Angewandte
Chemie
strikingly allows efficient, high-yielding, one-step
access to glycolipophilic compounds, such as the
glycoflavonoids 81–85 described herein, and includes
unique regioisomers of natural glycoflavonoids in a
manner not previously possible.
Experimental Section
GAR screening was performed as described by Yang et al.
[
24]
Briefly, in a 96-well plate, each well was filled with Tris-HCl
buffer solution (Tris = Tris(hydroxymethyl) aminomethane;
Figure 2. Observed Michaelis–Menten kinetics for Cel7B–E197S for the transfer
of lactosyl from LacF to quercetin (21), under pseudo-single-substrate condi-
tions of fixed [21] (a) and fixed [LacF] (b).
1
.0m m, pH 7.8, 150 mL), donor (10m m, 2 mL), acceptor
À1
(10m m, 2 mL), and enzyme (1 mgmL , 2 mL). The solutions
and the plate were incubated at 378C for 16 h, and the
solutions were analyzed for formation of product by LCMS
monitoring. Single-substrate concentration kinetics were
also performed by MS at 258C with both LacF fixed at
mm and the acceptors fixed at 100 mm. LCMS was used to monitor
the formation of product through the appearance of corresponding
ions and relating this to the total ion count with concentration
determined through internal calibration. Pseudo-single-substrate
kinetics were also performed by MS at 258C with the acceptor fixed
at 100 mm and the donor varied at different concentrations from 50–
2
droxylation in the C2 aryl substituent (O4’, O5’), suggests that
the aryl ring of 2 effectively mimics a monosaccharide residue
[
28]
in the + 1 subsite of Cel7B–E197S. This substrate mimicry
[
23,27]
is consistent with a mode of recognition often observed
in
such subsites that utilizes a hydrophobic “sandwich” coupled
with equatorial hydrogen bonding. Here, this motif simulta-
neously engages O5, O6, and O7 and p faces. Alternatively,
the more electron-rich aryl phenols may have more suitable
pKa values and/or nucleophilicities. Consistent with this
possibility, variation of pH from 7 to 9 led to a threefold
increase in the rate of glycosylation of 21 by Cel7B–E197S by
using LacF as a donor (see the Supporting Information;
1500 mm (i.e., up to 3.1 times K ); or LacF was fixed at 2000 mm and
M
the acceptor typically varied from 20–150 mm. Product formation was
monitored by LCMS through the appearance of corresponding ions
and relating this to the total ion count with concentration determined
through the internal standard glucose (500 mm).
Received: October 12, 2006
Published online: February 16, 2007
“
baseless” mutants of Cel7B–E197S are currently also being
investigated). Regardless, this functional “glycomimicry” in
the biocatalysis shown here by the polyhydroxylated-2-phenyl
of flavonoids gives rise to an unusual activity and otherwise
inaccessible regiospecificity (O4’ and O6) in biocatalytic
flavonoid glycosylation. Furthermore, the catalyst used here
allows the unusual transfer of a disaccharide rather than
monosaccharide motif.
Keywords: biocatalysis · carbohydrates · flavonoids ·
glycosyltransferases · glycosynthases
.
[
[
[
1] C. E. Ruefer, C. Gerhaeuser, N. Frank, H. Becker, S. E. Kulling,
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Finally, highly selective representative hydrolysis of 83
using E. coli b-galactosidase allowed access to the monogly-
cosylated glucoflavonoid natural product 85 in 90% yield.
Cel7B–E197S showed no activity with the monosaccharide
donor, a-glucosyl fluoride (GlcF). Therefore, this sequential
two-step biocatalytic procedure allowed unique and ready
access to unusual regioisomers of natural glucoflavonoids in a
striking overall yield (65%) without the need for protecting
groups. Spiraeoside (85), synthesized before in only 6%
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5] B. Henrissat, P. M. Coutinho, G. J. Davies, Plant Mol. Biol. 2001,
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[
[
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[30]
yield, shows powerful cytotoxic activity in oral cancers
and in human trials showed clear inhibition of platelet cell
[
9] S. Walker, M. J. Sofia, R. Kakarla, N. A. Kogan, L. Wierichs,
C. B. Longley, K. Bruker, H. R. Axelrod, S. Midha, S. Babu, D.
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signaling and thrombus formation.
This highly selective
one-pot tandem biocatalytic method also outstrips the yields
of other glucoflavonoid syntheses such as that of the quercetin
[
[
10] H. Matsuda, T. Wang, H. Managi, M. Yoshikawa, Bioorg. Med.
Chem. 2003, 11, 5317.
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H. Mohan, Bioorg. Med. Chem. 2003, 11, 2677.
[
32–34]
glucoside isoquercitrin obtained from chemical syntheses
(
< 37%) and even those of fermentor and whole-cell
[
35]
glycosylations (< 41%) with GTs.
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4
In summary, a non-natural glycosidase mutant (Cel7B–
E197S glycosynthase) has been identified that has novel
regiospecificity (O4’, O6) and activity (disaccharide transfer
to flavonoids) with catalytic efficiencies comparable with
those of natural glycosyltransferase counterparts. The mutant
can be used as a component in sequential biocatalysis and
[13] B. G. Davis, J. Chem. Soc. Perkin Trans. 1 2000, 2137.
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Angew. Chem. Int. Ed. 2007, 46, 3885 –3888
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