Synthesis of triazolyl-linked polysialic acids

Article abstract of DOI:10.1016/j.tet.2014.10.028

A new approach for the synthesis of triazolyl-linked α-(2-9) oligosialic acids by iterative copper(I)-catalyzed alkyne-azide cycloaddition (CuAAC) and desilylation has been developed. By using propargyl α-sialoside 5 and trimethylsilylated-propargyl 9-azido-α-sialoside 3 as building blocks, triazolyl-linked α-(2-9) di- to octa-sialic acids were constructed in good to moderate yields. Finally, the pseudo-α-(2-9)-oligosialic acids were obtained very smoothly via O-deprotection. The oligosialic acids with a triazolyl-linkage represent a new type of pseudooligosialic acids. This protocol may find applications in the preparation of oligosialic acid analogues with biological importance.

Full text of DOI:10.1016/j.tet.2014.10.028

Accepted Manuscript
Synthesis of triazolyl-linked polysialic acids
De-Cai Xiong, Yichuan Zhou, Yuxin Cui, Xin-Shan Ye
PII:
S0040-4020(14)01455-0
10.1016/j.tet.2014.10.028
TET 26098
DOI:
Reference:
To appear in: Tetrahedron
Received Date: 14 September 2014
Revised Date: 8 October 2014
Accepted Date: 14 October 2014
Please cite this article as: Xiong D-C, Zhou Y, Cui Y, Ye X-S, Synthesis of triazolyl-linked polysialic
acids, Tetrahedron (2014), doi: 10.1016/j.tet.2014.10.028.
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ACCEPTED MANUSCRIPT
Synthesis of triazolyl-linked polysialic acids
De-Cai Xiong+, Yichuan Zhou+, Yuxin Cui, Xin-Shan Ye*
State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical
Sciences, Peking University, Xue Yuan Road No. 38, Beijing, 100191, China
*Corresponding author. Fax.: +86 10 8280 2724; E-mail: xinshan@bjmu.edu.cn
+These authors contributed equally to this work.
Abstract:
A new approach for the synthesis of triazolyl-linked α-(2-9) oligosialic acids by iterative
copper(I)-catalyzed alkyne-azide cycloaddition (CuAAC) and desilylation has been developed. By
using propargyl α-sialoside 5 and trimethylsilylated-propargyl 9-azido-α-sialoside 3 as building
blocks, triazolyl-linked α-(2-9) di- to octa-sialic acids were constructed in good to moderate yields.
Finally, the pseudo-α-(2-9)-oligosialic acids were obtained very smoothly via O-deprotection. The
oligosialic acids with a triazolyl-linkage represent a new type of pseudooligosialic acids. This
protocol may find applications in the preparation of oligosialic acid analogues with biological
importance.
Keywords: polysialic acid; click reaction; triazole; cycloaddition; carbohydrate
Graphical abstract:
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1. Introduction
Protein or lipid glycosylation is an omnipresent and life-governing process. Sialic acids, a
family of sugar units with a nine-carbon backbone that are typically attached to the outermost ends
of sugar chains,1 exert a multitude of biological and pathological effects such as cellular adhesion,
inflammatory
response,
cell
signaling,
and
cell
differentiation.
2
Polysialic acid (PSA) is a type of linear homopolymer of sialic acid. Three kinds of polysialic
acids have been identified in nature, these include α(2-8), α(2-9), and alternating α(2-8) and α(2-9)
linkages,3 as shown in Figure 1.
Figure 1. Structures of polysialic acids.
Due to its outermost location, PSA plays a vital role in biological processes such as
embryogenesis, neural cell growth, differentiation, cell-cell mediating, and membrane transport.4
In addition, many studies suggest that polysialic acids, present on the surface of N. meningitidis B
and C,5 human embryonal carcinoma cells,6 mouse neurobrastoma cells,7 and sea urchin sperm
flagella et al.,8 serve as bacterial or cancer cell shields against the attack systems. Moreover, PSA
can also be used in biomedical engineering and tissue engineering.9 Therefore, polysialic acids
are considered either good targets for the development of vaccines or tools for probing the
interactions between polysaccharides and their receptors.10 However, polysialic acids are often
heterogeneous or contaminated from natural sources and susceptible to degradation by chemical
2

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hydrolysis or glycosyl hydrolases.10b,11 The establishment of an effective method to synthesize
pure polysialic acids/pseudopolysialic acids with the well-defined structure will not only simplify
the complexity of polysialic acid-based vaccines, but also benefit a better understanding of the
functions of polysialic acids.
Considerable efforts have been aroused in the synthesis of both PSA and PSA analogues.
Some methods have been developed for the synthesis of homooligosialic acids with α(2-8), α(2-9),
and alternating α(2-8)/α(2-9) intersialyl linkages.12 S-Linked α(2-8) and alternating α(2-8)/α(2-9)
oligosialic acids resistant to hydrolysis have also been prepared.13 On the other hand, the [3+2]
Huisgen cyclization of an azide with an alkyne to form a triazole has recently emerged as a
powerful conjugate strategy for the preparation of neoglycoconjugates with biomedical interest,14
in which the formed triazole ring can improve the solubility in water and take part in hydrogen
bonding and dipole interactions. 15 By using the iterative copper(I)-catalyzed azide-alkyne
cycloaddition (CuAAC) reaction strategy,16 herein we report the synthesis of triazolyl-linked
α(2-9) oligosialic acids.
2. Results and discussion
To achieve triazolyl-linked α(2-9) oligosialic acids, sialic acid building blocks 5 and 3 were
chosen. As shown in Scheme 1, compound 5 is coupled with 3 using CuAAC reaction to afford
the pseudodisialic acid. The temporarily silyl protective group can be removed to yield the
desilylated product, which is followed by the cycloaddition reaction to produce the pseudotrisialic
acid. The repetition of cycloaddition-desilylation reactions allows rapid assembly of
triazolyl-linked oligosialic acids. In this synthetic strategy, O-peracetylated propargyl α-sialoside
5 and O-peracetylated trimethylsilylated-propargyl 9-azido-α-sialoside 3 are the key building
blocks. The trimethylsilyl protection of alkyne was a prerequisite devoid of polymerization. The
use of O-acetyl protection is to facilitate the preparation of the building blocks, the coupling
reactions, and the isolation of the coupling products. Ethynyl α-sialoside might be the most proper
building blocks in view of the similarity and resistance to chemical and enzymatic degradation,
but it is difficult to obtain. Thus, a longer propargyl linker, which might not impair the functions
of polysaccharides,14f was used.
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Scheme 1. The synthetic strategy of triazolyl-linked α(2-9) oligosialic acids.
The synthesis of building block 5 and intermediate 11 followed the reported procedure with
some modifications (Scheme 2).17 The peracetylated sialic acid 718 was treated with p-thiocresol
to give thiosialoside 8 in 87% yield. Promoted by NIS/TfOH, glycosyl donor 8 underwent the
sialylation reaction with propargyl alcohol smoothly to afford compound 5αβ in 89% yield (α/β =
2/1, inseparable mixture). Deacetylation of 5αβ obtained the desired α-isomer 9 after column
chromatography, which was acetylated to provide 5. Treatment of 9 with TsCl, which was
followed by the azido-substitution with NaN3 generated compound 10. Acetylation of 10 gave
compound 11 in 75% yield.
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Scheme 2. Reagents and conditions: a) Pyr., Ac2O, DMAP, r.t., 95%; b) BF3•Et2O, TolSH, CH2Cl2,
r.t. overnight, 87%; c) NIS/TfOH, propargyl alcohol, CH2Cl2, 4 Å MS, 89%; d) NaOMe/MeOH,
r.t. 47%; e) TsCl, Pyr., 4 Å MS, 0 oC; f) NaN3, 15-crown-5, DMF, H2O, 65 oC, 42% over two steps;
g) Pyr., Ac2O, DMAP, r.t., 75% for 11, 85% for 5.
Next, we turn our attention to the synthesis of silylalkyne 3. There are many methods for
silylation of terminal alkynes.19 Due to the presence of ester group in compound 11, mild
trimethylsilylation conditions are needed. The silylation conditions varying both the reagents and
solvents were investigated at different temperatures, and the results were summarized in Table 1.
When TMSOTf/Et3N16b (Entry 1) or Et2NTMS20 (Entry 2) was employed in THF using ZnCl2 as
the catalyst, there was no reaction observed. When trifluoromethyltrimethylsilane (TMSCF3)/KF
or CsF (Entries 3 and 4) was used,21 the silylalkyne 3 was obtained in low yield (< 25%). We
inferred that the fluoride could react with SiO2. Therefore, when Teflon tube was used instead of
glassware, the yield was improved significantly (70%, Entry 5). Similarly, 4Å molecular sieves
also made this reaction not occur in Teflon tube (Entry 6). With the increase of the reaction
temperature, the yield was decreased (< 50%, Entry 7), resulting from the decomposition of
silylalkyne 3 at higher temperatures. So we got the optimized silylation reaction conditions:
TMSCF3/CsF/anhydrous 1,4-dioxane/30 oC in Teflon tube.
Table 1. Preparation of 3 and optimization of the silylation conditions of 11.
Entry
Reagent
Solvent
T (oC)
Yield
5

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1
ZnCl2/TMSOTf/
Et3N
THF
r.t. to 60
no reaction
2
3
4c
5a,c
ZnCl2/Et2NTMS
TMSCF3/KF
TMSCF3/CsF
TMSCF3/CsF
THF
r.t. to 60
r.t.
no reaction
< 25%
< 20%
70%
DMF
1,4-dioxane
1,4-dioxane
r.t.
30
6a,b,c
7a,c
TMSCF3/CsF
TMSCF3/CsF
1,4-dioxane
1,4-dioxane
30
50
no reaction
< 50%
aIn Teflon tube. bWith 4Å molecular sieves. cConditions: TMSCF3 (3.0 equiv.), CsF (0.4 equiv.),
1,4-dioxane (2.0 mL).
With these building blocks in hand, the copper(I)-catalyzed azide-alkyne cycloaddition
reaction of propargyl α-sialoside 5 and trimethylsilylated-propargyl 9-azido-α-sialoside 3 was
investigated (Table 2). When CuSO4/sodium ascorbate/DMF was used, the reaction failed to
provide the desired product 4a (Entry 1). Under the catalysis of CuSO4/sodium ascorbate in
t-BuOH/CH2Cl2/H2O or DMF/H2O, the thin layer chromatography (TLC) monitoring showed that
most of the starting materials disappeared and some polymeric products generated (Entries 2 and
3). This is because the silylalkynes 3 and 4a, which are water-sensitive, were desilylated under
these conditions. When using CuI (0.2 eq.)/DIPEA/DMF at 50 oC, the reaction proceeded and the
dimeric product 4a was isolated in 48% yield (Entry 4). The yield was improved as the solvent
was changed to acetonitrile (Entry 5, 65% yield). However, the microwave irradiation provided no
gains in the yield (Entry 6, 54% yield). With the decrease of the reaction temperature, the yield
was further increased (Entry 7, 78% yield).
Table 2. CuAAC reaction of monosaccharide building blocks 5 and 3.
Entry
1
Catalyst
CuSO4(0.15 eq.)/sodium
ascorbate
Solvent
DMF
Temperature
50 oC
Yield
no
reaction
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2
3
CuSO4 (0.15 eq.)/sodium
DMF/H2O
50 oC
50 oC
polymers
polymers
ascorbate
CuSO4(0.15 eq.)/sodium
ascorbate
t-BuOH/CH2Cl2/H2O
4
5
6
7
CuI (0.2 eq.)/DIPEA
CuI (0.2 eq.)/DIPEA
CuI (0.2 eq.)/DIPEA
CuI (0.2 eq.)/DIPEA
DMF
MeCN
MeCN
MeCN
50 oC
50 oC
50 oCꢀMW 150W
30 oC
48%
65%
54%
78%
Subsequently, we optimized the reaction conditions of desilylation. After surveying the
efficiency of reaction with TBAF/THF/MeCN,22 AgF/Et3N/MeCN23 or AgNO3/H2O/acetone,24 we
were pleased to discover that the treatment of dimer 4a with AgNO3 in H2O and acetone at 40 °C
afforded the desired desilylated pseudodisaccharide 2a in quantitative yield. As the coupling
reaction and the desilylation reaction were merged in one-pot, some higher oligomers could be
produced, resulting from the further reaction of the desilylated product with the residual
azido-containing building block. Therefore, the desired oligomeric product needed to be purified
by column chromatography.
Table 3. Iterative CuAAC reaction and desilylation to produce pseudo-α(2-9)-oligosialic acids.
Cycle
n
Cycloaddition and Desilylation
Deprotection
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Product
2a
Yield
78%
76%
60%
77%
68%
56%
47%
Product
1a
yield
78%
89%
88%
99%
87%
94%
96%
1
2
3
4
5
6
7
0
1
2
3
4
5
6
2b
1b
2c
1c
2d
1d
2e
1e
2f
1f
2g
1g
Thus, the same cycloaddition-desilylation sequence was repeated over several times (up to
seven cycles) with gradually decreased efficiency. This is because higher
a
trimethylsilylated-propargyl oligomer (with one more sialic acid than the desired) was formed due
to the further cycloaddition reaction of the partially desilylated product with 3. As the sugar chain
becomes longer, the side-product (higher oligomer) would be produced in an increased amount.
Oligomers 2a-g were deprotected smoothly under the basic conditions to afford the corresponding
triazolyl-linked α-(2-9) di- to octa- oligosialic acids 1a-1g. Based on this protocol, all synthetic
compounds 2a-g and 1a-g were obtained in excellent to moderate yields. The isolated yield of
each cycle was summarized in Table 3. The structures of 2a-g and 1a-g were unambiguously
identified by their 1H NMR, 13C NMR, and high-resolution mass spectra.
3. Conclusion
In conclusion, we have developed an efficient way to synthesize α-(2-9)-oligosialic acid
analogues linked by a triazole ring using the Cu(I)-catalyzed azide-alkyne cycloaddition strategy.
By using propargyl α-sialoside 5 and trimethylsilylated-propargyl 9-azido-α-sialoside 3 as
building blocks, triazolyl-linked α-(2-9) di- to octa- oligosialic acids were assembled in good to
moderate yields via the iterative cycloaddition-desilylation operation. The subsequent
O-deprotection afforded the pseudo-α-(2-9)-oligosialic acids in high yields and with high purity.
To the best our knowledge, the triazolyl linkage was introduced by us for the assembly of
pseudooligosialic acids for the first time. The disclosed protocol may find applications in the
preparation of α(2-8) and alternating α(2-8)/α(2-9) oligosialic acid analogues which will facilitate
the understanding of functions of polysialic acids and the related drug discovery.
8

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4. Experimental
4.1. General
All chemicals were purchased as reagent grade and used without further purification, unless
otherwise noted. Dichloromethane (CH2Cl2), pyridine, and acetonitrile were distilled over calcium
hydride (CaH2). Methanol was distilled from magnesium. DMF and 1,4-dioxane were stirred with
calcium hydride (CaH2) and distilled under reduced pressure. All reactions were carried out under
anhydrous conditions with freshly distilled solvents, unless otherwise noted. Reactions were
monitored by analytical thin-layer chromatography on silica gel 60-F254 precoated on alunminum
plates (E. Merck.). Spots were detected under UV (254 nm) and/or by staining with acidic ceric
ammonium molybdate. Solvents were evaporated under reduced pressure and below 40 oC (bath).
Column chromatography was performed on silica gel (200-300 mesh, TsingDao Ocean or 230-400
mesh, Merck), reversed phase column chromatography was performed on LiChroprep RP-18
(40-63 ꢀm, Merck). 1H NMR spectra were recorded on a Bruker 400M or 600M spectrometers at
o
25 C. Chemical shifts (in ppm) were referenced to tetramethylsilane (δ = 0 ppm) in deuterated
chloroform. 13C NMR spectra were obtained by using the same NMR spectrometers and were
calibrated with CDCl3 (δ = 77.00 ppm). Mass spectra were recorded using a Bruker APEX IV
FT-MSꢁ7.0 Tꢂspectrometer.
4.2. General procedure for cycloaddition-desilylation reactions
To a mixed solution of 5 or 2a-f (1.0 eq.) and 3 (1.0-1.1 eq.) in dried MeCN, CuI (0.2 eq.)
and DIPEA (2.0 eq.) was added. The reaction mixture was stirred at 30 oC. After the
disappearance of 5 or 2a-f (detected by TLC) the solvent was evaporated. The silylalkyne
products 4a-g were purified by column chromatography on silica gel (CH2Cl2/MeOH, 25:1).
Products 4a-g were dissolved in acetone (3.0 mL) and water (0.1 mL). To the reaction mixture
was added AgNO3 (0.5 eq.), and the mixture was stirred for 1 h at 40 °C. Subsequently, the
reaction was quenched by saturated aq. NH4Cl and the solvent was evaporated. The residue was
purified by column chromatography on silica gel (CH2Cl2/MeOH) to afford 2a-g.
4.3. General procedure for deprotection
To a solution of 2a-2g in methanol (1.0 mL) was added NaOMe (81.0 mg, 1.5 mmol). After
stirring for 0.5 h, the reaction mixture was added H2O (0.1 mL) and stirred overnight at 50 oC. The
resulting solution was neutralized with hydrochloric acid (1 M) to pH = 4-5, and evaporated to
9

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dryness. The residue was purified by reversed-phase column chromatography to afford the
deprotected products 1a-g.
4.4.
Methyl
(2-(3-trimethylsilyl-2-propynyl)
5-acetamido-4,7,8-tri-O-acetyl-9-azido-3,5,9-trideoxy-D-glycero-α-D-galacto-2-nonulopyrano
sid)onate (3)
To a solution of 1117 (600.0 mg, 1.17 mmol) in dry 1,4-dioxane in a Teflon tube was added
TMSCF3 (0.6 mL) and cesium fluoride (20.0 mg) under N2 atmosphere at 30 oC. After stirring for
0.5 h, the mixture was evaporated, and the residue was purified by column chromatography on
silica gel (petroleum ether/acetone, 3:1) to afford compound 3 as a white solid (476.0 mg, 70%
yield). [α]30 -0.81 (c 2.0, CHCl3); H NMR (400 MHz, CDCl3): δ 5.34-5.30 (m, 2H, H-7, H-8),
1
D
5.15 (d, J = 9.4 Hz, 1H, NH), 4.85 (ddd, J = 12.0, 9.9, 4.7 Hz, 1H, H-4), 4.43 (d, J = 15.6 Hz, 1H,
HC≡CCH2), 4.12-4.03 (m, 3H, HC≡CCH2, H-5, H-6), 3.82 (s, 3H, OCH3), 3.59-3.56 (m, 1H,
H-9b), 3.30-3.25 (m, 1H, H-9a), 2.64 (dd, J = 12.8, 4.6 Hz, 1H, H-3b), 2.18 (s, 3H, COCH3), 2.16
(s, 3H, COCH3), 2.04 (s, 3H, COCH3), 2.01 (t, J = 12.6 Hz, 1H, H-3a), 1.89 (s, 3H, NHCOCH3),
0.17 (s, 9H, (CH3)3Si); 13C NMR (100 MHz, CDCl3): δ 170.98, 170.25, 170.19, 170.12, 167.74,
100.26, 98.22, 91.57, 72.92, 69.75, 68.80, 67.88, 53.60, 52.88, 50.88, 49.33, 37.81, 23.16, 21.00,
20.81, -0.27. HRMS (ESI) Calcd. for C24H40N5O11Si ([M + NH4]+): 602.2488; found: 602.2486.
4.5. Protected disialic acid (2a)
The reaction of 517 (27.2 mg, 51.4 ꢀmol, 1.0 eq.) and 3 (30.0 mg, 51.4 ꢀmol, 1.0 eq.) was
performed for 2 h as described in the general procedure for cycloaddition-desilylation, affording
2a (elution with CH2Cl2/MeOH (25/1); 41.6 mg, 78% yield; Rf = 0.48, MeOH/EtOAc, 1:8, v/v) as
white solids. [α]30 -0.93 (c 3.0, CHCl3); H NMR (400 MHz, CDCl3): δ 7.73 (s, 1H, NCH),
1
D
5.48-5.43 (m, 2H, H-8, H-8’), 5.36 (d, J = 9.8 Hz, 1H, NH), 5.33 (dd, J = 8.5, 2.0 Hz, 1H, H-7’),
5.29 (dd, J = 6.4, 1.6 Hz, 1H, H-7), 5.24 (d, J = 9.6 Hz, 1H, NH), 4.91-4.83 (m, 4H, H-4, H-4’,
OCH2C), 4.53 (d, J = 12.0 Hz, 1H, H-9b), 4.43 (dd, J = 15.7, 2.4 Hz, 1H, HC≡CCH2), 4.40 (dd, J
= 14.6, 7.9 Hz, 1H, H-9b’), 4.34 (dd, J = 12.4, 2.7 Hz, 1H, H-9a’), 4.23 (dd, J = 15.7, 2.2 Hz, 1H,
HC≡CCH2), 4.16-4.06 (m, 5H, H-9a, H-5, H-5’, H-6, H-6’), 3.83 (s, 3H, OCH3), 3.79 (s, 3H,
OCH3), 2.67 (dd, J = 12.8, 4.7 Hz, 1H, H-3e), 2.62 (dd, J = 12.8, 4.5 Hz, 1H, H-3e’), 2.46 (t, J =
2.4 Hz, 1H, HC≡C), 2.21 (s, 3H, COCH3), 2.17 (s, 3H, COCH3), 2.16 (s, 3H, COCH3), 2.07 (s, 3H,
10

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COCH3), 2.04 (s, 6H, 2COCH3), 2.03 (s, 3H, COCH3), 2.01-1.94 (m, 2H, H-3a, H-3a’), 1.89 (s,
3H, NHCOCH3), 1.88 (s, 3H, NHCOCH3); 13C NMR (100 MHz, CDCl3): δ 170.96, 170.93,
170.71, 170.67, 170.30, 170.21, 170.14, 170.08, 169.86, 168.17, 167.69, 143.97, 124.02, 98.63,
98.37, 78.84, 74.69, 73.23, 72.60, 70.17, 68.93, 68.66, 68.41, 67.35, 62.49, 58.50, 52.98, 52.89,
50.08, 49.39, 49.34, 37.90, 37.68, 23.16, 23.11, 21.10, 20.89, 20.81, 20.74; HRMS (ESI) Calcd.
for C44H60N5O24 ([M + H]+): 1042.3623; found: 1042.3657.
4.6. Protected trisialic acid (2b)
The reaction of 2a (33.9 mg, 32.6 ꢀmol) and 3 (24.6 mg, 35.8 ꢀmol, 1.1 eq.) was performed
for 4 h as described in the general procedure for cycloaddition-desilylation, affording 2b (elution
with CH2Cl2/MeOH (20/1); 38.4 mg, 76% yield; Rf = 0.48, MeOH/EtOAc, 1:8, v/v) as white
solids. [α]30 –1.04 (c 3.1, CHCl3); 1H NMR (400 MHz, CDCl3): δ 7.75 (s, 1H), 7.72 (s, 1H), 5.68
D
(d, J = 9.0 Hz, 1H), 5.49-5.28 (m, 8H), 4.95-4.84 (m, 7H), 4.61 (d, J = 12.0 Hz, 1H), 4.53 (d, J =
12.0 Hz, 1H), 4.45-4.39 (m, 3H), 4.35 (dd, J = 12.3, 1.6 Hz, 1H), 4.24 (dd, J = 15.7, 2.0 Hz, 1H),
4.18-4.06 (m, 7H), 3.81 (s, 3H), 3.79 (s, 3H), 3.76 (s, 3H), 2.69-2.59 (m, 3H), 2.46 (br.s, 1H), 2.22
(s, 3H), 2.20 (s, 3H), 2.16 (s, 6H), 2.07-2.02 (m, 18H), 2.00-1.94 (m, 3H), 1.88-1.87 (m, 9H); 13
C
NMR (100 MHz, CDCl3): δ 170.94, 170.79, 170.73, 170.69, 170.62, 170.34, 170.29, 170.25,
170.10, 169.91, 169.78, 168.17, 168.12, 167.73, 143.90, 143.81, 124.16, 123.97, 98.81, 98.58,
98.34, 78.86, 74.71, 73.23, 72.60, 70.38, 70.15, 68.93, 68.74, 68.53, 68.47, 68.32, 67.34, 62.45,
58.42, 52.96, 52.87, 50.10, 49.39, 49.19, 37.88, 37.64, 23.14, 23.10, 23.06, 21.10, 20.91, 20.88,
20.80, 20.74; HRMS (ESI) Calcd. for C65H88N9O35 ([M + H]+): 1554.5377; found: 1554.5416.
4.7. Protected tetrasialic acid (2c)
The reaction of 2b (38.4 mg, 24.7 ꢀmol) and 3 (15.9 mg, 27.2 ꢀmol, 1.1 eq.) was performed
for 8 h as described in the general procedure for cycloaddition-desilylation, affording 2c (elution
with CH2Cl2/MeOH (15/1); 30.4 mg, 60% yield; Rf = 0.48, MeOH/EtOAc, 1:7, v/v) as white
solids. [α]30 -0.27 (c 3.0, CHCl3); 1H NMR (400 MHz, CDCl3): δ 7.75 (s, 1H), 7.74 (s, 1H), 7.72
D
(s, 1H), 5.76 (br.d, J = 9.4 Hz, 1H), 5.58 (br.d, J = 9.6 Hz, 1H), 5.51-5.43 (m, 4H), 5.37-5.27 (m,
6H), 4.95-4.85 (m, 10H), 4.63-4.53 (m, 3H), 4.46-4.38 (m, 4H), 4.35 (dd, J = 12.5, 2.7 Hz, 1H),
4.24 (dd, J = 15.7, 2.4 Hz, 1H), 4.16-4.06 (m, 9H), 3.80 (s, 3H), 3.78 (s, 3H), 3.76 (s, 3H), 3.75 (s,
3H), 2.69-2.56 (m, 4H), 2.46 (t, J = 2.4 Hz, 1H), 2.21- 2.20 (m, 9H), 2.16 (s, 6H), 2.05-2.03 (m,
11

ACCEPTED MANUSCRIPT
21H), 2.00-1.93 (m, 4H), 1.88 (br.s, 12H); 13C NMR (150 MHz, CDCl3): δ 170.99, 170.90, 170.86,
170.80, 170.71, 170.69, 170.41, 170.34, 170.18, 170.12, 169.96, 169.92, 169.88, 168.16, 168.14,
168.12, 167.78, 143.92, 143.82, 143.71, 124.25, 124.18, 124.05, 98.80, 98.78, 98.57, 98.34, 78.92,
74.79, 73.23, 73.16, 72.59, 70.47, 70.41, 70.16, 68.91, 68.85, 68.78, 68.75, 68.46, 68.44, 68.32,
67.32, 62.46, 58.46, 58.40, 58.37, 53.01, 52.90, 50.12, 50.08, 50.02, 49.37, 49.26, 49.13, 37.86,
37.65, 29.66, 23.15, 23.11, 23.08, 23.06, 21.13, 21.03, 20.93, 20.91, 20.83, 20.77; HRMS (ESI)
Calcd. for C86H116N13O46 ([M + H]+): 2066.7132; found: 2066.7181.
4.8. Protected pentasialic acid (2d)
The reaction of 2c (40.5 mg, 19.6 ꢀmol) and 3 (12.0 mg, 20.6 ꢀmol, 1.05 eq.) was performed
for 12 h as described in the general procedure for cycloaddition-desilylation, affording 2d (elution
with CH2Cl2/MeOH (12/1); 38.9 mg, 77% yield; Rf = 0.39, MeOH/EtOAc, 1:3, v/v) as white
solids. [α]30 –1.15 (c 2.3, CHCl3); 1H NMR (600 MHz, CDCl3): δ 7.75-7.72 (m, 4H), 5.87 (d, J =
D
9.2 Hz, 1H), 5.79 (d, J = 9.1 Hz, 1H), 5.70 (d, J = 9.2 Hz, 1H), 5.57-5.44 (m, 6H), 5.37-5.29 (m,
6H), 4.92-4.86 (m, 14H), 4.63-4.53 (m, 4H), 4.45-4.42 (m, 5H), 4.35 (dd, J = 12.4, 2.6 Hz, 1H),
4.24 (dd, J = 15.7, 2.4 Hz, 1H), 4.20-4.07 (m, 12H), 3.80-3.74 (m, 15H), 2.68-2.59 (m, 5H), 2.47
(t, J = 2.4 Hz, 1H), 2.21-2.20 (m, 12H), 2.16 (s, 6H), 2.05-2.02 (m, 30H), 2.00-1.97 (m, 5H),
1.88-1.87 (m, 15H); 13C NMR (150 MHz, CDCl3): δ 170.99, 170.88, 170.84, 170.80, 170.72,
170.70, 170.67, 170.43, 170.34, 170.18, 170.11, 169.94, 169.89, 168.17, 168.14, 168.12, 167.79,
143.92, 143.71, 124.20, 124.04, 98.80, 98.77, 98.58, 98.34, 78.88, 74.78, 73.22, 73.16, 72.59,
70.45, 68.91, 68.85, 68.78, 68.76, 68.47, 68.44, 68.34, 67.32, 62.47, 60.39, 58.47, 58.37, 54.03,
53.01, 52.91, 52.89, 50.11, 49.38, 49.28, 49.23, 49.13, 37.87, 37.65, 23.16, 23.11, 23.08, 21.14,
21.03, 20.95, 20.92, 20.86, 20.83, 20.77. HRMS (ESI) Calcd. for C107H144N17O57 ([M + H]+):
2578.8886; found: 2578.8902.
4.9. Protected hexasialic acid (2e)
The reaction of 2d (32.1 mg, 12.4 ꢀmol) and 3 (7.3 mg, 12.4 ꢀmol, 1.0 eq.) was performed
for 12 h as described in the general procedure for cycloaddition-desilylation, affording 2e (elution
with CH2Cl2/MeOH (10/1); 26.2 mg, 68% yield; Rf = 0.35, MeOH/EtOAc, 1:3, v/v) as white
solids. [α]30 -0.73 (c 3.6, CHCl3); H NMR (600 MHz, CDCl3): δ 7.77 (m, 5H), 6.00-5.89 (m,
1
D
4H), 5.66-5.44 (m, 8H), 5.35-5.30 (m, 6H), 4.92-4.88 (m, 16H), 4.62-4.35 (m, 12H), 4.26-4.07 (m,
12

ACCEPTED MANUSCRIPT
14H), 3.80-3.74 (m, 18H), 2.69-2.60 (m, 6H), 2.51 (s, 1H), 2.21-2.20 (m, 15H), 2.15 (s, 6H),
2.07-1.98 (m, 42H), 1.88(s, 18H); 13C NMR (150 MHz, CDCl3): δ 170.97, 170.96, 170.82, 170.76,
170.68, 170.54, 170.50, 170.41, 170.24, 170.11, 170.00, 169.96, 168.14, 168.12, 167.77, 143.78,
124.25, 98.79, 98.76, 98.56, 98.32, 74.84, 73.20, 73.13, 72.57, 70.53, 70.19, 68.89, 68.85, 68.77,
68.47, 68.42, 68.32, 67.32, 62.46, 58.44, 58.33, 53.00, 52.92, 52.87, 50.13, 49.31, 49.16, 49.06,
37.83, 37.62, 23.11, 23.06, 23.02, 21.12, 20.92, 20.89, 20.84, 20.81, 20.75; HRMS (ESI) Calcd.
for C128H171N21O68K2 ([M + 2K]2+): 1564.5089; found: 1564.5160.
4.10. Protected heptasialic acid (2f)
The reaction of 2e (25.6 mg, 8.3 ꢀmol) and 3 (4.9 mg, 8.3 ꢀmol, 1.0 eq.) was performed for
12 h as described in the general procedure for cycloaddition-desilylation, affording 2f (elution
with CH2Cl2/MeOH (8/1); 16.8 mg, 56% yield; Rf = 0.48, MeOH/EtOAc, 2:5, v/v) as white solids.
[α]30 -0.31 (c 3.8, CHCl3); H NMR (400 MHz, CDCl3): δ 7.76 (m, 6H), 5.93-5.84 (m, 5H),
1
D
5.59-5.44 (m, 8H), 5.33-5.30 (m, 7H), 4.93-4.88 (m, 19H), 4.61-4.52 (m, 5H), 4.44-4.34 (m, 9H),
4.26-4.07 (m, 17H), 3.80-3.74 (m, 21H), 2.68-2.63 (m, 7H), 2.50 (s, 1H), 2.20 (m, 18H), 2.15 (m,
6H), 2.07-1.96 (m, 49H), 1.88 (br.s, 21H); 13C NMR (150 MHz, CDCl3): δ 170.95, 170.83, 170.78,
170.75, 170.66, 170.46, 170.40, 170.33, 170.16, 170.07, 169.90, 168.14, 167.77, 143.82, 132.25,
130.88, 129.97, 129.67, 128.79, 124.25, 98.75, 98.56, 98.32, 74.83, 73.20, 73.14, 72.57, 70.40,
68.90, 68.87, 68.77, 68.49, 68.41, 68.35, 67.31, 65.53, 62.46, 58.34, 53.01, 52.88, 50.17, 49.34,
49.20, 49.09, 37.85, 37.64, 23.14, 23.09, 23.06, 22.63, 21.13, 20.95, 20.90, 20.85, 20.81, 20.76.
HRMS (ESI) Calcd. for C149H201N25O79 ([M + 2H]2+): 1802.1239.; found: 1802.2267.
4.11. Protected octasialic acid (2g)
The reaction of 2f (49.0 mg, 13.6 ꢀmol) and 3 (8.3 mg, 14.3 ꢀmol, 1.05 eq.) was performed
for 12 h as described in the general procedure for cycloaddition-desilylation, affording 2g (elution
with CH2Cl2/MeOH (8/1); 26.1 mg, 47% yield; Rf = 0.39, MeOH/EtOAc, 2:5, v/v) as white solids.
[α]30 -0.97 (c 3.3, CHCl3); 1H NMR (400 MHz, CDCl3): δ 7.76-7.73 (m, 7H), 5.98-5.85 (m, 6H),
D
5.48-5.43 (m, 8H), 5.35-5.29 (m, 9H), 4.93-4.85 (m, 22H), 4.62-4.60 (m, 6H), 4.47-4.33 (m, 10H),
4.27-4.06 (m, 19H), 3.80-3.73 (m, 24H), 2.64-2.62 (m, 8H), 2.49 (s, 1H), 2.21-2.15 (m, 27H),
2.07-1.97 (m, 56H), 1.88 (br.s, 27H); 13C NMR (150 MHz, CDCl3): δ 170.97, 170.80, 170.76,
170.67, 170.50, 170.45, 170.36, 170.19, 170.09, 169.93, 168.16, 167.78, 167.70, 143.74, 132.26,
13

ACCEPTED MANUSCRIPT
130.89, 129.98, 129.68, 128.80, 124.24, 98.79, 98.76, 98.57, 98.33, 74.82, 73.19, 72.58, 70.43,
70.15, 68.88, 68.78, 68.44, 68.35, 67.33, 65.54, 62.47, 58.34, 53.01, 52.91, 52.87, 50.14, 49.34,
49.19, 49.09, 37.64, 23.05, 20.94, 20.91, 20.85, 20.82, 20.77. MS (MALDI-TOF) m/z Calcd. for
C170H227N29O90Na [M + Na]+ : 4138.40; found: 4139.24.
4.12. Triazolyl-linked α(2-9)-disialic acid (1a)
The deprotection of 2a (25.4 mg, 24.4 ꢀmol) was performed as described in the general
procedure for deprotection, affording 1a as white solids (13.7 mg, 78% yield). [α]30 -0.15 (c 2.6,
D
1
H2O); H NMR (400 MHz, D2O): δ 8.06 (s, 1H), 4.90 (d, J = 12.0 Hz, 1H), 4.84-4.74 (m, 1H),
4.61 (d, J = 12.0 Hz, 1H), 4.54 (dd, J = 14.6, 8.0 Hz, 1H), 4.33 (d, J = 15.6 Hz, 1H), 4.25 (dd, J =
9.0, 2.4 Hz, 1H), 4.20 (d, J = 15.6 Hz, 1H), 3.94-3.80 (m, 4H), 3.75-3.58 (m, 6H), 3.43 (dd, J =
9.0, 1.4 Hz, 1H), 2.77-2.69 (m, 2H), 2.03 (s, 3H), 2.02 (s, 3H), 1.71-1.64 (m, 2H); 13C NMR (100
MHz, D2O): δ 175.10, 175.03, 173.23, 172.73, 143.77, 126.07, 100.79, 100.67, 72.69, 72.54,
71.68, 70.03, 69.25, 68.25, 68.99, 62.63, 57.34, 52.92, 52.58, 51.84, 51.76, 40.27, 22.04; HRMS
(ESI) Calcd. for C28H42N5O17 ([M + H]+): 720.2576; found: 720.2569.
4.13ꢃTriazolyl-linked α(2-9)-trisialic acid (1b)
The deprotection of 2b (21.2 mg, 13.6 ꢀmol) was performed as described in the general
procedure for deprotection, affording 1b as white solids (13.6 mg, 89% yield). [α]30 -3.23 (c 2.9,
D
H2O); 1H NMR (600 MHz, D2O): δ 8.07 (s, 1H), 8.05 (s, 1H), 4.90 (d, J = 12.0 Hz, 1H), 4.88 (d, J
= 12.0 Hz, 1H), 4.82-4.75 (m, 2H), 4.62-4.52 (m, 4H), 4.33 (dd, J = 15.7, 2.3 Hz, 1H), 4.26-4.19
(m, 3H), 3.88-3.81 (m, 5H), 3.76-3.62 (m, 7H), 3.59 (d, J = 10.0 Hz, 1H), 3.43-3.38 (m, 2H),
2.76-2.70 (m, 3H), 2.03 (s, 3H), 2.01 (s, 6H), 1.67 (t, J = 12.0 Hz, 3H); 13C NMR (150 MHz, D2O):
δ 175.83, 175.80, 175.76, 173.98, 173.92, 173.48, 144.49, 126.98, 126.88, 101.54, 101.50, 101.41,
80.35, 76.07, 73.42, 73.28, 73.13, 72.40, 70.71, 70.45, 69.96, 69.93, 69.01, 68.98, 68.84, 68.75,
63.34, 58.07, 53.63, 53.54, 53.35, 52.58, 52.50, 41.03, 39.45, 22.76; HRMS (ESI) Calcd. for
C42H61N9O25Na ([M + Na]+): 1114.3671; found: 1114.3677.
4.14. Triazolyl-linked α(2-9)-tetrasialic acid (1c)
The deprotection of 2c (29.4 mg, 14.2 ꢀmol) was performed as described in the general
procedure for deprotection, affording 1c as white solids (18.4 mg, 88% yield). [α]30 -6.68 (c 1.6,
D
1
H2O); H NMR (600 MHz, D2O): δ 8.07-8.06 (m, 3H), 4.91-4.87 (m, 3H), 4.84-4.75 (m, 3H),
14

ACCEPTED MANUSCRIPT
4.62-4.52 (m, 6H), 4.33 (d, J = 15.7 Hz, 1H), 4.26-4.20 (m, 4H), 3.88-3.81 (m, 6H), 3.77-3.63 (m,
9H), 3.59 (d, J = 10.4 Hz, 1H), 3.44-3.37 (m, 3H), 2.76-2.70 (m, 4H), 2.03 (s, 3H), 2.02 (s, 3H),
2.01 (s, 3H), 2.01 (s, 3H), 1.68 (t, J = 12.0 Hz, 4H); 13C NMR (150 MHz, D2O): δ 175.83, 175.81,
175.77, 173.98, 173.93, 173.49, 144.47, 127.01, 126.88, 101.54, 101.50, 101.41, 79.92, 73.43,
73.28, 73.12, 72.41, 70.74, 70.41, 69.97, 69.89, 69.01, 68.99, 68.83, 68.76, 63.35, 58.07, 53.64,
53.51, 53.34, 52.59, 52.50, 41.02, 22.78. HRMS (ESI) Calcd. for C56H82N13O33 ([M + H]+):
1464.5132; found: 1464.5173.
4.15. Triazolyl-linked α(2-9)-pentasialic acid (1d)
The deprotection of 2d (32.9 mg, 12.8 ꢀmol) was performed as described in the general
procedure for deprotection, affording 1d as white solids (23.4 mg, 99% yield). [α]30 -4.89 (c 3.0,
D
H2O); 1H NMR (600 MHz, D2O): δ 8.10 (m, 4H), 4.92-4.89 (m, 4H), 4.84-4.75 (m, 4H), 4.64-4.54
(m, 8H), 4.35 (d, J = 15.7 Hz, 1H), 4.28-4.22 (m, 5H), 3.90-3.64 (m, 18H), 3.61 (d, J = 9.3 Hz,
1H), 3.46-3.37 (m, 4H), 2.78-2.72 (m, 5H), 2.06-2.02 (m, 15H), 1.70 (t, J = 12.0 Hz, 5H); 13C
NMR (150 MHz, D2O): δ 175.86, 175.83, 175.79, 173.93, 173.48, 171.80, 144.62, 127.10, 101.57,
79.97, 73.44, 73.29, 73.13, 72.48, 72.43, 70.83, 70.76, 70.53, 70.47, 69.99, 69.96, 69.92, 69.00,
68.83, 68.76, 63.40, 58.11, 53.69, 53.58, 53.44, 53.37, 53.30, 52.64, 52.61, 41.03, 22.91, 22.87,
22.82; HRMS (ESI) Calcd for C70H101N17O41Na2 ([M + 2Na]2+): 940.8068; found: 940.8051.
4.16. Triazolyl-linked α(2-9)-hexasialic acid (1e)
The deprotection of 2e (33.6 mg, 10.9 ꢀmol) was performed as described in the general
procedure for deprotection, affording 1e as white solids (20.9 mg, 87% yield). [α]30 -11.39 (c 2.5,
D
1
H2O); H NMR (600 MHz, D2O): δ 8.08-8.07 (m, 5H), 4.91-4.88 (m, 5H), 4.84-4.75 (m, 5H),
4.63-4.53 (m, 10H), 4.40 (d, J = 15.7 Hz, 1H), 4.27-4.20 (m, 5H), 3.89-3.59 (m, 23H), 3.45-3.36
(m, 5H), 2.77-2.72 (m, 6H), 2.04-2.01 (m, 18H), 1.68 (t, J = 12.0 Hz, 6H). 13C NMR (150 MHz,
D2O): δ 182.22, 175.85, 175.82, 175.78, 173.99, 173.94, 173.49, 171.80, 144.47, 127.04, 126.89,
101.49, 79.94, 73.43, 73.29, 73.12, 72.43, 70.77, 70.45, 70.37, 69.98, 69.90, 69.83, 69.09, 68.99,
68.83, 68.76, 63.37, 58.07, 53.66, 53.53, 53.48, 53.38, 52.61, 52.52, 41.02, 22.81. HRMS (ESI)
Calcd. for C84H121N21O49Na2 ([M + 2Na]2+): 1126.8698; found: 1126.8745.
4.17. Triazolyl-linked α(2-9)-heptasialic acid (1f)
The deprotection of 2f (35.0 mg, 9.7 ꢀmol) was performed as described in the general
15

ACCEPTED MANUSCRIPT
procedure for deprotection, affording 1f as white solids (23.6 mg, 94% yield). [α]30 -10.10 (c 3.1,
D
1
H2O); H NMR (400 MHz, D2O): δ 8.13 (m, 6H), 4.90-4.74 (m, 14H), 4.62 (m, 10H), 4.36-4.20
(m, 8H), 3.90-3.59 (m, 25H), 3.45-3.36 (m, 6H), 2.81-2.72 (m, 7H), 2.04-2.01 (m, 21H), 1.72-1.70
(m, 7H); 13C NMR (150 MHz, D2O): δ 175.81, 175.77, 173.88, 144.83, 143.25, 130.19, 127.18,
126.08, 103.39, 101.58, 73.43, 73.29, 73.12, 72.42, 70.74, 70.40, 70.31, 69.98, 69.89, 69.80, 69.01,
68.99, 68.83, 68.76, 63.36, 58.09, 53.67, 53.53, 53.47, 53.35, 52.61, 52.51, 41.02, 22.80. MS
(MALDI-TOF) m/z Calcd. for C98H139N25O57 [M - 2H]2- : 1288.94; found: 1288.55.
4.18. Triazolyl-linked α(2-9)-octasialic acid (1g)
The deprotection of 2g (35.3 mg, 8.58 ꢀmol) was performed as described in the general
procedure for deprotection, affording 1g as white solids (24.4 mg, 96% yield). [α]30 -12.45 (c 3.2,
D
1
H2O); H NMR (400 MHz, D2O): δ 8.08 (br.s, 7H), 4.92-4.89 (m, 7H), 4.80-4.75 (m, 2H),
4.64-4.55 (m, 15H), 4.37-4.21 (m, 10H), 3.91-3.60 (m, 31H), 3.47-3.37 (m, 7H), 2.79-2.74 (m,
8H), 2.05-2.02 (m, 24H), 1.70 (t, J = 12.0 Hz, 8H); 13C NMR (150 MHz, D2O): δ 175.80, 173.99,
173.93, 173.49, 144.39, 127.04, 101.54, 101.46, 101.40, 73.40, 73.26, 73.09, 70.86, 70.42, 70.19,
70.04, 69.91, 69.83, 69.72, 69.00, 68.83, 68.76, 63.44, 63.28, 58.09, 53.54, 53.44, 53.32, 52.68,
52.54, 41.12, 41.02, 40.91, 23.06, 22.88, 22.70, 22.53. MS (MALDI-TOF) m/z Calcd. for
C112H162N29O65 [M + H]+ : 2953.02; found: 2953.00.
Acknowledgements
This work was financially supported by the grants from the Ministry of Science and
Technology of China (2012CB822100, 2012ZX09502001-001), the National Natural Science
Foundation of China (21102008, 21232002), and Beijing Higher Education Young Elite Teacher
Project (YETP0063).
Supplementary data
Copies of 1H NMR and 13C NMR spectra of the reported compounds. Supplementary data related
to this article can be found at http://dx.doi.org/10.1016/j.tet.2014.07.099.
References and notes
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Fazio, F.; Bryan, M. C.; Blixt, O.; Paulson, J. C.; Wong, C.-H. J. Am. Chem. Soc. 2002, 124, 14397–14402; (c)
Dondoni, A. Chem. Asian J. 2007, 2, 700-708.
16. (a) Cheshev, P.; Marra, A.; Dondoni, A. Org. Biomol. Chem. 2006, 4, 3225-3227; (b) Conte, M. L.; Chambery,
A.; Marra, A.; Dondoni, A. Synlett, 2009, 16, 2679-2681; (c) Polakova, M.; Belanova, M.; Mikusova, K.; Lattova,
E.; Perreault, H. Bioconjugate Chem. 2013, 22, 289-298; (d) dos Anjos, J. V.; Sinou, D.; de Melo, S. J.; Srivastava,
R. M. Synthesis, 2007, 2647-2652; (e) Lo Conte, M.; Marra, A.; Chambery, A.; Gurcha, S. S.; Besra, G. S.;
Dondoni, A. J. Org. Chem. 2010, 75, 6326-6336.
17. Shelke, S. V.; Cutting, B.; Jiang, X. H.; Ernst, B. Angew. Chem. Int. Ed. 2010, 49, 5721-5725.
18. (a) Marra, A.; Sinay, P. Carbohydr. Res. 1989, 187, 35-42; (b) Ye, X.-S.; Huang, X.; Wong, C.-H. Chem.
Commun. 2001, 974–975.
19. Al-Huniti, M. H.; Lepore, S. D. Org. Lett. 2014, 16, 4154-4157.
20. Andreev, A. A.; Konshin, V. V.; Komarov, N. V.; Gevorgyan, V. Org. Lett. 2004, 6, 421-424.
21. Ishizaki, M.; Hoshino, O. Tetrahedron 2000, 56, 8813-8819.
22. Fustero, S.; Sancho, A. G.; Acena, J. L. Sanz-Cervera, J. F. J. Org. Chem. 2009, 74, 6398-6401.
23. Hwang, S.; Bae, H.; Kim, S.; Kim, S. Tetrahedron 2012, 68, 1460-1465.
24. (a) Orsini, A.; Viterisi, A,; Bodlenner, A.; Pale P. Tetrahedron Lett. 2005, 46, 2259-2262; (b) Viterisi, A.; Orsini,
A.; Weibel, J. M.; Pale, P. Tetrahedron Lett. 2006, 47, 2779-2781.
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Electronic Supplementary Information
Synthesis of triazolyl-linked polysialic acids
De-Cai Xiong, Yichuan Zhou, Yuxin Cui, Xin-Shan Ye*
State Key Laboratory of Natural and Biomimetic Drugs, School of
Pharmaceutical Sciences, Peking University, Xue Yuan Road No. 38,
Beijing 100191, China
*Corresponding author. Tel.: +86 10 8280 5736; Fax.: +86 10 8280 2724; E-mail:
xinshan@bjmu.edu.cn
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Contents
General procedures..........................................................................................S-3
1H NMR spectra of 7 and 8…………………………………………………….S-9
1H NMR spectra of 5 and 9……………………………………………………S-10
1H NMR spectra of 9-Ts and 10…………………………………………….....S-11
1H NMR and 13C NMR spectra of 11…………………………………..……...S-12
1H NMR and 13C NMR spectra of 3…………………………………..……….S-13
1H NMR and 13C NMR spectra of 2a………………………………..………...S-14
1H NMR and 13C NMR spectra of 2b...…………………………………….....S-15
1H NMR and 13C NMR spectra of 2c………………………………………....S-16
1H NMR and 13C NMR spectra of 2d………..………………………….…….S-17
1H NMR and 13C NMR spectra of 2e………..………………………………..S-18
1H NMR and 13C NMR spectra of 2f………..………………………….……..S-19
1H NMR and 13C NMR spectra of 2g………..………………………………..S-20
1H NMR and 13C NMR spectra of 1a…………………………………….…...S-21
1H NMR and 13C NMR spectra of 1b...…………………………………….....S-22
1H NMR and 13C NMR spectra of 1c………………………………………....S-23
1H NMR and 13C NMR spectra of 1d………..………………………….…….S-24
1H NMR and 13C NMR spectra of 1e………..………………………………..S-25
1H NMR and 13C NMR spectra of 1f………..………………………………..S-26
1H NMR and 13C NMR spectra of 1g………..………………………………..S-27
2

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General procedures. All chemicals were purchased as reagent grade and used
without further purification, unless otherwise noted. Dichloromethane (CH2Cl2),
pyridine, and acetonitrile were distilled over calcium hydride (CaH2). Methanol was
distilled from magnesium. DMF and 1,4-dioxane were stirred with calcium hydride
(CaH2) and distilled under reduced pressure. All reactions were carried out under
anhydrous conditions with freshly distilled solvents, unless otherwise noted.
Reactions were monitored by analytical thin-layer chromatography on silica gel
60-F254 precoated on alunminum plates (E. Merck.). Spots were detected under UV
(254 nm) and/or by staining with acidic ceric ammonium molybdate. Solvents were
o
evaporated under reduced pressure and below 40 C (bath). Column chromatography
was performed on silica gel (200-300 mesh, TsingDao Ocean or 230-400 mesh,
Merck), reversed phase column chromatography was performed on LiChroprep RP-18
1
(40-63 μm, Merck). H NMR spectra were recorded on a Bruker 400M or 600M
spectrometers at 25 oC. Chemical shifts (in ppm) were referenced to tetramethylsilane
13
(δ = 0 ppm) in deuterated chloroform. C NMR spectra were obtained by using the
same NMR spectrometers and were calibrated with CDCl3 (δ = 77.00 ppm). Mass
spectra were recorded using a Bruker APEX IV FT-MS（7.0 T）spectrometer.
c
e
M
O
A
CO2
c
c
OA
OA
c
OA
e
M
H
O
CO2
c
c
A O
OA
b
H
c
A O
O
H
O
a
o
T
S
l
O
e
M
CO2
O
H
O
c
A HN
O
c
A HN
c
A HN
c
A O
c
A O
d
8
7
H
O
6
e
M
CO2
H
O
H
O
H
O
c
e
M
CO2
OA
c
OA
c
c
A O
g
O
O
c
A HN
5
O
O
c
A HN
H
O
c
A O
9
α
5
b
e
f
c
e
M
CO2
OA
e
M
CO2
H
O
c
A
g
O
H
O
N3
N3
O
O
O
c
A HN
O
c
A HN
c
A O
H
O
10
11
3

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Methyl
(5-acetamido-2,4,7,8,9-penta-O-acetyl-3,5-dideoxy-D-glycero-α-D-galacto-2-nonu
lopyranosid)onate (7).
To a solution of 6 (1.0 g) in dry pyridine (4.0 mL) was added Ac2O (3.0 mL) and
DMAP (50.0 mg) at 0 oC. After stirring at room temperature for 3 h, the solvents were
evaporated under reduced pressure. The residue was dissolved in CH2Cl2 (100 mL)
and washed with H2O (20 mL), saturated aq. NaHCO3 and brine (20 mL). The organic
layer was dried over Na2SO4. After filtration, the solution was evaporated to afford 7
as a yellow foam (1.5 g, 95%). α isomer: 1H NMR (400 MHz, CDCl3): δ 5.38 (dd, J =
5.2, 1.8 Hz, 1H, H-7), 5.30-5.22 (m, 2H, H-8, NH), 5.07 (ddd, J = 6.6, 5.3, 2.6 Hz, 1H,
H-4), 4.49 (dd, J = 12.4, 2.5 Hz, 1H, H-9b), 4.14-4.09 (m, 3H, H-5, H-6, H-9a), 3.80
(s, 3H, OCH3), 2.55 (dd, J = 13.5, 4.9 Hz, 1H, H-3e), 2.18 (s, 3H, COCH3), 2.15 (s,
3H, COCH3), 2.14-2.10 (m, 1H, H-3a), 2.07 (s, 3H, COCH3), 2.04 (s, 3H, COCH3),
1
2.04 (s, 3H, COCH3), 1.90 (s, 3H, NHCOCH3). The H NMR data is consistent with
the literature.[1]
Methyl
(2-p-methylphenyl
5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero-α-D-galacto-2-thio-non
ulopyranosid)onate (8).
To a solution of 7 (16.90 g, 31.7 mmol) and p-methylthiophenol (7.50 g, 60.4 mmol,
2.0 eq.) in dry CH2Cl2 (50 mL) was added BF3·Et2O (6.5 mL, 1.5 eq.). The resulting
solution was stirred at r.t. overnight. The mixture was diluted with CH2Cl2 (200 mL)
and washed with H2O (20 mL), saturated aq. NaHCO3 and brine (20 mL). The organic
layer was dried over Na2SO4. After filtration, the solution was evaporated. The
residue was purified by column chromatography on silica gel (petroleum ether/ethyl
acetate, 3/1 to 0/1) to afford 8 as a white foam (16.5 g, 87%). α isomer: 1H NMR (400
MHz, CDCl3): δ 7.39 (d, J = 7.6 Hz, 2H, ArH), 7.14 (d, J = 7.6 Hz, 2H, ArH),
5.30-5.24 (m, 2H, H-7, H-8), 5.09 (d, J = 9.7 Hz, 1H, NH), 4.84 (td, J = 10.5, 3.8 Hz,
1H, H-4), 4.41 (d, J = 12.1 Hz, 1H, H-9b), 4.21 (dd, J = 12.7, 6.0 Hz, 1H, H-9a), 3.97
1
Marra, A. and Sinay, P. Carbohydr. Res. 1989, 187, 35-42
4

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(q, J = 10.8 Hz, 1H, H-5), 3.87 (d, J = 11.0 Hz, 1H, H-6), 3.61 (s, 3H, OCH3), 2.78
(dd, J = 12.3, 4.5 Hz, 1H, H-3e), 2.36 (s, 3H, ArCH3), 2.14 (s, 3H, COCH3), 2.06 (s,
3H, COCH3), 2.01 (s, 3H, COCH3), 2.00-1.94 (m, 1H, H-3a), 1.86 (s, 3H,
NHCOCH3). The 1H NMR data is consistent with the literature.[2]
Methyl
(2-propynyl
5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero-α-D-galacto-2-nonulop
yranosid)onate (5αb).
To a suspension of 4 Å molecular sieves (3.0 g), compound 8 (16.50 g, 27.6 mmol)
and propargyl alcohol (6.2 mL) in dry CH2Cl2 (30 mL) was added NIS (14.30 g, 63.5
mmol, 2.3 eq.) and TfOH (0.93 mL, 11.0 mmol, 0.3 eq.) at 0 oC. After stirring for 1 h
o
at 0 C, the mixture was filtered through a pad of celite and the celite was washed
thoroughly with CH2Cl2. The filtrate was washed with saturated aq. NaHCO3,
saturated aq. Na2S2O3 and brine. The organic layer was dried over Na2SO4 and
concentrated. The residue was purified by column chromatography on silica gel
(petroleum ether/acetone, 3:1) to afford 5αb as white foams (13.0, 89%). The α
isomer is compound 5：1H NMR (400 MHz, CDCl3): δ 5.43-5.40 (m, 1H, H-8), 5.31
(d, J = 9.0 Hz, 1H, NH), 5.14-5.12 (m, 1H, H-7), 4.91-4.84 (m, 1H, H-4), 4.41 (dd, J
= 15.7, 2.4 Hz, 1H, HC≡CCH2), 4.29 (dd, J = 12.4, 2.7 Hz, 1H, H-9b), 4.17 (dd, J =
15.6, 2.4 Hz, 1H, HC≡CCH2), 4.11-4.05 (m, 3H, H-5, H-6, H-9a), 3.82 (s, 3H, OCH3),
2.64 (dd, J = 12.8, 4.6 Hz, 1H, H-3e), 2.44 (t, J = 2.4 Hz, 1H, HC≡C), 2.16 (s, 3H,
COCH3), 2.15 (s, 3H, COCH3), 2.05 (s, 3H, COCH3), 2.04 (s, 3H, COCH3), 1.99 (t, J
= 12.4 Hz, 1H, H-3a), 1.88 (s, 3H, NHCOCH3). The 1H NMR data is consistent with
the literature.[3]
Methyl
(2-propynyl
5-acetamido-3,5-dideoxy-D-glycero-α-D-galacto-2-nonulopyranosid)onate (9).
To a solution of 5αb (11.1 g, 21.0 mmol) in dry MeOH (100 mL) was added 1 M
2
Ye, X.-S.; Huang, X. and Wong, C.-H. Chem. Commun. 2001, 974–975.
Shelke, S. V.; Cutting, B.; Jiang, X. H.; Ernst, B. Angew. Chem. Int. Ed. 2010, 49, 5721-5725.
3
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NaOMe/MeOH (20 mL). The resulting solution was stirred at room temperature for
0.5 h. The reaction mixture was neutralized with Amberlyst 15 (H+) ion-exchange
resin and filtered through a pad of celite. The celite was thoroughly washed with
MeOH, and the combined filtrates were concentrated under vacuum. The residue was
purified by column chromatography on silica gel (CH2Cl2/MeOH, 14:1) to afford pure
α isomer 9 as white solids (3.54 g, 47%). 1H NMR (400 MHz, MeOD): δ 4.37 (ABq,
J = 12.0, 2.5 Hz, 2H, HC≡CCH2), 3.86 (s, 3H, OCH3), 3.85-3.77 (m, 2H, H-8, H-9b),
3.71-3.64 (m, 2H, H-4, H-9a), 3.60 (dd, J = 10.4, 1.4 Hz, 1H, H-6), 3.52 (dd, J = 9.0,
1.1 Hz, 1H, H-7), 2.88 (t, J = 2.0 Hz, 1H, HC≡C), 2.72 (dd, J = 12.8 ,4.6 Hz, 1H,
H-3e), 2.02 (s, 3H, NHCOCH3), 1.77 (t, J = 12.4 Hz, 1H, H-3a). The 1H NMR data is
consistent with the literature.[3]
Methyl [2-propynyl 5-acetamido-3,5-dideoxy-9-O-(4-toluenesulfonyl)-D-glycero-
α-D-galacto-2-nonulopyranosid]onate (9Ts).
To a solution of 4Å molecular sieves (3.0 g) and 9 (3.24 g, 8.98 mmol) in dry pyridine
(15 mL) was added p-TsCl (2.91 g, 15.3 mmol, 1.7 eq.) and DMAP (200 mg) at 0 oC.
After stirring for 2 h, the reaction was quenched with methanol (20 mL). After
removal of the sieves and solvent, the residue was purified by column
chromatography on silica gel (CH2Cl2/MeOH, 30:1) to afford the product as white
solids (5.50 g). 1H NMR (400 MHz, MeOD): δ 7.82 (d, J = 8.3 Hz, 2H, ArH), 7.46 (d,
J = 8.1 Hz, 2H, ArH), 4.33 (dd, J = 8.2, 1.7 Hz, 1H, H-9b), 4.29 (ABq, J = 11.3, 2.4
Hz, 2H, HC≡CCH2), 4.11 (dd, J = 9.9, 6.0 Hz, 1H, H-9a), 4.02-3.98 (m, 1H, H-8),
3.83 (s, 3H, OCH3), 3.72 (t, J = 10.1 Hz, 1H, H-5), 3.66 (td, J = 10.5, 4.5 Hz, 1H,
H-4), 3.57 (d, J = 10.4 Hz, 1H, H-6), 3.47 (d, J = 8.7 Hz, 1H, H-7), 2.86 (t, J = 2.1 Hz,
1H, HC≡C), 2.69 (dd, J = 12.9, 4.6 Hz, 1H, H-3e), 2.47 (s, 3H, ArCH3), 2.01 (s, 3H,
NHCOCH3), 1.72 (t, J = 12.1 Hz, 1H, H-3a). The 1H NMR data is consistent with the
literature.[3]
Methyl
(2-propynyl
5-acetamido-9-azido-3,5,9-trideoxy-D-glycero-α-D-galacto-2-nonulopyranosid)on
6

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ate (10).
To a solution of 9-Ts (5.50 g) in DMF (25 mL) was added water (1 mL), 15-crown-5
o
(0.2 mL) and NaN3 (2.10 g, 5eq.). The resulting solution was stirred at 65 C
overnight. The mixture was filtered through a pad of celite and the filtrate was
evaporated to dryness. Purification by column chromatography on silica gel
1
(CH2Cl2/MeOH, 25:1) afforded 10 as white foams (1.46 g, 42% from 9). H NMR
(400 MHz, MeOD): δ 4.37 (ABq, J = 10.7, 2.4 Hz, 2H, HC≡CCH2), 4.02-3.98 (m, 1H,
H-8), 3.86 (s, 3H, OCH3), 3.79 (t, J = 10.2 Hz, 1H, H-5), 3.68 (dd, J = 11.7, 4.6 Hz,
1H, H-4), 3.63 (dd, J = 10.5, 1.4 Hz, 1H, H-6), 3.56 (dd, J = 12.8, 2.6 Hz, 1H, H-9b),
3.48 (dd, J = 9.0, 1.2 Hz, 1H, H-7), 3.41 (t, J = 6.2 Hz, 1H, H-9a), 2.86 (t, J = 2.4 Hz,
1H, HC≡C), 2.71 (dd, J = 12.8, 4.6 Hz, 1H, H-3e), 2.03 (s, 3H, NHCOCH3), 1.75 (t, J
= 12.0 Hz, 1H, H-3a). The 1H NMR data is consistent with the literature.[3]
Methyl
(2-propynyl
5-acetamido-4,7,8-tri-O-acetyl-9-azido-3,5,9-trideoxy-D-glycero-α-D-galacto-2-no
nulopyranosid)onate (11).
To a solution of 10 (1.46 g, 37.8 mmol) in dry pyridine (10 mL) was added Ac2O (7.5
o
mL) and DMAP (50.0 mg) at 0 C. The reaction mixture was stirred at room
temperature for 3 h. The solvents were evaporated under reduced pressure. The
residue was purified by column chromatography on silica gel (petroleum
ether/acetone, 3:1) to afford 11 as white foams (1.47 g, 75%). [α]30 -0.29 (c 2.1,
D
CHCl3); 1H NMR (400 MHz, CDCl3): δ 5.36-5.30 (m, 2H, H-7, H-8), 5.15 (d, J = 9.6
Hz, 1H, NH), 4.87 (ddd, J = 12.0, 9.9, 4.5 Hz, 1H, H-4), 4.40 (dd, J = 15.6, 2.5 Hz,
1H, HC≡CCH2), 4.16 (dd, J = 15.6, 2.4 Hz, 1H, HC≡CCH2), 4.11-4.06 (m, 2H, H-5,
H-6), 3.83 (s, 3H, OCH3), 3.57 (dd, J = 13.2, 2.2 Hz, 1H, H-9b), 3.28 (dd, J = 13.4,
5.2 Hz, 1H, H-9a), 2.64 (dd, J = 12.8, 4.6 Hz, 1H, H-3e), 2.46 (t, J = 2.5 Hz, 1H,
HC≡C), 2.19 (s, 3H, COCH3), 2.17 (s, 3H, COCH3), 2.04 (s, 3H, COCH3), 1.99 (t, J =
12.6 Hz, 1H, H-3a), 1.89 (s, 3H, NHCOCH3); 13C NMR (100 MHz, CDCl3): δ 170.98,
170.26, 170.19, 170.11, 167.75, 98.12, 78.81, 74.56, 72.86, 69.48, 68.71, 67.78, 52.90,
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52.81, 50.90, 49.28, 37.79, 23.16, 21.02, 20.84, 20.82. The 1H NMR data is consistent
with the literature.[3]
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12