Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Reexamination Certificate
2000-03-31
2002-04-16
Marx, Irene (Department: 1651)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
C435S132000, C435S252500, C435S147000, C435S148000, C435S170000
Reexamination Certificate
active
06372458
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to the microbial biotransformation of colchiconic compounds into derivative compounds, which are glycosylated exclusively at the C-3 position of the six-member ring. The process of the present invention provides the 3-O-glycosyl derivatives in high yields and purity.
BACKGROUND ART
A number of efforts using either chemical reactions or biotransformations have been made to obtain highly regiospecific glycosydated derivatives of compounds of formula (I), which is shown below, and related colchicinoid compounds.
For example, the chemical reaction route consists of sequences of complex, non-specific, non-selective reactions involving different molecular sites, which produce a mixture of glycosydated derivatives. Thus, the conversion yields of the desired effective or active product, which is specifically glycosydated at the C-3 position of the aromatic ring, are very low.
The biological approach substantially relates to the biotransformation of colchicinoid compounds such as colchicine and thiocolchicine, which are indirectly related with the colchicone compounds. For example, a known transformation, which is accomplished by a culture of
Centella asiatica,
yields derivatives, which are monoglycosydated at the C-2 and at C-3 positions of the aromatic ring (Solet, J. M., et al., Phytochemistry, 33, 4, 817-820, 1993). Thus, the transformation is not highly selective and also provides poor yields and productivity.
Other efforts to biotransform colchicinoid compounds have yielded simple demethylations of the methoxy groups bound to the aromatic ring at the C-2 and C-3 positions. These transformations are also characterized by limited yields, limited productivity, and by poor regioselectivity.
Attempts have been made to transform colchicine and its derivatives into the corresponding 3-demethylated derivatives using
Streptomyces griseus
and/or
Streptomyces spectabilis
(Hufford C. D. et al. J. Pharm. Sc., 68, 10, 1239-1242, 1979). Other workers have attempted the same biotransformation using different strains of Streptomyces and of other species of bacteria and fungi (Bellet P. et al. GB-923421, 1959). These results, however, confirm that these known microbial enzymes non-selectively produce the C-2, C-3, or C-10 derivatives of the alkaloid molecule. Moreover, the productivity of these catalytic systems are rather poor due to the low conversion yields, a requirement for reduced substrate concentrations, and frequent degradation of the tropolone ring.
More recently, Poulev et al. J. Ferment. Bioeng. 79, 1, 33-38, 1995 have obtained a specific demethylation using bacterial microorganisms, however, the demethylation also occurs with generally poor yields and productivity.
Enzymes from microorganisms similar to the above mentioned microorganisms, such as, for example, Streptomyces, Bacillus, have been applied to biotransform compounds, such as maytansinoids (U.S. Pat. No. 4,361,650 to Asai et al. and Izawa, M., et al., J. Antibiotics, 34, 12, 1587-1590, 1981). In these references, however, the catalyzed reaction consists exclusively of a demethylation characterized by low conversion yields and productivity.
Brumm, P. J., et al. (Starch, 43, 8, 319-323, 1991) have described the glycosyl transferase activity of an &agr;-amylase enzyme, which was derived from strains of
Bacillus megaterium
and has particularly high acceptor specificities for glucose or glucosides. For example, starting from starch, cyclodextrin-glucosyl transferases, produced by the same microbial source, catalyze the &agr;-1,4-transglucosylation of rubusoside (13-0-&bgr;-D-glucosyl-steviol &bgr;-D-glucosyl ester). It has been reported (Darise, M., et al., Agric. Bioel. Chem., 48, 10, 2483-2488, 1984), that, in this bioconversion, the acceptor of the transferase reaction is the substrate glucide fraction. Cyclodextrin-glycosyl transferases have also been used for preparing the cyclodextrins G6 and G8 from starch (Kitahata, S., Okada, S., Agric. Biol. Chem., 38, 12, 2413-2417, 1974).
These examples demonstrate the high substrate specificity toward glucosyl acceptors of glycosyl transferase enzymes expressed by
Bacillus megaterium.
Given this specificity, reactions toward substrates or metabolites having a different, complex molecular structure such as colchicones are entirely unexpected. In fact, no examples of the use of these microorganisms for the enzyme conversion of colchicone compounds to 3-glycosyl derivatives are known.
Now, it has been found that strains of
Bacillus megaterium
capable of growing in the presence of high concentrations of colchicone (R
1
=—OCH
3
, R
2
=—OCH
3
in formula I below), 3-demethylcolchicone, or thio derivatives thereof, have exceedingly high, very specific activities for biotransforming such substrate compounds into derivative compounds, which are glycosydated exclusively at C-3 of the aromatic ring. The transformation takes place in very short times and is characterized by surprisingly high yields.
SUMMARY OF THE INVENTION
One aspect of the present invention relates to a process for the preparation of a compound of formula (I)
which process comprises contacting a compound of formula (II)
with
Bacillus megaterium
or a mutant thereof or an enzyme isolated from
Bacillus megaterium
or a mutant thereof, under conditions sufficient to effect a biotransformation of the formula II compound to the formula I compound, wherein R
1
is a glycoside residue, R
2
is C
1
-C
6
alkoxy or C
1
-C
6
thioalkyl, and R
3
is OH or methoxy. The process further comprises recovering the compound of formula (I).
In a preferred embodiment, R
1
is an 0-glucoside residue. Preferably, the compound of formula II is glycosylated exclusively at the C-3 position of aromatic ring A to obtain a 3-O-glycosylcolchicone compound.
In one embodiment, the process of the present invention comprises culturing the
Bacillus megaterium
in a medium comprising the compound of formula II in an amount sufficient to provide a recoverable amount of the compound of formula I, preferably from about 0.1 to 3 g/l. The medium may comprise water.
The
Bacillus megaterium
strain used in the process of the invention may be selected for the ability to grow in contact with the compound of formula II in an amount sufficient to produce recoverable amounts of the compound of formula I.
The medium may comprise at least one organic nitrogen source, which is preferably selected from the group consisting of meat extract, peptone, tryptone, casein hydrolysates, or corn-step water. In another embodiment, the medium comprises at least one carbon source, which is preferably selected from the group consisting of glucose, fructose, or glycerol. In yet another embodiment, the medium comprises at least one inorganic salt selected, which is preferably selected from the group consisting of K
+
, Na
+
, Mg
++
, or NH
4
+
. The pH of the medium of the present invention is preferably from about 5 to 8, more preferably from about 6 to 7.
The culturing step is preferably carried out at a temperature ranging from about 20 to 45° C., more preferably from about 28 to 40° C. The culturing step is preferably, carried out at a maximum aeration level from about 1 to 2 liters of air per liter of culture per minute (vvm), more preferably from 1.5 to 2 vvm.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
The compounds obtained by the biotechnological process of the invention, particularly thiocolchicosone (3-O-glucosylthiocolchicone, i.e., with reference to formula (I), R
1
=—OCH
3
e=—SCH
3
), are active principles of remarkable pharmacological importance, mainly for the preparation of new antitumor therapeutics.
Bacillus megaterium
is a Gram-positive spore generating bacterium with a cell diameter greater than about 1.0 &mgr;m.
Bacillus megaterium
is capable of growing aerobically on or within a number of culture media, is catalase-positive, and hydrolyzes gelatin.
Strains of
Bacillus megaterium
that are useful according to the invention grow satisfacto
Bombardelli Ezio
Ponzone Cesare
Indena S.p.A.
Marx Irene
Pennie & Edmonds LLP
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