Technical Field
The present invention relates to process for the preparation of optically active
3-acyloxy-gamma-butyrolactone repesented by the general formula 5 and optically
active 3-hydroxy-gamma-butyrolactone represented by the general formula 6 in
scheme 1 from racemic 3-acyloxy-gamma-butyrolactone by enzymatic method. In
more detail, racemic 4-chloro-3-hydroxybutyronitrile(2), racemic
3-hydroxy-gamma-butyrolactone(3), racemic 3-acyloxy-gamma-butyrolactone(4) is
prepared from racemic epichlorohydrin(l) in turn, racemic
3-acyloxy-gamma-butyrolactone is hydrolyzed stereospecifically by lipases or lipase-
producing microorganisms in aqeous phase or organic Dphase containing water for the
preparation of optically active 3-hydroxy-gamma-butyrolactone and optically active
3-acyloxy-gamma-butyrolactone. Optically active 3-acyloxy-gamma-butyrolactone can
be converted to optically active 3-hydroxy-gamma-butyrolactone by deacylation, and
is used for precursor of other useful substances. According to this invention, it is
possible to transform 3-acyloxy-gamma-butyrolactone with low optical purity into
3-acyloxy-gamma-butyrolactone or 3-hydroxy-gamma-butyrolactone with high optical
purity.
[Scheme 1]


(R=C H , n=l~8)
n 2n+I
Optically active 3-hydroxy-gamma-butyrolacotne and
3-acyloxy-gamma-butyrolactone are important intermediates. Particularly they are
used in preparing pharmaceuticals such as L-carnitine, hypertension drug and hyper-
lipidemia drug.
Background Art
There are several methods to prepare optically active
3-hydroxy-gamma-butyrolactone and optically active 3-acyloxy-gamma-butyrolactone.
Yuasa et al.(Liebigs ann. /Recuei/. 1997. 1877-1879) obtained ethyl
(S)-4-chloro-hydroxybutanoate(94 %e.e) using Ru-(R)-p-tolyl-BINAP catalyst from
ethyl 4-chloro-3-oxobutanoate, and (S)-3-hydroxy-gamma-butyrolactone is
synthesized using HC1 from above compound(yield 83%, 94%e.e).
In another case, Cheon et al.(KP 10-1999-0030062, KP 10-1999-0030065)
produced (S)-3,4-dihydroxybutyric acid from amilopectin and this is converted to
(S)-3-hydroxy-gamma-butyrolactone by cyclization. In addition, optically active
3-hydroxy-gamma-butyrolactone can be produced in high yield from starch or mal-
tooligosaccharide, in this condition other similar compounds are produced and it is not
easy to seperate impurities by the conventional method because of the structural
similarity. And this process includes many complicated steps.
Also, Gwak et al. (KP 2003-0004902) obtained
(S)-3-hydroxy-gamma-butyrolactone by cyclization of dihydroxybutyric acid methyl
ester produced from malic acid. But this method needs high temperature and high
pressure during the reaction.
Hollingworth et al.(USP 5808107) preparated (S)-3-hydroxy-gamma-butyrolactone

from malic acid. Sodium borohydride used as a reducing agent in this process is too
expensive, so this method is unsuitable for production on the large scale. Eur et al.(KP
2002-0073751) obtained (S)-3-acetoxy-gamma-butyrolactone by reduction of
(S)-3-acetoxy-succinic acid produced from L-malic acid.
Lee et al.(KP 2003-0065192) obtained (S)-4-chloro-3-hydroxybutyronitrile from
(S)-epichlorohydrin and (S)-3-hydroxy-gamma-butyrolactone was produced from
(S)-4-chloro-3-hydroxybutyronitrile. This method has a disadvantage of high cost
because (S)-epichlorohydrin is too expensive.
On the other hand, Suzuki et al.(Enzyme Microbiology and Technology, 1999, 24,
13-20) produced (S)-hydroxy-garnma-butyrolactone(92.4%e.e) from racemic ethyl
4-chloro-3-hydroxybutyrate using dechlorinase-producing microorganism.
Daicel, Ltd. produced optically active 3-hydroxy-gamma-butyrolactone(93.9%e.e)
from optically active ethyl 4-chloro-3-hydroxybutyrate using microorganism, but this
method is not useful for industrial use because substrate concentration is low(JP
2002-204699). And in the same way, they produced optically active
3-hydroxy-gamma-butyrolactone from racemic ethyl 4-chloro-3-hydroxybutyrate, but
they obtained the compound with low optical purity.
Miyazawa et al.(USP 5084392) obtained chiral 3-hydroxy-gammma-butyrolactone
by transesterification. After reaction (S)-3-hydroxy-gamma-butyrolactone with low
optical purity is re-esterified with the result of 85%e.e(yield 40 %).
The present invention relates to process for the preparation of optically active
3-acyloxy-gamma-butyrolactone and optically active 3-hydroxy-gamma-butyrolactone
from racemic 3-acyloxy-gamma-butyrolactone by hydrolysis using lipases or lipase-
producing microorganisms, and this method is a new method which was not reported
yet.
Disclosure of Invention
Technical Problem
With this in mind, the inventors synthesized 4-chloro-3-hydroxybutyronitrile from
epichlorohydrin(JP 5-310671) and 4-chloro-3-hydroxybutyronitrile was converted to
racemic 3-hydroxy-gamma-butyrolactone by well-known method(KP 2003-0065192).
Racemic 3-acyloxy-gamma-butyrolactone was obtained by acylation from racemic
3-hydroxy-gamma-butyrolactone. And the inventors obtained optically active
3-acyloxy-gamma-butyrolactone and optically active 3-hydroxy-gamma-butyrolactone
from racemic 3-acyloxy-gamma-butyrolactone by enzymatic hydrolysis.
This method is a new process for preparing optically active
3-hydroxy-gamma-butyrolactone. Also, it is easy to seperate optically active
compound from reaction mixture after reaction.

Technical Solution
This invention is explained in more detail as follows. As previously stated, this
invention includes the process for preparing 3-hydroxy-gamma-butyrolactone from
epichlorohydrin one by one, and 3-acyloxy-gamma-butyrolactone is obtained by
acylation. Racemic 3-acyloxy-gamma-butyrolactone is subjected to produce optically
active 3-acyloxy-gamma-butyrolactone and optically active
3-hydroxy-gamma-butyrolactone by hydrolysis using lipases or lipase-producing mi-
croorganisms.
In preparing optically active compound, lipases such as CAL B(Novozym 435,
Novozym) or PS-D(Amano) or lipase-producing microorganisms were used as bio-
catalysts.
After reaction, reactants and products were analyzed as belows.
Racemic 3-hydroxy-gamma-butyrolactone is determined using HP-FFAP
column(Agilent, Inc., 30 m X 0.53 mm). The oven temperature was maintained
initially at 100 °C for 5 min and then raised at the rate of 20 °C/min to 220 °C, and
maintained for 10 min. Helium gas was used as carrier and compounds were detected
using FID at 220 °C. In this condition the typical retention time of the compounds was
as follows:
racemic 3-acetoxy-gamma-butyrolactone -12.42 min
racemic 3-butoxy-gamma-butyrolactone -13.04 min
racemic 3-hydroxy-gamma-butyrolactone -17.83 min
Optically active 3-hydroxy-gamma-butyrolactone and optically active
3-acyloxy-gamma-butyrolactone were determined by HPLC(LAB Alliance, Model
201) equipped with chiral column AD-H(Daicel, 0.46 cm X 25 cm). Hexane and
isopropyl alcohol mixture(90:10) used as mobile phase and flow rate was 0.7 ml/min,
and the absorbance was 220nm. The typical retention time of the compounds in this
invention was as follows:
(R)-3-acetoxy-gamma-butyrolactone -20.30 min
(S)-3-acetoxy-gamma-butyrolactone - 21.39 min
(R)-3-butoxy-gamma-butyrolactone - 16.35min
(S)-3-butoxy-gamma-butyrolactone -18.19 min
(S)-3-hydroxy-gamma-butyrolactone -23.05 min
(R)-3-hydroxy-gamma-butyrolactone -28.14 min
The following specific examples are intended to be illustrative of the invention and
should not be construed as limiting the scope of the invention as defined by appended

claims.
Example 1. Preparing of racemic 3-acetoxy-gamma-butyrolactone
Pyridine(1.8 g) and acetyl chloride(1.57 g) was added to 20 ml of chloroform
containing racemic 3-hydroxy-gamma-butyrolactone at 0 °C and stirred at room
temperature for 2 hours. The reaction mixture was extracted with organic solvent and
concentrated to afford 1.83 g of racemic 3-acetoxy-gamma-butyrolactone. And this
compound was confirmed by nuclear magnetic resonance.
1H-NMR(3OOMHz, CDC1) : 2.06(s, 3H), 2.5~2.8(m, 2H), 4.3~4.5(m, 2H), 5.4(m,
1H) ppm
Example 2. Hydrolysis of racemic 3-acetoxy-gamma-butyrolactone
Racemic 3-acetoxy-gamma-butyrolactone(5%(v/v)) prepared from Example 1 was
added to 4.75 ml 0.2 M potassium phosphate buffer(pH 7.0) and the reaction was
carried out at 30 °C after adding lipase CAL B. The reaction mixture was extracted
with ethyl acetate and (R)-3-acetoxy-gamma-butyrolactone and
(S)-3-hydroxy-gamma-butyrolactone was analyzed by above-mentioned method. The
results are shown in Table 1.

Example 3. Preparation of racemic 3-butoxy-gamma-butyrolactone
Pyridine(3.5g) and butyryl chloride(4.7 g) was added to 100 ml of chloroform
containing racemic 3-hydroxy-gamma-butyrolactone at 0 °C and stirred at room
temperature. The reaction mixture was extracted with organic solvent and concentrated
to afford racemic 3-butoxy-gamma-butyrolactone. And this compound was confirmed
by nuclear magnetic resonance.
1H-NMR(300MHz, CDCy : 0.9(t, 3H), 1.5~1.7(dd, 2H), 2.2~2.3(t, 2H),
2.5~2.9(m, 2H), 4.3~4.5(m, 2H), 5.4(m, 1H) ppm

Example 4-5. Hydrolysis of racemic 3-butoxy-gamma-butyrolactone
Instead of racemic acetoxy-gamma-butyrolactone used in Example 2,
3-butoxy-gamma-butyrolactone synthesized in example 3 was used as a reactant. After
reaction the reaction mixture was extracted with ethyl acetate and
(R)-3-butoxy-gamma-butyrolactone and (S)-3-hydroxy-gamma-butyrolactone were
analyzed by above-mentioned method. The results are shown in Table 2.

Advantageous Effects
As aforementioned, racemic 3-acyloxy-gamma-butyrolactone is hydrolyzed to
optically active 3-acyloxy-gamma-butyrolactone and optically active
3-hydroxy-gamma-butyrolactone, also it is easy to seperate products from reaction
mixture. Optically active 3-acyloxy-gamma-butyrolactone produced according to this
invention can be converted to optically active 3-hydroxy-gamma-butyrolactone by
deacylation. Therefore, this method is an useful process on the industrial scale for
making optically active 3-acyloxy-gamma-butyrolactone or optically active
3-hydroxy-gamma-butyrolactone used as pharmaceutical intermediates.

We Claim:-
A process for preparing optically active 3-acyloxy-gamma-butyrolactone(5) and
3-hydroxy-gamma-butyrolactone(6) from racemic or
3-acyloxy-gamma-butyrolactone with low optical purity by hydrolysis using
enzymes or enzyme-producing microorganisms.
[Sheme 1]

A process of preparing optically active 3-acyloxy-gamma-butyrolactone wherein
racemic epichlorohydrin represented by the general formula 1 in scheme 1 is
subjected to prepare racemic 4-chloro-3-hydroxybutyronitrile, racemic
3-hydroxy-gamma-butyrolactone and racemic 3-acyloxy-gamma-butyrolactone
in turn, and racemic 3-acyloxy-gamma-butyrolactone is hydrolyzed stere-
ospecifically using enzymes or enzyme-producing microorganisms in the aqeous
phase or organic phase containing water.
The process for preparing optically active 3-acyloxy-gamma-butyrolacotne(5)
and 3-hydroxy-gamma-butyrolactone(6) according to claim 1 and claim 2,
wherein biocatalysts were lipase, esterase, protease or these enzyme-producing
microorganisms.

The present invention relates to the process for preparing optically active 3-acyloxy-gamma-
butyrolactone represented by the general formula 5 and optically active 3-hydroxy-gamma-
butyrolactone represented by the general formula 6 in scheme 1 from racemic 3-acyloxy-
gamma-butyrolactone represented by the general formula 4 by enzymatic method. In more
detail, this invention relates to the process for the preparation of optically active 3-acyloxy-gamma-butyrolactone and optically active 3-hydroxy-gamma-butyrolactone wherein racemic
epichlorohydrin represented by the general formula 1 is subjected to produce racemic 4-
chloro-3-hydroxybutyronitrile, racemic 3-hydroxy-gamma-butyrolactone and racemic 3-acyloxy-gamma-butyrolactone in turn and racemic 3-acyloxy-gamma-butyrolactone is hydrolyzed sterospecifically using lipases or lipase-producing microorganisms in the aqueous
phase or organic phase containing water. This method is useful in the practical process
because production and separation of compounds with high optical purity are easy comparing with other reported process.