Introducing N-glycans into natural products through a chemoenzymatic approach

Article abstract of DOI:10.1016/j.carres.2008.08.033

The present study describes an efficient chemoenzymatic method for introducing a core N-glycan of glycoprotein origin into various lipophilic natural products. It was found that the endo-β-N-acetylglucosaminidase from Arthrobactor protophormiae (Endo-A) had broad substrate specificity and can accommodate a wide range of glucose (Glc)- or N-acetylglucosamine (GlcNAc)-containing natural products as acceptors for transglycosylation, when an N-glycan oxazoline was used as a donor substrate. Using lithocholic acid as a model compound, we have shown that introduction of an N-glycan could be achieved by a two-step approach: chemical glycosylation to introduce a monosaccharide (Glc or GlcNAc) as a handle, and then Endo-A catalyzed transglycosylation to accomplish the site-specific N-glycan attachment. For those natural products that already carry terminal Glc or GlcNAc residues, direct enzymatic transglycosylation using sugar oxazoline as the donor substrate was achievable to introduce an N-glycan. It was also demonstrated that simultaneous double glycosylation could be fulfilled when the natural product contains two Glc residues. This chemoenzymatic method is concise, site-specific, and highly convergent. Because N-glycans of glycoprotein origin can serve as ligands for diverse lectins and cell-surface receptors, introduction of a defined N-glycan into biologically significant natural products may bestow novel properties onto these natural products for drug discovery and development.

Full text of DOI:10.1016/j.carres.2008.08.033

Carbohydrate Research 343 (2008) 2903–2913
Contents lists available at ScienceDirect
Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
Introducing N-glycans into natural products
through a chemoenzymatic approach
*
Wei Huang, Hirofumi Ochiai, Xinyu Zhang, Lai-Xi Wang
Institute of Human Virology and Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, 725 West Lombard Street, Baltimore, MD 21201, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 29 June 2008
Received in revised form 23 August 2008
Accepted 31 August 2008
Available online 9 September 2008
The present study describes an efficient chemoenzymatic method for introducing a core N-glycan of gly-
coprotein origin into various lipophilic natural products. It was found that the endo-b-N-acetylglucosa-
minidase from Arthrobactor protophormiae (Endo-A) had broad substrate specificity and can
accommodate a wide range of glucose (Glc)- or N-acetylglucosamine (GlcNAc)-containing natural prod-
ucts as acceptors for transglycosylation, when an N-glycan oxazoline was used as a donor substrate.
Using lithocholic acid as a model compound, we have shown that introduction of an N-glycan could be
achieved by a two-step approach: chemical glycosylation to introduce a monosaccharide (Glc or GlcNAc)
as a handle, and then Endo-A catalyzed transglycosylation to accomplish the site-specific N-glycan
attachment. For those natural products that already carry terminal Glc or GlcNAc residues, direct
enzymatic transglycosylation using sugar oxazoline as the donor substrate was achievable to introduce
an N-glycan. It was also demonstrated that simultaneous double glycosylation could be fulfilled when
the natural product contains two Glc residues. This chemoenzymatic method is concise, site-specific,
and highly convergent. Because N-glycans of glycoprotein origin can serve as ligands for diverse lectins
and cell-surface receptors, introduction of a defined N-glycan into biologically significant natural prod-
ucts may bestow novel properties onto these natural products for drug discovery and development.
Ó 2008 Elsevier Ltd. All rights reserved.
Dedicated to Professor Yongzheng Hui on
the occasion of his 70th birthday
Keywords:
Natural product
Glycosylation
Enzymatic transglycosylation
Endoglycosidase
Sugar oxazoline
1. Introduction
pharmacological properties. However, pure chemical synthesis of
complex sugar-containing natural products is still a difficult task,
Glycosylation constitutes an elegant strategy that nature takes
to expand the structural diversity and functions of biological
molecules. For example, attachment of sugars to lipophilic natural
products such as steroids and flavonoids represents a common
mechanism by which living organisms modulate the physico-
chemical properties and biological activities of these com-
pounds.1–4 On the other hand, it is well known that the aspara-
gine-linked (N-linked) glycans of glycoproteins can serve as
specific ligands for lectins and/or cell-surface receptors to
participate in many important biological processes such as cell
adhesion, host–pathogen interaction, development, and immune
response.5–9 As the sugars found in small-molecule natural
products are usually different from the conserved N-glycans of
glycoproteins, we reasoned that transforming the sugars in glycos-
ylated natural products with typical N-glycan of glycoprotein ori-
gin, or introducing N-glycan into non-glycosylated secondary
metabolites, would bestow novel properties onto these natural
products, for example, enhanced solubility, glycan-mediated cellu-
lar targeting, altered mechanism of action, and overall improved
as stepwise chemical glycosylation would involve tedious protect-
ing group manipulations and lengthy synthetic schemes. Direct
glycosylation of natural products by glycosyltransferases or glycos-
idases provides an attractive alternative that can greatly simplify
the synthetic scheme.10–14 We have previously reported an effi-
cient chemoenzymatic method for introducing N-glycans into Glc-
NAc-tagged polypeptides to make homogeneous glycopeptides and
glycoproteins.15–19 This method is based on the transglycosylation
activity of endo-b-N-acetylglucosaminidases and the use of syn-
thetic sugar oxaozlines as the activated donor substrates. Notably,
the Endo-A from Arthrobactor protophormiae20,21 and Endo-M from
Mucor hiemalis22–24 could accept a range of modified and truncated
oligosaccharide oxazolines as donor substrates for transglycosyl-
ation.15–19,25,26 In addition, by using a novel endoglycosynthase
EndoM-N175A, a full-size natural N-glycan can be efficiently
introduced into polypeptide to form a natural N-glycopeptide.27
In contrast to the glycosyltransferase approach that adds monosac-
charides one at a time, the unique advantage of this endoglycos-
idase-catalyzed transglycosylation is the single-step, site-specific
attachment of a pre-assembled oligosaccharide into the acceptor
in a stereospecific manner to form a natural glycosidic linkage,
without the need for any protecting groups.19 We describe in this
* Corresponding author. Tel.: +1 410 706 4982; fax: +1 410 706 5068.
E-mail address: LWang3@ihv.umaryland.edu (L.-X. Wang).
0008-6215/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2008.08.033

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W. Huang et al. / Carbohydrate Research 343 (2008) 2903–2913
paper an extension of this chemoenzymatic approach to the
synthesis of novel glycosylated small-molecule natural products
with defined N-glycans. We have found that Endo-A has a broad
substrate specificity and is able to accommodate different types
of glucose (Glc)- or N-acetylglucosamine (GlcNAc)-tagged natural
products as acceptor substrates for transglycosylation, making it
possible to introduce a defined N-glycan into natural products in
a highly convergent fashion.
The synthesis of the GlcNAc-containing lithocholic acid deriva-
tive (8) is summarized in Scheme 1. Lithocholic acid (1) was cou-
pled with phenylalanine tert-butyl ester (2) using DCC as the
coupling reagent to give compound 3 in 95% yield. The conjugate
3 was then glycosylated with 3,4,6-tri-O-acetyl-2-deoxy-2-phthl-
amido-b-D
-glucopyranosyl trichloroimidate (4)36 in the presence
of BF3ꢀEt2O to afford the b-glycoside 5 in 45% yield. Treatment of
5 with ethylenediamine to remove the phthaloyl group with simul-
taneous O-deacetylation, followed by selective N-acetylation of the
resulting amino group, gave the GlcNAc-containing compound 6 in
88% yield over two steps. Initial attempts to remove the tert-butyl
group by treatment of 6 with 20% trifluoroacetic acid (TFA) in
CH2Cl2 at rt gave a product, compound 7, in which the tert-butyl
group was selectively removed. But, surprisingly, 2D NMR analysis
of 7 revealed that the resulting glycoside was converted to the
2. Results and discussion
2.1. Introduction of a monosaccharide handle into natural
products
Previous studies have implicated that the endoglycosidases
Endo-A and Endo-M were able to transfer an N-glycan to some
monosaccharides, including Glc, GlcNAc, and their derivatives, to
form a new glycosidic linkage.28–30 An interesting application of
this enzymatic transglycosylation for synthesizing novel glycosyl-
ated cyclodextrin was recently reported.31 But enzymatic glycosyl-
a-isomer (H-1 of the GlcNAc, d 4.72 ppm, J1,2 = 3.6 Hz). Milder
conditions (5–10% TFA in CH2Cl2) resulted in incomplete removal
of the tert-butyl group, but yet still led to significant formation of
the
a-anomer. Clearly, a TFA-promoted isomerization of the
b-glycosidic bond was difficult to avoid in this case. Finally, a
successful deprotection of the tert-butyl group was achieved by
treatment of 6 with formic acid without anomeric isomerization,
giving the desired b-glycoside 8 in excellent yield (H-1 of the
GlcNAc in 8, d 4.32 ppm, J1,2 = 8.0 Hz) (Scheme 2).
To introduce a glucose moiety into the lithocholic acid, the lith-
ocholamide derivative 3 was glycosylated with the O-benzoylated
glucopyranosyl bromide 9 in the presence of AgOTf and 2,6-di-tert-
butylpyridine to afford compound 10, in which the newly formed
glycosidic bond was determined to be the desired b-linkage. After
de-O-benzoylation, the tert-butyl group in 10 was successfully
removed by treatment with 20% TFA in CH2Cl2 to give glycoconju-
gate 11 in 80% yield over two steps. In contrast to the GlcNAc-con-
taining compound 6, which resulted in the isomerization of the
glycosidic linkage upon treatment with 20% TFA, the b-glucoside
linkage in 10 could withstand 20% TFA treatment without isomeri-
zation. In comparison, it appears that the 2-acetamido group in 6
ation of
a
broad range of natural products using this
chemoenzymatic approach has not been investigated. For those
natural products that do not carry sugars, monosaccharide moie-
ties such as Glc and GlcNAc need to be introduced, which would
serve as a handle for the transglycosylation to introduce a defined
N-glycan. Either chemical glycosylation or enzymatic glycosyla-
tion11,14,32–34 could be applied for introducing monosaccharide
tags. Here we took lithocholic acid as a model natural product to
introduce a Glc and GlcNAc tag to its 3-hydroxyl group by chemical
glycosylation. Lithocholic acid is a bile acid derivative that has a
free hydroxyl group at the 3-position and a carboxyl group at C-
24. Its metabolic form includes amidation at the 24-carboxyl with
glycine or taurine and glycosylation at 3-hydroxyl with GlcNAc, Glc
or glucuronic acid.35 To facilitate its UV detection during transfor-
mations, we replaced the glycine moiety in the native conjugate by
a phenylalanine.
promotes the b- to
a
-glycosidic conversion under the action of TFA.
H
N
COOt-Bu
Ph
OH
a
O
H2N
HCl
COOt-Bu
Ph
O
+
H
H
H
H
HO
HO
H
H
1
2
3
OAc
O
H
N
H
N
COOt-Bu
Ph
COOtBu
Ph
AcO
AcO
O
CCl3
NH
NPhth
O
O
c,d
4
OH
O
OAc
O
H
H
H
H
AcO
AcO
HO
HO
O
b
O
H
H
NPhth
NHAc
5
6
f
e
H
N
H
COOH
Ph
N
COOH
Ph
OH
O
O
O
HO
HO
OH
H
H
H
H
NHAc
O
O
HO
HO
O
H
H
NHAc
8
7
Scheme 1. Synthesis of GlcNAc-bile acid phenylalanine conjugate 7. Reagents and conditions: (a) DCC, NHS, DIPEA, CH2Cl2, rt, overnight, 95%; (b) BF3ꢀEt2O, 4 Å MS, CH2Cl2,
ꢁ20 °C, 2 h, 45%; (c) ethylene diamine, n-BuOH, 80 °C, 3 h; (d) Ac2O, 0.1 M NaHCO3, rt, 1 h, 88% for 2 steps; (e) 20% TFA, CH2Cl2, rt, 2 h, 70%; (f) HCO2H, rt, 5 h, 90%.

W. Huang et al. / Carbohydrate Research 343 (2008) 2903–2913
2905
H
N
COOt-Bu
Ph
OBz
O
a
BzO
BzO
O
3
+
OBz
OBz
H
H
Br
O
BzO
BzO
O
H
9
OBz
10
H
N
COOH
Ph
O
b,c
OH
O
H
H
HO
HO
O
H
OH
11
Scheme 2. Synthesis of Glc-bile acid phenylalanine conjugate 11. Reagents and conditions: (a) 2,6-di-tert-butylpyridine, AgOTf, 4 Å MS, CH2Cl2, rt, overnight, 56%; (b) NaOMe,
MeOH, rt, 1 h; (c) 20% TFA in CH2Cl2, rt, 3 h, 80% for 2 steps.
2.2. Chemoenzymatic transglycosylation to GlcNAc- and
Glc-containing lithocholamides
(pH 7.0) at 23 °C, and the reaction was monitored by analytical
RP-HPLC. It was found that compound 8 was a good substrate for
Endo-A and, after 2 h under the above-mentioned conditions, the
transglycosylation product 13 was obtained in 75% yield (Scheme
3).
Previous studies on substrate specificity of Endo-A have shown
that some glucose-containing compounds such as p-nitrophenyl
glucoside, as well as free glucose and GlcNAc, could be acceptors
for Endo-A catalyzed transglycosylation.28–30 However, it was not
clear whether the Glc or GlcNAc in context of a steroid could effi-
ciently serve as an acceptor substrate for Endo-A. To examine the
feasibility of these Glc- and GlcNAc-containing compounds for
enzymatic transglycosylation, we have chosen the Man3GlcNAc
oxazoline (12) as donor substrate, as this tetrasaccharide oxazoline
was shown to be an excellent donor substrate for the Endo-A cat-
alyzed transglycosylation.15,16 Thus, the GlcNAc-containing litho-
cholic acid derivative (8) and the sugar oxaozline 12 (donor/
acceptor, 2:1) were incubated with Endo-A in a phosphate buffer
The transglycosylation product was characterized by ESI-MS
and NMR spectroscopic analysis. The observed data [m/z =
1416.70 for [M+H]+] was in good agreement with the calculated
molecular mass (M = 1415.68), indicating that the product is the
adduct of the tetrasaccharide oxazoline and the acceptor 8. On
the other hand, 1H NMR analysis revealed
a doublet at d
4.38 ppm with a relatively large coupling constant (J1,2 = 8.0 Hz)
assigned for the H-1 of the second GlcNAc, suggesting that
the transferred glycan was attached in the desired b-glycosidic
linkage. It should be noted that the signals for H-1 and H-10
were almost overlapping at d 4.38–4.39 ppm as doublets. The
H
OH
HO
HO
HO
N
COOH
Ph
O
O
O
OH
OH
O
HO
OH
O
O
H
H
O
O
O
O
HO
HO
HO
HO
HO
H
NHAc
NHAc
O
OH
13
Endo-A
phosphate buffer
8
75%
OH
HO
HO
HO
O
OH
OH
O
O
O
HO
O
O
HO
HO
HO
HO
O
N
O
OH
12
Endo-A
phosphate buffer
11
H
OH
HO
HO
HO
N
COOH
Ph
O
55%
O
O
OH
OH
OH
HO
O
H
H
O
O
O
O
O
O
HO
HO
HO
HO
HO
H
NHAc
OH
O
OH
14
Scheme 3. Transglycosylations on GlcNAc/Glc-bile acid phenylalanine conjugates by Endo-A.

2906
W. Huang et al. / Carbohydrate Research 343 (2008) 2903–2913
b-(1?4)-glycosidic linkage type of the newly formed glycosidic
bond was confirmed by the apparent NOE correlation between
H-10 and H-4 by the NOESY analysis of compound 13 (Fig. 1).
1H NMR and NOESY analysis (Fig. 2). A strong NOE between the
H-10 and H-4 protons clearly suggest that the GlcNAc and the Glc
moieties were linked through the expected b-(1?4)-glycosidic
bond. Comparison of the enzymatic transglycosylation yields with
compounds 7 and 11 indicates that the GlcNAc derivative was a
relatively better acceptor substrate for Endo-A than the Glc-deriv-
ative. These experiments substantiated a broad substrate specific-
ity of Endo-A, implicating its feasibility for glycosylating a wide
range of sugar-containing natural products.
Interestingly, it was found that the
a-linked GlcNAc derivative
(7) was also an acceptor substrate of Endo-A. Thus, incubation of
7 with oxazoline 12 and Endo-A gave the corresponding transgly-
cosylation product in 48% yield [ESI-MS: calculated molecular
mass, M = 1415.68; found, 1416.87 [M+H]+]. These experiments
suggest that the enzyme has a flexibility to accommodate both
the
a- and b-linked GlcNAc moiety for transglycosylation. Simi-
larly, incubation of the Glc-containing compound 11 and oxazoline
12 (donor/acceptor, 2:1) with Endo-A in a phosphate buffer (pH
7.0) for 2 h gave the transglycosylation product 14 in 55% yield
(Scheme 3). Again, the b-(1?4)-glycosidic linkage for the newly
formed glycosidic bond in compound 14 was confirmed by the
2.3. Chemoenzymatic transglycosylation of other natural
products containing a Glc moiety
Encouraged by the positive results from the enzymatic transgly-
cosylation with the synthetic GlcNAc- and Glc-containing steroids,
Figure 1. 1H NMR (A) and NOESY (B) of the transglycosylation product 13.

W. Huang et al. / Carbohydrate Research 343 (2008) 2903–2913
2907
Figure 2. 1H NMR (A) and NOESY (B) of the transglycosylation product 14.
we next examined the enzymatic reactions with an array of com-
mercially available natural products, which already contain a sugar
moiety in the structure. First, we tested a few flavonoid glycosides
(Scheme 4). Flavonoids occur widely in plants and have been impli-
cated as potential therapeutics.37–40 Daidzin (15),41 glycitin (17),42
and puerarin (19)43 are three flavonoid glucosides extracted from
Pueraria, which exhibit the activities on reduction of bone loss
and lipid metabolism abnormality.44 All the enzymatic reactions
were performed in a phosphate buffer at 23 °C on an analytical
scale using the tetrasaccharide oxazoline 12 as the donor substrate,
and a fixed molar ratio (2:1) of donor/acceptor was applied. The
reaction was monitored by reverse-phase HPLC for 2 h and the
transglycosylation product was isolated and was subjected to
ESI-MS analysis.
It was found that all the three flavonoid glucosides (15, 17, and
19) were substrates for the Endo-A-catalyzed transglycosylation,
giving the corresponding transglycosylation products 16, 18, and
20, respectively (Scheme 4). In comparison, daidzin 15 achieved
a higher yield of transglycosylation (82%) than glycitin (17) (56%)
and puerarin (19) (60%) under the same reaction conditions. Inter-
estingly, the only difference between 15 and 17 is the presence of
an extra methoxyl group at the 6-position of the benzene ring in
glycitin (17). A plausible explanation is that the 6-methoxy group
proximal to the 7-glucoside may exert partially steric hindrance for
enzyme recognition, thus making glycitin (17) a less active sub-
strate for transglycosylation. Puerarin (19) is actually a C-linked
glucoside. Yet it could also serve as a substrate for the Endo-A
catalyzed transglycosylation. The relatively low transglycosylation

2908
W. Huang et al. / Carbohydrate Research 343 (2008) 2903–2913
OH
HO
HO
HO
OH
OH
O
OH
OH
O
O
O
O
12
O
OH
OH
O
OH
O
HO
O
OH
O
Endo-A
phosphate buffer
82%
O
O
O
O
HO
HO
HO
HO
O
HO
HO
HO
NHAc
OH
O
OH
OH
15
16
OH
HO
HO
O
O
O
O
HO
O
O
12
MeO
OH
OH
O
MeO
HO
O
OH
OH
O
Endo-A
phosphate buffer
56%
O
O
O
HO
O
O
HO
HO
HO
HO
HO
HO
O
NHAc
OH
O
OH
OH
17
18
OH
HO
HO
HO
O
O
HO
OH
O
12
HO
OH
OH
O
O
HO
OH
O
HO
HO
O
O
Endo-A
phosphate buffer
60%
O
O
O
HO
O
OH
HO
HO
HO
HO
NHAc
O
OH
O
OH
OH
19
20
OH
Scheme 4. Transglycosylations on flavonoids natural products with a terminal glucose moiety.
yield of 19 in comparison with that of 15 might be again attributed
to the presence of a proximal hydroxyl group at the 7-position, but
not necessarily due to the nature of C-linked glycosidic bond.
C-linked GlcNAc and Glc-derivatives as acceptors for endoglycos-
idase-catalyzed transglycosylation were previously reported.21,30,45
These results further demonstrate the wide substrate tolerance of
Endo-A.
In addition to the flavonoid glucosides, we also tested three
other plant extracts: Geniposide (21)46 was extracted from Garde-
nia jaminoides, a Chinese herb; Rhaponticin (23)47 was extracted
from rhubarb; and Paeoniflorin (25)48 was extracted from Paeonia
lactiflora. All the three natural products have shown diverse biolog-
ical activities. It was found that these three natural product gluc-
osides were substrates for Endo-A, and yet a different reactivity
COOMe
H
COOMe
H
HO
HO
HO
OH
O
O
12
OH
O
O
OH
O
HO
HO
O
H
HO
H
HO
O
H
Endo-A
phosphate buffer
100%
O
O
O
H
HO
O
HO
HO
HO
HO
O
O
HO
HO
NHAc
O
OH
HO
OH
OH
21
22
OMe
OH
OMe
OH
HO
OH
O
HO
HO
12
OH
O
OH
O
O
OH
O
HO
OH
O
Endo-A
O
O
O
O
O
HO
phosphate buffer
HO
HO
HO
HO
HO
HO
NHAc
OH
O
OH
85%
OH
OH
O
OH
23
24
HO
HO
HO
OH
O
12
PhCOO
PhCOO
OH
O
O
O
O
OH
O
O
OH
O
OH
O
HO
Endo-A
O
O
O
O
O
HO
HO
HO
phosphate buffer
HO
HO
HO
HO
NHAc
HO
HO
OH
O
OH
OH
60%
26
25
Scheme 5. Transglycosylations on more natural products with glucose.

W. Huang et al. / Carbohydrate Research 343 (2008) 2903–2913
2909
for these structurally distinct compounds were observed (Scheme
3.5
3
2.5
2
1.5
1
0.5
0
1
a
b
c
d
5). Interestingly, while the geniposide (21) could quickly achieve
a quantitative conversion to the transglycosylation product 22,
rhaponticin 23 and paeoniflorin 25 gave a 85% and 60% yield of
the transglycosylation product 24 and 26, respectively, within 2 h
incubation with Endo-A. The relatively low transglycosylation
yield for 25 might be attributed to the steric hindrance of the
acceptor, whereas the reason for the unusual high reactivity of
21 is not clear. Probably the proximal primary hydroxyl group in
the aglycon portion may assist enzyme recognition and/or
facilitate the substrate–enzyme binding. Further testing of an
expanded library of compounds with diverse structures may
provide an answer to this interesting question.
-0.5
6
8
10
12
14
16
18
20
0.3
0.25
0.2
2
3
0.15
0.1
1
1
0.05
0
6
8
10
12
14
16
18
20
2.4. Double transglycosylation to luteolin 30,7-diglucoside
0.3
3
0.25
Luteolin 30,7-diglucoside (27) is a flavonoid extracted from
Reseda luteola that shows antioxidant activity.49,50 Compound 27
contains two b-glucoside moieties. Therefore, it would be inter-
esting to test its behavior in the Endo-A catalyzed transglycosyl-
ation. The Endo-A catalyzed reaction between 27 and oxazoline
12 was performed under the same conditions as described above
for the other natural product glycosides (Scheme 6). The enzy-
matic reaction was monitored by reverse-phase HPLC, and the
new products were isolated for ESI-MS analysis. It was observed
that when compound 27 was incubated with 2 mol equiv of the
oxazoline 12 in the presence of Endo-A for 2 h, the mono-trans-
glycosylated product (Fig. 3, peak 2) was formed as the major
product (about 60%) as validated by ESI-MS (data not shown),
together with the doubly transglycosylated product (Fig. 2, peak
3) (about 25%). However, when an excess of donor substrate
(12) (4 mol equiv in total) was supplemented to the reaction
and the mixture was incubated for 4 h, further conversion could
be achieved and the doubly transglycosylated product 28 was iso-
lated in 70% yield (Fig. 3, peak 3). The identity of the transglyco-
sylation product 28 was confirmed by its HR-MS: calculated,
[M+H]+ = 1989.6385; found, 1989.6369 [M+H]+. These results
suggest that the enzymatic transglycosylation may be equally
efficient for simultaneous transfer of multiple N-glycans to a
natural product when more than one monosaccharide tags, for
example, terminal glucose moieties, are present in the structure.
We assume that the transferred glycans were attached to the
glucose moieties in the expected b-(1?4)-glycosidic linkages, as
clearly demonstrated for the glycosylated lithocholic acid deriva-
tives 13 and 14. This assumption was also based on previous
studies, indicating that when a Glc or GlcNAc moiety was used
as an acceptor, the Endo-A demonstrated a strict stereospecificity
to form a new b-(1?4)-glycosidic linkage, which was determined
by detailed NMR analysis and enzymatic transformations of the
transglycosylation products.15,17,18,51
0.2
0.15
0.1
2
0.05
0
6
6
10
12
14
16
18
20
0.5
0.45
0.4
3
0.35
0.3
0.25
0.2
0.15
0.1
0.05
0
-0.05
6
8
10
12
14
16
18
20
Retention Time (min)
Figure 3. HPLC monitoring of Endo-A catalyzed transglycosylation with luteolin
30,7-di-O-glucoside (27). A solution of oxazoline 12 and luteolin glucoside 27
(donor/acceptor 2:1) in a phosphate buffer (50 mM, pH 7.0) was incubated with
Endo-A. The reaction progress was monitored by analytical HPLC: (a) 0 min; (b) 2 h
incubation; (c) another 2 h incubation with an addition of 2 mol equiv of donor
substrate; and (d) purified glycosylated product 28. Peak 1, compound 27; peak 2,
mono-transglycosylated product; peak 3, doubly transglycosylated product 28.
3. Conclusion
An efficient enzymatic transglycosylation for introducing a core
N-glycan into various lipophilic natural products was described.
The present study reveals a broad acceptor substrate specificity
of the endoglycosidase Endo-A, making it possible to attach an
N-glycan to a range of natural products in a site-specific and highly
convergent fashion. N-Glycans of glycoprotein origin can serve as
ligands for diverse lectins and cell-surface receptors. Thus, intro-
duction of a defined N-glycan into biologically significant natural
O
OH
OH
O
HO
HO
HO
HO
OH
O
OH
O
O
OH
O
O
OH
O
O
HO
O
O
O
O
HO
HO
OH
HO
12
HO
HO
HO
NHAc
OH
O
HO
HO
HO
OH
O
OH
O
O
HO
HO
O
Endo-A
OH
O
O
OH
O
phosphate buffer
OH
OH
OH
HO
HO
O
O
HO
O
O
O
70%
O
OH
HO
HO
HO
HO
HO
NHAc
OH
27
O
OH
28
Scheme 6. Transglycosylations on doubly substituted natural product 27.

2910
W. Huang et al. / Carbohydrate Research 343 (2008) 2903–2913
products may bestow novel properties onto these natural products,
for example, leading to enhanced solubility, specific cellular target-
ing, altered mechanism of action, and overall improvement of
pharmacological properties. Together with our previous results
that Endo-A could accommodate various selectively modified
sugar oxazolines as donor substrates, the broad acceptor substrate
specificity revealed by the present study should facilitate a quick
generation of an expanded glyco-randomized natural product
library valuable for drug discovery and development.
4.4. 3-(3,4,6-Tri-O-acetyl-2-deoxy-2-phthalimido-b-D-
glucopyranosyl)-N-(L-phenylalanine-tert-butyl ester)-
lithocholamide (5)
A solution of compound 3 (100 mg, 0.172 mmol) and 3,4,6-tri-
O-acetyl-2-deoxy-2-phthalimido-b- -glucopyranosyl trichloroace-
D
timidate 4 (200 mg, 0.345 mmol) in CH2Cl2 (5 ml) containing acti-
0
vated 4 ÅA molecular sieves (300 mg) was stirred under an
atmosphere of argon at room temperature for 0.5 h. After cooling
to ꢁ20 °C, BF3ꢀEt2O (10
lL, 80 lmol) was added and the resulting
mixture was stirred at ꢁ20 °C for 2 h. The mixture was filtered
through a Celite pad and the filtrate was poured into saturated
NaHCO3. Then the mixture was extracted with CH2Cl2. The organic
layer was washed with brine, dried over MgSO4, and filtered. The
filtrate was concentrated and the residue was subject to column
chromatography on silica gel using EtOAc–hexanes (1:2, v/v) as
eluent to provide 5 (77 mg, 45%) as a white solid. 1H NMR (CDCl3,
400 MHz): d 7.86 (m, 2H, Phthaloyl-ArH), 7.75 (m, 2H, Phthaloyl-
ArH), 7.28 (m, 3H, Ph-ArH), 7.14 (m, 2H, Ph-ArH), 5.85 (d, 1H,
J = 7.6 Hz, NH), 5.76 (t, 1H, J = 9.6 Hz, H-3 of Glc), 5.46 (d, 1H,
J = 9.6 Hz, H-1 of Glc), 5.14 (t, 1H, J = 9.6 Hz, H-4 of Glc), 4.75 (dd,
4. Experimental
4.1. Materials
Lithocholic acid (1), 2,3,4,6-tetra-O-benzoyl-a-D-glucopyran-
osyl bromide (9), daidzin (15), glycitin (17), peurarin (19), and
rhaponticin (23) were purchased from Sigma–Aldrich and used
as received. Geniposide (21) and paeoniflorin (25) were purchased
from AXXORA Life Sciences Inc. Luteolin-30,7-diglucoside (27) was
purchased from ChromaDex Corporate. The recombinant wild type
Endo-A was overproduced inEscherichia coli and purified by affinity
chromatography according to the literature.52 All other reagents
were purchased from Sigma–Aldrich and used as received.
1H, J = 6.0 Hz, 14.0 Hz, Phe-aH), 4.30 (m, 2H, H-2, H-6 of Glc),
4.15 (dd, 1H, J = 2.8 Hz, 11.6 Hz, H-6 of Glc), 3.84 (m, 1H, H-5 of
Glc), 3.53 (m, 1H, 3-H of lithocholamide), 3.07 (d, 2H, J = 6.0 Hz,
Phe-bH), 2.09 (s, 3H, Ac), 2.02 (s, 3H, Ac), 1.85 (s, 3H, Ac), 1.39 (s,
9H, t-Bu), 0.85 (d, 3H, J = 6.8 Hz, 21-CH3), 0.83 (s, 3H, 19-CH3),
0.56 (s, 3H, 18-CH3); 13C NMR (CDCl3, 100 MHz) d 173.0, 170.9,
170.8, 170.3, 169.6, 136.3, 134.3, 131.5, 129.6, 128.4, 127.0,
123.6, 97.1, 82.4, 80.5, 71.7, 71.0, 69.2, 62.4, 56.3, 56.0, 55.0,
53.3, 42.7, 42.2, 40.2, 40.0, 38.1, 35.8, 35.4, 35.1, 34.6, 34.2, 33.5,
31.7, 29.7, 28.2, 28.0, 27.2, 27.0, 26.5, 24.2, 23.3, 20.8, 20.7, 20.5,
18.4, 12.0; ESI-MS: calculated for C57H76N2O13, M = 996.53, found,
997.86 [M+H]+.
4.2. Analytical methods
Analytical RP-HPLC was performed on a Waters 626 HPLC
instrument with
TM
a
Symmetry300
C18 column (5.0 lm,
4.6 ꢂ 250 mm) at 40 °C. The column was eluted with a linear gra-
dient 0–60% MeCN containing 0.1% trifluoroacetic acid (TFA) for
20 min at a flow rate of 1 mL/min (Method A) or a linear gradient
0–90% MeCN containing 0.1% trifluoroacetic acid (TFA) for
10 min at a flow rate of 1 mL/min and then isocratic 90% MeCN
containing 0.1% TFA for 20 min (Method B). Preparative HPLC was
performed on a Waters 600 HPLC instrument with a preparative
C18 column (Symmetry300, 19 ꢂ 300 mm). The column was
eluted with a suitable gradient of water–acetonitrile containing
0.1% TFA. NMR spectra were measured with JEOL ECX 400 MHz.
The chemical shifts were assigned in ppm. ESI-MS Spectra were
measured on a micromass ZQ-4000 single quadruple mass spec-
trometer. High-resolution mass spectra (HR-MS) were measured
on a QSTAR/Pulsar (Applied Biosystems/MDS Sciex) ESI mass
spectrometer.
4.5. 3-(2-Acetamido-2-deoxy-b-D-glucopyranosyl)-N-
(L-phenylalanine-tert-butyl ester)-lithocholamide (6)
A solution of compound 5 (50 mg, 50 lmol) in n-BuOH (2 mL)
containing ethylenediamine (2 mL) was heated at 80 °C with stir-
ring for 3 h. The solution was concentrated and the residue was
subject to preparative HPLC to afford the product with free amino
and hydroxyl groups. The obtained intermediate was dissolved in
a mixed solution of MeOH (2 mL) and 0.1 M NaHCO3 (5 mL), to
which Ac2O (50
lL) was added. The mixture was stirred at rt
for 1 h. The solution was concentrated and the residue was sub-
4.3. N-(L-Phenylalanine-tert-butyl ester)-lithocholamide (3)
ject to preparative HPLC to give the N-acetyl derivative
6
(34.5 mg, 88% for 2 steps) as a white solid. 1H NMR (DMSO-
d6 + 5% D2O, 400 MHz): d 7.22–7.12 (m, 5H, Ph-ArH), 4.32 (d,
To a solution of lithocholic acid (1) (376 mg, 1.0 mmol) and
phenylalanine tert-butyl ester hydrochloride (2) (300 mg,
1.2 mmol) in CH2Cl2 (10 mL) were added N,N0-dicyclohexylcarbodi-
imide (DCC) (340 mg, 1.65 mmol), N-hydroxysuccimide (NHS)
(160 mg, 1.39 mmol), and N,N-diisopropylethylamine (DIPEA)
(0.5 mL, 2.87 mmol). The mixture was stirred at rt overnight. The
precipitate was filtered out and the filtrate was concentrated under
vacuum. The residue was subject to column chromatography on
silica gel using EtOAc–hexanes (1:2, v/v) as eluent to give the cou-
pling product 3 (550 mg, 95%) as a white powder. 1H NMR (CDCl3,
400 MHz): d 7.30–7.12 (m, 5H, Ph-ArH), 6.00 (d, 1H, J = 8.0 Hz, NH),
1H, J = 8.8 Hz, H-1 of GlcNAc), 4.25 (m, 1H, Phe-aH), 3.62 (m,
1H, H-6, of GlcNAc), 3.38 (m, 2H, 3-H of lithocholamide, H-6 of
GlcNAc), 3.25 (m, 2H, H-3, H-4 of GlcNAc), 2.99 (m, 2H, H-5 of
GlcNAc, Phe-bH), 2.87 (m, 1H, H-2 of GlcNAc), 2.78 (m, 1H, Phe-
bH), 1.75 (s, 3H, Ac), 1.24 (s, 9H, t-Bu), 0.80 (s, 3H, 19-CH3),
0.78 (d, 3H, J = 8.0 Hz, 21-CH3), 0.51 (s, 3H, 18-CH3); 13C NMR
(DMSO-d6 + 5% D2O, 100 MHz)
d 173.5, 173.3, 170.4, 138.0,
129.6, 128.4, 126.8, 95.7, 77.6, 73.0, 71.0, 70.9, 61.2, 56.6, 56.2,
56.0, 54.2, 53.7, 42.8, 41.6, 37.1, 35.8, 35.5, 35.3, 34.8, 32.6,
32.5, 32.4, 32.0, 28.5, 28.2, 28.0, 27.2, 26.6, 24.3, 23.6, 23.0,
20.9, 18.7, 18.5, 12.3; ESI-MS: calculated for C45H70N2O9,
M = 782.51, found, 783.78 [M+H]+.
4.74 (dd, 1H, J = 6.4 Hz, 13.6 Hz, Phe-aH), 3.62 (m, 1 H, 3-H of
lithocholamide), 3.06 (d, 2H, J = 6.8 Hz, Phe-bH), 1.39 (s, 9H,
t-Bu), 0.89 (s, 3H, 19-CH3), 0.87 (d, 3H, J = 6.8 Hz, 21-CH3), 0.61
(s, 3H, 18-CH3); 13C NMR (CDCl3, 100 MHz) d 173.4, 170.9, 136.2,
129.6, 128.4, 127.0, 82.4, 71.9, 56.5, 56.0, 53.4, 42.8, 42.1, 40.5,
40.2, 38.1, 36.4, 35.9, 35.5, 35.4, 34.6, 33.5, 31.7, 30.5, 28.2, 28.0,
27.2, 26.5, 24.2, 23.4, 20.9, 18.4, 12.1; ESI-MS: calculated for
C37H57NO4, M = 579.43, found, 580.56 [M+H]+.
4.6. 3-(2-Acetamido-2-deoxy–D-glucopyranosyl)-
N-( -phenylalanine)-lithocholamide (7)
L
A solution of compound 6 (20 mg, 25
lmol) in 20% TFA in
CH2Cl2 (3 mL) was stirred at rt for 2 h. The solution was concen-

W. Huang et al. / Carbohydrate Research 343 (2008) 2903–2913
2911
trated and the residue was subject to preparative HPLC to afford 7
MS: calculated for C71H83NO13
,
M = 1157.59, found, 1158.81
(13 mg, 70%) as a white solid. 1H NMR (DMSO-d6 + 5% D2O,
400 MHz): d 7.23–7.12 (m, 5H, Ph-ArH), 4.72 (d, 1H, J = 3.6 Hz,
[M+H]+.
H-1 of
a-GlcNAc), 4.33 (m, 1H, Phe-aH), 3.55 (m, 2H, H-2, H-6 of
4.9. 3-b-D-Glucopyranosyl-N-(L-phenylalanine)-lithocholamide
GlcNAc), 3.40 (m, 4H, 3-H of lithocholamide, H-3, H-5, H-6 of Glc-
NAc), 3.08 (t, 1H, J = 8.0 Hz, H-4 of GlcNAc), 3.00 (dd, 1H, J = 2.8 Hz,
13.6 Hz, Phe-bH), 2.78 (dd, 1H, J = 8.8 Hz, 13.6 Hz, Phe-bH), 1.80 (s,
3H, Ac), 0.83 (s, 3H, 19-CH3), 0.78 (d, 3H, J = 7.2 Hz, 21-CH3), 0.58
(s, 3H, 18-CH3); 13C NMR (DMSO-d6 + 5% D2O, 100 MHz) d 173.7,
173.5, 170.4, 138.1, 129.6, 128.6, 126.8, 96.3, 77.6, 73.1, 71.2,
70.9, 61.2, 56.6, 56.2, 56.0, 54.2, 53.7, 42.8, 41.8, 37.1, 35.8, 35.5,
35.3, 34.8, 32.6, 32.5, 32.3, 32.0, 28.5, 28.1, 27.2, 26.6, 24.3, 23.6,
23.2, 20.9, 18.7, 18.4, 12.3; ESI-MS: calculated for C41H62N2O9,
M = 726.45, found, 727.78 [M+H]+.
(11)
To a solution of compound 10 (100 mg, 86
(5 mL) was added a solution of NaOMe in MeOH (0.5 M, 0.1 mL,
lmol) in MeOH
50 lmol). The solution was stirred at rt for 1 h. The base was neu-
tralized by Dowex 50 W-X8 (H+ form), and the solution was filtered
and the filtrate was concentrated. The residue was purified by pre-
parative HPLC to give N-(
mide-3-b- -glucoside. The obtained intermediate was treated with
20% TFA in CH2Cl2 at rt for 3 h. The solution was then concentrated
and the residue was subject to preparative HPLC to provide 11
(47 mg, 80% for 2 steps) as a white solid. 1H NMR (DMSO-d6 + 5%
D2O, 400 MHz): d 7.16 (m, 5H, Ph-ArH), 4.32 (q, 1H, J = 4.8 Hz,
L-phenylalanine t-butyl ester)-lithochola-
D
4.7. 3-(2-Acetamido-2-deoxy-b-
D-glucopyranosyl)-
N-( -phenylalanine)-lithocholamide (8)
L
Phe-aH), 4.18 (d, 1H, J = 7.6 Hz, H-1 of Glc), 3.57 (m, 2H, H-6 of
A solution of compound 6 (34.5 mg, 44
l
mol) in formic acid
Glc, 3-H of lithocholamide), 3.38 (dd, 1H, J = 5.6 Hz, 12.0 Hz, H-6
of Glc), 3.10 (t, 1H, J = 8.4 Hz, H-3 of Glc), 2.99 (m, 3H, H-5, H-4
of Glc, Phe-bH), 2.87 (t, 1H, J = 8.4 Hz, H-2 of Glc), 2.77 (dd, 1H,
J = 9.6 Hz, 13.6 Hz, Phe-bH), 0.80 (s, 3H, 19-CH3), 0.76 (d, 3H,
J = 6.4 Hz, 21-CH3), 0.50 (s, 3H, 18-CH3); 13C NMR (DMSO-d6 + 5%
D2O, 100 MHz) d 173.9, 173.6, 138.0, 129.5, 128.7, 127.0, 100.7,
77.5, 76.7, 73.7, 70.3, 61.4, 56.5, 55.9, 55.8, 53.8, 42.7, 42.3, 41.8,
40.4, 37.0, 35.7, 35.1, 34.6, 34.2, 32.5, 31.9, 28.1, 27.1, 26.7, 26.5,
24.2, 23.5, 20.8, 18.7, 12.2; ESI-MS: calculated for C39H59NO9,
M = 685.42, found, 686.74 [M+H]+.
(5 mL) was stirred at rt for 5 h. The solution was then added to a
cold NaHCO3 (1 M) solution and the solution was stirred at rt for
3 h. The mixture was concentrated and the residue was subject
to preparative HPLC to afford 8 (12 mg, 90% based on a small recov-
ery of starting material. 1H NMR (DMSO-d6 + 5% D2O, 400 MHz): d
7.22–7.10 (m, 5H, Ph-ArH), 4.32 (d, 1H, J = 8.0 Hz, H-1 of GlcNAc),
4.23 (m, 1H, Phe-aH), 3.63 (m, 1H, H-6 of GlcNAc), 3.40 (m, 2H,
3-H of lithocholamide, H-6 of GlcNAc), 3.25 (m, 2H, H-3, H-4 of Glc-
NAc), 3.00 (m, 3H, H-5, H-2 of GlcNAc, Phe-bH), 2.78 (m, 1H, Phe-
bH), 1.75 (s, 3H, Ac), 0.81 (s, 3H, 19-CH3), 0.74 (d, 3H, J = 6.8 Hz, 21-
CH3), 0.52 (s, 3H, 18-CH3); 13C NMR (DMSO-d6 + 5% D2O, 100 MHz)
d 173.7, 173.4, 170.5, 138.1, 129.6, 128.6, 126.8, 95.9, 77.6, 73.1,
71.1, 70.9, 61.2, 56.6, 56.2, 56.0, 54.2, 53.7, 42.8, 41.7, 37.1, 35.8,
35.5, 35.3, 34.8, 32.6, 32.5, 32.4, 32.0, 28.5, 28.1, 27.2, 26.6, 24.3,
23.6, 23.0, 20.9, 18.7, 18.5, 12.3; ESI-MS: calculated for
C41H62N2O9, M = 726.45, found, 727.70 [M+H]+.
4.10. Transglycosylation with GlcNAc-containing
lithocholic acid derivative (8). Preparation of glycosylated
lithocholic acid derivative (13) carrying a core
N-pentasaccharide
A solution of compound 8 (1 mg, 1.37
l
mol) and oxazoline 12
L)
(2 mg, 2.9 mol) in a phosphate buffer (50 mM, pH 7.0, 150
l
l
4.8. 3-(2,3,4,6-Tetra-O-benzoyl-b-
D
-glucopyranosyl)-N-
containing 20% DMSO was incubated with Endo-A (60 mU) at
23 °C. The reaction was monitored by analytic HPLC (method B).
After 2 h, the reaction solution was directly subject to preparative
HPLC to afford 13 (1.46 mg, 75%). 1H NMR (DMSO-d6 + 5% D2O,
(L
-phenylalanine-tert-butyl ester)-lithocholamide (10)
A solution of compound 3 (100 mg, 0.172 mmol), 2,3,4,6-tetra-
O-benzoyl- -glucopyranosyl bromide 9 (235 mg, 0.346 mmol),
and 2,6-di-t-butylpyridine (120 L, 0.544 mmol) in CH2Cl2
a-
D
400 MHz): d 7.20–7.11 (m, 5H, Ph-ArH), 4.81 (s, 1H, H-1 of
0
l
Man4), 4.61 (s, 1H, H-1 of Man4 ), 4.47 (m, 1H, H-1 of Man3), 4.39
0
(5 mL) containing activated 4 ÅA molecular sieves (300 mg) was
stirred under an atmosphere of argon at rt for 0.5 h. Then, AgOTf
(80 mg, 0.311 mmol) was added and the resulting mixture was
stirred at rt overnight. The mixture was filtered through a Celite
pad and the filtrate was poured into a saturated NaHCO3 solution.
The solution was extracted with CH2Cl2. The organic layer was
washed with brine, dried over MgSO4, and filtered. The filtrate
was concentrated and the residue was subject to column chroma-
tography on silica gel using EtOAc–hexanes (1:2, v/v) as eluent to
provide 10 (112 mg, 56%) as a white solid. 1H NMR (CDCl3,
400 MHz): d 8.01–7.82 (m, 8H, Bz-ArH), 7.56–7.22 (m, 15H, Bz-
ArH, Ph-ArH), 7.14 (m, 2H, Ph-ArH), 5.89 (t, 1H, J = 9.6 Hz), 5.60
(t, 1H, J = 9.6 Hz), 5.48 (t, 1H, J = 9.6 Hz), 4.92 (d, 1H, J = 8.0 Hz,
(d, 1H, J = 8.0 Hz, H-1 of GlcNAc1), 4.38 (d, 1 H, J = 8.0 Hz, H-1 of
GlcNAc2), 4.27 (m, 1H, Phe-
aH), 2.98 (dd, 1H, J = 4.8 Hz, 13.6 Hz,
Phe-bH), 2.76 (dd, 1H, J = 7.2 Hz, 13.6 Hz, Phe-bH), 1.81 (s, 3H,
Ac), 1.74 (s, 3H, Ac), 0.79 (s, 3H, 19-CH3), 0.75 (d, 3H, J = 8.8 Hz,
21-CH3), 0.50 (s, 3H, 18-CH3); HR-MS (ESI): calculated for
C67H106N3O29, [M+H]+ = 1416.6912; found, 1416.7057; Analytical
RP-HPLC (method B), tR = 11.86 min.
4.11. Transglycosylation with the Glc-containing
lithocholic acid derivative (11). Preparation of glycosylated
lithocholic acid derivative (14) carrying a novel Man3GlcNAcGlc
glycan
H-1 of Glc), 4.75 (dd, 1H, J = 6.0 Hz, 14.0 Hz, Phe-
aH), 4.59 (dd,
A solution of compound 11 (1 mg, 1.46
lmol) and oxazoline 12
1 H, J = 3.2 Hz, 12.4 Hz, H-6 of Glc), 4.51 (dd, 1H, J = 6.4 Hz,
12.4 Hz, H-6 of Glc), 4.15 (m, 1H, H-5 of Glc), 3.61 (m, 1H, 3-H
of lithocholamide), 3.08 (d, 2 H, J = 6.8 Hz, Phe-bH), 1.40 (s, 9H,
t-Bu), 0.85 (d, 3H, J = 6.8 Hz, 21-CH3), 0.83 (s, 3H, 19-CH3), 0.57
(s, 3H, 18-CH3); 13C NMR (CDCl3, 100 MHz) d 173.1, 170.9,
166.2, 165.9, 165.4, 165.2, 136.3, 133.5–133.1 (m), 130.2–129.6
(m), 129.0, 128.9, 128.5–128.3 (m), 127.0, 100.4, 82.4, 81.3,
73.1, 72.3, 72.2, 70.2, 63.5, 56.2, 55.9, 55.0, 53.3, 42.7, 42.3,
40.6, 40.2, 38.1, 35.8, 35.4, 35.2, 34.6, 34.3, 33.5, 31.7, 29.7,
28.2, 28.0, 27.2, 27.1, 26.4, 26.3, 24.2, 23.3, 20.9, 18.4, 12.0; ESI-
(2 mg, 2.9 mol) in a phosphate buffer (50 mM, pH 7.0, 150 l
l
L)
containing 20% DMSO was incubated with Endo-A (60 mU) at
23 °C. After 2 h, the reaction mixture was subject directly to pre-
parative HPLC to afford 14 (1.1 mg, 55%). 1H NMR (DMSO-d6 + 5%
D2O, 400 MHz): d 7.18–7.11 (m, 5H, Ph-ArH), 4.80 (s, 1H, H-1 of
0
Man4), 4.60 (s, 1H, H-1 of Man4 ), 4.47 (m, 1H, H-1 of Man3), 4.35
(d, 1H, J = 8.4 Hz, H-1 of GlcNAc1), 4.24 (d, 1H, J = 8.8 Hz, H-1 of
Glc2), 4.22 (m, 1H, Phe-
aH), 2.99 (dd, 1H, J = 4.8 Hz, 13.6 Hz, Phe
-bH), 2.76 (dd, 1H, J = 8.8 Hz, 13.6 Hz, Phe-bH), 1.81 (s, 3H, Ac),
0.80 (s, 3H, 19-CH3), 0.75 (d, 3H, J = 7.2 Hz, 21-CH3), 0.50 (s, 3H,

2912
W. Huang et al. / Carbohydrate Research 343 (2008) 2903–2913
18-CH3); HR-MS (ESI): calculated for C65H102N2O29Na,
[M+Na]+ = 1397.6466; found, 1397.6548; Analytical RP-HPLC:
method B, tR = 11.87 min.
were measured in the Johns Hopkins University School of Medi-
cine’s mass spectrometry/proteomics facility. This work was sup-
ported by the National Institutes of Health (NIH Grants
GM073717 and GM080374).
4.12. Transglycosylation with natural products 15, 17, 19, 21,
23, and 25. A general procedure
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lmol) and the
l
l
4.12.1. Daidzin-GlcNAcMan3 (16)
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4.13. Double transglycosylation with the luteolin 30,7-di-O-
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