Novel bisubstrate uridine-peptide analogues bearing a pyrophosphate bioisostere as inhibitors of human O-GlcNAc transferase

Article abstract of DOI:10.1016/j.bioorg.2021.104738

Protein O-linked β-D-N-acetylglucosamine (O-GlcNAc) modification (O-GlcNAcylation), an essential post-translational as well as cotranslational modification, is the attachment of β-D-N-acetylglucosamine to serine and threonine residues of nucleocytoplasmic proteins. An aberrant O-GlcNAc profile on certain proteins has been implicated in metabolic diseases such as diabetes and cancer. Inhibitors of O-GlcNAc transferase (OGT) are valuable tools to study the cell biology of protein O-GlcNAc modification. In this study we report novel uridine-peptide conjugate molecules composed of an acceptor peptide covalently linked to a catalytically inactive donor substrate analogue that bears a pyrophosphate bioisostere and explore their inhibitory activities against OGT by a radioactive hOGT assay. Further, we investigate the structural basis of their activities via molecular modelling, explaining their lack of potency towards OGT inhibition.

Full text of DOI:10.1016/j.bioorg.2021.104738

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Contents lists available at ScienceDirect
Bioorganic Chemistry
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Short communication
Novel bisubstrate uridine-peptide analogues bearing a pyrophosphate
bioisostere as inhibitors of human O-GlcNAc transferase
,
b
,
,b,c,*
Philip Ryana c, Yun Shid, Mark von Itzsteind, Santosh Rudrawara
a Menzies Health Institute Queensland, Griffith University, Gold Coast, QLD 4222, Australia
b School of Pharmacy and Pharmacology, Griffith University, Gold Coast, QLD 4222, Australia
c School of Chemistry, The University of Sydney, NSW 2006, Australia
d Institute for Glycomics, Griffith University, Gold Coast, QLD 4222, Australia
A R T I C L E I N F O
A B S T R A C T
Keywords:
Protein O-linked β-D-N-acetylglucosamine (O-GlcNAc) modification (O-GlcNAcylation), an essential post-
translational as well as cotranslational modification, is the attachment of β-D-N-acetylglucosamine to serine
and threonine residues of nucleocytoplasmic proteins. An aberrant O-GlcNAc profile on certain proteins has been
implicated in metabolic diseases such as diabetes and cancer. Inhibitors of O-GlcNAc transferase (OGT) are
valuable tools to study the cell biology of protein O-GlcNAc modification. In this study we report novel uridine-
peptide conjugate molecules composed of an acceptor peptide covalently linked to a catalytically inactive donor
substrate analogue that bears a pyrophosphate bioisostere and explore their inhibitory activities against OGT by
a radioactive hOGT assay. Further, we investigate the structural basis of their activities via molecular modelling,
explaining their lack of potency towards OGT inhibition.
OGT inhibitor
Pyrophosphate bioisostere
Bisubstrate analogue as inhibitor
O-GlcNAc transferase (OGT)
1. Introduction
inhibitors [17].
UDP, the by-product of O-GlcNAcylation, is a potent feedback in-
The ubiquitously expressed O-GlcNAc transferase (OGT) enzyme
boasts numerous vital cellular roles within eukaryotes. It is solely
responsible for transferring O-linked N-acetylglucosamine (O-GlcNAc)
onto the serine and threonine residues of acceptor substrate proteins
(Fig. 1). OGT employs the universal donor substrate sugar nucleotide
(UDP-GlcNAc) to modify myriads of proteins, modulating their func-
tions, processing and associated signalling pathways [1]. Indeed, more
than 4000 proteins have been identified as O-GlcNAcylation (O-GlcNAc
modification) candidates. O-GlcNAcylation roles are numerous and
range from coupling metabolic status to the regulation of an extensive
variety of cellular signalling pathways [2–5]. Understandably, dysre-
gulation of protein O-GlcNAcylation is linked to the pathological pro-
gression of various chronic metabolic diseases including diabetes [6,7],
cancers [8–10] to cardiovascular [11] and neurodegenerative disorders
[12–14]. The consequences of cellular hyper-O-GlcNAcylation, via both
knockout and chemical inhibition of O-GlcNAc hydrolase (OGA) [15],
the counterpart O-GlcNAc cleavage enzyme, have been extensively
studied [12,16]. The investigation of the precise biological functions as
well as implications of cellular hypo-O-GlcNAcylation via OGT inhibi-
tion has been significantly impeded due to a marked lack of OGT
hibitor of OGT and has been observed to occupy the same UDP-GlcNAc
binding pocket in OGT competitively [18]. Many of the more potent
OGT inhibitors have been designed to engage the UDP binding site
where they exploit the native binding mode of the donor substrate UDP-
GlcNAc [19,20]. The most widely employed inhibitor to date, per-O-
acetylated 5-thio-N-acetylglucosamine, hijacks the hexosamine biosyn-
thetic pathway (HBP) to yield the donor substrate analogue UDP-5S-
GlcNAc [21]. As UDP is commonly employed as donor substrate by
other enzymes, including oxidoreductases, transferases, hydrolases, ly-
ases and isomerases, UDP analogues suffer limited selectivity for OGT.
The structure of ternary hOGT complexes (OGT-UDP-substrate pep-
tide derived from the casein kinase II) have revealed that glycosyl-
transfer takes place via an ordered bi-bi kinetic mechanism, wherein
UDP-GlcNAc binds prior to the polypeptide substrate [18]. The way in
which OGT targets specific sites on a limited subset of intracellular
proteins remains largely unknown however. Furthermore, the involve-
ment of a catalytic base in the OGT-catalysed glycosyl transfer reaction
is currently debated [22–24] and the precise catalytic mechanism of
OGT remains to be discovered. As a member of Glycosyltransferase
Family 41 (GT41), OGT possesses a GT-B fold and the glycosyl transfer
* Corresponding author.
E-mail address: s.rudrawar@griffith.edu.au (S. Rudrawar).
https://doi.org/10.1016/j.bioorg.2021.104738
Received 12 September 2020; Received in revised form 4 February 2021; Accepted 5 February 2021
Available online 13 February 2021
0045-2068/© 2021 Elsevier Inc. All rights reserved.

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P. Ryan et al.
Bioorganic Chemistry 110 (2021) 104738
reaction proceeds with inversion of stereochemistry at the anomeric
center. Bisubstrate mimics of the transition state structure have emerged
as promising, selective inhibitors. These compounds are designed to
couple the high affinity of the donor substrate and high selectivity of the
acceptor substrate (Fig. 1). Bisubstrate analogue antagonists that are
constructed from covalently-linked bridging donor and acceptor sub-
strates, may allow each substrate to occupy their natural orientations
within their respective binding sites, affecting the antagonists potency
[25].
A number of bisubstrate analogue inhibitors of OGT have been re-
ported. Goblin1 is a UDP-peptide conjugate bearing sequence VTPVS(O-
propyl-UDP)TAtethered via an ether linkage [26]. Goblin1 was found to
inhibit glycosylation of an acceptor substrate in vitro in a dose-
dependent manner (IC50 = 18 µM). Goblin1 was unable to efficiently
penetrate cells however, likely as it possessed pyrophosphate in its
structure. A second series of bisubstrate analogue inhibitors, bearing
neutral aliphatic linker units in place of the natural substrate’s pyro-
phosphate moiety, produced only modest inhibitors of OGT activity
[27]. Finally, van Aalten and co-workers produced bisubstrate inhibitors
such as the thiol-linked VPTVC(S-propyl-UDP)TA conjugate, function-
alised with cell-penetrant peptide sequences, specifically Penetratin and
TAT [28,29], to promote cellular uptake [30]. The Goblin bisubstrate
Fig. 2. (a) The structures of the squaramide and phosphate functional groups;
(b) An expanded region of a modified oligodeoxynucleotide. The central
thymidine dimer has been modified with a squaryldiamide linkage (TsqT). (c)
The generalised structure for the squaramide-linked bisubstrate analogue OGT
inhibitors described here.
cell penetrating conjugate displayed hOGT inhibition (IC50 = 5 μM) in
vitro as a parent compound, however conjugates could not target cyto-
solic hOGT due to entrapment in the early endosomes [30].
Pyrophosphate units tends to confer an increase in potency and a
decrease in cell-permeability when implemented into the bisubstrate
scaffold. Use of a pyridine unit as a pyrophosphate surrogate has pro-
vided compounds with moderate in vitro inhibition hOGT [31]. High-
throughput screens have found that compounds mimicking pyrophos-
phate exhibit exquisite inhibition of OGT [32,33]. A series of compounds
bearing a five heteroatom dicarbamate core were found to occupy py-
rophosphate’s position within the OGT enzyme’s catalytic site. Investi-
gation into the mechanism of OGT inhibition suggested that the
dicarbamate scaffold maintains the potential to deactivate a wide
assortment of enzymes that also utilise pyrophosphate-based substrates.
Pyrophosphate groups augment potential substrates with limited
bioavailability, physiological instability, inability to permeate mem-
branes and also complicate synthetic methods. We were interested to
explore if employing a bioisosteric replacement for the pyrophosphate
moiety in the design of novel bisubstrate inhibitors of OGT would bypass
these issues and afford potency. The diketocyclobutane diamine, or
‘squaramide’, function has become a well-established phosphate bio-
isostere owing to its remarkable chemical stability, hydrogen-bonding
capability and high double acidity with it’s resonance-stabilised dia-
nion (Fig. 2a) [34]. Secondary squaramides offer the potential to
hydrogen-bond to acceptors, donors and mixed acceptor-donor groups,
strengthened by an increase in their aromaticity on complexing [35,36].
Indeed, squaramides have been used to replace phosphate as inter-
nucleotidic linkers in the structures of sugar-nucleotide analogues
suggested for use as antiviral and anticancer agents (Fig. 2b) [37,38].
The inhibitors here employ the squaramide bioisostere to exploit pyro-
phosphate’s role in establishing stabilising interactions during the
transfer reaction within the active site of OGT (Fig. 2c). Here we disclose
the rational design, synthesis and evaluation of some novel bisubstrate
analogue inhibitors against the hOGT enzyme (Fig. 3).
2. Materials and methods
2.1. General
1H NMR spectra were recorded using Bruker Avance 300, Avance III
400 and Avance III 500 spectrometers at frequencies of 300 MHz, 400
MHz and 500 MHz respectively. The spectra are reported as parts per
million (ppm) downfield shift using the solvent peak as internal refer-
ence. The data are reported as chemical shift (δ), multiplicity, relative
integral, coupling constant (J Hz) and assignment where possible. Low
resolution mass spectra were recorded on a Finnigan LCQ Deca ion trap
mass spectrometer (ESI). High resolution mass spectra were recorded on
a Bruker 7 T Fourier Transform Ion Cyclotron Resonance Mass Spec-
trometer (FTICR). Analytical reverse-phase HPLC was performed on a
Shimadzu LCMS 2020 separations module with an LC-20AD pump and
SPD-20A photodiode array detector. A Waters Sunfire C18 5 μm, 2.1 ×
150 mm column was used at a flow rate of 0.2 mL minꢀ 1 using a mobile
Fig. 1. OGT catalysed O-GlcNAcylation and bisubstrate analogue as OGT inhibitor.
2

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P. Ryan et al.
Bioorganic Chemistry 110 (2021) 104738
previously reported [39].
2.2.2. Synthesis of 2′,3′-O-isopropylidene-5′-O-toluenesulfonyluridine
To 2′,3′-O-isopropylideneuridine (2.50 g, 8.79 mmol) dissolved in
pyridine (15.0 mL) at 0 ◦C over an hour. After, 4-toluenesulfonyl chlo-
ride (4.67 g, 24.6 mmol) and DMAP (0.247 g, 2.02 mmol) were added
and mixed at room temperature over 48 h. The mixture was diluted with
water (100 mL) and extracted with ethyl acetate (3 × 100 mL). The
combined organic fractions were washed with 0.1 M hydrochloric acid
(50.0 mL) before water (100 mL) and finally brine (100 mL) before
drying (Na2SO4) and concentrating under reduced pressure. The crude
material was a yellow oil and was purified by flash chromatography (1:5
v/v Hex/EtOAc) to yield the title compound (2.85 g, 74%) as a pale
yellow foam; mp 93 ◦C; 1H NMR (500 MHz, CDCl3): δ 9.93 (s, 1H) 7.71
(d, J = 8.2 Hz, 2H), 7.28 (d, J = 8.0 Hz, 2H), 7.22 (d, J = 8.1 Hz, 1H),
5.66 (d, J = 8.0 Hz, 1H), 5.6 (d, J = 2.0, 1H), 4.90 (dd, J = 1.9, 6.4 Hz,
1H), 4.74 (dd, J = 3.8, 6.4 Hz, 1H), 4.25–2.33 (m, 1H), 4.21–4.28 (m,
2H) 2.29 (s, 3H) 1.44 (s, 3H), 1.23 (s, 3H); 13C NMR (100 MHz, CDCl3): δ
163, 150, 145, 143, 133, 130, 128, 115, 103, 95.0, 85.2, 84.4, 80.9,
69.5, 27.1, 25.2, 21.7. Data are in agreement with that previously re-
ported [40].
Fig. 3. Antagonistic activity was evaluated by a radioactive hOGT assay using
recombinant nup62-MBP fusion construct as the protein acceptor and [3H]-
UDP-GlcNAc in UDP-GlcNAc as competitive donor substrate. Reactions were
performed in duplicate with inhibition measured as a percentage of relative
OGT activity in absence of inhibitor. All compounds assayed at 20
concentration.
μM
2.2.3. Synthesis of 5′-azido-5′-deoxy-2′,3′-O-isopropylideneuridine
To a stirred solution of 2′,3′-O-isopropylidene-5′-O-toluenesulfony-
luridine (3.76 g, 8.00 mmol) in DMF (15.0 mL) was added sodium azide
(0.779 g, 12.0 mmol) under argon at room temperature. The mixture
was heated to 80 ◦C and stirred for 24 h. The mixture was diluted with
water (100 mL) and extracted with ethyl acetate (3 × 100 mL). The
combined organic layers were washed with brine before drying
(Na2SO4) and concentrating under reduced pressure. The crude material
was purified by flash chromatography (2:3 v/v Hex/EtOAc) to yield the
phase of 0.1% TFA in water (Solvent A) and 0.1% TFA in acetonitrile
(Solvent B) and a linear gradient of 2–50% B over 30 min. The results
were analysed with Waters Empower software. Preparative reverse-
phase HPLC was performed using a Waters 600 Multisolvent Delivery
System and Waters 500 pump with 2996 photodiode array detector or
Waters 490E Programmable wavelength detector operating at 214 and
254 nm. A Waters Sunfire 5 μm, 19 × 150 mm column was used at a flow
title compound (2.30 g, 97%) as an amorphous yellow foam; νmax
3
rate of 7 mL minꢀ 1 using a mobile phase of 0.1% TFA in water (Solvent
A) and 0.1% TFA in acetonitrile (Solvent B) using a linear gradient of
one of either i) 2–80% B over 60 min, ii) 2–60% B over 60 min or 2–40%
over 60 min. LC-MS was performed on a Shimadzu LCMS 2020 system
coupled with an LC-20AD pump and SPD-20A detector on a Waters
(film)/cmꢀ 1 219, 2985, 2106, 1677, 1538, 1461, 1375, 1260, 1154,
1091; 1H NMR (300 MHz, CDCl3): δ 8.76 (s, 1H), 7.30 (d, J = 8.0 Hz,
1H), 5.75 (d, J = 8.0 Hz, 1H), 5.65 (d, J = 2.1 Hz, 1H), 4.99 (dd, J = 2.0,
6.5 Hz, 1H), 4.81 (dd, J = 4.2, 6.5 Hz, 1H), 4.21–4.24 (m, 1H) 3.60 (dd,
J = 5.2 Hz, 2H), 1.45 (s, 3H), 1.24 (s, 3H); 13C NMR (100 MHz, CDCl3): δ
164, 150, 143, 115, 103, 94.9, 86.0, 84.4, 81.6, 52.4, 27.1, 25.3. Data
are in agreement with that previously reported [40].
Sunfire C18 5
μ
m, 2.1 × 150 mm column at a flow rate of 0.2 mL minꢀ 1
coupled to a Thermoquest Finnigan LCQ Deca mass spectrometer (ESI)
operating in positive mode. Separations involved a mobile phase of 0.1%
formic acid in water (Solvent A) and 0.1% formic acid in acetonitrile
(Solvent B) using a linear gradient of 2–50% B over 30 min. Analytical
thin layer chromatography (TLC) was performed on commercially pre-
pared silica plates (Merck Kieselgel 60 0.25 mm F254). Flash column
chromatography was performed using 230–400 mesh Kieselgel 60 silica
eluting with analytical grade solvents. Commercial materials were used
a received unless otherwise noted. Amino acids, coupling reagents and
resins were obtained from Novabiochem. Dichloromethane and meth-
anol were distilled from calcium hydride. DMF was obtained as peptide
synthesis grade from Auspep or Labscan.
2.2.4. Synthesis of 5′-amino-5′-deoxy-2′,3′-O-isopropylideneuridine (4)
To
a
stirred
solution
of
5′-azido-5′-deoxy-2′,3′-O-iso-
propylideneuridine (2.75 g, 9.64 mmol) in methanol (50.0 mL) was
added Pd/C (0.275 g, 10% w/w) under argon at 10 ◦C. The mixture was
then bubbled with hydrogen gas at room temperature and the progress
of the reaction was monitored closely by TLC analysis. After completion,
the reaction mixture was filtered through a Celite® bed, washed with
cold methanol, and concentrated under reduced pressure to afford the
title compound (2.39 g, 95%) as a white powder which was not purified
further; mp. 85 ◦C; 1H NMR (300 MHz, DMSO‑d6): δ 7.85 (d, J = 8.1 Hz,
1H), 5.78 (d, J = 2.8 Hz, 1H), 5.63 (d, J = 8.0 Hz, 1H), 4.94 (dd, J = 6.4,
2.8 Hz, 1H), 4.74 (dd, J = 6.5, 3.9 Hz, 1H), 3.94 (m, 1H), 3.19 (br, s, 2H),
2.75 (d, J = 5.5 Hz, 2H), 1.44 (s, 3H), 1.26 (s, 3H). Data are in agreement
with that previously reported [40].
2.2. Synthesis of bisubstrate conjugates
2.2.1. Synthesis of 2′’,3′-O-isopropylideneuridine
To a stirred solution of uridine (3, 10.0 g, 41.0 mmol) and p-tolue-
nesulfonic acid (0.779 g, 4.10 mmol) in acetone (100 mL) was added
2,2-dimethoxypropane (6.40 g, 61.4 mmol) under argon. The mixture
was stirred at 60 ◦C for 24 h before being concentrated under reduced
pressure. The crude material was purified by flash chromatography (1:9
v/v MeOH/DCM) to yield the title compound (11.2 g, 96%) as a white
foam; mp. 169 ◦C; 1H NMR (500 MHz, CD3OD): δ 7.78 (d, J = 8.1 Hz,
1H), 5.86 (d, J = 2.8 Hz, 1H), 5.50 (d, J = 8.0 Hz, 1H) 4.96 (dd, J = 2.8,
6.3 Hz, 1H), 4.63 (dd, J = 3.4, 6.3 Hz, 1H), 4.22 (m, 1H), 3.75 (dd, J =
3.6, 11.9 Hz, 1H), 3.72 (dd, J = 4.5, 11.9 Hz, 1H), 1.54 (s, 3H), 1.30 (s,
3H); 13C NMR (100 MHz, CD3OD): δ 166.8, 153.1, 144.5, 113.1, 101.6,
92.5, 88.5, 85.6, 82.4, 62.1, 26.7, 25.5. Data are in agreement with that
2.2.5. Synthesis of Boc-6-aminocaproic acid
To a solution of 6-aminocaproic acid (1, 3.00 g, 22.9 mmol) and
NaOH (0.930, 25.2 mmol) in dioxane/H2O (90.0 mL, 2:1) was added di-
◦
tert-butyl dicarbonate (5.49 g, 25.2 mmol) at 0 C in three equal por-
tions. The reaction mixture was stirred for 16 h before being concen-
trated under reduced pressure. The residue was redissolved in H2O (150
mL) and washed with ethyl acetate (2 × 90.0 mL) before being acidified
to pH 2 with aqueous 1 M HCl. The aqueous phase was then extracted
with ethyl acetate (3 × 150 mL) after which the organic phase was dried
with Na2SO4, filtered and concentrated under reduced pressure to yield
1
the title compound (5.29 g, quant.) as a colourless oil; H NMR (300
3

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P. Ryan et al.
Bioorganic Chemistry 110 (2021) 104738
MHz, CDCl3): δ 3.09 (m, 2H), 2.34 (t, J = 7.5 Hz, 2H), 1.65 (m, 2H), 1.49
(m, 2H), 1.46 (s, 9H), 1.37 (m, 2H). Data are in agreement with that
previously reported [41].
DIPEA (25.3 mmol, 3.55 mL) with stirring at room temperature. The
reaction mixture was left for 48 h after which it had formed a thick white
precipitate. The mix was concentrated under reduced pressure, and the
impurities were decanted with ethyl acetate to yield the title compound
(4.28 g, 87%) as a white foam; νmax (solid)/cmꢀ 1 3162, 2942, 1734,
1712, 1689, 1637, 1571, 1487, 1441, 1304, 1071; 1H NMR (500 MHz,
DMSO‑d6): δ 11.4 (s, 1H), 7.70 (d, J = 7.8 Hz, 1H), 7.35 (m, 5H), 5.80 (s,
1H), 5.62 (d, J = 7.8 Hz, 1H) 5.07 (s, 2H) 5.05 (d, J = 1.5 Hz, 1H), 4.75
(t, J = 5.1 Hz, 1H), 4.07 (dd, J = 5.05 Hz, 1H), 3.86 (br, 1H), 3.70 (m,
1H), 3.46 (d, J = 5.1 Hz, 2H), 2.36 (t, J = 7.2 Hz, 2H) 1.53 (m, 4H), 1.47
(s, 3H), 1.28 (m, J = 8.4 Hz, 2H), 1.28 (s, 3H); 13C NMR (126 MHz,
DMSO‑d6): δ 182.7, 182.2, 172.7, 167.5, 163.2, 150.2, 143.1, 136.2,
128.4, 127.9, 127.9, 113.5, 101.9, 92.2, 85.5, 83.4, 80.9, 65.3, 45.1,
43.1, 33.3, 30.3, 26.9, 25.5, 25.2, 24; HRMS (ESI) 605.22131 ([M +
Na]+), calcd. for C29H34N4NaO+9 605.22180.
2.2.6. Synthesis of benzyl-6-(Boc-N-amino)hexanoate
To a solution of Boc-6-aminocaproic acid (5.42 g, 23.4 mmol) in
DCM (150 mL) was added EDC (4.93 g, 25.7 mmol), benzyl alcohol
◦
(30.4 mmol, 3.16 mL) and DMAP (0.286 g, 2.34 mmol) at 0 C. The
reaction mixture was allowed to warm to room temperature and was
stirred for 16 h before being quenched by addition of saturated NH4Cl
(100 mL) solution. The mixture was extracted with ethyl acetate (3 ×
150 mL), washed with brine (25 mL) and dried over Na2SO4 before being
concentrated under reduced pressure and purified by flash chromatog-
raphy (1:3 EtOAc/Hex) yielding the title compound (7.51 g, quant.) as a
yellow oil; 1H NMR (300 MHz, CDCl3): δ 7.32 (m, 5H), 5.08 (s, 2H), 4.63
(br, 1H), 3.20 (m, 2H) 2.35 (t, J = 7.4 Hz, 2H), 1.64 (m, 4H), 1.56 (s,
9H), 1.37 (m, 2H). Data are in agreement with that previously reported
[42].
2.2.11. Synthesis of 6-[(2-(5′-amino-5′-deoxy-2′,3′-O-
isopropylideneuridine)-3,4-dioxo-1-cyclobuten-1-yl)amino]hexanoic acid
(6)
2.2.7. Synthesis of benzyl-6-aminohexanoate
To a
solution of benzyl 6-[(2-(5′-amino-5′-deoxy 2′,3′-O-iso-
To a mixture of benzyl-6-(Boc-N-amino)hexanoate (7.50 g, 23.4
mmol) in DCM (120 mL) was added TFA (60.0 mL) at 0 ◦C with stirring.
After 2 h, the mixture was concentrated under reduced pressure, after
which the residue was redissolved in saturated NaHCO3 solution (100
mL) and extracted with ethyl acetate (3 × 120 mL). The mixture was
concentrated under reduced pressure to yield the title compound (5.16
propylideneuridine)-3,4-dioxo-1-cyclobuten-1-yl)amino]hexanoate
5
(2.00 g, 3.44 mmol) in methanol (30.0 mL) was added 1 M sodium
hydroxide solution (20.0 mL) and stirred at rt. The reaction was kept at
pH greater than 14 and was monitored by TLC. Upon completion, the
reaction was treated with Amberlite® IR 120H+ resin until pH 4. The
mixture was filtered and concentrated under reduced pressure. To this
dry mixture was suspended into ethyl acetate and once settled the su-
pernatant was decanted and re-dried to yield the title compound (1.46 g,
quant.) as a pale yellow powder; νmax cmꢀ 1 2939, 1648, 1564, 1433,
1270, 1061; 1H NMR (500 MHz, DMSO‑d6): δ 7.65 (d, J = 8.0 Hz, 1H),
5.77 (s, 1H), 5.59 (d, J = 7.9 Hz, 1H), 5.59 (d, J = 7.9 Hz, 1H), 4.72 (s,
1H), 4.07 (app, 1H), 3.86 (app, 1H), 3.70 (app, 1H), 3.46 (app, 2H), 2.17
(t, J = 7.2 Hz, 2H), 1.47 (m, 4H), 1.44 (3H), 1.25 (m, 4H), 1.24 (s, 1H);
13C NMR (126 MHz, DMSO‑d6): δ 183.2, 182.7, 175.2, 168.5, 164.0,
150.9, 143.8, 114.3, 102.5 92.7, 86.3, 84.0, 81.5, 45.7, 43.8, 34.1,31.0,
27.5, 25.7, 25.4, 24.7; HRMS (ESI) 449.18933 ([M + Na]+), calcd. for
1
g, quant.) as a yellow wax requiring no further purification; H NMR
(300 MHz; CD3OD): δ 7.36 (m, 5H), 5.13 (s, 2H), 2.91 (t, J = 1.6 Hz, 2H)
2.42 (t, J = 7.3 Hz, 2H), 1.64 (m, 4H), 1.56 (s, 9H), 1.37 (m, 2H). Data
are in agreement with that previously reported [42].
2.2.8. Synthesis of 2-diethoxy-1-cyclobutene-3,4-dione
To a stirred solution of 3,4-dihydroxycyclobut-3-ene-1,2-dione (3.00
g, 26.4 mmol) in anhydrous ethanol (150 mL) was added triethyl
orthoformate (72.2 mmol, 12.0 mL) at room temperature. The mixture
was heated to reflux at 80 ◦C and left for 48 h before being concentrated
under reduced pressure. The crude yellow oil was purified by flash
chromatography (DCM) to yield the title compound (3.57 g, 97%) as an
intensely yellow oil which was not purified further; 1H NMR (300 MHz,
CDCl3): δ 4.72 (m, 4H), 1.44 (t, J = 87.9 Hz, 6H). Data are in agreement
with that previously reported [43].
C
20H30N2NaO+8 449.18944.
2.2.12. Synthesis of 1-(2-bromoethyl)-2-acetamido-2-deoxy-
α-D-
glucopyranoside
To 2-bromoethanol (30.0 mL) was added acetyl chloride (46.1 mmol,
3.30 mL) dropwise at 0 ◦C with mixing under argon. To this mixture, N-
acetylglucosamine (7, 3.00 g, 13.6 mmol) was added before heating to
70 ◦C for 4 h. After, solid sodium bicarbonate was added until pH 7 and
the suspension was filtered through a celite bed and washed with
methanol. The mixture was concentrated under reduced pressure before
purification by flash chromatography (1:5 MeOH/DCM) to give the title
compound (2.618 g, 55%) as a light brown powder; 1H NMR (500 MHz,
CD3OD): δ 4.82 (d, J = 4.0 Hz, 1H), 3.98 (m, 1H), 3.91 (dd, J = 13.5, 4.5
Hz, 1H), 3.79 (m, 2H), 3.66 (m, 2H), 3.61 (m, 2H), 3.37 (m, 1H), 3.31 (t,
J = 2.1 Hz, 1H), 2.01 (s, 3H); 13C NMR (126 MHz, CD3OD): δ 172, 97.8,
72.6, 71.7, 68.4, 60.9, 54.2, 47.5, 29.9, 21.0. Data are in agreement with
that previously reported [44].
2.2.9. Synthesis of benzyl 6-[(2-ethoxy-3,4-dioxo-1-cyclobuten-1-yl)
amino]hexanoate (2)
To a stirred solution of 1,2-diethoxy-1-cyclobutene-3,4-dione (3.40
g, 20.0 mmol) in ethanol (70 mL) was portionally added benzyl-6-
aminohexanoate (4.42 g, 20.0 mmol) on ice. After 30 min, DIPEA
(20.0 mmol, 2.80 mL) was added and the mixture was allowed to reach
room temperature and the reaction was monitored by TLC. Upon
completion, the reaction mixture was concentrated under reduced
pressure before being purified by flash chromatography (1:1 v/v Hex/
EtOAc) to yield the title compound (6.62 g, 96%) as a pale-yellow wax;
mp. 52 ◦C; νmax (film)/cmꢀ 1 3264, 2938, 1803, 1793, 1594, 1523, 1493,
1454, 1414, 1383, 1337, 1235, 1057; 1H NMR (500 MHz, CDCl3): δ 7.32
(m, 5H), 5.09 (s, 2H), 4.74 (q, J = 6.9, 6.3 Hz, 2H), 3.4 (d, J = 6.8 Hz,
1H), 2.35 (t, J = 7.4 Hz, 2H), 1.64 (m, 4H), 1.42 (t, J = 7.0 Hz, 3H), 1.37
(m, 2H); 13C NMR (126 MHz, CDCl3): δ 189.25 , 182.37 , 176.86 , 172.99
, 172.36 , 135.73 , 128.23 , 127.88 , 127.81 , 69.30 , 65.82 , 44.25 ,
33.71 , 29.82 , 25.47 , 24.04, 15.55; HRMS (ESI) 368.14710 ([M +
Na]+), calcd. for C19H23NNaO+5 368.14684.
2.2.13. Synthesis of 1-(2-azidoethyl)-2-acetamido-2-deoxy-
α-D-
glucopyranoside
To a solution of 1-(2-bromoethyl)-2-acetamido-2-deoxy-α-D-gluco-
pyranoside (2.13 g, 6.10 mmol) in acetone/H2O (1:1 v/v, 20 mL) was
added sodium azide (2.379 g, 36.60 mmol) and tetrabutylammonium
iodide (2.275 g, 6.16 mmol) under argon. The mixture was then stirred
at 60 ◦C for 24 h, before being concentrated under reduced pressure and
purified by flash chromatography (1:5 v/v MeOH/DCM) to give the title
compound (0.956 g, quant.) as an amorphous yellow foam; 1H NMR
(400 MHz, CD3OD): δ 4.88 (d, J = 3.6 Hz, 1H), 3.91 (m, 2H), 3.84 (dd, J
= 11.9, 2.5 Hz, 1H), 3.73 (m, 2H), 3.63 (m, 2H) 3.44 (m, 2H), 3.33 (m,
1H), 2.01 (s, 3H); 13C NMR (101 MHz, MD3OD): δ 172.37, 97.42, 72.51,
2.2.10. Synthesis of benzyl 6-[(2-(5′-amino-5′-deoxy-2′,3′-O-
isopropylideneuridine)-3,4-dioxo-1-cyclobuten-1-yl)amino]hexanoate (5)
To a solution of benzyl 6-[(2-ethoxy-3,4-dioxo-1-cyclobuten-1-yl)
amino]hexanoate 2 (2.91 g, 8.45 mmol) and 5′-amino-5′-deoxy-2′,3′-
O-isopropylideneuridine 4 (2.39 g, 8.45) in ethanol (25 mL) was added
4