Part F | 30.4
740 Part F Marine Derived Metabolites
the antiviral and immunomodulatory activity of tilorone
congeners: 9-fluorenone carboxyhydroxyesters (AG series Fig. 30.5) and 9-fluorenone carboxyhydroxamides
(MG series Fig. 30.5). The ˛-glucosidase provided several ˛-O-glucosides of both model acceptors. The easy
biocatalytic access to these polyglucosylated derivatives is very interesting for the quick screening of the
pharmaceutical profiles of these drugs as modified by
carbohydrate(s) moieties with respect to their parental
aglycones [30.50].
The homogenate from the hepatopancreas of
Aplysia fasciata contained a ˇ-galactosidase activity,
which after purification was shown to be a homodimer with a molecular mass of 164 kDa as estimated
by gel-filtration and 78 kDa by SDS-PAGE [30.51].
ˇ-D-Galactosidase activity measured as a function of
pH revealed that the activity increases going from pH
5:52:3 and drastically drops at lower pH. At pH 3:7
a maximum activity around 75
ı C was observed. The
enzyme completely retains its activity after 23 h incubation at pH 4:7 in K-acetate buffer, while it retains 81%
of its activity when incubated at pH 3:9 in Na-citrate
buffer for 23 h. At pH 2:8 in glycine-HCl, the half-life
time was 0:6 h. At 50
ı C in K-acetate buffer pH 4:5 the
half-life was 15 h. This enzyme is highly specific for
pNP-ˇ-D-galactopyranoside (pNP-ˇ-Gal) and is completely inactive on other substrates; it is also able to
hydrolyze the glycosidic linkage present in lactose, although to a much lesser extent than those of pNP-ˇ-Gal
and oNP-ˇ-Gal. In transgalactosylation reactions this
enzyme showed interesting characteristics. Due to the
specificity of the acceptor site of most galactosidases
for compounds with phenyl groups [30.52], the yields
obtained in the reactions using free or methyl derivative of xylose and methyl ˇ-galactopyranoside and Dgalactose are interestingly high. The uncommon ˇ-1,3
selectivity is also of interest; using free xylose or its ˇallyl or methyl derivative, the ˇ-1,3 isomer was always
selectively formed. The same result was obtained by
using methyl ˇ-D-galactopyranoside and glucal. With
ˇ-aryl linked aglycones for both xylose and galactose, this ˇ-1,3 selectivity is again expressed, although
it is lost with ˛-anomers. However the influence of
aryl groups as aglycones is not limited to the regioselectivity of reaction but also to the yield as shown
when comparing the results of the reactions using the
p-nitrophenyl and benzyl xylopyranosides [30.53]. oNitrophenyl ˇ-D-galactopyranoside (oNP-ˇ-Gal) was
used as glycosyl donor in reactions with 5 equivalents
of nucleoside acceptors (uridine, fluorouridine, thymidine, adenosine, cytidine, 5-chlorocytosine arabinoside,
and 3
0 azido-3
0 -deoxythymidine) in the presence of this
enzyme. The regioselectivity was extremely high, in all
reactions only the product of galactosylation in 5
0 position of the nucleoside was observed. Reaction yields
were satisfactory in most cases, and very high for
uridine derivatives. In particular, 5
0 -O-ˇ-galactosyl-5fluorouridine, the galactosylated derivative of the anticancer drug fluorouridine, was synthesized with a 60%
yield, and 5
0 -O-ˇ-galactosyl-3
0 -azido-3
0 -deoxythymidine, the derivative of the anti-HIV drug, was obtained
in 43% yield [30.54]. This was the first report dealing
with a glycoside hydrolase used for the modification
of nucleosides with such convenient yields. Nucleoside
analogs play an important role in antiviral and anticancer therapy. In addition, it has been found that many
natural antibiotics possessing significant antitumor and
antiviral activities have the structure of a nucleoside
connected to oligosaccharides [30.55].
There are at least three main points stating the
importance of enzymes acting on mannose-based carbohydrates. The first is that the ˇ-mannopyranoside
linkage is one of the most difficult glycosidic bonds to
synthesize using chemical strategies [30.56], because
the steric and polar effects favor the formation of ˛anomer. Hence, alternative biological methodologies
based on ˇ-mannosidases are very attractive. However,
natural examples of new ˇ-mannosidase are needed
also for the advancement of conformational studies of
the enzymatic transition state and for the possibility
of genetic manipulation of such enzymes to produce
efficient synthetic catalysts (mannosynthase) [30.57].
As a third point it could be stated that ˇ-mannosidases and ˇ-mannanases are of interest for the reutilization of waste materials. In this context, both the
degradation of ˇ-1,4-mannan to simple sugars and the
direct ˇ-mannosidase-catalyzed synthesis of alkyl ˇD-mannosides from mannobiose by ˇ-mannosidase are
possible [30.58]. ˇ-D-Mannosidase activity was identified in the hepatopancreas of Aplysia fasciata and
with higher specific activity in the visceral mass homogenate [30.38]. The apparent molecular mass for
the purified protein was 229 kDa, while the molecular
mass as determined by SDS-PAGE was 130 kDa, indicating that the purified enzyme is a homodimer. It
showed an optimal activity at pH 4:5 and 45
ı C [30.59].
At optimal pH the enzyme showed a half-life of 23 h.
The enzyme retained most of its activity (82%) after 24 h incubation in the presence of 10% DMSO,
while in the presence of other co-solvents the activity
quickly dropped down (a half-life of 1 h was estimated in the presence of 10% acetonitrile, while in
740 Part F Marine Derived Metabolites
the antiviral and immunomodulatory activity of tilorone
congeners: 9-fluorenone carboxyhydroxyesters (AG series Fig. 30.5) and 9-fluorenone carboxyhydroxamides
(MG series Fig. 30.5). The ˛-glucosidase provided several ˛-O-glucosides of both model acceptors. The easy
biocatalytic access to these polyglucosylated derivatives is very interesting for the quick screening of the
pharmaceutical profiles of these drugs as modified by
carbohydrate(s) moieties with respect to their parental
aglycones [30.50].
The homogenate from the hepatopancreas of
Aplysia fasciata contained a ˇ-galactosidase activity,
which after purification was shown to be a homodimer with a molecular mass of 164 kDa as estimated
by gel-filtration and 78 kDa by SDS-PAGE [30.51].
ˇ-D-Galactosidase activity measured as a function of
pH revealed that the activity increases going from pH
5:52:3 and drastically drops at lower pH. At pH 3:7
a maximum activity around 75
ı C was observed. The
enzyme completely retains its activity after 23 h incubation at pH 4:7 in K-acetate buffer, while it retains 81%
of its activity when incubated at pH 3:9 in Na-citrate
buffer for 23 h. At pH 2:8 in glycine-HCl, the half-life
time was 0:6 h. At 50
ı C in K-acetate buffer pH 4:5 the
half-life was 15 h. This enzyme is highly specific for
pNP-ˇ-D-galactopyranoside (pNP-ˇ-Gal) and is completely inactive on other substrates; it is also able to
hydrolyze the glycosidic linkage present in lactose, although to a much lesser extent than those of pNP-ˇ-Gal
and oNP-ˇ-Gal. In transgalactosylation reactions this
enzyme showed interesting characteristics. Due to the
specificity of the acceptor site of most galactosidases
for compounds with phenyl groups [30.52], the yields
obtained in the reactions using free or methyl derivative of xylose and methyl ˇ-galactopyranoside and Dgalactose are interestingly high. The uncommon ˇ-1,3
selectivity is also of interest; using free xylose or its ˇallyl or methyl derivative, the ˇ-1,3 isomer was always
selectively formed. The same result was obtained by
using methyl ˇ-D-galactopyranoside and glucal. With
ˇ-aryl linked aglycones for both xylose and galactose, this ˇ-1,3 selectivity is again expressed, although
it is lost with ˛-anomers. However the influence of
aryl groups as aglycones is not limited to the regioselectivity of reaction but also to the yield as shown
when comparing the results of the reactions using the
p-nitrophenyl and benzyl xylopyranosides [30.53]. oNitrophenyl ˇ-D-galactopyranoside (oNP-ˇ-Gal) was
used as glycosyl donor in reactions with 5 equivalents
of nucleoside acceptors (uridine, fluorouridine, thymidine, adenosine, cytidine, 5-chlorocytosine arabinoside,
and 3
0 azido-3
0 -deoxythymidine) in the presence of this
enzyme. The regioselectivity was extremely high, in all
reactions only the product of galactosylation in 5
0 position of the nucleoside was observed. Reaction yields
were satisfactory in most cases, and very high for
uridine derivatives. In particular, 5
0 -O-ˇ-galactosyl-5fluorouridine, the galactosylated derivative of the anticancer drug fluorouridine, was synthesized with a 60%
yield, and 5
0 -O-ˇ-galactosyl-3
0 -azido-3
0 -deoxythymidine, the derivative of the anti-HIV drug, was obtained
in 43% yield [30.54]. This was the first report dealing
with a glycoside hydrolase used for the modification
of nucleosides with such convenient yields. Nucleoside
analogs play an important role in antiviral and anticancer therapy. In addition, it has been found that many
natural antibiotics possessing significant antitumor and
antiviral activities have the structure of a nucleoside
connected to oligosaccharides [30.55].
There are at least three main points stating the
importance of enzymes acting on mannose-based carbohydrates. The first is that the ˇ-mannopyranoside
linkage is one of the most difficult glycosidic bonds to
synthesize using chemical strategies [30.56], because
the steric and polar effects favor the formation of ˛anomer. Hence, alternative biological methodologies
based on ˇ-mannosidases are very attractive. However,
natural examples of new ˇ-mannosidase are needed
also for the advancement of conformational studies of
the enzymatic transition state and for the possibility
of genetic manipulation of such enzymes to produce
efficient synthetic catalysts (mannosynthase) [30.57].
As a third point it could be stated that ˇ-mannosidases and ˇ-mannanases are of interest for the reutilization of waste materials. In this context, both the
degradation of ˇ-1,4-mannan to simple sugars and the
direct ˇ-mannosidase-catalyzed synthesis of alkyl ˇD-mannosides from mannobiose by ˇ-mannosidase are
possible [30.58]. ˇ-D-Mannosidase activity was identified in the hepatopancreas of Aplysia fasciata and
with higher specific activity in the visceral mass homogenate [30.38]. The apparent molecular mass for
the purified protein was 229 kDa, while the molecular
mass as determined by SDS-PAGE was 130 kDa, indicating that the purified enzyme is a homodimer. It
showed an optimal activity at pH 4:5 and 45
ı C [30.59].
At optimal pH the enzyme showed a half-life of 23 h.
The enzyme retained most of its activity (82%) after 24 h incubation in the presence of 10% DMSO,
while in the presence of other co-solvents the activity
quickly dropped down (a half-life of 1 h was estimated in the presence of 10% acetonitrile, while in