Chemo-enzymatic synthesis of p-nitrophenyl β-D-galactofuranosyl disaccharides from Aspergillus sp. fungal-type galactomannan

Article abstract of DOI:10.1016/j.carres.2019.01.005

β-D-Galactofuranose (Galf) is a component of polysaccharides and glycoconjugates. There are few reports about the involvement of galactofuranosyltransferases and galactofuranosidases (Galf-ases) in the synthesis and degradation of galactofuranose-containing glycans. The cell walls of filamentous fungi in the genus Aspergillus include galactofuranose-containing polysaccharides and glycoconjugates, such as O-glycans, N-glycans, and fungal-type galactomannan, which are important for cell wall integrity. In this study, we investigated the synthesis of p-nitrophenyl β-D-galactofuranoside and its disaccharides by chemo-enzymatic methods including use of galactosidase. The key step was selective removal of the concomitant pyranoside by enzymatic hydrolysis to purify p-nitrophenyl β-D-galactofuranoside, a promising substrate for β-D-galactofuranosidase from Streptomyces species.

Full text of DOI:10.1016/j.carres.2019.01.005

CarbohydrateResearch473(2019)99–103
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Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
Chemo-enzymatic synthesis of p-nitrophenyl β-D-galactofuranosyl
disaccharides from Aspergillus sp. fungal-type galactomannan
Ryo Otaa,1, Yumi Okamotoa,1, Christopher J. Vavrickab, Takuji Okac, Emiko Matsunagad,
Kaoru Takegawad, Hiromasa Kiyotaa, Minoru Izumia,∗
a Graduate School of Environmental and Life Science, Okayama University, Tsushima-naka 1-1-1, Okayama, 700-8530, Japan
b Graduate School of Science, Technology and Innovation, Kobe University, 1-1 Rokkodai, Nada, Kobe, 657-8501, Japan
c Department of Applied Microbial Technology, Faculty of Biotechnology and Life Science, Sojo University, Ikeda, 4-22-1, Kumamoto 860-0082, Japan
d Department of Bioscience and Biotechnology, Faculty of Agriculture, Kyushu University, Hakozaki 6-10-1, Fukuoka, 812-8581, Japan
A R T I C L E I N F O
A B S T R A C T
Keywords:
β-D-Galactofuranose (Galf) is a component of polysaccharides and glycoconjugates. There are few reports about
the involvement of galactofuranosyltransferases and galactofuranosidases (Galf-ases) in the synthesis and de-
gradation of galactofuranose-containing glycans. The cell walls of filamentous fungi in the genus Aspergillus
include galactofuranose-containing polysaccharides and glycoconjugates, such as O-glycans, N-glycans, and
fungal-type galactomannan, which are important for cell wall integrity. In this study, we investigated the
synthesis of p-nitrophenyl β-D-galactofuranoside and its disaccharides by chemo-enzymatic methods including
use of galactosidase. The key step was selective removal of the concomitant pyranoside by enzymatic hydrolysis
Galactofuranose
Chemo-enzymatic synthesis
p-Nitrophenyl galactofuranoside
Galactosidase
to purify p-nitrophenyl β-D-galactofuranoside,
Streptomyces species.
a promising substrate for β-D-galactofuranosidase from
1. Introduction
Galf and galactofuranose biosynthesis, as well as for the development of
specific antigens. p-Nitrophenyl glycosides are important and well
known as enzyme substrates, however, there are few reports on the
synthesis and use of p-nitrophenyl β-D-galactofuranoside (1) [12–14].
One major synthetic obstacle is the inability to use catalytic hydro-
genation for deprotection of benzyl groups in the presence of a p-ni-
trophenyl group. p-Nitrophenyl β-D-galactofuranoside is recently com-
mercially available, however, it is necessary to develop a convenient
and large-scale synthesis to enable medicine and agrochemical studies.
The present paper delineates the synthesis of p-nitrophenyl β-D-ga-
lactofuranoside and its disaccharides by chemo-enzymatic methods
including use of galactosidase.
β-D-Galactofuranose (Galf) is a component of polysaccharides and
glycoconjugates. There are few reports about the involvement of ga-
lactofuranosyltransferases and galactofuranosidases (Galf-ases) in the
synthesis and degradation of galactofuranose-containing glycans.
Recently, it has been revealed that ORF1110 from Streptomyces encodes
a Galf-specific Galf-ase, and that GfsA from Aspergillus fumigatus acts as
a
UDP-α-D-galactofuranose (β-D-galactofuranoside β1,5-galactofur-
anosyltransferase) in the biosynthetic pathway of galactomannans
[1–3]. The cell walls of filamentous fungi in the genus Aspergillus in-
clude galactofuranose-containing polysaccharides and glycoconjugates,
such as O-glycans, N-glycans, and fungal-type galactomannan (Fig. 1),
which are important for cell wall integrity [4,5]. Although Galf-glycans
have been broadly used as an indicator of pulmonary aspergillosis in
clinical settings, and its importance in fungal cell growth is recognized,
information pertaining to the galactofuranosyltransferase involved in
the biosynthesis of Galf-glycans is still sparse [6–9]. Galf metabolism is
a promising target for chemotherapy because Galfs are not biosynthe-
sized and/or utilized in mammals [10,11]. In this context, Galf-con-
taining oligosaccharides are valuable tools for the characterization of
The synthesis of some Galf derivatives have already been reported
[15–21]. With exception to sugars possessing the arabino (2,3,4-
trans,cis) configuration, monosaccharides prefer to exist in a pyranose
form [22]. Therefore, isomerization from a stable galactopyranose to
galactofuranose is important. To date, a method for D-galactose iso-
merization followed by protection with benzoyl groups has been re-
ported [12,15,16], however, it is troublesome to scale up the procedure
as benzoyl chloride is a fuming liquid with an irritating odor and a high
boiling point. In this paper, we used acetic anhydride for acetylation
∗ Corresponding author.
E-mail address: mizumi@okayama-u.ac.jp (M. Izumi).
1 These authors contributed equally to this work.
https://doi.org/10.1016/j.carres.2019.01.005
Received 18 December 2018; Received in revised form 10 January 2019; Accepted 10 January 2019
Availableonline11January2019
0008-6215/©2019ElsevierLtd.Allrightsreserved.

R. Ota et al.
CarbohydrateResearch473(2019)99–103
material, p-nitrophenyl β-D-galactofuranoside (1), was purified by si-
lica-gel column chromatography (chloroform (CHCl3):CH3OH, 4/1)
(Scheme 2). Additionally, 1 could be hydrolyzed by β-D-galactofur-
anosidase from Streptomyces species [1], while 1p was not hydrolyzed.
Our method was applied to the synthesis of a disaccharide with a
β(1,5) linkage due to its ease of donor and acceptor preparation. As a
donor, a mixture of 4 and 4p was treated with ammonium carbonate
((NH4)2CO3) in dimethylformamide (DMF)/H2O to give a mixture
of 6 and 6p. The reaction of the mixture of 6 and 6p with tri-
chloroacetonitrile (CCl3CN) in the presence of cesium carbonate
(Cs2CO3) obtained imidates 7 and 7p. Next, in order to prepare the
acceptor, regioselective isopropylidenation of 1 with acetone was cat-
alyzed by H2SO4 to produce 5,6-O-isopropylidenated product 8 in 77%
yield. Acetylation and subsequent deprotection of the 5,6-O-iso-
propylidene group under acidic conditions gave 10 in 92% yield over
two steps. In the presence of benzoyl chloride with pyridine, the pri-
mary hydroxy group was regioselectively benzoylated to give 6-O-
benzoylated compound 11 in 64% yield (Scheme 3).
Fig. 1. Galactofuranose-containing fungal-type galactomannan and p-ni-
trophenyl β-D-galactofuranoside (1).
To a solution of 11, 7 and 7p in CH3CN was added TMSOTf at 0 °C
followed by stirring at 23 °C for 24 h to produce a mixture of 12 and
12p. After deprotection under basic conditions using a solution of aq.
NH3 in CH3OH, the resulting mixture of 13 and 13p dissolved in 50 mM
phosphate buffer (pH 7.2) was treated with β-galactosidase (from
Escherichia coli, FUJIFILM Wako Pure Chemical Corporation) with
stirring at 37 °C for 24 h. After the selective hydrolysis of the pyranoside
13p to 1 and 2 being confirmed by TLC, the reaction mixture was
concentrated in vacuo. Since the TLC mobility of 13 was different from
those of 1 and 2, target material was easily purified by silica-gel column
chromatography (CHCl3:CH3OH, 4/1) to give p-nitrophenyl 5-O-(β-D-
galactofuranosyl)-β-D-galactofuranoside (pNP-Galf-β(1,5)-Galf, 13)
(Scheme 4) [2,14]. When a trialkylsilyl group was used to protect the 6-
position instead of a benzoyl group, glycosylation did not proceed.
In summary, we demonstrated the synthesis of p-nitrophenyl β-D-
galactofuranoside and its disaccharides by chemo-enzymatic methods
involving galactosidase. p-Nitrophenyl glycosides are important and
well known as enzyme substrates, so this work provides a solid and
reliable new approach for synthesizing galactofuranosyl-containing
oligosaccharides. Certainly, these studies present a broad scope to ac-
cess an orthogonal protected galactofuranosyl precursor of synthetic
relevance. Further developments of the present protocol are currently
being pursued in our laboratory.
instead of benzoylation in order to simplify the workup including
purification.
2. Results and discussion
D-Galactopyranose (2) was initially transformed into the corre-
sponding furanose (3) in pyridine with heating and then subjected to
subsequent per-O-acetylation in one-pot. After systematically exploring
reaction conditions (temperature and time) for the selective synthesis
and purification of the furanose, it was found that the reaction pro-
ceeded more rapidly and selectively at 70 °C. After isomerization at
70 °C in pyridine, one-pot acetylation with addition of acetic anhydride
at the same temperature afforded a mixture of per-O-acetylated D-ga-
lactofuranose (4) and D-galactopyranose (4p) in 96% yield. As esti-
mated by 1H NMR peak integration, the ratio of furanose to pyranose
was approximately two to three. It was difficult to separate the furanose
and pyranose species by silica-gel column chromatography, therefore
the resulting mixture was treated with p-nitrophenol in the presence of
boron trifluoride diethyl etherate (BF3·Et2O) to give a mixture of p-ni-
trophenyl 2,3,5,6-tetra-O-acetyl-β-D-galactofudanoside (5) and its pyr-
anoside derivative (5p) in 84% yield. Glycosyaltion proceeded β-se-
lectively because of the neighbouring group participation. The acetyl
groups of 5 (with 5p) were removed using a solution of aqueous am-
monia (aq. NH3) and methanol (CH3OH) to give a mixture of p-ni-
trophenyl β-D-galactofuranoside (1) and its pyranoside derivative (1p)
quantitatively (Scheme 1).
3. Experimental
3.1. General
Compounds were analyzed using the Varian 400 MHz systems at the
Okayama University Collaboration Center. 1H NMR (operating at
400 MHz) and 13C NMR (operating at 100 MHz) spectra were measured
in CDCl3 or D2O. NMR chemical shifts (δ) are provided in parts per
million (ppm), and coupling constants (J) are listed in Hz. Residual
peaks of chloroform (7.26 ppm) or H2O (4.63 ppm) were used as 1H
NMR references. The melting points were obtained on Yamato melting
point MP-21 apparatus. Optical rotations values were measured with a
JASCO P-2200 apparatus (20 °C, sodium discharge lamp). Elemental
analyses were measured at the Division of Instrumental Analysis,
Okayama University. TLC was carried out on Merck Kieselgel 60 PF254
It was not easy to separate 1 and 1p by silica-gel column chroma-
tography as both compounds have very similar mobility. We hypothe-
sized that a galactosidase should selectively hydrolyze pyranosides over
furanosides and this produced a simple strategy for isolation of fur-
anosides. On the basis of the hypothesis, we performed the following
experiment. To a solution of a mixture of 1 and 1p in 50 mM phosphate
buffer (pH 7.2) was added β-galactosidase (from Escherichia coli, FUJ-
IFILM Wako Pure Chemical Corporation) followed by stirring at 37 °C
for 24 h. After the selective pyranoside hydrolysis being checked by
TLC, the reaction mixture was concentrated in vacuo. The target
Scheme 1. Synthesis of p-nitrophenyl galacto-
furanoside (1). Reagents and conditions: (a)
pyridine, 70 °C, 1 h and then (b) Ac2O, 70 °C,
1 h, 96% over two steps; (c) p-NO2PhOH,
BF3·Et2O, CH3CN, 23 °C, 24 h, 84%; (d) aq. NH3,
CH3OH, 23 °C, 24 h, quant.
100

R. Ota et al.
CarbohydrateResearch473(2019)99–103
Scheme 2. Selective hydrolysis of 1p by β-galactosidase for isolation of 1.
3.2. Isomerization and acetylation to obtain per-acetylated galactofuranose
(4)
while stirring at 70 °C. After 1 h, aceticDa-nghayladcrtiodsee(6(1.0.801mgL,, 6103..05 mmmmooll))
To pyridine (30 mL) was added
was added into a reaction mixture at 70 °C and the mixture was stirred
for 1 h. Then the reaction mixture was cooled to 23 °C, stirred for 24 h,
and then concentrated in vacuo. The resulting syrup was diluted with
EtOAc (100 mL), washed with water (100 mL), 1 M aq. HCl (100 mL),
and brine, followed by drying over MgSO4. Concentration in vacuo gave
a mixture of per-acetylated galactofuranose (4) and per-acetylated ga-
lactopyranose (4p) in 96% yield (3.75 g, 9.60 mmol).
1H NMR (CDCl3): δ5.73–5.70 (m, 0.6H, H-14p), 5.43–5.41 (m, 0.4H,
H-14), 5.35–5.28 (m, 1H, H-3), 5.08–5.02 (m, 1H, H-2), 4.37–4.31 (m,
1H, H-4), 4.18–4.16 (m, 1H, H-5), 4.13–4.08 (m, 2H, H-6), 2.16 (s, 3H,
Ac), 2.12 (s, 3H, Ac), 2.10 (s, 3H, Ac), 2.04 (s, 3H, Ac), 1.99 (s, 3H, Ac);
13C NMR (CDCl3): δ 170.3, 170.1, 169.9, 169.8, 169.4, 169.0, 99.1,
93.0, 92.1, 89.7, 82.1, 80.6, 79.0, 76.3, 75.3, 73.3, 71.7, 70.8, 70.3,
69.2, 68.7, 67.8, 67.4, 67.3, 66.8, 66.4, 62.5, 62.1, 61.2, 61.0, 21.0,
20.9, 20.8, 20.7, 20.6, 20.5, 20.4; Anal. Calc. for: C16H22O11: C, 49.23;
H, 5.68. Found: C, 49.28; H, 5.87.
3.3. p-Nitrophenyl 2,3,4,6-tetra-O-acetyl-β-D-galactofuranoside (5)
To a solution of 4 and 4p (2:3, 3.72 g, 9.55 mmol) with MS4A in
CH3CN (50 mL) was added p-nitrophenol (3.99 g, 28.7 mmol) and
BF3·Et2O (3.63 mL, 28.67 mmol) at 0 °C. The reaction mixture was
stirred at the same temperature for 1 h and then at 23 °C for 24 h. Next
the mixture was filtered through a Celite pad and the filtrate was
concentrated in vacuo. The residue was diluted with EtOAc (100 mL),
washed with sat. aq. NaHCO3 solution (100 mL x 8) to remove excess p-
nitrophenol as a sodium salt and brine (50 mL), dried over MgSO4, and
concentrated in vacuo to dryness, producing a mixture of p-nitrophenyl
2,3,4,6-tetra-O-acetyl-β-D-galactofuranoside (5) and p-nitrophenyl
2,3,4,6-tetra-O-acetyl-β-D-galactopyranoside (5p) as a brown syrup
(3.77 g, 8.02 mmol, 84%). The mixture of 5 and 5p was used for de-
protection and hydrolysis without any purification.
Scheme 3. Preparation of glycosyl donor 7 and glycosyl acceptor 11. Reagents
and conditions: (a) (NH4)2CO3, DMF, H2O, 23 °C, 24 h, 68%; (b) CCl3CN, DCE,
23 °C, 3 h, quant; (c) conc. H2SO4, acetone, 23 °C, 3 h, 77%; (d) Ac2O, pyridine,
23 °C, 12 h, quant.; (e) 70% aq. AcOH, 23 °C, 12 h, 92%; (f) BzCl, pyridine,
23 °C, 12 h, 64%.
(0.25 mm thickness). Column chromatography was performed using
Merck Kieselgel 60 (0.063–0.200 mm). All chemicals used in this study
were commercially available, and all of the organic solvents used in
this study were dried over appropriate drying agents and distilled
prior to use.
1H NMR (CDCl3): δ 8.21 (m, 2H, Jo,m = 9.2 Hz, Ph(m)), 7.19 (d, 2H,
J
o,m = 9.2 Hz, Ph(o)), 5.88 (d, 1H, J1,2 = 4.0 Hz, H-1), 5.57–5.54 (m,
1H, H-4), 5.53–5.52 (m, 1H, H-5), 5.31 (dd, 1H, J2,3 = 4.0 Hz,
Scheme 4. Synthesis of β(1,5) linked disaccharide 13. Reagents and conditions: (a) TMSOTf, CH3CN, −15 °C then 23 °C, 24 h; (b) aq. NH3, CH3OH, 23 °C, 24 h; (c) β-
galactosidase (form E. Coli), phosphate Buffer, 37 °C, 24 h, 35% over three steps.
101

R. Ota et al.
CarbohydrateResearch473(2019)99–103
J3,4 = 10.8 Hz, H-3), 4.25 (t, 1H, J1,2 = J2,3 = 4.0 Hz, H-2), 4.13–4.03
(m, 2H, H-6), 2.18 (s, 3H, Ac), 2.08 (s, 3H, Ac), 2.04 (s, 3H, Ac), 1.93 (s,
3H, Ac); 13C NMR (CDCl3): δ 170.4, 170.3, 170.2, 170.1, 170.0, 169.9,
169.6, 169.3, 160.7, 142.8, 126.2, 125.8, 116.5, 103.7, 98.6, 94.7,
89.7, 81.8, 81.2, 76.2, 69.1, 68.7, 67.8, 67.2, 66.4, 62.4, 61.3, 61.2,
20.9, 20.8, 20.7, 20.6, 20.5; Anal. Calc. for: C20H23NO12+H2O: C,
47.81; H, 4.81; N, 2.79. Found: C, 47.59; H, 4.89; N, 2.67.
1H, H-2), 4.40–4.31 (m, 1H, H-4), 4.18–4.07 (m, 3H, H-5 and H-6),
2.14 (s, 3H, Ac), 2.09 (s, 3H, Ac), 2.04 (s, 3H, Ac), 1.98 (s, 3H, Ac); 13
C
NMR (CDCl3): δ 173.1, 170.6, 170.4, 170.3, 170.1, 96.7, 86.8, 68.2,
67.2, 61.8, 20.8, 20.7, 20.6, 20.5.
3.7. p-Nitrophenyl 5,6-O-isopropylidene-β-D-galactofuranoside (8)
To a solution of 1 (3.01 g, 10.0 mmol) in acetone (100 mL) was added
conc. H2SO4 (1.0 mL) followed by stirring at 23 °C for 3 h. The mixture
was concentrated in vacuo, and then the residue was diluted with CHCl3
(100 mL), washed with sat. aq. NaHCO3 solution (100 mL x 3) and brine
(50 mL), dried over Na2SO4, and concentrated in vacuo. Residue was
purified by silica-gel column chromatography (CHCl3:CH3OH, 9/1) to
give p-nitrophenyl 5,6-O-isopropylidene-β-D-galactofuranoside (8) as a
brown syrup (2.63 g, 7.70 mmol, 77%).
3.4. p-Nitrophenyl β-D-galactofuranoside (1)
To a solution of 5 and 5p (2.07 g, 4.41 mmol) in CH3OH (10 mL)
was added 28% aq. NH3 solution (10 mL) at 0 °C. The resulting solution
was stirred at the same temperature for 1 h and then at 23 °C for 24 h.
The reaction solution was concentrated using a rotary evaporator
(35 °C), and the resulting syrup was crystalized with Et2O to afford the
mixture of p-nitrophenyl β-D-galactofuranoside (1) and p-nitrophenyl β-
D-galactopyranoside (1p) as a brown solid (1.32 g, 4.39 mmol, 99%).
To a solution of the above mixture of 1 and 1p (0.58 g, 1.9 mmol) in
50 mM phosphate buffer (pH 7.2, 20 mL) was added β-galactosidase from
Escherichia coli (FUJIFILM Wako Pure Chemical Corporation, > 600
Units/mg, 1.0 mg) followed by stirring at 37 °C for 24 h. After confirming
selective pyranoside hydrolysis with TLC analysis, the reaction mixture
was washed with CHCl3 (20 mL x 3), and then concentrated in vacuo.
Target material was purified by silica-gel column chromatography
(CHCl3:CH3OH, 4/1) to give p-nitrophenyl β-D-galactofuranoside (pNP-
Galf, 1) as a colorless solid (0.25 g, 0.83 mmol, 43%).
[α]D −29.3 (c 0.26, CHCl3); 1H NMR (CDCl3): δ 8.20 (d, 2H,
Jo,m = 9.6 Hz, Ph(m)), 7.15 (d, 2H, Jo,m = 9.6 Hz, Ph(o)), 5.82 (s, 1H,
H-1), 5.51 (s, 1H, PhCH), 4.73–4.70 (m, 1H, H-2), 4.45–4.41 (m, 1H, H-
3), 4.33–4.25 (m, 1H, H-4), 4.13–4.07 (m, 1H, H-5), 3.46–3.43 (m, 2H,
H-6), 1.43 (s, 3H, Me), 1.37 (s, 3H, Me); 13C NMR (CDCl3): δ 160.9,
142.7, 125.8, 116.5, 110.4, 107.3, 86.6, 79.5, 78.6, 75.4, 65.5, 25.6,
25.5; Anal. Calc. for: C15H19NO8+H2O: C, 50.14; H, 5.89; N, 3.90.
Found: C, 50.37; H, 5.51; N, 3.73.
3.8. p-Nitrophenyl 2,3-di-O-acetyl-5,6-O-isopropylidene-β-D-
[α]D −19.9 (c 0.1, H2O), lit.12 [α]D −203 (c 1, MeOH); mp
153–154 °C, lit.12 mp 153–155 °C; 1H NMR (D2O): δ 8.26 (d, 2H,
galactofuranoside (9)
To a solution of 8 (0.47 g, 1.37 mmol) in pyridine (20 mL) was
added Ac2O (0.52 mL, 5.47 mmol) followed by stirring at 23 °C for 24 h.
The mixture was concentrated in vacuo, and then the residue was di-
luted with AcOEt (100 mL), washed with 1 M aq. HCl (100 mL x 3) and
brine (50 mL), followed by drying over Na2SO4. The organic layer was
concentrated in vacuo to give p-nitrophenyl 2,3-di-O-acetyl-5,6-O-iso-
J
o,m = 9.0 Hz, Ph(m)), 7.24 (d, 2H, Jo,m = 9.0 Hz, Ph(o)), 5.82 (s, 1H,
H-1), 4.42 (d, 1H, J2,3 = 6.0 Hz, H-2), 4.23 (dd, 1H, J2,3 = 6.0 Hz,
J
3,4 = 4.0 Hz, H-3), 4.12 (dd, 1H, J3,4 = 4.0 Hz, J4,5 = 5.6 Hz, H-4),
3.71–3.67 (m, 2H, H-6), 3.89–3.87 (m, 1H, H-5); 13C NMR (D2O): δ
175.0, 162.1, 125.2, 116.3, 106.3, 84.2, 82.0, 76.7, 70.7, 62.8; Anal.
Calc. for: C12H15NO8: C, 47.84; H, 5.02; N, 4.65. Found: C, 47.59; H,
5.09; N, 4.67.
propylidene-β-D-galactofuranoside (9) as
a colorless solid (0.58 g,
1.37 mmol, quant.).
[α]D −101 (c 0.42, CHCl3); 1H NMR (CDCl3): δ 8.20 (d, 2H,
Jo,m = 9.6 Hz, Ph(m)), 7.15 (d, 2H, Jo,m = 9.6 Hz, Ph(o)), 5.78 (d, 1H,
3.5. 2,3,4,6-Tetra-O-acetyl-D-galactofuranose (6)
J
1,2 = 0.4 Hz, H-1), 5.33 (dd, 1H, J1,2 = 0.4 Hz, J2,3 = 1.2 Hz, H-2),
To a solution of 4 and 4p (3.90 g, 10.0 mmol) in DMF (50 mL) and
H2O (5 mL) was added (NH4)2CO3 (4.80 g, 50.0 mmol), and the mixture
was stirred at 23 °C for 24 h. Then the solution was filtered through a
Celite pad and the filtrate was concentrated in vacuo. The residue was
diluted with EtOAc (100 mL), washed with 50% brine (100 mL) and
brine (100 mL), dried over MgSO4, and driend in vacuo to afford a
mixture of 2,3,4,6-tetra-O-acetyl-D-galactofuranose (6) and 2,3,4,6-
tetra-O-acetyl-D-galactopyranose (6p) as a colorless syrup (2.37 g,
6.80 mmol, 68%).
5.19 (dd, 1H, J2,3 = 1.2 Hz, J3,4 = 4.8 Hz, H-3), 4.41–4.36 (m, 1H, H-
5), 4.22 (t, 1H, J3,4 = J4,5 = 4.8 Hz, H-4), 4.07 (dd, 1H, J5,6b = 7.2 Hz,
J6a,6b = 8.8 Hz, H-6b), 3.89 (dd, 1H, J5,6a = 6.0 Hz, J6a,6b = 8.8 Hz, H-
6a), 2.15 (s, 3H, Ac), 2.16 (s, 3H, Ac), 1.43 (s, 3H, Me), 1.37 (s, 3H,
Me); 13C NMR (CDCl3): δ 170.0, 169.7, 160.8, 142.7, 125.8, 116.6,
110.1, 103.8, 84.2, 81.3, 74.8, 65.4, 26.3, 25.1, 20.8, 20.7, 14.2; Anal.
Calc. for: C19H23NO10: C, 53.65; H, 5.45; N, 3.29. Found: C, 53.25; H,
5.50; N, 2.99.
1H NMR (CDCl3): δ 5.39–5.38 (m, 1H, H-3), 5.07–5.05 (m, 1H, H-2),
4.73 (m, 1H, H-1), 4.37–4.31 (m, 1H, H-4), 4.18–4.16 (m, 1H, H-5),
4.16–4.07 (m, 2H, H-6), 2.14 (s, 3H, Ac), 2.09 (s, 3H, Ac), 2.04 (s, 3H,
Ac), 1.98 (s, 3H, Ac); 13C NMR (CDCl3): δ 170.6, 170.4, 170.3, 170.1,
90.7, 68.2, 67.2, 61.8, 20.8, 20.7, 20.6, 20.5; Anal. Calc. for:
3.9. p-Nitrophenyl 2,3-di-O-acetyl-β-D-galactofuranoside (10)
To a solution of 70% aq. acetic acid (30 mL) was added 9 (0.91 g,
2.32 mmol) and the resulting mixture was stirred at 23 °C for 12 h. The
solution was diluted with CHCl3 (100 mL), washed with sat. aq.
NaHCO3 solution (100 mL x 3) and brine (50 mL), followed by drying
over Na2SO4. The organic layer was concentrated in vacuo to give p-
nitrophenyl 2,3-di-O-acetyl-β-D-galactofuranoside (10) as a colorless
solid (0.89 g, 2.13 mmol, 92%) without any purification.
C
14H20O10+H2O: C, 48.28; H, 5.79. Found: C, 47.19; H, 6.31.
3.6. 2,3,4,6-Tetra-O-acetyl-β-D-galactofuranosyl imidate (7)
To a solution of 6 and 6p (1.16 g, 3.12 mmol) in DCE (100 mL) was
added CCl3CN (1.56 mL, 15.6 mmol), MS4A and Cs2CO3 (0.70 g,
1.56 mmol) followed by stirring at 23 °C for 3 h. Then the mixture was
filtered through a Celite pad and the filtrate was concentrated in vacuo
to dryness to afford 2,3,4,6-tetra-O-acetyl-D-galactofuranosyl imidate
(7) and 2,3,4,6-tetra-O-acetyl-D-galactopyranosyl imidate (7p) as a
colorless liquid (1.54 g, 3.12 mmol, quant.). The mixture of 7 and 7p
was used for glycosylation as a donor without any purification because
most of imidate sugar was especially sensitive to moisture.
[α]D −150 (c 0.1, CHCl3); mp 124–126 °C; 1H NMR (CDCl3): δ 8.21
(d, 1H, Jo,m = 9.2 Hz, Ph(m)), 7.12 (d, 2H, Jo,m = 9.2 Hz, Ph(o)), 5.80
(d, 1H, J1,2 = 0.8 Hz, H-1), 5.39 (dd, 1H, J1,2 = 0.8 Hz, J2,3 = 1.2 Hz,
H-2), 5.31 (dd, 1H, J2,3 = 1.2 Hz, J3,4 = 5.2, H-3), 4.25 (dd, 1H,
J3,4 = 5.2 Hz, J4,5 = 4.0 Hz, H-4), 3.98–3.96 (m, 1H, H-5), 3.77–3.69
(m, 2H, H-6), 2.16 (s, 3H, Ac), 2.15 (s, 3H, Ac); 13C NMR (CDCl3): δ
170.6, 169.7, 160.7, 142.8, 125.8, 116.4, 103.7, 84.6, 81.1, 70.4, 63.7,
20.8, 20.7; Anal. Calc. for: C16H19NO10+CH3CO2H: C, 44.91; H, 5.65;
N, 2.91. Found: C, 44.56; H, 6.02; N, 2.54.
1H NMR (CDCl3): δ 5.42–5.34 (m, 2H, H-1 and H-3), 5.08–5.05 (m,
102

R. Ota et al.
CarbohydrateResearch473(2019)99–103
3.10. p-Nitrophenyl 2,3-di-O-acetyl-6-O-benzoyl-β-D-galactofuranoside
3), and then concentrated in vacuo. The target material was purified by
silica-gel column chromatography (CHCl3:CH3OH, 4/1) to give p-ni-
trophenyl 5-O-(β-D-galactofuranosyl)-β-D-galactofuranoside (pNP-Galf-
β(1,5)-Galf, 13) (0.070 g, 0.14 mmol, 35%) as a colorless syrup.
[α]D −132 (c 0.082, H2O); 1H NMR (D2O): δ 8.19 (d, 2H,
Jo,m = 9.2 Hz, Ph(m)), 7.18 (d, 2H, Jo,m = 9.2 Hz, Ph(o)), 5.78 (d, 1H,
(11)
To a solution of 10 (0.50 g, 1.40 mmol) in pyridine (15 mL) were
added BzCl (0.23 mL, 2.0 mmol) and DMAP (0.01 g, 0.07 mmol) at 0 °C,
and the reaction was stirred at 23 °C for 12 h. The mixture was diluted
with CHCl3 (100 mL), washed with sat. aq. NaHCO3 solution (100 mL x
3) and brine (50 mL), followed by drying over Na2SO4 and then con-
centration in vacuo. The residue was purified by silica-gel column
chromatography (hexane:AcOEt, 3/1) to give p-nitrophenyl 2,3-di-O-
J
J
J
1,2 = 1.6 Hz, H-1), 5.22 (d, 1H, J1′,2’ = 2.0 Hz, H-1′), 4.40 (dd, 1H,
1,2 = 1.6 Hz, J2,3 = 3.2 Hz, H-2), 4.26 (dd, 1H, J2,3 = 3.2 Hz,
3,4 = 6.0 Hz, H-3), 4.19 (dd, 1H, J3,4 = 6.0 Hz, J4,5 = 3.6 Hz, H-4),
4.13–4.19 (m, 1H, H-2′), 4.04–4.12 (m, 1H, H-3′), 4.03–3.97 (m, 1H, H-
5), 3.88–3.81 (m, 1H, H-5′), 3.61–3.77 (m, 4H, H-6 and H-6’); 13C NMR
(D2O) δ (ppm) 162.0, 142.2, 125.2, 116.3, 107.7, 106.1, 83.5, 83.3,
82.2, 81.5, 77.3, 76.7, 75.6, 70.8, 62.7, 62.0; Anal. Calc. for:
acetyl-6-O-benzoyl-β-D-galactofuranoside (11) as
a colorless syrup
(0.44 g, 0.90 mmol, 64%).
[α]D −101 (c 0.1, CHCl3); 1H NMR (400 MHz, CDCl3): δ 8.21 (d,
1H, Jo,m = 9.2 Hz, Ph(m)), 8.08–7.94 (m, 2H, Bz(m)), 7.59–7.55 (m,
1H, Bz(p)), 7.48–7.39 (m, 2H, Bz(o)), 7.12 (d, 2H, Jo,m = 9.2 Hz, Ph(o)),
5.79 (s, 1H, H-1), 5.41 (dd, 1H, J1,2 = 0.4 Hz, J2,3 = 2.0 Hz, H-2), 5.38
(dd, 1H, J2,3 = 2.0 Hz, J3,4 = 4.4 Hz, H-3), 4.55 (dd, 1H, J5,6b = 6.4 Hz,
J6a,6b = 11.6 Hz, H-6b), 4.39 (dd, 1H, J5,6a = 4.4 Hz, J6a,6b = 11.6 Hz,
C
18H25NO13+H2O: C, 44.91; H, 5.65; N, 2.91. Found: C, 45.26; H, 6.01;
N, 3.27.
Acknowledgements
H-6a), 4.32–4.29 (m, 2H, H-4,5), 2.15 (s, 3H, Ac), 2.13 (s, 3H, Ac); 13
C
The authors are grateful to Dr. Kei Matsumoto at the Division of
Instrumental Analysis, Okayama University for carrying our NMR
spectral measurements. The authors are also grateful to Ms. Megumi
Kosaka and Mr. Motonari Kobayashi at the Division of Instrumental
Analysis, Okayama University for the measurements of elemental ana-
lyses. Financial support from KAKENHI (No. 17K07772) and Wesco
Scientific Promotion Foundation are acknowledged.
NMR (CDCl3): δ 170.5, 169.7, 166.4, 160.6, 142.7, 133.3, 129.6, 128.4,
125.7, 116.4, 103.8, 84.0, 81.2, 68.4, 65.8, 60.4, 20.8, 20.7, 14.2; Anal.
Calc. for: C23H23NO11: C, 56.44; H, 4.74; N, 2.86. Found: C, 56.17; H,
4.87; N, 2.96.
3.11. p-Nitrophenyl 5-O-(β-D-galactofuranosyl)-β-D-galactofuranoside
(pNP-Galf-β(1,5)-Galf, 13) via p-nitrophenyl 5-O-(2′,3′,4′,6′-tetra-O-
acetyl-β-D-galactofuranosyl)-2,3-di-O-acetyl-6-O-benzoyl-β-D-
galactofuranoside (12)
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103