Effect of β-O-glucosylation on L-Ser and L-Thr diamides: A bias toward α-helical conformations

Article abstract of DOI:10.1002/chem.200600128

β-D-O-glucosylation produces a remarkable effect on the peptide backbone of the model peptides derived from serine and threonine. Consequently, this type of glycosylation is responsible for the experimentally observed shift from extended conformations (mo

Full text of DOI:10.1002/chem.200600128

DOI: 10.1002/chem.200600128
Effect of b-O-Glucosylation on l-Ser and l-Thr Diamides: A Bias toward
a-Helical Conformations
Francisco Corzana,[a] Jesffls H. Busto,[a] Søren B. Engelsen,[b] Jesffls JimØnez-Barbero,[c]
Juan L. Asensio,[d] Jesffls M. Peregrina,[a] and A. Avenoza*[a]
Abstract: b-d-O-glucosylation produces
a remarkable effect on the peptide
backbone of the model peptides de-
rived from serine and threonine. Con-
sequently, this type of glycosylation is
responsible for the experimentally ob-
served shift from extended conforma-
tions (model peptides) towards the
folded conformations (model glycopep-
tides). The conclusion has been solidly
assessed by a combined NMR/MD pro-
tocol. Interestingly, the MD (molecular
dynamics) results for the glycopeptides
point towards the existence of water-
bridging molecules between the sugar
and peptide moieties, which could ex-
plain the stabilization of the folded
conformers in aqueous solution.
Keywords: conformation analysis ·
glycopeptides · molecular dynamics ·
NMR spectroscopy · peptides
Introduction
such as the b-O-linked attachment of d-Glc to Ser/Thr (O-
glucosylation) are less frequent and have been found in the
epidermal growth factor (EGF) domains of different serum
proteins[4a–d] and the Notch receptor.[4e–g] The role of the glu-
cose in these systems is unknown and remains controversial.
In addition, in the field of unnatural glycopeptides as thera-
peutics, a glucosylated form of enkephalin Tyr-Thr-Gly-Phe-
Leu-Ser peptide, in which a b-d-Glc has been appended to
the Ser residue, produces analgesic effects similar to mor-
phine.[5] On the one hand, it is well appreciated that in
nature, the a-O-glycosylation with GalNAc and higher oli-
gosaccharides have a profound organizational effect on the
underlying peptide backbone, forcing it into an extended
conformation.[6] On the other hand, other authors have sug-
gested that glycosylation per se has no effect on the peptide
backbone conformation, in the absence of amide groups at
the sugar moiety.[7]
Thus, taking into account these features, it seems essential
to improve our perception of the mechanisms that allow the
carbohydrate to modify the conformational equilibrium of
the peptide backbone to properly understand the key bio-
logical processes in which glycopeptides are involved, such
as enzymatic catalysis, hormonal control, transport, cell ad-
hesion or cell–cell recognition.[8]
The physical and biological properties that O-glycosylation
confers to the protein to which the sugars are attached are
well described in several reviews on the biosynthesis, struc-
tures and functions of O-glycosylation.[1] Logically, these
properties include conformational alterations of the protein
structures. The most common O-glycosylations involve the
a-O-glycosidic linkage of GalNAc attached to a Ser/Thr-rich
domain (mucin-type)[2] or the b-O-glycosidic linkage of
GlcNAc linked to a Ser/Thr residue of cytoplasmic and nu-
clear proteins, which play a regulatory role in protein func-
tion.[3] In contrast, some specific types of O-glycosylation,
[a] Dr. F. Corzana, Dr. J. H. Busto, Dr. J. M. Peregrina, Dr. A. Avenoza
Departamento de Química, Universidad de La Rioja
UA-CSIC. 26006 LogroÇo (Spain)
Fax : (+34)941-299-655
E-mail: alberto.avenoza@dq.unirioja.es
[b] Dr. S. B. Engelsen
The Royal Veterinary and Agricultural University
Rolighedsvej 30, 1958 Frederiksberg C (Denmark)
[c] Prof. J. JimØnez-Barbero
Centro de Investigaciones Biológicas (C.S.I.C.)
Ramiro Maeztu 9, 28040 Madrid (Spain)
On this basis, as a first step to evaluate the implications
that b-O-glucosylation has on the peptide conformation, we
decided to conduct the synthesis and the structural (NMR
and modelling) analysis of the l-Ser diamide (Ac-l-Ser-
NHMe, 1) and l-Thr diamide (Ac-l-Thr-NHMe, 2) as refer-
ence model peptides, as well as the simplest glycopeptide
[d] Dr. J. L. Asensio
Instituto de Química Orgµnica (C.S.I.C.)
Juan de la Cierva 3, 28006 Madrid (Spain)
Supporting information for this article is available on the WWW
under http://www.chemeurj.org/ or from the author.
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Pagination corrected Oct. 17, 2006

FULL PAPER
derivatives Ac-l-Ser
A
(b-
only two cases, the structural studies were carried out in
water solution.[11c,d]
d-Glc)-NHMe (2g, Figure 1). In this context, it is important
to note that computational studies on these systems are not
trivial. Indeed, the currently used all-atom force fields that
Results and Discussion
Synthesis: Derivatives 1 and 2 were easily obtained from the
corresponding natural amino acids as described in the Sup-
porting Information.
Glycopeptides 1g and 2g were obtained by using the stan-
dard conditions of the Koenigs–Knorr glycosylation. There-
fore, compounds 1 and 2 were treated with 2,3,4,6-tetra-O-
benzoyl-a-d-glucopyranosyl bromide in presence of silver
triflate and dichloromethane. Under these conditions, the
reaction gave exclusively the corresponding b-anomers in a
moderate yield. Finally, the hydrolysis of the benzoyl groups
with sodium methoxide in methanol gave the desired com-
pounds 1g and 2g (Scheme 1).
Geometry of the backbone: Our conformational study of
compounds 1, 2, 1g and 2g has involved the use of NOE-
based distance information and homonuclear coupling con-
stants, which have been interpreted with the assistance of
MD-tar (molecular dynamics with time-averaged restrains)
simulations.[12] Under these conditions, the conformational
Figure 1. a) Structure of model peptides 1 and 2 and glycopeptides 1g
and 2g. b) 3D structure of 1 and 1g, including the atomic and dihedral
labels employed in this work.
predict reasonable conforma-
tional dynamics for larger pep-
tides or proteins fail to repro-
duce the measured conforma-
tional distribution for di- and
tripeptide systems and vice
versa.[9] As a consequence, the
conformational study of small
model glycopeptides reported
Scheme 1. Synthetic routes to 1g and 2g.
to date is rather limited. More-
over, these studies[6,7,10,11] in-
clude mainly small glycopepti-
des, with a-d-GalNAc or b-d-GlcNAc. Investigation of gly-
copeptides containing Glc is limited to a few studies and, in
regions sampled by the simulations (within the sterically al-
lowable regions) are mainly determined by the experimental
constrains and the fine details of the simulation are not criti-
cal.
As a first step in the structural study of the model pep-
tides 1 and 2 and glycopeptides 1g and 2g, selective
1D NOESY experiments in D2O (see Supporting Informa-
tion) and 2D NOESY experiments (Figure 2) in H2O/D2O
9:1 were carried out for all the compounds. In addition,
Abstract in Spanish: La b-d-O-glucosilación de pØptidos
modelo derivados de serina y treonina afecta notablemente a
la estructura de dichos pØptidos. De hecho, esta glicosilación
es responsable del cambio, experimentalmente observado, de
conformaciones extendidas (pØptidos modelo) a conforma-
ciones plegadas (glicopØptidos modelo). A esta conclusión se
ha llegado combinando experimentos de RMN con cµlculos
de dinµmica molecular. Es importante destacar que los resul-
tados obtenidos mediante estos cµlculos apuntan hacia la
existencia de molØculas de agua que enlazan las partes peptí-
dica y carbohidrato del glicopØptido. Dichas molØculas de
agua podrían explicar la estabilización de las conformaciones
plegadas observadas en disolución acuosa.
3J
A
N
sured for the peptides and glycopeptides.
The exhaustive study of the 2D NOESY spectra corre-
sponding to the model peptides 1 and 2 in H2O/D2O 9:1 re-
veals that they present similar patterns of NOEs. Moreover,
the strong d
A
sence of the d(NH1,NH2) one, suggests the existence of ex-
A
tended conformations in the peptide backbone[13] (Fig-
ure 2a). However, in model glycopeptides 1g and 2g, the
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7865

A. Avenoza et al.
sion of ff99 (ff99’)[20] force fields were also tested with com-
pounds 1 and 1g, obtaining similar results with the three
force fields (see Supporting Information). In addition, 10 ns
unrestrained MD simulations in explicit water (MD-free)
were performed for compounds 1 and 1g (see Tables 1 and 2
and Supporting Information).
Table 1. Comparison of the experimental (NMR) and simulated (MD)
3
distances and J couplings for 1 and 2.[a]
peptide 1
peptide 2
Exptl[b] MD-free ff94 MD-tar Exptl[b] MD-tar
d
d
d
C
absent
(>3.0)
s
2.4
3.1
2.9
3.0
2.4
2.9
absent
(>3.0)
s
(2.3)
m
3.2
2.4
2.9
A
ACHTREUNG
A
m
A
N
3J
3J
C
10.7/3.4
6.5
5.4
6.5
4.2
7.3
4.1
7.0
[a] Distances are given in Š and 3J coupling in Hz. [b] w=weak, m=
medium and s=strong NOE. [c] Experimentally observed as a pseudo-
triplet and estimated by using the Karplus equation given in referen-
ce [15a] [d] Estimated by using the Karplus equation given in referen-
ce [15b].
Table 1 gathers all the relevant NOE-derived distances
3
and J values, together with those obtained from unrestrain-
ed (MD-free) and MD-tar simulations for the model pep-
tides 1 and 2.
As shown in Table 1, the unrestrained MD simulations
Figure 2. Section of the 800 ms 2D NOESY spectra (400 MHz) in H2O/
D2O 9:1 at 258C of model peptide 1 (a), and model glycopeptide 1g (b),
showing the amide cross-peaks.
suggest folded conformations (d
C
G
medium d
A
3
medium d(NH1,NH2) NOE, suggest the coexistence of ex-
G
data (distances and J
A
tended and folded conformers (Figure 2b).
torily reproduce the conformation of the peptide backbone
of 1. On the other hand, the distances and 3J calculated
values from the MD-tar simulations are in very good agree-
ment with the experimental ones.
To get an experimentally derived ensemble, 80 ns MD-tar
simulations were carried out by inclusion of the experimen-
3
3
ACHTREUNG
tal distances and J(NH2,Ha) and J(Ha,Hb), as time-aver-
A
aged restraints. The experimentally determined distances
were derived from the corresponding NOE build-up
curves[14] (see Supporting Information). Additionally, distan-
ces involving NH protons were
Figure 3a shows the F/Y distributions for the peptide
backbone in 1 and 2 obtained from the MD-tar simulations.
It can be observed that according to the NOE experiments
semiquantitatively determined
3
Table 2. Comparison of the experimental and MD simulations derived distances and J couplings for glycopep-
tides 1g and 2g.[a]
by integrating the volume of
the corresponding cross-peaks.
The 3J coupling constants de-
rived from the simulations
were estimated by using the
appropriate Karplus equa-
tion.[15]
The MD-tar were performed
by using AMBER version
6.0.[16] The ff94[17] force field
was implemented with the new
GLYCAM 04 parameters,[18] to
accurately simulate the confor-
mational behaviour around the
sugar moiety. For comparison,
the ff99[19] and a modified ver-
glycopeptide 1g
MD-free ff94
glycopeptide 2g
Exptl[b]
MD-tar
Exptl[b]
MD-tar
d
d
d
C
m (2.7)
s (2.3)
m (2.6)
m (2.9)
m (2.8)
2.6
2.6
2.6
2.3
4.6
2.2
3.2
2.9
2.8
2.9
2.6
2.5
2.6
2.5
2.5
2.9
2.7
2.8
2.5
2.5
2.6
2.4
5.1
6.8
s (2.3)
m (2.5)
m (2.8)
m (2.9)
m[e]
2.2
2.5
2.9
2.9
3.0
2.5
2.9
2.3
3.2
3.5
7.5
d(Hbpro-S,NH2)
d(Hbpro-R,NH2)
d(Hbpro-S,Ha)
d(Hbpro-R,Ha)
d(Hbpro-S,H1)
d(Hbpro-R,H1)
m
m[e]
2.6
2.4
6.4/4.4
6.9
2.4Æ0.5[e]
3J
3J
N
3.4
N
6.9
7.5
3
[a] Distances are given in Š and J coupling in Hz. [b] The w=weak, m=medium and s=strong NOE. [c] Ex-
perimentally observed as a pseudotriplet and estimated by using the Karplus equation given in reference [15a].
[d] Estimated by using the Karplus equation given in reference [15b]. [e] In compound 2g, H
substituted by b-Me of Thr.
A
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b-O-Glucosylation
FULL PAPER
conformations towards folded conformations (a-helix and
G). This finding is, as commented above, experimentally cor-
roborated by the medium-size NOE for d(NH1,NH2).
G
Geometry of the side chain: Concerning the side chain c1
torsional angle, the model peptide 1 mainly adopts the three
lowest energy staggered rotamers, denoted as gauche(À) or
g(À) (c1 ~À608), gauche(+) or g(+) (c1 ~608) and anti or t
(c1 ~À1808), with a similar population in aqueous solution
(Figure 4a).
The methyl group at the b-position of the threonine deriv-
ative constrains the lateral chain and, consequently, in
model peptide 2, c1 torsional angle exhibits mainly the g(+)
conformation with a significant decrement of the g(À) rota-
mer. This result is in good agreement with the experimental-
ly observed J(Ha,Hb). Thus, the larger J found in 1 (5.7 Hz
U
in comparison to 4.2 Hz) suggests more flexibility for the
side chain.[15a] Moreover, the major g(+) conformer found in
2 is in accordance with the NOE observed between Meb
and NH2 (see Supporting Information). This behaviour of
the side chain is somehow similar to that found by Hruby
et al.[23] for serine and threonine in regular proteins.
Another consequence of the b-O-glucosylation is related
to the lateral chain of the amino acid moiety (Figure 4b).
Thus, while in glycopeptide 1g the c1 dihedral shows a simi-
lar distribution to that of its parent peptide 1, glycopeptide
2g differs significantly from 2. Consequently, in 2g, c1
mainly adopts the g(+) (44%) and the t (47%) conforma-
tions, while in derivative 2 the g(+) conformer is mainly
populated (72%).
Figure 3. F/Y distributions obtained from the MD-tar simulations for the
peptide backbone of peptides 1 and 2 (a), and glycopeptides 1g and 2g
(b).
Geometry of the glycosidic linkage: With regard to the
sugar moiety, Figure 5 shows the Fs/Ys distribution for the
glycosidic linkage of the model glycopeptides 1g and 2g ob-
tained from the MD-tar simulations. The most significant
feature is that the glycosidic linkage is rather rigid in both
compounds. As can be seen, Fs exhibits a value close to
À608 in both molecules, which is in accordance with the
exoanomeric effect,[24] while Ys takes mainly a value close to
1808 in 1g, while in 2g, and due to the Meb, the value is
slightly different (around 1408).
mentioned above, the F/Y dihedral values (backbone) of
model peptides 1 and 2 are similar to those typical for ex-
tended conformations, such as PPII (~60%) and b-sheet
ACHTREUNG
A
found in aqueous solution for other small peptides.[21]
The next step was to investigate the conformational pref-
erences of the target model glycopeptides 1g and 2g in
water. Thus, Table 2 gathers the relevant NOE-derived dis-
Anisotropic hydration of glycopeptide 1g: Finally, to shed
some light on the factors that govern the experimentally ob-
served shift to the folded conformations in the model glyco-
peptides, we carried out an extensive theoretical study on
glycopeptide 1g. Therefore, and assuming that the unre-
strained MD simulation (MD-free ff94, Table 2) is somehow
representative of folded conformations in aqueous solutions,
the first step was to investigate the hydrogen bonds between
the sugar and the peptide moieties. As result, no significant
hydrogen bonds were detected between the peptide and
sugar moieties over the course of the MD-free simulation,
which is in accordance with the MD-tar simulations. In fact,
the most persistent one was present only 4% of the trajecto-
ry time and involves oxygens O6 and O8 (O···O distance >
or=3.3 Š).
tances and J values, together with those obtained from un-
restrained (MD-free) and MD-tar simulations for these
model glycopeptides.
As can be seen in Table 2, although the MD-free simula-
tions on 1g reproduce most of the experimental data, a non-
realistic high percentage of folded conformers (a-helix and
[22]
Gconformers)
ed large d
is predicted (as deduced from the comput-
(NH1,NH2) distances).
(Ha,NH1) and small d
ACHTREUNG
Figure 3b shows the Fp/Yp distributions obtained for the
backbone of glycopeptides 1g and 2g from the MD-tar sim-
ulations. Interestingly, the b-O-glucosylation strongly per-
turbs the peptide backbone of the model glycopeptides de-
rived from natural amino acids. Consequently, for glycopep-
tides 1g and 2g, there is an important shift from extended
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A. Avenoza et al.
Figure 4. c1 distributions obtained from the MD-tar simulations for model peptides 1 and 2 (a), and model glycopeptides 1g and 2g (b).
To gain detailed knowledge about the influence of water
upon the folded conformations, we initially calculated the
number of water molecules in the first hydration shell. This
number was 18.3 or 5.0, depending on the distance consid-
ered between the solute and water molecules, 3.5 or 2.8 Š,
respectively. These values are similar, for example, to that
found in disaccharide methyl-a-d-maltoside.[25a] The next
stage was to inspect the anisotropic hydration of the solute
in these free trajectories. In this sense, and taking into ac-
count that the GLYCAM 04 force field exhibits reasonably
structured solute–water interactions,[25] (see Supporting In-
formation), the normalised two-dimensional radial pair dis-
tributions[26] were calculated for all possible shared water
density sites (Os1···Ow···Os2 and Os···Ow···N), in which Ow
is the water oxygen and Os and N are solute oxygen or ni-
trogen, respectively.
Figure 5. Fs/Ys distributions obtained from the MD-tar simulations for
the glycosidic linkage of glycopeptides 1g and 2g (F=O5-C1-O1-Cb,
Y=C1-O1-Cb-Ca).
The most populated intraresidue water bridge found for
1g was accommodated between atoms O2···N2, which is
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b-O-Glucosylation
FULL PAPER
present about 24% of the time. The maximum density of
this shared water site was 4.2 times the bulk density
(Figure 6), producing maximum and average residence times
Figure 7. a) B3LYP/6-31+G(d) geometry of the minimum-energy confor-
mation of 1g. b) Fp/Yp distributions obtained from the MD-tar simula-
tions for Ac4-1g in water (right-up) and chloroform (right-down), and the
gL-turn conformer of Ac4-1g obtained from the MD-tar simulation in
chloroform (left-down).
Figure 6. Two-dimensional radial pair distribution functions of different
water-bridging situations found in the unrestrained 10 ns MD simulations
for glycopeptide 1g.
Moreover, to corroborate the influence of water on the
conformational preferences of the glycopeptides, we also
synthesised the peracetylated derivative of 1g, compound
Ac4-1g (see Supporting Information). Interestingly, this
compound is totally soluble not only in chloroform but also
in water, which makes it an attractive candidate for our pur-
pose. The conformational study of this compound in both
solvents, employing the NMR/MD protocol described
above, revealed that compound Ac4-1g exhibits similar con-
formational behaviour to 1g in water (see Figures 3b
and 7b). This fact has been previously described in the liter-
ature[30] for different glycopeptides. Thus, regarding struc-
ture stabilization, the hydroxyl groups of the sugar moiety
in 1g can only act as hydrogen bonding acceptors, which is
consistent with the existence of water-bridging molecules.
On the other hand, as deduced from the NMR/MD data
(Figure 7b), the conformational preferences of compound
Ac4-1g in chloroform considerably differs from those found
in aqueous solution. In fact, in chloroform an important
population of b-sheet coexists with a moderate population
of gL-turn, which is predicted by DFT calculations in vacuo
for 1g. The gL-turn conformer is mainly stabilised by intra-
molecular hydrogen bonding.
of 10.0 and 1.3 ps, respectively. The average distance
O2···N2 was 4.9 Š, ranging from 6.9 to 2.7 Š. As can be
seen in Figure 6, the water-bridging molecules are present in
both folded conformers. Furthermore, when this water
bridge is present, c1 dihedral exhibits a value close to 608,
which is the most populated in both the MD-free and MD-
tar of 1g. Additionally, other less significant water bridges
(present about 10% of the time) were found between
O5···N2, which is mainly found in the a-helix conformer,
and another one in the Gconformation between atoms
O6···O8. While in the carbohydrates field the existence of
these water-bridging molecules have been considered re-
sponsible for their conformational preferences,[25a,h] to the
best of our knowledge, this is the first time that these struc-
tural water molecules have been found in glycoamino acid
derivatives.
We also studied the conformational behaviour of these
relatively small systems by quantum mechanical approaches.
Therefore, to obtain the optimised geometry of 1g, its con-
formational space was deeply explored by means of semiem-
pirical methods (AM1).[27] As a result, the lowest energy
structure found was fully optimised at the B3LYP/6-31G(d)
level[28] in vacuo. The backbone of this global minimum
structure corresponds to a gL-turn motif, which is in accord-
ance with the most stable ab initio conformer of a-d-
GalNAc-Ac-l-Ser-NHMe previously calculated by Csonka
et al (Figure 7a).[29] Nevertheless, this conformation is not
experimentally observed in aqueous solution as deduced
from our NMR/MD protocol. Therefore, it is important to
note that great care must be taken when the conformational
behaviour of these flexible molecules are studied by using
quantum mechanical methods in which the solvent is not
taken into account.
Conclusion
b-d-O-Glucosylation produces a remarkable effect on the
conformational behaviour of the peptide backbone of the
model peptides investigated in this work. Indeed, it seems
responsible for the observed shift from extended conforma-
tions (model peptides) towards the folded conformations
(model glycopeptides). This conclusion has been solidly as-
sessed by a combined NMR/MD protocol. Interestingly, the
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A. Avenoza et al.
Calculations
MD results for the glycopeptides, together with those ob-
tained for a peracetylated derivative, point towards the exis-
tence of water-bridging molecules between the sugar and
peptide moieties that could explain the stabilization of a-
helix and Gconformers in aqueous solution. Furthermore,
application of these approaches to larger systems will be
considered in future work.
MD-tar simulations (dielectric constant=80): NOE-derived distances (see
Tables 1 and 2) were included as time-averaged distance constraints, and
scalar coupling constants J as time-averaged coupling constraints. A<
À1/6
rÀ6
>
average was used for the distances and a linear average was used
for the coupling constants. Final trajectories were run by using an expo-
nential decay constant of 8000 ps and a simulation length of 80 ns.
Molecular modelling in explicit water: The solute molecule was immersed
in a bath TIP3P water molecules[31] with the LEAP module.[32] The simu-
lation was performed by using periodic boundary conditions and the par-
ticle-mesh Ewald approach[33] to introduce long-range electrostatic ef-
fects. The SHAKE algorithm[34] for hydrogen atoms was employed and a
9 Š cutoff was applied to Lennard–Jones interactions. The 10 ns unre-
strained MD trajectories were collected at constant pressure (1 atm) and
temperature (300 K) and analysed by using the CARNAL module.[35]
Experimental Section
General procedures: Solvents were purified according to standard proce-
dures. Analytical TLC was performed by using Polychrom SI F254 plates.
Column chromatography was performed by using silica gel 60 (230–400
mesh). 1H and 13C NMR spectra were recorded on a Bruker ARX 300
and Bruker Avance 400 spectrometer. 1H and 13C NMR spectra were re-
corded in CDCl3 and D2O with TMS as the external standard by using a
coaxial microtube (chemical shifts are reported in ppm on the d scale,
coupling constants in Hz). Melting points were determined on a Büchi B-
545 melting point apparatus and are uncorrected. Optical rotations were
measured on a Perkin–Elmer 341 polarimeter. Microanalyses were car-
ried out on a CE Instruments EA-1110 analyser and are in good agree-
ment with the calculated values.
DFT calculations: The calculations were carried out by means of the
B3LYP hybrid functional.[28] Full optimizations, by using the 6-31+G(d)
basis set, were carried out with the Gaussian 03 package.[36] Analytical
frequencies were calculated at the B3LYP/6-31+G(d) level to determine
the nature of the optimised geometries.
Acknowledgements
Ac-l-Ser(b-d-Glc)-NHMe (1g): Silver triflate (975 mg, 3.60 mmol) was
G
This work was supported by the Ministerio de Educación y Ciencia and
FEDER (project CTQ2005–06235/BQU and Ramón y Cajal contracts of
F.C. and J.H.B.), the Universidad de La Rioja (project API-05/B01) and
the Gobierno de La Rioja (ANGI-2004/03 and ANGI-2005/01 projects).
The authors thank CESGA for computer support. F.C. and J.H.B. have
contributed equally to this work.
added to a suspension of Ac-l-Ser-NHMe (1, 350 mg, 2.18 mmol) and
powdered molecular sieves (4 Š, 1 g) in dichloromethane (5 mL) under
an inert atmosphere. The mixture was stirred at À308C and then 2,3,4,6-
tetra-O-benzoyl-a-d-glucopyranosyl bromide (2 g, 3.03 mmol) in di-
chloromethane (2 mL) was added. The mixture was stirred at À308C for
1 h and then was warmed at 258C and stirred for 14 h. The crude was fil-
tered, concentrated and purified by silica gel column chromatography,
with dicloromethane/methanol 95:5 as the eluent to yield 821 mg (51%)
of Ac-l-Ser(b-d-Bz4Glc)-NHMe as a white solid. A solution of this com-
pound (355 mg, 0.48 mmol) in methanol (10 mL) was treated with a 0.5m
solution of sodium methoxide in methanol (0.1 mL). After stirring for
3 h, the mixture was neutralised with Dowex 50-X8, filtered and concen-
trated. Purification of the residue with C18 reverse-phase sep-pak car-
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= +2.7 (c=0.53, CH3OH);
1H NMR (400 MHz, D2O) d=2.04 (s, 3H), 2.71 (s, 3H), 3.25 (dd, 1H, J=
8.1 Hz, J=9.2 Hz), 3.31–3.36 (m, 1H), 3.39–3.48 (m, 2H), 3.68 (dd, 1H,
J=5.9 Hz, J=12.3 Hz), 3.81–3.90 (m, 2H), 4.19 (dd, 1H, J=5.2 Hz, J=
10.6 Hz), 4.42 (d, 1H, J=7.9 Hz), 4.48 ppm (t, 1H, J=4.6 Hz); 13C NMR
(100 MHz, D2O): d=21.8, 26.0, 53.9, 60.7, 68.7, 69.5, 73.0, 75.6, 75.9,
102.2, 171.8, 174.5 ppm; elemental analysis calcd for C12H22N2O8: C 44.72,
H 6.88, N 8.69; found: C 44.64, H 6.76, N 8.56.
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Ac-l-Thr
ACHTREUNG
1g, compound 2g (101 mg, 27%) was obtained from
2
0.68 mmol). [a]2D5 =À2.8 (c=1.35, CH3OH): 1H NMR (400 MHz, D2O):
d=1.23 (d, 3H, J=6.3 Hz), 2.10 (s, 3H), 2.74 (s, 3H), 3.23 (dd, 1H, J=
8.2, J=9.2 Hz), 3.34–3.50 (m, 3H), 3.71 (dd, 1H, J=5.4, J=12.3 Hz),
3.88 (dd, 1H, J=1.6, J=12.3 Hz), 4.35 (d, 1H, J=3.4 Hz), 4.39–4.46 (m,
1H), 4.50 ppm (d, 1H, J=7.9 Hz); 13C NMR (100 MHz, D2O): d=15.7,
21.7, 25.9, 58.4, 60.6, 69.5, 72.9, 73.3, 75.6, 75.7, 99.6, 172.2, 174.9 ppm; el-
emental analysis calcd for C13H24N2O8: C 46.42, H 7.19, N 8.33; found: C
46.37, H 7.10, N 8.42.
2D NMR experiments: NMR spectroscopic experiments were recorded
on a Bruker Avance 400 spectrometer at 298 K. Magnitude-mode ge-
2D COSY spectra were recorded with gradients and by using the co-
sygpqf pulse program with 908 pulse width. Phase-sensitive ge-2D HSQC
spectra were recorded by using z filter and selection before t1 removing
the decoupling during acquisition by use of the invigpndph pulse program
with CNST2 J(H,C)=145 Hz. 2D NOESY experiments were made by
G
using phase-sensitive ge-2D NOESY for CDCl3 spectra and phase-sensi-
tive ge-2D NOESY with WATERGATE for H2O/D2O 9:1 spectra. Selec-
tive ge-1D NOESY experiments were carried out by using the 1D-
DPFGE NOE pulse sequence.
7870
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Chem. Eur. J. 2006, 12, 7864 – 7871

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Received: January 30, 2006
Revised: May 11, 2006
Published online: July 19, 2006
Chem. Eur. J. 2006, 12, 7864 – 7871
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7871