An Improved Helferich Method for the α/β-Stereoselective Synthesis of 4-Methylumbelliferyl Glycosides for the Detection of Microorganisms

An improved Helferich method is presented. It involves the glycosylation of 4-methyl-umbelliferone with glycosyl acetates in the presence of boron trifluoride etherate combined with triethylamine, pyridine, or 4-dimethylaminopyridine under mild conditions, followed by deprotection to give fluorogenic 4-methylumbelliferyl glycoside substrates. Due to the use of base, the glycosylation reaction proceeds more easily, is uncommonly α- or β-stereoselective, and affords the corresponding products in moderate to excellent yields (51%–94%) under appropriate conditions.


Introduction
The detection and identification of microorganisms in food, such as prevalent pathogens, e.g., Salmonella and enterohemorrhagic Escherichia coli, is essential for realizing and managing microbiological risks to ensure food safety, a worldwide public health concern [1][2][3]. New techniques that are faster and simpler, based on synthetic enzymatic substrates, have been developed and drawn wide attention in recent decades. Synthetic enzymatic substrates are powerful tools in biochemistry that can produce an easily measured output, such as variation of absorbance or fluorescence, to facilitate the detection of enzymatic activities [4][5][6][7][8][9]. For example, 4-methylumbelliferyl glycosides have been widely exploited in diagnostic microbiology [4][5][6]9], in newborn screening of lysosomal storage disorders (LSDs) [10][11][12][13], for the prediction of glycan structures and potential bioactivities of bovine milk [14], characterizing and identifying vegetables [15], and investigating the molecular mechanisms involved in sperm-oocyte binding and gamete-oviductal epithelium interactions [16], by monitoring the specific cellular glycosidases activities to the substrates. These compounds have low toxicity, are stable under physiological conditions, and are easily hydrolyzed by corresponding glycosidase  Table 2 also suggests that other products were generated in the reactions, as indicated by the fact that the total combined yield of 3a, yield of recovered 1 and yield of by-product 5 ( Figure 1) ranged from 65% to 75%. Herein, lots of small equivalent tests were performed in order to more conveniently and more quickly know the main glycosylation product and its yield and investigate multiple but different factors to improve the yield. Considering that the original Helferich procedure with methyl tetra-O-acetyl-β-D-glucopyranuronate was stereochemically reliable, giving only β-D-glucuronide (3a) [36], we did not search for the other anomer glycosylation product, perhaps generated but in a low yield below the limits of our detection, and other by-products.
Molecules 2015, 20, page-page 5 Table 2 also suggests that other products were generated in the reactions, as indicated by the fact that the total combined yield of 3a, yield of recovered 1 and yield of by-product 5 ( Figure 1) ranged from 65% to 75%. Herein, lots of small equivalent tests were performed in order to more conveniently and more quickly know the main glycosylation product and its yield and investigate multiple but different factors to improve the yield. Considering that the original Helferich procedure with methyl tetra-O-acetyl-β-D-glucopyranuronate was stereochemically reliable, giving only β-D-glucuronide (3a) [36], we did not search for the other anomer glycosylation product, perhaps generated but in a low yield below the limits of our detection, and other by-products.

Synthesis of Other Protected Glycosides 3b-3f
Based on the abovementioned success, the glycosylation of 4-MU with some other peracetyl sugar donors: peracetyl glucopyranose (2b), peracetyl galactopyranose (2c), peracetyl mannopyranose (2d), peracetyl xylopyranose (2e) [42] and peracetyl ribofuranose (2f), was also investigated (Scheme 3). In view of the differences between the different peracetyl sugars in aspects such as reactivity and stability, we speculated that the abovementioned optimal conditions may not be optimal for other peracetyl sugars. Hence multiple conditions including a TEA or pyridine to BF3·OEt2 ratio of 0.2 referred to Lee et al.'s method [37] were used to study the glycosylation as follows.
As summarized in Table 3, all reactions proceeded smoothly and gave the glycoside products in moderate to excellent yields. In the presence of a relatively lower amount of BF3·OEt2 and DMAP, the glycosidation of 1 with 2b afforded mainly the protected β-D anomer product (3b1) in 36% yield, and the use of a higher amount of pyridine gave 3b1 in 42% yield (entries 1 and 2).

Synthesis of Other Protected Glycosides 3b-3f
Based on the abovementioned success, the glycosylation of 4-MU with some other peracetyl sugar donors: peracetyl glucopyranose (2b), peracetyl galactopyranose (2c), peracetyl mannopyranose (2d), peracetyl xylopyranose (2e) [42] and peracetyl ribofuranose (2f), was also investigated (Scheme 3). In view of the differences between the different peracetyl sugars in aspects such as reactivity and stability, we speculated that the abovementioned optimal conditions may not be optimal for other peracetyl sugars. Hence multiple conditions including a TEA or pyridine to BF 3¨O Et 2 ratio of 0.2 referred to Lee et al.'s method [37] were used to study the glycosylation as follows.
As summarized in Table 3, all reactions proceeded smoothly and gave the glycoside products in moderate to excellent yields. In the presence of a relatively lower amount of BF 3¨O Et 2 and DMAP, the glycosidation of 1 with 2b afforded mainly the protected β-D anomer product (3b 1 ) in 36% yield, and the use of a higher amount of pyridine gave 3b 1 in 42% yield (entries 1 and 2). When a relatively high amount of BF3·OEt2 and DMAP was used, the reaction gave 3b1 in 45% yield; In addition, the protected α-D anomer product (3b2) was unexpectedly obtained in a low yield of 7% (entry 3). The glycosidation of 1 with 2b in the absence of base afforded no products by TLC analysis (entry 4). We expected the glycosylation procedure to be β-stereoselective for the peracetyl sugars, but this was changed by the results obtained with 2c. Compound 2c afforded mainly the protected α-D-galactopyranoside product (3c) in 54% yield at 60 °C, and 17% yield at 0 °C in the presence of TEA, and 56% yield at 60 °C in the presence of DMAP, respectively (entries 5-7). In view of the facts that an excess of peracetyl sugar donors causes purification difficulties and an excess of 4-MU could be removed easily by aqueous sodium hydroxide solution, and that 2d should have relatively high reactivity, we investigated the glycosylation of excess of 4-MU with 2d as a mixture of α-and β-D anomers in the presence of pyridine. This case gave mainly the protected α-D-mannopyranoside product 3d in 59% yield that was very pure after three recrystallizations (entry 8). Using an excess of 2d in the presence of DMAP gave 3d in a slight higher yield of 62% (entry 9). these two 4-methylumbelliferyl α-D-pyranosides were also obtained by the early Helferich method: in 1970 Vervoot et al. reported that the condensation of 4-MU with α-D-mannose pentaacetate by a method using fusion treatment and a zinc chloride catalyst under diminished pressure gave the protected α-D-mannopyranoside (3d) in 37% yield [28]. In 1978 Courtin-Duchateau et al. reported that the condensation of 4-MU with α-D-galactose pentaacetate (2c) in boiling xylene in the presence of zinc chloride gave a mixture of protected α-D-galactopyranoside, protected β-D-galactofuranoside and protected β-D-galactopyranoside in a ratio of 3:5:7 in 30% total yield; and condensation of the O-trimethylsilyl derivative of 4-MU with β-D-galactose pentaacetate (2c) in the presence of stannic chloride gave a mixture of α and β-Dgalactopyranoside in a ratio of 15:4 in low total yield of 19%. The protected α-D-mannopyranoside 3d and α-D-galactopyranoside 3c could also be obtained stereoselectively by condensation of the sodium salt of 4-MU with the corresponding O-acetylated glycosyl chlorides in hexamethylphosphoric triamide, but the reaction time was long (a few days) and the products were obtained in only 30% and 47% yields, respectively [27]. Compared with the reported methods mentioned above, the improved method for 3d and 3c here shows higher efficiency, higher yields and high stereoselectivity. 4-Methylumbelliferyl β-D-xylopyranoside (4e) was prepared in 1965 by De Bruyne et al., who reported a 35% yield by the Michael condensation of acetobromoxylose with 4-MU in acetone/water [43]. Here the improved Helferich condensation of β-D-xylose tetraacetate (2e) with 4-MU at room temperature (20-27 °C) When a relatively high amount of BF 3¨O Et 2 and DMAP was used, the reaction gave 3b 1 in 45% yield; In addition, the protected α-D anomer product (3b 2 ) was unexpectedly obtained in a low yield of 7% (entry 3). The glycosidation of 1 with 2b in the absence of base afforded no products by TLC analysis (entry 4). We expected the glycosylation procedure to be β-stereoselective for the peracetyl sugars, but this was changed by the results obtained with 2c. Compound 2c afforded mainly the protected α-D-galactopyranoside product (3c) in 54% yield at 60˝C, and 17% yield at 0˝C in the presence of TEA, and 56% yield at 60˝C in the presence of DMAP, respectively (entries 5-7). In view of the facts that an excess of peracetyl sugar donors causes purification difficulties and an excess of 4-MU could be removed easily by aqueous sodium hydroxide solution, and that 2d should have relatively high reactivity, we investigated the glycosylation of excess of 4-MU with 2d as a mixture of αand β-D anomers in the presence of pyridine. This case gave mainly the protected α-D-mannopyranoside product 3d in 59% yield that was very pure after three recrystallizations (entry 8). Using an excess of 2d in the presence of DMAP gave 3d in a slight higher yield of 62% (entry 9). these two 4-methylumbelliferyl α-D-pyranosides were also obtained by the early Helferich method: in 1970 Vervoot et al. reported that the condensation of 4-MU with α-D-mannose pentaacetate by a method using fusion treatment and a zinc chloride catalyst under diminished pressure gave the protected α-D-mannopyranoside (3d) in 37% yield [28]. In 1978 Courtin-Duchateau et al. reported that the condensation of 4-MU with α-D-galactose pentaacetate (2c) in boiling xylene in the presence of zinc chloride gave a mixture of protected α-D-galactopyranoside, protected β-D-galactofuranoside and protected β-D-galactopyranoside in a ratio of 3:5:7 in 30% total yield; and condensation of the O-trimethylsilyl derivative of 4-MU with β-D-galactose pentaacetate (2c) in the presence of stannic chloride gave a mixture of α and β-D-galactopyranoside in a ratio of 15:4 in low total yield of 19%. The protected α-D-mannopyranoside 3d and α-D-galactopyranoside 3c could also be obtained stereoselectively by condensation of the sodium salt of 4-MU with the corresponding O-acetylated glycosyl chlorides in hexamethylphosphoric triamide, but the reaction time was long (a few days) and the products were obtained in only 30% and 47% yields, respectively [27]. Compared with the reported methods mentioned above, the improved method for 3d and 3c here shows higher efficiency, higher yields and high stereoselectivity. 4-Methylumbelliferyl β-D-xylopyranoside (4e) was prepared in 1965 by De Bruyne et al., who reported a 35% yield by the Michael condensation of acetobromoxylose with 4-MU in acetone/water [43]. Here the improved Helferich condensation of β-D-xylose tetraacetate (2e) with 4-MU at room temperature (20-27˝C) gave mainly the protected β-D-xylopyranoside (3e) in 73% and 69% yields in the presence of TEA and DMAP, respectively, which is more than about twice as high as De Bruyne et al.'s method (entries 10 and 11); it should also be noted that the reaction gave a very low yield at 50-60˝C by comparative TLC analysis. 4-Methyl-umbelliferyl β-D-ribofuranoside (4f) was prepared in 1997 by Schramm et al., who reported just a 25% yield using a variant of the Koenigs-Knorr condensation of O-benzoylated ribofuranosyl chloride with the silver salt of 4-MU in toluene under reflux [44]. In this study, the condensation of β-D-ribofuranose tetraacetate (2f) with 4-MU at room temperature (20-27˝C) afforded mainly the protected β-D-ribofuranoside 3f in much higher yields of 94% and 77% in the presence of TEA and DMAP, respectively (entries 12 and 13). Moreover, the method used here was far simpler. The use of DMAP gave lower yields than of TEA for glycosylation with 3e and 3f, which suggested that optimal conditions of glycosylation of 4-MU with different glycosyl acetate donors should be different. And here comparison between multiple conditions used was necessary and helpful.
An additional noteworthy detail was the following: the cooled reaction mixtures were directly quenched and separated by column chromatography (silica gel, 200-300 mesh) with a view to preventing 4-MU from dissolving in water or aqueous alkaline solution to determine its recovery yield. This reaction post-processing was relative simpler, however, it may be affected by different silica gels of different brands, due to the fact that the single use of Macklin silica gel unexpectedly gave a remarkable lower yield of 3a compared with any of numerous examples using Haiyang silica gel, all other conditions being basically equal.
An explanation for the above stereoselective glycosylation is presented as follows: it is reported that base can abstract the proton quickly from the adduct intermediate cation generated via phenol attack on the acetyloxonium ion intermediate in the glucosidation and prevent anomerization of the β-anomer to the α-anomer [45]. Here base should also have a role in preventing anomerization in the glycosylation of 4-MU with glycosyl acetates. Besides, one of the most powerful principles of the enforced 1,2-trans glycosylation is neighboring group participation by the acyl group at C-2 (generation of the intermediate acyloxonium ion) [46,47] that can be applied to further explain the selective formation of the protected glycosides 3a, 3b 1 , 3d, 3e, and 3f. However, how can the selective formation of the protected α-D-galactopyranoside 3c that is a 1,2-cis glycoside be explained? It is reported that the acyl group at C-4 of a galactosyl donor with a non-participating substitute at C-2 can effect a remote participation effect that is beneficial for the formation of 1,2-cis galactosidic bonds [46,48,49]. Therefore, the selective formation of 3c may be attributed to the participation of the acetyl group at C-2 followed by participation of the one at C-4 due to the fact the reactivity of 4-MU may be weaker compared to the acetyl groups at C-2 and C-4 of β-D-galactose pentaacetate, beside its role as base.

Deprotection of the Protected Glycosides 3a-3f
The deprotection step for glucuronides is different from that of other glycosides. In particular, there are more problems that need to be considered in order to obtain the free glucuronic acid. Due to the possibility of elimination as a side reaction, generating ∆ 4,5 -(dehydro) glucuronide, and opening of the umbelliferone lactone, many deprotection methods including chemical and enzymatic treatments have been used, as well as the hydrolysis of Na 2 CO 3 in aqueous MeOH, followed by desalting using a cation exchange resin or acidification followed by reverse-phase column chromatography treatment [34,36,[50][51][52][53][54]. Here, a modified method for the deprotection of 3a was used (Scheme 4). Namely, using excess of Ba(OH) 2 hydrate in aqueous MeOH in an ice water bath, the barium salt of 4a was obtained, that was insoluble in MeOH. This was then acidified using H 2 C 2 O 4 hydrate in fresh MeOH, which can improve the practicality of the procedure by avoiding the difficulty of evaporating the product solution containing a lot of water.
The entries 3, 7, 9, 11 and 13 were performed according to the procedure in Table 2, and the recovery yield of 1 and the yield of by-product 5 were determined by isolation and HPLC analysis; b Isolated yield; c The main anomer was determined by TLC analysis and isolation; d The reaction gave α-D anomer product (3b 2 ) and by-product 5 in 7% and 16% yields, respectively, and the recovery yield of 1 was 8%; e None; f The reaction gave by-product 5 in 15% yield, and the recovery yield of 1 was 9%; g The reaction gave by-product 5 in 12% yield, and the recovery yield of 1 was 8%; h The reaction gave by-product 5 in 3% yield, and the recovery yield of 1 was 13%; i The reaction gave no by-product 5, and the recovery yield of 1 was 20%.

General Information
Methyl tetra-O-acetyl-β-D-glucopyranuronate (2a), and β-D-xylopyranose tetraacetate (2e) were prepared according to the references [38,42], respectively. α/β-D-Mannopyranose pentaacetate (2d) was prepared as specified in this section. Other reagents and all organic solvents were purchased from commercial sources and were of analytical reagent grade or contained the desired chemical in a purity of more than 97%. Those that were used as reaction solvents were dried prior to use. Petroleum ether (PE) refers to the fraction boiling in the 60-90 °C range. TLC was performed using silica gel GF-254 plates (purchased from Qingdao Haiyang Chemical Co., Ltd., Qingdao, China) with detection by iodine fumigation or 15% H2SO4-EtOH/heating or UV (254 nm and 365 nm) (Shanghai JiaPeng Technology Co., Ltd., Shanghai, China), or charring with 20% H2SO4 in EtOH. Column chromatography was performed on silica gel (200-300 mesh, purchased from Qingdao Haiyang Chemical Co., Ltd., Qingdao, China) using a PE-EtOAc system as eluent. Organic solutions were distilled on a rotary evaporator at 35-40 °C or 40-45 °C. 1 H-and 13 C-NMR spectra were recorded on 300 and 500 MHz NMR spectrometers (Bruker (Beijing) Scientific Technology Co. Ltd., Beijing, China). 1 H-NMR spectra were recorded at 300 or 500 MHz in CDCl3 or DMSO-d6 solvent. 13

General Information
Methyl tetra-O-acetyl-β-D-glucopyranuronate (2a), and β-D-xylopyranose tetraacetate (2e) were prepared according to the references [38,42], respectively. α/β-D-Mannopyranose pentaacetate (2d) was prepared as specified in this section. Other reagents and all organic solvents were purchased from commercial sources and were of analytical reagent grade or contained the desired chemical in a purity of more than 97%. Those that were used as reaction solvents were dried prior to use. Petroleum ether (PE) refers to the fraction boiling in the 60-90˝C range. TLC was performed using silica gel GF-254 plates (purchased from Qingdao Haiyang Chemical Co., Ltd., Qingdao, China) with detection by iodine fumigation or 15% H 2 SO 4 -EtOH/heating or UV (254 nm and 365 nm) (Shanghai JiaPeng Technology Co., Ltd., Shanghai, China), or charring with 20% H 2 SO 4 in EtOH. Column chromatography was performed on silica gel (200-300 mesh, purchased from Qingdao Haiyang Chemical Co., Ltd., Qingdao, China) using a PE-EtOAc system as eluent. Organic solutions were distilled on a rotary evaporator at 35-40˝C or 40-45˝C. 1 H-and 13 C-NMR spectra were recorded on 300 and 500 MHz NMR spectrometers (Bruker (Beijing) Scientific Technology Co. Ltd., Beijing, China). 1 H-NMR spectra were recorded at 300 or 500 MHz in CDCl 3 or DMSO-d 6 solvent. 13 C-NMR spectra were recorded at 75 MHz in CDCl 3 or DMSO-d 6 solvent. HRMS spectra were recorded on an ultrahigh-resolution quadrupole time-of-flight (UHR-Q-TOF) mass spectrometer (Bruker (Beijing) Scientific Technology Co. α/β-D-Mannopyranose pentaacetate (2d) Acetic anhydride (25 mL) and pyridine (20 mL) were added successively under magnetic stirring to D-(+)-mannose (5.00 g, 27.75 mmol) in an ice-water bath. After approximately 4 h, a clear and colorless liquid was obtained and stirring in an ice-water bath was continued for 20 h. Distilled water (55 mL) was then added. After stirring for approximately 5 h, the mixture was left to stand at 4 °C in a refrigerator for 24 h, then was extracted with CH2Cl2 (45 mL). The organic phase was washed successively with dilute HCl (1 M), saturated aqueous NaHCO3, distilled water and saturated aqueous NaCl, dried with anhydrous Na2SO4. After removing the solvent under reduced pressure at 35-40 °C, a crude product was obtained and further dried in a vacuum desiccator. The constant weight crude product was a clear, colorless and sticky syrup (10.51 g, 97%), and it was used directly for glycosylation.

General Procedure for the Glycosylation Step
To a mixture of 4-MU (1.0-6.0 mmol) and glycosyl acetate (1.0-4.0 mmol) under an argon atmosphere, dry solvent was added successively, followed by the corresponding molar equivalents of base and Lewis acid. The mixture was stirred for a set amount of time at a certain temperature (as shown in Tables 1-3). Then, an equal volume of CH2Cl2 was added to dilute the reaction mixture, and the reaction was quenched with saturated aqueous NaHCO3. The organic phase was washed with diluted aqueous NaOH (1 M) until the aqueous phase was a light brownish-yellow or almost colorless, then washed successively with distilled water, saturated aqueous NaCl, dried with anhydrous Na2SO4, and the solvent was removed under reduced pressure. The crude product was purified by flash column chromatography (silica gel, 200-300 mesh, PE/EtOAc, 5/2), then the desired product was crystallized from anhydrous ethyl ether and dried, or the crude product was purified by several recrystallizations from ethanol. α/β-D-Mannopyranose pentaacetate (2d) Acetic anhydride (25 mL) and pyridine (20 mL) were added successively under magnetic stirring to D-(+)-mannose (5.00 g, 27.75 mmol) in an ice-water bath. After approximately 4 h, a clear and colorless liquid was obtained and stirring in an ice-water bath was continued for 20 h. Distilled water (55 mL) was then added. After stirring for approximately 5 h, the mixture was left to stand at 4 °C in a refrigerator for 24 h, then was extracted with CH2Cl2 (45 mL). The organic phase was washed successively with dilute HCl (1 M), saturated aqueous NaHCO3, distilled water and saturated aqueous NaCl, dried with anhydrous Na2SO4. After removing the solvent under reduced pressure at 35-40 °C, a crude product was obtained and further dried in a vacuum desiccator. The constant weight crude product was a clear, colorless and sticky syrup (10.51 g, 97%), and it was used directly for glycosylation.

General Procedure for the Glycosylation Step
To a mixture of 4-MU (1.0-6.0 mmol) and glycosyl acetate (1.0-4.0 mmol) under an argon atmosphere, dry solvent was added successively, followed by the corresponding molar equivalents of base and Lewis acid. The mixture was stirred for a set amount of time at a certain temperature (as shown in Tables 1-3). Then, an equal volume of CH2Cl2 was added to dilute the reaction mixture, and the reaction was quenched with saturated aqueous NaHCO3. The organic phase was washed with diluted aqueous NaOH (1 M) until the aqueous phase was a light brownish-yellow or almost colorless, then washed successively with distilled water, saturated aqueous NaCl, dried with anhydrous Na2SO4, and the solvent was removed under reduced pressure. The crude product was purified by flash column chromatography (silica gel, 200-300 mesh, PE/EtOAc, 5/2), then the desired product was crystallized from anhydrous ethyl ether and dried, or the crude product was purified by several recrystallizations from ethanol.  α/β-D-Mannopyranose pentaacetate (2d) Acetic anhydride (25 mL) and pyridine (20 mL) were added successively under magnetic stirring to D-(+)-mannose (5.00 g, 27.75 mmol) in an ice-water bath. After approximately 4 h, a clear and colorless liquid was obtained and stirring in an ice-water bath was continued for 20 h. Distilled water (55 mL) was then added. After stirring for approximately 5 h, the mixture was left to stand at 4 °C in a refrigerator for 24 h, then was extracted with CH2Cl2 (45 mL). The organic phase was washed successively with dilute HCl (1 M), saturated aqueous NaHCO3, distilled water and saturated aqueous NaCl, dried with anhydrous Na2SO4. After removing the solvent under reduced pressure at 35-40 °C, a crude product was obtained and further dried in a vacuum desiccator. The constant weight crude product was a clear, colorless and sticky syrup (10.51 g, 97%), and it was used directly for glycosylation.

General Procedure for the Glycosylation Step
To a mixture of 4-MU (1.0-6.0 mmol) and glycosyl acetate (1.0-4.0 mmol) under an argon atmosphere, dry solvent was added successively, followed by the corresponding molar equivalents of base and Lewis acid. The mixture was stirred for a set amount of time at a certain temperature (as shown in Tables 1-3). Then, an equal volume of CH2Cl2 was added to dilute the reaction mixture, and the reaction was quenched with saturated aqueous NaHCO3. The organic phase was washed with diluted aqueous NaOH (1 M) until the aqueous phase was a light brownish-yellow or almost colorless, then washed successively with distilled water, saturated aqueous NaCl, dried with anhydrous Na2SO4, and the solvent was removed under reduced pressure. The crude product was purified by flash column chromatography (silica gel, 200-300 mesh, PE/EtOAc, 5/2), then the desired product was crystallized from anhydrous ethyl ether and dried, or the crude product was purified by several recrystallizations from ethanol. α/β-D-Mannopyranose pentaacetate (2d) Acetic anhydride (25 mL) and pyridine (20 mL) were added successively under magnetic stirring to D-(+)-mannose (5.00 g, 27.75 mmol) in an ice-water bath. After approximately 4 h, a clear and colorless liquid was obtained and stirring in an ice-water bath was continued for 20 h. Distilled water (55 mL) was then added. After stirring for approximately 5 h, the mixture was left to stand at 4˝C in a refrigerator for 24 h, then was extracted with CH 2 Cl 2 (45 mL). The organic phase was washed successively with dilute HCl (1 M), saturated aqueous NaHCO 3 , distilled water and saturated aqueous NaCl, dried with anhydrous Na 2 SO 4 . After removing the solvent under reduced pressure at 35-40˝C, a crude product was obtained and further dried in a vacuum desiccator. The constant weight crude product was a clear, colorless and sticky syrup (10.51 g, 97%), and it was used directly for glycosylation.

General Procedure for the Glycosylation Step
To a mixture of 4-MU (1.0-6.0 mmol) and glycosyl acetate (1.0-4.0 mmol) under an argon atmosphere, dry solvent was added successively, followed by the corresponding molar equivalents of base and Lewis acid. The mixture was stirred for a set amount of time at a certain temperature (as shown in Tables 1-3). Then, an equal volume of CH 2 Cl 2 was added to dilute the reaction mixture, and the reaction was quenched with saturated aqueous NaHCO 3 . The organic phase was washed with diluted aqueous NaOH (1 M) until the aqueous phase was a light brownish-yellow or almost colorless, then washed successively with distilled water, saturated aqueous NaCl, dried with anhydrous Na 2 SO 4 , and the solvent was removed under reduced pressure. The crude product was purified by flash column chromatography (silica gel, 200-300 mesh, PE/EtOAc, 5/2), then the desired product was crystallized from anhydrous ethyl ether and dried, or the crude product was purified by several recrystallizations from ethanol.     4′-Methylumbelliferyl 2,3,4,6-tetra-O-acetyl-β-D-glucopyranoside (3b1) Compound 3b1 was prepared according to the general procedure for the glycosylation step using 4-MU (176 mg, 1.0 mmol), glycosyl acetate (781 mg, 2.0 mmol, 2.0 equiv.), dry ClCH2CH2Cl (3 mL), corresponding molar equivalents of pyridine and BF3·OEt2. The mixture was stirred for 5 h at 60 °C (as shown in Table 3     4′-Methylumbelliferyl 2,3,4,6-tetra-O-acetyl-α-D-galactopyranoside (3c) Compound 3c was prepared according to the general procedure for the glycosylation step using 4-MU (352 mg, 2.0 mmol), β-D-galactose pentaacetate (1561 mg, 4.0 mmol, 2.0 equiv.), dry ClCH2CH2Cl (6 mL), TEA (700 μL, 5.0 mmol, 2.5 equiv.) and BF3·OEt2 (3218 μL, 25 mmol, 12.5 equiv.). The mixture was stirred for 5 h at 60 °C (as shown in Table 3). The crude product was purified by flash column chromatography.   Table 3). The crude product was purified by flash column chromatography. White powder; yield: 548 mg (54%); mp: 176-180˝C; 0.75mmol, 3 equiv) in MeOH (6 mL) and distilled water (2.4 mL) in an ice-water bath under an argon atmosphere was added compound 3a (123 mg, 0.25 mmol). The mixture was stirred for 4 h in an ice-water bath, then glacial acetic acid was added carefully into to adjust the pH value to 7.5-8.0. The straw yellow solid precipitation was filtered off, washed with MeOH, then further purified by recrystallization using MeOH, and dried. The obtained barium salt product (105 mg) was added to fresh MeOH (5 mL) in an ice-water bath again, acidified with H2CO4·2H2O (20 mg, 0.16 mmol). After stirring for 0.5 h, the mixture was filtered. The filter residue was washed with 2-3 mL MeOH, and the merged filtrate was evaporated to a syrup under reduced pressure. The final product was crystallized from anhydrous ether, washed with a small account of cold acetone, and dried. White 4-Methylumbelliferyl β-D-glucopyranosiduronic acid (4a) To a stirred suspension of Ba(OH) 2¨H2 O (142 mg, 0.75mmol, 3 equiv) in MeOH (6 mL) and distilled water (2.4 mL) in an ice-water bath under an argon atmosphere was added compound 3a (123 mg, 0.25 mmol). The mixture was stirred for 4 h in an ice-water bath, then glacial acetic acid was added carefully into to adjust the pH value to 7.5-8.0. The straw yellow solid precipitation was filtered off, washed with MeOH, then further purified by recrystallization using MeOH, and dried. The obtained barium salt product (105 mg) was added to fresh MeOH (5 mL) in an ice-water bath again, acidified with H 2 CO 4¨2 H 2 O (20 mg, 0.16 mmol). After stirring for 0.5 h, the mixture was filtered. The filter residue was washed with 2-3 mL MeOH, and the merged filtrate was evaporated to a syrup under reduced pressure. The final product was crystallized from anhydrous ether, washed with a small account of cold acetone, and dried. White powder; yield: 42 mg (47%); mp: 140-144˝C;