Rational optimization of the mannoside building block for automated electrochemical assembly of the core trisaccharide of GPI anchor oligosaccharides

Article abstract of DOI:10.1016/j.carres.2017.08.009

We have developed a carbohydrate building block of mannosides based on DFT calculations, electrochemical analysis, and automated solution-phase synthesis. The optimized building block in hand was used to prepare the core trisaccharide of GPI anchor oligos

Full text of DOI:10.1016/j.carres.2017.08.009

Accepted Manuscript
Rational optimization of the mannoside building block for automated electrochemical
assembly of the core trisaccharide of GPI anchor oligosaccharides
Sujit Manmode, Takumi Sato, Norihiko Sasaki, Ikumi Notsu, Shuichi Hayase, Toshiki
Nokami, Toshiyuki Itoh
PII:
S0008-6215(17)30589-X
10.1016/j.carres.2017.08.009
CAR 7434
DOI:
Reference:
To appear in: Carbohydrate Research
Received Date: 13 July 2017
Revised Date: 9 August 2017
Accepted Date: 9 August 2017
Please cite this article as: S. Manmode, T. Sato, N. Sasaki, I. Notsu, S. Hayase, T. Nokami, T. Itoh,
Rational optimization of the mannoside building block for automated electrochemical assembly of
the core trisaccharide of GPI anchor oligosaccharides, Carbohydrate Research (2017), doi: 10.1016/
j.carres.2017.08.009.
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ACCEPTED MANUSCRIPT
Rational Optimization of the Mannoside Building Block for Automated
Electrochemical Assembly of the Core Trisaccharide of GPI Anchor
Oligosaccharides
Sujit Manmode,a Takumi Sato,a Norihiko Sasaki,a Ikumi Notsu,a Shuichi Hayase,a
Toshiki Nokami,*a,b and Toshiyuki Itoh,*a,b
aDepartment of Chemistry and Biotechnology, Graduate School of Engineering, Tottori University
4-101 Koyamacho-minami, Tottori City, 680-8552 Tottori, Japan
bCenter for Research on Green Sustainable Chemistry, Faculty of Engineering, Tottori University
E-mail addresses: tnokami@chem.tottori-u.ac.jp (T. Nokami), titoh@chem.tottori-u.ac.jp (T. Itoh)
Keywords: Automated synthesis; Electrochemical oxidation; Glycosylation; Mannoside;
Neighboring group participation
Abstract
We have developed a carbohydrate building block of mannosides based on DFT calculations,
electrochemical analysis, and automated solution-phase synthesis. The optimized building block in
hand was used to prepare the core trisaccharide of GPI anchor oligosaccharides.
Introduction
Mannosides are abundant monosaccharides found in organisms including bacteria. For example,
tuberculosis (TB) is a fatal disease caused by Mycobacterium tuberculosis, as the Global Burden of
Disease Study 2013 (GBD 2013) indicates that TB caused 9.2 million deaths in 2013 [1]. The
mycobacterial cell wall consists of a highly dense network of glycoproteins including
arabinogalactan (AG), lipoarabinomannan (LAM), mannose capped lipoarabinomannan (ManLAM),
trehalose, glycosylphosphatidylinositol (GPI) and phosphatidylinositol mannoside (PIM). Glycans
on the cell surface play a vital role in sustaining the lives of many fundamental processes [2,3].
Among these glycans PIM, GPI and LAM are found to be the most potent molecules for host
immune response [4]. In the last few decades these oligosaccharides have become the molecules of
particular interest for many chemists and biologists.
Oligomannosides are one of the most abundant oligosaccharides having the repeating unit of
mannose residue with 1,2- or 1,6-α-glycosidic linkages and these oligosaccharides have been target
structures to demonstrate benefits of novel strategy for oligosaccharide synthesis [5-7]. Automated
solid-phase synthesis of oligomannosides up to 30mer has already been achieved, however, the
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quantity of the target oligosaccharides is limited [8]. Although iterative one-pot synthesis of
oligosaccharides based on pre-activation of glycosyl donors has already been applied for the
synthesis of lipomannans, a tetramannoside with the same repeating structure is, to date, the only
example of this method [9,10].
We have already reported automated electrochemical assembly of carbohydrate building blocks
for the synthesis of the potential precursor of TMG-chitotriomycin tetrasaccharide by rational
optimization of carbohydrate building blocks based on DFT calculation, and electrochemical
measurements of their oxidation potentials [11-13]. Although α-glycosidic linkages of mannosides
are thermodynamically favorable and can be selectively constructed by the neighboring group
participation, it is important to optimize the carbohydrate building blocks which can be converted to
the corresponding storable glycosylation intermediates with reasonable reactivity for the subsequent
glycosylation with a free OH of glycosides. Here we report optimization of carbohydrate building
blocks for electrochemical automated solution-phase synthesis of oligomannosides with α-glycosidic
linkages and its application to the synthesis of GPI anchor oligosaccharides.
Results and Discussion
We initiated our study on the evaluation of carbohydrate building blocks by preparing a variety
of building blocks equipped with a neighboring group such as pivaloyl and acetyl groups at the
hydroxyl group of C-2 (2-OH) and/or C-6 (6-OH) (Figure 1). Building blocks 1a-c with a
neighboring group at 2-OH were easily accessible from the anomeric orthoester (See Supporting
Information), whereas other building blocks 2-5 were synthesized by the conventional manipulations
of protecting groups. Oxidation potentials of thus-obtained building blocks were measured by a
standard technique of linear sweep voltammetry (LSV) using a rotation-desk electrode (RDE) (Table
1). These oxidation potentials were compared with that of 4-fluorophenyl
2,3,4,6-tetra-O-benzyl-α-D-thiomannoside 2. Derivatization with an acetyl group at 2-OH increased
the oxidation potential about 0.09 V. On the other hand, in the case of pivaloyl and phosphate,
oxidation potentials were increased about 0.08 V and 0.11 V, respectively. Introduction of a pivaloyl
group to 6-OH showed comparatively less change in oxidation potential (1.64 V), whereas pivaloyl
functionalization of 2-OH as well as 6-OH increased the oxidation potential about 0.11 V. These
results suggest that a structurally similar protecting group causes slightly different electronic
behavior which influences the electrochemical oxidation. To get the a better understanding of this
phenomenon we performed DFT calculations (B3LYP/6-31G(d)). Statistical data obtained by these
calculations showed good correlation with oxidation potentials of glucosamine derivatives, however
potential differences did not completely fit those obtained by measurements, especially in building
blocks equipped with an acetyl or pivaloyl group at 2-OH. Therefore, further investigation is
required for better prediction of oxidation potentials based on theoretical calculations.
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Table 1. Oxidation potentials of building blocks.
building
oxidation potential Eox
(V vs SCE)
∆Eox (V)
∆EHOMO (eV)a
block
1.69
1.70
1.73
1.62
1.64
1.73
1.68
1.62
+0.07
+0.08
+0.11
0.00
+0.25
+0.21
+0.13
0.00
1a
1b
1c
2
+0.02
+0.11
+0.06
0.00
+0.11
+0.15
+0.07
+0.03
3
4
5a
5b
aB3LYP/6-31G(d)
To confirm the stereoselectivity in glycosylation, we performed disaccharide synthesis based on
the electrochemical pre-activation protocol (Table 2) [14]. A building block was placed in the anodic
chamber of a divided glass cell in the presence of Bu4NOTf as an electrolyte (0.1 M in CH2Cl2).
o
Anodic oxidation was performed with a stoichiometric amount of electricity (1.0 F/mol) at -80 C
o
and the subsequent glycosylation with a glycosyl accepter was carried out at -50 C for 1 h (see
Supporting Information for details). As a result, the building blocks 1a-c equipped with a
neighboring group at 2-OH afforded the corresponding disaccharides with desired α-glycosidic
linkage in reasonable yields.
Depending upon the reaction outcome, we hypothesized that there is a formation of glycosyl
triflate which becomes more accumulated during the course of anodic oxidation. As the reaction
progresses, the triflate is displaced by the neighboring participating group to give acyloxonium ion,
followed by the nucleophilic attack of a glycosyl acceptor at the anomeric carbon to secure
α-selectivity in glycosylation. To check the validity of our hypothesis, comparative study was
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performed where the same reaction was carried out with a building block 2 without a participating
group and a building block 3 equipped with a pivaloyl group at 6-OH. The building block without
any participating group did not afford the corresponding disaccharide at all. Disappearance of the
glycosyl donor spot on Thin Layer Chromatography (TLC) and the maximum amount of acceptor
recovered after reaction suggested that the corresponding glycosyl triflate intermediate was not
stable under the standard reaction condition and hence decomposed. Further investigation may be
required, because this result was not consistent with those of pre-activation of 4-methylphenyl
2,3,4,6-tetra-O- benzyl-α-D-thiomannoside [14 ,15]. On the contrary, the building block equipped
with a O-pivaloyl group at 6-OH afforded the corresponding disaccharide with complete
α-selectivity. The neighboring group participation of the protecting group at 6-OH has already been
suggested by Kim [16]; however, this was the first example that clearly suggests the stabilizing
effect of the protecting group at 6-OH of the glycosyl triflate intermediate.
Table 2. Pre-activation and the subsequent coupling with methylglycoside as an acceptor.
R1
Bn
Bn
Bn
Bn
Piv
Piv
R2
Ac
product
8a
selectivity (α:β)
building block
yield
78%
60%b
71%b
tracec
1a
1b
1c
2
α only
α only
4:1
Piv
Phosa
Bn
8b
8c
-
9
Bn
45% (41%)b
47% (58%)b
3
10
α only
α only
Piv
4
11
aPO(OCH2CMe2CH2O). bNMR yields. cdetected by MS.
We extended the scope of our methodology for synthesis of 1,6-α-linked trimannosides, where
we choose substrate 1a as a terminal building block (Fig. 1). The schedule of trisaccharide synthesis
is also shown in Figure 2. The first cycle of the process was initiated by anodic oxidation of building
block 1a at -80 oC, followed by the coupling with building block 5a (R = Bz) or 5b (R = Piv) which
has a free 6-OH. The second cycle was performed in one pot and the subsequent purification of the
crude product by preparative gel-permeation chromatography (GPC) afforded desired trisaccharides
14a and 14b in 15% and 24% yield, respectively.
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HO
BnO
OR
O
BnO
OAc
O
BnO
BnO
BnO
BnO
OAc
O
BnO
OAc 5a (R = Bz)
anodic
oxidation
SAr
5b
(R = Piv)
O
BnO
BnO
O
OR
O
BnO
BnO
BnO
BnO
Bu4NOTf
CH2Cl2, -80 oC
SAr
OTf
12a
12b
1a
6a
(R = Bz)
(R = Piv)
SAr
Ar = 4-FC6H4
BnO
OAc
O
HO
OR
O
BnO
BnO
OAc
O
BnO
BnO
BnO
BnO
5a
BnO
O
OR
O
anodic
oxidation
(R = Bz)
SAr
5b (R = Piv)
O
OR
O
BnO
BnO
BnO
BnO
Bu4NOTf
CH2Cl2, -80 oC
O
OR
O
OTf
13a (R = Bz)
13b (R = Piv)
14a 15% (R = Bz)
BnO
14b 24% (R = Piv) BnO
SAr
Fig. 1. Synthesis of trimannoside.
To explore the efficacy of this method, we finally performed automated synthesis of the core
trisaccharide of GPI anchor oligosaccharides (Fig. 2). In this case, we chose 1a as the first building
block, because installation of acid labile 2-O-acetyl group may enable us to perform selective
cleavage of the group and elongation from trisaccharide 16. The second building block 5b, equipped
with an orthogonal protecting group was added with exactly a 1.0 equivalent to prevent formation of
undesired by-products. The third building block 15 having an azido group, which has frequently
been chosen to perform α-selective glycosylation with an inositol as a glycosyl acceptor, was used in
this synthesis, and finally, synthesis of core trisaccharide 16 present in GPI anchor oligosaccharides
was accomplished with 40% overall yield.
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Fig. 2. Synthesis of the core trisaccharide of GPI anchor oligosaccharides.
Conclusion
In summary, we have optimized the monosaccharide building block of mannosides for automated
electrochemical solution-phase synthesis of oligosaccharides and exploited its application in the
synthesis of a GPI anchor core trisaccharide. In order to synthesize biologically important
oligomannosides on a preparative scale further optimization of reaction conditions using the
automated synthesizer is in progress in our laboratory.
Acknowledgements
This work was supported by JSPS KAKENHI Grant Number JP15H05844 on Middle Molecular
Strategy. T. N. acknowledges support of the discretionary budget of the President of Tottori
University. All of the calculations with Gaussian09 were performed using the Research Center for
Computational Science (RCCS) in Okazaki (Nagoya, Japan). The authors thank the RCCS for their
permission to access their computer resources.
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Highlights of this work
The following points should be stressed:
(1) We optimized a carbohydrate building block for α-glycosidic linkage of mannosides based
on DFT calculation, measurement of oxidation potentials, and disaccharide synthesis.
(2) A pivaloyl protecting group not only at the C-2 hydroxyl group of a mannoside (2-OH) but
also at 6-OH was effective to accumulate the glycosyl triflate intermediate and control the
stereochemistry of the α-glycosidic linkage.
(3) The target trisaccharide, which is the core structure of GPI anchor oligosaccharides, was
synthesized by the automated electrochemical assembly of optimized building blocks.