A facile synthesis of mannose tri- and tetrasaccharide repeating units of fungal cell-wall polysaccharide from Microsporum and Trychophyton species

Article abstract of DOI:10.1081/SCC-120003613

A facile synthesis of the trisaccharide α-D-mannopyranosyl-(1→2)-α-D-mannopyranosyl-(1→6) -α-D-mannopyranose and the tetrasaccharide α-D-mannopyranosyl-(1→2)-α-D-mannopyranosyl-(1→6) -α-D-mannopyranosyl-(1→6)-D-mannopyranose, the repeating units of fungal cell-wall polysaccharide from Microsporum gypseum and Trychophyton, was achieved using α(1→2)-linked disaccharide imidate as the donor. The disaccharide imidate was prepared from the self-condensation of 3,4,6-tri-O-benzoyl-1,2-O-allyloxyethylidene-β-D-mannopyranose.

Full text of DOI:10.1081/SCC-120003613

SYNTHETIC COMMUNICATIONS, 32(8), 1219–1226 (2002)
A FACILE SYNTHESIS OF MANNOSE
TRI- AND TETRASACCHARIDE
REPEATING UNITS OF FUNGAL
CELL-WALL POLYSACCHARIDE
FROM MICROSPORUM AND
TRYCHOPHYTON SPECIES
*
Yuliang Zhu and Fanzuo Kong
Research Center for Eco-Environmental Sciences,
Academia Sinica, P.O. Box 2871, Beijing 100085, China
ABSTRACT
A facile synthesis of the trisaccharide a-D-mannopyranosyl-
(1!2)-a-D-mannopyranosyl-(1!6)-a-D-mannopyranose and
the tetrasaccharide a-D-mannopyranosyl-(1!2)-a-D-manno-
pyranosyl-(1!6)-a-D-mannopyranosyl-(1!6)-D-mannopyranose,
the repeating units of fungal cell-wall polysaccharide from
Microsporum gypseum and Trychophyton, was achieved
using a-(1!2)-linked disaccharide imidate as the donor.
The disaccharide imidate was prepared from the self-conden-
sation of 3,4,6-tri-O-benzoyl-1,2-O-allyloxyethylidene-b-D-
mannopyranose.
*Corresponding author.
1219
Copyright & 2002 by Marcel Dekker, Inc.
www.dekker.com

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ZHU AND KONG
Dermatophytes are a group of fungi that parasitise animals and
human beings, causing superficial cutaneous infections on the keratinised
tissues of the epidermis, nails and pilosebaceous follicles. The cell wall of
dermatophytes contains antigenically relevant mannan consisting of
branched oligosaccharide repeating units.1 Further studies on the mannan
of cell wall from Microsporum and Trychophyton species revealed that there
was an idealized general skeleton for the mannan, being, approximately
m ¼ 0 for M. gypseum, T. rubrum and T. soudannense; n ¼ m for M. canis
and M. cookei.2 Now we report the synthesis of the tri- and tetrasaccharide
repeating units in these skeletons of mannan.
Although the desired tri- and tetrasaccharide structures are not
very complex, orthogonal protective groups and multisteps will be needed
if traditional methods are used. Our previous communications3,4 revealed
new methods for construction of a-(1!6)- and a-(1!2)-mannopyranosyl
linkages using unprotected or partially protected sugars as the acceptors
via Schmidt’s coupling. Based on these methods, a concise and efficient
synthesis of the title oligosaccharides was accomplished. Thus 3,4,6-tri-O-
acetyl-1,2-O-allyloxyethylidene-b-D-mannopyranose (1) was chosen as the
starting material that was obtained by treatment of acetobromomannose
with allyl alcohol in the presence of 2,4-lutidine.5 Deacetylation with catalytic
sodium methoxide in methanol (!2) followed by benzoylation6 with
benzoyl chloride in pyridine furnished 3,4,6-tri-O-benzoyl-1,2-O-allyloxy-
ethylidene-b-D-mannopyranose (3) in high yield. Transformation of 3
with catalytic TMSOTf afforded the self-condensation product,4 allyl 2-O-
acetyl-3,4,6-tri-O-benzoyl-a-D-mannopyranosyl-(1!2)-3,4,6-tri-O-benzoyl-a-
D-mannopyranoside (5), as the major one in satisfactory yield together
with the rearrangement product allyl 2-O-acetyl-3,4,6-tri-O-benzoyl-a-D-
mannopyranoside (4) as the minor one. Thus the preparation of (1!2)
linked disaccharide by self-condensation of orthoester 3 became a concise
and effective method, being much simpler than the stepwise glycosyla-
tion from a monosugar donor and acceptor. Deallylation of 5 with PdCl2
in NaOAc-containing acetic acid followed by treatment with CCl3CN

TRI- AND TETRASACCHARIDE REPEATING UNITS
1221
Scheme.
and DBU7 gave 2-O-acetyl-3,4,6-tri-O-benzoyl-a-D-mannopyranosyl-(1!2)-
3,4,6-tri-O-benzoyl-a-D-mannopyranosyl trichloracetimidate (6) in satis-
factory yield.
With the disaccharide donor 6 in hand, the tri- and tetrasaccharides
were concisely synthesized by coupling of 6 with allyl 2,3,4-tri-O-benzoyl-a-
D-mannopyranoside (11) and with allyl a-D-mannopyranosyl-(1!6)-2,3,4-
tri-O-benzoyl-a-D-mannopyranoside (7)3 respectively. Deacylation afforded

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ZHU AND KONG
the corresponding trisaccharide (13) and tetrasaccharide (10) respectively,
and their bioassay is in process.
In summary, here we present a facile and effective method for the
synthesis of mannose tri- and tetrasaccharide repeating units of fungal
cell-wall polysaccharides from Microsporum and Trychophyton species.
The key disaccharide was prepared from the self-condensation of manno-
pyranose allyl orthoester directly.
EXPERIMENTAL
Melting points were determined with a ‘Mel-Temp’ apparatus. Optical
rotations were determined with a Perkin-Elmer model 241-MC automatic
1
polarimeter for solutions in a 1-dm, jacketed cell. H NMR spectra were
recorded with Varian XL-400 and Varian XL-200 spectrometers, for solu-
tions in CDCl3 with tetramethylsilane (Me4Si) as the internal standard.
Chemical shifts are expressed in ppm downfield from the internal Me4Si
absorption. Mass spectra were recorded with a VG PLATFORM mass spec-
trometer using the ESI technique to introduce the sample. Thin-layer chro-
matography (TLC) was performed on silica gel HF, detection being affected
by charring with 30% (v/v) sulfuric acid in methanol or sometimes by UV
detection. Column chromatography was conducted by elution of a column
(8/100 mm, 16/240 mm, 18/300 mm, 35/400 mm) of silica gel (100–200 mesh)
and EtOAc/petroleum ether (b.p. 60–90ꢀC) as the eluent. Analytical LC was
performed with a Gilson HPLC consisting of a pump (model 306), stainless
steel packed with silica gel (Spherisorb SiO2, 1 0Â 300 mm or 4.6 Â 250 mm),
differential refractometer (132-RI Detector), UV/VIS detector (model 118),
and EtOAc/petroleum ether (b.p. 60–90ꢀC) was used as the eluent at a flow
rate of 1–4 ml/min. Solutions were concentrated at a temperature of <60ꢀC
under diminished pressure.
3,4,6-Tri-O-Benzoyl-1,2-O-allyloxyethylidene-b-D-mannopyranose (3):
3,4,6-Tri-O-Acetyl-1,2-O-allyloxyethylidene-b-D-mannopyranose 1 (776 mg,
2 mmol) was suspended in freshly distilled MeOH (10 ml), a solution of
sodium methoxide (2 M, 0.1ml) was added, and the mixture was stirred
overnight at room temperature. TLC showed that the reaction was com-
plete, and the resulting solution was de-ionized with Amberlite IR-120 (Hþ)
anion-exchange resin, filtered, concentrated, and then benzoylated with
BzCl/pyridine to furnish 3 in quantitative yield. m.p. 141–144ꢀC; [a]D
À11.5ꢀ (c 1.1, CHCl3); H NMR d 8.03–7.26 (m, 15H, Bz-H), 5.92 (t, 1H,
1
J3,4 9.8 Hz, H-4), 5.90 (m, 1H, CH2¼CH-CH2), 5.66 (d, 1H, J1,2 2.6 Hz,
H-1), 5.56 (dd, 1H, J2,3 3.9 Hz), 5.30–5.20 (m, 2H, CH2¼CH-CH2), 4.86
(dd, 1H, H-2), 4.62 (dd, 1H, H-6), 4.46 (dd, 1H, H-6), 4.30 (m, 1H,

TRI- AND TETRASACCHARIDE REPEATING UNITS
1223
CH2¼CH-CH2), 4.10–4.05 (m, 2H, H-5, CH2¼CH-CH2). Anal. calcd for
C32H30O10: C, 66.90; H, 5.23. Found: C, 66.93; H, 5.21.
Allyl 2-O-acetyl-3,4,6-tri-O-benzoyl-a-D-mannopyranosyl- (1!2)-3,4,6-
tri-O-benzoyl-a-D-mannopyranoside (5): 3,4,6-Tri-O-Benzoyl-1,2-O-allyloxy-
ethylidene-b-D-mannopyranoside 3 (1148 mg, 2 mmol) were dried under
high vacuum for 2 h, then dissolved in anhydrous dichloromethane
(60 ml). TMSOTf (30 ml, 0.08 equiv.) was added dropwise at À42ꢀC with
N2 protection. The reaction mixture was stirred for 3 h. Then the mixture
was neutralized with triethylamine and concentrated. Further purification
by column chromatography (2/1petroleum ether/ethyl acetate) gave
5
(692 mg, 66%) as a colorless solid. m.p. 148–150ꢀC; [a]D þ 3.0ꢀ (c 1.3,
CHCl3); 1H NMR d 8.07–7.33 (m, 30H, Bz-H), 5.99 (t, 1H, J 9.9 Hz),
5.90–5.85 (m, 4H), 5.70 (dd, 1H, J 1.7 Hz, J 2.7 Hz), 5.28–5.18 (m, 2H,
CH2¼CH-CH2), 5.16 (d, 1H, J 1.5 Hz), 5.11 (d, 1H, J 1.8 Hz), 4.61–4.47
(m, 5H), 4.39–4.36 (m, 2H), 4.15–4.13 (m, 1H), 3.95–3.92 (m, 1H), 2.03 (s,
3H, CH3CO). Anal. calcd for C59H52O18: C, 67.56; H, 4.96. Found: C,
67.60; H, 4.92.
2-O-Acetyl-3,4,6-tri-O-benzoyl-a-D-mannopyranosyl-(1Þ2)-3,4,6-tri-O-
benzoyl-a-D-mannopyranosyl trichloroacetimidate (6): Allyl 2-O-acetyl-3,4,6-
tri-O-benzoyl-a-D-mannopyranosyl-(1!2)-3,4,6-tri-O-benzoyl-a-D-manno-
pyranoside 5 (524 mg, 0.5 mmol) in 90% acetic acid (10 ml) containing
sodium acetate (293 mg, 3 mmol) was added PdCl2 (89 mg, 0.5 mmol) and
the mixture was stirred for 12 h, at the end of which time TLC (2/1 petro-
leum ether/ethyl acetate) indicated that the reaction was complete. The
mixture was diluted with dichloromethane (30 ml), washed with water and
a saturated aqueous solution of sodium bicarbonate. The organic layer was
concentrated under reduced pressure and the obtained residue was passed
a short silica gel column with 2/1petroleum ether/ethyl acetate as the
eluent to give crude 2-O-acetyl-3,4,6-tri-O-benzoyl-a-D-mannopyranosyl-
(1!2)-3,4,6-tri-O-benzoyl-a-D-mannopyranose (456 mg), which was dis-
solved in dichloromethane (20 ml), then CCl3CN (0.1ml, 2 mmol) and
DBU (14 ml, 0.18 mmol) were added. The reaction mixture was stirred for
2 h, at the end of which time TLC (2/1petroleum ether/ethyl acetate) indi-
cated that the reaction was complete. Concentration of the reaction mixture
followed by purification on a silica gel column with 2/1petroleum ether/
ethyl acetate as the eluent furnished the disaccharide donor 6 as crystals in a
good yield (473 mg, 90% for two steps). m.p. 139–142ꢀC; [a]D À1.5ꢀ (c 1.1,
CHCl3); 1H NMR d 8.64 (s, 1H, C¼NH), 8.02–7.26 (m, 30H, Bz-H), 6.59 (d,
1H, J1,2 1.9 Hz, H-1), 6.09 (t, 1H, J3,4 9.8 Hz, H-4), 5.95–5.87 (m, 3H), 5.70
(dd, 1H, J1 ,2 2.8 Hz, J1 ,2 2.1Hz, H-2 0), 5.20 (d, 1H, H-10), 4.68–4.48 (m,
8H), 2.03 (s, 3H, CH3CO). Anal. calcd for C58H48O18Cl3N: C, 60.31; H,
4.16. Found: C, 60.34; H, 4.15.
0
0
0
0

1224
ZHU AND KONG
Allyl 2-O-acetyl-3,4,6-tri-O-benzoyl-a-D-mannopyranosyl-(1 Þ 2)-3,4,6-
tri-O-benzoyl-a-D-mannopyranosyl-(1 Þ 6)-a-D-mannopyranosyl-(1 Þ 6)-
2,3,4-tri-O-benzoyl-a-D-mannopyranoside (8): Compound (461mg,
6
0.4 mmol) and allyl a-D-mannopyranosyl-(1!6)-2,3,4-tri-O-benzoyl-a-D-
mannopyranoside 7 (278 mg, 0.4 mmol) were dried together under high
vacuum for 2 h, then dissolved in anhydrous dichloromethane (30 ml).
TMSOTf (8 ml, 0.10 equiv.) was added dropwise at À42ꢀC with N2 protec-
tion. The reaction mixture was stirred for 3 h, at the end of which time TLC
(1/1.5 petroleum ether/ethyl acetate) indicated that the reaction was com-
plete. Then the mixture was neutralized with triethylamine, concentrated
under reduced pressure to dryness. Further purification by column chroma-
tography (1/1.5 petroleum ether/ethyl acetate) gave 8 (505 mg, 75%) as a
colorless solid. m.p. 131–134ꢀC; [a]D þ21.5ꢀ (c 1.0, CHCl3); H NMR d
1
8.04–7.27 (m, 45H, Bz-H), 6.22 (t, 1H, J 10.1 Hz), 6.03–6.00 (m, 3H),
5.88–5.80 (m, 4H), 5.58 (dd, 1H, J 1.4 Hz, J 2.8 Hz), 5.27 (dd, 1H,
CH2¼CH-CH2), 5.21(dd, H1 , C H2¼CH-CH2), 5.21(d, H1 ,
J 1.5 Hz),
5.18 (d, 1H, J 1.4 Hz), 4.95 (d, 1H, J 1.5 Hz), 4.80 (d, 1H, J 1.6 Hz),
4.61–4.58 (m, 2H), 4.54–4.50 (m, 1H), 4.46–4.40 (m, 3H), 4.37–4.28 (m, 6H),
4.29–4.24(m,2H),3.87–3.834(m,3H),3.39–3.37(m,1H),2.03(s,3H,CH3CO).
Anal. calcd for C92H84O31: C, 65.56; H, 4.99. Found: C, 65.52; H, 5.02.
Allyl 2-O-acetyl-3,4,6-tri-O-benzoyl-a-D-mannopyranosyl-(1 Þ 2)-3,4,6-
tri-O-benzoyl-a-D-mannopyranosyl-(1 Þ 6)-2,3,4-tri-O-benzoyl-a-D-manno-
pyranosyl-(1 Þ 6)-2,3,4-tri-O-benzoyl-a-D-mannopyranoside (9): Tetrasac-
charide 8 (337 mg, 0.2 mmol) was dissolved in pyridine (10 ml), to the
solution was added dropwise benzoyl chloride (0.15 ml, 15 mmol), and the
mixture was stirred overnight at room temperature. TLC (2/1petroleum
ether/ethyl acetate) indicated that the reaction was complete. The mixture
was concentrated under reduced pressure to dryness. Further purification by
column chromatography (2/1petroleum either/ethyl acetate) gave 9 (391mg,
98%) as a colorless solid. m.p. 133–136ꢀC; [a]D þ38.5ꢀ (c 1.1, CHCl3);
1H NMR d 8.08–7.26 (m, 60H, Bz-H), 6.22 (t, 1H, J 10.0 Hz), 6.05–6.02 (m,
4H), 5.89–5.81(m, 6H), 5.59 (dd, 1H, J 1.4 Hz, J 2.7 Hz), 5.29 (dd, 1H,
CH2¼CH-CH2), 5.20 (dd, 1H, CH2¼CH-CH2), 5.22 (d, 1H, J 1.5 Hz), 5.20
(d, 1H, J 1.3 Hz), 4.98 (d, 1H, J 1.4 Hz), 4.83 (d, 1H, J 1.6 Hz), 4.61–4.58 (m,
1H), 4.54–4.49 (m, 1H), 4.44–4.41 (m, 2H), 4.39–4.31 (m, 5H), 4.28–4.22 (m,
2H), 3.89–3.83 (m, 3H), 3.39–3.37 (m, 1H), 2.03 (s, 3H, CH3CO). 13C NMR d
169.19(CH3CO),166.10,165.82,165.68,165.52,165.52,165.43,165.36,165.36,
165.28, 165.17, 165.09, 164.85 (12C6H5CO), 118.37 (CH2¼CH-CH2-), 99.62,
98.00, 97.86, 96.83 (C-1I–IV), 77.60 (C-2), 70.51, 70.46, 70.42, 70.35, 70.33,
69.57, 69.57, 69.51, 69.51, 68.72, 68.72, 68.55, 67.12, 66.78, 66.71, 66.67,
66.51, 65.37, 63.33, 62.89 (CH2¼CH-CH2-, C-2,3,4,5,6I–IV); Anal. calcd
for C113H96O34: C, 67.94; H, 4.81. Found: C, 67.98; H, 4.78.

TRI- AND TETRASACCHARIDE REPEATING UNITS
1225
Allyl
a-D-mannopyranosyl-(1 Þ 2)-a-D-mannopyranosyl-(1 Þ 6)-a-D-
mannopyranosyl-(1 Þ 6)-a-D-mannopyranoside (10): Tetrasaccharide
9
(399 mg, 0.2 mmol) was suspended in freshly distilled MeOH (5 ml), a solu-
tion of sodium methoxide (2 M, 0.1ml) was added, and the mixture was
stirred overnight at room temperature. TLC showed that the reaction was
complete, and the resulting solution was de-ionized with Amberlite IR-120
(Hþ) anion-exchange resin, filtered, and concentrated. The tetrasaccharide
10 was finally obtained after chromatography on sephadex G-25 column
(H2O) as an amorphous powder (120 mg, 85%) after freeze-drying;
1H NMR d 5.93–5.84 (m, 1H, CH2¼CH-CH2), 5.26 (dd, 1H, CH2¼CH-
CH2), 5.18 (dd, 1H, CH2¼CH-CH2), 4.96 (s, 1H), 4.94 (s, 1H), 4.81 (s,
1H), 4.79 (s, 1H), 4.11–3.60 (m, 26H). ESMS for C27H46O21 (706.25):
706.50.
Allyl 2-O-acetyl-3,4,6-tri-O-benzoyl-a-D-mannopyranosyl-(1 Þ 2)-3,4,6-
tri-O-benzoyl-a-D-mannopyranosyl-(1 Þ 6)-2,3,4-tri-O-benzoyl-a-D-manno-
pyranoside (12): Disaccharide donor 6 (461mg, 0.4 mmol) and allyl 2,3,4-tri-
O-benzoyl-a-D-mannopyranoside 11 (213 mg, 0.4 mmol) were dried together
under high vacuum for 2 h, then dissolved in anhydrous dichloromethane
(30 ml). TMSOTf (8 ml, 0.10 equiv.) was added dropwise at À42ꢀC with N2
protection. The reaction mixture was stirred for 3 h, at the end of which time
TLC (1/1.5 petroleum ether/ethyl acetate) indicated that the reaction was
complete. Then the mixture was neutralized with triethylamine, concen-
trated under reduced pressure to dryness. Further purification by column
chromatography (1/1.5 petroleum ether/ethyl acetate) gave 12 (548 mg,
90%) as a colorless solid. m.p. 138–140ꢀC; [a]D À11.5ꢀ (c 1.3, CHCl3);
1H NMR d 8.12–7.26 (m, 45H, Bz-H), 6.05–5.85 (m, 7H), 5.74 (dd, 1H, J
1.6 Hz, J 3.2 Hz), 5.60 (dd, 1H, J 1.7 Hz, J 3.2 Hz), 5.50–5.34 (m, 2H,
CH2¼CH-CH2), 5.20 (d, 1H, J 1.6 Hz), 5.15 (d, 1H, J 1.5 Hz), 4.95 (d,
1H, J 1.9 Hz), 4.51–4.31 (m, 9H), 4.23–4.17 (m, 1H), 4.02–3.96 (m, 1H),
3.64–3.60 (m, 1H), 2.00 (s, 3H, CH3CO). 13C NMR d 169.24 (CH3CO),
166.17, 165.95, 165.61, 165.61, 165.51, 165.49, 165.39, 165.23, 164.92
(9C6H5CO), 118.46 (CH2¼CH-CH2-), 99.75, 98.41, 96.81 (C-1I–III), 77.50
(C-2), 70.74, 70.69, 70.23, 69.65, 69.65, 69.59, 69.59, 68.87, 68.79, 67.12,
67.11, 67.00, 66.41, 63.49, 63.16 (CH2-CH¼CH2, C-2,3,4,5,6I–III); Anal.
calcd for C86H74O26: C, 67.80; H, 4.86. Found: C, 67.83; H, 4.83.
Allyl
a-D-mannopyranosyl-(1 Þ 2)-a-D-mannopyranosyl-(1 Þ 6)-a-D-
mannopyranoside (13): Trisaccharide 12 (304 mg, 0.2 mmol) was suspended
in freshly distilled MeOH (5 ml), a solution of sodium methoxide (2 M,
0.1ml) was added, and the mixture was stirred overnight at room tem-
perature. TLC showed that the reaction was complete, and the resulting
solution was de-ionized with Amberlite IR-120 (Hþ) anion-exchange resin,
filtered, and concentrated. The trisaccharide 13 was finally obtained after

1226
ZHU AND KONG
chromatography on sephadex G-25 column (H2O) as an amorphous powder
1
(97 mg, 89%) after freeze-drying; H NMR d 5.99–5.91(m, 1H, CH ¼CH-
2
CH2), 5.37 (dd, 1H, CH2¼CH-CH2), 5.27 (dd, 1H, CH2¼CH-CH2), 4.91(s,
1H), 4.85 (s, 1H), 4.80 (s, 1H), 4.02–3.62 (m, 20H).
ACKNOWLEDGMENTS
This work was supported by The Chinese Academy of Sciences
(Projects KJ952J1510 and RCEES9904) and by The National Natural
Science Foundation of China (Projects 29802009, 30070815 and 39970864).
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Received in the UK January 4, 2001