Synthesis and conformational analysis of glycomimetic analogs of thiochitobiose

Article abstract of DOI:10.1016/j.tet.2009.03.067

The synthesis of six analogs of N,N′-diacetylchitobiose is reported, including a novel transglycosylation reaction for the preparation of S-aryl thioglycosides. The conformations of the compounds were studied by a combination of NMR spectroscopy and molec

Full text of DOI:10.1016/j.tet.2009.03.067

Tetrahedron 65 (2009) 4356–4366
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Tetrahedron
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Synthesis and conformational analysis of glycomimetic analogs of thiochitobiose
*
Anja Fettke, Dirk Peikow, Martin G. Peter, Erich Kleinpeter
Chemisches Institut, Universita¨t Potsdam, Karl-Liebknecht-Straße 24-25, D-14476 Potsdam (Golm), Germany
a r t i c l e i n f o
a b s t r a c t
The synthesis of six analogs of N,N0-diacetylchitobiose is reported, including a novel transglycosylation
reaction for the preparation of S-aryl thioglycosides. The conformations of the compounds were studied
by a combination of NMR spectroscopy and molecular modeling, using force field calculations. In the case
of the S-aryl thioglycosides with exclusively S-glycosidic linkages, dihedral angles of the disaccharidic S-
Article history:
Received 18 February 2009
Received in revised form 20 March 2009
Accepted 23 March 2009
Available online 1 April 2009
glycosidic bonds, F0 and J0 and of the S-arylglycoside bonds,
F and J, were found to be similar, whereas
they were different in mixed glycosides and in a thiazoline derivative. An adequate correlation between
the calculated H,H-distances of the local minima and the measured NOE contacts was achieved by ap-
plying population-weighted averages over participating conformers based on weighted relative energies.
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
Carbohydrates
S-Glycosides
Thiochitobiose
Conformational analysis
Molecular modeling
1H NMR spectroscopy
NOE
1. Introduction
molecular modeling, using force field calculations. On the one hand
the NMR spectroscopy is the method of choice for assessing the
Oligosaccharides composed of
b
-(1,4)-linked N-acetylglucos-
conformation of oligosaccharides in aqueous solutions as well as in
the protein bound state.33 But on the other hand the limiting problem
in determining the solution state conformation is that oligosaccha-
rides populate several conformations at ambient temperature34 with
respect to the glycosidic linkages. As a consequence of this limitation,
H,H-coupling constants and NOE enhancements prove to be
weighted averages of this unknown number of conformers. In order
to accompany and support the experimental conformational NMR
study, computational methods have to be incorporated. Especially
force field methods afforded useful and widely suitable results; hence
the oligosaccharides 1–6 (Scheme 1) were studied by molecular
mechanical calculations using the AMBER force field. Internuclei
distances in the conformers, that participates in the conformational
equilibria were examined and compared with the experimental
NOEs. Because of the disagreement of the experimental NMR data
and the theoretically obtained global minimum structure, several
conformations had to be considered as Boltzmann population-
weighted averages subject to their relative energies.
amine, so called chitooligosaccharides, are formed in nature by en-
zymatic degradation of chitin. Increasing evidence indicates that
these compounds function as signaling molecules in some funda-
mental biological processes, such as pattern formation invertebrates1
and synthesis of hyaluronic acid (for reviews, see Refs. 2 and 3). In this
context, the enzymes involved in the formation of chitooligo-
saccharides are studied intensively.4–8 Within our work on the
mechanism and inhibition of chitinases,9–18 we report here on the
synthesis and conformation of four novel O- and S-glycosides of N,N0-
diacetylthiochitobiose 2–5, respectively, as well as the known analog
1.19 As sulfur is less basic than oxygen, S-glycosides with respect to the
O-analogs would be more resistant to acid catalyzed enzymatic hy-
drolysis20–26 and thus may represent competitive chitinase and/or
hexosaminidase inhibitors. In addition, we have prepared the thi-
azoline derivative 6, which mimics the corresponding oxazoline de-
rivative that occurs as an intermediate during the hydrolysis of chitin
by family 18 glycosyl hydrolases,27 including also chitinases and N-
acetylhexosaminidases. Thus, as an analog of the intermediate,28–32 6
could act as an inhibitor of family 18 chitinolytic enzymes.
2. Results and discussion
2.1. Synthesis
The conformational behavior of compounds 1–6 (Scheme 1) has
been investigated by the combination of NMR spectroscopy and
Conversion of 2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-a-D-glu-
copyranosyl chloride (7) or pentaacetylglucosamine (8) to methyl
* Corresponding author. Tel.: þ49 331 977 5210; fax: þ49 331 977 5064.
E-mail address: kp@chem.uni-potsdam.de (E. Kleinpeter).
glucosaminide 919 and the corresponding p-methoxyphenyl
0040-4020/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2009.03.067

A. Fettke et al. / Tetrahedron 65 (2009) 4356–4366
4357
OH
O
OH
O
NHAc
NHAc
S
HO
HO
HO
HO
S
S
HO
OMe
O
HO
1
O
OH
NHAc
2
OH
NHAc
OH
O
NHAc
OH
O
NHAc
HO
HO
S
S
HO
S
HO
HO
O
OH
S
HO
O
NHAc
NHAc
4
OH
3
Me
CH3
SMe
OMe
S
OH
O
NHAc
OH
O
NH
O
S
HO
HO
S
HO
HO
HO
O
O
HO
6
NHAc
OH
5
OH
S
N
CH3
Scheme 1. Structures of studied compounds.
OH
OAc
OR1
OBz
O
OBz
O
O
O
HO
HO
HO
BzO
AcO
AcO
OR
R1
OR
OR2
BzO
NHAc
9: R = Me
NHAc
11: R = Me
NHAc
2
R2
NHAc
13: R = H, R = Me
i
v
v
iv
iv
1
2
1
7: R = H, R = Cl
ii, iii
1 2
8: R = OAc, R = H
1 2
14: R = H, R = PMP
10: R = PMP
16: Ar = C6H5
12: R = PMP
1 2
15: R = Ac, R = PMP
vi
16
17: Ar = p-C6H4SCH3
17
18
OTf
OBz
18: Ar = p-C6H4CH3
O
OH
OAc
vii
OBz
OBz
BzO
R
O
O
15
BzO
SAr
SAr
NHAc
BzO
ix
NHAc
22: R = OMe
23: R = OPMP
24: R = SC6H5
13
14
NHAc
ix
ix
viii
19: Ar = C6H5
16: Ar = C6H5
viii
viii
ix
ix
17: Ar = p-C6H4SCH3
18: Ar = p-C6H4CH3
20: Ar = p-C6H4SCH3
21: Ar = p-C6H4CH3
25: R = p-SC6H4SCH3
26: R = p-SC6H4CH3
Scheme 2. Synthesis of 4-O-Tf-galactoside acceptors. (i): NaOMe, MeOH, CH2Cl2, rt, 2 h (76%); (ii): p-methoxyphenol, BF3$Et2O, CH2Cl2, rt, 72 h (87%); (iii): LiOH, MeOH, rt, 4–8 h
(94%); (iv): BzCl, Py, ꢀ60 ꢁC/rt, 16 h (11: 87%; 12: 78%); (v): Tf2O, Py, CH2Cl2, ꢀ15 ꢁC, 1 h, then NaNO2, DMF, rt, 16 h (13: 86%, 14: 58%); (vi): Ac2O, Py, 0 ꢁC/rt, 24 h (96%); (vii): aryl-
SH, BF3$Et2O, molecular sieves 4 Å, CHCl3, 60 ꢁC, 24 h (16: 79%, 17: 68%, 18: 67%);(viii): AcCl, MeOH, CHCl3, 0 ꢁC/rt, 24–48 h (19: 56%, 20: 54%, 21: 17%); (ix): Tf2O, Py, CH2Cl2, 0 ꢁC,
2 h (22: 99%, 23: 99%, 24: 99%, 25: 99%, 26: 99%).
glycoside 10,35,36 respectively, was carried out by standard pro-
cedures (Scheme 2). Regioselective benzoylation of 9 and 10
afforded the 3,6-di-O-benzoyates 11 and 12, respectively, which
were epimerized19 to the corresponding galactosaminides 13 and
14. Acetylation of 14 afforded the 4-O-acetate 15. The S-glycosides
16, 17, and 18 were obtained from 15 by a novel transglycosylation
with appropriate arylthiols in the presence of BF3$OEt2. Selective 4-
O-deacetylation of 16–18 gave the 4-OH galactosides 19–21, re-
spectively. The 4-O-trifluoromethanesulfonyl acceptors 22–26
were prepared by 4-O-sulfonylation of galacto acceptors 13, 14, and
19–21, respectively, in nearly quantitative yield.
experiments. Anomeric protons were useful entry points into
analysis of spin systems. From the HMBC spectra both quaternary
carbons and interresidue connectivities, could be unequivocally
assigned by examining the H-10, C-4 cross-peaks (Scheme 4); 1H, 13C
chemical shifts and vicinal coupling constants between H-1 and
H-2, H-5 and H-6proS, and H-5 and H-6proR, respectively, for
compounds 1–6 are given in Tables 1 and 2. Due to the lower
3
0
0
electronegativity of sulfur, the coupling constants JH1 –H2 of the
trans diaxial protons of the S-glycosidic nonreducing end GlcNAc
residues were ca. 10.5 Hz, which is 1–2 Hz higher than in the cor-
responding
chitooligosaccharides
possessing
O-glycosidic
Coupling of the acceptors with the thio donor 2737,38 in the
presence of NaH and 15-crown-5 (cf. Ref. 19) afforded the corre-
sponding protected N,N0-diacetylthiochitobiosides 28–32 (Scheme
3). Finally, Zemplen cleavage39 of the O-acetyl and O-benzoyl groups
gave the free pseudotrisaccharides 1–3, 5, and 6 in excellent yields.
Thionation of 32 with Lawesson reagent40,41 afforded, instead of the
expected bis-thioacetamide, the mono-thioacetamidoglucosyl
linkages.10,13,42
In the reducing end GlcNAc units, the corresponding values
3JH1–H2 were 7.0–10.5 Hz, dependent on the nature of the glycosidic
3
atom (Table 1). The small JH1–H2 value of 7.0 Hz in the thiazoline
residue is consistent with the cis pseudo equatorial orientation of
H-1 and H-2 in the five-membered heterocyclic ring, confirmed by
the NOE enhancement between H-1 and H-2.
a
-gluco-tetrahydropyranothiazoline 33, which was deprotected
under Zemplen conditions to give the free pseudo-disaccharide 4
(Scheme 3).
2.2. Conformational analysis
The structures of 1–6 were confirmed by 1H and 13C NMR
spectroscopy, employing COSY, TOCSY, NOESY, HMQC, and HMBC
The detailed assignment of both 1H and 13C spectra is based on
NOESY measurements by examining inter- and intraresidual NOEs.

4358
A. Fettke et al. / Tetrahedron 65 (2009) 4356–4366
OBz
OAc
O
NHAc
O
O
AcO
AcO
S
AcO
AcO
R
BzO
SH
NHAc
iii
iii
iii
OAc
NHAc
27
i
ii
27 + 22
27 + 24
27 + 25
27 + 26
27 + 23
28: R = OMe
29: R = SC6H5
1
2
ii
ii
ii
30: R = p-SC6H4SCH3
31: R = p-SC6H4CH3
32: R = OPMP
3
4
5
iii
iii
CH3
S
iii
OBz
iv
NH
O
6
32
O
S
AcO
AcO
BzO
OAc
S
N
33
CH3
Scheme 3. Synthesis of thiochitobiose derivatives. (i): NaH, DMF, 0 ꢁC/rt, 5–16 h
(57%); (ii): NaH, 15-crown-5, THF, rt, 6 h (28: 57%, 29: 31%, 30: 28%, 31: 33%, 32: 47%);
(iii): NaOMe, MeOH, rt, 16 h (87–97%); (iv): Lawesson reagent, toluene, 80 ꢁC, 72 h
(56%).
Therefore compounds 1–6 were subjected to 2D-NOESY experi-
ments using a general mixing time of 500 ms, which was applied in
all NOESY-experiments (at this mixing time, tentative intensity
build up curves proved NOEs to attain maximal values). Under these
conditions, distances rij between the corresponding protons were
calculated from a known distance rkl by using the accepted re-
1/6
lationship rij¼rkl(skl
/sij)
(sij and skl are cross-relaxation rates be-
tween protons i and j and between protons k and l, respectively).43 It
was not attempted to calculate precise distances because of severe
signal overlap and the uncertainty in the dynamic properties of
carbohydrate and heterocyclic parts of the compounds. Only upper
and lower limits of interproton distances were estimated by cali-
bration against the known distances between two adjacent aro-
matic protons (e.g., H-10 and H-11; 2.48ꢂ0.01 Å) in 2–5 or between
H-1 and H-5 of the pyranose ring (2.24ꢂ0.01 Å) in 1 and 6 of the
nonreducing residue of N-acetylglucosamine. The intensities of the
NOE cross-peaks in NOESY spectra were determined and compared
to the intensities of the NOEs between H-10 and H-11 and H-1 and
H-5, respectively, and assessed as very strong (dH,H<2.8 Å), strong
(2.8 Å<dH,H<3.2 Å), medium (3.2 Å<dH,H<3.6 Å), and weak
(3.6 Å<dH,H<4.0 Å).10,13
As further NMR parameters, to determine solution conforma-
tions, both homonuclear H,H and heteronuclear H,C coupling
constants, in particular the trans-glycosidic 1H–13C J-coupling
constant, could be potentially employed. However, because of the
3
very low quantity of compounds 1–6 only the JH,H-couplings in
terms of CH2OH rotamers could be consulted.
Since the NOEs determined represent the population-weighted
averages of the NOEs of all conformers participating in the con-
formational equilibrium, NMR data alone can rarely define the
conformational composition of oligosaccharides unambiguously.
S
6' OH
CH3
78
12
13
3
5
NHAc
ω'
9
4'
O
1
HO
Φ'
11
2
S
HO
HO
10
5'
3'
S
2'
Ψ
1'
Ψ'4
Φ
O
ω
NHAc
6
7'8'
OH
Φ ’ = H1‘ - C1‘ - S1‘ - C4 Ψ ’ = C1‘ - S1‘ - C4 - H4
ω = O5 - C5 - C6 - O6 ω' = O5’ - C5’ - C6’ - O6’
Scheme 4. Denomination of atoms, bonds, and torsional angles.

A. Fettke et al. / Tetrahedron 65 (2009) 4356–4366
4359
Therefore, the conformational analysis of these highly flexible oli-
gosaccharides requires additional assistance of molecular modeling
to acquire conformational information. For this reason, population-
weighted average distances were calculated considering the con-
formers of 1–6 and were compared to the experimentally observed
NOE data at room temperature. Temperature dependency of the
equilibria was not studied.
Even though the AMBER force field described by Woods et al.44
has fully compatible parameters for common carbohydrates, pa-
rameters involving the S-glycosidic linkage and the thioacetamide
moiety are not included. Hence, the AMBER99-GLYCAM04 param-
eter set had to be completed for the studied compounds 1–6 and
the monomer database, included in the SYBYL program package
(vide infra) was accumulated with the new carbohydrate and aro-
matic monomers (see also Experimental section). In Figure 1, the
torsional angle of the S-glycosidic linkage (O–C1–S–C4), obtained by
the modified AMBER force field, has been compared with the result
of the ab initio calculation. Excellent results were obtained and the
thus freshly paramerized force field was employed for the grid
search calculations of 1–6.
Each molecule was exposed to an extensive systematic confor-
mational search. Grid search simulations in 10ꢁ increments from
0 to 350ꢁ were run to calculate relaxed potential energy ( F0
F/J, /
J
0) maps with the expanded modified AMBER force field. The
torsional angles at the glycosidic linkages are commonly defined as:
(H10–C10–S–C4) and (C10–S–C4–H4) for 1 and 6; 0 (H10–C10–S–
C4) and J0 (C10–S–C4–H4) for 2–5; and
(H1–C1–X–C9) and
(C1–X–C9–C10) for 2–5 with X¼O or S (Scheme 4).
F
J
F
F
J
Population-weighted averages were then calculated from the
global and local conformational minima, thus obtained, employing
weighting factors based on Boltzmann distributions,45 which were
calculated from the relative energies. The experimentally observed
interresidue H,H-distances, which characterize the conformation of
the sugar-sugar-(pyranosyl)-glycosidic linkage of compounds 1–6
are displayed in Table 3 together with the calculated H,H-distances
of the minimum states A–D and the population-weighted in-
ternuclear proton–proton distances of all conformational minima,
the minimum states A and the sum of minimum states A and B,
respectively.
The accurate determination of the NOEs in D2O was limited due
to existing signal overlap. The observed interglycosidic NOEs
measured at ambient temperature indicate at least for compounds
2 and 5 more than one solution conformation. The other com-
pounds reveal strong signal overlap in the regions of interglycosidic
NOEs. The presence of the H10–H3 NOE in 2 and 5 indicates that the
8.00
ab initio HF/6-32G*
fitted force field
7.00
6.00
5.00
4.00
3.00
2.00
1.00
0.00
0
50
100
interglycosidic torsional angle
150
200
250
300
350
Figure 1. Comparison of ab initio and force field calculated interglycosidic torsional
angle (O–C1–S–C4).

4360
A. Fettke et al. / Tetrahedron 65 (2009) 4356–4366
Table 3
Comparison of intensities of F0
/
J
0-interresidual NOEs from experimental NOESY spectra with internuclear distances for low-energy conformers of 1–6 as obtained from force
field calculations together with population-weighted average values
H–H
Theoretical distances
Obsd signal-intensity
p-w average
distance [Å]
overall conf.
p-w average
distance [Å]
over A
p-w average
distance [Å]
over AþB
respecting F0 J0
/
A
B
C
D
1
2
3
4
5
6
H10–H4
4.6
3.8
4.3
4.4
5.9
2.7
3.8
5.1
5.1
3.9
5.7
2.7
2.3
4.7
4.2
2.9
4.3
2.8
3.9
2.1
3.7
4.7
5.8
2.7
w
3.9
4.0
4.4
4.1
5.6
2.7
4.6
3.8
4.3
4.4
5.9
2.7
4.3
4.2
4.5
4.2
5.8
2.7
H10–H3
overlaid
n.d.
w
n.d.
vs
H10–H5
H10–H6pR
H10–H6pS
H1–OMe
H10–H4
3.8
5.1
5.1
3.9
5.6
4.5
2.3
4.7
4.3
2.9
4.2
4.5
4.6
3.9
4.4
4.4
5.9
4.4
3.9
2.1
3.6
4.7
5.7
4.4
s
3.4
4.7
4.7
3.7
5.2
4.5
3.7
5.1
5.1
3.9
5.6
4.5
3.2
5.0
4.8
3.6
5.1
4.5
H10–H3
w
w
w
n.d.
m
H10–H5
H10–H6pR
H10–H6pS
H1–HoPhen
H10–H4
3.8
5.1
5.1
3.9
5.6
4.5
2.3
4.7
4.2
2.9
4.2
4.5
3.9
2.1
3.6
4.7
5.7
4.4
4.6
3.9
4.3
4.3
5.9
4.4
s
3.3
4.6
4.6
3.6
5.1
4.5
3.8
5.2
5.2
3.9
5.7
4.5
3.2
5.1
4.8
3.5
5.1
4.6
H10–H3
w-m
w
w
n.d.
n.d.
H10–H5
H10–H6pR
H10–H6pS
H1–HoPhen
H10–H4
3.8
5.1
5.1
3.9
5.6
4.5
2.3
4.7
4.3
3.0
4.3
4.5
3.9
2.1
3.6
4.7
5.7
4.4
4.6
3.9
4.3
4.4
5.9
4.4
m-s
w-m
n.d.
n.d.
w
3.3
4.6
4.5
3.7
5.1
4.5
3.8
5.2
5.2
3.9
5.7
4.5
3.1
5.0
4.7
3.5
5.0
4.5
H10–H3
H10–H5
H10–H6pR
H10–H6pS
H1–HoPhen
n.d.
H10–H4
4.6
3.8
4.3
4.4
5.9
3.3
3.8
5.1
5.1
3.9
5.6
3.4
3.9
2.1
3.7
4.7
5.8
3.3
2.3
4.7
4.2
2.9
4.2
3.3
w
w
w
w
n.d.
s
4.0
4.0
4.4
4.1
5.6
3.4
4.5
3.7
4.2
4.2
5.7
3.2
4.2
4.2
4.5
4.1
5.6
3.3
H10–H3
H10–H5
H10–H6pR
H10–H6pS
H1–HoPhen
H10–H4
2.3
4.7
4.2
3.1
2.5
3.7
5.2
5.0
4.7
3.8
3.8
2.1
2.6
5.1
4.1
4.6
3.9
4.3
5.9
4.5
s
n.d.
w
n.d.
w
3.2
4.3
4.1
4.2
3.3
2.3
4.7
4.0
3.0
2.4
2.9
4.9
4.4
3.7
3.0
H10–H3
H10–H5
H10–H6pR
H10–H6pS
syn
F
/anti
J
minimum is heavily populated while the presence of
predominantly strong H10–H4 NOEs, and the compounds with
mixed glycosidic linkages (1 and 5), which exhibit only weak H10–
H4 NOEs. Thus, the type of the adjacent glycosidic linkage proves to
be of considerable conformational influence.
the H10–H4 NOE as well as the H10-H-6proR/S-NOEs in 1–6 indicate
the presence of the syn
H3 NOE can theoretically describe the anti
whereas the H20-H4 NOE describes the anti
Due to the strong signal overlap of the ring protons and the vicinity
of the potential H20-H5/H20-H3 NOEs to the peaks of the diagonal it
F/synJ
conformer. The H20-H5 and the H20-
F
/anti
/syn
J
conformation
conformation.
F
J
Another conformational flexibility of oligosaccharides exists for
the conformation of the exocyclic hydroxymethyl group. In this
work, only gg and gt orientations had to be taken into account; the
population of the tg conformer is low because the vicinal coupling
constants of H-5 and H-6proS and H-5 and H-6proR, respectively, in
1–6 show a rotameric distribution that tends to ggꢃgt[tg as
concluded from the experimental values by employing Eqs. I–III in
Table 4.46–49 These results are in accordance with studies on other
glucosaminides,10,13 e.g., by Nishida et al.,50 who found the rota-
meric distribution of N-acetylglucosamine in aqueous solution to
be gg:gt:tgz60:40:0; thus, four combinations for the disaccharide
units were considered exemplary (gg–gg, gg–gt, gt–gg, gt–gt). It
was found that the shape of the potential energy surface was in-
dependent of the initial conformation of the hydroxymethyl group
as reported previously.51 Therefore, we used subsequently the
hydroxymethyl group orientations u
and u0 as calculated from the
vicinal coupling constants of H-5/H-6proS/proR; they are given in
Table 4.
For all calculations, a distance-dependent dielectric constant of
78 was used to implicitly consider the presence of water as solvent
because of the strong tendency of carbohydrates to form inter- and
intramolecular hydrogen bonds.
was difficult to conclude whether the anti
anti conformation were present.
F/synJ or the antiF/
J
The population-weighted averaged H,H-distances from the
force field, given in Table 3, are in sufficient agreement with ex-
perimentally obtained H,H-distances in terms of obtained NOE
enhancements. The agreement is already good if conformations A
and B were taken into account, in 1 both conformers contribute 71%
(A: 49%), in 2 84% (A: 54%), in 3 84% (A: 44%), in 4 82% (A: 44%), in 5
74% (A: 46%), and in 6 72% (A: 43%) to the overall population.
Consideration of all conformational minima reveals similar values
as for AþB only due to their high contributions. The averaged H,H-
distances of AþB for the F0
/J
0-interglycosidic linkages correspond
to two different conformers for 1 and 6, to 12 different conformers
for 2–4 and to 8 different conformers for 5, since with increasing
glycosidic linkages the number of potential solution conformations
increases as well.
Both the experimental and calculated H,H-distances, given
in Table 3, show clearly the differences between the compounds
with exclusively S-glycosidic linkages (2–4, 6), which exhibit

A. Fettke et al. / Tetrahedron 65 (2009) 4356–4366
4361
Table 4
For the S-pyranosylglycosidic linkages between two sugar resi-
dues four local minimum conformer families were obtained (Fig. 2).
The minimum conformation and the corresponding population
distribution, proved to be similar to those reported for N,N0-
diacetyl-4-thiochitobiose by Munoz et al.,52 using the AMBER force
field as well. Three main minimum conformations were found for
the reducing end 4-thio-GlcNAc residue (A: 58/192, B: 52/13, C:
173/1). For compound 1, the closest analog to N,N0-diacetyl-4-thio-
chitobiose, very similar minimum conformations A–C and, additi-
onally the antiF/antiJ conformer as global minimum were
obtained while for the three structurally similar compounds 2–4
the conformational maps proved to be nearly identical; the global
/
minimum conformation A is located around 170/5 for F0 J0 and
Determination of rotameric distributions of the CH2OH group in 1–6 [D2O] em-
ploying Eqs. I–III45–48
I
II
III
1.3gg + 2.7gt + 11.7tg = JH5-H6proS
1.3gg + 11.5gt + 5.8tg = JH5-H6proR
gg + gt + tg = 1
Monomer
Hz
Rotameric distribution in %
3JH5–H6pS
3JH5–H6pR
gg
gt
tg
1 GlcNAc-OMe
GlcNAc0
2.1
2.2
5.5
5.6
57
57
40
40
3
3
2 GlcNAc-S-Phenyl
GlcNAc0
2.1
1.8
5.3
5.6
60
58
37
42
3
0
3 GlcNAc-S-Phenyl-SMe
GlcNAc0
2.0
2.3
5.1
5.9
62
53
36
43
2
4
180/170 for
nonreducing sugar residue from GlcNAc to NAG-thiazoline in 6
influences the dihedral angles and of the global minimum
structure A. In contrast, the replace from S-aryl to the O-aryl in 5
influences but not in the global minimum A compared to 2–4.
F/J (Table 5). Compared to 2–4, the variation of the
4 GlcNAc-S-Phenyl-Me
GlcNAc0
2.0
2.1
5.0
5.8
63
56
35
43
2
1
F
J
5 GlcNAc-O-Phenyl-OMe
GlcNAc0
2.2
2.1
4.8
5.6
63
56
32
41
5
3
J
F
The determination of the three-dimensional structures of
S-glycosides and the comparison with those of O-glycosides is
important for evaluating the S-isologs as prospective chitinase
inhibitors. To determine the differences between the glycosidic
isologs, we also investigated the potential energy surface for the
analogs 1a and 5a where the S-pyranosylglycosidic linkages of 1
and 5 were replaced by O-pyranosylglycosidic linkages; similar
results were obtained (Fig. 3). For the global minimum conforma-
6 NAG-Thiazolin
GlcNAc(S)0
2.3
2.0
6.8
5.8
44
54
52
42
4
4
The torsional angles between the
and the -(1-9)-S- and -(1-9)-O-arylglycosidic linkages, re-
b
-(10-4)-S-glycosidic linkages
b
b
spectively, and the aromatic moiety strongly influence the dynamic
tion of the
b-(1-4)-O-pyranosylglycosidic linkage isologs F/J
values around 350/320 are reported.10,13,53–56 Due to the structural
difference of the pyranosylglycosidic linkage isologs (Table 6) the
replace of the glycosidic linkage from O- to S-isologs increase dis-
tinctly the conformational flexibility and hence influences clearly
but not drastically the whole energy surface and hence, the location
of the global minimum, number of local minima, and their depth.
conformational space of compounds 1–6. Figure 2 displays the
conformational maps of the S-glycosidic intersugar
linkage, respectively, in these compounds. Conformational maps of
the -(1-9)-S- and -(1-9)-O-arylglycosidic linkages between
F/J /
or F0 J0
b
b
F/J
the middle sugar residue and the aromatic moiety in 2–5 are dis-
played in Figure 4 and will be discussed subsequently.
Figure 2. Contour plots for 1–6 concerning the interglycosidic torsional angles
F
and
J
(1, 6) or F0 and J0 (2–5), respectively. Population densities are indicated by contour lines;
A represents the global minimum, B–D are local minima.

4362
A. Fettke et al. / Tetrahedron 65 (2009) 4356–4366
Table 5
Torsional angles about glycosidic linkages and the corresponding relative energies of the conformational families of 1–6
F0 and J0 (min)
F0 and J0 (ꢁ)
Rel energy
(kcal/mol)
Type of F0 and J0
F
and
(min)
F
and
J
(ꢁ)
Rel energy
(kcal/mol)
Type of
and J
J
F
1
2
d
d
d
d
A
B
C
D
183/204
174/3
49/4
0
anti/anti
anti/syn
syn/syn
syn/anti
0.46
0.62
0.83
61/201
A
B
C
D
174/4
48/5
59/199
183/203
0
anti/syn
syn/syn
syn/anti
anti/anti
E
E0
F
F0
G
G0
179/170
179/350
57/178
57/357
320/194
320/14
0.00–1.42
0.00–1.42
0.90–2.11
0.90–2.11
5.67–6.85
5.67–6.85
anti/anti
anti/syn
syn/anti
syn/syn
non-exo-anti
non-exo-syn
0.35
1.25
1.42
3
4
A
B
C
D
174/5
49/3
58/199
184/202
0
anti/syn
syn/syn
syn/anti
anti/anti
E
179/170
179/350
57/177
57/357
318/196
318/16
0.00–1.45
0.00–1.45
0.92–2.03
0.92–2.03
5.57–6.78
5.57–6.78
anti/anti
anti/syn
syn/anti
syn/syn
non-exo-anti
non-exo-syn
0.07
0.84
1.45
E0
F
F0
G
G0
A
B
C
D
174/5
49/3
58/199
184/203
0
anti/syn
syn/syn
syn/anti
anti/anti
E
E0
F
F0
G
G0
179/171
179/351
57/178
57/357
319/196
319/15
0.00–1.47
0.00–1.47
0.92–2.03
0.92–2.03
5.61–6.81
5.61–6.81
anti/anti
anti/syn
syn/anti
syn/syn
non-exo-anti
non-exo-syn
0.06
0.85
1.47
5
6
A
B
C
D
183/204
174/3
61/202
47/5
0
anti/anti
anti/syn
syn/anti
syn/syn
E
44/184
44/5
192/120
192/298
0.00–0.77
0.00–0.77
2.06–2.67
2.06–2.67
syn/anti
syn/syn
anti/anti
anti/syn
0.28
0.62
0.77
E0
F
F0
d
d
d
d
A
B
C
D
31/154
176/353
178/188
49/55
0
syn/anti
anti/syn
anti/anti
syn/syn
0.05
0.44
0.78
The conformational maps of 1a and 5a are very similar. In 1a and
positions of the local minima differ only slightly from the positions
of the minima of the adjacent torsional angles F0 J0 (Table 5), but
5a, we found one low-energy conformation with dominating
population and one additional energetically less stable conformer
as found previously (Fig. S4 in Supplementary data).10,13,53–56
The global minima structures of 1a and 5a differ slightly con-
/
the potential energy surface as a whole varies clearly because of
the structural difference between the sugar and aromatic residue,
respectively. Furthermore, we found two additional and energet-
ically equal minima G/G0 of higher energy (>5.5 kcal/mol) around
320/20 and 320/190 for 2–4. Compared to 5 the contributions of
which are less than 1% at 298 K and can hence be neglected. In 5
however the arylglycosidic global minimum E/E0 is located around
40/0 and 40/180 similar to the pyranosylglycosidic minima C and D
of compound 5. Thus, there is a clear but not drastic difference in
the conformational maps between S- and O-arylglycosidic linkages
concerning the location of the global minimum, number of local
minima and their depth.
To sum up the grid search calculation results, four low-energy
conformers were found for compounds 1 and 6, 16 for compound 5,
and 24 (also 16 without the negligible G/G0-minima) for com-
pounds 2–4 dependent on both the number of monomer entities
and type of glycosidic linkage. The S-glycosides are conformation-
ally more flexible in comparison to the O-glycosides and populate
heavier the anti-conformations. The non-exo-anti and non-exo-syn
minima are only minor populated and can be neglected.
A critical comparison of computed and experimentally de-
termined H,H-distances in the global energy minimum conformer
alone gives unsatisfying results (Table 3). If the NOEs, which result
from internuclei distances averaged over the A and B conformers,
are considered, they fit in with the experimental data. Although the
distances between the interresidual protons are dominated by the
global minimum, the fit is better, when also local minima are taken
into account. This can be verified with the experimentally observed
NOE contacts. The found NOEs between H-10 and H-4 as well as the
NOEs between H-10 and H-6proR/S-NOEs indicate the existence of
cerning the
F angle. Differences are caused only by the different
aglycons on the disaccharidic residue. The global minimum of
compounds 1a and 5a were found as local minimum B in 2–4, as
local minimum C in compound 1 and as local minimum D in
compounds 5 and 6 in this work (Table 5). The minima concerning
the O-arylglycosidic linkage in 5 are dominated by exo-anomeric
conformations about syn
F (like the minima A–C in 1a and 5a) as
obtained similarly by Espinosa et al.51 for the N-acetylglucosamine
part and additionally by the crystal structure analysis of chito-
biose;56 the S-arylglycosidic linkage is however dominated by the
anti
Each of the disaccharidic S-pyranosylglycosidic low-energy
conformers A–D,
0 and 0, of 2–5 were set as starting point for the
next run of grid search calculations about the dihedral angles,
and , of the S- or O-arylglycosidic bond, respectively. The con-
formational maps, thus obtained, for and of 2–5 are displayed
F conformers.
F
J
F
J
F
J
in Figure 4. For the S-arylglycosidic linkage in 2–4, six local mini-
mum conformer families were obtained, whereas for the O-aryl-
glycosidic bond in 5, four local minimum conformer families were
obtained. As expected, the
are similar both show low-energy-syn
F
/
J
conformational maps of 5 and 5a
-conformations (data for 5a
F
not shown). Because of the symmetry of the aromatic residue in 2–
4 and 5, the number of conformational families reduces to three
and two, respectively.
For the three structurally analog compounds 2–4, also the
conformational maps are very similar. The global minimum E/E0 is
located at ca.180/170 and 180/350 (anti
). The local minima F/F0
are located at ca. 60/180 and 60/300 (syn ). In general, these
F/J-
F
F
synF/synJ-conformers in solution and can be compared with

A. Fettke et al. / Tetrahedron 65 (2009) 4356–4366
4363
1
5
350
300
250
200
150
100
50
350
300
250
200
A
B
A
B
150
100
50
C
D
C
D
0
0
0
50
100 150 200 250 300 350
0
50
100 150
200 250 300 350
Ψ
Ψ
1a with O-glycosidic linkage
5a with O-glycosidic linkage
350
300
250
200
150
100
50
350
300
250
200
150
100
50
A
AA
A
A
B
B
0
0
0
50
100 150 200 250
300 350
0
50
100 150 200 250 300 350
Ψ
Ψ
Figure 3. Comparison of S- and O-glycosidic linkages in 1 and 5 and in 1a and 5a, respectively (plots above represent S-glycosidic linkages, the plots below O-glycosidic linkages).
conformers B in 2–4 (Table 7), conformer C in 1 and conformers D in
5 and 6. In addition the NOEs between H-10 and H-3 militate in
favor of high population of the synF/antiJ-conformation and can
conformational influence. With regard to the conformational maps,
this different NOE-behavior is caused by the different location of
the global minimum conformers.
be compared with conformers C in 2–5 (cf. in Table 7), conformer D
in 1 and conformer A in 6. Even though the conformational be-
havior in solution is dominated by up to eight (neglecting G/G0)
conformers, there are still many other local minimum conformers
of lower population that could slightly contribute to the averaged
results. The eight dominating low-energy conformations of com-
pound 2 are shown as examples in Table 7; all other conformations
contribute less than 4%. The conformers concerning minimum A
2.3. Enzymatic studies
The chitothiooligosaccharides 1–6 were investigated with re-
spect to their ability as potential inhibitors of an insect cell line
chitinase and a N-acetylhexosaminidase of Chironomus tentans, as
well as with two bacterial chitinases, i.e., isoenzymes ChiA and ChiB
from Serratia marcescens (for details, see Ref. 42). None of the
compounds showed appreciable inhibition of the hydrolysis 4-
methylumbelliferyl N,N0-diacetylchitobioside (GlcNAc2-MU) by the
according to the disaccharidic linkage F0 0, with ca. 170/5, are
/J
calculated to dominate the solution state with a population of more
than 50%.
insect chitinase and ChiA (IC50ꢃ960
m
m
M), while 2 and 6 where poor
M, respectively). The hydro-
As mentioned above, the experimental and calculated H,H-dis-
tances given in Table 3 show clearly the differences between the
compounds that have exclusively S-glycosidic linkages (2–4, 6) and
the compounds with mixed glycosidic linkages (1 and 5). We found
that the type of an adjacent glycosidic linkage has an evident
inhibitors of ChiB (IC50 560 and 380
lysis of GlcNAc-MU by the insect N-acetylhexosaminidase was
inhibited by 2, 3, 4, and 5 (IC50 110–150 mM) while the IC50 of the
inhibition of the hydrolysis of GalNAc-MU by this enzyme was
60 mM.
Table 6
Comparison of calculated C–X–C bond angles [ꢁ] and C–X distances [Å] for F0 J0 and
/
3. Conclusions
F
/J
in 5a, 5, and 3 with X¼O, S
2D-NOESY NMR experiments and force field calculations show
that the oligosaccharides studied adopt more than one conforma-
tion in aqueous solution. Congruent results were only reached by
assessing population-weighted averaged conformers according to
the Boltzmann function deduced from their calculated relative en-
ergies. These S-glycosidic oligosaccharides differ from the O-gly-
cosidic isologs clearly but not drastically and hence, reflect the
Angle/distance
5a (O0, O)
5 (S0, O)
3 (S0, S)
C40–X–C1
C4–X–C9
C40–X
X–C1
C4–X
114.63
123.30
1.42
1.42
1.42
103.03
123.20
1.82
1.83
1.42
102.50
118.70
1.82
1.82
1.81
X–C9
1.33
1.33
1.78

4364
A. Fettke et al. / Tetrahedron 65 (2009) 4356–4366
Figure 4. Contour plots for 2–5 with respect to torsional angles
and
F and J F
. Population densities are indicated by contour lines; E and E0 represent the global minimum concerning
J
; F, F0 and G, G0 are other local minima.
higher conformational flexibility caused by the small energy bar-
riers between the different regions of thiochito-oligosaccharidic
conformational maps. Thus, minimum conformations different
from the global minimum conformer may be bound to carbohy-
drate-active enzymes or sugar-binding proteins without eminent
energy conflicts. This will be investigated in future with trNOESY
and STD experiments.
Due to structural variations of the investigated pseudo-di- and
trisaccharides 1–6, we found that the type of glycosidic linkage, the
replace of a sugar residue by an aromatic residue and the change
from an N-acetylglucosamine to a NAG-thiazoline monomer in-
duces changes of the conformational equilibria. These variations
have only a slight influence on the location of the minima, but
a clear influence on the potential energy surface affecting the
location of the global minimum, the number of local minima and
their depth. S-Glycosides are conformationally much more flexible
than the O-isologs. Additionally, we found experimentally and
theoretically that the conformation of a glycosidic linkage is evi-
dently influenced by the type of the adjacent glycosidic linkage.
The enzyme assays show that compounds 2–5 reveal a low in-
hibition of the N-acetylhexosaminidase of C. tentans and of 2 and 6
of chitinase B of S. marcescens. These results lead to the conclusion
that at least three monomer residues are necessary for a distinct
inhibition, e.g., as the well known inhibitor allosamidine. Further-
more, an aromatic residue can take the place of a sugar unit but
does not induce stronger inhibition, as compared with allosamidin;
the same effect was found for the variation of the glycosidic linkage
to a S-type analog. As the results of the molecular modeling cal-
culations show, the change to the conformationally more flexible S-
glycosidic linkage has no dramatically effect on the conformation
but a distinct influence on the population. Thus, no decisive in-
fluence on the inhibition due to conformational differences can be
expected, however, the effect of lower basicity of the S-glycosidic
linkage should be considered.
Table 7
Torsional angles
in %
F,J,
F0 and J0 of the eight preferred conformers of 2; population
2 Conformer C10–S-C4 dihedral angle C1–S–C9 dihedral angle Population in %
F0
J0
F
J
1 (A/E)
2 (A/E0)
3 (B/E)
4 (B/E0)
5 (A/F)
6 (A/F0)
7 (C/E)
8 (C/E0)
172
172
49
3
4
4
4
4
4
179
179
180
179
58
58
180
180
350
170
351
170
357
177
170
350
22
22
12
12
5
5
4
4
4. Experimental section
4.1. Synthesis
49
174
174
59
Compounds 1–6 were obtained by de-O-acylation of 28–33,
respectively. The synthesis of 28–33 is described in Supplementary
data, see also Ref. 42.
199
199
59

A. Fettke et al. / Tetrahedron 65 (2009) 4356–4366
4365
27
4.2. General procedure for de-O-acetylation and de-O-
benzoylation of 28–33
MeOH, 7:3). [
a
]
þ25 (c 0.4, H2O). 1H and 13C NMR, see Tables 1 and
D
2. IR:
n
¼3330 (br ss), 3059 (s), 2928 (s), 1621 (m), 1556 (m), 1385 (br
s), 1277 (m), 1172 (s), 1051 (br ss) cmꢀ1. HR ESI MS: C16H27N2O7S3,
calcd [MþH]þ: 455.0980; found 455.0996. C16H26N2O7S3 (454.59):
calcd C 42.28, H 5.77, N 6.16, S 21.16; found C 42.02, H 5.95, N 5.98, S
21.32.
The educt was added to a 0.4 M solution of NaOMe in MeOH,
and the reaction mixture was stirred for 16 h at rt. Neutralization
was followed by filtration, washing of the resin with MeOH, and
concentration of the combined filtrate and washing solutions. The
residue was suspended in H2O and the mixture was freeze-dried.
4.3. NMR spectroscopy
4.2.1. Methyl 2-acetamido-4-S-(2-acetamido-2-deoxy-
b
-
D
-
Spectra were recorded at ambient temperature in D2O partly
without sample spinning using the HDO signal as internal reference
(4.70 ppm). Data acquisition was performed using X-WIN NMR 3.5
software, whereas processing was performed using the Topspin 1.3
software. For the assignment of the 1H and 13C signals a combina-
tion of 1H and 13C NMR spectroscopy, COSY, HMQC, HMBC, TOCSY,
and NOESY (mixing times: 250, 500, and 800 ms) experiments with
HDO suppression if necessary were recorded with a relaxation
delay of 2 s. For the calculation of the rotameric population of gg-,
gt-, and tg-conformers of the exocyclic hydroxymethyl group, ad-
ditional Jres spectra were recorded; for the analysis the Eqs. I–III in
Table 4 were employed.
glucopyranosyl)-2-deoxy-4-thio-
b-D
-glucopyranoside (1)19
From 28 (503 mg, 0.38 mmol) in NaOMe/MeOH (20 mL). Col-
orless solid, yield: 160 mg (92%) [Ref.: 19 85%], mp 285.6 ꢁC [Ref.: 19
200 ꢁC], Rf¼0.14 (CHCl3/MeOH, 7:3). 1H and 13C NMR, see Tables 1
and 2. IR (KBr):
n
¼3271 (br ss), 2940 (m), 2863 (m), 1650 (ss), 1553
(ss), 1377 (s), 1315 (s), 1104 (br ss), 616 (s) cmꢀ1
.
4.2.2. Phenyl 2-acetamido-4-S-(2-acetamido-2-deoxy-
b-D-
glucopyranosyl)-2-deoxy-1,4-dithio- -glucopyranoside (2)
b-D
From 29 (78 mg, 0.09 mmol) in NaOMe/MeOH (10 mL). Color-
less solid, yield: 46 mg (97%), mp 280 ꢁC (decomp.), Rf¼0.30
27
(CHCl3/MeOH, 7:3). [
a]
–26 (c 0.3, DMSO). 1H and 13C NMR, see
D
Tables 1 and 2. IR:
n
¼3386 (br ss), 2928 (s), 1654 (ss), 1553 (ss), 1374
4.4. Molecular modeling
(s), 1310 (s), 1056 (br ss), 610 (m) cmꢀ1. HR ESI MS: C22H33N2O9S2,
calcd [MþH]þ: 533.1627; found 533.1627. C22H32N2O9S2 (532.64):
calcd C 49.61, H 6.06, N 5.26, S 12.04; found C 49.72, H 6.14, N 5.31,
S 12.06.
Force field calculations were performed on a workstation, the ab
initio calculations were performed on a PC-Linux cluster. The cal-
culations were prepared and analyzed with the Sybyl 7.0 program
package.57 Force field calculations were run with a modified AM-
BER58–60 force field implemented in the Sybyl 7.0 program package.
The force field was expanded with additional GLYCAM0461 pa-
rameters (based on GLYCAM93 parameterized by Woods et al. for
carbohydrates44) and a new set of parameters developed to be
constituent with AMBER. The new atom types and parameters for
bond lengths, bond angles, torsional angles, improper torsions, and
van der Waals parameters are shown in Tables S1–S6 in Supple-
mentary data.
Most of the new parameters were achieved by the well estab-
lished method by deducing from known similar parameters. Ad-
ditionally, some torsional angles had to be determined by fitting the
force field function on an ab initio grid search function (OS-CG-SS-
CG, OS-CG-SS-CA, OS-CG-OS-CA) (see Supplementary data, S1–S3),
4.2.3. 4-Methylthiophenyl 2-acetamido-4-S-(2-acetamido-2-
deoxy-b-D-glucopyranosyl)-2-deoxy-1,4-dithio-b-D-
glucopyranoside (3)
From 30 (45 mg, 0.05 mmol) in NaOMe/MeOH (10 mL). Colorless
solid, yield: 28 mg (98%), mp 197.0–199.7 ꢁC, Rf¼0.53 (CHCl3/
MeOH, 7:3). [
Tables 1 and 2. IR:
a]
28 –67 (c 0.4, H2O/50% DMSO). 1H and 13C NMR, see
D
n
¼3281 (br ss), 2925 (s), 1654 (ss), 1562 (ss), 1407
(br s), 1374 (s), 1105 (s), 1057 (br s), 812 (w), 613 (m) cmꢀ1. HR ESI
MS: C23H35N2O9S3, calcd [MþH]þ: 579.1505; found 579.1514.
C23H34N2O9S3 (578.73): calcd C 47.74, H 5.92, N 4.84, S 16.62; found
C 47.65, H 5.99, N 4.76, S 16.53.
4.2.4. 4-Methylphenyl 2-acetamido-4-S-(2-acetamido-2-deoxy-
b-
D
-glucopyranosyl)-2-deoxy-1,4-dithio- -glucopyranoside (4)
b
-
D
which were calculated at the HF/6-31G level of theory with the
*
From 31 (62 mg, 0.11 mmol) in NaOMe/MeOH (10 mL). Colorless
opt¼z-matrix keyword and a systematic variation of the observed
torsional angles in 10ꢁ increments from 0 to 350ꢁ as a scan calcu-
lation. On the other hand, the monomer database of the biopolymer
sugar dictionary included in Sybyl had to be expanded to create
compounds 1–6. The new monomers were first of all geometry
solid, yield: 57.1 mg (95%), mp 320 ꢁC, Rf¼0.51 (CHCl3/MeOH, 7:3).
28
[
a]
ꢀ16 (c 0.12, MeOH). 1H and 13C NMR, see Tables 1 and 2. IR:.
D
n
¼3277 (br s), 1652 (ss), 1545 (s), 1493 (m), 1373 (ss), 1314 (m), 1032
(br ss), 945 (s), 876 (m), 805 (m) cmꢀ1. HR ESI MS: C23H35N2O9S2,
calcd [MþH]þ: 547.1784; found 547.1773. C23H34N2O9S2 (546.4),
calcd C 50.54, H 6.27, N 5.12, S 11.73; found C 50.31, H 6.19, N 4.99, S
11.68.
optimized with the opt keyword at the HF/6-31G
without restrictions using the Gaussian 03 suite of programs.62
Afterward, HF/6-31G single point calculations were run with the
*
level of theory
*
keyword pop¼mk iop(6/33¼2) to get electrostatic points in a form
that the RESP program understands. RESP is a freely downloadable
program, to assist and automatize the process of calculating RESP
charges (prepared by A. Pigache, P. Cieplak, and F.-Y. Dupradeau).
With these electrostatic points for every monomer, the 2-stage-
restraint-multiple-conformation-ESP-charges (RESP charges) were
assigned, that consider several conformations, and not only the
low-energy conformations, as well as corresponding molecular
symmetry63,64 and added for every new monomer together with
the z-matrix, the atom types and the connectivity information in
the sybyl biopolymer sugar dictionary.
4.2.5. 4-Methoxyphenyl 2-acetamido-4-S-(2-acetamido-2-deoxy-
b-D-glucopyranosyl)-2-deoxy-4-thio-b-D-glucopyranoside (5)
From 32 (113 mg, 0.13 mmol) in NaOMe/MeOH (20 mL). Color-
less solid, yield: 69 mg (98%), mp 240.3–241.0 ꢁC, Rf¼0.25 (CHCl3/
MeOH, 7:3). [
2. IR:
a]
23 –13 (c 0.2, H2O). 1H and 13C NMR, see Tables 1 and
D
n
¼3347 (br ss), 2930 (m), 1658 (ss), 1548 (s), 1507 (ss), 1381
(s), 1219 (s), 1058 (br ss), 752 (m) cmꢀ1. HR ESI MS: C23H35N2O11S,
calcd [MþH]þ: 547.1962; found 547.1967. C23H34N2O11S (546.60):
calcd C 50.54, H 6.27, N 5.13, S 5.87; found C 50.38, H 6.31, N 5.09, S
5.92.
Two different calculations were considered with either anti- or
syn-orientation for the H2–C2–NH torsion angle of the acetamide
group. Additionally, the experimentally found u
and u0 hydroxy-
4.2.6. 2-Methyl 4-S-[(2-deoxy-2-thioacetamido-
b-D-gluco-
pyranosyl)]-(1,2-dideoxy- -glucopyrano)-[2,1-d]-2-thiazoline (6)
a-D
From 33 (70 mg, 0.09 mmol) in NaOMe/MeOH (20 mL). Color-
less solid, yield: 39 mg (98%), mp 146.0–148.0 ꢁC, Rf¼0.55 (CHCl3/
methyl group orientations were used. Following, the force field grid
search calculations of the oligosaccharides were run in 10ꢁ

4366
A. Fettke et al. / Tetrahedron 65 (2009) 4356–4366
increments from 0 to 350ꢁ and the local minima were relaxed af-
terward. 1,4-Interactions were scaled by a factor 0.5. A cut off the
nonbonding interaction at 12 Å and a dielectric constant of 78 was
used to implicitly consider the presence of water as solvent. The
conformational plots were created with the OriginPro7.0 software.
All calculations according to the Boltzmann function were done at
298 K.
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Acknowledgements
This work was supported by the Deutsche For-
schungsgemeinschaft (DFG) KL 754/8 (to E.K.) and Pe 264/18 (to
M.G.P.). We thank Prof. K.-D. Spindler and Mr. Wagemann, Uni-
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