Selective Alkoxycarbonylation of A-Ring Precursors of Vitamin D Using Enzymes in Organic Solvents. Chemoenzymatic Synthesis of 1α,25-Dihydroxyvitamin D3 C-5 A-Ring Carbamate Derivatives

Article abstract of DOI:10.1021/jo970133b

A-Ring modification of 1α,25-dihydroxyvitamin D₃ [2, 1α,25-(OH)₂-D₃] is an important area of analog studies to investigate biological activity of vitamin D-related structures. An efficient synthesis of 1α,25-(OH)₂-D₃ C-5 A-ring carbamate derivatives 19 and amino acid derivatives 21 was developed by applying a two-step chemoenzymatic strategy, involving the enzymatic synthesis of carbonates followed by reaction with amino derivatives. Accordingly, we began the studies of enzymatic alkoxycarbonylation of 1α,25-(OH)₂-D₃ A-ring precursor 7. Candida antarctica lipase (CAL) was found to be the best catalyst in toluene. Regioselective alkoxycarbonylation occurred only at the C-5-(R) hydroxyl group. Good to excellent yields were achieved by chemical reaction of these carbonates with amino derivatives. The procedure provided convenient synthesis of carbonates 19 and 21 under mild reaction conditions.

Full text of DOI:10.1021/jo970133b

4358
J . Org. Chem. 1997, 62, 4358-4363
Selective Alk oxyca r bon yla tion of A-Rin g P r ecu r sor s of Vita m in D
Usin g En zym es in Or ga n ic Solven ts. Ch em oen zym a tic Syn th esis
of 1r,25-Dih yd r oxyvita m in D3 C-5 A-Rin g Ca r ba m a te Der iva tives1
Miguel Ferrero, Susana Ferna´ndez, and Vicente Gotor*
Departamento de Quı´mica Orga´nica e Inorga´nica, Universidad de Oviedo, 33071 Oviedo, Spain
Received J anuary 24, 1997X
A-Ring modification of 1R,25-dihydroxyvitamin D3 [2, 1R,25-(OH)2-D3] is an important area of analog
studies to investigate biological activity of vitamin D-related structures. An efficient synthesis of
1R,25-(OH)2-D3 C-5 A-ring carbamate derivatives 19 and amino acid derivatives 21 was developed
by applying a two-step chemoenzymatic strategy, involving the enzymatic synthesis of carbonates
followed by reaction with amino derivatives. Accordingly, we began the studies of enzymatic
alkoxycarbonylation of 1R,25-(OH)2-D3 A-ring precursor 7. Candida antarctica lipase (CAL) was
found to be the best catalyst in toluene. Regioselective alkoxycarbonylation occurred only at the
C-5-(R) hydroxyl group. Good to excellent yields were achieved by chemical reaction of these
carbonates with amino derivatives. The procedure provided convenient synthesis of carbamates
19 and 21 under mild reaction conditions.
Ch a r t 1
In tr od u ction
Vitamin D3 (1), through its hormonally active form
1R,25-dihydroxyvitamin D3 [2, 1R,25-(OH)2-D3], plays an
important role in the endocrine system (Chart 1). Be-
cause of the biological profile of 2 and the promising
clinical application of some analogs, which have been
investigated for treatment of diverse diseases such as
psoriasis, renal osteodystrophy, rickets, osteoporosis,
several cancer types, AIDS, and Alzheimer’s disease,2 the
synthesis of potential chemotherapeutic vitamin D drugs
has been one of the most interesting aims of vitamin D
research.3
The classical biological responses associated with
vitamin D actions are calcitropic, which induces intesti-
nal calcium absorption (ICA) and bone calcium mobiliza-
tion (BCM). However, it has been shown that 1R,25-
(OH)2-D3 induces differentiation and affects cellular
proliferation, opening its use in the treatment of certain
cancers and skin disorders.4 The clinical utility of 1R,25-
(OH)2-D3 is limited because therapeutically effective
doses induce hypercalcemia through its normal action in
stimulating ICA and BCM.5 This has prompted discovery
of more selective analogs, specifically directed toward
derivatives with high cell differentiating ability and low
calcitropic action (ICA and BCM).
One of the strategies entails studies of analogs in terms
of their ability to bind to various receptors and other
proteins. For example, the binding to the plasma protein
(DBP) is a crucial factor determining the half-life and
uptake of the analogs in different tissues by regulating
their free or available concentration.6 In the vitamin D
field, the indirect affinity-labeling approach might still
be the only practical method to determine the topography
of the binding sites of the hormone-specific proteins (DBP
or VDR) as long as a reasonable quantity of ligand bound
complex is difficult to obtain.7
X Abstract published in Advance ACS Abstracts, J une 1, 1997.
(1) (a) Note that R (down) and â (up) have their usual definitions
for the structures of vitamin D3 and its analogs. Because vitamin D is
typically drawn in its more stable nonsteroidal, 6-s-trans conformation,
the R’s (up) and â’s (down) are reversed in orientation for its A-ring
only. Analogously, the numbering of the vitamin D and derivatives
are the same as steroidal structures, but note that A-ring alone has
different numbering, compare Chart 1 with Scheme 2. Thus, carbons
1 and 3 in vitamin D structures correspond with carbons 3 and 5 in
the A-ring, respectively. (b) For a preliminary account of part of this
study, see: Ferrero, M.; Ferna´ndez, S.; Gotor, V. New Frontiers in
Screening for Microbial Biocatalysts; Ede: The Netherlands Dec 15-
18, 1996, commun. 18 (to be published).
A-ring modification of 1R,25-(OH)2-D3 is the second
most extensive area of analog studies next to side chain
modifications. The two hydroxyl groups at C-1 and C-25
in 1R,25-(OH)2-D3 are known to be essential for optimum
(2) Bouillon, R.; Okamura, W. H.; Norman, A. W. Endocrine Rev.
1995, 16, 200-257.
(3) (a) Zhu, G.-D.; Okamura, W. H. Chem. Rev. 1995, 95, 1877-
1952. (b) Dai, H.; Posner, G. H. Synthesis 1994, 1383-1398.
(4) (a) Honma, Y.; Hozumi, M.; Abe, E.; Konno, K.; Fukushima, M.;
Hata, S.; Nishii, Y.; DeLuca, H. F.; Suda, T. Proc. Natl. Acad. Sci.
U.S.A. 1983, 80, 201-204. (b) MacLaughlin, J . A.; Gange, W.; Taylor,
D.; Smith, E.; Holick, M. F. Proc. Natl. Acad. Sci. U.S.A. 1985, 82,
5409-5412.
(6) (a) Bishop, J . E.; Collins, E. D.; Okamura, W. H.; Norman, A.
W. J . Bone Miner. Res. 1994, 9, 1277-1288. (b) Dusso, A. S.; Negrea,
L.; Gunawardhana, S.; Lopez-Hilker, S.; Finch, J .; Mori, T.; Nishii,
Y.; Slatopolsky, E.; Brown, A. J . Endocrinology 1991, 128, 1687-1692.
(c) Honda, A.; Nakashima, N.; Mori, Y.; Katsumata, T.; Ishizuka, S.
J . Steroid Biochem. Mol. Biol. 1992, 41, 109-112. (d) Bouillon, R.;
Allewaert, K.; Xiang, D. Z.; Tan, B. K.; Van Baelen, H. J . Bone Miner.
Res. 1991, 6, 1051-1057. (e) Vanham, G.; Van Baelen, H.; Tan, B. K.;
Bouillon, R. J . Steroid Biochem. Mol. Biol. 1988, 29, 381-386.
(7) Verboven, C. C.; De Bondt, H. L.; De Ranter, C.; Bouillon, R.;
Van Baelen, H. J . Steroid Biochem. Mol. Biol. 1995, 54, 11-14.
(5) (a) Hibberd, K. A.; Norman, A. W. Biochem. Pharmacol. 1969,
18, 2347-2355. (b) Figarede, B.; Norman, A. W., Henry, H. L.; Koeffler,
H. P.; Zhou, J .-Y.; Okamura, W. H. J . Med. Chem. 1991, 34, 2452-
2463.
S0022-3263(97)00133-3 CCC: $14.00 © 1997 American Chemical Society

Alkoxycarbonylation of A-Ring Precursors of Vitamin D
J . Org. Chem., Vol. 62, No. 13, 1997 4359
Sch em e 1
Sch em e 2
catalyzed alkoxycarbonylation in organic solvents of the
1R,25-(OH)2-D3 A-ring precursors to obtain very interest-
ing intermediates that provide us with access to new and
very promising A-ring synthons of vitamin D analogs,
including 19 and 21.
Resu lts a n d Discu ssion
binding to the VDR, but 6-fluorovitamin D3 (3, Chart 1)
is the first analog lacking these hydroxyls to be observed
to bind significantly to the n-VDR in vitro.8 It is the
purpose of this article to provide several A-ring precur-
sors which could be converted by known chemical strate-
gies to analogs of steroid hormone 1R,25-(OH)2-D3. Two
of the major synthetic routes utilized in recent years to
synthesize the hormone 1R,25-(OH)2-D3 and its various
analogs are depicted in Scheme 1. The cross-coupling
approach, wherein type 5 dienynes are semihydrogenated
to a previtamin structure which undergoes rearrange-
ment to the corresponding vitamin D analogs, was
developed by Lythgoe.9 A main contribution to this
method was the synthesis Okamura developed for 4,
starting with (S)-carvone.10 The vinylallene approach11
involves the production of 6 from 4 and the subsequent
rearrangement of the former using either heat or a
combination of metal-catalyzed isomerization followed by
sensitized photoisomerization.12
The synthetic potential of enzymes in organic solvents
has been well documented in the last few years.13
Esterification reactions have been commonly used whereas
the alkoxycarbonylation reaction has scarcely been in-
vestigated.14 Previously we reported regioselective en-
zymatic transformations of several A-ring precursors
using lipase-mediated reactions in organic solvents.15
Here we report a systematic study of the enzyme-
The studies of enzymatic alkoxycarbonylation were
focused on the A-ring fragment 7 (Scheme 2),10,11 which
possesses the natural enyne stereochemistry (3S,5R) used
to prepare certain 1R,25-(OH)2-D3 analogs. Initial ex-
periments concerned screening Chromobacterium visco-
sum lipase (CVL), Pseudomonas cepacia lipase (PSL), and
Candida antarctica lipase (CAL) as catalysts to deter-
mine which enzyme gives the best regioselectivity for
alkoxycarbonylation. For the initial studies (vinyloxy)-
carbonyl oxime 8 was used. In principle, alkoxycarbon-
ylation could occur in two ways, since the leaving group
could be the acetone oxime moiety16 or the vinyloxy
group.17 We first focused our attention on CVL in THF
using a ratio of carbonate 8 to diol 7 of 10:1 because this
gave the best results in previous studies of acylation.15
At 30 °C the starting material was recovered, while at
60 °C the reaction proceeded with poor conversion of the
hydroxyl group at the C-5 position, the acetone oxime
moiety acting as leaving group (see entries 1 and 2; Table
1). Recognizing that significant changes in enzymatic
properties18 can be affected simply by changing the
solvent utilized in the reaction, we investigated the effect
of other solvents on this lipase-catalyzed alkoxycarbon-
ylation process. Different behavior was observed de-
pending on solvent. When 1,4-dioxane or toluene was
used, the conversion was higher. In both cases, traces
of 10, which is the bis-vinyloxycarbonylation product,
were observed by gas chromatography (GC) analysis.
In order to find the best enzyme-solvent system for
alkoxycarbonylation of the C-5-(R) hydroxyl group, with
total conversion and good yield, other enzymes were
tested. Negative results were observed when PSL was
used as catalyst and THF as solvent at 30 °C or 60 °C
(entries 5 and 6; Table 1), when no reaction was pro-
duced. The change from THF to 1,4-dioxane or toluene
showed an improvement in the process. The combination
(8) (a) Dauben, W. G.; Greenfield, L. J . J . Org. Chem. 1992, 57,
1597-1600. (b) Dauben, W. G.; Kohler, B.; Roesle, A. J . Org. Chem.
1985, 50, 2007-2010.
(9) Harrison, R. G.; Lythgoe, B.; Wright, P. W. J . Chem. Soc., Perkin
Trans. 1 1974, 2654-2657.
(10) Aurrecoechea, J . M.; Okamura, W. H. Tetrahedron Lett. 1987,
28, 4947-4950.
(11) Okamura, W. H.; Aurrecoechea, J . M.; Gibbs, R. A.; Norman,
A. W. J . Org. Chem. 1989, 54, 4072-4083.
(12) VanAlstyne, E. M.; Norman, A. W.; Okamura, W. H. J . Am.
Chem. Soc. 1994, 116, 6207-6216.
(13) (a) Davies, H. G.; Green, R. G.; Kelly, D. R.; Roberts, S, M.
Biotransformations in Preparative Organic Chemistry; Academic
Press: New York, 1989. (b) Wong, C.-H.; Whitesides, G. M. Enzymes
in Synthetic Organic Chemistry; Elsevier Science Ltd.: Oxford, 1994.
(c) Faber, K. Biotransformations in Organic Chemistry; 2nd ed.;
Springer-Verlag: Berlin, 1995.
(16) (a) Gotor, V.; Mor´ıs, F. Synthesis 1992, 626-628 and literature
cited therein. (b) Ferna´ndez, S.; Mene´ndez, E.; Gotor, V. Synthesis
1991, 713-716. (c) Gotor, V.; Pulido, R. J . Chem. Soc., Perkin Trans.
1 1991, 491-492.
(17) (a) Wang, Y.-F.; Chen, S.-T.; Liu, K.-C.; Wong, C.-H. Tetrahe-
dron Lett. 1989, 30, 1917-1920. (b) Degueil-Castaing, M.; de J eso, B.;
Drouillard, S.; Maillard, B. Tetrahedron Lett. 1987, 28, 953-954.
(18) (a) Klibanov, A. M. Trends Biochem. Sci. 1989, 14, 141-144.
(b) Dordick, J . S. Enzyme Microb. Technol. 1989, 11, 194-211.
(14) (a) Garc´ıa-Alles, L. F.; Gotor, V. Tetrahedron 1995, 51, 307-
316. (b) Gotor, V.; Mor´ıs, F.; Garc´ıa-Alles, L. F. Biocatalysis 1994, 10,
295-305, and literature cited therein.
(15) Ferna´ndez, S.; Ferrero, M.; Gotor, V.; Okamura, W. H. J . Org.
Chem. 1995, 60, 6057-6061.

4360 J . Org. Chem., Vol. 62, No. 13, 1997
Ta ble 1. Rea ction of Diol 7 w ith (Vin yloxy)ca r bon yl Oxim e 8 Ca ta lyzed by En zym esa
Ferrero et al.
entry
enzyme
solvent
THF
T (°C)
t (h)
conv (%)b
7 (%)b
9 (%)b
10 (%)b
1
2
3
4
5
6
7
8
9
CVL
CVL
CVL
CVL
PSL
30
60
30
30
30
60
30
30
30
60
30
30
94.5
87
94
80
94.5
87
94
23
94.5
87
94
0
38
52.4
80.7
0
100
62
-
38
51.5
78.9
-
-
43.5
80
-
82
-
-
THF
1,4-dioxane
toluene
THF
47.7
19.3
100
100
51.1
-
100
18
16.6
-
0.9
1.8
-
PSL
PSL
PSL
THF
0
-
1,4-dioxane
toluene
THF
THF
1,4-dioxane
toluene
49.0
100
0
82
83.4
100
5.5
20
-
CALc
CALc
CALc
CALc
10
11
12
-
4.0
-
79.4
4
100d
a
b
d
These processes were carried out using a ratio of 1:10 (7:8). Calculated by GC analysis. c SP 435L. 98% isolated yield.
Sch em e 3
of PSL and toluene gave a higher conversion of 9 (80%),
although an important amount of diprotected product 10
was also obtained (20%) (entry 8; Table 1).
The best results were obtained when CAL was used
as catalyst. In the initial screening no reaction was
observed in THF at 30 °C. But when the temperature
was raised, the process took place with good conversion
and generated a unique C-5-(R) regioisomer. When 1,4-
dioxane was used, the percentage of conversion was also
high, but in this case only a small amount of product 10
was formed (entry 11; Table 1). Of the enzyme-solvent
systems studied, CAL-toluene gave the best result. It
catalyzed vinyloxycarbonylation with total conversion in
4 h at 30 °C. Only the C-5-(R) hydroxyl group was
protected and with excellent yield (98% of isolated
product) (entry 12; Table 1). It is noteworthy that no C-3
alkoxycarbonylation products were observed in any of
these processes, and alkoxycarbonylation did not take
place in the absence of enzyme even if stronger conditions
were used. However, a great difference in reaction time
was observed when the solvent was changed from 1,4-
dioxane to toluene in all three cases. Especially signifi-
cant was when CAL was used as catalyst. Thus, after
94 h in 1,4-dioxane the reaction did not evolve further
(83.4% conversion) because the enzyme became inactive,
whereas in toluene CAL catalyzed the alkoxycarbonyla-
tion process with 100% of conversion in 4 h.
The above-mentioned structures were determined by
means of their spectral data. With regard to the 1H-
NMR, the more significant change occurs on the multiplet
corresponding to H5 (Scheme 2), which changes from 4.11
ppm in 7 to 5.07 ppm in 9. In addition, 13C-NMR
spectrum presents a shift of ca. 8 ppm on the C-5 of
carbonate 9 (from 62.9 ppm in 7 to 71.3 ppm in 9).
However, dicarbonate 10 displays a downfield shift of
both protons corresponding to the triplet of H3 (from 4.26
ppm to 5.37 ppm) and the multiplet of H5 (from 4.11 ppm
to 5.06 ppm), and in 13C-NMR spectrum a shift of 6 ppm
on the C-3 of 10 (from 68.8 ppm in 7 to 74.8 ppm in 10)
plus ca. 8 ppm on the C-5 of 10 (from 62.9 ppm in 7 to
70.5 ppm in 10). Complete 1H- and 13C-NMR spectral
data are given in the Experimental Section, as well as
microanalyses and high resolution mass spectra (HRMS).
We subsequently examined a variety of alkoxycarbon-
ylating agents to study the utility of CAL in toluene and
take advantage of the fact that CAL shows excellent
vinyloxycarbonylation selectivity and yield (Scheme 3).
Reactions were run at 30 °C using a ratio of carbonates
11-14 to diol 7 of 10:1. As Scheme 3 indicates, good to
excellent yields of carbonates 15-17 were achieved in 6
to 72 h. Alkoxycarbonylation occurred selectively at the
C-5-(R) hydroxyl in all cases. Thus, the methoxycarbonyl
moiety was incorporated into 7 when methoxycarbonyl
oxime 11 was used, obtaining 15 exclusively. When we
used smaller ratio of carbonates (1:5), longer reaction
times were required and a slight decrease in yield was
observed. In both cases, after purification of compound
15 by flash chromatography, carbonate 11 was recovered.
When acetone O-[(benzyloxy)carbonyl]oxime (12) was
used this process proved to be of great utility in the one-
step regioselective protection (through introduction of
Cbz) of the enynediol 7 in mild conditions and with high
yield (85%). With the aim of testing the possible different
reactivity between oxime carbonates and vinyl carbon-
ates, reactions with acetone O-(phenoxycarbonyl)oxime
(13) and phenyl vinyl carbonate (14) were run. Carbon-
ate 17 was obtained with excellent yield (90%) when the
process was carried out with carbonate 13. On the other
hand, if carbonate 14 was used, the starting material
reacted completely after 24 h, but phenyl carbonate 17
and vinyl carbonate 9 were obtained as a mixture, 1.4:1,
respectively (by GC).
In order to demonstrate the synthetic utility of these
A-ring fragment derivatives, the carbamates 19 were
synthesized from carbonates 9. Four kinds of compounds
have been tested, as a proof of the versatility of the
procedure: (a) ammonia, (b) amines (primary and sec-
ondary aliphatic and primary aromatic), (c) amino alco-
hols, and (d) diamines. In the first place, ammonolysis
of 9 was carried out in THF, bubbling NH3 through at 0
°C for 30 min, and then leaving to react for 18 h at room
temperature, until the disappearance of the starting

Alkoxycarbonylation of A-Ring Precursors of Vitamin D
J . Org. Chem., Vol. 62, No. 13, 1997 4361
Sch em e 4
Sch em e 5
Ta ble 2. Rea ction of Ca r bon a te 9 w ith Am in o
Der iva tives 18a
compound 18
entry
R1
R2
ratio 9:18
T (°C) t (h) 19 (%)b
1
2
3
4
5
6
7
8
9
H
H
H
H
H
H
H
c
25
30
60
60
60
reflux
18
7
100
-
Bu
Bu
Bu
Ph
Ph
Pr
1:2.5
1:2.5
1:10
1:3
39
24
96
72
90
48
48
48
-
78
-
-
-
80
82
76
excess 18c
Pr excess 18d reflux
HO(CH2)2
HO(CH2)6
H2N(CH2)3
H
H
H
1:2.5
1:2.5
1:2.5
60
60
60
10
a
b
These processes were carried out in THF. Isolated yield.
c Bubbling at 0 °C on the reaction solution.
H3 (3J CH), and with the multiplet at 5.17 ppm which
belongs to the H5 (3J CH). The full assignment of the H-
1
carbonate (TLC monitoring) (Scheme 4). Carbamate 19a
was formed in quantitative yield, as the sole product
(entry 1; Table 2), which was shown to be essentially pure
by TLC and spectroscopic analysis.
and 13C-NMR spectra is given in the Experimental
Section.
Su m m a r y
The next step was the reaction of primary aliphatic
amines with vinyl carbonate 9. Aminolysis reaction
between butylamine and carbonate 9 required heating
at 60 °C in THF with excess amine 18b (10:1) for 24 h to
provide carbamate 19b (entries 2-4; Table 2). In the
case of aromatic amines, no reaction was observed even
when a big excess of aniline was used at reflux in THF
(entries 5 and 6; Table 2). Secondary amine, dipropyl-
amine in excess in THF at reflux over 90 h gave no
reaction (entry 7; Table 2).
Interesting products to synthesize are A-ring carbam-
ate derivatives reached from linear amino alcohols or
diamines. Carbonate 9 was allowed to react with 2-amino-
1-ethanol (18e), 6-amino-1-hexanol (18f), and 1,3-pro-
panediamine (18g) at 60 °C in THF for approximately 2
days to provide carbamates 19e-g in good yields (entries
8-10; Table 2).
A chemoenzymatic procedure has been shown, involv-
ing the enzymatic transesterification of carbonates and
the subsequent transformation with ammonia, amines,
amino alcohols, diamines, and amino acids to obtain the
corresponding 1R,25-(OH)2-D3 A-ring C-5 carbamates
synthons. Evaluation of the enzymatic alkoxycarbon-
ylation of the vitamin D A-ring enyne 7 revealed that
CAL is the best enzyme for effecting practical levels of
regioselectivity. The leaving group was always the
acetone oxime moiety (except for carbonate 14), giving
rise to regioselective formation of the corresponding C-5-
(R) carbonate derivatives. Direct application of these
carbonate derivatives provided convenient formation of
carbamates 19 and amino acid derivatives 21, useful
precursors of analogs of 1R,25-(OH)2-D3 for pharmaceuti-
cal research.
We extended the aforementioned methodology to syn-
thesize some amino acid A-ring precursors. The best
results were obtained submitting the corresponding
vinyloxycarbonylated C-5-(R) 1R,25-(OH)2-D3 A-ring pre-
cursor 9 to reaction with the amino acid sodium salts
20a ,b in DMF at 60 °C to provide 21a ,b (Scheme 5).
Generally, sonication of the amino acid salts was per-
formed to improve dissolution.
The structural assignment of the compounds described
in this paper is based on the analysis of their 1H- and
13C-NMR spectra. Additional DEPT experiments and the
correct assignment were confirmed by 1H-13C hetero-
nuclear correlation experiments. Thus for example,
heteronuclear correlation of compound 19g showed, for
the methyl protons at 2.16 ppm, the cross peaks with the
carbon atoms at 68.95, 115.21, and 144.54 ppm corre-
sponding to the C3 (3J CH), C1 (3J CH), and C2 (2J CH),
respectively.19 The signal at 115.21 ppm further cor-
relates with the double doublets at 2.33 and 2.74 ppm
arising from the methylene diasterotopic protons H6
(2J CH), with the triplet at 4.38 ppm corresponding to the
Exp er im en ta l Section 19
Gen er a l. Chromobacterium viscosum lipase, CVL, was a
gift from Genzyme Co.; Pseudomonas cepacia lipase, PSL, was
obtained from Amano Pharmaceutical Co. and Candida ant-
arctica lipase, CAL SP 435L, was donated by Novo Nordisk
Co. All other reagents were purchased from Aldrich. Solvents
were distilled over an adequate desiccant under nitrogen.
A-ring synthon 7 was obtained according to the method of
Okamura.10,11 Vinyl carbonate 14, oxime carbonates 8 and 11-
13 were synthesized as has been previously reported.16b Amino
acid sodium salts were prepared dissolving the corresponding
L-amino acids in water adding NaOH (1 N) and evaporating
under vacuum.
(19) Structures of the products are numbered as follows:

4362 J . Org. Chem., Vol. 62, No. 13, 1997
Ferrero et al.
En zym a tic Alk oxyca r bon yla tion of Diol 7 w ith Ac-
eton e O-[(Vin yloxy)ca r bon yl]oxim e (8). Syn th esis of
(3S,5R)-1-Eth yn yl-3-h yd r oxy-2-m eth yl-5-[(vin yloxy)ca r -
bon yl]-1-cycloh exen e (9). To a solution of 7 (10 mg, 0.066
mmol) in 2.5 mL of solvent (THF, 1,4-dioxane, or toluene) was
added one of the following enzymes: 200 mg of PSL, 10 mg of
CVL, or 45 mg of CAL and carbonate 8 (94.5 mg, 0.66 mmol).
The suspension was shaken at 30 °C or 60 °C, and the progress
of the reaction was followed by TLC and GC analysis until no
further reaction was apparent. After removal of the enzyme
by filtration and evaporation of the solvent and 1H-NMR
analysis, the residual mixture was purified by HPLC (Spher-
isorb W, 1 × 25 cm, 5 µm silica gel 60 column, 15% ethyl
acetate/hexanes, 4 mL/min) to give monovinyloxycarbonylation
product 9 (or 9 and bis(vinyl carbonate) 10 depending on
conditions). All these data are summarized in Table 1. 9: 1H-
NMR (CDCl3, 300 MHz): δ 2.01 (s, 3H, H9), 2.05 (m, 2H, H4),
H, 6.34. Found: C, 71.5; H, 6.6. HRMS (m/ z) Calcd for
C17H18O4: 286.1205. Found: 286.1211.
(3S,5R)-1-Eth yn yl-3-h yd r oxy-2-m eth yl-5-(p h en oxyca r -
bon yl)-1-cycloh exen e (17). 1H-NMR (CDCl3, 200 MHz): δ
1.90 (br s, 1H, OH), 2.05 (s, 3H, H9), 2.11 (m, 2H, H4), 2.41
2
3
2
(dd, 1H, H6, J HH 17.2, J HH 7.0 Hz), 2.76 (dd, 1H, H6, J HH
3
17.2, J HH 5.0 Hz), 3.14 (s, 1H, H8), 4.34 (br s, 1H, H3), 5.14
(m, 1H, H5), 7.25 (m, 3H, 2Hm+Hp), and 7.40 (m, 2H, Ho); 13C-
NMR (CDCl3, 50.3 MHz): δ 18.32 (C9), 34.78 (C4), 36.12 (C6),
68.13 (C3), 71.39 (C5), 80.92 (C8), 82.44 (C7), 113.68 (C1), 120.93
(C12), 126.00 (C14), 129.42 (C13), 142.82 (C2), 150.92 (C11), and
152.97 (C10). Anal. Calcd (%) for C16H16O4: C, 70.56; H, 5.93.
Found: C, 70.6; H, 6.1. HRMS (m/ z) Calcd for C16H16O4:
272.1049. Found: 272.1050.
Syn th esis of Ca r ba m a tes 19: Gen er a l P r oced u r e. Car-
bonate 9 (15 mg, 0.068 mmol) and ammonia (bubbled during
30 min at 0 °C), amines (0.680 mmol), amino alcohols (0.170
mmol), or diamines (0.170 mmol) were dissolved in 5 mL of
THF. The solution was stirred under nitrogen atmosphere at
60 °C (30 °C for ammonolysis reaction) until no starting
material 9 remained (18-48 h). Then, solvent was evaporated
under reduced pressure, and the residue subjected to flash
chromatography.
2
3
2.16 (br s, 1H, OH), 2.31 (dd, 1H, H6, J HH 17.2, J HH 7.5 Hz),
2.67 (dd, 1H, H6, 2J HH 17.2, 3J HH 3.7 Hz), 3.10 (s, 1H, H8), 4.29
3
2
(br s, 1H, H3), 4.58 (dd, 1H, H12-cis, J HH 6.4, J HH 2.1 Hz),
3
2
4.91 (dd, 1H, H12-trans, J HH 13.8, J HH 2.1 Hz), 5.07 (m, 1H,
3
3
H5), and 7.06 (dd, 1H, H11, J HH 13.8, J HH 6.4 Hz); 13C-NMR
(CDCl3, 75.5 MHz): δ 18.32 (C9), 34.66 (C4), 35.99 (C6), 68.03
(C3), 71.29 (C5), 80.93 (C8), 82.37 (C7), 97.88 (C12), 113.56 (C1),
142.37 (C11), 142.81 (C2), and 152.00 (C10). Anal. Calcd (%)
for C12H14O4: C, 64.84; H, 6.35. Found: C, 64.7; H, 6.4.
HRMS (m/ z) Calcd for C12H14O4: 222.0892. Found: 222.0895.
(3S,5R)-1-Eth yn yl-2-m eth yl-3,5-bis[(vin yloxy)car bon yl]-
(3S,5R)-5-(Car bam oyloxy)-1-eth yn yl-3-h ydr oxy-2-m eth -
yl-1-cycloh exen e (19a ). 1H-NMR (MeOH-d4, 200 MHz): δ
2
2.13 (m, 2H, H4), 2.17 (s, 3H, H9), 2.35 (dd, 1H, H6, J HH 17.1,
3J HH 7.0 Hz), 2.75 (dd, 1H, H6, 2J HH 17.1, 3J HH 3.4 Hz), 3.66 (s,
3
1H, H8), 4.38 (t, 1H, H3, J HH 4.9 Hz), and 5.16 (m, 1H, H5);
1
13C-NMR (MeOH-d4, 50.3 MHz): δ 18.86 (C9), 36.85 (C4), 37.94
1-cycloh exen e (10): H-NMR (CDCl3, 300 MHz): δ 1.97 (s,
3H, H9), 2.13 (m, 1H, H4), 2.25 (m, 1H, H4), 2.35 (dd, 1H, H6,
(C6), 68.56 (C3), 68.98 (C5), 82.17 (C8), 84.17 (C7), 115.26 (C1),
2J HH 16.6, J HH 6.4 Hz), 2.75 (dd, 1H, H6, J HH 16.6, J HH 3.4
3
2
3
144.51 (C2), and 159.66 (C10). Anal. Calcd (%) for C10H13
-
3
2
Hz), 3.18 (s, 1H, H8), 4.60 (dd, 1H, H12-cis, J HH 6.0, J HH 2.1
Hz), 4.62 (dd, 1H, H15-cis, 3J HH 6.2, 2J HH 1.9 Hz), 4.93 (dd, 1H,
H12-trans, 3J HH 13.8, 2J HH 2.1 Hz), 4.94 (dd, 1H, H15-trans, 3J HH
NO3: C, 61.51; H, 6.72; N, 7.18. Found: C, 61.3; H, 6.8; N,
7.0. HRMS (m/ z) Calcd for C10H13NO3: 195.0895. Found:
195.0898.
2
3
14.0, J HH 1.9 Hz), 5.06 (m, 1H, H5), 5.37 (t, 1H, H3, J HH 4.7
(3S,5R)-5-[(N-Bu tylcar bam oyl)oxy]-1-eth yn yl-3-h ydr oxy-
3
3
2-m eth yl-1-cycloh exen e (19b). 1H-NMR (CDCl3, 300 MHz):
Hz), 7.07 (dd, 1H, H11, J HH 12.5, J HH 6.9 Hz), and 7.09 (dd,
1H, H14, 3J HH 12.5, 3J HH 6.9 Hz); 13C-NMR (CDCl3, 75.5 MHz):
δ 18.23 (C9), 32.87 (C4), 34.52 (C6), 70.51 (C5), 74.77 (C3), 81.64
(C7), 82.23 (C8), 98.06 (C12), 98.17 (C15), 116.88 (C1), 137.90 (C2),
142.33 (C11), 142.43 (C14), 151.84 (C10), and 152.33 (C13). Anal.
Calcd (%) for C15H16O6: C, 61.62; H, 5.52. Found: C, 61.7; H,
5.3. HRMS (m/ z) Calcd for C15H16O6: 292.0947. Found:
292.0951.
δ 0.91 (t, 3H, H14
2H, H12), 1.97 (m, 2H, H4), 2.01 (s, 3H, H9), 2.17 (br s, 1H,
,
3J HH 7.3 Hz), 1.33 (m, 2H, H13), 1.45 (m,
2
3
OH), 2.19 (m, 1H, H6), 2.60 (dd, 1H, H6, J HH 17.0, J HH 2.2
3
Hz), 3.08 (s, 1H, H8), 3.15 (q, 2H, H11, J HH 6.6 Hz), 4.23 (br s,
1H, H3), 4.65 (br s, 1H, NH), and 5.04 (m, 1H, H5); 13C-NMR
(CDCl3, 75.5 MHz): δ 13.65 (C14), 18.27 (C9), 19.81 (C13), 31.91
(C12), 35.34 (C4), 36.57 (C6), 40.60 (C11), 66.74 (C5), 68.28 (C3),
80.47 (C8), 82.84 (C7), 113.90 (C1), 143.11 (C2), and 155.87 (C10).
Anal. Calcd (%) for C14H21NO3: C, 66.89; H, 8.43; N, 5.58.
En zym a tic Alk oxyca r bon yla tion of 7 w ith oth er Ca r -
bon a tes. In a typical procedure, C. antarctica lipase (90 mg)
was added to a solution of 7 (20 mg, 0.131 mmol) and
carbonates 11-14 (0.657 mmol or 1.314 mmol) in 5 mL of
toluene as summarized in Scheme 3. The suspension was
shaken at 30 °C, and the progress of the reaction was followed
by TLC and GC analysis. At 100% conversion the mixture
was filtered, and the solvent was removed under reduced
pressure. After 1H-NMR analysis, the crude material was
subjected to HPLC (Kromasil 60, 2 × 25 cm, 7 µm silica gel
column, 20% ethyl acetate/hexanes for 15 and 16 and 15%
EtOAc/hexanes for 17, 8 mL/min) to give compounds 15-17.
(3S,5R)-1-E t h yn yl-3-h yd r oxy-5-(m et h oxyca r b on yl)-2-
m eth yl-1-cycloh exen e (15). 1H-NMR (CDCl3, 300 MHz): δ
2.01 (s, 3H, H9), 2.02 (m, 2H, H4), 2.11 (br s, 1H, OH), 2.28
Found: C, 66.7; H, 8.5; N, 5.4. HRMS (m/ z) Calcd for C14H21
-
NO3: 251.1521. Found: 251.1522.
(3S,5R)-1-E t h yn yl-3-h yd r oxy-5-[[N-(2-h yd r oxyet h yl)-
ca r ba m oyl]oxy]-2-m eth yl-1-cycloh exen e (19e). 1H-NMR
(MeOH-d4, 300 MHz): δ 2.13 (m, 2H, H4), 2.17 (s, 3H, H9),
2.35 (dd, 1H, H6, 2J HH 17.2, 3J HH 6.4 Hz), 2.76 (d, 1H, H6, 2J HH
3
17.2 Hz), 3.39 (t, 2H, H11, J HH 5.7 Hz), 3.67 (s, 1H, H8), 3.76
3
(t, 2H, H12
,
3J HH 5.7 Hz), 4.39 (t, 1H, H3, J HH 4.8 Hz), and
5.19 (m, 1H, H5); 13C-NMR (MeOH-d4, 75.5 MHz): δ 18.86 (C9),
36.90 (C4), 37.96 (C6), 44.40 (C11), 62.18 (C12), 68.74 (C3), 68.97
(C5), 82.24 (C8), 84.17 (C7), 115.23 (C1), 144.53 (C2), and 158.89
(C10). Anal. Calcd (%) for C12H17NO4: C, 60.22; H, 7.17; N,
5.86. Found: C, 60.3; H, 7.0; N, 6.0. HRMS (m/ z) Calcd for
C12H17NO4: 239.1158. Found: 239.1147.
2
3
2
(dd, 1H, H6, J HH 17.0, J HH 7.1 Hz), 2.65 (dd, 1H, H6, J HH
3
17.0, J HH 3.7 Hz), 3.09 (s, 1H, H8), 3.77 (s, 3H, H11), 4.27 (br
(3S,5R)-1-E t h yn yl-3-h yd r oxy-5-[[N-(6-h yd r oxyh exyl)-
ca r ba m oyl]oxy]-2-m eth yl-1-cycloh exen e (19f). 1H-NMR
(CDCl3, 300 MHz): δ 1.36 (m, 4H, H12 + H13), 1.53 (m, 4H,
H14 + H15), 1.83 (br s, 1H, OH), 1.98 (m, 2H, H4), 2.02 (s, 3H,
s, 1H, H3), and 5.01 (m, 1H, H5); 13C-NMR (CDCl3, 50.3 MHz):
δ 18.31 (C9), 34.83 (C4), 36.16 (C6), 54.68 (C11), 68.17 (C3), 70.44
(C5), 80.79 (C8), 82.49 (C7), 113.76 (C1), 142.76 (C2), and 155.03
(C10). Anal. Calcd (%) for C11H14O4: C, 62.83; H, 6.72.
Found: C, 63.0; H, 6.7. HRMS (m/ z) Calcd for C11H14O4:
210.0892. Found: 210.0900.
2
3
H9), 2.19 (dd, 1H, H6, J HH 17.3, J HH 4.9 Hz), 2.39 (br s, 1H,
2
OH), 2.60 (d, 1H, H6, J HH 17.3 Hz), 3.09 (s, 1H, H8), 3.15 (m,
3
2H, H11), 3.63 (t, 2H, H16, J HH 6.5 Hz), 4.22 (m, 1H, H3), 4.73
(t, 1H, N-H, J HH 5.8 Hz), and 5.03 (m, 1H, H5); 13C-NMR
3
(3S,5R)-5-[(Ben zyloxy)ca r bon yl]-1-eth yn yl-3-h yd r oxy-
2-m eth yl-1-cycloh exen e (16). 1H-NMR (CDCl3, 300 MHz):
δ 1.78 (br s, 1H, OH), 2.01 (s, 3H, H9), 2.03 (m, 2H, H4), 2.30
(CDCl3, 50.3 MHz): δ 18.29 (C9), 25.15 (C13), 26.22 (C14), 29.81
(C15), 32.40 (C12), 35.34 (C6), 36.56 (C4), 40.61 (C11), 62.54 (C16),
66.85 (C5), 68.21 (C3), 80.47 (C8), 82.86 (C7), 113.80 (C1), 143.21
(C2), and 156.00 (C10). Anal. Calcd (%) for C16H25NO4: C,
65.05; H, 8.54; N, 4.74. Found: C, 65.2; H, 8.6; N, 5.0. HRMS
(m/ z) Calcd for C16H25NO4: 295.1784. Found: 295.1787.
(3S ,5R )-5-[[N -(3-Am in o p r o p y l)c a r b a m o y l]o x y ]-1-
et h yn yl-3-h yd r oxy-2-m et h yl-1-cycloh exen e (19g). 1H-
NMR (MeOH-d4, 400.1 MHz): δ 1.82 (p, 2H, H12, 3J HH 7.0 Hz),
2
3
2
(dd, 1H, H6, J HH 17.1, J HH 7.3 Hz), 2.67 (dd, 1H, H6, J HH
3
3
17.1, J HH 4.5 Hz), 3.10 (s, 1H, H8), 4.28 (t, 1H, H3, J HH 4.7
Hz), 5.04 (m, 1H, H5), 5.15 (s, 2H, H11), and 7.37 (m, 5H, ArH);
13C-NMR (CDCl3, 75.5 MHz): δ 18.31 (C9), 34.80 (C4), 36.12
(C6), 68.14 (C3), 69.58 (C11), 70.54 (C5), 80.78 (C8), 82.49 (C7),
113.74 (C1), 128.36 (C13), 128.55 (C12+C14), 134.96 (C15), 142.77
(C2), and 154.39 (C10). Anal. Calcd (%) for C17H18O4: C, 71.30;

Alkoxycarbonylation of A-Ring Precursors of Vitamin D
J . Org. Chem., Vol. 62, No. 13, 1997 4363
2
2.12 (m, 2H, H4), 2.16 (s, 3H, H9), 2.33 (dd, 1H, H6, J HH 16.9,
1H-NMR (MeOH-d4, 300 MHz): δ 2.13 (m, 2H, H4), 2.17 (s,
3J HH 6.4 Hz), 2.74 (dd, 1H, H6, 2J HH 16.9, 3J HH 3.5 Hz), 2.85 (t,
2H, H13, J HH 6.1 Hz), 3.34 (t, 2H, H11, J HH 6.8 Hz), 3.66 (s,
1H, H8), 4.38 (t, 1H, H3, J HH 4.8 Hz), and 5.17 (m, 1H, H5);
2
3H, H9), 2.31 (s, 3H, H14), 2.37 (d, 1H, H6, J HH 16.7 Hz), 2.76
3
3
2
3
(d, 1H, H6, J HH 16.7 Hz), 2.99 (dd, 1H, H13,
3J HH 13.7, J HH
3
7.5 Hz), 3.19 (dd, 1H, H13, 3J HH 13.7, 3J HH 4.2 Hz), 3.67 (s, 1H,
H8), 4.41 (m, 2H, H3 + H11), and 5.19 (m, 1H, H5); 13C-NMR
(MeOH-d4, 50.3 MHz): δ 16.35 (C14), 18.89 (C9), 36.88 (C4),
37.94 (C6), 38.82 (C13), 57.11 (C11), 68.79 (C3), 68.94 (C5), 82.19
(C8), 84.20 (C7), 115.23 (C1), 144.52 (C2), 158.02 (C10), and
178.38 (C12). Anal. Calcd (%) for C14H18NNaO5S: C, 50.14;
H, 5.41; N, 4.18. Found: C, 50.2; H, 5.5; N, 4.1; MS (FAB-,
glycerol): m/ z 312 (M- - Na, 21%), 275 (36), 183 (100), and
92 (41).
13C-NMR (MeOH-d4, 100.6 MHz): δ 18.87 (C9), 33.98 (C12),
36.93 (C6), 37.97 (C4), 39.20 (C11), 39.87 (C13), 68.65 (C5), 68.95
(C3), 82.25 (C8), 84.17 (C7), 115.21 (C1), 144.54 (C2), and 158.92
(C10). Anal. Calcd (%) for C13H20N2O3: C, 61.87; H, 7.99; N,
11.11. Found: C, 62.0; H, 7.9; N, 11.3. HRMS (m/ z) Calcd
for C13H20N2O3: 252.1474. Found: 252.1475.
Syn th esis of Am in o Acid Der iva tives 21a ,b. Amino acid
sodium salts 20 (0.170 mmol) were suspended in 4 mL of DMF
and sonicated in order to dissolve the amino acid faster.
Carbonate 9 (15 mg, 0,068 mmol) was added, and the mixture
was stirred at 60 °C until there was no more starting carbonate
remaining in solution. The solvent was then evaporated under
vacuum and the residue subjected to flash chromatography
(30% MeOH/EtOAc).
(3S,5R)-N-[[(1-Eth yn yl-3-h yd r oxy-2-m eth yl-1-cycloh ex-
en -5-yl)oxy]ca r bon yl]glycin e (21a ). 1H-NMR (MeOH-d4,
300 MHz): δ 2.12 (m, 2H, H4), 2.17 (s, 3H, H9), 2.37 (dd, 1H,
H6, J HH 17.1, J HH 6.6 Hz), 2.76 (d, 1H, H6, J HH 17.1 Hz),
3.65 (s, 1H, H8), 3.85 (s, 2H, H11), 4.40 (t, 1H, H3, J HH 4.6
Ack n ow led gm en t. We express our appreciation to
Genzyme Co. for the generous gift of the CVL, as well
as Novo Nordisk Co. for the generous donation of the
lipase CAL. Financial support from CICYT (Spain;
Project BIO95-0687) is gratefully acknowledged. M.F.
and S.F. also thank the Ministerio de Educacio´n y
Ciencia (Spain) and II PRI (Asturias, Spain), respec-
tively, for their postdoctoral fellowships.
2
3
2
3
Hz), and 5.19 (m, 1H, H5); 13C-NMR (MeOH-d4, 75.5 MHz): δ
18.88 (C9), 31.09 (C11), 36.91 (C4), 37.98 (C6), 68.76 (C3), 68.97
(C5), 82.19 (C8), 84.21 (C7), 115.31 (C1), 144.53 (C2), 158.61 (C10),
and 177.67 (C12). Anal. Calcd (%) for C12H14NNaO5: C, 52.35;
H, 5.13; N, 5.09. Found: C, 52.4; H, 5.0; N, 5.1; MS (FAB-,
glycerol): m/ z 252 (M- - Na, 12%), 183 (100), and 92 (42).
(3S,5R,11S)-N-[[(1-E t h yn yl-3-h yd r oxy-2-m et h yl-1-cy-
cloh exen -5-yl)oxy]ca r bon yl]-S-m eth yl-L-cystein e (21b).
Su p p or tin g In for m a tion Ava ila ble: Spectral and ana-
lytical data (14 pages). This material is contained in libraries
on microfiche, immediately follows this article in the microfilm
version of the journal, and can be ordered from the ACS; see
any current masthead page for ordering information.
J O970133B