Field of the invention a ~ E P 4 The present invention relates to a process for the preparation of optically active
(R)-(-)-3-Quinuclidinofl rom 3-quinuclidinone mediated by whole cell biocatalysts.
Particularly, the present invention provides a process for the preparation of
optically active (R)-(-)-3-Quinuclidinol from 3-quinuclidinone using Mucor such as
M. hiemalis with 100%ee.
Background of the invention
Biocatalysis employing enzymes has been attracting attention as an eco-friendly
synthetic method and is being increasingly used in pharmaceutical and chemical
industries. Enzymes have the ability to catalyze reactions with strict enantioand
regio-selectivities under favorable conditions and omit complicated protection and
deprotection steps which are'common in conventional organic synthesis and also
prevents the fossil fuel wastage by avoiding high-temperature and high-pressure
conditions. Thus, biocatalysts are more satisfactory than conventional chemical
catalysts from economic and environmental points of view. Both isolated enzymes
and whole cells are being industrially used as catalysts. The use of pure isolated
enzymes is limited as they cannot be reused and the cofactors used are very
expensive.
Chiral compounds are valuable precursors for pharmaceuticals and chemicals
including chemical catalysts, liquid crystals, flavors, agrochemicals and drugs.
Mostly, only single isomer exhibits the desired activity and thus important to
develop routes to produce optically pure compounds. Many chiral alcohols can be
produced by biocatalytic. methods either by kinetic resolution of racemates or
through asymmetric reduction from prochiral compounds (Griffin D.R.; Gainer J.L.;
Carta G. ; Biotechnol. Prog., 2001, 1 7,304-3 10). Biocatalyst mediated asymmetric
reduction of prochiral carbonyl compounds is a promising method for the
production of chiral alcohols as they carry out the chemical reactions under mild
conditions and with specific chemo-, regio- and stereo-selectivities ( Kataoka M.;
Kita K.; Wada M.; Yasohara Y.; Hasegawa J.; & Shimizu S., Appl. Microbiol.
Biotechnol., 2003, 62, 437-445)(R)-3-Quinuclidinol 2 (R- 1 -azabicyclo[2.2.2 Joctan-
3-01) is an important precursor for the syntheses of muscarinic receptor ligands,
2
including solifenacin3 (M3 receptor antagonist), revatropate4 (M3 receptor
antagonist), and talsaclidine5 (MI receptor ag~nist)'~'~T'a. lsaclidinefumarate6
(WAL 2014 FU), cevimeline HCl , 7 and YM 905 8 have shown potential in the
treatment of Alzheimer's disease, Sjijgren syndrome, and urinary incontinence,
respectively. (R)-3-quinuclicinol has also been used as an important intermediate
to produce a variety of physiologically active or pharmacologically active agents,
for example, in therapeutic agents for arteriosclerosis, which have the activity of
inhibiting squalene synthase, bronchodilators having antagonistic action to the
muscarinic receptor, agents suppressing gastrointestinal motility, etc.
(Unexamined Published Japanese Patent Application No. (JP-A) Hei8-134067; EP-
404737A2; EP-42402 1 A1 ; W092 104346; and W093 106098).
Carbamoyl derivatives of 3-quinuclidinol such as 9 and 10 are known to inhibit the
enzyme acetyl cholinesterase, which i s responsible for the degradation of the
neurotransmitter acetylcholine. The (R)-enantiomer of 9 and the racemic 10 have
been shown to be protective against organophosphate poisoning in guinea pigs and
mice respectively. In Organic synthesis, (R) - or (S)-3-quinuclidinol i s used as a as a
catalytic base in the Baylis-Hillman reaction (Basavaiah D.; Rao P.D.; Hyma R.S.,
Tetrahedron, 1996, 52, 8001 ).
To produce (R)-(-)-3-Quinuclidinol of such pharmacological importance, many 4 approaches have been developed till date, each however, suffering from a
practical drawbacks. The number of prior art approaches and the drawbacks
involved in the same are listed below:
Kinetic resolution of a racemic mixture of N-butyl-3-quinuclidinol through
asymmetric hydrolysis with Aspergillus melleus protease has been used for
industrial production. (Nomoto F.; hirayama Y.; lkunaka M.; lnoue T.; Otsuka
K., Tetrahedron, 2003, 14, 1871 - 1877)
Asymmetric reduction of 3-Quinuclidinone with 3-Quinuclidinone reductase from
Rhodotorula rubra which requires cofactors (Uzura A.; Nomoto F.; Sakoda A.;
Nishimoto Y.; Kataoka M.; Shimizu S., Appl. Microbial. Biotechnol., 2009, 83,
61 7-626)
Kinetic resolution through diastereomeric salt formation of (*)-3-Quinuclidinol
with (1 S)-camphorsulphonic acid (Sternbach L. H. ; Kaiser S., J. Am. Chem. Soc.
1952, 74, 2215)
Kinetic resolution through enantioselective hydrolysis of (R)-quinuclidin- 3-yl
butyrate by horse serum butyrylcholine esterase (Primoz'ic' I.; Hrenar T.;
Tomic' 5.; Meic Z., Eur. J. Org. Chem., 2003, 295-301)
Kinetic resolution via enantioselective hydrolysis of (9-3-
butyryloxyquinuclidinium butyrate by substilisin Carlsberg (US Patent
5,215,918)
A process which comprises reducing the starting material imine, which is
prepared from a 3-quinuclidinone and an optically active phenethylamine, with
the use of a sodium borohydrate (Langlois M.; Meyer C.; Soulier J. L., Synth.
Commun., 1992, 22(13), 1895-1 91 1).
Using the racemate of 3-quinuclidinol as a raw material, the R form i s obtained
by converting, with subtilisin protease, only the S form to (S)-3-quinuclidinyl
butyrate (German Patent 1971 5465)
A process for producing optically active 3-quinuclidinol by asymmetric
hydrogenation of 3-quinuclidinone, adduct with a Lewis acid, ammonium salt
thereof or the like in the present of a rhodium-optically active phosphine
complex (US Patent 5,744,606)
A process for producing optically active 3-quinculidinol by asymmetric
hydrogenation of 3-quinuclidinone in the presence of a basic compound, a
complex of a transition metal in Groups 8 to 10 with an optically active
bidentate ligand and an optically active diamine (European Patent 1650207) . A process for producing optically active 3-quinuclidinol by asymmetric reduction
of 3-quinculidinone using tropinone reductase (US Patent 7,645,599)
. Asymmetric reduction of 3-quinuclidinone to (R)-3-quinuclidinol by the
microorganisms belonging to the genus Nakazawaea(92% ee), the genus Candida
(49% ee) and the genus Proteus (80% ee). The microorganisms capable of producing
(S)-3-quinuclidinol include the genus Arthrobacter(95% ee), the genus
Pseudomonas (76% ee) and the genus Rhodosporidium (4% ee) (European Patent
0863 2 1 2)
Asymmetric hydrogenation of 3-quinuclidinone with a Ruthenium(l1) complex to
yield (R)-3-Quinuclidinol with 88-90% ee (Tsutsumi K.; Katayama T.; Utsumi N.;
Murata K.; Arai N.; Kurono N.; Ohkuma T., Organic Process Research and
development, 2009, 13, 625-628)
0 Granted Japanese patent JP4818507 discloses a method for producing optically
active 3-quinuclidinol with 82%eeby asymmetrical reduction reaction using an
effective microbiological catalyst i.e. Mucor sp 14021 .
However, the product obtained by these production methods either have very low
optical purity or involve very complicated steps with expensive reagents or
enzymes and cofactors. Thus, most of the methods mentioned above cannot be
treated as a method to produce (R)-3-quinuclidinol in a simple, economical and an
eco-friendly way.
Objectives of the invention
The main object of the present invention is to provide a process for producing an
optically active quinuclidinol [(R)-(-)-3-quinuclidinol] which is simple, economical
and eco- friendly.
5
I b r 9 $1 pL
(,R'&c: ' ' o , sc? @
Another object of the present invention is to provi r the preparation 0 of optically active (R)-(-)-3-Quinuclidinol from 3-quinuclidinone using Mucor such
as M. hiemalis.
Another object of the present invention is to provide a process for the preparation
of optically active (R)-(-)-3-Quinuclidinol from 3-quinuclidinone using Mucor such
as M. hiemalis with 100% ee.
Another object of the present invention is to provide a process for preparation of
an optical active quinuclidinol [(R)- (-)-3-quinuclidinol] efficiently with good
conversion using an asymmetric reduction by a whole cell microorganism.
Summary of the invention:
Accordingly, the present invention provides a process for asymmetric reduction of
3-quinuclidinone hydrochloride to give (R)-(-)-3- quinuclidinol with 100% ee
comprising:
a) inoculating spore suspensions of Mucor hiemalis, in Czapek Dox medium
followed by incubating at 28" to 30°C on a shaking incubator at 180 to 200
rpm for 24-36 hours;
b) adding the substrate, 3-quinuclidinone hydrochloride to the culture medium
as obtained in step (a) followed by incubation for an additional period of 9
days to 12 days to obtain (R)-(-)-3-quinuclidinol hydrochloride;
c) adjusting the pH of (R)-(-)-3-quinuclidinol hydrochloride as obtained in
step (b) to an alkaline pH 11-12 using base solution to obtain (R)-(-)-3-
quinculidinol ;
d) extracting free (R)-(-)-3-quinuclidinol with an organic solvent; and
e) purifying (R)-(-)-3-quinuclidinol.
In one embodiment of the present invention the concentration of 3-quinuclidinone
hydrochloride added to the culture medium i s in the range of 1 - 2gIL.
In an embodiment of the present invention the base used in step (c) i s sodium
hydroxide.
In another embodiment of the present invention the concentration of sodium
@ hydroxide is in the range of 25-30%.
In another embodiment of the present invention the organic solvent used in step
(d) is selected from the group consisting of dichloroethane, dichloromethane,
methylene dichloride and chloroform.
Description of drawings:
Fig 1 depicts biotransformation kinetics of 3-quinuclidinone hydrochloride.
Fig 2 depicts GC profile of asymmetrically reduced (R)-(-)-3-quinculidinol, which
was confirmed by co-injecting with the commercially available standards.
Fig 3 shows 'HNMR of (R)-(-)-3-quinculidinol produced according to the process of
the invention.
Fig 4 depicts I3c NMR of (R)-(-)-3-quinculidinol produced according to the process
of the invention.
Fig 5 shows DEPT (DistortionlessEnhancement by Polarization Transfer) spectra of
(R)-(-)-3-Quinuclidinol produced according to the process of the invention.
Fig 6 shows IR of (R)-(-)-3-quinculidinol produced according to the process of the
invention.
Detailed description of the invention:
The present invention discloses a method for producing (R)-(-)-3-quinuclidinol, an
important chiral precursor for various pharmaceuticals. After extensive screening,
the present inventors have found that an optically active quinuclidinol [(R)-(-)-3-
quinuclidinol] can efficiently and economically be produced from a quinuclidinone
(e.g. 3-quinuclidinone) through asymmetric reduction by microorganisms belonging
to the family Mucoraceae, genus Mucor such as M. hiemalis, as depicted in scheme
1. Scheme 1
Mtrcor species
0""P N - a"" N -
CI H'+ CI
Formula 1 Formula 2
Thus the present invention discloses a process for producing an optically active 4 quinuclidinol which comprises, utilization of a microorganism for the production of
(R)-(-)-3-quinuclidinol and the method for the extraction of the product. The
microorganisms used in this study are fungal strains, belonging to the family
Mucoraceae, genus Mucor such as M. hiemalis.
The present invention provides an efficient, economical and eco-friendly process
for the production of (R)-(-)-3-Quinuclidinol quantitatively with 100% ee through
asymmetric reduction using whole cell microorganism.
The inventors of the present invention conducted extensive screening of the
microorganisms which can efficiently produce 3-quinuclidinols through stereoselective
reduction of 3-quinuclidinone.
A wide range of microorganisms were screened against the substrate, 3-
Quinuclidinone hydrochloride, for the efficient and quantitative transformation
into i t s reduced metabolite, 3-Quinuclidinol. All the strains were grown in 500 ml
Erlenmeyer flasks containing 100 ml of sterile modified Czapek D O X ~m~e dium.
After a period of 24 h, 80 mg of 3-Quinuclidinonk hydrochloride was added to the
well grown cultures. Incubation was continued for 5 days and then extracted with
dichloromethane at pH 12 and analyzed the conversion by TLC and GC.
As a result, the inventors of the present invention found that the fungal strains,
Mucor hiemalis (From NCIM, NCL, Pune, Cat No. 873) was efficiently reduced 3-
quinuclidinone to (R)-(-)-3-quinuclidinol with quantitative yield.
The invention provides a process for producing an optically active 3-quinculidinol
comprising asymmetric reduction of 3-quinuclidinone (1 -Aza- bicyclo[2.2.2]octan- 3-
one) by whole cell microorganisms, wherein 3- quinuclidinol with 100% ee i s
achieved.
The optically active alcohol according to the process of the invention i s (R)-(-)-3-
quinculidinol ( I -ha-bicyclo[2. 2.2loctan-3-01).
The whole cell microorganisms according to the process of the invention are
@ selected from the fungal strains belonging to the family Mucoraceae, of the genus
Mucor species having asymmetric reduction capability.
The process for asymmetric reduction of 3-quinuclidinone to give 3-quinuclidinol is
depicted in Scheme1 as follows:
Scheme 1:
hfucor 0"-"hfuc or species N - GN O- H
CI CI
Formula 1 Formula 2
The present invention provides a process for asymmetric reduction of 3-
quinuclidinone hydrochloride to give 3-quinuclidinol with 100% ee comprising the
steps of:
a) propagating and maintaining the fungal strain, Mucor hiernalis on potato
dextrose agar slants;
b)inoculating spore suspensions of Mucor hiemaiis is in Czapek Dox medium
followed by incubating at 30°C on a shaking incubator at 200 rpm for 24-36
hours;
c) adding the substrate, 3-quinuclidinone hydrochloride to the culture medium
followed by incubating for an additional period of 9 days; and
d) monitoring the course of the reaction through TLC and GC analysis to obtain
the desired (R)-(-)-3-quinuclidinol hydrochloride by maximum conversion.
e) adjusting the pH of (R)-(-)-3-quinuclidinol hydrochloride solution alkaline to
obtain (R)-(-)-3-quinculidinol by altering the pH to 12 using base solution;
f)extracting free (R)-(-)-3-quinuclidinol with an organic solvent; and
8) Purifying (R)-(-)-3-quinuclidinol.
Accordingly, in a sterile modified Czapek Dox medium asymmetric reduction of the
substrate i.e.3-quinuclidinone hydrochloride i s performed by inoculating the sterile
medium respectively with spore suspensions from a 2 day old, well sporulated
fungal strains, Mucor hiernolis followed by incubating at 3 0 ' ~ on a shaking 6 incubator at 200 rpm for 24-36 hours.
The composition of the modified Czapek dox medium comprises glucose (30 g L-I),
sodium nitrate (3 g L-I), dipotassium hydrogen phosphate (1 g I ) , magnesium
sulphate (hydrated) (0.5 g L"), potassium chloride (0.5 g L-I), Yeast extract (0.5 g
L-I), corn steep liquor (5 mL L") in distilled water maintained at pH 5.8. The pH of
the medium was adjusted with 1M K2HP04.
The concentration of 3-quinuclidinone hydrochloride added to the culture medium
is in the range of 1 - 2glL.
The inorganic salts and analytical grade solvents were obtained from Merck,
Mumbai. The media ingredients were procured from Himedia, Mumbai and used
deionized milliQ water.
The pH is adjusted to 12 using a base solution. The base i s selected from the group
consisting of sodium hydroxide, potassium hydroxide.
The base used to alter the pH of (R)-(-)-3-quinuclidinol hydrochloride obtained in
step (a) is sodium hydroxide solution in the range of 25-30%.
The solvent used for extraction is selected from the group consisting of chlorinated
solvents such as dichloro-ethane, dichloromethane, methylene chloride,
chloroform etc.
The purification step includes removal of moisture content from the extract. The
crude extract is analyzed through TLC and GC and passed through a filter column
to obtain the product, which is the characterized product, (R)-(-)-3-quinuclidinol
through NMR, IR, optical rotation, melting point and HRMS analysis.
The purification of the product, (R)-(-)-3-quinuclidinol was performed by
subjecting the crude extract to column chromatography using basic alumina as the
stationary phase and methano1:dichloromethane (1:99 to 5:95) as the mobile
phase.
The Thin-layer chromatography (TLC) was performed on silica gel G-coated plates
(0.25 mm for analytical) developed with MethanollDichloromethane (95:5).
10
Compounds were visualized by spraying with a solution of 3.0 % anisaldehyde, 2.8% 4 ti2So4, 2% acetic acid in ethanol followed by heating at 80°C for 1-2 min or the
compounds can be visualized by charring with Iodine.
The Gas chromatography analysis were carried out on an Agilent 7890 instrument
equipped with a hydrogen flame ionization detector and HP-5 Chiral capillary
column (30 m X 0.32 mm X 0.25 pm, J 8 W Scientific). Nitrogen was used as the
carrier gas at a constant pressure of 3.885 psi. Initially, the column temperature
was maintained at 60 "C, followed by a temperature gradient from 60°C to 140 "C
at 2 "C min" and held constant for 25 min at 140 "C, then finally raised to a
temperature of 200 'C with a 10 "C min-' rise and maintained for 5 min at 200 "C.
The injector and detector temperatures were maintained at 230 O C and operated
in split mode (split ratio 1 : 10).
The 'H and 13c NMR spectra were recorded in D20 on a Bruker DRX-500
spectrometer at 500.13 and 125.78 MHz respectively. Chemical shifts are given in
&values relative to TMS (tetramethylsilane) as an internal standard. IR spectra
were recorded on Shimadzu 8400 series FTlR instrument and values are reported in
cm-' units. Optical rotations ([aID) have been recorded using Jasco, P-2000
polarimeter and are reported in deg dm-'.
The process according to the invention for the biotransformation of 3-
quinuclidinone to 3-quinculidinol i s depicted in the scheme (2) as shown below:
Scheme 2
go M-- s~ecles , ~ o H e n r a n i O n * TLC and GCiMS analysis
N of analytical quantities
3-Quinuclidinone (R)9-Quinuclidinol
hydrochloride hydrochloride 1 Column chromatography
purification of
preparative amounts
To collect the analytical data;
NMR, IR, melting point,
HRMS, optical rotation
The biotransformation kinetics and time course study of 3-Quinuclidinone (2gIL):
3-Quinuclidinone (A) and (R)-(-)-3-Quinuclidinol (0) is shown in fig 1.
11
The GC profile of asymmetrically reduced (R)-(-)-3-quinculidinohas confirmed by 4 co-injecting with the commercially available standards as in fig 2.
The NMR, IR, optical rotation, melting point details are as follows;
'H NMR (500 MHz, D20); 6(ppm): 1.45-1.52 (2H, m, H-5a & H-8a), 1.7-1.8 (2H, m,
H-56 & H-86), 1.84 (1 H, m, H-4), 2.44-2.47 (1 H, m, H-26), 2.57-2.67 (2H, m, H-6),
2.74 (2H, m, H-7), 3.07 (IH, m,H-2a), 3.94 (IH, m, H-3). (Figure 3)
"C NMR(125 MHz, D20): d(ppm): 55.44 (C-2), 67.30 (C-3), 26.90 (C-4), 17.78 (C-
5), 45.01 (C-6), 46.04 (C-7), 23.48 (C-8).(Figure 4)
IRV m (~cm -I): 3270.76, 3020.78, 2972.14, 1465.01, 1371.39, 13 1 2.45, 1286.49,
1 1 19.39, 1044.23, 896.12, 829.36, 775.46, 640.82. (Figure 6)
Optical Rotation: [a]d5 is -44.42 (c 2.0, 1M HCl) which i s in agreement with the
reported value of -44.9 (c 2.0, 1M HCI).
According to the present invention with the aid of a microorganism, an optically
active 3-quinuclidinol [especially (R)-(-)-3-quinuclidinol)] can be produced from a
3-quinuclidinone with commercial advantages in a simple and easy manner.
Furthermore, an optically active 3-quinclidinol [especially(R)-(-)-3-quinuclidinol)]
can efficiently be produced by using an asymmetric reduction .caused by a wild
type microorganism.
Industrial applicability:
According to the present invention, an optically active (R)-(-)-3-quinuclidinol with
100% ee can be produced through asymmetric reduction by the fungal strains,
Mucor hiemalisand. The present invention discloses a green, efficient, economical,
simple, mild and a convenient method for the production of (R)-(-)-3-Quinuclidinol
with a substrate concentration of 2glL with conveniently maintainable fungal
strains, Mucor hiemalis and, thereby making the present invention extremely @ viable for industrial production.
Examples:
The following examples are given by way of illustration of the working of invention
in actual practice and should not be construed to limit the scope of the present
invention in any way.
Example 1:
Cultivation of Mucor spp.and screening of microorganisms against the substrate
A wide range of microorganisms were screened against the substrate i.e. 3-
Quinuclidinone hydrochloride for the efficient and quantitative transformation into
i t s reduced metabolite,3-Quinuclidinol. All the strains were grown in 500 ml
Erlenmeyer flasks containing 100 ml of sterile modified Czapek Dox medium and
incubated for a period of 24 hrs. After incubating the cultures for a period of 24
hours, 80 mg of the substrate, i.e. 3-Quinuclidinone hydrochloride was added to
the cultivated cultures. Incubation was continued for 5 days and then extracted
with chlorinated organic solvents such as dichloromethane at a pH 12 and the
conversion was analyzed by Thin Layer chromatography (TLC) and
Gaschromatography (GC).
Mucor species selected from Mucor was used as biocatalyst in the conversion of 3-
Quinuclidinone to 3-quinuclidinol.
Example 2:
Process of.biocatalysis of 3-Quinuclidinone to 3-quinuclidinol by Mucor spp.
Each of the fungal strains of Mucor hiemalis was individually propagated and
maintained on potato dextrose agar slants.
The process of asymmetric reductionof 3-Quinuclidinone was performed in a sterile
modified Czapek dox medium by inoculating the sterile medium with spore
suspensions from a 2 day old, well sporulated fungal strain of either Mucor
hiemalis followed by incubating at 30°C on a shaking incubator at 200 rpm for 36
hours.
13
The substrate comprising 3-quinuclidinone hydrochloride (80mg)was added to the 0 culture medium (50 ml) followed by incubating for an additional period of 9 days
and monitoring the course of the reaction through TLC and GC analysis to obtain
the desired (R)-(-)-3-quinuclidinol hydrochloride by maximum conversion of 95 %
and 100 % ee.
The extraction process further comprises adjusting the pH of (R)-(-)-3-quinuclidinol
hydrochloride solution to an alkaline pH to obtain (R)-(-)-3-quinculidinol by altering
the pH to 12 using base solution. The free (R)-(-)-3-quinuclidinol was extracted
with an organic solvent andwas further subjected to purification procedures.
Example 3:
Analysis of 3-quinuclidinol
i. Thin-layer chromatography (TLC) was performed on silica gel G-coated plates
(0.25 mm for analytical) developed with MethanollDichloromethane (95:5). 10
to 20 pl of the sample was spotted on the TLC plates. Compounds were
visualized by spraying with 3% anisaldehyde, 2.8% HzS04, 2%acetic acid in
ethanol followed by heating at 80°C for 1 min or visualizing the compounds by
charring with Iodine.
ii. Gas chromatography analysis was carried out on an Agilent 7890 instrument
equipped with a hydrogen flame ionization detector and HP-5 Chiral capi llary
column (30 m X 0.32 mm X 0.25 pm, J & W Scientific). Nitrogen was used as the
carrier gas at a constant pressure of 3.885 psi. Initially, the column
temperature was maintained at 60°C, followed by a temperature gradient from
60°C to 140°C at 2 'C min" and held constant for 25 min at 140 "C, then finally
raised to a temperature of 200 "C with a 10°C min*' rise and maintained for 5
min at 200 "C. 1pl of the samplelanalyte was injected into the "entrance"
(head) of the column, usually using a microsyringe. The injector and detector
temperatures were maintained at 230 "C and operated in split mode (split ratio
1:IO).
iii. The 'H and I3c NMR spectra were recorded in D20 on a Bruker DRX-500
spectrometer at 500.13 and 125.78 MHz respectively. Chemical shifts were
given in &values relative to TMS (tetramethylsilane) as an internal standard. IR 4 spectra were recorded on Shimadzu 8400 series FTlR instrument and values are
reported in cm-' units. Optical rotations ([aID) were recorded using Jasco, P-
2000 polarimeter and were reported in deg dm-'.
Advantages of the present invention:-
Present invention describes conversion of 3-quinuclidinone to (R)-3-quinuclidinol
using whole cell fungus Mucor hiemalis. The culture has ability to reduce 3-
quinuclidinone with high enantiomeric excess (almost 100 %). The organism can
transform substrate up to 2 g/L concentration with 94 to 96 % conversion. Thus.
this transformation process can be scaled up for the industrial production of (R)-3-
quinuclidinol.
We claim, 4 I. A process for asymmetric reduction of 3-quinuclidinone hydrochloride to give
(R)-(-)-3- quinuclidinol with 100% ee comprising:
a) inoculating spore suspensions of Mucor hiemalis, in Czapek Dox medium
followed by incubating at 28" to 30°C on a shaking incubator at 180 to 200
rpm for 24-36 hours;
b) adding the substrate, 3-quinuclidinone hydrochloride to the culture medium
as obtained in step (a) followed by incubation for an additional period of 9
days to 12 days to obtain (R)-(-)-3-quinuclidinol hydrochloride;
c) adjusting the pH of (R)-(-)-3-quinuclidinol hydrochloride as obtained in
step (b) to an alkaline pH 11 -12 using base solution to obtain (R)-(-)-3-
quinculidinol ;
d) extracting free (R)-(-)-3-quinuclidinol with an organic solvent; and
e) purifying (R)-(-)-3-quinuclidinol.
2. The process according to claim 1, wherein the concentration of 3-
quinuclidinone hydrochloride added to the culture medium is in the range of 1
- 2glL.
3. The process according to claim 1, wherein the base used in step (c) is sodium
hydroxide.
4. The process according to claim 3, wherein the concentration of sodium
hydroxide is in the range of 25-30%.
5. The process according to claim 1, wherein the organic solvent used in step (d)
is selected from the group consisting of dichloroethane, dichloromethane,
methylene dichloride and chloroform.