Triazole pyrimidine nucleosides as inhibitors of Ribonuclease A. Synthesis, biochemical, and structural evaluation

Article abstract of DOI:10.1016/j.bmc.2012.09.067

Five ribofuranosyl pyrimidine nucleosides and their corresponding 1,2,3-triazole derivatives have been synthesized and characterized. Their inhibitory action to Ribonuclease A has been studied by biochemical analysis and X-ray crystallography. These compounds are potent competitive inhibitors of RNase A with low μM inhibition constant (K_i) values with the ones having a triazolo linker being more potent than the ones without. The most potent of these is 1-[(β-d-ribofuranosyl)-1,2,3-triazol-4-yl]uracil being with K_i = 1.6 μM. The high resolution X-ray crystal structures of the RNase A in complex with three most potent inhibitors of these inhibitors have shown that they bind at the enzyme catalytic cleft with the pyrimidine nucleobase at the B₁ subsite while the triazole moiety binds at the main subsite P₁, where P-O5′ bond cleavage occurs, and the ribose at the interface between subsites P₁ and P₀ exploiting interactions with residues from both subsites. The effect of a susbsituent group at the 5-pyrimidine position at the inhibitory potency has been also examined and results show that any addition at this position leads to a less efficient inhibitor. Comparative structural analysis of these RNase A complexes with other similar RNase A - ligand complexes reveals that the triazole moiety interactions with the protein form the structural basis of their increased potency. The insertion of a triazole linker between the pyrimidine base and the ribose forms the starting point for further improvement of these inhibitors in the quest for potent ribonucleolytic inhibitors with pharmaceutical potential.

Full text of DOI:10.1016/j.bmc.2012.09.067

Bioorganic & Medicinal Chemistry 20 (2012) 7184–7193
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Triazole pyrimidine nucleosides as inhibitors of Ribonuclease A. Synthesis,
biochemical, and structural evaluation
Vanessa Parmenopoulou a, , Demetra S.M. Chatzileontiadou , Stella Manta , Stamatina Bougiatioti ,
a,
a
a
a
a
a
a
Panagiotis Maragozidis , Dimitra-Niki Gkaragkouni , Eleni Kaffesaki , Anastassia L. Kantsadi ,
Vassiliki T. Skamnaki , Spyridon E. Zographos , Panagiotis Zounpoulakis , Nikolaos A.A. Balatsos ,
a
b
b
a,
⇑
a,
⇑
a,
⇑
Dimitris Komiotis , Demetres D. Leonidas
a
Department of Biochemistry and Biotechnology, University of Thessaly, 26 Ploutonos St., 41221 Larissa, Greece
Institute of Biology, Medicinal Chemistry & Biotechnology, National Hellenic Research Foundation, 48 Vas. Constantinou Avenue, 11635 Athens, Greece
b
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 24 July 2012
Revised 21 September 2012
Accepted 24 September 2012
Available online 16 October 2012
Five ribofuranosyl pyrimidine nucleosides and their corresponding 1,2,3-triazole derivatives have been
synthesized and characterized. Their inhibitory action to Ribonuclease A has been studied by biochemical
analysis and X-ray crystallography. These compounds are potent competitive inhibitors of RNase A with
low
ones without. The most potent of these is 1-[(b-
= 1.6 M. The high resolution X-ray crystal structures of the RNase A in complex with three most
potent inhibitors of these inhibitors have shown that they bind at the enzyme catalytic cleft with the
lM inhibition constant (K
i
) values with the ones having a triazolo linker being more potent than the
D
-ribofuranosyl)-1,2,3-triazol-4-yl]uracil being with
K
i
l
Keywords:
Ribonuclease A
Inhibition
X-ray crystallography
Triazole nucleosides
Structure-assisted inhibitor design
pyrimidine nucleobase at the B
P-O5 bond cleavage occurs, and the ribose at the interface between subsites P
1
subsite while the triazole moiety binds at the main subsite P
1
, where
0
1
and P exploiting inter-
0
actions with residues from both subsites. The effect of a susbsituent group at the 5-pyrimidine position at
the inhibitory potency has been also examined and results show that any addition at this position leads to
a less efficient inhibitor. Comparative structural analysis of these RNase A complexes with other similar
RNase A—ligand complexes reveals that the triazole moiety interactions with the protein form the struc-
tural basis of their increased potency. The insertion of a triazole linker between the pyrimidine base and
the ribose forms the starting point for further improvement of these inhibitors in the quest for potent
ribonucleolytic inhibitors with pharmaceutical potential.
Ó 2012 Elsevier Ltd. All rights reserved.
2
1
. Introduction
diseases. Eosinophil ribonucleases, eosinophil derived neurotoxin
(
EDN) and eosinophil cationic protein (ECP) are both involved in
3,4
Ribonucleases (RNases) are endonucleases that control post-
the immune response system and inflammatory disorders. The
biological activity of all these RNase A homologues is linked to
their enzymatic activity. Therefore, these proteins are pharmaceu-
tical targets for the rational design of specific inhibitors to suppress
their activity and hence modulate their pathological actions.
Several subsites exist within the central catalytic groove of
transcriptionally the RNA population in cells. RNases and in
particular RNase A, have proven to be excellent model systems
for the study of protein structure, folding and stability, and enzyme
catalysis. In the last decade several homologues of the RNase A
superfamily have been found to play an important role in human
1
pathologies. Human angiogenin (Ang), a potent inducer of neovas-
RNases, where substrate RNA, binds. These are defined as P
o n
...P ,
cularization in vivo is an important target for many angiogenesis
dependent diseases such as tumors and other non-neoplastic
R
o
...R , and B ...B according to the phosphate, ribose and base of
n
o
n
RNA that bind respectively, (n indicates the position of the group
with respect to the cleaved phosphate phosphodiester bond where
5
n = 1). The active site of all RNases is highly conserved in terms of
Abbreviations: RNase A, bovine pancreatic Ribonuclease A; PEG, poly(ethylene
glycol); U5P, deoxyuridine 5 -phosphate; ppA-3 -p, 5 -diphosphoadenosine
-phosphate.
Corresponding authors. Tel.: +30 2410 565261; fax: +30 2410 565290
N.A.A.B.); tel.: +30 2410 565285; fax: +30 2410 565290 (D.K.); tel.: +30 2410
65278; fax: +30 2410 565290 (D.D.L.).
E-mail addresses: balatsos@bio.uth.gr (N.A.A. Balatsos), dkom@bio.uth.gr
D. Komiotis), ddleonidas@bio.uth.gr (D.D. Leonidas).
These two authors contributed equally to this work.
0
0
0
sequence and structural architecture. However, specific differences
in their peripheral RNA binding sites give rise to variations in their
specificity towards different substrates and may provide clues for
the rational design of specific ligands for each RNase. In the past,
we have initiated structure assisted inhibitor design studies using
as a template RNase A, and studied a variety of substrate
analogues, mainly mono and diphosphate (di)nucleotides with
0
3
⇑
(
5
(
0
968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.09.067

V. Parmenopoulou et al. / Bioorg. Med. Chem. 20 (2012) 7184–7193
7185
0
0
adenine at the 5 -position, and cytosine or uridine at the 3 -posi-
Most of the more recent studies try to replace the pyrophos-
phate linker of the most potent inhibitors with a non-phosphoryl
tion of the scissile bond.6
number of polar groups (pyrophosphates, phosphates or sulfates)
which may lead to cell impermeability and problems in their
bioavailability. Especially pyrophosphate groups could be hydro-
lysed by enzymes like Ap
them ineffective and possibly toxic (themselves or degraded prod-
ucts).1 Hence, we have turned our attention towards compounds
that did not have phosphate groups.
In the recent years, aminonucleosides have been identified as a
new class of potent inhibitors of RNase A and angiogenin with K
values in the mid M range while the binding of the most potent of
this class has been studied by X-ray crystallography. The binding
a series of uridine and thymidine analogues that had a morpholino,
piperidino, or pyrrolidino group at the 5 -position and K
RNase A in the mid
dibasic acid and nucleoside-amino acid conjugates
also shown to be inhibitors of similar potency. Furthermore, two
–10
All these compounds had significant
1
5,16,18,21
group.
Thus, following the structure-guided inhibitor
design approach we wanted to study the effect of the molecular
length in the inhibitory potency and more specifically the effect
of the distance between the nucleotide base and the ribose. This
way we could correlate this distance to their potency for RNase
A. Furthermore, since the 5-position of pyrimidine is amenable to
additions we also wanted to explore the limitations as to the
molecular size of any putative chemical group addition to this po-
sition. Herein, we have explored the binding effect of nucleoside
analogs lacking any phosphate groups with the insertion of a tria-
zole group between the ribose and the base moieties, in order to
provide a variation in the inhibitor length. Our results show that
the insertion of a triazole between the sugar and the base moieties
increases the inhibitory potency significantly while any substitu-
tion at the 5-pyrimidine proton with halogens or a methyl group
partially released the inhibitory effect of the novel nucleosides.
1
1
2,13
3
A and Ap
4
A hydrolases1
rendering
4
1
5
i
l
16
0
i
values for
17
lM range has been also studied. Nucleoside-
18–20
have been
0
0
16
3
-N-piperidine-4-carboxyl-3 -deoxy-ara-uridine molecules and
9
three molecules of IMP bind in the catalytic site cleft of RNase
A. More recently dinucleosides connected by either polar or non-
polar linkers have been shown to inhibit RNase A and angiogenin
2. Results
2.1. Chemistry
i
with K values in the areas of mid-lM and upper-lM range, respec-
2
1
tively Despite the number of synthetic inhibitors examined thus
Our first approach was focused on the synthesis of the 1,2,3-tri-
azole 5-substituted uracil furanonucleosides 4a–e (Scheme 1).
Copper-catalyzed azide-alkyne cycloaddition (CuAAC) was utilized
to couple the protected azido ribofuranose 1 with propargyl ura-
cil (2a), thymine (2b), 5-bromouracil (2c), 5-chlorouracil (2d) and
0
far, the best ribonucleolytic inhibitor is pdUppA-3 p with K
i
values
of 27 nM, 180 nM, and 360
l
M for RNase A, EDN and Ang, respec-
7
,22,23
24
tively,
genuine transition states.
whereas transition state theory predicts pM values for
1
Scheme 1. (i) propargyl pyrimidine base, THF, H
2
O, CuSO
4
ꢀ5H
2
O, sodium ascorbate; (ii) methanolic ammonia.

7
186
V. Parmenopoulou et al. / Bioorg. Med. Chem. 20 (2012) 7184–7193
5
-fluorouracil (2e),25 in the presence of catalytic amounts of
CuSO O and sodium ascorbate. The Cu(I)-catalyzed [2 + 3]
Conclusively, the previous results show that the presence of the tri-
4
ꢀ5H
2
azole group between the sugar and the base of the inhibitor is of
primary importance for efficient inhibition, while adding a substi-
tuent at
dipolar cycloaddition reaction occurred with full regioselectivity,
resulting in the formation of the corresponding 1,4-disubstituted
1
,2,3-triazoles 3a–e. Finally, the acetyl groups of the protected
nucleosides 3a–e were cleaved using NH /MeOH solution to afford
the corresponding free analogues 4a–e, in very good yields (74–
3%).
The current study also describes an effective and high-yield ap-
5-position of the base decreases potency.
3
2.3. X-ray crystallography
8
To elucidate the structural basis of inhibition, we have deter-
mined the crystal structure of RNase A in complex with the three
most potent compounds, 4a, 4b, and 4e. In the monoclinic crystal
form of RNase A used in the experiments there are two protein
proach for the preparation of C-5 substituted uridines 6a–e using a
domestic microwave oven and adapting the typical two step
Vorbruggen coupling into a 3 min one pot reaction. Therefore,
condensation of the commercially available 1,2,3,5-tetra-O-acet-
2
6
1
6
molecules in the asymmetric unit. All three ligands were found
bound only in one of these molecules since binding to the active
yl-b-D-ribofuranose with 5-fluorouracil, 5-chlorouracil, 5-bromo-
uracil, uracil and thymine furnished the protected furanonucle
site of the other RNase A molecules is impeded by crystal packing
contacts. This has been also observed in previous studies8
,16,17
and
osides 5a–e, in the presence of trimethylsilyl trifluoromethane-
2
6
0
sulfonate, as catalyst. The participation of 2 -acetoxy group led
to the exclusive formation of the b-anomers 5. Finally, removal of
all O-acetyl protecting groups of 5a–e with saturated methanolic
ammonia, afforded the target unprotected nucleosides 6a–e, in
quantitative yields (85–92%).
provided us with two structures one free and one inhibitor com-
plexed, from the same crystal, facilitating thus a comparative
structural analysis of the binding of each ligand to RNase A. The
structures of the 4a, 4b, and 4e complexes are determined at 1.7,
1.8 and 1.9 Å resolution and contain 334, 246, and 324 water
molecules, respectively. Upon binding to RNase A, all inhibitor
molecules adopt similar conformations and bind similarly at the
All new compounds were well-characterized by NMR and UV
spectroscopy, mass spectrometry and elemental analysis. The
structure elucidation of the newly synthesized 1-(b-
D
-ribofurano-
active site by anchoring the uracil at the B
1
subsite like previous
5
,7,8,10
syl)-1,2,3-triazole nucleosides, was made on the basis of their
pyrimidine inhibitors.
The primary functional component of
1
spectroscopic data. According to H NMR spectra of the ‘click’ prod-
this subsite is Thr45, forms two hydrogen bonds with pyrimidine:
its main-chain NH donates a hydrogen to O2 of the uracil while its
ucts the newly formed triazole proton was observed at 7.91–
7
.94 ppm for analogues 3a–e and 8.27–8.35 ppm for analogues
Oc1 donates a hydrogen to N3 of uracil (Fig. 2). The phenyl group
4
a–e.
of Phe120 and His12 C 1 on one side, and Val43 and Asn44 on the
e
opposite side of the pyrimidine, lie in van der Waals contact dis-
tance from the ring stabilizing its binding (Fig. 2). The triazole moi-
ety is involved in an extensive hydrogen bond interactions network
2
.2. Enzyme activity
We have evaluated the effect of the novel nucleoside analogs
with protein residues His12, Lys41 and Gln11 (Fig. 2) at subsite P
(Table 2) while the ribose binds at the far end of subsite P hydro-
gen-bonding to His119, Gln11 and Lys7 (subsite P ). The 5-methyl
group of 4b is involved in van der Waals interactions with Asp121.
The 5-fluorine of 4e forms halogen bonds with NZ of Lys66 (subsite
1
,
bearing a triazole-ring between the ribose and the sugar moieties
and also lacking any phosphate groups (Fig. 1). Kinetic analysis re-
vealed that compound 4a could efficiently inhibit RNase A in a
1
2
competitive mode with K
the triazole group, 6a, inhibited the enzyme following again
competitive kinetics, but the K value was 28.5 M. This result
i
= 1.6 lM. The same nucleoside without
0
P ) and the carbonyl oxygen of Asp121 (Table 2; Fig. 2).
i
l
Upon binding to RNase A, inhibitor molecules become partly
shows that the distance between the sugar and the base moieties
of the nucleoside is important for inhibitory potency, as the inser-
tion of a triazolo group ameliorated the inhibitory effect of the
nucleoside. We further designed and synthesized nucleoside ana-
logs with substitutions at the 5-position of the base moiety. This
position is important for activity modulation and the substitution
with halogens has been widely used in modern medicinal chemis-
try. We therefore substituted the proton at 5-position with Br, Cl
or F resulting to compounds 4c, 4d or 4e, respectively. Detailed
kinetic analysis of the inhibitory potencies of 4d or 4e against
buried. The solvent accessibilities of the free ligand molecules 4a,
2
4b, and 4e are 488, 507, and 517 Å , respectively. When bound
2
these molecular surfaces shrink to 134, 165, and 154 Å , respec-
tively. This indicates that approximately 70% of the ligand
molecules surfaces become buried. Compounds 4a, 4b, and 4e on
binding to RNase A, make a total of 14, 12 and 15 hydrogen bonds,
respectively and 72, 55 and 61 van der Waals interactions, respec-
tively (4a:11 nonpolar/nonpolar, 45 polar/nonpolar, 16 polar/po-
lar; 4b:8 nonpolar/nonpolar, 36 polar/nonpolar, 11 polar/polar;
4e:10 nonpolar/nonpolar, 37 polar/nonpolar, 14 polar/polar) (see
Supplementary data).
2
7
RNase A revealed that the K
i
values were, 94.76 ± 8.73 and
M of 4c produced 20%
3
0.85 ± 4.44 M, respectively, while 50 l
l
inhibition of the enzymatic activity (Table 1). The analysis showed
that the halogen substitution resulted to partial release of the inhi-
3. Discussion
bition, and the bulkier the halogen the greater the K
less efficient the inhibition. When the same experiments were
performed with the nucleoside analogs lacking the triazole ring,
i
value and the
The hydrogen bond and the van der Waals interactions of the tri-
azole moiety seem to form the structural basis of the increased po-
tency of these compounds in comparison to the ones that lack this
moiety (Table 1). Structural comparison of the three triazole pyrim-
idine nucleoside protein complexes with the RNase A–U5P com-
(
i
compounds 6a–e), the calculated K values were following the
same potency pattern as the triazole-bearing ones (Table 1). We
also substituted the 5-proton with a methyl group, which is less
polar than the halogens, in both triazole and non-triazole nucleo-
1
0
plex (Fig. 3) reveals that although they bind with their uracil at
the B subsite in a similar structural mode the rest of the molecules
1
sides (4b and 6b, respectively). In this case, the K
i
value for 4b
follow a different binding pattern, which could be attributed to the
introduction of the triazole moiety in the pyrimidine nucleosides.
was 25.8 ± 2.4 M, while for 6b was 44.1 ± 6.4 M, showing again
l
l
1
0
that the triazole group is important for efficient inhibition. For
either the triazole or non-triazole methyl-substituted nucleoside,
Thus while in the RNase A–U5P complex the phosphoribose moi-
ety binds away from subsite P pointing to the solvent, the triazole
nucleosides bind at the core of the enzyme active site (subsites B
-P ). There, they exploit more protein interactions than U5P and
1
the K
i
values were higher than the unsubstituted nucleosides
1
-
(Table 1), but lower than almost all the 5-halogen substituted ones.
R
1
1

V. Parmenopoulou et al. / Bioorg. Med. Chem. 20 (2012) 7184–7193
7187
Figure 1. Pyrimidine and triazole pyrimidine nucleosides are competitive inhibitors of RNase A. Double reciprocal plots of enzyme assays are shown for nucleosides 4a (A),
b (B), 4e (C) and 6e (D). 6e is a pyrimidine nucleoside without a triazole ring. The nucleoside concentrations are 0 (s), 1 (d), 10 (h) and 50 (j) M, except (A) and (C) where
j) is 100 M. RNase A concentration is 10 nM and substrate tRNA concentrations vary from 0–6 M. Calculated K values are listed in Table 1. K
calculation of K values are shown on the right of each panel. Representative plots of at least three independent experiments.
4
(
l
app
l
l
i
M
versus [I] plots for the
i
hence they are more potent inhibitors with K
i
values an order of
providing a possible structural explanation for 4a potency. It is
10
0
magnitude lower than that of U5P (K
parison of the binding of 4a to that of ppA-3 -p (the most potent
mononucleoside inhibitors thus far with K
= 240 nM28) to RNase
i
= 4 mM). Structural com-
however the interactions of the adenosine of ppA-3 -p with resi-
0
6
0
dues of the B
2
subsite which lead to the higher potency of ppA-3 -
i
p in comparison to 4a. This is also supported by the fact that UDP
1
0
A reveals that the ribose moiety of 4a superposes onto the
with a K
Earlier kinetic studies
nucleotides bind to RNase A with K
i
value of 650
l
M
is a weaker inhibitor of RNase A.
0
23,29,30
pyrophosphate group in the RNase A–ppA-3 -p complex with its hy-
had demonstrated that thymidine
values comparable with those
droxyl groups onto the position of the pyrophosphate oxygen atoms
i

7
188
V. Parmenopoulou et al. / Bioorg. Med. Chem. 20 (2012) 7184–7193
0
0
Table 1
base and the ribose (as opposed between the 3 -and the 5 posi-
tions of the two ribose molecules of a dinucleotide) produced more
potent ribonuclease inhibitors. This encourages following this
route in our structure assisted inhibitor design approach. Compar-
ative structural analysis of the RNase A in complex with 4a, 4b, and
Kinetic parameters and structures of the inhibitors
Triazole nucleoside
Nucleoside
O
O
N
X
X
NH
NH
4
e with other RNase A–ligand complexes, suggests ways to
improve their potency and this study is the starting point for the
design of better inhibitors. Thus, we are currently aiming to intro-
HO
O
N
O
O
0
duce an additional triazole ring at the 3 -position of the ribose of
N
N
4
a, to link a second heterocyclic base with the nucleosidic moiety,
N
OH
OH
yielding to more potent inhibitors.
HO
O
4
4
. Experimental section
.1. Chemistry. Materials and general methods
Thin layer chromatography (TLC) was performed on Merck pre-
OH
OH
5
-Substitute Compound
K
i
(l
M)
Compound
i
K (lM)
(
-X)
coated 60F254 plates. Reactions were monitored by TLC on silica
gel, with detection by UV light (254 nm) or by charring with sulfu-
ric acid. Flash column chromatography was performed using silica
gel (240–400 mesh, Merck). UV–Vis spectra were recorded on a PG
T70 UV–VIS spectrometer and mass spectra were obtained with a
H
Me
Br
4a
4b
4c
1.6 ± 0.2
25.8 ± 2.4
20% Inhibition at
50 lM
94.8 ± 8.7
30.8 ± 4.4
6a
6b
6c
28.5 ± 4.5
44.1 ± 6.4
69.1 ± 2.1
Cl
F
4d
4e
6d
6e
44.5 ± 4.6
33.6 ± 2.6
1
Micromass Platform LC (ESIMS). H NMR spectra were recorded
III
13
at 300 MHz on a Bruker AVANCE 300 spectrometer and
NMR spectra at 75.5 MHz on the same spectrometer, using respec-
tively CDCl and dimethylsulfoxide-d (DMSO-d ) with internal
C
of uridine inhibitors. Our results with compounds 6a–e are in
agreement with these studies since uracil (6a) and thymine (6b)
compounds have similar K values (Table 1). Furthermore, halogen
i
atoms do not seem to improve significantly the inhibitory potency
with the fluorine compound (6e) being slightly better than thy-
mine (6b) which in turn is almost equipotent with the chlorine
compound (6d) while the bromine compound (6c) is the less
potent of all, but only 2.4 times less potent than the best inhibitor
3
6
6
tetramethylsilane (TMS). Chemical shifts (d) were given in ppm
measured downfield from TMS, and spin–spin coupling constants
are in Hz. UV–Vis spectra were recorded on a PG T70 UV–VIS spec-
trometer and mass spectra were obtained on a Thermo Quest
Finnigan AQA Mass Spectrometer (electrospray ionization). Optical
rotations were measured using an Autopol I polarimeter. Acetoni-
trile was distilled from calcium hydride and stored over 3E molec-
ular sieves. Microwave assisted reactions were carried out in a
domestic microwave oven (Samsung-LCE 2733 GXTL) for realistic
control of the microwaves operating at 850 W generating
2450 MHz frequency throughout the required time.
(
6a). However, with the triazole compounds it seems that the
diversity of the 5-pyrimidine substituent has an impact on RNase
A inhibition since the potency of these compounds ranges from
1
.6 to 94.8
lM and beyond (50 lM of compound 4c cause only
2
0% inhibition). In addition the triazole compound with uracil is
With the exception of CuAAC reactions, all reactions sensitive to
moisture or oxygen were carried out under nitrogen environment.
Azide 1 as well as the corresponding propargyl bases 2a–e
more potent than that with a thymine. The structural superposi-
tion of the 4a, 4b, and 4e complexes (Fig. 4) reveals that the addi-
tion of a methyl group in the 5-position of pyrimidine causes a tilt
of the pyrimidine ring by ꢁ10° since it is moving away from the
amide nitrogen of the Lys66 side chain which also moves to a
new position with respect to its position in 4a and 4e complexes
2
4
25
were prepared according to the procedures described in literature.
4.2. General procedure for preparation of the protected
nucleosides 3a–e
(
Fig. 4). As a result the entire inhibitor molecule is shifted by
0.7 Å and hydroxyl O5 of the ribose cannot form hydrogen bond
2
4
0
ꢁ
To a stirred mixture of azide 1 (6.6 mmol) and the corre-
2
5
interaction with the side chain of Lys7 (Table 2). This together with
the fact that 4a forms 72 van der Waals interactions with the pro-
tein while 4b forms 55 can offer a structural explanation for the
greater potency of 4a with respect to 4b. Comparison of the 4a
and 4e complexes reveals that the fluorine atom of 4e engages in
halogen bond interactions with the side chain amide of Lys66
sponding propargyl base 2a–e (7.6 mmol) in THF (41.6 mL),
was added solution of sodium ascorbate (3.5 mmol) and
CuSO O (0.84 mmol) in H O (41.6 mL). The reaction mixture
ꢀ5H
a
4
2
2
was refluxed for 2 h and then cooled. After being treated with
MeOH and concentrated, the residue was purified by flash chroma-
tography (EtOAc) to give the desired analogues 3a–e as white
solids.
(Table 2); interactions which are not observed in the 4a complex.
Furthermore, 4e participates in 61 van der Waals interactions with
the protein, 11 less than 4a. It seems that these 11 van der Waals
interactions are enough to counterbalance the halogen bond inter-
actions of 4e and the latter is 20 times less potent than 4a. These
observations show that even small modifications of the inhibitor’s
chemical structure can generate profound alterations in its inhibi-
tory potency for the target protein. Nevertheless, the main conclu-
sion is that any substitution of the 5-pyrimidine proton leads to a
decrease in inhibitory potency.
0
0
0
4.2.1. 1-[(2 ,3 ,5 -Tri-O-acetyl-b-D-ribofuranosyl)-1,2,3-triazol-4-
yl]uracil (3a)
2
2
Yield 75%; R
265 nm ( 9241); mp: 107–109 °C; H NMR (300 MHz, CDCl
8.97 (br s, 1H, NH), 7.94 (s, 1H, Triazole H), 7.50 (d, 1H,
5,6 = 7.9 Hz, H-6), 6.14 (d, 1H, J1;2 = 3.7 Hz, H-1 ), 5.79 (dd, 1H,
J = 3.9 and 5.1 Hz, H-2 ), 5.72 (d, 1H, H-5), 5.59 (t, 1H, H-3 ), 5.02,
f
= 0.28 (EtOAc); ½
a
ꢂ
3
+2 (c 0.20, CHCl ); kmax
D
1
e
3
): d
0
0
0
J
0
0
0
4.96 (q, AB-system, 2H, J = 15.1 Hz, CH
2
), 4.48 (m, 1H, H-4 ), 4.40
0
Although previous studies with dinucleosides synthesized by
(dd, 1H, J = 12.4 and 3.1 Hz, H-5 ), 4.24 (dd, 1H, J = 12.4 and
2
1
0
13
linking two nucleosides with either a polar or a non-polar linker
led to potent inhibitors with K values between 59 and 544 M for
RNase A. However, the introduction of a polar linker between the
4.5 Hz, H-5 ), 2.12, 2.11, 2.09 (3s, 9H, 3OAc). C NMR (75.5 MHz,
CDCl ) d 170.4, 169.4, 169.2, 163.2, 150.7, 144.2, 123.3, 102.8,
90.2, 81.0, 74.3, 70.6, 62.8, 43.1, 29.7, 20.7, 20.5, 20.4; ESIMS m/z:
i
l
3

V. Parmenopoulou et al. / Bioorg. Med. Chem. 20 (2012) 7184–7193
7189
Figure 2. Stereo diagrams of the interactions between RNase A and molecule 4a (A), 4b (B) and 4e (C). The side chain atoms of protein residues involved in ligand binding are
shown as ball-and-stick models. Bound waters are shown as black spheres. Hydrogen bond interactions are represented as dashed lines.
+
4
1
52.42 [M+H] . Anal. Calcd for C18
H
21
N
5
O
9
: C, 47.90; H, 4.69; N,
1H, NH), 7.91 (s, 1H, Triazole H), 7.33 (s, 1H, H-6), 6.13 (d, 1H,
0
0
0
0
5.52. Found: C, 47.73; H, 4.93; N, 15.83.
J
(
= 3.7 Hz, H-1 ), 5.80 (dd, 1H, J = 3.9 and 5.1 Hz, H-2 ), 5.59
1
;2
0
t, 1H, H-3 ), 5.00, 4.92 (q, AB-system, 2H, J = 15.0 Hz, CH
2
), 4.48
0
0
0
0
0
4
.2.2. 1-[(2 ,3 ,5 -Tri-O-acetyl-b-
D
-ribofuranosyl)-1,2,3-triazol-4-
(m, 1H, H-4 ), 4.40 (dd, 1H, J = 12.4 and 3.0 Hz, H-5 ), 4.24 (dd,
1H, J = 12.4 and 4.5 Hz, H-5 ), 2.12, 2.11, 2.09 (3s, 9H, 3OAc), 1.91
(s, 3H, CH ). C NMR (75.5 MHz, CDCl ) d 170.4, 169.4, 169.2,
3 3
0
yl]thymine (3b)
Yield 70%; R
= 0.3 (EtOAc); ½
2
D
2
13
f
aꢂ
ꢃ4 (c 0.20, CHCl
3
); kmax 271 nm
): d 8.64 (br s,
1
(e
8030); mp: 101–102 °C; H NMR (300 MHz, CDCl
3
163.9, 150.8, 142.4, 140.1, 123.2, 111.4, 90.3, 81.0, 74.3, 70.6,

7
190
V. Parmenopoulou et al. / Bioorg. Med. Chem. 20 (2012) 7184–7193
Table 2
150.2, 143.5, 141.6, 123.5, 97.2, 90.3, 81.0, 74.3, 70.6, 62.8, 43.3,
Potential hydrogen bond interactions of the inhibitors when bound to RNase A in the
crystal
+
2
C
0.7, 20.5, 20.4; ESIMS m/z: 531.30 [M+H] . Anal. Calcd for
20BrN : C, 40.77; H, 3.80; N, 13.21. Found: C, 41.02; H,
18
H
5 9
O
Inhibitor atoma
Protein atom
4a
4b
4e
3.53; N, 12.94.
0
O2
His119 ND1
Wat215
Wat306
His119 ND1
Wat22
Lys7 NZ
2.3
2.2
3.1
3.0
2.5
2.4
—
2.8
3.0
2.7
2.6
2.9
2.9
2.5
3.1
—
2.8
—
—
2.9
3.0
—
2.6
2.8
2.8
2.7
2.7
3.1
2.9
2.9
3.1
—
2.3
—
0
0
0
4.2.4. 1-[(2 ,3 ,5 -Tri-O-acetyl-b-
D-ribofuranosyl)-1,2,3-triazol-4-
3.3
3.2
2.8
2.4
-
2.8
3.0
2.9
2.7
3.3
2.9
2.7
2.9
3.3
3.1
15
yl]5-chlorouracil (3d)
0
O4
22
D
Yield 68%; R
279 nm (
8.77 (br s, 1H, NH), 7.94 (s, 1H, Triazole H), 7.76 (s, 1H, H-6),
f
= 0.39 (EtOAc); ½
a
ꢂ
ꢃ8 (c 0.20, CHCl
3
); kmax
1
0
3
e 9402); mp: 80–82 °C; H NMR (300 MHz, CDCl ): d
O5
Wat226
N3
N5
Thr45 OG1
His12 NE2
Lys41 NZ
His12 NE2
Lys41 NZ
Thr45 N
Wat186
Wat99
Asp121 O
Lys66 NZ
0 0
0
0
6
.15 (d, 1H, J = 3.6 Hz, H-1 ), 5.79 (dd, 1H, J = 4.1 and 5.0 Hz,
1;2
0
0
H-2 ), 5.58 (t, 1H, H-3 ), 5.00 (t, 2H, J = 15.7 Hz, CH
H-4 ), 4.40 (dd, 1H, J = 12.4 and 3.0 Hz, H-5 ), 4.25 (dd, 1H,
2
), 4.49 (m, 1H,
0
0
N6
0
13
J = 12.4 and 4.5 Hz, H-5 ), 2.12, 2.11, 2.10 (3s, 9H, 3OAc). C NMR
(75.5 MHz, CDCl ) d 170.4, 169.5, 169.3, 159.1, 149.9, 141.6,
41.0, 123.4, 109.5, 90.3, 81.0, 74.3, 70.6, 62.8, 43.3, 20.7, 20.5,
O2
O4
3
1
2
4
+
0.4; ESIMS m/z: 486.85 [M+H] . Anal. Calcd for C18
4.50; H, 4.15; N, 14.42. Found: C, 44.68; H, 3.92; N, 14.12.
5 9
H20ClN O : C,
F5A
—
14
—
12
Total
0
0
0
4
.2.5. 1-[2 ,3 ,5 -Tri-O-acetyl-b-D-ribofuranosyl)-1,2,3-triazol-4-
Water mediated interactions. Wat215 is hydrogen-bonded to Wat306, which in turn
forms hydrogen bonds with Wat221. Wat221 is hydrogen-bonded to Asp121 OD1.
Wat22 is hydrogen-bonded to Gln11 OE1, Wat16, which in turn forms hydrogen
bond interactions with Ala4 O, and Val118 O. Wat186 is hydrogen-bonded Ser123
OG, Thr45 OG1, and Asp83 OD1. Wat99 forms hydrogen bonds with Ser123 N and
Wat100. Wat100 is hydrogen-bonded to Ser123 O.
yl]5-fluorouracil (3e)
Yield 71%; R
= 0.5 (EtOAc); ½
7718); mp: 49–51 °C; H NMR (300 MHz, CDCl
NH), 7.93 (s, 1H, Triazole H), 7.61 (d, 1H, J6,F = 5.3 Hz, H-6), 6.15 (d,
2
D
2
f
a
ꢂ
+2 (c 0.20, CHCl
3
); kmax 271 nm
1
(e
3
): d 8.78 (br s, 1H,
0
0
a
1H, J
(
1
1
0
,2
0
= 3.5 Hz, H-1 ), 5.79 (dd, 1H, J = 3.9 and 4.9 Hz, H-2 ), 5.58
For ligand atom definitions see Table 1.
0
t, 1H, H-3 ), 5.00, 4.94 (q, AB-system, 2H, J = 15.2 Hz, CH
H, H-4 ), 4.40 (dd, 1H, J = 12.4 and 2.9 Hz, H-5 ), 4.25 (dd, 1H,
J = 12.5 and 4.5 Hz, H-5 ), 2.13, 2.12, 2.10 (3s, 9H, 3OAc). C NMR
75.5 MHz, CDCl ) d 170.4, 169.5, 169.3, 157.2, 149.5, 142.2,
41.6, 139.1, 128.7, 123.4, 90.3, 80.9, 74.3, 70.6, 62.8, 20.7, 20.4,
2
), 4.49 (m,
+
0
0
6
2.8, 42.9, 20.7, 20.5, 20.4, 12.3; ESIMS m/z: 466.39 [M+H] . Anal.
0
13
Calcd for C19
H
23
N
5
O
9
: C, 49.03; H, 4.98; N, 15.05. Found: C,
(
1
3
4
9.40; H, 4.74; N, 15.32.
+
0
0
0
20 5 9
20.3; ESIMS m/z: 470.39 [M+H] . Anal. Calcd for C18H FN O : C,
4
.2.3. 1-[(2 ,3 ,5 -Tri-O-acetyl-b-
D
-ribofuranosyl)-1,2,3-triazol-4-
4
6.06; H, 4.29; N, 14.92. Found: C, 46.45; H, 4.48; N, 14.67.
yl]5-bromouracil (3c)
Yield 65%; R
82 nm ( 8703); mp: 90–93 °C; H NMR (300 MHz, CDCl
.80 (br s, 1H, NH), 7.93 (s, 1H, Triazole H), 7.87 (s, 1H, H-6),
2
2
f
= 0.41(EtOAc); ½
a
ꢂ
D
ꢃ24 (c 0.20, CHCl
3
); kmax
1
4
.3. General procedure for the preparation of unprotected
2
8
6
2
e
3
): d
nucleosides 4a–e
0
0
0
.15 (d, 1H, J = 3.7 Hz, H-1 ), 5.79 (dd, 1H, J = 3.9 and 5.2 Hz, H-
), 5.58 (t, 1H, H-3 ), 5.04, 4.98 (q, AB-system, 2H, J = 15.1 Hz,
1
;2
0
0
The protected nucleosides 3a–e (1 mmol) were treated with
ammonia/MeOH (saturated at 0 °C, 41.8 mL) overnight at room
temperature. The solvent was evaporated under reduced pressure
to afford pure 4a–e, in 74–83% yields as white solids.
0
0
2
CH ), 4.48 (m, 1H, H-4 ), 4.40 (dd, 1H, J = 12.4 and 3.1 Hz, H-5 ),
0
4
3
.25 (dd, 1H, J = 12.4 and 4.5 Hz, H-5 ), 2.12, 2.11, 2.09 (3s, 9H,
OAc). 1 C NMR (75.5 MHz, CDCl
3
) d 170.4, 169.5, 169.3, 159.2,
3
0
10
0
Figure 3. A stereo diagram of the RNase A—4a complex structure (grey) superimposed onto the RNase A—U-5 -P complex structure (white). Ligand molecules, 4a and U-5 -
P, are in grey and white, respectively.

V. Parmenopoulou et al. / Bioorg. Med. Chem. 20 (2012) 7184–7193
7191
Figure 4. A stereo diagram of the with the superimposed structures of the three RNase A inhibitor complexes at the active site. Coloring scheme used for the complexes is 4a
grey, 4b white, and 4e black.
4
.3.1. 1-[(b-
Yield 82%; R
MeOH); max 264 nm
300 MHz, DMSO-d
D
-Ribofuranosyl)-1,2,3-triazol-4-yl]uracil (4a)
4.3.4. 1-[(b-
(4d)
D
-Ribofuranosyl)-1,2,3-triazol-4-yl]5-chlorouracil
22
f
= 0.25 (EtOAc/MeOH 8.5:1.5); ½
a
ꢂ
D
ꢃ4 (c 0.20,
1
22
D
k
(e
10255); mp: 97–99 °C;
H
NMR
Yield 79%; R
MeOH); max 277 nm
(300 MHz, DMSO-d
f
= 0.38 (EtOAc/MeOH 8.5:1.5); ½
aꢂ
ꢃ4 (c 0.20,
1
(
6
): d 10.97 (br s, 1H, NH), 8.30 (s, 1H, Triazole
k
(
e
7550); mp: 103–105 °C;
H NMR
0
H), 7.75 (d, 1H, J5,6 = 7.9 Hz, H-6), 5.91 (d, 1H, J
1
0
,2
0
= 4.8 Hz, H-1 ),
6
): d 10.94 (br s, 1H, NH), 8.32 (s, 1H, Triazole
0
0
0
5
4
3
8
.58 (m, 2H, H-5 and 3 -OH), 5.22 (d, 1H, J
2
0
-
O
H
,
2
0
= 5.1 Hz, 2 -OH),
H), 8.28 (s, 1H, H-6), 5.91 (d, 1H, J
1
0
,2
0
= 4.8 Hz, H-1 ), 5.57, 5.23 (2
0
0
0
0
.95 (m, 3H, CH
2
and 5 -OH), 4.36 (dd, 1H, J = 4.8 and 10.2 Hz, H-
br s, 2H, 3 -OH and 2 -OH), 4.96 (m, 3H, CH
2
and 5 -OH), 4.36 (t,
0
0
0
0
0
), 4.11 (dd, 1H, J = 4.6 and 9.2 Hz, H-2 ), 3.96 (dd, 1H, J = 4.2 and
1H, H-3 ), 4.10 (t, 1H, H-2 ), 3.96 (dd, 1H, J = 4.2 and 8.4 Hz, H-4 ),
0
0
13
0
13
.4 Hz, H-4 ), 3.63–3.42 (m, 2H, H-5 ).
C NMR (75.5 MHz,
3.62–3.44 (m, 2H, H-5 ). C NMR (75.5 MHz, DMSO-d
6
) d 159.4,
DMSO-d
6
) d 171.4, 163.6, 150.6, 145.4, 142.6, 122.0, 101.2, 91.9,
149.8, 142.8, 142.6, 142.3, 122.2, 106.4, 91.9, 85.7, 74.8, 70.3,
+
+
8
5.8, 74.9, 70.3, 61.2; ESIMS m/z: 326.27 [M+H] . Anal. Calcd for
: C, 44.31; H, 4.65; N, 21.53. Found: C, 44.19; H, 4.86;
42.8; ESIMS m/z: 360.71 [M+H] . Anal. Calcd for C12
5 6
H14ClN O : C,
C
12
15 5 6
H N O
40.07; H, 3.92; N, 19.47. Found: C, 39.97; H, 4.19; N, 19.16.
N, 21.13.
4
.3.5. 1-[(b-
(4e)
Yield 83%; R
MeOH); kmax 271 nm (e 3645); mp: 63–65 °C; H NMR (300 MHz,
D
-Ribofuranosyl)-1,2,3-triazol-4-yl]5-fluorouracil
4
.3.2. 1-[(b-
Yield 78%; R
MeOH); kmax 270 nm (
D-Ribofuranosyl)-1,2,3-triazol-4-yl]thymine (4b)
22
22
f
= 0.29 (EtOAc/MeOH 8.5:1.5); ½
aꢂ
D
ꢃ8 (c 0.20,
f
= 0.32 (EtOAc/MeOH 8.5:1.5); ½
aꢂ
D
ꢃ4 (c 0.20,
1
1
e
5184); mp: 76–78 °C; H NMR (300 MHz,
DMSO-d
6
): d 11.08 (br s, 1H, NH), 8.28 (s, 1H, Triazole H), 7.63 (s,
DMSO-d
6
): d 10.73 (br s, 1H, NH), 8.31 (s, 1H, Triazole H), 8.19
0
0
1
H, H-6), 5.91 (d, 1H,
J
1
0
,2
0
= 4.7 Hz, H-1 ), 5.56 (d, 1H,
(d, 1H, J6,F = 6.6 Hz, H-6), 5.91 (d, 1H, J
5.24 (2 br s, 2H, 3 -OH and 2 -OH), 4.96, 4.91 (2s, 3H, 5 -OH and
1
0
,2
0
= 4.3 Hz, H-1 ), 5.58,
0
0
0
0
0
J
3
0
-
O
H,3
0
= 5.8 Hz, 3 -OH), 5.22 (d, 1H, J
2
0
-
O
H
,
2
0
= 4.9 Hz, 2 -OH), 4.96
0
0
0
0
(
t, 1H, J = 5.0 Hz, 5 -OH), 4.91 (s, 2H, CH
2
), 4.36 (dd, 1H, J = 4.3
CH
3.62–3.46 (m, 2H, H-5 ). C NMR (75.5 MHz, DMSO-d
152.5, 145.6, 143.5, 142.0, 125.4, 95.0, 89.0, 78.2, 73.5, 64.4, 45.8;
2
), 4.35 (m, 1H, H-3 ), 4.10 (m, 1H, H-2 ), 3.95 (m, 1H, H-4 ),
0
0
0
13
and 9.0 Hz, H-3 ), 4.09 (d, 1H, H-2 ), 3.96 (dd, 1H, J = 3.4 and
6
) d 160.5,
0
0
13
7
.4 Hz, H-4 ), 3.63-3.45 (m, 2H, H-5 ), 1.76 (s, 3H, CH
3
). C NMR
+
(
75.5 MHz, DMSO-d ) d 164.2, 150.6, 142.7, 141.1, 122.0, 108.8,
6
5 6
ESIMS m/z: 344.29 [M+H] . Anal. Calcd for C12H14FN O : C, 41.99;
H, 4.11; N, 20.40. Found: C, 42.35; H, 3.92; N, 20.69.
+
9
1.9, 85.8, 74.9, 70.3, 61.2, 22.4, 11.9; ESIMS m/z: 340.33 [M+H] .
: C, 46.02; H, 5.05; N, 20.64. Found: C,
17 5 6
Anal. Calcd for C13H N O
4
6.35; H, 4.88; N, 20.52.
4.4. Preparation of uridines 6a–e
4
.3.3. 1-[(b- -Ribofuranosyl)-1,2,3-triazol-4-yl]5-bromouracil
D
A mixture of commercially available b-D-ribose tetraacetate
(
4c)
Yield 74%; R
MeOH); max 280 nm
300 MHz, DMSO-d
(6.28 mmol) with pyrimidine base (8.8 mmol), 30.8 mL of acetoni-
trile, hexamethyldisilazane (10.9 mmol, 1.24 equiv), saccharine
(0.4 mmol, 0.046 equiv) and trimethylsilyl trifluoromethane-
sulfonate (8.8 mmol, 1.4 equiv) was taken in a Erlenmeyer flask.
The Erlenmeyer flask was placed in a microwave oven and irradi-
ated under at low power (100 W) for 3 min. The reaction mixture
was cooled at room temperature, neutralized with aqueous sodium
2
D
2
f
= 0.4 (EtOAc/MeOH 8.5:1.5); ½
aꢂ
ꢃ4 (c 0.20,
1
k
(
e
9785); mp: 176–178 °C;
H NMR
(
6
): d 11.65 (br s, 1H, NH), 8.35 (s, 1H, Triazole
0
H), 8.31 (s, 1H, H-6), 5.91 (d, 1H, J
1
0
,2
0
= 4.7 Hz, H-1 ), 5.56 (d, 1H,
0
0
J
3
0
-
O
H,3
0
= 6.1 Hz, 3 -OH), 5.22 (d, 1H, J
2
0
-OH,2
0
= 5.2 Hz, 2 -OH), 4.97
0
0
(
4
8
m, 2H, CH
2
and 5 -OH), 4.37 (dd, 1H, J = 5.2 and 10.6 Hz, H-3 ),
0
.11 (dd, 1H, J = 4.9 and 9.5 Hz, H-2 ), 3.96 (dd, 1H, J = 4.3 and
2
bicarbonate, and extracted with CH Cl2. The organic extract was
0
0
13
.4 Hz, H-4 ), 3.62–3.45 (m, 2H, H-5 ).
C NMR (75.5 MHz,
dried over anhydrous sodium sulfate, filtered and evaporated to
dryness. The residue was purified by column chromatography elut-
ing with AcOEt/hexane 7:3, resulting in the desired nucleosides
5a–e, in 75–82% yields. Finally, the protected nucleosides 5a–e
(1 mmol) were treated with ammonia/MeOH (saturated at 0 °C,
DMSO-d
6
) d 159.6, 150.0, 145.1, 142.3, 122.1, 94.9, 91.9, 85.8,
+
7
C
3
4.9, 70.3, 61.2, 42.7; ESIMS m/z: 405.19 [M+H] . Anal. Calcd for
14BrN : C, 35.66; H, 3.49; N, 17.33. Found: C, 35.91; H,
.25; N, 17.53.
12
H
5 6
O

7
192
V. Parmenopoulou et al. / Bioorg. Med. Chem. 20 (2012) 7184–7193
Table 3
Crystallographic statistics
source diffractometer with a 135 mm Atlas CCD area detector
using Nova microfocus Cu-K radiation source (k = 1.54178 Å) at
a
RNase complex
4a
4b
4e
100 K using a cryprotectant solution of the crystallization medium
supplemented with 20% v/v 2-Methyl-2,4-pentanediol. Data
Experiment
Resolution (Å)
Outermost shell (Å)
Reflections measured
10 mM (3 h)
30–1.7
1.79–1.70
95436
10 mM (19 h)
30–1.80
1.9–1.8
157333
22087
0.100 (0.441)
99.7 (99.7)
12.4 (2.8)
0.199 (0.241)
0.257 (0.332)
246
10 mM (19 h)
30–1.90
2.0–1.9
72335
18814
0.072 (0.312)
99.6 (98.9)
12.7 (2.8)
0.175 (0.206)
0.236 (0.271)
324
Pro39
processing was performed with CrysAlis
and scaled using SCA-
4
0
LA from CCP4 program suite. The crystal structure of the free
16
RNase A was used a starting model, building was performed with
Unique reflections
25105
41
a
COOT , and refinement using the maximum likelihood target
R
symm
0.051 (0.195)
96.6 (78.1)
15.1 (3.2)
0.187 (0.229)
0.237 (0.295)
334
4
2
a
function as implemented in the program PHENIX.
molecules were modeled using the Dundee PRODRG server
http://davapc1.bioch.dundee.ac.uk/programs/prodrg/) and they
Inhibitor
Completeness (%)
a
<
R
R
I/
r
I>
a
cryst
(
a
free
were fitted in the electron density maps by adjustment of their
torsion angles during the final stages of the refinement. Details of
data processing and refinement statistics are provided in Table 3.
The stereochemistry of the protein residues was validated by
No of solvent molecules
r.m.s. deviation from ideality
in bond lengths (Å)
in angles (°)
Average B factor
Protein atoms (Å )
0.007
1.3
0.006
1.2
0.007
1.2
43
MolProbity. Hydrogen bonds and van der Waals interactions
2
16.5/18.0
16.7/16.9
17.7/18.1
were calculated with the program CONTACT as implemented in
(
molecule
4
0
CCP4 applying a distance cut off 3.3 and 4.0 Å, respectively.
Solvent accessible areas were calculated with the program
A/molecule B)
Solvent molecules (Å )
Ligand atoms (Å )
PDB entry
2
24.3
30.1
4G8V
22.9
30.2
4G8Y
25.5
42.0
4G90
2
44
45
NACCESS. Figures were prepared with the program Pymol.
a
Values in parentheses are for the outermost shell.
Acknowledgments
4
1.8 mL) overnight at room temperature. The solvent was evapo-
We would like to thank S. Karoulias, for preliminary work with
the inhibitors. This work was supported by the Hellenic General
Secretariat for Research and Technology (GSRT), through a Joint
Research and Technology project between Greece and France
rated under reduced pressure to give compounds 6a–e, in 85–
9
2% yields. Chemical and physical properties of the ribofuranosyl
31–35
nucleosides were in agreement with previous data.
(
2010–2012) and in part by the Postgraduate Programmes ‘Bio-
technology-Quality assessment in Nutrition and the Environment’,
Application of Molecular Biology-Molecular Genetics-Molecular
4
.5. Enzyme kinetics
‘
Bovine pancreatic RNase A (type XII-A), yeast tRNA and other
chemicals were obtained from Sigma–Aldrich (Athens, Greece).
Concentrations of the RNase solutions were determined
spectrophotometrically using absorption coefficient
Markers’, Department of Biochemistry and Biotechnology, Univer-
sity of Thessaly.
A
e
278.5 = 9800
ꢃ1
ꢃ136
Supplementary data
M
cm
The Ribonuclease A activity assay used was adapted
37
from the procedure described by Anfinsen et al. and Slifman
3
8
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2012.09.067.
et al. Briefly, yeast tRNA was solubilized in a 50 mM sodium
phosphate buffer, pH 7.4 at concentrations varying from 2 M to
M. The final volume of the reaction was 300 l. tRNA solutions
l
7
l
l
were incubated at 30 °C for 10 min. 10 nM RNase A diluted in 0.1 M
sodium acetate/acetic acid buffer, pH 6.0, and added to the reaction
mixture. The reaction was stopped one minute later by the addi-
tion of 300 ll of fresh ice-cold solution of 40 mM lanthanum
nitrate, 6% v/v perchloric acid. Stopped reactions were maintained
on ice for 15 min, and insoluble tRNA was removed by centrifuga-
tion at 4 °C for 15 min at 14.500 rpm on a bench centrifuge. 50 ll of
the supernatant was taken and diluted to 1 ml. The amount of
cleaved tRNA was determined from the ultraviolet absorbance at
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