Synthesis and biological evaluation of 5′-glycyl derivatives of uridine as inhibitors of 1,4-β-galactosyltransferase

Article abstract of DOI:10.1016/j.bioorg.2014.11.001

New 5′-glycyl derivatives of uridine containing fragments of varying lipophilicity were synthesized as analogues of natural peptidyl antibiotics. One of the studied compounds, 5′-O-(N-succinylglycyl)-2′,3′-O-isopropylideneuridine (A4), showed moderate inhibition against 1,4-β-galactosyltransferase. However, additional studies showed that the observed inhibitory effect was due to binding to bovine serum albumin, which was used in assays as a stabilizer.

Full text of DOI:10.1016/j.bioorg.2014.11.001

Bioorganic Chemistry 58 (2015) 18–25
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Synthesis and biological evaluation of 50-glycyl derivatives of uridine
as inhibitors of 1,4-b-galactosyltransferase
a
a
a
a
a
_
Jadwiga Paszkowska , Katarzyna Kral , Tadeusz Bieg , Karolina Zaba , Katarzyna We˛grzyk ,
a
b
b
a,
⇑
´
Natalia Jaskowiak , Antonio Molinaro , Alba Silipo , Ilona Wandzik
a Department of Organic Chemistry, Bioorganic Chemistry and Biotechnology, Silesian University of Technology, B. Krzywoustego 4, 44-100 Gliwice, Poland
b Department of Chemical Sciences, University of Naples Federico II, Via Cintia 4, 80126 Naples, Italy
a r t i c l e i n f o
a b s t r a c t
New 50-glycyl derivatives of uridine containing fragments of varying lipophilicity were synthesized as
analogues of natural peptidyl antibiotics. One of the studied compounds, 50-O-(N-succinylglycyl)-20,30-
O-isopropylideneuridine (A4), showed moderate inhibition against 1,4-b-galactosyltransferase. However,
additional studies showed that the observed inhibitory effect was due to binding to bovine serum
albumin, which was used in assays as a stabilizer.
Article history:
Received 15 August 2014
Available online 10 November 2014
Keywords:
Uridine
Peptidyl nucleoside
Glycosyltransferase inhibitors
Ó 2014 Elsevier Inc. All rights reserved.
1. Introduction
proper binding of natural substrates like uridinediphospho-sugars
(UDP-sugar).
Among numerous types of nucleoside antibiotics found in nat-
ure, peptidyl pyrimidine-containing antibiotics have been shown
to exhibit activity against a broad variety of microbes [1,2]. There
is a large group of uridyl peptide antibiotics, like mureidomycins,
pacidamycins, liposidomycins, muramycins, caprazamycins and
capuramycin, targeting the bacterial enzyme translocase, MraY,
which is involved in the lipid-linked cycle of peptidoglycan biosyn-
thesis [3,4]. Tunicamycin exhibits antimicrobial activity, and is a
well-known inhibitor of replication in yeast, fungi, protozoa and
enveloped virus [5]. Unfortunately, tunicamycin is also a potent
inhibitor of mammalian phospho-GlcNAc transferase, which pre-
cludes its use as a candidate for drug development [6]. The other
examples of peptidyl antibiotics are polyoxins and nikkomycins,
which are competitive inhibitors of chitin synthases that demon-
strate antifungal, insecticidal and acarcidal activities [7].
Several unnatural uridine conjugates were designed and syn-
thesized as analogues of natural active compounds or substrate
analogues of GTs [8–11], but only a few of conjugates were strong
inhibitors of GTs. Among the latter, derivatives featuring modifica-
tion of uridine at the C-5 position inhibit galactosyl transfer by sev-
eral different galactosyltransferases. For example, incorporation of
a 5-formylthien-2-yl group onto the uracil of UDP-Gal yielded an
efficacious micromolar inhibitor of GTB and a-1,3- and a-1,4-GalT
[12,13]. Similarly, introducing a trifluoromethyl group at the C-5
position in the uracil part of UDP-Glc and UDP-Gal led to discovery
of inhibitors of b-1,4-GalT and LgtC [14]. The other examples of
well-studied b-1,4-GalT inhibitors are compounds with modifica-
tion of the carbohydrate part of UDP-Gal. The strongest inhibitors
in this group contain a naphthalene substituent linked with 6-O-
galactose (Ki 1.86 lM) [15] or a carbacyclic analogue of galactose
Despite favorable activity in vitro, these inhibitors are not good
drug candidates, because they are charged, and do not readily
transport across the cell membrane. The design of cell-permeable
inhibitors of protein glycosylation is therefore of great interest.
The common motif of the aforementioned natural products is uri-
dine nucleoside. Because glycosyltransferases (GTs) are usually tar-
gets of these natural antibiotics, it can be hypothesized that the
uridine component is essential for recognition and proper binding
at the active site of the enzymes, similar to the recognition and
[16]. An example of a pyrophosphate analogue as a micromolar
inhibitor is C-glycosyl ethyl phosphonophosphate [17]. Some
potent inhibitors were designed as bisubstrate analogues. Interest-
ing examples include the first tricomponent inhibitor with robust
inhibition of b-1,4-GalT [18].
Several uridine derivatives were synthesized and tested against
chitin synthase, because this fungal glycosyltransferase is absent in
mammalian cells and is, therefore, a valuable therapeutic target
[19]. The 1,2,3-triazoyl-linked uridine derivatives, synthesized by
click reaction approach, were identified as inhibitors of chitin syn-
thase activities, with some having inhibition strength comparable
with that of nikkomycin [20]. Other strong inhibitors include
⇑
Corresponding author.
E-mail address: ilona.wandzik@polsl.pl (I. Wandzik).
http://dx.doi.org/10.1016/j.bioorg.2014.11.001
0045-2068/Ó 2014 Elsevier Inc. All rights reserved.

J. Paszkowska et al. / Bioorganic Chemistry 58 (2015) 18–25
19
nikkomycin Z analogues that were synthesized by condensation of
uridinyl aldehyde with various carboxylic acids and isocyanides in
a combinatorial synthesis approach [21] or by introducing the aro-
matic groups at the terminal amino acid starting from uracil poly-
oxin C [22].
In this report, we describe the synthesis and biological evalua-
tion of a series of 50-O-glycyl-and 50-N-glycyl uridine derivatives
(Fig. 1). These compounds were tested against commercially avail-
able b-1,4-GalT, which plays a role in the biosynthesis of blood
group antigens and is involved in some pathological conditions,
such as arthritis and cancer.
deprotection of the acetonide group can lead to rupture of the ester
bond. We, therefore, synthesized amide analogs B1–B4, which are
more resistant to hydrolysis. The synthetic strategy relies on the
coupling of relevant uridine substrates 3 [31] or 9 [27] with com-
mercially available glycine derivatives 1 and 2 (Schemes 1 and 2,
respectively). This approach has been demonstrated in works of
Alargov et al. [25], who used dicyclohexylcarbodiimide (DCC) as
the condensing agent. We used diisopropylcarbodiimide (DIC) as
the condensing agent because, unlike DCC, DIC is believed to be
non-allergenic. All reactions were carried out in the presence of
4-dimethylaminopyridine (DMAP) as a catalyst, affording products
A1, A2 and B1, B2 in excellent yields. No side reactions (e.g., acyl-
ation of uracil or cyclization caused by the Michael addition of a 50-
amino group to the double bond of uracil) were observed. To
obtain compounds A3, A4 and B3, B4, intermediate substrates 6
and 14 were synthesized by removal of N-benzyloxycarbonyl
groups of glycine in A1 and B1, respectively, which was accom-
plished by catalytic hydrogenation with cyclohexene as the hydro-
gen donor and Pd(OH)2/C as the catalyst. The next step was
coupling of compounds 6 and 14 with benzoyl chloride or succinyl
anhydride in the presence of base (NEt3 or pyridine) without any
coupling agent. Reactions proceeded smoothly to give new com-
pounds containing the ester (A3, A4) or amide (B3, B4) linkages.
The latter were subjected to deprotection of ribose 20 and 30
hydroxyls. Attempts to remove the acetonide group in the ester
series in the presence of acetic acid, trifluoroacetic acid, Amberlyst
15 or AcCl failed due to the acid hydrolysis of the ester linkage.
Removal of tert-butyldimethylsilyl (TBDMS) groups from deriva-
tives B2 and B4 was achieved by treatment with tetrabutylammo-
nium fluoride in THF as a solvent, whereas deprotection of N-acetyl
derivatives B1 and B3 was accomplished by treatment with AcCl in
methanol.
2. Results and discussion
2.1. Chemistry
Amino acids can act as ligands in metal complexes, and incorpo-
ration of amino acid residues into nucleosides have been reported.
There are many studies describing the synthesis and biological
evaluation of uridine-containing analogues [23–28]. With the goal
of broadening the library of uridine derivatives that could be active
against glycosyltransferases, we have synthesized new 50-glycyl
derivatives of uridine, in which the glycine is attached via an ester
or an amide linkage to the 50-position of uridine (series A and B
respectively, Fig. 1).
Relevant lipophilicity is a crucial parameter for transport across
the cell membrane, and some highly polar compounds cannot be
used in therapeutic applications because they cannot cross the cell
membrane. We have designed compounds that are significantly
less polar than peptidyl antibiotics, according to calculated XLogP3
parameter [29], [data not shown]. This algorithm has shown con-
siderable agreement with experimentally determined logP values
in the library of uridine derivatives [30]. Hydrophobic fragments,
like benzyloxycarbonyl or benzoyl groups, were incorporated into
target structures to ensure higher lipophilicity.
All new compounds from series A and B were purified by
column chromatography, and their structures were elucidated
with the aid of 1H and 13C NMR spectroscopy data (including
two-dimensional DQCOSY, HMQC and HMBC experiments and
simulation analysis) and mass spectrometry analysis (for details
see Section 4 and Supplementary materials).
The proposed amino acid derivatives of uridine are divided into
two series according to (1) the type of linkage between uridine and
glycine and (2) the compounds in series A, which contain an iso-
propylidene group as a protecting group of C-20 and C-30 hydroxyls
of a ribose unit, and compounds in series B, which contain a totally
deprotected uridine unit. Although compounds A1–A4 can be
easily synthesized from 20,30-isopropylideneuridine (3) by coupling
2.2. Biological evaluation
The important part of this work was the biological evaluation of
the synthesized compounds from series A and B. Bovine milk b-1,4-
GalT I was chosen as the target enzyme. It is one of the most well
with
a relevant glycine derivative, these compounds are
acid sensitive, limiting their biological applicability. Moreover,
O
N
O
N
O
N
O
N
NH
NH
NH
NH
O
O
O
O
O
z
Cb
c
A
Bz
H
O
O
O
O
O
N
H
N
H
N
H
N
H
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
A2
A1
A3
A4
O
N
O
N
O
N
O
N
NH
NH
NH
NH
O
O
O
O
O
H
N
H
N
H
N
H
N
z
Cb
c
A
Bz
H
O
N
H
N
H
N
H
N
H
O
O
O
O
O
O
O
O
O
H
O
H
O
H
O
H
O
H
O
H
O
H
O
H
O
B1
B2
B4
B
3
Cbz - benzyloxycarbonyl; Ac - acetyl; Bz - benzoyl
Fig. 1. C-50-glycyl derivatives of uridine: series A and B.

20
J. Paszkowska et al. / Bioorganic Chemistry 58 (2015) 18–25
O
O
N
NH
NH
N
O
O
i
+
H
O
H
O
O
R1HN
R1HN
O
O
O
O
O
O
O
O
4/A1 R1= Cbz
5/A2 R1= Ac
1 R1= Cbz
2 R1= Ac
3
O
N
NH
O
O
O
N
H
O
O
O
O
iii
O
O
7/A3
NH
NH
O
N
O
N
O
ii
O
N
O
O
v
i
H2N
N
H
O
O
O
NH
O
O
O
O
O
O
O
O
H
O
O
6
4/A1
N
H
O
O
O
O
O
8/A4
Scheme 1. Synthesis of 50-O-glycyl derivatives of uridine. Reagents and conditions: (i) DIC (1.5 equiv.), DMAP (0.1 equiv.), DMF, 0 °C ? rt, 24 h; (ii) 20% Pd(OH)2/C,
cyclohexene, MeOH, reflux, 20–30 min, (iii) BzCl (1.0 equiv.), pyridine (10 equiv.), DMF, rt, 1 h; (iv) succinyl anhydride (1.0 equiv.), THF, rt, 4 h.
O
N
O
N
O
N
NH
NH
NH
O
O
O
H
N
H
N
ii
i
+
H
O
H2N
R1HN
R1HN
R1HN
O
O
O
O
O
O
TBDM
TBDM
O S
SO
TBDM
SO
TBDM
z
H
H
O
S
O
O
1
1
1
1
=
10 R
11 R
=
=
z
c
Cb
=
=
z
b
c
1 R
2 R
9
Cb
A
/
12 B1
R
B2
C
A
1
1
=
c
A
1
3/
R
O
N
NH
O
O
H
N
N
H
O
O
R
R
O
O
v
i
O
N
O
=
R TBDM
15
S
NH
O
NH
ii
O
N
1
B
=
O
6/ 3 R H
H
N
H
N
iii
N
H
H2N
O
O
N
O
O
v
O
O
NH
TBDM
SO
TBDM
S
O
TBDM
SO
TBDM
S
O
O
O
H
N
H
O
14
10
N
H
O
O
O
R
R
O
O
TBDMS - tert-butyldimethylsilyl
=
R TBDM
S
17
ii
=
18/B4 R H
Scheme 2. Synthesis of 50-N-glycyl derivatives of uridine. Reagents and conditions: (i) DIC (1.5 equiv.), DMAP (0.1 equiv.), CH2Cl2, 0 °C ? rt, 15 min – 3 h; (ii) AcCl
(2.0 equiv.), MeOH, rt, 5–24 h; or TBAF (1 M solution in THF, 3.0 equiv.), THF, rt, 1.5–3.0 h; (iii) 20% Pd(OH)2/C, cyclohexene, MeOH, reflux, 4 h; (iv) BzCl (1.1 equiv.), Et3N
(1.1 equiv.), CH2Cl2, 0 °C, 10 min; (v) succinyl anhydride (1.0 equiv.), Et3N (1.0 equiv.), THF, rt, 30 min.

J. Paszkowska et al. / Bioorganic Chemistry 58 (2015) 18–25
21
Table 1
The stronger effect was observed when HSA was used, regardless
of the buffer applied. The highest BSA antidenaturation activity
was exhibited with compound A4 in (4-(2-hydroxyethyl)-1-pipe-
razineethanesulfonic acid) (HEPES) buffer. Antidenaturation activ-
ity of some fatty acids [34] and nonsteroidal anti-inflammatory
drugs [32,33,35] was reported. For comparison, effect of known
anti-inflammatory drug aspirin at 1.1 mM was included in our
experiments as a positive control.
The higher antidenaturation effect of examined compounds
towards HSA than to BSA suggests that binding specificities of
these proteins are different. Serum albumins serve as carriers for
molecules of low water solubility and possess many binding pock-
ets. In addition, the distribution of BSA and HSA binding sites is not
the same [36–38].
BSA is essential for predictive and repeatable activity of b-1,4-
GalT I. We observed a twofold decrease of activity when enzy-
matic reactions were carried out without BSA. However, the use
of BSA as a stabilizer in enzymatic assays should be treated with
caution, because some compounds may bind to BSA, decreasing
the activity of a specific enzyme and leading to false positive
results.
To further examine the interaction, the binding of A4 to BSA
was investigated using STD-NMR spectroscopy [39]. STD-NMR
has been previously proven to be extremely informative in the
examination of protein-substrate interactions since it allows map-
ping of ligand epitopes in close contact with proteins [40]. A qual-
itative comparison of the 1H NMR spectrum of A4 and the STD
spectrum (Fig. 2) using a saturation time of 2 s revealed changes
in the relative areas of signals. A quantification of STD signals
allowed to define the epitope mapping. Thus, beside the interact-
ing proton signals from the lateral chain, the region comprising
ribose and uracil were both in contact to the BSA and contributed
to the overall binding.
Inhibition of serum albumins denaturation by 50-glycyl derivatives of uridine at
1.8 mM and aspirin at 1.1 mM at 70 °C in 30 min.
Protein Buffer
Inhibition of denaturation
(%)
A1
A4
B1
Aspirin
BSA
HSA
50 mM Hepes (pH 5.3)
100 mM Phosphate (pH 5.3) 61
35
68
36
42
25
76
<20
50 mM Hepes (pH 5.3)
100 mM Phosphate (pH 5.3) 86
57
69
78
57
84
>97
83
studied enzymes among glycosyltransferases with commercial
availability. We used the fluorescence competition assay with
b-1,4-GalT I, using the fluorescent acceptor b-GlcNAc-O-(CH2)6-dansyl
as a substrate [17]. Among the tested compounds, activity was
observed only for derivative A4 (IC50 = 2.0 mM). However,
additional experiments showed that the observed inhibitory effect
was not due to direct interaction of compound A4 with b-1,4-GalT
I. It was observed that some compounds had an antidenaturation
effect on bovine serum albumin (BSA), which was used in assays
as a stabilizer. According to the procedure, when the reaction
was completed, proteins were denatured in a boiling water bath.
No denaturation was observed when compound A4 was present
in the reaction mixture. It was hypothesized that binding of some
compounds to BSA disrupted stabilization of target b-1,4-GalT I. To
confirm this theory, studies on the antidenaturation activity of the
examined compounds from series A and B were carried out in the
presence of BSA or human serum albumin (HSA) at varying pH at
70 °C. Samples of 0.2% albumins (control) and albumins with
studied compounds were incubated for 30 min. Next, turbidity
was measured spectrophotometrically at 660 nm, and percent of
inhibition of protein denaturation was calculated [32,33]. Samples
were incubated in various buffers in different pH. The strongest
inhibition of denaturation was observed at pH 5.3. Three
compounds, A1, A4 and B1, showed significant antidenaturation
activity against HSA and BSA in 1.8 mM concentration (Table 1).
The observed ability of some compounds to inhibit thermally
induced protein denaturation is of interest, because denaturation
of proteins is
a well-documented cause of inflammation.
Compounds that inhibit denaturation can be useful in studying
O
5
42%
60%
NH
O
99%
C
73%
A
6
HO
B
O
N
O
37%
N
H
100%
5'
4'
O
31%
1'
37%
H
O
H
H
O
24%
21%
3'
2'
B
H
O
O
95%
CH3
A
A
1’
B
C
5’
6
2’ 3’
5
4’
7.5
6.5
5.5
4.5
3.5
2.5
1.5
ppm
Fig. 2. NMR analysis of the binding of A4 to BSA. (A) 1H NMR spectrum of A4 in the presence of BSA. Signals are indicated as reported in the figure. (B) STD spectrum. An on-
resonance irradiation frequency at 0 ppm and a saturation time of 2 s was used, with a 1:75 BSA-ligand ratio.

22
J. Paszkowska et al. / Bioorganic Chemistry 58 (2015) 18–25
anti-inflammation processes, similar to known nonsteroidal anti-
inflammatory drugs [32,33].
After completion of reaction the mixture was concentrated. Crude
product was purified by flash chromatography (CHCl3 ?
CHCl3:MeOH 100:1 ? 20:1) to obtain 5/A2 (0.374 g, 98%) as a
white foam. ½a 2D0
ꢁ
¼ 13:2 (CHCl3, c = 1.00); 1H NMR (600 MHz,
3. Conclusions
CDCl3): d 1.35 (s, 3H, C(CH3)2), 1.56 (s, 3H, C(CH3)2), 2.04 (s, 3H,
CH3), 4.04, 4.09 (qABd, 2H, JAB = 18.3 Hz, J = 5.4 Hz, NH–CH2–C(O)),
4.31 (ddd, 1H, J = 3.7, J = 4.3, J = 5.9 H-40), 4.38 (dd, 1H, J = 5.9,
J = 11.9 Hz, H-50b), 4.45 (dd, 1H, J = 3.7, J = 11.9 Hz, H-50a), 4.88
(dd, 1H, J = 4.3, J = 6.4 Hz, H-30), 5.08 (dd, 1H, J = 1.8, J = 6.4 Hz, H-20),
5.56 (d, 1H, J = 1.8 Hz, H-10), 5.74 (dd, 1H, J = 1.7, J = 8.0 Hz, H-5),
6.42 (t, 1H, J = 5.4 Hz, C(O)NHCH2), 7.27 (d, 1H, J = 8.0 Hz, H-6),
9.67 (s, 1H, NHur), 13C NMR (150 MHz, CDCl3): d 22.82 (CH3C(O)),
25.37 (C(CH3)2), 27.09 (C(CH3)2), 41.33 (NH–CH2–C(O)), 64.46
(C-50), 80.81 (C-30), 84.26 (C-20), 85.34 (C-40), 95.57 (C-10), 102.60
(C-5), 114.60 (C(CH3)2), 142.95 (C-6), 149.99 (C-2), 163.50 (C-4),
169.83 (C(O)), 170.57 (C(O)); ESI-LRMS: calcd for C16H21N3O8Na
([M+Na]+): m/z 406.1, found: 406.2.
Two series of compounds, 50-O-glycyl and 50-N-glycyl deriva-
tives of uridine, were synthesized in simple coupling reactions of
relevant uridine (3 and 9) and glycine (1 and 2) substrates followed
by incorporation of lipophilic (benzoyl, benzyloxycarbonyl) or
hydrophilic (succinyl) groups at the terminal nitrogen of glycine.
Compounds were tested as b-1,4-GalT I inhibitors, but only com-
pound A4 exhibited moderate activity (IC50 = 2.0 mM). Accurate
assays proved that with the conditions used, the inhibition effect
was caused by binding of A4 to BSA and subsequent lack of enzyme
stabilization. Moreover it was observed that compounds A1, A4
and B1 exhibit a dose-dependent ability to inhibit thermally
induced BSA and HSA denaturation. Binding of A4 into BSA was
additionally confirmed by STD NMR experiment, which showed
that the highest saturation received lipophilic parts of the
compound: ethylene and isopropylidene groups.
4.2.2. 50-O-(N-benzoyl-glycyl)-20,30-O-isopropylideneuridine (7/A3)
A cooled suspension of N-benzyloxycarbonyl-glycine 1 (0.627 g,
3 mmol) and DIC (0.235 mL, 1.5 mmol) in DMF (4 mL) was stirred
for 30 min at 0 °C. Then 3 (0.284 g, 1 mmol) and DMAP (0.012 g,
0.1 mmol) were added and the mixture was stirred at rt for 24 h.
After completion of reaction the mixture was diluted with AcOEt
(20 mL), washed with 5% solution of NaHCO3 (15 mL) and water
(2 ꢀ 15 mL). Organic layer was dried (MgSO4) and concentrated.
Crude product was purified by flash chromatography (CHCl3 ?
CHCl3:MeOH 24:1) to obtain 4/A1 (0.461 g, 97%) as a white foam.
In the next step 4 (0.461 g, 0.97 mmol) was dissolved in MeOH
(11 mL). Then 20% Pd(OH)2/C (0.092 g) and cyclohexene (2.2 mL)
were added and the mixture was heated for 30 min. After removal
of the catalyst by filtration the reaction mixture was concentrated
to give 6. Crude product 6 was dissolved in DMF (10 mL) and BzCl
(0.113 mL, 0.97 mmol) and pyridine (0.783 mL, 9.7 mmol) were
added. The reaction mixture was stirred for 1 h at rt, diluted with
CH2Cl2 (50 mL) and washed with H2O (3 ꢀ 50 mL). Organic layer
was dried (MgSO4) and concentrated. Crude product was purified
by flash chromatography (hexane:AcOEt 1:9) to obtain 7/A3
4. Experimental
4.1. General methods
Optical rotations were measured with a JASCO P-2000 polarim-
eter using a sodium lamp (589 nm) at room temperature. NMR
spectra were recorded with a Varian spectrometer at a frequency
of 600 MHz. Mass spectra were recorded with a WATERS LCT Pre-
mier XE system (high resolution mass spectrometer with TOF ana-
lyzer) using electrospray-ionization (ESI) technique. Reactions
were monitored by TLC on precoated plates of silica gel 60 F254
(Merck) and visualized using UV light (254 nm). Crude products
were purified using column chromatography performed on silica
gel 60 (70–230 mesh, Fluka) or flash chromatograph (Isolera™,
Biotage) equipped with UV detector (254 nm, flow rate 10–
25 mL/min). Hexane/EtOAc or CHCl3/MeOH were used as solvent
systems. All evaporation were performed under diminished pres-
sure at 50 °C. Reversed phase HPLC analyses were performed using
JASCO LC 2000 apparatus equipped with a reverse column (Nucle-
(0.145 g, 32% overall yield) as a white foam; ½a D20
¼ 1:00 (CHCl3,
ꢁ
c = 1.00); 1H NMR (600 MHz, CDCl3): d 1.32 (s, 3H, CH3), 1.55 (s,
3H, CH3), 4.24–4.28 (qABd, 2H, JAB = 18.2, J = 5.3 Hz, NH–CH2–
C(O)), 4.32 (ddd, 1H, J = 3.8, J = 4.3, J = 5.7 Hz, H-40), 4.39 (dd, 1H,
J = 5.7, J = 11.9 Hz, H-50b), 4.49 (dd, 1H, J = 3.8, J = 11.9 Hz, H-50a),
4.87 (dd, 1H, J = 4.3, J = 6.5 Hz, H-30), 5.03 (dd, 1H, J = 1.9,
J = 6.5 Hz, H-20), 5.56 (d, 1H, J = 1.9 Hz, H-10), 5.70 (d, 1H,
J = 8.1 Hz, H-5), 7.03 (t, 1H, J = 5.3 Hz, NH–CH2), 7.26 (d, 1H,
J = 8.1 Hz, H-6), 7.42 (m, 2H, Har-m), 7.50 (tt, 1H, J = 1.3, J = 7.4 Hz,
1H, Har-p), 7.82 (dd, 2H, J = 1.3, J = 8.3 Hz, Har-o), 9.56 (s, 1H, NHur);
13C NMR (150 MHz, CDCl3): d 25.20 (CH3), 27.09 (CH3), 41.82 (NH–
CH2–C(O)), 64.47 (C-50), 80.78 (C-30), 84.23 (C-20), 85.24 (C-40),
95.35 (C-10), 102.65 (C-5), 114.66 (C(CH3)2), 127.14 (2 Car-o),
128.60 (Car-p), 131.88 (2 Car-m), 133.52 (Car-IV), 142.82 (C-6),
150.03 (C-2), 163.45 (C-4), 167.68, 169.82 (2 C(O)); ESI-HRMS:
Calcd for C21H24N3O8 ([M+H]+): m/z 446.1563, found: 446.1562.
osil 100 C18, 5
l
m, 25 ꢀ 0.4 cm; mobile phase: H2O/MeCN 73:27,
flow rate 1 mL/min) with a fluorescence detector. Fluorescence of
acceptor substrate and product was read at 385 nm excitation/
540 nm emission.
Bovine milk b-1,4-galactosyltransferase I (b-1,4-GalT I, 1 U/mg),
BSA (A7638), HSA (A1653) and other chemicals were purchased
from Sigma–Aldrich Co. LLC. and were used without purification.
20,30-O-isopropylideneuridine (3) was prepared according to the
published procedure [31]. 50-amino-50-deoxy-20,30-bis-O-(tert-
butyldimethylsilyl)-uridine (9) was prepared in three step
synthesis by direct conversion of unprotected uridine to 50-azido-
50-deoxyuridine [41], followed by TBDMS protection and
hydrogenation [27]. 50-(N-(benzyloxycarbonyl)-glycyl)-20,30-O-iso-
propylideneuridine (4/A1) was prepared by coupling of N-benzyl-
oxycarbonyl-glycine (1) with 20,30-O-isopropylideneuridine (3)
according to procedure described by Algarov et al. [25] with slight
modification: as coupling agent DIC was used instead of DCC.
4.2.3. 50-O-(N-succinyl-glycyl)-20,30-O-isopropylideneuridine (8/A4)
Crude product 6 (synthesised as described in Section 4.2.2) was
dissolved in THF (18 mL), succinyl anhydride (0.097 g, 0.97 mmol)
was added and the mixture was stirred for 4 h at rt. Then the mix-
ture was concentrated and crude product was purified by flash
chromatography (CHCl3:MeOH 100:1 ? 25:1) to obtain 8/A4
4.2. Synthesis of 50-O-glycyl uridine derivatives (A2–A4)
4.2.1. 50-O-(N-acetyl-glycyl)-20,30-O-isopropylideneuridine (5/A2)
A cooled suspension of N-acetyl-glycine 2 (0.351 g, 3.0 mmol)
and DIC (0.235 mL, 1.5 mmol) in DMF (4 mL) was stirred for
30 min at 0 °C. Then 3 (0.284 g, 1.0 mmol) and DMAP (0.012 g,
0.1 mmol) were added and the mixture was stirred at rt for 24 h.
(0.171 g, 38% overall yield) as yellowish foam; ½a D20
¼ 2:13 (MeOH,
ꢁ
c = 1.00); 1H NMR (600 MHz, CD3OD): d 1.35 (s, 3H, CH3), 1.54 (s,
3H, CH3), 2.53, 2.59 (qAB, 2H, JAB = 12.5 Hz, CH2–CH2), 2.53, 2.59
(qAB, 2H, JAB = 8.7 Hz, CH2–CH2), 3.95, 3.97 (qAB, 2H, JAB = 17.6 Hz,
NH–CH2–C(O)), 4.29–4.39 (m, 3H, H-40, H-50a, H-50b), 4.86

J. Paszkowska et al. / Bioorganic Chemistry 58 (2015) 18–25
23
(m, 1H, H-30), 5.05 (dd, 1H, J = 2.2, J = 6.4 Hz, H-20), 5.71 (d, 1H,
J = 8.0 Hz, H-5), 5.77 (d, 1H, J = 2.2 Hz, H-10), 7.62 (d, 1H,
J = 8.0 Hz, H-6); 13C NMR (150 MHz, CD3OD): d 24.12 (CH3), 26.06
(CH3), 29.04, 30.00 (CH2–CH2), 40.70 (NH–CH2–C(O)), 64.20
(C-50), 80.97 (C-30), 84.20 (C-20), 84.69 (C-40), 93.90 (C-10), 101.48
(C-5), 114.06 (C(CH3)2), 143.07 (C-6), 150.50 (C-2), 164.76 (C-4),
169.63, 173.77 (2 C(O)), 175.16 (COOH); ESI-HRMS: Calcd for
1H, J = 5.6 Hz, H-10), 7.61 (d, 1H, J = 8.0 Hz, H-6), 8.08 (t, 1H,
J = 5.9 Hz, CH3–C(O)–NH–CH2), 8.16 (t, 1H, J = 5.9 Hz, CH2–C(O)–
NH–CH2); 13C NMR (150 MHz, DMSO): d 22.96 (CH3), 41.20
(C-50), 42.54 (NH–CH2–C(O)), 71.17 (C-30), 72.96 (C-20), 82.78
(C-40), 88.99 (C-10), 102.49 (C-5), 141.55 (C-6), 151.98 (C-2),
164.70 (C-4), 169.79 (CH2–C(O)–NH), 170.04 (CH3–C(O)–NH);
ESI-HRMS: Calcd for C13H18N4O7Na ([M+Na]+): m/z 365.1073,
found: 365.1079.
C
18H22N3O10 ([MꢂH]ꢂ): m/z 440.1305, found: 440.1296.
4.3. Synthesis of 50-N-glycyl uridine derivatives (B1–B4)
4.3.3. 50-(N-benzoyl-glycyl)-50-amino-50-deoxyuridine (16/B3)
Compound 10 (0.635 g, 0.96 mmol, synthesised as described in
Section 4.3.1) was dissolved in MeOH (45 mL). Then 20% Pd(OH)2/C
(0.092 g) and cyclohexene (1.55 mL) were added and the mixture
was heated for 4 h. After removal of the catalyst by filtration the
reaction mixture was concentrated. Crude product was purified
by flash chromatography (CHCl3:MeOH 100:1 ? 75:1) to obtain
14 (0.400 g, 79%). In the next step 14 (0.400 g, 0.76 mmol) was dis-
solved in CH2Cl2 (23 mL) and the solution was cooled in ice bath to
0 °C. Then BzCl (0.097 mL, 0.84 mmol) and Et3N (0.116 mL,
0.84 mmol) were added and the mixture was stirred at 0 °C for
10 min and concentrated. Crude product was purified by column
chromatography (CHCl3 ? CHCl3:MeOH 20:1) to obtain 15
(0.460 g, 96%). For desilylation, derivative 15 (0.460 g, 0.728 mmol)
was dissolved in MeOH (9 mL) and AcCl (0.104 mL, 1.456 mmol)
was added. The reaction mixture was stirred at rt for 5 h. After
completion of reaction the mixture was neutralised by Amberlyst
21 and concentrated. Crude product was purified by column chro-
matography (CHCl3:MeOH 20:1 ? 10:1) to obtain 16/B3 (0.144 g,
4.3.1. 50-(N-benzyloxycarbonyl-glycyl)-50-amino-50-deoxyuridine (12/
B1)
A
cooled suspension of N-benzyloxycarbonyl-glycine (1)
(0.627 g, 3.0 mmol) and DIC (0.235 mL, 1.5 mmol) in CH2Cl2
(14 mL) was stirred for 30 min at 0 °C. Then 9 (0.471 g, 1.0 mmol)
and DMAP (0.012 g, 0.1 mmol) were added and the mixture was
stirred at rt for 15 min. After completion of reaction the mixture
was diluted with CH2Cl2 (70 mL), washed with 5% solution of
NaHCO3 (80 mL) and water (2 ꢀ 80 mL). Organic layer was dried
(MgSO4) and concentrated. Crude product was purified by flash
chromatography (CHCl3:MeOH 100:1 ? 96:1) to obtain 10
(0.635 g, 96%) as a white foam. For desilylation, derivative 10
(0.635 g, 0.96 mmol) was dissolved in MeOH (13 mL) and AcCl
(0.137 mL, 1.92 mmol) was added. The reaction mixture was stir-
red at rt for 24 h. After completion of reaction the mixture was
neutralised by Amberlyst 21 and concentrated. Crude product
was purified by column chromatography (CHCl3:MeOH
20:1 ? 13:1) to obtain 12/B1 (0.288 g, 69%) as a white foam;
49%); ½a 2D0
ꢁ
¼ ꢂ27:5 (MeOH, c = 1.00); 1H NMR (600 MHz, DMSO):
½
a 2D0
ꢁ
¼ ꢂ9:33 (MeOH, c = 1.00); 1H NMR (600 MHz, CD3OD) d 3.52
d 3.30 (ddd, 1H, J = 5.9, J = 6.4, J = 14.1 Hz, H-50b), 3.43 (ddd, 1H,
J = 4.5, J = 5.9, J = 14.1 Hz, H-50a), 3.83 (ddd, 1H, J = 4.5, J = 5.7,
J = 6.4 Hz, H-40), 3.86–3.91 (m, 3H, NH–CH2–CO, H-30), 4.05 (ddd,
1H, J = 5.7, J = 5.7, J = 5.7 Hz, H-20), 5.15 (d, 1H, J = 5.2 Hz, 30-OH),
5.36 (d, 1H, J = 5.7 Hz, 20-OH), 5.58 (d, 1H, J = 8.1 Hz, H-5), 5.73
(d, 1H, J = 5.7 Hz, H-10), 7.47 (m, 2H, Har-m), 7.54 (tt, 1H, J = 1.3,
J = 7.5 Hz, Har-p), 7.66 (d, 1H, J = 8.1 Hz, H-6), 7.88 (m, 2H, Har-o),
8.09 (t, 1H, J = 5.9 Hz, C(O)–NH), 8.73 (t, 1H, J = 5.9 Hz, C(O)NHCH2
C(O)); 11.33 (s, 1H, NHur), 13C NMR (150 MHz, DMSO): d 41.24
(C-50), 43.13 (NH–CH2–C(O)), 71.10 (C-30), 72.84 (C-20), 83.04
(C-40), 88.51 (C-10), 102.44 (C-5), 127.80 (Car-o), 128.69 (Car-m),
131.77 (Car-p), 134.47 (Car-IV), 141.74 (C-6), 151.20 (C-2), 163.45
(C-4), 166.96 (C(O)Bz), 169.77 (CH2–C(O)–NH); ESI-HRMS: Calcd for
C18H20N4O7Na ([M+Na]+): m/z 427.1230, found: 427.1236.
(dd, 1H, J = 4.3, J = 14.2 Hz, H-50a), 3.56 (dd, 1H, J = 5.6,
J = 14.2 Hz, H-50b), 3.77, 3.78 (qAB, 2H, JAB = 16.9 Hz, NH–CH2–
C(O)), 3.98 (ddd, 1H, J = 4.3, J = 5.4, J = 5.6 Hz, H-40), 4.01 (dd,1H,
J = 5.4, J = 5.6 Hz, H-30), 4.21 (dd, 1H, J = 4.3, J = 5.6 Hz, H-20), 5.09,
5.10 (qAB, 2H, JAB = 12.4 Hz, CH2-Ph), 5.71 (d, 1H, J = 8.1 Hz, H-5),
5.75 (d, 1H, J = 4.3 Hz, H-10), 7.26–7.36 (m, 5H, Har), 7.65 (d, 1H,
13
J = 8.1 Hz, H-6); C NMR (150 MHz, CD3OD) d 41.82 (C-50), 45.01
(NH–CH2–CO), 67.87 (CH2-Ph), 83.92, 74.61, 72.10 (C-20, C-30,
C-40), 92.24 (C-10), 103.01 (C-5), 138.05 (Car-IV), 128.89–129.47
(Car), 143.36 (C-6), 152.30 (C-2), 159.06 (C(O)), 166.05 (C-4),
172.70 (C(O)); ESI-HRMS: Calcd for C19H22N4O8Na ([M+Na]+): m/z
457.1335, found: 457.1326.
4.3.2. 50-(N-acetyl-glycyl)-50-amino-50-deoxyuridine (13/B2)
A cooled suspension of N-acetyl-glycine (2) (0.351 g, 3.0 mmol)
and DIC (0.235 mL, 1.5 mmol) in CH2Cl2 (14 mL) was stirred for
30 min at 0 °C. Then 9 (0.471 g, 1.0 mmol) and DMAP (0.012 g,
0.1 mmol) were added and the mixture was stirred at rt for 3 h.
After completion of reaction the mixture was diluted with CH2Cl2
(80 mL), washed with 5% solution of NaHCO3 (80 mL) and water
(2 ꢀ 80 mL). Organic layer was dried (MgSO4) and concentrated.
Crude product was purified by flash chromatography (CHCl3:MeOH
100:1 ? 90:1) to obtain 11 (0.520 g, 91%) as a white foam. For
desilylation, derivative 11 (0.520 g, 0.91 mmol) was dissolved in
THF (27 mL) and TBAF (2.73 mL, 1 M solution in THF, 3 equiv.)
was added. The reaction mixture was stirred at rt for 1.5 h. After
completion of reaction Amberlyst 15 was added and the mixture
was left to stir for 3 h at rt. Then the mixture was filtered and con-
centrated. Crude product was purified by column chromatography
(CHCl3:MeOH 5:1) to obtain 13/B2 (0.104 g, 30% overall yield);
4.3.4. 50-(N-succinyl-glycyl)-50-amino-50-deoxyuridine (18/B4)
Compound 14 (0.400 g, 0.76 mmol, synthesised as described in
Section 4.3.3) was dissolved in THF (15 mL) and succinyl anhydride
(0.758 g, 0.76 mmol) and Et3N (0.105 mL, 0.76 mmol) were added.
The mixture was stirred at rt for 30 min and concentrated. Crude
product was purified by flash chromatography (CHCl3:MeOH
20:1) to obtain 17 (0.213 g, 45%). For desilylation, derivative 17
(0.213 g, 0.34 mmol) was dissolved in THF (10 mL) and TBAF
(1.02 mL, 1 M solution in THF, 3.0 equiv.) was added. The reaction
mixture was stirred at rt for 3 h. After completion of reaction
Amberlyst 15 was added and the mixture was left to stir for 3 h
at rt. Then the mixture was filtered and concentrated. Crude prod-
uct was purified by column chromatography (CHCl3:MeOH 2:1) to
obtain 18/B4 (0.046 g, 33%); ½a D20
ꢁ
¼ ꢂ6:73 (MeOH, c = 1.00); 1H
NMR (600 MHz, DMSO): d 2.37 (m, 2H, CH2–CH2), 2.42 (m, 2H,
CH2–CH2), 3.26 (ddd, 1H, J = 5.9, J = 6.2, J = 13.9 Hz, H-50b), 3.39
(ddd, 1H, J = 4.6, J = 5.9, J = 13.9 Hz, H-50a), 3.67 (d, 2H, J = 5.8 Hz,
NH–CH2–C(O)), 3.81 (ddd, 1H, J = 4.3, J = 4.6, J = 6.2 Hz, H-40), 3.86
(dd, 1H, J = 4.3, J = 5.4 Hz, H-30), 4.04 (dd, 1H, J = 5.4, J = 5.6 Hz,
H-20), 5.63 (d, 1H, J = 8.1 Hz, H-5), 5.71 (d, 1H, J = 5.7 Hz, H-10),
7.66 (d, 1H, J = 8.1 Hz, H-6), 7.98 (t, 1H, J = 5.8 Hz, C(O)–NH–CH2–
C(O)), 8.14 (t, 1H, J = 5.9 Hz, C(O)–NH), 11.33 (s, 1H, NHur); 13C
½
a 2D0
ꢁ
¼ 3:53 (DMSO, c = 1.00); 1H NMR (600 MHz, DMSO): d 1.85
(s, 3H, CH3), 3.28 (ddd, 1H, J = 5.9, J = 6.3, J = 14.0 Hz, H-50b), 3.39
(ddd, 1H, J = 4.5, J = 5.9, J = 14.0 Hz, H-50a), 3.66, 3.65 (qABd, 2H,
JAB = 16.7, J = 5.9 Hz, NH–CH2–C(O)), 3.80 (ddd, 1H, J = 4.5, J = 4.5,
J = 6.3 Hz, H-40), 3.87 (dd, 1H, J = 4.5, J = 5.5 Hz, H-30), 4.04 (dd,
1H, J = 5.5, J = 5.6 Hz, H-20), 5.59 (d, 1H, J = 8.0 Hz, H-5), 5.70 (d,

24
J. Paszkowska et al. / Bioorganic Chemistry 58 (2015) 18–25
NMR (150 MHz, DMSO):
d
22.22, 22.18 (CH2–CH2), 40.69
Data acquisition and processing were performed with TOPSPIN
software.
(NH–CH2–C(O)), 64.10 (C-50), 81.13, 72.64, 69.68 (C-20, C-30, C-40),
88.36 (C-10), 102.14 (C-5), 140.71 (C-6), 150.63 (C-2), 163.00
(C-4), 169.87 (C(O)); ESI-HRMS: Calcd for C15H19N4O9 ([MꢂH]ꢂ):
m/z 399.1152, found: 399.1147.
Acknowledgments
Publication part-financed by the European Union within the
European Regional Development Fund (POIG. 01.01.02-14-102/
09). KK received scholarships under the project ‘DoktoRIS-Scholar-
ship Program for Innovative Silesia’. AS and IW acknowledge the
Executive Programme of Scientific and Technological Cooperation
between the Italian Republic and the Replubic of Poland, project
code: PL13MO6.
4.4. Biological evaluation
4.4.1. Bovine milk b-1,4-galactosyltransferase I assay
b-1,4-GalT I activity was assayed using UDP-Gal as glycosyl
donor and b-GlcNAc-O-(CH2)6-dansyl as glycosyl acceptor [17].
Assays were performed in a total volume of 100
mixtures contained reagents in the following final concentrations:
50 mM Hepes buffer, 10 mM MnCl2, 2.0 mg/mL BSA, 200 M b-Glc-
NAc-O-(CH2)6-dansyl, 40 M UDP-Gal and compounds A1–A4 and
lL. The reaction
l
Appendix A. Supplementary material
l
B1–B4 at a range of concentrations from 0 mM (control) to 2.0 mM.
The enzymatic reactions were started by the addition of 0.2 mU
b-1,4-GalT I and incubated at 30 °C for 14 min. Inactivation was
quickly done by immersion of the reaction solutions for 2 min in
a boiling water bath. The solutions were diluted with water
Supplementary data associated with this article can be found,
in the online version, at http://dx.doi.org/10.1016/j.bioorg.2014.
11.001.
References
(200 lL) and centrifuged for 10 min, and the supernatant was
injected into RP-HPLC system. The percentage of inhibition was
evaluated from the fluorescence intensity of the peaks referring
to product (Galb-1,4-GlcNAcb-O-(CH2)6-dansyl).
[1] K. Isono, J. Antibiot. 41 (1988) 1711–1739.
[2] K. Isono, Pharmacol. Therapeut. 52 (1991) 269–286.
[3] M. Winn, R.J. Goss, K. Kimura, T.D. Bugg, Nat. Prod. Rep. 27 (2010) 279–304.
[4] C.T. Walsh, W. Zhang, ACS Chem. Biol. 6 (2011) 1000–1007.
[5] A. Varki, R.D. Cummings, J.D. Esco, H.H. Freeze, P. Stanley, C.R. Bertozzi, G.W.
Hart, M.E. Etzler, Essentials of Glycobiology, second ed., CSHL Press, 2009.
[6] A. Heifetz, R.W. Keenan, A.D. Elbein, Biochemistry 18 (1979) 2186–2192.
[7] K.E. Jackson, P.K. Pogula, S.E. Patterson, Heterocycl. Commun. 19 (2013) 375–
386.
4.4.2. In vitro antidenaturation studies
Assays were performed in 96-well microtiter plates (Costar
3596, Corning) in total volume of 100 lL. The reaction mixtures
contained reagents in the following final concentrations: 0.2%
W/V of BSA or HSA, 100 mM phosphate buffer (pH 5.3) or 50 mM
Hepes buffer (pH 5.3) and compounds A1–A4 and B1–B4 at a range
of concentrations from 0 mM (control) to 2.0 mM. The plates were
heated at 70 °C for 30 min and then cooled for 10 min. The absor-
bance of these solutions was determined by using spectrophotom-
eter at a wavelength of 660 nm. The %inhibition of precipitation
(denaturation of the protein) was determined on a% basis relative
to the control using the following formula:
[8] T.M. Gloster, D.J. Vocadlo, Nat. Chem. Biol. 8 (2012) 683–694.
[9] R. Roychoudhury, N.L.B. Pohl, Curr. Opin. Chem. Biol. 14 (2010) 168–173.
[10] T. Kajimoto, M. Node, Synthesis (2009) 3179–3210.
[11] S. Wang, S. Vidal, Recent Design of Glycosyltransferase Inhibitors, in: A.P.
Rauter, T. Lindhorst (Eds.), Carbohydrate Chemistry, vol. 39, RSC Publishing,
London, 2013, pp. 78–101.
[12] K. Descroix, T. Pesnot, Y. Yoshimura, S.S. Gehrke, W. Wakarchuk, M.M. Palcic,
G.K. Wagner, J. Med. Chem. 55 (2012) 2015–2024.
[13] T. Pesnot, R. Jørgensen, M.M. Palcic, G.K. Wagner, Nat. Chem. Biol. 6 (2010)
321–323.
[14] A. Evitt, L.M. Tedaldi, G.K. Wagner, Chem. Commun. 48 (2012) 11856–11858.
[15] K. Takaya, N. Nagahori, T. Furuike, N. Miura, K. Monde, Y.C. Lee, S.-I. Nishimura,
J. Med. Chem. 48 (2005) 6054–6065.
[16] H. Yuasa, M.M. Palcic, O. Hindsgaul, Can. J. Chem. 73 (1995) 2190–2195.
[17] S. Vidal, I. Bruyère, A. Malleron, C. Augé, J.-P. Praly, Bioorg. Med. Chem. 14
(2006) 7293–7301.
[18] H. Hashimoto, T. Endo, Y. Kajihara, J. Org. Chem. 62 (1997) 1914–1915.
[19] J.-B. Behr, Chimia 65 (2011) 1–5.
[20] P.M. Chaudhary, S.R. Chavan, F. Shirazi, M. Razdan, P. Nimkar, S.P. Maybhate,
A.P. Likhite, R. Gonnade, B.M. Hazara, M.V. Deshpande, S.R. Deshpande, Bioorg.
Med. Chem. 17 (2009) 2433–2440.
%Inhibition of denaturation ¼ ðAcontrol ꢂ AtreatedÞ=Acontrol ꢀ 100
4.4.3. STD NMR experiments
Experiments were recorded on
a Bruker 600-MHz DRX
equipped with triple resonance (1H, 13C and 15N) probe head with
a cryo probe at 303 K. Spectra were calibrated with internal [D4]
(trimethylsilyl)propionic acid sodium salt (TSP, 10 lM). All the
[21] A. Suda, A. Ohta, M. Sudoh, T. Tsakuda, N. Shimma, Heterocycles 55 (55) (2001)
1023–1028.
samples were dissolved in deuterated PBS buffer at pH 6.5. For
[22] K. Obi, J.-I. Uda, K. Iwase, O. Sugimoto, H. Ebisu, A. Matsuda, Bioorg. Med.
Chem. Lett. 10 (2000) 1451–1454.
[23] D.K. Alargov, Z. Naydenova, K. Grancharov, P.S. Denkova, E.V. Golovinsky,
Monatsh. Chem. 128 (1997) 725–732.
[24] D.K. Alargov, Z. Naydenova, K. Grancharov, P.S. Denkova, E.V. Golovinsky,
Monatsh. Chem. 129 (1998) 755–760.
[25] D.K. Alargov, R.G. Gugova, P.S. Denkova, G. Müller, E.V. Golovinsky, Monatsh.
Chem. 130 (1999) 937–943.
[26] N.I. Howard, T.D.H. Bugg, Bioorg. Med. Chem. 11 (2003) 3083–3099.
[27] A.E. Trunkfield, S.S. Gurcha, G.S. Besra, T.D.H. Bugg, Bioorg. Med. Chem. 18
(2010) 2651–2663.
[28] K. Vembaiyan, J.A. Pearcey, M. Bhasin, T.L. Lowary, W. Zou, Bioorg. Med. Chem.
19 (2011) 58–66.
[29] I.V. Tetko, J. Gasteiger, R. Todeschini, A. Mauri, D. Livingstone, P. Ertl, V.A.
Palyulin, E.V. Radchenko, N.S. Zefirov, A.S. Makarenko, V.Y. Tanchuk, V.V.
Prokopenko, J. Comput. Aid. Mol. Des. 19 (2005) 453–463 (VCCLAB, Virtual
Computational Chemistry Laboratory <http://www.vcclab.org>).
[30] J. Paszkowska, B. Kania, I. Wandzik, J. Liq. Chromatogr. RT 35 (2012) 1202–
1212.
[31] A.M. Bello, E. Poduch, M. Fujihashi, M. Amani, Y. Li, I. Crandall, R. Hui, P.I. Lee,
K.C. Kain, E.F. Pai, L.P. Kotra, A. Potent, J. Med. Chem. 50 (2007) 915–921.
[32] Y. Mizushima, M. Kobayashi, J. Pharm. Pharmacol. 20 (1968) 169–173.
[33] L. Saso, G. Valentini, M.L. Casini, E. Grippa, M.T. Gatto, M.G. Leone, B. Silvestrini,
Arch. Pharm. Res. 24 (2001) 150–158.
the acquisition of STD NMR spectra, the protein–ligand molar sat-
uration times at 2 s was used; a T1q filter (50 ms spin-lock pulse)
to eliminate the unwanted broad resonance signals of the protein
was used. The on resonance frequency was set at 0 ppm and the
off-resonance frequency was maintained at 40 ppm. The on reso-
nance irradiation frequency of 0 ppm falls in a region in which
ligand signals are completely absent, as necessary to avoid false
positives in the STD spectrum. A train of 40 Gaussian-shaped
pulses of 50 ms each (1 ms delay between pulses, field strength
of 21 Hz) was employed. Saturated and reference spectra were
acquired simultaneously by creating a pseudo-2D experiment. Ref-
erence experiments were carried out to assure the absence of
direct irradiation of the ligand. STD spectra were performed with
16 K and 32 K data points. The original FID was zero-filled to
64 k and Fourier transformation with use of an exponential win-
dow function was applied. In order to determine the magnitude
of the STD effects, the intensity of the signal in the STD NMR spec-
trum were compared with the signal intensities of a reference
spectrum (off-resonance). The STD signal with the highest inten-
sity was set to 100% and the others were normalized to this signal.
[34] P.D. Boyer, F.G. Lum, G.A. Ballou, J.M. Luck, R.G. Rice, J. Biol. Chem. 162 (1946)
181–198.

J. Paszkowska et al. / Bioorganic Chemistry 58 (2015) 18–25
25
[35] L.V. Nargund, G.R. Reddy, V. Hariprasad, J. Pharm. Sci. 83 (1994) 246–248.
[36] J. Ghuman, P.A. Zunszain, I. Petitpas, A.A. Bhattacharya, M. Otagiri, S. Curry, J.
Mol. Biol. 353 (2005) 38–52.
[39] F. Squeglia, R. Marchetti, A. Ruggiero, R. Lanzetta, D. Marasco, J. Dworkin, M.
Petoukhov, A. Molinaro, R. Berisio, A. Silipo, J. Am. Chem. Soc. 133 (2011)
20676–20679.
[37] P. Bourassa, S. Dubeau, G.M. Maharvi, A.H. Fauq, T.J. Thomas, H.A. Tajmir-Riahi,
Eur. J. Med. Chem. 46 (2011) 4344–4353.
[38] B. Sekula, K. Zielinski, A. Bujacz, Int. J. Biol. Macromol. 60 (2013) 316–324.
[40] B. Meyer, T. Peters, Angew. Chem. Int. Ed. Engl. 42 (2003) 864–890.
[41] J. Park, S. Bhuniya, H. Lee, Y.-W. Noh, Y.T. Lim, J.H. Jung, K.S. Hong, J.S. Kim,
Chem. Commun. 48 (2012) 3218–3220.