Chemoenzymatic synthesis of α2-3-sialylated carbohydrate epitopes

SCIENCE CHINA

Chemistry

January 2011 Vol.54 No.1: 117–128

doi: 10.1007/s11426-010-4175-9

• ARTICLES •

· SPECIAL TOPIC · The Frontiers of Chemical Biology and Synthesis

Chemoenzymatic synthesis of 2–3-sialylated

carbohydrate epitopes

HUANG ShengShu, YU Hai & CHEN Xi^*

Department of Chemistry, University of California, One Shields Avenue, Davis, California 95616, USA

Received July 31, 2010; accepted September 24, 2010

Sialic acids are common terminal carbohydrates on cell surface. Together with internal carbohydrate structures, they play im-

portant roles in many physiological and pathological processes. In order to obtain 2–3-sialylated oligosaccharides, a highly

efficient one-pot three-enzyme synthetic approach was applied. The P. multocida 2–3-sialyltransferase (PmST1) involved in

the synthesis was a multifunctional enzyme with extremely flexible donor and acceptor substrate specificities. Sialyltransferase

acceptors, including type 1 structure (Gal1–3GlcNAcProN₃), type 2 structures (Gal1–4GlcNAcProN₃and 6-sulfo-Gal1–

4GlcNAcProN₃), type 4 structure (Gal1–3GalNAcProN₃), type 3 or core 1 structure (Gal1–3GalNAcProN₃) and human

milk oligoscaccharide or lipooligosaccharide lacto-N-tetraose (LNT) (Gal1–3GlcNAc1–3Gal1–4GlcProN₃), were chemi-

cally synthesized. They were then used in one-pot three-enzyme reactions with sialic acid precursor ManNAc or ManNGc, to

synthesize a library of naturally occurring 2–3-linked sialosides with different internal sugar structures. The sialylated oligo-

saccharides obtained are valuable probes for their biological studies.

carbohydrate, chemoenzymatic synthesis, sialic acid, sialylation, sialyltransferase

4GlcNAc- (type 2 glycan), Gal1–3GalNAc- (core 1 or

type 3 glycan, TF or T antigen), Gal1–3GalNAc- (type 4

glycan), and Gal1–4Glc- (type 5 glycan). Sialic acids are

also commonly seen 2–6-linked to the terminal galactose

residue in glycans containing disaccharides such as Gal1–

3GlcNAc- (type 1 glycan), Gal1–4GlcNAc- (type 2

glycan), and Gal1–4Glc- (type 5 glycan). 2–6-Sialylated

GalNAc- (Sia2–6GalNAc-, STn antigen) has also been

found in the O-GalNAc glycans on glycoproteins of certain

types of cancer cells and is believed to be a potent cancer

marker (Figure 1(A)) [6–8]. Sialylated lipooligosaccharide

(LOS) structures have been found in many bacteria, such as

Haemophilus influenzae [9], possibly Pasteurella multocida

[10], Escherichia coli K1 and K92 [11]. The core structures

for sialylation in LOS are lacto-N-tetraose (LNT) and

lacto-N- neotetraose (LNnT). They both can be sialylated

and their corresponding sialosides are called sialyllacto-N-

tetraose (LST) a, b, c and d (Figure 1(B)) [12, 13]. In addition,

1 Introduction

In nature, terminal sialic acids are commonly 2–3- or

2–6-linked to galactose (Gal) or N-acetyl-galactosamine

(GalNAc) [1]. Sialic acid residues can also be 2–8- or

2–9-linked to each other [2]. Currently, more than 50 dif-

ferent sialic acid forms have been identified in nature. The

presentation of different forms is species and tissue specific.

They are developmentally regulated and are believed to be

closely related to their biological functions [3–5]. In addi-

tion to the naturally existing diversity of sialic acids and

sialyl linkages, the internal carbohydrates are quite different

which provide additional complexity of sialic acid-containing

structures. For example, sialic acids can be 2–3- to the

terminal galactose residue in glycans containing disaccha-

rides such as Gal1–3GlcNAc- (type 1 glycan), Gal1–

*Corresponding author (email: chen@chem.ucdavis.edu)

chem.scichina.com

www.springerlink.com

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Huang SS, et al. Sci China Chem January (2011) Vol.54 No.1

homogenous sialosides [31–34].

Herein we report the synthesis of a library of 2–3-sia-

lylated glycans using an efficient one-pot three-enzyme

chemoenzymatic approach. Sialyltransferase acceptors, in-

cluding type 1 (Gal1–3GlcNAcProN₃), type 2 (Gal1–

4GlcNAcProN₃and 6-sulfo-Gal1–4GlcNAcProN₃), type

3 or core 1 (Gal1–3GalNAcProN₃), and type 4 (Gal1–

3GalNAcProN₃) disaccharides as well as type 1 tetrasac-

charide (Gal1–3GlcNAc1–3Gal1–4GlcProN₃) were

chemically synthesized and used in the one-pot threeen-

zyme system containing a sialic acid aldolase, a CMP-sialic

acid synthetase, and an 2–3-sialyltransferase for the pro-

duction of a library of natural occurring 2–3-linked sialoside

epitopes. Two different sialic acid precursors, N-acetylmanno-

samine (ManNAc) and N-glycolylmannosamine (ManNGc),

were used to produce 2–3-linked sialosides containing

N-acetylneuraminic acid (Neu5Ac), the most abundant

sialic acid form, and N-glycolylneuraminic acid (Neu5Gc),

a common non-human animal sialic acid form, respectively.

Figure 1 Common sialic acid-containing structures in nature.

sialyl Lewis^xand sialyl Lewis^aare two of the most impor-

tant sialosides that have been broadly studied. They both

contain a sialic acid 2–3-linked to a trisaccharide com-

posed of galactose, GalNAc and fucose (Figure 1(C)), with

or without sulfation at galatose or N-acetylglucosamine

(GLcNAc) [14].

2 Experimental

The interaction of sialoside and sialic acid-binding pro-

teins is dependent on the diversity of sialoside structures

including sialic acid forms, sialyl linkages, and internal

carbohydrate structures [4, 15]. Other than well documented

importance of sialic acids and sialyl linkages in host-virus

interactions [16–21], the internal carbohydrate structures of

sialosides have also been reported to be important for Siglec

binding in the immune system [22–25]. For example,

among CD-33 related Siglecs, Siglec-8 in human binds

strongly only to sialyl Lewis^xstructures with O-sulfation at

C-6 of galactose. Sialyl Lewis^x, sialyl LacNAc, and sialyl

Lewis^xwith O-sulfation at C-6 of GlcNAc did not bind to

Siglec-8 at all [26–28]. However, Siglec-9 favored sialyl

Lewis^xstructures with O-sulfation at the C-6 of GlcNAc

instead of galactose [29]. Another example of the impor-

tance of sialic acid form was shown by preferred binding of

Siglec-10 to Neu5Gc2–6LacNAc rather than to Ne5Ac2–

6LacNAc [25].

To better understand the biological significance of sialic

acid-containing glycans, homogenous sialosides with different

forms of sialic acids and various internal carbohydrates are

needed. Chemical sialylation is usually more challenging

and more time consuming compared to other chemical gly-

cosylation processes due to the structural complexity of

sialic acid [30] including a sterically hindered tertiary ano-

meric center, lacking of a stereo-directing group adjacent to

the anomeric position, and the presence of an elec-

tron-withdrawing carboxyl group in sialyl donors. Isolation

of sialosides from natural sources is also difficult due to

their low abundance, libility, and the complication of di-

verse sialic acid forms, different linkages, and various in-

ternal glycans. To overcome these difficulties, chemoenzy-

matic methods were developed in our laboratory to obtain

2.1 General

¹H and ¹³C NMR spectra were recorded on a Varian Mer-

cury-300, a Varian Inova-400, or a Varian Inova-600 spec-

trometer. Chemical shifts were reported in (ppm) unit using

¹³C and residual H signals from deuterated solvents as ref-

1

erences. Assignment of H NMR spectra was achieved us-

ing 2D methods (COSY) when necessary. Low and high

resolution electrospray ionization (ESI) mass spectra were

obtained at the Mass Spectrometry Facility at the Ohio State

University. Silica gel 60 Å (40–63 m, Sorbent technolo-

gies) was used for flash column chromatography. Analytical

thin layer chromatography was performed on silica gel

plates 60 GF₂₅₄(Sorbent technologies). Anisaldehyde stain

was used for detection. Gel filtration chromatography was

performed on a column (100 cm × 2.5 cm) packed with

BioGel P-2 Fine resins (Bio-Rad, Hercules, CA). All re-

agents were of analytical grade and were used as supplied

without further purification unless specified. Solvents used

in chemical reactions were distilled under an inert argon

atmosphere.

2.2 Chemical synthesis of sialyltransferase acceptors

2.2.1 3-Azidopropyl -D-galactopyranosyl-(1–3)-2-acetamido-

2-deoxy--D-glucopyranoside (Gal1–3GlcNAcProN₃, 1)

Compound 7 [35] (0.90 g, 1.62 mmol) was dissolve in dry

MeOH (20 mL), NaOMe (100 mg) was then added. The

reaction mixture was stirred for overnight, neutralized by

Dowex-50 (H⁺) resin, and evaporated to dryness. This dried

product was dissolved in 10 mL of anhydrous DMF, fol-

lowed by the addition of PhCH(OMe)₂(1 mL, 6.57 mmol).

Huang SS, et al. Sci China Chem January (2011) Vol.54 No.1

119

The pH of the solution was adjusted to 2–4 by adding

D(+)-10-camphorsulfonic acid and the mixture was heated

at 60 C under reducing pressure at 150 mbar. After 1 h, the

reaction mixture was condensed in vacuo. The product was

purified by flash column chromatography (Hexane: EtOAc

= 3:2, by volume) to afford a white foam 8 (0.73 g, 87%).

¹H NMR (600 MHz, CDCl₃) 7.83 (dd, 1 H, J=5.4 Hz and

3.0 Hz), 7.70 (dd, 1 H, J = 6.0 and 3.3 Hz), 7.50–7.49 (m, 2

H), 7.37–7.36 (m, 3 H), 5.56 (s, 1 H), 5.23 (d, 1 H, J = 8.4

Hz), 4.64 (t, 1 H, J = 9.6 Hz), 4.37 (dd, 1 H, J=10.5 and 4.5

Hz), 4.21 (dd, 1 H, J = 10.8 and 2.4 Hz), 3.94–3.91 (m, 1 H),

3.81 (t, 1 H, J = 10.5 Hz), 3.64–3.56 (m, 3 H), 3.28–3.19 (m,

2 H), 1.94–1.90 (m, 1 H), 1.82–1.76 (m, 1 H). ¹³C NMR (75

MHz, CDCl₃)  137.21, 134.43, 131.85, 129.60, 128.62,

126.57, 102.14, 99.33, 83.38, 68.88, 68.67, 67.48, 66.36,

56.82, 41.48, 32.25.

refluxed at 100 C for 3 h, the reaction mixture was directly

dried in vacuo. To the dried white powder was added pyri-

dine (10 mL) and acetic anhydride (2 mL). After overnight

stirring, the mixture was concentrated and purified by flash

column chromatography (hexane:acetone = 1:1, by volume).

The purified white foam product was dissolved in dry

MeOH (10 mL), and NaOMe (50 mg) was added. The reac-

tion was stirred for overnight, neutralized by Dowex-50 (H⁺)

resin, and evaporated to dryness to afford Gal1–3GlcNAc-

1

ProN₃(1) (262 mg, 84%). H NMR (600 MHz, D₂O) 

4.51 (d, 1 H, J = 8.4 Hz), 4.39 (d, 1 H, J = 7.8 Hz),

3.96–3.92 (m, 1 H), 3.90–3.87 (m, 2 H), 3.80–3.59 (m, 8 H),

3.51–3.44 (m, 3 H), 3.35–3.32 (m, 2 H), 2.00 (s, 3 H), 1.80

(pentalet, 2 H, J = 6.6 Hz). ¹³C NMR (75 MHz, D₂O)

174.73, 103.64, 101.04, 82.48, 75.44, 75.38, 72.56, 70.77,

68.81, 68.61, 67.26, 61.13, 60.80, 54.66, 47.88, 28.21,

22.32. HRMS (ESI) m/z calcd for C₁₇H₃₀N₄O₁₁(MH)

465.1828, found 465.1833.

A solution of compound 8 (0.69 g, 1.32 mmol) and ga-

lactosyl trichloroacetimidate 9 [36] (0.79 g, 1.60 mmol) in

anhydrous CH₂Cl₂(10 mL) was stirred with activated 4 Å

molecular sieves (1.20 g) under argon atmosphere for 30

min. The reaction mixture was cooled to 20 °C and TMSOTf

(20 L) was added drop wisely. The reaction mixture was

quenched with Et₃N (0.5 mL) after 2 h and was filtered

through Celite. The filtrate was concentrated in vacuo. The

residue was purified by flash column chromatography

(toluene:EtOAc = 3:1, by volume) to give the glycosylation

2.2.2 2.3-Azidopropyl -D-galactopyranosyl-(1–4)-2-acetam-

ido-2-deoxy--D-glucopyranoside (Gal1–4GlcNAcProN₃, 2)

To a solution of compound 7 (2.0 g, 3.91 mmol) in methanol

(30 mL) was added NaOMe (100 mg). After stirring at rt for

2 h, TLC (Hexane:Acetone = 1:2, by volume) showed the

completion of the reaction. The reaction mixture was neu-

tralized by Dowex-50 (H⁺) resin, filtered, and evaporated to

dryness. The dried product was then dissolved in anhydrous

CH₂Cl₂, followed by the addition of imidazole (531 mg,

7.82 mmol) and TBSCl (718 mg, 4.69 mmol). After over-

night stirring, the mixture was condensed and purified by

flash column chromatography (hexane:EtOAc = 2:1, by

1

product 10 (0.60 g, 53%). H NMR (600 MHz, CDCl₃) 

7.86 (dd, 2 H, J = 5.4 Hz and 3.0 Hz), 7.75 (dd, 2 H, J=5.4

Hz and 3.0 Hz), 7.48–7.46 (m, 2 H), 7.36–7.35 (m, 3 H),

5.56 (m, 1 H), 5.17 (dd, 1 H, J = 3.3 and 0.9 Hz), 5.13 (d, 1

H, J = 9.0 Hz), 4.97 (dd, 1 H, J = 10.2 and 7.8 Hz),

4.77–4.72 (m, 2 H), 4.53 (d, 1 H, J = 8.4 Hz), 4.36 (dd, 1 H,

J = 10.2 Hz and 4.8 Hz), 4.27 (dd, 1 H, J =10.2 and 8.4 Hz),

4.01 (dd, 1 H, J = 10.8 and 7.8 Hz), 3.91–3.88 (m, 1 H),

3.84 (t, 1 H, J = 10.2 Hz), 3.81–3.77 (m, 2 H), 3.65–3.62 (m,

1 H), 3.56–3.53 (m, 1 H), 3.49–3.46 (m, 1 H), 3.23–3.20 (m,

1 H), 3.17–3.13 (m, 1 H), 1.94–1.90 (m, 1 H), 1.89 (s, 3 H),

1.82 (s, 3 H), 1.81–1.75 (m, 1 H), 1.54 (s, 3 H). ¹³C NMR

(150 MHz, CDCl₃)  170.47, 170.26, 170.24, 169.09,

137.23, 134.54, 131.86, 129.50, 128.58, 126.24, 101.68,

100.73, 99.17, 81.16, 75.80, 71.20, 70.51, 69.38, 68.90,

67.35, 66.85, 66.51, 60.99, 55.54, 41.48, 32.25, 20.82,

20.76, 20.65, 20.31.

The protected disaccharide 10 (600 mg, 0.70 mmol) was

dissolved in 10 mL of methanol and 100 mg Pd/C was

added. The mixture was shaken under H₂(4 Bar) for over-

night, filtered, and dried. The dried product, TBAI (100 mg,

0.27 mmol), and sodium azide (228 mg, 3.5 mmol) were

dissolved in 10 mL of anhydrous DMF. After stirred at 60 °C

for 3 hours, the reaction mixture was poured into water (50

mL) and extracted by ethyl acetate (30 mL). The organic

layer was dried to afford product 11 (485 mg, 95%), which

was dissolved in ethanol (5 mL), followed by the addition

of hydrazine hydrate (64% hydrazine, 10 mL). After being

1

volume) to afford a white foam 12 (1.64 g, 84%). H NMR

(600 MHz, CDCl₃)  7.83 (dd, 2 H, J = 5.4 Hz and 3.0 Hz),

7.71 (dd, 2 H, J = 5.4 Hz and 3.0 Hz), 5.20 (d, 1 H, J = 8.4

Hz), 4.36 (dd, 1 H, J = 11.4 Hz and 9.0 Hz), 4.09 (dd, 1 H,

J = 10.8 Hz and 4.8 Hz), 3.98 (dd, 1 H, J = 10.8 Hz and 4.8

Hz), 3.91–3.86 (m, 2 H), 3.63–3.56 (m, 2 H), 3.54–3.51 (m,

1 H), 3.40–3.34 (m, 2 H), 1.96–1.90 (m, 1 H), 1.85–1.80 (m,

1 H), 0.91 (s, 9 H), 0.12 (s, 3 H), 0.11 (s, 3 H).

A solution of acceptor 12 (0.75 g, 1.50 mmol) and tri-

chloroacetimidate donor 9 (0.89 g, 1.80 mmol) in anhydrous

CH₂Cl₂(20 mL) was stirred with activated 4 Å molecular

sieves (2 g) under argon atmosphere for 30 min. The reac-

tion mixture was cooled to 40 °C and TMSOTf (15 L)

was added drop wisely. The reaction mixture was quenched

with Et₃N (0.5 mL) after 10 h, and filtered through celite.

The filtrate was concentrated in vacuo. The residue was

purified by flash column chromatography (Hexane:EtOAc=

2:1, by volume) to afford the coupled product 13 (0.98 g,

1

79%). H NMR (600 MHz, CDCl₃) 7.75–7.73 (m, 2 H),

7.64–7.62 (m, 2 H), 5.27 (d, 1 H, J = 3.0 Hz), 5.14 (dd, 1 H,

J=10.2 Hz and 8.4 Hz), 5.11 (d, 1 H, J=8.4 Hz), 4.90 (dd, 1

H, J = 10.5 Hz and 3.3 Hz), 4.53 (d, 1 H, J = 7.8 Hz), 4.31

(dd, 1 H, J = 10.8 Hz and 8.4 Hz), 4.02–3.94 (m, 4 H),

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Huang SS, et al. Sci China Chem January (2011) Vol.54 No.1

3.92–3.89 (m, 1 H), 3.82–3.77 (m, 2 H), 3.68 (dd, 1 H, J =

11.4 Hz and 3.6 Hz), 3.56 (t, 1 H, J = 9.0 Hz), 3.51–3.47 (m,

1 H), 3.42 (dd, 1 H, J = 7.8 Hz and 2.4 Hz), 3.35–3.28 (m, 2

H), 2.03 (s, 3 H), 2.00 (s, 3 H), 1.88 (s, 3 H), 1.80 (s, 3 H),

1.88–1.70 (m, 2 H), 0.83 (s, 9 H), 0.03 (s, 3 H), 0.00 (s, 3

H). ¹³C NMR (150 MHz, CDCl₃)  170.59, 170.18, 169.43,

134.27, 131.89, 101.92, 98.19, 82.12, 74.90, 71.35, 71.08,

69.81, 68.91, 66.99, 65.91, 61.75, 61.70, 56.23, 41.61,

32.52, 26.08, 20.90, 20.76, 20.70, 20.45, 18.48, 4.69,

5.00.

2.12 (s, 3 H), 2.02 (s, 3 H), 1.95 (s, 3 H), 1.89 (s, 3 H),

1.70–1.67 (m, 2 H). ¹³C NMR (75 MHz, CDCl₃)  170.61,

170.45, 170.36, 170.11, 169.46, 134.66, 134.47, 123.78,

101.23, 98.16, 75.79, 75.03, 71.56, 71.18, 70.70, 69.40,

66.91, 66.47, 61.00, 60.67, 55.15, 48.08, 29.03, 20.95,

20.91, 20.87, 20.81.

Compound 15 (434 mg, 0.51 mmol) and pyridine sulfur

trioxide (182 mg, 1.02 mmol) were dissolved in pyridine

(10 mL) and stirred at room temperature. The reaction was

completed in 1.5 h. Purification by flash column chromatog-

raphy (hexane:EtOAc = 1:1, by volume) afforded the sul-

fated product. After this sulfated product was treated simi-

larly as described for compound 11, the sulfated disaccha-

ride Gal1–4GlcNAc6SProN₃(3) was obtained as a white

foam (184 mg, 66%). ¹H NMR (600 MHz, D₂O) 4.54 (d, 1

H, J = 8.4 Hz), 4.52 (d, 1 H, J = 7.8 Hz), 4.38 (dd, 1 H, J=

11.1 and 1.5 Hz), 4.31 (dd, 1 H, J = 11.1 and 4.5 Hz),

3.97–3.94 (m, 1 H), 3.91 (d, 1 H, J = 3.6 Hz), 3.81–3.64 (m,

9 H), 3.51 (dd, 1 H, J = 7.8 and 1.8 Hz), 3.37–3.34 (m, 2 H),

2.04 (s, 3 H), 1.84–1.81 (m, 2 H). ¹³C NMR (75 MHz, D₂O)

 174.64, 102.65, 101.32, 77.48, 75.49, 72.67, 72.61, 72.40,

71.13, 68.75, 67.41, 66.39, 61.20, 55.22, 47.92, 28.26,

22.32. HRMS (ESI) m/z calcd for C₁₇H₂₉N₄O₁₄SNa (MNa)

545.1407, found 545.1398

Compound 13 (0.98 g, 1.18 mmol) was treated by TBAF

(4 mL, 1 M in THF) in THF (20 mL) for 2 hours. The TLC

(hexane:EtOAc = 1:2, by volume) showed the TBS group

was removed. After flash column chromatography purification,

the product was dissolved in DMF (10 mL). Sodium azide

(325 mg, 5 mmol) and TBAI (50 mg, 0.14 mmol) were

added. The mixture was heated at 70 °C for 4 h, condensed in

vacuo, and purified by flash column chromatography

(hexane:EtOAc = 1:2, by volume) to afford compound 14

(647 mg, 76%). Deprotection and N-acetylation of compound

14 (647 mg, 0.89 mmol) were achieved by using a similar

approach as described for compound 11 to give Gal1–

1

4GlcNAcProN₃(2) (343 mg, 82%) as a white foam. H

NMR (600 MHz, D₂O)  4.51 (d, 1 H, J = 7.8 Hz), 4.45 (d,

1 H, J = 7.8 Hz), 3.98–3.95 (m, 2 H), 3.90 (d, 1 H, J = 3.6

Hz), 3.81 (dd, 1 H, J = 12.3 Hz and 5.1 Hz), 3.76–3.67 (m, 7

H), 3.65 (dd, 1 H, J = 9.6 Hz and 3.6 Hz), 3.59–3.56 (m, 1

H), 3.52 (dd, 1 H, J = 9.6 Hz and 7.8 Hz), 3.37–3.34 (m, 2

H), 2.03 (s, 3 H), 1.82 (pentalet, 2 H, J = 6.6 Hz). ¹³C NMR

(150 MHz, D₂O)  174.62, 103.01, 101.24, 78.56, 75.48,

74.88, 72.63, 72.50, 71.09, 68.68, 67.26, 61.16, 60.18,

55.22, 47.90, 28.24, 22.31. HRMS (ESI) m/z calcd for

C₁₇H₃₀N₄O₁₁(MH) 465.1828, found 465.1840.

2.2.4 3.3-Azidopropyl -D-galactopyranosyl-(1–3)-2-acetam-

ido-2-deoxy--D-galactopyranoside (Gal1–3GalNAcProN₃,

4)

To a solution of compound 16 [37] (2.5 g, 5.74 mmol) in

3-chloro-1-propanol (15 mL) was added acetyl chloride

(1 mL). The solution was heated to 90 °C. 4 h later, TLC

showed the disappearance of the starting material. The

mixture was cooled to room temperature and transferred

directly to silica gel column for purification (hexanes:EtOAc =

1:1 followed by EtOAc, and then hexanes:acetone = 1:1, by

volume). According to the proton NMR spectroscopy, the

purified product was an / mixture without any O-acety-

lation. The product was then dissolved in DMF (20 mL),

followed by the addition of PhCH(OMe)₂(2 mL).

D(+)-10-Camphorsulfonic acid was added to adjust the pH

to 2–4. The reaction mixture was heated at 50 °C at 150

mBar for 30 min, condensed, and purified by flash column

chromatography (hexane:EtOAc = 3:2, by volume) to afford

two products, compound 17 (0.57 g, 21%) and 18 (1.55 g,

2.2.3 3-Azidopropyl -D-galactopyranosyl-(1–4)-2-acetam-

ido-2-deoxy-6-O-sulfo--D-glucopyranoside (Gal1–4GlcNAc-

6SProN₃, 3)

To a solution of compound 13 (500 mg, 0.60 mmol) in

pyridine (10 mL) was added acetic anhydride (1 mL). After

overnight stirring, the reaction mixture was diluted by

EtOAc (100 mL) and washed sequentially with 2 N HCl

(100 mL), saturated NaHCO₃(100 mL), and brine (100 mL).

The organic layer was dried by MgSO₄and evaporated to

produce a yellow syrup, which was treated by TBAF and

sodium azide (with TBAI) sequentially to give compound

15 (434 mg, 85%) with a free hydroxyl group at the C-6 of

GlcNAc. ¹H NMR (600 MHz, CDCl₃)  7.86–7.81 (m, 2 H),

7.74–7.71 (m, 2 H), 5.72 (dd, 1 H, J = 10.8 Hz and 9.0 Hz),

5.39 (d, 1 H, J = 8.4 Hz), 5.32 (dd, 1 H, J=3.3 and 0.9 Hz),

5.11 (dd, 1 H, J = 10.8 and 7.8 Hz), 4.98 (dd, 1 H, J = 10.2

and 3.6 Hz), 4.65 (d, 1 H, J=7.8 Hz), 4.15 (dd, 1 H, J=10.8

and 8.4 Hz), 4.09–4.00 (m, 3 H), 3.94–3.92 (m, 1 H),

3.89–3.86 (m, 2 H), 3.80–3.76 (m, 1 H), 3.59–3.57 (m, 1 H),

3.54–3.51 (m, 1 H), 3.23–3.19 (m, 1 H), 3.16–3.12 (m, 1 H),

1

57%). For compound 17, H NMR (600 MHz, CDCl₃) 

7.79 (dd, 2 H, J = 5.4 Hz and 3.0 Hz), 7.60 (dd, 2 H, J =

5.0 Hz and 3.0 Hz), 7.54 (dd, 2 H, J = 7.2 Hz and 1.5 Hz),

7.39–7.35 (m, 3 H), 5.61 (s, 1 H), 5.44 (dd, 1 H, J=11.4

Hz and 3.6 Hz), 5.04 (d, 1 H, J = 3.0 Hz), 4.65 (dd, 1 H, J=

11.4 Hz and 3.6 Hz), 4.37 (d, 1 H, J=4.2 Hz), 4.29 (dd, 1 H,

J = 12.6 Hz and 1.2 Hz), 4.11 (dd, 1 H, J = 12.6 Hz and 1.2

Hz), 3.88–3.84 (m, 2 H), 3.42–3.39 (m, 1 H), 1.94–1.88 (m,

2 H). ¹³C NMR (150 MHz, CDCl₃)  168.85, 137.70,

134.28, 131.92, 129.50, 128.53, 126.65, 123.45, 101.59,

Huang SS, et al. Sci China Chem January (2011) Vol.54 No.1

121

98.84, 75.92, 69.72, 64.40, 63.34, 54.27, 41.90, 31.93. For

compound 18, ¹H NMR (600 MHz, CDCl₃)  7.83–7.80 (m,

2 H), 7.71–7.69 (m, 2 H), 7.55–7.52 (m, 2 H), 7.40–7.36 (m,

3 H), 5.60 (s, 1 H), 5.25 (d, 1 H, J = 8.4 Hz), 4.52 (dd, 1 H, J

= 11.1 Hz and 3.9 Hz), 4.42–4.36 (m, 2 H), 4.28 (d, 1 H, J=

3.6 Hz), 4.12 (dd, 1 H, J = 12.0 Hz and 1.8 Hz), 3.98–3.95

(m, 1 H), 3.64–3.59 (m, 2 H), 3.42–3.34 (m, 2 H),

1.98–1.93 (m, 1 H), 1.85–1.81 (m, 1 H). ¹³C NMR (150

MHz, CDCl₃)  168.85, 137.57, 134.33, 129.57, 128.54,

126.71, 101.73, 98.74, 75.34, 69.45, 68.05, 67.00, 66.14,

54.94, 41.70, 32.37.

Compound 17 (570 mg, 1.20 mmol) was coupled to tri-

chloroacetimidate 9 (710 mg, 1.44 mmol) using a similar

approach as described for compound 10. Purification by

flash column chromatography (hexane:EtOAc = 1:1, by

volume) gave product 19 (461 mg, 62%) as a white foam.

¹H NMR (600 MHz, CDCl₃)  7.85–7.81 (m, 2 H), 7.72–7.68

(m, 2 H), 7.59 (d, 2 H, J = 7.2 Hz), 7.39–7.33 (m, 3 H), 5.60

(s, 1 H), 5.36 (dd, 1 H, J=11.7 Hz and 3.3 Hz), 5.32 (d, 1 H,

J = 3.6 Hz), 5.06 (dd, 1 H, J = 10.5 Hz and 2.1 Hz), 5.00 (d,

1 H, J = 3.6 Hz), 4.93–4.89 (m, 2 H), 4.81 (d, 1 H, J = 8.4

Hz), 4.48 (d, 1 H, J = 3.0 Hz), 4.28 (d, 1 H, J = 12.6 Hz),

4.19 (dd, 1 H, J = 11.7 Hz and 5.7 Hz), 4.10–4.06 (m, 2 H),

3.92 (t, 1 H, J = 6.6 Hz), 3.88–3.85 (m, 1 H), 3.82 (s, 1 H),

3.60–3.53 (m, 2 H), 3.43–3.39 (m, 1 H), 2.10 (s, 3 H), 2.04

(s, 3 H), 1.95–1.88 (m, 2 H), 1.86 (s, 3 H), 1.15 (s, 3 H). ¹³C

NMR (150 MHz, CDCl₃)  170.62, 170.55, 168.88, 168.48,

137.98, 134.50, 134.36, 132.57, 131.20, 129.09, 128.41,

126.54, 123.58, 123.53, 101.82, 100.98, 98.96, 76.50, 72.51,

71.37, 70.87, 69.69, 68.98, 67.26, 64.44, 63.57, 61.63,

51.31, 41.83, 31.88, 21.00, 20.95, 20.73, 19.64.

2.2.5 3-Azidopropyl -D-galactopyranosyl-(1–3)-2-acetam-

ido-2-deoxy--D-galactopyranoside (Gal1–3GalNAcProN₃,

5)

Compound 18 (1.0 g, 2.11 mmol) was coupled to trichlor-

oacetimidate 9 (1.25 g, 2.53 mmol) using a similar approach

as described for compound 10. Purification by flash column

chromatography (Hexane:EtOAc = 1:1, by volume) gave

product 20 (812 mg, 48%) as a white foam. ¹H NMR (600

MHz, CDCl₃)  7.84–7.82 (m, 2 H), 7.73–7.71 (m, 2 H),

7.57 (d, 2 H, J = 7.8 Hz), 7.38–7.31 (m, 3 H), 5.57 (s, 1 H),

5.27 (d, 1 H, J = 3.6 Hz), 5.08–5.05 (m, 2 H), 4.80 (dd, 1 H,

J = 10.5 Hz and 3.3 Hz), 4.75 (dd, 1 H, J = 11.4 Hz and 3.0

Hz), 4.68 (dd, 1 H, J = 11.4 Hz and 8.4 Hz), 4.54 (d, 1 H,

J = 8.4 Hz), 4.36 (d, 1 H, J = 3.0 Hz), 4.33 (d, 1 H, J=12.6

Hz), 4.10–4.02 (m, 3 H), 3.95–3.92 (m, 1 H), 3.80 (t, 1 H,

J = 6.6 Hz), 3.57 (s, 1 H), 3.56–3.52 (m, 1 H), 3.35–3.32 (m,

1 H), 3.30–3.26 (m, 1 H), 2.09 (s, 3 H), 2.00 (s, 3 H),

1.97–1.92 (m, 1 H), 1.87 (s, 3 H), 1.81–1.72 (m, 1 H), 1.39

(s, 3 H). ¹³C NMR (150 MHz, CDCl₃)  170.48, 170.31,

169.40, 168.96, 167.53, 137.93, 134.60, 134.49, 131.79,

129.10, 128.38, 126.59, 123.82, 123.53, 101.68, 101.08,

98.94, 75.77, 75.04, 71.18, 71.00, 69.44, 68.81, 67.09,

66.96, 65.81, 61.51, 51.93, 41.66, 32.13, 20.95, 20.91,

20.70, 19.99.

Compound 22 (664 mg, 91%) was obtained from com-

pound 20 (812 mg, 1.01 mmol) using a similar approach as

described for compound 21. Deprotection and N-acetylation

of compound 22 (664 mg, 0.92 mmol) were achieved using

a similar approach as described for compound 11 to give

Gal1–4GalNAcProN₃(5) (326 mg, 76%) as the final

product. ¹H NMR (600 MHz, D₂O)  4.51 (d, 1 H, J = 8.4

Hz), 4.45 (d, 1 H, J = 7.8 Hz), 4.19 (d, 1 H, J = 3.0 Hz),

4.02–3.97 (m, 2 H), 3.92 (d, 1 H, J = 3.6 Hz), 3.88 (dd, 1 H,

J = 10.8 Hz and 3.0 Hz), 3.83–3.65 (m, 7 H), 3.62 (dd, 1 H,

J = 9.6 Hz and 3.0 Hz), 3.53 (dd, 1 H, J = 10.2 Hz and 7.8

Hz), 3.41–3.38 (m, 2 H), 2.04 (s, 3 H), 1.86 (pentalet, 2 H, J

= 6.6 Hz). ¹³C NMR (150 MHz, D₂O)  174.88, 105.00,

101.57, 80.05, 75.15, 74.91, 72.65, 70.77, 68.76, 68.17,

67.19, 61.18, 61.11, 51.47, 47.99, 28.31, 22.45. HRMS (ESI)

m/z calcd for C₁₇H₃₀N₄O₁₁(MH) 465.1828, found

465.1835.

Compound 19 (461 mg, 0.57 mmol) was dissolved in

DMF (10 mL). Sodium azide (195 mg, 3.0 mmol) and

TBAI (30 mg, 0.08 mmol) were added. The mixture was

heated at 70 °C for 4 h, condensed in vacuo, diluted by wa-

ter, and extracted by CH₂Cl₂. The organic layer was evapo-

rated to dryness and 80% HOAc (15 mL) was added to dis-

solve it. The solution was then heated at 60 °C for 3 hrs to

remove the benzylidene protecting group. After removal of

the solvent, compound 21 (387 mg, 94%) was obtained

without purification. Deprotection and N-acetylation of

compound 21 (387 mg, 0.54 mmol) were achieved using a

similar approach as described for compound 11 to give

Gal1–4GalNAcProN₃(4) (214 mg, 85%) as the final

2.2.6 3-Azidopropyl -D-galactopyranosyl-(1–3)-2-acetam-

ido-2-deoxy--D-glucopyranosyl-(1–3)--D-galactopyranos

yl-(1–4)--D-glucopyranoside (Gal1–3GlcNAc1–3Gal1–

4GlcProN₃, 6)

To a solution of compound 23 [38] (1.00 g, 2.09 mmol) in

MeOH (20 mL) was added NaOMe (100 mg). The reaction

mixture was stirred for overnight, neutralized by Dowex-50

(H⁺) resin, and evaporated to dryness. This dried product

was dissolved in 10 mL of anhydrous DMF followed by the

addition of PhCH(OMe)₂(1 mL, 6.57 mmol). The pH of the

solution was adjusted to 2–4 using D(+)-10-camphorsulfonic

acid, and was then heated at 60 C under reducing pressure

1

product. H NMR (600 MHz, D₂O)  4.90 (d, 1 H, J = 3.6

Hz), 4.48 (d, 1 H, J =7.8 Hz), 4.35 (dd, 1 H, J=10.8 Hz and

3.6 Hz), 4.26 (d, 1 H, J = 2.4 Hz), 4.04 (dd, 1 H, J=10.8 Hz

and 3.0 Hz), 4.00 (t, 1 H, J = 6.0 Hz), 3.92 (d, 1 H, J = 3.0

Hz), 3.83–3.74 (m, 5 H), 3.68–3.63 (m, 2 H), 3.58–3.45 (m,

4 H), 2.04 (s, 3 H), 1.92 (quintet, 2 H, J=6.6 Hz). ¹³C NMR

(150 MHz, D₂O)  174.70, 104.89, 97.41, 77.40, 75.18,

72.71, 70.81, 68.92, 68.79, 65.13, 61.39, 61.19, 48.87,

48.41, 28.17, 22.22. HRMS (ESI) m/z calcd for

C₁₇H₃₀N₄O₁₁(MH) 465.1828, found 465.1829.

122

Huang SS, et al. Sci China Chem January (2011) Vol.54 No.1

at 150 mbar. After 1 h, the reaction mixture was condensed

and purified by flash column chromatography (hexane:

EtOAc = 3:2, by volume) to afford a white foam 24 (0.76 g,

3 H), 3.81–3.78 (m, 1 H), 3.74–3.58 (m, 5 H), 3.51–3.46 (m,

2 H), 3.37 (t, 1 H, J=6.6 Hz), 3.25 (dd, 1 H, J=11.4 Hz and

7.8 Hz), 3.17–3.10 (m, 2 H), 1.99 (s, 3 H), 1.83 (s, 3 H),

1.73 (s, 3 H), 1.72–1.60 (m, 2 H), 1.37 (s, 3 H). ¹³C NMR

(150 MHz, CDCl₃)  177.19, 170.29, 170.03, 168.73,

166.02, 165.99, 165.61, 165.57, 165.34, 164.21, 137.33,

133.54–132.88, 130.10–128.45, 128.05, 126.24, 101.55,

101.34, 100.85, 100.27, 99.15, 80.63, 77.41, 75.60, 75.11,

73.25, 72.75, 72.14, 71.88, 71.74, 71.19, 70.46, 69.84,

69.32, 68.64, 66.90, 66.72, 66.52, 62.54, 62.14, 61.00,

55.29, 48.10, 29.81, 20.61, 20.59, 20.46, 20.01.

Compound 27 (428 mg, 0.24 mmol) was dissolved in

80% acetic acid (10 mL) and heated to 60 °C. After 3 hours,

the solvent was removed and the residue was dissolved in

ethanol (5 mL). Hydrazine hydrate (10 mL, 66.7% in water)

was added. The reaction mixture was refluxed at 100 °C for

3 h, cooled to room temperature and evaporated to dryness.

To the dry product in pyridine (10 mL) was added acetic

andydride (1 mL). After overnight stirring, the mixture was

condensed and applied to flash column chromatography

(hexanes:Acetone = 1:1, by volume) to give the O-acetyl/

benzoyl protected tetrasaccharide. The O-acetyl and O-

benzoyl were then removed by treating with NaOMe (50

mg) in methanol (10 mL) to afford Gal1–3GlcNAc1

–3Gal1–4GlcProN₃(6) as a white foam (102 mg, 54%).

¹H NMR (600 MHz, D₂O)  4.74 (d, 1 H, J = 8.4 Hz), 4.50

(d, 1 H, J = 7.8 Hz), 4.45 (d, 2 H, J = 7.8 Hz), 4.16 (d, 1 H,

J = 3.6 Hz), 4.03–3.98 (m, 2 H), 3.93–3.89 (m, 3 H),

3.84–3.71 (m, 10 H), 3.67–3.46 (m, 11 H), 3.34–3.30 (m,

1 H), 2.04 (s, 3 H), 1.92 (pentalet, 2 H, J = 6.6 Hz). ¹³C

NMR (75 MHz, D₂O)  175.19, 103.72, 103.18, 102.75,

102.36, 82.37, 82.22, 78.69, 75.52, 75.44, 75.13, 75.00,

74.62, 73.03, 72.73, 70.93, 70.24, 68.78, 68.72, 68.55, 67.61,

62.81, 61.26, 61.18, 60.78, 60.36, 54.94, 48.14, 28.48, 22.49.

HRMS (ESI) m/z calcd for C₂₉H₅₀N₄O₂₁(MH) 789.2884,

found 789.2875.

1

83%). H NMR (600 MHz, CDCl₃)  7.87–7.85 (m, 2 H),

7.72–7.71 (m, 2 H), 7.50–7.48 (m, 2 H), 7.38–7.36 (m, 3 H),

5.57 (s, 1 H), 5.40 (d, 1 H, J = 10.8 Hz), 4.65 (t, 1 H, J=9.6

Hz), 4.39 (dd, 1 H, J = 10.2 Hz and 4.8 Hz), 4.31 (t, 1 H, J=

10.5 Hz), 3.80 (t, 1 H, J = 10.2 Hz), 3.71–3.67 (m, 1 H),

3.60 (t, 1 H, J = 9.3 Hz), 2.72–2.63 (m, 2 H), 1.19 (t, 3 H,

J = 7.5 Hz). ¹³C NMR (150 MHz, CDCl₃)  168.00, 167.38,

137.17, 134.46, 130.00, 129.61, 129.23, 128.62, 126.55,

102.17, 82.33, 82.10, 70.60, 69.76, 68.86, 55.73, 24.43,

15.11.

Compound 24 (0.76 g, 1.73 mmol) was coupled to tri-

chloroacetimidate 9 (1.02 g, 2.08 mmol) using the same

approach as described for compound 10. Purification by

flash column chromatography (hexane:EtOAc = 1:1, by

volume) gave product 25 (1.03 g, 77%) as a white foam. ¹H

NMR (600 MHz, CDCl₃)  7.90–7.77 (m, 4 H), 7.49–7.47

(m, 2 H), 7.40–7.35 (m, 3 H), 5.57 (s, 1 H), 5.28 (d, 1 H, J=

10.8 Hz), 5.19 (d, 1 H, J = 3.6 Hz), 4.99 (dd, 1 H, J = 10.2

Hz and 7.8 Hz), 4.77 (t, 1 H, J = 10.2 Hz), 4.74 (dd, 1 H, J=

10.2 Hz and 3.6 Hz), 4.55 (d, 1 H, J=7.8 Hz), 4.42–4.37 (m,

2 H), 4.03 (dd, 1 H, J = 10.8 Hz and 7.8 Hz), 3.85–3.80 (m,

3 H), 3.72–3.69 (m, 1 H), 3.48 (dd, 1 H, J = 7.8 Hz and 6.6

Hz), 2.71–2.62 (m, 2 H), 2.07 (s, 3 H), 1.91 (s, 3 H), 1.84 (s,

3 H), 1.55 (s, 3 H), 1.17 (t, 3 H, J = 7.2 Hz). ¹³C NMR (150

MHz, CDCl₃)  170.49, 170.29, 170.27, 169.11, 137.22,

129.53, 128.60, 126.31, 126.25, 101.67, 100.64, 81.93,

91.15, 76.64, 71.20, 70.87, 70.52, 69.40, 68.90, 66.85,

61.02, 54.40, 24.09, 20.85, 20.79, 20.67, 20.33, 15.01.

A solution of glycosyl donor 25 (350 mg, 0.46 mmol)

and glycosyl acceptor 26 [39] (400 mg, 0.38 mmol) in an-

hydrous CH₂Cl₂(20 mL) was stirred with activated 4 Å

molecular sieves (0.80 g) under argon for 30 min. The reac-

tion mixture was cooled to 20 °C and NIS (368 mg, 1.64

mmol) was added. After 10 min, TfOH (10 L) was added.

The reaction mixture was quenched with Et₃N (0.5 mL)

after 1 h and filtered through celite. The filtrate was con-

centrated in vacuo. The residue was purified by flash col-

umn chromatography (hexanes:EtOAc = 2:1, by volume) to

afford tetrasaccharide 27 as an amorphous solid (428 mg,

2.3 One-pot three-enzyme synthesis of 2–3-linked

sialosides

General procedure

1

A prospective acceptor for a multifunctional Pasteurella

multocida 2–3-sialyltransferase PmST1 (1–6, 0.1 mmol), a

sialic acid precursor (ManNAc or ManNGc, 1.2 equiv),

sodium pyruvate (1.8 equiv), and CTP (1.2 equiv) were dis-

solved in water. Stock solutions of Tris-HCl buffer (1 M,

pH 8.8, 1 mL) and MgCl₂·6H₂O (0.2 M, 1 mL) were added.

After the addition of a recombinant E. coli sialic acid aldolase

[40] (1.96 mg), an N. meningitidis CMP-sialic acid syn-

thetase [40] (1.08 mg), and PmST1 [31] (0.62 mg), water

was added until the total volume of the reaction mixture

reached 10 mL. The reaction was carried out by incubating

the solution in an isotherm incubator for 2 h at 37 C (or for

15 h at room temperature) with agitation at 140 rpm. The

64%). H NMR (600 MHz, CDCl₃)  8.10 (dd, 1 H, J =8.4

Hz and 1.2 Hz), 7.99 (dd, 1 H, J = 8.4 Hz and 1.2 Hz),

7.90–7.86 (m, 4 H), 7.74 (dd, 1 H, J = 8.4 Hz and 1.2 Hz),

7.65–7.60 (m, 3 H), 7.55–7.31 (m, 20 H), 7.25–7.17 (m,

4 H), 6.87 (d, 2 H, J = 7.8 Hz), 5.61 (t, 1 H, J=9.6 Hz), 5.51

(d, 1 H, J = 3.0 Hz), 5.45 (s, 1 H), 5.35–5.31 (m, 2 H), 5.20

(d, 1 H, J = 8.4 Hz), 5.10 (dd, 1 H, J = 3.6 Hz and 1.2 Hz),

4.80 (dd, 1 H, J = 10.2 Hz and 1.8 Hz), 4.61 (dd, 1 H, J =

10.2 Hz and 3.6 Hz), 4.57 (d, 1 H, J = 7.8 Hz), 4.55 (d, 1 H,

J = 7.8 Hz), 4.51 (dd, 1 H, J = 10.2 Hz and 9.0 Hz),

4.35–4.33 (m, 2 H), 4.28 (dd, 1 H, J = 10.8 Hz and 4.8 Hz),

4.23 (dd, 1 H, J = 12.6 Hz and 4.5 Hz), 4.07 (dd, 1 H, J =

10.2 Hz and 8.4 Hz), 4.04 (t, 1 H, J = 9.6 Hz), 3.97–3.93 (m,

Huang SS, et al. Sci China Chem January (2011) Vol.54 No.1

123

product formation was monitored by TLC developed with

O₁₉Na (MNa) 756.2782, found 756.2789.

Neu5Gc2–3Gal1–4GlcNAcProN₃(33)

EtOAc: MeOH:H₂O:HOAc = 5:2:1:0.2 (by volume, for sub-

strates 1, 2, 4, 5) or 4:2:1:0.2 (by volume, for substrates 3

and 6). TLC plates were stained with p-anisaldehyde sugar

stain. The reaction was quenched by adding the same vol-

ume (10 mL) of ice-cold EtOH and incubation at 4 C for

30 min. The mixture was then centrifuged to remove pre-

cipitates. The supernatant was concentrated and passed

through a BioGel P-2 gel filtration column with water as the

eluant to obtain the crude product. Further purification was

performed using flash column chromatography to afford the

desired 2–3-linked sialosides.

From disaccharide 2 (46.6 mg, 0.1 mmol), compound 33

was obtained as a white foam (64 mg, 80%). ¹H NMR (600

MHz, D₂O)  4.53 (d, 1 H, J = 7.8 Hz), 4.50 (d, 1 H, J=8.4

Hz), 4.11 (dd, 1 H, J = 9.3 Hz and 3.3 Hz), 4.10 (s, 2 H),

3.99–3.54 (m, 20 H), 3.38–3.34 (m, 2 H), 2.75 (dd, 1 H, J=

12.6 Hz and 4.8 Hz), 2.02 (s, 3 H), 1.82 (pentalet, 2 H, J=

6.0 Hz), 1.80 (t, 1 H, J =12.6 Hz). ¹³C NMR (75 MHz, D₂O)

 175.90, 174.19, 174.06, 102.68, 101.27, 99.92, 78.36,

75.57, 75.28, 74.86, 72.71, 72.45, 71.94, 69.51, 68.21,

68.12, 67.57, 67.24, 62.64, 61.08, 60.12, 55.20, 51.49,

47.88, 39.79, 28.22, 22.29. HRMS (ESI) m/z calcd for

C₂₈H₄₆N₅O₂₀Na (MNa) 772.2731, found 772.2730.

Neu5Ac2–3Gal1–3GlcNAcProN₃(30)

From disaccharide 1 (46.6 mg, 0.1 mmol), compound 30

was obtained as a white foam (66 mg, 85%). ¹H NMR (600

MHz, D₂O)  4.53 (d, 1 H, J = 8.4 Hz), 4.47 (d, 1 H, J=7.8

Hz), 4.06 (dd, 1 H, J = 9.6 Hz and 3.0 Hz), 3.97–3.45 (m, 20

H), 3.37–3.34 (m, 2 H), 2.73 (dd, 1 H, J = 12.3 Hz and 4.5

Hz), 2.01 (s, 3 H), 2.00 (s, 3 H), 1.82 (pentalet, 2 H, J=6.3

Hz), 1.76 (t, 1 H, J = 12.3 Hz). ¹³C NMR (75 MHz, D₂O) 

175.08, 174.70, 174.04, 103.57, 101.02, 99.75, 82.63, 75.72,

75.49, 75.21, 72.92, 71.96, 69.20, 68.84, 68.51, 68.14,

67.36, 67.27, 62.56, 61.14, 60.83, 54.56, 51.77, 47.90,

39.87, 28.22, 22.43, 22.18. HRMS (ESI) m/z calcd for

C₂₈H₄₆N₅O₁₉Na (MNa) 756.2782, found 756.2783.

Neu5Ac2–3Gal1–4GlcNAc6SProN₃(34)

From disaccharide 3 (56.8 mg, 0.1 mmol), compound 34

was obtained as a white foam (77 mg, 88%). ¹H NMR (400

MHz, D₂O)  4.61 (d, 1 H, J = 7.6 Hz), 4.54 (d, 1 H, J=8.0

Hz), 4.42–4.32 (m, 2 H), 4.12 (dd, 1 H, J = 9.8 Hz and 3.0

Hz), 3.99–3.53 (m, 18 H), 3.39–3.54 (m, 2 H), 2.74 (dd, 1 H,

J = 12.4 Hz and 4.4 Hz), 2.04 (s, 3 H), 2.03 (s, 3 H), 1.84

(pentalet, 2 H, J = 6.2 Hz), 1.80 (t, 1 H, J = 12.0 Hz). ¹³C

NMR (75 MHz, D₂O)  175.09, 174.67, 174.21, 102.27,

101.36, 99.89, 77.29, 75.47, 75.24, 72.99, 72.66, 72.37,

71.64, 69.60, 68.59, 68.21, 67.62, 67.44, 66.40, 62.65,

61.21, 55.21, 51.86, 47.94, 39.74, 28.26, 22.34, 22.24.

HRMS (ESI) m/z calcd for C₂₈H₄₅N₅O₂₂SNa₂(MNa)

858.2174, found 858.2151.

Neu5Gc2–3Gal1–3GlcNAcProN₃(31)

From disaccharide 1 (46.6 mg, 0.1 mmol), compound 31

was obtained as a white foam (65 mg, 83%). ¹H NMR (600

MHz, D₂O)  4.53 (d, 1 H, J = 8.4 Hz), 4.48 (d, 1 H, J=7.8

Hz), 4.10 (s, 2 H), 4.07 (dd, 1 H, J = 9.6 Hz and 3.0 Hz),

3.98–3.44 (m, 20 H), 3.37–3.34 (m, 2 H), 2.75 (dd, 1 H, J=

12.3 Hz and 4.5 Hz), 2.02 (s, 3 H), 1.82 (pentalet, 2 H, J=

6.3 Hz), 1.78 (t, 1 H, J =12.3 Hz). ¹³C NMR (75 MHz, D₂O)

 175.87, 174.71, 174.08, 103.59, 101.04, 99.77, 82.63,

75.73, 75.51, 75.22, 72.64, 72.04, 69.21, 68.84, 68.25,

68.07, 67.36, 67.28, 62.53, 61.15, 60.85, 54.58, 51.48,

47.92, 39.97, 28.24, 22.44. HRMS (ESI) m/z calcd for

C₂₈H₄₆N₅O₂₀Na (MNa) 772.2731, found 772.2728.

Neu5Gc2–3Gal1–4GlcNAc6SProN₃(35)

From disaccharide 3 (56.8 mg, 0.1 mmol), compound 35

was obtained as a white foam (74 mg, 82%). ¹H NMR (400

MHz, D₂O)  4.60 (d, 1 H, J = 7.6 Hz), 4.53 (d, 1 H, J=7.6

Hz), 4.40–4.31 (m, 2 H), 4.12 (dd, 1 H, J = 9.6 Hz and 2.4

Hz), 4.10 (s, 2 H), 3.98–3.52 (m, 18 H), 3.38–3.32 (m, 2 H),

2.75 (dd, 1 H, J = 12.6 Hz and 4.6 Hz), 2.03 (s, 3 H), 1.83

(pentalet, 2 H, J = 6.0 Hz), 1.80 (t, 1 H, J = 12.6 Hz). ¹³C

NMR (100 MHz, D₂O)  175.90, 174.69, 174.23, 102.36,

101.38, 99.95, 77.46, 75.53, 75.26, 72.73, 72.42, 71.70,

69.63, 68.34, 68.21, 67.66, 67.45, 66.44, 62.64, 61.22,

55.24, 51.63, 47.96, 39.85, 27.27, 22.38. HRMS (ESI) m/z

calcd for C₂₈H₄₅N₅O₂₃SNa₂(MNa) 874.2124, found

874.2107.

Neu5Ac2–3Gal1–4GlcNAcProN₃(32)

From disaccharide 2 (46.6 mg, 0.1 mmol), compound 32

was obtained as a white foam (65 mg, 84%). ¹H NMR (600

MHz, D₂O)  4.53 (d, 1 H, J = 7.8 Hz), 4.50 (d, 1 H, J=7.8

Hz), 4.09 (dd, 1 H, J = 9.9 Hz and 3.3 Hz), 3.99–3.93 (m,

3 H), 3.88–3.81 (m, 4 H), 3.73–3.53 (m, 13 H), 3.37–3.33

(m, 2 H), 2.73 (dd, 1 H, J = 12.6 Hz and 4.2 Hz), 2.02 (s,

3 H), 2.01 (s, 3 H), 1.82 (pentalet, 2 H, J = 6.3 Hz), 1.78 (t,

1 H, J = 12.6 Hz). ¹³C NMR (75 MHz, D₂O)  175.12,

174.61, 174.03, 102.69, 101.28, 99.93, 78.37, 75.58, 75.28,

74.86, 73.00, 72.46, 71.89, 69.50, 68.48, 68.20, 67.59,

67.25, 62.68, 61.15, 60.13, 55.20, 51.80, 47.89, 39.74,

28.23, 22.29, 22.17. HRMS (ESI) m/z calcd for C₂₈H₄₆N₅-

Neu5Ac2–3Gal1–3GalNAcProN₃(36)

From disaccharide 4 (46.6 mg, 0.1 mmol), compound 36

was obtained as a white foam (68 mg, 87%). ¹H NMR (600

MHz, D₂O)  4.88 (d, 1 H, J = 3.6 Hz), 4.52 (d, 1 H, J=7.8

Hz), 4.29 (dd, 1 H, J = 10.8 Hz and 3.6 Hz), 4.22 (d, 1 H,

J = 3.0 Hz), 4.05 (dd, 1 H, J=10.8 Hz and 3.6 Hz), 4.02 (dd,

1 H, J = 10.8 Hz and 3.0 Hz), 3.97 (t, 1 H, J=6.0 Hz), 3.91

(d, 1 H, J = 3.6 Hz), 3.87–3.41 (m, 17 H), 2.73 (dd, 1 H, J=

124

Huang SS, et al. Sci China Chem January (2011) Vol.54 No.1

12.3 Hz and 4.5 Hz), 2.01 (s, 3 H), 1.88 (pentalet, 2 H, J=

6.3 Hz), 1.76 (t, 1 H, J = 12.3 Hz). ¹³C NMR (150 MHz,

D₂O)  175.12, 174.71, 174.05, 104.60, 99.85, 97.31, 77.52,

75.78, 74.90, 72.93, 71.96, 70.76, 69.22, 68.70, 68.51,

68.17, 67.52, 65.04, 62.63, 61.35, 61.11, 51.78, 48.81,

48.30, 39.84, 28.10, 22.18. HRMS (ESI) m/z calcd for

C₂₈H₄₆N₅O₁₉Na (MNa) 756.2782, found 756.2801.

Neu5Ac2–3Gal1–3GlcNAc1–3Gal1–4GlcProN₃(40)

From tetrasaccharide 6 (30 mg, 0.038 mmol), compound 40

was obtained as a white foam (37 mg, 88%). ¹H NMR (600

MHz, D₂O)  4.71 (d, 1 H, J = 8.4 Hz), 4.49 (d, 1 H, J=7.8

Hz), 4.46 (d, 1 H, J = 7.8 Hz), 4.42 (d, 1 H, J=8.4 Hz), 4.13

(d, 1 H, J = 3.6 Hz), 4.06 (dd, 1 H, J = 9.6 Hz and 3.0 Hz),

4.00–3.43 (m, 32 H), 3.30–3.27 (m, 1 H), 2.73 (dd, 1 H, J=

12.6 Hz and 4.8 Hz), 2.01 (s, 6 H), 1.89 (pentalet, 2 H, J=

6.3 Hz), 1.76 (t, 1 H, J=12.6 Hz). ¹³C NMR (75 MHz, D₂O)

 175.08, 174.05, 103.51, 103.06, 102.65, 102.23, 99.75,

82.24, 82.05, 78.46, 75.71, 75.31, 75.21, 75.02, 74.89,

74.48, 72.90, 71.97, 70.12, 69.20, 68.51, 68.41, 68.14,

67.48, 67.36, 62.71, 62.56, 61.08, 60.61, 60.14, 54.70,

51.77, 47.97, 39.88, 28.35, 22.42, 22.16. HRMS (ESI) m/z

calcd for C₄₀H₆₆N₅O₂₉Na (MNa) 1080.3849, found

1080.3827.

Neu5Gc2–3Gal1–3GalNAcProN₃(37)

From disaccharide 4 (46.6 mg, 0.1 mmol), compound 37

was obtained as a white foam (60 mg, 76%). ¹H NMR (600

MHz, D₂O)  4.88 (d, 1 H, J = 3.6 Hz), 4.52 (d, 1 H, J=8.4

Hz), 4.30 (dd, 1 H, J = 11.1 Hz and 3.9 Hz), 4.22 (d, 1 H,

J = 3.0 Hz), 4.10 (s, 2 H), 4.06 (dd, 1 H, J = 9.6 Hz and 3.6

Hz), 3.97 (t, 1 H, J = 6.0 Hz), 3.93–3.41 (m, 18 H), 2.75 (dd,

1 H, J = 12.6 Hz and 4.8 Hz), 2.01 (s, 3 H), 1.88 (pentalet, 2

H, J=6.3 Hz), 1.78 (t, 1 H, J=12.3 Hz). ¹³C NMR (75 MHz,

D₂O)  175.98, 174.79, 174.13, 104.67, 99.98, 97.42, 77.60,

75.91, 75.03, 72.78, 72.12, 70.87, 69.34, 68.80, 68.34,

68.24, 67.64, 65.21, 62.74, 61.45, 61.23, 51.62, 48.93,

48.44, 40.05, 28.19, 22.30. HRMS (ESI) m/z calcd for

C₂₈H₄₆N₅O₂₀Na (MNa) 772.2731, found 772.2758.

Neu5Gc2–3Gal1–3GlcNAc1–3Gal1–4GlcProN₃(41)

From tetrasaccharide 6 (30 mg, 0.038 mmol), compound 41

was obtained as a white foam (35 mg, 88%). ¹H NMR (600

MHz, D₂O)  4.71 (d, 1 H, J = 8.4 Hz), 4.49 (d, 1 H, J=8.4

Hz), 4.46 (d, 1 H, J = 7.8 Hz), 4.42 (d, 1 H, J=7.8 Hz), 4.13

(d, 1 H, J = 3.6 Hz), 4.10 (s, 2 H), 4.07 (dd, 1 H, J=9.6 Hz

and 3.0 Hz), 3.99–3.51 (m, 29 H), 3.47–3.43 (m, 3 H),

3.30–3.27 (m, 1 H), 2.75 (dd, 1 H, J = 12.6 Hz and 4.8 Hz),

2.01 (s, 3 H), 1.89 (pentalet, 2 H, J = 6.6 Hz), 1.78 (t, 1 H,

J = 12.6 Hz). ¹³C NMR (75 MHz, D₂O)  175.91, 175.09,

174.10, 103.55, 103.11, 102.67, 102.28, 99.83, 99.77, 82.29,

82.10, 78.56, 75.71, 75.33, 75.25, 75.03, 74.94, 74.48,

72.95, 72.69, 72.07, 70.18, 69.26, 68.57, 68.48, 68.25,

68.15, 67.49, 62.59, 61,13, 60.62, 60.18, 54.76, 51.54,

47.98, 39.97, 28.40, 22.48. HRMS (ESI) m/z calcd for

C₄₀H₆₆N₅O₃₀Na (MNa) 1096.3787, found 1096.3783.

Neu5Ac2–3Gal1–3GalNAcProN₃(38)

From disaccharide 5 (46.6 mg, 0.1 mmol), compound 38

was obtained as a white foam (63 mg, 81%). ¹H NMR (600

MHz, D₂O)  4.53 (d, 1 H, J = 7.8 Hz), 4.52 (d, 1 H, J=8.4

Hz), 4.19 (d, 1 H, J =3.0 Hz), 4.08 (dd, 1 H, J=10.2 Hz and

3.0 Hz), 4.04–3.98 (m, 2 H), 3.90 (d, 1 H, J = 2.4 Hz),

3.87–3.54 (m, 16 H), 3.40 (t, 2 H, J =6.6 Hz), 2.77 (dd, 1 H,

J = 12.3 Hz and 4.5 Hz), 2.05 (s, 3 H), 2.04 (s, 3 H), 1.86

(pentalet, 2 H, J = 6.0 Hz), 1.80 (t, 1 H, J = 12.3 Hz). ¹³C

NMR (75 MHz, D₂O)  175.24, 174.95, 174.14, 104.80,

101.64, 99.95, 80.24, 75.82, 74.98, 73.04, 72.80, 72.04,

69.26, 68.59, 68.32, 68.12, 67.64, 67.27, 62.89, 62.77,

61.19, 51.93, 51.43, 48.07, 39.99, 28.36, 22.56, 22.31.

HRMS (ESI) m/z calcd for C₂₈H₄₆N₅O₁₉Na (MNa)

756.2782, found 756.2783.

3 Results and discussion

3.1 Synthesis of sialyltransferase acceptors

Six sialyltransferase acceptors (1–6) including type 1 (Gal1–

3GlcNAcProN₃, 1), type 2 (Gal1–4GlcNAcProN₃, 2 and

6-sulfo-Gal1–4GlcNAcProN₃, 3), type 3 or core 1 (Gal1–

3GalNAcProN₃, 4), and type 4 (Gal1–3GalNAcProN₃,

5) disaccharides as well as type 1 tetrasaccharide (Gal1–

3GlcNAc1–3Gal1–4GlcProN₃, 6) were chemically syn-

thesized.

Disaccharide Gal1–3GalNAcProN₃(1) was prepared

as shown in Scheme 1. Deacetylation of glucosamine de-

rivative 7 with NaOMe/MeOH [35] followed by 4,6-O-

benzylidene protection with benzaldehyde dimethyl acetal

produced 8 (87%), which was coupled with 2,3,4,6-O-ace-

tyl--D-galactopyranosyl trichloroacetimidate donor 9 [36]

in the presence of TMSOTf to afford disaccharide 10 in a

moderate 53% yield. Removal of benzylidene protection

group from 10 by hydrogenation followed by S_N2 substitution

Neu5Gc2–3Gal1–3GalNAcProN₃(39)

From disaccharide 5 (46.6 mg, 0.1 mmol), compound 39

was obtained as a white foam (63 mg, 79%). ¹H NMR (600

MHz, D₂O)  4.53 (d, 1 H, J = 7.8 Hz), 4.52 (d, 1 H, J=8.4

Hz), 4.19 (d, 1 H, J = 3.6 Hz), 4.14 (s, 2 H), 4.10 (dd, 1 H,

J = 9.6 Hz and 3.0 Hz), 4.04–3.55 (m, 19 H), 3.41 (t, 2 H,

J = 6.3 Hz), 2.79 (dd, 1 H, J = 12.3 Hz and 4.5 Hz), 2.05 (s,

3 H), 1.87 (pentalet, 2 H, J = 6.3 Hz), 1.82 (t, 1 H, J = 12.3

Hz). ¹³C NMR (150 MHz, D₂O)  175.92, 174.92, 174.16,

104.79, 101.60, 99.88, 80.21, 75.76, 74.97, 74.93, 72.70,

72.07, 69.19, 68.31, 68.17, 68.06, 67.54, 67.20, 62.64,

61.17, 61.14, 51.56, 51.35, 47.99, 39.99, 28.30, 22.50.

HRMS (ESI) m/z calcd for C₂₈H₄₆N₅O₂₀Na (MNa)

772.2731, found 772.2729.

Huang SS, et al. Sci China Chem January (2011) Vol.54 No.1

125

aride Gal1–4GlcNAc6SProN₃(3) in 66% overall yield.

To prepare disaccharides 4 and 5, GalNAc derivative 16

[37] was treated with 3-chloro-1-propanol and acetyl chlo-

ride followed by 4,6-O-benzylidenation to produce a mix-

ture of 17 (21%) and 18 (57%). Coupling of trichloroace-

timidate 9 with 17 and 18, respectively, using TMSOTf as a

promoter produced disaccharides 19 and 20 in 62% and

48% yields, respectively. Substitution of the chlorine by

azide using sodium azide and subsequent removal of ben-

zylidene using acetic acid afforded 21 (94%) and 22 (91%).

Deprotection of 21 and 22 as described for 11 afforded di-

saccharides Gal1–4GalNAcProN₃(4) and Gal1–4GalNAc-

ProN₃(5) in 85% and 76% yields, respectively (Scheme 3).

For the synthesis of type 1 LNT tetrasaccharide 6, com-

pound 23 [38] was deacetylated and selectively protected with

benzylidene to produce 24 (88%) (Scheme 4). Disaccharide

donor 25 was synthesized by coupling the monosaccharide

24 with trichloroacetimidate donor 9 in the presence of

TMSOTf with 77% yield. Glycosylation of 25 with a

Scheme 1 Reagents and yields: (a) (i) NaOMe, MeOH; (ii) PhCH(OMe)₂,

CSA, DMF, 87%; (b) TMSOTf, CH₂Cl₂, 53%; (c) (i) Pd/C, H₂; (ii) NaN₃,

TBAI, DMF, 95%; (d) (i) N₂H₄-H₂O, EtOH; (ii) Ac₂O, pyridine; (iii)

NaOMe, MeOH, 84%.

of the chlorine by azido group gave 11 in 95% yield. Re-

moval of the phthalimido group by treating with hydrazine,

followed by acetylation and deacetylation, the disaccharide

Gal1–3GlcNAcProN₃(1) was produced in 84% yield

over three steps.

Disaccharides 2 and 3 were synthesized as shown in

Scheme 2. Monosaccharide acceptor 12 was synthesized

from 7 in 84% yield by deacetylation followed by selective

silylation at O-6 with tert-butyldimethylsilyl chloride. Cou-

pling of trichloroacetimidate 9 with acceptor 12 was per-

formed in dichloromethane using TMSOTf as a promoter to

give 13 (79%). Removal of the tert-butyldimethylsilyl and

transformation of chlorine to azide afforded 14 in 76% yield.

Deprotection of 14 as described for 11 afforded disaccha-

ride Gal1–4GlcNAcProN₃(2) in 82% yield over three

steps. Acetylation of 13 with Ac₂O in pyridine, followed by

removal of TBS group and transformation of chlorine to

azide produced 15 (85%). Sulfation at C-6 of 15 with

SO₃·Py in pyridine, removal of the 2-N-phthalyl group, fol-

lowed by acetylation and deacetylation produced disacch-

Scheme 3 Reagents and yields: (a) (i) AcCl, 3-chloro-1-propanol; (ii)

PhCH(OMe)₂, CSA, DMF, 17:21%; 18: 57%; (b) 9, TMSOTf, CH₂Cl₂,

19:62%; 20: 48%; (c) (i) NaN₃, TBAI, DMF; (ii) 80% HOAc, 21:94%; 22:

91%; (d) (i) N₂H₄-H₂O, EtOH; (ii) Ac₂O, pyridine; (iii) NaOMe, MeOH,

4:85%; 5: 76%.

Scheme 2 Reagents and yields: (a) (i) NaOMe, MeOH; (ii) TBSCl, imi-

dazole, CH₂Cl₂, 84%; (b) 9, TMSOTf, CH₂Cl₂, 79%; (c) (i) TABF, THF;

(ii) NaN3, TBAI, DMF, 76%; (d) (i) Ac₂O, pyridine; (ii) TABF, THF; (iii)

NaN₃, TBAI, DMF, 85%; (e) (i)) N₂H₄-H₂O, EtOH; (ii) Ac₂O, pyridine; (iii)

NaOMe/MeOH, 82%; (f) (i) Py-SO₃, pyridine; (ii) N₂H₄-H₂O, EtOH; (iii)

Ac₂O, pyridine; (iv) NaOMe, MeOH, 66%.

Scheme 4 Reagents and yields: (a) (i) NaOMe, MeOH; (ii) PhCH(OMe)₂,

CSA, 88%; (b) 9, TMSOTf, CH₂Cl₂, 77%; (c) NIS, TfOH, CH₂Cl₂, 64%; (d)

(i) 80% HOAc; (ii) N₂H₄-H₂O, EtOH; (iii) Ac₂O, pyridine; (iv) NaOMe,

MeOH, 54%.

126

Huang SS, et al. Sci China Chem January (2011) Vol.54 No.1

Figure 2 One-pot three-enzyme synthesis of sialosides containing different sialic acid forms and various internal glycans. Abbreviations: ManNAc,

N-acetylmannosamine; ManNGc, N-glycolylmannosamine; CTP, cytidine 5′-triphosphate. Enzymes: E. coli aldolase, Escherichia coli K12 sialic acid aldolase;

NmCSS, Neisseria meningitidis CMP-sialic acid synthetase; PmST1, Pasteurella multocida sialyltransferase for the formation of 2–3-linked sialosides.

Table 1 2–3-Linked sialosides synthesized using the one-pot three-enzyme system shown in Figure 2

Entry

Acceptor

Sialic acid precursor

　2–3-Linked sialosides (yield %)

a

30 (85%)

ManNAc, 28

1

b

c

d

e

f

ManNGc, 29

31 (83%)

32 (84%)

28

2

3

4

29

28

29

28

33 (80%)

34 (88%)

35 (82%)

36 (87%)

g

h

29

37 (76%)

38 (81%)

i

j

28

29

28

29

5

39 (79%)

40 (86%)

k

l

6

41 (81%)

Huang SS, et al. Sci China Chem January (2011) Vol.54 No.1

127

disaccharide acceptor 26 [39] using NIS–TfOH as the pro-

moters produced tetrasaccharide 27 (64%). Global depro-

tection led to the production of the desired LNT tetrasaccha-

ride Gal1–3GlcNAc1–3Gal1–4GlcProN₃(6) in 54%

yield.

naturally occurring diverse sialic acid-containing carbohydrate

epitopes.

This work was supported by NIH grants R01GM076360 and U01CA128442.

X. Chen is an Alfred P. Sloan Research Fellow, a Camille Dreyfus

Teacher-Scholar, and a UC- Davis Chancellor’s Fellow.

3.2 One-pot three-enzyme approach for the synthesis

of sialylated glycans

1

2

Tsuji S, Datta AK, Paulson JC. Systematic nomenclature for

sialyltransferases. Glycobiology, 1996, 6: v–vii

With chemically synthesized glycan acceptors 1–6 in hands,

the preparation of 2–3-linked sialyl oligosaccharides was

carried out using an efficient one-pot threeenzyme approach

[32] (Figure 2) developed in our laboratory. In this system,

N-acetylmannosamine (ManNAc) or N-glycolylmannosamine

(ManNGc) was coupled with pyruvate to give sialic acids

Neu5Ac or Neu5Gc by a recombinant Escherichza coli K12

sialic acid aldolase-catalyzed reaction [40]. The sialic acid

formed was then activated by a recombinant Neisseria

meningitidis CMP-sialic acid synthetase [40], and trans-

ferred to a suitable sialyltransferase acceptor by a Pas-

teurella multocida multifunctional 2–3-sialyltransferase

[31] for the formation of sialosides.

As shown in Table 1, all six oligosaccharides 1–6 pre-

pared were very good acceptor substrates for Pasteurella

multocida 2–3-sialyltransferase. Sialic acids (Neu5Ac or

Neu5Gc) were successfully transferred to the galactose (Gal)

residue through an 2–3-sialyl linkage by the one-pot three-

enzyme reaction system. Twelve sialosides representing co-

mmon naturally occurring sialic acid-containing carbohydr-

ate epitopes including 2–3-linked sialyl type 1 Neu5Ac2–

3Gal1–3GlcNAcProN₃(30, 85%) and Neu5Gc2–3Gal1–

3GlcNAcProN₃(31, 83%); type 2 Neu5Ac2–3Gal1–

4GlcNAcProN₃(32, 84%), Neu5Gc2–3Gal1–4GlcNAc-

ProN₃(33, 80%), Neu5Ac2–3Gal1–4GlcNAc6SProN₃

(34, 88%), and Neu5Gc2–3Gal1–4GlcNAc6SProN₃(35,

82%); type 3 Neu5Ac2–3Gal1–3GalNAcProN₃(36,

87%) and Neu5Gc2–3Gal1–3GalNAcProN₃(37, 76%);

type 4 Neu5Ac2–3Gal1–3GalNAcProN₃(38, 81%), and

Neu5Gc2–3Gal1–3GalNAcProN₃(39, 79%) glycans

as well as 2–3-linked sialyllacto-N-tetraose Neu5Ac2–

3Gal1–3GlcNAc1–3Gal1–4GlcProN₃(40, 86%) and

Neu5Gc2–3Gal1–3GlcNAc1–3Gal1–4GlcProN₃(41,

81%) were obtained in high yields.

Fukuda MN, Dell A, Oates JE, Fukuda M. Embryonal lactosaminoglycan.

The structure of branched lactosaminoglycans with novel disialosyl

(sialyl alpha 2–9 sialyl) terminals isolated from PA1 human

embryonal carcinoma cells. J Biol Chem, 1985, 260: 6623–6631

Schauer R. Achievements and challenges of sialic acid research.

Glycoconj J, 2000, 17: 485–499

Angata T, Varki A. Chemical diversity in the sialic acids and related

alpha-keto acids: An evolutionary perspective. Chem Rev, 2002, 102:

439–469

3

4

5

6

Chen X, Varki A. Advances in the biology and chemistry of sialic

acids. ACS Chem Biol, 2010, 5: 163–176

Tomiya N, Narang S, Lee YC, Betenbaugh MJ. Comparing N-glycan

processing in mammalian cell lines to native and engineered

lepidopteran insect cell lines. Glycoconj J, 2004, 21: 343–360

Betenbaugh MJ, Tomiya N, Narang S, Hsu JT, Lee YC. Biosynthesis

of human-type N-glycans in heterologous systems. Curr Opin Struct

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Article Doi

Chemoenzymatic synthesis of α2-3-sialylated carbohydrate epitopes

DOI: 10.1007/s11426-010-4175-9

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Full text of DOI:10.1007/s11426-010-4175-9

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