Highly efficient α-sialylation by virtue of fixed dipole effects of N-phthalyl group: Application to continuous flow synthesis of α(2-3)-and α(2-6)-Neu5Ac-Gal motifs by microreactor

Article abstract of DOI:10.1080/07328300701634796

Highly α-selective sialylation of sialic acid N- phenyltrifluoroacetimidate with various galactose and lactose acceptors has been achieved by introducing the C-5 N-phthalyl group on the donor. The "fixed dipole effect" of the N-phthalyl group was proposed to explain the high reactivity and α-selectivity. The microfluidic system was applied to the present α-sialylation, which is amenable to large-scale synthesis. The N-phthalyl group was removed by treatment with methylhydrazine acetate, for which protocol can be readily applied to the synthesis of a variety of sialic acid-containing oligosaccharides. (Chemical Equation Presented). Copyright Taylor & Francis Group, LLC.

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Highly Efficient α‐Sialylation by Virtue of
Fixed Dipole Effects of N‐Phthalyl Group:
Application to Continuous Flow Synthesis
of α(2‐3)‐and α(2‐6)‐Neu5Ac‐Gal Motifs by
Microreactor
Shin‐ichi Tanaka a , Takashi Goi a , Katsunori Tanaka a & Koichi Fukase a
a Department of Chemistry, Graduate School of Science, Osaka University,
Toyonaka, Osaka, Japan
Version of record first published: 07 Nov 2007.
To cite this article: Shin‐ichi Tanaka , Takashi Goi , Katsunori Tanaka & Koichi Fukase (2007): Highly Efficient
α‐Sialylation by Virtue of Fixed Dipole Effects of N‐Phthalyl Group: Application to Continuous Flow Synthesis
of α(2‐3)‐and α(2‐6)‐Neu5Ac‐Gal Motifs by Microreactor, Journal of Carbohydrate Chemistry, 26:7, 369-394
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Journal of Carbohydrate Chemistry, 26:369–394, 2007
Copyright # Taylor & Francis Group, LLC
ISSN: 0732-8303 print 1532-2327 online
DOI: 10.1080/07328300701634796
Highly Efficient a-Sialylation
by Virtue of Fixed Dipole
Effects of N-Phthalyl Group:
Application to Continuous
Flow Synthesis of a(2-3)-
and a(2-6)-Neu5Ac-Gal
Motifs by Microreactor
Shin-ichi Tanaka, Takashi Goi, Katsunori Tanaka,
and Koichi Fukase
Department of Chemistry, Graduate School of Science, Osaka University, Toyonaka,
Osaka, Japan
Highly a-selective sialylation of sialic acid N-phenyltrifluoroacetimidate with various
galactose and lactose acceptors has been achieved by introducing the C-5 N-phthalyl
group on the donor. The “fixed dipole effect” of the N-phthalyl group was proposed to
explain the high reactivity and a-selectivity. The microfluidic system was applied to the
present a-sialylation, which is amenable to large-scale synthesis. The N-phthalyl group
was removed by treatment with methylhydrazine acetate, for which protocol can be
readily applied to the synthesis of a variety of sialic acid-containing oligosaccharides.
Received February 6, 2007; Accepted July 24, 2007
Address correspondence to Koichi 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
369

S.-i. Tanaka et al.
370
Keywords a-Sialylation, N-Phthalyl group, Fixed dipole effects, Microreactor,
Oligosaccharides
N-Acetylneuramic acid (Neu5Ac), the most abundant sialic acid congener in
nature, is found at the termini of glycoproteins and glycolipids on mammalian
cell surfaces, usually linked with galactose or N-acetylgalactosamine through
a(2-3) or a(2-6) sialoglycoside bonds. Since Neu5Ac on the cell surfaces plays
diverse and important roles in cell/cell interaction processes,[1] such as
pathogen/host recognition, tumor metastasis, and cell differentiation/prolifer-
ation, much effort has been devoted to the development of efficient and stereo-
selective synthesis of a(2-3) and a(2-6)-Neu5Ac-Gal units in order to further
investigate their biological functions.[2,3] However, an efficient and general sia-
lylation with high a-selectivity still has not been fully realized, because (1) the
presence of the electron-withdrawing carboxylic acid groups at C-2 of the sialyl
acid donors deactivates the oxocarbenium ions, thus electronically and steri-
cally interfering with efficient sialylation. This leads to the formation of a sig-
nificant amount of the dehydrated byproduct, 2,3-glycal. In addition, (2) the
absence of the neighboring participation group at C-3 cannot ensure the stereo-
chemical outcome for the a-selective sialylation. This inherent reactivity of the
sialyl donor renders the a-selective sialylation as one of the most difficult[4] and
challenging topics in the field of oligosaccharide synthesis.[5]
Recently, Cai and Yu have successfully enhanced the reactivity of sialic acid
donors by utilizing phenyltrifluoroacetimidate as a leaving group, and achieved
efficient sialylation with a variety of acceptors in 59% to 90% yields: while (2-3)-
Neu5Ac-Gal synthesis has been realized in 81% yield (a:b ¼ 3:1), the corre-
sponding a-selective (2-6)-sialylation was achieved in 61% yield.[6] Kiso and
coworkers[7] and Takahashi and coworkers[8] have used the C-5-N-Troc-protected
thiophenyl derivatives as the sialic acid donors and achieved good a-selectivity,
namely, a:b ¼ 54:10 for the (2-3)-sialylation case.[7] Apparently, one of the most
exciting achievements in this field is the a-(2-8)-linkage formation between the
sialosides, quite recently realized by Kiso’s[9a] and Takahashi’s[9b] groups.
Especially, Takahashi’s group has utilized the 4-O,5-N-oxazolidinone-protecting
group both on the sialoside donor and the acceptor and realized both a-(2-9)-
and a-(2-8)-oligosialoside synthesis with excellent selectivity.[9b]
In our program directed toward the establishment of a general and practical
synthesis of N-linked oligosaccharides and other neuramic acid-containing
natural products, highly yielding and a-selective sialylation is essential. The
recent successful precedents mentioned above led us to utilize the phenyl-
trifluoroacetimidate donor and to pursue the possibility of increasing the
a-selectivity by modifying the C-5-N-protecting groups of the sialic acid donors
(i.e., the amide groups of which dipole moments direct the stereochemical
course of glycosylation and/or the carbonyls take part in the neighboring group

Highly Efficient a-Sialylation
371
participation). In this paper, we disclose in detail[10] highly efficient sialylation
with excellent a-selectivity by using the N-phthalyl group, which led to a
general synthesis of the a(2-3)- and a(2-6)-Neu5Ac-Gal moieties. The method is
applied to microfluidic a-sialylation in pursuit of a scaled-up synthesis of
neuramic acid-containing compounds. The possible mechanisms for the
enhanced a-selectivity based on the conformational analysis and electronic
property calculations are also described.
For the sialic acid donors, we planned six differently protected derivatives
on the C-5 nitrogen, namely, acetyl (2a), Troc (2b), bis-acetyl (2c), dimethylma-
leoyl (DMM, 2d), tetrachlorophthalyl (TCP, 2e), and phthalyl (Pht, 2f) (Sch. 1).
These donors were easily prepared from thioglycoside 1,[11] according to
Higuchi’s procedure:[3e] (1) deacetylation, (2) protection of the C-5 nitrogen,
(3) peracetylation of the remaining hydroxyls, (4) hydrolysis of the thioglyco-
side, and (5) phenyltrifluoroacetimidate formation.
a-Selective (2-6)-sialylation trials using donors 2a–2f thus prepared and
1-O-allyl-2,3-O-benzoylgalactose 3, appropriately protected for further func-
tional group manipulation, are shown in Table 1. First, the reactivity of the
sialy donors 2a–2f was screened using 50 mg of each sample (Table 1). All reac-
tions were performed at 2788C in the presence of TMSOTf as the Lewis acid
activator, using 1.5 equivalents of acceptor 3 with respect to the donors. Propio-
nitrile was used as the optimal solvent, by taking advantage of “nitrile solvent
effect.”[13] The reaction of N-mono-acetyl donor 2a with acceptor 3 provided the
corresponding disacchalide 4a in good yield (entry 1, 93%) and with moderate
selectivity (a:b ¼ 77:23). It is noteworthy that, when N-Troc derivative 2b was
used (entry 2), the reaction was significantly accelerated (6 h for N-Ac and
Scheme 1: Preparation of sialic acid donors 2a–f bearing a variety of N-protected groups.

S.-i. Tanaka et al.
372
Table 1: a-Selective (2-6)-sialylation Using 2a–f.a
Entry
Donor
Solvent
Temp
Time
Product
Yield (%)b
a:bc
1
2
3
4
5
6
7
8
2a
2b
2c
2d
2e
2f
EtCN
EtCN
EtCN
EtCN
EtCN
EtCN
CH2CI2
EtCN
2788C
2788C
2788C
2788C
2788C
2788C
rt
6 h
4a
4b
4c
4d
4e
4f
93
84
75
94
77:23
92:8
72:28
96:4
96:4
a only
9:91
30 min
,5 min
,5 min
,5 min
30 min
,5 min
30 min
87
92d
87
2f
2f
4f
4f
2788C
60e
a only
aAll sialylations were performed using 1.5 equiv of acceptor 3 relative to donors 2a–2f. The
mixture of anomeric stereoisomers for imidates 2a–2f was used.
bIsolated yields.
cThe a/b- ratio was determined by NMR analysis: a- and b-isomers were identified based on the
empirical rule for their characteristic proton chemical shifts.[12]
dYield at 50 mg- scale of 3.
eYield at 100 mg- scale of 3.
30 min for N-Troc), and a-selectivity also increased up to a:b ¼ 92:8. This
observation is in accordance with that of Kiso and coworkers.[7]
Encouraged by these promising results, we then examined the N-bis-
acylated donors 2c–2f. Bis-N-acetyl derivative 2c gave the corresponding dis-
acchalide 4c in moderate yield and with moderate a-selectivity (75% and
a:b ¼ 72:28, entry 3), similar to that observed for monoacylate 2a (entry 1).
However, we were glad to find that, when two acyl groups on the C-5
nitrogen were fixed within the five-membered rings, the a-selectivity
increased dramatically. Thus, N-DMM- and N-TCP-protected donors 2d and
2e successfully provided disacchalides 4d and 4e in 94% and 87% yields,
respectively, and both with excellent a-selectivity (a:b ¼ 96:4, entries 4 and
5). Furthermore, the utilization of the N-Pht-protected donor 2f resulted in
perfect a-selectivity and excellent yield (92% on 50-mg scale, entry 6). It is
worthwhile mentioning that, when the solvent was exchanged from propioni-
trile to the noncoordinating dichloromethane, the stereoselectivity was
reversed (entry 7, a:b ¼ 9:91), indicating the importance of the “nitrile
solvent effect” in order to obtain good a-selectivity. However, when the scale
of the reaction was increased by only two, the product yield markedly
decreased, although the a-selectivity remained high. Thus, the 100-mg

Highly Efficient a-Sialylation
373
scale reaction of 3 gave only 60% of a-sialoside accompanied by a significant
amount of a glycal byproduct (entry 8).
The decrease in sialylation efficiency observed in entry 8 might be due to
the high reactivity of 2f. For such a case, precise reaction control is very diffi-
cult under the conventional batch process conditions, especially when the
reaction is scaled up. Thus, the disorder of the reaction factors in the scaled-
up batch reaction, that is, (1) precise temperature control; (2) mixing efficiency
between acceptor, donor, and Lewis acid; and (3) reaction time might lead to the
glycal production. In order to circumvent these problems, we used a continuous
flow microreactor, which is reported to realize efficient mixing and fast heat
transfer and, therefore, is recognized as innovative technology in recent
organic synthesis.[14] Once the reaction conditions are optimized for the
small-scale reaction, the same conditions are directly applied to a large-scale
synthesis, since the reaction is conducted under the flow process conditions.[15]
An application of the microfluidic system to the glycosylation reaction was first
reported by Seeberger and coworkers on a-mannosylation.[16a] We also have
established an efficient microfluidic glycosylation in combination with the
affinity separation method.[16b]
For the present microfluidic sialylation, a propionitrile solution of sialyl
donor 2f and acceptor 3 with various concentrations was mixed with
TMSOTf solution in dichloromethane at 2788C using an IMM micromixer[17]
at the flow rate of 1.0 mL/min (Table 2). After the reaction mixture was allowed
to flow at 2788C for additional 47 sec through a reactor tube (F ¼ 1.0 mm,
Table 2: Optimization of a-(2-6)-sialylation between donor 2f and acceptor 3 using
microreactor.
Donor
2f (M)
Acceptor
3 (M)
TMSOTf
(M)
Yield of
4f (%)
Entry
a:ba
1
2
3
0.15
0.15
0.2
0.1
0.1
0.1
0.08
0.15
0.15
14
88
.99
a only
a only
a only
aBased on 1H NMR analysis.

S.-i. Tanaka et al.
374
l ¼ 1.0 m), the mixture was quenched by another flow of excess triethylamine
dissolved in dichloromethane by using a T-shaped mixer at 2788C. CH2Cl2
was used as a cosolvent for the microfluidic sialylation in order to avoid
blockages during the micromixing. By using the mixed solvent system of
EtCN/CH2Cl2 (1:1), the same yield and a-selectivity in batch sialylation was
observed as in Table 1, entry 6. When the concentrations of the donor 2f,
acceptor 3, and TMSOTf were adjusted to 0.15 M, 0.1 M, and 0.08 M, respect-
ively, disaccharide 4f was obtained in only 14% yield and an excess amount of
acceptor 3 was recovered (entry 1). However, we were pleased to find that the
yield of 4f dramatically increased (88%) when the concentration of the Lewis
acid was increased up to 0.15 M (entry 2). Finally, the desired a-sialoside 4f
was obtained in quantitative yield by increasing the concentration of the
donor 2f to 0.2 M (entry 3). The use of excess amounts of donor 2f or
TMSOTf under the batch reaction conditions did not improve the sialylation
yield, but rather resulted in a large amount of glycal production. It is noted
that, by this continuous microflow reaction, single a-sialoside 4f was reprodu-
cibly obtained without any decrease of the yield. The microfluidic reaction suc-
cessfully controlled the high reactivity of the sialyl donor 2f for a-sialylation,
and efficient and large-scale procedures have now been realized.
The excellent a-selectivity achieved for a(2-6)-sialoglycosidation using the
N-Pht donor 2f was also applied to the (2-3)-sialylation cases (Table 3).
Although poor selectivity (a:b ¼ 65:35) was obtained by the reaction of 2f
with the sterically hindered 4,6-benzylidene-protected monosaccharaide
acceptor 5 (entry 1), excellent a-selectivity (97:3) was achieved by the
reaction with the 1-O-allyl-2-O-benzoyl-6-O-benzyl-protected acceptor 6
(entry 2). A slightly lower yield of 77%, compared with the (2-6)-sialylation
case, is due to the decreased reactivity of the C-3 hydroxyl of acceptor 6,
thus giving rise to the 2,3-glycal production (16%). In order to prevent the
glycal formation for such a (2-3)-sialylation case, we further examined the
more reactive 2-benzyl-protected acceptor 7 (entry 3). Although the reaction
rapidly proceeded and the glycal byproduct could not be detected from
the crude mixtures, both (2-3)-and (2-4)-sialoglycosides 13a and 13b
(13a:13b ¼ 2:1) were produced in 85% total yield as their single a-isomers.
The C2-a-configuration in (2-3)-and (2-4)-sialoglycosides 13a and 13b was
assigned from the NOEs between methyl protons of the ester and H-4 and/or
H-6 in the neuramic acid moiety.
The method was also applied to the lactose-derived disaccharide acceptors,
providing the corresponding trisaccharides with excellent a(2-3)-selectivity
and moderate yields (entries 4–6). The reaction of 2f with the perbenzoyl-pro-
tected lactoses 8 and 9 gave a(2-3)-trisaccahride derivatives 14 and 15 as the
single stereoisomers in 38% and 43% yields, respectively (entries 4 and 5).
The yield was increased up to 50% by the reaction with more reactive perben-
zylated acceptor 10, although the a-selectivity slightly decreased (entry 6,

Table 3: a-Selective (2-3)-sialylation between 2f and a variety of acceptors.a
Entry
Acceptor
Condition
Product
Yield (%)b
a:bc
65:35
1
EtCN, 2788C,
84%
5 min
2
EtCN, 2788C,
5 min
77%
85%
97:3
3d,e
EtCN, 2788C,
5 min
a only both
for a(2-3)
and a(2-4)
4
EtCN, 2788C,
38%
a only
15 min
(continued)

Table 3: Continued.
Product
Entry
Acceptor
Condition
Yield (%)b
a:bc
a only
5
EtCN, 2788C,
43%
15 min
6
EtCN, 2788C,
50%
93:7
1 h
aAll sialylations were performed using 1.5 equiv of acceptor 5–10 relative to donor 2f.
bIsolated yields.
cThe a/b-ratio was determined by NMR analysis.[12]
dProducts ratio of a(2-3) and b(2-4) sialosides was 2:1. Their structures were analyzed after benzoylation.
ePNP ¼ p-Nitrophenyl.

Highly Efficient a-Sialylation
377
Table 4: Optimization of a-(2-3)-sialylation between donor 2f and acceptor 9 using
microreactor.
Donor
2f (M)
Acceptor
9 (M)
TMSOTf
(M)
Yield of
15 (%)
Entry
a:ba
1
2
3
0.1
0.3
0.3
0.15
0.1
0.1
0.05
0.15
0.3
21
50
62
a only
a only
a only
aBased on 1H NMR analysis.
a:b ¼ 93:7). The main byproduct of the sialylation using the disaccharide accep-
tors 8 and 9 was the glycal derivative of 2f. The sterically bulkier disaccharides
would not react smoothly with the donor cation derived from 2f, but instead the
3H proton of the donor cation was subtracted to give rise to the glycal.
In anticipation of the efficient mixing effects between donor, acceptor, and
acid, the microfluidic system was again applied to this case (Table 4). The per-
benzoylated lactose 9 was used for the microfluidic sialylation trials, since the
functional group manipulation after the glycosylation was easier than the cor-
responding perbenzylated acceptor 10. After the optimization of the micromix-
ing conditions, trisaccharide 15 was obtained in 62% yield as a single
a-stereoisomer (entry 3), when 0.3 M of the donor 2f, 0.1 M of the acceptor 9,
and 0.3 M of TMSOTf were combined by the IMM micromixer. This is about a
20% increase in the yield compared with that of the batch reaction (43% yield
in Table 3, entry 5). By taking the results obtained in Table 2 and Table 4
together, the microfluidic reaction enabled a-selective sialylation for both
a-(2-6) and a-(2-3) cases in higher yields than the conventional batch process.
In order to understand the good reactivity and a-selectivity observed for
the C-5-N-phthalyl- and the other protected donors, conformational analysis
of the intermediary oxocarbenium ions 17a–17f, derived from the imidates
2a (N-Ac), 2b (N-Troc), 2c (N-Ac2), and 2f (N-Pht), was performed based on
the molecular mechanics calculations with the MMFF94s/MonteCarlo
method using Spartan 02 software (Fig. 1a–d).[18,19] The boat-like folded con-
formations were found as the lowest-energy conformations for both

S.-i. Tanaka et al.
378
Figure 1: Optimized structures of oxocarbenium ions 17a–17f by molecular mechanics
calculation with MMFF94s/MonteCarlo Method Using Spartan 02 Software. The most stable
conformations are shown in (a–d): Based on the experimental results in Table 1, the
calculation was mainly performed to provide a rationale for the electronic effects of 5N-
substitutents on oxocarbenium ion. Accordingly, the C-6 alkyl side chain was replaced by a
simple methyl group in order to simplify the conformational analysis. Among the optimized
conformations of 17c, the trans-diacetyl conformation was obtained as the second lowest
structure at 0.1 kcal/mol higher energy level.
monoacylates 17a (N-Ac) and 17b (N-Troc) (Fig. 1a, b). For these energy-mini-
mized conformations, both N-acetyl and N-Troc carbonyls are thought to be
participating in the electrostatic interaction with the oxocarbenium ions,
thus stabilizing the intermediates. Although the observed a-selectivity is
mainly caused by the nitrile solvent effects as exemplified by both Kiso’s and
Takahashi’s experiments with 5-N-Troc derivatives,[7,8] the additional confor-
mational preferences of Figures 1a and 1b may also lead to the attack of the
acceptor from the backside of the molecule and thus high a-selective sialylation
would result. The carbonate (O-C55O) group would be involved in a stronger
interaction with the carbenium ion than the acyl (C55O) group; thereby, the
higher a-selectivity of Troc-protected 2b would result.
On the other hand, chair-like extended conformations were obtained for
both N-bis-acyl derivatives 17c (N-Ac2) and 17f (N-Pht) as their energy-mini-
mized conformations (Fig. 1c, d). Since the N-Ac2 and N-Pht moieties deter-
mine the totally different reactivity and selectivity toward the glycosylation

Highly Efficient a-Sialylation
379
Figure 2: Optimized structures and dipole moments of simplified N-Pht and N-Ac2 moieties
by DFT/BLYP/6-31Gꢀ level calculation using Gaussian 03W software.
observed in Table 1, their local conformations and electronic properties were
further investigated (Fig. 2). Thus, the geometries of the simplified Me-NPht
and Me-N-Ac2 models were optimized at the DFT/BLYP/6-31Gꢀ level, and
on these obtained minima, the dipole moments (m) were then calculated by
using Gaussian 03W Software.[20]
While one minimum was found, as expected, for the conformationally rigid
Me-NPht (Figure 2a, the dipole moment shown with a red arrow), two absolute
minima were found for Me-N-Ac2, where the trans isomer is more stable than
the cis isomer by 3.57 kcal/mol (Fig. 2b, c). In the trans isomer, the dipole
moment is directed from the nitrogen atom toward one of the carbonyls while
it is aligned toward the N-Me direction in the cis isomer.
Therefore, in the oxocarbenium ion intermediate 17f (Fig. 1d), the fixed
dipole of the N-Pht may possibly facilitate the nitrile solvent participation
from the b-face of the molecule. Namely, the fixed dipole moment in N-Pht
aligned on the same plane as the sugar six-membered ring would interact
with the dipoles of the attacking nitrile solvent due to the favorable dipole/
dipole arrangement in space. It is noted that the rotation around the C-N
bond in 17f does not change the direction of the dipole of N-Pht (see yellow
arrow in Fig. 1d); thus, the strong dipole/dipole effects may be anticipated.
Furthermore, the fixed dipole may also stabilize the cation intermediate 17f
by interacting with the dipole of oxocarbonium ion. This stabilized cation can
be trapped by the nitrile solvent from the b-side of the molecule, namely, due
to the stereoelectronic effect, which in turn is attacked by the acceptor from
the a-face of the molecule via SN2-type substitution reaction. The significant
solvent effects observed in Table 1 (entries 6 and 7) support this rationale.
A similar dipole stabilized carbocation intermediate has also been proposed
by Wong and coworkers in order to explain the highly a-selective sialylation
using the C 5-N-azide sialoside donor.[2p]

S.-i. Tanaka et al.
380
On the other hand, no such strong dipole/dipole interactions can be
expected for the conformationally flexible bis-N-acylate oxocarbenium ion
17c. As predicted from the dipole orientations of N-Ac2 in Figures 2b and 2c,
when two acetyl groups on the nitrogen and the N-C bond rotate freely (see
yellow arrows in Fig. 1c), the dipoles are randomized; that is, the dipole is
not fixed as parallel to the pyrane plane. This offset of the dipole orientations
of N-Ac2 might therefore decrease the reactivity and a-selectivity of 17c,
compared with that of 17f. Although many other effects, including the solvation
and temperature effects, operate the total reactivity and stereochemical results
on the sialylation, the conformational and “fixed dipole” analysis performed on
17c and 17f supported the experimentally observed high a-selectivity of 2f, the
5-N phthalyl-protected sialoside donor.
Finally, removal of the N-phthalyl group of di- and trisaccharides 4f, 12, 15,
and 16 was attempted (Table 5). Hydrazine acetate, a common reagent for the
deprotection of the N-phthalyl group, unexpectedly hydrogenated the 1-O-allyl
moiety of a(2-6)-disaccharide 4f into the O-propyl group. The problem was cir-
cumvented by using the corresponding methylhydrazine acetate. The reaction
of 4f with excess methylhydrazine acetate in ethanol at 808C provided a
Table 5: Removal of N-phthalyl group.
Yield
Entry
Substrate
Product
(%)
1
84
2
3
73a
45
aCorresponding hydrazone was obtained in 11% yield as a byproduct.

Highly Efficient a-Sialylation
381
complex mixture of the products consisting of partially deacetylated com-
pounds, which without the purification was acetylated with Ac2O in pyridine
to give N-Ac derivative 18 in 84% yield (entry 1). Similarly, the treatment of
a(2-3)-disaccharide and trisaccharides 12 and 15 yielded the corresponding
N-Ac derivatives 19 and 20 in 73% and 45% yields, although the corresponding
hydrazone derivatives were obtained as the main byproducts for these a(2-3)-
sialoside cases.
In summary, we have achieved highly efficient a-selective sialylation
toward the synthesis of the a(2-6)- and a(2-3)-Neu5Ac-Gal units by tuning
the electronic properties of the C-5 nitrogen-protecting groups. We proposed
the fixed dipole moment concept in order to explain the high reactivity and
excellent a-selectivity observed when the N-phthalyl group was utilized.
Furthermore, the microfluidic system was applied to the present a-sialylation,
and an efficient route to large-scale synthesis was established. The C5-N-
phthalyl group utilized for a-selective sialylation was readily removed (i.e.,
by treatment with methylhydrazine acetate), thereby enabling efficient and
general procedures for easy access to the sialic acid-containing library or
complex N-linked glycans. Research directed along this line is now in
progress in our laboratory.
EXPERIMENTAL
All commercially available reagents were used without further purification.
Dichloromethane and propionitrile were refluxed over and distilled from
CaH2. Preparative separation was usually performed by column chromato-
graphy on silica gel (FUJI silysia LTD, BW-200 and BW-300) and by thin
layer chromatography on silica gel (Merck, 20 ꢁ 20 cm, Silica gel 60 F254
,
1
1 mm). H NMR spectra were recorded on a JEOL a-500 spectrometer and
chemical shifts were represented as d values relative to the internal
standard TMS. ESI-MS was measured on an Applied Biosystems Mariner.
High-resolution mass spectra (HRMS) were measured on a JEOL JMS-
T100LC mass spectrometer.
Representative Procedure for N-protected Sialyl Donors
Methyl 4,7,8,9-Tetra-O-acetyl-3,5-dideoxy-5-phthalimido-D-glycero-D-galacto-
2-nonulopyranosylonate-2-N-phenyltrifluoroacetimidate (2f):
To a solution of methyl (phenyl 5-acetamide-4,7,8,9-tetra-O-acetyl-3,5-
dideoxy-2-thio-D-glycero-D-galacto-2-nonulopyranosid)onate (1.5 g, 2.6 mmol)
in MeOH (25 mL) was added methanesulfonic acid (190 mL, 2.6 mmol) and
the resulting mixture was stirred at 608C for 24 h. After the solution was
cooled to rt, the mixture was concentrated in vacuo. Without purification of
the intermediary amine derivative, NaOMe (530 mL, 2.6 mmol) was added to

S.-i. Tanaka et al.
382
a crude material dissolved in anhydrous THF (25 mL) to neutralize at rt under
Ar atmosphere. After being stirred at rt for 10 min, phthalic anhydride (760 mg,
5.1 mmol) and Et3N (720 mL, 5.1 mmol) were added and the reaction mixture
was stirred at 708C overnight. After the solution was cooled to rt, the mixture
was concentrated in vacuo. The residue was dissolved in EtOAc; washed with
saturated aqueous NaHCO3, 1 M HCl, and brine; dried over Na2SO4; filtered;
and concentrated in vacuo to give the crude product, which was roughly
purified by column chromatography on silica gel (6% MeOH in chloroform) to
give the intermediate N-Phth derivative as colorless needles (798 mg, 62%).
1H NMR (500 MHz, CD3OD) d 7.93–7.91 (m, 1H, SPh), 7.66–7.64 (m, 2H,
Phth), 7.62–7.60 (m, 1H, SPh), 7.57–7.52 (m, 2H, Phth), 7.45–7.40 (m, 3H,
SPh), 4.77 (d, J ¼ 10.5 Hz, 1H, H-6), 4.33 (ddd, J ¼ 4.70, 9.80, 11.2 Hz, 1H,
H-4), 4.18 (brt, J ¼ 10.3 Hz, 1H, H-5), 4.13 (brd, J ¼ 8.25 Hz, 1H, H-7), 3.91–
3.85 (m, 3H, H-8, H-9a, H-9b), 3.60 (s, 3H, CO2CH3), 2.76 (dd, J ¼ 4.55,
13.5 Hz, 1H, H-3eq), 2.05 (dd, J ¼ 11.7, 13.4 Hz, 1H, H-3ax); ESI-MS m/z
calcd for C24H25NO9SNa (M þ Na)þ 526.11, found 526.11.
To a solution of N-Phth derivative obtained above (280 mg, 560 mmol) in
pyridine (1 mL) was added Ac2O (1 mL) at rt under Ar atmosphere. After the
solution was stirred at rt overnight, the mixture was concentrated in vacuo
and the residue was coevaporated with toluene three times. The residue was
dissolved in EtOAc, and the organic layer was washed with saturated
aqueous NaHCO3, 1 M HCl solution, and brine; dried over Na2SO4; filtered;
and concentrated in vacuo to give the crude product. The residue was
purified by silica-gel column chromatography (17% ethyl acetate in toluene)
to give N-Phth tetraacetate as colorless needles (61% for three steps): 1H
NMR (500 MHz, CDCl3) d 7.79 (m, 2H, Phth), 7.68–7.66 (m, 2H, Phth),
7.53–7.51 (m, 2H, SPh), 7.32–7.28 (m, 3H, SPh), 5.89 (td, J ¼ 5.15, 10.8,
10.8 Hz, 1H, H-4), 5.71 (dd, J ¼ 2.45, 10.5 Hz, 1H, H-6), 5.20 (dd, J ¼ 2.45,
3.65 Hz, 1H, H-7), 4.93 (ddd, J ¼ 2.85, 7.00, 8.23 Hz, 1H, H-8), 4.36 (dd,
J ¼ 2.60, 12.3 Hz, 1H, H-9a), 4.14 (t, J ¼ 10.4 Hz, 1H, H-5), 3.96 (dd,
J ¼ 7.75, 12.4 Hz, 1H, H-9b), 3.62 (s, 3H, CO2CH3), 2.72 (dd, J ¼ 5.50,
13.8 Hz, 1H, H-3eq), 2.04 (s, 3H, Ac), 1.90 (s, 3H, Ac), 1.87 (s, 3H, Ac), 1.75
(s, 3H, Ac), 2.03 (dd, J ¼ 11.3, 14.4 Hz, 1H, H-3ax); ESI-MS m/z calcd for
C32H33NO13SNa (M þ Na)þ 694.16, found 694.16.
To a solution of N-Phth tetraacetate obtained above (50 mg, 74 mmol) in
acetone were added NBS (27 mg, 150 mmol) and water (4.0 mg, 220 mmol) at
rt. After the solution was stirred at rt for 1 h, the mixture was quenched by
10% aqueous Na2S2O3 and extracted with EtOAc. The organic layer was
washed with saturated aqueous NaHCO3 and brine, dried over Na2SO4,
filtered, and concentrated in vacuo to give the crude product. The residue
was purified by column chromatography on silica gel (chloroform) to give the
1
hydrolyzed derivative as colorless needles (33 mg, 75%): H NMR (500 MHz,
CDCl3) d 7.88–7.80 (m, 2H, Phth), 7.74–7.72 (m, 2H, Phth), 5.82 (ddd,

Highly Efficient a-Sialylation
383
J ¼ 5.63, 10.5, 10.5 Hz, 1H, H-4), 5.40 (ddd, J ¼ 2.58, 6.17, 8.12 Hz, 1H, H-8),
5.26 (dd, J ¼ 2.01, 10.5 Hz, 1H, H-6), 5.13 (dd, J ¼ 2.04, 7.92 Hz, 1H, H-7),
4.24 (dd, J ¼ 2.64, 12.5 Hz, 1H, H-9a), 4.22 (t, J ¼ 10.5 Hz, 1H, H-5), 4.03
(dd, J ¼ 6.22, 12.4 Hz, 1H, H-9b), 3.89 (s, 3H, CO2CH3), 2.36 (dd, J ¼ 5.57,
13.0 Hz, 1H, H-3eq), 2.32–2.29 (m, 1H, H-3ax), 2.17 (s, 3H, Ac), 2.12 (s, 3H,
Ac), 2.12 (s, 3H, Ac), 1.95 (s, 3H, Ac); ESI-MS m/z calcd for C26H29NO14Na
(M þ Na)þ 602.15, found 602.16.
To solution of the compound obtained above (55 mg, 95 mmol) in acetone
(1.0 mL) were added N-phenyltrifluoroacetoimidoylchloride (200 mg,
950 mmol) and K2CO3 (39 mg, 290 mmol) at rt under Ar atmosphere. After
being stirred at rt for 3 h, the mixture was filtered and the filtrate was concen-
trated in vacuo to give the crude product. The residue was purified by column
chromatography on silica gel (17% ethyl acetate in toluene) to give 2f as a color-
less needles (70 mg, 99%). Since the imidate product was labile under the acidic
condition, after the identification by ESI-MS, it was immediately used for the
sialylation: ESI-MS m/z calcd for C32H33NO13SNa (M þ Na)þ 773.18, found
773.14.
Methyl 4,7,8,9-Tetra-O-acetyl-3,5-dideoxy-5-dimethylmaleimido-D-glycero-D-
galacto-2-nonulopyranosylonate-2-N-phenyltrifluoroacetimidate (2d):
ESI-MS m/z calcd for C32H35F3N2O14Na (M þ Na)þ 751.19, found 751.18.
Data for precursor of imidate: 1H NMR (500 MHz, CDCl3) d 5.66 (ddd,
J ¼ 5.53, 10.5, 10.5 Hz, 1H, H-4), 5.35 (ddd, J ¼ 2.44, 6.41, 8.03 Hz, 1H, H-8),
5.09–5.05 (m, 2H, H-6, H-7), 4.26 (dd, J ¼ 2.49, 12.4 Hz, 1H, H-9a), 4.03 (dd,
J ¼ 6.43, 12.3 Hz, 1H, H-9b), 3.99 (t, J ¼ 10.6 Hz, 1H, H-5), 3.87 (s, 3H,
CO2CH3), 2.30 (dd, J ¼ 5.51, 13.0 Hz, 1H, H-3eq), 2.25–2.21 (m, 1H, H-3ax),
2.14 (s, 3H, Ac), 2.10 (s, 3H, Ac), 2.00 (s, 3H, Ac), 1.90 (s, 3H, Ac), 1.95 (s, 6H,
CH3CH55CHCH3); ESI-MS m/z calcd for C24H31NO14Na (M þ Na)þ 580.16,
found 580.17.
Methyl 4,7,8,9-Tetra-O-acetyl-3,5-dideoxy-5-tetrachlorophthalimido-D-glycero-
D-galacto-2-nonulopyranosylonate-2-N-phenyltrifluoroacetimidate (2e):
ESI-MS m/z calcd for C24H29Cl4F3N2O14Na (M þ Na)þ 909.02, found
909.04. Data for precursor of imidate: 1H NMR (500 MHz, CDCl3) d 5.69
(ddd, J ¼ 6.13, 10.4, 10.4 Hz, 1H, H-4), 5.31 (ddd, J ¼ 2.41, 5.93, 8.18 Hz, 1H,
H-8), 5.18 (dd, J ¼ 2.06, 10.5 Hz, 1H, H-6), 5.05 (dd, J ¼ 2.23, 8.04 Hz, 1H,
H-7), 4.20 (dd, J ¼ 2.52, 12.4 Hz, 1H, H-9a), 4.12 (t, J ¼ 10.4 Hz, 1H, H-5),
3.95 (dd, J ¼ 6.03, 12.4 Hz, 1H, H-9b), 3.83 (s, 3H, CO2CH3), 2.27 (dd,
J ¼ 6.11, 12.9 Hz, 1H, H-3eq), 2.26–2.23 (m, 1H, H-3ax), 2.05 (s, 3H, Ac),
2.04 (s, 3H, Ac), 1.91 (s, 3H, Ac), 1.80 (s, 3H, Ac); ESI-MS m/z calcd for
C26H25Cl4NO14Na (M þ Na)þ 737.99, found 738.00.

S.-i. Tanaka et al.
384
Representative Procedure of Sialylation
To a solution of the donor 2f (50 mg, 67 mmol), acceptor 3 (45 mg, 100 mmol),
and MS4A (70 mg) in propionitrile (1.0 mL) was added TMSOTf (2.5 mL,
13 mmol) at –788C under Ar atmosphere. After the mixture was stirred for
30 min at this temperature, the reaction was quenched by a saturated
NaHCO3 solution. After MS4Awas removed by filtration, filtrate was extracted
with ethyl acetate, washed with brine, dried over Na2SO4, filtered, and con-
centrated in vacuo to give the crude product. The residue was purified by
column chromatography on silica gel (chloroform) to afford the a-sialoside 4f
(61 mg, 92%).
Allyl 2,3-Di-O-benzoyl-6-O-(methyl 4,7,8,9-tetra-O-acetyl-3,5-dideoxy-
5-phthalimido-D-glycero-a-D-galacto-2-nonulopyranosylonate)-a-D-
galactopyranoside (4f):
(a-isomer) 1H NMR (500 MHz, CDCl3) d 7.97–7.92 (m, 4H, aromatic),
7.76–7.75 (m, 2H, Phth), 7.66–7.65 (m, 2H, Phth), 7.45–7.42 (m, 4H,
aromatic), 7.32–7.28 (m, 2H, aromatic), 5.87 (ddd, J ¼ 5.51, 10.7, 22.3 Hz,
1H, OCH2CH55CH2), 5.71 (m, 2H, H-2,3), 5.53 (ddd, J ¼ 5.03, 10.6, 11.1 Hz,
1H, H-40), 5.45 (dd, J ¼ 2.71, 5.55, 8.42 Hz, 1H, H-80), 5.34 (m, 1H, H-4), 5.31
(dd, J ¼ 1.73, 17.2 Hz, 1H, OCH2CH55CH2), 5.18 (dd, J ¼ 2.42, 8.31 Hz, 1H,
H-70), 5.15 (dd, J ¼ 1.44, 10.4 Hz, 1H, OCH2CH55CH2), 5.10 (dd, J ¼ 2.34,
10.6 Hz, 1H, H-60), 4.43 (s, 1H, H-1), 4.29 (dd, J ¼ 2.73, 9.76 Hz, 1H, H-90a),
4.27 (dd, J ¼ 5.09, 13.2 Hz, 1H, OCH2CH55CH2), 4.23 (t, J ¼ 10.6 Hz, 1H,
H-50), 4.19 (t, 1H, H-5), 4.00 (dd, J ¼ 6.00, 13.3 Hz, 1H, OCH2CH55CH2),
4.05–4.02 (m, 2H, H-90b, H-6a), 3.91 (s, 3H, CO2CH3) 3.87 (dd, J ¼ 6.50,
10.0 Hz, 1H, H-6b), 3.03 (s, 1H, OH), 2.80 (dd, J ¼ 5.14, 12.9 Hz, 1H, H-30eq),
2.15 (s, 3H, Ac), 2.13 (s, 3H, Ac), 1.88 (s, 3H, Ac), 1.84 (s, 3þH, Ac), 2.01
(m, 1H, H-30ax); HRMS m/z calcd for C49H51NO21Na (M þ Na) 1012.2851,
found 1012.2873. The a-configuration of 4f was confirmed by converting to
1
the corresponding N-Ac derivatives. (b-isomer) H NMR (500 MHz, CDCl3) d
8.17–8.15 (m, 2H), 8.00–7.95 (m, 4H), 7.72–7.69 (m, 2H), 7.49–7.43 (m, 2H),
7.37–7.29 (m, 2H), 5.86–5.76 (m, 1H), 5.69–5.65 (m, 1H), 5.46–5.44 (m, 1H),
4.83 (s, 1H), 4.65 (dd, J ¼ 2.50, 12.5 Hz, 1H), 4.52–4.43 (m, 2H), 4.23–3.65
(m, 7H), 3.78 (s, 3H), 3.75–3.763 (1H, m), 2.60 (dd, J ¼ 5.50, 12.5 Hz, 1H),
2.05 (s, 3H, Ac), 2.00 (s, 3H, Ac), 1.92 (s, 3H, Ac).
Allyl 2,3-Di-O-benzoyl-6-O-(methyl 4,7,8,9-tetra-O-acetyl-3,5-dideoxy-
5-dimethylmaleimido-D-glycero-a-D-galacto-2-nonulopyranosylonate)-
a-D-galactopyranoside (4d):
1H NMR (500 MHz, CDCl3) d 8.03–7.98 (m, 4H, aromatic), 7.52–7.48
(m, 2H, aromatic), 7.39–7.35 (m, 4H, aromatic), 5.86 (m, 1H,
OCH2CH55CH2), 5.70 (m, 2H, H-2,3), 5.42 (ddd, J ¼ 2.83, 6.14, 8.45 Hz, 1H,

Highly Efficient a-Sialylation
385
H-80), 5.36 (ddd, J ¼ 5.12, 10.2, 10.2 Hz, 1H, H-40), 5.34–5.33 (m, 1H, H-4), 5.31
(dd, J ¼ 1.65, 15.6 Hz, 1H, OCH2CH55CH2), 5.12 (dd, J ¼ 2.41, 8.24 Hz, 1H,
H-70), 5.15–5.13 (m, 1H, OCH2CH55CH2), 4.92 (dd, J ¼ 2.16, 10.8 Hz, 1H,
H-60), 4.41 (s, 1H, H-1), 4.31 (dd, J ¼ 2.87, 12.5 Hz, 1H, H-90a), 4.26 (dd,
J ¼ 5.13, 13.3 Hz, 1H, OCH2CH55CH2), 4.17 (t, 1H, H-5), 4.07–3.97 (m, 4H,
H-50, H-6a, H-90b, OCH2CH55CH2), 3.87 (s, 3H, CO2CH3), 3.85 (dd, J ¼ 6.74,
10.0 Hz, 1H, H-6b), 2.75 (dd, J ¼ 5.13, 12.9 Hz, 1H, H-30eq), 2.12 (s, 3H, Ac),
1.94 (s, 3H, Ac), 1.93 (s, 3H, Ac), 1.91, (s, 3H, Ac), 1.94 (m, 1H, H-30ax), 1.94
(s, 6H, CH3CH55CHCH3); ESI-MS m/z calcd for C47H53NO21Na (M þ Na)þ
990.30, found 990.37.
Allyl 2,3-Di-O-benzoyl-6-O-(methyl 4,7,8,9-tetra-O-acetyl-3,5-dideoxy-
5-tetrachlorophthalimido-D-glycero-a-D-galacto-2-nonulopyranosylonate)-
a-D-galactopyranoside (4e):
1H NMR (500 MHz, CDCl3) d 8.03–7.98 (m, 4H, aromatic), 7.51–7.50
(m, 2H, aromatic), 7.39–7.35 (m, 4H, aromatic), 5.87 (m, 1H,
OCH2CH55CH2), 5.71 (m, 2H, H-2,3), 5.49–5.43 (m, 2H, H-40, H-80), 5.34
(m, 1H, H-4), 5.31 (dd, J ¼ 1.77, 15.5 Hz, 1H, OCH2CH55CH2), 5.18 (dd,
J ¼ 2.49, 8.18 Hz, 1H, H-70), 5.15 (dd, J ¼ 1.66, 10.6 Hz, 1H,
OCH2CH55CH2), 5.05 (dd, J ¼ 2.65, 10.8 Hz, 1H, H-60), 4.42 (d, J ¼ 0.90 Hz,
1H, H-1), 4.32 (dd, J ¼ 2.91, 12.5 Hz, 1H, H-9a), 4.27 (dd, J ¼ 5.15, 13.3 Hz,
1H, OCH2CH55CH2), 4.23 (t, J ¼ 10.5 Hz, 1H, H-50), 4.19 (t, J ¼ 6.33 Hz, 1H,
H-5), 4.07 (dd, J ¼ 6.07, 13.3 Hz, 1H, OCH2CH55CH2), 4.03 (dd, J ¼ 5.51,
12.6 Hz, 1H, H-90b), 4.07–4.02 (m, 1H, H-6a), 3.90 (s, 3H, CO2CH3) 3.86 (dd,
J ¼ 6.76, 10.1 Hz, 1H, H-6b), 2.80 (dd, J ¼ 5.22, 13.0 Hz, 1H, H-30eq), 2.14
(s, 3H, Ac), 2.13 (s, 3H, Ac), 1.92 (s, 3H, Ac), 1.88 (s, 3H, Ac), 2.03 (m, 1H,
H-30ax); ESI-MS m/z calcd for C49H47Cl4NO21Na (M þ Na)þ 1148.13, found
1148.13.
Allyl 4,6-O-Benzylidene-3-O-(methyl 4,7,8,9-tetra-O-acetyl-3,5-dideoxy-
5-phthalimido-D-glycero-a-D-galacto-2-nonulopyranosylonate)-
a-D-galactopyranoside (11):
1H NMR (500 MHz, CDCl3) d 7.83–7.81 (m, 2H, Phth), 7.72–7.71 (m, 2H,
Phth), 7.50–7.48 (m, 2H, aromatic), 7.34–7.31 (m, 3H, aromatic), 5.99–5.92
(m, 1H, OCH2CH55CH2), 5.50 (ddd, J ¼ 4.94, 10.5, 11.8 Hz, 1H, H-40), 5.44
(s, 1H, PhCH), 5.41 (ddd, J ¼ 2.92, 4.84, 9.13 Hz, 1H, H-80), 5.34 (dd,
J ¼ 1.61, 17.2 Hz, 1H, OCH2CH55CH2), 5.18 (dd, J ¼ 1.8,3 10.6 Hz, 1H,
OCH2CH55CH2), 5.16 (dd, J ¼ 1.93, 9.23 Hz, 1H, H-70), 5.11 (d, J ¼ 3.55 Hz,
1H, H-1), 5.04 (dd, J ¼ 1.91, 10.9 Hz, 1H, H-60), 4.65 (dd, J¼ 3.35, 10.4 Hz,
1H, H-2), 4.27 (dd, J ¼ 5.05, 13.2 Hz, OCH2CH55CH2), 4.24–4.18 (m, 2H,
H-6a, H-90a), 4.20 (t, J ¼ 10.4 Hz, 1H, H-50), 4.16–4.12 (m, 3H, H-3, H-4,
H-6b), 4.09 (dd, J ¼ 5.53, 13.3 Hz, 1H, OCH2CH55CH2), 4.05 (dd, J ¼ 4.92,
12.5 Hz, 1H, H-90b), 3.75 (s, 1H, H-5), 3.71 (s, 3H, CO2CH3), 2.61 (s, 1H, OH),

S.-i. Tanaka et al.
386
2.87 (dd, J ¼ 5.08, 13.0 Hz, 1H, H-30eq), 2.17 (s, 3H, Ac), 2.15 (s, 3H, Ac), 1.90
(s, 3H, Ac), 1.80 (s, 3H, Ac), 2.02 (dd, J ¼ 11.8, 13.1 Hz, 1H, H-30ax); ESI-MS m/
z calcd for C42H47NO19Na (M þ Na)þ 892.81, found 892.26.
Allyl 2-O-Benzoyl-6-O-benzyl-3-O-(methyl 4,7,8,9-tetra-O-acetyl-3,5-dideoxy-
5-phthalimido-D-glycero-b-D-galacto-2-nonulopyranosylonate)-a-D-
galactopyranoside (12):
1H NMR (500 MHz, CDCl3) d 8.13–8.12 (m, 4H, PhCO-), 7.83–7.82 (m, 2H,
Phth), 7.74–7.73 (m, 2H, Phth), 7.59–7.56 (m, 4H, PhCO-), 7.47 (m, 2H, PhCO-
), 7.37–7.32 (m, 5H, PhCH2-), 5.90 (ddd, J ¼ 4.56, 9.73, 22.1 Hz, 1H, -
OCH2CH55CH2), 5.52 (td, J ¼ 5.00, 15.9 Hz, 1H, H-40), 5.43–5.39 (m, 2H,
H-80, H-2), 5.31 (m, 1H, -OCH2CH55CH2), 5.24 (d, J ¼ 2.64 Hz, 1H, H-1),
5.16–5.12 (m, 2H, H-70, -OCH2CH55CH2), 5.02 (dd, J ¼ 1.72, 10.9 Hz, 1H,
H-60), 4.76 (dd, J ¼ 3.58, 10.3 Hz, 1H, H-90a), 4.64 (d, J ¼ 8.53 Hz, 1H,
PhCH2-), 4.62 (d, J ¼ 8.51 Hz, 1H, PhCH2-), 4.21 (m, 1H, OCH2CH55CH2),
4.21 (m, 1H, H-3), 4.17 (m, 1H, H-50), 4.19 (m, 1H, H-5), 4.06 (dd, J ¼ 6.01,
13.3 Hz, 1H, -OCH2CH55CH2), 4.00 (dd, J ¼ 5.43, 12.4 Hz, 1H, H-90b), 3.89–
3.78 (m, 2H, H-6), 3.82 (s, 3H, -COOCH3), 2.95 (brs, 1H, -OH), 2.62 (dd,
J ¼ 4.93, 13.3 Hz, 1H, H-30eq), 2.12 (s, 3H, Ac), 2.00 (s, 3H, Ac), 1.91 (s, 3H,
Ac), 1.80 (s, 3H, Ac), 2.07 (m, 1H, H-30ax); ESI-MS m/z calcd for
C49H53NO20Na 998.31 (M þ Na)þ, found 998.34.
p-Nitrophenyl 4-O-Benzoyl-2,6-di-O-benzyl-3-O-(methyl 4,7,8,9-tetra-
O-acetyl-3,5-dideoxy-5-phthalimido-D-glycero-a-D-galacto-2-
nonulopyranosylonate)-b-D-galactopyranoside (Benzoyl Derivative of 13a):
1H NMR (500 MHz, CDCl3) d 8.20–8.18 (m, 2H, aromatic), 8.07–8.05
(m, 2H, aromatic), 7.83–7.82 (m, 2H, Phth), 7.73–7.72 (m, 2H, Phth), 7.62–
7.59 (m, 1H, aromatic), 7.50–7.47 (m, 2H, aromatic), 7.40–7.39 (m, 2H,
aromatic), 7.31–7.22 (m, 8H, aromatic), 7.11–7.09 (m, 2H, aromatic), 5.59–
5.57 (m, 2H, H-4, H-80), 5.49 (ddd, J ¼ 5.00, 10.5, 11.6 Hz, 1H, H-40), 5.33 (dd,
J ¼ 3.32, 10.1 Hz, 1H, H-70), 5.26 (d, J ¼ 3.51 Hz, 1H, H-1), 5.20 (dd,
J ¼ 2.22, 8.53 Hz, 1H, H-3), 4.90 (d, J ¼ 12.5 Hz, 1H, CH2Ph), 4.78–4.75
(m, 2H, H-2, H-60), 4.65 (d, J ¼ 12.7 Hz, 1H, CH2Ph), 4.54 (d, J ¼ 11.9 Hz,
1H, CH2Ph), 4.50 (t, J ¼ 6.02 Hz, 1H, H-5), 4.46 (d, J ¼ 11.9 Hz, 1H, CH2Ph),
4.29 (dd, J ¼ 2.81, 12.6 Hz, 1H, H-90a), 4.14 (dd, J ¼ 4.31, 12.7 Hz, 1H,
H-90b), 4.00–3.96 (t, 1H, H-50), 3.96 (s, 3H, CO2CH3), 3.57–3.55 (m, 2H,
H-6a,b), 2.52 (dd, J ¼ 5.02, 12.6 Hz, 1H, H-30eq), 2.18 (s, 3H, Ac), 2.15 (s, 3H,
Ac), 1.90 (s, 3H, Ac), 1.73 (s, 3H, Ac); ESI-MS m/z calcd for C59H58N2O22Na
(M þ Na)þ 1169.34, found 1169.32.

Highly Efficient a-Sialylation
p-Nitrophenyl 3-O-Benzoyl-2,6-di-O-benzyl-4-O-(methyl 4,7,8,9-tetra-
387
O-acetyl-3,5-dideoxy-5-phthalimido-D-glycero-a-D-galacto-2-
nonulopyranosylonate)-b-D-galactopyranoside (Benzoyl Derivative of 13b):
1H NMR (500 MHz CDCl3) d 8.19–8.17 (m, 2H, aromatic), 8.07–8.05
(m, 2H, aromatic), 7.82–7.80 (m, 2H, Phth), 7.73–7.72 (m, 2H, Phth), 7.60–
7.57 (m, 1H, aromatic), 7.47–7.44 (m, 2H, aromatic), 7.31–7.05 (m, 12H,
aromatic), 5.70 (ddd, J ¼ 2.83, 5.04, 9.41 Hz, 1H, H-80), 5.51 (ddd, J ¼ 5.02,
10.5, 11.7 Hz, 1H, H-40), 5.45 (dd, J ¼ 3.33, 13.0 Hz, 1H, H-2), 5.02–4.97
(m, 1H, H-3), 4.99 (d, J ¼ 12.6 Hz, 1H, CH2Ph), 4.73 (dd, J ¼ 12.6, 10.6 Hz,
1H, H-60), 4.73 (d, J ¼ 12.6 Hz, 1H, CH2Ph), 4.55 (d, J ¼ 11.9 Hz, 1H,
CH2Ph), 4.47 (d, J ¼ 11.9 Hz, 1H, CH2Ph), 4.30 (dd, J ¼ 2.71, 12.5 Hz, 1H,
H-90a), 4.12 (t, J ¼ 6.21 Hz, 1H, H-5), 4.03–3.97 (m, 2H, H-50H-90b), 4.03
(s, 3H, CO2CH3), 3.66 (dd, J ¼ 6.31, 9.93 Hz, 1H, H-6a), 3.59 (dd, J ¼ 6.22,
9.93 Hz, 1H, H-6b), 2.57 (dd, J ¼ 5.01, 12.6 Hz, 1H, H-30eq), 2.21 (s, 3H, Ac),
2.15 (s, 3H, Ac), 1.94 (s, 3H, Ac), 1.74 (s, 3H, Ac); ESI-MS m/z calcd for
C59H58N2O22Na (M þ Na)þ 1169.34, found 1169.32.
2-Azidoethyl 2,3,6-O-Benzoyl-4-O-[2,6-benzoyl-4-hydroxyl-3-O-(methyl 4,7,8,
9,-tetra-O-acetyl-3,5-dideoxy-5-phthalimido-D-glycero-a-D-galacto-2-
nonulopyranosylonate)-b-D-galactopyranosyl]-b-D-glucopyranoside (14):
1H NMR (500 MHz, CDCl3) d 8.10–8.08 (m, 4H), 7.99–7.97 (m, 2H), 7.93–
7.91 (m, 4H), 7.84–7.82 (m, 2H), 7.75–7.73 (m, 2H), 7.65–7.63 (m, 1H), 7.58–
7.33 (m, 14H), 5.73 (t, J ¼ 9.46 Hz, 1H), 5.46–5.36 (m, 3H), 5.27–5.23 (m, 1H),
5.09 (dd, J ¼ 1.53 Hz, 9.46 Hz, 1H), 4.79 (dd, J ¼ 1.53 Hz, 10.9 Hz, 1H), 4.73
(d, J ¼ 7.78 Hz, 1H), 4.64 (dd, J ¼ 1.98 Hz, 13.0 Hz 1H), 4.61 (d, J ¼ 7.93 Hz,
1H), 4.49 (dd, J ¼ 4.43 Hz, 12.1 Hz, 1H), 4.39 (d, J ¼ 2.75 Hz, 1H), 4.21
(t, J ¼ 9.46 Hz, 1H), 4.19 (t, J ¼ 10.7 Hz, 1H), 4.11 (dd, J ¼ 3.05 Hz, 12.7 Hz,
1H), 3.98 (dd, J ¼ 4.58 Hz, 12.7 Hz, 1H), 3.95–3.91 (m, 2H), 3.85–3.80
(m, 1H), 3.81 (s, 3H), 3.67–3.61 (m, 3H), 2.83 (dd, 1H, J ¼ 5.04 Hz, 13.0 Hz),
2.09 (s, 3H, Ac), 2.03 (s, 3H, Ac), 2.02–1.98 (m, 1H), 1.92 (s, 3H, Ac), 1.82
(s, 3H, Ac).
Phenyl 2,3,6-O-Benzoyl-4-O-[2,6-benzoyl-4-hydroxyl-3-O-(methyl 4,7,8,9-
tetra-O-acetyl-3,5-dideoxy-5-phthalimido-D-glycero-a-D-galacto-2-nonu-
lopyranosylonate)-b-D-galactopyranosyl]-1-thio-b-D-glucopyranoside (15):
1H NMR (500 MHz, CDCl3) d 8.17 (m, 2H), 8.03 (m, 2H), 7.98–7.94
(m, 5H), 7.77–7.30 (m, 17H), 7.18 (t, J ¼ 7.35, 1H), 7.09–7.05 (m, 2H), 5.68
(t, J ¼ 9.30 Hz, 1H), 5.57 (ddd, J ¼ 2.20 Hz, 4.73 Hz, 9.73 Hz, 1H), 5.40
(t, J ¼ 9.80 Hz, 1H), 5.35–5.25 (m, 2H), 5.03 (dd, J ¼ 2.55 Hz, 9.63 Hz, 1H),
4.87–4.84 (m, 3H), 4.67 (d, J ¼ 11.3 Hz, 1H), 4.50–4.47 (m, 2H), 4.22 (dd,
J ¼ 2.30 Hz, 13.0 Hz, 1H), 4.14 (t, J ¼ 9.65 Hz, 1H), 4.03–4.00 (m, 1H), 3.95
(dd, J ¼ 4.85 Hz, 12.6 Hz, 1H), 3.89 (t, J ¼ 10.4 Hz, 1H), 3.82-3.79 (m, 1H),

S.-i. Tanaka et al.
388
3.70 (s, 3H), 3.65–3.56 (m, 2H), 3.44–3.43 (m, 1H), 2.69 (dd, J ¼ 5.05 Hz,
12.6 Hz, 1H), 1.93 (3H, s, Ac), 1.93 (3H, s, Ac), 1.77 (3H, s, Ac), 1.48 (3H, s,
Ac); HRMS m/z calcd for C79H73NO28SNa (M þ Na)þ 1538.3937, found
1538.3953.
Phenyl 2,3,6-O-Benzyl-4-O-[2,6-benzyl-4-hydroxyl-3-O-(methyl 4,7,8,9,-
tetra-O-acetyl-3,5-dideoxy-5-phthalimido-D-glycero-a-D-galacto-2-
nonulopyranosylonate)-1-thio-b-D-galactopyranosyl]-b-D-
glucopyranoside (16):
1H NMR (500 MHz, CDCl3) d 7.82 (bs, 2H), 7.73–7.72 (m, 2H), 7.56–7.54
(m, 2H), 7.41–7.16 (m, 14H), 5.53–5.47 (m, 2H), 5.17 (dd, J ¼ 2.30 Hz,
8.78 Hz), 5.06–5.03 (m, 2H), 4.82–4.69 (m, 5H), 4.62 (d, J ¼ 9.75 Hz, 1H),
4.61 (d, J ¼ 7.60 Hz, 1H), 4.51 (bs, 2H), 4.45 (d, J ¼ 12.1 Hz, 1H), 4.36
(t, J ¼ 11.9 Hz, 1H), 4.23–4.16 (m, 3H), 4.02 (t, J ¼ 9.50 Hz, 1H), 3.95 (dd,
J ¼ 5.20 Hz, 12.6 Hz, 1H), 3.86–3.84 (bs, 1H), 3.85 (s, 3H), 3.70–3.67
(m, 1H), 3.61 (t, J ¼ 8.85 Hz, 1H), 3.57–3.49 (m, 3H), 3.44 (t, J ¼ 9.60 Hz,
1H), 3.40–3.37 (m, 1H), 2.73 (dd, J ¼ 5.00 Hz, 13.0 Hz, 1H), 2.11 (s, 3H, Ac),
1.93 (s, 3H, Ac), 1.88 (s, 3H, Ac), 1.82 (s, 3H, Ac), 2.12–1.98 (m, 1H); ESI-MS
m/z calcd for C79H83NO23SNa (M þ Na)þ 1468.50, found 1468.51.
Deprotection Procedure of N-phthalyl Group
To a solution of 4f (1.0 mg, 1.0 mmol) in anhydrous EtOH (200 mL) were
added methylhydrazine (3.2 mL, 61 mmol) and AcOH (3.5 mL, 61 mmol) at rt
under Ar atmosphere. After the solution was stirred 24 h at 808C and cooled
to rt, the mixture was concentrated in vacuo. A crude product obtained was dis-
solved in Ac2O and pyridine at rt. After being stirred overnight, the mixture
was concentrated in vacuo and coevaporated with toluene three times. The
residue was dissolved in EtOAc; washed with saturated aqueous NaHCO3
solution, 1 M HCl solution, and brine; dried over Na2SO4; filtered; and concen-
trated in vacuo to give the crude product. The residue was purified by silica-gel
column chromatography (from 0 to 2% MeOH in chloroform) to give 18 as color-
less needles (800 mg, 84%).
Allyl 4-O-Acetyl-2,3-di-O-benzoyl-6-O-(methyl 5-acetamido-4,7,8,9-tetra-O-
acetyl-3,5-dideoxy-D-glycero-a-D-galacto-2-nonulopyranosyloate)-a-D-
galactopyranoside (18):
1H NMR (500 MHz, CDCl3) d 7.96–7.91 (m, 4H), 7.45–7.41 (m, 4H), 7.32–
7.26 (m, 4H), 5.89–5.81 (m, 1H), 5.39–5.28 (3H, m), 5.17–512 (m, 2H), 4.93–
4.88 (m, 1H), 4.40–4.37 (2H, m), 4.27–4.23 (2H, m), 4.18–4.02 (5H, m), 3.93
(dd, J ¼ 5.50, 9.50 Hz, 1H), 3.83–3.80 (m, 1H), 3.82 (s, 3H), 2.17 (s, 3H, Ac),
2.18 (s, 3H, Ac), 2.13 (s, 3H, Ac), 2.02 (s, 3H, Ac), 1.95 (s, 3H, Ac), 1.88 (s, 3H,
Ac), 2.06–2.03 (1H, m); ESI-MS m/z calcd for C45H53NO21Na (M þ Na)þ
966.30, found 966.43.

Highly Efficient a-Sialylation
389
Allyl 4-O-Acetyl-2-O-benzoyl-6-O-benzyl-3-O-(methyl 5-acetamido- 4,7,8,9-
tetra-O-acetyl-3,5-dideoxy-D-glycero-a-D-galacto-2-nonulopyranosyloate)-
a-D-galactopyranoside (19):
1H NMR (500 MHz, CDCl3) d 8.12-8.10 (m, 2H), 7.62–7.19 (m, 8H), 5.86–
5.79 (m, 1H), 5.41–5.38 (m, 1H), 5.29 (dd, J ¼ 2.50, 7.50 Hz, 1H), 5.25–5.12
(m, 4H), 5.02 (t, J ¼ 10.1 Hz, 1H), 5.02 (dd, J ¼ 5.50, 10.0 Hz, 1H), 4.88–4.82
(m, 1H), 4.50 (d, J ¼ 11.5 Hz, 1H), 4.42 (d, J ¼ 11.5 Hz, 1H), 4.33 (dd,
J ¼ 2.50, 12.5 Hz, 1H), 4.25 (brt, J ¼ 6.00 Hz, 1H), 4.20–4.17 (m, 1H), 4.09
(dd, J ¼ 2.50, 12.5 Hz, 1H), 4.02 (q, J ¼ 10.0 Hz, 1H), 3.98–3.94 (m, 1H), 3.68
(dd, J ¼ 2.00, 10.8 Hz, 1H), 3.44 (dd, J ¼ 2.50, 6.00 Hz, 2H), 2.42 (dd,
J ¼ 5.00, 12.8 Hz, 1H), 2.03 (3H, s, Ac), 1.99 (3H, s, Ac), 1.96 (3H, s, Ac), 1.89
(3H, s, Ac), 1.82 (3H, s, Ac), 1.79 (3H, s, Ac), 1.73 (t, J ¼ 12.5 Hz, 1H) ESI-
MS m/z calcd for C45H55NO20Na 952.32 (M þ Na)þ, found 951.54.
Phenyl 2,3,6-O-Benzoyl-4-O-[2,6-benzoyl-4-O-acetyl-3-O-(methyl 6-acetoamide-
4,7,8,9-tetra-O-acetyl-3,5-dideoxy-5-acetoamido-D-glycero-a-D-galacto-2-
nonulopyranosylonate)-b-D-galactopyranosyl]-1-thio-b-D-glycopyranoside
(20):
1H NMR (500 MHz, CDCl3) d 7.95–7.92 (m, 2H, Bz), (m, 6H, Bz), 7.74–7.73
(m, 2H, Bz), 7.54–7.16 (m, 15H, Bz), 7.11–7.06 (m, 4H, SPh), 6.97
(t, J ¼ 8.00 Hz, 2H, SPh), 5.70 (t, J ¼ 9.00 Hz, 1H, H-3), 5.48–5.43 (m, 2H,
H-20, H-40), 5.40–5.37 (m, 1H, H-800), 5.32–5.26 (m, 2H, H-2, H-30), 5.23 (dd,
J ¼ 1.00, 8.75 Hz, 1H, H-900a), 4.89 (d, J ¼ 8.00 Hz, 1H, H-10), 4.86
(d, J ¼ 10.0 Hz, 1H, H-1), 4.80–4.75 (m, 1H, H-800), 4.58 (dd, J ¼ 2.00,
11.8 Hz, 1H, H-6a), 4.50 (dd, J ¼ 6.50 Hz, 12.0 Hz, 1H, H-6b), 4.33 (dd,
J ¼ 3.00 Hz, 12.0 Hz, 1H, H-700), 4.18 (t, J ¼ 10.0 Hz, 1H, H-4), 4.06–4.03
(m, 2H, H-900b, H-600), 3.96–3.90 (m, 1H, H-500), 3.80–3.77 (m, 1H, H-60a), 3.71
(s, 3H, CO2Me), 3.44 (dd, J ¼ 5.50 Hz, 10.8 Hz, 1H, H-60b), 2.76–2.72 (m, 1H,
H-50), 2.37 (dd, J ¼ 4.50 Hz, 12.8 Hz, 1H, H-300eq), 2.14 (s, 3H, Ac), 2.11
(s, 3H, Ac), 2.02 (s, 3H, Ac), 1.94 (s, 3H, Ac), 1.86 (s, 3H, Ac), 1.83 (s, 3H, Ac),
1.77–1.72 (m, 1H, H-300ax); ESI-MS m/z calcd for C73H73NO27SNa 1492.41
(M þ Na)þ, found 1492.56.
Representative Procedure of Microfluidic Reaction
A solution of TMSOTf (54 mL, 0.30 mmol, 0.15 M) in CH2Cl2 (2.0 mL) was
injected in advance to the micromixer by using a syringe pump at the flow
rate of 1.0 mL/min. Subsequently, a solution of donor 2f (150 mg, 0.20 mmol,
0.20 M) and the acceptor 3 (43 mg, 0.10 mmol, 0.10 M) dissolved in EtCN
(1.0 mL) was also injected to the IMM micromixer by another syringe pump
at the flow rate of 1.0 mL/min and mixed at 2788C. After the reaction
mixture was allowed to flow at 2788C for an additional 47 sec through a stain-
less reactor tube (F ¼ 1.0 mm, l ¼ 1.0 m), the mixture was quenched by

S.-i. Tanaka et al.
390
another flow of an excess triethylamine dissolved in CH2Cl2 at –788C. It takes
about 2 to 3 min to consume 43 mg of the acceptor 3 under the above conditions.
The mixture was extracted with ethyl acetate, washed with brine, dried over
Na2SO4, filtered, and concentrated in vacuo to give the crude product. The
excess glycal obtained as a byproduct was removed by preparative TLC on
1
silica gel (4% MeOH in chloroform) and the yield of 4f was analyzed by H
NMR. The conditions established herein can be readily applicable to the
scale-up synthesis simply by preparing the stock solutions of substrate
and reagents and pumping them continuously into the micromixer (i.e.,
a-sialylation using 2.3 g of the acceptor 3 under the present microfluidic
system has been realized for 10 min).
Computational Methods
The conformational distribution search for 17a–17c and 17f in Figure 1
has been performed by the Spartan02 package[18,19] using the MMFF94s mol-
ecular mechanics force field. The most stable conformations are shown in
Figure 1. Similarly, the preliminary conformational distribution search for sim-
plified N-Pht and N-Ac2 moieties in Figure 2 was first performed by the
Spartan02 package using the MMFF94s molecular mechanics force field. The
real minimum energy conformers found by molecular mechanics have been
further fully optimized at the DFT/B3LYP/6-31Gꢀ level as implemented in
the Gaussian03 package.[20] These calculations gave one minima for N-Pht
and two absolute minima for N-Ac2 (trans isomer is more stable than cis
isomer by 3.57 kcal/mol). The dipole moment (m) was calculated on these
obtained three absolute minima by means of DFT methods using the hybrid
B3LYP functional and the 6-31Gꢀ basis set as available within Gaussian03.
ACKNOWLEDGMENT
The present work was financially supported in part by Grant-in-Aid for Scien-
tific Research No. 17310128, No. 18850014, and No. 19681024 from the Japan
Society for the Promotion of Science and also by the Mitsubishi Foundation,
Research Grants in Natural Sciences.
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