Aromatic thioglycoside inhibitors against the virulence factor LecA from Pseudomonas aeruginosa

Article abstract of DOI:10.1039/c3ob41422a

Three small families of hydrolytically stable thioaryl glycosides were prepared as inhibitors of the LecA (PA-IL) virulence factor corresponding to the carbohydrate binding lectin from the bacterial pathogen Pseudomonas aeruginosa. The monosaccharidic arylthio β-d-galactopyranosides served as a common template for the major series that was also substituted at the O-3 position. Arylthio disaccharides from lactose and from melibiose constituted the other two series members. In spite of the fact that the natural ligand for LecA is a glycolipid of the globotriaosylceramide having an α-d-galactopyranoside epitope, this study illustrated that the β-d-galactopyranoside configuration having a hydrophobic aglycon could override the requirement toward the anomeric configuration of the natural sugar. The enzyme linked lectin assay together with isothermal titration microcalorimetry established that naphthyl 1-thio-β-d-galactopyranoside (11) gave the best inhibition with an IC ₅₀ twenty-three times better than that of the reference methyl α-d-galactopyranoside. In addition it showed a K_D of 6.3 μM which was ten times better than that of the reference compound. The X-ray crystal structure of LecA with 11 was also obtained.

Full text of DOI:10.1039/c3ob41422a

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Biomolecular Chemistry
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Aromatic thioglycoside inhibitors against the virulence
factor LecA from Pseudomonas aeruginosa†
Cite this: Org. Biomol. Chem., 2013, 11,
6906
Jacques Rodrigue,a Géraldine Ganne,b Bertrand Blanchard,b Catherine Saucier,a
Denis Giguère,a Tze Chieh Shiao,a Annabelle Varrot,b Anne Imberty*b and
René Roy*a
Three small families of hydrolytically stable thioaryl glycosides were prepared as inhibitors of the LecA
(PA-IL) virulence factor corresponding to the carbohydrate binding lectin from the bacterial pathogen
Pseudomonas aeruginosa. The monosaccharidic arylthio β-D-galactopyranosides served as a common tem-
plate for the major series that was also substituted at the O-3 position. Arylthio disaccharides from
lactose and from melibiose constituted the other two series members. In spite of the fact that the natural
ligand for LecA is a glycolipid of the globotriaosylceramide having an α-D-galactopyranoside epitope, this
study illustrated that the β-D-galactopyranoside configuration having a hydrophobic aglycon could over-
ride the requirement toward the anomeric configuration of the natural sugar. The enzyme linked lectin
assay together with isothermal titration microcalorimetry established that naphthyl 1-thio-β-D-galactopyr-
anoside (11) gave the best inhibition with an IC50 twenty-three times better than that of the reference
methyl α-D-galactopyranoside. In addition it showed a KD of 6.3 μM which was ten times better than that
of the reference compound. The X-ray crystal structure of LecA with 11 was also obtained.
Received 10th July 2013,
Accepted 12th August 2013
DOI: 10.1039/c3ob41422a
www.rsc.org/obc
Introduction
Infection by pathogens is generally initiated by crucial steps of
recognition and adhesion on host epithelial surfaces. Very fre-
quently, the strategy used by micro-organisms involves
binding to host glycoconjugates by sugar-binding proteins,
lectins, which are specific for the target tissue. The depen-
dence between pathogen receptors and host glycans leads to
the concept of “glycoecology”.1 Pseudomonas aeruginosa is an
opportunistic pathogen implicated in the development of lung
infections in cystic fibrosis (CF) patients through these sugar–
protein binding. These infections are triggered by the abnor-
mal abundance of mucus in CF patients, thus offering an
accumulation of anchoring sites for bacteria. Pseudomonas
aeruginosa’s mechanism of action is managed by the adhesion of
proteins called lectins LecA (PA-IL) and LecB (PA-IIL) specific
to the galactose and fucose subunit, respectively.2–4
Fig. 1 Structure of selected P. aeruginosa LecA inhibitors. (A) Natural ligand:
globotriaosylceramide (αGal(1–4)βGal(1–4)βGlc-Cer);15 (B) synthetic C-galacto-
pyranoside;8 (C) naphthyl sulfone lactoside;20 and (D) α-L-fucoside and β-D-galacto-
pyranoside dendrimer hybrid21 allowing the inhibition of both LecA and LecB.
LecA, the galactoside-binding lectin, has been demon-
strated to be involved in adhesion to lung tissues and leads to
alveolar destruction during the first steps of bacterial infection
in a mice model,5 and treatment with galactose or N-acetyl-
galactosamine (GalNAc) has a protective effect. Therefore, the
design and synthesis of galactose-based inhibitors for LecA is
an attractive strategy for new anti-infectious compounds.
Several LecA inhibitors have been reported in the literature6–14
and some of them are displayed in Fig. 1, but none of them
aPharmaQAM – Department of Chemistry, Université du Québec à Montréal, P.O.
Box 8888, Succ. Centre-ville, Montréal, Québec, Canada H3C 3P8.
E-mail: roy.rene@uqam.ca; Fax: +1 514 987 4054
bCentre de Recherche sur les Macromolécules Végétales (CERMAV)-CNRS, Université
Grenoble 1 and Institut de Chemie Moléculaire de Grenoble BP53, F-38041, Grenoble
cedex 9, France. E-mail: imberty@cermav.cnrs.fr
†Electronic supplementary information (ESI)
available. See DOI:
10.1039/c3ob41422a
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have given rise to a well-established treatment against nosoco-
mial infections.
The lectin crystal structure is tetrameric and one calcium
ion is present in each binding site.2 The natural ligand of LecA
in the lung has been proposed to be glycolipids terminated
with the αGal(1–4)Gal epitope (globotriaosylceramide).15
Indeed, the lectin has a strong specificity for αGal terminating
oligosaccharides and glycoconjugates, while lactose or other
βGal oligosaccharides are not bound. However, β-D-galactopyra-
nosides possessing an aromatic aglycon were reported to be
efficient ligands for LecA.16 The affinity of LecA for p-nitrophe-
nyl β-D-galactopyranoside (PNP-β-Gal, Kd = 14 μM)12 or a larger
O-phenyl derivative (Kd = 6 μM)12 is stronger than that for
αMeGal or αGal disaccharides (50 to 100 μM).15 The recent
crystal structure of the LecA/PNP-β-Gal complex demonstrated
the stabilizing interaction between the aromatic ring and a his-
tidine residue.10 It is therefore of interest to further investigate
the role played by hydrophobic aglycones together with anome-
ric configurations in binding to LecA.
To this end, a series of galactopyranoside (β-D-Gal), lacto-
side (β-D-Gal(1–4)β-D-Glc), and melibioside (α-D-Gal(1–6)β-D-Glc)
Scheme 1 Syntheses of three families of thioglycosides under PTC conditions.
derivatives was prepared. These derivatives were synthesized Reagents and conditions: (i) HSR1 (see Table 1 for details), TBAHS, Na2CO3 1.0 M,
EtOAc, 12 h, r.t. (ii) NaOMe 1.0 M, MeOH, o.n., r.t.
keeping in mind their potential usage as therapeutic agents
and were thus prepared as hydrolytically stable S-glycosides.
Additionally, given the possible common binding modes
transfer catalyzed (PTC) reaction on the readily and commer-
cially available glycosyl bromides 1–3. The desired arylthio gly-
cosides were all obtained in good to excellent yields (Table 1).
The mild conditions and complete anomeric stereoselectivity
of the PTC reaction gave access to pure β-anomers without
having to use various protecting groups or harsher reaction
conditions, such as Lewis acid catalyzed conditions that might
damage the anomeric integrity of the desired thioglycosides.25
The anomeric configurations of all molecules have been
between the P. aeruginosa bacterial lectin LecA and the family
of galectins toward aryl β-D-galactopyranosides,17–19 we
included the syntheses of 3-O-substituted analogs, a modifi-
cation that was beneficial for the human galectin series.20
Results and discussion
Chemistry
1
Previous studies by our group18–20 and others22 have uncovered
that S-aryl galactopyranosides were potent inhibitors for
human galectins. Moreover, recent biological evaluations have
proven that the use of S-naphthyl group at the anomeric posi-
tion led to positive adhesion of the inhibitors to the target
lectin.20 Synthesis of a family of inhibitors based on the
S-naphthyl moiety could be easily achievable through the phase
transfer catalysis (PTC) reaction developed in our research labo-
ratories.23,24 As an initial step towards the synthesis of potent
LecA inhibitors, we focused our work on the syntheses of two
families of inhibitors. The first family relied on modifications
to the aglyconic part of the three sugars discussed above using
the PTC reaction. The second family was focusing on the
functionalization at the O-3 position of the glycosides using the
Sonogashira and Glaser–Hay coupling reactions. The purpose of
the second library family was to narrow the complexity of the
oligosaccharide moiety by keeping only the key galactopyrano-
side in direct contact with the LecA receptors. These modifi-
cations were encouraged by the modeling analysis carried out
on the naphthylsulfonyl lactoside C described in Fig. 1.
proven by H NMR and correlation experiments (COSY) as all
JH1,2 coupling constants were in the range of 8–10 Hz, thus
establishing the 1,2-trans relationship at the anomeric centers.
For each of the three family members 1–3 (galactoside (1), lacto-
side (2), and melibioside (3)), 2-naphthylthiol, 4-fluorobenze-
nethiol, and 7-mercapto-4-methylumbelliferone were used to
afford thioglycosides 4–6, 7–8, and 9–10, respectively
(Scheme 1, Table 1). For comparison purposes, the commer-
cially available fluorogenic substrates 3-carboxyumbelliferyl β-D-
galactopyranoside (CUG) and resorufin β-D-galactopyranoside
(RG) (Scheme 1) were added to the panel of thioglycosides.
Based on previous biological data15 and on molecular mod-
eling, the second family of analogs was derived from naphthyl
1-thio-β-D-galactopyranoside (4) (Scheme 2). The main purpose
of this series was to further functionalize the O-3 position to
increase potential binding interactions between the sugar
inhibitor and the receptor by taking advantage of additional
contacts from the lectin’s binding site. This series of inhibitors
was synthesized using three different routes starting from a
regiochemically installed 3-O-propargyl ether on 4 to afford
intermediate 18 using tin chemistry. The common intermedi-
ate 18 was prepared from compound 4 by classical Zemplén
The stepwise synthesis of the targeted three thioglycoside
families is described in Scheme 1. It started with a phase
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Table 1 Thioglycosides as potential inhibitors of LecA synthesized by PTC
reaction
Entry R1
1
Producta
Yieldb,c (%)
91 (88)
2
3
88 (>95)
94 (88)
4
5
85 (86)
51 (90)
6
7
67 (92)
Scheme 2 Functionalization on the O-3 position of 2-naphthyl 1-thio-β-D-
galactopyranoside. Reagents and conditions: (a) NaOMe 1.0 M, MeOH, 24 h, r.t.;
(b) (i) Bu2SnO, MeOH, reflux, 3 h, (ii) TBAI, propargyl bromide, dioxane, reflux
3 h, (iii) Ac2O, pyridine, 24 h, r.t.; (c) CuSO4·5H2O, sodium ascorbate, THF–H2O, r.
t.; (d) propargyl alcohol, CuCl, TMEDA, DMF, O2, 40 °C; (e) Ar–I, Pd(PPh3)2Cl2,
CuI, Et3N, DMF, 3 h, r.t.; (f) Pd/C, H2, MeOH, 5 h, r.t.
56 (>95)
Secondly, by sequentially using Glaser–Hay27 alkyne oxi-
dative homo- and cross-coupling of 18 with propargyl alcohol,
heterodimer 21 followed by homodimer 22 were prepared.
Toward this second series, propargylic ether 18 was treated
with propargyl alcohol under Glaser–Hay coupling conditions
(CuCl, TMEDA, DMF, O2, 40 °C)29 to provide 21 in 57% yield.
The homodimer was similarly obtained under the above con-
ditions (without propargylic alcohol) to give compound 22 in
77% yield.
The third series was prepared using palladium catalyzed
Sonogashira28 cross coupling with various aryl groups at the
propargylic carbon after which intermediates 30–36 could be
obtained in good yields after deprotection. To this end, com-
pound 18 was subjected to the usual coupling conditions with
diverse aryl iodides (Pd(PPh3)2Cl2, CuI, Et3N, DMF, 3 h, r.t.) to
afford compounds 25–29 in yields ranging from 54 to 77%. Of
these, compounds 30 and 33 were subjected to hydrogenation
under a hydrogen atmosphere to provide analogs 35 and 36 in
order to give further flexibility to the side chain of the newly
formed molecules (Scheme 2).
a Numbers in parentheses correspond to de-O-acetylated products.
b PTC reaction yields. c Deprotection yields are shown in parentheses.
de-O-acetylation (NaOMe, MeOH) to provide unprotected thio-
galactoside 11 in 88% yield. Compound 11 was then treated
with dibutyltin oxide in methanol under reflux to give the
intermediate tin ketal (not isolated) which was then heated
under reflux in the presence of propargyl bromide and tetra-
butylammonium iodide in dioxane. The resulting crude
product was peracetylated in pyridine and acetic anhydride to
afford key intermediate 18 in 88% yield over three steps.
Then, “Click Chemistry”26 was used in order to attach a tria-
zole moiety at the O-3 position of the galactoside 18 to provide
derivative 19. For this, compound 18 was treated with methyl
azidoacetate under standard conditions (CuSO4·5H2O, sodium
ascorbate, THF–H2O, r.t.) to give the expected adduct 19 in
68% yield which upon deprotection under Zemplén conditions
(NaOMe, MeOH) gave compound 20. The low yield (13%) for
this simple deprotection may be accounted for by the presence
of traces of water in the methanolic solution, hence providing
the corresponding acid directly (vide infra).
Lectin inhibition
The ability of the molecules described above to inhibit the
binding of LecA lectin to a galactosylated surface was deter-
mined by an enzyme-linked lectin assay (ELLA) using its bio-
tinylated form. Related IC50 values are listed in Table 2
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Table 2 Inhibition of LecA binding to polymeric α-Me-Gal tested by ELLA.
Methyl α-D-galactopyranoside was used as the reference to provide the relative
inhibitory potency (RIP)
Inhibitora
Mean IC50 (μM)
RIP
α-MeGal
11
12
70
3
20
5
1
23
3.5
14
13
14
16
17
1490
170
50
0.05
0.4
1.4
23
24
30
31
32
33
34
35
380
190
1490
1450
1670
810
1520
1510
1670
0.18
0.37
0.05
0.05
0.04
0.09
0.05
0.05
0.04
37
a Thioaryl aglycones alone and compounds 15, 20, and 36 gave no
inhibition at the tested concentration.
Fig. 2 Microcalorimetry data. The ITC plot (measured by VP-ITC, Microcal)
obtained for the titration of LecA with compound 11. The plot in the lower
panel shows the total heat released as a function of total ligand concentration
for the titration shown in the upper panel. The solid line represents the best
least-square fit to experimental data using a one site model.
together with inhibitory potency relative to αMeGal. When
looking at disaccharides, lactose-based compound (14) was not
a good inhibitor, while melibiose-containing molecules (16
and 17) with αGal(1–6)Glc linkage were slightly better than the
monosaccharide. These data are in good agreement with a pre-
vious analysis of specificity toward LecA performed by inhi-
bition of hemaglutination30 or by a glycan array analysis.15
LecA is indeed described as an αGal binding lectin with a pre-
ference for 1–6, 1–4, and 1–3 linkages. Our present results
indicate that in most cases, LecA binding to these disacchar-
ides was not affected by the presence of a large aromatic group
at the anomeric position. Substitutions on position 3 of the
galactose ring also showed a negative effect on the relative
inhibitory potency. Only compound 24 with two galactose
rings bridged by their O3 oxygen retained some activity, albeit
lower than the monosaccharide standard. All β-D-galactopyra-
noside derivatives with an aromatic ring at the anomeric posi-
tion were good inhibitors, the best one being compound 11
with an IC50 of 3 μM, a 23-fold enhancement over the reference
αMeGal. Compound 13, which contains a large thiocoumaryl
group at C1, had a similar inhibitory potency (5 μM, 14-fold
enhancement). These results confirm the previous observation
that galactose derivatives with aromatic groups in the β-con-
figuration represent very efficient ligands toward LecA.12,16
Interestingly, while compounds 11 and 13 were rather
similar in terms of aglycon, and have close affinity for LecA,
the contributions to the thermodynamics of binding were
different. In both cases the interaction was driven by enthalpy,
but the entropy cost of binding was significantly higher for 13
(−TΔS = 17.1 kJ mol−1) than for 11 (−TΔS = 3.8 kJ mol−1). This
could be correlated to the presence of oxygen atoms in the
aglycon of compound 13 that could act as hydrogen bond
acceptors and therefore stabilize the conformation of some
protein side chains. Commercial compounds with the aro-
matic group in β-configuration of galactose such as p-nitrophe-
nyl β-D-galactopyranoside (βPNPGal), 3-carboxyumbelliferyl
β-D-galactopyranoside (CUG) and resorufin β-D-galactopyrano-
side (RG) were also tested (Table 3). All were good ligands for
LecA, although the observed dissociation constants were
Table 3 Microcalorimetry data for higher affinity compounds binding to LecA.
The experiments were realized in duplicate at 298 K unless otherwise stated
−ΔG
−ΔH
TΔS
Isothermal titration calorimetry
Ligand
KD (μM)
(kJ mol−1
)
(kJ mol−1
)
(kJ mol−1
)
In order to evaluate the affinity of the best compounds in solu-
tion, titration microcalorimetry experiments (ITC) were run on
the highest affinity monosaccharide derivatives 11 and 13 and
disaccharide derivative 17. A typical thermogram of LecA inter-
acting with 11 is given in Fig. 2. An analysis of the Kd values
indicated that both compounds 11 and 13 were high affinity
ligands for LecA with dissociation constants of 5 to 6 μM. This
is 10 times better than the affinity observed for galactose,
methyl galactoside or digalactoside.15,31
αMeGal
βMeGal
βPNPGal
11
50.0 0.7
55.7 3.6
26.1 0.4
6.3 0.4
5.4 0.3
19.6 0.3
11.4c
24.6
24.3
26.2
29.7
30.1
26.9
28.2
29.3
40.9 0.3
19.0 0.5
44.6 0.3
33.5 3.3
47.2 1.5
33.4 0.3
37.4
16.3
−5.3
−18.4
−3.8
−17.1
−6.5
−9.2
−0.2
13
17
CUGa
RGb
7.3c
28.5
a CUG: 3-carboxyumbelliferyl β-D-galactopyranoside. b RG: resorufin
β-D-galactopyranoside. c Only one experiment.
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weaker than those obtained with compounds 11 and 13. The
melibioside-based compound 17 was slightly less efficient with
a KD of 19 μM.
X-Ray data
A crystal of LecA complexed with compound 11 was obtained
by co-crystallization, and diffraction data were collected at a
resolution of 2.15 Å (Table S1†). The asymmetric unit con-
tained one tetramer of LecA together with four calcium ions,
four molecules of 11 and 235 water molecules (Fig. 3). Each
monomer of LecA adopts the β-sandwich fold that was
described previously.2 The tetrameric association is the same
as the one described in previous crystal structures.2,12,15,31 The
overall structure of the lectin will therefore not be further
described herein. Each binding site contains one calcium ion
and one ligand that coordinates the calcium ion through
oxygens O3 and O4, and establishes hydrogen bonds between
the protein and all its hydroxyl groups as described in Fig. 4.
One water molecule bridges hydroxyl O6 to the main chain
and its importance in maintaining the O6 conformation and
in the binding energy has been previously emphasized.31
While the galactose moiety was bound in the same orien-
tation in the four sites of the tetramer, the naphthyl moiety
displayed two different conformations in sites A and D in com-
parison to sites B and C. As displayed in Fig. 4E, the aromatic
ring was much closer to the protein surface in chain B than in
chain A. Such an orthogonal interaction between the aromatic
Fig. 4 Comparison of the two binding modes of 11 by LecA. A and B: com-
ring of the aglycon and His50 has been observed in the crystal pound 11 observed in the binding site of chains A and B of LecA respectively
with a hydrogen bond network. C and D: the same molecules with the represen-
structure of LecA complexed with p-nitrophenyl β-galactopyra-
tation of the accessible surface for the protein. E: superimposition of the
noside and has been described as T-stack interaction.12 In
binding sites of chain A (green) and chain B (blue) with the representation of
chains B and C, a distance of 3.1 Å is measured between Cε1
the two different conformations of Gln53. F: superimposition of the binding
of His50 and the center of mass of the first 6-member ring,
sites of LecA complexed with 11 (chain B) and PNP-Gal (PDB code 3ZYF).
which is shorter than that in chains A and D (3.8 Å).
The difference in the naphthyl group orientation between
chain A/D and chain B/C is due to changes in both Φ and Ψ
torsion angles at the glycosidic linkage (Φ = −84.0 3.3°, Ψ =
−166.6 0.4° for chain A/D and Φ = −54.6 6.3°, Ψ = +170.5 0.1°
for chain B/C). This change in the ligand conformation
was correlated to a change in orientation of the Gln53 side
chain. In chains B and C, the Gln53 side chain is oriented
towards the ligand and establishes a hydrogen bond with the
O6 hydroxyl of galactose, while in chains A and D, it is
oriented towards the solvent. In contrast, His50 does not
adopt different conformations in the different chains and the
hydrogen bond between the His50 Nε2 atom and O6 of galac-
tose is maintained. Comparison with the crystal structure of
the LecA/PNPGal complex (PDB code 3ZYF)12 indicates that the
naphthyl group in chains B and C comes closer to the protein
surface than the phenyl group in the PNP-Gal complex (a dis-
tance of 3.5 Å between Cε1 of His50 and the center of the aro-
matic group). As shown in Fig. 4F, this is due to the difference
of conformation for the Φ torsion angle of the glycosidic
linkage (Φ = −81.6 3.8°, Ψ = +168.9 6.0° for PNP-Gal), and
also to the smaller value of the C–S–C valence angle (Θ = 104.3
1.6°) compared to the C–O–C one (Θ = 109.7 1.3°).
Conclusions
The search for potent inhibitors of P. aeruginosa adhesion to
human tissue is an active field of research. Most synthesized
compounds that target LecA efficiently are multimeric,32–36
Fig. 3 Crystal structure of LecA complexed with compound 11. Each protein
chain is represented by a ribbon with different colors. Calcium ions are depicted
by green spheres and the ligand by sticks.
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taking advantage of the closely located four galactose binding EtOAc (1 mL per 100 mg of compound). Then, the nucleophile
sites on the lectin.37 Such compounds display high avidity for (2.0 eq.) was added, followed by TBAHS (1.5 eq.). Finally, the
the lectins but they should result in lectin-mediated aggrega- 1.0 M sodium carbonate solution (1 mL per 100 mg) was
tion of bacteria. The resulting formation of microcolonies may added to the reaction. The yellowish mixture was stirred for
be counterproductive in terms of antimicrobial strategy. There- 24 h at r.t. Upon reaction completion, the reaction mixture was
fore, the design and synthesis of a monovalent and specific diluted with EtOAc (50 mL), washed twice with a saturated
LecA ligand is a target of paramount interest. The aromatic solution of sodium carbonate, washed once with brine, dried
thioglycosides described herein constitute an innovative class on anhydrous magnesium sulfate, filtered and concentrated.
of inhibitors for LecA that present two advantages: the high The crude oil was purified by silica gel chromatography (3/7
affinity (low micromolar range) provided by the aromatic EtOAc and hexanes as the eluent) to provide the desired
aglycon and the chemical stability in biological medium pro- product.
vided by the glycosidically stable thio linkage. This illustrates
the strength of using intensive QSAR in glycobiology in the
search for potent glycomimetic therapeutics, as we demon-
strated previously for LecB. A complex trisaccharide used as a
natural ligand could be replaced by a simple monosaccharide
derivative.38–40 In addition, the present investigation opens the
door to seriously reconsideration of the limitations that the
community has imposed on itself to strictly obey the anomeric
linkage observed for naturally occurring oligosaccharide
ligands while typical medicinal chemistry pharmacophores
have the potential to overcome such criteria. During the cre-
ation of this manuscript, further work was published.47
General procedure for the Zemplén de-O-acetylation (Method B).
Acetylated compounds (1.0 eq.) were dissolved in methanol
(0.2 M) under a nitrogen atmosphere. Sodium methoxide
1.0 M (0.1 eq.) was added until pH 9. The reaction mixture was
stirred for 18 h at r.t. Upon reaction completion, the reaction
mixture was neutralized to pH 7 using Dowex 50X8 H+ resin,
filtered and concentrated to obtain the desired product.
General procedure for O-3 selective propargylation (Method
C). The thiogalactoside (1.0 eq.) was dissolved in MeOH
(0.2 M) under a nitrogen atmosphere. Dibutyltin oxide (1.1 eq.)
was added and the reaction was heated to reflux for 3 h. Then,
the solvent was removed and the crude product was dissolved
in dioxane (0.2 M) under a nitrogen atmosphere. Tetrabutyl-
ammonium iodide (0.4 eq.) and propargyl bromide (5.0 eq.)
were added to the mixture which was heated to reflux for 5 h.
Upon reaction completion, the solvent was removed and the
crude material was dissolved in pyridine (0.2 M) under a nitro-
gen atmosphere. Acetic anhydride (0.2 M) was added and the
reaction mixture was stirred at r.t. for 18 h. The reaction
mixture was diluted with EtOAc (25 mL) and washed twice
with a 1.0 M HCl solution, washed once with brine, dried over
anhydrous magnesium sulfate, filtered and concentrated. The
crude material was purified by silica gel chromatography using
EtOAc and hexanes in a 3/7 ratio as the eluent to obtain the
desired product.
General procedure for the click chemistry (Method D). To a
solution of 18 (1.0 eq.) in THF (0.2 M) was added copper(II)
sulfate pentahydrate (0.4 eq.) dissolved in water (0.2 M).
Sodium ascorbate (0.2 eq.) and methyl azidoacetate (2.0 eq.)
were then added to the solution. The reaction mixture was
stirred for 24 h at room temperature. Once the reaction ended,
as judged by tlc, the reaction mixture was diluted with dichlor-
omethane (10 mL) and the solution was washed with 5% EDTA
and once with brine. The organic layer was dried over anhy-
drous magnesium sulfate, filtered and concentrated under
vacuum. The crude product was purified by silica gel flash
chromatography using a 1/9 (v/v) mixture of methanol and
dichloromethane to provide the desired product.
Experimental section
General methods
All reactions in organic media were carried out under a nitro-
gen atmosphere using freshly distilled solvents. After work-up,
organic phases were dried over anhydrous Na2SO4. The evol-
ution of reactions was monitored by analytical thin-layer
chromatography using silica gel 60 F254 precoated plates
(E. Merck). Purifications by column chromatography were per-
formed using silica gel 60 (40–63 μm) with the indicated
eluent. Optical rotations were measured using a JASCO P-1010
polarimeter. Melting points were measured using a Fisher
Jones apparatus. Roman numerals in ascending order are
given to the residues from the reducing end. NMR spectra
were recorded using Varian Gemini 300 and Gemini 500 spec-
trometers. Proton and carbon chemical shifts (δ) are reported
in ppm downfield from TMS and/or with the internal reference
of residual solvents. Coupling constants (J) are reported in
hertz (Hz), and the following abbreviations are used: singlet
(s), doublet (d), doublet of doublets (dd), triplet (t), multiplet
(m), and broad (b). Analysis and assignments were made using
COSY, DEPT, and HSQC experiments. Low-resolution (MS)
and high-resolution mass spectra (HRMS) were recorded by
Dr Alexandra Furtos and Karine Venne (Mass Spectrometry
Laboratory, Université de Montréal, Québec, Canada).
General procedure for the Glaser–Hay coupling (Method E).
Propargyl ether 18 (1.0 eq.) and copper(I) chloride (0.3 eq.)
were dissolved in DMF (0.2 M). Tetramethylethylenediamine
(TMEDA, 0.6 eq.) was added followed by propargyl alcohol
(4.0 eq.) (for the heterocoupling leading to 21). Then, the reac-
tion mixture was stirred for 4 h at 40 °C under a positive
pressure of oxygen. Once the reaction has terminated, as
Material. 3-Carboxyumbelliferyl
β-D-galactopyranoside
(CUG) and resorufin β-D-galactopyranoside (RG) were pur-
chased from Marker Gene Technologies (Eugene, OR).
Chemistry
General procedure for phase transfer catalyzed reaction
(Method A). The glycosyl bromides (1.0 eq.) were dissolved in
This journal is © The Royal Society of Chemistry 2013
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judged by tlc, 10 mL of ethyl acetate and 1.0 mL of hexanes 86.3, 74.4, 71.93, 7710, 61.5, 20.8, 20.7, 20.6; 19F NMR
were added. The solution was washed once with a 5% aqueous (540 MHz, CDCl3) δ −115.5; HRMS (ESI+) C20H23FO9S: Calcd:
solution of EDTA, twice with water, and once with brine. The 481.0955 [M + Na]+; Found: 481.0950 [M + Na]+.
organic layer was dried over anhydrous magnesium sulfate, fil-
4-Methylumbellifer-7-yl
2,3,4,6-tetra-O-acetyl-1-thio-β-D-
tered, and concentrated under vacuum. The crude product was galactopyranoside (6): Method A: Starting with compound 1
purified by silica gel flash column chromatography using a (100 mg, 0.256 mmoles) afforded 6 (125 mg, 94%) as a white
1/1 mixture of ethyl acetate and hexanes to afford the pure foam. [α]2D0 = −21.3 (c = 0.81 CHCl3); 1H NMR (300 MHz,
product.
CDCl3) δ 7.50–7.57 (m, 2H), 7.28–7.32 (m, 1H), 6.28 (bs, 1H),
5.48 (d, 1H, J = 3.3 Hz, H4), 5.33 (t, 1H, J = 9.9 Hz, H2), 5.11
(dd, 1H, J = 9.9 Hz, J = 3.3 Hz, H3), 4.84 (d, 1H, J = 9.9 Hz, H1),
4.20 (d, 2H, J = 5.9 Hz, H6a+b), 4.04 (t, 1H, J = 5.8 Hz, H5), 2.43
(s, 3H, CH3), 2.19 (s, 3H, AcO), 2.13 (s, 3H, AcO), 2.09 (s, 3H,
AcO), 1.99 (s, 3H, AcO); 13C NMR (75 MHz, CDCl3) δ 170.5,
170.1, 169.9, 169.3, 153.5, 151.8, 138.4, 125.9, 124.5, 118.9,
117.8, 115.0, 85.2, 74.8, 71.7, 67.1, 66.6, 61.8, 20.7 (2×), 20.6,
20.5, 18.5; HRMS (ESI+) C24H26O11S: Calcd: 545.1093 [M + Na]+;
Found: 545.1092 [M + Na]+.
General procedure for the Sonogashira coupling (Method F).
Dichlorobis(triphenylphosphine)palladium (Pd(PPh3)2Cl2, 0.05
eq.), copper(I) iodide (0.025 eq.) and the corresponding aryl
iodide (2.0 eq.) were dissolved in DMF (0.2 M) under a nitro-
gen atmosphere using a pretreatment of 5 min under ultra-
sound. Propargyl ether 18 (1.0 eq.) was then added, followed
by triethylamine (5.0 eq.). The reaction mixture was stirred for
3 h at room temperature. Once the reaction ended, the solvent
was evaporated under vacuum and the residue was dissolved
in ethyl acetate (10 mL). The solution was washed once with a
saturated solution of ammonium chloride, twice with water,
and once with brine. The organic layer was dried over anhy-
drous magnesium sulfate, filtered and concentrated under
vacuum. The crude product was purified by silica gel flash
column chromatography using a 3/7 (v/v) mixture of ethyl
acetate and hexanes to give the series of products 25–29.
General procedure for the hydrogenolysis (Method G). Com-
pound 30 or 33 (1 eq.) was dissolved in MeOH to which was
added 10% palladium on charcoal (10% w/w). Hydrogen gas
was bubbled into the reaction mixture until starting materials
disappeared as judged by tlc. The reaction mixture was filtered
over a celite pad and the filtrate was concentrated under
vacuum. The crude products were purified by silica gel flash
column chromatography using a mixture of MeOH and DCM
(1 : 9).
2-Naphthyl
2′,3′,4′,6′,2,3,6-hepta-O-acetyl-1-thio-β-D-lacto-
pyranoside (7): Method A: Starting with compound 2 (2.0 g,
2.94 mmoles) gave 7 (1.94 g, 85%) as a white foam; [α]D20
=
1
−17.2 (c = 1, CHCl3); mp. 126.3–127.0 °C; H NMR (300 MHz,
CDCl3) δ 7.97 (bs, 1H), 7.69–7.89 (m, 3H), 7.47–7.57 (m, 3H),
5.33 (d, 1H, J = 3.5 Hz), 5.23 (tapp, 1H, J = 9.1 Hz), 5.06–5.12 (m,
1H), 4.89–4.97 (m, 2H), 4.74 (d, 1H, J = 9.8 Hz), 4.44 (m, 2H),
4.06–4.13 (m, 3H), 3.82–3.87 (m, 1H), 3.62–3.77 (m, 2H),
2.11–2.14 (m, 6H), 2.01–2.08 (m, 12H), 1.95 (s, 3H); 13C NMR
(75 MHz, CDCl3) δ 170.4, 170.3, 170.1, 169.8, 169.7, 169.2,
133.5, 132.9, 130.4, 128.8, 128.5, 127.8, 126.8, 126.7, 101.1,
85.4, 76.8, 76.2, 73.9, 71.1, 70.8, 70.4, 69.2, 66.8, 62.2, 61.0,
20.9, 20.7, 20.6. MS (ESI+) C36H42O17S1: Calcd: 801.2 [M + Na]+;
Found: 801.3 [M + Na]+.
4-Fluorophenyl 2′,3′,4′,6′,2,3,6-hepta-O-acetyl-1-thio-β-D-lac-
topyranoside (8): Method A: Starting with compound
2
2-Naphthyl 2,3,4,6-tetra-O-acetyl-1-thio-β-D-galactopyrano-
(150 mg, 0.221 mmoles) gave 9 (83 mg, 51%) as a white foam.
[α]2D0 = −1.76 (c = 1.4 acetone); 1H NMR (300 MHz, CDCl3) δ
7.46–7.51 (m, 2H), 6.98–7.05 (m, 2H), 5.33 (d, 1H, J = 3.2 Hz,
H4gal), 5.20 (t, 1H, J = 9.0 Hz, H3glu), 5.09 (dd, 1H, J = 7.8 Hz,
H2gal), 4.94 (dd, 1H, J = 10.4 Hz, J = 3.3 Hz, H3gal), 4.82 (t, 1H,
J = 9.5 Hz, H2glu), 4.51–4.58 (m, 1H, H1glu), 4.46 (d, 1H, J = 7.8 Hz,
side (4): Method A: Starting with compound
7.69 mmoles) gave 5 (3.44 g, 91%) as a white foam. H NMR
(600 MHz, CDCl3) δ 7.97 (s, 1H), 7.73–7.78 (m, 3H), 7.53 (d,
1 (3.0 g,
1
1H, J = 8.3 Hz), 7.44–7.46 (m, 1H), 5.38 (bs, 1H, H4), 5.24 (tapp
,
1H, J = 10.0 Hz, H2), 5.03 (dd, 1H, J = 8.7 Hz, H3), 4.76 (d, 1H,
J = 10.0 Hz, H1), 4.15–4.18 (m, 1H, H6a), 4.07–4.10 (m, 1H, H6b),
3.91 (tapp, 1H, J = 6.3 Hz, H5), 2.08 (s, 3H), 2.03 (s, 3H), 1.97 (s,
3H), 1.93 (s, 3H); 13C NMR (125 MHz, CDCl3) δ 170.3, 170.1,
169.9, 169.3, 133.3, 132.6, 131.8, 129.6, 129.5, 128.3, 127.6,
127.5, 126.5, 86.5, 74.4, 71.9, 67.2, 61.6, 20.8, 20.6, 20.5.
The spectral data corresponded to those reported in the
litterature.41
H1gal), 4.02–4.16 (m, 4H, H6a+b
+ H6a+b glu), 3.85 (m, 1H,
gal
H5gal), 3.71 (t, 1H, J = 9.4 Hz, H5glu), 3.56–3.63 (m, 1H, H4glu),
2.15 (s, 3H), 2.11 (s, 3H), 2.10 (s, 3H), 2.04 (m, 9H), 1.96 (s,
3H); 13C NMR (75 MHz, CDCl3) δ 204.7, 170.33, 170.18, 170.10,
169.7, 169.5, 168.9, 136.5, 116.0, 101.0, 85.0, 76.7, 75.9, 73.7,
70.9, 70.6, 70.0, 69.0, 66.5, 61.8, 60.6, 20.83, 20.7 (2×), 20.6,
20.5, 20.4; 19F NMR (540 MHz, CDCl3) δ −115.2; HRMS (ESI+)
C32H39FO17S; Calcd: 769.1789 [M + Na]+; Found: 769.1789
[M + Na]+.
4-Fluorophenyl 2,3,4,6-tetra-O-acetyl-1-thio-β-D-galactopyra-
noside (5): Method A: Starting with compound 1 (150 mg,
0.384 mmoles) gave 6 (155 mg, 88%) as a white foam. [α]D20
=
2-Naphthyl 2′,3′,4′,6′,2,3,4-hepta-O-acetyl-1-thio-β-D-melibio-
pyranoside (9): Method A: Starting with compound 3 (154 mg,
1
15.5 (c = 0.8 acetone); H NMR (300 MHz, CDCl3) δ 7.46–7.51
(m, 2H), 6.95–7.01 (m, 2H), 5.35 (d, 1H, J = 3.3 Hz, H4), 5.13
(tapp, 1H, J = 9.8 Hz, H2), 4.99 (dd, 1H, J = 3.3 Hz, J = 6.6 Hz,
H3), 4.56 (d, 1H, J = 9.8 Hz, H1), 4.02–4.17 (m, 2H, H6a,b), 3.87
(tapp, 1H, J = 6.2 Hz, H5), 2.07 (s, 3H), 2.05 (s, 3H), 2.00 (s, 3H),
1.93 (s, 3H); 13C NMR (75 MHz, CDCl3) δ 203.8, 170.3, 170.1,
170.0, 170.0, 169.4, 164.7, 161.4, 135.9, 135.8, 116.0, 115.7,
0.220 mmoles) gave 9 (115.4 mg, 67%) as a white foam. [α]D20
=
1
49.4 (c = 1.1 CHCl3); H NMR (300 MHz, CDCl3) δ 7.94 (s, 1H),
7.78–7.87 (m, 3H), 7.48–7.56 (m, 3H), 5.23–5.33 (m, 3H), 5.15
(d, 1H, J = 3.5 Hz), 4.98–5.01 (m, 3H), 4.85 (d, 1H, J = 9.8 Hz)′
4.15 (tapp, 1H, J = 6.2 Hz), 3.92–4.04 (m, 2H), 3.71–3.79 (m,
2H), 3.57(d, 1H, J = 8.9 Hz), 2.12 (s, 3H), 2.11 (s, 3H), 2.08 (s,
6912 | Org. Biomol. Chem., 2013, 11, 6906–6918
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3H), 2.03 (s, 3H), 1.99 (s, 6H), 1.98 (s, 3H); 13C NMR (75 MHz, 86%) as a white foam. [α]2D0 = −26.9 (c = 1, DMSO); mp:
1
CDCl3) δ 170.2, 170.1, 170.0, 169.8, 169.7, 169.3, 169.2, 151.9, 217.5–218.2 °C; H NMR (600 MHz, DMSO-d6) δ 7.48–7.50 (m,
137.9, 125.2, 117.6, 115.0, 96.2, 76.8, 73.6, 69.5, 68.4, 67.9, 2H), 7.10–7.13 (m, 2H), 5.40 (d, 1H, J = 5.2 Hz), 5.03 (bs, 1H),
67.8, 67.3, 66.5, 66.4, 61.5, 20.7, 20.6, 18.5; HRMS (ESI+) 4.72 (bs, 1H), 4.58 (bs, 1H), 4.54–4.56 (m, 2H), 4.46 (bs, 1H),
C36H42O17S; Calcd: 778.2147 [M + H]+; Found: 801.2039 4.16 (d, 1H, J = 7.0 Hz), 3.69–3.72 (m, 1H), 3.57 (bs, 3H),
[M + Na]+.
3.39–3.47 (m, 5H), 3.24–3.34 (m); 13C NMR (125 MHz, DMSO-
4-Methylumbellifer-7-yl 2′,3′,4′,6′,2,3,4-hepta-O-acetyl-1-thio- d6) δ 162.9, 161.3, 134.0, 129.8, 116.5, 104.3, 87.4, 80.7, 79.4,
β-D-melibiopyranoside (10): Method A: Starting with compound 76.8, 76.1, 73.8, 72.6, 71.1, 68.7, 61.0; MS (ESI+) C22H28O10S;
3 (150 mg, 0.221 mmoles) gave 10 (99.6 mg, 56%) as a white Calcd: 484.1 [M]+; Found: 507.3 [M + Na]+.
1
foam. [α]2D0 = 19.9 (c = 0.73 CHCl3); H NMR (300 MHz, CDCl3)
4-Fluorophenyl 1-thio-β-D-lactopyranoside (15): Method B:
δ 7.63 (d, 1H, J = 8.6 Hz), 7.31–7.34 (m, 2H), 6.27 (bs, 1H), Starting with compound 8 (53 mg, 0.071 mmoles) afforded 15
5.26–5.32 (m, 3H), 4.99–5.14 (m, 4H), 4.90 (d, 1H, J = 10.0 Hz), (28.7 mg, 90%) as a white foam. [α]2D0 = −17.8 (c = 0.4 MeOH);
4.22 (tapp, 1H, J = 6.4 Hz), 4.04 (d, 2H, J = 7.0 Hz), 3.77–3.87 (m, 1H NMR (600 MHz, DMSO-d6) δ 7.48–7.50 (m, 2H), 7.10–7.13
2H), 3.57–3.62 (m, 1H), 2.44 (s, 3H), 2.13 (s, 3H), 2.12 (s, 3H), (m, 2H), 5.40 (d, 1H, J = 5.2 Hz), 5.03 (bs, 1H), 4.72 (bs, 1H),
2.08 (s, 3H), 2.06 (s, 3H), 2.01 (s, 3H), 2.00 (s, 3H), 1.97 (s, 3H); 4.58 (bs, 1H), 4.54–4.56 (m, 2H), 4.46 (bs, 1H), 4.16 (d, 1H, J =
13C NMR (75 MHz, CDCl3, δ, ppm); 170.6, 170.5, 170.4, 170.3, 7.0 Hz), 3.69–3.72 (m, 1H), 3.57 (bs, 3H), 3.39–3.47 (m, 5H),
169.8, 169.5, 133.6, 132.9, 132.1, 129.5, 129.2, 129.0, 128.0, 3.24–3.34 (m); 13C NMR (125 MHz, DMSO-d6) δ 162.9, 161.3,
127.7, 126.9, 96.5, 85.8, 76.8, 74.1, 70.3, 68.7, 68.2, 67.5, 66.9, 134.0, 129.8, 116.5, 104.3, 87.4, 80.7, 79.4, 76.8, 76.1, 73.8,
66.6, 61.8, 21.0, 20.9, 20.8; HRMS (ESI+) C36H42O19S; Calcd: 72.6, 71.1, 68.7, 61.0; 19F NMR (540 MHz, DMSO-d6) δ −115.7;
801.2044 [M + Na]+; Found: 801.2039 [M + Na]+.
HRMS (ESI+) C18H25FO10S; Calcd: 475.1049 [M + Na]+; Found:
2-Naphthyl 1-thio-β-D-galactopyranoside (11): Method B: 475.1055 [M + Na]+.
Starting with compound 4 (3.44 g, 7.02 mmoles) provided 11
2-Naphthyl 1-thio-β-D-melibiopyranoside (16): Method B:
(1.98 g, 88%) as a white foam. 1H NMR (600 MHz, pyridine-d5) Starting with compound 9 (87 mg, 0.112 mmoles) gave 16
δ 8.92 (s, 1H), 8.16 (m, 1H), 8.00–8.08 (m, 3H), 7.67 (m, 2H), (54 mg, 92%) as a white foam. [α]2D0 = 94.2 (c = 0.48 MeOH); 1H
5.61 (d, 1H, J = 9.5 Hz, H1), 4.85–4.89 (m, 2H, H2 + H4), 4.70 NMR (600 MHz, DMSO-d6) δ 7.91 (bs, 1H), 7.85 (d, 1H, J = 8.0
(dd, 1H, J = 6.8 Hz, H6a), 4.63 (dd, 1H, J = 5.2 Hz, H6b), 4.48 Hz), 7.81 (d, 2H, J = 8.8 Hz), 7.59 (d, 1H, J = 8.6 Hz), 7.42–7.47
(dd, 1H, J = 3.2 Hz, H3), 4.43 (tapp, 1H, J = 6.0 Hz, H5); 13C NMR (m, 2H), 5.35 (bs, 1H), 5.26 (bs, 1H), 5.17 (bs, 1H), 4.66 (d, 2H),
(125 MHz, pyridine-d5, δ, ppm); 134.5, 134.1, 132.6, 129.1, 4.47–4.50 9 m, 2H), 4.31 (bs, 1H), 3.56–3.65 (m, H), 3.41–3.47
128.9, 128.8, 128.3, 128.0, 127.1, 126.4, 90.0 (C1), 81.3 (C5), (m, 3H), 3.35–3.38 (m, 1H), 3.20 (tapp, 1H, J = 8.5 Hz), 3.06 (m,
76.7 (C3), 71.0 (C2), 70.4 (C4), 62.5(C6); HRMS (ESI+); Calcd: 2H); 13C NMR (125 MHz, DMSO-d6) δ 133.8, 133.0, 132.1,
322.0876; Found: 345.0768. Spectral and physical data corre- 128.9, 128.7, 128.6, 128.1, 128.0, 127.2, 126.5, 99.5, 87.7, 79.6,
spond to those reported in the litterature.20
78.8, 73.0, 71.5, 70.6, 70.2, 69.4, 69.0, 67.6, 61.2; HRMS (ESI+)
4-Fluorophenyl 1-thio-β-D-galactopyranoside (12): Method B: C22H28O10S; Calcd: 507.1303 [M + Na]+; Found: 507.1298
Starting with compound 5 (3.44 g, 7.02 mmoles) gave 12 [M + Na]+.
(78.8 mg, 100%) as a white foam. [α]2D0 = −39.1 (c = 0.76
4-Methylumbellifer-7-yl 1-thio-β-D-melibiopyranoside (17):
1
acetone); mp: 136.8–141.0 °C; H NMR (300 MHz, DMSO-d6) δ Method B: Starting with compound 10 (99.6 mg, 0.123 mmoles)
7.48–7.52 (m, 2H), 7.12–7.18 (m, 2H), 5.75 (bs, 1H), 5.13 (m, provided 17 (63.5 mg, 99%) as a white foam. [α]2D0 = 46.7 (c =
1H, H), 4.88 (bs, 1H, H4), 4.62 (bs, 1H, H1), 4.46–4.50 (m, 2H, 0.89 in MeOH); 1H NMR (600 MHz, DMSO-d6) δ 7.66 (d, 1H, J =
H2), 3.68 (m, 1H, H3), 3.39–3.48 (m, 4H); 13C NMR (75 MHz, 8.3 Hz), 7.43 (d, 1H, J = 8.4 Hz), 7.32 (bs, 1H), 6.29 (s, 1H,
pyridine-d5) δ 162.7, 161.1, 133.7, 133.2, 132.7 (2×), 131.3, Hallyl), 5.59 (bs, 1H), 5.41 (bs, 1H), 4.79 (d, 1H, J = 9.7 Hz,
128.0, 127.6, 127.5, 127.4, 127.1, 126.4, 125.7, 115.9, 115.8, H4Gal), 4.61 (bs, 1H), 4.50–4.53 (m, 2H), 4.38 (bs, 1H),
87.6, 86.4, 84.2, 81.8, 79.0, 68.1, 64.9, 60.4, 56.7; 19F NMR 3.48–3.61 (m, 8H), 3.37–3.39 (m, 1H), 3.22 (tapp, 1H, J = 8.7
(540 MHz, CDCl3) δ −114.8; HRMS (ESI+) C12H15FO5S; Calcd: Hz), 3.06–3.11 (m, 2H), 2.38 (s, 3H, Me), 1.56 (s, 1H); 13C NMR
313.0528 [M + Na]+; Found: 313.0524 [M + Na]+.
(125 MHz, DMSO-d6) δ 160.4, 153.9, 153.4, 141.6, 126.4, 124.8,
4-Methylumbellifer-7-yl 1-thio-β-D-galactopyranoside (13): 118.0, 115.8, 114.0, 99.5, 86.3, 79.6, 78.7, 73.1, 71.5, 70.5, 70.1,
Method B: Starting with compound 6 (83.5 mg, 0.159 mmoles) 69.4, 68.9, 67.4, 61.0, 18.7; HRMS (ESI+) C22H28O12S; Calcd:
gave 13 (49.8 mg, 88%) as a white foam. [α]2D0 = −54.9 (c = 0.33 539.1204 [M + Na]+; Found: 539.1200 [M + Na]+.
1
in CHCl3); H NMR (300 MHz, DMSO-d6); δ 7.62 (d, 1H, J = 8.6
2-Naphthyl 3-O-propargyl-2,4,6-tri-O-acetyl-1-thio-β-D-galac-
Hz), 7.38 (s, 1H), 7.31 (d, 1H, J = 8.2 Hz), 6.28 (s, 1H), 4.75 (d, topyranoside (18): Method C: Starting with compound 4
1H, J = 9.2 Hz), 3.67–3.69 (m, 1H), 3.41–3.56 (m, 5H), 3.29–3.36 (678 mg, 2.10 mmoles) gave 18 (892 mg, 88%) as a white foam.
(m, 4H), 2.44 (s, 3H); 13C NMR (75 MHz, DMSO-d6) δ 160.3, [α]2D0 = 43.1 (c = 0.6 CHCl3); 1H NMR (300 MHz, CDCl3) δ
153.7, 142.2, 126.0, 124.4, 117.7, 115.3, 113.9, 86.8, 79.9, 75.0, 8.02–8.00 (m, 1H), 7.82–7.75 (m, 3H), 7.59–7.56 (m, 1H),
69.5, 68.9, 61.1, 18.6; HRMS (ESI+) C16H18O7S; Calcd: 377.0673 7.50–7.46 (m, 2H), 5.42 (d, 1H, J = 3.4 Hz, H4), 5.14 (t, 1H, J =
[M + Na]+; Found: 377.0669 [M + Na]+.
9.8 Hz, H2), 4.80 (d, 1H, J = 9.8 Hz, H1), 4.23–4.12 (m, 4H, CH2
2-Naphthyl 1-thio-β-D-lactopyranoside (14): Method B: Start- + H6a+b), 3.92–3.85 (m, 2H, H5 + H3), 2.44 (t, 1H, J = 2.3 Hz,
ing with compound 7 (3.44 g, 7.02 mmoles) gave 14 (1.10 g, H–CC–CH2), 2.15 (s, 3H), 2.05 (s, 3H), 2.02 (s, 3H); 13C NMR
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(75 MHz, CDCl3) δ 171.2, 170.6, 170.5, 170.4 (2×), 170.3, 170.2, H6a+b + H6a,b′), 3.88 (t, 2H, J = 6.8 Hz, H5), 3.78 (dd, 2H, J = 9.4
169.5, 133.4, 132.6, 131.6, 129.9, 129.7, 129.6, 128.2, 127.6, Hz, H3), 2.15 (s, 6H, OAc(2×)), 2.04 (s, 6H, OAc(2×)), 2.00 (s,
127.5, 126.5, 126.4, 86.5, 78.9, 76.7, 76.5, 75.1, 74.6, 68.4, 65.6, 6H, OAc(2×)); 13C NMR (125 MHz, CDCl3) δ 170.4(2×), 169.5,
62.2, 56.4, 21.0, 20.6; IR (NaCl, cm−1); 3278, 3055, 2920, 2851, 133.4, 132.6, 131.7, 129.8, 129.6, 128.3, 127.6, 127.5, 126.5 (2×),
2118, 1747, 1372, 1228, 1068, 1050; HRMS (ESI+) C25H26O8S; 86.5, 77.3, 74.9, 74.5, 70.6, 68.3, 65.6, 62.1, 57.0, 20.9, 20.6; IR
Calcd: 509.1257 [M + Na]+; Found: 509.1252 [M + Na]+.
2-Naphthyl 3-(4-(hydroxymethyl)-1H-1,2,3-triazole-1-methyl HRMS (ESI+) C50H50O16S; Calcd: 993.2437 [M + Na]+; Found:
ester)-2,4,6-tri-O-acetyl-1-thio-β-D-galactopyranoside
(19): 993.2431 [M + Na]+.
(NaCl, cm−1); 3146, 3013, 2956, 1748, 1372, 1230, 1050, 752;
Method F: Starting with compound 18 (242 mg, 0.498 mmoles)
2-Naphthyl 3-O-hepta-2,4-diyn-1-ol-1-thio-β-D-galactopyrano-
gave 19 (205 mg, 68%) as a white foam. [α]2D0 = 42.3 (c = 1.9 side (23): Method B: Starting with compound 21 (30 mg,
1
CHCl3); H NMR (300 MHz, CDCl3) δ 7.95 (bs, 1H), 7.78–7.71 0.055 mmoles) gave 23 (23 mg, 99%) as a white solid. [α]D20
=
(m, 3H), 7.63 (s, 1H, Htriazole), 7.54–7.50 (m, 1H), 7.47–7.40 (m, 84.8 (c = 0.36 MeOH); 1H NMR (600 MHz, DMSO-d6) δ 8.02 (bs,
2H), 5.45 (d, 1H, J = 3.2 Hz, H4), 5.15–5.09 (m, 3H, H2 + CH2– 1H), 7.80–7.88 (m, 3H), 7.55–7.57 (m, 1H), 7.45–7.51 (m, 2H),
CO2Me), 4.74–4.59 (m, 3H, H1 + CH2–CvC–H), 4.19–4.08 (m, 5.46 (d, 1H, J = 6.3 Hz, H4), 4.72–4.78 (m, 3H, H2 + H1 + OH),
2H, H6a,b), 3.87–3.82 (m, 1H, H5), 3.78–3.71 (m, 4H, H3
+
4.40–4.49 (qapp, 2H, CH2), 4.18 (bs, 2H, OH), 3.94 (bs, 1H, OH),
OMe), 2.03–2.00 (m, 6H), 1.98 (s, 3H); 13C NMR (75 MHz, 3.60–3.64 (m, 1H, H3), 3.50–3.55 (m, 3H, H6a+b, OH), 3.40–3.42
CDCl3) δ 170.2, 170.1, 169.4, 166.4, 144.6, 133.2, 132.3, 131.0, (dd, 2H, J = 3.0 Hz, J = 5.8 Hz, H5); 13C NMR (125 MHz, DMSO-
129.9, 129.2, 128.0, 127.4, 127.3, 126.3, 126.2, 124.3, 86.1, d6) δ 133.9, 133.3, 132.0, 128.7, 128.3, 128.2, 127.8, 127.1,
77.69, 74.4, 68.5, 66.3, 62.7, 62.0, 52.7, 50.4, 20.6, 20.5, 20.4; IR 126.4, 88.2, 82.5, 80.7, 79.6, 77.1, 70.2, 68.7, 68.4, 65.4, 61.0,
(NaCl, cm−1); 3146, 3013, 2956, 1748, 1372, 1230, 1050, 752; 57.2, 49.9; IR (NaCl, cm−1); 3146, 3013, 2956, 1748, 1372, 1230,
HRMS (ESI+) C28H31N3O10S; Calcd: 602.1812 [M + H]+; Found: 1050, 752; HRMS (ESI+) C22H22O6S; Calcd: 437.1042 [M + Na]+;
602.1805 [M + H]+.
Found: 437.1037 [M + Na]+.
2-Naphthyl 3-(4-(hydroxymethyl)-1H-1,2,3-triazole-1-methyl
Deprotected dimer (24): Method B: Starting with compound
ester)-1-thio-β-D-galactopyranoside (20): Method B: Starting 22 (58 mg, 0.060 mmoles) gave dimer 24 (41 mg, 96%) as a
1
with compound 19 (189 mg, 0.314 mmoles) gave 20 (29 mg, white solid. [α]2D0 = 22.5 (c = 1.9 acetone); H (600 MHz, CDCl3)
13%) as a white foam. [α]2D0 = 15.4 (c = 0.6 MeOH); 1H NMR δ 7.98 (bs, 2H), 7.76–7.84 (m, 6H), 7.41–7.47 (m, 6H), 5.39 (d,
(300 MHz, CDCl3) δ 8.03 (bs, 2H), 7.81–7.90 (m, 3H), 7.46–7.55 2H, J = 6.2 Hz), 4.67–4.73 (m, 4H), 4.41 (m, 4H), 3.87–3.91 (m,
(m, 3H), 5.41 (s, 2H), 5.12 (tapp, 1H, J = 10.0 Hz), 40.97–5.00 2H), 3.56–3.60 (m, 2H), 3.46–3.51 (m, 8H), 3.37 (dd, 2H, J = 3.0
(m, 2H), 4.82 (bs, 1H), 4.72 (d, 1H, J = 12.2 Hz), 4.55 (d, 1H, J = Hz, 6.0 Hz); 13C NMR (75 MHz, CDCl3) δ 133.9, 133.3, 132.0,
12.3 Hz), 4.10 (bs, 1H), 3.58–3.70 (m, 7H); 13C NMR (75 MHz, 128.6, 128.3, 128.2, 127.8, 127.1, 126.4, 88.2, 82.6, 79.6, 77.6,
CDCl3) δ 169.9, 168.3, 144.8, 133.8, 132.3, 128.9, 128.6, 128.3, 70.0, 68.7, 65.4, 61.0, 57.2; IR (NaCl, cm−1); 3146, 3013, 2956,
127.9, 127.2, 126.6, 125.8, 85.5, 80.2, 79.9, 69.6, 65.1, 62.2, 1748, 1372, 1230, 1050, 752; HRMS (ESI+) C38H38O10S; Calcd:
61.0, 53.1, 50.8, 21.3; IR (NaCl, cm−1); 3146, 3013, 2956, 1748, 741.1801 [M + Na]+; Found: 741.1796 [M + Na]+.
1372, 1230, 1050, 752; HRMS (ESI+) C22H25N3O7S; Calcd:
476.1492 [M + H]+; Found: 476.1486 [M + H]+.
2-Naphthyl
3-(3-phenyl-2-propyn-1-hydroxy)-2,4,6-tri-O-
acetyl-1-thio-β-D-galactopyranoside (25): Method F: Starting
2-Naphthyl 3-O-hepta-2,4-diyn-1-ol-2,4,6-tri-O-acetyl-1-thio- with compound 18 (130 mg, 0.267 mmoles) afforded 25
1
β-D-galactopyranoside (21): Method E: Starting with compound (75 mg, 77%) as a white foam. [α]D20 = 60.5 (c = 0.5 CHCl3); H
18 (315 mg, 0.647 mmoles) gave 21 (198.6 mg, 57%) as a white NMR (300 MHz, CDCl3) δ 8.03 (bs, 1H), 7.83–7.76 (m, 3H),
foam. [α]2D0 = 58.6 (c = 1.1 CHCl3); 1H NMR (600 MHz, CDCl3) δ 7.61–7.57 (m, 1H0, 7.52–7.43 (m, 4H), 7.36–7.30 (m, 3H), 5.52
7.99 (bs, 1H), 7.73–7.79 (m, 3H), 7.53–7.56 (m, 1H), 7.45–7.47 (d, 1H, J = 3.3 Hz, H4), 5.18 (t, 1H, J = 9.3 Hz, H2), 4.83 (d, 1H,
(m, 2H), 5.37 (d, 2H, J = 3.5 Hz, H4), 5.10 (t, 1H, J = 10.0 Hz, J = 9.3 Hz), H1), 4.41 (s, 2H, O–CH2–CC), 4.23–4.16 (m, 2H, H6a,
H2), 4.75 (d, 1H, J = 10.0 Hz H1), 4.31 (bs, 2H, CH2)) 4.22 (s, b), 3.99–3.91 (m, 2H, H3 + H5), 2.13 (s, 3H), 2.08 (s, 3H), 2.02 (s,
2H, CH2), 4.11–4.17 (m, 2H, H6a+b), 3.86 (t, 2H, J = 6.9 Hz, H5), 3H); 13C NMR (75 MHz, CDCl3) δ 170.4, 170.3, 169.6, 133.4,
3.75 (dd, 2H, J = 9.4 Hz, H3), 2.13 (s, 3H, OAc), 2.02 (s, 3H, 132.5, 131.6, 131.4, 130.0, 129.5, 128.6, 128.4, 128.3, 128.2,
OAc), 1.98 (s, 3H, OAc); 13C NMR (125 MHz, CDCl3) δ 170.3, 127.6, 127.5, 126.5, 126.4, 122.2, 86.7, 86.4, 76.7, 74.6, 68.5,
169.5, 169.4 133.5, 132.7, 131.9, 129.8, 128.3, 127.6, 126.5, 65.8, 62.2, 57.2, 20.9, 20.7, 20.6; IR (NaCl, cm−1); 3063,
86.5, 74.9, 70.9, 70.7, 69.6, 68.6, 65.8, 62.2, 60.3, 57.1, 51.3, 2937, 2854, 1748, 1371, 1226, 1066, 1049, 757; HRMS (ESI+)
20.9, 20.5; IR (NaCl, cm−1); 3146, 3013, 2956, 1748, 1372, 1230, C31H30O8S; Calcd: 585.1566 [M + Na]+; Found: 585.1562
1050, 752; HRMS (ESI+) C28H28O9S; Calcd: 563.1347 [M + Na]+; [M + Na]+.
Found: 563.1342 [M + Na]+.
2-Naphthyl 3-(3-(2-pyridine)-2-propyn-1-hydroxy)-2,4,6-tri-O-
Dimer 22: Method E: Starting with compound 18 (100.2 mg, acetyl-1-thio-β-D-galactopyranoside (26): Method F: Starting
0.206 mmoles) gave dimer 22 (72 mg, 77%) as a white foam; with compound 18 (97.2 mg, 0.200 mmoles) gave 26 (82 mg,
1H NMR (600 MHz, CDCl3) δ 8.00 (bs, 2H), 7.75–7.81 (m, 6H), 73%) as a white foam. [α]D20 = 63.9 (c = 0.4 CHCl3); 1H NMR
7.56–7.58 (m, 2H), 7.47–7.49 (m, 2H), 5.39 (d, 2H, J = 2.8 Hz, (300 MHz, CDCl3) δ 8.01 (bs, 1H), 7.88–7.71 (m, 4H), 7.60–7.57
H4 + H4′), 5.12 (t, 2H, J = 9.8 Hz, H2 + H2′), 4.77 (dd, 2H, J = (m, 1H), 7.51–7.45 (m, 2H), 7.33–7.25 (m, 3H), 5.51 (d, 1H, J =
10.0 Hz H1 + H1′), 4.25 (bs, 4H, CH2(2×)), 4.10–4.20 (m, 4H, 3.3 Hz, H4), 5.17 (t, 1H, J = 10.0 Hz, H2), 4.82 (d, 1H, J =
6914 | Org. Biomol. Chem., 2013, 11, 6906–6918
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10.0 Hz, H1), 4.43 (s, 2H, CH2), 4.25–4.14 (m, 2H, H6a+b),
2-Naphthyl 3-(3-phenyl-2-propyn-1-hydroxy)-1-thio-β-D-galac-
3.99–3.87 (m, 2H, H3 + H5), 2.13 (s, 3H), 2.08 (s, 3H), 2.02 (s, topyranoside (30): Method B: Starting with compound 25
3H); 13C NMR (75 MHz, CDCl3) δ 170.4, 170.3, 169.5, 151.5, (51 mg, 0.091 mmoles) gave 30 (32 mg, 82%) as a white solid.
148.2, 139.3, 133.4, 132.6, 131.6, 129.9, 129.6, 128.3 (3×), 128.2, [α]2D0 = −13.5 (c = 0.4 MeOH); mp: 158.6–161.1 °C; 1H NMR
128.0, 127.6 (2×), 127.5, 127.4, 126.5, 126.4, 88.3, 86.5, 77.2, (300 MHz, DMSO-d6) δ 7.98 (bs, 1H), 7.75–7.84 (m, 3H),
76.5, 74.6, 68.5, 65.8, 62.2, 57.2, 21.0, 20.6; IR (NaCl, cm−1); 7.32–7.53 (m, 8H), 5.42 (d, 1H, J = 6.1 Hz, H4), 4.68–4.76 (m,
3009, 2907, 1748, 1371, 1227, 1066, 1048; HRMS (ESI+) 2H, H2 + H1), 4.49 (q, 2H, CH2), 3.96–3.99 (m, 1H, OH),
C30H29NO8S; Calcd: 564.1701 [M + H]+; Found: 564.1695 3.56–3.65 (m, 1H, H6a), 3.43–3.54 (m, 4H, H6b + H5 + OH(2×)).
[M + H]+.
2-Naphthyl
13C NMR (75 MHz, pyridine-d5) δ 152.2, 151.9, 151.5, 151.2,
3-(3-(4-methoxyphenyl)-2-propyn-1-hydroxyl)- 138.2, 137.8, 137.5, 137.2, 136.8, 136.3, 135.8, 134.4, 134.0,
2,3,4,6-tetra-O-acetyl-1-thio-β-D-galactopyranoside (27): Method 131.2, 130.9, 130.9, 130.5, 130.0, 129.8, 128.8, 128.1, 126.1,
F: Starting with compound 18 (150 mg, 0.308 mmoles) gave 27 125.8, 125.5, 125.2, 124.8, 91.9, 89.3, 88.2, 86.1, 83.1, 71.7,
(71.2 mg, 74%) as a white foam. [α]2D0 = 68.0 (c = 0.63 CDCl3). 69.4, 64.5, 60.4. HRMS (ESI+) C25H24O5S; Calcd: 459.1241
1H NMR (300 MHz, CDCl3) δ 7.97 (bs, 1H), 7.70–7.77 (m, 3H), [M + Na]+; Found: 459.1231 [M + Na]+.
7.51–7.55 (m, 1H), 7.41–7.46 (m, 2H), 7.33 (d, 2H, J = 8.6 Hz),
6.79 (d, 2H, J = 8.6 Hz), 5.46 (d, 1H, J = 3.4 Hz, H4), 5.12 (tapp
2-Naphthyl 3-(3-(2-pyridine)-2-propyn-1-hydroxy)-1-thio-β-D-
galactopyranoside (31): Method B: Starting with compound 26
,
1H, J = 9.8 Hz, H2), 4.78 (d, 1H, J = 9.8 Hz), 4.34 (s, 2H, CH2), (82 mg, 0.145 mmoles) provided 31 (30.5 mg, 66%) as a white
1
4.10–4.20 (m, 2H, H2a+b), 3.85–3.93 (m, 2H, H3 + H5), 3.74 (s, solid. [α]2D0 = 11.2 (c = 0.23 in MeOH); mp: 133.0–135.7 °C; H
3H, MeO), 2.07 (s, 3H), 2.03 (s, 3H), 1.97 (s, 3H); 13C NMR NMR (300 MHz, DMSO-d6) δ 8.61–8.62 (m, 1H), 8.51–8.53 (m,
(75 MHz, CDCl3) δ 170.4, 170.3, 169.6, 159.7, 133.3, 133.1, 1H), 7.97 (s, 1H), 7.74–7.87 (m, 4H), 7.37–7.53 (m, 4H), 4.74 (d,
132.5, 131.4, 130.0, 129.5, 128.2, 127.5, 127.4, 126.3, 113.9, 1H, J = 9.6 Hz), 4.40–4.59 (m, 3H), 3.98 (d, 1H, J = 2.8 Hz), 3.61
86.4, 82.8, 76.6, 74.6, 68.5, 65.9, 62.2, 57.3, 55.2, 20.9, 20.6; IR (t, 1H, J = 9.3 Hz), 3.43–3.53 (m, 4H); 13C NMR (125 MHz,
(NaCl, cm−1); 3078, 2922, 1747, 1520, 1344, 1226, 749; HRMS DMSO-d6) δ 151.4, 148.7, 138.8, 133.2, 132.7, 131.3, 128.0,
(ESI+) C32H32O9S; Calcd: 615.1651 [M + Na]+; Found: 615.1646 127.6, 127.5, 127.4, 127.1, 126.4, 125.7, 123.6, 119.1, 90.0, 87.6,
[M + Na]+.
82.1, 81.9, 79.0, 68.1, 64.9, 60.4, 56.7; HRMS (ESI+)
2-Naphthyl 3-(3-(4-nitrophenyl)-2-propyn-1-hydroxy)-2,4,6- C24H23NO5S; Calcd: 489.1347 [M + Na]+; Found: 489.1341
tri-O-acetyl-1-thio-β-D-galactopyranoside (28): Method F: Start- [M + Na]+.
ing with compound 18 (139 mg, 0.286 mmoles) gave 28
2-Naphthyl
3-(3-(4-methoxyphenyl)-2-propyn-1-hydroxyl)-
(173 mg, 61%) as a white foam. [α]2D0 = 55.6 (c = 1.0 CHCl3); 1H 1-thio-β-D-galactopyranoside (32): Method B: Starting with com-
NMR (300 MHz, CDCl3) δ 8.19–8.16 (m, 2H), 8.01 (s, 1H), pound 27 (68 mg, 0.114 mmoles) gave 32 (39 mg, 75%) as a
7.82–7.76 (m, 3H), 7.60–7.55 (m, 3H), 7.50–7.47 (m, 2H), 5.52 white foam. [α]2D0 = −17.0 (c = 0.23 CHCl3); H NMR (300 MHz,
1
(d, 1H, J = 3.2 Hz, H4), 5.17 (t, 1H, J = 10.0 Hz, H2), 4.84 (d, 1H, DMSO-d6) δ 7.98 (s, 1H), 7.79 (m, 2H), 7.28–7.56 (m, 3H), 6.88
J = 10.0 Hz, H1), 4.44 (s, 2H, CH2), 4.21–4.17 (m, 2H, H6a+b), (d, J = 7.6 Hz, 1H), 5.40 (d, 1H, J = 6.4 Hz, H4), 4.64–4.77 (m,
3.97–3.89 (m, 2H, H3 + H5), 2.13 (s, 3H), 2.09 (s, 3H), 2.02 (s, 3H, H1 + H2 + H3), 4.46 (q, 1H, J = 16.0 Hz, CH2), 3.96 (bs, 1H,
3H); 13C NMR (75 MHz, CDCl3) δ 170.4, 170.3, 169.4, 147.2, OH), 3.71 (s, 3H, OMe), 3.54–3.66 (m, 1H, H6a), 3.39–3.54 (m,
133.3, 132.3, 131.5, 129.5, 128.2, 127.5, 127.4, 126.5, 126.4, 4H, H6b + H5 + OH(2×)); 13C NMR (75 MHz, DMSO-d6) δ 160.0,
123.5, 89.7, 86.4, 84.7, 77.3, 76.5, 74.5, 68.5, 65.8, 62.2, 57.2, 133.9, 133.6, 133.4, 131.9, 128.6, 128.3, 128.2, 128.0, 127.7,
20.9, 20.6; IR (NaCl, cm−1); 3078, 2922, 1747, 1520, 1344, 1226, 127.1, 126.3, 114.9, 88.3, 85.7, 82.3, 79.7, 68.7, 65.6, 61.1, 57.5,
749; HRMS (ESI+) C31H29NO10S; Calcd: 630.1417 [M + Na]+; 55.8; HRMS (ESI+) C26H26O6S; Calcd: 489.1347 [M + Na]+;
Found: 630.1412 [M + Na]+.
Found: 489.1341 [M + Na]+.
2-Naphthyl 3-(3-(4-fluorophenyl)-2-propyn-1-hydroxy)-2,4,6-
2-Naphthyl 3-(3-(4-nitrophenyl)-2-propyn-1-hydroxy)-1-thio-
tri-O-acetyl-1-thio-β-D-galactopyranoside (29): Method F: β-D-galactopyranoside (33): Method B: Starting with compound
Starting with compound 18 (139 mg, 0.286 mmoles) gave 28 (105 mg, 0.172 mmoles) gave 33 (63.9 mg, 77%) as a white
1
29 (90 mg, 54%) as a white foam. [α]2D0 = 53.7 (c = 0.49 CHCl3); solid. [α]2D0 = −19.89 (c = 0.25 MeOH); mp: 175.5–180.2 °C; H
1H NMR (300 MHz, CDCl3) δ 7.97 (bs, 1H), 7.78–7.71 (m, 3H), NMR (300 MHz, DMSO-d6) δ 8.16–8.19 (m, 2H), 7.98 (bs, 1H),
7.56–7.53 (m, 1H), 7.45–7.35 (m, 4H), 7.00–6.94 (m, 2H), 7.75–7.83 (m, 3H), 7.65–7.68 (m, 2H), 7.41–7.53 (m, 3H), 5.44
5.46 (d, 1H, J = 2.6 Hz, H4), 5.13 (t, 1H, J = 9.7 Hz, H2), 4.79 (d, (d, 1H, J = 6.4 Hz, H4), 4.69–4.76 (m, 3H, H2 + H1 + H3), 4.56 (q,
1H, J = 9.7 Hz, H1), 4.35 (s, 2H, O–CH2–CC), 4.21–4.10 (m, 2H, CH2), 3.98 (bs, 1H, OH), 3.57–3.66 (m, 1H, H6a), 3.44–3.54
2H, H6a,b), 3.96–3.83 (m, 2H, H3 + H5), 2.08 (s, 3H), 2.03 (m, 4H, H6b + H5 + OH(2×)); 13C NMR (125 MHz, DMSO-d6) δ
(s, 3H), 1.97 (s, 3H); 13C NMR (75 MHz, CDCl3) δ 170.4 162.7, 161.1, 133.7, 133.2, 132.7(2×), 131.3, 128.0, 127.6, 127.5,
(2×), 170.3, 169.5, 164.2, 160.9, 133.5, 133.4, 133.3, 127.4, 127.1, 126.4, 125.7, 115.9, 115.8, 87.6, 86.4, 84.2, 81.8,
132.5, 131.4, 129.9, 129.5, 128.2, 127.5, 127.4, 126.4 (2×), 79.0, 68.1, 64.9, 60.4, 56.7; HRMS (ESI+) C25H23NO7S; Calcd:
115.7, 115.4, 86.3, 85.6, 84.0, 76.8, 74.5, 68.5, 65.8, 62.2, 504.1092 [M + Na]+; Found: 504.1090 [M + Na]+.
57.2, 20.9, 20.6; 19F NMR (540 MHz, CDCl3) δ −113.2; HRMS
2-Naphthyl
3-(3-(4-fluorophenyl)-2-propyn-1-hydroxyl)-
(ESI+) C31H29FO8S; Calcd: 603.1478 [M + Na]+; Found: 603.1473 1-thio-β-D-galactopyranoside (34): Method B: Starting with com-
[M + Na]+.
pound 29 (86 mg, 0.148 mmoles) gave 34 (50 mg, 75%) as a
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white solid. [α]2D0 = −14.0 (c = 0.74 CHCl3); mp: 157.1–159.1 °C; Binding assays
1H NMR (600 MHz, pyridine-d5) δ 8.01 (bs, 1H), 7.78–7.86 (m,
3H), 7.43–7.55 (m, 5H), 7.20 (t, 1H, J = 8.6 Hz), 5.41 (d, 1H, J =
ELLA (enzyme-linked lectin assay) experiments. ELLA tests
6.4 Hz, H4), 4.76 (d, 1H, J = 9.6 Hz, H1), 4.71 (t, 1H, J2,3 = 5.4 were monitored using 96-well microtitre plates (Nunc Maxi-
Hz, J1,2 = 9.6 Hz, H2), 4.69 (d, 1H, J = 5.4 Hz, H3), 4.51 (q, 2H, sorb) coated with 5 μg mL−1 of poly[N-(2-hydroxyethyl)acryla-
CH2), 3.98–4.00 (m, 1H, OH), 3.61–3.66 (m, 1H, H6a), 3.45–3.55 mide]α-D-galactopyranoside (Lectinity Holding, Inc.) diluted in
(m, 4H, H6b + H5 + OH(2×)); 13C NMR (125 MHz, pyridine-d5) δ carbonate buffer pH 9.6 (100 μL per well) for 1 hour at 37 °C.
162.7, 161.1, 133.7, 133.2, 132.7(2×), 131.3, 128.0, 127.6, 127.5, Blocking was performed for 1 h at 37 °C with 100 μL of 3%
127.4, 127.1, 126.4, 125.7, 115.9, 115.8, 87.6, 86.4, 84.2, 81.8, (w/v) BSA in PBS per well. Plates were then incubated at 37 °C
79.0, 68.1, 64.9, 60.4, 56.7; 19F NMR (540 MHz, pyridine-d5) δ for 1 h with 100 μL of 3 μg mL−1 of biotinylated LecA in the
−111.0; HRMS (ESI+) C25H23FO5S; Calcd: 477.1147 [M + Na]+; presence of serial dilutions of inhibitors diluted in 0.3% (w/v)
Found: 477.1137 [M + Na]+.
BSA in PBS. After washing with 0.05% Tween-PBS, 100 μL of
2-Naphthyl 3-(3-phenyl-1-hydroxy)-1-thio-β-D-galactopyrano- streptavidin–peroxidase conjugate (dilution 1 : 5000; Boehrin-
side (35): Method G: Starting with compound 30 (15 mg, ger-Mannheim) was added for 1 h at 37 °C. Signal recording
0.034 mmoles) gave 35 (12.5 mg, 85%) as a white solid. was made possible using 100 μL per well of 0.05 M phosphate/
[α]2D0 = −6.2 (c = 0.6 CHCl3); mp: 167.1–174.0 °C; 1H NMR citrate buffer containing O-phenylenediamine dihydrochloride
(300 MHz, DMSO-d6) δ 7.98 (s, 1H), 7.79 (m, 3H), 7.38–7.54 (0.4 mg mL−1) and urea hydrogen peroxide (0.4 mg mL−1
)
(m, 3H), 7.07–7.25 (m, 5H), 5.26 (d, 1H, J = 6.2 Hz, H4), 4.67 (Sigma-Aldrich). Reactions were stopped by the addition of
(d, 2H, J = 9.7 Hz, H2 + H1), 4.46 (d, 1H, J = 4.6 Hz, H), 3.88 50 μL of 30% H2SO4 and the absorbance was read at 490 nm
(s, 1H, OH), 3.44–3.62 (m, 6H), 3.39 (m, 2H), 3.15 (d, 1H, J = using a microtitre plate reader (Bio-Rad; model 680).
8.4 Hz), 2.60 (d, 2H, J = 9.2 Hz, CH2), 1.69–1.83 (m, 2H, CH2);
ITC (isothermal titration microcalorimetry) analysis. ITC
13C NMR (125 MHz, DMSO-d6) δ 142.7, 133.9, 133.5, 132.0, experiments were performed with a VP-ITC isothermal titration
129.0, 128.8, 128.6, 128.3, 128.2, 128.1, 127.8, 127.1, 126.4, microcalorimeter (Microcal; GE Healthcare) except for the
126.2, 88.4, 88.5, 79.8, 68.6, 65.5, 61.2, 32.2, 31.9; HRMS (ESI+) PNPGal ligand that was tested on ITC200 (Microcal; GE Health-
C25H28O5S; Calcd: 463.1554 [M + Na]+; Found: 463.1558 care). Experiments were carried out at 25 °C 0.1 °C. Purified
[M + Na]+.
2-Naphthyl
and lyophilized LecA and carbohydrate ligands were dissolved
3-(3-(aniline)-2-propyn-1-hydroxyl)-1-thio-β-D- in the same buffer, i.e. 0.1 M Tris-HCl pH 7.5, 5% DMSO
galactopyranoside (36): Method G: Starting with compound 33 (dimethyl sulfoxide) and 6 μM CaCl2 and degassed. Protein
(39.8 mg, 0.091 mmoles) gave 36 (41.4 mg, 100%) as a white concentrations in the microcalorimeter cell (1.4 mL) varied
solid. [α]2D0 = −31.8 (c = 0.16 MeOH); mp: 149.0–156.9 °C; from 0.05 to 0.16 mM (cell of 200 μM for the ITC200). Concen-
1H NMR (300 MHz, DMSO-d6) δ 7.97 (s, 1H), 7.74–7.83 (m, 3H), tration was checked by the measurement of optical density
7.39–7.52 (m, 3H), 6.80 (d, 2H, J = 8.0 Hz), 6.42 (m, 2H, J = 8.2 using a theoretical molarity extinction coefficient of 28 000
Hz), 5.23 (d, 1H, J = 6.2 Hz, H4), 4.75 (bs, 1H), 4.65–4.68 (m, (1 cm). A total of 30 injections of 10 μL of sugar solution at
2H, H2 + H1), 4.43 (d, 1H, J = 4.9 Hz, H), 3.85–3.88 (m, 1H, concentrations varying from 0.9 to 1.7 mM were added every
OH), 3.42–3.58 (m, 5H), 3.30–3.39 (m, 1H), 3.11–3.15 (m, 1H), 300 s at 310 rev min−1. Integrated heat effects were analyzed by
1.62–1.69 (m, 2H, CH2); 13C NMR (125 MHz, DMSO-d6) δ 146.2, non-linear regression using
a single-site binding model
133.2, 132.8, 131.1, 128.8, 128.6, 127.9, 127.6, 127.5, 127.4, (Origin 7.0). The experimental data fitted to a theoretical titra-
127.1, 126.4, 125.6, 113.9, 87.7, 82.8, 79.1, 68.0, 64.8, 60.5, tion curve gave the association constant (Ka) and the enthalpy
31.6, 30.7; IR (NaCl, cm−1); 3078, 2922, 1747, 1520, 1344, 1226, of binding (ΔH). Other thermodynamic parameters such as
749; HRMS (ESI+) C25H29NO5S; Calcd: 456.1845 [M + H]+; changes in free energy (ΔG) and entropy (ΔS) were calculated
Found: 456.1835 [M + H]+; Calcd: 478.1663 [M + Na]+; 478.1659 from the equation ΔG = ΔH − TΔS = −RT ln Ka, where T is the
[M + Na]+.
absolute temperature and R is the molar gas constant (8.314 J
2-Naphthyl 3-O-propargyl-1-thio-β-D-galactopyranoside (37): mol−1 K−1). All experiments were performed with c values of 10
Method C (without acetylation): Starting with compound 11 < c < 100.42
(250 mg, 0.775 mmoles) gave 37 (162.5 mg, 58%) as a white
Crystallography. LecA at 0.8 mM was incubated with 1 mM
solid. [α]2D0 = +1.7 (c = 0.72 DMSO); mp: 144.5–152.6 °C; 1H of compound 11 for 1 h prior to crystallization which was
NMR (600 MHz, pyridine-d5) δ 6.76–6.78 (m, 1H), 6.56–6.61 performed by the hanging drop vapor diffusion method using
(m, 2H), 6.39 (s, 1H), 6.20–6.24 (m, 1H), 6.03 (s, 1H), 4.44 (bs, 1 + 1 μL drops at 20 °C. Lozenge shaped crystals were obtained
4H). 4.21 (d, 1H, J = 9.6 Hz, H1), 3.53–3.62 (m, 4H, H2 + H4 + from solution 2 of the Clear Strategy Screen II (Molecular
CH2), 3.31–3.34 (m, 1H, H6a), 3.23–3.25 (m, 1H, H6b), 2.99 (t, Dimensions Ltd) containing 0.8
M lithium sulfate and
1H, J = 5.8 Hz, H5), 2.91 (dd, 1H, J = 3.1 Hz, J = 9.0 Hz, H3), 100 mM sodium acetate pH 4.6. 25% glycerol was added for
2.19 (t, 1H, J = 1.9 Hz, CH); 13C NMR (125 MHz, pyridine-d5) δ cryoprotection prior to mounting on a litholoop (Molecular
133.05, 132.60, 131.20, 127.9, 127.7, 127.3, 126.8, 126.5, 125.5, Dimensions Ltd) and freezing in liquid nitrogen. Data were
124.8, 88.6, 82.6, 80.3, 79.9, 74.7, 68.4, 66.1, 61.2, 56.5; HRMS collected on beamline BM14 from ESRF, Grenoble, France
(ESI+) C19H20O5S; Calcd: 383.0928 [M + Na]+; Found: 383.0930 using a MARCCD detector. The structure was solved by mole-
[M + Na]+.
cular replacement using the coordinates of tetramer from PDB
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1OKO and the Phaser program.43 The first model was updated 13 F. Pertici and R. J. Pieters, Chem. Commun., 2012, 48, 4008.
using ARP/wARP44 prior to subsequent cycles of TLS and 14 M. Reynolds, M. Marradi, A. Imberty, S. Penadés and
restrained refinement using REFMAC545 iterated with manual
S. Pérez, Chem.–Eur. J., 2012, 18, 4264.
rebuilding in COOT.46 Details of the model quality are given in 15 B. Blanchard, A. Nurisso, E. Hollville, C. Tétaud, J. Wiels,
Table 1. The coordinates and structure factor were deposited
in the Protein Data bank under the code PDB 4A6S.
M. Pokorná, M. Wimmerová, A. Varrot and A. Imberty,
J. Mol. Biol., 2008, 383, 837.
16 N. Garber, U. Guempel, A. Belz, N. Gilboa-Garber and
R. J. Doyle, Biochim. Biophys. Acta, 1992, 1116, 331.
17 D. Giguère, M.-A. Bonin, P. Cloutier, R. Patnam, C. St-
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7811.
Acknowledgements
R. R. is thankful to NSERC for a Canadian Research Chair in
Therapeutic Chemistry and for a Discovery grant. A. I.’s work 18 S. André, D. Giguère, T. K. Dam, C. F. Brewer, H.-J. Gabius
was supported by CNRS. A. I. and A. V. also acknowledge ANR and R. Roy, New J. Chem., 2010, 34, 2229.
grants PA-ANTIADH (ANR-09-JCJC-0047) and GLYCOASTERIX 19 D. Giguère, S. André, M.-A. Bonin, M.-A. Bellefleur,
(ANR-08-PCVI-0028). The project was also supported by Associ-
A. Provencal, P. Cloutier, B. Pucci, R. Roy and H.-J. Gabius,
ation Vaincre la Mucoviscidose, GDR Pseudomonas, COST
Bioorg. Med. Chem., 2011, 19, 3280.
action BM1003 and Labex ARCANE (ANR-11-LABX-003). Crystal 20 D. Giguère, S. Sato, C. St-Pierre, S. Sirois and R. Roy, Bioorg.
data were collected at the European Synchrotron Radiation Med. Chem. Lett., 2006, 16, 1668.
Facility and the authors are grateful for the assistance in using 21 I. Deguise, D. Lagnoux and R. Roy, New J. Chem., 2007, 31,
beamline BM14. The help of Yan Seguin in interaction analysis
is acknowledged.
1321.
22 C. T. Öberga, H. Leffler and U. J. Nilsson, Chimia, 2011, 65,
18.
23 R. Roy, in Handbook of phase transfer catalysis, ed. Y. Sasson
and R. Neumann, Chapman & Hall, London, 1997, ch. 7,
pp. 244–275.
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