Nucleoside-amino acid conjugates: An alternative route to the design of ribonuclease A inhibitors

Article abstract of DOI:10.1016/j.bmc.2009.06.002

Nucleoside-amino acid conjugates have been employed to inhibit the ribonucleolytic activity of ribonuclease A (RNase A) and affect the protonation/deprotonation equilibrium of its active site histidine residues. Agarose gel and precipitation assays indica

Full text of DOI:10.1016/j.bmc.2009.06.002

Bioorganic & Medicinal Chemistry 17 (2009) 4921–4927
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Nucleoside–amino acid conjugates: An alternative route to the design
of ribonuclease A inhibitors
*
*
Joy Debnath, Swagata Dasgupta , Tanmaya Pathak
Department of Chemistry, Indian Institute of Technology, Kharagpur, Kharagpur 721 302, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Nucleoside–amino acid conjugates have been employed to inhibit the ribonucleolytic activity of ribonu-
clease A (RNase A) and affect the protonation/deprotonation equilibrium of its active site histidine resi-
dues. Agarose gel and precipitation assays indicate inhibition of RNase A activity by these molecules with
a possible role of the polar side chains of the amino acids in RNase A inhibition. Kinetic experiments dem-
onstrated that the mode of inhibition is competitive in nature with inhibition constants (Ki) in the micro-
molar range. The nucleoside–serine conjugate occupies the active site of RNase A and preferential
perturbs the pKa value of His-119 by its ‘free amino group’ as found from 1H NMR studies. Docking studies
revealed that the free amino groups of the most active compounds are within hydrogen bonding distance
of His-119 in inhibitor–RNase A complexes.
Received 18 April 2009
Revised 1 June 2009
Accepted 2 June 2009
Available online 6 June 2009
Keywords:
Nucleoside–amino acid conjugate
Ribonuclease A inhibitor
Agarose gel
Ó 2009 Elsevier Ltd. All rights reserved.
Kinetics
Docking
1. Introduction
Ribonucleases become cytotoxic when they adsorb specifically
to certain cells, enter the cytosol, degrade RNA and consequently in-
hibit protein syntheses leading to cell death. Progress in ribonucle-
ase inhibition studies has increased over the years to limit the
nefarious biological activities of some of the ribonuclease super-
family proteins such as angiogenin,1 eosinophil-derived neurotoxin
(EDN),2 and bovine seminal RNase.3 All these proteins bear active
site homology with ribonuclease A (RNase A),4–6 a model protein
of this superfamily. The biological activities of these proteins are
also very much dependent on their ribonucleolytic activity.7 Thus
inhibitors which can inhibit RNase A by targeting its ribonucleolytic
site, can also lead to inhibition of other ribonuclease homologues.
Therefore ribonuclease inhibitors have been intensively sought
after for therapeutic purposes. The ribonucleolytic center of RNase
A is comprised of multiple subsites which bind the phosphate, base,
and sugar components of RNA.8,9 Among them, the P1 subsite is des-
ignated for cleavage of the phosphodiester bond. His-12, Lys-41 and
His-119 serve as the catalytic residues at the P1 subsite (Fig. 1). The
B1 and B2 sites recognize the nucleobases whose ribose sugar unit is
attached to the phosphate group via the 30- and 50-oxygen atom,
respectively. Generally the B1 site shows pyrimidine specificity
whereas the B2 site has purine affinity.10
Figure 1. Key residues of the active site of RNase A.
* Corresponding authors. Tel.: +91 3222 283306; fax: +91 3222 255303 (S.D.);
tel.: +91 3222 283342; fax: +91 3222 255303 (T.P.).
E-mail addresses: swagata@chem.iitkgp.ernet.in (S. Dasgupta), tpathak@chem.
iitkgp.ernet.in (T. Pathak).
Several nucleoside-based inhibitors of RNase A functionalized
with phosphate or pyrophosphate, which are substrate mimics in-
hibit the ribonucleolytic activity of the enzyme effectively.9,11
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.06.002

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J. Debnath et al. / Bioorg. Med. Chem. 17 (2009) 4921–4927
However, the major problem associated with these compounds is
their transport across biological membranes. The high negative
charge on the phosphate group makes them difficult to use
in vivo.12 In case of reported RNase A inhibitors, which are func-
tionalized with acidic groups (phosphate, carboxylic or sulfonic),
it is expected that they would exist in the deprotonated form at
physiological pH and would therefore disrupt the normal acid-base
equilibrium at the P1 site. Aminonucleosides as inhibitors of RNase
A have been studied13,14 since it is known that the pKa of His-12
and His-119 of RNase A changes from ꢀ5.22/6.78 for free enzyme
to ꢀ6.30/8.10 for enzyme–substrate complexes.15 Thus the intro-
duction of a primary amino group with a comparatively higher
pKa value should also disrupt the normal acid-base equilibrium
of active site.
In this report we have argued that nucleosides functionalized
with a ‘free amino group’, with pKa ꢀ 9–10 should perturb the
microenvironment of the histidine residues of RNase A present at
the P1 site. This in turn would diminish the ribonucleolytic activity
of RNase A in a different mode as exhibited by the presence of a
phosphate moiety. As His-12 and His-119 contribute significantly
to the stability of the RNase A-single stranded nucleic acid com-
plex, it is likely that stronger amino groups delivered to the P1 site
by an appropriately functionalized pyrimidine nucleoside, would
bind with RNase A and perturb the histidine residues responsible
for its catalytic activity.9 It is known that the 20-OH group contrib-
utes significantly towards the conformational preferences of the
furanose ring. Nevertheless, we selected thymidine derivatives as
model compounds for this study because of the easier synthetic
manipulations required for accessing the final conjugates due to
the absence of the 20-OH group.
have selected an amide linkage because of its easy synthesis and
known stability towards the ribonucleolytic activity of RNase A.
We also opined that the structurally varied nucleoside–amino acid
conjugates, which can be synthesized by coupling the amino group
of aminonucleosides and the carboxylic function of an amino acid,
would deliver the amino group embedded in a range of hydrophilic
and hydrophobic environments to the P1 site of RNase A. It is ex-
pected that the series of nucleoside–amino acid conjugates with
different side chains in the amino acid part of the molecule would
also help us in identifying the contributions of other factors such as
steric bulk, hydrophobicity and chirality around the amino group,
in proper positioning of the amino function near the histidine res-
idues at the P1 site.
The inhibition of the ribonucleolytic activity of RNase A was ini-
tially checked qualitatively by an agarose gel based assay, where
the degradation of tRNA by RNase A was monitored. The most in-
tense band observed in lane 1 is due to the presence of the control,
tRNA. The maximum possible degradation of tRNA by RNase A re-
sults in the faint intensity of the band observed in lane 2 (Fig. 2).
The differential intensity of bands in lanes 3–5 indicates the extent
of RNase A inhibition by the compounds at three different concen-
trations (higher to lower concentrations, respectively). These re-
sults qualitatively show better inhibition potency of Ser-aT, Tyr-
aT and Trp-aT compared to others.
The histogram of relative ribonucleolytic activities obtained
from a precipitation assay at a particular inhibitor concentration
(0.5 mM) gave us a clear idea about the comparative inhibitory
efficacy of the nucleoside–amino acid conjugates (Fig. 3). It was ob-
served that the compounds with polar amino acid side chains such
as Ser-aT, Tyr-aT and Trp-aT (except His-aT) were more efficient
inhibitors compared to those having hydrophobic side chains (dis-
cussed later). Since the active site of RNase A contains ionic resi-
dues,9 Ser-aT, Tyr-aT and Trp-aT with polar side chains are
expected to bind more tightly with the active site residues of
RNase A compared to others. Further kinetic experiments were
conducted with Ser-aT and Tyr-aT, which showed comparatively
good inhibitory activity both in the agarose gel based assay as well
2. Results and discussion
We report here for the first time the use of nucleoside–amino
acid conjugates as effective inhibitors (Scheme 1) of the ribonucle-
olytic activity of RNase A. Since our intention was to deliver a ‘free
amino group’ through a spacer to the P1 site of the enzyme, we
Scheme 1. Synthesis of nucleoside–amino acid conjugates. Reagents and conditions: (i) DCC, DMAP, DMF, 60 h, rt; (ii) TFA in DCM (7:3) 1 h, rt.

J. Debnath et al. / Bioorg. Med. Chem. 17 (2009) 4921–4927
4923
Figure 2. Agarose gel based assay for the inhibition of RNase A. Lane1: tRNA; Lane 2: tRNA and RNase A; Lanes 3, 4 and 5: tRNA and RNase A with decreasing concentration of
nucleoside–amino acid conjugates.
about the role of the amino acid side chain in binding interaction
with RNase A. Trp-aT was therefore selected as a compound for
monitoring the microenvironment of the fluorophoric indole moi-
ety of tryptophan. Gradual addition of excess RNase A (of same
concentration) to Trp-aT did not shift the kmax at 363 nm corre-
sponding to the emission band. Negligible quenching was observed
even after the addition of excess RNase A in which the final molar
ratio of RNase A to Trp-aT was over 3:1 (Fig. 5). This experiment
clearly indicated that the indole moiety of Trp-aT was not in close
proximity to the P1 site of RNase A, which would have led to a shift
in kmax of the fluorophore. However, the observed inhibition of the
enzymatic activity of RNase A by Trp-aT led us to conclude that the
amino group rather than the amino acid side chain played a crucial
role in ligand–protein complex. Docking studies further substanti-
ated these observations (discussed later).
Keeping these results in mind, we further proceeded to visualize
the protein–ligand complexes as obtained from docking studies.
Protein ligand docking studies provide an insight into the interac-
tion and mode of binding of the inhibitors with the proteins. Pos-
Figure 3. Ribonucleolytic inhibition of RNase A by nucleoside–amino acid conju-
sible hydrogen bonding distances of the ligands with the
interacting residues of the RNase A are given in Tables S1–S10 in
Supplementary data along with the docking poses (Figs. S1–S10).
For the nucleoside–amino acid conjugates having hydrophobic side
chains, we have proceeded from Gly-aT to Ala-aT, Val-aT, Leu-aT,
DLeu-aT and GABA-aT by concomitant increase in hydrophobicity.
In this series the ‘free amino group’ of Ala-aT and Val-aT was found
to be in the proximity of His-119. However, for the other com-
pounds in this series it was found that the ‘free amino group’
was not close to the active site histidine residues. The presence
of the long hydrophobic side chain of Leu-aT and DLeu-aT (irre-
spective of the chirality) results in an unfavorable hydrophobic-io-
nic interaction. For the series of compounds with polar side chains
Ser-aT, Tyr-aT and Trp-aT showed inhibition, His-aT being an
exception. The reason for this may be as follows. For the purine
base to be involved in a favorable stacking interaction with the
imidazole moiety of His-119, it is required that Gln-69 forms a
hydrogen bond with the nucleobase to position it properly. How-
ever, the imidazole moiety of His-aT was found to be within hydro-
gen bonding distance of Gln-69 of RNase A thus likely preventing
its free –NH2 group to interact with the active site His residues.
The docking poses indicate that the ‘free amino groups’ of Ser-aT,
Tyr-aT and Trp-aT are suitably positioned to interact with His-
119. This substantiates the experimental observation of a compet-
itive mode of inhibition from kinetic experiments. In comparison
gates. RNase A and compound concentrations are 0.2 lM and 0.5 mM, respectively.
Table 1
Inhibition constant values of 20-CMP, 30-CMP and synthesized compounds
Compounds
Experimental Ki (lM)
30-CMP
20-CMP
Ser-aT
Tyr-aT
10311a
711a
80
3
451
2
a
Ref. 11: Review for Ki values of 20-CMP, 30-CMP and other nucleotidic inhibitors.
as in the precipitation assay. The inhibition constant values ob-
tained for Ser-aT and Tyr-aT and those of 20-CMP and 30-CMP are
presented in Table 1. The nature of the Dixon plots for Ser-aT
and Tyr-aT (Fig. 4A and B, respectively) are indicative of a compet-
itive mode of inhibition. The order of values obtained for the inhi-
bition constants correlate well with those obtained from the
precipitation assay.
In order to shed light on the possible orientation of the amino
acid counterpart of the nucleoside–amino acid conjugates, a fluo-
rescence experiment was performed in which the fluorophoric
property of the Trp-aT conjugate was exploited. Since RNase A
lacks Trp, excitation at 295 nm would result in excitation and
emission from Trp-aT alone thus providing important information
to Ala-aT, Ser-aT showed an additional interaction involving the
1
side chain hydroxyl group of (Oc ) with the backbone of Val-118

4924
J. Debnath et al. / Bioorg. Med. Chem. 17 (2009) 4921–4927
Figure 4. Dixon plot for inhibition of RNase A by (A) Ser-aT: substrate concentrations: 0.80 mM (d), 0.64 mM (j) and 0.48 mM (N); (B) Tyr-aT: substrate concentrations:
0.20 mM (d), 0.14 mM (j) and 0.09 mM (N).
Figure 5. Change of fluorescence intensity of Trp-aT (5
lM) with addition of RNase
A (17.5 M). The arrow indicates the progress of titration.
l
(Fig. 6). This interaction with Val-118 brings the ‘free amino group’
closer to His-119. As a consequence, Ser-aT behaves as better
inhibitor than Ala-aT (found in agarose gel and precipitation assay)
because of the favorable interaction in the active site cleft. In case
of Trp-aT, it appears from the docking studies that the indole moi-
ety is not in close proximity to the P1 subsite. This supports the
fluorescence studies wherein we observed that the fluorescence
emission from the indole moiety of Trp-aT is not affected.
Figure 6. Docking poses of Gly-aT (cyan), Ala-aT (pink) and Ser-aT (orange) with
RNase A (1FS3).
they successfully inhibit the ribonucleolytic activity of RNase A.
These observations suggest that slight modifications in the chemi-
cal structures of the inhibitors can potentially alter the interaction
pattern with the target protein.
To get some insight into the binding pattern of the substrate
with the protein, we have compared the interaction of 20-CMP-
RNase A and 30-CMP-RNase A with the docking conformations of
the synthesized compounds. 20-CMP and 30-CMP are well known
inhibitors of RNase A. The crystal structures of 20-CMP (1JVU)16
and 30-CMP (1RPF)17 with RNase A show that the phosphate group
(for both complexes) makes two hydrogen bonds simultaneously
with His-12 and His-119. The corresponding distances from the
nearest oxygen atom of the phosphate moiety to the N2 of His-12
is 2.56 Å for 20-CMP and 2.68 Å for 30-CMP. The distances to the
Nd1 of His-119 are 2.59 Å for 20-CMP and 3.06 Å for 30-CMP. It is
also observed that the nucleobases are well recognized by the B1
subsite of RNase A indicating a binding pattern similar to the nor-
mal substrate of RNase A. However, the binding pattern of our
compounds is somewhat different than 20-CMP and 30-CMP as ob-
served from docking studies and substantiated by experimental
studies (discussed below). Despite their different mode of binding
To visualize the effect of the inhibitors on the local environment
of active site histidines, changes in pKa of His-12 and His-119 were
monitored on complexation of Ser-aT and Leu-aT with RNase A by
1H NMR experiment. The pKa values for His-12, His-105 and His-
119 were found to be 5.88 0.08, 6.59 0.06 and 6.22 0.03,
respectively for free RNase A (red curves in Fig. 7A–C). On addition
of Ser-aT to RNase A, the pKa values shifted to 5.91 0.02,
6.58 0.04 and 5.72 0.05, respectively (green curves in Fig. 7A–
C). This implies that Ser-aT interacted with the active site residues
of RNase A, preferentially near His-119 resulting in a distinct shift
of its pKa value. The pKa values of His-12 and His-105, on the other
hand remained essentially unaltered. From this observation, we
conclude that the normal protonation/deprotonation equilibrium
of the active site residues by the ‘free amino group’ was disrupted
on binding. The decrease in pKa of His-119 suggests that it was sur-
rounded by positively charged species. From the docking study it
was also found that the ‘free amino group’ of Ser-aT formed a

J. Debnath et al. / Bioorg. Med. Chem. 17 (2009) 4921–4927
4925
Figure 7. pH dependence of C-2 proton signals. (A) His-12, (B) His-105, (C) His-119. Chemical shifts for RNase A (j), for compound Leu-aT (d) and for compound Ser-aT (N).
hydrogen bond with His-119. Since the pKa value of His-119 is low-
er than the pKa of the ‘free amino group’ (ꢀ9 to 10), so it influenced
His-119 (behaves as acidic entity in catalysis mechanism) to disso-
or 5% ninhydrin in MeOH solution. 1H NMR (400 MHz) and 13C
NMR (100 MHz) spectra were recorded on a Bruker NMR spec-
trometer d scale). UV–Vis measurements were made using a Per-
kin Elmer UV–Vis spectrophotometer (Model Lambda 25).
Fluorescence measurements were recorded on a Spex Fluoro-
log-3 machine. Concentrations of the solutions were determined
spectrophotometrically using the following data: e278.5 (RNase
þ
ciated. Thereafter, the amino group got ionized to —NH3 ion and
formed a hydrogen bond with His-119. This observation is in con-
trast to that found in case of phosphate functionalized nucleotides
(e.g., increase of pKa value for 20-CMP and 30-CMP) as reported
earlier.18,19
A),20 e268 (20,30-cCMP)21 are 9800 and 8500 Mꢁ1 cmꢁ1
,
Phosphate esters resulted in a greater increase in the pKa of ac-
tive site histidine residues because of the better alignment of two
anionic oxygen atoms of the phosphate moiety. Their perfect align-
ment helps them to form strong hydrogen bond with active site
residues than the molecules reported here. To support our specula-
tion regarding the pKa perturbation, we chose Leu-aT, which did
not show any inhibition of RNase A. From the 1H NMR titration
of RNase A-Leu-aT complex we found that the pKa values of His-
12, His-105 and His-119 changed from 5.88 0.08, 6.59 0.06
and 6.22 0.03 (of RNase A) to 5.88 0.02 6.54 0.05 and
6.18 0.03, respectively (blue curve in Fig. 7A–C). Almost un-
changed pKa values implied that Leu-aT was not able to perturb
the pKa of the active site histidine residues in ligand–protein com-
plex. This may be due to the presence of the large hydrophobic
moiety of Leu-aT that prevented the entry into the polar active site
cleft, which is essentially hydrophilic in nature.
respectively.
3.2. Synthesis of nucleoside–amino acid conjugates
3.2.1. General procedure for the synthesis of compounds 2a–j
Compound 122,23 (1 mmol) and the N-BOC-protected amino
acids (1 mmol) were suspended in dry DMF (15 ml) and cooled
to ꢁ15 °C. Thereafter, DMAP (1 mmol) and DCC (1 mmol) were
added consecutively and the mixture was stirred for 48 h at room
temperature. After completion of the reaction, the mixture was fil-
tered and washed with ethyl acetate. The filtrate was stirred with
saturated NaHCO3 solution and extracted with ethyl acetate. Com-
bined organic layers were evaporated under reduced pressure.
Crude residues thus obtained were purified over silica gel to afford
compounds 2a–j. Synthesis of compounds 2a, 2b and 2g were re-
ported earlier.24
Nucleoside–amino acid conjugates are shown to inhibit the
ribonucleolytic activity of RNase A for the first time. Although
these compounds contain a ‘free amino group’ the inhibition prop-
erties were found to depend also on the side chain counterpart of
the amino acid moiety. Since the active site of the enzyme is ionic
in nature, polar conjugates such as Ser-aT and Tyr-aT were found
to be more effective inhibitors. Kinetic experiments indicate com-
petitive inhibition by Ser-aT which was also found to preferentially
perturb the microenvironment of His-119 during binding with
RNase A as observed from 1H NMR studies. The decrease in pKa val-
ues for His-119 is indicative of its interaction with the ‘free amino
group’ of the inhibitors. Docking studies further substantiated
these experimental observations.
3.2.2. General procedure for the synthesis of compounds 3a–j
Compounds 2a–j were stirred with 70% trifluoroacetic acid in
DCM for 1 h at room temperature. All volatile matters were re-
moved under reduced pressure. Crude residues thus obtained were
purified over silica gel to afford the required nucleoside–amino
acid conjugates, 3a–j.
3.2.2.1. 20,30-Dideoxy-30-glycylamino thymidine 3a (Gly-aT). Com-
pound 2a (0.1 g, 0.16 mmol) was converted to 3a (0.03 g, 64%) fol-
lowing the general procedure. 1H NMR: (D2O): d 1.9 (s, 3H), 2.41–
2.49 (m, 2H), 3.44 (bs, 2H), 3.74 (dd, J = 3.2, 12.4 Hz, 1H), 3.87 (m,
1H), 3.99 (bs, 1H), 4.53 (m, 1H), 6.25 (t, J = 5.2 Hz, 1H), 7.7 (s, 1H).
13C NMR (D2O + DMSO-d6): d 13.2, 38.0 (CH2), 44.5 (CH2), 50.1,
62.3 (CH2), 85.6, 86.2, 112.8, 139.1, 153.2, 168.1, 174.9. HRMS
(ESI+): m/z calcd for C12H19N4O5 [M+H]+: 299.1355; found:
299.1351.
3. Materials and methods
3.1. Materials
3.2.2.2. 20,30-Dideoxy-30-
L-alanylamino thymidine 3b (Ala-aT). Com-
Bovine pancreatic RNase A, yeast tRNA, 20,30-cCMP, 30-CMP,
human serum albumin (HSA), 3-trimethylsilylpropane sulfonic
acid (DSS) and D2O were from Sigma–Aldrich. All other reagents
were from SRL India. Column chromatographic separations were
performed using silica gel (60–120 and 230–400 mesh). Solvents
were dried and distilled following standard procedures. TLC was
carried out on precoated plates (Merck Silica Gel 60, f254), and
the spots visualized with UV light or by charring the plates
dipped in 5% H2SO4–MeOH solution or 5% H2SO4/vanillin/EtOH
pound 2b (0.32 g, 0.49 mmol) was converted to 3b (0.09 g, 59%) fol-
lowing the general procedure. 1H NMR: (DMSO-d6): d 1.12 (d,
J = 6.4 Hz, 3H), 1.78 (s, 3H), 2.12–2.24 (m, 2H), 3.27 (m, 1H), 3.53
(m, 1H), 3.62 (m, 1H), 3.77 (bs, 1H), 4.32 (bs, 1H), 6.19 (t, J = 6.4 Hz,
1H), 7.77 (s, 1H). 13C NMR (DMSO-d6): d 12.6, 21.6, 37.4 (CH2),
49.1, 50.4, 61.6 (CH2), 83.9, 85.3, 109.8, 136.6, 150.8, 164.1, 176.0.
HRMS (ESI+): m/z calcd for C13H21N4O5 [M+H]+: 313.1512; found:
313.1502.

4926
J. Debnath et al. / Bioorg. Med. Chem. 17 (2009) 4921–4927
3.2.2.3. 20,30-Dideoxy-30-
L
-valinylamino thymidine 3c (Val-
(0.04 g, 47%) following the general procedure. 1H NMR: (DMSO-
d6): d 1.76 (s, 3H), 2.02–2.16 (m, 2H), 2.81–2.9 (m, 1H), 3.03–3.08
(m, 1H), 3.40–3.44 (m, 1H), 3.52–3.59 (m, 3H), 4.31 (bs, 1H), 6.13
(t, J = 6.8 Hz, 1H), 6.93–7.06 (m, 2H), 7.13 (s, 1H), 7.32 (m, 1H),
7.55 (m, 1H), 7.75 (s, 1H). 13C NMR (DMSO-d6): d 12.7, 29.4
(CH2), 37.4 (CH2), 49.1, 55.7, 61.6 (CH2), 83.8, 85.2, 109.8, 111.7,
118.6, 118.9, 121.3, 124.2, 127.8, 136.6, 136.6, 136.7, 150.8,
164.1, 175.0. HRMS (ESI+): m/z calcd for C21H26N5O5 [M+H]+:
428.1934; found: 428.1934.
aT). Compound 2c (0.52 g, 0.49 mmol) was converted to 3c
(0.10 g, 52%) following the general procedure. 1H NMR: (DMSO-
d6): d 0.81 (dd, J = 6.8, 23.2 Hz, 6H), 1.77–1.86 (m, 4H), 2.09–2.23
(m, 2H), 2.89 (d, J = 5.2 Hz, 1H), 3.51–3.63 (m, 2H), 3.76 (bs, 1H),
4.34 (bs, 1H), 6.17 (t, J = 6.4 Hz, 1H), 7.76 (s, 1H). 13C NMR
(DMSO-d6): d 12.6, 17.8, 19.9, 32.2, 37.4 (CH2), 49.1, 60.3, 61.6
(CH2), 83.9, 85.4, 109.8, 136.6, 150.8, 164.1, 175.0. HRMS (ESI+):
m/z calcd for C15H25N4O5 [M+H]+: 341.1825; found: 341.1808.
3.2.2.4. 20,30-Dideoxy-30-
L
-leucylamino thymidine 3d (Leu-
3.2.2.10. 20,30-Dideoxy-30-(
c-aminobutyric acid)amino thymi-
aT). Compound 2d (0.50 g, 0.72 mmol) was converted to 3d
(0.20 g, 78%) following the general procedure. 1H NMR: (D2O): d
0.95 (m, 6H), 1.62–1.76 (m, 3H), 1.90 (s, 3H), 2.41–2.54 (m, 2H),
3.73 (dd, J = 4.4, 12.8 Hz, 1H), 3.86 (dd, J = 2.8, 12.8 Hz, 1H), 3.96–
4.02 (m, 2H), 4.56 (q, J = 7.6 Hz, 1H), 6.25 (q, J = 5.2 Hz, 1H), 7.70
(s, 1H). 13C NMR (D2O + DMSO-d6): d 13.2, 22.8, 23.3, 25.5, 37.8
(CH2), 41.5 (CH2), 50.5, 53.5, 62.3 (CH2), 85.4, 86.3, 112.9, 139.1,
153.1, 167.9, 172.0. HRMS (ESI+): m/z calcd for C16H27N4O5
[M+H]+: 355.1981; found: 355.1950.
dine 3j (GABA-aT). Compound 2j (0.10 g, 0.14 mmol) was con-
verted to 3j (0.03 g, 58%) following the general procedure. 1H
NMR: (D2O): d 0.96 (m, 3H), 1.83–1.91 (m, 5H), 2.43–2.52 (m,
2H), 3.72–3.78 (m, 2H), 3.87 (dd, J = 2.8, 12.8 Hz, 1H), 3.99–4.02
(m, 1H), 4.55 (m, 1H), 6.24 (q, J = 5.2 Hz, 1H), 7.70 (s, 1H). 13C
NMR (D2O + DMSO-d6): d 10.2, 13.2, 26.7 (CH2), 37.9 (CH2), 50.3,
56.2, 62.3 (CH2), 85.6, 86.3, 112.9, 139.1, 153.1, 167.9, 173.3. HRMS
(ESI+): m/z calcd for C14H23N4O5 [M+H]+: 327.1668; found:
327.1660.
3.2.2.5. 20,30-Dideoxy-30-
D
-leucylamino thymidine 3e (DLeu-
3.3. Agarose gel-based assay
aT). Compound 2e (0.25 g, 0.36 mmol) was converted to 3e
(0.11 g, 86%) following the general procedure. 1H NMR: (D2O): d
0.94 (m, 6H), 1.64–1.76 (m, 3H), 1.91 (s, 3H), 2.41–2.51 (m, 2H),
3.76 (dd, J = 4.4, 12.8 Hz, 1H), 3.85–3.9 (m, 1H), 3.96 (t, J = 7.2 Hz,
1H), 4.04–4.12 (m, 1H), 4.55 (q, J = 7.2 Hz, 1H), 6.25 (t, J = 6 Hz,
1H), 7.70 (s, 1H). 13C NMR (D2O + DMSO-d6): d 13.2, 22.8, 23.4,
25.5, 37.8 (CH2), 41.5 (CH2), 50.5, 53.5, 62.3 (CH2), 85.4, 89.3,
112.9, 139.1, 153.1, 167.9, 172.1. HRMS (ESI+): m/z calcd for
C16H27N4O5 [M+H]+: 355.1981; found: 355.1980.
Inhibition of RNase A by all nucleoside–amino acid conjugates
was checked qualitatively by the degradation of tRNA in an agarose
gel. In this method, 20
ll of RNase A (0.66 lM) was mixed with 10,
15 and 20 l of the compounds (0.92 mM) to a final volume of
l
50
20
20
l
l
l
l and the resulting solutions were incubated for 6 h at 37 °C.
l aliquots from incubated mixtures were then mixed with
l of tRNA solution (5.0 mg/ml) and 10 ll of sample buffer (con-
taining 10% glycerol and 0.025% bromophenol blue). The mixture
was then incubated for another 30 min. A volume of 15 l from
l
3.2.2.6. 20,30-Dideoxy-30-
L
-serinylamino thymidine 3f (Ser-
each solution were extracted and loaded onto a 1.1% agarose gel.
The gel was run in 0.04 M Tris-acetic acid-EDTA (TAE) buffer (pH
8.0). The undegraded tRNA was visualized by ethidium bromide
staining under UV light.
aT). Compound 2f (0.09 g, 0.13 mmol) was converted to 3f
(0.02 g, 45%) following the general procedure. 1H NMR: (D2O): d
1.79 (s, 3H), 2.32–2.39 (m, 2H), 3.44 (t, J = 5.2 Hz, 1H), 3.61–3.65
(m, 3H), 3.75 (dd, J = 2.8, 12.8 Hz, 1H), 3.90 (m, 1H), 4.42 (q,
J = 7.6 Hz, 1H), 6.15 (t, J = 5.2 Hz, 1H), 7.60 (s, 1H). 13C NMR
(D2O + DMSO-d6): d 12.2, 36.9 (CH2), 48.7, 56.6, 61.2 (CH2), 64.0
(CH2), 83.4, 84.9, 109.4, 136.2, 150.4, 163.7, 173.1. HRMS (ESI+):
m/z calcd for C13H21N4O6 [M+H]+: 329.1349; found: 329.1353.
3.4. Precipitation assay
Inhibition of the ribonucleolytic activity of RNase A was quanti-
fied by the precipitation assay as described by Bond.25 In this
method 10
nucleoside–amino acid conjugate (1 mM) to a final volume of
100 l and incubated for 2 h at 37 °C. A 20 l aliquot of the result-
ing solutions from the incubated mixtures were then mixed with
40 l of tRNA (5 mg/ml), 40 l of Tris–HCl buffer of pH 7.5 contain-
ing 5 mM EDTA and 0.5 mg/ml HSA. After incubation of the reac-
tion mixture at 25 °C for 30 min, 200 l of ice-cold 1.14 (N)
perchloric acid containing 6 mM uranyl acetate was added to
quench the reaction. The solution was then kept in ice for another
30 min and centrifuged at 4 °C at 12000 rpm for 5 min. A 100 ll
aliquot of the supernatant was taken and diluted to 1 ml. The
change in absorbance at 260 nm was measured and compared to
a control set. Based on the results obtained, further experiments
were carried out with the compounds showing substantial inhibi-
tion potency.
ll of RNase A (2 lM) was mixed with 50 ll of each
3.2.2.7. 20,30-Dideoxy-30-
L-tyrosylamino thymidine 3g (Tyr-
aT). Compound 2g (0.18 g, 0.24 mmol) was converted to 3g
(0.05 g, 51%) following the general procedure. 1H NMR: (DMSO-
d6): d 1.76 (s, 3H), 2.04–2.19 (m, 2H), 2.49–2.75 (m, 2H), 3.28 (t,
J = 6.4 Hz, 2H), 3.45–3.62 (m, 2H), 4.30 (bs, 1H), 6.14 (t, J = 6.4 Hz,
1H), 6.63 (m, 2H), 6.95 (m, 2H), 7.74 (s, 1H). 13C NMR (DMSO-
d6): d 12.7, 37.4 (CH2), 40.9 (CH2), 49.0, 56.8, 61.6 (CH2), 83.8,
85.3, 109.8, 115.3, 128.8, 130.5, 136.6, 150.8, 156.1, 164.1, 174.8.
HRMS (ESI+): m/z calcd for C19H25N4O6 [M+H]+: 405.1742; found:
405.1731.
l
l
l
l
l
3.2.2.8. 20,30-Dideoxy-30-
L-histidinylamino thymidine 3h (His-
aT). Compound 2h (0.16 g, 0.22 mmol) was converted to 3h
(0.05 g, 57%) following the general procedure. 1H NMR: (D2O): d
1.87 (s, 3H), 2.31–2.48 (m, 2H), 3.08–3.21 (m, 2H), 3.56–3.59 (m,
1H), 3.63–3.74 (m, 2H), 4.03–4.20 (m, 1H), 4.36–4.43 (m, 1H),
6.08–6.16 (m, 1H), 7.12 (s, 1H), 7.64 (s, 1H), 7.95 (s, 1H). 13C
NMR (D2O + DMSO-d6): d 13.6, 17.4, 42.0 (CH2), 50.5 (CH2), 54.4,
57.9, 66.3 (CH2), 88.7, 89.9, 114.6, 140.3, 141.3, 155.5, 168.9,
173.2, 173.3. HRMS (ESI+): m/z calcd for C16H23N6O5 [M+H]+:
379.1730; found: 379.1698.
3.5. Inhibition kinetics
The inhibition of RNase A by Ser-aT and Tyr-aT was assessed
individually by a spectrophotometric method as described by
Anderson and co-workers.21 The assay was performed in oligovinyl-
sulfonic acid free26 0.1 M Mes-NaOH buffer, pH 6.0 containing 0.1 M
NaCl using 20,30-cCMP as the substrate. For Ser-aT, the inhibitor con-
3.2.2.9. 20,30-Dideoxy-30-
(Trp-aT). Compound 2i (0.15 g, 0.19 mmol) was converted to 3i
L
-tryptophanylamino thymidine 3i
centration was ranged from 0 to 20
lM and the substrate concentra-
tion was used from 0.48 to 0.80 mM. For Tyr-aT, the inhibitor

J. Debnath et al. / Bioorg. Med. Chem. 17 (2009) 4921–4927
4927
concentration was ranged from 0 to 100
trations from 0.09 to 0.20 mM. The RNase A concentration was
12 M. The inhibition constants (Ki) were determined from initial
velocity data. The reciprocal of initial velocity was plotted against
the inhibitor concentration (Dixon Plot) according to the equation:
l
M with substrate concen-
where, D1 and D2 are the chemical shift changes for the acid pH
inflection (pK1) and the basic pH inflection (pK2), respectively.
Eq. 1 was used for the calculation of pKa for His-105 and Eq. 2
was used for better fit of the calculated curve of the experimental
points obtained for His-12 and His-119, if the existence of an acid
inflection is accepted. The titration curve His-48 which is inacces-
sible to solvent shows anomalous behavior with pH, preventing
determination of its pKa value as was reported earlier.30 Using
Eq. 2 pK1 and pK2 are obtained. The large error in the estimated
pK1 values is generally encountered in all cases due to the uncer-
tainty in the experimental points which is close to the width of
the acid inflection as reported earlier.31 Hence for our discussion
of pKa only pK2 was considered.
l
ꢀ
ꢁ
1
v
Km
Vmax½SꢂKi
1
Vmax
Km
½Sꢂ
¼
½Iꢂ þ
1 þ
where,
v is the initial velocity, [S] the substrate concentration, [I]
the inhibitor concentration, Km the Michaelis constant, Ki the inhi-
bition constant and Vmax the maximum velocity.
3.6. Fluorescence study
Acknowledgments
Fluorimetric titrations were performed in a 1 cm quartz cuvette
using an excitation wavelength of 295 nm. 2.5 ml of 5 lM Trp-aT in
20 mM phosphate buffer pH 7.0 containing 50 mM NaCl was ti-
trated by successive addition of RNase A to reach a final protein
concentration of 17.5 lM. The emission spectra were recorded
J.D. thanks CSIR, India for a fellowship. S.D. and T.P. are grateful
to the Department of Science and Technology (DST), New Delhi, In-
dia, for financial support. J.D. thanks Dr. Kalyan S. Ghosh for his
help in some of the experiments.
from 305 to 450 nm with a slit width of 5 nm for both the excita-
tion and emission beams.
Supplementary data
3.7. Docking studies of the nucleoside–amino acid conjugates
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2009.06.002.
The crystal structure of RNase A (PDB entry 1FS3) was down-
loaded from the Protein Data Bank.27 The 3D structures of the
nucleoside–amino acid conjugates were generated by SYBYL6.92
(Tripos Inc., St. Louis, USA) and their energy-minimized conforma-
tions were obtained with the help of the TRIPOS force field using
Gasteiger-Hückel charges with a gradient of 0.005 kcal/mol. The
FlexX software as part of the SYBYL suite was used for docking of
the nucleoside–amino acid conjugates with RNase A. PyMol28
was used for visualization of the docked conformations.
References and notes
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3.8. 1H NMR Study for proton association–dissociation
equilibrium
Exchangeable hydrogen atoms of RNase A were replaced by
deuterium to ease the pH titration for the 1H NMR study. To ex-
change hydrogen by deuterium the protein was dissolved in D2O
and incubated at 50 °C for 20 min and then lyophilized following
the method of Quirk and Raines.29 This was repeated three times.
The lyophilized protein was dissolved in D2O containing 0.17 M
NaCl and 0.5 mM DSS. DCl and NaOD solutions were used to adjust
the pH* for the range of 3–8, where pH* is the direct measure of the
pH which was not corrected for a deuterium isotope effect. The li-
gand to enzyme molar ratio was 5:1, which was maintained for
both compounds Leu-aT and Ser-aT. 1H NMR data were recorded
on a Bruker 400 MHz spectrometer at 22.5 °C. The acquisition time
was 2 s with 100 scans. The observed d value was recorded with re-
spect to DSS. The values of dobs for histidine C-2 protons at different
pH were fitted in the following equations:
7. Shapiro, R.; Riordan, J. F.; Vallee, B. L. Biochemistry 1986, 25, 3527.
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(a)
D
ꢃ 10ðpKꢁpHÞ
dobs ¼ d0 þ
ð1Þ
1 þ 10ðpKꢁpHÞ
26. Smith, B. D.; Soellner, M. B.; Raines, R. T. J. Biol. Chem. 2003, 278, 20934.
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Shindyalov, I. N.; Bourne, P. E. Nucleic Acids Res. 2000, 28, 235.
28. DeLano, W. L. The PyMOL Molecular Graphics System, DeLano Scientific, San
Carlos, CA, 2004. USA. http://pymol.sourceforge.net/.
29. Quirk, D. J.; Raines, R. T. Biophys. J. 1999, 76, 1571.
30. Markley, J. L. Biochemistry 1975, 14, 3554.
31. Arús, C.; Paolillo, L.; Llorens, R.; Napolitano, R.; Parés, X.; Cuchillo, C. M.
Biochim. Biophys. Acta 1981, 660, 117.
where, dobs is the observed chemical shift, d0 is the chemical shift of
the unprotonated form and
protonation.
D is the chemical shift change upon
(b) In the cases where an acid inflection was present the equation:
2ꢁpHÞ
D1 ꢃ 10ðpK
D2 ꢃ 10ðpK
ꢁpHÞ þ
ð2Þ
1
dobs ¼ d0 þ
1ꢁpHÞ
2ꢁpHÞ
1 þ 10ðpK
1 þ 10ðpK