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Liquid-Phase and Ultrahigh-Frequency-Acoustofluidics-Based Solid-Phase Synthesis of Biotin-Tagged 6′/3′-Sialyl-N-Acetylglucosamine by Sequential One-Pot Multienzyme System

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6′/3′-Sialylated N-acetyllactosamine (6′/3′-SLN) is important for discrimination of the source (human or avian) of influenza virus strains. Biotinylated oligosaccharides have been widely used for analysis and quick detection. The development of efficient strategies to synthesize biotin-tagged 6′/3′-SLN have become necessary. Effective mixing is essential for enzymatic solid-phase oligosaccharide synthesis (SPOS). In the current study, newly developed technology ultrahigh-frequency-acoustofluidics (UHFA), which can provide a powerful source for efficient microfluidic mixing, solid-phase oligosaccharide synthesis and one-pot multienzyme (OPME) system, were used to develop a new strategy for oligosaccharide synthesis. Firstly, biotinylated N-acetylglucosamine was designed and chemically synthesized through traditional approaches. Secondly, biotinylated 6′- and 3′-sialyl-N-acetylglucosamines were prepared in solution through two sequential OPME modules in with a yield of ~95%. Thirdly, 6′-SLN was also prepared through UHFA-based enzymatic solid-phase synthesis on magnetic beads with a yield of 64.4%. The current strategy would be potentially used for synthesis of functional oligosaccharides.
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catalysts
Article
Liquid-Phase and
Ultrahigh-Frequency-Acoustofluidics-Based
Solid-Phase Synthesis of Biotin-Tagged
60/30-Sialyl-N-Acetylglucosamine by Sequential
One-Pot Multienzyme System
Mengge Gong 1,2, Tiechuan Li 3, Lina Wu 2, Zhenxing Zhang 4, Lishi Ren 2, Xuexin Duan 3,
Hongzhi Cao 5, Meishan Pei 1, *, Jian-Jun Li 2, * and Yuguang Du 2
1
School of Chemistry and Chemical Engineering, University of Jinan, Jinan 250022, China; GMengge@163.com
2National Key Laboratory of Biochemical Engineering, National Engineering Research Center
for Biotechnology (Beijing), Key Laboratory of Biopharmaceutical Production & Formulation Engineering,
PLA, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, China;
lnwu@ipe.ac.cn (L.W.); lsren@ipe.ac.cn (L.R.); ygdu@ipe.ac.cn (Y.D.)
3State Key Laboratory of Precision Measuring Technology and Instruments, Tianjin University,
Tianjin 300072, China; leetch@tju.edu.cn (T.L.); xduan@tju.edu.cn (X.D.)
4Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China; zzx080250140@163.com
5National Glycoengineering Research Center, State Key Laboratory of Microbial Technology,
Shandong University, Qingdao 266237, China; hzcao@sdu.edu.cn
*Correspondence: chm_peims@ujn.edu.cn (M.P.); jjli@ipe.ac.cn (J.-J.L.);
Tel./Fax: +86-531-8973-6800 (M.P.); +86-10-8254-5039 (J.-J.L.)
Received: 16 October 2020; Accepted: 15 November 2020; Published: 19 November 2020


Abstract:
6
0
/3
0
-Sialylated N-acetyllactosamine (6
0
/3
0
-SLN) is important for discrimination of the
source (human or avian) of influenza virus strains. Biotinylated oligosaccharides have been widely
used for analysis and quick detection. The development of ecient strategies to synthesize
biotin-tagged 6
0
/3
0
-SLN have become necessary. Eective mixing is essential for enzymatic
solid-phase oligosaccharide synthesis (SPOS). In the current study, newly developed technology
ultrahigh-frequency-acoustofluidics (UHFA), which can provide a powerful source for ecient
microfluidic mixing, solid-phase oligosaccharide synthesis and one-pot multienzyme (OPME)
system, were used to develop a new strategy for oligosaccharide synthesis. Firstly, biotinylated
N-acetylglucosamine was designed and chemically synthesized through traditional approaches.
Secondly, biotinylated 6
0
- and 3
0
-sialyl-N-acetylglucosamines were prepared in solution through
two sequential OPME modules in with a yield of ~95%. Thirdly, 6
0
-SLN was also prepared through
UHFA-based enzymatic solid-phase synthesis on magnetic beads with a yield of 64.4%. The current
strategy would be potentially used for synthesis of functional oligosaccharides.
Keywords:
biotinylated 6
0
/3
0
-sialyl-N-acetylglucosamine; one-pot multienzyme; solid-phase
oligosaccharide synthesis; influenza viruses; ultrahigh-frequency-acoustofluidics
1. Introduction
Proteins, nucleic acids, lipids, and carbohydrates are four main types of biomolecules forming
the basis of life. Like proteins, nucleic acids and lipids, carbohydrates play key roles in many
biological processes such as protein conformation [
1
], molecular recognition [
2
], cell proliferation and
dierentiation [
3
], and are closely related with occurrence and development of many diseases [
4
].
Catalysts 2020,10, 1347; doi:10.3390/catal10111347 www.mdpi.com/journal/catalysts
Catalysts 2020,10, 1347 2 of 12
However, compared with nucleic acids and proteins, studies toward carbohydrates are lagging behind.
In recent years, with the rapid progress in glycobiology, investigations into carbohydrates have received
more and more attention.
Sialic acid-containing glycans play important functions in many physiological and pathological
processes, including intercellular adhesion, signaling, microbial attachment, etc. [
5
], and most sialic
acid-related biological processes require specific sialic acid forms, glycosidic linkage and defined
underlying glycan chains [
6
]. Furthermore, sialic acid is generally located at the nonreducing ends of
the sugar chains [
7
]. This outmost position and ubiquitous distribution enable sialylated glycans to
be involved in numerous cellular processes. For example, studies have shown that hemagglutinin
(HA) of human influenza virus strains preferentially binds to oligosaccharides that terminate with
6
0
-
α
-sialyl-N-acetyllactosamine (6
0
-SLN), whereas HA of the avian influenza virus strains prefers
oligosaccharides that terminate with 3
0
-
α
-sialyl-N-acetyllactosamine (3
0
-SLN), thereby enabling an
interspecific barrier for virus transmission [
8
]. In addition, aberrant protein sialylation has been
closely correlated with various cancers. For instance, prostate cancer features an elevated expression of
α2-3-linked glycans [9], whereas breast cancer exhibits overexpression of α2-6-linked glycans [10].
Mutations in the viral HA binding site that switch selectivity from
α
2-3 to
α
2-6 sialoglycans are a
prerequisite for interspecies transfer and can indicate a newly acquired ability of avian viruses to infect
humans [
8
]. For example, several avian influenza virus subtypes (such as H5N1, H7 or H9N2) broke
through the species barrier and gained the ability to infect humans [
11
]. Therefore, screening tools
to identify changes in influenza glycan specificity have been employed for early diagnosis of virus
transmissibility and assessment of possible pandemic risks, and are also important for dierentiation of
the source of influenza virus strains [
12
]. Sialyllactosamine derivatives-based probes would potentially
achieve those goals. Some sialyllactosamine derivatives were synthesized and investigated for that,
including imidazolium-tagged 6
0
/3
0
-sialyllactosamine [
13
], multivalent 6
0
-sialyllactosamine-carrying
glyco-nanoparticles [14], and sialylglycopolymers-bearing 60-sialyllactosamine [15].
Biotin-avidin binding, with a very high anity (10
15
M
1
) [
16
], is the strongest known noncovalent
interaction in nature and has been extensively exploited for biological applications. That binding
can be used for controlled immobilization of biotinylated oligosaccharides onto streptavidin-coated
ELISA plates and beads and for tracing carbohydrate binding molecules. Therefore, biotinylated
oligosaccharides have been widely used for analysis and detection. For example, biotinylated
chondroitin sulfate tetrasaccharides were used for analyzing their interactions with the monoclonal
antibodies 2H6 and LY111 [
17
].The biotinylated derivatives of the oligo-
α
-(1
3)-D-glucosides
could be used to investigate glycan-protein interactions and cytokine induction associated
with the immune response to Aspergillus fumigatus [
18
]. Biotinylated N-acetyllactosamine- and
N,N-diacetyllactosamine-based oligosaccharides were used as novel ligands for human galectin-3 [
19
].
Biotinylated 6
0
/3
0
-sialyl-N-acetyllactosamine could be potentially used for early diagnosis of virus
transmissibility and assessment of possible pandemic risks too. In 2007, biotinylated 6
0
/3
0
-sialyl-N-
acetyllactosamine was prepared. p-Aminophenyl glycosides of 6
0
/3
0
-sialyl-N-acetyllactosaminide was
synthesized starting from p-nitrophenyl-N-acetyl-
β
-D-glucosaminide through three steps: synthesis
of p-nitrophenyl-N-acetyllactosaminide with
β
-D-galactosidase, followed by chemical reduction of
the p-nitrophenyl group and sialylation with recombinant rat
α
2,3-sialyltransferase and rat liver
α
2,6-sialyltransferase. Finally, the p-aminophenyl glycosides were biotin-labeled through the coupling
with biotinyl-6-aminohexanoic acid to aord biotinylated oligosaccharides. The biotin-labeled sugars
were shown to be useful for immobilization and assay of the carbohydrate-lectin interactions by surface
plasmon resonance (SPR). Due to low specificity and uncontrolled transglycosylation of
β
-galactosidase,
in addition to the target product-N-acetyllactosamine,
β
1,6-galactosyl N-acetylglucosamine,
trisaccharide and tetrasaccharide were also produced. Moreover, rat sialyltransferases were used for
sialylation [20].
Catalysts 2020,10, 1347 3 of 12
Compared to mammalian glycosyltransferases, bacterial ones are less sensitive to nucleotide
inhibition, and show broad substrate specificity, and are available in recombinant and soluble
form with high expression level. Therefore, bacterial glycosyltransferases are widely used in
oligosaccharide synthesis. Considering the fact that eective and economic synthesis oligosaccharide
is to combine the sugar nucleotide biosynthetic process with glycosyltransferase-catalyzed reactions,
highly ecient bacterial glycosyltransferases based one-pot multienzyme (OPME) [
21
] methods
starting from simple monosaccharides have been developed and used for synthesis of many structurally
complicated oligosaccharides.
In comparison with liquid-phase oligosaccharide synthesis, solid-phase oligosaccharide synthesis
(SPOS) oers advantages in several aspects: (1) only one purification step is needed in most cases at the
end of the reaction; (2) unwanted reagents and side products can be easily removed by washing and
filtering, and so excessive glycosyl donor can be used to ensure the high production yield. Enzymatic
SPOS, combining advantages of OPME and SPOS in particular, is more desirable since it could oer a
real simplification by combining the advantages of the OPME approach with those of the solid-phase
method [22].
A micro-fabricated solid-mounted thin-film piezoelectric resonator (SMR) with a frequency
of 1.54 GHz has been integrated into microfluidic systems. Experimental and simulation results
showed that UHF (ultrahigh frequency)-SMR triggers strong acoustic field gradients to produce
ecient and highly localized acoustic streaming vortices, providing a powerful source for microfluidic
mixing [
23
]. Ultrahigh frequency (~2.5 GHz) piezoelectric resonators as acoustic micromixers were
excited to produce turbulent flow in microdroplets for in situ, pumping-free, and highly ecient
mixing [
24
]. This ultrahigh-frequency-acoustofluidics (UHFA) was successfully used for classic
Diels-Alder reactions [25].
In this study, biotinylated 6
0
- and 3
0
-sialyl-N-acetyllactosamine were synthesized by the
liquid-phase and SPOS-based OPME approach. Given the fact that ecient mixing is important
for SPOS-based OPME synthesis, UHFA was applied for SPOS of biotinylated 6
0
- and 3
0
-sialyl-
N-acetyllactosamine due to its highly localized acoustic streaming vortices.
2. Results and Discussion
2.1. Design and Synthesis of Biotin-Tagged N-Acetylglucosamine (Biotin-GlcNAc)
The starting biotinylated monosaccharide acceptor-biotin-tagged N-acetylglucosamine (named as
Biotin-GlcNAc in this study) was designed and synthesized through well-known procedures shown in
Scheme 1(Supplementary materials). Compound
1
was synthesized from p-hydroxymethyl phenol
and 1,3-dibromo propane in the presence of K
2
CO
3
with medium yield. Compound
2
was prepared
from compound with large excess of NaN
3
at 95% yield. Compound
3
was synthesized through four
steps: glucosamine was first reacted with o-phthalic anhydride, then followed by peracetylation by
acetic anhydride and selective hydrolysis of C1-acetate, and finally trichloroacetimidate glycosyl donor
compound
4
was obtained stereo specifically. Compound
4
was produced through direct coupling
between compounds
2
and
3
. Compound
4
was then fully deprotected and followed by selective
acetylation at the amino group to give compound
5
. Compound
5
was reduced and condensed with
N-hydroxysuccinimide (NHS)-activated biotin to aord the final product-Biotin-GlcNAc (compound
6
),
which was purified by HPLC (High Performance Liquid Chromatography) (Figures S1 and S2
in Supplementary materials). Compound
6
was characterized by NMR and MS (Figure S3 in
Supplementary materials).
Catalysts 2020,10, 1347 4 of 12
Catalysts 2020, 10, x FOR PEER REVIEW 4 of 12
Scheme 1. Synthesis of biotin-labeled N-acetylglucosamine (Biotin-GlcNAc).
2.2. Sequential OPME Synthesis of Biotinylated 6′/3′-sialyl-N-Acetylglucosamine in Solution
With chemically prepared Biotin-GlcNAc (compound 6) in hand, a one-pot two-enzyme
β1,4-galactosylation system (OPME 1) was adopted for introducing β1,4-linked galactose to
Biotin-GlcNAc based on previous report (Scheme 2) [26]. Considering the fact that UDP-Gal (CAS
No. 137868-52-1, catalog No. 254155, $648/50 mg) (J&K Scientific Ltd., Beijing, China) was much
more expensive than UDP-Glc (CAS No.117756-22-6, catalog No. 542767, $30/50 mg) (J&K Scientific
Ltd., Beijing, China), UDP-Gal was selected as the starting material for OPME 1. Thus, in this
one-pot two-enzyme system (OPME 1), UDP-Glc was converted into UDP-Gal by an Escherichia coli
UDP-galactose isomerase (EcGalE) (Figure S4 in Supplementary materials). The in situ generated
UDP-Gal donor was then used by a Neisseria meningitides 1,4-galactosyltransferase (NmLgtB) to
afford Compound I during 16 h with the yield of 94.8% after convenient BioGel P-2 gel filtration
purification (Figure S5 in Supplementary materials) (Figure 1) (Table 1). Compound I (C I) was
purified by HPLC and confirmed by mass spectrometry with m/z 773.07 (predicted molecular weight
772.32) and NMR (Figures S9, S16 and S17 in Supplementary materials).
Table 1. Yields of three OPMEs by different approaches.
Different OPMEs
Yield
In Solution
UHFA-Based SPOS
OPME1
94.8%
64.4%
OPME2
95.4%
58.6%
OPME3
94.5%
ND 1
1 ND: Not done.
Scheme 1. Synthesis of biotin-labeled N-acetylglucosamine (Biotin-GlcNAc).
2.2. Sequential OPME Synthesis of Biotinylated 60/30-sialyl-N-Acetylglucosamine in Solution
With chemically prepared Biotin-GlcNAc (compound
6
) in hand, a one-pot two-enzyme
β
1,4-galactosylation system (OPME 1) was adopted for introducing
β
1,4-linked galactose to
Biotin-GlcNAc based on previous report (Scheme 2) [
26
]. Considering the fact that UDP-Gal (CAS
No. 137868-52-1, catalog No. 254155, $648/50 mg) (J&K Scientific Ltd., Beijing, China) was much
more expensive than UDP-Glc (CAS No.117756-22-6, catalog No. 542767, $30/50 mg) (J&K Scientific
Ltd., Beijing, China), UDP-Gal was selected as the starting material for OPME 1. Thus, in this
one-pot two-enzyme system (OPME 1), UDP-Glc was converted into UDP-Gal by an Escherichia coli
UDP-galactose isomerase (EcGalE) (Figure S4 in Supplementary materials). The in situ generated
UDP-Gal donor was then used by a Neisseria meningitides
β
1,4-galactosyltransferase (NmLgtB) to aord
Compound
I
during 16 h with the yield of 94.8% after convenient BioGel P-2 gel filtration purification
(Figure S5 in Supplementary materials) (Figure 1) (Table 1). Compound
I
(C I) was purified by HPLC
and confirmed by mass spectrometry with m/z773.07 (predicted molecular weight 772.32) and NMR
(Figures S9, S16 and S17 in Supplementary materials).
To fulfill the final sialylation step, a one-pot two-enzyme
α
2,6/2,3-sialylation system (OPME 2 or
OPME 3) was adopted for introducing
α
2,6/2,3-linked sialic acid (N-acetylneuraminic acid, Neu5Ac)
to the galactose unit of Compound
I
(Scheme 2). In this one-pot two-enzyme system (OPME 2 or
OPME 3), Neu5Ac was converted into CMP-Neu5Ac in the presence of cytidine 5’-triphosphate (CTP),
and a recombinant CMP-sialic acid synthetase from Neisseria meningitides (NmCSS), and the resulting
CMP-Neu5Ac as a donor was used by an
α
2,6-sialyltransferase (Pd2,6ST:
α
2,6-sialyltransferase from
Photobacterium damsela) and an
α
2,3-sialyltransferase (PmST1:
α
2,3-sialyltransferase from Pasteurella
multocida) for the formation of compound
II
and
III
in the yield of around 95% after convenient Bio-Gel
P-2 gel filtration purification respectively (Figure S6–S8, S10 and S12 in Supplementary materials)
(Table 1). Compounds
2
and
3
were characterized by MS and NMR (Figures S11, S13 and S18–S21 in
Supplementary materials).
Catalysts 2020,10, 1347 5 of 12
Catalysts 2020, 10, x FOR PEER REVIEW 5 of 12
Scheme 2. OPMEs synthesis of biotin-tagged 6′-and 3′-sialyl-N-acetyllactosamine. Biotinylated 6′-
and 3′-sialyl-N-acetyllactosamine would be synthesized by three enzymatic modules: OPME 1
consisting of GalE and NmLgtB, OPME 2 including NmCSS and Pd2,6ST, and OPME 3 comprising
NmCSS and PmST1. GalE: Escherichia coli UDP-galactose C4-epimerase; NmLgtB: Neisseria
meningitides β1,4-galactosyltransferase; NmCSS: Neisseria meningitidis CMP-sialic acid synthetase;
Pd2,6ST: Photobacterium damselae α2,6-sialyltransferase; PmST1 M144D: Pasteurella multocida
α2,3-sialyltransferase.
Figure 1. HPLC analysis of OPME 1-catalyzed synthesis of compound I (C I).
To fulfill the final sialylation step, a one-pot two-enzyme α2,6/2,3-sialylation system (OPME 2
or OPME 3) was adopted for introducing α2,6/2,3-linked sialic acid (N-acetylneuraminic acid,
Neu5Ac) to the galactose unit of Compound I (Scheme 2). In this one-pot two-enzyme system
(OPME 2 or OPME 3), Neu5Ac was converted into CMP-Neu5Ac in the presence of cytidine
5’-triphosphate (CTP), and a recombinant CMP-sialic acid synthetase from Neisseria meningitides
(NmCSS), and the resulting CMP-Neu5Ac as a donor was used by an α2,6-sialyltransferase
(Pd2,6ST: α2,6-sialyltransferase from Photobacterium damsela) and an α2,3-sialyltransferase (PmST1:
α2,3-sialyltransferase from Pasteurella multocida) for the formation of compound II and III in the
yield of around 95% after convenient Bio-Gel P-2 gel filtration purification respectively (Figure
S6S8, S10 and S12 in Supplementary materials) (Table 1). Compounds 2 and 3 were characterized
by MS and NMR (Figures S11, S13 and S18S21 in Supplementary materials).
Until now, biotin-labeled 6′/3′-sialyl-N-acetylglucosamine was only synthesized by Zeng et al.
[20]. However, galactosidase, which led to lower yield of targeted galactosylation, and rat
Scheme 2.
OPMEs synthesis of biotin-tagged 6
0
-and 3
0
-sialyl-N-acetyllactosamine. Biotinylated
6
0
- and 3
0
-sialyl-N-acetyllactosamine would be synthesized by three enzymatic modules: OPME
1 consisting of GalE and NmLgtB, OPME 2 including NmCSS and Pd2,6ST, and OPME 3
comprising NmCSS and PmST1. GalE: Escherichia coli UDP-galactose C4-epimerase; NmLgtB:
Neisseria meningitides
β
1,4-galactosyltransferase; NmCSS: Neisseria meningitidis CMP-sialic acid
synthetase; Pd2,6ST: Photobacterium damselae
α
2,6-sialyltransferase; PmST1 M144D: Pasteurella multocida
α2,3-sialyltransferase.
Catalysts 2020, 10, x FOR PEER REVIEW 5 of 12
Scheme 2. OPMEs synthesis of biotin-tagged 6′-and 3′-sialyl-N-acetyllactosamine. Biotinylated 6′-
and 3′-sialyl-N-acetyllactosamine would be synthesized by three enzymatic modules: OPME 1
consisting of GalE and NmLgtB, OPME 2 including NmCSS and Pd2,6ST, and OPME 3 comprising
NmCSS and PmST1. GalE: Escherichia coli UDP-galactose C4-epimerase; NmLgtB: Neisseria
meningitides β1,4-galactosyltransferase; NmCSS: Neisseria meningitidis CMP-sialic acid synthetase;
Pd2,6ST: Photobacterium damselae α2,6-sialyltransferase; PmST1 M144D: Pasteurella multocida
α2,3-sialyltransferase.
Figure 1. HPLC analysis of OPME 1-catalyzed synthesis of compound I (C I).
To fulfill the final sialylation step, a one-pot two-enzyme α2,6/2,3-sialylation system (OPME 2
or OPME 3) was adopted for introducing α2,6/2,3-linked sialic acid (N-acetylneuraminic acid,
Neu5Ac) to the galactose unit of Compound I (Scheme 2). In this one-pot two-enzyme system
(OPME 2 or OPME 3), Neu5Ac was converted into CMP-Neu5Ac in the presence of cytidine
5’-triphosphate (CTP), and a recombinant CMP-sialic acid synthetase from Neisseria meningitides
(NmCSS), and the resulting CMP-Neu5Ac as a donor was used by an α2,6-sialyltransferase
(Pd2,6ST: α2,6-sialyltransferase from Photobacterium damsela) and an α2,3-sialyltransferase (PmST1:
α2,3-sialyltransferase from Pasteurella multocida) for the formation of compound II and III in the
yield of around 95% after convenient Bio-Gel P-2 gel filtration purification respectively (Figure
S6S8, S10 and S12 in Supplementary materials) (Table 1). Compounds 2 and 3 were characterized
by MS and NMR (Figures S11, S13 and S18S21 in Supplementary materials).
Until now, biotin-labeled 6′/3′-sialyl-N-acetylglucosamine was only synthesized by Zeng et al.
[20]. However, galactosidase, which led to lower yield of targeted galactosylation, and rat
Figure 1. HPLC analysis of OPME 1-catalyzed synthesis of compound I(C I).
Table 1. Yields of three OPMEs by dierent approaches.
Dierent OPMEs
Yield
In Solution UHFA-Based SPOS Eppendorf Tube-Based SPOS
OPME1 94.8% 64.4% 49.5%
OPME2 95.4% 58.6% 45.3%
OPME3 94.5% ND 1ND 1
1ND: Not done.
Until now, biotin-labeled 6
0
/3
0
-sialyl-N-acetylglucosamine was only synthesized by Zeng et al. [
20
].
However, galactosidase, which led to lower yield of targeted galactosylation, and rat sialyltransferases
were used. Bacterial galactosyltransferase and sialyltransferases were used in the current study instead,
which led to highly ecient galactosylation and sialylation, respectively.
Catalysts 2020,10, 1347 6 of 12
2.3. Ultrahigh-Frequency-Acoustofluidics (UHFA)-Based Solid-Phase Sequential OPME Synthesis of
Biotinylated 60-sialyl-N-Acetylglucosamine
Considering the fact that ecient mixing is one of the main characteristics of ultrahigh-frequency-
acoustofluidics (UHFA), which is important for solid-phase oligosaccharide synthesis (SPOS)-based
enzymatic synthesis, UHFA-based solid-phase sequential OPME synthesis of biotinylated
60-sialyl-N-acetylglucosamine was attempted (Schemes 3and 4).
Catalysts 2020, 10, x FOR PEER REVIEW 6 of 12
sialyltransferases were used. Bacterial galactosyltransferase and sialyltransferases were used in the
current study instead, which led to highly efficient galactosylation and sialylation, respectively.
2.3. Ultrahigh-Frequency-Acoustofluidics (UHFA)-Based Solid-Phase Sequential OPME Synthesis of
Biotinylated 6′-sialyl-N-Acetylglucosamine
Considering the fact that efficient mixing is one of the main characteristics of
ultrahigh-frequency-acoustofluidics (UHFA), which is important for solid-phase oligosaccharide
synthesis (SPOS)-based enzymatic synthesis, UHFA-based solid-phase sequential OPME synthesis
of biotinylated 6′-sialyl-N-acetylglucosamine was attempted (Schemes 3 and 4).
Scheme 3. Ultrahigh-frequency-acoustofluidics (UHFA)-based solid-phase sequential OPME
synthesis of Compound I (C I).
Scheme 4. Ultrahigh-frequency-acoustofluidics (UHFA)-based solid-phase sequential OPME
synthesis of Compound II (C II).
Streptavidin magnetic beads (Dyna beads) from Invitrogen were used as solid-phase carriers.
GlcNAc-Biotin (compound 6) or Compound I was attached to the surface of streptavidin magnetic
beads through strong interaction between biotin and streptavidin. OPME 1- and 2-catalyzed
reactions were performed on surface of magnetic beads on the UHFA platform. Both reactionslasted
for 24 h. After enzymatic reactions were stopped, the product (Compound I or II) was released from
magnetic beads by reversibly disrupting biotin-streptavidin interaction through heating at 70 C [27]
on a metal bath, and analyzed by MS, demonstrating the feasibility of UHFA-based solid-phase
sequential OPME synthesis of compound I and II (Figures 2 and 3). Unfortunately, the yields of
two OPMEs-catalyzed reactions were only around 58.664.4% (Table 1). Despite attempts with
increases in amounts of enzymes, UDP-Glc, sialic acid and CTP, or reaction time, the yields of
UHFA-based synthesis of compound I and II through two OPMEs were not improved too much. In
addition, even washing of magnetic beads after coupling and repeated coupling with fresh enzymes
and nucleotide sugars (or double-coupling”) did not lead to better yields. The results were
consistent with previous ones of solid-phase-based enzymatic synthesis of oligosaccharides.
Obviously, there were unreactive acceptor sites present on the surface of magnetic beads. This is a
well-known phenomenon in chemical solid-phase synthesis, caused by, e.g., capping by an
Scheme 3.
Ultrahigh-frequency-acoustofluidics (UHFA)-based solid-phase sequential OPME synthesis
of Compound I(C I).
Catalysts 2020, 10, x FOR PEER REVIEW 6 of 12
sialyltransferases were used. Bacterial galactosyltransferase and sialyltransferases were used in the
current study instead, which led to highly efficient galactosylation and sialylation, respectively.
2.3. Ultrahigh-Frequency-Acoustofluidics (UHFA)-Based Solid-Phase Sequential OPME Synthesis of
Biotinylated 6′-sialyl-N-Acetylglucosamine
Considering the fact that efficient mixing is one of the main characteristics of
ultrahigh-frequency-acoustofluidics (UHFA), which is important for solid-phase oligosaccharide
synthesis (SPOS)-based enzymatic synthesis, UHFA-based solid-phase sequential OPME synthesis
of biotinylated 6′-sialyl-N-acetylglucosamine was attempted (Schemes 3 and 4).
Scheme 3. Ultrahigh-frequency-acoustofluidics (UHFA)-based solid-phase sequential OPME
synthesis of Compound I (C I).
Scheme 4. Ultrahigh-frequency-acoustofluidics (UHFA)-based solid-phase sequential OPME
synthesis of Compound II (C II).
Streptavidin magnetic beads (Dyna beads) from Invitrogen were used as solid-phase carriers.
GlcNAc-Biotin (compound 6) or Compound I was attached to the surface of streptavidin magnetic
beads through strong interaction between biotin and streptavidin. OPME 1- and 2-catalyzed
reactions were performed on surface of magnetic beads on the UHFA platform. Both reactionslasted
for 24 h. After enzymatic reactions were stopped, the product (Compound I or II) was released from
magnetic beads by reversibly disrupting biotin-streptavidin interaction through heating at 70 C [27]
on a metal bath, and analyzed by MS, demonstrating the feasibility of UHFA-based solid-phase
sequential OPME synthesis of compound I and II (Figures 2 and 3). Unfortunately, the yields of
two OPMEs-catalyzed reactions were only around 58.664.4% (Table 1). Despite attempts with
increases in amounts of enzymes, UDP-Glc, sialic acid and CTP, or reaction time, the yields of
UHFA-based synthesis of compound I and II through two OPMEs were not improved too much. In
addition, even washing of magnetic beads after coupling and repeated coupling with fresh enzymes
and nucleotide sugars (or double-coupling”) did not lead to better yields. The results were
consistent with previous ones of solid-phase-based enzymatic synthesis of oligosaccharides.
Obviously, there were unreactive acceptor sites present on the surface of magnetic beads. This is a
well-known phenomenon in chemical solid-phase synthesis, caused by, e.g., capping by an
Scheme 4.
Ultrahigh-frequency-acoustofluidics (UHFA)-based solid-phase sequential OPME synthesis
of Compound II (C II).
Streptavidin magnetic beads (Dyna beads) from Invitrogen were used as solid-phase carriers.
GlcNAc-Biotin (compound
6
) or Compound
I
was attached to the surface of streptavidin magnetic
beads through strong interaction between biotin and streptavidin. OPME 1- and 2-catalyzed reactions
were performed on surface of magnetic beads on the UHFA platform. Both reactionslasted for 24 h.
After enzymatic reactions were stopped, the product (Compound
I
or
II
) was released from magnetic
beads by reversibly disrupting biotin-streptavidin interaction through heating at 70
C [
27
] on a metal
bath, and analyzed by MS, demonstrating the feasibility of UHFA-based solid-phase sequential OPME
synthesis of compound
I
and
II
(Figures 2and 3). Unfortunately, the yields of two OPMEs-catalyzed
reactions were only around 58.6–64.4% (Table 1). Despite attempts with increases in amounts of
enzymes, UDP-Glc, sialic acid and CTP, or reaction time, the yields of UHFA-based synthesis of
compound
I
and
II
through two OPMEs were not improved too much. In addition, even washing
of magnetic beads after coupling and repeated coupling with fresh enzymes and nucleotide sugars
(or “double-coupling”) did not lead to better yields. The results were consistent with previous ones
of solid-phase-based enzymatic synthesis of oligosaccharides. Obviously, there were unreactive
acceptor sites present on the surface of magnetic beads. This is a well-known phenomenon in chemical
solid-phase synthesis, caused by, e.g., capping by an undesired chemical group during the coupling
procedure or steric factors. In our case, it was reasonable to assume that the low yields were caused not
by capping but rather by steric factors. The relatively short length of the linker (11 atoms, approximately
Catalysts 2020,10, 1347 7 of 12
13 Å in the most extended conformation) connecting the acceptor monosaccharide or disaccharide to
biotin and the size of enzymes such as glycosyltransferases (galactosyltransferase or sialyltransferase)
(diameter approximately 60 Å, assuming a globular form) should make at least some acceptor sites
“unapproachable” by the enzymes or lead to steric interference between enzymes and magnetic beads
and also less conformational flexibility [
28
]. Moreover, the binding of biotin to streptavidin on the
surface of magnetic beads would make that worse [
29
]. In addition, we think the low yields were
possibly due to heterogeneous reactions catalyzed by two OPMEs.
Catalysts 2020, 10, x FOR PEER REVIEW 7 of 12
undesired chemical group during the coupling procedure or steric factors. In our case, it was
reasonable to assume that the low yields were caused not by capping but rather by steric factors. The
relatively short length of the linker (11 atoms, approximately 13 Å in the most extended
conformation) connecting the acceptor monosaccharide or disaccharide to biotin and the size of
enzymes such as glycosyltransferases (galactosyltransferase or sialyltransferase) (diameter
approximately 60 Å , assuming a globular form) should make at least some acceptor sites
“unapproachable” by the enzymes or lead to steric interference between enzymes and magnetic
beads and also less conformational flexibility [28]. Moreover, the binding of biotin to streptavidin on
the surface of magnetic beads would make that worse [29]. In addition, we think the low yields were
possibly due to heterogeneous reactions catalyzed by two OPMEs.
Figure 2. MS analysis of UHFA-based solid-phase sequential OPME synthesis of Compound I (C I).
Figure 3. MS analysis of UHFA-based solid-phase sequential OPME synthesis of Compound II (C II).
Continuous UHFA-based solid-phase sequential synthesis of biotinylated
6′-sialyl-N-acetylglucosamine catalyzed by OPME 1 and OPME 2 was not attempted due to the
observation that yield of OPME 1-catalyzed UHFA-based solid-phase synthesis of Compound I was
below 50%.
The results of UHFA-based solid-phase sequential OPME synthesis of biotinylated
6′-sialyl-N-acetylglucosamine were also compared with those of traditional solid-phase sequential
OPME synthesis, which was carried out in Eppendorf tubes by using a tube rotator at 37 °C (Figures
S11 and S12 in Supplementary materials). It seems that yields of UHFA-based solid-phase approach
were about 15% higher than those of Eppendorf tube-based one (Table 1).
Figure 2. MS analysis of UHFA-based solid-phase sequential OPME synthesis of Compound I(C I).
Catalysts 2020, 10, x FOR PEER REVIEW 7 of 12
undesired chemical group during the coupling procedure or steric factors. In our case, it was
reasonable to assume that the low yields were caused not by capping but rather by steric factors. The
relatively short length of the linker (11 atoms, approximately 13 Å in the most extended
conformation) connecting the acceptor monosaccharide or disaccharide to biotin and the size of
enzymes such as glycosyltransferases (galactosyltransferase or sialyltransferase) (diameter
approximately 60 Å , assuming a globular form) should make at least some acceptor sites
“unapproachable” by the enzymes or lead to steric interference between enzymes and magnetic
beads and also less conformational flexibility [28]. Moreover, the binding of biotin to streptavidin on
the surface of magnetic beads would make that worse [29]. In addition, we think the low yields were
possibly due to heterogeneous reactions catalyzed by two OPMEs.
Figure 2. MS analysis of UHFA-based solid-phase sequential OPME synthesis of Compound I (C I).
Figure 3. MS analysis of UHFA-based solid-phase sequential OPME synthesis of Compound II (C II).
Continuous UHFA-based solid-phase sequential synthesis of biotinylated
6′-sialyl-N-acetylglucosamine catalyzed by OPME 1 and OPME 2 was not attempted due to the
observation that yield of OPME 1-catalyzed UHFA-based solid-phase synthesis of Compound I was
below 50%.
The results of UHFA-based solid-phase sequential OPME synthesis of biotinylated
6′-sialyl-N-acetylglucosamine were also compared with those of traditional solid-phase sequential
OPME synthesis, which was carried out in Eppendorf tubes by using a tube rotator at 37 °C (Figures
S11 and S12 in Supplementary materials). It seems that yields of UHFA-based solid-phase approach
were about 15% higher than those of Eppendorf tube-based one (Table 1).
Figure 3.
MS analysis of UHFA-based solid-phase sequential OPME synthesis of Compound
II
(C II).
Continuous UHFA-based solid-phase sequential synthesis of biotinylated 6
0
-sialyl-N-
acetylglucosamine catalyzed by OPME 1 and OPME 2 was not attempted due to the observation that
yield of OPME 1-catalyzed UHFA-based solid-phase synthesis of Compound Iwas below 50%.
The results of UHFA-based solid-phase sequential OPME synthesis of biotinylated 6
0
-sialyl-N-
acetylglucosamine were also compared with those of traditional solid-phase sequential OPME synthesis,
which was carried out in Eppendorf tubes by using a tube rotator at 37
C (
Figures S11 and S12
in
Supplementary materials). It seems that yields of UHFA-based solid-phase approach were about 15%
higher than those of Eppendorf tube-based one (Table 1).
Catalysts 2020,10, 1347 8 of 12
3. Materials and Methods
3.1. General Information
Unless otherwise stated, chemicals were purchased and used without further purification.
Gel filtration chromatography was performed using a column (100 cm
×
2.5 cm) packed with Bio-Gel
P-2 Fine resins (Bio-Rad, Hercules, CA, USA).
1
H and
13
C NMR spectra were recorded on Bruker
AVANCE-500 or Bruker AVANCE-400 spectrometer (Brucker, Bremen, Germany) at 25
C. Low and
high ESI (Electrospray ionization) (Thermo Fisher Scientific, Waltham, MA, USA) and MALDI-TOF
(Matrix-Assisted Laser Desorption/Ionization Time of Flight) (Brucker, Bremen, Germany) mass spectra
were obtained at Institute of Process Engineering, Chinese Academy of Sciences.
3.2. Enzymes Used in the Current Study
Neisseria meningitides CMP-sialic acid synthetase (NmCSS) [
30
,
31
], Photobacterium damselae
α
2,6-sialyltransferase (Pd2,6ST) [
32
,
33
], Escherichia coli UDP-galactose isomerase (GalE) [
31
,
34
], Neisseria
meningitides
β
1,4-galactosyltransferase (NmLgtB) [
26
,
31
], Pasteurella multocida
α
2,3-sialyltransferase
1 M144D mutant (PmST1 M144D) [
31
,
35
] were expressed and purified as reported in the literature
(Figures S3 and S4 in Supplementary materials).
3.3. OPME (One-Pot Multienzyme) 1-Catalyzed Reaction in Solution
OPME 1 includes GalE (UDP-galactose isomerase) and NmLgtB (
β
1,4-galactosyltransferase).
The enzyme-catalyzed reaction was carried out in a 10 mL centrifuge tube, including UDP-glucose
(UDP-Glc, 3.6 mM, 1.2 equiv), compound
6
(Biotin-GlcNAc, 3 mM, 1.0 equiv) (Supplementary materials),
Tris-HCl buer (100 mM, pH 7.5), MgCl
2
(20 mM), GalE (0.1 mg/mL) and NmLgtB (0.1 mg/mL). Reaction
was performed overnight at 37
C, 140 rpm, and was monitored by reversephase HPLC (wavelength:
230 nm, C
18
column) with the following conditions: flow rate at 1 mL/min; A: H
2
O, B: acetonitrile,
B increased from 5% to 50% within 30 min. When an optimal yield was achieved, the reaction
was stopped by adding the same volume of cold ethanol and kept at 4
C for 30 min, the mixture
was centrifuged at 12,000 rpm for 30 min and the precipitates were removed. The supernatant was
concentrated, and compound
I
(C I) was purified by preparative HPLC using the above HPLC conditions.
Compound
I
(CI) (yield: 94.8%), white solid after lyophilization.
1
H NMR400 MHz (MeOD/D
2
O)
δ
7.23 (d, J=8.0 Hz, 2H), 6.88 (d, J=8.0 Hz, 2H), 4.78 (d, J=12.0 Hz, 1H),
4.52 (d, J=12.0 Hz
, 2H),
4,45 (m, 2H),
4.37 (d, J=8.0 Hz, 1H), 4.24 (dd, J=8.0, 4.0 Hz, 1H),
4.0 (dd, J=8.0
,
4.0 Hz
, 2H), 3.93
(1H), 3.89 (d, J=4.0 Hz, 1H), 3.80 (d, J=4.0 Hz, 2H), 3.73 (d, J=8.0 Hz, 1H), 3.68 (d, J=4.0 Hz,
1H), 3.60 (dd, J=8.0, 4.0 Hz, 2H), 3.53 (d, J=8.0 Hz, 1H), 3.50 (s, 2H), 3.48 (d, J=4.0 Hz, 1H), 3.38
(
d, J=4.0 Hz, 1H
), 3.36 (d, J=4.0 Hz, 1H), 3.34 (d, J=4.0 Hz, 1H), 3.15 (m, 1H), 2.89 (dd, J=8.0, 4.0 Hz,
2H), 2.67 (d, J=4.0 Hz, 1H), 2.20 (dd, J=8.0, 4.0 Hz, 2H), 1.97 (t, J=8.0 Hz, 2H), 1.94 (s, 3H), 1.65 (m,
3H), 1.41 (m, 2H);
13
C NMR (400 MHz, MeOD/D
2
O)
δ
175.00, 172.5, 165.00, 158.75, 130.0, 129.5, 114.00,
103.9, 100.1, 79.9, 76.0, 75.5, 74.5, 73.0, 72.7, 71.5, 70.0, 69.0, 65.5, 62.5, 61.5, 60.4, 57.0, 56.0, 39.6, 36.0,
35.5, 28.0, 27.8, 27.6, 21.8, 16.5; ESI-MS m/zcalculated for C
34
H
52
N
4
O
14
S [M+H]
+
773.32, found 773.07.
3.4. OPME 2-Catalyzed Reaction in Solution
OPME 2 includes NmCSS (sialic acid synthase) and Pd2,6ST (
α
2,6-sialyltransferase). The enzyme-
catalyzed reaction was done in a 10 mL centrifuge tube at 37
C and 140 rpm, including compound CI
(1.0 equiv), sialic acid (1.2 equiv), CTP (1.5 equiv), Tris-HCl buer (100 mM, pH 8.5), MgCl2(20 mM),
NmCSS (0.1 mg/mL) and Pd2,6ST (0.18 mg/mL). Reaction was followed by reverse phase HPLC
(wavelength: 230 nm, C
18
column) with the same conditions as above. When an optimal yield was
achieved, the reaction was stopped by adding the same volume of cold ethanol and kept at 4
C for
30 min, the mixture was centrifuged at 12,000 rpm for 30 min and the precipitates were removed.
The supernatant was concentrated, and compound
II
(C II) was purified by BioGel P-2 column (eluted
with H
2
O). Compound
II
(C II) (yield: 95.4%), white solid after lyophilization.
1
H NMR400 MHz
Catalysts 2020,10, 1347 9 of 12
(MeOD/D
2
O)
δ
8.38 (br, 1H), 8.02 (d, J=8.0 Hz, 2H), 7.25 (d, J=8.0 Hz, 1H), 6.96 (d, J=8.0 Hz, 1H),
6.06 (d, J=8.0 Hz, 2H), 5.93 (br, 2H), 4.53 (br, 1H), 4.43 (br, 1H), 4.37 (br, 1H), 4.04–3.32 (m, 16H), 2.62
(m, 4H), 2.15 (m, 2H), 1.96 (br, 6H), 1.86 (d, J=16.0 Hz, 4H), 1.63 (t, J=16.0 Hz, 2H), 1.25 (m, 2H),
1.09 (br, 6H);
13
C NMR (400 MHz, MeOD/D
2
O)
δ
175.1, 171.6, 169.4, 166.8, 155.4, 142.0, 136.2, 124.8,
119.7(2C), 114.8 (2C), 97.2, 92.5, 89.2, 86.8, 83.5, 80.6, 75.4, 75.3, 74.4, 72.6, 71.7, 70.4, 69.7, 69.6, 68.4, 68.3,
65.8, 65.1, 62.9, 62.6, 62.5, 61.9, 60.6, 51.8, 51.7, 47.8, 40.1, 37.1, 36.9, 35.5, 25.2, 22.0, 21.0. HRMS (ESI) m/z
calculated for C45H69N5O22S [MH]1062.32, found 1062.40751.
3.5. OPME 3-Catalyzed Reaction
OPME3 includes NmCSS (sialic acid synthase) and PmST1 (
α
2,3-sialyltransferase M144D).
The enzyme-catalyzed reaction was done in a 10 mL centrifuge tube at 37
C and 140 rpm, including
compound CI (1.0 equiv), sialic acid (1.2 equiv), CTP (1.5 equiv), Tris-HCl buer (100 mM, pH 8.5),
MgCl
2
(20 mM), NmCSS (0.1 mg/mL) and PmST1 (0.2 mg/mL). Reaction was followed by reverse
phase HPLC (wavelength: 230 nm, C
18
column) with the same conditions as above. When an optimal
yield was achieved, the reaction was stopped by adding the same volume of cold ethanol and kept at 4
C for 30 min, the mixture was centrifuged at 12,000 rpm for 30 min and the precipitates were removed.
The supernatant was concentrated, and compound
III
(C III) was purified by BioGel P-2 column (eluted
with H
2
O). Compound
III
(C III)(yield: 94.5%), white solid after lyophilization.
1
H NMR400 MHz
(MeOD/D
2
O)
δ
8.37 (br, 1H), 8.02 (d, J=4.0 Hz, 2H), 7.24 (d, J=8.0 Hz, 1H), 6.95 (
d, J=8.0 Hz, 1H
),
6.06 (d, J=4.0 Hz, 2H), 5.92 (br, 2H), 4.53 (d, J=12.0 Hz, 1H), 4.47 (d, J=8.0 Hz, 1H), 4.42 (br, 1H),
4.04–3.43 (m, 16H), 2.64 (m, 4H), 2.16 (m, 2H), 1.96 (br, 6H),1.83 (br, 4H), 1.61 (t, J=16.0 Hz, 2H), 1.24
(m, 2H), 1.09 (br, 6H);
13
C NMR (400 MHz, MeOD/D
2
O)
δ
175.1, 171.8, 171.5, 166.3, 155.4, 141.8, 136.2,
122.3, 121.7(2C), 117.6 (2C), 99.8, 96.5, 89.2, 86.8, 83.5, 78.7, 75.4, 74.8, 74.2, 72.7, 71.7, 70.5, 69.7, 69.6,
68.3, 68.2, 65.8, 65.1, 62.9, 62.6, 62.5, 61.9, 60.6, 51.8, 51.7, 45.8, 44.5, 39.6, 35.5, 27.5, 25.2, 24.0, 22.0.
MALDI-TOF-MS m/zcalculated for C45H69N5O22S [MH]1062.32, found 1062.342.
3.6. Ultrahigh-Frequency-Acoustofluidics (UHFA)-Based Solid-Phase Sequential OPME Synthesis of
Biotinylated 60-sialyl-N-Acetylglucosamine
The fabrication process of the hypersonic device—Solid Mount Resonator (SMR) was performed
according to a published procedure [
36
]. The experimental set-up was very similar to the published
one [25,36].
Streptavidin magnetic beads were washed 4 times with buer 1 (PBS containing 0.01% Tween 20),
and were diluted to 5 mg/mL with buer 1 in a 2 mL Eppendorf tube. Compound
6
or compound
I
was added, and tube was rotated up and down for 30 min at room temperature to ensure that
compound
6
or compound
I
would be bound to streptavidin, which is equivalent to compound
6
or
compound
I
was immobilized onto magnetic beads. Then supernatant was separated from magnetic
beads with a magnet, and magnetic beads were washed 4 times with buer 1. Magnetic beads were
then resuspended in buer 1 containing 10 mg/mL BSA, and transferred into a plastic chamber which
was immobilized onto the top of the device. The components for OPME 1 or OPME 2-catalyzed
reaction as above excluding compound
6
or compound
I
were premixed, and added into the plastic
chamber. Finally, the chamber was covered with a lid, and 200 mW power was applied to the resonator.
The enzymatic reactions were lasted for 24 h at 37
C. Magnetic beads were separated from supernatant
with a magnet, and washed 4 times with PBS. Then magnetic beads were responded in 20
µ
L deionized
water, and heated on a metal bath at 70
C and 1000 rpm for 5 min. Finally, deionized water was
separated from magnetic beads, and used for MALDI-TOF-MS analysis.
3.7. Optimization of UHFA-Based Solid-Phase Sequential OPME Synthesis of Biotinylated
60-sialyl-N-Acetylglucosamine
To improve the yields of UHFA-based solid-phase OPME synthesis of biotinylated 6
0
-sialyl-N-
acetylglucosamine, the following conditions were tested: (1) increasing amounts of enzymes, UDP-Glc,
Catalysts 2020,10, 1347 10 of 12
sialic acid and CTP; (2) extending reaction time to 48 h; (3) washing magnetic beads after 24 h,
and adding a new batch of enzymes and substrates.
3.8. Traditional Solid-Phase Sequential OPME Synthesis of Biotinylated 60-sialyl-N-Acetylglucosamine
For comparison, traditional solid-phase sequential OPME synthesis of biotinylated 6
0
-sialyl-s-
acetylglucosamine was also carried out as above except OPME 1 and OPME 2-catalyzed reactions were
done in 2 mL of Eppendorf tubes by using a tube rotator at 37 C for 24 h.
4. Conclusions
In conclusion, using biotin-labeled N-acetylglucosamine (Biotin-GlcNAc) synthesized by
chemical method as the glycosyl receptor, the one-pot multi-enzyme (OPME) synthesis strategy
was successfully adopted to achieve the liquid-phase enzymatic synthesis of biotinlylated
6
0
/3
0
-sialyl-N-acetylglucosamine (6
0
/3
0
-SLN). Biotinylated 6
0
-sialyl-N-acetylglucosamine (6
0
-SLN) was
also prepared through ultrahigh-frequency-acoustofluidics (UHFA)-based solid-phase sequential
OPME system on magnetic beads. The biotinylated 6
0
/3
0
-sialyl-N-acetylglucosamine synthesized
here would be used to identify whether influenza viruses can infect humans or the source (human
or avian) of influenza virus strains. Alternatively, in order to reuse enzymes or to reduce production
cost of enzymes, immobilization of glycosyltransferases and/or related enzymes, which has been
successfully for oligosaccharide synthesis [37,38], could be used for synthesis of 60/30-SLN too. All in
all, this novel UHFA-based solid-phase synthetic strategy could be potentially applied toother organic
and enzymatic synthesis.
Supplementary Materials:
The following are available online at http://www.mdpi.com/2073-4344/10/11/1347/
s1, Synthetic procedures for biotin-tagged N-acetylglucosamine (Biotin-GlcNAc). Figure S1. Purification of
biotinylated N-acetylglucosamine (Biotin-GlcNAc) by reverse HPLC. Figure S2. HPLC analysis of purified
biotinylated N-acetylglucosamine (Biotin-GlcNAc). Figure S3. MS analysis of purified Biotin-GlcNAc. Figure S4.
SDS PAGE analysis of purified GalE. Figure S5. SDS PAGE analysis of purified NmLgtB. Figure S6. SDS PAGE
analysis of purified NmCSS. Figure S7. SDS PAGE analysis of purified Pd26ST. Figure S8. SDS PAGE analysis
of purified PmST1 (M144D). Figure S9. MS analysis of purified Compound
I
(C I). Figure S10. HPLC analysis
of OPME 2-catalyzed synthesis of compound
II
(C II). Figure S11. MS analysis of purified Compound
II
(C II).
Figure S12. HPLC analysis of OPME 3-catalyzed synthesis of compound
III
(C III). Figure S13. MS analysis of
purified Compound
III
(C III). Figure S14. MS analysis of traditional solid-phase sequential OPME synthesis of
Compound I (C I). Figure S15. MS analysis of traditional solid-phase sequential OPME synthesis of Compound
II
(C II). Figure S16.
1
H NMR analysis of purified Compound
I
(C I). Figure S17.
13
C NMR analysis of purified
Compound
I
(C I). Figure S18.
1
H NMR analysis of purified Compound
II
(C II). Figure S19.
13
C NMR analysis of
purified Compound
II
(C II). Figure S20.
1
H NMR analysis of purified Compound
III
(C III). Figure S21.
13
C NMR
analysis of purified Compound III (C III).
Author Contributions:
Conceptualization, J.-J.L., Y.D., M.P., X.D.; Methodology, validation, investigation,
formal analysis, data curation, M.G., L.W., T.L., Z.Z. and L.R.; Writing-original draft preparation, M.G. and J.-J.L.;
Writing-review and editing, M.G. and J.-J.L.; Supervision, J.-J.L., Y.D., M.P., H.C. and X.D.; Project administration,
J.-J.L.; Funding acquisition, Y.D. All authors have read and agreed to the published version of the manuscript.
Funding: This research was funded by National Natural Science Foundation of China (grant number 21877114).
Conflicts of Interest: The authors declare no conflict of interest.
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... Increasing the length of the carbon chain also reduces the steric hindrance of the biotin rings and facilitates access of the reactive linker, enabling further reactions used in the analysis or in the synthesis. The currently commercially available Ahx derivative in combination with biotin is Fmoc-Lys(biotinyl-ε-aminocaproyl)-OH, used in solid phase peptide synthesis [72][73][74][75][76][77]. ...
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