Gram-scale production of sugar nucleotides and their derivatives

Article abstract of DOI:10.1039/d1gc00711d

Here, we report a practical sugar nucleotide production strategy that combined a high-concentrated multi-enzyme catalyzed reaction and a robust chromatography-free selective precipitation purification process. Twelve sugar nucleotides were synthesized on a gram scale with a purity up to 98%.

Full text of DOI:10.1039/d1gc00711d

Green Chemistry
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Gram-scale production of sugar nucleotides and
their derivatives†
Cite this: Green Chem., 2021, 23,
2628
Shuang Li,‡a Shuaishuai Wang, ‡b Yaqian Wang,a Jingyao Qu,c Xian-wei Liu,a
Received 24th February 2021,
Accepted 12th March 2021
Peng George Wangd and Junqiang Fang
*
a
DOI: 10.1039/d1gc00711d
rsc.li/greenchem
Here, we report a practical sugar nucleotide production strategy phorylation and subsequent pyrophosphorylation by one or
that combined a high-concentrated multi-enzyme catalyzed reac- two enzymes.9 The salvage pathway (Scheme 1) has become
tion and a robust chromatography-free selective precipitation the predominant approach for enzymatic synthesis of sugar
purification process. Twelve sugar nucleotides were synthesized nucleotides and their derivatives as fewer steps and enzymes
on a gram scale with a purity up to 98%.
are needed.10–14
Various strategies have been developed for the preparation
of natural and unnatural sugar nucleotides, including chemi-
cal synthesis,6,15 in situ regeneration,16,17 one-pot multi-
enzyme system,18,19 metabolic engineering,20,21 and super-
Sugar nucleotides are essential intermediates for glycan
metabolism and glycoconjugate biosynthesis.1 They are
formed as either nucleoside diphosphate (NDP) or monophos-
phate (NMP) nucleoside derivatives. In mammalian cells, only
9 common sugar nucleotides are used as essential donors for
Leloir-type glycosyltransferases involved in most glycosylation
reactions.2 The affordable sugar nucleotides and their deriva-
tives are one of the prerequisites for cell metabolic labeling
and automated glycan synthesis.3–5
Chemical synthesis of sugar nucleotides has been well-
documented. But low yield, tedious process and hazardous
substances have restricted the application of this method.6,7 In
contrast, an enzyme-catalyzed synthetic reaction, which
mimics the biosynthetic pathway, is considered more attractive
with the advantages of strong regiospecificity and stereospeci-
ficity, high conversion rate, and mild reaction conditions.8
Biosynthesis pathways of sugar nucleotides fall into two
classes: de novo pathways and salvage pathways. In de novo
pathways, sugar nucleotides are generated via multi-enzyme
catalyzed reactions from monosaccharides or other sugar
nucleotides, while in salvage pathways, sugar nucleotides are
produced within 2 steps from monosaccharides via phos-
aNational Glycoengineering Research Center, Shandong Provincial Key Laboratory of
Glycochemistry and Glycobiology, Shandong University, Qingdao, Shandong 266237,
People’s Republic of China. E-mail: fangjunqiang@sdu.edu.cn
bDepartment of Chemistry, George State University, Atlanta, GA, 30302-4098, USA
cState Key Laboratory of Microbial Technology, Shandong University, Qingdao,
Shandong 266237, People’s Republic of China
dSchool of Medicine, Southern University of Science and Technology, Shenzhen,
Guangdong 518055, People’s Republic of China
†Electronic supplementary information (ESI) available. See DOI: 10.1039/
d1gc00711d
Scheme 1 Common salvage synthetic routes to generate sugar
‡These authors contributed equally to this work.
nucleotides.
2628 | Green Chem., 2021, 23, 2628–2633
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bead technologies.22 However, a practical strategy is still duction, we first explored the optimal substrate concentration
unavailable due to two major bottlenecks: low substrate con- of the multi-enzymatic cascade reaction with high catalytic
centration of enzymatic reactions and time-consuming purifi- efficiencies. The salvage biosynthesis of UDP-GlcNAc was used
cation process. Most reported in vitro sugar nucleotide syn- for optimization and various substrate concentrations (5 mM,
thetic approaches are typically performed at substrate concen- 10 mM, 50 mM, 100 mM, 200 mM and 300 mM) were assayed
trations ranging from μM to 5–10 mM,10–14,16–19 which lead to (Tables S1 and S2†). As depicted in Fig. S1,† the conversion
a huge reaction volume and extra chemical consumption once rates were all above 90% when the concentration of GlcNAc
scaled up. Additionally, due to similarities of molecular was less than 200 mM (44.2 g L−1). When the concentration
weights and polar characteristics between products, substrates, reached 300 mM, the conversion rate decreased dramatically
and byproducts, the current purification processes mainly rely to 30%, leaving large amounts of unreacted GlcNAc. The low
on chromatographic separation technologies, such as size conversion rate may be due to the low fluidity of the reaction
exclusion chromatography, ion-exchange chromatography, or system, as reaction mixture containing 400 mM GlcNAc exhibi-
HPLC.23,24 However, these methods could only be performed ted high viscosity, most likely hydrogel, leading to a low con-
on a research scale in a time-consuming manner. Studies on version rate. Thus, 200 mM was chosen as the optimal mono-
glycans and glycoconjugates have been hindered by the lack of saccharide concentration. With the application of this high-
efficient strategies to produce substantial amounts of sugar concentrated multi-enzyme cascade reaction system, the syn-
nucleotides and their derivatives.
thetic reaction could be conducted in a mini tube with an
To accelerate applications of environmentally friendly enzy- improved space–time yield around 40 times when compared
matic synthetic reactions and reduce waste accumulation, with the common 5 mM reaction system.
herein, we reported a practical and economical strategy for
A buffer system, which functions to resist changes in the
multi-gram scale production of sugar nucleotides (Scheme 2). hydrogen ion concentration, has been extensively used in enzy-
With the employment of a high-concentrated multi-enzyme matic reactions to mimic the biological environment.
cascade synthetic system and a robust chromatography-free However, huge amounts of chemicals in buffer systems
purification strategy, 12 sugar nucleotides and their derivatives increase difficulties for product analysis and purification. To
were generated from cheap and readily available monosacchar- simplify the reaction system and facilitate the final purification
ides in a condensed reaction system with a starting monosac- process, we further evaluated the necessity of a buffer system
charide up to 200 mM. More importantly, up to 2.35 g pure for a high-concentrated enzymatic reaction system. Two enzy-
product could be obtained from a 20 mL reaction system by a matic reactions with the same reactants for the synthesis of
selective precipitation process with a purity up to 98%. UDP-GlcNAc except the buffer system were carried out. As
Subsequently, the synthesized sugar nucleotides were verified shown in Fig. S2,† the synthetic reaction without the buffer
as glycosyl donors to generate the tumor-associated carbo- system could reach a comparable conversation rate to that with
hydrate antigen, Globo H and its trifluoroacetyl modified the buffer system, indicating that the effects of the buffer
derivative, in overall 85% yield via in vitro glycosyltransferase- system could be omitted by pre-adjusting the pH value with
catalyzed stepwise reactions.
the amounts of base solution directly, thus facilitating the sub-
As mentioned above, the low substrate concentration of the sequent purification process.
enzymatic reaction system was unsuitable for practical appli-
To reduce the cost-effectiveness, we also carried out the
cations. To improve the capabilities and efficiencies of the recovery of enzymes used for the synthesis of sugar nucleo-
multi-enzymatic cascade reaction for sugar nucleotide pro- tides. The enzymes were recovered by ultrafiltration and
reused for sugar nucleotide generation. As shown in Fig. S3,†
the recovered enzymes could catalyze the completion of the
reaction, which is consistent with
a
previous report.25
However, a longer reaction time was needed when compared
with the fresh enzymes, which may be due to the high-concen-
trated reaction system. Nevertheless, our results indicated that
the enzymes used in this work can be recovered and could still
catalyze the production of sugar nucleotides in high yields.
Having established the optimal reaction conditions for the
synthesis of sugar nucleotides, we turned our efforts to explore
an efficient strategy for the purification of sugar nucleotides.
Challenges are multiplied when purifying the desired sugar
nucleotides from a complex reaction mixture. Routine labor-
intensive and time-consuming purification processes cannot
guarantee large-scale purification under low-temperature con-
ditions, thus may cause product degradation. Barium chloride
precipitation26 and silver nitrate precipitation27,28 strategies
have been reported for the purification of phosphate-contain-
Scheme 2
purification on a multi-gram scale.
A practical strategy for sugar nucleotide synthesis and
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ing saccharides; however, the functions of metal ions in sugar characterized by X-ray Photoelectron Spectroscopy (XPS). C 1s
nucleotide purification were not fully elucidated. Herein, seven (284.8 eV) was used to calibrate the binding energies.
metal ions (Ag+, Ba2+, Cu2+, Zn2+, Ca2+, Mn2+, and Ni2+) that Characteristic peaks of Ba, P, O and C elements were clearly
can undergo precipitation with phosphate (PO43−) were tested observed in the full scan XPS spectra (Fig. 2A), which indicated
with a mixed solution containing ATP, UTP, and UDP-GlcNAc. that the precipitate contains the Ba element and corres-
As summarized in Fig. 1A, Ag+ and Ba2+ exhibited predomi- ponding nucleoside phosphates. The high resolution XPS spec-
nant efficiency than other metal ions. Subsequently, the selec- trum of Ba 3d demonstrates that the binding energy of Ba in
tive precipitation abilities of Ag+ and Ba2+ toward nucleoside the precipitate complex has shifted from 0.5 eV to a lower
triphosphates (ATP, UTP, GTP, and CTP) were further value compared to that in BaCl2 (Fig. 2B), which indicated that
measured. Both metal ions can precipitate nucleoside tripho- electrons are transferred from Ba to the phosphate groups.
sphates with over 98% efficiency (Fig. 1B).
These results imply that Ba2+ forms a coordinate covalent
Additionally, recovery rates of two metal ions toward bond with a terminal phosphate in ATP and UTP (Fig. 2C). It is
different sugar nucleotides were measured via the precipi- worth mentioning that two hydroxyl groups of the terminal
tation of pure sugar nucleotides with Ag+ and Ba2+, respect- phosphate and monosaccharide may play a critical role in the
ively. UDP-GlcNAc, GDP-Fuc, and sugar nucleotides with car- Ba2+ precipitation process as diphosphates on sugar nucleo-
boxylic acid (UDP-GlcA, UDP-GalA and CMP-Neu5Ac) were tides would not undergo precipitation with Ba2+, while nucleo-
assayed. As shown in Fig. 1C, Ag+ would precipitate sugar side triphosphates and nucleoside diphosphates could interact
nucleotides with carboxylic acid, which would lead to a low with Ba2+ to form precipitates. Furthermore, solid-state
recovery rate (20% to 40%), while Ba2+ gave over 95% recovery nuclear magnetic resonance (31P ssNMR) was applied to verify
rate for all tested sugar nucleotides. It is worth mentioning the XPS results. As shown in Fig. 3A, 31P in the precipitate
that although Ag+ has been used for the purification of complex (ATP + Ba2+ or UTP + Ba2+) has shifted from 1.29 or
UDP-GlcNAc and its derivatives,29 excess amount Ag+ needs to 0.29 ppm to a high field compared to that in ATP or UTP
be removed immediately by NaCl precipitation and size exclu- (Fig. 3B), indicating that Ba2+ forms bonds with the phosphate
sion chromatography due to its strong oxidizing properties, group existing in the nucleoside triphosphates.
thus leading to more labor and low efficiency. Moreover, Ag+
Finally, the practicability of the gram-scale sugar nucleotide
cannot be used to precipitate carboxyl-containing structures, synthetic strategy was systematically evaluated by the synthesis
such as CMP-Neu5Ac and UDP-GlcA. Thus, Ba2+ was chosen as of 12 different sugar nucleotides and their derivatives in a
the optimal metal ion for the selective precipitation purifi- 20 mL high-concentrated multi-enzyme cascade reaction, fol-
cation of sugar nucleotides.
lowed by the selective precipitation purification process. As
To further investigate the mechanisms underlying Ba2+ pre- shown in Table 1, the sugar nucleotide synthetic strategy devel-
cipitation, the composition of the formed precipitate was oped in this study exhibited excellent yields up to 70–97%
toward most sugar nucleotides except UDP-GalA. More impor-
tantly, 1.6–2.35 g final products with a purity up to 98% were
quickly generated within 1 h via selective precipitation purifi-
cation, demonstrating a powerful and robust separation proto-
col for glycobiological studies. It is worth mentioning that the
Fig. 1 Evaluation of metal ions for the precipitation of sugar nucleo-
tides. (A) Metal ions precipitate ATP, UTP and UDP-GlcNAc mixture solu-
tion; (B) Ag+ and Ba2+ precipitate nucleoside triphosphates; and (C) Ag+
and Ba2+ precipitate pure sugar nucleotides.
Fig. 2 XPS measurements of Ba2+ precipitation. (A) Full scan XPS
spectra of ATP, ATP + Ba2+, UTP + Ba2+, and BaCl2; (B) Ba 3d spectra of
ATP + Ba2+, UTP + Ba2+, and BaCl2; and (C) the supposed mechanisms
of Ba2+ precipitation.
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tively coupled plasma mass spectrometry (ICP-MS). Sugar
nucleotides were dissolved in a final concentration at 30 μg
mL−1 using deionized water. The AAS results (Table S3†)
showed that the concentration of Na+ is 2.22 μg mL−1, indicat-
ing that UDP-GlcNAc binds to Na+ to form a disodium salt,
and it failed to detect the concentrations of Ba+ and Mg2+, as
they were lower than the detection limit. ICP-MS (Table S4†)
showed that the concentrations of Ba2+ and Mg2+ are 44.77 ng
mL−1 and 13.39 ng mL−1, respectively, which is equal to 0.14%
and 0.04% (w : w), respectively. The trace amount of residual
metal ions would not affect the subsequent usage as lower
than 0.2% metal ion existed in sugar nucleotides.
Additionally, we explored the synthesis of the tumor-associ-
ated carbohydrate antigen Globo H (Fucα1-2Galβ1-3GalNAcβ1-
3Galα1-4Galβ1-4Glc), one of the most attractive biomarkers of
cancer cells and cancer stem cells (Scheme 3).30 Lactose
β-PEG-N3 was first extended with α1-4Gal using an α1-4-galac-
tosyltransferase (LgtC)31 in the presence of UDP-Gal. Then, the
trisaccharide 2 was elongated by β1-3GalNAc with the employ-
ment of UDP-GalNAc and β1-3-N-acetylgalactosyl-transferase
(LgtD) to afford compound 3.32 Interestingly, UDP-GalNTFA
Fig. 3 31P ssNMR measurements of Ba2+ precipitation. (A) 31P ssNMR
spectra of ATP and ATP + Ba2+ and (B) 31P ssNMR spectra of UTP and
UTP + Ba2+
.
Table 1 Gram-scale synthesis of sugar nucleotides and their derivatives
via a high-concentrated multi-enzyme cascade synthetic system
(UDP-N-trifluoroacetyl galactosamine),
a
C2 modified
Entry
Compound
Yielda (%)
Amountb (g)
UDP-GalNAc analog, can also be recognized by LgtD to provide
the tetrasaccharide derivative 4. With the further extension of
β1-3-Gal by using LgtD and UDP-Gal to provide 5 and 6, Globo
1
2
3
UDP-GlcNAc
UDP-GalNAc
UDP-Gal
84
80
71
68
97
76
69
52
78
78
81
79
2.04
2.00
1.60
1.60
2.35
1.87
1.62
1.23
2.06
2.05
2.10
2.05
H 7 (Fucα1-2Galβ1-3GalNAcβ1-3Galα1-4Galβ1-4Glcβ-PEG-N3,
4
5
GDP-Fuc
GDP-Man
140 mg, 85% overall yield) and its derivative 8 (Fucα1-2Galβ1-
3GalNTFAβ1-3Galα1-4Galβ1-4Glcβ-PEG-N3, 110 mg, 76%
overall yield) were assembled by using GDP-Fuc and an α1-2-
fucosyltransferase.33 All synthesized compounds were con-
6
7
CMP-Neu5Ac
UDP-GlcA
8
9
10
11
12
UDP-GalA
UDP-GlcNTFA
UDP-GalNTFA
UDP-GlcNAz
UDP-GalNAz
a Yields calculated from the extracted product and theoretical amount.
b Lyophilized pure product isolated from a 20 mL high-concentrated
multi-enzyme cascade reaction system.
yield of UDP-GalA decreased to 52%, partly due to poor cata-
lytic activities of BiGalK (galactokinase from Bifidobacterium
infantis) and AtUSP (UDP-sugar pyrophosphorylase from
Arabidopsis thaliana) toward the unnatural substrate GalA. All
synthetic sugar nucleotides were well characterized by ESI-MS
and NMR analysis, respectively (ESI†). Compared with the
routine sugar nucleotide purification strategies, our method
reduces environmental pollution as the reaction volume
decreased significantly by using the high-concentrated enzy-
matic reaction. The whole processing time was also signifi-
cantly reduced from 3–4 days to 1 day by the chromatography-
free selective precipitation purification process.23 Most impor-
tantly, as the existing technologies are not suitable for gram-
scale production, our strategy has exceptional potential for
sugar nucleotide production.
To verify the qualities of the formed sugar nucleotides, the
amount of residual metal ions in the final products was
measured by atomic absorption spectrometry (AAS) and induc- Scheme 3 Stepwise synthesis of Globo H and its derivative.
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firmed by ESI-MS and NMR analysis, respectively (ESI†).
Compared to the assembly of Globo H by a sugar nucleotide
regeneration system,32 the synthesis of Globo H by using
readily available sugar nucleotides simplifies the purification
process and increases the overall yield, which provides a feas-
ible way for the synthesis of oligosaccharides and their
derivatives.
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Conclusions
Sugar nucleotides are essential intermediates involved in
Leloir-type glycosyltransferase-catalyzed glycosylation pro-
cesses. Current synthetic strategies are still facing several chal-
lenges, which in turn hinder the illustrations of biological
roles of glycans and glycoconjugates. We herein reported a
practical and economical approach toward the production of
sugar nucleotides and their derivatives to meet the urgent
needs in the large-scale synthesis of complex glycans and gly-
coconjugates. This universal approach could produce sugar
nucleotides from the corresponding monosaccharides on a
multi-gram scale with a combination of a high-concentrated
multi-enzyme cascade reaction and a fast chromatography-free
purification process. 12 kinds of sugar nucleotides and their
derivatives were firstly synthesized on a multi-gram scale from
a 20 mL reaction mixture and purified by a robust Ba2+ selec-
tive precipitation process with a purity up to 98%. This meth-
odology breaks through the limitations of existing strategies
and provides new insights into the large-scale production of
sugar nucleotides. The synthesized sugar nucleotides facilitate
the in vitro synthesis of functional glycans, such as Globo H
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