Efficient Synthesis of Antigenic Trisaccharides Containing N-Acetylglucosamine: Protection of NHAc as NAc2

Article abstract of DOI:10.1002/ejoc.201901809

The antigenic trisaccharides, α-gal epitope and H antigen, containing N-acetyl-d-glucosamine (GlcNAc) were synthesized using a diacetyl strategy, in which NHAc is tentatively converted to NAc₂ during oligosaccharide construction. Acetylation of

Full text of DOI:10.1002/ejoc.201901809

A Journal of
Accepted Article
Title: Efficient synthesis of antigenic trisaccharides containing N-
acetylglucosamine: protection of NHAc as NAc2
Authors: Masato Tsutsui, Julinton Sianturi, Seiji Masui, Kento
Tokunaga, Yoshiyuki Manabe, and Koichi Fukase
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To be cited as: Eur. J. Org. Chem. 10.1002/ejoc.201901809
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10.1002/ejoc.201901809
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Efficient synthesis of antigenic trisaccharides containing N-
acetylglucosamine: protection of NHAc as NAc2
Masato Tsutsui,†[a] Julinton Sianturi, †§[a] Seiji Masui,[a] Kento Tokunaga,[a] Yoshiyuki Manabe,*[a, b] and
Koichi Fukase*[a, b] (†equal contribution)
Abstract: The antigenic trisaccharides, -gal epitope and H antigen,
containing N-acetyl-D-glucosamine (GlcNAc) were synthesized using
a diacetyl strategy, in which NHAc is tentatively converted to NAc2
during oligosaccharide construction. Acetylation of NHAc in GlcNAc
significantly improved the reactivity in glycosylation reactions. The
diacetyl strategy allowed to achieve the efficient synthesis of -gal
via a sequential one-pot and one-flow procedure. Meanwhile, H
antigen was synthesized by stepwise elongation from the reducing
end side of GlcNAc. The enhancement of the reactivity by NAc2
protection was observed in both glycosylations at proximal and distal
positions to GlcNAc. This diacetyl strategy is expected to be
applicable to the synthesis of a wide range of glycans.
gaster, intestine, and pancreas.[8] Meanwhile, -gal (Figure 1) is
another important antigenic glycan that is abundantly expressed
in nonprimate mammals, prosimians, and New World monkeys.
In contrast, humans and Old World monkeys do not have -gal
due to mutation of -1,3-galactosyltransferase (1,3GT) during
evolution.[9] Instead, humans produce a large amount of anti-Gal
antibodies, which specifically interact with -gal.[10] Thus, -gal
induces an acute immune response, i.e., hyperacute rejection
caused by xenotransplantation from pig to baboon.[11] These
findings prompted the use of -gal as an adjuvant in vaccine
therapy[12] or immune therapy using antibody-recruiting
molecules.[13] Herein, we describe a new approach for the
efficient synthesis of H antigen and -gal.
Introduction
Glycans are involved in various biological phenomena including
self and nonself recognition, viral and bacterial infection,
immunoregulation, cancer invasion, and cell development.[1]
Bacterial glycoconjugates such as lipopolysaccharide and
peptidoglycan are known to activate innate immunity.[2] More
than 60% of proteins are posttranslationally modified with
glycans, which control protein folding and quality,[3] in vivo
biological activity,[4] circulatory residence,[5] and receptor
function[6]. Since natural glycans have high diversity and
heterogeneity, the production of pure glycans by chemical
synthesis has been a powerful tool for the elucidation of the
biological function of glycans.
Many glycans act as antigens, among which ABO blood group
antigens stand out. ABO blood types are classified according to
the glycan structure on erythrocytes. O blood type individuals
express H antigen (Figure 1), which is also a precursor of ABO
blood antigens. The interaction between ABO blood antigens
and their natural antibodies causes blood agglutination.[7] They
are also closely related to various diseases, since these glycans
are widely expressed on various organs including epithelium,
Figure 1. Structure of H antigen and -gal.
Amino sugars, including N-acetylglucosamine (GlcNAc) and
sialic acid (Neu5Ac), are contained in many glycans such as H
antigen and -gal. When synthesizing glycan containing amino
sugars, the selection of the protecting group on amines is
important because it affects the glycosylation reactivity (Figure
2a).
Various
protecting
groups
including
2,2,2-
trichloroethoxycarbonyl,[14] allyloxycarbonyl,[15] trichloroacetyl,[16]
trifluoroacetyl,[17] and phthaloyl[18] have been reported for the
protection of amino sugars. The azide group is also a useful
precursor of the amine group.[19] On the other hand, since most
amino groups are acetylated in natural glycans, protected amino
groups have to be converted to acetamide (NHAc) by an
acetylation reaction. These steps can be omitted when glycans
bearing the NHAc group are directly used for the construction of
glycan chains. However, low reactivity of fragments having
NHAc in glycosylations has been reported,[20] with the exception
of the work by Tamura that describes that the formation of
interglycosidic O-imidates with NHAc in GalNAc increases the
glycosylation yields.[21] Crich et al. reported that intermolecular
hydrogen bonding formed by NHAc in GlcNAc acceptor
decreases the reactivity, which can be increased by converting
NHAc to NAc2.[20b] Auzanneau et al. observed by NMR
spectroscopy the presence of aggregation of a tetrasaccharide
through the formation of hydrogen bonds involving the NHAc
group in GlcNAc.[20f] Boons et al. reported that a NAc2 sialyl
donor showed significantly higher reactivity than the NHAc
donor.[20a] Moreover, the presence of NHAc hydrogen bonds was
detected in a Neu5Ac donor by Kononov et al. More recently,
the reactivity has been improved by protection of NHAc as NAc2
or addition of external amides/imides to cleave the mentioned
hydrogen bonds.[20c-e] We previously demonstrated that NAc2
[a]
Mr. M. Tsutsui, Dr. J. Sianturi, Dr. S. Masui, Mr. K. Tokunaga, Dr. Y.
Manabe, Prof. K. Fukase
Department of Chemistry, Graduate School of Science
Osaka University
Machikaneyama 1-1, Toyonaka
Osaka 560-0043 (Japan)
E-mail: koichi@chem.sci.osaka-u.ac.jp
manabey12@chem.sci.osaka-u.ac.jp
§
Present address : Max Planck Institute of Colloids and Interfaces,
Am Mühlenberg 1, 14424 Potsdam,Germany
Dr. Y. Manabe, Prof. K. Fukase
[b]
Core for Medicine and Science Collaborative Research and
Education, Project Research Center for Fundamental Science
Osaka University
Supporting information for this article is given via a link at the end of
the document.((Please delete this text if not appropriate))
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10.1002/ejoc.201901809
European Journal of Organic Chemistry
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protection of Neu5Ac can greatly improve the reactivity in
glycosylation at a position away from the Neu5Ac residue in the
glycosylation of glucosamine 1 or 2 with galactose 6 and fucose
7 (Figure 3). NAc2 protection enhanced the reactivity in both
glycosylation reactions at proximal and distal positions to
GlcNAc.
synthesis of
a
Neu5Ac-containing N-glycan and
a
tetrasaccharide having two Neu5Ac.[20g, 20h] On the basis of these
reports, we envisioned that a “diacetyl strategy,” in which the
NHAc group is tentatively protected as NAc2 during sugar
elongation to enhance the reactivity in glycosylation (Figure 2b).
Subsequent conversion of NAc2 to NHAc was readily
accomplished by simple removal of one Ac group under mild
basic conditions. This strategy avoids handling of polar and
reactive amine intermediates. Herein, we applied this strategy to
the synthesis of the GlcNAc-containing glycans -gal and H
antigen. Since GlcNAc is the most abundant amino sugar, this
study can greatly expand the applicability of diacetyl strategy.
Figure 3. Synthesis of -gal 5 and H antigen 8 using the diacetyl strategy.
Results and Discussion
We first synthesized glucosaminyl acceptors 1 having NHAc and
2 having NAc2 (Scheme 1). Synthesis was started from 9.[24]
Ozonolysis of the allyl group in 9 followed by Pinnick oxidation
and methyl esterification afforded 10 in 55% yield. Then,
selective cleavage of the benzylidene group gave 1 in 76% yield.
Meanwhile, NAc2 protection of 10 using AcCl proceeded in
excellent yield (95%). Obtained 11 was then converted to
glucosaminyl acceptor 2.
Figure 2. (a) Reactivity in glycosylations and conversion process to NHAc for
each amino sugar structure and (b) diacetyl strategy.
The outline of the synthesis for -gal 5 and H antigen 8 is shown
in Figure 3. The synthesis of -gal has been previously reported
by several groups including us.[13g, 22] In this study, -gal was
obtained via glycosylation between
3 and 4 followed by
glycosylation of 1 or 2 (Figure 3). In our previous -gal synthesis,
the low reactivity of 1 caused moderate yield in the 2nd
glycosylation. In the present study, the yield of the glycosylation
was improved by the diacetyl strategy. To demonstrate the
effectiveness of this approach, we investigated a one-pot and
one-flow glycosylation, in which the enhancement of the
reactivity by diacetylation was essential for the efficient
construction of -gal 5.
Scheme 1. Syntheses of acceptors 1 and 2.
The diacetyl strategy was also applied to the synthesis of H
antigen 8. After the first synthesis of B blood type antigen by
Lemieux et al., many groups have synthesized ABO blood
glycans.[23] In this study, we synthesized
H antigen by
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As described above, formation of intermolecular hydrogen bonds
between acceptors is known to decrease their reactivity.[20b] We
confirmed the presence of a hydrogen bond involving NHAc in 1
by NMR measurements (Figure 4). When the 1H-NMR spectrum
of 1 was measured in CD2Cl2 at the concentration of 1, 10, and
50 mM at 25 °C and −40 °C, respectively, the peak of the amide
proton was downfield shifted upon increasing the concentration
at each temperature (Figure 4a-(i–iii) and (iv–vi)). This downfield
shift was also observed as the temperature decreased. These
results suggested the existence of intermolecular hydrogen
bonding. Vasella et al. and Crich et al. previously reported
hydrogen bonding of GlcNAc derivatives. They observed
concentration and temperature dependent chemical shift of
amide proton of GlcNAc derivatives and concluded that GlcNAc
derivatives are likely to take dynamic situation, incorporating
both inter- and intramolecular hydrogen bonding.[20b, 25]
We also measured the diffusion-ordered two-dimensional
nuclear magnetic resonance spectra (DOSY)[26] of 1 and 2 to
gain further information on the hydrogen bonding formed by 1
(Figure 4b). In the DOSY spectrum, the NMR signals are
separated according to the self-diffusion coefficient of the
molecules. The DOSY spectra of 1 (red, Figure 4b) and 2 (black,
Figure 4b) were measured using 50 mM solutions in CD2Cl2 at
−40 °C. The effective volumes (V) of 1 and 2 were estimated by
Stokes–Einstein equation[27] using the self-diffusion coefficient
obtained from the DOSY spectra as 3.98×10−27 m3 (V1) and
3.31×10−27 m3 (V2), respectively. V1 was 1.2 times larger than V2,
indicating that 1 formed a supramolecular structure through
intermolecular hydrogen bonding, whereas 2 did not aggregate.
This difference between V1 and V2 was small but significant,
considering that the formation of a trimeric cluster of N-
methylacetamide was predicted under neat conditions by X-ray
and neutron scattering with density-functional theory
calculations.[28] Our results suggest that the hydrogen-bonding
network formed by acetamide 1 is dynamically rearranged at
NMR time scale (~10−4 sec). This is the first observation of the
supramolecular structure construction of GlcNAc by DOSY NMR
spectroscopy.
For the efficient synthesis of -gal, we investigated sequential
one-pot and one-flow glycosylations,[29] using thioglycosides as
glycosyl donors because they have sufficient stability upon
storage and can be smoothly activated during glycosylation to
realize high productivity.
One-pot glycosylation reactions can be divided into three types:
1) reactivity-based one-pot glycosylation,[30] 2) orthogonal
leaving group-based one-pot glycosylation,[31] and 3) pre-
activation-based one-pot glycosylation.[32] Here, we investigated
the reactivity-based one-pot glycosylation by applying the so-
called armed–disarmed strategy, using
a reactive (armed)
Figure 4. (a) Concentration and temperature dependence of the 1H-NMR
spectrum of acetamide acceptor 1, where downfield shifts of the amide proton
are observed upon increasing the concentration and decreasing the
temperature. (b) DOSY spectrum of N-acetylated 1 (red) and N,N-diacetylated
2 (black). The effective volume (V) of 1 and 2 estimated by Stokes–Einstein
equation was 3.98×10−27 m3 (V1) and 3.31×10−27 m3 (V2), respectively.
thioglycoside protected with electron-donating groups and a less
reactive (disarmed) thioglycoside protected with electron-
withdrawing groups.
We started the investigation on the one-pot glycosylation
procedure by evaluating the condition for each glycosylation.
The glycosylation of armed thioglycoside 3 (Scheme S1) with
disarmed thioglycoside 4[13h] was carried out by using NIS and
TfOH at −20 °C to give 12 in 80% with perfect -selectivity
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(Table 1, entry 1). However, when scaling up, the yield
decreased to 57% mainly due to overreaction of 12.
excellent yield (91%; Table 2, entry 2). These results indicate
that the formation of hydrogen bonding by NHAc in 1 decreases
its reactivity and demonstrate the effectiveness of the diacetyl
strategy in this glycosylation.
One-pot and one-flow glycosylations were then investigated
(Table 3). When the N-acetylated glucosaminyl acceptor 1 was
used for one-pot glycosylation, the yield of 13 was only 25% due
to the low reactivity of 1 in the 2nd glycosylation step (Table 3,
entry 1). The reactivity was dramatically enhanced by NAc2
protection: the 2nd glycosylation using 2 was completed in 5 min
at −40 ºC to give 14 in 86% (Table 3, entry 2). In the one-pot
glycosylation using the armed–disarmed strategy, the low
reactivity in the 2nd glycosylation is inevitable because the
disarmed donor should not be activated in the first step.
Therefore, highly reactive acceptors are preferred in the 2nd
glycosylation for an efficient one-pot glycosylation, which is
achieved in this study by diacetyl protection.
Table 1. The glycosylation between 3 and 4
Entry
1[a]
Method
Batch
Conditions
Time (min)
5
Products
NIS (1.5 eq),
TfOH (0.5 eq)
80%[c], 57%[d]
NIS (2.1 eq),
TfOH (0.8 eq)
2[b]
Microflow
1
94%
[a] 1 eq of 3 was used. [b] 1.5 eq of 3 was used. [c] 20 mg of 4 was
used. [d] 120 mg of 4 was used.
To overcome the scale-up limitation, we employed a microflow
system. Microflow synthesis is an innovative reaction control
system that enables efficient mixing, rapid heat transfer, and
precise residence time control, as well as good scalability.[33]
Scaled-up syntheses can be achieved under the same
conditions by continuous flow. These excellent features have
been utilized for glycosylation reactions. Thus, a microflow
glycosylation was first reported by Seeberger et al.[34] We also
achieved efficient and stereoselective -sialylation,[35] -
mannosylation,[36] and N-glycosylation[37] under microflow
conditions. The microflow system is obviously advantageous in
glycosylations using armed–disarmed strategy, since it allows
efficient mixing and accurate temperature control with the
concomitant enhancement of the selectivity in thioglycoside
activation. In addition, precise residence time control under
fluidic conditions prevents overreaction. Importantly, flow
reactions can overcome scalability and reproducibility limitations.
We applied the microflow system to the glycosylation between
3 and 4 (Table 1, entry 2). The reaction was completed within 1
min when using a slight excess amount of 3 and reagents. A
solution containing 3, 4, and NIS was mixed with TfOH in a T-
shape mixer at −20 ºC. After 1 min reaction, the reaction mixture
was quenched by adding to a solution of Et3N in CH2Cl2. As
expected, overreaction of 12 was suppressed, and target
disaccharide 12 was obtained in excellent yield (94%). Thus, the
scalable synthesis of 12 was achieved under microflow
conditions.
Table 2. Investigation of the reactivity in the [2+1] glycosylation using
N-acetylated acceptor
1 or N,N-diacetylated acceptor 2 for the
synthesis of protected -gal 13 or 14
Entry
Acceptor
Temp (°C)
Products
68%
1
2
1
2
0
−40
91%
Although the one-pot construction of -gal 14 was achieved,
reproducibility and scalability still remained unsolved because
the 1st glycosylation was difficult to control. As mentioned above
(Table 1), the 1st glycosylation requires precise control of
reaction temperature and time to suppress the overreaction of
disaccharide 12. To overcome these issues, we employed a
microflow system (Table 3, entry 3; Figure 5). Thus, a solution
containing 3, 4, and NIS was mixed with TfOH in a T-shape
mixer at −20 ºC. After 1 min reaction, N,N-diacetylated acceptor
2 and TfOH were successively added at −40 ºC. The resulting
mixture was collected in the flask and stirred for additional 5 min
at −40 ºC. Target trisaccharide 14 was then obtained in 82%
yield, which is comparable to the maximum yield of the one-pot
reaction under batch conditions (Table 3, entries 2 and 3).
Therefore, it is demonstrated that the one-flow reaction under
microflow conditions can overcome the issues of scalability and
reproducibility in the one-pot procedure. Although an orthogonal
leaving group-based one-flow glycosylation has been
reported,[38] to the best of our knowledge, this is the first report
on a one-flow glycosylation using the armed–disarmed strategy.
Having thiodisaccharide 12 in hand, we then investigated the
second glycosylation using glucosaminyl acceptors 1 or 2 to
evaluate the effectiveness of the diacetyl strategy (Table 2).
Glycosylation between 1 and 12 showed the low reactivity of 1;
Elevating the reaction temperature to 0 ºC was required, and the
yield of trisaccharide 13 was 68% (Table 2, entry 1). In contrast,
acetylation of the NHAc group in 2 improved the reactivity of the
acceptor. Accordingly, the reaction of N,N-diacetylated acceptor
2 with 12 proceeded at −40 °C to give trisaccharide 14 in
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We then verify the effectiveness
of the diacetyl strategy in the
synthesis of H antigen 5. We
employed stepwise construction
of glycan chain: fucose was
introduced to the 2-position of
galactose after reliable -
selective galactosylation using
neighboring group participation.
9-Fluorenylmethyloxycarbonyl
(Fmoc) group[39] was hence used
for the protection of the 2-
hydroxy group on the galactose
residue, since the -directing
neighboring group participation
of Fmoc has been previously
reported. After the synthesis of
Table 3. Investigation of one-pot and one-flow glycosylation using N-acetylated acceptor 1 or N,N-diacetylated
acceptor 2
Entry
Acceptor
Method
One-pot
One-pot
One-flow
Temp (°C)
−20
Time
3 h
Products
25%
1
2
3
1
2
2
−40
5 min
5 min
86%[a]
82%
galactosyl donors 15 and
6
−40
(Scheme S2), we investigated
the glycosylation using N-
[a] Low reproducibility
acetylated
diacetylated
comparative
expected,
1
2
and
(Table 4) for
purposes. As
showed higher
N,N-
2
reactivity than 1. When 1 was
used as an acceptor for the
glycosylation of 15, elevation of
the reaction temperature to 0 ºC
was required, and the yield of
disaccharide 16 was only 7%
(Table 4, entry 1). Unexpectedly,
only the undesired  isomer was
obtained in this case even
though the 2-position of 15 was
Fmoc-protected. In contrast, the
reactivity was improved when
using N,N-diacetylated acceptor
2. The reaction proceeded at
−40 °C to give disaccharide 17 in
Figure 5. One-flow synthesis of protected -gal 14 under microfluidic conditions.
35% yield with an / selectivity of 1/1.7 (Table 4, entry 2). To
improve the -selectivity, the glycosyl donor was replaced with 6
because 4,6-O-benzylidene-protected galactose was reported to
block the -face leading to an increase of the -selectivity.[40]
Thus, when 6 was used as a galactosyl donor, NAc2 protection
improved the reactivity. Glycosylation of 1 showed low reactivity
and required a temperature increase to 0 °C to obtain 18 in 24%
yield (Table 4, entry 3). Conversely, glycosylation using 2
proceeded smoothly at −20 °C to give disaccharide 19 in 76%
yield with perfect -selectivity (Table 4, entry 4).
Global deprotection of obtained 14 gave -gal 5 (Scheme 2).
After cleavage of the benzylidene group, hydrogenation followed
by hydrolysis under basic conditions afforded -gal 5. It is
noteworthy that the removal of one acetyl group from NAc2
proceeded smoothly under basic conditions. Since basic
treatment is necessary for removal of acyl protection on hydroxy
groups, no extra steps were required for the conversion of NAc2
to NHAc.
Scheme 2. Deprotection of protected -gal 14.
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Table 4. Investigation of the reactivity in the glycosylation using N-
acetylated 1 or N,N-diacetylated acceptor 2
Entry
Donor
Acceptor
Temp (°C)
Products
7% ( only)
1
2
3
4
15
15
6
1
2
1
2
−40 to 0
−40
Table 5. Investigation of the reactivity using N-acetylated disaccharide
20 and N,N-diacetylated disaccharide 21
35% (: = 1:1.7)[a]
24% ( only)[b]
76% ( only)
−20 to 0
−20
Entry
Acceptor
Conditions
−30 °C to rt
−30 °C
Products
45%
6
1
2
20
21
[a] / selectivity was estimated by 1HNMR. [b] Product 18 can not be
83%
isolated. Thus, yield was estimated by 1HNMR.
We next compared the reactivity of the disaccharide acceptors
20 (N-acetylated) and 21 (N,N-diacetylated) in the [2+1]
glycosylation (Table 5). After the syntheses of 20 and 21 by
cleavage of the Fmoc group of 18 and 19, respectively,
glycosylations with fucosyl donor 7[30a] were investigated. In the
case of 20, the glycosylation did not proceed at −30 °C, and
trisaccharide 22 was obtained in 45% yield when the reaction
temperature was elevated to rt (Table 5, entry 1). In contrast,
glycosylation with 21 proceeded smoothly at −30 °C to give 23 in
83% yield (Table 5, entry 2). Both glycosylations proceeded with
perfect -selectivity, and diacetyl protection of NHAc in GlcNAc
enhanced the reactivity at proximal and distal positions to
GlcNAc in both cases. This remote effect strongly supports the
hypothesis that aggregation induced by NHAc hydrogen bond
decreases the reactivity. N,N-diacetyl protection can enhance
the reactivity even when the reaction site is far from the NAc2
group, indicating that the diacetyl strategy can be generally
applied to the synthesis of glycans containing GlcNAc.
Scheme 3. Deprotection of protected H antigen 23.
Conclusions
In summary, we have demonstrated the effectiveness of diacetyl
strategy in the synthesis of -gal 5 and H antigen 8. The
formation of hydrogen bond through NHAc in GlcNAc was
clearly shown by NMR analysis. Although the effective volume of
1 was 1.2 times larger than that of 2 according to the DOSY
spectra, this small difference indicates that hydrogen bonds
formed by NHAc were dynamically rearranged, which
significantly reduced the reaction rate of glycosylations.
Conversion of NHAc to NAc2 dramatically improved the reactivity
in all cases. The diacetyl strategy enabled the effective one-pot
and one-flow glycosylation using armed and disarmed donors.
Even though the glycosylation position was far from NHAc,
diacetyl protection enhanced the reactivity. In the synthesis of -
gal and H antigen, glycans were elongated from the nonreducing
and reducing ends, respectively. Since the reactivity in
glycosylations tends to decrease as the size of fragments
increases, the donor reactivity decrease in the former and the
Finally, global deprotection of 23 gave H antigen 8 (Scheme 2).
After removal of the TBS group with TASF, hydrolysis of the
acetyl group followed by hydrogenation afforded 8. In this case,
NAc2 was also smoothly converted to NHAc during hydrolysis.
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activated MS4A (ca. 40 mg) in CH2Cl2 (1 mL) was slowly added a
solution of TfOH (1.4 L, 15.48 mol) in CH2Cl2 (1 mL) at -40 ºC under Ar
atmosphere. After being stirred for 20 mins at -40 ºC, the reaction was
quenched with Et3N and sat. Na2S2O3 aq., and filtration using membrane
filter (Fluoro pore®). The filtrate was extracted with CH2Cl2, and the
organic layer was washed with brine, dried over Na2SO4, filtered and
concentrated in vacuo. The residue was purified by silica-gel column
chromatography (Toluene/EtOAc = 5/1 to 4/1) to give 14 (48.2 mg, 91%)
as a white powder.
acceptor reactivity decrease in the latter with the glycan
elongation. The reactivity of acceptors by the diacetyl strategy
proved to be enhanced in both cases. These results clearly
indicate that the diacetyl strategy can be a powerful tool for the
synthesis of GlcNAc-containing glycans. We also showed that
the reactivity was affected by the supramolecular structure
based on hydrogen bonding and can be controlled by slight
structure modification such as amide acetylation. Application of
this concept not only to other glycans but also to a wide range of
organic compounds is further important work.
1H NMR (500 MHz, CDCl3):  = 8.05-7.17 (m, 35H), 5.48 (d, J = 3.4 Hz,
1H), 5.41 (dd, J = 9.9, 8.0 Hz, 1H), 5.17 (s, 1H), 5.10 (d, J = 10.9 Hz, 1H),
5.00 (d, J = 3.4 Hz, 1H), 4.95 (d, J = 3.7 Hz, 1H), 4.74 (m, 2H), 4.69 (d, J
= 9.0 Hz, 1H), 4.66-4.59 (m, 4H), 4.56 (d, J = 12.3, 1H), 4.36 (dd, J =
11.2, 3.8 Hz, 1H), 4.32 (d, J = 12.2 Hz, 1H), 4.14-4.00 (m, 5H), 3.92 (dd,
J = 10.0, 3.5 Hz, 1H), 3.82 (d, J = 12.5 Hz, 1H), 3.73-3.67 (m, 3H), 3.64
(s, 3H), 3.62-3.61 (m, 2H), 3.56 (d, J = 3.2 Hz, 1H), 3.42 (dd, J = 11.1,
1.8 Hz, 1H), 3.37 (dd, J = 12.5, 1.6 Hz, 1H), 3.26 (s, 1H), 2.28 (s, 6H),
1.75, (s, 3H). 13C NMR (100 MHz, CDCl3):  =175.6, 170.1, 169.6, 166.0,
164.4, 139.2, 138.8, 138.7, 137.9, 137.7, 133.7, 133.3, 129.9, 129.7,
128.9, 128.8, 128.7, 128.6, 128.4, 128.2, 128.0, 127.7, 127.6, 127.3,
127.2, 126.3, 100.9, 99.9, 98.1, 96.6, 76.1, 76.0, 74.5, 74.5, 74.4, 74.0,
73.8, 73.3, 72.2, 71.8, 71.1, 71.0, 68.9, 67.6, 65.5, 63.7, 62.9, 61.8, 59.3,
51.9, 26.8, 20.4. HRMS (ESI-LTQ-Orbitrap) m/z [M+Na]+ calcd for
C76H79NO22, 1380.4986, found 1380.4985.
Experimental Section
Glycosylation between 3 and 4, Compound 12
Batch conditions: To a suspension of donor 3 (20 mg, 37.1 mol),
acceptor 4 (13.3 mg, 24.7 mol), N-iodosuccinamide (8.36 mg, 37.2
mol), and activated MS4A (ca. 20 mg) in CH2Cl2 (0.5 mL) was slowly
added a solution of TfOH (1.07 L, 12.39 mol) in CH2Cl2 (0.3 mL) at -20
ºC under Ar atmosphere. After being stirred for 5 mins at -20 ºC, the
reaction was quenched with Et3N and sat. Na2S2O3 aq., and filtered using
membrane filter (Fluoro pore®). The filtrate was extracted with CH2Cl2,
and the organic layer was washed with brine, dried over Na2SO4, filtered,
and concentrated in vacuo. The residue was purified by silica-gel column
chromatography (CH2Cl2/EtOAc = 60/1) to give 12 (19.2 mg, 80%) as a
white powder.
One-flow glycosylation using 3, 4, and 2, Compound 14
A microreactor system consisted of syringe (HAMILTON CO., RENO.
NEVADA, size 5 mL), syringe pump (HARVARD 11 plus single syringe
pump), T-Shaped micro mixer, and tubes (inner diameter Φ = 80 mm).
Preparation of the solutions were carried out at rt under Ar atmosphere.
A solution of 3 (78.41 mg, 0.15 mmol), 4 (43.39 mg, 80.9 mol), and NIS
(38.2 mg, 0.17 mmol) in CH2Cl2 (2.0 mL) in the syringe A was pumped at
the flow rate of 0.3 mL/min. A solution of TfOH (5.74 L, 64.64 mol) in
CH2Cl2 (2.0 mL) in the syringe B was pumped at the flow rate of 0.3
mL/min. These solutions were mixed at -20 ºC. After 1 min reaction, a
solution of 2 (34.2 mg, 66.3 mol) and NIS (32.7 mg, 0.145 mmol) in
CH2Cl2 (2.0 mL) in the syringe C was pumped at the flow rate of 0.3
mL/min, and mixed at -40 ºC. A solution of TfOH (5.74 L, 64.6 mol) in
CH2Cl2 (2.0 mL) in the syringe D was pumped at the flow rate of 0.3
mL/min, and mixed at -40 ºC. The reaction mixture was collected for 60 s
in a flask containing MS4A. After being stirred for 5 min at -40 ºC under
Ar atmosphere, the reaction mixture was diluted with CH2Cl2 (5 mL),
quenched with Et3N and sat. Na2S2O3 aq. at -40 ºC, and extracted with
CH2Cl2. The organic layer was washed with sat. NaHCO3 aq., brine,
dried over Na2SO4, filtrated, and concentrated in vacuo. The residue was
purified by silica-gel column chromatography (Toluene/EtOAc = 5/1→4/1)
to give 14 (10.7 mg, 82%).
Microflow conditions:
(HAMILTON CO., RENO. NEVADA, size
A
microreactor system consisted of syringe
mL), syringe pump
5
(HARVARD 11 plus single syringe pump), T-Shaped micro mixer, and
tubes (inner diameter Φ = 80 mm). A solution of donor 3 (56.85 mg, 0.11
mmol), accepor 4 (31.46 mg, 58.62 mol), and NIS (27.7 mg, 0.12 mmol)
in CH2Cl2 (1.45 mL) in the syring A was pumped at the flow rate of 0.3
mL/min. A solution of TfOH (4.16 L, 46.84 mol) in CH2Cl2 (1.45 mL) in
the syringe B was pumped at the flow rate of 0.3 mL/min. These
solutions were mixed at -20 ºC. The reaction mixture was collected for 60
s in a flask which was filled with a solution of Et3N in CH2Cl2 at -20 ºC.
Then, sat. Na2S2O3 aq. was added, and the reaction mixture was
extracted with CH2Cl2. The organic layer was washed with sat. NaHCO3
aq., brine, dried over Na2SO4, filtrated, and concentrated in vacuo. The
residue was purified by silica gel column chromatography (CH2Cl2/EtOAc
= 60/1) to give 12 (10.6 mg, 94%).
1H NMR (500 MHz, CDCl3):  = 8.07-6.90 (m, 29H), 5.62 (d, J = 3.0 Hz,
1H), 5.52 (m, 1H), 5.18 (s, 1H), 5.09 (d, J = 3.3 Hz, 1H), 4.79 (d, J = 10.1
Hz, 1H), 4.73-4.54 (m, 4H), 4.48-4.38 (m, 2H), 4.04-3.99 (m, 2H), 3.95
(dd, J = 10.2, 3.3 Hz, 1H), 3.82 (d, J = 11.5 Hz, 1H), 3.74 (dd, J = 10.2,
3.4 Hz, 1H), 3.57 (d, J = 3.3 Hz, 1H), 3.39 (d, J = 12.0 Hz, 2H), 1.90 (s,
3H), 1.80 (s, 3H). 13C NMR (100 MHz, CDCl3):  = 170.2, 166.0 (2x),
164.8, 138.8, 138.6, 138.1, 137.7, 133.5, 133.2, 129.8, 129.6, 128.8,
128.6, 128.4, 128.3, 128.1 (2x), 127.6, 127.5, 126.2, 100.8 (2x), 96.0,
87.5, 76.1, 75.2, 74.4, 74.3, 73.9 (2x), 72.1, 68.8, 65.8, 62.9, 21.1, 20.4.
HRMS (ESI-LTQ-Orbitrap) m/z [M+Na]+ calcd for C56H54O13S 989.3177,
found 989.3189.
Glycosylation between 6 and 2, Compound 19
To a suspension of donor 6, acceptor 2, N-iodosuccinimide (8.25 mg,
36.65 mol), and MS4A powder (ca. 32 mg) in CH2Cl2 (610 L) was
added trifluoromethanesulfonic acid (1.1 L, 12.21 mol) at -20 ºC under
Ar atmosphere. After being stirred for 20 mins at -20 ºC, the reaction
mixture was quenched with sat. NaHCO3 aq. and 10% Na2S2O3 aq. and
extracted with CHCl3. The organic layer was dried over Na2SO4, filtered,
and concentrated in vacuo. The residue was purified by preparative layer
chromatography (hexane/EtOAc = 1/1) to give 19 (25.4 mg, 76%) as a
colorless oil.
Glycosylation between 12 and 2, Compound 14
To a suspension of disaccharide donor 12 (37.5 mg, 38.7 mol), acceptor
2 (20.0 mg, 38.7 mol), N-iodosuccinamide (11.5 mg, 46.5 mol), and
This article is protected by copyright. All rights reserved.

10.1002/ejoc.201901809
European Journal of Organic Chemistry
FULL PAPER
1H-NMR (500 MHz, CDCl3):  = 7.79 (dd, J = 7.6, 2.6 Hz, 2H), 7.62 (dd, J
= 7.3, 2.6 Hz, 2H), 7.43 (t, J = 7.3 Hz, 2H), 7.36-7.19 (m, 12H), 5.06 (d, J
= 3.7 Hz, 1H), 5.01 (d, J = 11.0 Hz, 1H), 4.98 (d, J = 3.9 Hz, 1H), 4.74
(dd, J = 9.4, 8.1 Hz, 1H), 4.66 (d, J = 12.4 Hz, 1H), 4.63-4.56 (m, 3H),
4.48 (dd, J = 10.3, 6.2 Hz, 1H), 4.41 (dd, J = 11.3, 3.7 Hz, 1H), 4.35 (d, J
= 8.1 Hz, 1H), 4.29 (d, J = 12.1 Hz, 1H), 4.24 (t, J = 6.2 Hz, 1H), 4.16 (d,
J = 16.5 Hz, 1H), 4.11 (d, J = 16.5 Hz, 1H), 3.96 (t, J = 9.3 Hz, 1H), 3.88
(dd, J = 11.3, 6.6 Hz, 1H), 3.76-3.69 (m, 3H), 3.69 (s, 3H), 3.44-3.36 (m,
3H), 2.30 (s, 6H), 2.03 (s, 3H), 1.97 (s, 3H), 0.77 (s, 9H), 0.02 (s, 3H), -
0.06 (s, 3H). 13C-NMR (500MHz, CDCl3): = 175.49, 170.33, 169.95,
169.45, 154.12, 143.45, 143.40, 141.53, 141.38, 139.18, 137.90, 128.47,
127.93, 127.89, 127.85, 127.78, 127.34, 127.14, 127.12, 127.02, 124.82,
124.79, 120.07, 120.05, 100.08, 98.24, 77.90, 77.20, 76.61, 76.04, 73.22,
71.11, 70.91, 70.64, 69.45, 69.22, 67.39, 63.85, 61.46, 59.06, 51.84,
46.81, 26.72, 25.30, 20.62, 17.67, -4.93, -5.32. HRMS (ESI-LTQ-
Orbitrap) m/z [M+Na]+ calcd for C58H71NO18SiNa, 1120.4333, found
1120.4320.
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Glycosylation between 21 and 7, Compound 23
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1H-NMR (500 MHz, CDCl3) :  = 7.42-7.19 (m, 25H), 5.46 (d, J = 3.9 Hz,
1H), 5.04 (d, J = 10.6 Hz, 1H), 5.01-4.97 (m, 3H), 4.94 (d, J = 3.7 Hz, 1H),
4.74-4.68 (m,4H), 4.64 (d, J = 12.0 Hz, 1H), 4.53 (dt, J = 10.6, 5.0 Hz,
2H), 4.42-4.35 (m, 3H), 4.26 (d, J = 7.9 Hz, 1H), 4.18 (d, J = 16.5 Hz, 1H),
4.14-4.06 (m, 3H), 3.99-3.93 (m, 2H), 3.89-3.78 (m, 5H), 3.66 (s, 3H),
3.53 (dd, J = 9.3, 3.4 Hz, 1H), 3.43 (dd, J = 10.8, 1.5 Hz, 1H), 3.35 (t, J =
6.6 Hz, 1H), 2.29 (s, 6H), 2.00 (s, 3H), 1.92 (s, 3H), 1.28 (d, J = 6.6 Hz,
3H), 0.79 (s, 9H), 0.06 (s, 3H), 0.01 (s, 3H). 13C-NMR (500 MHz, CDCl3):
 = 175.49, 170.41, 170.16, 169.66, 139.02, 138.97, 138.80, 138.65,
137.90, 128.59, 128.36, 128.22, 128.14, 127.99, 127.97, 127.92, 127.79,
127.68, 127.57, 127.50, 127.31, 127.19, 127.15, 100.52, 98.74, 97.43,
79.99, 77.51, 76.27, 76.04, 75.91, 74.97, 74.26, 73.74, 73.52, 73.42,
73.32, 71.73, 71.27, 70.59, 70.33, 67.77, 66.53, 64.49, 61.95, 59.15,
51.81, 26.66, 25.75, 20.69, 20.65, 17.57, 16.70, -4.07, -4.57. HRMS
(ESI-LTQ-Orbitrap) m/z [M+Na]+ calcd for C70H89NO20SiNa 1314.5639,
found 1314.5661.
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Acknowledgments
This work was financially supported in part by JSPS KAKENHI
Grant Number 15H05836 in Middle Molecular Strategy, JSPS
KAKENHI Grant Number 16H01885, JSPS KAKENHI Grant
Number 16H05924, JSPS KAKENHI Grant Number 17K19201,
JSPS A3 Foresight Program.
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Keywords: glycosylation • protecting group • hydrogen bond •
glycan antigen • microflow
This article is protected by copyright. All rights reserved.

10.1002/ejoc.201901809
European Journal of Organic Chemistry
FULL PAPER
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This article is protected by copyright. All rights reserved.

10.1002/ejoc.201901809
European Journal of Organic Chemistry
FULL PAPER
Key Topic: Carbohydrate synthesis
FULL PAPER
Masato Tsutsui, Julinton Sianturi, Seiji
Masui, Kento Tokunaga, Yoshiyuki
Manabe,* and Koichi Fukase*
Page No.–Page No.
Efficient synthesis of antigenic
trisaccharides containing N-
acetylglucosamine: protection of
NHAc as NAc2
Diacetyl strategy: Acetamide (NHAc) is tentatively converted to diacetylimide
(NAc2) during oligosaccharide construction. Formation of hydrogen bonds through
NHAc in GlcNAc was confirmed by NMR measurements, and N,N-diacetyl
protection improved the reactivity in glycosylation to realize the efficient
construction of glycans.
This article is protected by copyright. All rights reserved.