Sequential One-Pot Multienzyme Chemoenzymatic Synthesis of Glycosphingolipid Glycans

Article abstract of DOI:10.1021/acs.joc.6b01905

Glycosphingolipids are a diverse family of biologically important glycolipids. In addition to variations on the lipid component, more than 300 glycosphingolipid glycans have been characterized. These glycans are directly involved in various molecular recognition events. Several naturally occurring sialic acid forms have been found in sialic acid-containing glycosphingolipids, namely gangliosides. However, ganglioside glycans containing less common sialic acid forms are currently not available. Herein, highly effective one-pot multienzyme (OPME) systems are used in sequential for high-yield and cost-effective production of glycosphingolipid glycans, including those containing different sialic acid forms such as N-acetylneuraminic acid (Neu5Ac), N-glycolylneuraminic acid (Neu5Gc), 2-keto-3-deoxy-d-glycero-d-galacto-nononic acid (Kdn), and 8-O-methyl-N-acetylneuraminic acid (Neu5Ac8OMe). A library of 64 structurally distinct glycosphingolipid glycans belonging to ganglio-series, lacto-/neolacto-series, and globo-/isoglobo-series glycosphingolipid glycans is constructed. These glycans are essential standards and invaluable probes for bioassays and biomedical studies.

Full text of DOI:10.1021/acs.joc.6b01905

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Article
Sequential One-Pot Multienzyme (OPME)
Chemoenzymatic Synthesis of Glycosphingolipid Glycans
Hai Yu, Yanhong Li, Jie Zeng, Vireak Thon, Dung M. Nguyen, Thao Ly, Hui Yu Kuang, Alice Ngo, and Xi Chen
J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.6b01905 • Publication Date (Web): 13 Oct 2016
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The Journal of Organic Chemistry
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Sequential One-Pot Multienzyme (OPME) Chemoenzymatic
Synthesis of Glycosphingolipid Glycans
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Hai Yu,a,b* Yanhong Li,a,b Jie Zeng,b,c Vireak Thon,b,† Dung M. Nguyen,b,‡ Thao Ly,b Hui Yu Kuang,b,
Alice Ngo,b Xi Chenb*
aGlycohub, Inc., 4070 Truxel Road, Sacramento, CA 95834, USA
bDepartment of Chemistry, University of California, One Shields Avenue, Davis, CA 95616, USA
cSchool of Food Science, Henan Institute of Science and Technology, Xinxiang, Henan 453003, China
KEYWORDS carbohydrate, ganglioside, glycosphingolipid, one-pot multienzyme (OPME), oligosaccharide
ABSTRACT: Glycosphingolipids are a diverse family of biologically important glycolipids. In addition to variations on the lipid
component, more than 300 glycosphingolipid glycans have been characterized. These glycans are directly involved in various mo-
lecular recognition events. Several naturally occurring sialic acid forms have been found in sialic acid-containing glycosphingoli-
pids, namely gangliosides. However, ganglioside glycans containing less common sialic acid forms are currently not available.
Herein, highly effective one-pot multienzyme (OPME) systems are used in sequential for high-yield and cost-effective production
of glycosphingolipid glycans, including those containing different sialic acid forms such as N-acetylneuraminic acid (Neu5Ac), N-
glycolylneuraminic acid (Neu5Gc), 2-keto-3-deoxy-D-glycero-D-galacto-nononic acid (Kdn), and 8-O-methyl-N-acetylneuraminic
acid (Neu5Ac8OMe). A library of 64 structurally distinct glycosphingolipid glycans belonging to ganglio-series, lacto-/neolacto-
series, and globo-/isoglobo-series glycosphingolipid glycans is constructed. These glycans are essential standards and invaluable
probes for bioassays and biomedical studies.
1. INTRODUCTION
sin binds to GM3 containing Neu5Gc, but not N-
acetylneuraminic acid (Neu5Ac).18 Neu5Gc-containing GM1
is a better ligand than Neu5Ac-containing GM1 for simian
virus 40 (SV40).19 In addition to being key components in cell
recognition, structurally diverse glycosphingolipids with dif-
ferent glycan structures are involved in cell signaling.20 There-
fore, obtaining pure glycosphingolipid oligosaccharides will
facilitate structure- activity studies of the glycan components
of glycosphingolipids at the molecular level.
Glycosphingolipids are essential components of human plasma
membrane. They are believed to be clustered in "lipid rafts"
which are spatial mammalian cell membrane microdomains
important for various biological processes including protein
sorting, signal transduction, membrane trafficking, viral and
bacterial infection, and cell-cell communications.1 Aberrant
expression of glycosphingolipids has been found to be asso-
ciated with glycosphingolipid storage diseases and cancer
progression.2,3 For example, increased expression of GD3 and
GM2 in melanoma, elevated levels of sialyl Lewis a and sialyl
Lewis x in gastrointestinal cancers have been reported.4 In
addition, a non-human sialic acid form, N-glycolylneuraminic
acid (Neu5Gc), is overexpressed on several types of human
tumor cells.5-7 Some cancer-associated gangliosides have been
developed as potential cancer markers, cancer vaccine candi-
dates,8,9 and immunosuppressants.10
Glycosphingolipids for functional studies have been tradi-
tionally purified from animal tissues by extraction.21,22 Hetero-
geneity inherited from these purification processes generates
complications in data analysis and identifying the ligand that
is responsible for protein/antibody/cell-binding. Releasing
glycans from glycosphingolipids purified from natural sources
chemically21,23 or enzymatically24 suffers similarly from poten-
tial contaminations. Additional challenges are limited access
to the structures that are less abundant in nature and the loss of
labile groups during purification and glycan cleavage
processes.25 Recently, significant progresses have been made
on the synthesis of glycosphingolipids and their glycans. Sev-
eral complex gangliosides have been synthesized by sophisti-
cated chemical approaches.26 Chemically synthesized stage-
specific embryonic antigen (SSEA-3 or Gb5) by pre-
activation-based one-pot approach followed by enzymatic
fucosylation and sialylation produced Globo-H and SSEA-4
(or V3Sia-Gb5) successfully.27 Globo-H has also been synthe-
sized by total chemical synthesis,28 programmable reactivity-
based one-pot strategy,29 and an enzymatic approach.30 Che-
moenzymatic synthesis of Neu5Ac-containing GD3, GT3,
GM2, GD2, GT2, GM1, and GD1a ganglioside glycans with a
2-azidoethyl linker has also reported.31 All of these glycans
Glycosphingolipids exhibit a large structural heterogeneity
with more than 300 different glycans characterized to date.
They are divided into several subfamilies including ganglio-,
lacto-, neolacto-, globo-, and isoglobo-series.11 The diverse
glycan structures on glycosphingolipids have been found to be
important for molecular recognition. Viruses and pathogenic
bacteria adhesins use glycosphingolipids on the host cell sur-
face to bind and invade epithelial cells,12 and the binding is
microbe-specific for the glycan structure.13,14 For example,
norovirus binds ganglioside GM1, but not other glycolipids.12
Cholera toxin also binds to GM1 on the cell surface.15,16 Botu-
linum toxin binds to GT1b and GQ1b.17 In addition, the bind-
ing of bacteria and viruses to gangliosides is specific to sialic
acid forms. For example, Escherichia coli K99 fimbrial adhe-
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obtained by chemical and enzymatic approaches either have a adhesins use gangliosides on the host cell surface for binding
and invasion.12 Lack of functional ganglioside metabolic genes
leads to rare genetic disorders such as lysosomal glycosphin-
golipid storage diseases.39 Aberrant expressing of gangliosides
is associated with cancer progression.2,3 Therefore, some can-
cer-associated gangliosides have been developed as potential
cancer markers, cancer vaccine candidates,8,9 and immunosup-
pressants.10 Here, four natural occurring sialic acid forms in-
cluding Neu5Ac, Neu5Gc, Kdn and Neu5Ac8OMe are intro-
duced into the structures of the target ganglioside glycans.
Both Neu5Ac (in humans and animals) and Neu5Gc (in ani-
mals and small amounts in humans) are common sialic acid
forms found in gangliosides.40,41 Kdn-containing gangliosides
have been found in the sperm,42 ovarian fluid,43 testis44 of
rainbow trouts as well as in yak milk45 and possibly in porcine
milk.46 Neu5Ac8OMe has been found in starfish as the com-
ponents of gangliosides47,48 and human erythrocyte mem-
brane.49 Its unique property of resistance to sialidases makes
the glycans containing Neu5Ac8OMe moiety interesting for
biofunctional studies.
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lipid aglycon or are tagged with a non-cleavable linker. More
recently, free reducing glycans have been released from gly-
cosphingolipids after treatment with ozone followed by heat-
ing in neutral aqueous buffer23 but the types of the glycans
produced by this method are limited as it relies on glycosphin-
golipids purified from natural sources. Despite the progresses
in chemical and enzymatic synthesis, sialic acid-containing
glycosphingolipids and the corresponding glycan head groups
containing naturally occurring sialic acid forms other than the
most abundant Neu5Ac are not readily available and some
have never been synthesized.
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Most of the earlier glycosyltransferase-catalyzed synthesis
of glycosphingolipid glycans27,29-32 relied on the use of expen-
sive and not readily accessible sugar nucleotides as donor sub-
strates. Here we report the use of highly efficient sequential
one-pot multienzyme (OPME) systems33 for high-yield syn-
thesis of complex glycosphingolipid glycans. In these systems,
simple monosaccharides or derivatives can be activated by one
or more enzymes to form desired sugar nucleotides for glyco-
syltransferase-catalyzed formation of target elongated glycans
in one pot. Each OPME process adds one monosaccharide or
derivative with a desired glycosidic linkage defined by the
glycosyltransferase used. Multiple OPME reactions can be
carried out to build up more complex glycan targets. As dem-
onstrated here, a library of free oligosaccharides found as the
glycan components of glycosphingolipids belonging to gan-
glio-series, lacto- and neolacto-series, as well as globo- and
isoglobo-series are successfully obtained in high yields from
lactose (Lac) using sequential OPME approaches (Scheme 1).
Synthesis of GM3 and GD3 glycans containing Neu5Ac,
Neu5Gc, Kdn, and Neu5Ac8OMe using OPME sialylation
systems. Sialic acid is a key component of gangliosides. Major
sialyl linkages in gangliosides are 2–3- and 2–8-linkages
although 2–6-sialyl linkage has also been found.50 We have
developed efficient OPME sialylation approaches for the syn-
thesis of 2–3/6/8-linked sialosides containing different sialic
acid forms and diverse underlying glycans.51-53 This approach
was tested and applied for the synthesis of GM3 and GD3
glycans containing different sialic acid forms including
Neu5Ac, Neu5Gc, Kdn, and Neu5Ac8OMe. For the ones with
Neu5Ac form, commercially available inexpensive Neu5Ac
was directly used for the synthesis in one-pot two-enzyme
systems containing a suitable sialyltransferase and a cytidine
5'-monophosphate sialic acid (CMP-Sia) biosynthetic enzyme
Neisseria meningitidis CMP-sialic acid synthetase (NmCSS).54
For the ones with other sialic acid forms including Neu5Gc,
Kdn, and Neu5Ac8OMe, one-pot three-enzyme systems were
used. In these systems, in addition to NmCSS and a sialyl-
transferase, Pasteurella multocida sialic acid aldolase (PmNa-
nA) was used to form the desired sialic acid forms from their
corresponding chemically synthesized precursors and pyru-
vate.
The most significant advantage of the OPME strategy is to
allow easy introduction of structurally modified monosaccha-
rides including challenging naturally occurring sialic acid
forms to the desired glycan structures. As shown here, gangli-
oside glycans containing one or two sialic acid residues se-
lected from four naturally occurring sialic acid forms, includ-
ing N-acetylneuraminic acid (Neu5Ac), N-glycolylneuraminic
acid (Neu5Gc), 2-keto-3-deoxy-D-glycero-D-galacto-nononic
acid (Kdn), and 8-O-methyl-N-acetylneuraminic acid
(Neu5Ac8OMe), have been successfully obtained. The access
to these structurally defined molecules will help to elucidate
the important function of glycosphingolipid glycans including
those containing naturally occurring sialic acid diversity which
is not currently feasible.
As shown in Scheme 2, GM3 trisaccharide containing
Neu5Ac (Neu5Ac2–3Lac, 1) was readily synthesized in an
excellent 98% yield from lactose as the acceptor substrate and
Neu5Ac as the donor precursor using a one-pot two-enzyme
system (OPME1) containing NmCSS and Pasteurella multo-
Scheme 1. Sequential One-Pot Multienzyme (OPME) Syn-
thesis of Ganglio-, Lacto-/Neolacto-, and Globo-/Isoglobo-
Series Glycosphingolipid Glycans.
cida 2–3-sialyltransferase
1 M144D mutant (PmST1
M144D)55 with decreased 2–3-sialidase and donor hydrolysis
activity. On the other hand, GM3 trisaccharides containing
Neu5Gc, Kdn, and Neu5Ac8OMe (Neu5Gc2–3Lac, 2;
Kdn2–3Lac, 3; and Neu5Ac8OMe2–3Lac, 4) were synthe-
sized from lactose and the corresponding sialic acid precursors
N-glycolylmannosamine (ManNGc), mannose (Man), and 5-
O-methyl-N-acetylmannosamine (ManNAc5OMe),56 respec-
tively, in excellent yields (93%, 95%, and 91%, respectively)
using a one-pot three-enzyme system (OPME2) containing
PmNanA, NmCSS, and PmST1 M144D.
2. RESULTS AND DISCUSSION
Chemoenzymatic synthesis of ganglioside glycans. Gan-
gliosides are a group of sialylated glycosphingolipids that are
presented in all tissues but are particularly abundant in the
nervous system34-36 where they affect neuronal plasticity dur-
ing development, adulthood, and aging.37 They regulate im-
munological function.38 Some viruses and pathogenic bacteria
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Scheme 2. Production of GM3 and GD3 Glycans Using
OPME 2–3- and 2–8-Sialylation Systems.
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2
Three
synthetic
GM3
trisaccharides
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4
5
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8
9
Neu5Ac/Neu5Gc/Kdn2–3Lac (1–3) were further used as
acceptor substrates for synthesizing nine GD3 tetrasaccharides
using
a
Campylobacter jejuni 2–3/8-sialyltransferase
(CjCstII)52-dependent one-pot two-enzyme (OPME3 when
Neu5Ac was used as the sialyltransferase donor precursor) or
a one-pot three-enzyme (OPME4 when ManNGc or Man was
used as the sialic acid precursor) 2–8-sialylation system.
From Neu5Ac2–3Lac (1), OPME3 and OPME4 produced
three GD3 glycans Neu5Ac2–8Neu5Ac2–3Lac (5),
Neu5Gc2–8Neu5Ac2–3Lac (6), and Kdn2–8Neu5Ac2–
3Lac (7) in good 85%, 84%, and 83% yields, respectively.
Similarly, from Neu5Gc2–3Lac (2), three GD3 glycans
Neu5Ac2–8Neu5Gc2–3Lac (8), Neu5Gc2–8Neu5Ac2–
3Lac (9), and Kdn2–8Neu5Ac2–3Lac (10) were synthe-
sized in good 86%, 83%, and 81% yields, respectively. From
Kdn2–3Lac (3), three GD3 glycans Neu5Ac2–Kdn2–
3Lac (11), Neu5Gc2–Kdn2–3Lac (12), and Kdn2–
Kdn2–3Lac (13), were synthesized in 82%, 78%, 81%
yields, respectively. Neu5Ac8OMe2–3Lac (4) has a O-
methyl group at C-8 of the terminal sialic acid and cannot be
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Scheme 3. Synthesis of GM2/GD2 and GM1/GD1b Glycans Containing Neu5Ac, Neu5Gc, Kdn, or Neu5Ac8OMe via
One-Pot Multienzyme (OPME) GalNAc and Gal Transfer Systems, Respectively.
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taining different sialic acid forms from the corresponding GD2
used for adding an additional 2–8-linked sialic acid. In addi-
tion, CMP-Neu5Ac8OMe (formed in situ in the OPME4 sys-
tem) was found as a poor donor substrate for CjCstII. There-
fore, the corresponding GD3 glycan containing a terminal
Neu5Ac8OMe was not produced.
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8
pentasaccharides (18, 19, 22, 23, and 25) were synthesized
similarly. Excellent yields were achieved using 1.1 equivalent
of galactose (Gal) as the donor precursor by incubating reac-
tion mixtures in Tris-HCl (100 mM, pH 7.5) at 37 C for 24
hours. It was found important not to add larger equivalents of
Gal. Otherwise, an additional Gal would be added to the de-
sired GM1 and GD1b products.
Synthesis of GM2 and GD2 glycans using an OPME 1–
4-GalNAc transfer system. The synthesis of GM2 and GD2
glycans involved the use of Campylobacter jejuni 1–
4GalNAcT (CjCgtA). The gene sequence of this enzyme was
reported before.57 A recombinant CjCgtA was used previously
for the synthesis of ganglioside oligosaccharides containing an
ethyl azido aglycon.31 In our attempts to obtain an active
CjCgtA and improve its expression level, a customer synthe-
sized synthetic gene based on the Campylobacter jejuni CgtA-
II protein sequence (GenBank accession number: AAL05993)
was used as a template for polymerase-chain reaction (PCR)
for cloning into pET22b(+) vector. In addition, series trunca-
tion of N-terminal sequence was carried out. Compared to the
full length construct and the constructs with N-terminal 10
amino acid (aa), 20 aa, or 25 aa truncation, the one with the N-
terminal 15 aa had a higher expression level (40 mg/L cul-
ture). Therefore, it was expressed and used for synthesis. The
purified CjCgtA samples were not stable for storage at 4 C. In
comparison, purified CjCgtA and lysates could be stored at -
20 C for over a year without significant loss of activity.
CjCgtA lysate was used directly in the enzymatic synthesis.
Scheme 4. Synthesis of Lc3, LNnT, and Sialylated LNnT
using OPME Glycosylation Systems.
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As shown in Scheme 3, four GM2 tetrasaccharides
Neu5Ac2–3(GalNAc1–4)Lac
(14),
Neu5Gc2–
3(GalNAc1–4)Lac (15), Kdn2–3(GalNAc1–4)Lac (16),
and Neu5Ac8OMe2–3(GalNAc1–4)Lac (17) were readily
obtained from four synthetic GM3 (1–4) trisaccharides in ex-
tremely high yields (95–99%) using an OPME 1–4-GalNAc
activation and transfer system (OPME5) containing CjCgtA
and uridine 5'-diphosphate N-acetylgalactosamine (UDP-
GalNAc) biosynthetic enzymes including Bifidobacterium
longum
NahK_ATCC55813),
N-acetylhexosamine-1-kinase
(BLNahK,
N-
Pasteurella multocida
Synthesis of lacto- and neolacto-series glycosphingoli-
pid glycans. Lacto- and neolacto-series glycosphingolipids
differ only by one galactosyl linkage: Gal1–3Lc3 for Lc4 in
the lacto-series and Gal1–4Lc3 for nLc4 in the neolacto-
series. Lc4 is a precursor for fucosyltransferase-catalyzed for-
mation of Lea and Leb. Taking advantage of PmST1 M144D
which was shown previously to be able to tolerate fucosylated
acceptors with or without further O-sulfation,59 direct 2–3-
sialylation of Lea can form sialyl Lea (sLea). While nLc4 is a
precursor for fucosyltransferase-catalyzed formation of Lex
and Ley, and 2–3-sialyation of Lex using PmST1 M144D can
form sialyl Lex (sLex). Neolacto-series glycosphingolipids
have been found on the surface of human hematopoietic cells
and are involved in the differentiation of hematopoietic cells.60
Lea, sLea, Lex, and sLex have been found to be overexpressed
on some cancer cell surface.61-63
acetylglucosamine uridyltransferase (PmGlmU), Pasteurella
multocida inorganic pyrophosphatase (PmPpA). All four en-
zymes were quite active in Tris-HCl buffer at pH 7.5.
The same OPME5 system (Scheme 3) was also used for
the synthesis of eight GD2 pentasaccharides (18–25) from
GD3
tetrasaccharides
(5–10,
12–13).
(18),
Neu5Ac2–
Neu5Gc2–
8Neu5Ac2–3(GalNAc1–4)Lac
8Neu5Ac2–3(GalNAc1–4)Lac (19), Kdn2–8Neu5Ac2–
3(GalNAc1–4)Lac
3(GalNAc1–4)Lac
(20),
(21),
Neu5Ac2–8Neu5Gc2–
Neu5Gc2–8Neu5Gc2–
3(GalNAc1–4)Lac (22), Kdn2–8Neu5Gc2–3(GalNAc1–
4)Lac (23), Neu5Gc2–8Kdn2–3(GalNAc1–4)Lac (24),
and Kdn2–8Kdn2–3(GalNAc1–4)Lac (25), were obtained
in excellent yields (nearly quantitative conversion).
Synthesis of GM1 and GD1b glycans using an OPME
1–3-galactosylation system. As shown in Scheme 3, the syn-
thesis of GM1 pentasaccharides (26–29) from GM2 tetrasac-
charides (14–17) was achieved using a one-pot four-enzyme
galactose-activation and transfer system (OPME6) containing
Campylobacter jejuni 1–3-galactosyltransferase (CjCgtB)
and uridine 5'-diphosphate galactose (UDP-Gal) biosynthetic
enzymes including Escherichia coli galactokinase (EcGalK),58
Bifidobacterium longum UDP-sugar pyrophosphorylase
(BLUSP), and PmPpA. GD1b hexasaccharides (30–34) con-
As shown in Scheme 4, LNnT Gal1–4GlcNAc1–3Lac
(36) was synthesized from lactose using a sequential two-step
OPME33 process similar to that was reported previously.64
Briefly, Lc3 trisaccharide GlcNAc1–3Lac 35 was synthesized
from lactose (Lac) and N-acetylglucosamine (GlcNAc) in a
94% yield using a one-pot four-enzyme GlcNAc activation
and transfer system (OPME7) containing Neisseria meningiti-
dis 1–3-N-acetylglucosaminyltransferase (NmLgtA) and
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uridine 5'-diphosphate N-acetylglucosamine (UDP-GlcNAc) tion system with Neu5Ac, ManNGc, Man, or ManNAc5OMe
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4
5
6
7
8
biosynthetic enzymes (the same set of enzymes for UDP-
GalNAc biosynthesis in OPME5) including BLNahK
as the sialyltransferase donor precursor.
Scheme 6. Synthesis of Lex Pentasaccharide and Its Sialy-
lated Forms using OPME Glycosylation Systems.
(NahK_ATCC55813),
PmGlmU,
PmPpA.
Lacto-N-
neotetraose (LNnT) tetrasaccharide Gal1–4GlcNAc1–
3Gal1–4Glc 36 was then synthesized from Lc3 (35) and ga-
lactose in an excellent (99%) yield using a OPME galactose
activation and transfer system (OPME8)64 containing Neisse-
ria meningitidis 1–4-galactosyltransferase (NmLgtB) and
UDP-Gal biosynthetic enzyme including EcGalK, BLUSP,
and PmPpA (the same set of UDP-Gal biosynthetic enzymes
in OPME6).
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14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
With LNnT in hand, sialylated LNnT pentasaccharides
containing Neu5Ac, Neu5Gc, Kdn, and Neu5Ac8OMe (37–
40) were successfully synthesized using OPME1 sialylation
system with Neu5Ac as the donor precursor or OPME2 sialy-
lation system with ManNGc, Man, or ManNAc5OMe as the
sialic acid precursor.
Similarly, sialylated lacto-N-tetraose (LNT) pentasaccha-
rides containing Neu5Ac, Neu5Gc, Kdn, and Neu5Ac8OMe
(42–45) were obtained via OPME1 or OPME2 sialylation sys-
tem using commercial available LNT (41) as the acceptor sub-
strate and Neu5Ac, ManNGc, Man, and ManNAc5OMe, re-
spectively, as donor precursors (Scheme 5).
Scheme 5. Synthesis of Sialylated LNT Using OPME Sialy-
lation Systems.
Synthesis of globo- and isoglobo-glycosphingolipid gly-
cans. The globo (Gb) and isoglobo (iGb) series glycosphingo-
lipid glycans are built, respectively, on trisaccharides Gb3
(Gal1–4Lac) and iGb3 (Gal1–3Lac) that differ by only one
terminal Gal linkage. Globo-series glycosphingolipids are
used as receptors by Shiga toxin,66 verotoxins, and HIV adhe-
sin gp120.67 They have also attracted much attentions due to
their overexpression in cancer68 and accumulation in Fabry’s
disease.69 Tumor-associated Globo H antigen was initially
identified from human breast cancer cell line MCF-770 and
was later found in several human cancers. Globo H-based syn-
thetic vaccines have shown promising results in clinical trials
for breast and prostate cancers.71-74
Although sialylated Lex pentasaccharides 47–50 can be
synthesized by fucosylation of sialylated LNnT 37–40, purifi-
cation of the product from starting materials in these fucosyla-
tion reactions was found difficult due to their similarity in
sizes and polarity. To simplify the production and purification
processes, fucosylated LNnT 46 was synthesized and used as
the acceptor substrate for PmST1 M144D-catalyzed OPME
2–3-sialylation. This was made feasible by a single mutation
M144D introduced to PmST1 which made the 2–3-
sialylation of fucosylated acceptors efficient by reducing do-
nor hydrolysis and 2–3-sialidase activity of PmST1.59 As
shown in Scheme 6, Lex pentasaccharide Gal1–4(Fuc1–
3)GlcNAc1–3Lac (46) was synthesized in a preparative-scale
(500 mg) in an excellent 94% yield from LNnT tetrasaccha-
ride (36), using a one-pot three-enzyme fucose activation and
transfer system (OPME9) containing Helicobacter pylori 1–
3-fucosyltransferase (Hp1–3FT)55 and guanosine 5'-
diphosphate fucose (GDP-Fuc) biosynthetic enzymes includ-
ing a bifunctional Bacteroides fragilis L-fucokinase and gua-
nidine 5'-diphosphate (GDP)-fucose pyrophosphorylase
(BfFKP)65 and PmPpA. Sialylated Lex pentasaccharides 47–50
were then synthesized from 46 via OPME1 or OPME2 sialyla-
As shown in Scheme 7, Gb3 trisaccharide Gal1–4Lac (51)
was readily obtained in an excellent 95% yield from Lac, Gal,
adenosine 5'-triphosphate (ATP), and uridine 5'-triphosphate
(UTP) using an OPME 1–4-galactosylation system
(OPME10)
containing
N.
meningitidis
1–4-
galactosyltransferase (NmLgtC)75,76 and UDP-Gal biosynthetic
enzymes including EcGalK, BLUSP, and PmPpA. On the
other hand, iGb3 trisaccharide Gal1–3Lac (52) was synthe-
sized in an outstanding 99% yield from Lac, Gal, ATP, and
UTP using an OPME 1–3-galactosylation system (OPME11)
containing a recombinant bovine 1–3GalT (B1–3GalT)77
and UDP-Gal biosynthetic enzymes including EcGalK,
BLUSP, and PmPpA.
A bifunctional Haemophilus influenzae 1–3GalT/1–
3GalNAcT (HiLgtD)78,79 was used to catalyze the transfer of
GalNAc from in situ generated UDP-GalNAc to Gb3 (51) and
iGb3 (52) in an OPME 1–3-GalNAc transfer system
(OPME12) containing HiLgtD and UDP-GalNAc biosynthetic
enzymes NahK, PmGlmU, and PmPpA to produce Gb4 (53,
92%) and iGb4 (54, 91%) tetrasaccharides, respectively, in
excellent yields.
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For the synthesis of Gb5 (55) and iGb5 (56) pentasaccha-
rides by adding a 1–3-linked Gal to Gb4 (53) and iGb4 (54)
tetrasaccharides, respectively, the bifunctional HiLgtD (having
both 1–3-Gal and 1–3-GalNAc transferase activities) was
initially tested. However, it was found that HiLgtD-catalyzed
reaction for forming pentasaccharides was very low. In com-
parison, CjCgtB was found to be able to catalyze the transfer
of Gal from UDP-Gal to Gb4 to form Gb5 in moderate yields.
Therefore, Gb5 (55) and iGb5 (56) pentasaccharides were syn-
thesized from Gb4 (53) and iGb4 (54) tetrasaccharides using
CjCgtB-containing OPME6 in 61% and 45% yields, respec-
tively.
sialylated Gb5 (57–60, 78–86%) and sialylated iGb5 (61–64,
1
2
3
4
5
6
7
8
71–85%) hexasaccharides containing Neu5Ac, Neu5Gc, Kdn,
and Neu5Ac8OMe sialic acid forms, respectively, with good
yields.
Enzymatic reaction conditions and purification
processes. A pH range of 8.0–8.5 was found to be optimal and
Tris-HCl buffer (100 mM, pH 8.5) was used in the OPME
sialylation systems for the synthesis of desired sialosides. In
comparison, a pH range of 7.5–8.0 was found to be more suit-
able and Tris-HCl buffer (100 mM, pH 8.0) was used in
NmLgtB-containing OPME reaction for the synthesis of LNnT
(36). On the other hand, Tris-HCl buffer (100 mM, pH 7.5)
was used in CjCgtA-containing OPME GalNAc-transfer sys-
tem for the production of GM2 and GD2, NmLgtA-catalyzed
GlcNAc-transfer system for the synthesis of Lc3 (GlcNAc1–
3Lac), and other OPME galactosylation (including CjCgtB-
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Gb5 (55) and iGb5 (56) pentasaccharides were then used as
the acceptor substrates in PmST1 M144D-containing OPME
2–3-sialylation (OPME1 or OPME2) systems to produce
Scheme 7. OPME Synthesis of Globo and Isoglobo-Series Glycans.
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The Journal of Organic Chemistry
manipulation. Developed TLC plates were visualized with
catalyzed production of GM1, GD1b, Gb5, and iGb5, B1–
1
anisaldehyde sugar stain and heat provided by a hotplate. Sil-
ica gel flash column chromatography was performed using
flash silica gel (40–63 m) and employed a solvent polarity
correlated with TLC mobility. Gel filtration chromatography
was performed with a column (100 cm × 2.5 cm) packed with
BioGel P-2 Fine resins.
3GalT/NmLgtC/HiLgtD-catalyzed OPME galactosylation for
the production of Gb3, iGb3, Gb4, and iGb4). OPME fucosyla-
tion of LNnT was also carried out at Tris-HCl buffer (100
mM, pH 7.5). Reactions were carried out at 37 C or at room
temperature and were completed in a time frame of 2–48 h.
The reaction progress was monitored by thin-layer chromato-
graphy (TLC) and mass spectrometry (MS).
2
3
4
5
6
7
8
9
The combinations of various columns were used to purify
target glycans from OPME reactions. A simple silica gel col-
umn followed by a final gel filtration column packed with Bio-
gel P2 resin were used to purify Gb3, iGb3, Gb4, and iGb4 gly-
cans (51–54). For purifying GM3 trisaccharides (1–4), Lc3
trisaccharide (35), nLc4 tetrasaccharide (36), and Lex penta-
saccharide (46), a Bio-gel P2 gel filtration column followed by
a silica gel column and a final gel filtration column for desalt-
ing were used. For purifying sialylated LNnT, LNT, Lex (37–
50) as well as Gb5 (55), iGb5 (56), and their sialylated glycans
(57–64), Bio-gel P2 gel filtration column followed by high-
performance liquid chromatography (HPLC) purification with
a reverse-phase C18 column was used. For purifying GD3
tetrasaccharides (5–13), sialyl Lex hexasaccharides (47–50),
Bio-gel P2 gel filtration column followed by silica gel column
and HPLC purification with a reverse-phase C18 column was
used. For purifying GM2 tetrasaccharides (14–17), GD2 pen-
tasaccharides (18–25), GM1 pentasaccharides (26–29), and
GD1 hexasaccharides (30–34), Bio-gel P2 gel filtration col-
umn followed by HPLC purification using an XBridge BEH
amide column was used. We have also found that the addition
of a commercially available alkaline phosphatase from bovine
intestinal mucosa to reaction mixture after glycosylation reac-
tions could efficiently break down nucleotides byproducts
(e.g. ADP, AMP, UDP, UMP, and GDP) byproducts and make
the purification procedures much easier.
Cloning of CjCgtA-His6. Synthetic DNA based on Cam-
pylobacter jejuni CgtA-II protein sequence (GenBank acces-
sion number: AAL05993) and optimized for Escherichia coli
was customer synthesized by Biomatik. It was used as a tem-
plate for target gene amplification of the full-length and N-
terminal truncated constructs by polymerase chain reactions
(PCRs) for cloning into pET22b(+) vector. The primers used
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
were
CAGCGTCGACTTTGATCTCACCCTGAAACTTC
TTCAG-3’ (SalI restriction site is underlined); full length
CgtA-His6 forward 5’-
GATCCATATGCTGAAAAAGATTATCAGCCTGT
ACAAG-3’ (NdeI restriction site is underlined); 10CgtA-
His6 forward 5’-
GATCCATATGCGCTACAGCATCAGCAAGAAAC
reverse
5'-
TGGTG-3’ (NdeI restriction site is underlined); 15CgtA-His6
forward
5’-
GATCCATATGAAGAAACTGGTGCTGGACAAC
GAGCAC-3’ (NdeI restriction site is underlined); 20CgtA-
His6 forward 5’-
GATCCATATGGACAACGAGCACTTTATTAAGG-3'
(NdeI restriction site is underlined). PCRs for amplifying the
target gene were each performed in a 50 L reaction mixture
containing plasmid DNA (10 ng), forward and reverse primers
(0.2 M each), 1 × Herculase buffer, dNTP mixture (0.2 mM),
and 5 U (1 L) of Herculase-enhanced DNA polymerase. The
reaction mixture was subjected to 30 cycles of amplification at
an annealing temperature of 55C. The resulted PCR product
was purified and double digested with NdeI and SalI restric-
tion enzymes. The purified and digested PCR product was
ligated with the predigested pET22b(+) vector and trans-
formed into E. coli DH5 electrocompetent cells. Selected
clones were grown for minipreps and characterized by restric-
tion mapping. Positive construct was transformed into E. coli
BL21 (DE3) chemical component cells.
3. CONCLUSIONS
In conclusion, we have successfully applied sequential one-pot
multienzyme (OPME) systems for high-yield and cost-
effective production of glycosphingolipid glycans including
those belonging to the ganglio-, lacto-, neolacto-, globo-, and
isoglobo-series. The OPME approaches allow easy introduc-
tion of naturally occurring structurally modified diverse sialic
acid forms to glycosphingolipid glycans. These glycans are
essential standards for glycan analysis and critical probes for
bioassays and biomedical studies for developing novel carbo-
hydrate-based diagnostics and therapeutics.
Expression and purification of enzymes involved in the
synthesis. This was carried out similarly to those reported
previously.54,55,80 Briefly, E. coli BL21 (DE3) strains harboring
the recombinant plasmid with target gene was cultured in 50
mL Luria-Bertani (LB) rich medium (10 g/L tryptone, 5 g/L
yeast extract, and 10 g/L NaCl) containing 0.1 mg/mL ampi-
cillin with rapid shaking (220 rpm) at 37 C overnight. Then
15 mL of the overnight cell culture was transferred into 1 L of
LB rich medium with 0.1 mg/mL ampicillin and incubated at
37 C. When the OD600nm of the cell culture reached 0.8–1.0,
isopropyl-1-thio--D-galactopyranoside (IPTG, 0.1 mM) was
added to induce the over-expression of the recombinant en-
zyme, which was followed by incubation at 20 C with shak-
ing (190–250 rpm) for 20 h. Cells were collected by centrifu-
gation at 4000 rpm for 2 h at 4 C. Harvested cells were resus-
pended with lysis buffer (100 mM Tris-HCl buffer, pH 8.0,
containing 0.1% Triton X-100). The cells were broken by so-
nication to obtain cell lysate which was centrifuged at 12,000
4. EXPERIMENTAL SECTION
Materials and general methods
All reagents were purchased from commercial sources and
1
used without further purification unless stated otherwise. H
and 13C spectra were measured in the solvent stated at 800
MHz, and 200 MHz, respectively. Chemical shifts are quoted
in parts per million (ppm) and coupling constants (J) are given
in Hertz (Hz). Multiplicities are abbreviated as br (broad), s
(singlet), d (doublet), t (triplet), q (quartet), and m (multiplet)
or combinations thereof. High resonance mass spectrometry
samples were analyzed by electrospray ionization mass spec-
trometry in positive mode or negative mode using flow-
injection analysis. Glass-backed TLC plates (Silica Gel 60
with a 254 nm fluorescent indicator) were used without further
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calcd for C21H35O19 (M-H) 591.1773, found 591.1782. NMR
rpm for 15 min at 4 C. The supernatant was collected and
loaded onto a Ni2+-NTA affinity column pre-equilibrated with
a binding buffer (50 mM, pH 7.5, Tris-HCl buffer, 5 mM im-
idazole, 0.5 M NaCl). The column was washed with 10 col-
umn volumes of binding buffer and 10 column volumes of
washing buffer (50 mM Tris-HCl buffer, pH 7.5, 20 mM im-
idazole, 0.5 M NaCl). The target protein was eluted using
Tris-HCl buffer (50 mM, pH 7.5) containing 200 mM of im-
idazole and NaCl (0.5 M).
data were consistent with those reported in the literature.53
1
2
3
4
5
6
7
8
Neu5Ac8OMe2–3Lac (4). 12 mg, yield 91%; white solid.
1H NMR (800 MHz, D2O)  5.21 (d, J = 3.2 Hz, 0.4H), 4.66
(d, J = 8.0 Hz, 0.6H), 4.49 (d, J = 8.0 Hz, 1H), 4.08–3.26 (m,
19H), 3.48 (s, 3H), 2.67 (dd, J = 12.0 and 4.8 Hz, 1H), 2.02 (s,
3H), 1.75 (t, J = 12.0 Hz, 1H); 13C NMR (200 MHz, D2O) 
174.84, 173.54, 102.65, 100.09, 95.66, 91.72, 80.20, 78.17,
78.02, 75.64, 75.12, 74.73, 74.17, 73.70, 72.71, 71.23, 71.05,
70.02, 69.27, 67.91, 67.59, 66.86, 60.92, 59.94, 59.80, 59.23,
57.40, 51.89, 39.70, 21.96; HRMS (ESI) m/z calcd for
C24H40NO19 (M-H) 646.2195, found 646.2191.
General procedures for OPME synthesis of GM3 gly-
cans (4 compounds). Lac (20 mM, 1 eq.), Neu5Ac or a sialic
acid precursor (ManNGc, mannose, or ManNAc5OMe, 1.5
eq.) with sodium pyruvate (7.5 eq.) were incubated at 37 C in
a Tris-HCl buffer (100 mM, pH 8.5) containing CTP (1.5 eq.),
MgCl2 (20 mM), NmCSS (0.15 mg/mL), PmST1 M144D (0.3
mg/mL), with or without PmNanA (0.2 mg/mL, omit if
Neu5Ac was used). The reaction was monitored by TLC with
a developing reagent constituted of i-PrOH:H2O:NH4OH=
5:2:1 (by volume) and stained with p-anisaldehyde sugar
Reactions were typically completed in 12–24 h. Upon comple-
tion, to the reaction mixture was added the same volume of
ethanol and incubated at 4 C for 30 min before the mixture
was centrifuged to remove precipitates. The supernatant was
concentrated and passed through a BioGel P-2 gel filtration
column and eluted with degassed water. The fractions contain-
ing the product were collected, concentrated, and further puri-
fied by silica gel column (EtOAc:MeOH:H2O, 4:2:1). The
collected fractions were concentrated and passed through the
gel filtration column again to obtain the desired GM3 glycans
(yield from 91% to 98%).
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
General procedures for OPME synthesis of GD3 gly-
cans (9 compounds). A GM3 glycan (20 mM, 1 eq.) as an
acceptor for the 2–8-sialyltransferase activity of CjCstII,
Neu5Ac or a sialic acid precursor (ManNGc, mannose, or
ManNAc5OMe, 1.2 eq.) with sodium pyruvate (7.5 eq.) were
incubated at 37 C in Tris-HCl buffer (100 mM, pH 8.5), CTP
(1.5 eq.), MgCl2 (20 mM), NmCSS (0.15 mg/mL), CjCstII
(0.35 mg/mL) with or without PmNanA (0.2 mg/mL, omit if
Neu5Ac was used). The reaction was carried out by incubating
the solution in an incubator shaker at 37 C for 2 h (or at room
temperature for overnight) with agitation at 140 rpm. The
product formation was monitored by LC-MS. When an optim-
al yield was achieved, the reaction was quenched by adding
the same volume of ice-cold ethanol and incubation at 4 C for
30 min. The mixture was centrifuged and the precipitates were
removed. The supernatant was concentrated, passed through a
BioGel P-2 gel filtration column and eluted with water to ob-
tain sialoside mixtures. The fractions containing the product
were collected and then purified by silica gel column
(EtOAc:MeOH:H2O, 5:3:2). The compound was further puri-
fied by a reverse-phase C18 column (10 m, 21.2 × 250 mm)
with a flow rate of 10 mL/min using a gradient elution of 0–
100% acetonitrile in water containing 0.05% formic acid over
20 minutes [Mobile phase A: 0.05% formic acid in water
(v/v); Mobile phase B: acetonitrile (v/v); Gradient: 0% B for 3
minutes, 0% to 100% B over 12 minutes, 100% B for 2 mi-
nutes, then 100% to 0% B over 3 minutes]. HPLC purification
was monitored by absorption at 210 nm, and glycan-
containing fractions were analyzed by TLC and MS. The frac-
tions containing the pure product were collected and concen-
trated to obtain the final pure GD3 glycans (yields 78–86%).
1
Neu5Ac2–3Lac (1). 2.1 g, yield 98%; white solid. H
NMR (800 MHz, D2O)  5.21 (d, J = 4.0 Hz, 0.4H), 4.65 (d, J
= 8.0 Hz, 0.6H), 4.52 (d, J = 8.0 Hz, 1H), 4.11–3.26 (m, 19H),
2.74 (dd, J = 12.0 and 4.8 Hz, 1H), 2.02 (s, 3H), 1.79 (t, J =
12.0 Hz, 1H); 13C NMR (200 MHz, D2O)  174.87, 173.77,
102.49, 99.66, 95.65, 91.70, 78.15, 78.01, 75.35, 75.05, 74.68,
74.20, 73.67, 72.74, 71.65, 71.26, 71.02, 69.97, 69.24, 68.24,
67.96, 67.33, 62.44, 60.91, 59.93, 59.78, 51.55, 39.51, 21.92.
HRMS (ESI) m/z calcd for C23H38NO19 (M-H) 632.2038,
found 632.2036. NMR data were consistent with those
reported in the literature.53
1
Neu5Gc2–3Lac (2). 360 mg, yield 93%; white solid. H
NMR (800 MHz, D2O)  5.21 (d, J = 4.0 Hz, 0.4H), 4.64 (d, J
= 8.0 Hz, 0.6H), 4.51 (d, J = 8.0 Hz, 1H), 4.10 (s, 2H), 4.10–
3.26 (m, 19H), 2.75 (dd, J = 12.0 and 4.8 Hz, 1H), 2.02 (s,
3H), 1.80 (t, J = 12.0 Hz, 1H); 13C NMR (200 MHz, D2O) 
175.65, 173.82, 102.51, 102.49, 99.68, 95.63, 91.72, 78.16,
78.01, 75.34, 75.05, 74.68, 74.20, 73.70, 73.66, 72.46, 71.74,
71.67, 71.02, 69.26, 69.24, 68.00, 67.96, 67.88, 67.37, 67.31,
62.44, 62.38, 60.94, 60.92, 60.86, 59.94, 59.80, 59.19, 51.29,
51.22, 39.58, 39.55; HRMS (ESI) m/z calcd for C23H38NO20
(M-H) 648.1987, found 648.1984. NMR data were consistent
with those reported in the literature.81
Neu5Ac2–8Neu5Ac2–3Lac (5). 1.4 g, yield 86%; white
1
solid. H NMR (800 MHz, D2O)  5.21 (d, J = 3.2 Hz, 0.4H),
4.66 (d, J = 8.0 Hz, 0.6H), 4.52 (d, J = 7.2 Hz, 1H), 4.16–4.07
(m, 3H), 3.99–3.25 (m, 23H), 2.77 (dd, J = 4.8 and 12.8 Hz,
1H), 2.67 (dd, J = 4.8 and 12.8 Hz, 1H), 2.06 (s, 3H), 2.02 (s,
3H), 1.73 (t, J = 12.0 Hz, 2H). 13C NMR (200 MHz, D2O) 
174.88, 174.86, 174.81, 173.39, 173.24, 102.57, 102.54,
100.41, 100.08, 100.07, 95.70, 95.66, 91.75, 91.70, 78.08,
77.97, 77.83, 75.33, 75.10, 74.71, 74.14, 73.89, 73.76, 73.71,
72.53, 71.69, 71.59, 71.24, 71.07, 70.06, 69.25, 69.18, 69.16,
68.36, 68.27, 68.02, 67.99, 67.80, 67.39, 67.30, 62.41, 61.49,
61.46, 61.43, 60.99, 59.96, 59.89, 59.81, 59.74, 52.24, 52.18,
52.15, 52.10, 51.66, 51.63, 51.58, 40.39, 40.35, 39.62, 39.53,
22.23, 21.96. HRMS (ESI) m/z calculated for C34H55N2O27
(M-H) 923.2992, found 923.2983. NMR data were consistent
with those reported in the literature.52
1
Kdn2–3Lac (3). 82 mg, yield 95%; white solid. H NMR
(800 MHz, D2O)  5.19 (d, J = 4.0 Hz, 0.3H), 4.63 (d, J = 8.0
Hz, 0.7H), 4.50 (d, J = 8.0 Hz, 1H), 4.07–3.24 (m, 19H), 2.67
(dd, J = 12.0 and 4.8 Hz, 1H), 1.72 (t, J = 12.0 Hz, 1H); 13C
NMR (200 MHz, D2O)  173.93, 170.45, 102.50, 99.65,
95.65, 91.69, 78.13, 77.99, 75.32, 75.05, 74.68, 74.20, 73.78,
73.68, 71.95, 71.25, 71.02, 70.12, 69.97, 69.61, 69.23, 67.58,
67.28, 62.49, 60.91, 59.93, 59.79, 39.16; HRMS (ESI) m/z
Neu5Gc2–8Neu5Ac2–3Lac (6). 52 mg, yield 84%;
1
white solid. H NMR (800 MHz, D2O)  5.19 (d, J = 4.0 Hz,
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0.4H), 4.64 (d, J = 8.0 Hz, 0.6H), 4.50 (d, J = 8.0 Hz, 1H), 77.89, 75.45, 75.21, 74.79, 74.21, 73.79, 73.69, 73.58, 72.02,
1
2
3
4
5
6
7
8
4.16 (m, 1H), 4.12 (m, 1H), 4.10 (s, 2H), 4.10 (s, 2H), 4.06
(m, 1H), 3.97–3.25 (m, 23H), 2.77 (dd, J = 12.0 and 4.8 Hz,
1H), 2.66 (dd, J = 12.0 and 4.8 Hz, 1H), 2.05 (s, 3H), 1.73 (t, J
= 12.0 Hz, 1H), 1.723 (t, J = 12.0 Hz, 1H); 13C NMR (200
MHz, D2O)  175.61, 174.85, 173.37, 173.26, 102.55, 102.53,
100.38, 100.05, 100.03, 95.67, 91.71, 78.10, 77.94, 77.78,
75.33, 75.10, 74.71, 74.14, 73.89, 73.71, 72.24, 71.68, 71.20,
71.06, 70.00, 69.16, 68.11, 67.91, 67.80, 67.32, 62.38, 61.44,
60.99, 60.84, 59.86, 52.14, 51.31, 40.42, 39.57, 22.19. HRMS
(ESI) m/z calculated for C34H55N2O28 (M-H) 939.2941, found
939.2920.
71.27, 71.14, 70.27, 70.09, 69.76, 69.23, 69.01, 67.74, 67.41,
62.59, 61.42, 61.08, 59.97, 59.82, 52.06, 48.64, 40.16, 39.70.
HRMS (ESI) m/z calculated for C32H52NO28 (M-H) 898.2676,
found 898.2668.
Neu5Ac2–8Kdn2–3Lac (11). 24 mg, yield 82%; white
1
solid. H NMR (800 MHz, D2O)  5.20 (d, J = 4.0 Hz, 0.4H),
4.64 (d, J = 8.0 Hz, 0.6H), 4.50 (d, J = 8.0 Hz, 0.4H), 4.49 (d,
J = 8.0 Hz, 0.6H), 4.18–3.25 (m, 26H), 2.76–2.61 (m, 2H),
2.01 (s, 3H), 1.80–1.70 (m, 2H); 13C NMR (200 MHz, D2O) 
174.83, 169.88, 102.54, 95.66, 91.71, 77.93, 77.83, 77.69,
75.38, 75.10, 74.90, 74.71, 74.15, 73.71, 73.54, 72.60, 71.59,
71.20, 71.06, 70.59, 70.40, 70.00, 69.46, 69.35, 69.17, 69.13,
68.77, 68.37, 68.31, 68.04, 67.86, 67.47, 67.38, 67.32, 62.46,
61.23, 61.03, 60.97, 59.90, 52.20, 51.60, 40.35, 39.78, 39.05,
22.19, 22.09. HRMS (ESI) m/z calculated for C32H52NO27 (M-
H) 882.2727, found 882.2715.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Kdn2–8Neu5Ac2–3Lac (7). 39 mg, yield 83%; white
1
solid. H NMR (800 MHz, D2O)  5.20 (d, J = 4.0 Hz, 0.4H),
4.64 (d, J = 8.0 Hz, 0.6H), 4.51 (d, J = 8.0 Hz, 0.4H), 4.50 (d,
J = 8.0 Hz, 0.6H), 4.16–4.06 (m, 3H), 3.97–3.26 (m, 23H),
2.70 (dd, J = 12.0 and 4.8 Hz, 1H), 2.66 (dd, J = 12.0 and 4.8
Hz, 1H), 2.05 (s, 3H), 1.72 (t, J = 12.0 Hz, 1H), 1.69 (t, J =
12.0 Hz, 1H); 13C NMR (200 MHz, D2O)  174.85, 173.45,
173.36, 143.29, 102.54, 100.90, 100.37, 100.06, 95.66, 91.71,
78.02, 77.96, 77.80, 75.33, 75.10, 74.70, 74.14, 73.88, 73.70,
73.52, 71.91, 71.20, 71.06, 70.27, 70.00, 69.69, 69.19, 69.15,
67.80, 67.63, 67.33, 62.49, 61.42, 60.98, 59.87, 59.72, 59.29,
52.14, 39.92, 39.55, 22.19. HRMS (ESI) m/z calculated for
C32H52NO27 (M-H) 882.2727, found 882.2719.
Neu5Gc2–8Kdn2–3Lac (12). 28 mg, yield 78%; white
1
solid. H NMR (800 MHz, D2O)  5.20 (d, J = 4.0 Hz, 0.4H),
4.64 (d, J = 8.0 Hz, 0.6H), 4.50 (d, J = 8.0 Hz, 0.4H), 4.49 (d,
J = 8.0 Hz, 0.6H), 4.21–3.26 (m, 28H), 2.76–2.61 (m, 2H),
1.78–1.66 (m, 2H); 13C NMR (200 MHz, D2O)  176.01,
175.64, 173.78, 173.57, 173.44, 102.61, 101.11, 100.09,
99.99, 95.75, 91.79, 78.51, 78.03, 77.86, 77.42, 75.51, 75.22,
74.80, 74.67, 74.22, 73.79, 73.59, 72.37, 72.27, 71.75, 71.28,
71.14, 70.70, 70.08, 69.48, 69.45, 69.24, 69.17, 68.24, 68.16,
68.11, 67.69, 67.41, 62.50, 61.38, 61.25, 61.09, 61.07, 60.93,
59.99, 52.12, 51.39, 40.60, 39.60. HRMS (ESI) m/z calculated
for C32H52NO28 (M-H) 898.2676, found 898.2663.
Neu5Ac2–8Neu5Gc2–3Lac (8). 121 mg, yield 86%;
1
white solid. H NMR (800 MHz, D2O)  5.19 (d, J = 4.0 Hz,
0.3H), 4.63 (d, J = 8.0 Hz, 0.7H), 4.49 (d, J = 8.0 Hz, 1H),
4.17–3.24 (m, 28H), 2.73 (dd, J = 4.8 and 12.8 Hz, 1H), 2.67
(dd, J = 4.8 and 12.8 Hz, 1H), 1.99 (s, 3H), 1.71 (t, J = 12.0
Hz, 2H). 13C NMR (200 MHz, D2O)  175.94, 174.86, 174.84,
174.60, 173.50, 173.29, 172.49, 170.61, 170.59, 102.60,
102.52, 101.94, 100.11, 100.04, 95.68, 95.65, 78.28, 75.37,
75.32, 75.08, 74.69, 74.12, 73.77, 73.69, 73.59, 72.68, 72.49,
71.77, 71.60, 71.06, 69.16, 68.98, 68.91, 68.28, 68.22, 67.98,
67.82, 67.46, 67.28, 62.50, 61.34, 61.30, 60.98, 59.18, 52.01,
51.88, 51.65, 40.42, 39.51, 39.10, 21.95, 21.91. HRMS (ESI)
m/z calculated for C34H55N2O28 (M-H) 939.2941, found
939.2935.
Kdn2–8Kdn2–3Lac (13). 24 mg, yield 81%; white
1
solid. H NMR (800 MHz, D2O)  5.19 (d, J = 4.0 Hz, 0.4H),
4.63 (d, J = 8.0 Hz, 0.6H), 4.49 (d, J = 8.0 Hz, 0.4H), 4.48 (d,
J = 8.0 Hz, 0.6H), 4.18–3.24 (m, 26H), 2.67 (dd, J = 12.0 and
4.8 Hz, 1H), 2.61 (dd, J = 12.0 and 4.8 Hz, 1H), 1.76 (t, J =
12.0 Hz, 1H), 1.68 (t, J = 12.0 Hz, 1H); 13C NMR (200 MHz,
D2O)  173.88, 173.45, 102.55, 95.66, 91.70, 77.97, 77.82,
77.70, 75.36, 75.11, 74.95, 74.70, 74.14, 73.70, 73.59, 71.84,
71.19, 71.05, 70.41, 70.31, 70.00, 69.66, 69.39, 69.34, 69.15,
67.70, 67.34, 62.50, 61.26, 60.96, 59.90, 59.75, 39.12. HRMS
(ESI) m/z calculated for C30H49O27 (M-H) 841.2461, found
841.2464.
Neu5Gc2–8Neu5Gc2–3Lac (9). 76 mg, yield 83%;
1
white solid. H NMR (800 MHz, D2O)  5.21 (d, J = 4.0 Hz,
0.4H), 4.64 (d, J = 8.0 Hz, 0.6H), 4.50 (d, J = 8.0 Hz, 0.4H),
4.49 (d, J = 8.0 Hz, 0.6H), 4.18 (d, J = 16.8 Hz, 1H), 4.16 (m,
1H), 4.13 (m, 1H), 4.10 (s, 2H), 4.09 (d, J = 16.8 Hz, 1H),
4.07 (m, 1H), 3.98–3.25 (m, 23H), 2.76 (dd, J = 12.0 and 4.8
Hz, 1H), 2.68 (dd, J = 12.0 and 4.8 Hz, 1H), 1.73 (t, J = 12.0
Hz, 2H); 13C NMR (200 MHz, D2O)  175.61, 174.85, 173.37,
173.26, 102.55, 102.53, 100.38, 100.05, 100.03, 95.67, 91.71,
78.10, 77.94, 77.78, 75.33, 75.10, 74.71, 74.14, 73.89, 73.71,
72.24, 71.68, 71.20, 71.06, 70.00, 69.16, 68.11, 67.91, 67.80,
67.32, 62.38, 61.44, 60.99, 60.84, 59.86, 52.14, 51.31, 40.42,
39.57, 22.19. HRMS (ESI) m/z calculated for C34H55N2O29
(M-H) 955.2890, found 955.2900.
General procedures for OPME synthesis of GM2 (4
compounds, 14–17) and GD2 glycans (8 compounds, 18–
25). A GM3 or GD3 glycan (10 mM, 1 eq.) as an acceptor
substrate, GalNAc (1.5 eq.), ATP (1.5 eq.), UTP (1.5 eq.), and
MgCl2 (20 mM) were incubated at 37 C in Tris-HCl buffer
(100 mM , pH 7.5) containing BLNahK (3 mg/mL), PmGlmU
(3 mg/mL), CjCgtA (lysate, 4.0 mg/mL), and PmPpA (2
mg/mL). The reaction was carried out by incubating the solu-
tion in an incubator shaker at 37 C for 2 days with agitation at
100 rpm. The product formation was monitored by LC-MS.
When an optimal yield was achieved, alkaline phosphatase
(10–20 mg) was added to the reaction mixture which was in-
cubated in an incubator shaker at 37 C for overnight with
agitation at 100 rpm. The reaction was then quenched by add-
ing the same volume of ice-cold ethanol and incubated at 4 C
for 30 min. The mixture was centrifuged and the precipitates
were removed. The supernatant was concentrated, passed
through a BioGel P-2 gel filtration column, and eluted with
water to obtain crude sialosides. The fractions containing the
product were collected, concentrated, and further purified by
Kdn2–8Neu5Gc2–3Lac (10). 62 mg, yield 81%; white
1
solid. H NMR (800 MHz, D2O)  5.20 (d, J = 4.0 Hz, 0.4H),
4.64 (d, J = 8.0 Hz, 0.6H), 4.50 (d, J = 8.0 Hz, 0.4H), 4.49 (d,
J = 8.0 Hz, 0.6H), 4.18 (d, J = 16.8 Hz, 1H), 4.15 (m, 1H),
4.12 (m, 1H), 4.08 (d, J = 16.8 Hz, 1H), 4.07 (m, 1H), 3.97–
3.26 (m, 23H), 2.68 (m, 2H), 1.73 (t, J = 12.0 Hz, 1H), 1.67 (t,
J = 12.0 Hz, 1H); 13C NMR (200 MHz, D2O)  176.06,
173.83, 173.38, 102.66, 100.10, 95.74, 91.79, 78.36, 78.04,
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 10 of 19
0.4H), 4.49 (d, J = 8.0 Hz, 0.6H), 4.18–3.25 (m, 32H), 2.75
HPLC over a XBridge BEH Amide Column (130Å, 5 m, 4.6
mm × 250 mm). Mobile phase A: 100 mM ammonium for-
mate, pH 3.46; Mobile phase B: acetonitrile; Gradient: 65% to
50% B over 25 minutes, 50% to 0% B over 1 minute, 0% B
for 2 minutes, 0% to 65% B over 2 minutes, 65% B for 5 mi-
nutes. HPLC purification was monitored by absorption at 210
nm, and glycan-containing fractions were analyzed by TLC
and MS. The fractions containing pure product were collected
and lyophilized to obtain the desired GM2 and GD2 glycans
(yields 90–99%).
1
2
3
4
5
6
7
8
(dd, J = 12.0 and 4.8 Hz, 1H), 2.66 (dd, J = 12.0 and 4.8 Hz,
1H), 2.05 (s, 3H), 2.03 (s, 3H), 2.02 (s, 3H), 1.76 (t, J = 12.0
Hz, 1H), 1.72 (t, J = 12.0 Hz, 1H); 13C NMR (200 MHz, D2O)
 174.95, 174.90, 174.82, 173.35, 173.28, 102.68, 100.50,
95.75, 95.73, 91.79, 91.76, 78.27, 78.23, 78.15, 75.87, 74.79,
74.48, 74.42, 74.21, 73.73, 73.68, 72.61, 71.73, 71.26, 71.08,
70.81, 70.09, 69.65, 69.27, 69.23, 68.44, 68.07, 67.70, 67.66,
62.52, 61.45, 60.91, 60.61, 59.97, 59.82, 59.30, 52.44, 52.33,
51.72, 40.41, 39.15, 22.53, 22.33, 22.03. HRMS (ESI) m/z
calcd for C42H68N3O32 (M-H) 1126.3786, found 1126.3770.
9
Neu5Ac2–3(GalNAc1–4)Lac (14). 211 mg, yield 99%;
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
1
Neu5Gc2–8Neu5Ac2–3(GalNAc1–4)Lac (19). 32 mg,
yield 97%; white solid. 1H NMR (800 MHz, D2O)  5.20 (d, J
= 4.0 Hz, 0.4H), 4.69 (d, J = 8.0 Hz, 0.4H), 4.68 (d, J = 8.0
Hz, 0.6H), 4.65 (d, J = 8.0 Hz, 0.6H), 4.50 (d, J = 8.0 Hz,
0.4H), 4.49 (d, J = 8.0 Hz, 0.6H), 4.19–4.12 (m, 3H), 4.10 (s,
2H), 4.03–3.24 (m, 29H), 2.76 (dd, J = 12.0 and 4.8 Hz, 1H),
2.66 (dd, J = 12.0 and 4.8 Hz, 1H), 2.05 (s, 3H), 2.04 (s, 3H),
1.76 (t, J = 12.0 Hz, 1H), 1.74 (t, J = 12.0 Hz, 1H); 13C NMR
(200 MHz, D2O)  175.62, 174.83, 174.75, 173.31, 173.22,
102.62, 102.60, 100.43, 95.66, 93.47, 93.44, 78.19, 78.16,
78.08, 75.82, 75.80, 74.71, 74.40, 74.34, 74.14, 73.65, 73.61,
72.25, 71.70, 71.57, 71.19, 71.01, 70.74, 70.02, 69.59, 69.16,
68.33, 68.12, 68.01, 67.92, 67.60, 67.29, 62.40, 61.37, 61.07,
60.86, 60.83, 60.53, 59.90, 52.36, 52.25, 51.33, 49.85, 49.81,
40.40, 22.44, 22.24, 21.94. HRMS (ESI) m/z calcd for
C42H68N3O33 (M-H) 1142.3735, found 1142.3749.
white solid. H NMR (800 MHz, D2O)  5.21 (d, J = 4.0 Hz,
0.4H), 4.73 (d, J = 8.0 Hz, 1H), 4.65 (d, J = 8.0 Hz, 0.6H),
4.52 (d, J = 8.0 Hz, 1H), 4.16–3.25 (m, 25H), 2.65 (dd, J =
12.0 and 4.8 Hz, 1H), 2.02 (s, 3H), 2.01 (s, 3H), 1.91 (t, J =
12.0 Hz, 1H); 13C NMR (200 MHz, D2O)  174.89, 174.71,
173.97, 102.63, 102.45, 102.41, 101.50, 95.63, 91.68, 78.45,
78.37, 77.05, 74.65, 74.59, 74.23, 74.20, 73.89, 73.61, 72.94,
72.16, 71.27, 71.14, 70.96, 69.94, 69.90, 68.58, 67.87, 67.65,
62.70, 61.04, 60.45, 59.98, 59.84, 52.21, 51.47, 36.82, 22.49,
21.94. HRMS (ESI) m/z calcd for C31H51N2O24 (M-H)
835.2832, found 835.2821. NMR data were consistent with
those reported in the literature.82
Neu5Gc2–3(GalNAc1–4)Lac (15). 114 mg, yield 99%;
1
white solid. H NMR (800 MHz, D2O)  5.21 (d, J = 4.0 Hz,
0.5H), 4.74 (d, J = 8.0 Hz, 1H), 4.65 (d, J = 8.0 Hz, 0.5H),
4.52 (d, J = 8.0 Hz, 1H), 4.15 (m, 1H), 4.11 (s, 2H), 3.96–3.25
(m, 25H), 2.67 (dd, J = 12.0 and 4.8 Hz, 1H), 2.01 (s, 3H),
1.93 (t, J = 12.0 Hz, 1H); 13C NMR (200 MHz, D2O)  175.64,
174.71, 173.99, 102.63, 102.45, 102.41, 101.51, 95.63, 78.45,
78.37, 77.03, 74.65, 74.59, 74.23, 74.21, 74.20, 73.90, 73.61,
72.66, 72.23, 71.27, 71.14, 70.96, 69.95, 69.90, 68.33, 67.79,
67.65, 62.66, 61.04, 60.87, 60.45, 59.98, 52.22, 51.18, 36.89,
22.50. HRMS (ESI) m/z calcd for C31H51N2O25 (M-H)
851.2781, found 851.2770.
Kdn2–8Neu5Ac2–3(GalNAc1–4)Lac (20). 16 mg,
yield 96%; white solid. 1H NMR (800 MHz, D2O)  5.20 (d, J
= 4.0 Hz, 0.4H), 4.68 (d, J = 8.0 Hz, 0.4H), 4.67 (d, J = 8.0
Hz, 0.6H), 4.64 (d, J = 8.0 Hz, 0.6H), 4.48 (d, J = 8.0 Hz,
0.4H), 4.47 (d, J = 8.0 Hz, 0.6H), 4.17–4.01 (m, 4H), 3.96–
3.24 (m, 28H), 2.70–2.65 (m, 2H), 2.05 (s, 3H), 2.03 (s, 3H),
1.75 (t, J = 12.0 Hz, 1H), 1.68 (t, J = 12.0 Hz, 1H); 13C NMR
(200 MHz, D2O)  174.89, 174.81, 173.57, 173.29, 160.99,
102.67, 100.49, 100.48, 95.74, 95.72, 78.19, 78.13, 75.84,
74.77, 74.47, 74.40, 74.19, 73.67, 73.60, 71.99, 71.06, 70.79,
70.35, 69.76, 69.63, 69.27, 69.22, 68.07, 67.67, 62.42, 61.42,
60.90, 60.58, 52.31, 39.98, 39.94, 34.17, 22.51, 22.31. HRMS
(ESI) m/z calcd for C40H65N2O32 (M-H) 1085.3520, found
1085.3508.
Kdn2–3(GalNAc1–4)Lac (16). 27 mg, yield 99%; white
1
solid. H NMR (800 MHz, D2O)  5.19 (d, J = 4.0 Hz, 0.4H),
4.72 (d, J = 8.0 Hz, 1H), 4.63 (d, J = 8.0 Hz, 0.6H), 4.49 (d, J
= 8.0 Hz, 1H), 4.01–3.23 (m, 25H), 2.59 (dd, J = 12.0 and 4.8
Hz, 1H), 1.99 (s, 3H), 1.85 (t, J = 12.0 Hz, 1H); 13C NMR
(200 MHz, D2O)  174.70, 174.12, 170.54, 102.60, 102.43,
102.39, 101.50, 95.62, 91.67, 78.38, 78.30, 76.95, 74.63,
74.58, 74.21, 74.14, 73.91, 73.85, 73.60, 72.41, 71.25, 71.09,
70.96, 70.45, 69.93, 69.87, 69.51, 67.64, 67.44, 62.76, 61.05,
60.38, 59.95, 52.20, 36.39, 22.47. HRMS (ESI) m/z calcd for
C29H48NO24 (M-H) 794.2566, found 794.2571.
Neu5Ac2–8Neu5Gc2–3(GalNAc1–4)Lac (21). 51 mg,
yield 94%; white solid. 1H NMR (800 MHz, D2O)  5.19 (d, J
= 4.0 Hz, 0.4H), 4.68 (d, J = 8.0 Hz, 0.4H), 4.67 (d, J = 8.0
Hz, 0.6H), 4.64 (d, J = 8.0 Hz, 0.6H), 4.48 (d, J = 8.0 Hz,
0.4H), 4.47 (d, J = 8.0 Hz, 0.6H), 4.18 (d, J = 16.8 Hz, 1H),
4.17–4.09 (m, 3H), 4.08 (d, J = 16.8 Hz, 1H), 4.03–3.24 (m,
29H), 2.73 (dd, J = 12.0 and 4.8 Hz, 1H), 2.68 (dd, J = 12.0
and 4.8 Hz, 1H), 2.02 (s, 3H), 2.00 (s, 3H), 1.77 (t, J = 12.0
Hz, 1H), 1.70 (t, J = 12.0 Hz, 1H); 13C NMR (200 MHz, D2O)
 177.01, 175.95, 174.87, 174.84, 174.73, 174.58, 173.58,
173.19, 172.49, 102.63, 102.61, 101.89, 100.44, 100.42,
100.05, 96.46, 95.65, 91.68, 78.40, 78.15, 78.06, 75.85, 75.82,
75.05, 74.70, 74.41, 74.32, 74.14, 74.13, 73.63, 73.33, 72.68,
72.48, 71.98, 71.64, 71.17, 70.98, 70.72, 70.12, 69.99, 69.57,
69.46, 68.88, 68.29, 68.28, 68.00, 67.84, 67.58, 67.52, 67.44,
66.81, 62.43, 61.27, 61.00, 60.81, 60.69, 60.50, 60.11, 59.88,
59.73, 52.34, 52.20, 52.06, 51.61, 51.53, 40.89, 40.46, 39.11,
39.02, 22.43, 22.04, 21.92, 21.91. HRMS (ESI) m/z calcd for
C42H68N3O33 (M-H) 1142.3735, found 1142.3755.
Neu5Ac8OMe2–3(GalNAc1–4)Lac (17). 6 mg, yield
95%; white solid. 1H NMR (800 MHz, D2O)  5.20 (d, J = 4.0
Hz, 1H), 4.65 (d, J = 8.0 Hz, 1H), 4.47 (d, J = 8.0 Hz, 1H),
4.15–3.24 (m, 25H), 3.47 (s, 3H), 2.62 (dd, J = 12.0 and 4.8
Hz, 1H), 2.03 (s, 3H), 2.01 (s, 3H), 1.79 (t, J = 12.0 Hz, 1H);
13C NMR (200 MHz, D2O)  174.84, 174.75, 173.43, 102.61,
102.55, 100.51, 95.63, 80.33, 78.23, 76.23, 74.73, 74.69,
74.43, 74.16, 74.13, 73.61, 72.43, 70.99, 69.74, 68.19, 67.59,
66.93, 60.87, 60.51, 59.34, 57.57, 52.37, 51.96, 38.76, 22.42,
21.96. HRMS (ESI) m/z calcd for C32H53N2O24 (M-H)
849.2988, found 849.2975.
Neu5Ac2–8Neu5Ac2–3(GalNAc1–4)Lac (18). 150 mg,
yield 99%; white solid. 1H NMR (800 MHz, D2O)  5.21 (d, J
= 4.0 Hz, 0.4H), 4.69 (d, J = 8.0 Hz, 0.4H), 4.68 (d, J = 8.0
Hz, 0.6H), 4.65 (d, J = 8.0 Hz, 0.6H), 4.50 (d, J = 8.0 Hz,
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The Journal of Organic Chemistry
General procedures for OPME synthesis of GM1 (4
Neu5Gc2–8Neu5Gc2–3(GalNAc1–4)Lac (22). 36 mg,
yield 96%; white solid. 1H NMR (800 MHz, D2O)  5.19 (d, J
= 4.0 Hz, 0.4H), 4.69 (d, J = 8.0 Hz, 0.4H), 4.68 (d, J = 8.0
Hz, 0.6H), 4.64 (d, J = 8.0 Hz, 0.6H), 4.49 (d, J = 8.0 Hz,
0.4H), 4.48 (d, J = 8.0 Hz, 0.6H), 4.18 (d, J = 16.8 Hz, 1H),
4.17–4.14 (m, 2H), 4.11 (m, 1H), 4.09 (s, 2H), 4.08 (d, J =
16.8 Hz, 1H), 4.03–3.24 (m, 29H), 2.75 (dd, J = 12.0 and 4.8
Hz, 1H), 2.68 (dd, J = 12.0 and 4.8 Hz, 1H), 2.02 (s, 3H), 1.77
(t, J = 12.0 Hz, 1H), 1.72 (t, J = 12.0 Hz, 1H); 13C NMR (200
MHz, D2O)  175.96, 175.61, 174.74, 173.62, 173.19, 102.63,
102.61, 100.45, 100.06, 95.65, 91.69, 78.40, 78.15, 78.07,
75.86, 74.70, 74.42, 74.32, 74.13, 73.63, 73.34, 72.20, 71.70,
70.99, 70.73, 70.00, 69.58, 69.46, 68.87, 68.03, 67.93, 67.59,
67.53, 62.40, 61.28, 61.01, 60.84, 60.81, 60.51, 59.89, 52.35,
52.07, 51.32, 40.53, 22.43. HRMS (ESI) m/z calcd for
C42H68N3O34 (M-H) 1158.3684, found 1158.3690.
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compounds, 26–29) and GD1 glycans (5 compounds, 30–
34). A GM2 or GD2 glycan (10 mM, 1 eq.) as an acceptor and
Gal (1.1 eq.) were incubated at 37 C in Tris-HCl buffer (100
mM, pH 7.5) containing ATP (1.2 eq.), UTP (1.2 eq.), MgCl2
(10 mM), EcGalK (3 mg/mL), BLUSP (3 mg/mL), CjCgtB
(2.5 mg/mL), and PmPpA (2 mg/mL). The reaction was car-
ried out by incubating the solution in an incubator shaker at 37
C for overnight with agitation at 100 rpm. The product for-
mation was monitored by LC-MS. When an optimal yield was
achieved, alkaline phosphatase (10–20 mg) was added to the
reaction mixture and the mixture was incubated in an incuba-
tor shaker at 37 C for overnight with agitation at 100 rpm.
The reaction was then quenched by adding the same volume of
ice-cold ethanol and incubated at 4 C for 30 min. The mixture
was then centrifuged and the precipitates were removed. The
supernatant was concentrated, passed through a BioGel P-2
gel filtration column, and eluted with water to obtain sialoside
mixtures. The fractions containing the product were collected,
concentrated, and further purified by HPLC with a XBridge
BEH Amide Column (130Å, 5 μm, 4.6 mm X 250 mm). Mo-
bile phase A: 100 mM ammonium formate, pH 3.46; Mobile
phase B: acetonitrile; Gradient: 65% to 50% B over 25 mi-
nutes, 50% to 0% B over 1 minute, 0% B for 2 minutes, 0% to
65% B over 2 minutes, 65% B for 5 minutes. HPLC purifica-
tion was monitored by absorption at 210 nm, and glycan-
containing fractions were analyzed by TLC and MS. The frac-
tions containing the pure product were collected and lyophi-
lized to produce the desired GM1 and GD1b glycans (yields
80–90%).
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Kdn2–8Neu5Gc2–3(GalNAc1–4)Lac (23). 46 mg,
yield 94%; white solid. 1H NMR (800 MHz, D2O)  5.19 (d, J
= 4.0 Hz, 0.4H), 4.68 (d, J = 8.0 Hz, 0.4H), 4.67 (d, J = 8.0
Hz, 0.6H), 4.64 (d, J = 8.0 Hz, 0.6H), 4.48 (d, J = 8.0 Hz,
0.4H), 4.47 (d, J = 8.0 Hz, 0.6H), 4.17 (d, J = 16.8 Hz, 1H),
4.16–4.13 (m, 2H), 4.08 (d, J = 16.8 Hz, 1H), 4.07 (m, 1H),
4.01 (m, 1H), 3.95–3.23 (m, 28H), 2.70–2.66 (m, 2H), 2.02 (s,
3H), 1.76 (t, J = 12.0 Hz, 1H), 1.66 (t, J = 12.0 Hz, 1H); 13C
NMR (200 MHz, D2O)  175.97, 174.73, 173.79, 173.18,
102.62, 100.42, 100.41, 100.05, 95.65, 78.35, 78.15, 78.07,
75.81, 74.70, 74.42, 74.32, 74.14, 74.12, 73.63, 73.50, 73.35,
71.94, 70.99, 70.72, 70.19, 69.68, 69.56, 69.46, 68.88, 67.64,
67.58, 67.52, 62.50, 61.27, 61.00, 60.83, 60.50, 52.35, 52.06,
48.58, 40.07, 25.99, 22.44. HRMS (ESI) m/z calcd for
C40H65N2O33 (M-H) 1101.3470, found 1101.3478.
Neu5Ac2–3(Gal1–3GalNAc1–4)Lac (26). 140 mg,
yield 90%; white solid. 1H NMR (800 MHz, D2O)  5.21 (d, J
= 4.0 Hz, 0.4H), 4.76 (d, J = 8.0 Hz, 1H), 4.65 (d, J = 8.0 Hz,
0.6H), 4.52 (d, J = 8.0 Hz, 1H), 4.51 (d, J = 8.0 Hz, 1H), 4.10
(s, 2H),4.15–3.24 (m, 31H), 2.66 (dd, J = 12.0 and 4.8 Hz,
1H), 2.00 (s, 3H), 1.92 (t, J = 12.0 Hz, 1H); 13C NMR (200
MHz, D2O)  174.97, 174.72, 174.07, 104.68, 102.51, 102.47,
101.58, 95.72, 91.77, 80.28, 78.51, 78.44, 77.10, 74.84, 74.72,
74.38, 74.31, 74.28, 74.03, 73.69, 73.02, 72.45, 72.23, 71.36,
71.04, 70.64, 70.02, 69.97, 68.65, 68.56, 68.53, 67.96, 67.85,
62.78, 61.07, 60.90, 60.60, 60.07, 59.92, 51.57, 51.14, 36.91,
22.56, 22.05.HRMS (ESI) m/z calcd for C37H61N2O29 (M-H)
997.3360, found 997.3349.
Neu5Gc2–8Kdn2–3(GalNAc1–4)Lac (24). 12 mg,
yield 90%; white solid. 1H NMR (800 MHz, D2O)  5.19 (d, J
= 3.2 Hz, 0.4H), 4.69 (d, J = 8.0 Hz, 0.4H), 4.68 (d, J = 8.0
Hz, 0.6H), 4.64 (d, J = 8.0 Hz, 0.6H), 4.49 (d, J = 8.0 Hz,
0.4H), 4.48 (d, J = 8.0 Hz, 0.6H), 4.28–3.24 (m, 34H), 2.75
(dd, J = 12.0 and 4.8 Hz, 1H), 2.68 (dd, J = 12.0 and 4.8 Hz,
1H), 2.03 (s, 3H), 1.74–1.69 (m, 2H); 13C NMR (200 MHz,
D2O)  175.57, 175.66, 174.44, 173.60, 103.61, 102.49,
102.43, 95.67, 91.72, 79.92, 78.29, 78.16, 77.81, 77.71, 75.73,
75.64, 75.17, 75.08, 74.75, 74.72, 74.59, 74.17, 73.71, 73.24,
72.23, 71.69, 71.31, 71.23, 71.06, 70.52, 70.10, 70.03, 69.37,
69.17, 69.11, 68.69, 68.02, 67.76, 67.64, 67.44, 62.45, 61.51,
61.07, 61.00, 60.84, 59.92, 59.83, 52.41, 51.96, 51.31, 51.22,
40.49, 38.96, 22.30. HRMS (ESI) m/z calcd for C40H65N2O33
(M-H) 1101.3470, found 1101.3455.
Neu5Gc2–3(Gal1–3GalNAc1–4)Lac (27). 51 mg,
yield 86%; white solid. 1H NMR (800 MHz, D2O)  5.20 (d, J
= 4.0 Hz, 0.4H), 4.76 (d, J = 8.0 Hz, 1H),4.65 (d, J = 8.0 Hz,
0.6H), 4.52 (d, J = 8.0 Hz, 1H), 4.51 (d, J = 8.0 Hz, 1H), 4.51
(d, J = 8.0 Hz, 0.6H), 4.16–3.25 (m, 31H), 2.65 (dd, J = 12.0
and 4.8 Hz, 1H), 2.02 (s, 3H), 1.99 (s, 3H), 1.92 (t, J = 12.0
Hz, 1H); 13C NMR (200 MHz, D2O)  175.71, 174.72, 174.09,
104.67, 102.49, 102.45, 102.41, 101.58, 95.71, 91.76, 80.28,
78.50, 78.42, 77.06, 74.83, 74.71, 74.29, 74.03, 74.02, 73.69,
73.66, 72.73, 72.43, 72.28, 71.02, 70.62, 69.97, 69.95, 68.56,
68.49, 68.41, 68.37, 67.85, 67.82, 62.73, 62.69, 61.05, 60.94,
60.89, 60.87, 60.58, 60.05, 59.91, 51.28, 51.24, 51.14, 36.98,
36.93, 22.56. HRMS (ESI) m/z calcd for C37H61N2O30 (M-H)
1013.3309, found 1013.3318.
Kdn2–8Kdn2–3(GalNAc1–4)Lac (25). 8 mg, yield
95%; white solid. 1H NMR (800 MHz, D2O)  5.20 (d, J = 4.0
Hz, 0.4H), 4.69 (d, J = 8.0 Hz, 0.4H), 4.68 (d, J = 8.0 Hz,
0.6H), 4.64 (d, J = 8.0 Hz, 0.6H), 4.47 (d, J = 8.0 Hz, 1H),
4.18–3.24 (m, 32H), 2.67 (dd, J = 12.0 and 4.8 Hz, 1H), 2.61
(dd, J = 12.0 and 4.8 Hz, 1H), 2.01 (s, 3H), 1.75 (t, J = 12.0
Hz, 1H), 1.72 (t, J = 12.0 Hz, 1H); 13C NMR (200 MHz, D2O)
 174.73, 173.96, 173.41, 135.18, 102.59, 100.68, 100.49,
95.64, 91.68, 78.10, 77.78, 75.96, 74.72, 74.69, 74.36, 74.32,
74.13, 73.62, 73.57, 71.86, 71.18, 70.98, 70.76, 70.50, 70.32,
70.24, 69.99, 69.67, 69.59, 69.46, 69.37, 67.70, 67.58, 63.44,
62.50, 61.21, 60.80, 60.55, 59.91, 52.34, 42.51, 39.15, 38.42,
34.11, 22.42. HRMS (ESI) m/z calcd for C38H62NO32 (M-H)
1044.3255, found 1044.3186.
Kdn2–3(Gal1–3GalNAc1–4)Lac (28). 5 mg, yield
87%; white solid. 1H NMR (800 MHz, D2O)  5.20 (d, J = 4.0
Hz, 0.4H), 4.76 (d, J = 8.0 Hz, 1H), 4.64 (d, J = 8.0 Hz, 0.6H),
4.52 (d, J = 8.0 Hz, 1H), 4.50 (d, J = 8.0 Hz, 1H), 4.17–3.23
(m, 31H), 2.59 (dd, J = 12.0 and 4.8 Hz, 1H), 1.98 (s, 3H),
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Page 12 of 19
1.86 (t, J = 12.0 Hz, 1H); 13C NMR (200 MHz, D2O)  174.73,
174.15, 104.61, 104.15, 102.42, 102.36, 95.62, 78.31, 76.93,
74.90, 74.74, 74.63, 74.22, 74.15, 73.93, 73.59, 72.40, 72.35,
71.25, 70.95, 70.90, 70.54, 70.46, 69.92, 69.87, 69.52, 68.44,
67.77, 67.45, 62.75, 60.99, 60.79, 60.44, 59.96, 36.37, 22.45.
HRMS (ESI) m/z calcd for C35H58NO29 (M-H) 956.3094,
found 956.3088.
Hz, 0.6H), 4.51 (d, J = 8.0 Hz, 1H), 4.49 (d, J = 8.0 Hz, 1H),
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4.20–3.24 (m, 40H), 2.68 (dd, J = 12.0 and 4.8 Hz, 2H), 2.01
(s, 3H), 1.78 (t, J = 12.0 Hz, 1H), 1.67 (t, J = 12.0 Hz, 1H);
13C NMR (200 MHz, D2O)  175.99, 174.71, 173.80, 173.26,
104.53, 102.61, 102.31, 95.65, 79.72, 78.34, 78.10, 74.79,
74.70, 74.42, 74.14, 73.98, 73.63, 73.49, 73.35, 72.33, 71.96,
71.19, 70.54, 70.19, 70.00, 69.69, 69.60, 68.85, 68.46, 67.66,
67.55, 62.52, 61.24, 61.00, 60.81, 60.76, 52.07, 40.08, 22.41.
HRMS (ESI) m/z calcd for C46H75N2O38 (M-H) 1263.3998,
found 1263.4009.
Neu5Ac8OMe2–3(Gal1–3GalNAc1–4)Lac (29). 2 mg,
yield 80%; white solid. 1H NMR (800 MHz, D2O)  5.20 (d, J
= 4.0 Hz, 1H), 4.65 (d, J = 8.0 Hz, 1H), 4.47 (d, J = 8.0 Hz,
1H), ), 4.42 (d, J = 8.0 Hz, 2H), 4.20–3.24 (m, 31H), 3.41 (s,
3H), 2.54 (dd, J = 12.0 and 4.8 Hz, 1H), 1.95 (s, 3H), 1.94 (s,
3H), 1.74 (t, J = 12.0 Hz, 1H); HRMS (ESI) m/z calcd for
C38H63N2O29 (M-H) 1011.3516, found 1011.3521.
9
Kdn2–8Kdn2–3(Gal1–3GalNAc1–4)Lac (34). 3 mg,
yield 83%; white solid. 1H NMR (800 MHz, D2O)  5.19 (d, J
= 4.0 Hz, 0.4H), 4.74 (d, J = 8.0 Hz, 1H), 4.65 (d, J = 8.0 Hz,
0.6H), 4.51 (d, J = 8.0 Hz, 1H), 4.49 (d, J = 8.0 Hz, 1H), 4.18–
3.24 (m, 38H), 2.65 (dd, J = 12.0 and 4.8 Hz, 2H), 2.01 (s,
3H), 1.75 (t, J = 12.0 Hz, 1H), 1.74 (t, J = 12.0 Hz, 1H). 13C
NMR (200 MHz, D2O) δ 174.70, 174.69, 173.98, 173.48,
104.56, 102.59, 102.30, 100.69, 95.65, 91.69, 79.78, 78.22,
78.15, 77.76, 76.06, 74.78, 74.73, 74.70, 74.36, 74.16, 73.99,
73.62, 73.57, 72.35, 71.87, 71.20, 70.98, 70.55, 70.52, 70.34,
69.99, 69.69, 69.63, 69.39, 68.47, 67.73, 62.53, 61.20, 60.81,
60.75, 60.54, 59.93, 51.20, 39.16, 38.35, 22.42. HRMS (ESI)
m/z calcd for C44H72NO37 (M-H) 1206.3783, found 1206.3774.
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Neu5Ac2–8Neu5Ac2–3(Gal1–3GalNAc1–4)Lac (30).
1
12 mg, yield 88%; white solid. H NMR (800 MHz, D2O) 
5.19 (d, J = 4.0 Hz, 0.4H), 4.73 (d, J = 8.0 Hz, 0.4H), 4.48 (d,
J = 8.0 Hz, 0.6H), 4.64 (d, J = 8.0 Hz, 0.6H), 4.50 (d, J = 8.0
Hz, 1H), 4.49 (d, J = 8.0 Hz, 0.4H), 4.49 (d, J = 8.0 Hz, 0.6H),
4.17–3.24 (m, 38H), 2.74 (dd, J = 12.0 and 4.8 Hz, 1H), 2.66
(dd, J = 12.0 and 4.8 Hz, 1H), 2.05 (s, 3H), 2.01 (s, 3H), 2.00
(s, 3H), 1.77 (t, J = 12.0 Hz, 1H), 1.71 (t, J = 12.0 Hz, 1H);
13C NMR (200 MHz, D2O)  174.86, 174.81, 174.71, 173.29,
173.28, 104.52, 102.58, 102.29, 95.65, 79.70, 78.17, 78.05,
75.85, 74.80, 74.70, 74.38, 74.14, 73.98, 73.59, 72.52, 72.33,
71.65, 71.20, 71.00, 70.54, 69.60, 69.14, 69.11, 68.47, 68.44,
68.36, 68.01, 67.98, 67.68, 62.43, 61.32, 60.81, 60.76, 60.55,
59.87, 52.23, 51.62, 51.21, 40.32, 22.41, 22.22, 21.91. HRMS
(ESI) m/z calcd for C48H78N3O37 (M-H) 1288.4314, found
1288.4320.
One-pot four-enzyme preparative-scale synthesis of
GlcNAc1–3Lac (35). Lactose (0.90 g, 2.63 mmol, 40.5
mM), GlcNAc (0.756 g, 3.42 mmol), ATP (1.88 g, 3.42
mmol), and UTP (1.99 g, 3.42 mmol) were dissolved in Tris-
HCl buffer (65 mL, pH 8.0) containing MgCl2 (20 mM).
BLNahK (19.0 mg), PmGlmU (8.0 mg), NmLgtA (6.0 mg),
and PmPpA (4–5 mg) were added. The reactions were carried
out by incubating the reaction mixture in an incubator shaker
at 37 C for 48 h. The product formation was monitored by
TLC (EtOAc:MeOH:H2O:HOAc = 4:2:1:0.2 and detected by
p-anisaldehyde sugar stain) and mass spectrometry (MS).
Upon completion, to the reaction was added the same volume
(65 mL) of ethanol and the mixture was incubated at 4 C for
30 min. After centrifugation, the supernatant was concentrated
and passed through a Bio Gel P-2 gel filtration column (water
was used as an eluant). The fractions containing the product
were collected, concentrated, and further purified by silica gel
column (EtOAc:MeOH:H2O, 5:2:1) to obtain trisaccharide
Neu5Gc2–8Neu5Ac2–3(Gal1–3GalNAc1–4)Lac
1
(31). 8 mg, yield 85%; white solid. H NMR (800 MHz, D2O)
 5.19 (d, J = 4.0 Hz, 0.4H), 4.74 (d, J = 8.0 Hz, 1H), 4.64 (d,
J = 8.0 Hz, 0.6H), 4.49 (d, J = 8.0 Hz, 1H), 4.48 (d, J = 8.0
Hz, 1H), 4.16–3.25 (m, 40H), 2.76 (dd, J = 12.0 and 4.8 Hz,
1H), 2.66 (dd, J = 12.0 and 4.8 Hz, 1H), 2.05 (s, 3H), 2.01 (s,
3H), 1.76 (t, J = 12.0 Hz, 1H), 1.73 (t, J = 12.0 Hz, 1H); 13C
NMR (200 MHz, D2O)  175.61, 174.81, 174.71, 173.29,
173.28, 104.53, 102.58, 102.29, 95.65, 79.71, 78.15, 78.06,
75.85, 74.80, 74.70, 74.39, 74.15, 73.99, 73.60, 72.34, 72.24,
71.71, 71.19, 71.00, 70.54, 69.61, 69.12, 68.46, 68.10, 68.02,
67.91, 67.69, 62.40, 61.32, 60.84, 60.56, 52.23, 51.33, 51.22,
40.38, 22.41, 22.22. HRMS (ESI) m/z calcd for C48H78N3O38
(M-H) 1304.4263, found 1304.4241.
1
GlcNAc1–3Lac (35) (1.35 g, 94%). H NMR (800 MHz,
D2O)  5.19 (d, J = 4.0 Hz, 0.4H), 4.66 (d, J = 8.0 Hz, 0.4H),
4.65 (d, J = 8.0 Hz, 0.6H), 4.64 (d, J = 8.0 Hz, 0.6H), 4.41 (d,
J = 8.0 Hz, 1H), 4.12 (d, J = 3.2 Hz, 1H), 3.93–3.24 (m, 17H),
2.01 (s, 3H). 13C NMR (200 MHz, D2O) -isomer:  174.87,
102.84, 102.75, 95.66, 81.87, 78.21, 75.57, 74.80, 74.71,
74.20, 73.71, 73.49, 70.03, 69.92, 68.26, 60.88, 60.41, 60.01,
56.58, 22.09. HRMS (ESI) m/z calculated for C20H36NO16
(M+H) 546.2034, found 546.2050. NMR data were consistent
with those reported in the literature.64
Neu5Gc2–8Neu5Gc2–3(Gal1–3GalNAc1–4)Lac
1
(32). 6 mg, yield 86%; white solid. H NMR (800 MHz, D2O)
 5.20 (d, J = 4.0 Hz, 0.4H), 4.74 (d, J = 8.0 Hz, 1H), 4.64 (d,
J = 8.0 Hz, 0.6H), 4.51 (d, J = 8.0 Hz, 1H), 4.49 (d, J = 8.0
Hz, 1H), 4.21–3.24 (m, 42H), 2.76 (dd, J = 12.0 and 4.8 Hz,
1H), 2.68 (dd, J = 12.0 and 4.8 Hz, 1H), 2.03 (s, 3H), 1.78 (t, J
= 12.0 Hz, 1H), 1.71 (t, J = 12.0 Hz, 1H); 13C NMR (200
MHz, D2O)  175.97, 175.63, 174.71, 173.63, 173.27, 104.53,
104.15, 102.60, 102.31, 95.65, 79.71, 78.37, 78.08, 75.92,
74.80, 74.70, 74.42, 74.15, 73.99, 73.64, 73.34, 72.34, 72.21,
71.73, 71.20, 71.00, 70.54, 70.00, 69.61, 68.87, 68.46, 68.02,
67.95, 67.69, 67.55, 62.42, 61.26, 61.01, 60.84, 60.81, 60.76,
60.55, 52.07, 51.33, 51.22, 40.52, 22.42. HRMS (ESI) m/z
calcd for C48H78N3O39 (M-H) 1320.4212, found 1320.4232.
One-pot four-enzyme preparative-scale synthesis of
Gal1-4GlcNAc1–3Lac (36). Trisaccharide GlcNAc1–
3Lac (1.0 g, 1.83 mmol, 22.9 mM), galactose (0.43 g, 2.38
mmol), ATP (1.40 g, 2.38 mmol), and UTP (1.58 g, 2.38
mmol) were dissolved in Tris-HCl buffer (80 mL, 100 mM,
pH 8.0) containing MgCl2 (20 mM), EcGalK (20.0 mg),
BLUSP (20 mg), NmLgtB (15 mg), and PpA (20 mg). The
reactions were carried out by incubating the reaction mixture
in an incubator shaker at 37 C for 30 h. The product forma-
tion was monitored by TLC (n-PrOH:H2O:NH4OH = 5:2:1 and
detected by p-anisaldehyde sugar stain) and mass spectrometry
Kdn2–8Neu5Gc2–3(Gal1–3GalNAc1–4)Lac (33). 3
1
mg, yield 85%; white solid. H NMR (800 MHz, D2O)  5.20
(d, J = 4.0 Hz, 0.4H), 4.74 (d, J = 8.0 Hz, 1H), 4.65 (d, J = 8.0
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The Journal of Organic Chemistry
(MS). When an optimal yield was achieved, to the reaction
and the mixture was incubated in an incubator shaker at 37 C
1
2
3
4
5
6
7
8
mixture was added the same volume (80 mL) of ethanol and
the mixture was incubated at 4 C for 30 min. The precipitates
were removed by centrifugation and the supernatant was con-
centrated and purified by a Bio Gel P-2 gel column (water as
eluent). Further purification was achieved by silica gel chro-
matography (EtOAc:MeOH:H2O = 5:3:1.5, by volume) to
for overnight with agitation at 100 rpm. The reaction was then
quenched by adding the same volume of ice-cold ethanol and
the mixture was incubated at 4 C for 30 min. The precipitates
were removed by centrifugation and the supernatant was con-
centrated, passed through a BioGel P-2 gel filtration column,
and eluted with water to obtain sialoside mixtures. The frac-
tions containing the product were collected, concentrated, and
further purified by HPLC using a reverse-phase C18 column
(10 m, 21.2 × 250 mm) with a flow rate of 10 mL/min using
a gradient elution of 0–100% acetonitrile in water containing
0.05% formic acid over 20 minutes. Mobile phase A: 0.05%
formic acid in water (v/v); Mobile phase B: acetonitrile (v/v);
Gradient: 0% B for 3 minutes, 0% to 100% B over 12 minutes,
100% B for 2 minutes, then 100% to 0% B over 3 minutes.
HPLC purification was monitored by absorption at 210 nm,
and glycan-containing fractions were analyzed by TLC and
MS. The fractions containing the pure product were collected
and concentrated to obtain the desired sialylated lacto- and
neolacto-series glycosphingolipid glycans (yields 80–94%).
1
obtain Gal1–4GlcNAc1–3Lac (36) (1.28 g, 99%). H NMR
(800 MHz, D2O)  5.17 (d, J = 4.0 Hz, 0.4H), 4.66 (d, J = 8.0
Hz, 0.4H), 4.65 (d, J = 8.0 Hz, 0.6H), 4.61 (d, J = 8.0 Hz,
0.6H), 4.43 (d, J = 7.2 Hz, 1H), 4.38 (d, J = 8.0 Hz, 1H), 4.11
(d, J = 3.2 Hz, 1H), 3.91–3.87 (m, 2H), 3.84–3.22 (m, 21H),
1.98 (s, 3H). 13C NMR (200 MHz, D2O) -isomer:  174.83,
102.79, 102.76, 102.73, 95.61, 81.82, 78.21, 78.11, 75.52
(2C), 74.76, 74.66, 74.22, 73.65, 73.42 (2C), 70.99, 69.88,
68.24, 68.22, 60.85, 60.84, 60.34, 59.93, 56.52, 22.03. HRMS
(ESI) m/z calculated for C26H46NO21 (M+H) 708.2562, found
708.2586. NMR data were consistent with those reported in
the literature.64
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
One-pot three-enzyme preparative-scale synthesis of
Gal1–4(Fuc1–3)GlcNAc1–3Lac (46). LNnT (160 mg,
0.23 mmol, 23 mM), L-fucose (74 mg, 0.45 mmol), ATP (250
mg, 0.45 mmol), and GTP (240 mg, 0.45 mmol) were dis-
solved in Tris-HCl buffer (10 mL, 100 mM, pH 7.5) contain-
ing MgCl2 (20 mM), FKP (3.0 mg), Hp1–3FT (2.5 mg), and
PmPpA (2 mg). The reactions were carried out by incubating
the reaction mixture in an incubator shaker at 37 C for 48 h.
The product formation was monitored by TLC (n-
PrOH:H2O:NH4OH = 4:2:1 and detected by p-anisaldehyde
sugar stain) and mass spectrometry (MS). When an optimal
yield was achieved, to the reaction mixture was added the
same volume (10 mL) of ethanol and the mixture was incu-
bated at 4 C for 30 min. The precipitates were removed by
the centrifuge and the supernatant was concentrated and puri-
fied by a Bio Gel P-2 gel column (water was used as an
eluant). Further purification was achieved by silica gel chro-
matography (EtOAc:MeOH:H2O = 5:3:2, by volume) to obtain
Neu5Ac2–3Gal1–4GlcNAc1–3Lac (37). 126 mg, yield
93%; white solid. 1H NMR (800 MHz, D2O)  5.16 (d, J = 3.2
Hz, 0.4H), 4.65 (d, J = 8.0 Hz, 0.4H), 4.64 (d, J = 8.0 Hz,
0.6H), 4.60 (d, J = 8.0 Hz, 0.6H), 4.50 (d, J = 8.0 Hz, 1H),
4.38 (d, J = 8.0 Hz, 1H), 4.10 (d, J = 3.2 Hz, 1H), 4.06 (dd, J
= 3.2 and 9.6 Hz, 1H), 3.91–3.21 (m, 29H), 2.70 (dd, J = 4.8
and 12.8 Hz, 1H), 1.97 (s, 6H), 1.74 (t, J = 12.0 Hz, 1H). 13C
NMR (200 MHz, D2O) -isomer:  174.86, 174.77, 173.76,
102.78, 102.69, 102.38, 99.65, 95,59, 81.90, 78.08, 77.77,
75.29, 75.00, 74.73, 74.37, 74.17, 73.59, 72.70, 71.96, 71.60,
71.22, 70.93, 69.81, 69.21, 68.20, 67.88, 67.28, 62.37, 60.87,
59.85, 59.72, 59.61, 56.03, 51.58, 39.92, 22.02, 21.89. HRMS
(ESI) m/z calculated for C37H61N2O29 (M-H) 997.3360, found
997.3364. NMR data were consistent with those reported in
the literature.64
Neu5Gc2–3Gal1–4GlcNAc1–3Lac (38). 28 yield 91%;
1
white solid. H NMR (800 MHz, D2O)  5.21 (d, J = 4.0 Hz,
1
Gal1–4(Fuc1–3)GlcNAc1–3Lac (46) (181 mg, 94%). H
0.4H), 4.70 (d, J = 8.0 Hz, 1H), 4.66 (d, J = 8.0 Hz, 1H), 4.55
(d, J = 8.0 Hz, 1H), 4.43 (d, J = 8.0 Hz, 1H), 4.15 (d, J = 3.2
Hz, 1H), 4.12 (dd, J = 3.2 and 9.6 Hz, 1H), 4.11 (s, 2H), 3.97–
3.28 (m, 29H), 2.77 (dd, J = 4.8 and 12.8 Hz, 1H), 2.02 (s,
3H), 1.81 (t, J = 12.0 Hz, 1H). 13C NMR (200 MHz, D2O) 
175.75, 174.88, 173.87, 102.87, 102.74, 102.51, 99.79, 95.71,
82.02, 78.23, 77.94, 75.43, 75.14, 74.86, 74.76, 74.51, 74.31,
72.56, 72.10, 71.08, 69.93, 69.35, 68.30, 68.03, 67.96, 61.00,
60.94, 39.64, 22.15. HRMS (ESI) m/z calculated for
C37H61N2O30 (M-H) 1013.3309, found 1013.3292.
NMR (800 MHz, D2O)  5.17 (d, J = 4.0 Hz, 0.3H), 5.08 (d, J
= 4.0 Hz, 1H), 4.66 (d, J = 7.2 Hz, 1H), 4.61 (d, J = 8.0 Hz,
0.7H), 4.42 (d, J = 8.0 Hz, 1H), 4.39 (d, J = 8.0 Hz, 1H), 4.11
(d, J = 3.2 Hz, 1H), 3.92–3.22 (m, 27H), 1.98 (s, 3H), 1.13 (d,
J = 6.4 Hz, 3H). 13C NMR (200 MHz, D2O) -isomer: 
174.57, 102.77, 102.43, 101.62, 98.48, 95.59, 81.91, 78.04,
74.95, 74.77, 74.65, 74.59, 74.19, 73.62, 72.87, 72.30, 71.75,
71.24, 70.88, 69.96, 69.81, 69.03, 68.20, 67.53, 66.55, 62.22,
61.37, 60.82, 59.45, 56.80, 22.08, 15.16. HRMS (ESI) m/z
calculated for C32H55NO25Na (M+Na) 876.2961, found
876.2965.
Kdn2–3Gal1–4GlcNAc1–3Lac (39). 27 mg, yield
92%; white solid. 1H NMR (800 MHz, D2O)  5.20 (d, J = 4.0
Hz, 0.4H), 4.69 (d, J = 8.0 Hz, 0.4H), 4.68 (d, J = 8.0 Hz,
0.6H), 4.64 (d, J = 8.0 Hz, 0.6H), 4.52 (d, J = 8.0 Hz, 1H),
4.42 (d, J = 8.0 Hz, 1H), 4.13 (d, J = 3.2 Hz, 1H), 4.07 (dd, J
= 3.2 and 9.6 Hz, 1H), 3.95–3.25 (m, 29H), 2.68 (dd, J = 4.8
and 12.8 Hz, 1H), 2.01 (s, 3H), 1.73 (t, J = 12.0 Hz, 1H). 13C
NMR (200 MHz, D2O)  174.85, 173.99, 102.75, 102.50,
99.75, 95.68, 82.00, 78.20, 77.90, 75.39, 75.13, 74.84, 74.75,
74.49, 74.29, 73.87, 73.73, 72.08, 72.01, 71.07, 70.20, 69.92,
69.68, 69.31, 68.28, 67.64, 62.57, 60.99, 60.93, 55.13, 39.22,
22.14. HRMS (ESI) m/z calculated for C35H58NO29 (M-H)
956.3094, found 956.3105.
General procedures for OPME synthesis of sialylated
LNnT, LNT, and Lex pentasaccharides (37–50). LNnT,
LNT, or Lex pentasaccharide (20 mM, 1 eq.), Neu5Ac or a
sialic acid precursor (ManNGc, mannose, or ManNAc5OMe,
1.5 eq.) with sodium pyruvate (7.5 eq.) were incubated at 37
C in a Tris-HCl buffer (100 mM, pH 8.5) containing CTP
(1.5 eq.), MgCl2 (20 mM), NmCSS (1.5 mg/mL), PmST1
M144D (3 mg/mL), with or without PmNanA (0.2 mg/mL,
omit if Neu5Ac was used). The reactions were carried out by
incubating the solution in an incubator shaker at 37 C for 1 or
2 days with agitation at 100 rpm. The product formation was
monitored by LC-MS. When an optimal yield was achieved,
alkaline phosphatase (10–20 mg) was added to the reaction
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The Journal of Organic Chemistry
Page 14 of 19
Neu5Ac8OMe2–3Gal1–4GlcNAc1–3Lac (40). 15 mg,
yield 83%; white solid. 1H NMR (800 MHz, D2O)  5.21 (d, J
= 4.0 Hz, 0.4H), 4.69 (d, J = 8.0 Hz, 0.4H), 4.68 (d, J = 8.0
Hz, 0.6H), 4.65 (d, J = 8.0 Hz, 0.6H), 4.51 (d, J = 8.0 Hz, 1H),
4.43 (d, J = 8.0 Hz, 1H), 4.21–3.25 (m, 31H), 3.49 (s, 3H),
2.68 (dd, J = 4.8 and 12.8 Hz, 1H), 2.01 (s, 3H), 1.74 (t, J =
12.0 Hz, 1H). 13C NMR (200 MHz, D2O)  174.86, 174.80,
173.52, 102.79, 102.70, 102.67, 102.59, 100.09, 95.63, 81.96,
80.20, 78.25, 78.14, 77.86, 76.26, 75.62, 75.12, 74.77, 74.68,
74.47, 74.23, 71.98, 71.00, 70.57, 69.85, 69.27, 69.07, 68.22,
67.88, 66.84, 60.91, 59.22, 57.40, 55.10, 43.72, 42.92, 39.71,
26.15, 25.70, 22.07, 21.98. HRMS (ESI) m/z calculated for
C38H63N2O29 (M-H) 1011.3516, found 1011.3514.
1.74 (t, J = 12.0 Hz, 1H). 13C NMR (200 MHz, D2O) 
1
2
3
4
5
6
7
8
181.39, 174.82, 174.74, 173.62, 161.98, 103.08, 102.82,
102.78, 102.43, 100.01, 95.65, 95.62, 81.82, 80.35, 78.17,
75.56, 75.06, 74.92, 74.78, 74.68, 74.24, 71.01, 69.89, 69.16,
68.36, 68.21, 67.95, 67.30, 66.82, 62.79, 62.78, 60.87, 59.32,
57.37, 57.36, 56.42, 51.94, 23.15, 22.22, 21.98. HRMS (ESI)
m/z calculated for C38H63N2O29 (M-H) 1011.3516, found
1011.3510.
Neu5Ac2–3Gal1–4(Fuc1–3)GlcNAc1–3Lac (47). 51
1
9
mg, yield 86%; white solid. H NMR (800 MHz, D2O)  5.22
(d, J = 4.0 Hz, 0.4H), 5.18 (d, J = 3.2 Hz, 0.4H), 5.12 (d, J =
4.0 Hz, 0.6H), 4.56 (d, J = 8.0 Hz, 0.6H), 4.53 (d, J = 8.0 Hz,
1H), 4.46 (d, J = 8.0 Hz, 0.4H), ), 4.44 (d, J = 8.0 Hz, 1H),
4.42 (d, J = 8.0 Hz, 0.6H), 4.16 (d, J = 3.2 Hz, 1H), 4.13–3.27
(m, 34H), 2.77 (dd, J = 4.8 and 12.0 Hz, 1H), 2.03 (s, 6H),
1.78 (d, J = 12.0 Hz, 1H), 1.17 (d, J = 6.4 Hz, 3H). 13C NMR
(200 MHz, D2O) -isomer:  174.93, 174.59, 173.77, 102.83,
102.44, 101.47, 99.56, 98.48, 95.64, 82.00, 78.27, 78.17,
77.92, 75.57, 75.07, 74.82, 74.42, 73.68, 72.94, 72.81, 71.81,
71.77, 71.03, 70.02, 69.87, 69.17, 69.08, 68.21, 68.00, 67.61,
67.21, 66.56, 62.50, 61.40, 60.89, 59.97, 59.42, 55.17, 51.60,
39.69, 22.13, 21.94, 15.18. HRMS (ESI) m/z calculated for
C43H71N2O33 (M-H) 1143.3939, found 1143.3920.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Neu5Ac2–3Gal1–3GlcNAc1–3Lac (42). 107 mg, yield
94%; white solid. 1H NMR (800 MHz, D2O)  5.35 (d, J = 3.2
Hz, 0.4H), 4.87 (d, J = 8.8 Hz, 1H), 4.63 (d, J = 8.0 Hz, 1H),
4.57 (d, J = 8.0 Hz, 1H), 4.26 (d, J = 3.2 Hz, 1H), 4.20 (dd, J
= 3.2 and 9.6 Hz, 1H), 4.09–3.39 (m, 29H), 2.89 (dd, J = 4.8
and 12.8 Hz, 1H), 2.15 (s, 6H), 1.90 (t, J = 12.0 Hz, 1H). 13C
NMR (200 MHz, D2O) -isomer:  175.07, 174.99, 173.88,
103.42, 102.99, 102.95, 99.76, 95,79, 82.29, 81.98, 78.53,
75.30, 74.73, 74.37, 74.17, 73.59, 72.70, 71.96, 71.60, 71.22,
70.93, 69.81, 69.21, 68.20, 67.88, 67.28, 61.07, 60.65, 60.21,
60.09, 54.66, 51.78, 39.87, 22.40, 22.11. HRMS (ESI) m/z
calculated for C37H61N2O29 (M-H) 997.3360, found 997.3368.
NMR data were consistent with those reported in the litera-
ture.83
Neu5Gc2–3Gal1–4(Fuc1–3)GlcNAc1–3Lac (48). 18
1
mg, yield 84%; white solid. H NMR (800 MHz, D2O)  5.21
(d, J = 4.0 Hz, 0.3H), 5.11 (d, J = 4.8 Hz, 0.7H), 5.09 (d, J =
4.8 Hz, 0.3H), 4.69 (d, J = 8.0 Hz, 1H), 4.65 (d, J = 8.0 Hz,
0.7H), 4.52 (d, J = 8.0 Hz, 1H), 4.44 (d, J = 8.0 Hz, 0.3H), ),
4.42 (d, J = 8.0 Hz, 0.7H), 4.42 (d, J = 8.0 Hz, 1H), 4.15 (d, J
= 3.2 Hz, 1H), 4.11 (s, 2H), 4.08 (dd, J = 3.2 and 9.6 Hz, 1H),
3.98–3.25 (m, 33H), 2.77 (dd, J = 4.8 and 12.0 Hz, 1H), 2.00
Neu5Gc2–3Gal1–3GlcNAc1–3Lac (43). 202 mg, yield
90%; white solid. 1H NMR (800 MHz, D2O)  5.35 (d, J = 3.2
Hz, 0.4H), 4.88 (d, J = 8.8 Hz, 1H), 4.64 (d, J = 8.0 Hz, 2H),
4.58 (d, J = 8.0 Hz, 0.6H), 4.27 (d, J = 3.2 Hz, 1H), 4.24 (s,
2H), 4.21 (dd, J = 3.2 and 9.6 Hz, 1H), 4.08–3.39 (m, 29H),
2.89 (dd, J = 4.8 and 12.8 Hz, 1H), 2.15 (s, 6H), 1.92 (t, J =
12.0 Hz, 1H). 13C NMR (200 MHz, D2O) -isomer:  175.80,
174.94, 173.90, 103.44, 102.97, 102.52, 99.78, 95,74, 82.04,
81.92, 78.54, 75.36, 74.81, 74.50, 74.25, 73.88, 71.98, 71.55,
71.54, 70.19, 69.15, 69.10, 68.33, 67.35, 67.10, 61.09, 61.01,
60.62, 60.08, 54.62, 51.49, 39.96, 22.41. HRMS (ESI) m/z
calculated for C37H61N2O30 (M-H) 1013.3309, found
1013.3318.
(s, 3H), 1.80 (t, J = 12.0 Hz, 1H), 1.15 (d, J = 6.4 Hz, 3H). 13
C
NMR (200 MHz, D2O)  175.66, 174.56, 173.78, 102.79,
102.46, 101.42, 99.54, 98.46, 95.60, 91.67, 81.97, 81.94,
78.21, 78.11, 75.52, 74.89, 74.79, 74.76, 74.67, 74.51, 74.22,
73.64, 72.89, 72.50, 72.12, 71.78, 71.26, 70.98, 69.98, 69.83,
69.14, 69.04, 68.16, 67.92, 67.88, 67.67, 67.57, 67.16, 66.53,
62.41, 61.37, 60.83, 59.93, 59.38, 51.40, 51.26, 48.72, 39.71,
22.11, 15.26, 15.15. HRMS (ESI) m/z calculated for
C43H71N2O34 (M-H) 1159.3888, found 1159.3898.
Kdn2–3Gal1–4(Fuc1–3)GlcNAc1–3Lac (49). 6 mg,
yield 83%; white solid. 1H NMR (800 MHz, D2O)  5.21 (d, J
= 4.0 Hz, 0.3H), 5.10 (d, J = 4.0 Hz, 0.7H), 5.09 (d, J = 4.0
Hz, 0.3H), 4.69 (d, J = 8.0 Hz, 1H), 4.65 (d, J = 8.0 Hz, 0.7H),
4.50 (d, J = 8.0 Hz, 1H), 4.44 (d, J = 8.0 Hz, 0.3H), ), 4.43 (d,
J = 8.0 Hz, 0.7H), 4.42 (d, J = 8.0 Hz, 1H), 4.14 (d, J = 3.2
Hz, 1H), 4.04 (dd, J = 3.2 and 9.6 Hz, 1H), 3.97–3.25 (m,
33H), 2.69 (dd, J = 4.8 and 12.0 Hz, 1H), 2.01 (s, 3H), 1.72 (t,
J = 12.0 Hz, 1H), 1.15 (d, J = 6.4 Hz, 3H). 13C NMR (200
MHz, D2O)  174.56, 173.91, 102.80, 102.77, 102.46, 101.45,
98.46, 95.60, 91.68, 81.97, 78.21, 78.11, 75.50, 74.90, 74.79,
74.76, 74.67, 74.51, 74.22, 73.81, 73.65, 72.91, 72.04, 71.77,
71.27, 70.99, 70.09, 69.99, 69.83, 69.73, 69.62, 69.12, 69.04,
68.17, 67.57, 67.14, 66.53, 62.51, 61.37, 60.85, 59.93, 59.39,
39.29, 22.11, 15.26, 15.15. HRMS (ESI) m/z calculated for
C41H68NO33 (M-H) 1102.3674, found 1102.3686.
Kdn2–3Gal1–3GlcNAc1–3Lac (44). 31 mg, yield
91%; white solid. 1H NMR (800 MHz, D2O)  5.20 (d, J = 4.0
Hz, 0.4H), 4.71 (d, J = 8.0 Hz, 0.4H), 4.70 (d, J = 8.0 Hz,
0.6H), 4.67 (d, J = 8.0 Hz, 0.6H), 4.47 (d, J = 8.0 Hz, 1H),
4.42 (d, J = 8.0 Hz, 1H), 4.12 (d, J = 4.0 Hz, 1H), 4.04 (dd, J
= 3.2 and 9.6 Hz, 1H), 3.94–3.24 (m, 29H), 2.68 (dd, J = 4.8
and 12.8 Hz, 1H), 2.00 (s, 3H), 1.70 (t, J = 12.0 Hz, 1H). 13C
NMR (200 MHz, D2O)  174.85, 174.00, 103.37, 103.34,
102.88, 102.84, 102.49, 102.43, 99.52, 95.70, 95.67, 91.78,
91.73, 82.01, 81.89, 81.87, 78.34, 78.22, 75.50, 75.48, 75.15,
75.04, 74.84, 74.74, 74.30, 73.78, 73.72, 72.11, 71.07, 70.20,
69.96, 69.66, 69.01, 69.00, 68.37, 68.27, 68.25, 67.61, 67.16,
67.09, 62.47, 62.41, 60.98, 60.92, 39.44, 39.38, 22.26. HRMS
(ESI) m/z calculated for C35H58NO29 (M-H) 956.3094, found
956.3106.
Neu5Ac8OMe2–3Gal1–3GlcNAc1–3Lac (45). 32 mg,
yield 83%; white solid. 1H NMR (800 MHz, D2O)  5.21 (d, J
= 4.0 Hz, 0.4H), 4.73 (d, J = 8.0 Hz, 0.4H), 4.72 (d, J = 8.0
Hz, 0.6H), 4.65 (d, J = 8.0 Hz, 0.6H), 4.48 (d, J = 8.0 Hz, 1H),
4.43 (d, J = 8.0 Hz, 1H), 4.15–3.25 (m, 31H), 3.48 (s, 3H),
2.67 (dd, J = 4.8 and 12.8 Hz, 1H), 2.02 (s, 3H), 2.01 (s, 3H),
Neu5Ac8OMe2–3Gal1–4(Fuc1–3)GlcNAc1–3Lac
(50). 2 mg, yield, 80%; white solid. 1H NMR (800 MHz, D2O)
 5.20 (d, J = 4.0 Hz, 0.4H), 5.10 (d, J = 4.0 Hz, 1H), 4.70 (d,
J = 8.0 Hz, 1H), 4.65 (d, J = 8.0 Hz, 0.6H), 4.45 (d, J = 8.0
Hz, 1H), 4.43 (d, J = 8.0 Hz, 1H) 4.41 (d, J = 8.0 Hz, 1H),
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The Journal of Organic Chemistry
4.14–3.25 (m, 35H), 2.65 (m, 1H), 2.01 (s, 6H), 1.68 (m, 1H), tion and the supernatant was concentrated and purified by
1.15 (d, J = 6.4 Hz, 3H). 13C NMR (200 MHz, D2O)  174.86,
174.84, 174.62, 173.70, 173.54, 102.81, 102.78, 101.68,
101.66, 100.91, 99.90, 98.53, 98.49, 95.61, 91.69, 81.98,
81.95, 80.44, 80.34, 78.24, 78.14, 75.81, 75.05, 74.97, 74.86,
74.77, 74.68, 74.23, 73.65, 73.21, 72.77, 72.74, 72.56, 72.13,
71.92, 71.79, 71.28, 71.00, 70.89, 70.00, 69.84, 69.13, 69.06,
69.00, 68.21, 68.18, 67.87, 67.83, 67.67, 67.58, 67.38, 67.03,
66.80, 66.54, 62.41, 61.36, 60.86, 59.94, 59.81, 59.65, 59.57,
59.23, 57.61, 57.45, 52.06, 51.89, 39.89, 22.12, 21.95, 15.25,
15.17. HRMS (ESI) m/z calculated for C44H73N2O33 (M-H)
1157.4096, found 1157.4084.
1
2
3
4
5
6
7
8
silica gel column (EtOAc:MeOH:H2O, 5:3:2) followed by a
Bio-gel P2 gel filtration column to afford the desired Gb4 or
iGb4 glycan.
GalNAc1–3Gal1–4Lac (53). 570 mg, yield 91%; white
1
solid. H NMR (800 MHz, D2O):  5.20 (d, J = 3.2 Hz, 0.4H),
4.89 (d, J = 3.2 Hz, 1H), 4.64 (d, J = 8.0 Hz, 0.6H), 4.60 (d,
J = 8.0 Hz, 1H), 4.49 (d, J = 8.0 Hz, 1H), 4.37 (m, 1H), 4.23
(bs, 1H), 4.01 (bs, 1H), 3.95–3.22 (m, 21 H), 2.02 (s, 3H); 13C
NMR (200 MHz, D2O)  175.09, 103.21, 103.15, 100.29,
95.60, 91.64 78.74, 78.55, 77.09, 77.03, 75.34, 75.32, 74.81,
74.72, 74.32, 73.83, 73.78, 71.98, 71.09, 70.77, 70.76, 70.65,
70.16, 70.12, 68.85, 68.81, 67.67, 67.59, 60.88, 60.26, 60.25,
60.20, 52.61, 22.16. HRMS: calculated for C26H46NO21 (M
+H), 708.2562, found 708.2598. NMR data were consistent
with those reported in the literature.78
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
General procedures for OPME synthesis of Gb3 and
iGb3 glycans. Lac (20 mM, 1 eq.), Gal (1.5 eq.) were incu-
bated at 37 C in 100 mM of Tris-HCl buffer (pH 7.5) contain-
ing ATP (1.5 eq.), UTP (1.5 eq.), MgCl2 (10 mM), MnCl2 (10
mM), EcGalK (4 mg/mL), BLUSP (4 mg/mL), B1–3GalT (6
mg/mL, for preparing iGb3) or NmLgtC (5 mg/mL, for prepar-
ing Gb3), and PmPpA (3 mg/mL). The reaction was carried out
by incubating the solution in an incubator shaker at 37 C for
overnight with agitation at 100 rpm. The product formation
was monitored by LC-MS. When an optimal yield was
achieved, the reaction was quenched by adding the same vo-
lume of ice-cold ethanol and the mixture was incubated at 4
C for 30 min. The precipitates were removed by centrifuga-
tion and the supernatant was concentrated and purified by
silica gel column (EtOAc:MeOH:H2O, 4:2:1) followed by a
Bio-gel P2 gel filtration column to obtain the desired Gb3 or
iGb3.
GalNAc1–3Gal1–3Lac (54). 340 mg, yield 92%; white
1
solid. H NMR (800 MHz, D2O):  5.20 (d, J = 4.0 Hz, 0.4H),
4.88 (d, J = 4.0 Hz, 1H), 4.64 (d, J = 8.0 Hz, 0.6H), 4.60 (d,
J = 8.8 Hz, 1H), 4.49 (d, J = 8.8 Hz, 0.4H), 4.48 (d, J = 8.0
Hz, 0.6H), 4.35 (m, 1H), 4.23 (bs, 1H), 4.00–3.24 (m, 22 H),
2.01 (s, 3H); 13C NMR (200 MHz, D2O)  175.08, 103.15,
100.29, 100.22, 99.91, 95.59, 95.54, 91.69, 91.61, 78.73,
78.55, 77.10, 76.98, 75.33, 74.78, 74.71, 74.31, 73.83, 73.75,
71.97, 71.35, 71.07, 70.75, 70.62, 70.16, 70.10, 68.86, 68.78,
67.71, 67.59, 67.53, 60.86, 60.31, 60.25, 60.21, 60.05, 59.80,
52.51, 52.34, 22.17, 22.14. HRMS: calculated for C26H46NO21
(M +H), 708.2562, found 708.2596. NMR data were
consistent with those reported in the literature.78
1
Gal1–4Lac (51). 850 mg, yield 95%; white solid. H
General procedures for OPME synthesis of Gb5 and
iGb5 glycans. Gb4 or iGb4 glycan (20 mM, 1 eq.) as an accep-
tor and Gal (1.1 eq.) were incubated at 37 C in Tris-HCl buf-
fer (100 mM, pH 7.5) containing ATP (1.2 eq.), UTP (1.2 eq.),
MgCl2 (10 mM), MnCl2 (10 mM), EcGalK (3 mg/mL),
BLUSP (3 mg/mL), CjCgtB (6 mg/mL), and PmPpA (2
mg/mL). The reaction was carried out by incubating the solu-
tion in an incubator shaker at 37 C for overnight with agita-
tion at 100 rpm. The product formation was monitored by LC-
MS. When an optimal yield was achieved, alkaline phospha-
tase (10–20 mg) was added to the reaction mixture and incu-
bated in an incubator shaker at 37 C for overnight with agita-
tion at 100 rpm. The reaction was quenched by adding the
same volume of ice-cold ethanol and the mixture was incu-
bated at 4 C for 30 min. The precipitates were removed by
centrifugation and the supernatant was concentrated, passed
through a BioGel P-2 gel filtration column, and eluted with
water to obtain sialoside mixtures. The fractions containing
the product were collected, concentrated, and further purified
by HPLC using a reverse-phase C18 column (10 m, 21.2 ×
250 mm) with a flow rate of 10 mL/min and a gradient elution
of 0–100% acetonitrile in water containing 0.05% formic acid
over 20 minutes. Mobile phase A: 0.05% formic acid in water
(v/v); Mobile phase B: acetonitrile (v/v); Gradient: 0% B for 3
min, 0% to 100% B over 12 min, 100% B for 2 min, then
100% to 0% B over 3 min. HPLC purification was monitored
by absorption at 210 nm, and glycan-containing fractions were
analyzed by TLC and MS. The fractions containing the pure
product were collected and concentrated to obtain the desired
Gb5 or iGb5 glycan.
NMR (800 MHz, D2O):  5.18 (d, J = 4.0 Hz, 0.4H), 4.90 (d, J
= 4.0 Hz, 1H), 4.63 (d, J = 8.0 Hz, 0.6H), 4.47 (d, J = 7.2 Hz,
1H), 4.31 (m, 1H), 4.00 (m, 2H), 3.92–3.23 (m, 15H); 13C
NMR (200 MHz, D2O):  103.41, 103.37, 100.46, 95.86,
91.94, 78.82, 78.71, 77.51, 75.58, 74.99, 74.56, 74.04, 72.30,
71.59, 71.35, 71.06, 70.96, 70.30, 69.28, 69.08, 68.71, 60.65,
60.53, 60.18, 60.06; HRMS: calculated for C18H32O16Na (M
+Na) 527.1588, found 527.1613. NMR data were consistent
with those reported in the literature.84
1
Gal1–3Lac (52). 790 mg, yield 99%; white solid. H
NMR (800 MHz, D2O):  5.21 (d, J = 3.2 Hz, 0.4 H), 5.13
(d, J = 3.2 Hz, 1H), 4.65 (d, J = 8.0 Hz, 0.6 H), 4.51 (d, J = 8.0
Hz, 1H), 4.17 (m, 2H), 4.00–3.27 (m, 16 H); 13C NMR (200
MHz, D2O)  102.71, 102.68, 95.65, 95.31, 95.30, 91.70,
78.53, 78.41, 77.07, 77.05, 74.93, 74.65, 74.31, 73.66, 71.37,
71.00, 70.70, 69.95, 69.46, 69.16, 69.00, 68.09, 64.71, 64.69,
60.90, 60.88, 60.80, 60.03, 59.89. HRMS: calculated for
C18H32O16Na (M +Na), 527.1588, found 527.1583. NMR data
were consistent with those reported in the literature.85
General procedures for OPME synthesis of Gb4 and
iGb4 glycans. Gb3 or iGb3 glycan (20 mM, 1 eq.) as an accep-
tor and GalNAc (1.5 eq.) were incubated at 37 C in Tris-HCl
buffer (100 mM, pH 7.5) containing ATP (1.5 eq.), UTP (1.5
eq.), MgCl2 (20 mM), NahK (3 mg/mL), PmGlmU (3 mg/mL),
HiLgtD (6 mg/mL), and PmPpA (2 mg/mL). The reaction was
carried out by incubating the solution in an incubator shaker at
37 C for 2 days with agitation at 100 rpm. The product for-
mation was monitored by LC-MS. When an optimal yield was
achieved, the reaction was quenched by adding the same vo-
lume of ice-cold ethanol and the mixture was incubated at 4
C for 30 min. The precipitates were removed by centrifuga-
Gal1–3GalNAc1–3Gal1–4Lac (55). 124 mg, yield,
60%; white solid. 1H NMR (800 MHz, D2O):  5.20 (d, J = 3.2
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The Journal of Organic Chemistry
Page 16 of 19
Hz, 0.4H), 4.89 (d, J = 4.0 Hz, 1H), 4.67 (d, J = 8.0 Hz, 1H), 99.55, 95.57, 79.67, 78.64, 78.58, 78.52, 77.02, 75.42, 75.32,
1
2
3
4
5
6
7
8
4.64 (d, J = 8.0 Hz, 0.6H), 4.50 (d, J = 8.0 Hz, 1H), 4.43 (d,
J = 8.0 Hz, 1H), 4.36 (m, 1H), 4.23 (bs, 1H), 4.16–3.24 (m, 28
H), 2.00 (s, 3H); 13C NMR (200 MHz, D2O)  175.01, 104.69,
103.16, 103.13, 102.87, 102.84, 100.27, 95.60, 91.67, 79.46,
78.67, 78.63, 78.58, 78.52, 77.05, 75.32, 74.87, 74.71, 74.47,
74.32, 73.80, 72.31, 71.97, 71.35, 71.09, 70.74, 70.46, 70.13,
70.01, 68.81, 68.44, 67.86, 67.49, 60.87, 60.82, 60.23, 60.18,
59.92, 59.79, 51.34, 22.14. HRMS: calculated for
C32H55NNaO26 (M +Na), 892.2910, found 892.2898.
74.71, 74.65, 74.48, 74.32, 73.80, 72.66, 71.98, 71.71, 71.35,
71.11, 70.75, 70.14, 70.02, 68.89, 68.81, 68.28, 67.92, 67.72,
67.49, 67.23, 62.35, 60.86, 60.25, 60.18, 51.53, 51.21, 39.59,
22.21, 21.92. HRMS (ESI) m/z calculated for C43H71N2O34
(M-H) 1159.3888, found 1159.3907.
Neu5Gc2–3Gal1–3GalNAc1–3Gal1–4Lac (58).
9
mg, yield 83%; white solid. 1H NMR (800 MHz, D2O)  5.20
(d, J = 4.0 Hz, 0.4H), 4.89 (d, J = 4.0 Hz, 1H), 4.67 (d, J = 8.0
Hz, 1H), 4.65 (d, J = 8.0 Hz, 0.6H), 4.50 (d, J = 8.0 Hz,
1H), 4.49 (d, J = 8.0 Hz, 1H), 4.36 (m, 1H), 4.23 (bs, 1H),
4.15 (d, J = 3.2 Hz, 1H), 4.09 (s, 2H), 4.07–3.25 (m, 34 H),
2.75 (dd, J = 4.8 and 12.0 Hz, 1H), 2.01 (s, 3H), 1.78 (t, J =
12.0 Hz, 1H); 13C NMR (200 MHz, D2O)  175.60, 174.98,
173.84, 104.45, 103.16, 103.13, 102.84, 100.25, 99.55, 95.57,
91.65, 79.65, 78.64, 78.58, 78.52, 77.02, 75.41, 75.32, 74.71,
74.66, 74.47, 74.32, 73.80, 72.37, 71.97, 71.77, 71.35, 71.10,
70.74, 70.14, 70.02, 68.89, 68.80, 68.00, 67.84, 67.73, 67.49,
67.21, 62.31, 60.84, 60.24, 60.18, 59.92, 51.21, 39.66, 22.21.
HRMS (ESI) m/z calculated for C43H71N2O35 (M-H)
1175.3837, found 1175.3874.
9
Gal1–3GalNAc1–3Gal1–3Lac (56). 110 mg, yield
45%; white solid. 1H NMR (800 MHz, D2O):  5.20 (d, J = 4.0
Hz, 0.4H), 5.09 (d, J = 4.0 Hz, 1H), 4.68 (d, J = 8.0 Hz, 1H),
4.64 (d, J = 8.0 Hz, 0.6H), 4.50 (d, J = 8.0 Hz, 1H), 4.43 (d,
J = 8.8 Hz, 1H), 4.21–3.26 (m, 30 H), 2.00 (s, 3H); 13C NMR
(200 MHz, D2O)  175.01, 104.69, 102.76, 102.65, 95.63,
95.51, 91.68, 79.48, 78.70, 78.43, 78.30, 77.05, 74.90, 74.87,
74.64, 74.50, 74.30, 73.65, 72.31, 71.36, 70.98, 70.46, 70.26,
69.94, 69.49, 68.91, 68.44, 67.85, 67.13, 64.72, 60.87, 60.79,
60.66, 60.00, 59.86, 51.39, 22.15. HRMS: calculated for
C32H55NNaO26 (M +Na), 892.2910, found 892.2930.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Kdn2–3Gal1–3GalNAc1–3Gal1–4Lac (59). 8 mg,
yield 85%; white solid. H NMR (800 MHz, D2O)  5.21
General procedures for OPME synthesis of sialylated
Gb5 and iGb5 glycans. Gb5 or iGb5 (20 mM, 1 eq.) and
Neu5Ac or a sialic acid precursor (ManNGc, mannose, or
ManNAc5OMe, 1.5 eq.) with sodium pyruvate (7.5 eq.) were
incubated at 37 C in a Tris-HCl buffer (100 mM, pH 8.5)
containing CTP (1.5 eq.), MgCl2 (20 mM), NmCSS (3
mg/mL), PmST1 M144D (4 mg/mL), with or without PmNa-
nA (1.5 mg/mL, omit if Neu5Ac was used). The reactions
were carried out by incubating the solution in an incubator
shaker at 37 C for 1 or 2 days with agitation at 100 rpm. The
product formation was monitored by LC-MS. When an optim-
al yield was achieved, alkaline phosphatase (10–20 mg) was
added and the reaction mixture was incubated in an incubator
shaker at 37 C for overnight with agitation at 100 rpm. The
reaction was then quenched by adding the same volume of ice-
cold ethanol and the mixture was incubated at 4 C for 30 min.
The precipitates were removed by centrifugation and the su-
pernatant was concentrated, passed through a BioGel P-2 gel
filtration column, and eluted with water to obtain sialoside
mixtures. The fractions containing product were collected,
concentrated, and further purified by HPLC with a reverse-
phase C18 column (10 m, 21.2 × 250 mm) with a flow rate of
10 mL/min using a gradient elution of 0–100% acetonitrile in
water containing 0.05% formic acid over 20 min. Mobile
phase A: 0.05% formic acid in water (v/v); Mobile phase B:
acetonitrile (v/v); Gradient: 0% B for 3 min, 0% to 100% B
over 12 min, 100% B for 2 min, then 100% to 0% B over 3
min. HPLC purification was monitored by absorption at 210
nm, and glycan-containing fractions were analyzed by TLC
and MS. The fractions containing the pure product were col-
lected and concentrated to obtain the desired sialylated lacto-
and neolacto-series glycosphingolipid glycans (yields 71–
86%).
1
(d, J = 3.2 Hz, 0.4H), 4.90 (d, J = 4.0 Hz, 1H), 4.67 (d, J = 8.0
Hz, 1H), 4.65 (d, J = 8.0 Hz, 0.6H), 4.50 (d, J = 8.0 Hz,
1H), 4.49 (d, J = 8.0 Hz, 1H), 4.37 (m, 1H), 4.24 (bs, 1H),
4.15 (d, J = 3.2 Hz, 1H), 4.07–3.26 (m, 34 H), 2.68 (dd, J =
4.8 and 12.0 Hz, 1H), 2.01 (s, 3H), 1.72 (t, J = 12.0 Hz, 1H);
13C NMR (200 MHz, D2O)  175.06, 174.05, 104.56, 103.24,
102.92, 100.33, 99.53, 95.65, 79.66, 78.72, 78.66, 78.60,
77.10, 75.45, 75.40, 74.79, 74.76, 74.56, 74.39, 73.87, 73.77,
72.08, 72.05, 71.42, 71.17, 70.82, 70.21, 70.10, 69.66, 68.93,
68.88, 67.82, 67.63, 67.57, 67.20, 62.47, 61.34, 60.91, 60.32,
60.26, 60.00, 51.30, 39.38, 24.41, 22.29. HRMS (ESI) m/z
calculated for C41H68NO34 (M-H) 1118.3623, found
1118.3629.
Neu5Ac8OMe2–3Gal1–3GalNAc1–3Gal1–4Lac
1
(60). 7 mg, yield 78%; white solid. H NMR (800 MHz, D2O)
 5.20 (d, J = 4.0 Hz, 0.4H), 4.89 (d, J = 4.0 Hz, 1H), 4.66
(d, J = 8.0 Hz, 1H), 4.64 (d, J = 8.0 Hz, 0.6H), 4.49 (d, J = 8.0
Hz, 1H), 4.48 (d, J = 8.0 Hz, 1H), 4.36 (m, 1H), 4.23–3.25 (m,
36 H), 3.44 (s, 3H), 2.63 (dd, J = 4.8 and 12.0 Hz, 1H), 2.01
(s, 3H), 2.00 (s, 3H), 1.80 (t, J = 12.0 Hz, 1H); 13C NMR (200
MHz, D2O)  176.36, 174.94, 174.81, 174.64, 173.75, 173.65,
100.48, 100.26, 100.00, 96.19, 95.56, 80.28, 79.89, 78.53,
74.71, 74.60, 74.46, 74.30, 71.96, 70.75, 70.16, 69.75, 69.45,
69.15, 69.03, 67.93, 67.06, 66.81, 64.92, 62.31, 61.70, 60.82,
60.23, 59.78, 57.74, 57.44, 57.35, 39.49, 21.98, 19.94. HRMS
(ESI) m/z calculated for C44H73N2O34 (M-H) 1173.4045, found
1173.4046.
Neu5Ac2–3Gal1–3GalNAc1–3Gal1–3Lac (61). 40
1
mg, yield 85%; white solid. H NMR (800 MHz, D2O)  5.16
(d, J = 4.0 Hz, 0.4H), 5.05 (d, J = 4.0 Hz, 1H), 4.65 (d, J = 8.0
Hz, 1H), 4.61 (d, J = 8.0 Hz, 0.6H), 4.47 (d, J = 8.0 Hz,
1H), 4.46 (d, J = 8.0 Hz, 1H), 4.26–3.21 (m, 37 H), 2.68 (dd, J
= 4.8 and 12.0 Hz, 1H), 1.97 (s, 3H), 1.96 (s, 3H), 1.72 (t, J =
12.0 Hz, 1H); 13C NMR (200 MHz, D2O)  175.07, 174.92,
173.87, 104.48, 103.20, 102.88, 100.29, 99.60, 95.61, 79.72,
78.68, 78.63, 78.57, 77.06, 75.47, 75.36, 74.75, 74.69, 74.52,
74.35, 73.84, 72.66, 71.98, 71.71, 71.35, 71.11, 70.75, 70.14,
70.02, 68.89, 68.81, 68.28, 67.92, 67.72, 67.49, 67.23, 62.35,
Neu5Ac2–3Gal1–3GalNAc1–3Gal1–4Lac (57). 26
1
mg, yield 86%; white solid. H NMR (800 MHz, D2O)  5.21
(d, J = 4.0 Hz, 0.4H), 4.89 (d, J = 4.0 Hz, 1H), 4.67 (d, J = 8.0
Hz, 1H), 4.65 (d, J = 8.0 Hz, 0.6H), 4.51 (d, J = 8.0 Hz,
1H), 4.50 (d, J = 8.0 Hz, 1H), 4.37 (m, 1H), 4.24–3.25 (m, 36
H), 2.73 (dd, J = 4.8 and 12.0 Hz, 1H), 2.01 (s, 3H), 2.00 (s,
3H), 1.77 (t, J = 12.0 Hz, 1H); 13C NMR (200 MHz, D2O) 
174.99, 174.83, 173.82, 104.44, 103.16, 102.84, 100.26,
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The Journal of Organic Chemistry
60.86, 60.25, 60.22, 51.56, 51.24, 39.61, 22.23, 21.94. HRMS
(ESI) m/z calculated for C43H71N2O34 (M-H) 1159.3888, found
1159.3896.
ACKNOWLEDGMENT
1
2
3
4
5
6
7
8
This work was supported by the National Cancer Institute, Na-
tional Institutes of Health, Department of Health and Human Ser-
vices, under Contract No. HHSN261201300041C. The plasmid
for expressing HiLgtD was a kind gift from Dr. Peng G. Wang in
Georgia State University.
Neu5Gc2–3Gal1–3GalNAc1–3Gal1–4Lac (62).
6
1
mg, yield 84%; white solid. H NMR (800 MHz, D2O)  5.20
(d, J = 4.0 Hz, 0.4H), 5.09 (d, J = 4.0 Hz, 1H), 4.68 (d, J = 8.0
Hz, 1H), 4.64 (d, J = 8.0 Hz, 0.6H), 4.50 (d, J = 8.0 Hz,
2H), 4.21 (d, J = 4.0 Hz, 0.4H), 4.18 (m, 1H), 4.15 (d, J = 4.0
Hz, 0.6H), 4.09 (s, 2H), 4.07–3.25 (m, 35 H), 2.75 (dd, J = 4.8
and 12.0 Hz, 1H), 2.00 (s, 3H), 1.78 (t, J = 12.0 Hz, 1H); 13C
NMR (200 MHz, D2O)  175.61, 174.99, 173.84, 104.44,
102.75, 102.66, 99.56, 95.63, 95.54, 79.66, 78.68, 78.45,
78.32, 77.09, 75.41, 74.90, 74.66, 74.64, 74.50, 74.30, 73.65,
72.37, 71.77, 70.99, 70.27, 69.48, 68.89, 68.00, 67.84, 67.72,
67.13, 62.31, 60.87, 60.83, 60.67, 51.25, 39.66, 22.22. HRMS
(ESI) m/z calculated for C43H71N2O35 (M-H) 1175.3837, found
1175.3865.
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60
Kdn2–3Gal1–3GalNAc1–3Gal1–3Lac (63). 7 mg,
1
yield 82%; white solid. H NMR (800 MHz, D2O)  5.20
(d, J = 3.2 Hz, 0.4H), 5.09 (d, J = 4.0 Hz, 1H), 4.68 (d, J = 8.0
Hz, 1H), 4.65 (d, J = 8.0 Hz, 0.6H), 4.50 (d, J = 8.0 Hz,
1H), 4.49 (d, J = 8.0 Hz, 1H), 4.23–3.26 (m, 37 H), 2.68 (dd, J
= 4.8 and 12.0 Hz, 1H), 2.00 (s, 3H), 1.71 (t, J = 12.0 Hz, 1H);
13C NMR (200 MHz, D2O)  174.99, 173.98, 104.48, 102.75,
102.67, 99.48, 95.63, 95.54, 79.61, 78.68, 78.46, 78.33, 77.09,
75.38, 74.91, 74.69, 74.65, 74.51, 74.30, 73.70, 73.65, 73.46,
73.37, 73.06, 72.01, 71.36, 70.99, 70.28, 70.14, 69.94, 69.80,
69.59, 69.48, 68.91, 68.86, 67.74, 67.55, 67.14, 64.74, 62.55,
62.40, 60.88, 60.81, 60.68, 51.28, 39.30, 22.23. HRMS (ESI)
m/z calculated for C41H68NO34 (M-H) 1118.3623, found
1118.3622.
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Neu5Ac8OMe2–3Gal1–3GalNAc1–3Gal1–3Lac
1
(64). 1 mg, yield 71%; white solid. H NMR (800 MHz, D2O)
 5.21 (d, J = 3.2 Hz, 0.5H), 4.89 (d, J = 4.0 Hz, 1H), 4.67
(d, J = 8.8 Hz, 1H), 4.65 (d, J = 8.0 Hz, 0.5H), 4.50 (d, J = 7.2
Hz, 1H), 4.49 (d, J = 8.0 Hz, 1H), 4.37–3.25 (m, 37 H), 3.45
(s, 3H), 2.64 (dd, J = 4.8 and 12.0 Hz, 1H), 2.02 (s, 3H), 2.00
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for C44H73N2O34 (M-H) 1173.4045, found 1173.4025.
ASSOCIATED CONTENT
1
Supporting Information. H and 13C NMR spectra as well as
HRMS chromatographs of synthesized glycans. This material is
available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
* E-mail: hyu@glycohubusa.com
* E-mail: xiichen@ucdavis.edu
Present Addresses
†Current address: Laboratory of Bacterial Polysaccharides, Food
and Drug Administration, Bethesda, MD 20892, USA
‡Current address: Center for Neuroscience, University of Califor-
nia, Davis, CA 95616, USA
Current address: College of Pharmacy, Touro University, Valle-
jo, CA 94592, USA
Notes
HY, YL, and XC are co-founders of Glycohub, Inc., a company
focused on the development of carbohydrate-based reagents, di-
agnostics, and therapeutics.
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