Chemoenzymatic Synthesis of Campylobacter jejuni Lipo-oligosaccharide Core Domains to Examine Guillain-Barré Syndrome Serum Antibody Specificities

Article abstract of DOI:10.1021/jacs.0c08583

Guillain-Barré syndrome is often caused by Campylobacter jejuni infection that has induced antibodies to the lipo-oligosaccharide (LOS) that cross-react with gangliosides at peripheral nerves causing polyneuropathy. To examine fine specificities of anti-ganglioside antibodies and develop a more robust platform for diagnosis and disease monitoring, we developed a chemoenzymatic approach that provided an unprecedented panel of oligosaccharides composed of the inner-core of the LOS of C. jejuni extended by various ganglioside mimics. The compounds and corresponding ganglio-oligosaccharides were printed as a microarray to examine binding specificities of lectins, anti-ganglioside antibodies, and serum antibodies of GBS patients. Although lectins and anti-ganglioside antibodies did not differentiate the ganglio-oligosaccharides and mimics, the patient serum samples bound much more strongly to the ganglioside mimics. The data indicate that antibodies have been elicited to a foreign epitope that includes a heptosyl residue unique of bacterial LOS and that these antibodies subsequently cross-react with lower affinity to gangliosides. The microarray detected anti-GM1a antibodies with high sensitivity and will be attractive for diagnosis, disease monitoring, and immunological research.

Full text of DOI:10.1021/jacs.0c08583

pubs.acs.org/JACS
Article
Chemoenzymatic Synthesis of Campylobacter jejuni Lipo-
́
oligosaccharide Core Domains to Examine Guillain−Barre Syndrome
Serum Antibody Specificities
Tiehai Li, Margreet A. Wolfert, Na Wei, Ruth Huizinga, Bart C. Jacobs, and Geert-Jan Boons*
Cite This: https://dx.doi.org/10.1021/jacs.0c08583
Read Online
sı
*
Metrics & More
Article Recommendations
Supporting Information
ACCESS
́
ABSTRACT: Guillain−Barre syndrome is often caused by
Campylobacter jejuni infection that has induced antibodies to the
lipo-oligosaccharide (LOS) that cross-react with gangliosides at
peripheral nerves causing polyneuropathy. To examine fine
specificities of anti-ganglioside antibodies and develop a more
robust platform for diagnosis and disease monitoring, we
developed a chemoenzymatic approach that provided an
unprecedented panel of oligosaccharides composed of the inner-
core of the LOS of C. jejuni extended by various ganglioside
mimics. The compounds and corresponding ganglio-oligosacchar-
ides were printed as a microarray to examine binding specificities
of lectins, anti-ganglioside antibodies, and serum antibodies of
GBS patients. Although lectins and anti-ganglioside antibodies did not differentiate the ganglio-oligosaccharides and mimics, the
patient serum samples bound much more strongly to the ganglioside mimics. The data indicate that antibodies have been elicited to
a foreign epitope that includes a heptosyl residue unique of bacterial LOS and that these antibodies subsequently cross-react with
lower affinity to gangliosides. The microarray detected anti-GM1a antibodies with high sensitivity and will be attractive for diagnosis,
disease monitoring, and immunological research.
resembling the saccharide moieties of gangliosides. In
particular, LOSs of C. jejuni that are associated with GBS
often produce structures mimicking the oligosaccharide
moieties of GM1a and GD1a.11−13 In addition, strains have
been isolated that express GD3, GM2, GM3, and GT1a
mimics.14 Clinical and serologic data support a model in which
the LOS of specific C. jejuni strains elicit antibodies that
recognize both bacterial molecules and gangliosides, and
recognition of the latter biomolecules, which are abundantly
expressed in the nervous system where they are involved in
neurotransmission, causes neurological disfunction.11,15 Anti-
GM1a antibodies are the most frequently observed antibodies
in GBS and associated with a severe and pure motor clinical
phenotype.16,17
INTRODUCTION
■
́
Guillain−Barre syndrome (GBS) is a potentially life-threat-
ening disease of the peripheral nervous system characterized by
rapid and progressive limb weakness.1 The clinical course and
severity of GBS are variable, and approximately a quarter of the
patients develop respiratory failure or severe autonomic
dysfunction.2,3 GBS often occurs after a respiratory or
gastrointestinal tract infection, and previous studies have
shown some of these infections induce antibodies that cross-
react with gangliosides at peripheral nerves thereby causing
polyneuropathy.4 The type of infection and the specificity of
the resulting anti-ganglioside antibodies are important
determinants of the subtype and clinical course of GBS.5
The most common pathogen causing the antecedent infection
of GBS is Campylobacter jejuni (C. jejuni), which is associated
with acute motor axonal neuropathy subtype of GBS and a
severe clinical course.6 Other infections that have been
associated with GBS include Mycoplasma pneumoniae, hepatitis
E virus, cytomegalovirus, Epstein−Barr virus, and Zika
virus.7−9
Although there is strong scientific support for the
involvement of anti-ganglioside antibodies in the pathogenesis
of GBS,2 molecular mechanisms by which immunotolerance is
broken leading to autoimmune-like responses are not well
Received: August 10, 2020
C. jejuni is a Gram-negative, non-spore-forming bacterium
that is a common cause of gastroenteritis and is transmitted to
humans through ingestion of insufficiently cooked poultry,
contaminated milk, and water.10 It produces a lipo-
oligosaccharide (LOS) that often terminates in a structure
https://dx.doi.org/10.1021/jacs.0c08583
J. Am. Chem. Soc. XXXX, XXX, XXX−XXX
© XXXX American Chemical Society
A

Journal of the American Chemical Society
pubs.acs.org/JACS
Article
Figure 1. Synthetic ganglioside mimics from C. jejuni core oligosaccharides and normal ganglio-oligosaccharide library by a chemoenzymatic
approach: (A) synthetic ganglioside mimic and ganglioside oligosaccharide library; (B) chemically synthesized inner core hexasaccharide 1 and
enzymatic extension of 1 to afford compounds such as GT1a ganglioside mimic 6.
understood. Furthermore, the role of anti-ganglioside antibod-
ies in diagnosis is fraught with difficulties, and in particular the
frequency and specificity of anti-ganglioside antibodies are low
leading to false negative results. The positive predictive value
of anti-ganglioside antibodies, especially those of the IgM class,
is also compromised because these can occur in other diseases.
Detection of anti-ganglioside antibodies is mainly performed
by ELISA using gangliosides,18 usually obtained by isolation
from natural sources. These compounds are often not
homogeneous, and only a limited number of structures are
readily available, which is impeding comprehensive analysis of
structure−binding relationships and mechanisms by which
they promote nerve damage.
and the ganglioside structural analogs. To test this mode of
immune recognition, we synthesized a large panel of
oligosaccharides composed of the inner core oligosaccharide
of the LOS of C. jejuni extended by various ganglioside mimics.
Compound 1 resembles the inner core oligosaccharide of C.
jejuni, and compounds 2−13 are derived from this structure
but extended by the oligosaccharide mimics of GM1 (2), GM2
(3), GM1a (4), GD1a (5), GT1a (6), GD3 (7), GD2 (8),
GD1b (9), GT1b (10), GA1 (11), GM1b (12), and GD1c
(13). We also prepared the corresponding ganglioside
oligosaccharides 14−25 (Figure 1). Compounds 2−13 were
obtained by a chemoenzymatic strategy in which inner core
oligosaccharide 1 was prepared chemically and then extended
using appropriate microbially derived glycosyltransferases to
install ganglio-mimetics. The ganglioside oligosaccharides 14−
25 were prepared starting from spacer modified lactose which
was enzymatically extended to provide the target compounds.
During natural infections, the immune system is primed by
LOS of C. jejuni strains,19 and thus we hypothesized that anti-
ganglioside antibodies elicited during C. jejuni infection are
directed to epitopes that straddle the inner core region of LOS
B
https://dx.doi.org/10.1021/jacs.0c08583
J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

Journal of the American Chemical Society
pubs.acs.org/JACS
Article
a
Scheme 1. (A) Chemical Synthesis of Inner Core Hexasaccharide 1 and (B) Synthesis of Kdo2 Glycosyl Acceptor 29
a
Reagents and conditions: (a) HO(CH2)5NHCbz, BF3·Et2O, CH2Cl2, 3 Å molecular sieves, 0 °C to rt, 2.5 h, 75%; (b) lauroyl peroxide,
ClCH2CH2Cl/cyclohexane 1:7, reflux at 90 °C, 2 h, 83% for 32, 87% for 29; (c) NaOCH3/CH3OH, rt, 4 h, 92%; (d) diphosgene, sym-collidine,
THF, −25 °C, 30 min, 76%; (e) BF3·Et2O, CH2Cl2, 3 Å molecular sieves, 0 to 15 °C, 2 h, 80%. (C) Assembly of hexasaccharide 1, reagents and
conditions: (a) TMSOTf, CH2Cl2, 4 Å molecular sieves, −20 to −10 °C, 30 min, 89%; (b) DDQ, CH2Cl2/PBS buffer (100 mM, pH 7.4) 10:1, rt,
1.5 h, 78%; (c) TMSOTf, CH2Cl2, 4 Å molecular sieves, 0 to 5 °C, 70 min, 80%; (d) thiourea, sym-collidine, CH2Cl2/CH3OH 2:3, 70 °C, 24 h,
85%; (e) TMSOTf, CH2Cl2, 4 Å molecular sieves, −10 to 0 °C, 60 min, 90%; (f) (i) PdCl2, CH2Cl2/MeOH 1:5, rt, 2.5 h, 86%; (ii) CF3C(NPh)Cl,
Cs2CO3, CH2Cl2, rt, 4 h, 85%; (g) TMSOTf, CH2Cl2, 5 Å molecular sieves, rt, 2 h, 40%; (h) (i) 1 M NaOH(aq)/CH3OH/dioxane 1:1:3, rt, 16 h,
(ii) H2, Pd(OH)2/C, t-BuOH/H2O/HOAc 3:2:0.02, rt, 18 h, 42% over 2 steps.
The oligosaccharides were printed as a glycan microarray,
which was used to investigate the binding reactivities of a range
of lectins, anti-ganglioside antibodies, and serum antibodies of
normal and GBS patients. Although the lectins and anti-
ganglioside antibodies recognized a ganglio-oligosaccharide
and corresponding mimic equally well, the patient serum
samples bound much more strongly to the mimics. This
observation indicates that GBS patients have not generated
primary autoreactive antibodies but cross-reactive antibodies,
and most likely epitopes targeted by serum antibodies include
C
https://dx.doi.org/10.1021/jacs.0c08583
J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

Journal of the American Chemical Society
pubs.acs.org/JACS
Article
a heptosyl residue unique of bacterial LOS. Such an epitope
will be perceived as foreign for mammalian immune cells,
thereby providing a rationale for breaking immune-tolerance. It
was also observed that the microarray platform detects with
greater sensitivity anti-GM1a antibodies compared to tradi-
tional ELISA using bovine extracted gangliosides, and this will
be attractive for diagnosis, disease monitoring, and immuno-
logical research.
in high yield. Dehalogenation of the latter compound was
achieved using standard conditions providing the desired
dimeric Kdo acceptor 29.
Next, attention was focused on the preparation of 3,4-
branched heptosyl donor 42 (Scheme 1C). Thus, a TMSOTf
promoted glycosylation of 36 with 37 gave disaccharide 26 as
only α-anomer (JC1−H1 = 176 Hz) due to neighboring group
participation by the acetyl ester at C-2 of the donor. To give
38, the Nap ether of 26 was oxidatively removed using 2,3-
dichloro-5,6-dicyano-1,4-benzoquinone (DDQ)29 in a mixture
of CH2Cl2 and phosphate buffer (pH 7.4) without affecting
any other protecting groups. A glycosylation of the latter
acceptor with glycosyl donor 27, using TMSOTf (1.3 equiv) as
the activator, afforded the 3,4-branched trisaccharide 39. A
relatively large quantity of TMSOTf was required to avoid the
formation of an orthoester byproduct (Figure S1). Further-
more, the use of per-benzyolated glucosyl trichloroacetimidate
as donor did not provide the corresponding trisaccharide
probably due to steric hindrance.23 Next, the chloroacetyl
(ClAc) ester30,31 of 39 was selectively cleaved by treatment
with thiourea in the presence of sym-collidine to give
trisaccharide 40, which was glycosylated with glycosyl donor
28 using a catalytic amount of TMSOTf as promotor to afford
tetrasaccharide 41 in 90% yield. Trifluoro-N-phenylacetimidate
42 was prepared by a two-step procedure involving selective
removal of the ally ether of 41 using PdCl2 to give a lactol,
which was reacted with N-phenyltrifluoroacetimidoyl chloride
in the presence of Cs2CO3.32,33
Having glycosyl acceptor 29 and donor 42 in hand, attention
was focused on the preparation of inner core hexasaccharide 1.
Preactivated of glycosyl acceptor 29 using a catalytic amount of
TMSOTf followed by slow addition of glycosyl donor 42 at
room temperature resulted in the formation of hexasaccharide
43. Inverse glycosylation34,35 at room temperature was
essential, and standard glycosylation conditions gave only
trace amounts of 43, and in this case mainly decomposition of
the glycosyl donor and acceptor was observed. The low
reactivity of the sterically hindered C-5 hydroxyl of 29
probably causes a mismatch in reactivity with glycosyl donor
42 resulting in a challeging glycosylation. Finally, global
deprotection of 43 to give hexasaccharide 1 was accomplished
by saponification of the esters and carbonate using sodium
hydroxide, followed by catalytic hydrogenolysis over Pd-
(OH)2/C to remove the benzyl ethers. The NMR data of 1
confirmed the anomeric configurations of all glycosidic
linkages.
RESULTS AND DISCUSSION
■
Chemical Synthesis of Inner Core Oligosaccharide
(1). The chemical synthesis of the inner core hexasaccharide 1
is challenging due to the low acceptor reactivity of the C5-
hydroxyl of Kdo-α-(2→4)-Kdo acceptor 2920−22 and the need
to install glycosidic bonds to form a highly crowded 3,4-
branched heptoside.23,24 We envisaged that fully protected
hexasaccharide 1 could be synthesized by a convergent and
stereocontrolled [4 + 2] approach (Scheme 1) relying on the
use of flexible disaccharide intermediate 26, which is modified
by the orthogonal protecting groups 2-naphthylmethyl (Nap)
ether, chloroacetyl (ClAc) ester, and allyl (All) ether. The
ability to remove the Nap ether and ClAc ester protecting
groups in different orders made it possible to establish the
optimal glycosylation sequence, using glycosyl 27 and 28, to
prepare tetrasaccharide 41 having a crowded 3,4-branched
heptoside. Removal of the anomeric allyl ether of 41 and
conversion of the resulting lactol into a donor followed by
glycosylation with 29 would install the challenging α(1→5)-
linked Hep-Kdo glycosidic linkage affording the protected
hexasaccharide 43. Global deprotection of the latter compound
would provide the required inner core hexasaccharide 1.
First, α(2→4)-linked Kdo dimer 29 was prepared which has
a free hydroxyl at C5 for further glycosylation at this position
(Scheme 1B). Glycosylation using a Kdo donor with an iodide
at C3 as a stereodirecting group is attractive because it
provides only α-anomeric products.25 Thus, coupling of donor
30 with benzyloxycarbonyl protected 5-aminopentanol in the
presence of BF3·Et2O (2 equiv) as the promoter gave ketoside
31 in a yield of 75% as only the 2,3-trans-isomer. Only a small
amount of the glycal byproduct (8%) was isolated due to a
competing elimination reaction. The 3-iodo-substituent of 31
was readily removed by hydrogen atom transfer from
cyclohexane induced by lauroyl peroxide in 1,2-dichloroethane
to afford spacer modified α-linked Kdo 32.26 1H NMR analysis
of 32 showed a low-field shift of H-4 (δ = 5.26 ppm), which
was typical for an α-anomeric Kdo linkage in 4-O-acylated Kdo
derivatives.27,28 Treatment of 32 with a catalytic amount of
sodium methoxide in methanol resulted in removal of the
acetyl esters to give tetraol 33. The heteronuclear coupling
constant of C-1 with the axial proton at C-3 position (3JC1−H3ax
< 1 Hz) further confirmed the α-anomeric configuration of
Kdo.27 The 7,8-diol of 33 was selectively protected as a cyclic
carbonate by treating with diphosgene (1 equiv) in the
presence of sym-collidine at −25 °C to give 34 having a free
diol at C-4,5. Careful control of the reaction conditions
including the equivalence of diphosgene and temperature was
important to avoid further reaction of the 4,5-O-diol. The
equatorial C4 hydroxyl of 34 has higher glycosyl acceptor
reactivity compared to the axial hydroxyl at C5, and thus it was
expected that 34 is an appropriate acceptor for glycosylation at
C4 without a need for further protecting group manipulations.
Indeed, a BF3·Et2O promoted glycosylation of 30 with 34
resulted in the selective formation of α(1,4)-linked ketoside 35
Previously, we36 and others23,37,38 have employed a 7,8-O-
isopropylidene protected Kdo acceptor for glycosylations to
increase the reactivity of the axial C5-hydroxyl. However, the
use of such an acceptor proved incompatible with BF3·Et2O
mediated glycosylation and resulted in the formation of a
complex mixture of products probably due to isopropylidene
cleavage (Figure S2). Our studies demonstrate that a 7,8-
carbonate is an attractive protecting group for Kdo containing
oligosaccharides being extended at the anomeric center and C-
4 and C-5. We also explored an alternative glycosylation
sequence for the preparation of 1 by constructing the
challenging α-Hep-(1→5)-Kdo glycosidic linkage to give α-
Hep-(1→5)-Kdo-α-(2→4)-Kdo trisaccharide that was con-
verted into a glycosyl acceptor for coupling with a β-Gal-(1→
3)-Hep donor to form a properly protected pentasaccharide for
further glucosylation (Figure S3). Although the pentasacchar-
ide was detected by MALDI-MS, it was difficult to purify and
D
https://dx.doi.org/10.1021/jacs.0c08583
J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

Journal of the American Chemical Society
pubs.acs.org/JACS
Article
a
Scheme 2. Enzymatic Extension of 1 to Install Various Outer Core Ganglioside Epitopes
a
Reagents and conditions: (a) PmST1, CMP-Neu5Ac, 100 mM Tris-HCl (pH 8.0), 37 °C, 81% for 2, 75% for 5, 74% for 10, 76% for 12; (b)
CgtA, UDP-GalNAc, 10 mM MgCl2, 50 mM Tris-HCl (pH 7.5), 37 °C, 92% for 3, 88% for 8; (c) CgtB, UDP-Gal, 10 mM MgCl2, 50 mM Tris-
HCl (pH 7.5), 37 °C, 83% for 4, 80% for 9; (d) CstII, CMP-Neu5Ac, 100 mM Tris-HCl (pH 8.0), 37 °C, 76% for 6, 77% for 7, 74% for 13; (e)
α2-3,6,8,9 neuraminidase A from Arthrobacter ureafaciens, 5 mM CaCl2, 50 mM sodium acetate (pH 5.5), 37 °C, 94%.
the yield was low (<10%). Probably steric hindrance at the C-
3″ hydroxyl of α-Hep-(1→5)-Kdo-α-(2→4)-Kdo trisaccharide
led to failure of the glycosylation.
First, inner-core hexasaccharide 1 was sialylated with PmST1
and cytidine-5′-monophospho-N-acetylneuraminic acid
(CMP-Neu5Ac) to give GM3 mimic 2. The progress of the
reaction was carefully monitored by ESI-MS because PmST1
has residual hydrolytic activity and, after consumption of
CMP-NeuAc, can hydrolyze the product.45 Oligosaccharide 2
was smoothly converted into GM2 mimic 3 using CgtA and
uridine 5′-diphospho-N-acetylgalactosamine (UDP-GalNAc).
Next, the synthesis of GM1a mimic 4 was accomplished by
treatment of 3 with CgtB in the presence of uridine 5′-
diphosphogalactose (UDP-Gal). To accomplish a high yield, it
was critical to employ only 1 equiv of UDP-Gal because an
excess of this reagent resulted in further galactosylation of 4.
α2,3-Sialylation of 4 with PmST1 and CMP-Neu5Ac resulted
in the formation of GD1a mimic 5, which was further sialylated
using α(2,8)-sialyltransferase CstII in the presence of CMP-
Neu5Ac and calf intestine alkaline phosphatase (CIAP) to
Enzymatic Synthesis of Ganglioside Oligosaccharides
and Ganglioside Mimics. Having inner core hexasaccharide
1 in hand, attention was focused on enzymatic extension to
give ganglioside mimics 2−13 (Scheme 2). Glycosyltrans-
ferases from microbes such as C. jejuni and Pasteurella
multocida have been identified that can assemble ganglio-series
oligosaccharides. These include the α2,3-sialyltransferase
PmST139 from Pasteurella multocida that can form GM3; the
β1,4-N-acetylgalactosaminyltransferase (CgtA) from C. jejuni
which can synthesize GM2; β1,3-galactosyltransferase
(CgtB)40 from C. jejuni that can prepare GM1; and the
bifunctional sialyltransferase CstII41 from C. jejuni that has
both α2,3-sialyltransferase (GM3 oligosaccharide synthase)
and α2,8-sialyltransferase (GD3 oligosaccharide synthase)
activity. Starting from lactose, these enzymes have been used
to prepare several oligosaccharide moieties of gangliosides
including GM3, GM2, GM1a, GD3, GD2, and GD1b.42 The
enzyme toolbox has, however, not made it possible to prepare
a number of other ganglio-oligosaccharides including GA1,
GM1b, and GD1c. Here, we introduce a synthetic strategy that
addressed the latter deficiency and made it possible to prepare
an unprecedented collection of ganglio-oligosaccharides (14−
25). It was expected that the galactosyl moiety of inner-core
oligosaccharide 1 would provide a proper starting structure for
extension by the glycosyltransferases24,42−44 and was amenable
to our strategy to give access to ganglioside mimics 2−12.
1
provide GT1a mimic 6 (Scheme 2A). H NMR analysis of 6
showed a downfield shift of the H-8 (from 3.73 to 4.02 ppm)
compared to the same proton of the terminal α(2,3)-Neu5Ac-
II residue of 5. In addition, a NOESY spectrum revealed a
correlation between the H-3ax (δ = 1.580) of terminal α(2,8)-
Neu5Ac-III and H-8 (δ = 4.016) of α(2,3)-Neu5Ac-II43
(Figure S16), further confirming that the α(2,8)-Neu5Ac
moiety was attached to the terminal α(2,3)-Neu5Ac. Thus, the
results demonstrate that CstII only extends the terminal α2,3-
linked sialoside.
In parallel, 2 was converted into GD3 mimic 7 using CstII
and CMP-Neu5Ac (Scheme 2B). The amount of CMP-
Neu5Ac was carefully controlled to avoid further α(2,8)-
E
https://dx.doi.org/10.1021/jacs.0c08583
J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

Journal of the American Chemical Society
pubs.acs.org/JACS
Article
Figure 2. Microarray results of the synthetic ganglioside library at 100 μM with (A) RCA I (20 μg/mL), (B) SBA (20 μg/mL), (C) MAL II (20
μg/mL), (D) WGA (10 μg/mL), (E) anti-GD1a mAb, clone GD1a-1 (2 μg/mL), and (F) anti-GD2 mAb, clone 14.G2a (2 μg/mL). Bars represent
the mean SD.
sialylation.44 Furthermore, it was observed that the rate of
α(2,8)-sialylation of 2 is faster than for 6. Next, 7 was
subjected to CgtA in the presence of UDP-GalNAc to form
GD2 mimic 8. Treatment of the latter compound with CgtB
and 1 equiv UDP-Gal resulted in the formation of GD1b
mimic 9, which was further sialylated by PmST1 to provide
GT1b mimic 10.
The corresponding ganglioside oligosaccharides 14−25
(Figure S11) were prepared by a similar strategy starting
from lactose modified by an anomeric aminopropyl linker. The
preparation of GA1, GM1b, and GD1c did not require
expensive α2-3,6,8,9 neuraminidase, and in this case, treatment
of 16 with 2 M acetic acid resulted in clean removal of the
internal Neu5Ac to give GA1 oligosaccharide 23 (Figure
S11),47 which was further extended using appropriate
glycosyltransferases to provide GM1b and GD1c oligosacchar-
ides 24 and 25, respectively. It is important to note that
treatment of 4 with acetic acid results in cleavage of the Kdo
glycosidic linkages.
The enzymatic conversions were monitored by ESI-MS, and
products were purified by Bio-Gel P-2 or P-4 size exclusion
chromatography and HPLC using a HILIC column (XBridge
Amide 5 μm, 10 mm × 250 mm, Waters), and the structural
identity of each compound was confirmed by NMR spectros-
copy (1D and 2D) and HR-MS.
Glycan Recognition of Lectins, mAbs, and Serum
Antibodies Using a Glycan Micoarray Platform. The
synthetic oligosaccharides 1−25 were employed to examine
specificities of serum antibodies of patients in the acute stage
of GBS following a C. jejuni infection, and the results were
compared to healthy control serum samples. For this purpose,
a glycan microarray was constructed by piezoelectric non-
contact printing of the aminopentyl containing glycans onto N-
hydroxysuccinimide (NHS) ester activated glass in replicates
The synthesis of GA1, GM1b, and GD1c mimics 11, 12, and
13 is challenging because previous studies have shown that
CgtA46 requires an α(2,3)-linked sialic acid at the terminal
galactoside to install a β(1,4)-GalNAc moiety, and thus
compound 1 cannot be extended by this enzyme and the
enzyme toolbox does not provide entry into 11−13. To
address this difficulty, we opted to treat 4 with a sialidase to
give 11 which can then be further elaborated with the
glycosyltransferases to provide 12 and 13. Commercially
available α2-3,6,8 neuraminidases from Clostridium perfringens
and Vibrio cholerae were not able to cleave the internal and
branched Neu5Ac moiety. Gratifyingly, α2-3,6,8,9 neuramini-
dase A from Arthrobacter ureafaciens could remove the Neu5Ac
residue of 4 to give GA1 mimic 11. A relatively high
concentration of this neuraminidase and a prolonged
incubation time were required to cleave the internal Neu5Ac
residue. Sialylation of 11 using PmST1 and CMP-Neu5Ac
afforded GM1b mimic 12, which could be further sialylated
using CstII to provide GD1c mimic 13 (Scheme 2C).
F
https://dx.doi.org/10.1021/jacs.0c08583
J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

Journal of the American Chemical Society
pubs.acs.org/JACS
Article
Figure 3. Microarray results of the synthetic ganglioside library (1−25) at 100 μM with serum samples (1:500) (A) S005, (B) S038, (C) S002,
(D) S012, (E) S010, and (F) S023. For each sample, the graph at the top shows in blue IgG responses and the graph at the bottom in green IgM
responses. Bars represent the mean SD. C indicates blank control. Assays are performed in the same session, and results are depicted at the same
scale.
G
https://dx.doi.org/10.1021/jacs.0c08583
J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

Journal of the American Chemical Society
pubs.acs.org/JACS
Article
of six. To validate the array, the slides were examined for
binding by the plant lectins RCA I, SBA, MAL II, and WGA.
The subarrays were also probed for binding by a number of
commercially available anti-ganglioside antibodies.
against GA1 (23); however, the clinical significance of this
observation needs validation. As expected, on slide treatment
of 14 and 19 with neuraminidase to create lactose abolished
binding confirming lactose is not recognized by the serum
antibodies.
RCA I binds galactose, and as expected, all compounds
bearing such a terminal residue (16/4 GM1a/GM1a mimic;
21/9 GD1b/GD1b mimic; 23/11 GA1/GA1 mimic) were
recognized (Figure 2A). SBA preferentially binds GalNAc but
also recognizes Gal residues although with much lower
affinity.48 Binding to SBA was observed for compounds having
a terminal GalNAc including compounds 15 (GM2), 20
(GD2), and their corresponding mimics 3 and 8 (Figure 2B).
Weak binding was detected for compounds 23/11 (GA1/GA1
mimic) having a terminal Gal residue, and no binding was
observed for 16/4 (GM1a/GM1a mimic) and 21/9 (GD1b/
GD1b mimic) due to the inhibitory effect of Neu5Ac.49 MAL-
II binds Neu5Ac(α2−3)Gal(β1−4)GlcNAc/Glc moieties,50
and as anticipated, compounds 14 and 19, having this epitope,
were bound by this lectin (Figure 2C).49 The corresponding
ganglioside mimics 2 and 7, respectively, were not recognized
most likely because the glucose moiety of the binding epitope
is replaced by heptose. Finally, we examined WGA, which
preferentially binds GlcNAc moieties and also interacts with
glycoproteins via terminal sialic acid residues. As expected,
only the ganglioside oligosaccharides having a terminal
α(2,3)Neu5Ac moiety (14, 17, 22, 24) and the corresponding
mimics (2, 5, 10, 12) showed binding (Figure 2D).
Next, binding profiles of several anti-ganglioside antibodies
were determined. As expected, an anti-GD1a mAb (Figure 2E)
only showed binding to 17 (GD1a) and 5 (GD1a mimic), and
an anti-GD2 mAb (Figure 2F) only bound to 20 (GD2) and 8
(GD2 mimic). A polyclonal GM1 antibody (Figure S12A)
exhibited some promiscuity and recognized compounds 16/4
(GM1a/GM1a mimic), 21/9 (GD1b/GD1b mimic), and 23/
11 (GA1/GA1 mimic), indicating the terminal Gal(β1,3)-
GalNAc moiety of these compounds is recognized.51 The
subarrays were also treated with neuraminidase A (N-A),
which cleaves terminal α2,3-, α2,6-, and α2,8-linked sialic acid
residues, and then reprobed by this antibody, showing the
expected binding patterns (Figure S12B). Importantly, the
antibodies recognized the corresponding ganglio-oligosacchar-
ides and ganglio-mimics with similar intensities indicating that
the underlying inner-core oligosaccharide of the mimics does
not influence recognition.
It was observed that there is a significant correlation
between the recognition of specifc ganglio-oligosaccharides
and the corresponding mimic indicating cross-reactivity (see
Figure S14 for 4 vs 16). However, for almost all of the
examined samples, the binding of the ganglioside mimics (2−
13) was much stronger than that of the corresponding
ganglioside oligosaccharides (14−25). Furthermore, in most
cases, no or a low response was observed for the inner-core
oligosaccharide (1). These observations indicate that the GBS
patients had elicited antibodies mainly to the outer core region
of LOS of C. jejuni. The finding that the ganglioside
oligosaccharides are not as well recognized as the mimics
indicates that gangliosides are suboptimal ligands for the serum
antibodies. In this respect, the ganglio-oligosaccharides have a
lactosyl (Galβ(1,4)Glc) moiety at the reducing end, whereas
the mimics have a Galβ(1,3)Hep epitope at this position.
Thus, it is likely that the serum antibodies recognize an epitope
that encompasses the heptosyl residue of the inner-core and
the ganglio-oligosaccharide of the outer core. A heptoside
poorly mimics the glucosyl moiety of gangliosides, thereby
providing a rationale for the differences in responsiveness of
ganglio-oligosaccharides and mimics. Thus, it appears that
antibodies elicited during C. jejuni infection leading to GBS are
directed to epitopes that straddle the inner- and outer-core
region of LOS that can cross-react with autologous ganglio-
sides. This finding is in accordance with the clinical
observation that GBS has a monophasic course in which
patients tend to recover after clearing of the infection.
Although commonalities were observed in the antibody
responses, also differences were noted, which probably is due
to the clinical heterogeneity of GBS. First, the magnitude of
the antibody responses differed considerably between the
various serum samples. For one patient, mainly IgG antibodies
were observed (S005), whereas for another one predominantly
IgM antibodies were detected (S038). However, most patients
had elicited IgM and IgG antibodies, but in these cases also
differences in binding patterns were observed. For several
patients (S010, S014, S023, S035, S039) IgM and IgG
antibodies were detected that bound to the ganglio-
oligosaccharides as well as to the mimics. Other samples
(S002, S007, S012) showed strong IgG antibody responses to
the ganglioside mimics (2−14) with little- or no binding to the
corresponding ganglio-oligosaccharides. In these cases, IgM
antibodies were present that bound the ganglioside-oligosac-
charides. Probably, class switching and antibody affinity
maturation resulted in IgG antibodies with high affinity for
the mimics but a low affinity for the gangliosides. It can,
however, not be excluded that antibodies binding potently to
gangliosides have been scavenged by nerves leaving antibodies
to the mimics behind.53
Having established the glyco-microarray is appropriate for
examining specificities of anti-ganglioside antibodies, attention
was focused on determining IgG and IgM antibodies binding
profiles of 12 serum samples from patients that suffered from
́
Guillain−Barre syndrome and had an antecedent C. jejuni
infection (Table S2) and showed positive serological responses
in a traditional ELISA for anti-ganglioside antibodies (Table
S3).
Representative array results are presented in Figure 3, and
the data for all patient serum samples are shown in Figure
S13A. In each sample, IgM and/or IgG antibodies were
observed that bound one or more of the ganglioside
oligosaccharides (14−25). Anti-GM1a (16), anti-GM1b
(24), and anti-GD1a (17) antibodies have been associated
with the motor form of GBS after C. jejuni infection.11,12,52 The
array data showed that the majority of the patients had elicited
IgM and/or IgG antibodies targeting GM1a. Anti-GM1b and
GD1a antibody responses were observed in only a few serum
samples. Interestingly, many patients had elicited antibodies
We also examined five serum samples (S017, S019, S024,
S033, S037) of patients that had suffered GBS but for whom
no anti-ganglioside antibodies had been detected by traditional
ELISA (Table S3). In three of these samples (S017, S024,
S033), the microarray detected anti-GM1a IgM antibody
indicating the new platform is more sensitive to detect such
antibodies (Figures S13B and S15). In addition, we analyzed
antibody responses of 10 control serum samples, and as
H
https://dx.doi.org/10.1021/jacs.0c08583
J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

Journal of the American Chemical Society
pubs.acs.org/JACS
Article
expected, no or very low responses were measured (Figure
S13C).
IgM antibodies targeting GM1a. This may be due to the
covalent binding of the antigens allowing the use of detergents
during washing steps reducing nonspecific binding. Further-
more, a good correlation was found between the responsive-
ness of GM1a and its mimic (Figure S14), and thus the
ganglioside mimics, which were recognized much more
potently, may be attractive for the detection of serum
antibodies associated with GBS. A glycan microarray has
many advantages for the development of the next generation
platform to examine the presence of anti-ganglioside antibodies
to facilitate GBS diagnoses and disease monitoring. It requires
minute quantities of precious oligosaccharides that can be
printed in a format that allows in one operation the
examination of a large number of compounds and samples.
The assay is robust because compounds are chemically
immobilized, and it is more sensitive and has a greater
dynamic range compared to traditional ELISA. Antibodies that
recognize ganglioside complexes have been observed in GBS
patients.55 A microarray platform makes it possible to print
many different combinations of ganglio-oligosaccharides
allowing the detection of such antibodies. To establish it as a
platform for GBS diagnoses and monitoring of disease activity,
future efforts will focus on the preparation of additional
ganglioside mimics to determine the minimal epitope for
binding by serum antibodies. In particular, attention will be
focused on compounds having truncated inner core
oligosaccharides to determine to what extent it is recognized
by the serum antibodies. In addition, a large number of serum
samples will be screened to establish threshold values of
clinically relevant antibody responses.
CONCLUSIONS
■
It is widely accepted that antibodies elicited to the outer core
oligosaccharide of the LOS of C. jejuni, which structurally
resembles the oligosaccharide moiety of gangliosides, cause
neural injury and clinical symptoms of GBS.1−3 Despite their
importance in the pathogenesis of GBS, the fine specificities of
anti-ganglioside serum antibodies have been difficult to
examine, and for example it is not known if elements of the
inner core are part of antigenic epitopes. It is the expectation
that knowledge of the fine specificities of anti-ganglioside
antibodies elicited by GBS patients will provide a more
comprehensive understanding of the microbial factors
important for the pathogenesis of GBS and give opportunties
to develop more robust platforms for diagnosis and monitoring
of disease activity. Such investigations require a comprehensive
set of well-defined and pure lipo-oligosaccharides of C. jejuni in
which the conserved inner core is extended by various
ganglioside mimetics. It also necessitates a complementary
range of ganglio-oligosaccharides to systematically compare
antigenic responses with the ganglio-mimics. To obtain a
comprehensive range of ganglioside mimics, we drew
inspiration from enzymatic approaches to prepare ganglio-
oligosaccharides by extension of lactose by various microbial
glycosyl transferases41,42,44,54 and envisaged that an inner core
terminating in galactose would be also an appropriate
precursor for these enzymes. We devised an efficient synthetic
approach for C. jejuni inner core hexasaccharide based on a
convergent and stereo−controlled [4 + 2] oligosaccharide
assembly. It employed orthogonally protected building blocks
that offered flexibility in the assembly of the hexasaccharide.
Enzymatic extension of the hexasaccharide by a panel of
glycosyltransferases afforded core oligosaccharides having
various outer core ganglioside mimics. In parallel, we
developed a chemoenzymatic approach for the gangliosides
including the previously inaccessible GA1, GM1b, and GD1c
by chemical or enzymatic removal of the sialoside of GM1a
followed by enzymatic extension. The synthetic efforts provide
an unprecedented collection of ganglio-oligosaccharides and
their mimics including GM1, GM2, GM1a, GD1a, GT1a,
GD3, GD2, GD1b, GT1b, GA1, GM1b, and GD1c.
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/jacs.0c08583.
■
sı
Synthetic protocols, microarray procedures, compound
characterization, and NMR and LC−MS spectra (PDF)
AUTHOR INFORMATION
Corresponding Author
■
Geert-Jan Boons − Complex Carbohydrate Research Center
and Department of Chemistry, University of Georgia, Athens,
Georgia 30602-4712, United States; Department of
Chemical Biology and Drug Discovery, Utrecht Institute for
Pharmaceutical Sciences, and Bijvoet Center for Biomolecular
Research, Utrecht University, 3584 Utrecht, The
Netherlands; orcid.org/0000-0003-3111-5954;
Email: g.j.p.h.boons@uu.nl, gjboons@ccrc.uga.edu
The oligosaccharides were printed as a microarray to
examine fine specificities of serum antibodies of GBS patients
that had suffered an antecedent C. jejuni infection. The results
showed that the majority of GBS patients had elicited IgM
and/or IgG antibodies targeting GM1a (16). Interestingly, in
almost all cases the GM1a mimic (4) was bound much more
strongly indicating that in addition to the outer core, which
mimics GM1a, part of the inner core is also recognized. The
inner core (1) by itself gave low or no responsiveness
highlighting that the outer core contains the critical antigenic
component. Collectively, these observations indicate that
antibodies elicited during the preceding C. jejuni infection
leading to GBS are primarily raised to an epitope on the
pathogen (LOS) that cross-react to the autologous ganglio-
sides. Thus, although the outer core resembles a self-structure,
it is attached to heptosyl residue thereby creating a foreign
antigen for mammalian immune cells. It was also observed that
the microarray platform can more sensitively detect anti-GM1a
antibodies compared to a traditional ELISA format using
bovine extracted gangliosides, which was especially the case for
Authors
Tiehai Li − Complex Carbohydrate Research Center,
University of Georgia, Athens, Georgia 30602-4712, United
States
Margreet A. Wolfert − Complex Carbohydrate Research
Center, University of Georgia, Athens, Georgia 30602-4712,
United States; Department of Chemical Biology and Drug
Discovery, Utrecht Institute for Pharmaceutical Sciences, and
Bijvoet Center for Biomolecular Research, Utrecht University,
3584 Utrecht, The Netherlands; orcid.org/0000-0003-
4864-0026
Na Wei − Complex Carbohydrate Research Center, University
of Georgia, Athens, Georgia 30602-4712, United States
I
https://dx.doi.org/10.1021/jacs.0c08583
J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

Journal of the American Chemical Society
pubs.acs.org/JACS
Article
Furukawa, K.; Plomp, J. J.; Willison, H. J. Overexpression of GD1a
ganglioside sensitizes motor nerve terminals to anti-GD1a antibody-
mediated injury in a model of acute motor axonal neuropathy. J.
Neurosci. 2005, 25, 1620−1628.
(13) Godschalk, P. C.; Kuijf, M. L.; Li, J.; St. Michael, F.; Ang, C.
W.; Jacobs, B. C.; Karwaski, M. F.; Brochu, D.; Moterassed, A.; Endtz,
H. P.; van Belkum, A.; Gilbert, M. Structural characterization of
Campylobacter jejuni lipooligosaccharide outer cores associated with
Ruth Huizinga − Department of Immunology, Erasmus MC,
University Medical Center Rotterdam, 3015 GD Rotterdam,
The Netherlands; orcid.org/0000-0002-9563-4860
Bart C. Jacobs − Department of Immunology and Department
of Neurology, Erasmus MC, University Medical Center
Rotterdam, 3015 GD Rotterdam, The Netherlands;
orcid.org/0000-0002-8985-2458
́
Guillain-Barre and Miller Fisher syndromes. Infect. Immun. 2007, 75,
1245−1254.
Complete contact information is available at:
https://pubs.acs.org/10.1021/jacs.0c08583
(14) Gilbert, M.; Karwaski, M. F.; Bernatchez, S.; Young, N. M.;
Taboada, E.; Michniewicz, J.; Cunningham, A. M.; Wakarchuk, W. W.
The genetic bases for the variation in the lipo-oligosaccharide of the
mucosal pathogen, Campylobacter jejuni. Biosynthesis of sialylated
ganglioside mimics in the core oligosaccharide. J. Biol. Chem. 2002,
277, 327−337.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
(15) Nachamkin, I.; Allos, B. M.; Ho, T. Campylobacter species and
The authors thank Dr. J. Glushka for assisting with NMR
experiments, A. P. Tio-Gillen and W. van Rijs for technical
assistance with ELISAs, and Dr. L. Liu for assisting with
microarray printing and calculations. This research was
supported by the National Institute of General Medical
Sciences from the U.S. National Institutes of Health (Grant
U01GM120408 to G.-J.B.) and the Dutch Prinses Beatrix
Spierfonds (Grant W.OR09-04 to B.C.J), cofinanced by
Stichting Spieren voor Spieren.
́
Guillain-Barre syndrome. Clin. Microbiol. Rev. 1998, 11, 555−567.
(16) Yuki, N.; Yoshino, H.; Sato, S.; Miyatake, T. Acute axonal
polyneuropathy associated with anti-GM1 antibodies following
Campylobacter enteritis. Neurology 1990, 40, 1900−1902.
(17) Jacobs, B. C.; van Doorn, P. A.; Schmitz, P. I.; Tio-Gillen, A. P.;
Herbrink, P.; Visser, L. H.; Hooijkaas, H.; van der Meche, F. G.
Campylobacter jejuni infections and anti-GM1 antibodies in Guillain-
́
Barre syndrome. Ann. Neurol. 1996, 40, 181−187.
(18) Ravindranath, M. H.; Ravindranath, R. M. H.; Morton, D. L.;
Graves, M. C. Factors affecting the fine specificity and sensitivity of
serum antiganglioside antibodies in ELISA. J. Immunol. Methods 1994,
169, 257−272.
REFERENCES
■
́
(1) Goodfellow, J. A.; Willison, H. J. Guillain-Barre syndrome: a
(19) van Belkum, A.; van den Braak, N.; Godschalk, P.; Ang, W.;
Jacobs, B.; Gilbert, M.; Wakarchuk, W.; Verbrugh, H.; Endtz, H. A
Campylobacter jejuni gene associated with immune-mediated neuro-
pathy. Nat. Med. 2001, 7, 752−753.
(20) Pokorny, B.; Kosma, P. First and stereoselective synthesis of an
alpha-(2→5)-linked disaccharide of 3-deoxy-D-manno-oct-2-ulosonic
acid (Kdo). Org. Lett. 2015, 17, 110−113.
(21) Kong, L.; Vijayakrishnan, B.; Kowarik, M.; Park, J.; Zakharova,
A. N.; Neiwert, L.; Faridmoayer, A.; Davis, B. G. An antibacterial
vaccination strategy based on a glycoconjugate containing the core
lipopolysaccharide tetrasaccharide Hep2Kdo2. Nat. Chem. 2016, 8,
242−249.
(22) Trattnig, N.; Farcet, J. B.; Gritsch, P.; Christler, A.; Pantophlet,
R.; Kosma, P. Synthesis of a pentasaccharide fragment related to the
inner core region of rhizobial and agrobacterial lipopolysaccharides. J.
Org. Chem. 2017, 82, 12346−12358.
century of progress. Nat. Rev. Neurol. 2016, 12, 723−731.
(2) Willison, H. J.; Jacobs, B. C.; van Doorn, P. A. Guillain-Barre
syndrome. Lancet 2016, 388, 717−727.
(3) Kondziella, D. Autonomic dysfunction in Guillain-Barre
syndrome puts patients at risk. Neurocrit. Care 2020, 32, 86−87.
(4) Kaida, K.; Ariga, T.; Yu, R. K. Antiganglioside antibodies and
́
́
́
their pathophysiological effects on Guillain-Barre syndrome and
related disorders - a review. Glycobiology 2009, 19, 676−692.
(5) van den Berg, B.; Walgaard, C.; Drenthen, J.; Fokke, C.; Jacobs,
́
B. C.; van Doorn, P. A. Guillain-Barre syndrome: pathogenesis,
diagnosis, treatment and prognosis. Nat. Rev. Neurol. 2014, 10, 469−
482.
(6) Rees, J. H.; Soudain, S. E.; Gregson, N. A.; Hughes, R. A. C.
́
Campylobacter jejuni Infection and Guillain-Barre syndrome. N. Engl.
J. Med. 1995, 333, 1374−1379.
(7) Jacobs, B. C.; Rothbarth, P. H.; van der Meche, F. G.; Herbrink,
P.; Schmitz, P. I.; de Klerk, M. A.; van Doorn, P. A. The spectrum of
(23) Bernlind, C.; Oscarson, S. Synthesis of a branched heptose- and
KDO-containing common tetrasaccharide core structure of Haemo-
philus influenzae lipopolysaccharides via 1,6-anhydro-L-glycero-beta-
D-manno-heptopyranose intermediate. J. Org. Chem. 1998, 63, 7780−
7788.
́
antecedent infections in Guillain-Barre syndrome: a case-control
study. Neurology 1998, 51, 1110−1115.
(8) van den Berg, B.; van der Eijk, A. A.; Pas, S. D.; Hunter, J. G.;
Madden, R. G.; Tio-Gillen, A. P.; Dalton, H. R.; Jacobs, B. C.
(24) Yoshida, F.; Yoshinaka, H.; Tanaka, H.; Hanashima, S.;
Yamaguchi, Y.; Ishihara, M.; Saburomaru, M.; Kato, Y.; Saito, R.;
Ando, H.; Kiso, M.; Imamura, A.; Ishida, H. Synthesis of the core
oligosaccharides of lipooligosaccharides from Campylobacter jejuni: A
́
Guillain-Barre syndrome associated with preceding hepatitis E virus
infection. Neurology 2014, 82, 491−497.
(9) Cao-Lormeau, V. M.; Blake, A.; Mons, S.; Lastere, S.; Roche, C.;
Vanhomwegen, J.; Dub, T.; Baudouin, L.; Teissier, A.; Larre, P.; Vial,
A. L.; Decam, C.; Choumet, V.; Halstead, S. K.; Willison, H. J.;
Musset, L.; Manuguerra, J. C.; Despres, P.; Fournier, E.; Mallet, H. P.;
Musso, D.; Fontanet, A.; Neil, J.; Ghawche, F. Guillain-Barre
syndrome outbreak associated with Zika virus infection in French
Polynesia: a case-control study. Lancet 2016, 387, 1531−1539.
(10) Young, K. T.; Davis, L. M.; DiRita, V. J. Campylobacter jejuni:
molecular biology and pathogenesis. Nat. Rev. Microbiol. 2007, 5,
665−679.
(11) Yuki, N.; Susuki, K.; Koga, M.; Nishimoto, Y.; Odaka, M.;
Hirata, K.; Taguchi, K.; Miyatake, T.; Furukawa, K.; Kobata, T.;
Yamada, M. Carbohydrate mimicry between human ganglioside GM1
and Campylobacter jejuni lipooligosaccharide causes Guillain-Barre
syndrome. Proc. Natl. Acad. Sci. U. S. A. 2004, 101, 11404−11409.
(12) Goodfellow, J. A.; Bowes, T.; Sheikh, K.; Odaka, M.; Halstead,
S. K.; Humphreys, P. D.; Wagner, E. R.; Yuki, N.; Furukawa, K.;
́
putative cause of Guillain-Barre syndrome. Chem. - Eur. J. 2019, 25,
796−805.
(25) Pokorny, B.; Kosma, P. Synthesis of chlamydia lipopolysac-
charide haptens through the use of alpha-specific 3-iodo-Kdo fluoride
glycosyl donors. Chem. - Eur. J. 2015, 21, 305−313.
(26) Pokorny, B.; Kosma, P. Synthesis of 5-O-oligoglucosyl extended
alpha-(2→4)-Kdo disaccharides corresponding to inner core frag-
ments of Moraxellaceae lipopolysaccharides. Carbohydr. Res. 2016,
422, 5−12.
́
(27) Unger, F. M.; Stix, D.; Schulz, G. The anomeric configurations
of the two ammonium (methyl 3-deoxy-d-manno-2-octulopyranosid)-
onate salts (methyl α- and β-ketopyranosides of KDO). Carbohydr.
Res. 1980, 80, 191−195.
(28) Kosma, P.; Strobl, M.; Allmaier, G.; Schmid, E.; Brade, H.
Synthesis of pentasaccharide core structures corresponding to the
́
J
https://dx.doi.org/10.1021/jacs.0c08583
J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

Journal of the American Chemical Society
pubs.acs.org/JACS
Article
genus-specific lipopolysaccharide epitope of Chlamydia. Carbohydr.
Res. 1994, 254, 105−132.
hydrolysis and reduced sialidase activities for directly sialylating
Lewisx. ACS Chem. Biol. 2012, 7, 1232−1240.
(46) Wen, L. Q.; Zheng, Y.; Jiang, K.; Zhang, M. Z.; Kondengaden,
S. M.; Li, S. S.; Huang, K.; Li, J.; Song, J.; Wang, P. G. Two-step
chemoenzymatic detection of N-acetylneuraminic acid-alpha(2-3)-
galactose glycans. J. Am. Chem. Soc. 2016, 138, 11473−11476.
(47) Varki, A.; Diaz, S. The release and purification of sialic acids
from glycoconjugates: methods to minimize the loss and migration of
O-acetyl groups. Anal. Biochem. 1984, 137, 236−247.
(29) Xia, J.; Abbas, S. A.; Locke, R. D.; Piskorz, C. F.; Alderfer, J. L.;
Matta, K. L. Use of 1,2-dichloro 4,5-dicyanoquinone (DDQ) for
cleavage of the 2-naphthylmethyl (NAP) group. Tetrahedron Lett.
2000, 41, 169−173.
(30) Masaki, M.; Kitahara, T.; Kurita, H.; Ohta, M. A new method
for the removal of chloroacetyl groups. J. Am. Chem. Soc. 1968, 90,
4508−4509.
(48) Rao, V. S. R.; Lam, K.; Qasba, P. K. Three dimensional
structure of the soybean agglutinin Gal/GalNAc complexes by
homology modeling. J. Biomol. Struct. Dyn. 1998, 15, 853−860.
(49) ’t Hart, I. M. E.; Li, T.; Wolfert, M. A.; Wang, S.; Moremen, K.
W.; Boons, G. J. Chemoenzymatic synthesis of the oligosaccharide
moiety of the tumor-associated antigen disialosyl globopentaosylcer-
amide. Org. Biomol. Chem. 2019, 17, 7304−7308.
(50) Goldstein, I. J.; Winter, H. C.; Poretz, R. D. In New
Comprehensive Biochemistry; Elsevier, 1997; Vol. 29, Part B, pp
403−474.
́
̌
(31) Kovac, P.; Yeh, H. J. C.; Glaudemans, C. P. J. Synthesis of
methyl O-(3-deoxy-3-fluoro-β-d-galactopyranosyl)-(1→6)-β-d-galac-
topyranoside and methyl O-(3-deoxy-3-fluoro-β-d-galactopyranosyl)-
(1→6)-O-β-d-galactopyranosyl-(1→6)-β-d-galactopyranoside. Carbo-
hydr. Res. 1985, 140, 277−288.
(32) Yu, B.; Tao, H. C. Glycosyl trifluoroacetimidates. Part 1:
Preparation and application as new glycosyl donors. Tetrahedron Lett.
2001, 42, 2405−2407.
(33) Zhu, X. M.; Schmidt, R. R. New principles for glycoside-bond
formation. Angew. Chem., Int. Ed. 2009, 48, 1900−1934.
(34) Toepfer, A.; Schmidt, R. R. An efficient synthesis of the Lewis
X (Lex) antigen family. Tetrahedron Lett. 1992, 33, 5161−5164.
(35) Liu, M.; Yu, B.; Hui, Y. First total synthesis of 25(R)-
ruscogenin-1-yl β-D-xylopyranosyl-(1→3)-[β-D-glucopyranosyl-(1→
2)]-β-D-fucopyranoside, an ophiopogonis saponin from the tuber of
Liriope muscari (Decne.). Tetrahedron Lett. 1998, 39, 415−418.
(36) Boltje, T. J.; Zhong, W.; Park, J.; Wolfert, M. A.; Chen, W.;
Boons, G. J. Chemical synthesis and immunological evaluation of the
inner core oligosaccharide of Francisella tularensis. J. Am. Chem. Soc.
2012, 134, 14255−14262.
(51) Steck, A.; Yuki, N.; Graus, F. Antibody testing in peripheral
nerve disorders. Handb. Clin. Neurol. 2013, 115, 189−212.
(52) Kusunoki, S.; Iwamori, M.; Chiba, A.; Hitoshi, S.; Arita, M.;
Kanazawa, I. GM1b is a new member of antigen for serum antibody in
́
Guillain-Barre syndrome. Neurology 1996, 47, 237−242.
(53) Cunningham, M. E.; McGonigal, R.; Meehan, G. R.; Barrie, J.
A.; Yao, D.; Halstead, S. K.; Willison, H. J. Anti-ganglioside antibodies
are removed from circulation in mice by neuronal endocytosis. Brain
2016, 139, 1657−1665.
(54) Meng, X.; Yao, W.; Cheng, J.; Zhang, X.; Jin, L.; Yu, H.; Chen,
X.; Wang, F.; Cao, H. Regioselective chemoenzymatic synthesis of
ganglioside disialyl tetrasaccharide epitopes. J. Am. Chem. Soc. 2014,
136, 5205−5208.
(55) Rinaldi, S.; Brennan, K. M.; Kalna, G.; Walgaard, C.; van
Doorn, P.; Jacobs, B. C.; Yu, R. K.; Mansson, J. E.; Goodyear, C. S.;
Willison, H. J. Antibodies to heteromeric glycolipid complexes in
(37) Yang, Y.; Martin, C. E.; Seeberger, P. H. Total synthesis of the
core tetrasaccharide of Neisseria meningitidis lipopolysaccharide, a
potential vaccine candidate for meningococcal diseases. Chem. Sci.
2012, 3, 896−899.
(38) Huang, W.; Zhou, Y.; Pan, X.; Zhou, X.; Lei, J.; Liu, D.; Chu,
Y.; Yang, J. Stereodirecting effect of C5-carboxylate substituents on
the glycosylation stereochemistry of 3-deoxy-d-manno-oct-2-ulosonic
acid (Kdo) thioglycoside donors: Stereoselective synthesis of α- and
β-Kdo glycosides. J. Am. Chem. Soc. 2018, 140, 3574−3582.
(39) Yu, H.; Chokhawala, H.; Karpel, R.; Yu, H.; Wu, B.; Zhang, J.;
Zhang, Y.; Jia, Q.; Chen, X. A multifunctional Pasteurella multocida
sialyltransferase: a powerful tool for the synthesis of sialoside libraries.
J. Am. Chem. Soc. 2005, 127, 17618−17619.
́
Guillain-Barre syndrome. PLoS One 2013, 8, No. e82337.
(40) Bernatchez, S.; Gilbert, M.; Blanchard, M.-C.; Karwaski, M.-F.;
Li, J.; DeFrees, S.; Wakarchuk, W. W. Variants of the β1,3-
galactosyltransferase CgtB from the bacterium Campylobacter jejuni
have distinct acceptor specificities. Glycobiology 2007, 17, 1333−1343.
(41) Yu, H.; Cheng, J.; Ding, L.; Khedri, Z.; Chen, Y.; Chin, S.; Lau,
K.; Tiwari, V. K.; Chen, X. Chemoenzymatic synthesis of GD3
oligosaccharides and other disialyl glycans containing natural and
non-natural sialic acids. J. Am. Chem. Soc. 2009, 131, 18467−18477.
(42) Yu, H.; Li, Y.; Zeng, J.; Thon, V.; Nguyen, D. M.; Ly, T.;
Kuang, H. Y.; Ngo, A.; Chen, X. Sequential one-pot multienzyme
chemoenzymatic synthesis of glycosphingolipid glycans. J. Org. Chem.
2016, 81, 10809−10824.
(43) Gilbert, M.; Brisson, J. R.; Karwaski, M. F.; Michniewicz, J.;
Cunningham, A. M.; Wu, Y.; Young, N. M.; Wakarchuk, W. W.
Biosynthesis of ganglioside mimics in Campylobacter jejuni OH4384.
Identification of the glycosyltransferase genes, enzymatic synthesis of
model compounds, and characterization of nanomole amounts by
600-MHz 1H and 13C NMR analysis. J. Biol. Chem. 2000, 275, 3896−
3906.
(44) Blixt, O.; Vasiliu, D.; Allin, K.; Jacobsen, N.; Warnock, D.; Razi,
N.; Paulson, J. C.; Bernatchez, S.; Gilbert, M.; Wakarchuk, W.
Chemoenzymatic synthesis of 2-azidoethyl-ganglio-oligosaccharides
GD3, GT3, GM2, GD2, GT2, GM1, and GD1a. Carbohydr. Res.
2005, 340, 1963−1972.
(45) Sugiarto, G.; Lau, K.; Qu, J.; Li, Y.; Lim, S.; Mu, S.; Ames, J. B.;
Fisher, A. J.; Chen, X. A sialyltransferase mutant with decreased donor
K
https://dx.doi.org/10.1021/jacs.0c08583
J. Am. Chem. Soc. XXXX, XXX, XXX−XXX