Synthesis, structure and conformation of oligo- and
polysaccharides
E. Andreas Larsson
Department of Organic Chemistry
Arrhenius Laboratory
Stockholm University
Stockholm, Sweden 2004
ii
Doctoral Dissertation 2004
Department of Organic Chemistry
Stockholm University
Abstract
Carbohydrates are a complex group of biomolecules with a high structural
diversity. Their almost omnipresent occurrence has generated a broad field of
research in both biology and chemistry. This thesis focuses on three different
aspects of carbohydrate chemistry, synthesis, structure elucidation and the
conformational analysis of carbohydrates.
The first paper describes the synthesis of a penta- and a tetrasaccharide related to
the highly branched capsular polysaccharide from Streptococcus pneumoniae type
37. In the second paper, the structure of the O-antigenic repeating unit from the
lipopolysaccharide of E. coli 396/C1 was determined along with indications of the
structure of the biological repeating unit. In addition, its structural and
immunological relationship with E. coli O126 is discussed. In the third paper,
partially protected galactopyranosides were examined to clarify the origin of an
intriguing 4JHO,H coupling, where a W-mediated coupling pathway was found to
operate. In the fourth paper, the conformation of methyl α-cellobioside is studied
with a combination of molecular dynamics simulations and NMR spectroscopy. In
addition to the expected syn-conformation, detection and quantification of anti-φ
and anti-ψ conformers was also possible.
 Andreas Larsson 2004
ISBN 91-7265-872-X, pp 1-40
Akademitryck AB, Edsbruk
iii
iv
Contents
List of papers
vi
List of Abbreviations
vii
1
General introduction
1.1 Synthesis of oligosaccharides
1.2 Experimental methods for structure determination of carbohydrates.
1.3 Conformational analysis of carbohydrates
2
1
2
4
8
2.1
2.2
2.3
2.4
2.5
2.6
Synthesis of oligosaccharides related to the repeating unit of Streptococcus
pneumoniae type 37 (paper I)
Introduction
Strategy for the synthesis
Synthesis of tetrasaccharide 1
Synthesis towards hexasaccharide 2 - an abandoned approach
Synthesis of pentasaccharide 13
Future possibilities
13
13
15
15
17
18
21
3.1
3.2
3.3
3.4
Structural and immunochemical relationship between the O-antigenic
polysaccharides from enteroaggregative Escherichia coli strain 396/C1 and
Escherichia coli O126 (paper II)
Introduction
The E. coli 396/C1 O- antigen repeating unit
Structural and immunological relationship with E. coli O126
The biological repeating unit
22
22
22
23
23
4.1
4.2
4.3
4.4
4.5
Analysis of NMR J couplings in partially protected galactopyranosides (paper
III).
Introduction
The model substances and their NMR spectra
Optimization and analysis of the different rotamers
W-coupling
Comparison of experimental and calculated properties
25
25
25
27
29
29
5.1
5.2
5.3
5.4
5.5
Determination of the conformational flexibility of methyl α-cellobioside in solution
by NMR spectroscopy and molecular simulations (paper IV)
33
Introduction
33
1D DPFGSE T-ROESY
33
Deuterium isotope effects
34
Computer simulations
35
Population analysis
35
3
4
5
Acknowledgements
36
References
37
v
List of papers
This thesis is based on the following papers and manuscripts, referred to by their
Roman numerals I-IV.
I
Synthesis of oligosaccharides related to the repeating unit of the
capsular polysaccharide from Streptocuccus pneumoniae type 37
E. A. Larsson, M. Sjöberg and G. Widmalm
In manuscript
II
Structural and
immunochemical relationship between the O-
antegenic polysaccharides from the enteroaggregative Escherichia
coli strain 396/C1 and Escherichia coli O126#
E. A. Larsson, F. Urbina, Z. Yang, A. Weintraub and G. Widmalm
Carbohydr. Res. 2004, article in press
III
Analysis
of
NMR
J
couplings
in
partially
protected
galactopyranosides*
E. A. Larsson, J. Ulicny, A. Laaksonen and G. Widmalm
Org. Lett. 2002, 4, 1831-1834
IV
Determination of conformational flexibility of methyl α-cellobioside
in solution by NMR spectroscopy and molecular simulations*
E. A. Larsson, M. Staaf, P. Söderman, C. Höög and G. Widmalm
J. Phys. Chem. A 2004, 108, 3932-3937
Reprinted with kind permission of Elsevier# and ACS publications.*
vi
List of Abbreviations
AgOTf
silver trifluoromethanesulfonate (triflate)
CPS
capsular polysaccharide
DDCCR
dipole dipole cross correlated relaxation
DDQ
2,3-dichloro-5,6-dicyano-1,4-benzoquinone
DMTST
dimethyl(methylthio)sulfonium triflate
DPFGSE
double pulse field gradient spin echo
EAEC
enteroaggregative E. coli
h
hydrogen bond mediated J-coupling
J
HSEA
hard sphere exo anomeric
ISPA
isolated spin pair approximation
LPS
lipopolysaccharide
MALDI-TOF
matrix assisted laser desorption ionization – time of flight
MC
monte carlo
MD
molecular dynamics
MM
molecular mechaniscs
NIS
N-iodosuccinimide
PMB
p-methoxybenzyl
QM
quantum mechanics
R2
transverse relaxation rate, 1/T2
sym-collidine
2,4,6-trimethylpyridine
TFA
trifluoroacetic acid
TfOH
trifluoromethanesulfonic acid (trifflic acid)
T-ROESY
transverse rotating frame Overhauser effect spectroscopy
vii
viii
1
General introduction
Carbohydrates form a complex group of biomolecules with diverse biological
functions. These include roles such as being the primary energy source for
biological systems, the main structural elements of plants in the form of cellulose
and the K- and O-antigens – an information carrying immunologically active
identifier tag, coating cell walls of bacteria in the form of capsular and
lipopolysaccharides.[1]
The possibility of forming a large number of possible structures with just a few
monosaccharide units gives rise to an unsurpassed information density in
polysaccharides, as compared to DNA. This makes it possible to form unique
combinations of small repeating units, consisting of very few sugars. However, the
same structural diversity makes both the analysis and synthesis of carbohydrate
structures challenging.
To be able to understand the carbohydrates themselves or the interactions involving
carbohydrates and other biomolecules, pure oligosaccharides are needed. Such
substances can be obtained, either by isolation from natural sources or by chemical
or enzymatic synthesis. If the aim is to use analogues or isotopically labelled
versions of the native structure, chemical synthesis is usually the only option.
Biological interactions such as molecular recognition are based on the threedimensional shape of molecules. This shape is in turn determined by the chemical
structure and the conformation of the molecule. Determining the structure of
carbohydrates includes several steps: finding out which monosaccharides are
present, elucidating the actual linkage positions between the monosaccharide units
and determining the stereochemistry in the structure. This information corresponds
to the sequence of amino acids in proteins, with the additional complication that it
does not necessarily have to be linear and that all linkages have two possible
stereoisomers. This analysis is usually performed with a variety of NMR
experiments. In addition, fragment analysis with mass spectrometry lends further
support to the proposed structures. The conformation is typically determined by a
combination of computational techniques and NMR spectroscopy but X-ray
crystallography is also useful if crystals can be grown.[2]
In paper I, the synthesis of penta- and tetrasaccharides structurally related to the
highly branched capsular polysaccharide from S. pneumoniae type 37 is described.
In Paper II, the structure of the O-antigenic lipopolysaccharide repeating unit of E.
coli 396/C1 was determined. In addition, its structural and immunological
1
relationship with E. coli O126 is discussed. In paper III, partially protected
galactopyranosides are examined to clarify the origin of an observed 4JHO,H
coupling. In paper IV, the conformation of methyl α-cellobioside is studied with a
combination of molecular mechanics and NMR. In addition to the expected synconformation, detection and quantification of anti-φ and anti-ψ conformers was
also possible.
1.1 Synthesis of oligosaccharides
1.1.1
The glycosylation reaction
A glycosylation is described as the reaction between the hydroxyl group of a
glycosyl acceptor and the anomeric centre of a glycosyl donor (Figure 1.1). The
glycosyl donor carries an anomeric leaving group, which reacts under the influence
of a promoter, commonly in a SN1 fashion.
PO
PO
Promoter
O
PO
+
OP
OP
- LG−
PO
PO
HO-R
PO
LG
Glycosyl donor
O
Oxocarbenium ion
-H+
PO
PO
PO
O
PO
P = Non participating protecting group
LG = Leaving group
Figure 1.1
α−
+
OR
PO
PO
PO
O
OR
PO
and β−glycosides
Typical mechanism of a glycosylation.
The oxocarbenium intermediate (Figure 1.1) is accessible from both the α and the β
face, typically generating a mixture of anomers. A weak stereoelectronic effect
termed the anomeric effect[3, 4] stabilizes products with axially oriented electron
withdrawing substituents. By employing equilibrating conditions,[5] the
thermodynamically stable α-anomer can often be the dominant product in the
2
reaction mixture[6]. In addition, the influence of the solvent[7,
effect on both the yield and stereochemistry.
1.1.2
8]
can also have an
Neighbouring group participation
The presence of a participating protecting group (i.e., a protecting group with an
electron rich carbonyl oxygen which reversibly can stabilize the positive charge at
the anomeric centre) influences the stereoselectivity greatly. The acyloxonium
intermediate, (see Figure 1.2) shields one of the faces from incoming nucleophiles
and thereby directs the stereochemistry of the reaction. If present on the vicinal 2position, a high degree of selectivity can be achieved, producing 1,2-trans
glycosides. In addition, participating protecting groups in the more distant 4position of galactose can also influence the anomeric selectivity.[9]
OP
OP
O
O
PO
PO
LG
Promoter
O
R
O
- LG−
PO
PO
+
PO
PO
O
O
O
R
+
OP
O
HO-R
O
R
-H+
PO
PO
PO
OR
PO
P = Protecting group
LG = Leaving group
Figure 1.2
O
1,2-trans glycosides
Glycosylation in presence of a participating protecting group.
Protecting groups may also affect the reaction in a more passive way. Steric
interactions can make the approach to one side less favourable, and thus alter the
reaction path.[10]
In the case of mannose configured pyranoses, neither the anomeric effect nor
participation of protecting groups work in favour of the formation of β-glycosides.
Synthesis of such glycosidic linkages requires different methods.[11-13]
A wide range of leaving group/promoter systems have been developed.[5, 14-20] A
general disadvantage of most of the leaving groups is their low stability towards
many of the conditions involved in protecting group manipulations. One of the few
3
exceptions is thioglycosides[21] which in general are relatively inert, but reacts in
the presence of soft electrophiles and oxidizers. Thus they can be used as an
anomeric protecting group as well.
1.2 Experimental methods for structure determination of
carbohydrates.
1.2.1
Introduction
To define the structure of a carbohydrate polymer, several aspects must be
determined. Three techniques dominate the field of structure determination: X-ray
crystallography, mass spectroscopy and NMR spectroscopy. X-ray crystallography
is a very powerful technique, invaluable for protein and inorganic chemists.
However, it has so far found a relatively limited use with carbohydrates, mainly
due to difficulties obtaining crystals. Mass spectroscopy is also a very useful
technique which has grown in importance for bio-molecules since the development
of soft ionization techniques such as MALDI[22] and electrospray.[23] However, the
amount of information that can be extracted is relatively limited when compared to
NMR. In addition, carbohydrate polymers can be difficult to ionize. This is
particularly true for lipopolysaccharides.
NMR spectroscopy has for many years been an important method for structural
analysis of carbohydrates.[24] With the aid of highfield magnets, sensitive cryoprobes and modern pulse sequences, NMR structural determination is becoming
more and more the tool of choice.[25] Sensitivity, the major drawback of NMR is
gradually becoming less of an issue. However, solubility, polymer heterogeneity[26]
and polymer size[27] can still complicate the procedure considerably.
In principal, before employing physical methods (e.g. NMR), chemical methods are
used to gather information about the identity and quantity of present
monosaccharides components[28] and their absolute configuration.[29]
1.2.2
NMR spectroscopy
First a handle is identified for all spin systems (residues). In most cases, the
anomeric protons serve well since they often are sufficiently resolved. The next
4
task is to assign the individual sugar residues. When all atoms in all residues
(optimally) are assigned, the inter-glycosidic connectivities may be established.
During this process, a range of mainly 2D-experiments are used. I have chosen to
divide the description of these experiments in two sections: those mainly useful for
determining intra-glycosidic correlations and those deducing the glycosylation
pattern, respectively.
A typical structure determination starts by acquiring a proton spectrum. Usually,
the amount of information that can be extracted is limited: However, some
important aspects can be addressed. Impurities or heterogeneity may be visible in
the one dimensional proton spectra. In addition, the number of unique anomeric
protons and the CH3 protons of N- and O-acetyl groups and 6-deoxy sugars may
also be determined (see Figure 1.3). Following this, a 1H,13C correlated HSQC
(Heteronuclear Single-Quantum Correlation)[30] spectrum is run and the anomeric
proton/carbon atom pairs can be detected. It also provides the 1JCH coupling
constants which, in combination with the 3JHH coupling constants extractable from
the proton spectrum, are invaluable in determining the anomeric configuration.[31-33]
The 1H,13C HSQC spectrum commonly resolves the majority of the 1H,13C pairs.
Although not assigned, they provide a “pool” of 1H,13C pairs which can be
expected to be found in later experiments.
6.0
5.5
5.0
4.5
4.0
3.5
1
3.0
2.5
2.0
1.5
1.0
H (ppm)
1
Figure 1.3
H NMR of the E. coli 396/C1 polysaccharide. The signals from the
anomeric, N-acetyl and H6 protons from the fucosyl residues protrude from the bulk
region at ~3.2-4.4 ppm where most protons are found. The “hump” at ~4.6 ppm is the
residual HDO peak after pre-saturation and post-processing.
5
1.2.3
Intra-glycosidic correlations
Using an anomeric proton as a reference point, a 1H,1H-COSY (COrrelated
SpectroscopY)[34] provides access to H2. In a 1H,1H-TOCSY (TOtal Correlation
SpectroscopY)[35, 36] a spin lock allows the detection of correlations to protons
which do not couple directly to H1, i.e. H3-H6. How far into the spin system that
correlations will be detected depends on the mixing time and the size of the
coupling constants. For example, in the case of β-glucose, where all the coupling
constants are large, a mixing time of 90 ms typically provides correlations to all
protons in the residue. In contrast, for galactose, transfer to protons beyond H4 is
rarely observed, due to the smaller coupling constants associated with H4.
Performing multiple TOCSY spectra with increasing mixing times (typically 30100 ms) consequently allows the user to “walk through” the spin systems starting
from H1.
A complement to 1H,1H-COSY and 1H,1H-TOCSY, which are J-coupling mediated
experiments, 1H,1H-NOESY (Nuclear Overhauser Effect SpectroscopY)[37] can also
be useful, in particular for systems containing protons with very small coupling
constants such as H1-H2 in β-mannosides. In contrast to COSY and TOCSY, the
correlations in a NOESY experiment arise from through space interactions of two
protons. Hence, care must be taken, to avoid confusing intra- and inter-residue
interactions (see Figure 1.4). NOESY and J-coupled spectra well complement each
other and when combined, most proton resonances can be assigned. In cases of
overlap in the 1H-dimension, combinations of the above mentioned techniques such
as HSQC-TOCSY[38] can greatly facilitate the assignment. Although, accompanied
with some loss of sensitivity, the fact that it transfers the correlations to the usually
much less crowded 13C dimension can be hugely beneficial.
1.2.4
Inter-glycosidic correlations
At this point in the analysis, all 1H,13C pairs of the individual residues are usually
assigned. Then, information on the sequence of the involved residues is gathered.
Performing this task, one mainly relies upon two techniques: HMBC
(Heteronuclear Multiple-Bond Correlation)[39] and NOESY.
Most often, the anomeric proton is in the proximity of the proton of the substituted
position on the other side of the glycosidic linkage. Hence, a crosspeak can be
observed in a NOESY experiment. However, care must be taken since occasionally
6
more than one inter-glycosidic correlation can be found and sometimes to more
than one residue (Figure 1.4). These multiple NOEs can provide an insight on the
conformation of the polymer. In contrast, HMBC produces correlations of longrange J-coupled proton and carbon atoms. Generally, with a mixing time of ~50
ms, 2JCH and 3JCH correlations are observed, such as those found at inter-glycosidic
sites.
For very large polymers, (many thousands of repeating units) a mixing time of 50
ms is not feasible. The R2 increases with the size of the polymer, and when
sufficiently large, the magnetization decays too rapidly for use of the HMBC
experiment. Until recently, in such cases one had to rely on NOESY experiments
from which, if spectral overlap was present in the proton dimension, sequence
information is difficult to obtain. The DDCCR (Dipole Dipole Cross Correlated
Relaxation)[40] which was designed to counter these problems needs a mixing time
of only 10-15 ms to produce a spectrum similar to HMBC (see Figure 1.4).
13
C (ppm)
68
70
3.7
E2
B2
B5
3.9
B3
72
4.1
H (ppm)
66
1
64
74
76
E2
4.3
78
D2
b
a
80
5.07
5.07
1
c
4.5
5.07
H (ppm)
Figure 1.4
(a) HMBC, (b) DDCCR, and (c) NOESY showing the
correlations from H1 of the α-L-fucosyl residue B at 5.07 ppm in the polysaccharide
from E. coli 396/C1 (see Section 3). Connectivity to position 2 of residue E is
established with all methods. Note the additional NOE correlation to residue D which
is not linked to residue B.
7
1.3 Conformational analysis of carbohydrates
Conformational analysis is performed to gain access to information about the three
dimensional shape of a molecule. The goal of a conformational analysis is to be
able to highlight significantly populated conformers. In the case of a carbohydrate
oligomer, the main degrees of freedom are found at the exocyclic bonds (see Figure
1.5 and Section 5). The flexibility of the ring itself is inconsequential for the most
commonly occurring pyranoses.[41] However, if more detailed information is
required, the hydroxyl torsions should also be monitored (see Figure 1.3 and
Section 4).
ω
a
H
OMe
OH
O
C1'
O
φ ψ
H1'
C4
O
b
θ
H
O
C
O
H4
Figure 1.5
A picture of two simplified systems showing the principal degrees of
freedom for carbohydrates: (a) φ (phi), ψ (psi) and ω (omega) and (b) the rotation of a
hydroxyl group θ (theta). The glycosidic torsion angles are in this work defined as
φ = H1´-C1´-O4-C4 and ψ = C1´-O4-C4-H4 and the hydroxyl torsion as θ = HO4O4-C4-H4.
1.3.1
Factors influencing the conformation of carbohydrates
The 3D structure of carbohydrates is influenced by a number of factors. One of the
most important is the exo-anomeric effect.[3] It is the same type of stereo-electronic
effect that influences the stereoselectivity of glycosylations. The exo-anomeric
effect stabilizes particular conformations of the φ-torsion. This leads to a
conformational preference for α- and β-glycosides at about −60° and +60°,
respectively.[42] But as demonstrated in paper IV, other conformations are also
possible.
Steric interactions also restrict the conformational space. In addition, inter-residue
hydrogen bonds can also stabilize specific conformers and thereby affect the
conformation.[43] Solvent interactions can also be important to the conformational
preference at the glycosidic linkage.[44]
8
1.3.2 Computational methods for conformational analysis
Two conceptually very different methods, Quantum Mechanics (QM) and
Molecular Mechanics (MM) are commonly employed in conformation analysis.
Which one to implement is based on which properties are being studied and the
available computing power. QM is much more calculation intensive and as such it
limits the size of the system that can be studied. Despite the ongoing development
in the speed of computers, QM calculations can still be very time consuming. QM
calculations generate information about electronic interactions within a molecule.
From the results of such calculations, intrinsic physical properties can be described,
such as NMR J-coupling constants, IR spectra etc. (see Section 4 and paper III).
When properties not directly dependent on the orientation of orbitals or electron
densities are studied, MM is a more efficient method to use. MM is less computer
intensive and allows for a more generous inclusion of specific solvent molecules
which can prove more valuable than the precision of QM. The basis of MM is a
force field,[45] a set of equations from which, a potential energy can be calculated.
The force field simulates the forces that act upon molecules by optimizing the
potential energy of bond angles, bond lengths, torsions etc. Upon minimization of
the potential energy the molecule relaxes to lower potential energy conformers.
A number of force fields exist. Some are optimized for use with carbohydrates.[46,
47]
One example is HSEA[48] (Hard Sphere Exo Anomeric), which essentially treats
the pyranose moiety as a rigid sphere. As the name explains, it is parametrized for
the exo-anomeric effect.
Perhaps, the most important technique is Molecular Dynamics (MD) simulations.
By adding heat (in the form of kinetic energy), the simulated molecule is allowed to
stochastically pass over energy barriers and over time, optimally populate all low
energy conformations accordingly. The results are often viewed as trajectories of a
distance or angle of interest plotted against time (see paper IV).
1.3.3
Conformational analysis employing NMR spectroscopy
The cycle time for a typical NMR experiment is on the order of seconds. Processes
such as molecular motion and conformational dynamics usually equilibrate at a
timescale several orders of magnitude shorter (i.e. in the nano- to milli-second
range). The enormous difference in timescale between sampling and the processes
observed in most cases means that the data extracted from the NMR experiments
9
represents a population weighted average of the different possible states a certain
molecule can possess.
Under such conditions, it is not possible to study or measure the presence of
different conformations directly from NMR data, since the proportions and
composition of the different states are usually not known. However, if NMR is used
in combination with modelling techniques, the information from this averaged data
can be converted into populations.
1.3.4
NOE derived distances
NOEs are transmitted through space and provide distance related information on
nearby protons.[49] By determining the inter-proton cross-relaxation rates, σij,
distances can be calculated. The derived distances are time averaged, but are very
useful in the study of the three-dimensional structure of carbohydrates.
The sign and the magnitude of the NOE is dependent on the magnetic field and the
correlation time and thus of the size of the molecule. As a consequence, for certain
combinations of the correlation time and the magnetic field, the NOE is minuscule.
In such conditions, experiments based on ROE (Rotating frame Overhauser effect)
or T-ROE[50, 51] (Transverse Rotating frame Overhauser effect), which give positive
and significant ROE or T-ROE for all combinations of correlation-time and
magnetic field, can be employed. Both ROESY and T-ROESY utilize a spin-lock.
In a ROESY experiment the spin-lock can give rise to unwanted TOCSY-transfer.
T-ROESY however, does not suffer from such problems.
A convenient way to obtain the relaxation rates is to monitor the build-up of NOE
with time. The NOE build-up can be mapped by a series of experiments with a
range of mixing-times. By fitting this data to a second-order polynomial the crossrelaxation rates can be determined.
Cross-relaxation rates can be interpreted with the Isolated Spin Pair Approximation
(ISPA)[52]. ISPA requires the knowledge of a reference distance rref which can be
obtained from molecular mechanics simulations. The distance of the unknown spinpair rij, can then be calculated as rij = rref (σref / σij)1/6.
10
If the correlation time and the dynamics of the molecule are known, the crossrelaxation rates can be interpreted with Lipari and Szabo’s model-free approach[53,
54]
.
1.3.5
NMR Spin-spin couplings
For two nuclei to be able to exchange information in the form of scalar or indirect
coupling their electrons must be able to interact.[55] Theoretically, it is usually
described as a presence of electron density at the bond critical point, that is, the
point where the orbitals of the two nuclei meet. Covalent bonds obviously fulfill
this criterion, and normally scalar couplings can be considered to be bond
mediated.
The 3J spin-spin coupling constants are sensitive to the bond geometry and can be
interpreted by a Karplus-type relationship. The relationship between coupling
constants and geometry was formulated in an equation by Karplus in 1959.[56] The
first equation was designed for 3J H-C-C-H, H-C-C-F and F-C-C-F systems, but
other variants of the equation have been developed such as that for H-O-C-H
torsion angles[57] (shown in the equation below and in Figure 1.6), which was
suitable for the HO4-O4-C4-H4 torsion investigated in Section 4.
3
J HCOH = 10.4 cos 2 θ − 1.5 cos θ + 0.2
This is a useful tool but one must use the relation with caution. Due to the
periodicity of the function, at least two, but normally four conformations are
possible for every value of the coupling constant with the exception of the trans
conformation.
11
Figure 1.6
The Karplus relationship for H-O-C-H torsion angles.
In addition, these J spin-spin coupling constants will be time averaged and one
must realize that the measured coupling constant is an average of all conformers,
weighted by their relative population.
Similar equations exist for many other systems including the trans-glycosidic C-OC-H torsion.[58] In that case 3JCH long range couplings provide information on the
conformational preference of the glycosidic linkage.
1.3.6
Hydrogen bond mediated J-couplings
As mentioned in Section 1.3.5, J-coupling is seen as bond mediated. There are
however other possibilities. Fluorine nuclei, in close proximity in space, but
separated by multiple bonds can sometimes be seen to couple to each other (an
effect of overlap of their electrons).[59] There are also examples in which hydrogen
bonds have mediated couplings, apparently when sufficient electron density
between the hydrogen bond donor and acceptor is found. Hydrogen bond mediated
scalar couplings have recently been described by several groups, and are known to
exist in between the base pairs of DNA and RNA[60-62] as well as in structures
resembling carbohydrates.[63] The hJ couplings are usually small, but are extremely
valuable in the refinement of protein and nucleic acid structures.[55, 64-66]
12
2
Synthesis of oligosaccharides related to the repeating
unit of Streptococcus pneumoniae type 37 (paper I)
2.1 Introduction
Streptococcus pneumoniae is a serious human pathogen. It can among other things,
cause community-acquired pneumonia and meningitis.[67] The surface of the
bacterium is covered with capsular polysaccharides (CPS), which play an important
role in its interaction with its host. The presence of a capsule has been shown to be
an important virulence factor,[68, 69] in particular for disease development after
primary infection.[70] The capsule offers protection for the bacterium against
phagocytosis, and might also be involved in adhesion during infection.[70]
2.1.1
Streptococcus pnemoniae type 37
Of the 90 known types of S. pneumoniae, type 37 is the only one whose CPS is a
homopolymer, containing only glucose residues. The genetic regulation for the
biosynthesis of the type 37 capsule differs from all other known species of S.
pneumoniae in another aspect; only one gene directs the synthesis of the capsular
polysaccharide.[67, 71] Interestingly, it is located far from the clusters where these
genes normally reside.[72]
2.1.2
Structural features of the capsular polysaccharide
The type 37 capsular homopolymer of S. pneumoniae is built from repeating units
of →3)[β-D-Glcp-(1→2)]-β-D-Glcp-(1→ (see Figure 2.1). Earlier studies have
indicated that this comb-like structure is most likely to be very sterically
crowded.[73]
The proton NMR chemical shifts of the CPS deviate considerably from what are
expected compared to non-branched analogues. This indicates that O2 of a
neighboring residue is in close proximity with the anomeric proton, since the
calculated chemical shift[74] should be observed at 4.82 ppm in a non-perturbed
structure compared to 5.12 ppm in the native CPS.[73]
13
HO
HO
O
O
O
HO
O
HO OH
Figure 2.1
OH
n
The repeating unit of the CPS from S. pneumoniae type 37.
A number of structurally related trisaccharides have previously been investigated
by our group with interesting results. The trisaccharide β-D-Glcp-(1→2)-β-D-Glcp(1→3)-α-D-Glcp-OMe showed an increased flexibility at the (1→2)-linkage.[75]
Oligomers of the repeating unit of the type 37 CPS might also show an interesting
tertiary structure and serve as a representative model of the native polymer in NMR
studies.
2.1.3
Target substances
The question of how large an oligosaccharide needs to be to attain the tertiary
structure of its native polymer has recently been addressed, both with
spectroscopic[76] and immunologic methods. Such knowledge is of great importance
for example in the development of polysaccharide vaccines.[77] The epitope
involved in biological interactions may require that the saccharide adopts
conformations not populated by an oligomer with to few repeats.
In the case of the type 37 related structures, the synthesis has also been performed
to generate substances with interesting structural features. Hence, a dimer,
tetrasaccharide 1 is also an interesting target. However in terms of mimicking the
steric pressure of the polymer, a hexasaccharide 2 is probably needed (Figure 2.2).
The choice of an α-configured methyl glycoside as anomeric protecting group for
the reducing end of the oligosaccharide is to provide a spectroscopic handle and
reduce spectral overlap as much as possible.
14
HO
HO
HO
HO
HO
O HO
O
O
O
O
O
HO
OH
OH
HO
O
HO
HO
HO
HO
O
HO
HO
O
O
OMe
OH
HO
OH
OH
O
HO
OH
HO
O
O
O
OH
O
HO
1
Figure 2.2
HO
O HO
O
OH
HO
O
HO
OMe
OH
OH
2
The two initial target substances.
2.2 Strategy for the synthesis
The type 37 polymer is a (1→3)-linked β-D-glucopyranosyl backbone, substituted
with β-(1→2)-linked residues. A strategy based on simultaneous introduction of the
β-(1→2)-linked residues to a (1→3)-linked diol/triol acceptor therefore seemed an
efficient and logical protocol. If successful, it could potentially be extended for
synthesis of larger analogues. The type 37 structures are not only homopolymers,
they contain exclusively β-linkages as well. Consequently, participating and
temporary protecting groups were needed at the 2-positions of the donors. These
protecting groups could then be removed simultaneously to form an acceptor. In
addition, an orthogonal protecting group at position 3 is needed for the backbone
residues to facilitate its elongation.
Although it worked for the synthesis of 1, the strategy was unsuccessful for the
synthesis of 2, which is discussed in more detail in Section 2.4.
2.3 Synthesis of tetrasaccharide 1
2.3.1
Formation of the disaccharide acceptor
In our initial efforts to synthesize the first β-(1→3)-linkage, a NIS/TfOHpromoted[19] regioselective glycosylation of donor 3[78] and methyl 4,6-Obenzylidene-α-D-glucopyranoside[79] was employed (Scheme 2.1). This sequence
reduces the number of protecting group manipulations. The regio- and
stereoselectivity was good. Similar results have been reported by Zeng and co15
workers.[80] However, solubility and separation problems were a major drawback
for this route when scaled up.
Ph
O
O
O
BnO
Ph
SEt
AcO
+
O
O
HO
O
HO
Ph
i
O
O
BnO
Ph
O
O
O
HO
AcO
OMe
3
O
O
OMe
(73%)
Scheme 2.1
Regioselective glycosylation. i. NIS/TfOH, CH2Cl2, −35 °C.
Acceptor 4[81] is accessible in one step from methyl 4,6-O-benzylidene-α-Dglucopyranoside utilizing tin-activated benzoylation in almost quantitative yield.[81]
NIS/TfOH-promoted glycosylation of 3 and 4 gave 5 in 83% yield (Scheme 2.2). In
addition, this route was more effective if more labile protecting groups such as allyl
and p-methoxybenzyl (PMB) ethers was present at the 3-postiton. The ester
protecting groups were removed with sodium methoxide in methanol to form
disaccharide acceptor 6.
Ph
O
O
O
BnO
AcO
3
Ph
SEt
+
O
O
HO
O
BzO
i
Ph
O
O
BnO
Ph
O
R 1O
OMe
4
ii
O
O
O
O
R 2O
OMe
5 R1 = Ac, R2 = Bz (83%)
6 R1 = H, R2 = H (99%)
Scheme 2.2
2.3.2
i. NIS/TfOH, CH2Cl2, 25 °C; ii. NaOMe, MeOH.
Formation and deprotection of tetrasacharide 1
Diglycosylation of 6 with thioglycoside donors employing NIS/TfOH or DMTSTpromoted[15] conditions resulted in poor yields due to incomplete glycosylation
forming a mixture of tri- and tetrasaccharides. However, the tetrasaccharide 8 could
be produced by a AgOTf-promoted[20] glycosylation (Scheme 2.3) with 4.5
equivalents of 2,3,4,6-tetra-O-benzoyl-α-D-glucosyl bromide 7 in the presence of
of sym-collidine at −55 °C.
16
Ph
O
O
BnO
Ph
6
O
O
O
Ph
O
O
HO
OH
+
OMe
i
O
O
BnO
O
O
O
O
O
O
O
BzO
OBz
Ph
O
BzO
OBz
O
OMe
OBz
O
BzO
BzO
BzO
BzO OBz
BzO OBz
Br
7
ii, iii
8 (84%)
1 (79%)
Scheme 2.3
i. AgOTf, CH2Cl2, sym-collidine, −55 °C, 5 h; ii. NaOMe, MeOH ,
25 °C, 12 h; iii. H2, Pd/C, MeOH/HOAc/H2O: (6/1/1), 100 psi, 12 h.
Repeated additions of donor 7 and AgOTf throughout the glycosylation pushed the
yield to 83%. However, the use of larger excesses of donor and promoter
eventually resulted in the removal of the 4,6-benzylidene groups on the acceptor so
the reaction must be carefully monitored. Subsequent deprotection of 8 by sodium
methoxide and catalytic hydrogenation gave the deprotected tetrasaccharide 1 in
79% yield.
2.4 Synthesis towards hexasaccharide 2 - an abandoned
approach
Encouraged by the success for the synthesis of 1, we tried to synthesize the
hexasaccharide trimer with the polyglycosylation strategy. Hence, an analogous
trisaccharide triol acceptor was constructed. By exchanging the benzyl ether at
position 3 of donor 3 for a PMB group, elongation of the backbone is possible.
Glycosylation of such a donor in analogy with the synthesis of 5, disaccharide 9
could be produced. By removing the PMB protecting group with DDQ[82] in water
saturated chloroform, acceptor 10 is formed (Scheme 2.4). NIS/TfOH-promoted
glycosylation of 3 with 10 followed by O-deacetylation with sodium methoxide in
methanol gave triol 12.
17
Ph
O
O
PMBO
Ph
O
O
OAc
O
Ph
O
i
(87%)
O
9
AcO
O
O
HO
Ph
O
O
OAc
OMe
O
O
+ 3
O
AcO
10
OMe
ii
(97%)
Ph
O
O
BnO
Ph
O O
OR
iii
Scheme 2.4
MeOH.
O
Ph
O
O
O
O
O
O
OR
RO
OMe
11 R= Ac
12 R= H (99%)
i. DDQ, CHCl3-H2O; ii. NIS/TfOH, −30 °C, CH2Cl2; iii. NaOMe,
The tri-glycosylation of 12 proved to be difficult. A number of glycosyl donors,
promoters and conditions were explored leading only to either incomplete
glycosylation or removal of the 4,6-O-benzylidene protecting groups on the
acceptor. An O-benzylated analogue of acceptor 12 was synthesized, with the idea
that a more durable acceptor could solve these problems. However, glycosylations
employing this acceptor rendered similar results. Polyglycosylation proved
unsuccessful for the synthesis of 2.
2.5 Synthesis of pentasaccharide 13
We decided to try a reversed order of glycosylation and introduce a β-(1→3) linked
glucose residue to generate a pentasaccharide. Such a derivative, 13 (Figure 2.3),
could be structurally interesting in and of it self. In addition, with proper protecting
groups it could also provide a path to larger compounds such as hexasaccharide 2.
18
HO
HO
HO
HO
O HO
O
HO
HO
O
HO
O HO
O
O
O
O
OH
HO
O
OMe
OH
HO OH
HO OH
13
Figure 2.3
2.5.1
Synthesis of tetrasaccharide acceptors
Suitably protected acceptors were prepared by O-deacetylation of 9 and its 3´-Oallyl analogue. However, the results from the glycosylations of these acceptors
were not satisfactory. NIS/TfOH-promoted conditions resulted in low yields and
low reproducibility. In addition, AgOTf-promoted conditions which were
successfully used in the formation of tetrasaccharide 8 were even worse and led
mainly to side reactions.
Instead, we turned to a strategy of reprotecting 8 at tetrasaccharide level. If the 4,6O-benzylidene acetals were replaced with suitable protecting groups, the 3´-Obenzyl ether could be used as an orthogonal protecting group (Scheme 2.5).
The change of protecting groups from benzoyl to acetyl esters on the two terminal
residues is not without reason. Initially, benzoyl protecting groups were introduced
directly after the removal of the 4,6-O-benzylidenes. However, upon treatment with
H2, Pd-C, only small amounts of tetrasaccharide acceptor were formed. Reactions
involving heterogeneous catalysts can be sensitive to steric interactions. As a
consequence, a method employing NaBrO3/Na2S2O4 that should be more
compatible with steric hindrance was also explored.[83] However, yields were low.
Similar results from both methods were also obtained with the 4,6,4´,6´-tetra-Oacetylated analog. In addition, none of the substances formed during these
reactions were functional as glycosyl acceptors.
Eventually, The 4,6-O-benzylidene acetals in 8 were found to be efficiently cleaved
with 90% TFA and treatment with sodium methoxide in methanol removed the
benzoyl protecting groups. Subsequent O-acetylation gave 14 in 73% overall yield
19
(Scheme 2.4). On 14 however, the 3´-O-benzyl group was readily removed by
treatment with H2, Pd-C producing acceptor 15 in 95% yield.
Ph
O
O
BnO
Ph
O
O
O
O
O
BzO
O
BzO
OBz
BzO OBz
AcO
AcO
RO
O
O
O
i,ii,iii
OMe
AcO
OBz
BzO OBz
O
AcO OAc
8
iv
AcO
O AcO
O
O
O
O
AcO
OAc
O
OMe
OAc
AcO OAc
14 R = Bn (73%)
15 R = H (95%)
Scheme 2.5
i. 90% TFA 0.1 h; ii. NaOMe, MeOH; iii. Ac2O, pyridine, DMAP,
CH2Cl2, 20 h; iv. H2, 5% Pd/C, THF, 100 psi, 30 h.
2.5.2
Synthesis and deprotection of the pentasaccharide
More importantly, when 15 were used as an acceptor in a NIS/TfOH-promoted
glycosylation with a small donor, 16[84] the per-O-acetylated pentasaccharide 17
was formed in 58% yield (Scheme 2.6). Deprotection utilizing sodium methoxide
in methanol gave 13 in 93% yield.
20
AcO
AcO
AcO
O
SEt
+
i
15
AcO
O AcO
O
AcO
AcO
AcO
AcO
AcO
AcO
O
16
AcO OAc
ii
HO
HO
HO
HO
O HO
O
HO
HO
O
HO
O HO
O
O
O
O
OH
HO
O
AcO
O AcO
O
O
O
O
AcO
OAc
O
OMe
OAc
AcO OAc
17 (58%)
OMe
OH
HO OH
HO OH
13 (93%)
Scheme 2.6
i. NIS/TfOH, MS 4Å, CH2Cl2, 25°C; ii. NaOMe, MeOH.
2.6 Future possibilities
Evidently, the choice of small protecting groups is important. If a donor with a
suitable orthogonal protecting group can be introduced instead of 16, a
pentasaccharide acceptor is within reach, possibly allowing the synthesis of
hexasaccharide 2.
Whether or not these structures display structurally interesting properties in general
or as mimics for the CPS of S. pneumoniae type 37 are left for future investigators.
21
3
Structural and immunochemical relationship between
the O-antigenic polysaccharides from enteroaggregative
Escherichia coli strain 396/C1 and Escherichia coli O126
(paper II)
3.1 Introduction
396/C1 is an E. coli strain which belongs to the enteroaggregative E. coli (EAEC)
virotype. EAEC strains are believed to cause persistent diarrhea among infants,
especially in the developing world[85]. Like many EAEC strains E. coli 396/C1
autoagglutinates. This initially precluded serotyping by standard methods such as
slide agglutination. In such cases, characterization must be done by determination
of the structure of the O-antigenic polysaccharide.
3.2 The E. coli 396/C1 O- antigen repeating unit
The 1H NMR spectrum of the polysaccharide from E. coli 396/C1 showed five
significant resonances in the anomeric region. Assignment of 1H and 13C nuclei for
the five residues were accomplished using an array of 1H,1H and 1H,13C 2D NMR
techniques (see Section 1.2.4 and paper II). The sequence and glycosylation pattern
i.e., the repeating unit, was determined by 1H,13C-HMBC and corroborated by
1
H,13C-DDCCR and 1H,1H-NOESY (see Section 1.2.5).
With the NMR data and a determination of the absolute configuration, the 396/C1
polysaccharide repeating unit was found to be a pentasaccharide (Figure 3.1).
→2)-β-D-Manp-(1→3)-β-D-Galp-(1→3)-α-D-GlcpNAc-(1→3)-β-D-GlcpNAc-(1→
│
α-L-Fucp-(1→2)
Figure 3.1
The repeating unit of the polysaccharide from E. coli 396/C1.
Component and methylation analysis were also performed and were in agreement
with the NMR data.
22
3.3 Structural and immunological relationship with E. coli
O126
Eventually, by employing a different agglutination method (see paper II) slide
agglutination tests were possible. The result showed that E. coli 396/C1 was
positive towards monospecific O126 serum and no selectivity was found in
competitive tests using purified LPS from O126 reference strains and E. coli
396/C1. When submitted to the International Escherichia and Klebsiella Centre
(WHO), the E. coli 396/C1 sample was found to be E. coli O126:K+:H27.
The structure of the O-antigen from O126 has previously been described as a
structurally very similar tetrasaccharide repeating unit (see Figure 3.2).[86]
→2)-β-D-Manp-(1→3)-α-D-Galp-(1→3)-β-D-GlcpNAc-(1→
│
β-L-Fucp-(1→2)
Figure 3.2
The previously described repeating unit for E. coli O126.
A brief inspection of the 1H NMR spectrum of the reference O126 LPS showed
five anomeric resonances with very similar chemical shifts and coupling constants
as compared to the E. coli 396/C1 sample. In addition, other protruding features
such as NHAc and fucose-H6 resonances were also present. The reason for the
discrepancy in the identification of the repeating unit of the E. coli O126 strains is
yet to be explained.
3.4 The biological repeating unit
A more careful analysis of the 1H spectrum revealed additional signals of low
intensity in the anomeric region. In combination with data primarily from TOCSY
and HMBC, it was probable that these signals belonged to unsubstituted fucosyl
and mannosyl[87] residues located at the terminus of the polymer. Consequently, we
can deduce that the biological repeating unit of E. coli 396/C1 is initiated with a
GlcpNAc residue (Figure 3.1). In many other cases if GlcpNAc is found, it often
forms the first residue of the biological repeating unit[88].
23
B
a
b
B´
5.2
5.1
1H
5.0
(ppm)
Figure 3.3
(a) Part of the 1H NMR spectrum of E. coli 396/C1 PS showing the
anomeric proton resonance of fucosyl residue B and the tentatively assigned terminal
fucosyl residue here denoted as B’. (b) SDS/PAGE of an LPS isolate of E. coli
396/C1.
In addition, integration of the two anomeric fucosyl signals B and B´ allow us to
comment on the length of the polymer. A ratio of 12:1 was found suggesting a
length of ~13 repeating units. SDS/PAGE Gel electrophoresis was also performed
on the E. coli 396/C1 LPS. The length of the polymer was found to be fairly
dispersed, however a majority of the population contained 10-15 repeats; consistent
with the NMR data (Figure 3.3).
24
4
Analysis of NMR J couplings in partially protected
galactopyranosides (paper III).
4.1 Introduction
4.1.1
Synthesis
As mentioned in Section 1.1.2, protecting groups are of great importance in the
stereochemical outcome of glycosylation reactions. For donors in the gluco- and
manno-series the O2 protection groups are able to participate and thus control the
anomeric configuration in the product. Galactose however, which has an axial
hydroxyl at C4, is different since the protection group on O4 can also interfere.[9] A
free hydroxyl group at this position can also have a directing effect.[89] In addition,
galactosyl acceptors show a different reactivity depending on their anomeric
configuration.[90] An interaction between the hydroxyl proton and the ring oxygen
was judged to be important in both of these cases.
4.1.2
Spectroscopy
A 1H,1H-COSY crosspeak between HO4 and H5 was observed for several galactose
derivatives.[89] The authors stated that the origin of this crosspeak is a hydrogen
bond mediated scalar coupling from the hydroxyl proton via the ring oxygen (O5).
We found these results interesting and they prompted us to investigate the role of
the hydroxyl proton further. We were able to confirm a 1H,1H-COSY crosspeak
between HO4 and H5 in two model substances, but the interpretation was not
straightforward.[91]
4.2 The model substances and their NMR spectra
Two partially protected galactosides were selected as model substances: ethyl
2,3,6-tri-O-benzyl-1-thio-β-D-galactopyranoside 18[92] and methyl 2,3,6-tri-Obenzyl-α-D-galactopyranoside 19.[93] (Figure 4.1) They were synthesized via
reductive benzylidene opening of the corresponding 2,3-di-O-benzyl-4,6-Obenzylidene derivatives in analogy with previously described methods.[93]
25
H
H
H
O
O
O
O
H
S
O
O
O
O
O
O
O
MeO
18
H
H
θ
O
O
19
O
O
20
O
O
O
O
H
O
O
H
21
O
O
O
O
θ
O
O
H
22
Figure 4.1
The structure of the investigated molecules. The torsion angle θ =
HO4-O4-C4-H4.
4.2.1
Evaluation of the experimental J-coupling constants
1D 1H and 2D 1H,1H-COSY NMR spectra were collected for both 18 and 19 in
CDCl3 solution. (See Figure 4.2 for an example) The 1D spectra clearly showed a
complex coupling pattern for the H4, HO4 and H5 resonances and a crosspeak
between HO4 and H5 was unmistakably present in the 1H,1H-COSY spectrum. The
hydroxyl proton in 18 for example, was a dd with JH4,HO4 = 2.6 Hz and JHO4,H5 =
1.0 Hz. (Figure 4.3)
Figure 4.2
The 1H NMR spectrum of 18 in CDCl3 at 25°C. The HO4 resonance
is present at 2.50 ppm.
26
Using the relevant Karplus relationship, gauche rotamers show a reasonable
agreement with the measured value of about 2 Hz, whereas if the trans rotamer
(θ ≈ 180°, with the hydroxyl proton pointing towards the ring oxygen) was present
to a large extent, a much higher value of the coupling constant would be expected.
(Compare in Figure 1.6)
In a freely rotating hydroxyl group, all angles of the θ torsion will be equally
populated and hence averaged to a JH4,HO4 value of about 5 Hz. Thus, the low
experimental value of this coupling (shown below) suggests that a gauche
conformation is preferred and that a structure similar to 20t (see Figure 4.4) is not
predominant in solution.
Figure 4.3
Part of the 1H NMR spectrum at 25 °C showing the HO4 resonance,
δ 2.50, with JH4,HO4 = 2.6 Hz and JHO4,H5 = 1.0 Hz.
4.3 Optimization and analysis of the different rotamers
To map the source of the observed J coupling between HO4 and H5, we decided to
calculate the properties for three static rotamers of 20, a simplified system more
suitable for computational calculations. We used the three staggered rotamers of
the hydroxyl group for optimization, namely, trans with the hydroxyl proton
oriented towards the ring oxygen O5, gauche+ and gauche–. These rotamers are
referred to as 20t, 20g+ and 20g– (see Figure 4.4 for the optimized structures of 20t,
20g+) as defined by the torsion angle θ = HO4-O4-C4-H4 (Figure 4.1). The ab
initio method used was Density Functional Theory calculations (at the B3LYP/631G* level of theory).
27
By analyzing the electron density at the bond critical point for a selected interaction
of an optimized structure such as 20t, information on whether a hydrogen bond
mediated coupling pathway is likely or not can be obtained[55]. When structure 20t
was analyzed with the Atoms In Molecules package,[94] no proof in terms of
electron density was found supporting a hydrogen bond mediated coupling
pathway. This is also supported by the large equilibrium interatomic distance of
about 2.3 Å between the ring oxygen and the hydroxyl proton, too long to favour a
strong hydrogen bond interaction.
Looking at the two gauche structures, 20g+ and 20g–, the latter rotamer repeatedly
converged to the 20g+ rotamer, despite the fact that several starting conformations
were used. Interestingly, the relative energy of structure 20g+ was 3.5 kcal/mole
lower than 20t due to more favourable hydrogen bonding of the hydroxyl group to
O3 rather than to O5 (the ring oxygen). This suggests that 20g+ should be a major
conformation also in solution.
Figure 4.4
(right).
The three optimized structures 20t (left), 20g+ (middle) and 22
To validate our results from the calculations above, a molecule for which
experimental 3JH,HO couplings have been reported[95] was selected (21). The NMR
data strongly suggested a trans conformation of the H4-C4-O4-HO4 torsion – as
opposed to our results on galactopyranosides 18-20. A simplified model - 22 (see
Figures 4.1 and 4.4) was then created. The computational results showed good
agreement with the spectroscopic data, predicting trans geometry to be dominating
in this case.
28
4.4 W-coupling
Four bond spin-spin couplings are normally small and therefore seldom observed.
But if two protons, separated by four bonds are arranged as a W, then these
couplings are often significant.[96, 97] Most organic chemists have seen examples of
this as “meta-couplings” when interpreting spectra of aromatic compounds.
However, 4J-couplings may also exist in nonaromatic compounds such as αbromocyclohexanone (shown in Figure 4.5) as long as it fulfils the above
mentioned criteria.[96]
Br
Ha
a
O
H
O
Hc
O
b
Hb
H
Figure 4.5
(a) α-Bromocyclohexanone where Jab, Jac and Jbc are all about 1.1
Hz. (b) Schematic fragment of the investigated galactopyranosides highlighting the
atoms in the W-arrangement which mediate the coupling between HO4 and H5.
The four atoms defining the torsion O4-C4-C5-H5 in galactopyranosides are
practically anti-periplanar. As a result, when the hydroxyl proton is oriented in the
gauche+ conformer, a W geometry exists between HO4 and H5. This pathway
rather than a hydrogen bond mediated one is a plausible explanation for the
observed couplings and crosspeaks in the 1H-1H-COSY spectra of our model
substances and the related structures that initiated our interest in this subject.[89]
4.5 Comparison of experimental and calculated properties
4.5.1
IR spectra
The IR absorption frequencies of hydroxyl protons are very sensitive to hydrogen
bonding. Variation in the O-H stretching frequencies is a classic indication of the
presence of such interactions. IR can therefore provide additional information
which can help in determining geometry and the direction of hydrogen bonds. In
particular, since IR spectra can be calculated via ab initio methods for distinct
conformers, the magnitudes of red- or blue shift, i.e., the decrease or increase,
29
respectively, of the O-H absorption frequencies can be compared to experimental
data.
H2O
5-
6-membered ring
Figure 4.6
Selected region of the IR spectrum of 18 in CH2Cl2 at ambient
temperature with relevant absorptions marked.
IR spectra were recorded for 18 (Figure 4.6) and 19. Both showed a distinct O-H
stretching band at 3565 cm-1 in CH2Cl2. This suggests that a cis five-membered
ring is present to a large extent. The shoulder around 3500 cm-1 indicates that a sixmembered ring also is present.[97]
From the NMR JH5,H6 couplings the geometry of the hydroxymethyl group can be
estimated. The population of a gg conformation of ω (O5–C5–C6–O6) required for
a six-membered hydrogen bonded ring is low as predicted with IR spectroscopy.
This explains the shoulder at 3500 cm-1. IR spectra for the different conformers of
22 and 20 were calculated. As expected, the two supposedly hydrogen-bonded
conformers, i.e., 20g+ and 22t showed the largest relative redshift as compared to
the the other conformers. However, for the two analyzed conformers of 20, the
difference is small. As a result, it is difficult to experimentaly differentiate 20g+
from 20t via IR.
4.5.2
NMR J couplings
Spin-spin coupling constants were calculated for 20g+ and 20t at the UB3LYP/6311G** level of theory. The results are compiled in Figure 4.7 together with the
30
corresponding experimental data of 18. Excellent agreement is observed for JH4,H5
as well as for the postulated large value of JH4,HO4 in 20t. It is shown that JHO4,H5 is
significant in 20g+ but only marginal in 20t, thereby supporting our line of
reasoning.
Despite the fact that only static conformers are evaluated, i.e., no conformational
averaging is accounted for as in NMR data, the agreement between simulation and
experiment is quite satisfactory.
H
O
OR
O
2.6
1.0
H
SEt
RO
1.0
H
OR
18
H
O
1.8
H
12.2
OR
O
0.04
-0.4
RO
1.6
H
OR
OMe
H
O
OR
O
RO
1.6
20g+
H
H
OR
OMe
20t
Figure 4.7
A comparison of the J coupling constants from the recorded NMR
spectrum of 18 (R = Bn) and the calculated coupling constants from the static
rotamers of 20 (R = Me).
31
4.5.3
Conclusion
We do not find it necessary to invoke a “virtual” bond and strong hydrogen
bonding via the ring oxygen as proposed by Kwon and Danishefsky.[89] A
straightforward interpretation of the observed JHO4,H5 as a classic W-mediated
coupling is to be preferred. In essence, due to these five points:
• There is no support for electron density at the hydrogen bond critical point as
deduced by ab initio calculations
• The relative energies between conformations
• The magnitude of 3JHO4,H4
• The likelihood of significant 4J couplings when W arrangements are present,
also in saturated systems
• The calculated J couplings, in particular 4JHO4,H5
The discussion of these galactopyranosides concerns their conformation in solution.
The picture may however be different for the high-energy transition state species
formed as new glycosidic bonds are constructed. Even so, the JH4,HO4 coupling
cannot be considered as an argument for a strong hydrogen bond interaction via the
ring oxygen.
32
5
Determination of the conformational flexibility of methyl
α-cellobioside in solution by NMR spectroscopy and
molecular simulations (paper IV)
5.1 Introduction
The cellobiose disaccharide is a fragment from the nearly omnipresent cellulose
polymer. As a consequence conformational studies of cellobiosides have
reoccurred many times in the literature.[98-103] In an earlier investigation, an
increased flexibility of the ψ-torsion was found.[99] In a recent study, employing ab
initio calculations of β-cellobiose, hydrogen bonds were found to have a great
impact on the conformation.[100, 101] Interestingly, in this case the anti-φ
conformation was found to be the most stable. It has however, not before been
confirmed by NMR spectroscopy.
HO
HO
HO
OH
O
HO
HO
O
φ ψ
H
R
OMe
O
OH
23 R = H
23-d R = D
Figure 5.1
Methyl α-cellobioside (23) and its deuterated analogue (23-d).
In the present investigation, the conformational flexibility in solution was studied
with 1H,1H T-ROESY experiments in combination with molecular dynamics
simulations. Detection and quantification of anti-φ and anti-ψ conformations was
performed.
5.2 1D DPFGSE T-ROESY
Employing 1D DPFGSE T-ROESY[104] with a selective excitation of H1´ generated
the proton-proton cross relaxation rates to H3, H4 and H5 in D2O and DMSO-d6.
The cross relaxation rates were interpreted using both ISPA[52] and the model-free
33
approach[53, 54], both methods generating very similar distances. In addition, via
excitation of H2´, rH2´,H4 were also calculated using rH2´,H4´ as reference distance. To
avoid possible misinterpretation of the H1´-H3 and H1´-H5 relaxation rates,
deuterium labeling was performed[105] (23-d) to suppress three-spin effects between
H1´ and H4.
HO
HO
HO
OH
O
HO
HO
O
H
H
OMe
HO
HO
OH
O
HO
O
OH
O
HO
H
H
OH
O
HO
OMe
H
HO
HO
O
OMe
OD
H
O
O
OH
HO
anti-ψ
syn
HO
OH
anti-φ
Figure 5.2
The three conformations and the key inter-glycosidic proton-proton
pairs for the determination of their relative populations.
5.3 Deuterium isotope effects
In addition to the measurement of inter-glycosidic proton-proton distances, methyl
α-cellobioside was also screened for deuterium isotope effects.[106, 107] In aprotic
solvents such as DMSO, observation of hydroxyl-protons is possible.
DO
DO
OD
O
DO
O
O H O
D
OD
O
DO
DO
OMe
OD
O
D
23
OD
O
O H O
DO
O
OMe
24
Figure 5.3
Methyl α-cellobioside 23 and β-D-Galp-(1→3)-β-D-Glcp-OMe, 24.
Partially deuterium exchanged (H/D 2/1) samples were prepared in DMSO-d6.
Substance 24 displayed deuterium isotope effects whereas none were observed for 23.
Partially deuterium exchanged samples can, if a hydrogen-bond is present, give rise
to deuterium isotope effects and thereby act as a sensor for specific conformations.
We were able to reproduce the results of Dabrowski and co-workers[106, 107] on β-DGalp-(1→3)-β-D-Glcp-OMe, 24 but could not detect such effects for 23.
34
5.4 Computer simulations
To provide support for the interpretation of the NMR data and to investigate the
conformational space of 23, computer simulations were performed. Following
initial Monte Carlo (MC) simulation using the HSEA approach, a molecular
dynamics simulation employing the PARM22 force field with explicit water as
solvent was performed. In addition to the syn conformation, the simulation also
revealed the presence of anti-φ and anti-ψ conformations.
5.5 Population analysis
The T-ROE derived distances from the experiments performed in D2O were
evaluated in combination with data from the MD simulation with respect to the
three conformations (see table 5.5). Employing a three-state analysis, relative
populations of 93%, 5% and 2% of the syn, anti-ψ and anti-φ conformer
respectively were present.
Table 5.5
experiments.
Distances obtained from simulation, minimization and NMR
Proton pair
<r>syn
<r>anti-ψ
ranti-φ
rexp
H1',H4
2.39
3.58
3.53
2.29
H1',H3
4.45
2.26
4.45
3.58
H2',H4
4.41
4.71
2.22
3.90
<r>syn and <r>anti-ψ are average distances extracted from the MD simulation. ranti-φ is
obtained by energy minimization. rexp is derived from NMR data with ISPA. All
distances are given in Å.
35
Acknowledgements
Many people have helped me in various ways since I started here back in ´99. Hence, I would
like to acknowledge:
My supervisor, Professor Göran Widmalm for the knowledge, encouragement, and patience.
Professor Jan Erling Bäckvall and professor emeritus Per J. Garegg for kind intrest in our work.
Andrei Weintraub for good collaboration.
Past and present members of the Widmalm group; Mange, Tina, Classe, Micke, Chrille, Peter,
Glen, Malin, Torgny, Smühl (for the “Berlin” wall), Rob, Jennie, Ulrika and Tara.
Tjockisarna Mia, Peter and Rolle For nice lunch company and stimulating discussions of
chemistry, football and other important things.
Micke, Classe, Tina, Smühl and Jennie for the crash course in NMR
The former habitants of Lab 7.
Glen for linguistic improvement.
And the rest of the department for providing a pleasant environment to work in!
Dr. Jan Lindberg and Professor Peter Konradsson for the introduction to carbohydrate synthesis.
Britt, Pia, Kristina, Robin and Olle for help in various ways.
All off-campus friends
My family.
Anna
I would also like to acknowledge the financial support from: Nils Löfgrens fond, Svenska
kemistsamfundet, Wallenbergsstiftelsens jubileumsfond and C. F. Liljevalch Jr:s stipendiefond.
36
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