Chemoenzymatic Synthesis of Glycoconjugates Mediated by Regioselective Enzymatic Hydrolysis of Acetylated 2-Amino Pyranose Derivatives

Article abstract of DOI:10.1002/ejoc.201900382

Highly regioselective deprotection of a series of 2-amino pyranose building blocks was achieved by enzymatic hydrolysis. These monodeprotected intermediates were successfully used in the synthesis of a variety of glycoconjugate derivatives with a core of glucosamine or galactosamine, including neo-glycoproteins and glycosphingolipids. The hydrolysis catalyzed by acetylxylan esterase from Bacillus pumilus (AXE) is suitable for the synthesis of neo-glycoproteins with an N-acetyl glucosamine core. The hydrolysis catalyzed by Candida rugosa lipase (CRL) was successfully applied in the preparation of new sialylated glycolipids starting from glucosamine building blocks protected as phthalimide. This chemoenzymatic approach can be used for the preparation of new glycoconjugate products with anticancer activity.

Full text of DOI:10.1002/ejoc.201900382

A Journal of
Accepted Article
Title: Chemoenzymatic Synthesis of Glycoconjugates Mediated by
Regioselective Enzymatic Hydrolysis of Acetylated 2-Amino
Pyranose Derivatives
Authors: Changping Zheng, Teodora Bavaro, Sara Tengattini, Andrea
Gualla Mascherpa, Matthieu Sollogoub, Yongmin Zhang, and
Marco Terreni
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To be cited as: Eur. J. Org. Chem. 10.1002/ejoc.201900382
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10.1002/ejoc.201900382
European Journal of Organic Chemistry
Chemoenzymatic Synthesis of Glycoconjugates Mediated by
Regioselective Enzymatic Hydrolysis of Acetylated 2-Amino Pyranose
Derivatives
Changping Zheng,[a,§] Teodora Bavaro,[b,§] Sara Tengattini,[b] Andrea Gualla Mascherpa,[c] Matthieu
Sollogoub,[a] Yongmin Zhang,*[a,d] and Marco Terreni*[b]
[a]
CNRS, UMR 8232, IPCM, Sorbonne Université, 4 Place Jussieu, 75005 Paris, France.
yongmin.zhang@upmc.fr
[b]
Drug Sciences Department, University of Pavia, Viale Taramelli 12, 27100 Pavia, Italy.
marco.terreni@unipv.it
[c]
School of Physical and Chemical Sciences, University of Canterbury, Private Bag 4800, 8140
Christchurch, New Zealand.
[d]
Institute for Interdisciplinary Research, Jianghan University, Wuhan Economic and
Technological Development Zone, 430056 Wuhan, China.
§ These two authors contributed equally to this work.
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10.1002/ejoc.201900382
European Journal of Organic Chemistry
Abstract: Highly regioselective deprotection of a series of 2-amino pyranose building blocks was
achieved by enzymatic hydrolysis. These monodeprotected intermediates were successfully used in
the synthesis of a variety of glycoconjugated derivatives with a core of glucosamine or galactosamine,
including neo-glycoproteins and glycosphingolipids. The hydrolysis catalyzed by Acetyl xylan
esterase from Bacillus pumilus (AXE) is suitable for the synthesis of neo-glycoproteins with an N-
acetyl glucosamine core. The hydrolysis catalyzed by Candida rugosa lipase (CRL) was successfully
applied in the preparation of new sialylated glycolipids starting from glucosamine building blocks
protected as phthalimide. This chemoenzymatic approach can be used for the preparation of new
glycoconjugated products with anticancer activity.
Introduction
Glycoproteins and glycolipids are involved in many physiological and pathological processes.[1]
Consequently, synthetic non-natural glycoconjugates are largely investigated for developing new
therapeutic strategies,[2] including the use of glycolipids with anticancer activity[3] or carbohydrate-
based vaccines.[4-7]
In particular, sialylated oligosaccharides are involved in cell-cell adhesion processes[8] and many of
these oligosaccharides are considered as tumor associated carbohydrate antigens (TACAs) because
overexpressed in cancer cells. Specific glycosphingolipid types, which are detectable in normal cells,
are more highly expressed in tumors. The high level of expression on the surface of tumour cells
causes an antibody response towards these glycosphingolipids, which are therefore considered as
targets for cancer therapy[9]. For example the lactosyl-ganglioside GM3 (NeuAcα3Galβ4Glcβ1Cer),
is recognized as an important melanoma-associated antigen, and may have a role in metastasis.[10,11]
Similarly, Sialyl-LeX, sialyl-α-1,6-galactosamine (Sialyl-Tn antigen), and other sialylated TACAs
with a core of N-acetyl glucosamine or galactosamine, are over-expressed in a wide variety of human
cancers.[12]
Glycoconjugated lipid analogues of GM3, obtained by sialylation of glucose at C-6, recently have
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European Journal of Organic Chemistry
been reported as a new class of potential anticancer agents.[3] Semi-synthetic glycoproteins (neo-
glycoproteins) can instead provide immunogens from which therapeutic agents can be derived.[13,14]
In particular, neo-glycoproteins have been largely investigated as therapeutic
anticancer vaccines[15,16] based on TACAs. Sialyl-Tn has been proposed as therapeutic vaccine
against prostate cancer, and its assembly with other TACAs has been proposed for developing
multivalent glycoconjugate vaccines.[17] However, the study of glycoconjugates is limited by the
complex procedures required for the preparation of protected building blocks bearing free hydroxyl
groups in the desired positions. In presence of glucosamine or galactosamine, the synthesis of
oligosaccharides is currently performed protecting the amino as acetyl-amide,[18,19] as naturally
existing in the TACAs structure. However, protection as phthalimide is often preferred.[20] At the
anomeric position of the acceptor, different groups can be used, including thio-alkyl/aryl leaving
groups, because after glycosylation of the free hydroxyl group, these products can be used as sugar
donors for a further glycosylation reaction.[21] In addition, for the synthesis of neo-glycoproteins
should be done the preparation of building blocks with an appropriate reactive linker at anomeric
position, required for conjugation with the protein carrier. One of the most used strategies implies the
use of 2-iminomethoxyethyl thioglycosides (IME) that selectively reacts with lysines on the protein
surface.
The use of enzymatic regioselective hydrolysis of peracetylated sugar has been proposed as an
important tool for developing simple and efficient synthesis of sugar building blocks.[22] This strategy
was employed for the preparation of mannopyranose glycans with the anomeric thiocyanomethyl
group, that were used for protein glycosylation after activation as anomeric IME-linker.[23,24]
Peracetylated monodeprotected galactopyranoses have also been used for the preparation of sialylated
glycoconjugate lipids.[25]
The most used biocatalyst for regioselective deacetylation is the lipase from Candida rugosa (CRL).
However, other enzymes were also used. In particular, Acetyl xylan esterase from Bacillus pumilus
(AXE), has been successfully employed in the preparation of advanced building blocks by
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European Journal of Organic Chemistry
regioselective deacetylation of acetylated disaccharides.[26] The use of this chemoenzymatic approach
has never been investigated for the preparation of glyco-derivatives (neo-glycoproteins and
glycolipids) using TACAs with a core of glucosamine or galactosamine, such as the STn antigen.
Consequently, in this work, we have studied the regioselective enzymatic hydrolysis of 2-amino-
pyranoside derivatives (Scheme 1), exploring the effect on the enzyme activity and selectivity
induced by the presence of different reactive groups in anomeric position and by the protection of the
2-amino function.
Scheme 1. Regioselective hydrolysis of acetylated glucosamine and galactosamine derivatives 1-11 catalyzed by
immobilized CRL and AXE (Table 1).
The results obtained demonstrated that this approach is versatile and can be conveniently used for the
preparation of different building blocks bearing only a free hydroxyl group at C-6 with presenting
different functional groups at anomeric position, including the thio-cyanomethyl group currently
employed for protein glycosylation. These intermediates can be conveniently glycosylated with
different sugar donors, allowing the preparation of glycans used for the synthesis of glycoconjugated
derivatives (neo-glycoproteins and glycosphingolipids) including sialylated products such as the STn
antigen.
Results and Discussion
Enzymatic hydrolysis of different substrates
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European Journal of Organic Chemistry
In this work, two immobilized enzymes have been compared (CRL and AXE) for the preparation of
various glucosamine and galactosamine building blocks. A screening has been performed at low
substrate concentration (10 mM) and in presence of 20% of acetonitrile in order to ensure the
complete solubility and hydrolysis of the substrates (Table 1).
Table 1. Enzymatic Hydrolysis of Different Derivatives of Glucosamine and Galactosamine
Substrates
Products
12
CRL: yield (h)
94% (24)
80% (24)
70% (24)
68% (48)
<10% (48)a
<10% (48)a
76% (6)
AXE: yield (h)
1: R,R4=OAc; R1,R3=H; R2=NHAc
10% (3)a
1b: R,R4=OAc; R1,R3=H; R2=NHAc
2: R,R3=H; R1=OMe; R2=NHAc R4=OAc
3: R,R3=H; R1=SCH2CN; R2=NHAc; R4=OAc
4: R,R3=H; R1=SEt; R2=NHAc; R4=OAc
5: R,R3=H; R1=SPh; R2=NHAc; R4=OAc
6: R,R4=OAc; R1,R3=H; R2=NPhth
-
12
94% (24)
90% (24)
85% (12)
65% (6)
<10% (24)a
12% (8)a
30% (96)a
<10% (194)a
-
13
14
15
16
17
7: R,R3=H; R1=OMe; R2=NPhth; R4=OAc
8: R,R3=H; R1=SEt; R2=NPhth; R4=OAc
9: R,R3=H; R1=SPh; R2=NPhth; R4=OAc
10c: R,R3=OAc; R1,R4=H; R2=NHAc
11: R,R4=H; R1=SEt; R2=NPhth; R3=OAc
85% (4)
18
64% (24)
38% (24)
70% (30)
80% (5)
19
20
21
22
-
aYield evaluated by HPLC analysis without isolation of the product.
bα/β=5/1. cα/β=85/15.
Experimental conditions: substrate 10 mM, volume 15 mL, phosphate buffer 50 mM,
acetonitrile 20% (for substrates 6 and 7, 25% of acetonitrile was used), pH 5, r.t., enzyme 1 g
of CRL-OD (1200 UI/g) or AXE (90 UI/g).
The esterase AXE was used immobilized by covalent attachment on epoxy carrier since covalent
immobilization generally ensures the stabilization of enzymes such as esterases or proteases. In the
case of CRL, this enzyme was used immobilized by adsorption on hydrophobic carrier because this
method is appropriate to obtain good stability and activity of lipases.[22]
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Except for 2-deoxy-2-acetamido-1,3,4,6-tetra-O-acetyl-α-D-glucopyranoside 1 (since AXE was
reported to catalyze hydrolysis the anomeric position of pyranoses with acetoxy group),[27]
considering the other N-acetylated substrates 2-5, the esterase AXE allowed best performances. In
fact, the pure α-anomer of 1, with the anomeric acetyl leaving group, can be used as substrate for
immobilized CRL according to the procedure previously reported.[28,29] In this work, we used CRL
immobilized on Sepabeads-OD (a C18 hydrophobic carrier) and the obtained results (Table 1) were
similar (94% of 2-deoxy-2-acetamido-1,3,4-tri-O-acetyl-α-D-glucopyranose 12) to those previously
reported using CRL immobilized on octyl-agarose (a C8 hydrophobic carrier). However, CRL-
Sepabeads-OD permitted much higher reaction rate providing almost quantitative hydrolysis of 1 in
24 hours. When the same reaction condition was used, the CRL immobilized on C8 agarose provided
only 75% of the substrate hydrolyzed in 48 hours. The enzymatic hydrolysis of 1 was performed up
to 20 mM concentration (about 8 g/L) and 92% yield of 12 was obtained after 24 hours. In addition,
the hydrolysis of 1 with CRL was also tested using the α and β mixture (5/1) avoiding the separation
of the two anomers after the chemical synthesis of this substrate (Table 1). The enzyme can be
completely selective to provide the pure α anomer of 12 in 80% yield, corresponding to almost
complete hydrolysis of the α-form of 1. In this case, the β anomer remains completely unreacted and
can be easily separated from the α-12.
Using AXE, methyl 2-deoxy-2-acetamido-3,4-di-O-acetyl-β-D-glucopyranoside 13 was prepared in
94% yield (Table 1), much better compared with the result obtained by means of CRL (70% yield).
Similarly, yields ranging from 65% to 90% of various sugar building blocks have been obtained with
AXE, starting from N-acetyl glucosamine derivatives with an anomeric S-alkyl group, including the
thio-cyanomethyl reactive linker used for the glycosylation of proteins. In fact, cyanomethyl 2-deoxy-
2-acetamido-3,4,-di-O-acetyl-1-thio-β-D-glucopyranoside 14 was prepared in 90% yield. This
reaction was scaled up to 50 mM concentration (about 20 g/L) of substrate 3 (see Table S1 in
Supporting Information).
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Similar results have been obtained in the hydrolysis of ethyl 2-deoxy-2-acetamido-3,4,6-tri-O-acetyl-
1-thio-β-D-glucopyranoside 4 providing ethyl 2-deoxy-2-acetamido-3,4-di-O-acetyl-1-thio-β-D-
glucopyranoside 15 (85% yields). Lower yield (Table 1) was obtained in the hydrolysis of phenyl 2-
deoxy-2-acetamido-3,4,6-tri-O-acetyl-1-thio-β-D-glucopyranoside 5 (65% yield of product 16).
Also in the hydrolysis of the 2-deoxy-2-phthalimido glucopyranoside derivatives 6-9, good or very
good yields were obtained, but in these cases CRL provided the best results. Starting from the 2-
deoxy-2-phthalimido-1,3,4,6-tetra-O-acetyl-α-D-glucopyranose 6, 76% yield of 2-deoxy-2-
phthalimido-1,3,4-tri-O-acetyl-α-D-glucopyranose 17 (with the anomeric α-O-acetyl group) was
obtained in 6 h of reaction (Table 1). As observed for other substrates, also in this case the reaction
catalyzed by the Octyl-agarose derivative of CRL proceeded much slower (18 hours for complete
hydrolysis of 6) compared with the CRL-OD derivative. This reaction was performed by increasing
the substrate concentration up to 10 g/L (see Table S2 Supporting Information).
Products with one free hydroxyl group at C-6 in good yields have also been obtained in the hydrolysis
of methyl 2-deoxy-2-phthalimido-3,4,6-tri-O-acetyl-β-D-glucopyranoside 7, (85% of product 18),
and ethyl 2-deoxy-2-phthalimido-3,4,6-tri-O-acetyl-1-thio-β-D-glucopyranoside 8, (64% of product
19). About 90% of these two substrates, characterized by a little O- or S-alkyl anomeric group, were
hydrolyzed in 4 and 24 hours, respectively. Lower yield was obtained in the hydrolysis of phenyl 2-
deoxy-2-phthalimido-3,4,6-tri-O-acetyl-1-thio-β-D-glucopyranoside 9, because this reaction
proceeded slowly (38% yield of product 20 in 24 hours).
Finally, the enzymatic hydrolysis of galactosamine derivatives was also investigated. The hydrolysis
of 2-deoxy-2-acetamido-1,3,4,6-tetra-O-acetyl-α-D-galactopyranose 10 (Table 1), obtained by
chemical synthesis in α and β mixture (85/15), has been performed using CRL. This reaction provided
2-deoxy-2-acetamido-1,3,4-tri-O-acetyl-α-D-galactopyranose 21 (with the anomeric O-acetyl group)
with 70% yield in pure α form (the β anomer is not hydrolyzed by CRL) and was scaled up by
improving the substrate concentration up to 10 g/L (70% yield after purification). The hydrolysis of
the ethyl 2-deoxy-2-phthalimido-3,4,6-tri-O-acetyl-1-thio-β-D-galactopyranoside 11 proceeded
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European Journal of Organic Chemistry
much faster compared with the fully acetylated galactosamine 10, and ethyl 2-deoxy-2-phthalimido-
3,4-di-O-acetyl-1-thio-β-D-galactopyranoside 22 was obtained in very good yield (80% after
purification) after only 5 hours of reaction (Table 1).
Synthesis of disaccharides and glycoconjugated derivatives
The different building blocks prepared by enzymatic hydrolysis have been then used as intermediates
for the preparation of disaccharides. Starting from 14, products 23 and 24 (with the anomeric thio-
cyanomethyl reactive linker) have been synthesized in almost pure anomeric form with good yields
(Scheme 2).
Scheme 2. (i) a) 14, Gal-TCA, BF3∙Et2O, CH2Cl2, N2, 0 °C, (23, 68% yield; β>95%); b) 14, Man-TCA, BF3∙Et2O,
CH2Cl2, N2, 0°C, (24, 63% yield; α>95%); (ii) a) MeONa/MeOH, r.t., 24 h, 50% yield IME-glycans; b) sodium
tetraborate buffer, pH 9.5, 25 °C, molar ratio IME-glycan/ribonuclease A 100:1, 6 h; 23a yield 81%; 24a yield>98%;
(iii) a) 14, sialyl xanthate, NIS, TfOH, CH3CN; -40 °C; 25 yield 55% (α/β 3/2); b) 12, sialyl xanthate, PhSCl, AgOTf,
CH3CN and CH2Cl2 (2/1), N2; -68 °C; 26 yield 60% (α/β 2/1); c) 21, sialyl xanthate, PhSCl, AgOTf, CH3CN and
CH2Cl2 ( 2/1), N2; -68 °C; 27 yield 64% (α/β 1/1).
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These compounds, after activation to obtain the corresponding deprotected iminomethoxyethyl
thioglycosides, (see Supporting Information), have been used for the preparation of neo-glycoproteins
23a and 24a in 80% and 100% yields respectively, as resulting by MS analysis (Figure 1) considering
the % of glycosylated protein (RNase-A selected as model of carrier protein). These results
demonstrate that the proposed approach, mediated by regioselective hydrolysis catalyzed by AXE, is
suitable for the preparation of neo-glycoproteins with glycans having a glucosamine core.
However, when the building block 14 was used for the preparation of sialylated glyco-derivatives
(potentially advantageous for the preparation of sialylated neo-glycoprotein), the glycosylation with
sialyl xanthate afforded product 25 (55% yield) in 3/2 α/β mixture (Scheme 2). Similarly, sialylation
of N-acetyl glucosamine and galactosamine building blocks 12 and 21, allowed preparation of the
disaccharides 26 and 27 as a mixture of α/β anomers.
The difficulties intrinsic in the stereo-controlled synthesis of α-sialosides have been largely reported
and complex procedures are required in order to obtain pure or almost pure anomers.[30] In fact, α/β
mixtures are obtained in different ratio using monodeprotected sugar acceptors, regardless the
protection used for the C-2 amino group of the pyranose acceptor or using precursors with the 2-azido
group.[31,32] In our work, this effect was observed regardless the kind of sialyl-donor used in the
glycosylation of 2-deoxy-2-acetamido glucose or galactose building blocks. In addition, the
sialylation of 21 was investigated in different conditions (including the use of sialyl chloride donor),
but in all cases similar yields and an anomeric mixtures of 27 was obtained (see Supporting
Information).
Surprisingly, high stereoselectivity was obtained when N-phthalimido glucosamine or galactosamine
intermediates were considered for the sialylation after enzymatic deprotection at C-6. Accordingly, it
was possible to perform the preparation of sialyl-disaccharides with different reactive groups at C-1,
in pure or almost pure α-form (Scheme 3). Product 28 (with 1-α-acetoxy) was obtained in 70% yield
as almost pure α anomer, allowing results much better compared with those previously reported.[31]
Similarly, pure α anomer of 29 (with 1-β-methoxy) was obtained in 64% yield.
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European Journal of Organic Chemistry
Figure 1. MS spectra obtained in the glycosylation of RNase A with the glycans IME-23 and IME-24 to obtain
neo-glycoproteins 23a (A) (average number of incorporated saccharides: 2.3) and 24a (B) (average number of
incorporated saccharides: 4.1).
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Scheme 3. (i) a) sialyl xanthate, PhSCl, AgOTf, CH3CN and CH2Cl2 (2/1), N2, -68°C; 28 yield 70% (>98%); 29
yield 64% (>98%); 30 yield 32% (>98%); 31 yield 30% (>98%); b) sialyl chloride, AgOTf, CH2Cl2, N2, -40 °C; 30
yield 65% (95%); 31 yield 70% (95%). (ii) 28, NH2CH2CH2NH2, AcOH, THF, r.t., 3 h, 32 (75% crude). (iii) 32,
CCl3CN, DBU, CH2Cl2, -5 °C, 2 h, 33 (60%, α/β 4/1). (iv) 33, 3-O-benzoyl-azidosphingosine, BF3∙Et2O, CH2Cl2, -
30 °C, 1.5 h, 34 (61%).
Much poorer performances were observed when sialyl xanthate was used as donor for sialylation of
N-phthalimido- glucosamine 19 and galactosamine 22 (with 1-β-thioethyl), probably as consequence
of the activation of the anomeric S-alkyl group in the reaction condition used that causes the formation
of by-products. In fact, starting from N-phthalyl-glucosamine 19, pure 30 was obtained only in 32%
yield, and the sialyl-Tn derivative 31 was produced in 30% yield from N-phthalyl-galactosamine 22
(see Supporting Information). For improving the yield in the sialylation of N-phthalimido building
blocks, the sialyl chloride was investigated as donor in the glycosylation of intermediates 19 and 22
(Scheme 3). In this condition, 65% and 70% yields were obtained respectively for product 30 and
sialyl-Tn precursor 31 (in both cases as 95/5 mixture of α/β isomers).
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These results suggest that the presence of an acetamido group at position C-2, unlike the 2-
phthalimido group, could be responsible for the formation of the β-anomer during sialylation. In
particular, the 2-acetamido group could be involved in the formation of a hydrogen bond with the
carboxy-ester of the sialic acid donor. Accordingly, this effect was not observed in the glycosylation
of N-acetamido-glucosamine intermediate 14 with the Gal-TCA or Man-TCA. In fact, disaccharides
23 and 24 were obtained in pure β and α-form, respectively (Scheme 2). Consequently, the use of 2-
phthalimido pyranoses should be preferred for the synthesis of sialyl derivatives.
Finally, starting from the sialylated disaccharide 28 (Scheme 3), after removing the anomeric acetyl
group (yielding 75% of the crude product 32) and activation as trichloroacetimidate (yield in 60% of
33 after purification), the glycosylation of the 3-O-benzoyl-azidosphingosine building block provided
the sialylated glycosphingolipid 34 in 61% yield.
Conclusions
In summary, a new chemoenzymatic synthetic strategy has been investigated and optimized for the
preparation of neo-glycoproteins and glycolipids with a core of glucosamine or galactosamine, which
are interesting as potential anticancer agents. A regioselective enzymatic hydrolysis can be
conveniently used for the synthesis of 2-amino-pyranose building blocks bearing free hydroxyl group
in primary position, starting from different acetylated 2-deoxy-2-acetamido or 2-deoxy-2-
phthalimido gluco- and galactopyranoses. However, depending on the product desired, the
appropriate starting material (substrate) and enzyme should be selected.
Thus, AXE can be used for the synthesis of neo-glycoproteins with an N-acetyl glucosamine core. In
fact, the hydrolysis catalyzed by this enzyme provided different intermediates with a β-1-thio-(S-
alkyl) group in very good yields (from 65% to 95%). For example, cyanomethyl 2-deoxy-2-
acetamido-3,4,-di-O-acetyl-1-thio-β-D-glucopyranoside 14 was prepared in 90% yield and, after
glycosylation, two disaccharides with the thiocyanomethyl reactive linker at anomeric position were
obtained and used for the preparation of neo-glycoproteins.
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CRL provides C-6 monodeprotected 2-N-phthalyl pyranose building blocks in good yields (from 65%
to 85%) with different groups at anomeric position that can be used for the preparation of new
sialylated glycoderivatives. Accordingly, the synthesis of protected Sialyl-Tn antigen 31 has been
performed in two steps (56% yield) while the glycosphingolipid 34 has been prepared in 3 steps in
40% yield.
The monodeprotected compounds obtained in this work can be used as intermediates for the
preparation of different products as potential new anticancer agents. Different glycosphingolipids will
be prepared using this synthetic route and investigated as anticancer GM3 analogues.
Experimental Section
Materials and general experimental methods
2-acetamido-1,3,4,6-tetra-O-acetyl-2-deoxy-α-D-glucopyranoside (1) and 2-acetamido-2-deoxy-D-
galactopyranoside, Candida rugosa lipase were purchased from Sigma-Aldrich (Italy, Milan). The
octadecyl-SepabeadsTM was kindly supplied by Resindion (Italy). Immobilized AXE was kindly
donated by ACS Dobfar (Italy). All chemicals were purchased as reagent grade and used without
further purification. All chemical reactions were carried out under N2 atmosphere and anhydrous
conditions with freshly distilled solvents, unless otherwise noted. Reactions were monitored by thin-
layer chromatography (TLC) on a pre-coated plate of silica gel 60 F254 (Merck) and detection by
staining with sulfuric acid. Solvents were evaporated under reduced pressure and below 40 °C (water
bath). Column chromatography was performed on silica gel 60 (230-400 mesh, Merck). 1H NMR and
13C NMR spectra were recorded at 400 MHz with Bruker AVANCE DRX 400 spectrometer. The
chemical shifts were referenced to the solvent peak, 7.26 ppm (1H) and 77.16 ppm (13C) for CDCl3 at
25 °C, and coupling constants were given in Hz. High-resolution mass spectra (HRMS) were recorded
with a Bruker micro-TOF spectrometer in electrospray ionization (ESI) mode, using Tuning-Mix as
reference. All the new compounds were fully characterized by 1H and 13C NMR, as well as HRMS.
All the known products were characterized only by 1H NMR by comparison with literatures.
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Synthesis of substrates 1-11
Synthesis of substrates 1-11 has been performed using conventional chemical procedures.
Experimental detail and analytical characterization are reported in the Supporting Information.
Immobilization of Candida rugosa lipase (CRL)
Immobilization of CRL was performed slightly modifying the procedure previously reported.[33]
Briefly, the crude extract of CRL (2 g) was diluted in 25 mM phosphate buffer pH 7 (32 mL) and
kept stirring for 30 min. 5 g of octadecyl-SepabeadsTM, previously washed with the same buffer, was
added and the suspension was stirred at r.t. for 3 h. The enzyme derivative (CRL-OD) was then
filtered and washed with distilled water.
The activity of the enzyme was measured at pH 7.0 and r.t. in the hydrolysis of tripropionin according
to the literature.[34]
Enzymatic hydrolysis
The enzymatic hydrolysis of substrates 1-11 (10 mM) was carried out in 50 mM phosphate buffer (15
mL) containing 20% acetonitrile (for 6 and 7, 25% of acetonitrile for complete solubility of the
substrates) under mechanical stirring. The reaction started after the addition of 1 g of immobilized
enzyme CRL-OD (1200 UI/g) or AXE (90 UI/g). During the reaction, the pH of the solution was
maintained by automatic titration. The course of the hydrolysis reaction was monitored by HPLC
(HPLC analysis: 30-50% acetonitrile in phosphate buffer (10 mM) at pH 4, flow rate 1.0 mL/min, λ
= 210 nm) and TLC. After complete or almost-complete consumption of the substrate, the reaction
was stopped by biocatalyst filtration, and the obtained products were isolated by extraction with ethyl
acetate followed by purification by flash chromatography (product 12, 13 and 14: CH2Cl2-MeOH,
95:5, product 15: CH2Cl2-Et2O, 7:3, product 16: EtOAc-Hexane, 4:1, product 17: Toluene-EtOAc,
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3:2, product 18 and 21: Hexane-EtOAc, 1:1, product 19, 20 and 22: Hexane-EtOAc, 3:2) and
characterized by NMR spectra and mass spectrometry.
Analytical data of enzymatic products 12-22 (1H, 13C NMR and MS)
2-acetamido-1,3,4-tri-O-acetyl-2-deoxy-α-D-glucopyranoside (12). Rf = 0.36 (CH2Cl2-MeOH, 95:5).
The 1H NMR is accordance with the literature previously reported.[28] 1H NMR (400 MHz, CDCl3):
δ 6.30 (d, J=3.3 Hz, 1H, H-1), 5.64 (d, J=8.2 Hz, 1H, NH), 5.32 (t, J=9.8 Hz, 1H, H-3), 5.18 (t, J=9.6
Hz, 1H, H-4), 4.49 (m, 1H, H-2), 3.84 (m, 1H, H-5), 3.60, 3.74 (dd, 2H, H-6), 2.22 (s, 3H, OAc), 2.11
(s, 3H, OAc), 2.05 (s, 3H, OAc), 1.96 (s, 3H, NAc).
Methyl-2-acetamido-3,4-di-O-acetyl-2-deoxy-β-D-glucopyranoside (13). The 1H NMR is in
accordance with the literature previously reported.[35] Rf = 0.30 (CH2Cl2-MeOH, 95:5). 1H NMR (400
MHz, CDCl3): δ 5.90 (d, J=8.4 Hz, 1H, NH), 5.30 (dd, J=10.2, 9.8 Hz, 1H, H-3), 5.10 (t, J=9.8 Hz,
1H, H-4), 4.60 (d, J=8.2 Hz, 1H, H-1), 4.22 (dd, J=12.6, 4.0 Hz, 1H, H-6a), 4.16 (dd, J=12.4, 2.4 Hz,
1H, H-6b), 3.82 (dd, J=10.3, 8.4 Hz, 1H, H-2), 3.70-3.78 (m, 1H, H-5), 3.50 (s, 3H, OCH3), 2.08 (s,
3H, OAc), 2.02 (s, 3H, OAc), 1.98 (s, 3H, NAc).
Cyanomethyl 2-acetamido-3,4-di-O-acetyl-2-deoxy-1-thio-β-D-glucopyranoside (14). Rf = 0.28
1
(CH2Cl2-MeOH, 95:5). The H NMR is accordance with the literature previously reported.[36] 1
H
NMR (400 MHz, CDCl3): δ 5.80 (d, J=9.5 Hz, 1H, NH), 5.20 (t, J=9.5 Hz, 1H, H-3), 5.13 (t, J=9.5
Hz, 1H, H-4), 4.74 (d, J=10.3 Hz, 1H, H-1), 4.20 (td, J=10.3, 9.5 Hz, 1H, H-2), 3.74 (d, J=17.3 Hz,
1H, CH2CN), 3.80-3.71 (m, 1H, H-6a), 3.66-3.61 (m, 1H, H-5), 3.60-3.58 (m, 1H, H-6b), 3.32 (d,
J=17.3 Hz, 1H, CH2CN), 2.07 (s, 3H, OAc), 2.06 (s, 3H, OAc), 1.97 (s, 3H, NAc).
Ethyl-2-acetamido-3,4-di-O-acetyl-2-deoxy-1-thio-β-D-glucopyranoside (15). The product is as a
white solid, m.p.: 65-68 ℃. Rf = 0.36 (CH2Cl2-Et2O, 7:3). 1H NMR (400 Hz, CDCl3): δ 5.85 (d, J=11.4
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Hz, 1H, NH), 5.20 (t, J=8.5 Hz, 1H, H-3), 5.03 (t, J=8.5 Hz, 1H, H-4), 4.61 (d, J=10.4 Hz, 1H, H-1),
4.10 (dd, J=19.3, 10.4 Hz, 1H, H-2), 3.76-3.52 (m, 3H, H-5, H-6), 2.76-273 (m, 2H, SCH2CH3), 2.04
(s, 3H, OAc), 2.03 (s, 3H, OAc), 1.95 (s, 3H, NAc), 1.25 (t, J=7.4 Hz, 3H, SCH2CH3). 13C NMR (100
Hz, CDCl3): δ 14.87 (SCH2CH3), 20.78, 20.84 (OAc), 23.38 (NHAc), 24.12 (SCH2CH3), 53.33 (C-
2), 61.74 (C-6), 68.92 (C-4), 73.95 (C-3), 78.44 (C-5), 84.43 (C-1), 170.12, 170.29, 171.27 (C=O).
ESI-HRMS (m/z) calcd for C14H23NNaO7S [M+Na]+: 372.1093, found: 372.1078.
Phenyll-2-acetamido-3,4-di-O-acetyl-2-deoxy-1-thio-β-D-glucopyranoside (16). The product is as a
white solid, m.p.: 88-91 ℃. Rf = 0.30 (EtOAc-Hexane, 4:1). 1H NMR (400 Hz, CDCl3): δ 7.48-7.46
(m, 2H, ArH), 7.33-7.30 (m, 3H, ArH), 5.58 (d, J=9.4 Hz, 1H, NH), 5.24 (t, J=8.8 Hz, 1H, H-3), 5.01
(t, J=9.8 Hz, 1H, H-4), 4.88 (d, J=10.2 Hz, 1H, H-1), 4.04 (dd, J=20.3, 10.4 Hz, 1H, H-2), 3.74-3.69
(m, 1H, H-5), 3.62-3.52 (m, 2H, H-6), 2.04 (s, 3H, OAc), 2.03 (s, 3H, OAc), 1.98 (s, 3H, NAc). 13C
NMR (100 Hz, CDCl3): δ 20.78, 20.84 (OAc), 23.49 (NHAc), 53.70 (C-2), 61.88 (C-6), 68.78, 73.76,
78.47 (C-3, C-4, C-5), 86.70 (C-1), 129.24, 132.50 (aromatic C), 170.06, 170.11, 171.23 (C=O). ESI-
HRMS (m/z) calcd for C18H23NNaO7S [M+Na]+: 420.1093, found: 420.1069.
1,3,4-tri-O-acetyl-2-phthalimido-2-deoxy-α-D-glucopyranoside (17). Rf = 0.36 (Toluene-EtOAc,
3:2). The 1H NMR is accordance with the literature previously reported.[37] 1H NMR (400 Hz, CDCl3):
δ 7.87-7.83 (m, 2H, phthalimido protons), 7.75-7.72 (m, 2H, phthalimido protons), 6.60 (dd, J=11.5,
9.1 Hz, 1H, H-3), 6.29 (d, J=3.3 Hz, 1H, H-1), 5.15 (t, J=9.6 Hz, 1H, H-4), 4.71 (dd, J=11.6, 3.3 Hz,
1H, H-2), 4.15-4.08 (m, 1H, H-5), 3.82-3.62 (2m, 2H, H-6), 2.09 (s, 3H, OAc), 2.08 (s, 3H, OAc),
1.88 (s, 3H, OAc).
Methyl-3,4-di-O-acetyl-2-phthalimido-2-deoxy-β-D-glucopyranoside (18). The product is as
1
colorless oil. Rf = 0.40 (Hexane-EtOAc, 1:1). H NMR (400 Hz, CDCl3): δ 7.88-7.84 (m, 2H,
phthalimido protons H), 7.75-7.73 (m, 2H, phthalimido protons), 5.83 (dd, J=9.8, 1.6 Hz, 1H, H-3),
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5.34 (d, J=9.6 Hz, 1H, H-1), 5.12 (t, J=9.2 Hz, 1H, H-4), 4.28 (dd, J=8.8, 2.6 Hz, 1H, H-2), 3.84-3.81
(m, 1H, H-5), 3.71-3.64 (m, 2H, H-6), 3.45 (s, 3H, OCH3), 2.07 (s, 3H, OAc), 1.87 (s, 3H, OAc). 13C
NMR (100 Hz, CDCl3): δ 20.67 (OAc), 20.92 (OAc), 50.65 (C-2), 60.52 (OMe), 61.21 (C-6), 66.45
(C-4), 70.17 (C-3), 72.80 (C-5), 90.63 (C-1), 124.07, 134.99 (aromatic C), 167.60-171.29 (C=O).
ESI-HRMS (m/z) calcd for C19H21NNaO9 [M+Na]+: 430.1114, found: 430.1126.
Ethyl-3,4-di-O-acetyl-2-phthalimido-2-deoxy-1-thio-β-D-glucopyranoside (19). Rf = 0.41 (Hexane-
1
EtOAc, 3:2). The H NMR is accordance with the literature previously reported.[38] 1H NMR (400
MHz, CDCl3): δ 7.75-7.95 (m, 4H, phthalimido protons), 5.90 (t, J=8.8 Hz, 1H, H-3), 5.52 (d, J=10.5
Hz, 1H, H-1), 5.16 (t, J=8.6 Hz, 1H, H-4), 4.40 (t, J=9.5 Hz, 1H, H-2), 4.31 (dd, J=11.7, 5.1 Hz, 1H
, H-5), 3.72 (m, 1H, H-6a), 3.68 (m, 1H, H-6b), 2.60-2.82 (m, 2H, SCH2CH3), 2.15 (s, 3H, OAc),
1.89 (s, 3H, OAc), 1.22 (t, J=7.4 Hz, 3H, SCH2CH3).
Phenyl-3,4-di-O-acetyl-2-phthalimido-2-deoxy-1-thio-β-D-glucopyranoside (20). Rf = 0.38 (Hexane-
1
EtOAc, 3:2). The H NMR is accordance with the literature previously reported.[38] 1H NMR (400
MHz, CDCl3): δ 7.94-7.75 (m, 4H, phthalimido protons), 7.48-7.26 (m, 5H, Ph), 5.85 (dd, J=9.5 Hz,
1H, H-3), 5.79 (d, J=10.6 Hz, 1H, H-1), 5.14 (t, J=9.6 Hz, 1H, H-4), 4.39 (t, J=10.6 Hz, 1H, H-2),
3.84–3.61 (m, 3H, H-5, H-6), 2.05 (s, 3H, OAc), 1.87 (s, 3H, OAc).
2-acetamido-1,3,4-tri-O-acetyl-2-deoxy-α-D-galactopyranoside (21). Rf = 0.30 (EtOAc-Hexane,
1
1:1). The H NMR is accordance with the literature previously reported.[28] 1H NMR (400 MHz,
CDCl3): δ 6.25 (d, J=3.6 Hz, 1H, H-1), 5.58 (bd, 1H, NH), 5.45 (bdd, 1H, H-4), 5.30 (dd, J=8.7, 3.1
Hz, 1H, H-3), 4.80 (ddd, J=11.0, 9.8, 3.6 Hz, 1H, H-2), 4.08 (t, J=12.8 Hz, 1H, H-5), 3.50-3.64 (m,
2H, H-6), 2.23 (s, 3H, OAc), 2.18 (s, 3H, OAc), 2.08 (s, 3H, OAc), 1.96 (s, 3H, NAc).
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Ethyl-3,4-di-O-acetyl-2-phthalimido-2-deoxy-1-thio-β-D-galactopyranoside (22). The product is as
a white solid, m.p.: 78-80 ℃. Rf = 0.35 (Hexane-EtOAc, 3:2). 1H NMR (400 Hz, CDCl3): δ 7.87-7.85
(m, 2H, phthalimido protons), 7.76-7.74 (m, 2H, phthalimido protons), 5.85 (dd, J=10.6, 3.2 Hz, 1H,
H-3), 5.50 (d, J=3.0 Hz, 1H, H-4), 5.48 (d, J=10.4 Hz, 1H, H-1), 4.63 (t, J=10.5 Hz, 1H, H-2), 3.98-
3.95 (m, 1H, H-5), 3.78, 3.55 (2m, 2H, H-6), 2.75-2.67 (m, 2H, SCH2CH3), 2.22 (s, 3H, OAc), 1.86
13
(s, 3H, OAc), 1.21 (t, J=7.2 Hz, 3H, SCH2CH3). C NMR (100 Hz, CDCl3): δ 15.11 (SCH2CH3),
20.67 (OAc), 20.92 (OAc), 24.71 (SCH2CH3), 50.65 (C-2), 61.21 (C-6), 67.97 (C-4), 69.07 (C-3),
77.69 (C-5), 81.85 (C-1), 123.77, 123.86, 134.42, 134.47, 134.54 (aromatic C), 167.60, 168.11,
169.80, 171.29 (C=O). ESI-HRMS (m/z) calcd for C20H23NNaO8S [M+Na]+: 460.1042, found:
460.1025.
Synthesis of disaccharides 23-31
Cyanomethyl-(2’,3’,4’,6’-tetra-O-acetyl-β-D-galactopyranosyl)-(1→6)-2-acetamido-3,4-di-O-
acetyl-2-deoxy-1-thio-β-D-glucopyranoside-(23).
2,3,4,6-Tetra-O-acetyl-α-D-galactopyranose
trichloroacetimidate (0.137 g, 0.278 mmol) and compound 14 (0.05 g, 0.139 mmol) were co-
evaporated with toluene (2×10 mL) and dried under reduced pressure. The mixture was then dissolved
in dry CH2Cl2 (25 mL) in presence of 4Å molecular sieves and cooled at 0 °C. The BF3·Et2O (0.034
mL, 0.278 mmol) was added and the reaction allowed to warm to r.t. After 2.5 h, the reaction mixture
was quenched with Et3N, filtered and concentrated in vacuo. The residue was purified by flash column
chromatography (EtOAc-Et2O, 3:2) to afford disaccharide 23 as a white solid (0.065 g, 68 %). Rf =
0.37 (EtOAc-Et2O, 3:2). m.p.: 106-108 ℃. 1H NMR (400 MHz, CDCl3): δ 5.71 (d, J=10.0 Hz, 1H,
NH), 5.21-5.10 (m, 2H, H-4’, H-3), 5.01 (dd, J=10.5, 3.0 Hz, 1H, H-2’), 4.94 (t, J=9.1 Hz, 1H, H-
3’), 4.64 (d, J=10.1 Hz, 1H, H-1), 4.56 (d, J=7.8 Hz, 1H, H-1’), 4.21-4.09 (m, 4H, H-2, H-4, H-6a,
H-6a’), 3.93-3.87 (m, 2H, H-6b, H-6b’), 3.79-3.73 (m, 2H, H-5, CHCN), 3.65-3.60 (m, 1H, H-5’),
13
3.27 (d, J=17.3 Hz, 1H, CHCN), 1.96-2.14 (7s, 21H, OAc). C NMR (100 MHz, CDCl3): δ 14.55
(SCH2CN), 20.39-29.57 (7CH3), 52.70 (C-2), 61.38 (C-6), 67.14 (C-6’), 68.50 (C-5’), 68.81 (C-4),
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68.85 (C-2’), 70.88, 70.89 (C-3’, C-4’), 73.39 (C-3), 77.77 (C-5), 83.48 (C-1), 101.44 (C-1’), 116.86
(SCH2CN), 169.56-171.40 (7C=O). ESI-HRMS (m/z) calcd for C28H38N2NaO16S [M+Na]+:
713.1840, found: 713.1859.
Cyanomethyl-(2’,3’,4’,6’-tetra-O-acetyl-α-D-mannopyranosyl)-(1→6)-2-acetamido-3,4-di-O-
acetyl-2-deoxy-1-thio-β-D-glucopyranoside
(24).
2,3,4,6-tetra-O-acetyl-α-D-mannopyranose
trichloroacetimidate (0.20 g, 0.417 mmol) and compound 14 (0.075 g, 0.208 mmol) were co-
evaporated with toluene (2×10 mL) and dried under reduced pressure. The mixture was then dissolved
in dry CH2Cl2 (25 mL) in presence of 4Å molecular sieves and cooled at 0 °C. The BF3·Et2O (0.058
mL, 0.417 mmol) was added and the reaction allowed to warm to r.t. After 2.5 h, the reaction mixture
was quenched with Et3N, filtered and concentrated in vacuum. The residue was purified by flash
column chromatography (EtOAc-Et2O, 3:2) to afford disaccharide 24 as a white solid (0.1 g, 70 %).
Rf = 0.39 (EtOAc-Et2O, 3:2). m.p.: 99-102 ℃. 1H NMR (400 MHz, CDCl3): δ 5.92 (d, J=9.6 Hz,
1H, NH), 5.31-5.01 (m, 5H, H-4’, H-3’, H-1’, H-4, H-4’, H-5’), 4.66 (d, J=10.6 Hz, 1H, H-1), 4.58
(dd, J=13.6, 2.1 Hz, 1H, H-3), 4.28-4.03 (m, 5H, H-2, H-2’, H-6, Ha-6’), 3.86 (t, J=9.6 Hz, 1H, H-
5), 3.71-3.65 (m, 2H, CHCN, Hb-6’), 3.30 (d, J=17.6 Hz, 1H, CHCN), 2.14, 2.13, 2.11, 2.09, 2.04,
13
1.98, 1.96 (7s, 21H, OAc). C NMR (100 MHz, CDCl3): δ 14.72 (SCH2CN), 20.97-20.77 (6CH3,
OAc), 23.21 (CH3, NAc), 52.96 (C-2), 62.33 (C-6, C’-6) , 62.62 (C’-2), 65.73, 68.67, 69.70, 70.13
(C-3, C’-3, C’-4, C’-5, C-4), 75.30 (C-5), 83.09 (C-1), 100.06 (C’-1), 116.17 (SCH2CN), 169.71-
171.80 (7C=O). ESI-HRMS (m/z) calcd for C28H38N2NaO16S [M+Na]+: 713.1840, found:
713.1828.
Cyanomethyl O-(methyl 5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero-α/β-D-galacto-2-
nonulopyranosylonate)-(2→6)-2-acetamido-3,4-di-O-acetyl-2-deoxy-β-D-glucopyranose (25). The
sialyl xanthates (248 mg, 0.42 mmol) and compound 14 (100 mg, 0.28 mmol) were dissolved in the
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CH3CN (4.5 mL) stirred for 1 h with 4Å powdered molecular sieves. Then it cooled to -40 °C, and
NIS (187 mg, 0.83 mmol) and TfOH (36 µL, 0.46 mmol) were added. After 2 h, the reaction was
quenched with Et3N. The mixture was diluted with CH2Cl2, filtered through celite, washed with 20%
aqueous Na2S2O3 solution, dried over MgSO4, and concentrated under reduced pressure. The residue
was purified by column (Cy-EtOAc, 1:4) to obtain the disaccharide 25 (127 mg, 55%) as α and β
1
1
isomer in the ratio of 3:2 from H NMR. Rf = 0.35 (Cy-EtOAc 1:4, twice). H NMR (400 MHz,
CDCl3): δ 4.63, 4.62, H-1’; 3.81, 3.79, COOCH3; 3.51, 3,25, SCH2CN; 2.64, 2.48, H-3eq; 2.20-1.89,
13
OAc, NAc. C NMR (100 MHz, CDCl3): δ 171.93-167.49, C=O; 115.18, CN; 98.42, 98.32, C-2;
83.53, 83.50, C-1’; 52.94, 52.77, COOCH3; 51.12, C-2’; 49.33, C-5; 37.59, C-3; 23.22-20.61, NAc,
OAc; 14.20, SCH2CN. ESI-HRMS (m/z) calcd for C34H47NO22Na [M+Na]+: 870.2579, found:
870.2571.
O-(methyl-5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero-α/β-D-galacto-2-
nonulopyranosylonate)-(2→6)-2- acetamido-1,3,4-tri-O-acetyl-2-deoxy-α-D-glucopyranose (26).
The method in the literature[39] was used with some modifications: A mixture of sialyl xanthates (137
mg, 0.23 mmol) and compound 12 (40 mg, 0.12 mmol) with 4Å powdered molecular sieves were
dissolved in the dry CH3CN (2.4 mL) and CH2Cl2 (1.2 mL) stirring at r.t. for 1 h. Further, AgOTf (50
mg, 0.19 mmol) and DTBP (46 µL, 0.21 mmol) were added, and the mixture was cooled to -68 oC
and kept protected from light. Then PhSCl (22 µL, 0.19 mmol) in the dry CH2Cl2 (0.12 mL) was
added by running the solution down the cold wall of the reaction flask. Finally, the mixture was stirred
for 2.5 h at -68 oC. After that, the mixture was diluted with a suspension of silica gel (0.5 g) in EtOAc
(3 mL), filtered through celite, washed with saturated aqueous NaHCO3 and water, dried with MgSO4,
and concentrated under reduced pressure. The residue was chromatographed (MeOH-EtOAc 1:20 to
1:5) to give disaccharide 26 (58 mg, 60%) as α and β isomer in the ratio of 2:1 from 1H NMR. Rf =
1
0.29 (MeOH-EtOAc 1:10). H NMR (400 MHz, CDCl3): δ 6.22, 6.16, H-1’; 3.81, 3.79 COOCH3;
2.63, 2.47, H-3eq; 2.20-2.05, OAc; 1.94-1.89 NAc. 13C NMR (100 MHz, CDCl3): δ 171.93-167.49,
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C=O; 98.42, 98.32, C-2; 90.82, 90.68, C-1’; 52.94, 52.77, COOCH3; 51.21, 51.12, C-2’; 49.33, C-5;
37.59, C-3; 23.22-23.08, NAc; 21.18-20.61, OAc. ESI-HRMS (m/z) calcd for C34H47NO22Na
[M+Na]+: 857.2804, found: 857.2800.
O-(Methyl-5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero-α/β-D-galacto-2-
nonulopyranosylonate)-(2→6)-2-acetamido-1,3,4-tri-O-acetyl-2-deoxy-α-D-galactopyranose (27). A
mixture of sialyl xanthates (343 mg, 0.58 mmol) and compound 21 (40 mg, 0.29 mmol) with 4Å
powdered molecular sieves were dissolved in the dry CH3CN (6.0 mL) and CH2Cl2 (3.0 mL) stirring
at r.t. for 1 h. Further, AgOTf (123 mg, 0.47 mmol) and DTBP (115 µL, 0.53 mmol) were added, and
the mixture was cooled to -68 oC and kept protected from light. Then PhSCl (54 µL, 0.47 mmol) in
the dry CH2Cl2 (0.3 mL) was added by running the solution down the cold wall of the reaction flask.
Finally, the mixture was stirred for 3 h at -68 oC. After that, the mixture was diluted with a suspension
of silica gel (1.25 g) in EtOAc (7.5 mL), filtered through celite, washed with saturated aqueous
NaHCO3 and water, dried with MgSO4, and concentrated under reduced pressure. The residue was
chromatographed (MeOH-EtOAc 0:1 to 1:9) to give disaccharide 27 (150 mg, 64%) as α and β isomer
in the ratio of 1:1 from 1H NMR. Rf = 0.26 (EtOAc, twice). 1H NMR (400 MHz, CDCl3): δ 6.18, H-
13
1’; 3.78, 3.76 COOCH3; 2.50, 2.43, H-3eq; 2.18-1.85, OAc, NAc. C NMR (100 MHz, CDCl3): δ
172.06-167.15, C=O; 98.84, 98.58, C-2; 90.32, C-1’; 53.12, 52.03, COOCH3; 49.33, C-2’; 48.00, C-
5; 37.88, 36.57, C-3; 23.26-20.81, NAc, OAc. ESI-HRMS (m/z) calcd for C34H47NO22Na [M+Na]+:
857.2804, found: 857.2815.
O-(methyl-5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero-α-D-galacto-2-
nonulopyranosylonate)-(2→6)-1,3,4-tri-O-acetyl-2-phthalimido-2-deoxy-α-D-glucopyranoside (28).
A mixture of sialyl xanthates (912 mg, 1.54 mmol) and acceptor 17 (370 mg, 0.85 mmol) with 4Å
powdered molecular sieves were dissolved in the dry CH3CN (17.2 mL) and CH2Cl2 (8.6 mL) stirring
at r.t. for 1 h. Further, AgOTf (360 mg, 1.38 mmol) and DTBP (337 µL, 1.55 mmol) were added, and
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the mixture was cooled to -68 oC and kept protected from light. Then PhSCl (337 µL, 1.38 mmol) in
the dry CH2Cl2 (0.86 mL) was added by running the solution down the cold wall of the reaction flask.
Finally, the mixture was stirred for 3 h at -68 oC. After that, the mixture was diluted with a suspension
of silica gel (3.6 g) in EtOAc (22.5 mL), filtered through celite, washed with saturated aqueous
NaHCO3 and water, dried with MgSO4, and concentrated under reduced pressure. The residue was
chromatographed (Cy-EtOAc 1:3) to give disaccharide 28 (540 mg, 70%). Rf = 0.31 (Cy-EtOAc 1:3,
1
twice). H NMR (400 MHz, CDCl3): δ 7.84-7.81 (m, 2H, phthalimido protons), 7.73-7.71 (m, 2H,
phthalimido protons), 6.52 (dd, J=9.2, 2.2 Hz, 1H, H-3’), 6.24 (d, J=3.9 Hz, 1H, H-1’), 5.35-5.15 (m,
3H, H-4’, H-7, H-8), 4.89-4.82 (m, 1H, H-4), 4.68 (dd, J=8.2, 3.6 Hz, 1H, H-2’), 4.28-4.23 (m, 2H,
H-5’, H-9a), 4.03-3.99 (m, 4H, Ha-6’, H-5, H-6, H-9b), 3.80 (s, 3H, COOCH3), 3.40 (dd, J=11.4, 1.6
Hz, 1H, Hb-6’), 2.65 (dd, J=12.7, 4.4 Hz, 1H, H-3eq), 2.14 (s, 3H, OAc), 2.13 (s, 3H, OAc), 2.09 (s,
3H, OAc), 2.05 (s, 3H, OAc), 2.03 (s, 3H, OAc), 2.02 (s, 3H, OAc), 2.01 (s, 3H, OAc), 1.96 (t, J=3.6
Hz, 1H, H-3ax), 1.85 (s, 3H, NAc). 13C NMR (100 MHz, CDCl3): δ 171.17, 170.73, 170.40, 170.32,
170.22, 170.18, 169.83, 169.56, 169.52, 167.79 (C=O), 134.51, 123.81 (aromatic C), 98.46 (C-2),
90.80 (C-1’), 72.47, 70.74, 69.26, 69.14, 68.01, 67.50, 67.33 (C-6, C-5’, C-4’, C-4, C-8, C-3’, C-7),
62.60 (C-9), 62.30 (C-6’), 53.67 (C-2’), 53.05 (COOCH3), 49.50 (C-5), 37.89 (C-3), 23.33 (CH3,
NAc), 21.29, 21.15, 20.99, 20.96, 20.92, 20.85, 20.81 (CH3, OAc). ESI-HRMS (m/z) calcd for
C34H47NO22Na [M+Na]+: 931.2596, found: 931.2590.
Methyl
O-(methyl-5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero-α-D-galacto-2-
nonulopyranosylonate)-(2→6)-3,4-di-O-acetyl-2-phthalimido-2-deoxy-β-D-glucopyranoside (29). A
mixture of sialyl xanthates (117 mg, 0.20 mmol) and acceptor 18 (40 mg, 0.10 mmol) with 4Å
powdered molecular sieves were dissolved in the dry CH3CN (1.66 mL) and CH2Cl2 (0.83 mL)
stirring at r.t. for 1 h. Further, AgOTf (50 mg, 0.20 mmol) and DTBP (45 µL, 0.20 mmol) were added,
and the mixture was cooled to -68 oC and kept protected from light. Then PhSCl (24 µL, 0.21 mmol)
in the dry CH2Cl2 (0.13 mL) was added by running the solution down the cold wall of the reaction
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10.1002/ejoc.201900382
European Journal of Organic Chemistry
o
flask. Finally, the mixture was stirred for 3 h at -68 C. After that, the mixture was diluted with a
suspension of silica gel (0.42 g) in EtOAc (2.5 mL), filtered through celite, washed with saturated
aqueous NaHCO3 and water, dried with MgSO4, and concentrated under reduced pressure. The
residue was chromatographed (Cy-EtOAc 1:3) to give disaccharide 29 (55 mg, 64%). Rf = 0.47 (Cy-
EtOAc 1:3, twice). 1H NMR (400 MHz, CDCl3): δ 7.88-7.86 (m, 2H, phthalimido protons), 7.76-7.74
(m, 2H, phthalimido protons), 5.78 (dd, J=9.1, 1.2 Hz, 1H, H-3’), 5.44 (d, J=10.9 Hz, 1H, H-1’),
5.32-5.17 (m, 3H, H-4’, H-7, H-8), 4.94-4.85 (m, 1H, H-4), 4.68 (dd, J=9.2, 3.6 Hz, 1H, H-2’), 4.28-
4.23 (m, 2H, H-5’, H-9a), 4.06-4.01 (m, 4H, Ha-6’, H-5, H-6, H-9b), 3.80 (s, 3H, COOCH3), 3.40
(dd, J=11.4, 1.6 Hz, 1H, Hb-6’), 3.31 (s, 3H, OCH3), 2.65 (dd, J=12.7, 4.4 Hz, 1H, H-3eq), 2.13 (s,
3H, OAc), 2.09 (s, 3H, OAc), 2.05 (s, 3H, OAc), 2.03 (s, 3H, OAc), 2.02 (s, 3H, OAc), 2.01 (s, 3H,
13
OAc), 1.96 (t, J=3.6 Hz, 1H, H-3ax), 1.86 (s, 3H, NAc). C NMR (100 MHz, CDCl3): δ 171.17,
170.72, 170.39, 170.33, 170.22, 170.18, 169.83, 169.56, 169.51, 167.80 (C=O), 134.51, 123.81
(aromatic C), 98.46 (C-2), 91.90 (C-1’), 72.47, 70.74, 69.26, 69.14, 68.01, 67.50, 67.33 (C-6, C-5’,
C-4’, C-4, C-8, C-3’, C-7), 62.60 (C-9), 62.30 (C-6’), 60.53 (OCH3), 53.67 (COOCH3), 53.05 (C-2’),
49.50 (C-5), 37.89 (C-3), 23.33 (CH3, NAc), 21.29, 21.15, 20.99, 20.96, 20.92, 20.85 (CH3, OAc).
ESI-HRMS (m/z) calcd for C34H47NO22Na [M+Na]+: 903.2647, found: 903.2658.
Ethyl-O-(methyl-5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero-α-D-galacto-2-
nonulopyranosylonate)-(2→6)-3,4-di-O-acetyl-2-phthalimido-2-deoxy-1-thio-β-D-glucopyranoside
(30). The method in the literature was used[40] with some modifications: The acceptor 19 (20 mg, 0.046
mmol) and acetochloroneuraminate methyl ester (70 mg, 0.138 mmol) in the dry CH2Cl2 (4.6 ml)
were stirred with 4Å powdered molecular sieves for 1 h at r.t.. Then it was cooled to -45oC and kept
protected from light, and AgOTf (83 mg, 0.322 mmol) was added. The mixture was gradually warmed
to r.t. during 12 h, and further stirred at r.t. for 3 h. At last, filtered by celite and concentrated under
reduced pressure. The residue was chromatographed (DCM-MeOH 9:1) to give disaccharide 30 (27
1
mg, 65%). Rf = 0.41 (Cy-EtOAc 1:3, twice). H NMR (400 MHz, CDCl3): δ 7.88-7.85 (m, 2H,
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European Journal of Organic Chemistry
phthalimido protons), 7.76-7.74 (m, 2H, phthalimido protons), 5.81 (dd, J=9.1, 1.2 Hz, 1H, H-3’),
5.45 (d, J=10.9 Hz, 1H, H-1’), 5.30-5.17 (m, 3H, H-4’, H-7, H-8), 4.94-4.85 (m, 1H, H-4), 4.51 (dd,
J=9.2, 3.6 Hz, 1H, H-2’), 4.36-4.30 (m, 2H, H-5’, H-9a), 4.17-4.02 (m, 5H, H-6’, H-5, H-6, H-9b),
3.80 (s, 3H, COOCH3), 2.73-2.55 (m, 3H, H-3eq, SCH2CH3), 2.13 (s, 3H, OAc), 2.09 (s, 3H, OAc),
2.05 (s, 3H, OAc), 2.03 (s, 3H, OAc), 2.02 (s, 3H, OAc), 2.01 (s, 3H, OAc), 1.96 (t, J=3.6 Hz, 1H,
H-3ax), 1.85 (s, 3H, NAc), 1.23 (t, J=1.9 Hz, 3H, SCH2CH3). 13C NMR (100 MHz, CDCl3): δ 172.06,
172.00, 170.99, 170.56, 170.36, 170.33, 170.09, 170.06, 169.61, 167.61 (C=O), 134.66, 123.08
(aromatic C), 98.49 (C-2), 81.12 (C-1’), 76.19, 71.24, 70.15, 69.37, 68.96, 68.28, 67.34 (C-5’, C-6,
C-4’, C-4, C-8, C-3’, C-7), 62.24 (C-9), 62.07 (C-6’), 53.81 (COOCH3), 51.89 (C-2’), 49.14 (C-5),
37.88 (C-3), 23.97 (SCH2CH3), 21.31 (CH3, NAc), 19.34, 19.31, 19.29, 19.25, 19.21 (CH3, OAc),
14.16 (SCH2CH3). ESI-HRMS (m/z) calcd for C34H47NO22Na [M+Na]+: 933.2575, found: 933.2585.
Ethyl-O-(methyl-5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero-α-D-galacto-2-
nonulopyranosylonate)-(2→6)-3,4-di-O-acetyl-2-phthalimido-2-deoxy-1-thio-β-D-
galactopyranoside (31). The acceptor 22 (20 mg, 0.046 mmol) and acetochloroneuraminate methyl
ester (70 mg, 0.138 mmol) in the dry CH2Cl2 (4.6 ml) were stirred with 4Å powdered molecular sieves
for 1 h at r.t.. Then it was cooled to -45 oC and kept protected from light, and AgOTf (83 mg, 0.322
mmol) was added. The mixture was gradually warmed to r.t. during 12 h, and further stirred at r.t. for
3 h. At last, filtered by celite and concentrated under reduced pressure. The residue was
chromatographed (DCM-MeOH 9:1) to give disaccharide 31 (29 mg, 70%). Rf = 0.38 (Cy-EtOAc
1
1:3, twice). H NMR (400 MHz, CDCl3): δ 7.87-7.85 (m, 2H, phthalimido protons), 7.76-7.73 (m,
2H, phthalimido protons), 5.87 (dd, J=9.4, 2.9 Hz, 1H, H-3’), 5.72-5.70 (m, 2H, NH, H-4’), 5.52 (d,
J=10.9 Hz, 1H, H-1’), 5.37-5.35 (m, 1H, H-8), 5.27-5.20 (m, 1H, H-4), 5.14-5.10 (m, 1H, H-7), 4.72
(dd, J=12.2, 3.6 Hz, 1H, H-6), 4.60 (t, J=10.2 Hz, 1H, H-2’), 4.19-4.05 (m, 3H, H-5, H-5’, H-9a),
3.83 (s, 3H, COOCH3), 3.66-3.59 (m, 2H, Ha-6’, H-9b), 3.44 (t, J=7.2 Hz, 1H, Hb-6’), 2.74-2.63 (m,
1H, SCH2CH3), 2.47 (dd, J=12.7, 4.4 Hz, 1H, H-3eq), 2.35 (s, 3H, OAc), 2.16 (s, 3H, OAc), 2.11 (s,
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European Journal of Organic Chemistry
3H, OAc), 2.04 (s, 3H, OAc), 2.00 (s, 3H, OAc), 1.89 (s, 3H, OAc), 1.86 (s, 3H, NAc), 1.83 (t, J=10.4
Hz, 1H, H-3ax), 1.20 (t, J=9.6 Hz, 3H, SCH2CH3). 13C NMR (100 MHz, CDCl3): δ 172.13, 170.71,
170.67, 170.65, 170.43, 170.17, 169.56, 168.06, 167.61, 167.06 (C=O), 134.57, 134.40, 131.76,
131.39, 123.83 (aromatic C), 98.70 (C-2), 81.94 (C-1’), 75.10, 72.18, 72.08, 69.04, 69.01, 68.15,
67.70 (C-5’, C-6, C-4, C-3’, C-8, C-4’, C-7), 62.50 (C-9), 60.55 (C-6’), 53.13 (COOCH3), 50.76 (C-
2’), 48.32 (C-5), 37.06 (C-3), 24.66 (SCH2CH3), 23.33 (CH3, NAc), 21.43, 21.23, 21.07, 20.95, 20.65
(CH3, OAc), 15.03 (CH3). ESI-HRMS (m/z) calcd for C34H47NO22Na [M+Na]+: 933.2575, found:
933.2580.
Synthesis and analysis of neo-glycoproteins
Neo-glycoproteins 23a and 24a, have been prepared according to the method previously reported[23]
by activation of the disaccharides 23 and 24 (Figure S1), followed by glycosylation of the protein
RNase A.
Synthesis of glycosphingolipid derivative 34
(Methyl-5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero-α-D-galacto-2-
nonulopyranosylonate)-(2→6)-1,3,4-tri-O-acetyl-2-phthalimido-2-deoxy-β-D-
glucopyranoside(1→1)-(2S,3R,4E)-2-azido-3-O-benzoyl-4-octadecene-l,3-diol-(34).
The
NH2CH2CH2NH2 (18 uL) in the dry THF (5.2 mL) was added AcOH (18 uL) dropwise. Then
compound 28 (200mg, 0.22 mmol) was added, and the mixture was further stirred at r.t. for 3 h. The
solvent was evaporated and the residue was taken into CH2Cl2 and washed progressively with 1.0 M
HCl, saturated aqueous NaHCO3 and water. The organic layer was dried over MgSO4 and
concentrated to give the crude intermediate 32 with the yield of 75%. Then the intermediate 32 (110
mg, 0.13 mmol) was dissolved in the dry CH2Cl2 (4.2 mL), cooled to -5 oC, and trichloroacetonitrile
(420 µL) and 42 µL of DBU were added at -5 oC, and the mixture was stirred at -5 oC for 2 h. After
concentration, the residue was purified by flash column chromatography (Cy-EtOAc 1:4) to yield the
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10.1002/ejoc.201900382
European Journal of Organic Chemistry
trichloracetimidate 33 with α and β (4:1) mixture as pale yellow foam (77 mg, 60%). Rf = 0.45
(EtOAc). The 33 (60 mg, 0.059 mmol) and 3-O-benzoyl-azidosphingosine (50 mg, 0.12 mmol) in 2.8
mL of dry CH2Cl2 were stirred with 4Å powdered molecular sieves (500 mg) at r.t. for 1 h. The
mixture was then cooled to -30 oC, and BF3∙Et2O (60 µL, 0.48 mmol) was added dropwise, stirred for
another 1.5 h at -30 oC, and then filtered through celite. Thefiltrate was washed with saturated aqueous
NaHCO3 and water, dried over MgSO4 and concentrated under reduced pressure. The residue was
applied to a flash chromatography eluted with Cy-EtOAc 1:5 to give 34 (46 mg, 61%) as an
amorphous solid. Rf = 0.58 (EtOAc). m.p.: 158-161 ℃. 1H NMR (400 MHz, CDCl3): δ 7.98-7.95
(m, 2H, Ar-H), 7.86-7.82 (m, 2H, Ar-H), 7.73-7.71 (m, 2H, Ar-H), 7.57-7.53 (m, 1H, Ar-H), 7.44-
7.40 (m, 2H, Ar-H), 5.83-5.64 (m, 1H, H-5’’), 5.58-5.46 (m, 2H, H-3’’, H-4’’), 5.41-5.30 (m, 6H, H-
8, H-7, H-5’, H-3’, NH, H-2’), 4.87-4.84 (m, 1H, H-4), 4.34-4.28 (m, 2H, H-1’, Ha-9), 4.12-4.01 (m,
6H, Hb-9, H-5, H-6, H-2’’, Ha-6’, Ha-1’’), 3.84-3.81 (m, 2H, Hb-1’’, Hb-6’), 3.79 (s, 3H, COOCH3),
3.58 (dd, J=5.7, 2.5 Hz, 1H, H-4’), 2.57 (dd, J=12.8, 4.6 Hz, 1H, H-3eq), 2.14 (t, J=1.5 Hz, 9H,
3OAc), 2.04 (s, 6H, 2OAc), 2.03 (d, J=2.1 Hz, 2H, H2-6’’), 2.02 (s, 3H, OAc), 1.93 (t, J=5.9 Hz, 1H,
H-3ax), 1.87 (s, 3H, NAc), 1.24 (m, 22H, 11×CH2), 0.87 (t, J=6.9 Hz, 3H, CH3). 13C NMR (100 MHz,
CDCl3): δ 171.02 (C=O), 170.67 (C=O), 170.28 (C=O), 170.24 (C=O), 170.17 (2C=O), 170.15
(C=O), 170.03 (C=O), 168.55 (C=O), 167.70 (C=O), 164.87 (C=O), 138.92 (C-5’’), 134.22 (C, CH
aromatic), 133.13 (C, CH aromatic), 131.44 (C, CH aromatic), 129.70 (C, CH aromatic), 128.39 (C,
CH aromatic), 123.60 (C-4’’), 98.76 (C-1’), 98.49 (C-2), 74.78, 72.61, 72.44, 70.96, 69.13, 69.03,
68.58, 68.12, 67.37 (C-3’’, C-3’, C-4’, C-6, C-4, C-5’, C-1’’, C-8, C-7), 63.35 (C-2’’), 62.38 (C-6’),
60.79 (C-9), 54.46 (C-2’), 52.65 (COOCH3), 49.51 (C-5), 38.19 (C-3), 31.91 (CH2, C-6’’), 29.68,
29.66, 29.64, 29.57, 29.39, 29.34, 29.15, 28.62 (CH2), 23.20 (CH3, NAc), 21.09 (CH3, OAc), 20.85
(CH3, OAc), 20.84 (CH3, OAc), 20.82 (CH3, OAc), 20.76 (CH3, OAc), 20.70 (CH3, OAc), 14.10
(CH3). ESI-HRMS (m/z) calcd for C57H82N4O23Na [M+Na]+: 1300.5377, found: 1300.5365.
Acknowledgments
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10.1002/ejoc.201900382
European Journal of Organic Chemistry
Authors would like specially to acknowledge Dr. Roberto Pavesi (ACS DOBFAR; Italy) that kindly
provided immobilized AXE. Authors also thank the China Scholarship Council (CSC) for a Ph.D.
fellowship to Changping Zheng. Financial supports from the Centre National de la Recherche
Scientifique (CNRS) and the Sorbonne Université in France are gratefully acknowledged.
Keywords: enzymatic hydrolysis • sialyl-Tn antigen • glycosphingolipids • neo-glycoproteins
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