Chemical O-Glycosylations: An Overview
Rituparna Das* and Balaram Mukhopadhyay*
[a]
Dedicated to Prof. Dr. Richard R. Schmidt, University of Konstanz, Germany
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DOI: 10.1002/open.201600043
1. Introduction
Carbohydrates, or sugars, have been deemed an important
weapon in solving many of nature’s mysteries. They are the
most diverse and the most abundant biomolecules on earth.
Apart from their energy-storage functions, they were only as-
sumed to serve as the structural and protective elements in
the plant, bacterial, and animal cell walls.
[1]
It was only in the
later part of the 20th century that the involvement of carbohy-
drates in various other biological processes
[2]
became apparent.
The renaissance witnessed over the past few years in the field
of glycobiology shows that glycoconjugates have enormous
significance in various biochemical proce sses, such as molecu-
lar recognition, cell–cell interaction, immunological recogni-
tion, transmission of biological information, and so forth.
[3]
Proper scientific knowledge of the mechanism of these biologi-
cal processes, in turn, leads to the study of various new appli-
cations in biomedical research. These applications invariably
lead to the advancement of modern day science, thereby in-
creasing its fundamental interest to scientists.
[4]
Thus, the
demand for homogenous carbohydrate samples and their d e-
rivatives for biological research have increased extensively over
the past decade.
In nature, carbohydrates exist as polysaccharides, glycocon-
jugates, or glycosides. But, the isolation of pure carbohydrate
samples in sufficient amounts is difficult and cumbersome. In
such cases, chemical syntheses of the relevant glyco-structures
become pertinent.
[5]
For the chemical synthesis of complex car-
bohydrate molecules, the main challenge is to build glycosidic
linkages connecting the monomeric units with proper stereo-
and regiochemical orientation. Over the last few decades,
many new and sophisticated procedures have been estab-
lished for the successful synthesi s of complex oligosaccharides.
Thus, development of new methodologies for chemical glyco-
sylation has emerged as an active area of research. More and
more intriguing glycosidic bond syntheses are being standar-
dized. However, achieving complete stereocontrol over glyco-
sylation remains an eluding area of carbohydrate chemistry.
[6]
Hence, optimizing the reaction conditions has long been the
basic theme in carbohydrate synthesis.
Various types of novel glycosyl donors for glycosylation reac-
tions have been synthesized and implemented.
[7,8]
Different ac-
tivation strategies have been developed for the successful as-
sembly of oligosaccharides from protected building blocks.
[9]
Solid-phase oligosaccharide synthesis has also made significant
progress and is of high interest, as the process avoids the ne-
cessity to purify the intermediates.
[10]
Automated oligosacchar-
ide synthesis has also been developed, which has given a new
edge in the field of synthetic oligosaccharide chemistry.
[11]
Fundamental concepts of glycosylation have been covered
in a wide range of Review articles.
[7]
In this Review, we aim to
indicate all of the different types of donors and their activation
protocols, as well as the different types of O-glycosylation pro-
cesses developed after the onset of the 21st century. We will
primarily stick to all newly developed methodologies of O-gly-
coside formation reported in the 21st century.
2. General Aspects
2.1. Historical Background
Glycosylation, with all of its complexity, has long been a topic
of research. The end of the 19th century witnessed the chemi-
cal formation of the first aryl and alkyl glycosidic bond formu-
lated by Michael
[12]
(p-methoxy phenyl b-d-glucopyranoside)
and Fischer
[13]
(methyl a-d-glucopyranoside), respectively. Fol-
lowing their lead, Knoenigs and Knorr formulated a more con-
trolled and modified glycosylation protocol.
[14]
New methodol-
ogies are still being developed with more advanced variations
in the mechanistic pathways. Later Zmpln and Gerecs,
[15]
and
subsequently Helferich and Wedermeyer,
[16]
illustrated the prin-
ciple of the metal(II) salt-induced removal of leaving groups.
[17]
With the general standardization of glycosyl protocols, efforts
were made to control the stereochemical outcome of glycosy-
lations. The requirement for the efficient syntheses of the two
anomeric stereoisomers, 1,2-cis and 1,2-trans glycoside led to
more advanced and detailed studies to introduce variation in
the mechanistic pathways. The works of Lemieux et al.
[18]
as
well as Ness and Fletcher
[19]
instigated the importance of the
nature of the protecting groups, especially at the C-2 position
for its correlation with the stereoselective outcome of the gly-
cosylations. This observation was further documented by Ha-
shimoto et al and Fraser-Reid et al.,
[20]
whereby the concept of
the ‘armed–disarmed’ approach was initiated, claiming ether
linkage at the C-2 position to be arming, leading to the 1,2-cis
The development of glycobiology relies on the sources of par-
ticular oligosaccharides in their purest forms. As the isol ation
of the oligosaccharide structures from natural sources is not
a reliable option for providing samples with homogeneity,
chemical mean s become pertinent. The growing demand for
diverse oligosaccharide structures has prompted the advance-
ment of chemical strategies to stitch sugar molecules with pre-
cise stereo- and regioselectivity through the formation of gly-
cosidic bonds. This Review will focus on the key developments
towards chemical O-glycosylations in the current century. Syn-
thesis of novel glycosyl donors and acceptors and their unique
activation for successful glycosylation are discussed. This
Review concludes with a summary of recent developments
and comments on future prospects.
[a] Dr. R. Das, Dr. B. Mukhopadhyay
Department of Chemical Sciences
Indian Institute of Science Education and Research (IISER) Kolkata
Mohanpur, Nadia 741246 (India)
E-mail: sugarnet73@hotmail.com
ritu_iiser@yahoo.com
2016 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.
This is an open access article under the terms of the Creative Commons
Attribution-NonCommercial License, which permits use, distribution and
reproduction in any medium, provided the original work is properly
cited and is not used for commercial purposes.
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glycoside, whereas an acyl linkage at the same position yielded
the 1,2-trans glycoside by virtue of the disarming property and
neighboring group participation. Roy et al. introduced the
‘active’ and ‘latent’ thioglycoside donors that further expanded
the concept of selective anomeric reactivity.
[21]
The latent-
active glycosylation strategy was further utilized by Boons and
Isles to form trisaccharide libraries, using vinyl and allyl donors
with selective reactivity.
[22]
Furthermore, Ogawa and co-workers
investigated the possibility of using orthogonal glycosyl
donors and implemented the ‘orthogonal glycosylation strat-
egy’ in the synthesis of various oligosaccharides.
[23]
The search to find the ideal conditions for an effective glyco-
side formation revealed various modifications in the already es-
tablished protocols. Varied promoter systems were implement-
ed, newer activating groups established, and more versatile
protecting groups were introduced. With further advanced
control on the glycosidic linkages and reaction conditions,
one-pot glycosylation also came into vogue.
[24]
More advances
in computational chemistry and subsequent kinetic studies
aided in solving the mystery behind the mechanism of glyco-
sylation to a considerable degree. But, despite all of these ef-
forts in the refinement of reaction conditions, the recognition
of a single general procedure to describe chemical glycosyla-
tion in its entirety is yet to be accomplished.
[6]
2.2. Anomeric Effect
It was between 1955 and 1958 that Edwar d and Lemieux first
defined the ‘anomeric effect ’ , based on the stereochemistry of
the C-1 carbon of the pyranose ring, that is, the anomeric
carbon.
[25,26]
It is also referred to as the Edward–Lemieux effect.
It was originally defined as the tendency of an electronegative
substituent attached to the anomeric carbon to reside in the
axial position. However, further studies have revealed various
physical interpretations behind it.
A popular and widely accepted theory is based on molecular
orbital interaction, employing the hyperconjugation of the
nonbonding electron pair on the ring oxygen atom with the
vacant s* orbital of the CX bond, thereby stabilizing the axial
configuration
[27]
(Figure 1 A). However, extensive computational
studies revealed that the energy due to hyperconjugation is
not the bulk contributor in establishing the energy difference
between the axial and equatorial configuration.
[28]
Further,
dipole moment theory
[26]
says that, in the equatorial isomer,
the dipoles of the heteroatoms are partially aligned, thereby
repelling each other. But, in the axial configuration, the oppo-
site orientation of the dipoles stabilizes the system by implicat-
ing a lower energy barrier (Figure 1 B). Studies have, thus, led
to the hypothesis that the dipole– dipole interaction and elec-
trostatic interaction form the bulk of the reasoning behind the
conformational preferences of the carbohydrates; whereas, the
hyperconjugation is only a minor factor. In view of the explan-
ations, the axial stereoisomer represents the thermodynamical-
Rituparna Das was born in 1987 in India.
She studied Chemistry at St. Xavier’s College
(Kolkata, India), where she obtained her
B.Sc. in 2009. She then entered the Integrat-
ed Ph.D. at IISER Kolkata (India) and ob-
tained her Ph.D. in 2015 under the supervi-
sion of Dr. Balaram Mukhopadhyay, working
in the field of synthetic oligosaccharide
chemistry. Currently, she is a Postdoctoral
Fellow in the research group of Dr. Mukho-
padhyay, working with glyco-dendrimers focusing on multivalent
carbohydrate–protein interactions.
Balaram Mukhopadhyay obtained his Ph.D.
in 2001 from the IACS, Jadavpur (India)
under the supervision of Prof. Nirmolendu
Roy, working in the field of synthetic carbo-
hydrate chemistry. He then continued his
Postdoctoral studies at the University of
East Anglia (Norwich, UK; 2001–2005) with
Prof. Robert A. Field. He started his inde-
pendent career at CDRI, Lucknow (India) as
Scientist-C in 2005. In 2008, he moved to
IISER Kolkata (India) as Assistant Professor and became Associate
Professor in 2012. His research interests include the synthesis of
oligosaccharides, glyco-nanoparticles, and supramolecular chemis-
try of carbohydrates.
Figure 1. A) Explanation based on molecular orbital theory; B) explanation
based on the dipole moment theory; C) the thermodynamically and kineti-
cally controlled product.
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ly controlled product, whereas the equatorial isomer repre-
sents the kinetically controlled product (except for sugars with
axial 2-OH groups like d-mannose and l-rhamnose, where the
axial product is both kinetically and thermodynamically con-
trolled) (Figure 1 c).
2.3. Mechanistic Pathways
For proper, methodical chemical synthesis of glycosides, the
most important phenomenon involved is called glycosylation.
Chemical glycosylation is the coupling of the two monomeric
sugar units, with each other or with other aglycons, through
the formation of a new linkage, known as the glycosidic bond.
The linkage may be of different types depending on the link-
ing heteroatom. Herein, we primarily deal with O-glycosides.
However, other heteroatomic glycosides such as C, S, and N
glycosides are also widely known in literature. Complete cate-
gorization of chemical glycosylation as an S
N
1orS
N
2 reactions
was tedious, as each theory brought forth many evidences in
their support.
[29]
For a more generalized mechanistic pathway (Scheme 1),
the donor 7 is first pre-activated with a leaving group attached
to its anomeric hydroxyl group. The addition of an electrophilic
promoter activates the leaving group of the donor to form
complex 8. The next step initiates the formation of oxacarbeni-
um ion 9 in its flattened half-chair conformation. After this, nu-
cleophilic attack by acceptor 10 occurs, thereby leading to the
required glycoside formation 11. However, the attack of the ac-
ceptor can occur via two pathways, owing to the structural
property of the oxacarbenium intermediate. The attack of the
glycosyl acceptor from the bottom of the sugar ring leads to
the formation of alpha (a ) or 1,2-cis glycoside 11 b ; attack of
the glycosyl acceptor from the top of the sugar ring yields
beta (b) or 1,2-trans glycoside 11 a. Furthermore, manipulation
of the stability of the intermediate oxacarbenium ion remains
responsible for the anomerization of the compound.
[30]
Presence of a participating acyl group in the C-2 position
governs the stereochemistry of the glycosidic bond formed.
The oxacarbenium ion formed owing to the departure of the
leaving group further (Scheme 2) interacts with the acyl group
in the C-2 position to form the acyloxonium ion complex 14
by virtue of neighboring group participation.
[31]
This enables
the acceptor 10 to attack the anomeric carbon from only one
side, that is, the 1,2-trans side to form the 1,2-trans glycoside
15.
For the formation of 1,2-cis glycoside 5, the presence of
a non-participating group is required in the C-2 position of the
donor molecule. Thus, increasing the stability of the oxacarbe-
nium ion and its various electronic and steric factors may lead
to the formation of the more thermodynamically co ntrolled
product, caused by the action of the anomeric effect described
above (Figure 1 C).
Thus, starting from Michael’s vision of glycosylation, where
the glycosyl acceptor was converted into its corresponding
salt,
[12]
to Fischer’s use of hemiacetals as the donor,
[13]
we have
come a long way via Fraser-Reid’s idea of ‘armed–disarmed’
glycosylation.
[20b]
However, the details of the newer and the
more developed concepts in decoding the mystery behind the
mechanism of chemical glycosylations are beyon d the scope
of this Review.
[32]
3. Glycosylations
3.1. Glycosyl Halides
3.1.1. Glycosyl Bromides and Chlorides
In 1901, Koenigs and Knorr
[14]
and Fischer and Armstrong
[33]
in-
dependently in troduced the concept of glycosyl halides as
donors, where glycosyl chlorides and bromides were reacted
with alcohols in the presence of AgCO
3
or Ag
2
O. Since then,
there have been extensive studies on the preparation of glyco-
syl halides,
[34]
including the treatment of free sugar units with
AcBr
[35]
(AcBrAcOH),
[36]
treatment of per-acetylated sugars
with HBrAcOH
[37]
(BiBr
3
Me
3
SiBr),
[38]
and subsequent conver-
sion of the sugar hemiacetals under modified Mitsunobu con-
ditions with PPh
3
/N-halosuccinimide
[39]
or PBr
3
.
[40]
However, the
harsh conditions in the established protocols often cause diffi-
culties in extensive oligosaccharide synthesis, where acid-labile
protecting groups in substrates are a general phenomenon.
Scheme 1. Mechanistic pathway depicting glycosylation.
Scheme 2. Mechanism for the formation of 1,2-trans glycoside.
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Hence, in the search for more environmentally friendly and
greener methodologies, photocatalytic synthesis came into
vogue.
[41]
Likewise, while Mizuno and co-workers established
the photo-irradiative phase-vanishing method for a-glycosyl
bromide synthesis
[42]
by applying UV light (irradiation at
352 nm), Xue and co-workers introduced the more versatile
visible-light-mediated halide synthesis.
[43]
However, in the latter
case, an additional photocatalyst, tris(2,2’-bipyridyl)ruthen-
ium(II) chloride [Ru(bpy)
3
Cl
2
], was essential. Various protocols
for the activation of glycosyl bromides are in use for the syn-
thesis of 1,2-cis glycosides with the help of Ag salts (AgOTf,
Ag
2
CO
3
, AgClO
4
, etc.) or Hg salts [Hg(CN)
2
, HgBr
2
, HgCl
2
,
etc.].
[44]
The versatile use of Ag
2
O for glycosyl bromide activa-
tion was shown by Beejmohun et al. in the synthesis of mono-
lignol glucosides.
[45]
Owing to the restrictions offered by glycosyl halides in
terms of their moisture stability and handling, glycosylations
with glycosyl halides as donors require optimized low temper-
atures and inert conditions. Requirement of such conditions
was usually managed with various limitations. However, the
present century saw the emergence of room-temperature ionic
liquids (RTILs), which came as a ready solution for all of the
constraints. Malhotra and co-workers
[46]
implemented a series
of RTILs and molten halide salts for the glycosylation of ace-
tabromo-a-d-galactose with p-nitrophenol. By virtue of their
unique ionic combination and their probable role in the glyco-
sylation pathway, ILs holds the promise of being an extensively
used solvent and activator in the future. It has been shown
that the activation of glycosyl bromides neat in ILs in the pres-
ence of AgCO
3
gave b-products in moderate yields.
In terms of reactivity, glycosyl bromides have lower reactivity
than glycosyl iodides.
[47]
However, glycosyl bromides exhibit
less stability than glycosyl chlorides. In 2006, Oscarson and co-
workers reported the total synthesis of a tetrasaccharide O139
through the activation of glycosyl bromide over its corre-
sponding thioethyl glycoside acceptor by using AgOTf as the
promoter.
[48]
This selective activation process, in turn, marks
the initial steps towards more convenient one-pot oligosac-
charide synthesis. This method also promotes the selective ac-
tivation of glycosyl chlorides with the help of AgOTf in the
presence of thioglycosides.
[49]
Recently, Nitz and co-workers re-
ported the activation of unprotected glycosyl chloride donors
by using p-toluene sulfonylhydrazide and it has been proven
to be more reactive than its protected counterpart.
[50]
3.1.2. Glycosyl Iodides
The limited shelf-life of glycosyl iodides has always rendered
them disadvantageous as donors for glycosylation. This restric-
tion requires the in situ formation of glycosyl iodides and their
subsequent involvement in glycosylation. In this context, many
research groups have demonstrated reliable methods for gen-
erating glycosyl iodides from per-O-acetylated sugars through
treatment of trimethylsilyl iodide (TMSI)
[51]
or HI equivalents.
[52]
The instability of TMSI was addressed by Koreeda and co-work-
ers who utilized the possibility of the synthesis of anhydrous
HI by the reaction of solid iodine with a thiol component in an
organic solvent.
[52a]
In line with this, Field and co-workers also
generated TMSI in situ by reacting hexamethyldisilane (HMDS)
with iodine.
[53]
The same group further modified their estab-
lished procedures in 2004, by presenting the synthesis of per-
O-acetylated glycosyl iodides 16 from unprotected free sugars
15 in significant yields (Scheme 3).
[54]
This protocol, equipped
with a no-solvent system and inexpensive reagents, prove d
a versatile method, enabling the effective formation of glycosyl
iodides.
Field and co-workers later studied the stability, reactivi ty,
and characterization of glycosyl iodides as donors.
[55]
Over ex-
tensive studies, it has been observed that, although glycosyl
iodides are usually less effective for glycosylations, many re-
search groups have illustrated its unique advantages over
other glycosyl halides in terms of efficiency and stereospecific-
ity.
[56]
Thus, by using these glycosyl iodide donors, various gly-
cosides have been synthesized . Here, the work of Gervay-
Hague and group deserve special mention. They have illustrat-
ed how glucosyl, galactosyl, and mannosyl iodides can react
with oxa (20) and thio cycloalkanes to yield O-glycosides, 21
and 22, respectively, with high b-selectivity (Scheme 4).
[57]
They
also demonstrated the selectivity with decreasing temperature,
where the b-isomer was dominant at lower temperatures
(Table 1).
[58]
The most noteworthy point in this study was the
realization that reactions did not require pre-activation of the
donor, which is exclusive for glycosyl iodides.
Glycosyl iodides can also be selectively activated in the pres-
ence of other donor systems. In this respect, Gervay-Hague
and co-workers demonstrated iterative oligosaccharide synthe-
Scheme 3. Preparation of glycosyl iodide from unprotected free sugars.
Scheme 4. Synthesis of oxacycloalkane with glycosyl iodides.
Table 1. Dependence of b-selectivity with decreasing temperature.
No. Donor Temp [8C] Time Yield [%] a/b ratio
11840 2h 82 1:2
2 18 0 5 h 76 1:5
31860 3 days 76 1:29
4 19 40 5 min 83 1:4
5 19 0 30 min 74 1:9
61960 12 h 73 1:50
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