Glycoconjugate probes containing a core-fucosylated N-glycan trisaccharide for fucose lectin identification and purification

Article abstract of DOI:10.1016/j.carres.2017.07.011

Glyco-PAMAM dendrimers and glyco-agarose beads bearing a core-fucosylated N-glycan trisaccharide GlcNAcβ1,4(Fucα1,6)GlcNAc or a non-fucose disaccharide GlcNAcβ1,4GlcNAc were successfully synthesized and characterized by monosaccharide analysis with HPAEC-PAD technique. These glycoconjugates as fucose lectin probes were applied in fucose-specific lectin detection and purification. The model fucose lectin AAL indicated binding activity with the FITC-labeled PAMAM carrying core-fucose trisaccharide. An affinity chromatography column stuffed with the agarose beads carrying core-fucosylated trisaccharide exhibited a good specificity in purification of AAL than non-fucose disaccharide agarose beads. These novel glycoconjugates bearing the precise and simplified core-fucose N-glycan structure provided a potential application for core-fucose-specific lectin discovery.

Full text of DOI:10.1016/j.carres.2017.07.011

Carbohydrate Research 449 (2017) 143e152
Contents lists available at ScienceDirect
Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
Glycoconjugate probes containing a core-fucosylated N-glycan
trisaccharide for fucose lectin identification and purification
,
,
,
Deqin Cai a b, Chaochao Xun a c, Feng Tang a b, Xiaobo Tian a, Liyun Yang a, Kan Ding a,
Wenzhe Li d **, Zhiping Le c, Wei Huang a
,
, b, *
a CAS Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 Zuchongzhi Rd, Pudong, Shanghai,
201203, China
b University of Chinese Academy of Sciences, No.19A Yuquan Road, Beijing, 100049, China
c Department of Chemistry, Nanchang University, 999 Xuefu Avenue, Nanchang, 330031, China
d College of Basic Medical Sciences, Dalian Medical University, 9-Western Section, Lvshun South Road, Dalian, Liaoning, 116044, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Glyco-PAMAM dendrimers and glyco-agarose beads bearing a core-fucosylated N-glycan trisaccharide
GlcNAcb1,4(Fuca1,6)GlcNAc or a non-fucose disaccharide GlcNAcb1,4GlcNAc were successfully synthe-
Received 23 June 2017
Received in revised form
27 July 2017
Accepted 27 July 2017
Available online 3 August 2017
sized and characterized by monosaccharide analysis with HPAEC-PAD technique. These glycoconjugates
as fucose lectin probes were applied in fucose-specific lectin detection and purification. The model fucose
lectin AAL indicated binding activity with the FITC-labeled PAMAM carrying core-fucose trisaccharide.
An affinity chromatography column stuffed with the agarose beads carrying core-fucosylated trisac-
charide exhibited a good specificity in purification of AAL than non-fucose disaccharide agarose beads.
These novel glycoconjugates bearing the precise and simplified core-fucose N-glycan structure provided
a potential application for core-fucose-specific lectin discovery.
Keywords:
Core-fucose
Glycoconjugate
Glyco-dendrimer
Glyco-agarose
Fucose-specific lectin
© 2017 Elsevier Ltd. All rights reserved.
1. Introduction
GlcNAc which linked to Asn could be fucosylated and this core-
fucose correlates with certain important biological process of gly-
Protein glycosylation as the most common form of post-
translational modification associates with protein folding [1], virus
invasion [2], cell adhesion [3], immunomodulation [4e6], and
many other biological events [7,8]. The precise structure and
component of glycan, the oligosaccharide moiety of glycoproteins,
sensitively affected their biological functions [9e12]. Fucose is a
monosaccharide ordinarily attached on both N-linked glycans and
O-linked glycans. And, fucose is the essential sugar in human ABO
blood group [13] and Lewis antigens [14] which appear in complex
N-glycans and extended O-glycan cores. Sialyl Lewis X antigen al-
ternations and overexperession were reported in various cancers
[15,16]. In N-glycan core pentasaccharide Man3GlcNAc2, the first
coproteins. For instance, the core-fucosylation of therapeutic anti-
body dramatically reduced the antibody-dependent cell-mediate
cytotoxicity (ADCC) effect by hindering the binding between anti-
body Fc domain and Fc receptors on immune cells [17,18]. Alpha-
fetoprotein [19], a core-fucosylated glycoprotein, is used in clinic
as the diagnostic biomarker of hepatocellular carcinoma, and the
fucosyltransferase level also increased in the plasma of patients
with hepatocellular carcinoma [20]. Abnormal fucosylation was
also reported in other diseases such as rheumatoid arthritis [21]
and prostate cancer [22].
Although scientists have recognized the importance of fucosy-
lated glycoproteins in disease diagnosis and prognosis, the further
functional studies on fucose involved biological events are always
hampered by difficulty in identification and purification of in vivo
fucose-binding proteins. So far, some fucose lectins derived from
plants or bacteria were discovered, including AAL (Aleuria aurantia
lectin) [23], LCA (Lens culinaris agglutinin-A) [24], AOL (Aspergillus
oryzae L-fucose-specific lectin) [25], LecB from Pseudomonas aeru-
ginosa [26], etc. Meanwhile, it is seldom reported on fucose-specific
* Corresponding author. CAS Key Laboratory of Receptor Research, Shanghai
Institute of Materia Medica, Chinese Academy of Sciences, 555 Zuchongzhi Rd,
Pudong, Shanghai, 201203, China.
** Corresponding author. College of Basic Medical Sciences, Dalian Medical Uni-
versity, 9-Western Section, Lvshun South Road, Dalian, Liaoning, 116044, China.
E-mail addresses: liwenzhe@dlmedu.edu.cn (W. Li), huangwei@simm.ac.cn
(W. Huang).
http://dx.doi.org/10.1016/j.carres.2017.07.011
0008-6215/© 2017 Elsevier Ltd. All rights reserved.

144
D. Cai et al. / Carbohydrate Research 449 (2017) 143e152
binding proteins or lectins isolated from mammalian animals. A
suitable probe for fucose lectin identification and enrichment is an
urgent need for mammalian fucose lectin discovery.
fucose glycosylation and de-phthalylation of amino groups under
basic condition. With this design, we synthesized monosaccharide
intermediate 3 and 9 as shown in Scheme 1.
Carbohydrate probes or glycomimics [27e29] were often used
for detection of lectin-glycoconjugate interaction. Fucose-
containing glycoconjugates such as fucose dendrimers containing
the monosaccharide of fucose have been previously applied in
studies of fucose-specific lectins from bacteria [30,31]. Meanwhile,
more specific glycoconjugate probe for lectins recognizing N-glycan
core fucose is not yet reported. Here, we sought to develop a gly-
coconjugate probe with potential application in core-fucose lectin
identification and purification. We chose an asparagine-linked N-
The trichloroacetimidate 3 was synthesized from 1,3,4,6-tetra-
O-acetyl-2-deoxy-2-phthalimido-D-glucopyranose (1) by selective
deacetylation on 1-hydroxy and successive trichloroacetimidate
formation following the reported procedures [33]. Then we pre-
pared the intermediate 9 from compound 1. Azidation of 1 was
achieved using TMS-N3 in the presence of SnCl4. The obtained 4
was deacetylated and converted to 6 carrying 4,6-benzylidene
protection following the literature [34]. Then, Bz protection on 3-
OH of 6 was performed with BzCl and Py (pyridine) to give com-
pound 7. The benzylidene acetal ring was opened using TsOH to
afford the diol 8. Thereafter, selective protection of TBDMS was
introduced at the 6-OH as the temporary protecting group for late-
stage fucosylation. The target glycosyl acceptor 9 was achieved
with the exposed 4-OH for glycosylation with the donor substrate
3.
glycan trisaccharide GlcNAcb1,4(Fuca1,6)GlcNAc as the core struc-
ture for the glycoconjugate probe. This trisaccharide motif as a
simplified N-glycan does not contains other sugar moieties of
mannose, galactose, sialic acid, etc. which may lead to cross-
reactivity with other lectins. Combined this trisaccharide with
GlcNAc
specific detection on core-fucose motif might be achieved. With
this idea, we synthesized an asparagine-linked GlcNAc 1,4(-
Fuc 1,6)GlcNAc trisaccharide and GlcNAc 1,4GlcNAc disaccharide
b1,4GlcNAc disaccharide and fucose monosugar probes,
With the glycosyl donor 3 and the acceptor 9 in hands, we
started to prepare the disaccharide azide 12 and the trisaccharide
azide 15 (Fig. 2). The glycosylation reaction of 3 and 9 using BF3-
Et3O as the promoter gave the disaccharide 10 in a good yield of
85%. The proton NMR of compound 10 indicated a doublet peak at
5.53 ppm with a coupling constant of 8.0 Hz suggesting the beta-
1,4-linkage of nearly formed glycosidic bond. Then, the 6-O-TBDMS
of compound 10 was removed in presence of TBAF to afford com-
pound 11. One-step global deprotection of phthalyl, acetyl, and
benzoyl of 11 was performed using ethylenediamine and followed
full acetylation of the free hydroxyls and amines with acetic an-
hydride gave disaccharide azide 12. Meanwhile, using 11 as the
glycosyl acceptor, the fucose unit was introduced to its 6-OH
employing an in situ TMS-fucose iodide (13) as the fucosylation
donor which was prepared following reference approach [35]. The
b
a
b
respectively and conjugated them onto a PAMAM (polyamido-
amine) dendrimer and an agarose bead. A fucose-specific lectin was
supposed to bind the core-fucosylated glycoconjugates but not the
non-fucosylated glycoconjugates. In a test model, fucose lectin AAL
indicated distinguishing binding affinity to the core-fucose PAMAM
and was feasibly isolated through the core-fucose agarose beads.
Although AAL is not a core-fucose-specific lectin, these results
implicated our novel glycoconjugates as fucose probes could be
practically applied in fucose-specific lectin investigation.
2. Results and discussion
resulted trisaccharide 14 was characterized by 1H, 13C, 1H-1H-13
C
2.1. Synthesis of fully acetylated trisaccharide GlcNAc
b
1,4(Fuca1,6)
HSQC and HMBC (see supplementary data). The coupling constant
(3.2 Hz) of 4.83 ppm in the proton NMR confirmed the alpha-
linkage of newly formed glycosidic bond. The global deprotection
of 14 and successive acetylation gave the fully acetylated trisac-
charide azide 15 following the same procedures for compound 12
(see Scheme 2).
GlcNAc
b
1-azide (12) and disaccharide GlcNAc
b
1,4GlcNAc 1-azide
b
(15)
In previous literature [32], an N-glycopeptide bearing trisac-
charide GlcNAc 1,4(Fuc 1,6)GlcNAc was synthesized. We prepared
b
a
the trisaccharide building block 15 according to their method with
an optimized protecting strategy. Benzyl protection used in previ-
ous article on both of fucose motif and the Asn-linked GlcNAc 3-
hydroxy group was avoided to simplify the deprotection proced-
ures. Instead, TMS-protected fucose iodide (13) and 3-benzoyl-
protected intermediate 9 were employed, therefore the depro-
tection of TMS and benzoyl (Bz) could be easier during the steps of
2.2. Synthesis of the PAMAM glycoconjugates (21e22) and agarose
glycoconjugates (23e24) bearing core-fucosylated trisaccharide
and non-fucosylated disaccharide
Next, we employed the disaccharide 12 and trisaccharide 15 to
Scheme 1. Synthesis of building blocks 3 and 9. (a) hydrazine acetate, DMF, 85%; (b) CCl3CN, DBU, DCM, 78%; (c) TMS-N3, SnCl4, DCM, molecular sieves 4A, 80%; (d) NaOMe, MeOH,
88%; (e) benzaldehyde dimethyl acetal, 2,4,6-trichloro-1,3,5-triazine (TCT), acetonitrile, 76%; (f) BzCl, Py, DCM, 86%; (g) p-TsOH, H2O, DCM, MeOH, 94%; (h) TBDMS-Cl, Py, 70%.

D. Cai et al. / Carbohydrate Research 449 (2017) 143e152
145
Scheme 2. Synthesis of disaccharide 12 and trisaccharide 15. (a) BF3-Et2O, DCM, ꢀ16 ꢁC, 85%; (b) TBAF, THF, rt, 70%; (c) ethylenediamine, 2-butanol, 100 ꢁC; (d) Ac2O, pyridine, 88%;
(e) 2,6-di-tert-butylpyridine, DCM, 86%.
prepare corresponding glyco-dendrimers and glyco-agaroses as
shown in Scheme 3. Firstly, the azido on anomeric position of 12 or
15 was reduced to amine by Staudinger reaction using PMe3 in
MeOH containing small amount water. Then, the resulted amine
was coupled with the side-chain carboxylic acid of Fmoc-Asp-OtBu
to afford Asn-linked disaccharide or trisaccharide, respectively. The
deprotection of Fmoc and Ac groups was achieved with K2CO3 in
MeOH/H2O to give the compound 16 and 17 which indicated
identical NMR profiles as reported in reference [32]. After removing
tert-butyl of 16 or 17, a bi-functional linker DSG (di-N-succinimidyl-
Scheme 3. Synthesis of glyco-dendrimers 21/22 and glyco-agaroses 23/24. (a) PMe3, H2O, MeOH; (b) Fmoc-Asp(OH)-OtBu, HATU, DIPEA, DMF; (c) K2CO3, H2O, MeOH, 56% for 3
steps; (d) 98% HCOOH; (e) di-N-succinimidyl-glutarate (DSG); (f) 0.1 M NaOAc pH7.5, MeCN.

146
D. Cai et al. / Carbohydrate Research 449 (2017) 143e152
glutarate) was coupled onto the amino group of Asn to give the in
situ intermediate 18 and 19. The excessive DSG was removed by
DCM extraction and the in situ 18 or 19 bearing an activated suc-
cinimide ester reacted with the amino groups of PAMAM or agarose
in an aqueous NaOAc buffer. Using a FITC-labeled PAMAM (20) as
conjugates 21 and 22 were found carrying 10.3% and 18.6% (w/w)
carbohydrate substrates and the glyco-agarose 23 and 24 carried
0.1 and 0.16 mg carbohydrates per mL agarose beads, respectively.
2.4. AAL binding assay with the PAMAM glycoconjugate probes and
its isolation through the agarose glycoconjugate beads
the
GlcNAc
conjugation
carrier,
glyco-dendrimer
21
a
bearing
1,6)GlcNAc
b
1,4GlcNAc and 22 bearing GlcNAc
b
1,4(Fuc
were obtained respectively. While, using an amino-agarose as the
carrier, glyco-agarose beads 23 and 24 bearing the disaccharide and
trisaccharide motifs were also achieved.
The FITC-labeled PAMAM glycoconjugate probe (22) bearing the
core-fucosylated trisaccharide enables the fucose lectin detection.
We employed an ELISA-like assay to determine the binding of the
probe 22 with a reference fucose lectin AAL. The non-fucosylated
disaccharide glycoconjugate 21 and non-sugar FITC-PAMAM (20)
2.3. Characterization of the glycoconjugate probes by carbohydrate
analysis
were also tested for comparison. AAL (20 mg/mL) was coated to a
96-well ELISA plate and BSA (1 mg/mL) was used as the blocking
protein to occupy the rest coating capacity. Then, FITC-PAMAM
probes 20e22 in 1.0, 0.5, 0.25, and 0.125 mM concentrations were
incubated with the AAL-displayed plate. After washed with the
binding buffer to remove unbound probes, the plate was treated
with 500 mM fucose to release the bound probes in solution for
fluorescent detection as summarized in Fig. 2. Under low concen-
The glycoconjugates 21e24 were characterized on their loading
degree of sugar components by acidic hydrolysis and followed
monosaccharide analysis. To measure the mass percentages of
carbohydrate on the PAMAM or agarose carriers, portions of the
glycoconjugate samples were treated with 2 M TFA and heated to
95 ꢁC to release the monosaccharide units from the conjugates. For
glyco-dendrimer 21 and 22, the acidic hydrolysis was performed for
4 h to completely digest the sugars and removed the N-acetyl of
GlcNAc, giving monosaccharide fucose and glucosamine. While,
glyco-agarose 23 and 24 was treated with 2 M TFA at 95 ꢁC for
30 min to release the glycan, then the supernatant was separated
from the agarose beads to avoid the agarose decomposition and
continued the acidic hydrolysis for additional 3.5 h for complete
hydrolysis. The monosugar fucose and glucosamine residues
released from these glycoconjugates were then subject to quanti-
fication by HPAEC-PAD (high-performance anion-exchange chro-
matography with pulsed amperometric detection) which is a high-
sensitive analytical approach for monosaccharide and glycan
detection and analysis [36e38].
tration (0.125 mM), both trisaccharide probe 22 and disaccharide
probe 21 indicated slightly increased fluorescent intensities
compared with the non-sugar control (20) that may due to the non-
fucose-specific binding. While, in higher concentrations
(0.25e1.0 mM), fluorescent intensity of 20 and 21 increased poorly
but fucosylated probe 22 demonstrated a dramatically enhanced
binding affinity. These data implicated that the fucosylated trisac-
charide glycoconjugate 22 is a good probe for fucose lectin detec-
tion after eliminating the non-sugar and non-fucose-specific
background. Meanwhile, these data also implicated that AAL may
weakly bind GlcNAc-bearing glycoconjugates as well. And, as a
promising perspective, if distinguishing lectin binding affinity
could be detected on the core-fucose probe 22 from the mono-
fucose glycoconjugates, core-fucose specific lectin discovery could
be achieved.
As shown in the HPAEC-PAD profile (Fig. 1), the fucose and
glucosamine units were well separated from other monosugars and
indicated two peaks at retention times of 2.64 min (fucose) and
3.70 min (glucosamine) respectively. The quantification was based
on the peak areas of these sugars compared with the standard
curves. After monosaccharide analysis, the glyco-PAMAM
Next, we examined the application of glyco-agarose 24 in af-
finity chromatography isolation for fucose-specific lectins as
shown in Fig. 3. The schematic procedures for fucose lectin affinity
chromatography were indicated in Fig. 3A. The core-fucose
Fig. 1. Representative HPAEC-PAD profile of carbohydrate analysis of trisaccharide glyco-dendrimer 22.

D. Cai et al. / Carbohydrate Research 449 (2017) 143e152
147
Fig. 2. Fucose lectin binding assay with FITC-labeled glyco-dendrimers. AAL was coated on a 96-well ELISA plate. Then FITC-labeled PAMAM (20), FITC-PAMAM-disaccharide (21),
and FITC-PAMAM-trisaccharide (22) in 1.0, 0.5, 0.25, and 0.125
M concentrations were added to wells and incubated at 37 ꢁC for 2 h. After washed with the binding buffer and
m
eluted with 500 mM fucose, the plate was subject to fluorescent detection with a BioTek fluorescent microplate reader.
Fig. 3. Affinity chromatography for fucose lectin purification with the glyco-agarose beads. Panel A: schematic flow diagram of fucose lectin purification through an affinity column
stuffed with the glyco-agarose 24. Panel B and C: SDS-PAGE profiles of AAL binding and elution in the affinity columns containing trisaccharide-agarose 24 (panel B) or disaccharide-
agarose 23 (panel C). Lane 1:AAL standard sample; Lane 2: flow through; Lane 3: wash with a 10 mM pH 8.0 Hepes buffer containing 0.1 mM Ca2þ; Lane 4: elution with a 100 mM
fucose solution in above Hepes Buffer; Lane 5: elution with a 250 mM fucose solution in above Hepes buffer; Lane 6: elution with 500 mM Fucose; Lane 7: elution with a 1.0 M
fucose solution in above Hepes buffer; Lane 8: protein marker.
agarose beads (24) were stuffed in an empty affinity column and a
fucose lectin (AAL) was loaded to the column followed by wash
with a Hepes binding buffer containing Ca2þ and elution with
fucose solution in various concentrations. In comparison, we also
prepared a column using the non-fucosylated disaccharide agarose
23 to compare the retention behavior of AAL on these two

148
D. Cai et al. / Carbohydrate Research 449 (2017) 143e152
different columns. The flow-through, wash, elution fraction sam-
ples were analyzed by SDS-PAGE as shown in Fig. 3B (from gly-
coconjugate 24 column) and 3C (from glycoconjugate 23 column).
Flow-through fractions of AAL after loading to both columns
indicated no detectable AAL in SDS-PAGE (lane 2 in Fig. 3B and C),
implicating AAL may have non-specific binding or weak binding
glycoconjugates provided new probes for fucose specific lectin
discovery with more precise recognition on sugar moieties. The
core-fucose-containing agarose beads demonstrated good poten-
tials in specific fucose lectin purification combined with other non-
fucose or mono-fucose agaroses.
with GlcNAcb1,4GlcNAc motif that caused its retention on the 23-
stuffed column. In wash fractions, major of AAL was released from
the 23-stuffed column but only a small-portion AAL was released
from the 24-stuffed column based on the SDS-PAGE analysis (lane
3). This result confirmed the non-specific or weak interaction of
AAL and non-fucose beads 23 could be less stable and was easily
washed out with binding buffers. On the contrary, AAL binds 24
more strongly and remains its major portion in the column during
the wash. SDS-PAGE analysis of the elution fractions with 0.1, 0.25,
0.5, 1.0 M fucose (lane 4e7 of Fig. 3B and C) more clearly indicated
the difference of AAL retention in these two columns. For non-
fucose column, only few AAL was left in the column after wash
and it was completely eluted with low concentration of fucose
(0.1 M). While for fucose-containing column, a part of AAL was
eluted by 0.1 M fucose and higher concentrations of 0.25 and
0.5 M fucose were required to complete the elution, implying the
tighter binding of AAL with 24 agarose. It is noteworthy to
mention that each step of the wash and elution volume was set up
to 2 mL (2 column volumes) for comparison in our experiment,
and more specific purification may be achieved by further opti-
mization on wash and elution conditions. These data clearly
illustrated the core-fucose agarose beads could be very useful for
fucose-specific lectin clarification.
4. Experimental section
4.1. General
Chemical reagents and solvents were purchased from Sino-
pharm Chemical Reagent Co. (Shanghai, China) and used without
further purification. Fluorescein isothiocyanate (FITC) and the
PAMAM (polyamidoamine) dendrimer (ethyleneamine core, gen-
eration 5.0) were purchased from Sigma-Aldrich (USA). The in-
termediate building block 3 and 9 were synthesized as described in
supplementary data. Amino agarose beads and fucose lectin AAL
(Aleuria Aurantia Lectin) were purchased from Shanghai Seebio
Biotech. Inc. Lectin AOL (Aspergillus oryzae lectin) was purchased
from TOKYO CHEMICAL INDUSTRY CO. LTD. Reactions were
monitored by TLC on glass Silica Gel HSGF254 plates with UV
254 nm detection. TLC staining for carbohydrate samples was
performed by dipping the plates into 10% H2SO4 in methanol and
drying with a heat gun. Anhydrous dichloromethane (DCM) was
freshly distilled from calcium hydride under nitrogen prior to use.
Molecular sieves 4A powder was purchase from Sigma-Aldrich Co.
LLC (USA) and was activated by heating at 200 ꢁC in vacuum for
2
h. HPLC grade acetonitrile was purchased from Meryer
The AAL binding assay with glyco-PAMAM probes and the lectin
purification with affinity column stuffed with glyco-agarose beads
presented the application of these newly synthesized core-fucose-
containing glycoconjugates in fucose lectin detection and purifi-
cation. From these assays, AAL indicated specific binding affinity
with fucose-containing structures and also weaker or non-specific
binding with the GlcNAcb1,4GlcNAc disaccharide moiety. With
these probes, a potential application for discovery of more specific
fucose lectins could be enabled.
(Shanghai, China). Nuclear magnetic resonance (NMR) spectra
were measured on a Varian-MERCURY Plus-400 instrument. The
chemical shifts were assigned in ppm and the coupling constants
in Hz. ESI-HRMS spectra were measured on an Agilent 6230 LC-
TOF MS spectrometer. The fluorescence of 96-well ELISA plate
was measured on a BioTek Synergy™ 2 Multi-Mode Microplate
Reader with a fluorescent excitation filter of 485 nm and an
emission filter of 528 nm.
2.5. Isolation of AOL from a mixed protein sample with glyco-
agarose beads
4.2. High-performance liquid chromatography (HPLC)
Analytical RP-HPLC was performed on an Agilent 1260 UHPLC
system with a C18 column (5.0 mm, 4.6 ꢂ 250 mm) at 40 ꢁC. The
column was eluted with a linear gradient of 2%e90% acetonitrile
containing 0.1% TFA in 30 min at a flow rate of 1 mL/min. Prepar-
ative HPLC was performed on a Beijing ChuangXinTongHeng
LC3000 (preparative) instrument with a preparative C18 column
(10 mm, 20 ꢂ 300 mm). The column was eluted with a suitable
gradient of aqueous acetonitrile containing 0.1% TFA at a flow rate
of 10 mL/min.
To test whether these glyco-agarose beads could be used for
isolation of fucose lectins from other proteins, we employed a
mixture protein sample containing another fucose lectin AOL
(Aspergillus oryzae lectin) and a non-related protein BSA (Bovine
Serum Albumin) in the column chromatography. As shown in Fig. 4,
in the washing fractions, BSA was firstly washed out (lane 3 in both
beads) since its poor interaction with neither glyco-agarose 23 nor
24. While, AOL can be washed out from the disaccharide column
(panel B lane 4) but not from fucosylated trisaccharide column
(panel A lane 4). The elution of AOL from trisaccharide column
required 200 mM fucose as the competitive eluent (panel A lane 5).
These data demonstrated the isolation of fucose lectins from mixed
protein samples could be achieved using the glyco-agarose
columns.
4.3. High-performance anion-exchange chromatography with
pulsed amperometric detection (HPAEC-PAD)
HPAEC-PAD was performed on a DIONEX ICS5000 chromatog-
raphy system (ThermoFisher, USA) equipped with an electro-
chemical detector (ED50) and an anion exchange column (CarboPac
PA1, 4.6 ꢂ 250 mm). An isocratic mobile phase (flow rate, 1.0 mL/
min) of 18 mM NaOH was used. The released fucose and glucos-
amine from the acid-hydrolyzed glycoconjugate samples were
measured and quantified by the peak areas at respective retention
time 2.64 and 3.70 min after compared with the standard curves of
fucose and glucosamine standard samples.
3. Conclusion
A
core-fucose N-glycan trisaccharide GlcNAc
b1,4(Fuc a1,6)
GlcNAc and non-fucose disaccharide GlcNAc 1,4GlcNAc were suc-
b
cessfully synthesized and conjugated to a FITC-labeled PAMAM
dendrimer and an amino-agarose beads. Applications of these
glycoconjugate probes in fucose lectin detection and purification
were achieved. The structural design on these novel

D. Cai et al. / Carbohydrate Research 449 (2017) 143e152
149
Fig. 4. Isolation of AOL from a mixed protein sample with the glyco-agarose beads. Panel A and B: SDS-PAGE profiles of the mixture sample (AOL/BSA) and its binding and elution in
the affinity columns containing trisaccharide-agarose 24 (panel A) or disaccharide-agarose 23 (panel B). Lane 1:the mixture sample of AOL (the lower band around 35 KDa) and BSA
(the upper band around 60 KDa); Lane 2: flow through; Lane 3 and 4: wash with a 10 mM pH 8.0 Hepes buffer containing 0.1 mM Ca2þ; Lane 5 and 6: elution with a 200 mM fucose
solution in above Hepes buffer; Lane M: protein marker.
4.4. Synthesis of 3-O-benzoyl-6-O-tert-butyldimethylsilyl-2-deoxy-
2-phthalimido-4-O- (3,4,6-tri-O-acetyl-2-deoxy-2-phthalimido-
d-glucopyranosyl)- -d-glucopyranosyl azide (10)
3H), 3.85e3.67 (m, 3H), 3.50e3.44 (m, 2H), 3.25e3.18 (m, 1H), 2.00
(s, 3H, Ac), 1.91 (s, 3H, Ac), 1.82 (s, 3H, Ac). ESI-HRMS: m/z: calcd for
C
b-
b
41H37N5O16: 855.2235; found: 878.2201 [MþNa]þ.
Compound 3 (1.0, 1.82 mmol) and compound 9 (350 mg,
0.63 mmol) were dissolved in anhydrous DCM (5 mL). The freshly
activated 4 Å MS(molecular sieves) powder were added, and the
mixture was stirred at ꢀ16 ꢁC for 20 min under N2 protection. Then,
4.6. Synthesis of 2-acetamido-2-deoxy-3,6-di-O-acetyl-4-O-(2-
acetamido-2-deoxy-3,4,6- tri-O-acetyl-b-d-glucopyranosyl)-b-d-
glucopyranosyl azide (12)
BF3-Et2O (32 mL, 0.25 mmol) was added and the residue was stirred
Compound 11 (200 mg, 0.23 mmol) was dissolved in 2-butanol
(5 mL). Ethylenediamine (5 mL) was added and the mixture was
refluxed at 100 ꢁC for 12 h. The solution was concentrated to dry-
ness in vacuum then acetic anhydride (10 mL) and pyridine (10 mL)
were added. The residue was stirred at rt overnight. After removal
of the solvent, the crude product was purified by silica gel column
chromatography eluted with DCM/MeOH 30:1. The compound 12
was obtained as a white powder (yield 88%). 1H NMR (400 MHz,
at ꢀ16 ꢁC for 1 h then warmed to the rt and stirred for additional 2 h
until TLC indicated the completion of the reaction. Et3N (100 mL)
was added to quench the reaction. The solid was filtered and the
liquid was concentrated in vacuum and purified by silica gel col-
umn chromatography eluted with PE/EA/DCM 3.5:1:2.5. The com-
pound 10 was obtained as a white powder (yield 85%). 1H NMR
(400 MHz, CDCl3):
d
7.94e7.37 (m, 13H, Ar), 6.01 (t, J ¼ 8.0 Hz, 1H,
H-3), 5.66 (t, J ¼ 8.0 Hz, 1H, H-30), 5.53 (t, J ¼ 8.0 Hz, 2H, H-1, H-10),
4.48 (t, J ¼ 12 Hz, 1H, H-40), 4.25e4.09 (m, 3H, H-4, H-2, H-20),
3.78e3.68 (m, 3H, H-6a, H-60a, H-60b), 3.58e3.46 (m, 3H, H-5, H-50,
H-6b), 2.00 (s, 3H, Ac), 1.93 (s, 3H, Ac), 1.78 (s, 3H, Ac), 0.93 (s, 9H, t-
CDCl3):
d
6.01 (d, J ¼ 9 Hz, 1H), 5.84 (d, J ¼ 8 Hz, 1H), 5.21e5.01 (m,
3H), 4.55 (m, 2H), 4.42e4.24 (m, 3H), 4.07e3.83 (m, 3H), 3.78e3.60
(m, 3H) 2.15e2.01 (m, 15H, 5Ac), 1.98 (s, 3H, NHAc), 1.94 (s, 3H,
NHAc). ESI-HRMS: m/z: calcd for C26H37N5O15 659.2286; found
660.2364 [MþH]þ.
Bu), 0.08 (d, 6H, Si-Me2).13C NMR (100 MHz, CDCl3):
d 170.6, 170.1,
169.4, 165.2, 163.6, 134.5, 133.3, 131.3, 129.7, 129.3, 128.5, 123.7, 97.0,
85.0, 77.2, 73.7, 71.7, 71.2, 70.5, 68.7, 61.9, 60.9, 54.8, 54.5, 25.9, 20.8,
20.6, 20.4, 18.4, 5.2, 5.2. ESI-HRMS: m/z: calcd for C47H51N5O16Si:
969.3100; found: 992.3011 [MþNa]þ.
4.7. Synthesis of 3-O-benzoyl-2-deoxy-2-phthalimido-4-O-(3,4,6-
tri-O-acetyl-2-deoxy-2- phthalimido-b-d-glucopyranosyl)-6-O-(a-
l-fucopyranosyl)- -d-glucopyranosyl azide (14)
b
4.5. Synthesis of 3-O-benzoyl-2-deoxy-2-phthalimido-4-O-(3,4,6-
tri-O-acetyl-2-deoxy-2- phthalimido-b-d-glucopyranosyl)-b-d-
glucopyranosyl azide (11)
To a solution of 1,2,3,4-tetra-TMS-fucose (0.96 g, 2.12 mmol) in
anhydrous DCM (3 mL) were added TMS-I (0.4 mL, 2.12 mmol).
After stirring at rt for 2 h with aluminium foil, a solution of com-
pound 11 (250 mg, 0.29 mmol) and 2,6-di-tert-butylpyridine
(0.62 mL, 2.9 mmol) in DCM (3 mL) was added to the reaction. The
mixture was stirred at rt overnight then was quenched by Et3N
(0.1 mL). The reaction solution was diluted with DCM, washed with
brine, and dried over anhydrous Na2SO4. After removing the solid
by filtration, the residue was concentrated and purified by silica gel
column chromatography eluted with PE/EA 1:1 then DCM/MeOH
20:1. The compound 14 was obtained as a pale yellow powder
To a solution of compound 10 (1.38 g, 1.43 mmol) in THF (20 mL)
was added tetrabutylammonium fluoride (TBAF, 1.0 M in THF so-
lution, 4 mL), and the mixture was stirred at rt overnight. The so-
lution was diluted with ethyl acetate and then washed with
saturated aqueous ammonium sulfate and brine successively. The
organic phase was dried over anhydrous Na2SO4. After removal of
the solid by filtration, the liquid was concentrated and purified by
silica gel column chromatography eluted with EA/PE 1:1. The
compound 11 was obtained as a white powder (yield in 70%). 1H
(yield 86%). 1H NMR (400 MHz, CDCl3):
d 7.96e7.37 (m, 13H, Ar),
6.03 (dd, J ¼ 8.8 Hz 10.4 Hz, 1H, H-3), 5.70 (d, J ¼ 8.4 Hz, 1H, H-10),
5.65 (dd, J ¼ 9.2 Hz 10.4 Hz, 1H, H-30),5.57 (d, J ¼ 9.2 Hz, 1H, H-1),
4.85 (m, 1H, H-40), 4.83 (d, J ¼ 3.2 Hz, 1H, H-100), 4.30e4.24 (m, 2H,
NMR (400 MHz, CDCl3):
1H), 5.69 (m, 2H), 5.63 (m, 1H), 5.03 (t, J ¼ 12 Hz, 1H), 4.29e4.19 (m,
d
7.99e7.39 (m, 13H, Ar), 6.13 (t, J ¼ 8 Hz,

150
D. Cai et al. / Carbohydrate Research 449 (2017) 143e152
H-2, H-4), 4.19 (dd, J ¼ 8.4 Hz, 10.4 Hz, 1H, H-20), 4.14 (q, J ¼ 6.8 Hz,
1-H, H-500), 3.97 (m, 2H, H-200, H-300), 3.91e3.81 (m, 4H, H-400, H-6a,
H-50, H-6a’), 3.76 (m, 1H, H-5), 3.65 (m, 1H, H-6b’), 3.31 (dd,
J ¼ 3.2 Hz 10.8 Hz, 1H, H-6b), 2.02 (s, 3H, Ac), 1.95 (s, 3H, Ac), 1.80 (s,
3H, Ac), 1.33 (d, J ¼ 6.6 Hz, 3H, H-600). 13C NMR (400 MHz, CDCl3):
follow the procedure described above for preparation of compound
16. 1H NMR (400 MHz, D2O):
d
4.98 (d, J ¼ 9.6 Hz, 1H, GlcNAc [1]-
H1), 4.81 (d, J ¼ 3.2 Hz, 1H, Fuc-H1), 4.56 (d, J ¼ 8.0 Hz, 1H, GlcNAc
[2]-H1), 4.25 (m, 1H), 4.06 (q, J ¼ 6.6 Hz, 1H, Fuc-H5), 3.85e3.30 (m,
15H), 2.92e2.78 (m, 2H, Asn-bH), 2.03 (s, 3H, Ac), 1.98 (s, 3H, Ac),
d
170.6, 170.2, 169.5, 165.2, 134.7, 133.3, 131.1, 129.8, 129.3, 128.4,
1.41 (s, 9H, tBu), 1.18 (d, J ¼ 6.6 Hz, 3H, Fuc-CH3). ESI-HRMS: calcd
123.7, 98.1, 96.9, 85.1, 75.6, 74.2, 71.8, 71.0, 70.9, 70.5, 69.3, 69.2,
66.2, 64.4, 62.3, 54.8, 54.6, 25.9, 20.79, 20.55, 20.3, 16.0.
for C30H52N4O17, 740.3328; found 741.3421 [MþH]þ.
4.11. Synthesis of FITC-PAMAM (20)
4.8. Synthesis of 2-acetamido-2-deoxy-3-O-acetyl-4-O-(2-
acetamido-2-deoxy-3,4,6-tri-O- acetyl-
(2,3,4-tri-O-acetyl-a-l-fucopyranosyl)-b-d-glucopyranosyl azide
b
-d-glucopyranosyl)-6-O-
To a solution of amino-PAMAM (50 mg) in a 0.1 M NaOAc buffer
(pH 7.5, 2 mL) was added FITC (Fluorescein isothiocyanate) (0.5 mg,
(15)
1.3 mmol, in 0.5 mL acetonitrile). The solution was stirred with an
aluminium foil at rt for 7 h. The mixture was subject to gel filtration
via a Sephadex G-25 column eluted by water. The fractions con-
taining FITC-PAMAM dendrimer was combined and lyophilized to
give product 20 as an oil (48 mg). The FITC labeling of compound 20
was confirmed by fluorescent detection with a BioTek microplate
reader.
To a solution of compound 14 (200 mg, 0.21 mmol) in 2-butanol
(5 mL) was added ethylenediamine (5 mL) and the reaction mixture
was refluxed at 100 ꢁC for 12 h. Then the solvent was removed in
vacuum. Acetic anhydride (10 mL) and pyridine (10 mL) were added
and the residue was stirred at rt overnight. The solution was
concentrated to dryness and the crude product was purified by
silica gel column chromatography eluted with DCM/MeOH 30:1.
The compound 15 was obtained as a white powder (yield 85%). 1H
4.12. Synthesis of FITC-PAMAM-Asn(GlcNAcb1,4GlcNAc)
(glycodendrimer 21)
NMR (400 MHz, CDCl3):
d
6.28e6.24 (m, 1H), 5.90 (d, J ¼ 8 Hz, 1H),
5.41e5.27 (m, 2H), 5.22e4.92 (m, 4H), 4.63 (d, J ¼ 9.0 Hz, 1H),
4.46e4.37 (m,1H), 4.33e4.25 (m,1H), 4.14e4.03 (m,1H), 4.00e3.85
(m, 3H), 3.84e3.61 (m, 4H), 3.59e3.50 (dd, J ¼ 8.0 Hz, 10.2 Hz, 1H),
3.46e3.35 (m, 1H), 2.17 (s, 3H, Ac), 2.15 (s, 3H, Ac), 2.12 (s, 3H, Ac),
2.07 (s, 3H, Ac), 2.06 (s, 3H, Ac), 2.00 (s, 3H, Ac), 1.99 (s, 3H, Ac), 1.98
Compound 16 (3 mg, 5 mmol) was dissolved in 98% HCOOH
(2 mL) and the mixture was stirred at rt overnight. The solution was
concentrated to dryness and the residue was dissolved in a sodium
acetate buffer (0.1 M, pH 7.5, 1 mL). The mixture was slowly added
into another solution of di-N-succinimidyl-glutarate (DSG, 19 mg,
(s, 3H, Ac), 1.90 (s, 3H, Ac). ESI-HRMS: calcd for C36H51N5O21
,
58 mmol) in acetonitrile (0.5 mL). The mixture was stirred at rt for
889.3077; found 890.3182 [MþH]þ.
2 h and monitored by HPLC and LC-MS. The in situ product 18 was
observed in MS determination indicating a m/z peak of 750.2696
[MþH]þ which is in good agreement with the calculated molecular
weight (C29H43N5O18, 749.2603). The residue was washed with
dichloromethane to remove the excessive DSG. To the resulted
aqueous layer containing the crude product 18, FITC-labeled
amino-PAMAM (20) (10 mg) was added and the mixture was stir-
red at rt overnight. The mixture was subject to gel filtration via a
Sephadex G-25 column eluted by water. The fractions containing
glycodendrimer was combined and lyophilized to give product 21
as an oil (12 mg). To determine the loading degree of
4.9. Synthesis of Nw-[2-acetamido-2-deoxy-4-O-(2-acetamido-2-
deoxy-b-d- glucopyranosyl)-b-d-glucopyranosyl]-l-asparagine tert-
butyl ester (16)
To a solution of compound 12 (200 mg, 0.37 mmol) in Methanol
(8 mL) was added trimethylphosphine (PMe3) (1.0 M in THF, 800 L)
and water (400 L). The mixture was stirred at rt for 1.5 h until TLC
m
m
showed the completion of the reaction. The solution was concen-
trated in vacuum and co-evaporated with toluene to complete
dryness. Then, the residue was dissolved in DMF (8.4 mL) and to the
solution was added Na-(9-Fluorenylmethyloxycarbonyl)-L-aspartic
acid tert-butyl ester (Fmoc-Asp(OH)-OtBu, 160 mg, 0.39 mmol),
HATU (400 mg,1.0 mmol), and DIPEA (1.0 mL). The reaction mixture
was stirred at rt for 2.5 h. The residue was diluted with DCM and
washed with brine. The organic layer was combined and dried over
anhydrous sodium sulfate. After removing the solid by filtration,
the liquid was concentrated in vacuum to dryness giving the crude
product as a pale yellow powder which was directly used in next
step. The above powder was dissolved in methanol (20 mL) and
water (10 mL). Potassium carbonate (200 mg) was added and the
reaction mixture was stirred at rt overnight. The residue was
concentrated and subject to preparative HPLC purification. The
compound 16 was obtained as a white solid (yield 56% for 3 steps).
GlcNAcb1,4GlcNAc disaccharide motif on PAMAM product, 21 was
treated with 2 M trifluoroacetic acid and heated at 95 ꢁC for 4 h to
completely release the glucosamine. Then quantification of
released glucosamine was performed by HPAEC-PAD (High-per-
formance anion-exchange chromatography with pulsed ampero-
metric detection) analysis on a DIONEX ICS5000 system following
the method described above. Based on glucosamine quantification,
the GlcNAcb1,4GlcNAc disaccharide loaded on PAMAM glycoden-
drimer 21 was 10.3% w/w (average 8.4 disaccharides per PAMAM).
4.13. Synthesis of FITC-PAMAM-Asn[GlcNAcb1,4(Fuca1,6)GlcNAc]
(glycodendrimer 22)
A solution of compound 17 (3.7 mg, 5 mmol) was dissolved in
1H NMR (400 MHz, D2O):
4.60 (d, J ¼ 8.2 Hz, 1H, GlcNAc [2]-H1), 4.30 (m, 1H), 3.90e3.25 (m,
12H), 2.95e2.70 (m, 2H, Asn- H), 2.06 (s, 3H, Ac), 2.01 (s, 3H, Ac),
d
5.03 (d, J ¼ 9.6 Hz, 1H, GlcNAc [1]-H1),
98% HCOOH (2 mL) and the mixture was stirred at rt overnight. The
solution was concentrated to dryness and the residue was dissolved
in a sodium acetate buffer (0.1 M, pH 7.5, 1 mL). The mixture was
slowly added into another solution of added into DSG (19 mg,
b
1.41 (s, 9H, tBu). ESI-HRMS: calcd for C24H42N4O13, 594.2748; found
595.2835 [MþH]þ.
58 mmol) in acetonitrile (0.5 mL). The reaction mixture was stirred
at rt for 2 h and monitored by HPLC and LC-MS. The in situ product
19 was observed in MS determination indicating a m/z peak of
896.3298 [MþH]þ which is in good agreement with the calculated
4.10. Synthesis of Nw-[2-acetamido-2-deoxy-4-O-(2-acetamido-2-
deoxy-b-d- glucopyranosyl)-6-O-(a-l-fucopyranosyl)-b-d-
glucopyranosyl]-l-asparagine tert-butyl ester (17)
molecular weight (C35H53N5O22
, 895.3182). The residue was
washed with dichloromethane to remove the excessive DSG. To the
Compound 17 (18 mg) was synthesized from compound 15
resulted aqueous layer containing the crude product 19, FITC-

D. Cai et al. / Carbohydrate Research 449 (2017) 143e152
151
labeled amino-PAMAM (20) (10 mg) was added and the mixture
solution were washed with chloroform (500
aqueous layer was taken and diluted with 800
m
L ꢂ 3), and 200
mL
was stirred at rt overnight. The mixture was subject to gel filtration
via a Sephadex G-25 column eluted by water. The fractions con-
taining glycodendrimer was combined and lyophilized to give
product 22 as an oil (12.5 mg). To determine the loading degree of
m
L water. The sam-
ples were subject to HPAEC-PAD analysis for monosaccharide
quantification. And the concentrations of released fucose and
glucosamine from respective glycoconjugates were calculated from
integral area of corresponding peaks (see the example in Fig. 1)
based on the standard quantification curves of fucose and
glucosamine.
GlcNAcb1,4(Fuca1,6)GlcNAc trisaccharide motif on PAMAM prod-
uct, 22 was treated with 2 M trifluoroacetic acid and heated at 95 ꢁC
for 4 h to completely release the monosaccharides glucosamine and
fucose. Then quantification of released glucosamine and fucose was
performed by HPAEC-PAD analysis on a DIONEX ICS5000 system.
4.17. Fucose lectin AAL binding assay
Based on monosaccharide quantification, the GlcNAcb1,4(Fuca1,6)
GlcNAc trisaccharide loaded on PAMAM glycodendrimer 22 was
18.6% w/w (average 12.0 disaccharides per PAMAM).
AAL (20 mg/mL, 100 mL) was coated on a 96-well ELISA plate at
4
ꢁC overnight. Then, the plate was washed with a Hepes buffer
(10 mM, pH 8.0) containing 0.1 mM Ca2þ and was incubated with a
blocking protein BSA (1 mg/mL) at 37 ꢁC for 2 h. The plate was
washed again with the above Hepes buffer. Then, FITC-PAMAM
(20), FITC-PAMAM-disaccharide (21), and FITC-PAMAM-
4.14. Preparation of Agarose-Asn(GlcNAcb1,4GlcNAc)
(glycoconjugate 23)
The in situ compound 18 prepared from 16 following above
procedure in sodium acetate butter (0.1 M, pH 7.5) was mixed with
amino-agarose (10 mL) and the mixture was shaken at rt overnight.
The beads was loaded to an empty plastic column with a filter pad
and washed with water and sodium acetate buffer. The obtained
glycoconjugate 23 was then suspended in 10 mL sodium acetate
buffer (0.1 M, pH 7.5) for storage. To determine the loading degree
trisaccharide (22) in concentrations of 1.0, 0.5, 0.25, and 0.125 mM
were added in respective wells and the plate was incubated at 37 ꢁC
for 2 h. After washed with above Hepes buffer, 500 mM fucose was
added and the plate was incubated at rt for 0.5 h then was subject to
fluorescent detection with a BioTek Synergy™ 2 microplate reader.
The recorded fluorescent intensity data were summarized in Fig. 2.
of GlcNAc
portion of 23 (100
b
1,4GlcNAc disaccharide motif on the agarose beads,
L) was treated with 2 M trifluoroacetic acid and
4.18. Affinity chromatography purification of AAL with glyco-
agarose beads
m
heated at 95 ꢁC for 0.5 h then the supernatant was taken out to be
heated at 95 ꢁC for additional 3.5 h. Then quantification of released
glucosamine was performed by HPAEC-PAD (High-performance
anion-exchange chromatography with pulsed amperometric
detection) analysis on a DIONEX ICS5000 system following the
method described above. Based on glucosamine quantification, the
The glyco-agarose beads (23 and 24) were respectively stuffed
into empty columns with filter pads (~1 mL volume of the beads).
The columns were pre-washed with a Hepes buffer (10 mM
pH ¼ 8.0) containing 0.1 mM Ca2þ. AAL (50
mg) in above Hepes
buffer (500 mL) was loaded to the agarose columns respectively and
GlcNAcb1,4GlcNAc disaccharide loaded on glyco-agarose 23 was
the columns was kept at 4 ꢁC for 30 min. Then, the columns was
washed with above Hepes buffer (2 mL) and eluted with 2 mL of
100 mM, 250 mM, 500 mM, and 1.0 M Fucose in above Hepes buffer
successively. The flow-through, wash, and elution fractions were
0.1 mg/mL.
4.15. Preparation of Agarose-Asn[GlcNAcb1,4(Fuca1,6)GlcNAc]
(glycoconjugate 24)
respectively concentrated to about 200 mL by ultrafiltration with a
10 kDa cut-off filter and were subject to SDS-PAGE analysis as
The in situ compound 19 prepared from 17 following above
procedure in sodium acetate butter (0.1 M, pH 7.5) was mixed with
amino-agarose (10 mL) and the mixture was shaken at rt overnight.
The beads was loaded to an empty plastic column with a filter pad
and washed with water and sodium acetate buffer. The obtained
glycoconjugate 24 was then suspended in 10 mL sodium acetate
buffer (0.1 M, pH 7.5) for storage. To determine the loading degree
shown in Fig. 3.
4.19. Isolation of AOL from a mixed protein sample with glyco-
agarose beads
The glyco-agarose beads (23 and 24) were respectively stuffed
into empty columns with filter pads (~1 mL volume of the beads).
The columns were pre-washed with a Hepes buffer (10 mM
pH ¼ 8.0) containing 0.1 mM Ca2þ. The mixture of AOL (Aspergillus
of GlcNAcb1,4(Fuc
a1,6)GlcNAc trisaccharide motif on the agarose
beads, portion of 24 (100
mL) was treated with 2 M trifluoroacetic
acid and heated at 95 ꢁC for 0.5 h then the supernatant was taken
out to be heated at 95 ꢁC for additional 3.5 h. Then quantification of
released glucosamine was performed by HPAEC-PAD (High-per-
formance anion-exchange chromatography with pulsed ampero-
metric detection) analysis on a DIONEX ICS5000 system following
the method described above. Based on glucosamine quantification,
oryzae lectin, TOKYO CHEMICAL INDUSTRY CO. LTD) (30
mg) and
BSA (50
mg) in above Hepes buffer (500 mL) was loaded to the
agarose columns respectively and the columns was kept at 4 ꢁC for
30 min. Then, the columns was washed with above Hepes buffer
(2 mL) and eluted with 800 mL of 200 mM L-Fucose (s-9007, Vector
Laboratories,Inc.) in above Hepes buffer successively. The flow-
the GlcNAcb1,4(Fuc
a1,6)GlcNAc trisaccharide loaded on glyco-
through, wash, and elution fractions were respectively concen-
agarose 24 was 0.16 mg/mL.
trated to about 500 mL by ultrafiltration with a 3 kDa cut-off filter
and were subject to SDS-PAGE analysis as shown in Fig. 4.
4.16. Monosaccharide analysis of glycoconjugates 21e24
Acknowledgments
For glycodendrimer 21 or 22, 100
solved in 420 L water, then TFA (77 L, 1 mmol) was added and the
mixture was heated at 95 ꢁC for 4 h. For glyco-agarose 23 or 24,
mg of the samples was dis-
m
m
This work was supported by the National Natural Science
Foundation of China (NNSFC, No.21372238, 21572244, and
31570797), the SIMM institute fund (CASIMM0120153004), and the
“Personalized Medicines: Molecular Signature-based Drug Discov-
ery and Development”, Strategic Priority Research Program of the
Chinese Academy of Sciences, Grant No. XDA12020311.
100
(77
mL of the samples was diluted with 320 mL water, then TFA
m
L, 1 mmol) was added and the mixture was heated at 95 ꢁC for
0.5 h. The supernatant was taken out and heated at 95 ꢁC for
additional 3.5 h. The hydrolyzed monosaccharides in the aqueous

152
D. Cai et al. / Carbohydrate Research 449 (2017) 143e152
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