DEVELOPMENT OF A NOVEL PROMOTER SYSTEM
FOR THIOGLYCOSIDE
ACTIVATION
and its application in the synthesis of a GD3 bis-lactam
Andréas Meijer
Bioorganic Chemistry
2003
Akademisk avhandling som för avläggandet av teknisk doktorsexamen vid
tekniska fakulteten vid Lunds Universitet kommer att offentligen försvaras
å Kemicentrum, sal K:B, torsdagen den 18 december 2003, kl. 13.30.
A doctoral thesis at a university in Sweden is produced as a monograph or as a collection
of papers. In the latter case, the introductory part constitutes the formal thesis, which
summarizes the accompanying papers. These have either already been published or are
manuscripts at various stages (in press, submitted, or in manuscript).
© Andréas Meijer
Department of Bioorganic Chemistry
Center for Chemistry and Chemical Engineering
Lund University
P.O. Box 124
SE-221 00 LUND
SWEDEN
Cover printed on Conqueror 250 gr
Body printed on Munken Pure 100 gr
Title font : Adobe Garamond
Headline font : Frutiger 45 Light
Body font : Georgia
Printed by KFS AB, Lund
ISBN 91-628-5924-2
PREFACE
I wrote this thesis based on my view of synthetic carbohydrate chemistry. The results and
discussions underlying the publications and manuscripts are therefore not presented in
any specific order, but rather fragmentized all over the various chapters. The mix of my
contributions with that of others will hopefully supply the reader with a homogenous
experience.
Chapter 1 is an introduction to carbohydrates and a brief explanation of their
importance. Chapter 2 deals with the fundamentals of carbohydrate chemistry and
introduces terminology and nomenclature necessary for the forthcoming chapters. Some
observations of more general synthetical value are also reported. Chapter 3 is a
condensation of some of the theories that are circulating in contemporary mechanistical
carbohydrate chemistry. These theories represent the foundation underlying the
mechanistical discussions leading to the development of the novel promoter system
reported in chapter 4, which is the core of this thesis. In chapter 4 observations related to
glycosylations are also discussed. Chapter 5 is a brief introduction to the biological
background of the lactam project, which motivates the choice of target compounds of
which the promoter system described in chapter 4 is evaluated. Chapter 6 describes the
use of the developed promoter system in the synthesis of the bis-lactam of GD3.
i
ACKNOWLEDGEMENTS
I would like to express my gratitude to the following persons:
Ulf Ellervik, my supervisor, without whom I would not have been able to complete this
project.
Göran Magnusson, who started the lactam project and accepted me as a PhD student, but
unfortunately left us too soon.
Olov Sterner for managing the department in an excellent way even when times are bad.
Maria Levin for always being helpful and for proofreading this thesiss.
Teddy Ercégovic for deciding to use that bottle of ICl in a glycosylation instead of throwing
it away, and for introducing me to sialic acids.
Jörgen Ohlsson for sharing his great knowledge in name reactions.
Mårten Jacobsson, Anders Wirén, Richard Johnsson for allowing me to hijack Ulf the last
month.
Anna Andersson and Eva Welinder for helping me out in the lab when the idéas
outnumbered my synthetic capabilities.
Karl-Erik Bergquist, Einar Nilsson and Anders Sundin for techical assistance.
My former and present roommates; Karolina Larsson, Johan Tejler, Bader Salameh, Ian
Cumpstey, Anette Svensson, Anders Bergh, Nafizal Hossain and Tapes Bhattacharyya.
The fysikalorgan-team: Magnus Berglund, Jörgen Toftered, Johan Billing, Daniel Röme,
Hans Grundberg and Johan Ericsson, for indirectly teaching me how to control my anger
when confronted in infected debates (I still believe that it is the Field effect that is
responible for the difference in pKa, even though the chlorine atom is far away……).
Ulf Lindström for cheering for the right team. Next year……
Ulf Nilsson for helping me out with chemicals and carbohydrate chemistry in general.
All former and present members of Bioorganic Chemistry, for creating a great department.
ii
Mette my wonderful whife, for taking care of me while writing this thesis and at the same
time taking care of our newborn son Albert. I will get up at nights from now on, promise….
iii
LIST OF PAPERS
This thesis summarizes the following papers, which are referred to in the text by the
roman numerials I-IV. Papers I, II and IV are reprinted with kind permission from the
publishers.
I
Teddy Ercegovic; Andréas Meijer; Göran Magnusson; Ulf Ellervik;
Iodine Monochloride/Silver Trifluoromethanesulfonate (ICl/AgOTf) as a
convenient Promoter System for O-Glycoside Synthesis
Org. Lett. 2001, 3, 913
II
Andréas Meijer; Ulf Ellervik;
Study of Interhalogens/Silver Trifluoromethanesulfonate as Promoter
System for High-Yielding Sialylations
J. Org. Chem. 2002, 67, 7407
III
Andréas Meijer; Ulf Ellervik;
Interhalogens (ICl/IBr) and AgOTf in thioglycoside activation; application
in the synthesis of bis-lactam analogs of ganglioside GD3
Preliminary manuscript
IV
Andréas Meijer; Ulf Ellervik;
Direct aromatic iodination and bromination using interhalogens and
Lewis acids
Submitted
v
ABBREVIATIONS
Ac
acetyl
AcOH
acetic acid
AgOTf
silver trifluoromethanesulfonate
Bn
benzyl
Bz
benzoyl
Cr
cresyl
e.g.
for example
Et
ethyl
equiv
equivalents
IBr
iodine monobromide
ICl
iodine monochloride
i.e.
that is
LG
leaving group
N3
azide
pMP
para-methoxyphenol
MSB
methyl sulfenyl bromide
NIS
N-iodisuccinimide
NMR
nuclear magnetic resonance
Ph
phenyl
R
any group
r.t.
room temperature
Troc
2,2,2-trichlororethoxycarbamate
TFA
trifluoroacetyl
TfOH
trifluoromethanesulfonic acid
TMSEt
trimethylsilylethyl
TfO
trifluoromethanesulfonate
X
any halogen
vii
CONTENTS
PREFACE ...................................................................................................................i
ACKNOWLEDGEMENTS............................................................................................ ii
LIST OF PAPERS......................................................................................................... v
ABBREVIATIONS ..................................................................................................... vii
1. INTRODUCTION ................................................................................................... 1
2. BASIC CARBOHYDRATE CHEMISTRY ................................................................... 5
3. ADVANCED CARBOHYDRATE CHEMISTRY ........................................................ 25
4. NOVEL PROMOTER SYSTEM............................................................................... 41
5. CARBOHYDRATE CANCER VACCINES................................................................ 65
6. SYNTHESIS OF A GD3 BIS-LACTAM.................................................................... 71
7. SUMMARY AND FUTURE PERSPECTIVES ............................................................ 83
8. SUPPLEMENTARY INFORMATION ....................................................................... 85
9. REFERENCES ...................................................................................................... 91
10. APPENDIX........................................................................................................ 97
1
1 INTRODUCTION
For a layman the title “Development of a Novel Promoter System for Thioglycoside
Activation; and its application in the synthesis of a GD3 bis-lactam” might seem quite
intimidating. However the reader will be directed, over the next 90 pages, through the
work leading to this title and hopefully it will appear more obvious.
1.1 Carbohydrates
The term carbohydrate actually stems from french; hydrate de carbone and was originally
applied only to monosaccharides, in recognition of the fact that their empirical
composition can be expressed as Cn(H2O)n. However the term is now used generically in a
wider sense to include structures containing several monosaccharide fragments, i.e.
oligosaccharides (Figure 1-1).
OH
O
HO
HO
OH
OH
OH
HO
OH
O
OH
O
HO
HO
HO
OH
OH
O
OH
O
HO
O
HO
OH
OH
O
OH
OH
monosaccharides
oligosaccharide
Figure 1-1 Mono- and oligosaccharide fragments. Different types
of glycosidic bonds (i.e. equatorial or axial) are stressed by arrows.
1
The smallest denominator of a carbohydrate is the monosaccharide structure
(Figure 1-1). There are several different monosaccharide structures available in nature and
they can all be linked to form oligosaccharides (Figure 1-1). This linkage, a glycosidic
bond, can be formed by any of the available hydroxyl groups and in addition the bond can
be placed either axially or equatorially, generating a great diversityi1 of oligosaccharide
structures (Figure 1-1).
One of the great pioneers in organic chemistry, Emil Hermann Fischerii, was also
an outstanding carbohydrate chemist. He elucidated the stereochemical configuration of
all the known monosaccharide fragments, beginning with glucose in 1891,2
and is
responsible for the systemized carbohydrate nomenclature that we use today. Despite this
early introduction of carbohydrates in organic chemistry, the synthesis of carbohydrates
did initially not gain any greater interest in the scientific community. This lack of interest
was due to the unawareness of the importance of carbohydrates in biological systems.
Until the 1960´s, carbohydrates were only thought of as an energy source, such as starch,
or as a structural material, such as cellulose.3 When carbohydrate structures eventually
were identified on the surface of cells they were believed to protect the cell membrane by
functioning as a mechanical protection. It has now been shown that the carbohydrates are
involved in more subtle interactions than that. The carbohydrates have been identified to
play important roles in many biological signalling systems, where the great diversity and
complexity of carbohydrates permits them to carry out a wide range of functions. The
newfound importance of carbohydrates in biological systems have brought them into the
spotlight of the pharmaceutical industry, and therefore research on carbohydrates is now
growing considerable and promises to be a major source of drug discovery leads in the
future.4
i
As an example; more than 11 000 000 different oligosaccharide isomers can be formed by the
combination of three monosaccharide fragments.1
ii
Emil Hermann Fischer (1852-1919), PhD 1874, Nobel prize 1902.
2
Figure 1-2 Cartoon of the cellular processing of carbohydrates.i5
Monosaccharide fragments enter the cell by the action of specific
carbohydrate recognizing proteins (A). The monosaccharides are
processed (B ) to form activated building blocks that can be
assembled, in an intracellular compartment called the Golgi
apparatus, into oligosaccharides (C). These oligosaccharides can
then be transported to the cell surface where they are involved in
cell-cell interactions (D).
The biological synthesis of oligosaccharide fragments is conducted by cells, which
are able to absorb various monosaccharide fragments and then process them, by the aid of
enzymes, to form oligosaccharides (Figure 1-2), which can then be used in different
biological functions such as cell-cell interactions (D ). The chemical synthesis of
oligosaccharides also use monosaccharide fragments obtained from biological sources.
However, the differentiation of the various hydroxyl groups and the formation of either an
axial or an equatorial glycosidic bond (Figure 1-1), that was accomplished by enzymes in
the cell, must now be achieved by chemical means. This chemical synthesis can be quite
troublesome, and a large variety of different strategies for the synthesis of glycosidic
bonds have evolved and new strategies and methods are still being developed, indicating
the need for better ways of constructing oligosaccharides. It is thus the objective of this
thesis to describe the development of a new method for the chemical construction of
glycosidic bonds.
i
The cartoon is adapted from a published image.5
3
2
2 BASIC CARBOHYDRATE CHEMISTRY
The following chapter will give a brief introduction to the fundamentals of carbohydrate
chemistry, introducing the reader to terminology and general concepts routinely used in
the synthesis of oligosaccharides. Also some different strategies for the construction of the
glycosidic bond is reported together with a brief overview of the reaction conditions
normally employed in their synthesis.
2.1 Terminology
The nomenclature of carbohydrates differ from that of general organic compounds. Below
are some terminology essential in the discussion of carbohydrates described, together with
an explanation of the nomenclature of the stereocenter at the anomeric position.
2.1.1 Basic Nomenclature and Numbering
The nomenclature of carbohydrates is too complex to be fully treated in this brief
introduction,i6 however some concepts essential for the forthcoming discussions in this
thesis are presented. Some of the systematic names derived from the nomenclature
guidelines are too complex for everyday use, so trivial names have evolved, which are now
routinely used. The monosaccharides discussed in this thesis are shown in Figure 2-1,
together with their trivial and systematic names.
i
The reader is referred to the IUPAC nomenclature for a full description.6
5
OH
HO
OH
O
OH
O
HO
HO
OH
OH
OH
galactose
D-galactopyranose
OH OH
HO
AcHN
O
OH
glucose
D-glucopyranose
CO2Me
OH
O
HO
HO
HO
OH
OH
HO
sialic acid
5-acetamido-3,5-dideoxy-Dglycero-D-galacto-non-2ulopyranosonate
mannose
D-mannopyranose
Figure 2-1 Common monosaccharides.
The monosaccharides can be unfolded to form a straight carbon chain consisting of an
aldehyde (or ketone) group and hydroxyl groups. This open chain is not as stable as the
cyclized form and the carbohydrate cyclize spontaniously in solution (Figure 2-2).
Monosaccharides are numbered along the carbon sequence of the open chain
(Figure 2-2). The numbering extends to the oxygens connected to the carbons, thus the 3hydroxyl of glucose is the hydroxyl group at the carbon atom numbered as 3 (C-3).
6
O
4
1
2
OH
OH
HO
3
OH
4
HO
O
HO
HO
OH
OH
5
1
3
6
HO
HO
1
O
2
O
OH
3
9
4
HO
5
NHAc
7
OH
HO
OH
O
OH
HO
AcHN
1
HO
6
HO
O
OH
4
2
8
9
HO
Figure 2-2 Numbering of carbohydrates. Some numbers are
omitted for clarity.
6
2.1.2 Anomeric configuration
The anomeric position is the stereogenic center formed in the cyclization of the open chain
monosaccharide (Figure 2-3). The aldehyde (or ketone) functionality is attacked by an
appropriate hydroxyl functionality forming a six membered ring.i The hydroxyl group of
the anomeric position can equilibrate between the axial and the equatorial position,
illustrating how reactive the anomeric position is compared to the other locations of the
carbohydrate.
HO
HO
HO
HO
HO
HO
O
HO
HO
HO
OH
OH
H
O
OH
HO
HO
HO
OH
H
O
HO
HO
HO
O
H
O
OH O
OH
HO
HO
HO
O
HO
OH
O
HO
H
OH
Figure 2-3 Equilibrium of glucose.
The two stereoisomers, formed from the cyclization, are referred to as anomers,
designated a or b according to the configurational relationship between the anomeric
center and a specified anomeric reference atom. If the carbohydrate contains less than five
stereocenters, anomeric position excluded, the anomeric reference atom coincides with
the configurationalii atom and is defined as the highest numbered centre of chirality. If the
carbohydrate contains more than four chiral centers the anomeric reference atom no
longer coincides with the configurational atom, and its definition is more complex.iii In the
a anomer the oxygen of the reference atom is cis to the anomeric oxygen and in the b
anomer the relationship is trans.
i
Other ring sizes can be formed as well, but only the six membered rings (pyranosides) are
discussed in this thesis.
ii
The configurational atom decides the D or L series placement. If the hydroxy group (or oxygen
bridge) of the configurational atom is directed to the right in a Fischer projection, the sugar belongs
to the D-series.
iii
For a full description of the definition of anomeric reference atoms of carbohydrates containing
more than four stereocenters the reader is referred to the IUPAC definitions.6
7
In Figure 2-4 the determination of the anomeric configuration of galactose and
sialic acid is exemplified. The carbohydrate must be drawn in a Fischer projection in order
to examine the relationship between the anomeric hydroxyl group and that of the
anomeric reference atom. The Fischer projection is obtained by arranging the carbon
chain vertically so that the C-1 carbon is placed at the top and the carbon atoms of the ring
in a smooth arc. The array of atoms are viewed from the convex side and projected on the
plane of the paper as a vertical line. To illustrate the cyclic form, a long bond can be drawn
between the oxygen involved in the ring formation and the anomeric carbon. The
transformation of conformationally correct structure into a Fischer projection is simplified
by first drawing a Haworth projection, which is a structure devoid of all conformational
information.
Conformationally
correct structure
OH
HO
Haworth projection
CH2OH
O OH
HO
OH
O
H
OH H
OH
H
OH
HO
H
H
OH
HO
H
H
Fischer projection
OH
H
HO
H
H
O
CH2OH
H
OH OH
HO
AcHN
O
OH
O OH
AcHN
R
H
CO2OH
H
H
HO
CO2OH
R:
OH
H
OH
CO2OH
H
H
H
OH
AcHN
OH H
H
HO
H
O
H
H
OH
H
OH
galacto
glycero
CH2OH
CH2OH
Configurational atom
Anomeric reference atom
Figure 2-4 Determination of anomeric configuration of
carbohydrates.
For a galactose substrate with an equatorial hydroxyl group the Fischer projection
depicts that the oxygen of the anomeric reference atom and the anomeric hydroxyl group
are in a trans relationship, thus the carbohydrate should be denoted b (Figure 2-4). For
sialic acid, which contains more than four stereocenters, the anomeric reference atom is
defined to C-7. In the Fischer projection the axial hydroxyl group is in a trans relationship
with the anomeric reference atom, designating the anomeric configuration to be b (Figure
2-4).
8
2.2 Stereoelectronic effects
There is a stereoelectronic effect in carbohydrates that renders them quite unique
compared to other cyclic compounds. This effect has consequences not only for the
structure of carbohydrates but also for the chemical reactivity. Below is this electronic
effect described along with a related controversial effect.
2.2.1 Anomeric effect
The anomeric effect is a stereoelectronic effect which is defined as the tendency to form
gauche conformations around the C-Y bond in the X-C-Y-C system where X and Y are
electronegative heteroatoms with free electron pairs. In carbohydrate systems the
anomeric effect is manifested as the tendency to position electronegative aglycons axially,
thus countering the normal trend of avoiding axial interactions by placing the aglyconi
equatorially (Figure 2-5).7
AcO
AcO
OAc OAc
O
Cl
CHCl3
OAc
OAc
O
OAc
2%
Cl
OAc
98%
Figure 2-5 Anomeric effect.
The anomeric effect must be explained by two theories,8 since no theory alone is capable
of explaining the observations associated with the effect. The first theory explains the
effect as a stabilization of the antibonding orbital of the C-Y bond by the lone pairs of atom
X, or in a carbohydrate system; the interactions of the lone pairs of the cyclic oxygen with
the antibonding orbital of the C-1-aglycon bond (Figure 2-6). This interaction is so strong
that the oxygen changes hybridization from pure sp3 to more sp2 character. This change in
hybridization also changes the bond lengths, thus shortening the oxygen-C-1 bond and
lengthening the C-1-aglycon bond, which has been proved experimentally.
i
The term aglycon is used for a substituent on the anomeric position.
9
Oxygen lone pairs
X
O
Antibonding orbital
C
Y
O
C
O
Figure 2-6 Orbital interactions responsible for the anomeric
effect.
The second theory explains the anomeric effect as a minimization of unfavorable dipole
interactions. The interaction is minimized when the anomeric substituent is placed axially
(Figure 2-7). This theory accounts for the solvent dependency of the anomeric effect,
which is more pronounced in nonpolar solvents.
O
O
vs
O
O
Figure 2-7 Favorable dipole interactions with an axial aglycon.
There is, of course, also an anomeric effect in the opposite direction called the exo
anomeric effect, where the lone pairs of the aglycon oxygen interacts with the antibonding
orbital of the C-1-ring oxygen bond (Figure 2-8). This effect, that is present in both axial
and equatorial aglycons, is however more difficult to observe and is therefore not so
widely recognized.
X
O
O
Antibonding orbital
C
Y
C
O
Oxygen lone pairs
Figure 2-8 Exo anomeric effect.
2.2.2 Reverse anomeric effect
For a long time there was a debate whether there existed a reverse anomeric effect (RAE),
where a positively charged aglycon would be stereoelectronically favored in an equatorial
position. The debate was initiated by the observation that a positively charged aglycon
obtained an equatorial conformation (Figure 2-9).9 Over the years various attempts to
10
theoretically explain this (and other) observations have been published but none has
gained any support in the scientific community.
AcO
AcO
OAc OAc
O
AcO
OAc
O
N
OAc
N
OAc
Figure 2-9 The so called reverse anomeric effect (RAE)
Recent publications have stated that there exist no stereoelectronic forces, like the one
operating in the anomeric effect, that supports RAE, and the explanation for the previous
observations supporting RAE is either misinterpretation of data or solvent effects. The
solvent effect is believed to originate from solvent molecules arranging around the
charged atom, increasing the steric bulk of the aglycon and therefore amplifying the
tendency for an equatorial positioning.10,11,12
2.3 Basic carbohydrate synthesis
In the synthesis of oligosaccharides the formation of the glycosidic bond is the most
crucial step. The fundamental mechanism is outlined below, together with essential
terminology.
2.3.1 General mechanism
In a glycosylation reaction two carbohydrate fragments are coupled together. The
carbohydrate substrate containing the hydroxyl functionality is termed acceptor and the
substrate containing the anomeric carbon to be coupled is termed donor. The donor is
equipped with a latent leaving group that is chemoselectively activated by a promoter
system (depicted as P in Figure 2-10), generating an oxocarbenium ion which can react
with the acceptor alcohol, thus forming a glycosidic bond (Figure 2-10). One must realize
that the oxocarbenium ion is very unstable and will react with other nucleophiles than the
acceptor (e.g. moisture) if possible, and if the acceptor is of low nucleophilicity the
activated donor can eliminate to form a C-1-C-2 unsaturated carbohydrate. In either way
the reactive donor intermediate is terminated and the glycosylation will be low yielding.
11
Another complicating factor is the acid produced in the condensation step, which can
degrade sensitive compounds if not taken care of.
O
O
LG
P
O
HO
P
O
LG
O
O
P LG
H
O
O
Figure 2-10 Generalized glycosylation reaction.
The newly formed bond, depicted as a wavy bond in Figure 2-10, can assume either an
axial or an equatorial position since the attack of the alcohol group of the acceptor can
take place either from above or from below the plane of the oxocarbenium ion.
2.4 The Acceptor
The various hydroxyl groups of the acceptor must be distinguished in order to construct
the correct glycosidic bond in the glycosylation reaction. For this purpose protective
groups that are compatible with the reaction conditions employed in carbohydrate
chemistry have been developed. Below are some protective groups used in the synthetic
work leading to papers I-III presented.
2.4.1 Protective group patterni
The acceptor molecules are normally protected in a suitable way so that one hydroxyl
group is preferentially glycosylated in the coupling reaction. Some hydroxyl groups are of
such low reactivity that their protection may be omitted. Acceptor 1 was selectively
glycosylated at the equatorial hydroxyl group, leaving the axial group unreacted. Another
example is the 7-hydroxyl group of sialoside 2 which was left unreacted when the
8-hydroxyl group was glycosylated. The omission of protection of unreactive hydroxyl
groups can be very beneficial, especially when the hydroxyl groups are in close proximity,
due to the minimization of steric crowding in the transition state of the coupling reaction.
i
Experimental data for reactions mentioned in this chapter can be found in paper III
12
If the acceptor carries equally reactive hydroxyl groups, extensive protecting group
manipulations are necessary, as in compound 3 (Figure 2-11).
OH
HO
OBn
O BnO
O
NHTroc
N3
OBn
1
OH
HO
TFAHN
O OpMP
OBn
CO2Me
O
OTMSEt
HO
BnO
BzO 2
OBn
O
3
OpMP
OBn
Figure 2-11 Protective group strategies for various acceptors.
2.4.2 Protective groups
The groups employed for hydroxyl group protection can roughly be organized into the
following categories; ethers, esters and acetals (Figure 2-12). Among ether protection
groups the benzyl groups are most commonly used. Benzyl ethers are stable in a multitude
of reaction conditions and can be selectively removed by hydrogenolysis.
O
O
O
O
Benzyl
O
Benzoate
O
O
Acetate
O
O
O
Benzylidene
O
Isopropylidene
Figure 2-12 Common protective groups.
The esters are good temporary protective groups since they can be cleaved off easily and in
high yields using a large variety of reaction conditions. Acetates and benzoates are the
most commonly used but variants with electron withdrawing groups for altered chemical
properties are available.i Acetals are used as protective groups but can also be used to
selectively protect hydroxyl groups with special geometric properties. Benzylidene acetals
preferentially forms six-membered cyclic acetals since the bulky benzene ring can be
placed in an equatorial position minimizing sterical interactions. This allows selective
i
The electron withdrawing properties of ester protective groups can be tuned to affect parameters
such as selectivity of deprotection and stereoselectivity of glycosylations.
13
protection of the 4- and 6-hydroxyl groups of hexopyranosides. Isopropylidene acetals
preferentially form five-membered cyclic acetals since the six-membered acetal suffers
from 1,3-diaxial interactions with one of the two methyl groups. In the more flexible
five-membered ring the two methyl groups can obtain pseudo equatorial conformations.
The formation of isopropylidene acetals are basically limited to cis diols, since trans rings
are too strained, invoking further means of selectivity between the hydroxyl groups.
2.4.3 Anomeric protective groups
The anomeric position is protected throughout the whole synthesis of the oligosaccharide,
and must thus be protected by a group that can withstand all reaction conditions normally
employed in carbohydrate synthesis, and at the same time be removed using mild reaction
conditions.
O
O
O
SiMe3
O
pMP
TMSEt
OMe
Figure 2-13 Anomeric protective groups.
The trimethylsilylethyl group (TMSEt) has been a popular anomeric protecting system due
to its simple deprotection and chemical inertness to many reaction conditions.13 However
the sensitive nature of the TMSEt glycoside towards hard Lewis acids such as H+ renders
many glycosylation conditions low yielding without the proper use of acid scavengers. Also
the flexibility of the aglycon induces an aversion towards crystallization, often rendering
the pure compound as an oil. The para-methoxyphenyl (pMP) group is less reactive than
the TMSEt group and not as sensitive towards hard Lewis acids.14 It is easily attached and
induces crystallinity in the compounds it protects.
2.5 The Donor
There are fundamental differences of the protective group pattern and choice of aglycon
for donors compared to acceptors. These differences are highlighted and the difficulties in
glycosylation using a sialic acid donor is explained.
14
2.5.1 Protective group strategy
In the design of protective group patterns of donors, different groups are employed so that
positions can be deprotected selectively after glycosylation, to form a new acceptor
molecule for further elongation with a second donor fragment (Figure 2-14).i The most
common protective groups for selective deprotection are esters and acetals since their
deprotection can be performed using mild conditions. The ether groups are normally
chosen as “permanent” protective groups since they are more stable under a range of
reaction conditions.
AcO
OAc OBn
O
SEt
OH
OBn
O
HO
BnO
OpMP
HO
OBn
4 NHTroc
NHTroc 1
3
Acceptor
Donor
OBn
O BnO
O
OBn
O OpMP
OBn
Acceptor
Figure 2-14 Protective group strategy for donor fragments.
2.5.2 Leaving group
There is a large variety of leaving groups, and for every type of leaving group there are
several promoter systems, generating an even larger amount of leaving group/promoter
systems available. An optimal leaving group should be stable both on the shelf and in a
reaction mixture, it should be easily preparedii and be of high reactivity. The criterion of
high stability and reactivity can be difficult to combine, so a solution is either to use a very
reactive promoter system together with a stable donor or to attach a reactive leaving group
just prior to activation in the glycosylation reaction.
O
O
O
1
SR
O
O
O
OAc
S
R2
O
CCl3
NH
X
X : F, Cl, Br
Figure 2-15 Various leaving groups employed in carbohydrate
synthesis.
i
ii
Reaction conditions can be found in paper III.
Some scientists claim that the odor of the reagents involved is an important factor when choosing
the type of leaving group employed in a synthesis.
15
The most common leaving group in carbohydrate chemistry is the thioglycoside, being
employed in almost 50% of all published carbohydrate syntheses in 2000.i Other common
leaving groups are acetates, sulfoxides, trichloroacetimidates and halides (Figure 2-15).15
2.5.3 Sialic acid donors16
The sialic acid donors deserve special attention, due to their defiantness in carbohydrate
synthesis. The carbonyl at C-1 destabilizes the anomeric position therefore augmenting the
need of a reactive leaving group or a powerful promoter.ii17 However the C-1 carbonyl also
promotes elimination, by forming a glycal 5, therefore dictating the demand of a stable
leaving group. Some leaving groups, such as chloride, bromide and sulfoxide, are therefore
of minor practical use together with sialic acid. Sialic acid bromide is so unstable that it
decomposes under vacuum in a matter of hours, leaving its preparation troublesome.iii
Sialic acid chloride is slightly more stable but decomposes upon silica purification.iii Sialic
acid sulfoxide derivatives have not even been isolated due to their decomposition into
glycal (Figure 2-16).18
OAc
OAc OAc 1
R
AcO
AcHN
AcO
O
OAc
O
AcO
AcHN
CO2Me
CO2Me
AcO
5
R1 : Cl, Br, S(O)R2
Figure 2-16 Some sialic acid donors decompose to a glycal
derivative.
The carbonyl group is also partly responsible for the sterical hindrance around the tertiary
anomeric carbon, which impedes the coupling reaction with the acceptor, leading to an
increased preference for the elimination into the glycal derivative 5. The deoxy position at
C-3 prevents the use of stereodirecting groupsiv which further complicates synthesis since
the thermodynamic product is the unnaturalv axial glycoside.
i
Oral communication by Peter Fügedi at Eurocarb XI, Lissabon 2001.
ii
It has been shown that a sialic acid donor increases its reactivity by a factor of 1000 by reducing
the C-1 carbonyl.17
iii
Paper II
iv
The use of stereodirecting groups is discussed in greater detail on page 26.
v
The axial sialoside is extremely rare in nature.
16
2.6 Promoters for thioglycoside activation
The primary role of the promoter is to activate the leaving group of the donor as
chemoselectively and as mildly as possible. Other aspects such as avoiding heavy metals,
stability of reagents, commercial availability and economical factors, have stimulated the
search for viable alternatives. The promoter systems most commonly used in thioglycoside
activation can be divided into three groups; iodonium-, sulfonium- and triflic anhydride
based.
2.6.1 Iodonium based
The iodonium ion speciesi can be formed in situ in the reaction mixture (1)ii (2)19,20 (Figure
2-17), by the addition of an activator and a source of iodine (Figure 2-17). The iodonium
species can also be precipitated in a separate reaction to form a reactive salt (3)21 which
can then be dissolved in the glycosylation reaction mixture. The iodonium ion is chosen
for its soft (HSAB) electronic character that is well matched to the soft sulfur atom of the
thioglycoside, rendering the reactive species chemoselective.
(1)
I
AgOTf
X
OTf
I
AgX
X = Cl, Br, I
O
(2)
O
N
TfOH
I
OTf
I
N H
O
O
N
N
(3)
I
OTf
*2
I
OTf
2
Figure 2-17 Promoter systems activating via an iodonium species.
i
Since the degree of ionization is unclear the word species is used.
ii
Paper I-III
17
2.6.2 Sulfonium based
The sulfonium ion based promoter systems are similar to the iodonium based systems and
can be prepared either in situ (1)22 or dissolved as a somewhat stable salt (2)23 (Figure
2-18). These promoter systems were developed as alternatives to the reactive but
unselective electrophilesi that was used for activation in the early development of
thiogycoside chemistry.
(1)
(2)
AgOTf
MeS Br
MeS S
MeS
Me
OTf
Me
AgBr
OTf
Me
OTf
MeS S
Me
Figure 2-18 Promoter systems activating via a sulfonium species.
2.6.3 Triflic anhydride based
The search for environmentally safe promoter systems has led to the triflic anhydride and
sulfoxideii based methods. The triflic anhydride is generating the thiophilic species by
reacting with the sulfoxide. The reactivity of these systems is easily tunable by varying the
substituents of the sulfoxide; (1)24,25, (2)26, (3)24 (Figure 2-19). The donor has to be
activated by the promoter system prior to addition of the acceptor, probably to avoid
triflation of the hydroxy function of the acceptor by triflic anhydride.
O
S
OTf
R
S
Tf2O
R
OTf
(1) R : S-4-MeOPh
(2) R : 1-piperidino
(3) R : Ph
Figure 2-19 Promoter systems activating via a sulfoxide
derivative.
i
Activation included the use of methyl trifluoromethanesulfonate (MT) which is a rather
unselective electrophile that can alkylate other positions than the sulfur-containing leaving group.
MT is also an carcinogenic compound, which complicates its handling.
ii
The derivatives presented in Figure 2-19 are not really sulfoxides but a thiosulfinate (1) and a
sulfinylpiperidino (2) compound, but the term sulfoxide is used for simplicity.
18
2.7 Reaction Conditions
A glycosylation reaction could be meticulously planned by choosing the right acceptor,
donor and promoter system, and although fail miserably. The reaction is dependent on
several factors such as choosing the right temperature and solvents but also practical
parameters such as stirring and the addition of base can affect the yield. Below are some of
the most common parameters described with actual observations made during
glycosylations.
2.7.1 Temperature
Glycosylation reactions can be performed at almost any temperature. The factors that
determine at what temperature to run a glycosylation reaction are the stability and the
reactivity of the components. For unreactive donor/promoter systems a high reaction
temperature is necessary, whereas for a more reactive system, a lower temperature is
sufficient for complete activation. In general, lower temperatures suppress unwanted side
reactions, like the cleavage of protective groups, often mediated by the acid formed in the
condensation reaction, and reactions between the promoter system and the product or
acceptor molecule. The temperature can also influence the stereoselectivity of the
reaction. A low temperature usually favors the equatorial kinetic product, whereas a
higher temperature favors the thermodynamic axial product.
2.7.2 Solvent
The choice of solvent in a glycosylation reaction is based on a few key abilities; solubility,
reactivity and stereoselectivity. The solvent system should of course be able to solubilize
both the acceptor and the donor at an acceptable concentration. The reactants should not
be too diluted since the rate of the glycosylation is proportional to the concentration and a
prolonged coupling time will increase any side reactions. Sialylations in particular often
perform badly in diluted coupling reactions, due to competing elimination of the activated
donor.
The polarity of the solvent generally determines the reaction speed, since the
glycosylation mechanism normally proceeds via a charged intermediate. Thus choosing a
polar solvent for the glycosylation will increase the reaction speed.27
The stereoselectivity of a glycosylation reaction can be controlled by the
appropriate choice of solvent. Acetonitrile generally promotes the formation of an
19
equatorial glycosidic bond through the so called nitrile effect.28 The nitrile effect facilitates
the formation of an equatorial glycosidic bond through the generation of a nitrilium ion,
stabilized by the anomeric effect (Figure 2-20). This nitrilium ion is attacked in an SN2
manner by the acceptor to form an equatorial bond. Since acetonitrile freezes at –44°C,
dichloromethane is normally added as an antifreezing agent, when a glycosylation is to be
performed at lower temperatures.
ROH
O
O
N C CH3
O
OR
N
C
CH3
O
N
C
CH3
Figure 2-20 The nitrile effect.
Etheral solvents, on the other hand, promote formation of an axial glycosidic bond. This
affinity is explained by an SN2 like attack of the acceptor on a b-ether-oxonium ion
intermediate (Figure 2-21). The preference for the ether molecule to obtain an equatorial
position was previously explained by the reverse anomeric effect29 but since the scientific
community abandoned this effect, the increased affinity for the equatorial position is
believed to originate from a greater tendency to place bulky substituents equatorially, as
the ethers normally are bulkier than the linear nitrile functionality. Another explanation
could be the low polarity of the ether solvents which enhance the anomeric effect and thus
the formation of the thermodynamic axial product.i
O
O
Et
O
Et
Et
O
Et
Et
O
O
Et
ROH
O
OR
Figure 2-21 The so called ether effect.
i
Solvents of low polarity will enhance the dipole interactions and thus the anomeric effect. Diethyl
ether has an e of 4.3, and acetonitrile has an e of 38.
20
2.7.3 Base
Due to the acid formation in the condensation reaction, sensitive groups can be cleaved off
during the glycosylation. In order to minimize problems like these, a hindered base can be
added to the reaction before activation of the donor. This addition is however not always
beneficial. It has been shown that the formation of orthoester, a semistable byproduct, is
promoted under neutral or basic conditions.i The addition of base can also affect the
reaction rate, since most promoter systems consist of an electrophilic reagent, which will
complex with added Lewis base and therefore slow down the activation.
2.7.4 Molecular sieves
Molecular sieves are crystalline metal aluminosilicates having uniform cavities which
selectively can absorb molecules of a specific size. The 3Å size molecular sieves are
commonly used in glycosylation reactions to remove moisture. In order to remove
moisture efficiently the sieves have to be activated prior to use. Activation involves heating
(500°C for 20 min) with a heating gun under high vacuum.ii Besides from acting as a
dehydrant the sieves are also capable of acting as a Brønstedt base, neutralizing a reaction
mixture.30,31 The basicity of molecular sieves has led to the development of acid washed
molecular sieves. These, with acid “saturated” molecular sieves, are utilized in reactions
where a catalytic amount of acid is used.
2.7.5 Stirring
The importance of efficient stirring in a heterogeneous reaction mixture or a reaction
mixture containing substances of low solubility, should not be ignored. The effects of
insufficient stirring are most pronounced in upscaled reactions. One example is the
glycosylation reaction of donor 4 with acceptor 3 to form disaccharide 6 which proceeded
in an excellent yield of 97% (0.14 mmol donor) (Scheme 2-1).iii When the reaction was
performed under identical conditions at a larger scale (2.37 mmol donor) the yield
dropped to 49%.
i
Orthoester formation will be discussed in greater detail on page 26.
ii
It should be noted that storage of molecular sieves in an oven (120°C) prior to exposure to vacuum
only results in molecular sieves that are saturated with water since the sieves are capable of
absorbing water even at the elevated temperature in the oven.
iii
Paper III
21
AcO
OAc OBn
O
SEt
OBn
O
HO
BnO
4 NHTroc
OAc OBn
O BnO
O
AcO
OpMP
3 OBn
NHTroc 6
OBn
O OpMP
OBn
Scheme 2-1 Reaction conditions; 1.5 equiv donor, ICl/AgOTf,
CH2Cl2, -45°C.
After analysis of the reaction mixture it was concluded that the low yield was due to the
insufficient dispersion of one of the components of the promoter system. The component,
silver trifluoromethanesulfonate, is insoluble in the reaction medium, dichloromethane,
and had not been properly stirred/grounded by the stirring bar. The stirring bar and the
round bottom flask had formed a compartment in which chunks of silver triflate was left
undispersed throughout the reaction (Figure 2-22).
Solvent
Stirring bar
AgOTf
a)
b)
Figure 2-22 Cartoon of a) round bottom reaction flask and b) flat
bottom reaction flask.
Another example is the sialylation of donor 7 with trimethylsilylethanol to produce
the acid labile trimethylsilylglycoside 8.i When the reaction was scaled up from 0.16 mmol
to 3.14 mmol donor the yield dropped from 93% to 15%. The low yield was due to the
formation of sialic acid hemiacetal 9 , which is formed from the acid mediated
decomposition of 8 (Scheme 2-2).
i
Paper III
22
OAc OAc
SPh
AcO
TFAHN
O
AcO
SiMe3
HO
CO2Me
OAc OAc
CO2Me
AcO
TFAHN
O
AcO
7
OTMSEt
8
[H+]
OAc OAc
OH
AcO
TFAHN
O
OAc OAc
CO2Me
AcO
TFAHN
CO2Me
AcO
O
AcO
9
O
OTf
SiMe3
H
Scheme 2-2 Reaction conditions; 2 equiv acceptor, IBr/AgOTf,
CH2Cl2, CH3CN, -40°C.
The reason for the stability of compound 8 under identical conditions in the small scale
reaction is probably due to the higher stirring efficiency in the smaller reaction vessel.
Since the acid formed in the condensation reaction, is neutralized by the molecular sieves
in a heterogeneous reaction, vigorous stirring is necessary for an efficient reaction. When
the stirring was optimized, by changing to a flat bottom reaction vessel (Figure 2-22), the
yield of the large scale reactions rose to 86% (0.78 mmol) and 72% (7.85 mmol).i
i
The stirring bar used in the flat bottom flask must have a pivot ring, in order to minimize the risk
for the stirring bar to jam against the flask walls.
23
3
3 ADVANCED CARBOHYDRATE CHEMISTRY
The reactivity and stereoselectivity of a glycosylation reaction can be greatly influenced by
the protective group pattern of the donor and external nucleophiles present in the
reaction. The following chapter will emphasize some methodologies and theories and
provide the reader with an image of the complex and enigmatic mechanism of the
glycosylation reaction.
3.1 Influence of protective groups on donor reactivity
The reactivity of a donor is greatly influenced by the protective groups employed. Both
protective groups on the hydroxyl groups and on the aglycon will influence the rate of
activation.
3.1.1 Armed-Disarmed
The armed-disarmed concept is based on the observation that donor molecules with
electron donating groups, called armed, can be selectively activated in the presence of
donor molecules protected with electron withdrawing groups, i.e. disarmed.32 The main
reason for deactivation of the disarmed sugar is the instability of the developing charge of
the intermediate oxocarbenium ion due to the proximity of an electron withdrawing group
at C-2. The presence of electron withdrawing groups at other positions also promote the
loss of reactivity, albeit at a much lower degree due to the greater distance to the
developing charge. The armed-disarmed concept has been used in one-pot syntheses,
25
where a donor has been selectively activated in the presence of other donors of lesser
reactivity. After complete activation a new acceptor is added to the reaction and the less
reactive donor is activated, either by a more powerful promoter system24 or by adding a
more polar solvent,33 hence increasing the reaction speed (Figure 3-1). The difference in
the activation rates of donors with similar structural features can be difficult to assess,
therefore the relative reactivities of a great number of donors have been mapped in an
empirical way. By activating a mixture of several types of donor molecules with
substoichiometric amounts of promoter and then analyzing the degree of activation, sets
of relative reactivities can be constructed.34
O
1
SR
O
HO
a) promoter
O
SR2
b)
O
HO
O
O
O
O
OR3
OR3
c) promoter/solvent
Figure 3-1 One pot glycosylation.
3.1.2 Leaving group manipulations
By varying the leaving group aglycon the reactivity of the donor can be altered, and an
effect similar to that of the armed-disarmed concept can be achieved. The reactivity of the
aglycon can be altered electronically by manipulating it with electron donating or
withdrawing groups.35,36 The reactivity can also be altered by varying the bulkiness of the
aglycon.37
3.2 Influence of protective groups on stereoselectivity
The protective groups can interfere with the formed oxocarbenium ion of the donor, and
due to their geometric restrictions result in stereodirecting effects. Some common
protective groups, and their interaction with the oxocarbenium ion are discussed below.
3.2.1 Esters
Protecting the donor with an ester group is one of the most common ways of controlling
the stereoselectivity of a glycosylation reaction. The ability of the carbonyl oxygen of the
acetate to stabilize the charge of the activated donor will bring about a possibility of
26
controlling the stereochemistry of the glycosidic bond, by occupying one of the faces of the
oxocarbenium ion (Figure 3-2).
O
O
O
O O
O
O
O
O
O
O
O
Figure 3-2 Participating groups.
The incoming acceptor will preferentially attack from the side of the oxocarbenium ion
that is not blocked by the ester group. The stereodirecting effect is of course dependent on
the stability of the cyclized acyloxonium ion, therefore the stereodirecting effect is most
prominent when an ester group at the C-2 position is involved, since a stabilized five
membered ring is formed. Participation from the other positions (C-3, C-4 and C-6) are
also possible but their impact is strongly influenced by the ability of the sugar ring to
adopt a low energy conformation with the cyclized acyloxonium ion (Figure 3-2). The
electron density of the carbonyl oxygen will also influence the degree of interaction (i.e.
esters with electron withdrawing groups are less prone to interact).38 If the acyloxonium
ion is formed to a lesser extent there are no pronounced sterical factors directing the
incoming acceptor and a mixture of stereoisomers is formed (A and B in Figure 3-3). If an
acyloxonium ion is formed, the acceptor can either attack the anomeric carbon in an SN2
manner and form a 1,2-trans glycosidic bond (B), or attack the acyloxonium ion directly.
The product of an attack on the acyloxonium ion is called orthoester and is normally a
quite unstable byproduct (C in Figure 3-3).
O
O
O
O O
O
O
O
O
O
OR
O
OR
O
O O
O
A
B
Figure 3-3 Orthoester formation.
27
OR
C
The formation of orthoester has been extensively studied,39,40 and several factors
promoting its formation have been identified. The presence of a base in the reaction
mixture promotes the accumulation of orthoester since the acid catalyzed decomposition
into acyloxonium ion is retarded. The use of benzoates instead of acetates also retards the
formation of orthoester due to the greater stability, and thus lesser reactivity, of the
intermediate bridged cation of the benzoate.41
3.2.2 Amides and carbamates
Amides and carbamates are normally used to protect amine functionalities, and they affect
the stereochemistry of a glycosylation in a manner similar to that of ester groups. The
amides are however rarely used to protect the C-2 of a donor, since they form stable
oxazoline derivatives by an elimination reaction of the bridged cation (Figure 3-4).
O
-H
O
NH
O
HN O
N O
O
Figure 3-4 Oxazoline formation.
In order to minimize the formation of this stable byproduct, amine derivatives less prone
to eliminate into oxazoline derivatives have been developed (Figure 3-5). The various
protective groups all share the 1,2-trans directing effect but utilize different deprotection
methods and are stable under different reaction conditions.
O
O
N
O
O
O
N
O
O
O
NH
O
N,N-diacetyl
NAc2
Phthalimido
NPhth
CCl3
Trichlororethoxycarbamate
NHTroc
Figure 3-5 Common amine protective groups.
The trichloroethoxycarbamate (Troc) protective group was developed to minimize
the formation of oxazoline byproduct and maintaining the stereodirecting properties.42
28
However when the Troc donor 4 was used in a glycosylation reaction using acetonitrile as
solvent more than 50% of the donor formed the cyclized product 10 instead of coupling to
the acceptor (Figure 3-6). When the solvent was changed to pure dichloromethane the
glycosylation was performed in near quantitative yield.i The intermediate nitrilium ion has
been trapped in studies of the nitrilium effect43 and in the synthesis of N-acylated
glycosylamines.44 Also an intramolecular reaction with acetonitrile, similar to the one
presented here, has been reported in the literature.45 One could speculate that the
intramolecular reaction of acetonitrile is a general reaction for all amine protective groups
containing an NH fragment, and care should be taken when choosing acetonitrile as
solvent.
AcO
OAc OBn
O
SEt
AcO
OAc OBn
O
NH CCl
3
NH
O
AcO
OAc OBn
O
CCl3
O
O
NH
O
O
N
C
CH3
AcO
OAc OBn
O
-[H]
CCl3
O
N
N
CH3
CCl3
O
O
10
4
Figure 3-6 Formation of dehydroimidazole byproduct.
3.2.3 Thioauxiliaries
Deoxysugars without a “handle” to attach a participating group for directing a
glycosylation reaction, can utilize a thioauxiliary. The sulfur atom will interact with a
neighboring oxocarbenium ion forming an episulfonium ion that will direct the incoming
acceptor (Figure 3-7). The thioether can then be removed using reductive conditions,
reforming the deoxysugar. This approach is elaborative since extra steps adding and
removing the stereodirecting group are necessary. The main area of application has been
in sialylation reactions where the formation of equatorial glycosidic bonds are notoriously
difficult.ii
i
For further details regarding the reaction conditions see page 80.
ii
For a complete discussion of the problems associated with sialylations see page 16.
29
OAc OAc
CO2Me
AcO
AcHN
AcO
O
LG
SR1
OAc OAc
CO2Me
AcO
AcHN
AcO
O
LG
OAc OAc
CO2Me
-LG
R2OH
OAcOAc
OAc OAc
AcO
AcHN
AcO
O
AcO
AcHN
AcO
R1
O
O
OR2
SR1
OAc OAc
CO2Me
S
CO2Me
S
R1
AcO
AcHN
AcO
AcO
CO2Me AcHN
AcO
O
OR2
Figure 3-7 Thioauxiliaries in sialic acid chemistry.
3.2.4 Ethers
Ether protective groups are unable to participate and stabilize a proximate oxocarbenium
ion so the stereochemical outcome of the glycosylation reaction is determined by other
factors such as solvent interactions, anomeric effect and transient intermediates. Solvent
interactions such as the nitrile and the ether effects will induce a stereochemical
preference for the formed glycosidic bond by interaction with the oxocarbenium ion.i An
uncoordinating solvent of low polarity will enhance the anomeric effect and increase
formation of the axial glycosidic bond. Transient intermediates can be subtle in their
actions and the use of ether protective groups avoids any override of their effects. ii
3.3 Influence of external nucleophiles on stereoselectivity
Nucleophiles other than the acceptor can interact with the activated donor. This
interaction can influence the stereochemical outcome of the glycosylation. Below are two
of the most common external nucleophiles and their influence on the stereoselectivity
discussed.
i
The nitrile and the ether effect are discussed in greater detail on page 19.
ii
The transient intermediates are discussed in greater detail on page 30.
30
3.3.1 Halide
BnO
BnO
OBn
O
BnO
ROH
BnO
BnO
QBr
Br
OBn
O
BnO
OR
Figure 3-8 In situ anomerization method.
Halide ions have been used to direct the stereoselectivity of glycosylation reactions for a
long time and the generalized effect has been named the in situ anomerization method.46
The stereocontrol is believed to originate from the greater reactivity of the equatorial
halide (compound B Figure 3-9), which is in equilibrium with the more stable axial
epimer D.i The greater reactivity of the equatorial halide is proposed to be derived from
the, by the anomeric effect, stabilized transition state, in the SN2 like attack by the
nucleophile. The axial halide does not benefit from any stabilization of the transition state,
in the low energy chair conformation D, and a conformational change into substrate E is
not viable due to the high energy of this boat conformation. The stability of the halide
derivatives, towards nucleophilic displacement by an acceptor, renders this approach
viable only at higher temperatures (i.e. room temperature).
O
OR
X
O
O
O
LG
X
O
B
X
C
X
A
O
O
D X
E
O
OR
Figure 3-9 Illustration of the various reaction pathways available
in glycosidic bond formation when transient intermediates are
available.
i
The anomeric effect renders the axial halide more stable.
31
X
For highly unreactive halide derivatives, Lewis acids such as HgBr2 can be added to
catalyze the epimerisation reaction.47 The activation must however be finely tuned, as a
too reactive Lewis acid can form the oxocarbenium derivative A , overriding the
stereocontrol. The reactivity can also be increased by exchanging the glycosyl bromide for
the more reactive glycosyl iodide, however the instability of glycosyl iodides renders them
hard to handle, and therefore they have been generated in situ by adding iodide ions to an
activated donor (i.e. derivative A).48,49
3.3.2 Triflate
Triflate ions have long been used in reactions due to their chemical inertness and are often
considered as spectator ions. However the triflate intermediate corresponding to
compound D (Figure 3-9), formed from the reaction of triflate ion and oxocarbenium ion
A, has been characterized using NMR spectroscopy at low temperatures, and is postulated
to affect the stereochemical outcome of the glycosylation reaction. The formation of the
triflate intermediate is crucial for stereocontrol as activation of donor with acceptor
present results in poor stereocontrol, whereas addition of acceptor to an activated donor
results in good stereocontrol. The stereodirecting effect was originally found by Crich in a
glycosylation reaction using a 4,6-benzylidene protected mannose donor (reaction 1,
Figure 3-10).50 The acetal protecting group seems to influence the stereocontrol as the
corresponding mannose donor without a benzylidene group (2) was unable to glycosylate
with any stereocontrol, under the same reaction conditions.50 This variance was attributed
to the torsional restrains of the benzylidene, favoring intermediate D by destabilizing the
oxocarbenium ion A (Figure 3-9).51
(1)
Ph
O
O
BnO
OTBDMS
O
R1OH
Ph
O
O
BnO
OTBDMS
O
OR1
LG
(2)
BnO
BnO
BnO
OBn
O
R2OH
BnO
BnO
BnO
OBn
O
OR2
LG
(3)
Ph
O
O
BnO
R3OH
O
BnO
Ph
O
O
BnO
O
BnO
LG
OR3
Figure 3-10 Reactions used in the study of triflate intermediates.
32
Without the benzylidene acetal the glycosylation probably proceeds via an SN1 mechanism
by an attack on the oxocarbenium ion A (Figure 3-9), but with torsional restrains the
oxocarbenium ion will be destabilizedi52and react via an SN2 mechanism on the axial
triflate compound D.ii The discrepancy of the proposed reaction pathway with that of the
in situ anomerization was troublesome at first, but when a glycosylation reaction with a
benzylidene protected glucose showed contrasting behavior, some light was shed.53 The
axial stereoselectivity of the glycosylation reaction using a benzylidene protected glucose
donor (reaction 3, Figure 3-10) implied that the reaction pathway proceeded via an SN2
mechanism on the equatorial triflate intermediate B, in accordance with the in situ
anomerization theory. The inverse stereocontrol of the analogous mannose derivative was
contributed to a greater anomeric effect due to the axial silylether functionality on the C-2
carbon.iii It was speculated that the greater anomeric effect of mannose “shuts off” the
Curtin-Hammettiv kinetic scheme leading to the axial product via the minor equatorial
triflate B.v The summoning of the anomeric effect is ambiguous since it operates in “two
directions”. It promotes the equatorial product by shifting the anomerization equilibrium
towards the axial triflate D, and promotes the axial product by stabilizing the transition
state of the SN2 attack on compound B. If the postulate is viable the increased anomeric
effect affects the equilibrium between B and D far more than the stabilization of the
transition state with compound B and the acceptor. Other factors such as a greater
tendency for the mannose derivative to flip into the high energy boat conformation Evi, in
which the anomeric effect can stabilize the transition state and yet form an equatorial
bond, should be considered.54
i
The planar nature of the oxocarbenium ion forces the carbohydrate into a sofa (4E) conformation.
With torsional restrictions this conformational change is very high in energy.52
ii
Stereoselective glycosylations involving triflate intermediates, albeit with furanosides, has been
reported in an excellent publication by Lowary.54 The need of torsional restraints (i.e. 2,3-epoxide)
of the donor for a stereoselective coupling, although not mentioned in the publication, have been
disclosed in an oral communication.
iii
iv
The axial group at C-2 induces favorable dipole interactions.
The Curtin-Hammett principle: the product composition is controlled by the difference in
standard free energies of the respective transition states, and is not in direct proportion to the
relative concentrations of the conformational isomers in the substrate.
v
Quote from publication: “Thus it seems perfectly reasonable that the more significant preference
for the a-triflate in the mannose series shuts off any Curtin-Hammett kinetic scheme leading to the
a-glycoside via the minor b-triflate, whereas the reduced anomeric effect in the glucose series
permits such a scheme to operate”53
vi
The boat conformation of mannose is more stable than that of glucose due to the axial substituent
at C-2 which will obtain an equatorial position in the boat conformer.
33
Recently Schmidt55 postulated that the stereoselectivity in 4,6-benzylidene
protected mannosylations should not be honored the triflate species D (Figure 3-9), but
rather the twist boat conformation of the activated oxocarbenium derivative (Figure 3-11).
The benzylidene acetal, previously postulated to be responsible for the torsional
stabilization of the triflate intermediate D by disfavoring the flattening of the chair to the
preferred sofa conformation, is now postulated to favor the twist boat over the chair
conformation of the intermediate oxocarbenium ion (Figure 3-11). In the twist boat
conformation the attacking nucleophile will be directed to attack from the axial
orientation due to the anomeric effect, generating an equatorial glycosidic bond when the
product changes back to the chair conformation.
R2
O
O
O
R1
Ph
Ph
O
O
R1
R2
O
OR3
LG
R3OH
-LG
Ph
O
O
R1
Ph
O
O
O
R1
O
R2
R2
Figure 3-11 Dipole interactions postulated by Schmidt to be
responsible for the conformational change of the oxocarbenium
ion.
In the Schmidt model, an electron withdrawing group at the C-2 position would
stabilize the twist boat conformation, by favorable dipole interactions, making the
benzylidene acetal obsolete. This theory was proven in the reactions shown in Figure 3-12
where high stereoselectivity was obtained.55
It is disturbing that the two theories use contradicting terms when proving their
models. Crich claims that the benzylidene group is destabilizing the planar oxocarbenium
ion by disfavoring the sofa conformation, whereas Schmidt asserts that the benzylidene
group is stabilizing the oxocarbenium ion in a flattened twist boat conformation.i It should
be noted that the sofa and the flattened twist boat conformation are quite similar, as can
be seen in Figure 3-11.
i
The flattened twist boat conformation is nonstandard and the structure in Figure 3-11 is a
resemblance from that of the publication.55
34
(1)
BnO
BnO
BnO
R1
O
R2OH
BnO
BnO
BnO
R1
O
OR2
LG
(2)
Ph
O
O
AllO
R1
O
R2OH
Ph
O
O
AllO
R1
O
OR2
LG
1
R : O(SO2)Bn
Figure 3-12 Reactions used to asset the stereodirecting properties
of electron withdrawing groups.
There is, however, one possibility that the Schmidt glycosylations actually are
proceeding via intermediate D (Figure 3-9). Since all donors evaluated have the leaving
group localized axially, the glycosylation could proceed via an SN2 mechanism on the
activated leaving group either via a tight ion pair or a charged aglycon. The torsional
stabilization of the benzylidene group will hinder formation of the oxocarbenium ion A,
and the improved stereoselectivity, due to the electron withdrawing groups at C-2, could
be explained as a further deactivation of the oxocarbenium ion, thus increasing the
probability of an SN2 attack of the acceptor.i56 The influence of electron withdrawing
groups at C-2 has recently been investigated by Crich and stereoselectivity, via a
postulated triflate species D in absence of benzylidene groups, has been reported.57
3.4 Influence of external nucleophiles on reaction outcome
The reactivity of the activated donor can be hampered by the presence of nucleophiles
other than the acceptor. The oxocarbenium ion can interact with nucleophiles forming
stabilized species that are too unreactive to react with the acceptor. These interactions can
vary in strength, being either transient or permanent.
i
It should be noted that a donor with an equatorial group is used by Schmidt. No yield or a/b ratio
is given for the reaction, but the correlation with an identical reaction utilizing a donor with an
axial group is reported. The low a/b ratio obtained (1:3.6) in the reaction renders it a poor model
system.56
35
3.4.1 Transient influence
The addition of Lewis bases to glycosylation reactions will affect the reaction rate. Firstly
the Lewis base will complex with the electrophilic promoter system reducing the
concentration of actual promoter. Secondly the Lewis base will interact with the
oxocarbenium ion of the activated donor stabilizing the developed charge, thus lowering
the reactivity. The interaction of pyridine bases on activated donors has been monitored
using low temperature NMR and it was concluded that pyridinium intermediates were
slowly formed from the corresponding triflate intermediates upon addition of pyridine
base.58 The anomeric configuration of the pyridinium intermediates did not correspond to
that of the glycoside product formed upon addition of alcohol, implying that the
pyridinium intermediates are not involved in any stereodirecting SN2 mechanism. It has
also been shown that Lewis base fragments, generated in the glycosylation mechanism,
can hamper the reactivity of the activated donor to the point that the glycosylation stops
due to the downregulation of the reactive donor. Kahne has shown that the reactive
oxocarbenium B ion formed from the activation of a sulfoxide donor A (Figure 3-13) with
triflic anhydride will react with an unactivated sulfoxide donor forming intermediate C
which collapses into sulfenate D and an oxocarbenium species B, completing its catalytic
circle.59 For some donors the sulfenate intermediate is so stable that it can be isolated,
whereas others are more reactive and forms the wanted glycosidic product, albeit at a
higher temperature than the oxocarbenium species A.59
Tf2O
O
S Ph
A
O
O
TfO
PhSOTf
B
TfO
S Ph
O
O
A
O
B
S Ph
O
O
S Ph
O
O
O
C
Figure 3-13 Sulfenate formation from sulfoxide donors.
36
D
This is just a few examples but one could anticipate that the problem is general for most
glycosylation reactions, but due to a tendency in some published synthetic works allowing
the glycosylation reaction to slowly rise to room temperature before quenching and
working up, these downregulations are difficult to estimate.
3.4.2 Permanent influence
Sometimes external nucleophiles can react to form an intermediate that is so stable that it
will lower the yield of the glycosylation. There have been several reports of the formation
of succinimidyl byproducts in the glycosylation reactions using NIS/TfOH as promoter
system (Figure 3-14)34,60 where the succinimide is originating from the N-iodosuccinimide
of the promoter system. The formation of the succinimidyl byproduct is mainly a problem
when unreactive acceptors are used.
O
N H
O
I
O
SR
O
O
O
N
O
Figure 3-14 Formation of succinimide byproducts in NIS
promoted glycosylations.
Halides have also been observed forming stable byproducts. Kihlberg61 discovered
the glycosyl bromide 12 when activating the corresponding thioglycoside 11 with
MSBi/AgOTf and the sialic acid chloride was isolated when activating thioglycoside 13
with ICl/AgOTf (Figure 3-15).ii The corresponding bromide and iodide of 1 3 was not
isolated when IBr/AgOTf and I2/AgOTf was used as promoter system, probably due to
their instability. However the ethyl sialoside 14 was isolated when ethanol was added
during purification of the reaction mixture, trapping a reactive intermediate believed to be
the sialic acid bromide and iodide.ii
i
The glycosyl bromide was postulated to have been formed from unreacted Br2 from the
preparation of the MSB solution, but even though the MSB preparation was improved, glycosyl
bromide was still formed in the reaction, although to a lesser extent.
ii
Paper II
37
OAc
O
N3
BnO
MSB/AgOTf
O
O
N3
BnO
11
OAc
O
N3
BnO
O
O
N3
BnO
12
SEt
Br
OAc OAc
X
OAc OAc
SPh
AcO
AcHN
AcO
O
13
IX/AgOTf
CO2Me
X : Cl, Br, I
AcO
AcHN
AcO
O
CO2Me
OAc OAc
OEt
AcO
AcHN
O
CO2Me
AcO
14
Figure 3-15 Formation of glycosyl halides in MSB and IX
promoted reactions.
3.5 Influence of donor-structure in interactions with nucleophiles
Up to now the donor has been depicted as a quite promiscuous electrophile, interacting
with any nucleophile present in the reaction mixture. This behavior is not compatible with
some observations made, and theories regarding the structural influence of the donors’
interaction with nucleophiles have emerged.
3.5.1 Compact-Diffuse
It is well established that the hydroxyl groups of carbohydrate derivatives react
regioselectively with acylating and alkylating reagents. The myo-inositol compounds 15
and 16 in Figure 3-16 were subjected to various alkylating and acylating reagents and
selectivities originating from the structure of the electrophile were observed.62 The same
regioselectivity was observed when the myo-inositol derivatives were glycosylated with
donors carrying participating or non-participating protecting groups at the C-2 position
(Figure 3-16).
38
BnO OH
OBn
BnO
OBn
R1O
acylating
agents
alkylating
BnO OH
OBn
BnO
reagents
OBn
HO
2
BnO OR
OBn
BnO
OBn
HO
15
BnO OH
OBn
R3O
OBn
BnO
acylating
agents
BnO OH
HO
BnO
OBn
OBn
alkylating
reagents
4
BnO OR
HO
BnO
OBn
OBn
16
Figure 3-16 Regioselective alkylation or acylation of myo-inositol
compounds.
It was rationalized that the electrophilic pattern of the acylating and alkylating reagents
correlated with those of the donors (i.e. with or without participating groups), described
either as compact or diffuse depending on the degree of charge delocalization in the
transition state.63,64 The theory was used to explain unexpected regioselectivities of a
number of glycosylation reactions.
3.5.2 HSAB
While evaluating a new promoter system it was found that one reaction gave very low yield
compared to the other glycosylations in the study.i The low yield was traced down to a
competing reaction with chloride ions of the promoter system. The difference in chloride
affinity of the activated donor structures was explained by the charge delocalization
pattern of the donors. In all the successful glycosylations, donors with a participating
group were employed, whereas in the unsuccessful glycosylation a non-participating group
was present. The high chloride affinity for one of the donor types but not for the other, was
rationalized as a greater correlation of the hard chloride ion with the hard oxocarbenium
ion, than with the acyloxonium ion, according to the HSAB65 theory (Figure 3-17). Thus
the extent of charge delocalization of the activated donor determined the interaction with
competing nucleophiles.
i
Paper I
39
Hard
O
Cl
O
Br
O
O
O
O
O
O O
O
I
O
Soft
Figure 3-17 The HSAB relationship between halides and activated
donors.
By changing the competing counterion from chloride to bromide, less glycosyl halide
species were formed and the yield increased.i
i
Paper II
40
4
4 NOVEL PROMOTER SYSTEM
The MSB/AgOTf promoter system has been routinely used for thioglycoside activation at
our department. However the preparation of the MSB solution is elaborativei and the
quality of the prepared solution can vary.61 In addition the instability of the prepared MSB
solution renders it less suitable for long time storage. A further complication is that the
only way of assessing the quality of the solution is to perform a glycosylation reaction.
These drawbacks inspired us to search for an alternative way of activating thioglycosides.
Due to the availability of an ICl solution, acquired for a different purpose, we evaluated its
potential as a promoter and found it to be able to activate thioglycosides in high yields.
After a survey of the use of interhalogens in carbohydrate chemistry we found that they
already had been reported to activate thioglycosides.66,67 However these reports did not
combine the interhalogens with silver triflate, a Lewis acid essential for the success of the
glycosylation. Without the use of AgOTf the interhalogens are unable to glycosylate any
but the most simple carbohydrate fragments, due to the decomposition of the
carbohydrates at the high temperatures required for activation.
4.1 ICl/AgOTf
Iodine monochloride (ICl) and silver trifluoromethanesulfonate (AgOTf) are both
commercially available. ICl as a 1 M solution in dichloromethane and AgOTf as a
crystalline salt. It was anticipated that the combination of iodine monochloride with silver
i
The preparation involves distillation of molecular bromine and the handling of dimethyldisulfide,
an extremely foul-smelling chemical.
41
triflate would generate an iodonium species, capable of activating thioglycosides, together
with insoluble silver chloride.
4.1.1 Evaluation
The ICl/AgOTf promoter system was evaluated in a number of glycosylation reactions
(Scheme 4-1).i The protective group pattern of the carbohydrates involved were varied in
order to demonstrate the mildness of the promoter system. Of special interest was the
robustness of the aromatic functional groups, since the iodination of aromatic compounds
has been reported using interhalogens and silver salts.68 To our great relief no iodination
of the protective groups was detected, even the activated aromatic ring of the pMP group
(compounds 23, 6 and 25) was left unreacted.
OAc
O
(1) AcO
AcO
SEt
NHTroc
17
OAc OBn
O
SPh
(2) AcO
20 OAc
18
18
(4)
AcO
4
N3
(5)
AcO
OBn
O
3
OAc OAc
CO2Me
(6) AcO
O
AcHN
AcO
26
OBn
O
HO
BnO
S
EtO
OH
HO
OBz
O
N3
S
76%
OpMP
b)
97%
OpMP
OAc
HO
O
O
AcO
AcO
NHTroc
OAc
b)
27
O OTMSEt
OBn
21
NHTroc
AcO
OTMSEt
NPhth
NTCP
O OpMP
OBn
OAc OBn
O BnO
O
AcO
c)
35-46%
19
OAc 23
98%
OBn
O OTMSEt
OBn
OAc OBn
HO
O
O
AcO
N3
OpMP
NPhth
OAc OBn
HO
O
O
AcO
c)
OBn
3
24 OAc
b)
NTCP
OBn
O
HO
BnO
SCr
78%
OTMSEt
OBn
O
22
NHTroc
a)
NPhth
HO
HO
SEt
82%
OTMSEt
NPhth
OBn
O
HO
HO
OAc OBn
O
SPh
(3) AcO
20 OAc
OAc OBn
O
OBn
O
HO
HO
OBn
O OpMP
OBn
6
OBn
O BnO
O
OBn
O OpMP
OBn
OAc 25
OAc OAc
CO2Me OH
AcO
AcHN
AcO
O
a/b 8:1
O
28
OBz
O
OTMSEt
N3
Scheme 4-1 Reaction conditions; 1.5 equiv donor, ICl/AgOTf;
a) CH3CN, CH2Cl2, -45°C; b) CH3CN, CH2Cl2, -72°C; c) CH2Cl2,
-45°C.
i
Experimental data for entries 1-3 and 6 can be found in paper I; experimental data for entries 4
and 5 can be found in paper III.
42
The yields of the reported glycosylation reactions were all comparable to those reported
using other promoter systems, except for the sialylation reaction (6) which gave low and
unpredictable yields.i We now routinely use ICl/AgOTf as a promoter system for
thioglycoside activation.
4.1.2 Investigationii
The low yieldingiii69reaction using sialic acid xanthate 2670 was unexpected since all other
reactions gave yields comparable to those reported. When the reaction mixture was
analyzed a complex mixture of sialic acid byproducts was encountered (Figure 4-1).
OAc OAc
OH
AcO
AcHN
AcO
OAc
O
OAc OAc
OEt
CO2Me
29
AcO
AcHN
AcO
OAc
O
AcO
AcHN
AcO
O
CO2Me
14
OAc OAc
Cl
CO2Me
AcO
AcHN
O
AcO
5
CO2Me
30
Figure 4-1 Byproducts formed in the sialylation reaction.
The sialic acid hemiacetal 2971 is a known byproduct when moisture is present in the
glycosylation reaction, however since the reaction was performed under dry conditionsiv
its appearance was unexpected. The expected identification of glycal 5, which is a known
byproduct, especially when hindered acceptors are sialylated, was in contrast to the
unexpected isolation of the ethyl sialoside 1472 since no ethanol was present in the
i
The reason for the low yield and the solution to this problem will be discussed in the next
chapters.
ii
Paper II
iii
The reported yield for the same reaction using MSB/AgOTf as promoter system was 61%.69
iv
Acceptor, donor, activated molecular sieves and AgOTf were kept at 10 mbar overnight to remove
traces of moisture. All syringes used in the transfer of solvents and reagents were stored in
dessicator over night. All solvents were distilled immediately (minutes) before use. The reactions
were performed under an argon atmosphere.
43
glycosylation reaction.i The ethanol was eventually traced to the solvents (ethanol:toluene)
used in the purification of the quenchedii and concentrated reaction mixture. Apparently a
stable intermediate is formed in the sialylation reaction which then decomposes to the
ethyl sialoside upon purification. When a less nucleophilic solvent system
(acetone:toluene) was used, no ethyl sialoside was detected. Instead the chloride
derivative 30, together with glycal and hemiacetal which are probably partly formed from
decomposing chloride derivative, could be characterized. The formation of the chloride
derivative 30 was quite unexpected since silver ions were present in the reaction mixture.
Firstly there should be no chloride nucleophiles available due to the insolubility of the
AgCl salt formed from the postulated iodonium formation (reaction 1, Figure 4-2),
secondly compound 30 has been reported to be activated by silver salts and thus should
not be stable in the reaction mixture.73
(1)
I
Cl
Ag
(2)
I
Cl
Ag
(3)
2 I
Cl
AgCl (s)
I
AgCl (s)
LB
ICl2
I
LB
I
Figure 4-2 Reactions of iodine monochloride.
A simple experiment was carried out in order to investigate the reactivity of the ICl/AgOTf
system. To a solution of AgOTf in acetonitrile and dichloromethane was added ICl, and to
our big surprise no AgCl salt precipitated (entry 1; Figure 4-2). It was only upon addition
of a Lewis base such as diisopropylamine or xanthate donor 26 that a precipitate was
formed (entry 2; Figure 4-2).iii Silver is thus incapable of forming the AgCl salt by itself.
The iodonium species is though capable of formation from ICl alone (entry 3; Figure 4-2)
as shown by spectroscopic investigations74,75 and in practice by Field.iv To further
i
The characterization of the inseparable ethyl sialosides was delayed since they initially were
thought to be derivatives of the xanthate donor, which also contains an ethoxy fragment in the
xanthate aglycon.
ii
Diisopropylamine is added to the reaction mixture in order to neutralize the acid formed and to
trap the excess iodonium species.
iii
The experiment was conducted both at room temperature and at the same temperature as the
sialylation reaction investigated, -72°C .
iv
Iodine monochloride has been shown to activate thioglycosides without the aid of silver ions,
albeit at a higher temperature.66,67
44
investigate the role of silver ions we repeated the sialylation of acceptor 27 by xanthate
26, but this time without any AgOTf present. Interestingly we were only able to detect
traces of product 28 together with chloride 30 and some glycal 5, indicating that silver
plays a major role both in the activation of ICl and in the abstraction of chloride
nucleophiles, at -72°C.i
4.1.3 Donor-structureii
The reason for the low yield of the sialylation reaction (reaction 6, Scheme 4-1) was traced
down to the interference of chloride nucleophiles, but the question of why only the sialic
acid donor is affected remains. Since all the donors that performed well in the
glycosylation reactions carried a participating group next to the anomeric center and the
sialic acid donor 26 did not, we now hypothesized that the interaction of the participating
groups with the anomeric position of the donor influenced the reaction outcome. Their
interaction might result in one of the following scenarios; (a) the glycosyl chloride formed
from a donor carrying a participating group is unstable and reacts to form product, or (b)
glycosyl donors carrying a participating group are less prone to be transformed into
chlorides. To examine scenario (a) i.e. if the corresponding chloride derivatives of the
donors of reactions (1) to (5) (Scheme 4-1) were unstable intermediates, donor 3176, the
chloride derivative of donor thioglycoside 20, was subjected to standard glycosylation
conditions together with acceptor 18 (reaction 1, Scheme 4-2).iii Interestingly no product
was formed and the chloride donor 31 could be recovered, indicating that chloride donor
31 not is a reactive intermediate in the glycosylation of thiodonor 20 and acceptor 18
(entry 2; Scheme 4-1) and thus that the donor is less prone to react with chloride
nucleophiles.
i
Iodine monochloride has been shown to activate thioglycosides without the aid of silver ions,
albeit at a higher temperature.66,67
ii
iii
Paper II
Donor 31 has been used in a glycosylation reaction with AgOTf as promoter, albeit at a higher
temperature (-45°C to room temperature)76
45
(1) AcO
OAc OBn
O
31
Cl
32
HO
HO
OAc
OAc OAc
O
SCr
(2) AcO
OBn
OBn
O
18
OH
AcO
OTMSEt
NPhth
OBn
O
33
OAc OBn
O BnO
O
AcO
OAc
OTMSEt
0-35%
AcO
AcO
OAc
OAc
O
21
NPhth
O OTMSEt
OBn
BnO
NPhth
BnO O
34
O
OBn
OTMSEt
Scheme 4-2 Reaction conditions; 1.3 equiv, ICl/AgOTf, CH3CN,
CH2Cl2, -72°C.
To further test hypothesis (b) donor 3277, without a participating group, was subjected to
standard reaction conditions. Only traces of product 3477 were isolated, and all donor had
been converted to the corresponding chloride derivative.
4.1.4 HSAB
We now wondered why there was such a large discrepancy between the interactions of the
chloride nucleophiles with the two types of donori. One explanation could be the character
of the electronic charges, according to the Hard Soft Acid Base (HSAB) theory,65 of the
activated donors. The donors without a participating group stabilize the developed charge
on two atoms, the anomeric carbon and the ring oxygen, forming an oxocarbenium ion
(Figure 4-3). Whereas the donors with a participating group delocalize the charge on an
additional atom, the carbonyl carbon of the participating ester group. The oxocarbenium
ion has been reported to be a hard electrophile,78 in contrast to the softer, i.e. more
delocalized, charge of the acyloxonium ion. We now speculate that the hard chloride
nucleophile is more matched to the harder electrophile of the donors without a
participating group, and thus form the glycosyl chloride byproduct to a greater extent. The
donors with a participating group form an electrophile with a softer character, not as well
matched to the hard chloride nucleophile.
i
i.e. donors with or without a participating group next to the anomeric center.
46
Hard
O
Cl
O
Br
O
O
O
O
O
O O
I
O
O
Soft
Figure 4-3 The HSAB correlation of activated donors with halide
ions.
The different selectivities of donors with or without participating groups have been
noted by others. Fraser-Reid has reported glycosylation reactions where the presence or
absence of a participating group in the donor controlled the regioselectivity. The
selectivity could not be explained by purely steric factors. Instead, a model describing the
electronic properties of the carbocations (depicted as Compact and Diffuse) was used to
explain the reaction outcome.i
4.1.5 IX/AgOTfii
With the proposed mechanism for the formation of the byproduct at hand, the obvious
way of minimizing its formation would be to change the counterion of the promoter
system to a softer one.
OAc OAc
CO2Me
AcO
AcHN
AcO
O
26
S
EtO
OH
HO
S
OBz
O
27
N3
IX/AgOTf
OTMSEt
ICl
IBr
I2
OAc OAc
CO2Me OH
AcO
AcHN
AcO
35-46%
74%
67%
O
O
a/b 8:1 28
OBz
O
OTMSEt
N3
Scheme 4-3 Reaction conditions; 1.5 equiv donor, IX/AgOTf,
CH3CN, CH2Cl2, -72°C.
The sialylation reaction in Scheme 4-3 was performed with the two softer promoter
systems, IBr/AgOTf and I2/AgOTf, and indeed the yields were improved. The IBr system
i
The theory of the Compact-Diffuse model is discussed in greater detail on page 38.
ii
Paper II
47
gave an excellent yield of 74% whereas molecular iodine performed slightly worse at 67%,
but the recovery of unactivated donor 26 indicated that the lower yield was due to lower
reactivity of the promoter system, and not to a greater extent of halide interaction with the
donor. i Besides from being less reactive, molecular iodine is not commercially available in
solution and thus has to be prepared,ii increasing the unattractiveness of its usage. When
analyzing the remains of the activated donor no sialic acid bromide or iodide was
encountered, only the glycal 5 and hemiacetal 29 derivatives could be characterized. Since
these derivatives could have been formed directly in the reaction we could not assess if the
activated donor still experienced problems with halide interaction. However when we
added ethanol to the purification system we were again able to trap the reactive
intermediates as the ethyl sialoside 14, indicating the presence of sialic acid bromide and
iodide in the respective reactions. In order to rule out the possibility of the increased yield
of the IBr/AgOTf promoted reaction to be due to a more labile halide intermediate, i.e. if
sialic acid bromide is formed to the same extent as the chloride derivative but is activated
by silver ions to form product, the sialic acid bromide was synthesized and subjected to
standard glycosylation conditions (Scheme 4-4). Since no product at all could be detected,
and the ethyl sialoside 14 could be isolatediii we felt confident that the increased yield of
the IBr/AgOTf promoted reaction was due to a decreased interaction of halide ions with
the activated donor.
OAc OAc
Br
AcO
AcHN
O
AcO
35
OH
CO2Me
HO
27
OBz
O
OAc OAc
CO2Me OH
OTMSEt
AcO
AcHN
AcO
N3
O
O
28
OBz
O
OTMSEt
N3
Scheme 4-4 Reaction conditions; 1.5 equiv donor, IBr/AgOTf,
CH3CN, CH2Cl2, -72°C.
i
The corresponding salts of the IBr and I2 promoted reactions are more insoluble than that of the
ICl promoted reaction (i.e. AgBr and AgI vs. AgCl), thus a lowered concentration of halide ions
could also be a factor in improving the glycosylation yield. However the different reactivities,
correlated with the donor-structures, towards halide nucleophiles can not be explained by
variances in halide concentration.
ii
It is advantageous to handle the interhalogens as a liquid reagent since the transfer and
measurement can be performed in syringes. Molecular iodine has to be purified by sublimation and
the weighing and transfer then introduces a risk of contamination.
iii
Due to the instability of the bromide donor 35 (it decomposes within hours even under vacuum)
we believe that the unreacted donor decomposes to a great extent upon workup and the indirect
detection through the ethyl sialoside 14 is a good evidence of the bromides stability in the reaction
mixture.
48
In order to investigate whether other donor types not carrying a participating
group would benefit from changing to a softer counterion, the galactosylation that
previously was reported to not yield any product when performed with ICl/AgOTf
(reaction 2, Scheme 4-2) was repeated with IBr/AgOTf as promoter. To our satisfaction we
were now able to obtain a product,i although in a modest yield, indicating that the change
in counterion resulted in less unfavourable interactions with the activated donor.
We also investigated the IBr/AgOTf and I2/AgOTf systems on donors with
participating groups to see if the change to a more matched counterion would result in a
yield lower than that obtained with the ICl/AgOTf system. The glycosylation of donor 20
and acceptor 22 (reaction 3, Scheme 4-1) which had been performed in 76% yield using
ICl/AgOTf gave a slightly lower yield of 71% when using IBr/AgOTf under otherwise
identical conditions.ii This result indicates that donors with a participating group are not
as sensitive to halide nucleophiles as the ones without, however ICl/AgOTf is still superior
to IBr/AgOTf in activating donors with participating groups. Molecular iodine and silver
triflate did not activate the donor to any greater extent,iii and the material decomposed
when the reaction temperature gradually was increased to room temperature, again
showing the low reactivity of the I2/AgOTf system.
4.1.6 Optimization of the IX/AgOTf systemiv
Since we were able to detect the presence of the bromide intermediate 35 even in the high
yielding sialylation using IBr/AgOTf, there are room for further optimizations. By
increasing the equivalents of AgOTf from 1 to 1.5 (with respect to IBr), thus further
suppressing the concentration of nucleophilic halide species, we were able to increase the
yield from 74% to an excellent 89% (Scheme 4-5).
i
The yield has increased from 0% (ICl) to 35% (IBr) but is still modest. We wish to point out that
the reaction conditions are not optimized but chosen for best comparison with the other
glycosylations performed in the study.
ii
Unpublished results.
iii
Less than 10% of the donor was activated after 2h at –72°C according to TLC analysis.
iv
Paper II
49
OAc OAc
CO2Me
AcO
AcHN
AcO
O
OH
HO
R
OBz
O
IBr/AgOTf
OTMSEt
AcO
AcHN
N3
O
AcO
27
R : Xanthate
R : SPh
OAc OAc
CO2Me OH
O
OTMSEt
N3
a/b 8:1 28
R : Xanthate
R : SPh
26
36
OBz
O
89%
97%
Scheme 4-5 Reaction conditions; 1.5 equiv donor, IBr/AgOTf,
CH3CN, CH2Cl2, -72°C.
To investigate the importance of the leaving group, we examined the glycosylation
properties of thiophenol donor 36. To our great surprise the already optimized yield was
further increased to 97%. Since we estimated that the interactions of the halide
nucleophiles had been eradicated by the change to the softer IBr system and the increase
of AgOTf equivalents, the increased yield had to depend on other factors. We therefore
performed an investigation of the reaction kinetics of the two donors. Small-scale
reactions were conducted and stopped at different times (using cyclohexene and
diisopropylamine) and then analyzed by NMR. Interestingly, the xanthate donor 26 seems
to follow a faster initial reaction rate than the thiophenyl donor 36. After 1 min 50% of the
xanthate donor is consumed, compared to 15% of the thiophenyl donor (Figure 4-4).
100
80
Unreacted donor / %
60
40
20
0
0
5
Time / min
10
15
Figure 4-4 Plot showing the amount of unreacted donor left in
the reaction mixture as a function of time; (-g-) xanthate donor
26; (-n-) thiophenyl donor 36.
50
However, while the thiophenyl donor is consumed in about 10 min, the activation of the
xanthate donor slows down and trace amounts of unreacted donor can be isolated even
after 1 h. We therefore speculate that the higher yield using the thiophenyl donor
compared to the xanthate donor is due to the different rates of consumption (i.e.
formation of reactive species). By assuming that the rate determining step is not the
formation of the oxocarbenium species,i but in its subsequent reaction, either to the glycal
5 or the glycoside, the fast initial activation of the xanthate donor which gives rise to a
higher concentration of oxocarbenium species, will favor the unimolecular elimination
over the bimolecular coupling with the acceptor (k1>k2>>k3=k4; Figure 4-5).ii79
OAc
OAc
O
AcO
AcHN
k3
OAc OAc
CO2Me
AcO
AcHN
AcO
O
OAc OAc
k1
LG
k2
AcO
AcHN
AcO
AcO
CO2Me
5
+
-H
CO2Me
O
ROH
k4
OAc OAc
CO2Me
AcO
AcHN
O
OR
AcO
Fast
Slow
Figure 4-5 Postulated reaction kinetics of sialylation reactions.
The complex reaction kinetics of the xanthate donor could be explained by the
Lewis base character of the formed xantenyl iodide (structure A, Figure 4-6), which can
accommodate a second iodonium ion forming the unstable compound B. This buffering
effect will lower the activation rate of the xanthate donor and give rise to complex reaction
kinetics.iii By increasing the equivalents of iodonium species (e.g. IBr/AgOTf) from 1 to 2
the buffering effect should be overridden. However the better performance of the
thiophenol donor 36 made us abandon any optimization of the xanthate donor, besides an
increased activation rate would benefit the glycal over the glycoside formation.
i
The term species is used to emphasize that the activated donor is stabilized either by the solvent or
by Lewis bases present.
ii
Bimolecular nucleophilic substitution reactions that proceed through carbocation reaction
intermediates are not common, however they have been observed in the literature.79
iii
This equilibrium will affect the k2 reaction constant in Figure 4-5.
51
A
B
I
S
O
AcO
26
S
EtO
OAc OAc
CO2Me
AcO
AcHN
I
I
I
S
S
OEt
S
EtO
S
I
OAc OAc
AcO
AcHN
AcO
CO2Me
O
Figure 4-6 Xantenyliodide capable of buffering iodonium species.
4.2 IBr/AgOTf
After a thorough investigation we were able to increase the yield of the model reaction
(Scheme 4-5) from an unpredictable 35-46% using ICl/AgOTf to excellent 97% using
IBr/AgOTf and an alternate leaving group. We now felt confident enough to test the
optimized promoter/leaving group system in more challenging reactions, to fully evaluate
its potential.
4.2.1 Evaluation
The novel IBr/AgOTf promoter system was evaluated in a number of sialylation reactions
(Scheme 4-6). The yields were all better or much better than those reported for similar
reactions using other promoter systems. Sialylation of acceptor 37 was performed in 44%
yield,i this is in great contrast to previous attempts with MSB/AgOTf as promoter system
and xanthate 26 as donor, where no product at all could be detected.ii The diol 39 was
sialylated in 88% yieldi, also in great contrast to the published yields ranging from 31 to
78%.80 The hindered acceptor 2 was sialylated in excellent 59% yield with the axial
thiophenol donor 7. iii The stereoselectivity of all sialylation reactions performed is
comparable to the ones reported, indicating that the promoter system and the leaving
i
Paper II
ii
Unpublished results by Ulf Ellervik.
iii
Paper III
52
groups employed are of minor importance. We now routinely use IBr/AgOTf as a
promoter system in sialylation reactions.
BnO
(1) N3
HO
OAc OAc
OBn
AcO
AcHN
OBn
O
O
OH
O
O
OBn
37
O
AcO
NHAc
OBn
O
OpMP
36
44%
a)
BnO
CO2Me
OBn
SPh
OBn
O
N3
CO2Me
OAc OAc
O
O
AcO
AcHN
38
AcO
NHAc
OBn
O
O
O
O
OBn
OH
OpMP
a/b/lactone 66:17:17
OH
(2) HO
OBn
O BnO
O
OBn
39
OBn
O
OBn
OH
OBn
OAc OAc CO2Me
O BnO
O
O
O
88% AcO
OBn
40
b) AcHN
AcO
OBn
36
OTMSEt
O
OBn
OTMSEt
a/b 95:5
OAc OAc
(3)
N3 OH
CO2Me
HO
TFAHN
O
BzO
AcO
TFAHN
OTMSEt
2
O
AcO
SPh
7
AcO
TFAHN
59%
N3
CO2Me
OAc OAc
CO2Me
a)
O
AcO
CO2Me
O
TFAHN
41
HO
O
OTMSEt
OBz
Scheme 4-6 Reaction conditions; IBr/AgOTf, CH3CN, CH2Cl2,
-72°C; a) 3 equiv donor; b) 1.5 equiv donor.
4.3 Optimization
During the investigations leading to the interhalogen promoter systems we have been able
to isolate dixantogen81,82 (the disulfide of ethyl xanthate) and phenyl disulfide83 in the
respective donor reaction mixtures. The isolation of disulfides indicates that an
electrophilic sulfur species is present in the reaction mixture.80 We postulate that the
species responsible are the sulfenyl iodides formed from the reaction between iodonium
ion (from IX/AgOTf) and thio donor.19 Thus, the role of the sulfur leaving group is not
only to be reactive enough to be activated by the thiophilic species but also to form a
“second” thiophilic species in the reaction mixture.
53
4.3.1 Equivalents of IXi
In order to study the postulate that a second thiophilic species, formed from the
thioaglycon, was taking part in the activation of donor molecules, we subjected a high
yielding model reaction (Scheme 4-7) to various equivalents of ICl together with a
unimolecular amount of acceptor, donor and AgOTf.
AcO
OAc OBn
O
SEt
OBn
O
HO
BnO
NHTroc
4
3
OAc OBn
O BnO
O
AcO
OpMP
OBn
NHTroc
OBn
6
O OpMP
OBn
Scheme 4-7 Reaction conditions; 1 equiv donor, ICl/AgOTf,
CH2Cl2, -45°C.
Several small scale reactions were run and then quenched at various time intervals after
which the conversion of donor into product was determined by NMR, using the pM P
group as internal standard. A graph was constructed illustrating the substoichiometric
need of ICl for complete conversion of donor 4 into product 6 (Figure 4-7). Apparently
only 0.5 equivalents of ICl is sufficient for almost complete conversion, and 0.25
equivalents of ICl is capable of almost 50% conversion. These results indicate that 1
equivalent of IClii is capable of activating 2 equivalents of donor.
Conversion (%)
100
1.5 eq
75
0.5 eq
50
0.25 eq
25
0
0
10
20
30
40
50
60
70
Time (min)
Figure 4-7 Graph showing the substoichiometric requirements of
ICl in a glycosylation reaction.
i
Paper III
ii
The ICl:AgOTf stoichiometry is 1:2.
54
Inspired by these findings we constructed a mechanistic scheme capable of illustrating the
proposed reaction pathway (Figure 4-8). We suggest that the interhalogen forms an
activated complex, depicted as an iodonium ion in Figure 4-8, with the silver ion which
then reacts with a thioglycoside A forming an activated complex B. This reactive complex
then collapses into an oxocarbenium ion C, which can proceed to form the product, and a
sulfenyl iodide compound. The latter can be activated by a second silver ion forming a
reactive sulfonium ion species, which can activate a second thioglycoside A, forming the
reactive intermediate D. This reactive complex collapses into an oxocarbenium ion C and
a disulfide compound.
I X
AgX
O
Ag
S
R
A
"
" I
R "
"
S
R
S
I
O
S
R
O
AgI
Ag
S
R
D
B
I
R
S
S
O
S
R
R
C
Figure 4-8 The proposed reaction pathway of an IX/AgOTf
promoted thioglycoside activation.
The mechanistic scheme in Figure 4-8 explains several observations made while
performing glycosylations using interhalogens. Firstly the stoichiometry of the added
interhalogen needed for donor activation is explained. Secondly, the formation of
disulfides using substoichiometric amounts of interhalogen can be explained. Thirdly, we
have observed that fractions obtained from the column purification of the reaction
mixture slowly darkens over time. When the fractions have been analyzed the
corresponding disulfide of the donor aglycon has been identified. We now believe that it is
the sulfenyl iodide species that decomposes forming disulfide and molecular iodine,
55
responsible for the strong color. Due to the lesser reactivity of the sulfonium species
compared to the iodonium species, there will be an accumulation of sulfenyl iodide in the
reaction mixture, when equimolar amounts of interhalogen is used. The lower reactivity of
the sulfonium ion has consequences for unreactive donors. When performing a sialylation
of acceptor 2 using donor 7 (reaction 3, Scheme 4-6) a substoichiometric amount of IBr
failed to activate all donor. The low reactivity of donor 7 is due to the electron withdrawing
trifluoroacetyl group (TFA) on the C-5 nitrogen.
A similar reaction pathway has recently been reported by Wong, where a
substoichiometric amount of promoter is used to activate thioglycosides. Both the
BSP/Tf2O promoter protocol84 and a modified NIS-TfOH-AgOTf system85 are
demonstrated as viable promoter systems. Unfortunately no mechanistical data, or
characterization of byproducts from the promoter system (such as the disulfide in Figure
4-8) supporting the postulate, is presented. However these findings together with the
results presented above, indicates that the reaction pathway in Figure 4-8 is a general one.
4.4 Alternative Lewis Acids
After evaluating the different interhalogens in the IX/AgOTf promoter system we now
turned to the Lewis acid part, which has been concluded to be of great importance in a
previous chapter (4.1.2), where it was shown that the Lewis acid must be capable of both
activating the interhalogen and complexing the halide nucleophiles. In order to find
alternative Lewis acids we therefore scanned a variety of metal triflate salts, investigating
their capabilities in the two areas; nucleophilicity and electrophilicity.
4.4.1 Electrophilicity
In order assess the different Lewis acids capabilities of generating an iodonium species
from ICl and IBr, an electrophilicity model was constructed.
NHAc
NHAc
L.A. / IX
CH3CN
42
I
43
Figure 4-9 Model reaction for asserting the electrophilicity of the
complex formed between IX and various Lewis acids.
56
All the Lewis acids were scanned by performing an electrophilic aromatic substitution on
acetanilide with ICl or IBr (Figure 4-9). The results are presented in the two charts; Figure
4-10 and Figure 4-11.i The Lewis acids used were; HOTf, AgOTf, LiOTf, Cu(OTf)2,
Mg(OTf)2, Yb(OTf)2, Sc(OTf)2, Zn(OTf)2, Hg(OTf)2, Sn(OTf)2, Al(OTf)3, In(OTf)3.
100%
75%
43
42
50%
25%
0%
H
Sc
Sn
Al
Yb
No
LA
Mg
Mn
Li
Cu
Ag
Zn
In
Hg
Figure 4-10 The conversion of acetanilide 42 to iodoacetanilide
43 mediated by various Lewis acids.
The iodination reaction using ICl indicated that mercuric triflate was by far the most
reactive Lewis acid. This was not surprising since mercury is a known halophile, however
the environmental aspects of the use of mercury salts, render mercuric triflate of little
practical use. More interesting was the activating capability of indium triflate, exceeding
that of silver triflate. The other activating Lewis acid, zinc, matched the activating
capabilities of silver. Interestingly hydrogen, scandium and tin all quenched the iodination
reaction. Aluminum, ytterbium, magnesium, mangane, lithium and copper, all matched
the blank iodination (only ICl added to reaction) thus not affecting the reactivity of ICl.
i
See paper IV for experimental details.
57
100%
75%
44
43
42
50%
25%
0%
Sn
H
Sc
Yb
Al
Mn
Li
Zn
--
Mg
Cu
In
Ag
Hg
Figure 4-11 The conversion of acetanilide 42 to iodoacetanilide
43 and bromoacetanilide 44 mediated by various Lewis acids.
The iodination reaction using IBr was more complex due to a competing
bromination reaction. The bromonium ion originated from molecular bromine formed
from the dissociation of IBr (Figure 4-12).i As can be seen from Figure 4-11, the
bromination reaction is much faster than the iodinating reaction, and in the uncatalyzed
reaction only the brominated product is formed. Only silver, indium and mercury are
capable of inducing an iodonium species reactive enough to form iodoacetanilide.
I
Br
Slow
NHAc
NHAc
43
I
Slow
I
42
I
NHAc
Br Br
Fast
44
Br
Figure 4-12 The equilibrium of IBr, generating molecular
bromine responsible for brominating acetanilide.
i
For a complete investigation of the bromination of acetanilide using IBr see supplementary
information on page 86.
58
The equilibrium forming molecular bromine in Figure 4-12 is probably very slow at
the low temperatures used in the glycosylation reactions reported previously. Since the
low reactivity of molecular iodine, formed in equimolar amounts compared to molecular
bromine, would not be able to activate all donor in the model reaction (Scheme 4-3) due to
the low reactivity. However the formation of bromosugars from thioglycosides by the
reaction of IBr at room temperature can proceed via molecular bromine.86 In summary,
based on the electrophilic properties, the triflate salts of indium and zinc have emerged as
viable options for the substitution of silver triflate in ICl promoted reactions, and indium
alone has emerged as a substitute for silver triflate in IBr promoted reactions.
4.4.2 Nucleophilicityi
The interhalogens readily dissociate into ionic fragments, according to measurements of
electrical conductivity (Figure 4-13).87 The triatomic anion IX2- has been studied by
spectroscopical methods and it is anticipated that it is the major anionic species in the
complex equilibrium of interhalogens.74,75,88
3I
X
I2
X
I
X2
X : Cl, Br
Figure 4-13 Equilibrium postulated to be responsible for the
formation of nucleophilic halide species.
We therefore speculate that the nucleophile responsible for the conversion of activated
donor into the corresponding glycosyl halide is the IX2- ion. In order to scan the Lewis
acids ability of complexing the nucleophile we conducted a spectroscopic titration of ICl
and IBr solutions respectively. Since no absorptivity constants are available for the
triatomic anions, we were not able to obtain any numerical values of the equilibrium
constants, however the data can be related to that of the silver titrations, since we were
searching for a Lewis acid with similar or better complexing capabilities. The ability of the
Lewis acids to hamper the nucleophilicity of the ICl and IBr systems was thus investigated
by titrating a solution of either ICl or IBr with a solution of a Lewis acid and measuring the
difference in absorbance of the ICl2- and IBr2- ions respectively.ii The resulting variance in
i
Unpublished results
ii
See appendix on page 95 for further details.
59
absorption of the various Lewis acids is presented in Figure 4-14 and Figure 4-15
respectively.
Absorbance
1.5
1
Li
Mg
Ag
Yb
Al
Mn
0.5
0
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
Equivalents
Figure 4-14 The absorbance of ICl solution at 227 nm titrated
with various Lewis acids.
Based on the ability of suppressing the absorption at 227 nm of an ICl solution, the
Lewis acids can be divided into four groups; 1. lithium and magnesium; 2. silver; 3.
ytterbium, aluminium and mangane; 4. hydrogen, indium, zinc and scandium. The Lewis
acids in group 1 did not affect the absorbance of the ICl2- ion, the one in group 2
suppressed the nucleophile only after 1 equivalent of Lewis acid had been added. The ones
in group 3 were able to suppress the absorbance of the nucleophile even at
substoichiometric amounts of Lewis acid. The ones in group 4 were able to totally
suppress the absorbance of the nucleophile even at substoichiometric amounts (omitted
from Figure 4-14 for clarity). Mercury, tin and copper were all excluded from the
evaluation due to the formation of complex absorption spectra.i
i
See appendix on page 95 for absorbtion spectra.
60
1.5
Li
Absorbance
Mg
Mn
1
Al
Yb
H
Sc
Ag
0.5
Cu
Zn
In
0
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
Equivalents
Figure 4-15 The absorbance of IBr solution at 256 nm titrated
with various Lewis acids.
Based on the ability of suppressing the absorption at 256 nm of an IBr solution, the
Lewis acids can be divided into four groups; 1. lithium, magnesium, mangane, aluminum
and ytterbium; 2. hydrogen and scandium; 3. silver; 4. copper, zinc and indium. The Lewis
acids in group 1 did not affect the absorbance of the IBr2- ion to any greater extent, the
ones in group 2 suppressed the absorbance to a slightly greater extent. The one in group 3
was able to suppress the absorbance of the nucleophile even at substoichiometric amounts
of Lewis acid. The ones in group 4 were able to totally suppress the absorbance of the
nucleophile even at substoichiometric amounts. Mercury and tin were excluded from the
evaluation due to the formation of complex absorption spectra.i
In summary several Lewis acids were able to suppress the absorption of the ICl2ion, only lithium and magnesium performed worse than silver. The best Lewis acids were
hydrogen, indium, zinc and scandium. It is interesting that hydrogen, which is formed in a
glycosylation reaction, was able to totally suppress the absorption. The IBr2- ion was not as
promiscuous in its interactions with the Lewis acids, and only copper, zinc and indium
outperformed silver.
i
See appendix on page 95 for absorbtion spectcra.
61
4.4.3 Evaluationi
The most promising candidates from the nucleophilicity and electrophilicity investigations
were, for ICl, indium and zinc and for IBr, indium and copper. Of these indium and copper
were evaluated in a number of glycosylations in order to assess their potential as a
promoter system (Scheme 4-8).
OAc OAc
CO2Me
(1) AcO
AcHN
AcO
O
(2)
AcO
OAc OBn
O
4
HO
R
R : Xanthate
R : SPh
OH
26
36
SEt
NHTroc
27
HO
BnO
OBz
O
OTMSEt
N3
OBn
O
3
OpMP
OAc OAc
CO2Me OH
AcO
AcHN
AcO
O
O
a/b 8:1 28
OAc OBn
O BnO
O
AcO
OBn
NHTroc
OBz
O
OTMSEt
N3
OBn
6
O OpMP
OBn
Scheme 4-8 Reaction conditions; see Table 1.
The sialylation of acceptor 27 was performed using the promoter systems and
donors listed in Table 1. Without any Lewis acid the activation of donor 26 is slow, for
both ICl and IBr, and no product at all can be detected (entries 1 and 4). The addition of
Cu(OTf)2 to ICl increases the reactivity of the promoter system and all donor is consumed.
Copper is however not so efficient in removing the nucleophiles and no product at all can
be detected (entry 2). When Cu(OTf)2 is added to the IBr promoted reaction the reactivity
is not increased, and the same amount of donor can be recovered, but the nucleophilicity
of the halide species is slightly retarded, giving product in modest 10% yield (33% based
on activated donor; entries 5 and 6). Indium, together with ICl, was previously proved to
be the most promising Lewis acid, but the ICl/In(OTf)3 promoted reaction did not give any
product at all (entry 3). The failure of the Lewis acids to complex the nucleophilic species
forced us to choose a different model system, less sensitive towards chloride nucleophiles.
i
Unpublished results.
62
Table 1 Reaction conditions for Scheme 4-8
Entry
Donor
Acceptor
Promoter
Yield
Donor recovery
1
26
27
ICl a
0%
50%
2
36
27
ICl/Cu(OTf)2a,f
0%
0%
0%
0%
3
36
27
ICl/In(OTf)3a,d
4
26
27
IBr
0%
70%
10%
70%
10%
70%
0%
20%
33%
0%
a
5
26
27
IBr/Cu(OTf)2a,f
6
36
27
IBr/Cu(OTf)2a,f
7
4
3
ICl
8
4
3
ICl/AgOTf
9
4
3
ICl/In(OTf)3b,d
40%
0%
10
4
3
ICl/AgOTf c,g
97%
0%
3
ICl/In(OTf)3c,d
16%
50%
3
ICl/In(OTf)3c,e
16%
50%
11
12
4
4
b
b,f
Donor:acceptor 1.5:1; a) CH3CN, CH2Cl2, -72°C; b) CH3CN, CH2Cl2,
-45°C; c) CH2Cl2, -45°C; d) 0.4 equiv Lewis acid; e) 1.2 equiv Lewis
acid; f) 1.0 equiv Lewis acid; g) 2.0 equiv Lewis acid
The donor 4 with a participating group, hence not as sensitive towards chloride
nucleophiles, was activated with a number of promoter systems. The activation with ICl
alone gives no product at all and 20% of the donor could be recovered (entry 7),
illuminating the need for a Lewis acid. The addition of silver increases the reactivity and
the disaccharide 6 can be isolated in 33% yield (entry 8). The yield is very low and it was
later found that the low reactivity was due to the acetonitrile solvent.i More interesting
was the fact that the In(OTf)3 promoted glycosylation produced the disaccharide 6 in 40%
yield, also suffering from interactions with the nitrile solvent (entry 9). When we later
were able to increase the yield to 97%, excluding the acetonitrile, using AgOTf as promoter
system (entry 10) we anticipated that In(OTf)3 would perform equally well. However the
yield dropped to a disappointing 16%, and 50% of the donor could be recovered (entry 11).
We explain this result with the low solubility of the triflate salts in dichloromethane.
AgOTf, also suffering from low solubility, does however form an insoluble precipitate of
AgCl which drives the equilibrium towards the consumption of silver ions. In(OTf)3, which
does not form a solid precipitate but rather a soluble complex with the chloride
i
The incompatibility of the NTroc donor with nitrile solvents is discussed in greater detail on page
28.
63
nucleophile, suffers from the insolubility. This was exemplified by the fact that tripling the
equivalents of In(OTf)3 results in a reaction mixture (entry 12) identical to the one
obtained when using 0.4 equiv In(OTf)3 (entry 11; Table 1).
A new model system, based on the following criteria;
•
the donor should have a participating group next to the anomeric center;
•
the solvent must be capable of solubilizing the Lewis acids used;
•
the activated donor should be “inert” to the solvents used;
should be constructed, however due to time restraints, this project will be continued by
others.
4.4.4 Summary
In summary we have been unable to find an alternative for AgOTf in the novel IX/AgOTf
promoter system. The model reactions used for scanning the large amount of Lewis acids
have provided us with conclusions of varying quality. The data received from the
measurements in electrophilicity seems to correlate well with the actual performance in
the glycosylation reaction.i The data received from the nucleophilicity measurements have
not correlated equally well since the indium promoted activation of donor 36 (entry 3)
failed to remove “any” of the chloride nucleophiles resulting in no product at all. Since
only two Lewis acids capable of lowering the halide concentrationii were tested in the
sialylation it is hard to fully evaluate the model systems ability to predict the halide ion
interaction with the activated donor, but one could speculate if the decrease in absorption
of the IX2- ion has any correlation with the nucleophilicity in the glycosylation reaction.
Perhaps the glycosyl halides are formed from another nucleophilic species, or perhaps the
equilibrium complexing the halide species is very fast, providing the reaction mixture with
a low but constant concentration of nucleophilic species (Figure 4-16).
L.A.
I
L.A.
X2
I
X2
X : Cl, Br
Figure 4-16 Lewis acids complexing the nucleophilic halide
species.
i
ii
Cu(OTf)2 does not activate IBr but ICl and In(OTf)3 is capable of activating both ICl and IBr.
Cu(OTf)2 was able to lower the IBr2- absorption and In(OTf)3 to lower both IBr2- and ICl2-
absorption.
64
5
5 CARBOHYDRATE CANCER VACCINES
In recent years carbohydrates have been found to play important roles in many biological
systems and subsequently the interest in using carbohydrates in drug development has
increased. Carbohydrates have especially been anticipated to play an important role in the
development of cancer vaccines, due to the tendency of tumor cells to dramatically alter
their glycosylation pattern compared to that of the corresponding normal cell (i.e. a way of
distinguishing the tumor cells from the normal cells when directing the immune system).
Carbohydrate structures that are overexpressed on the surface of tumor cells have been
identified and several research groups are working on cancer vaccines based on these
structures.
5.1 Cancer vaccines
The intention of a cancer vaccine is to teach the immune system to recognize antigens that
it, for some reason, is unable to detect. A cancer vaccine normally consists of three
components;
•
an antigen that the immune system is to be directed against.
•
a carrier that will transport the antigen to antigen presenting cells (APC) in the
patient.
•
i
an adjuvanti that will boost the immune response in a nonspecific way.
In general any additive that enhances the actions of a medical treatment is termed adjuvant.
65
The choice of antigen is the key element and can be further divided into the following
categories and subcategories;
•
•
Multiple antigens
o
Autologous
o
Allogeneic
Specific antigens
o
anti-idiotype
o
direct immunization
The cancer vaccine strategies based on multiple antigens normally relies on tumor cells as
a source of antigens for the immunization of patients. The antigens are obtained either
from the patient to be treated (autologous antigens) or from another patient (allogeneic
antigens). Two new cancer vaccines have reached the market; Melacine89 and M-Vax90,
which both rely on antigens derived from tumor cells. The vaccines are very expensive and
the survival rate of the patients is not greatly increased compared to other methods of
fighting cancer. However the improvement in quality of life, compared to patients
undergoing chemotherapy, justifies the use of these vaccines. The low immunogenicity of
tumor cells has led to strategies where specific antigenic structures are injected into the
patient together with various adjuvants boosting the immunogenicity. The antigenic
structures can either be specific antigens that are presented to the immune system
directly, or anti-idiotype antibodies that present the antigen indirectly (i.e. the antibodies
carry an imprint of the antigen). The anti-idiotype antibody strategy is normally used
when the antigens are difficult to obtain in sufficient quantities, since the antibodies can
be produced by cell lines instead of being synthesized or extracted from an antigen source
such as a tumor.
5.2 Carbohydrate antigens
Carbohydrates have been used as antigens in several cancer vaccine studies. However the
low immunogenicity initiated in the model systems rendered their use quite limited. Now
several methods of enhancing the immunogenicity of carbohydrates have been developed,
opening the field towards cancer vaccines based on carbohydrate antigens. Livingston has
developed a system consisting of an immunogenic protein (KLH), to which the
carbohydrate antigen is covalently linked and an adjuvant (QS-21) that is capable of
immunizing patients.91 Estevez has developed a transport system that is able to deliver
66
carbohydrate antigens without binding them covalently to a carrier. Apparently the low
immunogenicity obtained in previous investigations was due to the carrier blocking the
antigenic epitopes of the carbohydrate.92
5.2.1 Gangliosides
Gangliosides are acidic glycosphingolipids containing at least one sialic acid residue. They
are overexpressed on the cell surface of tumor cells, which makes them interesting targets
for cancer vaccines. The gangliosides’ systematic names are complex and abbreviations are
used, all starting with the letter G for ganglioside.93,i94No vaccines based on a carbohydrate
antigen have yet reached the market but there are clinical trials ongoing. Livingston has
been able to immunize patients with vaccines consisting of ganglioside structures GD3 or
GM2, with clinical trials still ongoing.95 Gomez has used an animal model for
immunization with ganglioside structure GM3,96 and clinical trials on patients are
ongoing.97
OH OH CO HOH OH
2
O
HO
O
O
AcHN
OH
HO
OH
O
O
HO
OpMP
OH
45
-H2O
HO
HO
OH OH
O
HO O
O
O
O
OH
O
O
HO
OpMP
OH
46
AcHN OH
Figure 5-1 Example of lactonization equilibrium of gangliosides.
i
The gangliosides are named according to the Svennerholm abbreviations, where the ganglioside
family is indicated by the letter G, the number of sialic acids by the letters M-mono, D-di, T-tri or Q
for tetrasialoglycosides. A number is then assigned at the end of the name. The number is
correlated to the number of saccharide structures in the compound but originally referring to the
migration order of the individual compounds in a certain chromatographic system.94
67
5.3 Lactam analogs
Due to the low pH environment of tumor cells, gangliosides are thought to transform into
their corresponding lactone form (Figure 5-1). The lactones' equilibrium changes the
overall three-dimensional shape of the ganglioside in a dramatic way. However,
immunizations using lactonized gangliosides as antigen will result in antibodies of low
specificity due to the hydrolytical instability of the lactone. Despite its instability, the
corresponding lactone of a ganglioside has been used to immunize patients, and it was
concluded that the lactonized form is a stronger immunogen than the open form.95
The need for hydrolytically stable analogs of ganglioside lactones has led to the
development of two strategies. An ether analog of GM3 has been synthesized (Figure
5-2).98 The ether functionality induces stability towards hydrolysis but the conformational
change induced by the reduction of the carbonyl group renders this cyclized ganglioside
less of a structural analog.
HO
OH OH
O
HO O
O
O
HO
O
HO
OH
O
OTMSEt
OH
47
AcHN OH
Figure 5-2 Ether analog of GM3.
The monolactams GM2, GM3 and GM4 and the disialoside monolactam GD3
(Figure 5-4) have been synthesized.69,99 The lactam functionality is hydrolytically stable
under the conditions encountered in the body, and the structural similarities of lactones
and lactams have been shown both using computational methods and NMR
investigations.100,101
HO
HO
OH OH
O
HO O
O
NH
O
O
HO
OH
O
OH
48
AcHN OH
Figure 5-3 Lactam analog of GM3.
68
OTMSEt
The lactam analogs have been tested in biological systems only in preliminary
studies.102 To complete the growing arsenal of lactam analogs of gangliosides (Figure 5-4),
we decided to synthesize bis-lactam analogs of GD3.
HO
OH OH
O
HO O
O
NH
O
HO
HO
OH
O
O
HO
OH OH
O
HO
OTMSEt
O
NHAc
HO O
O
48
NH
O
HO
OH
OH
O
OH
O
O
HO
OTMSEt
OH
49
AcHN OH
AcHN OH
GM2
GM3
O
OH OH
HO
AcHN
O
HO
H
N
O
OH OH
O
HO O
O
O
O
O
HO
OH
O
OH
50
HO
OTMSEt
HO
OH OH
O
OTMSEt
HO O
O
NH
O
51
AcHN OH
AcHN OH
GM4
GD3
Figure 5-4 Synthesized lactam analogs of various gangliosides.
69
6
6 SYNTHESIS OF A GD3 BIS-LACTAM
In chapter 4 we introduced the ICl/AgOTf and IBr/AgOTf promoter systems for
thioglycoside activation, and showed the relevance of the donor-structure for the choice of
promoter system. We now demonstrate the generality of the promoter system by the
combined use of IBr and ICl, together with AgOTf, in the synthesis of a biologically
important carbohydrate. We selected two isomers of the GD3 bis-lactam as target
compounds (Figure 6-1), since a retrosynthetic analysis revealed donor-structures suitable
for both IBr and ICl activation. This chapter presents IX/AgOTf as a general promoter
system in the synthesis of a GD3 bis-lactam.
6.1 Ganglioside synthesis
The gangliosides have been synthesized by several different research groups and various
strategies have emerged. Some strategies are discussed below, followed by a retrosynthetic
evaluation and a report of the total synthesis.
6.1.1 Target compounds
Two bis-lactam analogs of the corresponding bis-lactone variants of GD3 were chosen as
target compounds. The 1’’-2’;1’’’-9’’-bis-lactam 5 2 and the 1’’-4’;1’’’-9’’-bis-lactam 53
(Figure 6-1). The pMP group was chosen as an anomeric protecting group for several
reasons. Firstly it is stable under the reaction conditions normally employed in
carbohydrate synthesis. Secondly the pMP group can be transformed into a leaving group
71
for conjugation with linkers, necessary for biological studies. Thirdly the pMP groups
absorption in the UV region facilitates the detection of compounds 52 and 53 in HPLC
purification.
OH
O
OH
OH
HO
AcHN
O
HO
OH
HO
AcHN
O
OH
O
HO
H
N
O
O
O
HO
AcHN
H
N
O
OH
O
HO
O
NH
OH
OpMP
O
OH
O
52
OH
NH
O
O
AcHN
HO HO
O
53
OH
O
HO
O
OH
OH
O
OH
OpMP
Figure 6-1 Target compounds.
6.1.2 Approaches in ganglioside lactam synthesis
The disialoside-ganglioside family has been systematically synthesized in a number of
publications. Hasegawa103, Goto104 and Boons105 used block syntheses to synthesize GD3,
whereas Hossain99 and Schmidt106 used a linear approach. When consulting these
publications it is clear that the main synthetic challenge of the target compounds lies in
the construction of the sialic acid glycosidic bonds, especially the 2-8-bis-sialo bond.
Hasegawa relied on commercial sources of disialic acid and thus avoided the construction
of this bond, whereas the other groups relied on participating groups or stereoelectronic
effects in its construction. The use of thiophenol as an auxiliary group for the induction of
stereoselectivity, is used in three of the four syntheses of the 2-8-bis-sialic bond
(reactions 1, 2 and 3 in Figure 6-2).i The fourth reaction is utilizing a novel protective
group strategy that induces stereoselectivity from a remote position. The introduction of a
trifluoroacetyl (TFA) group on the amide functionality will dramatically influence the
stereoselectivity of the sialylation. No explanation for this effect has yet been presented,
but the introduction of an electron withdrawing group at the C-5 position has been
suggested to increase the stabilization of the intermediate axial nitrilium ion, responsible
for the stereodirecting nitrile effect,ii allowing the acceptor to interact with the activated
donor in an SN2 like reaction.17
i
The use of auxiliary groups are discussed in greater detail on page 29.
ii
The nitrile effect is discussed in greater detail on page 19.
72
The nitrogen atom of the acetamido functionality can undergo reaction with
electrophilic reagents from the promoter system. In order to minimize unwanted side
reactions the amine functionality can be manipulated in different ways. Donor 54 was bisacetylated and donor 60 and acceptor 61 were TFA protected. These electron withdrawing
protective groups render the nitrogen atom less nucleophilic.
OAc OAc
CO2Me
(1)
AcO
Ac2N
O
AcO
O
(2)
AcO
AcHN
BnO
N3
HO
AcHN
BzO
SEt
SPh
54
O
P(OEt)2
O
OH
CO2Me
SPh
CO2Me
O
56
O
O BnO
OBn OBn
55
CO2Me
O
57
OAc OAc
Cl
AcO
O
AcHN
AcO 58
(3)
(4)
O
AcO
O
OH
OBn
OBn
O OBn
O
O BnO
OBn
CO2Me
SPh
AcO
AcHN
AcO
CO2Me
F
O
59
O
AcO
60
54%
OPiv
OBn OH
AcO
TFAHN
50%
OBn
OAc OH
OAc OAc
SMe
AcO
TFAHN
OBn
O OTMSEt
O
OTBS
OH
HO
AcHN
TBSO
OBn
CO2Me
SPh
CO2Me
49%
55%
OTMSEt
61
Figure 6-2 Literature procedures to form a 2-8 bis-sialo bond.
The research-groups using the block synthesis strategy all experienced low yields
when glycosylating with the bis-sialic acid donors (Figure 6-3). The yields ranging from 31
to 42% are remarkably lower than those reported with monosialic acid donors and the
same acceptors. Acceptor 39 which was sialylated in 31% with donor 66 (entry 3; Figure
6-3) was for instance, sialylated in 88% yield using monosialic acid donor 36 (entry 2;
Scheme 4-6)
73
OAc
AcO
(1) AcHN
O
AcO
62
OAc
(2)
AcO
TFAHN
OAc
SPh O
AcHN
AcO
AcHN
AcO
OAcOAc
66
OH
OH
SPh
O
O
TFAHN
AcO
AcO
64
O
HO
CO2Me
SPh
O
OPiv
HO
O
O
OPiv
39%
O OMe
OPiv
63
OBn
OAc CO2Me
O
OAc
OH
F
O
AcO
(3)
OAc
OAc CO2Me
HO
CO2Me
OBn
O BnO
O
OBn
42%
OBn
O OMe
OBn
65
O
OH
SPh
O
O
AcHN
AcO AcO
OBn
O BnO
O
HO
CO2Me
OBn
39
OBn
31%
O OTMSEt
OBn
Figure 6-3 Reported sialylations, using bis-sialic acid donors, in
the synthesis of GD3.
The formation of ganglioside lactams has been reported in the literature.
Ercégovic100 and Hossain99 used an azido functionality to form the 2-8-bis-sialo lactam
(Figure 6-4). Wilstermann69 and
Ellervik107
used
either
an
azido
or
a
trichloroethoxycarbamate group to form the lactam at the non-sialic portion of the
ganglioside.
OH
HO
AcHN
N3
OH CO2Me
CO2Me
O
HO
O
SPh O
AcHN
67
HO HO
Ph3P,
THF,
H2O
87%
OH
HO
AcHN
O
OH
H
N
CO2Me
O
HO
OTMSEt
O
SPh
AcHN
68
HO
O
OTMSEt
HO
Figure 6-4 Reported synthesis of 2-8-bis-sialo lactam.
74
6.1.3 Retrosynthetic analysis
O
O
H
N
SR1
O
O
CO2Me
A
O
HO
NH2
O
O
NH2
OpMP
HO
O
E
HO
O
O
OpMP
F
O
2
SR
HO
O
NH2
B
D
NH2
OpMP
HO
O
SR3
C
Figure 6-5 Carbohydrate fragments obtained from a retrosynthetic
analysis of the target compounds.
Due to the structural resemblance of the two target compounds a convergent block
synthesis was planned. A bis-sialic fragment containing the lactam functionality and an
appropriate leaving group at the anomeric position would be a suitable donor molecule for
the synthesis of both target compounds (Figure 6-5; compound A ). The galactose
structure differs (i.e. by the NH or OH groups) in the two target compounds. In target
compound 52 an amino functionality at the galactose C-2 position will serve as the lactam
handle B, whereas the galactose structure in target compound 5 3 requires an amino
functionality at the C-4 position of compound C. Both galactose structures then share the
structural requirements of a hydroxyl group at the C-3 position, for coupling with
fragment A, and a leaving group at the anomeric position. The glucose fragment D is the
same in the synthesis of both target compounds and requires a hydroxyl group at the C-4
position, for coupling with the galactose fragment B or C, and a pMP group for protection
of the anomeric position. The B and C fragments will then combine with fragment D to
form the disaccharide fragments E and F respectively. The E and F fragments can then
combine with disialoside A to form the corresponding tetrasaccharides.
75
6.2 Synthesis of GD3 bis-lactami
Based on the retrosynthetic observations vide supra the two GD3 bis-lactam derivatives
were constructed using a convergent block synthesis strategy. Unfortunately time was
running short and the final deprotections leading to the target compounds have not been
performed at the time of printing this thesis. However all the glycosylating steps have
been performed using the IX/AgOTf system, thus fulfilling the initial goal of
demonstrating the capabilities of the novel promoter system.
6.2.1 Fragment A
OAc
H
N
O
OAc
AcO
TFAHN
O
AcO
SPh
O
TFAHN
AcO BzO
O
CO2Me
69
Figure 6-6 Fragment A.
Compound 6 9 was chosen as the retrosynthetic equivalent of fragment A . The
introduction of TFA protected amide functionalities will ensure high stereoselectivity
according to Boons.105 The TFA group will be removed in the deprotection steps of the
final compound. Compound 8105 was synthesized from the known donor 7108 (Scheme
6-1). By changing to the IBr/AgOTf promoter system the published yield (75%)105 could be
increased to 93%.ii It should be noted that donor 7 is too unreactive to be activated with in
situ generated phenyl sulphenyl iodide,iii and thus equimolar amounts of IBr must be
used. Compound 8 was then de-O-acetylated using standard procedures to give
compound 70105 which was used without purification in the next step.
i
Paper III
ii
Due to the acid lability of compound 8 the yield is lowered when the reaction is performed at
larger scale. The upscaling problems of this reaction are discussed in greater detail at page 21.
iii
The mechanistic scheme leading to the formation of the second thiophilic species and its
influence on the equivalents of promoter added to the glycosylation, is discussed in greater detail
on page 54.
76
OAc OAc
SPh
AcO
TFAHN
O
AcO
7
OH OH
HO
TFAHN
O
HO
O
BzO
CO2Me
OTMSEt
70
OH OH
HO
TFAHN
CO2Me
CO2Me
OTMSEt
72-93%
a)
OAc OAc
CO2Me
AcO
TFAHN
O
AcO
O
78%
c)
O
HO
TFAHN
HO
e)
HO
TFAHN
72
87%
OTMSEt
71
OH
O
BzO
b)
a:b 88:12
CO2Me
O
N3
78%
8
98%
OTMSEt
d)
CO2Me
OTMSEt
2
Scheme 6-1 Reaction conditions; a) IBr/AgOTf, CH3CN, -40°C,
1 h; b) NaOMe/MeOH; c) acetone, 2,2-dimethoxypropane, pTSA,
30 min; d) (i) pyridine, BzCl, 0°C, 30 min, (ii) HOAc, MeOH, reflux
2 h; e) (i) TsCl, CH2Cl2, pyridine, 16 h, (ii) NaN3, DMF, 60°C, 20 h.
The introduction of the azide functionality at the C-9 position was more
problematic than anticipated. Ercégovic100 had previously been able to selectively tosylate
the C-9 position on the acetamide analog of compound 70 in an excellent yield of 91%.
When the tetraol 70 was subjected to the same the reaction conditions the reaction was
too slow to be of practical value. The regio-selectivity was also poor, the 4- and 9-hydroxyl
groups seemed to be equally reactive, resulting in a reaction mixture consisting of
unreacted 70 together with the products of mono and bis tosylation of the C-4 and C-9
position. Increasing the reaction temperature and changing the solvent from a
dichloromethane:pyridine 2:1 mixture to pure pyridine improved the reactivity and
selectivity somewhat, but not enough to make the procedure a viable route to 2. Since the
4- and 9-hydroxyl groups are almost equally reactive we decided to use an acetal to
selectively protect the 9-hydroxyl group. Both isopropylidene109 and benzylidene acetal105
have been used on similar substrates. Compound 7 0
was treated with
2,2-dimethoxypropane in acetone under acidic conditions to give the a anomer of 71 in an
excellent 78%i yield. The 4-hydroxyl group was then selectively benzoylated using 1.2
equiv benzoylchloride in pyridine at 0°C for 30 minutes, and then the isopropylidene
acetal was removed by refluxing in methanol and acetic acid, to give compound 72 in 87%
yield. The 7-hydroxyl group was deliberately left unreacted since Ercégovic has shown that
acetate groups at the C-7 position are prone to migrate to the C-8 position under azide
i
Since the starting material consisted of an 88:12 anomeric mixture the theoretical yield of the a
anomer would be 78/88 = 89%.
77
substitution conditions.100 It was also shown that the 7-hydroxyl group is of such low
reactivity that the 7,8-hydroxy-sialic acid acceptor would selectively be sialylated at the
C-8 position. With the reactive 4-hydroxyl group protected as a benzoate, the selective
tosylation proceeded in high yield and total regioselectivity even with five equiv of tosyl
chloride in dichloromethane and pyridine at room temperature for 16 h. The crude
tosylation mixture was subjected to sodium azide in DMF to give acceptor 2 in 78% yield.
OAc
7
59%
2
AcO
TFAHN
a)
OAc
AcO
TFAHN
O
OAc
O
AcO
AcO
CO2Me
O
CO2Me
O
H
N
O
TFAHN
HO BzO
73
N3
OAc CO2Me
O
TFAHN
HO BzO
41
OAc
73%
OTMSEt c)
AcO
TFAHN
O
OAc
O
AcO
70%
d)
O
86%
b)
OTMSEt
H
N
R
O
TFAHN
AcO BzO
R = OAc
74
R = SPh
75
O
CO2Me
Scheme 6-2 Reaction conditions; a) 3 equiv donor, IBr/AgOTf,
CH2Cl2, CH3CN, -72°C; b) Ph3P, THF, H2O; c) first: TFA, CH2Cl2;
then: pyridine, Ac2O; d) PhSH, CH2Cl2, BF3Et2O.
Acceptor 2 was then coupled with 3 equiv of donor 7, using IBr and AgOTf as
promoter system, to form disialoside 41 in an excellent yield of 59%, as a single
diastereomer. The high yield and total stereoselectivity is attributed to the TFA groups of
both the donor and acceptor. The azido functionality of compound 41 was reduced using
H2 on Pd/C under high pressure,i but no lactamized product was formed at all. Only when
the hydrogenated compound was de-O-acetylated, using NaOMe/MeOH, did
lactamization take place, and then only in poor yield. When compound 41 was subjected
to triphenylphosphine in THF and water at 60°C, the compound cyclized to form lactam
73 in 86% yield. Whether it was the increased temperature or a higher nucleophilicity of
the intermediate phosphinimide, compared to a free amine, that facilitated the
lactamization is not known. However when azide 41 was reduced at room temperature
using triphenylphosphine in THF and water an unknown product which had not
undergone lactamization was formed. This unknown product lactamized and formed a
product identical to the one obtained by high-pressure hydrogenation vide supra. Lactam
73 was then converted to donor 7 5 via the acetate 74 using standard conditions. The
reaction converting the acetate 74 to the thiol 75 is extremely sensitive towards unpurified
i
100 bar in a Parr high-pressure apparatus.
78
reagents. Conversions of carbohydrate acetates to thioglycosides are generally not so
sensitive, but when standard (unpurified) reagentsi were used a low and irreproducible
yield of about 40% was obtained, and compound 7 4 had reacted to form the
corresponding glycal and hemiacetal derivatives. When all reagents were distilled the yield
was improved to 70% (a:b 84:16). The anomeric mixture could be separated using flash
chromatography.
6.2.2 Fragment B
AcO
OAc OBn
O
SEt
4 NHTroc
Figure 6-7 Fragment B.
The known compound 4110 was chosen as the retrosynthetic equivalent of fragment B. The
Troc group at the C-2 position will ensure a b-coupling with fragment D, and will be
deprotected to form the amine functionality. The acetates will be deprotected after
glycosylation, forming a 3,4-diol which will react selectively at the more reactive
3-position.110
6.2.3 Fragment C
N3
OBn
O
AcO
24
SCr
OAc
Figure 6-8 Fragment C.
The known compound 24110 was chosen as the retrosynthetic equivalent of fragment C.
The azido group can be reduced to form the amine functionality. The acetate at the C-2
position ensures a b-coupling with fragment D. The acetates can easily be deprotected
after formation of the glycosidic bond, and the resulting 2,3-diol will react selectively at
the more reactive 3-position.110
i
The same reagents were used before and after to transform other carbohydrate acetates to
thioglycosides in acceptable yields.
79
6.2.4 Fragment D
O
O
HO
Ph
O
OpMP
76 OH
O
O
BnO
94% Ph
a)
O
OpMP
88%
b)
77 OBn
OBn
O
HO
BnO
OpMP
3 OBn
Scheme 6-3 Reaction conditions; a) BnBr, NaH, DMF;
b) NaCNBH3, HCl, THF.
Compound 3 was chosen as the retrosynthetic equivalent of fragment D. The synthesis
starts from the known compound 76111 which was benzylated, using standard conditions,
to form compound 77. Compound 77 was then subjected to NaCNBH3, under acidic
conditions, to form compound 3.
6.2.5 Assembly of fragments B and D
4
3
97%
a)
OAc OBn
O BnO
O
AcO
OBn
O OpMP
OBn
NHTroc
6
OH
75%
b)
HO
OBn
O BnO
O
OBn
O OpMP
OBn
NHTroc
1
Scheme 6-4 Reaction conditions; a) 1.5 equiv donor, ICl/AgOTf,
CH2Cl2, -45°C; b) Guanidinium nitrate/NaOMe
The first attempts to glycosylate acceptor 3 with donor 4 using ICl/AgOTf in CH3CN and
CH2Cl2 gave only low yields of approximate 50%. After analyzing the reaction products, a
dehydroimidazole compound was isolated.i Apparently the activated donor reacted with
the solvent, trapping the intermediate b-nitrilium ion by an attack of the carbamate
nitrogen and forming byproduct 10. When acetonitrile was left out and the reaction was
performed in neat dichloromethane, the yield rose to excellent 97%.ii Disaccharide 6 was
then de-O-acetylated using a guanidinium nitrate buffer112 in 75% yield to give compound
1 (fragment E).
i
ii
The formation of the dehydroimidazole byproduct is discussed in greater detail on page 28.
Since AgOTf is poorly soluble in dichloromethane it was anticipated that the reaction would form
large amounts of galactosyl chloride, which is formed when the reaction is run in the absence of
AgOTf, however no chloride derivatives were found.
80
6.2.6 Assembly of fragments C and D
N3
98%
24
3
a)
AcO
OBn
O BnO
O
OAc
25
OBn
N3
90%
O OpMP
OBn
HO
b)
OBn
O BnO
O
OH
78
OBn
O OpMP
OBn
Scheme 6-5 Reaction conditions; a) 1.5 equiv donor, ICl/AgOTf,
CH2Cl2, -45°C; b) NaOMe/MeOH
Donor 24 and acceptor 3 was glycosylated using reaction conditions identical to that of
the reaction in Scheme 6-4. To our great satisfaction the disaccharide 25 was obtained in
near quantitative yield (98%). The omission of acetonitrile in this reaction was not due to
the fear of the activated donor forming a byproduct similar to the dehydroimidazole
derivative 10 of donor 4, but merely to its redundancy since we now knew that CH2Cl2 is
capable of mediating the promoter system by its own despite the insolubility of AgOTf in
this solvent. The disaccharide 25 was de-O-acetylated using standard reaction conditions
to give the acceptor building block 78 in 90% yield (fragment F).
6.2.7 Assembly of fragments A and E
OAc
69
1
40%
AcO
TFAHN
O
OAc
O
AcO
H
N
CO2Me
O
TFAHN
AcO BzO
O
O
79
OH
NHTroc
O
O BnO
OBn
OBn
O OpMP
OBn
Scheme 6-6 Reaction conditions; 1 equiv donor, IBr/AgOTf,
CH2Cl2, CH3CN, -72°C.
The tetrasaccharide 79 was obtained in 40% yield from the glycosylation of unimolecular
amounts of acceptor 1 and bis-sialic acid donor 69. The yield, albeit low, is comparable to
other sialylations using bis-sialic acid donors (Figure 6-3).
81
6.2.8 Assembly of fragments A and F
OAc
69
78
44%
AcO
TFAHN
H
N
O
OAc
O
AcO
CO2Me
O
TFAHN
AcO BzO
O
O
80
N3
OH
O
O BnO
OBn
OBn
O OpMP
OBn
Scheme 6-7 Reaction conditions; 1 equiv donor, IBr/AgOTf,
CH2Cl2, CH3CN, -72°C.
The tetrasaccharide 80 was obtained in 44% yield from the glycosylation of unimolecular
amounts of acceptor 78 and bis-sialic acid donor 69. The yield, albeit low, is comparable
to other sialylations using bis-sialic acid donors (Figure 6-3).
6.2.9 Lactamization
OAc
80
47%
AcO
TFAHN
AcO
O
OAc
O
H
N
OBn
O
O
TFAHN
AcO BzO
NH
OBn BnO
O
O
O
O
OpMP
O
OBn
OH
81
Scheme 6-8 Reaction conditions; Ph3P, THF, H2O; 72 h, 60°C.
Tetrasaccharide 80 was lactamized in 47% yield, by stirring with Ph3P in THF and water
for 72 h at 60°C.i
Due to time restrictions no further synthetic steps towards the GD3 bis-lactam
analogs can be presented in this thesis.
i
For experimental details see Supplementary information on page 85.
82
7
7 SUMMARY AND FUTURE PERSPECTIVES
To summarize we have developed a novel promoter system for thioglycoside activation
and evaluated its potential in a number of glycosylations. The importance of the Lewis
acid (i.e. AgOTf) has been investigated in model studies, where the electrophilic and
nucleophilic properties have been quantified. No viable substitute for AgOTf was found,
however some promising results were obtained using In(OTf)3 and ICl. The results
indicate that In(OTf)3 is an alternative to AgOTf in ICl mediated glycosylations using
donors with participating groups in acetonitrile solvents.
The two promoter systems ICl/AgOTf and IBr/AgOTf are optimized towards
different donor-types, and their combined use was demonstrated in the synthesis of two
GD3 bis-lactam analogs.
Unfortunately I was not able to complete the synthesis of the GD3 target
structures, however the capability of the IX/AgOTf system in promoting the glycosylations
leading to the two tetrasaccharide fragments was demonstrated, which was the main
objective of the synthesis. The deprotection of the two tetrasaccharides as well as
biological testing will be performed in near future.
83
8
8 SUPPLEMENTARY INFORMATION
8.1 Experimental data for bis-lactam analog of GD3 81
4-Methoxyphenyl (4,7,8,9-tetra-O-acetyl-3,5-dideoxy-5-trifluoroacetamido-Dglycero-a-D-galacto-non-2-ulopyranosyl)-(2Æ8)-(7-O-acetyl-9-amino-4-Obenzoyl-3,5,9-trideoxy-5-trifluoroacetamido-D-glycero-a-D-galacto-non-2ulopyranosyl)onate)-(2Æ3)-(4-amino-6-O-benzyl-4-deoxy-b-Dgalactopyranosyl)-(1Æ4)-2,3,6-tri-O -benzyl-b- D -glucopyranoside 1’’’–9’’,
1’’–4’-bis-lactam (81).
Compound 80 (33 mg, 0.018 mmol) was dissolved in THF (1.5 mL) and water (0.4 mL),
followed by addition of Ph3P (15 mg, 0.057 mmol). The stirred reaction mixture was then
heated to 60 °C in a sealed vessel for 72 h. The reaction mixture was then concentrated
and purified on sephadex LH-20, and a short plug of silica (1:3 toluene-EtOAc) to give 81
(15 mg, 47%). 1H NMR (CDCl3) d 8.45 (d, 1H, J = 10.3), 8.01-7.96 (m, 2H), 7.53-7.80 (m,
1H), 7.20-7.46 (m, 22H), 7.05-7.13 (m, 3H), 6.83-6.88 (m, 2H), 5.95-6.01 (m, 1H), 5.75
(td, 1H, J = 5.2, 10.8), 5.45-5.51 (m, 1H), 5.20-5.30 (m, 3H), 5.10 (dd, 1H, J = 2.3, 10.7),
5.05 (d, 1H, J = 7.9), 4.96-5.02 (m, 1H), 4.97, 4.72 (ABq, 1H each, J = 10.4), 4.83, 4.75
(ABq, 1H each, J = 12.2), 4.83, 4.38 (ABq, 1H each, J = 12.2), 4.50-4.60 (m, 2H), 4.57,
4.38 (ABq, 1H each, J = 12.1), 4.20-4.35 (m, 3H), 4.08-4.18 (m, 5H), 4.00 (d, 1H, J =
10.2), 3.74-3.86 (m, 2H), 3.80 (s, 3H), 3.64-3.70 (m, 1H), 3.50-3.60 (m, 3H), 3.33-3.42
(m, 2H), 3.15-3.23 (m, 1H), 2.97 (dd, 1H, J = 6.9, 14.2), 2.88 (dd, 1H, J = 5.4, 13.0), 2.40
(t, 1H, J = 12.6), 2.19 (s, 3H), 2.14 (s, 3H), 2.08 (s, 3H), 1.98-2.06 (m, 2H), 2.06 (s, 3H),
2.03 (s, 3H).
13C
NMR (CDCl3) d 172.19, 171.31, 171.02, 170.56, 169.94, 166.02, 163.87,
85
155.18, 151.64, 139.69, 138.60, 138.51, 138.07, 133.71, 130.13, 129.72, 128.96, 128.93,
128.91, 128.60, 128.48, 128.35, 128.24, 128.13, 128.06, 127.99, 127.76, 117.03, 115.03,
114.58, 101.20, 100.59, 97.35, 95.07, 82.96, 81.96, 76.19, 75.63, 75.58, 74.75, 74.51, 74.16,
73.92, 73.85, 72.67, 71.92, 71.33, 69.93, 68.39, 68.28, 66.91, 66.75, 62.95, 60.85, 56.02,
50.83, 50.23, 50.17, 41.19, 40.80, 39.11, 32.31, 30.13, 21.49, 21.23, 21.02, 20.95, 20.83,
14.63, 14.56 LRMS calcd for C86H92F6N4O30Na (M+Na): 1797.6, found: 1797.
8.2 Determination of the anomeric configuration of sialic acid
The stereoselectivity in sialylations can not be determined by standard methods (i.e. by
measuring the 3JH-1,
H-2
coupling frequency) since there is no proton on the anomeric
carbon. The anomeric configuration of sialic acids are instead determined by the 3JC-1 - H-3ax
coupling constant (Figure 8-1).113
OAc OAc
CO2Me
AcO
AcHN
AcO
O
AcO
AcHN
AcO
R
Heq
Hax
C1
180O
OAc OAc
R
C4
60o
Hax
R
C4
Heq
O
CO2Me
Heq
O
Heq
60o
O
R
C1
Hax
Hax
60o
Figure 8-1 Determination of anomeric configuration of sialic acid.
In an a (equatorial) sialoside the 3JC-1 - H-3ax coupling will be large due to the 180° angle
between C-1 and Hax, whereas in the b (axial) sialoside the coupling of C-1 and the protons
at C-3 will be small due to the 60° angle. The 3JC-1 - H-3ax coupling of a-sialosides is normally
around 6 Hz whereas the b-sialoside has a smaller coupling constant around 1 Hz. The
protons of the ester methyl group must be decoupled, since they will complicate the
coupling pattern.
8.3 Bromination of acetanilide using IBr
The bromination of aromatics using IBr has been extensively studied and the electrophilic
bromine species has been derived to molecular bromine formed from IBr (Figure
86
8-2).114,115 The low reactivity of molecular iodine renders the iodination of aromates quite
slow, and often only the brominated product is isolated.
I
Br
NHAc
Slow
NHAc
43
I
I
Slow
I
NHAc
42
Br Br
Fast
44
Br
Figure 8-2 The equilibrium of IBr, forming molecular bromine.
As can be seen in Figure 4-11 the four Lewis acids capable of activating IBr show
unexpected variances in reaction outcome. The Hg(OTf)2 activated reaction only produced
the iodinated aromatic compound, whereas In(OTf)3 and AgOTf produced a mixture of
brominated an iodinated compounds. The Cu(OTf)2 activated reaction on the other hand
produced the brominated product alone. Thus it seems as if we can control the reaction
pathway in Figure 8-2 by adding the appropriate Lewis acid (Figure 8-3). This kind of
selective activation of interhalogens has not been reported in literature, and we therefore
decided to further investigate the Cu(OTf)2 activated bromination.
NHAc
NHAc
I
42
43
Hg(OTf)2
I
Br
NHAc
44
Cu(OTf)2
Br
Figure 8-3 Lewis acids capable of influencing the electrophilic
atom of IBr.
The bromination of aromatic compounds using IBr was studied using a different promoter
system more suitable for NMR investigations.i Firstly the reaction kinetics using various
i
Acetanilide was excluded as a model system due to the incapabilities of obtaining reproducible
NMR spectra of the reaction mixture. Apparently the acetamido functionality of acetanilide is very
sensitive towards the pH, rendering it a poor model system for the monitoring of reaction kinetics.
87
amounts of Cu(OTf)2 was investigated (Scheme 8-1), the results are presented in Figure
8-4.i To our great surprise we found that 1 equiv of IBr was able to brominate more than
0.5 equiv of the aromatic model compound (Scheme 8-1). This stoichiometric relationship
is impossible if the bromination proceeds via the accepted reaction pathway (Figure 8-2).
Br
I
Br
Cu(OTf)2
Scheme 8-1 Reaction conditions; 1 equiv IBr, X equiv Cu(OTf)2,
CD3CN, RT.
100
Conversion (%)
75
1.0 eq CuOTf
0.75 eq CuOTf
0.50 eq CuOTf
0.25 eq CuOTf
0 eq CuOTf
50
25
0
0
50
100
150
200
Time (min)
Figure 8-4 Graph showing the Cu(OTf)2 dependency of the
bromination reaction.ii
Firstly the thought of an umpolung of the IBr molecule induced by Cu(OTf)2 tickled our
minds, but the problem turned out to have a much less exotic explanation. After searching
i
Representative procedure for the NMR reactions with mesitylene:
To 0.4 ml CD3CN was added 0.0025 ml (0.018 mmol) mesitylene followed by 1.7 mg (0.0045
mmol) Cu(OTf)2. The reaction was initiated by the addition of 0.018 ml IBr (0.018mmol).
ii
The time interval is minimized for clarity, after 8000 min the reaction promoted by 1.0 equiv
Cu(OTf)2 has reached 92% conversion.
88
the literature we found that Cu(II) ions are capable of acting as an oxidant, converting
iodide ions into molecular iodine and Cu(I) derivatives (Figure 8-5).116
2 Cu 2+
4I -
2 CuI + I2
Figure 8-5 Oxidative regeneration of molecular iodine from
iodide ions, by the action of Cu(II) ions.
We therefore assumed that Cu(II) was converting the bromine ions into molecular
bromine in a similar way. The oxidizing effects of Cu(II) was demonstrated by the
reactions in Figure 8-6. IBr is capable of brominating mesitylene by itself (entry 1; Figure
8-6), the addition of soluble bromine ions to molecular iodine does not initiate any
bromination (entry 2; Figure 8-6), indicating that IBr is not formed by bromine ions and
molecular iodine alone. The addition of a Cu(II) source to a mixture of molecular iodine
and bromine ions however initiates the formation of brominated mesitylene (entry 3;
Figure 8-6), indicating the presence of an oxidative process, mediated by Cu(II).
Br
(1)
(2)
I
Br
I
I
QBr
No reaction
Br
(3)
I
QBr
I
Cu(OTf)2
Figure 8-6 Bromination of mesitylene.
89
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Magnusson, G.; Ding, K.; Ray, A. K.; Rosen, A.; Sjögren, H. O.; Complex Carbohydrates in Drug
Research, 1994, 36, 89
103
Hasegawa, A.; Ishida, H.; Kiso, M.; J. Carbohydr. Chem. 1993, 12, 371
104
Kondo, T.; Tomoo, T.; Abe, H.; Isobe, M.; Goto, T.; J. Carbohydr. Chem. 1996, 15, 857
105
De Meo, C.; Demchenko, A.; Boons, G. J.; J. Org. Chem. 2001, 66, 5490
106
Castro-Palomino J. C.; Simon, B.; Speer, O.; Leist, M.; Schmidt, R. R.; Chem. Eur. J. 2001, 7,
2178
107
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108
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109
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110
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111
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112
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113
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114
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94
115
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116
Skoog, D. A.; West, D. M.; Holler, F. J.; Fundamentals of Analytical Chemistry, 6ed, p864
95
10
10 APPENDIX
10.1 Spectroscopic titrations of IX with different Lewis acids
Representative experimental procedure; 1 ml of a 0.1 mM solution of ICl in acetonitrile
was titrated with a 10 mM solution of AgOTf in acetonitrile. Spectroscopical data was
obtained using a Varian Cary 100 Bio UV-Vis Spectrophotometer.
Spectra for ICl titrations
1
1.2
0 eq
0 eq
0.8
0.6
1
0.8
0.6
0.4
0.4
0.2
0.2
0
180
200
220
240
260
280
300
0 eq
0.1 eq
0.2 eq
0.3 eq
0.4 eq
0.5 eq
1.0 eq
1.5 eq
1.4
Absorbance
1.2
Absorbance
Hg
0 eq
0.1 eq
0.2 eq
0.3 eq
0.4 eq
0.5 eq
1.0 eq
1.5 eq
H
1.4
0
180
320
200
220
240
260
A
Wavelength
A
Wavelength
97
280
300
320
Cu
1
1.2
1
0 eq
0 eq
0.8
0.6
0.8
0.6
0.4
0.4
0.2
0.2
0
180
200
220
240
260
280
300
0
180
320
200
220
A
Wavelength
0 eq
0.1 eq
0.2 eq
0.3 eq
0.4 eq
0.5 eq
1.0 eq
1.5 eq
1.2
1
0 eq
0.6
0.6
0.4
0.2
0.2
280
300
0
180
320
200
220
A
Wavelength
0 eq
0.1 eq
0.2 eq
0.3 eq
0.4 eq
0.5 eq
1.0 eq
1.5 eq
1.2
1
0 eq
0.6
320
0.8
0.6
0.4
0.4
0.2
0.2
260
300
280
300
0 eq
0.1 eq
0.3 eq
0.2 eq
0.4 eq
0.5 eq
1.0 eq
1.5 eq
1.2
0 eq
Absorbance
1
240
280
Li
0.8
220
260
1.4
Absorbance
In
200
240
A
Wavelength
1.4
0
180
320
0.8
0.4
260
300
0 eq
0.1 eq
0.2 eq
0.3 eq
0.4 eq
0.5 eq
1.0 eq
1.5 eq
1.2
0.8
240
280
Ag
0 eq
Absorbance
1
220
260
1.4
Absorbance
Al
200
240
A
Wavelength
1.4
0
180
0 eq
0.1 eq
0.2 eq
0.3 eq
0.4 eq
0.5 eq
1.0 eq
1.5 eq
1.4
Absorbance
1.2
Absorbance
Blank
0 eq
0.1 eq
0.2 eq
0.3 eq
0.4 eq
0.5 eq
1.0 eq
1.5 eq
1.4
0
180
320
A
Wavelength
200
220
240
260
A
Wavelength
98
280
300
320
0 eq
0.1 eq
0.2 eq
0.3 eq
0.4 eq
0.5 eq
1.0 eq
1.5 eq
1
1.2
1
0.8
0 eq
0 eq
Absorbance
1.2
0.6
0.8
0.6
0.4
0.4
0.2
0.2
0
180
200
220
240
260
280
300
0
180
320
200
220
0.6
0.8
0.6
0.4
0.4
0.2
0.2
280
300
0
180
320
200
220
Mn
0.8
0.6
1
320
0.8
0.6
0.4
0.4
0.2
0.2
260
300
280
300
0 eq
0.1 eq
0.3 eq
0.2 eq
0.4 eq
0.5 eq
1.0 eq
1.5 eq
1.2
0 eq
0 eq
Absorbance
1
240
280
1.4
Absorbance
1.2
220
260
Mg
0 eq
0.1 eq
0.2 eq
0.3 eq
0.4 eq
1.0 eq
1.5 eq
0.5 eq
1.4
200
240
A
Wavelength
A
Wavelength
0
180
320
0 eq
0.1 eq
0.2 eq
0.4 eq
0.3 eq
0.5 eq
1.0 eq
1.5 eq
1
0.8
260
300
1.2
0 eq
0 eq
Absorbance
1
240
280
1.4
Absorbance
1.2
220
260
Sc
0 eq
0.1 eq
0.2 eq
0.3 eq
0.4 eq
0.5 eq
1.0 eq
1.5 eq
Sn
1.4
200
240
A
Wavelength
A
Wavelength
0
180
0 eq
0.1 eq
0.2 eq
0.3 eq
0.4 eq
1.0 eq
0.5 eq
1.5 eq
Yb
1.4
Absorbance
Zn
1.4
0
180
320
A
Wavelength
200
220
240
260
A
Wavelength
99
280
300
320
Spectra for IBr titrations
1
1.2
0.8
0.6
1
0.8
0.6
0.4
0.4
0.2
0.2
0
180
200
220
240
260
280
300
0
180
320
200
220
1.2
0.8
0.6
0.4
0.4
0.2
0.2
280
300
0
180
320
200
220
Wavelength (nm)
Wavelength
Mn
0 eq
0.6
320
0.8
0.6
0.4
0.2
0.2
280
300
0 eq
0.1 eq
0.2 eq
0.3 eq
0.5 eq
0.4 eq
1.0 eq
1.5 eq
1
0.4
260
300
1.2
0.8
240
280
1.4
Absorbance
0 eq
Absorbance
1
220
260
Mg
0 eq
0.1 eq
0.2 eq
0.3 eq
0.4 eq
0.5 eq
1.0 eq
1.5 eq
1.2
200
240
A
Wavelength
1.4
0
180
320
0 eq
0.1 eq
0.2 eq
0.3 eq
0.4 eq
0.5 eq
1.0 eq
1.5 eq
1
0.6
260
300
1.2
0 eq
Absorbance
Absorbance
1
240
280
1.4
0.8
220
260
Sc
0 eq
0.1 eq
0.2 eq
0.3 eq
0.4 eq
0.5 eq
1.0 eq
1.5 eq
Absorbance
Sn
1.4
200
240
A
Wavelength
A
Wavelength
0
180
0 eq
0.1 eq
0.2 eq
0.3 eq
0.4 eq
0.5 eq
1.0 eq
1.5 eq
1.4
0 eq
0 eq
1.2
Absorbance
Yb
0 eq
0.1 eq
0.2 eq
0.3 eq
0.4 eq
0.5 eq
1.0 eq
1.5 eq
Absorbance
Zn
1.4
0
180
320
A
Wavelength
200
220
240
260
A
Wavelength
100
280
300
320
0 eq
0.1 eq
0.2 eq
0.3 eq
0.4 eq
0.5 eq
1.0 eq
1.5 eq
H
1.4
1.2
1
Absorbance
1
0.8
0.6
Absorbance
0 eq
Absorbance
1.2
0.8
0.6
0.4
0.4
0.2
0.2
0
180
200
220
240
260
280
300
0
180
320
200
220
1.2
1
0.6
0.6
0.4
0.2
0.2
280
300
0
180
320
200
220
1.2
0.8
0.6
0.4
0.4
0.2
0.2
280
300
0
180
320
200
220
In
1
0 eq
0.8
0.6
0.4
0.4
0.2
0.2
240
260
280
300
320
0 eq
0.1 eq
0.2 eq
0.3 eq
0.4 eq
0.5 eq
1.0 eq
1.5 eq
1
0.6
220
300
1.2
0.8
200
280
1.4
Absorbance
0 eq
Absorbance
1.2
260
Li
0 eq
0.1 eq
0.2 eq
0.3 eq
0.4 eq
0.5 eq
1.0 eq
1.5 eq
1.4
240
Wavelength
Wavelength (nm)
A
Wavelength
0
180
320
0 eq
0.1 eq
0.2 eq
0.3 eq
0.4 eq
0.5 eq
1.0 eq
1.5 eq
1
0.6
260
300
1.2
0.8
240
280
1.4
Absorbance
0 eq
Absorbance
1
220
260
Ag
0 eq
0.1 eq
0.2 eq
0.3 eq
0.4 eq
0.5 eq
1.0 eq
1.5 eq
Absorbance
Al
1.4
200
240
Wavelength (nm)
Wavelength
Wavelength (nm)
Wavelength
0
180
320
0.8
0.4
260
300
0 eq
0.1 eq
0.2 eq
0.3 eq
0.4 eq
0.5 eq
1.0 eq
1.5 eq
1.2
0.8
240
280
1.4
0 eq
Absorbance
Absorbance
1
220
260
Blank
0 eq
0.1 eq
0.2 eq
0.3 eq
0.4 eq
0.5 eq
1.0 eq
1.5 eq
Absorbance
Cu
1.4
200
240
Wavelength (nm)
Wavelength
A
Wavelength
0
180
0 eq
0.1 eq
0.2 eq
0.3 eq
0.4 eq
0.5 eq
1.0 eq
1.5 eq
Hg
1.4
0
180
320
A
Wavelength
200
220
240
260
A
Wavelength
101
280
300
320