S-Michael additions to chiral dehydroalanines as an entry to glycosylated cysteines and a sulfa-tn antigen mimic

Article abstract of DOI:10.1021/ja411522f

Stereoselective sulfa-Michael addition of appropriately protected thiocarbohydrates to chiral dehydroalanines has been developed as a key step in the synthesis of biologically important cysteine derivatives, such as S-(β-d-glucopyranosyl)-d-cysteine, which has not been synthesized to date, and S-(2-acetamido-2-deoxy-α-d-galactopyranosyl)-l-cysteine, which could be considered as a mimic of Tn antigen. The corresponding diamide derivative was also synthesized and analyzed from a conformational viewpoint, and its bound state with a lectin was studied.

Full text of DOI:10.1021/ja411522f

Article
pubs.acs.org/JACS
S‑Michael Additions to Chiral Dehydroalanines as an Entry to
Glycosylated Cysteines and a Sulfa-Tn Antigen Mimic
Carlos Aydillo, Ismael Companon, Alberto Avenoza,* Jesus H. Busto, Francisco Corzana,
́
́
̃
Jesus M. Peregrina,* and María M. Zurbano
́
́
Departamento de Química, Universidad de La Rioja, Centro de Investigacion en Síntesis Química, E-26006 Logrono, Spain
̃
S
* Supporting Information
ABSTRACT: Stereoselective sulfa-Michael addition of appropriately
protected thiocarbohydrates to chiral dehydroalanines has been developed
as a key step in the synthesis of biologically important cysteine derivatives,
such as S-(β-D-glucopyranosyl)-D-cysteine, which has not been synthesized
to date, and S-(2-acetamido-2-deoxy-α-D-galactopyranosyl)-L-cysteine, which
could be considered as a mimic of Tn antigen. The corresponding diamide
derivative was also synthesized and analyzed from a conformational
viewpoint, and its bound state with a lectin was studied.
modifications by the conjugate addition of thiols.13 In this field,
we have reported a biomimetic synthetic approach to
lanthionine (Lan) by using an asymmetric sulfa-Michael
addition of appropriately protected L- and D-Cys to a new
chiral Dha derivative.14
INTRODUCTION
■
Glycoproteins are ubiquitous components of cellular surfaces
where their oligosaccharide moieties are involved in an
extensive variety of molecular recognition events.1 To study
these binding interactions, the synthesis of structurally defined
glycopeptides is of significant current interest.2 As a result,
chemical access to covalently modified proteins is a quickly
expanding area in chemical biology.3 In naturally occurring
glycopeptides, the carbohydrate moiety and the peptide
backbone are most commonly joined by an O-glycosidic
linkage. However, these natural products present, in general, a
low chemical stability, and, consequently, their use as
therapeutics is hampered in many cases. Because of this, recent
research has focused on the efficient synthesis of glycopeptide
mimics with a stable glycosidic linkage as a fundamental tool for
biological research.4 In particular, S-linked glycopeptides may
avoid this disadvantage due to their enhanced chemical stability
and enzymatic resistance.5
RESULTS AND DISCUSSION
■
Herein, we report a new stereocontrolled entry to S-glycosyl
cysteines, as building blocks for solid phase synthesis of
glycopeptides, exploiting the high nucleophilicity of the
sulfhydryl group of thiosugars in the diastereoselective Michael
addition to chiral Dha 1 (see Table 1). The synthesis was
previously communicated15 and the absolute configuration of
their stereogenic centers further established and corroborated
by X-ray analysis.14 Additionally, derivatives 1′ and 1″ and the
enantiomers ent-1 and ent-1′ have been synthesized in a gram-
scale (see the Supporting Information) and tested in the
Michael reactions (see Table 1). Adequate monocrystals of
compound 1′ were obtained and examined by X-ray diffraction
analysis.
Cysteine S-glycosylation6 has recently emerged as a new
post-translational modification found in glycopeptide bacter-
iocins glyocin F7 and sublancin,8 which contain a β-N-
acetylglucosamine (β-GlcNAc) and a β-glucose (β-Glc),
respectively. This glycosylation is essential for the antimicrobial
activity.
Because of the emerging relevance of S-glycopeptides, a
variety of glycosylation methods6,2f have been applied. These
methods generally use protected carbohydrates as electrophiles
and Cys derivatives as nucleophiles. Alternatively, nucleophilic
thiocarbohydrate derivatives9 have been used in the 1,4-
conjugate addition to dehydroalanine (Dha) and dehydrobu-
tyrine (Dhb) containing peptides with poor results in
diastereoselectivity.10 Particularly, Dha is an unsaturated
amino acid residue of both biological and synthetic interest,11
found in the initial stages of the biosynthesis of lanthionine-
containing antibiotic peptides (lantibiotics).12 Dha is also a
useful chemical precursor to a range of post-translational
The asymmetric Michael reaction of Dha derivatives 1, 1′,
and 1″ with different anomeric thioglycosylates in the presence
of 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) as a base and
tetrahydrofuran (THF) as a solvent (Figure 1 and Table 1) was
assayed. Under these conditions, mutarotation was not
observed.16
This methodology was generalized to both α-thiosugars and
β-thiosugars. The reaction with mono- and disaccharides, as
well as with N-substituted 2-amino-2-deoxy-D-thiosugars, was
also examined. Therefore, seven different thiosugars were used,
tetra-O-acetyl-1-thio-β-D-glucose (2a), tetra-O-acetyl-1-thio-β-
D-galactose (2b), tri-O-acetyl-2-benzyloxycarbonylamino-2-
deoxy-1-thio-β-D-glucose (2c), tetra-O-acetyl-β-D-galactosyl-
Received: November 14, 2013
Published: December 27, 2013
© 2013 American Chemical Society
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Table 1. Asymmetric Michael Reactions of Chiral Dehydroalanines 1, 1′, or 1″ and Thiosugars 2a−g
a
b
entry
dehydroalanine
R
thiosugar
R1
R2
R3
yield (%)
Michael adducts
diastereoselectivity (dr)
1
2
1
Me
Me
Me
Me
Me
Me
Me
tBu
tBu
Bn
2a
2b
2c
2d
2e
2f
H
OAc
H
OAc
84
82
56
78
79
67
80
86
74
71
3a/4a
3b/4b
3c/4c
3d/4d
3e/4e
3f/4f
>5:95
>5:95
>5:95
>5:95
23/77
>5:95
>5:95
>5:95
>5:95
15:85
1
OAc
H
OAc
3
1
OAc
NHCbz
OAc
4
1
H
β-D-(OAc)4Gal
5
1
H
OAc
H
OAc
6
1
OAc
H
NHAc
NHAc
NHAc
NHAc
NHAc
7
1
2g
2f
OAc
H
3g/4g
3′f/4′f
3′g/4′g
3″g/4″g
8
1′
1′
1″
OAc
H
9
2g
2g
OAc
OAc
10
H
b
a
Yield of products after column chromatography. See ref 18.
corresponding protected S-glycosyl cysteine β-anomers (4a−d)
or S-glycosyl cysteine α-anomers (4f,g and 4′f,g) in good yields
(56−86%) (Table 1).
In the case of product 4a, representative of β-glycosidic
derivatives, the absolute configuration of the new stereocenter
was determined by transformation of this compound into the
corresponding hydrochloride of the thioglycoamino acid S-β-D-
Glc-D-Cys 5a. This transformation was carried out in an
aqueous 6 N HCl solution at 60 °C (Scheme 1). For this
compound, we obtained a specific optical rotation value of +43.
The reported value19b,c for S-β-D-Glc-L-Cys is −40, indicating
Scheme 1. Synthesis of S-Glycosyl Amino Acids 5a, 5f, and
5g
Figure 1. Thiosugars 2a−g used as nucleophiles in the Michael
additions to Dha derivatives.
(1→4)-tri-O-acetyl-1-thio-β-D-glucose or hepta-O-acetyl-1-thio-
β-D-lactose (2d), tetra-O-acetyl-1-thio-α-D-glucose (2e), tri-O-
acetyl-2-acetamido-2-deoxy-1-thio-α-D-galactose (2f), and tri-
O-acetyl-2-acetamido-2-deoxy-1-thio-α-D-glucose (2g) (Figure
1). These protected thiosugars were prepared according to
literature procedures.17
It is important to highlight that, in general, most of the tested
thiosugars gave good yields and the diastereoselectivities
obtained were excellent in all cases with a dr > 95:5,18 except
for the α-anomers 2e19a (entry 5 in Table 1) and 2g (entry 10
in Table 1) for which 23/77 and 15/85 lower diastereomeric
ratios were obtained, respectively. For the rest of the
compounds, purification of the crude reaction mixture by
column chromatography gave exclusively one product, the
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that the new stereogenic center created in the Michael reaction
shows an S-configuration (Scheme 1).
To access the L-Cys-containing glycopeptides, we assayed the
asymmetric Michael reaction of thiosugars 2a and 2f with the
acceptors ent-1 and ent-1′, enantiomers of chiral Dha
derivatives 1 and 1′, respectively, using the aforementioned
conditions (Scheme 3). The best results provided 7a and 7′f
with complete diastereoselectivity and in 79% and 76% yield,
respectively. After chromatographic purification, compounds 7a
and 7′f were hydrolyzed to the corresponding hydrochlorides
S-β-D-Glc-L-Cys 8a19c and S-α-D-GalNAc-L-Cys 8f, respectively
(Scheme 3).
Glycoamino acids 8a and 8f were transformed into the
corresponding building blocks 9a and 9f, which are ready to use
in solid-phase synthesis. First, the free amine in compound 8a
was protected with Fmoc employing Fmoc−OSu in a basic
medium. This compound then was treated with acetic
anhydride (Ac2O) in the presence of phosphoric acid to
acetylate the hydroxyl groups of the carbohydrate moiety,
giving compound 9a.8c,20b Compound 8f was converted into
the building block 9f following four steps: (a) Fmoc protection
of the amino group with Fmoc−OSu, (b) transformation of the
acid group into the corresponding allyl ester, (c) acetylation20a
of the carbohydrate hydroxyls with Ac2O and pyridine (Py),
and (d) conversion of the allyl ester group into the
corresponding carboxylic acid by treatment with Pd(PPh3)4
in morpholine (Scheme 4).
Similarly, the representative α-glycosyl compounds 4f and 4g
were transformed into the hydrochlorides S-α-D-GalNAc-D-Cys
5f and S-α-D-GlcNAc-D-Cys 5g, respectively, using the
conditions described above. Unfortunately, these S-glycoamino
acids were formed together with the corresponding hydrolysis
products of the acetamido group, precluding the purification of
the target compounds. This issue was overcome by using softer
acid hydrolysis conditions and the tert-butyl derivatives 4′f and
4′g as starting materials, diminishing considerably the
hydrolysis of acetamido group. Surprisingly, compounds 5a,
5f, and 5g19c have not been synthesized to date (Scheme 1). In
fact, it is important to highlight that, to the best of our
knowledge, this is the first time that this type of
glycoconjugates, in which both the carbohydrate and the
cysteine belong to D-series, is described. Taking into account
that neither S-α-D-GalNAc-L-Cys nor S-α-D-GlcNAc-L-Cys are
described, and to determine the absolute configuration of the
Michael adducts, compounds 5f and 5g were converted into
protected derivatives 6f and 6g, respectively (Scheme 2). These
Scheme 2. Determination of the Absolute Configuration of
Michael Adducts
Scheme 4. Synthesis of Building Blocks 9a and 9f
conversions involved three steps: (a) protection of the amino
group as Cbz (6g) or Fmoc (6f) carbamate derivatives, (b) acid
group conversion to formation of allyl (6f) or benzyl (6g) ester
derivatives, and (c) acetylation of hydroxyl groups (exper-
imental details in the Supporting Information). The compar-
ison of the spectroscopic data of these compounds with those
described5b,c,20a for the well-kown derivatives Fmoc-L-Cys(α-D-
(OAc)3GalNAc)-OAllyl and Cbz-L-Cys(α-D-(OAc)3GlcNAc)-
OBn confirmed that the new stereocenter created in the
Michael reaction shows also an S-configuration.
Starting from building block 9f, we synthesized the
corresponding diamide 10f, which can be regarded as the
shortest S-glycopeptide analogue of the O-glycopeptide (Figure
2). We previously synthesized and analyzed the conformational
properties in water of Ac-L-Ser(α-D-GalNAc)-NHMe and Ac-L-
Thr(α-D-GalNAc)-NHMe as model glycopeptides.21 Notably,
Scheme 3. Asymmetric Michael Reactions of Chiral Dehydroalanines and Thiosugars 2a and 2f
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Figure 2. Tn antigen derivatives and compound 10f conformationally
studied in water.
we found that water molecules between the carbohydrate and
the underlying amino acid contribute to stabilize defined
conformations, which may be relevant for important biological
features. Moreover, Ac-L-Thr/Ser(α-D-GalNAc)-NHMe are
particularly important because they include the substructure
of Tn antigen, which can be found in cancer cells (Figure 2)
and, consequently, attract attention in developing Tn-based
vaccines and other therapeutic approaches based on Tn
expression.22
Figure 3. 2D-NOESY for Ac-L-Cys(α-D-GalNAc)-NHMe (10f) in
H2O/D2O (9:1) at 400 MHz (pH 5.5, 298 K).
To understand the bioactivity of glycomimic motifs, it is
crucial to determine their conformational preferences and
dynamics. The presentation mode of the carbohydrate moiety
is important for the recognition by the corresponding
molecular entities. On this basis, we compared the conforma-
tional features in water of Ac-L-Ser(α-D-GalNAc)-NHMe and
Ac-L-Thr(α-D-GalNAc)-NHMe with the sulfa-mimic Ac-L-
Cys(α-D-GalNAc)-NHMe (10f) to understand the differences
induced by the sulfur atom (Figure 2).
Target compound 10f was synthesized stating from building
block 9f (Scheme 5). Initially, the acid group was transformed
Scheme 5. Synthesis of Ac-L-Cys(α-D-GalNAc)-NHMe 10f
Figure 4. Distributions for the peptide backbone, glycosidic linkage,
and side-chain obtained from the MD-tar simulations for Ac-L-Ser(α-
D-GalNAc)-NHMe, Ac-L-Thr(α-D-GalNAc)-NHMe, and Ac-L-Cys(α-
D-GalNAc)-NHMe (10f).
into the corresponding methyl amide using O-(benzotriazol-1-
yl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate (TBTU)
as a coupling agent. Deprotection of Fmoc with piperidine,
followed by acetylation of the amino group with Ac2O in
pyridine, gave the corresponding diamide derivative, which was
treated with sodium metoxide (MeONa) to deacetylate the
hydroxyl groups of the carbohydrate moiety (Scheme 5).
We performed then the conformational analysis in aqueous
solution on compound 10f through NMR experiments and
molecular dynamics (MD) simulations. The relevant proton−
proton distances obtained from 2D-NOESY experiments
(Figure 3) were then used as restraints in MD simulations
with time-averaged restraints (MD-tar)23 to obtain a distribu-
tion of low-energy conformers able to quantitatively reproduce
the NMR data (see the Experimental Section).
for the glycosidic linkage and for the side-chain. A comparison
to the natural Tn antigens is also shown.21
For compound 10f, the peptide backbone exhibited mainly
folded (helix-like) conformations, corroborated by the NOE
intensities observed for the crosspeaks NH2−NH1, NH1−Hα,
and NH2−Hα (Figure 3). A small population of extended
conformations (about 10% of the total trajectory time) was also
observed. Conversely, and as described previously by us, both
natural Tn antigens prefer extended conformations for the
underlying amino acid. Concerning the glycosidic linkage, the
ϕs (O5−C1−S−Cβ) dihedral angle is quite rigid, in accordance
with the exoanomeric effect. However, significant differences
Figure 4 shows the conformer distribution obtained from
these simulations for the backbone of compound 10f, as well as
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are observed in the ψs (C1−S−Cβ−Cα) torsional angle when
compared to the natural derivatives (Figure 4).
For Tn serine derivative, this angle is close to 180°, while the
threonine derivative shows an “eclipsed” conformation (ψs ≈
120°). However, for the S-glycosyl derivative 10f, a major
conformation with ψs around 60° was observed. It is important
to note that this conformer lies at one of the local minima
calculated for methyl 4-thio-α-maltoside.24 Consequently, using
different underlying amino acids (serine, threonine, or
cysteine), it is possible to modulate the values of ψs, ranging
from 60° to 180°. With respect to the side chain, defined by the
χ1 (X−Cβ−Cα−N) torsion angle, the simulations indicated
that the side chain of compound 10f is rather rigid, exhibiting
only the gauche(+) conformer, similarly to Ac-L-Thr(α-D-
GalNAc)-NHMe. Through all of these observations, it can be
concluded that the conformational behavior of compound 10f
is closely related to Tn antigen containing threonine as the
underlying amino acid. In fact, as can be observed in Figure 5,
Figure 6. (a) Comparison between the inter-residue water pockets
deduced from the MD-tar simulations of Ac-L-Thr(α-D-GalNAc)-
NHMe and Ac-L-Cys(α-D-GalNAc)-NHMe (10f). (b and c) Radial
pair distribution function (RDF) and two-dimensional RDF for N2
and N3 found in the 16 ns MDH2O-tar simulations of 10f.
Figure 5. Representative conformations for natural Tn antigens, along
with calculated ensemble obtained for compound 10f from MD-tar
simulations.
The binding affinities of the sulfa-mimic 10f and the natural
Tn antigen serine derivative to Soybean agglutinin (SBA) lectin
were studied by an enzyme-linked lectin assay (ELLA). We
selected SBA as the binding target because it is a well-known,
readily available, and stable lectin with high affinity toward
GalNAc, Tn antigen, and other mimics.27h,31 The X-ray
structure of this protein bound to galactose has been
described.32 To compare the relative affinities of 10f and Ac-
L-Ser(α-D-GalNAc)-NHMe to SBA lectin, we used a com-
petitive ELLA (Table 2 and Supporting Information). There-
while in serine derivative the carbohydrate moiety is almost
parallel to the peptide backbone, in both Tn bearing threonine
and 10f compounds, the GalNAc unit adopts a perpendicular
disposition.
The 3D-disposition of compound 10f allows the formation of
a water pocket between the NHAc group of GalNAc (N3) and
the NH of the backbone (N2). This is similar to that previously
reported21b for compound Ac-L-Thr(α-D-GalNAc)-NHMe, and
it is characterized by a density of 3.7 times the bulk density
(Figure 6). This spatial disposition of 10f is corroborated by a
medium NOE peak between the NH2 proton of the acetyl
group of the amino acid and the H1 proton of the carbohydrate
(NH2−H1, Figures 3 and 6). The presence of these structured
water molecules may contribute to rigidify the molecule.
Recent developments in carbohydrate-based cancer vaccines
have addressed the concept of introducing analogues into
tumor-associated carbohydrate antigens to improve their
immunogenicity.25 Different strategies, including incorporation
of fluorine atoms,26 synthesis of C-glycoside27 and S-glycoside
derivatives,28 and the use of homoserine and β3-homothreonine
conjugates,29 have been reported to prepare mucin-like
glycopeptides antigen analogues. Recently, we contributed to
this field by synthesizing a conformationally restricted Tn
antigen mimic.27h However, S-glycoside analogues of Tn
antigen have been scarcely used to develop anticancer
vaccines.28b,30 Because the O-glycosidic linkage of the Tn
antigen can be chemically or enzymatically cleaved, the higher
stability of the sulfa-Tn vaccine has been attributed to be the
source of its superior activity at lower doses when compared to
the natural Tn.
Table 2. Inhibition Ratio (%) of Compounds Ac-L-Ser(α-D-
GalNAc)-NHMe and Ac-L-Cys(α-D-GalNAc)-NHMe (10f) at
Different Concentrations Employing 100 nmol of Ala-Pro-
Asp-Thr(α-D-GalNAc)-Arg Bound to SBA Lectin
nmol
Ac-L-Ser(α-D-GalNAc)-NHMe
10f
50
100
150
72.2
91.5
95.3
54.4
83.9
91.3
fore, Ala-Pro-Asp-Thr(α-D-GalNAc)-Arg glycopeptide (100
nmol per well) was covalently linked to a 96-well plate, and a
fixed amount of SBA lectin was then added. The competitive
assay was carried out at three different concentrations of 10f
and the natural Tn serine derivative, as summarized in Table 2.
The results obtained from these assays are represented as the
inhibition ratio (%). This ratio is defined as the optical density
(OD) observed at 450 nm in the presence of each Tn derivative
(10f or the natural Tn antigen) divided by the OD in the
absence of the corresponding Tn derivative. From these
experiments, it can be inferred that both compounds present
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similar inhibition ratios, although slightly higher values were
observed for the natural Tn serine-derivative antigen. Of note,
these results agree with those of a recently reported
comparative binding study of S- and O-glycoconjugates to
different lectins, which showed that both types of derivatives
exhibited similar affinities.33
To explain these experimental results, a putative 3D model of
the complex of 10f with SBA was established using MD
simulations (Figure 7a). As expected, these calculations indicate
been studied through NMR experiments and MD simulations.
The conformational properties and the first hydration shell of
this compound were analyzed and compared to those of the
natural O-glycosyl analogues. The 3D spatial disposition of the
sulfa-Tn mimic is closely related to the natural Tn carrying
threonine as the underlying residue. In fact, both molecules
expose the GalNAc moiety perpendicularly to the amino acid
backbone and accommodate a similar water pocket between the
peptide and the sugar moieties. Finally, the binding affinity of
the sulfa-mimic Tn antigen toward Soybean agglutinin (SBA)
lectin was studied through enzyme-linked lectin assays and
compared to that showed by the natural Tn serine-derivative,
demonstrating that both molecules have similar preferences for
this lectin. This methodology is currently being expanded in
our lab to obtain longer glycopeptides of biological relevance.
EXPERIMENTAL SECTION
■
Reagents and General Procedures. Commercial reagents were
used without further purification. Solvents were dried and redistilled
prior to use in the usual way. All reactions were performed in oven-
dried glassware with magnetic stirring under an inert atmosphere
unless noted otherwise. Analytical thin layer chromatography (TLC)
was performed on glass plates precoated with a 0.25 mm thickness of
silica gel. The TLC plates were visualized with UV light and by
staining with Hanessian solution (ceric sulfate and ammonium
molybdate in aqueous sulfuric acid) or sulfuric acid−ethanol solution.
Column chromatography was performed on silicagel (230−400 mesh).
Optical rotations (OR) were measured with a polarimeter at a
concentration (c) expressed in g/100 mL. H and 13C NMR spectra
1
were measured with a 400 MHz spectrometer with TMS as the
internal standard. Multiplicities are quoted as singlet (s), broad singlet
(br s), doublet (d), doublet of doublets (dd), triplet (t), or multiplet
(m). Spectra were assigned using COSY and HSQC. All NMR
chemical shifts (δ) were recorded in ppm, and coupling constants (J)
were reported in Hz. The results of these experiments were processed
with MestreNova software. Melting points were determined on a
Figure 7. (a) Snapshot taken from the 25 ns MD simulations of
compound 1of bound to SBA lectin. (b) Ligand interaction diagram
for SBA:10f complex obtained using Maestro 9.3.5 software. (c)
Intensity diagram for protons of compound 10f obtained from the
saturation-transfer difference (STD) NMR experiments of a sample
containing 10f and SBA lectin.
Buchi melting-point apparatus and are uncorrected. Optical rotations
̈
were measured on a polarimeter from solutions in 1.0 dm cells of
capacity 1.0 or 0.3 mL. High-resolution electrospray mass (ESI)
spectra were recorded on a microTOF spectrometer; accurate mass
measurements were achieved by using sodium formate as an external
reference.
that compound 10f interacts with the lectin mainly through the
carbohydrate moiety. Hydrogen bonds between the sugar and
the lectin are observed (Figure 7b). For instance, OH-3 and
OH-4 interact with Asp88, OH-6 participates in a hydrogen
bond with Asp215, and OH-3 interacts with Asn130. These
interactions are similar to those found in the crystal structure of
SBA bound to galactose.32 This model was experimentally
corroborated by studying the interactions of compound 10f
with SBA lectin through saturation transfer difference (STD)-
NMR experiments34 (Experimental Section). Unequivocal clear
STD signals were detected for the carbohydrate moiety (with
STD effects >85%) of compound 10f (Figure 7c), which prove
the predicted specific interactions with SBA lectin. Therefore,
these experiments corroborated the binding epitope previously
proposed by the MD simulations.
NMR Experiments. NMR experiments were performed on a 400
spectrometer at 298 K. Magnitude-mode ge-2D COSY spectra were
acquired with gradients by using the cosygpqf pulse program with a
pulse width of 90°. Phase-sensitive ge-2D HSQC spectra were
acquired by using z-filter and selection before t1 removing the
decoupling during acquisition by use of the invigpndph pulse program
with CNST2 (JHC) = 145. Phase-sensitive ge-2D NOESY experiments
were performed. NOE intensities were normalized with respect to the
diagonal peak at zero mixing time. The saturation-transfer difference
(STD) NMR experiments34 (Supporting Information) were achieved
using standard conditions on a 55 μM/2.75 mM lectin/ligand sample
(molar ratio 1:50) in D2O at 298 K at 400 MHz. The saturation time
was 2s, at an on-resonance frequency of δ 0.370 ppm. No spin-lock
filter and no water suppression schemes were employed.
X-ray Diffraction Analysis. ORTEP diagrams are presented in the
Supporting Information. The SHELXL97 program35 was used for the
refinement of crystal structures, and hydrogen atoms were fitted at
theoretical positions. CIF files in the Supporting Information contain
the supplementary crystallographic data for this Article.
Unrestrained Molecular Dynamics Simulations. All of the
molecular dynamics simulations were carried out on the Finis-Terrae
CONCLUSION
■
We have described a novel and straightforward methodology
for the stereoselective synthesis of different S-glycosyl cysteine
derivatives. This strategy is based on the double asymmetric
sulfa-Michael addition reaction of adequately protected
thiosugars to chiral dehydroalanines. These compounds were
easily transformed into the corresponding glycosyl amino acid
building blocks, which are ready to use in phase solid synthesis.
The conformational analysis of a sulfa-Tn antigen mimic has
cluster belonging to the Centro de Supercomputacion de Galicia
́
(CESGA), Spain. The starting geometries for the complexes were
generated from the available data deposited in the Protein Data Bank
(pdb code 1SBF) and modified accordingly. Each model complex was
immersed in a 10 Å-sided cube with pre-equilibrated TIP3P water
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molecules. To equilibrate the system, we followed a protocol
consisting of 10 steps. First, only the water molecules are minimized,
and then heated to 300 K. The water box, together with Na+ ions, was
then minimized, followed by a short MD simulation. At this point, the
system was minimized in the four following steps with positional
restraints imposed on the solute, decreasing the force constant step by
step from 20 to 5 kcal mol−1. Finally, a nonrestraint minimization was
performed. The production dynamics simulations were accomplished
at a constant temperature of 300 K (by applying the Berendsen
coupling algorithm for the temperature scaling) and constant pressure
(1 bar). The Particle Mesh Ewald Method, to introduce long-range
electrostatic effects, and periodic boundary conditions were also used.
The SHAKE algorithm for hydrogen atoms, which allows using a 2 fs
time step, was also employed. Finally, a 9 Å cutoff was applied for the
Lennard-Jones interactions. MD simulations were performed with the
sander module of AMBER 11.0 (parm99 force field),36 which was
implemented with GAFF parameters37 to accurately simulate
compound 10f. A simulation length of 25 ns and the trajectory
coordinates were saved each 0.5 ps. The data processing of the
generated trajectories was done with the ptraj module of Amber 11.0
and with the Carnal module of Amber 6.0.
MD Simulations with Time-Averaged Restraints (MD-tar).
MD-tar simulations were performed with AMBER 11 (parm99 force
field), which was implemented with GAFF parameters. Distances
derived from NOE interactions were included as time-averaged
distance restraints. A ⟨r−6⟩−1/6 average was used for the distances. Final
trajectories were run using an exponential decay constant of 2000 ps
and a simulation length of 16 ns in explicit TIP3P water molecules.
General Procedure for the Sulfa-Michael Additions. The
corresponding α,β-dehydroamino acid (1 equiv) was dissolved in dry
THF (concentration of 10 mg/mL), and the resulting solution was
introduced in a Schlenk tube under an argon atmosphere. This
solution was stirred and cooled to −78 °C. The corresponding
thiocarbohydrate (2a−g, 1.2 equiv) dissolved in THF was introduced
into the Schlenk tube by a syringe, and DBU (1.2 equiv) was then
added slowly by another syringe. After the mixture was stirred at this
temperature for 3 h, a saturated NH4Cl solution (the same volume as
THF) was added. This mixture was stirred vigorously while it was
allowed to warm to room temperature. After that, the reaction crude
was diluted with ethyl ether, and the aqueous phase was extracted with
ethyl acetate. The combined organic phases were washed with brine
and dried with anhydrous Na2SO4. The solvent was filtered and
evaporated, and the residue was purified by silica gel column
chromatography to give the corresponding sulfa-Michael adduct.
Methyl (2S)-2-((4S,5R)-4,5-Dimethyl-4-hydroxy-5-methoxy-2-ox-
ooxazolidin-3-yl)-3-(tetra-O-acetyl-β-D-glucosylthio)propanoate
(4a). Compound 4a was obtained from α,β-dehydroamino acid 1 (550
mg, 2.24 mmol) and tetra-O-acetyl-1-thio-β-D-glucose 2a (980 mg,
2.69 mmol) following the procedure described for sulfa-Michael
additions, using DBU (403 μL, 2.69 mmol) as a base. After column
chromatography (ethyl acetate/hexane, 1:1), compound 4a was
obtained as a white foam (1.147 g, 1.88 mmol, 84%). [α]2D5 = −34.5
(c 1.03, CHCl3). 1H NMR (400 MHz, CDCl3): δ (ppm) 1.40 (s, 3H;
CH3), 1.57 (s, 3H; CH3), 1.99 (s, 3H; Ac), 2.02 (s, 3H; Ac), 2.04 (s,
3H; Ac), 2.05 (s, 3H; Ac), 3.31 (dd, J = 5.3, 14.3, Hz, 1H; CHCH2),
3.43 (s, 3H; OCH3), 3.49−3.62 (m, 1H; CHCH2), 3.76 (s, 3H;
CO2CH3), 3.70−3.78 (m, 1H; H-5), 4.08−4.19 (m, 2H; H-6a; H-6b),
4.19−4.29 (m, 1H; CHCH2), 4.64 (d, J = 10.1 Hz, 1H; H-1), 4.95−
5.09 (m, 2H; H-2; H-4), 5.21 (t, J = 9.3 Hz, 1H; H-3). 13C NMR (100
MHz, CDCl3): δ (ppm) 15.6 (CH3), 19.8 (CH3), 20.4, 20.5, 20.6, 20.7
(Ac), 30.7 (CHCH2), 50.8 (OCH3), 52.9 (CO2CH3), 55.8 (CHCH2),
62.0 (C-6), 68.0 (C-4), 70.0 (C-2), 73.4 (C-3), 76.2 (C-5), 84.9 (C-1),
90.0 (CNCH3OH), 108.4 (CCH3OCH3), 154.5 (NCO2), 169.3,
169.4, 169.5, 170.0, 170.4 (4×Ac; CO2CH3). HRMS ESI+ (m/z):
calcd for C24H35NO15SNa+ [M + Na]+ 632.1620, found 632.1626.
Methyl (2S)-2-((4S,5R)-4,5-Dimethyl-4-hydroxy-5-methoxy-2-ox-
ooxazolidin-3-yl)-3-(tetra-O-acetyl-β-D-galactosylthio)propanoate
(4b). Compound 4b was obtained from α,β-dehydroamino acid 1 (200
mg, 0.82 mmol) and tetra-O-acetyl-1-thio-β-D-galactose 2b (327 mg,
0.82 mmol) following the procedure for sulfa-Michael additions, using
DBU (136 μL, 0.89 mmol) as a base. After column chromatography
(ethyl acetate/hexane, 1:1), 4b was obtained as a white amorphous
solid (406 mg, 0.67 mmol, 82%). [α]2D5 = −24.6 (c 1.00, CHCl3). Mp:
64−66 °C. 1H NMR (400 MHz, CDCl3): δ (ppm) 1.43 (s, 3H; CH3),
1.60 (s, 3H; CH3), 1.99 (s, 3H; Ac), 2.04 (s, 3H; Ac), 2.08 (s, 3H; Ac),
2.19 (s, 3H; Ac), 3.36 (dd, J = 5.0, 14.8 Hz, 1H; CHCH2), 3.44 (s, 3H;
OCH3), 3.53−3.59 (m, 2H; CHCH2; OH), 3.77 (s, 3H; CO2CH3),
3.98 (t, J = 6.31 Hz, 1H; H-5), 4.07−4.17 (m, 2H; H-6a; H-6b), 4.21−
4.25 (m, 1H; CHCH2), 4.59 (d, J = 9.9 Hz, 1H; H-4), 5.03−5.06 (m,
1H; H-2), 5.24 (t, J = 9.9 Hz, 1H; H-3), 5.43 (d, J = 3.2 Hz, 1H; H-1).
13C NMR (100 MHz, CDCl3): δ (ppm) 15.5 (CH3), 19.8 (CH3), 20.6,
20.7, 20.8, 20.9 (Ac), 30.2 (CHCH2), 50.8 (OCH3), 53.0 (CO2CH3),
55.9 (CHCH2), 61.8 (C-6), 67.0 (C-3), 67.2 (C-1), 71.6 (C-2), 75.1
(C-5), 85.0 (C-4), 90.1 (CNCH3OH), 108.5 (CCH3OCH3), 154.5
(NCO2), 169.4, 169.7, 170.0, 170.3, 170.4 (4×Ac; CO2CH3). HRMS
ESI+ (m/z): calcd for C24H35NO15SNa+ [M + Na]+ 632.1620, found
632.1606.
Methyl (2S)-2-((4S,5R)-4,5-Dimethyl-4-hydroxy-5-methoxy-2-ox-
ooxazolidin-3-yl)-3-(tri-O-acetyl-2-benzyloxycarbonylamino-2-
deoxy-β-D-glucosylthio)propanoate (4c). Compound 4c was ob-
tained from α,β-dehydroamino acid 1 (22 mg, 0.09 mmol) and tri-O-
acetyl-2-benzyloxycarbonyl-2-deoxy-1-thio-β-D-glucose 2c (45 mg,
0.10 mmol) following the procedure for sulfa-Michael additions,
using DBU (15 μL, 0.10 mmol) as a base. After column
chromatography (ethyl acetate/hexane, 1:1), 4c was obtained as a
white amorphous solid (36 mg, 0.05 mmol, 56%). [α]2D5 = −27.4 (c
1.00, CHCl3). Mp: 68−70 °C. 1H NMR (400 MHz, CDCl3): δ (ppm)
1.40 (s, 3H; CH3), 1.59 (s, 3H; CH3), 1.93 (s, 3H; Ac), 2.02 (s, 3H;
Ac), 2.06 (s, 3H; Ac), 3.31 (dd, J = 4.5, 14.9 Hz, 1H; CHCH2), 3.44
(s, 3H; OCH3), 3.63−3.72 (m, 3H; CHCH2; H-5; H-4), 3.76 (s, 3H;
CO2CH3), 4.10−4.23 (m, 2H; H-6a; H-6b), 4.28 (d, J = 6.1 Hz, 1H;
CHCH2), 4.84 (d, J = 10.0 Hz, 1H; H-1), 4.95 (d, J = 8.8 Hz, 1H;
NHCbz), 5.01−5.13 (m, 3H; H-2; OCH2Ph), 5.20 (d, J = 9.2 Hz, 1H;
H-3), 7.29−7.43 (m, 5H; Ph). 13C NMR (100 MHz, CDCl3): δ (ppm)
15.7 (CH3), 19.9 (CH3), 20.6, 20.7, 20.7 (Ac), 30.8 (CHCH2), 51.0
(OCH3), 53.1 (CO2CH3), 55.6 (CHCH2), 56.0 (C-4), 62.3 (C-6),
67.2 (CH2Ph), 68.5 (C-2), 73.2 (C-3), 76.3 (C-5), 86.0 (C-1), 90.3
(CNCH3OH), 108.6 (CCH3OCH3), 128.2, 128.4, 128.7 (Ph), 154.8
(NCO2), 155.9 (NCO2Bn), 169.6, 169.6, 170.7, 170.8 (3×Ac;
CO2CH3). HRMS ESI+ (m/z): calcd for C30H40N2O15SNa+ [M +
Na]+ 723.2042, found 723.2037.
Methyl (2S)-2-((4S,5R)-4,5-Dimethyl-4-hydroxy-5-methoxy-2-ox-
ooxazolidin-3-yl)-3-(hepta-O-acetyl-β-D-lactosylthio)propanoate
(4d). Compound 4d was obtained from α,β-dehydroamino acid 1 (200
mg, 0.82 mmol) and hepta-O-acetyl-1-thio-β-D-lactose 2d (535 mg,
0.82 mmol) following the procedure for sulfa-Michael additions, using
DBU (136 μL, 0.89 mmol) as a base. After column chromatography
(ethyl acetate/hexane, 1:1), 4d was obtained as a white amorphous
solid (574 mg, 0.64 mmol, 78%). [α]2D5 = −21.0 (c 1.00, CHCl3). Mp:
97−99 °C. 1H NMR (400 MHz, CDCl3): δ (ppm) 1.36 (s, 3H; CH3),
1.53 (s, 3H; CH3), 1.92 (s, 3H; Ac), 2.00 (s, 3H; Ac), 2.01 (s, 3H; Ac),
2.02 (s, 3H; Ac), 2.03 (s, 3H; Ac), 2.05 (s, 3H; Ac), 2.11 (s, 3H; Ac),
3.24 (dd, J = 4.6, 14.9 Hz, 1H; CHCH2), 3.40 (s, 3H; OCH3), 3.55
(dd, J = 10.3, 14.9 Hz, 1H; CHCH2), 3.60−3.68 (m, 2H; HGlc-5; OH),
3.69−3.78 (m, 4H; CO2CH3; HGlc-4), 3.85 (t, J = 6.8 Hz, 1H; HGal-5),
3.98 (dd, J = 5.9, 12.1 Hz, 1H; HGlc-6a), 4.04−4.11 (m, 2H; HGal-6a;
HGal-6b), 4.19 (dd, J = 4.5, 10.3 Hz, 1H; CHCH2), 4.44 (d, J = 7.9 Hz,
1H; HGal-1), 4.51 (dd, J = 1.5, 12.0 Hz, 1H; HGlc-6b), 4.59 (d, J = 10.0
Hz, 1H; HGlc-1), 4.85−4.95 (m, 2H; HGlc-2; HGal-3), 5.05 (dd, J = 7.9,
10.4 Hz, 1H; HGal-2), 5.16 (t, J = 9.1 Hz, 1H; HGlc-3), 5.30 (d, J = 3.0
Hz, 1H; HGal-4). 13C NMR (100 MHz, CDCl3): δ (ppm) 14.2 (CH3),
15.6 (CH3), 19.8, 20.5, 20.6, 20.6, 20.7, 20.7, 21.0 (Ac), 30.6
(CHCH2), 50.9 (OCH3), 52.9 (CO2CH3), 55.8 (CHCH2), 60.9 (CGal
-
6), 62.1 (CGlc-6), 66.7 (CGal-4), 69.1 (CGal-2), 70.3 (CGlc-2), 70.8
(CGal-5), 71.0 (CGal-3), 73.3 (CGlc-3), 75.9 (CGlc-4), 77.1 (CGlc-5), 84.8
(CGlc-1), 90.0 (CNCH3OH), 101.0 (CGal-1), 108.5 (CCH3OCH3),
154.5 (NCO2), 169.1, 169.3, 169.6, 169.6, 170.1, 170.1, 170.3, 170.4
(7×Ac; CO2CH3). HRMS ESI+ (m/z): calcd for C36H51NO23SNa+
[M + Na]+ 920.2465, found 920.2470.
795
dx.doi.org/10.1021/ja411522f | J. Am. Chem. Soc. 2014, 136, 789−800

Journal of the American Chemical Society
Article
Methyl 2-((4S,5R)-4,5-Dimethyl-4-hydroxy-5-methoxy-2-oxooxa-
zolidin-3-yl)-3-(tetra-O-acetyl-α-D-glucosylthio)propanoate, Mix-
ture (3e/4e). Diastereomeric mixture 3e/4e was obtained from α,β-
dehydroamino acid 1 (101 mg, 0.41 mmol) and tetra-O-acetyl-1-thio-
α-D-glucose 2e (151 mg, 0.41 mmol) following the procedure for sulfa-
Michael additions, using DBU (67 μL, 0.45 mmol) as a base. After
column chromatography (ethyl acetate/hexane, 1:1), diastereomeric
mixture 3e/4e was obtained as a colorless syrup (197 mg, 0.32 mmol,
79%) in a diastereomeric ratio 23/77, as determined by H NMR. H
NMR data of major compound (400 MHz, CDCl3): δ (ppm) 1.46 (s,
3H), 1.59 (s, 3H), 2.00 (s, 3H), 2.06 (s, 3H), 2.12 (s, 3H), 2.16 (s,
3H), 3.27−3.41 (m, 3H), 3.44 (s 3H), 3.78 (s, 3H), 4.08−4.24 (m,
2H), 4.26−4.37 (m, 2H), 5.19−5.36 (m, 4H). 13C NMR (100 MHz,
CDCl3): δ (ppm) 15.7, 19.8, 20.6, 20.7, 20.9, 29.9, 50.9, 53.1, 53.4,
62.2, 66.1, 69.2, 69.6, 70.8, 81.3, 90.6, 108.3, 154.4, 169.5, 169.7, 169.8,
170.1, 170.7. HRMS ESI+ (m/z): calcd for C24H35NO15SNa+ [M +
Na]+ 632.1620, found 632.1617.
Ac), 2.07 (s, 3H; Ac), 2.14 (s, 3H; Ac), 3.29 (d, J = 7.6 Hz, 2H;
CHCH2), 3.41 (s, 3H; OCH3), 4.00 (t, J = 7.6 Hz, 1H; CHCH2),
4.06−4.19 (m, 3H; H-6a; H6-b; OH), 4.53 (t, J = 6.3 Hz, 1H; H-5),
4.62−4.72 (m, 1H; H-2), 5.06 (dd, J = 3.2, 11.7 Hz, 1H; H-3), 5.39 (d,
J = 2.6 Hz, 1H; H-4), 5.72 (d, J = 5.4 Hz, 1H; H-1), 6.14 (d, J = 7.5
Hz, 1H; NH). 13C NMR (100 MHz, CDCl3): δ (ppm) 15.4 (CH3),
19.8 (CH3), 20.6, 20.7, 20.7, 23.1 (Ac), 27.8 (C(CH3)3), 28.7
(CHCH2), 48.6 (C-2), 50.7 (OCH3), 54.1 (CHCH2), 61.9 (C-6), 67.2
(C-4), 67.7 (C-5), 68.4 (C-3), 82.6 (C(CH3)3), 83.1 (C-1), 90.3
(CNCH3OH), 108.2 (CCH3OCH3), 154.6 (NCO2), 168.0, 170.2,
1
1
t
170.7, 171.1, 171.2 (4xAc; CO2 Bu). HRMS ESI+ (m/z): calcd for
C27H42N2O14SNa+ [M + Na]+ 673.2249, found 673.2243.
tert-Butyl (2S)-2-((4S,5R)-4,5-Dimethyl-4-hydroxy-5-methoxy-2-
oxooxazolidin-3-yl)3-(tri-O-acetyl-2-acetamido-2-deoxy-α-D-
glucosylthio)propanoate Ester (4′g). Compound 4′g was obtained
from α,β-dehydroamino acid 1′ (504 mg, 1.75 mmol) and tri-O-acetyl-
2-acetamido-2-deoxy-1-thio-α-D-glucose 2g (765 mg, 2.10 mmol)
following the procedure for sulfa-Michael additions, using DBU (288
μL, 1.92 mmol) as a base. After column chromatography (CHCl3/
MeOH, 19:1), 4′g was obtained as a white amorphous solid (845 mg,
1.30 mmol, 74%). [α]2D5 = +59.5 (c 0.99, CHCl3). Mp: 71−73 °C. 1H
NMR (400 MHz, CDCl3): δ (ppm) 1.39 (s, 3H; CH3), 1.42 (s, 9H;
tBu), 1.53 (s, 3H; CH3), 1.92 (s, 3H; Ac), 1.99 (s, 3H; Ac), 2.00 (s,
Methyl (2S)-2-((4S,5R)-4,5-Dimethyl-4-hydroxy-5-methoxy-2-ox-
ooxazolidin-3-yl)-3-(tri-O-acetyl-2-acetamido-2-deoxy-α-D-
galactosylthio)propanoate (4f). Compound 4f was obtained from
α,β-dehydroamino acid 1 (424 mg, 1.73 mmol) and tri-O-acetyl-2-
acetamido-2-deoxy-1-thio-α-D-galactose 2f (690 mg, 1.90 mmol)
following the procedure for sulfa-Michael additions, using DBU
(310 μL, 2.07 mmol) as a base. After column chromatography
(CHCl3/MeOH, 95:5), 4f was obtained as a white amorphous solid
(704 mg, 0.64 mmol, 67%). [α]2D5 = +76.6 (c 1.10, CHCl3). Mp: 179−
3H; Ac), 2.07 (s, 3H; Ac), 3.19−3.33 (m, 2H; CHCH2), 3.39 (s, 3H;
OCH3), 4.00 (dd, 1H; J = 5.9, 9.0 Hz, CHCH2), 4.08 (d, 1H; J = 12.2
Hz; H-6a), 4.20−4.30 (m, 2H; H-6b; OH), 4.30−4.37 (m, 1H; H-5),
4.44 (dd, 1H; J = 2.6, 5.3 Hz; H-2), 5.13−4.97 (m, 2H; H-3; H-4),
5.56 (d, J = 5.4 Hz, 1H; H-1), 6.15 (d, J = 7.8 Hz, 1H; NHAc). 13C
NMR (100 MHz, CDCl3): δ (ppm) 15.4 (CH3), 19.7 (CH3), 20.6,
20.7, 20.7, 23.1 (Ac), 27.8 (C(CH3)3), 29.4 (CHCH2), 50.7 (OCH3),
52.6 (C-2), 54.2 (CHCH2), 61.9 (C-6), 68.2 (C-4), 68.6 (C-5), 71.2
(C-3), 82.8 (C(CH3)3), 83.1 (C-1), 90.4 (CNCH3OH), 108.2
(CCH3OCH3), 154.7 (NCO2), 168.0, 169.3, 170.8, 170.9, 171.7
1
181 °C. H NMR (400 MHz, CDCl3): δ (ppm) 1.41 (s, 3H; CH3),
1.57 (s, 3H; CH3), 1.97 (s, 3H; Ac), 2.00 (s, 3H; Ac), 2.07 (s, 3H; Ac),
2.14 (s, 3H; Ac), 3.31−3.35 (m, 2H; CHCH2), 3.43 (s, 3H; OCH3),
3.77 (s, 3H; CO2CH3), 4.01−4.22 (m, 4H; CHCH2; H-6a; H-6b;
OH), 4.52 (t, J = 6.3 Hz, 1H; H-5), 4.61−4.71 (m, 1H; H-2), 5.06 (dd,
J = 3.2, 11.7 Hz, 1H; H-3), 5.40 (d, J = 2.8 Hz, 1H; H-4), 5.78 (d, J =
5.4 Hz, 1H; H-1), 6.16 (d, J = 7.2 Hz, 1H; NH). 13C NMR (100 MHz,
CDCl3): δ (ppm) 15.8 (CH3), 19.9 (CH3), 20.7, 20.7, 20.8, 23.1 (Ac),
28.8 (CHCH2), 48.7 (C-2), 50.9 (OCH3), 53.0 (CO2CH3), 53.1
(CHCH2), 62.0 (C-6), 67.2 (C-4), 67.6 (C-5), 68.4 (C-3), 82.5 (C-1),
90.6 (CNCH3OH), 108.3 (CCH3OCH3), 154.6 (NCO2), 169.6,
170.2, 170.6, 171.1, 171.3 (4×Ac; CO2CH3). HRMS ESI+ (m/z):
calcd for C24H36N2O14SNa+ [M + Na]+ 631.1785, found 631.1788.
Methyl (2S)-2-((4S,5R)-4,5-Dimethyl-4-hydroxy-5-methoxy-2-ox-
ooxazolidin-3-yl)-3-(tri-O-acetyl-2-acetamido-2-deoxy-α-D-
glucosylthio)propanoate (4g). Compound 4g was obtained from α,β-
dehydroamino acid 1 (143 mg, 0.58 mmol) and tri-O-acetyl-2-
acetamido-2-deoxy-1-thio-α-D-glucose 2g (234 mg, 0.64 mmol)
following the procedure for sulfa-Michael additions, using DBU
(105 μL, 0.70 mmol) as a base. After column chromatography
(CHCl3/MeOH, 95:5), 4g was obtained as a white foam (284 mg,
0.46 mmol, 80%). [α]2D5 = +54.7 (c 1.14, CHCl3). 1H NMR (400 MHz,
CDCl3): δ (ppm) 1.38 (s, 3H; CH3), 1.53 (s, 3H; CH3), 1.91 (s, 3H;
Ac), 1.98 (s, 3H; Ac), 1.99 (s, 3H; Ac), 2.06 (s, 3H; Ac), 3.23−3.35
(m, 2H; CHCH2), 3.39 (s, 3H; OCH3), 3.72 (s, 3H; CO2CH3), 4.05−
4.28 (m, 3H; H-6a; H6-b; CHCH2), 4.28−4.38 (m, 1H; H-5), 4.38−
4.47 (m, 1H; H-2); 4.98−5.09 (m, 2H; H-3; H-4), 5.57 (d, J = 5.4 Hz,
1H; H-1), 6.19 (d, J = 8.0 Hz, 1H; NHAc). 13C NMR (100 MHz,
CDCl3): δ (ppm) 15.6 (CH3), 19.8 (CH3), 20.6, 20.7, 20.7, 23.0 (Ac),
29.8 (CHCH2), 50.9 (OCH3), 52.5 (C-2), 53.0 (CO2CH3), 53.3
(CHCH2), 62.0 (C-6), 68.3 (C-4), 68.7 (C-5), 71.1 (C-3), 83.2 (C-1),
90.7 (CNCH3OH), 108.3 (CCH3OCH3), 154.7 (NCO2), 169.4,
169.5, 170.7, 171.0, 171.6 (4xAc; CO2CH3). HRMS ESI+ (m/z): calcd
for C24H36N2O14SNa+ [M + Na]+ 631.1785, found 631.1780.
t
(4×Ac; CO2 Bu). HRMS ESI+ (m/z): calcd for C27H42N2O14SNa+ [M
+ Na]+ 673.2249, found 673.2258.
Benzyl (2S)-2-((4S,5R)-4,5-Dimethyl-4-hydroxy-5-methoxy-2-ox-
ooxazolidin-3-yl)-3-(tri-O-acetyl-2-acetamido-2-deoxy-α-D-
glucosylthio)propanoate (4″g). Compound 4″g was obtained from
α,β-dehydroamino acid 1″ (271 mg, 0.85 mmol) and tri-O-acetyl-2-
acetamido-2-deoxy-1-thio-α-D-glucose 2g (337 mg, 0.92 mmol)
following the procedure for sulfa-Michael additions, using DBU
(161 μL, 1.00 mmol) as a base. After column chromatography
(CHCl3/MeOH, 19:1), 4″g was obtained as a white amorphous solid
(411 mg, 0.60 mmol, 71%) with the presence of minor compound 3″g
in a ratio 3″g/4″g of 15:85. [α]2D5 = +60.4 (c 1.03, CHCl3). Mp: 46−48
°C. 1H NMR (400 MHz, CDCl3): δ (ppm) 1.36 (s, 3H; CH3), 1.52 (s,
3H; CH3), 1.91 (s, 3H; Ac), 1.99 (s, 3H; Ac), 2.00 (s, 3H; Ac), 2.00 (s,
3H; Ac), 3.26−3.48 (m, 5H; OCH3; CHCH2), 4.01−4.12 (m, 1H; H-
6a), 4.14−4.23 (m, 2H; H-6b; CHCH2), 4.28−4.36 (m, 1H; H-5),
4.38−4.47 (m, 1H; H-2), 5.01−5.09 (m, 2H; H-3; H-4), 5.15 (s, 2H;
CH2Ph), 5.59 (d, J = 5.4 Hz, 1H; H-1), 6.23 (d, J = 7.8 Hz, 1H;
NHAc), 7.30 (m, 5H; Ph). 13C NMR (100 MHz, CDCl3): δ (ppm)
15.5 (CH3), 19.7 (CH3), 20.6, 20.6, 20.7, 23.0 (Ac), 29.7 (CHCH2),
50.7 (OCH3), 52.6 (C-2), 54.5 (CHCH2), 62.0 (C-6), 67.8 (CH2Ph),
68.3 (C-4), 68.7 (C-5), 71.2 (C-3), 83.2 (C-1), 90.6 (CNCH3OH),
108.4 (CCH3OCH3), 128.1, 128.4, 128.6, 135.0 (Ph), 154.7 (NCO2),
168.9, 169.3, 170.7, 171.0, 171.6 (4×Ac; CO2Bn). HRMS ESI+ (m/z):
calcd for C30H40N2O14SNa+ [M + Na]+ 707.2092, found 707.2090.
S-(β-D-Glucosyl)-D-cysteine Hydrochloride (5a). Compound 4a
(220 mg, 0.36 mmol) was introduced in a flask with 6 N HCl (5 mL).
The mixture was stirred overnight at 60 °C. The solvent was removed
in vacuo, and the crude was dissolved in water (3 mL) and extracted
with ethyl acetate (3 mL). Aqueous layer was evaporated to give 5a (S-
β-D-Glc-D-Cys·HCl) as a colorless syrup (108 mg, 0.34 mmol, 94%).
tert-Butyl (2S)-2-((4S,5R)-4,5-Dimethyl-4-hydroxy-5-methoxy-2-
oxooxazolidin-3-yl)-3-(tri-O-acetyl-2-acetamido-2-deoxy-α-D-
galactosylthio)propanoate (4′f). Compound 4′f was obtained from
α,β-dehydroamino acid 1′ (720 mg, 2.51 mmol) and tri-O-acetyl-2-
acetamido-2-deoxy-1-thio-α-D-galactose 2f (1.00 g, 2.73 mmol)
following the procedure for sulfa-Michael additions, using DBU
(310 μL, 2.07 mmol) as a base. After column chromatography
(CHCl3/MeOH, 95:5), 4′f was obtained as a white amorphous solid
(1.40 g, 2.15 mmol, 86%). [α]2D5 = +89.3 (c 1.00, CHCl3). Mp: 184−
[α]2D5 = +43.0 (c 0.56, H2O). H NMR (400 MHz, D2O): δ (ppm)
1
3.21−3.55 (m, 6H; CHCH2; H-2; H-4; H-3; H-5), 3.71 (dd, J = 6.3,
12.2 Hz, 1H; H-6a), 3.80−3.97 (m, 1H; H-6b), 4.31−4.40 (m, 1H;
CHCH2), 4.62 (d, J = 9.7 Hz, 1H; H-1). 13C NMR (100 MHz, D2O):
δ (ppm) 31.1 (CHCH2), 53.2 (CHCH2), 60.9 (C-6), 69.4 (C-4), 72.0
1
186 °C. H NMR (400 MHz, CDCl3): δ (ppm) 1.41 (s, 3H; CH3),
t
1.44 (s, 9H; Bu), 1.56 (s, 3H; CH3), 1.96 (s, 3H; Ac), 1.99 (s, 3H;
796
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(C-2), 76.9 (C-3), 79.9 (C-5), 85.8 (C-1), 174.0 (CO2H). HRMS ESI
+ (m/z): calcd for C9H18NO7S+ [M]+ 284.0798, found 284.0799.
S-(2-Acetamido-2-deoxy-α-D-galactosyl)-D-cysteine Hydrochlor-
ide (5f). Compound 4′f (1.40 g, 2.15 mmol) was introduced in a
flask with 4 N HCl (25 mL). The mixture was stirred overnight at 40
°C. The solvent was removed in vacuo, and the crude was dissolved in
water (20 mL) and extracted with ethyl acetate (20 mL). Aqueous
layer was evaporated, and the crude was purified with a LC-18 SPE
tube to give 5f (S-α-D-GalNAc-D-Cys·HCl) as a colorless syrup (567
5.58 (d, J = 5.2 Hz, 1H; H-1), 5.66 (d, J = 8.6 Hz, 1H; NHAc), 5.80
(d, J = 7.8 Hz, 1H; NHFmoc), 5.92 (ddd, J = 6.0, 11.2, 16.5 Hz, 1H;
CHCH2), 7.37 (dd, J = 6.5, 13.5 Hz, 2H; Fmoc), 7.40−7.50 (m,
2H; Fmoc), 7.60−7.64 (m, 2H; Fmoc), 7.80 (d, J = 7.4 Hz, 2H;
Fmoc). 13C NMR (75 MHz, CDCl3): δ (ppm) 20.4, 20.7, 20.7, 23.2
(4xAc), 33.5 (CHCH2), 47.0 (CHFmoc), 48.1 (C-2), 53.7 (CHCH2),
62.0 (C-6), 66.6 (CH2Allyl), 66.6 (CH2Fmoc), 67.1 (C-4), 67.5 (C-5),
68.2 (C-3), 85.6 (C-1), 119.6 (CHCH2), 120.0, 124.8, 124.8, 127.1,
127.1, 127.7 (Fmoc), 131.1 (CHCH2), 141.3, 141.3, 143.6, 143.7
(Fmoc), 155.7 (NCO2), 169.7, 170.2, 170.3, 170.5, 170.9 (4×Ac;
CO2allyl). HRMS ESI+ (m/z): calcd for C35H40N2O12SNa+ [M +
Na]+ 735.2194, found 735.2202.
1
mg, 1.57 mmol, 73%). [α]D25 = +141.6 (c 1.00, H2O). H NMR (400
MHz, D2O): δ (ppm) 2.02 (s, 3H; Ac), 3.10 (dd, J = 7.8, 14.9 Hz, 1H;
CHCH2), 3.34 (dd, J = 4.6, 14.9 Hz, 1H; CHCH2), 3.69−3.81 (m, 2H;
H-6a; H-6b), 3.84 (dd, J = 3.1, 11.4 Hz, 1H; H-3), 3.99 (d, J = 3.1 Hz,
1H; H-4), 4.23 (dd, J = 4.8, 7.2 Hz, 1H; H-5), 4.29 (dd, J = 4.6, 7.6
Hz, 1H; CHCH2), 4.36 (dd, J = 5.5, 11.4 Hz, 1H; H-2), 5.59 (d, J =
5.5 Hz, 1H; H-1). 13C NMR (100 MHz, D2O): δ (ppm) 21.9 (Ac),
30.5 (CHCH2), 50.0 (C-2), 52.9 (CHCH2), 61.1 (C-6), 67.4 (C-3),
68.3 (C-4), 72.1 (C-5), 84.3 (C-1), 170.2 (Ac), 174.8 (CO2H). HRMS
ESI+ (m/z): calcd for C11H21N2O7S+ [M]+ 325.1064, found 325.1056.
S-(2-Acetamido-2-deoxy-α-D-glucosyl)-D-cysteine Hydrochloride
(5g). Compound 4′g (845 mg, 1.30 mmol) was introduced in a
flask with 4 N HCl (20 mL). The mixture was stirred overnight at 40
°C. The solvent was removed in vacuo, and the crude was purified by a
LC-18 SPE tube to give 5g (S-α-D-GlcNAc-D-Cys·HCl) as a colorless
syrup (447 mg, 95%). This compound was obtained along with a small
impurity (83/17 ratio by NMR) corresponding to the hydrolysis of the
N-acetyl group of carbohydrate moiety, which was not possible to
S-((Tri-O-acetyl)-2-acetamido-2-deoxy-α-D-glucosyl)-N-Cbz-D-
cysteine Benzyl Ester (6g). (1) Compound 5g (244 mg, 0.68 mmol)
was dissolved in water (2.5 mL), and NaHCO3 (114 mg, 1.36 mmol)
was added with stirring. The resulting solution was cooled at 0 °C, and
Cbz-Cl (145 μL, 1.02 mmol) was added as a solution in 1,4-dioxane
(2.5 mL). The resulting mixture was stirred at 0 °C for 1 h and
allowed to warm to room temperature overnight. Water was then
added, and the mixture was acidified to a pH of 1 with HCl 2 N. The
aqueous layer was extracted with AcOEt (3 × 10 mL) and
CHCl3/iPrOH (3:1) (4 × 10 mL). The organic layers were combined
and evaporated, and the crude was purified by LC-18 SPE tube to give
the N-Cbz-protected derivative (91 mg, 0.20 mmol, 29%). (2) Cbz-D-
Cys(α-D-GlcNAc)-OH was introduced as a solution in dry DMF (2
mL), under argon atmosphere, in a Schlenk tube previously charged
with 3 Å molecular sieves and Cs2CO3 (77 mg, 0.24 mmol). Benzyl
bromide (38 μL, 0.32 mmol) was then added by a syringe, and the
mixture was stirred overnight at room temperature. Next, the solvent
was evaporated, and the crude was dissolved in AcOEt and filtered.
Evaporation of the solvent gave the benzyl ester derivative Cbz-D-
Cys(α-D-GlcNAc)-OBn (60 mg, 0.11 mmol, 55%). (3) Treatment
with Ac2O and pyridine (1:2, 1.5 mL) at room temperature for 1 h
yielded compound 6g (42 mg, 0.063 mmol, 57%), which was purified
by a silica gel column chromatography (hexane/AcOEt 3:7) affording
6g as a colorless syrup. The spectroscopic data of this compound were
found different from those of its diastereoisomer Cbz-L-Cys(α-D-
(OAc)3GlcNAc)-OBn reported in the literature.5c [α]D25 = +71.0 (c
1
separate. H NMR (400 MHz, D2O): δ (ppm) 1.96 (s, 3H; Ac), 3.09
(dd, J = 6.9, 14.9 Hz, 1H; CHCH2), 3.27 (dd, J = 4.7, 14.9 Hz, 1H;
CHCH2), 3.42 (t, J = 9.4 Hz, 1H; H-4), 3.60 (dd, J = 8.8, 10.9 Hz H-
3), 3.68−3.82 (m, 2H; H-6a; H-6b), 3.91 (ddd, J = 2.3, 5.1, 10.1 Hz
1H; H-5), 4.04 (dd, J = 5.3, 11.0 Hz, 1H; H-2), 4.28 (dd, J = 4.8, 6.9
Hz, 1H; CHCH2), 5.45 (d, J = 5.3 Hz, 1H; H-1). 13C NMR (100
MHz, D2O): δ (ppm) 21.9 (Ac), 30.5 (CHCH2), 52.6 (CHCH2), 53.7
(C-2), 60.3 (C-6), 70.0 (C-4), 70.6 (C-3), 72.9 (C-5), 84.2 (C-1),
169.9 (Ac), 174.5 (CO2H). HRMS ESI+ (m/z): calcd for
C11H21N2O7S+ [M]+ 325.1064, found 325.1059.
S-(Tri-O-acetyl-2-acetamido-2-deoxy-α-D-galactosyl)-N-Fmoc-D-
cysteine Allyl Ester (6f). (1) Compound 5f (567 mg, 1.57 mmol) was
dissolved in water (10 mL), and NaHCO3 (263 mg, 3.14 mmol) was
added with stirring. Fmoc-OSu (793 mg, 2.35 mmol) was then added
as a solution in acetonitrile (20 mL). The resulting mixture was stirred
overnight at room temperature. Water was then added, and the
mixture was acidified to a pH of 1 with HCl 2 N. Next, acetonitrile was
evaporated, and the mixture was filtered. Fmoc-protected derivative
was collected and dried as a white solid (608 mg, 1.11 mmol, 70%).
(2) Fmoc-D-Cys-(α-D-GalNAc)-OH (518 mg, 0.94 mmol) was
introduced as a solution in dry DMF (10 mL), under argon
atmosphere, in a Schlenk previously charged with 3 Å molecular
sieves and Cs2CO3 (370 mg, 1.13 mmol). Allyl bromide (131 μL, 1.51
mmol) was then added by a syringe, and the mixture was stirred
overnight at room temperature. After that, the solvent was evaporated,
and the crude was dissolved in AcOEt and filtered. Evaporation of the
solvent yielded the compound Fmoc-D-Cys(α-D-GalNAc)-OAllyl (217
mg, 0.37 mmol, 37%). (3) Treatment with Ac2O in pyridine (1:2, 5
mL) at room temperature for 1 h produced compound 6f (249 mg,
0.35 mmol, 94%), which was purified by a silica gel column
chromatography (CHCl3/AcOEt 1:1) affording 6f as a colorless
syrup. Its diastereoisomer Fmoc-L-Cys(α-D-(OAc)3GalNAc)-OAllyl
was synthesized for comparative purposes by the method reported in
1
1.01, CHCl3). H NMR (400 MHz, CDCl3): δ (ppm) 1.94 (s, 3H;
Ac), 2.03 (s, 3H; Ac), 2.03 (s, 3H; Ac), 2.05 (s, 3H; Ac), 3.01 (dd, J =
5.6, 13.9 Hz, 1H; CHCH2), 3.21 (dd, J = 4.5, 13.9 Hz, 1H; CHCH2),
4.05 (d, J = 10.5 Hz, 1H; H-6a), 4.15−4.28 (m, 2H; H-6b; H-5), 4.47
(ddd, J = 11.0, 8.7, 5.4 Hz, 1H; H-2), 4.61−4.71 (m, 1H; CHCH2),
4.98 (dd, J = 11.1, 9.4 Hz, 1H; H-3), 5.03−5.21 (m, 5H; H-4;
2×CH2Ph), 5.44 (d, J = 5.3 Hz, 1H; H-1), 5.64 (d, J = 7.7 Hz, 1H;
NHCbz), 5.76 (d, J = 8.4 Hz, 1H; NHAc), 7.36 (m, 10H; 2xPh). 13C
NMR (100 MHz, CDCl3): δ (ppm) 20.6, 20.7, 20.7, 23.2 (4×Ac), 34.6
(CHCH2), 52.5 (C-2), 54.0 (CHCH2), 61.8 (C-6), 67.3 (CH2Ph),
67.9 (CH2Ph), 68.0 (C-4), 68.9 (C-5), 71.2 (C-3), 85.8 (C-1), 128.1,
128.3, 128.6, 128.6, 128.8, 128.8, 134.8, 136.0 (2×Ph), 155.7 (NCO2),
169.2, 169.9, 170.1, 170.7, 171.6 (4×Ac; CO2Bn). HRMS ESI+ (m/z):
calcd for C32H38N2O12SNa+ [M + Na]+ 697.2038, found 697.2035.
Methyl (2R)-2-((4R,5S)-4,5-Dimethyl-4-hydroxy-5-methoxy-2-ox-
ooxazolidin-3-yl)-3-(tetra-O-acetyl-β-D-glucosylthio)propanoate
(7a). Compound 7a was obtained from α,β-dehydroamino acid ent-1
(2.20 g, 8.97 mmol) and tetra-O-acetyl-1-thio-β-D-glucose 2a (3.57 g,
9.85 mmol) following the procedure for sulfa-Michael additions, using
DBU (1.60 mL, 10.50 mmol) as a base. After column chromatography
(ethyl acetate/hexane, 1:1), 7a was obtained as a white amorphous
solid (4.32 g, 7.09 mmol, 79%). [α]2D5 = +11.3 (c 1.00, CHCl3). Mp:
54−56 °C. 1H NMR (400 MHz, CDCl3): δ (ppm) 1.42 (s, 3H; CH3),
1.57 (s, 3H; CH3), 2.02 (s, 3H; Ac), 2.03 (s, 3H; Ac), 2.07 (s, 3H; Ac),
2.09 (s, 3H; Ac), 3.37 (dd, J = 10.0, 14.9, Hz, 1H; CHCH2), 3.44 (s,
3H; OCH3), 3.54−3.65 (m, 1H; CHCH2), 3.71−3.83 (m, 1H; H-5),
3.79 (s, 3H; CO2CH3), 4.09−4.26 (m, 2H; H-6a; CHCH2), 4.26−4.35
(m, 1H; H-6b), 4.56 (d, J = 10.0 Hz, 1H; H-1), 5.03−5.15 (m, 2H; H-
2; H-4), 5.22 (t, J = 9.5 Hz, 1H; H-3). 13C NMR (100 MHz, CDCl3):
δ (ppm) 15.4 (CH3), 19.6 (CH3), 20.6, 20.6, 20.7, 20.7 (Ac), 30.1
(CHCH2), 50.8 (OCH3), 53.0 (CO2CH3), 55.7 (CHCH2), 61.4 (C-
6), 67.7 (C-4), 69.3 (C-2), 73.6 (C-3), 76.3 (C-5), 84.7 (C-1), 90.1
the literature,20a and their H NMR spectra were compared, finding
1
that these spectroscopic data are different (see the Supporting
Information). [α]2D5 = +70.8 (c 1.00, CHCl3). H NMR (400 MHz,
1
CDCl3): δ (ppm) 1.94 (s, 3H; Ac), 1.99 (s, 3H; Ac), 2.05 (s, 3H; Ac),
2.18 (s, 3H; Ac), 3.07 (dd, J = 5.8, 14.0 Hz, 1H; CHCH2), 3.27 (dd, J
= 4.5, 13.8 Hz, 1H; CHCH2), 3.95−4.17 (m, 2H; H-6a; H-6b), 4.27 (t,
J = 6.2 Hz, 1H; CHFmoc), 4.36 (t, J = 6.1 Hz, 1H; H-5), 4.40−4.56 (m,
1H; CH2Fmoc), 4.56−4.62 (m, 1H; CH2Fmoc), 4.63−4.72 (m, 3H;
CHCH2; CH2Allyl), 4.78 (ddd, J = 5.4, 8.5, 11.9 Hz, 1H; H-2), 5.00
(dd, J = 3.1, 11.7 Hz, 1H; H-3), 5.26−5.46 (m, 3H; H-4; CHCH2),
797
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(CNCH3OH), 108.4 (CCH3OCH3), 154.7 (NCO2), 169.2, 169.4,
169.4, 170.0, 170.6 (4×Ac; CO2CH3). HRMS ESI+ (m/z): calcd for
C24H35NO15SNa+ [M + Na]+ 632.1620, found 632.1630.
solution of NaHCO3 (30 mL). The solution was acidified to pH = 1
with 2 N HCl and then extracted with ethyl acetate (3 × 15 mL). This
crude compound was purified by flash silica gel chromatography
(CH2Cl2/MeOH, 95:5) to give building block 9a (566 mg, 0.84 mmol,
tert-Butyl (2R)-2-((4R,5S)-4,5-Dimethyl-4-hydroxy-5-methoxy-2-
oxooxazolidin-3-yl)-3-(tri-O-acetyl-2-acetamido-2-deoxy-α-D-
galactosylthio)propanoate (7′f). Compound 7′f was obtained from
α,β-dehydroamino acid ent-1′ (764 mg, 2.66 mmol) and tri-O-acetyl-
2-acetamido-2-deoxy-1-thio-α-D-galactose 2f (1.06 g, 2.93 mmol)
following the procedure for sulfa-Michael additions, using DBU
(529 μL, 3.19 mmol) as a base. After column chromatography
(CHCl3/MeOH, 19:1), 7′f was obtained as a white amorphous solid
(1.31 g, 2.02 mmol, 76%). [α]2D5 = +141.8 (c 1.00, CHCl3). Mp: 140−
142 °C. 1H NMR (400 MHz, CDCl3): δ (ppm) 1.45 (s, 12H;
C(CH3)3; CH3), 1.57 (s, 3H; CH3), 1.96 (s, 3H; Ac), 1.99 (s, 3H; Ac),
2.03 (s, 3H; Ac), 2.14 (s, 3H; Ac), 3.33 (dd, J = 7.3, 10.5 Hz, 2H;
CHCH2), 3.41 (s, 3H; OCH3), 4.05−4.18 (m, 3H; H-6a; H-6b;
CHCH2), 4.52 (t, J = 6.4 Hz, 1H; H-5), 4.60−4.77 (m, 1H; H-2), 5.02
(dd, J = 3.0, 11.6 Hz, 1H; H-3), 5.37 (d, J = 2.4 Hz, 1H; H-4), 5.64 (d,
J = 5.3 Hz, 1H; H-1), 5.95 (d, J = 8.1 Hz, 1H; NHAc). 13C NMR (100
MHz, CDCl3): δ (ppm) 15.3 (CH3), 19.9 (CH3), 20.7, 20.7, 20.8, 23.2
(Ac), 27.9 (C(CH3)3), 30.8 (CHCH2), 48.6 (C-2), 50.7 (OCH3), 56.4
(CHCH2), 61.8 (C-6), 67.2 (C-4), 67.7 (C-5), 68.4 (C-3), 83.3
(C(CH3)3), 85.5 (C-1), 90.5 (CNCH3OH), 108.1 (CCH3OCH3),
47%) as a colorless syrup. [α]2D5 = −6.2 (c 1.00, CHCl3). H NMR
1
(400 MHz, CDCl3): δ (ppm) 2.01 (s, 3H; Ac), 2.04 (s, 3H; Ac), 2.06
(s, 3H; Ac), 2.10 (s, 3H; Ac), 3.10 (dd, J = 4.3, 14.2 Hz, 1H; CHCH2),
3.30 (d, J = 10.7 Hz, 1H; CHCH2), 3.62−3.69 (m, 1H; H-5), 4.07−
4.30 (m, 3H; CHCH2Fmoc, H-6a; H-6b), 4.35−4.58 (m, 3H;
CHCH2Fmoc, H-1), 4.59−4.67 (m, 1H; CHCH2), 4.99 (t, J = 9.7
Hz, 1H; H-2), 5.05 (m, 1H; H-4), 5.22 (t, J = 9.3 Hz, 1H; H-3), 5.94
(d, J = 7.1 Hz, 1H; NHFmoc), 7.32 (t, J = 7.4 Hz, 2H; Fmoc), 7.40 (t, J
= 7.4 Hz, 2H; Fmoc), 7.55−7.64 (m, 2H; Fmoc), 7.77 (d, J = 7.5 Hz,
2H; Fmoc). 13C NMR (100 MHz, CDCl3): δ (ppm) 20.6, 20.6, 20.7,
20.7 (Ac), 33.0 (CHCH2), 47.2 (CHCH2Fmoc), 53.8 (CHCH2), 62.4
(C-6), 67.2 (CHCH2Fmoc), 68.6 (C-4), 69.7 (C-2), 73.5 (C-3), 75.9
(C-5), 84.2 (C-1), 120.0, 125.1, 125.3, 127.2, 127.8 (Fmoc), 141.3,
143.7, 143.7 (Fmoc), 156.1 (NCO2), 169.4, 169.6, 170.2, 171.1, 176.7
(4×Ac; CO2H). HRMS ESI− (m/z): calcd for C32H34NO13S− [M −
H]− 672.1756, found 672.1778.
S-(Tri-O-acetyl-2-acetamido-2-deoxy-α-D-galactosyl)-N-Fmoc-L-
cysteine (9f). Compound 8f (670 mg, 1.85 mmol) was treated
following the same methodology for the synthesis of Fmoc-D-Cys(α-D-
(OAc)3GalNAc)-OAllyl (6f). (1) Fmoc-OSu, NaHCO3, H2O/
acetonitrile (1:2), room temperature, overnight (738 mg, 1.35
mmol, 73%). (2) AllylBr, Cs2CO3, DMF, 3 Å molecular sieves,
overnight (277 mg, 0.47 mmol, 35%). (3) Ac2O, pyridine, 1 h (298
mg, 0.42 mmol, 89%). All physical and spectroscopic properties of
Fmoc-L-Cys(α-D-(OAc)3GalNAc)-OAllyl were identical to those
reported in the literature.20a (4) Fmoc-L-Cys(α-D-(OAc)3GalNAc)-
OAllyl (298 mg, 0.42 mmol) was dissolved in dry THF (10 mL) under
argon atmosphere. Pd(PPh3)4 (5.2 mg, 4.5 × 10−3 mmol) and
morpholine (182 μL, 2.1 mmol) were added, and the resulting
solution was stirred at room temperature for 1 h. Next, the solvent was
removed, and the crude was dissolved in CHCl3/iPrOH (3:1, 10 mL).
The solution was washed with 1 N HCl (2 × 5 mL), and the
combined aqueous phases were extracted with more CHCl3/iPrOH (2
× 5 mL). The organic layers were combined, dried with anhydrous
Na2SO4, and solvent was removed. The crude product was purified by
column chromatography (ethyl acetate/hexane 7:3 to CH2Cl2/MeOH
9:1) to give building block 9f (268 mg, 0.40 mmol, 95%) as a yellow
amorphous solid. [α]2D5 = +120.3 (c 1.00, CHCl3). Mp: 102−104 °C.
1H NMR (400 MHz, CDCl3): δ (ppm) 2.01 (s, 3H; Ac), 2.02 (s, 3H;
Ac), 2.04 (s, 3H; Ac), 2.20 (s, 3H; Ac), 3.08 (d, J = 13.8 Hz, 1H;
CHCH2), 3.36 (d, J = 12.4 Hz, 1H; CHCH2), 4.04−4.13 (m, 1H; H-
6a), 4.20−4.30 (m, 2H; H-6b; CHCH2Fmoc), 4.33−4.44 (m, 1H;
CHCH2Fmoc), 4.45−4.56 (m, 2H; CHCH2Fmoc; H-5), 4.73−4.89 (m,
2H; H-2; CHCH2), 4.94−5.03 (m, 1H; H-3), 5.40 (s, 1H; H-4), 5.58
(s, 1H; H-1), 6.42 (s, 1H; NHFmoc), 6.64 (s, 1H; NHAc), 7.31−7.46
(m, 4H; Fmoc), 7.60−7.68 (m, 2H; Fmoc), 7.79 (d, J = 7.4 Hz 2H;
Fmoc). 13C NMR (100 MHz, CDCl3): δ (ppm) 20.6, 20.7, 20.7, 23.1
(Ac), 36.4 (CHCH2), 47.1 (CHCH2Fmoc), 48.4 (C-2), 54.6 (CHCH2),
61.6 (C-6), 67.0 (CHCH2Fmoc), 67.2 (C-4), 68.0 (C-5), 68.3 (C-3),
87.4 (C-1), 120.0, 125.1, 125.1, 127.1, 127.8, 128.6, 128.7 (Fmoc),
141.3, 141.4, 143.7, 143.9 (Fmoc), 155.9 (NCO2), 170.4, 170.6, 171.2,
171.4, 174.7 (4×Ac; CO2H). HRMS ESI− (m/z): calcd for
C32H35N2O12S− [M − H]− 671.1916, found 671.1904.
S-(2-Acetamido-2-deoxy-α-D-galactosyl)-N-(acetyl)-L-cysteine
Methylamide (10f). Fmoc-L-Cys(α-(OAc)3-D-GalNAc)-OH (9f, 268
mg, 0.40 mmol) was dissolved in dry acetonitrile (10 mL) in the
presence of 3 Å molecular sieves. TBTU (350 mg, 1.09 mmol) was
added in one portion, followed by DIEA (732 μL, 4.2 mmol). After the
mixture was stirred for 5 min, MeNH2·HCl (170 mg, 2.52 mmol) was
added under argon atmosphere. The reaction was stirred overnight,
the mixture was then filtered off over a Celite pad, and brine was
added to the remaining solution. Aqueous layer was extracted with
CH2Cl2 (2 × 5 mL), and organic layers were combined and washed
with 1 N HCl (2 × 5 mL) and NaHCO3 (2 × 5 mL) saturated
solution. After evaporation of the solvents, the reaction crude was
purified by column chromatography (CH2Cl2/MeOH, 95:5) yielding
t
154.8 (NCO2), 168.3, 170.2, 170.7, 170.7, 171.1 (4×Ac; CO2 Bu).
HRMS ESI+ (m/z): calcd for C27H42N2O14SNa+ [M + Na]+ 673.2249,
found 673.2257.
S-(β-D-Glucosyl)-L-cysteine Hydrochloride (8a). Compound 7a
(4.32 g, 7.09 mmol) was introduced in a flask with 6 N HCl (100
mL). The mixture was stirred overnight at 60 °C. The solvent was
removed in vacuo, and the crude was dissolved in water (25 mL) and
extracted with ethyl acetate (25 mL). Aqueous layer was evaporated to
give 8a (S-β-D-Glc-L-Cys·HCl) as a colorless syrup (2.13 g, 6.66 mmol,
94%). [α]2D5 = −37.4 (c 0.63, H2O). H NMR (400 MHz, D2O): δ
1
(ppm) 3.14 (dd, J = 8.0, 15.3 Hz, 1H; CHCH2), 3.36−3.47 (m, 3H;
CHCH2; H-2; H-4), 3.47−3.56 (m, 2H; H-3; H-5), 3.72 (dd, J = 5.8,
12.4 Hz, 1H; H-6a), 3.88−3.99 (m, 1H; H-6b), 4.02 (dd, J = 3.7, 7.8
Hz, 1H; CHCH2), 4.56 (d, J = 9.7 Hz, 1H; H-1). 13C NMR (100
MHz, D2O): δ (ppm) 30.4 (CHCH2), 54.5 (CHCH2), 60.9 (C-6),
69.4 (C-4), 71.7 (C-2), 77.0 (C-3), 80.1 (C-5), 84.7 (C-1), 172.2
(CO2H). HRMS ESI+ (m/z): calcd for C9H18NO7S+ [M + H]+
284.0798, found 284.0787. A small percentage of mutarotation was
16
1
observed in the H NMR spectrum.
S-(2-Acetamido-2-deoxy-α-D-galactosyl)-L-cysteine Hydrochlor-
ide (8f). Compound 7′f (1.31 g, 2.02 mmol) was introduced in a
flask with 4 N HCl (25 mL). The mixture was stirred overnight at 40
°C. The solvent was removed in vacuo, and the crude was dissolved in
water (10 mL) and extracted with ethyl acetate (10 mL). Aqueous
layer was evaporated to give 8f (S-α-D-GalNAc-L-Cys·HCl) as a
colorless syrup (670 mg, 1.85 mmol, 92%). [α]2D5 = +144.9 (c 1.00,
1
H2O). H NMR (400 MHz, D2O): δ (ppm) 2.05 (s, 3H; Ac), 3.09−
3.19 (m, 1H; CHCH2), 3.34 (dd, J = 6.2, 15.0 Hz, 1H; CHCH2), 3.7
4−3.87 (m, 3H; H-6a; H-6b; H-3), 4.00 (s, 1H; H-3), 4.04−4.11 (m,
1H; CHCH2), 4.26−4.36 (m, 1H; H-5), 4.40 (dd, J = 5.4, 11.4 Hz,
1H; H-2), 5.54 (d, J = 5.3 Hz, 1H; H-1). 13C NMR (100 MHz, D2O):
δ (ppm) 21.9 (Ac), 33.1 (CHCH2), 49.9 (C-2), 54.4 (CHCH2), 61.2
(C-6), 67.4 (C-3), 68.4 (C-4), 72.4 (C-5), 86.2 (C-1), 172.3 (CO2H),
174.6 (Ac). HRMS ESI+ (m/z): calcd for C11H21N2O7S+ [M]+
325.1064, found 325.1061.
S-(Tetra-O-acetyl-β-D-glucosyl)-N-Fmoc-L-cysteine (9a). Com-
pound 8a (975 mg, 3.05 mmol) was dissolved in water (25 mL),
and NaHCO3 (511 mg, 6.08 mmol) was added with stirring. Fmoc-
OSu (1.54 g, 4.57 mmol) was then added as a solution in acetonitrile
(50 mL). The resulting mixture was stirred at room temperature
overnight. Water was then added, and the mixture was acidified to pH
= 1 with 2 N HCl. Next, acetonitrile was evaporated, and the mixture
was filtered. Fmoc-protected derivative, Fmoc-L-Cys(β-D-Glc)-OH,
was collected and dried as a white amorphous solid (902 mg, 1.79
mmol, 59%). This compound was suspended in Ac2O (10 mL), and a
few drops of H3PO4 (85 wt % in water) were added. The reaction was
stirred for 2 h at room temperature and then quenched with saturated
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Journal of the American Chemical Society
Article
Fmoc-L-Cys(α-D-(OAc)3GalNAc)-NHMe (158 mg, 0.23 mmol, 57%).
This Fmoc-derivative was treated with piperidine (20% in acetonitrile,
10 mL) for 30 min at room temperature. The solvent was evaporated
and the crude purified by flash column chromatography (CH2Cl2/
MeOH, 90:10) yielding L-Cys(α-D-(OAc)3GalNAc)-NHMe (102 mg,
0.22 mmol, 98%). This amine was reacted with Ac2O in the presence
of pyridine (1:2, 5 mL) for 1 h at room temperature. The solvent was
evaporated, and the reaction crude was purified by column
chromatography (CH2Cl2/MeOH, 95:5) yielding Ac-L-Cys(α-D-
(OAc)3GalNAc)-NHMe (96 mg, 0.19 mmol, 87%). This peracetylated
carbohydrate was treated with 0.5 M NaOMe solution in MeOH (2
mL) for 1.5 h. After neutralization with Dowex 50WX8, the solution
was evaporated, and the crude was purified by a LC-18 SPE tube to
give Ac-L-Cys(α-D-GalNAc)-NHMe 10f as a white foam (58 mg, 0.15
mmol, 78% and 38% overall yield from 9f). [α]2D5 = +73.4 (c 0.50,
H2O). 1H NMR (400 MHz, D2O): δ (ppm) 2.04 (s, 3H; AcN2), 2.06
(s, 3H; AcN3), 2.75 (s, 3H; NHMe), 2.97−3.10 (m, 2H; CHCH2),
3.79 (d, J = 6.0 Hz, 2H; H-6a; H-6b), 3.84 (dd, J = 2.4, 11.2 Hz, 1H;
H-3), 4.00 (s, 1H; H-4), 4.26 (t, J = 6.0 Hz, 1H; H-5), 4.36 (dd, J =
5.5, 11.4 Hz, 1H; H-2), 4.53 (t, J = 6.4 Hz, 1H; CHCH2), 5.55 (d, J =
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1
5.5 Hz, 1H; H-1). H NMR (400 MHz, D2O/H2O, 9:1): δ (ppm)
7.96−8.02 (m, 1H; NH1), 8.15 (d, J = 8.0 Hz, 1H; NH3), 8.28 (d, J =
7.5 Hz, 1H; NH3). 13C NMR (100 MHz, D2O): δ (ppm) 21.8 (Ac),
21.8 (Ac), 25.8 (NHMe), 31.7 (CHCH2), 50.0 (C-2), 54.0 (CHCH2),
61.0 (C-6), 67.4 (C-3), 68.4 (C-4), 71.6 (C-5), 84.6 (C-1), 172.5
(CONHMe), 174.3 (Ac), 174.5 (Ac). HRMS ESI+ (m/z): calcd for
C14H26N3O7S+ [M + H]+ 380.1486, found 380.1471.
ASSOCIATED CONTENT
■
(11) Bonauer, C.; Walenzyk, T.; Konig, B. Synthesis 2006, 1.
̈
S
* Supporting Information
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Experimental and computational details, X-ray data, and
spectroscopic characterization of all new compounds. This
material is available free of charge via the Internet at http://
pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Authors
alberto.avenoza@unirioja.es
jesusmanuel.peregrina@unirioja.es
(14) Aydillo, C.; Avenoza, A.; Busto, J. H.; Jimen
Peregrina, J. M.; Zurbano, M. M. Org. Lett. 2012, 14, 334.
(15) (a) Aydillo, C.; Avenoza, A.; Busto, J. H.; Jimenez-Oses
Peregrina, J. M.; Zurbano, M. M. Tetrahedron: Asymmetry 2008, 19,
2829. (b) Aydillo, C.; Jimenez-Oses, G.; Busto, J. H.; Peregrina, J. M.;
́
ez-Oses
́
, G.;
Notes
́
́
, G.;
The authors declare no competing financial interest.
́
́
Zurbano, M. M.; Avenoza, A. Chem.Eur. J. 2007, 13, 4840.
ACKNOWLEDGMENTS
■
(16) For a recent study on mutarotation of 1-thiocarbohydrates, see:
́
We thank the Ministerio de Economia y Competitividad
(project CTQ2012-36365/FEDER). We also thank CESGA for
computer support. We appreciate the helpful comments of Dr.
Caraballo, R.; Deng, L.; Amorim, L.; Brinck, T.; Ramstrom, O. J. Org.
̈
Chem. 2010, 75, 6115.
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Gonzalo Jimenez-Oses.
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