A new approach for the synthesis of hyperbranched N-glycan core structures from locust bean gum

Article abstract of DOI:10.1021/ol403140h

A novel protocol for the synthesis of general N-glycan core structures was established by means of Manβ(1→4)Man peracetate derived from a naturally abundant locust bean gum as a key starting material. Phenyl (2-O-benzyl-4,6-O-benzylidine-β-d-mannopyranosyl)-(1→4)-3, 6-di-O-benzyl-2-azido-2-deoxy-1-thio-β-d-glucopyranoside facilitated the synthesis of key intermediates leading to hyperbranched N-glycan core structures.

Full text of DOI:10.1021/ol403140h

ORGANIC
LETTERS
2013
Vol. 15, No. 24
6278–6281
A New Approach for the Synthesis
of Hyperbranched N‑Glycan Core
Structures from Locust Bean Gum
Ravi Kumar H. V.,† Kentaro Naruchi,‡ Risho Miyoshi,‡ Hiroshi Hinou,‡ and
Shin-Ichiro Nishimura*,‡
Division of Drug Discovery Research, Faculty of Advanced life Science and Graduate
School of Life Science, Hokkaido University, N21, W11, Kita-ku, Sapporo 001-0021,
Japan, and Medicinal Chemistry Pharmaceuticals, Co. Ltd., N21, W12, Kita-ku,
Sapporo 001-0021, Japan
shin@sci.hokudai.ac.jp
Received October 31, 2013
ABSTRACT
A novel protocol for the synthesis of general N-glycan core structures was established by means of Manβ(1f4)Man peracetate derived from a
naturally abundant locust bean gum as a key starting material. Phenyl (2-O-benzyl-4,6-O-benzylidine-β-D-mannopyranosyl)-(1f4)-3,6-di-O-benzyl-
2-azido-2-deoxy-1-thio-β-D-glucopyranoside facilitated the synthesis of key intermediates leading to hyperbranched N-glycan core structures.
Asparagine-linked type oligosaccharides (N-glycans) of
glycoproteins play essential roles in the maintenance and
regulation of many functions such as cell differentiation,
cell adhesion, and homeostatic immune balance.1 Recent
studies on large-scale N-glycan analysis have revealed
the importance of structural alteration of human serum/
cellular N-glycans during disease progression. Many such
glycans serve as potent biomarkers for the early diagnosis
of disease and as new agents for therapeutic antibodies.2 In
addition, as expression levels of highly branched N-glycans
are enhanced distinctly during cancer cell proliferation and
metastasis,3,4 it is clear that the construction of N-glycan
compound libraries is of growing importance for gaining
insights into the functions of cell surface glycoproteins.
However, the relationship between structure and function
of N-glycans remains mostly unclear due to the structural
complexity and heterogeneity in the dynamic posttransla-
tional modification at the potential N-glycosylation sites.5
Recently, chemical and enzymatic syntheses of glycopep-
tides and glycoproteins having N-glycans have been accel-
erated conspicuously by using various endo-β-N-acetyl-
D-glucosaminidases and oligosaccharide oxazolines as
† Hokkaido University.
‡ Medicinal Chemistry Pharmaceuticals, Co. Ltd.
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r
10.1021/ol403140h
Published on Web 11/21/2013
2013 American Chemical Society

sophisticated donor substrates.6 However, it is thought that
the feasibility of this potential approach depends strongly
on the availability of the complex N-glycans found ubiqui-
tously in Nature. Although extensive efforts have been
devoted to the synthesis of complicated hyperbranched
N-glycans,7 the syntheses generally entail tedious proce-
dures for the preparation of many designated monosacchar-
ide synthons and multistep stereoselective glycosidation
reactions. Especially, it should be noted that formation of
the Manβ(1f4)GlcNAc unit present in the N-glycan core
moiety is one of the most difficult steps in stereoselective
glycoside synthesis8 due to both the anomeric effect and
neighboring group participation in the mechanism of
glycosidation reactions using D-mannosyl donors, which
are not beneficial for the stereoselective generation of the
β1,4-mannoside linkages.9
Scheme 1. Synthesis of Hyperbranched N-Glycan Core
Structures Using Galactomannan As a Key Starting Material
We hypothesized that the β1,4-mannobiose derivative 2,
distributed often in some natural polysaccharides (1),
might become an ideal starting material10 for the synthesis
of a key intermediate 3, phenyl (2-O-benzyl-4,6-O-benzy-
lidine-β-D-mannopyranosyl)-(1f4)-3,6-di-O-benzyl-2-azido-
2-deoxy-1-thio-β-D-glucopyranoside, to facilitate the con-
struction of tri- and tetra-antennary derivatives 7 and
8 when employed in combination with compounds
(4, 5, and 6) as outlined in Scheme 1. To demonstrate the
feasibility of this synthetic strategy, we decided to establish
the standardized synthetic protocols for accessing com-
pounds 2ꢀ6 and to assess their potential for the synthesis
of the above-mentioned hyperbranched N-glycan core
structures.
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Scheme 2. Synthetic Route to the Key Intermediate 3 from
Locust Bean Gum 1
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Our previous finding that the β1,4-mannobiose octa-
acetate 2,11 obtainable through the acid hydrolysis of guar
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Org. Lett., Vol. 15, No. 24, 2013
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Scheme 3. Synthesis of Compounds 17 and 7 from 3, 4, and 5
Scheme 5. Synthesis of Oxazoline Derivative 23 from 8
Scheme 4. Synthesis of Compound 8 from 3, 5, and 6
conditions as listed in Table S1. Consequently, we discov-
ered that the degradation of locust bean gum, by treatment
with pectinase from Aspergillus aculeatus at 50 °C for
48 h in 50 mM acetate buffer (pH 5.0), gave dominantly
a
disaccharide Manβ(1f4)Man, and subsequent
per-O-acetylation of the crude product facilitated the
isolation of β1,4-mannobiose octaacetate 2 by silica gel
chromatography in 32% overall yield (51 g from 100 g of
crude locust bean gum) (Scheme 2).
As anticipated, per-O-acetate 2 allowed for the highly
efficient synthesis of the key intermediate 3 by a general
procedure as shown in Scheme 2. 2-Azido derivative 10
obtained readily by azidonitration of glycal 9 was treated
with tetrabutylammonium chloride in acetonitrile to
make the purification of the gluco configured chloride 11
possible. Next, β-thioglycoside 12 derived from the nucleo-
philic substitution of 11 with thiophenol was subjected to de-
O-acetylation, benzylidenation, and benzylation. It afforded
regioselectively protected disaccharide 14 as a mixture
of stereoisomers at the 20,30-O-benzylidene group. However,
it was revealed that a reductive ring-opening reaction of
compound 14 occurred at the 20,30-O-benzylidine ring12
gum, can be converted into compounds corresponding to
the disaccharide unit, Manβ(1f4)GlcNAc, encouraged us
to improve and optimize the procedure for the preparation
of this important compound 2 from the most suited
material among some abundant polysaccharides contain-
ing the common formula of galactomannan shown in
Scheme 1. In the present study, we investigated the degra-
dation products of two abundant polysaccharides, guar
gum and locust bean gum, under various hydrolytic
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Org. Lett., Vol. 15, No. 24, 2013

Scheme 6. Synthesis of EPO-Related Peptide (20ꢀ28) Having
Tetra-antennary N-Glycan 25 Using 23 as a Glycosyl Donor
Figure 1. EPO-related peptide having tetra-antennary N-glycan
25 characterized by HPLC (A) and MALDI-TOF-MS (B).
oxazoline 23 from 8. After galactosylation of partially
protected 20 by GalT with UDP-Gal, dodecasaccharide
22 was converted readily into the oxazoline 23 under con-
ditions reported previously.16 As shown in Figure 1, it was
demonstrated, for the first time, that the reaction between 23
and 24 conducted by recombinant endo-M (N175Q)6b
proceeded significantly and afforded the desired glycopep-
tide 25 in moderate yield (Scheme 6). Further optimization
will be needed for the practical, large-scale synthesis.
In conclusion, we developed a novel synthetic strategy
toward various multivalent N-glycan core structures by
using β1,4-mannobiose octaacetate 2 derived efficiently
from abundant locust bean gum as a key starting material.
Versatility of the intermediate 3 was demonstrated by
synthesizing bi-, tri-, and tetra-antennary N-glycan core
derivatives 17, 7, and 8 in high yields. It should be
emphasized that the present strategy should allow for the
synthesis of a variety of glycopeptides and glycoproteins
having homogeneous and highly complicated N-glycans
when combined with endoglycosidase-catalyzed trans gly-
cosylation protocols.6
selectively in the endo isomer in the presence of DIBAL and
gave the desired disaccharide intermediate 3 having a 30-OH
group in high yield.
We tested the feasibility of the intermediate 3 for the
synthesis of di-, tri-, and tetra-branched derivatives. To
achieve a systematic and streamlined synthesis of such
hyperbranched N-glycan structures from disaccharide ac-
ceptor 3, we prepared glycosyl donors 4, 5, and 6 by
conventional procedures13ꢀ15 (see Supporting Information).
As indicated in Schemes 3 and 4, an advantage of the
present strategy is evident because tetrasaccharide
15/16 derived by coupling 3 with 4 could be employed
directly for the synthesis of key intermediates 17 and 7
using a suited donor 4 or 5, respectively. Similarly, the
pentasaccharide 18/19 obtained by coupling 3 with 6
becomes the glycosyl acceptor for the coupling with the
imidate 5 to lead efficiently to the targeted octasaccha-
ride derivative 8 in high yield.
Next, our interest was focused on the feasibility of using
8 in the synthesis of glycopeptides via an endoglycosidase-
catalyzed reaction with peptides carrying a GlcNAc resi-
due. Scheme 5 indicates a process for the preparation of
Acknowledgment. This work was partly supported by a
grant for “Innovation COE Project for Future Medicine
and Medical Research” from the Ministry of Education,
Culture, Science, and Technology, Japan and JSPS
KAKENHI Grant Number 25220206.
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Res. 2008, 343, 2675.
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Terrak, M.; Herdewijn, P. J. Am. Chem. Soc. 2012, 134, 9343–9351.
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Res. 1967, 4, 432–434. (b) Kovac, P. Carbohydr. Res. 1986, 153, 168–170.
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S.-i. J. Org. Chem. 2009, 74, 2210–2212.
Supporting Information Available. General procedure,
characterization data of all new compounds, and 1H and
13C NMR spectra. This material is available free of charge
via the Internet at http://pubs.acs.org.
The authors declare no competing financial interest.
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