Biosynthetic production of 7-[1',2',6',-7',8',8a'(R)-hexahydro-2'(S),6'(R)-dimethyl-8'(S)-hydroxy-1'( S)-naphthyl]-3(R),5(R)-dihydroxyheptanoic acid, "triol acid", is accomplished by enzymatic hydrolysis of lovastatin acid or a salt thereof, by treating it with Clonostachys compactiuscula ATCC 38009 or ATCC 74178, or mutants thereof, or a cell-free extract derived therefrom, or a hydrolase derived therefrom. The triol acid and its lactone form are both inhibitors of HMG-CoA reductase and thus useful as anti-hypercholesterolemic agents, and may also serve as intermediates for preparation of other HMG-CoA reductase inhibitors. Also, in the synthesis of simvastatin by direct methylation of lovastatin, selective hydrolysis of residual lovastatin salt by treatment with Clonostachys compactiuscula ATCC 38009 or ATCC 74178 or mutants thereof or a cell-free extract derived therefrom, or a hydrolase derived therefrom yields the "triol" salt which can be easily separated from simvastatin.

BRIEF SUMMARY OF THE INVENTION 
The present invention relates to biosynthetic production of 
7-[1',2',6',7',8',8a'(R)-hexahydro-2'(S),6'(R)-dimethyl-8'(S)-hydroxy-1'(S 
)-naphthyl]-3(R),5(R)-dihydroxyheptanoic acid "triol acid" by 
microbiological hydrolysis of lovastatin acid, a fermentation product, 
using the filamentous fungus, Clonostachys compactiuscula, or a hydrolase 
derived therefrom. This invention also relates to the use of this process 
in the synthesis of simvastatin from lovastatin to facilitate the 
separation and isolation of simvastatin from unreacted lovastatin starting 
material. 
The triol acid and its lactone form are old compounds, i.e., ones known in 
the art, and they are inhibitors of 3-hydroxy-3-methylglutaryl-coenzyme A 
(HMG-CoA) reductase, an enzyme involved in cholesterol biosynthesis. As 
inhibitors of that enzyme, they are useful as antihypercholesterolemic 
agents. They find further usefulness as intermediates for the preparation 
of other antihypercholesterolemic agents, especially those having various 
side chains at the 8'-position of the polyhydronaphthyl ring. For example, 
simvastatin, which has a 2,2-dimethylbutyryloxy side chain at the 
8'-position, may be prepared using the lactone form of the triol acid as a 
starting material, in accordance with known procedures. 
The selective conversion of lovastatin salt to the triol salt would be 
useful in the separation of simvastatin from unreacted lovastatin in the 
production of simvastatin from lovastatin. Lovastatin acid has a 
2-methylbutyryloxy side chain in the 8'-position and is difficult to 
separate from the newly formed simvastatin acid which has a 
2,2-dimethyl-butyryloxy side chain at the 8'-position. Applicants have now 
found that selective cleavage of the 2-methylbutyryloxy side chain from 
lovastatin acid salt using the process of this invention employing a 
hydrolase enzyme from Clonostachys compactiuscula (ATCC 38009 or ATCC 
74178) to yield the triol salt, results in a more easily separable mixture 
and greater purity of the simvastatin produced. 
The present invention also relates to a substantially pure form of a 
hydrolase enzyme produced by Clonostachys compactiuscula ATCC 38009 or 
ATCC 74178, and mutants thereof, which is capable of hydrolysing 
lovastatin acid or a salt thereof to triol acid or a salt thereof in 
accordance with the process of the present invention. 
The present invention further relates to mutant strains of Clonostachys 
compactiuscula, ATCC 38009 or ATCC 74178, which are able to produce a 
hydrolase capable of hydrolysing lovastatin acid or a salt thereof to 
triol acid or a salt thereof. 
The present invention also relates to a process in which the triol acid 
produced by treating lovastatin acid or a salt thereof with Clonostachys 
compactiuscula ATCC 38009 and ATCC 74178, or mutants thereof, or a 
hydrolase derived therefrom, is thereafter converted to its lactone form. 
BACKGROUND OF THE INVENTION 
The present invention is in the field of inhibitors of HMG-CoA reductase 
which are useful as antihypercholesterolemic agents. It is now well 
established that hypercholesterolemia is a significant risk factor in the 
development of cardiovascular disease, particularly atherosclerosis. 
Compounds which are able to inhibit the HMG-CoA reductase enzyme interfere 
with and limit the biosynthesis of cholesterol, and in that way function 
as antihypercholesterolemic agents. Such compounds, especially the natural 
fermentation products compactin and mevinolin, are now well known. There 
is a continuous search, nevertheless, for additional analogs which will 
give improved antihypercholesterolemic performance. The triol acid 
produced by enzymatic hydrolysis of lovastatin acid using an enzyme 
derived from Clonostachys compactiuscula in accordance with the 
biosynthetic process of the present invention provides quantities of a 
starting material for the preparation and production of such semisynthetic 
analogs. 
##STR1## 
The process of this invention may also be conducted starting with 
pravastatin, which differs from lovastatin in that the 6-.alpha.-methyl 
group on the hexahydronaphthyl ring is replaced with a 6-.beta.-hydroxyl 
group. Treatment of pravastatin with Clonostachys compactiuscula in 
accordance with the biosynthetic process of the present invention provides 
the corresponding pravastatin triol acid below. 
##STR2## 
As already described above, the triol acid and its lactone form are old 
compounds. The triol acid in its lactone form, for example, is described 
in Endo, published Japanese Pat. Appln. 86-13798 (1986), where its 
production by fermentation of Monascus ruber and a demonstration of its 
ability to reduce blood cholesterol levels is also set out. The triol acid 
in its lactone form, as well as the triol acid itself, are also described 
in Willard U.S. Pat. No. 4,293,496 (1981). However, in Willard, these 
compounds are prepared by chemical hydrolysis to remove the 
8-(.alpha.-methylbutyryloxy) ester side chain of lovastatin, the starting 
material which is a fermentation product of a particular strain of 
Aspergillus terreus. There is no suggestion that such hydrolysis might be 
carried out biochemically or microbiologically. 
Lovastatin and simvastatin are also compounds known in the art as HMG-CoA 
reductase inhibitors. The two compounds differ in that lovastatin has a 
2-methylbutyryloxy side chain at the 8'-position and simvastatin has a 
2,2-dimethylbutyryloxy side chain. 
##STR3## 
Although simvastatin has been formed from lovastatin, it has been difficult 
to separate and purify simvastatin from a mixture of simvastatin and 
lovastatin. The similarity in structure between the two compounds (the two 
compounds differ by only one methyl group) makes high pressure liquid 
chromatography (HPLC) separation difficult because the compounds have such 
similar retention times. One methodology used to isolate simvastatin from 
a mixture of simvastatin and lovastatin is to convert the unreacted 
lovastatin to the triol acid or the diol lactone using base hydrolysis 
with, for example, sodium hydroxide (NaOH) or lithium hydroxide (LiOH). 
However, this base hydrolysis hydrolyzes only a percentage of the 
lovastatin, leaving unreacted lovastatin as a contaminant of the final 
simvastatin product. An additional problem with the base hydrolysis is 
partial hydrolysis of the simvastatin, thus reducing the yield of the 
desired simvastatin product. The present invention provides for a process 
of isolating simvastatin from mixtures of simvastatin and lovastatin in 
greater purity and without concomitant yield losses. 
Komagata et al., J. Antibiotics, 39, 1574-77 (1986), describes enzymatic 
hydrolytic conversion of compactin (ML-236B) to the 8-hydroxy analog 
(ML-236A) in which the same side chain is removed as in the present 
invention. Of 1600 fungal strains investigated, 59 were found to be 
effective in catalyzing the hydrolytic reaction, and Emericella unguis 
showed the most potent activity. However, C. compactiuscula is not 
disclosed. 
Endo, published Japanese Pat. Appln. 85-176595 (1985) describes the same 
conversion as Komagata et al. above, but additionally includes conversion 
of "monacolin K" (which is lovastatin) to "monacolin J", (which is the 
triol acid in the present invention). Especially useful are said to be the 
molds Mortierella isabellina, Emericella unguis, Diheterospora 
chlamydosporia, Humicola fuscoatra, Dichotomomyces cejpii, Neocosmospora 
africana, Xylogone sphaerospora, Torulomyces ragena, and Thielavia fimeti. 
However, the highest conversion rate is 78% for a starting material 
concentration of 0.5 mg/ml, compared to 90-100% with the present 
invention. And, there is an indication in the related Komagata et al. 
paper that at higher concentrations, such as the 2.5 mg/ml employed in the 
present invention, there is a significant drop-off in efficiency of the 
enzyme. Thus, there is no suggestion in the prior art of the improved 
microbiological hydrolysis which can be achieved using Clonostachys 
compactiuscula. 
Lovastatin can be converted to a more active HMG-CoA reductase inhibitor by 
C-methylation of the natural 2(S)-methylbutyryloxy side chain to obtain 
simvastatin. C-methylation may be accomplished by any known process 
amenable to the functionalities of the molecule. 
One process for direct C-methylation of the 2(S)-methylbutyryloxy side 
chain is described in U.S. Pat. No. 4,582,915. This process is detailed in 
Scheme I and in the description which follows. 
##STR4## 
wherein: M is an alkali metal salt, preferably potassium; 
X is halo, such as chloro, bromo or iodo, preferably bromo or iodo; 
M.sub.1.sup.+ is a cation derived from lithium, sodium or potassium, 
preferably lithium; and 
R.sup.1 and R.sup.2 are 
1) independently C.sub.1-3 alkyl, or 
2) R.sup.1 and R.sup.2 joined together form a 5- or 6-membered heterocycle 
such as pyrrolidine or piperidine with the nitrogen to which they are 
attached, preferably pyrrolidine. 
In the process of forming simvastatin by the direct methylation of 
lovastatin, the lovastatin lactone compound is first converted to an 
alkali metal salt, preferably a potassium salt of the 
dihydroxycarboxylate. Although any conceivable method preparing a dry salt 
would suffice, it is convenient to add a substantially stoichiometric 
amount of aqueous potassium hydroxide to a solution of the lactone 
starting material in a hydrocarbon solvent such as benzene, toluene or 
cyclohexane containing a small amount of a C.sub.1-3 alkanol, preferably 
isopropanol, ethanol or methanol, or alternatively in tetrahydrofuran 
(THF) with or without added alkanol, stirring for a few minutes to about 
an hour and finally concentrating to dryness in vacuo. The residue is 
subjected to rigorous drying such as by azeotropic distillation with 
cyclohexane, toluene or dry tetrahydrofuran, preferably extremely (less 
than 0.08 mg H.sub.2 O/mL) dry tetrahydrofuran. 
The dry alkali metal salt is dissolved in an ethereal solvent such as 
tetrahydrofuran, diethyl ether, 1,2-dimethoxyethane, cooled to about 
-80.degree. C. to -25.degree. C., preferably -35.degree. C. to -30.degree. 
C. and treated with an excess of a strong base such as an alkali metal 
amide, wherein the alkali metal is lithium, sodium or potassium, 
preferably lithium, and the amide is diethylamide, pyrrolidide, 
dimethylamide or diisopropyl amide in an ethereal solvent in a dry, inert 
environment. After about 2 to 8 hours, preferably about two hours at 
-80.degree. to -25.degree. C., preferably -35.degree. to -30.degree. C., a 
methyl halide, such as methyl bromide, methyl chloride or methyl iodide, 
preferably methyl bromide or methyl iodide, is added to the mixture while 
maintaining the low temperature. Treatment with the strong base and methyl 
halide as described can be repeated if appreciable amounts of starting 
material remain. After 0.5 to about 3 hours following final addition of 
methyl halide, the reaction mixture is quenched by adding to it excess 
water. 
Following this direct methylation, attempts to convert unreacted lovastatin 
to the triol acid or the diol lactone for final product purification 
purposes were made using NaOH or LiOH. However, this base hydrolysis 
hydrolyzed only a small percentage of the lovastatin. Thus, unreacted 
lovastatin remained as a contaminant of the final simvastatin product. 
Furthermore, the base hydrolysis also hydrolyzed simvastatin, thus 
reducing yields of the desired simvastatin product. Following hydrolysis, 
the open ring acid form of simvastatin or a salt form thereof was then 
converted to the lactone by either heat or acid-catalyzed lactonization, 
and separated and purified by crystallization. 
DETAILED DESCRIPTION OF THE INVENTION 
The present invention is concerned with biosynthetic production of 
6(R)-[2-(8(S)-hydroxy-2(S), 
6(R)-dimethyl-1',2',6',7',8',8a'(R)-hexahydronaphthyl)-ethyl]-4(R)-hydroxy 
-3,4,5,6-tetrahydro-2H-pyran-2-one, the triol acid (2), or a salt form 
thereof by treating lovastatin acid (1) or a salt thereof with 
Clonostachys compactiuscula or mutants thereof, or a cell-free extract 
derived therefrom, or a hydrolase derived from Clonostachys 
compactiuscula. The triol acid may be subsequently converted by known 
chemistry to its lactone form (3). 
The term "mutant" refers to an organism in which some gene (or its 
regulatory region of DNA) on the genome is modified, leaving the gene or 
genes responsible for the organism's ability to hydrolyze lovastatin acid 
to the triol acid functional and heritable. Mutants within the scope of 
this invention have essentially the same characteristics as those of the 
parent strain, ATCC 38009. 
The starting material for the method of the present invention is lovastatin 
acid (1), the open-ring form of lovastatin, or a salt thereof. The acid 
form is the material produced by fermentation of Aspergillus terreus in 
accordance with culturing methods known in the art. Lovastatin itself is 
too insoluble in aqueous systems to be a useful starting material in the 
method of the present invention; and those solvents in which it is soluble 
are generally incompatible with the method of the present invention. 
The lovastatin acid starting material will typically be employed in the 
salt form. Unless otherwise specified, the terms "acid", "open ring acid" 
and "acid form", when applied to the starting materials, intermediates and 
final products of the present invention, include any suitable salt form 
thereof as well. Any salt which permits good solubility and which will not 
interfere with the other conditions encountered in carrying out the 
particular reaction is permissible. For example, the alkali metal salts, 
such as lithium, sodium and potassium; alkaline earth metal salts, such as 
calcium or magnesium; or salts with other metals such as aluminum, iron, 
zinc, copper, nickel or cobalt; amino acid salts formed from basic amino 
acids, such as arginine, lysine, .alpha.,.beta.-diaminobutyric acid and 
ornithine; amine salts such as t-octylamine, dibenzylamine, 
ethylenediamine, morpholine, and tris(hydroxymethyl)aminomethane; or the 
ammonium salt may be employed. The alkali metal salts (Li, Na, and K) and 
the ammonium salt forms of the lovastatin acid may be employed and are 
preferred. Especially preferred are the potassium and ammonium salt forms. 
For convenience, the structural formulas for lovastatin acid, the triol, 
acid, and its lactone form, are set out below as Formulas 1, 2, and 3, 
respectively: 
##STR5## 
wherein: M.sup.3 is selected from the group consisting of 
a) H, 
b) an alkali metal salt such as Li, Na or K, 
c) an alkaline earth metal salt such as Ca or Mg, 
d) a salt with other metals such as Al, Fe, Zn, Cu, Ni or Co, 
e) an amino acid salt formed from a basic amino acid such as arginine, 
lysine, .alpha.,.beta.-diaminobutyric acid, or ornithine, 
f) an amine salt such as t-octylamine, dibenzylamine, ethylenediamine, 
morpholine, or tris(hydroxy-methyl) aminomethane, and 
g) the ammonium salt. 
The basic mechanism of biosynthetic production of triol acid in accordance 
with the present invention is though to be enzymatic hydrolysis of 
lovastatin acid whereby an enzyme produced by Clonostachys compactiuscula 
ATCC 38009 or ATCC 74178, or mutants thereof, catalyzes removal of the 
8-(.alpha.-methylbutyryloxy) ester side chain of lovastatin to give the 
triol acid. As already explained, for reasons of solubility in aqueous 
systems, it has been found most desirable to use the lovastatin starting 
material in its open ring or acid form, and for this purpose the ammonium, 
potassium, sodium and lithium salt forms of lovastatin acid are preferred. 
The enzyme produced by Clonostachys compactiuscula ATCC 38009 or ATCC 74178 
or a mutant thereof may be brought into contact with the lovastatin acid 
starting material in any number of ways, all of which will be apparent to 
the person of ordinary skill in this art. All of these are within the 
definition of the term "treating" as defined in this invention. For 
example, whole cells may be used, and in accordance with this procedure, a 
fermentation culture of Clonostachys compactiuscula is produced to which 
the lovastatin acid starting material is simply added and the triol acid 
final product recovered. 
A variation of this whole cell procedure is one in which a fermentation 
culture of Clonstachys compactiuscula as described above is produced, but 
a small concentration (0.5 to 2.5 g/L, preferably 1.0 to 2.0 g/L) of 
lovastatin acid is added for the purpose of inducing hydrolytic activity. 
The cell mass is then harvested by centrifugation or filtration and 
recovered as pellets or as a hyphal mat which can be used immediately or 
frozen for later use. These may be added to the lovastatin acid starting 
material where the latter is present in the fermentation culture in which 
it has been produced, e.g., by fermentation of Aspergillus terreus. 
Alternatively, the lovastatin acid may be separated from its culture 
medium and then brought into contact with the frozen pellets of 
Clonostachys compactiuscula described above. 
It is not necessary that the whole cells of Clonostachys compactiuscula be 
alive as described above. It is also possible to employ dead cells, e.g., 
those which have been acetone-dried. 
As an alternative to whole cells, it is possible to use crude homogenates 
derived from these whole cell cultures. It is also possible to isolate the 
hydrolytic enzyme itself from the crude homogenates and employ the 
substantially purified enzyme. 
The process of bringing the Clonostachys compactiuscula enzyme into contact 
with the lovastatin acid starting material may be carried out batch-wise, 
or it may be carried out in a continuous manner. The contacting of these 
reactants themselves may be modified in various ways in keeping with 
advances in process technology. Thus, an immobilized enzyme column may be 
employed for the Clonostachys compactiuscula enzyme with the lovastatin 
acid starting material being passed through the column. Another example of 
such process technology is that relating to membrane reactors. Another 
alternative process for contacting of the reactants would be to culture 
the Clonostachys compactiuscula ATCC 38009 or ATCC 74178, or mutants, in 
the same fermentation broth used to produce the lovastatin. It would also 
be possible to modify that fermentation broth, if necessary, in order to 
support growth of Clonostachys compactiuscula once the lovastatin acid is 
produced, by adding culture media elements and then introducing the 
Clonostachys compactiuscula ATCC 38009 or ATCC 74178, or mutants thereof, 
and culturing it to produce the triol acid. This approach, however, is not 
likely to produce optimum yields. The preferred methods of contacting the 
reactants is by way of the immobilized enzyme column described above or by 
using a purified enzyme preparation. 
Working examples set out further below describe the method currently 
employed to demonstrate the enzymatic hydrolysis of lovastatin acid. 
However, the methods in those working examples would not necessarily be 
suggestive of methods which would be utilized for commercial production. 
The use of the process of this invention to separate and purify simvastatin 
from mixtures of simvastatin and lovastatin is shown in Scheme II. 
The mixture of the simvastatin and lovastatin lactones is converted to a 
mixture of the corresponding open-ring acids, preferably by treatment with 
an essentially stoichiometric aqueous alkali hydroxide such as potassium 
hydroxide or sodium hydroxide in a hydrocarbon solvent such as benzene, 
toluene or cyclohexane containing a small amount of a C.sub.1-3 alkanol, 
preferably isopropanol, ethanol or methanol, stirring for a few minutes to 
about an hour. The substrate is then extracted into an aqueous medium, 
such as TRIS (Tris(hydroxymethyl)aminomethane), glycine, TES 
(N-tris[Hydroxymethyl)methylamino]-2-hydroxy-propane-sulfonic acid), 
sodium phosphate, MOPSO (3-[N-Morpholino]-2-hydroxypropanesulfonic acid), 
BIS-TRIS PROPANE (1,3-bis[tris(Hydroxymethyl)-methylamino]propane), BES 
(N,N-bis[2-hydroxyethyl]-2-aminoethanesulfonic acid), MOPS 
(3-[N-Morpholino]propanesulfonic acid), HEPES 
(N-[2-Hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid]), DIPSO 
(3-[N,N-bis(2-Hydroxyethyl)amino]-2-hydroxypropanesulfonic acid), TAPSO 
(3-[N-tris(Hydroxymethyl)methylamino]-2-hydroxypropanesulfonic acid), 
HEPPSO (N-[2-Hydroxyethyl]piperazine-N'-[2-hydroxypropanesulfonic acid]), 
POPSO (Piperazine-N,N'-bis[2-hydroxypropane sulfonic acid]), EPPS 
(N-[2-Hydroxyethyl]piperazine-N'-[3-propanesulfonic acid], TEA 
(N-tris[Hydroxymethyl]methyl-2-aminoethanesulfonic acid), TRICINE 
(N-tris[Hydroxymethyl]-methylglycine), BICINE 
(N,N-bis[2-Hydroxyethyl]-glycine), TAPS 
(N-tris[Hydroxymethyl]methyl-3-aminopropanesulfonic acid), AMPSO 
(3-[(1,1-Dimethyl-2-hydroxyethyl)amine]2-hydroxypropanesulfonic acid) or 
CHES (2-[N-Cyclohexylamino]-2-hydroxypropanesulfonic acid) buffers, pH 
7-10, 25 mM to 1M; distilled water, or one of the aqueous media listed 
above supplemented with up to 20% (vol./vol.) of a water-miscible solvent 
such as methanol, ethanol, propanol, butanol, tetrahydrofuran, or acetone. 
Preferred are TRIS, glycine, TES and sodium phosphate buffers, pH 7.5-9.5, 
25 mM to 75 mM. The dissolved substrate is then treated with Clonostachys 
compactiuscula, (ATCC 38009 or ATCC 74178) or a mutant thereof or a 
cell-free extract derived therefrom or a hydrolase derived from 
Clonostachys compactiuscula or the substrate is converted to the ammonium 
salt and treated with Clonostachys compactiuscula, or a mutant thereof or 
a cell-free extract derived therefrom or a hydrolase derived therefrom. 
The aqueous system may be added prior to or simultaneous with the addition 
of Clonostachys compactiuscula, mutants thereof, or the cell-free extract 
derived therefrom or the hydrolase derived from Clonostachys 
compactiuscula. 
Lactonization by either acid-catalyzed or heat-catalyzed methods, for 
example, by stirring in isopropylacetate (I) containing 7 mM methane 
sulfonic acid for two hours at room temperature follows. The resulting 
simvastatin lactone and diol lactone are separable by high pressure liquid 
chromatography (HPLC) or by crystallization to obtain substantially pure 
simvastatin. 
Reversed-phase HPLC is conducted using as a mobile phase an organic-aqueous 
mixture with the aqueous component being 0.01 to 1.0% phosphoric acid or 
trifluoroacetic acid or other suitable acid and suitable organic 
components include acetonitrile, methanol and ethanol. 
##STR6## 
Simvastatin may also be purified by crystallization from ethyl acetate, 
isopropyl acetate and methanol. 
The enzymatic hydrolysis of lovastatin acid to the triol acid can also be 
employed in the process for making simvastatin by direct methylation of 
lovastatin. This overall process is shown in Scheme III. 
In the process of forming simvastatin by the direct methylation of 
lovastatin, the lovastatin lactone compound is first converted to an 
alkali metal salt, preferably potassium salt of the dihydroxycarboxylate. 
Although any conceivable method of preparing a dry salt would suffice, it 
is convenient to add a substantially stoichiometric amount of aqueous 
potassium hydroxide to a solution of the lactone starting material in a 
hydrocarbon solvent such as benzene, toluene or cyclohexane containing a 
small amount of a C.sub.1-3 alkanol, preferably isopropanol, ethanol or 
methanol, or alternatively employing tetrahydrofuran (THF), with or 
without the added alkanol, stirring for a few minutes to about an hour and 
finally concentrating to dryness in vacuo. The residue is subjected to 
rigorous water removal such as by azeotropic distillation with 
cyclohexane, toluene, or dry tetrahydrofuran, preferably extremely (less 
than 0.08 mg H.sub.2 O/mL) dry tetrahydrofuran. 
The dry alkali metal salt is dissolved in an ethereal solvent such as 
tetrahydrofuran, diethyl ether, 1,2-dimethoxyethane or the like, cooled to 
about -80.degree. C. to -25.degree. C., preferably -35.degree. C. to 
-30.degree. C. and treated with an excess of a strong base such as an 
alkali metal amide, wherein the alkali metal is lithium, sodium or 
potassium, preferably lithium, and the amide is diethylamide, pyrrolidide, 
dimethylamide or diisopropyl amide in an etheral solvent in a dry inert 
environment. After about 2 to 8 hours, preferably about two hours at 
-80.degree. to -25.degree. C., preferably -35.degree. to -30.degree. C., a 
methylhalide, such as methyl bromide, methyl chloride or methyl iodide, 
preferably methyl bromide or methyl iodide, is added to the mixture while 
maintaining the low temperature. Treatment with the strong base and methyl 
halide as described can be repeated if appreciable amounts of starting 
material remain. After 0.5 to about 3 hours following final addition of 
methyl halide, the reaction mixture is quenched by adding to it excess 
water. 
##STR7## 
The mixture of lovastatin acid salt and simvastatin acid salt is then 
preferably converted to the corresponding ammonium salt by ammonium 
hydroxide-methanol in ethyl acetate and treating with Clonostachys 
compactiuscula, or a mutant thereof or a hydrolase derived therefrom. 
Alternatively the Clonostachys compactiuscula enzyme is added directly to 
the mixture of lovastatin salt and simvastatin salt following the removal 
of residual organics by distillation. 
The resulting mixture of simvastatin acid and triol acid may be converted 
to the corresponding mixture of lactones by a suitable method, for 
example, heat-catalyzed or acid-catalyzed lactonization. Simvastatin is 
separable from the resulting mixture of simvastatin and diol lactone by 
HPLC or crystallization. Alternatively, the simvastatin acid may be 
separated from the triol acid by HPLC or crystallization, followed by 
conversion of the pure simvastatin acid to simvastatin lactone. If the 
simvastatin acid is to be isolated and purified by crystallization, it is 
preferred to convert the simvastatin acid to the ammonium salt prior to 
lactonization. 
The present invention is also directed to mutants of the particular strain 
of Clonostachys compactiuscula, ATCC 38009 or ATCC 74178, which are 
capable of converting lovastatin acid to triol acid. There are techniques 
well known in the fermentation art for improving the yields of desired 
products produced by various strains of microorganisms. For example, a 
given producing strain may be irradiated or exposed to other stimuli known 
to greatly increase the ongoing mutation of the genetic material of the 
microorganism. By using a sensitive screen, it is then possible to select 
from the many mutations thus produced only those which result in an 
enhanced production of the desired product. In this way, it is usually 
possible to continually improve the output of a producing strain through 
its various selected descendants. A biologically pure culture of a mutant 
is a culture that consists substantially of one strain of the mutant. With 
regard to the present invention, similar improvements in output of 
lovastatin acid hydrolase by selected mutants of Clonostachys 
compactiuscula ATCC 38009 or ATCC 74178, may be achieved. A satisfactory 
screen for this purpose is the use of high performance liquid 
chromatography (HPLC) which can detect the enzymatic cleavage products at 
very low concentrations, thus clearly establishing that triol acid has 
been produced by any particular mutant in question. 
Culture Medium 
The fermentation of Clonostachys compactiuscula is carried out in aqueous 
media such as those employed for the production of other fermentation 
products. Such media contain sources of carbon, nitrogen and inorganic 
salts assimilable by the microorganism. 
In general, carbohydrates such as sugars, for example, lactose, glucose, 
fructose, maltose, mannose, sucrose, xylose, mannitol and the like and 
starches such as grains, for example, oats, ryes, cornstarch, millet, corn 
meal and the like can be used either alone or in combination as sources of 
assimilable carbon in the nutrient medium. The exact quantity of the 
carbohydrate source or sources utilized in the medium depends in part upon 
the other ingredients of the medium but, in general, the amount of 
carbohydrate usually varies between about 1% and 6% by weight of the 
medium. These carbon sources can be used individually, or several such 
carbon sources may be combined in the medium. In general many 
proteinaceous materials may be used as nitrogen sources in the 
fermentation process. Suitable nitrogen sources include for example, yeast 
hydrolysates, primary yeast, soybean meal, cottonseed flour, hydrolysates 
of casein, corn steep liquor, distiller's solubles or tomato paste and the 
like. The sources of nitrogen either alone or in combination, are used in 
amounts ranging from about 0.2% to 6% by weight of the aqueous medium. 
Among the nutrient inorganic salts which can be incorporated in the culture 
media are the customary salts capable of yielding sodium, potassium, 
ammonium, calcium, phosphate, sulfate, chloride, carbonate, and like ions. 
Also included are trace metals such as cobalt, manganese, iron and 
magnesium. In addition, if necessary, a defoaming agent such as 
polyethylene glycol or silicone may be added, especially if the culture 
medium foams seriously. 
It should be noted that the media described in the Examples are merely 
illustrative of the wide variety of media which may be employed, and are 
not intended to be limitative. Specifically, the carbon sources used in 
the culture media include dextrose, dextrin, oat flour, oatmeal, molasses, 
citrate, soybean oil, glycerol, malt extract, cod liver oil, starch, 
ethanol, figs, sodium ascorbate and lard oil. Included as nitrogen sources 
were peptonized milk, autolyzed yeast, yeast RNA, tomato paste, casein, 
primary yeast, peanut meal, distillers solubles, corn steep liquor, 
soybean meal, corn meal, NZ amine, bean extract, aspargine, cottonseed 
meal an ammonium sulfate. The major ionic components are CaCO.sub.3, 
KH.sub.2 PO.sub.4, MgSO.sub.4.7H.sub.2 O and NaCl and small amounts of 
CoCl.sub.2.6H.sub.2 O and traces of Fe, Mn, Mo, B, Co and Cu were also 
present. 
Lactonization 
Treatment of lovastatin acid with Clonostachys compactiuscula ATCC 38009 or 
ATCC 74178, or mutants thereof, or a cell-free extract derived therefrom, 
or a hydrolase derived therefrom, in accordance with the process of the 
present invention provides the triol acid as the predominant product. 
However, it is also desirable to obtain the lactone form of this compound, 
since it is also useful as an antihypercholesterolemic agent or as an 
intermediate for preparing such agents. Lactonization of triol acid is 
carried out using standard procedures, i.e., either heat or acid catalyzed 
lactonization. Procedures for acid-catalyzed lactonization of lovastatin 
acid-related compounds are known and described in U.S. Pat. No. 4,916,239. 
For simvastatin acid and the triol acid, lactonization has been carried 
out by stirring in isopropyl acetate containing 7 mM methane sulfonic acid 
for 2 hours at room temperature.

EXAMPLE 1 
Biotransformation of lovastatin acid to triol acid by whole cells of 
Clonostachys compactiuscula 
Clonostachys compactiuscula ATCC 38009 was grown in a 2L airlift fermentor 
with 1.8L working volume in medium EN (glucose 1%; peptone 0.2%; beef 
extract 0.1%; yeast extract 0.1%; and corn steep liquor 0.3%), at 
29.degree. C., at an aeration rate of 1.25 vvm, for 48-72 hrs. Lovastatin 
ammonium salt was added (0.5 g/L final concentration) to induce hydrolytic 
activity. The fermentation was harvested 24-72 hrs. after addition of the 
lovastatin ammonium salt by straining through a sieve and washing the 
pellets with buffer (20 mM Tris, pH 8.5). The cell pellets were frozen 
until ready to use. 
For the biotransformation, Clonostachys compactiuscula pellets (17 g wet 
weight) from an airlift fermentation were contacted with 20 ml of crude 
lovastatin acid (@20 g/L) in carbonate buffer harvested from an 
Aspergillus terreus fermentation. The biotransformation was carried out in 
a 250 ml Erlenmeyer flask at 27.degree. C. and 160 rpm. After 17 hrs. 
approximately 60% of the lovastatin acid was converted to triol acid. 
In an additional experiment, Clonostachys compactiuscula pellets from an 
airlift fermentation (5 g wet weight) were contacted with 10 ml crude 
lovastatin acid (3.5 g/L) extracted from an A. terreus fermentation by 
methanol. The final concentration of methanol in the biotransformation 
mixture was 25%. The bioreaction was carried out in a 250 ml Erlenmeyer 
flask at 27.degree. C. and 160 rpm. After 2 hrs. the biotransformation 
employing Clonostachys compactiuscula converted nearly 100% of the 
lovastatin acid to triol acid, as measured by thin layer chromatography. 
EXAMPLE 2 
Biotransformation of lovastatin acid to triol acid by crude homogenate of 
Clonostachys compactiuscula. 
Clonostachys compactiuscula ATCC 38009 was grown in 250 ml shake flasks 
containing 12 ml of medium EN at 29.degree. C. for 3 days. Lovastatin 
ammonium salt was added to give a concentration of 2.5 g/L and 
fermentation was continued for 2 additional days. To prepare the crude 
homogenate, the culture was harvested by centrifugation at 3000 rpm for 10 
minutes, after which it was washed with 50 mM of 
N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid (TES) buffer, pH 
7.7. The culture medium was again centrifuged and the cell mass was 
chilled on ice and then subjected to grinding in a mortar and pestle 
containing glass fragments and powdered dry ice. The contents of 1 shake 
flask was resuspended in 2.0 ml of 50 mM TES buffer and centrifuged at 
6000 rpm for 10 minutes to remove cell debris and glass fragments. The 
supernatant was used as the source of crude homogenate with protein 
concentration of approximately 0.5 mg/ml. 
In order to carry out the biotransformation, one volume of crude homogenate 
was combined with an equal volume of lovastatin acid ammonium salt (5 
g/L), and the mixture was incubated at 29.degree. C. Using this method, 
80-90% conversion of lovastatin acid to triol acid was observed within 2 
hrs. 
EXAMPLE 3 
Purification of the lovastatin hydrolyzing enzyme from C. compactiuscula 
cells 
A hydrolytic enzyme which carries out the biotransformation of lovastatin 
acid to triol acid was purified by Fast Protein Liquid Chromatography 
(FPLC*) employing a MONO Q.RTM. anion exchange column to near homogeneity 
from homogenates of Clonostachys compactiuscula employing the procedures 
described below. 
The supernatant from the 6,000 rpm centrifugation as in Example 2 above, 
but where 50 mM of tris(hydroxymethyl)aminomethane (TRIS) buffer (pH 7.8) 
is substituted for 50 mM TES buffer, was centrifuged at 15,000 rpm for 20 
minutes and the resulting supernatant filtered through a 0.45 mm filter. 
Batches (10 mL) of filtrate containing 0.3-0.5 mg/mL protein were then 
applied at a rate of 1.0-2.0 mL/minute to a Pharmacia MONO Q.RTM. (HR 5/5) 
anion exchange column connected to a Pharmacia Fast Protein Liquid 
Chromatography (FPLC) system. 
After allowing binding of the anionic proteins to the column matrix, the 
hydrolase was specifically eluted by the application of a linear gradient 
of sodium chloride (0-500 mM) in 20 mM TRIS, pH 7.8. Eluted protein was 
collected in 1 mL fractions and assayed either using lovastatin ammonium 
salt (in which case percent hydrolysis was estimated by TLC (thin-layer 
chromatography) and densitometry or HPLC), or a colorimetric substrate 
(ortho-nitrophenyl butyrate, o-NPB) towards which the enzyme had been 
shown to have hydrolytic activity. When the latter substrate was used, the 
hydrolytic reaction was monitored spectrophotometrically at 410 nm 
essentially as described by Lawrence, R. C. et al. in J. Gen. Microbiol. 
(1967) 48, 401-418. Both assay methods revealed that the hydrolase was 
eluted when the NaCl concentration approached 300 mM. 
Sodium dodecyl sulfate-polyacrylamide (SDS) gel electrophoresis revealed 
the peak lovastatin acid hydrolase-containing fractions to contain a 
prominent band of molecular weight approximately 45,000 Da. 
Using the purified enzyme preparation, the biotransformation was carried 
out in accordance with the procedures described above in Examples 1, 2, 4 
and 6, and an estimate was made of the hydrolase's Km and specific 
activity with lovastatin ammonium salt as substrate. The value for Km 
obtained was 4.14 mM and under saturating substrate conditions the enzyme 
was found to have a specific activity of 0.04 mmol lovastatin ammonium 
salt hydrolyzed/mg protein per minute. 
EXAMPLE 4 
Biotransformation of lovastatin acid to triol acid by purified hydrolase 
from Clonostachys compactiuscula 
A hydrolytic enzyme which carries out the biotransformation of lovastatin 
acid to triol acid was purified by Fast Protein Liquid Chromatography 
(FPLC*) employing a MONO Q.RTM. anion exchange column to near homogeneity 
from homogenates of Clonostachys compactiuscula employing the procedures 
described below. 
A supernatant from the 6,000 rpm centrifugation as in Example 2 above, but 
where 20 mM of tris(hydroxymethyl)aminomethane (TRIS) buffer is 
substituted for 50 mM TES buffer, was centrifuged at 15,000 rpm and the 
resulting supernatant filtered through a 0.45 micrometer filter. Batches 
(10 ml) of filtrate containing 0.3-0.5 mg/ml protein were then applied to 
a Pharmacia MONO Q.RTM. anion exchange column connected to a Pharmacia 
Fast Protein Liquid Chromatography (FPLC) system. 
After allowing binding of the anionic proteins to the column matrix, the 
hydrolase was specifically eluted by the application of a linear gradient 
of sodium chloride (0-500 mM). Eluted protein was collected in 1 ml 
fractions and assayed either using lovastatin ammonium salt (in which case 
percent hydrolysis was estimated by TLC and densitometry or HPLC), or a 
colorimetric substrate (ortho-nitrophenyl butyrate o-NPB) towards which 
the enzyme had been shown to have hydrolytic activity. When the latter 
substrate was used, the hydrolytic reaction was monitored 
spectrophotometrically at 410 nm essentially as described by Lawrence, R. 
C. et al. in J. Gen. Microbiol. (1967) 48, 401-418. Both assay methods 
revealed that the hydrolase was eluted when the NaCl concentration 
approached 300 mM. 
Sodium dodecyl sulfate-polyacrylamide (SDS) gel electrophoresis revealed 
the peak lovastatin acid hydrolase-containing fractions to contain a 
prominant band of molecular weight approximately 45,000 Da. 
Using the purified enzyme preparation, the biotransformation was carried 
out in accordance with the procedures described above in Examples 1 and 2, 
and an estimate was made of the hydrolase's Km and specific activity with 
lovastatin ammonium salt as substrate. The value for Km obtained was 4.14 
mM and under saturating substrate conditions the enzyme was found to have 
a specific activity of 0.11 mmol lovastatin ammonium salt/mg protein per 
hour. 
EXAMPLE 5 
Biotransformation of lovastatin ammonium salt in the presence of excess 
simvastatin ammonium salt 
Forty-five grams of frozen Clonostachys compactiuscula (ATCC 38009) cells, 
which had been grown in medium EN as detailed in Example 5 (and washed 
with 50 mM Tris buffer, pH 7.8, prior to freezing) was homogenized with 
glass fragments and dry ice using a mortar and pestle. The resulting 
homogenized, frozen powder was transferred to a suitable tube and the 
material remaining in the mortar washed into the same tube using a minimal 
volume of 50 mM Tris, pH 7.8. The mixture was then allowed to thaw and 
then centrifuged at 6000 rpm for 10 minutes to remove large cell debris 
and glass. 
The 6000 rpm supernatant was used as a crude source of hydrolase and 0.8 mL 
was mixed with 0.2 mL methanol and 1.0 mL of a solution of simvastatin 
(18.6 mM and lovastatin (1.4 mM) ammonium salts in 50 MM Tris, pH 7.8.) 
The reaction mixture was incubated at 29 C. and sampled after 1 h, 2 h, 
and 17 h by removing 0.1 mL and diluting with 0.9 mL methanol. The samples 
were then subjected to analysis by HPLC using a Whatman C-8 column as 
stationary phase and a 60:40 mixture of acetonitrile: 0.5% phosphoric acid 
as mobile phase; under these conditions the respective retention times for 
simvastatin, lovastatin and triol ammonium salts are 4.4 min., 3.8 min., 
and 2.5 min. After 17 h the area percent of the lovastatin peak had been 
reduced from 23.2% to 0.7%, representing a greater than 99% conversion. 
Greater than 96% of the initial simvastatin ammonium salt remained intact 
over this same contact period. 
EXAMPLE 6 
Biotransformation of residual lovastatin acid to triol acid following the 
synthesis of simvastatin acid from lovastatin acid by direct methylation. 
Step 1: Preparation of Lovastatin Potassium Salt 
A solution of lovastatin (99% pure; 25 g; 60.57 mmol) in 325 mL 
tetrahydrofuran (THF) was prepared under nitrogen then cooled to 5.degree. 
C. An aqueous solution (6.1 ml) of 10.01M potassium hydroxide was added 
over 15 min then the mixture was warmed to 25.degree. C. and aged, with 
stirring, until complete (&gt;99%) conversion to the potassium salt (by HPLC 
analysis) had occurred. 
Step 2: Preparation of Simvastatin Potassium Salt 
The lovastatin potassium salt solution prepared in Step 1 was heated to 
reflux, distilling a total of 500-700 mL THF through a 10 in. Vigreaux 
column while maintaining a minimum pot volume of 215 mL with sieve-dried 
THF. The water content of the lovastatin potassium salt solution was thus 
reduced to a level of &lt;0.1 mg/mL. This solution was then diluted with 150 
mL of sieve-dried THF (water content &lt;0.1 mg water/mL) to give a total 
volume of 365 mL. Sieve-dried pyrrolidine (5.81 g; 81.7 mmol; water 
content &lt;0.2 mg/ml) was added as a single batch and the reaction cooled in 
a dry ice/acetone bath to -78.degree. C. Next, 117 mL of 1.6M 
n-butyllithium in hexane was added over a one hour period, sub-surface, 
while maintaining rapid agitation and an internal temperature below 
-70.degree. C. 
The lovastatin potassium salt solution, now containing the lithium 
pyrrolidide intermediate, was warmed to -35.degree. C. using a dry 
ice-acetonitrile bath and aged for 2 hours. After recooling to -45.degree. 
C., 13.32 g of sieve-dried methyl iodide (93.0 mmol; density 2.89 g/mL) 
was added in one portion and the mixture aged at -30.degree. C. (internal 
temperature following methyl iodide addition) for 30 minutes. The mixture 
was quenched with 200 mL water and the phases allowed to separate in a 
separating funnel. The lower, aqueous, layer was diluted to a volume of 
1250 mL by the further addition of water and then cooled to below 
10.degree. C. The pH was adjusted to 6 using 6M hydrochloric acid then 250 
mL ethyl acetate was added and the pH further adjusted to 2.0 (again using 
HCl). Phase separation was again allowed to occur then the aqueous layer 
was re-extracted with 175 mL cold (5.degree.-10.degree. C.) ethyl acetate. 
The two organic (ethyl acetate) layers were pooled and then washed with 
150 mL water before drying the final organic layer over sodium sulfate (to 
&lt;10 mg/ml water) and filtering. Next, 112.3 mL methanol was charged into 
the (425 mL) dry, filtered mixture at 25.degree. C. and then 1.3 mL of a 
methanol:aqueous ammonium hydroxide (3:1) solution was added over a 5 
minute period. The mixture was seeded with simvastatin ammonium salt (SAS) 
and aged for 10 minutes then a further 35.9 mL of the methanol:aqueous 
ammonium hydroxide (3:1) solution was added dropwise over 1 hour. The 
mixture was then cooled to -10.degree. C. over 2.5 hours and aged for an 
additional 1 hour. The product was filtered and washed with 25 mL cold 
(0.degree. C.) methanol and the resulting white crystals were dried in 
vacuo to give simvastatin ammonium salt as white needles (87% pure SAS 
containing 10% residual lovastatin as the ammonium salt). 
Step 3: Biotransformation of residual lovastatin acid (as the ammonium 
salt) to triol acid 
Clonostachys compactiuscula esterase was purified from 57 g mycelial cells 
which had been grown up in medium EN using the methods detailed in 
Examples 1 and 3. The use of a Pharmacia HR 10/10 MONO Q.RTM. column 
allowed the application of 85 mL of crude cell-free extract per 
purification run. In total 0.89 mg of purified esterase was obtained (in a 
volume of 10 mL) which was then concentrated to 0.175 mg protein/mL by 
ultrafiltration using a 10,000 molecular weight cut-off CENTRIPREP.RTM. 
device (AMICON.RTM.). 
Samples of the esterase were then incubated with the simvastatin ammonium 
salt prepared by direct methylation of lovastatin; final concentrations of 
protein were 0.4, 4.0 and 40 microgram/mL and simvastatin concentrations 
used were 10, 35 and 50 mM. Other conditions which were varied were pH 
(7.8 and 9.5 were assessed) and methanol concentration (0, 10 and 20% 
[v/v, final concentration]). The reactions were buffered by the inclusion 
of either 100 mM TRIS (in the case of reactions carried out at pH 7.8) or 
100 mM glycine (pH 9.0). Greater than 90% hydrolysis of residual 
lovastatin acid to triol acid was obtained within 16 h under the following 
conditions: 
______________________________________ 
Enzyme conc. 
Simvastatin conc. Methanol conc. 
(microgram/ml) 
(mM) pH (% v/v) 
______________________________________ 
4.0 10 7.8 0 
4.0 10 7.8 10 
4.0 10 9.5 0 
4.0 10 9.5 10 
4.0 10 9.5 20 
4.0 35 9.5 10 
40.0 35 7.8 0 
40.0 35 7.8 10 
40.0 35 9.5 0 
40.0 35 9.5 10 
40.0 35 9.5 20 
______________________________________ 
EXAMPLE 7 
Biotransformation of residual lovastatin acid to triol acid following the 
synthesis of simvastatin acid from lovastatin acid by direct methylation. 
Step 1: Preparation of Simvastatin Ammonium Salt 
Starting with 5 g lovastatin, the potassium salt solution in THF is 
prepared according to Example 6, Step 1. A solution of sieve-dried 
pyrrolidine (2.48 mL; 2.4 equivalents; 29.67 mmoL; &lt;0.2 mg water/ml) in 
12.3 mL sieve-dried THF) is cooled to -20.degree. C. in a dry 
ice/acetonitrile bath. Then a solution of 1.6M butyllithium in hexane 
(18.2 mL; 2.35 equivalents) is added at such a rate as to keep the 
temperature below -10.degree. C. After the addition is complete the 
lithium pyrrolidide/THF solution is aged at -20.degree. C. for 15 minutes. 
The dry solution of lovastatin potassium salt in THF is cooled to 
-35.degree. C. in a dry ice/acetonitrile cooling bath. The lithium 
pyrrolidide/THF solution at -20.degree. C. is added to the rapidly 
agitated mixture at such a rate as to maintain the internal temperature 
below -30.degree. C. at all times throughout the addition. The mixture is 
aged at - 35.degree. C. for 2 hours then, following cooling to -40.degree. 
C., 1.16 ml (18.67 mmol; 1.5 equivalents) methyl iodide is added to the 
solution in a single batch which causes the internal temperature of the 
mixture to rise (to approximately -20.degree. C.); the internal 
temperature is brought back to -30.degree. C. and aged for 1 hour, then 
warmed to -10.degree. C. and aged for 30 minutes. 
The mixture is quenched with 40 mL water and the phases allowed to separate 
in a separating funnel. The lower, aqueous, layer is diluted to a volume 
of 250 mL by the further addition of water and then is cooled to below 
10.degree. C. The pH is adjusted to 6 using 6M aqueous hydrochloric acid 
then 50 mL ethyl acetate is added and the pH further adjusted to 2.0 
(again using HCl). Phase separation is again allowed to occur then the 
aqueous layer was re-extracted with 35 mL cold (5.degree.-10.degree. C.) 
ethyl acetate. The two organic (ethyl acetate) layers are pooled and then 
washed with 30 mL water before drying the final organic layer over sodium 
sulfate and filtering. Next, 22.5 mL methanol is charged into the dry, 
filtered mixture at 25.degree. C. and then 0.26 mL of a methanol:aqueous 
ammonium hydroxide (3:1) solution is added over 5 minutes. The mixture is 
seeded with simvastatin ammonium salt and aged for 10 minutes then a 
further 7.2 mL of the methanol/ammonium hydroxide is added dropwise over 1 
hour. The mixture is then cooled to -10.degree. C. over 2.5 hours and aged 
for an additional 1 hour. The product is filtered and washed with 5 mL 
cold (0.degree. C.) methanol and the resulting white crystals are dried in 
vacuo to give simvastatin ammonium salt. 
Step 2: Biotransformation of residual lovastatin acid (as the ammonium 
salt) to triol acid 
Biotransformation is conducted according to the procedures in Example 6, 
Step 3. 
EXAMPLE 8 
Lactonization of Simvastatin Ammonium Salt and Crystallization and 
Isolation of Pure Simvastatin Lactone 
Step 1: Lactonization of Simvastatin Ammonium Salt 
Distilled water (20 mL) glacial acetic acid (40 mL) and butylated 
hydroxyanisole (BHA, 50 mg) were charged to a 250 ML 3-neck round bottom 
flask under a nitrogen atmosphere. The batch temperature was adjusted to 
20.degree.-25.degree. C. and simvastatin ammonium salt (12.5 g, 27.56 
mmoles) was added and agitated at 20.degree.-25.degree. C. for 15 min. or 
until dissolved. Methane sulfonic acid (70%, 4.35 g, 30.8 mmoles, 1.118 
equiv) was added and the mixture was aged at 20.degree.-25.degree. C. for 
2 hours until the lactonization reaction was more than 75% complete. 
Percent conversion was determined by HPLC following the conditions in 
Preparation A. Percent conversion was calculated as follows: 
##EQU1## 
Step 2: Crystallization and Isolation of Pure Simvastatin 
The batch was seeded with crude Simvastatin seed crystals (60 mg) and aged 
at 20.degree.-25.degree. C. for 0.5 hour. Distilled water (22.5 mL) was 
added over 3 hours (0.13 mL/min.) and a second distilled water charge (35 
mL was added over one hour (0.58 mL/min.). The batch was aged at 
20.degree.-25.degree. C. for one hour and then treated dropwise with 28 
w/w % ammonium hydroxide (4.0 mL). 
The batch was aged at 20.degree.-25.degree. C. for one hour and filtered to 
collect the Simvastatin crude crystals. The Simvastatin crude wet cake was 
washed with 2:1) distilled water:acetic acid (50 mL), distilled water (50 
mL) and 1:1 methanol:distilled water (50 mL). The product was dried 
overnight in vacuo with a nitrogen purge at 25.degree.-30.degree. C. to 
give the Simvastatin crude as white needles (10.38 g HPLC assay 98 w/w %). 
EXAMPLE 9 
Crystallization and Isolation of Pure Simvastatin 
Crude Simvastatin (10 g, 23.89 mmoles) and butylated hydroxyanisole (50 mg) 
were charged to a flask containing 126.4 mL degassed methanol under a 
nitrogen atmosphere. The batch temperature was adjusted to 
20.degree.-25.degree. C. and agitated for 15 minutes until solids 
dissolved. The solution was filtered through a bed of activated carbon, 
such as ECOSORB C.RTM. which is composed of: water, activated carbon, 
cellulose fiber, styrene divinyl benzene and anion exchange resin (91.5 g 
of methanol (50 mL) washed ECOSORB C.RTM.) and the carbon cake is washed 
with 40 mL of degassed methanol. The combined methanol solution was 
transferred to a 250 mL 3 neck round bottom flask and heated to 
38.degree.-40.degree. C. under a nitrogen atmosphere. Degassed distilled 
water (83.3 mL) was added subsurface over 30 minutes (2.78 mL/min.) and 
aged at 38.degree.-40.degree. C. for 30 minutes. The batch was cooled to 
25.degree. C. over 1 hour. Degassed distilled water (83.3 mL was charged 
subsurface over 1 h (1.38 mL/min.) at 25.degree. C. and cooled to 
10.degree.-15.degree. C. over 1 hour. 
The slurry was filtered and the wet cake was washed with 50 mL of 50% 
methanol/distilled water (vol./vol.) at 10.degree. C. The product was 
dried overnight in vacuo with a nitrogen purge at 35.degree.-40.degree. C. 
to give pure simvastatin as white needles (9.49 g HPLC assay=99 w/w %). 
PREATION A 
HPLC Weight Percent Assay for Dry Simvastatin Crude 
30 mg of standard or sample were accurately weighed into a 100 mL 
volumetric flask and were diluted to the mark with 60:40 acetonitrile: 
0.01M KH.sub.2 PO.sub.4 (pH=4.0). 
Column: PERKIN-ELMER.RTM. C.sub.18, 3 cm length, 3 micron particle size, 
reversed-phase column 
Temperature: 25.degree. C. 
Flow rate: 3.0 mL/min 
Detection: uv 238 nm 
Injection: 5 microliters 
Mobile phase: 50:50 acetonitrile: 0.1% H.sub.3 PO.sub.4 (aq) 
______________________________________ 
Retention Time: 
Time (min) Identity 
______________________________________ 
1.80 1. Simvastatin ammonium salt 
2.20 2. Lovastatin and epimer 
3.44 3. Simvastatin crude 
______________________________________ 
The weight % is calculated as follows: 
##EQU2##