Substrate flexibility of vicenisaminyltransferase VinC involved in the biosynthesis of vicenistatin

Article abstract of DOI:10.1021/ja0685250

A glycosyltransferase VinC is involved in the biosynthesis of antitumor β-glycoside antibiotic vicenistatin. It catalyzes a glycosyl transfer reaction between dTDP-α-D-vicenisamine and vicenilactam. Previous identification of its broad substrate specifici

Full text of DOI:10.1021/ja0685250

Published on Web 03/28/2007
Substrate Flexibility of Vicenisaminyltransferase VinC
Involved in the Biosynthesis of Vicenistatin
Atsushi Minami† and Tadashi Eguchi*,‡
Contribution from the Department of Chemistry, and Department of Chemistry and Materials
Science, Tokyo Institute of Technology, 2-12-1, O-okayama, Meguro-ku, Tokyo 152-8551, Japan
Received November 28, 2006; E-mail: eguchi@cms.titech.ac.jp
Abstract: A glycosyltransferase VinC is involved in the biosynthesis of antitumor â-glycoside antibiotic
vicenistatin. It catalyzes a glycosyl transfer reaction between dTDP-R-D-vicenisamine and vicenilactam.
Previous identification of its broad substrate specificity toward various glycosyl acceptors enabled us to
explore the potential of VinC for glycodiversification. In vitro study of the substrate specificity toward several
dTDP-sugars with vicenilactam established that VinC displayed activities with R-anomers of several dTDP-
2-deoxy-D-sugars such as mycarose, digitoxose, olivose, and 2-deoxyglucose to afford respective
â-glycosides. Notably, â-anomers of dTDP-2-deoxy-D-sugars also appeared to be accepted by VinC to
form R-glycosides. Furthermore, VinC is capable of catalyzing glycosyl transfer reactions from both the
R-anomer and â-anomer of dTDP-L-mycarose, respectively, into â-glycoside and R-glycoside. These results
indicate that VinC is a unique glycosyltransferase possessing broad substrate specificity. The mechanism
of this axially oriented glycosidic bond formation from the equatorially oriented dTDP-sugar might be
explained by conformational change of dTDP-sugar to a boat conformation during the glycosyl transfer
reaction. To apply these features of VinC for glycodiversification, 22 sets of structurally diverse glycosides
were constructed using unnatural glycosyl donors and acceptors.
Introduction
natural glycosides, further identification of new glycosyltrans-
ferases exhibiting a broad substrate specificity toward both sugar
Glycosyltransferase, which catalyzes a transfer of sugar in
the form of nucleoside diphosphosugar (NDP-sugar) to the
respective aglycons, is an important enzyme in the biosynthesis
of biologically active secondary metabolites because the pres-
ence of sugar is, in most cases, necessary for exerting their
biological activities.1 It is therefore a current subject of active
research in the field to exploit the usefulness of these glyco-
syltransferases for structurally diverse glycoside synthesis. In
fact, chemoenzymatic glycoside syntheses using glycosyltrans-
ferases have been applied to change the sugar fraction of natural
glycosides.2-7 Structural changes of the sugar component in
these examples include substitution of the functional group and
configurational change. In addition, recent studies have shown
examples of change in the aglycon part of natural glycosides
using glycosyltransferases.6-11 Although these examples indicate
the importance of a chemoenzymatic approach to create un-
and aglycon components is highly desirable for development
of a glycoside library because the success of this approach
depends mainly on the substrate specificity of glycosyltrans-
ferases themselves.
Vicenistatin (1), an antitumor â-glycosidic antibiotic produced
by Streptomyces halstedii HC 34, comprises amino sugar
vicenisamine and 20-membered macrocyclic vicenilactam (Fig-
ure 1A).12 We identified the whole vicenistatin biosynthetic gene
cluster (Vin) and confirmed that VinC is a vicenisaminyltrans-
ferase catalyzing the transfer of vicenisamine from dTDP-
vicenisamine (4) to vicenilactam (3) in the last step of
vicenistatin biosynthesis (Figure 1B).13 We recently demon-
strated that VinC accepts structurally diverse aglycons to form
respective vicenisaminides.9,11 The broad substrate specificity
toward the glycosyl acceptor showed that VinC is an attractive
glycosyltransferase for glycodiversification. On the other hand,
the substrate specificity of VinC toward a glycosyl donor
remains unclear, even though previous isolation of vicenistatin
M (2) indicated the possibility of D-mycarose transfer to
† Department of Chemistry.
‡ Department of Chemistry and Materials Science.
(1) Weymouth-Wilson, A. C. Nat. Prod. Rep. 1997, 14, 99.
(2) Griffith, B. R.; Langenhan, J. M.; Thorson, J. S. Curr. Opin. Biotechnol.
2005, 16, 622.
(3) Losey, H. C.; Jiang, J.; Biggins, J. B.; Oberthu¨r, M.; Ye, X.-Y.; Dong, S.
D.; Kahne, D.; Thorson, J. S.; Walsh, C. T. Chem. Biol. 2002, 9, 1305.
(4) Lu, W.; Leimkuhler, C.; Oberthu¨r, M.; Kahne, D.; Walsh, C. T. Biochem-
istry 2004, 43, 4548.
(5) Oberthu¨r, M.; Leimkuhler, C.; Kruger, R. G.; Lu, W.; Walsh, C. T.; Kahne,
D. J. Am. Chem. Soc. 2005, 127, 10747.
(6) Borisova, S. A.; Zhang, C.; Takahashi, H.; Zhang, H.; Wong, A. W.;
Thorson, J. S.; Liu, H.-W. Angew. Chem., Int. Ed. 2006, 45, 2748.
(7) Zhang, C.; Griffith, B. R.; Fu, Q.; Albermann, C.; Fu, X.; Lee, I.-K.; Li,
L.; Thorson, J. S. Science 2006, 313, 1291.
(8) Feel Meyers, C. L.; Oberthu¨r, M.; Anderson, J. W.; Kahne, D.; Walsh, C.
T. Biochemistry 2003, 42, 4179.
(9) Minami, A.; Uchida, R.; Eguchi, T.; Kakinuma, K. J. Am. Chem. Soc. 2005,
127, 6148.
(10) Yang, M.; Proctor, M. R.; Bolam, D. N.; Errey, J. C.; Field, R. A.; Gilbert,
H. J.; Davis, B. G. J. Am. Chem. Soc. 2005, 127, 9336.
(11) Minami, A.; Kakinuma, K.; Eguchi, T. Tetrahedron Lett. 2005, 46,
6187.
(12) Shindo, K.; Kamishohara, M.; Okagawa, A.; Matsuoka, M.; Kawai, H. J.
Antibiot. 1993, 46, 1076.
(13) Ogasawara, Y.; Katayama, K.; Minami, A.; Otsuka, M.; Eguchi, T.;
Kakinuma, K. Chem. Biol. 2004, 11, 79.
9
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J. AM. CHEM. SOC. 2007, 129, 5102-5107
10.1021/ja0685250 CCC: $37.00 © 2007 American Chemical Society

Substrate Flexibility of Vicenisaminyltransferase VinC
A R T I C L E S
Figure 1. (A) Structure of vicenistatin (1) and vicenistatin M (2). (B) Glycosylation between dTDP-R-D-vicenisamine (4) and vicenilactam (3) catalyzed
by VinC.
Table 1. Results of the VinC Reaction with NDP-Sugars (4-21)a
vicenilactam (Figure 1A).14 For further exploitation of VinC
for glycodiversification, more detailed information about the
relative
substrate
product
yield (%)
activity
substrate specificity toward the glycosyl donor, especially in
the hexose moiety, is indispensable.
Herein, we report the biochemical characterization and in vitro
study of the substrate specificity for glycosyl donor in VinC
reaction using structurally varied dTDP-sugars including both
anomers of D-sugars and L-sugars.
4
5
6
7
8
9
dTDP-R-D-vicenisamine
dTDP-â-D-vicenisamine
UDP-R-D-vicenisamine
UDP-â-D-vicenisamine
ADP-R-D-vicenisamine
ADP-â-D-vicenisamine
â-glycoside 85
no reaction
â-glycoside 61
no reaction
â-glycoside 49
no reaction
1
1.8 × 10-3
2.2 × 10-5
10 dTDP-R-D-mycarose
11 dTDP-â-D-mycarose
12 dTDP-R-D-digitoxose
13 dTDP-â-D-digitoxose
14 dTDP-R-D-olivose
15 dTDP-â-D-olivose
16 dTDP-R-D-2-deoxyglucose â-glycoside 69
17 dTDP-â-D-2-deoxyglucose R-glycoside 41
18 dTDP-R-L-mycarose
19 dTDP-â-L-mycarose
20 dTDP-R-D-glucose
21 dTDP-R-D-glucosamine
â-glycoside 55
R-glycoside 12
â-glycoside 54
5.7 × 10-2
6.1 × 10-8
3.3 × 10-3
3.4 × 10-7
1.1 × 10-4
6.4 × 10-7
5.6 × 10-6
6.2 × 10-7
5.2 × 10-5
5.6 × 10-5
Results and Discussion
NDb
18
Biochemical Properties of VinC. Heterologous expression
of VinC in Escherichia coli was described in our previous
paper.13 The expressed VinC was purified using ammonium
sulfate precipitation and DEAE Sepharose fast flow column
chromatography. VinC was obtained as an electrophoretically
homogeneous state, and the molecular mass was estimated as
46 kDa using SDS-PAGE analysis (Figure S1, see Supporting
Information). The molecular weight was confirmed as 46 064
Da (Figure S2) from LC-ESI-MS analysis, which is consistent
with the estimated molecular weight (46 066.5 Da) from the
amino acid sequence. In addition, native-PAGE analysis results
suggest the apparent molecular mass as 88 kDa (data not shown),
which strongly indicates that the overexpressed VinC exists in
a homodimer.
The highest VinC activity with dTDP-vicenisamine and
vicenilactam was observed with 50 mM Tris-HCl buffer, pH
8.0, at ca. 25 °C. The effect of divalent metal cations to VinC
activity was eliminated because VinC activity was not changed
in the presence of various divalent metal cations (1 mM of
Mg2+, Mn2+, Co2+, Cu2+, Zn2+, or Ca2+) or EDTA (1 mM).
No metal requirement for catalytic activity is apparent in other
glycosyltransferases such as GtfB and GtfE, which are involved,
respectively, in the biosynthesis of chloroeremomycin and
vancomycin.15
â-glycoside 75
R-glycoside 61
â-glycoside 12
R-glycoside 40
no reaction
no reaction
a Yields of this table were calculated using the equation, % yield ) [Ap/
(Ap + Ar)], where Ap represents the integration of the product peak, and Ar
represents the integration of the unreacted aglycon peak. Relative activity
is the difference of the initial formation rate of glycoside, which was adjusted
by the concentration of VinC, between dTDP-sugar and dTDP-R-D-
vicenisamine. b ND ) Stereochemistry is not determined.
nisamine (4-9) were prepared using chemical synthesis fol-
lowed by separation of anomers by preparative HPLC with a
ODS-AQ column. These glycosyl donors were subjected
separately to VinC reaction with vicenilactam. Subsequent
HPLC analysis showed clearly that these R-anomers were
accepted by VinC to form vicenistatin. Comparison of their
relative activities, which were estimated from the initial forma-
tion rate of 1 adjusted by the concentration of VinC, indicated
that dTDP-vicenisamine was the best among those tested and
that UDP-vicenisamine was also an active substrate, as shown
in Table 1. In contrast, ADP-vicenisamine represented low
activity among those three glycosyl donors. On the other hand,
â-anomers (5, 7, 9) were not accepted at all, even in the presence
of high concentration of VinC (0.2 mM). It was apparent,
therefore, that VinC originally catalyzes a stereospecific glycosyl
Specificity toward Nucleoside Diphosphate Moiety of
NDP-Vicenisamine. To gain additional information about
the substrate specificity of VinC toward the NDP moiety,
R-anomers and â-anomers of dTDP-, UDP-, and ADP-vice-
(14) Matsushima, Y.; Nakayama, T.; Fujita, M.; Bhandari, R.; Eguchi, T.; Shindo,
(15) Losey, H. C.; Peczuh, M. W.; Chen, Z.; Eggert, U. S.; Dong, S. D.; Pelczer,
K.; Kakinuma, K. J. Antibiot. 2001, 54, 211.
I.; Kahne, D.; Walsh, C. T. Biochemistry 2001, 40, 4745.
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A R T I C L E S
Minami and Eguchi
transfer reaction between dTDP-R-D-vicenisamine (4) and
vicenilactam (3) to form vicenistatin (1) in the inversion
mechanism (Figure 1B).
The recognition mechanism toward the nucleoside part has
been well-demonstrated from crystal structures such as GtfA
and GtfD, which are, respectively, homologous to VinC with
32 and 23% homogeneity at the amino acid level.16,17 Regarding
the recognition mechanism toward the thymine or uracil moiety,
the carbonyl oxygen and ring nitrogen atoms of the pyrimidine
base make contacts through hydrogen-bonding interaction with
the main-chain amide and the carbonyl group of the highly
conserved valine residue (294V in GtfD, 278V in GtfA) within
glycosyltransferases recognizing pyrimidinyl diphosphosugar.
This valine residue is also conserved in VinC (305V); therefore,
this valine residue might play an important role in dTDP
recognition of VinC by these hydrogen-bonding interactions.
Specificity toward the Hexose Moiety of dTDP-Sugar.
Chemoenzymatic glycoside synthesis, such as glycorandom-
ization, is a powerful method for glycoside synthesis, especially
in the sugar component alteration of biologically active glyco-
sides. Previous identification of vicenistatin M from the same
strain indicated the possibility of VinC to accept dTDP-
mycarose. Therefore, glycosylation between dTDP-D-mycarose
and vicenilactam was first investigated.
The R-anomer of dTDP-D-mycarose (10), prepared by chemi-
cal synthesis from methyl R-D-mannopyranoside followed by
HPLC separation, was subjected to VinC reaction. The product
was analyzed using HPLC equipped with a photodiode array
detector. Compared to the authentic sample,14 the naturally
occurring â-anomer of vicenistatin M was identified (Figure
2A). To our surprise, the â-anomer of dTDP-D-mycarose (11)
was also accepted, and the R-anomer of vicenistatin M was
produced (Figure 2B).
This stereospecific glycosylation from both the R-anomer and
â-anomer of dTDP-mycarose to form respective glycosides in
the inversion mechanism is unique. Although substrate specifici-
ties of several glycosyltransferases toward a glycosyl donor have
been investigated,3-8,10,15,18 to our knowledge, no report has
described that glycosyltransferase stereospecifically catalyzes
the glycosyl transfer reaction from both anomers of the glycosyl
donor. Therefore, we set out to investigate this unique phe-
nomenon of VinC reaction in detail.
Figure 2. HPLC profiles of the VinC reaction products with (A) R-anomer
(10), (B) â-anomer (11). Top line, co-injection with authentic sample; middle
line, enzyme reaction products; bottom line, control experiment without
dTDP-mycarose.
structurally diverse dTDP-2-deoxysugar to afford both R-gly-
cosides and â-glycosides, respectively, from â-glycosyl and
R-glycosyl donors in the inversion mechanism.
For further investigation of this unique stereospecific glyco-
sylation of VinC, several dTDP-sugars (12-21) were tested for
VinC reaction (Figure 3). Results indicated that R-anomers and
â-anomers of dTDP-D-digitoxose (12, 13), dTDP-D-olivose (14,
15), dTDP-2-deoxy-D-glucose (16, 17), and dTDP-L-mycarose
(18, 19) were accepted by VinC to yield respective vicenistatin
analogues (Table 1, Figure S3). Generated glycosides, except
for 13, were isolated from large-scale VinC reactions, and the
stereochemistries of the anomeric position were determined
Comparison of the relative activities between these accepted
dTDP-D-sugars showed that the R-anomer of dTDP-vice-
nisamine (4) was the best substrate for VinC. The reactivity
decreased in order of mycarose (10), digitoxose (12), olivose
(14), and 2-deoxyglucose (16). In contrast, glucose (20) and
glucosamine (21) were not accepted at all. The fact that dTDP-
2-deoxyglucose was accepted and that neither dTDP-glucose
nor dTDP-glucosamine was accepted indicates that the steric
bulkiness or the hydrophobic interaction at the C2 position of
the sugar component is probably important for VinC recognition.
In contrast, VinC tolerates the modification at C3, C4, and C6
functional groups within D-series sugars. Furthermore, VinC
recognized L-mycarose (18, 19) as well as D-mycarose to afford
their respective glycosides, but the marked difference of
reactivity between these two glycosyl donors indicates that VinC
prefers the D-series sugars as a glycosyl donor. In contrast,
1
using H NMR, which showed clearly that â-glycosides were
produced from R-glycosyl donors and that R-glycosides were
generated from â-glycosyl donors. Therefore, it appears that
VinC was able to catalyze the stereospecific glycosylation with
(16) Mulichak, A. M.; Losey, H. C.; Lu, W.; Wawrzak, Z.; Walsh, C. T.;
Garavito, R. M. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 9238.
(17) Mulichak, A. M.; Lu, W.; Losey, H. C.; Walsh, C. T.; Garavito, R. M.
Biochemistry 2004, 43, 5170.
(18) Zhang, C.; Albermann, C.; Fu, X.; Thorson, J. S. J. Am. Chem. Soc. 2006,
128, 16420.
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Substrate Flexibility of Vicenisaminyltransferase VinC
A R T I C L E S
Figure 3. Tested dTDP-sugars for VinC reaction.
Figure 4. Glycosylation mechanism of (A) â-glycoside formation from the R-glycosyl donor, (B) R-glycoside formation from the â-glycosyl donor in the
direct inversion mechanism, and (C) R-glycoside formation from the â-glycosyl donor passing through a conformational change of the hexose part.
â-anomers of dTDP-D-sugar exhibited decreased activity com-
pared to respective R-anomers, which implies that VinC
preferentially recognized the R-configuration of dTDP-D-sugar.
Reaction Mechanism for Glycoside Formation. Generally,
regioselectivity and stereoselectivity are strictly controlled in
the glycosyl transfer reaction by glycosyltransferase. For gly-
cosylation with D-series sugars, the R-anomer of the relevant
NDP-sugar is used as a glycosyl donor. The reactions involving
simple inversion and double inversion (retention) at the anomeric
position give rise, respectively, to â-glycoside and R-glycoside.
On the basis of its amino acid sequence, VinC belongs to
glycosyltransferase family 1.19-21 Furthermore, in the VinC
reaction with the natural substrate dTDP-vicenisamine, the
R-anomer is only accepted to form the â-glycoside, which
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A R T I C L E S
Minami and Eguchi
oxygen and the σ* orbital of C1-O1 bond and facilitates the
formation of the oxocarbenium ionic intermediate. A boat
conformation or a twisted-boat conformation seems to be
suitable for the intermediate structure because this conformation
resembles the transition-state conformation. Therefore, when the
NDP substituent occupies a pseudoaxial position, VinC reaction
proceeds smoothly via the oxocarbenium ionic intermediate,
followed by a nucleophilic attack to afford R-glycoside, ac-
cording to the general mechanism of inverting glycosyltrans-
ferases (Figure 4C, steps 2 and 3).
Similar examples have already been found for glycosylation
catalyzed by DesVII, which originally catalyzes D-desosamine
transfer in the inverting mechanism. In addition, dTDP-â-L-
rhamnose is reportedly transferred to 12-membered and 14-
membered macrolactones to give the respective R-glycosides
by DesVII.25 Although details of the L-rhamnose transfer have
not been discussed, similar conformational change might also
occur as in the dTDP-L-â-mycarose transfer by VinC. An
additional example can be found in a glycosyl transfer reaction
by UrdGTII.26 These examples present the possibility that some
glycosyltransferases catalyze stereospecific glycosylation to form
both anomers.
Figure 5. Glycodiversification between glycosyl donors (10-19) and
acceptors (22-26).
indicates that VinC originally catalyzes the glycosyl transfer
reaction in the inversion mechanism, just as other family 1
glycosyltransferases do.
Construction of Glycoside Library. A goal of chemoen-
zymatic glycoside synthesis, such as glycorandomization, is
construction of a glycoside library with structurally diverse
scaffolds. Although glycoside diversification has been partially
achieved by changing either the sugar or aglycon part, few
examples are known to demonstrate changes of both parts
simultaneously.6,7,10 The VinC has a relaxed specificity toward
both parts and catalyzes a unique stereospecific glycosylation.
Therefore, we set out to explore the potential of VinC for
glycodiversification.
This reaction is considered to proceed via the oxocarbenium
ionic intermediate. Formation of the oxocarbenium ionic
intermediate is facilitated by the interaction between the
unshared electron pair of the endocyclic oxygen and the σ*
orbital of the C1-O1 bond. The reaction is completed by a
nucleophilic attack of a hydroxy group to the anomeric carbon
of the intermediate (Figure 4A). However, when altered dTDP-
sugars were subjected to VinC reaction, the unexpected R-gly-
coside formations from â-anomers of dTDP-D-sugars were
observed. In these cases, such an interaction cannot occur in a
4
stable C1 chair conformation having an equatorially oriented
As an effort toward glycodiversification, 50 sets of VinC
reaction using dTDP-sugars (10-19) and aglycons including
neovicenilactam (22), R-zeararenol (23), â-zeararenol (24),
â-estradiol (25), and brefeldinA (26) were carried out; the VinC
reaction products were analyzed using HPLC-PDA and LC-
MS. Figure 5 shows that, when R-anomers of dTDP-mycarose,
dTDP-digitoxose, and dTDP-olivose were subjected to VinC
reaction, the respective glycosides were observed in 12 of 15
reactions, but the R-anomer of dTDP-2-deoxyglucose was
transferred only to neovicenilactam. These results indicate that
2,6-dideoxysugar is a more suitable substrate for glycodiversi-
fication with structurally diverse glycosides in this case. In
contrast, regarding glycosylation from the â-anomer of dTDP-
D-sugar, the expected glycosides were observed only when
neovicenilactam 22 was used as a glycosyl donor and the other
aglycons were not accepted at all. In addition, the respective
glycosides were obtained in six reactions from VinC reaction
with dTDP-L-mycarose. In all, formation of 22 glycosides was
achieved from 50 sets of VinC reactions. These results indicate
that VinC is an attractive glycosyltransferase for glycodiversi-
fication.
dTDP group. The 4C1 conformation for these sugars in solution
was confirmed by their 1H NMR data (see Supporting Informa-
tion), which indicates that the direct inversion mechanism, as
shown in Figure 4B from the â-anomer of dTDP-D-sugar, seems
implausible.
The mechanism for these R-glycoside formation reactions is
explainable according to the mechanism of â-glycosidase, which
catalyzes a cleavage of the â-glycosidic bond. In the â-gly-
cosidase reaction, the conformational change of the hexose part
in the active site is generally required for inducing the scissile
bond to pseudoaxial orientation to fulfill the stereoelectronic
requirements described above.22-24 This conformational change
permits the oxocarbenium ion formation, followed by a nucleo-
philic attack of water, thereby leading to the cleavage of the
â-glycosidic bond. Therefore, it seems likely that the NDP
moiety of dTDP-â-sugar occupies a pseudoaxial position, as in
the case of â-glycosidase (Figure 4C, step 1). This conforma-
tional change of dTDP-â-sugar in the active site causes the
interaction between the unshared electron pair of the endocyclic
(19) Campbell, J. A.; Davies, G. J.; Bulone, V.; Henrissat, B. Biochem. J. 1997,
326, 929.
Conclusion
(20) Coutinho, P. M.; Deleury, E.; Davies, G. J.; Henrissat, B. J. Mol. Biol.
2003, 328, 307.
This in vitro study of substrate specificity toward glycosyl
donors in VinC reaction showed that VinC is a promiscuous
(21) http://afmb.cnrs-mrs.fr/CAZY/index.html.
(22) Sulzenbacher, G.; Driguez, H.; Henrissat, B.; Schu¨lein, M.; Davies, G. J.
Biochemistry 1996, 35, 15280.
(23) Vocadlo, D. J.; Davies, G. J.; Laine, R.; Withers, S. G. Nature 2001, 412,
835.
(25) Yamase, H.; Zhao, L.; Liu, H.-W. J. Am. Chem. Soc. 2000, 122, 12397.
(26) Hoffmeister, D.; Dra¨ger, G.; Ichinose, K.; Rohr, J.; Bechthold, A. J. Am.
Chem. Soc. 2003, 125, 4678.
(24) Vasella, A.; Davies, G. J.; Bo¨hm, M. Curr. Opin. Chem. Biol. 2002, 6,
619.
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Substrate Flexibility of Vicenisaminyltransferase VinC
A R T I C L E S
glycosyltransferase to accept various dTDP-sugars as a substrate.
In fact, VinC displayed activities with several dTDP-2-deoxy-
D-sugars in addition to dTDP-L-mycarose. Moreover, VinC was
capable of catalyzing the unexpected â-glycoside and R-gly-
coside formations, respectively, from R-anomers and â-anomers
of dTDP-sugars. The reaction mechanism for axially oriented
glycoside formation from the equatorially oriented dTDP-sugar
might be explained using the process involving conformational
change to a boat or a twisted-boat conformation followed by a
nucleophilic attack by a hydroxy group. To apply this feature
of VinC, 22 glycosides were constructed using unnatural
glycosyl donors and acceptors. These results strongly suggest
the potential of VinC for construction of glycoside libraries.
Acknowledgment. Research Fellowships for Young Scientists
support to A.M. from JSPS is gratefully acknowledged. This
work was supported in part by the Naito Foundation.
Supporting Information Available: Experimental procedures
and preparation of NDP-sugars and vicenistatin analogues. This
material is available free of charge via the Internet at
http://pubs.acs.org.
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