Novel chiral precursors of 6-s-cis locked 1α,25-dihydroxyvitamin D3 analogues through selective enzymatic acylation

Article abstract of DOI:10.1016/S0957-4166(02)00149-0

The syntheses of selectively modified chiral A-ring precursors for the preparation of 1α,25-dihydroxyvitamin D₃ analogues by regioselective enzymatic acylation are described. Candida antarctica lipase B (CAL-B) catalyzes the acylation of 1α,25-dihydroxy-19-nor-previtamin D₃ trans A-ring precursors 4 and 5 with high selectivity. The opposing regioselectivities observed for each pair of enantiomers is noteworthy: whereas CAL-B acylates the C-3 hydroxyl groups for derivatives of (3S,5R)-configuration, it catalyzes acylation at the C-5 hydroxyl group for substrates which possess (3R,5S)-stereochemistry. In relation to stereoisomer 4b, Chromobacterium viscosum lipase (CVL) showed opposite behavior to CAL-B, catalyzing acylation at the C-5 hydroxyl group with acceptable selectivity. In the enzymatic acylation of cis A-ring synthons 6 and 7, CVL gave total selectivity for acylation of the C-5 hydroxyl group of (3S,5S)-6 and the C-3 hydroxyl group of (3R,5R)-7. CAL-B also exhibits high selectivity towards the acylation of the C-3 hydroxyl in 19-nor-A-ring precursors with (3R,5R)-configuration.

Full text of DOI:10.1016/S0957-4166(02)00149-0

TETRAHEDRON:
ASYMMETRY
Pergamon
Tetrahedron: Asymmetry 13 (2002) 539–546
Novel chiral precursors of 6-s-cis locked 1a,25-dihydroxyvitamin
D3 analogues through selective enzymatic acylation
Mo´nica D´ıaz, Miguel Ferrero, Susana Ferna´ndez and Vicente Gotor*
Departamento de Qu´ımica Orga´nica e Inorga´nica, Facultad de Qu´ımica, Universidad de Oviedo, 33071 Oviedo, Spain
Received 26 February 2002; accepted 21 March 2002
Abstract—The syntheses of selectively modified chiral A-ring precursors for the preparation of 1a,25-dihydroxyvitamin D3
analogues by regioselective enzymatic acylation are described. Candida antarctica lipase B (CAL-B) catalyzes the acylation of
1a,25-dihydroxy-19-nor-previtamin D3 trans A-ring precursors 4 and 5 with high selectivity. The opposing regioselectivities
observed for each pair of enantiomers is noteworthy: whereas CAL-B acylates the C-3 hydroxyl groups for derivatives of
(3S,5R)-configuration, it catalyzes acylation at the C-5 hydroxyl group for substrates which possess (3R,5S)-stereochemistry. In
relation to stereoisomer 4b, Chromobacterium viscosum lipase (CVL) showed opposite behavior to CAL-B, catalyzing acylation at
the C-5 hydroxyl group with acceptable selectivity. In the enzymatic acylation of cis A-ring synthons 6 and 7, CVL gave total
selectivity for acylation of the C-5 hydroxyl group of (3S,5S)-6 and the C-3 hydroxyl group of (3R,5R)-7. CAL-B also exhibits
high selectivity towards the acylation of the C-3 hydroxyl in 19-nor-A-ring precursors with (3R,5R)-configuration. © 2002
Elsevier Science Ltd. All rights reserved.
1. Introduction
1) which are unable to undergo rearrangement to the
respective vitamin form because of the absence of the
C(19) methyl group, and are therefore potential tools
for exploring the biological significance of the previta-
min form. The synthesis of A-ring analogues of these
The efficacy of 1a,25-dihydroxyvitamin D3 [1a,25-
(OH)2-D3] 1 (Scheme 1) analogues as chemotherapeutic
agents in a variety of human diseases is highly promis-
ing.1 This secosteroid, the bioactive metabolite of vita-
min D3, can generate biological responses via both
genomic2 and non-genomic3 pathways by interaction
with vitamin D receptors placed in the nucleus (n-
VDR) and/or in the cellular membrane (m-VDR).
25
OH
H
1a,25-(OH)2-D3 exists as two conformationally
interequilibrating forms (6-s-trans 1 and a minor form
6-s-cis 1%), which are present in slow chemical equi-
librium (5–10%) with 1a,25-dihydroxyprevitamin D3
[1a,25-(OH)2-pre-D3, 2]. Due to the spontaneous iso-
merization of previtamin 2 back to the thermally more
stable vitamin 1, biological evaluation of 2 is very
difficult. 6-s-cis-1a,25-(OH)2-D3 was proposed as the
active conformer in eliciting non-genomic effects, with
1a,25-(OH)2-pre-D3 2 simply behaving as an excellent
analogue of this conformer.4
6
HO
19
OH
H
HO
3
1
OH
HO
1'
1
6-s-cis-1α,25-(OH)2-D3
6-s-trans-1α,25-(OH)2-D3
HO
OH
In our laboratory we have synthesized5 the A-ring
diastereoisomers of 1a,25-(OH)2-19-nor-pre-D3 (3, Fig.
H
HO
2
1α,25-(OH)2-pre-D3
* Corresponding author. Tel.: +34 98 510 3448; fax: +34 98 510 3448;
e-mail: vgs@sauron.quimica.uniovi.es
Scheme 1.
0957-4166/02/$ - see front matter © 2002 Elsevier Science Ltd. All rights reserved.
PII: S0957-4166(02)00149-0

540
M. D´ıaz et al. / Tetrahedron: Asymmetry 13 (2002) 539–546
or methyl ester derivatives 4b–7b. Different lipases such
as Candida antarctica B (CAL-B), C. viscosum (CVL)
and Pseudomonas cepacia (PSL) are used to obtain the
monoacylated derivatives in a highly regioselective way.
HO
OH
2
H
HO
OH
3
5
HO
3
2. Results and discussion
4a−7a
6-s-cis locked analogues
Diols 4–7 were obtained through deprotection of the
corresponding silylether derivatives,5 previously
described, with hydrochloric acid in methanol.
2
2.1. Acylation of A-ring precursors 4a–b
HO
OH HO
3
OH HO
OH HO
OH
5
CVL catalyzes the acylation of A-ring 8 at the C-5
hydroxyl group with high selectivity when vinyl acetate
is used as both the solvent and acylation reagent.11
When similar conditions were applied to 19-nor-A-ring
4a (Scheme 2), which has the same stereochemistry but
lacks the methyl group at the C(2) position, no selectiv-
ity was observed with this lipase at 30 or 40°C (entries
1 and 2, Table 1). However, when CAL-B was used as
catalyst, reasonably high regioselectivity towards the
C-3 position was achieved, with the monoacetate 12a
being obtained as the major product after 16 h at 30°C
(entry 3, Table 1). If THF is used as solvent, with a
10:1 ratio of vinyl acetate to diol, longer reaction times
were required and no improvement in the selectivity
8
9
10
11
Figure 1.
derivatives is a difficult task since the A-ring synthon
possesses two stereogenic centers and two hydroxyl
groups of similar reactivity, as a result of which it is
very difficult to discern between these two groups from
a chemical point of view. An important and syntheti-
cally relevant transformation mediated by lipases6 is the
selective modification of polyfunctionalized com-
pounds, such as carbohydrates,7 steroids,8 and
nucleosides.9 In this way, we described recently the
enzymatic alkoxycarbonylation processes of 1a,25-
(OH)2-D3 and 1a,25-(OH)2-19-nor-pre-D3 A-ring syn-
thons.10 Previously, we reported the regioselective
enzymatic acylation of 1a,25-(OH)2-D3 A-ring precur-
sors 8–11 (Fig. 1) catalyzed by Chromobacterium visco-
sum lipase (CVL).11 Whereas CVL selectively catalyzes
the acylation of the C-5 hydroxyl of the three stereoiso-
meric vitamin D A-ring precursors 8–10, only the C-3
hydroxyl group of the fourth stereoisomer 11 is
acylated under these conditions in organic solvent. Here
we report the synthesis of novel chiral 19-nor-A-ring
precursors selectively modified at C-3 or C-5 through
selective enzymatic acylation of ethynyl synthons 4a–7a
R1
R1
Enzyme
AcOCH=CH2
HO
OH
3S
R3O
OR2
4a−b
12a−b, R2= Ac; R3= H
13a−b, R2= H; R3= Ac
14b, R2= R3= Ac
a, R1= C CH
b, R1= CO2Me
Scheme 2.
Table 1. Enzymatic acylation of 19-nor-A-ring synthons 4a–b
Entry
Substrate
Enzyme
T (°C)
t (h)
Conv. (%)a
C-3 (%)b
C-5 (%)b
C-3,5 (%)b
1
2
3
4
5
6
7
8
4a
4a
4a
4a
4a
4a
4b
4b
4b
4b
4b
4b
CVL
CVL
CAL-B
CAL-Bc
PSL-C
CRL
CVL
CVL
CAL-B
CAL-Bd
PSL
30
40
30
30
30
30
30
40
30
30
30
30
240
96
16
41
40
91
93
42
48
81
80
64
65
16
9
74
80
48
50
49
45
19
20
36
35
76
80
20
20
32
50
100
100
100
100
92
93
94
100
98
100
24
240
192
50
96
43
4
9
10
11
12
18
CRL
60
a Calculated by GC.
b Ratio of regioselectivity at position C-3, C-5 or C-3,5 calculated by 1H NMR.
c THF as solvent and 10 equiv. of vinyl acetate.
d Isopropenyl acetate as both the solvent and acylating reagent.

M. D´ıaz et al. / Tetrahedron: Asymmetry 13 (2002) 539–546
541
was observed (entry 4, Table 1). Other lipases such as
PSL-C (immobilized P. cepacia lipase) and CRL (Can-
dida rugosa lipase) exhibited lower selectivities than
CAL-B (entries 5 and 6, Table 1).
regioselectivity (entry 2, Table 2). This selectivity
decreased when THF was used as the solvent (entry 3,
Table 2).
It is worth noting the opposing selectivity observed
with CAL-B for both enantiomers: that is, (3S,5R)-4a
showed preference for acylation of the C-3 hydroxyl
group meanwhile (3R,5S)-5a was acylated at C-5. PSL
showed low selectivity in this process (entry 4, Table 2).
With the aim of determining the influence of the substi-
tution at C(1), the methyl ester derivative 4b was also
studied. Thus, the acylation of diol 4b with CVL at
30°C is very slow but shows moderate regioselectivity
toward the C-5 hydroxyl group (entry 7, Table 1).
Shorter reaction times and a slight increase in the
selectivity for the formation of 13b is observed if the
process is carried out at 40°C (entry 8, Table 1). CVL
displayed higher regioselectivity for ester 4b than for
the ethynyl analogue 4a. The enzymatic acylation using
CAL-B is much faster and still maintains high selectiv-
ity toward the C-3 position, just the opposite of CVL,
when vinyl acetate is the solvent and the acylating agent
(entry 9, Table 1). If isopropenyl acetate is used, instead
of vinyl acetate, the process is slower but slightly more
regioselective (entry 10, Table 1). When other lipases,
such as PSL or CRL, are used the enzymatic reaction
takes place without selectivity (entries 11 and 12, Table
1).
Replacement of the ethynyl group by the methyl ester
functionality at C-1 gives rise to a decrease in the
selectivity of the acylation with CVL although this
lipase still maintains preference towards the same posi-
tion (entry 5, Table 2). However, CAL-B shows higher
selectivity with the methyl ester 5b than the ethynyl
analogue 5a catalyzing the formation of 16b in 87%
yield after 1 h. When THF was used as the solvent, no
improvement in the selectivity was achieved (entry 7,
Table 2). PSL did not show preference for any of the
hydroxyl groups in 5b (entry 8, Table 2). For example,
in the enyne enantiomers, CAL-B catalyzed the
acylation of 4b and 5b with opposite selectivity,
whereas CVL preferentially acylated the C-5 position of
both stereoisomers.
2.2. Acylation of A-ring precursors 5a–b
2.3. Acylation of A-ring precursors 6a–b
Table 2 summarizes the results obtained for the
acylation of (3R,5S)-5a at 30°C, shown in Scheme 3.
The enzymatic acylation of 6a with CVL and vinyl
acetate takes place with total selectivity towards the C-5
OH (Scheme 4), isolating the monoacetate 19a exclu-
sively after 50 h (entry 1, Table 3). CAL-B displayed a
slight preference for acylation at C-3 instead of C-5 for
this isomer (entry 2, Table 3).
R1
R1
Enzyme
AcOCH=CH2
30 °C
HO
OH
3R
R3O
OR2
5a−b
15a−b, R2= Ac; R3= H
16a−b, R2= H; R3= Ac
17b, R2= R3= Ac
1
C CH
a, R =
R1
R1
b, R1= CO2Me
Enzyme
AcOCH=CH2
HO
OH
3S
R3O
OR2
Scheme 3.
30 °C
6a−b
18a−b, R2= Ac; R3= H
19a−b, R2= H; R3= Ac
20a−b, R2= R3= Ac
In this case, CVL exhibited moderate preference for the
C-5 position when vinyl acetate was used as solvent and
acylating agent (entry 1, Table 2). The best results are
obtained when the process is catalyzed by CAL-B,
giving rise after 2 h to the monoacetate 16a with 80%
1
C
CH
a, R =
b, R1= CO2Me
Scheme 4.
Table 2. Enzymatic acylation of 19-nor-A-ring synthons 5a–b
Entry
Substrate
Enzyme
t (h)
Conv. (%)a
C-3 (%)b
C-5 (%)b
C-3,5 (%)b
1
2
3
4
5
6
7
8
5a
5a
5a
5a
5b
5b
5b
5b
CVL
18
2
36
2
26
1
6
100
100
100
100
97
100
100
100
30
20
31
40
32
7
70
80
69
60
65
87
82
47
CAL-B
CAL-Bc
PSL
CVL
CAL-B
CAL-Bc
PSL
6
8
10
53
4
a Calculated by GC.
b Ratio of regioselectivity at position C-3, C-5 or C-3,5 calculated by 1H NMR.
c THF as solvent and 10 equiv. of vinyl acetate.

542
M. D´ıaz et al. / Tetrahedron: Asymmetry 13 (2002) 539–546
Table 3. Enzymatic acylation of 19-nor-A-ring synthons 6a–b
Entry
Substrate
Enzyme
t (h)
Conv. (%)a
C-3 (%)b
66
C-5 (%)b
C-3,5 (%)b
1
2
3
4
5
6
7
8
6a
6a
6a
6a
6b
6b
6b
6b
CVL
CAL-B
PSL
50
18
8
18
40
13
8
97
91
100
100
100
93
97
9
16
10
90
100
100
48
3
PSLc
CVL
CAL-B
PSL
40
5
95
38
98
99
PSLc
20
61
a Calculated by GC.
b Ratio of regioselectivity at position C-3, C-5 or C-3,5 calculated by 1H NMR.
c Isopropenyl acetate as both the solvent and acylating reagent.
R1
R1
Surprisingly, PSL also catalyzed the acylation process
at C-5 with excellent regioselectivity (entry 3, Table 3).
This selectivity is complete if the reaction is carried out
with isopropenyl acetate as both the solvent and
acylating reagent (entry 4, Table 3).
Enzyme
AcOCH=CH2
R3O
OR2
HO
OH
3R
30 °C
21a−b, R2= Ac; R3= H
22a−b, R2= H; R3= Ac
23a−b, R2= R3= Ac
7a−b
1
C
CH
a, R =
When methyl ester 6b was used as substrate, CVL
maintained the total selectivity at the C-5 hydroxyl
group (entry 5, Table 3). However, CAL-B and PSL
were not suitable biocatalysts for this reaction (entries
6, 7 and 8, Table 3).
b, R1= CO2Me
Scheme 5.
21b exclusively after 8 h with 100% conversion (entry 5,
Table 4). Also, the opposing regioselectivity was
obtained for methyl ester enantiomers 6b and 7b if CVL
was used.
2.4. Acylation of A-ring precursors 7a–b
Among all A-ring stereoisomers of 1a,25-(OH)2-D3,
A-ring synthon 11 was the only one acylated at the C-3
allylic hydroxyl group with CVL as biocatalyst. Simi-
larly, CVL catalyzed the synthesis of the C-3
monoacetylated derivate 21a from diol 7a (Scheme 5)
with total regioselectivity using vinyl acetate at 30°C
(entry 1, Table 4). The opposite selectivity achieved for
the cis enantiomers 6a and 7a is noteworthy.
3. Summary
Several chiral monoacylated precursors for the prepara-
tion of 6-s-cis locked analogues of 1a,25-dihydroxyvita-
min D3 have been synthesized. trans A-ring precursors
4 and 5 have been acylated toward S-configuration
positions (at C-3 in 4 and at C-5 in 5) with high
selectivity using C. antarctica lipase B (CAL-B), inde-
pendently of the functionality at C-1 (ethynyl or methyl
ester). The opposing regioselectivities shown by each
couple of enantiomers is noteworthy. In relation to
stereoisomer 4b, C. viscosum lipase (CVL) showed
opposite behavior to CAL-B, catalyzing the acylation
at C-5 position with acceptable selectivity.
The total selectivity seen with CVL could not be
obtained with CAL-B, although this enzyme gives rise
to a mixture of the regioisomers 21a and 22a in a ratio
of approximately 10:1 (entry 2, Table 4). PSL was less
selective than CVL or CAL-B and provided diacetyl-
ated 23a as the major product (entry 3, Table 4).
Methyl ester 7b was acylated at the same position with
excellent regioselectivities when CVL, CAL-B, or PSL
were used (entries 4, 5 and 6, Table 4). The process
catalyzed by CAL-B is of note, forming monoacetate
For cis enyne and methyl ester 19-nor-A-ring precur-
sors 6 and 7, CVL gave the excellent regioselectivity
Table 4. Enzymatic acylation of 19-nor-A-ring synthons 7a–b
Entry
Substrate
Enzyme
t (h)
Conv. (%)a
C-3 (%)b
C-5 (%)b
C-3,5 (%)b
1
2
3
4
5
6
7a
7a
7a
7b
7b
7b
CVL
CAL-B
PSL
CVL
CAL-B
PSL
22
26
10
52
8
99
98
100
99
100
100
99
89
32
95
100
90
9
4
68
10
1.5
a Calculated by GC.
b Ratio of regioselectivity at position C-3, C-5 or C-3,5 calculated by 1H NMR.

M. D´ıaz et al. / Tetrahedron: Asymmetry 13 (2002) 539–546
543
shown with A-ring isomers 10 and 11. Thus, the
regioselectivity shown by each couple of enantiomers
was opposite and independent of the functionality at
the C-1 or C-2 positions. From the results of the
acylation it is clear that for cis compounds with stereo-
chemistry (3S,5S), C-5-(S) position are selectively
acylated. However, if the substrates possess (3R,5R)
configuration, the enzyme chooses the hydroxyl group
at C-3-(R). CAL-B also shows high selectivities toward
the C-3 position in 19-nor-A-ring precursors with
configuration (3R,5R). Moreover, PSL was an ade-
quate biocatalyst to prepare monoacetate derivatives
19a and 21b. In general, the enzymatic processes with
cis synthons take place with higher selectivities than for
trans stereoisomers.
(M+, 1%), 351 (2), 309 (19), 285 (4), 234 (88), 178 (28),
151 (73) and 147 (49); HRMS calcd for C8H10O2 (M+):
138.0681. Found: 138.0684. 4a: [h]2D0=−111.1 (c 0.80,
MeOH). Anal. calcd (%) for C8H10O2: C, 69.55; H, 7.3.
Found: C, 69.6; H, 7.4. 5a: [h]2D0=+113.9 (c 0.44,
MeOH). Anal. calcd (%) for C8H10O2: C, 69.55; H, 7.3.
Found: C, 69.4; H, 7.4.
4.1.2. (3S,5S)- and (3R,5R)-1-Ethynyl-3,5-dihydroxycy-
clohex-1-ene 6a and 7a. Rf (80% EtOAc/hex): 0.2; IR
1
(Nujol): w 3320 and 1648 cm−1; H NMR (MeOH-d4,
2
200 MHz): l 1.65 (ddd, 1H, H4a, ꢀ JHHꢀ 11.6, JHH 11.6,
2
3
9.5 Hz), 2.26 (dddd, 1H, H6a%, ꢀ JHHꢀ 16.7, JHH 9.2,
JHH 3.1, 3.1 Hz), 2.41 (m, 1H, H4e), 2.56 (m, 1H, H6e%,
2
3
ꢀ JHHꢀ 16.7, JHH 5.4 Hz), 3.45 (s, 1H, H8), 4.01 (m, 1H,
H5), 4.50 (m, 1H, H3) and 6.20 (m, 1H, H2); 13C NMR
(MeOH-d4, 75.5 MHz): l 39.5 and 41.5 (C4, C6), 66.3
and 67.7 (C3, C5), 78.2 (C8), 84.8 (C7), 120.5 (C1) and
139.3 (C2); MS (EI, m/z): 366 (M+, 1%), 351 (2), 309
(19), 285 (4), 234 (88), 178 (28), 151 (73) and 147 (49);
HRMS calcd for C8H10O2 (M+): 138.0681. Found:
138.0685. 6a: [h]2D0=+49.3 (c 0.50, MeOH). Anal. calcd
(%) for C8H10O2: C, 69.55; H, 7.3. Found: C, 69.3; H,
7.4. 7a: [h]2D0=−50.0 (c 0.30, MeOH). Anal. calcd (%)
for C8H10O2: C, 69.55; H, 7.3. Found: C, 69.6; H, 7.2.
4. Experimental12
Candida antarctica lipase B (CAL-B, 7300 PLU/g) and
C. viscosum lipase (CVL, 4918 U/mg protein) were a
gift from Novo Nordisk Co. and Genzyme, respec-
tively. Pseudomonas cepacia lipase (PSL, 30000 U/g)
and immobilized P. cepacia lipase (PSL-C, 783 U/g)
were purchased from Amano Pharmaceutical Co. Can-
dida rugosa lipase (CRL, 4570 U/mg protein) was pur-
chased from Sigma. Gas chromatography (GC) was
carried out with flame ionization detection (FID) and a
HP-1 capillary column (25 m×0.2 mm×0.33 mm) coated
with methylsilicone gum, with nitrogen as carrier gas
and following a method with injector and detector
temperatures set at 300°C, press head column 15 psi
and split 40:1, column initial temperature 150°C (3
min), rate 10°C/min until 228°C and then at 15°C/min
until 270°C (3 min). Acetanilide (as internal standard)
appeared at 4.91 min; 4a–5a at 4.27 min; 6a–7a at 4.29
min; 4b–5b at 6.89 min; 6b–7b at 6.78 min. Conversion
of the reaction was calculated by disappearance of the
starting material with respect to the internal standard.
4.2. Syntheses of methyl 3,5-dihydroxycyclohex-1-ene-
carboxylate 4b–7b
Using the same procedure as that described for 4a–7a
yielded 4b–7b from the corresponding silyl ether deriva-
tives previously described.5
4.2.1. Methyl (3S,5R)- and (3R,5S)-3,5-dihydroxycyclo-
hex-1-enecarboxylate 4b and 5b. Rf (90% EtOAc/hex):
0.1; IR (NaCl): w 3383, 1718 and 1648 cm−1; 1H NMR
2
(MeOH-d4, 300 MHz): l 1.95 (ddd, 1H, H4a, ꢀ JHH
ꢀ
3
2
13.2, JHH 6.4, 2.8 Hz), 2.09 (ddd, 1H, H4e, ꢀ JHHꢀ 13.2,
2
3
3JHH 8.1, 5.3 Hz), 2.35 (m, 1H, H6a%, ꢀ JHHꢀ 17.8, JHH
2
6.1 Hz), 2.79 (m, 1H, H6e%, ꢀ JHHꢀ 17.8 Hz), 3.94 (s, 3H,
4.1. Syntheses of 1-ethynyl-3,5-dihydroxycyclohex-1-ene
4a–7a
H8), 4.32 (m, 1H, H5), 4.66 (m, 1H, H3) and 7.06 (m,
1H, H2); 13C NMR (MeOH-d4, 75.5 MHz): l 34.2 and
39.5 (C4, C6), 52.7 (C8), 65.1 and 65.3 (C3, C5), 130.4
(C1), 140.9 (C2) and 169.2 (C7); MS (EI, m/z): 172
(M+, 1%), 155 (3), 154 (29), 140 (36), 126 (45) and 95
(100); HRMS calcd for C8H8O2 (M+−2H2O): 136.0524.
Found: 136.0495. 4b: [h]2D0=−106.0 (c 0.38, MeOH).
Anal. calcd (%) for C8H12O4: C, 55.81; H, 7.02. Found:
C, 55.9; H, 7.1. 5b: [h]2D0=+103.0 (c 0.45, MeOH).
Anal. calcd (%) for C8H12O4: C, 55.81; H, 7.02. Found:
C, 55.7; H, 7.1.
To
a solution of the corresponding 3,5-di[(tert-
butyldimethylsilyl)oxy]-1-ethynylcyclohex-1-ene previ-
ously described5 (500 mg, 1.36 mmol) in MeOH (10
mL) was added three drops of HClconc. The reaction
mixture was stirred at rt during 3 h and then solid
NaHCO3 was added. Solvent was evaporated and the
residue was dissolved in EtOAc. After separation of the
solid by filtration, the filtrate was evaporated to afford
diols 4a–7a (yields >90%).
4.2.2. Methyl (3S,5S)- and (3R,5R)-3,5-dihydroxycyclo-
hex-1-enecarboxylate 6b and 7b. Rf (90% EtOAc/hex):
0.1; IR (NaCl): w 3391, 2953, 2252, 1712 and 1652 cm−1;
1H NMR (MeOH-d4, 200 MHz): l 1.68 (ddd, 1H, H4a,
4.1.1. (3S,5R)- and (3R,5S)-1-Ethynyl-3,5-dihydroxycy-
clohex-1-ene 4a and 5a. Rf (80% EtOAc/hex): 0.2; IR
1
(Nujol): w 3320 and 1648 cm−1; H NMR (MeOH-d4,
2
3
200 MHz): l 1.99 (dd, 2H, 2H4, JHH 6.5, 6.5 Hz), 2.23
ꢀ JHHꢀ 11.8, JHH 11.8, 9.8 Hz), 2.25 (dddd, 1H, H6a%,
2
2
3
(dddd, 1H, H6a%, ꢀ JHHꢀ 17.3, 3JHH 6.8, JHH 1.8, 1.8 Hz),
ꢀ JHHꢀ 17.0, JHH 9.2, JHH 3.6, 2.7 Hz), 2.45 (m, 1H,
2
3
2
3
2.63 (m, 1H, H6e%, ꢀ JHHꢀ 17.3, JHH 4.8 Hz), 3.46 (s,
1H, H8), 4.27 (m, 1H, H5), 4.55 (m, 1H, H3) and 6.29
(m, 1H, H2); 13C NMR (MeOH-d4, 75.5 MHz): l 39.1
and 39.8 (C4, C6), 64.5 and 65.5 (C3, C5), 78.1 (C8),
85.1 (C7), 121.5 (C1) and 137.4 (C2); MS (EI, m/z): 366
H6e%, ꢀ JHHꢀ 17.0, JHH 5.6, JHH 3.2, 1.7 Hz), 2.85 (m,
2
3
1H, H4e, ꢀ JHHꢀ 11.8, JHH 4.0 Hz), 3.93 (s, 3H, H8),
4.03 (dddd, 1H, H5, 3JHH 9.8, 5.9, 3.3, 2.1 Hz), 4.58 (m,
1H, H3) and 7.06 (m, 1H, H2); 13C NMR (MeOH-d4,
75.5 MHz): l 34.8 and 41.5 (C4, C6), 52.7 (C8), 66.7

544
M. D´ıaz et al. / Tetrahedron: Asymmetry 13 (2002) 539–546
and 67.7 (C3, C5), 129.9 (C1), 142.6 (C2) and 168.9
(C7); MS (EI, m/z): 172 (M+, <1%), 154 (1) and 43
(100); HRMS calcd for C8H12O4 (M+): 172.0735.
Found: 172.0739. 6b: [h]2D0=+29.0 (c 0.81, MeOH).
Anal. calcd (%) for C8H12O4: C, 55.81; H, 7.02. Found:
C, 55.7; H, 6.9. 7b: [h]D20=–27.0 (c 1.05, MeOH). Anal.
calcd (%) for C8H12O4: C, 55.81; H, 7.02. Found: C,
55.8; H, 7.1.
H8), 4.44 (m, 1H, H3), 5.20 (m, 1H, H5) and 6.21 (m,
1H, H2); 13C NMR (CDCl3, 75.5 MHz): l 21.1 (C10),
34.2 and 35.2 (C4, C6), 64.2 and 66.8 (C3, C5), 77.1
(C8), 83.2 (C7), 119.5 (C1), 136.4 (C2) and 170.5 (C9);
MS (EI, m/z): 180 (M+, <1%), 165 (3), 162 (34), 151
(17), 137 (34), 120 (93), 102 (19), 92 (38) and 91 (100);
HRMS calcd for C10H12O3 (M+): 180.0786. Found:
180.0794. 13a: Anal. calcd (%) for C10H12O3: C, 66.65;
H, 6.71. Found: C, 66.4; H, 6.8. 16a: Anal. calcd (%)
for C10H12O3: C, 66.65; H, 6.71. Found: C, 66.5; H, 6.6.
4.3. Enzymatic acylation of 4–7. Syntheses of acetates
12–23
4.3.4. Methyl (3S,5R)- and (3R,5S)-5-acetoxy-3-hydroxy-
i
In a typical procedure, a lipase (15 mg of CAL-B, CRL,
or PSL; or 9 mg of CVL) was added to a solution 0.055
M of diol 4–7 (4a–7a: 15 mg, 0.11 mmol; 4b–7b: 19 mg,
0.11 mmol) in vinyl acetate (2 mL, acetanilide is present
as internal standard in 0.013 M) under nitrogen. Tem-
perature, other solvents or acylating agent and reaction
time are given in Tables 1–4. The suspension was
shaken at 250 rpm and the progress of the reaction was
followed by GC analysis. Close to 100% conversion the
mixture was filtered and the solvent was removed under
cyclohex-1-enecarboxylate 13b and 16b. Rf (90% PrOH/
1
hex): 0.3; IR (NaCl): w 3391, 1712 and 1652 cm−1; H
2
NMR (CDCl3, 200 MHz): l 1.80 (ddd, 1H, H4a, ꢀ JHH
ꢀ
3
13.0, JHH 7.3, 2.8 Hz), 2.04 (s, 3H, H10), 2.07–2.77 (m,
2
2H, H4e+H6), 2.68 (m, 1H, H6, ꢀ JHHꢀ 18.2 Hz), 3.76 (s,
3H, H8), 4.55 (m, 1H, H3), 5.26 (m, 1H, H5) and 6.95
(m, 1H, H2); 13C NMR (CDCl3, 75.5 MHz): l 21.2
(C10), 29.7 and 35.0 (C4, C6), 52.0 (C8), 64.1 and 67.3
(C3, C5), 128.7 (C1), 139.2 (C2), 166.9 (C7) and 170.4
(C9); MS (EI, m/z): 214 (M+, <1%), 182 (7), 172 (14),
154 (88), 139 (32), 126 (32), 111 (20) and 95 (100);
HRMS calcd for C8H12O4 (M+): 214.0841. Found:
214.0843. 13b: Anal. calcd (%) for C10H14O5: C, 56.07;
H, 6.54. Found: C, 56.0; H, 6.6. 16b: Anal. calcd (%)
for C10H14O5: C, 56.07; H, 6.54. Found: C, 56.1; H, 6.4.
1
reduced pressure. After H NMR analysis, the crude
material was subjected to flash chromatography (gradi-
ent eluent 5–30% EtOAc/hexane) to give compounds
12–23.
4.3.1. (3S,5R)- and (3R,5S)-3-Acetoxy-1-ethynyl-5-
i
hydroxycyclohex-1-ene 12a and 15a. Rf (5% PrOH/hex):
4.3.5. Methyl (3S,5R)- and (3R,5S)-di(acetoxy)cyclohex-
1
0.4; IR (NaCl): w 3391, 1712 and 1652 cm−1; H NMR
i
1-enecarboxylate 14b and 17b. Rf (5% PrOH/hex): 0.8;
(CDCl3, 200 MHz): l 1.55–1.95 (m, 2H, 2H4), 2.04 (s,
1
IR (NaCl): w 1722 and 1657 cm−1; H NMR (CDCl3,
2
3H, H10), 2.18 (m, 1H, H6), 2.56 (m, 1H, H6, ꢀ JHH
ꢀ
2
3
200 MHz): l 1.90 (ddd, 1H, H4a, ꢀ JHHꢀ 13.6, JHH 6.7,
3
17.4, JHH 4.8 Hz), 2.94 (s, 1H, H8), 4.15 (m, 1H, H5),
5.46 (m, 1H, H3) and 6.15 (m, 1H, H2); 13C NMR
(CDCl3, 75.5 MHz): l 21.2 (C10), 35.6 and 37.8 (C4,
C6), 63.5 and 67.4 (C3, C5), 77.6 (C8), 83.2 (C7), 122.5
(C1), 131.7 (C2) and 170.3 (C9); MS (EI, m/z): 180
(M+, <1%), 165 (3), 162 (22), 151 (8), 137 (12), 120
(100) and 91 (97); HRMS calcd for C10H12O3 (M+):
180.0786. Found: 180.0779.
3.1 Hz), 2.03–2.20 (m, 1H, H4e), 2.06 (s, 3H, H10), 2.09
2
3
(s, 3H, H10%), 2.35 (m, 1H, H6, ꢀ JHHꢀ 18.5, JHH 4.9
2
3
Hz), 2.74 (m, 1H, H6, ꢀ JHHꢀ 18.5, JHH 5.3 Hz), 3.78 (s,
3H, H8), 5.25 (m, 1H, H5), 5.60 (m, 1H, H3) and 6.85
(m, 1H, H2); 13C NMR (CDCl3, 75.5 MHz): l 21.0 and
21.1 (C10, C10%), 29.7 and 31.9 (C4, C6), 52.0 (C8), 66.5
and 66.8 (C3, C5), 130.8 (C1), 134.9 (C2), 166.4 (C7)
and 170.3 (C9, C9%); MS (ESI+, m/z): 279 [M+Na]+.
4.3.2. Methyl (3S,5R)- and (3R,5S)-3-acetoxy-5-hydroxy-
i
4.3.6. (3S,5S)- and (3R,5R)-3-acetoxy-1-ethynyl-5-
cyclohex-1-enecarboxylate 12b and 15b. Rf (90% PrOH/
i
1
hex): 0.3; IR (NaCl): w 3391, 1712 and 1652 cm−1; H
hydroxycyclohex-1-ene 18a and 21a. Rf (5% PrOH/hex):
1
0.4; IR (NaCl): w 3391, 1712 and 1652 cm−1; H NMR
NMR (CDCl3, 200 MHz): l 1.97 (m, 1H, H4), 2.06 (s,
3H, H10), 2.07–2.40 (m, 2H, H4%+H6), 2.75 (m, 1H,
2
(CDCl3, 200 MHz): l 1.82 (ddd, 1H, H4a, JHH 13.1,
3JHH 8.7, 6.9 Hz), 2.07 (s, 3H, H10), 2.22 (m, 1H, H4e),
2
H6, ꢀ JHHꢀ 18.2 Hz), 3.74 (s, 3H, H8), 4.20 (m, 1H, H5),
2
5.58 (m, 1H, H3) and 6.84 (m, 1H, H2); 13C NMR
(CDCl3, 75.5 MHz): l 21.0 (C10), 32.9 and 35.1 (C4,
C6), 51.9 (C8), 64.3 and 67.2 (C3, C5), 131.4 (C1),
134.6 (C2), 166.8 (C7) and 170.3 (C9); MS (EI, m/z):
214 (M+, <1%), 182 (7), 172 (7), 154 (90), 139 (68), 126
(67), 122 (70), 111 (42) and 95 (100); HRMS calcd for
C9H10O4 (M+−MeOH): 182.0579. Found: 182.0576.
12b: Anal. calcd (%) for C10H14O5: C, 56.07; H, 6.54.
Found: C, 56.1; H, 6.6.
2.28 (m, 1H, H6a%, ꢀ JHHꢀ 17.3, JHH 6.8 Hz), 2.49 (m,
2
1H, H6e%, ꢀ JHHꢀ 17.3, JHH 5.0 Hz), 2.95 (s, 1H, H8),
4.06 (m, 1H, H5), 5.45 (m, 1H, H3) and 6.12 (m, 1H,
H2); 13C NMR (CDCl3, 75.5 MHz): l 21.1 (C10), 35.3
and 37.6 (C4, C6), 64.3 and 67.6 (C3, C5), 77.7 (C8),
83.1 (C7), 121.5 (C1), 136.5 (C2) and 170.2 (C9); MS
(EI, m/z): 180 (M+, <1%), 165 (1), 162 (10), 151 (3), 137
(4), 120 (100), 102 (21), 92 (22) and 91 (81); HRMS
calcd for C8H12O4 (M+): 180.0786. Found: 180.0781.
21a: Anal. calcd (%) for C10H12O3: C, 66.65; H, 6.71.
Found: C, 66.8; H, 6.6.
4.3.3. (3S,5R)- and (3R,5S)-5-Acetoxy-1-ethynyl-3-
i
hydroxycyclohex-1-ene 13a and 16a. Rf (5% PrOH/hex):
1
0.4; IR (NaCl): w 3391, 1712 and 1652 cm−1; H NMR
4.3.7. Methyl (3S,5S)- and (3R,5R)-3-acetoxy-5-hydroxy-
2
i
(CDCl3, 200 MHz): l 1.83 (m, 1H, H4, ꢀ JHHꢀ 13.8, JHH
cyclohex-1-enecarboxylate 18b and 21b. Rf (90% PrOH/
1
6.4, 3.6 Hz), 1.93–2.32 (m, 2H, H4%+H6), 2.04 (s, 3H,
hex): 0.3; IR (NaCl): w 3391, 1712 and 1652 cm−1; H
2
H10), 2.57 (m, 1H, H6, ꢀ JHHꢀ 17.9 Hz), 2.90 (s, 1H,
NMR (CDCl3, 200 MHz):
l
1.78 (ddd, 1H,

M. D´ıaz et al. / Tetrahedron: Asymmetry 13 (2002) 539–546
545
2
3
H4a, ꢀ JHHꢀ 12.6, JHH 9.8, 7.8 Hz), 2.09 (s, 3H, H10),
3JHH 10.0, 7.7 Hz), 2.08 (s, 3H, H10), 2.11 (s, 3H,
H10%), 2.20–2.80 (m, 2H, H4e+H6), 2.74 (m, 1H, H6,
2
2.70 (m, 1H, H4e, ꢀ JHHꢀ 12.6, JHH 6.3 Hz), 2.33 (m,
2
2
1H, H6a%, ꢀ JHHꢀ 17.6, JHH 7.6 Hz), 2.69 (m, 1H, H6e%,
ꢀ JHHꢀ 17.7, JHH 3.6 Hz), 3.75 (s, 3H, H8), 5.15 (m,
2
ꢀ JHHꢀ 17.6, JHH 5.2 Hz), 3.76 (s, 3H, H8), 4.06 (m,
1H, H5), 5.57 (m, 1H, H3) and 6.80 (m, 1H, H2); 13C
NMR (CDCl3, 75.5 MHz): l 20.9 and 21.0 (C10,
C10%), 29.8 and 32.3 (C4, C6), 52.0 (C8), 66.6 and
67.0 (C3, C5), 130.3 (C1), 135.6 (C2), 166.2 (C7) and
170.1 and 170.2 (C9, C9%); MS (ESI+, m/z): 279 [M+
Na]+.
1H, H5), 5.53 (m, 1H, H3) and 6.80 (m, 1H, H2); 13C
NMR (CDCl3, 75.5 MHz): l 21.0 (C10), 33.2 and
35.8 (C4, C6), 52.0 (C8), 64.8 and 67.8 (C3, C5),
130.9 (C1), 135.4 (C2), 166.6 (C7) and 170.2 (C9); MS
(EI, m/z): 214 (M+, <1%), 182 (13), 172 (33), 154
(100) and 93 (85); HRMS calcd for C10H14O5 (M+):
214.0841. Found: 214.0843. 21b: Anal. calcd (%) for
C10H14O5: C, 56.07; H, 6.54. Found: C, 56.0; H, 6.6.
Acknowledgements
4.3.8. (3S,5S)- and (3R,5R)-5-Acetoxy-1-ethynyl-3-
i
hydroxycyclohex-1-ene 19a and 22a. Rf (5% PrOH/
We express our appreciation to Novo Nordisk Co.
and Genzyme for the generous gift of the lipase CAL-
B and CVL, respectively. Financial support from Prin-
cipado de Asturias (Spain; Project GE-EXP01-03) and
MCYT (Spain; Project PPQ-2001-2683) is gratefully
acknowledged. S.F. also thanks MCYT (Spain) for a
personal grant (Ramo´n y Cajal Program).
hex): 0.4; IR (NaCl): w 3391, 1712 and 1652 cm−1; H
NMR (CDCl3, 200 MHz): l 1.90 (ddd, 1H, H4a,
1
2
3
ꢀ JHHꢀ 13.8, JHH 8.4, 6.1 Hz), 2.06 (s, 3H, H10), 2.13
2
2
(m, 1H, H4e, ꢀ JHHꢀ 13.8 Hz), 2.32 (m, 1H, H6, ꢀ JHH
ꢀ
2
17.8 Hz), 2.48 (m, 1H, H6, ꢀ JHHꢀ 17.8 Hz), 2.92 (s,
1H, H8), 4.34 (m, 1H, H3), 5.12 (m, 1H, H5) and
6.24 (m, 1H, H2); 13C NMR (CDCl3, 75.5 MHz): l
21.2 (C10), 34.0 and 35.1 (C4, C6), 64.5 and 67.2 (C3,
C5), 77.3 (C8), 83.2 (C7), 118.5 (C1), 136.8 (C2) and
170.1 (C9); MS (EI, m/z): 180 (M+, 1%), 162 (2), 149
(2), 137 (3), 121 (30), 120 (98), 102 (28), 92 (89) and
91 (100); HRMS calcd for C8H12O4 (M+): 180.0786.
Found: 180.0782. 19a: Anal. calcd (%) for C10H12O3:
C, 66.65; H, 6.71. Found: C, 66.5; H, 6.8.
References
1. (a) Vitamin D; Feldman, D.; Glorieux, F. H.; Pike, J. W.,
Eds.; Academic Press: New York, 1997; (b) Ettinger, R.
A.; DeLuca, H. F. Adv. Drug Res. 1996, 28, 269–312; (c)
Bouillon, R.; Okamura, W. H.; Norman, A. W.
Endocrine Rev. 1995, 16, 200–257.
2. (a) Lowe, K. E.; Maiyar, A. C.; Norman, A. W. Crit.
Rev. Eukar. Gene Exp. 1992, 2, 65–109; (b) Pike, J. W.
Annu. Rev. Nutr. 1991, 11, 189–216; (c) Vitamin D: Gene
Regulation, Structure–Function Analysis and Clinical
Application; Norman, A. W.; Bouillon, R.; Thomasset,
M., Eds.; Walter de Gruyter: Berlin, 1991; (d) Norman,
A. W. Vitamin D: The calcium Homeostatic Steroid Hor-
mone; Academic Press: New York, 1979.
3. (a) Norman, A. W.; Nemere, I.; Zhou, L. X.; Bishop, J.
E.; Lowe, K. E.; Maiyar, A. C.; Collins, E. D.; Taoka,
T.; Sergeev, I.; Farach-Carson, M. C. J. Steroid Biochem.
Mol. Biol. 1992, 41, 231–240; (b) Deboland, A. R.;
Nemere, I.; Norman, A. W. Biochem. Biophys. Res. Com-
mun. 1990, 166, 217–222.
4.3.9. Methyl (3S,5S)- and (3R,5R)-3-acetoxy-5-
hydroxycyclohex-1-enecarboxylate 19b and 22b. Rf
i
(90% PrOH/hex): 0.3; IR (NaCl): w 3391, 1712 and
1652 cm−1; H NMR (CDCl3, 200 MHz): l 1.90 (ddd,
1
2
3
1H, H4a, ꢀ JHHꢀ 13.0, JH2H 8.9, 6.7 Hz), 2.05 (s, 3H,
H10), 2.17 (m, 1H, H4e, ꢀ JHHꢀ 13.0 Hz), 2.45 (m, 1H,
2
2
H6, ꢀ JHHꢀ 18.0, JHH 6.5 Hz), 2.60 (m, 1H, H6, ꢀ JHH
ꢀ
18.0, JHH 5.2 Hz), 3.76 (s, 3H, H8), 4.45 (m, 1H, H3),
5.15 (m, 1H, H5) and 6.95 (m, 1H, H2); 13C NMR
(CDCl3, 75.5 MHz): l 21.2 (C10), 29.7 and 35.3 (C4,
C6), 52.0 (C8), 64.8 and 67.5 (C3, C5), 128.2 (C1),
139.2 (C2), 166.8 (C7) and 170. 1 (C9); MS (ESI+,
m/z): 237 [M+Na]+. 19b: Anal. calcd (%) for
C10H14O5: C, 56.07; H, 6.54. Found: C, 56.0; H, 6.5.
4. Okamura, W. H.; Midland, M. M.; Norman, A. W.;
Hammond, M. W.; Dormanen, M. C.; Nemere, I. Ann.
N.Y. Acad. Sci. 1995, 761, 344–348.
5. (a) D´ıaz, M.; Ferrero, M.; Ferna´ndez, S.; Gotor, V. J.
Org. Chem. 2000, 65, 5647–5652; (b) D´ıaz, M.; Ferrero,
M.; Ferna´ndez, S.; Gotor, V. Tetrahedron Lett. 1999, 41,
775–779.
6. (a) Carrea, G.; Riva, S. Angew. Chem., Int. Ed. Engl.
2000, 39, 2226–2254; (b) Faber, K. Biotransformations in
Organic Chemistry, 4th ed.; Springer-Verlag: Berlin, 2000;
(c) Bornscheuer, U. T.; Kazlauskas, R. J. Hydrolases in
Organic Synthesis: Regio- and Stereoselective Biotransfor-
mations; Wiley-VCH: Weinheim, 1999.
4.3.10. (3S,5S)- and (3R,5R)-di(acetoxy)-1-ethynylcy-
i
clohex-1-ene 20a and 23a. Rf (5% PrOH/hex): 0.8; IR
1
(NaCl): w 1718 and 1662 cm−1; H NMR (CDCl3, 200
2
3
MHz): l 1.93 (ddd, 1H, H4a, ꢀ JHHꢀ 13.1, JHH 9.5,
7.2 Hz), 2.07 (s, 6H, H10+H10%), 2.15–2.40 (m, 2H,
2
H4e+H6), 2.56 (m, 1H, H6, ꢀ JHHꢀ 17.2 Hz), 2.91 (s,
1H, H8), 5.14 (m, 1H, H5), 5.45 (m, 1H, H3) and
6.09 (m, 1H, H2); 13C NMR (CDCl3, 75.5 MHz): l
21.2 (C10, C10%), 32.0 and 34.2 (C4, C6), 66.3 and
66.7 (C3, C5), 77.3 (C8), 83.2 (C7), 118.7 (C1), 132.1
(C2) and 170.3 (C9, C9%); MS (ESI+, m/z): 245 [M+
Na]+.
7. (a) Gijsen, H. J. M.; Qiao, L.; Fitz, W.; Wong, C.-H.
Chem. Rev. 1996, 96, 443–473; (b) Drueckhammer, D. G.;
Hennen, W. J.; Pederson, R. L.; Barbas, C. F., III;
Gautheron, C. M.; Krach, T.; Wong, C.-H. Synthesis
1991, 499–525.
4.3.11. Methyl (3S,5S)- and (3R,5R)-5-di(acetoxy)
i
cyclohex-1-enecarboxylate 20b and 23b. Rf (5% PrOH/
1
hex): 0.4; IR (NaCl): w 1715 and 1657 cm−1; H NMR
2
(CDCl3, 200 MHz): l 1.90 (ddd, 1H, H4a, ꢀ JHHꢀ 12.6,

546
M. D´ıaz et al. / Tetrahedron: Asymmetry 13 (2002) 539–546
8. Ferrero, M.; Gotor, V. In Stereoselective Biocatalysis;
11. Ferna´ndez, S.; Ferrero, M.; Gotor, V.; Okamura, W. H.
J. Org. Chem. 1995, 60, 6057–6061.
12. Structures of the products are numbered as follows:
Patel, R. N., Ed.; Marcel Dekker: New York, 2000;
Chapter 20, pp. 579–631.
9. Ferrero, M.; Gotor, V. Chem. Rev. 2000, 100, 4319–4347.
10. (a) D´ıaz, M.; Gotor-Ferna´ndez, V.; Ferrero, M.; Ferna´n-
dez, S.; Gotor, V. J. Org. Chem. 2001, 66, 4227–4232; (b)
Gotor-Ferna´ndez, V.; Ferrero, M.; Ferna´ndez, S.; Gotor,
V. J. Org. Chem. 1999, 64, 7504–7510; (c) Ferrero, M.;
Ferna´ndez, S.; Gotor, V. J. Org. Chem. 1997, 62, 4358–
4363.
8
8
7
7
CO2Me
1
1
2
2
6
6
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O
O
O
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O
O
3
5
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9
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9'
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