Facile synthesis of the pentasaccharide repeating unit of the exopolysaccharide from Cryptococcus neoformans serotype D

Article abstract of DOI:10.1002/hlca.201400297

Abstract β-D-GlcpA-(1→2)-[β-D-Xylp-(1→2)-α-D-Manp-(1→3)]-α-D-Manp-(1→3)-α-D-Manp, the repeating unit of the exopolysaccharide from Cryptococcus neoformans serotype D, was synthesized as its 4-methoxyphenyl glycoside. The approach presented here also provi

Full text of DOI:10.1002/hlca.201400297

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Helvetica Chimica Acta – Vol. 98 (2015)
Facile Synthesis of the Pentasaccharide Repeating Unit of the
Exopolysaccharide from Cryptococcus neoformans Serotype D
by Xiao-Bin Zhuoa)1), Yan Zoub)1), Sheng-Jun Huangb), Jun Liaob), Hong-Gang Hub),
Qing-Jie Zhao*a)b), and Qiu-Ye Wu*a)b)
a) School of Pharmacy, Yantai University, No. 30 Qingquan Road, Yantai 264005, P. R. China
b) Department of Organic Chemistry, School of Pharmacy, Second Military Medical University, No. 325
Guohe Road, Shanghai 200433, P. R. China
(phone/fax: þ 86-21-81871228; e-mail: qjzhao@smmu.edu.cn (Q.-J. Z.), wuqysmmu@sohu.com (Q.-Y. W.))
b-d-GlcpA-(1 ! 2)-[b-d-Xylp-(1 ! 2)-a-d-Manp-(1 ! 3)]-a-d-Manp-(1 ! 3)-a-d-Manp, the re-
peating unit of the exopolysaccharide from Cryptococcus neoformans serotype D, was synthesized as
its 4-methoxyphenyl glycoside. The approach presented here also provides a route to the synthesis of
more complex repeating units of glucuconoxylomannan (GXM) of C. neoformans serotypes A – C.
Introduction. – Cryptococcus neoformans is an opportunistic fungal pathogen with
worldwide distribution that can cause a life-threatening disease, especially for those
who suffer from AIDS, renal transplant recipients, and patients undergoing immuno-
suppressive therapy. Furthermore, the disease has also been distributed in healthy
individuals, resulting in 600,000 deaths annually [1 – 4]. The virulence factors of C.
neoformans include the presence of a polysaccharide capsule of which glucuronox-
ylomannan (GXM) is the major constituent [5 – 7]. Based on the genetic characteristics
and serologic properties of capsular polysaccharides (CPS), four major serotypes have
been defined as A – D (Fig.), in which serotype D owns the simplest pentaose structure,
while type C has the most complex octaose structure. All four serotypes are composed
of a linear a-(1 ! 3)-linked mannosyl backbone with b-glucopyranosyluronic acid, b-
xylopyranosyl, and 6-O-Ac substituents [8][9].
Syntheses of the repeating units of the polysaccharides from C. neoformans
serotypes A – D have been already reported [10 – 13]. In these syntheses, multiple steps
and orthogonal masking groups were involved, rendering the procedure rather complex
and tedious. Herein, we report the synthesis of the repeating unit of the serotype D in
an efficient way, and the strategy presented here also provides a route to the synthesis
of more complex repeating units of GXM of C. neoformans serotypes A – C.
Results and Discussion. – Our strategy for the synthesis of the pentaose repeating
unit involves the construction of the acceptors 7 and 8 (Scheme 1), and donors 13 and
14 (cf. Schemes 2 and 3). To obtain the pentaose, b-d-Xylp-(1 ! 2)-a-d-Manp and b-d-
GlcpA-(1 ! 2)-a-d-Manp, the two key middle disaccharide blocks, were first synthe-
sized, then coupling of them (2 þ 2) and extension of the tetrasaccharide chain thus
1
)
These two authors contributed equally to this work.
ꢀ 2015 Verlag Helvetica Chimica Acta AG, Zürich

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619
Figure. Model structures of GXM of Cryptococcus neoformans serotypes A – D
obtained with a monosaccharide acceptor on the reducing end (4 þ 1) were carried out
at the final stage.
As shown in Scheme 1, the 4-methoxyphenyl (MP) group (Scheme 1) was
introduced to 1,2,3,4,6-penta-O-acetyl-d-mannopyranose (1) to afford 4-methoxyphen-
yl 2,3,4,6-tetra-O-acetyl-a-d-mannopyranoside (2) [14][15] in high yield (90%), using
TBDMSOTf as promoter. Cleavage of the acetates and then reaction with 2,2-
dimethoxypropane under catalysis by TsOH afforded the diacetal 3 as an oil [16].
Treatment with TsOH · H2O (cat.) in acetone/H2O 20 :1 at 508 for 10 min afforded the
2,3-O-isopropylidene derivative 4 predominantly [17]. Compound 4 was benzoylated
[18] to give 4-methoxyphenyl 4,6-di-O-benzoyl-2,3-O-isopropylidene-a-d-mannopyr-
anoside (5), which exhibited characteristic signals in its 1H-NMR spectrum. Removal
of isopropylidene group [19] from 5 was followed by selective 3-O-acetylation [20] of
the resulting diol 6 with AcCl in the presence of pyridine. In addition to the expected
Scheme 1
a) 4-MeOÀC6H4 – OH (MP – OH), tBu(Me2)Si OSO2CF3 (TBDMSOTf), CH2Cl2, r.t. b) MeONa,
MeOH, then MeC(MeO)2Me, TsOH · H2O (cat.), acetone, r.t. c) TsOH · H2O (cat.), acetone/H2O 20 :1,
508, 10 min. d) BzCl, Pyridine. e) 90% CF3COOH (TFA), r.t. f) AcCl, pyridine, CH2Cl2, 80% (for 7),
10% (for 8), 5% (for 9).

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acceptor 4-methoxyphenyl 3-O-acetyl-4,6-di-O-benzoyl-a-d-mannopyranoside (7;
80%), small portions of 4-methoxyphenyl 2-O-acetyl-4,6-di-O-benzoyl-a-d-manno-
pyranoside (8; 10%) and 4-methoxyphenyl 2,3-di-O-acetyl-4,6-di-O-benzoyl-a-d-
1
mannopyranoside (9; 5%) were also obtained. This was confirmed by the H-NMR
spectrum in which compound 7 gave rise to a characteristic singlet at d(H) 2.03 (3-Ac),
while the singlet at d(H) 2.21 (2-Ac) was attributed to 8. When 9 was treated with 1%
AcCl/MeOH [10], for the first few hours, compound 9 was only transformed to 8, but if
this reaction was continued for ca. 2 d, a total conversion of 9 to 6 was observed. Based
on all these findings, it can be concluded that the 3-OH is more reactive than 2-OH.
This selective 3-O-acetylation was the key step in the synthesis.
The synthesis of the donor 2,3,4-tri-O-benzoyl-d-xylopyranosyl trichloroacetimi-
date (13) is shown in Scheme 2. Starting from xylose, we applied N,O-dimethylhy-
droxylamine as the anomeric protecting group [20], and, in subsequent benzoylation
and deprotection of the hydroxylamine, a solution of NCS in THF/H2O 9 :1 [21] was
used at 50 – 608 to furnish 12, followed by a reaction with CCl3CN in the presence of
traces of DBU [22] to afford donor 13. The advantage of this method is that N,O-
dimethylhydroxylamine can be directly introduced in unprotected xylose, reducing the
number of synthesis steps when compared with previously reported methods. The
trichloroacetimidate donor 14 was easily available from the commercial d-glucofur-
anurono-6,3-lactone in four steps [23][24].
The synthesis of the pentasaccharide b-d-GlcpA-(1 ! 2)-[b-d-Xylp-(1 ! 2)-a-d-
Manp-(1 ! 3)]-a-d-Manp-(1 ! 3)-a-d-Manp was outlined in Scheme 3. 4-Methoxy-
phenyl 3-O-acetyl-4,6-di-O-benzoyl-a-d-mannopyranoside (7) was chosen as the
starting material, and glycosylation [25] of 7 with 13 and methyl 2,3,4-tri-O-benzoyl-
1-O-(2,2,2-trichloroethanimidoyl)-d-glucopyranuronate (14) furnished the b-(1 ! 2)-
linked disaccharides 15 and 17, respectively. Subsequent removal of the MP group by
oxidative cleavage [26] of 15 with (NH4)2Ce(NO3)6 (CAN) and trichloroacetimidate
formation gave the b-(1 ! 2)-linked disaccharide donor 16. Selective deacetylation of
17 with AcCl, MeOH, and CH2Cl2, accompanied with some decomposition, perhaps
caused by breaking of xylosyl linkage, furnished the b-(1 ! 2)-linked disaccharide
acceptor 18. The condensation of 16 with 18, promoted by TMSOTf, went smoothly to
afford the required tetrasaccharide 19 in a satisfactory yield (60%). Oxidative cleavage
of MP group led to the tetrasaccharide donor 20, along with a trichloroacetimidate
formation. Then, the protected pentasaccharide 21 was obtained by coupling 8 with the
tetrasaccharide trichloroacetimidate in good yield (71%). Deprotection [27] of 21 was
carried out in MeOH with MeONa for 96 h, affording the target pentasaccharide 22 as
foamy solid.
Scheme 2
a) HN(Me)OMe · HCl, AcONa, H2O. b) BzCl, Pyridine. c) N-Chlorosuccinimide (NCS), THF/H2O 9 :1.
d) Cl3CCN, 1,8-Diazabicyclo[5.4.0]undec-7-ene (DBU), CH2Cl2.

Helvetica Chimica Acta – Vol. 98 (2015)
621
Scheme 3
a) Me3SiOSO2CF3 (TMSOTf), CH2Cl2, À 208 to r.t. (dry). b) (NH4)2Ce(NO3)6 (CAN), MeCN/H2O
4 :1, r.t. c) Cl3CCN, DBU, CH2Cl2. d) 1% AcCl/MeOH, 08 to r.t. e) MeONa/MeOH, r.t., 96 h; then H2O,
r.t., 2 h.
In summary, we presented here a facile synthesis of the pentasaccharide repeating
unit of GXM of C. neoformans serotype D. This strategy offers a route to obtain higher
oligosaccharides with similar structures such as the repeating units of GXM of C.
neoformans serotypes A – C.

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We are grateful to the National Natural Science Foundation of China (No. 20902109), the Shanghai
Leading Academic Discipline Project (B906), the State Project for Essential Drug Research and
Development (2012ZX09502-001-005), and the Instrumental Analysis Center of our university for NMR
spectroscopic and mass spectrometric analyses.
Experimental Part
General. Anh. solvents were obtained from a solvent purification system, while commercial anh.
reagents were used without further purification. TLC: Silica gel (SiO2) GF254 plates; visualization under
UV light or by spraying with a 5% H2SO4 soln. in EtOH. Column chromatography (CC): SiO2 (200 –
1
300 mesh, 10 – 40 mm; Qingdao Marine Chemical Factory). H- and 13C-NMR spectra: Bruker Avance;
300 and 600 (1H), 75, and 150 MHz (13C) spectrometers; d in ppm rel. to Me4Si as internal standard, J in
Hz. ESI-MS and HR-QTOF-MS: Finngan LCQ Deca XP Max instrument and Agilent 6538 UHD
Accurate Mass Q-TOF LC/MS mass spectrometer, resp.; in m/z.
General Procedure for the Glycosylations. A mixture of the donor and acceptor was dried together
under high vacuum for 2 h, then, dissolved in anh. CH2Cl2, TMSOTf (0.05 equiv.) was added dropwise at
À 308 under N2. The mixture was stirred for 3 h, during which time the temp. was gradually increased to
r.t. The mixture was then neutralized with Et3N. Concentration of the mixture, followed by purification
on a silica-gel column, gave the desired products.
4-Methoxyphenyl 2,3,4,6-Tetra-O-acetyl-a-d-mannopyranoside (2). To a cold (08) soln. of 1 (4.33 g,
11.1 mmol) and 4-methoxyphenol (2.065 g, 16.65 mmol) in dry CH2Cl2 (75 ml) was added TBDMSOTf
(2.80 ml, 12.21 mmol) under Ar. After 12 h, TLC (petroleum ether (PE)/AcOEt 5 :4) indicated that the
reaction was complete, and the mixture was diluted with CH2Cl2, and washed with H2O, sat. aq. NaHCO3
soln., and brine. The org. phase was dried (Na2SO4), filtered, and concentrated under reduced pressure.
1
Purification of the crude product by CC (PE/AcOEt 3 :1) gave 2 (4.54 g, 90%). White solid. H-NMR
(300 MHz, CDCl3): 7.02 (d, J ¼ 9.1, 2 arom. H); 6.83 (d, J ¼ 9.1, 2 arom. H); 5.55 (dd, J ¼ 10.0, 3.4, 1 H);
5.44 – 5.41 (m, 2 H); 5.36 (t, J ¼ 9.9, 1 H); 4.28 (dd, J ¼ 11.7, 4.9, 1 H); 4.16 – 4.06 (m, 2 H); 3.77 (s, MeO);
2.19, 2.09, 2.06, 2.05 (4s, 4 Â Ac). ESI-MS: 477.13 ([M þ Na]þ).
4-Methoxyphenyl 2,3 :4,6-Di-O-isopropylidene-a-d-mannopyranoside (3). A soln. of 2 (91 mg,
0.20 mmol) in MeOH (2 ml) was treated with MeONa (2.16 mg, 0.04 mmol) and stirred for 2 h at r.t. The
mixture was then neutralized with Amberlite Hþ resin, filtered, and concentrated. The residue was dried
under high vacuum and dissolved in acetone (2 ml), TsOH · H2O (3.8 mg, 10%) was added, and the
resulting soln. was then treated with 2,2-dimethoxypropane (0.15 ml, 1.2 mmol). After the stirring
overnight, solid Na2CO3 was added, and the mixture was filtered and concentrated. The residue was then
separated by CC (PE/AcOEt 5 :2) to afford 3 (59 mg, 80%, over two steps). Light-yellow oil. 1H-NMR
(300 MHz, CDCl3): 6.96 (d, J ¼ 9.0, 2 arom. H); 6.83 (d, J ¼ 9.0, 2 arom. H); 5.65 (s, 1 H); 4.40 (d, J ¼ 5.6,
1 H); 4.34 – 4.27 (m, 1 H); 3.85 – 3.71 (m, HÀC(5), CH2(6), MeO); 1.59, 1.53, 1.43, 1.40 (4s, 2 Me2C). ESI-
MS: 389.17 ([M þ Na]þ).
4-Methoxyphenyl 2,3-O-Isopropylidene-a-d-mannopyranoside (4). Compound
3 (200 mg,
0.545 mmol) was dissolved in acetone/H2O 20 :1 (2 ml), TsOH (9 mg) was added, and the mixture
was stirred at 508 for 10 min; then the reaction was quenched with Et3N, and the mixture was
concentrated. The residue was subjected to CC (PE/AcOEt 1:1) to give 4 (150 mg, 84%). White solid.
1H-NMR (300 MHz, CDCl3): 7.07 (d, J ¼ 9.0, 2 arom. H); 6.85 (d, J ¼ 9.0, 2 arom. H); 5.61 (s, 1 H); 4.36
(dd, J ¼ 5.6, 0.7, 1 H); 4.19 (t, J ¼ 6.1, 1 H); 3.75 (s, MeO); 3.71 (dd, J ¼ 6.9, 3.1, 2 H); 3.67 (d, J ¼ 2.5, 2 H);
1.52, 1.39 (2s, Me2C). ESI-MS: 349.14 ([M þ Na]þ).
4-Methoxyphenyl 4,6-Di-O-benzoyl-2,3-O-isopropylidene-a-d-mannopyranoside (5). Compound 4
(1.63 g, 5.0 mmol) was dissolved in dry CH2Cl2 (20 ml) containing pyridine (3.58 ml, 50 mmol), then,
under N2 protection and stirring, a soln. of BzCl (0.83 ml, 5.0 mmol) in anh. CH2Cl2 (10 ml) was added
dropwise within 30 min at 08. The mixture was slowly warmed to r.t. and stirred for 2 h, when TLC
indicated the completion of the reaction. The mixture was diluted with CH2Cl2, washed with H2O, 1n
HCl, and dried (Na2SO4). The soln. was concentrated, and purification by CC (CH2Cl2/MeOH 400 :1)
gave 5 (2.40 g, 90%). White solid. 1H-NMR (300 MHz, CDCl3): 8.08 (d, J ¼ 8.3, 2 arom. H); 7.91

Helvetica Chimica Acta – Vol. 98 (2015)
623
(d, J ¼ 8.3, 2 arom. H); 7.70 – 7.73 (m, 6 arom. H); 7.07 (d, J ¼ 9.0, 2 arom. H); 6.75 (d, J ¼ 9.0, 2 arom. H);
5.58 (s, 1 H); 5.45 (t, J ¼ 9.5, 1 H); 4.58 – 4.26 (m, 5 H); 3.75 (s, MeO). ESI-MS: 535.56 ([M þ H]þ).
4-Methoxyphenyl 4,6-Di-O-benzoyl-a-d-mannopyranoside (6). Compound 5 (30 mg, 0.056 mmol)
was dissolved in CH2Cl2 (2 ml) containing 90% aq. CF3COOH (TFA ; 0.5 ml), and the soln. was stirred at
r.t. for 2 h, when TLC (CH2Cl2/MeOH 100 :1) indicated that the reaction was complete. Evaporation of
the solvent gave 6 (25 mg, 90%) as white solid, which was directly used for next step without further
purification. 1H-NMR (300 MHz, CDCl3): 8.07 (d, J ¼ 8.3, 2 arom. H); 7.90 (d, J ¼ 8.3, 2 arom. H); 7.69 –
7.32 (m, 6 arom. H); 7.06 (d, J ¼ 9.0, 2 arom. H); 6.75 (d, J ¼ 9.0, 2 arom. H); 5.58 (s, 1 H); 5.44 (t, J ¼ 9.5,
1 H); 4.57 – 4.26 (m, 5 H); 3.74 (s, MeO). ESI-MS: 517.16 ([M þ Na]þ).
4-Methoxyphenyl 3-O-Acetyl-4,6-di-O-benzoyl-a-d-mannopyranoside (7). Compound 6 (2.18 g,
4.4 mmol) was dissolved in dry CH2Cl2 (20 ml) containing pyridine (3.52 ml, 44 mmol), then, under N2
protection and stirring, a soln. of AcCl (0.8 ml, 4.84 mmol) in anh. CH2Cl2 (10 ml) was added dropwise
within 30 min at 08. The mixture was slowly warmed to r.t. and stirred for 2 h, when TLC (CH2Cl2/MeOH
100 :1) indicated that the reaction was complete. The mixture was diluted with CH2Cl2, washed with H2O
and 1n HCl, and dried (Na2SO4). The soln. was concentrated, and purification by CC (CH2Cl2/MeOH
400 :1) gave 7 (1.90 g, 80%), 8 (236 mg, 10%), and 9 (127mg, 5%) as white solid.
Data of 7. 1H-NMR (300 MHz, CDCl3): 8.02 (d, J ¼ 7.9, 2 arom. H); 7.91 (d, J ¼ 7.9, 2 arom. H); 7.58 –
7.33 (m, 6 arom. H); 7.08 (d, J ¼ 9.0, 2 arom. H); 6.76 (d, J ¼ 9.0, 2 arom. H); 5.75 (s, 1 H); 5.55 (t, J ¼ 2.9,
1 H); 4.53 – 4.35 (m, 4 H); 3.74 (s, MeO); 2.75 (s, 2-OH); 2.03 (s, MeCO). ESI-MS: 559.16 ([M þ Na]þ).
4-Methoxyphenyl 2-O-Acetyl-4,6-di-O-benzoyl-a-d-mannopyranoside (8). 1H-NMR (300 MHz,
CDCl3): 8.05 (d, J ¼ 7.9, 2 arom. H); 7.94 (d, J ¼ 7.9, 2 arom. H); 7.63 – 7.37 (m, 6 arom. H); 7.07 (t, J ¼
10.7, 2 arom. H); 6.75 (t, J ¼ 10.7, 2 arom. H); 5.53 (s, 1 H); 5.39 – 5.29 (m, 1 H); 4.64 – 4.33 (m, 4 H);
3.74 (s, MeO); 2.21 (s, MeCO). ESI-MS: 559.16 ([M þ Na]þ).
N-Methoxy-N-methyl-b-d-xylopyranosylamine (10). Xylose (15.01 g, 0.1 mmol) was dissolved in
H2O (150 ml) in a round-bottomed flask. N,O-Dimethylhydroxylamine hydrochloride (10.73 g,
0.11 mmol) and AcONa (9.02 g, 0.11 mmol) were dissolved in ca. 10 ml of H2O, and the soln. was
added slowly to the xylose soln. at 08. The reaction was allowed to proceed for 20 h at r.t., when TLC
analysis showed conversion of the starting material to a faster-moving product (CH2Cl2/MeOH 5 :1).
H2O was evaporated from the reaction in vacuo, and the product was purified by CC (CH2Cl2/MeOH
1
8 :1) to give 10 (16.4 g, 85%). White solid. H-NMR (600 MHz, D2O): 4.02 (d, J ¼ 8.5, 1 H); 3.49 (s,
MeO); 3.38 – 3.47 (m, 3 H); 3.24 – 3.34 (m, 2 H); 2.76 (s, MeN). ESI-MS: 216.10 ([M þ Na]þ).
2,3,4-Tri-O-benzoyl-N-methoxy-N-methyl-b-d-xylopyranosylamine (11). To the soln. of 10 (2.90 g,
15 mmol) in anh. CH2Cl2 (30 ml), containing pyridine (7.24 ml), a soln. of BzCl (7.8 ml) in 20 ml of anh.
CH2Cl2 was added dropwise at 08 under N2, and the mixture was stirred overnight at r.t. TLC (PE/AcOEt
5 :1) indicated that the reaction was complete. Ice-water was added, and the mixture was diluted with
CH2Cl2, washed with 1n HCl, H2O, and sat. aq. NaHCO3. The org. layer was combined, dried, and
concentrated. Purification of the crude product by CC gave 11 (6.8 g, 90%). White solid. 1H-NMR
(600 MHz, CDCl3): 7.98 – 7.30 (m, 15 arom. H); 5.92 (t, J ¼ 9.6, 1 H); 5.64 (t, J ¼ 9.4, 1 H); 5.40 (dt, J ¼
10.1, 5.5, 1 H); 4.51 – 4.46 (m, 2 H); 3.61 – 3.55 (m, 1 H); 3.46 (s, MeO); 2.79 (s, MeN). ESI-MS: 528.17
([M þ Na]þ).
2,3,4-Tri-O-benzoyl-d-xylopyranose (12). Compound 11 (505.52 mg, 1 mmol) was dissolved in THF/
H2O 9 :1 (15 ml), and NCS (267.06 mg, 2 mmol) was added. The soln. was warmed to 508 for 2 h when
TLC analysis showed complete conversion to a slower-moving product (PE/AcOEt 4 :1). The solvent
was evaporated, and the crude oil was purified by CC (PE/AcOEt 4 :1) to furnish 12 (416 mg, 90%).
White solid. 1H-NMR (300 MHz, CDCl3): 8.02 – 7.30 (m, 15 arom. H); 6.22 (t, J ¼ 9.5, 1 H); 5.69 (t, J ¼
3.5, 1 H); 5.42 (d, J ¼ 7.1, 1 H); 5.31 (t, J ¼ 4.9, 1 H); 4.14 (d, J ¼ 7.8, 2 H); 3.25 (s, 1 H). ESI-MS: 485.13
([M þ Na]þ).
2,3,4-Tri-O-benzoyl-1-O-(2,2,2-trichloroethanimidoyl)-d-xylopyranose (13). Compound 12 (230 mg,
0.5 mmol) was dried under high vacuum for 2 h, then dissolved in CH2Cl2 (10 ml), and CCl3CN (0.5 ml,
5 mmol) and DBU (7 ml, 0.05 mmol) were added in an ice bath. The mixture was stirred for 2 h, when
TLC (PE/AcOEt 4 :1) indicated that the reaction was complete. Concentration of the mixture, followed
by purification of the crude product on CC (SiO2; PE/AcOEt 4 :1), gave 13 (243 mg, 80%). Foamy solid.
1H-NMR (300 MHz, CDCl3): 8.70 (s, C¼NH); 8.05 – 7.31 (m, 15 arom. H); 6.83 (d, J ¼ 3.0, 1 H); 6.13 (t,

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Helvetica Chimica Acta – Vol. 98 (2015)
J ¼ 9.6, 2 H); 5.88 (s, 1 H); 5.53 (dd, J ¼ 10.0, 3.5, 1 H); 4.42 – 4.18 (m, 2 H). ESI-MS: 629.84 ([M þ
Na]þ).
4-Methoxyphenyl 2,3,4-Tri-O-benzoyl-b-d-xylopyranosyl-(1 ! 2)-3-O-acetyl-4,6-di-O-benzoyl-a-d-
mannopyranoside (15). As described in the General Procedure, 13 (88 mg, 0.145 mmol) and 7 (65 mg,
0.121 mmol) were coupled, and the product was purified by CC (SiO2; PE/AcOEt 3 :1) to give 15
(103 mg, 86.8%). White solid. 1H-NMR (600 MHz, CDCl3): 8.12 – 7.29 (m, 25 arom. H); 6.91 (d, J ¼ 9.1, 2
arom. H); 6.72 (d, J ¼ 9.1, 2 arom. H); 6.05 (d, J ¼ 4.7, 1 H); 5.71 (dd, J ¼ 5.0, 1 H); 5.67 (dd, J ¼ 10.0,
1 H); 5.50 (dd, J ¼ 10.0, 3.3, 1 H); 5.37 (dd, J ¼ 5.0, 3.7, 1 H); 5.28 – 5.19 (m, 1 H); 4.97 (d, J ¼ 3.7, 1 H);
4.61 (dd, J ¼ 12.5, 3.2, 1 H); 4.34 (dd, J ¼ 11.8, 3.0, 1 H); 4.26 (dd, J ¼ 3.3, 1.4, 1 H); 4.22 – 4.16 (m, 2 H);
4.08 – 3.97 (m, 1 H); 3.65 (s, MeO); 1.97 (s, MeCO). 13C-NMR (150 MHz, CDCl3): 169.8; 165.3; 165.0;
164.8; 164.7; 164.4; 154.6; 149.2; 133.0; 132.9; 132.8; 132.6; 132.3; 129.5; 129.4; 129.3; 129.1; 129.1; 128.8;
128.7; 128.5; 128.4; 128.3; 128.2; 128.1; 128.0; 127.9; 127.7; 127.7; 127.6; 117.1; 114.0; 99.0; 95.8; 75.4; 69.6;
69.0; 68.6; 68.5; 67.8; 66.7; 63.1; 60.1; 55.0; 20.3. HR-QTOF-MS: 1003.2784 ([M þ Na]þ, C55H48NaO1þ7
;
calc. 1003.2789).
3-O-Acetyl-4,6-di-O-benzoyl-2-O-(2,3,4-tri-O-benzoyl-b-d-xylopyranosyl)-1-O-(2,2,2-trichloroetha-
nimidoyl)-a-d-mannopyranose (16). To a soln. of 15 (100 mg, 0.1 mmol) in MeCN/H2O 4 :1 (5 ml) was
added (NH4)2Ce(NO3)6 (CAN; 164 mg, 0.3 mmol), and the mixture was stirred at r.t. for 30 min, when
TLC (PE/AcOEt 2 :1) indicated that the reaction was complete. The mixture was extracted with CH2Cl2
and washed with H2O. The org. layer was concentrated, and the crude hemiacetal was purified by CC
(SiO2; PE/AcOEt 2 :1) to afford white solid. After drying under high vacuum for 2 h, the solid was
dissolved in CH2Cl2 (5 ml), and CCl3CN (0.1 ml, 1 mmol) and DBU (2 ml, 0.01 mmol) were added in an
ice bath. The mixture was stirred for 2 h, at the end of which time TLC (PE/AcOEt 2 :1) indicated that
the reaction was complete. Concentration of the mixture, followed by purification of the crude product
1
on a SiO2 column (PE/AcOEt 2 :1), afforded 16 (66 mg, 68%, for two steps). Yellow solid. H-NMR
(600 MHz, CDCl3): 8.15 (s, NH); 8.09 – 7.23 (m, 25 arom. H); 5.69 – 5.65 (m, 2 H); 5.52 (dd, J ¼ 10.1, 3.2,
1 H); 5.31 (dd, J ¼ 11.0, 5.9, 2 H); 5.23 (dd, J ¼ 8.4, 5.0, 1 H); 4.94 (d, J ¼ 3.6, 1 H); 4.58 (dd, J ¼ 12.5, 3.2,
1 H); 4.41 – 4.34 (m, 1 H); 4.29 (dd, J ¼ 12.0, 3.4, 1 H); 4.26 – 4.23 (m, 1 H); 4.10 – 4.01 (m, 2 H); 3.78 –
3.74 (m, 2 H); 3.32 (s, 1 H); 1.93 (s, MeCO). HR-QTOF-MS: 1040.2839 ([M þ Na]þ, C50H42Cl3NaO1þ6
;
calc. 1040.1569).
4-Methoxyphenyl 3-O-Acetyl-4,6-di-O-benzoyl-2-O-(2,3,4-tri-O-benzoyl-6-methyl-b-d-glucopyra-
nuronosyl)-a-d-mannopyranoside (17). Donor 14 (74 mg, 0.111 mmol) was coupled with 7 (50 mg,
0.09 mmol) as described in the General Procedure, and the product was purified by CC (SiO2; PE/AcOEt
3 :1) to give 17 (80 mg, 85%). Foamy solid. 1H-NMR (600 MHz, CDCl3): 8.00 – 7.28 (m, 25 arom. H); 6.93
(d, J ¼ 9.1, 2 arom. H); 6.70 (d, J ¼ 9.1, 2 arom. H); 6.09 (d, J ¼ 4.8, 1 H); 5.75 (t, J ¼ 9.9, 1 H); 5.64 (t, J ¼
2.8, 1 H); 5.53 (dd, J ¼ 10.1, 3.4, 1 H); 5.47 (dd, J ¼ 7.6, 1.5, 1 H); 5.37 (d, J ¼ 1.7, 1 H); 4.78 – 4.75 (m,
1 H); 4.45 (dd, J ¼ 11.7, 2.0, 1 H); 4.25 (ddt, J ¼ 8.2, 5.8, 2.9, 3 H); 4.14 (dd, J ¼ 3.3, 2.0, 1 H); 3.68 (s,
MeO); 3.58 (s, COOMe); 1.92 (s, MeCO). 13C-NMR (150 MHz, CDCl3): 169.3; 167.8; 165.8; 165.5; 165.1;
165.0; 164.8; 154.7; 149.2; 133.0; 132.9; 132.8; 132.7; 132.4; 129.5; 129.3; 129.3; 129.3; 129.2; 128.5; 128.4;
128.3; 128.2; 128.1; 128.0; 127.9; 127.9; 127.8; 127.7; 117.2; 114.1; 97.6; 90.0; 75.0; 71.0; 70.8; 69.5; 69.0;
68.8; 68.7; 68.6; 68.1; 66.7; 62.8; 55.0; 52.3; 20.2. HR-QTOF-MS: 1061.2839 ([M þ Na]þ, C57H50NaO1þ9
;
calc. 1061.2946).
4-Methoxyphenyl 4,6-Di-O-benzoyl-2-O-(2,3,4-tri-O-benzoyl-6-methyl-b-d-glucopyranuronosyl)-a-
d-mannopyranoside (18). To a soln. of 17 (100 mg, 0.1 mmol) in anh. CH2Cl2 (2 ml) was added anh.
MeOH (0.8 ml). AcCl (20 ml) was then added to the mixture at 08. The soln. was stirred in a stoppered
flask at r.t., until TLC (PE/AcOEt 2 :1) showed that the starting material had disappeared. The soln. was
neutralized with Et3N, then concentrated to dryness. The residue was passed through a short SiO2 column
to give 18 (80.05 mg, 80%). Foamy solid. 1H-NMR (600 MHz, CDCl3): 8.15 – 7.82 (m, 10 arom. H); 7.56 –
7.26 (m, 15 arom. H); 7.09 (d, J ¼ 9.0, 2 arom. H, MeOC6H4O); 6.77 (d, J ¼ 9.0, 2 arom. H, MeOC6H4O);
6.27 (t, J ¼ 9.6, 1 H); 5.86 (t, J ¼ 3.6, 1 H); 5.79 – 5.63 (m, 2 H); 5.58 – 5.47 (m, 1 H); 5.36 – 5.33 (m, 1 H);
4.88 (d, J ¼ 9.9, 1 H); 4.56 – 4.40 (m, 2 H); 4.26 (t, J ¼ 6.7, 1 H); 3.73 (s, MeO); 3.63 (s, COOMe).
13C-NMR (150 MHz, CDCl3): 169.5; 168.0; 165.2; 165.1; 165.1; 164.8; 149.2; 133.1; 133.0; 133.0; 132.9;
132.9; 132.8; 132.7; 132.5; 132.4; 129.4; 129.3; 129.3; 129.2; 129.2; 128.5; 128.3; 128.2; 128.1; 128.0; 127.9;

Helvetica Chimica Acta – Vol. 98 (2015)
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127.8; 127.8; 127.7; 127.7; 117.4; 117.2; 114.1; 97.7; 90.0; 73.0; 72.4; 71.8; 71.1; 69.5; 68.9; 68.0; 66.7; 62.8;
62.8; 55.0; 52.3. ESI-MS: 997.18 ([M þ H]þ).
4-Methoxyphenyl 2,3,4-Tri-O-benzoyl-6-methyl-b-d-glucopyranuronosyl-(1 ! 2)-[2,3,4-tri-O-benzo-
yl-b-d-xylopyranosyl-(1 ! 2)-3-O-acetyl-4,6-di-O-benzoyl-a-d-mannopyranosyl-(1 ! 3)]-4,6-di-O-ben-
zoyl-a-d-mannopyranoside (19). Compound 16 (102 mg, 0.10 mmol) and 18 (50 mg, 0.05 mmol) were
coupled under the same conditions as those used for the preparation of 17 from 7 and 14, to give 19
(56 mg, 60%). Foamy solid. 1H-NMR (600 MHz, CDCl3): 8.07 – 7.28 (m, 50 arom. H); 6.93 (d, J ¼ 9.1, 2
arom. H, MeOC6H4O); 6.76 (d, J ¼ 9.1, 2 arom. H, MeOC6H4O); 6.24 (t, J ¼ 9.9, 1 H); 5.80 – 5.71 (m,
4 H); 5.70 – 5.60 (m, 2 H); 5.54 (ddd, J ¼ 13.7, 10.2, 3.5, 3 H); 5.41 (dd, J ¼ 5.7, 4.1, 1 H); 5.33 (d, J ¼ 1.8,
1 H); 5.32 – 5.24 (m, 2 H); 5.01 (d, J ¼ 4.1, 1 H); 4.67 (d, J ¼ 10.0, 1 H); 4.59 (dd, J ¼ 12.5, 3.4, 1 H); 4.51
(dd, J ¼ 3.3, 1.8, 1 H); 4.30 (d, J ¼ 9.7, 1 H); 4.00 (ddd, J ¼ 11.2, 6.3, 2.9, 1 H); 3.86 – 3.79 (m, 2 H); 3.72 (s,
MeO); 2.03 (s, MeCO). 13C-NMR (150 MHz, CDCl3): 171.7; 169.0; 167.4; 167.1; 167.0; 166.8; 166.7; 166.6;
166.5; 166.5; 166.4; 166.2; 156.6; 150.7; 135.0; 134.9; 134.8; 134.7; 134.3; 134.2; 131.4; 131.4; 131.3; 131.2;
131.2; 131.2; 131.1; 131.1; 130.9; 130.8; 130.7; 130.6; 130.5; 130.4; 130.4; 130.3; 130.2; 130.1; 129.9; 129.9;
129.8; 129.7; 129.6; 129.6; 129.5; 119.1; 116.0; 100.5; 100.1; 98.5; 95.1; 93.3; 77.21; 76.6; 76.3; 71.7; 71.7;
71.3; 71.2; 71.2; 71.1; 71.0; 70.9; 70.7; 70.6; 70.3; 69.7; 69.4; 69.3; 69.0; 68.1; 65.3; 64.9; 64.4; 61.9; 61.4;
56.9; 54.4; 31.1. HR-QTOF-MS: 1853.5664 ([M þ H]þ, C103H89O3þ3 ; calc. 1853.5286).
2,3,4-Tri-O-benzoyl-6-methyl-b-d-glucopyranuronosyl-(1 ! 2)-[2,3,4-tri-O-benzoyl-b-d-xylopyrano-
syl-(1 ! 2)-3-O-acetyl-4,6-di-O-benzoyl-a-d-mannopyranosyl-(1 ! 3)]-4,6-di-O-benzoyl-1-O-(2,2,2-tri-
chloroethanimidoyl)-a-d-mannopyranose (20). To a soln. of 19 (56 mg, 0.03 mmol) in MeCN/H2O 4 :1
(5 ml) was added CAN (50 mg, 0.09 mmol), and the mixture was treated as described for 16 to afford 20
(28 mg, 50%, over two steps). Yellow solid. 1H-NMR (600 MHz, CDCl3): 8.30 (s, NH); 8.02 – 7.23 (m, 50
arom. H); 6.72 (d, J ¼ 3.6, 1 H); 6.03 (d, J ¼ 3.6, 1 H); 5.87 – 5.84 (m, 3 H); 5.78 (dd, J ¼ 10.4, 3.6, 1 H);
5.61 (dd, J ¼ 10.4, 8.0, 1 H); 5.57 (dd, J ¼ 10.4, 8.0, 1 H); 5.44 (dd, J ¼ 5.7, 4.1, 1 H); 5.33 (d, J ¼ 1.8, 1 H);
5.32 – 5.24 (m, 2 H); 5.01 (d, J ¼ 4.1, 1 H); 4.80 (d, J ¼ 8.0, 1 H); 4.67 (d, J ¼ 10.0, 1 H); 4.59 (dd, J ¼ 12.5,
3.4, 1 H); 4.51 (dd, J ¼ 3.3, 1.8, 1 H); 4.32 (d, J ¼ 9.7, 1 H); 4.16 (dd, J ¼ 11.2, 6.0, 1 H); 4 .14 – 4.03 (m,
3 H); 3.79 – 3.70 (m, 2 H); 3.68 (s, MeO) 2.03 (s, MeCO). HR-QTOF-MS: 1912.5709 ([M þ Na]þ,
C98H82Cl3NaOþ32 ; calc. 1912.3783).
4-Methoxyphenyl 2,3,4-Tri-O-benzoyl-6-methyl-b-d-glucopyranuronosyl-(1 ! 2)-[2,3,4-tri-O-benzo-
yl-b-d-xylopyranosyl-(1 ! 2)-3-O-acetyl-4,6-di-O-benzoyl-a-d-mannopyranosyl-(1 ! 3)]-4,6-di-O-ben-
zoyl-a-d-mannopyranosyl-(1 ! 3)-2-O-acetyl-4,6-di-O-benzoyl-a-d-mannopyranoside (21). Donor 20
(28 mg, 0.015 mmol) was coupled with acceptor 8 (80 mg, 0.15 mmol) as described in the General
Procedure, and the product was purified by CC (PE/AcOEt 3 :1) to give 21 (24 mg, 71%). Foamy solid.
1H-NMR (600 MHz, CDCl3): 8.10 – 7.25 (m, 60 arom. H); 6.83 (d, J ¼ 9.1, 2 arom. H, MeOC6H4O); 6.59
(d, J ¼ 9.1, 2 arom. H, MeOC6H4O); 5.83 (dd, J ¼ 9.0, 1 H); 5.73 – 5.69 (m, 3 H); 5.65 – 5.57 (m, 2 H);
5.53 – 5.48 (m, 3 H); 5.41 (dd, J ¼ 5.7, 4.1, 1 H); 5.33 (d, J ¼ 1.8, 1 H); 5.32 – 5.24 (m, 2 H); 5.01 (d, J ¼ 4.1,
1 H); 4.91 (d, J ¼ 7.5, 1 H); 4.67 (d, J ¼ 10.0, 1 H); 4.59 (dd, J ¼ 12.5, 3.4, 1 H); 4.52 (dd, J ¼ 3.3, 1.8, 1 H);
4.44 (dd, J ¼ 5.9, 12.0, 1 H); 4.27 (d, J ¼ 9.7, 1 H); 4.21 – 4.17 (m, 3 H); 4.04 – 3.93 (m, 6 H); 3.86 – 3.79 (m,
2 H); 3.67 (s, MeO); 2.08 (s, MeCO); 1.97 (s, MeCO). 13C-NMR (150 MHz, CDCl3): 171.9; 171.7; 169.8;
168.1; 167.9; 167.5; 167.1; 167.0; 166.9; 166.8; 166.8; 166.5; 166.4; 166.3; 156.7; 151.2; 135.0; 134.8; 134.6;
134.5; 134.3; 131.3; 131.3; 131.2; 131.1; 131.0; 130.9; 130.8; 130.6; 130.5; 130.4; 130.3; 130.2; 129.9; 129.8;
129.7; 129.7; 119.3; 116.0; 100.5; 100.3; 100.1; 97.7; 91.9; 76.3; 74.9; 74.5; 74.4; 73.7; 73.0; 71.7; 71.5; 71.3;
71.2; 71.1; 71.0; 70.9; 70.7; 70.4; 70.0; 70.0; 69.7; 69.2; 68.1; 64.7; 61.9; 56.9; 54.2; 31.1; 22.3. HR-QTOF-
MS: 2287.2782 ([M þ Na]þ; C125H108NaO4þ1 ; calc. 2287.6264).
4-Methoxyphenyl b-d-Glucopyranuronosyl-(1 ! 2)-[b-d-xylopyranosyl-(1 ! 2)-a-d-mannopyrano-
syl-(1 ! 3)]-a-d-mannopyranosyl-(1 ! 3)-a-d-mannopyranoside (22). Compound 21 (490 mg,
0.2 mmol) was dissolved in a sat. MeONa/MeOH soln. (50 ml). After 36 h at r.t., H2O (1.0 ml) was
added to the mixture to cleave the methyl ester. After stirring at r.t. for 5 h, the mixture was concentrated
and separated by CC (SiO2; MeOH), to affording 22 (126 mg, 63.0%). Foamy solid. 1H-NMR (600 MHz,
CDCl3): 6.83 (d, J ¼ 9.1, 2 H); 6.59 (d, J ¼ 9.1, 2 H); 5.22 (d, J ¼ 9.0, 1 H); 5.20 (d, J ¼ 1.7, 1 H); 4.75 (d,
J ¼ 7.5, 1 H); 4.48 (d, J ¼ 7.8, 1 H); 4.38 (d, J ¼ 4.1, 1 H); 4.26 – 3.97 (m, 5 H); 3.95 (m, 1 H); 3.92 – 3.74,
3.67 – 3.64 (m, 12 H); 3.91 (dd, J ¼ 3.5, 1 H); 3.67 (d, J ¼ 5.4, 1 H); 3.67 (s, MeO); 3.65 (d, J ¼ 11.1, 1 H);
3.57 (d, J ¼ 9.2, 1 H); 3.55 (d, J ¼ 10.0, 1 H); 3.49 (dd, J ¼ 9.2, 1 H); 3.44 (t, J ¼ 9.2, 1 H); 3.38 (d, J ¼ 9.2,

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Helvetica Chimica Acta – Vol. 98 (2015)
1 H); 3.34 (d, J ¼ 7.7, 1 H); 3.29 (d, J ¼ 11.1, 1 H). 13C-NMR (150 MHz, CDCl3): 176.6; 166.8; 166.8;
166.5; 166.4; 166.3; 156.7; 103.6; 102.9; 76.42; 76.3; 74.4; 74.1; 73.6; 73.4; 72.6; 70.5; 70.4; 70.1; 68.5; 67.2;
67.; 66.2; 61.8; 61.5; 61.1; 55.7. HR-QTOF-MS: 941.2830 ([M þ Na]þ, C36H54NaO2þ7 ; calc. 941.2750).
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Received September 18, 2014