Rational Design and Synthesis of Methyl-β- d -galactomalonyl Phenyl Esters as Potent Galectin-8 N Antagonists

Article abstract of DOI:10.1021/acs.jmedchem.0c00602

Galectin-8 is a β-galactoside-recognizing protein having an important role in the regulation of bone remodeling and cancer progression and metastasis. Methyl β-d-galactopyranoside malonyl aromatic esters have been designed to target and engage with particular amino acid residues of the galectin-8N extended carbohydrate-binding site. The chemically synthesized compounds had in vitro binding affinity toward galectin-8N in the range of 5-33 μM, as evaluated by isothermal titration calorimetry. This affinity directly correlated with the compounds' ability to inhibit galectin-8-induced expression of chemokines and proinflammatory cytokines in the SUM159 breast cancer cell line. X-ray crystallographic structure determination revealed that these monosaccharide-based compounds bind galectin-8N by engaging its unique arginine (Arg59) and simultaneously cross-linking to another arginine (Arg45) located across the carbohydrate-binding site. This structure-based drug design approach has led to the discovery of novel monosaccharide galactose-based antagonists, with the strongest-binding compound (Kd 5.72 μM) holding 7-fold tighter than the disaccharide lactose.

Full text of DOI:10.1021/acs.jmedchem.0c00602

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Article
Rational design and synthesis of methyl-#-D-galactomalonyl
phenyl esters as potent galectin-8N antagonists
Brijesh Patel, Chandan Kishor, Todd A. Houston, Hadaz Shatz-
Azoulay, Yehiel Zick, Yaron Vinik, and Helen Blanchard
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.0c00602 • Publication Date (Web): 18 Aug 2020
Downloaded from pubs.acs.org on August 18, 2020
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Rational design and synthesis of methyl-β-D-
galactomalonyl phenyl esters as potent galectin-8N
antagonists
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†‡
Brijesh Patel, † Chandan Kishor,
Todd. A. Houston, † Hadas
Shatz-Azoulay, § Yehiel Zick, § Yaron Vinik, §* and Helen
†‡*
Blanchard
†Institute for Glycomics, Griffith University, Gold Coast Campus, Queensland 4222, Australia.
‡School of Chemistry and Molecular Bioscience, and Molecular Horizons, University of
Wollongong, Wollongong, New South Wales 2522, Australia.
§Department of Molecular Cell Biology, Weizmann Institute of Science, Rehovot 76100, Israel.
Corresponding authors
* E-mail: Helen_Blanchard@uow.edu.au
*E-mail: yaron.vinik@weizmann.ac.il
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ABSTRACT
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Galectin-8 is a β-galactoside recognizing protein having an
important role in the regulation of bone remodeling, cancer
progression and metastasis. Methyl-β-D-galactopyranoside malonyl
aromatic esters have been designed to target and engage with
particular amino acid residues of the galectin-8N extended
carbohydrate-binding site. The chemically synthesized compounds
had in vitro binding affinity towards galectin-8N in the range of
5-33 μM, as evaluated by isothermal titration calorimetry. This
affinity directly correlated with the compounds’ ability to
inhibit galectin-8-induced expression of chemokines and
proinflammatory cytokines in SUM159 breast cancer cell line. X-
ray crystallographic structure determination revealed that these
monosaccharide-based compounds bind galectin-8N by engaging its
unique arginine (Arg59), and simultaneously cross-linking to
another (Arg45) located across the carbohydrate-binding site. This
structure-based drug design approach has led to discovery of novel
monosaccharide galactose-based antagonists, with the strongest
binding compound (Kd 5.72 µM) being 7-fold tighter than the
disaccharide lactose.
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INTRODUCTION
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Galectin-8 is a member of the galectin family that comprises 15 mammalian lectins that bind β-
galactosides through their carbohydrate recognition domains (CRD).1 Based on their structural
features, galectins are classified into three categories, termed proto type (galectin-1, -2, -5, -7, -10,
-11, -13, -14, -15) that mainly exist as homodimers; tandem-repeat (galectin-4, -6, -8, -9, -12) that
have two distinct CRDs joined by a variable length of polypeptide linker; and the chimera type
galectin (galectin-3 (sole member)) which has only one CRD located at the C-terminus of the
protein.2-4 Galectin-8, like other galectins, is secreted by various cell types and act as matricellular
proteins that ligate cell surface receptors and extracellular matrix (ECM) proteins to affect a wide
range of biological functions including cell adhesion, migration, cell growth, apoptosis, and
autophagy. Accordingly, galectin-8 expression is upregulated in cancerous tissue including lung,
kidney, prostate, and breast.5,6 Galectin-8-treated osteoblasts enhance expression of the
osteoclastogenic factor receptor activator of nuclear factor kappa-B ligand (RANKL), which binds
to the RANK receptor on osteoclasts and promotes osteoclastogenesis.7,8 Galectin-8 plays
important roles in rheumatic, autoimmune and inflammatory disorders, bone loss and
osteoporosis.7-10 It was recently reported that the galectin-8 treated primary cultures of murine
osteoblasts trigger ~5-60 fold gene expression of several cytokines and chemokines including
RANKL, monocyte chemoattractant protein-1 (MCP-1), stromal cell-derived factor 1 (SDF-1),
interferon-γ-inducible protein 10 (IP-10), interleukin-6 (IL-6), interleukin-1β (IL-1β), and tumor
necrosis factor-α (TNF-α).11 Galectin-8 also increases ~4-fold protein levels of cytokines and
chemokines, such as SDF-1 and MCP-1, in the culture medium of galectin-8 treated osteoblasts.11
Galectin-8 increases SDF-1, MCP-1 gene expression in a number of cell types which promotes
migration of cancer cells toward cells treated with this lectin.11 Galectin-8, being an important
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player in so many diseases, promoted us to design potent galectin-8 antagonists. Several galactose-
based compounds, endogenous glycans have shown promising galectin-8N binding affinity such
as fused tricyclic carbohydrate-benzene hybrid molecules (Kd 180 µM)12, galactose-quinoline
based compounds (Kd ~ 2 µM)13, wild-type erythropoietin (EPO) glycoforms14 produced in
Chinese hamster ovary (CHO) cells (Kd 60 nM). Galectin-8’s N-terminal ("galectin-8N") and C-
terminal ("galectin-8C") CRDs share an overall amino acid sequence identity of 37%, but the
amino acid residues Trp86, His65, Asn67, Arg69, Asn79, and Glu89 that are on the S4-S6 β-
strands and form the carbohydrate-binding site region that recognizes the galactose portion of
ligands, being identical in both CRDs. Important sequence differences do occur, most notably
Arg45, Gln47, and Tyr141 in galectin-8N are replaced by Ser255, Asn257, and Asn348,
respectively, in galectin-8C.15 Between them, there are some important distinct characteristics,
notably the presence of a unique arginine, Arg59, in galectin-8N which is absent in the galectin-
8C binding site.15 Galectin-8N and galectin-8C show different binding affinities for particular
carbohydrate sequences.16 For instance, galectin-8N demonstrated a 2.7 µM affinity for 3′-
sialyllactose (3′-SiaLac) whereas galectin-8C has >1000 µM affinity.17 An X-ray crystallographic
study led to the explanation that the strong affinity with galectin-8N is due to the presence of its
unique carbohydrate-binding site Arg59.18 This finding prompted us to aim for a direct
engagement with this amino acid as an approach to design potent galectin-8N antagonists that will
serve as modulators or inhibitors of galectin-8 function. We have previously investigated methyl-
3‐O‐[1‐carboxyethyl]‐β‐D‐galactopyranoside binding affinity and binding interactions with each
CRD domain individually. This galactose-based compound showed 32 µM binding affinity with
galectin-8N and no significant binding affinity with galectin-8C.19 In this manuscript, we continue
our drug design campaign and report the design and synthesis of galactose based methyl-β-D-
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galactomalonyl phenyl esters, their binding affinity with galectin-8N CRD and their inhibition on
galectin-8 mediated chemokines and proinflammatory cytokines expression in SUM159 breast
cancer cells. Finally, protein X-ray crystallography was used to provide a structural basis for the
mode of inhibition of galactomalonic acid derivatives at CRD of galectin-8N.
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The galectin-8N CRD comprises two anti-parallel β-sheets forming a β-sandwich structure
(Figure 1A).18 The concave side of the CRD is formed by six β-strands (S1-S6) and contains amino
acids that form the carbohydrate binding site, with the S4-S6 β-strands possessing amino acids that
recognize the galactose portion of ligands. Galectin-8N preferentially recognizes oligosaccharides
substituted with an anionic functional group such as 3ʹ-sulfolactose and 3′-SiaLac.17 This selective
anionic recognition is due to the presence of the unique Arg59 on the S3-S4 loop (Figure 1A). The
3ʹ-SiaLac carboxylic side chain forms a salt bridge interaction with Arg59, and a hydrogen bond
with Gln47 (Figure 1B). Of further note, the S2 β-strand Tyr141 was identified as an important
amino acid of the galectin-8N extended binding site because it forms van der Waals interaction
with Lacto-N-fucopentaose-III ( LNF-III) in the galectin-8N-LNF-III crystal structure.18
A
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Figure 1. (A) Galectin-8N CRD. The illustration emphasizes the S1-S6
β-strands forming the concave face and the amino acids of the
primary and extended carbohydrate binding sites. The Arg45 and
Arg59 (in sticks; carbon atoms orange) are situated directly
opposite to each other, highlighting their important positioning
with respect to antagonist design. (B) The binding conformation of
3′-SiaLac (carbon atoms yellow) within the galectin-8N carbohydrate
binding site (carbon atoms green) (PDB ID: 3AP7).17
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The design of monosaccharide-based galectin-8N antagonists that we recently initiated, resulted
in a galactose-based compound that interacts with Arg59 of galectin-8N with a binding affinity of
32 µM, equivalent to that of the disaccharide lactose.19 Here we aim to design an antagonist which
could cross-link both this arginine (Arg59), with that located across the binding site groove
(Arg45), thus anchoring it from both sides. Further, we explore the enhancement of interactions
with the extended binding site. To cross-link both arginines, we propose that two electron-rich
functional groups to engage with the positively charged guanido groups of arginine would be
needed. In light of this hypothesis we focused on the diketo functional group of malonic acid,
which possess two electron rich carbonyl oxygens. Several synthetic and biological studies on
diketo functional group-based compounds have been reported such as integrase20,21, ribonuclease
H22 (RNase H), Hepatitis C Virus (HCV) RNA polymerase23, malate synthetase24, histone
deacetylase (HDAC) inhibitor.25,26 In these studies, it was observed that diketo oxygens of these
compounds form a strong complex with the metal ion of the above enzymes and proved to be better
inhibitors. Based on the knowledge of the three-dimensional structure of the carbohydrate binding
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site of galectin-8N CRD, we have designed aromatic ring substituted galactomalonic acid in which
it is supposed that the galactose portion would be recognized by the conserved amino acids of the
binding site, the malonic oxygens are postulated to be able to cross-link the two arginines, and the
aromatic ring is anticipated to make van der Waals interaction with tyrosine residue Tyr141.
Malonic acid undergoes keto-enol tautomerism by the movement of its alpha hydrogens and the
shifting of π-bonding electrons (Figure 2). Because of tautomerism, there is the possibility to
generate stabilized enolate ions which have the ability to bind positively charged guanido group
of arginines. This group offers a diverse “shape-shifting” array of hydrogen bond acceptors near
the arginine residue.
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Figure 2. Keto-enol tautomerism of galactomalonyl aromatic ester.
OH
OH
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RESULTS AND
In Silico Analysis
Compounds with
DISCUSSION
Interactions
R
O
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OH
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of
of
Designed
(I)
Galectin-8N
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OH
OH
(II)
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(IV)
(III)
Following the rationale of incorporating a malonic acid group to develop potent galectin-8
antagonists, eight compounds of galactomalonic acid and derivatives were designed. A molecular
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docking investigation was undertaken for these compounds with galectin-8N (PDB ID: 3AP7).18
The key potential binding interactions that were consistent for these compounds with the galectin-
8N carbohydrate binding site were revealed, as exemplified by the compound that was later
evaluated to have the best binding affinity, the galactomalonyl aromatic ester 10 (Scheme 1, Page
10) (Figure 3).
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Figure 3. Docking conformation of galactomalonyl ester 10 within the galectin-8N binding site
(PDB ID: 3AP7).17
The planar ring of the galactose portion was predicted to make van der Waals interactions with
the aromatic tryptophan (Trp86) ring, whereas the free hydroxyls of the galactose interact with
other conserved amino acids of the binding site. In this docked model, the malonic acid chain
interacts with amino acids of the extended binding site in which there is feasibility for one carbonyl
oxygen to make a potential salt-bridge (if ionized) or else a hydrogen-bonding interaction with
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Arg59 (3.2 Å distance), and the second carbonyl oxygen potentially interacts with Arg45 and
Gln47, with 3.3 Å and 2.9 Å distances respectively. In addition, the aromatic ring of the
galactomalonyl ester 10 is predicted to make van der Waals interactions with Tyr141 as well as
engage in a potential cation-π stacking with the Arg59 guanidinium group. The galectin-8N-
galactomalonyl ester 10, in its docked confirmation, shows potential for making the two important
key interactions that we desired. One is the interaction of a negatively charged moiety with the
unique Arg59, akin to that of carboxylic acid group of the 3ʹ-siaLac in galectin-8N-3ʹ-siaLac (PDB
ID=3AP7);18 and the second is the interaction with Tyr141 of the extended binding site, as
exemplified by galactose of LNF-III in galectin-8N-LNF-III (PDB ID = 3AP9).18
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Chemistry
The docking results supported that the designed compounds had potential to bind and engage
with the unique Arg59. Steglich esterification was undertaken using either of two common
coupling reagents N,N'-dicyclohexylcarbodiimide (DCC) and N-(3-dimethylaminopropyl)-N′-
ethylcarbodiimide hydrochloride (EDCI), individually to synthesize the aromatic substituted
galactose malonic esters 2-7 (Scheme 1) from methyl 4,6-di-O-benzylidene-2-O-methoxymethyl-
β-D-galactopyranoside 1 (synthesis described previously19 ).
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Ph
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O
O
OH
O
HO
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O
a
b
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O
HO
O
O
O
O
OH
O
O
1
Ar
Ar
O
O
O
O
O
O
2-7
8-13
Ar=phenyl (83%)
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Ar=phenyl (87%)
Ar=4-methoxyphenyl (86%)
Ar=4-fluorophenyl (85%)
Ar=2,4-dichlorophenyl (89%)
Ar=3,5-dimethoxyphenyl (90%)
Ar=benzyl (95%)
Ar=4-methoxyphenyl (90%)
Ar=4-fluorophenyl (81%)
Ar=2,4-dichlorophenyl (90%)
Ar=3,5-dimethoxyphenyl (90%)
Ar=benzyl (90%)
Scheme 1. Reagents and conditions: (a) DCC, aryl malonic acid 24-28, 30 (scheme S1), DMAP,
DCM, -20 °C to room temperature, overnight (b) 60% aqueous acetic acid solution, 60 °C reflux
for 4 h.
From reaction monitoring, it was observed that use of EDCI resulted in low conversion and
low yield, whereas DCC acted as a better coupling reagent and completed the reaction in good
yield. Different substituted phenyl malonic acids 24-28, 30 (synthesis, described in scheme S1)
were prepared by reacting various substituted phenols with Meldrum’s acid27 and then these acids
were used in a Steglich coupling reaction with the free 3-OH group of protected
galactopyranoside.28,29 Deprotection of ester intermediates was carried out by refluxing in 60%
glacial acetic acid solution at 60 °C for 3 hours to give phenyl ring substituted galactomalonates
8-13.30 In the synthesis of galactomalonic acid 15, the intermediate 7 was debenzylated by
palladium catalyst with hydrogen gas to yield the malonic acid substituted galactopyranoside 14.31
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Deprotection of intermediate 14 in the presence of 60% acetic acid gave the malonic acid
monoester of methyl β-galactopyranoside 15 (Scheme 2).
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Ph
Ph
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O
O
OH
O
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HO
O
O
O
a
b
O
O
O
O
O
O
O
O
OH
HO
O
HO
O
O
O
O
O
O
O
O
14
15
7
Scheme 2. Reagents and conditions: (a) 10% Pd/C, H2 gas, anhydrous methanol (b) 60% aqueous
acetic acid solution, 60 °C reflux for 4 h.
While this has the potential to decarboxylate at high temperature, malonate monoesters are
stable to mildly acidic conditions at temperatures 60-70 °C.32 The protected galactomalonyl methyl
ester 16 was synthesized by nucleophilic substitution of methyl malonyl chloride with
galactopyranoside 1 in the presence of organic base. Reaction was performed with three different
bases: triethylamine (TEA), N,N'-diisopropylethylamine (DIPEA) and N,N,N',N'-tetramethyl-1,2-
ethylenediamine (TMEDA). Results showed that TEA, DIPEA gave lower conversion and lower
yield compare to TMEDA. Deprotection of intermediate 16 was done by refluxing in 60% glacial
acetic acid solution at 60 °C that gives methyl malonyl galactopyranoside ester 17 (Scheme 3).
Ph
Ph
O
O
OH
O
O
HO
O
O
O
a
b
O
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O
O
HO
O
O
O
OH
O
O
O
O
O
O
O
O
16
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1
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Scheme 3. Reagents and conditions: (a) TMEDA, methyl malonyl chloride, DCM, -20 °C to room
temperature (b) 60% aqueous acetic acid solution, 60 °C reflux for 4 h.
Thermodynamic Analysis
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Eight novel compounds were designed and synthesized, these are the galactomalonic acid 15
and its derivatives with aromatic substitution (compounds 8-13), as well as an aliphatic methyl
ester 17 (Figure 4).
OH
OH
OH
HO
O
HO
O
HO
O
O
O
O
O
O
O
OH
OH
OH
HO
O
O
Ar
O
O
O
O
O
O
17
15
8-13
8
9
10
11
12
13
Ar=phenyl (83%)
Ar=4-methoxyphenyl (86%)
Ar=4-fluorophenyl (85%)
Ar=2,4-dichlorophenyl (89%)
Ar=3,5-dimethoxyphenyl (90%)
Ar=benzyl (95%)
Figure 4. Methyl-β-D-galactomalonic acid 15 and its derivative 8-13, 17.
These compounds were evaluated for in vitro galectin-8N binding affinity by isothermal titration
calorimetry (Table 1, Figure 5).
Table 1. Binding affinity and estimated thermodynamic parameters from ITC experiments
Compound
Kd (µM)
12.84 ±0.66
9.47 ±0.43
5.72 ±0.27
11.89 ±0.56
11.02 ±0.56
9.17 ±0.67
33.56 ±1.25
24.85 ±0.92
37.92 ±1.27
2.3 ±0.20
ΔH
-TΔS
-13.29
-16.93
-16.30
-18.54
-16.32
-17.26
-31.6
ΔG
n
8
9
-14.63 ±0.4
-11.75 ±0.3
-13.63 ±0.2
-9.56 ±0.4
-11.98 ±0.2
-12.40 ±0.4
6.08 ±0.4
-27.92
-28.68
-29.93
-28.11
-28.30
-28.75
-25.5
0.81 ±0.02
0.87 ±0.02
0.89 ±0.02
0.89 ±0.01
0.81 ±0.05
0.96 ±0.05
0.89 ±0.13
0.80 ±0.15
0.98 ±0.03
0.83 ±0.01
10
11
12
13
15
17
-7.068 ±0.6
-4.7 ±0.6
-19.22
-20.5
-26.28
-25.2
Lac
3’-SL
-51.8 ±0.8
19.64
-32.1
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D Galectin-8N-Cpd 12
C Galectin-8N-Cpd 9
Time (s)
A Galectin-8N-Cpd 10
B Galectin-8N-Cpd 13
Time (s)
Time (s)
Time (s)
2000
2000
2000
4000
6000
6000
2000
6000
10
11
12
13
14
15
16
17
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2
2
2
2
0
0
0
0
0
0
0
0
Kd= 11.02
Kd= 9.17
Kd= 5.72
Kd= 9.47
-15
-15
-15
-15
1
0
2
0
2
0
2
1
1
0
1
2
Molar Ratio
Molar Ratio
Molar Ratio
Molar Ratio
E Galectin-8N-Cpd 11
H Galectin-8N-Cpd 15
G Galectin-8N-Cpd 17
F Galectin-8N-Cpd 8
Time (s)
Time (s)
Time (s)
Time (s)
6000
2000
4000
2000
6000
2000
5000
2000
2
2
2
2
0
0
0
0
0
0
0
0
Kd= 11.89
1
Kd= 12.84
1
Kd= 24.85
1
Kd= 33.56
1 2
Molar Ratio
-15
-15
-15
-15
0
2
0
2
0
2
0
Molar Ratio
Molar Ratio
Molar Ratio
Figure 5. Isothermal titration calorimetric analysis. Binding isotherm for titration of 0.5 mM
galactomalonyl ester 8-13, 17 (A-G) and 1mM galactomalonic acid 15 (H) with 100 µM galectin-
8N in 20 mM Tris buffer at pH 7.5 containing 100 mM NaCl and 4 mM BME.
Galectin-8N shows 33.56 µM binding affinity for the monosaccharide galactomalonic acid 15,
which is slightly stronger than which we measured with the disaccharide lactose (37.92 µM). It
shows a typical endothermic binding isotherm with unfavourable enthalpy difference in isothermal
titration calorimetry (ITC) analysis (Figure 5H). When the electron-donating methyl group is
introduced to the malonyl side chain this increased the affinity of the galactomalonic acid
derivative 17 to 24.85 µM. From this, we can assume that the electron-donating group strengthens
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the electron cloud associated with the carbonyl oxygens. Aromatic phenyl substitution of the
galactomalonic acid derivative 8 shows almost three times better affinity (12.84 µM) than the
parent galactomalonic acid 15, and two times better than the methyl galactomalonyl ester 17,
giving strong evidence that the aromatic phenyl ring significantly enhances protein-ligand binding.
This improvement may be due to interactions of the aromatic ring with the binding site for example
by van der Waals interactions with Tyr141, cation-π stacking interaction with Arg59 and/or by
making the oxygens of the malonyl group stronger nucleophiles. Introduction of fluoro substitution
at the 4-position of the phenyl ring shows approximately 6-fold stronger binding affinity than
galactomalonyl ester 15. The 4-fluorophenyl substitution of galactomalonic acid (compound 10,
Kd 5.72 µM) presents the strongest binding monosaccharide galactose-based toward galectin-8N
to date, with almost 7 times stronger binding affinity than lactose (37.92 µM) (Figure S1A).
Addition of a methylene spacer group between the oxygen of malonyl and the aromatic phenyl
ring produces the 9.17 µM affinity inhibitor 13, which is slightly better than compound 8. When
an electron-donating 4-methoxy functional group is introduced to the para position on the phenyl
ring, this increased binding affinity of galactomalonyl phenyl ester 9 to 9.47µM. Disubstitution on
the phenyl ring shows 3-fold improved binding affinity over the galactomalonic acid 15. For
example, the 2,4-dichlorophenyl galactomalonyl ester 11, containing electron-donating dichloro
substitution, exhibits a 11.89 µM affinity and the 3,5-dimethoxyphenyl galactomalonyl ester 12,
containing dimethoxy substitution, shows 11.02 µM affinity, which suggests that disubstitution on
the phenyl ring does not diminish the affinity of the overall compound and instead gives a slight
enhancement over the phenyl substituted galactomalonic acid 8. In addition to that, the different
electronic effects of both dichloro and dimethoxy substituents to the phenyl ring do not create a
significant difference in binding affinity of compound 11 and 12. A molecular docking study of
10
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disubstituted phenyl ring containing compounds shows that these substitutions do not create any
steric hindrance in the binding site which could worsen the binding affinity of these compounds
(Figure S2B and S2D). It also suggests that there is possibility of hydrogen bond interactions
between the substituent of these compounds with Arg59 as represented (Figure S2D), which might
be the reason for the slight improvement in binding affinity compared to the unsubstituted phenyl
ring containing compound 8.
10
11
12
13
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17
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Cytokine and Chemokine Expression Analysis
Galectin-8 upregulates cytokine and chemokine expression such as SDF-1, TNF-α, RANKL,
IL-1β, MCP-1, IL-6 in osteoblasts extracted from calvariae of newborn CD1 mice.11 A cell culture
study was performed to determine the effects of the antagonists on galectin-8 induced expression
of chemokines and inflammatory cytokines (IL-8, IL-1β, and IL-6) (Figure 6).
SFM
GAL8
IL1β
IL8
IL6
8
6
4
2
0
6
4
2
0
6
4
2
0
*
*
*
*
*
***
**
*
**
*
*
*
N
15
17
13
10
NO INH 15
17
13
10
NO INH 15
17
13
10
Figure 6. Effect of weak binding affinity compound 15, 17 and strong binding affinity compound
13, 10 on IL8, IL1β and IL6 expression of SUM 159 breast cancer cell line in absence of galectin-
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8 (SFM - red block) and presence of 50nM galectin-8 (green block). Results shown are means ±
SEM of experiments done in duplicates (*p<0.05; **p<0.01; ***p<0.001).
7
8
9
SUM159 breast cancer cells were treated with or without 50 nM galectin-8, in the presence or
absence of our weakest (15, 17) or strongest (13, 10) antagonists. As expected, treatment of
SUM159 breast cancer cells with galectin-8 induced ~ 5-fold increased expresion of IL-8
(interleukin-8), IL-1β, and IL-6. Inclusion of the galectin-8 antagonists inhibited its effects, in
correlation with the antagonists’ affinity. Compounds with the strongest binding affinity (10, 13)
showed more pronounced inhibition compared to the compounds having a weaker affinity (15,
17). Compound 10 showed 5-fold, 1.5-fold and 13-fold greater IL-8, IL-1β and IL-6 expression
inhibition respectively than compound 15. In comparison of weaker binding affinity compounds,
compound 17 shows 1.5-fold IL-8, IL-6 expression inhibition than compound 15. We further
performed the dose-response relationship study of compound 10 and 13 at four different
concentrations (1 µM, 10 µM, 100 µM and 500 µM) and observed that the lower concentration (1
µM) of both these compounds was able to inhibit galectin-8 induced IL-8 and IL-6 gene expression
in SUM159 breast cancer cell line (Figure S3). It is clear from our analysis that in the absence of
inhibitors the IL-6 and IL-8 expression is high, in the presence of weak inhibitors their expression
is moderated, and in the presence of the best inhibitors (compounds 13 and 10) expression is
significantly lower.
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Crystal Structure of Galectin-8N CRD in Complex with 13
Our X-ray crystallographic studies investigated binding interactions of 13 to galectin-8N
(Figure 7). The galectin-8N-13 complex structure was determined at 1.59 Å resolution (Table 2).
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After initial refinement of galectin-8N CRD model, the difference electron density revealed
unambiguous placement of the galactose ring portion of 13 facing the Trp86, and the galactose
C3-hydroxy substituted benzyl malonic side chain within the galectin-8N extended binding site.
The positive difference electron density that appears near the anomeric methoxy group and the C6
10
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13
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60
hydroxy group of galactose represents water molecules W1 and W2 respectively.
Figure 7. Stereo diagram of electron density map (green mesh) 2│Fo│- │Fc│ αc contoured at 1σ,
for 13 (in sticks; carbon in yellow, oxygen in red) in complex with galectin-8N (gray cartoon)
(PDB ID 6W4Z). Interacting side chain of amino acid residues of galectin-8N denoted as green
sticks and the water molecules interacting with compound 13 indicated as red sphere.
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Table 2. Crystallographic data and refinement statistics for the galectin-8N-13 structure
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
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29
30
31
32
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Data
Galectin-8N-13 complex
Indexing
Crystal system, Space group
Unit cell
Monoclinic, C2
a= 110.39, b= 40.00, c= 70.32;
α=89.96, β=98.22, γ=90.11
Merging and scaling
Resolution [Å]
Total observations
Unique observations
Multiplicity
46.31-1.59
280344 (13417) [a]
41190 (1987)
6.8 (6.8)
Completeness [%]
I/σ
99.9 (99.7)
8.8 (2.5)
Rmerge [%]
11.6 (153.8)
0.997 (0.805)
CC (1/2) [%]
Refinement
Rfactor [%]
Rfree [%]
19.25
22.55
Number of atoms
Protein
Ligand
Water
1211
26
58
Root mean square deviations
Bond length [Å]
Bond angle [°]
0.015
1.90
Ramchandran plot statistics
Favoured [%]
Allowed [%]
98.67
1.33
Average B-factor [Å2]
Protein
Ligand
Water
20.7
43.6
27.0
PDB ID
6W4Z
[a] Values in parenthesis are for the highest-resolution shell.
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Overall, the binding mode observed for the galactose portion of 13 is identical to the reported
galectin-8N-lactose complex crystal structure in which the O4-hydroxy of galactose makes
hydrogen bonds with His65, Asn67, Arg45, and Arg69, whereas the O6-hydroxy group engages
in hydrogen bonding with Asn79 and Glu89. Two carbonyl groups of extended benzyl malonic
acid cross link the two positively charged arginine Arg59 and Arg45, which is located across the
binding site groove. This cross-linking interaction with arginine enables the inhibitor to be held
from both sides. One carbonyl oxygen hydrogen-bonds with Trp86 and Arg59, whereas the second
carbonyl oxygen hydrogen bonds with Arg45. This binding interaction validates the measured
binding affinity of the parent compound 15 and its improved derivative structure 17 in which the
electron-donating methyl group increased its binding affinity by increasing the nucleophilicity of
the two carbonyl groups. The X-ray crystal structure of galectin-8N-13 reveals two important
interactions of the galectin-8N extended binding site amino acids which increase galactomalonyl
phenyl esters binding affinity sharply from 33.56 μM to 12.84 µM. One is van der Waals
interactions of Tyr141 with the phenyl ring, and the other is a cation-π stacking interaction of
Arg59 with the phenyl ring. The galectin-8N-13 X-ray crystal structure validates our design
hypothesis and the predicted binding conformation from molecular docking, for compound 13.
The X-ray crystallographic structure reveals the binding characteristics of galactomalonyl
esters, which was able to explain molecular docking predicted binding energies of all other
compounds. For example, galactomalonic acid 15 exhibits -5 kcal/mol and methyl ester 17 shows
-5.4 kcal/mol, this small difference is probably due to the electron-donating effect of the methyl
group which makes the two carbonyl oxygens of malonic acid stronger nucleophiles and observed
to cross link with Arg45 and Arg59 in the crystal structure. When the aromatic phenyl ring was
introduced to the malonyl substitution, these molecules show -6 to -7 kcal/mol binding energies
9
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indicating stronger binding. From our X-ray crystallography study, it is very clear that the higher
binding energies (tighter binding) of these compounds is due to the van der Waals interactions of
the aromatic phenyl ring with Tyr141 as well as cation-π stacking with the Arg59.
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CONCLUSIONS
We have designed a series of novel monosaccharide based galactomalonyl esters, subsequently
chemically synthesized, and their binding affinities toward galectin-8N were analyzed. In addition,
the binding mode and interactions of 18 with galectin-8N were elucidated based on their atomic
structure, revealed by X-ray crystallography. This structure-based drug design approach has led to
the discovery of novel monosaccharide galactose-based antagonists with single digit micromolar
binding affinity (Kd 5.72-12.83 μM) with the strongest binding molecule (10) being 7-fold tighter
than the disaccharide lactose. The malonyl substitution to the monosaccharide galactose generates
compounds with a binding affinity almost equivalent to the disaccharide β-lactose, thus giving a
novel scaffold for developing galectin-8 antagonists. Further, we discover that the aromatic phenyl
ring substitution to the galactose malonic acid is the key factor in enhancing affinity. From the
study of galectin-8’s biological activities (assessed here by its stimulatory effects on
cytokine/chemokine expression in cultured cell line) we concluded that the inhibitory effects of
the antagonists directly correlate with their order of binding affinity to galectin-8N. Atomic
structure, experimental binding affinities, and the cell culture results show that it is the aromatic
malonyl substitution on the methyl β-D-galactopyranoside that leads to a sharp increase in binding
affinity. The malonyl linker offers binding interactions that provide a novel approach to engineer
potent galectin-8N antagonist.
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EXPERIMENTAL SECTION
5
6
7
8
9
Chemistry: All reagents and solvents were dried prior to use according to standard methods.
Commercial reagents were used without further purification. Analytical thin layer chromatography
(TLC) was performed using on silica gel 60 F254 (Merck) with detection by UV absorption and/or
by charring following immersion in a 5% ethanolic solution of sulfuric acid. Reaction products
were purified using flash chromatography with silica gel 60 (0.040-0.063 mm). 1H and 13C NMR
spectra were recorded at 298K using an Avance (400 MHz and 100 MHz respectively),
spectrometer (Bruker Biospin) in solutions of deuterated chloroform (CDCl3) or deuterated
methanol (MeOH-D4). Chemical shifts are reported to two decimal places in parts per millions
(ppm) relative to tetramethylsilane (0 ppm). Electrospray ionization (ESI) low resolution mass
spectrometry (LRMS) was performed using a Bruker Daltronics esquire 3000 Ion-Trap
instruments, using Bruker Daltronics esquire control 5.0 software. All spectra were recorded in
positive-ion mode at a concentration of 0.1-0.3 mg/mL. High Resolution Mass Spectra (HRMS)
were measured by maXis II ETD to evaluate precise mass for novel synthesized derivatives. The
purity of all final compounds was analysed by Thermo Fisher Scientific high performance liquid
chromatography (HPLC), Accucore C18 column 2.1*150 mm, 2.6 micromolar. The detailed
analytical condition is available in the supporting information. The purity of all analyzed
compounds was found greater than 95%.
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General Procedure for Preparation of Intermediates 2-7. A mixture of Methyl 4,6-di-O-
benzylidene-2-O-methoxymethyl-β-D-galactopyranoside 1 (100 mg, 0.31 mmol, 1 equiv), aryl
(Ar) malonic acid 24-28, 30 (0.46 mmol, 1.5 equiv), DCC (95 mg, 0.46 mmol, 1.5 equiv), and 4-
dimethylaminopyridine (DMAP) (25 mg) in 15 mL dichloromethane (DCM) was stirred overnight
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at -20 °C to room temperature. The reaction mixture was filtered and freeze to precipitate side
product. Precipitated side product was filtered, and the filtrate was purified by column
chromatography on silica gel (hexanes: ethyl acetate 3:1) to give intermediates 2-7.
Methyl 4,6-di-O-benzylidene-2-O-methoxymethyl-3-O-[(3′-O-phenyl)malonyl]-β-D-gala-
ctopyranoside (2) Ar = phenyl, yield: 132 mg, 87%. 1H NMR (CDCl3, 400 MHz): δ 3.31 (s, 3H,
H3C-O-C1), 3.44 [s, 1H, H2C(CO)2], 3.50 (s, 3H, H3C-O-CH2), 3.60-3.62 [m, 2H, H2C(CO)2, H-
6), 3.92-3.95 (m, 1H, H-5), 3.96-4.0 (m, 1H, H-2), 4.25 (d, J = 4.2 Hz, 1H, H-6), 4.30 (d, J = 8.3
Hz, 1H, H2C-O-CH3), 4.34 (d, J = 4.2 Hz, 1H, H-4), 4.62 (d, J = 8.2 Hz, 1H, H-1), 4.78 (d, J =
8.3 Hz, 1H, H2C-O-CH3), 4.94 (dd, J = 8.2 Hz, 4.3 Hz, 1H, H-3), 5.43 (s, 1H, HC-Ph), 6.95-6.97
(m, 2H, Ar), 7.13-7.15 (m, 1H, Ar), 7.19-7.21 (m, 2H, Ar), 7.23-7.25 (m, 3H, Ph), 7.39-7.42 (m,
2H, Ph). 13C NMR (CDCl3,400 MHz): δ 41.5 [CH2(C=O)2], 55.9 (CH3-O-C1), 56.9 (CH3-O-CH2),
66.0 (C-5), 68.9 (C-6), 72.7 (C-3), 73.4 (C-2), 74.6 (C-4), 97.2 (CH2-O-CH3), 101.1 (C-1), 103.9
(CH-Ph), 121.3 (2Ar), 126.2 (2Ar), 126.4 (2Ar), 128.1 (2Ar), 129.0 (2Ar), 129.5 (2Ar), 137.5 (Ar),
150.3 (Ar), 164.6 (C=O), 166.0 (C=O). ESIMS m/z [M + Na]+ calcd for [C25H28O10Na]+: 511.2,
found : 511.2.
10
11
12
13
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17
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Methyl 4,6-di-O-benzylidene-2-O-methoxymethyl-3-O-[(3′-O-{4-methoxyphenyl})malon-
1
yl]-β-D-galactopyranoside (3) Ar = 4-methoxyphenyl, yield: 145 mg, 90%. H NMR (CDCl3,
400 MHz): δ = 3.40 (s, 3H, H3C-O-C1), 3.52 [d, J = 4.3 Hz, 1H, H2C(CO)2], 3.60 (s, 3H, H3C-O-
CH2), 3.67 [d, J = 4.2 Hz, 2H, H2C(CO)2, H-6], 3.79 (s, 3H, H3C-O-Ar), 4.01-4.05 (m, 1H, H-5),
4.08 (dd, J = 12.3 Hz, 4.1 Hz, 1H, H-2), 4.34 (d, J = 4.2 Hz, 1H, H-6), 4.39 (d, J = 12.3 Hz, 1H,
H2C-O-CH3), 4.43 (d, J = 4.2 Hz, 1H, H-4), 4.71 (d, J = 4.3 Hz, 1H, H-1), 4.88 (d, J = 4.3 Hz,
1H, H2C-O-CH3), 5.05 (dd, J = 16.3 Hz, 4.2 Hz, 1H, H-3), 5.52 (s, 1H, HC-Ph), 6.76-6.79 (m, 2H,
Ar), 6.94-6.96 (m, 2H, Ar), 7.33-7.35 (m, 3H, Ph), 7.49-7.52 (m, 2H, Ph). 13C NMR (400 MHz,
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CDCl3): δ 41.5 [CH2(C=O)2], 55.5 (CH3-O-C1), 55.9 (CH3-O-CH2), 56.9 (CH3-O-Ar), 66.1 (C-5),
68.9 (C-6), 72.7 (C-3), 73.5 (C-2), 74.6 (C-4), 97.2 (CH2-O-CH3), 101.1 (C-1), 103.9 (CH-Ar),
114.4 (2Ar), 122.1 (2Ar), 126.4 (2Ar), 128.1 (2Ar), 129.0 (Ar), 137.5 (Ar), 143.8 (Ar), 157.4 (Ar),
165.0 (C=O), 166.0 (C=O). ESIMS m/z [M + Na]+ calcd for [C26H30O11Na]+: 541.2, found : 541.3.
Methyl 4,6-di-O-benzylidene-2-O-methoxymethyl-3-O-[(3′-O-{4-fluorophenyl})malonyl]-
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β-D-galactopyranoside (4) Ar = 4-fluorophenyl, yield: 127 mg, 81%. H NMR (CDCl3, 400
MHz): δ 3.30 (s, 3H, H3C-O-C1), 3.44-3.46 [m, 1H, H2C(CO)2], 3.51 (s, 3H, H3C-O-CH2), 3.58
[d, J = 4.2 Hz, 2H, H2C(CO)2, H-6], 3.91-3.95 (m, 1H, H-5), 3.99 (dd, J = 8.3 Hz, 4.2 Hz, 1H, H-
2), 4.26 (s, 1H, H-6), 4.29 (d, J = 4.2 Hz, 1H, H2C-O-CH3), 4.34 (d, J = 4.1 Hz, 1H, H-4), 4.61
(d, J = 4.3 Hz, 1H, H-1), 4.78 (d, J = 8.2 Hz, 1H, H2C-O-CH3), 4.94 (dd, J = 16.2 Hz, 4.3 Hz, 1H,
H-3), 5.42 (s, 1H, HC-Ph), 6.84-6.89 (m, 4H, Ar), 7.24-7.26 (m, 3H, Ph), 7.39-7.41 (m, 2H, Ph).
13C NMR (400 MHz, CDCl3): δ 41.5 [CH2(C=O)2], 55.9 (CH3-O-C1), 57.0 (CH3-O-CH2), 66.0
(C-5), 68.9 (C-6), 72.7 (C-3), 73.4 (C-2), 74.7 (C-4), 97.3 (CH2-O-CH3), 101.2 (C-1), 103.9 (CH-
Ph), 116.0 (2Ar), 122.7 (2Ar), 126.1 (2Ar), 128.1 (2Ar), 130.0 (Ar), 137.4 (Ar), 146.0 (Ar), 161.5
(Ar), 164.6 (C=O), 165.8 (C=O). ESIMS m/z [M + Na]+ calcd for [C25H27FO10Na]+: 529.2, found
: 529.2.
Methyl 4,6-di-O-benzylidene-2-O-methoxymethyl-3-O-[(3′-O-{2,4-dichlorophenyl})malo-
nyl]-β-D-galactopyranoside (5) Ar = 2,4-dichlorophenyl, yield: 162 mg, 90%. 1H NMR (CDCl3,
400 MHz): δ 3.30 (s, 3H, H3C-O-C1), 3.44-3.46 [m, 1H, H2C(CO)2], 3.51 (s, 3H, H3C-O-CH2),
3.64 [d, J = 4.3 Hz, 2H, H2C(CO)2, H-6], 3.93 (dd, J = 16.2 Hz, 4.3 Hz, 1H, H-2), 3.99 (dd, J =
16.2 Hz, 4.3 Hz, 1H, H-5), 4.26 (d, J = 12.3 Hz, 1H, H-6), 4.29 (d, J = 4.2 Hz, 1H, H2C-O-CH3),
4.35 (d, J = 4.3 Hz, 1H, H-4), 4.61 (d, J = 4.2 Hz,1H, H-1), 4.78 (d, J = 4.3 Hz, 1H, H2C-O-CH3),
4.92-4.96 (m, 1H, H-3), 5.42 (s, 1H, HC-Ph), 6.87-6.95 (m, 2H, Ar), 7.21-7.27 (m, 3H, Ph), 7.32
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(s, 1H, Ar), 7.33-7.37 (m, 2H, Ph). 13C NMR (400 MHz, CDCl3): δ 41.1 [CH2(C=O)2], 55.9 (CH3-
O-C1), 57.0 (CH3-O-CH2), 66.0 (C-5), 68.9 (C-6), 72.7 (C-3), 73.4 (C-2), 74.7 (C-4), 97.2 (CH2-
O-CH3), 101.0 (C-1), 103.9 (CH-Ar), 124.3 (Ar), 126.4 (Ar), 127.5 (Ar), 128.0 (3Ar), 128.1 (Ar),
129.1 (Ar), 130.0 (Ar), 132.3 (Ar), 137.5 (Ar), 145.2 (Ar), 163.5 (C=O), 165.4 (C=O). ESIMS m/z
[M + Na]+ calcd for [C25H26Cl2O10Na]+: 579.2, found : 579.2.
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Methyl 4,6-di-O-benzylidene-2-O-methoxymethyl-3-O-[(3′-O-{3,5-dimethoxyphenyl})ma-
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lonyl]-β-D-galactopyranoside (6) Ar = 3,5-dimethoxyphenyl, yield: 160 mg, 90%. H NMR
(CDCl3, 400 MHz): δ 3.30 (s, 3H, H3C-O-C1), 3.44-3.46 [m, 1H, H2C(CO)2], 3.50 (s, 3H, H3C-
O-CH2), 3.59 [d, J = 4.2 Hz, 2H, H2C(CO)2, H-6], 3.65 (s, 6H, 2H3CO-Ar), 3.93 (dd, J = 16.3 Hz,
4.2 Hz, 1H, H-5), 3.99 (dd, J = 16.2 Hz, 4.3 Hz, 1H, H-2), 4.26 (d, J = 4.2 Hz, 1H, H-6), 4.29 (d,
J = 8.3 Hz, 1H, H2C-O-CH3), 4.35 (d, J = 4.2 Hz, 1H, H-4), 4.62 (d, J = 8.3 Hz, 1H, H-1), 4.78
(d, J = 8.2 Hz, 1H, H2C-O-CH3), 4.93 (dd, J = 16.3 Hz, 4.2 Hz, 1H, H-3), 5.43 (s, 1H, HC-Ph),
6.18-6.19 (m, 2H, Ar), 6.25-6.26 (m, 1H, Ar), 7.22-7.24 (m, 3H, Ph), 7.38-7.41 (m, 2H, Ph). 13C
NMR (400 MHz, CDCl3): δ 41.4 [CH2(C=O)2], 55.4 (2CH3O-Ar), 55.9 (CH3-O-C1), 57.0 (CH3-
O-CH2), 66.0 (C-5), 68.9 (C-6), 72.6 (C-3), 73.4 (C-2), 74.7 (C-4), 97.2 (CH2-O-CH3), 98.7 (Ar),
99.9 (2Ar), 101.0 (C1), 103.9 (CH-Ar), 126.3 (2Ar), 128.0 (2Ar), 128.9 (Ar), 137.4 (Ar), 151.8
(Ar), 161.1 (2Ar), 164.4 (C=O), 166.0 (C=O). ESIMS m/z [M + Na]+ calcd for [C27H32O12Na]+:
571.2, found : 571.2.
Methyl 4,6-di-O-benzylidene-2-O-methoxymethyl-3-O-[(3′-O-benzyl)malonyl]-β-D-galac-
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topyranoside (7) Ar = benzyl, yield: 147 mg, 90%. H NMR (MeOH-D4, 400 MHz): δ 3.19 (s,
3H, H3C-O-C1), 3.45 (s, 3H, H3C-O-CH2), 3.51 [s, 1H, H2C(CO)2], 3.71-3.76 [m, 1H, H2C(CO)2],
4.01 (d, J = 12.2 Hz, 1H, H-2), 4.10 (d, J = 12.2 Hz, 1H, H-5), 4.29 (d, J = 4.2 Hz, 1H, H2C-O-
CH3), 4.32 (d, J = 12.3 Hz, 1H, H-4), 4.49 (d, J = 8.3 Hz, 1H, H-1), 4.66 (d, J = 8.3 Hz, 1H, H2C-O-
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CH3), 4.91 (d, J = 4.3 Hz, 1H, H2C-Ar), 4.93 (d, J = 8.2 Hz, 1H, H2C-Ar), 4.99 (d, J = 12.3 Hz,
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1H, H-3), 5.37 (s, 1H, HC-Ph), 7.21-7.25 (m, 5H, Ar), 7.26-7.28 (m, 3H, Ph), 7.35-7.37 (m, 2H,
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Ph). C NMR (MeOH-D4, 400 MHz): δ 33.3 (CH2C=O), 54.9 (CH3-OC1), 55.9 (CH3-O-CH2),
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66.0 (C-5), 66.7 (CH2O), 68.5 (C-6), 72.7 (C-3), 73.4 (C-2), 74.0 (C-4), 100.7 (CH2-O-CH3), 103.6
(C-1), 103.7 (CH-Ar), 126.0 (2Ar), 127.6 (2Ar), 127.7 (2Ar), 127.9 (2Ar), 128.1 (2Ar), 128.5 (Ar),
135.6 (Ar), 138.0 (Ar), 166.2 (C=O), 166.3 (C=O). ESIMS m/z [M + Na]+ calcd for
[C26H30O10Na]+: 525.2, found : 525.2.
Methyl 4,6-di-O-benzylidene-2-O-methoxymethyl-3-O-malonyl-β-D-galactopyranoside (14)
Methyl 4,6-di-O-benzylidene-2-O-methoxymethyl-3-O-[(3′-O-benzyl)malonyl]-β-D-galactopyra-
noside 7 (180 mg, 0.36 mmol), was dissolved in anhydrous MeOH followed by addition of 10%
Pd on activated carbon (60 mg) and stirred at room temperature in argon environment. After 5 min,
hydrogen gas was passed continuously through balloon for 2 h. After 2 h, reaction mixture was
filtered through celite as filtering agent and purified on silica column by using ethyl acetate: hexane
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(3:7) to yield product 14 (120 mg, 80%) as solids. H NMR ( CDCl3, 400 MHz): δ 3.29 (s, 3H,
H3C-O-C1), 3.37 [d, J = 4.0 Hz, 2H, H2C(CO)2, H-6], 3.41 [s, 1H, H2C(CO)2], 3.48 (s, 3H, H3C-
O-CH2), 3.88 (t, J = 8.3 Hz, 1H, H-2), 3.97 (dd, J = 12.3 Hz, 4.2 Hz, 1H, H-5), 4.24 (d, J = 4.3 Hz,
1H, H-6), 4.26 (d, J = 4.3 Hz, 1H, H2C-O-CH3), 4.28 (d, J = 4.3 Hz, 1H, H-4), 4.62 (d, J = 8.3
Hz, 1H, H-1), 4.75 (d, J = 4.2 Hz, 1H, H2C-O-CH3), 4.87 (dd, J = 12.2 Hz, 4.3 Hz, 1H, H-3), 5.40
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(s, 1H, HC-Ph), 7.26-7.28 (m, 3H, Ar), 7.40-7.43 (m, 2H, Ar). C NMR (400 MHz, CDCl3): δ
40.9 [CH2(C=O)2], 55.8 (CH3-O-C1), 56.9 (CH3-O-CH2), 66.0 (C-5), 68.8 (C-6), 72.8 (C-3), 73.7
(C-2), 74.5 (C-4), 100.9 (CH2-O-CH3), 103.8 (C-1), 103.8 (CH-Ar), 126.3 (2Ar), 128.1 (2Ar),
129.0 (Ar), 137.5 (Ar), 166.6 (C=O), 169.8 (C=O). ESIMS m/z [M + Na]+ calcd for
[C19H24O10Na]+: 435.2, found : 435.3.
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Methyl 4,6-di-O-benzylidene-2-O-methoxymethyl-3-O-[(3′-O-methyl)malonyl]-β-D-galacto-
pyranoside (16) Methyl 4,6-di-O-benzylidene-2-O-methoxymethyl-β-D-galactopyranoside 1
(100 mg, 0.306 mmol, 1 equiv), was dissolved in anhydrous DCM followed by addition TMEDA
(229 µL, 1.53 mmol, 5 equiv) and stirred at -20 °C in argon environment. After 5 min, methyl
malonyl chloride (98 µL, 0.918 mmol, 3 equiv) was added dropwise and stirred overnight room
temperature. After 24 h, reaction mixture was filtered purified on silica column by using ethyl
acetate: hexane (3:7) to yield product 16 (120 mg, 80%) as solids. 1H NMR (CDCl3, 400 MHz): δ
3.31 (s, 3H, H3C-O-C1), 3.37 [d, J = 4.2 Hz, 2H, H2C(CO)2], 3.42 (d, J = 4.2 Hz, 1H, H-6), 3.49
(s, 3H, H3C-O-CH2), 3.58 (s, 3H, H3C-OCO), 3.88 (t, J = 8.2 Hz, 1H, H-2), 3.99 (dd, J = 12.2 Hz,
4.3 Hz, 1H, H-5), 4.25 (d, J = 4.2 Hz, 1H, H-6), 4.28 (d, J = 4.2 Hz, 1H, H2C-O-CH3), 4.33 (d, J
= 4.0 Hz, 1H, H-4), 4.61 (d, J = 8.2 Hz, 1H, H-1), 4.77 (d, J = 8.2 Hz 1H, H2C-O-CH3), 4.85 (dd,
J = 8.2 Hz, 4.1 Hz, 1H, H-3), 5.43 (s, 1H, HC-Ph), 7.28-7.29 (m, 3H, HC-Ph), 7.42-7.45 (m, 2H,
HC-Ph). 13C NMR (400 MHz, CDCl3): δ 41.2 [CH2(C=O)2], 52.5 (CH3-COO), 55.8 (CH3-O-C1),
56.9 (CH3-O-CH2), 66.0 (C-5), 68.9 (C-6), 72.6 (C-3), 73.3 (C-2), 74.5 (C-4), 97.2 (CH2-O-CH3),
101.0 (C-1), 103.9 (CH-Ar), 126.4 (2Ar), 128.1 (2Ar), 129.0 (Ar), 166.3 (C=O), 166.4 (C=O).
ESIMS m/z [M + Na]+ calcd for [C20H26O10Na]+: 449.2, found : 449.3.
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General Procedure for Final Compounds 8-13, 15, and 17. Sugar 2-7, 14, 16 (100 mg) were
dissolved in 60% aqueous acetic acid (6 mL) and refluxed at 60 °C. Reaction was quenched by
addition of excess water, acetic acid was removed by toluene-chloroform mixture, concentrated
and purified by column chromatography (DCM : MeOH, 97:3) to give 8-13, 15, 17.
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Methyl 3-O-[(3′-O-phenyl)malonyl]-β-D-galactopyranoside (8). Yield: 60 mg, 83%. H
NMR (CDCl3, 400 MHz): δ 3.53 (s, 3H, H3C-O-C1), 3.55-3.57 [m, 1H, H2C(CO)2], 3.66 (s, 1H,
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H-4), 3.74 [s, 1H, H2C(CO)2], 3.78-3.81 (m, 1H, H-5), 3.82-3.84 (m, 1H, H-2), 3.87-3.91 (m, 2H,
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H-2, H-6), 4.22 (d, J = 4.2 Hz, 1H, H-6), 4.24 (d, J = 4.2 Hz, H-1), 4.84 (dd, J = 12.4 Hz, 4.2 Hz,
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1H, H-3), 7.04-7.07 (m, 2H, Ar), 7.22 (s, 1H, Ar), 7.31-7.33 (m, 2H, Ar). C NMR (400 MHz,
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CDCl3): δ 41.5 [CH2(C=O)2], 57.3 (CH3-O-C1), 62.8 (C-6), 67.8 (C-5), 68.9 (C-2), 73.7 (C-3),
104.1 (C-1), 121.2 (2Ar), 126.5 (Ar), 129.6 (2Ar), 150.0 (Ar), 165.5 (C=O), 166.4 (C=O). HRMS
m/z [M + Na]+ calcd for [C16H20O9Na]+: 379.1005, found: 379.0994.
Methyl 3-O-[(3′-O-{4-methoxyphenyl})malonyl]-β-D-galactopyranoside (9). Yield: 64 mg,
86%. 1H NMR (CDCl3, 400 MHz): δ 3.53 (s, 3H, H3C-O-C1), 3.55-3.56 [m, 1H, H2C(CO)2], 3.64
(s, 1H, H-4), 3.71 [s, 1H, H2C(CO)2], 3.73 (s, 3H, H3C-O-CH2), 3.81 (dd, J = 12.2 Hz, 4.3 Hz, 1H,
H-5), 3.88-3.89 (m, 1H, H-6), 3.90-3.92 (m, 1H, H-2), 4.17 (d, J = 4.3 Hz, 1H, H-6), 4.22 (d, J =
8.3 Hz, H-1), 4.82 (dd, J = 12.3 Hz, 4.2 Hz, 1H, H-3), 6.81-6.83 (m, 2H, 2H-Ar), 6.96-6.98 (m,
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2H, 2H-Ar). C NMR (400 MHz, CDCl3): δ 41.4 [CH2(C=O)2], 55.6 (CH3-O-Ar), 57.3 (CH3-
OC1), 62.7 (C-6), 67.7 (C-5), 68.9 (C-2), 73.7 (C-3), 104.1 (C-1), 114.6 (2Ar), 122.0 (2Ar), 143.6
(Ar), 157.7 (Ar), 165.6 (C=O), 166.8 (C=O). HRMS m/z [M + Na]+ calcd for [C17H22O10Na]+:
409.1110, found: 409.1101.
Methyl 3-O-[(3′-O-{4-fluorophenyl})malonyl]-β-D-galactopyranoside (10). Yield: 63 mg,
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85%. H NMR (CDCl3, 400 MHz): δ 3.54 (s, 3H, H3C-O-C1), 3.62 [s, 1H, H2C(CO)2], 3.68 (s,
1H, H-4), 3.72 [s, 1H, H2C(CO)2], 3.84-3.85 (m, 1H, H-5), 3.86-3.88 (m, 1H, H-6), 3.89-3.91 (m,
1H, H-2), 4.18 (d, J = 4.3 Hz, 1H, H-6), 4.22 (d, J = 8.2 Hz, H-1), 4.84 (dd, J = 12.3 Hz, 4.2 Hz,
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1H, H-3), 6.81-6.83 (m, 2H, Ar), 6.96-6.98 (m, 2H, Ar). C NMR (400 MHz, CDCl3): δ 41.4
[CH2(C=O)2], 57.4 (CH3-O-C1), 62.8 (C-6), 67.9 (C-5), 68.9 (C-2), 73.7 (C-3), 104.1 (C-1), 116.2
(2Ar), 122.7 (Ar), 122.8 (2Ar), 160.0 (Ar), 165.4 (C=O), 166.2 (C=O). HRMS m/z [M + Na]+ calcd
for [C16H19FO9Na]+: 397.0910, found: 397.0901.
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Methyl 3-O-[(3′-O-{2,4-dichlorophenyl})malonyl]-β-D-galactopyranoside (11). Yield: 68
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mg, 89%. H NMR (MeOH-D4, 400 MHz): δ 3.26 (s, 3H, H3C-O-C1), 3.43-3.44 [m, 3H,
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H2C(CO)2, H-4], 3.48 (dd, J = 4.2 Hz, 2.3 Hz, 1H, H-5), 3.62-3.65 (m, 1H, H-6), 3.70-3.75 (m, 1H,
H-4), 3.98 (dd, J = 16.2 Hz, 2.3 Hz, 1H, H-2), 4.24 (t, J = 8.3 Hz, 1H, H-6), 4.55 (dd, J = 12.2
Hz, 4.3 Hz, H-1), 4.71 (dd, J = 8.3 Hz, 4.2 Hz, 1H, HO-C6), 4.80 (dd, J = 8.3 Hz, 4.2 Hz, 1H, H-
3), 6.76-6.78 (m, 1H, H-Ar), 7.0-7.02 (m, 1H, H-Ar), 7.18-7.21 (m, 1H, H-Ar), 7.28-7.31 (m, 1H,
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H-Ar), 7.48-7.49 (m, 1H, H-Ar). C NMR (MeOH-D4, 400 MHz): δ 54.7 [CH2(C=O)2], 55.8
(CH3-O-C1), 60.6 (C-6), 66.2 (C-4), 73.2 (C-2), 74.6 (C-3), 76.3 (C-5), 96.6 (C-1), 104.1 (Ar),
117.1 (Ar), 121.1 (Ar), 127.4 (Ar), 128.9 (Ar), 152.1 (Ar), 166.4 (C=O), 167.3 (C=O). HRMS m/z
[M + Na]+ calcd for [C16H18Cl2O9Na]+: 447.0225, found: 447.0247.
Methyl 3-O-[(3′-O-{3,5-dimethoxyphenyl})malonyl]-β-D-galactopyranoside (12). Yield: 68
mg, 90%. 1H NMR (CDCl3, 400 MHz): δ 3.53 (s, 3H, H3C-O-C1), 3.54-3.56 [m, 1H, H2C(CO)2],
3.64 (s, 1H, H-4), 3.71 (s, 6H, 2H3CO-Ar), 3.72 [s, 1H, H2C(CO)2], 3.81 (dd, J = 12.3 Hz, 4.2 Hz,
1H, H-5), 3.86-3.87 (m, 1H, H-6), 3.88-3.91 (m, 1H, H-2), 4.17 (d, J = 4.3 Hz, 1H, H-6), 4.23 (d,
J = 8.2 Hz, 1H, H-1), 4.83 (dd, J = 8.2 Hz, 4.3 Hz, 1H, H-3), 6.22-6.23 (m, 2H, 2H-Ar), 6.28-6.30
(m, 1H, H-Ar). 13C NMR (400 MHz, CDCl3): δ 41.5 [CH2(C=O)2], 55.5 (2CH3-O-Ar), 57.3 (CH3-
O-C1), 62.7 (C-6), 67.8 (C-5), 68.9 (C-2), 73.7 (C-3), 98.7 (C-1), 99.9 (2Ar), 104.1 (Ar), 151.6
(Ar), 161.2 (2Ar), 165.5 (C=O), 166.1 (C=O). HRMS m/z [M + Na]+ calcd for [C18H24O11Na]+:
439.1216, found: 439.1201.
1
Methyl 3-O-[(3′-O-benzyl)malonyl]-β-D-galactopyranoside (13). Yield: 70 mg, 95%. H
NMR (CDCl3, 400 MHz): δ 3.56 (s, 3H, H3C-O-C1), 3.57-3.59 [m, 1H, H2C(CO)2], 3.72-3.77 (m,
3H, H-2, H-5, H-6), 4.06 (d, J = 4.3 Hz, 1H, H-6), 4.26 (d, J = 8.3 Hz, 1H, H-1), 4.79 (dd, J = 12.2
13
Hz, 4.2 Hz, 1H, H-3), 5.20 (s, 2H, H2C-Ar), 7.33-7.42 (m, 5H, 5H-Ar). C NMR (400 MHz,
29
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