a factor of 3,7 and also to the observations
made with the simple ethynyl ketones (7a–k), increasing the
size of the ester group results in faster reaction rates in
the 4-oxohex-5-ynoate series (5a–c) and has no effect on the
rates in the 5-oxohept-6-ynoate series (5d and e). Although
methyl 5-oxohept-6-ynoate was not examined, it would be
expected to show similar ee values to the ethyl and isopropyl
esters (5d and e). Elongation of the tether also seems to have no
marked effect on the rate. The higher enantioselectivities with
which the ethynylketoesters are reduced compared to the simple
ethynyl ketones and especially the high enantioselectivity, high
Experimental
General
UV-Vis spectra and enzyme assays were recorded on a Varian
Cary 1E spectrophotometer equipped with a thermoelectric
6 × 6 cell cuvette changer. Capillary GC was performed on a
Varian 3300 gas chromatograph with FI detection (Supelco
β-Dex 120 chiral column, 30 m × 0.250 mm id, 0.25 µm film
thickness) programmed between 40 and 175 ЊC. 1H and 13C
NMR spectra were taken on a Bruker AC-250 or AC-300 spec-
trometer, respectively, using the residual CHCl3 signal (δ 7.26
ppm) as internal reference. Optical rotations were measured on
a Rudolph Autopol IV polarimeter. IR spectra were obtained
with NaCl plates on a Perkin-Elmer Paragon 1000PC FT-IR
spectrometer. Pet. ether refers to the fraction boiling in the
range 35–60 ЊC.
Enzyme assay
SADH was assayed following the increase in absorbance at 340
nm due to the formation of NADPH (∆ε340 = 6.22 mMϪ1 cmϪ1
)
at 50 ЊC. The assay mixture contained 200 mM PriOH and 1.25
mM NADP in 50 mM Tris, pH 8.0. One unit (U) is the amount
of enzyme required to reduce 1 µmol of NADP per minute.
Purification of SADH
E. coli cells expressing SADH were grown as described.9 The
wet cells (150 g) were suspended in 50 mM tris, pH 8.0, (375
cm3) containing 0.1 mM dithiothreitol, sonicated, and centri-
fuged (30 min at 10000 × g, all of the following centrifugations
were done under these conditions). The supernatant was heated
in a capped bottle at 70 ЊC for 1 h, 100 cm3 buffer was added
and the thick suspension again centrifuged. (NH4)2SO4 (48.0 g,
20% saturation) was added to the supernatant in portions, and
the mixture was stirred for 1 h at room temperature. After
removal of the precipitate by centrifugation, an additional 110
g (NH4)2SO4 (60% saturation) were added and stirring was
J. Chem. Soc., Perkin Trans. 1, 2000, 2821–2825
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continued for 90 min. The mixture was centrifuged, the pellet
was dissolved in Tris (50 cm3), and the resulting solution was
dialyzed against 50 mM Tris, pH 8.0, (3 × 4 dm3). The solution
was then heated at 70 ЊC for 1 h, centrifuged, and lyophilized
yielding 2.12 g of SADH as a tan powder with the specific
activity of 27 U mgϪ1 solid.
(R)-Hept-1-yn-3-ol (8h). Yield 30.3 mg (27%), 42% ee, Rf 0.19
(10% ether–pet. ether), [α]D20 ϩ3.8 (c = 5.8, CHCl3) (lit.16 [α]D20
1
Ϫ5.5 (c = 0.9, CHCl3), 70% ee for (S)-enantiomer), H NMR
(250 MHz, CDCl3) δ 4.37 (dt, J = 7.3 Hz, 2.1 Hz, 1H), 2.47 (d,
J = 2.1 Hz, 1H), 1.87 (br s, 1H), 1.72 (m, 2H), 1.41 (m, 4H), 0.92
(t, J = 7.0 Hz, 3H).
Determination of minimum enzyme concentration required for
complete conversion
(R)-6-Methylhept-1-yn-3-ol (8k). Yield 61.8 mg (49%), 80%
ee, Rf 0.21 (10% ether–pet. ether), [α]D20 ϩ9.2 (c = 1.2, CHCl3),
ϩ13.8 (c = 2.0, dioxane), H NMR (300 MHz, CDCl3) δ 4.35
(dt, J = 6.6 Hz, 1.9 Hz, 1H), 2.47 (d, J = 1.9 Hz, 1H), 2.03 (br s,
1H), 1.71 (m, 2H), 1.57 (m, 1H), 1.33 (m, 2H), 0.89 (d, J = 6.5
Hz, 6H).
1
SADH (2.0, 13.5, 27, 54, 81, 108 U) and NADP (0.1 mg, 0.12
µmol) were dissolved in 50 mM Tris, pH 8.0, (0.85 cm3). After
pre-incubating the solution for 10 min at 50 ЊC, a solution of 5c
(16.8 mg, 0.10 mmol) or 7c (9.6 mg, 0.10 mmol) in PriOH (0.15
cm3) was added in one portion. The reactions were monitored
by extracting an aliquot (40 mm3) with CH2Cl2 (40 mm3)
followed by GC analysis (40–125 ЊC, 2 ЊC minϪ1 for 7c, 40–
175 ЊC, 2 ЊC minϪ1 for 5c). The presence or absence of enzyme
activity was assessed by incubating an aliquot (0.2 cm3) with
pentan-2-one (1.7 mg, 0.02 mmol) for 10 min and checking for
the presence of pentan-2-ol by GC.
(R)-Methyl 4-hydroxyhex-5-ynoate (6a). Yield 49.8 mg (35%),
82% ee, Rf 0.20 (20% ethyl acetate–hexanes), [α]D20 ϩ9.9 (c = 1.8,
CHCl3), 1H NMR (250 MHz, CDCl3) δ 4.49 (dt, J = 6.0 Hz, 2.0
Hz, 1H), 3.69 (s, 3H), 2.55 (m, 2H), 2.49 (d, J = 2.1 Hz, 1H),
2.21–1.98 (m, 3H).
(R)-Ethyl 4-hydroxyhex-5-ynoate (6b). Yield 79.6 mg (51%),
90% ee, Rf 0.18 (15% ethyl acetate–hexanes), [α]D20 ϩ17.9 (c = 2.0,
Enzymatic reduction of ethynyl ketones and ethynylketoesters
with SADH
1
CHCl3), IR (neat) νmax 3443, 3291, 2112, 1732 cmϪ1; H NMR
(300 MHz, CDCl3) δ 4.49 (dt, J = 6.1 Hz, 2.1 Hz, 1H), 4.14 (q,
J = 7.1 Hz, 2H), 2.52 (m, 2H), 2.48 (d, J = 1.7 Hz, 1H), 2.21 (br
s, 1H), 2.04 (m, 2H), 1.26 (t, J = 7.1 Hz, 3H); 13C NMR (75
MHz, CDCl3) δ 173.9, 97.6, 84.0, 73.4, 61.3, 60.7, 32.1, 29.8,
14.1.
SADH (1000 U) and NADP (1 mg, 1.2 µmol) were dissolved in
50 mM Tris, pH 8.0, (8.5 cm3). After pre-incubation for 10 min
at 50 ЊC, the appropriate ketone (5 or 7) (1.0 mmol) was added
in one portion and the mixture was kept at 50 ЊC. When com-
plete conversion was obtained (GC), the reaction mixture was
saturated with NaCl and extracted with Et2O (3 × 4 cm3). Due
to their tendency to form emulsions, the extractions had to be
centrifuged (10 min at 4000 rpm) prior to separation. The com-
bined extracts were dried with Na2SO4, the solvent was removed
in vacuo, and the residue chromatographed.
(R)-Isopropyl 4-hydroxyhex-5-ynoate (6c). Yield 150 mg
(88%), >98% ee, Rf 0.21 (15% ethyl acetate–hexanes), [α]D20 ϩ13.1
(c = 2.0, CHCl3), IR (neat) νmax 3442, 3293, 2114, 1731 cmϪ1; 1H
NMR (250 MHz, CDCl3) δ 5.02 (m, 1H), 4.49 (dt, J = 6.1 Hz,
2.0 Hz, 1H), 2.51 (m, 3H), 2.03 (m, 2H), 1.24 (d, J = 6.4 Hz,
6H); 13C NMR (75 MHz, CDCl3) δ 173.2, 84.0, 73.3, 68.1, 61.2,
32.2, 30.2, 21.7.
Determination of optical purity of the alcohols
The alcohol (0.02 mmol) was dissolved in CDCl3 (0.3 cm3) in an
NMR tube and a 50 mM solution of Eu(hfc)3 in CDCl3 (0.5
(R)-Ethyl 5-hydroxyhept-6-ynoate (6d). Yield 129 mg (76%),
97% ee, Rf 0.22 (20% ethyl acetate–hexanes), [α]D20 ϩ16.9
(c = 1.66, CCl4), ϩ7.8 (c = 3.8, CHCl3) (lit.17 [α]D20 Ϫ16.4
1
cm3) was added in portions of 0.1 cm3. A H NMR spectrum
was taken after each addition, and the two signals correspond-
ing to the diastereotopic methanol protons were integrated. If
only one signal was visible, the enantiomeric excess was
assumed to be greater than 98%.
1
(c = 3.30, CCl4), 96% ee for (S)-enantiomer); H NMR (300
MHz, CDCl3) δ 4.38 (dt, J = 6.2 Hz, 1.8 Hz, 1H), 4.12
(q, J = 7.2 Hz, 2H), 2.46 (d, J = 2.2 Hz, 1H), 2.35 (t, J = 7.0
Hz, 2H), 2.24 (br s, 1H), 1.78 (m, 4H), 1.24 (t, J = 7.1 Hz,
3H).
Spectral data of isolated products
(S)-Pent-1-yn-3-ol (8b). Yield 30.3 mg (36%), 80% ee, Rf 0.20
(10% ether–pet. ether), [α]D20 Ϫ19.2 (c = 12, dioxane) (lit.15 [α]D25
ϩ23.15 (c = 2, dioxane), 86% ee for (R)-enantiomer), 1H NMR
(300 MHz, CDCl3) δ 4.32 (dt, J = 6.5 Hz, 2.0 Hz, 1H), 2.46 (d,
J = 1.8 Hz, 1H), 1.74 (m, 3H), 1.02 (t, J = 7.4 Hz, 3H).
(R)-Isopropyl 5-hydroxyhept-6-ynoate (6e). Yield 139 mg
(76%), >98% ee, Rf 0.24 (20% ethyl acetate–hexanes), [α]D20 ϩ10.3
(c = 2.6, CHCl3), IR (neat) νmax 3433, 3291, 2112, 1727 cmϪ1; 1H
NMR (300 MHz, CDCl3) δ 5.01 (m, 1H), 4.40 (dt, J = 6.0 Hz,
2.6 Hz, 1H), 2.47 (d, J = 2.6 Hz, 1H), 2.33 (t, J = 6.9 Hz, 2H),
2.03 (br s, 1H), 1.78 (m, 4H), 1.23 (d, J = 6.2 Hz, 6H); 13C NMR
(75 MHz, CDCl3) δ 173.0, 84.5, 73.0, 67.7, 61.7, 36.7, 34.0, 21.8,
20.4.
(S)-4-Methylpent-1-yn-3-ol (8c). Yield 49.1 mg (50%), >98%
ee, Rf 0.22 (10% ether–pet. ether), [α]D20 Ϫ15.7 (c = 13, dioxane)
(lit.15 [α]D20 ϩ13.8 (c = 2, dioxane), 86% ee for (R)-enantiomer),
1H NMR (300 MHz, CDCl3) δ 4.17 (dd, J = 5.7 Hz, 2.0 Hz,
1H), 2.45 (d, J = 2.3 Hz, 1H), 1.89 (m, 2H), 1.01 (m, 6H).
(R)-Methyl 6-hydroxyoct-7-ynoate (6f). Yield 115 mg (68%),
>98% ee, Rf 0.22 (20% ethyl acetate–hexanes), [α]D20 ϩ8.8 (c = 2.2,
(S)-Hex-1-yn-3-ol (8e). Yield 27.5 mg (28%), 51% ee, Rf 0.22
(10% ether–pet. ether), [α]D20 Ϫ4.5 (c = 1.5, CHCl3) (lit.15 [α]D20
CHCl3), IR (neat) νmax 3434, 3289, 2111, 1734 cmϪ1 1H
;
NMR (300 MHz, CDCl3) δ 4.36 (dt, J = 6.5 Hz, 2.1 Hz, 1H),
3.65 (s, 3H), 2.45 (d, J = 2.5 Hz, 1H), 2.32 (t, J = 7.4 Hz, 2H),
2.22 (br s, 1H), 1.79–1.60 (m, 4H), 1.50 (m, 2H); 13C NMR
(75 MHz, CDCl3) δ 174.1, 84.8, 73.0, 62.0, 51.6, 37.1, 33.9,
24.5 (2×).
1
ϩ9.0 (c = 1.0, CHCl3), 75% ee for (R)-enantiomer), H NMR
(250 MHz, CDCl3) δ 4.39 (dt, J = 6.5 Hz, 2.2 Hz, 1H), 2.47 (d,
J = 2.1 Hz, 1H), 1.71 (m, 3H), 1.50 (m, 2H), 0.97 (t, J = 7.0 Hz,
3H).
(R)-5-Methylhex-1-yn-3-ol (8f). Yield 22.4 mg (20%), 50% ee,
Rf 0.20 (10% ether–pet. ether), [α]D20 ϩ18.3 (c = 1.2, dioxane)
(lit.15 [α]D25 ϩ28.8 (c = 3, dioxane), 88% ee for (R)-enantiomer),
1H NMR (300 MHz, CDCl3) δ 4.42 (dt, J = 7.4 Hz, 2.3 Hz, 1H),
2.46 (d, J = 2.1 Hz, 1H), 1.87 (m, 1H), 1.60 (m, 3H), 0.95 (d,
J = 6.7 Hz, 3H), 0.93 (d, J = 6.8 Hz, 3 H).
Acknowledgements
We thank Professor J. Gregory Zeikus and Dr Douglas S.
Burdette for generously providing the microorganisms express-
ing SADH.
2824
J. Chem. Soc., Perkin Trans. 1, 2000, 2821–2825

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