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Chemical Synthesis of Oligosaccharides related to the Cell Walls of Plants and Algae

Kinnaert, Christine; Daugaard, Mathilde; Nami, Faranak; Clausen, Mads Hartvig

Published in:Chemical Reviews

Link to article, DOI:10.1021/acs.chemrev.7b00162

Publication date:2017

Document VersionPeer reviewed version

Link back to DTU Orbit

Citation (APA):Kinnaert, C., Daugaard, M., Nami, F., & Clausen, M. H. (2017). Chemical Synthesis of Oligosaccharides relatedto the Cell Walls of Plants and Algae. Chemical Reviews, 117(17), 11337-11405.https://doi.org/10.1021/acs.chemrev.7b00162

https://doi.org/10.1021/acs.chemrev.7b00162

https://orbit.dtu.dk/en/publications/1bb38b29-e095-4bec-97ea-b4332cd5099b

https://doi.org/10.1021/acs.chemrev.7b00162

1

Chemical Synthesis of Oligosaccharides related to the Cell Walls of Plants and

Algae

Christine Kinnaert, Mathilde Daugaard, Faranak Nami, Mads H. Clausen*

Center for Nanomedicine and Theranostics, Department of Chemistry,

Technical University of Denmark, Kemitorvet, Building 207, 2800 Kgs. Lyngby, Denmark

[email protected]

Abstract

Plant cell walls are composed of an intricate network of polysaccharides and proteins that varies

during the developmental stages of the cell. This makes it very challenging to address the functions

of individual wall components in cells, especially for highly complex glycans. Fortunately,

structurally defined oligosaccharides can be used as models for the glycans, to study processes such

as cell wall biosynthesis, polysaccharide deposition, protein-carbohydrate interactions, and cell-cell

adhesion. Synthetic chemists have focused on preparing such model compounds, as they can be

produced in good quantities and with high purity. This review contains an overview of those plant and

algal polysaccharides, which have been elucidated to date. The majority of the content is devoted to

detailed summaries of the chemical syntheses of oligosaccharide fragments of cellulose,

hemicellulose, pectin, and arabinogalactans, as well as glycans unique to algae. Representative

synthetic routes within each class are discussed in detail and the progress in carbohydrate chemistry

over recent decades is highlighted.

2

Contents

Introduction ........................................................................................................................... 3 Architecture and composition of plant cell walls ............................................................... 6

2.1 Polysaccharides in plant and algal cell walls ................................................ 8 2.2 The study of cell wall composition, function and evolution ....................... 10

Cellulose ............................................................................................................................... 12 Hemicellulose ....................................................................................................................... 18

4.1 Xyloglucans ................................................................................................. 20 4.2 β-(1→3)-D-Glucans ..................................................................................... 28 4.3 Mixed-linkage [β-(1→3), β-(1→4)] glucans .............................................. 35 4.4 Xylans (arabinoxylans, glucuronoxylans and glucurono-arabinoxylans) ... 38 4.5 Mannans ...................................................................................................... 47

Pectin .................................................................................................................................... 56 5.1 Homogalacturonan ...................................................................................... 59 5.2 Xylogalacturonan ........................................................................................ 66 5.3 Apiogalacturonan ........................................................................................ 66 5.4 Rhamnogalacturonan I ................................................................................ 70

RG-I backbone ............................................................................................................... 71 RG-I side chains ............................................................................................................. 80

5.5 Rhamnogalacturonan II ............................................................................... 81 RG-II side chain A ......................................................................................................... 83 RG-II side chain B ......................................................................................................... 89 RG-II side chain C ......................................................................................................... 94 RG-II side chain D ......................................................................................................... 95 RG-II side chain E/F ...................................................................................................... 95

(Arabino)galactans and arabinans .................................................................................... 96 6.1 Type I AGs and galactans ........................................................................... 97 6.2 Type II AGs ............................................................................................... 107 6.3 Arabinans .................................................................................................. 119

Polysaccharides exclusive to algae ................................................................................... 124 7.1 Glucuronans .............................................................................................. 124 7.2 Carrageenans ............................................................................................. 125 7.3 Agarans ...................................................................................................... 127 7.4 Porphyrans ................................................................................................. 127 7.5 Alginates .................................................................................................... 128 7.6 Fucans ........................................................................................................ 137 7.7 Ulvans ........................................................................................................ 150

Conclusion .......................................................................................................................... 151 References ................................................................................................................................ 154 TOC Graphic ........................................................................................................................... 183

3

Introduction Plant cell walls are highly sophisticated fiber composite structures. They are dynamic systems, which

have evolved to fulfill the wide range of biological functions necessary to support plant life. The wall

is the outer layer of the cell, providing a strong and protective supporting boundary, and defining the

size and shape of the cell. Beyond its structural function, the cell wall also plays an important role in

cell expansion during plant growth, and in plant-microbe interactions, in particular the defense

response against pathogens.1,2 The cell wall is not just a boundary between plant cells, but forms the

interface to neighboring cells, contributing to intercellular communication.

Plant cell walls are highly heterogeneous, with a wide range of constituent components. Table 1

compares the monosaccharides found in mammalian cells with the ones occurring in plant and/or algal

cell wall, together with the symbols used here and in the CFG nomenclature. The diversity can make

it challenging to distinguish the function of individual components and how they interact. Researchers

have therefore used well-defined oligosaccharides as model compounds to represent domains from

the larger, more complex polysaccharides. Many groups have succeeded in synthesizing such

oligosaccharides using different strategies. This review will introduce the glycans found in plant cell

walls and describe all relevant chemical oligosaccharide synthesis, up to and including February 2017.

The material is organized in tables showing the structures according to glycan nomenclature given in

Figure 1. With the exception of some rare disaccharide combinations found in pectin, we present only

trisaccharides and longer structures, and only oligosaccharides that have been deprotected

successfully. Representative syntheses are discussed in detail, in order to include different approaches

to plant cell wall glycan assembly. Some of the syntheses described here have been covered in

4

previous reviews; we are endeavoring to provide a comprehensive overview of the field, and therefore

have chosen not to exclude these.

Table 1. Comparison between mammalian monosaccharides and plant/ algae cell wall monosaccharides.

Monosaccharide CFG symbol (existing or suggested)

Symbol used here Plant/algal cell wall glycans

Mammalian glycans

Glucose

D D

Galactose

D and L D

N-Acetyl-glucosamine

D

N-Acetyl-galactosamine

D

Mannose

D D

Galacturonic acid

D D

Xylose

D D

Fucose

L L

Sialic acid

D

Iduronic acid

L L

Rhamnose

L

Gulose

L

Arabinose

L

5

Apiose

D

3-Deoxy-lyxo-heptulosaric acid

(Dha)

D

2-Keto-3-deoxy- manno-

octulosonic acid (Kdo)

D

Aceric acid

Me

L

L-gulose

D-glucose

D-xylose

D-mannose

D-galactose L-arabinose

L-rhamnose OAc

COOH

SO3H

β-notation

α-notation

= O1

23

46

L-fucoseCOOMe

D-apiose

L-aceric acid less common enantiomer (D/L)

D-3,6-anhydrogalactose

D-2-Keto-3-deoxy-D-manno-octulosonic acid (Kdo)

D-3-deoxy-D-lyxo-heptulosaric acid (Dha)

OMe

Example:

=

OCOOHHO

HOHO

OMeO

OHO

OOH

HO OH

O

= α-D-GalpA-(1→2)-[α-L-Araf-(1→4)]-β

-L-RhapOMe

L-iduronic acid

Me

Figure 1. Glycan notation.

6

Architecture and composition of plant cell walls As a simple model, plant cell walls can be grouped in two types: the primary and the secondary cell

wall. The primary cell wall is a thin and flexible structure deposited when the cell is growing, while

the secondary cell wall, much thicker and stronger, is deposited on certain cells when they have ceased

to grow. Primary cell walls are typically composed of around 70% water. The dry material is made up

of polysaccharides (approximately 90%) and (glyco)proteins (~ 10%). In the primary cell wall,

cellulose microfibrils are cross-linked by non-cellulosic polysaccharides (often referred to as

hemicellulose), and these are intertwined with pectic polysaccharides. The chemical composition of

the main polysaccharides found in land plants is summarized in Table 2.

There is strong evidence that hemicellulose, mostly xyloglucans, bind to the cellulose microfibrils via

two different mechanisms: being entwined in the microfibril during crystallization, which occurs

immediately after synthesis; and by hydrogen bonding between the glycan chains.3–5 The

polysaccharide network in the cell wall helps the plant resist turgor pressure and other deformations.

Less is known about the interaction of pectic polysaccharides with the other wall components, but

there is evidence that neutral glycans side chains also interact with cellulose, a factor that is usually

not accounted for in models describing plant cell wall architecture.6–8 Pectic polysaccharides are

usually divided into four different domains (HG, RG-I, RG-II, and XGA / AGA, see Table 1), which

are believed to be covalently linked to form the pectin macromolecule. Evidence suggests that some

pectic polysaccharides are also crosslinked to hemicelluloses, phenolic compounds and wall proteins,

which provides an even higher degree of complexity in the structure and function of the wall.5,9 A

2009 paper detailing the structure, function and biosynthesis of plant cell wall pectic polysaccharides

by Mohnen & Caffall provides a good overview.6

7

Table 2. Cellulose, hemicellulose and pectin structures. Cellulose β-(1→4)-Glup Hemicellulose Xyloglucans (XyG) β-

(1→4)-Glup with mainly Xylp branching

Glucans β-(1→3)-Glup with β-(1→6) branching

Mixed-linkage glucans (MLGs) β-(1→3)-β-(1→4)-Glup

Xylans β-(1→4)-Xylp (potentially with Araf and GlupA branching)

Mannans β-(1→4)-Manp and β-(1→4)-Glup/Manp with Galp branching

Pectin Homogalacturonan (HG) α-(1→4)-GalpA

Rhamnogalacturonan I (RG-I) α-(1→4)-GalpA-α-(1→2)-Rhap backbone with side chains of Galp and Araf

Rhamnogalacturonan II (RG-II) α-(1→4)-GalpA with conserved side chains of mainly rare sugars

Xylogalacturonan (XGA) α-(1→4)-GalpA with Xylp branching

Apiogalacturonan (AGA) α-(1→4)-GalpA with Apif branching

In addition to polysaccharides, the primary cell wall also contains about 10% protein, which is mostly

glycosylated. Arabinogalactan proteins (AGPs) are some of the most abundant and most studied

glycoproteins in plant cell walls and they are thought to be involved in recognition and signaling

events at the cell surface.10 However, as the focus of this review is on polysaccharides, we will not

describe the plant cell wall proteome. We invite the reader to look at some comprehensive reviews

covering this subject.11,12

In secondary cell walls, the embedding material is not pectin, but rather the phenolic polymer lignin,

which constitutes up to 30% of the dry weight of the wall.13 The secondary cell wall also contains

cellulose and a network of connecting hemicellulosic polysaccharides with a lower degree of

backbone branching (mainly xylan and mannans). The deposition of lignin results in dehydration of

the wall, rendering it more hydrophobic and less dynamic. Other components such as the aliphatic

polyesters cutin and suberin can also be found in the plant cell wall as minor components.14 Plant cell

walls display a considerable degree of diversity in their composition and molecular architecture

depending on plant species but also related to cell type and cell wall microstructure.15 An interesting

8

review of the heterogeneity in the chemistry, structure and function of plant cell walls has been

published by Fincher and co-workers recently.15

2.1 Polysaccharides in plant and algal cell walls

The primary cell walls of seed plants and especially angiosperms (flowering plants) are by far the

most studied. However, since land plants have algal ancestors, the cell walls of algae can be interesting

from an evolutionary point of view. Land plants originate from freshwater green algae of the

Charophytes class, which emerged from their aquatic habitat approximately 450 million years

ago.16,17,18 As shown in the phylogenetic tree of eukaryotic organisms (Figure 2), land plants

(Embryophytes), red algae (Rhodophytes) and green algae (Chlorophytes) all belong to the

Archaeplastida clade, while brown algae (Phaeophytes) belong to the separate Chromalveolata clade.

The phylogenetics can explain why the cell walls of brown algae share polysaccharides with both

plants (cellulose) and animals (sulfated fucans), but also with some bacteria (alginates).19

9

Figure 2. Phylogenetic tree of eukaryotic organisms.

The nature of the different cell wall components from the major plant and algal families has been the

topic of several reviews and is summarized in Table 3.16,20,21 The unique presence of different sulfated

polysaccharides in the Rhodophytes and Phaeophytes is noteworthy. Agar, carrageenan and porphyran

are the most important sulfated polysaccharides found in the red algae, while brown algae produce

homofucans. The structure of these polysaccharides will be described in detail later in this review.

10

Table 3. Major cell wall polymers present in different plant and algal taxa.22

Taxa Polymer

Chloroplastida Rhodophytes

(red algae)

Phaeophytes

(brown algae) Embryophytes (land plants)

Charophytes (green algae)

Chlorophytes (green algae)

Crystalline polysaccharides

cellulose cellulose cellulose

cellulose mannan xylan

(1→3)-β-D-xylan

cellulose

Hemicellulose

xyloglucan mannans xylans MLG

glucan (callose)

xyloglucan mannans xylans

glucan (callose)

xyloglucan mannans

glucuronan glucan (callose)

mannan sulfated MLG

(1→3), (1→4)-β-D-xylan

sulfated xylofucoglucan

sulfated xylofuco- glucuronan

glucan (laminarin) Matrix

carboxylic polysaccharides

pectins pectins ulvans alginates

Matrix sulfated

polysaccharides ulvans

agars carrageenans

porphyran fucans

2.2 The study of cell wall composition, function and evolution

The study of the plant cell wall requires a multi-disciplinary approach and there are many challenges

in trying to elucidate cell wall architecture, function, and evolution. The field is being advanced by

technologies such as atomic force microscopy, immunofluorescence microscopy and the development

of molecular probe sets.23 In a 2012 review, Willats and co-workers discussed a multiple-level strategy

for correlating the occurrence of cell wall polysaccharides with genetic diversity in order to get an

insight into underlying evolutionary mechanisms.24 Many of the most efficient techniques employ

molecular probes that interact specifically with glycans, such as monoclonal antibodies (mAbs) and

carbohydrate-binding modules (CBMs). Well-defined oligosaccharides provide the highest resolution

possible when characterizing protein-carbohydrate interactions, and they contribute significantly to

11

the toolkit available for plant research. Figure 3 shows an example of in situ mapping of cellulose,

rhamnogalacturonan I and methyl esterified homogalacturonan in seed coat epidermal cells and

mucilage in Arapidopsis thaliana.

Figure 3. Labelling of polysaccharidic components of seed coat epidermal cells and mucilage in Arabidopsis thaliana. (A) Composite image of a section through whole seed and mucilage. Scale bar = 100 µm. (B) Magnification of region in (A). Scale bar = 50 µm. Blue: calcofluor-labeled cellulose; red: INRA-RU1-immunolabelled rhamnogalacturonan; green: JIM7-immunolabelled highly methyl esterified homogalacturonan.23 Images used with permission from Adeline Berger.

A B

12

Cellulose

Figure 4. Structure of cellulose.

Cellulose is the main component of the primary cell wall in plants. It is the most abundant organic

compound on earth and consists of an unbranched homopolymer of β-(1→4)-glucan units (Figure 4).

The biopolymer is currently used in a wide variety of applications including the production of biofuels,

paper products, and textile fibers.25 Cellulosic chains are held together tightly by both intra- and

intermolecular hydrogen bonds facilitated by the conformation of the polymer. The chain length

depends on the source of the cellulose; the degree of polymerization (DP) is believed to reach up to

8,000 glucose residues in primary cell walls and 15,000 in secondary cell walls. This makes cellulose

one of the longest bio-macromolecules known.

Table 4 shows all chemically synthesized β-(1→4)-glucans with at least three glucose residues. It is

worth noting that the majority of the reports on the preparation of β-(1→4)-glucans detail enzymatic

procedures.

Table 4. The cellulose fragments synthesized chemically.

Structure R Year Reference

OR

Me

H

(CH2)3(CH)2O

1981

1983

1983

1990

Takeo et al.26

Takeo et al.27

Takeo et al.27

Rodriguez and Stick28

13

CH2CH2C10H7

(CH2)5NHCOO(CH2)3C8F17

(CH2)5NH2

1999

2010

2016

Xu and Vasella29

Chang et al.30

Dallabernardina et al.31

OR

Me

(S)-CH-CH3CH2OH

(R)-CH-CH3CH2OH

(S)-CH2CH-OHCH3

(R)-CH2CH-OHCH3

1983

1983

1986

1983

1986

1983

1985

1986

1983

1985

1986

Takeo et al.27

Sugawara et al.32

Sugawara et al.33

Sugawara et al.32

Sugawara et al.34

Sugawara et al.32

Nicolaou et al.33

Sugawara et al.34

Sugawara et al32

Nicolaou et al.33

Sugawara et al.34

OR

H

(CH2)5NHCOO(CH2)3C8F17

CH2CH2C10H7

1983

1996

2010

1999

Takeo et al.27

Nishimura and Nakatsubo35

Chang et al.30

Xu and Vasella29

OR (S)-CH2CH-OHCH3

(R)-CH2CH-OHCH3

1985

1985

Nicolaou et al.33

Nicolaou et al.33

OR2

(CH2)5NH2 2016 Dallabernardina et al.31

14

OR2

(S)-CH2CH-OHCH3

(R)-CH2CH-OHCH3

1985

1985

Nicolaou et al.33

Nicolaou et al.33

OR5

H 1996

1999

Nishimura and Nakatsubo35

Xu and Vasella29

In 1981 Takeo and co-workers reported the synthesis of β-methylcellotrioside using a Koenigs-Knorr

glycosylation36 between building blocks 1 and 2 (Scheme 1).26 This produced the trisaccharide in 39%

yield alongside unreacted 1 and 2. The trisaccharide was converted into the bromide 4 before

methanolysis in the presence of mercuric cyanide, which gave 84% of 5. Zemplén deacetylation37 then

resulted in the targeted cellotrioside 6 in 92% yield.

15

Scheme 1. Synthesis of β-methyl cellotrioside reported by Takeo and co-workers in 1981.26

AcO OOAc

AcOAcO

O OOAc

AcOAcO

Br

HO OOAc

AcOAcO OAc

1 2

+

O

AcOAcO O O

OAc

AcOAcO

O OOAc

AcOAcO

AcOOAc

OAc

3

O

AcOAcO O O

OAc

AcOAcO

O OOAc

AcOAcO

AcOOAc

Br4

O

AcOAcO O O

OAc

AcOAcO

O OOAc

AcOAcO

AcOOAc

OCH3

5

O

OHHO O O

OH

HOOH

O OOH

OHHO

HOOH

OCH3

6

AgOTf(CH3)2NCON(CH3)2

CH2Cl2,

39%

HBr in AcOH

84%

Hg(CN)2

CH3OH:C6H6 1:10

84%

NaOMe

MeOH

92%

Nishimura and Nakatsubo published the first synthesis of cellooctaose using a convergent synthetic

strategy in 1996.35 The synthesis is depicted in Scheme 2. The tetrasaccharide 8 (derived from allyl

3-O-benzyl-4-O-(p-methoxybenzyl)-2,6-di-O-pivaloyl-β-D-glucopyranoside 7 using a linear

synthetic method described by Nishimura et al.38) was used as a common precursor for the

tetrasaccharide acceptor 9 and Schmidt39 donor 11. These two were coupled under standard conditions

(BF3⋅OEt2) giving the octasaccharide 12 in 95% yield. The glycosylation was conducted in a

high-vacuum system allowing for better control of the anhydrous reaction conditions. Protecting

group manipulations afforded the fully protected oligosaccharide 14. The allyl group was removed

using a one-pot procedure first reported by Kariyone and Yazawa40 before the anomeric position was

acetylated. Catalytic hydrogenolysis followed by acetylation yielded the fully acetylated

16

octasaccharide 18. After purification, deacetylation afforded cellooctaose 19 in 87% yield. In a similar

sequence, tetrasaccharide 8 was converted into cellotetraose 25 (see Scheme 2). More recently,

Pfrengle and co-workers produced linear β-(1→4)-glucans using solid-phase oligosaccharide

synthesis.31 This work was part of a larger study of xyloglucan synthesis and will be described in

section 4.1.

17

Scheme 2. Synthesis of cellotetraose and cellooctaose published by Nishimura and Nakatsubo.35

O

PivOBnO O O

OPiv

BnOPivO

O OOPiv

PivOBnO

OOPiv

OAllAcO OOPiv

BnOPivO

PMBO OOPiv

PivOBnO OAll

O

PivOBnO O O

OPiv

BnOPivO

O OOPiv

PivOBnO

OOPiv

OAllHO OOPiv

BnOPivO

O

PivOBnO O O

OPiv

BnOPivO

O OOPiv

PivOBnO

OOPiv

ORAcO O

OPiv

BnOPivO

10: R = H11: R = C(NH)CCl3

9

7 8

O

ROBnO O O

OR

BnORO

O OOR

ROBnO

OOR

OAllRO OOR

BnORO

O

AcOR2O O O

OAc

R2OAcO

O OOAc

AcOR2O

OOAc

OR1AcO O

OAc

R2OAcO

O

OHHO O O

OH

HOOH

O OOH

OHHO

OOH

OHHO O

OH

HOOH

25

O

PivOBnO O O

OPiv

BnOPivO

O OOPiv

PivOBnO

OOPiv

OAllAcO OOPiv

BnOPivO

12

O

ROBnO O O

OR

BnORO

O OOR

ROBnO

OOR

OAllRO OOR

BnORO

13: R = H14: R = Ac

O

AcOR2O O O

OAc

R2OAcO

O OOAc

AcOR2O

OOAc

OR1AcO O

OAc

R2OAcO

15: R1 = H, R2 = Bn16: R1 = Ac, R2 = Bn17: R1 = Ac, R2 = H18: R1 = R2 = Ac

3

3

3

O

OHHO O O

OH

HOOH

O OOH

OHHO

OOH

OHO O

OH

HOOH

19

O

OHHO O O

OH

HOOH

O OOH

OHHO

OOH

HO OOH

HOOH

SeO2, AcOH, dioxane

79%

DBU, CCl3CN, CH2Cl2, 96%

DBU, MeOH

65%

BF3·OEt2,

CH2Cl2

95%

NaOMeMeOH

78%

Ac2O, py, 50 oC, quant.

SeO2AcOH, dioxane

77%

Ac2O, py, quant.Pd(OH)2/C, H2

, THF

Ac2O, py, 64%

DBUMeOH:CH2Cl2

1:4

87%

NaOMeMeOH

quant.

20: R = H21: R = Ac

Ac2O, py, 82%

SeO2AcOH, dioxane

54%

22: R1 = H, R2 = Bn23: R1 = R2 = H24: R1 = R2 = Ac

Pd/C, H2, EtOH:AcOH 4:1

92%Ac2O, py, 72%

DBUMeOH:CH2Cl2

1:4

75%

18

Hemicellulose Hemicellulose was first described in 1891 by Ernst Schulze, as the components of the cell wall that

are easily dissolved in hot diluted mineral acids.41 Such a definition based on extractability was not,

however, found to be useful, and these days the term is commonly used to refer to a group of non-

starch, non-pectic polysaccharides found in association with cellulose in the cell walls of higher plants.

Based on current knowledge, hemicellulose is generally divided into four classes of structurally

different cell-wall polysaccharides: xylans, mannans, xyloglucans and glucans including mixed

linkage glucans (β-(1→3),(1→4)-glucans, MLGs) as shown in Figure 5.42 These are the types of

polysaccharides described in most reviews about hemicellulose, but Peter Ulvskov and Henrik

Scheller, in their review of hemicellulose, further narrow down the definition to glycans sharing a β-

(1→4)-linked backbone with an equatorial configuration at the C-4 position. This excludes MLGs,

callose and laminarin (β-(1→3)-linked glucans) from the hemicelluloses.4,42 Other polysaccharides,

such as galactans, arabinans, and arabinogalactans are sometimes included in this group as well, but

these will be discussed separately in section 6.42

19

X X S

G L X F G

Xyloglucans β-1,3-Glucans

n = 0,1,2

Mixed Linkage Glucans (MLGs)

MeO MeO

Glucuronoxylan (GX)

MeO

Glucuronoarabinoxylan (GAX)

Arabinoxylan (AX) Galactomannans

Galactoglucomannans

Xylans Mannans

L-gulose

D-glucose

D-xylose

D-mannose


L-rhamnose OAc

COOH

SO3H

β-notation

α-notation

= O1

23

46

L-fucoseCOOMe

D-apiose

L-aceric acid less common enantiomer (D/L)




L-iduronic acid

Me

Figure 5. Overview of the different families of hemicellulose polysaccharides.

20

4.1 Xyloglucans

X X S

G L X F G

Figure 6. Structure of XyGs with the most common side chains.

Xyloglucans (XyGs) constitute the most abundant class of hemicellulose in the primary cell wall of

higher plants. They have been found in every species of land plant that has been analyzed, except

charophytes.4 An exemplary structure of XyG with the different side chains is shown in Figure 6.

However, this schematic representation does not indicate the frequency of each side chain and many

variations in this general pattern can be observed. XyGs have a backbone of β-D-(1→4)-glucans

substituted by α-D-(1→6)-linked xylose residues. A special one-letter code is used to label the

different XyG side chains. G denotes an unbranched Glcp residue, while X corresponds to an α-D-

Xylp-(1→6)-Glcp. The xylosyl residues can be further branched at O-2 with α-L-Araf (S) or β-Galp

(L), which can be further substituted at O-2 with α-L-Fucp (F). The galactose residues can be

acetylated. The branches dictate the physical properties of the molecules; the fewer the branches, the

less soluble the XyG. Only a few syntheses of XyGs with substituted xylose (side chains S, L or F)

have been reported to date, including an LG fragment produced by Jacquinet and co-workers43 and

an XXFG fragment synthesized by Ogawa and co-workers.44 In 2016, Pfrengle and co-workers

achieved the automated solid phase synthesis of a variety of XyG oligomers without xylose

substitution.31 Table 5 shows all the XyG fragments synthesized to date.

21

Table 5. The synthetic XyG fragments.


OR


OR

Pr 1990 Wotovic et al.43

OR


OR


OR

H 1990 Sakai et al.44

OR

Me 2000 Watt et al.45

OR

H 1990 Sakai et al.44

OR


OR


22

In 1990, Jacquinet and co-workers synthesized tetrasaccharide LG (38) by condensation of the two

key disaccharides galactoxylose and cellobiose 33 and 36 respectively, as shown in Scheme 3. The

building blocks 33 and 36 were synthesized through standard protection group manipulations and

glycosylation reactions. Coupling of donor 33 and diol acceptor 36 afforded tetrasaccharide 37 in 50%

yield as a single regio- and stereoisomer. However, the authors showed that steric hindrance around

the O-6’ position of the acceptor had a dramatic impact on the regioselectivity as well as the yield of

the reaction. Indeed, when using a diol acceptor with benzyl protecting groups in lieu of acetyl groups,

only 10% of the undesired β-linked tetrasaccharide could be isolated, while none of the desired

stereoisomer was observed.

23

Scheme 3. Synthesis of LG tetrasaccharide by Jacquinet and co-workers.43

OAcO

OAllOAc

AcOO

HO

OAcO

AcOHO

O

OAc

BnO

BnO

OBn

O

ClBnO

BnOO

OAcO

OAllOAc

AcOO

O

OAcO

AcOHO

O

OAc

BnO

BnO

OBn

OBnOBnO

O

OHO

OPrOH

HOO

O

OOH

HOHO

O

OH

HO

HO

OH

OHOHO

O

OAcOAcO

OO

OEtMe

OBnOBnO

OO

OEtMe

OBnOBnO

OHOAll

OBnO

OO

OMeMe

BnO OBn

OBnO

AcOCl

BnO OBn

O

OAc

BnO

BnO

OBn

OOAllBnO

BnOO

1) 95% aq. AcOH2) Ac2O, py

3) BnNH2, Et2O4) (COCl)2

, DMF, CH2Cl2

57%

AgOTfClCH2CH2Cl

83%

AgOTfClCH2CH2Cl

50%

1) NaOMe, MeOH

2) Pd/C, H2

88%

OAcO

OAllOAc

AcO

OAcO

OAcO

AcOAcO O

HO

OAllOH

HO

OOO

OHHOO

Ph1) NaOMe, MeOH

2) benzaldehyde dimethylacetal DMF

75%

1) Ac2O, py

2) 60% aq. AcOH

85%

26 27 28

29 30

31 33

34 35 36

3633

37 38 (LG)

1) NaOMe, MeOH

2) NaH, BnBr, DMF

79%

1) 2,6-dimethylpyridinium perchlorate,ClCH2CH2Cl, AllBr, PhCl

2) NaOMe, MeOH

74%

3028 1) NaOMe, MeOH

2) KOtBu, DMSO

80%

O

OH

BnO

BnO

OBn

OOBnO

BnOO

32

1) Ac2O, pyridine2) HgCl2/HgO

acetone/water

3) (COCl)2

ClCH2CH2Cl, DMF

70%

The same year, Ogawa and co-workers44 reported the synthesis of both the heptasaccharide XXXG

(48) and the branched nonasaccharide XXFG (57) using a convergent approach as shown in Scheme

4. To reach the two targets, they used the key glycohexaosyl acceptor 46 (XXGG) as a common

precursor. This resulted from a coupling between fragment 43 (XX) and cellobiose acceptor 44 (GG).

Glycosylation with xylopyranose donor 40 and hexose acceptor 46 afforded the heptasaccharide 47

in 64% yield as a 4:3 ratio of separable α:β stereoisomers. Finally, saponification and hydrogenolysis

24

afforded the desired unprotected XXXG target 48. In the same manner, coupling between

trisaccharide donor 55 and 46 afforded the nonasacharide 56 as a 3:1 mixture of stereoisomers which,

after separation and deprotection, gave the XXFG xyloglucan oligosaccharide 57. Even though the

crucial glycosylation steps were plagued by low yields and poor stereoselectivity, both XyG targets

could be reached by way of a common advanced intermediate using this convergent strategy.

25

Scheme 4. Synthesis of XXXG and XXFG oligomers by Ogawa and co-workers.44

OHO

OBnOBn

BnO

OHO

OBnO

BnOBnO

OOR

ORRO

OO

ORO

RORO

O

OBn

BnOBnO SMe

O

RO

RORO

O

O

RO

RORO

41: R = Bn42: R = H

OO

OAcAcO

OO

OAcO

AcOAcO

O

AcO

AcOAcO

O

O

AcO

AcOAcO

CuBr2, Bu4NBr, HgBr2CH3NO2

62%

1) Ac2O, py2) NH2NH2·AcOH, DMF

3) CCl3CN, DBU, CH2Cl2

72%NH

CCl3

OBnO

OBnOBn

BnO

OPMBO

OBnO

BnOHO

39

40

43

43BF3·OEt2

52% OO

OAcAcO

OO

OAcO

AcOAcO

O

AcO

AcOAcO

O

O

AcO

AcOAcO

4445

R = PMB46

R = H

OBnO

OBnOBn

BnO

ORO

OBnO

BnO

40, CuBr2, Bu4NBr, HgBr2

CH3NO2

64% O

OOAc

AcO

OO

OAcO

AcOAcO

O

AcO

AcOAcO

O

O

AcO

AcOAcO

47

OBnO

OBnOBn

BnO

O

O

OBnO

BnO

O

AcO

AcOAcO

α:β 4:3 - 25% α isolated

1) NaOMe, MeOH

2) H2, Pd/C, MeOH

63%

OO

OHHO

OO

OOH

HOHO

O

HO

HOHO

O

O

HO

HOHO

48 (XXXG)

OHO

OHOH

HO

O

O

OOH

HO

O

HO

HOHO

O

OH

BnO OAllBnOO

OBnBnO

BnOAcOO NH

CCl3

OOBnBnO

BnORO

O

O

BnO OAllBnOO

OAcAcO

AcO

O

O

AcOAcO

51: R = Ac52: R = H

BF3·OEt2

73%NaOMe, MeOH

94%

MeOTf

81%

OOBnBnO

BnOO

O

O

BnO OAllBnOSMe

7 steps

31%

49 54 55

55

OO

OAcAcO

OO

OAcO

AcOAcO

O

AcO

AcOAcO

O

O

AcO

AcOAcO

OBnO

OBnOBn

BnO

OO

OBnO

BnO

CAN, CH3CN, H2O56%

OOAc

OAcOAc

O

OO

OAcAcO

1) NaOMe, MeOH

2) H2, Pd/C, MeOH, H2O

54%

46, CuBr2, Bu4NBr, HgBr2 Et2O 26%

56α:β 3:1 - 20% α isolated

57 (XXFG)

O SMeOBn

OBnBnO

Me

53

O OBnOBnBnO

Me O OAcOAcAcO

Me

O

O

OAc

AcOAcOMe

50

OO

OHHO

OO

OHO

HOHO

O

HO

HOHO

O

O

HO

HOHO

OHO

OHOH

HO

O

O

OHO

HO

OO

HOHO

OOH

HO OHO

O

OH

Me OHOH

26

More recently, Pfrengle and co-workers reported an automated glycan assembly of five XG

oligosaccharides using monosaccharide 58 and disaccharide 59 as building blocks (Scheme 5).31 This

solid phase synthesis also enabled the production of linear cellotriose and cellopentaose (see Table 4).

The authors used a Merrifield resin46 with a photocleavable linker47 and two phosphate glycosyl

donors48 with either Fmoc or a Lev groups as temporary O-4 protection. Using this strategy, the five

XyG oligomers (60–64) were produced in overall yields of 2–16%.

27

Scheme 5. Automated glycan assembly of five XyG oligosaccharides by Pfrengle and co-workers.31

HO

O

ON

O2N

O

OBnO

BnOOBz

OP

OBuOBu

O

FmocOO

O

BnOOBz

OP

OBuOBu

O

LevO

OBnO

OBnO

BnO

OR2O

BnOOBn

OHO

n

R2 = Bn, α-xylopyranosyl

OR2O

BnOOBz

OR1O

n

R1 = Fmoc, LevR2 = Bn, α-xylopyranosyl

OHO

HOOH

HO O OO

HOOH

O OO

HOOH

O OHO

HOOH

O

5859

Start

1a) 1 or 2 × 3.7 equiv. 58,

TMSOTf, CH2Cl2 or1b)

2 or 3 ×

3.7 equiv. 59,

TMSOTf, CH2Cl2

2a) 3 × 20% Et3N in DMF or

2b) N2H4·HOAc py, AcOH, H2O

Cleavage and global deprotection3) hν, CH2Cl24) NaOMe, MeOH, THF5) Pd/C, H2, EtOAc, MeOH, H2O, HOAc

OHO

HO

HOO

HOHO

HO

NH23

OHO

HOOH

HO O OO

HOOH

O OHO

HOOH

O NH23

OHO

HO

HO

OO

HOOH

O O OO

HOOH

O OHO

HOOH

O NH23

OHO

HOOH

HO O OO

HOOH

OHO

HO

HOO

HOHO

HOO

HOHO

HO

OHO

HOOH

O O OO

HOOH

O OHO

HOOH

O NH23

OHO

HOOH

HO O OO

HOOH

OHO

HO

HOO

HOHO

HOO

O

HOOH

O O OHO

HOOH

O OHO

HOOH

O NH23

HO OO

HOOH

OHO

HO

HOO

HOHO

HO

60: 15% (GXG)

61: 16% (GXXG) 62: 2% (GXXXG)

63: 10% (GXGXG) 64: 9% (XXGG)

HO

Merrifieldresin

28

4.2 β-(1→3)-D-Glucans

Figure 7. Structure of β-(1→3)-D-glucan (callose).

β-(1→3)-D-Glucans with various short β-(1→3)-D-glucan chains branched at the 6-position of the

backbone are found in the cell walls of land plants, algae and also fungi.49 Callose is in this class of

polysaccharide and has been isolated from all multicellular green algae as well as some higher plants

(embryophytes).50 This polymer plays an important role in maintaining cell wall integrity as well as

in plant defense. In response to pathogen attack, callose is deposited between the plasma membrane

and the pre-existing cell wall at the sites of attack.51 Another member of this class, laminarin, has been

found in brown algae. As certain types of β-glucans containing (1→3) and (1→6) linkages exhibit

biological activity by stimulating the innate immune system in man, their synthesis has attracted much

interest.52 Several groups have successfully accomplished syntheses of linear as well as branched

oligosaccharides mostly with the motivation of synthesizing fungal glycans. The synthesized

fragments are summarized in Table 6.

Table 6. The β-(1→3)-D-glucan fragments synthesized to date.


OR

Me

H

1993

2011

2005

Takeo et al.53

Adamo et al.54

Jamois et al.55

29

(CH2)3S(CH2)2NH2

Cy-(C5H9)

2011

2013

Adamo et al.54

Jong et al.56

OR

Me 2011 Adamo et al.54

OR

All 2003 Zeng et al.57

OR

2

Me

H

1993

2003

2005

Takeo et al.53

Zeng et al.57

Jamois et al.55

OR

3

Me

CH2CHOCH2

H

1993

2005

2005

Takeo et al.53

Huang et al.58

Jamois et al.55

2

OR

(CH2)3NH2 2016 Yashunsky et al.59

OR

4

Me

H

(CH2)3S(CH2)2NH2

(CH2)5COOCH3

(CH2)2NHCO(CH2)3COOSu

1993

2009

2011

2015

2015

Takeo et al.53

Mo et al.60

Adamo et al.54

Elsaidi et al.61

Liao et al.62

OR

5

Me 1993 Takeo et al.53

30

OR

6

Me

(CH2)6NH2


1993

2010

2015

Takeo et al.53

Tanaka et al.63

Liao et al.62

3

OR

3

n = 0, 2

n

(CH2)2NH2 2015 Liao et al.64

OR

n = 4, 8, 12

(CH2)6NH2 2010 Tanaka et al.63

ORn = 1, 4, 8

(CH2)5NH2 2017 Weishaupt et al.65

OR

8

(CH2)2NHCO(CH2)3COOSu 2015 Liao et al.62

OR

10

(CH2)6NH2

(CH2)5NH2


2010

2013

2015

Tanaka et al.63

Weishaupt et al.66

Liao et al.62

OR

12

(CH2)6NH2 2010 Tanaka et al.63

OR

14

Me

(CH2)6NH2

2010

2010

Tanaka et al.63

Tanaka et al.63

Several strategies have been developed to produce both linear and branched β-(1→3)-D-glucan

oligosaccharides through solution phase synthesis. For oligosaccharides up to the pentasaccharide

31

level, an iterative approach has been used, which has enabled the synthesis of tri-, tetra- and

pentasaccharides in the same sequence, as exemplified by the preparation of β-(1→3)-D-glucan

oligosaccharides reported by Vetvicka and co-workers (Scheme 6).55,56 The group used an iterative

deprotection-glycosylation approach with monosaccharide 65 to elongate the chain. This key

monosaccharide could act successfully as both donor and acceptor to afford linear glucans 66–69. In

common with most other reported syntheses of linear β-(1→3)-D-glucans, the glucoside building

block 65 carried a benzoyl group at the 2-position to invoke neighboring group participation and a

4,6-benzylidene acetal as a permanent protecting group. The short length oligosaccharides were easily

deprotected using Zemplén conditions and catalytic hydrogenolysis to afford the corresponding

reducing oligosaccharides.

Scheme 6. Synthesis of protected di, tri, tetra- and penta-β-D-(1→3)-glucans by Vetvicka and co-workers.55

OOOPh

SEtOBz

NAPO

65

1) NIS, BnOH, TESOTf 89%

2) DDQ, CH2Cl2, MeOH

85%

3) 65, NIS, TESOTf 83%

OOOPh

OBzNAPO

OOOPh

OBnOBz

O

OOOPh

OBzNAPO

OOOPh

OBzO

OOOPh

OBnOBz

O

n

66

67 : n = 1

68 : n = 2

69 : n = 3


86%






For the synthesis of longer β-D-glucan oligosaccharides in solution, the use of convergent strategies

employing di-53,59–62,64, tri-54,57–59,64, tetra-52,64 and pentasaccharide52,59 building blocks has been

demonstrated to be advantageous, as it reduces the number of steps. This type of approach has been

32

used to synthesize long linear52,62 and branched52,59,64 oligosaccharides. In 2015, Guo and co-workers62

reported a convergent synthesis of tetra-, hexa-, octa-, deca- and dodeca-β-(1→3)-D-glucans, which

they achieved through pre-activation-based iterative glycosylation with p-tolylthioglycosides as

donors and disaccharide 70 as the key building block. The latter was used as a donor to elongate the

chain from disaccharide to tetra-, hexa-, octa-, deca- and finally dodecasaccharides.

Scheme 7. Convergent linear synthesis of tetra-, hexa-, octa-, deca-, and dodecasaccharides by Guo and co-workers.62

OOOPh

OBzNAPO

OOOPh

STolOBz

O

70

OOOPh

OBzHO

OOOPh

OOBz

O

N3

1) 70, AgOTf, TTBP, p-TolSCl,

CH2Cl2, - 78 οC

2) DDQ

CH2Cl2, H2O

90%

OOOPh

BzOO

OOOPh

OOBz

O

N3

OOOPh

BzOHO

7271

2

70OO

OPh

BzOO

OOOPh

OOBz

O

N3

OOOPh

BzOHO

n

73a-d n = 4, 6, 8, 10

OHOHO

OHO

OHOHO

OOH

O

H2N

OHOHO

OHHO

n

74a-d n = 4, 6, 8, 10

1) 2-azidoethanol, AgOTf, TTBP

p-TolSCl, CH2Cl2, - 78 οC

2) DDQ, CH2Cl2, H2O

91%

Using another highly convergent and efficient synthesis, the same group prepared two branched

oligosaccharides consisting of a nonaglucan backbone carrying a β-(1→3)-D-glucodiose or -tetraose

at the 6’’’’-position.64 The backbone was assembled in a [4 + 1 + 4] fashion, in which each

tetrasaccharide building block was generated using an iterative one-pot glycosylation as shown in

Scheme 8.

33

Scheme 8. Retrosynthesis of branched glucans by Guo and co-workers.64

OOOPh

STolOBz

HOOO

OPhSTol

OBzNAPO

Preactivation-based iterative one-pot glycosylation

OOOPh

BzOO

OOOPh

OOBz

O

N3

OOOPh

BzOHO

2

OOOPh

BzOO

OOOPh

STolOBz

OOOOPh

BzOBzO

2

Preactivation-based iterative one-pot glycosylation

OOOPh

STolOBz

HOOO

OPhSTol

OBzBzO

OBnO STolOBz

HO

OLev

OOOPh

BzOO O

4

OBnO

OBzO

OH

OOOPh

BzOOBz

4

N3

OHOHO

OHO O

4

OHO

OHOOHO

HO

OHOH

4

NH2

RO

R =

1) 2 glycosylations2) selective removal of Lev

1) glycosylation of the branch R2) Zemplén deprotection and hydrogenolysisOHO

HO

OHHO

OHOHO

OHO

OHOHO

OHO

n = 0, 2

75a-b

76

77 78 79

80 81 82 81

+ +

Takahashi and co-workers achieved the impressive syntheses of linear hexadeca- and branched

heptadeca-saccharides by conducting a convergent synthesis employing a tetrasaccharide as the key

building block.52 In this case, 2,3-diol glycosides with a 4,6-O-benzylidene protecting group were

effective as glycosyl acceptors, made possible by the higher reactivity of the 3-position over the 2-

position. This afforded the desired (1→3)-linked regioisomer in each glycosylation reaction.

As shown by these examples, the use of pre-assembled building blocks is efficient but only gives

access to glucans of limited chain lengths. In 2013, the group of Seeberger developed an iterative

automated solid-phase synthesis of linear β-D-glucan oligosaccharides giving great flexibility in terms

of the length of the targeted oligosaccharides. They showed the efficiency of the approach by

synthesizing a linear β-(1→3)-D-glucan dodecasaccharide, as shown in Scheme 9.66 Glycosyl

34

phosphate building block 83 in combination with the resin-linker construct 84 (see also Scheme 5)

proved to be a suitable choice for the automated synthesis of linear glucans. The efficiency of this

glycosylation sequence was proven by the average glycosylation yield over 12 cycles: 89%. In 2017,

the group further optimized the conditions to synthesize a series of branched β-(1→3)-D-glucans.65

Scheme 9. Automated solid-phase synthesis of β-glucan dodecasaccharide 87 by Seeberger and co-workers.66

OBnO

FmocOOPiv

OP

OBuOBu

O

Merrifieldresin

83

12 ×3 ×

3 equiv. of 83

TMSOTf, CH2Cl2, dioxane3 ×

piperidine, DMF

O

O2N

NCBzO

BnO

OBnO

OOPiv

BnOOBnO

OOPiv

BnOOBnO

HOOPiv

BnO

10

hν, CH2Cl2

NHCBzOOBnO

OOPiv

BnOOBnO

OOPiv

BnOOBnO

HOOPiv

BnO

10

86

OOHO

OOH

HOOHO

OOH

HOOHO

HOOH

HO

10

1) NaOMe, MeOH, CH2Cl22) Pd(OH)2

, H2, THF, H2O, AcOH

NH2

87

85

O

O2N

NCBzHO

84

35

4.3 Mixed-linkage [β-(1→3), β-(1→4)] glucans

n = 0,1,2

Figure 8. Structure of MLGs.

Mixed-linkage glucans (MLGs) are usually composed of short stretches of β-(1→4)-glucans

connected through β-(1→3)-linkages. They are a type of hemicellulose characteristically found in

both the primary and secondary cell walls of grasses. These glucans have also been isolated from

bryophytes67 and from the horsetail plant (Equisetum).68 More recently, in 2000, Lahaye and co-

workers discovered MLG 6-sulfate in the cell wall of the red alga Kappaphycus alvarezii. No chemical

synthesis of MLG oligosaccharides had been reported before the automated synthesis achieved by

Pfrengle and co-workers in 2017.69

Table 7. The synthetic MLG fragments prepared by Pfrengle and co-workers.

Structure R Reference

OR

(CH2)5NH2 Dallabernardina et al.69

OR


OR

2


OR


36

OR

2 (CH2)5NH2 Dallabernardina et al.69

OR


OR

2


This group performed the automated synthesis of seven MLG oligosaccharides using glucose building

blocks 58 and 88 as shown in Scheme 10. After optimizing the conditions for trisaccharide 89, they

concluded that two glycosylation cycles were required for the formation of β-(1→3)-linkages while

one coupling sufficed to form the β-(1→4)-linkages. After completion of the solid-phase synthesis,

the oligomers were cleaved from the resin in a continuous flow reactor, and deprotected under

standard conditions to afford the targets (89–95).

37

Scheme 10. Automated synthesis of seven MLG oligosaccharides by Pfrengle and co-workers.69

OBnO

FmocOOBz

OP

OBuOBu

O

O

O2N

NCBzHO

84

BnOOBnO

BnOOBz

OP

OBuOBu

O

FmocO

58 88

StartCleavage and global deprotection3) hν, CH2Cl24) NaOMe

MeOH, THF5) Pd/C, H2,

EtOAc

MeOH, H2O, AcOH

1a) 2 × 3.7 equiv. of 58

TMSOTf, CH2Cl2

or1b) 1 ×

3.7 equiv. of 88

TMSOTf, CH2Cl2

2) 3 × 20% Et3N

DMF

OHO

HOOH

OHO O

HO

OHO

HO OHO

HOOH

O NH23

OHO

HOOH

OO

HO

OHO

HO OHO

HOOH

O NH23

OHO

HOOH

OHO

OHO

HOOH

OO

HO

OH

HOOHO

HOOH

OOO NH2

3

OHO

HOOH

OHO O

HO

OHO

HO OHO

HOOH

O OHO

HOOH

O NH23

OHO

HOOH

OO O

HO

OH

HO OO

HO

OHO

HO OHO

HOOH

OHO

HOOH

HO

OHO

HOOH

OO

HO

OHO

HO OHO

HOOH

O NH23

OHO

HOOH

OO

OHO

HOOH

OO

HO

HOOH

HO

OHO

HOOH

OO

HO

OH

HOO O NH23

OHO

HOOH

OO

HO

OH

HOOHO

HOOH

OO

OHO

HOOH

HO

OHO

HOOH

OO

HO

OH

HOOHO

HOOH

OOO NH2

3

O OHO

HOOH

OHO

HOOH

OO O

HO

OH

HOOHO

HOOH

OHO O

HO

OH

HO

89: 13%

90: 26%

91: 12%

92: 12%

93: 34%

94: 18%

95: 23%

= Merrifield resin

84

38

4.4 Xylans (arabinoxylans, glucuronoxylans and glucurono-

arabinoxylans)

MeO MeO

Glucuronoxylan (GX)

MeO

Glucuronoarabinoxylan (GAX)

Arabinoxylan (AX)

Figure 9. Schematic representation of different common heteroxylans.

Xylan, the most prevalent non-cellulosic polysaccharide in plant cell walls, binds to cellulose

microfibrils and to lignin in order to strengthen the cell wall.4 Recently, Dupree and co-workers used

13C solid-state magic-angle spinning (MAS) nuclear magnetic resonance (NMR) spectroscopy to

show that xylans, which form a threefold helical screw in solution, flatten into a twofold helical screw

ribbon to bind intimately to cellulose microfibrils in the plant cell wall.70 Xylans have a backbone of

β-(1→4) linked D-xylose and they are known to have highly complex and heterogeneous structures

due to the number, distribution and differentiation of side chains. Xylans are usually classified into

homoxylans and heteroxylans, where the latter includes arabinoxylans (AX), glucuronoxylans (GX)

and glucuronoarabinoxylans (GAX) as shown in Figure 9. Arabinoxylans present L-arabinofuranosyl

substituents α-(1→2) and/or α-(1→3) linked to the xylose residues of the backbone. Additionally, it

39

has been found that in grasses, the O-5 position of arabinose can be esterified with for example ferulic

acid that can form covalent bonds to lignin.71,72 In glucuronoxylans, 4-O-methyl-D-glucuronic acid

and/or D-glucuronic acid residues are generally α-(1→2)-linked to xylose substituents, with an

average substitution ratio of 1:10.73 Finally, GAX has 4-O-methyl-D-glucuronic acid in the O-2-

postitions and arabinofuranosides in the O-3-position with a ratio of xylan:substituents of

approximately 10:1.4.73 In addition to these substituents, most xylans are acetylated to a varying

degree. Esterification is mainly found in the O-3 position, and more rarely in the O-2 position. An

overview of the xylan fragments that have been synthesized is presented in Table 8.

Table 8. Synthetic xylan fragments.


OR H 1982 Hirsch et al.74

OR

2 Me

H

Bn

(CH2)5NH2

1981

1982

1995

2015

Kováč et al.75

Hirsch et al.74

Takeo et al.76

Schmidt et al.77

OR

3 H

Bn

1982

1995

Hirsch et al.74

Takeo et al.76

OR

4 (CH2)5NH2

Bn

2015

1995

Schmidt et al.77

Takeo et al.76

OR

5 Bn 1995 Takeo et al.76

40

OR

6 (CH2)5NH2

Bn

2015

1995

Schmidt et al.77

Takeo et al.76

OR


OR


OR

MeO

H 2001 Oscarson et al.78

OR

Me 1978 Kováč et al.79

OR

(CH2)5NH2 2015 Schmidt et al.77

OR

(CH2)5NH2 2017 Senf et al.80

OR

MeO

Me 1980 Kováč et al.79

OR


OR


OR


OR


41

OR


OR


OR


OR


OR


OR


OR


OR


The chemical synthesis of well-defined homoxylans has been reported over recent decades by several

groups using a range of synthetic strategies.74–77 Fewer have described the synthesis of heteroxylans,

77–79 probably due to the difficulty in producing differentially-protected xylose building blocks needed

to introduce side chains. Xylobiose was first synthesized by Myrhe and Smith in 1961, and the reaction

was carried out via Helferich coupling81 between benzyl 2,3-di-O-benzyl-D-xylopyranoside and a

peracetylated xylosyl bromide in the presence of Hg(CN)2.82 Twenty years later, Hirsch and Kováč

synthesized a series of oligoxylans in the same fashion by using a sequential approach to elongate the

42

chain and reach up to a pentaxylan as shown in Scheme 11.74,75 1,2,3-Tri-O-acetyl-4-O-benzyl-β-D-

xylopyranose 96 was used as common building block to form both the bromide donor 97 and acceptor

98. Mercury cyanide-mediated coupling of the two afforded the disaccharide product as a 1:1.5

mixture of α:β stereoisomers, which could be separated by column chromatography to afford 23% of

the desired β-(1→4)-linked disaccharide 99. Subsequent hydrogenolysis afforded the 4’-OH

disaccharide acceptor 100 ready for coupling with donor 97. Repetition of the two last steps enabled

the researchers to reach tri-, tetra- and pentaxylans (103a-c) after saponification. Using this procedure,

the yields of the glycosylation reactions were all quite low (< 25%). The same iterative strategy was

subsequently employed to obtain a series of methyl β-xylosides up to a xylohexaoside.83

Scheme 11. Hirsch and Kováč strategy for the synthesis of oligohomoxylans.75

OBnO

OAcOAcAcO

OBnO

AcOAcO

96

97 Br

OHO

OAcOAcAcO

98

Hg(CN)2

MeCN

23%

OO

OAcOAcAcO

99: R = Bn100: R = H

ORO

AcOAcO

1) 97, Hg(CN)2

2) Pd/C, MeOH acetone OO

AcOAcO

ORO

AcOAcO OO

OAcOAcAcO

n = 1,2,3

R = Bn 101a-cR = H 102a-c

OO

HOHO

OHO

OHHO OO

HO OHHO

103a-c n = 1,2,3

NaOMe

MeOH

HBrtoluene CH2Cl2

Pd/CMeOH

acetone

Pd/CMeOH, acetone

Pd/CMeOH, acetone

More recently, Takeo and co-workers developed a blockwise approach to a series of linear xylo-

oligosaccharides up to xylodecaose using suitably protected thio-xylobiosides as building blocks and

43

N-iodosuccinimide (NIS) in combination with silver triflate as promoting agent. This strategy afforded

glycosylation yields of around 80%.76

The first synthesis of heteroxylan oligosaccharides was reported by Hirsch and Kováč in 1982. 75,79

The fragment was a xylotriose backbone bearing a β-(1→3)-linked xylose unit and an α-(1→2)-linked

glucuronic acid. The researchers started a stepwise synthesis from methyl 2,3-anhydro-β-D-

ribopyranoside 104 as shown in Scheme 12. This epoxide was coupled with a xylosyl bromide donor

105 and the resulting disaccharide 106 was then selectively deprotected at positions O-3 and O-4 and

further glycosylated with xylosyl bromide 108 to afford the tetrasaccharide 109 in 40% yield. The

epoxide was then opened selectively with benzyl alcohol following the Fürst-Plattner rule84, in order

to produce the xylose tetrasaccharide 112. The free hydroxyl group in position 2 was then glycosylated

with glucoronate donor 113 in the presence of silver perchlorate and 2,4,6-collidine affording the

desired α-branched pentasaccharide in 57% yield.

Scheme 12. Synthesis of a branched 4-O-methyl-α-D-glucuronic acid-containing xylotetraose by Hirsch and Kováč.75,79

OAcO

BnOAcO

Br105

104

ORO

BnORO

106: R = Ac107: R = H

OAcO

AcOAcO

Br108

OO

BnOO

109: R = Ac110: R = H

ORO

RORO

ORO

RORO

Hg(CN)2

MeCN

62%

Hg(CN)2

MeCN

40%

BnBrNaH

DMF

83%

OO

BnOO

111

OBnO

BnOBnO

OBnO

BnOBnO

OO

BnOO

OBnO

BnOBnO

OBnO

BnOBnO

O

OHBnO OMeOBnONa

BnOH

OO

OHO

OHOOH

HOOHO

OHHO

112

OOHO

HOO

OHO

MeOMeOOC

HO

OBnO

MeOMeOOC

BnO Cl

1) AgClO4, Et3N CH2Cl2

, 57%

2) H2, Pd/C, H2O

acetone

113

114

NaOMe, MeOH 98%

NaOMe, MeOH 98%

OHO

O

OMe OO

O

OMe OO

O

OMe

OO

O

OMe

44

In 2001, Oscarson and Svahnberg reported the synthesis of two glucuronoxylan fragments based on

xylobiose. Using dimethyl(methylthio)sulfonium trifluoromethane sulfonate (DMTST) in diethyl

ether, they were able to couple the glucuronic acid donor 116 with complete α-selectivity to the

disaccharide acceptor 115 (Scheme 13).78

Scheme 13. Synthesis of a glucuronoxylan trisaccharide by Oscarson and Svahnberg.78

O

OBzBzO OPMBO

O

OBnBnO SEtMeO

OMeO

O

OBzBzO OPMBO

OOO

OMe

OMe

O

O

BnOBnO

MeO

OMeO

O

OHHO OHO

OHOHO

O

O

HOHO

MeO

OHO

DMTSTEt2O

89%

deprotection

32%

115 117

116

118

OOO

OMe

OMe

OH

Recently, Pfrengle and co-workers developed an automated synthesis of arabinoxylans.77,80 They first

reported the synthesis of a set of xylan oligosaccharides up to an octasaccharide decorated in different

positions with either one or two α-(1→3)-linked arabinofuranosides as shown in Scheme 14.65

45

Scheme 14. Automated synthesis of arabinoxylan fragments by Pfrengle and co-workers.76 O

BnOOBz

OP

OBuOBu

O

FmocO

119

ONapO

OBzO

POBu

OBuO

FmocO

120

OOBz

OBzBzO

SEt

121

OOBz

OFmocBzO

SPh

122

OO

HOHO

OHO

OHHO OO

OHHO

2 123 : 27%

O NH23

OO

HOHO

OHO

OHHO OO

OHHO

4

O NH2

3

124 : 20%

OO

HOHO

OHO

OHHO OO

OHHO

6

O NH2

3

125 : 43%

OO

OHO

OHO

OHHO OO

OHHO O NH2

3OOH

OHHO 126 : 23%OO

OHHO

OHO

OHO OO

OHHO O NH2

3O

OH

OHHO 127 : 19%

OO

OHO

OHO

OHHO OO

OHHO O NH2

3OOH

OHO 128 : 42%

OO

OHO

OO

OHHO

OOH

OHHO 129 : 8%

OHO

OHHO OO

OHHO OO

OHHO O NH2

3

OO

OHHO

OO

OHO

OO

OHHO OO

OHHO O NH2

3OOH

OHHO 130 : 15%

OO

OHO

OHO

OHHO

OOH

OHHOOO

OHO

OO

OHO OO

OHHO O NH2

3OOH

OHHO 131 : 21%

OO

OHHO

OHO

OHHO

OOH

OHHO

OO

OHO

OO

OHHO OO

OHHO O NH2

3OOH

OHHO 132 : 7%

OO

OHHO

OO

OHO

OOH

OHHO

OHO

OHHO

O

HO OH

OH

Automated solid-phase synthesis

Coupling:

TMSOTf, CH2Cl2 (119, 120)

NIS, TfOH, CH2Cl2, dioxane (121, 122)

Deprotection:

20% Et3N in DMF (Fmoc) DDQ, ClCH2CH2Cl (NAP)

Capping:

Ac2O, pyCleavage:

hν, CH2Cl2

Deprotection:

NaOMe, CH2Cl2, MeOH Pd/C, H2,

EtOAc, MeOH, H2O, AcOH

To achieve their synthesis, two xylosides 119 and 120, differentially protected at their O-3 position,

were used as building blocks for the assembly of the linear xylan backbone, while perbenzoylated and

2-O-Fmoc-L-arabinofuranosides 121 and 122 were used for substitution. The sequential synthesis

provided, within short timeframes and with overall yields of 7-43%, a collection of arabinoxylan

46

fragments (123–132) as shown in Scheme 14. Very recently, they optimized this strategy in order to

supplement this set of α-(1→3)-substituted glycans with α-(1→2)-substituted arabinoxylan

oligosaccharides 135 to 139 (see Figure 10). By using the 2-O-(azidomethyl)benzoyl (Azmb)-

protected donor 133 the team could make oligosaccharides containing monosubsituted xylose

residues. With the inclusion of the orthogonally protected donor 134, synthesis of the disubstituted

arabinoxylan 135 was possible.

OBnO

AzmbOO

POBu

OBuO

FmocO

133

ONapO

AzmbOO

POBu

OBuO

FmocO

134

OO

OO

OHO

OHHO OO

OHHO O NH2

3OOH

OHHO135 : 4%

OOH

OHHO

OO

OHO

OHO

OHHO OO

OHHO O NH2

3

137 : 13%

OOH

OHHO

OO

OHHO

OHO

OHO OO

OHHO O NH2

3

139 : 41%

OOH

OHHO

OO

OHO

OO

OHHO

136 : 7%

OHO

OHHO OO

OHHO OO

OHHO O NH2

3

OO

OHHO

OOH

OHHO

OO

OHHO

OO

OHHO

138 : 4%

OHO

OHO OO

OHHO OO

OHHO O NH2

3

OO

OHHOO

OH

OHHO

OO

OHO

OO

OHHO

OHO

OHHO OO

OHHO OO

OHHO O NH2

3

OO

OHHO

O

HOOH

HO 140 : 11%

Figure 10. A set of substituted (arabino)xylan oligosaccharides synthesized in 2017 by Pfrengle and co-workers.80 The strategy is presented in Scheme 14.

47

4.5 Mannans

Galactomannans

Galactoglucomannans Figure 11. Schematic representation of different mannans.

Mannans are widely distributed and the main hemicellulose in Charophytes. 67 They have been

intensively studied due to their role as seed storage compounds, but they are found in variable amounts

in all cell walls.4 The mannan class of polysaccharides in higher plants can be divided into two

categories: galactomannans and glucomannans. Galactomannans have a homologous backbone made

up of β-(1→4)-linked mannose residues only, and can be substituted at some of the O-6 positions by

β-D-galactose units. The glucomannan backbone is composed of β-(1→4)-linked glucose and

mannose units assembled in a random pattern with a higher number of mannose than glucose

monosaccharides. Galactoglucomannans are further substituted with galactose at the O-6 positions of

some backbone mannose residues. Furthermore, mannans are often acetylated at C-2, C-3 and more

rarely at C-6 positions depending on the plant species.85 Mannans and glucomannans have been highly

conserved throughout plant evolution, however their role and function in the plant cell wall remain

largely unclear. The exception is the galactoglucomannans which are often described as seed storage

compounds.4 The formation of β-linked mannosides is an exceedingly difficult glycosylation reaction

since neighboring group participation, the steric hindrance from a protecting group at the O-2 position

and the anomeric effect uniformly favor the formation of α-D-mannopyranosides. Nevertheless,

48

several groups have developed strategies to circumvent the challenges and have successfully

synthesized certain mannan fragments, as shown in Table 9. Numerous reviews dealing with advances

in the construction of β-D-mannosides have been published. Among those, a review by Gridley and

co-workers86 deals with most of the strategies. A more recent one, by Lichtenthaler, presents the use

of 2-oxoglycosyl donors in assembling oligosaccharides with β-D-mannose, β-L-rhamnose, N-acetyl-

β-D-mannosamine, and N-acetyl-β-D-mannosaminuronic acid residues.87

Table 9. Synthetic mannan fragments.


OR Bn 2003 Twaddle et al.88

OR2

Me 2008 Kim et al.89

OR4

Me

H

Pr

2004

2013

2015

Crich et al.90

Ohara et al.91

Tang et al.92

OR

H 2013 Ohara et al.91

OR


OR5


OR

2 H 2013 Ohara et al.91

OR

2 H 2013 Ohara et al.91

49

OR6

C6H11

H

2004

2013

Crich et al.90

Ohara et al.91

OR

2 2 H 2013 Ohara et al.91

OR Me 2012 Ekholm et al.93

OR

Me 2012 Ekholm et al. 93

In 2004, Crich and co-workers developed a direct approach for the synthesis of β-mannosides using

4,6-O-benzylidene-directed glycosylation via in situ generation of α-mannosyl triflates from their

thio- or sulfoxidoglycoside precursors. They employed this strategy in combination with a three step

iterative glycosylation protocol to synthesize a β-linked hexamannoside as shown in Scheme 15.90

Benzylidene deprotection, followed by selective Piv protection of the primary alcohol in each case

provided the acceptor ready for the glycosylation with 4,6-O-benzylidene-thiomannoside 141. High

glycosylation yields were obtained in each case (71–82%) and very good β-selectivity was achieved

(α:β ~1:10).

50

Scheme 15. Three-step iterative glycosylation protocol to synthesize hexamannoside 146d by Crich and co-workers.90

OOO

Ph

BnO

OBn

SPh

SO

N

BSP, Tf2OTTBP

MeOH

82%

OOO

Ph

BnO

OBnOMe

1) neopentyl glycol, CSA,CH2Cl2 89%

2) PivCl, DMAP, Et3N, CH2Cl2 98%

OHOBnO

OBnOMe

OPiv

141 142 143

141, BSPTf2O, TTBP

CH2Cl2

80%

OOBnO

OBnOMe

OPivOO

OPh

BnO

OBn

144

3-step protocol: A) removal of benzylideneB) Piv protectionC) glycosylation with donor 141

OOBnO

OBnOMe

OPivOO

BnO

OBnOOO

Ph

BnO

OBnOPiv

n145a-d n = 1, 2, 3, 4

OOHO

OHOMe

OHOO

HO

OHOHOHO

OHOH

4

OH

OOBnO

OBnOMe

OPivOO

BnO

OBnOOO

Ph

BnO

OBnOPiv

4145d 146d

1) NaOMe, MeOH 94%

2) H2, Pd/C, MeOH

87%

N

N

2-benzenesulfonyl piperidine

(BSP)

2,4,6-tri-tert-butylpyrimidine

(TTBP)

In 2008, Jeon and co-workers reported an efficient one-pot, three-step glycosylation strategy starting

from a reducing sugar. A 4,6-O-benzylidene mannopyranose was treated with phthalic anhydride in

the presence of DBU, and the α-mannosyl phthalate intermediate was further activated with triflic

anhydride to generate a mannosyl triflate. Like Crich and co-workers, the group used 4,6-O-

benzylidene protected mannose to impart stereoselectivity and form the desired β-mannosidic

linkage.89

Another approach was taken by Pohl and co-workers in an automated solution phase synthesis of a β-

(1→4)-linked hexamannoside, shown in Scheme 16.92 Inspired by the work of van der Marel and co-

workers in their formation of β-mannosidic linkages using a mannuronate donor (section 7.5), Pohl

and co-workers used a β-directing C-5 carboxylate strategy and synthesized the protected β-(1→4)-

hexamannoside 149 in an average yield of 75% per step. Post-assembly, the mannuronic acid residues

were reduced to mannans with DIBAL-H.

51

Scheme 16. Automated solution phase synthesis of β-hexamannoside by Pohl and co-workers.92

OMeO2C

TBSOOBn

BnOO

NH

CCl3

R = H

148, TMSOTf CH2Cl2

TBAF, Et3N · 3HFDMSO, THFReduction

DIBAL-H, CH2Cl2, toluene

1) TMSOTf, 148 CH2Cl2

2) TBAF, Et3N⋅3HF

DMSO, THF

R = TBS

Start Coupling

Deprotection

148

OMeO2CO

OBn

BnO O O

C8F17R

n

OMeO2CO

OBn

BnO O O

C8F17R

n

HO O

C8F17

OOOBn

BnO O O

C8F17H

6

HO

149

OOOH

HO OPrH

6

HO

150

1) Grubbs' cat. 2nd gen. ethylene, CH2Cl2

2) H2, Pd/C, MeOH

73%

147

Indirect approaches requiring epimerization at C-2 in readily available β-glucosides have been used

by other groups to synthesize linear β-(1→4)-linked mannans.88,91 Nikolaev and co-workers accessed

β-(1→4)-mannotriose from commercially available and cheap D-cellobiose octa-acetate 151 via an

epimerizing SN2 reaction as shown in Scheme 17.88 After some protecting group manipulations

required to reach benzyl 4’,6’-O-benzylidene-cellobioside diol 153, simultaneous epimerization of C-

2 and C-2’ was performed via triflation followed by reaction with Bu4NOBz, affording β-(1→4)-

linked mannobiose 154 in 76% yield. Mannobiose 156 was further coupled to glucose donor 157,

forming a trisaccharide, which was then epimerized to give mannotriose 160 in a reasonable overall

yield.

52

Scheme 17. Synthesis of β-mannotriose through C-2 epimerization of glucoside by Nikolaev and co-workers.88

O

OAc

AcO

OAcOAcO O

OAcAcO

OAc

OAc

151

O

OH

HO

OHOHO O

OHHO

OH

OBn

1) HBr, AcOH, CH2Cl2

2) BnOH, Hg(OAc)2

3) NaOMe, MeOH

70%

OO

OHOBzO O

OHBzO

OBz

OBnOPh

1) PhCH(OMe)2,

TsOH·H2O, DMF 68%

2) BzCN, Et3N 33%

1) Tf2O, py CH2Cl2

2) Bu4NOBz,

toluene

76%

OO OBzO OBzO

OBz

OBnOPh

OBz

OBz

OHO OBzO OBzO

OBz

OBnOBz

OBz

OH

OHO OBzO OBzO

OBz

OBnOBz

OBz

OBzBzCN, Et3N, MeCN, CH2Cl2

92%

AcOH

76%

O

OAc

AcO

AcOAcO

O NH

CCl3

TMSOTf, CH2Cl247%

OO OBzO OBzO

OBz

OBnOBz

OBz

OBzO

OAc

AcO

AcOAcO

OO OBzO OBzO

OBz

OBnOBz

OBz

OBzOO

OHBzO

OPh1) HCl,

MeOH, 61%

2) PhCH(OMe)2,

TsOH·H2O, MeCN

3) BzCN, Et3N MeCN 65% (two steps)

1) Tf2O, py CH2Cl2

2) Bu4NOBz,

toluene

88%

OO OBzO OBzO

OBz

OBnOBz

OBz

OBzOO

BzO

OPhOBz

OO OHO OHO

OH

OBnOH

OH

OHOHO

HO

OHOH

1) TFA, H2O, CH2Cl2

2) NaOMe, MeOH, THF

70%

152 153

154 155 156

157

158 159

160 161

A combination of direct and indirect strategies was used by Wong and co-workers in their synthesis

of β-(1→4)-linked hexa- to octamannans, where some of the targets were further acetylated at one or

two positions along the backbone.91 An example of the convergent synthetic strategy taken to the three

hexamannan targets 162, 163, 164 is presented in Scheme 18. The three oligosaccharides can be

derived from a common protected hexamannan intermediate 165. As direct β-selective mannosylation

remains challenging, the group chose to synthesize the hexamannoside 165 by an indirect method

involving β-selective glucosylation of trimannoside acceptor 167 with mannoglucose donor 166. The

subsequent epimerization of the glucose C-2 hydroxyl group was achieved via an oxidation-reduction

sequence affording the desired β-mannoside. This methodology simultaneously allowed for

53

acetylation at the designated positions. The trisaccharide fragments 166 and 167 could be obtained

from monosaccharides 168 and 171 respectively by repeating direct β-selective mannosylations using

169 or 170 as the donors.

Scheme 18. Retrosynthetic analysis of hexamannans 162–164 by Wong and co-workers.91

O

OH

HOHO

OHO O

OHOH

HO O

OH

OHO

OR1

O OOR2

OH

HO O

OH

OHO

OHO O

OHOH

HOOH

162: R1 = R2 = H163: R1 = Ac, R2 = H164: R1 = R2 = Ac

OOBnO

OBnO O

OBnOBn

BnO O

OBn

OBnO

OHO O

OPMBOBn

BnO O

OBn

OBnO

OBnO O

OBnOBn

BnOOBn

165

OPh

OOBnO

OBnO O

OBnOBn

BnO O

OBn

OBnO

OPh

BzO O

Cl3C NH

+ HO OOPMB

OBn

BnO O

OBn

OBnO

OBnO O

OBnOBn

BnOOBn

166 167

OOBnO

OROPh

SPh

O

OBn

HOBnO

BzOOAll O

OBnOBn

BnOOBn

HO

168 171169: R = Bn170: R = PMB

The authors used the same strategy, with a late β-glucosylation in [4 + 3] and [4 + 4] couplings

followed by epimerization to get the hepta- and octamannans shown in Figure 12.

54

O

OH

OHO

OHO O

OHOH

HO O

OH

OHO

OR1

O OOR2

OH

HO O

OH

OHO

OHO O

OHOH

HOOH

172: R1 = R2 = H173: R1 = Ac, R2 = H174: R1 = R2 = Ac

HO OOH

OH

HO

O

OH

OHO

OHO O

OHOH

HO O

OH

OHO

ORO O

OHOH

HO O

OH

OHO

OHO O

OHOH

HOHO O

OHOH

HO O

OH

OHO

OH

OH

175: R = H176: R = Ac

Figure 12. Hepta- and octamannans synthesized by Wong and co-workers.91

To date, only one synthesis of a galactoglucomannan fragment has been reported. The tetrasaccharide

fragment 185 was synthesized by Leino and co-workers in 2012, using a linear synthesis involving a

β-selective mannosylation with 4,6-O-benzylidene thiomannosyl donor 177, Scheme 19.93 A β-

selective glucosylation afforded trisaccharide 181, which was further selectively deprotected at the C-

6’’ position. Finally, and following Ando and co-workers’ discovery,94 α-selective galactosylation

with di-tert-butylsilylene galactoside donor 183 afforded tetrasaccharide 184 ready for deprotection

to reach the target 185.

55

Scheme 19. Synthesis of galactoglucomannan tetrasaccharide by Leino and co-workers.93

OHO

OAcAcO SPhBnO

OOO

Ph

BnO SPh

OBnBSP, TTBP, Tf2O

CH2Cl2

53%177

178

OOO

Ph

BnO

OBnOO

OAcAcO SPh

BnO

179

OBnOHOBnO

OBn

OMe180NIS, TMSOTf,

CH2Cl2

77%

OOO

Ph

BnO

OBnOO

AcOAcO

BnOO

BnOO

BnO

OBn

OMe

OHOBnO

BnO

OBnOO

AcOAcO

BnOO

BnOO

BnO

OBn

OMe

Cu(OTf)2, BH3

·THFCH2Cl2

85%

OOBnO

BnO

OBnOO

AcOAcO

BnOO

BnOO

BnO

OBn

OMe

O

BzOBzO

OOSiO

OBzBzO

OOSi

SPh

NIS, TMSOTf,CH2Cl2

68%

181

182 184

183

OOHO

HO

OHOO

HOHO

HOO

HOOHO

OH

OMe

O

HOHO

HOOH

185

1) HF·py, THF, 68%2) NaOMe, MeOH, THF, 96%

3) H2, Pd/C, MeOH 96%

56

Pectin Pectin was first discovered in 1790 by Vauquelin,95 being first described as ‘pectin’ in 1825 by

Braconnot.96 The name derives from the Greek word pektikos, meaning ‘congeal, solidify or curdle’,

and accurately describes the main characteristic of this group of polysaccharides. As a result of their

ability to form gels, pectic polysaccharides have many industrial applications. They are used as gelling

and stabilizing ingredient in a wide range of food products. They also fulfil important biological roles,

including imparting mechanical strength and flexibility to the cell wall. Pectin therefore has multiple

functions in plant growth, morphology, development, and defense.97 Pectic polysaccharides also play

an important role in cell-cell adhesion.6

Pectins make up around 35% of the dry weight of the primary cell wall in dicots and non-graminaceous

monocots, 2–10% in grasses and other commelinoid primary walls, and up to 5% in woody tissue.98

Although pectin is structurally and functionally the most complex and heterogeneous class of

polysaccharides in nature, it is relatively well-defined, being characterized by a high content of

galacturonic acid (see Figure 13). It is widely believed that four to five pectic domains, namely,

homogalacturonan (HG), rhamnogalacturonan I (RG-I), rhamnogalacturonan II (RG-II), and

xylogalacturonan (XGA) and/or apiogalacturonan (AGA) are covalently linked to one another to form

the pectin complex in muro. As yet, there is no sufficiently compelling evidence, as to how the

different blocks are assembled in the macromolecule. Therefore, the fine-structure of pectin is still a

matter of controversy and several models have been proposed. A review by Yapo in 2011 describes

the two traditional models, one with alternating ‘smooth’ (HG) and ‘hairy’ (XGA, RG-I/RG-II)

regions, the other proposing a backbone of RG-I, with side chains of HG, XGA and RG-II. The author

argues that in the face of more recent analysis, the traditional models become less and less convincing

57

as comprehensive descriptions of pectin.99 Irrespective of which model of polysaccharide structure

one subscribes to, it is undisputed that pectins consist of distinct domains. HG and RG-I represent the

two major kinds of backbone in pectin. While HG is a linear homopolymer of partially methyl

esterified and/or acetylated α-(1→4)-linked D-galacturonic acid, the RG-I backbone is made up of

alternating galacturonic acids and L-rhammnose monosaccharides, with various L-arabinan, D-

galactan and arabinogalactan side chains. Three more domains of pectin, xylogalacturonan (XGA),

apiogalacturonan and rhamnogalacturonan II (RG-II) can be seen as structurally modified HG as they

share the same backbone. While 25–75% of the galacturonic acid residues of XGA bear xylose

substitution, RG-II is a complex branched HG having four different side chains, with a total of up to

29 different monosaccharides, including a number of rare sugars.100 More detail on the structure of

the five pectic domains will be given further on in this section. Only few pectic oligosaccharides are

available from the chemical or enzymatic degradation of polysaccharides. Although degradation

techniques have been widely used for elucidating the structure of pectin (especially RG-I), the

oligosaccharide fragments prepared in this way required extensive purification and were generally

only obtained in analytical quantities. Furthermore, this approach has mostly been limited to isolation

of linear backbone fragments since the techniques generally involve the removal of side chains.97 This

fact has prompted chemists to devise synthetic approaches to pectic oligosaccharides, albeit to date

only for a limited number of structures. A review by Nepogodiev and co-workers summarized the

fragments of HG, RG-I and RG-II, which had been synthesized before 2011.101 These are included in

the following discussion.

58

Rhamnogalacturonan I Homogalacturonan

XylogalacturonanRhamnogalacturonan II

OMe

MeO

L-gulose

D-glucose

D-xylose

D-mannose


L-rhamnose OAc

COOH

SO3H

β-notation

α-notation

= O1

23

46L-fucose COOMe

D-apiose

L-aceric acidless common enantiomer (D/L)




L-iduronic acid

Me

Figure 13. Schematic representation of the four domains of pectin.

59

5.1 Homogalacturonan

Figure 14. Homogalacturonan (HG) structure.

Homogalacturonan (HG), which is referred to as the ‘smooth region’ of pectin constitutes

approximately 65% of the macromolecule. HG consists of a linear chain of α-(1→4)-linked

galacturonic acid, which can be methyl esterified or acetylated at the C-2 and C-3 positions depending

on the plant as well as the type of tissue. HG has been shown to be present in stretches of

approximately 100 GalpA residues.102 It is believed that HG is synthesized as a highly methyl

esterified form in the Golgi apparatus and then de-esterified enzymatically, once it has been deposited

in the cell wall.98,103,104 The degree of methylation is well known to influence gelling capability as

well as other physical properties of pectin. C-6 carboxylates of HG residues can interact with calcium

ions to form a stable gel when more than 10 consecutive un-esterified galacturonic acid residues are

present.6 Furthermore, the methyl esterified GalpA residues of HG can associate through hydrophobic

interactions.100

Efforts to synthesize different fragments of HG, in some cases with a distinct pattern of methyl

esterification have provided a variety of oligomers ranging from tri- to dodecasaccharides, presented

in Table 10.

Table 10. Overview of the synthesized homogalacturonan fragments.


60

OR H 1990 Nakahara et al.105

OR Pr 2011 Pourcelot et al.106

OR H 1999

2001

Kester et al.107

Clausen et al.108

OR H 1999

2001

Kester et al.107

Clausen et al.108

OR H 1999

2001

Kester et al.107

Clausen et al.108

OR H 2003 Clausen and Madsen.109





OR8

Pr 1989 Nakahara et al.110

10OR

H 1990 Nakahara et al.111

61

As is typical for the synthesis of glycuronides, two distinct approaches have been employed to reach

oligomers of α-(1→4)-linked D-GalpA: a post-glycosylation oxidation and a pre-glycosylation

oxidation. A comprehensive microreview by van der Marel and co-workers explains the rational

behind these two strategies.112 With post-glycosylation oxidation, often the preferred approach, the

backbone of the target is assembled from aldose building blocks, followed by oxidation of the primary

alcohols to carboxylic acids prior to or after global deprotection. The alternative pre-glycosylation

oxidation strategy involves the glycosylation between protected uronic acid donors and acceptors.

Due to the electron-withdrawing carboxylic acid group, galacturonic acid building blocks are

substantially less reactive than the corresponding galactose derivatives, which makes this second

approach challenging. In fact, only two syntheses of trigalacturonides have been reported using the

pre-glycosylation oxidation method, and no oligomer longer than this has been produced.

The first was performed by Anker and co-workers in 1997 using thiophenyl galacturonides as glycosyl

donors, as shown in Scheme 20.113 Good stereoselectivity and a high yield was obtained for the first

glycosylation affording disaccharide 188. However, attempts to elongate the chain to a trisaccharide

by coupling the thiogalacturonide 190 with the disaccharide acceptor 189 only afforded the target

molecule in 45% yield. The trisaccharide 191 was not deprotected. Similar results were obtained by

Kramer et al., also by glycosylating a disaccharide acceptor with a thiogalacturonide donor. Despite

slight variations in the protecting group pattern, this group obtained the targeted trisaccharide in a

similarly disappointing 48% yield.114

62

Scheme 20. Protected trigalacturonide synthesized through a pre-glycosylation oxidation strategy by Anker and co-workers.113

OCO2BnPMBO

BnO SPhOBn

OCO2BnHO

BnO OBnOBn

78% α:β > 95:5

NIS, TfOHCH2Cl2

OCO2BnO

BnO OBnOBn

OCO2BnPMBO

BnOBnO

OCO2BnO

BnO OBnOBn

OCO2BnHO

BnOBnO

DDQCH2Cl2

, H2O

86% OCO2BnO

BnO OBnOBn

OCO2BnO

BnOBnO

OCO2MeBnO

BnO SPhOBn

45% α:β = 95:5

OCO2MeBnO

BnOBnO

186

187

188 189

190

191

NIS, TfOHCH2Cl2

These results encouraged other groups to turn their attention to the post-glycosylation oxidation

approach. Galactopyranose-based building blocks used for the synthesis of the neutral α-(1→4)-linked

galactan backbone require a temporary protecting group at the C-6 position, which can release the

primary alcohol prior to oxidation at a late stage of the synthesis. This method includes early work

done by Nakahara and Ogawa in the 1990s.105,110,111 Among their elegant syntheses, the pinnacle

remains the total synthesis of dodecagalacturonic acid using a highly convergent approach as shown

in Scheme 21.111

They chose galactosyl fluorides as donors, which afforded highly stereoselective glycosylations and

gave good yields (62–95%). Acetyl groups were used as temporary protecting groups for all the

primary alcohols, which were then selectively unmasked to afford the α-(1→4)-linked dodecagalactan

102. The alcohols were further oxidized to the corresponding carboxylic acids using a Swern115

followed by a Lindgren-Kraus-Pinnick oxidation116–118 affording the dodecagalacturonic acid product

in 50% yield. Final TBDPS deprotection and hydrogenolysis gave the target (203) in a good yield

overall.

63

Scheme 21. Synthesis of dodecagalacturonic acid by Nakahara and Ogawa.111

OO

BnOBnO

OBnO

BnOBnO

OAc

F

OO

BnOBnO

OHO

BnOBnO

OAc

OTBDPS

OAc

OAc

SnCl2,

AgClO4, Et2O

92% OO

BnOBnO

OO

BnOBnO

OAc

R

OAc

OO

BnOBnO

OBnO

BnOBnO

OAc

OAc

194: R = OTBDPS

195: R = OH (90%)

196: R = F (97%)

TBAF, AcOH, THF

Et2NSF3, MeOH

OO

BnOBnO

OHO

BnOBnO

OAc

OTBDPS

OAc

OO

BnOBnO

OO

BnOBnO

F

OAc

O

SnCl2,

AgClO4, Et2O

77% OO

BnOBnO

OO

BnOBnO

OAc

OTBDPS

OAc

OO

BnOBnO

OO

BnOBnO OAc

O

198

192

193

197

193 1) TBAF, AcOH, THF 2) Et2NSF3

, MeOH

93%

OO

BnOBnO

OO

BnOBnO

OAc

OAc

OO

BnOBnO

OO

BnOBnO OAc

O

OO

BnOBnO

OO

BnOBnO

OAc

OAc

OO

BnOBnO

OHO

BnOBnO OAc

F199

200OTBDPS

OAc

1) 80% aq. AcOH2) AcCl, py

86%

199

1) 200, SnCl2,

AgClO4 Et2O

2) 80% aq. AcOH 62%

3) AcCl, py 82%

O

O

BnOBnO

OHO

BnOBnO

OAc

O

O

BnOBnO

OAc

OTBDPS

OAc

6

O

O

BnOBnO

OBnO

BnOBnO

OH

O

O

BnOBnO

OH

OTBDPS

OH

1) 196, SnCl2,

AgClO4 Et2O 64%

2) NaOMe, MeOH 81%

O

O

HOHO

OHO

HOHO

COOH

O

O

HOHO

COOH

COOH1) Me2SO, (COCl)2, i-Pr2EtN

CH2Cl2

2) NaClO2, 2-methyl-2-butene tBuOH, NaH2PO4, H2O

50%3) TBAF, AcOH, THF

75%

4) 10% Pd/C in 80% aq. MeOH

75%

OH201 202 203

10 10

64

In order to synthesize some partially methyl-esterified hexagalacturonates, Madsen and co-workers

introduced two orthogonal protecting groups at the C-6 positions of D-galactose: acetyl and para-

methoxyphenyl (PMP). This enabled them to oxidize the C-6 position to the carboxylic acid or to the

methyl ester selectively, and thereby synthesize tri- and hexagalacturonans with different methylation

patterns.108,119 Their methodology was based on the repeated coupling of galactose mono- and

disaccharide donors onto a galactose acceptor to produce hexagalacturonan backbones. They chose n-

pentenyl donors, which provided good yields of the desired α-anomers. An example of their synthesis

of two partially-esterified hexamers is shown in Scheme 22. Tetrasaccharide acceptor 211, formed

from disaccharide 208 by sequential glycosylation with pentenyl donor 209 (ClAc = chloroacetate)

was coupled with disaccharide donor 207 to form the pure α-(1→4)-linked hexagalactoside, which

was further deacetylated to afford 212, the hexamer precursor of both targets. The oxidation to esters

was carried out in a one-pot three-step procedure. The primary alcohols were first oxidized by Dess-

Martin periodinane120 to the aldehydes, which underwent a sodium chlorite oxidation to afford the

corresponding carboxylic acids. The resulting acids were then esterified with either TMSCHN2

affording 213 or with PhCHN2 giving 214. The benzyl protection masked the positions destined to

become carboxylic acids, and aided the purification of the intermediates. To complete the synthesis,

the PMP-ethers were removed with cerium(IV) ammonium nitrate (CAN) and the primary alcohols

were oxidized and esterified using the same one-pot three-step procedure, affording both benzyl and

methyl esters. Hydrogenolysis afforded the two targets 215 and 216.

65

Scheme 22. Example of two partially methyl esterified hexagalacturonic acids prepared by Madsen and co-workers.109

O

OAcBnO

BnOOBn

OO

OAcBnO

BnOBnO F

NBS, Et2NSF3CH2Cl2

74%

O

OAcBnO

BnOBnO

O

OAcHO

BnOOBn

O

O

OAcO

BnOOBn

O

SnCl2,

AgClO4CH2Cl2

55%

204 205

206

207

O

OPMPHO

BnOBnO

O

OAcO

BnOOBn

OBn

O

OPMPClAcO

BnOOBn

O

1) 209, NIS, TESOTf CH2Cl2

2) thiourea, NaHCO3,

Bu4NI, THF 88%

209

O

OPMPO

BnOBnO

O

OAcO

BnOOBn

OBn

O

OPMPHO

BnOBnO

O

OPMPO

BnOBnO

O

OAcO

BnOOBn

OBn

O

OPMPO

BnOBnO

O

OPMPHO

BnOBnO

208 210 211

OCOORBnO

BnOBnO

OCOORO

BnOBnO

O

OPMPO

BnOBnO

OCOORO

BnOOBn

OBn

O

OPMPO

BnOBnO

O

OPMPO

BnOBnO

a: 213: R = Me (44%)b:

214: R = Bn (59%)

a) Dess-Martin periodinane, CH2Cl2then NaClO2

, NaH2PO4, THF, tBuOH, H2Othen TMSCHN2

, THF, MeOH

or

b) Dess-Martin periodinane, CH2Cl2then NaClO2

, NaH2PO4, THF, tBuOH, H2Othen PhCHN2

, EtOAc

OCOOR1HO

HOHO

OCOOR1O

HOHO

OCOOR2O

HOHO

OCOOR1O

HOHO OH

OCOOR2O

HOHO

OCOOR2O

HOHO

1) CAN, MeCN, H2O

2a) Dess-Martin periodinane, CH2Cl2then NaClO2

, NaH2PO4, THF, tBuOH, H2Othen TMSCHN2

, THF, MeOH (for 214)

or

2b) Dess-Martin periodinane, CH2Cl2then NaClO2

, NaH2PO4,THF, tBuOH, H2O

then PhCHN2, EtOAc (for 213)

3) H2, Pd/C, THF, MeOH, H2O 215: R1 = Me, R2 = H (48%)

216: R1 = H, R2 = Me (30%)

O

OHBnO

BnOBnO

O

OHO

BnOBnO

O

OPMPO

BnOBnO

O

OHO

BnOOBn

OBn

O

OPMPO

BnOBnO

O

OPMPO

BnOBnO1) 207, NIS, TESOTf

CH2Cl2

2) NaOMe, MeOH, THF 71%

212

212

1) 209, NIS, TESOTf CH2Cl2

2) thiourea, NaHCO3,

Bu4NI, THF 90%

66

5.2 Xylogalacturonan

Figure 15. Example of XGA structure.

Xylogalacturonan (XGA) is similar to HG except that the backbone is substituted with single β-

(1→3)-linked xylose residues or short β-(1→4)-linked xylan side chains as shown in Figure 15.121

XGA-based glycans have not yet received attention from synthetic chemists.

5.3 Apiogalacturonan

Figure 16. Example of AGA structure showing various types of branching points.

Apiogalacturonan (AGA) is found in the cell walls of aquatic plants such as duckweeds (Lemnaceae)

and marine seagrasses (Zosteraceae).122 In AGA, β-D-apiofuranosyl residues are attached to the 2-

and/or 3-positions of selected GalpA monosaccharides in the HG chain in the form of mono- or

sometimes short oligo-saccharides, as shown in Figure 16.6 While several groups have focused on the

synthesis of apiose-containing oligosaccharides found in the RG-II region of pectin (see section 5.5),

only two groups have reported the synthesis of oligosaccharides related to apiogalacturonan (see Table

11).

67

Table 11 Three different synthetic AGA oligosaccharides.


OR

Me 2004

2011

Buffet et al.123

Nepogodiev et al.124

OR

Me 2011 Nepogodiev et al.124

OR


As D-apiose is a rare sugar, all syntheses of D-apiose-containing oligosaccharides start with the

preparation of the appropriate apiose derivative, usually from D-mannose, L-arabinose or D-xylose.125–

128 Reimer and co-workers chose a pre-glycosylation oxidation approach to synthesize AGA

disaccharide 225 as shown in Scheme 23. They started by preparing 2,3-O-isopropylidene

apiofuranose 219 from D-mannose in five steps.126,127 Four additional transformations afforded

thioapiofuranoside donor 222. Glycosylation of the 2-OH galacturonate acceptor 223 proceeded

smoothly, giving disaccharide 224 in 83% yield. Deprotection afforded the disaccharide 225 in 76%

yield.

68

Scheme 23. Synthesis of disaccharide 225 by Reimer and co-workers.123

O

O

OH

O

HO

O OHOOO

O

O OHOOO

O

OH

37% aq. HCOH K2CO3

, MeOH

86%

H2SO4, aq. MeOH

then NaBH4then NaIO4

79%

O

O

OBn

O

BnOBnBr, NaHDMF

92%

1) 80% HCOOH2) Ac2O, py

81%

O

OAc

R

AcO

BnO

221: R = OAc222: R = STol

217 218 219 220 TolSH, BF3.Et2O

90%

OO

O

CO2Me

OMeHO

222, NIS, AgOTfCH2Cl2

84%

OO

O

CO2Me

OMeOO

OAcAcO

BnO

223 224

1) Pd(OAc)2, H2

EtOAc, MeOH, AcOH 90%

2) NaOMe, MeOH3) H2O, Amberlite H+

4) NaOH (aq.) 85% (three steps)

OHO

HO

CO2H

OMeOO

OHHO

HO

225

Some years later, in 2011, Field and co-workers synthesized the AGA fragments 225, 234 and 237

shown in Scheme 24.124 Like Reimer and co-workers, they prepared apiofuranoside donor 226 from

D-mannose according to previously published procedures. Treatment of arylthio glycoside 226 with

90% TFA followed by acetylation afforded triacetate 227 as an anomeric mixture. Coupling between

227 and 223 afforded the β-linked disaccharide 228 exclusively in 79% yield, which was further

deprotected to give the target disaccharide 225. Galacturonate acceptors with unprotected 3-OH or

both 2- and 3-OH groups (239 and 235, respectively) were prepared and coupled with 227, leading to

di- and tri-saccharides 233 and 236 in good yields. Cleavage of the ester protecting groups afforded

the targeted apiogalacturonans 234 and 237.

69

Scheme 24. Synthesis of di- and trisaccharide AGA fragments by Field and co-workers.124

O

OO

AcO STol

O

OAcAcO

AcOSTol

1) 90% TFA2) Ac2O, DMAP

py

76%

OCO2MeO

OHOOMe

226 227

223

NIS, HClO4/SiO2CH2Cl2

79% O

OAcAcO

AcO

OCO2MeO

OOOMe

O

OHHO

HO

OCO2HHO

HOOOMe

1) 80% aq. AcOH 2) NaOMe, MeOH3) NaOH, H2O

80%

228 225

OCO2MeHO

HOHOOMe

OCO2MeO

OBzOOMe

MeC(OEt)3, TsOH

THF

73%

EtO OCO2MeAcO

HOBzOOMe

80% AcOH

79%

BzCl, py

78%

OCO2MeO

OHOOMe

EtO

OCO2MeAcO

HOHOOMe

80% AcOH95%

227, NIS, HClO4/SiO2, CH2Cl2

84%

227, NIS, HClO4/SiO2

CH2Cl2

74%

OCO2MeAcO

OOOMe

O

OAcAcO

AcO

O

OAcAcO

AcOO

CO2HHO

OOOMe

O

OHHO

HO

O

OHHO

HO1) NaOMe, MeOH2) Et3N, MeOH, H2O

73%

OCO2MeAcO

OBzOOMeO

OAcAcO

AcO

OCO2HHO

OHO

OMeO

OHHO

HO

1) NaOMe, MeOH2) NaOH, H2O, MeOH

66%

229 230 231 232 233

234

235

236 237

70

5.4 Rhamnogalacturonan I

Figure 17. Representation of RG-I structure.98

RG-I (Figure 17) makes up 20–35% of pectin. Its average length has not been determined, since

isolated RG-I domains are generally accompanied by HG. However, the RG-I backbone has generally

been found to be shorter than HG. Furthermore, the RG-I backbone is predicted to have a three-fold

helix conformation.129 RG-I polysaccharides have repeating disaccharide units of (1→2)-α-L-

rhamnopyranosyl-(1→4)-α-D-galactopyranuronic acid. The GalpA residues may be O-acetylated on

C2-O and/or C3-O as seen for HG, but no methyl esterification has been observed.98 The diversity of

RG-I stems from the variety of side chains that can decorate the C-4 positions of rhamnose. Depending

on the plant source and the method of isolation, 20–80% of the rhamnosyl residues are found to be

substituted. These side chains consist of three different types of branched oligosaccharides of varying

length (1-20 residues): galactans, arabinogalactans (AG), and arabinans.98,130 Pectic arabinogalactans

are usually divided into two main groups.131 Type I arabinogalactan (AG-I) is the most prevalent

neutral pectic glycan and is especially abundant in citrus fruits and apples. It is characterized by a

71

linear β-(1→4)-linked D-Galp chain, which can be substituted at the C-3 position by L-Araf residues

and occasionally by arabinosyl oligosaccharides. It sometimes also carries galactan at the C-6

position. Type II arabinogalactan (AG-II) are highly branched polysaccharides composed of a

backbone of β-(1→3) or (1→6)-linked D-galactan, which can be (arabino)galactan-substituted at the

C-6 or C-3 positions, respectively.97 Galactans are simply β-(1→4)-linked D-galactan chains and have

been observed in lengths of up to 40 residues. Finally, the arabinans consist of α-(1→5)-linked L-

Araf residues with some degree of (1→3)-branching. The glycosyl residues α-L-fucosyl, β-D-

glucuronosyl and 4-O-methyl β-D-glucuronosyl may also be present.98 The (arabino)galactans and

arabinans will be discussed separately in section 6.

RG-I backbone

Figure 18. RG-I backbone structure.

Several syntheses of fragments of the RG-I backbone (Figure 18) have been carried out and an

overview of the different oligosaccharides produced is shown in Table 12.

Table 12. Synthetic fragments of RG-I backbone.


72

OR

Pr 2013 Ma et al.132

OR

Pr

H

2000

2008

2010

Maruyama et al.133

Nemati et al.134

Scanlan et al.129

OR

Me

2008 Reiffarth & Reimer135

OR


OR

H 2013 Zakharova et al.136

OR


As with HG fragment syntheses, and more generally all oligosaccharides containing uronic acids, both

pre-glycosylation oxidation and post-glycosylation oxidation approaches have been investigated by

different groups. The former approach was chosen by Reiffarth & Reimer135, also Vogel and co-

73

workers134 and Yu and co-workers.132 In 2008, Reiffarth and Reimer135 used a block synthesis

approach, coupling two disaccharide repeating units derived from the same disaccharide precursor, to

form their target rhamnogalacturonan tetrasaccharide 244 as shown in Scheme 25. The C-4 positions

of the rhamnosyl residues were orthogonally protected with an allyl group in order to allow potential

introduction of RG-I side chains. An acetyl group was used to protect their C-2 positions to enable

elongation of the backbone. Rhamnosyl thioglycoside donor 237 and methyl galacturonate acceptor

238 were coupled to form the key disaccharide intermediate 239. This was then further transformed

in a three-step sequence to generate a new disaccharide acceptor 240, by removal of the 2-acetyl

group, and a new trichloroacetimidate disaccharide donor 241. However, glycosylation of 240 with

donor 241 turned out to be more difficult than expected. Tetrasaccharide 242 was obtained in only

39% yield and attempts to optimize the reaction by changing the promoter and donor types were

unsuccessful. The fully deprotected tetrasaccharide methyl ester 243 was then reached over four steps

in 33% overall yield and ester hydrolysis using NaOH followed by acidification afforded the desired

rhamnogalacturonic acid tetrasaccharide 244 in 77% yield.

74

Scheme 25. Synthesis of an RG-I backbone tetrasaccharide fragment by Reiffarth & Reimer.135

O

SEt

OAc

AllOBzO

OCOOMeHO

BnOBnOOMe

NIS, TfOHCH2Cl2

78%237

238

OCOOMeO

BnOBnOOMe

O

OAcBzOAllO

OCOOMeO

BnOBnOOMe

O

OHBzOAllOHCl,

MeOH, CH2Cl2

80%

239 240

OCOOMeO

BnOBnO

O

OAcBzOAllO

1) H2SO4, AcOH, Ac2O (81%)

2) 4Å MS, CH2Cl2,

MeOH (89%)

3) CCl3CN, DBU, CH2Cl2 (81%)

241

240 + 241

AgOTfCH2Cl2

39%

O CCl3

NH

OCOOMeO

BnOBnO

O

OAcBzOAllO

OCOOMeO

BnOBnOOMe

O

OBzOAllO

242

OCOOMeO

HOHO

O

OHHOHO

OCOOMeO

HOHOOMe

O

OHOHO

OCOOHO

HOHO

O

OHHOHO

OCOOHO

HOHOOMe

O

OHOHO

1) Rh(PPh3)3Cl, DABCO EtOH, toluene, H2O

2) HgO, HgCl2 acetone, H2O

3) NaOMe, MeOH

4) H2,

Pd(OAc)2 EtOAc, MeOH, AcOH

33% (4 steps)

243 244

aq. NaOH, thenaq. HCl

77%

In 2008, Vogel and co-workers134 used a similar synthetic approach. Unlike Reiffarth & Reimer,

trimethylsilyl trifluoromethanesulfonate-promoted glycosylation with a similar trichloroacetimidate

disaccharide donor and disaccharide acceptor (O-3’ and O-4’ protection was modified compared to

240 and 241),provided them with the desired tetrasaccharide in 60% yield. More recently, in 2013,

Yu and co-workers132 reported the synthesis of tri-, penta-, and heptasaccharide fragments (251, 254

and 257) of the RG-I backbone using the pre-glycosylation oxidation approach with an iterative

elongation of the chain, using disaccharide building block 248 (Scheme 26). The syntheses feature

highly stereoselective formation of the α-D-GalpA-linkage, with very good yields, when using N-

phenyltrifluoroacetimidates as donors.

75

Scheme 26. Synthesis of tri-, penta- and heptasaccharide fragments of RG-I backbone by Yu and co-workers.132

OCOOMeAcO

BnOBnO

O

BnOBnO

O

OCOOMeO

BnOOBn

OAll

1) LiOH MeOH, H2O, 87%

2) H2,

Pd(OH)2/C MeOH, EtOAc, 92%

OCOOHHO

HOHO

O

HOHO

O

OCOOHO

HOOH

OPr

250 251

NaOMeMeOH

87%

OCOOMeHO

BnOBnO

O

BnOBnO

O

OCOOMeO

BnOOBn

OAll

252

OCOOMeAcO

BnOBnO O CF3

NPh

245

O

OAll

OHBnOBnO

TBSOTfCH2Cl2

68%

246O

COOMeAcO

BnOBnO

O

BnOBnO

O

OAll247

OCOOMeAcO

BnOBnO

O

BnOBnO

O

O

NPh

CF3

248

OCOOMeHO

BnOOBn

OAll

2491) PdCl2 THF, MeOH

2) PhN=C(Cl)CF3 K2CO3,

acetone

87%

248TBSOTftoluene

99%

OCOOMeO

BnOBnO

O

BnOBnO

O

OCOOMeO

BnOOBn

OAll

OCOOMeAcO

BnOBnO

O

BnOBnO

O

OCOOHO

HOHO

O

HOHO

O

OCOOHO

HOOH

OPr

OCOOHHO

HOHO

O

HOHO

O


2) H2,


253 254

OCOOMeO

BnOBnO

O

BnOBnO

O

OCOOMeO

BnOOBn

OAll

OCOOMeHO

BnOBnO

O

BnOBnO

O

NaOMeMeOH

86%

248TBSOTftoluene

92%

OCOOMeO

BnOBnO

O

BnOBnO

O

OCOOMeO

BnO OAll

OCOOMeO

BnOBnO

O

BnOBnO

O

OCOOMeAcO

BnOBnO

O

BnOBnO

O

OBn

TBSOTfCH2Cl2

88%

OCOOHO

HOHO

O

HOHO

O

OCOOHO

HO OPr

OCOOHO

HOHO

O

HOHO

O

OCOOHHO

HOHO

O

HOHO

O

OH


2) H2,


255 256 257

76

The three other groups who have synthesized RG-I backbone fragments used the post-glycosylation

approach, i.e. synthesizing a neutral backbone and oxidizing the primary hydroxyl groups to the

desired carboxylic acid functionality afterwards. 129,133,136 In 2000, Takeda and co-workers prepared

the unprotected propyl RG-I backbone tetrasaccharide 269 by employing trichloroacetimidate

glycosyl donors (see Scheme 27).133 Selected rhamnose residues bore PMB protective groups at C-4,

allowing for potential introduction of RG-I side chains.

77

Scheme 27. Synthesis of fully unprotected propyl glycosyl tetrasaccharide of RG-I backbone by Takeda and co-workers.133

OOAll

OBnHO

BnOOBn

O

O

NH

CCl3

AcOAcO OAc

258

259AgOTfCH2Cl2

97% OOAll

OBnO

BnOOBn

O

ORRORO

260: R = Ac261: R = H

NaOMe, MeOH70%

OOAll

OBnO

BnOOBn

O

OORO2,2-dimethoxypropane

TsOH

93%

262: R = H263a: R = PMB

PMBCl, NaH, DMF65%

OOAll

OBnO

BnOOBn

O

OR2R3O

R1O

80% AcOH

7a 67%7b

94%

264a: R1 = PMB, R2 = H, R3 = H

265a: R1 = PMB, R2 = H, R3 = Bn266a: R1 = PMB, R2 = Ac, R3 = Bn

1) Bu2SnO, benzene2) BnBr, TBABr

93%Ac2O, py, 71%

OOBnO

BnOBnO

O

OAcBnORO

262: R = H263b

R = BnBnBr, NaH, DMF

82%

1) Bu2SnO, benzene2) BnBr, TBABr

91%Ac2O, py, 99%

264b: R1 = Bn, R2 = H, R3 = H

265b: R1 = Bn, R2 = H, R3 = Bn266b: R1 = Bn, R2 = Ac, R3 = Bn

1) PdCl2, NaOAc,

90% aq. AcOH

2) CCl3CN, DBU CH2Cl2 O CCl3

NH267a: R = PMB (17%)267b: R = Bn (30%)

265a + 267b

AgOTfCH2Cl2

67%

OOBnO

BnOBnO

O

OAcBnOBnO

OOBnO

BnOOBn

O

OBnOPMBO

OAll

1) NaOMe, MeOH, 93%2) Pd/C, H2

, MeOH, 74%

3) TEMPO, KBr, NaClO aq. NaHCO3

, 50%

OCOOHO

HOBnO

O

OHHOHO

OCOOHO

HOOH

O

OHOHO

OPr

268 269

AgOTf-catalyzed glycosylation between allyl galactoside acceptor 258 and trichloroacetimidate

donor 259 afforded the α-linked disaccharide 260 in 97% yield as a single stereoisomer. Protecting

group manipulations afforded four differently protected disaccharides: two trichloroacetimidate

donors 267a and 267b (PMB and Bn protected at the Rhap C-4, respectively) and two 2’-OH acceptors

265a and 265b. One noteworthy aspect is that the two-step conversion into trichloroacetimidate

donors 267a and 267b resulted in modest yields (17% and 30%, respectively). Coupling of 265a and

267b gave the α-linked tetrasaccharide 268 in 67% yield. Deprotection followed by selective oxidation

78

of the primary hydroxyl groups with 2,2,6,6-tetramethylpiperidin-1-oxyl (TEMPO), KBr and NaClO

afforded the desired unprotected propyl tetrasaccharide 269 in 34% yield over the three steps.

In the same year, Davis and co-workers used an orthogonal approach, combined with a late-stage

oxidation strategy, to synthesize a fully unprotected RG-I backbone tetrasaccharide 281 and its

corresponding methyl ester (see Scheme 28).129 Surprisingly, initial attempts to couple a

galactorhamnosyl disaccharide donor to Galp C4’-OH of a disaccharide acceptor failed. This was

probably due to low reactivity of the acceptor, and forced the authors to change strategy and assemble

the RG-I tetrasaccharide through galactosylation instead of rhamnosylation. The group used both

trichloroacetimidates and ethylthioglycosides as donors, affording good glycosylation yields and α-

stereoselectivity.

79

Scheme 28. Davis and co-workers’ synthesis of an RG-I backbone tetrasaccharide 281.129

O

O

NH

CCl3

OAcBnOBnO

OSEt

OBzHO

BnOOBn

270

271TMSOTfCH2Cl2

65% OSEt

OBzO

BnOOBn

O

OAcBnOBnO

272

O

OAcBnOBnO

SEt

OOBn

OBzHO

BnOOBn

274NIS, TMSOTf

CH2Cl2

75%

273

OOBn

OBzO

BnOOBn

O

ORBnOBnO

275: R = Ac276: R = H

NaOMe, MeOH, THF84%

272

276NIS, TMSOTf

CH2Cl2

83%

OOBn

R2O

BnOOBn

O

OBnOBnO

OR2O

BnOBnO

O

OR1BnO

BnO

OOH

COOHO

HOOH

O

OHOHO

OCOOHO

HOHO

O

OHHOHO

Pd/C, H2, H2O

62%

277: R1 = Ac, R2 = CH2OBz278: R1 = H, R2 = CH2OH279: R1 = H, R2 = COOH280: R1 = H, R2 = COOBn

281

NaOMe, MeOH, THF 78%

Cs2CO3, BnBr, DMF

72% over 2 steps

TEMPO, NaClO2, NaClO

In 2013, Clausen and co-workers synthesized a fully unprotected hexasaccharide backbone of RG- I

using a strategy relying on iterative coupling of a common pentenyl disaccharide glycosyl donor

284.136 The latter was prepared by an efficient chemoselective armed-disarmed coupling of a

thiophenyl rhamnoside donor 282, with a pentenyl galactoside acceptor 283 bearing the strongly

electron-withdrawing pentafluorobenzoyl ester (PFBz) protective group, as shown in Scheme 29. Two

iterative glycosylations using pentenyl disaccharide 284 afforded hexasaccharide 289 in reasonable

yields. Removal of the PFBz groups under Zemplén conditions afforded the Galp primary alcohols,

80

which were oxidized with Dess-Martin periodinane to the aldehydes and then with NaClO2 to the

desired carboxylic acids. As in many other cases, the carboxylic acids were protected with benzyl

esters in order to facilitate purification. Palladium catalyzed hydrogenolysis gave the fully unprotected

hexasaccharide 291.

Scheme 29. Fully unprotected hexasaccharide backbone of RG-I synthesized by Clausen and co-workers.136

O

BnOBnO

ONAP

SPh

OHO OPFBz

BnOOBn

O

NIS, TESOTfEt2O

78%

282

283O

BnOBnO

ONAP

OO OPFBz

BnOOBn

O

284

O

BnOBnO

OR

OO OPFBz

BnOBnOOBn

1) Br2, CH2Cl2

2) BnOH, TBABr

90%

285: R = NAP286: R = H

DDQ, H2O, CH2Cl2, MeOH

74%

284NIS, TESOTf

Et2O

71% O

BnOBnO

O

OO OPFBz

BnOBnOOBn

O

BnOBnO

OR

OO OPFBz

BnOBnO

287: R = NAP288: R = H

DDQ, H2O, CH2Cl2, MeOH

76%

O

BnOBnO

O

OO OH

BnOBnOOBn

O

BnOBnO

O

OO OH

BnOBnO

O

BnOBnO

ONAP

OO OH

BnOBnO

1) 284, NIS, TESOTf, Et2O

2) MeONa, MeOH

40%

O

BnOBnO

O

OOCOOBn

BnOBnOOBn

O

BnOBnO

O

OOCOOBn

BnOBnO

O

BnOBnO

ONAP

OOCOOBn

BnOBnO1) DMP, CH2Cl2

2) NaClO2, NaH2PO4

2-methyl-2-butene tBuOH, H2O, THF

3) PhCHN2, EtOAc

60%

O

HOHO

O

OOCOOH

HOHO

O

HOHO

O

OOCOOH

HOHO

O

HOHO

OH

OOCOOH

HOHO

H2, Pd/C, MeOHTHF, H2O

95%

OH289 290 291

RG-I side chains

As explained in the introduction to rhamnogalacturonan I (section 5.4), RG-I has three main types of

side chain: galactans, arabinogalactans and arabinans. Since (arabino)galactans are also found in

81

arabinogalactan proteins (AGPs), oligosaccharides related to these structures will be discussed

separately in Chapter 6.

5.5 Rhamnogalacturonan II

OMe

MeO

A B

CD

D-apiose

L-aceric acid

D-2-keto-3-deoxy-D-manno-octulosonic acid (Kdo)


OHOOC

OH

OH

O

OHHO

HOOH

OH

Me

OHO

HO COOHOH

OHOCOOH

HO COOHOH

OHOH

Rare sugar structures:

α-stereochemistry discovered in 2017

Figure 19. Schematic representation of RG-II based on the findings of Gilbert and co-workers.137

Rhamnogalacturonan II (RG-II) constitutes ~10% of pectin in the cell wall of higher plants and is a

structurally complex branched pectic polysaccharide.97 It is present in above-average quantities in

wine and other beverages obtained by fermentation of fruits and vegetables (one liter of red wine

contains between 100 and 150 mg of RG-II while around 20 mg is present in a liter of white wine).138

Therefore, red wine has been used as a source of RG-II in several studies of this polysaccharide.139

Unlike RG-I, the RG-II structure has been regarded to have remarkably little variation and to be

evolutionarily conserved in all vascular plants. Variations are minor and are limited to methylation

and/or acetylation of the side chains.140 However, recent discoveries have shown evidence of variation

also in the monosaccharide composition of the side chains.141 RG-II has an HG backbone, made up of

82

seven to nine residues, and substituted by four different side chains (A–D), which can contain up to

29 monosaccharides, including a number of rare sugars (see Figure 19).100,101,142 These comprise L-

aceric acid (AcefA; 3-C-carboxy-5-deoxy-L-xylose), 2-keto-3-deoxy-D-manno-octulosonic acid

(Kdop), 2-keto-3-deoxy-D-lyxo-heptulosaric acid (Dhap), D-apiose (Apif; 3-C-(hydroxymethyl)-β-D-

erythrose), L-galactose, 2-O-methyl-D-xylose and 2-O-methyl-L-fucose. The monosaccharides are

connected by over 20 different glycosidic linkages. Side chains A and B are the largest and have been

described as highly ramified octa- and heptasaccharides, respectively.143,144 The C and D side chains

are disaccharides, and two extra side chains E and F, composed of a single L-arabinose residue linked

to a mono- or di-substituted galacturonic acid residue respectively, have been isolated from red wine

RG-II recently.100,137,145

Before 2000, knowledge of the primary structure of RG-II was mostly based on the determination of

the glycosidic linkage composition (using methylation analysis) and identification of various

oligosaccharide fragments by mass spectroscopy (after acid and/or enzymatic hydrolysis of RG-II).

Recently, several NMR solution studies have helped determine the fine- and three-dimensional

structure of the oligosaccharide.144,146,147 In 2000, Vidal and co-workers published a convincing NMR

study of an RG-II fragment, which contained a hexasaccharide homogalacturonan backbone

substituted by side chains B and D.146 Their structural studies showed that the original configurations

postulated for the anomeric centers of two residues in the B-chain were incorrect. This led some other

groups to look in more detail at the structure of RG-II. In 2003, Pérez and co-workers reported a

thorough NMR investigation of RG-II. They reassigned most of the RG-II primary structure and used

NOESY data to propose a conformational model of the oligosaccharide in solution.145 Nevertheless,

several NMR assignments remained ambiguous. In 2017, Gilbert and co-workers published a revised

83

model of the RG-II structure based on an impressive work including crystal structure of side chain B

in complex with a glycosyl hydrolase combined with enzymatic degradation studies. This revised

structure is the one shown in Figure 19.137 The main difference compared to previously proposed

structures of RG-II is highlighted by pink circles: the L-Rhap-D-Apif linkage in chains A and B is α

and not β. It has been observed that more than 90% of the RG-II in the cell wall self-assembles into a

covalently cross-linked dimer in the presence of boron. RG-II is dimerized by borate ester formation

involving the apiosyl residues of side chain A belonging to two different RG-II monomers, which

results in networks of pectic polysaccharides.100 This secondary structure has been confirmed by 11B-

NMR.148 Several studies support the idea that RG-II and its borate crosslinking support cell adhesion

and wall mechanical strength and are therefore significant for plant growth and development.149,150

All the knowledge about RG-II structure and function accumulated before 2004 is summarized in a

comprehensive review by Darvill and co-workers.100

Most synthetic efforts have focused on side chain A and B, which also constitute most of the RG-II

structure, as mentioned previously. Since many of the synthesized fragments are only disaccharides,

the goal of synthesizing them was often not so much to study RG-II function, but rather to develop

synthetic methods. Furthermore, as the chemical synthesis of some of the rare sugars present in RG-

II is not straightforward, we will refer the reader to selected articles reporting methods that have been

developed for their preparation.

RG-II side chain A

Despite the fact that RG-II side chains were thought to be highly conserved in the plant kingdom,

some variations in the composition of side chain A were reported by Léonard and co-workers in

84

2013.141 They showed that in wild-type Arabidopsis plants, up to 45% of L-fucose was replaced by L-

galactose (see Figure 20). The study also provided evidence that a large proportion of the GlcpA

residues were methyl esterified and some of the β-GalpA residues were O-methylated at positions 3

and 4 (these variations are highlighted in red on Figure 20). Furthermore, until the work published by

Gilbert and co-workers in 2017, it was believed that the Rhap-Apif linkage (shown in pink-dot circle

on Figure 20) was a β-linkage, which was proved to be the wrong stereochemistry by Gilbert’s group.

MeO

A

MeO

AOMe

MeO

Or COOMe

OMe

MeO

Or COOMe


Figure 20. Schematic representation of side chain A including the variations across different plant species observed by Léonard and co-workers.141,145

Side chain A is attached to the homogalacturonan backbone residue via a D-Apif residue.151,152 One-

and two-dimensional 1H NMR spectroscopic analysis showed that apiose is β-(1→2)-linked to

GalpA.146 The synthesized fragments belonging exclusively to side chain A are reported in Table 13,

while fragments found in both side chain A and B are presented in Table 14.

Table 13. Synthetic fragments found exclusively in RG-II side chain A.


OR Me 1990 Backman et al.153

85

OR

Me 2005 Chauvin et al.154

OR


OR


Jansson and co-workers synthesized a Fucp-Rhap disaccharide by MeOTf-promoted glycosylation

between 2,3,4-tri-O-benzyl-1-thio-β-L-fucopyranoside 292 and methyl 2,3-isopropylidene-α-L-

rhamnopyranoside 293 affording the desired α-linked disaccharide 294 in 77% yield as shown in

Scheme 30.153

Scheme 30. α-L-Fucp(1→2)-α-L-RhapOMe fragment synthesized by Jansson and co-workers.153

O SEtOBn

OBnBnO

Me

O

O

Me

OMe

OHO

292

293MeOTfEt2O

77% OOBn

OBnBnO

Me

O

O

MeOMe

OO

294

90% aq. AcOHthen H2

, Pd/C

72% OOH

OHHO

Me

O

HO

MeOMe

OHO

295

In 2005, Nepogodiev, Field and co-workers achieved the synthesis of branched tetrasaccharide

fragment 308 following the route shown in Scheme 31. As this tetrasaccharide contains a combination

of two 6-deoxy sugar residues (Rhap and Fucp) and two uronic acids (GalpA), they chose a post-

86

glycosylation oxidation strategy in order to introduce the carboxylic acids in the very last step of their

synthesis. The method was based on the use of the differentially protected rhamnoside 299. Sequential

glycosylation afforded the neutral tetrasaccharide 307 in reasonable yields and stereoselectivities

overall. Following deprotection of 307, the primary hydroxyl groups were successfully oxidized using

TEMPO affording the target 308 in 47% yield.

Scheme 31. Synthesis of a branched tetrasaccharide fragment from side chain A by Nepogodiev, Field and co-workers.154

O

OMe

OHHOHO

Me O

OMe

OORO

Me

OEtMe

297: R = H298: R = PMB

296

MeC(OEt)3

TsOH

MeCNO

OMe

OAcHOPMBO

Me80% aq. AcOH

299 (70% 3 steps)

PMBCl, NaH, MeCN

O

OBzBzO

OBzBzO SMe

300NIS, TfOH

CH2Cl2

70%

O

OMe

OROPMBO

Me

OOBz

BzOOBz

BzO

301: R = Ac302: R = H

0.4 M HCl in MeOH45%

O

OMe

OORO

Me

OOBz

BzOOBz

BzO

O

OBnBnO

OBnBnO SMe

303NIS, TMSOTf

CH2Cl2

87%

O

OBn

OBnBnOBnO

304: R = PMB305: R = H

CAN, MeCN, H2O93%

OOBn

Me

BnO

OBn

306NIS (2 equiv.),TfOH (2 equiv.)

Et2O, CH2Cl2

87%

O

OMe

OO

Me

OOBzBzO OBz

BzO

O

OBn

OBnBnOBnO

O

OOBn

Me

BnO

OBnSPh

OOH

Me

HO

OH

O

OMe

OO

Me

ORHOOH

HO

OR

OHHOHO

O1) 0.05% NaOMe, MeOH

then H2, Pd/C, EtOH

61%

2) TEMPO, NaOCl, KBr H2O 47%

307 308: R = COOH

The linear trisaccharide α-L-Rhap-(1→3’)-β-D-Apif-(1→2)-α-D-Galp is a fragment that is common to

both side chain A and B. However, since the linkage between the rhamnose and the apiose residues

was believed to be β until 2017, chemists focused on synthesizing fragments with this configuration.

Reimer and co-workers123 and Nepogodiev and co-workers155 both reported the synthesis of

disaccharide elements of the fragment in 2004 (β-D-Apif(1→2)-α-D-Galp and β-L-Rhap-(1→3’)-β-D-

Apif, respectively). In 2011, Nepogodiev, Field and co-workers124 reported the synthesis of the full

structure (see Table 14).

87

Table 14. Synthetic fragments found in both side chain A and side chain B of RG-II.


OR

Me

Me

2004

2011

Buffet et al.123

Nepogodiev et al.124

OR


OR


All these syntheses required access to the rare sugar apiose. As explained in section 5.3, D-apiose

derivatives are usually produced from D-mannose, L-arabinose or D-xylose.125–128 The approach by

Reimer and co-workers was described earlier (see Scheme 23). The two other fragments synthesized,

required the challenging β-rhamnosylation to form β-L-Rhap-(1→3)-β-D-Apif disaccharide. Unlike

for β-mannosylation, only few methods for β-rhamnosylation have been reported.156,157 The first

report by Hodosi and co-workers in 1997 described the SN2 coupling between a sugar triflate and an

unprotected 1,2-O-stannylene acetal of L-rhamnopyranose.157 In 2003, Crich and Picione reported a

second method enabling the direct formation of a β-L-rhamnoside by means of thiorhamnoside donors

protected with a 2-O-sulfonate ester and, ideally, a 4-O-benzoyl ester.156 After attempting in vain to

apply Hodosi’s method for the synthesis of β-L-Rhap-(1→3)-β-D-Apif, Nepogodiev and co-workers

decided to use a cyclic 2,3-carbonate rhamnosyl bromide donor as shown in Scheme 32.124 It is worth

mentioning that the synthesis of thiophenyl apiofuranoside residue 310 required extensive

88

optimization and was achieved through a tedious 9-steps synthesis from L-arabinose. The authors did

test simpler protecting group schemes, but these strategies were unsuccessful. Koenigs-Knorr β-

rhamnosylation between 313 and apiogalacturonate acceptor 312 afforded the desired core

trisaccharide 314 in 59% yield, which was deprotected in four steps to reach the target molecule. In

2012, Yasomanee and Demchenko demonstrated a third approach by forming a β-rhamnoside

employing a picoloyl protecting group in the 3-position of a thioethyl rhamnose donor.158

Scheme 32. Synthesis of trisaccharide β-L-Rhap-(1→3’)-β-D-Apif(1→2)-α-D-Galp by Nepogodiev, Field and co-workers.124

O

Br

OOAcO

Me

O

O

OO

HPh

ClAcO

310

STol

OCOOMeO

OHOOMe

309

NIS, TfOHCH2Cl2

62%

OCOOMeO

OO

OMeO

OO

HPh

RO

311: R = ClAc312: R = H

Ag2OCH2Cl2

59%

313O

COOMeO

OO

OMeO

OO

HPh

OO

OOAcO

Me

O

OCOOHHO

HOO

OMeO

OHHO

OO

OHHOHO

Me

314 315NaOMe, MeOH, 96%

1) H2, Pd/C, EtOAc

2) 80% aq. AcOH3) NaOMe, MeOH

56%

4) Et3N, H2O, MeOH

78%

89

RG-II side chain B

OMe

B

not conserved


Figure 21. Side chain B of RG-II.

The most common structure of side chain B is represented in Figure 21. However, some variations

have been observed depending on the plant species, which mainly involve the presence or absence of

substituents at the O-2 and/or O-3 positions of the Arap residue.100 The different sugar substituents,

which have been observed at these two positions by Pérez and co-workers, are summarized in Table

15. Furthermore, mono-O- or di-O-acetylation can occur at the O-3 position of the AcefA residue as

well as the O-3/O-4 positions of 2-O-Me-Fucp.145

Table 15. Plant dependent variations observed by Pérez and co-workers for side chain B of RG-II.145

Glycose linked to Arap Plant Source

O-2 O-3

β-L-Araf-(1→2)-α-L-Rhap α-L-Rhap Grape (wine)

Ginseng

Sycamore

3-O-Me α-L-Rhap 3-O-Me α-L-Rhap P. bifurcatum

P. nudum

90

α-L-Rhap 3-O-Me α-L-Rhap C. thalictroides

L. tristachyum

P. bifurcatum

α-L-Rhap OH A. thaliana

E. hyemale

S. kraussiana

The synthetic fragments of side chain B that have been reported to date are summarized in Table 16

and Table 14.

Table 16. Synthetic fragments found exclusively in RG-II side chain B.


OMe

OR

C8H17

All

2000

2001

Mukherjee et al.159

Amer et al.160

OR

(CH2)3NH2 2008 Rao et al.161

OMe

OR

(CH2)3NH2 2007 Rao & Boons162

91

OMe

OR

(CH2)3NH2 2007 Rao & Boons162

Although aceric acid (AcefA) has only been found in RG-II, and the development of synthetic routes

to this unusual sugar started over five years ago, no RG-II fragments including aceric acid have yet

been reported. Two main approaches have been developed for its synthesis: a 4-step approach from

L-xylose163 and a 6-step synthesis from D-arabinose.164 Both Hindsgaul and co-workers159 and Kosma

and co-workers160 reported the synthesis of 2-O-Me-α-L-Fucp(1→2)-β-D-Galp early in the 21st

Century. The synthetic route employed by Hindsgaul and co-workers159 afforded the desired β-linked

disaccharide 321 in a straightforward manner (Scheme 33).

Scheme 33. Synthesis of 2-O-Me-α-L-Fucp(1→2)-β-D-Galp by Hindsgaul and co-workers159

OBnO

BnOOH

OBn

O(CH2)7CH3

O SEtOBnBnO

OPMB

OBnO

BnOO

OBn

O(CH2)7CH3

OOBnBnO

OR

318: R = PMB319: R = H320: R = Me

DMTST, DTBMPCH2Cl2

67%

316

317

CAN, MeCN, H2O, 70%MeI, NaH, DMF, 86%

OHO

HOO

OH

O(CH2)7CH3

OOHHO

OMe

H2,

Pd(OH)2

MeOH

89%

321

In 2007 Rao & Boons reported the synthesis of two highly challenging targets: tetra- and

hexasaccharide portions of side chain B containing the central L-Arap (338 and 337, respectively,

Scheme 34).162 The tetrasaccharide 338 was chosen as a second target because some plant species,

92

such as Arabidopsis, lack the β-L-Araf-(1→3)-L-Rhap substituent on the 2-position of the L-Arap unit.

This chemical synthesis was challenging because it involved a number of unusual monosaccharides,

due to the steric hindrance around the central arabinopyranoside as well as the presence of both

(1→2)-cis-linked 2-O-methyl-α-L-fucoside and β-L-arabinofuranoside residues, which are difficult to

install stereoselectively. The authors first attempted a convergent [2+2+2] approach. However, the

final glycosylation between tetrasaccharide acceptor 331 and a β-L-Araf-(1→3)-L-Rhap donor gave

an unacceptably low yield (8%). The strategy was therefore revised and the final two monosaccharides

were added sequentially as shown in Scheme 34.

Glycosylation between 2-O-methyl thioethylfucoside donor 322 and galactosyl acceptor 323 carrying

an electron withdrawing benzoyl ester group at the C-3 position afforded the desired α-(1→2)-linked

disaccharide 324 in high yield and with high α-selectivity. Reductive opening of the benzylidene

acetal followed replacement of the ester protecting groups with benzyl ethers, in order increase the

reactivity of acceptor 326.

93

Scheme 34. Tetra- and hexasaccharide portions of side chain B synthesized by Rao & Boons.162

O SEtOMe

OAcAcO

OO

OPh

O(CH2)3N3OH

BzO

NIS,TfOHCH2Cl2

, Et2O

88%

323

322

OO

OPh

O(CH2)3N3O

R1O

O OMeOR2

R2O

324: R1 = Bz, R2 = Ac325: R1 = R2 = Bn (80%)

OHO OBn

O(CH2)3N3O

BnO

O OMeOBnBnO

1) NaOMe, MeOH2) NaH, BnBr, TBAI, DMF

NaCNBH3, THF

2 M HCl, Et2O

85%

326

O O

OAcAcOAcO

NH

CCl3

OOBz

SPhOLev

HO

328

TMSOTf, CH2Cl2

91%

OOBz

SPhOLev

O

O

OAcAcOAcO

329

326NIS, TfOH

CH2Cl2

71%

OO OBn

O(CH2)3N3O

BnO

O OMeOBnBnO

OOBz

ORO

O

OAcAcOAcO

330: R = Lev331: R = H (85%)

NH2NH2⋅AcOH

CH2Cl2, MeOH

O

OBzLevOBzO

SPh

332NIS, TfOH

CH2Cl2

60%

OO OBn

O(CH2)3N3O

BnO

O OMeOBnBnO

OOBz

OO

O

OAcAcOAcO

O

OBzROBzO

333: R = Lev334: R = H (90%)

NH2NH2⋅AcOH

CH2Cl2, MeOH

OO

OOBn

O CCl3

NHtBu2Si

335TMSOTf, CH2Cl2

92%

OO OBn

O(CH2)3N3O

BnO

O OMeOBnBnO

OOBz

OO

O

OAcAcOAcO

O

OBz

BzOO

O

OOBn

tBu2Si

O

OO OH

O(CH2)3NH2O

HO

O OMeOHHO

OOH

OO

O

OHHOHO

O

OH

HOO

OH

OHO

330

OO OH

O(CH2)3NH2O

HO

O OMeOHHO

OOH

OHO

O

OHHOHO

HO

1) TBAF, THF

2) NaOMe, MeOH3) Pd/C, H2

, tBuOH, AcOH, H2O

51%336

337

338

1) NaOMe, MeOH2) Pd/C, H2

, tBuOH, AcOH, H2O

65%

327

Chemoselective TMSOTf-promoted glycosylation between rhamnopyranosyl trichloroacetimidate

donor 327 and thiophenyl arabinopyranoside 328 gave the desired α-(1→3)-linked disaccharide donor

94

329 in 91% yield, with high stereoselectivity, thanks to neighboring group participation from the

acetyl ester of 327. This disaccharide was coupled with acceptor 326 using thiophilic promoter

NIS/TfOH, affording the desired β-(1→3)-linked tetrasaccharide 330 in 71% yield. Selective removal

of the Lev group, followed by coupling with rhamnopyranoside donor 332 afforded α-(1→2)-linked

pentasaccharide 333. After Lev removal, the pentasaccharide was glycosylated with the

conformationally-constrained trichloroacetimidate arabinofuranosyl donor 335 affording

hexasaccharide 336 in 91% yield and, gratifyingly, exclusively as the β-anomer. Deprotection of both

tetrasaccharide 330 and hexasaccharide 336 proceeded smoothly to afford the two target

oligosaccharides.

RG-II side chain C

C

RG II backbone

Figure 22. Side chain C of RG-II.

The C side chain of pectin, consisting of the disaccharide α-L-Rhap-(1→5)-D-KDOp, was first isolated

and characterized in 1985.165 The disaccharide is linked to C3-O of a GalpA residue from the RG-II

backbone (see Figure 22). Synthesis of this side chain has not yet been reported. However, the rare

eight-carbon sugar α-D-KDOp is found in non-plant glycans, and therefore various synthetic

procedures have been developed.165–168

95

RG-II side chain D

D

RG-II backbone

Figure 23. Side chain D of RG-II.

RG-II side chain D, consisting of disaccharide β-L-Araf-(1→5)-D-Dhap, was first isolated from pectin

plant cell wall by Albersheim and co-workers in 1988.169 As for side chain C, this disaccharide is

attached to the C-3 position of a galacturonic acid in the backbone (see Figure 23), and has not been

synthesized either. However a procedure to synthesize the rare sugar 3-deoxy-D-lyxo-heptulosaric

acid (Dhap) was reported in 1995 by Banaszek.170

RG-II side chain E/F

E RG-II backbone

Figure 24. Side chain E of RG-II.

Several independent studies suggest that a single α-L-Araf residue is linked to O-3 of one of the GalpA

backbone residues of RG-II (see Figure 24).147,151,171 However, it has not yet been proven, whether

this residue is linked to O-3 or O-2 of GalpA and no synthesis of this linkage has been reported.

96

(Arabino)galactans and arabinans Arabinogalactans, galactans and arabinans are the three main types of side chain in the pectin RG-I

domain. However, arabinogalactans are also present in many plants as a constituent of hemicellulose

and in arabinogalactan proteins.172

Type II AGs (three examples of possible structures)

Type I AGs Galactans

Figure 25. Exemplary structures of arabinogalactans (AGs) and galactans.

Arabinogalactans (AGs) are cell wall polysaccharides having high proportions of galactose and

arabinose. They are widely present in the plant kingdom and occur as variable complex structures,

whose composition is highly dependent on the plant and even cells from which they originate. They

are not found to be well conserved. Arabinogalactans are usually divided into two groups: type I and

type II, which are schematically represented in Figure 25.131 Type I AGs are characterized by a linear

β-(1→4)-linked D-Galp chain, which may be substituted with α-(1→3)-linked single L-Araf, short α-

(1→3)- or α-(1→5)-linked arabinan oligosaccharides, or with β-(1→6)-linked galactose residues.97

97

Type I AGs are found in pectic complexes in seeds, bulbs and leaves and are mostly covalently bound

to the RG-I backbone.172 Galactans are simply β-(1→4)-linked D-galactan chains. They have been

observed in lengths of up to 40 residues.173 Type II AGs are highly branched polysaccharides

composed of a β-(1→3) or (1→6)-linked D-galactan backbone frequently substituted at C-3 and/or C-

6 with β-galactan or arabinogalactan branching, and occasionally carrying C-2 substitution.97 This

second type of AG exists in various plant tissues, including coniferous woods, mosses as well as gums,

saps and exudates of angiosperms.131 Type II AGs make up the carbohydrate part of arabinogalactan

proteins (AGPs) and account for over 90% of the molecular mass of these complexes. Although the

structure of arabinogalactans appears to be well established, new structural diversity is found in this

class of polysaccharides occasionally. Huisman et al., for example, reported the presence of arabinose

residues in the galactan backbone as well as a terminal L-Arap residue in the side chains of type I AG

from soybean pectin.174 NMR analysis has clearly demonstrated that type I AGs are covalently bound

to rhamnose in RG-I and this is the main type of pectic arabinogalactan.172,175 However, Cordeiro and

co-workers analyzed pectin from starfruit and found evidence of both type I and II AGs among the

RG-I side chains.176 The next section will give an overview of the AG fragments prepared by chemical

synthesis.

6.1 Type I AGs and galactans

β-(1→4)-linked galactan synthesis is challenging, due to the low accessibility and reactivity of the

axially disposed C4-OH. There is a requirement for protecting groups at both C-3 and C-6, which

increases steric hindrance around the nucleophile. Until 2016, only four groups had reported syntheses

of β-(1→4)-D-galacto-oligosaccharides, of which, three only reached the trisaccharide level.177-178

98

Lately a small library of oligogalactans including linear tetra-, penta-, hexa-, and heptasaccharides

have been synthesized by Clausen and co-workers.179 As commented on by Sakamoto and Ishimura

in 2013, the low availability of pure type I AG oligosaccharides has hindered the study of

galactonolytic enzymes.180 No synthesis of type I AGs had been reported until early 2017, when

Pfrengle and co-workers produced a set of type I AGs and determined the substrate specificities of

three β-(1→4)-endogalactanases.181 The galactans and type I AGs that have so far been synthesized

are presented in Table 17.

Table 17. Synthetic β-(1→4)-linked galactans including type I AGs.


OR Pr

Me

H

1984

1987

2001

El-Shenawy & Schuerch177

Kováč & Taylor182

Lichtenthaler et al.183

OR2

PMP

(CH2)5NH2

2001

2017


Bartetzko et al.181

OR3

PMP

H

2001

2016


Andersen et al.179

OR4

PMP

H

(CH2)5NH2

2001

2016

2017


Andersen et al.179

Bartetzko et al.181

OR5

H 2016 Andersen et al.179

99

OR

(CH2)5NH2 2017 Bartetzko et al.181

OR


OR


OR


OR


OR4


OR


OR


OR


OR


OR


100

The first reported synthesis of a linear β-(1→4)-D-galactan trisaccharide was achieved by Schuerch

& El-Shenawy177 and is shown in Scheme 35.

Scheme 35. Synthesis of protected β-(1→4)-D-galactotriose 16 by Schuerch & El-Shenawy.177

OAcO

BzOBnO

OBn

OAll

1) Rh(PPh3)3Cl iPr2NEt, EtOH

2) HgO, HgCl2 acetone, H2O

89%

OAcO

BzOBnO

OBn

OH

339

1) Ac2O, py

2) TiBr4 EtOAc, CH2Cl2

78%

OAcO

BzOBnO

OBn

Br340 341

OHO

BzOBnO

OBn

OAll342

AgOTresCH2Cl2 65%

OAcO

BzOBnO

OBn

OO

BzOBnO

OBn

OAll343

1) Rh(PPh3)3Cl iPr2NEt, EtOH

2) ZnCl2, ZnO

acetone, H2O

85%

OAcO

BzOBnO

OBn

OO

BzOBnO

OBn

344OH

OAcO

BzOBnO

OBn

OO

BzOBnO

OBn

345 Cl

1) PhNCO N,N'-diphenylurea CH2Cl2

2) HCl (g), CH2Cl2

80%

OAcO

BzOBnO

OBn

OO

BzOBnO

OBn

OO

BzOBnO

OBn

OAll346

AgOTresCH2Cl2

85%

342

1) KCN in 95% EtOH

2) Pd/C, H2 acetone, H2O

70%

OHO

HOHO

OH

OO

HOHO

OH

OO

HOHO

OH

OPr347

The key building block 339 carried an allyl group as an exchangeable protecting group at the anomeric

center and an acetyl group for temporarily blocking the 4-position. A benzoyl group at the C-2 position

ensured neighboring group participation to form the desired β-linkage. Finally, benzyl groups were

used to block the remaining hydroxyl groups. Acceptor 342 was coupled to glycosyl bromide 340 in

a silver tresylate (2,2,2-trifluoroethanesulfonate, OTres) catalyzed glycosylation reaction providing

the disaccharide 343 in 65% yield. This disaccharide was then converted into the corresponding

glycosyl chloride 345, over four steps, with an overall yield of 69%. A second glycosylation of

acceptor 342 afforded the trisaccharide 346 in 85% yield, and this could easily be deprotected to afford

the target 347. Further selective deacetylation of the di- and trisaccharide did not proceed well, and

this strategy did not enable the authors to elongate the chain in a convergent way as they had hoped.

101

To improve the efficiency of this galactotriose synthesis, Kovác & Taylor suggested another approach

starting from acceptor 348.182 This was converted to the glycosyl chloride 349 in two steps (Scheme

36). Coupling of acceptor 348 and donor 349 promoted by silver trifluoromethanesulfonate gave the

desired disaccharide 350 in 62% yield. The bromoacetyl group was selectively removed with thiourea

to give acceptor 351, which was once again glycosylated with glycosyl chloride 349. However, in this

case, the glycosylation went slower and the trisaccharide 352 could only be obtained in 35% yield.

Further elongation was thus not feasible in this case either.

Scheme 36. Synthesis of β-(1→4)-D-galactriose by Kovác & Taylor.182

OHO

BzOBzO

OBz

OMeO

BzOBzO

OBz

Cl

BrAcO+ O

OBzBzO

OBzRO

350: R = AcBr351: R = H (85%)

348 349

O

OBzBzO

OBzO

OMe

O

OHHO

OHO

O

OHHO

OHO

OMe

O

OHHO

OHHO

352

1) 1,1,3,3-tetramethylurea BrAcBr 1,2-dimethoxyethane

2) ZnCl2, Cl2HCOMe

80%

O

OBzBzO

OBzO

O

OBzBzO

OBzO

OMe

O

OBzBzO

OBzBrAcO349, AgOTf

CH2Cl2

35%

AgOTfCH3NO2

, toluene

62%

thiourea, MeOH

NaOMe, MeOHtoluene

82%

353

Lichtenthaler and co-workers published the first synthetic route by which longer β-(1→4)-linked

galactans, up to a hexasaccharide, could be produced (Scheme 37).183 An iterative block strategy and

easy transformation of the PMP acceptors into thioglycoside donors really did make this a convergent

synthesis. Furthermore, the protecting group pattern was judiciously designed to minimize steric

hindrance around the unreactive galactosyl-4-OH. Hence, allyl and/or benzyl groups were chosen for

C-3 and C-6 in order to enhance the nucleophilicity of the acceptor. In this case, a pivaloyl ester was

chosen for C-2 protection. The acetyl group could then be used for blocking C-4, as mild Zemplén

102

conditions will not remove the Piv ester. MeOTf-mediated glycosylation of thioglycoside building

block 354 and acceptor 355 afforded the disaccharide 356 in a good yield (79%). This could then be

turned both into a new disaccharide donor 357 (by glycosylation with TMSSPh) or a new disaccharide

acceptor 358 (by deacetylation). Two rounds of glycosylation then afforded the hexasaccharide 361

on a gram scale. In comparison with the two previous studies, this strategy afforded remarkably high

glycosylation yields, considering the low reactivity of the Galp C-4 position. However, the removal

of the protecting groups, especially the allyl group, turned out to be challenging, and a five-step

procedure was necessary to reach the target 362.

103

Scheme 37. Synthesis of β-(1→4)-D-hexagalactoside 362 by Lichtenthaler and co-workers.183

OAcO OBn

AllOOPiv

SPhO

HO OBn

AllOOPiv

OPMP+

MeOTf, DTBMPCH2Cl2

79%

354 355

OO OBn

AllOOPiv

OPMP

OAcO OBn

AllOOPiv

NaOMeMeOH85%

OO OBn

AllOOPiv

OPMP

OHO OBn

AllOOPivO

O OBn

AllOOPiv

SPh

OAcO OBn

AllOOPiv

PhSSiMe3BF3

.OEt2

92%

356

357 358

357 + 358

MeOTf 2,6-di-tert-butyl-4-

methylpyridineCH2Cl2

76%

OO OBn

AllOOPiv

ORO OBn

AllOOPiv

OO OBn

AllOOPiv

OPMP

OO OBn

AllOOPiv

359 R = Ac

360 R = H

357, MeOTf2,6-di-tert-butyl-4-

methylpyridineCH2Cl2

78%

OO OBn

AllOOPiv

OO OBn

AllOOPiv

OO OBn

AllOOPiv

OPMP

OO OBn

AllOOPiv

OO OBn

AllOOPiv

OAcO OBn

AllOOPiv

361NaOMe,MeOH

86%

OO OH

HOOH

OO OH

HOOH

OO OH

HOOH

OO OH

HOOH

OO OH

HOOH

OHO OH

HOOH

OPMP

1) LiOH, MeOH, 81%2) DIBAL, [NiCl2(dppp)], THF, Et2O3) Ac2O, DMAP, py4) H2

, Pd/C, AcOH5) NaOMe, MeOH

54% (4 steps)

362

Clausen and co-workers achieved the synthesis of a set of galactooligosaccharides with a β-(1→4)-

linked backbone and the potential for (1→6)-branching. The group conducted a convergent block

synthesis, using a disaccharide building block to create linear oligosaccharides of varying lenghts.

TMSOTf-mediated coupling of trifluoroacetimidate donor 363 and acceptor 364 afforded the key

pentenyl disaccharide 365 in 83% yield (Scheme 38).179

104

Scheme 38. Synthesis of linear β-(1→4)–D-penta- and heptasaccharides by Clausen and co-workers.179

OClAcO

OPivBnO

OBn

O CF3

NPh

OHO

OPivBnO

OBn

O

363 364

+

OClAcO

OPivBnO

OBn

OO

OPivBnO

OBn

O

365

TMSOTfCH2Cl2

83%

OHO

OBnBnO

OBn

OBn

366

OO

OBnBnO

OBn

OBn

ORO

OPivBnO

OBn

OO

OPivBnO

OBnNIS,TESOTfMeCN,CH2Cl2

85%

367: R = ClAc368: R = H

NIS,TESOTfMeCN, CH2Cl2

83%

OO

OBnBnO

OBn

OBn

OO

OPivBnO

OBn

OO

OPivBnO

OBn

369

365O

ClAcO

OPivBnO

OBn

OO

OPivBnO

OBn

NIS,TESOTfMeCN,CH2Cl2

80%

365

OO

OBnBnO

OBn

OBn

OO

OPivBnO

OBn

OO

OPivBnO

OBn

O

OPivBnO

OBn

OO

OPivBnO

OBn

OClAcO

OPivBnO

OBn

OO

OPivBnO

OBn

O

372

NaOMe, MeOH91%

NaOMe, MeOH93%

OO

OHHO

OH

OH

OO

OHHO

OH

OO

OHHO

OH

O

OHHO

OH

OO

OHHO

OH

OHO

OHHO

OH

OO

OHHO

OH

O1) Et4NOH, MeOH, THF2) H2,Pd(OH)2/C, THF, MeOH, H2O

75%

373

OO

OHHO

OH

OH

OO

OHHO

OH

OO

OHHO

OH

OHO

OHHO

OH

OO

OHHO

OH

OO

OBnBnO

OBn

OBn

OO

OPivBnO

OBn

OO

OPivBnO

OBn

OHO

OPivBnO

OBn

OO

OPivBnO

OBn

1) Et4NOH, MeOH, THF2) H2,Pd(OH)2/C, THF, MeOH, H2O

78%

370

371

NIS-TESOTf-mediated glycosylation of the monosaccharide acceptor 366, with disaccharide 365,

afforded the trisaccharide 367. Selective deprotection of the chloroacetyl group, followed by coupling

with 365, enabled the synthesis both of a linear penta- and heptasaccharide (369 and 372, respectively)

105

in high yields. Finally, de-esterification by treatment with Et4NOH, followed by hydrogenolysis gave

the targets 370 and 373 in 78% and 75% yield, respectively.

In 2017, Pfrengle and co-workers reported the synthesis of a small library of type I AG

oligosaccharides through an automated solid phase sequence, using the six monosaccharide building

blocks 121, 374–378 shown in Scheme 39. Fmoc-protected galactose building blocks 374 and 375

were required for the synthesis of the β-(1→4)-linked backbone. A temporary Lev protecting group

was used at the C-3 position of building block 375, allowing for substitution. A third galactose

building block (376) with an Fmoc group in the C-3 position was used to prepare a mixed-linkage

galactan (383). Both benzyl ethers and benzoyl esters were used as permanent protecting groups for

the remaining positions. Finally, the three L-arabinofuranosides 121, 377 and 378 were used for the

side chains.

106

Scheme 39. Automated assembly of type I AGs by Pfrengle and co-workers.181

OBnO

OBzO

POBu

OBuO374

OBnFmocOO

LevOOBz

OP

OBuOBu

O375

OBnFmocOO

FmocOOBz

OP

OBuOBu

O376

OBnBnO

OOBz

BzO OBz

SEt

121

OFmocO

BzO OBz

SEt

377

OOBz

FmocO OBz

SEt

378

OO

HOHO

OH

OO

HOHO

OH

OO

OHHO

OH

O NH23

OHO

HOHO

OH

OO

HOHO

OH

OO

HOHO

OH

OO

OHHO

OH

O NH23

OO

HOHO

OHOO

HOHO

OHOHO

HOHO

OHO

HO

HOHO

OH

OO

HOO

OH

OO

OHHO

OH

O NH23

OOH

HO OH

OO

HOHO

OH

OO

HOHO

OH

OO

OHHO

OH

O NH23

OHO

HOO

OH

OOH

HO OH

OO

HOO

OH

OO

HOHO

OH

OO

OHHO

OH

O NH2

OO

HOHO

OHOHO

HOHO

OH

OOH

HO OH

OHO

HOHO

OH

OO

HOO

OH

OO

OHHO

OH

O NH23

OO

HOOH

OOH

HO

HO

O

HOO

OH

OO

OH

O OH

HO

NH23

OOH

O OHO

OH

HO OH 3

379: 15% 381: 2%380: 6%

382: 7% 384: 8%

385: 38% 386: 5%

OHO

HOHO

OH

OO

HO

OH

OO

HO

HO

OH

OO

HOHO

OH

OOH

NH23

383: 16%


Coupling:

TMSOTf, CH2Cl2 (374, 375, 376)

NIS, TfOH, CH2Cl2, dioxane (121, 377, 378)

Deprotection:

20% Et3N in DMF (Fmoc) N2H4

, py, AcOH, H2O (Lev)Capping:

BzCl, DMAP, py

Cleavage:

hν, CH2Cl2Deprotection:

NaOMe, THF Pd/C, H2

, EtOAc, MeOH, H2O, AcOH

107

6.2 Type II AGs

Various different type II arabinogalactans have been produced by several groups and the synthesized

fragments are presented in Table 18 and Table 19. For the most part, arabinogalactans with a β-(1→6)-

galactan backbone have been synthesized (Table 19), but two groups reported the synthesis of some

fragments containing a β-(1→3)-galactan backbone, which is mostly found in arabinogalactan

proteins (AGPs, Table 18).184,185

Table 18. Synthetic type II AG fragments containing a β-(1→3)-linked backbone.


OR

Me 2002 Yang & Du185

OR


OR


OR


OR


108

OR


OR


OR


OR


OR


OR


OR


109

OR


The only solution phase synthesis of β-(1→3)-linked type II arabinogalactan was achieved by Yang

& Du in 2002 and is presented in Scheme 40.185 Regioselective glycosylation of diol galactoside 388

with arabinose donor 387 afforded the desired α-linked disaccharide 389 in 76% yield. Standard

protecting group manipulations gave acceptor 391, which could be further selectively coupled with

trichloroacetimidate donor 392, affording protected trisaccharide 393 in 94% yield. Deprotection gave

the desired trisaccharide 394.

Scheme 40. Synthesis of type-II arabinogalactan trisaccharide 394 by Yang & Du.185

OOBz

BzO OBz

OC(NH)CCl3

OOHO

O

OMeHO

OO

OBz

OBzBzO

OR3O

R2O

OMeR1O

TMSOTf, CH2Cl2

76%

387

388

389: R1 = H , R2, R3 = (CH3)2C390: R1 = Ac , R2, R3 = (CH3)2C391: R1 = Ac , R2 = R3 = H

Ac2O, py

90% TFA

OOBzBzO

BzO

OC(NH)CCl3BzO

TMSOTf, CH2Cl2

94%

392

OO

OBz

OBzBzO

OHO

O

OMeAcO

OOBzBzO

BzOBzO

OO

OH

OHHO

OHO

O

OMeHO

OOHHO

HOOH

393 394

NH3MeOH

74%

In 2015, Pfrengle and co-workers published the synthesis of a small library of 14 type II AGPs through

an automated solid phase approach using the four monosaccharide building blocks 121, 376, 395 and

396 (Scheme 41).184 Both linear and branched oligomers were synthesized as well as arabinogalactan

fragments that had either a β-(1→3)- or a β-(1→6)-galactan backbone.

110

Scheme 41. Examples of the type II AG fragments obtained by Pfrengle and co-workers.184

OFmocO

OBzO

POBu

OBuO376

OBnBnOO

FmocOOBz

OP

OBuOBu

O395

OLevBnOO

BnOOBz

OP

OBuOBu

O396

OFmocBnOO

OBz

BzO OBz

SEt

121

OHO

OHO

OOHHO

OOH

OO

OH

OHHOOH

O NH23

OOHHO

OOHO

OH

HO OH

OHO

OHO

OHO

OOH

OH

O NH23

OOHHO

HOOH

O

OHO

OHO

OHO

OOH

OH

O NH23

OOHHO

HOOH

OOH

OHHO

O

OH

OHHO

O

O

OHO

OHO

OHO

OOH

OH

O NH23

OOHHO

HOOH

OOH

OHHO

O

OH

OHO

OO HO

OH

OHHO

O

OHO

OHO

OHO

OOH

OH

O NH23

OOHHO

HOOH

O

OH

OHO

OHO O

OH

OHO

OOH

OHHO

O

HOHO

OH

OH

OOHHO

OOH

OOHO

OOHO

OH

HO OH

O NH2

3

O

OH

OHHO

OH

OOHO

HOOH

O NH23

O

OHO

HO

OO

OH

OHHO

OH

OOH

HO

HO397:17% 398: 17%

399: 10% 400: 16%

401: 16% 402: 11% 403: 9%


Coupling:

TMSOTf, CH2Cl2 (376, 395, 396)

NIS, TfOH, CH2Cl2, dioxane (121)

Deprotection:

20% Et3N in DMF (Fmoc) N2H4

, py, AcOH, H2O (Lev)Capping:

BzCl, DMAP, py

Cleavage:

hν, CH2Cl2Deprotection:

NaOMe, THF Pd/C, H2

, EtOAc, MeOH, H2O, AcOH

Table 19. Synthetic type II AG fragments containing a β-(1→6)-linked backbone.


OR


111

OR

H

Me

2002

2010

Ning et al.186

Kaji et al.187

OR

(CH2)11CO2Me

C12H25

1998

2000

2003

Timmers et al.188

Du et al.189

Ning et al.190

OR

(CH2)11CO2Me

H

C12H25

1998

2002

2003

Timmers et al.188

Csávás et al.191

Ning et al.190

OR

(CH2)3NH2

(CH2)5NH2

2009

2013

2015

Fekete et al.192

Liang et al.193

Bartetzko et al.184

OR

(CH2)11CO2Me

C12H25

1998

2003

Timmers et al.188

Ning et al.190

OR

H

PMP

2002

2003

Csávás et al.191

Zeng et al.194

OR

(CH2)3NH2 2009 Fekete et al.192

112

OR

C12H25 2003 Ning et al.190

OR

PMP 2003 Zeng et al.194

OR

Me

H

2001

2002

Gu et al.195

Ning et al.186

OR

PMP 2005 Li et al.196

OR

(CH2)3NH2

2014 Huang et al.197

OR


113

OR


OR


OR


OR

2


The first synthesis of a tetrameric arabinogalactan with a β-(1→6)-linked backbone was achieved in

1998 by van Boom and co-workers, based on 1,2-anhydrosugar building blocks, as shown in Scheme

114

42.188 The group synthesized three different tetrasaccharide analogues, differing in the position of the

arabinofuranosyl side chain, which was linked either to the 2-, 2’- or 2’’-position of the trisaccharide

backbone. To afford the three targets, an oxidative coupling of galactals was undertaken. This

methodology involved a two-step procedure starting with stereoselective epoxidation of a suitably-

protected galactal 404 with 3,3-dimethyldioxirane, followed by ZnCl2-catalyzed condensation of the

resulting 1,2-anhydrosugar with a galactal acceptor. This glycal approach afforded an unprotected 2-

hydroxyl function, ready for glycosylation with an arabinosyl donor in order to introduce the

branching.

115

Scheme 42. Example of one of the three tetrameric arabinogalactans containing a β-(1→6)-linked backbone prepared by van Boom and co-workers.188

OOTBDPSBnO

BnOO

OTBDPSBnO

BnO

O

OOBz

BzO OBz

SEt

3,3-dimethyldioxiraneCH2Cl2

, acetone

quant.

HO OMe

O

10

OBnO

BnO O(CH2)11CO2MeOH

OTBDPS OBnO

BnO O(CH2)11CO2MeO

OR

OOBz

BzO OBz

OOTBDPSBnO

BnO

O

OBnO

BnO O(CH2)11CO2MeO

OOBz

BzO OBz

OBnO

BnOOH

OR

O

ZnCl2, THF

78%

NIS, TfOHClCH2CH2Cl, THF

91%

408: R = TBDPS409: R = H

410: R = TBDPS411: R = H

OBnO

BnO O(CH2)11CO2MeO

OOBz

BzO OBz

OBnO

BnOOH

O

OBnO

BnOOH

OTBDPS

O

OHO

HO O(CH2)11CO2MeO

OOH

HO OH

OHO

HOOH

O

OHO

HOOH

OH

O

ZnCl2, THF

68%

404 405

406

407

121

405

412

413

TBAF, THF, 80%

TBAF, THF, 76%

1) TBAF, THF2) tBuOK, CH2Cl2

, MeOH3) H2

, Pd/C, tBuOH, H2O

64%

OOTBDPSBnO

BnO

O

ZnCl2, THF

60%

405

Kong and co-workers later reported several syntheses of well-defined arabinogalactans carrying

different lengths of arabinan side chain.189,194–196,198,199 They started by producing a tetrasaccharide in

2000, and by 2005, they had reached a chain length of up to 20 residues. As one example of their

work, the synthesis of octaarabinogalactan 429 is presented in Scheme 43.198 This target consists of a

β-(1→6)-linked pentagalactan carrying arabinofuranosyl and diarabinofuranosyl branches at its 2’’’-

and 2’-positions, respectively.

116

Scheme 43. Synthesis of octaarabinogalactan by Kong and co-workers.198

O

OH

OPMPOAc

BzO

BzO

O

OBz

BzOBzO

BzO

OC(NH)CCl3

O

OBz

BzOBzO

BzO

OOROAc

BzO

BzOO

415: R = PMP (β)416: R = C(NH)CCl3

(α)

392

414TMSOTf, CH2Cl24-methoxyphenol

81%

1) CAN, H2O, CH3CN2) CCl3CN, K2CO3

, CH2Cl2 79%

O

OH

OPMPOBz

BzO

BzO

417

TMSOTf, CH2Cl24-methoxyphenol

80%

O

OBz

BzOBzO

BzO

O

ROBzO

BzOO

OOPMP

OBzBzO

BzOO

418: R = Ac419: R = H (79%)

1% MeCOCl, MeOHCH2Cl2

OOBz

BzO OBz

OC(NH)CCl3

O

OBz

BzOBzO

BzO

O

OBzO

BzOO

OOROBz

BzO

BzOO

387

TMSOTf, CH2Cl2

78%

OOBz

BzO OBz

420: R = PMP (β)421: R = C(NH)CCl3

(α)

1) CAN, H2O, CH3CN2) CCl3CN, K2CO3

, CH2Cl2 81%

O

OH

OPMPOBz

BzO

BzO

417

O

OAc

AcOBzO

BzO

OC(NH)CCl3422

TMSOTf, CH2Cl24-methoxyphenol

81% OOPMP

OBzBzO

BzO

O

OR

AcOBzO

BzO

O

423: R = Ac424: R = H (82%)

0.1% MeCOClMeOH, CH2Cl2

�421+ 424

O

OBz

BzOBzO

BzO

O

OBzO

BzOO

O

BzOBzO

BzOO

OOBz

BzO OBz

OOPMP

OBzBzO

BzO

O

ROBzO

BzO

O

O

425: R = Ac426: R = H (72%)

1% MeCOClMeOH, CH2Cl2

TMSOTf, CH2Cl2

75%

OOBz

BzO OBz427

TMSOTf, CH2Cl2

75%

OOBz

O OBz

OC(NH)CCl3O

OR

RORO

RO

O

ORO

ROO

O

RORO

ROO

OOR

RO OR

OOPMP

ORRO

RO

O

ORO

RO

O

O

OOR

RO OR

OOR

O OR

428: R = Bz429: R = H (84%)

NH3, MeOH

Branching at both the C-2 and C-3 positions is a well-known characteristic for AGs. In order to

evaluate differences in immunomodulating activity between C-2 and C-3 branched variants, the same

group synthesized a series of model AGs in 2005. To illustrate their method, the retrosynthetic analysis

of the arabinogalactan 20-mer is shown in Scheme 44.196 The synthesis of the key tetrasaccharide

building blocks 431–433 was achieved from the monosaccharides 387, 417, 422, 434 and 435.

117

Through coupling reactions between the tetrasaccharides, the authors reached arabinogalactans of

varying lengths, up to the longest (430), with twenty monosaccharides.

Scheme 44. Retrosynthetic analysis of AG 430 synthesized by Kong and co-workers.196

OBzO

OBzO

O

OAcBzO

BzOBzO

O

OBzO

BzOBzO

O

OC(NH)CCl3

OOBz

BzO OBz

OBzO

BzOO

O

OAcBzO

BzOBzO

O

OBzO

BzOBzO

O

OC(NH)CCl3

OOBz

BzO OBz

O

OAcBzO

BzOAcO

OC(NH)CCl3

OBzO

OBzO

O

OHBzO

BzOBzO

O

OBzO

BzOBzO

O

OPMPO

OBz

BzO OBz

O

OHBzO

AllOOBz

SiPrO

OAcBzO

BzOBzO

OC(NH)CCl3

OBzO

BzOOBz

OPMP

OHO

OBz

BzO OBz

OC(NH)CCl3

OHO

HOO

OHO

HOHO

O

OHO

HOHO

O

OOH

HO OH

OHO

OHO

OHO

HOHO

O

OHO

HOHO

O

OPMPO

OH

HO OH

O

OHO

OHO

OHO

HOHO

O

OHO

HOHO

O

OOH

HO OH

O

O

H

2

434 435 417 387 422

431 432 433

430

118

Separately, the groups of Yang and Zhang have developed one-pot glycosylation strategies for the

synthesis of 3,6-branched tetra- and hexaarabinogalactan. Both groups relied on the same

galactopyranosyl thioglycoside diol 436 as a key glycosylating agent, as shown in Scheme 45. Zhang

and co-workers, achieved the synthesis of the hexasaccharide 441 from the assembly of two

disaccharides and two monosaccharides, in a one-pot procedure involving three glycosylation

reactions, with an overall yield of 60%.197

OBzO

BzOBzO

O

OBzBzO

BzOBzOO

BzO

HOOBz

SPh

OHO

OBz

BzO OBz

SPhO

OC(NH)CCl3

OBzO

BzOOBz

O

OHBzO

BzOBzO

O

O(CH2)3N3

436 439437 438

OBzO

HOOBz

SPh

OH

436

1) 437, TMSOTf, CH2Cl22) 438, NIS, TfOH, CH2Cl23) 439, NIS, TfOH,

CH2Cl2

60% over 3 steps

OBzO

OBzO

OBzO

BzOBzO

O

OBzBzO

BzOBzO

O

OBzO

BzOOBz

OBzO

BzOBzO

O

O(CH2)3N3

O

O

OOBz

BzO OBz

OHO

OHO

OHO

HOHO

O

OHHO

HOHO

O

OHO

HOOH

OHO

HOHO

O

O(CH2)3NH2

O

O

OOH

HO OH

440 441

1) NaOMe, MeOH, 88%

2) Pd/C, H2, MeOH, 81%

Scheme 45. One-pot synthesis to reach a hexaarabinogalactan by Zhang and co-workers.197

In the same manner and starting from the same key monosaccharide building block 436, Yang and

co-workers achieved the synthesis of their tetrasaccharide in a one-pot manner, reaching the target in

52% yield over three glycosylation steps.

119

6.3 Arabinans

linear arabinan

branched arabinan Figure 26. Example of linear and branched arabinan structures.

Arabinans consist of α-(1→5)-linked L-Araf residues, with some degree of α-(1→3) and/or α-(1→2)

branching, as shown in Figure 26. In many schematic representations of pectin, the arabinan side

chains are shown as being attached directly to the rhamnose units of the RG-I backbone. 6,99,200–202

Very limited evidence for this claim has been reported, however. Most arabinan has been observed as

being linked to galactose. A great deal of work has been carried out on synthesizing oligomers of D-

arabinofuranosides from bacterial cell walls highlighted by a highly efficient assembly of a 92-mer

polysaccharide published in 2017 by Xin-Shan and co-workers.203 Significantly fewer syntheses of L-

arabinofuranosides have been reported and they are presented in Table 20.

Table 20. Synthetic α-L-arabinofuranosides.


OR


OR

Me 1995 Kaneko et al.205

120

OR

6 All 1999 Du et al.206,207

The first synthesis of a diarabinofuranoside was carried out by Arnd & Graffi in 1976.208 Ten years

later, Nepogodiev and co-workers reported the synthesis of a linear α-(1→3)-linked L-arabinotrioside

as shown in Scheme 46.204 TrClO4-mediated coupling of disaccharide 444 with monosaccharide 445

afforded trisaccharide 446 in 85% yield. This was further deesterified to obtain the target triarabinan

447.

Scheme 46. Synthesis of a linear α-(1→3)-linked L-arabinotrioside by Nepogodiev and co-workers.204

+TrClO4

, CH2Cl2

85%O

OBz

BzO OBz

BrO

OH

AcO OOCN

OO

AcO OOCN

OOBz

OBzBzO

OOTr

AcO OAc

OMe

OO

AcO OAc

OMeOOBz

BzO OBz

O

OAcAcO

O

OO

HO OH

OMeOOH

HO OH

O

OHHO

O

442 443 444

445

446 447

4Å MSMeCN

44%

NaOMe, MeOH

64%

In 1995, Kusakabe and co-workers synthesized all three α-L-diarabinofuranosides as well as the

trisaccharide shown in Table 20.205,209 Koenigs-Knorr glycosylations promoted by AgOTf afforded

the four targets. The synthesis of the trisaccharide also involved simultaneous couplings onto the 3-

and 5-positions of the reducing end monosaccharide. A year later, Kong and co-workers reported the

synthesis of a linear octaarabinan, bearing an allyl group at the anomeric position, as shown in Scheme

47.206 They were able to perform a regioselective glycosylation between the perbenzoylated

trichloroacetimidate donor 387 and the unprotected allyl α-L-arabinofuranoside 448 to obtain partially

protected disaccharide 449 in 78% yield. The latter was converted both to the diol disaccharide 450

and the trichloroacetimidate disaccharide donor 451, which were further coupled in a selective manner

121

to form arabinotetrasaccharide 452 in 79% yield. This could then be converted into both a new

acceptor 453, holding five free hydroxyl groups and a new donor 454. By coupling these, an

octasaccharide was obtained, which, after deprotection, afforded the desired target 455 in 71% over

the four last steps.

122

Scheme 47. Synthesis of linear allyl octa-α-(1→5)-L-arabinofuranoside by Kong and co-workers.206

OOBz

OOH

BzO OBz

O CCl3

NH

HO OH

OAll

TMSOTfCH2Cl2

78% OOBz

BzO OBz

OOH

O OH

OAll 1) BnOC(NH)CCl3

TMSOTf

2) NaOMe, MeOH

70% OOH

HO OH

OOBn

O OBn

OAll

OOBz

BzO OBz

OOBz

O OBz

O

1) BzCl, py2) PdCl2

, NaOAc, HOAc3) Cl3CCN, K2CO3

, CH2Cl2 70%

NH

CCl3

387

448

449 450

451

451

450, TMSOTfCH2Cl2

79% OOH

O OH

OOBn

O OBn

OAll

OOBz

BzO OBz

OOBz

O OBz 452

1) BnOC(NH)CCl3

TMSOTf

2) NaOMe, MeOH 67%

OOBn

O OBn

OOBn

O OBn

OAll

OOH

HO OH

OOH

O OH

1) Pd(OH)2/C, H2, EtOAc

2) Ac2O, py3) PdCl2

, NaOAc, HOAc4) Cl3CCN, K2CO3

, CH2Cl2 65%

OOAc

O OAc

OOAc

O OAc

O

OOBz

BzO OBz

OOBz

O OBz

NH

CCl3

453

454

454

1) 453, TMSOTf, CH2Cl22) Pd(OH)2/C, H2

, EtOAc3) BzCl, py4) NaOMe, MeOH

71% OOH

O OH

OOH

O OH

OOH

HO OH

OOH

O OH

OOH

O OH

OOH

O OH

OAll

OOH

O OH

OOH

O OH

455

123

Most recently, Yang and co-workers have synthesized a fully-protected pentaarabinan composed of a

linear α-(1→5)-linked arabinotrioside substituted with an α-L-arabinose unit at the 3- and 3’-positions

through an elegant four-component one-pot synthesis.210

124

Polysaccharides exclusive to algae This section focuses on the most common polysaccharides found exclusively in algal cell walls:

glucuronans, carrageenans, agarans, alginates, fucans and ulvans. β-(1→3)-D-Xylans have also been

isolated from the cell wall of green algae.211,212 So far, only one paper by Kong and Chen reports the

synthesis of such structures.213 A few other, more complex and highly branched sulfated

heteropolysaccharides (xylofucoglucans, xylofucoglucuronans, etc.) have been identified as minor

components in polysaccharide isolates from brown algae. However, their structures have not yet been

fully elucidated, and they have never been the subject of chemical synthesis, therefore they will not

be discussed further in this section.214

7.1 Glucuronans

Figure 27. Glucuronan structure.

Glucuronans are β-(1→4)-D-polyglucuronic acids, i.e. anionic homopolymers as shown in Figure 27.

These polysaccharides have been extracted from the cell wall of green algae such as Ulva and

Enteromorpha compressa.215,216 The biological activity of the glucuronans has only been considered

recently, and no efforts to isolate them were reported before the year 2000.217 In 2009, Redouan and

co-workers reported a rapid process to extract glucuronans selectively in large amounts from the green

algae Ulva lactura, using enzymatic depolymerization. The process could potentially be used on an

industrial scale to produce bioactive glucuronic acid oligosaccharides.218 In 1998, Isogai and co-

125

workers prepared some glucuronan oligosaccharides from cellulose using TEMPO-mediated

oxidation, work which could open up a new field of cellulose exploitation.219 No chemical synthesis

of a glucuronan longer than a disaccharide has been reported to date, and these disaccharides were

produced to optimize the TEMPO-oxidation of cellobiose.

7.2 Carrageenans

The carrageenans are a family of highly-sulfated D-galactans found in the cell walls of certain red

seaweeds of the Rhodophyte class. This family of polysaccharides has the ability to form thermo-

reversible gels or viscous solutions in the presence of salts. They are organized into ten different types

of carrabiose repeating units that differ by their degree of sulfation, and the presence or absence of

3,6-anhydrogalactose residues (see Figure 28).

126

β-carrageenan

α-carrageenanδ-carrageenan

µ-carrageenan κ-carrageenan

ν-carrageenan ι-carrageenan

λ-carrageenan θ-carrageenan

Enzymesor

KOH

γ-carrageenan

Figure 28. Schematic representation of the ten types of idealized disaccharide repeating units of carrageenans.

Natural carrageenans are non-homologous polysaccharides. This diverse family of glycans shares a

common galactan backbone (carrabiose) of alternating 3-linked-β-D-galactopyranose and 4-linked-α-

D-galactopyranose. It has been shown that hot alkaline treatment of the carrageenan having a 4-linked-

α-D-galactopyranose residue sulfated at the 6 position leads to cyclization, forming the 3,6-anhydro

rings.220,221 During carrageenan biosynthesis, this transformation is catalyzed by enzymes. No

synthesis of this type of algal polysaccharide has been reported to date.

127

7.3 Agarans

Enzymesor

KOH

Figure 29. Agarobiose backbone units.

The agarans are another family of sulfated galactans found in red seaweeds. As with carrageenans,

agarans have linear chains of alternating 3-linked β-galactopyranosyl residues and 4-linked-α-

galactopyranosyl residues. They differ from carrageenans in that the latter are L-Galp. As in

carrageenans, these residues can have 3,6-anhydro bridges (Figure 29). In 1991, Lahaye reported that

at least eleven different agarobiose structures could be identified depending on the type of red alga.222

The common modifications observed were mainly the presence of sulfate substituents, pyruvate,

urinate and methoxy groups. Red seaweeds producing agaran were named agarophytes, while the ones

producing carrageenans were called carrageenophytes. However, it has recently been shown that some

carrageenophytes also synthesize considerable amounts of agaran or DL-hybrid galactans.223 No

organic synthesis of agaran has yet been reported.

7.4 Porphyrans

OMe OMe

Enzymesor

KOH

Figure 30. Porphyran repeating unit.

128

The porphyrans are polysaccharides found in the red alga Porphyra umbilicalis, which was

characterized by Rees in 1961.224 This family of glycans shares the same backbone as agarans, but are

substituted with 6-O-methylation of the D-galactose units as shown in Figure 30.225 Similar to

carrageenans, Rees and co-workers showed that suitably linked L-galactose 6-sulphate units in

porphyrans can be converted quantitatively by alkaline treatment into 3,6-anhydrogalactose.224 This

type of oligosaccharide has not received attention from synthetic chemists yet.

7.5 Alginates

The alginates are a family of polysaccharides present in both brown algae and some gram-negative

bacteria. They are composed of two uronic acids: D-mannuronic acid (M) and L-guluronic acid (G,

the C5 epimer of M). The monomers are connected by (1→4) glycosidic linkages, with a 1,2-cis

configuration. The residues are arranged in either homopolymeric (polymannuronate -MM-, or

polyguluronate -GG-) or heteropolymeric (-MG-) segments, as shown in Figure 31.226,19

-MM--MG--GG-

Figure 31. Block composition of alginates.

These anionic polysaccharides can be isolated from marine brown algae. They have widespread

application in the biomaterial and food industries due to their gelling properties. It has been shown

that G-block-rich alginates increase gel strength by forming links with calcium, bridging two

129

antiparallel chains, as described earlier for homogalacturonan (see section 5.1).227 Alginates also offer

putative medical benefits, so there is considerable interest in generating chemically well-defined

alginate fragments. The assembly of mixed alginate sequences -MG- is particularly challenging, as it

requires the synthesis of β-D-mannuronic acid and α-L-guluronic acid, both of which have 1,2-cis

linkages. Only one synthesis of mixed sequence alginate oligosaccharides has in fact been reported,

by Codée and co-workers in 2015.228 Table 21 gives an overview of all the alginate fragments

synthesized to date.

Table 21. Synthetic alginates fragments.


OR (CH2)3NH2 2006

2008

van den Bos et al.229

Codée et al.230

OR2

(CH2)3NH2

Pr

2008

2012

Codée et al.230

Walvoort et al.231

OR3

(CH2)3NH2 2008 Codée et al.230

OR4

Pr 2015 Tang et al.92

OR6

Pr 2012 Walvoort et al.231

OR10

Pr 2012 Walvoort et al.231

OR (CH2)3NH2 2008 Dinkelaar et al.232

130

Pr 2009 Chi et al.233

OR2

Pr 2009 Chi et al.233

ORn = 1,2,3

(CH2)2NHAc 2015 Zhang et al.228

OR

n = 1,2

(CH2)2NHAc 2015 Zhang et al.228

OR (CH2)2NHAc 2015 Zhang et al.228

As mentioned earlier (section 4.5), forming the β-mannosidic linkage is considered one of the biggest

challenges in carbohydrate chemistry. Lately, syntheses of –MM- fragments up to dodecamers have

been published. In 2006, van der Marel and co-workers discovered the use of 1-thiomannuronate ester

donors in the stereocontrolled formation of 1,2-cis mannuronic linkages.229 They proved that the β-

selectivity of the mannuronate ester comes from the stereoelectronic influence of the C-5 ester and

demonstrated the efficiency of this strategy in the generation of longer alginate oligosaccharides (up

to a pentasaccharide) using a block coupling strategy (Scheme 48).230 Thioglycosyl donors were pre-

activated prior to coupling, and it was shown that judicious choice of promoter was crucial in ensuring

high yields as the oligomers grew in length. Excellent β-selectivity was maintained in all glycosylation

reactions and was not compromised by the size of the coupling partners.

131

Scheme 48. Assembly of mannuronate ester oligomers (-MM-) by van der Marel and co-workers.230

OMeO2CRO

OBn

BnOO

SPh

MeO2CO

OBn

BnO

O

X

OBnMeO2CROBnO

OOBnMeO2C

ROBnO O

CO2H

OOBnMeO2C

HOBnO SPh

456: X = β-SPh, R = Lev457: X = α-SPh, R = Bn

458: R = Lev (66%)459: R = Bn (65%)

4601) benzyl 2-

(hydroxymethylbenzoate) Ph2SO, TTBP, CH2Cl2

2) H2, Pd/C, NH4OAc, EtOAc, MeOH

461: R = Lev (65%, α:β = 1:10) 462: R = Bn (65%, α:β = 1:10)

OMeO2CHO

OBn

BnOO

O

MeO2CO

OBn

BnO

N3O

OBnMeO2CHOBnO O

N3

463 464

461 + 463 OMeO2C

OOBn

BnOO

O

MeO2CO

OBn

BnO

N3O

OBnMeO2CLevO

BnO

a) Ph2SO, TTBP, CH2Cl2 28% (α:β = 1:10)

or

b) BSP, Tf2O, TTBP, CH2Cl2

74% (α:β = 1:10)

Ph2SO, TTBP, CH2Cl2

BSP, Tf2O, TTBP, CH2Cl2

67% (α:β = 1:10)

OMeO2C

OOBn

BnOO

O

MeO2CO

OBn

BnO

N3O

OBnMeO2CBnO

BnO

OMeO2C

OOBn

BnOO

O

MeO2CO

OBn

BnO

N3O

OBnMeO2CBnO

BnO

BSP, Tf2O, TTBP, CH2Cl2

69% (α:β = 1:10)

2

3

467

468

OMeO2C

OOBn

BnOO

O

MeO2CO

OBn

BnO

N3O

OBnMeO2CHOBnO

H2NNH2py, HOAc

78%

465 466

OMeO2C

OOBn

BnOO

O

MeO2CO

OBn

BnO

N3O

OBnMeO2CROBnO

n

464: n = 0, R = H466: n = 1, R = H467: n = 2, R = Bn468: n = 3, R = Bn

OHO2CO

OH

HOO

O

HO2CO

OBn

HO

H2NO

OHHO2CHO

HO

469: n = 0 (59%)470: n = 1 (86%)471: n = 2 (65%)472: n = 3 (76%)

n

1) KOH, THF, H2O

2) H2, Pd/C, MeOH, HOAc

(tBuOH, H2O)

462 + 464

462 + 466

Mannuronic acid donors were also used by Codée and co-workers in an automated solid-phase

synthesis, to build alginates up to a dodecasaccharide. Trifluoroacetimidate donors were used as

building blocks, since pre-activation of donors was not possible on their solid phase synthesizer and

because the nature of the linker prohibited the use of soft electrophiles required for the activation of

132

thioglycosides. The stereodirecting effect of the C-5 carboxylate again provided the desired (1→2)-

cis glycosides with excellent stereoselectivity.

The synthesis of short L-guluronic acid oligomers (-GG- blocks up to a tetrasaccharide) has been

achieved by only two groups, Hung and co-workers233 and van der Marel, Codée and co-workers.232

As L-gulose and L-guluronic acid are rare sugars, they are not commercially available and need to be

synthesized prior to use, as shown in Scheme 49.

Scheme 49. Synthesis of L-gulose.232,233

OOH

OH

OH

HOOH

L-gulose476

OOHOH

O

OHHO

L-gulonic acidγ-lactone

474100 g scale

OOO

OH

OO

1) H2SO4 dimethoxypropane acetone

2) DIBAL-H toluene

84%

20% TFA in H2O

quant.

OOHOH

O

OHHOL-ascorbic acid

Hung and co-workers

van der Marel, Codée and co-workers

473

475

In contrast to D-mannopyranose, L-gulopyranose has an intrinsic preference for the formation of 1,2-

cis-glycosidic bonds without the need to incorporate stereodirecting groups. Furthermore, Codée and

co-workers demonstrated that L-guluronic acid donors are less stereoselective in glycosylation

reactions than L-gulose donors.232 Therefore, a post-glycosylation oxidation strategy has been the

preferred approach for the synthesis of -GG- block oligosaccharides.

133

After screening the efficiency of several gulose donors, Codée and co-workers found that a

dehydrative glycosylation234 with hemiacetal 479 gave the best yield, and α-selectivity, in the

glycosylation step. They achieved the synthesis of a guluronic acid trisaccharide, as shown in Scheme

50.232

Scheme 50. Synthesis of guluronic acid trisaccharide (-GGG-) by Codée and co-workers.232

O ROBn

OBn

OO

SitBu

tBu

O SPhOH

OH

OH

HO

OOBn

OBn

OHTBSO

ON3

OOBn

OBn

OO

478: R = SPh479: R = OH

O OBnOBn

OTBSO

ON3Ph2SO, TTBP, Tf2O

CH2Cl2

84% (α:β = 10:1)

481

O OBnOBn

OR1O

OOBn

OBn

OR1O

ON3

O OBnOBn

OR2R2O

484: R1, TBS, R2 = Si(tBu)2485: R1, R2 = H

O OHOH

HOOC

O

O OHOH

HOOC

O

ONH2

O OHOH

HOOC

OH

NIS, TFA, CH2Cl2

95%

477

480

TBAF, THF83%

1) tBuOH, H2O, TEMPO, BAIB

2) tBuOH, Pd/C, H2

85%

OOBn

OBn

OR3R1O

O OBnOBn

OR2O

ON3

TBAF, THF

87%

SitButBu

482: R1, R2 = R3 = H483: R1, R2 = TBS, R3 = H

TBSCl, imidazole, DMF78%

479, Ph2SO, TTBP, Tf2O, CH2Cl2then 483

42% only α

486

Hung and co-workers employed 1,6-anhydrogulose synthons to lock the C4-OH in a more accessible

environment and increase the reactivity of the nucleophile.233

As mentioned above, only one synthesis of alginate sequences containing both M and G residues has

been reported, by Codée and co-workers in 2015.228 They decided on an approach employing GM

building blocks, thus featuring a mannuronic acid donor, and pre-glycosylation oxidation in order to

134

minimize functional-group manipulation at a late stage of the synthesis. The preparation of the

building blocks required for the assembly of the longer oligosaccharides is presented in Scheme 51.

Glycosylation between the silylene-protected gulose imidate donor 487 and mannuronic acid acceptor

488 or 463 led to GM dimers 489 and 495, respectively, in good yields and with high

stereoselectivities. A desilylation/oxidation/methyl ester formation sequence afforded GM acceptor

building blocks 490 and 496. The trifluoroacetimidate GM donor 493 was then obtained from 490 in

an 83% yield over three steps.

Scheme 51. GM donor and acceptor building blocks for the synthesis of mixed alginate oligosaccharides by Codée and co-workers.228

O OBnOBn

OO

SitBu

tBu

OCF3

NPhO

OBnMeO2CHOBnO

STol

O OBnOBn

MeO2C

OOBn

OBn

O

STol

CO2Me

OR

490: R = H491: R = Lev

TMSOTf, CH2Cl2

91%

OOBnMeO2C

HOBnO

O OBnOBn

OO

SitBu

tBu

OCF3

NPhO

N3

O OBnOBn

MeO2C

OH

O OOBnMeO2C

BnOO

N3

O OBnOBn

MeO2C

OLev

O OOBnMeO2C

BnOOR

492: R = H493: R = C(=NPh)CF3

487

488

487

463

O OBnOBn

MeO2C

OH

O N3

494

1) HF·py, THF2) TEMPO, PhI(OAc)2

tBuOH, THF, H2O3) MeI, K2CO3

DMF

83%

O OBnOBn

OOBn

OBn

O

STol

CO2Me

OO

SitBu

tBu

489

O OBnOBn

O

O OOBnMeO2C

BnOO

N3

OSitBu

tBu

TMSOTf

CH2Cl2

-78 οC to -20 οC

58%495 496

NIS, TFA, CH2Cl291%

1) HF·py, THF2) TEMPO, PhI(OAc)2

tBuOH, THF, H2O3) MeI, K2CO3

DMF

84%

LevOH, EDCI, DMAPCH2Cl2

, 92%

F3CC(=NPh)Cl, K2CO3acetone, 98%

135

With the GM donors and acceptors in hand, the assembly of larger -GM- oligomers could proceed.

This was successfully achieved for structures ranging from the GMG trisaccharide to a GMGMGMG

heptasaccharide (Scheme 52).

136

Scheme 52. Assembly of -GM- oligomers up to heptasaccharide by Codée and co-workers.228

OOBn

OBnMeO2C

OR

O OOBnMeO2C

BnO

OOBn

OBnMeO2C

O

O N3

497: R = Lev498: R = H

493 + 494

TBSOTfCH2Cl2

84%α:β

> 20:1

GMG

N2H4, H2O

AcOH, py, 89%

493TBSOTf, CH2Cl2

31%

493TBSOTf, CH2Cl2

34%

OOBn

OBnMeO2C

O

O OOBnMeO2C

BnO

OOBn

OBnMeO2C

O

O N3

GMGMG

R

2

OOBn

OBnMeO2C

O

O OOBnMeO2C

BnO

OOBn

OBnMeO2C

O

O N3

Lev

3

501

GMGMGMG

493 + 496

TBSOTf CH2Cl2

26%α:β

> 20:1

493TBSOTf, CH2Cl2

30%

GMGM

OOBn

OBnMeO2C

O

O OOBnMeO2C

BnOO

N3

Lev

3OOBn

OBnMeO2C

O

O OOBnMeO2C

BnOO

N3

R

2

GMGMGM

493 + 490

TBSOTf CH2Cl2

91%α:β

> 20:1O

OBn

OBnMeO2C

OLev

O OOBnMeO2C

BnO O

OOBn

OBnMeO2C

OOBn

OBn

O

R

CO2Me

505: R = α-STol506: R = O(C=NPh)CF3

(92%)

505

1) NIS, TFA, CH2Cl22) F3CC(=NPh)Cl, K2CO3

, acetone

GMGM

OOBn

OBnMeO2C

O

O OOBnMeO2C

BnO O

OOBn

OBnMeO2C

OOBn

OBn

O

R

CO2Me

490TBSOTf, CH2Cl2

73%α:β

> 20:1

Lev

2

GMGMGM

499: R = Lev500: R = H

N2H4, H2O

AcOH, py, 98%

503: R = Lev504: R = H

N2H4, H2O

AcOH, py, 78%

507: R = α-STol508: R = O(C=NPh)CF3

(80%)

1) NIS, TFA, CH2Cl22) F3CC(=NPh)Cl, K2CO3

, acetone

OOH

OHNaOOC

O

O OOHNaOOC

HO

OOH

OHNaOOC

O

O NHAc

H

3

1) N2H4, H2O, AcOH, py

2) LiOH, H2O2, H2O, THF

3) Pd/C, H2, tBuOH, THF, H2O

4) Ac2O, NaHCO3,THF, H2O

60%

502

GMGMGMG

Surprisingly, the nature of the reducing end anomeric center of the GM acceptor had a tremendous

effect on the efficiency of glycosylation by influencing the conformation of the acceptor. The rigid

137

GM acceptor 496, bearing a β-linked anomeric azidopropyl spacer, led to lower glycosylation yields

(26–34%), whereas the conformationally-flexible GM-α-STol acceptor 490 gave much better yields

(73–91%).235 The GMGGMG hexasaccharide was synthesized using a [2+3+1] approach with

mannuronic acid donors, as summarized in Error! Reference source not found..

Scheme 53. Convergent assembly of GMGGMG hexasaccharide by Codée and co-workers.228

GGMreducing end

acceptor

GM donor:493

GMGGM GMGGM donor

G acceptor:494

87% 43%GMGGMG

All the oligomers were deprotected by removal of the levulinoyl groups, saponification of the methyl

esters, debenzylation plus azide reduction and finally acetylation of the spacer amine group as shown

for heptamer 502 in Scheme 52.

7.6 Fucans

In 1913, Kylin reported the first extraction of “fucoidin” from marine brown algae.236 Most of the

fucose containing sulfated polysaccharides (FCSPs) found in brown algae are fucoidans, which are

heteropolymers of sulfated fucans. These polysaccharides can account for more than 40% of the dry

weight of isolated cell walls in brown algae.237 Fucoidans have been found to have two types of

homofucose backbone chains as shown in Figure 32. Type I consists of (1→3)-linked α-L-

fucopyranoside residues, while type II is made up of alternating α-(1→3)- and α-(1→4)-L-

fucopyranoside residues.238 The complexity and heterogeneity of fucoidans is due to the sulfation

pattern, which differs depending both on the type of alga as well as between cell types. Sulfation can

138

occur at the O-2, O-3, and/or O-4 position of the fucose residues. Acetylation has also been observed

at various positions of the backbone, as has the presence of other sugar residues such as α-D-

glucuronopyranosyl or α-L-fucopyranosyl.237

Type I Type II

Figure 32. The two types of homofucose backbone chains found in brown seaweed fucoidans with examples of sulfation patterns.

It has been known for some time that fucoidans act as modulators of coagulation.239 But how their

sulfation pattern and conformation influence biological activity has not been studied. Early in the 21st

Century, synthetic chemists began showing interest in these targets, aiming to synthesize certain well-

defined oligofucans and help elucidate their structure-activity relationship. An overview of the type I

and type II fucan and fucoidan fragments synthesized are shown in Table 22 and Table 23,

respectively.

Table 22. Synthetic type I fucan/fucoidan fragments.


OR

Pr

Pr

2003

2011

Gerbst et al.240

Krylov et al.241

139

OR

Pr 2003 Gerbst et al.240

OR

Pr 2016 Vinnitskiy et al.242

OR


OR


OR


OR

2

C8H17

Pr

2004

2015

2006

2011

Hua et al.243

Kasai et al.244

Ustyuzhanina et al.238

Krylov et al.241

140

OR

2

Pr 2006 Ustyuzhanina et al.238

OR

2

C8H17

2010

2015

Zong et al.245

Kasai et al.244

OR

2

Pr

C8H17

2006

2010


Zong et al.245

OR

2

C8H17 2015 Kasai et al.244

OR

2

Pr

2011

Krylov et al.241

OR

2

C8H17

2004

2015

Hua et al.243

Kasai et al.244

OR

3

C8H17 2010 Zong et al.245

141

OR

3


OR

4

Pr

2006

2011


Krylov et al.241

OR

4

Pr

C8H17

2006

2010


Zong et al.245

OR

4


OR

4


OR

5


OR

6

Pr 2011 Krylov et al.241

142

OR

6

Pr

C8H17

2006

2010


Zong et al.245

OR

6


OR

10


OR

10


OR

14


OR

14


Table 23. Synthetic type II fucan/fucoidan fragments.


OR Pr 2011 Krylov et al.241

143

OR


OR

Pr

C8H17

2006

2011

2014

2015


Krylov et al.241

Arafuka et al.246

Kasai et al.244

OR


OR

C8H17

2014

2015

Arafuka et al.246

Kasai et al.244

OR

C8H17

2014

2015

Arafuka et al.246

Kasai et al.244

OR

C8H17

2014

2015

Arafuka et al.246

Kasai et al.244

OR

Pr

C8H17

2011

2014

2015

Krylov et al.241

Arafuka et al.246

Kasai et al.244

OR

2

Pr

2006

2011


Krylov et al.241

2

OR


144

OR

2


In 2003, Nifantiev and co-workers reported the first synthesis of the two type I fucoidan trisaccharides

515 and 516 (Scheme 54).240 Both fucotriosides were prepared by use of the readily accessible

disaccharide acceptor 509.247,248 Regio- and stereoselective glycosylation with fucosyl bromide donor

510, promoted by mercury salts afforded the protected α-linked trisaccharide 511 in 65% yield.

Saponification and subsequent catalytic hydrogenolysis provided fucotrioside target 515 in a good

yield overall. Protecting group manipulations and sulfation gave trisaccharide 514. The latter was

deprotected to afford the sulfated fucotrioside target 516.

Scheme 54. First type I fucan and fucoidan synthesized by Nifantiev and co-workers.240

OMe

HO

OBnO

OAll

OMe

HO

OBnOH

OMe

BzO

OBnOBz

Br

Hg(CN)2,

HgBr2CH2Cl2

65%

509

510

OMe

HO

OBnO

OAll

OMe

HO

OBnO

OMe

RO

OBnOR

511: R = Bz512: R = H (90%)

OMe

HO

OBnO

OAll

OMe

HO

OBnO

OMe

HO

OBnOBz

NaOMe, MeOH 513

Bu2SnO, toluenethen BzCl

62%

H2, Pd/C, MeOH

75%

OMe

HO

OHO

OPr

OMe

HO

OHO

OMe

HO

OHOH

515

SO3.py

DMF

80%

OMe

NaO3SO

OBnO

OAll

OMe

NaO3SO

OBnO

OMe

NaO3SO

OBnOBz

1) H2, Pd/C, MeOH

2) NaOMe, MeOH

78%

OMe

NaO3SO

OHO

OPr

OMe

NaO3SO

OHO

OMe

NaO3SO

OHOH

516

514

145

By applying a stepwise elongation strategy with thioglycoside donors, Du and co-workers

subsequently reported the synthesis of a fully sulfated tetrasaccharide with a β-configured octyl group

at the reducing end.243 In 2006, Nifantiev and co-workers developed a method using [2+2], [2+4] and

[4+4] coupling reactions to produce oligofucoidans of both type I and type II, employing

trichloroacetimidates as donors.238 Inspired by these syntheses and in order also to generate type I

oligofucoidans of uneven length, Li and co-workers decided to apply a [2+2] coupling strategy to

produce the tetrasaccharide, followed by an iterative assembly of the remaining targets, as shown in

Scheme 55.245 They used 4-O-benzoyl protecting groups, claiming this improved the α-

stereoselectivity of the glycosylation steps through remote participation, in combination with 3-O-

acetyl and 2-O-benzyl groups. This way, they could produce two different sulfation patterns from

each oligofucoidan precursor.

Scheme 55. Retrosynthetic analysis of sulfated tetra- to octafucosides by Li and co-workers.245

OMe

R2O

OR1

O

OC8H17

OMe

R2O

OR1

O

OMe

R2O

OR1

O

OMe

R2O

OR1

OR3

n

OMe

BzO

OBnO

OC8H17

OMe

BzO

OBnO

OMe

BzO

OBnO

OMe

BzO

OBnOAc

n

517a-e n = 1-5 518b-e n = 2-5

OMe

BzO

OBnO

OC8H17

OMe

BzO

OBnO

OMe

BzO

OBnO

OMe

BzO

OBnOH 518a

OMe

BzO

OBnOAc

OC(NH)CCl3

+

iterative strategy

OMe

BzO

OBnO

OC8H17

OMe

BzO

OBnOH

OMe

BzOOBn

O

OMe

BzO

OBnOAc

SEt

+

OMe

BzOOBn

OAc

SEt

2 + 2assembling

strategy

R1 = SO3Na, R2 = R3 = H

or R1 = H, R2 = R3 = SO3Na

519

520

521

522

Per-O-sulfation of organic compounds containing many hydroxy groups can require lengthy reaction

times (of up to several days) and high temperatures, as well as substantial excess of the sulfating

146

reagent. Even so, one may still produce mixtures of partially sulfated products. This happened to

Nifantiev and co-workers as they tried to synthesize a fully sulfated tetrafucoside. As a result, they

developed an improved TfOH-promoted per-O-sulfation protocol with sulfur trioxide/pyridine,

enabling the reaction to take place at 0 °C and over a shorter period of time.249 They then adapted this

method for use in their previously developed block strategy, synthesizing per-O-sulfated type I and II

oligofucoidans from two to sixteen fucose residues. To their surprise, when using TfOH to promote

the sulfation, they observed an unusual rearrangement of the reducing pyranose residue into the

corresponding furanose. As this pyranoside-into-furanoside rearrangement is of great interest in the

synthesis of furanoside containing oligosaccharide targets, the group later explored its mechanism and

optimized this reaction.250 To avoid the rearrangement, they exchanged TfOH for TFA and this

produced the desired targets without by-products.

In 2014 and 2015, Toshima and co-workers designed a series of type I and type II fucoidan

tetrasaccharides with different sulfation patterns.246,251 An orthogonal deprotection strategy utilizing

the common key intermediates 528 and 531 (Scheme 56), carrying three types of protecting groups

(Bn, Bz and PMB) enabled an effective synthesis of the eight targets. The synthesis of the

tetrafucoside precursor 528 for the type II fragments is presented in Scheme 56.

147

Scheme 56. Key tetrasaccharides precursor 528 and 531 by Toshima and co-workers.246

OMe

BzOOBn

O

OMe

O

OBnOPMB

OMe

O

OBnOPMB

OC8H17

OMe

BzO

OBnOH

OMe

HO

OBnOPMB

S

OMe

BzOOBn

OClAc

OC(NH)CCl3

OMe

O

OBnOPMB

S

OMe

BzOOBn

OClAc

OMe

O

OBnOPMB

OMe

BzOOBn

OClAc

+

523

524

OC(NH)CCl3

Yb(OTf)3, Et2O

99%

525

1) NIS, Sc(OTf)3 H2O, MeCN 82%

2) DBU, CCl3CN CH2Cl2 76%

526

1) Yb(OTf)3, 1-octanol

CH2Cl2, 89%

2) thiourea, 2,6-lutidine DMF, 94%

OMe

O

OBnOPMB

OMe

BzOOBn

OH

OC8H17

527

1) 527, TMSOTf, Et2O2) thiourea, 2,6-lutidine

DMF

81% (2 steps)

528

OMe

BzOOBn

O

OMe

BzO

OBnO

OMe

BzO

OBnO

OMe

BzO

OBnOPMB

OC8H17

OMe

BzO

OBnOH

S

OMe

BzOOBn

OPMB

OC(NH)CCl3

529

530531

(type II precursor) (type I precursor)

Chemoselective glycosylation of 523 with 524, promoted by Yb(OTf)3, provided the desired α-

(1→4)-linked disaccharide 525 with very good stereoselectivity. The latter could be converted into a

trichloroacetimidate donor 526 and a new disaccharide acceptor 527 in two steps. Condensation of

527 with 1-octanol using Yb(OTf)3 afforded the corresponding octyl fucoside with surprisingly high

β-stereoselectivity. TMSOTf-promoted coupling of the two difucosides, followed by deprotection of

the ClAc group provided the key intermediate 528 as a single isomer in 81% yield over two steps. For

the five type II fucoidan targets, the deprotection and sulfation steps are presented in Scheme 57.

148

Scheme 57. Deprotection and sulfation of 528 to afford five type II tetrafucoidans 532–536.246

1) Pd(OH)2/C, H2

MeOH, AcOEt 88%

2) NaOMe, MeOH 97%

3) SO3.NEt3

, DMF

then 3 M aq. NaOH 81%

1) NaOMe, MeOH

80%2) DDQ, CH2Cl2

phosphate buffer 80%

3) SO3.NEt3

, DMF

then 3 M aq. NaOH

87%4) Pd(OH)2/C, H2

MeOH, H2O 99%

1) Pd(OH)2/C, H2

MeOH, AcOEt 98%

2) SO3.NEt3

, DMF

then aq. NaOH 97%

529

OMe

NaO3SO

OSO3NaO

OMe

O

OSO3NaOSO3Na

OMe

O

OSO3NaOSO3Na

OC8H17

OMe

NaO3SO

OSO3NaOSO3Na

OMe

HOOSO3Na

O

OMe

O

OSO3NaOSO3Na

OMe

O

OSO3NaOSO3Na

OC8H17

OMe

HO

OSO3NaOSO3Na

OMe

NaO3SOOH

O

OMe

O

OHOSO3Na

OMe

O

OHOSO3Na

OC8H17

OMe

NaO3SO

OHOSO3Na

OMe

NaO3SOOH

O

OMe

O

OHOH

OMe

O

OHOH

OC8H17

OMe

NaO3SO

OHOSO3Na

OMe

HOOH

O

OMe

O

OHOH

OMe

O

OHOH

OC8H17

OMe

HO

OHOH

533 534 535 536 537

1) NaOMe, MeOH

80%2) SO3

.NEt3

, DMF

then 3 M aq. NaOH

quant.3) Pd(OH)2/C, H2

MeOH, H2O 84%

1) Pd(OH)2/C, H2 MeOH, AcOEt 88%

2) NaOMe, MeOH 97%

A recent discovery showed that the fucoidan from the brown seaweed Chordaria flagelliformis

contains an α-(1→3)-linked polyfucopyranoside backbone with one third of the fucopyranosyl

residues bearing α-(1→2)-linked D-glucuronic acid substituents, and with half of the uronic acids

carrying α-(1→4)-L-fucofuranosides.252 In 2016, Nifantiev and co-workers achieved the synthesis of

a selectively O-sulfated branched pentasaccharide of this type (548) as shown in Scheme 58.242

149

Scheme 58. Synthesis of two branched type I fucoidan pentasaccharides by Nifantiev and co-workers.242

OCOOMe

TBSOBnO

BnO OC(NH)CCl3

O OC(NH)CCl3OBz

OBnMe OBn

OMe

BzO

OBnO

OAll

OMe

BzO

OPMBOClAc

OMe

BzO

OBnO

OAll

OMe

BzO

OHOClAc

OMe

BzO

OBnO

OAll

OMe

BzO

OOClAc

O COOMeOR

OBnBnO

540: R = TBS (α:β = 4:1)541: R = H (α

only)

537 538

539

OMe

BzO

OBnO

OAll

OMe

BzO

OOR

O COOMeO

OBnBnO

O

OBz

BnOMe OBn

543: R = ClAc 544: R = H

542

OMe

ClAcO

OBnOClAc

OC(NH)CCl3OMe

BzO

OBnO

OAll

OMe

BzO

OO

O COOMeO

OBnBnO

O

OBz

BnOMe OBn

OMe

ClAcO

OBnOClAc

OMe

HO

OHO

OPr

OMe

HO

OO

O COONaO

OHHO

O

OH

HOMe OH

OMe

HO

OBnOH

OMe

NaO3SO

OHO

OPr

OMe

NaO3SO

OO

O COONaO

OHHO

O

OSO3Na

HOMe OH

OMe

NaO3SO

OHOSO3Na

TFA (90%), CH2Cl2

96%

TMSOTf, CH2Cl2

87%

HF, MeCN, 77%

TMSOTf, CH2Cl2

79%

thiourea, MeOH, 80%

545

TMSOTf, CH2Cl2

86%

1) H2, Pd/C

THF, EtOAc, EtOH2) NaOH 2 M (aq.), MeOH

82%

546 547

548

1) LiOH 2 M (aq.), THF 2) Bu4NOH, THF 3) SO3·py, DMF4) H2

, Pd/C, THF, EtOAc, EtOH

76%

The assembly of both pentasaccharide targets 547 and 548 started with glycosylation between

difucopyranoside acceptor 538 and glucuronate donor 539, affording the trisaccharide 540 as a 4:1

α:β-mixture. The pure α-isomer 540 could be isolated after removal of the TBS group. This acceptor

was then glycosylated with fucofuranosyl donor 542 to give a mixture of α- and β-linked

tetrasaccharides in a ratio of 10:1. The desired α-anomer (isolated in 79% yield) was deprotected and

150

subsequently coupled with fucopyranoside donor 545, giving the core pentasaccharide 546 in 86%

yield. The target 547 was obtained after removal of all protecting groups. Alternatively, saponification

of 546, followed by O-sulfation and hydrogenolysis afforded the partially sulfated target 548.

7.7 Ulvans

Type A

Type B

= L-iduronic acid =OHO

HOOH OHHOOC

Rare sugar structure:

Figure 33. The two major types of disaccharide repeating units in ulvans.

Ulvans are water-soluble sulfated heteropolysaccharides, which have been extracted from the cell

walls of green marine algae. They represent between 8 and 29% of the dry weight of the cell wall.253,254

A review dealing with the structure and functional properties of ulvans was written by Lahaye and

Robic in 2007.254 The determination of the sugar sequences in ulvans represents a big challenge since

the polysaccharide composition varies widely and is dependent on algal source and ecophysiological

variation.254 However, acid hydrolysis and / or enzymatic degradation of cell walls from different

green seaweeds has shown the presence of L-rhamnose, L-xylose, D-glucuronic and L-iduronic acid.

These sugars are mostly distributed in disaccharide repeating units. Among these, the two most

frequent are ulvanobiuronic acid 3-sulfate Type A (β-D-GlcpA-(1→4)-α-L-Rhap3S-(1→4)) and Type

B (α-L-IdopA-(1→4)-α-L-Rhap3S-(1→4)), represented in Figure 33. Other less frequently occurring

units containing glucuronic acid on O-2 of rhamnose 3-sulfate or partially sulfated xylose replacing

151

the uronic acid have been found.255 No chemical synthesis of this type of oligosaccharide has been

undertaken to date.

Conclusion Plant cell wall polysaccharides include some of the most complex macromolecules found in nature.

For this reason, synthetic chemists have focused their attention on synthesizing well-defined

oligosaccharide fragments, as set out in this review. Not only are these targets extremely valuable in

studying protein-carbohydrate interactions, but they also help advance the development of new

methodologies in carbohydrate chemistry. Interest in this area is increasing, as witnessed by the many

contributions over the past twenty years. The more recent progress in automated synthesis holds

particular promise, as it is well suited to producing libraries of closely-related fragments of larger

polysaccharides. There is clearly no lack of unexplored structures to serve as inspiration for synthetic

chemists, especially within the marine polysaccharides. As this review makes clear, there is no

consensus as to the types of aglycone chosen for the targets: everything from reducing sugars over

simple alkyl groups to linkers predisposed to immobilization and / or conjugation is represented in the

review. More homogeneity in this choice, along with the establishment of a central repository for

synthetic oligosaccharides, represent areas, where we believe there is room for benefiting future

research. Nonetheless, we remain convinced that synthetic glycans will continue to provide

breakthroughs in the understanding of plant cell wall biology and physiology.

Author Information

152

Corresponding Author

E-mail: [email protected]

ORCID

Mads H. Clausen: 0000-0001-9649-1729

Notes

The authors declare no competing financial interest.

Biographies

Christine Kinnaert graduated from the Ecole Nationale des Industries Chimiques. Her thesis research

was carried out at Glaxo Smith-Kline and encompassed conformational analysis of polysaccharides.

She obtained her Ph. D. from the group of Prof. Mads H. Clausen at the Technical University of

Denmark in 2017. The subject of her thesis research was chemical synthesis of oligosaccharides

related to algal cell wall glycans. Christine is currently pursuing postdoctoral research at DTU.

Mathilde Daugaard obtained her M. Sc. (2011) and Ph. D. (2016) from the Technical University of

Denmark under the supervision of Prof. Thomas E. Nielsen and Prof. Mads H. Clausen, respectively.

The Ph. D. thesis research involved carbohydrate synthesis, more specifically preparation of α-L-

arabinans from rhamnogalacturonan I. She is currently employed by the Danish biopharmaceutical

company Nuevolution.

Faranak Nami obtained her M. Sc. in 2012 from Aarhus University under the supervision of Associate

Professor Henrik H. Jensen. The work centered on the synthesis of ibogaine analogues inducing an

153

inward conformation of the human serotonin transporter. Her Ph. D. was obtained in 2016 from the

Technical University of Denmark under the supervision of Professor Mads H. Clausen. Faranak

worked on oligosaccharide synthesis with the aim of carrying out enzymatic studies.

Mads H. Clausen obtained his Ph.D. from the Technical University of Denmark in 2002 under the

supervision of Prof. Robert Madsen. He was a postdoctoral fellow at Harvard University from 2002–

2004 in the group of Prof. Andrew G. Myers, where he worked on total synthesis of enediyne

antibiotics. In 2004, he started his independent career at the Department of Chemistry, Technical

University of Denmark, where he has been Professor of Chemical Biology since 2014. His research

interests center on chemical biology and bioorganic chemistry and include medicinal chemistry,

oligosaccharide and lipid synthesis and developing methods for library synthesis.

Acknowledgments

We are highly grateful to Ms. Adeline Berger and Dr. Helen North for providing the image used for

Figure 3. We acknowledge financial support from the Danish Council for Independent Research “A

biology-driven approach for understanding enzymatic degradation of complex polysaccharide

systems” (Grant Case no.: 107279), the Carlsberg Foundation, the Danish Strategic Research Council

(GlycAct and SET4Future projects), the Villum Foundation (PLANET project) and the Novo Nordisk

Foundation (Biotechnology-based Synthesis and Production Research).

154

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