Fully deprotected β-(1→2)-mannotetraose forms a contorted α-helix in solution: Convergent synthesis and conformational characterization by NMR and DFT

Article abstract of DOI:10.1039/b9nj00702d

β-(1→2)-linked oligomannosides, found in the cell wall of Candida albicans, are promising structures for the development of C. albicans vaccines. Considerable effort in recent years has been devoted to the synthesis of these carbohydrate structures. As a result, several successful synthetic methodologies based on linear approaches have emerged. Here, we demonstrate that a fully deprotected β-(1→2)-linked mannotetrasaccharide can also be conveniently constructed by the convergent direct coupling of two appropriately protected disaccharides. This improved approach offers several advantages over previously published methods. The number of steps needed to reach larger oligosaccharides is decreased, while high selectivity is retained when the crucial glycosylation step is performed with two disaccharides, herein providing β-(1→2)-mannotetraose in 4.2% overall yield over 16 steps starting from d-mannose. Additionally, the complete structural characterization of the products by NMR spectroscopy is reported. In the conformational study of the final product, 2D-NOESY was used in combination with spectral simulations performed using PERCH software. The experimental results obtained confirm the contorted α-helical structure predicted earlier for these oligosaccharides in solution. As a culmination of the conformational study, a model was constructed by molecular modeling using DFT, and the minimum energy conformation was found to be in full agreement with the experimental results. This is the first study to date where the conformation of a fully deprotected mannotetraose has been reported. The Royal Society of Chemistry and the Centre National de la Recherche Scientifique.

Full text of DOI:10.1039/b9nj00702d

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www.rsc.org/njc | New Journal of Chemistry
Fully deprotected b-(1-2)-mannotetraose forms a contorted a-helix
in solution: convergent synthesis and conformational characterization
by NMR and DFTw
Filip S. Ekholm,a Jari Sinkkonenb and Reko Leino*a
Received (in Montpellier, France) 24th November 2009, Accepted 3rd December 2009
First published as an Advance Article on the web 20th January 2010
DOI: 10.1039/b9nj00702d
b-(1-2)-linked oligomannosides, found in the cell wall of Candida albicans, are promising
structures for the development of C. albicans vaccines. Considerable effort in recent years has
been devoted to the synthesis of these carbohydrate structures. As a result, several successful
synthetic methodologies based on linear approaches have emerged. Here, we demonstrate that a
fully deprotected b-(1-2)-linked mannotetrasaccharide can also be conveniently constructed by
the convergent direct coupling of two appropriately protected disaccharides. This improved
approach offers several advantages over previously published methods. The number of steps
needed to reach larger oligosaccharides is decreased, while high selectivity is retained when the
crucial glycosylation step is performed with two disaccharides, herein providing b-(1-2)-
mannotetraose in 4.2% overall yield over 16 steps starting from D-mannose. Additionally, the
complete structural characterization of the products by NMR spectroscopy is reported. In the
conformational study of the final product, 2D-NOESY was used in combination with spectral
simulations performed using PERCH software. The experimental results obtained confirm the
contorted a-helical structure predicted earlier for these oligosaccharides in solution. As a
culmination of the conformational study, a model was constructed by molecular modeling using
DFT, and the minimum energy conformation was found to be in full agreement with the
experimental results. This is the first study to date where the conformation of a fully deprotected
mannotetraose has been reported.
from ester protective groups at C-2 will lead to formation of
the a-mannoside.4 Despite this challenge, several successful
methodologies have been developed to overcome these
Introduction
The yeast Candida albicans, an opportunistic pathogen found
difficulties.4a,5 Although the development of selective methodo-
in the mucous membrane of the gut, skin, gastrointestinal tract
and mouth, is considered to be part of the normal human
logies for the synthesis of complex carbohydrates can be
flora. In immunocompromised patients, C. albicans can grow
considered important, the full spectroscopic analysis of such
out of control, a condition termed candidiasis, thereby result-
ing in severe infections.1 Through studies on the composition
structures is of equal importance.
The cell wall composition of C. albicans has been the subject
of several structural studies.2 Special attention has been drawn
to the b-(1-2)-linked mannans, not only due to the immuno-
chemical activity shown by portions of these polymers but also
because such structures have been predicted to display interest-
ing conformational properties.6 Delicate conformational studies
by Bundle and colleagues on propyl-capped glycosides have
revealed that these carbohydrates form contorted a-helical
structures in solution.6b Studies on fully deprotected oligomanno-
sides have, however, not been reported previously.
of C. albicans’ cell wall, an epitope consisting of b-(1-2)-
linked mannans, isolated from the acid labile fraction of
phosphopeptidomannan, was identified as important for the
inhibition of such infections.1c,2 To date, several biological
studies have confirmed the protective role of b-(1-2)-linked
mannans and furthermore implicated their involvement in the
stimulation of TNF-a production from macrophages.1c,3
There are several synthetic challenges associated with the
construction of b-linked mannosides. Both thermodynamic
and kinetic conditions lead to formation of the a-anomer. In
addition to these effects, neighbouring group participation
In light of the extensive and elegant work previously carried
out in this field, we present here an improved convergent
synthetic protocol for the synthesis of b-(1-2)-mannotetraose,
in part derived and modified from the earlier linear approaches
developed by Crich and Mallet et al., for the synthesis of
b-(1-2)-linked oligomannosides.5d,e The advantages gained
from the enhanced convergent approach presented reduces the
number of reaction steps while maintaining a high selectivity
a
˚
Laboratory of Organic Chemistry, Abo Akademi University,
˚
FI-20500, Abo, Finland. E-mail: reko.leino@abo.fi;
Fax: +358 2-2154866; Tel: +358 2-2154132
b Laboratory of Organic Chemistry and Chemical Biology, University
of Turku, FI-20014, Turku, Finland
w Electronic supplementary information (ESI) available: Complete
spectral data for structures 3 and 4 (in both MeOD and D2O), along
with molecular modelling data for structure 4. See DOI: 10.1039/
b9nj00702d
1
in the crucial glycosylation. In addition, the full H and 13C
NMR spectroscopic analysis and signal assignments of both
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the protected and deprotected mannotetraoses, including spec-
tral simulations and accurate determinations of coupling
constants, are presented here for the first time. The experi-
mental data obtained was further utilized in the conforma-
tional analysis of the final product, the minimum energy
conformation of which was also verified by molecular model-
ling using density functional theory.
its linear counterpart, the most important being, in most cases,
a shortened synthetic route.9
The disaccharide building blocks 1 and 2 (Fig. 1) were
synthesized according to the slightly modified literature methodo-
logies developed by Crich et al. and previously also utilized
in our laboratory.10 The reaction route employed in our
previous study for the preparation of donor 1 was shortened
by four steps by the direct dibenzylation of phenyl 4,6-O-
benzylidene-1-thio-a-D-mannopyranoside, followed by
a
coupling with the thio acceptor at ꢁ78 1C, thereby giving
disaccharide donor 1 in 76% yield (b : a 9.9 : 1) over two steps.
Donor 1 was coupled with acceptor 2 in a similar fashion to
obtain protected tetrasaccharide 3 (Scheme 1).11 After a sub-
sequent work-up and purification, compound 3 was isolated in
55% yield (b : a 10 : 1). In comparison with the earlier reported
linear approach,5e the selectivity of the crucial glycosylation
was more than two times better when the convergent protocol
was tested. The yield was only slightly lower than those
reported earlier by Crich and co-workers5e (53% yield, starting
from the disaccharide acceptor and monosaccharide donor)
but slightly higher than the yield reported by Bundle et al.5b
(31% yield, starting from the disaccharide acceptor and
monosaccharide donor). In contrast with the convergent
approach of Mallet et al.,5d the deprotection of the b-(1-2)-
linked mannotetrasaccharide can here be achieved by a one
step hydrogenolysis instead of the three step deprotection
sequence required by the earlier approach. Therefore, the
improved convergent approach developed and utilized in this
paper can be considered a feasible alternative when construct-
ing larger b-(1-2)-mannans.
The final deprotection of 3 by hydrogenolysis under 2.8 bar
H2 in methanol proceeded smoothly to provide 4. The yield
was, however, highly dependent on the deprotection condi-
tions. When using 2.5 weight equiv. of Pd/C (10% Pd) with a
substrate concentration of 10 mg mlꢁ1 methanol, a reduced
tetrasaccharide containing mannitol at the reducing end was
obtained in 40% yield. The formation of this byproduct was
verified by both HRMS (m/z = 691.2281 [M + Na]+) and
NMR spectroscopy, showing three anomeric signals in the 1H
NMR spectrum at 4.89, 4.82 and 4.81 ppm, and in the 13C
NMR spectrum at 101.1, 100.5 and 99.7 ppm, respectively.
Formation of this undesired side-product was, however,
strongly suppressed when 2 weight equiv. Pd/C was used
and the concentration of 3 was 20 mg mlꢁ1 methanol, thereby
increasing the yield of the desired product to 75% (for
purification details, see the Experimental section).
Results and discussion
Synthesis of b-(1-2)-mannotetraose
Previous work at our laboratory has focused on the synthesis
of fully deprotected b-(1-2)-linked mannotriose and some of
its close analogues by the utilization of a linear glycosylation
strategy.7 With this protocol, the efficient and highly selective
synthesis of b-(1-2)-linked mannosides is accomplished by
activation of a thioglycoside (or mannopyranosyl sulfoxide) at
low temperatures with 1-(phenylsulfinyl) piperidine (BSP),
2,4,6-tri-tert-butyl pyrimidine (TTBP) and Tf2O, thereby
forming an a-D-mannopyranosyl triflate, which in turn reacts
with an acceptor in an SN2 fashion. Further notable features
of this method involve the influence of protecting groups,
especially the 4,6-O-benzylidene acetal, on the outcome of
glycosylation.8 This direct highly b-selective mannosylation
protocol, originally developed in the group of Crich, was, in
their work, successfully utilized for the synthesis of a cyclo-
hexyl-capped, b-(1-2)-linked mannooctasaccharide.5e As
noted by Crich and co-workers,5e when progressing from the
trisaccharide stage towards the tetrasaccharide, a significant
decrease in selectivity, from b : a 9.9 : 1 to 3.9 : 1, of the
individual coupling reaction is observed. Despite the decrease
in selectivity, a high efficiency is maintained throughout the
entire synthetic pathway.
Mallet et al. earlier reported a convergent approach based
on a similar protective group strategy, where two disaccha-
rides were coupled to obtain a tetrasaccharide.5d However, in
their work, neither the selectivity of the crucial coupling step
nor NMR spectroscopic data for the compounds prepared was
reported. A major drawback of their approach was the use of
three different types of protective groups, requiring a three
step deprotection sequence in order to obtain the fully depro-
tected b-(1-2)-linked mannotetrasaccharide. Another suc-
cessful approach, developed by Bundle and co-workers, has
likewise received considerable attention recently.5b,5f The key
features of this method consist of glycosylation utilizing a
ulosyl donor, followed by epimerization at C-2 with L-selec-
tride. This strategy was successfully applied to the synthesis of
b-(1-2)-linked oligomannosides with a degree of polymeriza-
tion (DP) from two to six.6b Despite the high selectivities
throughout the synthesis, the yields of the individual glyco-
sylation reactions declined when approaching the tetra-
saccharide, from 65% in the trisaccharide step to 48% in the
tetrasaccharide step.
Characterization by NMR spectroscopy
In order to verify the linkages and fully characterize the
products, several NMR spectroscopic methods were utilized.
Considering the general advantages of the previously reported
attempts, we set out to prepare fully deprotected b-(1-2)-
mannotetraose by
a modified convergent approach. In
general, the convergent approach has several advantages over
Fig. 1 The disaccharide building blocks 1 and 2.
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powerful tool and was utilized to overcome the complications.
Compound 4 gave rise to well separated signals corresponding
to the H-1 and H-2 protons on the different residues of both
anomers. These signals were also targeted with 1D-TOCSY. It
was observed that the separation is highly influenced by the
NMR solvent used (MeOD vs. D2O). In MeOD, an enhanced
separation of the crucial signals was observed. From a bio-
logical standpoint, however, D2O is of more interest and,
accordingly, NMR spectroscopic data was recorded in both
solvents. The importance of improved signal separation is
significant, and thus the following discussion is based on
NMR spectra recorded in MeOD. By narrowing the excitation
range of the 1D-TOCSY, even signals separated by only 6 Hz
were successfully excited to give a 1H NMR spectrum of a
single residue. The 1D-TOCSY spectra of all the residues are
shown in Fig. 3. To determine the order of the residues, the
13C NMR chemical shifts for the reducing end anomers were
chosen as the starting point. These signals appear at a con-
siderably lower frequency (94.1 ppm for C-1a and 95.5 ppm
for C-1b) compared to the other anomeric carbons (between
103.3–101.1 ppm). In HMBC, the long range coupling
between H-2–C-10 was clearly visible. The inverse coupling,
i.e., H-10–C-2, was also observed. By iteration of this approach,
the different monomers were determined in order, starting
from the reducing end residue. H-50 0 0 was shown to give a
signal at a higher frequency in both anomers (3.36 ppm for
H-50 00a and 3.39 ppm for H-50 0 0b) than the remaining H-5b
protons (between 3.25–3.20 ppm). This also indicates that the
residues were assigned correctly. It was noticed, however, that
in D2O, this effect does not occur, thereby all the H-5b protons
appear at a similar chemical shift. Furthermore, the 2D-
NOESY spectrum shows correlations through space between
Scheme 1 (i) (a) TTBP, BSP, Tf2O, CH2Cl2, ꢁ60 1C, 0.5 h; (b) 2, ꢁ78
1C, 3 h, 55%. (ii) Pd/C, H2 (2.8 bar), MeOH, rt, 19.5 h, 75%.
1
In the H NMR spectrum of 3, chemical shifts for the three
H-5b protons were observed at low frequencies (3.42 ppm for
H-500, 3.31 ppm for H-50 and 3.30 ppm for H-50 00) in compar-
ison to the chemical shift of H-5a (3.86 ppm for H-5), thereby
suggesting that the b-anomer was indeed formed. A similar
difference was witnessed in the 13C NMR spectrum, where the
three C-5b carbons (67.9 ppm for C-500, 67.8 ppm for C-50 and
67.5 ppm for C-50 00) appear at a slightly higher frequency than
the C-5a carbon (64.2 ppm for C-5). These results are in
accordance with the general view on the chemical shifts of
a- and b-mannosides.5e,7a,12 Furthermore, an upfield shift of
the three H-3b protons (3.64 ppm for H-30, and 3.53 ppm for
H-30 0 0 and H-300) was observed, further indicating a different
linkage to the one existing in the first residue (4.01 ppm for
H-3). In addition, the coupling constants obtained by simula-
1
tion of the H NMR spectrum clearly indicated a difference
between the H-1–H-2 coupling constants for the a-linked
residue (J1,2 = 1.6 Hz for H-1) and the b-linked residues
H-1n+ and H-2n (where n = 0, and 00), which are in
agreement with the assignment based on the HMBC spectrum.
The information gained by 2D-NOESY will be further dis-
cussed below when assessing the conformation of this mole-
cule. With the information received from these standard NMR
methods (1H, 13C, DQF-COSY, HSQC, HMBC, 1D-TOCSY
and 2D-NOESY), the spectrum was simulated using PERCH.
In order to simplify matters, the individual 1D-TOCSY spectra
were first simulated, followed simulating the complete spectrum,
containing both anomers (Fig. 4).
(J1 ,2 = 0.3 Hz for H-100, J1
= 0.8 Hz for H-100 0 and
0
0
00 00
0 0 0,20 0 0
J1 ,2 = 0.7 Hz for H-10), providing further support for the
assigned stereochemistry. Ultimately, the linkages were proven
by the use of coupled HSQC, showing large differences in the
0
0
0 0 0,H-10 0 0
JC,H coupling constants (JC-1
= 158.3 Hz for C-10 00
,
JC-1 ,H-1 = 158.6 Hz for C-100, JC-1 ,H-1 = 156.0 Hz for C-10
00
00
0
0
and J
= 167.8 Hz for C-1).
C-1,H-1
In general, the full spectral characterization of complex
oligosaccharides is one of the most demanding tasks in
carbohydrate chemistry.13 For this purpose, 1D-TOCSY
(1D-TOtal Correlation SpectroscopY) proved to be a valuable
tool.14 Well separated signals corresponding to the different
ring units were targeted by selective excitation, and a spinlock
time of 500 ms was found to be suitable for assuring magne-
tization spreading to the entire ring system.
Conformational analysis
The b-(1-2)-linked oligomannosides were first predicted to
form interesting 3D structures, contorted structures folding
back upon themselves, more than 30 years ago.6c The earlier
work by Bundle and co-workers on a b-(1-2)-linked manno-
pentasaccharide revealed these assumptions to be correct.6b
NOE contacts between non-contiguous residues were
witnessed, thus suggesting that a contorted a-helical structure
is indeed formed. These studies were, however, conducted on a
propyl-capped glycoside instead of a free b-(1-2)-linked
oligomannoside. Further evidence of a contorted structure
was reported by Crich et al.6a During the synthesis of an
octameric cyclohexyl-capped oligomannoside, they were able
to crystallize a fully protected tetrasaccharide. In the present
study, the conformational analysis was performed with 4,
The use of 1D-TOCSY reduced the spectral complexity to a
monosaccharide level. The order of the residues and the
positions of the protecting groups were determined by HMBC.
The full spectral assignment was finally carried out using
PERCH (PEAk ResearCH) NMR software, the results of
which are shown in Fig. 2.15
In order to fully assign the 1H NMR spectrum of 4, a similar
set of NMR spectroscopic methods were needed. The NMR
spectrum of a tetrasaccharide alone is, in many cases,
problematic to solve as such. Here, due to mutarotation, an
a : b ratio of 7 : 2 for 4 was observed, thereby complicating the
assignment even further. Again, 1D-TOCSY proved to be a
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Fig. 2 A spectral simulation of the 1H NMR spectrum of 3 (5.3–3.2 ppm region) using PERCH NMR software; top: simulated spectrum, bottom:
observed spectrum.
than the H-4–H-100 and H-4–H-100 0 correlations. This informa-
tion confirms the contorted structure predicted several years
ago, and witnessed by Bundle et al.6b and Crich et al.6a
In addition, a correlation between H-1–H-10 and H-1–H-20
was observed here for the first time. The lack of this correlation
in the study performed by Bundle and co-workers may have
been partly due to the use of a propyl-capped b-linked manno-
glycoside. Furthermore, 2D-NOESY correlations between
H-1n+0 and H-2n (where n = 0, 0 and 00) were witnessed, thereby
suggesting steric interactions to place the residues in a specific
order. In addition, correlations between H-1–H-2, H-1–H-3 and
H-1–H-5 of the b-linked residues, along with the accurate
coupling constants obtained using PERCH, suggest the differ-
4
ent residues exist in the C1 chair conformation.
To further investigate and verify the conformation sug-
gested by NMR, computational methods were utilized to
model the preferred structure. The initial structure was first
sketched by quick modelling using molecular mechanics with
Fig. 3 1D-TOCSY spectra of the different ring units in 4 (in MeOD).
From the top: b, b0, b00, b0 0 0, a, a0, a00, a0 0 0 and the entire spectrum.
Chem3D Pro 11.0 software. The geometry of the achieved
structure was further optimized by the DFT method B3LYP
using the 6-31G(d,p) basis set. This method has been found to
give good results in reasonable time for fairly large mole-
cules.16 Since the potential energy surface of relatively flexible
molecules may consist of several minima, the first attempt did
not find the structure that would correspond to all of the
NMR spectroscopic (especially NOE) results. Therefore, the
torsion angles were modified in order to obtain a starting
structure that was in agreement with the observed NOE
correlations. When this structure was optimized, a stationary
point was found that was in full agreement with the experi-
mental results. Frequency calculations were performed in
order to prove that the stationary point found is indeed a
minimum energy point. The optimized structure is presented
in Fig. 6 and Fig. 7, and is reported in Cartesian coordinates in
bearing no protective groups. Therefore, the observed con-
formation should only result from the steric and electronic
interaction displayed by the oligosaccharide itself. In contrast
to the previous studies, where b-linked glycosides were used,
the discussion below is based on information from the dom-
inating a-anomer.
Luckily, H-4 was well separated (dd at 3.63 ppm) in the 1H
NMR spectrum of 4 recorded in MeOD. This was not the case
in D2O. From the 2D-NOESY spectrum, the correlations also
witnessed by Bundle and co-workers were confirmed.6b The
most important correlations, between the non-contiguous
residues, are H-4–H-200, H-4–H-100 and H-4–H-10 0 0 (Fig. 5
and Fig. 6). The H-4–H-200 correlation was, however, stronger
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Fig. 4 Spectral simulation of the 1H NMR spectrum of 4 (5.2–3.1 ppm region) using PERCH NMR software; top: simulated spectrum, bottom:
observed spectrum.
Fig. 5 The 2D-NOESY spectrum of 4 in MeOD, displaying correlations between protons that are close in space. The most important correlations
are highlighted with red circles.
the ESI.w The distances between the protons in the optimized
structure for the key NOE connectivities were found to be 3.19 A
(H-4–H-200), 4.13 A (H-4–H-100) and 3.65 A (H-4–H-10 00).
These results were in good agreement with the peak intensities
witnessed in the NOESY spectrum. All of the mannose
moieties were found to exist in the 4C1 conformation as
expected. The optimized results also exhibit the contorted
a-helical structure of the molecule (Fig. 7).
It was previously suggested that the glycosidic linkages are
hidden in the middle of the contorted a-helical structure, thus
creating a hydrophobic interior, and that the hydroxyl groups
are oriented outwards, further creating
a hydrophilic
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study and complete NMR spectroscopic characterization of a
fully deprotected b-(1-2)-linked mannotetrasaccharide was
executed based on the information obtained from different
NMR spectroscopic techniques. By using molecular model-
ling, a model in good agreement with the experimental results
was obtained, thereby providing further evidence of the con-
formation adopted by these molecules in solution. The NMR
spectroscopic information presented here will, in the future, be
used as a starting point when studying interactions between
4 and receptors on macrophages. This could potentially
provide information about the crucial functionalities needed
for binding to the receptor and may result in the development
of simpler structures for C. albicans vaccines.
Fig. 6 The B3LYP/6-31G(d,p) optimized structure of the a-anomer,
showing the most important NOESY correlations.
Experimental section
Instrumentation and general experimental details
Reaction solvents were dried and distilled prior to use when
necessary. All reactions containing moisture or air sensitive
reagents were carried out under an argon atmosphere.
NMR spectra were recorded using Bruker Avance NMR
spectrometers operating at 500.13 MHz (1H: 500.13 MHz, 13C:
125.77 MHz) or 600.13 MHz (1H: 600.13 MHz, 13C: 150.90
MHz). Both spectrometers had a similar basic configuration
with a lock channel (2H), and a high frequency (1H–19F) and
broad band frequency channel (109Ag–31P). Both instruments
were equipped with a Z gradient facility and carried inverse
double resonance probes with a Z-axis gradient. Each spectro-
meter had two waveform generators for work with shaped
pulses. The probe temperature during the experiments was
kept at 25 1C. All products were fully characterized by utilizing
1H, 13C and TOCSY 1D techniques, in combination with
DQF-COSY, NOESY, HSQC (both coupled and decoupled)
and HMBC 2D techniques, by using pulse sequences provided
by the manufacturer.
Fig. 7 The B3LYP/6-31G(d,p) optimized structure of the a-anomer,
displaying the contorted a-helical nature of the molecule.
exterior.6b A similar pattern can be witnessed for the fully
deprotected tetrasaccharide, as displayed in Fig. 7. These
structures may have increased stability towards hydrolysis as
the glycosidic bonds are hidden in the middle of the helix. Due
to the helical nature of these molecules, they could be used in
the site selective modification of proteins to insert helical
fragments of well defined structure (as a structural motif) into
a protein backbone.17
1H NMR spectra were acquired using the zg30 pulse
program with spectral widths of 12 kHz consisting of 64 K
data points zero-filled to 256 K prior to Fourier transforma-
tion. A single-pulse excitation with a 301 flip angle was used.
13C NMR spectra were acquired using the zgpg30 pulse
program with spectral widths of 36 kHz consisting of 65 K
data points zero-filled to 128 K prior to Fourier transforma-
tion. A single-pulse excitation with a 301 flip angle was used.
Furthermore, an exponential window function was used when
processing the FID (lb 1 Hz). TOCSY 1D spectra were
acquired using either the selmlzf or selmlgp.2 pulse program
with a spin lock time of 500–700 ms. Spectral widths of 10–12
kHz consisting of 32 K data points zero-filled to 64 K prior to
FFT. Gradient versions of the 2D techniques were used. DQF-
COSY spectra were measured using the cosygpmfqf pulse
program. HSQC spectra were measured using the hsqcetgpsi
Conclusions
To summarize, an improved protocol for the synthesis of
b-(1-2)-linked oligomannosides has been developed and
utilized in the synthesis of a fully deprotected b-(1-2)-manno-
tetraose. The key features of this method involve a crucial
coupling being performed between a disaccharide donor and
acceptor. A careful design strategy for the protective group
allows the deprotection to be carried out in one step. Further-
more, NMR spectroscopic methods widely applicable to the
characterization of complex oligosaccharides were investi-
gated and presented in detail. These methods were utilized in
the full spectroscopic assignment and accurate determination
of coupling constants for both the protected and deprotected
mannan tetrasaccharides. In addition, the first conformational
1
pulse program. In order to obtain JC,H coupling constants,
the decoupling pulse power was turned to zero (120 dB),
resulting in a proton-coupled HSQC spectrum. HMBC spectra
were acquired using the hmbcgplpndqf pulse program with
n
1JH,C = 145 Hz and JH,C = 2–10 Hz. NOESY 2D spectra
were acquired using the noesygpph pulse program with a
mixing time between 0.5–1.0 s.
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Chemical shifts are expressed on the d scale (in ppm) using
TMS (tetramethylsilane), residual chloroform, acetone, H2O or
methanol as internal standards. Coupling constants are given in
Hz and coupling patterns are given as s: singlet, d: doublet,
t: triplet, etc. When signals appear at similar chemical shifts, if
possible, additional decimals are used to determine the order of
the signals. Computational analysis of the 1H NMR spectra of
compounds 3 and 4 was achieved using PERCH NMR soft-
ware, with starting values and spectral parameters obtained
from the various NMR techniques used.15
1H NMR (600.13 MHz, CDCl3, 25 1C): d = 7.48–7.07 (m, 50
H, Ar-H), 5.57 (s, 1 H, CH00 0Ph), 5.52 (s, 1 H, CHPh), 5.43 (s, 1
H, CH0Ph), 5.41 (s, 1 H, CH00Ph), 5.24 (d, 1 H, J1 ,2 = 0.3 Hz,
00 00
H-100), 5.05 (d, 1 H, J1
= 0.8 Hz, H-100 0), 5.00 and 4.83
0 0 0,20 0 0
(each d, each 1 H, J = ꢁ12.5 Hz, 20 0 0-CH2Ph), 4.89 (d, 1 H, J1,2
= 1.6 Hz, H-1), 4.72 and 4.495 (each d, each 1 H, J =
ꢁ11.9 Hz, 1-CH2Ph), 4.71 and 4.68 (each d, each 1 H,
J = ꢁ12.4 Hz, 3-CH2Ph), 4.69 and 4.62 (each d, each 1 H,
J = ꢁ12.3 Hz, 30-CH2Ph), 4.63 and 4.45 (each d, each 1 H, J =
ꢁ12.0 Hz, 300-CH2Ph), 4.496 and 4.39 (each d, each 1 H,
J = ꢁ11.8 Hz, 30 00-CH2Ph), 4.61 (d, 1 H, J1 ,2 = 0.070 Hz, H-10),
0
0
HRMS were recorded using a Bruker Micro Q-TOF instru-
ment with ESI (electrospray ionization) operating in positive
mode. Optical rotations were measured at 23 1C with a Perkin-
Elmer polarimeter equipped with a sodium lamp (589 nm).
TLC was performed on aluminium sheets pre-coated with
silica gel 60 F254 (Merck). Preparative TLC was performed
on aluminium sheets pre-coated with silica gel 60 F254 (0.5 cm,
Merck). Flash chromatography was carried out on silica gel 60
(0.040–0.060 mm, Merck). Spots were visualized by UV,
followed by charring with 1 : 10 H2SO4 : MeOH and heating.
Compound 4 was purified by high-pH anion exchange chro-
00 00
00 00
4.53 (dd, 1 H, J1 ,2 = 0.3 Hz, J2 ,3 = 3.1 Hz, H-2 ), 4.45 (dd,
1 H, J1 ,2 = 0.7 Hz, J2 ,3 = 3.2 Hz, H-20), 4.41 (dd, 1 H,
0
0
0
0
= 3.2 Hz, H-20 0 0), 4.34 (dd, 1 H,
0 0 0,20 0 0
0 0 0,30 0 0
J1
= 0.8 Hz, J2
J5 ,6 a = 4.7 Hz, J6 a,6 b = ꢁ10.2 Hz, H-600a), 4.29 (dd, 1 H,
00 00
00
00
0
0
J1,2 = 1.6 Hz, J2,3 = 3.5 Hz, H-2), 4.29 (dd, 1 H, J5 ,6 a
=
4.7 Hz, J6 a,6 b = ꢁ10.3 Hz, H-60a), 4.22 (dd, 1 H, J5,6a = 4.8 Hz,
0
0
0 0 0,50 0 0
J6a,6b = ꢁ10.3 Hz, H-6a), 4.182 (dd, 1 H, J4
= 9.3 Hz,
= 4.7 Hz,
a
J3
J6
= 9.8 Hz, H-40 00), 4.177 (dd, 1 H, J5
0 0 0,40 0 0
0 0 0,60 0 0
b = ꢁ10.3 Hz, H-60 0 0a), 4.06 (dd, 1 H, J4 ,5 = 9.5 Hz,
0 0 0a,60 0 0
00 00
J3 ,4 = 9.6 Hz, H-400), 4.01 (dd, 1 H, J2,3 = 3.5 Hz, J3,4
=
00 00
matography (HPAEC) using
a
Dionex DX600 system
10.0 Hz, H-3), 3.92 (dd, 1 H, J4,5 = 9.5 Hz, J3,4 = 10.0 Hz,
H-4), 3.91 (dd, 1 H, J4 ,5 = 9.4 Hz, J3 ,4 = 9.9 Hz, H-40),
0
0
0
0
equipped with an amperometric detector.
The initial structure for modelling was drawn using Chem-
BioDraw Ultra 11.0 and modelled by Chem3D Pro 11.0 using
a MM2 molecular mechanics method. All higher quality
calculations were performed by Gaussian 03 software using
the density functional method B3LYP as a hybrid functional
and 6-31G(d,p) as the basis set.18 The results were examined
using the GaussView 4.1.2 program.
3.864 (ddd, 1 H, J5,6a = 4.8 Hz, J4,5 = 9.5 Hz, J5,6b = 10.3 Hz,
0 0 0,60 0 0
0 0 0a,60 0 0
H-5), 3.862 (dd, 1 H, J5
b = 10.3 Hz, J6
b = ꢁ10.3 Hz,
00
H-60 00b), 3.78 (dd, 1 H, J5 ,6 b = 10.0 Hz, J6 a,6 b = ꢁ10.2 Hz,
00 00
00
H-600b), 3.75 (dd, 1 H, J5 ,6 b = 10.1 Hz, J6 a,6 b = ꢁ10.3 Hz,
0
0
0
0
H-60b), 3.69 (dd, 1 H, J5,6b = 10.3 Hz, J6a,6b = ꢁ10.3 Hz,
H-6b), 3.64 (dd, 1 H, J2 ,3 = 3.2 Hz, J3 ,4 = 9.9 Hz, H-30), 3.53
0
0
0
0
(dd, 1 H, J2
= 3.2 Hz, J3
= 9.8 Hz, H-30 00), 3.53 (dd,
0 0 0,30 0 0
0 0 0,40 0 0
1 H, J2 ,3 = 3.1 Hz, J3 ,4 = 9.6 Hz, H-300), 3.42 (ddd, 1 H,
00 00
00 00
J
5 ,6 a = 4.7 Hz, J4 ,5 = 9.5 Hz, J5 ,6 b = 10.0 Hz, H-500), 3.31
00 00 00 00 00 00
Synthetic procedures
0
0
0
0
0
0
(ddd, 1 H, J5 ,6 a = 4.7 Hz, J4 ,5 = 9.9 Hz, J5 ,6 b = 10.1 Hz,
H-50) and 3.30 (ddd, 1 H, J5
a = 4.7 Hz, J4 = 9.3 Hz,
0 0 0,60 0 0
0 0 0,50 0 0
Benzyl 2,3-di-O-benzyl-4,6-O-benzylidene-b-D-mannopyranosyl-
(1-2)-3-O-benzyl-4,6-O-benzylidene-b-D-mannopyranosyl-(1-2)
-3-O-benzyl-4,6-O-benzylidene-b-D-mannopyranosyl-(1-2) -3-O-
benzyl-4,6-O-benzylidene-a-D-mannopyranoside (3). To a solu-
tion containing 1 (76 mg, 0.086 mmol) in dry dichloromethane
(2.5 ml) was added at ꢁ60 1C (acetone + dry ice) BSP (22 mg,
0.10 mmol, 1.2 equiv.), TTBP (32 mg, 0.13 mmol, 1.5 equiv.)
and Tf2O (19 ml, 0.12 mmol, 1.3 equiv.). The resulting mixture
was stirred for 0.5 h, followed by cooling to ꢁ78 1C and the
addition of 2 (78 mg, 0.10 mmol, 1.15 equiv.) dissolved in
dichloromethane (1.5 ml) over a time period of 15 min. The
reaction mixture was stirred for 2 h and quenched by the
addition of triethylphosphite (68 ml). The reaction mixture was
then stirred for 1 h at ꢁ78 1C, brought to RT, diluted with
dichloromethane (30 ml) and washed with a saturated NaHCO3
solution. The water phase was separated and extracted with
dichloromethane (3 ꢂ 20 ml). The combined organic phase
was washed with brine (30 ml), dried with Na2SO4, filtered and
concentrated. The crude product was purified by preparative
TLC (hexane : ethyl acetate 3 : 2). The spots containing the
product were scraped off the plate and dissolved in EtOAc
(50 ml). After 2 h of stirring, the mixture was filtered and
concentrated to give the title compound as a colorless oil
(73 mg, b : a 10 : 1, 55%).
J5
= 10.3 Hz, H-50 00) ppm.
0 0 0,60 0 0
b
13C NMR (150.90 MHz, CDCl3, 25 1C): d = 139.4–126.1
(Ar-C), 103.5 (C-100 0, JC-1
101.88 (C0HPh), 101.87 (C-100, JC-1 ,H-1 = 158.6 Hz), 101.5
= 158.3 Hz), 101.91 (CHPh),
0 0 0,H-10 0 0
00
00
(C00HPh), 101.2 (C0 0 0HPh), 100.2 (C-10, JC-1 ,H-1 = 156.0 Hz),
96.9 (C-1, J C-1,H-1 = 167.8 Hz), 79.0 (C-300 0, C-4), 78.5 (C-40),
78.3 (C-40 00), 78.2 (C-400), 76.9 (C-300), 76.34 (C-30), 76.27
(C-200), 75.9 (C-20 0 0), 74.8 (C-2), 74.63 (20 00-CH2Ph), 74.60
(C-3), 74.2 (C-20), 72.2 (3-CH2Ph), 72.1 (300 0-CH2Ph), 71.2
(30-CH2Ph), 70.8 (300-CH2Ph), 69.5 (1-CH2Ph), 68.87 (C-600),
68.85 (C-6), 68.72 (C-60 00), 68.66 (C-60), 67.9 (C-500), 67.8
(C-50), 67.5 (C-500 0) and 64.2 (C-5) ppm.
0
0
HRMS: m/z calc. for C94H95O21Na [M + Na]+ 1582.6264;
found 1582.6207. m/z calc. for C94H95O21K [M + K]+
1598.6003; found 1598.5932.
b-D-Mannopyranosyl-(1-2)-b-D-mannopyranosyl-(1-2)-b-
D-mannopyranosyl-(1-2)-D-mannopyranose (4). To a solution
containing 3 (0.1 g, 0.064 mmol) in dry MeOH (5.5 ml) was
added Pd/C (10% Pd, 0.2 g, 2 weight equiv.). The reaction
mixture was placed inside a reactor and the H2 pressure set to
2.8 bar (40 psi). After 19.5 h, the reaction mixture was diluted
with MeOH (3 ml), filtered through Celite and concentrated.
The crude product was purified by HPAEC (CarboPac
PA 1 column; 9 mm ꢂ 250 mm) with the following gradient
TLC: Rf = 0.40 (hexane : ethyl acetate 3 : 2). [a]D = ꢁ25.4
(c, 0.5, CHCl3).
ꢀc
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New J. Chem., 2010, 34, 667–675 | 673

View Article Online
H-600b), 3.68 (dd, 1 H, J5 ,6 b = 6.2 Hz, J6 a,6 b = ꢁ11.8 Hz,
0
0
0
0
elution: 50 mM NaOH over 0–10 min, followed by a linear
gradient from 50 to 150 mM NaOH over 10–30 min and finally
a linear gradient from 0 to 200 mM NaOAc in 150 mM NaOH
over 30–60 min. The product was collected and the fractions
neutralized by the addition of 1 M aqueous acetic acid. The
sample was de-salted using a column of successive cation and
anion exchange resins (Bio-Rad AG 50W-8X Resin 200–400
mesh, hydrogen form and Bio-Rad AG 1-X8 Resin 200–400
mesh, acetate form), and concentrated to give the title com-
pound (32 mg, b : a 7 : 2, 75%).
H-60b), 3.65 (dd, 1 H, J5
b = 7.4 Hz, J6
b = ꢁ12.3 Hz,
0 0 0,60 0 0
0 0 0a,60 0 0
00
H-60 00b), 3.54 (dd, 1 H, J2 ,3 = 2.3 Hz, J3 ,4 = 9.5 Hz, H-3 ),
00 00
00 00
00
00 00
3.53 (dd, 1 H, J3 ,4 = 9.5 Hz, J4 ,5 = 9.5 Hz, H-4 ), 3.52 (dd,
00 00
1 H, J3,4 = 9.7 Hz, J4,5 = 9.7 Hz, H-4), 3.50 (dd, 1 H, J2,3 =
0 0 0,30 0 0
2.4 Hz, J3,4 = 9.7 Hz, H-3), 3.50 (dd, 1 H, J2 = 2.6 Hz,
= 9.0 Hz, H-30 00), 3.48 (dd, 1 H, J2 ,3 = 3.2 Hz,
J3 ,4 = 9.7 Hz, H-30), 3.45 (dd, 1 H, J3 ,4 = 9.7 Hz, J4 ,5
9.7 Hz, H-40), 3.44 (dd, 1 H, J4
H-40 00), 3.39 (ddd, 1 H, J5
= 8.9 Hz, H-50 00), 3.24 (ddd, 1 H, J5 ,6 a = 2.3 Hz,
J5 ,6 b = 6.2 Hz, J4 ,5 = 9.7 Hz, H-50), 3.23 (ddd, 1 H,
0 0
0 0 0,40 0 0
J3
0
0
0
0
0
0
=
0 0 0,50 0 0
0 0 0,40 0 0
= 8.9 Hz, J3
0 0 0,60 0 0
= 2.4 Hz, J5
= 9.0 Hz,
= 7.4 Hz,
b
0 0 0,60 0 0
a
0 0
0 0 0,50 0 0
J4
Data for the a-anomer. 1H NMR (600.13 MHz, CD3OD,
0
0
0
0
25 1C): d = 5.17 (d, 1 H, J1,2 = 1.8 Hz, H-1), 4.93 (d, 1 H,
J5 ,6 a = 2.5 Hz, J5 ,6 b = 5.9 Hz, J4 ,5 = 9.5 Hz, H-500)
00 00 00 00 00 00
00 00
0 0 0,20 0 0
J1
= 0.9 Hz, H-100 0), 4.84 (d, 1 H, J1 ,2 = 0.8 Hz, H-100),
and 3.20 (ddd, 1 H, J5,6a = 2.1 Hz, J5,6b = 5.3 Hz, J4,5 =
4.71 (d, 1 H, J1 ,2 = 0.7 Hz, H-10), 4.42 (dd, 1 H, J1 ,2 = 0.8 Hz,
9.7 Hz, H-5) ppm.
0
0
00 00
J2 ,3 = 3.1 Hz, H-200), 4.08 (dd, 1 H, J1 ,2 = 0.7 Hz, J2 ,3
2.8 Hz, H-20), 4.04 (dd, 1 H, J1
= 0.9 Hz, J2
=
13C NMR (150.90 MHz, CD3OD, 25 1C): d = 103.3 (C-100),
103.0 (C-10), 102.3 (C-100 0), 95.5 (C-1), 82.3 (C-2), 81.5 (C-20),
79.1 (C-200), 78.4 and 78.3 (C-5, C-50, C-500), 78.0 (C-50 0 0), 75.2
(C-30 0 0), 74.3 (C-30, C-300), 74.2 (C-3), 72.1 (C-200 0), 69.1 (C-40,
C-40 0 0), 68.8 (C-4), 68.6 (C-400), 63.6 (C-600 0) and 62.6 (C-6,
C-60, C-600) ppm.
00 00
0
0
0
0
0 0 0,20 0 0
0 0 0,30 0 0
=
3.1 Hz, H-20 0 0), 3.98 (dd, 1 H, J1,2 = 1.8 Hz, J2,3 = 3.1 Hz,
0 0 0,60 0 0
0 0 0a,60 0 0
H-2), 3.91 (dd, 1 H, J5
a = 2.2 Hz, J6
b = ꢁ12.2 Hz,
H-60 0 0a), 3.89 (dd, 1 H, J5 ,6 a = 2.3 Hz, J6 a,6 b = ꢁ12.0 Hz,
0
0
0
0
H-60a), 3.85 (dd, 1 H, J5 ,6 a = 2.5 Hz, J6 a,6 b = ꢁ11.7 Hz,
H-600a), 3.82 (dd, 1 H, J2,3 = 3.1 Hz, J3,4 = 9.6 Hz, H-3), 3.78
(dd, 1 H, J5,6b = 4.1 Hz, J6a,6b = ꢁ12.0 Hz, H-6b), 3.76 (dd,
1 H, J5,6a = 2.2 Hz, J6a,6b = ꢁ12.0 Hz, H-6a), 3.71 (dd, 1 H,
00 00
00
00
HRMS: m/z calc. for C24H24O21Na [M + Na]+ 689.2111;
found 689.2131.
J
J
5 ,6 b = 5.8 Hz, J6 a,6 b = ꢁ11.7 Hz, H-600b), 3.70 (ddd, 1 H,
5,6a = 2.2 Hz, J5.6b = 4.1 Hz, J4,5 = 9.7 Hz, H-5), 3.69 (dd,
00 00
00
00
Acknowledgements
1 H, J5 ,6 b = 5.8 Hz, J6 a,6 b = ꢁ12.0 Hz, H-60b), 3.65 (dd,
0
0
0
0
Financial support from the Academy of Finland (projects
#121334 and #121335) is gratefully acknowledged. The
authors thank Markku Reunanen (Abo Akademi University)
for HRMS analyses and Dr Jari Helin (Glykos Finland Ltd)
for the purification of compound 4. We also thank Dr
Johannes Savolainen, Kaisa Nieminen and Kaarina Ranta
(University of Turku), Mattias U. Roslund (Orion Pharma),
and Jonas Forsman (Abo Akademi University) for fruitful
discussions.
1 H, J5
= ꢁ12.2 Hz, H-60 00b), 3.63
0 0 0,60 0 0
b
0 0 0a,60 0 0
b
= 7.3 Hz, J6
(dd, 1 H, J3,4 = 9.6 Hz, J4,5 = 9.7 Hz, H-4), 3.53 (dd, 1 H,
J3 ,4 = 9.5 Hz, J4 ,5 = 9.7 Hz, H-400), 3.52 (dd, 1 H, J2 ,3
3.1 Hz, J3 ,4 = 9.5 Hz, H-300), 3.51 (dd, 1 H, J2 ,3 = 2.8 Hz,
00 00
00 00
00 00
=
00 00
0
0
J3 ,4 = 9.7 Hz, H-30), 3.50 (dd, 1 H, J2
= 3.1 Hz,
= 9.2 Hz,
0
0
0 0 0,30 0 0
J3
J3
= 9.5 Hz, H-30 0 0), 3.46 (dd, 1 H, J4
= 9.5 Hz, H-40 0 0), 3.45 (dd, 1 H, J4 ,5 = 9.3 Hz,
J3 ,4 = 9.7 Hz, H-40), 3.37 (ddd, 1 H, J5
0 0 0,40 0 0
0 0 0,50 0 0
0
0
0 0 0,40 0 0
0
0
0 0 0,60 0 0
= 2.2 Hz,
a
0 0 0,60 0 0
b
0 0 0,50 0 0
J5
= 7.3 Hz, J4
= 9.2 Hz, H-50 0 0), 3.25 (ddd, 1 H,
5 ,6 a = 2.3 Hz, J5 ,6 b = 5.8 Hz, J4 ,5 = 9.3 Hz, H-50) and 3.21
0
0
0
0
0
0
J
References
00 00
00 00
00 00
(ddd, 1 H, J5 ,6 a = 2.5 Hz, J5 ,6 b = 5.8 Hz, J4 ,5 = 9.7 Hz,
H-500) ppm.
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13C NMR (150.90 MHz, CD3OD, 25 1C): d = 103.3 (C-100),
102.3 (C-100 0), 101.1 (C-10), 94.1 (C-1), 81.8 (C-20), 81.5 (C-2),
79.1 (C-200), 78.3 (C-50, C-500), 78.1 (C-500 0), 75.2 (C-300 0), 74.3
(C-300), 74.2 (C-5), 74.1 (C-30), 72.1 (C-20 0 0), 71.2 (C-3), 69.1
(C-4, C-40 00), 69.0 (C-40), 68.6 (C-400), 63.5 (C-60 00), 62.5 (C-60,
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Data for the b-anomer. 1H NMR (600.13 MHz, CD3OD,
25 1C): d = 4.98 (d, 1 H, J1 ,2 = 0.8 Hz, H-100), 4.94 (d, 1 H,
J1
00 00
= 1.0 Hz, H-10 00), 4.84 (d, 1 H, J1,2 = 0.8 Hz, H-1),
0 0 0,20 0 0
4.76 (d, 1 H, J1 ,2 = 0.7 Hz, H-10), 4.40 (dd, 1 H, J1 ,2 = 0.8 Hz,
0
0
00 00
J2 ,3 = 2.3 Hz, H-200), 4.24 (d, 1 H, J1 ,2 = 0.7 Hz, J2 ,3
3.2 Hz, H-20), 4.04 (dd, 1 H, J1
= 1.0 Hz, J2
=
00 00
0
0
0
0
0 0 0,20 0 0
0 0 0,30 0 0
=
2.6 Hz, H-20 0 0), 4.02 (dd, 1 H, J1,2 = 0.8 Hz, J2,3 = 2.4 Hz,
0 0 0,60 0 0
0 0 0a,60 0 0
H-2), 3.91 (dd, 1 H, J5
a = 2.4 Hz, J6
b = ꢁ12.3 Hz,
H-60 0 0a), 3.89 (dd, 1 H, J5 ,6 a = 2.3 Hz, J6 a,6 b = ꢁ11.8 Hz,
0
0
0
0
H-60a), 3.86 (dd, 1 H, J5 ,6 a = 2.5 Hz, J6 a,6 b = ꢁ11.9 Hz,
H-600a), 3.83 (dd, 1 H, J5,6a = 2.1 Hz, J6a,6b = ꢁ12.1 Hz,
H-6a), 3.73 (dd, 1 H, J5,6b = 5.3 Hz, J6a,6b = ꢁ12.1 Hz,
00 00
00
00
4 For a review, see: (a) J. J. Gridley and H. M. I. Osborn, J. Chem.
Soc., Perkin Trans. 1, 2000, 1471–1491; (b) S. C. Ennis and H. M.
00 00
00
00
H-6b), 3.72 (dd, 1 H, J5 ,6 b = 5.9 Hz, J6 a,6 b = ꢁ11.9 Hz,
ꢀc
This journal is The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2010
674 | New J. Chem., 2010, 34, 667–675

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14 See for example: (a) J. B. Lambert and E. P. Mazzola, Nuclear
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10 For a more detailed discussion on the synthesis of compounds 1
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16 See, for example: C. J. Cramer, Essentials of Computational
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17 See, for example: S. I. van Kasteren, H. B. Kramer, D. P. Gamblin
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11 For experimental details, see the Experimental section.
12 (a) D. Crich and S. Sun, Tetrahedron, 1998, 54, 8321–8348;
(b) K. Bock and H. Thøgersen, in Annual Reports on NMR
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