Development and characterization of lysine based tripeptide analogues as inhibitors of Sir2 activity

Article abstract of DOI:10.1016/j.bmc.2009.10.003

Sirtuins are NAD⁺ dependent deacetylases that modulate various essential cellular functions. Development of peptide based inhibitors of Sir2s would prove useful both as pharmaceutical agents and as effectors by which downstream cellular alterations can be monitored. Click chemistry that utilizes Huisgen's 1,3-dipolar cycloaddition permits attachment of novel modifications onto the side chain of lysine. Herein, we report the synthesis of peptide analogues prepared using click reactions on Nε-propargyloxycarbonyl protected lysine residues and their characterization as inhibitors of Plasmodium falciparum Sir2 activity. The peptide based inhibitors exhibited parabolic competitive inhibition with respect to acetylated-peptide substrate and parabolic non-competitive inhibition with NAD⁺ supporting the formation of EI₂ and E·NAD⁺·I₂ complexes. Cross-competition inhibition analysis with the non-competitive inhibitor nicotinamide (NAM) ruled out the possibility of the NAM-binding site being the second inhibitor binding site, suggesting the presence of a unique alternate pocket accommodating the inhibitor. One of these compounds was also found to be a potent inhibitor of the intraerythrocytic growth of P. falciparum with 50% inhibitory concentration in the micromolar range.

Full text of DOI:10.1016/j.bmc.2009.10.003

Bioorganic & Medicinal Chemistry 17 (2009) 8060–8072
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Development and characterization of lysine based tripeptide analogues
as inhibitors of Sir2 activity
Subhra Prakash Chakrabarty a, Ramesh Ramapanicker b, , Roli Mishra b, Srinivasan Chandrasekaran b,
Hemalatha Balaram a,
*
a Molecular Biology and Genetics Unit, Jawaharlal Nehru Centre for Advanced Scientific Research, Jakkur, Bangalore 560064, India
b Department of Organic Chemistry, Indian Institute of Science, Bangalore 560012, India
a r t i c l e i n f o
a b s t r a c t
Sirtuins are NAD+ dependent deacetylases that modulate various essential cellular functions. Develop-
ment of peptide based inhibitors of Sir2s would prove useful both as pharmaceutical agents and as effec-
tors by which downstream cellular alterations can be monitored. Click chemistry that utilizes Huisgen’s
1,3-dipolar cycloaddition permits attachment of novel modifications onto the side chain of lysine. Herein,
Article history:
Received 1 August 2009
Revised 1 October 2009
Accepted 2 October 2009
Available online 9 October 2009
we report the synthesis of peptide analogues prepared using click reactions on Ne-propargyloxycarbonyl
protected lysine residues and their characterization as inhibitors of Plasmodium falciparum Sir2 activity.
The peptide based inhibitors exhibited parabolic competitive inhibition with respect to acetylated-pep-
tide substrate and parabolic non-competitive inhibition with NAD+ supporting the formation of EI2 and
EꢀNAD+ꢀI2 complexes. Cross-competition inhibition analysis with the non-competitive inhibitor nicotin-
amide (NAM) ruled out the possibility of the NAM-binding site being the second inhibitor binding site,
suggesting the presence of a unique alternate pocket accommodating the inhibitor. One of these com-
pounds was also found to be a potent inhibitor of the intraerythrocytic growth of P. falciparum with
50% inhibitory concentration in the micromolar range.
Keywords:
Sir2 activity
Peptide inhibitors
Carbohydrate conjugates
Click chemistry
Parabolic inhibition
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
Sirtuins have been implicated in various cellular processes such
as life span extension, gene silencing, DNA repair, apoptosis, and
Deacetylases are modulators of the functions of different his-
tone and non-histone proteins. The activities of these enzymes af-
fect the conformational state and thereby, functions of various
substrate proteins. There are three classes of deacetylases with
Class I and II being Zn2+ dependent. Class III deacetylases referred
to as sirtuins (Sir2-like protein), catalyze a NAD+ dependent reac-
tion that results in the formation of nicotinamide, deacetylated
substrate and O-acetyl ADP ribose (Scheme 1). SIR2, was the first
sirtuin gene discovered from S. cerevisiae.1 Subsequently, four addi-
tional genes, Hst1–4, were identified in S. cerevisiae with high
homology to the SIR2 gene.2 Although some prokaryotes lack Sir2
like proteins, numerous Sir2 homologues have been discovered in
many organisms ranging from bacteria to mammals, demonstrat-
ing that Sir2 is a highly conserved member of a large family of sir-
tuin genes.3,4 As in yeast, most organisms have multiple
homologues of sirtuins with different functions ascribed to them.
Apart from being deacetylases, sirtuins from bacteria, yeast, and
mammals also act as mono-ADP ribosyltransferases.4
different metabolic pathways, including adipogenesis, gluconeo-
genesis, and glucose homeostasis.5–7 In addition, sirtuins are in-
volved in regulation of several stress response factors, such as
p53 tumor suppressor protein,8 fork-head transcription factor,9
and NF-kB.10 Several important questions pertaining to the differ-
ent functions of sirtuin homologues remain to be answered and the
availability of different compounds that activate or repress sirtuins
may prove useful in this regard. A limited number of natural and
synthetic inhibitors of Sir2s are known, with the first reported syn-
thetic inhibitor being sirtinol.11 This was followed by the discovery
of splitomicin, which was found to be an inhibitor of yeast Sir212
but not of human sirtuin subtypes. Recently, various analogues of
splitomicin have been found to inhibit human SIRT2.13 Further,
the Sir2 inhibitor cambinol identified from a random screen is
the first inhibitor which has been tried on an animal model to dem-
onstrate its anti-cancer property.14 One of the known synthetic sir-
tuin inhibitors, bis(indoyl)maleimide derivative (12j), competes for
the NAD+ binding pocket and inhibits the activity of human
SIRT2.15 Two natural products gluttiferone G and hyperforin, iso-
lated from Garcinia cochinchinensis and Hypericum perforatum (St.
John’s wort), respectively, yielded high potency and modest selec-
tivity for SIRT1 over SIRT2. Aristoforin, a synthetic derivative of
hyperforin, exhibited similar potency and selectivity for SIRT1.16
* Corresponding author. Fax: +91 80 22082766.
E-mail address: hb@jncasr.ac.in (H. Balaram).
Present address: Department of Biochemistry and Organic Chemistry, Uppsala
University, Uppsala 75123, Sweden.
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.10.003

S. P. Chakrabarty et al. / Bioorg. Med. Chem. 17 (2009) 8060–8072
8061
Scheme 1. Schematic representation of the protein deacetylation reaction catalyzed by Sir2.
We have earlier reported on the inhibition of Plasmodium falcipa-
rum Sir2 (PfSir2) by surfactin, a depsipeptide isolated from Bacillus
licheniformis BC98.17
catalyzed variant of the classical Huisgen reaction between a ter-
minal alkyne and an azide, which furnishes a 1,4-substituted tria-
zole derivative has proven to be a powerful tool for the synthesis of
conjugates. We have exploited the use of propargyloxycarbonyl
Sir2 catalyzed reaction follows a sequential kinetic mechanism
where acetylated peptide binds first followed by the binding of
NAD+ leading to the formation of a ternary Michaelis–Menten com-
plex.18 The design of inhibitors for a bi-substrate reaction is often
aided by the understanding of the catalytic mechanism. A thioace-
tyl lysine peptide derived from the C-terminal sequence of p53 is a
competitive inhibitor with respect to the acetylated-peptide sub-
strate and forms a non-reactive ternary complex with enzyme
and NAD+.19 To avoid cross reaction with other NAD+ binding en-
zymes development of selective sirtuin inhibitors would require
preferential targeting of the acetylated peptide binding site. In a re-
cent report, the enol form of N-(5-benzyloxycarbonylamino-5-phe-
nylcarbamoylpentyl)malonamic acid ethyl ester (2k) has been
shown to inhibit the activity of human SIRT1 potently and selec-
tively over SIRT2 and SIRT3 by forming 2k–ADP-ribose conjugate.20
The affinity of Sir2 inhibitors can be increased by the design of
molecules that are covalently-linked analogues of both acetylated
peptide and NAD+. Suramin, a selective inhibitor of SIRT5, is an
example of an inhibitor that blocks the active site of the enzyme
through simultaneous binding to both NAD+ and acetylated pep-
tide binding sites.21 However, suramin has also been found to inhi-
bit several other non-Sir2 proteins due to its chemically
multifunctional nature.
One of the strategies for the synthesis of peptides containing
novel modifications on the lysine side chain has been through
the use of click chemistry which involves Huisgen’s versatile 1,3-
dipolar cycloaddition reaction. (E)-3-(1-Cinnamyl-1H-1,2,3-tria-
zol-4-yl)-N-ydroxyacrylamide (NSC746457), synthesized through
click chemistry was found to inhibit histone deacetylases 1
(HDAC1) with an IC50 value of 104 30 nM.22 In attempts to gener-
ate isozyme-specific HDAC inhibitors by exploiting the variability
in HDAC surface surrounding the active site, a set of triazolylphe-
nyl-based HDAC inhibitors were constructed. Octanedioic acid{4-
[1-(4-fluorobenzyl)-1H-[1,2,3]triazol-4-yl]phenyl}amide hydrox-
yamide was identified to be a selective inhibitor of HDAC6 over
HDAC1 by 51-fold and a potent inhibitor with an IC50 value of
1.9 nM.23
(Poc)24–26 modification on the
e-amino group of lysine as an alkyne
source for click reactions under the conditions reported by Sharp-
less27 (CuSO4ꢀ5H2O, sodium ascorbate, H2O–tBuOH) to prepare var-
ious peptide conjugates.28 The potency of these molecules was
evaluated on the activity of recombinant P. falciparum Sir2. The
parabolic nature of the secondary plots generated from the pri-
mary Lineweaver–Burk (LB) curves support the formation of EI2
and EꢀNAD+ꢀI2 complexes indicating the presence of an additional
inhibitor binding site apart from the peptide binding site. Multi-
inhibition studies rule out a competition between the NAM bind-
ing, D-site and the second inhibitor binding site, suggesting the
presence of a unique second pocket accommodating the inhibitor.
2. Results and discussion
2.1. Synthesis of lysine conjugates using click chemistry
The strategy used for the synthesis of various conjugates (9–
13), examined for the inhibition of Sir2, are detailed in Schemes
2–4. The peptides containing a lysine residue protected at the
e-amino group as a propargyloxycarbonyl (alkyne) derivative
were selectively functionalized with azide derivatives of sugars/
thymidine, using Cu(I) catalyzed click reactions. The peptide
derivatives required for the conjugation reactions were prepared
using standard solution phase peptide synthesis protocols. A
selectively protected derivative of lysine, Boc-Lys(Poc)-OH (1)
was condensed with valine methyl ester to get the dipeptide,
Boc-Lys(Poc)-Val-OMe (3) in very good yield. The dipeptide, 3
was treated with 50% TFA to remove the Boc group and the tri-
fluoroacetate salt (3a), thus obtained was treated with Boc-Phe-
OH to get the tripeptide Boc-Phe-Lys(Poc)-Val-OMe (4) in 82%
yield (Scheme 2). A benzyl ester derivative (2) of Ne-Poc lysine
was prepared by treating 1 with benzyl chloride (Scheme 2).
Standard procedures were used for the synthesis of the azido
derivatives, 5–8 of galactose,
D-methyl glucopyranoside, thymi-
dine, and mannose, respectively, from the corresponding halide
or tosyl derivatives using NaN3 in DMF (Scheme 3).29–31 Cu(I) cat-
alyzed click reactions were performed on the Poc derivatives 2
and 4 with the azide derivatives, 5–8 to get the conjugates
In this paper we report on the development and kinetic charac-
terization of peptide analogues containing novel sugar and nucleo-
side modifications as inhibitors of Sir2 activity. A copper(I)-

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S. P. Chakrabarty et al. / Bioorg. Med. Chem. 17 (2009) 8060–8072
Scheme 2. Synthesis of Boc-Lys(Poc)-OBn and tripeptide from Boc-Lys(Poc)–OH.
gates. The high stability of the Poc group to acids can be
exploited in sequential peptide synthesis. Placing the N -Poc lysine
e
residue at suitable positions in the sequence offers an efficient en-
try to the synthesis of designed polypeptides, which can be func-
tionalized using click chemistry approaches.
2.2. Synthetic peptide analogues inhibit PfSir2 deacetylase
activity
The synthetic peptide analogues were examined for their ability
to inhibit recombinant PfSir2 activity. The assays were carried out
using 3
expression system. The IC50 values for 10–13 were 151 16,
23 2, 33 2, and 34 M, respectively. The intermediate com-
lM recombinant PfSir2 protein purified from an E. coli
2
l
pounds 1 and 3 that are fragments of the parent molecules, and
9, a lysine–thymidine conjugate were also examined for their abil-
ity to inhibit PfSir2 activity. Even at a concentration of 500 lM,
these intermediates failed to inhibit PfSir2 activity suggesting the
need for the tripeptide as the minimum inhibitory unit. The com-
pounds 10–13 also inhibited human SIRT1 (CycLex, MBL Interna-
tional, Woburn, MA) (Fig. 1) indicating that the inhibition is a
common feature across the two Sir2 homologues.
Scheme 3. Synthesis of azides from the corresponding halides.
2.3. Mechanism of inhibition
9–13, with the lysine side chains modified with sugars/thymidine
(Scheme 4). The click reactions yielding the 1,4-disubstituted tri-
azole derivatives (9–13) proceeded well and the products could
be isolated in high yields and purity. All the products were puri-
fied by column chromatography and were characterized spectro-
scopically. Purity of the compounds used for the enzyme assays
were confirmed by reverse phase analytical HPLC.
The facile nature of these reactions suggests that the Poc group
functions well dually as a protection for the side chain amino
group of lysine during peptide synthesis and its alkyne functional-
ity facilitates click reactions for the preparation of peptide conju-
The kinetic mechanism of inhibition of PfSir2 activity by 10–13
was examined. Double reciprocal plots of 1/
m versus 1/[peptide]
and 1/
m
versus 1/[NAD+] at different fixed concentrations of the
inhibitors, 10 (Fig. 2A and B), 11 (Fig. 3A and B), 12 (Fig. 4A and
B) and 13 (Fig. 5A and B) yielded competitive and non-competitive
inhibition patterns, respectively. This indicates that though the
inhibitors contain sugar or nucleoside modifications they do not
compete for NAD+ binding site. The secondary plots for 10 were
linear (Fig. 2C–E) while the secondary plots of the slopes
(Figs. 3C, 4C, and 5C) and of slopes and intercepts (Figs. 3D–E,

S. P. Chakrabarty et al. / Bioorg. Med. Chem. 17 (2009) 8060–8072
8063
Scheme 4. Synthetic strategies for the preparation of peptide analogues and lysine thymidine analogue.
which varies with [I].32 Linear regression analyses using Eq. 1 were
performed on the plots of 1/Ksilope versus [I] (Fig. 6A and B) for Kislope
values obtained from both the non-competitive (with respect to
NAD+) and competitive (with respect to acetylated peptide) inhibi-
tion data.
1=Kislope ¼ 1=K2½Iꢁ þ 2=Ki
ð1Þ
i
Linearity of the curves in the 1/Ksilope versus [I] plots indicated
the presence of only two binding sites for the inhibitor molecules
on PfSir2 while presence of more than two binding sites would
have yielded non-linear (concave-up) plots. This suggests the pres-
ence of EI2 and EꢀNAD+ꢀI2 complexes in the reaction mixture. At sat-
urating substrate concentrations, no sigmoidicity was seen in
m
versus [I] plots, proving the absence of co-operativity in the bind-
ing of 11–13 to the two sites on PfSir2.
Figure 1. Inhibitory effect of 10–13 on human SIRT1 activity. Each bar indicates
percent residual activity and numbers above bars indicate concentrations of the
inhibitor in lM.
The initial velocity inhibition data for 11–13 fit to Eqs. 5 and 7
for single site inhibition yielded poorer statistics compared to their
fits to parabolic inhibition described by Eqs. 8 and 9. Table 1 sum-
marizes the Ki values obtained from fits of the primary plots to Eqs.
8 and 9 and, of the secondary plots to Eqs. 10 and 11. The Ki values
for 11–13 are apparent and represent an average Ki value for bind-
ing to both the sites on the enzyme. The linearity of the secondary
plots for 10 indicates a true Ki value and a single binding site on the
enzyme. The Ki values for the different inhibitors with respect to
peptide are largely similar, with the value for 10 being slightly
higher.
4D–E, and 5D–E) were all parabolic for 11–13. Parabolic secondary
plots in inhibition kinetics indicate the presence of more than one
binding site for the inhibitor.32 Also, the parabolic nature of the
Dixon plots of 1/m versus [inhibitor (I)] for 11–13 provided further
evidence for the presence of more than one binding site on the en-
zyme. The data indicate that apart from the peptide binding site,
11–13 have additional binding site/s on PfSir2.
To confirm the presence of EI2 and ESI2 (S corresponds to NAD+)
complexes with respect to the inhibitors, 11–13, the initial velocity
The number of binding sites on the enzyme for 11–13 was esti-
mated by generating plots of 1/Ksilope versus [I] where the values of
Ksilope are direct slope values of lines in double reciprocal plots of 1/
data were analyzed by constructing [S]/
m
versus [I] plots and fitting
to Eq. 2 (Fig. 7).33,34
m
versus 1/[peptide] or 1/[NAD+] at fixed saturating concentration
2
2
½Sꢁ=m
¼ a0 þ a1½Iꢁ þ a2½Iꢁ þ ½Sꢁðb0 þ b1½Iꢁ þ b2½Iꢁ Þ
ð2Þ
of the second substrate and fixed varying concentrations of the
inhibitor. It should be noted that Ksilope is not the Ki for the dissoci-
ation of an EI complex, but rather a more complex function of Ki
where a0, b0, a1, b1, a2, and b2 are the coefficients depending on
equilibrium constants and correspond to the presence of E, ES, EI,

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S. P. Chakrabarty et al. / Bioorg. Med. Chem. 17 (2009) 8060–8072
Figure 2. Lineweaver–Burk plots of PfSir2 inhibition by 10. (A) Compound 10 exhibits competitive inhibition pattern with respect to acetylated peptide. NAD+ concentration
was 70 M and [acetylated peptide] was varied from 1 to 50
M. (B) Compound 10 shows non-competitive inhibition with respect to NAD+. Acetylated peptide concentration
was fixed at 30 M. The micromolar concentration of 10 is indicated against each line. (C) Secondary plot of slope versus [10],
M while [NAD+] was varied from 2.5 to 70
l
l
l
l
generated from primary plot in (A). (D) and (E), secondary plots of slope versus [10] and intercept versus [10], respectively, generated from primary plot in (B). Solid line is the
curve generated from linear fit. Assays were done twice with two independently purified batches of enzyme.
Figure 3. Lineweaver–Burk plots of PfSir2 inhibition by 11. (A) Compound 11 shows competitive inhibition with respect to acetylated peptide. [NAD+] was held constant at
70
l
M and acetylated peptide concentration was varied from 1 to 50
l
M. (B) Compound 11 exhibits non-competitive inhibition with respect to NAD+. Acetylated peptide
concentration was fixed at 30
l
M while [NAD+] was varied from 2.5 to 70
lM. The micromolar concentration of 11 is indicated against each line. (C) Secondary plot of slope
versus [11], generated from primary plot in (A). (D) and (E), secondary plot of slope versus [11] and intercept versus [11], respectively, generated from primary plot in (B).
Solid line in panels (C–E) are curves generated from polynomial second order regression analysis. Assays were done twice with two independently purified batches of
enzyme.
ESI, EI2, and ESI2 enzyme species in the system. Non-zero values of
the coefficients that are statistically significant indicate the pres-
ence of a specific complex. The three inhibitors, 11–13 that showed
parabolic secondary plots in the LB data yielded non-zero, statisti-
cally significant values for the coefficients a2 and b2 for both sub-
strates supporting the presence of EI2 and ESI2 complexes.
Negative values were obtained for the coefficient associated with
EI and ESI complexes. Compound 10 which does not show parabolic
secondary plots yielded statistically significant and non-zero values
for the coefficients a2 and b1 only. However, the value for a2 was
100-fold lower than that obtained for 11–13, indicating the low lev-
els of EI2 complex and higher ESI levels in the reaction mixture.

S. P. Chakrabarty et al. / Bioorg. Med. Chem. 17 (2009) 8060–8072
8065
Figure 4. Lineweaver–Burk plots of PfSir2 inhibition by 12. (A) Compound 12 shows competitive inhibition with respect to acetylated peptide. [NAD+] was held constant at
70 M and acetylated peptide concentration was varied from 1 to 50
M. (B) Compound 12 exhibits non-competitive inhibition with respect to NAD+. Acetylated peptide
concentration was fixed at 30 M. The micromolar concentration of 12 is indicated against each line. (C) Secondary plot of slope
M while [NAD+] was varied from 2.5 to 70
l
l
l
l
versus [12], generated from primary plot in (A). (D) and (E), secondary plot of slope versus [12] and intercept versus [12], respectively, generated from primary plot in (B).
Solid line in panels (C–E) are curves generated from polynomial second order regression analysis. Assays were done twice with two independently purified batches of
enzyme.
Figure 5. Lineweaver–Burk plots of PfSir2 inhibition by 13. (A) Compound 13 shows competitive inhibition with respect to acetylated peptide. [NAD+] was held constant at
70
l
M and acetylated peptide concentration was varied from 1 to 50
l
M. (B) Compound 13 exhibits non-competitive inhibition with respect to NAD+. Acetylated peptide
concentration was fixed at 30
l
M while [NAD+] was varied from 2.5 to 70
lM. The micromolar concentration of 13 is indicated against each line. (C) Secondary plot of slope
versus [13], generated from primary plot in (A). (D) and (E), secondary plot of slope versus [13] and intercept versus [13], respectively, generated from primary plot in (B).
Solid line in panels (C–E) are curves generated from polynomial second order regression analysis. Assays were done twice with two independently purified batches of
enzyme.
2.4. Cross inhibition kinetics with NAM and 11
the location of the second site is not evident from the analysis.
Being a non-competitive inhibitor of NAD+, compound 11 does
not bind to NAD+ binding site. Studies on yeast Hst2, human SIRT2,
and yeast Sir2 have shown that NAM is a non-competitive inhibitor
with respect to both acetylated peptide and NAD+.35 A similar NAM
inhibition pattern was also seen with PfSir2.17 From the various
Inhibition kinetics presented above indicates the presence of
two binding sites for 11 on PfSir2. The competitive inhibition pat-
tern seen with respect to acetylated peptide indicates that one of
the binding sites for 11 is the acetylated peptide binding site while

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S. P. Chakrabarty et al. / Bioorg. Med. Chem. 17 (2009) 8060–8072
Figure 6. The plots of 1/Kislope versus [inhibitor 11 (h) or 12 ( ) or 13 (s)]. Solid lines in the two panels are curves generated from linear regression analyses. Kislope values for
D
panels (A) and (B) were derived from Lineweaver–Burk plots of competitive inhibition (with respect to peptide) in Figures 3A, 4A and 5A and non-competitive inhibition (with
respect to NAD+) in Figures 3B, 4B and 5B, respectively.
Table 1
Summary of the Ki values for compounds 10–13 obtained from different primary and secondary curve fits
Inhibitors
Type of inhibition
Ki from primary
plot ( M)
Ki from secondary plot of slope
Ki from secondary plot of intercept
l
versus [inhibitor] (
lM)
versus [inhibitor] (
lM)
Compound 10 versus Ac-peptidea
Compound 10 versus NAD+
Competitive
Non-Competitive
Competitive
Non-Competitive
Competitive
Non-Competitive
Competitive
85 11
154 11
80 10
NA
193
NA
119
NA
113
NA
172
60
5
6
7
7
7
Compound 11 versus Ac-peptidea
Compound 11 versus NAD+
60
134
60
124
58
7
7
7
5
7
6
6
90 11
64
115 11
62
90 12
Compound 12 versus Ac-peptidea
Compound 12 versus NAD+
5
Compound 13 versus Ac-peptidea
Compound 13 versus NAD+
5
Non-Competitive
122
125
a
Ac-peptide is acetylated peptide, NA—not applicable.
2
crystal structures of Sir2s complexed with ligands, the binding site
of the NAM group of NAD+ has been identified as C-site and found
to consist of conserved residues.36 Crystal structures of yeast Hst2
bound to an acetyl-lysine containing histone H4 peptide, adeno-
sine 50-diphosphate (hydroxymethyl) pyrrolidinediol (ADP-HPD),
and nicotinamide (NAM) (PDB ID 2OD9) show the presence of an
alternate binding site for nicotinamide.37 This binding site referred
to as the D-site has been proposed to be the structural basis for the
non-competitive inhibition of NAM with respect to NAD+. The res-
idues that form the D pocket are largely conserved with the excep-
tion of few substitutions, implying that this binding site may be
important for NAM mediated regulation of Sir2 activity. To exam-
ine the possibility of blocking NAM binding due to second site
occupancy by the inhibitors 11–13 the cross-competition method
of simultaneously investigating two inhibitors was adopted. This
method can provide information on whether enzyme inhibition
in the presence of two different inhibitors may be from simulta-
neous binding to independent sites or from mutually exclusive
binding to same or overlapping sites.32 Using this approach, the ef-
fect of binary combination of 11 and NAM was investigated. The
1=
m
¼ Km=VmaxK2½Sꢁð1 þ ½Xꢁ=bKxÞ½Iꢁ þ 1=Vmax½ð1 þ ½Xꢁ=
þ Km=½Sꢁð1 þ ½Xꢁ=KxÞꢁ
aKxÞ
i
ð4Þ
Replots of slope and intercept from the 1/v versus [NAM] Dixon
plot, against [11] would yield a straight line if the system follows
Eq. 3 while the curve would be concave-up in the case of Eq. 4.
We observe that both replots (Fig. 8C and D) are parabolic in nature
supporting the formation of Eꢀ(11)2ꢀNAM complex in the system.
The 1/v versus [11] Dixon plot at different fixed concentrations
of NAM is parabolic in nature (Fig. 8B). This observation also sup-
ports the presence of Eꢀ(11)2ꢀNAM complex in the system implying
the simultaneous binding of both inhibitors. The panel also shows
that at higher concentration of 40 lM of NAM, the curve is linear,
going to show that under this condition the PfSir2 enzyme is lar-
gely inhibited by NAM with low contribution from 11.
Taking together these results, the kinetic scheme for inhibition
of PfSir2 activity by 11 is shown in Scheme 5. The enzyme is inhib-
ited by 11 through binding of two molecules of inhibitors resulting
in the formation of both Eꢀ(11)2 and Eꢀ(11)2ꢀNAD+ dead-end com-
plexes. The scheme also shows the presence of the complexes
Eꢀ(11)2ꢀNAM and Eꢀ(11)2ꢀNAMꢀNAD+. Though the binding of 11 to
two sites on the PfSir2 does not exclude NAM binding, the data
cannot distinguish between the complexes Eꢀ(11)2ꢀNAM and
Eꢀ(11)2ꢀNAMꢀNAD+.
reciprocal of initial velocity, 1/m was plotted against [NAM] or
[11] to visualize the intersection pattern while the other inhibitor
was kept at different fixed concentrations. The cross-competition
pair formed an intersecting pattern in the second quadrant of Dix-
on plot when NAM was the titrant at different fixed concentrations
of 11 (Fig. 8A), indicating that the binding of 11 and NAM are not
mutually exclusive. For a two inhibitor system where inhibitors
(I and X) are not mutually exclusive and I is linear competitive
inhibitor while X is linear non-competitive inhibitor with respect
to substrate, the equation describing the initial velocity is,32
2.5. Effect of 11 on the growth of the intraerythrocytic P.
falciparum
We have investigated the effect of 11 on the intraerythrocytic
stages of P. falciparum in in vitro culture (Fig. 9). The IC50 value
for 11 is 9.8 1.7 lM for the killing of intraerythrocytic stages of
1=
m
¼ Km=VmaxKi½Sꢁð1 þ ½Xꢁ=bKxÞ½Iꢁ þ 1=Vmax½ð1 þ ½Xꢁ=
þ Km=½Sꢁð1 þ ½Xꢁ=KxÞꢁ
a
KxÞ
the parasite. Surfactin, a secondary metabolite from B. licheniformis
that inhibits PfSir2 activity by binding to the NAD+ site, is also an
inhibitor of parasite growth in in vitro cultures.17 NAM, a product
of the Sir2 catalyzed reaction and a non-competitive inhibitor with
respect to both acetylated peptide and NAD+, when added to
ð3Þ
While for the case, where I is parabolic competitive and X is lin-
ear non-competitive inhibitor with respect to the substrate, the
equation is,

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Figure 7. [S]/v versus [inhibitor] plots of PfSir2 inhibition by 11 (A, B), 12 (C, D) and 13 (E, F) at different fixed concentrations of peptide and NAD+, respectively. The
micromolar concentration of substrates (peptide and NAD+) is indicated against each line. The data were fit to Eq. 2 and compared with fit to the equation ([S]/
v = a0 + b0[S] + a1[I] + b1[S][I]) that describes a system for inhibitors having one binding site on the enzyme. For compounds 11–13 the preferred model is Eq. 2 and the nature
of the plots is parabolic suggesting the formation of EI2 and ESI2 complexes in solution while a linear profile with respect to both peptide and NAD+, would indicate the
formation of EI and ESI complex.
in vitro cultures of P. falciparum brought about a delay in parasite
growth and dispersed the localization of PfSir2 from the nucleus.38
Hyperforin, a natural product isolated from Hypericum perforatum
that inhibits human SIRT1 and SIRT2 activity, also exhibits anti-
plasmodial activity with an IC50 value in micromolar range.16,39
P. falciparum evades the host immune system by switching the
expression of the erythrocyte membrane protein (EMP) surface
antigens through the silencing of var gene expression. PfSir2 genes
(PF13_0152 and PF14_0489) have been implicated to be involved
in silencing of the var gene repertoire and also have been shown
to bind to telomeric ends of chromosomes in the intraerythrocytic
stages of the parasite.40,41 Halting the process of var gene silencing
through PfSir2 inhibition could serve to assist the process of para-
site clearance by the host immune system. Knock-out of the two P.
falciparum Sir2 genes, PF13_0152 and PF14_0489, has shown that
absence of either one of them is not lethal to the parasite and also
established their functional redundancy in the parasite.41 How-
ever, the effect of a simultaneous knock-out of both PfSir2 genes
has not been examined. The lethality that we see with 11 could
arise from simultaneous inhibition of both PfSir2 proteins suggest-
ing that apart from modulation of var gene expression, the Sir2
proteins in the parasite may have other essential roles. This, how-
ever, needs further experimental validation.
3. Conclusions
Sirtuins have been implicated in numerous biological processes
and availability of specific and potent inhibitors should prove to be
of enormous clinical value apart from their use as tools to probe
function and modulation of this important class of deacetylases.
Earlier studies on inhibitors of Sir2s have mainly focused on mole-
cules obtained from screens and hence, are largely unrelated to the
structures of the two substrates. Reports on the use of peptide ana-
logues and NAD+ analogues have indicated that these are indepen-
dently potent inhibitors of the enzyme. Our studies, for the first
time, show that the tripeptides containing lysine modified via a
1,4 triazole linker with sugars/thymidine derivatives are a novel
class of compounds inhibiting P. falciparum Sir2 activity through
competition for the peptide binding pocket. Also, the observed
inhibition of human SIRT1 by the peptide conjugates, along with
the high degree of sequence and structure conservation in the core
domain of Sir2s, suggest that these molecules may have the poten-
tial to inhibit this enzyme from other organisms. This further
agrees with the fact that catalytic mechanism of different Sir2s is
conserved. The utilization of click chemistry permits creation of
covalent links between diverse building blocks. Herein, we have
demonstrated the synthetic ease of click chemistry involving con-

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S. P. Chakrabarty et al. / Bioorg. Med. Chem. 17 (2009) 8060–8072
Figure 8. Cross-competition plots of PfSir2 inhibition between 11 and NAM. (A) NAM exhibits an intersecting inhibition pattern with respect to 11. NAD+ and acetylated
peptide concentrations were held constant at 70 and 30 M, respectively. (B) Compound 11 shows parabolic inhibition pattern with respect to NAM. The curve is linear at a
NAM concentration of 40 M. The micromolar concentrations of 11 and NAM are indicated against each line. (C) and (D), Slope and intercept obtained from reciprocal plot of
l
l
(A) versus [11], yielded a parabola, indicating the formation of Eꢀ(11)2ꢀNAM complex.
described here opens up the possibility of developing a novel class
of inhibitors specific to sirtuins.
4. Experimental
4.1. Materials
All bacterial growth media components were purchased from
HiMedia Laboratories (Mumbai, India). Substrates used for enzyme
assays and buffer components were purchased from Sigma Chem-
ical Company (St. Louis, MO, USA). All reagents were of analytical
grade and obtained from Aldrich (Steinheim, Germany). TLC was
performed on commercial plates coated with Silica Gel GF254
(0.25 mm). Silica gel (230–400 mesh) was used for column chro-
matography. Melting points determined are uncorrected. Yields re-
fer to chromatographically and spectroscopically pure compounds.
NMR spectra were recorded on 300 or 400 MHz instruments. The
following abbreviations explain the multiplicity in the spectra
s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet,
br = broad. Coupling constants are reported wherever necessary
in hertz (Hz). IR spectra were recorded on a FTIR spectrometer.
The frequencies are reported in wave number (cmꢂ1) and intensi-
ties of the peaks are denoted as s (strong), w (weak), m (medium)
and br (broad). High-resolution mass spectra (HRMS) were re-
corded on a Micromass Q-TOF mass spectrometer.
Scheme 5. Kinetic scheme for inhibition of PfSir2 activity by 11. Ac-P is the
acetylated peptide substrate.
4.2. Preparation of propargyloxycarbonyl chloride (PocCl)
To a stirred solution of triphosgene (2.23 g, 7.5 mmol) in anhy-
drous diethyl ether (30 mL), activated charcoal (0.05 g) was added
and stirred for 1 h at room temperature (28 °C). The solution was
cooled to 0 °C and propargyl alcohol (0.9 mL, 15 mmol) in anhy-
drous diethyl ether (10 mL) was added dropwise. The resultant
solution was stirred for 12 h and filtered. The diethyl ether layer
was concentrated under reduced pressure (100 mm) and the
remaining pale green liquid was used for reactions without any
further purification. Boiling point: 118–122 °C; density (28 °C):
1.215 g/cm3; FTIR (neat): 3303, 2982, 2131, 1777; dH (300 MHz,
Figure 9. Effect of 11 on uptake of [3H] hypoxanthine by parasitized erythrocytes in
culture. Culture was treated with 11 for 24 h, followed by the addition of 1 l
Ci [3H]
hypoxanthine to each well. The cells were harvested after another 12 h and [3H]
hypoxanthine was quantified by scintillation counting.
densation of propargyloxycarbonyl functionalized lysine with
azido derivatives of sugars/nucleosides that has resulted in excel-
lent yields of products with high purity. The synthetic strategy

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8069
CDCl3): 2.6 (t, J = 2.4 Hz, 1H), 4.7 (d, J = 2.4 Hz, 2H); dC (75 MHz,
CDCl3): 150.2, 77.6, 74.9, 58.3.
J1 = 9 Hz, J2 = 5 Hz, 1H), 4.07–4.15 (m, 1H), 3.75 (s, 3H), 3.17–3.23
(m, 2H), 2.48 (t, J = 2.4 Hz, 1H), 2.09–2.34 (m, 2H), 1.78–2.34 (m,
1H), 1.35–1.71 (m, 13H), 0.94 (d, J = 7.5 Hz, 3H), 0.91 (d,
J = 6.9 Hz, 3H); dC (75 MHz, CDCl3): 172.3, 172.1, 155.7, 155.6,
80.0, 78.3, 74.5, 57.1, 54.2, 52.3, 52.2, 40.3, 31.4, 30.9, 29.2, 28.2,
22.3, 18.9, 17.6; m/z (HRMS): calcd for C21H35N3O7+Na:
464.2373; found: 464.2367.
4.3. Synthesis of Boc-Lys(Poc)-OH (1)
Commercially available Boc-Lys-OH (1.25 g, 5 mmol) was dis-
solved in a solution of NaHCO3 (1 N, 20 mL) and NaOH (4 N,
1 mL). The solution was cooled to 0 °C, PocCl (0.55 mL, 5.5 mmol)
was added dropwise and the reaction mixture was stirred for 1 h.
It was allowed to come to room temperature and was stirred for
additional 3 h. The reaction mixture was acidified to pH 2–3 by
the slow addition of 1 N KHSO4 and was extracted with ethyl ace-
tate (3 ꢃ 25 mL). The organic layers were combined and washed
with saturated citric acid solution, water and then with brine.
The solvent was removed and the crude product was purified by
silica gel (100–200 mesh) column chromatography and eluted with
50–70% ethyl acetate in petroleum ether to get the desired com-
pound as colorless oil. Yield: 92%; FTIR (Neat): 3300 (br), 2128
(w), 1717 (s), 1709 (s), 1702 (s), 1692 (s); dH (3 00 MHz, CDCl3):
6.21 (br s, 1H), 5.35 (br d, J = 7.2 Hz, 1H), 5.20 (br s, 1H), 4.68 (d,
J = 1.8 Hz, 2H), 4.29 (br s, 1H), 3.16–3.21 (m, 2H), 2.49 (s, 1H),
1.84 (br s, 1H), 1.66–1.74 (m, 1H), 1.43–1.58 (m, 13H); dC
(75 MHz, CDCl3): 176.1, 156.8, 155.7, 80.1, 78.2, 74.6, 53.1, 52.4,
40.6, 31.8, 29.1, 28.3, 22.2; m/z (HRMS): calcd for C15H24N2O6+Na:
351.1532; found: 351.1522.
4.6. Preparation of the tripeptide Boc-Phe-Lys(Poc)-Val-OMe (4)
The dipeptide Boc-Lys(Poc)-Val-OMe, (3) (0.442 g, 1 mmol) was
dissolved in CH2Cl2 (5 mL) and TFA (5 mL) was added. The reaction
mixture was stirred at room temperature (28 °C) for 30 min. CH2Cl2
and TFA were removed under vacuum and the crude trifluoroacetate
salt (3a) was used without further purification. Boc-Phe-OH
(0.265 g, 1 mmol) and the trifluoroacetate salt (3a) were dissolved
in CH3CN (10 mL). N-Methyl morpholine (0.33 mL, 3 mmol) and N-
hydroxy benzotriazole (0.135 g, 1 mmol) was added to the solution
and the solution was cooled to 0 °C. EDCꢀHCl (0.288 g, 1.5 mmol)
were added to the solution in small portions, and the reaction mix-
turewas allowedto attain roomtemperature (28 °C) and, wasstirred
for 4 h. Acetonitrile was removed under vacuum and the reaction
mixture was extracted with ethyl acetate (30 mL), washed with sat-
urated citric acid solution (20 mL), saturated Na2CO3 (20 mL ꢃ 2),
and brine (20 mL). Ethyl acetate was removed under vacuum and
the tripeptide was purified by silica gel (100–200 mesh) column
chromatography eluting with a solution of ethyl acetate (30–40%)
in petroleum ether. The tripeptide (4) was obtained as white solid
in 82% yield. Mp: 89 °C; FTIR (Neat): 3302 (br), 2126 (w), 1709 (s),
1646 (s); dH (300 MHz, CDCl3): 7.17–7.30 (m, 5H), 6.99 (br d,
J = 7.2 Hz, 1H), 6.92 (br d, J = 8.4 Hz, 1H), 5.41 (br t, J = 5 Hz, 1H),
5.26 (br d, J = 7.8 Hz, 1H), 4.66 (d, J = 2.4 Hz, 2H), 4.44–4.54 (m,
3H), 3.75 (s, 3H), 3.00–3.18 (m, 4H), 2.64 (t, J = 2.4 Hz, 1H), 1.75–
1.87 (m, 1H), 1.26–1.70 (m, 15H), 0.92 (t, J = 7.5 Hz, 6H); dC
(75 MHz, CDCl3): 172.2, 171.7, 171.3, 155.6, 155.4, 136.5, 129.2,
128.4, 126.7, 80.0, 78.4, 74.4, 57.3, 55.5, 52.8, 52.2, 40.3, 38.1, 31.7,
30.7, 28.9, 28.1, 21.9, 18.9, 17.7; m/z (HRMS): calcd for C30H44N4O8+-
Na: 611.3057; found: 611.3059.
4.4. Preparation of Boc-Lys(Poc)-OBn (2)
Boc-Lys(Poc)-OH (1) (1.64 g, 5 mmol) was dissolved in DMF
(10 mL). The solution was stirred and cooled to 0 °C. K2CO3
(0.76 g, 5.5 mmol) was added to the cold solution and the stirring
was continued for 15 min. Benzyl bromide (0.65 mL, 5.5 mmol)
was added to the cold solution and stirring was continued for 1 h
at 0 °C. The reaction mixture was allowed to come to room temper-
ature and the stirring was continued for additional 2 h. DMF was
removed under vacuum and the compound was extracted with
CH2Cl2, filtered and concentrated. The crude product was purified
by silica gel (100–200 mesh) column chromatography and eluted
with 20–30% ethyl acetate in petroleum ether to get the Poc deriv-
ative (2) as colorless oil. Yield: 96%; FTIR (Neat): 3342 (br), 3307
(br), 2126 (w), 1713 (s), 1710 (s), 1706 (s), 1700 (s); dH
(300 MHz, CDCl3): 7.36 (br s, 1H), 5.10–5.19 (m, 3H), 4.91 (br s,
1H), 4.66 (d, J = 2.1 Hz, 2H), 4.29–4.36 (m, 1H), 3.10–3.15 (m,
2H), 2.47 (t, J = 2.1 Hz, 1H), 1.28–1.79 (m, 15H); dC (75 MHz,
CDCl3): 172.6, 155.5, 125.3, 128.6, 128.4, 128.3, 125.3, 79.9, 78.3,
74.5, 67.0, 53.2, 52.3, 40.6, 32.2, 29.2, 28.3, 22.2; m/z (HRMS): calcd
for C22H30N2O6+Na: 441.2002; found: 441.2009.
4.7. Preparation of galactose derived azide (5)
The azide derivative (5) of galactose was prepared by previously
reported procedure29 as a colorless solid in 92% yield. Mp: 101–
102 °C (lit: mp: 96 °C); dH (300 MHz, CDCl3): 5.40 (br s, 1H), 5.15
(t, J = 10.4 Hz, 1H), 5.01 (br s, 1H), 4.58 (d, J = 8.5 Hz, 1H), 4.13–
4.17 (m, 2H), 3.99 (t, J = 5.9 Hz, 1H), 2.10 (s, 3H), 2.09 (s, 3H),
2.09 (s, 3H), 2.04 (s, 3H); dC (75 MHz, CDCl3): 170.3, 170.2, 169.8,
169.3, 88.2, 72.8, 70.7, 68.0, 66.8, 61.2, 20.7, 20.7, 20.6, 20.5. m/z
(HRMS): calcd for C14H19N3O9+Na: 396.1019; found: 396.1016.
4.5. Preparation of the dipeptide Boc-Lys(Poc)-Val-OMe (3)
A solution of Boc-Lys(Poc)-OH (1) (0.656 g, 2 mmol), HClꢀH-Val-
OCH3 (0.334 g, 2 mmol), N-methyl morpholine (0.66 mL, 6 mmol)
and N-hydroxy benzotriazole (0.270 g, 2 mmol) in CH3CN (20 mL)
was cooled to 0 °C and EDCꢀHCl (0.576 g, 3 mmol) was added in
small portions. The reaction mixture was brought to room temper-
ature (28 °C) and stirred for 4 h. Acetonitrile was removed under
vacuum and the reaction mixture was extracted with ethyl acetate
(50 mL), washed with saturated citric acid solution (30 mL), satu-
rated Na2CO3 (30 mL ꢃ 2), and brine (30 mL). Ethyl acetate was re-
moved under vacuum and the dipeptide was purified by silica gel
(100–200 mesh) column chromatography eluting with a solution
of ethyl acetate (30–40%) in petroleum ether. The dipeptide (3)
was isolated as a white solid in 86% yield. Mp: 78 °C; FTIR (Neat):
3313 (br), 2126 (w), 1714 (s), 1699 (s), 1683 (s), 1667 (s); dH
(300 MHz, CDCl3): 6.73 (br s, J = 9 Hz, 1H), 5.26 (br d, J = 7.8 Hz,
1H), 5.19 (br t, J = 6.6 Hz, 1H), 4.67 (d, J = 2.4 Hz, 2H), 4.53 (dd,
4.8. Preparation of the glucose derived azide (6)
The azide derivative (6) of glucose was prepared by earlier re-
ported procedure.30 Yield: 62% (overall); mp: 129 °C; FTIR (Neat):
2104 (s), 1749 (s); dH (300 MHz, CDCl3): 5.40 (d, J = 3.6 Hz, 1H),
5.34 (dd, J1 = 10.8 Hz, J2 = 3.3 Hz, 1H), 5.16 (dd, J1 = 11 Hz,
J2 = 3.6 Hz, 1H), 5.03 (d, J = 3.3 Hz, 1H), 4.11–4.15 (m, 1H), 3.45 (s,
3H), 3.15 (dd, J1 = 12.9 Hz, J2 = 3.9 Hz, 1H), 2.17 (s, 3H), 2.10 (s,
3H), 1.99 (s, 3H); dC (75 MHz, CDCl3): 170.3, 170.1, 169.8, 97.2,
69.0, 68.0, 67.9, 67.5, 55.6, 50.8, 20.7, 20.5; m/z (HRMS): calcd for
C13H19N3O8+Na: 368.1070; found: 368.1067.
4.9. Preparation of the thymidine derived azide (7)
Thymidine (0.484 g, 2 mmol) was dissolved in pyridine (5 mL)
and the solution was cooled to 0 °C. Tosyl chloride (0.42 g,

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2.2 mmol) was added to the cold solution with stirring. Stirring
was continued for 6 h and acetic anhydride (1 mL) was added to
the reaction mixture. The solution was stirred for additional 8 h
and the solution was diluted with CH2Cl2 (30 mL). The solution
of the O-tosyl derivative, was washed with 1 N HCl (3 ꢃ 30 mL)
and then with brine (30 mL). The product was purified by silica
gel (100–200 mesh) column chromatography eluting with metha-
nol in chloroform.
3.16 (m, 4H), 1.88–2.22 (m, 13H), 1.23–1.40 (m, 16H), 0.88–0.93
(m, 6H); dC (75 MHz, CDCl3): 172.2, 171.5, 171.1, 170.2, 169.9,
169.7, 168.9, 156.2, 155.5, 144.1, 136.4, 129.2, 128.5, 126.8,
122.2, 80.2, 73.9, 70.7, 67.9, 66.8, 61.1, 60.3, 57.5, 57.3, 55.6,
52.1, 40.4, 38.0, 31.7, 30.8, 29.6, 29.0, 28.1, 22.0, 20.9, 20.5, 20.1,
18.9; m/z (HRMS): calcd for C44H63N7O17+Na: 984.4178; found:
984.4122.
The O-tosyl derivative of thymidine (0.438 g, 1 mmol) was dis-
solved in DMF (5 mL) and sodium azide (0.98 g, 1.5 mmol) was
added to the solution with stirring. The reaction mixture was al-
lowed to stir till the complete disappearance of starting material
in TLC. DMF was removed under vacuum and the products were
extracted with CH2Cl2 (25 mL). The solvent was removed under
vacuum and the product was purified by silica gel (100–200 mesh)
column chromatography eluting with CHCl3/MeOH and the azide
derivative (7) was isolated as a colorless liquid. Yield: 76% (over-
all); FTIR (Neat): 3186 (br), 2105 (s), 1737 (s), 1703 (s), 1693 (s);
dH (300 MHz, CDCl3): 9.78 (br s, 1H), 7.39 (s, 1H), 6.35 (dd,
J1 = 8.4 Hz, J2 = 5.6 Hz, 1H), 5.20 (d, J = 6 Hz, 1H), 4.11 (d, J = 2 Hz,
1H), 3.71–3.79 (m, 2H), 2.39–2.48 (m, 1H), 2.21–2.29 (m, 1H),
2.12 (s, 3H), 1.96 (s, 3H); dC (75 MHz, CDCl3): 170.6, 163.8, 150.6,
134.8, 111.8, 84.3, 74.5, 52.5, 36.9, 20.8, 12.6; m/z (HRMS): calcd
for C12H15N5O5+Na: 332.0971; found: 332.0968.
4.12.2. Compound 11
White solid; mp: 67.8 °C; yield: 88%; FTIR (Neat): 3313 (br), 1751
(s), 1720 (s), 1655 (s), 1648 (s); dH (300 MHz, CDCl3): 7.78 (s, 1H),
7.17–7.27 (m, 5H), 6.74 (br d, J = 6 Hz, 1H), 6.60 (br d, J = 8.1 Hz,
1H), 5.38–5.51 (m, 4H), 5.17 (s, 1H), 4.83–4.90 (m, 3H), 4.36–4.58
(m, 5H), 4.17 (t, J = 7.2 Hz, 1H), 3.7 (s, 3H), 3.11 (m, 6H), 2.95–3.00
(m, 1H), 2.15–2.18 (m, 1H), 2.09 (s, 3H), 2.06 (s, 3H), 2.01, (s, 3H),
1.75–1.80 (m, 1H), 1.60–1.64 (m, 1H), 1.46 (br s, 2H), 1.36 (s, 9H),
1.26–1.32 (m, 2H), 0.93 (d, J = 5.2 Hz, 3H), 0.91 (d, J = 5.2 Hz, 3H);
dC (75 MHz, CDCl3): 172.2, 171.6, 171.3, 170.0, 169.8, 156.2, 155.4,
143.5, 136.5, 129.2, 128.4, 126.7, 125.2, 96.4, 79.9, 70.6, 69.9, 69.6,
67.6, 57.5, 57.3, 55.3, 52.8, 52.1, 50.6, 40.3, 38.1, 31.7, 30.7, 29.0,
28.1, 22.0, 20.5, 18.9, 17.8; m/z (HRMS): calcd for C43H63N7O16+Na:
956.4229; found: 956.4233.
4.12.3. Compound 12
Yield: 86%; FTIR (Neat): 3788 (br), 3301 (br), 1703 (s), 1693 (s);
dH (400 MHz, CDCl3): 7.75 (s, 1H), 7.16–7.28 (m, 5H), 7.10 (m, 1H),
6.60 (br s, 1H), 6.16–6.18 (m, 1H), 5.09–5.35 (m, 5H), 4.73–4.79 (m,
2H), 4.68 (d, J = 12 Hz, 1H), 4.51 (m, 5H), 4.29 (d, J = 3 Hz, 1H), 4.11
(dd, J1 = 6 Hz, J2 = 9 Hz, 1H), 3.74 (s, 3H), 2.74–3.13 (m, 6H), 2.13 (s,
3H), 2.04 (s, 2H), 1.91 (s, 3H), 1.36–1.40 (m, 11H), 1.23–1.28 (m,
2H), 0.91 (t, J = 9 Hz, 6H); dC (100 MHz, CDCl3): 172.8, 172.5,
172.8, 172.1, 171.7, 171.5, 171.1, 170.5, 156.2, 155.4, 150.2,
136.5, 135.5, 135.3, 129.3, 128.6, 128.5, 126.9, 126.8, 126.1,
111.9, 81.7, 80.4, 80.2, 80.1, 60.3, 57.8, 57.3, 52.2, 50.8, 40.4, 38.2,
35.8, 30.9, 29.0, 28.2, 22.1, 21.0, 20.7, 18.9, 17.8, 14.1, 12.4. m/z
(HRMS): calcd for C42H58N8O14+Na: 921.3970; found: 920.4091.
4.10. Preparation of the mannose derived azide (8)
The azide derivative, 8 (Scheme 3) of mannose was prepared
using a previously reported method.31
4.11. Procedure for the synthesis of the lysine–thymidine
conjugate (9)
The Boc-Lys(Poc)-OBn derivative (2; Scheme 2) (0.5 mmol) and
the azide derivative of thymidine (7; Scheme 3) (0.5 mmol) were
dissolved in tert-butanol (5 mL) and H2O (10 mL). The solution
was stirred well and CuSO4ꢀ5H2O (2 mg, 0.005 mmol) and sodium
ascorbate (5 mg, 0.025 mmol) were added. The reaction mixture
was stirred at room temperature, till the complete disappearance
of the starting materials in TLC. Saturated brine (20 mL) was added
to the reaction mixture and the addition product was extracted
with ethyl acetate (3 ꢃ 35 mL). The solution was dried over anhy-
drous Na2SO4 and concentrated. The product was purified through
a column of silica gel (60–120 mesh) with ethyl acetate. White
crystalline solid; mp: 63.7 °C; Yield: 90%; FTIR (Neat): 3343 (br),
1702 (s), 1698 (s); dH (300 MHz, CDCl3): 10.07 (s, 1H), 7.75 (s,
1H), 7.35 (br s, 5H), 6.82 (s, 1H), 6.20 (br t, J = 6.0 Hz, 1H), 5.10–
5.30 (m, 8H), 4.96 (br s, 1H), 4.30–4.32 (m, 2H), 4.12 (q,
J = 7.5 Hz, 2H), 2.31–2.38 (m, 2H), 2.12 (s, 3H), 1.90 (s, 3H), 1.77
(br s, 1H), 1.64 (br s, 1H), 1.43 (br s, 13 H); dC (75 MHz, CDCl3):
172.6, 170.6, 163.4, 156.1, 155.4, 150.2, 146.8, 143.6, 135.3,
128.5, 128.4, 128.2, 125.7, 111.8, 85.0, 81.8, 79.9, 74.1, 66.9, 57.7,
53.2, 51.0, 40.5, 35.9, 32.1, 29.1, 28.2, 22.2, 20.7, 12.4; m/z (HRMS):
calcd for C34N45H7O11+Na: 750.3075; found: 750.3074.
4.12.4. Compound 13
Yield: 85%; FTIR (Neat): 3308 (br), 1714 (s), 1561 (s); dH
(300 MHz, CDCl3): 7.29 (s, 1H), 7.28–7.21 (m, 6H), 7.18 (br, 1H),
7.16 (br s, 1H), 6.09 (d, J = 3 Hz, 1H), 5.19 (s, 3H), 4.84–4.96 (m,
1H), 4.43–4.52 (m, 3H), 3.99–4.15 (m, 2H), 3.74 (s, 3H), 2.05–
2.17 (m, 1H), 1.55 (d, J = 6 Hz, 2H), 1.46 (s, 1H), 1.44 (s, 1H), 1.39
(s, 9H), 0.93–0.86 (m, 6H); dC (75 MHz, CDCl3): 172.2, 171.5,
171.2, 156.2, 155.4, 136.5, 129.8, 129.2, 128.5, 126.8, 113.8,
109.5, 88.8, 88.0, 79.6, 79.3, 79.1, 78.2, 76.5, 72.5, 69.5, 66.7,
60.3, 57.6, 57.4, 55.7, 52.9, 52.1, 40.4, 38.1, 31.6, 30.8, 29.6, 29.0,
28.9, 28.1, 26.9, 25.4, 25.0, 24.0, 22.0, 18.9, 17.8, 14.1; m/z (HRMS):
calcd for C42H63N7O13+Na: 896.4382; found: 896.4380.
4.13. Purification of recombinant P. falciparum Sir2 (PfSir2)
PfSir2 (PF13_0152) gene was cloned into pET 28b vector and ex-
pressed in BL21 (DE3) strain of E. coli as a C-terminal His-tagged fu-
sion protein. The protein was purified as reported earlier.17
Purification involved the incubation of cell lysate with Ni-NTA beads
followedby elution of bound PfSir2 with different imidazole concen-
trations. The fractions were checked on SDS–PAGE,42 pooled, dia-
lyzed against 10 mM Tris–HCl, pH 8.0, 20% glycerol, 500 mM NaCl
and 2 mM DTT and, concentrated. Protein concentration was esti-
mated by the method of Bradford using BSA as standard.43
4.12. Synthesis of the tripeptide conjugates (10–13)
The reactions between N-Poc lysine tripeptide derivative (4;
Scheme 2) and the azide derivatives (5–8; Scheme 3) were per-
formed as mentioned above.
4.12.1. Compound 10
Yield: 85%; FTIR (Neat): 3319 (br), 1747 (s), 1651 (s); dH
(300 MHz, CDCl3): 7.94 (s, 1H), 6.79–7.29 (m, 5H), 6.79–6.93 (m,
2H), 5.87 (d, J = 10 Hz, 1H), 5.55 (t, J = 9 Hz, 2H), 5.13–5.38 (m,
5H), 4.42–4.48 (m, 2H), 4.08–4.27 (m, 3H), 3.71 (s, 3H), 2.96–
4.14. Deacetylation assay
A FRET based assay was used to measure PfSir2 activity. A syn-
thetic peptide containing an acetylated lysine group and a donor–

S. P. Chakrabarty et al. / Bioorg. Med. Chem. 17 (2009) 8060–8072
8071
acceptor pair (EDANS–DABCYL) was used as substrate. Upon
deacetylation by Sir2, the peptide becomes a substrate for cleavage
by trypsin resulting in separation of the donor and acceptor pair
and thereby, an increase in the fluorescence intensity. For activity
plotted graphically against the inhibitor concentrations and linear
regression analyses were carried out using GraphPad Prism, ver-
sion 4. Both NAM and 11 concentrations were varied from 5 to
150 lM and, all assays were done at 37 °C temperature.
measurements, 100
Tris–HCl, pH 8.0, 10
l
l
L of a reaction mixture containing 5 mM
M trichostatin A, 75 ng trypsin and 3
lM
4.17. Cytotoxicity assay
PfSir2 were used. All assays were carried out at 37 °C on a Hitachi
F-2500 spectrofluorimeter (Hitachi, High-Technologies Corpora-
tion, Tokyo, Japan) fitted with a water circulated cell holder. The
temperature was maintained using JULABO F25 (JULABO Labor-
technik GmbH, Seelbach, Germany) water bath. Unit activity was
calculated by estimating total enhancement in fluorescence inten-
sity on complete digestion of peptide by treating the substrate
with Proteinase K, a non-specific protease. The purified enzyme
was found to have a specific activity of 47 nmol mgꢂ1 minꢂ1. To
monitor the effect of 1, 3, and 9–13 on PfSir2 activity, the enzyme
was preincubated with the required concentrations of the inhibi-
tors for 5 min prior to addition to the assay mixture.
The 3D7 strain of P. falciparum was cultivated in vitro using the
method described by Trager and Jensen.44 Parasites were main-
tained in human O+ erythrocytes isolated from blood collected
from healthy volunteers, at 5% hematocrit in RPMI 1640 (Sigma
Chemical Co., St. Louis, USA) containing 10% human serum. The
antiplasmodial activity was measured using previously reported
methods.45 Compound 11 was dissolved in dimethyl sulfoxide
(DMSO) and the compound serially diluted twofold over the con-
centration range 400 to 0.2 lM. Each well contained 250 lL of cell
suspension at 2% parasitemia and 2% hematocrit. Parasitized and
non-parasitized erythrocytes and solvent controls were incorpo-
rated in all the tests. The plates were incubated at 37 °C in a candle
4.15. Inhibition kinetics
jar. After 24 h, each well was pulsed with 10
lL of PBS containing
1.0
l
Ci of [3H] hypoxanthine, and the plates incubated for another
Inhibition patterns were determined by measuring the initial
reaction velocity at varying concentration of one substrate, fixed
concentration of the second substrate and different fixed concen-
trations of the inhibitor.32 To obtain values for the kinetic param-
eters the data were analyzed by non-linear regression analysis.
Lineweaver–Burk (LB) and related secondary plots were con-
structed for each inhibitor and variable substrate and, the pattern
examined. Eqs. 5–7 describe competitive, uncompetitive, and non-
competitive inhibition, respectively.
12 h. The contents of each well were then harvested onto glass fi-
ber filters using a Combi-12 automated cell harvester (Molecular
Devices, Sunnyvale, CA), washed extensively with distilled water
and dried. The radioactivity incorporated in the nucleotide pool
was measured as disintegrations per minute using a Wallac 1409
(Wallac Oy, Turku, Finland) liquid scintillation counter. The exper-
iment was done twice in duplicate. For test samples the percent
radioactivity incorporated with respect to the control was plotted
against the logarithm of the drug concentration. The concentration
causing 50% inhibition of radioisotope incorporation (IC50) was
determined by interpolation. A parallel experiment by microscopy,
using Giemsa-stained smears, was also conducted.
m
m
m
¼ Vmax½Sꢁ=ðKmð1 þ ½Iꢁ=KiÞ þ ½SꢁÞ
ð5Þ
ð6Þ
ð7Þ
¼ Vmax½Sꢁ=ðKm þ ½Sꢁð1 þ ½Iꢁ=KiÞÞ
¼ Vmax½Sꢁ=ðKmð1 þ ½Iꢁ=KiÞ þ ½Sꢁð1 þ ½Iꢁ=KiÞÞ
where Km is the Michaelis constant for the variable substrate, Ki is
the inhibition constant, [S] is the variable substrate concentration,
and [I] is the inhibitor concentration. The above equations are valid
only when enzyme binds to one molecule of inhibitor. When two
molecules of inhibitor are involved in the binding process with en-
zyme alone or with enzyme–substrate complex, the equations be-
come second order polynomial with respect to the inhibitor.32
Under these conditions, the equations for competitive and non-
competitive inhibition are,
Acknowledgements
This project was supported by grants from Council of Scientific
and Industrial Research (CSIR) and CSIR-NMITLI, Govt. of India. We
thank Y. K. Saikumari for providing us with the synthetic peptide
substrate. We acknowledge M. P. Bopanna for parasite assay and
Anubhab Roy (Engineering Mechanics Unit, JNCASR) for the gener-
ous help and clarifications on equations used for curve fitting. We
thank Bharath Srinivasan for the extensive time spent on
discussion.
2
m
m
¼ Vmax½Sꢁ=ðKmð1 þ ½Iꢁ=KiÞ þ ½SꢁÞ
¼ Vmax½Sꢁ=ðKmð1 þ ½Iꢁ=KiÞ2 þ ½Sꢁð1 þ ½Iꢁ=KiÞ Þ
ð8Þ
ð9Þ
2
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