Regioselective enzymatic syntheses of C-3 and C-5 carbonate A-ring stereoisomeric precursors of vitamin D

Article abstract of DOI:10.1016/j.tetasy.2004.07.052

The synthesis of selectively modified A-ring precursors for the preparation of 1α,25-dihydroxyvitamin D₃ analogues by enzymatic hydrolysis reaction of corresponding dicarbonates has been accomplished. Thus, Candida rugosa lipase (CRL) was found to hydrolyze with high selectivity the C-3 carbonate of stereoisomers 4a,b, and 4d, furnishing C-5 vinyloxycarbonates 5a,b, and 5d. On the other hand, Chromobacterium viscosum lipase exhibit opposite regioselectivity with cis enantiomers 4c and 4d, catalyzing hydrolysis at the C-5 carbonate for 4c and at C-3 position for 4d. In addition, CRL catalyzes the alkoxycarbonylation reaction at C-3 of diol 3d affording the monocarbonate complementary to the one obtained by the enzymatic hydrolysis process.

Full text of DOI:10.1016/j.tetasy.2004.07.052

Tetrahedron:
Asymmetry
Tetrahedron: Asymmetry 15 (2004) 2881–2887
Regioselective enzymatic syntheses of C-3 and C-5 carbonate
A-ring stereoisomeric precursors of vitamin D
´
´
Daniel Oves, Vicente Gotor-Fernandez, Susana Fernandez, Miguel Ferrero and
Vicente Gotor*
´ ´ ´ ´
Departamento de Quımica Organica e Inorganica, Facultad de Quımica, Universidad de Oviedo, 33071 Oviedo, Spain
Received 1 June 2004; accepted 1 July 2004
Available online 11 September 2004
Abstract—The synthesis of selectively modified A-ring precursors for the preparation of 1a,25-dihydroxyvitamin D3 analogues by
enzymatic hydrolysis reaction of corresponding dicarbonates has been accomplished. Thus, Candida rugosa lipase (CRL) was found
to hydrolyze with high selectivity the C-3 carbonate of stereoisomers 4a,b, and 4d, furnishing C-5 vinyloxycarbonates 5a,b, and 5d.
On the other hand, Chromobacterium viscosum lipase exhibit opposite regioselectivity with cis enantiomers 4c and 4d, catalyzing
hydrolysis at the C-5 carbonate for 4c and at C-3 position for 4d. In addition, CRL catalyzes the alkoxycarbonylation reaction
at C-3 of diol 3d affording the monocarbonate complementary to the one obtained by the enzymatic hydrolysis process.
Ó 2004 Elsevier Ltd. All rights reserved.
1.Introduction
25
To perform regio- and stereoselective transformations,
R
enzymes have become standard catalysts in organic syn-
thesis.1 Lipases in particular have received most atten-
tion because they are one of the most versatile classes
of biocatalysts acting with high efficiency in acylation
or hydrolysis as well as alkoxycarbonylation processes.2
In general, these catalysts are inexpensive and in many
cases able to adapt to a wide range of substrate struc-
tures. Moreover, lipases are ecologically beneficial
natural catalysts. Over the last few years, a bio-
transformation has sometimes proven to be the key
step in the synthesis of biologically active natural prod-
ucts and their analogues.2,3 Just as with enantioselectiv-
ity, the ability of the enzymes to catalyze the
regioselective modification of several functional groups
is also of great interest for organic chemists. Esterifica-
tion and hydrolysis reactions have been commonly used
whereas the alkoxycarbonylation process has scarcely
been investigated.
5
3
HO
OH
H
R
3a-d
Compound
Positions
1
3
S
R
S
R
HO
5
a
b
c
d
R
S
S
R
1, R = OH 1α,25-(OH)2-D3
2, R =H
Vitamin D3
Chart 1.
on normal and malignant cell types.4 Since 1a,25-
(OH)2-D3 itself is of limited therapeutic value due to
its hypercalcemic effects, interest has been focused on
the development of analogues having strong cell differ-
entiating and weak calcemic effects. The therapeutical
usefulness of 1a,25-(OH)2-D3 has led to the synthesis
of new vitamin D analogues.5 Most of the analogues
are altered in the side chain, although modifications in
the A-ring, less accessible synthetically, provide vitamin
D derivatives with unique biological profiles.6
1a,25-Dihydroxyvitamin D3 [1a,25-(OH)2-D3, 1, Chart
1], the hormonally active form of vitamin D3 2, is a
key calcium-regulating hormone but also displays
potent prodifferentiating and antiproliferative activities
*
In our ongoing research related to the synthesis of selec-
tively modified vitamin D3 analogues,7 appropriate
Corresponding author. Tel./fax: +34 98 510 3448; e-mail: vgs@fq.
uniovi.es
0957-4166/$ - see front matter Ó 2004Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetasy.2004.07.052

2882
D. Oves et al. / Tetrahedron: Asymmetry 15 (2004) 2881–2887
routes for the preparation of precursors are required.
Thus, we have described the selective enzymatic acyl-
ation of diols8 and hydrolysis9 of corresponding
diacylated derivatives of 1a,25-(OH)2-D3 and 1a,25-
(OH)2-19-nor-pre-D3 A-ring stereoisomeric synthons.
We have demonstrated the complementary regioselec-
tive behavior showed by enzymes in acylation and
hydrolysis processes to prepare the monoacylated deriv-
atives of the A-ring. Moreover, we have reported the en-
zyme-catalyzed regioselective alkoxycarbonylation
processes in organic solvents of the A-ring precursors
to obtain carbonate derivatives,10 very interesting inter-
mediates, which provide us the access to new A-ring syn-
thons of vitamin D analogues. Thus, Candida antarctica
lipase B (CAL-B) mainly allows the hydroxyl group at
C-5 of 3a–d to react giving place to C-5 carbonates.
As shown in entry 1 of Table 1, Chromobacterium vis-
cosum lipase (CVL) give rise to an approximately 1:1
mixture of C-3 and C-5 alkoxycarbonylated products,
indicating poor recognition. Similar behavior was ob-
served with Pseudomonas cepacia lipase (PSL-C) (Table
1, entry 4). In the case of CAL-B, no formation of the C-
5 carbonate derivative 5a was observed. However, sig-
nificant amounts of the diol 3a were isolated in addition
to the C-3 carbonate 6a (Table 1, entries 2 and 3). C.
antarctica lipase A (CAL-A), Chirazyme L2 (a lipase
from C. antarctica B immobilized in a different support),
and porcine pancreas lipase (PPL) did not show good
selectivities (Table 1, entries 5–8). Nevertheless, just
Chirazyme L2 was the biocatalyst that showed moderate
selectivity toward the C-5 position, obtaining carbonate
6a with 47% isolated yield (Table 1, entry 6). Interest-
ingly, excellent selectivity was found with Candida rugo-
sa lipase (CRL) as catalyst. This enzyme catalyzes the
hydrolysis at the C-3 carbonate group affording exclu-
sively the C-5 vinyloxycarbonyl derivative 5a (Table 1,
entries 9 and 10). However, low conversions were
achieved using a 58% buffer/dioxane even after longer
reaction times and using more amount of the biocata-
lyst. The conversion was further improved by increasing
buffer concentration up to 67%. Thus, after 90h, 96%
conversion was raised although selectivity decrease
slightly, 78% of 5a (62% isolated yield) being obtained
in addition to the C-3 carbonate derivative 6a and diol
3a (Table 1, entry 11). Higher concentrations of buffer
are not appropriate for this process due to the lack of
selectivity (Table 1, entry 12). We also tested an esterase
as catalyst (pig liver esterase, PLE), but not selectivity
was observed giving place to diol 3a as unique reaction
product (Table 1, entry 13).
Herein we report the enzyme mediated regioselective
hydrolysis of A-ring carbonate synthons as useful pre-
cursors for the synthesis of vitamin D analogues. This
study will allow us to have in hand all possible C-3
and C-5 monocarbonate A-ring stereosiomeric precur-
sors from diols 3a–d. Recently, the enzymatic resolution
of a carbonate intermediate has been disclosed as a use-
ful procedure for the preparation of (S)-(+)-Zoplicone.11
Also, Matsumoto has described the enantioselective
hydrolysis of various cyclic carbonates to obtain opti-
cally active 1,2-12 and 1,3-diol13 derivatives.
2.Results and discussion
In order to prepare carbonates as intermediates for the
introduction of other functionalities, it is desirable that
the substrate has a good leaving group. For this reason,
we chose vinyl carbonates 4a–d as starting materials.
For that, diols 3a–d were treated with vinyl chlorofor-
mate in the presence of pyridine and methylene chloride
as solvent.
The results of the enzymatic hydrolysis of dicarbonate
4b are summarized in Table 2. Among the enzymes
screened only CRL displayed high selectivity toward
the hydrolysis of the carbonate at the C-3 position
(Scheme 2). Under the conditions of 30°C and 58% buf-
fer/dioxane during 24h, the reaction proceeded to afford
carbonate 5b with total regioselectivity and 95% of
conversion (Table 2, entry 9). Longer reaction times
increased conversion up to 98%. In both cases,
compound 5b was isolated with >90% yield by flash
chromatography (Table 2, entries 9 and 10). All other
enzymes furnished exclusively diol 3b.
We first studied the enzymatic hydrolysis of dicarbonate
4a with several commercial lipases and esterases
(Scheme 1). Initially, the reactions were performed at
30°C in 0.1M phosphate buffer (pH7) using 1,4-dioxane
as co-solvent.
When the hydrolysis was performed on the cis-dicarbo-
nate 4c (Scheme 3 and Table 3), the preliminary screen-
ing with the eight enzymes was unsatisfactory. Changing
the reaction conditions did not have any significant
influence on the regioselectivity of the enzyme except
when CVL was the biocatalyst. The processes when car-
ried out with 83% buffer/dioxane, both at 30 or 10°C,
gave place to the total hydrolysis of the dicarbonate 4c
(Table 3, entries 1 and 2). However, a drastic reduction
of the amount of buffer (5%) led to a completely selec-
tive process. This lipase catalyzes the carbonate hydro-
lysis on the C-5 position in compound 4c with excellent
regioselectivity (Table 3, entry 3), yielding derivative 6c
(93% isolated yield). It is noteworthy that through a
hydrolysis reaction, CVL allows the synthesis of the
Hydrolase
O
O
buffer/1,4-dioxane
30oC
O
O
O
O
4a
+
+
HO
OH
RO
OH
HO
OR
6a
5a
3a
R = CO2CH=CH2
Scheme 1.

D. Oves et al. / Tetrahedron: Asymmetry 15 (2004) 2881–2887
Table 1. Enzymatic hydrolysis of A-ring synthon (3S,5R)–4aa
2883
Entry
Enzyme
Buffer (%)b
t (h)
Conv. (%)c
3a (%)c
5a (%)c
6a (%)c
1
CVLd
18
41
112
114
93
42
45
20
22
16
2
CAL-Be
29
3
4PSL-C
CAL-Be
83
58
6430
34
f
1463
48
28
35
5
CAL-Ag
Chirazyme L2h
Chirazyme L2h
PPLi
83
54
8
13
10
27
59
14
6
34216
58
41
77
7
37
114
48
98
15
15
46
96
31
11
15
52
4
8
9
CRLj
58
58
15
46
78
10
11
12
13
CRLk
216
90
CRLk
67
76
12
78
6
CRLk
2469
25
7
6
4
16
PLEl
83
78
a The reactions were carried out at 30°C, 250rpm, in 0.04M concentration with 17mg of 4a.
b Percentage of buffer solution (0.1M KH2PO4/KOH, pH7.0) in 1,4-dioxane; in all cases, total reaction volume was 1.5mL.
c Based on 1H NMR signal integration ( 3% error).
d Ratio 5.7:1 of 4a:CVL (w/w).
e Ratio 1:5.3 of 4a:CAL-B (w/w).
f Ratio 1:5.3 of 4a:PSL-C (w/w).
g Ratio 1:2.6 of 4a:CAL-A (w/w).
h Ratio 1:5.3 of 4a:Chirazyme L2 (w/w).
i Ratio 1:3.5 of 4a:PPL (w/w).
j Ratio 1:3.5 of 4a:CRL (w/w).
k Ratio 1:4.4 of 4a:CRL (w/w).
l Ratio 1:1.2 of 4a:PLE (w/w).
Table 2. Enzymatic hydrolysis of A-ring synthon (3R,5S)–4ba
Entry
Enzyme
Buffer (%)b
T (°C)
t (h)
Conv. (%)c
3b (%)c
5b (%)c
1
CVLd
CVLd
58
83
83
83
83
83
83
83
58
58
83
83
30
30
10
30
30
30
30
30
30
30
30
20
96
20
98
6
20
2
2498
246
48
3
4CAL-B
CAL-Be
e
77
22
46
28
53
77
22
46
5
PSL-Cf
CAL-Ag
Chirazyme L2h
PPLi
48
48
6
7
2428
42
8
53
95
9
CRLj
2495
48
10
11
12
CRLj
98
100
35
98
CRLj
48
3
100
35
PLEk
a The reactions were carried out at 250rpm in 0.04M concentration with 17mg of 4b.
b Percentage of buffer solution (0.1M KH2PO4/KOH, pH7.0) in 1,4-dioxane; in all cases, total reaction volume was 1.5mL.
c Based on 1H NMR signal integration ( 3% error).
d Ratio 5.7:1 of 4b:CVL (w/w).
e Ratio 1:5.3 of 4b:CAL-B (w/w).
f Ratio 1:5.3 of 4b:PSL-C (w/w).
g Ratio 1:2.6 of 4b:CAL-A (w/w).
h Ratio 1:5.3 of 4b:Chirazyme L2 (w/w).
i Ratio 1:3.5 of 4b:PPL (w/w).
j Ratio 1:3.5 of 4b:CRL (w/w).
k Ratio 1:1.2 of 4b:PLE (w/w).
A-ring carbonate precursor of 1a,25-(OH)2-D3, which is
complementary to that obtained with CAL-B by direct
alkoxycarbonylation reaction.
the opposite selectivity observed with CVL for both
enantiomers: that is, (3S,5S)-4c showed preference for
hydrolysis of the C-5 carbonate group meanwhile
(3R,5R)-4d was hydrolyzed at C-3. Similarly, CRL cat-
alyzes the synthesis of the C-5 monocarbonate deriva-
tive 5d (71% isolated yield) from dicarbonate 4d with
high regioselectivity using 67% buffer/dioxane at 30°C
(Table 4, entry 10). Other enzymes such as CAL-B,
PSL-C, CAL-A, Chirazyme L2, PPL, and PLE exhib-
ited lower selectivities than CVL and CRL. This process
As in the previous cis-dicarbonate 4c, CVL exhibits an
excellent selectivity in the hydrolysis reaction of its
enantiomer 4d (Scheme 4). Best results were obtained
at 20°C and 14% buffer/dioxane, with the monocarbo-
nate 5d being obtained as the major product after 15h
(81% isolated yield) (Table 4, entry 2). It is worth noting

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D. Oves et al. / Tetrahedron: Asymmetry 15 (2004) 2881–2887
Hydrolase
O
O
O
O
Hydrolase
buffer/1,4-dioxane
buffer/1,4-dioxane
O
O
O
O
O
O
O
O
4b
4d
+
+
+
HO
OH
RO
OH
HO
OR
RO
OH
HO
OH
5b
3b
3d
5d
6d
R = CO2CH=CH2
R = CO2CH=CH2
Scheme 2.
Scheme 4.
In view of the selectivity displayed by CRL with isomers
4a,b, and 4d and taking into account one of our aims to
prepare the C-3 monocarbonate A-ring precursors, we
tested this lipase in the alkoxycarbonylation reaction
of diols 3a,b, and 3d with acetone O-[(vinyloxy)carbon-
yl]oxime in similar conditions as previously described
for CAL-B (Scheme 5).10b The CRL-catalyzed enzy-
matic alkoxycarbonylation of trans-enantiomers 3a
and 3b took place without selectivity and a complex
mixture of monocarbonates and dicarbonates were ob-
tained. In contrast, CRL was a very efficient catalyst
for the regioselective transformation of substrate 3d.
This lipase catalyzed exclusively the alkoxycarbonyla-
tion in the C-3 position although a mixture of both
vinyloxy and oxime carbonates were obtained 6d and
7d, respectively. The conversions were lower in 1,4-diox-
ane or THF than in toluene (Table 5, entries 1–3). Com-
pounds 6d and 7d were formed even if the reaction was
carried out at lower temperature or with lesser amounts
of carbonylation agent (Table 5, entries 4–6). The fact to
obtain the mixture 6d/7d is not an inconvenience from
Hydrolase
buffer/1,4-dioxane
O
O
O
O
O
O
4c
+
+
RO
OH
HO
OR
HO
OH
5c
6c
3c
R = CO2CH=CH2
Scheme 3.
improves the synthesis of 5d, previously described for
ourselves,10b from diol 3d through alkoxycarbonylation
with CAL-B using acetone O-[(vinyloxy)carbonyl]oxime
as carbonylating agent.
Table 3. Enzymatic hydrolysis of A-ring synthon (3S,5S)-4ca
Entry
Enzyme
Buffer (%)b
T (°C)
t (h)
Conv. (%)c
3c (%)c
5c (%)c
6c (%)c
1
CVLd
CVLd
83
83
5
30
10
30
25
24
98
40
98
40
2
3
4CAL-B
CVLd
21
100
72
64
100
8
e
4
1
30
26
18
10
5
PSL-Cf
41
83
4
30
30
22
24
26
46
28
6
CAL-Ag
Chirazyme L2h
Chirazyme L2h
PPLi
46
7
1
30
70
35
22
13
7
8
83
76
67
67
30
30
30
30
2466
2435
72
66
35
9
10
11
CRLj
PLEk
32
6435
18
21
7
8
30
a The reactions were carried out at 250rpm in 0.04M concentration with 17mg of 4c.
b Percentage of buffer solution (0.1M KH2PO4/KOH, pH7.0) in 1,4-dioxane; in all cases, total reaction volume was 1.5mL.
c Based on 1H NMR signal integration ( 3% error).
d Ratio 5.7:1 of 4c:CVL (w/w).
e Ratio 1:5.3 of 4c:CAL-B (w/w).
f Ratio 1:5.3 of 4c:PSL-C (w/w).
g Ratio 1:2.6 of 4c:CAL-A (w/w).
h Ratio 1:5.3 of 4c:Chirazyme L2 (w/w).
i Ratio 1:3.5 of 4c:PPL (w/w).
j Ratio 1:3.5 of 4c:CRL (w/w).
k Ratio 1:1.2 of 4c:PLE (w/w).

D. Oves et al. / Tetrahedron: Asymmetry 15 (2004) 2881–2887
2885
with high selectivity the hydrolysis of the carbonate at
the C-3 position of A-ring dicarbonate precursors 4a,b,
and 4d to afford the corresponding C-5 monocarbonate
derivatives 5a,b, and 5d. However, this lipase was not
efficient in the hydrolysis of substrate 4c. On the other
hand, Chromobacterium viscosum lipase has shown
opposite selectivity with cis enantiomers 4c and 4d: the
hydrolysis occurs at the C-5 carbonate for 4c and at
the C-3 position in case of 4d. Additionally, CRL
allowed the selective synthesis of C-3 derivatives 6d
and 7d through direct alkoxycarbonylation reaction.
Although preparation of C-3 monocarbonates 6 was
not achieved in all cases with high selectivities, the
results obtained in this study could be useful in the selec-
tive deprotection of similar precursors of 1a,25-(OH)2-
D3 analogues.
O
N
O
O
CRL
HO
OH
3d
O
O
+
N
O
O
HO
O
O
HO
7d
6d
Scheme 5.
the synthetic point of view because both carbonates are
appropriate synthons to introduce additional
functionalities.
4.Experimental
Chromobacterium viscosum lipase, (CVL, 4100U/mg)
was a gift from Genzyme Co. C. antarctica lipase B
(CAL-B, Novozym 435, 10,000PLU/g), Chirazyme L-2
(lipase from C. antarctica B, 400U/g), and immobilized
Pseudomonas cepacia lipase (PSL-C, 904U/g) were
purchased from Novo Nordisk Co., Roche Diagnostics,
3.Summary
Several chiral monocarbonylated precursors for the
preparation of 1a,25-dihydroxyvitamin D3 analogues
have been synthesized. C. rugosa lipase has catalyzed
Table 4. Enzymatic hydrolysis of A-ring synthon (3R,5R)-4da
Entry
Enzyme
Buffer (%)b
T (°C)
t (h)
Conv. (%)c
3d (%)c
5d (%)c
6d (%)c
1
CVLd
CVLd
7
1420
18
67
29
67
58
67
67
67
67
30
73
87
8
11
76
2
15
98
90
3
4CAL-B
CVLd
20
30
30
30
30
30
30
30
30
37
19
17
69
17
70
25
46
25
100
25
91
35
49
8
3466
18
56
25
24
8
e
7
23
5
PSL-Cf
CAL-Ag
Chirazyme L2h
PPLi
12
10
6
7
25
8
9
CRLj
90
100
23
7
83
88
4
10
11
CRLj
12
145
PLEk
a The reactions were carried out at 250rpm in 0.04M concentration with 17mg of 4d.
b Percentage of buffer solution (0.1M KH2PO4/KOH, pH7.0) in 1,4-dioxane; in all cases, total reaction volume was 1.5mL.
c Based on 1H NMR signal integration ( 3% error).
d Ratio 5.7:1 of 4d:CVL (w/w).
e Ratio 1:5.3 of 4d:CAL-B (w/w).
f Ratio 1:5.3 of 4d:PSL-C (w/w).
g Ratio 1:2.6 of 4d:CAL-A (w/w).
h Ratio 1:5.3 of 4d:Chirazyme L2 (w/w).
i Ratio 1:3.5 of 4d:PPL (w/w).
j Ratio 1:3.5 of 4d:CRL (w/w).
k Ratio 1:1.2 of 4d:PLE (w/w).
Table 5. Enzymatic alkoxycarbonylation catalyzed by CRL of A-ring diol (3R,5R)-3da
Entry
Solvent
VCOb (equiv)
T (°C)
t (h)
Conv. (%)c
6d (%)c
7d (%)c
1
THF
10
10
10
30
10
5
40
40
40
1
96
96
46
47
20
19
26
65
40
39
26
28
74
2
1,4-Dioxane
Toluene
10
3
4Toluene
96
100
35
100
1.5
1.5
5
6
Toluene
Toluene
20
20
100
100
60
61
a The reactions were carried out at 250rpm in 0.03M concentration with 15mg of 3d, dissolved in 3.5mL of solvent; Ratio 1:6.7 of 3d:CRL (w/w).
b VCO, acetone O-[(vinyloxy)carbonyl]oxime.
c Based on 1H NMR signal integration (3% error).

2886
D. Oves et al. / Tetrahedron: Asymmetry 15 (2004) 2881–2887
20
has been previously described.10c ½aŠ ¼ À4 5 c( 0.35,
and Amano Pharmaceuticals, respectively. C. rugosa
lipase (CRL, 724U/mg), pig liver esterase (PLE,
3500U/mg), and porcine pancreas lipase (PPL, 46U/
mg) were purchased from Sigma. C. antarctica lipase
A (CAL-A, 50kU/19.24g) was purchased from Roche.
D
CHCl3).
4.2.2. (3R,5S)-1-Ethynyl-3-hydroxy-2-methyl-5-[(vinyl-
oxy)carbonyloxy]-1-cyclohexene 5b. Same data as for
20
D
its enantiomer, 5a. ½aŠ ¼ þ50 (c 0.58, CHCl3).
Solvents were distilled over an adequate desiccant under
nitrogen. A-ring synthons 3a and 3b were obtained
according to the method of Okamura et al.14 A-ring syn-
thons 3c and 3d were obtained through Mitsunobu
inversion.10a Acetone O-[(vinyloxy)carbonyl]oxime was
synthesized as previously reported.15
4.2.3. (3S,5S)-1-Ethynyl-3-hydroxy-2-methyl-5-[(vinyl-
oxy)carbonyloxy]-1-cyclohexene 5c. This compound
20
D
has been previously described.10b ½aŠ ¼ À4 8 c( 0.72,
CHCl3).
4.2.4. (3R,5R)-1-Ethynyl-3-hydroxy-2-methyl-5-[(vinyl-
oxy)carbonyloxy]-1-cyclohexene 5d. Same data as for
its enantiomer, 5c. ½aŠ ¼ þ51 (c 0.8, CHCl3).
4.1. General procedure for the syntheses of dicarbonates
4a–d
20
D
To a solution of diol 3 (197mg, 1.30mmol) in CH2Cl2
(15mL) and pyridine (0.6mL, 6.48mmol) at 0°C was
added dropwise vinyl chloroformate (0.62mL,
6.48mmol). The progress of the reaction was followed
by TLC (40% EtOAc/hexane). After consumption of
the starting material (1h) the solvent was removed under
reduced pressure, and the crude was extracted with
CH2Cl2. Then, the residue was subjected to flash chroma-
tography (20% EtOAc/hexane) to afford a colorless oil.
4.2.5. (3S,5R)-1-Ethynyl-5-hydroxy-2-methyl-3-[(vinyl-
c
20
D
oxy)carbonyloxy]-1-cyclohexene 6a. ½aŠ ¼ À104(
0.31, CHCl3); IR (NaCl): m 3441, 3288, 2934, 2094,
1
1756, and 1649cmÀ1; H NMR (CDCl3, 400.13MHz):
2
3
3
d 1.88 (ddd, 1H, H4, JHH 14.0, JHH 10.6, JHH
4.7Hz), 1.95 (s, 3H, 3H9), 2.17 (m, 2H, H4+H6), 2.60
2
3
(dd, 1H, H6, JHH 16.9, JHH 4.2Hz), 3.17 (s, 1H, H8),
4.12 (m, 1H, H5), 4.60 (dd, 1H, H12-cis
3JHH
3JHH
,
2
6.4, JHH 2.1Hz), 4.93 (dd, 1H, H12-trans
,
2
13.6, JHH 2.1Hz), 5.34(m, 1H, H 3), and 7.07 (dd,
1H, H11, JHH 13.6, JHH 6.4Hz); 13C NMR (CDCl3,
3
3
4.1.1. (3S,5R)-1-Ethynyl-2-methyl-3,5-bis[(vinyloxy)car-
bonyloxy]-1-cyclohexene 4a. Yield 87%. This com-
100.6MHz): d 18.4(C ), 36.6 (C4), 38.4(C ), 63.1 (C5),
9
6
20
D
pound has been previously described.10c ½aŠ ¼ À112
76.1 (C3), 81.8 (C8), 82.2 (C7), 98.0 (C12), 118.1 (C1),
137.6 (C2), 142.4 (C11), and 152.4(C 10); MS (FAB+,
m/z): 222 (M+, 4%), 204(11), 134(49), 116 (60), 115
(100), 105 (23), and 91 (61); Anal. Calcd (%) for
C12H14O4: C, 64.84; H, 6.35. Found: C, 64.8; H, 6.3.
(c 0.65, CHCl3).
4.1.2. (3R,5S)-1-Ethynyl-2-methyl-3,5-bis[(vinyloxy)car-
bonyloxy]-1-cyclohexene 4b. Yield 70%. Same data as
20
D
for its enantiomer, 4a. ½aŠ ¼ þ108 (c 0.85, CHCl3).
4.2.6. (3S,5S)-1-Ethynyl-5-hydroxy-2-methyl-3-[(vinyl-
4.1.3. (3S,5S)-1-Ethynyl-2-methyl-3,5-bis[(vinyloxy)car-
bonyloxy]-1-cyclohexene 4c. Yield 92%. This com-
20
D
oxy)carbonyloxy]-1-cyclohexene 6c. ½aŠ ¼ À39 (c
0.61, CHCl3); IR (NaCl): m 3385, 3298, 2921, 2855,
20
D
pound has been previously described.10b ½aŠ ¼ À4 1 c(
2095, 1756, and 1650cmÀ1
;
1H NMR (CDCl3,
0.57, CHCl3).
400.13MHz): d 1.96 (s, 3H, 3H9), 2.01 (ddd, 1H, H4,
3
3
2JHH 13.6, JHH3 7.9, JHH 6.1Hz), 2.25 (dddd, 1H,
H4, JHH 13.6, JHH 5.9, JHH 3.1, JHH 1.0Hz), 2.42
4.1.4. (3R,5R)-1-Ethynyl-2-methyl-3,5-bis[(vinyloxy)car-
bonyloxy]-1-cyclohexene 4d. Yield 81%. This com-
2
3
4
20
D
(m, 2H, 2H6), 3.18 (s, 1H, H8), 4.05 (m, 1H, H5), 4.61
(dd, 1H, H12-cis, JHH 6.2, JHH 2.1Hz), 4.93 (dd, 1H,
pound has been previously described.10b ½aŠ ¼ þ37 (c
3
2
0.6, CHCl3).
2
H12-trans
,
3JHH 13.9, JHH3 2.1Hz), 5.32 (m, 1H, H3),
and 7.09 (dd, 1H, H11, JHH 13.9, JHH 6.2Hz); 13C
NMR (CDCl3, 100.6MHz): d 17.9 (C9), 35.7 (C4), 38.3
(C6), 63.8 (C5), 75.1 (C3), 81.9 (C8), 82.3 (C7), 98.1
(C12), 116.8 (C1), 138.1 (C2), 142.4 (C11), and 152.4
(C10); MS (FAB+, m/z): 222 (M+, 3%), 204(6), 178
(5), 134(50), 116 (78), 115 (100), 105 (29), and 91 (89);
Anal. Calcd (%) for C12H14O4: C, 64.84; H, 6.35.
Found: C, 64.9; H, 6.2.
3
4.2. General procedure for the enzymatic hydrolysis of
dicarbonates 4a–d
In a typical procedure, to a solution of dicarbonate 4
(17mg, 0.058mmol) in 1.5mL of solvent (mixture of
1,4-dioxane and buffer) was added one of the following
enzymes: 90mg of CAL-B, PSL-C, or Chirazyme L2,
60mg of CRL or PPL, 45mg of CAL-A, 20mg of
PLE, or 3mg of CVL. The suspension was shaken
(250rpm) at 10, 20, or 30°C and the progress of the
reaction was followed by TLC (40% EtOAc/hexane).
After removal of the enzyme by filtration, the crude
was extracted with CH2Cl2. The organic layer was dried
over Na2SO4, evaporated, and the residue was analyzed
by 1H NMR. All these data are summarized in
Tables 1–4.
4.2.7. (3R,5R)-1-Ethynyl-5-hydroxy-2-methyl-3-[(vinyl-
oxy)carbonyloxy]-1-cyclohexene 6d. Same data as for
20
D
its enantiomer, 6c. ½aŠ ¼ þ4 2 c( 0.63, CHCl3).
4.3. General procedure for the enzymatic alkoxycar-
bonylation of diols 3
To a solution of 3a,b, or 3d (15mg, 0.098mmol) in
3.5mL of solvent (THF, 1,4-dioxane, or toluene) was
added 100mg of CRL and acetone O-[(vinyloxy)carbon-
4.2.1. (3S,5R)-1-Ethynyl-3-hydroxy-2-methyl-5-[(vinyl-
oxy)carbonyloxy]-1-cyclohexene 5a. This compound

D. Oves et al. / Tetrahedron: Asymmetry 15 (2004) 2881–2887
2887
M.; Gotor, V. Monatsh. Chem. 2000, 131, 585–616; (c)
Schoffers, E.; Golebiowski, A.; Johnson, C. R. Tetrahe-
dron 1996, 52, 3769–3826; (d) Theil, F. Chem. Rev. 1995,
95, 2203–2227.
yl]oxime (141mg, 0.986mmol or 70.5mg, 0.493mmol).
The suspension was shaken (250rpm) at 20, 30, or
40°C, and the progress of the reaction was followed
by TLC (50% EtOAc/hexane) until no further reaction
was apparent. After removal of the enzyme by filtration
4. (a) First International Conference on Chemistry and
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1994, 1383–1398.
6. (a) Tsugawa, N.; Nakagawa, K.; Kurobe, M.; Ohno, Y.;
Kubodera, N.; Ozono, K.; Okano, T. Biol. Pharm. Bull.
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4628.
1
and evaporation, the crude was analyzed by H NMR.
All these data are summarized in Table 5.
4.3.1. (3R,5R)-3-[(Acetonoxime)carbonyloxy]-1-ethynyl-
5-hydroxy-2-methyl-1-cyclohexene
20
7d. ½aŠ ¼ þ57
D
(c 0.5, CHCl3); IR (NaCl): m 3476, 3289, 2926, 2096,
1768, 1654, and 1378cmÀ1
;
1H NMR (CDCl3,
300MHz): d 1.98 (s, 3H, 3H12), 2.02 (s, 3H, 3H12),
2.04(s, 3H, 3H 9), 2.07 (m, 1H, 1H4), 2.27 (ddd,
3
3
2JHH 16.5, JHH 5.7, JHH 3.1Hz), 2.33–2.55 (m, 2H,
2H6), 3.18 (s, 1H, H8), 4.08 (m, 1H, H5), and 5.42
(m,1H, H3); 13C NMR (CDCl3, 75.5MHz): d 16.8
(C12), 17.9 (C12), 21.7 (C9), 35.7, 38.2 (C4 + C6), 63.8
(C5), 74.7 (C3), 81.7 (C8), 82.4(C ), 116.7 (C1), 138.4
7
(C2), 153.6 (C10), and 163.7 (C11). MS (EI, m/z):
251 (M+, 2%), 135 (M-OCO2NC3H6, 50), 133 (26), 105
(29), 91 (89) and 56 (100); Anal. Calcd (%) for
C13H17NO4: C, 62.14; H, 6.82; N, 5.57. Found: C,
62.0; H, 6.9; N, 5.6.
Acknowledgements
´
7. For recent papers: (a) Oves, D.; Ferrero, M.; Fernandez,
S.; Gotor, V. J. Org. Chem. 2003, 68, 1154–1157; (b) Dıaz,
´
´
M.; Ferrero, M.; Fernandez, S.; Gotor, V. J. Org. Chem.
2000, 65, 5647–5652; (c) Dıaz, M.; Ferrero, M.; Fernan-
dez, S.; Gotor, V. Tetrahedron Lett. 2000, 41, 775–779.
Financial support from Principado de Asturias (Spain;
Project GE-EXP01-03) and MCYT (Spain; Project
PPQ-2001-2683) is gratefully acknowledged. S.F. also
´
´
´
8. (a) Dıaz, M.; Ferrero, M.; Fernandez, S.; Gotor, V.
Tetrahedron: Asymmetry 2002, 13, 539–546; (b) Fernan-
´
´
thanks MCYT (Spain) for a personal grant (Ramon y
´
Cajal Program). D.O. thanks Principado de Asturias
(Spain) for a predoctoral fellowship.
dez, S.; Ferrero, M.; Gotor, V.; Okamura, W. H. J. Org.
Chem. 1995, 60, 6057–6061.
´
´
9. Gotor-Fernandez, V.; Ferrero, M.; Fernandez, S.; Gotor,
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