Conformational flexibility and dynamics of two (1→6)-Linked disaccharides related to an oligosaccharide epitope expressed on malignant tumour cells

Article abstract of DOI:10.1002/chem.200900507

The conformational flexibility and dynamics of two (1→6)-linked disaccharides that are related to the action of the glycosyl transferase GnT-V have been investigated. NMR NOE and T-ROE spectroscopy experiments, conformation-dependent coupling constants an

Full text of DOI:10.1002/chem.200900507

DOI: 10.1002/chem.200900507
Conformational Flexibility and Dynamics of Two (1!6)-Linked
Disaccharides Related to an Oligosaccharide Epitope
Expressed on Malignant Tumour Cells
Ulrika Olsson, Elin Sꢀwꢁn, Roland Stenutz, and Gçran Widmalm*[a]
Abstract: The conformational flexibili-
angle in a-d-Manp-(1!6)-a-d-Manp-
OMe for gt/gg/tg was equal to 45:50:5,
whereas in a-d-Manp-OMe it was de-
termined to be 56:36:8. The dynamic
model that was generated for b-d-
states in which the f torsion angle has
ty and dynamics of two (1!6)-linked
disaccharides that are related to the
action of the glycosyl transferase GnT-
V have been investigated. NMR NOE
and T-ROE spectroscopy experiments,
conformation-dependent coupling con-
stants and molecular dynamics (MD)
simulations were used in the analyses.
To facilitate these studies, the com-
the exo-anomeric conformation, the y
torsion angle an extended antiperipla-
nar conformation and the w torsion
angle a distribution of populations pre-
dominantly between the gauche–trans
and the gauche–gauche conformational
states (i.e., gt/gg/tg) is equal to 60:35:5,
respectively. The use of site-specific 13C
labelling in these disaccharides leads to
increased spectral dispersion, thereby
making NMR spectroscopy based con-
formational analysis possible that oth-
erwise might be difficult to attain.
GlcpNAc-(1!6)-a-d-Manp-OMe
MD simulations employing
PARM22/SU01
by
the
CHARMM-based
force field was in very good agreement
with experimental observations. b-d-
GlcpNAc-(1!6)-a-d-Manp-OMe is de-
scribed by an equilibrium of populated
pounds were synthesised as a-d-ACHTUNGTRNEUNG
[6-13C]-
Manp-OMe derivatives, which reduced
the 1H NMR spectral overlap and fa-
cilitated the determination of two- and
three-bond 1H,1H, 1H,13C and 13C,13C-
coupling constants. The population dis-
tribution for the glycosidic w torsion
Keywords:
enzymes
carbohydrates
molecular dynamics
·
·
·
NMR spectroscopy · torsion angles
Introduction
N-linked glycans are formed in a step-wise process in
which a Man5GlcNAc2-PP-dolicol precursor is synthesised in
the cytoplasm. Subsequently, it is ꢀflippedꢁ to the lumen of
the endoplasmic reticulum (ER) where it is extended to
Glc3Man9GlcNAc2-PP-dolicol.[4,5] The oligosaccharide is
then transferred to the nascent polypeptide chain by the oli-
gosaccharyltransferase complex. Following deglucosylation
reactions and protein-folding quality control, the newly syn-
thesised glycoprotein becomes available for glycosidase re-
action in the ER and the Golgi. The glycan part of the pro-
tein is trimmed to various degrees of mannosylation, fol-
lowed by structural diversification. In vertebrates, three N-
glycan subtypes are generated: 1) high mannose; 2) hybrid,
which contains one antenna with only mannosyl residues
and 3) complex types, which have N-acetylglucosaminyl and
fucosyl residues. The formation of multi-antennary struc-
tures in the complex-type of structures is the result of N-
acetylglucosylaminyl transferases, of which there are six (I–
VI). Glycosyl extension by, for instance, Gal, GlcNAc and
NeuAc residues can occur on the different antennae.
Protein glycosylation is ubiquitous and the glycoconjugates
are formed in several ways from different glycosyl residues
linked to various amino acids; this results in N-, O- and C-
glycosyl derivatives.[1] The structures expressed are the re-
sults of post-translational enzymatic modifications and an
estimate based on potential N- and O-glycosylation sites in-
dicate that more than half of all proteins in nature are glyco-
sylated.[2] The importance of the glycan moieties is exempli-
fied by the persistence time of proteins in serum and the re-
sistance to proteases, but also the fact that protein 3D struc-
ture can be stabilised by their presence.[3]
[a] Dr. U. Olsson, E. Sꢂwꢃn, Docent R. Stenutz, Prof. G. Widmalm
Department of Organic Chemistry
Arrhenius Laboratory, Stockholm University
106 91 Stockholm (Sweden)
Fax : (+46)815-4908
E-mail: gw@organ.su.se
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FULL PAPER
N-Acetylglucosylaminyl transferase V (GnT-V) is known
to be over-expressed in malignant cells.[6–12] It transfers a
GlcNAc residue to the 6-position of a Man residue, resulting
in increased branching of the N-glycan (Figure 1). GnT-V
between 3J
(H5,H6R) and 3J
ACHUTGTNRENNGU ACHTUNGTRNE(NUGN H5,H6S) to w are given by
Equations (1) and (2):[20]
3JðH5,H6RÞ ¼5:08þ0:47 cosðwÞþ0:90 sinðwÞ
ꢀ0:12 cosð2wÞþ4:86 sinð2wÞ
3JðH5,H6SÞ ¼4:92ꢀ1:29 cosðwÞþ0:05 sinðwÞ
þ4:58 cosð2wÞþ0:07 sinð2wÞ
ð1Þ
ð2Þ
In addition, the 2J(H5,C6) and 2J
ACTHUNTGRNENUG ACHTUNTGNER(NUGN C4,C6) coupling con-
stants can shed light on the conformational population dis-
tribution according to the relationships given in Equa-
tions (3) and (4):[21]
Figure 1. Schematic of a triantennary N-glycan structure. A trisaccharide
(bold), or slight modifications thereof, is required as a glycosyl acceptor
for GnT-V.
2JðH5,C6Þ ¼ ꢀ1:29þ1:53 cosðwÞꢀ3:68 sinðwÞ
2JðC4,C6Þ ¼ 0:02þ0:16 cosðwÞþ1:34 sinðwÞ
ð3Þ
ð4Þ
requires a trisaccharide as an acceptor in the reaction with
UDP-GlcNAc as a donor.[13] Chemically modified deriva-
tives of the acceptor residue, such as b-d-Man (b-d-Glc has
been used because it is a more readily accessible residue in
the chemical synthesis), have revealed the importance of
structural modifications at C4 as well as conformational re-
quirements at its w torsion angle,[13,14] that is, an acceptor
conformationally biased towards the gt rotamer is more
active, whereas one that is biased towards the gg rotamer is
less active than the non-perturbed acceptor. The syntheses
of related oligosaccharides[15] and of low-molecular-weight
compounds have been carried out with the goal of produc-
ing active inhibitors towards GnT-V.[16–18] Recently, an NMR
spectroscopy study[19] on the binding of both the glycosyl ac-
ceptor and the donor to GnT-V was described, in which a
model of the substrates in the active site of GnT-V was pro-
posed. The model describes the conformational arrange-
ments needed for a glycosyltransferase in which nucleophilic
displacement takes place by inversion of configuration at
the C1 atom of GlcNAc in the donor, thus forming a b-d-
GlcpNAc-(1!6)-a-d-Manp linkage.
3
In principle, it is possible to use J
A
sis;[21] however, we decided that its use was insufficient to
give any major input to the conformational analysis of the w
torsion angle and chose not to use this relationship in the
present analysis.
Proton–proton relaxation of disaccharides in solution is
governed by dipole–dipole cross-relaxation to adjacent
proton nuclei as well as 13C nuclei when present. The
strength of these interactions is dependent on both internal
dynamics and the overall rotation of the molecule, in con-
junction with their internuclear distances. If an appropriate
reference proton–proton pair distance of known separation
in the molecule is available and the dynamics of the differ-
ent proton pairs under considerations can be judged to be
similar, the isolated spin-pair approximation (ISPA)[22] can
be used by comparing the cross-relaxation rate of the refer-
ence spin pair, sref, to that of protons i and j, sij. The un-
known distance, rij, between the protons can subsequently
be obtained according to Equation (5):
We have investigated the conformational dynamics of b-
d-GlcpNAc-(1!6)-a-d-Manp-OMe and structurally related
mono- and disaccharides. Chemical synthesis enabled
[6-13C]-site-selectively labelled analogues to be prepared,
which subsequently were studied by using a range of NMR
spectroscopy experiments. Molecular dynamics (MD) simu-
lations were important in describing the conformational
flexibility and in generating a model that could be tested in
which comparisons are made to NMR spectroscopic obser-
vations, or variables derived thereof.
ꢀ
ꢁ
1
6
sref
sij
ð5Þ
rij ¼ rref
1
From 2D H,1H NOESY experiments the cross-relaxation
rates are possible to extract by different procedures.[23–26] As
detailed by Dixon et al.[27] the interproton cross-relaxation
rates can be obtained for sufficiently small tmix by using
Equation (6):
Ijðtmix
Þ
ð6Þ
¼ ꢀsij
½tmixIiꢁ
Theory
The population distribution at the w torsion angle in a hexo-
pyranosyl residue can be obtained from the homonuclear J-
ACHTUNGTRENNUNG(H,H) coupling constants between H5 and the two H6 pro-
tons, interpreted for the three conformational states gt (stag-
gered conformer at 608), gg (staggered conformer at ꢀ608)
and tg (staggered conformer at 1808). Pertinent relationships
in which Ij is the intensity of the signal at nucleus j, by fol-
lowing selective excitation of nucleus i with intensity Ii in a
1D 1H,1H M-GOESY experiment.
3
For the NOE measured in the laboratory frame,[28] the
off-diagonal elements (sij) of the relaxation matrix that de-
scribe the cross-relaxation rates are given by Equation (7):
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8887

G. Widmalm et al.
2
ðdHH
Þ
ð7Þ
sNOE
¼
½6Jð2wÞ ꢀ Jð0Þꢁ
4
in which J(w) is the spectral density function taken at differ-
ent combinations of the proton frequency. The dipolar cou-
pling constant, dHH =(m0/4p)gH2ꢀhrijꢀ3, is a measure of the
strength of the dipolar interaction in which m0 is the permea-
bility in vacuum, gH is the magnetogyric ratio for protons
and ꢀh is Planckꢁs constant divided by 2p. In analogy, for the
rotating-frame Overhauser effect (ROE),[28–30] the off-diago-
nal elements describing the cross-relaxation rates are then
given by Equation (8):
2
ðdHH
Þ
ð8Þ
sROE
¼
½3JðwÞ þ 2Jð0Þꢁ
4
In the multiple-pulse or transverse ROE (T-ROE) the
cross relaxation rate, sT-ROE, can be calculated as the mean
of sT-NOE and sT-ROE as described by Equation (9):[31–33]
Figure 2. The variation of sNOE/s as a function of teff in which s refers to
either sROE (dashed line) or sT-ROE (solid line) calculated for a spectrome-
ter frequency of 600 MHz. The insert shows short time behaviour up to
200 ps.
2
ðdHH
Þ
ð9Þ
sTꢀROE
¼
½6Jð2wÞ þ 3JðwÞ þ Jð0Þꢁ
8
When internal and overall correlation times cannot be de-
termined separately, an effective correlation time, teff, can
be used to describe the spectral density function as shown in
Equation (10):
ꢂ
ꢃ
2
5
teff
JðwÞ ¼
ð10Þ
2
1 þ ðwteff
Þ
This effective correlation time can be calculated from the
ratio of different cross-relaxation rates,[34] such as those
given in Equations (11) and (12):
sNOE
sROE
5 þ w2t2eff ꢀ 4w4te4ff
ð11Þ
ð12Þ
¼
5 þ 22w2t2eff þ 8w4te4ff
sNOE
10 þ 2w2t2eff ꢀ 8w4te4ff
¼
10 þ 23w2te2ff þ 4w4te4ff
sTꢀROE
The variations of these ratios as a function of teff are
shown in Figure 2 and from the graph it is possible to quick-
ly make an estimate of the effective correlation time. We
have utilised both 1D 1H,1H DPFGSE NOESY[35] and 1D
1H,1H DPFGSE T-ROESY[36] experiments to obtain effec-
tive interproton correlation times teff, and subsequently, the
effective proton–proton distances.
Figure 3. Schematic of compounds 1–3 with torsion angles denoted as f,
y and w.
Results and Discussion
The conformational flexibility at the w torsion angle has
been investigated for compounds 1–3 (Figure 3) in water so-
lution. Furthermore, in the (1!6)-linked disaccharide 3 con-
formational preferences were studied in greater detail, that
is, also at the f and y torsion angles. The conformational
analysis was facilitated by the use of the synthesised [6-13C]-
site-selective analogues 1c–3c (in which c indicates the
carbon-labelled compound). The syntheses were first carried
out for compounds 1–3. Compound 1 was made by Fischer
glycosylation with methanol. Disaccharides 2 and 3 were
formed by silver triflate and N-iodosuccinimide/silver tri-
flate promoted glycosylations, respectively,[37,38] of suitably
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Molecular Dynamics of Disaccharides
FULL PAPER
3
Table 1. 2J and J coupling constants and w rotamer distributions of the a-d-Manp-OMe residue in compounds 1–3 and structures present as part of gly-
coproteins in the solid state.[a]
Compound
3J
G
3J
N
2J
A
2J
E
gt [%]
gg [%]
tg [%]
a-d-Manp-OMe (1)
6.14 {0.06}[b]
5.12 {0.12}
6.48 {0.21}
2.23 {0.04}
1.96 {0.26}
1.91 {0.05}
ꢀ1.98 {0.03}
ꢀ1.69 {0.22}
ꢀ2.15 {0.04}
<0.5
<0.5
<0.5
55 (44)[c]
45
60 (67)
40
36 (38)
49
35 (28)
55
9 (18)
6
5 (5)
5
a-d-Manp-(1!6)-a-d-Manp-OMe (2)
b-d-GlcpNAc-(1!6)-a-d-Manp-OMe (3)
a-d-Manp-(1!6)-d-Manp-(1!?)[d]
[a] Coupling constants were determined from the 13C-site-specifically labelled analogues. [b] Standard deviations of the J values, determined at different
magnetic fields, are given in curly brackets. [c] Populations calculated from the MD simulations are given in parentheses. [d] Populations calculated from
the occurrences in the Protein Data Bank.
protected donors together with the acceptor methyl 2,3,4-tri-
O-benzyl-a-d-mannopyranoside. Syntheses of [6-13C]-site-se-
lective analogues 1c–3c were performed in an analogous
way.
ides.[44–55] Furthermore, the sign (again confirmed by the
1H,13C HSQC-HECADE experiment) and the magnitude of
2J
ACTHNUTRGNEUNG(H5,C6) in 2, reveal that the conformational distribution
at the w torsion angle should be similar to that of 1. Compu-
tational studies have been carried out for 2,[56,57] in which
the lowest potential energy conformation with respect to the
y torsion angle was observed for the antiperiplanar confor-
mation at around 1808, and one at around 908 that is
2.5 kcalmolꢀ1 higher in energy. For the w torsion angle, all
three conformational states were populated. From the
MM3(92)-based relaxed-residue Ramachandran maps,[56]
The 1H NMR spectroscopic assignments of the H6 pro-
tons to H6pro-R and H6pro-S have been reported for com-
pounds 1–3.[39–41] By using 3J
ACHTUNGTRENNUNG
w
ACHTUNGTRENNUNG
spin simulations of compounds 1c–3c and are presented in
Table 1 together with the populations of the three conforma-
tional states at the w torsion angle. In 1 the population dis-
tribution of the hydroxymethyl group is described by the gt
and the gg rotamers in approximately equal amounts,
whereas the tg rotamer is essentially absent, in agreement
with the gauche effect[42] and the Hassel–Ottar effect.[43] The
derived conformational distribution is in agreement with a
3J
complete adiabatic surface, giving 3J
ACHTUNGTRENNUNG
AHCTUNGTRENNUNG
mainly due to an exo-anomeric conformation with f~ꢀ458.
The three-bond heteronuclear coupling constant is in excel-
lent agreement with that determined herein from experi-
ment (3J
ACHTUNGTRENNUNG
vanishingly small 2J
ACHTUNGTRENNUNG
(C4,C6) and
a
negative sign of
ACHTUNGTRENNUNG
1
2J(H5,C6). The sign of the latter was confirmed by a H,13C-
HSQC-HECADE NMR spectroscopy experiment.
been reported in which it has also been possible to extract
conformational information of the carbohydrate part.[59–61]
Analysis of the crystal structures of N-linked glycoproteins
in the Protein Data Bank with a-d-Manp-(1!6)-d-Manp
linkages, in total 218 derived from structures determined
with a resolution of ꢂ2.5 ꢅ, revealed that there is a popula-
tion distribution (Table 1) very similar to that observed in
aqueous solution for compound 2. This result suggests that
inherent conformational preferences are retained in the
solid state.
1
In the a-(1!6)-linked disaccharide 2 (the H NMR spec-
trum of 2c is shown in Figure 4) the conformational distri-
bution is altered for the gt and the gg rotamers to favour the
The population distribution of the w torsion angle in b-d-
GlcpNAc-(1!6)-a-d-Manp-OMe (3) is 60:35:5 for the gt/
gg/tg rotamers (Table 1), in complete agreement with the
previous results.[41] For 3c the coupling constant J
0.5 Hz and as the tg rotamer is only marginally populated
these results support a conformational equilibrium between
G
2
Figure 4. The 1H NMR spectrum at 900 MHz of disaccharide 2c with the
HDO peak removed.
2
the gt and the gg rotamers. The vanishingly small J
(C4,C6)
2
latter slightly, whereas the tg rotamer is again not populated
to any great extent (Table 1). The results presented herein
underscore the fact that the gg rotamer is populated to a
higher extent in a-d-Manp-(1!6)-a-d-Manp-OMe, although
not as much as suggested previously.[40] With the tg rotamer
value and a negative sign of JACTHNGUTERNNU(G H5,C6) in 3, as for 1 and 2,
support the finding that the w torsion angle distributions are
similar, although not identical, in the three compounds ana-
lysed herein. For the w torsion angles in 1–3 (Table 1),
2
2J
C
G
having a low population, the vanishingly small 2J
N
be negative and positive, respectively.
again indicates a population equilibrium between the gt and
the gg conformational states at the w torsion angle in 2, sim-
ilar to those described for asparagine-linked oligosacchar-
Further analysis of b-d-GlcpNAc-(1!6)-a-d-Manp-OMe
employed coupling constants, NOEs and molecular dynam-
ics (MD) simulations. In compound 3c a trans-glycosidic
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8889

G. Widmalm et al.
heteronuclear coupling constant related to the f torsion
1
angle is readily extracted from the 1D H NMR spectrum
(namely, 3J
ACHTUNGTRENNUNG(H1’,C6)=4.1 Hz). The magnitude of this cou-
pling constant is similar to those observed in other oligosac-
charides[62] and indicates that the exo-anomeric conforma-
tion prevails at the f torsion angle. It was observed that
3J
than 2. However, a decrease for JACTHGUNTRNE(NUG C2’,C6) is anticipated be-
ACHTUNGTRENNUNG(C2’,C6)=2.9 Hz, which is lower in magnitude by 0.6 Hz
3
cause in 3 there is no in-plane terminal electronegative
group associated with the spin–spin coupling pathway.[63]
The fact that flexibility is present at this (1!6) linkage is
further supported by a study based on 13C nuclear spin re-
laxation of a Gal2GlcNAc2Man oligosaccharide, in which the
N-acetylglucosamine residue linked to the 6-position of Man
displayed a more extensive local motion than that attached
to the 2-position of the branching Man residue.[64]
The [6-13C]-site-selective analogue 3c resulted in in-
1
creased dispersion in H NMR spectrum (Figure 5), whereby
the H6pro-S resonances were still resolved and the one at the
Figure 6. Plots of Ij(tm)/ACHTNUTRGENUNG
[tmIi] versus tm for the 1D 1H,1H NOE (top) and
T-ROE (bottom) spectroscopy experiments applied to disaccharide 3c.
Selective excitation was carried out for the resonance from H1’ in 3c.
Cross-relaxation to H6pro-R (filled circle), H6pro-S (filled diamond) and H3’
(filled square) were investigated.
(Table 2) and very similar for the three proton pairs. Conse-
quently, it is valid to use the H1’–H3’ distance as a reference
in deriving the effective interproton distances H1’–H6pro-R
and H1’–H6pro-S that can be used to investigate conforma-
tional preferences at this glycosidic linkage, which has three
degrees of freedom, f, y and w. Effective interproton dis-
tances were derived from T-ROE data by ISPA in which the
reference distance was obtained from an MD simulation
(see below). The effective distance H1’–H6pro-R is shorter
than H1’–H6pro-S (Table 2), a result that will be used in the
subsequent analysis.
The conformational preferences and dynamics were ad-
dressed by MD simulations by using explicit water as sol-
vent and the recently developed CHARMM-based force
field for carbohydrates, termed PARM22/SU01. The force
field has been shown to give very good agreement with ex-
perimentally determined results for effective proton–proton
distances (based on NOEs) across glycosidic bonds. The
population distribution of the w torsion angle of glucopyra-
nosides was also quite reasonable, although it was not tested
extensively in previous MD simulations. Herein, MD simu-
1
Figure 5. Part of the 600 MHz H NMR spectrum of disaccharide 3c. Res-
onances from H6pro-R, H6pro-S, and H3’ are annotated.
higher chemical shift from H6pro-R was identifiable. In the
1H NMR spectrum the resonance from H3’ in the N-acetyl-
glucosamine residue was still resolved. 1D H,1H-DPFGSE
1
NOESY and 1D 1H,1H-DPFGSE T-ROESY experiments
with selective excitation at the resonance for H1’ of the N-
acetylglucosamine residue in 3c were carried out with
mixing times %200 ms. The results from the NMR spectra
with different mixing times tm were analysed as described
above. The cross-relaxation rates were obtained by extrapo-
lation to tm =0 (Figure 6) and
lations have been carried out for compounds 1 and 3. The
3
rotamer distribution in 1 was obtained from J
N
the results are presented in
Table 2. Because disaccharide 3
Table 2. Cross-relaxation rates of 3c obtained upon excitation of H1’ by using 1D NOE and T-ROE spectros-
is indeed flexible, we address
the use of different cross-relax-
ation rates by calculating effec-
tive correlation times teff for
each proton pair under study.
By using the sNOE/sROE ratios,
copy experiments at 600 MHz and subsequent analysis thereof.
Proton detected
sNOE
[ꢆ102 sꢀ1
sT-ROE
[ꢆ102 sꢀ1
sNOE/sT-ROE
teff
sij/sH3ꢁ
(T-ROE)
rij (NMR)
[ꢅ]
rij (MD)[b]
[ꢅ]
G
]
N
]
[ps]
G
H6pro-R
H3’
H6pro-S
6.67
4.01
3.08
10.1
6.06
4.51
0.660
0.662
0.683
124
124
119
1.67
1
0.744
2.33
2.39
2.54
2.74
2.54[a]
2.67
we observe that teff ~120 ps [a] Reference distance from the MD simulation of 25 ns. [b] 1/r=<rꢀ6
>
.
1/6
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Molecular Dynamics of Disaccharides
FULL PAPER
pling constants (see above) and it was readily obtained from
MD simulations, in total 50 ns (Table 1). The population dis-
tribution in 1 is in good agreement with that determined
from NMR spectroscopy experiments. Thus, the force field
describing the w torsion angle should also be suitable to use
for 3. The MD simulation of the latter, lasting 25 ns, showed
that all three w rotamers were populated (Figure 7). The gt
urements as well as by HSEA and HSEH potential energy
47–49
calculations.
The results from the NMR spectroscopy
experiments were consistent with the exo-anomeric confor-
mation, or a conformational region close to it, at the f tor-
sion angle of the b-(1!6) linkage. The y torsion angle of
the same linkage was from NMR spectroscopic data con-
fined, for the sterically allowed region, to a narrow range
centred approximately on 1908. However, the potential
energy calculations showed several local energy minima and
the results were interpreted as a conformational equilibrium
for y with two different values: around 908 and 2508. As a
result of this, the apparent conformation at around 1908 was
termed virtual and should, at best, only correspond to a
fraction of the populated states. It should be noted that for
the trisaccharide a local potential energy minimum was also
present at y~1908. At the w torsion angle the gt conforma-
tional state was judged to predominate. The presence of the
gt conformation was also addressed and discussed, in partic-
ular, from a potential energy point of view. What was previ-
ously concluded to be a virtual conformation at the b-(1!6)
linkage of 3, is from the experimental data and computa-
tional procedures described in this study the major confor-
mation, with excursions at the y torsion angle to adjacent
conformational regions that were the important ones in the
previous studies. Differentiation of these two models can, at
least in part, be performed on the basis of the absence of a
1
strong H,1H T-ROE between H1’ and H4,[41] which should
Figure 7. The time dependence of the glycosidic torsion angles f, y and
w of b-d-GlcpNAc-(1!6)-a-d-Manp-OMe from the MD simulation.
be present for the gg conformer with y ~2508.
conformer was favoured over the gg conformer, whereas the
tg conformer was only slightly populated. Notably, the popu-
lations of the three conformational states are in very good
agreement with experiment (Table 1).
The MD simulation for 3 showed an exo-anomeric confor-
mation with hfi=478 (rmsd=148), that is, predominantly
fluctuation in a single well with some transitions to confor-
Conclusion
For b-d-GlcpNAc-(1!6)-a-d-Manp-OMe a model was ob-
tained by MD simulations, in which dynamics play an impor-
tant part. It was found to be consistent with experimental
NMR spectroscopy data. In particular, both fluctuations and
conformational transitions were present during the simula-
tion period, which when analysed for effective trans-glycosi-
dic proton–proton distances was able to reproduce the cor-
mational regions transiently populated (Figure 7). The tor-
3
sion angle related to J
N
1
(rmsd=148). The y torsion angle is populated to >95% as
the antiperiplanar conformation (i.e., hyi=1788; rmsd=
248). Excursions to >2408 and <1208 are indeed present
(see below), but the torsion angle is centred on 1808. To as-
certain whether the described conformational dynamics are
reasonable, the effective interproton distances H1’–H6pro-R
and H1’–H6pro-S were calculated over the whole MD trajec-
responding distances from H,1H T-ROE experiments. Anal-
ysis of the rotamer distributions in a-d-Manp-OMe and b-d-
GlcpNAc-(1!6)-a-d-Manp-OMe reveals excellent agree-
ment between experiment and MD simulations that used
the PARM22/SU01 CHARMM force field. The present
study underscores the fact that site-specific 13C-labelling in
oligosaccharides is a powerful approach for increasing the
spectral dispersion and allowing experimental data for con-
formational analysis to be obtained, which would be difficult
by other means. By using site-specifically 13C-labelled oligo-
saccharides, in combination with computational procedures
as described herein, promising avenues for future studies of
structurally complex carbohydrate compounds as well as
their interaction with lectins and other proteins are provid-
ed. In addition, it has been shown that the w rotamer distri-
bution for a-d-Manp-(1!6)-a-d-Manp-OMe in solution is
essentially identical to that resulting from the analysis of
more than 200 crystal structures containing the correspond-
ꢀ6 1/6
tory as 1/reff =hrMD i , as well as for the H1’–H3’ reference
distance. From H1’ the distances to the other protons inves-
tigated were in the order H6pro-R <H3’<H1’–H6pro-S and the
quantitative analysis showed excellent agreement between
simulation and experiment (Table 2), that is, within the esti-
mated experimental error of ꢃ0.1 ꢅ. The MD simulation
model is thus in good agreement with observations from
NMR spectroscopy experiments.
Compound 3 and b-d-GlcpNAc-(1!6)[b-d-GlcpNAc-(1!
2)]-a-d-Manp-OMe were previously studied by steady-state
1H,1H NOE spectroscopic and 1H T1 spin relaxation meas-
Chem. Eur. J. 2009, 15, 8886 – 8894
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8891

G. Widmalm et al.
solved in MeOH (20 mL), and 1M NaOMe in MeOH was added until
the solution remained alkaline. After 1 h at room temperature, the reac-
tion was quenched by the addition of Dowex 50WX8 (16–40 mesh) in the
H+ form. The resin was removed by filtration and the solution was con-
centrated. After de-salting by size exclusion chromatography on a Bio-
Gel P-2 column, the disaccharide was obtained as a colourless powder in
67–73% yield. 1H and 13C NMR spectroscopy data were in complete
agreement with literature data.[41] HRMS of 3c: m/z calcd for
C1413CH27NO11Na: 421.1510 [M+Na]+; found 421.1525.
ing disaccharide fragment. The latter observation indicates
that conformational population distributions derived from
analysis of crystal structures may also be useful in relation
to solution-state conformational studies.
Experimental Section
NMR spectroscopy: Recording NMR spectra of protected compounds
made by organic synthesis was carried out on Varian 300 and 400 and
Bruker 400 MHz spectrometers at 258C. Chemical shifts were referenced
to internal TMS (dH =0.00 ppm) and to the solvent in CDCl3 (dC =
77.23 ppm). NMR spectroscopy experiments for conformational analysis
studies were performed at 378C in D2O on a Varian INOVA 600 MHz
spectrometer equipped with 5 mm PFG triple resonance probe. The 1D
1H NMR spectra that were used in combination with NMR spin simula-
tions were obtained at 378C in D2O by using a Bruker Avance 400 MHz
spectrometer, a Bruker Avance 500 MHz spectrometer equipped with a
cryoprobe, a Bruker Avance III 600 MHz spectrometer, a Bruker Avan-
ce III 700 MHz spectrometer equipped with a cyroprobe and a Varian
INOVA 900 MHz spectrometer equipped with a cold probe. Coupling
General: TLC was performed on precoated Merck 60 F254 plates and vi-
sualised with UV light or 8% H2SO4. For column chromatography, Ami-
cron silica gel 0.040–0.063 mm was used. High-resolution ESI-TOF MS
was carried out on a Bruker Daltonics micrOTOF instrument in the posi-
tive mode. Samples were dissolved in a mixture of MeCN/H2O (1:1) con-
taining 0.1% formic acid.
The atoms of a terminal residue in a disaccharide are designated with a
prime and those in an O-methyl residue are unprimed. The torsion
angles at a glycosidic linkage are defined as f=H1’-C1’-O6-C6 and y=
C1’-O6-C6-C5. The torsion angle w=O5-C5-C6-O6.
General procedure for synthesis of 2 and 2c: The donor 2,3,4,6-tetra-O-
benzoyl-a-d-glucopyranosyl bromide[65] (0.18–0.58 mmol) and the accept-
or methyl 2,3,4-tri-O-benzyl-a-d-mannopyranoside[66] (0.45–0.97 equiv)
were dissolved in CH2Cl2 (40 mL; distilled from CaH2) and the solution
was purged with argon for 5 min. Molecular sieves (3 ꢅ, powder) were
added and the suspension was stirred for 15 min. It was then cooled to
constants were determined by total lineshape analysis by using the
3
PERCH NMR spectroscopy software,[68] except for the J
G
which were determined from the peak-to-peak separation in resolution-
1
enhanced 1D 13C NMR spectra. In the PERCH analysis the 1D H NMR
ꢀ408C by using acetone/CO2 (s). AgOTf (3 equiv) and
a catalytic
spectrum was used to extract the spin–spin coupling constants and in
some cases also the 1D 13C-coupled NMR spectrum was used in a com-
amount 2,4,6-collidine (3 drops) were added. The mixture was stirred at
ꢀ40 to ꢀ208C for 3 h and then filtered and concentrated. The crude
product was purified by chromatography (silica gel, toluene/EtOAc 20:1)
to give the protected disaccharide in 70–75% yield.
1
bined manner for the iteration procedure. H,13C HSQC-HECADE NMR
spectroscopy experiments were carried out with a 10 ms DIPSI-2 spinlock
for the TOCSY transfer and a t1*/t1 scaling factor of unity.[69]
The protected disaccharide (0.04–0.18 mmol) was dissolved in MeOH
(30 mL), and 1M NaOMe in MeOH was added until the solution re-
mained alkaline. After 1 h at room temperature, the reaction was
quenched by adding Dowex 50WX8 (16–40 mesh) in the H+ form. The
resin was removed by filtration and the solution was concentrated. The
partially deprotected disaccharide was then dissolved in EtOH/EtOAc/
AcOH (20 mL; 6:6:1 v/v) and a catalytic amount of Pd(OH)2/C was
added. The suspension was hydrogenolysed under pressure (100 psi) at
room temperature overnight, filtered through Celite and concentrated.
To remove traces of acetic acid, the crude product was dissolved in
EtOH and concentrated at least three times. After de-salting by size ex-
clusion chromatography on a Bio-Gel P-2 column, the disaccharide was
obtained as a colourless powder (58–71%). 1H and 13C NMR spectrosco-
py data were in complete agreement with literature data.[40] HRMS of
2c: m/z calcd for C1213CH24O11Na: 380.1244 [M+Na]+; found: 380.1249.
Disaccharide 3c was treated with CHELEX 100 to remove any paramag-
netic ions. The sample was freeze dried and dissolved in D2O (0.7 mL) to
give a total concentration of 40 mM; it was transferred to a 5 mm NMR
tube and flame-sealed under vacuum after degassing by three freeze–
pump–thaw cycles. Proton–proton cross-relaxation rates in compound 3c
were measured by using 1D 1H,1H DPFGSE NOESY and 1D
1H,1H DPFGSE T-ROESY experiments.[35,36] Selective excitation at the
resonance for the anomeric proton of the N-acetylglucosamine residue
was enabled by using an i-Snob-2 shaped pulse of 50 ms duration. The
gradient durations in the initial DPFGSE part were 1 ms and the
strengths were 0.1 and 0.9 Gcmꢀ1. The T-ROESY spin lock was applied
with gB1/2p =3.3 kHz. Spectra were recorded by using a spectral width
of 3000 Hz and 16k data points, sampling 2048 transients at each mixing
time. The total relaxation delay between transients was 12 s, that is, it
was always >5ꢆT1. Five different cross-relaxation delays (mixing times)
of 50, 80, 110, 150 and 180 ms were used. Prior to Fourier transformation,
the FIDs were zero-filled once and multiplied with a 1 Hz exponential
line-broadening factor. Spectra were baseline corrected and integrated by
using the same integration limits at all mixing times. 1H,1H NOE and T-
ROE buildup peak areas were divided by the area of the selectively ex-
cited peak to produce the normalised buildup intensities that were used
to calculate the NOE and T-ROE buildup rates (s), respectively. The re-
sidual standard deviations of the plots that were used to obtain the cross-
relaxation rates were in the order of 2%. A second independent data set
of NOE and T-ROE buildup rates were measured, with five mixing times
in the range 40–200 ms to estimate experimental errors. Most of the
cross-relaxation rates of the second data set differed by only a few per-
cent relative to those of the data sets used for analysis. The experimental
error in s is estimated to be less than ꢃ20%, which corresponds to only
General procedure for synthesis of 3 and 3c: The donor ethyl 3,4,6-tri-O-
acetyl-2-deoxy-2-N-tetrachlorophthalimido-1-thio-b-d-glucopyranoside[67]
(0.17–0.26 mmol) and the acceptor methyl 2,3,4-tri-O-benzyl-a-d-manno-
pyranoside (1.1–1.5 equiv) were dissolved in CH2Cl2 (40 mL; distilled
from CaH2) and the solution was purged with argon for 5 min. Molecular
sieves (3 ꢅ, powder) were added and the suspension was stirred for
15 min, then N-iodosuccinimide (NIS, 1.5–2.0 equiv) was added, followed
by a sufficient amount of AgOTf to turn the solution dark red. Product
formation was instantaneous, as revealed by TLC. The mixture was fil-
tered and diluted with CH2Cl2 (75 mL) and further washed with an aque-
ous solution of Na2S2O3 (2ꢆ30 mL) to remove NIS and I2, then washed
with a saturated aqueous solution of NaHCO3 (2ꢆ30 mL), a saturated
aqueous solution of NaCl (2ꢆ30 mL) and finally twice with H2O
(30 mL). The organic phase was concentrated to dryness, and the residue
was purified by chromatography (silica gel, toluene/EtOAc 4:1) to give
the protected disaccharide in 83–95% yield.
ꢀ6
ꢃ3% in calculated proton–proton distances as a result of the rij de-
pendence, that is, it is on the order of ꢃ0.1 ꢅ in the present study.
The protected disaccharide (0.22–0.17 mmol) was dissolved in EtOH/
EtOAc/AcOH (3:2:1 v/v; 20 mL) and a catalytic amount of Pd(OH)2/C
was added. The suspension was hydrogenolysed under pressure (100 psi)
at room temperature overnight, filtered through Celite and concentrated.
To remove traces of acetic acid, the crude product was dissolved in
EtOH and concentrated at least three times. The residue was then dis-
Computer simulations: For the MD simulations, CHARMM[70] (parallel
version, C27b4) software was used by employing a CHARMM22-type of
force field[71] modified for carbohydrates and referred to as PARM22/
SU01.[72] Initial conditions were prepared by placing 1 and 3 in a previ-
ously equilibrated cubic water box 40.39 ꢅ in length containing 2197
modified TIP3P water[73] molecules, and removing the solvent molecules
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Chem. Eur. J. 2009, 15, 8886 – 8894

Molecular Dynamics of Disaccharides
FULL PAPER
that were closer than 2.5 ꢅ to any solute atom. This procedure resulted
in systems with solute molecules 1 and 3, and 2174 and 2158 water mole-
cules, respectively. Energy minimisation was performed with Steepest
Descent, 200 steps, followed by Adopted Basis Newton–Raphson until
the root-mean-square gradient was less than 0.01 kcalmolꢀ1 ꢅꢀ1. The sim-
ulations were carried out with the leap-frog algorithm,[74] a dielectric con-
stant of unity, a time step of 2 fs, and data were saved every 1 ps for anal-
ysis. SHAKE was used to restrain hydrogen–heavy-atom bonds[75] with a
tolerance gradient of 10ꢀ10. Periodic boundary conditions and the mini-
mum image convention were used. An atom-based force-shift method[76]
was used with a spherical cutoff scheme to truncate long-range electro-
static interactions and for the van der Waals interactions, an atom-based
shifting function was employed to truncate the nonbonded interactions at
a cut-off radius of 12 ꢅ. The nonbond list was generated to 14 ꢅ and up-
dated as soon as any atom had moved by >1 ꢅ since the last update.
The initial velocities were assigned at 105 K, followed by heating with
5 K increments during 8 ps to 310 K, in which the systems were equili-
brated for 500 ps. The production runs for 1 and 3 were performed for 50
and 25 ns, respectively, with the temperature scaled by using Berendsenꢁs
weak-coupling algorithm.[77]
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Received: February 24, 2009
Published online: July 27, 2009
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Chem. Eur. J. 2009, 15, 8886 – 8894