Rapid assembly of phosphate-bridged tetra-mannose by ionic liquid-supported oligosaccharide synthesis

Article abstract of DOI:10.1016/j.carres.2020.108209

An efficient ionic liquid-supported oligosaccharide synthesis (ILSOS) strategy was described for the synthesis of linear oligo-phosphomannan. A new cleavable benzyl carbamate-type IL supporter containing 5-aminopentanyl linker was designed as an acceptor IL tag to facilitate this synthesis. The chain elongation on IL tag was achieved by H-phosphonate chemistry, including condensation with α-mannosyl H-phosphonate, in situ oxidation reaction and subsequent deprotection. After four cycles, linear α-(1 → 6)-tetra-mannan phosphate was obtained with a total yield of 52.7% within 45 h. The IL tagged product exhibited a tunable solubility in polar and non-polar solvent systems that facilitate a chromatography-free purification in the assembly process. The IL tag could be easily removed after hydrogenolysis treatment after the final step, to afford an amine terminated linker at the reducing end of phosphoglycan for further conjugation with a carrier protein. This methodology offered an efficient and chromatography-free approach for the synthesis of phosphoglycan.

Full text of DOI:10.1016/j.carres.2020.108209

Carbohydrate Research xxx (xxxx) xxx
Contents lists available at ScienceDirect
Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
Rapid assembly of phosphate-bridged tetra-mannose by ionic
liquid-supported oligosaccharide synthesis
Guangyi Yang, Guodong Mei, Peng Shen, Haofei Hong**, Zhimeng Wu*
Key Laboratory of Carbohydrate Chemistry & Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, 214122, Wuxi, China
A R T I C L E I N F O
A B S T R A C T
Keywords:
An efficient ionic liquid-supported oligosaccharide synthesis (ILSOS) strategy was described for the synthesis of
linear oligo-phosphomannan. A new cleavable benzyl carbamate-type IL supporter containing 5-aminopentanyl
linker was designed as an acceptor IL tag to facilitate this synthesis. The chain elongation on IL tag was achieved
Ionic liquid-supported oligosaccharide synthe-
sis (ILSOS)
Phosphomannan
by H-phosphonate chemistry, including condensation with
α
-mannosyl H-phosphonate, in situ oxidation reaction
Synthesis
and subsequent deprotection. After four cycles, linear
α
-(1 → 6)-tetra-mannan phosphate was obtained with a
Chromatography-free purification
total yield of 52.7% within 45 h. The IL tagged product exhibited a tunable solubility in polar and non-polar
solvent systems that facilitate a chromatography-free purification in the assembly process. The IL tag could be
easily removed after hydrogenolysis treatment after the final step, to afford an amine terminated linker at the
reducing end of phosphoglycan for further conjugation with a carrier protein. This methodology offered an
efficient and chromatography-free approach for the synthesis of phosphoglycan.
1. Introduction
fluorinated tag [14] were used as supporters. Polymer-supported
methods allow the easy isolation and purification of products at the
Phosphoglycans are natural polysaccharides composed of multiple
glycosyl phosphate repeating units. One distinct feature of this class of
carbohydrates is that the glycosidic linkage is a phosphodiester formed
by a glycosyl phosphate and a hydroxyl group from another mono-
saccharide [1]. Phosphoglycans are widely found on the cell wall,
capsule or surface of bacterial, yeast and protozoa [2–4]. In many
pathogenic microorganisms, phosphoglycans are important virulence
factors that can be used as an antigenic determinant to develop glyco-
conjugate vaccines for preventing microbial infections [5,6]. The bio-
logical importance of phosphoglycans, such as those derived from the
pathogenic strain of Haemophilus influenzae type b (Hib) and Neisseria
meningitides serogroup A and X, have stimulated many efforts to develop
chemical methods to synthesize homogenous phosphoglycan fragments
for immunological studies [7–9]. For instance, step-wise or block-wise
chain elongation reaction in the solution phase is a traditional chemis-
try to prepare phosphoglycans [10,11]. However, time-consuming and
tedious chromatography purification was required after each step in this
approach. To simplify the purification process, supported carbohydrate
synthesis strategies were investigated for rapid assembly of phospho-
glycans, where solid [12]and soluble [13] polymers as well as
final step by a simple filtration, but usually large accesses of reagents are
required in the reaction process. In addition, the relatively low loading
capacity of polymer was another serious limitation. Alternatively, light
fluorous tag [15] was developed as a supporter to construct wall teichoic
acid fragment, a phosphoglycan drived from Enterococcus faecalis, in
multi-milligram scale. However, fluorous solid phase extraction was
required in each step to separate fluorous tagged products. Nevertheless,
developing a robust and cost-effective approach for the synthesis of
phosphoglycan is still a demanding challenge.
Ionic liquids (ILs) are a class of salts, typically composed of an
organic cation and an inorganic or organic anion. In the past decades,
applications of ILs in synthetic chemistry, as solvents, catalysts and re-
agents, have been intensively explored due to their unique physical and
chemical properties [16,17]. In carbohydrate synthesis, the use of ver-
satile of ILs as recyclable solvents and promoters in glycosylation was
well documented because of its favorable kinetic and thermodynamic
behaviors [18,19]. Furthermore, ILs displayed a tunable solubility in
polar and non-polar solvents, which made ILs be an excellent carrier in
carbohydrate synthesis. Accordingly, strategies based on ILs supported
oligocarbohydrate synthesis (ILSOS) were developed successfully. For
* Corresponding author.
** Corresponding author.
E-mail addresses: haofei@jiangnan.edu.cn (H. Hong), zwu@jiangnan.edu.cn (Z. Wu).
https://doi.org/10.1016/j.carres.2020.108209
Received 25 September 2020; Received in revised form 15 November 2020; Accepted 18 November 2020
Available online 23 November 2020
0008-6215/© 2020 Elsevier Ltd. All rights reserved.
Please cite this article as: Guangyi Yang, Carbohydrate Research, https://doi.org/10.1016/j.carres.2020.108209

G. Yang et al.
Carbohydrate Research xxx (xxxx) xxx
example, ester-linker IL support was applied to synthesize homolinear
mannose reacted with triphenylmethyl chloride in the presence of a
catalytic amount of 4-dimethylaminopyridine and then followed by
acetylation to give peracetylated compound 6. After selective removal of
the anomeric acetyl group in 6, hemiacetal 7 was afforded in a yield of
84% over three steps by only one final silica gel chromatographic pu-
α
-(1 → 6) linked tetra- and octa-mannoside [20]. Ionic Catch and
Release Oligosaccharide Synthesis (ICROS) is an effective strategy
capable of synthesizing β-(1 → 6) and β-(1 → 2)-linked oligosaccharides
library [21,22]. Li’s group demonstrated that benzyl carbamate-type IL
tag installed at the anomeric position of glycosylation acceptor could be
rification. The preparation of
α pure mannosyl H-phosphonate was
used to synthesize an
α
-linked nonamannoside [23]. Recently, the power
achieved by a modified two-step procedure. Firstly, hemiacetal 7 was
of ILSOS strategy was impressively proved by the synthesis of a complex
hetero-branched pentasaccharide on a 10 g scale [24]. However, up to
now, the application of ILSOS strategy for the synthesis of challenging
phosphoglycan has never been reported.
treated with diphenyl phosphite in the presence of pyridine to produce a
mixture of mannosyl-H-phosphonate derivative(α:β = 1:1). Then, the
α
/β-mannosyl H-phosphonate was further treated with excess phos-
phorous acid in anhydrous acetonitrile for 2 days to obtain -mannosyl
α
Here, we describe an ILs support approach for the synthesis of
H-phosphonate 3 (α:β = 8:1) as a major product in good yield, which was
phosphoglycan. A (1 → 6) phosphodiester
α
-linked tetra-mannose was
separable in silica gel column. The coupling constant of JC1-H1 (174.0
used as a model, which is a terminal immuno-dominant phosphomannan
fragment derived from yeasts S. cerevisiae and K. brevis [25,26]. In this
synthesis, a new benzyl carbamate-type IL support containing 5-amino-
pentanyl linker was designed and installed to the reducing end of
phosphoglycan, which exhibited a tunable solubility in a polar and
non-polar solvent system that facilitates a chromatography-free purifi-
cation in the assembly process. The IL tag could be easily removed by a
convenient hydrogenation condition in the final step while the linker
was kept at the reducing end of phosphoglycan, allowing for further
conjugation with a carrier protein for immunological studies.
Hz) in mannosyl H-phosphonate 3 unambiguously confirmed that
α-anomeric phosphate was formed [27].
Synthesis of benzyl carbamate-type IL tag 4 was shown in Scheme 2.
Briefly, 9 was synthesized following a reported procedure [23], which
further reacted with bis-(4-nitrophenyl) carbonate to give 10 in a yield
of 92%. Upon treatment with 5-amino-1-pentanol, 10 was transformed
to 4 in excellent yield, where the hydroxyl group will be used to couple
with glycosyl H-phosphate as an acceptor tag. The design of N-benzy-
loxycarbonyl group and linker in IL tag 4 allowed a mild hydrogenation
condition to remove the IL tag in the global deprotection step (see
Scheme 3).
2. Results and discussion
With building blocks in hand, IL tag 4 was firstly coupled with
α
-mannosyl H-phosphonate 3 in the presence of pivaloyl chloride. The
The target phosphomannan,
α-(1 → 6) phosphodiester tetra-mannose
condensation was completed smoothly in 5 h and in situ oxidation was
performed by adding iodine in moist pyridine. The crude IL tagged
product 11–1 was obtained by regular work-up procedure, such as
dichloromethane extraction and aqueous wash-up. The IL supported
product 11–1 was further purified by a convenient “dissolution-pre-
cipitation” procedure that was performed by dissolving crude product in
a small volume of MeOH (about 2 mL/g) and then precipitated the IL
tagged product by the addition of 6.0–8.0 equiv. volume of ether. The
isolated product 11–1 was treated with 5%TFA/DCM to remove trityl
protecting group and the resulting IL-tagged product 11–2 was isolated
by the same “dissolution-precipitation” procedure, which was charac-
terized by 1H-, 13C, 31P NMR and MS spectrum. The recovery yield of
11–2 is 95% over two steps and the purity was 98% as analyzed by
HPLC, indicating the “dissolution-precipitation” protocol is a highly
efficient method to separate IL tagged product.
1, and retrosynthetic analysis are presented in Fig. 1. IL tagged and full
protected tetra-saccharide was designed as 2, which was synthesized by
key intermediate 3 and 4. To assemble this mannosyl phospho-
saccharide, H-phosphate chemistry was chosen to form the phospho-
diester linkage, which enables the condensation of glycosyl H-
phosphonate with primary alcohol in monosaccharide and subsequent
oxidation in a one-pot procedure. In addition, mannosyl H-phosphonate
3 is quite stable compared with phosphoramidite method and
α pure
mannosyl H-phosphonate could be prepared from the corresponding α,
β-mannose hemiacetal derivative conventionally. Benzyl carbamate-
type IL tag 4 was designed to contain a 5-aminopentanyl linker and
served as an acceptor type tag.
Our synthesis started with preparing building blocks 3 as shown in
Scheme 1, where the triphenylmethyl (Trt) group is utilized as a tem-
porary protecting group for chain extension while the other three hy-
droxyl groups in mannose were protected by the acetyl group. D-
Next, we attempted to assembly
α-(1 → 6) phosphodiester linked
oligomannose using the above established protocol. IL-tagged
Fig. 1. Retrosynthetic analysis of phosphoglycan 1 and the design of intermediates.
2

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Carbohydrate Research xxx (xxxx) xxx
Scheme 1. Reagent and conditions: (a) TrtCl, DMAP, pyridine; (b) Ac2O, pyridine; (c) (NH4)2CO3, MeOH:THF = 2:1, 87% in 3 steps; (d) (1) diphenyl phosphite,
pyridine, room temperature, 2 h, TEA:H2O = 1:1, 1 h; (2) H3PO3, MeCN, room temperature, 4 d, 65% in 2 steps.
Scheme 2. Reagent and conditions: (a) HCl, toluene; (b) N-methylimidazole, KPF6, MeCN, reflux, overnight, 85% in 2 steps; (c) bis(4-nitrophenyl)carbonate,
pyridine, MeCN, 92%; (d) 5-amino-1-pentanol, MeCN, 95%.
Scheme 3. The assembly of α-(1 → 6)-oligomannan phosphate tetra-saccharide. Reagent and conditions: (a) PivCl, Py, then I2, Py:H2O = 9:1; (b) 5%TFA/CH2Cl2,
95% in 2 steps; (c) MeONa, MeOH, then 10% Pd(OH)2/C, H2, 82%.
monosaccharide 11–2 was used as an acceptor to further couple with
mannosyl H-phosphonate 3 under the same condition. The chain elon-
gation process was repeated three times. In each cycle, a similar IL tag
aided purification procedure was applied. Thus, di-, tri- and tetra-
saccharide were obtained efficiently, respectively. The results were
summarized in Table 1. Completing one cycle of carbohydrate chain
elongation totally took approximately 8 h, including one coupling re-
action, one deprotection reaction and two purification operations. The
IL tag aided isolation yield in each cycle is readily maintained ranging
from 89% to 95%. The isolated mono-, di-, tri- and tetra-
phosphomannan, naming 11-2, 12-2, 13-2 and 2, were fully character-
ized by 1H-, 13C, 31P NMR and HR-MS spectrum. And the purity of these
compounds was determined by HPLC analysis as well, which was 94, 96
and 87%, respectively (Fig. 2 and Table 1, entry 2–4). Finally, the final
Table 1
Rapid synthesis of target tetrasaccharide.
Entry
Operationa
Product
Timeb(min)
Yield(%)
Purity(%)
1
2
3
4
A,B,C,B
A,B,C,B
A,B,C,B
A,B,C,B
11–2
12–2
13–2
2
360 + 30+60 + 30
360 + 30+60 + 30
360 + 30+90 + 30
360 + 30+90 + 30
95
95
89
89
98
94
96
87
a
A: condensation and in situ oxidation. B: IL tag aided dissolution-
precipitation purification. C: removal of trityl group.
b
Typical time of condensation and oxidation is about 6 h; typical time of
centrifugal purification is 30 min. The removing trityl protection group is about
1 h.
´
global deprotection of 2 was achieved efficiently by the Zemplen
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Fig. 2. Determination the purity of IL-tagged product by HPLC analysis. HPLC conditions: gradient elution (MeCN(0.1% TFA): H2O (0.1% TFA) = 10 : 90–90 : 10 in
20 min; (1) retention time of compound 11-2: 15.57 min; purity 98%; (2) retention time of compound 12-2: 14.53 min; purity 94%; (3) retention time of 13-2: 6.01
min; purity 96%; (4) retention time of 2: 5.60 min; purity 87%.
transesterification reaction to remove the acetyl protecting group and
subsequent hydrogenation to remove the benzyl carbamate-type IL tag.
The crude product was further desalted by a Sephadex LH-20 column to
give 1 in an 82% yield after lyophilization. Its structure was fully
characterized by 1H-, 13C-, 31P- NMR and MS spectrum.
(MS) were obtained using electrospray ionization (ESI). Analytical thin
layer chromatography (TLC) was carried out on TLC plates pre-coated
with silica gel (250
μm layer thickness). Visualization was accom-
plished using either a UV lamp or sulfuric acid alcohol (5 mL H2SO4, 95
mL MeOH). Flash column chromatography was performed on silica gel
(300–400 mesh). Solvent mixtures used for TLC and flash column
chromatography are reported in v/v ratios. Commercially available re-
agents and solvents were used without further purification.
3. Conclusion
In summary, an efficient ionic liquid-supported oligosaccharide
synthesis (ILSOS) strategy was described for the synthesis of linear oligo-
phosphomannan. A new cleavable benzyl carbamate-type IL tag con-
taining 5-aminopentanyl linker was designed as a receptor type of IL tag
4.1.1. General ILSOS separation procedure
After filtration and concentration of the reaction mixture, the residue
was dissolved in a small volume of MeOH (about 2 mL/g), followed by
the addition of 6–8 equiv. volume of ether under shaking until no more
visible precipitate was generated. The precipitate was collected by
centrifugation (7000 rpm, 2 min, 3 times) to obtain IL tagged product as
a white powder.
to facilitate this synthesis. The assembly
α-(1 → 6) phosphodiester
linked oligomannose on IL tag was achieved by H-phosphonate chem-
istry, including condensation with α-mannosyl H-phosphonate, in situ
oxidation reaction and subsequent deprotection. The IL tag product was
separated conveniently by “dissolution-precipitation” procedure
without the need of chromatography purification. After four cycles,
4.1.2. General HPLC conditions
HPLC analyses were performed on a Waters 2695 infinite analyzer
linear α-(1 → 6)-tetra-mannan phosphate was obtained with a total yield
with a Welch Topsil CX-C18 column (5 μm, 4.6 × 250 mm), detection
of 52.7% and good purity. The acetyl protecting group and IL tag in the
wavelength of 254 nm, mobile phase of MeCN/H2O (0.1% TFA) and a
constant flow rate of 1 mL min-1.
´
final product was deprotected in two steps by Zemplen trans-
esterification reaction and subsequent hydrogenation condition,
respectively, to afford α-(1 → 6) phosphodiester linked tetra-mannose
4.1.3. 6-O-(triphenylmethyl)-2,3,4-triacetate-D-mannopyranose(7)
D-mannose (3 g, 16.7 mmol) and DMAP (0.18 g, 1.7 mmol) was
dissolved in anhydrous pyridine (30 mL) and triphenylmethyl chloride
(7.0 g, 25 mmol) was added. The mixture was stirred at 80 ◦C for 18 h.
After cooling down to 0 ◦C, 8.5 mL of Ac2O was added, and the solution
was stirred for overnight. The mixture was poured into ice-cold water
and extracted with CH2Cl2. The organic layer was dried over Na2SO4 and
concentrated under reduced pressure to give compound 6. To a solution
of the compound 6 (10 g, 16.9 mmol) in MeOH: THF(60 mL; 2 : 1 v/v)
was added ammonium carbonate (3.3 g, 33.8 mmol) at room tempera-
ture. The solution was allowed to stir at room temperature for 4 h. The
solvents were removed under reduced pressure. The residue was diluted
with CH2Cl2 (100 mL), washed with satd. H2O (100 mL), the combined
organic phase was collected and dried over Na2SO4. After the solvent
was removed, the crude product was purified by SiO2 column chroma-
tography eluting with CH2Cl2–CH3OH (10 : 1) to give pure compound 7
containing a 5-aminopentanyl linker. This methodology will provide
an efficient approach to rapidly synthesize other phosphoglycans with
biological importance for bacterial vaccine studies.
4. Experimental
4.1. General methods
Proton nuclear magnetic resonance (1H NMR) spectra were recorded
in deuterated solvents on a Bruker Avance III 400 MHz spectrometer.
Chemical shifts are reported in parts per million (ppm, δ) relative to
tetramethylsilane (0.00) for d-chloroform, or the residual protic solvent
peak for other solvents. 1H NMR splitting patterns with observed first
order coupling are designated as singlet (s), doublet (d), triplet (t), or
quartet (q). Carbon nuclear magnetic resonance (13C NMR) spectra were
recorded on a Bruker Avance III 400 MHz spectrometer. Mass spectra
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Carbohydrate Research xxx (xxxx) xxx
[28](7.6 g, 87%). 1H NMR (400 MHz, CDCl3) δ 7.38 (d, J = 7.3 Hz, 6H),
7.21 (t, J = 7.4 Hz, 6H), 7.18–7.11 (m, 4H), 5.31 (s, 1H), 5.29 (s, 1H),
5.18 (d, J = 2.3 Hz, 1H), 5.16 (s, 1H), 4.11–4.06 (m, 1H), 4.06–4.00 (m,
1H), 3.59 (s, 1H), 3.57 (d, J = 3.7 Hz, 1H), 3.19 (dd, J = 10.4, 2.2 Hz,
1H), 3.05 (dd, J = 10.4, 4.8 Hz, 1H), 2.35 (t, J = 7.2 Hz, 1H), 2.10 (s,
3H), 1.96 (s, 1H), 1.90 (s, 3H), 1.67 (s, 3H), 1.35 (s, 1H), 1.17 (t, J = 7.1
Hz, 2H), 0.89 (d, J = 6.7 Hz, 1H), 0.84–0.75 (m, 2H); MS (ESI) m/z:
Calcd. for C31H31O9, 547.20, found 548.10 [M+H]+
4.1.6. 4-((1-methylimidazol-3-yl)methyl)benzyl-(5-hydroxypentyl)
carbamate hexafluorophosphate salt (4)
To a solution of compound 10 (5.7 g, 17.1 mmol) in dry MeCN (40
mL) was added 5-amino-1-pentanol (2.4 mL, 25.65 mmol) in ice bath.
The reaction was kept for 4 h with TLC showing the absence of substrate.
The reaction mixture was warmed to room temperature, and purified via
the general ILSOS separation procedure, giving a yellowish syrup as 2
(4.9 g, 95%).1H NMR (400 MHz, MeOD) δ 7.44 (d, J = 5.8 Hz, 2H), 7.31
(s, 4H), 5.28 (s, 2H), 4.98 (s, 2H), 3.80 (s, 3H), 3.43 (t, 2H), 3.20 (s, 1H),
3.00 (t, 2H), 1.42 (m, 4H), 1.26 (m, 2H). 13C NMR (101 MHz, MeOD) δ
157.35, 138.60, 133.33, 128.46, 128.07, 123.81, 122.15, 65.19, 61.42,
52.35, 48.26, 48.05, 47.84, 47.63, 47.41, 47.20, 46.99, 40.38, 35.11,
31.84, 29.27, 22.70. HRMS (ESI) m/z: Calcd. for C18H26N3O3, 332.2008,
4.1.4. 6-O-(triphenylmethyl)-,2,3,4-triacetate-
α-D-mannopyranosyl
hydrogenphosphonate triethylammonium salt (3)
To a solution of the hemiacetal derivative 7 (7.6 g, 13.8 mmol) in
pyridine (50 mL) was added diphenyl phosphite (22.7 mL, 96.6 mmol)
at room temperature and it was allowed to stir at room temperature for
3 h. The reaction mixture was cooled to 0 ◦C, diluted with TEA: H2O (40
mL; 1 : 1 v/v) and it was stirred further for 30 min. The solvents were
removed under reduced pressure and co-evaporated with toluene (2 ×
50 mL). The residue was diluted with CH2Cl2 (100 mL), washed with
satd. NaHCO3 (100 mL), the combined organic phase dried over Na2SO4
and the solvent was removed under reduced pressure. The crude product
was purified by SiO2 column chromatography eluting with
CH2Cl2–CH3OH–TEA (95 : 5: 1) to furnish anomeric mixture of phos-
phonate derivative as pale yellow syrup (8.3 g, 98%). To a solution of the
obtained phosphonate derivative in anhydrous CH3CN (50 mL) was
added phosphorous acid (2.3 g, 27.6 mmol) and the solution was
allowed to stir at room temperature for 4 days. The reaction was
quenched by adding triethylamine (5 mL) at 0 ◦C, concentrated under
reduced pressure. The residue was diluted with CH2Cl2 (100 mL),
washed with satd. NaHCO3 (100 mL), the combined organic phase was
dried over Na2SO4 and the solvent was removed under reduced pressure.
The crude product was purified by SiO2 column chromatography eluting
with CH2Cl2–CH3OH–TEA (90 : 10: 1) to give compound 3 (6.3 g, 65%).
1H NMR (400 MHz, CDCl3) δ 7.81 (s, 0.5H), 7.38 (d, J = 7.4 Hz, 6H),
7.20 (t, J = 7.5 Hz, 7H), 7.13 (t, J = 7.2 Hz, 4H), 6.22 (s, 0.5H), 5.60 (dd,
J = 8.8, 1.8 Hz, 1H), 5.38–5.28 (m, 2H), 4.19–4.08 (m, 1H), 3.47 (q, J =
7.3 Hz, 1H), 3.16 (dd, J = 10.3, 2.3 Hz, 1H), 3.01 (dd, J = 10.3, 4.6 Hz,
1H), 2.86 (q, J = 7.3 Hz, 10H), 2.10 (s, 3H), 1.88 (s, 3H), 1.64 (s, 3H),
1.35 (t, J = 7.3 Hz, 1H), 1.19 (t, J = 7.3 Hz, 15H); 13C NMR (101 MHz,
CDCl3) δ 169.10, 109.08, 168.24, 142.80, 127.78, 126.71, 125.87,
92.01, 91.97, 85.39, 76.39, 76.08, 75.76, 69.98, 68.61, 65.39, 61.18,
52.44, 51.78, 44.77, 19.90, 19.75, 19.58, 8.29, 6.86; 31P NMR (162
MHz, CDCl3) δ 0.01; HRMS (ESI) m/z: Calcd. for C31H31O11P, 612.1760,
found 332.2050 [M-PF6]＋
.
4.1.7. 4-((1-methylimidazol-3-yl)methyl)benzyl (5-2,3,4-triacetate-
mannopyranosyl hydrogen phosphonate)pentyl)carbamate,
hexafluorophosphate salt (11-2)
α-D-
A mixture of compound 4 (1 g, 3.01 mmol) and H-phosphonate de-
rivative 3 (3.67 g, 6.02 mmol) were co-evaporated with anhydrous
pyridine under vacuum for two times. The mixture was dissolved in
anhydrous pyridine (30 mL) and pivaloyl chloride (0.73 mL, 6.02 mmol)
was added dropwisely at room temperature over 10 min under argon.
The reaction mixture was stirred further for 5 h. The reaction mixture
was cooled to 0 ◦C and was added a solution of I2 (1.53 g, 6.02 mmol) in
pyridine: water (10 mL; 9:1, v/v) over 15 min. The cooling was stopped
and the reaction mixture was stirred further for 1 h. The residue was
diluted with CH2Cl2 (100 mL), washed with saturated solution of
Na2S2O3⋅5H2O (50 mL). The combined organic layers dried over Na2SO4
and the solvent was removed. The reaction mixture was purified via the
general ILSOS separation procedure to obtain 11–1. Then residue 11–1
was dissolved in 5%TFA/DCM (30 mL) and the solution was allowed to
stir at room temperature for 1 h. The solvent removed under reduced
pressure and the reaction mixture was purified via the general ILSOS
separation procedure, giving a yellowish syrup as 11–2 (2.0 g, 95%). 1H
NMR (400 MHz, MeOD) δ 8.90 (s, 1H), 7.48 (d, J = 13.9 Hz, 2H), 7.33 (s,
4H), 5.46 (d, J = 7.1 Hz, 1H), 5.39 (s, 1H), 5.32 (s, 2H), 5.21 (d, J = 9.2
Hz, 2H), 4.98 (s, 1H), 3.83 (m, 3H), 3.6–3.4 (m, 2H), 3.20 (s, 3H), 3.02
(t, J = 4.2 Hz, 2H), 2.02 (s, 3H), 1.94 (s, 3H), 1.86 (s, 3H), 1.58–1.56 (m,
2H), 1.44–1.41 (m, 2H), 1.33–1.22 (m, 2H); 13C NMR (101 MHz, MeOD)
δ 170.22, 170.19, 157.26, 145.83, 138.80, 136.67, ]136.64, 133.39,
128.43, 128.02, 127.34, 127.04, 123.90, 122.33, 71.97, 69.27, 65.85,
65.08, 60.55, 52.40, 40.27, 35.19, 31.66, 29.88, 29.33, 29.01, 22.64,
22.32, 19.26, 19.19, 13.01.31P NMR (162 MHz, MeOD) δ ꢀ 3.26,
ꢀ 135.41, ꢀ 139.80, ꢀ 144.19, ꢀ 148.58, ꢀ 152.97. HRMS (ESI) m/z:
found 612.1740 [M+H]＋
.
4.1.5. 3-[4-benzyl (4-nitrophenyl) carbonate]-1-methylimidazolium
hexafluorophosphate (10)
Calcd. for C30H42N3O14P, 699.2399, found 699.2377 [M-PF6]＋
.
Compound 8 (3 g, 19.1 mmol) was dissolved in dry CH3CN (30 mL)
under argon, and it was added N-methylimidazole (3.2 mL, 38.2 mmol)
and KPF6 (3.5 g, 19.1 mmol). The reaction was refluxed overnight at
80 ◦C with stirring, and TLC showed complete conversion. The reaction
mixture was cooled to room temperature, filtered, and concentrated
under vacuum. The residue was dissolved in CH3CN (1 mL) and
precipitated with diethyl ether (10 mL) to obtain ILs support 9 (3.4 g,
88.0%) as a colorless oil: To a solution of 9 in dry MeCN (30 mL) was
added bis(4-nitrophenyl)carbonate (8.7 g, 28.6 mmol) and pyridine(1
mL). The reaction was stirring for 8 h and monitored by TLC. The re-
action mixture was purified via the general ILSOS separation procedure,
giving a yellow syrup as 10 (5.7 g, 92%).1H NMR (400 MHz, DMSO) δ
9.21 (s, 1H), 8.32 (d, J = 8.1 Hz, 2H), 7.75 (d, J = 28.1 Hz, 2H), 7.57 (d,
J = 8.1 Hz, 2H), 7.50 (dd, J = 24.7, 7.7 Hz, 4H), 5.45 (s, 2H), 5.33 (s,
2H), 3.86 (s, 3H), 2.50 (s, 2H); 13C NMR (101 MHz, DMSO) δ 155.71,
152.39, 145.69, 137.22, 135.79, 129.81, 129.44, 129.01, 125.90,
124.52, 123.04, 122.84, 70.30, 52.03, 40.64, 40.43, 40.22, 40.01,
39.80, 39.59, 39.38, 36.36; HRMS (ESI) m/z: Calcd. for C19H18N3O5,
4.1.8. 4-((1-methylimidazol-3-yl)methyl)benzyl (5-2,3,4-triacetate-α-D-
mannopyranosyl hydrophosphate)pentyl)carbamate-(1 → 6)-2,3,4-
triacetate-α-D-mannopyranosyl hydrophosphate hexafluorophosphate salt
(12-2)
A mixture of compound 11–2 (1 g, 1.43 mmol) and H-phosphonate
derivative 3 (1.75 g, 1.88 mmol) were co-evaporated with anhydrous
pyridine under vacuum for two times. The mixture was dissolved in
anhydrous pyridine (20 mL) and pivaloyl chloride (0.35 mL, 1.88 mmol)
was added drop wise at room temperature over 10 min under argon. The
reaction mixture was stirred further for 5 h. The reaction mixture was
cooled to 0 ◦C and to the cooled reaction mixture was added a solution of
I2 (0.72 g, 1.88 mmol) in Py:H2O (3 mL; 9:1 v/v) over 15 min. The
cooling was stopped and the reaction mixture was stirred further for 1 h.
The residue was diluted with CH2Cl2 (80 mL), washed with saturated
solution of Na2S2O3⋅5H2O (40 mL). the combined organic layers dried
over Na2SO4 and the solvent removed under reduced pressure. The re-
action mixture was purified via the General ILSOS separation procedure.
The residue was dissolved in 5%TFA/CH2Cl2 (15 mL) and the solution
was allowed to stir at room temperature for 1 h. The solvent removed
514.0967, found 514.0940 [M+H]＋
.
5

G. Yang et al.
Carbohydrate Research xxx (xxxx) xxx
under reduced pressure. and the reaction mixture was purified via the
general ILSOS separation procedure, giving a yellowish syrup as 12-2
(1.45 g, 95%).1H NMR (400 MHz, MeOD) δ 7.59 (d, J = 16.7 Hz, 2H),
7.41 (d, J = 8.2 Hz, 4H), 5.47 (d, J = 10.5 Hz, 1H), 5.42 (s, 3H),
5.38–5.26 (m, 3H), 5.10 (d, J = 14.8 Hz, 2H), 4.65–4.00 (m, 6H), 3.92
(m, 3H), 3.90–3.82 (m, 2H), 3.70–3.44 (m, 4H), 2.24–1.85 (m, 18H),
1.62 (m, 2H), 1.50 (m, 2H), 1.40 (m, 2H). 13C NMR (101 MHz, MeOD) δ
177.86, 170.24, 157.27, 147.45, 144.01, 143.23, 133.52, 130.30,
130.09, 128.49, 127.97, 127.63, 127.45, 124.27, 123.88, 122.25, 93.57,
71.90, 71.48, 71.17, 69.25, 65.97, 65.57, 65.13, 63.14, 60.57, 52.37,
52.10, 48.47, 46.23, 40.32, 35.24, 30.05, 29.07, 26.72, 26.14, 22.71,
21.91, 19.40, 19.32, 19.25, 19.23, 19.20.31P NMR (162 MHz, MeOD) δ
ꢀ 2.37, ꢀ 135.86, ꢀ 140.23, ꢀ 144.60, ꢀ 148.96, ꢀ 153.33. HRMS (ESI)
was removed under reduced pressure. The reaction mixture was purified
via the general ILSOS separation procedure. The residue was dissolved in
5%TFA/CH2Cl2 (8 mL) and the solution was allowed to stir at room
temperature for 1.5 h. The solvent was removed under reduced pressure
and the reaction mixture was purified via the general ILSOS separation
procedure, giving 2 as yellowish syrup (0.4 g, 80%). 1H NMR (400 MHz,
MeOD) δ8.55 (s, 1H), 7.59 (d, J = 17.8 Hz, 2H), 7.43 (s, 4H), 5.48–5.25
(m, 8H), 5.08 (s, 2H), 4.59 (d, J = 23.7 Hz, 4H), 4.29–4.17 (m, 3H), 4.12
(d, J = 15.9 Hz, 2H), 3.91 (d, J = 13.9 Hz, 6H), 3.63 (d, J = 11.1 Hz, 2H),
3.49 (dd, J = 13.2, 6.2 Hz, 2H), 3.12 (t, J = 5.2 Hz, 2H), 2.24–1.92 (m,
36H), 1.69–1.55 (m, 2H), 1.55–1.45 (m, 2H), 1.43–1.32 (m, 2H). 13C
NMR (101 MHz, MeOD) δ 177.23, 170.27, 128.57, 127.97, 127.83,
127.07, 127.07, 126.62, 71.87, 69.87, 48.58, 48.37, 48.15, 47.94,
47.73, 47.51, 47.30, 47.09, 39.08, 38.87, 38.66, 38.45, 38.24, 38.03,
37.82, 29.37, 21.98, 19.69, 19.59, 19.52.31P NMR (162 MHz, MeOD)
δ-0.44, ꢀ 1.64, ꢀ 136.32, ꢀ 140.69, ꢀ 145.07, ꢀ 149.44, ꢀ 153.32. HRMS
(ESI) m/z: Calcd. for C66H90N3O47P4, 1800.3690, found 1800.3693 [M-
m/z: Calcd. for C42H58N3O25P2, 1066.2829, found 1066.2806 [M-PF6]＋
.
4.1.9. 4-((1-methylimidazol-3-yl)methyl)benzyl (5-2,3,4-triacetate-α-D-
mannopyranosyl hydrophosphate)pentyl)carbamate-(1 → 6)-2,3,4-
triacetate-
α
-D-mannopyranosyl hydrophosphate-(1 → 6)-2,3,4-triacetate-
PF6]＋
.
α
-D-mannopyranosyl hydrophosphate, hexafluorophosphate salt (13-2)
A mixture of compound 12–2 (0.7 g, 0.65 mmol) and a-H-phospho-
4.1.11. 6-Aminopentyl-O-
mannopyranosylphosphate-(1 → 6)-
6)- -D-mannopyranoside (1)
The IL support 2 (0.2 g, 0.11 mmol) was dissolved in MeOH (5 mL), a
α
-D-mannopyranosylphosphate-(1 → 6)-
α-D-
nate derivative 3 (0.8 g, 1.3 mmol) were co-evaporated with anhydrous
pyridine under vacuum for two times. The mixture was dissolved in
anhydrous pyridine (10 mL) and pivaloyl chloride (0.15 mL, 1.3 mmol)
was added to it drop wise at room temperature over 10 min under argon.
The reaction mixture was stirred further for 5 h. The reaction mixture
was cooled to 0 ◦C and to the cooled reaction mixture was added a so-
lution of I2 (0.33 g, 1.3 mmol) in pyridine: water (1 mL; 9:1 v/v) over 15
min. The cooling was stopped and the reaction mixture was stirred
further for 1 h. The residue was diluted with CH2Cl2 (50 mL), washed
with satd. saturated solution of Na2S2O3⋅5H2O (25 mL). The combined
organic layers dried over Na2SO4 and the solvent removed under
reduced pressure. The reaction mixture was purified via the General
ILSOS separation procedure. The residue was dissolved in 5%TFA/DCM
(10 mL) and the solution was allowed to stir at room temperature for 1.5
h. The solvent removed under reduced pressure and the reaction mixture
was purified via the General ILSOS separation procedure, giving 13–2 as
a yellowish syrup (0.84 g, 89%). 1H NMR (400 MHz, MeOD) δ 8.54 (s,
1H), 7.59 (d, J = 18.6 Hz, 3H), 7.42 (s, 4H), 5.47 (d, J = 9.4 Hz, 1H),
5.45 (m, 1H), 5.41 (s, 2H), 5.32 (t, J = 13.3 Hz, 5H), 5.08 (s, 2H), 4.55
(m, 4H), 4.25 (t, J = 7.6 Hz, 3H), 4.11 (d, J = 8.8 Hz, 2H), 3.92 (m, 4H),
3.90–3.78 (m, 4H), 3.71–3.41 (m, 5H), 2.20–1.87 (m, 28H), 1.62 (m,
2H), 1.51 (m, 2H), 1.40 (m, 2H),. 13C NMR (101 MHz, MeOD) δ 171.00,
170.10, 157.27, 138.81, 130.58, 130.31, 130.10, 128.46, 128.23,
127.56, 126.82, 126.39, 123.85, 122.27, 93.61, 69.75, 69.68, 65.61,
65.06, 62.02, 55.97, 52.38, 48.03, 47.81, 48.55, 47.60, 47.49, 47.39,
47.17, 46.96, 40.27, 35.19, 30.07, 29.31, 29.03, 26.13, 25.82, 22.70,
19.28, 19.23, 19.16.31P NMR (162 MHz, MeOD) δ ꢀ 1.06,-2.39,-135.85,-
140.22,144.58,-148.95,-153.32. HRMS (ESI) m/z: Calcd. for
α
-D-mannopyranosylphosphate-(1 →
α
solution of NaOMe (0.1 M in MeOH) was added, and the mixture was
stirred for 1 h with TLC showing completion of de-acetylation. AcOH
was dropped to neutralize MeONa. The reaction mixture was filtered
and concentrated under reduced pressure to give the deacetylated
product. To the solution of the deacetylated product in MeOH: Formic
acid (5 mL; 1:1 v/v) was added 10% Pd(OH)2/C (10 mg) under inert
atmosphere at room temperature. The reaction mixture was allowed to
stir under H2 (4 atm) at room temperature for 9 h. The reaction mixture
was filtered through a Celite bed and the filtering bed was washed with
H2O (3 × 30 mL). The filtrate was concentrated under reduced pressure
and was dissolved in H2O and eluted through a column filled with
Sephadex LH-20. The eluate was concentrated to afford the target
compound 1 (0.1 g, 82%) as white powder. 1H NMR (400 MHz, D2O)
δ5.33 (d, J = 5.7 Hz, 3H), 5.06 (s, 1H), 3.94–3.51 (m, 27H), 3.06 (t, J =
5.0 Hz, 1H), 1.54 (dd, J = 12.7, 6.9 Hz, 2H), 1.44 (t, J = 14.0 Hz, 2H),
1.28 (dd, J = 7.1, 4.8 Hz, 2H); 13C NMR (101 MHz, D2O) δ 182.35,
96.16, 96.10, 95.15, 95.11, 73.88, 73.79, 73.00, 70.63, 70.55, 70.47,
70.10, 69.96, 69.92, 67.02, 66.46, 61.20, 60.85, 53.15, 53.09, 46.73,
39.33, 38.39, 29.50, 28.13, 26.59, 24.80, 22.35, 20.24; 31P NMR (162
MHz, D2O) δ ꢀ 2.16, ꢀ 2.28. HRMS (ESI) m/z: Calcd. for C29H53NO33P4,
1090.1348, found 1090.1366 [M+Na]＋
.
Declaration of competing interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
C
54H74N3O36P3, 1433.3259, found 1433.3209 [M-PF6]＋
.
4.1.10. 4-((1-methylimidazol-3-yl)methyl)benzyl(5-2,3,4-triacetate-α-D-
mannopyranosylhydrophosphate)pentyl)carbamate-(1 → 6)-2,3,4-
Acknowledgments
triacetate-α-D-mannopyra-nosylhydrophosphate-(1 → 6)-2,3,4-triacetate-
α
-D-mannopyranosylhydrophosph-ate-(1 → 6)-2,3,4-triacetate-
α-D-
This work was supported by the National Natural Science Foundation
of China [No. 21472070], project was partly funded by the 111 Project
[No. 111-2-06], the Open Foundation of Key Laboratory of Carbohy-
drate Chemistry & Biotechnology Ministry of Education (No. KLCCB-
KF201903) and National First-class Discipline Program of Food Science
and Technology (JUFSTR20180101).
mannopyranosylhydrophosphate hexafluorophosphate salt (2)
A mixture of compound 13–2 (0.4 g, 0.28 mmol) and H-phosphonate
derivative 3 (0.34 g, 0.56 mmol) were co-evaporated with anhydrous
pyridine under vacuum for two times. The mixture was dissolved in
anhydrous pyridine (8 mL) and pivaloyl chloride (0.07 mL, 0.56 mmol)
was added dropwisely at room temperature over 10 min under argon.
The reaction mixture was stirred further for 5 h. The reaction mixture
was cooled to 0 ◦C and a solution of I2 (0.14 g, 0.56 mmol) in Py:H2O (1
mL; 9:1 v/v) was added over 15 min. The cooling was stopped and the
reaction mixture was stirred further for 1 h. The residue was diluted with
CH2Cl2 (50 mL), washed with satd. saturated solution of Na2S2O3⋅5H2O
(25 mL). The combined organic layers dried over Na2SO4 and the solvent
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7