Chemoenzymatic approach for the preparation of asymmetric bi-, tri-, and tetra-antennary N-glycans from a common precursor

Article abstract of DOI:10.1021/jacs.6b12080

Progress in glycoscience is hampered by a lack of well-defined complex oligosaccharide standards that are needed to fabricate the next generation of microarrays, to develop analytical protocols to determine exact structures of isolated glycans, and to elu

Full text of DOI:10.1021/jacs.6b12080

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Article
A Chemoenzymatic Approach for the Preparation of Asymmetric
Bi-, Tri- and Tetra-Antennary N-Glycans from a Common Precursor
Ivan A Gagarinov, Tiehai Li, Javier Sastre Torano, Tomislav Caval, Apoorva
D Srivastava, John A. W. Kruijtzer, Albert J.R. Heck, and Geert-Jan Boons
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.6b12080 • Publication Date (Web): 21 Dec 2016
Downloaded from http://pubs.acs.org on December 25, 2016
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A Chemoenzymatic Approach for the Preparation of Asymmetric Bi-, Tri- and
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Tetra-Antennary N-Glycans from a Common Precursor
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Ivan A. Gagarinov,1 Tiehai Li,2 Javier Sastre Toraño,1 Tomislav Caval,4,5 Apoorva D. Srivastava,1
John A. W. Kruijtzer,1 Albert J.R. Heck,4,5 Geert-Jan Boons*,1,2,3
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1Department of Chemical Biology and Drug Discovery, Utrecht Institute for Pharmaceutical Sciences,
and Bijvoet Center for Biomolecular Research, Utrecht University, Universiteitsweg 99, 3584 CG
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Utrecht, The Netherlands, Complex Carbohydrate Research Center; Department of Chemistry,
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University of Georgia, 315 Riverbend Road, Athens, GA 30602, USA; and Biomolecular Mass
Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute for
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Pharmaceutical Sciences, Utrecht University, 3584 CH Utrecht, The Netherlands; Netherlands
Proteomics Centre, CH Utrecht, The Netherlands
Keywords: glycosylation, N-linked glycan, glycosyltransferase, carbohydrate standard
ABSTRACT: Progress in glycoscience is hampered by a lack of well-defined complex
oligosaccharide standards that are needed to fabricate the next generation of microarrays, to develop
analytical protocols to determine exact structures of isolated glycans, and to elucidate pathways of
glycan biosynthesis. We describe here a chemoenzymatic methodology that makes it possible, for the
first time, to prepare any bi-, tri-, and tetra-antennary asymmetric N-glycan from a single precursor. It
is based on the chemical synthesis of a tetra-antennary glycan that has N-acetylglucosamine
(GlcNAc), N-acetyllactosamine (LacNAc), and unnatural Galα(1,4)-GlcNAc and Manβ(1,4)-GlcNAc
appendages. Mammalian glycosyltransferases recognize only the terminal LacNAc moiety as a
substrate and thus this structure can be uniquely extended. Next, the β-GlcNAc terminating antenna
can be converted into LacNAc by galactosylation and can then be enzymatically modified into a
complex structure. The unnatural α-Gal and β-Man terminating antennae can sequentially be de-caged
by an appropriate glycosidase to liberate a terminal β-GlcNAc moiety, which can be converted into
LacNAc and then elaborated by a panel of glycosylransferases. Asymmetric bi- and tri-antennary
glycans could be obtained by removal of a terminal β-GlcNAc moiety by treatment with β-N-
acetylglucosaminidase and selective extension of the other arms. The power of the methodology is
demonstrated by the preparation of an asymmetric tetra-antennary N-glycan found in human breast
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carcinoma tissue, which represents the most complex N-glycan ever synthesized. Multistage mass
spectrometry of the two isomeric tri-antennary glycans uncovered unique fragment ions that will
facilitate identification of exact structures of glycans in biological samples.
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INTRODUCTION
Of all post-translational modifications of proteins, complex N-linked glycans are the most prominent
in terms of complexity and diversity.1 All eukaryotic N-linked glycans have a common
pentasaccharide core that can be modified by various N-acetyllactosamine (β-Gal(1,4)GlcNAc,
LacNAc) moieties, which in turn can be elaborated by glycosyltransferases to give highly complex
branched structures.2 The structural diversity of N-glycans arises from the degrees and patterns of
branching, various numbers of LacNAc repeating units, and further elaborations such as sialylation
and fucosylation.3 Structural studies have shown that the N-glycans are usually asymmetrically
substituted having a unique appendage at each branching point. Variations in N-glycan structures
occur during embryogenesis, cell activation, morphogenesis, cell cycle entry, and during oncogenesis.4
These differences are largely based on cell-specific expressions of collection of glycosyltransferases.
Furthermore, the degree of branching of N-glycans appears to be sensitive to the metabolic flux,5 and
it has been proposed that changes in branching cooperate with the regulation of cell proliferation and
differentiation.6
Diverse libraries of well-defined complex glycans are urgently needed for the fabrication of the
next generation of microarrays,7 as standards for the development of analytical protocols to determine
exact structures and quantities of isolated glycans, and for the elucidation of pathways of
glycoconjugate biosynthesis. Often well-defined oligosaccharides can only be obtained by chemical or
enzymatic approaches.7, 8 Despite that most complex N-glycans have asymmetrical architectures,
synthetic efforts have almost exclusively focused on the preparation of incomplete or symmetrically
branched structures.9
Recently, we reported a chemoenzymatic approach that for the first time can provide libraries of
complex asymmetrically branched N-glycans.10 It exploits chemically synthesized multi-antennary N-
glycans that at each antenna can selectively be extended by a panel of glycosyltransferases. The latter
was possible because specific antennae were temporarily rendered inactive for enzymatic extension by
acetylation of a GlcNAc and LacNAc appendage. At an appropriate stage of synthesis, the esters can
be removed to give substrates that can selectively be extended by appropriate glycosyltransferases.
The methodology made it possible to prepare a tri-antennary N-glycan having different complex
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oligosaccharide extensions at each antenna. Others laboratories are adopting this methodology for the
preparation of panels of complex N-glycans.11
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Although the chemoenzymatic approach gave entry into N-glycans of unprecedented complexity,
it requires a unique precursor for each type of branched oligosaccharide. Furthermore, we have
observed that acetyl esters are prone to hydrolysis during multiple enzymatic extensions, and could
not serve as an appropriate protecting group when complex tetra-antennary N-glycans were targeted.12
To address these difficulties, we describe here a more versatile chemoenzymatic methodology that
makes it possible to prepare any bi-, tri-, and tetra-antennary asymmetric N-glycans from a single
precursor (1) through enzymatic transformations (Figure 1). The universal precursor could be obtained
by sequential removal of the orthogonal protecting groups of core pentasaccharide 2 and glycosylation
with glycosyl donors 3-6. The resulting tetra-antennary glycan has a terminal GlcNAc, LacNAc, and
unnatural Gal-α(1,4)-GlcNAc and Manβ(1,4)-GlcNAc moiety. It was anticipated that relevant
glycosyltransferases would only modify LacNAc and not the other terminal structures allowing
selective extension of this arm. At an appropriate stage of the synthesis, the β-GlcNAc terminating
antenna can be converted into LacNAc by galactosylation using Gal-T1, which can then enzymatically
be extended into a complex structure. Next, the unnatural α-Gal and β-Man terminating antennae can
sequentially be de-caged by an appropriate glycosidase13 to liberate a terminal β-GlcNAc moiety,
which can then be converted into LacNAc and elaborated by our panel of glycosyltransferases. It was
also envisaged that asymmetric bi- and tri-antennary glycans could be obtained by removal of a
terminal β-GlcNAc moiety by treatment with β-N-acetylglucosaminidase. To demonstrate the power
of the methodology, we describe here the synthesis of one of the glycoforms of a tetra-antennary N-
glycan that was observed in human ductal invasive breast carcinoma tissue and has the potential to
serve as a biomarker.14 It represents the most complex N-glycan ever synthesized by chemoenzymatic
means. It is the expectation that the availability of such synthetic standard will allow quantitation of
the biomarker in biological samples by mass spectrometry. To further illustrate the flexibility of the
methodology, the common precursor 1 was employed to synthesize tri-antennary positional isomers 8
and 9 and a bi-antennary glycan 10. Analysis of the new compounds by multistage mass spectrometry,
including MS3, showed unique fragmentation patterns of the isomeric glycans. This data will facilitate
the assignment of positional isomers in complex biological samples.
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β4 β3 β4 β3 β4
β6
α2
α6
α3
LevO
BnO
BnO
OAlloc
O
β2
β4
Stepwise
Decaging-Extension
β4 β4
β3 β4
α3 β4
α3 β4
OH
O
HO
HO
HO
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OBn
O
OH
O
O
OBn
O
β2
OBn
O
O
HO
BnO
O
BnO
O
O
O
BnO
OBn
AcHN
PhthN
NPhth
OH
O
BnO
TBSO
BnO
α3 β4 β3 β4 β6
α3 β4
HO
HO
O
α6
2
O
β4 β4
AcHN
OFmoc
β4
Protecting Group
Manipulations
O
HO
HO
α3
β2
α3 β4
O
O
OH
O
8
OH
O
OH
O
HO
HO
Core Pentasaccharide
Full Arm
Deletion-Extension
OH
O
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HO
O
HO
OAc
O
O
HO
AcO
AcO
OBn
O
OAc
OH
O
AcHN
HO
OH
AcHN
O
PhthN
5
O
BnO
AcO
AcO
HO
O
O
HO
β4 β3 β4
α3
β6
β2
O
OAc
O
CF3
PhthN
O
CF3
NPh
AcHN
HO
α6
β4 β4
OH
HO
HO
1
NPh
OH
β4
α3
α3
3
O
α3
O
O
HO
O
OAc
O
β4 β2
AcO
BnO
HO
AcHN
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AcO
AcO
BnO
OBn
O
OBn
O
OBn
O
BnO
O
BnO
Asymmetric
Tetra-Antennary
Intermediate
O
BnO
O
CF3
NPh
β2
PhthN
β4
α3 β4
α3
α6
α3
O
CF3
NPh
PhthN
β4 β4
4
6
Double Arm
Deletion-Extension
β2
10
Building Blocks
Figure 1. Asymmetric bi-, tri, and tetra-antennary N-glycans from common intermediate 1
Scheme 1. Chemical assembly of common intermediate 1
LevO
OAlloc
O
LevO
BnO
BnO
OAlloc
O
BnO
BnO
OBn
O
O
OBn
O
OBn
O
OBn
O
O
OBn
O
BnO
OBn
O
3, TMSOTf, CH2Cl2
4, TMSOTf
CH2Cl2, -20 o
C
O
BnO
O
BnO
O
BnO
-20 o
C
O
BnO
OBn
O
O
BnO
PhthN
OBn
NPhth
PhthN
BnO
R
NPhth
BnO
TBSO
BnO
O
O
BnO
OBn
12. R = OTBS
13. R = OH
HF/Py, Py, rt,
44 h, (66%, two steps)
OAc
O
AcO
AcO
2. R = OFmoc
11. R = OH
Et3N, CH2Cl2
RT, 1h, (89%)
R
O
O
BnO
O
OAc
PhthN
OAc
O
AcO
AcO
R
OAlloc
O
O
R
O
PhthN
BnO
BnO
BnO
BnO
OBn
O
OBn
O
OAc
O
AcO
BnO
OBn
O
O
O
OBn
O
OBn
O
OAc
AcO
BnO
O
BnO
OBn
O
BnO
BnO
O
O
O
BnO
O
BnO
O
OBn
O
BnO
OBn
O
BnO
PhthN
OBn
OBn
O
BnO
NPhth
PhthN
5, TMSOTf, CH2Cl2,
-20 o
6, TMSOTf, CH2Cl2,
-20 oC (92%)
BnO
O
O
BnO
NPhth
BnO
O
O
BnO
C
O
O
PhthN
OAc
BnO
OBn
PhthN
OAc
N2H4*HOAc,
CH3OH:CH2Cl2 (1:1)
(91%, two steps)
Pd[PPh3]4, morpholine
THF:H2O (9:1)
(84%, two steps)
BnO
OBn
AcO
AcO
18. R = OAlloc
19. R = OH
14. R = OLev
15. R = OH
AcO
AcO
O
O
O
BnO
O
O
O
O
BnO
O
OAc
PhthN
OAc
PhthN
OBn
AcO
AcO
BnO
HO
OBn
O
HO
HO
HO
OH
O
O
O
O
BnO
O
HO
O
O
PhthN
OAc
O
AcHN
1. ethylenediamine,
n-BuOH, 90 o
OH
O
C
AcO
HO
HO
2. Ac2O, Py, DMAP
3. NaOMe, MeOH
AcO
O
O
PhthN
AcHN
O
BnO
BnO
O
HO
HO
4. Pd(OH)2, H2, MeOH
(66%, four steps)
OBn
OBn
O
O
OAc
O
O
OH
O
AcO
BnO
OH
O
OH
OBn
HO
HO
O
OH
O
BnO
O
O
BnO
O
HO
O
O
O
O
BnO
O
HO
OBn
OBn
O
HO
OH
O
BnO
PhthN
AcHN
HO
NPhth
OH
AcHN
BnO
O
O
BnO
HO
O
O
HO
O
O
PhthN
OAc
AcHN
OH
BnO
OBn
HO
OH
AcO
AcO
20
HO
HO
1
O
O
O
O
BnO
O
O
O
HO
O
OAc
HO
PhthN
AcHN
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RESULTS AND DISCUSSION
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Chemical synthesis of the common intermediate. Core pentasaccharide 2, which is modified by
the orthogonal protecting groups levulinoyl (Lev), fluorenylmethyloxycarbonyl (Fmoc),
allyloxycarbonyl (Alloc), and t-butyl-dimethylsilyl (TBS) at positions where branching points may
occur, was prepared from appropriate monosaccharide building blocks (see Scheme S1 for synthesis
of 2). Furthermore, N-phenyl trifluoroacetimidate-based15 glycosyl donors 3–6 were synthesized for
the stepwise extension of 2 to give target compound 1 (see Scheme S4 for synthesis of 3-6). Treatment
of 2 with the non-nucleophilic base triethylamine resulted in the selective removal of the Fmoc
protecting group without affecting the other base sensitive functionalities to give glycosyl acceptor 11
in an excellent yield of 89% (Scheme 1). Glycosylation of 11 with N-phenyl trifluoroacetimidate 3 in
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o
the presence of trimethylsilyl trifluoromethanesulfonate (TMSOTf) at –20 C gave heptasaccharide
12, which was immediately treated with HF/pyridine to remove TBS ether to give acceptor 13 in a
66% overall yield. A TMSOTf promoted glycosylation of 13 with donor 4 proceeded efficiently to
give, after purification by Biogel SX-1 (toluene:acetone, 1:1, v:v), the nonasaccharide 14 in a yield of
91 %. The latter glycosylation required a high concentration of donor and acceptor (>100 mM)
otherwise a significant quantity of the glycosyl acceptor was recovered resulting in a modest yield of
product. It was also found that the order of protecting group removal and glycosylation was critical for
the successful preparation of 14. First removal of the TBS ether of 2 followed by glycosylation with 4
gave the corresponding heptasaccharide in a low yield of 14% (see Scheme S2). Probably, the electron
withdrawing Fmoc protecting group at C-2 significantly reduces the glycosyl acceptor reactivity of the
C-4 hydroxyl of 16. The observations indicate that the C-4 hydroxyl has a much high reactivity when
the C-2 hydroxyl is glycosylated.
The Lev ester of 14 was selectively removed by treatment with hydrazine acetate to give alcohol
15 which was coupled with glycosyl donor 5 using TMSOTf as a promoter to provide the desired
decasaccharide 18. Next, the Alloc protecting group of 18 was removed by treatment with Pd[PPh3]4
and the resulting acceptor 19 was coupled with donor 6 using standard conditions to give
dodecasaccharide 20 in high yield. Global deprotection of 20 was accomplished in four steps by first
o
treatment with ethylenediamine in n-butanol at 90 C to remove the phthaloyl-protecting groups,
which was followed by re-acetylation of the resulting free amines and hydroxyls by acetic anhydride
in pyridine. Finally, the esters were selectively cleaved by catalytic sodium methoxide and
hydrogenation over Pd(OH)2 afforded the required tetra-antennary glycan 1. The orthogonal protecting
groups described here are more convenient than the previously employed set of protecting groups,10
and in particular the replacement of a 2-naphthylmethyl (NAP) by TBS ether was advantageous
because cleavage of the former by DDQ required great care and could result in side reactions such as
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cleavage of primary benzyl ethers. Also the use of phthaloyl instead of Troc for protection of amino
functions gave a more robust precursor.
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Enzymatic diversification of common intermediate. The stage was now set for regioselective
extension of each antenna of 1 to give entry into highly complex asymmetric tetra-antennary N-linked
glycans (Scheme 2). Compounds of such complexity have not been synthesized before and would
demonstrate the power of the new methodology. Towards this end, glycan 7 was selected as a target
which is a putative compound observed in a glycoproteomic study of human ductal invasive breast
carcinoma tissue samples and may be attractive for biomarker development for early disease
diagnosis.14
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Scheme 2. Synthesis of asymmetrical tetra-antennary glycan 7 from precursor 1
β4
β3 β4
β6
β6
β6
β6
ST3-Gal-IV
CMP-Neu5Ac
Gal-T1
UDP-Gal
β3-GnT-II
UDP-GlcNAc
α6
α3
α6
α3
α6
α3
α6
α3
β2
β4
β2
β4
β2
β4
β4
β4
β4
β2
β4
β4
β4
β4
β4
β4
α4
β4
β4
β4
α4
β4
α4
β4
β4
α4
1
21
22
23
β2
β2
β2
β2
α3
α3 β4
α3 β4
1. UDP-Gal
Gal-T1
2. β3-GnT-II
UDP-GlcNAc
β4 β3 β4 β3 β4
β6
β4 β3 β4 β3 β4
β4 β3 β4 β3 β4
β6
β6
Fut-I
GDP-Fuc
α2
α2
α6
α3
β2
β4
α-Gal-ase
α6
α3
α6
β4
β4
β2
β4
β2
β4
β4
α4
β4
β4 β4
β4
27
β4
α4
β4
3. UDP-Gal
Gal-T1
α3
26
28
β2
β2
β2
α3 β4
α3 β4
α3 β4
β4 β3 β4 β3 β4
β4 β3 β4 β3 β4
β6
β6
β4 β3 β4 β3 β4
β6
α2
α2
Gal-T1
UDP-Gal
α6
α3
ST3-Gal-IV
CMP-Neu5Ac
α6
β2
β4
α2
β2
β4
β4
β4
β4
β4
α6
α3
β4
α3 β4
α3 β4
β-Man-ase
β2
β4
β4
β4
29
β4
β4
α3
α3 β4
α3 β4
31
30
β2
β2
β2
α3 β4
β4 β3 β4 β3 β4
β6
Key:
β4 β3 β4 β3 β4
β6
α2
β3-GnT-II
UDP-GlcNAc
α2
α6
α3
Gal-T1
UDP-Gal
β2
β4
β4
Neu5Ac
Man
GlcNAc
β4
Gal
Fuc
β2
β4
β4
β3
β4
β4
β4
α3 β4
α3 β4
α3 β4
α3 β4
α3
7
32
β2
β2
The use of a bacterial α(2,3)-sialyltransferase (PmST1),16 cytidine-5’-monophospho-N-
acetylneuraminic acid (CMP-Neu5Ac), and calf intestine alkaline phosphatase (CIAP) resulted in
sialylation of the LacNAc moiety and terminal α-Gal moiety of the C-2 and C-3 arms. Fortunately,
this lack of selectivity could be addressed by using the mammalian α(2,3)-sialyltransferase ST3Gal-
IV17 and this enzyme exhibited absolute selectivity for the LacNAc antenna, and could selectively
convert compound 1 into mono-sialylated derivative 21. A downfield shift of the β-Gal H-1 supported
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the site of transfer of the Neu5Ac residue. The GlcNAc moiety of the β(1,6)-antenna of 21 was
converted into LacNAc by using recombinant mammalian β(1,4)-galactosyltransferase (Gal-T1),
uridine-5’-diphosphogalactose (UDP-Gal) and CIAP, and complete conversion to 22 was observed
after an incubation time of 10 h. Surprisingly, the use of bovine Gal-T1 led only to partial
galactosylation of 21. Treatment of 22 with β(1,3)-N-acetylglucosaminyltransferase (β3GnT-II),18
uridine-5’-diphospho-N-acetylglucosamine (UDP-GlcNAc) and CIAP resulted in the introduction of a
10
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17
18
19
20
21
22
23
24
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26
27
28
29
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32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
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54
55
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1
GlcNAc residue at the β(1,6)-branch to give 23. Full assignment of H NMR spectra (900 MHz)
combined with a band-selective 2D TOCSY experiments confirmed that only the terminal β-Gal of the
β(1,6)-arm was modified by GlcNAc. In this respect, only one new doublet at 4.69 ppm corresponding
to H-1 of the anomeric β-GlcNAc moiety was observed. In addition, an upfield shift of the H-1 and
the downfield shift of H-4 protons of the β-Gal residue (4.31 and 4.0 ppm, respectively) supported that
only the LacNAc moiety of the β(1,6)-arm was extended and that the α-Gal residue at the β(1,4)-arm
was unaffected by the enzymatic transformation. Permethylation of the product followed by mass
spectrometric (MS) analysis confirmed the attachment of a single GlcNAc moiety (m/z 3574.7880).
Controlled extension of the β(1,6)-arm to give a tri-LacNAc residue was accomplished by a repetition
of galactosylation by Gal-T1 and N-acetylglucosaminylation with β3GnT-II to give the
octadecasaccharide 26. After each step, the product was purified by size-exclusion chromatography,
and the progress of the reactions was monitored by MS of permethylated derivatives. If any starting
material was observed, the reaction was prolonged until a homogenous product was obtained.
Next, the terminal LacNAc moiety of the β(1,6)-arm was converted into an H-type antigen by
treatment with mammalian α(1,2)-fucosyltransferase (FuT-I)19 in the presence of GDP-Fuc to furnish
27. This intermediate was purified by semi-preparative HPLC using an amide HILIC column (10 x
250 mm, Waters Inc.) under isocratic conditions (55% CH3CN:100 mM ammonium formate, pH 3.4)
with the UV (210 nm) detection to give the homogeneous nonadecasaccharide 27 in a sufficient
quantity for further enzymatic modification of the β(1,2)- and β(1,4)-antennae.
At this point in the synthesis, the β(1,4)- and the β(1,2)-antenna were sequentially de-caged by
treatment with either α-galactosidase or β-mannosidase, respectively followed by elaboration of the
resulting terminal GlcNAc moieties into complex structures. Thus, the α-Gal moiety was removed by
treatment with the α-galactosidase from green coffee beans, which proceeded smoothly to give glycan
28. The resulting terminal GlcNAc residue of 28 could easily be converted into a sialyl LacNAc
moiety by subsequent treatment with Gal-T1 and ST3Gal-IV to give the di-sialylated glycan 30.
Finally, the β(1,2)-arm was elaborated by first removal of the capping β-mannoside using Helix
pomatia β-mannosidase. Mass spectrometric analysis of the product revealed that in addition to the β-
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mannoside, also the α-fucoside had been cleaved, which probably was due to contamination with a
fucosidase. Fortunately, the use of the inhibitor 1-deoxyfuconojirimycin abolished the loss of the
fucosyl residue to give reliable entry into glycan 31. Finally, the targeted asymmetric 21-mer 7 was
obtained by galactosylation of 31 with Gal-T1 to give 32, which was further extended by
β(1,3)GlcNAc unit by employing β3GnT-II. Homogeneity of glycan 7 was confirmed by LC-MS
using an amide HILIC column (55% CH3CN:100 mM ammonium formate, pH ± 3.4).
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40
41
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48
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55
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Scheme 3. Preparation of isomeric tri-antennary glycans 8 and 9 from common precursor 22
1. β-Man-ase +
β-GlcNAc-ase
2. α-Gal-ase
β4 β3 β4 β6
β6
β4
β4
1. β3-GnT-II
UDP-GlcNAc
α3 β4 β3 β4 β6
ST3-Gal-IV
CMP-Neu5Ac
α6
α3
α6
α3
α6
β4
β4
β4
β4
β4
β4
β4
α3
β4
β4
β4
β2
2. UDP-Gal
Gal-T1
α3
33
β2
β2
α3 β4
35
α3 β4
α3 β4
8
β4
β6
α6
α3
β2
β4
β4
β4
β4
α4
β2
α3 β4
β4
β4 β3 β4
α3
β4 β3 β4
β6
β2
β6
β2
22
β6
β2
1. β3-GnT-II
UDP-GlcNAc
ST3-Gal-IV
CMP-Neu5Ac
α6
α3
α6
α3
α6
β4
β4
β4
β4
β4
β4
β4
β4
α3
α3
α3
2. UDP-Gal
Gal-T1
β4 β2
α3
β4 β2
β4 β2
α3
1. α-Gal-ase +
β-GlcNAc-ase
2. β-Man-ase
36
9
38
Key:
Neu5Ac
Man
Gal
GlcNAc
Next, attention was focused on the synthesis of tri-antennary glycans from the universal precursor.
It was expected that selective removal of the terminal β-Man or α-Gal of the β(1,2)- or β(1,4)-antenna
of 22 followed by treatment with β-N-acetylglucosaminidase to remove the revealed terminal GlcNAc
moiety would provide tri-antennary structures 33 and 36, respectively (Scheme 3). The latter
compounds can then be elaborated to the tri-antennary glycans 8 and 9, which are positional isomers.
Thus, treatment of compound 22 with a mixture of α-galactosidase and β-N-acetylglucosaminidase
from Jack bean resulted in deletion of the β(1,4)-arm as shown by MS. Next, the reaction mixture was
heat deactivated and subsequent addition of β-mannosidase and incubation for additional 2 h afforded
homogenous tri-antennary glycan 36. Alternatively, exposure of 22 to β-mannosidase and β-N-
acetylglucosaminidase resulted in the removal of the β(1,2) appendage, and after the addition of the α-
galactosidase and further incubation for 2 h, glycan 33 was obtained. Detailed inspection of NMR data
as well as the permethylation followed by MALDI-TOF MS analysis confirmed that the trimming
process had proceeded with the expected regio-selectivity. For both compounds, the anomeric proton
for α-Gal (5.44 ppm) had disappeared while a similar signal for the β-Gal (4.47 ppm) was still present,
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which confirmed that the β(1,6)-arm had remained intact. The one-pot multiple-enzyme procedure16b
made the transformation very efficient.
6
7
8
9
Treatment of 33 and 36 with β3GnT-II and UDP-GlcNAc followed by bis-galactosylation with
Gal-T1 and UDP-Gal afforded tri-antennary glycans 35 and 38, respectively containing a terminal
β(1,6)-di-LacNAc moiety. Sialylation of 35 and 38 with ST3Gal-IV in the presence of CMP-Neu5Ac
gave the positional isomers 8 and 9, respectively.
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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
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60
Scheme 4. One-pot multiple-enzyme approach for the preparation of bi-antennary glycans from common
precursor 1
1. α-Gal-ase +
β6
β-GlcNAc-ase
ST3-Gal-IV
CMP-Neu5Ac
β2
β2
β2
β2
α6
α3
α6
α3
2. β-Man-ase
α6
α3
β4
β4
β4
β4
β2
β4
β4
β4
β4
α4
β4
β4
α3
39
40
β4
1
β2
+
+
Key:
Gal
β2
β4
β4
α3
α6
α3
β2
Gal-T1
UDP-Gal
α6
α3
Hpα1,3FT
GDP-Fuc
β4
β4
Neu5Ac
Man
GlcNAc
β4
β4
α3
α3
β4
β2
10
41
β2
α3
Fuc
The final goal was to synthesize a bi-antennary glycan from common tetra-antennary precursor 1
by a one-pot multiple-enzyme approach (Scheme 4). Sequential treatment of 1 with the α-
galactosidase and β-N-acetylglucosaminidase followed by heat-deactivation and further processing
with β-mannosidase lead to clean formation of bi-antennary glycan 39 (LC-MS on a HILIC column,
65% CH3CN:100 mM ammonium formate, pH±3.4). Sialylation of the LacNAc moiety of the β(1,2)-
arm with ST3Gal-IV resulted in the formation of glycan 40. Antenna-selective installation of a sialyl-
Lex moiety was easily accomplished by exposing the newly formed α(2,3)-sialyl LacNAc to GDP-Fuc
and Helicobacter pylori α(1,3)-fucosyltransferase (Hpα1,3FT)20 providing 41. The final step involved
galactosylation of the β (1,2)-arm of 41 by employing Gal-T1 and UDP-Gal to furnish bi-antennary
glycan 10.
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A"
*$
$Oxonium$ions$
$
*
657.23
100
80
60
40
20
0
*$PolyALacNAc$
$
6
7
8
9
Losses$corresponding$to$
oxonium$ions$
*$
1022.36
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21
22
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Y3α$
Y4β2+$
$
B4α$
Y3α/Y4β$ Y3α2+
$
B4α$
$
$
$
$
$
B4α/Y6
$
$
$
*$
366.14
1475.51
1295.46
1112.39
1184.41
1567.56
1641.59
7*49$ .28
1932.69
1770.64
$$$$$$Glycan$8"
*$
454.15
*$
819.28
$MW$=3244.14$
*$
102*2$.36
100
80
60
40
20
0
*$
657.23
Y3α$
Y3α2+
$
$
$
Y4β’2+
1112.39
$
Y3α/Y4β’$
B4α$
*$
1567.55
B4α$
1478.02
*$
366.14
1295.46
$$$$$$Glycan$9$
731.27
$
*$
45*4$.15
1932.68
1770.63
819.28
1405.50
MW$=3244.14$
1214.43
528.19
600
1641.59
$
400
800
1000
1200
1400
1600
1800
2000
m/z
B"
1114.41
100
80
657.23
60
40
20
Y3α!
1567.56
B4α!
Y3α/Y4β
749.28
!
B4α!
!
!
1475.51
!
!
! 1276.46
B4α/Y6
C4/Y5α/Y5α’!
366.14
454.15
967.35
1184.41
!!!!!!Glycan!8!Y4β!
!
!
!
819.28
587.23
1770.63
![M+2H]2+!=1112.39!
1641.59
0
657.23
100
80
60
40
20
0
1567.55
Y3α!
Y3α/Y4β’!
1405.50
1276.46
966.84
1112.89
366.14
!!!!!!Glycan!9!Y4β’!
819.28
800
![M+2H]2+!=1112.39!
454.15
1770.63
1800
400
600
1000
1200
m/z
1400
1600
2000
Figure 2. A. Multistage mass spectrometry can distinguish the isomer glycans 8 and 9. CID MS2 of the triply
charged precursor ion at m/z 1082.39. Fragment ions indicative for distinguishing the two isomers are color-
coded. B. CID-MS3. The MS2 fragment ion at m/z 1112.39 was subjected to MS3 to reveal the structural
differences between these two isobaric fragment ions. Fragment ions that can distinguish the two isomers are
color-coded.
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Multistage mass spectrometry of isomeric glycans. The use of MS in glycomics is driven by the
need to obtain quantitative and structural information of glycans of glycoproteins present in biological
samples to understand metabolic or disease processes and to discover and validate putative new
biomarkers.21 In general, glycans are enzymatically or chemically released from glycoproteins,
separated by chromatography and analyzed by MALDI-TOF or ESI-TOF MS. Although powerful,
such studies do often not provide exact structures of glycans because of their isobaric nature (i.e.
different structures with identical molecular weights). It is anticipated that the use of well-defined
glycan standards will make it possible to examine chromatographic behavior and fragmentation
patterns in multistage mass spectrometry, which may reveal unique features of glycan isomers that
may facilitate exact structure identification.22
[M+3H]3+ precursor ions corresponding to native/non-permethylated glycan 8 and glycan 9 were
isolated and subjected to CID to obtain fragments that may distinguish these two isomers. Oxonium
ions were observed in the low mass range as well as the corresponding fragments resulting from the
losses of oxonium ions from precursor ions in the higher mass range. A peak at m/z 1022 is indicative
of di-N-acetyllactosamine containing sialic acid (Neu5Ac-di-LacNAc), which can further lose Neu5Ac
as observed by the presence of peak at m/z 731. To confirm the branching structures of glycans 8 and
9, we searched for unique and structurally informative peaks in each spectrum, mainly the branching
specific B4α and Y3α fragment pairs. For glycan 8, MS2 CID branching specific fragments (Figure 2,
top panel, shaded in blue) were observed as B4α at m/z 1475 and its’ Y counterpart fragment Y3α2+ at
m/z 885 confirmed the presence of two Neu5Ac-LacNAc units on α(1,3)-branch and Neu5Ac-di-
LacNAc on α(1,6)-branch, respectively. The presence of two Neu5Ac-LacNAc units on the α(1,3)-
branch is further supported by the B4α/Y6 m/z 1184 peak (loss of one Neu5Ac from B4α). Doubly
protonated fragment Y4β2+ at m/z 1112 (shaded in red) resulting from the loss of Neu5Ac-di-LacNAc
from precursor ion. Y4β2+ can further lose the entire α(1,3)branch and give rise to a Y3α/ Y4β fragment
at m/z 749. Although Y3α2+ is barely visible in the spectra, it can still be confidently assigned with
signal to noise ratio (S/N) of 45. The low abundance thereof is due to the lability of Neu5Ac-di-
LacNAc unit in the gas phase resulting once again in Y3α/ Y4β fragment.
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The CID MS2 spectrum of glycan 9 provided two unique fragment peaks (Figure 2A, bottom
panel, shaded in green). A unique peak is observed for the Y3α2+ m/z 1214 fragment ion, resulting
from the loss of Neu5Ac-LacNAc-Man from α(1,3)-branch, which can be found as B4α fragment ion
at m/z 819. Loss of Neu5Ac-LacNAc-Man from Y4β’2+ m/z 1112 results in second unique peak
Y3α/Y4β’ found at m/z 1405. These two peaks corroborate glycan 9 branching pattern consisting of
one Neu5Ac-LacNAc unit on the α(1,3)-branch, and Neu5Ac-LacNAc Neu5Ac-di-LacNAc on the α6-
branch.
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In order to validate the annotation of the m/z 1112, which appears in both MS2 spectra, MS3 CID
analyses were performed for each glycan (Figure 2B). Fragmentation of the Y4β2+ m/z 1112 fragment,
belonging to glycan 8 MS2 CID (Figure 2B, top panel shaded in orange) produced B4α fragment at m/z
1475 as well as the Y3α/Y4β m/z 749 ion. These informative fragments can be formed if both Neu5Ac-
LacNAc blocks are present on the α(1,3) branch. In contrast, the MS3 CID fragmentation of the Y4β’2+
m/z 1112 ion isolated from glycan 9 MS2 CID (Figure 2B, bottom panel shaded in purple) resulted in a
distinct Y3α/Y4β’ m/z 1405 peak which can only be formed if the two Neu5Ac-LacNAc units are
found on different branches within 9. These results clearly demonstrate the importance of MS3 to
distinguish isobaric fragments of glycans that cannot be distinguished based on mass measurements
alone.
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Although it is possible to achieve richer fragmentation spectra of glycans by permethylation or
measuring them as adducts of alkali metals, the resulting spectra require expert knowledge of glycan
fragmentation for unambiguous annotation. We have measured glycans 8 and 9 as potassium and
sodium adducts in positive mode as well as chlorinated glycans in negative mode, and although the
resulting fragmentation spectra are rich in information, we have found that CID of protonated glycans
provides sufficient details to distinguish isobaric glycans 8 and 9 and result in a simple fragmentation
pathway consisting mainly of glycosidic bond cleavages and thus simplifying spectral annotation.
CONCLUSION
Although naturally occurring complex N-linked oligosaccharides are usually asymmetrically
substituted having a unique saccharide appendage at each branching point, previous synthetic efforts
have almost exclusively been directed to the preparation of simpler symmetrical structures.9a, 9d-j, l, m, o
This stems from the difficulties of controlling diversification at the various sites of branching,
especially when several different complex terminal structures need to be appended. To address this
challenge, we describe here a chemoenzymatic methodology that makes it possible to prepare any bi-,
tri-, and tetra-antennary asymmetric N-glycan from a common precursor (1). This latter precursor is a
tetra-antennary glycan that at positions where branching may occur is modified by LacNAc, GlcNAc
and unnatural α-Gal(1,4)GlcNAc and β-Man(1,4)GlcNAc. We have found that relevant mammalian
glycosyltransferases will only recognize the LacNAc containing antenna of the common precursor as a
substrate, therefore allowing unique extension of this arm. At an appropriate stage of synthesis, the
antenna containing a terminal GlcNAc residue can be “armed” by conversion into LacNAc by a
galactosyltransferase, which can then be further extended by our panel of glycosyltransferases into a
complex structure. The antenna that are modified by the unnatural hexoses α-Gal and β-Man can be
de-caged by an appropriate glycosidase to reveal a terminal β-GlcNAc moiety, and thus the process of
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conversion into LacNAc and extension by glycosyltransferases can be repeated to give entry into
asymmetrical tetra-antennary glycan. The use of unnatural distal hexoses to temporarily block an
antenna from enzymatic modification is much more robust than our previously reported approach10
based on acetylation of terminal GlcNAc or LacNAc residues. The new strategy made it possible, for
the first time, to prepare a tetra-antennary N-glycan that at each branching point has a different
appendage. We also describe that the common precursor can easily be converted into bi- and tri-
antennary glycans by deletion of an antenna by the action β-N-acetylglucosaminidase, which
selectively cleaves a terminal GlcNAc residue. This process can be performed by an efficient one-pot
multiple-enzyme procedure. The latter strategy made it possible to prepare two isomeric tri-antennary
N-glycans. As proof of principle to demonstrate the usefulness of the methodology, the isomers were
subjected to multistage mass spectrometry to discover diagnostic ions that can determine the
branching pattern of N-glycans in complex biological samples.
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ASSOCIATED CONTENT
Supporting Information
Synthetic protocols, compounds characterization of glycans, and NMR spectra. This material is
available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*gjboons@ccrc.uga.edu or G.J.P.H.Boons@uu.nl
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
This project was supported by the National Institute of General Medical Sciences (NIGMS) from the
US National Institutes of Health (NIH; P01GM107012) and TOP-PUNT grant (718.015.003),
Proteins@Work (184.032.201) and the Gravity Program Institute for Chemical Immunology by the
Netherlands Organisation for Scientific Research (NWO). We thank Dr. John Glushka (CCRC, UGA)
and Dr. Hans Wienk (UU) for assistance with NMR measurements.
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