glycosylation methods in oligosaccharide synthesis
TRANSCRIPT
GLYCOSYLATION METHODS
IN
OLIGOSACCHARIDE SYNTHESIS
by Inmaculada Robina
Department of Organic Chemistry. University of Seville
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GLYCOSYLATION METHODS IN OLIGOSACCHARIDE SYNTHESIS
Introduction
Glycoconjugates are biopolymers formed by an oligosaccharide moiety joined to a protein
(glycoproteins) or to a lipid moiety (glycolipids). These biopolymers together with proteins and
nucleic acids are mainly responsible of information transfer between cells, which is a
fundamental process of life and central to all cellular systems.
Nowadays it is well known that complex oligosaccharides in the form of glycolipids and
glycoproteins are present in the membranes of cells and can mediate a large number of diverse
and important biological functions. Oligosaccharides play a major role in inflammation, immune
response, metastasis, fertilization and many other important biomedical processes. Specific
carbohydrates cover different kinds of functions. For instance, they act as markers of certain
types of tumours, other act as signal molecules of symbiotic processes such as the symbiosis
between Rhizobium bacteria and legume plants; others are binding site for bacterial and viral
pathogens, etc…
The area of organic chemistry that deals with the study, preparation and biological role of
sugars, from monosaccharides to complex oligosaccharides and their analogues, is called
Glycobiology.
The important role of carbohydrates in Biology and Biomedicine has been a major incentive
for devising new methods for the chemical and enzymatic synthesis of this class of molecules.
The biological role of sugars depends on many factors. Compared with other biopolymers
such as nucleic acids, proteins and peptides, in which their biological activity depends on their
sequence of nucleotides or amino acids, in the case of oligosaccharides, the situation is more
complex. For oligosaccharides, besides the sequence of the monomeric structures, other aspects
such as the functional groups and their stereochemistry, the conformation of the sugars
ramification, the stereoselective formation of glycosidic linkages, etc… must be considered.
All these facts have made the area of oligosaccharide synthesis an ideal and challenging area
for the development and testing new synthetic methodologies.
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This course is divided in three lessons:
1. General Aspects of Oligosaccharide Synthesis
2. Different Procedures of Glycosylation Reactions by Direct Activation
3. Synthetic Strategies for the Assembly of Oligosaccharides
Bibliography (Books)
1.- Preparative Carbohydrate Chemistry, Ed. Stephen Hanessian. University of
Montreal, Canada. Marcel Dekker, Inc. New York, 1997
2.- Carbohydrate Chemistry, Ed. G. –J. Boons, Blackie Academic Professional,
1998
3.- Modern Methods in Carbohydrate Syntheses, Eds. S. H. Khan and R. A.
O`Neill. Haword Academic Press, 1996
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Lesson 1. General aspects of oligosaccharide synthesis
1. Formation of a glycosidic bond 2. General mechanistic pathway for glycosidic bond formation 3. Choices, challenges and problems of the glycosidic bond 4. Structure and reactivity of glycosyl donors and of glycosyl acceptors used in
oligosaccharide synthesis 5. Promoters, solvents and experimental conditions 6. Anomeric control in chemical glycosylations. Methods for stereoselective formation of
glycosidic linkages. 6.1. Preparation of 1,2-trans-glycosides by neighbouring group participation 6.2. In situ anomerization for the synthesis of α-glycosides (Lemieux) 6.3. Heterogeneous catalysis (Paulsen). 6.4. Stereoselective preparation of α- and β-glycosides by participation of the solvent 6.5. Intramolecular aglycone delivery approach
7. Common protecting groups used in oligosaccharide synthesis
1. Formation of a glycosidic bond
This bond is formed by a nucleophilic displacement of a leaving group (X) attached to the
anomeric carbon of a sugar moiety by an alcohol ROH, or by the OH group of a partially
protected sugar moiety. The compound that “gives” the glycosyl moiety, is called the glycosyl
donor, and the alcohol that receives it, is known as glycosyl acceptor. The reaction generally is
performed in the presence of an activator called “promoter”. The role of the promoter is to assist
the departure of the leaving group. Promoters are often used in catalytic amounts, although in
some instances they are used stoichiometrically. In some cases, other additives such as
molecular sieves or any base that may act as acid scavenger are used.
There are many methods available for glycosidic bond formation. In this course, we will
discuss the most important and the widely applicable ones.
The synthesis of disaccharides and oligosaccharides in general, involves the linking of two
polyfunctional compounds. It is much more complicated than the synthesis of other biopolymers
OX
ORG
O
RG
OOR'HO+
OOR'O
promotersolvent
glycosyl donor(electrophile)
glycosyl acceptor(nucleophile)
OX
ORG
O
RGHO-R'+ OR'promoter
solvent
Scheme 1
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such as peptides or nucleic acids because of the greater number of possibilities for the
combination of monomeric units and because the glycosidic linkages have to be introduced in a
stereospecific way.
2. General mechanistic pathway for glycosidic bond formation1
The General Mechanistic Pathways for Glycosidic Bond Formation is represented in Scheme
2. Over 90% of all the glycosylations reported, formally proceed via this general mechanistic
pathway. There are some exceptions such as in situ anomerization, intramolecular aglycon
delivery and the use of additives such as acetonitrile, which appears to react at the anomeric
center itself. These reactions will be discussed later on.
The timing of events heavily depends on the structures of the glycosyl donors, acceptors and
promoters. If the productive glycoside forming reactions proceed too slowly, numerous side
reactions imply the degradation of the labile glycosyl donor. However, under more vigorous
conditions, the acceptors can be also destroyed.
OX
OR
G
GO
OR
GH
OA
O
OR
OAG
O
RO O A
G
OX
O
R
O
GO
O O
R
GH
OA O
O
R
O
O AG
O
O
RO
O A
GO
O O
R
G
HO
A
OA
O
O O
R
promoterδ β
α
R = Non-participating group (benzyl, azido, etc.)
β
α
minor
major
A-OH = Glycosyl acceptor (A = Aglycone)
Glycosyl Donor
promoterβ
α
CO-R = participating group(R = alkyl, aryl, etc.)
βmajor
Glycosyl Donor
minor
orthoester(reversible)
(*)
Scheme 2
(*) Participation of the solvent has a strong influence on the stereoselectivity (See, p. 15)
3. Choices, challenges and problems of the glycosidic bond
The success of a coupling reaction between two sugars depends on the reactivity of the donor
and acceptor, on the promoter, on the kind of substituents on both saccharide units and, of
1 Barresi, F.; Hindsgaul, O. “Glycosylation methods in oligosaccharide synthesis” Modern Synthetic Methods, 1995, 7, 281-330.
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course, on the preferred selectivity of the reaction towards the α- or the β-anomeric form. The
experience of the person conducting the experiment also plays a role.
If we take the synthesis of a simple trisaccharide molecule as a target we can enumerate the
choices, challenges and potential problems listed in the following.
O
XROZ
OYHO
Z+
promotersolvent
ORO
Z
OYO
Zβ-linkage
ORO
Z OYO
Zα-linkage
or
Manipulate if needed
ORO
Z O
X
OZ
OYHO
Z+
promotersolvent
ORO
Z OO
Z OYO
Z
ORO
Z OO
Z
OYO
Z
α,α-linkage
α,β-linkage
or
X = leaving group R = protecting group
Y = potential leaving group
Z = participating or non-participating group
Scheme 3
Choices
1.- Choice of X and Z in the donor
2.- Choice of Y and Z in the acceptor
3.- Choice of the promoter or catalyst
4.- Choice of solvent and temperature
5.- Choice of protecting groups
Challenges and problems
1.- Anomeric selectivity for 1,2-cis or 1,2-trans linkages.
2.- Site selectivity and reactivity of acceptor OH groups (e.g. axial, equatorial, primary; D-
gluco, D-galacto, C-3, C-4, or others).
3.- Configuration, substituent, steric and electronic effect in the donor and acceptor (e. g. D-
glucopyranosyl and D-galactopyranosyl donors with identical substituents sometimes give
different α/β ratios with the same alcohol acceptor).
4.- Stoichiometry relative to the ratio donor:acceptor equivalents.
5.- Selective activation of anomeric groups (if X, Y are orthogonal groups that is have
different reactivities), Y can be activated in the presence of X.
6.- Iterative glycosylation in a stepwise manner or by block synthesis
7.- Minimum manipulation of protecting groups
8.- Prospects for solid-phase oligosaccharide and automated synthesis
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4.- Structures and reactivity of glycosyl donors and of glycosyl acceptors used in
oligosaccharide synthesis.
Structures of glycosyl donors
There are numerous glycosylation methods involving different glycosyl donors. The name of
the glycosylation method generally reflects the functionality of the glycosyl donor except for the
Fischer glycosylation that uses reducing sugars and the Köening-Knorr procedures that use
glycosyl halides as donors.
OL
(L = F, Cl, Br)Glycosyl halides
O
Trichloroacetimidates
OSR
Thioglycosides
CCl3
NH OSeAr
Selenoglycosides
OS
Glycosyl xantateS
SEtO
S
Glycosyl sulphoxide
Ar
O O
O
1,2-epoxide
OO
Glycosyl phosphorous(R = Alkyl, O-alkyl,X = O, S, lone pair)
PX
RR
OOH
Reducing sugars
OO
Pentenoyl Glycosides
O3
O
NN
Anomeric diaziridines
OO
Pentenyl Glycosides
OO
Anomeric acetateO
OO R
vinyl glycosides(R = H, Me)
O
Orthoester(R = OR', SR', CN)
OO
R
O
Glycals
3
Fig. 1 Structure of glycosyl donors used in oligosaccharide synthesis.
As a rule it is difficult to predict which glycosylation method will be the most suitable to
solve a certain problem. Nevertheless, there are some factors influencing the reactivity of
glycosyl donors that should be taken into account and that can be further used in the
optimization of an oligosaccharide synthesis.
Reactivity of Glycosyl Donors
The reactivity at the anomeric center depends to a large degree on the choice of the
protecting groups specially those on C-2. Glycosyl donors are then classified in two main
groups: armed donors (with an ether group on C-2) more reactive than disarmed donors (with
esters, amides on C-2).
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Ester groups induce some positive charge at the anomeric
center making the formation of the oxonium ion a slower
process.
When identical protecting groups patterns are desired,
reactivity may be controlled by different leaving groups.
Both the nature of the heteroatom X and substituent G of the leaving group will affect the
reactivity. The configuration of the glycoside also influences its reactivity. Another element of
control occurs via the use of different promoters P for leaving groups activation. Finally,
sterical/torsional factors also have an influence. Fused rings resist flattening of the pyranose ring
during oxonium ion formation). As examples, butanodione and ciclohexanedioneacetals (BDA
and CDA methodologies) on C-3 and C-4, also reduce reactivity.
A modern glycosyl donor must has the following characteristics:
Accessibility, high stability toward protecting group manipulations and mild activation
conditions.
Reactivity of Glycosyl Acceptors
With regard to the reactivity of the acceptor, this depends on the nucleophilicity of the
hydroxyl groups in partially protected carbohydrates that in turn depends on their nature (1º
more reactive than 2º), their spatial orientation (equatorial more reactive than axial), the
conformation of the sugar ring (4C1 or 1C4) and the presence of other protecting groups in the
molecule.2 It can be generalised that electron-withdrawing groups diminish the reactivity of the
acceptor. In addition, the steric hindrance of the groups has an influence i.e. bulky groups at C-6
such as OTBDPS or OTBDMS or OPiv reduce the yield of a 1→4 glycosylation to a large
extent.
5.- Promoters, Solvents and Experimental Conditions.
The nature of the promoter, generally a Lewis acid, has an influence in the sense that it
favours the departure of the leaving group. In addition, its nature classifies the reactions as
homogenous and heterogeneous and this has implications for the stereochemistry.
The solvent also has an influence on the overall rate of the process and on the stereochemistry,
especially in the case of non-participating glycosyl donors. Anhydrous solvents are required to
avoid competition from water. Solvents of low polarity, such as dichloromethane or ether are
frequently used. Sometimes polar aprotic solvent such as acetonitrile or nitromethane are used.
2 a) “Relative reactivities of hydroxy groups in carbohydrates”, Haines, A. H. Adv. Carbohydr. Chem Biochem. 1976, 33, 11-109. b) “Modulation of the relative reactivities of carbohydrate secondary hydroxyl groups. Modification of the hydrogen bond network”. Moitessier, N.; Chapleur, Y. Tetrahedron Lett. 2003, 44, 1731-1735.
OX-G
OBz
OSlowδ
OBz
OX-G
OBn
O
OBn
Fast
Fig. 2
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On the other hand, some solvents may also form complexes with the intermediate sugar
oxonium cations affecting the orientation of the incoming O-nucleophile. For example, diethyl
ether enhances the formation of α-glycosides while acetonitrile favours the accumulation of β-
anomers. This is explained by the formation of an exocyclic complex with the solvents that
hinder the β and α faces, respectively.
The influence of the combination promoter/solvents on the stereochemistry will be
commented later on.
O
BnOG
O
BnOG O
Et
Et
O
BnOG
MeN
α-glycosidation
β-glycosidation
Et-O-Et
Me-CN
Scheme 4
Experimental Conditions
The experimental conditions are very critical for the success of the reaction. Generally, the
use of extremely dry solvents, inert atmosphere and molecular sieves that can act as acid
scavenger are needed. Sometimes a non-nucleophilic base is also needed.
The order in which the reagents are added is also important in some cases.
The normal procedure of adding reagents (NP) is appropriate for less reactive disarmed
donors. The promoter (P) is added over a mixture of acceptor (A) and donor (D). For highly
reactive armed donors, the inverse procedure (IP) in which the donor is added over a mixture of
acceptor and promoter is the most convenient.
This can be rationalized as follows:
D + P + A
D.P
D.A
P.AP P
A D
APA
Decomp.
NP IP
Fig. 3
For a donor and acceptor with similar reactivities the NP procedure is commonly used. For a
termolecular reaction D + P +A, due to the nature of the reagents the reaction is expected to
occur through an association D.P and then interaction with A to obtain disaccharide D.A. For
highly reactive donors this strategy is less successful because the donor can decompose in the
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presence of P before interacting with A. The IP procedure in which the complex A.P is first
formed and then reacts with the donor, solves the problem.
Example:
Schmidt, R. R.; Toepfer, A. Tetrahedron Lett. 1991, 32, 3353.
O
AcOAcOAcO
AcO O
N3
HO
OBn
OTBSO
O
Me
BnOOBnOBn
CCl3
NH
O
O
AcOAcOAcO
AcO O
N3
OBn
OTBSO
O
O
OBnOBn
BnO
Me
Et2O, TMSOTfNP: 43%IP, 78%
Scheme 5
6. Anomeric control in chemical glycosylation. Methods for stereoselective formation of
glycosidic linkages
Types of anomeric linkages
The stereoselective introduction of the glycosidic linkage is one of the most challenging
aspects in chemical oligosaccharide synthesis. The anomeric linkages can be classified
according to the relative and absolute configuration at C-1 and C-2.
O
ZOR
O
ZOR O
ZOR
OH
ORH
O
HOR
OH
O
1,2-cis2-D-glycero
1,2-trans2-D-glycero
1,2-cis2-L-glycero
2-deoxy-glycosides
2-keto-3-deoxy-ulosonic acids
OZ
OR1,2-trans2-L-glycero
H
Fig. 4. Different types of glycosidic linkages
The 1,2-cis- and 1,2-trans-2-D-glycero series (allo-, gluco-, gulo- and galactopyranosides)
and the 1,2-cis and 1,2-trans-2-L-glycero series (altro-, manno-, ido- and talopyranosides). In
addition, some miscellaneous glycosidic linkages can be identified, including 2-deoxyglycosides
and 3-deoxy-2-keto-ulo(pyranosylic) acids.
6.1. Preparation of 1,2-trans-glycosides by neighbouring group participation
The nature of the protecting group at C-2 of the glycosyl donor is a major determinant of the
anomeric selectivity. A protecting group at C-2 that can perform neighbouring group
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participation (disarmed donors) during glycosylation will give 1,2-trans glycosidic linkages.
Nucleophilic attack of the alcohol at the anomeric center of the more stable oxonium cation 3
originated by participation of the neighbouring after departure of the leaving group X, results in
the formation of a 1,2-trans-glycoside 4. Glucosyl type donors will give β-linked products while
mannosides will give α-glycosides.
O
OXG
OR'
OH
OG
OR'
O
OORG
OR'
O
O O
R'
G
1 2 3 4
Scheme 6. Preparation of 1,2-trans-glycosides by neighbouring group participation
6.2. In situ anomerization for the synthesis of α-glycosides (Lemieux)
Lemieux and co-workers introduced this procedure in 1975 as a way of controlling the
anomeric selectivity in armed donors with non-assisting functionality at C-2. The reaction
conditions (e.g. solvent, temperature, and promoter) will determine the anomeric selectivity. The
in situ anomerization procedure results mainly in the formation of α-glycosides.
Scheme 7
Lemieux discovered that the α-haloglucopyranoside is in equilibrium with the more reactive
β-halide and that the equilibrium is catalysed by halide ions derived from tetraalkylammonium
halides, and the reaction proceeds with inversion of a highly reactive β-halide with the alcohol
component via nucleophilic substitution.
Scheme 8
This reaction is thought to proceed through several intermediates (Scheme 9).
At equilibrium the proportion of the α-halide is relatively high. The β-halide is less stable
because of the de-stabilization as a results of the anomeric effect but reacts more rapidly than
the α-halide with an O-nucleophile.
O
BnO Br
G ROH O
BnO OR
G major
Et4N Br
O
BnO Br
GO
BnO
BrG
1 2
Br
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Scheme 9. Preparation of α-glycosides by in situ anomerization
To allow substitution of the β-halide, the C-1-halide bond, in order to be broken, must be
antiperiplanar to the electron lone pair of the ring oxygen.3 To establish such an arrangement, a
conformational change to the highly reactive boat-like intermediate is required. This makes
reaction of the β-halide fast. In the case of the α-halide a conformational change is not required
since the C-1 halide bond is already anti-periplanar to the ring oxygen lone pair and the
substitution of the α-halide is slow. It is clear that the equilibrium rate must be fast enough to
ensure that sufficient β-halide is continuously present. If the difference in reaction rate between
the α- and β-halides with the alcohol is large enough, α-linked O-glycosides are obtained as
major compounds or exclusively.
The reaction requires very reactive glycosyl halides (armed) and long reaction times, in
particular when the originally tetra-alkyl ammonium bromides are used as catalysts.
The in situ anomerization procedure has proven to be very useful. The use of other liofilic
promoters such as mercuric bromide, silver perchlorate and silver triflate make it possible to
carry out the reaction with even less reactive halides. However, the stereoselective outcome of
the glycosylations is very dependent not only on the reactivity of the catalyst, but also on the
reactivity of both the halide and the acceptor. Careful adjustment of the reactivity of the two
different components is essential in order to obtain satisfactory results.
3 Kirby, A. J. The Anomeric Effect and Related Stereoelectronic Effects at Oxygen. Springer Verlag, Berlin, 1983
O
BnO
G O
BnO
G
O
BnO
ORGO
BnO OR
G
Et4NBr
7 8
O
BnO Br
GO
BnO
BrG
5 6
ROH
Br
ROHSlow
FastO
BnO
ORGO
BnO OR
G
O
BnOBr
G
ROHROH
O
BnO
G
Br
α-bromide β-bromide
β-glycoside α-glycoside
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6.3. Heterogeneous catalysis (Paulsen).
Glycosylation of α-halides in the presence of an insoluble silver salt proceeds mainly with
inversion of configuration and formation of the β-glycoside. In this case, the equilibration
between glycosyl halides is restricted because there is no nucleophile in the reaction mixture and
the reaction will therefore proceed with inversion of configuration. Silver silicate and silver-
silicate-aluminate have often been used as the heterogeneous catalyst. These catalysts have
proved to be valuable in the preparation of β-linked mannosides which can not be prepared by
neighbouring group participation or in situ anomerization.
OBnO
Br
GO
BnO
BrG O
BnOORG
9 10 11Ag
ROH
shielding α-face Scheme 10. Glycosylation by inversion of configuration
However, the method only works well with very reactive halides and sufficient reactive
alcohol components. With less reactive components, significant proportions of the α-isomers are
obtained. β-Glycosides from glucose, galactose or fucose can also be prepared by the Paulsen
method, but it is usually more convenient to come along with strategies involving neighbouring
group participation.
6.4. Stereoselective preparation of α- and β-glycosides by participation of the solvent
The choice of the combination promoter/solvent plays a crucial role for the anomeric
stereocontrol of a glycosylation, especially when a non-participating group is at C-2 position.
In general, if any participating group is present at C-2, the glycosylation reaction follows a
SN2 pathway in non-polar solvents. The influence of the solvent under SN1-type conditions has
been extensively studied for ethers and nitriles. O
BnOG
O
BnOG
α-glycosidation
O
BnO L+ ROH
Et-O-Etpromoter
Me-CNpromoter
OR
ORβ-glycosidation
(major compounds)
Scheme 11
Ethers such as diethyl ether or THF favour the α-linkage while with acetonitrile, β-glycosides
are commonly obtained.
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In diethyl ether, using strong acid promoters, the SN1-type reaction is favoured. Ethers
participate forming equatorial oxonium cations due to the reverse anomeric effect,4 which
favours thermodynamically α-glycosides.
The influence of nitriles, “The nitrile effect”, is more complex.5 Acetonitrile as polar solvent
favours an SN1 mechanism that implies the formation of an oxonium cation that is solvated with
preference at the α-face forming the kinetically controlled α-nitrilium-nitrile complex. This
complex finally renders the β-anomer by nucleophilic substitution by an alcohol.
On the other hand, the complex β-nitrilium-nitrile is thermodynamically more stable due to
the reverse anomeric effect, favouring the α-anomer. In any case, the complexation with the
nitrile increases the reactivity of the donor.
4 Lemieux, R. U. Pure. Appl. Chem., 1971, 25, 527. 5 Vankar, D.; Vankar, P. S.; Behrendt, M.; Schmidt, R. R. Tetrahedron 1992, 47, 9985
O
BnOG
O
BnOG
MeN
β-glycosidation
Me-CN
O
BnOG
L
O
BnOG Lpromoter promoter
O
BnOG OR
ROHO
BnOG
MeN
α-glycosidationROH
S
SS
S
kinetic control
O
BnOG
ORthermodynamic control
SN1
Scheme 12
Scheme 13
O
BnOG
O
BnO
OEt
EtG
Et-O-Et
O
BnO LG
O
BnOLG
O
BnO ORGROH
α-glycosidation
promoter promoter
reverse anomeric effect
SN1
- 16 -
“The Nitrile Effect”
Scheme 14
For quite some time, there has been controversy with respect to the absolute configuration of
the intermediate α-glycosyl nitrilium ion. Trapping the intermediate nitrilium ion by 2-
chlorobenzoic acid gave the corresponding amide with α-configuration, thus confirming α-
nitrilium ions.6
O
BnOG
MeN
O
BnOG
Me-CN O
BnOG O-R´R´-OH
O
BnOG
N
ClCOOH
Me
O
O Cl
O
BnOG
N
O ClAc
Scheme 15
Unfortunately this method gives low β-selectivity for mannosidases.
6 Ratcliffe, A. J.; Fraser-Raid, B. S. J. Chem. Soc., Perkin Trans I, 1990, 747.
- 17 -
6.5. Intramolecular aglycon delivery approach
This method has been applied with success to the synthesis of β-mannosides. In this method
the sugar alcohol (R´-OH) is first non-permanently linked to the C-2 position of a suitable
protected mannosyl donor via an acetal or silicon tether (Y = CH2 or SiMe2). Activation of the
mannose donor results in an intramolecular delivery of the alcohol in a concerted reaction
resulting in the formation of exclusively β-mannopyranosyl linkages.
OO
GO
HO
GR´-OHL
YOR´
L OO
G
YOR´
OO
G
YO R´
OHO
G OR´X-Y-X
Scheme 16 Examples:
Stork , G. and La Clair, J. J. J. Am. Chem. Soc. 1996, 118, 247.
OHO
S
BnOBnOBnO
PhO
O
NPhthBnOHO
OBn
OC8H17Me2SiCl2, imidazole+
DMAP 78%
OHO
BnOBnOBnO O
NPhthBnOO
OBn
OC8H17
OO
S
BnOBnOBnO
PhO
O
NPhthBnOO
OBn
OC8H17
Si
Tf-O-Tf
54%
Tf2O
Scheme 17
Barresi, F. Hindsgaul, O. J. Am. Chem. Soc. 1991,113, 9367 and Synlett 1992, 759.
OO
SEt
BnOBnOBnO
O
NPhthBnOHO
OBn
OC8H17+55%
OHO
BnOBnOBnO O
NPhthBnOO
OBn
OC8H17
OO
SEt
BnOBnOBnO
O
NPhthBnOO
OBn
OC8H17
51%
TsOH
I+
NIS
4-Me-DTBP
Scheme 18
- 18 -
8.- Common protecting groups used in oligosaccharide synthesis
Fig. 5
It is important to note, that in spite of the general approaches discussed above for
stereoselective control of the glycosidic linkage, other factors such as type of oligosaccharide,
leaving group at the anomeric center, protection and substitution pattern, promoter, solvent,
temperature, could have a major effect on the α/β selectivity.
It should be realized that there are no methods or strategies of general application for
oligosaccharide synthesis, which is one of its greatest difficulties. Nevertheless, convergent
multi-step synthetic sequences that give complex oligosaccharides consisting of up to 20
monosaccharide units are currently feasible by applying different strategies that will described
on Lesson 3.
- 19 -
Lesson 2. - Different procedures of glycosylation reactions by direct activation
1. Köenings-Knorr method and related. Glycosyl Fluorides (Mukaiyama) 2. n-Pentenyl glycoside method (Fraser-Reid) 3. S-Glycoside methods (Lönn, Garegg, van Boom) 4. Phenylselenoglycosides 5. O-Alkylation and the trichloroacetimidate method (Schmidt) 6. Glycosylation with glycals (Lemieux, Thiem, Danishefsky)
Introduction
From a chemical point of view, the synthesis of oligosaccharides still presents an important
challenge to synthetic chemists in spite of major advances in the area. In this lesson we will
briefly review the main synthetic methods available for glycoside bond formation. Although
some methods for glycoside synthesis are more popular than others, there is no universal
protocol that can be applied to any combinations of donors and acceptors without consideration
of their substitution patterns, configurations, or position of the hydroxyl groups. All the choices,
challenges and potential problems that have been commented on in Lesson 1, are mostly
applicable to the various glycosylation methods.
Strategies for the assembly of sugars will be discussed in the next lesson.
1. Köenings-Knorr and related methods.
The Köenings-Knorr method uses glycosyl bromides and chlorides as donors in the
glycosylation reaction. It was first performed in 1901 and up until the mid-1980s, the method
and its numerous variants have been extensively used to prepare a wide variety of O-glycosides.
Insoluble promoters such as Ag2O and Ag2CO3 were initially used. Soluble catalysts
including HgBr2 and Hg(CN)2 (Helferich-Weiss, 1956) and AgOTf (Hanessian-Banoub, 1977),
were exploited as promoters. In the latter case, the reactions were sometimes performed in the
presence of tetramethylurea as acid scavenger.
Examples:
Hanessian, H.; Banoub, J. Methods in Carbohydr. Chem. Vol. 8, Whistler, R. L.; BeMiller, J.
N. Eds. Academic Press, New York, 1980, 247.
O
BrAcOAcOAcOAcO
O
OMeAcHNHO
OO
Ph+ O
AcOAcOAcOAcO O
OMeAcHNO
OO
PhTfOAg, CH2Cl2Me2NCONMe2
82%(based on
consummed ROH) Scheme 1
- 20 -
Betaneli, V.; Ovchinnikov, M. V.; Backinowsky, Kotchekov, N. K., Carbohydr. Res. 1980,
84, 211-214.
O
Br
OAc
AcOAcOAcO
+ O
OOHO Me
O
OO
O
OO
Ph
Hg(CN)2
MeCN
O
OAc
AcOAcOAcO
O
OOO Me
O
OO
O
OO
Ph
81%(based on
consummed ROH)
O
OH
HOHOHO
O
OHHOO Me
O
HOO
OMe
HOHO
Scheme 2
In spite of the generality of the method there are several inconveniences that have limited its
use. The intrinsic instability of glycosyl halides, the requirement of at least an equimolar amount
(often up to 4 eq) of metal salts as promoters (frequently incorrectly termed as “catalyst”) and
problems concerning the disposal of waste material (e. g. mercury salts) have made the method
become less popular nowadays.
Other alternative methods of great interest have been developed.
1.1. Glycosyl fluorides (Mukaiyama)7
In 1981, Mukaiyama and co-workers introduced anomeric fluorides for the preparation of O-
glycosides. The introduction of fluorine as leaving group is a good alternative to the Köenings-
Knorr method due to the stability of the C-F bond. Glycosyl fluorides are easier to handle than
glycosyl chlorides or bromides. They are typically prepared from the anomeric acetates by
reaction with HF/py, from hemiacetals by reaction with DAST or from thioglycosides by
reaction with NBS/DAST.
Examples:
Hayashi, M.; Hashimoto, S.; Noyori, S. Chem. Lett. 1984, 1747.
OOAc
OBnBnOBnO
BnOO
BnOBnOBnO
BnOHF- py
-20ºC to 25ºC80%
F
α:β = 95:5
Scheme 3
7 For a review, Toshima, K. Carbohydr. Res. 2000, 327, 15-26.
- 21 -
Posner, G. H. Haines, S. R. Tetrahedron Lett. 1985, 26, 5-9.
OOH
OBnBnOBnO
BnOO
BnOBnOBnO
BnODAST, THF
-30ºC to 25ºC99%
F α:β = 1:7.7
Scheme 4
Nicolaou, K. C.; Dolle, R. E.; Papahatjis, D. P.; Randall, J. L. J. Am. Chem. Soc. 1984, 106,
4189.
OAcOAcO
AcONBS/DAST
CH2Cl2-0ºC to 25ºC
70%
100% α
SPh
OAcOAcO
AcO
F
Scheme 5
Because of the difference in halophilicity of this element compared with bromine and
chlorine, the glycosylation reactions require the use of other promoter systems besides silver
salts.
Mukaiyama and co-workers carried out the first reaction in 1981. In this case, 1,2-cis-α-
glycosides were predominantly obtained in high yields due to the anomeric effect.
Mukaiyama, T.; Murai, Y.; Shoda, S. Chem. Lett. 1981, 3, 431-432.
Scheme 6
Apart from SnCl2-AgClO4 (Mukaiyama, 1981), the following systems have been used:
TMSOTf (Hashimoto et al, 1984), BF3.Et2O (Kunz, 1985), Cp2ZrCl2-AgBF4 and Cp2HfCl2-
AgTfO/AgClO4 (Suzuki et al, 1989 and Mattheu et al, 1992), Cp2ZrCl2-AgClO4 (Matsumoto et
al, 1988), La(ClO4)3 (Kim et al, 1995 and LiClO4) (Böhm and Waldmann, 1995). The
promoters of wider application imply the use of lanthanide metals.
The glycosylations with anomeric fluorides follow the general principle as described for
bromides and chlorides. Apart from their enhanced stability, anomeric fluorides have not proven
to be superior to bromides or chlorides in terms of glycosylation efficacy.
- 22 -
Examples:
Mukaiyama, T.; Hashimoto, Y.; Shoda, S. Chem. Lett. 1983, 935-938.
Scheme 7
Takahashi, Y.; Ogawa, T. Carbohydr. Res. 1987, 164, 277-296.
Scheme 8
Wessel, H. P.; Ruiz, R. J. Carbohydr. Chem. 1991, 10, 901-910.
Scheme 9
Example:
In the total synthesis of NodRm-IV Factors:
Nicolaou, K. C.; Bockovich, N. J.; Carcanague, D. R.; Hummel, C. W. Even, L. F. J. Am.
Chem. Soc. 1992, 114, 8701-8702.
Nod Factors are the molecules signals involved in the symbiosis between legume plants and
bacteria of the genus Rhizobium. This symbiosis is responsible of the fixation of atmospheric
nitrogen in the roots of specific legume plants.
- 23 -
Structure and retrosynthetic analysis of Nod factors.
O FNPhth
PMBOPMBO
OTBDMS
O FNPhth
PMBOAcO
OMPO OMPNPhth
PMBOHO
OTBDMS
O
OH
O ONH
HOHO
OH
O ONHAc
HO
OH
O ONHAc
HO
OH
O OHNHAc
HO
OSO3-
O
a b c
d
Scheme 10
The key steps in the total synthesis imply glycosylation with glycosyl fluorides.
Scheme 11
Glycosyl fluorides are used together with thioglycosides in a double activation strategy. This
will be discussed in the next lesson.
2. n-Pentenyl glycoside method
This method, that uses pentenyl glycosides as glycosyl donors, was introduced by Fraser-
Reid in 1988. The activation of the leaving group is based on an electrophilic addition to the
double bond of the aglycone, followed by an intramolecular displacement by the ring oxygen
and eventual expulsion of the pentenyl chain to form an oxonium specie. Trapping with a
glycosyl acceptor, then leads to the desired glycoside.
O
NPhthPMBO
AcO
OMPO
NPhth
OBn
OMPOPBMOO
NPhthPMBO
AcO
OMP
FO
NPhth
OBn
OMPHOPBMO
N
O
NPhthPMBO
HO
OMPO
NPhth
OBn
OMPOPBMO
O
NPhthPMBO
AcO
OMP
O
NPhthPMBOO
OMPO
NPhth
OBn
OMPOPBMO
O
NPhthPMBO
AcO
OMP
F N
+ AgOTf, Cp2ZrCl2CH2Cl20º - 25º
56 %
AgOTf, Cp2HfCl2CH2Cl20º - 25º
60 %
NaOMe/MeOH
- 24 -
O
RG
E OO
RG
E E OO
RG
EO
RG
Sugar-OH
O
RG O-Sugar
Scheme 12
The promoter of choice is any source of halonium ion. NBS or NIS alone or activated by
Lewis acid. NIS/Et3SiOTf is commonly used. Sometimes TfOH is also used. When using
halosuccinimides alone, the reaction is very slow, and often requires hours or days for
completion. A promoter of intermediate potency is IDCP (iodonium dicollidone perchlorate).
OG
OG
X
OH promoter+
NPG Scheme 13
Scheme 14
Preparation of n-pentenyl glycosides (NPGs) may be carried out following standard
procedures for preparing alkyl glycosides, including Fischer or Koenigs-Knorr glycosylations
with 4-pentenol.
When using perbenzoylated glycosyl bromides, reaction with 4-pentenol gives n-pentenyl
1,2-orthoesters (NPOEs) which can also serve as glycosyl donors. NPOEs are transformed into
O
RO-SugarG
OO
OCOPh
G
O
PhOCOBrG
O
O O
PhO
G
H
I+
I+
O
RG
OH+ Sugar-OH
NPG
NPOE
2,6-lutidineBu4NI
- 25 -
NPGs through an acid-induced rearran-gement. The promoters of choice is NIS. Recently,8 an
efficient activation of NPOEs with NIS and lanthanide triflates (Yb(OTf)3) has been reported.
The advantage of using orthoesters is that they are stable to bases and so, several base-
promoted protecting group transformations can be carried out before the acid-induced
rearrangement that converts NPOE to NPG.
Basically, both donors proceed mechanistically in the same way. They generate the same
intermediate that leads to the oligosaccharide.9
Scheme 15
NPOEs have the advantage over NPGs of the high stereocontrol observed due to the effective
shielding of the α (for D-Man) and β (for D-Glc) faces. Thus the reaction of benzoyl bromides
with 4-pentenol gave the NPOEs that show a high stereocontrol in glycosidic linkage formation
shielding the β and α faces of D-mannose and D-glucose that lead to α- and β-glycosides,
respectively.
8 Jayaprakash, K. N.; Radhakrishnan, K. V.; Fraser-Reid, B. Tetrahedron Lett, 2002, 43, 6953-6955. 9 Macha, M.; Schlueter, U.; Mathew, F.; Fraser-Reid, B.; Hazen, K. C. Tetrahedron 2002, 58, 7345-7354.
- 26 -
Example: Macha, M.; Schlueter, U.; Mathew, F.; Fraser-Reid, B.; Hazen, K. C. Tetrahedron
2002, 58, 7345-7354.
Conditions: (i) PhCOCl, pyridine, DMAP; DCM; (ii) Ac2O, 30% HBr-AcOH(~85%); (iii) DCM, 2,6-lutidine, R′-OH or 4-
pentenol, Bu4NI; (iv) NaOMe, MeOH (89%); (v) NaH, BnBr, DMF (84%).
Scheme 16
Protecting groups influence the reactivity of pentenyl glycosides as donors. The so-called
armed-disarmed concept.
Example:
O OPentOBn
OBn
BnOBnOO OPentOAc
OH
AcO AcO+
Armed Disarmed
IDCP O
OBn
OBn
BnOBnO
O OPentOAc
AcO AcO
O
Scheme 17
Examples of glycosylations with NPOEs.
Recently, a strategy for Fully Inositol Acylated and Phosphorylated GPIs by the Synthesis of
a Malaria Candidate Glycosylphosphatidylinositol (GPI) Structure, has been reported using
NPOEs as donors.
Lu, J.; Jayaprakash, K. N.; Schlueter, U.; Fraser-Reid, B. J. Am. Chem. Soc. 2004, 126, 7540-
7547.
They are anchored to the cell membranes and are connected to proteins via a
phosphoethanolamine linker. Hundreds of GPI-anchored proteins have been identified in
organisms ranging from archeabacteria to humans. They occur in all mammalian cell types.
They have diverse functions, including hydrolytic enzymes, adhesion proteins, complement
regulatory proteins, receptors, prion proteins, and antigens.
- 27 -
Retrosynthetic analysis:
O OO
HOHO
HOPh
OBnOBn
OOHOBnO
V IV III II I
1 2
(Manα1) 2Manα1 2Manα1 6Manα1 4GlcNH2α1 6myoIno
D-mannose
myo-inositol
I
II
III
IV
V
O
OH2N
HOO
HO
OHOH
OCOR1HO
OOH
HOHO
O
OHOHO
HO
OOH
HOHO
OP
OOBn
O NH2
R2 O
O
O
OR3
O
PO
OH
O
O
Protein
Scheme 18
Synthesis:
O OO
BnOBnO
TBDMSOPh
O
ON3
BnOO
BnO
OBnOBn
OO
BnO
OOBn
BnOBnO
HO
O
O
ON3
BnOHO
BnO
OBnOBn
OBnO
I
II
III
O OO
BnOBnO
BnOPh
O
ON3
BnOO
BnO
OBnOBn
OO
BnO
OOBn
BnOBnO
O
OOR
BnOBnO
OBn
I
IIII
I
I
II
III
IV
O OO
AcOAcO
TrOPh
O
ON3
BnOO
BnO
OBnOBn
OO
BnO
OOBn
BnOBnO
O
OBnOBnO
BnO
I
II
III
IVO
OOBz
AcOAcO
TrOV
I
II
III
IV
V
O
OH2N
HOO
HO
OHOH
OCOR1HO
OOH
HOHO
O
OHOHO
HO
OOH
HOHO
OP
OOBn
O NH2
R2 O
O
O
OR3
O
PO
OH
O
O
O
O
OBnOPMBO
BnOPh
O
OBnOBn
OO
BnO1
, NIS/BF3.OEt2
(i)
(ii) manipulation of protecting groups
, NIS/BF3.OEt2
R = BzR = HNaOMe/MeOH
(i) deprotection(ii)introduction of aminophosphate moiety(iii)deprotection(iv)introduction of the fatty acid(v) reaction with glycerylphosphoamidite
(vi) reduction
Protein
R1, R2, R3 various fatty acyl groups
98%
(i) protection(ii) 2, NIS/Yb(OTf)3,
(i) change of Bz to Tf(ii) N3TMS(iii) deprotection
(i)
(ii) manipulation of protecting groups
, NIS/BF3.OEt2
Scheme 19
- 28 -
3. - S-Glycoside methods
There are several methods in which the anomeric carbon is activated by groups having
sulphur in place of the exocyclic hemiacetal oxygen. The best known example of this type of
protection/activation group is the alkyl(aryl)thio group (thioglycosides). Oxidized forms of
thioglycosides, such as sulfoxides can act as glycosyl donors as well as other derivatives like S-
xantates. We will focus our attention mainly on thioglycosides. Glycosyl sulfoxides will also be
considered.
3.1. Thioglycosides
The sulfur atom in a thioglycoside is a soft nucleophile and is able to react selectively with
soft electrophiles suchs as heavy metal cations, halogens, and alkylating or acylating reagents.
This fact make thioglycosides very versatile agents in carbohydrate chemistry. Additionally, the
hydroxy and ring oxygen atoms of carbohydrates are hard nucleophiles, which can be
functionalized with “hard” reagents, without affecting alkyl(aryl)thio function.
O OHHO
O SRHOO SRR'O
O OR"R'OR"OH
promoter
Scheme 20
An electrophile activates the thioglycoside by producing intermediate sulfonium ions, which
then give rise to glycosylating carbocationic intermediates that react with the alcohol giving the
glycoside.
O SR'OOBn
ER
O SR'OOBn
R
EO
R'O
OBn
OOR'O
OBnR
O SR'OO
ER
O SR'O R
EO
R'OO OR'O R
R
OO
R
O O O
R
ROH
ROH
O
R
O
Scheme 21
Although this possibility was known for a considerable time (Bonner, 1948; Ferrier, 1973), it
has been since 1984 that it has been extensively explored.
In 1984 Lönn first reported the use of methyl triflate as the first efficient general promoter for
direct glycosylation with thioglycosides. MeOTf has disadvantages because it is toxic and in the
- 29 -
presence of slow reacting glycosyl donors, it can give rise to methyl ethers in addition to
glycosides. For this reason, other thiophilic promoters have been developed.
For example dimethyl(methylthio)sulfonium triflate, DMTST (Fugedi, Garegg, 1986),
NOFB4 (Pozsgay, Jennings, 1987/88), MeSOTf, MeSBr (Dasgupta, Garreg, 1988), PhSeOTf
(Ogawa, 1989), MeI (Reddy, 1989), NIS, TfOH (van Boon, Konradsson, 1990), IDCP
(Veeneman, van Boom, 1990), TBPA (Sinaÿ, 1990).
N MeMeI ClO4
IDCP
MeS
S
MeMe OTf
DMTST
Br NH3
SbCl6
TBPA
N MeMeI TfO
IDCT
Fig. 2
Iodonium dicollidine perchlorate (IDCP) is better replaced by iodonium dicollidine triflate
(IDCT), which has similar reactivity and which does not require the use of AgClO4 in its
synthesis. MeOTf, DMTST, NIS-TfOH and in particular PhSeOTf are all most efficient
promoters that produce fast reactions. Tris(4-bromophenyl)ammoniumyl hexachloroantimonate
(TBPA) differs from others in that its cation is radical, and as such produces radical cationic
sulfonium ions as glycosylating species from thioglycosides.
Regarding stereochemistry, the glycosylations with thioglycosides follow the general
principle as described for bromides and chlorides.
With regards to the preparation of thioglycosides, they can be grouped into three categories:
A. Acid-promoted Displacement at the anomeric center. This implies the synthesis from a
sugar derivative of a thiol in the presence of a Lewis acid.
Example: Ferrier, R.; Furneaux, R. Methods, Carbohydr. Chem. 1980, 8, 251.
O OAcOAc
AcOAcO
OAcO SPhOAc
AcOAcO
OAcPhSHBF3/Et2O
71% Scheme 22
B. Base-promoted Displacement at the Anomeric Center. This implies the synthesis by S-
nucleophilic displacement at the Anomeric Center
Example: Tropper, F.; Andersson, F.; Grandmaitre, C.; Roy, R. Synthesis, 1991, 734.
O
BrAcO
AcOAcO
OAcO SPhOAc
AcOAcO
OAcPhS Na
Phase transfer catalysis
81% Scheme 23
- 30 -
C. Synthesis by preparation of a 1-thioglycoside followed by S-alkylation. Once prepared
the 1-thioglycoside, it is alkylated with an alkyl halide, often in situ. Although the total
number of steps is higher, the reagents are cheap and the yields are high throughout.
Example: Horton, D. Methods in Carbohydr. Chem. 1963, 2, 433.
O
BrAcO
AcOAcO
OAcO SOAc
AcOAcO
OAc
80%
H2N NH2
S
acetoneNH2
NH2 Br
O SHAcO
AcOAcO
OAc
K2CO3 aq.
O SMe
AcOAcOAcO
OAcMeI
Diisopropylethylamine
87%
100%
Scheme 24
There are many examples of glycosylations with thioglycosides.
Example: The synthesis of part of the carbohydrate structural component of a glycoprotein
isolated from fucosidosis patients. Lönn, H. Carbohydr. Res. 1985, 139, 115-121.
O SEtNPhth
O
OAc
O OBn
OBnBnO
Me
OOAcAcO
AcO OAcO
O
OBnOBn
BnO HOO
OBn
OBn
O
O
BnO
OOBn
OBnOBn
HO
O
OBnOBnBnO
OO
OBn
OBn
O
O
BnO
OOBn
OBnOBn
OO
NPhthO
OAc
O OBn
OBnBnO
Me
O
OAcAcO
AcO O
AcO
O
NPhthO
OAc
O OBn
OBnBnO
Me
O
OAcAcO
AcOO
AcO
MeOTf, Et2O61%
β-D-Galp(1 4)
α-L-Fucp(1 3)β-D-GlcpNAc(1 2)
α-L-Fucp(1 3)
β-D-Galp(1 4)β-D-GlcpNAc(1 2)
α-L-Manp(1 6)
α-L-Manp(1 3) D-Man
Scheme 25
- 31 -
Protecting groups influence the reactivity of thioglycosides:
Veeneman, G. H.; van Boom, J. H. Tetrahedron Lett. 1990, 31, 275
Scheme 26
3.2. Sulfinil glycosides: the sulfoxide method
The use of glycosyl sulfoxides as glycosyl donors, provides a new and powerful method for
chemical glycosylations, where a glycosyl sulfoxide (also called sulfinil glycosides) reacts with
a glycosyl acceptor in the presence of a promoter, to give a di- tri- or oligosaccharide.
O
S Ph
OO OGHO
O OGOpromoter+
promoters: Tf2O, TMSOTf, TfOHacid scavenger: DTBMP
O
Scheme 27
The promoter systems for these sulfinil glycosides are triflic anhydride (Tf2O) or
trimethylsilyl triflates in stoichiometric amount or triflic acid in catalytic amount. The reaction
is always carried out in the presence of an acid scavenger (diterc-butyl methyl pyridine).
Daniel Kahne first developed this method and was able to glycosylate very unreactive
hydroxyl groups as the C-7 hydroxyl group in a deoxycholic acid derivative.10 He used two
types of glycosyl donors with non-participant and participant protecting groups. Yields are good
with non-polar solvents. In the absence of a neighbouring group, the stereochemical outcome of
10 Kahne, D.; Walker, S.; Cheng, Y.; Van Engen, D. J. Am. Chem. Soc. 1989, 111, 6881-6882.
O SEt
OBnBnO
OBn
BnO
O SEt
OBzBzO
OBz
HO
+armed
disarmed
O
OBnOBnO
OBn
BnO
O SEt
OBzBzO
OBz
disarmed
IDCP
91%
O
OBnO
BnO
OBn
BnOO SEt
OBnBnO
OBn
armed
1. NaOMe2. NaH/BnBr/Bu4NI
O SEt
OBzBzO
OBz
HO
IDCP72%
disarmed
O
OBnOBnO
OBn
BnO
O
BnOBnO
OBn
OSEt
OBzBzO
OBz
O
- 32 -
the reaction is strongly influenced by the solvent: The yield of the β-glycoside increases with the
polarity of the solvent (nitrile effect). With a C-2 participating group, the final product is all β.
Me
OH
Me
EtOCO
COOMeMeO
SOBn
BnOBnO
OBn
Ph
O
OS
OPivPivOPivO
OPiv
Ph
O
Glycosyl acceptor Glycosyl donor Conditions Product ratio (yield)
dichloromethane all β (83%)
toluene α:β = 27:1 (86%)
CH2Cl2 α:β = 1:3 (80%)
acetonitrile α:β = 1:8 (50%)
Scheme 28
The sulfoxide-glycosylation method is highly efficient with rather unreactive nucleophiles,
has potential for chemoselective glycosylations and is applicable to the synthesis of
oligosaccharides on solid supports. However, the highly reactive donors used in this method
make it impractical in some cases due to their decomposition.
One advantage of the sulfoxide method is its flexibility and wide scope. It has been
demostrated that using a standard set of conditions, it is possible to construct families of
oligosaccharides. As an example, the syntheses of the Lewis blood group of antigens: Lewis a,
Lewis b and Lewis x (Lea, Leb and Lex).
Example: Yan, L.; Kahne, D. J. Am. Chem. Soc. 1996, 118, 9239-9248.
The synthesis of Lea begins at -78° C with the coupling of sulfoxide 1 and acceptor 2, the
promoter is triflate anhydride and di-tercbutylmethylpyridine as acid scavenger.
O
SPhN3
HOOOPh
O
OPivPivO S
Ph
OOPivPivO
O
OPivPivO
OPivPivOOO
O
SPhN3
PhO
O
OAcAcO
OAcAcOOHO
SPhN3
O
PivO
O
OAcAcO
OAcAcOO
SPhN3
O
PivOO
S
Me
OBnOBn
OBn
O Ph
OMeOBn
OBn
OBn
O
O
OHHO
OHHOO
AcHNO
HOOMeOH
OH
HO
OOMe
Tf2O
DTBMPCH2Cl2, -78°
Lea
+
+ Tf2O
DTBMPCH2Cl2, -78°
α(1→4)
65 4
1 2 3
β(1→3)
83%
95%
Lea
Scheme 29
- 33 -
A β(1→3) glycosidic bond is formed. The same reaction conditions were used in the
coupling of acceptor 4, obtained after normal manipulation of protecting groups, and
fucosylsulfoxide 5. An α(1→4) glycosyl bond is now formed. Subsequent transformation gives
the final molecule.
O
SPhN3
HOOOPh
O
OPivBnO S
Ph
OOBnBnO
O
OPivBnO
OBnBnOOO
O
SPhN3
PhO
O
OHBnO
OBnBnOOHO
SPhN3
O
PivO
O
OBnO
OBnBnOO
SPhN3
O
PivOO
S
Me
OBnOBn
OBn
O Ph
OMeOBn
OBn
OBn
O
O
OHO
OHHOO
N3O
HOOMeOH
OH
HO
OOMe
O OBnOBnBnO
Me
O OHOHHO
Me
Tf2O
DTBMPCH2Cl2, -78°C
α(1→4)α(1→2)
Tf2O
DTBMPCH2Cl2, -78°C
+
+
Leb
10
5
27
β(1→3)
8
9
77%
82%
Leb
Scheme 30
For the synthesis of Leb, the same reaction gave the β(1→3) linked disaccharide 8, that was
transformed into acceptor 9 with two unprotected hydroxy groups. Double glycosylation with
fucosylsulfoxide 5 gives tetrasaccharide 10 with two new α(1→4) and α(1→2) linkages.
Subsequent transformations gave the final compound. The yields are always very good, from 77
to 95%.
Lex contains the same three sugars as Lea but they are linked in a different manner: the
position of galactose and fucose are reversed. The first coupling reaction, with the formation of
a β(1→4) linkage, proceed in a slightly lower yield, probably because the HO-4 is greatly
hindered by the pivaloyl and para-methoxybenzyl groups. The reaction of 5 and 13, under the
same conditions, gave the α(1→3) new bond.
- 34 -
O
SPhN3PMBOHOPivO
O
OPivPivO S
Ph
OOPivPivO
O
PivOPivO
OPivPivO
O
SPhN3
O
S
Me
OBnOBn
OBn
O Ph
PMBOO
PivO
O
AcOAcO
OAcAcO
O
SPhN3
HOO
AcOO
AcOAcO
OAcAcO
O
SPhN3
O
AcO
O
O OBnOBnBnO
Me
O
HOHO
OHHO
O
NHAc
O
HO
O
O OHOH
HO
Me
OMe
14
+
+ Tf2O
DTBMPCH2Cl2, -78°C
α(1→3)
Tf2O
DTBMPCH2Cl2, -78°C
5
112
12
β(1→4)
Le x
13
65%
83%
Lex
Scheme 31
4. Phenylseleno glycosides
Anomeric phenylselenides are interesting glycosyl donors. The phenylseleno substituent
behaves largely like thioglycosides with respect to stability towards protecting group
manipulations and lability towards electrophilic reagents.
Scheme 32
Phenylseleno glycosides are more reactive than thioglycosides allowing chemoselective
glycosylations.
O OHHOO SeRHO
O SeRR'OO OR"R'O
R"OHpromoter
O SeR'OOBn
ER
O SeR'OOBn
R
EO
R'O
OBn
OOR'O
OBnR
O SeR'OO
ER
O SeR'O R
EO
R'OO OR'O R
R
OO
R
O O O
R
ROH
ROH
O
R
O
- 35 -
Example: Mehta, S.; Pinto, B. M. J. Org. Chem. 1993, 58, 3269-3276
O
OBnBnO
BnOOBn
SePhO
OH
BnOBnO
OBnSEt
O
OBnBnO
BnOBnOO
OBnOBnO
OBnSEt
+
O
OBzBzO
BzOOBz
SePh
NISTfOH79%
O
OBzBzO
BzOBzO
OO
OH
BzOBzO
OBzSEt
+ O
BnO OBn
OBnEtS
IDCP
(79% α/β :3/1)
Scheme 33
Both C-2 acylated and benzylated glycosyl donors can be activated with AgTfO. The
glycosylation is quenched with the presence of tetramethylurea or collidine. Thioglycosides are
usually stable towards AgOTf, so orthogonal glycosylations are feasible.
Example: Mehta, S.; Pinto, M. Tetrahedron Lett. 1993, 32, 4435.
O
OH
BnO BnOOBn
SEtAgTfOK2CO3 O
BnO BnOOBn
SEt
+OMe AcOAcO
OAc
SePh
OMe AcO
OAc
O
AcO85%
O
OH
BnO BnOOBn
SEtAgTfOK2CO3 O
BnO BnOOBn
SEt
OAcOAcO AcO
Phth
SePhOAcO
AcO AcOPhth
O+
Scheme 34
As AgTfO and bases such as tetramethylurea or collidine are frequently employed in
glycosylations with glycosyl halides, chemoselective glycosylations of glycosyl halides in the
presence of selenoglycosides are also possible.
Phenylseleno glycosides can be prepared from peracetylated sugars by reaction either with
phenylselenol, or from glycosyl halides by reaction with potassium phenyl selenoates or from
diglycopyranosyl diselenides by reaction with alkyl halides under reducing conditions.
Example: Mehta, S.; Pinto, B. M. J. Org. Chem. 1993, 58, 3269.
OMeAcOAcO
OAc
OAc
OMeAcOAcO
OAc
SePhPhSeOH
BF3.Et2O84%, α:β = 3.7:1
Scheme 35
- 36 -
Example: Benhaddou, R.; Czernecki, S.; Randriamandimby, D. Synlett, 1992, 967.
OBnOBnO BnO
OBnSe
2 NaBH3CN
BrOBnO
BnO BnOOBn
Se
Scheme 36 4. O-Alkylation and the trichloroacetimidate method (Schmidt)
4.1. O-Alkylation method
The anomeric oxygen of a sugar can be activated for a glycosylation not only by acids
(Fischer glycosylation) but also by bases. Upon treatment a hemiacetalic sugar with a base, the
generated anomeric oxide can be alkylated leading directly and irreversibly to a glycoside. This
process is called anomeric O-alkylation.
Schmidt, R. Angew. Chem. Int. Ed. Engl. 1986, 25, 212.
O
X
RORORO
Y
OH
OX
RORORO
Y
OO
X
RORORO
YO
O
X
RORORO
Y
O
H
Base
R'X
O
X
RORORO
YOR'
R'X
O
X
RORORO
Y
OR'
(thermodynamic control)(kinetic control)
Scheme 37
In this procedure, some inconveniences should be considered: The equilibrium between the
two anomeric forms and the open-chain form gives three sides of attack and also, a base
catalysed elimination in the open chain form could become an important side reaction.
Therefore, the yield, the regioselectivity and the stereoselectivity of the anomeric O-alkylation
was not expected to be outstanding.
However, Schmidt and co-workers have described several good examples of this method
including glycosylation of unprotected sugars.
- 37 -
Examples:
This method has been applied in the synthesis of lactosyl esphingolipid, by reaction of
hemiacetalic lactose with sphingosine triflate. The yield is moderate and the selectivity strongly
depends on the temperature.
W. Klotz, R. R. Schmidt, J. Carbohydr. Chem. 1994, 13, 1093.
O
OAc
OAcOOAc
OAcO
OAc
OAcAcO
OH
N3
OTBDMS
TfO
NaH 1,2-diethoxyethane
+
O
OAc
OAcOOAc
OAcO
OAc
OAcAcO
N3
OTBDMS
Or.t.
49%, β:α= 95:5
Scheme 38
Chelation control can also become a dominant factor in the determination of the α/β
selectivity. Example: Synthesis of KDO-α-glycosides of lipid A derivatives.
Rembold, H.; Schmidt, R. R. Carbohydr. Res. 1993, 246, 137-159.
Scheme 39
The anomeric hydroxyl group of KDO has a low reactivity because of the effect of the
carboxyl group. Formation of an amide that releases electrons and the formation of bulky
benzylidene acetals that promotes a boat-like conformation on the sugar ring make the reaction
of the anomeric oxygen with triflate 2 possible. The coupling is performed twice to give the
- 38 -
trisaccharide backbone that was further transformed into the lipid A analogue. The boat-like
conformation is stabilised by a chelating effect with the cation Na+ and the solvent.
4.2. The trichloroacetimidate method
Electron deficient nitriles are known to undergo direct and reversible base-catalysed addition
of alcohols to the triple bond system, providing O-alkyl imidates. The free imidates can be
directly isolated as stable adducts.
Scheme 40
The reaction of hemiacetalic sugars in the presence of a base with trichloroacetonitrile gives
the anomeric trichloroacetimidates. In this way, the anomeric oxygen atom has been
transformed into a good leaving group.11 O
OHRO BaseCl3C-C O
ORO CCl3
NH
N
Scheme 41
Taking into account the equilibrium between both anomers and the enhanced nucleophilicity
of equatorial oxygen atoms (owing to steric effects and to the stereoelectronic kinetic anomeric
affect), the equatorial (β)-trichloroacetimidate is generated with preference or even exclusively
in a very rapid and reversible reaction. However, this product anomerizes in a slow base-
catalysed reaction through retro-anomerization of the 1-oxide anion. Through a new
trichloroacetonitrile addition, the thermodynamically more stable axial (α)-trichloroacetimidate
is formed (thermodynamic anomeric effect).
O
OH
O OH
O
O
O O
Cl3C-C N
O
O CCl3
NH
O O
N
CCl3
H
Cl3C-C N
B
(thermodynamic control) (kinetic control)
RO RO
Base Base
RO RO + BHBH +
RO RO
Scheme 42
11 Schmidt, R. R.; Kinzy, W. Adv. Carbohydr. Chem. Biochem. 1994, 50, 21-123.
R3C-C N ROH+ base
ORR3C
NH
- 39 -
The equilibration between the two trichloroacetimidates can be speeded up by stronger bases. O
ORO
CCl3
NH
ORO OH
Cl3C-C N
NaH or DBU
O ORO
CCl3
NH
ORO OH
Cl3C-C N
K2CO3
R = BnO O
ROCCl3
NH
ORO OH
Cl3C-C N
NaH or DBUor K2CO3
R' = esters, amides, imides
OR' OR'
Scheme 43
Thus, with different bases both O-activated anomers can be obtained in pure form and high
yield. However, NaH is appropriate for axial trichloroacetimidates while weaker bases such as
K2CO3 is appropriate for equatorial trichloroacetimidates.
Concerning the glycosylation step, reaction of donor and acceptor under very mild acid
conditions leads to the corresponding glycoside in an irreversible manner. Acids, such as
BF3.OEt2 or TMSOTf are used in catalytic amounts. The proton liberated on the glycoside bond
formation reacts with the forming leaving group. This leads to a stable, non-basic
trichloroacetamide that provides the driving force of the reaction.
Example: Synthesis of lactosamine. Schmidt, R. R. University of Konstanz, unpublished
results.
O
O CCl3
NH
Cl3C-CONH2
OAcAcO
AcOAcO
O
OAc
HOAcO
AcHN
OR+O
OAcAcO
AcOAcO O
OAc
AcO AcHN
ORO
O
OHHO
HOHO O
OH
HO AcHN
OHO
leaving group + H+
deprotectionBF3.OEt2
Scheme 44
Fig. 3
- 40 -
The stereochemical requirements are the same as in other glycosylation methods.
Other mild activating species, such as, AgOTf, have also been used.
Example: Robina, I.; López-Barba, E; Fuentes, J. Tetrahedron 1996, 52, 10771-10784.
O
OAc
AcOAcO
NPhthO
NH
CCl3O
OBn
HOBnO
OBnO
OOBn
OBnBnO OMP+
AgOTf, Cl2CH2, 60%Stereoselectivity β, 100%Procedure (IP)
O
OAc
AcOAcO
NPhthO
OBn
OBnBnOO
OBn
OO OBnOBn
OMP
O
OAc
AcOAcO
NPhthO +
NH
CCl3
AgCF3SO3
O
OAc
AcOAcO
NPhth
AgHN
CCCl3
O
ROH
Glycoside
S. P. Douglas, D. M. Whitfield and J. J. Keprinsky, J. Carbohydr. Chem., 1993, 12, 131.
Scheme 45
For the synthesis of a tetrasaccharide derived from GlcNAc where the difference in reactivity
between donor and acceptor is high, AgOTf has proved to be convenient because it activates the
departure of the leaving group more slowly, thus minimizing decomposition of the donor.
Summary
Activation of the anomeric center with trichloroacetonitrile
• Convenient Base Catalyzed Trichloroacetimidate Formation • Controlled acces to α- and β-compounds by choice of the Base • Thermal stability of α- and β-trichloroacetimidates up to room temperature • If required, silica gel chromatography can be performed
Glycosyl transfer
• Catalysis by acids (mainly Lewis acids) under very mild conditions • Irreversible reaction • Other Glycosidic bonds are not affected • Usually High Chemical yield • Reactivity corresponds to the halogenose/silver triflate system • Stereocontrol of Glycoside Bond Formation is Mainly Good to Excellent: • Protecting groups with Neighbouring Group Participation: 1,2-trans-Glycopyranosides
β-Glycosides of: Glc, GlcN, Gal, GalN, Xyl, Mur, 2-deoxy-Glc α-Glycosides of: Man, Rha
• Protecting groups without Neighbouring Group Participation: • Catalyst BF3.OEt: Inversion of anomer configuration
β-Glycosides of: Glc, GlcN, Gal, GalN, Xyl, Mur, GlcUA α-Glycosides of: Man, Rha
• Catalyst TMSOTf : Thermodynamically more stable anomer α-Glycosides of: Glc, GlcN, Gal, GalN, Man, Fuc, Mur
- 41 -
The outstanding significance of the trichloroacetimidate method lies in the ability of glycosyl
trichloroacetimidates to act as strong glycosyl donors under relatively mild acid catalysis. This
has been demonstrated by its use in many laboratories all around the world. The efficiency of
the method makes it appropriate for use in solid-phase, as will be commented on in the next
lesson.
This method has not only been used in oligosaccharide synthesis, but also in the chemistry of
natural products where sugars are glycosylated to different moieties.
Example: Synthesis of Macroviracin D.
Mlynarski, J.; Ruiz-Caro, J.; Fürstner, A. Chem. Eur. J. 2004, 10, 2214-2222.
This is a new type of glycolipid with a rather intriguing structure isolated from Mycelicum
Streptomyces sp., that exhibits strong antiviral activity towards several viruses including HIV,
herpes, simple and varicella zoster. The synthesis of this compound implies three main reactions
that are indicated by A, B, C in the scheme.
Glycosylation with trichloroacetimidates in the presence of TMSOTf in MeCN gives the β-
anomer in all the cases, due to the participation of the solvent.
O
OH
HOHO
HO
OH
O
O
O
O
HOHO
HO OHO
HOOH
OH
O
OH
O
O
O
OHO
OH
OH
O
OH
O
O
OHOHO
HO
O
OHO
HO
OH
OH
O
O
OBn
BnOBnO
BnO O
NH
CCl31
OBn
Zn O
OH
O OButH
2 3
2
Scheme 46
- 42 -
6. Glycosylation with glycals (Lemieux, Thiem, Danishefsky)
Glycals in oligosaccharide synthesis were first used by Lemieux in 1960s, by Thiem in 1980s
and since then, by Danishefsky and co-workers.
Glycals can be used as glycosyl donors in two modalities.
O OOR
E
O
E
OX
E
transformation into a glycosyl donor
Glycosyl acceptor
Glycosyl acceptor
in situactivation
Scheme 47
In the 1st motif, in situ activation makes the glycal act as glycosyl donor by forming a non-
isolable intermediate. In the 2nd motif, the glycal is first converted into a glycosyl donor through
different types of reactions (epoxidation, azidonitration or sulfonamide glycosylation). That is,
the glycal is precursor of a defined glycosyl donor.
The pioneer experiments that used glycals as glycosyl donors, were done by Lemieux and
Thiem who used halonium-mediated coupling to suitable acceptors. This particular reaction has
the tendency to give a trans-diaxial addition and provides a crucial route to α-linked
disaccharides having an axial 2-iodo function at the non-reducing end.
OOP
PO POO
OP
PO POI
OOH
RO ROOR
OR
IO
PO
PO PO
O
ORO RO
OROR
I
Halo-glycosylation
Scheme 48
Because the displacement of an axial iodine atom has proven to be very difficult, aza-
glycosylation of glycals has been investigated with the idea of preparing glycosides of 2-
acylaminosugars.
Azidonitration with CAN/NaN3 was studied by Lemieux and constituted an important
advance at the time, nevertheless the conversion of the nitro-azido compounds into
oligosaccharides has not been fully optimized with regards to the yield and stereoselectivity.
- 43 -
OOP
PO PO
O
OP
PO PO
O
OPO PO
N3
ONO2
OP
OPO PO
I
NHSO2Ph
PO
OPO PONHAc
OR
OP
OPO PO
NSO2Ph
PO
OOH
PO PO
OPO POPhO2SHN
PO
O
O
PO PO
OOH
PO PO
OPO PO
HO
PO
O
O
PO PO
O
OOO
OOPO PO
HO
POO
[O]
CAN/NaN3
PhSO2NH2/IDCP
glycosyl donors
Base acceptor
acceptor
acceptor
Diisopropylidene-galactose
Sulfonamido-glycosylation
1,2-Anhydrosugar-glycosylation
Azidonitro-glycosylation
Aza-glycosylation
Scheme 49
Other procedures, such as iodo-sulfonamidation developed by Danishefsky, have been used
with more success for the synthesis of 2-acylamino oligosaccharides.
This method implies a trans-diaxial addition of an N-halobenzene sulfonamide to a glycal
followed by a base treatment that gives an intermediate that reacts with any acceptor, for
instance, another glycal, furnishing glycosides of benzenesulfonyl glucosamine derivatives:
sulfonamido-glycosylation.
While iodo-glycosylation and sulfonamido-glycosylation are rather good methods for the
conversion of glycals in various glycosides, the 1,2-anhydro sugar glycosylation provides a
general method for converting glycals into common oligosaccharides of glucose, mannose and
galactose in a high stereocontrolled manner. Once the glycal is converted into the 1,2-oxirane, it
may react with several acceptors leading to disaccharides. This method has been the most
widely used for the rapid assembly of oligosaccharides, and is appropriate for solid-phase
synthesis.
Protecting groups influence the reactivity of glycals as donors. The armed-disarmed concept
that prevails in pentenyl glycosides and thioglycosides is also applied here.
Example: Friesen, R. W.; Danishefsky, S. J. Tetrahedron 1990, 112, 8895
OOBn
BnOBnOO
OBz
HO BzOO
BnO
BnOBnOO
OBz
O BzO
II
OOBn
BnOBnOO
OBz
BzO HOO
BnO
BnOBnOO
OBz
BzOO
I
+
+I
58%
76%
Scheme 50
- 44 -
When a benzylated glycal is made to react with benzoylated glycal no self-condensation is
observed and only one product is obtained derived from the more reactive glycal acting as
donor.
With regards to 1,2-anhydro sugars, the method was able to be applied when it was
discovered that glycals react smoothly with 2,2-dimethyldioxirane prepared as a solution in
dichloromethane, giving 1,2-anhydro sugars in good yields. The stereoselectivity of the
epoxidation highly depends on the type of protecting groups and on the steric hindrance of the
substituents.
Examples: Danishefsky, S. J. ; Halcomb, R. I. J. Am. Chem. Soc. 1989, 111, 6661.
OOBn
BnOBnO OO
CH2Cl2
OOBn
BnOBnO
O
OOBn
BnOBnOOH
OMeMeOH
OOTBS
TBSO
TBSO OO
CH2Cl2
OOTBS
TBSO
TBSO
O
O
TBSO
OOPh O
TBSO
OOOPh
OO
CH2Cl2
O
TBSO
OO
Ph
OO
CH2Cl2
O
TBSO O
OO
Ph
α:β = 20:1
DMDO
α:β = 1:1β >>>α
only α
yields, 90 to 100% Scheme 51
The 3,4,6-tri-O-benzyl-D-glucal gives the epoxide in quantitative yield. Its solvolysis gave
the corresponding methyl glycoside with a stereoselectivity of 20:1 in favour of the α-isomer.
With resident acetyl protecting groups, the stereoselectivity of the epoxidation is much reduced.
TBS protecting groups or acetals also give high stereoselective epoxidations. Steric
hindrance also has an influence. Reaction of TBS-protected galactal gives stereoselectively the
α-epoxide, while the presence of an axial substituent at C-3 on the glycal promotes a quite
selective epoxidation from its β-face. On the other hand, the gulal configurated glycal with
hindering substituents on both faces of the double bond gave a 1:1 mixture of epoxides.
Examples:
Synthesis of Kijanimycin: Thiem. J.; Köpper, S. Tetrahedron 1990, 46, 113.
Halo-glycosylation has been mainly applied to the synthesis of 2-deoxy sugars due to the
inconveniences that the substitution of an iodine atom from the C-2 position generally offers.
NIS promoted glycosylation of glycals followed by reduction with H2/Pd and manipulation
of protecting groups furnished the desired oligosaccharide (Scheme 52).
- 45 -
OMeMPMO
OBz
OMeBnO
OH OBn
OMeMPMO
OBz
I
OMeBnO
O OBn
OMeAcO
OBnOMeAcO
OBn
O
MeMPMOO
MeBnO
O
BnO
O
I
OMeAcOOMPM
Cl
OMeAcO
OBn
O
MeHOO
MeBnO
O
BnO
O
I
DDQ
DDQ
AgOTf
OMeAcO
OMPM
OMeAcO
OBn
O
MeO
O
MeBnO
O
BnO
O
I
OMeHO
OH
OMeHO
OH
O
MeO
O
MeHO
O
HO
O
NIS
MeCN, r.t.
48% (α anomer)
+1. H2/Pd/C2. NaOMe
3. NIS, MeCN, r. t.
Kijanimycin
Scheme 52
A similar method has been applied for the synthesis of Avermectine
Example: Danishefshy S. J.; Selnick, H. G. ; Armistead, D. M.; Wincott, F.E. J. Am. Chem.
Soc. 1987, 109, 8119.
OMeHOOMe
OMe
OMeAcOOMe
OMeO
OMe
OMe
OMeAcOOMe
+ NIS
I
66% (α anomer)
OMeO
OMeOMeAcO
OMe
O
OMeAcOOMe
OMe
MeO
Me
OH
HO
O O
OMe
Me
MeH
OMe
MeMe
Avermectin 1α
1. NIS, 64% (α anomer)2. Bu4SnH-AIBN, 78%3. LiEt3BH, 97%
Scheme 53
Example: Total synthesis of Tumor-Related Antigens N3, isolated from human milk. Its
composition depends on the blood type of the lactating mother.
Kim, H. M.; Kim, I. J.; Danishefshy S. J. J. Am. Chem. Soc. 2001, 123, 35-48
- 46 -
Retrosynthesis:
O
OHHO
HOOH
O
OH
AcHN
O
OMe
HOOHOH
O
O
OHHO
HOHO
O
OH
NHAc
O O
OMe
HOOHOH
OHO
OOH
O
OH
OH
OHO
O
OH
O
OPPO
POOP
O
OP
PHN
O
OMe
POOPOH
O
O
OPPO
POHO
O
OP
NHP
O O
OMe
POOPOP
OPO
OOP
O
OP
O PO
O
O
OPPO
POHO
O
OP
NHP
O O
OMe
POOPOP
OPO
OOP
O
OP
O PO
OH
O
OPPO
POOP
O
OP
O
OMe
POOPOH
OO
OPPO
POHO
O
OP'
O O
OMe
POOPOP
OPO
HOOP
O
OP
O PO
OH
O
OPPO
POO
OP'
P"O P"O
OMe
POOPOP
F
Difucosyllacto-N-hexaose
Aza-glycosidation
Aza-glycosidation
P = Generalized Protecting GroupP'= C-6 Protecting Group
P" = P or H Scheme 54
Synthesis
OOHHO
HOOH
OOH
AcHN
O
OMe
HOOHOH
O
OOHHO
HOHO
OOH
NHAc
O O
OMe
HOOHOH
OHO
OOH
OOH
OH
OHO
O
OH
OTIPSO
O
O
OOTIPS
HOPh3SiO
OMeBnOOBn
OBn
F
OOTIPS
O HOOMe
BnOOBn
OBn
OTIPSO
O
OO
OTIPS
O
OMe
BnOOBn
OBn
OHO
OTIPSO
OO
OTIPSO
O
OMe
BnOOBn
OBn
OHO
I
NHSO2Ph
OTIPSO
O
O OOTIPS
HO BnO+O
LevHO
HOO
OTIPS
O BnOOTBS
NaMeO
MeOH
NaMeO
MeOH
OTIPSO
OO
OTIPSO
O
OMe
BnOOBn
OBn
OHO
NHSO2Ph
OOHHO
OO
OTIPS
O BnOOH
OOTIPS
HO HO
OMeBnOOBn
OBn
F OOTIPS
HO O
OMe
BnOOBn
OBn
OTIPSO
PMBO
OMe
BnOOBn
OBn
O
I
NHSO2Ph
OTIPSO
PMBO
OMe
BnOOBn
OBn
ONHSO2Ph
SEt
MeOTf
OTIPSO
HO
OMe
BnOOBn
OBn
OPhSO2NH
O
OOTIPSO
OO
OTIPSO
O
OMe
BnOOBn
OBn
OHO
NHSO2Ph
OHO
OO
OTIPS
O BnOOH
MeOTf
OTIPSO
OO
OHSEt
MeOTf
Difucosyllacto-N-hexaose
AgClO4
Fucosylation
DMDOCH2Cl2+
IDCPPhSO2NH2
MPG
DMDOCH2Cl2
MPG
MPG
1
2
3
4 5
6
7
8
7 + 8
AgClO4 MPG 12
9
11
IDCPPhSO2NH2
13MPG 14
EtSHLHMDS,DMF
149 + MPG MPG
aza-glycosylation
aza-glycosylation
MPG : manipulating Protecting groups
Scheme 55
- 47 -
Example: Synthesis of a branched oligosaccharide fragment of a complex Saponin:
Desgalactotigonin.
Randolph, J. T.; Danishefsky, S. J. J. Am. Chem. Soc. 193, 115, 8473-8474.
OHOHOOH
OOH
O
HOO
OHO
OHHO
OO
OHHO
OH
HO
O
Me
H
H
RO
Me
1: desgalactotigonin (R=tetrasaccharide)2: tigogenin (R=H)
The strategy consists on the preparation of a glycal epoxide that reacts as donor with a
glycosyl acceptor leading to a C(1)-O-sugar, with one hydroxyl group at C-2. This derivative
acts as glycosyl acceptor when it reacts with a glycosyl donor furnishing a branched
trisaccharide.
OOP
PO POO
OP
PO PO
O
O
OP
PO POOH
OSugarO
OP
PO POOSugar
OSugar
3 4
5
GA
6
GD
GA : Glycosyl acceptorGD: Glycosyl donor
This idea is exemplified in the following route:
OBnO BnOOBnO BnO
O
OOO
PhOBnO BnOOH
O
OHO
OO
Ph
ZnCl2THF
OOO
PhOBnO BnOOBn
OO
OOTIPSO
OO O
OTIPSO
OO
O
OOBnHO
BnOOBn
tigogenin
Zn(OTf)2
OOO
PhOBnO BnOOBn
OOH O
OBnO
BnOOBn
tigogenin
OOBn
BnOBnOOBn
F
Sn(OTf)2
OHOHOOH
OOH
O
HOO
OHO
OHHO
OO
OHHO
OH
HO
O
Me
H
H
O
Me
DMDOCH2Cl2
MPG
MPG : manipulating Protecting groups
DMDOCH2Cl2
tigogenin MPG
MPG
Scheme 57
Fig. 4
Scheme 56
- 48 -
- 49 -
Lesson 3. Synthetic Strategies for the Assembly of Oligosaccharides
1. The pioneer linear glycosylation strategy 2. Convergent block synthesis 3. Selective and two-Stage Activation and Orthogonal Glycosylation strategy 4. Chemoselective Glycosylation Reactions 5. One-pot multistep glycosylations 6. Solid-phase oligosaccharide synthesis
Introduction
In this lesson, we are going to comment on different strategies for the assembly of
oligosaccharides with the idea of achieving the most efficient total synthesis of a complex
oligosaccharide. We will consider several approaches that allow the convenient assembly of
complex oligosaccharides from properly protected building block units involving a minimum
number of synthetic steps.
1.-The pioneer linear glycosylation strategy
In the pioneer linear glycosylation strategy, monomeric glycosyl donors have to be added to
a growing saccharide chain. Each step requires manipulation of protecting and leaving groups
which increases the number of reaction steps considerably. This fact, together with its low
convergence, makes this linear strategy the least efficient for the synthesis of complex
oligosaccharides. It has been used with glycosyl halides that require drastic reaction conditions
for their preparation and, in consequence, is incompatible with complex oligosaccharides.
2. - Convergent block synthesis It is applicable for glycosylation methods in which the donors are formed under mild
conditions, are stable enough to be purified and stored for a considerable period of time, and are
able to undergo the glycosylation step also under mild conditions with high yield and high α/β
stereoselectivity. Trichloroacetimidates, thioglycosides, glycosyl fluorides and glycals have
been extensively used in block synthesis because they fulfil these requirements.
In a convergent glycosylation strategy most of the synthetic effort is directed towards the
preparation of monomeric glycosyl donors and acceptors. The assembly of these units to an
oligomer should involve the minimum number of synthetic steps and each synthetic step should
proceed with high stereoselectivity and high yield. Furthermore, an efficient synthetic
convergent strategy should make optimal use of common intermediates and oligosaccharide
building blocks.
- 50 -
Example: Several high-mannose and hybrid types of oligosaccharides have been recently
prepared as synthetic Carbohydrate-Based HIV Antigens using this strategy.
Dudkin, V. I.; Orlova, M.; Geng, X.; Mandal, M.; Olson, W. C.; Danishefsky, S. J. J. Am.
Chem. Soc. 2004, 126, 9560-9562
Gp120 carbohydrates can be used as antigens for eliciting broadly neutralizing immune
response. This idea has gained recognition after the structural determination of 2G12 antibody
epitope, isolated from long-term survivor of infection. This antibody is able to neutralize a wide
spectrum of HIV isolated in vitro and to protect macaques from SIV. The envelope glycoprotein
gp120 of HIV interacts sequentially with the cellular receptors CD4 and a member of the
chemokine co-receptor family.
O
OHOH
HO
OH
O
OHOHOH
O
O
O
OHOHOH
OHO
HO
OH
HO
OHO
HOHOO
O
HO
OH
HO
OH
O
HO
OHOH
O
OOHO O
OH
OO
HO
OH
OO
HOOHO
NHAc NHAc
OHO
OHOHN
Asn-Ile-Ser-Arg-NH2
O
SR
SR
OOH
HO
HOHO
OHO
O HO
O
NHAc
OHO
HOHO
O
OHO
HOHO
OH
O
HO
HOOH
HO
OOHO O
OH
OO
HO
OHO
HOOHO
NHAc NHAc
OHO
OHOHN
Asn-Ile-Ser-Arg-NH2
O
H2N-Cys-
High-mannose type glycopeptides
1
H2N-Cys-
Hybrid type glycopeptides
6
core D-mannose chitobiose trisasaccharide
D-mannose pentasaccharide branch
D-mannose trisaccharide branch
D-mannose trisasaccharide branch
D-lactose -D-mannose trisasaccharide branch
core D-mannose chitobiose trisasaccharide
Fig. 1
The synthesis of high mannose oligosaccharides has been carried out by a convergent block
synthesis using thioglycosides 2 and 3 as donors that were coupled to the core D-mannose
chitobiose trisaccharide acceptor through the stereoselective formation of α(1→6)
and α(1→3) linkages, respectively giving the free glycan (Man9(GlcNAc)2). On its side, the
synthesis of the core trisaccharide has been carried out from glycal 7 by iodosulfonamidation
and reaction with 3,4-di-O-benzylglycal, to give the glycal disaccharide 6 that gave 4 by
- 51 -
iodosulfonamidation, manipulation of protecting groups and glycosylation with phenyl sulfinil
glycoside 10. [Dudkin, V.Y.; Miller, J. S.; Danishefsky, S. J. Tetrahedron Lett. 2003, 44,
1791]. Formation of the corresponding glycosyl amine in glycan Man9(GlcNAc)2 followed by
aspartylation with 5 gave the target glycopeptide 1.
O
OHOH
HO
OH
O
OHOHOH
O
O
O
OHOHOH
OHO
HO
OH
HO
OHO
HOHOO
O
HO
OH
HO
OH
O
HO
OHOH
O
OOHO O
OH
OO
HO
OH
OO
HOOHO
NHAc NHAc
OHO
OHOHN
Asn-Ile-Ser-Arg-NH2
O
SR
OBnO
BnO
OAc
BnO
OBnO
BnO BnOO
O
BnO
OBn
BnO
OBn
O
BnO
OBnOBn
O
OOBnO O
OBn
SPh
OBnO
BnOOAc
BnO
OBnO
BnO BnO
O
O
OBnO
BnO BnO
SEt
OOBn
HOO
BnOOBnO
PhSO2NH PhSO2NH
OBnO
OBnO
OO
Ph
OTBS
Asn-Ile-Ser-Arg-NH2
SSBut
(A)
(B)
(B) (C)
(C)(D)
(D)
OBnO
BnOBnO
OOBn
PMBOO
OPh
SOPh
OBnO
BnO
O
BnOO
O
OBnO
O
O
BnOO
O
OBnO
BnOOBn
BnO
OBnO
HOBnOPhSO2NH
OBnO
OBnO
OBnO
AcOBnO
OBnO
AcOBnO
H2N-Cys-
High-mannose type glycopeptides
2
Fmoc-HN-Cys-
3
4
5
1
core D-mannose chitobiose trisasaccharide
D-mannose pentasaccharide branch
D-mannose trisaccharide branch
6
7
7
8
9
10
11
4
(i) glycosylation with 3(ii) deprotection(iii) glycosylation with 2(iv) global deprotection
Man9(GlcNAc)2 (free glycan)
(i) amination(ii) aspartylation with 5
1
Synthesis
7 iodosulfonamidation + 3,6-di-O-benzylglycal 6
(i) iodosulfonamidation(ii) Manipulation of P.G.(iii)Glycosylation with 9
Scheme 1
3.- Selective and Two-Stage Activation and Orthogonal Glycosylation strategies
Notwithstanding the attractive features of the above mentioned block synthesis, the
conversion of a common building block into a glycosyl donor requires several manipulations at
the anomeric center presenting the drawback of the removal of the anomeric protecting group
followed by the introduction of a leaving group, which can be a serious problem when
performed on larger fragments. The selective and two-stage activation strategy solves this
problem. In it, two types of anomeric leaving groups one obtained from the other, and one type
of activation is used.
In 1984, Nicolaou and co-workers described the glycosylation strategy that is outlined in
Scheme 2. Glycosylfluorides and thioglycosides are used. This two-stage strategy is convergent
- 52 -
and minimizes the number of manipulations, which have to be executed at the oligosaccharide
stage. Attractive features of the strategy are:
(i) The stability of thioglycosides under many different chemical conditions.
(ii) The ease of activation of thioglycosides by conversion into glycosyl fluorides.
(iii) The high efficiency of glycosyl fluorides in glycosidic bond formation.
(iv) The excellent behaviour of thioglycosides as glycosyl acceptors.
O
SPhOR1RO
OF
OR1ROO
SPhOR2HO
O
OR1ORO
OSPh
OR2
O
OR1OHO
OF
OR2O
OR1OHO
OSPh
OR2
DASTNBS
activation stage 1 activation
stage 2AgClO4SnCl2
oligosaccharide
Glycosyl donor Glycosyl acceptor
DASTNBS Deprotection
coupling
Higher oligosaccharide
Scheme 2
Example: Synthesis of Rhynchosporides III
Nicolaou, K. C.; Dolle, R. E.; Papahatjis, D. P. J. Am. Chem. Soc., 1984, 106, 4189-92.
O
BnO
AgClO4-SnCl2CH2Cl2. -15ºC
BnOAcO
OTPS
F
O
BnOBnOAcO
OH
SPh
O
BnOBnOAcO
OTPS
O
BnOBnOAcO
O
SPh
O
BnOBnOAcO
OTPS
O
BnOBnOAcO
O
F
O
BnOBnOAcO
OH
O
BnOBnOAcO
O
SPh
DAST-NBSCH2Cl2. 0º -15ºC 0º -15ºC
TBAF-THF
AgClO4-SnCl2CH2Cl2. -15ºC
85%
O
BnOBnOAcO
OTPS
O
BnOBnOAcO
O
OO
BnOBnOAcO
O
BnOBnOAcO
O
SPh
A
DAST-NBSCH2Cl2. 0º -15ºC, 85%
AgClO4-SnCl2CH2Cl2. -15ºC, 66%
A
1.-
O
BnOBnOAcO
OTPS
O
BnOBnOAcO
O
OO
BnOBnOAcO
O
BnOBnOAcO
O
O
2.-
O
BnOBnO
O
BnOBnOAcO
O
SPh
HO
Scheme 3
- 53 -
Example: Synthesis of LeX fluoride:
Nicolaou, K. C.; Dolle, R. E.; Papahatjis, D. P. J. Am. Chem. Soc. 1990, 112, 3693
O
AcOCAO
CAO OPiv
F
O
PhthNAllylOHO
OBn
SPh
O
AcOCAO
CAO OPiv
O
PhthNAllylOO
OBn
SPhO OBn
OBnBnO
Me
F
O
AcOCAO
CAO OPiv
O
PhthNO
O
OBn
SPh
O OBnOBn
BnO
Me
O
AcOCAO
CAO OPiv
O
PhthNO
O
OAc
F
O OAcOAc
AcO
Me
AgClO4-SnCl2CH2Cl2. -15ºC
72%2. AgClO4-SnCl2, Et2O. -30ºC, 87%
1. DAST-NBS, CH2Cl2, 0º -15ºC2. H2, Pd(OH)2/C, EtOH-EtOAc, 25ºC3. Ac2O, DMAP, 2,6-lutidine, 25 ºC, 84%, 2 steps
Scheme 4
1. H2Ru(PPh3)4, EtOH then TsOH, MeO, 25ºC, 86%
Another two-stage activation strategy reported employs anomeric sulphoxides as donors and
thioglycosides as acceptors. The latter can be converted into sulfinil glycosides by oxidation.
Example: Khiar N.; Martin-Lomas, M. J. Org. Chem. 1995, 60, 7017.
OOBzBzO
BzOOBz
SPh OOBzBzO
BzOOBz
SPhMCPBA
OOH
BzOBzOOBz
SPh
TMSOTfTEP
OOBzBzO
BzOOBz
O
OBzO BzOOBz
SPh
O
OOAcO
OOTBDMS
SPhO
OOHO
OOTBDMS
SPh
OOAcO
OOTBDMS
SPh
OOAcO
OTBDMSO
O
OO
OTBDMSO
SPh
OOAcO
OHO
O
OO
OHO
SPh
OOAcO
OO
O
OO
OO
SPh
OOAcO
OTBDMSO
OOAcO
OTBDMSO
OOAcO
OOTBDMS
SPhO
Scheme 5
[TEP, triethylphosphite, is required to trap the transiently formed phenylsulphenyl ester which may activate the
acceptor resulting in the formation of a 1,6-anhydro derivative].
In the examples discussed above, only one type of anomeric leaving group has been used.
However, for the successful preparation of complex oligosaccharides often a range of different
leaving groups needs to be examined. An orthogonal glycosylation strategy uses two set of
chemically distinct (orthogonal) glycosyl donors activated under different conditions. In 1994,
Ogawa and co-workers proposed this strategy that reduces the manipulation at the
oligosaccharide stage.
In this approach two anomeric leaving groups (X and Y) are used acting either as anomeric
protecting group or as leaving group, depending on the activation conditions.
- 54 -
Scheme 6
Example: Synthesis of chitotetraose oligosaccharide.
Kanie, O.; Ito, Y.; Ogawa, T. J. Am. Chem. Soc. 1994, 116, 12073.
NISAgOTf
O
NphthBnOAcO
OBn
SPh
O
NPhthBnOAcO
OBn
O
O
NphthBnOHO
OBn
F
O
NphthBnO
OBn
F1
2 R = Ac3 R = H
4
ONphth
BnOHOOBn
SPhCp2HfCl2AgClO4
3
O
NPhthBnOAcO
OBn
O O
NphthBnO
OBn
O
5
O
NphthBnO
OBn
SPh
O
NphthBnOHO
OBnFNIS
AgOTf
O
NPhthBnOAcO
OBn
O O
NphthBnO
OBn
O
6
O
NphthBnO
OBn
O O
NphthBnO
OBn
F
Scheme 7
4. - Chemoselective Glycosylation Reactions
This strategy uses the influence of the nature of the protecting groups on the reactivity of
donors and acceptors.
With respect to glycosyl donors, benzylated glycosyl donors (armed) are much more reactive
than acylated ones (disarmed). This difference makes chemoselective glycosylations possible,
OXRO
OYHO
OORO
OYPromotor-1
Glycosyl donor Glycosyl acceptor Disaccharide
OXHO
Glycosyl acceptorO
OROO
Oligosaccharide
OXO
Promotor-2
Promotor-1O
YHOGlycosyl acceptor O
OROO
Oligosaccharide
OO
OYO
- 55 -
the so-called Armed-Disarmed strategy. This strategy has been applied to several glycosyl
donors.
Armed-Disarmed strategy with NPGs
Benzylated pentenyl glycosides react faster than acylated ones.
O OPentOBn
OBn
BnOBnOO OPentOAc
OH
AcO AcO+
Armed Disarmed
IDCP O
OBn
OBn
BnOBnO
O OPentOAc
AcO AcO
O
O
OO
OHOONIS/TfOH
O
OBn
OBn
BnOBnOO OOAc
AcO AcO
O
O
OO
O
O
Scheme 8
O OOBn
O OOBz
O OOBn
XO OOBn
X
O
OBn
K1Fast
O O
XO O
X
OK1
Slowδ
OBz OBz
δ δ
OBz Scheme 9
IDCP is appropriate for the coupling of some reactive (armed) NPGs but is not potent enough
for use with unreactive (disarmed) NPGs. For this purpose, NIS/Et3SiOTf or NIS/TfOH must be
employed.
In the cases where the nature of the protecting groups does not allow the application of the
armed disarmed strategy, two NPGs can still be coupled by use of an intermediate dibromination
step. Thus, depending on how the reaction is carried out, one can obtain either the glycosyl
bromide or a vicinal bromide.
OO
RG
OO
R
Br
GO
OR
Br
GO
RG
Br2
Br
OO
R
BrBr
G
Zn
O
RBr
G
Et4NBr
Bu4NI
Br
Scheme 10
- 56 -
Example, synthesis of Glycophosphatidylinositol Membrane-Bound Protein Anchors (GPI)
Roberts, C.; Madsen, R.; Fraser-Reid, B. J. Am. Chem. Soc. 1995, 117, 1546-1553.
O
O
OH
BnOBnOBnO
O
O
OH
BnOBnOBnO
BrBr
OO
OO
BnOBnOAcO
Ph
O
O
O
BnOBnOBnO
BrBr
O
OO
BnOBnOAcO
O
O
O
BnOBnOBnO
BrBr
OOH
BnOBnOClAcO O
O
OBn
BnOBnOBnO
O
O
O
BnOBnOBnO
BrBr
OO
BnOBnOClAcO
OOBn
BnOBnOBnO
O
O
O
BnOBnOBnO
O
O
BnOBnOClAcO
OOBn
BnOBnO
BnO
Br2/Bu4NBr
NIS/Et3SiOTf
NIS/Et3SiOTf
Zn/Bu4NI
MPGGPI
Ph
Scheme 11
Armed-Disarmed strategy with thioglycosides
Protecting groups in the sugar ring and in the aglycone influence the reactivity of the donors.
Example: Veeneman, G. H.; van Boom, J. H. Tetrahedron Lett. 1990, 31, 275.
O SEt
OBnBnO
OBn
BnO
O SEt
OBzBzO
OBz
HO
+armed
disarmed
O
OBnOBnO
OBn
BnO
O SEt
OBzBzO
OBz
disarmed
IDCP
91%
O
OBnO
BnO
OBn
BnOO SEt
OBnBnO
OBn
armed
1. NaOMe2. NaH/BnBr/Bu4NI
O SEt
OBzBzO
OBz
HO
IDCP72%
disarmed
O
OBnOBnO
OBn
BnOO
BnOBnO
OBn
OSEt
OBzBzO
OBz
O
Scheme 12
The anomeric thio substituent also has an influence. Simple alkyl substituents such as
methyl, ethyl or isopropyl groups, show comparable reactivity towards thiophilic promoters.
However, a bulky alkyl substituent such as diciclohexylmethyl is much less reactive. This
allows the assembling of sugars in a chemoselective fashion.
- 57 -
Example: Boons, G. J.; Geurtsen, R.; Holmes, D. R. Tetrahedron Lett. 1995, 36, 6325.
O SEt
OBnBnO
OBn
BnO O S
OBnBnO
OH
BnO+
O
BnOBnO
OBn
BnO
O S
OBnBnO
O
BnO
IDCP
Scheme 13
Phenylthio groups are less reactive than alkyl groups, but for chemoselective glycosylation,
the reactivity of aryl thioglycosides must be further adjusted by incorporation of electron
withdrawing or donating substituents.
It is important to point out that “armed” thioglycosides can be readily activated with
moderate iodonium sources such as IDCP or NIS. Activation of “disarmed” thioglycosides
requires the presence of a more powerful iodonium source. The combined use of NIS (1 eq) and
catalytic TfOH (0.015 eq) was shown to be particularly effective for this purpose.
Armed-Disarmed strategy with selenoglycosides
Van Boom demonstrated that alkylated phenylseleno glycosides can also be activated by the
thiophilic promoter IDCP to give O-glycosides in a similar way to thioglycosides.
However, fully benzoylated phenylseleno glycosides are not completely inert towards IDCP.
In some instances, orthoesters were detected.
So acylated phenylseleno glycosides can be considered as “pseudo disarmed” substrates. On
the other hand, performances of the same coupling in the presence of the powerful iodonium
source NIS-TfOH smoothly yield the β-linked disaccharide in 91% yield.
Example: Zuurmond, H. M.; Veeneman, G. H.; van der Marel, G. A. and van Boom, J. H.
Tetrahedron Lett. 1992, 33, 2063.
O
OBnBnO
BnOOBn
SePhO
OHBzO
BzOOBz
SePh
O
OBnBnO
BnOBnOO
OBzO
BzOOBz
SePh
+IDCP
(87% α/β :4/1)
O
OBzBzO
BzOOBz
SePh
OOHBnO
BnOOBn
SePh
IDCP60%
O
OBzBzO
BzOO
O
O
OBnO
BnOOBn
SePh
Ph
O
OHBnO
BnOOBn
OMe+O
OBzBzO
BzOOBz
SePhO
BnO
OBn
OBnMeO
O
OBzBzO
BzOOBz
ONIS-TfOH
91%
Scheme 14
- 58 -
Armed-Disarmed strategy with glycals
Finally, glycals can be also selectively activated by varying the protecting groups.
Example: Friesen, R. W.; Danishefsky, S. J. J. Am. Chem. Soc. 1989, 111, 6656.
OBnOBnOBnO
OBzOHOBzO
+O
O
I
BnOBnOBnO
OBzO
BzOIDCP OH
O
OO
OO
IDCP
O
O
OO
O
O
O
O
I
BnOBnOBnO
OBzO
BzO I
O
O
OO
O
O
O
O
BnOBnOBnO
OBzO
BzO
Pr3SnHAIBN
Scheme 15
Tuning the glycosyl donor leaving group ability with a set of two groups, increases the
versatility of the armed–disarmed glycosylation strategy.
Chemoselective strategy with phenylseleno glycosides/thioglycosides
As expected, phenyl seleno glycosides are considerably more reactive than their thio
counterparts towards iodonium-ion mediated activation.
Example: Mehta, S.; Pinto, B. M. J. Org. Chem. 1993, 58, 3269-3276.
Scheme 16
These results indicate that the intrinsic higher reactivity of phenyl selenoglycosides with
respect to the sulphur congeners significantly increases the scope of the armed-disarmed
strategy.
S. Ley and co-workers have developed a chemoselective strategy for oligosaccharide
assembly by tuning the reactivity of glycosyl donors with a set of two leaving groups and by
ester groups and spiroketals.
O
OBnBnO
BnOOBn
SePhO
OH
BnOBnO
OBnSEt
O
OBnBnO
BnOBnOO
OBnOBnO
OBnSEt
+IDCP
(79% α/β :3/1)
O
OBzBzO
BzOOBz
SePh
NISTfOH79%
O
OBzBzO
BzOBzO
OO
OH
BzOBzO
OBzSEt
+ O
BnO OBn
OBnEtS
- 59 -
Donors and acceptors are grouped into four levels of reactivity (Fig. 2):
Fig.2
The general approach to the chemoselective synthesis of a trisaccharide by careful tuning of
glycosyl donor and glycosyl acceptor reactivity is outlined in Scheme 17.
O
XRRO
OOH
XRRO
High Reactive DonorA
Acceptor andIntermediate Reactive Donor
B
1 eq. NIS, cat. TfOH
(X = S or Se)
ORO
OO
XRRO
Intermediate Reactive Donor
AB
OOH
XRRO
Acceptor andLow Reactive Donor
C1 eq. NIS, cat. TfOH
(X = S or Se)
ORO
OO
XRRO
O
ORO
ABC Scheme 17
This methodology has been applied to the synthesis of high-mannose oligosaccharides.
OBnO
BnO BnO
OBn
SePh
O
OBnOBn
SePh
OO
MeO
MeO
RR
O
OTPSOH
SePh
OO
MeO
MeO
RR
O
OBzOH
SePh
OO
MeO
MeO
RR
O
OBzOH
SEt
OO
MeO
MeO
RR
O
OHOBz
SEt
HOBnO O
OTPSOBn
O(CH2)8COOMe
HOBnO
Level 1Most reactive glycosyl donor
Level 2electron-withdrawing groups and/or fused rings reduce
donor reactivity
Level 3change of Se to Sreduces reactivity
Level 4Not reactive
Oligosaccharide Assembly
R = Me, BDA R,R = -(CH2)4-, CDA
1
4
5
6
7
8
9
OBnFO
FBnOFBnO
OAc
SePh
2
OHO
BnO BnO
OBn
SePh
3
O
OTPSOH
OMe
OO
MeO
MeO
RR
10
- 60 -
Example: Grice, P.; Ley, S. V.; Pietruszka, J.; Osborn, H. M. I.; Priepke, H. W. M.;
Warriner, S. L. Chem. Eur. J. 1997, 3, 431-440.
Scheme 18
5. One-pot multistep glycosylations One-pot synthesis of oligosaccharides is often referred as a reactivity-based one-pot method
in which glycosyl donors with decreasing anomeric reactivities are allowed to react sequentially
in the same flask. This procedure, although is highly convenient because reduces the number of
steps considerably, has the inconvenience that the donor reactivities have to be carefully
adjusted which implies extensive protecting group manipulations.
Reactivity-based one-pot method
Tuning the reactivity of glycosyl donors by the influence of leaving and protecting groups,
together with the principle of orthogonal activation enabled a highly efficient tetrasaccharide
one-pot synthesis.
Example: Cheung, M.-K.; Douglas, N. L.; Berthold, H.; Ley, S. V.; Pannecoucke, X. M. Synlett 1997, 257.
O
OHOH
HO
OH
O
OHOHOH
O
O
O
OHOHOH
OHO
HOOH
HO
OHO
HOHOO
O
HOOH
HO
OH
O
HOOH
OH
O
OOHO O
OH
OO
HO
OH
O O(CH2)4COOMe
O
OHOBz
SEtHOBnO
OBnO
BnO BnO
OBn
SePh+
O
OBzOH
SePh
OO
MeO
MeO
OBnO
BnO BnO
OBn
OBzO O
SePh
OO
MeO
MeO
O
OBzOH
SEtOO
MeO
MeO
OBnO
BnO BnO
OBn
OBzO O
OO
MeO
MeO
OBzO
SEt
OO
MeO
MeO
O
O
OTPSOBn
O(CH2)8COOMeHOBnO
O
ROBn
O(CH
OBnO
OBnO
BnO BnO
OBn
OBzO O
OO
MeO
MeO
OBzO
OO
MeO
MeO
O
OBnO
BnO BnO
OBn
SePh+
O
OTPSOH
SePh
OO
MeO
MeO
OBnO
BnO BnO
OBn
OTPSO O
SePh
OO
MeO
MeO OOBz
SEt
BnO
OBnO
BnO BnO
OBn
OTPSO O
OOO
MeO
MeO
OBnO
BnO BnO
OBn
OTPSO O
OOO
MeO
MeO
High-mannose oligosaccharide
1
6
NIS, cat.TfOH
8NIS, cat.
TfOH
12, R = TPS13, R = H
AgOTf, Br2
1
5
NIS, cat.TfOH
5NIS, cat.
TfOH
14
13+ 14 NIS, cat.TfOH
Deprotection
6
11
- 61 -
OFBnO
FBnO FBnO
OAc
F
O
OHOBn
SePh
BnOBnO
OFBnO
FBnO FBnO
OAc
OOBn
SePh
BnOBnO
O
O
OBzOH
SePhOO
MeO
MeO
O
OTPSOH
OMe
OO
MeO
MeO
AgOTf
OBzO
SePh
OO
MeO
MeO
O
OFBnO
FBnO FBnO
OAc
OOBn
BnOBnO
O
OBzO
OO
MeO
MeO
O
OFBnO
FBnO FBnO
OAc
OOBn
BnOBnO
O
O
TPSOO
OMe
OO
OMe
MeO
3
2
6
NIS, cat.TfOH
10
(1.2 eq)
(1.0 eq)
CpHfCl24A MSCH2Cl2
+ (1.3 eq)
(1.6 eq)
NIS, cat.TfOH
15
overall yield, 21%
Scheme 19
Example: Synthesis of Cyclamycin 0
Raghavan, S.; Kahne, D. J. Am. Chem. Soc. 1993, 115, 1580-1581.
This is also a reactivity based one-pot procedure involving sulfinil glycosides. Groups in the
aglycon do the tuning of reactivity. This can be explained by taking into account that the
activation of sulfinil glycosides with Tf2O or TfOH begins with triflation of the sulfoxide.
OSO
OSO S
CF3
OF3C
SOY
O
R R
R NO2 < H < OMe+ YOrate limiting
Scheme 20
This step is rate limiting; therefore the reactivity of the glycosyl donor can be influenced by
manipulating the substituent in the para position of the phenyl ring in the order: NO2<H<OMe.
The reactivity difference between p-methoxyphenyl sulfinil donor and an unsubstituted phenyl
sulfinil glycosyl acceptor is large enough to permit selective activation. In addition, silyl ethers
are good glycosyl acceptors when catalytic TfOH is the activating agent because they react more
slowly than the corresponding alcohol. These features opened the way for one-pot synthesis of
- 62 -
Ciclamycin 0 trisaccharide in a stereoselective manner from the monosaccharide components in
one-step.
Scheme 21
The glycosylation takes place in a sequential manner, para-methoxyphenylsulfoxide 2 is
activated faster than phenyl sulphoxides 1, and 2 reacts preferentially with acceptor 3 using
triflic acid (TfOH) as promoter. In addition, while silyl ethers are stable to triflic anhydride
(Tf2O), they are good acceptors when the promoter is triflic acid; however, the HO-4 of 2 reacts
more slowly than the HO-4 of 3 because it has to be deprotected before reaction. In this way, the
reactivity of the reactants has been manipulated in order to obtain the trisaccharide in one-step.
Non-reactivity-based one-pot method
Recently Huang, Ye and co-workers have designed a general one-pot method independent of
differential glycosyl donors.
Example: Huang, X.; Huang, L.; Wang, H.; Ye, X.-S. Angew. Chem. Int. Ed. 2004, 43, 5221-
5224.
The method is achieved by pre-activating the donor, that generates a reactive intermediate
that reacts with the acceptor that contains the same reactive leaving group. The process can be
repeated in the same vessel allowing the rapid assembly of oligosaccharides (Scheme 22).
O
O
MeS
O
OMe
OMe
OBnO
S
O
OBn
Me
HO
OMe
OBnO
S
O
OBn
Me
O
O
O
Me
O
O
MeS
O
TfOHTfOH
OMe
OBnHO
S
O
OBn
Me
Me3SiO
SO
OH
OH O
O
OH
COOMe
OH
O
O
O
O
O
OHMe
MeOH
O
O
Me
+ +
+-70°
slow
-70°
fast
1 2 3
4
1
5
overall yield = 25%
Ciclamycin 0
+
- 63 -
Scheme 22
The general conditions were established by using p-tolyl thioglycosides as building blocks,
and as the stoichiometric promoter, p-toluenesulfenyl triflate (p-TolSOTf) formed in situ from
p-toluenesulfenyl chloride (p-TolSCl) and AgOTf.
O STol
RO + AgOTfp-TolSCl (1 eq)
Et2OAcceptor Product
OBzO
HO
BzOOBz
STolOBnO
BnO
BnO
OAc
STol OBnOBnO
BnO
OH
STol OAcOHO
AcOOAc
OAc
RT RT
OBnOBnO
BnO
OAc
OBnOBnO
BnO
O
OBzO
O
BzOBzO
OAcOO
AcOOAc
OAc
31 2 4
1 + AgOTf
p-TolSCl 2
5 min 15 min 15 min 5 min
p-TolSCl 3
15 min 15 min 5 min5 min
p-TolSCl 4
5 min 15 min
-60ºC -60ºC -60ºC
55% yield~ 2 hours
-20ºC
3
1
2
4
Scheme 23
The tetrasaccharide Man-α(1,2)-Man-α(1,6)-Glc-α(1,6)-Glc was assembled in this way in
55% overall yield and in less that two hours.
6. Solid-phase oligosaccharide synthesis
The solid-phase synthesis SPS (also called SPOS: Solid-Phase Organic Synthesis) is a
methodology that performs the synthesis of a target compound on insoluble supports.
It offers several advantages over solution phase reactions:
• Increased yields, because excess reagents can be used to drive the reaction to
completion.
• Easy and simple purification processes, because removal of the by-products and
excess of reagents can be done by simply washing the resin.
O STolRO
promoter ORO
Xreactive
intermediate
O STolHO
RO
ORO
O STolORO
promoter
ORO
O
X
ORO
reactive intermediate
O STolHO
RO
O STolORO
ORO
OO
RO
- 64 -
• Rapid overall process, purification of the reaction products is made at the end of the
synthesis minimizing the number of chromatographic steps required.
It is becoming a valuable alternative to traditional synthesis.
Bruce Merrifield was the chemist that in 1963, pioneered solid phase synthesis. For this
contribution, he earned the Nobel Prize of Chemistry in 1984.
The use of solid support for organic synthesis relies on three interconnected requirements:
Fig. 3
1. Solid support: A cross linked insoluble polymeric material that is inert to the conditions
of synthesis.
2. Linker: Some means of linking the functional group of the substrate to the solid phase
that permits selective cleavage of some or the entire product from the solid support
during synthesis to control the extent of the reaction, and finally, gives the desired
product.
3. Functional group: that requires a chemical protection/ deprotection strategy of the
reactive groups.
Merrifield developed a series of chemical reactions that were used to synthezise peptides
(Scheme 24). The carboxy terminal amino acid is anchored to a solid support. Then, the next
amino acid is coupled to the
first one. In order to prevent
further chain growth at this
point, the amino acid,
which is added, has its
amino group blocked. After
the coupling step, the
protecting group is removed
from the primary amino
group and the coupling
reaction is repeated with the
next amino acid. The process continues until the peptide or protein is completed. Then, the
molecule is cleaved from the solid support and any groups protecting amino acid side chains are
removed. Finally, the peptide or protein is purified to remove partial products and by-products.
Solid Support
Linker Functional Group
R. B. Merrifield, J. Am. Chem. Soc. 1963, 85, 2149
Merrifield Peptide Synthesis on Solid Phase
DCC
PS PS PS
PS
PS
ONH
OCbz
CH3 CH3
ONH
NH
NH
NH
CbzO
O
O
CH3 O
CH3
CH3
CH3CH3
ClNO2 NO2
NH
CO2NHEt3Cbz
CH3 CH3
ONH2
O
CH3 CH3NO2
NH
Cbz CO2H
ONH
NH
CbzO
OCH3
CH3
OHNH
NH
NH
NH2
O
O
O
CH3 O
CH3
CH3
CH3CH3
AttachmentDeprotection
Desattachment
Coupling
1) HBr/AcOH
Deprotection,
Neutralization Coupling
1) HBr/AcOH2) NaOH
L-leu-L-ala-gly-L-val
32) Et NNeutralization
R. B. Merrifield, J. Am. Chem. Soc. 1963, 85, 2149
Merrifield Peptide Synthesis on Solid Phase
DCC
PSPS PSPS PSPS
PS
PSPS
ONH
OCbz
CH3 CH3
ONH
NH
NH
NH
CbzO
O
O
CH3 O
CH3
CH3
CH3CH3
ClNO2 NO2
NH
CO2NHEt3Cbz
CH3 CH3
ONH2
O
CH3 CH3NO2
NH
Cbz CO2HNH
Cbz CO2H
ONH
NH
CbzO
OCH3
CH3
OHNH
NH
NH
NH2
O
O
O
CH3 O
CH3
CH3
CH3CH3
AttachmentDeprotection
Desattachment
Coupling
1) HBr/AcOH
Deprotection,
Neutralization Coupling
1) HBr/AcOH2) NaOH
L-leu-L-ala-gly-L-val
32) Et NNeutralization
Scheme 24
- 65 -
Merrifield’s Solid Phase synthesis concept, first developed for the synthesis of peptides, has
also been extensively used for other biopolymers such as oligonucleotides.
Additionally, it has spread into every field where organic synthesis is involved. Many
laboratories and companies focus on the discovery of new chemistry (new reagents, new
reactions) suitable for SPS. It has contributed to a spectacular advance which profoundly
changed the approach for new drugs, new catalysts or new natural discovery.
Many laboratories and companies focused on the development of technologies such as
automated solid-phase synthesis. This has been set up for peptides and oligonucleotides
SPS of oligosaccharides simplify considerably the synthesis of such complex structures and
has had an immense impact on the chemistry and biochemistry of oligosaccharides. However, it
implies more problems than the SPS of peptides or oligonucleotides, because the preparation of
a specific carbohydrate requires the stereospecific formation of each new glycosidic bond in
high yield. Such processes have been demonstrated to be very sensitive even to slight structural
or electronic variations in the glycosyl donor or acceptor.
However, important progress in the field is currently taking place and this will provide an
important and fundamental impulse in the field of Glycobiology.
Central Aspects of Solid-Phase Oligosaccharide Synthesis. 12
Points to be considered:
1. The design of an overall synthetic strategy with either the 'reducing' or the 'nonreducing'
end of the growing carbohydrate chain attached to the support.
2. Selection of a polymer and linker which has to be inert to all reaction conditions during
the synthesis but has to be cleaved smoothly and effectively when desired.
3. A protecting-group strategy consistent with the complexity of the desired oligosaccharide
4. Stereospecific and high-yielding glycosylation reactions
5. 'On-bead' analytical tools that facilitate reaction monitoring and enable a rational
development of efficient protocols.
With regard the 1st point there are three synthetic strategies (Scheme 25):
12 Seeberger, P. H; Haase, W.-C. Chem. Rev. 2000, 100, 4349-4393
- 66 -
In the donor-bound strategy, the glycosyl donor is bound to the solid support by a suitable
hydroxyl group, and then reacted with solution phase acceptors. In the acceptor-bound strategy
the acceptor is attached to the solid support usually at the anomeric center. In the 3rd , strategy
acceptor or donor can be attached to the polymer and elongated differentially.
With regard to the 2nd point, there are different polymer and linker systems that are used in
SPS of oligosaccharides. Merrifield resin is a polystyrene resin that has been extensively used. It
has high loading capacity (1.2 mmol/g), requires swelling by the solvents for efficient reaction
to occur, it has low price, but is limited to solvents such as DMF, CH2Cl2, THF and dioxane.
Recent developments includes the grafting of polyoxoethylene onto polystyrene crosslinked
resins such as Tentagel and related resins. These have better swelling properties and are
compatible with water, but have lower loading properties (0.2-0.3 mmol/g) and higher price.
Cl
Cl
Cl
ClCl
Cl
O
O
HO
n
O
O
HO
nO
O
HO
n
Merrifield 's resin Tentagel
Scheme 25
O X
OR1OR1
R1O
O D O
R2OOR2
OR2
OPHO O O
OR1OR1
R1O
O
O
R2OOR2
OR2
OPDA A
O
OR1OR1
R1O
OHO
A
O
X
R2OOR2
OR2
OPD O
OR1OR1
R1O
OO
O
R2OOR2
OR2
OPA
D
O
OR1OR1
O
OHY
A/D O
X
R2OOR2
OR2
OPD O
OR1OR1
O
YO
O
R2OOR2
OR2
OPD
A/D
O
R2OOR2
OR2
OPHO
AO
OR1OR1
O
OO
O
R2OOR2
OR2
OPO
PO
R2OOR2
OR2D
A/D
A
Donor-bound strategyremove Preiterate
Acceptor-bound strategy
remove Preiterate
Bi-directional strategyremove Preiterate
remove Preiterate
Fig. 4
- 67 -
With regard to the linkers, they must fulfil the following requirements:
a) Must be inert to all reaction conditions
b) Determine protecting groups and coupling possibilities
c) Can be viewed as a protecting group
d) Orthogonal method for effectively cleavage under mild conditions.
Linker systems are:
i. Silyl Ether Linkers
ii. Acid- and Base-Labile Linkers
iii. Thioglycoside Linkers
iv. Linkers cleaved by Oxidation
v. Linkers cleaved by Hydrogenation
vi. Photocleavable Linkers
vii. Linkers cleaved by olefin
Metathesis.
With regard to the protecting groups, the most commonly used are:
Benzyl ethers, base-labile and acid-labile protecting groups, silyl ethers and allyl protecting
groups or others, specifically 4-azido-3-chlorobenzyl (ClAzb).
With regard to stereospecific and high-yielding glycosylation reactions, the gycosylating
agents used for SPS of oligosaccharides are:
i. Glycosyl trichloroacetimidates
ii. Glycosyl sulfoxides
iii. 1,2-anhydrosugars
iv. Thioglycosides
v. Glycosyl Fluorides
vi. n-Pentenyl Glycosides
vii. Glycosyl Phosphates
Finally, 'on-bead' analytical tools that facilitate reaction monitoring and enable a rational
development of efficient protocols.
These methods have had an immense impact on the development of solid-phase
oligosaccharide synthesis by allowing direct reaction monitoring. NMR and IR spectroscopy
together with MS spectrometry have been adapted for use on polymeric supports. These allow
on-bead characterization of oligosaccharides and their intermediates. The techniques used for
this purpose are:
A. HR-MS
B. High-Resolution Magic Angle Spining NMR
C. Gated Decoupling 13C-NMR
D. FT-IR Microspectroscopy
- 68 -
Pioneering Studies were carried out during the 1970s and 1980s.
Different strategies (donor- vs acceptor-bound synthesis), linkers, temporary protecting
groups and glycosylating agents were explored.
Example: Synthesis of α-(1→6)-trisaccharide.
Fréchet, J. M. J.; Schuerch, C. J. Am. Chem. Soc. 1971, 93, 492-496.
This strategy was quite successful in the preparation of α-linked 1→6-oligomers.
Drawbacks: long reaction times and the failure to selectively synthesize β-linked glycosides.
OBnOBnO Br
OBn
O
ONO2
HO OBnOBnO
BnO
O
ONO2
O
OBnOBnO
BnO
OH
O
OBnOBnO
BnOO
OBnOBnO
BnO
O
ONO2
O
OBnOBnO
BnOO
OBnOBnO
BnOO
OBnOBnO
BnO
O
ONO2
O
OBnOBnO
BnOO
OH
1
2
2,6-lutidine, 2 d, 65 ºC, 96%3
MeONa/MeOH
quant.4
reiterative coupling/deprotection
90%
: Merrifield's resin
1) O3, -78 ºC 51-91%
2) SMe2, -78 ºC 79-95%
5 6
Scheme 26
Example: Synthesis of a chitobiose derivative.
Excoffier, G.; Gagnaire, D.; Utille, J.-P.; Vignon, M. Tetrahedron 1975, 31, 549-553
Scheme 27
OO
AcHN
OH
OBn
HO
O
O
Ph
OHO
AcHN
O
OBn
BzO
O
Cl
O
OAcO
AcHN
OAc
Cl
AcO
OO
AcHN
O
OBn
BzO
O
OAcO
AcHN
OAc
AcO
OO
AcHN
OAc
OBn
BzOOAcO
AcHN
OAc
AcO
1) pyridine, 7 d
2) PhCOCl, pyridine3) hydrazinium acetate,pyridine, AcOH, 50 ºC
20
21
22
23
24Hg(CN)2 85%
1) NaOMe, MeOH2) Ac2O, pyridine
2551%, based on 22
: "popcorn" polystyrene
- 69 -
Drawback of "popcorn" polystyrene: partial solubility and thus, considerable loss of
material during the synthesis, reduced overall yield.
Major advances (1990s up to now) in solid-phase oligosaccharide synthesis includes:
1. Development of more powerful glycosylating agents of improved selectivity.
2. Greater diversity of available protecting groups.
3. New analytical techniques.
4. Automatization.
This opens the window for the rapid future development which was briefly glanced at by the
pioneers.
Examples:
A. Donor-Bound Glycosylation Strategy
a) Danishefsky, S. J.; Bilodeau, M. T. Angew. Chem. Int. Ed. Engl. 1996, 35, 1380-1419.
b) Seeberger, P. H.; Bilodeau, M. T.; Danishefsky, S. J. Aldrichimica Acta 1997, 30, 75-92
1,2-anhydroglycal method.
OO
SiPh2OO DMDO O
O
SiPh2OO
O
OO
OHOO
OO
OO
ZnCl250 51
52
53
OO
OH
SiPh2OOO
OO
OH
SiPh2OOO
OO
OH
OOO
OO
OH
OOO
OBnO
BnO
TBAF, AcOH
OO
OH
OHOOO
OO
OH
OOO
OO
OH
OOO
OBnO
BnO54 55
32% overall
: Merrifield's resin Scheme 28
Drawback of the donor-bound strategy:
Most side reactions during glycosylations involve the glycosyl donor. Any side reaction in
the donor attached to the resin will provoke termination of chain elongation. The consequence is
a reduction of the overall yield.
However, an impressive array of complex oligosaccharides has been synthesized by
Danishefsky and co-workers using the glycal assembly method under this strategy.
- 70 -
B. Acceptor-Bound Glycosylation Strategy
Example.: Wang, Z.-G.; Douglas, S. P.; Krepinsky, J. J. Tetrahedron Lett. 1996, 39, 6985-6988.
Trichloroacetimidate method.
OBnO
PhthN
OBn
AcO O CCl3
NH
OBnO
PhthN
OBn
AcO ODOX-PEGM
OBnO
PhthN
OBn
HO ODOX-PEGMDBU
MeOH
OBnO
PhthN
OBn
O O CCl3
NH
OBnO
BnOBnO OAc
OBnO
PhthN
OBn
O O
OBnO
BnOBnO OAc
OBnO
PhthN
OBn
ODOX-PEGM
56
HODOX-PEGM,DBBOTf, 4 Å MS,-45 ºC
57
58
59
DBBOTf, 4 Å MS, -45 ºC, 95%
60 Scheme 29
Excess of donors are used and the overall yields are good and side products are washed away
after each coupling. For this reason, the acceptor-bound approach has generated an immense
interest in the solid-phase oligosaccharide synthesis.
C. Bidirectional Strategy
Elongation of the growing oligosaccharide in both directions requires two sets of orthogonal
glycosyl donors. Examples:
a) Ito, Y.; Kanie, O.; Ogawa, Y. Angew. Chem., Int. Ed. Engl. 1996, 35, 2510-2512.
b) Kanie, O.; Ito, Y.; Ogawa, T. J. Am. Chem. Soc. 1994, 116, 12073-12074.
OBnO
BnO
O
HO SEt
61
OOH
O
OBnO
BnO
O
HO SEt
63
O NH2
O
62
PyBOP, DIPEA
NH
O
O
O
O
OBnO
BnO
OBn
BnO
O CCl3
NH64
TMSOTf, 4Å MS
OBnO
BnO
OBn
BnO OBnO BnO
O
SEtO
65
OO
OH
O
O
O
66 OBnO
BnO
OBn
BnO OBnO BnO
O
OO
67
O
O
O
O
O
60% overall: TentaGel
NIS/TMSOTf, 4Å MS
Scheme 30
- 71 -
In the reaction scheme, first the acceptor containing a potential leaving group is bound to the
resin. Reaction with the donor is performed under different conditions. Then an acceptor is
made to react with the initial anomeric leaving group
Automated Solid-Phase Synthesis
P. Seeberger and co-workers have demonstrated that relatively simple carbohydrates can be
prepared on a machine that executes a coupling cycle, including steps for glycosylation and
deprotection.
The first automated solid-phase oligosaccharide synthesizer was used to prepare structures as
large as branched dodecamers within less than one day. A re-engineered peptide synthesizer
containing a coolable reaction vessel was used. As linker they used octenediol that can be
attached to the resin through either ester or ether linkage. Each monosaccharide has a protection
group pattern that permits the selective deprotection of a single hydroxyl group. As donors
glycosyl phosphates were used that are readily obtained by reaction with diphenylphosporyl
chloride following Sabesan’s method13. These donors are activated with a Lewis acid such as
TMSOTf and have reactivity similar to trichloracetimidates.
OOHRO
ClP OPh
O
OPhDMAP, CH2Cl2
OORO P
O
OPhOPh
R'OH, TMSOTf
MeCN, -78º
ORO
OR'
Scheme 31
The automated synthesis starts with glycosylation of a resin-bound acceptor producing a
coupling product that may be subsequently deprotected. Iteration of coupling and deprotection
cycles with phosphate donors followed by cleavage of the resin-bound oligosaccharides and
purification gives the products.
Fig. 5
Example: The Synthesis of Protected Tumor-Associated Antigen and Blood Group
Determinant Oligosaccharides 13 Sabesan, N.; Neira, S. Carbohydr. Res. 1992, 223, 6453
ProductsProducts
- 72 -
Routenberg K. L., Seeberger, P. H. Angew. Chem. Int. Ed, 2004, 43, 602-605
The Lewis blood group oligosaccharides are a family of fucosylated, ceramide-containing
glycoesphingolipids decorating the exterior of healthy and disease-derived cells.
Lewis type penta- and hexasaccharides are part of the inflammatory cascade and have been
implicated in bacterial and viral infection as well as in autoimmune diseases. The biological
importance of the Lewis antigens has made them targets of intense examination.
Bn=benzyl, Bu=butyl, Fmoc=9-fluorenylmethoxycarbonyl, Lev=levulinoyl, Piv=pivaloyl, TCA=trichloroacetamide
Scheme 32
Fmoc carbamate and levulinoyl ester were selected as temporary protecting groups because
both of them are completely orthogonal and are easily removed with piperidine and hydrazine,
respectively. As linker it was used octenediol that, in this case, reacted with carboxy-terminated
polystyrene resin resulting in an ester linkage, which was rapidly cleaved with a strong base at
the end of the synthesis. Glycosyl phosphates were used as donors.
Initial glycosylation of resin-bound acceptor 9 produces a coupling product that may be
subsequently deprotected. Iteration of coupling and deprotection cycles with phosphate donors
4-8 followed by cleavage of the resin-bound oligosaccharides and purification gives 1-3.
The automated synthesis of pentasaccharide 1, hexasaccharide 2, and nonasaccharide 3 on
the 25-mmol scale, is represented in Scheme 32. Each coupling is promoted with TMSOTf, in a
ratio 1:1 with the donor and is repeated 2 or 3 times. Washing with piperidine or with hydrazine
liberates the appropriate hydroxyl group. Finally treating with an excess of NaMeO/MeOH
several times, liberates the oligosaccharide from the resin.
Lewis X
Lewis Y
Lewis Y-Lewis X
monosaccharide building blocks 4-8.
- 73 -
Scheme 33
Example: Synthesis of a dodecasacharide
Plante, O. J. ; Palmacci, E. R.; Seeberger, P. H. Science 2001, 291, 1523; Bartolozzi, A.
Seeberger, P. H., Current Opinion in Structural Biology 2001, 11, 587.
Scheme 34
- 74 -
Each cycle involved the delivery and coupling of a building block to a growing, polymer-
bound oligosaccharide chain and the removal of a protecting group to expose a unique hydroxyl
group for attachment of the next carbohydrate. Stepwise coupling yields, greater than 94%, were
obtained in the assembly of linear and branched carbohydrates.
Finally, very recently P. Seeberger and co-workers have reported the use of a Microreactor
based method for performing glycosylation reactions very rapidly over a wide range of reaction
conditions.
Ratner, D. M.; Murphy, E. R.; Jhunjhunwala, M.; Snyder, D. A.; Jensen, K. F.; Seeberger, P.
H. J. Chem. Soc., Chem. Comm. 2005, 578-580.
The Silicon microfluidic microreactor (Fig. 6) was designed with three primary inlets to mix
and react glycosylating agents, acceptor and promoter. Once mixed the reactants, they enter a
reaction zone which is terminated by a secondary inlet used to quench the reaction, and after
that, the quenched reaction stream exits the reactor for collection and analysis.
Fig. 6
This method of optimization is currently under development and, together with
automatization, will probably have a tremendous impact on the progress of Glycochemistry.
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