Sequential one-pot enzymatic synthesis of oligo-N-acetyllactosamine and its multi-sialylated extensions

Article abstract of DOI:10.1039/c4cc01227e

A simple and efficient protocol for the preparative-scale synthesis of various lengths of oligo-N-acetyllactosamine (oligo-LacNAc) and its multi-sialylated extensions is described. The strategy utilizes one thermophilic bacterial thymidylyltransferase (RmlA) coupled with corresponding sugar-1-phosphate kinases to generate two uridine diphosphate sugars, UDP-galactose and UDP-N-acetylglucosamine. By incorporating glycosyltransferases, oligo-LacNAcs and their sialylated analogs were synthesized. the Partner Organisations 2014.

Full text of DOI:10.1039/c4cc01227e

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Sequential one-pot enzymatic synthesis
of oligo-N-acetyllactosamine and its
multi-sialylated extensions†
Cite this: Chem. Commun., 2014,
50, 5786
Received 17th February 2014,
Accepted 11th April 2014
Wei-Ting Chien,a Chien-Fu Liang,a Ching-Ching Yu,a Chien-Hung Lin,a Si-Peng Li,a
Indah Primadona,ab Yu-Ju Chen,bc Kwok Kong T. Mongd and Chun-Cheng Lin*ac
DOI: 10.1039/c4cc01227e
www.rsc.org/chemcomm
A simple and efficient protocol for the preparative-scale synthesis
The majority of oligo-LacNAc containing carbohydrates cannot
of various lengths of oligo-N-acetyllactosamine (oligo-LacNAc) and be isolated from natural sources. To gain better insight into
its multi-sialylated extensions is described. The strategy utilizes one carbohydrate-mediated biological mechanisms and to tackle such
thermophilic bacterial thymidylyltransferase (RmlA) coupled with inaccessible complex structures, many advanced chemical synthetic
corresponding sugar-1-phosphate kinases to generate two uridine strategies7 and enzymatic strategies8 have been developed to synthe-
diphosphate sugars, UDP-galactose and UDP-N-acetylglucosamine. size homogenous linear or biantennary oligo-LacNAc extensions and
By incorporating glycosyltransferases, oligo-LacNAcs and their intact glycoproteins bearing O- and N-glycans.9 Notably, a chemo-
sialylated analogs were synthesized.
enzymatic strategy was developed to synthesize asymmetrical multi-
antennary N-glycans.10 Because enzymatic routes are more efficient
for preparation of natural and non-natural glycans and glycoconju-
gates,11 many efforts are focussed on the availability of a variety of
sugar-nucleotides for GT-catalyzed oligosaccharide elongation.12
We herein report a simple and efficient method to produce
uridine diphosphate galactose (UDP-Gal) and uridine diphosphate
N-acetylglucosamine (UDP-GlcNAc) for the preparative-scale synthesis
of various lengths of oligo-LacNAc and its multi-sialylated extensions.
During the preparation of this manuscript, Paulson and coworkers
demonstrated the feasibility of a-2,6-sialylation at the internal Gal of
either linear or branched oligo-LacNAc containing complex sacchar-
ides by using a-2,6-sialyltransferase from Photobacterium damsela
(Pd2,6ST).13 With the oligo-LacNAc in hand, we further explored
the synthesis of multi-sialylated oligo-LacNAc extensions with a-2,3-
or a-2,6-linkage by using two bacterial STs, PmST14 from Pasteurella
multocida and Pd2,6ST, respectively. We found that different
degrees of a-2,6-sialylation of oligo-LacNAc by Pd2,6ST could be
achieved at varying concentrations of the sialic acid donor, cytidine
monophosphate-sialic acid (CMP-sialic acid).
N-Acetyllactosamine oligomers (oligo-LacNAc) are type-2 oligosac-
charides composed of repeating units with b(1 - 3) linked LacNAc
(Galb1 - 4GalNAc) residues.1 These oligomers are found in glyco-
proteins2 and glycolipids;3 serve as acceptor substrates for a series of
glycosyltransferases (GTs) in the production of various glycoconju-
gates, such as sialylated, fucosylated, and ABO blood antigens
decorated with oligo-LacNAc chains; and constitute glycan ligands
involved in specific biological activities.4 In addition, LacNAc exten-
sions are present in the branched core structures of glycans, some-
times with the linear assembly of several LacNAc units to form
so-called poly-LacNAc. It has been well-documented that LacNAc is
an essential structural unit recognized by common glycan-binding
proteins, such as galectins. Changes in binding specificity associated
with the multivalent LacNAc present in branched N-glycan or
repeated LacNAc chains have also been reported.5 Moreover,
60-sialylated compounds with different numbers of LacNAc repeat
units show significant differences in binding specificity and affinity to
the influenza-hemagglutinin receptor.6
To efficiently and quickly produce oligo-LacNAc, we used two
recombinant bacterial GTs, b-1,3-N-acetyl-glucosaminyltransferase
from Helicobacter pylori (HpGnT)15 and b-1,4-galactosyltransferase
from Neisseria meningitidis (NmGalT), overexpressed in E. coli.16
Defined lengths of oligo-LacNAc were synthesized using these
enzymes in the presence of the sugar donors UDP-Gal and UDP-
GlcNAc. To reduce cost and enable the simple production of the
UDP-sugars used in oligo-LacNAc synthesis, an enzymatic one-pot
synthesis of two UDP-sugars (UDP-Gal and UDP-GlcNAc) using one
thermophilic enzyme, RmlA (glucose-1-phosphate thymidylyltransfer-
ase, EC 2.7.7.24, from Aneurinibacillus thermoaerophilus DSM10155),17
a Department of Chemistry, National Tsing Hua University, 101, Sec. 2,
Kuang Fu Rd., Hsinchu, 30013, Taiwan. E-mail: cclin66@mx.nthu.edu.tw;
Fax: +886 3 5711082; Tel: +886 3 5753147
b Institute of Chemistry, Academia Sinica, 128, Sec. 2, Academia Rd., Nankang,
Taipei 11529, Taiwan
c Genomic Research Center, Academia Sinica, 128, Sec. 2, Academia Rd., Nankang,
Taipei 11529, Taiwan
d Applied Chemistry Department, National Chiao Tung University, 1001,
University Road, Hsinchu 30010, Taiwan
† Electronic supplementary information (ESI) available: Cloning and overexpres-
sion of enzymes and NMR spectral characterization of the synthesized oligo-
LacNAcs. See DOI: 10.1039/c4cc01227e
5786 | Chem. Commun., 2014, 50, 5786--5789
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was exploited. RmlA, which was found to synthesize thymidine sufficient to supply the glycoside transfer reaction in the synthesis of
diphosphate glucose (dTDP-Glc) in nature, has been reported to LacNAc, the reaction temperature was lowered to 25 1C. Thus, the
possess broad substrate tolerance toward sugar-1-phosphates remaining starting materials, ATP, UTP, and Gal, continued to be
(sugar-1-P) in the preparation of NDP-sugars.12a Additionally, we utilized to produce UDP-Gal while the hydrolysis of UDP-Gal was
explored its potential to produce UDP-Gal on a preparative scale, slowed. NmGalT is a thermo-unstable enzyme but retains good
which was further used in the multi-hundred milligram scale synth- catalytic activity below physiological temperatures, such as at 25 1C.
esis of LacNAc.17 Importantly, compared to other enzymes which can The addition of 0.2 mM (final concentration) dithiothreitol (DTT) and
produce UDP-sugars directly from the condensation reaction of 1 mg mLꢀ1 bovine serum albumin (BSA) can stabilize NmGalT and
uridine triphosphate (UTP) and sugar-1-P,12b–d the capability of maintain its activity.17 Furthermore, to prevent the hydrolysis of UDP-
producing both UDP-Gal and UDP-GlcNAc makes RmlA an ideal Gal and the decomposition of NmGalT at 55 1C, the above reaction
enzyme for the enzymatic oligo-LacNAc synthesis. The corresponding mixture was cooled to 25 1C, and NmGalT was then added. After
sugar-1-P required for RmlA, galactose-1-phosphate (Gal-1-P) and incubation for 4 h, the desired LacNAc (2) was obtained in 90% yield
N-acetylglucosamine-1-phosphate (GlcNAc-1-P) was produced by (46 mg). Thus, in this sequential addition one-pot synthesis, only the
using recombinant galactokinase (MtGalK, from Meiothermus purification of the final desired saccharide product is needed, which
taiwanensis sp. nov. WR-220)18 and N-acetylhexosamine kinase is more efficient and simpler in comparison with the purification of
(NahK, from Bifidobacterium longum),19 respectively. In addition to Gal-1-P and UDP-Gal.
RmlA, MtGalK is also a thermophilic enzyme and was coupled with
The procedure for the assembly of GlcNAc-LacNAc trisaccharide 3
RmlA in the one-pot synthesis of UDP-Gal in our group.18 Combining is similar to that described for the synthesis of 2. The transfer of a
RmlA with the corresponding kinases and GTs, the GlcNAc analog (1) GlcNAc moiety to LacNAc was catalyzed by HpGnT and required UDP-
was smoothly converted to defined lengths of oligo-LacNAc (2–8) by a GlcNAc as a donor substrate (Scheme 1). We found that RmlA could
sequential one-pot enzymatic synthesis starting from cheap materials also be used to prepare UDP-GlcNAc from the condensation reaction
(GlcNAc and Gal), as shown in Scheme 1.
of UTP and GlcNAc-1-P, and the latter was known to be obtained by
The studies were commenced from the preparation of LacNAc by enzymatic reaction using NahK.19 The optimal reaction condition for
using a 6-azidohexyl-GlcNAc acceptor (1, azido-GlcNAc);20 the azido NahK is 37 1C, and prolonged reaction times at elevated temperatures,
group could be employed for further modification. The following such as at 55 1C, degrade the enzyme activity. Thus, the one-pot UDP-
b-1,4-galactosidic linkage was constructed by NmGalT in the presence GlcNAc synthesis procedure was slightly modified, and the sequential
of UDP-Gal. Both MtGalK and RmlA are thermally stable and show addition of NahK and RmlA was employed (Scheme 1). GlcNAc
good activity at 55 1C under similar reaction conditions; thus, the one- (20 mM) and ATP (20 mM) were first dissolved in 100 mM Tris-HCl
pot synthesis of UDP-Gal is feasible using these two enzymes. As (pH 7.5) in the presence of 40 mM MgCl2 and NahK (1 mg mLꢀ1
)
shown in Scheme 1, in the one-pot synthesis of UDP-Gal, Gal at 37 1C. The progress of GlcNAc-1-P formation was monitored by
(20 mM), adenosine triphosphate (ATP, 20 mM), and uridine triphos- RP-HPLC, as indicated by the formation of ADP and the consumption
phate (UTP, 20 mM) were first dissolved in 100 mM Tris-HCl (pH 7.5) of ATP. The yield of GlcNAc-1-P reached 90% within 4 h. Then, the
in the presence of 40 mM MgCl2 and of MtGalK (10 mg mLꢀ1) and temperature of the above reaction mixture was increased to 55 1C,
RmlA (1 mg mLꢀ1) at 55 1C for 2 h. The formation of UDP-Gal was followed by the addition of UTP (20 mM) and RmlA (1 mg mLꢀ1).
monitored by RP-HPLC. However, due to the use of high reaction When the yield of UDP-GlcNAc reached 50% in 90 min as monitored
temperatures, longer incubation times resulted in the production of by the formation of UDP-GlcNAc using RP-HPLC, the above reaction
hydrolysis products of UDP-Gal, UMP, and 1,2-cyclic phosphate Gal. mixture was cooled to room temperature (25 1C), followed by the
To circumvent this problem and avoid the purification of UDP-Gal addition of HpGnT (40 mg mLꢀ1). After incubation for 4 h, the desired
from the mixture with ADP and UDP, two equivalents (to the sugar trisaccharide 3 was obtained in 99% yield (70 mg).
acceptor) of Gal, ATP, and UTP were used. When the yield of UDP-Gal
After the successful and stepwise assembly of Gal and GlcNAc
reached 50% within 90 min, at which point the UDP-Gal amount was on azido-GlcNAc 1, we extended our sequential addition one-pot
Scheme 1 Sequential synthesis of oligo-LacNAcs and sialylation of oligo-LacNAcs.
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Table 1 Sialylation of oligo-LacNAc by sialyltransferases
procedure to synthesize oligo-LacNAc, as shown in Scheme 1. Defined
lengths of oligo-LacNAc were synthesized by alternative addition of
pre-synthesized UDP-Gal and NmGalT and pre-synthesized UDP-
GlcNAc and HpGnT. The progress of each enzymatic elongation step
was monitored by TLC, as indicated by the complete conversion of
the corresponding acceptor substrates. As shown in Scheme 1, the
glycosylation yields of Gal in the non-reducing end GlcNAc (3 and 5)
acceptors by NmGalT were almost quantitative, yielding (LacNAc)2 4
and (LacNAc)3 6 in quantities of 84 mg (96%) and 62 mg (99%),
respectively. Likewise, the assembly of GlcNAc by HpGnT to the non-
reducing end Gal acceptor (4 and 6) also gave very good yields of the
desired products. The yields of GlcNAc-(LacNAc)2 5 and GlcNAc-
(LacNAc)3 7 were 89% (49 mg) and 93% (34 mg), respectively. Notably,
the synthetic yield of (LacNAc)4, 8 was significantly lower (43%,
15 mg). This low yield may be due to the poor substrate acceptance
of NmGalT to heptasaccharide acceptor 7 and to the low water
Entry
Acceptor
Donor (eq.)
Product
Yield (%)
PmST
1a
2a
3a
2
4
6
1.5
1.5
1.5
9
10
11
82
80
73
Pd2,6ST
4
5
6
2
3
4
1.5
1.5
1.5
12
13
14
15
15
16
17
17
18
99
57
72
26
99
32
56
12
85
7
8
4
6
2.5
2
9
6
2
a
Reaction for 4 h.
solubility of the octasaccharide 8 as the chain length increased. An 10 (80%) and 11 (73%), respectively (Table 1). Notably, all the
attempt to use buffer-DMSO co-solvent for the reaction was unsuc- acceptors provide a similar reaction rate in the PmST catalyzed
cessful due to the poor solubility of 8 in DMSO. Interestingly, the sialylation.
water solubility of GlcNAc terminated oligo-LacNAc was observed to
be better than that of the Gal terminated oligo-saccharides.
Pd2,6ST also displayed good catalytic efficiency toward oligo-
LacNAc. It was recently reported that Pd2,6ST cannot only transfer
We also investigated the potential one-pot synthesis of oligo- sialic acid to the terminal Gal but can also perform a-2,6-sialylation of
LacNAc by using MtGalK, NahK, and RmlA to prepare sugar donors internal Gals.11 In our approach, we controlled the amount of CMP-
at 55 1C followed by cooling the reaction temperature to 25 1C and Neu5Ac to produce different degrees of sialylated oligo-LacNAc, as
adding GlcNAc acceptor 1, NmGalT, and HpGnT simultaneously. shown in Table 1. Pd2,6ST was first applied to convert 2 and 3 to
However, the synthetic efficiency of UDP-GlcNAc was slightly higher sialylated product 12 (99%) and 13 (57%) in the presence of 1.5
than that of UDP-Gal. To precisely control the formation of each UDP- equivalents of CMP-Neu5Ac. When the same equivalent of CMP-
sugar, UDP-Gal and UDP-GlcNAc were prepared separately in two Neu5Ac was used in the Pd2,6ST catalyzed sialylation of (LacNAc)2 4,
vessels. When the formation of 90% of UDP-Gal and UDP-GlcNAc two products, mono-sialyl (LacNAc)2 14 and di-sialyl (LacNAc)2 15,
was achieved, as monitored by RP-HPLC, they were poured together were obtained with yields of 72% and 26%, respectively. Increasing
into one vessel that contained GlcNAc 1. After cooling the tempera- the CMP-Neu5Ac amount to 2.5 eq. gave a 99% yield of di-sialyl
ture of the vessel to 25 1C, NmGalT and HpGnT were added, and the (LacNAc)2 15. Although increasing the oligo-LacNAc length decreases
resulting mixture was stirred for 24 h. After the removal of the rest of its water solubility, the addition of sialic acid to oligo-LacNAc
the UDP-sugars and of the buffer salt by BioRad P-2 gel, the resulting significantly enhanced its water solubility. When CMP-Neu5Ac and
oligo-LacNAc products were analyzed by MALDI-MS. The results (LacNAc)3 6 (2 to 1 equivalents) were used, a 56% yield of mono-sialyl
revealed that compounds 2, 3, 4 and 5 were obtained, and no major (LacNAc)3 16 and a 32% yield of di-sialyl (LacNAc)3 17 were obtained.
product dominated (see ESI,† Fig. S1). Furthermore, the amounts Notably, no tri-sialylated product was obtained. The position of sialic
(based on the intensities of peaks in the MS spectrum) of the GlcNAc acid in sialyl-(LacNAc)3 16 was determined on the terminal Gal by MS/
terminated oligo-saccharides (3 and 5) were higher than those of the MS fragmentation analysis, while 17 had two sialic acids, including
Gal terminated oligo-saccharides (2 and 4), suggesting that HpGnT one attached to the terminal Gal and the other attached to an internal
may be more efficient for the conversion of corresponding acceptor Gal (see ESI,† Fig. S2). However, when 5.0 eq. of CMP-Neu5Ac was
substrates than NmGalT in this reaction system.
used, an 85% yield of tri-sialyl 18 and a 12% yield of di-sialyl 17 were
The sialic acids in the LacNAc chains of glycoproteins are typically obtained, while no mono-sialylated product was obtained. The results
linked a-2,3 or a-2,6 to the terminal (non-reducing end) Gal. These revealed that the internal Gal can be successfully a-2,6-sialylated and
sialylated oligo-LacNAcs were well-documented to interact with that the number of sialic acids attached to the oligo-LacNAc chains
glycan-binding proteins such as selectins, galectins, and siglecs (sialic can be controlled by using varying concentrations of CMP-Neu5Ac.
acid-binding immunoglobulin-type lectins) as well as with viral
In this study, we developed an economical and straightforward
hemagglutinins.21 To prepare a small library of sialylated glycans, strategy to prepare UDP-Gal and UDP-GlcNAc at a preparative scale by
two bacterial STs, a-2,3-ST and a-2,6-ST, were used to assemble a coupling the corresponding kinases with RmlA. In combination with
sialic acid moiety onto oligo-LacNAc at the C-3 position of the Leloir GTs, NmGalT and HpGnT, oligo-LacNAcs were synthesized. By
terminal Gal and at the C-6 positions of the Gals, respectively. Thus, using our newly developed enzymatic system, defined lengths of
recombinant PmST (an a-2,3-ST) and Pd2,6ST (an a-2,6-ST) were oligo-LacNAcs were synthesized in a sequential addition, one-pot
overexpressed in E. coli using an intein expression system,12 and the fashion. A panel of a-2,3- and a-2,6-sialylated oligo-LacNAcs were
donor, CMP-sialic acid (CMP-Neu5Ac), was prepared by CMP sialic further synthesized by PmST and Pd2,6ST. In addition, different
acid synthetase.22 PmST was used to perform a-2,3-sialylation of the degrees of a-2,6-sialylation of oligo-LacNAcs were achieved by con-
terminal Gal of 2, 4, and 6, yielding sialylated products 9 (82%), trolling the amount of CMP-Neu5Ac in the enzymatic reaction.
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LacNAcs for studying LacNAc associated biology.
This work was financially supported by National Tsing Hua
University, Academia Sinica, and Ministry of Science and
Technology, Taiwan.
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