Reinvestigation of the C5-acetamide sialic acid donor for α-selective sialylation: Practical procedure under microfluidic conditions

Article abstract of DOI:10.1039/c1ob06164j

Despite the previous literature describing the "low-to-modest" efficiency, the readily available C5-acetamide donor was reinvestigated for its use in α-sialylation under microfluidic conditions. The N-phenyltrifluoroacetimidate donor was efficiently mixed with an appropriate amount of TMSOTf to produce the α(2-6) and α(2-3)-sialylation products of galactose and glucosamine acceptors in excellent yields and with nearly perfect α-selectivity.

Full text of DOI:10.1039/c1ob06164j

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PAPER
Reinvestigation of the C5-acetamide sialic acid donor for a-selective
sialylation: practical procedure under microfluidic conditions†
Yosuke Uchinashi, Masahiro Nagasaki, Jiazhou Zhou, Katsunori Tanaka* and Koichi Fukase*
Received 14th July 2011, Accepted 22nd July 2011
DOI: 10.1039/c1ob06164j
Despite the previous literature describing the “low-to-modest” efficiency, the readily available
C5-acetamide donor was reinvestigated for its use in a-sialylation under microfluidic conditions. The
N-phenyltrifluoroacetimidate donor was efficiently mixed with an appropriate amount of TMSOTf to
produce the a(2–6) and a(2–3)-sialylation products of galactose and glucosamine acceptors in excellent
yields and with nearly perfect a-selectivity.
In contrast, the N-Ac derivatives have not been extensively
Introduction
studied for a-sialylation.3,10 These are considered to be the simplest
sialic acid donors because they are readily available from the
commercially available compounds and homologous to the natural
5N-Ac sialosides in that they do not require N-derivatization
after sialylation. N-Ac imidate shows high a-selectivity in a few
cases, such as a(2–6)-sialylation with the galactose acceptor,11
although most reports,3,10 including one of our own,9a have
described the sialylation using N-Ac donors resulted in the modest
yields and a-selectivities (~50% and a:b = ~3 : 1, respectively).
Recently, Kononov and co-workers has rationalized the “low-
to-modest” efficiency using the N-Ac donors by proving the
hydrogen-bonding network between the N- and O-acetyl moieties;
the supramolecular aggregation of the glycosyl donors, which is
sensitively affected by the reaction concentrations, attenuates the
nucleophilic attack and/or a-face approach of the acceptor.12
On the other hands, during a recent investigation of a-
sialylation under microfluidic conditions,9,13 we realized that in
the conventional flask reaction, the efficiency and reproducibility
of the reaction is quite sensitive to the heat generated during
syringe-addition of the Lewis acid to the donor and acceptor. We
hypothesized that this point has not been investigated to a great
depth for the N-Ac case. That is, the efficient sialylation could be
achieved by using the N-Ac donor, but the heat generated during
the inefficient mixing in the flask may lead to poor yield, selectivity,
and sometimes the reproducibility. Therefore, we reinvestigated the
use the most straightforward C5-acetamide donor in pursuit of a
practical route to a-sialylation.
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 and is usually linked to
galactose or N-acetylgalactosamine through a(2–3) or a(2–6)
sialoglycosidic bonds.1 Because the presence of Neu5Ac on cell
surfaces plays diverse and important roles, such as pathogen/host
recognition, tumor metastasis, regulation of immunosuppressive
signals, and cell differentiation/proliferation, much effort has
been devoted to the development of efficient and stereoselec-
tive synthetic methods for producing a(2–3) and Neu5Aca(2–
6)Gal units to investigate their biological functions.2 a-Sialylation
entails glycosylation at a sterically and electronically unfavorable
oxocarbenium ion in such a way that the neighboring groups do
not influence the stereochemical outcome. Although this reaction
is one of the most difficult in the field of oligosaccharide synthesis,
recent advances in glycosylation chemistry, e.g., the development
of new leaving and protecting groups, have enabled the efficient
construction of a-sialoside linkages.3
The novel a-sialoside linkages feature a variety of C5-
substituents, such as carbamates, on the sialic acid donors that
may be used to tune the reactivity of the oxocarbenium ions.
These substituents include N-Ac2,4 N-TFA,5,6c N-Troc,6 and azide7
groups and show improved yields and a-selectivities. Recently,
the 5,4-N,O-cyclic carbamates have been utilized especially for
the a(2–8)- and a(2–9)-sialylation case.8 We have developed
N-phenyltrifluoroacetimidate donors with C5-phthalimide9a,b or
azide9c groups to apply the fixed dipole moment effects, and we
have achieved quantitative sialylation with complete a-selectivity
under microfluidic conditions.
Results and discussion
We initially optimized the a-sialylation of the N-Ac imidate
111,14 using the C6-hydroxyl group of the galactose acceptor 29a
in the presence of TMSOTf as an activator in propionitrile
at -78 ◦C (Table 1). Assuming that temperature control was
critical for sialylation in the presence of the donor 1, the reaction
was initially performed on a small scale (50–100 mg scale) by
Department of Chemistry, Graduate School of Science, Osaka University,
Osaka, 560-0043, Japan. E-mail: ktzenori@chem.sci.osaka-u.ac.jp, koichi@
chem.sci.osaka-u.ac.jp; Fax: +81 06 6850 5419; Tel: +81 06 6850 5391
† Electronic supplementary information (ESI) available: Copies of NMR
spectra. See DOI: 10.1039/c1ob06164j
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Table 1 Optimization of the a-sialylation of N-Ac sialyl imidate using the C6-hydroxy of galactose
T/◦C
Apparatus
Yield (%)
a:b
c
Entry
1 (equiv)a
2 (equiv)b
LA (equiv)
1
2
3
4
5
6
7
8
2.0
2.0
2.0
2.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.5
1.5
1.5
1.5
1.5
1.5
0.1
0.5
1.0
1.0
0.2
0.4
1.0
1.0
1.0
0.2
-78
-78
-78
-78
-78
-78
-78
-78
-60
-20
Flask
Flask
Flask
Micro
Flask
Flask
Flask
Micro
Micro
Flask
Trace
<10
77
89
Trace
40
86
89
82
89
NA
99 : 1
96 : 4
94 : 6
NA
99 : 1
93 : 7
94 : 6
86 : 14
74 : 26
9
10
a Entries 1–4: 100 mM, entries 5–10: 50 mM. b Entries 1–4: 50 mM, entries 5–10: 75 mM. c Determined by 1HNMR.
Fig. 1 Practical a-sialylation under microfluidic conditions.
conventional syringe addition of the Lewis acid to a mixture
containing the donor and acceptor in a flask apparatus. One
goal was to verify that the syringe-addition procedure in the
flask did not affect the glycosylation outcome by introducing
heterogeneous mixing and local heating that would re-route the
reaction pathways away from the desired outcome. To achieve this
goal, the bulk reaction was compared with the reaction performed
under microfluidic conditions15,16 using a Comet X-01 apparatus,17
which produces efficient mixing and fast heat transfer.13,18–22
Another advantage to performing the reaction in the micromixer is
that the optimized conditions are directly applicable to large-scale
synthesis under flow. The optimal conditions may be appropriately
scaled up to yield a practical a-sialylation of oligosaccharides
(vide infra).
The reaction did not proceed significantly upon addition of
catalytic amounts of the acid, i.e., 0.1 equiv TMSOTf, to the
solution containing the acceptor 2 (1 equiv, 50 mM) and the donor
1 (2 equiv, 100 mM) (Table 1, entry 1). Increasing the acid to 0.5
equiv gave only a small amount of the disaccharide 3 (entry 2). The
reaction stopped after only 5 min, and the donor 1 was recovered
intact. Thus, the reaction yield was proportional to the amount of
Lewis acid present. We were gratified to find that 1.0 equiv of the
Lewis acid resulted in formation of the disaccharide 3 in 77% yield
with excellent a-selectivity (a : b = 96 : 4, entry 3). The conditions
in entry 3 were reproducible under the microfluidic conditions
(entry 4, 89% yield, a : b = 94 : 6, see the reaction system shown in
Fig. 1).
Although donor 1 exhibited excellent a-sialylation reactivity in
the presence of substoichiometric amounts of the acid under the
optimized conditions, the presence of excess donor (2 equiv relative
to the acceptor) inhibited purification of the sialoside 3 from the
glycal byproduct due to similar polarities on the silica gel. Such a
difficulty is incompatible with large-scale synthetic approaches.
Therefore, we optimized the conditions by reversing the ratio
between the donor and acceptor. One and one-half equivalents
of the acceptor 2 (75 mM) were used with donor 1 (50 mM,
entries 5–10). The observed trend mirrored the optimization
trend described above. Catalytic amounts of TMSOTf (0.2 equiv)
yielded essentially no product formation (entry 5). The use of
0.4 equiv acid gave 40% 3 (entry 6), and 1.0 equiv TMSOTf
successfully provided the a-sialoside 3 in 86% yield with good
selectivity (a : b = 93 : 7, entry 7). The yield and selectivity were
validated by performing the sialylation reaction described in entry
7 under microfluidic conditions (89%, a : b = 94 : 6, entry 8).
Concentrations of the substrates and Lewis acid did not affect
the outcome of the sialylation efficiency under the conditions
examined here;12 although the reactions at higher concentrations
than those performed in entry 8 could not be examined due to the
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◦
low solubility of the substrates in EtCN at -78 C, an excellent
Table 1, even catalytic amounts of the Lewis acid promoted these
glycosylation reactions. Hence, the amount of acid was optimized
in each case to maximize the glycosylation efficiency. The reaction
with the C6-hydroxy of N-Troc glucosamine 4 in the presence of
0.2 equiv TMSOTf provided the corresponding sialoside 5 in 95%
yield with perfect a-selectivity. Sialylation proceeded smoothly for
the more sterically demanding C3-hydroxy of galactose 6 in the
presence of 0.5 equiv of the acid, with an a-selectivity that yielded
7 in 80% yield. Reaction with the C9–OH of the sialic acid 8, a
hydroxyl group known for its poor reactivity, resulted in formation
of the glycal (9). This reaction illustrates the limitations associated
with using the N-Ac donor 1.
The pursuit of a sialylation protocol with practical applications
to large-scale synthesis led us to integrate microfluidic procedures
into the bulk procedures. The concentrations of the substrates
and Lewis acid in the final reaction mixture were scaled down
to half of those listed in Table 1, entry 8 to prevent solution
blockage problems.18 A propionitrile solution of the sialyl donor 1
(50 mM) and the acceptor 2 (75 mM) was mixed with TMSOTf in
propionitrile (50 mM) at -78 ◦C using a Comet X-01 micromixer
with a channel width of 500 mm at a flow rate of 1.0 mL min-1
(Fig◦. 1). The reaction mixture was allowed to flow with cooling at
-78 C for an additional 50 s through the reactor tube (f 3 = 1.0 mm,
l = 1.0 m). The mixture was then introduced into a flask apparatus
at -78 ◦C. Continuous stirring for another 1 hr at this temperature
successfully provided a gram-scale preparation of the disaccharide
3 without decreasing the efficiency (89%, a : b = 94 : 6).
reactivity of 1 was observed even in the diluted solutions (see Fig. 1
below). It should be noted that the mixing temperature affected
the a-selectivity in a non-trivial way. Mixing at -60 ◦C such that
the temperature was precisely adjusted by a micromixer yielded
the sialoside 3 in 82% yield but with substantially decreased a-
selectivity, a : b = 86 : 14 (entry 9).
As noted previously, most reported flask sialylation trials using
N-Ac sialyl donors resulted in unsatisfactory results (~50% yields,
a : b = ~3 : 1). We previously reported that microfluidic sialylation
under the conditions described in entry 5 in Table 1 proceeded
with good yields but with moderate selectivity (93%, a : b =
77 : 23).9a To resolve these contradictory results (current results:
<5% yield (entry 5)), we intentionally added 0.2 equiv acid to
the -20 ◦C solution containing the donor and acceptor in a
flask (entry 10). The sialoside 3 was rapidly obtained in 89%
yield with a : b = 74 : 26. The contradictory results may have
resulted from temperature heterogeneities and/or heat generation
during inefficient mixing in the flask, which promote the catalytic
reaction at the expense of a-selectivity. To counteract such effects,
one might add excess quantities of the Lewis acid in a small-
scale reaction. Considering that a slight increase in the mixing
temperature non-trivially affected the a-selectivity (entry 9), we
concluded that the low efficiency and reproducibility of the N-
Ac sialyl donors, reported by us and others, was not due to the
inherently reactivity of the N-Ac donors, but was derived from the
mixing inefficiencies associated with a flask apparatus.
After the NeuNAca(2–6)Gal 3 was found to be obtained
efficiently by using N-Ac donor 1 under the optimized conditions
listed in Table 1, the other important a-sialylation reactions of
the natural glycans, including a-sialylation of the C3- and C6-
hydroxyls of galactose and glucosamine acceptors, were examined
(Scheme 1). To our surprise, in contrast with the results reported in
Conclusion
In conclusion, we explored the utility of the most straightforward
C5-acetamide donor 1 for practical a-sialylation. The previously
reported poor and non-reproducible sialylation results may have
been due not to the inherent reactivity of this donor, but to
mixing artifacts in the flask apparatus, in addition to the aggregate
formation of the N-Ac donors under the certain concentrations.12
To achieve a large-scale synthesis, we used a microfluidic apparatus
to achieve rapid initial mixing, thereby establishing a practical
bulk a-sialylation procedure. Other reactive intermediates may
have been ignored simply due to technical issues associated
with the conventional flask apparatus. These results suggest that
several traditional reactions merit reinvestigation in microfluidic
devices, which have not been extensively utilized in organic
synthesis.
Experimental section
General
All commercially available reagents were used without further
purification. Propionitrile was refluxed over and distilled from
CaH2. Preparative separation was performed by column chro-
matography on silica gel (FUJI silysia LTD, BW-200 and BW-
1
300). H and 13C NMR spectra were recorded on either JEOL
JNM-LA 500 spectrometer and chemical shifts were represented
as d-values relative to the internal standard TMS. The melting
point was determined on a microscopic melting apparatus and un-
corrected. Optical rotation was measured on a PERKIN ELMER
models 241 polarimeter. IR spectra were recorded on a JASCO
Scheme 1 a-Selective sialylation with the C3- and C6-hydroxyls of
galactose and glucosamine acceptors.
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FT/IR-6100 Fourier Transform Infrared Spectrometer. MALDI-
TOF-mass spectra were measured on a PerSeptive Biosystems,
Voyager RP-DE/H mass spectrometer equipped with a nitrogen
laser (l = 337 nm).
the mixture was filtered and concentrated in vacuo. The residue
was directly purified by column chromatography on silica gel
(gradually from 50 to 83% of ethyl acetate in toluene) to give the
1
sialoside 5 as an a-isomer based on H NMR analysis (138 mg,
95%) as a white solid: [a]D20 +29.0 (c 0.1, CHCl3); mp 100–101 ◦C;
IR (neat, cm-1) 3388, 2955, 1747, 1518, 1452; MALDI-TOF-MS
m/z calcd for C47H55Cl3N2O21 (M+Na)+ 1111.2, found 1111.2; 1H
NMR (500 MHz, CDCl3) d 7.76 (d, J = 7.4 Hz, 2H, Fmoc), 7.60
(dd, J = 7.9, 9.4 Hz, 2H, Fmoc), 7.40 (t, J = 7.5 Hz, 2H, Fmoc),
7.32 (tdd, J = 7.3, 2.9, 1.2 Hz, 2H, Fmoc), 5.88 (ddd, J = 23.2, 10.3,
5.3 Hz, 1H, OCH2CH CH2), 5.37 (ddd, J = 11.2, 5.6, 2.6 Hz, 1H,
H-8), 5.37 (d, J = 9.7 Hz, 1H, TrocNH), 5.30 (dd, J = 10.0, 1.8 Hz,
1H, H-7), 5.30 (dd, J = 10.0, 1.8 Hz, 1H, AcHN), 5.29 (dq, J =
15.6, 1.5 Hz, 1H, OCH2CH CH2), 5.22 (dq, J = 10.3, 1.2 Hz, 1H,
OCH2CH CH2), 5.07 (dd, J = 10.5, 9.7 Hz, 1H, H-3¢), 5.03 (td,
J = 10.5, 4.7 Hz, 1H, H-4), 4.94 (d, J = 3.8 Hz, 1H, H-1¢), 4.61 (d,
J = 12.0 Hz, 1H, Cl3CCH2), 4.54 (d, J = 12.0 Hz, 1H, Cl3CCH2),
4.42 (dd, J = 10.3, 7.6 Hz, 1H, Fmoc), 4.33 (dd, J = 10.6, 7.6 Hz,
1H, Fmoc), 4.32 (dd, J = 12.0, 3.2 Hz, 1H, H-9a), 4.23 (dd, J =
15.0, 5.9 Hz, 1H, Fmoc), 4.23 (dd, J = 7.9, 2.1 Hz, 1H, H-6a¢),
4.18 (ddt, J = 12.6, 5.3, 1.5 Hz, 1H, OCH2CH CH2), 4.15 (dd,
J = 10.9, 1.8 Hz, 1H, H-6), 4.09 (dd, J = 12.0, 5.6 Hz, 1H, H-9b),
4.07 (dd, J = 10.3, 3.8 Hz, 1H, H-2¢), 4.05 (q, J = 10.6 Hz, 1H,
H-5), 4.00 (ddt, J = 12.9, 6.5, 1.2 Hz, 1H, OCH2CH CH2), 3.97
(dd, J = 10.0, 5.0 Hz, 1H, H-4¢), 3.81 (s, 3H, CO2Me), 3.77 (dd,
J = 10.9, 2.9 Hz, 1H, H-6b¢), 3.77 (ddd, J = 10.9, 4.4, 1.8 Hz, 1H,
H-5¢), 3.48 (d, J = 5.0 Hz, 1H, HO-4¢), 2.66 (dd, J = 13.5, 5.0 Hz,
1H, H-3eq), 2.13 (s, 3H, Ac), 2.11 (s, 3H, Ac), 2.07 (s, 3H, Ac),
2.06 (dd, J = 12.6, 10.0 Hz, 1H, H-3ax), 2.03 (s, 3H, Ac), 1.90 (s,
3H, Ac); 13C NMR (125 MHz, CDCl3) d 170.96, 170.63, 170.27,
170.11, 169.75, 168.21, 155.65, 154.23, 143.26, 143.18, 141.25,
133.16, 127.91, 127.21, 125.20, 120.04, 118.32, 97.92, 96.60, 95.31,
77.36, 74.57, 72.28, 71.09, 70.39, 68.78, 68.65, 68.57, 67.79, 67.30,
62.81, 62.3, 54.23, 52.88, 49.84, 46.62, 37.79, 30.86, 23.15, 21.05,
20.85, 20.69, 20.55; a-Confuguration of the sialoside linkage23 was
determined based on the 3JC1-3Hax of 13.9 Hz.
Allyl 2,3-Di-O-benzoyl-6-O-(Methyl 5-acetamide-4,7,8,9-
tetra-O-acetyl-3,5-dideoxy-D-glycero-a-D-galacto-2-
nonulopyranosylonate)-a-D-galactopyranoside 3
To a solution of the NAc donor 1 (30.0 mg, 45.3 mmol), the
acceptor 2 (29.1 mg, 68.0 mmol), and dry MS4A in EtCN (1.0 mL)
was added TMSOTf (8.2 mL, 45.3 mmol) at -78 ◦C, and the
resulting mixture was stirred at this temperature for 6 h. After
the reaction was quenched by adding the excess triethylamine
at -78 ◦C, the mixture was filtered and concentrated in vacuo.
The residue was directly purified by column chromatography on
silica gel from 67% to 100% of ethyl acetate in toluene) to give
the disaccharide 3 (35.1 mg and 86% (sum of a + b), a : b =
93 : 7). These a- and b-isomers were further separated by column
chromatography on silica gel (gradually from 67% to 100% of
ethyl acetate in toluene). Data for a-isomer (white solid): [a]D20
◦
+69.0 (c 0.1, CHCl3); mp 94–96 C; IR (neat, cm-1) 3377, 2957,
1742, 1538, 1452; MALDI-TOF-MS m/z calcd for C43H51NO20
(M+Na)+ 924.3, found 924.3; 1H NMR (500 MHz, CDCl3) d 8.01
(ddd, J = 16.3, 8.4, 1.3 Hz, 4H, Bz), 7.52–7.48 (m, 2H, Bz), 7.37
(td, J = 7.4, 3.6 Hz, 4H, Bz), 5.85 (ddd, J = 22.2, 10.5, 5.9 Hz, 1H,
OCH2CH CH2), 5.69 (dd, J = 2.1, 0.9 Hz, 1H, H-2¢), 5.69 (dd,
J = 2.1, 0.9 Hz, 1H, H-3¢), 5.37 (ddd, J = 13.7, 7.4, 2.7 Hz, 1H,
H-8), 5.34 (dd, J = 7.4, 2.0 Hz, 1H, H-7), 5.32 (d, J = 2.0 Hz, 1H,
H-1¢), 5.30 (ddd, J = 15.5, 3.2, 1.6 Hz, 1H, OCH2CH CH2), 5.21
(d, J = 9.7 Hz, 1H, AcHN), 5.14 (ddd, J = 10.6, 2.7, 1.1 Hz, 1H,
OCH2CH CH2), 4.90 (ddd, J = 12.0, 10.0, 4.7 Hz, 1H, H-4), 4.39
(bd, J = 2.9 Hz, 1H, H-4¢), 4.39 (dd, J = 12.3, 2.7 Hz, 1H, H-9a),
4.26 (ddt, J = 13.2, 4.6, 1.6 Hz, 1H, OCH2CH CH2), 4.17 (t, J =
6.2 Hz, 1H, H-5¢), 4.12 (dd, J = 10.7, 2.1 Hz, 1H, H-6), 4.06 (dd,
J = 12.3, 6.2 Hz, 1H, H-9b), 4.06 (q, J = 9.7 Hz, 1H, H-5), 4.05
(ddt, J = 13.2, 7.3, 1.6 Hz, 1H, OCH2CH CH2), 3.93 (dd, J =
9.7, 5.6 Hz, 1H, H-6a¢), 3.82 (s, 3H, CO2Me), 3.82 (dd, J = 9.7,
7.3 Hz, 1H, H-6b¢), 3.05 (d, J = 4.6 Hz, 1H, HO-4¢), 2.60 (dd, J =
12.7, 4.7 Hz, 1H, H-3eq), 2.14 (s, 3H, Ac), 2.11 (s, 3H, Ac), 2.03
(s, 3H, Ac), 2.01 (dd, J = 12.6, 7.2 Hz, 1H, H-3ax), 1.94 (s, 3H,
Ac), 1.88 (s, 3H, Ac); 13C NMR (125 MHz, CDCl3) d 170.91,
170.87, 170.21, 170.17, 168.08, 165.99, 165.91, 133.62, 133.13,
133.1, 129.81, 129.78, 129.72, 129.58, 128.33, 117.37, 98.73, 95.79,
72.90, 71.15, 69.25, 69, 68.92, 68.55, 68.26, 67.73, 67.48, 62.98,
62.60, 53.01, 49.43, 37.05, 23.15, 21.03, 20.8, 20.76, 20.58; a-
Confuguration of the sialoside linkage23 was determined based
on the 3JC1-3Hax of 6.9 Hz.
Allyl 2-O-Benzoyl-6-O-benzyl-3-O-(methyl 5-acetamide-
4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero-a-D-
galacto-2-nonulopyranosylonate)-a-D-galactopyranoside 7
To a solution of the donor 1 (30.0 mg, 45.3 mmol) and the acceptor
6 (28.2 mg, 68.0 mmol), and dry MS4A in◦EtCN (1.0 mL) was
added TMSOTf (4.1 ml, 22.7 mmol) at -78 C, and the resulting
mixture was stirred at this temperature for 6 h. After the reaction
was quenched by adding the excess triethylamine at -78 ◦C,
the mixture was filtered and concentrated in vacuo. The residue
was directly purified by column chromatography on silica gel
(gradually from 67% to 100% of ethyl acetate in toluene) to give
1
the disaccharide 7 essentially as an a-isomer based on H NMR
Allyl 2-Amino-2-deoxy-3-O-9-fluorenylmethoxycarbonyl-2-N-
2,2,2-trichloroethoxycarbonyl-6-O-(methyl 5-acetamide-
4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero-a-D-galacto-2-
nonulopyranosylonate)-a-D-glucopyranoside 5
analysis (32.3 mg, 80%) as a white solid: [a]2D0 +31.0 (c 0.1, CHCl3);
◦
mp 77–78 C; IR (neat, cm-1) 3375, 2928, 1748, 1537, 1453;
MALDI-TOF-MS m/z calcd for C43H53NO19 (M+Na)+ 910.3,
1
found 910.2; H NMR (500 MHz, CDCl3) d 8.09 (dd, J = 8.4,
To a solution of the donor 1 (88.0 mg, 133 mmol), the acceptor
4 (128 mg, 207 mmol), and dry MS4A in EtCN (1.0 mL) was
added TMSOTf (5.4 mL, 29.8 mmol) at -78 ◦C, and the resulting
mixture was stirred at this temperature for 6 h. After the reaction
was quenched by adding the excess triethylamine at -78 ◦C,
1.4 Hz, 2H, Bz), 7.57 (tt, J = 7.4, 1.5 Hz, 1H, Bz), 7.45 (t, J =
7.8 Hz, 2H, Bz), 7.36–7.32 (m, 4H, Bn), 7.27 (tt, J = 6.6, 2.3 Hz,
1H, Bn), 5.88 (ddd, J = 22.2, 10.5, 6.0 Hz, 1H, OCH2CH CH2),
5.40 (dd, J = 10.3, 3.7 Hz, 1H, H-2¢), 5.32 (dd, J = 5.6, 2.4 Hz,
1H, H-8), 5.31 (dd, J = 5.0, 2.4 Hz, 1H, H-7), 5.29 (dq, J = 17.2,
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1.6 Hz, 1H, OCH2CH CH2), 5.20 (d, J = 9.7 Hz, 1H, AcHN),
5.20 (d, J = 3.9 Hz, 1H, H-1¢), 5.12 (dq, J = 10.4, 1.4 Hz, 1H,
OCH2CH CH2), 4.89 (ddd, J = 11.9, 10.3, 4.7 Hz, 1H, H-4), 4.66
(dd, J = 10.3, 3.3 Hz, 1H, H-3¢), 4.64 (d, J = 11.9 Hz, 1H, PhCH2),
4.60 (d, J = 11.6 Hz, 1H, PhCH2), 4.32 (dd, J = 12.4, 2.4 Hz, 1H,
H-9a), 4.24 (ddt, J = 13.3, 5.2, 1.6 Hz, 1H, OCH2CH CH2), 4.14
(t, J = 6.0 Hz, 1H, H-5¢), 4.10 (bd, J = 2.0 Hz, 1H, H-4¢), 4.08
(q, J = 10.0 Hz, 1H, H-5), 4.05 (ddt, J = 13.5, 6.0, 1.4 Hz, 1H,
OCH2CH CH2), 4.02 (dd, J = 12.7, 5.3 Hz, 1H, H-9b), 4.00 (dd,
J = 10.9, 2.0 Hz, 1H, H-6), 3.84 (dd, J = 10.2, 5.3 Hz, 1H, H-6a¢),
3.77 (dd, J = 10.0, 6.3 Hz, 1H, H-6b¢), 3.71 (s, 3H, CO2Me), 2.89
(d, J = 1.0 Hz, 1H, HO-4¢), 2.38 (dd, J = 13.0, 4.7 Hz, 1H, H-
3eq), 2.14 (t, J = 12.5 Hz, 1H, H-3ax), 2.09 (s, 3H, Ac), 2.01 (s,
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3H, Ac), 2.01 (s, 3H, Ac), 2.00 (s, 3H, Ac), 1.88 (s, 3H, Ac); 13
C
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168.12, 165.73, 138.27, 133.85, 133.06, 130.03, 129.80, 128.35,
128.32, 127.58, 127.52, 117.30, 98.63, 95.75, 73.51, 72.89, 70.80,
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Procedure of microfluidic a-sialylation
A solution of TMSOTf (270 mL, 1.50 mmol, 50 mM) in EtCN
(30.0 mL) was injected to the Comet X-01 micromixer by using a
syringe-pump at the flow rate of 1.0 mL/min. At the same time, a
solution of the NAc donor 1 (994 mg, 1.50 mmol, 50 mM) and the
galactosyl acceptor 2 (964 mg, 2.25 mmol, 75 mM) dissolved in
EtCN (30.0 mL) was also injected to the Comet X-01 micromixer
by another syringe-pump flow at the rate of 1.0 mL min-1, and
two solutions were mixed at -78 ◦C in the cooling bath. The
resulting mixture was allowed to flow at -78 ◦C for additional 50 s
through a Teflon tube reactor (U = 1.0 mm, l = 1.0 m), and then
the solution was introduced into a flask, which contained the dry
MS4A and cooled down in advance at -78 ◦C. After the reaction
was stirred for another 1 h at this temperature, it was quenched by
adding the excess triethylamine -78 ◦C. The mixture was filtered
and concentrated in vacuo to gave the crude product, which was
purified by the same procedure described above, producing the
sialoside 3 (1.17 g, 89%, a : b = 94 : 6).
Acknowledgements
This work was supported in part by the Grants-in-Aid for
Scientific Research from the Japan Society for the Promotion
of Science, New Energy and Industrial Technology Development
Organization (NEDO), and Grants-in-Aid for Scientific Research
from the Ministry of Education, Culture, Sports, Science and
Technology (MEXT) of Japan.
Notes and references
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G. W. Hart and P. Sinay¨, ed.; Wiley-VCH: Weinheim, 2000; Vol. 3, pp.
227; (d) T. Ando, H. Ando and M. Kiso, Trends Glycosci. Glycotechnol.,
2001, 13, 573; (e) M. L. Allende and R. L. Proia, Curr. Opin. Struct.
Biol., 2002, 12, 587; (f) P. R. Crocker, Curr. Opin. Struct. Biol., 2002,
12, 609; (g) R. Kannagi, Curr. Opin. Struct. Biol., 2002, 12, 599.
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7248 | Org. Biomol. Chem., 2011, 9, 7243–7248
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The Royal Society of Chemistry 2011
©