An improved helferich method for the α/β-stereoselective synthesis of 4-methylumbelliferyl glycosides for the detection of microorganisms

Article abstract of DOI:10.3390/molecules201219789

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.

Full text of DOI:10.3390/molecules201219789

Article
An Improved Helferich Method for the
α/β-Stereoselective Synthesis of
4-Methylumbelliferyl Glycosides for
the Detection of Microorganisms
Xianhu Wei 1,2,3,4,5, Yanxia Ma 1,2,3,4,5, Qingping Wu 2,3,4,*, Jumei Zhang 2,3,4, Zhihe Cai 6
and Mianfei Lu 6
Received: 15 September 2015 ; Accepted: 26 November 2015 ; Published: 4 December 2015
Academic Editor: Vito Ferro
1
Guangzhou Institute of Chemistry, Chinese Academy of Sciences, Guangzhou 510650, China;
wxhu7508@163.com (X.W.); mayanxia880626@163.com (Y.M.)
State Key Laboratory of Applied Microbiology Southern China, Guangdong Institute of Microbiology,
2
Guangzhou 510070, China; zhangjm926@126.com
Guangdong Open Laboratory of Applied Microbiology, Guangdong Institute of Microbiology,
3
Guangzhou 510070, China
Guangdong Provincial Key Laboratory of Microbial Culture Collection and Application,
4
Guangdong Institute of Microbiology, Guangzhou 510070, China
University of Chinese Academy of Sciences, Beijing 100039, China
Guangdong Huankai Microbial Sci. & Tech. Co., Ltd., Guangzhou 510663, China;
5
6
caizhihe1971@163.com (Z.C.); lumianf@163.com (M.L.)
*
Correspondence: wuqp203@163.com; Tel./Fax: +86-20-8768-8132
Abstract: 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.
Keywords: fluorogenic substrates; 4-methylumbelliferyl glycosides; stereoselective glycosylation;
glycosyl acetates; peracetyl sugars
1. 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 [1–3]. 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 [
been widely exploited in diagnostic microbiology [
disorders (LSDs) [10 13], for the prediction of glycan structures and potential bioactivities of bovine
4–
9]. For example, 4-methylumbelliferyl glycosides have
4–6,9
], in newborn screening of lysosomal storage
–
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
Molecules 2015, 20, 21681–21699; doi:10.3390/molecules201219789
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Molecules 2015, 20, 21681–21699
catalysis, which induces strong fluorescence generated by the released 4-methylumbelliferone (4-MU;
7-hydroxy-4-methylcoumarin). As such, they are also considered as ideal molecular probes for
glycobiological studies in which a glycosidase activity needs to be assessed in vitro or in situ research
settings [17,18].
Although many such kinds of fluorogenic glycosidase substrates are commercially available, their
syntheses are not necessarily straightforward or highly efficient. According to the literature, they are
mainly produced by O-glycosylation followed by O-deacetylation. The methods of O-glycosylation
primarily include: (1) Michael-type glycosylation with acetylated glycosyl halides [19
glycosylation with glycosyl acetates via a fusion procedure, in boiling xylene in the presence of
zinc chloride, or using a stannic chloride catalyst, as in the O-trimethylsilylation of 4-MU [27 28];
(3) Koenigs-Knorr glycosylation with glycosyl halides [18 29 31]. As well as the methods mentioned
above, 4-methylumbelliferyl glucuronides can be produced via direct oxidation of the primary hydroxyl
group of 4-methylumbelliferyl glucopyranoside [27 32], indirect oxidation of the primary hydroxyl
group of 4-methylumbelliferyl-2,3,4-tri-O-acetyl-glucopyranoside followed by deprotection [33],
and Schmidt glycosylation with trichloroacetimidate [34 35]. However, these methods suffer from
drawbacks that limit their wider or practical application. For example, Michael-type glycosylations in
acetone/water often afford low yields [20 23]; and although relatively higher yields can be obtained
under biphasic conditions, as shown in our previous study on the synthesis of 4-methylumbelliferyl
-D-galactopyranoside [24], it is incompatible with glycosyl halides which easily transform into glycals
by hydrogen halide elimination under basic conditions, e.g., acetobromomethylglucuronate [26 36].
–27]; (2) Helferich
,
,
–
,
,
–
β
,
Silver compounds used as catalysts in Koenigs-Knorr glycosylation are relatively expensive. Compared
with other methods especially oxidation and Schmidt glycosylation, glycosyl acetates as glycosyl
donors or precursors in the Helferich procedure are more easily available. Therefore, an improved
Helferich procedure comprising other advantages, such as stereoselectivity, relatively high yield and
easy operation, would be extremely pertinent.
Lee et al. reported that the glycosylation of phenol and several substituted phenols with
penta-O-acetyl-
base afforded the products in both high yield and β-stereoselectivity [37]. Herein, we report a modified
Helferich procedure for the successful glycosylation of 4-MU with methyl tetra-O-acetyl- -D-
β-D-glucose in the presence of boron trifluoride etherate (BF3
¨
OEt2) and organic
β
glucopyranuronate and several other peracetylated sugars to afford the corresponding protected
glycosides with high stereoselectivity under mild conditions, followed by deprotection to produce
4-methylumberlliferyl glycosides products (Scheme 1).
CH3
CH3
O
Lewis acid,
organic base
O
OAc
O
+
O
O
O
OAc
HO
O
OAc
2
3
1
deprotection
CH3
O
O
O
O
OH
4
Scheme 1. Synthesis of 4-methylumbelliferyl glycosides.
2. Results and Discussion
Glucopyranuronides are usually more difficult to prepare than glucopyranosides. There is,
in fact, a glycosidation “league table” drawn up by Schmidt et al., ranking in increasing ease of
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Molecules 2015, 20, 21681–21699
glycosyl donation: glycuronates < aldoses < deoxy sugars < ketoses < 3-deoxy-2-glyculosonates [36].
Of all common sugars, glycuronates therefore require the highest activation for a given aglycone.
We envisioned that successful application of the Helferich procedure to the synthesis of
4-methyl-umbelliferyl glucopyranuronide, which has not been previously reported to the best of
our knowledge, could be more widely applied to this class of molecules.
2.1. Synthesis of the Protected β-D-Glucopyranuronide 3a
In view of this, we started our investigation by using methyl tetra-O-acetyl-β-D-glucopyranuronat
(2a) [38] as a model substrate in the glycosylation of 4-MU (Scheme 2).
e
Scheme 2. Synthesis of the protected β-D-glucopyranuronide.
As summarized in Table 1, glycosylation of 4-MU (1) with methyl tetra-O-acetyl-β-D-gluco-
pyranuronate (2a) under multiple conditions were investigated, and most of reactions gave mainly
the protected
β
-D-glucopyranuronide (3a). In the presence of BF3 OEt2 and TEA, glycosylation at
¨
room temperature (25–29 ˝C) and heating (60 ˝C) gave mainly the desired product 3a in the same
yield, but the latter case took a much shorter time (entries 1–2). However, 3a was not detected by
thin layer chromatography (TLC) in the absence of TEA (entry 4 vs. entry 3). Compared to the report
by Lee et al. [37] that a TEA to BF3
yield, the relatively higher ratio of TEA to BF3
closely related to the poor dissolution of 4-MU in dichloromethane and 1,2-dichloroethane (entry 2 vs.
entry 5). An excess of 4-MU and the glycosyl donor increased the yield, which was relatively higher
in the latter case (entries 5–7). A 4 Å molecular sieve (4 Å MS) is often applied to remove water in
¨
OEt2 ratio of more than 0.2 decreased the glycosylation rate and
OEt2 did not decreased the yield, which should be
¨
glycosylation reactions [39–41]. However, its presence here decreased the yield (entry 8). Other organic
bases, such as N,N-diisopropylethyl amine (DIPEA), pyridine, 4-dimethylaminopyridine (DMAP),
and 1,1,3,3-tetramethylurea (TMU) were also investigated. Under otherwise identical conditions,
glycosylation in the presence of pyridine or TEA afforded the protected β-D-glucopyranuronide in
23% yield, and in the cases of a hindered base (DIPEA) and a very weak base (TMU), the product was
obtained in slight lower and significantly lower yields, respectively (entries 7, 9–11). However, a higher
yield was obtained upon the use of DMAP, a nucleophilic base (28%, entry 12). In an attempt to achieve
complete conversion, we extended the reaction time from 5 to 10 h and increased the amounts of base
and Lewis acid. Unfortunately, this resulted in a lower yield of 19% (entry 13 vs. entry 10). Changing
the solvent from 1,2-dichloroethane to tetrahydrofuran (THF) and CH3CN was also unsuccessful,
giving no desired product and 3a in just 2% yield, respectively (entries 14 and 15). Other Lewis acids,
such as trimethylsilyl triflate (TMSOTf) and SnCl4, were investigated, but afforded 3a in less than 1%
yield and not at all, respectively (entries 16 and 17).
Based on the investigations mentioned above, further optimization was performed to improve
the yield of 3a by using BF3
appropriate increase of the additive amount of 2a, DMAP and BF3
When the amount of 2a, DMAP and BF3 OEt2 was increased to 2.0 equivalents, 4.0 equivalents and
12.5 equivalents, respectively, the yield of 3a increased remarkably to 51% (entry 9). Further but much
¨
OEt2/DMAP/ClCH2CH2Cl. As summarized in Table 2, further but
¨
OEt2 could gave 3a in higher yield.
¨
greater excess of DMAP or BF3
increase of the recovery yield of
a higher temperature (75 C) also caused a remarkable decrease of the yield of 3a, with a corresponding
obvious increase of the yield of by-product 5 (entry 11).
¨
OEt2 caused a remarkable decrease of the yield of 3a, with obvious
, or yield of by-product , respectively (entries 3, 4, 7, and 10). Besides,
1
5
˝
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Molecules 2015, 20, 21681–21699
Table 1. Glycosylation of 4-MU (1) with methyl tetra-O-acetyl-β-D-glucopyranuronate (2a).
The Main Anomer b
Entry
1 (Equiv.)
2a (Equiv.)
Organic Base (Equiv.)
Lewis Acid (Equiv.)
Reaction Conditions
Yield a of 3a
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
1.5
1.5
1.5
1.5
1.5
2.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.5
1.0
1.0
1.0
1.0
1.0
1.0
1.5
1.5
1.5
1.5
1.5
1.5
1.5
1.5
1.5
1.5
1.0
TEA (3.75)
TEA (3.75)
TEA (3.75)
BF3¨ OEt2 (10.0)
BF3¨ OEt2 (10.0)
BF3¨ OEt2 (15.0)
BF3¨ OEt2 (15.0)
BF3¨ OEt2 (10.0)
BF3¨ OEt2 (10.0)
BF3¨ OEt2 (10.0)
BF3¨ OEt2 (10.0)
BF3¨ OEt2 (10.0)
BF3¨ OEt2 (10.0)
BF3¨ OEt2 (10.0)
BF3¨ OEt2 (10.0)
BF3¨ OEt2 (15.0)
BF3¨ OEt2 (15.0)
BF3¨ OEt2 (15.0)
TMSOTf (4.0)
CH2Cl2, 25–29 ˝C, 72 h
ClCH2CH2Cl, 60 ˝C, 5 h
ClCH2CH2Cl, 60 ˝C, 5 h
ClCH2CH2Cl, 60 ˝C, 10 h
ClCH2CH2Cl, 60 ˝C, 5 h
ClCH2CH2Cl, 60 ˝C, 5 h
ClCH2CH2Cl, 60 ˝C, 5 h
ClCH2CH2Cl, 4A MS, 60 ˝C, 5 h
ClCH2CH2Cl, 60 ˝C, 5 h
ClCH2CH2Cl, 60 ˝C, 5 h
ClCH2CH2Cl, 60 ˝C, 5 h
ClCH2CH2Cl, 60 ˝C, 5 h
ClCH2CH2Cl, 60 ˝C, 10 h
THF, 60 ˝C, 10 h
17%
17%
19%
—
16%
18%
23%
13%
20%
23%
6%
28%
19%
—
2%
<1%
—
β
β
β
c
—
—
β
TEA (2.0)
TEA (2.0)
TEA (2.0)
β
β
TEA (2.0)
β
DIPEA (2.0)
Pyridine (2.0)
TMU (2.0)
DMAP (1.5)
Pyridine (3.0)
Pyridine (3.0)
Pyridine (3.0)
Pyridine (2.0)
DIPEA (3.0)
a
β
β
β
β
β
—
β
CH3CN, 60 ˝C, 10 h
ClCH2CH2Cl, 60 ˝C, 16 h
β
SnCl4 (5.0)
CH2Cl2, 25–29 ˝C, 48 h
—
Isolated yield; b The main anomer was determined by TLC analysis and isolation; c None.
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Molecules 2015, 20, 21681–21699
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.
Figure 1. 4-methylumbelliferyl acetate (by-product 5).
Table 2. Further optimization of conditions for synthesis of the protected
β-D-glucopyranuronide (3a).
Yield c
Main
1
2a
DMAP BF3¨ OEt2
Yield of
Entry a
Recovered 1 b
Anomer d
3a
5
(mmol) (mmol) (mmol)
(mmol)
1
2
3
4
5
6
7
8
9
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.5
1.5
1.5
1.5
1.5
1.5
3.0
2.0
2.0
2.0
2.0
3.0
4.5
6.0
5.0
4.0
3.5
8.0
5.0
4.0
4.0
4.0
10.0
10.0
10.0
10.0
10.0
10.0
20.0
12.5
12.5
15.0
12.5
21%
28%
44%
37%
24%
26%
22%
11%
9%
30%
32%
17%
23%
34%
33%
36%
49%
51%
41%
26%
14%
11%
9%
β
β
β
β
β
β
β
β
β
β
β
10%
12%
13%
14%
12%
15%
17%
26%
10
11
a
10%
15%
All the reactions were performed in 1,2-dichloroethane at 60 ˝C (except for entry 11 at 75 ˝C) for 5 h, then
the cooled reaction mixtures were directly separated by gradient-flash column chromatography (silica gel,
200–300 mesh, PE/EtOAc, 7/2, 3/1, 5/2); b,c The yield of recovered
1 and the yields of 3a and 5 refer to isolated
products or determined by isolation and HPLC analysis; d The main anomer was determined by TLC analysis
and isolation.
2.2. 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 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 with 2b afforded mainly the protected -D anomer product (3b1) in 36% yield, and
¨
1
β
the use of a higher amount of pyridine gave 3b1 in 42% yield (entries 1 and 2).
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Molecules 2015, 20, 21681–21699
Scheme 3. Synthesis of other protected glycosides.
When a relatively high amount of BF3
45% yield; In addition, the protected -D anomer product (3b2) was unexpectedly obtained in a low
yield of 7% (entry 3). The glycosidation of with 2b in the absence of base afforded no products
by TLC analysis (entry 4). We expected the glycosylation procedure to be -stereoselective for the
¨
OEt2 and DMAP was used, the reaction gave 3b1 in
α
1
β
peracetyl sugars, but this was changed by the results obtained with 2c. Co˝mpound 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
-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 -D-pyranosides were also obtained by the early Helferich method:
in 1970 Vervoot et al. reported that the condensation of 4-MU with -D-mannose pentaacetate by
a method using fusion treatment and a zinc chloride catalyst under diminished pressure gave the
protected -D-mannopyranoside (3d) in 37% yield [28]. In 1978 Courtin-Duchateau et al. reported that
the condensation of 4-MU with -D-galactose pentaacetate (2c) in boiling xylene in the presence of
zinc chloride gave a mixture of protected -D-galactopyranoside, protected -D-galactofuranoside
and protected -D-galactopyranoside in a ratio of 3:5:7 in 30% total yield; and condensation of the
O-trimethylsilyl derivative of 4-MU with -D-galactose pentaacetate (2c) in the presence of stannic
chloride gave a mixture of and -D-galactopyranoside in a ratio of 15:4 in low total yield of
19%. The protected -D-mannopyranoside 3d and -D-galactopyranoside 3c could also be obtained
α- and β-D anomers in the presence of pyridine. This case gave mainly the protected
α
α
α
α
α
α
β
β
β
α
β
α
α
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
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Molecules 2015, 20, 21681–21699
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 -D-xylose tetraacetate (2e
-D-xylopyranoside (3e) in 73%
β
)
˝
with 4-MU at room temperature (20–27 C) gave mainly the protected
β
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
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 -D-ribofuranose tetraacetate (2f) with
4-MU at room temperature (20–27 ˝C) afforded mainly the protected
-D-ribofuranoside 3f in much
β-D-ribofuranoside
(
β
β
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
formation of the protected glycosides 3a 3b1
of the protected -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,
,47] that can be applied to further explain the selective
,
,
3d 3e, and 3f. However, how can the selective formation
,
α
,
,
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.
2.3. 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 Na2CO3 in aqueous MeOH, followed by desalting
using a cation exchange resin or acidification followed by reverse-phase column chromatography
treatment [34,36,50–54]. 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 H2C2O4 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.
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Molecules 2015, 20, 21681–21699
Table 3. Glycosylation of 4-MU (1) with other peracetyl sugars (2b–2f).
Entry a
1 (Equiv.)
2 (Equiv.)
Organic Base (Equiv.)
BF3¨ OEt2 (Equiv.)
Reaction Conditions
The Main Anomer c
Yield b
1
2
1.0
1.0
1.0
1.0
1.0
1.0
1.0
2.0
1.0
1.0
1.0
1.0
1.0
2b (2.0)
2b (2.0)
2b (2.0)
2b (2.0)
2c (2.0)
2c (2.0)
2c (2.0)
2d (1.0)
2d (2.0)
2e (2.0)
2e (2.0)
2f (2.0)
2f (2.0)
DMAP (1.5)
Pyridine (4.5)
DMAP (4.0)
10.0
12.5
12.5
12.5
12.5
12.5
12.5
15.0
12.5
12.5
12.5
12.5
12.5
ClCH2CH2Cl, 60 ˝C, 5 h
ClCH2CH2Cl, 60 ˝C, 5 h
ClCH2CH2Cl, 60 ˝C, 5 h
ClCH2CH2Cl, 60–70 ˝C, 10 h
ClCH2CH2Cl, 60 ˝C, 5 h
ClCH2CH2Cl, 0 ˝C, 48 h
ClCH2CH2Cl, 60 ˝C, 5 h
ClCH2CH2Cl, 60 ˝C, 5 h
ClCH2CH2Cl, 60 ˝C, 5 h
CH2Cl2, 20–21 ˝C, 5 h
3b1 (36%)
3b1 (42%)
3b1 (45%)
—
3c (54%)
3c (17%)
3c (56%)
3d (59%)
3d (62%)
3e (73%)
3e (69%)
3f (94%)
3f (77%)
β
β
β
—
α
α
α
α
α
β
β
β
β
3 d
4
e
—
5
TEA (2.5)
TEA (2.5)
DMAP (4.0)
Pyridine (3.0)
DMAP (4.0)
TEA (2.5)
DMAP (4.0)
TEA (2.5)
DMAP (4.0)
6
7 f
8
9 g
10
11 h
12
13 i
a
ClCH2CH2Cl, 26–27 ˝C, 5 h
CH2Cl2, 20–21 ˝C, 3 h
ClCH2CH2Cl, 26–27 ˝C, 5 h
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
-D anomer product (3b2) and by-product
in 15% yield, and the recovery yield of
was 9%; g The reaction gave by-product
was 13%; i The reaction gave no by-product
, and the recovery
5
were determined by isolation and HPLC
in 7% and 16% yields,
in 12%
analysis; b Isolated yield; c The main anomer was determined by TLC analysis and isolation; d The reaction gave
respectively, and the recovery yield of
was 8%; e None; f The reaction gave by-product
α
5
1
5
1
5
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
5
yield of 1 was 20%.
21688

Molecules 2015, 20, 21681–21699
Using this unoptimized method, 4a was obtained in 47% yield. The deprotection of other
glycosides 3b1
,
3c
20–25 C; Scheme 5), afforded the desired products 4b1
79% yields, respectively. Due to little amount obtained, 3b2 was not deprotected.
,
3d
,
3e, and 3f via methanolysis catalyzed by KOH at room temperature (r.t.,
˝
,
4c 4d 4e, and 4f in 83%, 80%, 80%, 79%, and
,
,
Scheme 4. Deprotection of the protected β-D-glucopyranuronide 3a.
Scheme 5. Deprotection of the other protected glycosides 3b1–3f.
3. Experimental Section
3.1. 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. 1H- and 13C-NMR spectra were recorded on 300 and 500 MHz
1
NMR spectrometers (Bruker (Beijing) Scientific Technology Co. Ltd., Beijing, China). H-NMR spectra
were recorded at 300 or 500 MHz in CDCl3 or DMSO-d6 solvent. 13C-NMR spectra were recorded
at 75 MHz in CDCl3 or DMSO-d6 solvent. HRMS spectra were recorded on an ultrahigh-resolution
quadrupole time-of-flight (UHR-Q-TOF) mass spectrometer (Bruker (Beijing) Scientific Technology Co.
21689

Molecules 2015, 20, 21681–21699
Ltd., Beijing, China) equipped with an ESI source. Specific rotations were measured with an automatic
digital polarimeter (Sinoinstrument Co. Ltd., Guangzhou, China) at 19–21 ˝C. Melting points were
measured with a microscope melting point apparatus (Shanghai Precision & Scientific Instrument Co.,
Ltd., Shanghai, China).
Methyl tetra-O-acetyl-
β
-D-glucopyranuronate (2a). This compound was prepared according to a literature
procedure [38]; white powder; mp: 176–178 ˝C; {lit. [38] mp: 174.2–175.1 ˝C}. H-NMR (300 MHz,
1
CDCl3):
H-5), 3.77 (s, 3H, Me), 2.14 (s, 3H, OAc), 2.06, 2.06, 2.05 (3
= 169.88, 169.40, 169.16, 168.81, 166.79, 91.31 (C-1), 72.93, 71.77, 70.11, 68.88, 53.00, 20.75, 20.54, 20.51,
δ
= 5.78 (d, J1,2 = 7.7 Hz, 1H, H-1), 5.37–5.13 (m, 3H, H-2, H-3, H-4), 4.20 (d, J = 9.4 Hz, 1H,
ˆ
s, 9H, 3
ˆ
OAc). 13C-NMR (75 MHz, CDCl3):
δ
1
20.44. H- and 13C-NMR spectrograms are seen in the Supplement Materials.
β
-D-Xylopyranose tetraacetate (2e) This compound was prepared according to a literature procedure [42];
˝
˝
1
white acicular crystals; mp: 123–125 C; {lit. [42] mp: 122–124 C}. H-NMR (300 MHz, CDCl3):
δ = 5.73
(d, J1,2 = 6.9 Hz, 1H, H-1), 5.22 (t, J = 8.3 Hz, 1H, H-4), 5.10–4.96 (m, 2H, H-2, H-3), 4.17 (dd, J = 12.0,
5.0 Hz, 1H, H-5a), 3.54 (dd, J = 12.0, 8.4 Hz, 1H, H-5b), 2.13 (s, 3H, OAc), 2.08, 2.07, 2.07 (3
ˆ
s, 9H,
3
ˆ
OAc). 13C-NMR (75 MHz, CDCl3):
δ
= 169.76, 169.73, 169.23, 168.96, 91.96 (C-1), 70.90, 69.41, 68.24,
62.70, 20.73, 20.65, 20.59, 20.53. H- and 13C-NMR spectrograms are seen in the Supplement Materials.
1
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 Na˝Cl, 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.
3.2. 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,
21690

Molecules 2015, 20, 21681–21699
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.
Methyl (41-methylumbelliferyl 2,3,4-tri-O-acetyl-
β-D-glucopyranosid)urinate (3a) Compound 3a was
prepared according to the general procedure for the glycosylation step using 4-MU (176–352 mg,
1.0–2.0 mmol), glycosyl acetate (376–564 mg, 1.0–1.5 mmol), dry solvent (3 mL), 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 and 2). The crude product was purified by flash column
19
˝
˝
chromatography. White powder; mp: 187–190 C; rαsD ´98 (c = 0.40, CHCl3); Rf = 0.26 (PE–AcOEt,
20
˝
3:2); {lit. [29] mp: 189–190 C, rαsD
´
45˝ (c = 1, CHCl3); lit. [35] Rf = 0.2 (hexanes–EtOAc, 2:3)}.
1H-NMR (300 MHz, CDCl3):
δ
= 7.55 (d, J51,61 = 9.4 Hz, 1H, H-51), 6.97–6.93 (m, 2H, H-61, H-81), 6.22
(s, 1H, H-31), 5.43–5.24 (m, 4H, H-1, H-2, H-3, H-4), 4.29–4.22 (m, 1H, H-5), 3.76 (s, 3H, CO2Me), 2.43
1
(s, 3H, Me ), 2.09, 2.08, 2.09 (3
ˆ
s, 9H, 3
ˆ
OAc). 13C-NMR (75 MHz, CDCl3):
δ = 170.02, 169.37, 169.19
(3ˆ
OAc), 166.62 (C-6), 160.79 (C-21), 159.02 (C-71), 154.74, 152.22, 125.80, 115.65, 113.89, 113.22 (C-31,
C-41, C-51, C-61, C-4a1, C-8a1), 104.15 (C-81), 98.26 (C-1), 72.56 (C-5), 71.61, 70.79, 68.88 (C-2, C-3, C-4),
53.09 (CO2Me), 20.60, 20.60, 20.50 (3
ˆ
OAc), 18.69 (C-Me1). HRMS (ESI): m/z [M + Na]+ calcd for
C23H24NaO12: 515.1160; found: 515.1164. H- and 13C-NMR, and HRMS spectrograms are seen in the
1
Supplement Materials.
4-Methylumbelliferyl acetate (
5
) Yellowish white powder; mp: 148–150 ˝C; Rf = 0.35 (PE–AcOEt, 3:2).
δ = 7.54 (d, J5,6 = 8.7 Hz, 1H, H-5), 7.04 (d, J6,8 = 2.2 Hz, 1H, H-8), 7.01 (dd,
1H-NMR (300 MHz, CDCl3):
J5,6 = 8.7, J6,8 = 2.2 Hz, 1H, H-6), 6.20 (d, J3,Me = 1.2 Hz, 1H, H-3), 2.36 (d, J3,Me = 1.2 Hz, 3H, H-Me),
2.27 (s, 3H, OAc). 13C-NMR (75 MHz, CDCl3):
δ
= 168.78 (OAc), 160.51 (C-2), 154.16 (C-7), 153.04,
151.95, 125.41, 118.12, 117.85, 114.52, 110.47 (C-3, C-4, C-5, C-6, C-4a, C-8a), 21.13 (C-Me), 18.74 (C-Me).
1
HRMS (ESI): m/z [M + Na]+ calcd for C12H10NaO4: 241.0471; found: 241.0477. H- and 13C-NMR, and
HRMS spectrograms are seen in the Supplement Materials.
41-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). The crude
19
product was purified by flash column chromatography. White powder; mp: 142–143 ˝C; rαsD
´
35˝
20
(c = 0.40, CHCl3); Rf = 0.27 (PE–AcOEt, 3:2); {lit. [21] mp: 144 ˝C, rαsD
´40
˘
2˝ (c = 0.5, CHCl3);
21691

Molecules 2015, 20, 21681–21699
20
˝
lit. [25] mp: 144–145 C, rαsD
´
36˝ (c = 1, CHCl3), Rf = 0.26 (hexanes–AcOEt, 1:1); lit. [27] mp:
20
˝
143–144 C, rαsD
´
39˝ (c = 0.635, CHCl3), Rf = 0.18 (CHCl3–acetone, 19:1)}. 1H-NMR (300 MHz,
= 7.54 (d, J51,61 = 8.7 Hz, 1H, H-5 ), 6.98–6.93 (m, 2H, H-6 , H-8 ), 6.22 (d, J31,Me1 = 0.9 Hz, 1H,
1
1
1
CDCl3):
δ
1
H-3 ), 5.38–5.30 (m, 2H, H-1, H-3), 5.22–5.16 (m, 2H, H-2, H-4), 4.32 (dd, J6a,6b = 12.1, J5,6a = 5.5 Hz, 1H,
H-6a), 4.20 (dd, J6a,6b = 12.1, J5,6b = 1.7 Hz, 1H, H-6b), 3.93 (ddd, J4,5 = 7.4, J5,6a = 5.5, J5,6b = 1.7 Hz, 1H,
H-5), 2.43 (d, J31 Me1 = 0.9 Hz, 3H, Me1), 2.14, 2.09, 2.09, 2.06 (4
ˆ
s, 12H, 4
ˆ
OAc). 13C-NMR (75 MHz,
CDCl3):
δ
= 170.61, 170.17, 169.41, 169.26 (4
115.51, 113.98, 113.19 (C-31, C-41, C-51, C-61, C-4a1, C-8a1), 103.93 (C-81), 98.33 (C-1), 72.55, 72.40, 70.92,
68.06, 61.80 (C-2, C-3, C-4, C-5, C-6), 20.71, 20.62, 20.59, 20.59 (4
m/z [M + Na]+ calcd for C24H26NaO12: 529.1316; found: 529.1323. H- and 13C-NMR, and HRMS
ˆ
OAc), 160.74 (C-21), 159.17 (C-71), 154.82, 152.19, 125.74,
ˆ
OAc), 18.69 (C-Me1). HRMS (ESI):
1
spectrograms are seen in the Supplement Materials.
41-Methylumbelliferyl 2,3,4,6-tetra-O-acetyl-
α
-D-glucopyranoside (3b2) White powder; mp: 132–133 ˝C;
22
20
˝
˝
˝
rαsD +202 (c = 0.30, CHCl3); Rf = 0.31 (PE–AcOEt, 3:2); {lit. [27] mp: 131–132 C, rαsD +200 (c = 0.5,
1
CHCl3), Rf = 0.27 (CHCl3–acetone, 19:1)}. H-NMR (300 MHz, CDCl3):
δ = 7.56 (d, J51,61 = 8.7 Hz,
1H, H-51), 7.11 (d, J61,81 = 2.3 Hz, 1H, H-81), 7.06 (dd, J51,61 = 8.7, J61,81 = 2.4 Hz, 1H, H-61), 6.22 (s, 1H,
H-31), 5.82 (d, J1,2 = 3.3 Hz, 1H, H-1), 5.71 (t, J3,4 = 9.9 Hz, 1H, H-3), 5.18 (t, J3,4 = 9.9 Hz, 1H, H-4),
5.08 (dd, J2,3 = 10.2, J1,2 = 3.6 Hz, 1H, H-2), 4.28 (dd, J = 12.6, 4.8 Hz, 1H, H-6a), 4.13–4.01 (m, 2H,
H-5, H-6b), 2.43 (s, 3H, Me1), 2.08 (4s, 12H, 4
ˆ
OAc). 13C-NMR (75 MHz, CDCl3):
δ
= 170.51, 170.15,
1
1
1
170.11, 169.52 (4
ˆ
OAc), 160.75 (C-2 ), 158.61 (C-7 ), 154.84, 152.10, 125.85, 115.53, 113.32, 113.24 (C-3 ,
C-41, C-51, C-61, C-4a1, C-8a1), 104.58 (C-81), 94.37 (C-1), 70.18, 69.79, 68.40, 68.09, 61.45 (C-2, C-3, C-4,
C-5, C-6), 20.68, 20.62, 20.61, 20.57 (4
C24H26NaO12: 529.1316; found: 529.1321. H- and 13C-NMR, and HRMS spectrograms are seen in the
ˆ
OAc), 18.67 (C-Me1). HRMS (ESI): m/z [M + Na]+ calcd for
1
Supplement Materials.
41-Methylumbelliferyl 2,3,4,6-tetra-O-acetyl-
according to the general procedure for the glycosylation step using 4-MU (352 mg, 2.0 mmol),
-D-galactose pentaacetate (1561 mg, 4.0 mmol, 2.0 equiv.), dry ClCH2CH2Cl (6 mL), TEA (700 L,
5.0 mmol, 2.5 equiv.) and BF3 OEt2 (3218 L, 25 mmol, 12.5 equiv.). The mixture was stirred for 5 h at
α-D-galactopyranoside (3c) Compound 3c was prepared
β
µ
¨
µ
˝
60 C (as shown in Table 3). The crude product was purified by flash column chromatography. White
19
˝
˝
powder; yield: 548 mg (54%); mp: 176–180 C; rαsD +209 (c = 0.40, CHCl3); Rf = 0.32 (PE–AcOEt, 3:2);
20
˝
˝
1
{lit. [27] mp: 186.5–188.5 C, rαsD +210 (c = 0.281, CHCl3), Rf = 0.43 (CHCl3–acetone, 9:1)}. H-NMR
1
1
1
(500 MHz, CDCl3):
δ = 7.53 (d, J51,61 = 8.7 Hz, 1H, H-5 ), 7.06 (s, 1H, H-8 ), 7.00 (d, J = 8.7 Hz, 1H, H-6 ),
6.18 (s, 1H, H-31), 5.83 (d, J1,2 = 3.0 Hz, 1H, H-1), 5.55 (dd, J2,3 = 11.0 Hz, J1,2 = 3.0 Hz, 1H, H-2), 5.52
(d, J3,4 = 3.2 Hz, 1H, H-4), 5.29 (dd, J2,3 = 10.8 Hz, J3,4 = 3.2 Hz, 1H, H-3), 4.28 (t, J5,6a = 6.3 Hz, 1H,
H-5), 4.14–4.01 (m, 2H, H-6a, H-6b), 2.40 (s, 3H, Me1), 2.17 (s, 3H, OAc), 2.07 (s, 3H, OAc), 2.02 (s, 3H,
OAc), 1.94 (s, 3H, OAc). 13C-NMR (75 MHz, CDCl3):
δ
= 170.36, 170.31, 170.14, 170.06 (4
ˆ
OAc), 160.81
(C-21), 158.90 (C-71), 154.86, 152.13, 125.79, 115.45, 113.54, 113.20 (C-31, C-41, C-51, C-61, C-4a1, C-8a1),
104.59 (C-81), 95.04 (C-1), 67.60, 67.52, 67.51, 67.32, 61.30 (C-2, C-3, C-4, C-5, C-6), 20.73, 20.68, 20.64,
21692

Molecules 2015, 20, 21681–21699
20.57 (4
ˆ
OAc), 18.70 (C-Me1). HRMS (ESI): m/z [M + Na]+ calcd for C24H26NaO12: 529.1316; found:
1
529.1323. H- and 13C-NMR, and HRMS spectrograms are seen in the Supplement Materials.
41-Methylumbelliferyl 2,3,4,6-tetra-O-acetyl-
according to the general procedure for the glycosylation step using 4-MU (1057 mg, 6.0 mmol),
-D-mannose pentaacetate (1171 mg, 3.0 mmol, 0.5 equiv.), dry ClCH2CH2Cl (9 mL), pyridine (728 L,
L, 45 mmol, 7.5 equiv.). The mixture was stirred for 5 h at
α-D-mannopyranoside (3d) Compound 3d was prepared
β
µ
9.0˝mmol, 1.5 equiv.) and BF3
¨
OEt2 (5792 µ
60 C (as shown in Table 3). The crude product was purified by three recrystallizations in EtOH. White
20
and slightly yellow acicular crystals; yield: 899 mg (59%); mp: 161.5–163.5 ˝C; rαsD +104˝ (c = 0.35,
22
˝
˝
CHCl3); Rf = 0.28 (PE–AcOEt, 3:2); {lit. [27] mp 161–163 C, rαsD +106.0 (c = 0.321, CHCl3), Rf = 0.54
22
(CH2Cl2–acetone, 23:2); lit. [28] mp: 160–161 ˝C, rαsD +136.0˝ (c = 2, CHCl3)}. H-NMR (300 MHz,
1
1 1
δ = 7.56 (d, J51,61 = 8.7 Hz, 1H, H-5 ), 7.13 (d, J61,81 = 2.1 Hz, 1H, H-8 ), 7.04 (dd, J51,61 = 8.8, J61,81
CDCl3):
= 2.4 Hz, 1H, H-61), 6.21 (s, 1H, H-31), 5.60–5.36 (m, 4H, H-1, H-2, H-3, H-4), 4.30 (dd, J = 12.6, 6.0 Hz,
1H, H-6a), 4.09–4.03 (m, 2H, H-6b, H-5), 2.43 (s, 3H, Me1), 2.24 (s, 3H, OAc), 2.07, 2.06, 2.06 (3
ˆ
s, 9H,
1
3
ˆ
OAc). 13C-NMR (75 MHz, CDCl3):
δ
= 170.48, 169.94, 169.93, 169.63 (4
ˆ
OAc), 160.73 (C-2 ), 158.10
(C-71), 154.81, 152.14, 125.81, 115.44, 113.40, 113.19 (C-31, C-41, C-51, C-61, C-4a1, C-8a1), 104.29 (C-81),
95.84 (C-1), 69.49, 68.99, 68.62, 65.68, 61.98 (C-2, C-3, C-4, C-5, C-6), 20.85, 20.67, 20.65, 20.62 (4
ˆ
OAc),
1
1
18.66 (C-Me ). HRMS (ESI): m/z [M + Na]+ calcd for C24H26NaO12: 529.1316; found: 529.1322. H- and
13C-NMR, and HRMS spectrograms are seen in the Supplement Materials.
41-Methylumbelliferyl 2,3,4-tri-O-acetyl-
to the general procedure for the glycosylation step using 4-MU (352 mg, 2.0 mmol),
β
-D-xylopyranoside (3e) Compound 3e was prepared according
-D-xylose
β
tetraacetate (1273 mg, 4.0 mmol, 2.0 equiv.), dry CH2Cl2 (6 mL), TEA (700 L, 5.0 mm˝ol, 2.5 equiv.)
µ
and BF3 OEt2(3218 µL, 25.0 mmol, 12.5 equiv.). The mixture was stirred for 5 h at 20–21 C (as shown
¨
in Table 3). The crude product was purified by flash column chromatography. White powder; yield:
20
˝
633 mg (73%); mp: 148–153 C; rαsD
´
49˝ (c = 1.00, CHCl3). Rf = 0.33 (PE–AcOEt, 3:2); {lit. [43
]
22
mp: 158 ˝C, rαsD
´
45˝ (c = 2, CHCl3), Rf = 0.65 (AcOEt–benzene, 3:7)}. H-NMR (300 MHz, CDCl3):
1
δ
= 7.53 (d, J51,61 = 8.7 Hz, 1H, H-51), 6.97–6.92 (m, 2H, H-61, H-81), 6.19 (d, J32,Me1 = 1.2 Hz, 1H, H-31),
5.31–5.17 (m, 3H, H-1, H-2, H-3), 5.02 (td, J4,5b = 7.1, J4,5a = 4.5 Hz, 1H, H-4), 4.23 (dd, J5a,5b = 12.3,
J4,5a = 4.5 Hz, 1H, H-5a), 3.60 (dd, J5a,5b = 12.3, J4,5b = 7.2 Hz, 1H, H-5b), 2.41 (d, J32,Me1 = 1.2 Hz,
3H, Me1), 2.11, 2.11, 2.11 (3
ˆ
s, 9H, 3
ˆ
OAc). 13C-NMR (75 MHz, CDCl3):
δ = 169.84, 169.83, 169.36
(3ˆ
OAc),1160.88 (C-21), 158.99 (C-71), 154.83, 152.22, 125.76, 115.36, 113.59, 113.08 (C-31, C-41, C-51,
1
1
C-6v, C-4a , C-8a ), 104.18 (C-8 ), 97.77 (C-1), 70.15, 69.59, 68.09, 61.81 (C-2, C-3, C-4, C-5), 20.80, 20.75,
21693

Molecules 2015, 20, 21681–21699
20.71 (3
ˆ
OAc), 18.70 (C-Me1). HRMS (ESI): m/z [M + Na]+ calcd for C21H22NaO10: 457.1105; found:
1
457.1112. H- and 13C-NMR, and HRMS spectrograms are seen in the Supplement Materials.
1
4 -Methylumbelliferyl 2,3,5-tri-O-acetyl-
the general procedure for the glycosylation step using 4-MU (352 mg, 2.0 mmol),
(1273 mg, 4.0 mmol, 2.0 equiv), dry CH2Cl2 (6 mL), TEA (700 µL, 5.0 mmol, 2.5 equiv.) and BF3¨ OEt2
(3218
L, 25 mmol, 12.5 equiv.). The mixture was stirred for 3 h at 20–21 ˝C (as shown in Table 3).
β
-D-ribofuranoside (3f) Compound 3f was prepared according to
β-D-ribose tetraacetate
µ
The crude product was purified by flash column chromatography. Colorless syrup; yield: 817 mg
(94%); Rf = 0.31 (PE–AcOEt, 3:2). Little purer colorless syrup obtained by HPLC separation was
analyzed by NMR spectrometers and HRMS. 1H-NMR (300 MHz, CDCl3):
H-5 ), 6.92–6.85 (m, 2H, H-6 , H-8 ), 6.10 (s, 1H, H-3 ), 5.63 (s, 1H, H-1), 5.47–5.42 (m, 2H, H-2, H-3), 4.38
δ = 7.45 (d, J = 8.7 Hz, 1H,
1
1
1
1
(dd, J = 9.0, 4.8 Hz, 1H, H-4), 4.31 (dd, J = 12.0, 3.6 Hz, 1H, H-5a), 3.98 (dd, J = 12.0, 4.7 Hz, 1H, H-5b),
2.33 (s, 3H, Me1), 2.10, 2.04, 1.87 (3
ˆ
s, 9H, 3
ˆ
OAc). 13C-NMR (75 MHz, CDCl3):
δ = 170.47, 169.75,
169.57 (3
ˆ
OAc), 161.04 (C-21), 158.71 (C-71), 154.80, 152.36, 125.71, 114.96, 113.27, 112.84 (C-31, C-41,
C-51, C-61, C-4a1, C-8a1), 103.95 (C-81), 102.90 (C-1), 79.57, 74.78, 70.71, 63.22 (C-2, C-3, C-4, C-5), 20.64,
20.58, 20.51 (3
ˆ
OAc), 18.67 (C-Me1). HRMS (ESI): m/z [M + Na]+ calcd for C21H22NaO10: 457.1105;
1
found: 457.1111. H- and 13C-NMR, and HRMS spectrograms are seen in the Supplement Materials.
4-Methylumbelliferyl
β
-D-glucopyranosiduronic acid (4a) To a stirred suspension of Ba(OH)2 H2O (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 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
21
powder; yield: 42 mg (47%); mp: 140–144 ˝C; rαsD
´
114˝ (c = 0.20, H2O); {lit. [27] mp: 139–145 ˝C,
20
20
˝
rαsD
´
108˝ (c = 0.25, pyridine); lit. [29] mp: 139–140 C, rαsD
´
114˝ (c = 0.25, H2O); lit. [32] mp:
22
20
˝
˝
˝
˝
1
139–140 C, rαsD
´
119 (c = 0.25, H2O); lit. [33] mp: 140–143 C, rαsD
´
116 (c = 0.25, H2O)}. H-NMR
(500 MHz, DMSO-d6):
H-3v), 5.23 (d, J1,2 = 6.0 Hz, 1H, H-1), 4.00 (d, J4,5 = 9.0 Hz, 1H, H-5), 3.39–3.29 (m, 3H, H-2, H-3,
δ
= 7.72 (d, J51,61 = 8.5 Hz, 1H, H-51), 7.16–7.03 (m, 2H, H-61, H-81), 6.26 (s, 1H,
H-4), 2.40 (s, 3H, Me). 13C-NMR (75 MHz, DMSO-d6):
δ
= 170.17 (C-6), 160.01 (C-21), 159.62 (C-71),
154.33, 153.26, 126.49, 114.17, 113.18, 111.75 (C-31, C-41, C-51, C-61, C-4a1, C-8a1), 102.98 (C-81), 99.25
(C-1), 75.70 (C-5), 75.19, 72.77, 71.26 (C-2, C-3, C-4), 18.09 (C-Me1). HRMS (ESI): m/z [M + Na]+ calcd
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Molecules 2015, 20, 21681–21699
1
for C16H16NaO9: 375.0687; found: 375.0692. H- and 13C-NMR, and HRMS spectrograms are seen in
the Supplement Materials.
4-Methylumbelliferyl
for 1 h at room temperature with KOH (1.0 M in anhydrous MeOH; 40
β
-D-glucopyranoside (4b1) Compound 3b1 (100 mg, 0.20 mmol) was O-deacetylated
L, 0.2 equiv.) in anhydrous
µ
MeOH (3 mL). Concentration of the solution gave a crystalline material that was filtered off, washed
with ethanol until the washings were no longer fluorescent, and dried. White crystal; yield: 56 mg
20
20
(83%); mp: 209–211 ˝C; rαsD
´
85˝ (c = 0.30, H2O); {lit. [21] mp: 211 ˝C, rαsD
´
89.5˝ (c = 0.5, H2O);
= 7.71
20
˝
lit. [27] mp: 211–213 C, rαsD
´
68˝ (c = 0.5, pyridine)}. 1H-NMR (300 MHz, DMSO-d6):
δ
(d, J51,61 = 9.3 Hz, 1H, H-51), 7.08–7.00 (m, 2H, H-61, H-81), 6.26 (d, J31,Me1 = 0.9 Hz, 1H, H-31), 5.42
(d, J1,2 = 4.8 Hz, 1H, H-1), 5.16 (d, J1,2 = 4.5 Hz, 1H, H-2), 5.09–5.03 (m, 2H), 4.61 (t, J = 5.4 Hz, 1H),
3.73–3.66 (m, 1H), 3.50–3.40 (m, 2H), 3.31–3.23(m,13H), 3.20–3.13 (m, 1H), 2.41 (s, 3H, Me1). 13C-NMR
1 1
δ = 160.10 (C-2 ), 160.08 (C-7 ), 154.35, 153.30, 126.37, 114.02, 113.34, 111.65 (C-3 ,
(75 MHz, DMSO-d6):
C-41, C-51, C-61, C-4a1, C-8a1), 103.15 (C-81), 99.93 (C-1), 77.10, 76.44, 73.09, 69.58, 60.60 (C-2, C-3, C-4,
C-5, C-6), 18.11 (C-Me ). HRMS (ESI): m/z [M + Na]+ calcd for C16H18NaO8: 361.0894; found: 361.0900.
1
1H- and 13C-NMR, and HRMS spectrograms are seen in the Supplement Materials.
4-Methylumbelliferyl
α
-D-galactopyranoside (4c) Compound 3c (400 mg, 0.79 mmol) was O-deacetylated
L, 0.3 equiv.) in dry
for 15 min at room temperature with KOH (1.0 M in anhydrous MeOH; 237
µ
tetrahydrofuran (8 mL) and anhydrous MeOH (4 mL). The white solid precipitate was filtered off,
washed with ethanol until the washings were no longer fluorescent, and dried. White powder; yield:
20
20
˝
˝
214 mg (80%); mp: 222–223 C; rαsD +238˝ (c = 0.30, H2O); {lit. [27] mp: 212–217 C; rαsD +135˝
1
1
(c = 1.37, pyridine)}. H-NMR (500 MHz, DMSO-d6):
δ = 7.70 (d, J51,61 = 9.0 Hz, 1H, H-5 ), 7.09–7.07 (m,
2H, H-61, H-81), 6.24 (d, J31,Me1 = 1.0 Hz, 1H, H-31), 5.60 (d, J1,2 = 3.0 Hz, 1H, H-1), 4.99 (d, J = 6.5 Hz,
1H), 4.79 (d, J = 5.0 Hz, 1H), 4.61 (d, J = 4.5 Hz, 1H), 4.53 (t, J = 5.7 Hz, 1H), 3.83–3.74 (m, 3H), 3.64
(t, J = 5.7 Hz, 1H, H-5), 3.52 (dt, J = 11.0, 6.2 Hz, 1H, H-6a), 3.38 (dt, J = 11.0, 6.2 Hz, 1H, H-6b),
2.40 (s, 3H, H-Me1). 13C-NMR (75 MHz, DMSO-d6):
δ
1 = 160.07 (C-21), 160.07 (C-71), 154.30, 153.32,
1
1
1
1
1
1
126.32, 113.92, 113.82, 111.56 (C-3 , C-4 , C-5 , C-6 , C-4a , C-8a ), 103.65 (C-8 ), 98.07 (C-1), 72.68, 69.30,
68.42, 67.71, 60.20 (C-2, C-3, C-4, C-5, C-6), 18.09 (C-Me1). HRMS (ESI): m/z [M + Na]+ calcd for
C16H18NaO8: 361.0894; found: 361.0897. H- and 13C-NMR, and HRMS spectrograms are seen in the
1
Supplement Materials.
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Molecules 2015, 20, 21681–21699
4-Methylumbelliferyl
for 45 min at room temperature with KOH (1.0 M in anhydrous MeOH; 119
α
-D-mannopyranoside (4d) Compound 3d (200 mg, 0.39 mmol) was O-deacetylated
L, 0.3 equiv.) in anhydrous
µ
MeOH (2 mL). The beige solid precipitate was filtered off, washed with ethanol until the washings
20
were no longer fluorescent, and dried. Beige powder; yield: 106 mg (80%); mp: 213–214 ˝C; rαsD
20
+154˝ (c = 0.35, H2O); {lit. [27] mp: 220–224 ˝C, rαsD +157˝ (c = 0.4, H2O); lit. [28] mp: 222–225 ˝C,
22
rαsD +178.2˝ (c = 2, MeOH)}. H-NMR (500 MHz, DMSO-d6):
δ
= 7.69 (d, J51,61 = 9.0 Hz, 1H, H-51),
1
1
1
1
7.11–7.07 (m, 2H, H-6 , H-8 ), 6.24 (s, 1H, H-3 ), 5.53 (s, 1H, H-1), 5.13 (d, J = 2.0 Hz, 1H), 4.90–4.84 (m,
2H), 4.51 (t, J = 5.5 Hz, 1H), 3.85 (s, 1H), 3.68 (t, J = 4.0 Hz,1H), 3.60–3.57 (m, 1H), 3.52–3.42 (m, 2H),
1
1 1
δ = 160.04 (C-2 ), 159.07 (C-7 ), 154.30,
3.32 (s, 1H), 2.40 (s, 3H, H-Me ). 13C-NMR (75 MHz, DMSO-d6):
1
1
1
1
1
1
1
153.29, 126.41, 114.08, 113.64, 111.68 (C-3 , C-4 , C-5 , C-6 , C-4a , C-8a ), 103.61 (C-8 ), 98.72 (C-1), 75.31,
70.51, 69.74, 66.53, 60.91 (C-2, C-3, C-4, C-5, C-6), 18.09 (C-Me ). HRMS (ESI): m/z [M + Na]+ calcd for
1
C16H18NaO8: 361.0894; found: 361.0900. H- and 13C-NMR, and HRMS spectrograms are seen in the
1
Supplement Materials.
4-Methylumbelliferyl β-D-xylopyranoside (4e) Compound 3e (200 mg, 0.46 mmol) was O-deacetylated for
25 min at room temperature with KOH (1.0 M in anhydrous MeOH; 138 µL, 0.3 equiv.) in anhydrous
MeOH (5 mL). The white solid precipitate was filtered off, washed with ethanol until the washings
20
˝
˝
65
were no longer fluorescent, and dried. White powder; yield: 112 mg (79%); mp: 217–218 C; rαsD
´
22
(c = 0.10, H2O); {lit. [43] mp: 213–214 ˝C, rαsD
´
42˝ (c =10.10, 1H2O)}. H-NMR 1(300 MHz, DMSO-d6):
1
1
δ
= 7.70 (d, J51,61 = 9.3 Hz, 1H, H-5 ), 7.04–7.00 (m, 2H, H-6 , H-8 ), 6.25 (s, 1H, H-3 ), 5.45 (d, J1,2 = 4.8 Hz,
1H, H-1), 5.19 (t, J =2.3 Hz, 1H), 5.12 (d, J = 4.2 Hz, 1H), 5.06 (t, J = 3.6 Hz, 1H), 3.80–3.71 (m, 1H),
3.43–3.33 (m, 2H), 3.31–3.21 (m, 2H), 2.40 (s, 3H, Me1). 13C-NMR (75 MHz, DMSO-d6):
= 160.03
δ
(C-21), 159.83 (C-71), 154.33, 153.27, 126.44, 114.07, 113.26, 111.71 (C-31, C-41, C-51, C-61, C-4a1, C-8a1),
103.06 (C-81), 100.24 (C-1), 76.27, 72.89, 69.21, 65.68 (C-2, C-3, C-4, C-5), 18.09 (C-Me1). HRMS (ESI):
m/z [M + Na]+ calcd for C15H16NaO7: 331.0788; found: 331.0793. 1H- and 13C-NMR, and HRMS
spectrograms are seen in the Supplement Materials.
4-Methylumbelliferyl β-D-ribofuranoside (4f) Compound 3f (200 mg, 0.46 mmol) was O-deacetylated for
25 min at room temperature with KOH (1.0 M in anhydrous MeOH; 138 µL, 0.3 equiv.) in anhydrous
MeOH (5 mL). The white solid precipitate was filtered off, washed with ethanol until the washings
20
were no longer fluorescent, and dried. White powder; yield: 112 mg (79%); mp: 167–169 ˝C; rαsD
´
145˝ (c = 0.15, H2O). 1H-NMR (300 MHz, DMSO-d6): = 7.67 (d, J51,61 = 8.7 Hz, 1H, H-51), 7.00–6.96
δ
1
1
1
(m, 2H, H-6 , H-8 ), 6.22 (d, J31,Me1 = 1.2 Hz, 1H, H-3 ), 5.60 (s, 1H, H-1), 5.43 (d, J = 4.5 Hz, 1H), 5.08 (d,
J = 6.3 Hz, 1H), 4.71 (t, J = 5.4 Hz, 1H), 4.08–4.00 (m, 2H), 3.93 (td, J = 6.0, 3.6 Hz, 1H), 3.55 (ddd, J = 11.7,
5.4, 3.6 Hz, 1H), 3.38–3.29 (m, 1H), 2.38 (d, J31,Me1 = 0.9 Hz, 3H, Me1). 13C-NMR (751MHz, DMSO-d6):
1 1 1 1 1 1
= 160.02 (C-2 ), 159.31 (C-7 ), 154.26, 153.25, 126.41, 113.81, 113.32, 111.54 (C-3 , C-4 , C-5 , C-6 , C-4a ,
δ
C-8a1), 105.06 (C-81), 103.26 (C-1), 84.82, 74.47, 70.41, 62.52 (C-2, C-3, C-4, C-5), 18.07 (C-Me1). HRMS
21696

Molecules 2015, 20, 21681–21699
1
(ESI): m/z [M + Na]+ calcd for C15H16NaO7: 331.0788; found: 331.0791. H- and 13C-NMR, and HRMS
spectrograms are seen in the Supplement Materials.
4. Conclusions
We have developed a useful method for the synthesis of 4-methylumbelliferyl glycosides.
Notable benefits, such as the easy availability of glycosyl acetates, α-/β-stereoselectivity, moderate
to excellent yields (51%–94%), and easy operation, make this method a practical approach for
obtaining 4-methylumbelliferyl glycosides. These compounds have been often applied to detect
the activities of glycosidases from different sources, particularly microorganisms. For example,
4-methy-lumbelliferyl
are widely used for detection of Escherichia coli and enterococci, respectively, and 4-methylumbelliferyl
-D-galactopyranoside can be used to help differentiate between streptococci and enterococci [ 18].
β-D-glucopyranosiduronic acid and 4-methylumbelliferyl β-D-glucopyranoside
α
4,
The search for novel glycosidase substrates with better performance or new functions is also of
continuing interesting for biochemists. Herein we hope that this improved Helferich method will be
beneficial for this objective.
Supplementary Materials: Supplementary materials can be accessed at: http://www.mdpi.com/1420-3049/20/
12/19789/s1.
Acknowledgments: This work was supported by the Science & Technology Program of Guangdong Province
(2012A080203014) and Science & Technology Program of Guangzhou City (201300000074, 201508020037).
Author Contributions: Xianhu Wei, Yanxia Ma, Qingping Wu and Jumei Zhang conceived and designed the
experiments; Xianhu Wei and Yanxia Ma performed the experiments; Xianhu Wei and Qingping Wu analyzed the
data; Zhihe Cai and Mianfei Lu contributed reagents/materials/analysis tools; Xianhu Wei wrote the paper.
Conflicts of Interest: The authors declare no conflict of interest.
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