Synthesis and conformational analysis of neoglycoconjugates derived from O- and S-glucose

Article abstract of DOI:10.1016/j.carres.2013.02.013

Using olefin metathesis as a key step, four neoglycoconjugates incorporating α-O-glucose, α-S-glucose or β-S-glucose as a carbohydrate unit and l-serine or l-cysteine as an amino acid moiety have been synthesized. The four-atom carbon spacer allows the carbohydrate to explore a wide-ranging conformational space, which may have important implications for the molecular recognition of these molecules.

Full text of DOI:10.1016/j.carres.2013.02.013

Carbohydrate Research 373 (2013) 1–8
Contents lists available at SciVerse ScienceDirect
Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
Synthesis and conformational analysis of neoglycoconjugates
derived from O- and S-glucose
⇑
⇑
Víctor Rojas, Javier Carreras, Francisco Corzana, Alberto Avenoza, Jesús H. Busto , Jesús M. Peregrina
Departamento de Química, Universidad de La Rioja, Centro de Investigación en Síntesis Química, C/Madre de Dios, 51, 26006 Logroño, La Rioja, Spain
a r t i c l e i n f o
a b s t r a c t
Article history:
Using olefin metathesis as a key step, four neoglycoconjugates incorporating
b-S-glucose as a carbohydrate unit and L-serine or L-cysteine as an amino acid moiety have been synthe-
sized. The four-atom carbon spacer allows the carbohydrate to explore a wide-ranging conformational
space, which may have important implications for the molecular recognition of these molecules.
Ó 2013 Elsevier Ltd. All rights reserved.
a
-O-glucose,
a
-S-glucose or
Received 22 January 2013
Received in revised form 27 February 2013
Accepted 28 February 2013
Available online 13 March 2013
Keywords:
Carbohydrates
Glycoconjugates
Cross-coupling
NMR spectroscopy
Conformation analysis
1. Introduction
with a protected cysteine (Cys) residue has been the most widely
exploited.
O-Glycans are usually attached to serine (Ser) and threonine
(Thr) residues of the polypeptide and are structurally diverse. They
are involved in a plethora of biological processes, which usually
depend on the type of O-glycan unit directly attached to the
polypeptide chain.1–3 Although the structural features of each type
of O-glycan have been reviewed,4–8 it is important to note that no
From structural viewpoint, precise knowledge of the preferred
conformations adopted by natural glycopeptides as well as their
dynamics (flexibility or rigidity of carbohydrate or peptide back-
bone moieties) is desirable to explain the binding affinity and
selectivity towards different biological targets. Moreover, the syn-
thesis of glycopeptides analogous to natural ones but structurally
modified, named as neoglycopeptides, is an emerging field. These
compounds can help us to complete our understanding of different
biochemical processes, and also offer a battery of new candidates
to biological targets. One interesting approach to provide flexibility
to the carbohydrate moiety is the use of linkers. Therefore, a vari-
ety of unnatural spacers between the reducing end of the sugar and
the aglycon peptide have been reported, including the use of spac-
ers with appropriate functional groups, to perform selective liga-
tions..13,31–34 In this line of work, while in naturally occurring
glycopeptides, the interactions of the sugar unit with the biological
targets are, to some extend, limited by its restricted presentation
mode imposed by the peptide backbone, different or extra interac-
tions can be modulated by the nature of the underlying amino
acid.35–42
references concerning the
Nature have been reported. On the contrary, methyl
–Me-
-O-Glc– is recognized by several lectins9–11 and some
-glucosyl ceramides have been synthesized and evaluated due
a-O-Glc linked to a Ser/Thr residue in
a-O-glucose
a
a
to their ability to stimulate the activation and expansion of human
distinctive class of T cells known as invariant Natural Killer T
(iNKT) cells.12
On the other hand, the particular case of S-glycosylation13–15 is
a really uncommon post-translational modification in Nature, but
S-linked glycans have emerged as a useful class of unnatural glycan
linkages that can improve characteristic features of a native
linkage, including increased in vivo stability and enzymatic resis-
tance.16–23 On this basis, much effort has been made to synthesize
S-linked glycopeptides and more recently S-linked glycopro-
teins.24–28 Among the various synthetic methods for S-glycosyl
amino acids,29,30 the coupling of an activated glycan derivative
In an effort to combine the aforementioned subjects, we have
performed the synthesis and the conformational analysis in aque-
ous solution of the derivatives shown in Figure 1. These com-
pounds, derived from glucose, present
between the sugar and the peptide moieties and incorporate both,
a four-atom spacer
⇑
Corresponding authors. Tel.: +34 941 299654; fax: +34 941 299621 (J.M.P.); tel.:
+34 941 299668; fax: +34 941 299621 (J.H.B.).
O- and S-glycosidic linkages.
E-mail addresses: hector.busto@unirioja.es (J.H. Busto), jesusmanuel.peregri-
na@unirioja.es (J.M. Peregrina).
0008-6215/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.carres.2013.02.013

2
V. Rojas et al. / Carbohydrate Research 373 (2013) 1–8
carbohydrate based antigens as potential vaccines against different
OH
O
OH
O
OH
types of cancer.54,55 In general, the olefin partners employed in the
amino acid moiety were allylglycine derivatives.56,57 In agreement
with the empirical model for the prediction of CM selectivity58
developed by Grubbs and co-workers, the synthesis of carbohy-
drate based structures using CM reactions depends on the reactiv-
ity of both partners, glycosyl moiety and aglycon moiety (amino
acid derivative in this case). Regarding unsaturated glycosyl moie-
ties, reports dealing with reactions of allyl and higher homologues
of O-glycosides have appeared.59,60
HO
HO
HO
HO
O
HO
HO
S
HO
HO
O
S
HO
ζ
ε
δ
HN
O
γ
X
HN
O
HN
O
α
β
NH
O
O
O
O
NH
O
NH
1a
: X = O
With this background, we selected the tetra-O-benzoyl pro-
2α
2β
and 2b.
1b: X = S
tected allyl
to carry out the CM reactions with O-allyl
and S-allyl -Cys (4b), whose amino and carboxylic acid groups
a
-O-glucopyranoside61 3, as O-glycosyl olefin partner
L-Ser derivative (4a)
Figure 1. Target neoglycoconjugates 1a, 1b, 2
a
L
were protected as N-Boc and benzyl esters, respectively.62,63 Both
CM reactions were performed in toluene at 80 °C using Grubbs
and Hoveyda–Grubbs second generation catalysts, obtaining the
best results for compounds 5a,b with the latter catalyst.
The yields in CM reactions with 4a and 4b substrates were mod-
erate due to the presence of by-products. In the case of compound
4a, the major by-product corresponded to the olefin self-metathe-
sis (SM) of this amino acid derivative while, in the second case, the
major by-product corresponded to the SM of the glycosyl partner.
In both cases, SM of amino acid derivative and glycosyl partner by-
products was observed.
2. Results and discussion
2.1. Synthesis
Compounds 1a, 1b, 2a and 2b were synthesized following the
reactions shown in Scheme 1. The amino and carboxylic acid
groups were transformed into amides to simulate a peptide back-
bone. In the first case, the carbohydrate
of Ser (1a) or Cys (1b) by a linker that involves four carbon atoms.
In the second case, thioglycoside -S-Glc or b-S-Glc and Ser units
are separated by the same linker (2 ,b) (Fig. 1).
The choice of neoglycopeptides with sulfur to replace oxygen at
the glycosidic linkage was based on the existence of well-docu-
mented examples that state that, in general, this atom confers a
similar conformational preference about the thioglycosidic and
aglyconic bonds both when in solution and when complexed with
a protein.43–47
We envisioned the synthesis of these target compounds by
using the cross metathesis (CM) process as a key step, because it
has been used to synthesize other different neoglycoconjugates
and it is well implemented in carbohydrate chemistry.48–53 It is
important to note that similar compounds with different linkers
have been synthesized and used in the rational design of new
a-O-Glc is linked to a unit
a
a
In order to access the target compounds 2
and b-S-glycosidic linkages, we selected the mixture of
anomers of tetra-O-acetyl protected allyl S-glucopyranoside64
,b, as S-glycosyl olefin partner, to carry out the CM reaction with
O-allyl serine derivative 4a. Taking into account the difficulty to
separate 8 ,b, we assayed the CM reaction with this mixture of
compounds following the aforementioned conditions. Again, the
best yield (47%) for compounds 9 ,b involved the use of Hov-
a
,b with both the
a-
a- and b-
8a
a
a
eyda–Grubbs catalyst. Olefin self-metathesis (SM) of the Ser and
carbohydrate derivatives was also observed. Especially noteworthy
is the yield obtained for compounds 9a,b (47%) because, in general,
thioglycosides display poor or no reactivity in CM reactions.49
OBz
OBz
OBz
O
OR
O
O
BzO
BzO
O
BzO
BzO
BzO
RO
RO
BzO
BzO
BzO
BzO
RO
O
O
O
O
3
1) H2/Pd-C,
MeOH, rt
+
[Ru],
1) TFA,
2) MeNH2,
TBTU, DIEA,
CH3CN, rt
toluene,
80 °C
X
CH2Cl2, rt
X
X
X
2) Ac2O, Py, rt
CO2Bn
CO2Bn
NHAc
CO2Bn
NHBoc
CONHMe
NHAc
R = Bz
4a
4b
: X = O
NHBoc
: X = S
5a
: X = O (55%)
6a: X = O (72%)
6b: X = S (98%)
7a: X = O (71%)
5b: X = S (49%)
MeONa,
N
N
7b
MeOH, rt
: X = S (60%)
Cl
Ru
R = H
Cl
1a: X = O (72%)
O
1b
: X = S (51%)
[Ru]
OR
OAc
O
OAc
O
O
RO
RO
AcO
AcO
S
AcO
AcO
S
S
RO
AcO
AcO
1) H2/Pd-C,
MeOH, rt
OAc
O
1) TFA,
4a, [Ru],
toluene,
80 ºC
O
AcO
AcO
2) MeNH2, TBTU,
DIEA, CH3CN, rt
CH2Cl2, rt
O
O
S
2) Ac2O, Py, rt
CONHMe
CO2Bn
AcO
CO2Bn
NHAc
10α,β
41%
86%
47%
NHAc
NHBoc
9α,β
11α,β: R = Ac
2α,β: R = H
8α,β
MeONa, MeOH, rt
44%
Scheme 1. Synthesis of the neoglycoconjugates 1a,b and 2a,b incorporating a-O-Glc, a-S-Glc or b-S-Glc and L-Ser or L-Cys.

V. Rojas et al. / Carbohydrate Research 373 (2013) 1–8
3
In fact, they have been described as fast reactive for the self-
metathesis reactions,51 although sometimes similar yields (40–
50%) in CM reactions have been observed. For instance, the CM
reaction between protected S-allyl cysteine and an O-allyl carbohy-
drate derivative with a b-O-glycosidic linkage gave a moderate
yield of the corresponding glycoconjugate.51
Compounds 5a,b and 9a,b obtained from the CM reactions can
be regarded as neoglycosylamino acid building blocks adequately
protected and ready to use for the synthesis of new neoglycopep-
tides. Removal of Boc protecting groups using trifluoroacetic acid
(TFA) in methylene chloride, followed by acetylation with acetic
anhydride in pyridine (Py), gave the corresponding products 6a,b
and 10
a
,b, respectively. These products were transformed into
,b, following the next
the required target compounds 1a,b and 2
a
three steps. Firstly, hydrogenation of the double bond and conver-
sion of the benzyl ester into carboxylic acid group were performed
in one step, using hydrogen in the presence of palladium on carbon
as a catalyst. Secondly, transformation of the carboxylic acid into
the methyl amide group was carried out using methylamine as a
nucleophile and O-(benzotriazol-1-yl)-N,N,N0,N0-tetramethyluroni-
um tetrafluoro-borate (TBTU) as a coupling agent in the presence
of diisopropylethylamine (DIEA) as a base, obtaining compounds
7a,b and 11a,b, respectively. Thirdly, deprotection of hydroxyl
groups (protected as benzoates or acetates) of the carbohydrate
moiety was carried out by treatment with sodium methoxide.
The mixture of compounds 2
parative reverse phase HPLC analysis, using a Phenomenex Luna
10
m C18 (2) 100 A AXIA Packed 250 ꢀ 21.2 mm column.
a,b could be separated by semi pre-
Figure 2. Inter-hydrogen bonds between the sugar and peptide moieties in
compounds 1a, 1b and 2a, obtained from the MD-tar simulations.
l
thioglucosides,44,46 and are consistent with observations that the
thio-linkage provides a high degree of flexibility between glycosyl
units and that these molecules possess more conformers than their
O-analogues.
2.2. Conformational analysis
Once the four target compounds 1a, 1b, 2a and 2b were ob-
tained in their enantiomerically pure forms, we performed the con-
formational analysis in aqueous solution,65 by NMR and molecular
modelling of these simplest model neoglycopeptides. In a first step,
we carried out 2D-NOESY experiments in H2O/D2O (9/1) at 25 °C
and pH 5.2 to experimentally obtain the relevant proton-proton
distances. These distances were then used as restraints in molecu-
lar dynamics simulations with time-averaged restraints (MD-
tar),66 with the objective of obtaining a distribution of low-energy
conformers (dependent on the molecular mechanics force field)
that was able to quantitatively reproduce the NMR data. This pro-
cedure provides a simple and robust way to consider the high flex-
ibility of these glycopeptides in the interpretation of the NMR data
and it has been applied to successfully describe the conformational
analysis of flexible molecules in solution.35,36 Fittingly, it is impor-
tant to mention that, in all the cases, the matching between the
experimental and theoretical distances was more than satisfactory.
The peptide backbone exhibited both helix-like and extended con-
formations, except for 2b, which displays mainly the extended con-
former (see Supplementary data). This result is in good agreement
with the medium NOE contact between the NH groups observed
exclusively in glycopeptide 2b. The stabilization of the helix con-
It is important to note that, due to signal overlapping in the 1H
NMR, we could not determine accurately the percentage of each
conformer and some of them may be overestimated by the General
Amber Force Field (GAFF) employed in these MD-tar simulations.71
Figure 3 shows the ensembles obtained for all the compounds from
the MD-tar simulations.
Interestingly, both the combination of different heteroatoms (O/
S) and the four-carbon chain spacer allow a wide range of spatial
dispositions for carbohydrate moiety with respect to the backbone,
which could have important implications in molecular recognition
processes. Moreover, the extra-flexibility exhibited by S-glycopep-
tides, together with the emergence of this kind of S-glycosidic link-
age,22 will encourage us to develop a new research line oriented to
the design of novel S-glycopeptides that are able to mimic impor-
tant natural O-glycopeptides. In fact, different carbohydrates
linked to cysteine are currently being incorporated into biologi-
cally relevant peptides.72
3. Conclusions
To the best of our knowledge, this is the first time that a confor-
mational study of neoglycopeptides, in which the glucose and the
underlying amino acid of peptide backbone are separated by a
four-carbon chain, has been carried out. The spacer allows the car-
bohydrate to explore a more wide conformational space.
formation present in glycopeptides 1a, 1b and 2a, with a-glyco-
sidic linkages, may be attributed to inter-hydrogen bonds
between the sugar and peptide moieties (see Supplementary data
and Fig. 2). Concerning the glycosidic linkage, us (O5-C1-O/S-C)
was rather rigid in all the compounds, in accordance with the
exo-anomeric effect.67–69 In contrast, significant differences were
observed in ws (C1-O/S-C–C) torsional angle. Thus, while for com-
pounds with O-glycosidic linkage (1a and 1b) this angle showed
values close to 180°, for the S-glycosyl derivatives (compounds
4. Experimental section
4.1. General procedures
2
a
and 2b) three different conformations were populated, with val-
ues of
s around ꢁ60, 60 and 180° (see Supplementary data). These
three major conformers found for 2 and 2b lie at the local minima
previously calculated for methyl 4-thio-
-maltoside70 and other
Solvents were purified according to standard procedures. Col-
umn chromatography was performed using silica gel 60 (230–
400 mesh). 1H and 13C NMR spectra as well as NOESY, ge-COSY
w
a
a

4
V. Rojas et al. / Carbohydrate Research 373 (2013) 1–8
Figure 3. Calculated ensembles for compounds 1a (a), 1b (b), 2a (c) and 2b (d) obtained from the MD-tar simulations, showing the root-mean-square deviation (rmsd) in Å
for heavy atom superimposition.
and ge-HSQC experiments were recorded on 400 MHz spectrome-
ter. Coupling constants (J) and chemical shift (d) are expressed in
Hz and ppm, respectively. IR spectra were recorded on a Nicolet
Nexus FT-IR spectrometer from water solution. Melting points
were determined on a melting point apparatus and are uncor-
rected. Electrospray mass spectra were recorded on a microQTof
spectrometer; accurate mass measurements were achieved using
sodium formate as an external reference.
(ppm): 63.0, 68.1, 69.2, 69.8, 70.6, 72.1, 95.4, 118.2, 128.4, 128.5,
128.6, 129.1, 129.2, 129.4, 129.8, 129.9, 130.0, 130.1, 133.2,
133.3, 133.4, 133.5, 133.5, 165.5, 165.9, 165.9, 166.3; HRESIMS:
Cald for C37H32O10Na ([M+Na]+) 659.1888. Found: 659.1893.
4.3. Boc-L-Ser(allyl)-OBn (4a)
Benzyl alcohol (1.2 mL, 11.56 mmol) and 4-dimethylaminopyr-
idine (377 mg, 3.08 mmol) were added to a solution of Boc- -Ser(-
L
4.2. Allyl 2,3,4,6-tetra-O-benzoyl-a-D-glucopyranoside (3)
allyl)-OH (945 mg, 3.85 mmol) in dry CH2Cl2 (50 mL) under an
inert atmosphere, and cooled in an ice bath to 0 °C and stirred
for 5 min. Dicyclohexylcarbodiimide (874 mg, 4.24 mmol) was
added to the solution and this mixture was stirred for 24 h at rt.
The precipitated dicyclohexylurea was removed by filtration, the
solution was concentrated and the residue was purified by column
chromatography using hexane/EtOAc (7:3) as an eluent, to give
Benzoyl chloride (3.0 mL, 21.80 mmol) was added to a solution
of allyl -glucopyranoside (800 mg, 3.63 mmol) and triethylamine
D
(2.5 mL, 21.80 mmol) in CH2Cl2 (50 mL). The solution was stirred
at rt for 12 h. The dissolution was washed with HCl 2 N
(2 ꢀ 50 mL) and brine (2 ꢀ 50 mL). The organic layer was dried, fil-
tered and concentrated and the residue was purified by column
chromatography using hexane/EtOAc (7:3) as an eluent, to give
816 mg (35%) of compound 3 as a white solid, mp: 103–105 °C;
1.19 g (92%) of compound 4a as a colourless oil. ½a D20
ꢁ6.6 (c
ꢂ
1.00, CHCl3); 1H NMR (400 MHz, CDCl3) dH (ppm): 1.44 (s, 9H),
3.64 (dd, J = 9.4, 3.2 Hz, 1H), 3.82–3.96 (m, 3H), 4.47 (dd, J = 5.5,
3.2 Hz, 1H), 5.08–5.29 (m, 4H), 5.49 (d, J = 8.6 Hz, 1H), 5.75 (ddd,
J = 22.6, 10.7, 5.5 Hz, 1H), 7.23–7.40 (m, 5H); 13C NMR (100 MHz,
CDCl3) dC (ppm): 28.2, 54.0, 66.9, 69.8, 72.0, 79.7, 117.2, 128.0,
128.1, 128.4, 133.9, 135.5, 155.4, 170.4; HRESIMS: Cald for
½
a 2D0 +73.7 (c 1.00, CHCl3); 1H NMR (400 MHz, CDCl3) dH (ppm):
ꢂ
4.11 (dd, J = 13.1, 6.0 Hz, 1H), 4.30 (dd, J = 13.1, 5.2 Hz, 1H), 4.43–
4.52 (m, 2H), 4.56–4.65 (m, 1H), 5.16 (dd, J = 10.4, 1.0 Hz, 1H),
5.26–5.35 (m, 2H), 5.39 (d, J = 3.7 Hz, 1H), 5.69 (t, J = 9.4 Hz, 1H),
5.88 (ddd, J = 22.2, 10.8, 5.6 Hz, 1H), 6.22 (t, J = 9.9 Hz, 1H), 7.60–
7.24 (m, 12H), 7.84–8.08 (m, 8H); 13C NMR (100 MHz, CDCl3) dC
C
18H25NO5Na ([M+Na]+) 358.1625. Found: 358.1626.

V. Rojas et al. / Carbohydrate Research 373 (2013) 1–8
5
4.4. Boc-
L
-Cys(allyl)-OBn (4b)
4.7. (S)-2-Acetamido-3-(4-(
a
-D
-tetra-O-benzoylglucosyloxy)-(E)-
but-2-enyloxy)propanoate benzyl ester (6a)
Benzyl alcohol (731
lL, 7.03 mmol) and 4-dimethylaminopyri-
dine (229 mg, 1.87 mmol) were added to a solution of Boc-
L
-Cys
Trifluoroacetic acid (2.0 mL, 26.0 mmol) was added to a solution
of 5a (93 mg, 0.10 mmol) in CH2Cl2 (8 mL). The solution was stirred
for 4 h at rt and the solvent was removed under reduced pressure.
The residue was dissolved in Et2O (20 mL) and then evaporated.
This operation was repeated several times to obtain the corre-
sponding compound, which was used directly in the next reaction.
Acetic anhydride (1.5 mL) and pyridine (3 mL) were added to pre-
vious compound, and the solution was stirred for 12 h at rt. The
solvent was removed under reduced pressure and the residue
was purified by column chromatography using hexane/EtOAc
(2:8) as an eluent, to give compound 6a (63.0 mg, 72%) as a white
(allyl)-OH (612 mg, 2.34 mmol) in dry CH2Cl2 (30 mL) under inert
atmosphere, and cooled in an ice bath to 0 °C and stirred for
5 min. Dicyclohexylcarbodiimide (532 mg, 2.58 mmol) was added
to the solution and this mixture was stirred for 24 h at rt. The pre-
cipitated dicyclohexylurea was removed by filtration, the solution
was concentrated and the residue was purified by column chroma-
tography using hexane/EtOAc (7:3) as an eluent, to give 752.0 mg
(91%) of compound 4b as a colourless oil. ½a D20
ꢂ
+5.9 (c 1.00, CHCl3);
1H NMR (400 MHz, CDCl3) dH (ppm): 1.44 (s, 9H), 2.85 (dd, J = 13.7,
5.4 Hz, 1H), 2.94 (dd, J = 13.8, 4.5 Hz, 1H), 3.02–3.14 (m, 2H), 4.56
(d, J = 6.6 Hz, 1H), 5.02–5.11 (m, 2H), 5.19 (m, 2H), 5.34 (d,
J = 6.7 Hz, 1H), 5.65–5.77 (m, 1H), 7.27–7.41 (m, 5H); 13C NMR
(100 MHz, CDCl3) dC (ppm): 28.4, 33.0, 35.3, 53.5, 67.5, 80.2,
118.0, 128.5, 128.6, 128.7, 133.8, 135.3, 155.2, 171.1; HRESIMS:
Cald for C18H25NO4SNa ([M+Na]+) 374.1397. Found: 374.1400.
solid, mp: 42–44 °C; ½a D20
ꢂ
+33.8 (c 1.00, CHCl3); 1H NMR (400 MHz,
CDCl3) dH (ppm): 2.05 (s, 3H), 3.59 (dd, J = 9.5, 3.1 Hz, 1H), 3.78–
3.85 (m, 3H), 4.03–4.16 (m, 1H), 4.20–4.28 (m, 1H), 4.42–4.50
(m, 2H), 4.60–4.68 (m, 1H), 4.75–4.81 (m, 1H), 5.11 (d,
J = 12.3 Hz, 1H), 5.23 (d, J = 12.3 Hz, 1H), 5.28–5.32 (m, 1H), 5.38
(d, J = 3.7 Hz, 1H), 5.66 (m, 2H), 5.73 (t, J = 9.7 Hz, 1H), 6.21 (t,
J = 9.9 Hz, 1H), 6.53 (d, J = 8.2 Hz, 1H), 7.27–7.59 (m, 17H), 7.85–
8.09 (m, 8H); 13C NMR (100 MHz, CDCl3) dC (ppm): 23.2, 52.8,
63.2, 67.3, 68.0, 68.2, 69.5, 70.1, 70.5, 71.0, 72.1, 95.4, 128.1,
128.2, 128.4, 128.5, 128.6, 128.7, 128.8, 128.9, 129.0, 129.2,
129.7, 129.78, 129.8, 123.0, 133.3, 133.5, 133.6, 135.5, 165.4,
165.8, 165.9, 166.3, 170.2, 170.3; HRESIMS: Cald for C50H47NO14Na
([M+Na]+) 908.2889. Found: 908.2875.
4.5. Allyl 2,3,4,6-tetra-O-acetyl-1-thio-a,b-D-glucopyranoside
(8a,b)
Allyl bromide (306
clo[5.4.0]undec-7-ene (525
of 2,3,4,6-tetra-O-acetyl-1-thio-b-
l
l
L, 3.51 mmol) and 1,8-diazabicy-
L, 3.51 mmol) was added to a solution
-glucopyranoside (1.0 g,
D
2.93 mmol) in THF (50 mL). The resulting solution was stirred for
12 h at rt and concentrated. The residue was purified by column
chromatography using hexane/EtOAc (6:4) as an eluent, to give
4.8. (S)-2-Acetamido-N-methyl-3-(4-(a-D-tetra-O-
1.13 g (95%) of compounds 8
a
,b as a colourless oil with a propor-
benzoylglucosyloxy)butoxy)propanamide (7a)
tion 1:9 (
a
:b). NMR data of major compound 8b: 1H NMR
(400 MHz, CDCl3) dH (ppm): 1.88–2.00 (m, 12H), 3.13 (dd,
J = 13.5, 6.0 Hz, 1H), 3.29 (dd, J = 13.5, 8.4 Hz, 1H), 3.58 (ddd,
J = 9.9, 5.1, 2.2 Hz, 1H), 4.00–4.06 (dd, J = 12.3, 2.1 Hz, 1H), 4.13
(dd, J = 12.3, 5.2 Hz, 1H), 4.40 (d, J = 10.1 Hz, 1H), 4.92–5.00 (m,
2H), 5.01–5.16 (m, 3H), 5.77–5.64 (m, 1H); 13C NMR (100 MHz,
CDCl3) dC (ppm): 20.4, 20.5, 20.6, 20.7, 32.7, 62.1, 68.3, 69.8, 73.8,
75.6, 81.7, 117.8, 133.3, 169.2, 169.3, 170.0, 170.4; HRESIMS: Cald
for C17H24O9SNa ([M+Na]+) 427.1033. Found: 427.1015.
A solution of compound 6a (63.0 mg, 0.07 mmol) in MeOH
(5 mL) was hydrogenated under atmospheric pressure at rt, using
Pd/C 50% as a catalyst (30 mg). This mixture reaction was stirred
for 24 h. The solution was filtered over celite and after evaporation,
the compound was used directly in the next reaction. This residue
was dissolved in dry CH3CN (8 mL), under an inert atmosphere, and
methylamine hydrochloride (6.2 mg, 0.09 mmol) and TBTU
(29.6 mg, 0.09 mmol) were added in the presence of DIEA (62 lL,
0.36 mmol). The reaction was stirred at rt for 24 h. After evapora-
tion, the residue was purified by a silica gel column chromatogra-
phy, eluting with MeOH/EtOAc (2:98) to give compound 7a
4.6. (S)-2-(tert-Butoxycarbonylamino)-3-(4-(a-D-tetra-O-
benzoylglucosyloxy)-(E)-but-2-enyloxy)propanoate benzyl
ester (5a)
(41.0 mg, 71%) as a yellow oil. ½a D20
ꢂ
+59.6 (c 1.00, CHCl3); 1H NMR
(400 MHz, CDCl3) dH (ppm): 1.46–1.63 (m, 4H), 1.96 (d,
J = 13.0 Hz, 3H), 2.72 (dd, J = 4.8, 1.2 Hz, 3H), 3.24–3.38 (m, 3H),
3.40–3.48 (m, 1H), 3.61–3.81 (m, 2H), 4.33–4.45 (m, 3H), 4.53–
4.60 (m, 1H), 5.15–5.25 (m, 1H), 5.29 (t, J = 4.1 Hz, 1H), 5.61 (td,
J = 9.7, 3.2 Hz, 1H), 6.12 (t, J = 9.9 Hz, 1H), 6.36–6.53 (m, 2H),
7.17–7.54 (m, 12H), 7.75–8.03 (m, 8H); 13C NMR (100 MHz, CDCl3)
dC (ppm): 23.5, 26.0, 26.1, 26.3, 52.5, 52.6, 63.0, 63.1, 67.8, 67.9,
68.4, 69.5, 69.6, 69.7, 69.8, 70.4, 70.5, 70.9, 71.1, 72.1, 72.2, 95.9,
128.3, 128.4, 128.5, 128.9, 129.0, 129.1, 129.6, 129.7, 129.9,
133.2, 133.5, 165.3, 165.8, 165.9, 166.2, 170.2, 170.3, 170.6; HRE-
A solution of Boc-
lyl 2,3,4,6-tetra-O-benzoyl-
0.24 mmol) and second generation Hoveyda–Grubbs catalyst
(7.7 mg, 0.012 mmol) in dry toluene (3 mL) was stirred at 80 °C
for 24 h. After that, another 10% of catalyst (7.7 mg, 0.012 mmol)
L
-Ser(allyl)-OBn (4a) (41.0 mg, 0.12 mmol), al-
a-D-glucopyranoside (3) (156.0 mg,
and 2 equiv of allyl
a-D-glucopyranoside (156.0 mg, 0.24 mmol)
were added and the resulting mixture was stirred for another
9 h. The solvent was removed under reduced pressure, and the res-
idue was purified by column chromatography using hexane/EtOAc
(6:4) as an eluent, to give compound 5a (63.0 mg, 55%) as a white
SIMS: Cald for
833.2875.
C
44H46N2O13Na ([M+Na]+) 833.2892. Found:
solid, mp: 47–49 °C. ½a D20
ꢂ
+57.4 (c 1.00, CHCl3); 1H NMR (400 MHz,
CDCl3) dH (ppm): 1.44 (s, 9H), 3.56 (dd, J = 9.4, 3.2 Hz, 1H), 3.75–
3.83 (m, 3H), 4.01–4.07 (m, 1H), 4.23–4.28 (m, 1H), 4.41–4.50
(m, 3H), 4.58–4.64 (m, 1H), 5.10 (d, J = 12.3 Hz, 1H), 5.24 (d,
J = 12.4 Hz, 1H), 5.28–5.42 (m, 3H), 5.64–5.73 (m, 3H), 6.20 (t,
J = 9.9 Hz, 1H), 7.26–7.58 (m, 17H), 7.84–8.07 (m, 8H); 13C NMR
(100 MHz, CDCl3) dC (ppm): 28.4, 54.2, 63.1, 67.2, 68.1, 69.7, 70.3,
70.6, 71.0, 72.10, 80.1, 95.4, 128.0, 128.2, 128.3, 128.4, 128.5,
128.6, 128.7, 128.9, 129.0, 129.3, 129.7, 129.8, 129.9, 130.0,
130.1, 133.3, 133.4, 133.5, 133.6, 135.7, 155.7, 165.4, 165.9,
166.0, 166.30, 170.7; HRESIMS: Cald for C53H53NO15Na ([M+Na]+)
966.3307. Found: 966.3307.
4.9. (S)-2-Acetamido-N-methyl-3-(4-(
glucosyloxy)butoxy)propanamide (1a)
a-D-
To a solution of compound 7a (30.0 mg, 0.04 mmol) in MeOH
(2 mL), MeONa (0.5 M, 1.5 mL) was added. The reaction was stirred
at rt for 2 h. DowexÒ (60 mg) was then added to the reaction and it
was stirred for 5 min. After that, it was filtered and evaporated. The
residue was dissolved in H2O (2 mL) and eluted through a reverse-
phase Sep-pak C18 cartridge to obtain, after evaporation of the
H2O, compound 1a (10.5 mg, 72%) as a colourless oil. ½a D20
ꢂ
+40.7

6
V. Rojas et al. / Carbohydrate Research 373 (2013) 1–8
(c 1.00, H2O); IR mmax (H2O): 3345 (br), 1643 cmꢁ1
;
1H NMR
4.12. (S)-2-Acetamido-N-methyl-3-(4-(a-D-tetra-O-
(400 MHz, D2O) dH (ppm): 1.57–1.69 (m, 4H, Hd, ), 2.05 (s, 3H,
e
benzoylglucosyloxy)butylthio)propanamide (7b)
COCH3), 2.73 (s, 3H, NCH3), 3.34–3.43 (m, 1H, H-4), 3.46–3.60
(m, 4H), 3.62–3.77 (m, 6H), 3.84 (dd, J = 12.2, 2.2 Hz, 1H), 4.44 (t,
A solution of compound 6b (110.0 mg, 0.12 mmol) in MeOH
(5 mL) was hydrogenated under atmospheric pressure at rt, using
Pd/C 70% as a catalyst (75 mg). This mixture reaction was stirred
for 36 h. The solution was filtered over celite and after evaporation,
the compound was used directly in the next reaction. This residue
was dissolved in dry CH3CN (20 mL), under an inert atmosphere,
and methylamine hydrochloride (25.0 mg, 0.37 mmol) and TBTU
(52.0 mg, 0.15 mmol) were added in the presence of DIEA
J = 5.3 Hz, 1H,
(100 MHz, D2O) dC (ppm): 21.8 (COCH3), 25.3, 25.4 (Cd,
(NCH3), 53.9 (C ), 60.6 (C-6), 67.8 (Cf), 69.2 (Cb), 69.6 (C-4), 71.0
(C ), 71.3 (C-2), 71.8 (C-5), 73.2 (C-3), 98.1 (C-1), 172.1, 174.5
Ha
), 4.89 (d, J = 3.8 Hz, 1H, H-1); 13C NMR
e
), 25.9
a
c
(CO); HRESIMS: Cald for
C
16H30N2O9Na ([M+Na]+) 417.1844.
Found: 417.1829.
(150 lL, 0.86 mmol). The reaction was stirred at rt for 24 h. After
evaporation, the residue was purified by a silica gel column chro-
4.10. (S)-2-(tert-Butoxycarbonylamino)-3-(4-(a-D-tetra-O-
benzoylglucosyloxy)-(E)-but-2-enylthio)propanoate benzyl
ester (5b)
matography, eluting with MeOH/CH2Cl2 (2:98) to give compound
7b (30.8 mg, 60%) as a yellow oil. ½a D20
ꢂ
+51.7 (c 1.00, CHCl3); 1H
NMR (400 MHz, CDCl3) dH (ppm): 1.67–1.83 (m, 4H), 2.05 (d,
J = 5.4 Hz, 3H), 2.62 (dd, J = 15.3, 7.0 Hz, 2H), 2.72–2.81 (m, 1H),
2.85 (t, J = 4.5 Hz, 3H), 2.89–2.98 (m, 1H), 3.54 (dd, J = 10.1,
5.8 Hz, 1H), 3.85–3.92 (m, 1H), 4.46–4.55 (m, 3H), 4.64–4.70 (m,
1H), 5.31–5.36 (m, 1H), 5.38 (m, 1H), 5.73 (t, J = 9.5 Hz, 1H), 6.22
(td, J = 9.8, 4.1 Hz, 1H), 6.57 (d, J = 4.3 Hz, 1H), 6.64 (d, J = 6.0 Hz,
1H), 7.28–7.67 (m, 12H), 7.86–8.14 (m, 8H); 13C NMR (100 MHz,
CDCl3) dC (ppm): 23.2, 26.3, 26.4, 26.6, 28.3, 28.4, 32.2, 32.3, 33.9,
34.0, 52.8, 52.9, 63.2, 67.9, 68.0, 68.4, 68.5, 69.7, 70.7, 70.8, 72.1,
72.2, 96.1, 96.2, 128.5, 128.6, 128.7, 129.0, 129.1, 129.2, 129.8,
129.9, 130.0, 133.3, 133.4, 133.6, 165.4, 165.8, 165.9, 166.2,
A solution of Boc-
allyl 2,3,4,6-tetra-O-benzoyl-
0.26 mmol) and second generation Hoveyda–Grubbs catalyst
(8.2 mg, 0.013 mmol) in dry toluene (3.5 mL) was stirred at 80 °C
for 24 h. After that, another 10% of catalyst (8.2 mg, 0.013 mmol)
L
-Cys(allyl)-OBn (4b) (46.0 mg, 0.13 mmol),
-glucopyranoside (3) (167.0 mg,
a-D
and 2 equiv of allyl
a-D-glucopyranoside (167.0 mg, 0.26 mmol)
were added and the reaction was stirred for 9 h. The solvent was
removed under reduced pressure and the residue was purified by
column chromatography using hexane/EtOAc (6:4) as an eluent,
to give compound 5b (61.0 mg, 49%) as a white solid, mp: 48–
50 °C; ½a 2D0
ꢂ
+50.8 (c 1.00, CHCl3); 1H NMR (400 MHz, CDCl3) dH
166.4, 170.5, 170.9; HRESIMS: Cald for
C44H46N2O12SNa
([M+Na]+) 849.2664. Found: 849.2682.
(ppm): 1.42 (s, 9H), 2.76–2.87 (m, 2H), 2.92–3.03 (m, 2H), 4.02
(dd, J = 12.9, 5.8 Hz, 1H), 4.24 (dd, J = 13.0, 4.8 Hz, 1H), 4.42–4.55
(m, 3H), 4.61 (m, 1H), 5.11–5.22 (m, 2H), 5.28–5.38 (m, 3H),
5.50–5.64 (m, 2H), 5.70 (t, J = 9.7 Hz, 1H), 6.17–6.24 (m, 1H),
7.26–7.56 (m, 17H), 7.85–8.08 (m, 8H); 13C NMR (100 MHz, CDCl3)
dC (ppm): 28.4, 33.2, 33.8, 53.5, 63.1, 67.5, 67.9, 68.1, 69.7, 70.5,
72.0, 80.2, 95.1, 128.3, 128.4, 128.5, 128.6, 128.7, 128.8, 129.0,
129.1, 129.3, 129.8, 129.9, 130.0, 130.1, 133.3, 133.5, 135.2,
155.20, 165.4, 165.8, 165.9, 166.2, 171.0. HRESIMS: C53H53NO14SNa
([M+Na]+) 982.3079. Found: 982.3074.
4.13. (S)-2-Acetamido-N-methyl-3-(4-(
a-D-
glucosyloxy)butylthio)propanamide (1b)
To a solution of compound 7b (20.0 mg, 0.02 mmol) in MeOH
(2 mL), MeONa (0.5 M, 1.5 mL) was added. The reaction was stirred
at rt for 2 h. DowexÒ (40 mg) was added to the reaction and it was
stirred for 5 min. After that, it was filtered and evaporated. The res-
idue was dissolved in H2O (0.5 mL) and the compound was purified
by semipreparative HPLC analysis using the following procedure:
0.5 mL injection loop was conducted on a Waters Delta Prep
4000 reverse phase HPLC and Waters 2987 Dual Absorbance Detec-
4.11. (S)-2-Acetamido-3-(4-(a-D-tetra-O-benzoylglucosyloxy)-
(E)-but-2-enylthio)propanoate benzyl ester (6b)
tor, using
a
Phenomenex Luna C18(2) column (10 l,
250 mm ꢀ 21.2 mm), 5% (v/v) CH3CN in H2O (containing 0.1% v/v
Trifluoroacetic acid (2.0 mL, 26.0 mmol) was added to a solution
of 5b (120 mg, 0.12 mmol) in CH2Cl2 (8 mL). The solution was stir-
red for 3 h at rt and the solvent was removed under reduced pres-
sure. The residue was dissolved in Et2O (20 mL) and then
evaporated. This operation was repeated several times to obtain
the corresponding compound, which was used directly in the next
reaction. Acetic anhydride (1.5 mL) and pyridine (3 mL) were
added to the previous compound, and the solution was stirred at
rt for 12 h. The solvent was removed under reduced pressure and
the residue was purified by column chromatography using hex-
ane/EtOAc (2:8) as an eluent, to give compound 6b (110.0 mg,
TFA) gradient to 20% CH3CN (t = 30 min), 10 mL/min, k = 212 nm
to furnish 1b (5.1 mg, 51%, tR 18.7 min) as a colourless oil. ½a D20
ꢂ
+37.7 (c 1.00, H2O); IR mmax (H2O): 3374 (br), 1644 cmꢁ1 1H
;
NMR (400 MHz, D2O) dH (ppm): 1.63–1.77 (m, 4H, Hd,
e), 2.06 (s,
3H, COCH3), 2.62 (t, J = 6.5 Hz, 2H, H ), 2.75 (s, 3H, NCH3), 2.87
c
(dd, J = 14.0, 8.1 Hz, 1H, Hb), 3.00 (dd, J = 14.0, 5.5 Hz, 1H, Hb),
3.40 (t, J = 9.6 Hz, 1H, H-4), 3.51–3.58 (m, 2H, H-2, Hf), 3.64–3.80
(m, 4H, H-6, H-3, H-5, Hf), 3.82–3.88 (m, 1H, H-6), 4.43 (dd,
J = 7.7, 5.8 Hz, 1H, H
(100 MHz, D2O) dC (ppm): 21.7 (COCH3), 25.5 (Cd), 25.9 (NCH3),
27.7 (C ), 31.3 (C ), 32.7 (Cb), 53.4 (C ), 60.5 (C-6), 67.6 (Cf),
69.6 (C-4), 71.3 (C-2), 71.8 (C-5), 73.1 (C-3), 98.1 (C-1), 172.8,
a
), 4.91 (d, J = 3.7 Hz, 1H, H-1); 13C NMR
e
c
a
98%) as a white solid, mp: 48–50 °C; ½a D20
ꢂ
+61.0 (c 1.00, CHCl3);
1H NMR (400 MHz, CDCl3) dH (ppm): 2.06 (s, 3H), 2.80–3.05 (m,
4H), 4.07 (dd, J = 13.0, 6.0 Hz, 1H), 4.28 (dd, J = 13.0, 4.9 Hz, 1H),
4.47–4.56 (m, 2H), 4.63–4.70 (m, 1H), 4.81–4.88 (m, 1H), 5.16–
5.26 (m, 2H), 5.35 (dd, J = 10.2, 3.7 Hz, 1H), 5.41 (d, J = 3.7 Hz,
1H), 5.52–5.70 (m, 2H), 5.75 (t, J = 9.8 Hz, 1H), 6.26 (t, J = 9.9 Hz,
1H), 6.42 (dd, J = 14.3, 7.7 Hz, 1H), 7.29–7.63 (m, 17H), 7.90–8.12
(m, 8H); 13C NMR (100 MHz, CDCl3) dC (ppm): 23.2, 33.0, 33.8,
52.0, 63.1, 67.6, 67.8, 68.1, 69.6, 70.5, 72.1, 95.1, 128.3, 128.4,
128.5, 128.6, 128.7, 128.8, 129.0, 129.1, 129.2, 129.6, 129.7,
129.8, 129.9, 130.0, 133.4, 133.5, 133.6, 135.1, 165.4, 165.8,
165.9, 166.3, 169.9, 170.8; HRESIMS: Cald for C50H47NO13SNa
([M+Na]+) 924.2660. Found: 924.2663.
174.4 (2CO); HRESIMS: Cald for
433.1615. Found: 433.1621.
C
16H30N2O8SNa ([M+Na]+)
4.14. (S)-2-(tert-Butoxycarbonylamino)-3-(4-(a/b-D-tetra-O-
acetylglucosylthio)-(E)-but-2-enyloxy)propanoate benzyl ester
(9a,b)
A solution of Boc-
L-Ser(allyl)-OBn (4a) (50.0 mg, 0.15 mmol), al-
lyl
2,3,4,6-tetra-O-acetyl-
a
/b- -1-thioglucopyranoside (8 ,b)
D
a
(120.0 mg, 0.30 mmol) and second generation Hoveyda–Grubbs
catalyst (9.4 mg, 0.015 mmol) in dry toluene (4 mL) was stirred
at 80 °C for 24 h. After that, another 10% of catalyst (9.4 mg,

V. Rojas et al. / Carbohydrate Research 373 (2013) 1–8
7
0.015 mmol) and 2 equiv of allyl 2,3,4,6-tetra-O-acetyl-
a
/b-
D
-1-
26.4, 26.7, 28.4, 29.4, 29.6, 52.8, 62.3, 68.4, 68.5, 69.7, 69.8, 69.9,
70.0, 74.0, 76.0, 83.4, 83.6, 169.6, 170.4, 170.8, 170.9, 171.0,
171.5; HRESIMS: Cald for C24H38N2O12SNa ([M+Na]+) 601.2038.
Found: 601.2047.
thioglucopyranoside (120.0 mg, 0.30 mmol) were added and the
resulting solution was stirred at the same temperature for 9 h.
The solvent was removed under reduced pressure and the residue
was purified by column chromatography using hexane/EtOAc (6:4)
4.17. Synthesis of compounds 2a and 2b
as an eluent, to give the mixture of compounds 9a,b (50.0 mg, 47%)
as a yellow oil. NMR data of major compound 9b: 1H NMR
(400 MHz, CDCl3) dH (ppm): 1.43 (s, 9H), 1.97–2.09 (m, 12H),
3.19 (dd, J = 13.2, 5.6 Hz, 1H), 3.35 (dd, J = 13.2, 7.5 Hz, 1H), 3.65
(m, 2H), 3.82–3.96 (m, 3H), 4.12 (dd, J = 12.3, 2.0 Hz, 1H), 4.23
(dd, J = 12.4, 5.0 Hz, 1H), 4.42–4.52 (m, 2H), 5.00–5.16 (m, 3H),
5.21–5.35 (m, 2H), 5.43 (d, J = 8.6 Hz, 1H), 5.51–5.65 (m, 2H),
7.28–7.41 (m, 5H); 13C NMR (100 MHz, CDCl3) dC (ppm): 20.6,
20.7, 20.8, 20.9, 28.4, 31.4, 54.2, 62.3, 67.2, 68.5, 70.1, 70.3, 71.2,
74.0, 75.8, 80.1, 82.0, 128.2, 128.4, 128.5, 128.7, 129.8, 135.7,
155.6, 169.5, 169.6, 170.3, 170.6, 170.7; HRESIMS: Cald for
To a solution of compounds 11a,b (34.5 mg, 0.06 mmol) in
MeOH (2 mL), MeONa (0.5 M, 1.5 mL) was added. The reaction
was stirred at rt for 2 h and DowexÒ (60 mg) was then added to
the reaction. After stirring for 5 min, the reaction mixture was fil-
tered and evaporated. The residue was dissolved in H2O (0.5 mL)
and the mixture of compounds was separated by semipreparative
HPLC analysis using the following procedure: 0.5 mL injection loop
was conducted on a Waters Delta Prep 4000 reverse phase HPLC
and Waters 2987 Dual Absorbance Detector, using a Phenomenex
C
33H45NO14SNa ([M+Na]+) 734.2453. Found: 734.2481.
Luna C18(2) column (10
in H2O (containing 0.1% v/v TFA) gradient to 20% CH3CN
(t = 30 min), 10 mL/min, k = 212 nm to furnish 2 (1 mg, 4%, tR
17.2 min) and 2b (10 mg, 40%, tR 16.1 min) as colourless oils.
l, 250 mm ꢀ 21.2 mm), 5% (v/v) CH3CN
a
4.15. (S)-2-Acetamido-3-(4-(
a/b-
D-tetra-O-acetylglucosylthio)-
(E)-but-2-enyloxy)propanoate benzyl ester (10
a
,b)
4.17.1. (S)-2-Acetamido-N-methyl-3-(4-(
glucosylthio)butoxy)propanamide (2
+133.0 (c 0.16, H2O); IR mmax (H2O): 3381 (br), 1644 cmꢁ1
1H NMR (400 MHz, D2O) dH (ppm): 1.58–1.73 (m, 4H, Hd,
), 2.06 (s,
3H, COCH3), 2.55–2.72 (m, 2H, Hf), 2.74 (s, 3H,NCH3), 3.39 (t,
J = 9.5 Hz, 1H, H-4), 3.51–3.60 (m, 3H, H ,H-3), 3.69–3.89 (m, 5H,
Hb, H-2, H-6), 4.03 (ddd, J = 9.9, 5.3, 2.2 Hz, 1H, H-5), 4.45 (t,
J = 5.4 Hz, 1H,
), 5.40 (d, J = 5.5 Hz, 1H, H-1); 13C NMR
(100 MHz, D2O) dC (ppm): 21.8 (COCH3), 25.5, 25.9 (NCH3), 27.6,
29.8 (Cf), 53.9 (C ), 60.6 (C-6), 69.2 (Cb), 69.7 (C-4), 70.8 (C ),
a-D-
To a solution of compounds 9a,b (120 mg, 0.17 mmol) in CH2Cl2
a)
(8 mL), trifluoroacetic acid (2.0 mL, 26.0 mmol) was added. The
solution was stirred at rt for 4 h and the solvent was removed un-
der reduced pressure. The residue was dissolved in Et2O (20 mL)
and then evaporated. This operation was repeated several times
to obtain the corresponding mixture of compounds, which was
used directly in the next reaction. Acetic anhydride (2 mL) and pyr-
idine (5 mL) were added to the previous mixture of compounds
and the solution was stirred at rt for 12 h. The solvent was re-
moved under reduced pressure and the residue was purified by
column chromatography using hexane/EtOAc (1:9) as an eluent,
½
a 2D0
ꢂ
;
e
c
H
a
a
c
71.0 (C-2), 72.3 (C-5), 73.7 (C-3), 85.5 (C-1), 172.1, 174.5 (CO);
HRESIMS: Cald for C16H30N2O8SNa ([M+Na]+) 433.1615. Found:
433.1619.
to give compounds 10a,b (94.3 mg, 86%) as a yellow oil. NMR data
of major compound 10b: 1H NMR (400 MHz, CDCl3) dH (ppm):
1.99–2.08 (m, 15H), 3.19 (dd, J = 13.3, 5.6 Hz, 1H), 3.37 (dd,
J = 13.2, 7.5 Hz, 1H), 3.62–3.69 (m, 2H), 3.80–3.96 (m, 3H), 4.13–
4.23 (m, 2H), 4.46 (d, J = 10.1 Hz, 1H), 4.81 (dt, J = 7.9, 2.7 Hz,
1H), 5.05–5.10 (m, 2H), 5.16 (d, J = 12.3 Hz, 1H), 5.22–5.32 (m,
2H), 5.50–5.65 (m, 2H), 6.51 (d, J = 8.0 Hz, 1H), 7.29–7.44 (m,
5H); 13C NMR (100 MHz, CDCl3) dC (ppm): 20.6, 20.7, 20.8, 20.9,
23.2, 31.4, 52.9, 62.4, 67.3, 68.6, 70.2, 70.3, 71.3, 73.9, 75.8,
81.98, 128.3, 128.5, 128.8, 129.6, 135.6, 169.5, 169.6, 170.2,
170.2, 170.3, 170.9; HRESIMS: Cald for C30H39NO13SNa ([M+Na]+)
676.2034. Found: 676.2046.
4.17.2. (S)-2-Acetamido-N-methyl-3-(4-(b-
glucosylthio)butoxy)propanamide (2b)
D-
½
a 2D0 (c 1.00, H2O): +37.7; IR mmax (H2O): 3413 (br), 1644 cmꢁ1
;
ꢂ
1H NMR (400 MHz, D2O) d: 1.47–1.60 (m, 4H, Hd,
COCH3), 2.57–2.71 (m, 5H, Hz, Hf, NCH3), 3.14–3.22 (m, 1H, H-2),
3.24–3.38 (m, 3H, H-3,H-4, H-5), 3.38–3.47 (m, 2H, H ), 3.57 (dd,
J = 12.4, 5.6, 1H, H-6), 3.61–3.67 (m, 2H, Hb), 3.76 (dd, J = 12.4,
1.9, 1H, H-6), 4.33 (t, J = 5.3, 1H, H ), 4.39 (d, J = 9.9, 1H, H-1).
13C NMR (100 MHz, D2O) d: 21.8 (COCH3), 25.9, 27.6, 29.7 (Cf),
53.9 (C ), 60.9 (C-6), 69.2 (Cb), 69.6 (C-4), 70.8 (C ), 72.3 (C-2),
77.2, 79.9, 85.4 (C-1), 172.1, 174.5 (CO); HRESIMS: Cald for
16H30N2O8SNa ([M+Na]+) 433.1615. Found: 433.1598.
e), 1.93 (s, 3H,
c
a
a
c
4.16. (S)-2-Acetamido-N-methyl-3-(4-(
a/b-
D-tetra-O-
C
acetylglucosylthio)butoxy)propanamide (11
a,b)
4.18. NMR experiments
A solution of compounds 10a,b (94.3 mg, 0.14 mmol) in MeOH
NMR experiments were recorded on a Bruker Avance 400 spec-
trometer at 293 K. Magnitude-mode ge-2D COSY spectra were re-
corded with gradients and using the cosygpqf pulse program
with 90° pulse width. Phase-sensitive ge-2D HSQC spectra were re-
corded using z-filter and selection before t1 removing the decou-
pling during acquisition by use of invigpndph pulse program
with CNST2 (JHC) = 145. 2D NOESY experiments were made using
phase-sensitive ge-2D NOESY for CDCl3 spectra and phase-sensi-
tive ge-2D NOESY with WATERGATE for H2O/D2O (9:1) spectra.
(5 mL) was hydrogenated under atmospheric pressure at rt, using
Pd/C 80% as a catalyst (76 mg). This mixture reaction was stirred
for 72 h. The solution was filtered over celite and evaporated and
this mixture of compounds was used directly in the next reaction.
This residue was dissolved in dry CH3CN (15 mL), under an inert
atmosphere,
0.43 mmol) and TBTU (60.2 mg, 0.19 mmol) were added in the
presence of DIEA (175 L, 1.01 mmol). The reaction was stirred at
and
methylamine
hydrochloride
(29.1 mg,
l
rt for 24 h. After evaporation, the residue was purified by a silica
gel column chromatography, eluting with MeOH/CH2Cl2 (2:98),
4.19. Molecular dynamics (MD) simulations
to give the mixture of compounds 11a,b (34.5 mg, 41%) as a yellow
oil. NMR data of major compound 11b: 1H NMR (400 MHz, CDCl3)
dH (ppm): 1.65 (m, 4H), 1.96–2.12 (m, 15H), 2.65–2.74 (m, 2H),
2.85 (d, J = 4.8 Hz, 3H), 3.41–3.54 (m, 3H), 3.70–3.79 (m, 2H),
4.12–4.28 (m, 2H), 4.45–4.57 (m, 2H), 4.95–5.12 (m, 2H), 5.23
(td, J = 9.4, 3.1 Hz, 1H), 6.59 (s, 1H), 6.74 (d, J = 6.6 Hz, 1H); 13C
NMR (100 MHz, CDCl3) dC (ppm): 20.6, 20.7, 20,8, 20.9, 23.1, 26.3,
MD-tar simulations were performed with AMBER73 6.0 and the
General Amber Force Field (GAFF)71 parameters. NOE-derived dis-
tances were included as time-averaged distance constraints. For
each restraint, six main constants (r1 to r4, rk2 and rk3) have to
be defined. These parameters define the shape of the restraining

8
V. Rojas et al. / Carbohydrate Research 373 (2013) 1–8
29. Taylor, C. M. Tetrahedron 1998, 54, 11317–11362.
potential. The values of r1 to r4 have to be specified in Å and rk2
and rk3 are defined in kcal molꢁ1 Åꢁ2. The experimental distances
(R) were implemented as structural restraints with a 10% margin
by using a flat well potential between r2 and r3. The violation en-
ergy is defined as a well with parabolic sides between r3–r4 and
r1–r2, and linear sides for values smaller or greater than r1 and
r4, respectively. The input-file used to start a MD-tar simulation
consists of two parts. The first part is an unrestrained energy min-
imization of 1000 steps of steepest-descent minimization followed
by 500 steps of conjugate-gradient minimization. The second part
contains an 80 ns molecular dynamics simulation at 300 K in
which the distance restraints are time-averaged according to Rꢁ6
30. Jobron, L.; Hummel, G. Org. Lett. 2000, 2, 2265–2267.
31. Miller, N.; Williams, G. M.; Brimble, M. A. Int. J. Pept. Res. Ther. 2010, 16, 125–
132.
32. Fiore, M.; Lo Conte, M.; Pacifico, S.; Marra, A.; Dondoni, A. Tetrahedron Lett.
2011, 52, 444–447.
33. Specker, D.; Wittmann, V. Top. Curr. Chem. 2007, 267, 65–107.
34. Nicotra, F.; Cipolla, L.; Peri, F.; La Ferla, B.; Redaelli, C. Adv. Carbohydr. Chem.
Biochem. 2007, 61, 353–398.
35. Corzana, F.; Busto, J. H.; Engelsen, S. B.; Jiménez-Barbero, J.; Asensio, J. L.;
Peregrina, J. M.; Avenoza, A. Chem. Eur. J. 2006, 12, 7864–7871.
36. Corzana, F.; Busto, J. H.; Jiménez-Osés, G.; Jiménez-Barbero, J.; Asensio, J. L.;
Peregrina, J. M.; Avenoza, A. J. Am. Chem. Soc. 2006, 128, 14640–14648.
37. Corzana, F.; Busto, J. H.; Jiménez-Osés, G.; García de Luis, M.; Jiménez-Barbero,
J.; Asensio, J. L.; Peregrina, J. M.; Avenoza, A. J. Am. Chem. Soc. 2007, 129, 9458–
9467.
38. Fernández-Tejada, A.; Corzana, F.; Busto, J. H.; Jiménez-Osés, G.; Peregrina, J.
M.; Avenoza, A. Chem. Eur. J. 2008, 14, 7042–7058.
39. Corzana, F.; Busto, J. H.; García de Luis, M.; Jiménez-Barbero, J.; Avenoza, A.;
Peregrina, J. M. Chem. Eur. J. 2009, 15, 3863–3874.
40. Corzana, F.; Busto, J. H.; Marcelo, F.; García de Luis, M.; Asensio, J. L.; Martín-
Santamaría, S.; Sáenz, Y.; Torres, C.; Jiménez-Barbero, J.; Avenoza, A.; Peregrina,
J. M. Chem. Commun. 2011, 47, 5319–5321.
by using a exponential memory function with
s equal to 8 ns. To
assure a smooth run of the calculation, restraints are introduced
slowly over the first 1 ns of the calculation. All the calculations
were performed using a dielectric constant of 80 to simulate the
water environment.
41. Corzana, F.; Busto, J. H.; Marcelo, F.; García de Luis, M.; Asensio, J. L.; Martín-
Santamaría, S.; Jiménez-Barbero, J.; Avenoza, A.; Peregrina, J. M. Chem. Eur. J.
2011, 17, 3105–3110.
Acknowledgments
42. Corzana, F.; Fernández-Tejada, A.; Busto, J. H.; Joshi, G.; Davis, A. P.; Jiménez-
Barbero, J.; Avenoza, A.; Peregrina, J. M. ChemBioChem 2011, 12, 110–117.
43. Bundle, D. R.; Rich, J. R.; Jacques, S.; Yu, H. N.; Nitz, M.; Ling, C.-C. Angew. Chem.,
Int. Ed. 2005, 44, 7725–7729.
44. Aguilera, B.; Jiménez-Barbero, J.; Fernández-Mayoralas, A. Carbohydr. Res. 1998,
308, 19–27.
We thank the Ministerio de Ciencia e Innovación and FEDER
(project CTQ2009-13814/BQU), Ministerio de Economia y Compet-
itividad (project CTQ2012-36365/FEDER), Gobierno de La Rioja
(project COLABORA 2010/05) and the Universidad de La Rioja (pro-
ject EGI11/62, pre-doctoral fellowship of VR.)
45. Weimar, T.; Kreis, U. C.; Andrews, J. S.; Pinto, B. M. Carbohydr. Res. 1999, 315,
222–233.
46. Montero, E.; Vallmitjana, M.; Pérez-Pons, J. A.; Querol, E.; Jiménez-Barbero, J.;
Cañada, F. J. FEBS Lett. 1998, 421, 243–248.
Supplementary data
47. Nilsson, U.; Johansson, R.; Magnusson, G. Chem. Eur. J. 1996, 2, 295–302.
48. Leeuwenburgh, M. A.; van der Marel, G. A.; Overkleeft, H. S. Curr. Opin. Chem.
Biol. 2003, 7, 757–765.
49. Aljarilla, A.; López, J. C.; Plumet, J. Eur. J. Org. Chem. 2010, 6123–6143.
50. López, J. C.; Plumet, J. Eur. J. Org. Chem. 2011, 1803–1825.
51. Lin, Y. A.; Chalker, J. M.; Davis, B. G. J. Am. Chem. Soc. 2010, 132, 16805–16811.
52. Nolen, E. G.; Kurish, A. J.; Potter, J. M.; Donahue, L. A.; Orlando, M. D. Org. Lett.
2005, 7, 3383–3386.
Supplementary data (spectroscopic characterization, including
copies of the 1H NMR and 13C NMR spectra of all compounds, as
well as computational details) associated with this article can be
found, in the online version, at http://dx.doi.org/10.1016/
j.carres.2013.02.013.
53. McGarvey, G. J.; Benedum, T. E.; Schmidtmann, F. W. Org. Lett. 2002, 4, 3591–
3594.
54. Keding, S. J.; Danishefsky, S. J. Proc. Natl. Acad. Sci. 2004, 101, 11937–11942.
55. Ragupathi, G.; Koide, F.; Livingston, P. O.; Cho, Y. S.; Endo, A.; Wan, Q.;
Spassova, M. K.; Keding, S. J.; Allen, J.; Ouerfelli, O.; Wilson, R. M.; Danishefsky,
S. J. J. Am. Chem. Soc. 2006, 128, 2715–2725.
56. Biswas, K.; Coltart, D. M.; Danishefsky, S. J. Tetrahedron Lett. 2002, 43, 6107–
6110.
57. Kaiser, J.; Kinderman, S. S.; van Esseveldt, B. C. J.; van Delft, F. L.; Schoemaker,
H. E.; Blaauw, R. H.; Rutjes, F. P. J. T. Org. Biomol. Chem. 2005, 3, 3435–3467.
58. Chatterjee, A. K.; Choi, T.-L.; Sanders, D. P.; Grubbs, R. H. J. Am. Chem. Soc. 2003,
125, 11360–11370.
References
1. Varki, A. Glycobiology 1993, 3, 97–130.
2. Spiro, R. G. Glycobiology 2002, 12, 43R–56R.
3. Buskas, T.; Ingale, S.; Boons, G. J. Glycobiology 2006, 16, 113R–136R.
4. Dwek, R. A. Chem. Rev. 1996, 96, 683–720.
5. Hang, H. C.; Bertozzi, C. R. Bioorg. Med. Chem. 2005, 13, 5021–5034.
6. Hanisch, F. G. Biol. Chem. 2001, 382, 143–149.
7. Zachara, N. E.; Hart, G. W. Chem. Rev. 2002, 102, 431–438.
8. Brocke, C.; Kunz, H. Bioorg. Med. Chem. 2002, 10, 3085–3112.
9. Bourne, Y.; Cambillau, C. Proteins 1990, 8, 365–376.
10. Harrop, S. J.; Helliwell, J. R.; Wan, T. C. M.; Kalb, A. J.; Tong, L.; Yariv, J. Acta Cryst.
1996, D52, 143–155.
11. Jeyaprakash, A. A.; Jayashree, G.; Mahanta, S. K.; Swaminathan, C. P.; Sekar, K.;
Surolia, A.; Vijayan, M. J. Mol. Biol. 2005, 347, 181–188.
12. Fan, G.-T.; Pan, Y.-S.; Lu, K.-C.; Cheng, Y.-P.; Lin, W.-C.; Lin, S.; Lin, C.-H.; Wong,
C.-H.; Fanga, J.-M.; Lin, C.-C. Tetrahedron 2005, 61, 1855–1862.
13. Gamblin, D. P.; Scanlan, E. M.; Davis, B. G. Chem. Rev. 2009, 109, 131–163.
14. Zhu, X.; Pachamuth, K.; Schmidt, R. R. J. Org. Chem. 2003, 68, 5641–5651.
15. Zhu, X.; Haag, T.; Schmidt, R. R. Org. Biomol. Chem. 2004, 2, 31–33.
16. Wilson, J. C.; Kiefel, M. J.; Angus, D. I.; von Itzstein, M. Org. Lett. 1999, 1, 443–
446.
17. Witczak, Z. J.; Chhabra, R.; Chen, H.; Xie, X.-Q. Carbohydr. Res. 1997, 301, 167–
175.
18. Driguez, H. ChemBioChem 2001, 2, 311–318.
19. Hasegawa, A.; Morita, M.; Kojima, Y.; Ishida, H.; Kiso, M. Carbohydr. Res. 1991,
214, 43–53.
20. Stepper, J.; Shastri, S.; Loo, T. S.; Preston, J. C.; Novak, P.; Man, P.; Moore, C. H.;
Havlicek, V.; Patchett, M. L.; Norris, G. E. FEBS Lett. 2011, 585, 645–650.
21. Venugopal, H.; Edwards, P. J. B.; Schwalbe, M.; Claridge, J. K.; Libich, D. S.;
Stepper, J.; Loo, T.; Patchett, M. L.; Norris, G. E.; Pascal, S. M. Biochemistry 2011,
50, 2748–2755.
59. Lu, J.; Fraser-Reid, B.; Gowda, C. Org. Lett. 2005, 7, 3841–3843.
60. Munasinghe, V. R. N.; Corrie, J. E. T.; Kelly, G.; Martin, S. R. Bioconjugate Chem.
2007, 18, 231–237.
61. Starting material 3 was obtained in a 35% yield from allyl
following the procedure described in Section 4.
62. Starting material 4a was obtained in a 92% yield from Boc-
following the procedure described in Section 4.
63. Starting material 4b was obtained in an 91% yield from Boc-
D
-glucopyranoside,
-Ser(allyl)-OH,
-Cys(allyl)-OH,
L
L
following the procedure described in Section 4.
64. Starting material 8
a,b was obtained in a 95% yield from 2,3,4,6-tetra-O-acetyl-
1-thio-b- -glucopyranoside, following the procedure described in Section 4.
D
65. Peters, T.; Pinto, B. M. Curr. Opin. Struct. Biol. 1996, 6, 710–720.
66. Pearlman, D. A. J. Biomol. NMR 1994, 4, 1–16.
67. Lemieux, R. U.; Pavia, A. A.; Martin, J. C.; Watanabe, K. A. Can. J. Chem. 1969, 47,
4427–4439.
68. Lemieux, R. U.; Koto, S. Tetrahedron 1974, 30, 1933–1944.
69. Thatcher, G. R. J. The Anomeric Effect and Associated Stereoelectronic Effects;
American Chemical Society: Washington, DC, 1993.
70. Mazeau, K.; Tvaroska, I. Carbohydr. Res. 1992, 225, 27–41.
71. Wang, J.; Wolf, R. M.; Caldwell, J. W.; Kollman, P. A.; Case, D. Comput. Chem.
2004, 25, 1157–1174.
72. Hsieh, Y. S. Y.; Wilkinson, B. L.; O’Connell, M. R.; Mackay, J. P.; Matthews, J. M.;
Payne, R. J. Org. Lett. 2012, 14, 1910–1913.
22. Oman, T. J.; Boettcher, J. M.; Wang, H. A.; Okalibe, X. N.; van der Donk, W. A.
Nat. Chem. Biol. 2011, 7, 78–80.
73. (a) Pearlman, D. A.; Case, D. A.; Caldwell, J. W.; Ross, W. R.; Cheatham, T. E., III;
DeBolt, S.; Ferguson, D.; Seibel, G.; Kollman, P. A. Comput. Phys. Commun. 1995,
91, 1–41; (b) Case, D. A.; Pearlman, D. A.; Caldwell, J. W.; Cheatham, T. E., III;
Ross, W. S.; Simmerling, C. L.; Darden, T. A.; Merz, K. M.; Stanton, R. V.; Cheng,
A. L.; Vincent, J. J.; Crowley, M.; Tsui, V.; Radmer, R. J.; Duan, Y.; Pitera, J.;
Massova, I.; Seibel, G. L.; Singh, U. C.; Weiner, P. K.; Kollman, P. A. AMBER 6;
University of California: San Francisco, 1999.
23. Hsieh, Y. S. Y.; Wilkinson, B. L.; O’Connell, M. R.; Mackay, J. P.; Matthews, J. M.;
Payne, R. J. Org. Lett. 2012, 14, 1910–1913.
24. Marcaurelle, L. A.; Bertozzi, C. R. Chem. Eur. J. 1999, 5, 1384–1390.
25. Pachamuthu, K.; Schmidt, R. R. Chem. Rev. 2006, 106, 160–187.
26. Zhu, X.; Schmidt, R. R. Chem. Eur. J. 2004, 10, 875–887.
27. Schips, C.; Ziegler, T. J. Carbohydr. Chem. 2005, 24, 773–788.
28. Davis, B. G. Chem. Rev. 2002, 102, 579–601.