A new chemoenzymatic synthesis of d-cloprostenol

Article abstract of DOI:10.1016/j.tetasy.2005.08.038

A new chemoenzymatic synthesis of d-cloprostenol based on the biocatalytical resolution of anti-2-oxotricyclo[2.2.1.0]heptan-7-carboxylic acid 1 has been developed. The resolution was attempted by different approaches: esterification or reduction of the a

Full text of DOI:10.1016/j.tetasy.2005.08.038

Tetrahedron:
Asymmetry
Tetrahedron: Asymmetry 16 (2005) 3279–3282
A new chemoenzymatic synthesis of D-cloprostenol
Andrea Romano,a Diego Romano,a Francesco Molinari,a,*
Raffaella Gandolfib and Francesca Costantinoc
a
`
Dipartimento di Scienze e Tecnologie Alimentari e Microbiologiche, Universita degli Studi di Milano, via Celoria 2,
20133 Milano, Italy
b
`
Istituto di Chimica Organica ꢀAlessandro Marchesiniꢁ, Universita degli Studi di Milano, 20133 Milano, Italy
cIndustriale Chimica, via Grieg 13, 21047 Saronno (VA), Italy
Received 18 August 2005; accepted 30 August 2005
Available online 28 September 2005
Abstract—A new chemoenzymatic synthesis of D-cloprostenol based on the biocatalytical resolution of anti-2-oxotricy-
clo[2.2.1.0]heptan-7-carboxylic acid 1 has been developed. The resolution was attempted by different approaches: esterification or
reduction of the acid and hydrolysis or reduction of the corresponding esters. The most efficient method proved to be the reduction
of the propyl esters of 1 catalysed by the yeast Kluyveromyces marxianus, which allowed for the recovery of the enantiomerically
pure ester of anti-2-oxotricyclo[2.2.1.0]heptan-(R)-7-carboxylic acid (R)-3 at 60% molar conversion of 3.0 g/l of racemic substrate
acid under optimised conditions. anti-2-Oxotricyclo[2.2.1.0]heptan-(R)-7-carboxylic acid was obtained by alkaline hydrolysis and
employed for the synthesis of D-cloprostenol.
ꢀ 2005 Published by Elsevier Ltd.
1. Introduction
2. Results and discussion
The synthesis of prostaglandins via bicyclo[2.2.1]hep-
tane derivatives is a well-established approach.1,2 anti-
2-Oxotricyclo[2.2.1.0]heptan-7-carboxylic acid 1 is a
useful chiral intermediate for prostaglandin synthesis
and can easily be synthesised from norbornadiene as a
racemic mixture,3 but its synthetic utility is limited to
the (7R)-stereoisomer. The resolution of (RS)-1 can be
achieved by precipitation with chiral amines, but the
process is characterised by low yields.4,5 The resolution
can also be achieved by the enzymatic hydrolysis of
the corresponding methyl ester with commercial
lipases.6,7
The direct esterification of 1 was first investigated with
ethanol, n-propanol and n-butanol using 22 commercial
enzymes and 50 lyophilised microbial cells as biocatalyst
in organic solvents.8,9 Although different reaction condi-
tions (temperature, reagent/biocatalyst ratio and organ-
ic solvent) were checked, the reaction rates always
remained very sluggish. The best results were obtained
after 7 days with lipase AH from Pseudomonas cepacia
in toluene (20% molar conversion and E = 8.0) and with
lyophilised mycelium of Rhizopus oryzae CBS 391.34 in
n-heptane (15% molar conversion and E = 8.5) starting
from 2.5 g/l of 1 and an equimolar amount of ethanol.
Transesterification of the methyl ester of 1 with different
alcohols was also evaluated, but no significant improve-
ment in the yield or enantioselectivity was observed.
Different biocatalytic approaches for the resolution of 1
or its esters (Scheme 1) have been investigated herein as
an alternative to commercial enzymes. The resulting
anti-2-oxotricyclo[2.2.1.0]heptan-(R)-7-carboxylic acid
(R)-1 was employed for the synthesis of D-cloprostenol,
carried out by standard synthetic methods.
Enzymatic hydrolysis of the methyl ester of (RS)-1
(R = Me) was investigated using various microorgan-
isms previously selected in our laboratory for carboxy-
lesterase activity.10,11
Whole cells of Bacillus coagulans NCIMB 936512 gave,
under the optimal conditions (40 ꢁC, phosphate buffer
pH 6.5 and 30 g/l of biocatalyst), the highest enantio-
selectivity (Table 1).
*
Corresponding author. Tel.: +39 0250316695; fax: +39
0250316694; e-mail: francesco.molinari@unimi.it
0957-4166/$ - see front matter ꢀ 2005 Published by Elsevier Ltd.
doi:10.1016/j.tetasy.2005.08.038

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A. Romano et al. / Tetrahedron: Asymmetry 16 (2005) 3279–3282
HOOC
COOR
COOH
+
HOOC
lipases/whole cells
organic solvent
O
O
a. Prins reaction
COOH
HOOC
+
b. Jones oxidation
O
O
S
R
microbial reduction
+
(RS)-1
HO
O
COOR
COOR
HOOC
ROOC
COOR
ROOC
+
lipases/whole cells
water
O
O
(R)-1
+
O
O
(R)-2
microbial reduction
(S)-2
+
HO
O
(R)-1
Scheme 1. Biocatalytical approaches for the resolution of anti-2-oxotricyclo[2.2.1.0]heptan-7-carboxylic acid (RS)-1.
Table 1. Hydrolysis of different esters of 1 with Bacillus coagulans NCIMB 9365
Ester
Molar conversion (%)
ee (%) of the remaining (R)-ester
E
Time (h)
Methyl
Ethyl
Propyl
Butyl
Pentyl
Cl-ethyl
Br-ethyl
Benzyl
54
60
63
68
67
65
59
74
75
84
>99
>99
95
93
70
70
10
9
25
12
9
9
3
4
24
24
24
24
24
3
3
8
molar conversion
enantiomeric excess
The propyl ester (RS)-2 (R = Pr) was hydrolysed with
the highest enantioselectivity and the enantiomerically
pure unreacted ester was recovered from the biotrans-
formation mixture by filtration and extraction with ethyl
acetate at an alkaline pH.
100
80
60
40
20
0
100
80
60
40
20
The biocatalytic reduction of the carbonyl group in 1
and its esters (RS)-2 (R = methyl, propyl and butyl)
was also investigated. A screening was performed with
70 yeasts from international collections belonging to dif-
ferent genera and species. Kluyveromyces marxianus
CBS 2235 resulted to be the most enantioselective: opti-
misation of the reaction parameters (biocatalyst and
substrate concentration, pH, temperature, type and con-
centration of co-substrate) showed that the use of 15 g/l
of biocatalyst at 28 ꢁC, in a phosphate buffer (pH 7.0,
0.1 M) in the presence of 25 g/L of glucose allowed for
60% conversion of 3.0 g/L of substrate within 90 min
with >99% ee of the desired unreacted stereoisomer
(Fig. 1). The use of K. marxianus for the enantioselective
reduction of carbonyls has already been reported for the
preparation of useful chiral intermediates.13–17
0
4
0
0.5
1
1.5
2
2.5
3
3.5
Time (h)
Figure 1. Molar conversion and enantiomeric excess of propyl ester
(RS)-2 (R = Pr) with Kluyveromyces marxianus CBS 2235; enantio-
meric excess is referred to the unreacted propyl ester of anti-2-
oxotricyclo[2.2.1.0]heptan-(R)-7-carboxylic acid (R)-2 (R = Pr).
The reduction was also performed on a larger scale
(50 L reactor) giving similar or improved results. The

A. Romano et al. / Tetrahedron: Asymmetry 16 (2005) 3279–3282
3281
crude extract, recovered after filtration of the biomass,
4.4. Enzymatic esterification of anti-2-oxotricyclo-
[2.2.1.0]heptan-(RS)-7-carboxylic acid (RS)-1
extraction with ethyl acetate and solvent evaporation,
was derivatised with a Girard reagent for easy separa-
tion of the alcohol produced. The enantiomerically pure
ester was hydrolysed and used for next steps aimed at
the production of D-cloprostenol.
Anti-2-oxotricyclo[2.2.1.0]heptan-7-carboxylic acid (RS)-
1 (10.0 mg) was dissolved in organic solvent (5.0 mL)
and dry biocatalyst (125 mg as lyophilised cells or
dry enzymes) was added; the mixture was stirred at
different temperatures for 7 days and the reaction fol-
lowed by TLC (ethyl acetate/acetic acid = 98:2). After
7 days, the reaction mixture was filtered and the organic
phase evaporated. The residue was purified by chroma-
tography (n-hexane/ethyl acetate = 1:1 for recovering
the formed ester product and then ethyl acetate/acetic
acid = 98:2 for recovering the substrate). The enantio-
meric composition of the methyl, n-propyl and n-butyl
ester was determined by gas chromatographic analysis
using a chiral capillary column (diameter 0.25 mm,
length 25 m, thickness 0.25 lm, DMePeBeta-CDX-
PS086, MEGA, Legnano, Italy) at 130 ꢁC; the enantio-
meric composition of 1 was determined by HPLC using
a chiral column (Chiralcel OD, 4.6 · 250 mm, Daicel
Chemical Industries Ltd., Tokio, Japan) mobile phase:
n-hexane/2-propanol/HCOOH 90:10:1, flow 0.5 mL/
min, temperature 28 ꢁC, detection UV 280 nm.
3. Conclusion
In conclusion, the resolution of the propyl ester of anti-
2-oxotricyclo[2.2.1.0]heptan-7-carboxylic acid (RS)-2
(R = Pr) was efficiently achieved by the hydrolysis of
the ester or by the reduction of the carbonyl group. In
the latter case, although the reactive site is quite remote
from the stereocentre involved in the resolution, the
rigid structure allowed for sufficient stereodiscrimination.
Stereoselective reduction with Kluyveromyces marxianus
CBS 2235 showed a few technical advantages, such as
inexpensive fermentation, high biomass production,
easy scale-up and the simple separation of the alcohol
by derivatisation as Girard reagent.
4. Experimental
4.1. General
4.5. Enzymatic hydrolysis of anti-2-oxotricyclo[2.2.1.0]-
heptan-(R,S)-7-carboxylic acid esters (RS)-2
Chemicals were of reagent grade and purchased form
Fluka, Milano, Italy.
The biocatalyst (125 mg of whole cells or commercial
enzyme) was added to 5 mL of phosphate buffer (1/
15 M, pH 6.8) and the reaction started by the addition
of the substrate (RS)-2 (12.5 mg). The reaction was fol-
lowed by chiral HPLC under the conditions already
described. When the biotransformation had reached the
desired degree of conversion and/or enantioselectivity,
the suspension was centrifuged, and acidified with an
aqueous solution of HCl (5%) and extracted with ethyl
acetate. The extracts were washed with brine, dried over
Na2SO4 and evaporated. The residue was purified by
flash chromatography as described for the enzymatic
esterification.
4.2. Microorganisms, media and culture conditions
The microorganisms were from CBS (Centraal Bureau
voor Schimmelcultures, Baarn, The Netherlands),
MIM (Microbiologia Industriale Milano, Italy),
NCIMB (National Collection of Industrial and Marine
Bacteria, Aberdeen, UK) and cultured as described
before.7 Bacillus coagulans NCIMB 9365 and Kluyver-
omyces marxianus CBS 2235 were cultured in 5.0 L
fermenters: Bacillus coagulans was grown at 45 ꢁC
using CYSP broth agitation speed 100 rpm (casitone
15 g Lꢀ1, yeast extract 5 g Lꢀ1, soytone 3 g Lꢀ1, peptone
2 g Lꢀ1, MgSO4Æ7H2O 15 mg Lꢀ1, FeCl3 115 mg Lꢀ1
,
MnCl2 20 mg Lꢀ1 and pH 7.0), while Kluyveromyces
marxianus with 1.0 L of malt broth pH 6.0 for 48 h, at
27 ꢁC and agitation speed 100 rpm.
4.6. Screening for the reduction of methyl, propyl and
butyl esters of anti-2-oxotricyclo[2.2.1.0]heptan-(RS)-7-
carboxylic acid on small scale (RS)-2
The yeasts were centrifuged and suspended in 5 mL of
phosphate buffer (1/15 M, pH 6.8) to reach a concentra-
tion of 15 mg/mL, glucose (125 mg) was added and the
mixture stirred at 28 ꢁC for 30 min. Racemic esters
(RS)-2 (R = methyl, propyl and butyl) (150 mg) were
added and the mixture stirred at 28 ꢁC. When the reac-
tion had reached the desired conversion, the cells were
separated by centrifugation and the aqueous phase
extracted with ethyl acetate, dried over Na2SO4 and
evaporated. The crude products were purified by flash
chromatography (CHCl3/ethyl acetate = 95:5) and ana-
lysed by gas chromatographic analysis using a chiral
capillary column (diameter 0.25 mm, length 25 m, thick-
ness 0.25 lm, DMePeBeta-CDX-PS086, MEGA,
Legnano, Italy) at 130 ꢁC.
4.3. Synthesis of anti-2-oxotricyclo[2.2.1.0]heptan-7-car-
boxylic acid esters (RS)-2
Anti-2-oxotricyclo[2.2.1.0]heptan-7-carboxylic acid (RS)-
1 (2.0 g) was dissolved in dry CH2Cl2 (30 mL) and
dimethylaminopyridine (DMAP, 150 mg) and the corre-
sponding alcohol (0.9:1 molar ratio with respect to the
acid) were added; the mixture was brought to 4 ꢁC and
dicyclohexylcarbodiimide (DCC, 2.45 g) was added
and the reaction mixture stirred for 3 h at room temper-
ature. The mixture was filtered and the filtrate evapo-
rated at 35 ꢁC under vacuum. The residue was purified
by flash chromatography (n-hexane/ethyl acetate = 1:1)
to give the desired ester with yields in the range of 80–
95%.

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A. Romano et al. / Tetrahedron: Asymmetry 16 (2005) 3279–3282
25
4.7. Reduction of the propyl ester of anti-2-oxotricyclo-
[2.2.1.0]heptan-(RS)-7-carboxylic acid (RS)-2 (R = Pr)
with Kluyveromyces marxianus and recovery as Girard
reagent
carboxylic acid (R)-1. ½aꢁD ¼ þ93:0 (c 1.0 dioxane),
25
½aꢁD ¼ þ81:1 (c 1.0 MeOH); 1H NMR (300 MHz,
CDCl3)
d 1.50 (t, J = 5.9 Hz, 1H), d 1.92 (d,
J = 10.8 Hz, 1H), d 1.98 (d, J = 10.8 Hz, 1H), d 2.15
(s, 1H), 2.16 (t, J = 5.9 Hz, 1H), 2.40 (t,
d
d
Kluyveromyces marxianus CBS 2235 (48 g dry weight)
was suspended in 3.2 L of phosphate buffer (1/15 M,
pH 6.8) containing 80.0 g of glucose and the mixture
stirred for 45 min at 28 ꢁC; (RS)-2 (R = Pr) (8.0 g) was
then added and the reaction followed by chiral GC.
After 2 h, the enantiomeric excess of the substrate was
100% with 64–65% molar conversion and the reaction
mixture was filtered; the aqueous phase was extracted
with ethyl acetate, dried over Na2SO4 and evaporated
giving 8.1 g of crude residue. The crude residue was dis-
solved in MeOH (240 mL) and acetic acid (12.8 mL) and
Girard T reagent (16.0 g) then added. The mixture was
refluxed for 15 h. The mixture was then cooled at
20 ꢁC and NaOH (20% aqueous solution) was added
until pH 7.0. MeOH was distilled under vacuum and
the remaining solution was extracted with CH2Cl2.
The organic extracts were washed with brine, dried over
Na2SO4 and evaporated giving 2.0 g of enantiomerically
J = 5.9 Hz, 1H), d 3.04 (s, 1H). HRMS calculated for
CHO 170 (M+).
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25
(R)-7-carboxylic acid (R)-2 (R = Pr). ½aꢁD ¼ þ23:1 (c
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