Synthesis and bioassay of β-(1,4)-D-mannans as potential agents against Alzheimer's disease

Article abstract of DOI:10.1038/aps.2013.104

Aim: Oligomannurarate 971 derived from a marine plant has shown neuroprotective effects. In this study we synthesized a series of truncated derivatives of the oligosaccharide, and investigated the effect of these derivatives against Aβ peptide toxicity in vitro. Methods: The sulfoxide method was applied to synthesize the derivatives. SH-SY5Y human neuroblastoma cells were treated with A β 1-40 (2 μmol/L), and the cell viability was detected using a CCK8 assay. Results: A series of β-(1,4)-D-mannosyl oligosaccharide, ranging from the disaccharide to the hexasaccharide, were synthesized. Addition of 10 μmol/L β-(1,4)-D-mannobiose 6, β-(1,4)-D-mannotriose 9 or P-(l,4)-D-mannotetraose 12 in SH-SY5Y cells significantly attenuated Aβ1-40-induced toxicity. The efficacies were similar to those caused by 10 μmol/L oligomannurarate 971 or alzhemed. Other oligosaccharides including oligomaltoses and oligocelluloses were less active. Conclusion: Synthetic homogeneous short chain β-(1,4)-D-mannans shows neuroprotective effect against Aβ peptide toxicity similar to that of heterogeneous oligomannurarate 971 and alzhemed.

Full text of DOI:10.1038/aps.2013.104

Acta Pharmacologica Sinica (2013) 34: 1585–1591
© 2013 CPS and SIMM All rights reserved 1671-4083/13 $32.00
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Original Article
Synthesis and bioassay of β-(1,4)-D-mannans as
potential agents against Alzheimer’s disease
*
Ru-wei JIANG, Xiao-guang DU, Xuan ZHANG, Xin WANG, Ding-yu HU, Tao MENG, Yue-lei CHEN, Mei-yu GENG , Jing-kang
*
SHEN
State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Shanghai 201203, China
Aim: Oligomannurarate 971 derived from a marine plant has shown neuroprotective effects. In this study we synthesized a series of
truncated derivatives of the oligosaccharide, and investigated the effect of these derivatives against Aβ peptide toxicity in vitro.
Methods: The sulfoxide method was applied to synthesize the derivatives. SH-SY5Y human neuroblastoma cells were treated with
Aβ1-40 (2 µmol/L), and the cell viability was detected using a CCK8 assay.
Results: A series of β-(1,4)-D-mannosyl oligosaccharide, ranging from the disaccharide to the hexasaccharide, were synthesized.
Addition of 10 µmol/L β-(1,4)-D-mannobiose 6, β-(1,4)-D-mannotriose 9 or β-(1,4)-D-mannotetraose 12 in SH-SY5Y cells significantly
attenuated Aβ1-40-induced toxicity. The efficacies were similar to those caused by 10 µmol/L oligomannurarate 971 or alzhemed.
Other oligosaccharides including oligomaltoses and oligocelluloses were less active.
Conclusion: Synthetic homogeneous short chain β-(1,4)-D-mannans shows neuroprotective effect against Aβ peptide toxicity similar to
that of heterogeneous oligomannurarate 971 and alzhemed.
Keywords: Alzheimer’s disease; Aβ peptide; oligosaccharide; β-(1,4)-D-mannan; oligomannurarate 971; alzhemed; neuroprotection;
medicinal chemistry
Acta Pharmacologica Sinica (2013) 34: 1585–1591; doi: 10.1038/aps.2013.104; published online 18 Nov 2013
ties and low toxicity[5–10]. Certain types of saccharides have
Introduction
Alzheimer’s disease (AD) is a neurodegenerative disease with also been shown to demonstrate neuroprotective effects[9-10]
,
multiple etiologies. As no effective treatments have yet been but the limited availability of pure saccharide compounds
developed, AD remains pandemic in the 21st century and restricts further research on their activity. However, oligo-
imposes enormous social and economic burdens on patients mannurarate 971 is a readily available, inexpensive and potent
and their families[1]. It is estimated that 5.4 million individuals neuroprotective drug. Derived from a marine plant, the acidic
in the United States suffer from the disease, with AD patients heterogeneous β-(1,4)-D-oligomannurarate 971 (Figure 1) is a
numbering as many as 30 million globally, and these numbers potent inhibitor of neurotoxicity induced by the Aβ peptide. It
continue to increase[2].
is believed that 971 penetrates the blood brain barrier with the
AD is characterized histopathologically by senile plaques, aid of the transporter GLUT1, and it exhibits better efficacy in
neurofibrillary tangles, reactive astrocytosis and neuronal cell animal models of dementia than does alzhemed (also known
loss. The Aβ peptide, the major component of senile plaques, as AZ, an anti-AD drug targeting the HHQK subregion at the
has been identified in numerous cases as a major causative N-terminus of Aβ1-40, which failed in a late stage Phase III clini-
factor in AD pathophysiology[3, 4]. The Aβ peptide has, con- cal trial)[11, 12]. Mannurarate 971 is currently undergoing Phase
sequently, become one of the most important targets for AD II clinical studies[12]. As synthetic medicinal chemists, we
therapy.
Carbohydrate drugs are widely employed in the treatment
of a number of major diseases owing to their varied bioactivi-
wondered if truncated derivatives of 971 would exhibit similar
*
To whom correspondence should be addressed.
E-mail mygeng@simm.ac.cn (Mei-yu GENG);
jkshen@mail.shcnc.ac.cn (Jing-kang SHEN)
Received 2013-04-14 Accepted 2013-07-17
1. The structure of oligomannurarate 971. n=2−8.
Figure

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activity. We focused specifically on β-(1,4)-D-mannans as sim- provided benzyl β-mannoside 2[19]. Removal of the benzyli-
plified 971 analogs. To the best of our knowledge, the neuro- dene acetal of 2 under acidic conditions yielded the intermedi-
protective effects of β-(1,4)-D-mannans have not been reported ate diol (structure not shown), which was acetylated regiose-
in the literature. This may be due to the limited availability of lectively at the 6-OH to give mannosyl acceptor 3. Coupling of
these compounds. While it can be very difficult to synthesize acceptor 3 with mannosyl donor 1 under Crich’s standard con-
pure β-(1,4)-D-mannans, an effective solution was reported ditions afforded the fully protected β-(1,4)-linked mannosyl
recently by Crich et al[13–17]. We followed the reported method disaccharide 4 in 70% yield (Scheme 1). The β-configuration
and prepared a series of β-(1,4)-D-mannans for biological of 4 was confirmed from the assignment of the H-5 chemical
studies.
shift at δ 3.09[18] in the 1H NMR spectrum. This peak appeared
as a doublet of triples, which is typical and diagnostic for the
β-configuration in 4,6-O-benzylidene-protected mannosides.
Materials and methods
Chemistry
Glycoside 4 also displayed two anomeric carbon signals at δ
The sulfoxide method[13–17] is one of the few approaches that 101.4 and δ 99.8 with JCH coupling constants of 162.5 and 155
can be used reliably to overcome the difficulties in control- Hz, respectively, a pattern that is consistent with the presence
ling anomeric stereochemistry and yield the desired β-(1,4)-D- of β-O-glycosides[20]. The anomeric stereochemistry in all sub-
mannosyl oligosaccharide series. We have used this method sequent coupling products was assigned by similar compari-
to prepare the target β-(1,4)-D-mannosyl oligosaccharide son of the H-5 chemical shift in the 1H NMR spectrum.
1
series, ranging from the disaccharide to the hexasaccharide,
The preparation of β-(1,4)-linked mannosyl disaccharide
via multistep sequences. The linear syntheses of β-mannans 4 from β-mannoside 2 represented the first glycosylation
commenced from the sulfoxide glycoside donor 1[18], which cycle of a two-stage iterative sequence[17]. This protocol was
was prepared in 6 steps from D-mannose (30% yield overall). repeated to provide the fully protected β-(1,4)-mannans 7, 10,
Activation of mannose donor 1 at -78°C with triflic anhydride 13, and 16. Deacetylation of the corresponding acceptor fol-
(Tf2O) in the presence of the hindered base 2,4,6-tri-tert-butyl- lowed by global debenzylation under catalytic hydrogenation
pyrimidine (TTBP)[17], followed by addition of benzyl alcohol, conditions furnished the corresponding β-(1,4)-D-mannans 6,
1. Synthesis of compound 4. Reagents and conditions: (a) Tf O, TTBP, BnOH; (b) TFA then Ac O, Et N; (c) Tf O, TTBP.
Scheme
2
2
3
2
2. Synthesis of compounds 6, 9, 12, 15, and 17. Reagents and conditions: (a) TFA, then Ac O, Et N; (b) K CO , MeOH then 20% Pd(OH) /C,
Scheme
2
3
2
3
2
H2. Compound 17 was obtained directly from 16 using conditions (b).
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9, 12, 15, and 17 (Scheme 2).
Drugs and reagents
The β-(1,4)-D-mannosyl oligosaccharide series was synthe-
sized in our laboratory. Oligomannurarate 971 was obtained
from Mei-yu GENG’s lab at the Shanghai Institute of Materia
Medica. Alzhemed (AZ) was purchased from Sigma-Aldrich
China and the maltose and cellulose series were purchased
from J&K Chemical Ltd (Shanghai, China).
Neuroprotective effect assay against Aβ peptide toxicity
SH-SY5Y human neuroblastoma cells were purchased from
the American Type Culture Collection (ATCC, Manassas, VA,
USA). These are stable cells that typically will not be differen-
tiated by exposure to small carbohydrates. Cells were plated
at 4000 cells per well in 96-well plates and cultured in Dul-
becco’s modified Eagle’s medium at 37°C in 5% CO2 (v/v) in a
humidified incubator. After 24 h, aged Aβ1–40 (incubated for 96
h) was added to cells in combination with various compounds
at different concentrations and the cells were incubated for 48
h. The final concentration of Aβ was 2 µmol/L. Cell viability
was measured in a CCK8 assay.
2. Neuroprotective effects of oligosaccharides against Aβ peptide
Figure
toxicity on SH-SY5Y cells (mean±SD, n=3). cP<0.01 vs control. eP<0.05
vs model. M2 M7 represent maltobiose-maltoheptaose, respectively.
-
C2–C6 represent cellobiose-cellohexose, respectively. The concentrations
of 6 17, M2 M7, and C2 C6 were 10 μmol/L; the concentration of 971
-
-
-
was 50 μg/mL (approximately 50 μmol/L); the concentration of AZ was 50
Statistical analysis
μmol/L.
±SD.
The data were expressed as mean
used for statistical analysis.
The Student's t-test was
OD=1.13; C6: OD=1.19) (Figure 2). The differences in activ-
Results and discussion
Neuroprotective effects of oligosaccharides against Aβ peptide ity between oligomannoses, oligomaltoses and oligocellulose
toxicity
indicate that the nature of the monosaccharide unit and the
The synthetic homogeneous β-(1,4)-D-mannosyl oligosaccha- configuration of the anomeric center exert significant influence
rides were assessed for activity against Aβ peptide neurotoxic- over the neuroprotective potency of oligosaccharides. We
ity. Control compounds included D-mannose, mannurarate believe that this preliminary finding reveals important infor-
971 and AZ. Commercially available oligomaltoses, including mation on the structural characteristics of oligosaccharides
maltobiose (M2), maltotriose (M3), maltotetraose (M4), malto- that exhibit neuroprotective effects. More data are required to
pentaose (M5), maltohexaose (M6), and maltoheptaose (M7) enable a detailed analysis of structure activity relationships.
were assessed for comparison with the synthetic compounds.
Oligocelluloses, including cellobiose (C2), cellotriose (C3), cel-
lotetraose (C4), cellopentaose (C5) and cellohexose (C6) were In this study, we applied the sulfoxide method to the prepara-
also assessed.
tion of a series of β-(1,4)-D-mannosyl oligosaccharides. Oli-
Conclusion
Mannurarate 971 (OD=1.31) and AZ (OD=1.37) were found gosaccharides ranging from disaccharide to hexasaccharide
to exhibit potent neuroprotective effects (Aβ model: OD=1.12, were synthesized in multistep sequences. The neuroprotective
control: OD=1.42±0.02) as expected. AZ was the most potent activity of synthetic mannans was assessed and compared
of the compounds tested. The synthetic homogeneous β-(1,4)- with that of mannose, 971, AZ and a series of commercially
D-mannosyl oligosaccharides 6 (OD=1.32), 9 (OD=1.35), 12 available oligomaltoses and oligocelluloses. Synthetic com-
6, β-(1,4)-D-mannotriose 9 and
(OD=1.27), 15 (OD=1.22), and 17 (OD=1.24) also showed pounds β-(1,4)-D-mannobiose
12 showed potency similar to that
neuroprotective potency. Compounds 6 and 9 were slightly β-(1,4)-D-mannotetraose
more active than 971 and were nearly as potent as AZ. This is of 971 (and were slightly less potent than AZ) as inhibitors of
a significant result in light of the structural simplicity of these toxicity induced by the Aβ peptide. Other oligosaccharides
compounds. D-mannose did not show any neuroprotective failed to show significant neuroprotective activity. Taken
activity. Oligocelluloses and oligomaltoses were also exam- together, these results demonstrate that the structure of 971
ined to shed further light on the neuroprotective activity of can be modified without a loss of activity. We have disclosed
oligosaccharides. Only maltoheptaose (M7) showed signifi- a new class of neuroprotective agents with potency against Aβ
cant activity (OD=1.27), while the others were largely inactive toxicity and gained insight that will enable development of
(M2: OD=1.23; M3: OD=1.17; M4: OD=1.17; M5: OD=1.19; potent agents for the treatment of Alzheimer’s disease. Fur-
M6: OD=1.22; C2: OD=1.17; C3: OD=1.11; C4: OD=1.15; C5: ther research is ongoing in our group.
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twice with CH2Cl2. The combined organic phases were dried
Experiment
Reagents (chemicals) were purchased from Acros (Geel, over anhydrous Na2SO4, filtered and concentrated in vacuo.
Belgium) and the Shanghai Chemical Reagent Company The residue was purified by silica gel column chromatography
(Shanghai, China) and were used without purification. Ana- to give the title compound (6.3 g, 46%) as a colorless oil. 1H
lytical thin-layer chromatography was performed on HSGF NMR (300 MHz, CDCl3) δ 7.44–7.21 (m, 15H, ArH), 4.99 (d,
254 plates (150–200 μm thickness; Yantai Huiyou Company, J=11.9 Hz, 2H, PhCH2), 4.80 (d, J=12.4 Hz, 1H, PhCH2), 4.61
Yantai, Shandong, China). 1H NMR (300 MHz or 400 MHz) (d, J=12.0 Hz, 1H, PhCH2), 4.51–4.39 (m, 4H, PhCH2, H1, H6a,
spectra were recorded on Varian Mercury-300 or 400 High H6b), 4.29 (d, J=11.8 Hz, 1H, PhCH2), 3.99–3.87 (m, 2H, H4,
Performance Digital FT-NMR(Varian, Fort Collins, CO, USA) H2), 3.40 (ddd, J=9.6, 4.9, 3.1 Hz, 1H, H5), 3.27 (dd, J=9.4, 3.0
instruments using TMS as the internal standard. 13C NMR Hz, 1H, H3), 2.55 (s, 1H, OH), 2.12 (s, 3H, OAc). 13C NMR (100
(100 MHz) spectra were determined using a Varian Mer- MHz, CDCl3): δ=171.3, 138.4, 137.5, 137.1, 128.4–127.4, 100.2,
cury-400 High Performance Digital FT-NMR. Chemical shifts 81.2, 74.2, 74.0, 73.1, 71.1, 70.7, 66.6, 63.8, 20.8. ESI-MS: m/z
are reported in parts per million (ppm, δ) downfield from 515.3 [M+Na]+. HRMS: calcd for C29H32O7Na 515.2046, found:
tetramethylsilane. Proton coupling patterns are described as 515.2040. [α]D21=-110, (c 0.5, CHCl3).
singlet (s), doublet (d), triplet (t), quartet (q), multiplet (m),
and broad (br). LC-MS analysis was carried out on a Thermo Benzyl 2,3-di-O-benzyl-6-acetyl-4-O-(2,3-di-O-benzyl-4,6-O-benzyl-
Finnigan LCQ Deca XP (Thermo Electron Corporation, San idene-β-D-mannopyranosyl)-β-D-mannopyranoside (4)
Jose, CA, USA) and HRMS was performed on a Finnigan MAT Coupling sulfoxide 1 with 3 under the standard sulfoxide
95 mass spectrometer (Thermo Fisher Scientific, Waltham, β-mannosylation conditions afforded the title compound in
Massachusetts, USA). Optical rotation values were deter- 65% yield as a colorless oil. 1H NMR (400 MHz, CDCl3) δ
mined using a PerkinElmer-341 (589 nm) polarimeter (Perki- 7.49–7.20 (m, 30H, ArH), 5.52 (s, 1H, PhCH), 5.01–4.86 (m, 3H,
nElmer, Waltham, Massachusetts, USA).
PhCH2), 4.84–4.72 (m, 3H, PhCH2), 4.68–4.55 (m, 4H, PhCH2,
H1), 4.50 (d, J=11.9Hz, 1H, PhCH2), 4.46 (s, 1H, H1’), 4.31 (m,
General procedure for preparation of β-mannosides by the 2H, H6a, H6b), 4.14–4.03 (m, 2H, H4, H6a’), 3.99 (dd, J=10.4,
sulfoxide method
4.8 Hz, 1H, H6b’), 3.90 (m, 2H, H2, H2’), 3.66 (t, J=10.2 Hz, 1H,
A dispersion of sulfoxide, TTBP (2.5 equiv.) and powdered H4’), 3.58–3.49 (m, 2H, H3, H3’), 3.49–3.43 (m, 1H, H5), 3.09
molecular sieves in CH2Cl2 (0.1 mol/L) was cooled to -78 °C (td, J=9.9, 4.9 Hz, 1H, H5’), 2.08 (s, 3H, OAc). 13C NMR (100
for 30 min. To this mixture was added a solution of Tf2O (1 MHz, CDCl3): δ=170.8, 138.6, 138.5, 138.4, 137.6, 137.1, 128.8,
mol/L in CH2Cl2, 1.1 equiv.) dropwise at -78°C. The accep- 128.4–127.1, 126.1, 101.7, 101.4, 99.8, 79.6, 78.6, 78.3, 75.7, 75.2,
tor (1.2 equiv.) was added slowly as a solution in CH2Cl2 (1.0 74.1, 74.0, 73.4, 72.6, 71.7, 70.8, 68.5, 67.4, 63.5, 21.0. ESI-MS:
mol/L). The reaction mixture was stirred at -78 °C for 1 h, m/z 945.6 [M+Na]+. HRMS: calcd for C56H58O12Na 945.3826,
then gradually warmed to -20°C while the stirring was contin- found: 945.3823. [α]D21=-58.6, (c 1, CHCl3).
ued. When TLC indicated complete consumption of the sulf-
oxide, the reaction was quenched with saturated aq. NaHCO3. Benzyl 2,3-di-O-benzyl-6-acetyl-4-O-(2,3-di-O-benzyl-6-O-acetyl-β-
The aqueous phase was extracted thrice with EtOAc. The D-mannopyranosyl)-β-D-mannopyranoside (5)
combined organic phases were washed with water and brine, The method used to prepare 3 was employed for the synthesis
dried over anhydrous Na2SO4, filtered and concentrated. The of the title compound (colorless oil, 70% yield) using com-
residue was purified by silica gel chromatography.
pound 4 as the starting material. 1H NMR (400 MHz, CDCl3)
δ 7.30 (ddd, J=25.2, 15.9, 7.7 Hz, 25H, ArH), 5.00–4.89 (m, 2H,
PhCH2), 4.86–4.71 (m, 3H, PhCH2), 4.56 (m, 5H, PhCH2), 4.44
Benzyl 2,3-di-O-benzyl-6-O-acetyl-β-D-mannopyranoside (3)
To a solution of compound 2 (14.86 g, 27.62 mmol) in CH2Cl2 (m, 4H, H1, H1’, H6a, H6b), 4.17 (m, 2H, H6a’, H6b’), 4.10 (t,
(200 mL) was added TFA/H2O (85.2 mL, 1:1 v/v) slowly at J=8.9 Hz, 1H, H4), 3.90 (m, 2H, H2, H2’), 3.83 (t, J=9.4 Hz, 1H,
0 °C. The mixture was stirred at room temperature for 1 h H4’), 3.62–3.53 (m, 2H, H3, H5), 3.20 (m, 1H, H3’), 3.10 (m, 1H,
until TLC showed complete consumption of 2. The mixture H5’), 2.49 (s, 1H, OH), 2.09 (s, 3H, OAc), 1.91 (s, 3H, OAc). 13C
was diluted with CH2Cl2, the reaction quenched with cold NMR (100 MHz, CDCl3): δ=171.1, 170.8, 138.6, 138.4, 138.2,
saturated aq. NaHCO3 and the organic layer was separated. 137.6, 137.1, 128.5–127.2, 100.9, 99.8, 81.3, 78.9, 75.0, 74.4, 74.3,
The aqueous phase was extracted thrice with CH2Cl2. The 74.1, 73.9, 73.8, 73.3, 71.3, 71.2, 70.7, 66.3, 63.6, 63.4, 20.9, 20.7.
combined organic phases were washed with saturated aq. ESI-MS: m/z 899.5 [M+Na]+. HRMS: calcd for C51H56O13Na
NaHCO3 and brine, dried over anhydrous Na2SO4 and concen- 899.3619, found: 899.3646. [α]D21=-87.3, (c 1, CHCl3).
trated. The residue was taken up in a mixture of CH2Cl2 (200
mL) and TEA (9.6 mL, 69.05 mmol), and to this solution was β-(1,4)-D-mannobiose (6)[21]
added Ac2O (6.5 mL, 552.42 mmol). The mixture was stirred at To a solution of compound 5 (79 mg, 0.1 mmol) in anhydrous
room temperature for 4 h. When TLC showed complete con- MeOH (2.5 mL) was added powdered K2CO3 (13.8 mg, 0.1
sumption of the starting material, the reaction was quenched mmol), and the mixture was stirred at room temperature
with saturated aq. NaHCO3 and diluted with CH2Cl2. The for 1 h. When TLC showed complete consumption of 5, the
phases were separated and the aqueous phase was extracted mixture was filtered to remove solids and concentrated. The
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residue was taken up in MeOH (5 mL), and to this solution using compound 8 as the starting material. 1H NMR (400
was added Pd(OH)2/C (16 mg, 20% Pd w/w). The mixture was MHz, D2O) δ 5.21 (s, 0.53H), 4.93 (s, 0.47H), 4.79 (s, 1H), 4.76
stirred under an atmosphere of H2 for 24 h. The mixture was (d, J=6.1 Hz, 1H), 4.16 (s, 1H), 4.09 (d, J=2.5 Hz, 1H), 4.05–3.87
filtered through a Celite pad and concentrated. The residue (m, 6H), 3.85 (d, J=6.1 Hz, 2H), 3.82–3.74 (m, 3H), 3.71 (dd,
was dissolved in water and purified by Sephadex LH20 gel J=16.6, 6.0 Hz, 2H), 3.59 (t, J=9.4 Hz, 2H), 3.49 (dd, J=20.6, 13.3
chromatography eluted with water. The collected fractions Hz, 1H). 13C NMR (100 MHz, D2O): δ=94.8, 94.7, 94.6, 88.5,
were lyophilized to give the title compound (18 mg, 58%) as 88.3, 71.8–71.1, 69.7, 69.4, 67.4, 66.3, 66.2, 66.1, 65.5, 65.2, 64.8,
a white amorphous solid. 1H NMR (400 MHz, D2O) δ 5.23 (s, 64.6, 64.1, 63.6, 61.3, 57.7, 57.0, 55.7, 55.4, 55.2, 55.1. ESI-MS:
0.68H), 4.96 (s, 0.32H), 4.79 (s, 1H), 4.11 (s, 1H), 4.08–3.89 (m, m/z 549.0 [M+HCOO]–. HRMS: calcd for C18H31O16 503.1612,
5H), 3.89–3.75 (m, 3H), 3.71 (d, J=9.5 Hz, 1H), 3.62 (t, J=9.7 found: 503.1605. [α]D21=-22.5, (c 0.4, H2O).
Hz, 1H), 3.50 (dd, J=19.7, 12.6 Hz, 1H). 13C NMR (100 MHz,
D2O): δ=102.6, 102.5, 96.2, 96.0, 79.2, 78.9, 78.8, 77.2, 75.2, 74.1, Benzyl 2,3-di-O-benzyl-4,6-O-benzylidene-β-D-mannopyranosyl-
73.3, 73.0, 72.9, 72.6, 71.3, 69.1, 63.4, 62.9. ESI-MS: m/z 386.6 (1→4)-2,3-di-O-benzyl-6-O-acetyl-β-D-mannopyranosyl-(1→4)-
[M+HCOO]–. HRMS: calcd for C12H21O11 341.1084, found: 2,3-di-O-benzyl-6-O-acetyl-β-D-mannopyranosyl-(1→4)-2,3-di-O-
341.1076. [α]D21=-2.8, (c 0.4, H2O).
benzyl-6-O-acetyl-β-D-mannopyranoside (10)
Sulfoxide 1 was coupled with compound 8 using the stan-
Benzyl 2,3-di-O-benzyl-4,6-O-benzylidene-β-D-mannopyranosyl- dard sulfoxide β-mannosylation protocol to afford the title
(1→4)-2,3-di-O-benzyl-6-O-acetyl-β-D-mannopyranosyl-(1→4)-2,3- compound in 68% yield as a colorless oil. 1H NMR (400 MHz,
di-O-benzyl-6-O-acetyl-β-D-mannopyranoside (7)
CDCl3) δ 7.32 (mz, 50H), 5.50 (s, 1H), 4.94 (dd, J=16.4, 12.2 Hz,
Sulfoxide 1 was coupled with compound 5 using the stan- 2H), 4.87–4.66 (m, 10H), 4.66–4.51 (m, 8H), 4.43 (m, 2H), 4.36–
dard sulfoxide β-mannosylation protocol to afford the title 4.25 (m, 2H), 4.10 (m, 6H), 4.03–3.91 (m, 4H), 3.88 (dd, J=9.1,
compound in 65% yield as a colorless oil. 1H NMR (400 MHz, 2.8 Hz, 2H), 3.82 (dd, J=7.1, 2.4 Hz, 2H), 3.60 (t, J=10.3 Hz, 1H),
CDCl3) δ 7.62–6.96 (m, 40H), 5.51 (s, 1H), 4.99–4.90 (m, 2H), 3.56–3.48 (m, 4H), 3.26 (m, 2H), 3.13–2.98 (td, J=9.9, 4.9 Hz,
4.88–4.68 (m, 7H), 4.65–4.48 (m, 7H), 4.44 (s, 1H), 4.33 (m, 2H), 1H), 2.06 (s, 3H), 1.89 (2s, 6H). 13C NMR (100 MHz, CDCl3):
4.14–3.92 (m, 6H), 3.91–3.82 (m, 3H), 3.61 (t, J=10.2 Hz, 1H), δ=171.0, 170.8, 170.7, 138.8, 138.7, 138.6, 138.5, 138.4, 137.6,
3.56–3.43 (m, 4H), 3.26 (m, 1H), 3.07 (td, J=9.7, 4.9 Hz, 1H), 137.2, 128.9, 128.5–127.1, 126.2, 102.0, 101.4, 101.3, 100.8, 99.9,
2.08 (s, 3H), 1.90 (s, 3H). 13C NMR (100 MHz, CDCl3): δ=170.8, 80.1, 79.9, 79.5, 78.6, 78.3, 75.7, 75.6, 75.5, 75.3, 75.2, 74.4, 74.3,
170.6, 138.7, 138.6, 138.5, 139.4, 139.3, 137.5, 137.1, 128.8, 74.1, 74.0, 73.4, 73.3, 73.2, 72.6, 72.1, 72.0, 71.5, 70.8, 68.5, 67.5,
128.4–127.1, 126.1, 101.9, 101.3, 100.8, 99.87, 79.8, 79.6, 78.50, 63.7, 63.2, 50.9, 21.0, 20.9, 20.8. ESI-MS: m/z 1713.3 [M+Na]+.
78.2, 77.2, 75.7, 75.4, 75.2, 74.3, 74.0, 73.9, 73.3, 73.1, 72.5, 72.0, [α]D21=-72.5, (c 1, CHCl3).
71.4, 70.7, 68.4, 67.4, 63.4, 63.1, 20.9, 20.7. ESI-MS: m/z 1329.7
[M+Na]+. HRMS: calcd for C78H82O18Na 1329.5399, found: Benzyl 2,3-di-O-benzyl-6-O-acetyl-β-D-mannopyranosyl-(1→4)-
1329.5417. [α]D21=-66.3, (c 1, CHCl3).
2,3-di-O-benzyl-6-O-acetyl-β-D-mannopyranosyl-(1→4)-2,3-di-O-
benzyl-6-O-acetyl-β-D-mannopyranosyl-(1→4)-2,3-di-O-benzyl-6-
Benzyl 2,3-di-O-benzyl-6-O-acetyl-β-D-mannopyranosyl-(1→4)- O-acetyl-β-D-mannopyranoside (11)
2,3-di-O-benzyl-6-O-acetyl-β-D-mannopyranosyl-(1→4)-2,3-di-O- The method used to prepare 3 was employed for the synthesis
benzyl-6-O-acetyl-β-D-mannopyranoside (8)
of the title compound (colorless oil, 80% yield) using com-
The method used to prepare 3 was employed for the synthesis pound 10 as the starting material. 1H NMR (400 MHz, CDCl3)
of the title compound (colorless oil, 81% yield) using com- δ 7.53–7.02 (m, 45H), 4.94 (dd, J=16.0, 12.2 Hz, 2H), 4.82–4.68
pound 7 as the starting material. 1H NMR (400 MHz, CDCl3) (m, 9H), 4.55 (m, 9H), 4.45–4.27 (m, 4H), 4.20–4.12 (m, 4H),
δ 7.41–7.19 (m, 35H), 4.99–4.89 (m, 2H), 4.83–4.68 (m, 6H), 4.11–3.91 (m, 5H), 3.87 (dd, J=9.4, 2.8 Hz, 2H), 3.85–3.78 (m,
4.64–4.50 (m, 6H), 4.48 (s, 1H), 4.46–4.28 (m, 4H), 4.21–4.10 (m, 3H), 3.50 (m, 4H), 3.35 (m, 1H), 3.28 (m, 1H), 3.21 (dd, J=9.4,
4H), 4.08–3.96 (m, 2H), 3.90 (m, 1H), 3.87–3.79 (m, 3H), 3.53 2.7 Hz, 1H), 3.14–3.08 (m, 1H), 2.49 (s, 1H), 2.06 (s, 3H), 1.90
(m, 3H), 3.35 (dd, J=8.1, 4.2 Hz, 1H), 3.21 (dd, J=9.4, 2.6 Hz, (s, 3H), 1.89 (s, 3H), 1.88 (s, 3H). 13C NMR (100 MHz, CDCl3):
1H), 3.16–3.08 (m, 1H), 2.55 (s, 1H), 2.07 (s, 3H), 1.91 (s, 3H), δ=171.3, 170.7, 170.6, 170.5, 138.7–138.3, 137.6, 137.1, 128.5–
1.90 (s, 3H). 13C NMR (100 MHz, CDCl3): δ=171.2, 170.7, 170.6, 127.2, 101.4, 101.2, 100.7, 99.9, 81.3, 79.9, 79.5, 79.4, 77.2, 75.5,
138.6, 138.5, 138.4, 138.3, 137.5, 137.0, 128.5–127.1, 101.3, 100.7, 75.3, 75.2, 75.0, 74.4, 74.3, 74.2, 74.1, 74.0, 73.9, 73.8, 73.3, 73.1,
99.8, 81.3, 79.5, 79.4, 77.2, 75.4, 75.2, 75.0, 74.4, 74.2, 74.1, 74.0, 71.9, 71.7, 71.4, 71.3, 70.7, 66.2, 63.5, 63.4, 63.1, 29.6, 20.9, 20.7,
73.9, 73.8, 73.3, 73.1, 71.7, 71.4, 71.3, 70.6, 66.2, 63.5, 63.4, 63.2, 20.6. ESI-MS: m/z 1667.8 [M+Na]+. [α]D21=-79.2, (c 1, CHCl3).
20.9, 20.7, 20.6. ESI-MS: m/z 1283.6 [M+Na]+. HRMS: calcd
for C73H80O19Na 1283.5192, found: 1283.5153. [α]D21=-79.5, (c 1, β-(1,4)-D-Mannotetraose (12)[23]
CHCl3).
The method used to prepare 9 was employed for the synthe-
sis of the title compound (white amorphous solid, 50% yield)
using compound 11 as the starting material. 1H NMR (400
β-(1,4)-D-Mannotriose (9)[22]
The method used to prepare 6 was employed for the synthe- MHz, D2O) δ 5.05 (s, 0.57H), 4.79 (s, 0.43H), 4.62 (d, J=13.3 Hz,
sis of the title compound (white amorphous solid, 63% yield) 3H), 4.00 (s, 2H), 3.94 (d, J=2.1 Hz, 1H), 3.89–3.83 (m, 2H), 3.80
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Jiang RW et al
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(d, J=13.1 Hz, 4H), 3.71 (d, J=20.3 Hz, 5H), 3.67–3.57 (m, 4H), 3.85 (dd, J=18.0, 8.6 Hz, 7H), 3.68 (dt, J=12.3, 10.6 Hz, 12H),
3.57–3.51 (m, 1H), 3.44 (t, J=9.1 Hz, 3H), 3.40–3.26 (m, 2H). 13C 3.56 (dd, J=14.3, 4.9 Hz, 2H), 3.45 (d, J=9.1 Hz, 4H), 3.35 (d,
NMR (100 MHz, D2O): δ=100.1–100.0, 93.8, 93.6, 76.9–76.3, J=7.2 Hz, 1H). 13C NMR (100 MHz, D2O): δ=100.1–100.0, 93.7,
74.9, 74.7, 72.7, 71.6, 71.4, 70.8, 70.6, 70.5, 70.4, 70.1, 70.0, 69.8, 93.6, 76.7, 76.6–76.3, 74.9, 72.7, 71.6, 71.4, 71.3, 70.8, 70.6, 70.4,
68.8, 66.6, 60.9, 60.5. ESI-MS: m/z 710.3 [M+HCOO]–. HRMS: 70.1, 69.9, 69.8, 69.7, 68.8, 66.6, 60.9, 60.5. ESI-MS: m/z 872.5
calcd for C24H42O21Na 689.2116, found: 689.2144. [α]D21=-18.7, [M+HCOO]–. HRMS: calcd for C30H51O26 827.2669, found:
(c 0.3, H2O).
827.2680. [α]D21=-27.0, (c 0.2, H2O).
Benzyl 2,3-di-O-benzyl-4,6-O-benzylidene-β-D-mannopyranosyl- Benzyl 2,3-di-O-benzyl-4,6-O-benzylidene-β-D-mannopyranosyl-
(1→4)-2,3-di-O-benzyl-6-O-acetyl-β-D-mannopyranosyl-(1→4)- (1→4)-2,3-di-O-benzyl-6-O-acetyl-β-D-mannopyranosyl-(1→4)-
2,3-di-O-benzyl-6-O-acetyl-β-D-mannopyranosyl-(1→4)-2,3-di-O- 2,3-di-O-benzyl-6-O-acetyl-β-D-mannopyranosyl-(1→4)-2,3-di-O-
benzyl-6-O-acetyl-β-D-mannopyranosyl-(1→4)-2,3-di-O-benzyl-6-O- benzyl-6-O-acetyl-β-D-mannopyranosyl-(1→4)-2,3-di-O-benzyl-6-O-
acetyl-β-D-mannopyranoside (13)
acetyl-β-D-mannopyranosyl-(1→4)-2,3-di-O-benzyl-6-O-acetyl-β-D-
Sulfoxide 1 was coupled with compound 11 using the stan- mannopyranoside (16)
dard sulfoxide β-mannosylation protocol to afford the title Sulfoxide 1 was coupled with compound 14 using the stan-
compound in 75% yield as a colorless oil. 1H NMR (400 MHz, dard sulfoxide β-mannosylation protocol to afford the title
CDCl3) δ 7.51–7.14 (m, 60H), 5.51 (s, 1H), 4.95 (dd, J=16.1, 12.2 compound in 60% yield as a colorless oil. 1H NMR (400 MHz,
Hz, 2H), 4.89–4.66 (m, 12H), 4.65–4.48 (m, 11H), 4.44 (d, J=7.2 CDCl3) δ 7.47–7.17 (m, 70H), 5.50 (s, 1H), 4.94 (dd, J=16.4,
Hz, 1H), 4.41–4.23 (m, 3H), 4.21–4.00 (m, 8H), 3.96 (m, 4H), 3.88 12.2 Hz, 2H), 4.87–4.76 (m, 2H), 4.76–4.67 (m, 13H), 4.64–4.47
(m, 2H), 3.85–3.78 (m, 3H), 3.61 (t, J=10.3 Hz, 1H), 3.57–3.40 (m, 14H), 4.43 (s, 1H), 4.39–4.26 (m, 2H), 4.17–4.03 (m, 9H),
(m, 6H), 3.34–3.21 (m, 3H), 3.07 (td, J=9.7, 4.9 Hz, 1H), 2.06 (s, 4.02–3.90 (m, 6H), 3.87 (dd, J=8.3, 3.1 Hz, 2H), 3.83–3.76 (m,
3H), 1.95–1.82 (3s, 9H). 13C NMR (100 MHz, CDCl3): δ=170.7, 4H), 3.60 (t, J=10.2 Hz, 1H), 3.55–3.43 (m, 7H), 3.33–3.21 (m,
170.6–170.5, 138.7, 138.5, 138.4, 138.3, 137.5, 137.1, 128.8, 128.4– 4H), 3.06 (td, J=9.7, 4.8 Hz, 1H), 2.05 (s, 3H), 1.88 (4s, 12H). 13C
127.0, 126.0, 101.8, 101.3, 101.1, 100.6, 99.8, 80.0, 79.9, 79.8, 79.4, NMR (100 MHz, CDCl3): δ=170.8, 170.7–170.6, 138.7, 138.5,
78.5, 78.2, 77.2, 75.6, 75.5, 75.4, 75.2, 75.1, 75.0, 74.3, 74.2, 74.0, 138.4, 138.3, 137.5, 137.1, 128.8, 128.4–127.0, 126.0, 101.8, 101.3,
73.8, 73.3, 73.1, 72.4, 72.0, 71.9, 71.8, 71.4, 70.7, 68.4, 67.3, 63.6, 101.1, 100.7, 100.5, 99.8, 80.0, 79.9, 79.8, 79.3, 78.4, 78.2, 77.2,
63.1, 29.7, 20.9, 20.7. ESI-MS: m/z 2099.6 [M+Na]+. [α]D21=-64.7, 75.6, 75.5–75.1, 74.3, 74.2, 74.0, 73.9, 73.3, 73.1, 72.4, 72.0, 71.9,
(c 1, CHCl3).
71.8, 71.4, 70.7, 68.4, 67.3, 63.5, 63.1, 29.6, 20.9, 20.7. ESI-MS:
m/z 2482.9 [M+Na]+. [α]D21=-68.8, (c 0.4, CHCl3).
Benzyl 2,3-di-O-benzyl-6-O-acetyl-β-D-mannopyranosyl-(1→4)-
2,3-di-O-benzyl-6-O-acetyl-β-D-mannopyranosyl-(1→4)-2,3-di-O- β-(1,4)-D-Mannoheptaose (17)
benzyl-6-O-acetyl-β-D-mannopyranosyl-(1→4)-2,3-di-O-benzyl-6-O- The method used to prepare 9 was employed for the synthe-
acetyl-β-D-mannopyranosyl-(1→4)-2,3-di-O-benzyl-6-O-acetyl-β-D- sis of the title compound (white amorphous solid, 35% yield)
mannopyranoside (14)
using compound 16 as the starting material. 1H NMR (400
The method used to prepare 3 was employed for the synthesis MHz, D2O) δ 5.06 (s, 0.60H), 4.79 (s, 0.40H), 4.62 (d, J=11.9
of the title compound (colorless oil, 65% yield) using com- Hz, 5H), 4.01 (s, 3H), 3.93 (t, J=10.6 Hz, 2H), 3.82 (dt, J=29.0,
pound 13 as the starting material. 1H NMR (400 MHz, CDCl3) 12.0 Hz, 8H), 3.75–3.57 (m, 14H), 3.54 (dd, J=16.2, 6.8 Hz, 2H),
δ 7.42–7.21 (m, 55H), 4.95 (dd, J=20.0, 12.2 Hz, 2H), 4.83–4.70 3.43 (d, J=9.0 Hz, 4H), 3.41–3.27 (m, 3H). 13C NMR (100 MHz,
(m, 12H), 4.56 (ddd, J=31.6, 16.6, 10.2 Hz, 11H), 4.47–4.28 (m, D2O): δ=100.1–100.0, 93.8, 93.6, 76.7, 76.6–76.3, 75.0, 72.7, 71.6,
5H), 4.21–4.10 (m, 7H), 4.08–4.04 (m, 1H), 4.01 (d, J=9.1 Hz, 71.5–71.3, 70.8, 70.6, 70.4, 70.2, 69.9–69.8, 68.9, 66.6, 60.9, 60.4.
1H), 3.99–3.93 (m, 2H), 3.89 (dd, J=13.4, 2.4 Hz, 2H), 3.86–3.78 ESI-MS: m/z 1034.7 [M+HCOO]–. HRMS: calcd for C36H61O31
(m, 4H), 3.57–3.46 (m, 5H), 3.39–3.33 (m, 1H), 3.33–3.26 (m, 989.3197, found: 989.3218. [α]D21=-15.5, (c 0.4, H2O).
2H), 3.22 (dd, J=9.4, 2.4 Hz, 1H), 3.15–3.09 (m, 1H), 2.50 (s, 1H),
2.07 (s, 3H), 1.96–1.85 (4s, 12H). 13C NMR (100 MHz, CDCl3):
δ=171.3, 170.7–170.5, 138.7–138.3, 137.6, 137.1, 128.5–127.1,
101.3, 101.2, 101.1, 100.5, 99.8, 81.3, 79.9, 79.8, 79.5, 79.3, 77.2,
Acknowledgements
This work was supported by the National Science & Technol-
ogy Major Project “Key New Drug Creation and Manufactur-
75.4, 75.3, 75.1, 75.0, 74.4, 74.3, 74.1, 74.0, 73.9, 73.8, 73.3, 73.1,
ing Program,” China (2012ZX09301001).
71.8, 71.7, 71.4, 71.3, 70.7, 66.2, 63.5, 63.4, 63.1, 29.6, 20.9, 20.7,
20.6. ESI-MS: m/z 2054.8 [M+Na]+. [α]D21=-67.0, (c 1, CHCl3).
Author contribution
Jing-kang SHEN and Mei-yu GENG designed research; Ru-
wei JIANG, Xiao-guang DU, Xuan ZHANG, Ding-yu HU,
Tao MENG, and Yue-lei CHEN performed research; Ru-wei
JIANG, Yue-lei CHEN, Mei-yu GENG, and Jing-kang SHEN
analyzed data; Ru-wei JIANG, Xiao-guang DU, Yue-lei CHEN,
Mei-yu GENG, and Jing-kang SHEN wrote the paper.
β-(1,4)-D-Mannopentose (15)[24]
The method used to prepare 9 was employed for the synthe-
sis of the title compound (white amorphous solid, 33% yield)
using compound 14 as the starting material. 1H NMR (400
MHz, D2O) δ 5.08 (s, 0.60H), 4.81 (s, 0.40H), 4.71 (d, J=6.4 Hz,
2H), 4.57 (d, J=6.4 Hz, 2H), 4.02 (s, 3H), 3.96 (d, J=3.1 Hz, 1H),
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Jiang RW et al
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14 Crich D, Sun SX. Direct synthesis of beta-mannopyranosides by the
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15 Crich D, Mataka J, Sun SX, Lam KC, Rheingold AL, Wink DJ. Stereo-
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16 Crich D, Mark S, Yao QJ, John P. 2,4,6-Tri-tert-butylpyrimidine (TTBP): A
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tert-butylpyridine and its 4-substituted derivatives in glycosylation and
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17 Crich D, Li WJ, Li HM. Direct chemical synthesis of the beta-mannans:
Linear and block syntheses of the alternating beta-(l→3)-beta-(l→4)-
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