Synthesis of monoacyl A-ring precursors of 1α,25-dihydroxyvitamin D3 through selective enzymatic hydrolysis

Article abstract of DOI:10.1021/jo010941+

An efficient synthesis of monoacylated 1α,25-dihydroxyvitamin D₃ A-ring precursors 15, 16, 18, and 19 has been described through an enzymatic hydrolysis process. Candida antarctica A lipase (CAL-A) hydrolyzes the C-5 acetate ester in trans stereoisomers 9 and 13, with complete and high selectivity, respectively. In the case of cis isomers 11 and 14, Chromobacterium viscosum lipase (CVL) is the enzyme of choice, exhibiting opposite selectivity for these two enantiomers. This lipase selectively catalyzes the hydrolysis at the C-3 acetate in diester 11 and at C-5 position in diester 14. It is noteworthy that through a hydrolysis reaction CAL-A and CVL allow the synthesis of the four A-ring monoacetylated precursors of 1α,25-dihydroxyvitamin D₃, precursors which are complementary to those obtained by the enzymatic acylation process. In addition, with excellent yield CVL selectively hydrolyzes the C-3 chloroacetate ester instead of the C-5 acetate in diester 22, a key intermediate in the synthesis of new A-ring modified 1α,25-dihydroxyvitamin D₃ analogues.

Full text of DOI:10.1021/jo010941+

1266
J . Org. Chem. 2002, 67, 1266-1270
Syn th esis of Mon oa cyl A-Rin g P r ecu r sor s of
1r,25-Dih yd r oxyvita m in D3 th r ou gh Selective En zym a tic
Hyd r olysis
Vicente Gotor-Ferna´ndez, Miguel Ferrero, Susana Ferna´ndez, and Vicente Gotor*
Departamento de Quı´mica Orga´nica e Inorga´nica, Facultad de Qu´ımica, Universidad de Oviedo,
33071-Oviedo, Spain
VGS@sauron.quimica.uniovi.es
Received September 24, 2001
An efficient synthesis of monoacylated 1R,25-dihydroxyvitamin D3 A-ring precursors 15, 16, 18,
and 19 has been described through an enzymatic hydrolysis process. Candida antarctica A lipase
(CAL-A) hydrolyzes the C-5 acetate ester in trans stereoisomers 9 and 13, with complete and high
selectivity, respectively. In the case of cis isomers 11 and 14, Chromobacterium viscosum lipase
(CVL) is the enzyme of choice, exhibiting opposite selectivity for these two enantiomers. This lipase
selectively catalyzes the hydrolysis at the C-3 acetate in diester 11 and at C-5 position in diester
14. It is noteworthy that through a hydrolysis reaction CAL-A and CVL allow the synthesis of the
four A-ring monoacetylated precursors of 1R,25-dihydroxyvitamin D3, precursors which are
complementary to those obtained by the enzymatic acylation process. In addition, with excellent
yield CVL selectively hydrolyzes the C-3 chloroacetate ester instead of the C-5 acetate in diester
22, a key intermediate in the synthesis of new A-ring modified 1R,25-dihydroxyvitamin D3 analogues.
Ch a r t 1
In tr od u ction
The potential of enzymes in organic chemistry is well
recognized.1 The chiral nature of enzymes results in the
stereo- and regiospecific formation of products with
remarkable rate acceleration. Substrates with several
functional groups of similar reactivity can be selectively
manipulated by enzymatic acylation and/or deacylation
processes. Direct chemical methods possess low regiose-
lectivities, and the procedures often necessarily involve
low temperatures and long reaction times.
In addition to its classical calciotropic effects,2 1R,25-
dihydroxyvitamin D3 [2, 1R,25-(OH)2-D3] (Chart 1), the
major active metabolite of vitamin D3 (1), has also been
shown to possess immunosuppressive activity,3 to inhibit
cellular proliferation, and induce cellular differentiation.4
As a result, considerable effort has been made toward
the synthesis of structurally related congeners that show
interesting pharmacological applications.5 Most of the
(1) (a) Koeller, K. M.; Wong, C.-H. Nature 2001, 409, 232-240. (b)
Klibanov, A. M. Nature 2001, 409, 241-246. (c) Roberts, S. M.; Turner,
N. J .; Willetts, A. J .; Turner, M. K. Introduction to Biocatalysis Using
Enzymes and Microorganisms; Cambridge University Press: New
York, 2000. (d) Faber, K. Biotransformations in Organic Chemistry;
4th ed.; Springer-Verlag: Berlin, 2000. (e) Carrea, G.; Riva, S. Angew.
Chem., Int. Ed. 2000, 39, 2226-2254. (f) Bornscheuer, U. T.; Kazlaus-
kas, R. J . Hydrolases in Organic Synthesis: Regio- and Stereoselective
Biotransformations; Wiley-VCH: Weinheim, 1999.
(2) (a) Reichel, H.; Koeffler, H. P.; Norman, A. W. N. Engl. J . Med.
1989, 320, 980-991. (b) DeLuca, H. F. FASEB J . 1988, 2, 224-236.
(c) J ones, G.; Vriezen, D.; Lohnes, D.; Palda, V.; Edwards, N. S. Steroids
1987, 49, 29-53. (d) Ikekawa, N. Med. Res. Rev. 1987, 7, 333-366. (e)
DeLuca, H. F.; Schnoes, H. K. Annu. Rep. Med. Chem. 1984, 19, 179-
190.
analogues prepared have modifications on the upper side
chain, whereas A-ring manipulation is rare because of
difficulties in synthesis. These metabolites possess sev-
eral hydroxyl groups of similar reactivity, and as result
it is very difficult to discern between them from a
chemical point of view.
In our ongoing research related to the synthesis of
selectively modified vitamin D3 analogues, appropriate
routes for the preparation of the corresponding precursors
are required. In this way, we have described the selective
enzymatic acylation6 and alkoxycarbonylation7 of A-ring
(3) Casteels, K.; Bouillon, M.; Mathieu, W. C. Curr. Opin. Nephrol.
Hypertens. 1995, 4, 313-318.
(4) (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.
(5) (a) Zhu, G.-D.; Okamura, W. H. Chem. Rev. 1995, 95, 1877-
1952. (b) Dai, H.; Posner, G. H. Synthesis 1994, 1383-1398.
(6) Ferna´ndez, S.; Ferrero, M.; Gotor, V.; Okamura, W. H. J . Org.
Chem. 1995, 60, 6057-6061.
10.1021/jo010941+ CCC: $22.00 © 2002 American Chemical Society
Published on Web 01/26/2002

J . Org. Chem., Vol. 67, No. 4, 2002 1267
Sch em e 1a
Sch em e 2
Ta ble 1. En zym a tic Hyd r olysis of A-Rin g Syn th on
(3S,5R)-9
entry enzyme t (h) conv (%)a 3 (%)a 8 (%)a 15 (%)a
1
2
3
4
5
6
7
CVL
262
335
70
336
155
270
31
91
74
99
24
100
89
69
8
9
3
38
4
1
65
51
7
8
16
21
1
CAL-B
PSL-C
PPL
CRL
PLE
39
14
54
69
91
a
CAL-A
91
(a) Reference 8; (b) Ac2O, DMAP, Py, CH2Cl2, rt, 12 h (92%);
(c) AcOH, PPh3, DEAD, toluene, rt, 2 h (36% conv); (d) PNBA,
PPh3 DEAD, THF, rt, 1 h (87%); (e) NaOMe, MeOH, 2.5 h, 0 °C
(89%); (f) Ac2O, DMAP, Py, CH2Cl2, rt, 12 h (96%).
a
Calculated by 1H NMR and GC.
Sch em e 3
stereoisomeric synthons of 1R,25-(OH)2-D3 and 1R,25-
(OH)2-19-nor-pre-D3. With respect to the acylation reac-
tion6 of A-ring precursors 3-6 (Chart 1), Chromobac-
terium viscosum lipase (CVL) has selectively catalyzed
the acylation with vinyl esters of the C-5 hydroxyl of the
three stereoisomeric vitamin D A-ring synthons 3-5.
However, the stereoisomer 6 was acylated at the C-3
hydroxyl under the same conditions. Taking into account
the complementary behavior shown by enzymes in acyl-
ation and hydrolysis processes, here we report the lipase-
catalyzed hydrolysis of the corresponding diacyl sub-
strates 9, 11, 13, and 14 to prepare the monoacylated
derivatives of the A-ring, which are complementary to
those mentioned above.
from the diol 3, our aim was to find an enzyme that
hydrolyzes exclusively the ester group at the C-5 position
of 9 in order to obtain 15. The enzymatic hydrolysis
reactions were carried out at 30 °C using 0.1 M KH2PO4
(pH 7) in 1,4-dioxane as cosolvent (Scheme 2). In these
conditions, CVL showed low selectivity, isolating diol 3
as the major compound (entry 1, Table 1). When Candida
antarctica B lipase (CAL-B) was used, high regioselec-
tivity toward the hydrolysis of C-3 acetate was achieved,
with 8 being obtained (entry 2, Table 1). The long reaction
time required to reach moderate conversion in the
enzymatic hydrolysis made the enzymatic acylation
process more suitable from a synthetic point of view.
Other lipases such as immobilized Pseudomonas cepacia
(PSL-C), porcine pancreas (PPL), or Candida rugosa
(CRL) did not show any improvement in the hydrolysis
process (entries 3, 4, and 5, Table 1). Better results were
obtained when the reaction was carried out with porcine
liver esterase (PLE). This enzyme gave rise to a mixture
of regioisomers 15 and 8 in a ratio of approximately 4.3:
1, and traces of diol (entry 6, Table 1). However, total
selectivity toward the hydrolysis of the C-5 ester was
exhibited by Candida antarctica A lipase (CAL-A). This
lipase catalyzed exclusively the formation of monoacetate
15 after 31 h and 91% conversion (entry 7, Table 1).
En zym a tic Hyd r olysis of Dia ceta te 13. Reaction of
13 with phosphate buffer/1,4-dioxane in the presence of
CVL gave, after 130 h, diol 4 (Scheme 3) as the unique
reaction product (entry 1, Table 2). CAL-B, PSL-C, and
PPL showed poor reactivity, and no selectivity was
observed (entries 2, 3, and 4, Table 2). CRL and PLE
Resu lts a n d Discu ssion
First, diacetates 9, 11, 13, and 14 were synthesized.
Derivative 9 was obtained by acetylation of monoacetate
8, an intermediate en route to 1R,25-(OH)2-D3 A-ring
precursor 3 from (S)-(+)-carvone described by Okamura
et al.8 (Scheme 1). It was possible to prepare cis diester
11 directly, through the inversion of the allylic alcohol 8
under Mitsunobu9 conditions using acetic acid. Several
solvents, phosphines, and equivalents of reagents were
tried. However, although the process gave the desired
inversion, the yield of 11 was very poor, the highest
conversion achieved only reaching 36%. An alternative
approach to isomer 11 implies inversion of configuration
at C-3 in 8 using p-nitrobenzoic acid (PNBA) affording
p-nitrobenzoate ester 10 with total inversion of the
configuration and high yield. Deprotection of both ester
groups with NaOMe in MeOH, and subsequent acetyla-
tion with acetic anhydride, gave diacetate 11. Similarly,
diacyl derivatives 13 and 14 were obtained starting from
(R)-(-)-carvone.
(7) (a) Ferrero, M.; Ferna´ndez, S.; Gotor, V. J . Org. Chem. 1997,
62, 4358-4363. (b) Gotor-Ferna´ndez, V.; Ferrero, M.; Ferna´ndez, S.;
Gotor, V. J . Org. Chem. 1999, 64, 7504-7510. (c) D´ıaz, M.; Gotor-
Ferna´ndez, V.; Ferrero, M.; Ferna´ndez, S.; Gotor, V. J . Org. Chem.
2001, 66, 4227-4232.
(8) (a) Aurrecoechea, J . M.; Okamura, W. H. Tetrahedron Lett. 1987,
28, 4947-4950. (b) Okamura, W. H.; Aurrecoechea, J . M.; Gibbs, R.
A.; Norman, A. W. J . Org. Chem. 1989, 54, 4072-4083.
(9) Mitsunobu, O. Synthesis 1981, 1-28.
En zym a tic Hyd r olysis of Dia ceta te 9. Since CVL
catalyzes with excellent selectivity the formation of 8

1268 J . Org. Chem., Vol. 67, No. 4, 2002
Gotor-Ferna´ndez et al.
Ta ble 2. En zym a tic Hyd r olysis of A-Rin g Syn th on
Sch em e 5
(3R,5S)-13
entry enzyme T (° C) t (h) conv (%)a 4 (%)a 16 (%)a 17 (%)a
1
2
3
4
5
6
7
8
9
CVL
30
60
30
30
30
30
30
30
40
130
264
222
336
4
9
48
63
23
97
30
5
45
100
90
85
91
92
97
10
5
22
77
50
5
CAL-B
PSL-C
PPL
CRL
PLE
CAL-A
CAL-Ab
CAL-A
9
11
23
10
11
74
75
79
13
29
6
4
5
Ta ble 4. En zym a tic Hyd r olysis of A-Rin g Syn th on
(3R,5R)-11
12
8
entry enzyme T (°C) t (h) conv (%)a 6 (%)a 19 (%)a 20 (%)a
1
2
3
4
5
6
CVL
CVL
CVL
CAL-A
CAL-A
CAL-A
30
40
20
30
40
20
29
10
43
74
32
95
94
94
78
87
69
15
48
17
23
21
21
77
40
74
3
3
1
3
6
3
52
63
47
a
b
Calculated by 1H NMR and GC. Enzyme was added in two
portions: 55 mg at the beginning and 35 mg after 18 h of reaction.
Sch em e 4
144
a
Calculated by 1H NMR and GC.
Sch em e 6a
Ta ble 3. En zym a tic Hyd r olysis of A-Rin g Syn th on
(3S,5S)-14
entry enzyme
T (°C)
t (h) conv (%)a 5 (%)a 18 (%)a
1
2
3
4
CVL
30
30
25
52
130
81
97
31
96
93
3
94
31
88
86
CAL-A
CAL-Ab 30f40
8
7
CAL-A
40
a
b
Calculated by 1H NMR and GC. An extra fraction of 45 mg
was added at 52 h; after 74 h, temperature was increased up to
40 °C.
exhibited moderate regioselectivity toward the hydrolysis
of C-3 ester at low conversions; however, as the reaction
evolves, diol 4 was the major compound (entries 5 and 6,
Table 2). The best results were observed with CAL-A.
Thus, after 48 h, 85% conversion was achieved with a
selectivity of 74% for 16 (entry 7, Table 2). To enhance
that selectivity, the influence of temperature and the
amount of lipase were studied. Since the enzyme displays
Michaelis-Menten kinetics, it possesses its highest
activity at the beginning of the reaction. For this reason,
CAL-A was added in two portions (entry 8, Table 2)
during the process. Although a slight rise conversion was
obtained, the percentage of derivative 16 was the same.
When the process took place at 40 °C, a shorter reaction
time was observed for high conversion, in addi-
tion to a slight increase in the regioselectivity exhibited
by CAL-A, the ratio of C-3 monoacetate 16 being 79%
(entry 9, Table 2). CAL-A showed selectivity toward the
C-5 position for both enantiomers 9 and 13, although 9
displayed total selectivity and higher reactivity than 13.
En zym a tic Hyd r olysis of Dia ceta te 14. As in the
acylation process, CVL still maintained excellent selec-
tivity toward the C-5 position in the hydrolysis reaction
(Scheme 4). As shown in entry 1 of Table 3, when the
reaction was run at 30 °C, hydrolysis of the C-5 ester
occurred with almost complete regioselectivity, and only
trace amounts of diol 5 was observed. CAL-A also
revealed total selectivity toward the acetate at the C-5
position (entry 2, Table 3), but to get a higher conversion,
a temperature of 40 °C and more enzyme were used
(entries 3 and 4, Table 3). Under these conditions,
approximately 95% conversions were achieved with high
selectivities, although CVL still offered the best result.
a
(a) 0.1 M KH2PO4 (pH 7)/1,4-dioxane (10:2), 30 °C, CVL or
PSL-C; (b) ClCH2CO2H, PPh3, DEAD, THF, rt, 1 h (93%); (c) 0.1
M KH2PO4 (pH 7)/1,4-dioxane (10:2), 30 °C, CVL, 5 h (94%).
En zym a tic Hyd r olysis of Dia ceta te 11. Table 4 and
Scheme 5 summarize similar hydrolysis studies of the
cis stereoisomer 11. CVL catalyzed the hydrolysis of 11
at 30 °C with high selectivity toward the acyl group at
the C-3 position (entry 1, Table 4), in complete contrast
to its enantiomer 14, and in the same way as the
acylation reaction of the corresponding diol 6. When the
process was performed at 40 °C or 20 °C, the enzyme did
not exhibit any improvement in the selectivity (entries
2 and 3, Table 4). However, the opposite regioselectivity,
somewhat favoring the hydrolysis of the C-5 ester group,
was achieved when CAL-A was used as catalyst, although
conditions for selective hydrolysis at C-5 could not be
defined (entries 4, 5, and 6, Table 4).
Im p r oved Syn th esis of Mon oa ceta te 19. In the
synthesis of new 1R,25-(OH)2-D3 analogues carried out
in our laboratory, cleavage of p-nitrobenzoate ester 10
in the presence of acetate ester was necessary. As
indicated above, CVL catalyzed the hydrolysis of the C-3
ester in the corresponding diacetate derivative 11, with
high regioselectivity. When the same process was carried
out with diacyl 10, CVL hydrolyzed exclusively the
acetate group, isolating 21 as the unique reaction product
(Scheme 6). The enzyme probably cannot accommodate
a large group as p-nitrobenzoyl in the active site. The
same result was obtained with PSL-C, and because of
this drawback, we designed a new route to A-ring
synthon 19 through the inversion of 8 under Mitsunobu

J . Org. Chem., Vol. 67, No. 4, 2002 1269
organic phase was washed with 1 N HCl (3 × 5 mL). Solvent
was evaporated, and the residue was purified by flash chro-
matography (10% EtOAc/hexane) to afford 167 mg (92%) of
diacetate 9 as an oil. 1H NMR (CDCl3, 200 MHz): δ 1.88 (s,
3H, H9), 1.86-1.99 (m, 2H, H4), 2.03 (s, 3H, H11), 2.07 (s, 3H,
conditions using chloroacetic acid. The process took place
with excellent yield in 1 h to obtain 22. Subsequent
enzymatic hydrolysis with CVL of diacyl derivative 22
gave exclusively monoacetate 19 with 94% yield.
2
3
H
13), 2.20 (dd, 1H, H6, J HH 17.3, J HH 7.4 Hz), 2.63 (dd, 1H,
2
3
Su m m a r y
H6, J HH 16.8, J HH 4.2 Hz), 3.14 (s, 1H, H8), 5.07 (m, 1H, H5),
and 5.45 (t, 1H, H3, J HH 4.7 Hz); MS (ESI+, m/ z): 259 [(M +
3
Candida antarctica A lipase has catalyzed with high
selectivity the hydrolysis of the ester at the C-5 position
of 1R,25-(OH)2-D3 A-ring diacetate precursors 9, 13, and
14. However, this enzyme has exhibited low regioselec-
tivity with stereoisomer 11. Better results were obtained
for cis synthons 11 and 14 with Chromobacterium vis-
cosum lipase. This enzyme maintains the opposite selec-
tivity shown with respective diol enantiomers 6 and 5 in
the enzymatic acylation process: the hydrolysis is per-
formed at the C-5 ester for 14 and at the C-3 position in
case of 11. It is noteworthy that through a hydrolysis
reaction, both enzymes (CAL-A for trans isomers 9 and
13, and CVL for cis isomers 11 and 14) allowed the
synthesis of the four A-ring monoacetylated precursors
of 1R,25-(OH)2-D3, which are complementary to those
obtained by the acylation reaction. From this exhaustive
study, the selective deprotection of diacyl derivative 22,
a key intermediate in the synthesis of new A-ring
modified 1R,25-(OH)2-D3 analogues, has been carried out.
CVL hydrolyzed the C-3 chloroacetate ester instead of
the C-5 acetate selectively and with excellent yield.
Na)+, 100%].
En zym a tic Hyd r olysis of 9. In a standard procedure, CVL
(10 mg), CAL-B (90 mg), CRL (180 mg), PSL-C (90 mg), PLE
(20 µL), PPL (90 mg), or CAL-A (90 mg) was added to a
solution of diacetate 9 (20 mg, 0.085 mmol) in 2 mL of 0.1 M
KH2PO4 (pH 7)/1,4-dioxane (10:2). The suspension was shaken
at 250 rpm, and the progress of the reaction was followed by
GC analysis (conversions and percentages of compounds are
summarized in Table 1). The mixture was filtered, and the
solution was extracted with EtOAc (3 × 5 mL). The crude was
purified by flash chromatography (10-60% EtOAc/hexane) to
give compounds 3,6 8,6 and 15.
(3S,5R)-3-Acetoxy-1-eth yn yl-5-h yd r oxy-2-m eth ylcyclo-
h ex-1-en e (15).11 1H NMR (CDCl3, 200 MHz): δ 1.76-1.86 (m,
2H, H4), 1.88 (s, 3H, H9), 1.97-2.19 (m, 2H, H4+H6), 2.07 (s,
2
3
3H, H11), 2.54-2.61 (dd, 1H, H6, J HH 16.9, J HH 4.0 Hz), 3.14
(s, 1H, H8), 4.06 (m, 1H, H5), and 5.45 (m, 1H, H3).
Syn th esis of (3R,5R)-5-Acetoxy-1-eth yn yl-2-m eth yl-3-
[(4-n itr op h en yl)ca r bon yloxy]cycloh ex-1-en e (10). This
compound was prepared as previously reported.7c
Syn th esis of (3R,5R)-3,5-Dia cetoxy-1-eth yn yl-2-m eth -
ylcycloh ex-1-en e (11). A solution of MeONa in MeOH,
prepared in situ by addition of Na (67 mg, 2.93 mmol) to MeOH
(2.5 mL), was added dropwise to a solution of 10 (430 mg, 1.25
mmol) in MeOH (14 mL) at 0 °C. The reaction was stirred at
this temperature for 2.5 h and then acidified with Dowex
50WX4-400 ion-exchange resin (200-400 mesh). After re-
moval of the resin by filtration, the solution was evaporated,
and the residue was purified by flash chromatography (50%
EtOAc/hexane) to afford 170 mg (89%) of diol 6 as a white solid.
To a solution of 6 (145 mg, 0.95 mmol) in CH2Cl2 (12 mL) were
added pyridine (308 mL, 3.81 mmol), DMAP (23 mg, 0.38
mmol), and Ac2O (180 µL, 3.81 mmol). The mixture was stirred
at room temperature for 12 h, after which it was poured into
water (10 mL), and the organic phase was washed with 1 N
HCl (3 × 5 mL). Solvent was evaporated, and the residue was
purified by flash chromatography (10% EtOAc/hexane) to
Exp er im en ta l Section 10
Gen er a l. Chromobacterium vicosum lipase (CVL, 3800
U/mg of solid) and Candida antarctica B lipase (CAL-B, 7300
PLU/g) were a gift from Genzyme and Novo Nordisk Co.,
respectively. Immobilized Pseudomonas cepacia lipase (PSL-
C, 783 U/g) was obtained from Amano Pharmaceutical Co.
Porcine pancreas lipase (PPL, type II, 46 U/mg of protein using
triacetin), Candida rugosa lipase (CRL, typeVII, 950 U/mg of
solid), and porcine liver esterase [PLE, 260 U/mg of protein,
3.2 M (NH4)2SO4 solution pH 8] were purchased from Sigma.
Candida antarctica A lipase (CAL-A, chirazyme L-5, c-f, lyo.,
1000 U/g using tributyrin) was obtained from Roche. Reagents
were purchased from Aldrich, Fluka, Merck, or Sigma. Sol-
vents were distilled over an appropriate desiccant under
nitrogen. Gas chromatography (GC) was carried out with flame
ionization detection (FID) and a HP-1 capillary column (25 m
× 0.2 mm × 0.2 µm) coated with methylsilicone gum, with
nitrogen as carrier gas. In this method the injector and detector
temperatures were set at 250 °C and 275 °C, respectively,
column initial temperature was 80 °C (3 min), rate was 10
°C/min until 170 °C (5 min) and then 18 °C/min, column final
temperature was 260 °C; 3 and 4 appeared at 11.65 min; 5
and 6 at 11.55 min; 8 and 17 at 13.59 min; 9 and 13 at 15.08
min; 11 and 14 at 15.72 min; 15 and 16 at 13.41 min; 18 and
20 at 13.12 min; and 19 at 13.01 min.
1
afford 216 mg (96%) of diacetate 11 as a white solid. H NMR
(CDCl3, 200 MHz): δ 1.86 (s, 3H, H9), 1.86-2.00 (m, 1H, H4),
2.01 (s, 3H, H11), 2.05 (s, 3H, H13), 2.14 (ddd, 1H, H4, 2J HH 14.4,
3J HH 7.4, 3J HH 2.3 Hz), 2.19-2.56 (m, 2H, H6), 3.14 (s, 1H, H8),
3
4.96 (m, 1H, H5), and 5.43 (t, 1H, H3, J HH 5.9 Hz); MS (ESI+,
m/z): 259 [(M + Na)+, 100%].
En zym a tic Hyd r olysis of 11. The same procedure as that
described for the enzymatic hydrolysis of 9 yielded compounds
6,6 19, and 206 (conversions, percentages of compounds, and
temperatures are summarized in Table 4).
(3R,5R)-5-Acetoxy-1-eth yn yl-3-h yd r oxy-2-m eth ylcyclo-
h ex-1-en e (19).11 1H NMR (CDCl3, 300 MHz): δ 1.98-2.17
(m, 8H, H9+H11+H4), 2.33-2.54 (m, 2H, H6), 3.11 (s, 1H, H8),
4.09 (m, 1H, H3), and 5.19 (m, 1H, H5).
Syn th esis of (3R,5S)-3,5-Dia cetoxy-1-eth yn yl-2-m eth -
ylcycloh ex-1-en e (13). The same procedure as that described
for 9 yielded 13 (91%). Spectral data are identical to that of 9
given above.
Syn th esis of (3S,5R)-3,5-Dia cetoxy-1-eth yn yl-2-m eth -
ylcycloh ex-1-en e (9). To a solution of 8 (150 mg, 0.77 mmol)
in CH2Cl2 (12.5 mL) under nitrogen were added pyridine (187
µL, 2.32 mmol), DMAP (14 mg, 0.23 mmol), and Ac2O (73 µL,
1.54 mmol). The mixture was stirred at room temperature for
12 h, after which it was poured into water (10 mL), and the
En zym a tic Hyd r olysis of 13. The same procedure as that
described for enzymatic hydrolysis of 9 yielded compounds 4,6
16, and 176 (conversions, percentages of compounds, and
temperatures are summarized in Table 2). The spectral data
of compound 16 were identical to that of 15.
(10) Structures of the products are numbered as follows:
Syn th esis of (3S,5S)-3,5-Dia cetoxy-1-eth yn yl-2-m eth -
ylcycloh ex-1-en e (14). The same procedure as that described
(11) This A-ring synthon was previously described in the Supporting
Information of ref 6 as a minor compound.

1270 J . Org. Chem., Vol. 67, No. 4, 2002
Gotor-Ferna´ndez et al.
for 11 yielded 14 (91%). Spectral data are identical to that of
11 given above.
En zym a tic Hyd r olysis of 14. The same procedure as that
described for enzymatic hydrolysis of 9 yielded compounds 56
and 18 (conversions, percentages of compounds, and temper-
atures are summarized in Table 3).
(135 mg, 0.52 mmol), and diethyl azodicarboxylate (80 µL, 0.52
mmol). The mixture was stirred for 1 h at room temperature
and then evaporated under reduced pressure to leave a residue
which was purified by flash chromatography (10% EtOAc/
hexane) to obtain 68 mg (97%) of 22 as a white solid. 1H NMR
(CDCl3, 200 MHz): δ 1.89 (s, 3H, H9), 1.89-2.10 (m, 1H, H4),
2
3
(3S,5S)-3-Acetoxy-1-eth yn yl-5-h yd r oxy-2-m eth ylcyclo-
2.02 (s, 3H, H11), 2.14-2.25 (ddd, 1H, H4, J HH 13.7, J HH 6.1,
h ex-1-en e (18).11 1H NMR (CDCl3, 300 MHz): δ 1.87 (m, 1H,
3J HH 3.1 Hz), 2.28-2.57 (m, 2H, H6), 3.18 (s, 1H, H8), 4.04 (s,
2
3
H4), 1.90 (s, 3H, H9), 2.09 (s, 3H, H11), 2.13 (ddd, 1H, H4, J HH
2H, H13), 5.00 (m, 1H, H5), and 5.50 (t, 1H, H3, J HH 5.8 Hz);
3
3
14.4, J HH 5.8, J HH 3.0 Hz), 2.17-2.53 (m, 2H, H6), 3.15 (s,
MS (ESI+, m/z): 309 [(M + K)+, 15%], 295 [(M(37Cl) + Na)+,
15], and 293 [(M + Na)+, 40].
3
1H, H8), 4.03 (m, 1H, H5), and 5.46 (t, 1H, H3, J HH 5.7 Hz).
Syn th esis of (3R,5R)-5-Acetoxy-1-eth yn yl-3-h yd r oxy-
2-m eth ylcycloh ex-1-en e (19) fr om 22. The same procedure
as that described for enzymatic hydrolysis of 9 yielded
compound 19 (94%). Spectral data are given above.
Ack n ow led gm en t. We express our appreciation to
Novo Nordisk Co. and Genzyme for the generous gift of
the lipases CAL-B and CVL, respectively. Financial
support from CICYT (Spain; Project BIO98-0770) is
gratefully acknowledged. S.F. also thanks the Ministerio
de Educacio´n y Cultura (Spain) for her postdoctoral
fellowship. We thank Robin Walker for his revision of
the final manuscript.
Syn th esis of (3R,5R)-1-Eth yn yl-5-h yd r oxy-2-m eth yl-3-
[(4-n itr oph en yl)car bon yloxy]cycloh ex-1-en e (21). The same
procedure as that described for enzymatic hydrolysis of 9
yielded compound 21. The crude was purified by flash chro-
matography (10-25% EtOAc/hexane) as an oil. 1H NMR
(CDCl3, 400 MHz): δ 1.61 (br s, 1H, OH), 1.88-2.05 (m, 1H,
H4), 1.96 (s, 3H, H9), 2.32-2.60 (m, 3H, H4+H6), 3.21 (s, 1H,
H8), 4.11 (t, 1H, H5, 3J HH 7.0 Hz), 5.74 (t, 1H, H3, 3J HH 6.1 Hz),
Su p p or tin g In for m a tion Ava ila ble: Complete 1H and
13C NMR spectral data in addition to mp, IR, microanalysis,
optical rotation, and MS data for the new compounds. The level
3
3
8.20 (d, 2H, H13, J HH 2.0 Hz), and 8.29 (d, 2H, H12, J HH 2.0
Hz); MS (ESI+, m/z): 340 [(M + K)+, 7%] and 324 [(M + Na)+,
69].
1
of purity is indicated by the inclusion of copies of H and 13C
Syn th esis of (3R,5R)-5-Acetoxy-3-(ch lor oa cetoxy)-1-
eth yn yl-2-m eth yl-cycloh ex-1-en e (22). To a solution of 8
(50 mg, 0.26 mmol) in THF (3.5 mL) under nitrogen atmo-
sphere were added chloroacetic acid (49 mg, 0.52 mmol), PPh3
NMR spectra. This material is available free of charge via the
Internet at http://pubs.acs.org.
J O010941+